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Bee Products Properties, Applications, and Apitherapy

Bee Products Properties, Applications, and Apitherapy

Edited by

Avshalom Mizrahi The Israeli College of Complementary Medicine Tel Aviv, Israel

and

Yaacov Lensky Triwaks Bee Research Center The Hebrew University Rehovot, Israel

Springer Science+Business Media, LLC

Library of Congress Catalog1ng-1n-PublIcatlon Data

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96-51895 CIP

Proceedings of an International Conference on Bee Products: Properties, Applications, and Apitherapy, held May 2 6 - 3 0 , 1996, in Tel Aviv, Israel

ISBN 978-1-4757-9373-4 DOI 10.1007/978-1-4757-9371-0

ISBN 978-1-4757-9371-0 (eBook)

© Springer Science+Business Media New York 1997 Originally published by Plenum Press, New York in 1997 Softcover reprint of the hardcover 1st edition 1997 http://www.plenum.com All rights reserved 109 8 7 6 5 4 3 2 1 No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

PREFACE

The nature .and diversity of presentations at the conference on: "Bee Products: Properties, Applications and Apitherapy" held at Tel-Aviv on May 26--30, 1996, emphasize the increasing interest of physicians, practitioners, scientists, herbalists, dieticians, cosmeticians, microbiologists, and beekeepers in different facets of bee products. This volume consists of a selection of 31 contributions presented at the conference and which provide information on the present status of our knowledge in this area. In spite of their diversity, they reflect the mainstream of the conference, namely: "Imported" Products (honey, pollen and propolis), Exocrine Secretions of Workers (venom, royal jelly). Toxicity and Contaminants, Quality Control, Marketing, Apitherapy, Cosmetics, etc. Since antiquity, honey as well as other bee products were used as food, as a cure for ailments of humans and animals, and as cosmetics. We hope that this volume will contribute to interdisciplinary studies on chemical composition, pharmacological effects, nutrition, and other aspects of bee products. Critical and unbiased experimental research may unravel the yet unknown composition and mode of action of bee products and elucidate many unanswered questions. The noteworthy features of this conference were the participants from all parts of the world and of different cultural backgrounds, who shared their keen interest and curiosity regarding honey bees and their products. We thank all of them for their personal contribution to the success of this conference. Avshalom Mizrahi Yaacov Lensky Editors

v

THE CONFERENCE ON BEE PRODUCTS

The Conference was organized by: The Israeli Honey Production and Marketing Board and The Israeli Beekeepers' Associations and in informal alliances with: • • • • • • •

American Apitherapy Society Apimondia - The International Federation of Beekeeping Association Asian Apicultural Association International Bee Research Association Israeli Dietetic Association Ministry of Agriculture, State ofIsrael Ministry of Tourism, State of Israel

Local Organizing Committee Avshalom Mizrahi, Ph.D. (Chairman) Yaacov Lensky, Ph.D. (Vice Chairman) Moshe Almaliah, M.Sc. Tsila Dvir, M.Sc. Abraham Hefez, Ph.D. Anatol Karakowsky, M.D. Yanay Sachs David Sadeh Yeshayahu Stem, M.Sc. Boris Yakobson, D.V.M. International Advisory Committee Stefan Bogdanov, Ph.D. (Switzerland) Raymond Borneck, President, Apimondia (France) Kate Chatot (U.S.A.) Theodore Cherbuliez, M.D., President AAS (U.S.A.) Zhibin Lin, M.D. (China) Charles Mraz (U.S.A.) Tetsuo Sakai, Ph.D., President, AAA (Japan) Mira Spitzer-Adir (Croatia) Artur Stojko, Ph.D. (Poland) Bradford S. Weeks, M.D. (U.S.A.) Siriwat Wangsiri, Ph.D. (Thailand)

vii

CONTENTS

1. The Past and Present Importance of Bee Products to Man Eya Crane 2. Bee Products: Chemical Composition and Application . . . . . . . . . . . . . . . . . . . . . Justin o. Schmidt

15

3. Honey as an Antimicrobial Agent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. C. Molan

27

4. Non-Peroxide Antibacterial Activity of Honey Stefan Bogdanov

39

5. Antioxidant Properties of Honey Produced by Bees Fed with Medical Plant Extracts ..................................................... Gennady Rosenblat, Stephane Angonnet, Alexandr Goroshit, Mina Tabak, and Ishak Neeman 6. Speeding Up the Healing of Burns with Honey: An Experimental Study with Histological Assessment of Wound Biopsies ........................ Th. J. Postmes, M. M. C. Bosch, R. Dutrieux, J. van Baare, and M. J. Hoekstra

49

57

7. The Effect of Honey on Human Tooth Enamel and Oral Bacteria S. R. Grobler and N. 1. Basson

65

8. Honey Contact with Teeth in Situ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Gedalia, S. R. Grobler, I. Grizim D. Steinberg, L. Shapira, I. Lewinstein, and Mo. Sela

73

9. Medicinal Herbs as a Potential Source of High-Quality Honeys. . . . . . . . . . . . . . Zohara Yaniv and Michal Rudich

77

10. The Unique Properties of Honey as Related to Its Application in Food Processing Tsila Dvir

83

11. Honey as a Clarifying and Anti-browning Agent in Food Processing and a New Method of Mead Production. .. .. . . .. .. . . .. . . . . . . . .. . .. . . . . . . . . . . ChangY. Lee

89

ix

x

Contents

12. Bee-Pollen: Composition, Properties, and Applications M. G. Campos, A. Cunha, and K. R. Markham

93

13. Clinical Evaluation ofa New HypoaUergic Formula of Pro polis in Dressings. . . W. Fierro Morales and 1. Lopez Garbarino

101

14. Present State of Basic Studies on Propolis in Japan. . . . . . . . . . . . . . . . . . . . . . . . Tsuguo Yamamoto

107

15. The Usage and Composition of Propolis Added Cosmetics in Korea Park Jong-Sung and Woo Kun-Suk

121

16. Eucalyptus Propolis Beverages with Their Composition and Effects Woo Kun-Suk and Park Jong-Sung

125

17. An Inhibitory Effect of Propolis on Germination and Cell Division in the Root Tips of Wheat Seedlings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Sorkun, S. Bozcuk, A. N. Gomiirgen, and F. Tekin

129

18. The Exocrine Glands of the Honey Bees: Their Structure and Secretory Products Pierre Cassier and Yaacov Lensky

137

19. Alarm Pheromones of the Queen and Worker Honey Bees (Apis mellifera L.) Yaacov Lensky and Pierre Cassier

151

20. Protein Traffic between Body Compartments of the Female Honey Bee (Apis melli/era L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yoseph Rakover and Yaacov Lensky

161

21. Effects of Feeding, Age of the Larvae, and Queenlessness on the Production of Royal Jelly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuray Sahinler and Osman Kaftanoglu

173

22. The Use of Royal Jelly during Treatment of Childhood Malignancies Osman Kaftanoglu and Atilla Tanyeli

179

23. The Role of Hymenopterous Venoms in Nature Eli Zlotkin .

185

24. Effect of Apamin and Melittin on Ion Channels and Intracellular Calcium of Heart Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. G. Bkaily, M. Simaan, D. Jaalouk, and P. Pothier

203

25. Bee Venom in Treatment of Chronic Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . .. Th. Cherbuliez

213

26. Apitherapy in Orthopaedic Diseases Franco Feraboli

221

27. The Monitoring of Possible Biological and Chemical Contaminants in Bee Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Boris A. Yakobson

227

Contents

28. Heavy Metals in Propolis: Practical and Simple Procedures to Reduce the Lead Level in the Brazilian Propolis ................................... Nivia Macedo Freire Alcici

xi

231

29. Acaricide Residues in Beeswax and Honey. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Bogdanov, V. Ki1chenmann, and A. Imdorf

239

30. Judging the Quality of Honey by Sensory Analysis. . . . . . . . . . . . . . . . . . . . . . .. Michel Gonnet

247

31. Methods for the Characterization of the Botanical and Geographical Origin of Some Bee Products and for Their Quality Control . . . . . . . . . . . . . . . . . . .. Giancarlo Ricciardelli D'Albore Index

253 263

Bee Products Properties, Applications, and Apitherapy

1

THE PAST AND PRESENT IMPORTANCE OF BEE PRODUCTS TO MAN Eva Crane International Bee Research Association Woodside House, Woodside Hill, Gerrards Cross Bucks SL9 9TE United Kingdom

1. THE BEES FROM WHICH PRODUCTS ARE HARVESTED At this Conference we are considering the products of social bees, which beekeepers harvest from them. Candidate bees (Table I, Figure I) are: first, all the honey bees: Apis melli/era from Europe, eastern Mediterranean lands and Africa; Apis cerana the hive bee in Asia, and Apis dorsata, Apis jlorea and related species in the tropics of Asia. Second, in the tropics of all continents there are stingless bees (Meliponinae), some 500 species in all. In addition, honey-but not wax-is produced by colonies of honey wasps (Vespidae) and honey ants (Formicinae) and is harvested from them. The wasps live in parts oftropical South America, and the ants in some dry areas of Australia and North America. What we now think of as bee products were essential to the bees for their survival and development during and after the evolutionary period: this was and is their function. Stingless bees and honey bees evolved roughly 100 million and 50 million years ago, respectively, whereas man has existed to use the products for only I or 2 million years-a tiny fraction as long as social bees. The earliest records of man's harvesting from bees' nests are in the Mesolithic rock art of Europe and Asia, painted not more than 8000 years ago (Figures 2 and 3). There are also rock paintings in Australia showing stingless bee nests. Man used bee products in many ways: beeswax in various technologies, and honey as food and also in medicine and as offerings to the gods he worshipped. Man also had ideas about the origins of the various bee products, and attributed certain properties to them. But their true origins were not known until a few centuries ago, and their detailed chemical compositions were determined only in the late 1900s. I shall say most about honey, and then deal with other products: beeswax, propolis, rollen, bee brood, bee venom and royal jelly. Finally, I shall discuss changes in the importance of the various bee products during the period when man has been harvesting them.

E. Crane

2

Table 1. Substances collected or produced by certain social insects Insect

Where native

Honey

Wax

Prop.

Pollen

xx xx xx xx xx x x

xx xx xx xx xx

xx

xx x x x x

Brood Venom

Rj

Honey bees (Apis) A. melli/era

Old World Europe & E. Mediterranean; Africa A. cerana Asia Asia, tropics A. dorsata A.jlorea Asia, tropics Stingless bees (Meliponinae) tropics S. America, tropics Honey wasps Parts of Australia & N. Honey ants America

x x x

xx x x x x x x

xx x x x

xx x x x

x Collected or produced by the insects. xx Known to be commercially harvested and marketed by man.

2. HONEY The earliest known written records' of the use of honey by man relate to religious sacrifices in various regions; indeed honey may well have been one of the earliest nonanimal sacrifices. It was sometimes offered together with milk, or butter or ghee, oil, or incense. According to inscriptions on clay cylinders from Sumer in Mesopotamia, when the foundations of a new temple for the god Ningirsu were laid about 2500 Be, Gudea the ruler of Lagash made offerings of honey and butter. Then, when the image of the god was finally erected, he offered honey with other foods. The use of honey as an offering probably had a still older origin, because other inscriptions show that it was already customary by Gudea's time. In Ancient Egypt much honey was sacrificed in religious ceremonies, and when Israelites later presented the first harvest of their produce to God, this included honey. For instance in Jerusalem at the time of Hezekiah in the late 700s Be, 'they gave generously from the first fruits of their corn and new wine, oil and honey, .. .' (II Chronicles 31.5). Honey had, however, been forbidden as a burnt offering around 1300 Be: 'You shall not burn any leaven or any honey as a food-offering to the Lord' (Leviticus 2.11). What may be the earliest recorded medical prescription that includes honey is also from Sumer, dated to about 2000 Be. Oil was to be spread over a preparation of river dust kneaded with honey, water, and other ingredients. This was presumably for external application, and many Ancient peoples used honey in this way2. In the Ebers papyrus compiled in Egypt about 1550 Be, I found honey in 147 prescriptions for external use, and in 102 for internal use, both out of a total of several hundred. For internal use, honey was sometimes included because of its own properties, sometimes as a binder, and often to disguise the taste of other, unpalatable ingredients. The Roman poet Lucretius (c. 99-55 BC) referred to this use of honey: Physician-like, who when a bitter draught Of wormwood is disgusted by a child To cheat his taste, he brims the nauseous cup With the sweet lure of honey. • Historial records cited will be detailed in a forthcoming book on the history of man's use of bees, to be published by Duckworth in London.

45·

0°

23}·

,

~

A

Figure 1. Natural distribution of spec ies of bees kept in hives' , - - - - Apis melli/era; - , - , -Apis cera lla; Shaded areas: Meliponinae,

./:'10% >10% >10% >10% >10%

34

P. C. Molan

opsies of gastric ulcers. All five isolates tested were found to be sensitive in an agar well diffusion assay to a 20% (v/v) solution of a manuka honey with an average level of nonperoxide activity, but none showed sensitivity to a 50% (v/v) solution of a honey in which the antibacterial activity was due primarily to its content of hydrogen peroxide. Assessment of the minimum inhibitory concentration by inclusion of manuka honey in the agar showed that the growth of all of a further seven isolates tested was completely inhibited over the incubation period of 72 h by the presence of 5% (v/v) honey.

3.5. Honey for the Treatment of Gastroenteritis Honey has been found to be effective in treating bacterial gastroenteritis in infants 43 . Used in place of glucose in an oral re-hydration fluid, it was found to be as effective as glucose in achieving re-hydration, whilst the antibacterial activity cleared the infection in bacterial diarrhoea. However, there is little information available on the sensitivity of the gastroenteritis-causing species of bacteria to the antibacterial activity of honey, and on which of the antibacterial factors in honey is most effective against them. Therefore honey was tested for its relative antibacterial potency against all the bacterial species that commonly cause gastroenteritis, comparing manuka honey and a honey with the usual hydrogen peroxide activity, also an artificial honey to assess how much of the antibacterial activity was due simply to the acidity and the osmotic effect of the sugar in hone/ 4 , With some of the species of bacteria the assessment was repeated with additional strains obtained from clinical isolates supplied by medical and animal health laboratories to see if there was any variation in sensitivity between different strains of a species. Cultures of the bacteria were streaked on nutrient agar plates containing various concentrations of the honeys, and the growth of the bacteria was assessed to find the concentration of honey that was necessary to prevent growth of the bacteria. The honeys used were a mixed pasture honey with an average level of activity due to hydrogen peroxide and no detectable non-peroxide activity, and a man uk a honey with an average level of non-peroxide activity. Honey concentrations were in a 5% (v/v) step dilution series initially and then with I % dilution steps, the honey being diluted with either sterile distilled water (for the pasture honey and artificial honey) or a sterile solution of 0.2% catalase (for the manuka honey). Plates where inhibition of growth was observed were swabbed with a loopful of sterile water and streaked onto freshly prepared nutrient agar plates which did not contain honey. The plates were then incubated to find any surviving bacteria growing into visible colonies if the initial inhibition had been due to prevention of growth (bateriostasis) rather than killing the bacteria (bactericidal activity). The results, summarised in Table 4, showed that honey with an average level of hydrogen peroxide activity is bacteriostatic at 4-8% (v/v) and bactericidal at 5-10% (v/v). The non-peroxide activity of an average manuka honey is bacteriostatic at 5-11 % (v/v) and bactericidal at 8-15% (v/v). Activity (just bacteriostatic) was not seen with artificial honey unless it was at 20-30% (v/v), clearly showing the importance of factors other than sugar and acidity,

3.5. Honey for the Treatment of Tineas Honey has been reported to have antifungal activity, but not many species of fungi have been tested. An important group of fungi which regularly infect humans are the dermatophytes (Deuteromycotina). Cutaneous or superficial mycoses, caused through host infection by these fungi, are one of the most common diseases of humans. Only a small

Honey as an Antimicrobial Agent

35

Table 4. Minimum inhibitory concentration of honeys in nutrient agar plates (% v/v) giving partial inhibition (PI), bacteriostatic activity (BS) and bactericidal activity(BC) against various strains of bacteria which cause gastroenteritis Manuka honey with catalase PI

Bacterial strain Escherichia coli 916 Escherichia coli ex AHL Escherichia coli K88+ Salmonella enteritis 3484 Salmonella hadar 326 Salmonella infantis 93 Salmonella typhimurium 298 Salmonella typhimurium 1739 Salmonella typhimurium ex WH Shigella boydii 2616 Shigella jlexneri 983 Shigella sonnei 86 Shigella sonnei ex WH Vibrio cholorae Vibrio paraheamolyticus Yersinia enterocolitica

6% 6% 6% 7% 6% 7% 6% 6% 6% 6% 6% 5% 5% 5% 10%

BS

7% 7% 7% 8% 7% 8% 7% 7% 5% 7% 7% 7% 6% 7% 6% 11%

Pasture honey

BC

10% 10% 10% 10% 8% 10% 8% 9% 10% 10% 10% 10% 10% 10% 10% 15%

BS

PI

5% 4% 6%

6% 7%

6% 6% 6% 5% 6% 7% 6% 6% 5% 5% 6% 5% 6% 7% 4% 8%

BC

6% 6% 6% 6% 6% 10% 8% 7% 10% 6% 6% 5% 10% 10% 6% 9%

number of species of these, from the genera Epidermophyton , Microsporum and Trichophyton, regularly infect humans 45 • Superficial fungal infections are amongst the most difficult diseases to successfully treat, antibiotics which successfully combat bacterial diseases being largely ineffective against fungi. A common predisposition to some fungal infections is poor host immunity, thus bacterial infections may also be present quite often. So a treatment which has both antifungal and antibacterial activities would be most beneficial. Therefore the effectiveness of honey against the dermatophyte species which most frequently cause superficial mycoses (tineas such as ringworm and athletes foot) was invest igated46 • For this investigation two sorts of natural honey were used: a mixed pasture honey with an average level of antibacterial activity due to hydrogen peroxide production, and a manuka honey with an average level of non-peroxide antibacterial activity. An artificial honey was also used, to assess how much of the antibacterial activity was due simply to the acidity and the osmotic effect. The honeys were tested against clinical isolates of seven species of dermatophytes. An agar well diffusion assay was used, the contents of the wells being replaced with freshly prepared honey solutions at 24 hour intervals over the 3 - 4 days of incubation. The honeys were diluted with either sterile distilled water or a sterile solution of 0.2% catalase, a 5% (v/v) step dilution series being used for testing. The results are summarised in Table 5. No inhibitory activity was detected with any of the seven species with the pasture honey at any concentration up to the highest tested, 50% (v/v), when catalase was present, nor with the artificial honey even at 100%. This showed that it was the the hydrogen peroxide in the pasture honey, and the non-peroxide activity in the manuka honey, that were inhibiting the growth of the fungi. Although the concentrations of honey needed to inhibit some of the dermatophytes are higher than needed to inhibit bacteria, less dilution of the honey is likely with a tinea than with infected wounds, bums and ulcers where there would be serum exudation. It could be that

36

P. C. Molan

Table 5. Minimum inhibitory concentration of honeys in agar wells (% v/v) giving a clear zone around the wells in an agar well diffusion assay against seven species of fungi which cause tineas Species

Epidermophyton jloccosum Microsporum canis Microsporum gypseum Trichophyton rubrum Trichophyton tonsurans T. mentagrophytes var. interdigitate T. mentagrophytes var. mentagrophytes

Pasture honey

Manuka honey

Manuka honey with catalase

5-10% 10-15% 15-20% 2.5-5% 15-20% 10-15% 10-15%

5-10% 20-25% 45-50% 5-10 20-25% 20-25% 15-20%

20-25% 20-25% 50-55% 15-20% 20-25% 40-45% 20-25%

manuka honey may be more effective, even though the dermatophytes are less sensitive to its activity than they are to hydrogen peroxide, if there is insufficient dilution of honey on tineas for the enzymic production of hydrogen peroxide to be activated. Which type of honey is most effective, and the practical usefulness of honey as a topical antifungal salve, will only be known if comparative clinical trials are conducted.

4. REFERENCES I. Dustmann J H. (I 979) Antibacterial Effect of Honey. Apiacta 14, 7-11. 2. Majno G: The Healing Hand. Man and Wound in the Ancient World. Harvard University Press Cambridge, Massachusetts. 1975. 3. Ransome H M: The Sacred Bee in Ancient Times and Folklore. George Allen and Unwin London. 1937. 4. Molan PC. (1992) The Antibacterial Activity of Honey. I. The Nature of the Antibacterial Activity. Bee World 73, 5-28. 5. Molan PC. (\992) The Antibacterial Activity of Honey. 2. Variation in the Potency of the Antibacterial Activity. Bee World 73,59-76. 6. Aristotle (350 B.C.). Translated by Thompson D'A W. Historia Animalium in: The Works of Aristotle (Smith J A, Ross W D editors) Oxford University Press Oxford 1910 Volume IV. 7. Gunther R T: The Greek Herbal of Dioscorides (Translated by Goodyear J, 1655). Hafner N. Y. 1934, reprinted 1959. 8. Allen K L, Molan PC, Reid G M. (\ 991) A Survey of the Antibacterial Activity of Some New Zealand Honeys. J. Pharm. Pharmacol. 43, 817-822. 9. Allen K L, Molan P C, Reid G M. (1991) The Variability of the Aantibacterial Activity of Honey. Apiacta 26, 114--121. 10. Zumla A, Lulat A. (\ 989) Honey - a Remedy Rediscovered. J. Royal Soc. Med. 82, 384--385. II. Bulman M W. (\ 955) Honey as a Surgical Dressing. Middlesex Hosp. J. 55, 188-189. 12. Hutton D J. (1966) Treatment of Pressure Sores. Nurs. Times 62,1533-1534. 13. Cavanagh D, Beazley J, Ostapowicz F. (1970) Radical Operation for Carcinoma of the Vulva. A New Approach to Wound Healing. J. Obstet. Gynaecol. Br. Cmwlth. 77. 1037-1040. 14. BIomfield R. (1973) Honey for Decubitus Ulcers. J. Am. Med. Assoc. 224, 905. 15. Burlando F. (1978) Sull'azione Terapeutica del Miele nelle Ustioni. Minerva Dermat.1l3, 699-706. 16. Armon P J. (1980) The Use of Honey in the Treatment ofInfected Wounds. Trop. Doct. 10,91. 17. Bose B. (1982) Honey or Sugar in Treatment ofInfected Wounds? Lancet i, 963. 18. Dumronglert E. (1983) A Follow-up Study of Chronic Wound Healing Dressing with Pure Natural Honey. J. Natl Res. Counc. Thail. 15,39-66. 19. Kandil A. Elbanby M, Abd-Elwahed K, Abou Sehly G, Ezzat N. (1987) Healing Effect of True Floral and False Nonfloral Honey on Medical Wounds. J. Drug Res. (Cairo) 17, 71-75. 20. Effem SEE. (1988) Clinical Observations on the Wound Healing Properties of Honey. Br. J. Surg. 75, 679-681. 21. Farouk A, Hassan T, KashifH, Khalid S A, Mutawali I, Wadi M. (1988) Studies on Sudanese Bee Honey: Laboratory and Clinical Evaluation. Int. J. Crude Drug Res. 26, 161-168.

Honey as an Antimicrobial Agent

22. 23. 24. 25. 26. 27.

28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

37

Green A E.(l988) Wound Healing Properties of Honey. Br. 1. Surg. 75, 1278. Mcinerney R J F. (1990) Honey - a Remedy Rediscovered. 1. Royal Soc. Med. 83, 127. Braniki F J. (1981) Surgery in Western Kenya. Ann. Royal CoIl. Surg. Engl. 63, 348-352. Branemark P-I, Ekholm R, Albrektsson B, Lindstrom J, Lundborg G, Lundskog J. (1967) Tissue Injury Caused by Wound Disinfectants. 1. Bone Joint Surg. Am. Vol. 49, 48-62. Knutson R A, Merbit L A, Creekmore M A, Snipes H G. (1981) Use of Sugar and Povidone-iodine to Eenhance Wound Healing: Five Years Experience. South. Med. J. 74. 1329-1335. Chi rife J, Herszage L, Joseph A, Koh E S. (1983) In Vitro study of Bacterial Growth Inhibition in Concentrated Sugar Solutions: Microbiological Basis for the Use of Sugar in Treating Infected Wounds. Antimicrab. Agents Chemother. 23, 766--773. Rahal F, Mimica I M, Pereira V, Athie E. (1984) Sugar in the Treatment aflnfected Surgical Wounds. Internal. Surg. 69, 308. Middleton K, Seal 0 V. (1985) Sugar as an Aid to Wound Healing. Pharm. J. 235, 757-758. Trouillet J L, Fagon J Y, Domart Y, Chastre J, Pierre J, Gibert C. (1985) Use of Granulated Sugar in Treatment of Open Mediastinitis after Cardiac Surgery. Lancet ii, 180··184. Shimamoto Y, Shimamoto H, Fujihata H, Nakamura H, Matsuura Y. (1986) Topical Application of Sugar and Povidone-iodine in the Management of Decubitus Ulcers in Aged Patients. Hiroshima J. Med. Sci. 35, 167-169. Lowbury E J L, Ayliffe G A J: Drug Resistance in Antimicrobial Therapy. Thomas Springfield, Illinois. 1974. Smith M, Enquist I F. (1967) A Quantitative Study of Impaired Healing Resulting from Infection. Surg. Gynecol. Obstet. 125,965--973. Willix 0 J, Molan P C, Harfoot C 1. (J 992) A Comparison of the Sensitivity of Wound-infecting Species of Bacteria to the Antibacterial Activity of Manuka Honey and Other Honey. 1. Appl. Bacteriol. 73,388-394. Hancock B M: Microbiology Dept., Waikato Hospital, Hamilton, New Zealand: unpublished findings. Mossel 0 A A. (1980) Honey for Necrotic Breast Ulcers. Lancet ii, 1091. Huhtanen C N, Knox 0, Shimanuki H. (1981) Incidence of Clostridium botulinum Spores in Honey. J. Food Prot. 44, 812-814. Molan PC, Russell K M. (1988) Non-peroxide Antibacterial Activity in Some New Zealand Honeys. 1. Apic. Res. 27, 62-67. Allen K L: University ofWaikato, Hamilton, New Zealand: unpublished findings. Molan PC, Allen K L. (1996) The Effect of Gamma-irradiation on the Antibacterial Activity of Honey. J. Pharm. Pharmacol. In press. Allen K L, Molan pc. The Sensitivity of Mastitis-causing Bacteria to the Antibacterial Activity of Honey. Submitted for publication. Al Somai N, Coley K E, Molan PC, Hancock BM. (1994) Susceptibility of Helicobacter pylori to the Antibacterial Activity of Manuka Honey. J. Royal Soc. Med. 87, 9-12. Haffejee I E, Moosa A. (1985) Honey in the Treatment of Infantile Gastroenteritis. Br. Med. 1. 290, 1866--1867. Brady N F, Molan P C. The Sensitivity of Enteropathogenic Bacteria to the Antibacterial Activity of Honey. Paper in preparation. Rademaker M. (1993). Superficial Dermatophyte Infections. N. Z. Med. J. 106, 14--16. Brady N F, Molan P C, Harfoot C G. The sensitivity of dermatophytes to the antimicrobial activity of honey. Submitted for publication.

4

NON-PEROXIDE ANTIBACTERIAL ACTIVITY OF HONEY Stefan Bogdanov Federal Dairy Research Institute Bee Department 3097 Liebefeld, Switzerland

1. INTRODUCTION Honey acts as an antibacterial agent against many bacteria (1). There are two sorts of antibacterial agents or so called "inhibines." One of them is heat- and light-sensitive and has its origin in the HP2 ' produced by honey glucose oxidase (2,3,4). Some workers believe that hydrogen peroxide is the main antibacterial agent (2,5,6). Other authors find that the non-peroxide activity is the more important one (7,8,9). The H 20 2 amount in honey is very small and it can be produced only after aerobic incubation of diluted honey solutions, which might mean that it is not very important for the antibacterial action of honey (10). The argumentations of the pro and contra peroxide side are based on the results with the specific antibacterial test used. However, a certain antibacterial test might be sensitive only to certain types of antibacterian substances. In a previous study from dur laboratory it was found that while in an agar disc diffusion test only the peroxide activity was measured, in a liquid medium test only the non-peroxide substances were active( 10).

1.1. Antibacterial Substances The main honey substances are sugars, which by their osmotic effect exert an antibacterial action (1). The antimicrobial tests used in different studies are mostly carried out at concentrations, where the sugars are not osmotically active. Many different antibacterial substances, which are more or less heat- and light-stable have been characterised. It has bee claimed, that honey contains lysozyme, a well known antibacterial agent (8). However, in an other study it was found that no lysozyme activity was present in honey (10). Honey contains a number of flavonoids (11,12), many of which are known to have an antibacterial action (13). The profile and the concentration of the different flavonoids have been correlated to the floral origin of honey (11,12) , but not to its antibacterial activity (14). From New Zealand honeys, mainly manuka and viper's bugloss honey, several aromatic acidic substances with antibacterial activity have 39

S.Bogdanov

40

been isolated (1). These substances were proved to have a floral origin. Only in viper's bugloss honey the antibacterial activity measured could be explained by the amount of the antibacterial substance present. Another investigation claimed, that the low honey pH was responsible for the antibacterial activity (15). Some workers have isolated volatile substances with antibacterial activity (17,18), but their contribution to the antibacterial action was not examined. Other antibacterial substances have also been chemically characterized, mainly in New Zealand honey (1). Also, other workers found non-peroxide activity of honey, extractable by organic solvents, but were not able to identify the chemical nature of the substances (18,19).

1.2. Origin of the Antibacterial Substances Several clearly identified substances were shown to have a floral origin (1). In one study it was found, that the non-peroxide activity in blossom and honeydew honeys was not significantly different (20). Another study claimed, that the non-peroxide, partly volatile antibacterial activity of honey, has bee origin (21). Summarising the results of the previous studies, it seems that only in very rare cases the antimicrobial activity was quantitatively correlated to the amounts of the antimicrobial substances present. Also, in most studies it was not clear, whether the activity had a bee or a plant origin. This is surely due to the heterogenous nature of non-peroxide activity. In our study we looked for answers to following questions: I. Which different honey substance groups are responsible for the antimicrobial activity? 2. Does the activity have a plant or a bee origin? 3. What is the effect of heat and storage of honey on the non-peroxide activity? Here we summarise the work done in our laboratory, dealing with the non-peroxide honey activity, using an antibacterial test, which was shown to reflect only the non-peroxide part of antimicrobial activity (10)

2. MATERIALS AND METHODS 2.1. Materials and Honey Samples Turbidity Test. For the turbidity test we used the following liquid medium: I % pepton, 1% Lab-Lemco (both Oxoid) and 0.1 % glucose Test strains: Staphylococcus aureus and Sarcina lutea were both used for inocculation of bacteria growth tests as suspensions with 0.2 absorption units at 520 nm The spectrometer for measuring the turbidity of the bacterial suspensions was a Spectronic 20, where 20 mi test tubes could be read directly at 520 nm Thermostatable shaking incubator 20 mi sterile test tubes A "honey-sugar" standard was a solution of 40% fructose, 35% glucose, 7% maltose, 0,2 gil 00 g KCI and mM lactic acid. Honey Fractionation. The columns used for honey fractionation were:

• C-18 1000 mg SPE (Solid Phase Extraction) Baker 7020 disposable columns

Non-peroxide Antibacterial Activity of Honey

41

• 2 cm 3 /column 50 mesh (Dowex 50 W x 8) strong acidic cation exchanger • 2 cm 3 /column 50 mesh (Dowex I x 8) strong basic anion exchanger The SPE columns were mounted on Baker-IO SPE extraction manifold with vacuum. Biorad polypropylene disposable columns Nr.73l-l550 were used for the ion exchange fractionation, without the use of vacuum. Reagents and Honey Samples. All reagents were of analytical purity grade. Destilled water was used for all dilution steps. The honey samples anlysed in this study were either market samples (of Swiss and foreign origin) or they were taken at different parts of Switzerland for the purpose of the present study.

2.2. Methods 2.2.1. Honey Analysis. The honey humidity, acidity, invertase- and diastase (Phadebas method) activities were all determined according to the Swiss Food Manual (22). The free acidity is expressed in maeq/kg, the invertase in Hadorn units (invertase number) and the diastase in Schade units (diastase number). HP2 production was determined as described (10). 2.2.2. Antibacterial Growth Test. • Mix 10 mlliquid growth medium with 10 ml20g/100g honey solution. Add one drop of bacteria suspension and mix well. • Incubate in the shaking water bath at a constant shake-speed for a maximal bacterial grwoth: 12 hours for Staphylococcus and 36 hours for Sarcina. Whole honeys were tsted only against Staph.aureus was tested, while honey fractions were tested against both strains • Read turbidity at 520 nm Control incubation: 10 ml liquid growth medium were mixed with 10 ml 20gll00g honey sugar standard. It was shown earlier that due to osmosis, this sugar concentration has an inhibitory effect of about 10--20 %, compared to the growth in the medium without the "honey sugar" standard. 2.2.3. Honey Fractions. 50 gllOO ml honey water solutions were used for all fractionation steps. The initial pH of each honey solution was measured. The antimicrobial tests were carried out with the initial honey solutions and with the honey solutions after the removal of the fraction by each fractionation step. Before the antibacterial test the honey concentration of the solution after each fractionation step was adjusted to 20 gil 00 ml and the pH of the solution was set at the pH ofthe honey solution before the fractionation. 2.3.3.1. Removal of Volatile Substances. 50 gllOO ml honey solution was heated at 60° C under vacuum (15 mg Hg) for 2 hours in order to remove all volatile substances. 2.3.3.2. Removal of Non-Polar Non-Volatile Substances. The Baker C-IS columns were activated with one volume of ethanol, followed by one volume of water. The honey solution was then passed through under constant vacuum. The honey filtrate was used for the antimicrobial test.

S.Bogdanov

42 • TEST INITIAL ANTIMICROBIAL ACTIVITY OF WHOLE HONEY

Honey Solution • FRACTIONATION ./ Destilation 1. Removal of volatile substances (2 h. 60°C under vacuum)

~

honey without volatiles

1

Columns Removal of 2. Non-polar, non-volatiles (C-18) 3. Acids (anion exchange) 4. Bases (cation exchange)

• TEST LOSS OF ANTIMICROBIAL ACTIVITY OF HONEY SOLUTIONS AFTER REMOVAL OF THE DIFFERENT FRACTIONS AND COMPARE TO INITIAL ANTIBACTERIAL ACTIVITY Figure 1. Scheme of the fractionation and testing of the different antimicrobial fractions.

2.3.3.3. Removal of Bases (Relatively Polar). The cation exchange column was converted into the H-form by passing 2 ml of 2 M Hel. Then it was washed with water until the eluate was neutral. The honey solution, where the bases at the acidic pH of honey are in protonized form, is passed through. The flitrate is then set at the pH of the initial honey solution. 2.3.3.3. Removal of Acids (Relativly Polar). The anion exchange column was converted into the OH-form by passing of2 ml of2 M NaOH. Then it was washed with water until the eluate was neutral. The honey solution is then set at pH 11. The honey solution, where the acids at this high pH are in their dissociated form, is passed through. The flitrate is then set at the pH of the initial honey solution. 2.2.4. Expression ofAntimicrobial Inhibition. 2.2.4.1. Whole Honey Solutions. Results were calculated by the turbidities of the incubation mixtures at the end of the bacterial test. They were expressed in % inhibition compared to the absorbance of the control (control = 0 % inhibition or 100 % growth).

2.2.4.2 Honey Fractions. The increased bacterial growth after the removal of a certain honey fraction (see below) was attributed to the removal of antibacterial substances by this fractionation step.

Non-peroxide Antibacterial Activity of Honey

43

Example: A honey has an initial bacterial inhibition of 90 %. The inhibition after removal of: volatiles: 80%, non-polars: 65%, bases: 40% and acids: 20%. The initial inhibition of 90% was set as 100 parts to represent the whole antibacterial capacity of this honey. The partial inhibition after the loss of the different fractions in relation to the antibacterial capacitys of the whole honey was calculated as: 80/90x I 00, 65/90x I 00 etc.= 89, 72, 44, 22. The inhibition capacity of each absorbed fraction is then: 100-89; 100-72 etc = 11,28, 56 and 78. Then the sum of the inhibition capacities of the fractions was set as 100 %, e.g. for the above example:

11+28+56+78 = 173 = 100 %. In table 2 the % inhibition, attributed to each fraction, is a relative inhibition number, e.g. the relative inhibition of the volatile fraction is 1lI173xl 00 = 6%. The average sum of the inhibition parts of each fraction of the ten honeys in table 2 was 119 for Staph.aureus and 223 for Sarcina lutea. If all antibacterial substances were fractionated by our procedure and if their action is additive a sum of 100 should be expected in the above example. The significantly higher percentage than 100 for the fractional inhibition of Sarcina might be due to interactions of the different antibacterial fractions, when they act as a whole.

3. RESULTS AND DISCUSSION 3.1. Relative Distribution of Antimicrobial Activity among Different Honey Fractions We fractionated honey in 4 basic substance groups: volatile, non-volatile and nonpolar, acidic and basic substances.

Table 1. Relative distribution of antibacterial activity in different honey fractions The sum of the antibacterial activities against Staph. aureus und Sarcina /utea, attributed to each fraction is set as 100 % (see Methods) % antibacterial activity in different fractions Acidic Honey

St.

Manuca N.Z. Sunflower It Rape CH Lavender Fr Mountain CH Blossom S. America Honeydew CH Honeydew CH Honeydew CH Honeydew Europe

100 58 25 25 24 62 45 32 43 43

Average Minimum Maximum

46 24 100

Non-polar

Basic

Say.

51.

Say.

St.

Say.

75 46 40 25 73 46 31 26 32

0 13 7 34 60 13 26 37 22 25

10 15 33 30 25 20 15 31 26 31

0 16 63 23 8 9 26 19 19 26

5 25 22 29 25 7 15 31 26 37

42 25 75

24 0 60

24 10

21 0 63

22 5 37

27

33

Volatile

St.

Sar.

0 13

10 15

5 18 8 16 2 12 15 6

14 24 0 24 6 23 0

10 0 18

12 0 24

44

S.Bogdanov

Table 2. Correlation between free acidity, diastase and invertase activity and bacterial inhibition of Slaph.aureus. r-correlation coefficient, p---probability level at 95 %. For units see Methods

p n

Free acidity vs. inhibition

Diastase number vs. inhibiton

Invertase number vs. inhibition

0.35 0.001 81

0.65 0.0005 37

0.58 0.001 37

In table I the relative distribution of antibacterial activity in these fractions against Staph.aureus and Sarcina lutea is summarised. The acidic fraction has the greatest inhibitory power, while the volatiles are the weakest bacterial inhibitors. The relative distribution of the antibacterial activity in the different fractions is the same against both bacterial strains tested. On the average, for both strains the following relative distribution of antibacterial activity was observed: acids: 44%, bases: 24%; non-polars: 21 % and 11: volatiles. If the differences between the distribution of activity among the different groups were tested by a t-test, only the difference between the volatile activity on one side and the activity in the acidic (p=O.OOO) and the basic fraction (p=0.05) proved to be significantly different. This is due to the variation of the distribution among the fractions of the different honey types. In the manuca honey 90% of the activity was found in the acidic fraction, in the rape honey the greatest part of the activity was in the non-polar fraction and in one Swiss blossom honey the basic fraction had the highest activity.

3.2. Origin of the Non-peroxide Bacterial Inhibition 3.2.1. Plant Origin. In fig .2 the bacterial inhibition of 9 unifloral and 2 mixed (different blossom and different honeydew origins) are shown. Rhododendron honey had the

80

70 60 c .250

-

::cs 40 c

.- 30 ~

20 10

o

o

a:::

ro

o

o

:c

ro

a:::

Figure 2. Non-peroxide activity of unifloral honey against Staph.aureus: The values (averages) are for: Rh Rhododendron, n=3 ; Eucalyptus, n=4; Orange, n=3 ; Chestnut, n=7, Blossom, n=30; Acacia, n=7; Sunflower, n=4; Lavender, Dandelion, n=2; Honeydew, n=IO; Rape, n=7.

Non-peroxide Antibacterial Activity of Honey

45

lowest, while rape honey had the highest inhibitory power. However, there is a considerable variation in each honey type. In order to prove for significant differences between the honey types, a greater number of honeys should be analysed. Together with the fact, that the relative distribution of the antibacterial activity is different in the various honey types, the data shown here suggest, that some of the non-peroxide activity is of floral origin. 3.2.2 Bee Origin. Honey acidity, distase and and invertase are known to have a bee origin. There is a highly significant correlation between the free acidity, the diastase and the invertase activity on one hand and the bacterial inhibition on the other (table 2). These results show, that a part of the non-peroxide antibacterial activity has a bee origin.

3.3. Influence of Heat and Storage The experiments were carried out with light blossom- and dark honeydew honeys. Heating of both honey types at 70° C for 15 minutes had no or very little effect on the non-peroxide actitivity (table 3.). Under the same conditions the peroxide accumulation capacity of blossom honeys is severely damaged (10). In a next experiment glass pots with blossom or honeydew honeys were stored in the light (day-light) or in the dark at room temperature (about 20-25° C). The results are summarised in fig. 3. After 5 months of storage the activity remains the same, while after 15 months there is a small drop of activity of about 20 %. The results were the same both for both types of honeys stored in the light or in the dark. Under the same storage conditions the peroxide accumulating capacity of honey is strongly reduced, especially when blossom honeys are stored in the light (10).

4. CONCLUSIONS 1. There are four different honey fractions, which account for the non-peroxide antibacterial activity. The antibacterial activity of these fractions is: acids> bases"" non- polars > volatiles. 2. There is evidence for the floral- and for the botanical origin of the non-peroxide antibacterial activity 3. The non-peroxide activity is only slightly affected by heat and by storage for 15 months in the light or in the dark.

Table 3. Effect of heat on non-peroxide activity Fresh honeys of floral or honeydew origin were heated for 15 minutes at 70° C. Values are mcans ± SEM and are expressed in % of the initial inhibition Honey

n

Bacterial inhibiton % of initial

Blossom (light) Honeydew (dark)

3 4

94 ± I

86 ±4

S.Bogdanov

46

120

.."'

100

~

.:; u

80

"'iii 60

~

c

0

~

0

40 20 0 blossom, light

blossom, dark

honeydew, light

honeydew, dark

3, 5 and 15 months Figure 3. Effect of storage on the non-peroxide activity The non-peroxide activity of 7 mixed blossom honeys and 5 honeydew honeys, stored in glass pots in the light and in the dark, was tested. The values are averages for the above honeys.

SUMMARY In honey there are two sorts of antibacterial agents or so called inhibines. One of them is heat- and light sensitive and has its origin in HP2 . The other consists of thermostable substances. In our study we tested the antibacterial activity by a turbidity test with 20% honey solutions with Staphylococcus aureus and Sarcina lutea strains. Under these conditions destroying all the HP2 with catalase had no effect on the antibacterial activity. Thus with this test only the non-peroxide antibacterial activity is measured. We used this test to measure the non-peroxide antibacterial activity of whole honeys and of different honey fractions. The results can be summarised as follows: I. By fractionation in different substance classes the non-peroxid antibacterial activity is distributed among 4 fractions with different chemical characteristics: acidic; basic (both relatively polar); non-volatile and non-polar; volatile. 2. The antibacterial activity of the different fractions , tested in 10 different honeys was: acids> bases

= non- polar, non-volatiles> volatiles.

This order was the same for both Staph. au reus and Sarcina lutea as test strains 3. The distribution of activity was, however, dependent on the honey type : In manuca honey almost the whole activity was found in the acidic fraction, in rape honey the greatest activity was in the non-polar fraction and in one Swiss mountain honey the basic fraction had the highest inhibitory power.

Non-peroxide Antibacterial Activity of Honey

47

4. There were differences between the antibacterial actIVIties of different honey types: rhododendron, eucalyptus and orange honeys had a relatively low, lavender, dandelion, honeydew and rape honeys had a relatively higher activity. This result, together with point 3. suggests, that some of the non-peroxide activity has a plant orign. 5. There is also a significant correlation between the acidity, diastase- and invertase activity, all of bee origin, on one hand, and the non-peroxide activity, on the other. Thus, a substantial part of the non-peroxide activity has also a bee origin. 6. The non-peroxide activity is not or only slightly affected by heat (15 minutes 70 C and by storage for 15 months at room temperature. 0

REFERENCES I. Molan. P. (1992) The antimicrobial activity of honey I. The nature of antibacterial activity. Bee world, 73. 5-28 2. White, J.w., Subers, M.H. and Schepartz A.I., (1963), The identification ofinhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in honey glucose-oxidase system. Biochim. Biophys. Acta n 57-70 3. White, 1. W. and Subers, M.H. (1964), Studies of honey inhibine, 3. The effect of heat. J.Apic.Res. 3 454-450 4. Dustmann, J.H. (1972) Ueber den Einfluss des Lichtes auf den Peroxid-Wert des Honigs. L.Lebensm.Unters.Forsch. 148,263-268 5. Dustmann.J.H. (1979) Antibacterial effect of honey. Apiacta 14, 7-11 6. Morse, R.A. (1986) The antibiotic properties of honey. Pan-Pacific Entomologist 62, 370-371 7. Gonnet, M. and Lavie, P. (1960) Influence du chaufage sur Ie facteur antibiotic du miel. Annales de I' Abeille (Paris) 3, 349-364 8. Mohrig, W. and Messner, R. (1968), Lysozym als antibacterielles Agens im Honig und Bienengift. Acta Biologica Medica Germanica 21, 85-95 9. Radwan S.S. EI-Essawy A.A. and Sarhan, M.M. (1984) Experimental evidence for thc occurrence in honey of specific substances active against micro-organisms. Zentralblalt Mikrobio!. 139,249-255 10. Bogdanov, S. (1984) Characterisation of antibacterial substances in honey. Lebensm. Wiss. Techno!., 17, 74-76 II. Sabaticr, S., Amiot, M.1., Tachini, M and Aubert. S. (1992) Identification offlavonoids in sunflower honey. I.Food Sci. 57, 773-774 12. Tomas Barberan, FA., Ferreres, F Garcia-Viguera, C. and Tomas-Lorente, F Flavonoids in honey of different geographical origin. (1993) Z.Lebensm.Untersuch.Forsch. 196,38-44 13. Metzner 1., Bekemeier, H .• Paintz, M and Scheidewand E. and (1975) Zur antimikrobiellen Wirksamkeit von Propolis und Propolisinhaltsstoffen. Pharmazie, 34, 97-102 14. Bogdanov S. (1989) Determination of pinocembrin in honey using HPLC. J.Apic.Res. 28, 55-57 15. Yatsunami, K. and Echigo, T. (1984) Antibacterial activity of honey and royal jelly. Honeybee Science 5, 125-130 16. Lavie, P. (1963) Sur I'identification des substances antibacteriennes presentes dans Ie mie!. C.R.Seanc.Acad.Sci. Paris. 256, 1858-1960 17. Toth, G., Lemberkovics, E. and Kutasi-Szabo (1987) The volatile components of some Hungarian honeys and their antimicrobial effects. 127,496-497 18. Roth, L.A., Kwan, S. and Sporns P. (1986) Use ofa disc assay to detect oxytetracycline residues of honey. I.Food Prot. 49, 436-441 19. Schuler, R and Vogel, R. (1956) Wirkstoffe des Bienenhonigs. Arzneimittel Forsch. 6, 661-663 20. Bogdanov, S. , Rieder, K. and Ruegg, M. Neue Qualitatskriterien bei Honiguntersuchungen. Apidologie, 18,267-278 21. Lavie, P. Proprietes antibactcriennes et action physiologique des produits de la ruche et des abeilles in: Traite de Biologie de l'Abeille (R.Chauvin, editor) Masson & Cie pp.2-115 22. Swiss Food Manual, Chapter 23 A, Honey, Bern. Eidgen6ssische Druck und Materialzentralle, 1995

5

ANTIOXIDANT PROPERTIES OF HONEY PRODUCED BY BEES FED WITH MEDICAL PLANT EXTRACTS Gennady Rosenblat,1 Stephane Angonnet,2 Alexandr Goroshit,3 Mina Tabak, I and Ishak Neeman l IDepartment of Food Engineering and Biotechnology Technion-Israel Institute of Technology Haifa 32000, Israel 2ENSIA Paris 91305, France 3Tzuf Laboratori es Ltd. P.O.B. 408, Kiryat Shmona, Israel

ABSTRACT Honey is known to exert beneficial effect on many pathological conditions. The full gamut of its biological activity has yet to be elucidated. In this study five type of honey (regular commercial honey, Chinese honey and three other honeys, namely, Laryngomel, Bronchomel, and Dermomel) were studied for their antioxidant effect. Unlike regular honey, three last honey species were a product of bees which have been fed on a mixture of several medico-herbal water extracts with regular honey. All the assessed honeys demonstrated prevention of j3-carotene degradation in linoleic acid emulsion and obviation of superoxide radical generation by xanthine/xanthine oxidase system. Laryngomel and Bronchomel were particularly effective in reactive oxygen species scavenging at a concentration range 7-50 J.lg/ml. The relationship between the antioxidant properties of honey and its physiological activity is discussed.

INTRODUCTION One of the common properties of many plant natural products is the considerable degree of protection afforded against oxidative attack. The spontaneous reaction of oxygen or oxygen containing radicals with organic compounds leads to cell and tissue damage. Radicals and the radical-generating process are normally neutralized by antioxidative defense mechanisms [1], but in certain situation such as aging, inflammation, etc., radical 49

50

G. Rosenblat et al.

generation may increase with a consequent acceleration of accumulating damage [2,3]. In such situation the use of antioxidants in medication or of plant extracts with antioxidant properties in food has been advocated to minimize any oxidative degradation of the target molecules [4,5]. The antioxidant potential of honey, which is a widely used natural product containing mostly floral compounds, has not been sufficiently evaluated. Most studies on the biological properties of honey have thus far focused on its antibacterial activity [6]. Yet, in " folk medicine" honey also is believed to exert a beneficial effect on ulcers, the nervous system, the heart, the liver and the digestion [7]. The full spectrum of honey biological activities is still unc1arified but evidently much extend beyond were antibacterial activity. The present investigation attempted to fill the gap by studying the antioxidant effect of several types of honey.

MATERIALS AND METHODS All solvents were obtained either from Frutarom (Haifa, Israel) or Biolab (Jerusalem, Israel). Specific compounds were all purchased from Sigma (St. Louis, USA) excepting linoleic acid (from Fluka Chemie, Buchs, Swizerland) and OPA (from Zymed, USA)

Honey Venue and Production Commercial Chinese honey was received from Cho Yung International, Israel. Regular commercial honey was produced by kibbutz Ayelet HASHAHAR (Israel). The commercial honeys Bronchomel, Laryngomel and Dermomel were produced by Tzuf Laboratories Ltd., Kiriat Shmona, Israel; unlike regular honey, these three honey species were a product of bees which have been fed on a mixture of several medico-herbal water extracts with regular honey. The taxonomical data on the medical herbs used in the mentioned extracts are given in Table 1. Glucose-fructose syrup, which was used in part of the experiments as a model solution, was prepared by adding 35 g of each sugar to 100 ml of hot bidistil1ed water. ~-carotene

Degradation Test

Antioxidant activity was assessed via emulsion with p-carotene and linoleic acid, whose degradation was determined by the method of Marco [8] with modifications. Briefly, 0.4 mg of p-carotene, 100 !-II of linoleic acid and 200 !-II of Tween 40 were dissolved in 20 ml of chloroform, which was then evaporated by nitrogen. The model emulsion was now prepared by adding 30 ml of water. One gram of honey was next dissolved in 2 ml of water (or of appropriate buffer) and mixed with 3 ml of the emulsion; 2 ml of 95 % ethanol was added to the mixture, which was finally incubated at 50°C for several time. The antioxidant activity of I g of honey in 7 ml of reaction solution was compared with that of 1 !-Ig/ml (5.5 10-3 mM) and 5 !-Ig/ml (2.8 10.2 mM) ofBHA. The degradation of honey protein in the different tests was achieved by autoc1aving for 30 min 2 ml of water (or buffer) containing 19 of honey at I atm. Citric buffer, 0.1 M, pH=2, or citric-phosphate buffer, 0.1 M, pH=4.5 or phosphate buffer, 0.1 M, pH=7 were used for the pH-change in the different tests.

Antioxidant Properties of Honey and Medical Plant Extracts

51

Table 1. Medicinal herbal are used in forming of honey Laryngomel , Bronchomel , and Dermomel and medical-biological characteristic of the honey Type of honey Laryngomel

Bronchomel

Dermomel

Family of the herbalLauraceae Asteraceae Apiaceae Labiatae Betulaceae Chenopodiaceae Rutaceae Liliaceae Mirtaceae Lamiaceae Lauraceae Pinaceae Apiaceae Cruciferaceae Rosaceae Umbelliferae Juglandaceae Labiatae Salicaceae

Medical- biological characteristic active against laringitis,tracheitis, glossitis

active against inflamation of the upper respiratory tracts

active against suppurative wounds and chronic ulcer

*All plants are used in conventional or alternative medicine

Inhibition of O 2- Production by Xanthine/Xanthine Oxidase System (X/XO) The production of superoxide anion was evaluated by chemoluminescence. Reaction was initiated at room temperature by adding 250 f.ll solution of xantine oxidase solution (0.65 units/ml) to 2-ml of Hanks buffer (pH=8.3) containing 100 f.lM of xanthine and 60 f.lM of lucigenin. The mix was put in a luminometer (model 597B, Technion Physics Department, Haifa, Israel). The light production remined constant throughout the 2 - 5 minutes following onset of the reaction, during which period 25 f.ll of the differently diluted (in water) honey was added dropwise. The chemoluminiescence was now measured directly in triplicate samples.

Xanthine Oxidase Activity Activity of xanthine oxidase was determined by uric acid production. Uric acid generation was monitored by absorption spectroscopy, at 290 nm, under the same condition as in experiment with superoxide anion determination.

RESULTS Effect of Honey on the Degradation of ~-Carotene in Emulsion with Linoleic Acid The oxidation of ~-carotene in emulsion with linoleic acid in the presence of various honey species is shown on Fig. 1. A change in the absorbency at 470 nm (expressed in

52

G. Rosenblat et al. 120

100

QJ

u

to

...'0"

,.Q rJl

E c:

'~ci " ..."

,.Q

0

•.r::

•

.2 0 -~ ""'0 0~

60

40

...

.

20

o w............ o 50

'

I ",

I I

I

100 150 200 250 300 350

Time [min] Figure 1. Effect of honey on p-carotene degradation in emulsion with linoleic acid.

percent of initial absorbency) is characteristic of 0-carotene degradation in course of the oxidation. As can be seen from the data the antioxidant activities of all the honey solutions in distillated water (142,8 mg/ml) at normal for honey pH (about 4.0 ) are comparable with that of BHA (which is a potent antioxidant employed in the food and chemical industries [9]) in a concentration of I /-!g/ml or 5 /-!g/ml, respectively . The antioxidant effect of honey in emulsion with linoleic acid was found to be pH dependent. The antioxidant effect of all species of honey is maximal at physiological pH (7) but reduces with decrease in the pH, so that at pH of human stomach (pH=2) the tested honey types do not show antioxidant activity excepting Laryngomel and Bronchomel which demonstrate a negligible antioxidant effect. The capacity of honey to protect against 0-carotene oxidation was not altered by heating of the honey solution in the protein- cleaving state, thus supporting our contention regarding the non-proteine nature of the honey antioxidant moiety (data not shown). Nevertheless, the possibility in this case that thermal inactivation of antioxidant protein is compensated for by products of Maillard reaction cannot be entirely excluded, and it has been observed repeatedly that various Maillard reaction products formed through the interaction of protein and carbohydrates during the heat-processing of food do exert antioxidant activity [1OJ-

Inhibition of 02- Production by Xanthine / Xanthine Oxidase Xanthine / xanthine oxidase system is one of the two natural system of superoxide anion generation . Xanthine oxidase-derived superoxide anion has been linked to postischemic tissue injury and the generation of neutrophil chemotaxis [II] . Hence curtailment of 0 2' generation by this enzymatic pathway would be beneficial in the case of ischemia. Honey demonstrates a significant inhibitory effect on radical generation by xanthine/xanthine oxidase system. Indeed extintion of the chemoluminescence was observed in the presence of the regular honey (159-900 /-!g/ml), while an even greater effect was shown

Antioxidant Properties of Honey and Medical Plant Extracts

53

12° ~1

100

o

20

o

o

675

1450

2 25

3000

Concentration, IJ.gfml Figure 2. Effect of honey on superoxide anion production by a xanthine/xanthine oxidase enzymatic system.

by Dermomel and Chinese honey in the same concentration range. Strong effect on superoxide amount was found for Bronchomel and Laryngomel at a concentration of 7- 50 Jlg/ml (Fig 2. but data for Bronchomel is not shown). This effect was not abnogated by heating the honey samples at 100 °C and I atm for 30 min , thus supporting the supporting nonproteine nature of the inhibiting compounds. No significant inhibitory effect on the superoxide anion generation by xanthine/xanthine oxidase was displayed by glucose-fructose syrup even in markedly concentrations. To justify whether this activity was due to an inhibitory effect on the enzyme itself, a control experiment was carried out, in which production of uric acid was measured by UV spectroscopy at 290 nm. Under the same experimental condition it was not demostrated an inhibition of uric acid production by xanthine oxidase even in presence of markedly concentrations of honey.

DISCUSSION Honey contains various minerals and organic compounds but is comprised mainly of sugars (about 80 %) and water (about 20%) [12]. In addition, honey is known to contain a number of enzymes such as diastase, invertase, saccharase, catalase, glucose oxidase. Depending on the source from which bees obtain the material for honey production, the honey will acqure its specific aroma, color and test. Apparently , the medicinal and the nutritional properties of honey depend in part, also from the chemical composition of the flowers from which bees collect nectar. For this regard, there is growing interest in the honey created by bees that are nourished on medical herbs for the properties of the final product are enhanced during processing of the natural nectar in the bee's body. Using various plant extract that are well known in alternative and conventional medicine, it have been able to develope honey substitutes for easing cough and bronchitis (Bronchomel),

G. Rosenblat et al.

54

sore and inflamed throat (Laryngomel ), or for treatment of wounds (Dermomel). This and natural honey studied by us have all demonstrated antioxidant activity, particularly an inhibitory effect on superoxide radical production by xanthine Ixanthine oxidase. The inhibition of 02- production is manly due to scavenger properties since the tested honey are not xanthine oxidase inhibitors. Regular honey was found to be a weaker than the other samples tested by us. Presumably the protective properties of manufactured honey against reactive oxygen species reside mainly in the substances extracted from the medicinal herbs. Although butulated hydroxyanisole was used by us as the standard for assesing the antioxidant properties of honey it would be incorrect to compare BHA and honey antioxidant activity quantitatively, for obviously honey contains the biologically active ingradients in very low concentration. Nevertheless a simple calculation (using data from the ~-carotene degradation test) shows that active dose of natural honey which is recommended for medicinal use (one teaspoonful, which is about 7 g ) contains an amount of antioxidant which is equal to 50-250 Ilg of the potent antioxidant BHA. Turning now to the mechanism undrelying honey biological activity, accumulating evidence supports the assumption that the protective effect of honey against inflammation of whatever sourse is associated with its antioxidant moiety. Indeed, it has been shown that synthesis of inflammation mediators like leukotrienes and prostaglandins is involves the formation of arachidonic acid lipoperoxides [13]. Furthermore, in the wake of many types injury, including trauma, cells are known to rupture and release their contents, which include transition metal ions that can rapidly catalyze radical-mediated transformation and tissue injury [2]. Macrophage activation and the disruption of mitochondrial function may also result in the formation of excess reactive oxygen species [14,15]. Human phagocytes destroy bacteria or virus-infected cells throughout an oxidative burst of nitric acid NO", hypochlorite CIO' and superoxide 02- , but unfortunately the damage produced by radicals liberated from the phagocytic cells can sometime extend beyond the intended target and injure also surrounding tissue [16 ]. Uniquely vulnerable to such oxidative damage are epithelial cells lining the respiratory airways [17]. A part from potential oxidant exposure owing to normal cellular metabolism, the respiratory epithelium is exposed to relatively high oxygen tension and often also exposed to air pollutants, phagocytes, catalase-negative bacteria, and reactive xenobiotic-drug metabolites. From all the above, we can conjecture that the significant protective effect of honey (particularly Bronchomel and Laryngomel) against respiratory airway inflammation is explanable, in part, by its antioxidant properties. In conclusion, the in vitro antioxidant effect of honey revealed by the present study seems to comprise an is important attribute of honey, especially for such as was produced by bees nourished on mediI' ina I herbs.

REFERENCES I. Reiter R.J. (1995). Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J.

9,526-533 2. Kehrer J.P., Smith c.v. Free radicals in biology: sourse, reactivities, and roles in the etiology of human diseases in: Natural antioxidants in human health and disease (Balz Fri Editor.), Academic Press, Foreword, 1994, pp.25--62, 3. Harman D. (1993). Free radical involvement in aging. Pathophysiology and therapeutic implication. Drags Aging. 3, 60-80 4. Bermond P. Biological effects of food antioxidants in: Food antioxidant, (Hudson BJ.F. Editor), Elsevier Science Publishing Co., Inc. N.Y. 1990, pp. 193-251 5. Sies H., (1993). Strategies of antioxidant defense. Eur. J. Biochemistry. 215, 213-219 6. Molan P.c. (1992). The antibacterial activity of honey. Bee World. 73,5- 26

Antioxidant Properties of Honey and Medical Plant Extracts

55

7. Loyrish N: Bees and people. Mir Publishers, Moskow. 1977 8. Marco, G.J. ( 1968). A rapid method for evaluation of antioxidants. JAm Oil Chem.Soc .. 45, 594-598 9. Kukagawa K., Kunugi A., Kurechi T. Chemistry and implications of degradation of phenolic antioxidants in Food antioxidant. (Hudson B.1.F, Editor), Elsevier Science Publishing Co. Inc. N.V. 1990 10. Eichner, K. (1981). Antioxidative effects of Maillard reaction intermediates. Prog.Food Nut/:Sci .. 5, 441-451 11. Cotelle, N., bernier, J.L., Henichart, J.P., Catteau, J.P., Gaydou, E.. Wallet, 1. C. (1992). Scavenger and antioxidant properties of ten synthetic flavones. Free Rad.Biol.Med .. 13,211-219 12. White, I.w., Jr. (1978). Honey. Adv. Food Res., 24, 288--374 13. Borgeat,P., Samuelson, B. (1979). Proc. Natl.A cad. Sci. USA. 76.3213-3217 14. Kehrer, J.P.(l993). Free radicals as mediators of tissue injury and disease. Crit.Rev. Toxicol .. 23,21-48 15. Rosen, G.M., Pou S., Ramos c.L., Cohen M.S., Britigan B.E.(1995). Free radicals and phagocytic cells, FASEB J. 9. 200--209 16. Wright D.T., Cohn L.A., Li H., Fisher B., Li C.M., Adler K.B. (1994). Interaction of oxygen radicals with airway epithelium, Environ. Health Penpect. 102 (Suppl 10),85--90 17. Meyer A.S., Isaksen A. (1995). Application of enzymes as food antioxidants. Trend. FoodSci. Technol. 6, 300304

6

SPEEDING UP THE HEALING OF BURNS WITH HONEY An Experimental Study with Histological Assessment of Wound Biopsies

Th. 1. Postmes 1: M. M. C. Bosch 2 , R. Dutrieux 3, 1. van Baare 4 , and M. 1. Hoekstra 4 IDepartment ofInternal Medicine Academic Hospital Maastricht 2Leiden Cytology and Pathology Laboratory 3Dcpartment of histopathology Academic Hospital, Utrecht 4Burns Unit Red Cross Hospital Beverwijk, The Netherlands.

ABSTRACT In a pilot-study deep dermal burns, identical in depth and extent, were made at each flank of Yorkshire pigs. Wound healing characteristics and measurements of dermal thickness were investigated by comparing biopsies histologically in pairs. Wounds were treated with either honey of a defined antibacterial activity, or sugar, or silver sulfadiazine (SSD). Biopsies were taken on post burn days 7,14,21,28,35 and 42. Wounds treated with SSD were fully epithelialized after 28-35 days, whereas those treated with honey and sugar were closed within 21 days. In 5 out of 6 wounds the neodermis of the sugar treated burns was thicker than the neodermis of those treated with honey. In all honey experiments, on day 21, wounds were best microscopically characterized by (i) a quiet granulation tissue, (ii) an inconspicuous inflammation and (iii) a decrease of actine staining of myofibroblasts. In contrast, sugar treated wounds appeared ditTerent especially on day 21 and later. Furthermore, we observed in various sections signs of inflammation which were linkcd to pcrivascular infiltrates and well-stained myofibroblasts. These results suggest a difference between sugar and honey treatment. If antibacterial activity and anti-inflammatory activity count in wound treatment, then honey has to be prefered above sugar. * Correspondence should be addressed to: Dr. Th.Postmes, St.Servaasklooster 22; 6211 TE Maastrcht.

57

S8

Th. J. Postmes et al.

INTRODUCTION Epidermal regeneration of a wound is a complex process in which residual epithelial cells proliferate in an integrated manner into intact epidermis. In deep second degree burns re-epithelialisation primarily takes place from the wound margins; apparently most epithelial cells from hair follicles in the thermal injured area are not viable anymore. Granulation tissue is formed on the base of vessels and fibroblasts in the residual dermis and from the fatty tissue septa. Since the choreography of wound healing factors is still in the dark, an evaluation of the efficacy of current wound dressings remains worthwhile. Topical application of honey to open wounds has been recognized for centuries to be effective in controlling infection and producing a clean granulating wound bed. Both honey and sugar have been highly praised and recommended as proper treatments for traumatic wounds, burns, decubitis and ulcera l - 5 • Though honey is a complex medium it is safe and non-toxic 3- s. Moreover it is able to disinfect infected burn wounds, but it can also prevent colonization of non-infected burn wounds by bacteria until complete epithelization3 • According to Condon (1993) honey acts mainly as a hyperosmolar medium that precludes bacterial growth and as such does not differ from simple sugar6. Thus the real explanation of the antimicrobial effect of honey lies in its physical properties, not in its chemical composition 6 • However, the antibacterial effect after dilution up to 4% proves that honey must have intrinsic antibacterial properties quite distinct from sugar 7,R. Moreover the presence of e.g. traces of zinc, vitamins, amino acids and a large number of organic substances may contribute to and support the nutrition of the injured tissue. In spite of the numerous publications on honey and sugar as topicals for wound treatment, not a single clinical study comparing both substances has been published. In this pilot experiment, which is part of a larger evaluation on topical treatments, we wish to report some observations on the healing of deep second degree burns after treatment with honey, sugar or silver sulfadiazine.

MATERIALS AND METHODS A standard burn wound model, published by Hoekstra et al. (1993)., was strictly followed as described 9 • In brief:

Animals Experiments were performed in accordance with the Dutch Law on Animal Experimentation and the experimental protocol employed was approved by the Animal Welfare Committee of the University of Amsterdam. Three Yorkshire pigs of approximately 14 weeks (with a body weight of 25-35 kg) were thermally injured with a brass block of 6.7 x 6.7 cm, weighing 450 g. Twelve areas of 7 x 7 cm ( six on each flank) were marked in a symmetrical way, using the processus spinosi as an anatomical landmark. The block was heated up to 170°C and applied during 20 s without exerting pressure. The epidermal remnants of the burned skin were removed shortly after thermal injury. The total burned surface area amounted to not more than 10 % of the total body surface area.

Speeding Up the Healing of Burns with Honey

59

The details of the control of the animal's wellbeing, housing, food, general anaesthesia, daily wound treatment and covering were all similar to Hoekstra et aI., 1993.

Topical Agents Experiment (a), pig no 120, Silver sulfadiazine 1% cream vs honey. SSD ( Flammazine R , Duphar, Weesp, The Netherlands), as a topical agent and the most commonly used ointment for treating burns, was compared to unprocessed honey with a known antibacterial activity. The total inhibine (i) score of this particular lime honey was 15 as described previouslylo. Experiment (b), pig no 64. Honey vs honey. The honey was the same as in exp.(a). Experiment (c), pig 137. Honey vs sugar (artificial honey). The honey was the same as in expo (a). The osmolarity of the sugar paste containing 40% glucose, 40% fructose, 10% saccharose and 10% water was approximately that of honey.

Biopsy and Histology Each week (on post burn days 7, 14, 21,28, 35 and 42) biopsies were taken symmetrically from wounds on the left and right flanks. Biopsies included the wound bed as well as the healthy skin of the wound margins. Tissue fixing and staining were as described previousl/. An additional staining was included to detect myofibroblasts by an anti-Alpha smooth muscle actin stain.

RESULTS In this pilot study the wound healing pattern of the silver sulfadiazine treated flank of pig study no 120 was similar to the pattern reported earlier9 . Macroscopically it is almost impossible to assess the grade of epithelialization due to the presence of the crust. The time of a hundred percent epithelialization and the measurements of the dermal thickness of experiments a, band c, as found microscopically, are summarized in table I. An uninterrupted epidermis was found on day 21 for both honey (experiments a,b and c) and sugar (experiment c). Placed side by side the histology of the biopsies turned Table 1. Some data related to deep second degree burns treated with silver sulfadiazine (SSD), honey and sugar (artificial honey) Exp. no

Pig

a.

120

b.

64

c.

137

Thickness of the dermis I after day:

Treatment

100% epithelia1ised

14

21

28

35

42

honey vs SSD honey vs honey sugar vs honey

21-28 days' 28-35 days 21 days 21 days 21 days 21 days

1.7 1.0 3.3 2.5 1.8 1.3

3.3 1.1 1.5 3.3 3.5 2.5

2.7 2.7 2.0 1.8 2.7 1.5

2.9 3.3 2.0 1.5 2.3 1.3

3.0 2.g 1.7 1.7 2.2 2.4

'The thickness of the dermis is measured in the middle of:he biopsy. All data are given as a ratio of thickness of the burn wound versus that of normal skin. The pig's skin is normally 3 to 4 mm thick and in many ways it resembles the human skin. 'On day 21 the wound is almost fully epitheiialised, the next biopsy was on day 28, so the 100% point lies somewhere between day 21 and 28.

60

Th. J. Postmes et a!.

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:

,

.. .

,<

.

" ,

'",;

•

or

-

,

,

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•

Figure I. On day 21 , in sugar treated burns, deeper layers still show perivascular infiltration (Papanicoulaou staining, x25 ).

out to be quite different. All sections of honey treated burns were, more or less, similar. On post burn day 21 the general picture was primarily a quiet granulation tissue and a light degree of inflammation was seen. In contrast, the sugar treated burns showed more inflammation as revealed by the many perivascular infiltrations. According to the stained sections of the biopsies on day 28 the dermal thickness of the honey treated pig wounds was 5.1 mm, whereas the neodermal layer sugar of the treated wounds of the contralateral side was much thicker (7 .6 mm), see fig 2. On days 14, 21, 28 and 35 the honey treated burns showed a variation in dermal thickness between 4.0 mm and 9.5 mm while healed bum wounds in the contralateral sugar side varied between 5.5 and 10.5 mm. Part of the swelling of the derm is can be put down to local oedema in the first week of wound healing. In the honey treated wounds the typical actin staining in myofibroblasts appeared to be weak or diminished whereas sugar treated burns still showed a positive actin stain in many myofibroblasts. The honey treated burns were covered with a translucent layer of possibly non absorbed honey. This layer, however, could not be observed in the sugar experiments. In both honey and sugar treated wounds a few isolated bacterial colonies were found. These were less frequently seen in the honey than in the sugar wounds. A few micro-pustules were found in the neo-epidermis of the sugar treated wounds and less in the contralateral control side treated with honey. Bacteria were seen in the eschars of the sugar and the honey treated bums, though less in the latter and none in those of the SSD treated burns.

Speeding Up the Healing of Burns with Honey

61

Figure 2. (a): Normal pig's skin with subdermal fat and muscle tissue in the depth. (Papanicoulaou staining, x 12.5). (b). Pig number 137: honey treated side on day 28, the epidermis is uninterrupted and thus the wound is full y epilheliali sed. Clearly the dermis is thicker than normal. Staining and enlargement as in (a). (c): Pig number 137: sugar treated contralateral side on day 28. Staining and enlargement as in (a). Note: the dermis is 2.5 times thicker than normal.

DISCUSSION Honey and sugar paste, in comparable hyperosmolar concentrations, are non-toxic substances. The outgrowth of epithelial cells of the hair follicles is most likely inhibited by the uptake of either silver or SSDII. For the speeding up of wound healing by honey and sugar there are at least two explanations. First, in deep second degree burns, there are still a number of epithelial cells which might survive. Some belong to hair follicles , and others to sweat glands. Second, simple sugars in honey and sugar create a moist environment, which, as we know today, is a conditio sine qua non for quicker wound healing. Our study confirms the clinical study with honey vs SSD of Subrahmanyan (1991). In his study, honey turned out to be much better than SSD, which was proved by a significant reduction of the number of days spent in hospital 5 Today, in terms of re-epithelialisation, honey scores better than SSD (Flammazine R) and also seems to be better than sugar. In fig. 2 the most striking difference between sugar and honey is the thickness of the dermis. At cellular level the latter coincides mainly with a quantitative difference in inflammatory reaction and myofibroblast expression, both being less in the honey treated wounds. During the process of wound healing myofibroblasts are supposed to playa role in wound contraction. However, when contraction stops and the wound is fully epithelialized, these myofibroblasts which contain alpha myofibroblast smooth muscle actin disappear. The scar classically becomes less cellular probably as a result of apoptosis. It is then composed of typical fibroblasts with well-developed rough endoplasmatic reticulum but with no more actin filaments. In hypertrophic scars, on the other hand, the expression of alpha-smooth muscle actin in myofibroblasts persists l2 . Actually, in our study a persisting actin staining was seen in the sugar treated bums of pig 137 (table I).

62

Th. J. Postmes et al.

Hence, the "early" disappearance of myofibroblasts in the honey treated bums suggests a more advanced phase of wound healing than that of the sugar treated wound on the contralateral side. Granulation tissue, as required for the formation of the neodermis, is clearly suppressed initially by SSD. The effect of sugar on dermal thickness, as illustrated in fig. 2, is an interesting observation. Apparently, the large variation in dermal thickness in experiment b, where honey was applied on both flanks, implies that more data are needed to reach a final conclusion in the honey vs sugar inquiry. Unprocessed honey, unlike sugar, generates hydrogen peroxide when it becomes diluted. Hydrogen peroxide has been used for decades as an antibacterial agent with great success. Mainly due to the so-called Fenton reaction it can easily produce free hydroxyl (OB) radicals which are very bactericidal indeed. In vitro, hydrogen peroxide has also a biphasic effect on fibroblasts. Within a concentration of 10-8 and 10--{i molliiter it stimulates cell proliferation, whereas at higher concentrations an inhibiting effect becomes prominent I 3. In vivo, data are lacking. Nevertheless there is no reason to assume that fibroblasts in situ would react otherwise. The hydrogen peroxide concentration provided by honey in an open wound may have a growth inhibiting effect on the fibroblast. According to White et al. (1963), a 14 % honey solution may generate in one hour 0.0-2.12 mmollliter 7• Of the 90 samples investigated the one hour mean value was 0.47 mmolll ± 0.55 (s.d.Y4. In bleeding wounds or in the presence of traces of catalase, hydrogen peroxide breaks down almost instantly. This and other micro-environmental conditions make it almost impossible to predict the concentration of hydrogen peroxide at the interface of honey and wound bed.

CONCLUSION Honey is an ideal topical therapeutic agent, for it does not adhere to the wound surface. In comparison to silver sulfadiazine it is definitely superior because of its quick reepithelialization and its absence of a sustained inflammatory reaction, which in SSD is seen, even long after a complete epithilialization. Honey is also better than sugar paste, for it has a natural antibacterial activity, which is lacking in sugar paste of similar osmolarity. In addition it provides and maintains an environment in which healing can take place at an optimal rate. Honey treatment of burns is certainly cost effective, because it shortens the duration of treatment with ca. 25%, as shown in this study, and it certainly reduces, beyond all question, the duration of hospitalizations.

ACKNOWLEDGMENT We wish to thank the Dutch Burns Foundation for its financial and personal support of this study.

REFERENCES I. Knutson, R.A., Merbitz, L.A., Creekmore, M.A. and Snipes HG. (1981). Use of sugar and povidone -iodine to enhance wound healing:five years' experience. Southern Medical Journal 74, 1329--1335.

Speeding Up the Healing of Burns with Honey

63

2. Orouet, N. (1983) Utilisation du sucre et du miel dans Ie traitement des plaies infectees. Nouv Press Med 12,2355-2356. 3. Efem, S.E. (1988). Clinical observations on the wound healing properties of honey. British Journal of Surgery 75, 679--{i81. 4. Efem, S.E. (1993) Recent advances in the management of Fournier's gangrene: Preliminary observations. Surgery 113, 200 -204 5. Subrahmanyam, M. (1991). Topical application of honey in treatment of bums. British Journal of Surgery 78,497-498. 6. Condon, R.E. (1993). Curious interactions of bugs and bees. Surgery 113, 234-235. 7. White). W., Subers, M.H. and Shepartz AI. (1963). The identification of inhibine, the antibacterial factor in honey,as hydrogen peroxide and its origin in a honey glucose-oxidase system. Biochemica Biophysica Acta 73,57-70. 8. Molan, P.c. and Russel, K.M. (1988). Non-peroxide antibacterial activity in some New Zealand honeys. Journal of Apicultural Research 27, 62 -67. 9. Hoekstra, MJ, Hupkens, P., Outrieux, R.P., Bosch, M.M.C and Kreis, R.W. (1993). A comparative burn wound model in the New Yorkshire pig for the histo- pathological evaluation of local therapeutic regimens: silver sulfadiazine cream as a standard. British Journal of Plastic Surgery 46,585--589. 10. Postmes, L Bogaard van den A.E. and Hazen, M. (1993). Honey for wounds, ulcers, and skin graft preservation. Lancet 341,756--757. II. Teepe, R.G.C., Koebrugge, E.l, L6wik, C.W.G.M., Petit, P.L.c.P., Bosboom, R.W., Twiss, I.M., Boxma, H., Vermeer, BJ. and Ponec, M. (1993). Cytotoxic effects of topical antimicrobial and antseptic agents of human kerationocytes in vitro. The Journal of Trauma 35, 8-19. 12. Schmitt-Graff., Oesmouliere A. and Gabbiani G. (1994) Heterogeneity of myofibro blastic cell plasticity. Virchows Archive 425, 3--24. 13. Schmidt RJ., Chung, L.Y., Andrews, A.M. and Turner, T.O. (1992). Hydrogen is a murine (L 929) fibroblast cell proliferant at micro- and nanomolar concentrations. In:Second European Conference in advances in wound management. Proc. Int. Conf.Center Harrogate Oct. 20th-23th p. 117-121. 14. White, lW. and Subers, M.H. (1963). Studies on honey inhibine. 2. A chemical assay. Journal of Apicultural Research 2, 93-100.

7

THE EFFECT OF HONEY ON HUMAN TOOTH ENAMEL AND ORAL BACTERIA S. R. Grobler' and N. J. Basson Oral and Dental Research Institute Faculty of Dentistry University of Stellenbosch Private Bag Xl 7505 Tygerberg, South Africa.

SUMMARY Various fruit juices with relatively low pH values are known to have erosive effects on human tooth enamel in a reasonably short time (Grobler et al.1989! Clin Prev Dent, 11:23-28; 12:5--8). Honey, however, with a relatively low pH, could do the same. The honey sample consisted mainly of nectar gathered from the blossoms of Eucalyptus trees. The honey used did not contain any artificial preservatives or dilutents, neither had it been heated by any artificial method. Sixteen human incisor crowns were ultimately ground wet using 1200-grade silicon carbide paper. Each surface was divided into five segments and each segment exposed to pure honey and diluted honey as well as to artificial honey for different periods of time. The enamel segments were then investigated for their hardness as well as for any etch pattern, by scanning electron microscopy. Scanning electron microscopy showed no erosion of enamel by natural honey over a period of thirty minutes neither did Knoop microhardness tests show any deterioration of the enamel structure even in a 4 times diluted honey solution. However, the theoretical solubility and ion product values can be linked to the results obtained by the SEM study for the undiluted as well as for the four times diluted artificial honey sample. The absence of any effect by pure honey could only be partially attributed to the normal building blocks of enamel, namely calcium, phosphorus and fluoride levels. Seven different oral Streptococcus species, a Candida albicans strain and a Staphylococcus aureus strain were tested for antibacterial sensitivity towards the honey. Minimal inhibitory concentrations (MIC) were determined with a broth dilution method. The MIC was the lowest concentration of the honey which yielded no growth. * Correspondence address: S.R. Grobler, Oral and Dental Research Institute, University of Stellenbosch, Private Bag XI, Tygerberg, 7505, South Africa.

65

66

S. R. Grobler and N. J. Basson

The oral streptococci as well as the C. albicans strain were relatively resistant to Bluegum honey. However, the two species Streptococcus anginosus and Streptococcus oratis were inhibited at 17% and 12% respectively.

INTRODUCTION Honey has been used by man for many centuries. Written records date back to 3000 BC, which describe the practice of loading beehives onto small boats and moving them up and down the Nile River, in Egypt, depending on the season, to enable the bees to collect nectar where it was most abundant. Honey was also used by the Assyrians about 4000 years ago to embalm the bodies of their dead. Today honey is used extensively as a sweetening agent and is also considered as a health food, for example, it is used as a sweetener in cough mixtures. In a recent study in the British Journal of Surgery (1991)2, it was reported that honey not only renders burn wounds sterile within 7 days. but that 87% of burn wounds also healed within 15 days, against 10% in the control group. Large expansion of fruit farming practices took place in such a way that additional pollination became a necessity for flowering crops and orchards. Here hived honey-bees are employed to help with the pollination process. These small hard worker-bees are perfect pollinators which beekeepers can manipulate to supply in large quantities at appointed times. Two sub-species of the true honey-bee are found in South Africa. namely, the striped African honey-bee and the Cape honey-bee l . The pH of honey produced by these bees is slightly low, with an average value of about 3.9 for South Africa. It is interesting to note that the average value reported is the same as that for the USA 4. The demineralizing effects of excessive intake of fruit drinks, especially of citrus fruits and other acidic beverages, are well documented because of their low pH values s- 7 • Furthermore, it was reported that the highest dental caries incidence was found in farm workers in a citrus producing group in comparison to a control group. Published results 7 on different soft drinks showed the following degree of enamel demineralization: Pepsi Cola = orange juice> apple juice> Diet Pepsi Cola. Therefore, owing to the low pH values reported for honey one could presume that it might have a potential to cause erosion when it comes into contact with enamel. At the same time acid produced from dietary sugars through the metabolism of oral bacteria are also responsible for enamel erosion and dental caries 8 • Several workers 9- 1J investigated the antibacterial properties of honey and found it effective against organisms such as Staphylococcus aureus, Salmonella typhi, Shigella species, Escherichia coli and other pathogenic species. However, little information is available with regard to the antimicrobial effect of honey on the oral bacteria. Therefore, the purpose of this study was to determine the erosive effect of honey on human tooth enamel and to evaluate honey for its antimicrobial activity on certain oral bacteria.

MATERIAL AND METHODS The honey samples used in this experiment were harvested in the Western Cape of South Africa and consisted mainly of nectar gathered from the blossoms of Bluegum (Eucalyptus) trees. In order to be 100% certain that the honey sample collected were not

Human Tooth Enamel, Honey, and Bacteria

67

diameter

~

3 mm

Figure 1. The division of the enamel surface into four segments as used in subsequent phases of the study.

contaminated it was harvested by the researchers and was not heated by any artificial method. Sixteen human incisor crowns were polished up to 1200 grit fineness with silicon carbide paper before being exposed to the different honey concentrations for different periods of time. The surfaces were ground until an area of enamel approximately 3 m in diameter at the mid-central region had been smoothed. This was necessary to obtain a very smooth enamel surface which would highlight the slightest signs of enamel erosion, should any take place. The polished areas were washed and covered with a circular plastic adhesive tape (Fig. 1) and the tape then cut with a surgical scalpel into four segments.

Phase 1 Seven crowns were used in the first phase. The first specimen was prepared by removing the tape from one of the four segments from a crown. Honey was poured into a Petri dish and the crown with the one exposed segment immersed in the honey and continuously agitated for 10 minutes. A second outer segment of tape was then removed and the specimen again agitated in the honey for 10 minutes. Similarly, the third segment was removed and agitated for 10 minutes. The fourth segment was not exposed to honey and served as the control. In this way the four segments were exposed for 30, 20, 10 and 0 minutes. The effect of the honey treatments of these specimens were evaluated by scanning electron microscopy.

Phase 2 In this phase three crowns were exposed to a solution of 50% honey to 50% distilled water by volume, while three were exposed to a solution of 25% honey to 75% water. Again the exposure periods were 0, 10, 20, and 30 minutes per segment.

Phase 3 Six crowns were also divided into segments as explained above. In this investigation the surfaces were exposed to artificial undiluted honey (3 crowns) and to a solution containing 25% artificial honey and 75% distilled water (3 crowns). In this phase the immersion times were 0, 0, 30 and 30 minutes per segment. The artificial honey contained: 38%

68

S. R. Grobler and N. J. Hasson

Table 1. The minimal inhibitory concentration of honey and ofa carbohydrate control for different oral Streptococcus species (% vol/vol) 50% Organism (NCTC) Streptococcus mutans (10449) Streptococcus salivarius (8618) Streptococcus sanguis (7864) Streptococcus anginosus (10708) Streptococcus gordonii (3165) Streptococcus oralis (11427) Streptococcus sobrinus (10921) Candida albicans (NCPF 3118) Staphylococcus aureus (8530)

H

C

30%

25%

21%

H

H

H C

C

+ +

C

+ +

+ + + + + + +

+ + + + + + + + +

17% H

C

+ + + + + + + + + - + + + + + + +

12% H

+ + + + +

C

+ + + + + + + + + + + +

6%

H

C

+ + + + + + + + + + + + + + + + + +

3% H

C

+ + + + + + + + + + + + + + + + + +

H-honey, C~ontrol, + growth, - no growth NCPF-National Collection of Pathogenic Fungi (London)

D-fructose, 31 % D-glucose, 7% maltose, 1.5% sucrose, 100 mg Call, 308 mg PII, 0.7mg FII and the pH adjusted to that of the natural honey sample with HCI 4,14. All the segments in all the phases were evaluated for erosion by SEM. The honey was tested for its antimicrobial activity against a control solution with a sugar content similar to that of natural honey namely the abovementioned artificial honey. Eight different oral microbial species, obtained from the National Collection of Type Cultures (NCTC) (London), were used in the study (Table I). A strain of Staphylococcus aureus was included as a reference organism. The cultures were maintained on Brain Heart Infusion (BRI) agar (Oxoid) and subcultured weekly. The Minimal Inhibitory Concentration (MIC) of the honey (expressed as a vollvol percentage) were determined with the broth dilution method described by Ericsson and Sherris 15 • In short, two-fold dilutions of natural honey and the control solution were aseptically prepared in double strength sterile BHI broth (Oxoid) to give final volumes of 5 ml in each tube. The inocula used were prepared from overnight growth cultures in BHI broth. One drop of a 1I 100th dilution of a culture was used to inoculate 5 ml of the test solution. The tubes were incubated at 37°C for 24 hours and examined for growth. The MIC was noted as the lowest concentration of honey which yielded no growth.

RESULTS Fig I gives a diagrammatical outlay of the shape of the circular plastic adhesive tape which covered the smoothed enamel surface. The circular area was divided into 4 different segments by cutting it with a scalpel. All the segments in phase 1 and 2 showed no sign of enamel erosion under the scanning electron microscope (5l00x magnification) as a result of exposure to the different honey concentration for 0, 10, 20 and 30 minutes. Figure 2 gives a scanning electron photomicrograph (5100x magnification) of a typical polished enamel surface which was not exposed to any honey solution. In phase 3, Figure 3 (5100x) shows a high degree of enamel erosion when the enamel was exposed for 30 minutes to the 4 times diluted honey sample (25% artificial honey and 75% water). A lower degree of enamel erosion was also observed when the enamel was exposed for 30 minutes to undiluted artificial honey.

Human Tooth Enamel, Honey, and Bacteria

69

Figure 2. Scanning electron micrograph of the control enamel section, which was not exposed to honey. x5100 magnification.

Figure 3. Scanning electron micrograph of enamel section exposed to 4 times diluted artificial honey for 30 min. x5100 magnification.

70

S. R. Grobler and N. J. Basson

The MIC of the natural honey and of the control solution is shown in Table I. Except for the yeast species, all the organisms tested failed to grow at honey and sugar concentrations higher than 21 %. The two oral strains Streptococcus anginosus and Streptococcus oralis were more sensitive to the antimicrobial activity of honey than the S. aureus strain and failed to grow at concentrations of 17% and 12% respectively. The oral yeast Candida albicans was more resistant to honey than the bacteria and was able to grow at concentrations up to 30%.

DISCUSSION The natural honey was analysed l4 and the artificial honey prepared to contain the same amounts of different carbohydrates (namely fructose, glucose, maltose, sucrose), calcium, phoshorous and fluoride. This was done to investigate the effect of these species (which form the building blocks of enamel) on the solubility of enamel in honey, BECAUSE we soon observed that enamel does not dissolve in pure honey even when diluted up to 4 times. However, a low degree of erosion was already observed for the undiluted artificial honey in contrast to pure honey. while the 4x diluted artificial honey illustrated a higher degree of enamel erosion, again in contrast to 4 x diluted pure honey (30 min. 5100x enlargement). The fact that enamel dissolves in artificial honey and not in pure honey can be attributed to factors other than the concentrations of the elements that are normally associated with the solubility of enamel (calcium, phosphorous and fluoride). In order to establish why enamel dissolves more in diluted artificial honey than in undiluted artificial honey we have to consider the composition of pure honey and artificial honey at different dilutions. We looked mainly at the concentrations of the elements which are also found in the apatite crystal because these elements will have an affect on the solubility and the solubility product of enamel. What was very interesting to note is that honey also contains F (this sample 0.7 ppm). The question arose as to whether any correlation exists between concentrations of fluoride, calcium and phosphorous in the honey and the water used in its processing. Bees use water extensively, in the making of honey and to cool their hives, especially in very hot weather4 • The rapid movement of their wings produces draughts which cause evaporation, thus cooling the hives as well as condensing the honey. However, in an extensive study by Grobler et aI 16 ., on the analysis of honey samples from various areas and of the water collected from each source used by the bees for their water supply, it was concluded that the elemental composition of water does not contribute substantially towards the levels of calcium, phosphorous or fluoride of honey. On the other hand, it is possible that the relatively high concentrations of the above mentioned elements in honey relative to that in water could mask such a possible relationship. The pH of the different honey solutions is similar, namely 4.24 regardless of the dilution. Under a normal situation this pH is low enough to dissolve enamel quite rapidlyl7. This is due to the fact that the buffering capacity of honey is quite high-4x higher than that of saliva. This means that a small amount of saliva will not decrease the acidicity of honey to a harmless level of about 6.8. The joint effect can be summarised by calculating the ionic products of the honey solutions for hydroxyapatite (HAP) and fluoroapatite (F AP) as well as the solubility products of hydroxy-apatite and fluoro-apatite I4 . From these values it can be seen that the ionic product value for artificial honey with respect to HAP (63.2) is higher than the solubility

Human Tooth Enamel, Honey, and Bacteria

71

product value for HAP (54.6), which means that the 100% honey solution is under saturated with respect to HAP and HAP will dissolve but not FAP. On the other hand the ionic product of honey with respect to FAP (57.9) is lower than the solubility product (59.6) with respect to F AP and the honey solution is supersaturated with respect to F AP and FAP will not dissolve in the 100% honey. This explains why enamel which consists of both HAP and FAP will dissolve only slightly in 100% artificial honey, but will dissolve more readily in the 4x diluted honey solution as was found in this study. It has been shown convincingly that honey contain antibacterial activity and that this activity can be ascribed to much more than just the high sugar content of honei 8 • However, it is obvious from our results that the high sugar content can mask the real antibacterial activity of honey at honey concentrations of 25% and higher. Our results also show that except for S. anginosus and S. oratis, the oral streptococci are relatively resistant to the true antimicrobial activity of honey.

REFERENCES I. Grobler S.R .• Senekal P.J.c. and Van Wyk Kotze T.1. (1989) The degree of enamel erosion by five different kinds of fruit. c/in. Prev. Dent. 11,23-28. 2. Subrahmanyam M. (1991) Topical application of honey in treatment of burns. Br. 1. Surg. 78,497-498. 3. Anderson R.H., Buys B. and Johannsmeier M.F. (1983) Beekeeping in South AJrica. 2nd edn, Bulletin No. 394. p. 144. Department of Agriculture. 4. Root AI. (1983) ABC and XYZ oj Bee Culture. A.1. Root Pub!. Co. Medina, Ohio. 5. Grobler S.R. and Van der Horst G. (1982) Biochemical analysis of various cool drinks with regard to enamel erosion, de- and remineralization. 1. Dent. Assoc. South Afi-ica 37, 681-684. 6. Grobler S.R., Jenkins G.N. and Kotze D. (1985) The effects of the composition and method of drinking of soft drinks on plaque pH. & Dent. 1. 158,293-296. 7. Grabler S.R., Senekal P.J.c. and Laubser J.A. (1990) In vitro demineralization by orange juice, apple juice, Pepsi Cola and Diet Pepsi Cola. Clin. Prevo Dent. 12. 5--8. 8. Silverstone L.M., Johnson N.W., Hardie 1.M. and Williams R.A.D. (1981) Dental caries: Aetiology, pathology and prevention. MacMillan Press Ltd. London. 9. Cavanagh, D, Beazley, J & Ostapowicz. F (I 970) Radical operation for carcinoma of the vulva: a new approach to wound healing. Journal o{ Ohstetrics and Gynaecologv o{ the British Common Wealth, 77, 1037-1040. 10. Dold, H, Du, DH & Dziao, ST (1937) Nachweis antibakterieller, hitzc-und lichtempfindlicher hemmungsstoffe (inhibine) im naturhonig (Bliitenhonig). Zeitschrifi (iir Hygiene und Infektions-Krankheiten, 120,155-167. 11. Ibrahim A.S.( 1981). Antibacterial action of honey. Proceedings of the First International Conference on Islamic Medicine. Kuwait: Minister of Health, 363- 365. 12. Jeddcr A.,Kharsany A.,Ramsaroop U.G., Bhamjee A., Haffejee I.E. and Moosa A. (1985). The antibacterial action of honey. An in vitro study. S. Aji: Med. 1. 67. 257-258. 13. Zumla A. and Lulat A. (1989). Honey a remedy rediscovered. 1. Roy. Soc. Med. 82,384-385. 14. Grobler S.R., Du Toit 1.1. and Basson N.J. (J 994) The etlect of honey on human tooth enamel in vitro observed by electron microscopy and microhardness measurements. Arch. Oral Bioi. 39. 147-153. 15. Ericsson H.M. and Sherris J.e. (1971) Antibiotic sensitivity testing. Report of an international collaborative study. Acta Path Microbial Scand 79B, Suppl 217, 1-82. 16. Du Toit 1.1., Grobler S.R., Van Wyk Kotze TJ. and Basson N.J. (1995) Fluoride, calcium and phosphorus levels in bee honey and water. S. AJr. 1. Sci. 91, 391-392. 17. Driessens F.e.M. (1982) Mineral aspects oj dentistry (Edited by Meyers H.M.) Vol. 10, p. 117. S. Karger, London. 18. Molan P.c. (1992) The antibacterial activity of honey. 1. The nature of the antibacterial activity. Bee Wold 73,5-28.

8

HONEY CONTACT WITH TEETH IN SITU 1. Gedalia: S. R. Grobler, 1. Grizim D. Steinberg, L. Shapira, I. Lewinstein, and Mo. Sela

Hebrew University-Hadassah School of Dental Medicine Jerusalem, Israel; and Faculty of Dentistry University of Stellenbosch Tygerberg, South Africa.

SUMMARY Honey is a sweetening agent affecting dental caries like sucrose. It contains also a solubility-reducing agent, an organic phosphorus ester that is degradable by salivary emzymes. In the experimental design the changes of microhardness in prepared enamel surfaces from extracted human teeth were monitored by measurements of the tooth enamel microhardness at baseline and after intra-oral exposure, during a certain time period, to honey. Normal and salivary' flow deficient subjects volunteered for the study. pH measurements of saliva were carried out at baseline, during and after exposure of the enamel specimens in the mouth to the honey. The pH of the saliva (close to 7.0 at start) mixed up with that of the honey (3.9), decreased from about 6 to 4 in the saliva-honey mixture. After swallowing the mixture the pH returned to the baseline value. The microhardness of the surface enamel did not change in subjects with almost complete lack of saliva flow (dry-mouth subjects), as opposed in the subjects with a regular flow of saliva.

INTRODUCTION It was observed in rats that tooth destruction increased at a high rate with the addition of honey to their basal bread did. In vitro tests showed that pure honey with a relatively low pH (3.9) does not exert an erosive effect on human tooth enamel 2 • An organic phosphorus ester was suggested as the solubility reducing agent in honey, that is degradable by salivary enzymes 3 * Correspondence to: Prof. I. Gedalia, Oral Biology-Dental Research, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.

73

74

I. Gedalia et al.

The effect of honey consumption on human dental enamel was investigated in drymouth subjects.

MATERIALS AND METHODS Honey samples harvested in the southern part of Israel (Yad Mordechai) during autumn, consisting of nectar from the blossoms of wild flowers, were used. Ca, P and F were analyzed according to previous examinations in honey 4-7 . Changes of microhardness in prepared tooth enamel surfaces were monitored by measurements of the tooth enamel micro hardness at baseline and after intraoral exposure to honey, during a similar time period. Normal and salivary flow deficient subjects (xerostomic) volunteered for the study g.9. pH measurements were taken of the saliva at baseline, during and after exposure of the enamel specimens in the mouth to the honey-saliva mixture by means of indicator paper. Significance of the differences in microhardness between the baselines of the tested groups was determined by the Student t-test. Paired t-test was used to determine the significance of changes in microhardness before and after honey consumption within each subject group .

RESULTS The results of the Ca, P and F composition of the honey are: 166 ppm Ca ± SD 5, 410 ppm P ± SD 15 and 0.05 ppm F ± SD 0.01. The salivary pH levels and the microhardness levels are presented in Figs. 1 and 2. The pH of the honey-saliva mixture decreased from about 6 to 4 in the normal and the salivary-flow deficient subject groups (p < 0.05), returning to the baseline pH after the mixture was swallowed (p < 0.05), Fig. 1. The initial microhardenss of the surface of the enamel specimens decreased significantly (p < 0.000 I) after the honey consumption in the subjects with a regular flow of saliva, whereas in the dry-mouth subjects no enamel microhardness decrease took place (Fig.2).

8,----------------------,

o

7

•

Normal Irradiated

I

0.6

1\1

.~

iij 5

en

4

3~--------------------~

Figure I. Mean saliva pH values (±SE) at baseline, in the mouth, during consumption of the honey and at completion of swallowing the honey-saliva mixture.

7S

Honey Contact with Teeth in Situ

Z 300

:r:

>

-; 250

=

Before

=

After

,--!-

Ul

III C

"E

III

r--

200

:r: Qi 150 E III

Figure 2. Mean microhardness (±SE) expressed in Vickers hardness numbers before and after exposure to honey in the mouth of human enamel specimens.

C UJ

100

DISCUSSION A pH below 5.5 at the tooth enamel surface leads to mineral loss 10. 11 • The results of the present study indicate that tooth enamel decalcification occurred during consumption of the acidic honey in subjects with normal saliva secretion (Fig. 2). It is known that even trace amounts, 0.01 ppm of F in solution, decrease the rate of enamel solubility 12 • The F concentration was very low in the honey used in our study , 0.05 ppm, which probably explains why it did not suppress bacterial fermentation or exert a remineralizing effect (return oflost Ca and P to the tooth enamel surface). No erosive defects on in vitro exposed enamel surface to pure honey at a pH 4.24 was observed 2• The difference between the in vitro2 and the present in situ results regarding erosive effects on tooth enamel from honey, may be in part explained by the suggested solubility-reducing agent in honey, the phosphate ester, that is degradable by salivary enzymes 3 . In the dry-mouth subjects, the expected solubility-reducing agent in the honey was probably active protecting the tooth enamel when the honey-saliva mixture was below the pH level of 5.5 10• 11 , Fig. I.

A-CKNOWLEDGMENT The authors are indebted to Prof. I Roman, Dept. of Applied Science and Applied Physics, Hebrew University, Jerusalem, for his first-rate cooperation in this study. Thanks to Mrs. Shula Konig for her secretarial assistance.

REFERENCES I. Koenig K.G. ( 1967) Caries Induced in Laboratory Rats. Br. Dent. J. 123,585- 589. 2. Grobler R.S .. Du Toit I.J., Basson N.J. (1994) The Effect of Honey on Human Tooth Enamel In Vitro Observed by Electron Microscopy and Microhardness Measurements. Arch. Oral BioI. 39. 147- 153. 3. Edgar W.M .• Jenkins G.N. (1974) Solubility-Reducing Agents in Honey and Partly-Refined Crystalline Sugar. Br. Dent. J. 136. 7- 14. 4. Chakrabarti C .. L. ( 198 1) Progress in Analytical Atomic Spectroscopy. 2, 207. 5. Havezov I., Russeva E., Jordanov N . (1979) Fl ameless Atomic Absorption Determination of Phosphorus Using ZrC Coated Graphite Atomizer Tubes. Fresenius Z. Anal. Chern. 296, 125- 127. 6. Nicholson K. , DuffE.J. (1981) Fluoride Determination in Water. Anal. Lett. 14, 493- 517.

76

I. Gedalia et al. 7. du Toit I., Grobler S.S., v Kotze W.TJ., Basson, NJ. (1996) Fluoride, Calcium and Phosphorus Levels in Bee Honey and Water. S.Afr. J. of Science 91,391-392. 8. Gedalia I., Davidov I., Lewinstein I., Shapira L. (1992) Effect of Hard Cheese Exposure, With and Without Fluoride Prerinse, on the Rehardening of Softened Human Enamel. Caries Res. 26,290-292. 9. Sela Mo., Gedalia I., Shah L., Skobe Z., Kashket S., Lewinstein I. (1994) Enamel Rehardening with Cheese in Irradiated Patients. Amer. 1. Dent. 7, 134-136. 10. Grobler R.S., Jenkins G.N., Kotze D. (1985) The Effect of the Composition and Method of Drinking of Soft Drinks on Plaque pH. Br. Dent. J. 158,293--296. II. Gedalia I., Dakuar A., Shapira L., Lewinstein I., Goultschin J., Rahamim E. (1991) Enamel Softening with Coca-Cola & Rehardening with Milk or Saliva. Am. J. Dent. 4, 120-122. 12. White OJ. (1987) Reactivity of Fluoride Dentifrices with Artificial Caries. I. Effects of Early Lesions: F Uptake, Surface Hardening and Remineralization. Caries Res. 21, 126--140.

9

MEDICINAL HERBS AS A POTENTIAL SOURCE OF HIGH-QUALITY HONEYS Zohara Yaniv and Michal Rudich ARO The Volcani Center Bet Dagan, Israel

The use of plants for therapeutic purposes dates back to the beginning of time. Ancient civilizations, such as Egypt, China, India and the Incas of the Americas knew well the medicinal properties of herbs and used them in the prevention and treatment of diseases. Through the writings of great physicians, such as Hippocrates and Dioscorides, much of this ancient art had survived to our times. Nowadays we are witnessing the revival of the herbal tradition. There is a great interest in using this tradition in order to develop and define new, natural drugs for human use. Honey, too, has its important place in human nutrition since prehistoric time. It was considered not only as a sweetener but also as a high-quality food, with curative properties. The appreciation of honey is expressed in old manuscripts and documents, legends and mythologies. Honeybees feed on plants nectar, and honey is produced. Its qualities reflect the source of nectar which was used,so that the properties of the nectar and its chemical content are of great importance. In fact, the quality of the honey is directly related to the source of nectar. Therefore, there is a very great potential for using medicinal plants and nectars with very specific chemical compositions to produce honeys in the future with curative properties above and beyond those known hitherto. The taste, aroma and color of the honey is directly affected by the source of the plant nectar. (Table I). Examples include eucalyptus honey, which is dark, aromatic and spicy; thyme honey, light and scented; carrot honey, reddish and spicy, and so on. These examples indicate a direct transfer of components from the nectar to the honey. The use of honeys produced from diverse sources of nectar for specific therapeutical purposes is widely practised in France. Table 2 illustrates some examples of honeys, their source of nectar and their special medicinal indications. It is clear that the honeys and their source of nectar are used for identical medicinal treatments. Examples include lavender honey for diseases of the respiratory system, tilia honey, for spasms, insomnia and as a tranquilizer; acacia honey as an intestinal regulator, etc. (Table 2). It should be noted that this practice falls within the framework of folk medicine. 77

78

Z. Yaniv and M. Rudich Table 1. Colors and aromas of honeys Source of nectar Eucalyptus Thistle Carrot Onion Red Clover Cotton Sage Rosmary Thyme Savory

Color

Aroma and taste

dark dark green tone reddish brown bright yellow light light light light light

spicy spicy spicy onion flavor delicate flavor non·scented delicate flavor delicate flavor delicate scent delicate flavor

Scientific evidence for a transfer of active metabolites from the nectar to the honey produced is provided by studies performed in several laboratories. Some important topics are summarized as follows:

THE EFFECT OF PLANT ORIGIN ON THE ANTIBACTERIAL PROPERTIES OF HONEYS The antibacterial properties of honey have been known for a long time (Molan et ai, 1988). Scientists in various parts of the world have noticed that the intensity of these antibacterial properties depends on the plant source of the honey. In Brazil, Cortopassi-Laurino and Gelli showed that in Apis-bee honeys, the strongest antibacterial properties were found in honeys produced from mimosa and eucalyptus pollen. Mimosa was found to be also the best source of antibacterial honey produced by Melipona subnitida and Plebeiasp bees. (Cortopassi-Laurino and Gelli, 1991). Indeed, the use of mimosa and eucalyptus is known in phytotherapy and aromotherapy as antiseptic and antiflammatory agents. A study was performed in Poland recently on the antibacterial properties of honeys againsts everal strains of bacteria. In this study, too, it was found that various types of

Table 2. Honeys: sources and curative properties (folk medicine in France) Source of nectar

Curative properties

Acacia Erica

Intestinal regulator. Antiseptic (urinary tract), Diuretic.

Chestnut, forest flowers, sunflower

Stimulates blood circulation.

Lavender

Antiseptic, Anti-inflammatory (respiratory system), Anti-spasmodic. Anti-anemic,Antiseptic Anti-inflammatory (respiratory system),Diuretic. Anti spasmodic, Tranquilizer

Oak sap, fir tree

Tilia

Particular indications Good for intestinal stasis in infants. Urinary tract infections and kidney insufficiency. Improves blood circulation in general and varicose veins in particular. Diseases of the respiratory system. Diseases of the arteries. Certain types of anemia

Spasms of diverse origin, restlessness, insomnia and epilepsy.

Medicinal Herbs as a Potential Source of High-Quality Honeys

79

honey differ substantially in their antibacterial activity, depending on the plant source. The most active ones were honeydew and lime honey (Leszczynska Fik and Fik, 1993). This difference could be due to the compositions or amounts of essential oils and other components which were transferred from the plant to the honey. The direct effect of feeding medicinal plant extracts to honeybee colonies, on the antimicrobial activity of the honey produced, was studied in Egypt (Mishref et ai, 1989). Extracts from geranium, chamomile and majoram were fed to honeybee colonies once during an 8-week period. At the end of this period, honey in the combs was extracted and its antimicrobial activity was determined. Results showed that the honeys from colonies fed with medicinal plant extracts showed greater antibacterial activities than honey from control colonies, in the order chamomile> geranium> majoram. These findings correlate with the actual use of geranium, majoram and chamomile, as antiseptic and antimicrobial agents in folk medicine. The essential oils of geranium and majoram are widely used as antiseptic components of aromatherapic mixtures.

THE PRESENCE OF IDENTICAL FLAVONOIDS, CAROTENOIDS AND GLUCOSIDES IN BOTH HONEY AND THE NECTAR OF ITS HONEYBEES The following examples demonstrate the possibility that diverse secondary metabolites, besides essential oils, can be transferred by the honeybee, from the nectar to the honey, thus creating a honey with a specific chemical composition: 1. Sunflower honey was shown to be a very rich source of flavonoids. (Sabatier et ai, 1992; Sabatier et ai, 1988).The flavonoids were identified and their structures could provide an index of floral origin. 2. Analysis of many experimental and commercial citrus honey samples revealed the presence of the flavanone hesperetin in all samples. This flavanone was not detected in any non-citrus honey samples. The analysis of the flavonoids present in orange nectar revealed that the flavanone hesperidin was the major flavonoid detected. Hesperetin is probably produced by hydrolysis of hesperidin by the bee enzymes present in honey (Ferreres et ai, 1993). 3. Of a similar nature is the study of Czeczuga (Czeczuga, 1985), showing the presence of identical carotenoids in working bees and in the flowers being visited by the bees. The author suggested that some of the flower carotenoids are converted to more highly oxygenated forms in the honeybee. The total content and composition of carotenoids in foraging bees was related to the flower species visited. 4. The presence of the glucoside, arbutin in both bitter honey and in the nectar of the strawberry tree, Arbustus unedo, confirmed this tree to be the main source of the honey. (Floris and Prota, 1989). In Sardinia, this bitter honey is considered as having therapeutic value.

THE DANGER IN TOXIC HONEY We should be aware of the possibility of the presence of plant toxins in honey. Nectars from plants such as azalea, andromeda and rhododendrom (Ericaceae) are known to

80

Z. Yaniv and M. Rudich

contain gayanotoxins. Cases of intoxication, following the ingestion of such honey have been reported. Symptoms are similar to aconitine intoxication: progressive paralysis from the extremities to the diaphragm. (Alcaraz and Rios, 1991). The more recent report of the presence ofpyrrolizidine alkaloids in honey, may indicate a new health hazard (Deinzer et ai, 1977): these authors found that the hepatotoxic alkaloids known to occur in tansy ragwort (Senecio jacobaea L.) are also present in honey produced from the nectar of this species. These alkaloids (six were identified), are potentially carcinogenic, mutagenic and teratogenic, and may pose health hazards to the human consumer. However, due to the fact that ragwort honey is very bitter in taste and off-color, it is not likely that the individual consumer would consume enough honey to suffer acute effects.

FUTURE PRODUCTION OF HIGH QUALITY, THERAPEUTICAL HONEY Since the source of plant nectar can affect the composition and properties of the honey, future production of therapeutical honeys can be envisaged. Nectar-rich medicinal plants with desired therapeutical properties could be used as the source of nectar. Table 3 contains a list of some medicinal plants which could be used as a source of active compounds. Some of these plants have nectar-rich flowers, visited by the honeybee. Examples include aromatic plants from the Labiatae family, known for their high content of essential oils. Salvia ojJicinalis and S. Fruticosa; Coridothymus capitatus and Majorana syriaca. All three species are known for the treatment of colds, indigestion and external inflammations. Crataegus oxycantha is prescribed as a cardiac depressant and as a hypotensive. Relama raelam, a desert plant, is used for rheumatic pain and external wounds and has a potential application in chemotherapy against cancer. Echinacea angustifolia stimulates and strengthens the immune system and Cassia senna is a very popular natural laxative. Plants could also be used by feeding the bees with a sweetened extract of plant parts, such as leaves. roots, stems, on flowers. These plant parts should be selected on the basis of a high content of active compounds. The sweet extracts would then serve as a rich source for the production of medicinal honey. We hope that in the future the list of curative and medicinal honeys will be unlimited. This direction will offer a new avenue into natural medicine.

Table 3. Potential medicinal qualities of honeys Plant origin Salvia officinalis (Sage) Coridothymus capitatus (thyme) Majorana syriaca (Majoram) Crataegus oxycantha (Hawthorn) Retama raetam (White Broom) Echinacea angustifolia Cassia senna

Medicinal potential Stimulates bile secretion. wound and acne treatment, regulates menstruation. Antiseptic, expectorant and anti-spasmodic Expectorant, toothache and gum infection, indigestion, heart problems. Vasodilator, regulates blood pressure. Relieves abdominal and rheumatic pain, and wound remedial. Anti-carcinogenic activity. Anti-inflammatory, strengthens and stimulates the immune system. Laxative.

Medicinal Herbs as a Potential Source of High-Quality Honeys

81

REFERENCES Alcaraz, M.J. and Rios, J.L (1991). Pharmacology ofDiterpenoids. in: Ecological Chemistry and Biochemistry of Plant Terpenoids. (Harborne, 1.8. and Tomas-Barberan, F.A. eds.) p 230-263. Clarendon Press Oxford. Cortopassi-Laurino, M. and Gelli, D.S. (1991). Pollen analysis, physico-chemical properties and antibacterial action of Brazilian honeys from Africanized honeybees (Apis mellifera ) and stingless bees. Apidologie. 22. 61-73. Czeczuga, B. 1985. Investigations on carotenoids in insects. VII. Contents of carotenoids in worker bees feeding on flowers of different plants. Zoologica Poloniae 32. 183--190. Deinzer, M.L. Thomson, P.A Burgett, D.M. and Isaacson, D.L. 1977. Pyrro1izidine alkaloids: their occurrence in honey from tansy ragwort (Seneciojacobaea L.) Science, 195497-499. Ferreres, F. Garcia-Viguera, C. Tomas-Lorents, F. and Tomas-Barberan, F.A. 1993. Hesperetin: a marker of the floral origin of citrus honey. J. Sci. Food. Agric. Sussex. 61. 121-123. Floris I. and"Prota, R. 1989. The bitter honey of Sardinia. Apicoitore-Moderno. 80. 55--{)7. Leszczynska Fik, A. and Fik, M. (1993). Antibacterial properties of various types of honey and the effect of honey heating on antibacterial activity. Medycyna- Weterynaryjna. 49.415-419. Mishref, A. Magda, S.A. and Ghazi, I.M. 1989. The effect of feeding medicinal plant extracts to honeybee colonies on the antibacterial activity of the honey produced. Proceedings o[the Fourth International Conference on Apiculture in Tropical Climates cairo, Egypt. 80-87. Molan, P.C Smith, I.M. and Reid, G.M. 1988 . A comparison of the antibacterial activities of some New Zealand honeys. J. of Apicultural Research. 27. 252-256. Sabatier, S. Amiot, M.J.Aubert, S.Tacchini, M.and Gonnet, M. 1988. Importance of flavonoids in sunflower honeys. BuUetin-Technique-Apicole. 15. 171-178. Sabatier, S.Amiot. M.J. Tacchini, M. and Aubert, S. 1992. Identification of flavonoids in sunflower honey. J. Food. Sci. off. Publ. Ins!. Food. Techno!. Chicago. ILL. The Institute. 57. 773--774.

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THE UNIQUE PROPERTIES OF HONEY AS RELATED TO ITS APPLICATION IN FOOD PROCESSING Tsila Dvir Human Nutrition Unit Ministry of Agriculture 12, Aranya St., Tel Aviv, Israel

1. ABSTRACT Honey has a wide range of characteristics which differentiate it from sugar and other sweeteners. Some of the unique features of honey are the following: • Honey is a natural ingredient. Nowadays, when natural foods are so valued, this is very significant. • As opposed to the common belief, honey is not particularly calorie rich. • Honey contains many important nutritional values, like minerals and vitamins. • Honey has an important role in natural health care. It has potential in prevention and treatment of certain diseases. • Honey can serve many functions in cooking like browning (especially with microwave). It is an excellent bonding material and an ideal base for sauces. A strategy aimed at increasing the consumption of honey by the private consumers must emphasis these qualities and highlight honey's advantages over "competing" ingredients. Furthermore, these features should be attractive to food manufacturers because of their appeal to the consumer.. Such strategy can include the following components: • Bold and detailed labeling of products that contain honey. The label should list the nutritional values of the honey. • Presentation of honey and honey products in separate sections and on many shelves that are devoted to specific categories, like health food, honey products, spreads, sauces, cake decorations etc. • Marketing honey in new forms which are more convenient to use, like spread instead of liquid honey. • Unique and unusual containers that emphasis the natural aspects of honey. 83

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2. HONEY CONSUMPTION-A BRIEF HISTORY In ancient times honey was the common and unquestionable sweetener, and also a symbol to good life and wealth. All this was completely changed with the industrial revolution, the massive growth of sugar cane in the Americas and the knowledge to cheaply extract sugar from cane and sugar beets. This was an unavoidable development; there was no way by which honey could possibly supply the rapidly growing population which coincided with the rise of the standard of living; among other things this meant an enormous demand for sweeteners. The unfortunate thing that happened alongside this development was that gradually the wonderful food called bee-honey lost its glory: people regarded it as not much different from jam or cane sugar. In the last few decades the honey suffered two additional blows. First, the western world has become more calorie aware; and with the massive campaign against overweight all the natural sweeteners were among the first to be blamed as "the enemy", "the danger". Needless to say, the consumption of honey has suffered greatly. As a "natural" result, people started to shift to artificial sweeteners. This forum is not the place to analyze the consequences of this shift to artificial sweeteners, or to put it more boldly-the health problems which directly result from the exaggerated use of these non-natural substitutes. However, I feel that this matter is of critical importance to the honey future and therefore I will take the liberty to give at least a few clues: • Aspartame sugar substitutes cause symptoms from memory loss to brain tumors. It is sought to be one of the most dangerous substance [1]. • Saccharin, found in many "diet" drinks is considered a possible cancer hazard [2]. • The real amount of sugar substitute consumed in out diet exceeds by far the daily amounts approved as "safe" by USA FDA [1]. • It appears that many of the health problems that the US soldiers contracted in the Golf war are a result of the huge quantities of diet drink that they have consumed [3]. As if the damage caused by artificial sweeteners substitutes was not enough, another problem evolved. the public, and many professionals, got the idea the actually honey is almost the same as sugar. Consequently, housewives, cooks, institute mangers and industrialists said: if so, why bother with honey since sugar is cheaper and readily available? I am not sure how much of this basic misconception was due of somebody claiming that "honey equals sugar" or simply that there was nobody to clearly emphasize the differences between them. Whatever the reason-the damage is very real.

3. HONEY VS. SUGAR To show the full scope of the difference we should compare honey with sugar, item by item. To sum it up: honey is very different-and clearly superior-to sugar, when compared by almost all criteria.

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Table 1. The differences between honey and sugar [4,5] Property Source Health Qualities

Honey

A natural, unprocessed product Mild antibiotic Curing sore throat Avoiding constipation Curing diarrhea in babies Nutritional Properties: Water Content 20% Calories 304 cal per 100 gr. 13 kinds of sugars (glucose 33%, fructose Sugar Kinds 40%, other II kinds 5%) Vitamins (mg per 100 gr.) 0.004-0.006 Thaine B1: B6 Pyridoxine 0.008--0.032 0.1l..{J.36 Niacin 2.2-2.4 VitaminC mg per 100 gr. Minerals 0.4-3 Calcium 0.1-3.4 Iron Potassium 1.0-47.0 Also: Iodine, Copper, Magnesium, and traces of other Gourmet Qualities Rich taste Rich aroma Rich color Translucence Smooth texture Syrupy quality Used "as is" Cooking and Baking Qualities Adds color Browning Binding Preserves freshness & moisture Adds market value Symbolized tradition and "roots" Special Qualities Relates to the "back to nature" trends

Refined Sugar Industrially processed None

1% 375 cal per 100 gr only sucrose None

None

None

None

None

These two misconceptions--that honey, like sugar, is Calorie rich, and that sugar is an equivalent substitute to honey, have discouraged many potential honey consumers. The potential lose is estimated by perhaps hundreds of millions of consumers that would have used considerable amounts of honey in there daily diet. So we must find ways to reverse the trend-that is, to re-introduce wide sectors of the public and food industry with honey and its properties. Most of the data listed above is probably known to you. Furthermore, I will tell you a secret. Just a few days ago I went over note of a lecture I have delivered some 8 years ago. Surprise--it was almost all there. Now, the fact that we all knew the benefits of honey and the misconceptions about it is not the important issue. The real problem is that the users--I mean the public and the food industry as well as the professionals-are either ignorant of the facts or, as in the industry case, simply prefer to ignore them because of economical and other practical reasons.

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4. A STRATEGY TO INCREASE HONEY CONSUMPTION The honey industry has to form a strategy to face this problem. I would like to mention some of my thoughts on the subject.

4.1. General Finding Let me start with some general findings relating to the issue: • A "Back to Nature" fad is growing in Israel • The amount of health groups has doubled since 1987 • Confusion exists as to the difference between health foods and dietetic/low Calorie foods. • Women pay attention more than men to the food they eat. • Men think that health food/dietetic food is for sick people

4.2. Honey Drawbacks I then ask myself why honey is consumed less than other sweet spreads: • • • •

Availability-not always available Price--more expensive Variety-less then other Authenticity-how to tell real honey apart from imitations?

4.3. Honey vs. Sugar We should start a campaign to show that honey is not sugar-All facts listed in Table 1. We should emphasize nutritional value of honey-Again, see table 1.

4.4. Honey Is Much Safer We must convey the fact that honey is much safer then the artificial sweeteners. To do so we may have to point out what is reported about the dangers: Aspartame sugar substitutes cause worrying symptoms from memory loss to brain tumours. But despite USA FDA approval as a "safe" food additive, aspartame is one of the most dangerous substance ever to be foisted upon an unsuspecting public (Nexus Magazine, ref. 1).

4.5. Honey Uses Then we should talk about honey uses • • • • •

as a natural alternative to refined sugar barbecue sauce and dips in hot beverages, such as tea in cold beverages, such as ice tea, ice coffee, ice water+lemon and honey in manufactured foods: breads, cereals, sauces, bottled and canned beverages, fruit and carbonated drinks, milk products and candies

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4.6. Honey in Cooking and Baking Honey can and should be inserted into many home cooking and baking recipes. Honey can be used as food softener, for browning (good idea for micro-oven users), as a binding element, and as sweetener. It would serve well in many recipes, like: • • • • • • • • •

salads & vegetables spreads & butter breads, muffins and rolls marmalades and sauces cakes (traditional cakes, cheese cakes, oriental cakes, fruit cakes) cookies deserts and pies candies & snacks main entree

4.7. Honey Promotion And finally, promotion of honey consumption in all sectors is important. For example, honey should be placed in different sectors throughout supermarkets. Such sectors are spreads, cake decorating, sauces & salad dressings, Chinese food, natural and health foods [6,7]. Marketing honey in new forms which are more convenient to use, like spread instead ofliquid honey. Unique and unusual containers that emphasis the natural aspects of honey.

5. CONCLUSIONS Ifwe take these actions, I believe that 4 years from today, in 2000 Honey congress, the chairman or chairwoman will probably rise and point to a graph titled "Honey consumption 199Cr2000". It will show a dramatic increase.

REFERENCES I. 2. 3. 4. 5. 6. 7.

Gold M. D. (95), The Bitter Truth about Artificial Sweeteners, Nexus magazine, Vol. 2 # 28 Ames and Gold (I 987),Ranking Possibly Carcinogenic Hazards, Science pp. 236-271 Woodrow C M, Aspartame, Methanol & Public Health in: Journal of Applied Nutrition, 36 (I), pp. 42-53 Crane E., Honey, Heinemann London 1976, p. 264 Gebhards S. and Mathews R. Nutritive Values Foods, USA department of Agriculture, 1991 Hayes G. W. (1985), Lets Promote Honey, American Bee Journal, Feb 1985 Thomas and Payne (1988), A Leak at Japanese Market, American Bee Journal, June 1988

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HONEY AS A CLARIFYING AND ANTI-BROWNING AGENT IN FOOD PROCESSING AND A NEW METHOD OF MEAD PRODUCTION ChangY Lee Department of Food Science and Technology Cornell University Geneva, New York 14456

Today's consumers demand foods more natural with less food additives. We found honey to have very useful characteristics in food processing such as a fruit juice clarifying agent, an anti-browning agent among others. Honey can replace some of the conventional food additives in fruit juice or wine production and it can also be processed into a wide range of new beverage products. The following describes some of our research on honey that has been carried out for over ten years in our laboratory. Honey consists primarily of carbohydrates with an average concentration of about 80%. Water is the second major component at around 17%. The other important component, although its concentration is relatively low, is protein. The protein content was reported to have a wide range of 58-786 mgllOOg of honey with a mean value of 169 mgll OOg (1). In spite of low concentrations, honey protein is an important constituent because it influences many properties of honey and honey products (2). One of the unique properties of honey protein is that it readily interacts with phenolic polymers, including tannins, and forms macromolecule complexes (3). It is known that the reaction between proteins and tannins produces hazes in most natural fruit juices, and that sediments of fruit juices consist largely of phenolic materials mixed with protein (4). When a protein such as gelatin is added to a hazy juice, it entraps the particles and coagulates them by hydrogen bonds between the hydroxy groups of polyphenolic tannins and the carbonyl groups of the proteins (5,6). In a model solution of honey protein with tannic acid, it was found that the maximum rate of interaction occurred at the ratio of 1:2-3, tannic acid:honey protein (3). We employed this characteristic of honey protein in apple juice processing and found that the honey protein acts as gelatin-like in that it entraps the hazy particles and clarifies the juice. The optimum clarification conditions were found to be 4-5% honey concentration by weight at pH 3--4 at room temperature. Clarification oc-

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curred over a wide temperature range with the rate increasing rapidly as the temperature was raised (7). This process has been used commercially for several years. In order to find the derivation of this unique honey protein, honey samples from various floral sources, including citrus, dandelion, locust, basswood, clover, goldenrod, and honey from sucrose-fed bees were collected from various regions and compared for their juice clarification characteristics and electrophoretic patterns. There are reports that the honey protein could be originated in either the plant nectarines or the bees and that honey protein consisted of 4-7 components (8,9). We found that all honey samples we studied, regardless of their origin, contained the specific protein fraction that is responsible for clarifying apple juice and that is originated from honey bees (Apis melli/era) (10). In order to compare the protein component among honeys from different bee species. honey from A. melli/era bees from Cornell University, honey from A. laboriosa bees from Chhomrong, Nepal at the altitude of about 2,000 m and honey from A. cerana, common Indian honey bees from Kapre Chhap, Nepal at about 1,200 m from sea level were analyzed for their protein component. The electrophoretic pattern showed that protein fractions from the three diverse bee species were different and only Apis meli!fera and A. cerana bees produced the protein fraction with the apple juice clarifying activity, but A. laboriosa bees did not (11). Honey also has an inhibitory activity on polyphenol oxidase which is the major cause of enzymatic browning in fruit and vegetable products. In model solutions of polyphenols and polyphenol oxidase, added honey prevented browning reactions (12). We found this inhibitory effect of honey on Drowning in preparation of several fruit juices and wines and dehydrated fruit products. Grape juice prepared with added honey exhibited a similar color to commercial juice that was treated with ascorbic acid and enzyme. Table 1 shows effect of honey added to Niagara grape juice on color. Juice prepared with no treatment (control) was dark-brown with a higher absorbency at 420 nm and lower Hunter "L" value, but juices treated with honey showed no brown color with a low absorbency and higher Hunter "L" value. As early as in 1935, honey was added to fruit juices to make fermented products as a supplementary sweetening agent (13). We added honey to apple cider to bring soluble solids to 20° Brix and fermentation was carried out in the normal procedure at room temperature, 18-22°C for 30 days. The final products were analyzed for sensory quality and we found that apple wine treated with mild honey (orange blossom or clover-locust) was much better than apple wine made with added sugar (14). We also used honey in white grape wine production with no added sulfur dioxide. Several white grape cultivars were made into wines by conventional and new honey-treated (ameliorated with 22% honey solution) methods. We observed that wines made by the honey treatment with no S02 were very close to the conventionally prepared wines (commercial) in overall sensory quality. It appeared that honey acted as an anti-browning agent similar to S02.

Table 1. Application of honey in Niagara grape juice production Juice preparation

pH

Absorbency (420 nm)

Hunter"L"

Control (no treatment) Commercial" Honey treated#

3.78 3.11 3.11

0.71 0.07 0.07

47.30 58.41 58.36

*Treated with ascorbic acid and enzyme. #Added honey (3% by weight).

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In order to apply honey as an anti-browning agent in minimal processing of fruits, apple slices were treated by dipping them into a 3-5 % honey solution, packaged under the modified atmosphere conditions and then stored for 2-3 week at 3-5°C. We found that apple slices treated with honey were lighter in color and firmer in texture compared to those of apple slices prepared with commercial anti-browning acidulants. We also applied honey to various fruit slices for dehydrated products. Sensory quality of the dehydrated fruits (apple and pear slices, raisin) treated with honey was much better than those prepared by a conventional method with added S02' Although mead (honey wine) has been produced for many years, it has never been as popular as grape wines in the U. S. One of several reasons is that conventional mead lacks the overall quality of a typical alcoholic beverage. Due to the hazing problem in mead caused by honey protein, traditional mead-making requires boiling a honey solution for 30-{:)0 minutes before fermentation. This heat process is so excessive that it develops undesirable flavors, such as harshness and bitterness. To mask these undesirable flavors, a high residual sugar content and a long aging process are required. In order to overcome these problems and improve the mead quality, honey was passed through an ultra-filtration (UF) membrane with various molecular weight cut-offs (l0-50K) to remove haze forming proteins and then fermented in the usual manner. A complete fermentation and bottling process were accomplished within 3 weeks so there was no need for a long clarification and stabilization period. Sensory quality and stability of the UF-treated mead are superior to mead made by the conventional method. Several commercial firms are producing products by using this new method. This UF -treatment of honey opened the door for a wide range of honey application in food processing and there are already many new products being produced commercially using UF-treated honey.

REFERENCES 1. White, J. w.; Rudyj, O. N. (1978) The protein content of honey. J. Apic. Res. 17.234-238. 2. Paine, H. S.; Gertler, S. I.; Lothrop. R. E. (1934) Colloidal constituents of honey. Ind. Engng. Chem. Analyt. Edn. 26, 73-81. 3. Lee, C. Y. (1984) Interaction of honey protein and tannic acid. J. Apic. Res. 23, 106-109. 4. Johnson, G.; Donelly, B. J.; Johnson, D. K. (1968) The chemical nature and precursors of clarified apple juice sediment. 1. Food Sci. 33, 254-257. 5. Calderon, P.; Van Buren, J.; Robinson, W. B. (1968) Factors influencing the formation of precipitates and hazes by gelatine and condensed and hydrolyzable tannins. J. Agric. Food Chem. 16,479-482. 6. Gustavson, K. H. (1954) Interaction of vegetable tannins with polyamides as proof of the dominant function of the peptide bond of collagen for its binding of tannins. J. Polymer Sci. 12,317-324. 7. Lee, C. Y.; Kime, R. W. (1984) The use of honey for clarifying apple juice. J. Apic. Res. 23, 45-49. 8. White, J. W.; Kushnir, I. (1967) Composition of honey. VII. Proteins. 1. Apic. Res. 6, 163-178. 9. Bergner, K. G.; Diemair, S. (1975) Protein in honey. II. Gel-chromatography, enzymatic activity and origin of honey proteins. Z. Lebensm. Forsch. 157.7-13. 10. Lee, C. Y.;Smith, N. L.; Kime, R. w.; Morse, R. A. (1985) Source ofthe honey protein responsible for apple juice clarification. J. Apic. Res. 24, 190-194. 11. Lee, C. Y.; Smith, N. L.; Underwood, B. A.; Morse, R. A. (1990) Honey protein from different bee species in relation to apple juice clarification activity. Amer. Bee 1. 130,478-479. 12. Oszmianski, J.; Lee, C. Y. (1990) Inhibition of polyphenol oxidase activity and browning by honey. J. Agric. Food Chem. 38, 1892-1895. 13. Fabian, F. W. (1935) The use of honey in making fermented drinks. The Fruit Prod. J.I4, 363-365. 14. Kime, R. W.; Lee, C. Y. (1987) The use of honey in apple wine making. Amer. Bee J. 127,270-271.

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BEE-POLLEN Composition, Properties, and Applications

M. G. Campos,] A. Cunha,l and K. R. Markham 2 ILaborat6rio de Farmacognosia da Faculdade de Farmacia Universidade de Coimbra 3000 Coimbra, Portugal 2New Zealand Institute for Industrial Research and Development POBox 31310, Lower Hutt, New Zealand.

1. INTRODUCTION During ancient times, people throughout the world commonly used pollen, praising it for its goodness and medicinal properties. Some of the reasons the ancients used beepollen are why wc use it today. To date no scientific evidence has been cited to disprove the claimed properties of bee-pollen. One claim, attributes to bee-pollen the ability to reduce the rate of ageing. It is said that this product has a special factor that can improve health, and increase vital energy. The basis for this claim is the fact that the queen bee, and only the queen bee, can live five or six ycars on a diet of royal jelly, that the bees do with bee-pollen. Other bees only eat this diet for the first two days of their life and after that, they eat honey. They live for only a few weeks. An undeniable fact is that, this product can't be synthesised in the laboratory, can't be easy adulterated and has been taken by people over thousands of years, without manipulation or recorded side effects. It is fortunate that a number of scientists, world wide are earring out research in many different areas, out of interest, to test and prove the nutritive and curative properties claimed for the bee-pollen that we "steal" from the hive.

2. NUTRITIVE VALUE Concerning the value of bee pollen as a natural health food for people, it is well known that bees collect pollen because of its high content of protein (average 35%). Approximately half of the "protein" fraction is in the form of free amino acids which can be assimilated immediately by the body. 93

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It has been established by Peris (1984)1, that in a daily dose of 15 g (about a soup spoon) of bee-pollen provides the minimum amount of amino acids that the human body needs. Further, at least 40% of the content is made up of various forms of sugar. Huidobro et al. (1986,1987)2,3 reported a value of 61%, but they included in this fraction not only carbohydrates like reducing sugars (fructose, glucose), maltose, sucrose and polysaccharides, but also starch and other polysaccharides that can't be absorbed such as cellulose, hemicellulose and lignin, esporopolenin, etc" We have also verified that about 50% (Campos et ai, 1994)4 of the weight of the bee-pollen corresponds to a material that can't be extracted with organic or aqueous solvents. Lipids and minerals (carriers of calcium, phosphorus, magnesium, iron, copper and manganese, etc.) represent 5 and 3% respectively. Most (60%) of the fatty acids aTe in the free form. Bound fatty acids, which reflect the compositional profile of pollen, were characterised by a high level of a-linolenic acid (70%) and by small amounts of linoleic and oleic acid. Palmitic acid is the most abundant saturated fatty acid (Seppanen et al.,l989f Vitamins in bee-pollen include not only vitamin B complex and ascorbic acid, but also vitamins A, D and E. The levels vary between the pollen species and with season. For example, Herbert et aI., (1987)6 established that the seasonal thiamine (vitamin B 1) levels in bee-collected pollen varied greatly depending on the floral source and the time of year. It is important not to forget that in biological samples, vitamin B 1 activity is due not only to thiamine but also to the mono, di and triphosphate derivatives of thiamine. Thiamine and its mono and diphosphate forms are normally the most prevalent. Pollen apparently supplies the honey bees requirement for this vitamin since there is no real evidence that it can be synthesised by insects. Thiamine is relatively stable in acid pH, and pollen provides an acid environment. In stored pollen the vitamin has been shown to exist for up to 4 years (Hagedorn and Burger, 1968l Xie et al. (1994), in China, have studied the effect of bee pollen on maternal nutrition and foetal growth. They state that plant pollen collected by the honeybee is a natural nutrient. They studied the effects of the bee pollen from Brassica campestres L. on maternal nutrition and foetal growth using pregnant Sprague-Dawley rats. Pollen-fed dams had greater body weight and higher levels of haemoglobin, total protein, serum iron and albumin while the foetuses of pollen-fed dams had greater body weight and lower death rate. No gross external, visceral or skeletal malformation was observed in the foetuses. These results were interpreted as indicating that bee-pollen could improve maternal nutrition without affecting normal foetal development. Bee pollen was therefore considered a practical and effective nutrient during pregnancy.s

3. ANTIBIOTIC ACTIVITY Gillian et al., 1989 isolated one-hundred and forty-eight moulds from the following samples of almond, Prunus du/cis. pollen: floral pollen collected by hand; corbicular pollen from pollen traps placed in colonies of honeybees, Apis mellifera, in the almond orchard; and bee bread stored in comb cells for one, three and six weeks. They verified that corbicular pollen (pelletized bee pollen) contained 99,8% almond pollen when collected from pollen traps placed in bee colonies in an almond orchard. If micro-organisms are responsible for the fermentation and accompanying chemical changes in pollen stored in comb cells by honeybees, the moulds maybe a component of a required microbial content. It was considered, for example that they could produce antibiotics, organic acids and enzymes, products for which there are uses in industry. These compounds may limit the

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growth of deleterious micro-organisms in stored pollen and provide enzymes for utilisation of nutrients. If so, pollen moulds must produce enzymes involved in protein, lipid, and carbohydrate metabolism. Indeed, most moulds from all pollen sources were shown to produce caprylate esterase-lipase, leucine aminopeptidase, acid phosphatase, phosphoamidase, ~-glucosidase and N-acetyl-~-glucosamidase. A high percent of the isolates (50 %) from all sources also gave positive reactions for alkaline phosphatase. The majority of moulds identified were Aspergilli (17%), Mucorales (21%) and Penicillium (32%). We have also found a predominance of Penicillium in samples of Eucalyptus corbicular pollen collected in Portugal9 • In general, the number of isolates decreased in pollen following collection and storage by the bees. Moulds that may have been introduced by bees during collection and storage of pollen include Aureobasidium pullulans, Penicillium corylophilum, Penicillium crustosum and Rhizopus nigricans.. Mucor sp., the dominant moulds in floral pollen, were not found in corbicular pollen and bee bread. Thus, as with yeast and Bacillus ssp, the mould flora found in corbicular pollen and bee bread may be the result of microbial inoculations by bees. Chemical changes in pollen therefore may result both from additions by bees of secretions of glands during regurgitation of honey sac contents and from microbial fermentation. Such modifications may allow some species to survive but not others. Even though moulds were more numerous than yeast or Bacillus ssp in the samples, pollen was rarely overgrown by moulds. Potential microbial spoilage of stored pollen thus may be controlled by antibiotic substances produced by the normal microflora of bees or by those naturally present in pollen and/or honey. These results may assist in the understanding of the antibiotic power of bee pollen. We must emphasis that this is our interpretation of the research work referred to. The antibiotic power of flavonoids that has already been proved for propolis activity (Houghton et aI., 1995)\0 must also be considered a factor. 39 Chauvin and Lavie (1956)11 in a study of the antibiotic activity of bee-pollen, found a "factor" with activity against Spullorum. S. gallinarum. S. Dublin, E. coli, Proteus vulgaris, S. subtilis Caron and B. pyocyaneus. We have followed the extraction procedure that they used to extract this factor and it seems that this factor could be the flavonoids. We are currently checking the activity of flavonoids from extracts of bee-pollens from different floral origins. Chauvin and Lavie also verified that different floral sources of bee pollen gave different levels of activity. In our research we have found that flavonoid type varies according to the family of pollen plant source (Campos et aI., 1996).12

4. ANTIATHEROSCLEROTIC ACTIVITY Another property accredited to well known species of bee-pollen when consumed as a medicine is the lipid-lowering effect on serum. Literature relating to bee pollen however was not found in our literature search. We therefore quote below, research carried out on pollen generally which may help to provide an understanding of this activity. Some groups have revealed that extracts of pollen have beneficial properties, lowering serum lipid levels (Samochowiec et Wojcicki, 1981; Wojcicki et Samochowiec, 1984)13., reducing atherosclerosis plaque intensity (Wojcicki et aI., 1986)1\ and decreasing platelet aggregation both in vitro (Kosmider et al. 1983)15 and in vivo (Wojcicki et Samochowiec, 1984)16. These findings have been confirmed in humans (Wojcicki et aI., 1983)17. Additionally, studies in humans suggest that a diet supplemented with polyunsaturated fatty acids (as found in pollen extracts) decreases whole blood viscosity and re-

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duces triglyceride and cholesterol levels in patients with cardiovascular disease (Saynor et al. 1984)18. With these studies in mind Seppanen et al. (1989),19 analysed the fatty acid composition of the fat-soluble pollen extract (Cernitin GBX) by gas chromatography in order to account for the anti-atherosclerotic activity. The analyses revealed that most (more than 60%) of the fatty acids were in the free form, characterised by a high content oflinolenic acid (18:3n-3, a-LLA) (70%) If fatty acids are involved in the beneficial effects referred to, the role of a-linolenic acid as a precursor of eicosapentenoic acid (20: 5n-3, EPA) is significant, since EPA is considered to be responsible for reduced platelet aggregation. EPA in vivo is incorporated into platelet phospholipids, to some extent replacing arachidonic acid and exerting an antithrombotic effect either by competing with remaining arachidinic acid for cyclo-oxygenase and lip oxygenase or by being converted to less proagreggatory PGH 3 and TXA 3 (Moncada et Vane, 1984).20

5. ANTI-NEOPLASTIC ACTIVITY The same extract of pollen from AB Cernelle, Vegeholm, Sweden (Cernitin GBX), was analysed by Zhang et aI., 1995 21 , who isolated and characterised a cyclic hydroxamic acid [2,4-dihydroxy-2H-I,4-Benzoxazin-3(4H)-one] from a pollen extract, which inhibits cancerous cell growth in vitro. This hydroxamic acid is the active compound in the pollen extract which might be responsible for the symtomatic relief in patients with benign prostate hyperplasia. Seventy-nine patients with this disease, aged from 62 to 89 years, were treated with pollen extract and showed a mild beneficial effect on prostate volume and on urination (Habib et aI, 1995; Yasumoto et aI., 1995). Interestingly, one of the major beneficial qualities attributed to bee-pollen by the ancients was its usefulness in the treatment of prostatitis.

6. POLLEN PHENOLICS AND ANTI-OXIDANT AND FREE RADICAL SCAVENGING ACTIVITY As evident from the above, many chemical, biochemical and microbiological studies have been carried out with a wide variety of compounds from pollen, but only recently have scientists focused on a special group, the phenolic compounds. In fact, these exist in low quantities in bee pollen and other plant parts (Campos et aI., 1990)22 (Markham, 1982)23, (Markham and Campos, 1996)24, as is common for non-nutrient compounds, which are important for life. Because in pollen we can find that many groups of compounds differ during the year or from one species to another, we have developed a method that can identify the corbicular pollen species with precision using the phenolic compounds. This method offers reproducible conditions that produce a consistent profile (Campos et aI., 1996)13. We have verified that in bee pollen, and in pollen in general these compounds are reliable species indicators when. mature pollen is used. A special type of flavonoid glycoside was commonly found in bee-pollen. After comparison with the literature we verified that these glycosides often had the same glycosidic linkage. Because these pollens attract insects these glycosides may have a special taste which honeybees recognize as indicating a suitable food, as been observed with other

Bee-Pollen

97

insects (Harborne, 1994)25. Certain types of flavonoids are known to have a distinctive taste that insects are attracted to. The profile of phenolic compounds in pollen is different from one species to another thus providing a species-specific character for pollen identification. In our studies we have verified that in the bee-pollen collected from the Central Coast of Portugal, the floral species were Eucalyptus globulus, Ranunculus sardous, Salix atrocinerea, Taraxacum sp and Ulex europaeus. Between the Northern and Southern regions we found the Cistus ladanifer and in the Central Interior Erica australis was the major species collected for the bees. Salix atrocinerea and Rhaphanus raphanistrum were minor species in the samples examined by us. New Zealand samples were found to contain the same species as those from the Central Portuguese Coast and the Central Interior. This may be due to the climate and because the samples were also collected close to the coast. Only the Eucalyptus was not found in these samples. In contrast the Eucalyptus bee-pollen was commonly found in samples of bee pollen from Australia. The results of Godinho et Nansen, 1995 26 suggest, as do ours, that weeds are preferred by the bee, effectively competing with the cultivated plants. After analysis of the free-radical and anti-oxidant activity of bee-pollens, it was evident that only the phenolic compounds are active. The derivatives of cinnamic acid are the most effective phenolics, as evidenced by the loss of much activity when they are absent. This activity is relevant to the claim that bee-pollen has regenerative properties for the body and long lives are often attained by bee-pollen users. The literature records much research work that proves that flavonoids, and in general, phenolic compounds, have an antioxidant / radical scavenging effect in the human body and that they can be given to prevent and cure some diseases (Pathak et aI., 1991)27. It is probable that active free radicals, together with other factors are responsible for cellular ageing and can cause biological imbalance that in extreme conditions can be responsible for premature death. Ageing is a concern in all stories of human life and the story of the search for the elixir o/youth, continues to this day. There are many theories presented in literature to explain the ageing process. These include the "Genetic Theory"-that ageing and long life are genetically programmed ("Programmed Ageing") (Hayflick, 1965 28 ; Warner et aI., 1987 29 ; Holliday et al. 1985 3°). The Stochastic Theory-this theory claims that ageing is the result of a series of destructive events that affect all levels cellular organisation ("Random ageing"), e. g. Error catastrophe theory (Orgel, 1963)31 and Free radical theory (Harman, 1956)32. All these events cumulatively induce ageing and cellular death. A free radical is a neutral or charged structure that possesses an unpaired electron and is represented by the symbol R·. Our body produces active forms of oxygen and oxygen free radicals in the course of normal metabolism. These radical species are very reactive, produce secondary free radicals, inter and intramolecular bridges, oxidation, halogenations and molecular fragmentation. Cell membrane lipids are vulnerable to free radicals. "Peroxidation" of polyunsaturated fatty acids gives many derivatives that are indicative of the intensity of the phenomena of cellular oxidation: for example, lipid hydroperoxides, aldehydes (malonylaldehyde-MDA and 4-0H nonenal), conjugated dialdehydes, hydrocarbons and fluorescent conjugates (lipofuscins). Some of these derivatives have biological activities (chemostatic action, cellular division effect). Other deleterious effects of free radicals result from their action on polysaccharides (hialuronic acid de-polymerization), proteins (chemical modification of the crucial aminoacids for the enzymatic functions, fragmentation of the peptide chain), and nucleic acids (chromosome bridges).

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M. G. Campos et aI.

The free radical theory of ageing became more credible after the discovery that active free radicals are involved in cellular degradation process, such as cardiovascular diseases, arthritis, cancer, diabetes, etc .. They have also been implicated in Parkinson disease and Alzheimer disease (Feher et al., 1986)33,34. Bee-pollen has been used for centuries to protect the body from diseases and especially to slow the process of ageing. In studying this, Dudov et Starodub (1994)35, fed rats with bee pollen for one month and studied the resulting state of the erythrocyte redox system. It was established that the content of glutathione, total SH-groups, as well as activities of glutathione peroxidase and glutathione reductase in these animals were increased in comparision with the control group. Simultaneously a decrease of malondialdehyde and dienic conjugates in erythrocytes was demonstrated. It was concluded that the antioxidative system is non-specifically activated and oxidative processes blocked in erythrocytes of rats fed on bee pollen. In another study, primary and secondary humoral immune response (the level of specific IgM and IgG) as well as the intensity of delayed-type hypersensitivity of sheep erythrocytes were investigated in rabbits fed with bee pollen for one month. It was shown that bee pollen, acts as an immunomodulator in that it stimulated humoral immune response and changed the reaction of delayed-type hypersensitivity (Dudov et al.,1994).36 The free radical scavenging activity of different floral species of bee pollens were recently studied by us. The results show a big difference between the species containing derivatives of phenolic acids (which seem to be the more effective scavengers) and those containing only flavonoids (Campos et aI, 1994)37 (Campos et al., 1996 b)38. Returning to the story of life and ageing, lipid oxidation in the presence of oxygen, causes rancidity, a problem recognised since ancient times in relation to the preservation of oils and fats. Oxidation of vegetable oils and animal fats, produces organoleptic changes (colour, smell and taste), as well as changes in density, viscosity and solubility. Surprisingly, it was not until 1950 that scientists started to recognize the significance of lipid oxidation in Biology and Medicine (Dinis, 1995).39 Other evidence of the benefits of bee-pollen comes from Olympic athletes that eat this product as a supplement. The coach concluded that bee pollen increased the athletes crucial recovery time after stressed performance and enabled them to actually improve their performance the second time around (Fischer, 1986)40. This may be explained by the anti-oxidant theory. Physical exercise with aerobic characteristics Clln impose on the organism a supplementary consumption of oxygen that in humans can amount to ten times the level of basal metabolism. The concept of "oxidative stress" implies a disruption of the precarious equilibrium between the production and quenching of oxygen free radicals. With physical exercise this "stress" is brought about by excessive production of such radicals. Paradoxically, people involved in sport regularly live in a situation of "permanent oxidative stress" with the consequent biological cost. Today scientists know that fitness training, like an adaptive mechanism, increases the anti-oxidant defences. Thus more oxygen is consumed without negative consequences. Nevertheless, in studies carried out at "Faculty of Sport Sciences and at the Centre of Experimental Cytology of University of Porto", by Silva (1993)41, it has been verified that a fit athlete when exercised to exhaustion, suffers dramatically high oxidative injury. These results suggest that anti-oxidant defences could be insufficient in an extreme situation like this one. It was also shown in this study that those athletes with a high level of oxidative injury, who recovered quickest (within one hour after the exercise) produced high plasma levels of vit.E. This suggests that there is a systemic anti-oxidant response to the physical exercise.

Bee-Pollen

99

It is tempting to speculate about the anti-oxidant protection provided by bee pollen in these cases, because of the amount of phenolic compounds in the product given to the Olympic athletes. It is known today, for example, that some flavonoids can protect vit C, because when taken together the flavonoids are oxidized first. On the basis of this and other factors discussed above we can rationalize the benefits of bee pollen in the diet (Bors et aI., 1995)42. Diet has long been linked to Medicine and good health, and the use of bee products, including bee pollen, is frequently recorded. For example, about 487-380 b.c. (before Christ), Herodicos de Selymbrie, master of gymnastics, inaugurated a diet method, the "Grand Arte". This formed the basis of the Medicine, that Hippocrate de Cos (460-377 a.c.) established in his Diet Treatise, The Food and the Man Nature (Debry, 1991).43 Even then, in an empirical way, they knew that the utilisation of a correct diet could have a prophylactic effect in delaying or preventing the onset of some diseases. Research of the biological activity of compounds isolated from natural sources and used in folk medicine has established a relationship between chemical composition and pharmacological activity of many such natural drugs. With bee pollen, the results ofinvestigations of its therapeutic activity, add rationale to the "magic" properties attributed to it in the past. Bee pollen it seems, can be more than a prophylatic and has a place in treatment alongside other natural or synthetic products. More scientists are now beginning to give credence to the potential of folk remedies, and bee-pollen is one of them. "We need to ponder the present, to survive in the future, while learning from the past."

REFERENCES I. Peris J. (1984), "Produccion y comercio de los produtos apicolas en Espana". El Campo del Banco de Bilbao. Apicultura 93. Bilbao. 2. Huidobro J.F., Simal J., Muniategui S. (1986) "El polen: Determinacion del contenido en agua" Offarm 5 (3), 73-77. 3. Huidobro J.F., Simal J., Muniategui S. (1987) "El polen apicola: Determinacion del contenido em glucidos" Offarm 6 (5) 57-71. 4. Campos M.G., Cunha A. Rauter A. (1994) "Portuguese bee-pollen as a source of flavonoids" Acta Horticulturae,429-432. 5. Sepp"nen T., Laakso 1., Wojcicki J., Samochowiec L., (1989). An Analytical study on fatty acids in pollen extract. Phytotheraphy Research, 3 (3) 115-116. 6. Herbert E. W. Jr., Vanderslice J.T., Meis-Hsia Huang, Higgs D. J. (1987) Levels of thiamine and its esters in bee collected pollen using liquid chromatography and robotics. Apidologie 18 (2), 129-136. 7. Hagedorn H. H. and Burger M. (1967) Effect of the age of pollen used in pollen supplements on their nutritive value for the honeybee. II. Effect of vitamin content on pollens. J. Apic. Res .• 7,97-101. 8. Xie Y., Wan B., Li W. (1994) Effect of bee-pollen on maternal nutrition and foetal growth. Hua-Hsi-I-KoTa-Hsueh-Hsueh-Pao. 25 (4) 434-437. 9. Results not published 10. Houghton P., Woldemariam T., Basar A., Lau C. (1995) Quantification of pinocembrin content of propolis by densitometry and high performance liquid chromatography. Phytochemical Analysis, 6, 207-2\0. II. Chauvin R., Lavie P. (1956) Recherches sur la substance antibiotique du pollen. Annales de I'Institute Pasteur 90 (4) 523-527. 12. Campos M.G., Markham K., Mitchell K., Cunha A. (1996). An approach to the characterisation of bee pollens via their flavonoid/phenolic profiles. Phytochemical Analysis to be submited 13. Samochowiec L., Wojcicki J., (l981) Effect of pollen on serum and liver lipids in rats fed on a high-lipid diet. Herba Polon. 27, 33 Wojcicki J., Samochowiec L., (1984) Further studies in Cernitins: screening of the hypolipidemic activity in rats. Herba Pol on. 30, 115.

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14. W6jcicki J., Samochowiec L., Bartlomowicz B., Hinek A., laworska M., Gawronska-Szklarz B., (1986). Effect of atherosclerosis in rabbits. Atherosclerosis 62, 39. IS. Kosmider K., W6jcicki 1., Samochowiec L.. Woyke M., G6rnik w., (1983). Effect ofCernilton on platelet aggregation in vivo. Herba Polon. 29, 237. 16. W6jcicki 1., Samochowiec L (1984). Further studies on Cernitins: screening of the hypolipidemic activity in rats. Herba Polon. 30, lIS. 17. Wojcicki J., Kosmider K., Samochowiec L., Woyke M. (1983) Clinical evaluation of Cernilton as lipidlowering agent. Herba Polon. 29, 55. 18. 19. Sepp"nen T., Laakso I., Wojcicki J., Samochowiec L., (1989). An Analytical study on fatty acids in pollen extract. Phytotheraphy Research, 3 (3) 115--116. 20. Moncada S., Vane J. R., (1984) Prostacyclin and its clinical applications. An. Clin. Res. 16,241. 21. Zhang-X., Habib F.K., Ross M., Burger U., Lewenstein A., Rose K.. Jaton 1.e. (1995) Isolation and characterisation of a cyclic hydroxamic acid from a pollen extract, which inhibits cancerous cell growth in vitro. 1. Med. Chern. 38 (4) 735--738. 22. Campos M.G., Sabatier S., Amiot M., Aubert S. (1990) Characterisation of flavonoids in three hive products: Bee-pollen, propolis and honey. Planta Medica 56 (7) 580--581. 23. Markham K. R. Techniques of Flavonoid Identification. London, Academic Press. 1982, 24. Markham K., Campos (1996) 7- and 8-0-methylherbacetin-3-0-sophorosides from bee-pollens and some structure/activity observations. Phytochemistry in press 25. Harborne J. in The Flavonoids: advances in research since 1986. (1. Harborn Editor) Chapman & Hall. 1994 26. Godinho J. et Nansen e. (1995) Estrategia alimentar da abelha domestica (Apis melliferaj como polinizador da cultura da meloa (Cucumis melo) em estufa. 0 Apicultor 3 (8) 25--29. 27. Pathak D., Pathak K., Singla A. (1991). Flavonoids as medicinal agents--recent advances. Fitoterapia 28. Hayflick (1965) The limit in vitro lifetime of human diploid strains. Exp. Cell Res. 37, 614-636. 29. Warner H.T., Butler R.N., Sprott R.L., Schneider E.L.. Modem theories of ageing. Ageing vol. 31 (Raven Press N. Y.) 1987 30. Holliday R.. Kirkwood T.B.L., Cuzin F. (l985}--EMBO Workshop on "Oncogenes, immortalization and cellular Ageing" Grignon, 3-7 Setembro. 31. Orgel L.E .. (1963) The maintenance of the accuracy of protein synthesis and its relevance 5' to ageing. Prod. Natl. Acad. Sci., 49, 517-521. 32. Harman D. (I 956}--Thefree radical theory of ageing in "Free Radicals in Biology, (A. Pryor Editor) Academic Press, 1984,5,255--275. 33. Feher J., Csmos, Vereckci A. (1986) Clinical importance of free radical reactions and their role in the pathogenesis of various human diseases. in Free Radical Reactions in Medicine, 48-147. 34. Various Comunications--IQ Congresso de Radicais Livres em Quimica, Biologia e Medicina. Instituto Superior Tecnico, Lisboa. Portugal 21-23 de Junho de 1993. 35. Dudov LA., Starodub N.F., (1994). Antioxidant system of rat erythrocytes under conditions of prolonged intake honeybee{lower pollen load. Ukr. Biokhim. Zh.66 (6) 94-96. 36. Dudov LA., Morenets A.A., Artiukh v.P.. Starodub N.F., (1994). 1mmunomodulatory effect of honeybee flower pol/en load. Ukr. Biokhim. Zh. 66 (6) 91-93. 37. Campos M.G., Cunha A., Navarro M.e., Utrilla M.P. (1994). Free radical scavenging activity of bee pollen. Bull. Group Polyphenols. 17,415-416. 38. Campos M. Markham K., Mitchell K., Veiga J. Cunha A., Paredes F., Frazao L. (1996 b) Therapeutic activity of bee pollen-Preliminary essays. International Conference on: Bee-products: properties. applications and apitherapy. May 26--30. Tel-Aviv, Israel. 39. Dinis, T. (1995). Peroxida,iio /ipidica membranar. Actividade antioxidante de farmacos fen6licos (acetaminofeno, salicilato e 5-aminosalicilato). Disserta9ao de doutoramento apresentada it Faculdade de Farmacia da Universidade de Coimbra. pp 13-14. 40. Fischer W. L. How to fight cancer & win. 1986 41. Silva P. C. 1993 Radicais livres de oxigenio em Medicina Desportiva, I Q Congresso de Radicais Iivres em Quimica, Biologia e Medicina 42. Bors w., Michel e., Schikora S., (1995) interaction offlavonoids with ascorbate and determination of their univalent redox potentials: A pulse radiolysis study. Free Radic. BioI. Med. 19 (I) 45--52. 43. Debry G. (1991). Evolution des concepts en nutrition humain. Cah. Nutr. Diet. 26 (6),435-442.

13

CLINICAL EVALUATION OF A NEW HYPOALLERGIC FORMULA OF PROPOLIS IN DRESSINGS w. Fierro Morales and 1. Lopez Garbarino Department of Surgery--Outpatient Service Hospital "G. Saint Bois" Montevideo, Uruguay

ABSTRACT A new hypoallergic formula of propolis in dressings was evaluated against the standard formula. Patients (229) with wounds of diferent types that required ambulatory care were included. The new formula presented the same therapeutic action than the standard one with a notorious disminution of signs oflocal intolerance (l ,8% vs.18%) The therapeutics properties of pro polis were demostrated : anti-intlamatory, anti-microbian and stimulant of wound healing by a faster grow and initiation of granulation tissue. Other properties comprobated were the analgesic effect mainly in bums, the easier handling of the ambulatory patient and the posibility of avoid hurting the granulation tissue. The wounds and bum were completed healed in an average of I I days, the septic wounds en 17,5 days, and a complete reparation in the 67% of the ulcers in 36 days. In this paper a methodology for the use of this dressing is described, pointing the importance of acomplishing it by the medical personnel.

INTRODUCTION The complexity of the composition of propolis and the sinergic action among its diferent componcnts was well established by researchers of the Oxford University in 1990 (1,2).

At the local level a group of actions are important: anti-microbian, anti-inflamatory (by inhibition of the hydrofolate reductase(3), minimizing the prostaglandin production) and anti-oxidant(4) (neutralizing the nocive effects of the free radicals at the local level). These properties complemented each other, explain the therapeutic benefits obtained. Other benefit obtained is the analgesic effect at the locallevel(5). 101

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The utilization of propolis in the treatment of cutaneous lesions of diferent nature such as burns, wounds and ulcers is positive in the reparation process, shortening the healing of wounds and reducing the risk ofinfections(6), but in some cases it is observed allergic dermititis by contact. In 1990 it was published en Acta Chir. Plast.(7) a paper studying the beneficial effects of propolis in the treatment of burns against other products. The treatment of burns is an old issue not exent of contradictions(8). The utilization of topic anti-microbians it has become generalizated in spite of the lack of alleatory research. In the cutaneous burns it is increased the local production of prostaglandins and free radicals. The liberation of proteolitic enzimes and the aditional production of oxigen radicals contributes to the production of aedema, controlling these factors limits the convertion of the burns of partial thickness in complete burns(9). These phenomenae also are presents in the phisiophatology of the wouds. wich in the case of a terrain with vascular insufficiency or metabolic anomalies. are lessening or stopping the repairing process. Carefully pathologic studies were done in rats and explain the anti-inflamatory and healing properties of propolis : minimizing the acute inflamatory exudate (by inhibition of the degranulation of basofiles), estimulating the macrophagic activity, promoting the colagen production and stimulating the epitelization ( incremente of the number of mitosis of the basal layer and favoring the queratinization) Diaz, P.Prof of Histology, School of Medical Sciences of La Habana (oral comunication); (10,11,12).

OBJECTIVES To evaluate the tolerance and efficacy of the new hypo allergic formula of propolis in dressings against the standard formula.

MATERIAL AND METHODS The present research was done in the period from Sept./91 to Aug.l93 at the Emergency - Dept.of Surgery- Hosp. "G.Saint Bois" Hospital (Montevideo) with ambulatory patients. In the initial months were progressively evaluated 4 formulae named A, B, C, and S, and the best results were obtained with the type C (or NF ); 229 patients were in-

Table 1. Population of study and days of treatment. Propo Ii s type No.of cases

Bums Wounds Infected Wounds Ulcers

Days oftreatm.

Age average

NF(C),

B'

NF(C)

B

61 29 53 22

15 10 30 9

25 41 48 63

35 29 46 70

'Propolis C=Ncw Fonnula. 2% of pro polis in hydrosoluble cream. 'Propolis B=Standard formula. 8% of propolis in hydrosoluble cream.

NF(C)

B

II

16,5

11 17,5 36

11,2

16

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103

c1uded, 115 females and 114 males, from I year old to 92 years old, with varied pathology classified as follow: a) bums, b) wounds, c) infected wounds, and d) ulcers.

METHODOLOGY OF TREATMENT • Wounds. Washing with SF an Benzalconiun c1orure, applying the propolis dressing and closing over it with simple cotton dressing (oclusive treatment)(l3,14). Each other 48 hs.- 72 maximum - washing and replacement of the propolis dressing (burns excepted). • Burns. Flictenae resection, washing with SF, drying with warm air, applying the propolis dressing that remains there until complete granulation when fall by itself. Clinical controls each 48-72 hs., allowing moisture the dressing with propolis solution. • Infected wounds, ahcesses and ulcers. After bacteriological study it was made a surgical cleaning and/or drainage of the abcess, washing with SF and benzalconiun c1orure. Applying the propolis dressing and simple cotton dressing. In some cases it was used propolis lotion in order to get a better penetration. In 13 cases the area was drained with dressing. Clinical control and replacement each 48-72 hs. or whenever is indicated. In patients with ulcers were indicated to remain in bed with an elevated leg to favour the venous pressure.

RESULTS AND DISCUSSION 1. Among the 165 patients treated with propolis NF it were observed 3 cases

(1,8%) presenting local signs of intolerance and occurring in an average of 16 days after the treatment was initiated. The signs were prurite, eriteme and exudation and they promtly disappeared after the treatment was suspended. Unlikeness, the group treated with the propolis B registered a 18% of local intolerance and appearing at an average of the 9th. day of the initiated the treatment. 2. Only 4 patients had antecedents of local intolerance against the propolis B but they did not registered any intolerance with treatment of propolis NF. 3. After this initial period it was confirmed the good tolerance of propolis NF, so from that moment the study continued only with propolis NF. 4. There were included 61 patients with burns according the following age categorization: less of 10 years 19 cases; from II to 40 years 25 cases; from 40 and more years 17 cases. It was obtained good reparation and analgesic in an average of 11 days in the 3 groups, but we had the clinical impression that the younger group got a faster evolution. In 36% of the cases it was indicated ATB. The bums included were only those that did not required an inpatient treatment, most of them of 2nd. degree, with a few hours of evolution, and produced by agents as water, foods, bitumen and some cases by a direct contact with fire or hot metals. Those bums with several hours of evolution and signs of infection were included in the group of infected wounds and treated as such. It was observed, the same as other authors( 15), the propolis has the following beneficial effects: Easy handling of the ambulatory patient; less nursing care by replacing

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fewer times the dressing, analgesic effect, minimized pain with no replacing and only moisture the dressing, it has a revitalizing power, because the non replacement it has no trauma effect in the granulation tissue. 5. A group of 83 patients with infected wounds and abcesses was formed(ages from 3 to 84 years). In 18 of them it was found the following germs : estaf.aureus (5 cases) estrepto B hemoliticcus (2 cases), B.piocianic (2 cases) and only a case of nuemococcus, proteus and serratia. In 6 patients no pathogenic germ was isolated. In 85% of cases it was indicated a simultaneous treatment of ATB and propolis. In all but one case was observed good tolerance to propolis. A cure average of 17,5 days was obtained. 6. Non infected wounds with a few hours of evolution formed a group of 39 patients. Only in 41 % of the cases were indicated ATB. In 3 cases the wounds were drained with a dressing. In 28 cases the treatement with propolis NF was effective and it was obtained the cure in II days average. 7. The repairing process also was evaluated in 31 patients with ulcers, 22 were treated with propolis NF (age X = 65 r=I7-92 y.), 12 cases older than 62 years. Localization all of them in legs: 8 maleol.intern., 7 maleol.extern., 2 bi-maleol.; 16 had a terrain of veinstasis, 8 had arteriolar compromise, 5 post-trauma ulcers, and 2 diabetic patients. In 10 cases a pathogen germ was isolated: estafil.aureus in 4 cases, estreptoc. in 2, piocianic en 3, and proteus in I. ATB was indicated in 62% of the cases. All the skin layers were compromised by the wound and in 2 cases reached the aponeurotic level. The size of the ulcers was somewhere among 2 and 5 cms. A patient 17 years old with a postthrauma ulcer of 10 cms.of 45 days of evolution and already including the aponeurotic layer was healed in 28 days. The cicatrization was obtained in 68% of the cases in an average of 36 days. Similar results were obtained by Rodriguez in Cuba(16) testing the propolis in 80 patients. In those cases treated with propolis B, the treatment was suspended due to allergic contact dermatitis. The venous estasis subjacent was responsible for the slow evolution and the lack of cicatrizal response in most of them (17,18). The anti-inflamatory and regenerative effect of propolis was shown in the cicatrization of those wounds with a previous history of non tendency to the cicatrization. 8. In 16 cases of wounds drained with the propolis dressing, it was shown the fast regression of the inflamatory process and the supuration (anti-bacterian action). We must stress that the dressing was putted after a surgical cleaning and that the same was performed as many times as necessary (19). In all cases the cicatrization was obtained in an average of 22 days.

CONCLUSIONS I) 229 patient with wounds of varied nature were evaluated and it was shown the good tolerance to the new formula of propolis dressing with only a 1,8% of the cases with signs of local intolerance. There is no quantitative research in this field in Uruguay, according to our previous experiences the results obtained with the treatment of propolis NF in the present research, as a measure of the allergic reaction to the propolis, were significantly lower than those observed with the propolis standard formula. 2) A very satisfactory evolution and cicatrization was obtained in c~ses of wounds with and without

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105

infection and burns. A fast cure, shorter treatment period and less septic complications were obtained. In 67% of the cases with ulcers (trophics and non trophics) the cicatrization was obtained. The anti-inflamatory property was demostrated by the fast reduction of aedema of the limits of the wound. The cicatrizing action was evident by the early formation of the granulation tissue(between 4th. and 5th.day) being more notorius in those with a torpid evolution. The anti-microbian capacity was evident by a fast regression of the septic component of the supurated wounds. 3) Other positive elements to be quoted are: the importance of carefully follow the methodology, to be a natural product, easy application, cheap, and produced in this country by standard pharmaceutical procedures.

REFERENCES 1. Villanueva V. Barbier N. Gonnet M, Lavie P. Les flavonoids de la propolis isolement de une nouvelle substance bacteriostatique: Ie pinocembrine(dihidroxy-5,7flavonona). Annales del, Institute Pasteur 1970; 118( 1):84--7. 2) Greenaway W, Scaysbrook T and Whatley FR. The composition and plant origins of propolis: A report of work at Oxford Bee World 1990;71 (3): I 07-18. 3. Strehl E. Vol pert R, Eistner E, Biochemical Activities of propolis extracts. Inhibition of Dihydrofolate Reductase. Zeitschrift Fur Naturforschung C-A Journal of Biosciences 1994; 49 (1-2):39--43. 4. Pascual C, Torricella RG, Gonzalez R. Scavengig action of propolis extract against oxigen radicals. J. Ethnopharmacology 1994;41 (1-2):9-13. 5. Silvestre N, Stranieri G. y Bezerque P. Anestesia troncular de propolcos comparado con lidocaina. International Dental Research 1984. 6. Ponce de Leon R. y Benitez P. Estudio morfologico comparativo del efecto de la propolina, el alcohol y el balsamo de Shostakovski como agentes cicatrizantes. Investigaciones Cubanas sobre el propoleo. I Simposio sobre Propoleos. Varadero-Cuba 1988:269-71. 7. Troshev K. and others. Regulation of traumatic skin wound healing by influencing the process in the regnbouring bone of wound. Acta Chir. Plast.1990;32(3): 152-163. 8. Glastrup H, Knudsen L. y Mazanti C. Tratamiento de las quemaduras, nuevos enfoques de accidentes y catastrofes. Hezagono 1980;7(8): 1-10 9. Muller MJ, y Hernodon ON. Cirugia. EI reto de las quemaduras. The Lancet 1994;24(6):360-364. 10. Magro F. Application of propolis to dental sockets and skin wounds. Univ. Sch. Dent. 1990;32( I): 4--13 11. Bunta S. Efecto anti-inflamatorio de las pomadas con propoleos I1ISimposio Intemacional de Apiterapia. APIMONDIA 1978:94-7. 12. Neychev H. and others. Inmunomodulatory action of pro polis. Acta Microbiol. Bulg. 1988;23:58--62 13. Boswick J. Quemados. Clin. Quinirg. Norteamerica. 1987; I. 14. Lepore G. y Giuria H. Quemaduras menores, tratamiento de urgencia y seguimiento ambulatorio. Catedra de Cirugia Plastica y Quemados. ProfJ. Hornblas. 15. Ramirez M.Propoleos en el tratamiento de quemados. I' Jornadas Nacionales de asistencia integral del nino quemado. 1989. 16. Rodriguez A. y col. Resultados de la aplicacion del propoleos en ulceras y gangrenas de las extremidades inferiores. Investigaciones Cubanas sobre el propoleos. I Simposio sobre Propoleos. Varader-Cuba 1988:171-74 17. Mescon H.y col. Dermatitis por estasis 0 eczema varicoso. In: Robbins. Patologia estructural y funciona!. Interamericana Mexico. 1975: 1331-2. 18. Apositos para las ulceras de las piemas. Drug and Therapeutics Bulletin. 1094;24(3): 172-5. 19. Temesio P. Nuevo metodo del tratamiento local con prop6leos. Investigacion clinica. Policlinica de diabetes. Hosp.Maciel 1983.

14

PRESENT STATE OF BASIC STUDIES ON PROPOLIS IN JAPAN Tsuguo Yamamoto Nihon Natural Foods Co., Ltd. 6-26-12 Nishishinjuku, Shinjuku-ku, Tokyo, Japan

I. INTRODUCTION In October 1985, the 30th International Apicultural Congress held in Nagoya first introduced propolis into Japan, when the latest reports on basic studies on propolis and the clinical application of propolis to the intractable diseases were presented by the researchers and the medical doctors from various countries abroad. Together with the introduction of a crude propolis by the Brasilian beekeepers, the name of 'propolis' became known instantly with the possibility of future promising substance. However during first five years since 1985 propolis was regarded only as health foods and folk remedies and was not paid much attention to by scientists. Propolis studies made public in 1987 related only to general findings, introduction of overseas literatures, and distribution of flavonoid components I). In 1991 at the 50th Japan Cancer Society Meeting, Matsuno of the National Institute of Health reported three compounds with tumor killing activities in propolis from his experience curing terminal-stage uterine cancer with propolis2) With this as momentum, many of pharmaceutical companies and research institutes have started studying propolis with the prospect of its use more promising. Among them Hayashibara Biochemical Laboratories Inc. in the course of his interferon research, took an interest in the BRM (biological response modifiers)-like effects in propolis, such as the antivirus effect 3 ) and immune activating effect 4 ) and has been studying propolis these ten years, and published a fairly number of remarkable research results. This paper describes representative studies on the antimicrobial effect and cytotoxic effect of propolis from 1985 up to now in Japan.

II. ANTIMICROBIAL ACTIVITY OF PRO POLIS The strong antimicrobial activity of Propolis is often compared to a "natural antibiotic". Its antimicrobial characteristic against various microbes has recently been studied throughout the world. 107

108

T. Yamamoto

In Japan, Aga et a1. 5), 6) Matsun0 71, ltoh et a1. 8), and Nakano et a1. 9 ) have studied and reported the effects of propolis and antimicrobial substances isolated from propolis on various types of fungi, yeast's and bacteria, as well as on specific pathogenic microbes, such as Helicobacter pylori, and Methicillin-resistant Staphylococcus aureus (MRS A) between 1992 and 1995.

1. Antimicrobial Activity of Brazilian Propolis 5) In 1992, Aga et al. of Hayashibara Biochemical Laboratories Inc. published their research results on the antimicrobial activity of an ethanol aqueous solution extract of Brazilian propolis against 8 strains of fungi, 4 strains of yeast, and 42 strains of bacteria, including Enterobacter and Actinomyces. In their study, the propolis extract showed strong antimicrobial activity against Micrococcus lysodeikticus (MIC: 15.6 /-tg/ml), Bacillus cereus, Enterobacter aerogenes (MIC: each 31.3 /-tg/ml), Corynebacterium equi, Mycobacterium phlei, Thermoactinomyces intermedius, Arthroderma benhamiae & Microsporum gypseum (MIC: each 62.5 /-tg/ml), but showed very weak antimicrobial activity against Enterobacter such as Bifidobacterium, Lactobacillus, Eubacterium, and Bacteroides. This antimicrobial spectrum agreed with the results of German propolis studied by 1. Metzner et al. 10) and of American propolis studied by L. A. Lindenfelser lll in which antimicrobial activity was recognized against 7 strains of bacteria such as Micrococcus lysodeikticus & Bacillus cereus, and Arthroderma benhamiae. The MIC values of Brazilian propolis show about 3 times more antimicrobial activity against Bacillus subtilis, Staphylococcus aureus and Arthroderma benhamiae than that shown by German propolis. This result suggests a difference in antimicrobial substance contents and the presence of stronger antimicrobial substances. Accordingly, it is expected that isolation and identification of these substances and further elucidation of the mechanism of the antimicrobial action will be conducted.

2. Isolation and Identification of Antimicrobial Compounds in Propolis 6) In a later study, Aga et a1. isolated and identified three antimicrobial compounds from Brazilian propolis. As shown in Fig.l, they identified these compounds as 3,5diprenyl-4-hydroxy cinnamic acid (as Compound I), 3-prenyl-4-dihydro cinnamoloxy cinnamic acid (as Compound 2), and 2,2-dimethyl-6-carboxy ethenyl-2H-l-benzopyran (as Compound 3). The results of this study were published in 1994. Table I gives the antimicrobial activity (MIC values) of these compounds against three types of microbes selected as specimens to be studied. As shown in Table 2, compared with the original propolis extract, 3,5-diprenyl-4-hydroxycinnamic acid (Artepillin C) had stronger antimicrobial activity against cutaneous fungi (ex. Microsporum, Arthroderma), putrefying bacteria (ex. Bacillus), Corynebacterium, and pyogenic bacteria (ex. Pseudomonas). It has long been said that propolis is effective for the treatment of dermal disorder and burns. This characteristic of propolis suggests that Artepillin C plays a leading role in the antimicrobial and anti-inflammatory activities of pro polis. Later, Kimoto et a1. disclosed that Artepillin C played a principal role not only antimicrobial activity, but also in "anticancer action" of Brazilian propolis to be introduced below. 121

Present State of Basic Studies on Pro polis in Japan

109

o OH

R'=H, R 2 =CH 2CH=C(CH 3 )2 Compound 1 (3,5-diprenyl-4-hydroxycinnamic acid) (ArLepillin C) R'=CO(CH 2 )2 Ph , R2=H Compound 2 (3-prenyl-4-dihydrocinnamoloxycinnamic acid)

o OH Compound 3 (2,2-dimeLhyl-6-carboxyeLhenyl-2H-l-benzopyran)

Figure 1.

3. Anti-Helicobacter pylori Substances in Propolis

8)

Itoh et a1. of the Zenyaku Kogyo Co., Research Institute examined the antimicrobial activity of Chinese, Argentine and Brazilian propolis against Heliocobacter pylori, whose connection to gastritis and gastric ulcer was suspected. Their research results were published in 1994. According to these results, Argentine propolis showed the highest antimicrobial activity with an MIC value of 50 f..\g/ml, followed by Chinese propolis at 100 Ilg/ml, and Brazilian propolis at 200 Ilg/m1. They reported that each propolis showed an anti-Heliocobacter pylori effect.

Table 1. Antimicrobial activity of isolated compounds 1_3 6 )

MIC ( '-' g/ml)

Composilion'

B.cereus

Compound

5.296

15.6

31. 3

compound 2

2.396

31. 3

62.5

compound 3

0.896

Crude propolis

125 31. 3

E.aerogenes

125 31. 3

A.benhamiae 15.6 ,250 62.5 125

, as a percenlage relalive lo lhe dry solid crude propolis

T. Yamamoto

110

Table 2. Antimicrobial activity of artepillin C6) MlC Slrains

g/ml) Propolis

Microsporum gypseum (lFO 8231) Arlhroderma benhamiae (JCM 1885)

(/I.

Arlepillin C 7.8

62. S

15.6

62. S

Bacillus cereus (lFO 3466)

15.6

31. 3

Bacillus sublilis (ATCC 6633)

31. 3

31. 3

Corynebaclerium equi (lFO 3730)

31. 3

62. S

Micrococcus lysodeiklicus (lFO 3333)

31. 3

Pseudomonas aeruginosa (lFO 3453)

31. 3

Enlerobacler aerogenes (lFO 3321)

31. 3

Mycobaclerium smegmalis (JCM 5866 T )

31. 3

Mycobaclerium phlei (JCM 5865 T )

62.5

Slaphylococcus aureus (ATCC 6538P)

62.5

2S0

Slaphylococcus epidermidis (ATCC 12228)

62.5

SOO

Thermoaclinomyces inlermedius (JCM 3312 T )

62.S

lS.6 12S 31. 3

SOO 62. S

62.5

Micrococcus luleus (lAM 1099)

125

2S0

Propionibaclerium acnes (JCM 6425 T )

12S

SOO

Flavobaclerium meningoseplicum (lFO 12535)

2S0

2S0

Kloeckera apiculala (JCM 5947)

SOO

SOO

Saccharomyces cerevisiae (lFO 0214)

SOO

2S0

HO

HO OH 0

OH 0

pinocembrin

compound

galangin (R=OH), chrysin (R=H)

MIC

( /.1

g/ml)

pinocembrin

12.5

galangin

25

chrysin

25

Figure 2.

111

Present State of Basic Studies on Propolis in Japan

They further isolated fractions that had anti-Heliocobacter pylori activity from propolis by column chromatography, and identified these fractions as pinocembrin (MIC: 12.5 Ilg/mi), galangin and chrysin (MIC: each 25 Ilg/ml). The anti-Heliocobacter pylori activity of pinocembrin showed an antimicrobial activity equal to that of Lansoprazole, which was used as a control (Fig.2). However, pinocembrin showed a lower antimicrobial activity against another microbes other than Heliocobacter pylori (MIC: 50 ->200 Ilg/ml). This suggested that one of the factors contributing to propolis's anti-ulcer effect was specific anti-Heliocobacter pylori activity due to flavonoids such as pinocembrin.

4. Anti-MRSA Compound Isolated from Brazilian Propolis9 ) In 1995, Nakano et al. of Hayashibara Biochemical Laboratories Inc. studied active substances in Brazilian propolis to determine whether Brazilian propolis exhibits antiMRSA activity. This substance was 3-prenyl-4-dihydrocinnamoloxycinnamic acid having a chemical structure shown in Fig.3. As illustrated in Fig.3, only 2 Ilg/ml of this substance evaluated by the MIC method had an anti-MRSA activity 100 to 400 times higher than that of each component of the propolis ethanol extract and propolis. In 1994, this substance had been isolated from Brazilian propolis by Aga et a1. 6) at Hayashibara Biochemical Laboratories as an antimicrobial substance together with Artepillin C. At the time, however, no study was made on anti-MRSA activity. Regarding studies of anti-MRS A activity of propolis made so far, Grange et al. 13 ) reported on the relatively strong anti-MRS A activity of the extract of French propolis in 1990. Activity of the same degree as that in this report was recognized in Brazilian propolis. The anti-MRSA activity of propolis is considered due to the synergetic effect of complex components in propolis. During their work of isolation, the fraction containing this 3-prenyl-4-dihydro-cinnamoloxycinnamic acid was the most active, so Nakano et al.

o

o

~O

OH

3-prenyl-4-dihydrocinnamoloxycinnamic acid

co.pound

IIIC( JL9.m1)

compound

IIIC( JL 9.111)

200

9a1an9in

)aoo

caffeic acid

)800

Kaemferol

>800

coumarie acid

)800

ArLepi11in C

cinnallic acid

)800

anLi-KRSA compound

propolis exLracL

Figure 3.

200 2

112

T. Yamamoto

estimated that this substance was the main component of the anti-MRSA activity of Brazilian propolis. Findings on the anti-microbial activity of propolis and isolated substances against specific microbes in Japan have been described here in. It is interesting that, in addition to the known effect of flavonoids, both Artepillin C reported by Aga et al. and New Clerodane Diterpenoid founded by Matsuno as an anti-tumor substance have strong anti-microbial activities. We expect that new substances with antimicrobial activity against specific pathogenic microbes will be discovered from propolis, and through basic research, application studies for clinical use will be soon to follow.

III. IMMUNE ACTIVATING EFFECT AND CYTOTOXIC EFFECT OFPROPOLIS Commercially available books and European papers l4) describe that propolis and its components are effective in inhibiting human malignant tumors and cancer cells. However, there had been no papers objectively evaluating the mechanism or degree of their antitumor effects before 1990. In Japan, Matsun0 2 )?) of the National Institute of Health, Arai et al. 15 ). Kimoto et al. 12 ) of Hayashibara Biochemical Laboratories Inc., and Suzuki et al. 16 ) of the Suzuka College of Technology published results of studies on propolis, and on some of its isolated or fractionated active components between 1991 and 1996.

1. Cytotoxic Effect of New Clerodane Diterpenoid Isolated from

Brazilian Pro polis 7)

In 1990, Dr. Matsuno of the National Institute of Health found that the ethanol extract of Brazilian propolis transformed human hepatic carcinoma cells and uterine carcinoma cells cultured in vitro, and that it inhibited their growth. Afterwards, he orally administered a large quantity of a propolis drink to one of his relatives, who had cancer of uterine cervix and was unable to undergo surgery because of her poor physical strength. He also continuously applied the propolis ethanol extract directly to the affected part. As a result, the lesion became a burn-like scar several weeks later, and her cancer of uterine cervix disappeared2 ). Since then, he started to study the purification and isolation of the antitumor active substances contained in propolis, and observed the cytotoxic effect of fractionated substances on human hepatocellular carcinoma, HuHI3. As a result, he found this effect in the following three substances that is, the known substances of quercetin, and caffeic acid phenethylester, and a new compound belonging to clerodane diterpenoid. He reported his findings at the 50th Japanese Cancer Association Congress in 1991. Clerodane diterpenoid, in particular, was very active in destroying tumor cells, especially human cervical carcinoma cells (HeLa cells) and Burkitt's Lymphoma cells in addition to HuHI3. His findings suggested that this substance showed selective toxicity to tumor cells, stopped the cell growth cycle in the gene synthesis phase (phase S), changed the properties of the cell membranes, and killed cells by disturbing their ionic permeability.

Present State of Basic Studies on Propolis in Japan

Rl

113 100

R2

;e e....

~

Q) (.)

50

"iii >

• primary rabbi t. kidney ... !luman diploid f1brcb1ast. • 1IIIH13 (t.umor)

.~

:::l

II)

0

1

100

clerodane diterpenoid

(~g

1000

I ml)

Figure 4.

Although the details of the mechanism are still being analyzed, this substance acts on tumor cells in phase S, when tumor cells are growing more actively than normal cells and synthesizing genes rapidly. Therefore, Dr. Matsuno assumes that tumor cells are ultimately destroyed because they are growing at a different speed. The effects of clerodane diterpenoid on HuH 13 and two normal cells ( untransformed primary rabbit kidney cells and human diploid fibroblast cells) were examined. As a result, as is shown in FigA, a large difference in the effective concentrations of this substance was found, and it was proved that an appropriate concentration of this substance could kill tumor cells without affecting normal cells. These results emphasized the possibility that a new treatment, which could kill tumor cells exclusively without damaging normal cells, could be developed by determining an appropriate concentration and administration method for this substance. Dr. Matsuno confirmed its treatment effects on cancer patients, and made public the details of his experience at the 51 st Japanese Cancer Association Congress.

2. Biological Effects of Propolis on Macrophage Function and Tumor Metastasis 15) 17) Dr. Arai et al. of Hayashibara Biochemical Laboratories Inc. has begun to study the biological activity of Brazilian propolis from various perspectives since 1990. In order to make the propolis easy to use, they powdered the propolis extract using anhydrous maltose, and tried to confirm the effectiveness and mechanism of a BRM-like substance, aiming at isolating and identifying this substance. This propolis powder was dispersive in water, was free of endotoxin that macrophage activates, and contained 13.8% propolis-derived solids, so that concentrations during the experiment were all expressed in terms of propolis powder. They discovered a macrophage activation phenomenon related to the immune function of living organisms, then in 1993 published the results of their minute studies on the effects of propolis on macrophage spreading, phagocytosis, motility, and cytokine production!7). In 1994, they made public its inhibitory effect on lung metastasis in mice!5). After adding a culture medium containing propolis powder solution to the abdominal macrophage obtained from the BALB/c mouse, stretching as shown in Fig.5 was observed. This phenomenon correlated to propolis concentration and time, and a dose reaction was recognized.

114

T. Yamamoto

Figure 5.

Similarly, the effects of this propolis on fowl phagocytosis and motility, TNF production by LPS (lipopolysaccharide; pyrogen) coexistence. cytotoxic factor NO (nitrogen oxides) production inhibition were studied. As a result, it was revealed in vitro that its effects depended on concentration and time, or the presence or absence of an LPS stimulus. Moreover, in vivo, though TNF production in mouse blood was increased by an LPS stimulus, by administering 0.2 mg and 2.0 mg propolis Imouse 3 hours before an LPS stimulus, ten times more TNF could be produced. Propolis as one of BRM (biological response modifiers)-like substance activated macrophages by, Propolis by itself did not produce cytokine in vivo; however cytokine production was sharply accelerated by an LPS stimulus. These results suggest the activation effect of immune cells which produce cytokines. Prior to a test on the inhibition of tumor metastasis by this propolis, its effects on the growth of mouse colon carcinoma cell Colon 26 were studied. As shown in Fig.6, this propolis inhibited the growth of the cells in a concentration- dependent way, and had a direct effect. Next, 40 f.lg of this propolis was administered to BALB/c mice (7-week-old females), on to which Colon 26 cells were then transplanted. Various concentrations of this propolis were continuously administered for 6 days, and the metastatic lesions in the lung were counted 14 days later. As shown in Table 3, the dose of propolis was optimum. Lung metastases were reduced to 80% in the group receiving 0.1 mg propolis powder, 57% in the 0.2 mg group, and 70% in the 0.4 mg group. Based on these results, they assumed the following . The dose of propolis administered to the mice was too small to directly inhibit cell growth in vivo. Administration of propolis activated immune cells, mainly macrophages, and inhibited and removed the implantation of metastatic tumor cells as foreign matter to lung tissues, leading to a reduction in the number of metastatic lesions.

Present State of Basic Studies on Propolis in Japan

115

120

100L---------..'"

•

Maltose (Base)

•

Propolis POwder

80

~

60

3

o '"

40

20

o

0.4

0.2

0.6

concentrat ion

0.8

1.0

(mg/ml)

Figure 6.

3. Cytotoxic Effect of Artepillin C Isolated from Brazilian Propolis t2 ) Three substances isolated and identified from Brazilian propolis by Aga et aI., especially Artepillin C, have been shown to have a stronger antimicrobial activity than the crude propolis61. The above-mentioned study on the antitumor effect of propolis made by Dr. Arai et al. suggested the presence of a substance in propolis that was effective in killing tumor cells. This study also suggested that c1erodane diterpenoid, an antitumor substance in

Table 3. Inhibition lung metastasis of mouse colon carcinoma (colon 26) by propolis l51 dose Group Propolis powder

Anhydrous maltose Physiological saline

1. v. /mous

Colonies numbers

Average ± SE

O. 1mg/O. 2ml

13

O.2mg/O.2ml

13

66. P±

O.4mg/0.2ml

13

81. 0 ± 13.3

O.4mg/O.2ml

13

134.0 ±

O.2ml

13

115.9 ± 15.4

grafted cells: 5xlO'/mous, . ; p quadrate plates and Koschewnikow glands > venom glands and venom sac > Koschewnikow glands > Dufour gland and pure venom.

Table I. Secretory activity of the tarsal glands. Quantitative data. (Lensky et coil., 1984; Hyams, 1988) Categories 3-day-old queens 6-month-old queens 18-24-month-old queens Workers Drones

Number n of insects 5 8 8 70 70

Mg / hour / bee x ± SD 0.2180 ± 0.6500 ± 0.4660 ± 0.0718 ± 0.0630 ±

0.009 0.051 0.038 0.020 0.015

140

P. Cassier and Y. Lensky

Alarm: sting sheaths> setaceous membrane> venom gland and venom sac. Stinging: setaceous membrane> sting sheaths> quadrate plates and Koschewnikow glands> venom sacs. The setaceous membrane is directly connected to the sting sheaths and is disposed all around the base of the sting. The fine structure of the epithelium of the setaceous membrane of workers and queens is similar and does not show any characteristic of an exocrine gland. The flexible, untanned cuticle (4--5 !lm) rests on a flattened, inactive epithelium (3.5-4.5 !lm). It is abundantly covered with cuticular infoldings and multiforked bristles which, in the vicinity of the sting sheaths are embedded in an electron densc substance as on the sting sheaths. These cuticular expansions increase the surface of the evaporation area; so, the outer surface of the setaceous membrane is ideally suited for the discharge of pheromonal secretions from the sting sheaths and the Koschewnikow glands. The function of the setaceous membrane is to serve as a platform for the discharge of alarm pheromones that are secreted elsewhere and accumulate on its surface. I. 4. The wax gland complex (15, 16) of the honey bee workers consists of three cell types: epithelial cel1s, oenocytes and adipocytes, which act synergistical1y to secrete wax, a complex mixture of hydrocarbons, fatty acids and proteins (lipophorins). The hydrocarbons coming from the oenocytes and the proteins (17) from the haemolymph transit across the epithelium via the large smooth endoplasmic reticulum cisternae connected to extracellular spaces and through the mirror cuticular plates along a wel1 developed extracellular and pore canal filamentous system linked to wax canal filaments of the epicuticle. There is no excretory ducts or intermediate cel1s. The height of the epithelial cells is agedependent; in freshly emerged workers « one week old) the epidermis is a flat epithelium. At the height of wax secretion (2 week-old) the lengthened cells have a fibrillated appearance. In fact, the secretion of wax is a constant process even in overwintering workers. During the ageing of the workers, the fat body cells and the oenocytes show the volumetric variations similar to the epithelial cells. The wax may act as a slow release substance for pheromones secreted by other exocrine glands. The volatile and odoriferous part of the wax secretion may also act as a pheromonal component implies in the regulation of the wax secretion or building?

II. THE SECRETORY EXOCRINE GLANDS OF THE TYPE III In the type III (Fig. 3) exocrine glands (2, 3), each glandular cell shows a central reservoir lined by the invaginated plasma membrane; this membrane forms a10 () numerous cristae and microvilli; the content of the secretory vesicles is poured between them. The glandular cell is caped by a tubular cell named the duct cell because it secrete, in the central cavity, a thin duct edged by an epicuticular wall. In the reservoir of the glandular cell, the proximal part of the duct (end-apparatus) has a porous structure permeable to the secretory products. So, each glandular cell and its associated duct cell form a glandular unit. The ducts of the glandular units are connected either directly to the external cuticle (ex. : Nassanov, Renner or Koschewnikow glands), or, in the most developed conditions, to a general excretory canal (ex. : mandibular glands). Glandular cells involved in the secretion of non-proteinic pheromones contain numerous mitochondria and a well developed endoplasmic. During the secretory process mitochondria undergo maturation (swelling, disappearance of cristae and inner membrane, sticking to endoplasmic membranes, accumulation of macromolecular substances), and generate secretory vesicles. Rough en-

Exocrine Glands of the Honey Bees

141

Figure 3. Type III exocrine gland. BL : basal laminae; EC : epithelial cell; Ep : epicuticle; D :desmosome: DC : duct cell : GC : glandular cell ; Mt : microtubule; MVB : multi vesicular body; N :nucleus; Pc : procuticle; RER : rough endoplasmic reticulum: SD : spetate desmosome ; Tr : tracheole.

doplasmic reticulum and Golgi apparatus are implied in the synthesis of the proteinic part of the secretory products. II. I. The Nassanov gland or abdominal scent gland of the workers of the honey bee (18, 19) is a complex structure where the glandular units are associated with oenocytes and fat body cells; the respective part of these cells to the secretory activity of the Nassanof gland is to be elucidated using labelled precursors of the pheromonal or protein components (20). The Nassanov scent is used for orientation, particularly at the nest entrance, in swarm clustering, at water collection sites and possibly at flowers. The proteins may act as pheromone carriers, enzymes for pheromone degradation, slow release substances, caste or sex specific modulators of pheromone activity; they enhance the attractiveness of the volatile fraction. They may also form part or all of the surface cuticular proteinic repertoire . The pheromones produced by the Nassanov gland have been chemically identified; they are composed of the following terpenoids : geraniol, nerolic acid, geranic acid, (E)citral, (Z)-citral, (E-E)-famesol, nero!. If the geraniol is the major compound, the most attractive and efficient components are (E)-citral and geranic acid (18). II. 2. The Renner's glands or tergal glands (Figs. 4-10) of the queen located near the rear of tergites III, IV and V, (2, 21 , 22, 23, 24). The secretion of the tergal glands attracted drones to queens on their mating flights from distances of less than 30 cm and increased their mating activities. Queens with paraffin-coated tergites were about 16 % less attractive than queens with no coating (25). As the mandibular gland secretion, the tergal gland products are also attractive to workers at distances of several centimeters; so, in the queen's retinue the honey bee workers lick her with their tongues or antennates her abdomen. This behaviour stabilizes the court once it has been formed and likely participate in the inhibition of the ovaries of the workers. The general organization of the Renner gland of the queen bee is similar to the Nassanov glands of the workers although they are located in different segments. So, the type III glandular units are intimately associated with fat body cells and oenocytes. The coexistence of rough and smooth endoplasmic reticulum in the glandular cells suggests that they are able to synthesize two kinds of products: the non-volatile macromolecular protein components and the volatile, small molecular, phero-

142

P. Cassier and Y. Lensky

Figure 4. Tergal glands of the queen bee. Scanning electron microscopy. Dorsal view of a tergite (T) and of an articular membrane (AM) showing glandular pores (frame).

monal components. Although, Renner and Baumann (1964) and Sanford (1977) suggested that the gland is active throughout a queen's lifetime, there is no convincing evidence concerning the age-dependence of tergal gland secretion. The various analysis of the tergal gland secretions are only preliminary ones. De Hazan et al. (26), after wiping of the tergal gland areas, have identified by GC-MS 14 compounds (Table II). Six compounds are specific for tergal gland secretion: benzoic acid, 9-hexade-

Figure 5. Tergal glands of the queen bee. Scanning electron microscopy. Detail of the articular membrane showing cuticular scales (Sc) and ovoid or ci rcul ar glandular pores (arrows).

Exocrine Glands of the Honey Bees

143

Figure 6. Tergal glands of the queen bee. Scanning electron microscopy. Inner view of the cuticular membrane with glandular cell (GIC), ducts (d) connected to cuticular holes (arrow). Oe : oenocytes.

cenoic acid, 9, 12-octadecadienoic acid, 2-dodecanol, 5-methyl-cyclo-hexanal, 2-decanal. The others compounds are also present in other exocrine glands as follows: 1- mandibular glands : diethyl-I ,2-benzene dicarboxylate, 3-methyl-2,6-dioxo-4-hexenoic acid, hexadecanoic acid, tetradecanoic acid, tetratetracontane and 1,2-dodecadiene (27, 28); 2- tarsal glands: hexadecanoic acid and 3-methyl-2,6-dioxo-4-hexenoic acid (8); 3- Koschewnikow glands of 4-dayold queens: tetradecanoic acid and hexadecanoic acid (29). These results are not consistent with those of Espelie et al.. (30) who prepared the extracts for GC-MS analysis from dissected tergites of 1- to 4-day-old italian queen bees.

Figure 7. Tergal glands of the queen bee. Scanning electron microscopy. Detail of a group of glandular cells (GlC) with their duct (d). Tergal glands of the queen bee. The glandular unit.

Figure 8. Tergal gland of Ihe queen bee. Eleclron microscopy. General view of a glandular cell (GlC) under Ihe cUlicular inlima (CUI) of Ihe arlicular membrane. BL : basal laminae; M : milochondria; N :nucleus; n : nucleolus; R : reservoir; SV : secrelory vesicle .

'" Q

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:- setaceous membrane> venom gland and venom sac. • Stinging: setaceous membrane > sting sheaths > quadrate plates and Koschewnikow glands> venom sacs. Of all the organs tested, the setaceous-membrane volatiles elicited the strongest defence reaction of the guards (20). The secretions present on the surface of the setaceous membrane contained isoamyl acetate, isoamyl alcohol, hexyl acetate, nonanol, benzyl acetate, benzyl alcohol (13). The setaceous membrane (tergum IX) is directly connected to the sting sheaths and is disposed all around the base of the sting. It is abundantly covered with cuticular infoldings and multiforked bristles, which considerably increase the surface of the evaporation area.The function of the setaceous membrane is to serve as a platform for the discharge of alarm pheromones that are secreted elsewhere and accumulate on its surface (56).

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ACKNOWLEDGMENTS This review was prepared within the framework of the Agreement for Scientific Cooperation between the Universite Pierre et Marie Curie (Paris VI) and the Hebrew University of Jerusalem.

REFERENCES 1. Farrar e.L. (1953) Two queen colony management. Bee World 34, 189--194. 2. Loubel de I'Hoste E. (1959) La biruche. Laubet de l'Hoste, Toulouse. 3. Holzberstein 1.w. Jr (1955) Some whys and hows at two queen management. Glean. Bee Cult. 83, 344-347. 4. Wafa A. K. (1956) Two queen colonies for a plentiful yield of honey. safe wintering. means of propagation and swarming control. Bull. Fac. Agr. Cairo Univ. 98, 22. 5. Wallrebenstein W. (1955) Mein Beitrag zum Mehrmutterverfahren. 17th Int. Beekeep. Congr. 6. Darchen R. and Lensky Y. (1963) Etude preliminaire des facteurs favorisant la creation des societes polygynes d' Apis mellifica var. ligustica. Ann. Abeille 6,69-73. 7. Darchen R. and Lensky Y. (1962) Les societes polygynes de Reines (Apis melli/iea var. ligustica). e. R. Acad. Sci .. Paris, 255, 2300-2302. 8. Lensky Y.. Darchen R. and Levy R. (1970) L'aggressivite des reines entre e11es et des ouvrieres vis-a-vis de la creation des societes polygynes d'Apis mellifica L. Rev. Compo Animal. 4, 50-62. 9. Darchen R. (1960) L'ablation du dard des reines et des ouvrieres d'Apis mellifiea. e. R. Acad. Sci., Paris, 250, 934-936. 10. Gary N.E. (1962) Antagonistic reactions of workers honeybees to mandibular gland contents of the queen bee. Bee World 42, 14-17. 11. Yadava R.R.S. and Smith M. Y. (1971) Aggressive behavior of Apis melli/era L. workers towards introduced queens II. Role of mandibular gland content of the queen and releasing aggressive behaviour. Can. 1. Zool. 49, 1179-1183. 12. Grandperrin D. and Cassier P. (1983) Anatomy and ultrastructure of the Koschewnikow's gland of the honeybee, Apis melli/era L. (Hymenoptera: Apidae). Int. 1. Insect Morphol. & Embryol. 12,25-42. 13. Mauchamp B. and Grandperrin D. (1982) Chromatographie en phase ga7euse des composes volatils des glandes a pheromones des Abeilles: methode d'analyse directe. Apidologie 13, 29-37. 14. Butler e.G. and Simpson 1. (1965) Pheromones of the honeybee (Apis melli/era L.). An olfactory pheromone from the Koschewnikow gland of the queen. Ved. Pr. Wyz. Us. Vcel. Dole 4, 33-36. 15. Robinson G.E. (1984) Worker and queen honeybee behaviour during foreign queen introduction. Insectes Soc. 31, 254-263. 16. De-Hazan M., Lensky Y. and Cassier P. (191\9) Effects of quecn honeybee (Apis melli/era L.) ageing on her attractiveness towards workers. Compo Biochem. Physiol. 93A, 777-783. 17. Yadava R.R.S. and Smith M.Y.(1971b) Aggressive behaviour of Apis melli/era L. workers towards introduced queens. 111. Relationship between the attractiveness of the queen and worker aggression. Can. J. Zool. 49, 1359--1362. 18. Yadava R.R.S. and Smith M.Y. (J 971 c) Aggressive behavior of Apis melli/era L. workers towards introduced queens I. Behavioural mechanisms involved in the release of worker aggression. Behaviour 39, 211-236. 19. Cassier P., Finkel A. and Lcnsky Y (199 I) The chemical composition of tarsal gland secretions of honeybee, Apis melli/e.·a (L.) queens, workers and drones. (personal communication). 20. Lensky Y, Cassier P., Rosa S. and Grandperrin D. (1991) Induction of balling in worker honeybees (Apis melli/era L.) by stress pheromone from Koschewnikow glands of queen bees: behavioural, structural and chemical study. Compo Biochem. Physiol. 100A, 3. 585-594. 21. Boch R., Shearer D.A. and Stone B.C. (1962) Identification of isoamyl acetate as an active component in the sting pheromone of the honeybee. Nature 195, 1018-1020. 22. Pickett J.A., Williams I.H. and Martin A.P. (1982) (2)-II-Eicoson-I-ol, an important new pheromonal component from the sting of the honeybee, Apis melli/era L. (Hymenoptera, Apidae). J. Chern. Ecol. 8, 163-175. 23. Free J.B., Ferguson A.W. and Simpkins J.R. (1988) Honeybee responses to chemical components from the worker sting apparatus and mandibular glands in field tests. 1. Apic. Res. 28, 7-2 I.

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24. De-Hazan M. (1986) The attractiveness, composition and structure of mandibular glands of the queen honeybee (Apis melli/era L.). M.Sc. Thesis. Hebrew University of Jerusalem. 25. Finkel A. (1983) Biological effects and chemical characterization of the foot-print substance in the honeybee (Apis melli/era L.). M.Sc. Thesis. Hebrew University of Jerusalem. 26. Hyams Y. (1988) Characterization of pheromonal components of the tarsal and mandibular glands of the queen honeybee (Apis melli/era L.). M.Sc. Thesis. Hebrew University of Jerusalem. 27. Lensky Y, Cassier P. and Tel-Zur (1995) The setaceous membrane of the honey bee (Apis mellifera L.) workers sting apparatus and alarm pheromone distribution. J. Insect Physiol. 21, 589-595. 28. Winston M.L. (1987) The biology of the honey bee. Harvard University Press, Cambridge, 281 pp. 29. Crane E. (1990) Bees and beekeeping: science, practice and world resources. Heinemann Newnes, Oxford, UK,614p. 30. Collins A.M. and Blum M.S. (1983) Alarm responses caused by newly identified compounds derived from the honeybee sting. J. Chern. Ecol., 9, I, 57~5. 31. De Hazan M., Hyams J., Lensky Y. and Cassier P. (1989) Ultrastructure and ontogeny of the mandibular glands of the queen honey bee, Apis mellifera L. (Hymenoptera, Apidae). Int. J. Morph. Embryol. 18, ~, 311-320. 32. Blum M.S. (1992) Honey bee pheromones. In Graham, I. M.(ed.) The hive and the honey bee. Dadant & Sons; Hamilton, pp 373-400. 33. Cassier P., Tel Zur, D. and Lensky Y. (1994) The sting sheaths of honey bee workers (Apis melli{era L.) : structure and alarm pheromone secretion. J. Insect Physiol. 40, 233. 34. Tel-Zur D. (1993) Alarm pheromones of the sting apparatus of the honey bee (Apis mellifera L. var. ligusfica) PhD Thesis, Hebrew University of Jerusalem. Israel, II Op. 35. Boch R. and Shearer D.A. (1967) 2-heptanone and 10 hydroxy frans-dec-2-enoic acid in the mandibular glands of worker honeybees of different ages. Zeitsch. Vergl. Physiol. 54, I-II 36. Boch R., Shearer D.A. and Petrasovits A. (1970) Efficacies of two alarm substances of the honey bee. J. Insect Physiol. 16, 17-24. 37. Maschwitz U.W (1964) Gefarhrenalarmstoffe und Gefaharenalarmierung bei sozialen Hymenopteren. Z. Vergl. Physiol. 47, 59~55. 38. Shearer D.A. and Boch R. (1965) 2-heptanone in the mandibular gland secretion of the honey-bee. Nature 206,530. 39. Free lB., Simpson l (1968) The alerting pheromones of the honey bees. Z. Vergl. Physio. 61, 361-365. 40. Melksham K.J., Jacobsen N. and Rhodes J. (1988) Compounds which affect the behaviour of the honeybee, Apis melli{era L.: a review. Bee World 69,104-124. 41. Simpson J. (1966) Repellency of the mandibular gland scent of worker honey bees. Nature 209, 531-532. 42. Rieth J.P., Wilson WI. and Levin M.D. (1986) Repelling honeybees from insecticide-treated flowers with 2-heptanone. l Apicult. Res. 25, 78-84. 43. Vallet A., Cassier P. and Lensky Y. (1991) Ontogeny of the fine structure of the mandibular glands of the honeybee (Apis mellifera L.) workers and the pheromonal activity of 2-heptanone. l Insect Physiol. 37, 789-804. 44. Cassier P. and Lensky Y. (1991) Evolution du titre de I'hormone juvenile III, des ecdysterojdes et d'une pheromone, la 2-heptanone, en relation avec Ie polyethisme des ouvrieres de I'abeille domestique, Apis mellifera L. (Hymenoptera, Apididae). e. R. Acad. Sci. , Paris, III , 312, 343--348. 45. Crewe R.M. (1976) Aggressiveness of honey bees and their pheromone production. South Afr. J. Sc. 72, 7, 209-212. 46. Crewe R.M. and Hastings H. (1976) Production of pheromones by workers of Apis mellifera adansonii. J. Apicult. Res. 15, 149-154. 47. Kalmus H., Ribbands e.R. (1952) The origin of the odours by which honeybees distinguish their companions. Proc. Roy. Soc. B 140, 5Q.-59. 48. Butler e.G. (1966) Mandibular gland pheromone of worker honeybees. Nature 212, 530. 49. Free J.B., Pickett J.A., Ferguson A.W., Simpkins J.R. and Smith M. (1985) Repelling foraging honeybees with alarm pheromones. J. Agr. Sc. 105,255--260. 50. Robertson G.E. (1968) A morphological and functional study of the venom apparatus in representatives of some major groups of Hymenoptera. Austral. J. Zool. 16, 133--166. 51. Snodgrass R.E. (1956) Anatomy of the honey bee. Cornell University Press, Comstock Publishing Associates, Ithaca, NY, 334 pp. 52. Giufra M. and Nunez J.A. (1992) Honeybees mark with scent and reject recently visited flowers. Oecologia 89, 113-117. 53. Cassier P. and Lensky Y. (1992) Structure et role social de quelques glandes exocrines a secretion pheromonale chez I'abeille domestique, Apis mellifera L. (Hymenoptera, Apididae). Annee BioI. 31, 61-95.

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54. Ghent R.L. and Gary N.E. (1962) A chemical alarm releaser in honey bee stings (Apis mellifera L.). Psyche, 69, I, 1--6. 55. Collins A.M. and Blum M. S. (1982) Bioassay of compounds derived from the honeybee sting. J. Chern. Ecol. 8, 463-470. 56. Lensky Y. and Cassier P. (1995) The alarm pheromones of queen and worker honeybees. Bee World 76, 3, 119-129.

20

PROTEIN TRAFFIC BETWEEN BODY

COMPARTMENTS OF THE FEMALE HONEY

BEE (APIS MELLIFERA L.)

Yoseph Rakover 1 and Yaacov Lensky 2 IOtorhinolaryngology Department Central Emek Hospital Afula, Israel 2Triwaks Bee Research Center Hebrew University of Jerusalem, Faculty of Agriculture Rehovot, Israel

ABSTRACT Several "anatomical compartments" of the female honey bee body have bee studied: I. : Exocrine compartments (the venom gland and the head glands). 2. : Internal organs (ovaries and fat body). 3. : The haemolymph that bathes the above mentioned compartments. To explore the protein traffic from the haemolymph to the exocrine or external body compartments immunological and electrophoretic methods were used. The studies did not show any immunological identity between the proteins of larval food, venom and haemolymph but most of the haemolymph antigens were common with those of ovaries and fat body. A tentative model of the protein traffic between honey bee body compartments is proposed: a. Some compartments are enveloped by a cellular layer (venom and head glands) which prevents the macromolecular traffic between these compartments and the haemolymph. b. Some other compartments are lined by a cellular layer (fat body and ovaries), allowing macromolecular traffic between organs through the haemolymph. The traffic of macromolecules between the body compartments of the female honey bee may serve as a biological model and help in understanding the process in medical and pharmacological disciplines.

1. INTRODUCTION Protein traffic between body compartments is one of the most important process in animals to maintain the biological balance or the homeostatic status and to trigger defense 161

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Fat body

Postcerebral gland

Ovary Hypopharyngeal gland

Oocyte

Thoracic gland

Mid-gut

: Sting Venom Venom gland sac

Figure I. Longitudinal, schematic section of a worker honey bee, showing the exocrine glands and the internal organs (modified after Ribbands, 1953)(2).

Figure 2. The collection of haemolymph from the dorsal vessel of a worker with a fine glass capillary.

Protein Traffic between Body Compartments of the Female Honey Bee

163

mechanisms. So, a comprehensive study of this process is important particularly in physiological, pathophysiological and medical disciplines. In mammals, numerous investigations have been done about protein traffic between body compartments, for example traffic between the cerebrospinal fluid and the blood (1). Whereas in mammals the blood flows in a close system, in insects the "blood" (haemolymph) flows in an open system, baths tissues and organs which suggests a different model to study the protein traffic. The female honey bee, a new biological model with a clear anatomical compartmentalization, allows to study the protein traffic (Fig. 1). So, the protein components of three body compartments of the female honey bee are compared by immunological and electrophoretic methods: 1. The exocrine glands: the venom gland, the venom sac and the sting apparatus that secrete and drain venom; the hypopharyngeal and mandibular glands that drain their secretions through ducts and groves via the mouthparts to the exterior as a larval food. 2. The internal organs: ovaries and fat bodies. The vitellogenins are synthesized in the fat body, released to the haemolymph, selectively taken up by the oocyte through several cellular and acellular envelopes (tunica propria, external sheaths), then deposited as vitellin platelets. The oocyte membranes are secreted by the follicle cells (3). 3. The haemolymph that bathes the above mentioned compartments.

We examined the existence of separate body compartments with specific proteins and the possible uptake of haemolymph proteins by the body compartments. This biological model may help in understanding more about the protein traffic or its blocking between body compartments.

2. MATERIALS AND METHODS 2.1 Preparation of Samples for Analyses Italian honey bee (Apis mellifera L. vaL ligustica) adult workers were obtained from the apiary of the Triwaks Bee Research Center, Rehovot. Haemolymph was collected from the dorsal vessel of workers with a fine glass capillary (Fig 2.). Mandibular and hypopharyngeal glands were dissected from head capsules of nurse workers. Ovaries and fat bodies were removed from laying workers. Guardians were captured at hive entrances. The sting apparatus, venom gland and venom sac were removed fro IT their bodies. The venom sac membrane was punctured at several sites and the venom oozed out. Royal jelly was collected from queen cells after the removal of 3 to 4 day-old larvae.

2.2 Preparation of Antisera Antisera were prepared in rabbits against haemolymph and venom of adult workers, royal jelly, and ovaries oflaying queens (4, 5).

2.3 Immunological Analyses We used double diffusion and immunoelectrophoresis methods for the immunological analysis (5).

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2.4 Electrophoretic Analyses Polyacrylamide gradient slab gel electrophoresis with SDS (SDS-PAGE) and isoelectric focusing were used for the electrophoretic analysis (4).

3. RESULTS 3.1 Are Haemolymph Proteins Incorporated into the Secretory Products of Exocrine Glands? To establish whether haemolymph proteins participate in the composition of proteinaceous secretions of the exocrine glands we analyzed them using electrophoretic and immunological methods. SDS-PAGE. The electrophoretic separations of the peptide components of the royal jelly, venom and haemolymph on SDS-PAGE slabs are recorded in Fig. 3 (samples Nos. 5, 4, 3, respectively). Royal jelly was separated into 33 bands, venom into 10 bands and haemolymph into 36 bands. Although several minor bands in the three samples shared similar Rjs , no major haemolymph proteins were present either in royal jelly or in venom.

Isoelectric Focusing. Samples of haemolymph, royal jelly and venom were separated on PAG-plates (Fig. 4 samples Nos 5, 4, 3, respectively). Haemolymph proteins were separated into 29 bands within pI range of 4.0-7.2; royal jelly into 26 bands within pI range of 4.7-9.2 and venom into 3 bands within pI above 11.0. pI values of venom fractions corresponded neither to haemolymph nor to royal jelly. Several fractions of haemolymph and royal jelly have similar pI's. The results of electrophoretic analyses indicate that most of the protein constituents of the three samples were not shared in common. A small number of bands had similar R, or pI values. Which of these bands represented identical proteins is considered in the next section, using immunological methods and specific antisera. Immunoelectrophoresis. Samples of royal jelly, venom and haemolymph were analyzed by immunoelectrophoresis using antisera against royal jelly, venom and haemolymph (Fig. 5). Royal jelly (well 2) formed 16 precipitation lines following absorption with antiserum against royal jelly (A) and one diffuse line with antiserum against haemolymph (B), which seems to show a reaction of non-identity, as demonstrated below by double-diffusion analysis. Venom (well 3) formed 6 precipitation lines with antiserum against venom (C) and 2 lines (Nos 7 and 8) with antiserum against haemolymph (b). The position of these two lines neither corresponded to venom nor to haemolymph precipitation lines. This may indicate a reaction of partial- or non-identity. Haemolymph (well 1) formed 24 precipitation lines with antiserum against haemolymph (B). Haemolymph proteins were not precipitated by antiserum neither against royal jelly (A) nor against venom (C). Double Diffusion. Samples of haemolymph, royal jelly and venom were absorbed with antiserum against royal jelly, haemolymph and venom. With the antiserum against royal jelly (central well A) five precipitation lines were formed with royal jelly (well No.2) and one line of non-identity, forming a spur precipitated with haemolymph (well

Protein Traffic between Body Compartments of the Female Honey Bee

165

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-

-

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Figure 3. SDS-PAGE (5- 15% gradient) protein separation of ovaries (I), fat body (2), haemolymph (3), venom (4), royal jelly (5), ovalbumin (6), Rnase (7) and phosphorylase b (8).

No. I) (Fig. 6). With the antiserum against haemolymph (central well B) 15 precipitation lines were formed with haemolymph (wells No. I). This antiserum precipitated neither the antigens of royal jelly (wells No.2), nor the antigens of venom (wells No.3) (Fig. 7).Then, with the antiserum against venom (central well C) three precipitation lines were formed with venom (well No.3), but none with haemolymph (well No.1) (Fig. 8). The immunoelectrophoresis and double diffusion results demonstrated that no identical antigens were shared by royal jelly, venom and haemolymph. These data confirm and

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Figure 4. Isoelectric focusing of fat body (I), ovaries (2), venom (3), royal jelly (4) and haemolymph (5). PAGplates, pH range 3.5-9.5.

extend the results of the electrophoretic analyses. It is suggested that the head glands, the venom glands and the haemolymph are separate compartments with regard to macromolecular traffic.

3.2 Are Proteins Shared in Common by Haemolymph, Fat Body and Ovaries? To determine the presence of haemolymph proteins in the fat body and ovaries of laying workers, electrophoretic and immunological methods were used.

SDS-PAGE (Fig. 3, Samples 1, 2 and 3 Respectively). Ovaries were separated into 22 bands, fat body into 21 bands and haemolymph into 36 bands. Both ovaries and fat body shared, each, 17 bands of similar Rf s in common with haemolymph. Thirteen of these bands were common to all three samples. Ovaries and fat body shared in common 20 bands. Isoelectric Focusing (Fig. 4, Samples 2, 1 and 5, Respectively). Ovarian proteins were separated into 18 bands within pI range of 4.5-7.8; fat body into 19 bands within pI range of 4.5-7.2 and haemolymph into 29 bands within pI range 4.0-7.2.

Protein Traffic between Body Compartments of the Female Honey Bee

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Figure 5. Immunoelectrophoretic analysis of haemolymph (l), royal jelly (2), and venom (3) . Absorbed with antisera against royal jelly (A), haemolymph (8) and venom (C).

Figure 6. Double-diffusion analysis of haemolymph (I) and royal jelly (2) prepared in the surrounding wells. Absorbed with antisera against royal jelly (Al in the center well.

Figure 7. Double-diffusion analysis of haemolymph (I), royal jelly (2) and venom (3) prepared in the surrounding wells. Absorbed with antisera against haemolmph (B) in the center well.

Figure 8. Double-diffusion analysis of haemolymph (I 1 and venom (3) prepared in the surrounding wells. Absorbed with antisera against venom (e) in the center well.

Protein Traffic between Body Compartments of the Female Honey Bee

169

In general, the results confirmed those of SDS-PAGE, with small differences in the number of common bands between the three samples. The results of electrophoretic separations show that most (about 14) of the proteins of ovaries, fat body and haemolymph are shared in common. The identity of the proteins of the three samples was further analyzed by immunological methods using specific antisera. Immunoelectrophoresis. The identity of haemolymph, fat body and ovaries proteins was examined by Immunoelectrophoresis using antisera against haemolmph (B) and ovaries (A) (Fig. 9). Haemolymph formed 26 precipitation lines with antiserum against haemolymph and 12 lines with antiserum against ovaries. Ovaries formed 16 lines with antiserum against haemolymph and 7 lines with antiserum against ovaries. Fat body formed 14 lines with antiserum against haemolymph and 7 lines with antiserum against ovaries. It also emerges from the results that: haemolymph and fat body shared 14 lines precipitated by anti haemolymph serum and 5 lines in common when precipitated with antiserum against ovaries. Haemolymph and ovaries formed 5 common lines with anti haemolymph serum and 6 common lines with antiserum against ovaries. Double-Diffusion. Samples of ovaries, fat body and haemolymph were absorbed with antisera against ovaries or haemolymph. With the antiserum against ovaries (central well A) most of the lines formed by ovaries (well 2) and haemolymph (well I) merged, except for line No.5 which formed a spur. The major line (Nos 2 and 3) was not formed by the fat body (well 3) (Fig. 10). With the antiserum against haemolymph (central well B) 16 lines were formed by haemolymph (well I) which had in common with ovaries (well 2) 7 lines and 6 lines with fat body (Fig. 11). The results of immunoelectrophoretic and double diffusion analyses confirm the identity of several protein components of haemolymph, fat body and ovaries, which was indicated by the electrophoretic analysis. The data suggest that contrary to the above described separate compartments of exocrine glands, the ovaries and fat body cannot be considered as such, because of their association with haemolymph proteins.

DISCUSSION Since neither antiserum against venom precipitated.haemolymph proteins nor antihaemolymph serum formed precipitates with venom, no bands with similar Rf or pI values could be detected, it is concluded that the venom gland and the venom sac are separate compartments. The venom contains pharmacologically active components (6), which are lethal whenever a honey bee is stung. When a drop of venom is removed with a glass capillary from the sting of a donor bee and immediately injected into its body cavity, the bee dies instantly (Rakover, unpublished observation). It seems that the venom gland and sac walls serve as a macromolecular barrier at least with regards to the traffic of venom macromolecules. The fact that no identical antigens were shared by royal jelly, venom and haemol-ymph indicates that the head and venom glands are separate protein compartments from one another and from haemolymph proteins. It seems therefore that the glandular excretory proteins are not taken up from the haemolymph , except for their precursors, but that they are synthesized in situ. Contrary to the exocrine gland' proteins, the immunochemical and the electrophoretic separations of haemolymph, fat body and ovaries revealed that most of their proteins were identical. As a tentative model of protein traffic, the body compartments of the honey bee seem composed of two main parts:

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Figure 9. Immunoelectrophoretic analysis of haemolymph (I), ovaries (2), and fat body (3). Absorbed with antiserum against ovaries (A) and haemolymph (8).

a. Compartments enveloped by a cellular layer which acts as a protein barrier and prevents macromolecular traffic between the venom glands or the head glands and the haemolymph. b. Compartments lined with a cellular layer which allows a macromolecular traffic between one organ (the fat body or the ovaries) and the haemolymph. Separate fluid compartments have been described in the honey bee pupal molting space ( 7) and in the queen bee spermatheca (8).

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Figure 10. Double-diffusion analysis ofhaemolymph (I) , ovaries (2) and fat body (3) charged in the surrounding wells. Absorbed with antiserum against ovaries (A) in the center well.

Figure 11. Double-diffusion analysis of haemolymph (l), ovaries (2) and fat body (3) charged in the surrounding wells. Absorbed with antiserum against haemolymph (B) in the center well.

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The barrier to protein traffic in some insect tissues is related with the presence of separate and scalariform junctions (9). In mammals, the protein traffic between compartments is also composed of two main parts: a. A barrier and prevention of macromolecule traffic by tight junction. For example the blood brain barrier (10) or the pancreatic duct epithelium (11, 12). b. Compartment which are lined with a cellular layer, permitting macromolecular traffic between one organ and another such as between liver epithelial cells (13) or in the vestibular labyrinth (14). The macromolecular\ traffic can be done via gap junction or by receptor mediated protein transport (15).

REFERENCES I. Van-Deures B., Moller M., Amtorp O. (1978) Uptake of horseradish peroxidase from C.S.F into the choroid plexus of the rat with special reference to transepithelial transport. Cell. Tiss. Res. 233 , 215--234 2. Ribbands R. (1953) The Behaviour and Social Life of Honey Bees. pp. 55-63; Bee Res. Assoc., London. 3. Engelmann F. (1980) Insect vitellogenin: identification, biosynthesis and role in oogenesis. [n : Advances in [nsecs Physiology. Ed : Trehence JE., Berridge MJ. and Wigglesworth VB. Academic Press, London, 14, pp.49-108. 4. Lensky Y, Skolnik H. (1980) [mmunochemical and electrophoretic identification of the vitellogenin proteins ofthe queen bee (Apis mellifera L.). Compo Biochem. Physiol. 66B, 185-193. 5. Lensky Y., Rakover Y (1983) Separate protein body compartments of the worker honeybee (Apis mellifera L.). Compo Biochem. Physiol. 75B: 607-615. 6. O'Connor R., Peck ML. (1978) Venom of Apidae. In: Handbook of Experimental Pharmacology. Ed: Bettini S. Springer, Berlin. 48, pp. 613-615. 7. Lensky Y., Rakover Y (1972) Resorption of moulting fluid during the ecdysis of the honeybee. Compo Biochern. Physiol. 41B, 521-531. 8. Lensky Y, Alumot E. (1969) Proteins in the spermathecae and haemolymph of the queen Bee. Compo Biochern. Physiol. 30, 569-575. 9. Noirot-Timothee c., Noirot C. (1980) Separate and scalariformjunctions in arthropods. [nt. Rev. Cytol. 63, 97-140. 10. Lane NJ. (1991) Morphology of glial blood-brain barriers. Ann. N.Y Acad. Sci. 633, 348-362. II. Arendt T. (1991) Penetration of lanthanum through the main pancreatic duct epithelium in cats following exposure to infected human bile. Dig. Dis. Sci. 36, 75-81. 12. Satir P., Gilula NB. (1973) The fine structure of membranes and intercellular communication in insects. In : Ann. Rev. Entomol. Ed: Smith RF., MittlerTE.VoI18, p. 143-166. 13. Mesnil M., Asamoto M., Piccoli C., Yamasaki H. (1994) Possible molecular mechanism ofloss ofhomologous and heterologous gap junctional intercellular communication in rat liver epithelial cell lines. Cell Adhes. Commun. 2, 377-384. 14. Kikuchi T., Adams JC., Paul DL.. Kimura RS. (1994) Gap junction systems in the rat vestibular labyrinth: immunohistochemical and ultrastructural analysis. Acta Otolaryngol. (Stockh.) 114, 520-528. 15. Gitlin JD., Gitlin D. (1974) Protein binding by specific receptors on human placenta, murine placenta and suckling murine intestine in relation to protein transport across these tissues. J. Clinic. Invest. 54, 1155--1166.

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EFFECTS OF FEEDING, AGE OF THE LARVAE, AND QUEENLESSNESS ON THE PRODUCTION OF ROYAL JELLY Nuray Sahinler 1 and Osman Kaftanoglu 2 lMustafa Kemal University Faculty of Agriculture Hatay-Turkey 2Cukurova University Faculty of Agriculture 01330 Adana-Turkey

ABSTRACT The effects of feeding, the age of the larvae and queenlessness on the acceptance rates and royal jelly production were studied. The average acceptance rates were 65.0±0.82 % in queenright cell builders and 87.1±1.08 % in queenless cell builders. Feeding colonies with pollen substitute increased the acceptance rates significantly (P0.05). The age of the larvae was also important on the acceptance of the cells. The acceptance rates of I or 2 days old larvae were higher than that of3 days old larvae in both queenless and queenright colonies. In queenright cell builders the average royal jelly yields were 153.7±4.27 mg per cell when they were fed with sugar syrup and 185.3±5.68 mg when pollen substitute was given besides sucrose syrup. In the queenless cell builders the average yields were 189.3±9.11 mg in the sugar syrup fed and 225.6±14.52 mg in the pollen substitute fed colonies. In general royal jelly yield was much higher in queenless cell builders than that of qucenright. Feeding colonies with pollen substitutes in addition to sucrose syrup increased the royal jelly yield by 36 % in queenright colonies and 40 % in queenless colonies. The best result were obtained by grafting one day old larvae in queenless cell builders that were fed with pollen substitute and sucrose syrup.

1. INTRODUCTION Royal jelly is the whitish cream like secretion from the food glands of the young worker honey bees. It is used to feed the queen bee and young larvae in the colony. It 173

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shortens the developmental period of the larvae and causes the differentiation of the worker larvae into queen. It prolongs the life of the queen, stimulates cell divisions and promotes tissue regeneration. There is no genetical difference between the worker bees and queen bees raised from the same colony. The only difference comes from nutrition of the larvae during the development. The queen larvae are fed with royal jelly throughout the larval stage, while the older worker larvae are fed with the mixture of nectar and pollen. As a result queen bees develop within 16 days, they are bigger, their ovaries and the spermathecae are fully developed. They can live up to 2-3 years and can lay 1500-2000 eggs per day. On the other hand worker bees develop within 21 days, live 35-40 days, their ovaries are not developed and their spermathecae are not functional. Royal jelly has become a very popular bee product for the last 5--6 years. There is a big demand for fresh and good quality royal jelly in the country. It has been used for the healthy growth of children and for many maladies as folk medicine by elderly people, by impotent couples, by patients suffering from cancer and other diseases in order to increase appetite, stimulate the immune system and keep the body stronger. Most of royal jelly is imported from China and other countries. There are over 3.5 million colonies and 40.000 beekeepers in Turkey. We have a great potential in producing good quality bee products such as honey, royal jelly, pollen, propolis, bee venom etc., having rich flora, suitable climate and genetic richness of bees in the country. However most of the beekeepers produce only honey and do not know how to produce royal jelly. Therefore we have started a program to train beekeepers to produce quality bee products. Since the value of royal jelly is much higher than honey and other bee products, it is an excellent source of income for the beekeepers. We decided to work on the factors affecting the royal jelly production in order to initiate and spread the royal jelly production in the country. We have studied the effects of feeding and the age of the larvae on the grafting rates and royal jelly yield in queenless and queenright colonies. We are currently studying the effects of different genotypes and season on the production of royal jelly in subtropical climate.

2. MATERIALS AND METHODS This study was conducted at the Cukurova Region in Turkey, during july-September of 1995. Total of8 cell builders were used during the experiment. One half of the colonies were queenless and the other half were queenright. They were also divided into 2 groups and they were either fed with syrup or with syrup and pollen substitute. Pollen substitute was prepared by mixing 4 parts of soybean flour, I part dried skim milk and making a cake with sugar syrup. About 250 grams of pollen substitute was placed on top of the frames and they were replaced with the fresh one every week. Queenless starter colonies were prepared by dequeening the colonies and rearranging the frames in the brood chamber as; honey, sealed brood, open brood, open space for larvae transfer, open brood, sealed brood, honey and feeder. The supers and extra frames were removed from the hives and the bees were shaken to the brood chambers in order to have strong one-story free flying starter colonies. All the queenless cell builders were inspected regularly and the natural queen cells were removed. In order to strengthen the colonies adult worker bees and/or frames of sealed brood were added to the queenless cell

Production of Royal Jelly

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builders or new queenless colonies were prepared every 15 days. Grafting was repeated every 2 days, and royal jelly was harvested with 48 hour intervals. Queenright starter colonies were prepared by placing a queen excluder above the brood chamber confining the queen and rearranging the frames as in the queenless cell builder. Empty frames were exchanged with the brood frames between the brood chambers and supers as the brood emerged. Queen cell cups were made according to Laidlaw (I). Pure beeswax was melted in a double-jacketed tray. The wax was dipped first into a soap-solution, and the excess water was shaken off. The stick was then dipped into the melted wax 3 or 4 times to a depth of about 8-10 mm. After the last dip, the formed cups were fixed to a grafting bar and submerged in clean and cold water, where the cell cups were separated from the mold. By this way 15 cell cups were prepared on a grafting bar. Grafting was done in a tent or a room near the apiary. One drop of diluted royal jelly was placed to the bottom of the queen cell cups when the new cell cups were used (2). The larvae were lifted gently with some royal jelly from the cells and transferred to the bottom of the cell cups by using a grafting needle. A clean wet towel was placed over the cell cups in order to prevent the larvae from drying. One frame with three bars of cells was put into the space in the cell builders as soon as the grafting was finished. One day old larvae were grafted to the top bar, 2 days old larvae to the middle bar and 3 days old larvae to the lowest bar. All the frames were removed from the cell builders 2 days after grafting. The accepted cells were counted and the acceptance rates were determined. Later all the larvae in queen cells were removed by using a pair of fine forceps. Royal jelly was harvested by using a special plastic royal jelly collecting spoon and they were placed in a separate vial, weighed on an electronic balance and the average royal jelly yield was determined.

3. RESULTS AND DISCUSSION 3.1. Acceptance Rates 3.1.1. Queenright Cell Builders. The acceptance rates In queenright cell builders were summarized in Table 1. The acceptance rates in the queenright colonies were rather low. The average acceptance rates were 65.1±1.23 % in the syrup fed and 64.9±1.I0 % in the pollen substitutes fed colonies. Addition of pollen substitute did not increase the acceptance rates in queen-

Table 1. Acceptancc ratcs (%) in quecnright colonies

Fed with syrup Age of larvae

N

I Day old 2 Days old 3 Days old Total/Average

150 150 150 450

x± S; 68.7 66.7 60.0 65.1

± ± ± ±

1.96 1.68 1.81 1.23

Fed with syrup and pollen substitute Max.- Min.

N

67~80

150 150 150 450

67~73

53--67 53~80

'Different letters indicate significant differences among the means (PO.OS); however, the acceptance rates of 3 days old larvae were lower than that of young ( I or 2 days old) larvae (P