JMPR- 15 August, 2012 Issue

Journal of

Medicinal Plants Research Volume 6 Number 31 15 August, 2012 ISSN 1996-0875

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Prof. Parveen Bansal Department of Biochemistry Postgraduate Institute of Medical Education and Research Chandigarh India.

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Dr. Ravichandran Veerasamy AIMST University Faculty of Pharmacy, AIMST University, Semeling – 08100, Kedah, Malaysia.

Dr. Jianxin Chen Information Center, Beijing University of Chinese Medicine, Beijing, China 100029, China.

Dr. Sayeed Ahmad Herbal Medicine Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, 110062, India.

Dr. Hassan Sher Department of Botany and Microbiology, College of Science, King Saud University, Riyadh Kingdom of Saudi Arabia.

Dr. Cheng Tan Department of Dermatology, first Affiliated Hospital of Nanjing Univeristy of Traditional Chinese Medicine. 155 Hanzhong Road, Nanjing, Jiangsu Province, China. 210029

Dr. Jin Tao Professor and Dong-Wu Scholar, Department of Neurobiology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, P.R.China. Dr. Pongsak Rattanachaikunsopon Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand.

Dr. Naseem Ahmad Young Scientist (DST, FAST TRACK Scheme) Plant Biotechnology Laboratory Department of Botany Aligarh Muslim University Aligarh- 202 002,(UP) India. Dr. Isiaka A. Ogunwande Dept. Of Chemistry, Lagos State University, Ojo, Lagos, Nigeria.

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Dr. Arash Kheradmand Lorestan University, Iran.

Prof. Dr. Muradiye Nacak Department of Pharmacology, Faculty of Medicine, Gaziantep University, Turkey.

Prof Dr Cemşit Karakurt Pediatrics and Pediatric Cardiology Inonu University Faculty of Medicine, Turkey.

Dr. Sadiq Azam Department of Biotechnology, Abdul Wali Khan University Mardan, Pakistan.

Samuel Adelani Babarinde Department of Crop and Environmental Protection, Ladoke Akintola University of Technology, Ogbomoso Nigeria.

Kongyun Wu Department of Biology and Environment Engineering, Guiyang College, China. Prof Swati Sen Mandi Division of plant Biology, Bose Institute India. Dr. Ujjwal Kumar De Indian Vetreinary Research Institute, Izatnagar, Bareilly, UP-243122 Veterinary Medicine, India.

Dr.Wafaa Ibrahim Rasheed Professor of Medical Biochemistry National Research Center Cairo Egypt.

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International Journal of Medicine and Medical Sciences Journal of Medicinal Plants Research

Table of Contents:

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Volume 6

Number 31

15 August, 2012

ARTICLES

Research Articles Chemical composition and antibacterial activity of essential oils from six Moroccan plants Ahmed Talbaoui, Naoual Jamaly, M’hamed Aneb, Abdelkader Il Idrissi, Mohammed Bouksaim, Said Gmouh, Saaïd Amzazi, Mohammed El Moussaouiti, Abdelaziz Benjouad and Youssef Bakri

In vitro reversal of deformity and inhibition of aggregation of sickle red blood cells by two Congolese herbal medicines Marie Miezi Nsimba, José Nzunzu Lami, Chika Yamamoto, Toshiyuki Kaji, Matadi Mukengeshaie and Muhandisha Lufuluabo

Inhibition of angiogenesis and metastasis of uveal melanoma cells by astragaloside IV Zhang Wenjing, Ma Minwang, Wang Dong and Tang Dongrun

Analysis of anti-oxidant activity of medicinal plants according to the extracted parts Yu-Su Shin, Hyun-Jung Jo, Sang-Won Lee, Young-Ock Kim, Yoon-Pyo Hong and Kyung-Soo Chang

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Survey of herbal remedies used by Fulani herdsmen in the management of animal diarrhoea in Plateau State, Nigeria 4625 Nkechi Veronica Offiah, Christiana Joshua Dawurung, Olusola Olalekan Oladipo, Micah Shehu Makoshi, Sunday Makama, Ishaku Leo Elisha, Jurbe Gofwan Gotep, Ann Lohlum Samuel and David Shamaki

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15 August, 2012

ences ARTICLES In vitro anti-angiogenic activity fractions from hydroalcoholic extract of Elaeagnus angustifolia L. flower and Nepeta crispa L. arial part Badrhadad A., Piri Kh and Mansouri K.

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Glycyrrhizin and isoliquiritigenin production by hairy root culture of Glycyrrhiza glabra Zahra Shirazi, Khosro Piri, Asghar Mirzaie Asl and Tahereh Hasanloo

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Effects of irrigation and nitrogen (N) fertilization levels on yield, morphological traits and water use efficiency of chicory (Cichorium intybus L.) Seyyed Gholam Reza Moosavi

Chemical composition and antifungal, phytotoxic, brine shrimp cytotoxicity, insecticidal and antibacterial activities of the essential oils of Acacia modesta Bashir Ahmad, Ibrar Khan, Shumaila Bashir and Sadiq Azam

Role of auxins in the in vitro rooting and micropropagation of Holarrhena antidysenterica Wall., a woody aromatic medicinal plant, through nodal explants from mature trees Satyajit Kanungo, Chinmay Pradhan, Santi Lata Sahoo and Rajani Kanta Sahu

Antioxidant properties of free and bound phenolic extract of the leaves of Jatropha tanjorensis in vitro Atansuyi, K, Ibukun, E. O. and Ogunmoyole, T.

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Anti-hyperlipidaemic and antioxidant effect of aqueous and ethanolic extracts of Cassia italica leaves in streptozotocin-induced diabetes in rats 4675 Nadro, M. S. and Onoagbe, I. O.

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15 August, 2012

ences ARTICLES Antioxidant activities of different solvent extracts of leaves and root of Flabellaria paniculata Cav. (Malpighiaceae) Margaret Oluwatoyin Sofidiya and Oluwole Familoni

Evaluation of spasmolytic and analgesic activity of ethanolic extract of Chenopodium album Linn and its fractions Mansoor Ahmad, Omair Anwar Mohiuddin, Mehjabeen, NOOR JAHAN, MUNIR Anwar, Salman Habib, S. Mahboob Alam and Iftikhar Ahmed Baig

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South Siberian fruits: Their selected chemical constituents, biological activity, and traditional use in folk medicine and daily nutrition 4698 Pawel Pasko, Justyna Makowska-Was, Joanna Chlopicka, Marek Szlosarczyk, Malgorzata Tyszka-Czochara, Justyna Dobrowolska-Iwanek and Agnieszka Galanty

Journal of Medicinal Plants Research Vol. 6(31), pp. 4593-4600,15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR10.078 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Chemical composition and antibacterial activity of essential oils from six Moroccan plants Ahmed Talbaoui1, Naoual Jamaly1,2, M’hamed Aneb1, Abdelkader Il Idrissi3, Mohammed Bouksaim2, Said Gmouh4, Saaïd Amzazi1, Mohammed El Moussaouiti5, Abdelaziz Benjouad1 and Youssef Bakri1* 1

Laboratoire de Biochimie et Immunologie, Faculté des Sciences, Rabat, Université Mohammed V-Agdal, Morocco. 2 Laboratoire de Technologie agroalimentaire INRA Rabat –Morocco. 3 Laboratoire de Chimie des plantes et de synthèse organique et bio-organique, Faculté des Sciences Rabat, Université Mohammed V-Agdal, Morocco. 4 Plateforme Chimie Moléculaire UATRS, CNRST, Rabat- Morocco. 5 Laboratoire de Chimie Physique Générale, Faculté des Sciences, Rabat, Morocco. Accepted 16 May, 2012

The essential oils (EOs) of six plants (Artemisa herba alba, Rosmarinus officinalis, Ocimum basilicum, Lavandula officinalis, Mentha viridis and Mentha piperita) widely distributed in Morocco were isolated and their chemical composition was investigated by gas chromatography-mass spectrometry (GC/MS). These EOs were tested in vitro against four pathogenic bacterial strains (Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae and Streptococcus D) and we determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each EO. The oils of various plants showed high activity against all tested bacteria (MIC≤10 µL/ml), of which K. pneumoniae was the most sensitive strain (MIC≤5 µL/ml). In addition, the oil from M. viridis L. which contained high pulegone concentration (45%) exhibited a very interesting antibacterial activity against all the bacterial strains (MIC 2.5 µL/ml) and (MBC 2.5 µL/ml). The UV-visible study on the release of material absorbing at 260 nm showed significant leakage of cytoplasmic contents, indicating damage to the bacterial cell membrane integrity. Thus, these results indicate that the EOs represent a potential source of natural antibacterial substances that may be used against pathogenic systems. Key words: Artemisia herba alba, Rosmarinus officinalis, Ocimum basilicum, Lavandula officinalis, Mentha viridis, Mentha piperita, essential oil composition, pulegone, antibacterial activity.

INTRODUCTION Infectious diseases are the world’s leading cause of premature death, killing almost 50 000 people every day (Ahmad and Beg, 2001). In the recent years, development of microbial resistance to antibiotics is of a global concern, imposing the need for a permanent search and development of new drugs (WHO, 2003; Levy, 1984; Silver and Bostian, 1993). Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as microorganisms and plants. Therefore, pharmaceutical companies have

*Corresponding author. E-mail: [email protected]. Tel: +212537778012. Fax: +212 537775461.

been motivated to develop new antimicrobial drugs in recent years, especially due to the constant emergence of resistant micro-organisms to conventional antimicrobials all over the world (Piddock and Wise, 1989; Mulligen et al., 1993). The recently emerged resistant Escherichia coli in Europe is one of the frightening examples (Turner, 2011). The drug-resistant bacteria and fungal pathogens have further complicated the treatment of infectious diseases in immunocompromised, AIDS and cancer patients (Rinaldi, 1991; Diamond, 1991). Contrary to the synthetic drugs, antimicrobials of plant origin are not associated with many side effects and have an enormous therapeutic potential to heal many infectious diseases (Iwu et al., 1999). Aromatic and medicinal plants had acquired particular attention in the field of

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Table 1. Region and period of each plant collection studied.

Plant species Artemisia herba alba Ocimum basilicum Mentha viridis Rosmarinus officinalis Lavandula officinalis Mentha piperita

Region of collection Errachidia : Southeast Morocco Agadir : Southwest Morocco Marrakech : Southwest Morocco Rich : High Moroccan Atlas Azrou : Middle Moroccan Atlas Rabat : West Morocco

intensive research on the natural antimicrobial compounds. They constitute a constant source of active reagents against pathogen germs (Mahady, 2005). Among these products, essential oils (EOs) produced by aromatic plants as secondary metabolites, have gained a net interest by many investigators (Ismaiel and Pierson, 1990; Bauer and Garbe, 2001; Oumzil et al., 2002; Zenasni et al., 2008). EOs are volatile natural complex compounds characterized by strong odour (Bakkali et al., 2008), and represent very complex natural mixtures which may contain more than sixty individual components at quite different concentrations (Senatore, 1996). Major components can constitute up to 85% of the EOs, whereas other components are present only as trace (Bauer et al., 2001). It has been recognized that some EOs have antimicrobial, antifungal, anticancer and antioxidants properties (Sara, 2004; Hong et al., 2004; Bozin et al., 2006; Alzoreky and Nakhra, 2003; Baser et al., 2002; Sylvestre et al., 2006). Morocco has an enormous unexplored potential of medicinal plants that are used in traditional medicine, some of which are misused because of lack of scientific data. The heterogeneous ecologic conditions have favoured the proliferation of more than 42, 000 plant species (Tahraoui et al., 2007; Hmamouchi, 1999). Our previous studies have shown that essential oil from some plants such as Mentha suaveolens and Nepeta spp. produced remarkable antibacterial property against several pathogenic bacteria (Oumzil et al., 2002; Zenasni et al., 2008). In this context, we were interested in analyzing in the present work the chemical composition of EOs obtained from six endemic plants of Morocco (namely: Artemisia herba alba, Ocimum basilicum, Mentha viridis, Rosmarinus officinalis, Lavandula officinalis and Mentha piperita) and testing their antibacterial activity against four pathogenic bacterial strains including Enterococcus faecalis, E. coli, Klebsiella pneumoniae and Streptococcus D by disc diffusion method and dilution assay. We also attempted to elucidate the mechanism of action by testing the EOs’ capacity to disrupt the bacterial membrane structure in order to develop new type of disease control alternatives. The selection of medicinal plants is based on their traditional uses in Morocco (Hmamouchi, 1999; El-Hilaly et al., 2003; Bellakhdar et al., 1991).

Period of collection May 2009 May 2009 May 2009 June 2009 June 2009 June 2009

MATERIALS AND METHODS Whole plants were collected from different Moroccan regions where they are usually collected by herbalists for traditional use. Table 1 indicates the region and the period of each plant collection. Extraction of essential oils Fresh aerial parts of whole plants were desiccated at ambient temperature and 100 g of plant material were then subjected to steam distillation for 3 h. The extract recovered was subjected to successive ethyl acetate extractions (3 × 100 ml). After extraction, Na2SO4 (1 g Na2SO4 for 5 ml of EO) was added to the sample mixture to remove water. The sample mixture was then filtered on a filter membrane. The obtained EO was stored in sterile dark glass bottles in a freezer until use.

Analytical techniques Gas chromatography-mass spectrometry (GC/MS) analysis of the EO was performed on a TRACE GC ULTRA equipped with nonpolar VB5 (5% phenyl, 95% methylpolysiloxane) capillary column (30 m × 0.25 mm × 0.25 µM film thickness), directly coupled to a mass spectrometer (Polaris Q). The electron ionization energy was set at 70 eV. The temperature of injector and detector was set at 220 and 300°C, respectively. The oven temperature was programmed from 40 to 180°C at 4°C/min, then for 180 to 300°C at 20°C/min. The components of the oil were identified by comparison of their mass spectra with those in the Willey NIST 7th Edition Library of mass spectral data. The composition of the oil sample was calculated from GC-MS peak areas and given by percentages. The Kovats retention indices (KI) were calculated by using nalkanes C5 – C30 and the experimental values were compared with those reported in the literature (Adams, 2007). Preparation of bacterial strains The tested microorganisms included the following bacteria: E. faecalis, E. coli, K. pneumoniae and Streptococcus D. All pathogenic microorganisms isolated from patients were stored at the culture collection of the Microbiology Department (Microthec Unity) at the Institut National d’Hygiène (Rabat, Morocco). They were maintained in brain heart infusion (BHI) at -80°C. Prior to the experiment, cultures were prepared by subculturing 1 ml of each culture stock in 9 ml of BHI broth in order to obtain culture inoculates in an exponential growth phase of approximately 106 CFU/ml. Disc diffusion method The agar disc diffusion (ADD) method was employed for the

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determination of antimicrobial activities of the tested EO as described previously (Oumzil et al., 2002). Briefly, the test was performed in sterile Petri plates containing BHI agar. Sterile filter paper discs (6 mm in diameter) were impregnated with 6 µL of oil and were placed on the Petri plates previously inoculated with a sterile microbial suspension (one microorganism per Petri dish). All Petri plates were sealed with sterile laboratory films to avoid eventual evaporation of the test samples, and then incubated at 37°C for 24 h. The diameters of inhibition zones were measured in millimetres.

Determination of MIC and MBC We tested six serial concentrations of each EO (40, 20, 10, 5, 2.5, 1.25 and 0.625 µL/ml) diluted in BHI broth with 0.15% agar and strongly mixed for 2 min using a vortex. The MIC was assessed according to the procedure established by Canillac and Mourey (1995) and Oumzil et al. (2002). Briefly, 5 ml of culture medium was inoculated with 0.1 ml of a bacterium precultured in BHI at 37°C. The final concentration of bacteria was of 10 6 CFU/ml. The MIC is the lowest concentration of BHI (0.15% agar) for which no growth was detected after 24 h at 37°C (Canillac and Mourey, 1995). While for the determination of MBC, 0.1 ml of the cell suspensions from the tubes showing no growth were subcultured on nutrient agar plates and the Petri plates were incubated for 24 h at 37°C. The MBC was the highest dilution (lowest concentration) of the EO at which no growth occurred on the plates (Smith-Palmer et al., 1998).

Bactericidal activity E. coli were grown overnight at 37°C in 100 ml BHI broth. A series of increasing concentrations of each EO were prepared in the culture broth medium and 500 µL of viable bacteria were inoculated into each tube, shaken and incubated at 37°C for 24 h. The density of the each culture (designed as bacterial growth) was measured at a wavelength of 600 nm after each time point. To detect genetic material release, 1 ml sample of each tube were centrifuged at 1200 g for 5 min to remove all trace of bacteria. The supernatant was re-suspended in PBS and used to measure UV 260 nm absorption by Camspec UV/visible spectrophotometer at each time point. We used untreated bacteria as negative control and bacteria treated with penicillin-streptomycin (100 U/ml - 100 µg/ml) as positive control.

RESULTS Chemical composition of the essential oils The results obtained by GC-MS analysis of the EOs are presented in Table 2. A. herba alba contained eucalyptol (27.29%), camphor (23.42%) and chrysanthenone (21.76%) as the major compounds. The oils from R. officinalis are also characterized by a high percentage of eucalyptol (56.85%). Moreover, the main volatile components of the O. basilicum EO are linalol (53.98%) and methyl trans-cinnamate (15.33%). For L. officinalis EO, we found that linalyl acetate (44. 96%) and linalol (44.64%) were the main components. The chemical composition of EO of the two Mentha species (M. viridis and M. piperita) is qualitatively similar, although there are

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some differences in the concentrations of individual components; linalol (52%) and linalyl acetate (25.9%) are the predominant compounds in the oil of M. piperita, but the essential oil of M. viridis contained the largest amount of pulegone (45%).

Antibacterial activity of the essential oils The results of the disk diffusion test indicate that each EO showed different degree of growth inhibition (Figure 1). The maximum inhibition was recorded against E. coli with the EO of O. basilicum (20 mm) Figure 1A. Streptococcus D and Enterococcus faecalis were susceptible to L. officinalis EO with inhibition zone 12 mm and 17 mm respectively (Figure 1B and C). R. officinalis EO and L. officinalis exhibited significant activity against K. pneumoniae with similar inhibition zone of about 16 mm (Figure 1D). In addition, the antibacterial activity indicated that oils from A. herba alba, R. officinalis, O. basilicum, L. officinalis and M. piperita presented comparable activity against all strains of tested bacteria (MIC≤10 µL/ml) (Table 3). Furthermore, the EO of M. viridis was found to be more active at lower dilution against the chosen pathogenic bacterial strains (MIC = 2.5 µL/ml and MBC = 2.5 µL/ml; Table 3). In addition, K. pneumoniae was more sensitive to the oils studied (MIC≤5 µL/ml). In the case of O. basilicum, E. coli was the most sensitive (diameter of inhibition = 20 mm) (Figure 1A) and MIC was of about 5 µL/ml of oil dilution (Table 2).

Bactericidal activity and cell lysis The mechanism of action of EOs from our selected plants was studied by examining their bactericidal and cell lytic activity against E. coli, and comparing it with penicillinstreptomycin cocktails. The antibacterial activity of the EOs was determined using EO concentration corresponding to 2xCMI. We first tested antibacterial effect of all EOs against E. coli. EO treated bacteria showed growth arrest after 24 h. E. coli showed high sensitivity to all EO, with 75% of growth inhibition in the presence of EO from M. viridis and M. piperita as compared to untreated bacteria (T ) (Figure 2A). Bacteriostatic agents limit the growth of bacteria by interfering with protein production, DNA replication or other aspects of bacterial cellular metabolism. This is in contrast to bactericides which kill bacteria (Pankey and Sabath, 2004). Furthermore, leakage of cytoplasmic contents is an indicator of damage to the bacterial cytoplasmic membrane. Therefore, bacterial cell membrane integrity was examined by quantification of the released of material absorbing at 260 nm (DNA and RNA) after adding EOs at the indicated concentrations by spectrophotometry in the supernatant fluid. In the same time, the viable bacteria

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Table 2. Chemical composition (%) of six essential oils from Artemisia herba alba (AHA), Rosmarinus officinalis (RO), Ocimum basilicum (OB), Lavandula officinalis (LO), Mentha viridis (MV), Mentha piperita (MP).

Compounds α-pinene Camphene β-pinene Myrcene Eucalyptol Ocimene Linalyl acetate Linalool Thujone Alcohol fenchylique Chrysanthenone Camphor Menthone Borneol Limonene α-Terpineol Pulegone Carvone Geraniol Chrysanthenyl acetate Methyl trans-cinnamate Eugenol Eugenol methyl ether Trans- caryophyllene Cis-Caryophyllene Humulene Caryophyllene oxide Farnesol Others

AHA 3.40 3.87 27.29 3.83 1.76 21.76 23.42 4.02 3.14 2.30 2.66 2.55

RO 15.34 4.85 6.52 56.85 12.99 3.45 -

OB 9.33 1.70 54 1.99 2.85 15.3 1.89 3.43 2.63 4.56 2.32

Percentage (%) of compounds LO MV 1.5 2.08 2.5 44.96 0.5 44.64 2.66 33 2.51 5.5 45 0.5 4.5 7 3.15 -

MP 2.9 1.5 0.2 25.9 52 0.5 0.2 4.5 1.2 0.4 1.2 0.9 7.6

KI* 919 950 985 1000 1027 1038 1072 1089 1099 1108 1133 1141 1160 1164 1200 1229 1235 1249 1255 1277 1295 1358 1399 1404 1420 1438 1571 1695 -

KI** 939 953 980 991 1033 1040 1061 1098 1102 1112 1123 1143 1154 1165 1189 1237 1237 1242 1255 1262 1301 1356 1401 1404 1418 1440 1581 1697 -

I: Kovats retention indices; KI* experimental values and KI**: literature values.

decrease significantly after 24 h when compared to control. The release of material absorbing at 260 nm after 24 h in the treated cultures with EOs showed significant leakage compared to untreated bacteria (T ) (Figure 2B). When compared to E. coli treated with penicillinstreptomycin at the indicated concentration, EOs showed slightly lower capacity to damage bacterial membrane except for M. piperita which showed higher activity than the plant species (Figure 2B). This phenomenon was observed as soon as 1 h of incubation (data not shown), thus indicating membrane damage related to the addition of the EOs. For the first time, it was determined that Eos exhibit their activity by damaging the cell membrane and inducing leakage of the tested bacteria. The same results were confirmed at 48 h and five days of culture (data not shown). These results largely confirmed the strong antibacterial agents of the EOs studied.

DISCUSSION The results obtained by GC-MS analysis shows that each plant species has a specific quantitative and qualitative composition. The reasons of this variability can be due to different geographical sources, the genotype and the climate; all of this variability influences the chemical composition and the relative concentration of each constituent (Masotti et al., 2003; Angioni et al., 2006; Cosentino et al., 1999). For example, in our study, the oils from R. officinalis are characterized by a high percentage of Eucalyptol, although EO of this plant growing in Algeria belongs to 1,8-cineole chemotype (Boutekedjiret et al., 1998). In a previous study, it was shown that EO from Mentha suaveolens subspecies present variable chemical composition that is different from those of Mentha species studied here (Oumzil et al.,

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Essential oils

Essential oils

(D)

0

M.P

M.P

M.V

L.O

O.B

R.O

0

5

M.V

5

10

L.O

10

15

O.B

15

20

R.O

20

25

A.H.A

Diameter of inhibitio (mm)

25

A.H.A

Diameter of inhibition (mm)

(C)

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M.P

0

M.V

M.P

M.V

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O.B

R.O

0

4

L.O

5

8

O.B

10

12

R.O

15

16

A.H.A

20

Diameter of inhibition (mm)

(B)

25

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(A)

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Essential oils

Figure 1. Antibacterial activity* of EO from Artemisia herba alba (A.H.A), Rosmarinus officinalis (R.O), Ocimum basilicum (O.B), Lavandula officinalis (L.O), Mentha viridis (M.V) and Mentha piperita (M.P) against E. coli (A), Streptococcus D (B), E. faecalis (C) and K. pneumoniae (C). *: Mean zone of inhibition (Ø mm) and standard deviation.

Table 3. Minimal inhibitory concentrations (MIC) (µL/ml) and minimal bactericidal concentration (MBC) (µL/ml) of selected essential oils from Artemisia herba alba, Rosmarinus officinalis, Ocimum basilicum, Lavandula officinalis, Mentha viridis and Mentha piperita against four pathogenic bacteria.

Plant species Artemisia herba alba Rosmarinus officinalis Ocimum basilicum Lavandula officinalis Mentha viridis Mentha piperita

E. coli 10/10 10/10 5/5 10/10 2.5/2.5 10/10

Test organism (MIC/MBC) Streptococcus D E. faecalis 10/10 10/10 10/10 10/10 10/10 5/5 10/10 10/10 2.5/2.5 2.5/2.5 2.5/5 2.5/2.5

K. pneumoniae 2.5/2.5 5/5 5/5 5/5 2.5/2.5 2.5/2.5

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(A)

(B)

Bacterial growth

DNA release

Figure 2. Bacterial growth (measured as absorbance at 600 nm) registered after 24 h of exposure to each essential oils on E. coli (A) and leakage of material cytoplasmic (measured as absorbance at 260 nm) (B). T-: Untreated bacteria, T+: positive control (penicillinstreptomycin).

2002). The EO of M. viridis was found to be more active at lower concentration against all the pathogenic bacterial strains we used. These results could be due to differences in chemical composition of the oils as we have reported previously (Oumzil et al., 2002; Zenasni et al., 2008). Indeed, it has been reported that pulegone play an important role in antibacterial activity (Andersen and Jensen, 1984; Oumzil et al., 2002). Our findings may suggest the potential use of M. viridis oils in treatment of infections caused by those pathogenic germs. Moreover, we found that K. pneumoniae was the most sensitive germ to EO from M. viridis. In the case of O. basilicum, E. coli was the most sensitive; this oil contains eucalyptol which was reported to impart microbial effects on K. pneumoniae (Fabio et al., 2007). Thus, one may take into consideration that the inherent activity of an oil can be expected from the chemical configuration of the components, the proportions in which they are present and to interactions between them (Dorman and Deans, 2000). Considering the large number of different group of chemical compounds present in EOs, it is most likely that their antibacterial activity is not attributable to one specific mechanism of action, but there are several targets in the cells (Skandamis and Nychas, 2001; Carson et al., 2002) and the mechanisms of action have not been yet studied (Lambert et al., 2001; Oumzil et al., 2002). Our study showed that many essential oils possess important antibacterial activity against the four pathogenic bacteria species studied. Among the EO tested, M. viridis was the most active on all the bacteria tested with MIC≤2.5 µL/ml. In addition, O. basilicum was the most

active on E. coli. These findings suggest an interesting antibacterial potential of M. viridis and O. basilicum EO and the possible development of new drugs based on the EO components, in treating infections caused by pathogen germs with high morbidity and mortality worldwide. The measurement of growth inhibition of bacterial by disc diffusion method and dilution assay is not sufficient. Additional studies are required on the mode of action in pathogenic bacteria as effects on bacterial cell membranes. The disruption of the bacterial membrane structure has not yet been well characterized in term of the mode of action. Most antimicrobial agents may be categorized according to their principle mode of action. It is postulated that polymixin, hexachlorophene and chlorhehexidine exert their inhibitory effects by increasing bacterial membrane permeability, causing leakage of bacterial cell and they get partitioned into the lipid bilayer of the cell membrane, causing frequent fundamental changes in bacteria membrane and function, thus rendering it more permeable and provoke whole cell lysis (Hugo and Longworth, 2011; Joswick et al., 1971; Pankey and Sabath, 2004). Release of intracellular components is a good indicator of membrane integrity; small ions as potassium and phosphate tend to each out first, followed by large molecules such as DNA, RNA and other materials (Hugo and Longworth, 2011; Joswick et al., 1971). On the other hand, the mechanisms by which EOs can inhibit microorganisms involve different modes of action. The cytoplasmic cell membrane undoubtedly is the target of many antimicrobials agents. Although, the antibacterial properties of essential oils has been

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reviewed in the past. The mechanism of action has not been studied in great detail (Lambert et al., 2001). In line with this, EOs by penetrating through the cell wall and cytoplasmic membrane disrupt and permeabilize them and provokes leakage of cytoplasmic constituents: metabolites and ions (Cowan, 1999; Thoroski et al., 1989). This activity has been also demonstrated for essential oils from oregano and thyme (Horne et al., 2001). In addition chemical compounds from essential oils also act on cytoplasmic cell membrane (Knobloch et al., 1989). Carvacrol and thymol damaged cell membrane and increase its permeability (Lambert et al., 2001). When tested at concentration higher than their minimum inhibitory concentration, carvone and eugenol disintegrate the outer membrane (Thoroski et al., 1989; Oosterhaven et al., 1995). Leakage of cytoplasmic contents is an indicator of damage to the bacterial cytoplasmic membrane. The UV-visible study on the release of material absorbing at 260 nm showed significant leakage. This indicates membrane damage related to the addition of the essential oils studied here. To the best knowledge, no previous study was undertaken to provide comparative data on the mode action of EOs against pathogenic bacteria. For the first time, it was determined that M. piperita and M. viridis essential oils exhibit their activity by highly damaging the cell membrane of the tested bacteria in our experimental condition when compared to using antibiotics such as penicillin and streptomycin. Nevertheless, other studies should be aimed at determining the exact mechanism of action of each EO by comparison to the most potent antibiotics used in therapeutics and effects in vivo. Many bacteria can cause fatal diseases; in fact despite the existence of potent antibiotic agents, resistant or multi-resistant strains are continuously emerging. In an effort to discover new lead compounds, many research groups screen natural components to detect secondary metabolites with relevant antibacterial activities. In conclusion, the essential oils produced in Morocco offer a promising way for research of the phytochemical active principle in therapeutic indications (Hmamouchi, 1999). However, studies should be conducted to determine the mechanism of action of each EO and compare with the most potent antibiotics used in therapeutics.

ACKNOWLEDGEMENTS This work was supported by the Region Rabat-SaleZemmour-Zaer, Plan d’urgence program financed by Mohammed V University Agdal- Rabat, the Pole de Competence PHARCHIM and the convention CNRSTINSERM. REFERENCES Adams RP (2007). Identification of essential oil components by gas

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chromatography/mass spectrometry, 4th Ed. Allured Publishing corporation Carol Stream, Illinois, USA. Ahmad I, Z-Beg A (2001). Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-resistant human pathogens. J. Ethnopharmacol. 74:113-123. Alzoreky NS, Nakhra K (2003). Antibacterial activity of extracts from some edible plants commonly consumed in Asia. Int. J. Food Microbiol. 80:223-230. Andersen PH, Jensen NJ (1984). Mutagenic investigation of peppermint oil in the Salmonella/mammalian microsom test. Mutat. Res. 138:1720. Angioni A, Barra A, Coroneo V, Dessi S, Cabras P (2006). Chemical composition, seasonal variability, and antifungal activity of Lavandula stoechas L. ssp stoechas essential oils from stem/leaves and flowers. J. Agric. Food Chem. 54:4364-4370. Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008). Biological effects of essential oils- A review. Food Chem. Toxicol. 46:446-475. Baser KHC, Demirci B, Demirci F, Koçak S, Akinçi C, Malyer H, Güleryüz G (2002). Composition and antimicrobial activity of the essential oil of Achillea multifida. Planta Med. 68:941-943. Bauer K, Garbe D, Surburg H (2001). Common Fragrance and Flavor Materials : Preparations, Properties and uses. 2nd edn. Willey-VHC, Weinheim. Bellakhdar J, Claisse R, Fleurentin J, Younos C (1991). Repertory of herbal standard drugs in the Moroccan pharmacopoea. J. Ethnopharmacol. 35:123-143. Boutekedjiret C, Bentahar F, Belabbes R, Bessiere JM (1998). The essential oil from Rosmarinus officinalis L. in Algeria. J. Essent. Oil Res. 10:680-682. Bozin B, Mimica-Dukic N, Simin N, Anackov G (2006). Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oil. J. Agric. Food. Chem. 54:188-828. Canillac N, Mourey A (1995). Effect of fir and pine essential oil on some ripening microorganisms. M.A.N: Microbiol. Aliments Nutr., 13: 267273. Carson CF, Mee BJ, Riley TV (2002). Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage and salt tolerance assays and electron microscopy. Antimicrob. Agents Chemother. 46:1914-1920. Cosentino S, Tuberosa CIG, Pisano B, Satta M, Mascia V, Arzedi E, Palmas F (1999). In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Lett. Appl. Microbiol. 29:130-135. Cowan MM (1999). Plant products as antimicrobial agents. Clin. Microbiol. Rev., 12: 564-582. Diamond RD (1991). The growing problem of mycoses in patients infected with human Immunodeficiency virus. Rev. Infect. Dis., 13: 480-486. Dorman HJD, Deans SG (2008). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88:308316. El-Hilaly J, Hmamouchi M, Lyoussi B (2003). Ethnobotanical studies and economic evaluation of medicinal plants in Taounate province (Northern Morocco). J. Ethnopharmacol. 86:149-158. Fabio A, Cermelli C, Fabio G, Nicoletti P, Quaglio P (2007). Screening of the antibacterial effects of a variety of essential oils on microorganisms responsible for respiratory infections. Phytother. Res. 21:374-377. Hmamouchi M (1999). Medicinal and aromatic plants of Morocco (book). Imprimerie Fedala Morocco, pp. 11-29. Hong EJ, Na Kj, Choi IG, Choi KC, Jeung EB (2004). Antibacterial and antifungal effects of essential oils from coniferous trees. Biol. Pharm. Bull. 27:863-866. Horne DS, Holm M, Oberg C, Chaos S, young DG (2001). Antimicrobial effect of essential oil on Streptococcus pneumoniae. J. Essent. Oils. Res. 3:387-392. Hugo WB, Longworth R (2011). Some aspects of the mode of action of chlorhexidine. J. Pharm. Pharmacol. 16:655-662. Ismaiel A, Pierson MD (1990). Inhibition of growth and germination of C. botulinum 33A, 40 B, 1623, by essential oil of spices. J. Food. Sci. 55:1676-1678.

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Iwu MW, Duncan AR, Okunji CO (1991). New antimicrobials of plant origin. Int: Janick J. (Ed): Perspectives on new crops and New uses. ASHS. Press. Alexandria 5A:457-462. Joswick HL, Corner TR, Silvernale JN, Gerhardt P (1971). Antimicrobial actions of hexachlorophene, release of cytoplacmice material. J. Bacteriol. 108:492-500. Knobloch K, Pauli A, Ibert B (1989). Antibacterial and antifungal properties of essential oil components. J. Essent. Oil Res. 1(3):119128. Lambert RJW, Skandamis PN, Coote P, Nychas G (2001). A study of the minimum inhibitory concentration and mode of action of Oregano essential oil, thymol and carvacrol. J. Appl. Microbiol. 91:453-462. Levy SB (1984). The antibiotic paradox. How miracle drugs are destroying the miracle. Plenum Press. Mahady GB (2005). Medicinal plants for the prevention and treatment of bacterial infections. Curr. Pharm. D 11:2405-2427. Masotti V, Juteau F, Bessière JM, Viano J (2003). Seasonal and phenological variations of the essential oil from the narrow endemic species Artemisia molinieri and its biological activities. J. Agric. Food Chem. 51:7115-7121. Mulligen ME, Muny-Leisure KA, Ribner BS, Standiford HC, John JA, Kauffman CA, Yu VL (1993). Methicillin-resistant Staphylococcus aureus. Am. J. Med. 94:313-328. Oumzil H, Ghoulami S, Rhajaoui M, Ilidrissi A, Fkih-Tetouani S, Faid M, Benjouad A (2002). Antibacterial and antifungal activity of essential oils of Mentha suaveolens EHRH. Phytother. Res. 16:723-731. Oosterhaven K, Poolman B, Smid EJ (1995). S-carvone as natural potato sprout inhibiting fungistatic and bacteiostatic compound. Ind. Crops Prod. 4(1):23-31. Pankey GA, Sabath LD (2004). Clinical prevalence of bacteriostatic versus bactericidal mechanisms of action in the treatment of Gram + bacterial infections. Clin. Infect. Dis. 38(6):864-870. Piddock KJV, Wise R (1989). Mechanisms of resistances to quinolones and clinical perspectives. J. Antimicrob. Chemother. 23:475-483. Rinaldi MG (1991). Problems in the diagnostic of invasive fungal diseases. Rev. Infect. Dis., 13: 493-495. Sara B (2004). Essential oils: Their antibacterial properties and potential applications in foods. A review. Int. J. Food Microbiol. 94:223-253. Senatore F (1996). Influence of harvesting time on yield and composition of the essential oil of thyme (Thymus pulegioides L.) growing wild in Campania (Southern Italy). J. Agric. Food Chem. 44:1327-1332.

Silver LL, Bostian KA (1993). Discovery and development of new antibiotics: the problem of antibiotic resistance. Antimicrobiol. Agents. Chemother. 37:377-383. Skandamis PN, Nychas GJE (2001). Effect of Oregano essential oil microbiological and physico-chemical attributes of minced meat stored in air and modified atmospheres. J. Appl. Microbiol. 91:10111022. Smith-Palmer A, Stewart J, Fyfe L (1998). Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett. Food Microbiol. 26:118-122. Sylvestre M, Pichette A, longtin A, Nagau F, Legault J (2006). Essential oil analysis and anticancer activity of leaf essential oil of Croton flavens L. from Guadeloupe. J. Ethnopharmacol. 103:99-100. Tahraoui A, El Hilaly J, Israili ZH, Lyoussi B (2007). Ethnopharmacological survey of plants used in the traditional treatment of hypertension and diabetes in South-eastern Morocco (Errachidia province). J. Ethnopharmcol. 110:105-117. Thoroski J, Blank G, Billardis C (1989). Eugenol induced inhibition of extracellular enzyme production by Bacillus cereus. J. Food Prot. 52(6):397-403. Turner M (2011). German E. coli outbreak caused by previously unknown strain. Nature. doi: 10.1038/news 345. WHO (2003), world Health Report World Health Organization, Geneva, Switzerland. WHO. Publicat. office. pp. 1-50 Zenasni L, Bouidida H, Hancali A, Boudhane A, Amzal H, Ilidrissi A, El ouad R, Bakri Y, Benjouad A (2008). The essential oils and antimicrobial activity of four Nepeta species from Morocco. J. Med. Plants Res. 2:111-114.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4601-4608, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.376 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

In vitro reversal of deformity and inhibition of aggregation of sickle red blood cells by two Congolese herbal medicines Marie Miezi Nsimba1,2, José Nzunzu Lami2*, Chika Yamamoto1, Toshiyuki Kaji1, Matadi Mukengeshaie3 and Muhandisha Lufuluabo3 1

Organization for Frontier Research and Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan. 2 Laboratory of Bio-Organic Research, Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Kinshasa University, P. O. Box 212 Kinshasa XI, D. R. Congo. 3 Centre de Phytothérapie Moderne Nieca, Kinshasa, D. R. Congo. Accepted 9 May, 2011

The effects of aqueous extracts from the trunk bark and branches of Ceiba pentandra and from the root bark of Quassia africana Baill., which are claimed to overcome the clinical events of the sickle cell anemia (SCA) in Democratic Republic of Congo (DRC), were investigated in vitro on the red blood cells (RBC) deformity and aggregation. Blood samples from SCA patients and from healthy persons were treated with 2% sodium metabisulfite to induce hypoxia and sickling of erythrocytes, and then, were incubated with the drug extracts. It was found that extracts, used separately or together, reversed the induced deformity of RBC. On the other hand, aggregates of RBC were incubated with the plant extracts and the action was evaluated by microscope examination, which showed that cells became dispersed and isolated, while they remained stacked in the samples not treated. Sickling of RBC is a major factor among others, which are implicated for initiating the events of sickle cell crises as well as the increasing red blood cells adhesiveness observed in increased blood viscosity. These observations could support the use of the two medicinal drugs to deal with the clinical events of SCA. Key words: Sickle cell anemia (SCA), red blood cell (RBC), deformity, hypoxia, aggregation, hemoglobin S, hemoglobin A, blood viscosity, hemolysis, Ceiba pentandra, Quassia africana Baill.

INTRODUCTION Deformity of the red blood cells is the mechanism initiating pathologic manifestations, which are the source of several complications in sickle cell anemia (SCA). Red cells have a tendency of losing their elasticity and are

*Corresponding author. E-mail: [email protected]. Tel: +243 99 991 8243, +243 81 329 9876. Abbreviations: RBC, Red blood cell; SCA, sickle cell anemia; Hb-SS, related to sickle cell anemia subject with affected hemoglobin SS; Hb-AA, related to healthy subject with normal hemoglobin AA.

unable to flow through narrow capillaries, leading them to become stuck in blood vessels. This deprives the downstream tissues of oxygen and causes ischemia and infarction, which may lead to organ damage, such as stroke (Platt, 2000; Wikipedia, 2007; Lonergan et al., 2001). In addition, aggregation of red blood cells is another factor implicated in the pathophysiology in SCA and may influence the blood viscosity as a result (Martorana et al., 2007; Saldanha, 2002). Increased blood viscosity as well as alteration of membrane viscosity of red blood cell (RBC) are reported in SCA, playing a role on the disease defect and making worse the clinical complications (Chien et al., 1970; Wendell et al., 2000).

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SCA affects millions of people worldwide, mostly in Africa where the sickle trait frequency varies from 15 to 25% (Serjeant, 1994) and a greater number of people rely on traditional medicines to deal with the disease due to the high costs of lifelong modern treatments. The practice of traditional medicine is widespread in the world and a variety of activities and projects in medicinal plants are being conducted and promoted by several organizations worldwide (Hoareau and DaSilva, 1999). Many plants are being used as traditional medicines, but most of them have not yet been scientifically studied to prove or determine their efficacy. The drugs used in this study are prepared and administered since many years to SCA patients by the “Centre de Phytothérapie Moderne NIECA”, an officially recognized medical centre located in Kinshasa (DRC), named BEAT-SS for that prepared from Ceiba pentandra and DOCABE for Quassia africana (Baill). C. pentandra is a big tree generally found in rainforest tropical zones, and is widely used in herbal medicine in West and Central Africa, in South America, in West and South East Asian countries (Burkill, 1985). This plant is reported to have hypoglycemic (Ladeji et al., 2003; Dzeufiet et al., 2006, 2007), antidiarrheic (Abena et al., 2008), and antifungal (Nwachukwu et al., 2008) properties. Q. africana (Baill.) is a small tree of the lowland rainforest in the transition zone from evergreen to semi-deciduous forest (FAO, 1986); it has been found to have antiviral (Apers et al., 2002) and antipaludic (Lohombo et al., 2003) activities; in African folk medicine, the decoction of the bark and leaves is used for gastro-intestinal conditions and as a vermifuge, the root is used to treat bronchial illness, as a febrifuge and as anti-rheumatic (FAO, 1986). BEAT-SS is dispensed by the Centre NIECA to SCA patients to relieve the clinical manifestations. DOCABE is administered in association with BEAT-SS in case of painful crises and is considered to have an antiinflammatory effect. According to this centre, improvement of clinical symptoms has been observed during and after the treatment; especially referring to fatigue, acute pains, swelling in hands or feet, and skin pallor. Moreover, non-recourse to blood transfusion is done for a long period after cessation of medication. However, these activities have not yet been investigated. The aim of this work was to examine by in vitro experiments some scientific parameters in order to understand the use of these two traditional medicines as anti-sickling drugs in the management of SCA. MATERIALS AND METHODS Blood samples Blood samples from SCA patients used in this study were provided under the authorization of the Congolese ethic committee (N° d’approbation: ESP/CE/048/2009) by the “Centre de Phytothérapie Moderne NIECA” and the “Centre de Médécine Mixte et d’Anémie

SS,” located both in Kinshasa (DRC). Patients selected were those who did not receive blood transfusion two months before donating blood. Adults with known hemoglobin AA type (Hb-AA), mostly laboratory members, donated samples from healthy subjects. Blood was introduced in a plastic tube containing anticoagulant (Ethylenediaminetetraacetic acid (EDTA) 10%: 0.05 ml for 3 ml of blood). The samples were placed in an icebox for commuting to the laboratory and were kept at 4˚C before the experiment.

Extracts preparation Dried powdered plant materials were provided by the “Centre NIECA” and plant voucher specimens were identified at the “Institut National d’Etudes et des Recherches Agronomiques” (INERA) of Kinshasa University (DRC). 1000 ml of aqueous extracts from each plant were separately prepared according to the phytotherapist know-how, by boiling for 5 min in distilled water, respectively, 50 g of dried powder from the bark of trunk and branches of C. pentandra and 100 g of dried powder from the bark of roots of Q. africana. The prepared decoctions were filtrated and lyophilized to yield 3.5 and 4.3 g of dried powder for C. pentandra and Q. africana, respectively. The lyophilized substances from the two extracts were used to prepare, for each, a stock extract solution of 1 mg/ml using distilled water. The solutions were further filtrated on a sterile Millex HA filter Unit of 0.45 µm Millipore diameter and were kept at 4°C in a freezer for use in subsequent analysis.

Evaluation of RBC hemolysis after extracts addition Addition of extracts may induce the hemolysis of erythrocytes, which could be detected by reduction on the RBC number as compared to a control sample where extracts are not added. The effect of high concentrations of the two extracts on the presumed hemolysis of the RBC was evaluated by using erythrocytes count test. Blood samples from a sickle cell patient and from a healthy subject were diluted with Hayem’s solution 1/100 (Na2SO4, 2.5%; NaCl, 0.5%; and HgCl2, 0.25%), and then samples were incubated separately with the two extracts in different concentrations for 2 or 24 h. The number of erythrocytes from each sample was determined by cell count under microscope using Neubauer hemocytometer. The concentrations of the extracts used were 20, 100, 500, and 2500 µg/ml for BEAT-SS; and 5, 25, 125, and 625 µg/ml for DOCABE.

Inhibition of deformity of sickled RBCs Deformity or sickling of RBC is induced by hypoxia condition created by the addition of sodium metabisulfite (Na2S2O5, 2%) on the blood samples from SCA patients (Hb-SS) according to Emmel test (Murayama and Nalbandian, 1973). To evaluate the action of the extracts on the inhibition of deformity of sickled erythrocytes, the previous samples in hypoxia condition were treated separately with the two extracts in different concentrations. After incubation, sickled erythrocytes (elongated cells) will regain the normal round form: this activity was expressed as the inhibition of deformity or the normalization of sickled RBC. A determined volume of the blood sample from a SCA patient was mixed with an equivalent volume of sodium metabisulfite (2%). The mixture was incubated for 1 h at room temperature to complete the sickling. Observations under microscope confirmed that sickling of RBC begin to occur at least 30 min after the addition of sodium metabisulfite. Thus, the duration of 1 h of incubation was quite enough to complete sickling for the majority of RBC. This constituted the control sample of Hb-SS in hypoxia condition. Another control sample in oxygenated condition was observed

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before the addition of sodium metabisulfite to Hb-SS. After 1 h of incubation of blood with sodium metabisulfite, an equivalent volume of the extract in different concentrations was added to that of the previous mixture (blood with sodium metabisulfite) and incubated again for 30 or 120 min more at room temperature. A specimen was taken for microscopic examination and was pictured (LCD-microscope Bresser, GmbH & Co. KG; and Olympus microscope CKX41 with camera DP50). Cells were counted to determine the number of both sickled cells (distorted and elongated RBC) and normal cells (round shaped RBC). The total cells number was deducted by the summation of sickled and normal RBC. The percentage of normal RBC was obtained by calculation and it represents the ratio of normal rounded cells in the total cells numbered. The concentrations used were 10, 50, 100, 200, 400, and 500 µg/ml for BEAT-SS; and 1, 5, 10, 50, 100 and 200 µg/ml for DOCABE. Used in association, the two extracts were incubated together with blood samples in couples of concentrations as shown in Table 2. The same experiment was conducted on blood samples provided by healthy subjects.

Inhibition of red blood cells aggregation Blood samples were allowed to stand for 48 h at 4°C after their arrival in the laboratory. After this rest, aggregates of red RBC were observed on microscope for both Hb-SS (Figure 2A) and Hb-AA samples. Addition of an equivalent volume of physiological saline (NaCl, 0.9%) to blood was required to disperse these aggregates. Aggregates were not dispersed on samples from Hb-SS (Figure 2B), but this was not the case with samples from Hb-AA (Figure 2C). After these observations, a volume of Hb-SS blood sample with aggregates of RBC (Figure 2A) was incubated for 30 min or 120 min with an equivalent volume of the extracts in different concentrations, then they were examined under microscope. Two controls Hb-SS blood samples with aggregates of RBC were made up, as described earlier: one with blood alone and the other with blood mixed with the physiological saline. The extract’s concentrations in the sample tests were 100 and 200 µg/ml for BEAT-SS, 50 µg/ml for DOCABE, and in association of the two extracts, 200 µg/ml for BEAT-SS and 50 µg/ml for DOCABE. The same experiment was conducted on blood samples provided by a healthy subject. Furthermore, to verify if the dispersion of the aggregated RBC from Hb-AA after addition of saline (Figure 2C) occurred independently of the red blood cells density, the experiment was repeated using normal density of RBC from Hb-AA blood samples by avoiding dilution with saline solution. Extracts in different concentrations were added to blood samples in small volume (1 volume of extract for 19 volumes of blood), and then they were incubated for 2 h at room temperature. The extracts concentrations were 50, 100, and 200 µg/ml for BEAT-SS; and 5, 10, and 50 µg/ml for DOCABE. Statistical analysis Data were expressed as the mean ± standard deviation (SD) and were analyzed for statistical significance using analysis of variance (ANOVA) and Bonferroni’s multiple t-test. P-values of less than 0.05 were considered to be statistically significant.

RESULTS Effect of extract on the red blood cells hemolysis Table 1 reports the results of different concentrations of

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the two plants extracts action on the number of erythrocytes after incubation at room temperature with the blood samples from sickle cell patients and healthy subjects. In the case of BEAT-SS extract, a decrease on the number of erythrocytes was not noticed after incubation for 2 or 24 h of the blood samples with the extract from 20 to 500 µg/ml in the two groups of blood samples (HbSS and Hb-AA), denoting that hemolysis of RBC did not occur with addition of BEAT-SS at these concentrations. A significant decrease in the number of erythrocytes was noticed with the concentration of 2500 µg/ml in both blood samples (Hb-SS and Hb-AA) after 2 h of incubation and more again after 24 h, denoting that high concentration of BEAT-SS (2500 µg/ml) induced destruction (hemolysis) of a part of the population of RBC after 2 h of exposition, and more after 24 h. Also, concerning DOCABE extract, no decrease in the erythrocytes number was observed for the two groups of samples (Hb-SS and Hb-AA) after 2 or 24 h of incubation with the extract in the concentrations from 5 to 625 µg/ml, except for a significant decrease observed after 24 h of incubation with the highest concentration of 625 µg/ml, solely with the blood sample from Hb-AA. This denotes that DOCABE extract did not induce hemolysis of RBC in the used concentrations, except for 625 µg/ml, solely after 24 h of incubation for Hb-AA.

Effect of the extracts on the erythrocyte’s deformity Pictures taken from slides test of treated samples for the examination of the inhibition of deformity by the two extracts are as shown in Figure 1. After images examination, the total number of RBC, including sickled cells (SC or distorted RBC) and normal cells (NC or round shaped RBC), was determined by count and the percentage of normal RBC, compared to the total number of RBC in the sample, was deduced by calculation (Table 2). In the control samples from Hb-SS, it was observed that RBC did not exhibit the deformity on the shapes (Figure 1A1), when in the oxygenated condition (or before the addition of sodium metabisulfite on the blood sample from Hb-SS). Deformity of RBC was observed 1 h after hypoxia by the addition of sodium metabisulfite and most of the RBC lost the round shape and became distorted (Figure 1A2). Control RBC from Hb-AA did not display any shape’s change after addition of sodium metabisulfite in the same conditions (Figure 1A3). Furthermore, blood samples from Hb-SS which have been incubated with BEAT-SS extract after induction of hypoxia (sodium metabisulfite addition for 1 h) displayed an appreciable reversing effect on the induced deformity, as shown in Figure 1B1, B2, and B3, respectively for BEAT-SS 50, 100, and 200 µg/ml. It appeared that this reversing action depended on the concentration of the

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Table 1. Effect of the extracts on the erythrocytes number after extracts treatment for 2 or 24 h of incubation.

Extract concentration (µg/ml) Control BEAT-SS 20 BEAT-SS 100 BEAT-SS 500 BEAT-SS 2500 DOCABE 5 DOCABE 25 DOCABE 125 DOCABE 625

Hb- SS 2 h incubation 24 h incubation 3.4 4.0 3.6 3.7 3.8 3.2 3.6 3.3 2.1* 1.6** 3.9 4.1 3.7 3.4 3.9 3.9 3.9 3.8

2 h incubation 5.3 5.8 5.2 4.6 3.8* 5.7 5.0 5.1 5.0

Hb-AA 24 h incubation 4.5 4.7 4.8 4.7 3.2** 4.9 4.6 4.6 3.3**

To evaluate the effect of the extracts on the hemolysis of RBC, Hb-SS and Hb-AA blood samples were incubated with the extracts in different concentrations for 2 or 24 h, then the number of erythrocytes from each sample was determined by erythrocyte count test under microscope using Neubauer hemocytometer. Values are mean of four counts and are expressed as cell number ×106 mm-3 (Normal values are 4.5~6.5 × 106 mm-3 for male and 3.9-5.6 ×106 mm-3 for female). Significant difference from the corresponding control, *P < 0.05, **P < 0.01.

Table 2. Ratio of normal RBC after induction of hypoxia in Hb-SS blood samples and treatment with the extracts for 30 and 120 min.

Sample treatment (µg/ml)

30 min incubation NC Ratio-NC (%) 106 94.6 29 16.8 10 5.9 4 5.2 140 80.5 113 77.9 190 86.8 25 21.6

SC 6 131 84 72 41 24 44 54

120 min incubation NC Ratio-NC (%) 115 95.0 26 16.6 5 5.6 19 20.9 172 80.8 131 84.5 71 61.7 44 44.9

Control in oxygenated state Control in hypoxia (1) BEAT-SS 10 BEAT-SS 50 BEAT-SS 100 BEAT-SS 200 BEAT-SS 400 BEAT-SS 500

SC 6 144 160 73 34 32 29 91

Control in hypoxia (2) DOCABE 1 DOCABE 5 DOCABE 10 DOCABE 50 DOCABE 100 DOCABE 200

138 51 107 44 11 83 47

13 4 5 85 165 26 117

8.6 7.3 4.5 65.9 93.8 23.9 71.3

181 31 57 17 5 37 28

30 36 22 41 85 49 69

14.2 53.7 27.8 70.7 94.4 57.0 71.1

Control in hypoxia (3) BEAT-SS 20 µg/ml + DOCABE 1 BEAT-SS 100 µg/ml + DOCABE 5 BEAT-SS 200 µg/ml + DOCABE 50 BEAT-SS 400 µg/ml + DOCABE 100

106 191 198 35 66

9 8 11 153 45

7.8 4.0 5.3 81.4 40.5

150 53 28 16 39

7 17 4 91 34

4.5 24.3 12.5 85.0 46.6

Sodium metabisulfite (Na2S2O5) 2% was added to Hb-SS blood sample and set aside for 1 h to induce hypoxia and deformity of RBC. Then, extracts were added and incubated at room temperature for 30 or 120 min. Specimens were taken for microscope examination. Control in oxygenated state is the sample from Hb-SS not treated by sodium metabisulfite. Controls in hypoxia (1), (2), and (3) are samples where sodium metabisulfite was added without further incubation with the extracts. All other samples were incubated with the extracts in indicated concentrations, after a previous induction of hypoxia for 1 h by 2% sodium metabisulfite. SC represents the number of Sickled Cells (distorted shape RBC); NC represents the number of Normal Cells (rounded shape RBC) and Ratio-NC (%) is the ratio of Normal Cells, which is the percentage of normal RBC in the total numbered RBC (SC + NC) in the optical field. Ratio-NC (%) = [NC / (SC + NC)] × 100%.

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extract, but that the incubation time (30 or 120 min) of blood samples with the extract did not have an influence on the recovery from the RBC deformity. These observations could be later justified by cell count (Table 2) which indicated that the ratio of normal cells was 77.9% for 30 min incubation and 84.5% for 120 min with 200 µg/ml of BEAT-SS. Shape recovery was noticed with BEAT-SS extract beginning from 100 µg/ml of concentration. Images examination of the incubation of Hb-SS blood samples with DOCABE extract after hypoxia revealed that sickle RBC recovered from elongated form to the rounded one, starting from the concentration of 10 to 50 µg/ml (Figure 1C1, C2, and C3). At 50 µg/ml, this reversing action of DOCABE was maximal as confirmed by cell count reported on Table 2. Here, again, incubation time did not affect the result as observed with BEAT-SS. In the case of DOCABE, cell count confirmed that 93.8 and 94.4% of red blood cells were round-shaped after 30 and 120 min of incubation with 50 µg/ml of DOCABE extract respectively (Table 2). The two extracts used in association showed positive result in reversing the deformity of sickled RBC for the couple of concentration of 200 µg/ml for BEAT-SS and 50 µg/ml for DOCABE (Figure 1D3), and cell count indicated that the percentage of round-shaped RBC was 81.4 and 85% after incubation of the two extracts for 30 and 120 min respectively (Table 2). The other couples of concentrations did not exhibit satisfactory results for reversing the deformity of sickled RBC (Table 2 and Figure 1D1 and D2). The same experiments as described herein were equally conducted, too, with blood samples from Hb-AA subjects. Here, microscope images did not exhibit change on the RBC morphology after the same treatments; neither with sodium metabisulfite (Figure 1A3) nor with both drugs used separately or in association. Cells remained roundshaped in all cases (data not shown).

Inhibition of the aggregation of RBC After images examination on microscope, it was noticed that both Hb-SS blood samples and Hb-AA blood samples (Figure 2A) showed cloudy aggregates of RBC after 48 h of rest. The addition of an equivalent volume of physiological saline solution (NaCl, 0.9%) did not disperse these aggregates in the case of Hb-SS (Figure 2B), RBCs were still stacked and aggregates remained, whereas RBC aggregates from Hb-AA blood samples were dispersed in the same condition (Figure 2C). After addition of the extracts on the Hb-SS samples, which has displayed cloudy aggregates of RBC, it was observed that aggregations of RBC were dispersed after incubation with BEAT-SS 200 µg/ml (Figure 2D) and DOCABE 50 µg/ml (Figure 2E) used separately. The dispersion of RBC was most appreciated when DOCABE extract was used alone and in association with BEAT-SS

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extract (Figure 2F). Concerning Hb-AA samples, RBC remained dispersed in all cases after the aforementioned treatments (data not shown). On the other hand (Figure 3), Hb-AA samples in normal density showed aggregated RBC in the control sample before addition of the extracts (Figure 3A), but these aggregations were dispersed in the samples where BEAT-SS (Figure 3B) and DOCABE (Figure 3C) were added despite the high density of RBC.

DISCUSSION This study was conducted with the purpose to evaluate the in vitro action of two extracts prepared from C. pentandra and Q. africana Baill. on the deformity of sickled RBCs. Microscopic observations could suggest that the two extracts reversed the deformity of sickled erythrocytes in hypoxia conditions. The two extracts used, separately displayed a similar activity. It was estimated that 200 µg/ml for BEAT-SS and 50 µg/ml for DOCABE were suitable concentrations for reversing the deformity of sickled RBC. When the two extracts were incubated together with the sickled RBC in the aforementioned concentrations, the recovery from the sickling was not particularly different as compared to those of individual extracts alone. The sickling of RBC, which involves hemoglobin S polymerization, is a characteristic of SCA and this phenomenon leads to the pathophysiology of episodic acute pains. The inhibition of the deformity of sickled RBC is an important parameter, which may support in a preliminary stage, the use of these crude drugs in the management of the SCA. Abnormalities implication of the adherence of sickle RBC to endothelium have been reported by several studies (Mohandas and Evans, 1984; Hoover et al., 1979); these abnormalities may also include interactions between sickle RBCs, platelets, leukocytes, and plasma constituents (Harlan, 2000; Hebbel et al., 1980; Hebbel, 1977), since they could be strongly affected by plasma factors and membrane changes on the surface of sickle RBC. Enhanced RBC aggregation under physiological conditions could be noticed in low or non-flow conditions where RBC adheres face to face to form reversible cellto-cell contact leading to aggregations (Kavitha and Ramakrishnan, 2007). SCA is a disease among others in which, variation in blood viscosity is seen and is significantly higher than the normal viscosity. Sickle RBCs confer a major effect on the viscosity of blood in SCA because they are less deformable than normal red cells; and increase in blood viscosity is in part induced by the increase of erythrocytes aggregation (Thurston et al., 2004). In our experiment, this could be confirmed by the fact that the in vitro dispersion of the aggregated RBC by physiological saline was less obvious in the case of HbSS samples (Figure 2B) than in Hb-AA samples. This leads to the supposition that sickle RBC aggregates more

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Figure 1. Effect of extracts on the reversing action of induced deformity of sickled red blood cells. Pictures were taken from microscope observation of RBC before and after inducing hypoxia followed by the treatment with the extracts. Blood samples from SCA patients (Hb-SS) were incubated with 2%Na2S2O5 to induce hypoxia and sickling (deformity) of RBC. Samples were treated then with extracts in different concentrations. Group A displays control samples: A1 and A2, RBC from Hb-SS respectively before (oxygenated state) and after addition of Na2S2O5 (hypoxia); A3, RBC from healthy subjects (Hb-AA) after addition of Na2S2O5. Group B displays samples which have been incubated with BEAT-SS extract after deoxygenation and sickling of RBC, B1: 50 µg/ml, B2: 100 µg/ml, and B3: 200 µg/ml. Group C displays samples incubated with DOCABE extract after hypoxia as explained above, C1: 5 µg/ml, C2: 10 µg/ml, and C3: 50 µg/ml. Group D displays samples incubated with both extracts together, D1: BEAT-SS 20 µg/ml with DOCABE 1 µg/ml, D2: BEAT-SS 100 µg/ml with DOCABE 5 µg/ml and D3: BEAT-SS 200 µg/ml with DOCABE 50 µg/ml.

strongly each other than RBC from Hb-AA samples (Figure 2C). The effect of the two extracts on the aggregation of RBC in the present experiment showed that the two extracts might prevent RBC aggregation, either when they are used separately or together (Figures 2D, E, and F and 3B and C) for both cases (HB-SS and

Hb-AA); suggesting that the two extracts might contribute to reduce blood viscosity. Thus, the two drugs could be beneficial for preventing aggregations of RBC, which could be implicated in the abnormal adhesion of each other, a phenomenon among others influencing blood viscosity and initiating the cascade of micro vascular

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Figure 2. Effect of extracts on the inhibition of aggregated sickle red blood cells. Blood samples were allowed to rest for 48 h at 4°C, then equivalent volume of physiological saline NaCl 0.9% as control or extract diluted in physiological saline was added and incubated for 30 min. A: aggregates of RBC observed before saline addition for either Hb-SS or Hb-AA samples. B: aggregates of RBC from Hb-SS remaining after addition of equivalent volume of saline to blood sample. C: RBC from Hb-AA after addition of equivalent volume of saline to blood sample. D: RBC from Hb-SS after addition to blood of equivalent volume of BEAT-SS extract diluted in saline (200 µg/ml). E: RBC from Hb-SS after addition to blood of equivalent volume of DOCABE extract diluted in saline (50 µg/ml), and F: RBC from Hb-SS after addition to blood of equivalent volume of BEAT-SS extract and DOCABE extract in association.

occlusion. This study suggests that the two extracts might not affect the viability of the RBC due to the fact that hemolysis of RBC did not occur in the concentrations which are reported to produce satisfactory results in the present investigation; in this case, the concentration of 200 µg/ml for BEAT-SS and 50 µg/ml for DOCABE. Furthermore, high concentrations (2500 µg/ml for BEATSS and 625 µg/ml for DOCABE) give some indications about the toxic dose of the two extracts on the red blood cells. This study may be regarded as an exploratory analysis contributing to the safety profile of crude medicines from C. pentandra and Q. africana in the management of SCA. Hence, further investigations need to be carried out on

these plants to discover potential new lead compounds for the management of SCA.

ACKNOWLEDGEMENTS The authors thank Mr. Nyembwe Kadiata and his “Centre de Phytothérapie Moderne Nieca” as well as the “Centre de Medecine Mixte et d’Anémie SS” (Kinshasa) for respectively providing us with plant materials and their related information, and also for providing patient’s blood samples used in this study. The authors are indebted, too, to Belgian government CUD (Coopération Universitaire pour le Dévelopement) for its financial support in equipping partially the Bio-Organic Research

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Figure 3. Effect of extracts on the inhibition of aggregated Hb-AA RBC in normal density. Hb-AA blood sample was allowed to stay for 48 h and a specimen was taken for microscopic examination (A). Then, extracts were added to the blood samples without diluting the blood. Pictures show aggregated RBC in normal density after 48 h of blood stay (A), after addition of BEAT-SS 200 µg/ml (B), and DOCABE 50 µg/ml (C).

Laboratory of the Faculty of Pharmaceutical Sciences (Kinshasa University) where the first stage of this study was carried out. The authors are grateful to the Center for African Natural Products Research and Development (Kinshasa) for its assistance. The second stage of this work was primarily conducted at Hokuriku University (Kanazawa, Japan) and supported by the “Academic Frontier Research” Project (2005-2009), Funds to Private Universities from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan, to which the authors are grateful. REFERENCES Abena AA, Itou-Elion R, Sanogo R, Diallo D, Ouamba JM (2008). Effets anti-ulcéreux et anti-diarrhéiques de Ceiba pentandra (Gaertn) chez le Rat. Symposium (15ème colloque sur la Pharmacopée et la Médecine Traditionnelles Africaines). December 1st- 4th, 2008; Libreville (Gabon). Apers S, Cimanga K, Berghe DV, Meenen EV, Longanga AO, Foriers A, Vlietnick A, Pieters L (2002). Antiviral activity of Simalikalactone D, a quassinoid from Quassia africana. Planta Med. 68:20-24 Burkill HM (1985). The useful plants of west tropical Africa, Entry for Ceiba pentandra (Linn.) Gaertn. [family BOMBACACEAE]. p. 1. http://www.aluka.org/action/showMetadata?doi=10555?AL.Ap.UPWTA.1 -561&pgs (August 27th, 2009) Chien S, Usami S, Bertles F (1970). Abnormal rheology of oxygenated blood in sickle cell anemia. J. Clin. Invest. 49(4):623-634. Dzeufiet PDD, Tédong L, Asongalem EA, Dimo T, Sokeng SD, Kamtchouing P (2006). Hypoglycaemic effect of methylene chloride/methanol root extract of Ceiba pentandra in normal and diabetic rats. Indian J. Pharmacol. 38(3):194-197 Dzeufiet PDD, Ohandja DY, Tédong L, Asongalem EA, Dimo T, Sokeng SD, Kamtchouing P (2007). Antidiabetic effect of ceiba pentandra extract on streptozo-tocin-induced non-insulin- dependent diabetic (NIDDM) rats. Afr. J. Trad. Complim. Altern. Med. 4(1):47-54. FAO: Forest Resources Development Branch, Forest Resources Division, FAO Forestry Department (1986). Some medicinal forest plants of Africa and Latin America ISBN 92-5-1 02361-1. http://www.archive.org/stream/somemedicinalfor034648mbp/somemedi cinalfor034648mbp_djvu.txt , 173-178 (September 17th, 2009) Harlan JM (2000). Focus on hematology - Introduction: anti-adhesion therapy in sickle cell disease. Blood 95(2):365-367. Hebbel RP (1977). Adhesive Interactions of Sickle Erythrocytes with

Endothelium. J. Clin. Invest. 99(11):2561-2564. Hebbel RP, Boogaerts MA, Eaton JW, Steinberg MH (1980). Erythrocyte adherence to endothelium in sickle cell anemia. A possible determinant of disease severity. N. Eng. J. Med. 302(18):992-995. Hoareau L, DaSilva EJ (1999). Medicinal plants: a re-emerging health aid. Review article. Electron. J. Biotechnol. 2(2):56-70 Hoover R, Rubin R, Wise G, Warren R (1979). Adhesion of Normal and Sickle Erythrocytes to Endothelial Monolayer Cultures. Blood 54(4):872-876. Kavitha A, Ramakrishnan S (2007). Assessment of human red blood cell aggregation using image processing and wavelets. Meas. Sci. Rev. 7(5):44-51. Ladeji O, Omekarah I, Solomon M (2003). Hypoglycemic properties of aqueous bark extract of Ceiba pentandra in streptozotocin-induced diabetic rats. J. Ethnopharmacol. 84:139-142. Lohombo-Ekomba ML, Mvumbi-Lelo G, Kabongo C, Kasende OE (2003). Contribution à l'étude de l'activité antipaludique de Quassia africana Baill. La phytothérapie européenne ISSN 1628(6847):15:812. Lonergan GJ, Gline DB, Abbodanzo SL (2001). Sickle cell anemia. Radiographics 21(4):971-994. Martorana MC, Mojoli G, Cianciulli P, Tarzia A, Mannella E, Caprari P (2007). Sickle cell anemia: haemorheological aspects. Ann Ist Super Sanità 43(2):164-170. Mohandas N, Evans E (1984). Adherence of Sickle Erythrocytes to Vascular Endothelial Cells: Requirement for Both Cell Membrane Changes and Plasma Factors. Blood 64:282-287. Murayama M, Nalbandian RM (1973). Sickle cell hemoglobin, Molecule to Man. Copyright by Little, Brown and company(Inc), 1st edit. p. 135. Nwachukwu IN, Allison LN, Chinakwe EC, Nwadiaro P (2008). Studies on the effects Cymbopogon citratus, Ceiba pentandra and Loranthus bengwelensis extracts on species of dermatophytes. J. Am. Sci. 4(4):52-63. Platt OS (2000). Sickle cell anemia as an inflammatory disease. J. Clin. Invest. 106(3):337. Saldanha C (2002). Mini review on erythrocytes aggregation. Basic concepts and clinical repercussions. Bol. hemorreol. 2(2):1-10. Serjeant GR (1994). The geography of sickle cell disease: opportunities for understanding its diversities. Ann. Saudi Med. 14(3):237-246. Thurston GB, Henderson NM, Jeng M (2004). Effects of Erythrocytapheresis transfusion on the viscoelasticity of sickle cell blood. Clin. Hemorheol. Microcirc. 30:61-75. Wendell FR, Mohandas N, Petz LD, Steinberg MH (2000). New Views of Sickle Cell Disease. Pathophysiology and Treatment. Hematology 1:2-17. Wikipedia, the free encyclopedia (2007). Sickle cell disease. http://en.wikipedia.org/wiki/sickle_cell_disease (July 17th, 2007).

Journal of Medicinal Plants Research Vol. 6(31), pp. 4609-4614,15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.572 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Inhibition of angiogenesis and metastasis of uveal melanoma cells by astragaloside IV Zhang Wenjing1*, Ma Minwang1, Wang Dong2 and Tang Dongrun3 1

Affiliated Hospital of Medical College of Chinese People’s Armed Police Forces, Tianjin, China. 2 The 2nd Hospital of Tianjin Medical University, Tianjin, China. 3 Tianjin First Center Hospital, Tianjin, China. Accepted 7 September, 2011

Astragaloside IV (AS) has been recently shown to possess pharmacologic activities against cancer. Vascular endothelial growth factor (VEGF) plays a prominent role in the induction of physiological or pathophysiological processes of tumor angiogenesis. The present study focuses on the antiangiogenesis effects of AS in uveal melanoma cells. In this study, AS was demonstrated to exhibit higher anti-proliferation activity against cultured uveal melanoma cells compared with control. AS was also found to inhibit VEGF-a expression and secretion in human cells; functional assays also indicated inhibition of invasion and migration of the cells. This provides new information on a significant antitumor effect of AS. This saponin may be used as a novel therapeutic drug for the inhibition of tumor angiogenesis and metastasis. Key words: Astragaloside IV, anti-cancer, vascular endothelial growth factor, uveal melanoma.

INTRODUCTION Radix astragali (Huangqi) is one of the most widely prescribed Chinese herbs (Zhao et al., 2009; Xu et al., 2008). It has been widely used in Chinese medicine since ancient times. It is highly safe and demonstrates efficacy in the improvement of immune disorders and lung diseases (Zhang et al., 2006; Yuan et al., 2008; Jiang et al., 2008; Lv et al., 2010). The major active constituents of R. astragali are believed to be the total saponins and the total flavonoids. Astragaloside IV (AS) is a naturally occurring saponin isolated from R. astragali and is used for the quality evaluation of the herb, as listed in the 2005 edition of Pharmacopoeia of the People's Republic of China. Astragaloside IV is a major saponin of this herb. It has been recently shown to possess anti-inflammatory activities and pharmacologic activities against cancer, fatigue, and the coxsackie B virus Nalbantsoy et al., 2011; Chen et al., 2011; Shang et al., 2011). Vascular endothelial growth factor (VEGF) plays a

*Corresponding author. E-mail: [email protected]. Tel: 86022-60578775.

prominent role in the induction of physiological or pathophysiological processes of angiogenesis, vasculogenesis, arteriogenesis, and lymphangiogenesis, collectively termed as vascularization Dome et al., 2007). Although the evidence in the literature supports the idea that VEGF is a positive regulator of tumor growth, more reports indicate that VEGF also acts as a regulator that promotes tumor migration and invasion (Folkman, 1996; Dome et al., 2007; Xue et al., 2008; Siironen et al., 2006). In general, VEGF promotes angiogenesis by induction of the enzymes cyclooxygenase-2 (COX-2) and nitric oxide synthase. Over-expression of VEGF and COX-2 in cancerous tissues has been reported to be associated with poor prognosis. COX-2 is an inducible enzyme produced by many cell types in response to multiple stimuli (Shang et al., 2011). Recently, COX-2 overexpression has been detected in several types of human cancers such as those of the colon, breast, prostate, lung, pancreas, and blood (Toomey et al., 2009). It appears to control many cellular processes. Due to their roles in angiogenesis, carcinogenesis, and apoptosis, VEGF and COX-2 are excellent targets for developing new drugs.

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In this study, AS was demonstrated to exhibit higher antiproliferation activity against cultured uveal melanoma cells compared with controls. AS was also found to inhibit VEGF-a expression and secretion in human cells; functional assays also indicated inhibition of invasion and migration of the cells. This provides new information on a significant anti-tumor effect of AS. This saponin may be used as a novel therapeutic drug for the inhibition of tumor angiogenesis and metastasis. MATERIALS AND METHODS Cell cultures and AS treatment Human uveal melanoma cell-line OCM-1 was obtained from the American Type Culture Collection (Rochville, MD). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO 2. AS powder (> 98% assay by high-performance liquid chromatography) was purchased from the RuiQi Chemical Co. (Shanghai, China) and was dissolved in dimethylsulfoxide just before use. For the experiments on the influence of AS on OCM-1 cells, the cells were seeded onto flat-bottomed 96-well or 6-well plates (Costar) with or without AS in the medium at the time of seeding. Co-culture with HUVEC cells Indirect co-culture was established using cell culture inserts (0.4 μm pore size, 0.33 cm2, 24 wells; Transwell, Costar). Inserts were dehydrated and were loaded with OCM-1 melanoma cells while the bottom was inoculated with HUVEC cells. AS was added to the medium and cells were collected at certain times within a 24 h period for Western blot.

Proliferation, migration, and invasion assay The cell proliferation test involved the MTT assay. Cell invasion assay was performed using Transwell cell culture inserts (Invitrogen). The transfected cells were maintained for 48 h and allowed to migrate for another 24 h. The passed cells were stained with crystal violet solution and their absorbance at 570 nm was determined. Cell motility in the wound healing assays was assessed by measuring the movement of cells into a scrape. The speed of wound closure was monitored after 10 and 24 h by measuring the ratio of the distance of the wound to that at 0 h. Each experiment was done in triplicate.

Western blot RIPA lysate was used to lyse cells. After sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the lysed cells were transferred in the wet state onto PVDF film. Skimmed milk powder was used to block the reaction. Information on the primary and secondary antibodies is given in the Supplementary Data. Enhanced chemiluminescence method was used to determine protein expression.

concentration of VEGF-a in the medium, according to the manufacturer's instructions. The reaction between POD and ABTS at 405 nm was photometrically determined using a microplate reader. Statistical analysis All data in the study were evaluated using SPSS11.5 (SPSS Inc., USA). Differences were considered significant at p < 0.05. Significant results are marked with an asterisk (*).

RESULTS AS inhibits proliferation, migration, and invasion of uveal melanoma cells As shown in Figure 1, in vitro MTT analysis screening of AS demonstrated a strong inhibitory effect on OCM-1 cells. It showed Inhibition was dose-dependent and directly proportional to the AS concentration. At concentrations of 20 mg/L and above, AS killed more than 98% of the cells. The 50% inhibitory concentration (IC50) of AS was 4.028 mg/L. This indicates that AS was cytotoxic to uveal melanoma cells and an IC50 < 5 mg/L was cytotoxic to the cells. To determine if AS has a motility inhibitory effect, a wound healing assay was developed. OCM-1 cells were treated at various concentrations of AS for 10 and 24 h after being scratched (Figure 2). In the transwell invasion assay presented in Figure 3, around fourfold decrease in samples with the treated cells (compared the control group) occurred after exposure to 5 mg/L AS, following the Matrigel invasion assay. The results show that the cells proliferation, migration, and invasion were inhibited in a dose-dependent manner. AS inhibits VEGF-a expression and secretion in uveal melanoma cells To screen further the functional expression level of VEGF-a, we compared the level in cells treated with AS against those that were not exposed to AS. Western blot and ELISA of VEGF-a were done to analyze levels in the cytoplasm and those secreted into medium. As shown in Figure 4, the VEGF-a expression in the cytoplasm was not detected; this indicates a significant difference between the AS treatment and control groups. However, a significant difference was detected in media of the AS group and control. When treated by 5 to 20 mg/L AS, secretion of VEGF-a was completely inhibited compared with the control.

Enzyme-linked immunosorbent assay (ELISA)

AS inhibits VEGFR-2 expression in HUVEC cell coculture with uveal melanoma cells

The VEGF-a

Tumor cells can secrete VEGF-a to promote proliferation,

detection ELISA kit was used to detect the

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Figure 1. MTT assay analysis inhibitory effect of AS on OCM-1 cells. It showed inhibition was dose-dependent and directly proportional to the AS concentration. The 50% inhibitory concentration (IC50) of AS was 4.028 mg/L. This indicates that AS was cytotoxic to uveal melanoma cells.

Figure 2. Wound healing assay was developed to analysis the migration of OCM-1 cells were treated at various concentrations of AS for 10 and 24 h after being scratched. This indicates that AS was inhibited uveal melanoma cells migration.

migration, and division of endothelial cells. VEGF presents its function through the VEGF receptor on the cell membrane. In the current study, we developed a coculture system to detect the influence of AS on VEGFR expression in HUVEC cells co-cultured with OCM-1. The results show that VEGFR-2 expression levels decreased in HUVEC cells treated with AS, compared with the control. The treatment and control groups did not show a significant difference in VEGFR-1 expression levels.

DISCUSSION The anti-tumor activity of astragaloside IV was confirmed in the in vitro experiments by its suppression of VEGF-a secretion in the uveal melanoma cell-line OCM-1. More importantly, this activity was confirmed by inhibition of migration and invasion of the cells. Tumor metastasis is a multistep process by which a subset of cancer cells or individual cells disseminate from

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Figure 3. The transwell invasion assay was developed to analysis the invasion of OCM-1 cells were treated at various concentrations of AS. Fourfold decrease in samples with the treated cells (compared the control group) occurred after exposure to 5 mg/L AS. This indicates that AS was inhibited uveal melanoma cells invasion.

Figure 4. ELISA assay to screen the functional expression level of VEGF-a, we compared the level in cells treated with AS against those that were not exposed to AS. A significant difference was detected in media of the AS group and control. When treated by 5 to 20 mg/L AS, secretion of VEGF-a was completely inhibited compared with the control.

a primary tumor to distant secondary organs or tissues (McCawley and Matrisian, 2001). Tumor cells fulfill their metastatic potential after acquiring advantageous characteristics that allow them to escape from the primary tumor, migrate and invade surrounding tissues, enter the vasculature, circulate and reach secondary

sites, extravasate, and establish metastatic foci (Pietras and Ostman, 2010; Josson et al., 2010; Anton and Glod, 2009; Bertinet al., 2010). All these steps of the metastatic cascade require survival of tumor cells and communication among cells. During metastasis, tumor cells are involved in numerous interactions with the

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extracellular matrix (ECM). The tumor cells also interact with proteins, growth factors, and cytokines associated with the ECM, basement membranes, endothelial cell lining of the vasculature, blood cells in the circulation, and the microenvironment of the secondary site, where they eventually displace the normal tissue as they grow out and form metastatic foci (Josson et al., 2010; Peng and Wang, 2010). Several regulatory processes either are altered or are aberrant. This gives tumor cells the ability to accomplish all steps of the metastatic process, such as migration and invasion. Tumor angiogenesis is essential for tumor growth and metastasis. Without active angiogenesis, tumor diameters rarely exceed 2 to 3 mm. Angiogenesis is mediated by the release of angiogenic factors by tumor cells, cells in the tumor stroma and microenvironment, which include endothelial cells (Dome et al., 2007; Folkman, 2006). We report that AS decreases the synthesis and release of angiogenic factors by uveal melanoma cells and inhibits VEGF-a expression and secretion in melanoma cells. Endothelial cell (EC) migration, proliferation, and differentiation are essential to tumor angiogenesis. EC proliferation, in vitro tubulogenesis, and survival are known to be stimulated in large part by VEGF. Decreased VEGF levels or inhibition of receptor activation in ECs often correlate with decreased tumor size and metastatic potential. VEGF binds to the extracellular domain of VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1), induces receptor dimerization, and activates tyrosine kinases by autophosphorylation; this leads to angiogenesis, increased vascular permeability, and EC proliferation and survival (Bertin et al., 2010; Karaca et al., 2011). In general, VEGFR-2 is the major mediator of these effects. We found that AS decreases the expression of VEGFR-2 by HUVEC in co-culture with OCM-1. By reducing the VEGF-R2 levels, AS appears to have similar effects on tumor cells. VEGF also induces leakage within tumor vessels, and thereby allows tumor cells to infiltrate blood vessels and migrate into the blood stream. Hence, changes in angiogenic factors even early in tumor formation can affect metastasis. Spreading and inhibiting VEGF production by AS is expected to reduce the metastatic potential of tumor cells. Additionally, increased blood vessel permeability within the tumor may interfere with adequate delivery and retention of chemotherapeutic agents. Indeed, certain anti-angiogenic agents that prevent tumor vessel leakage (a phenomenon called vessel normalization) have been shown to enhance the delivery of chemotherapeutic agents into tumors. Thus, combined treatment with antiangiogenic factors and conventional chemotherapeutic agents may be superior to using the latter alone. Furthermore, as VEGF is likely required for migration and recruitment of ECs, AS-mediated VEGF reduction may decrease the number of blood vessels in the tumor. Our present findings suggest that AS may induce potent anti-angiogenic effects, and enhance their

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potential as a therapeutic option against cancer. Our data demonstrate that astragaloside IV has potent anti-tumor and anti-angiogenesis qualities and deserves to be further evaluated for the treatment of human uveal melanoma. Currently, astragaloside IV is under early development as an anti-tumor candidate. REFERENCES Zhao Z, Wang W, Wang F, Zhao K, Han Y, Xu W, Tang L (2009). Effects of Astragaloside IV on heart failure in rats. Chin. Med. 4:6. Xu XL, Chen XJ, Ji H, Li P, Bian YY, Yang D, Xu JD (2008). Astragaloside IV improved intracellular calcium handling in hypoxiareoxygenated cardiomyocytes via the sarcoplasmic reticulum CaATPase. Pharmacology 81:325-332. Zhang Y, Zhu H, Huang C, Cui X, Gao Y, Huang Y, Gong W (2006). Astragaloside IV exerts antiviral effects against coxsackievirus B3 by upregulating interferon-gamma. J. Cardiovasc. Pharmacol. 47:190195. Yuan W, Zhang Y, Ge Y, Yan M, Kuang R, Zheng X (2008). Astragaloside IV inhibits proliferation and promotes apoptosis in rat vascular smooth muscle cells under high glucose concentration in vitro. Planta Med. 74:1259-1264. Jiang B, Yang Y, Jin H, Shang W, Zhou L, Qian L, Chen M (2008). Astragaloside IV attenuates lipolysis and improves insulin resistance induced by TNFalpha in 3T3-L1 adipocytes. Phytother. Res. 22:14341439. Lv L, Wu SY, Wang GF, Zhang JJ, Pang JX, Liu ZQ, Xu W (2010). Effect of astragaloside IV on hepatic glucose-regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin. Phytother. Res. 24:219-224. Nalbantsoy A, Nesil T, Erden S, Calis I, Bedir E (2011). Adjuvant effects of Astragalus saponins Macrophyllosaponin B and Astragaloside VII. J. Ethnopharmacol. 134:897-903. Chen P, Xie Y, Shen E, Li GG, Yu Y, Zhang CB, Yang Y (2011). Astragaloside IV attenuates myocardial fibrosis by inhibiting TGFbeta1 signaling in coxsackievirus B3-induced cardiomyopathy. Eur. J. Pharmacol. 658:168-174. Shang L, Qu Z, Sun L, Wang Y, Liu F, Wang S, Gao H (2011). Astragaloside IV inhibits adenovirus replication and apoptosis in A549 cells in vitro. J. Pharm. Pharmacol. 63:688-694. Folkman J (2006). Angiogenesis. Annu. Rev. Med. 57:1-18. Folkman J (1996). Tumor angiogenesis and tissue factor. Nat Med. 2:167-168. Dome B, Hendrix MJ, Paku S, Tovari J, Timar J (2007). Alternative vascularization mechanisms in cancer: Pathology and therapeutic implications. Am. J. Pathol. 170:1-15. Xue Y, Religa P, Cao R, Hansen AJ, Lucchini F, Jones B, Wu Y (2008). Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome. Proc. Natl. Acad. Sci. USA. 105:18513-18518. Siironen P, Ristimaki A, Narko K, Nordling S, Louhimo J, Andersson S, Haapiainen R (2006). VEGF-C and COX-2 expression in papillary thyroid cancer. Endocr. Relat. Cancer 13:465-473. Toomey DP, Murphy JF, Conlon K (2009). COX-2, VEGF and tumour angiogenesis. Surgeon 7:174-180. McCawley LJ, Matrisian LM (2001). Tumor progression: defining the soil round the tumor seed. Curr. Biol. 11:R25-27. Pietras K, Ostman A (2010). Hallmarks of cancer: interactions with the tumor stroma. Exp. Cell Res. 316:1324-1331. Josson S, Matsuoka Y, Chung LW, Zhau HE, Wang R (2010). Tumorstroma co-evolution in prostate cancer progression and metastasis. Semin. Cell Dev. Biol. 21:26-32. Peng JY, Wang Y (2010). Tumor stroma: A determinant role in local recurrence of rectal cancer patients receiving total mesorectal excision? Med. Hypotheses. Anton K, Glod J (2009). Targeting the tumor stroma in cancer therapy. Curr. Pharm. Biotechnol. 10:185-191. Bertin S, Mohsen-Kanson T, Baque P, Gavelli A, Momier D, Anjuere F,

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Carle GF (2010). Tumor microenvironment modifications induced by soluble VEGF receptor expression in a rat liver metastasis model. Cancer Lett. 298:264-272. Folkman J (2001). A new family of mediators of tumor angiogenesis. Cancer Invest. 19:754-755.

Karaca Z, Tanriverdi F, Unluhizarci K, Ozturk F, Gokahmetoglu S, Elbuken G, Cakir I (2011). VEGFR1 expression is related to lymph node metastasis and serum VEGF may be a marker of progression in the follow-up of patients with differentiated thyroid carcinoma. Eur. J. Endocrinol. 164:277-284.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4615-4624, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.789 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Analysis of anti-oxidant activity of medicinal plants according to the extracted parts Yu-Su Shin1, Hyun-Jung Jo2, Sang-Won Lee1, Young-Ock Kim1, Yoon-Pyo Hong1 and Kyung-Soo Chang2* 1

Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, RDA, Chungbuk 369-873, Republic of Korea. 2 Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 609-757, Republic of Korea. Accepted 20 July, 2012

In this research, we compared and analyzed the anti-oxidant activity of ten medicinal plants using an oriental medicine and a folk remedy. Among them, the extracts from Oenothera odorata had the highest anti-oxidant effect. The extracts from the root, stem and flower of O. odorata were tested by 1,1diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity test, 3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide (MTT) cell viability assay, and lactate dehydrogenase (LDH) cytotoxicity assay. Stem and flower extracts of O. odorata were similar to the activity of quercetin, one of the most anti-oxidants in DPPH radical scavenging activity test, and the root extracts showed a weak DPPH radical scavenging activity. In MTT cell viability assay, the extracts from the flower, stem, and root were resistant against hydrogen peroxide (H2O2) treatment in order. The extracts from the root, stem and flower showed higher cell protection effect than those from quercetin against LDH cytotoxicity. And βsitosterol from the extracts of stem was isolated. These results suggest that the extracts from the flower, stem, and root of O. odorata might be a source of anti-oxidants. Key words: Anti-oxidants, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3-[4, 5-dimethylthiazol-2-yl]-2, diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) cytotoxicity, Oenothera odorata.

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INTRODUCTION Reactive oxygen species (ROS) such as superoxide anion (O2), hydroxy radical (-OH), hydrogen peroxide (H2O2) are produced as a result of mitochondria metabolism and immunization to external factors. ROS protects the body from pathogens by its strong sterilizing action (Chance et al., 1979). Human and animal bodies have self-protecting systems from damages such as ROS toxicity. Internal anti-oxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), and anti-oxidant components such as glutathione are known for deleting ROS produced in tissues (Evans

*Corresponding author. E-mail: [email protected]. Tel/Fax: +82(051) 5100565.

and Halliwell, 2001). When these internal protecting systems in our bodies have problems or exceed their ability, it causes oxidative stress (Arteel et al., 2000) associated with modern diseases like cancer by nucleic acid transformation, arteriosclerosis by physiological depres-sion, diabetes, cerebral apoplexy, nephritis, atopies and aging (Halliwell and Gutteridge, 1989; Halliwell and Aruoma, 1991; Kanno et al., 2003; Kuroki et al., 2003). Since various adult diseases are increased by oxidative stress, compounds that able to scavenge ROS, inhibit hyper-oxidant production and stimulate anti-oxidant enzyme are being actively developed. Mainly, vitamin C, vitamin E, carotene and polyphenols are known as antioxidants. These have fewer side effects and reduced cost because they are extracted from natural materials that

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are not synthesized components. Recently, many researches have been done to obtain anti-oxidants from natural materials. Some plants are already found having antioxidative effects such as see weeds, mushrooms, herbs and fruits. They contain much polyphenol groups like phloroglucinol and quercetin (Sung et al., 2000; Shin and Ihm, 2008; Kim and Chang, 2006; Yun et al., 2009; Park et al., 2005; Zia-Ul-Haq et al., 2008, 2011 a, b, 2012). The evening primrose (Oenothera odorata) is an annual plant that belongs to Onagraceae. They are classified into Oenothera biennis, Oenothera erythrosepala, Oenothera odorata and Oenothera laciniata. It is useful in treating fever and antiinflammation. The root is used in the treatment of sore throat and dermatitis, while the seed oil is effective in diabetes, high blood pressure and obesity. It is also applied to hyperlipidemia because it inhibits accumulation of lipid components like cholesterol. Especially, studies of the seed oil are progressing (Pellegrina et al., 2005; Senapati et al., 2008; Chenoy et al., 1994; Cameron et al., 1993). Recently, anti-oxidants from natural materials are being researched actively. In this study, we extracted natural material from medicinal plants and researched their antioxidant effect. Particularly, we used the extracts from the root, stem and flower of O. odorata which is discovered to contain high anti-oxidant activity. These materials were assayed for their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities. After we treated the extracts to liver cell producing anti-oxidant enzyme, we confirmed cyto-protective effect from oxidative stress by hydrogen peroxide (H2O2) on concentration and on time. The extracts from the root, stem and flower of O. odorata are therefore useful in developing natural anti-oxidant drug. MATERIALS AND METHODS Extraction and isolation from medicinal plants The ten medical plants cultivated by good agricultural practice (GAP) method of rural development area (RDA) were harvested at 2009 in Eumseong (GPS: E 128° 62´ N 36° 56´). The plan ts were extracted with methanol (MtOH) three times at room temperature (each time for 3 days). The combined MtOH extracts were then concentrated in vacuo at 40°C, respectively. Dried root extract of O. odorata was dissolved to be 1% in dimethyl sulfoxide (DMSO) and extracts of stem and flower was dissolved respectively to be 1% in MtOH. Other extracts were also dissolved to be 1% in MtOH or DMSO and used as test material. Quercetin (Sigma, USA), a known antioxidant, was dissolved to be 1% in MtOH. It was used as positive control (Table 1).

DPPH radical scavenging activities 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test was performed using the method of Lee et al. (2003). The extracts from various medical plants were diluted to 0, 0.001, 0.01, 0.05, 0.1 and 0.2% concentrations in each solvent. In brief, 200 µM DPPH (Aldrich) was dissolved in ethanol and 190 µL was added to 10 µL

of sample. After reacting at 37°C for 30 min, they are measured at an absorbance of 550 nm in the spectrophotometer.

Cell culture and cell treatment Human hepatoma cell lines (Huh7) were used to study cytoprotective effect of the extracts from medical plants against oxidative stress by H2O2. Huh7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing a 10% fetal bovine serum (FBS), 1% non-essential amino acids, 1% penicillin and streptomycin for proliferation at 37°C and 5% CO 2.

Thiazolyl blue tetrazolium bromide (MTT) cell viability assay We carried out the thiazolyl blue tetrazolium bromide (MTT) cell viability assay in Huh7 to determine anti-oxidant activity of natural extracts using test materials, which are effective to DPPH radical scavenging ability. In brief, 1×105/ml cells were put into each well and incubated for 24 h. The test materials were diluted to 0, 0.001, 0.01, 0.05, 0.1 and 0.2% final concentrations in DMEM. Then we added them into each well. After 48 h, 10 mM H2O2 was added for inducing oxidative stress in the treated group for 2 h. Next, 20 µL of 5mg/ml MTT solution (Amresco) was added and incubated more than 2 h at 37°C. Then we carefully removed the mediu m and added 200 µL DMSO per well to stop the reaction. After mixing for 5 min, we measured absorbance at 560 nm wavelength by Microplate ELISA reader. To investigate the hourly effects of extracts, extracts were diluted to be 0.05% final concentration in DMEM and then reacted for 0, 24 and 48 h after treatment.

LDH (Lactate dehydrogenase) cytotoxicity assay We carried out the LDH cytotoxicity assay in Huh7 to determine cyto-protective effect by anti-oxidant activity of natural extracts. 1X105/ml cells let in each well and incubated for 24 hours. Materials were diluted to 0, 0.001, 0.01, 0.05, 0.1 and 0.2% final concentrations in DMEM. Then they were added in each well. After 48 h, 10 mM H2O2 was added for inducing oxidative stress in the treated group for 2 h. Afterward, the 10 µL medium supernatant and 90 µL LDH substrate solution (LDH-cytotoxicity Assay kit II, BioVision) was reacted at room temperature for 30 min. To stop the reaction, a stop solution (1 M acetic acid) was added by 10 µL. Then, we measured absorbance at 560 nm wavelength by microplate ELISA reader. To investigate the hourly effects of materials, materials were diluted to 0.05% final concentration in DMEM and then reacted for 0, 24 and 48 h after treatment.

Extraction and isolation O. odorata (voucher no. OLS001001), which was a genetic resource of RDA, was harvested and collected at Eumseong (GPS: E 127° 45 ′ N 36° 56 ′) in Korea. The stem (ca. 2.3 kg) was extracted five times with 99% ethanol (EtOH, 2L) at room temperature for 24 h. The EtOH extracts were combined and concentrated under reduced pressures. The concentrated extracts (55.4 g) were successively separated on silica gel column chromatography (CC, Wakogel C-200) with developing solvents of n-hexane, nhexane/ethyl acetate (EtOAC) (HEA; 20:1, 10:1, 4:1, 2:1, 1:1, v/v), EtOAc, and EtOH, successively. By monitoring with thin layer chromatography (TLC) using the developing solvent (SGIII), the extractives were separated into 28 fractions. Crude β-sitosteol (I) was obtained from fractions Fr3 using a silica gel column (Wakogel C-200) with HEA (20:1. v/v) solvent. Finally, the purified β-sitosteol

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Table 1. Information of used medicinal plants.

Plants

Celosia cristata (Red)

Used parts Root Stem Flower Seed

Sample names Cec-1 Cec-2 Cec-3 Cec-4

Solvent MtOH MtOH DMSO DMSO

Celosia cristata (Yellow)

Root Stem Flower

Y-Cec-1 Y-Cec-2 Y-Cec-3

MtOH MtOH MtOH

Eupatorium chinensis var. simplicifolium

Root Stem Flower

Euc-1 Euc-2 Euc-3

MtOH MtOH DMSO

Eupatorium chinensis. spp

Root Stem Flower

R-Euc-1 R-Euc-2 R-Euc-3

MtOH MtOH MtOH

Oenothera odorata

Root Stem Flower

Evp-1 Evp-2 Evp-5

DMSO MtOH MtOH

Anethum graveolens

Root Stem Flower

Ang-1 Ang-2 Ang-3

MtOH MtOH MtOH

Cichorium intybus

Root Stem

Cii-1 Cii-2

DMSO MtOH

Abutilon theophrasti Medicus

Root Stem Bark of Seed

Aba-1 Aba-2 Aba-3

MtOH MtOH MtOH

Gomphrena globosa

Root Sterm Flower

Gog-1 Gog-2 Gog-3

MtOH MtOH MtOH

Chenopodium spp

Root Stem

Brc-1 Brc-2

MtOH MtOH

MtOH, Methanol; DMSO, dimethyl sulfoxide.

(I) (52 mg) was isolated. Compound I (β-sitosteol) was isolated as white powder. The NMR spectra were measured on a Varian FTNMR 500MHz using deuterated chloroform (CDCl3) as solvents and tetramethylsilane (TMS) as an internal standard. The 13C-NMR Spectral data were; 37.3(C-1), 32.0(C-2), 71.8(C-3), 42.3(C-4), 140.8(C-5), 121.5(C-6), 31.7(C-7), 31.9(C-8), 50.1(C-9), 36.5(C-10), 21.1(C-11), 39.8(C-12), 42.3(C-13), 56.8(C-14), 24.3(C-15), 28.2(C16), 56.1(C-17), 12.0(C-18), 19.4(C-19), 36.1(C-20), 18.8(C-21), 34.0(C-22), 26.0(C-23), 45.8(C-24), 29.1(C-25), 19.8(C-26), 19.0(C27), 23.1(C-28), 11.9(C-29)

RESULTS AND DISCUSSION High DPPH radical scavenging activities of the extracts from O. odorata We identified the anti-oxidant effect of the extracts from ten medical plants by measuring their DPPH radical scavenging activities. According to the result of screening test of various medicinal plants, Cec-2, Cec-3, Cii-1 and

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Table 2. Result of DPPH radical scavenging screening test.

Sample names Cec-1 Cec-2 Cec-3 Cec-4 Y-Cec-1 Y-Cec-2 Y-Cec-3 Euc-1 Euc-2 Euc-3 R-Euc-1 R-Euc-2 R-Euc-3 Evp-1 Evp-2 Evp-5 Ang-1 Ang-2 Ang-3 Cii-1 Cii-2 Aba-1 Aba-2 Aba-5 Gog-1 Gog-2 Gog-3 Brc-1 Brc-2 Quercetin PBS MtOH DMSO

Concentration (%) 0.01 0.360 0.380 0.382 0.382 0.408 0.400 0.299 0.385 0.354 0.395 0.385 0.347 0.381 * 0.371 * 0.336 * 0.280 0.357 0.350 0.373 0.387 0.387 0.399 0.383 0.382 0.352 0.357 0.381 0.395 0.354 0.280 -

0.1 0.326 0.403 0.382 0.358 0.367 0.365 0.300 0.349 0.316 0.347 0.360 0.318 0.347 * 0.299 * 0.104 * 0.048 0.310 0.310 0.321 0.381 0.379 0.320 0.321 0.357 0.393 0.332 0.370 0.388 0.320 0.046 0.386 0.358 0.338

*Effective materials.

Brc-1 did have not anti-oxidant effect. Although the flower extract of Celosia cristata (Yellow) has a little effective, it was so weak. The effect was greater in the extracts of O. odorata (Table 2). So we tested parts of O. odorata exactly. The result of DPPH radical scavenging activity confirmed the effectiveness of the roots, stem and flowers of O. odorata. At the 0.01% concentration, extract of the root showed similar DPPH scavenging activity to quercetin and higher radical scavenging activity than stem and flower extract. However, at the 0.1% concentration, the stem and flower extracts showed similar DPPH scavenging activity to that of quercetin and higher radical scavenging activity than the root extracts. And their effect had higher EC50 at 0.1% (Figure 1). Root

extract at low concentrations and the stem and flower extract at high concentrations represented a high antioxidant effect. Increase of cell viability by the extracts from O. odorata To determine anti-oxidant activity of natural extracts, we carried out the MTT cell viability assay in liver cell. To investigate anti-oxidant activity depending on concentrations, we diluted the extracts from O. odorata and treated cells. We confirmed the viability of the liver cell by H2O2 treatment. The root extract was effective at 0.1% in

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Figure 1. DPPH radical scavenging activities by concentration of each extracts from an evening primrose. A. Quercetin (positive control) B. Evp-1; extract from root of an O. odorata C. Evp-2; extract from stem of an O. odorata D. Evp-5; extract from flower of an O. odorata. Solvent; DMSO in B, and MtOH in A, C and D.

DPPH scavenging activity, although no noticeable effect observed. However, the cyto-protective effect of stem and flower extract was increased in order to concentrations. In particular, flower extract at the 0.1% concentration showed significant effects and the 0.05% concentration mostly showed a large effect. So there was similar or greater effects than the positive control group, quercetin. Stem extracts display lower effect than quercetin. But the pattern of increase is similar to quercetin (Figure 2). These results indicate that flower extract of the O. odorata is the most anti-oxidant effect on oxidative stress in cells. The anti-oxidant effect depending on the time, however, did not show remarkable effectiveness.

Decrease of LDH cytotoxicity by the extracts from O. odorata in dose dependant manner This study confirmed the cyto-protective effect by H2O2 treatment. Root, stem and flower extracts showed a protective effect at the 0.01% concentration. Stem and flower extract is EC50 and root extract goes over EC50 at 0.01%. And the higher a concentration, root and stem extracts increased the protective effect. However, quercetin and flower extracts did not have cyto-protective effect but had a lower protecting effect than root and stem extracts (Figure 3). These results indicate that roots and stem extracts are the most protective effect.

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Figure 2. MTT cell viability assay by concentration of the extracts from EVP-1, 2 and 5. (A) Quercetin (positive control). (B) Evp-1; extract from root of an O. odorata (C) Evp-2; extract from stem of an O. odorata. (D) Evp-5; extract from flower of an O. odorata. NT; Untreated Huh7cell. The data are expressed as mean ± S.D. (n = 3).

Decrease of LDH cytotoxicity by the extracts from O. odorata in time-dependent manner To determine the cyto-protective effect of the extracts from O. odorata in time-dependent manner, the 0.05% extracts were treated at 24-, 48- and 72-h interval. Roots

and stem cell extracts showed protective effects for 24 h like the untreated H2O2 group. Flower extracts showed a slight protective effect for 24 h. In general, all the treatment groups showed the highest cyto-protective effects for 48 h and a similar protective effect rate for 72 h. Quercetin, which is an anti-oxidant, protected the cell

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Figure 3. LDH cytotoxicity assay by concentration of the extracts from EVP-1, 2 and 5. (A) Quercetin (positive control). (B) Evp-1; extract from root of an O. odorata. (C) Evp-2; extract from stem of an O. odorata. (D) Evp-5; extract from flower of an O. odorata. NT; Untreated Huh7cell. The data are expressed as mean ± S.D. (n=3).

from damage for 24 h and showed similar cyto-protective effects independent on time (Figure 4). These results indicated that quercetin, which is one of anti-oxidant materials, has a cyto-protective effect depending on the concentration and not on the time. Moreover, O. odorata extracts contain anti-oxidant material and other substances like growth factors; so it has a high protective effect rather than anti-oxidants alone.

Structure of compound I Compound I was isolated from the Fr3 of stem extracts. The compound I(β-sitosterol) was identified in comparison with field desorption mass spectrometry (FD-MS), electron-impact ionization MS (EI-MS), and proton nuclear magnetic resonance (1H-NMR), respectively, 13 along with carbon nuclear magnetic resonance ( C-

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Time (h)

Time (h)

Time (h)

Time (h)

Figure 4. LDH cytotoxicity assay of 0.05% extracts by time after treatment. (A) Quercetin (positive control). (B) Evp-1; extract from root of an O. odorata. (C) Evp-2; extract from stem of an O. odorata. (D) Evp-5; extract from flower of an O. odorata. NC, Untreated Huh7 cell; NT-72, untreated test material and Huh7cell for 72 h.

NMR) spectral data previously published (Zhang et al., 2005) (Figure 5). DISCUSSION In this study, the stem and flower extracts have antioxidant effects. Particularly, flower extract had the greatest effect. Usually O. odorata was only used root or

seed oil. But flower has the most anti-oxidant effect. In the future, we hope to study the chemical structure of O. odorata flower whether it contains a polyphenol group or not. We also hope to check the anti-oxidant effect of detail sections in O. odorata flower, and through vivo test, determine the anti-oxidant enzyme (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx) and glutathione reductase (Gred) (Baynes and Thorpe, 1999)) activities and MDA (malondialdehyde)

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material. So far, though epicatechin, caffeic acid and vitamin E as well as quercetin have been used as antioxidants (Mahakunakorn et al., 2004); the extracts from each of O. odorata might be more cheap, effective and powerful anti-oxidants. Thus far, we studied 10 kinds of medicinal plants. Other medicinal plants are expected to have an anti-oxidant effect. Hence, recommend the study of the anti-oxidant effect of medicinal plants so as to ascertain their multi-functions. Conclusion

Figure 5. Chemical structure of compound I (β-sitosterol).

generation in liver cell. By checking their activity, we will be able to confirm the influence of anti-oxidant enzyme. For example, the biennial flower of Panax notoginseng has more anti-oxidant effect than other fractions of P. notoginseng. And a saponin of P. notoginseng promotes neural function (Choi et al., 2010). Chestnut flower extract has a phenolic and flavonoid contents. So it has antioxidant effect and it decreases melanin and tyrosinase activity (Sapkota et al., 2010). Jasminum lanceolarium are also known as having anti-inflammation and an antioxidant effect (Sun et al., 2007). Other medicinal plants have been known to possess anti-oxidant effect traditionally, although without scientific evidences. Basically, the roots of such plants are commonly used. There is need to confirm these effects of medicinal plants exactly, especially effects of stems and flower extracts. Oxidative stress causes several diseases. We can anticipate that anti-oxidants are effective against kinds of diseases. So the study about multi-function of antioxidants is proceeding. Terminalia chebula has been reported to possess effective anti-oxidative, anti-cancer, anti-diabetic, anti-mutagenic, anti-bacterial, anti-fungal and anti-viral activity (Cheng et al., 2003). Phloroglucinol is known as anti-oxidant, and it has wide range of biological activities in anti-cancer, anti-depressant, antimicrobial, anti-protozoal, anti-spasmodic, anti-viral and anti-inflammation. It is used in cosmetics, textiles, paints, dyeing industries as well as in biological activities (Singh et al., 2009; Crockett et al., 2008). The O. odorata extract is also known to possess anti-oxidant activity as well as anti-inflammatory, anti-cancer, anti-fungal, anti-bacterial, anti-viral, anti-allergy and nerve regeneration activities. The crude extract from flower of O. odorata has a similar anti-oxidation effect compared to that of quercetin, a representative commercial anti-oxidant which is composed of one compound. In the future, we hope to work on the separation and purification of the extracts from each parts of O. odorata for discovery of useful pure

In this study, we compared and analyzed the anti-oxidant activity of ten medicinal plants. Among them, the extracts from O. odorata had the highest anti-oxidant effect. The stem and flower extracts of O. odorata were similar to the activity of quercetin, one of the most anti-oxidants, in DPPH radical scavenging activity test, while the root extracts showed a weak DPPH radical scavenging activity. In MTT cell viability assay, the extracts from the flower, stem, and root were resistant against H2O2 treatment in order. The extracts from the root, stem and flower also showed higher cell protection effect than those from quercetin against LDH cytotoxicity. β-sitosterol from stem extracts, which also showed the highest antioxidant effect, was isolated. Further studies are in progress for isolation and chemical structure elucidation of the highest anti-oxidant compound.

REFERENCES Arteel GE, Schroede P, Sies H (2000). Reactions of peroxynitrite with cocoa procyanidin oligomers. J. Nutr. 130(8S Suppl):2100S-4S. Baynes JW, Thorpe SR (1999). Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 48(1):1-9. Cameron NE, Cotter MA, Dines KC, Robertson S, Cox D (1993). The effects of evening primrose oil on nerve function and capillarization in streptozotocin-diabetic rats: modulation by the cyclo-oxygenase inhibitor flurbiprofen. Br. J. Pharmacol. 109(4):972-979. Chance B, Sies H, Boveris A (1979). Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59(3):527-605. Cheng HY, Lin TC, Yu KH, Yang CM, Lin CC (2003). Antioxidant and free radical scavenging activities of Terminalia chebula. Biol. Pharm. Bull. 26(9):1331-1335. Chenoy R, Hussain S, Tayob Y, O'Brien PMS, Moss MY, Morse PF (1994). Effect of oral gamolenic acid from evening primrose oil on menopausal flushing. BMJ 308(6927):501-503. Choi RC, Jiang Z, Xie HQ, Cheung AW, Lau DT, Fu Q, Dong TT, Chen J, Wang Z, Tsim KW (2010). Anti-oxidative effects of the biennial flower of Panax notoginseng against H2O2-induced cytotoxicity in cultured PC12 cells. Chin. Med. 5:38. Crockett SL, Wenzig EM, Kunert O, Bauer R (2008). Anti-inflammatory phloroglucinol derivatives from Hypericum empetrifolium. Phytochem. Lett. 1(1):37-43. Evans P, Halliwell B (2001). Micronutrients: oxidant/antioxidant status. Br. J. Nutr., 85 Suppl. 2:S67-74. Halliwell B, Gutteridge JMC (1989). Oxygen is poisonous-An introduction to oxygen toxicity and free radicals. In Free radicals in biology and medicine (2nd ed.) Clarendon Press, Oxford, pp. 1-21. Halliwell B, Aruoma OI (1991). DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett.

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281(1-2):9-19. Kanno SI, Shouji A, Asou K, Ishikawa M (2003). Effects of naringin on hydrogen peroxide-induced cytotoxicity and apoptosis in P388 Cells. J. Pharmacol. Sci. 92:166-170. Kim DW, Chang CC (2006). Antioxidation Effect of berberine against paraquat toxicity in the liver of mice. Kor. J. Gerontol. 16(3):159-163. Kuroki T, Isshiki K, King GL (2003). Oxidative stress: The lead or supporting actor in the pathogenesis of diabetic complications. J. Am. Soc. Nephrol. 14:S216-S220. Lee SM, Na MK, An RB, Min BS, Lee HK (2003). Antioxidant activity of two phloroglucinol derivatives from Dryopteris crassirhizoma. Biol. Pharm. Bull. 26(9):1354-1356. Mahakunakorn P, Tohda M, Murakami Y, Matsumoto K, Watanabe H (2004). Antioxidant and free radical-scavenging activity of Choto-san and its related constituents. Biol. Pharm. Bull. 27(1):38-46. Park KE, Jang MS, Lim CW, Kim YK, Seo YW, Park HY (2005). Antioxidant activity on ethanol extract from boiled-water of Hizikia fusiformis J. Korean Soc. Appl. Biol. Chem. 48(4):435-439. Pellegrina CD, Padovani G, Mainente F, Zoccatelli G, Bissoli G, Mosconi S, Veneri G, Peruffo A, Andrighetto G, Rizzi C, Chignola R (2005). Anti-tumour potential of a gallic acid-containing phenolic fraction from Oenothera biennis. Cancer Lett. 226(1):17-25. Sapkota K, Park SE, Kim JE, Kim S, Choi HS, Chun HS, Kim SJ (2010). Antioxidant and antimelanogenic properties of chestnut flower extract. Biosci. Biotechnol. Biochem. 74(8):1527-1533. Senapati S, Banerjee S, Gangopadhyay DN (2008). Evening primrose oil is effective in atopic dermatitis: A randomized placebo-controlled trial. Indian J. Dermatol. Venereol. Leprol. 74(5):447-452. Shin CH, Ihm JH (2008). Effects of S-allylcysteine on oxidative stress in streptozotocin-induced diabetic rats. J. Korean Endocr. Soc. 23(2):129-136. Singh IP, Sidana J, Bansal P, Foley WJ (2009). Phloroglucinol compounds of therapeutic interest: global patent and technol. status. Expert. Opin. Ther. Pat. 19(6):847-866.

Sun JM, Yang JS, Zhang H (2007). Two new flavanone glycosides of Jasminum lanceolarium and their anti-oxidant activities. Chem. Pharm. Bull. 55(3):474-476. Sung H, Nah J, Chun S, Park H, Yang SE, Min WK (2000). In vivo antioxidant effect of green tea. Eur. J. Clin. Nutr. 54(7):527-529. Yun MJ, Oh SI, Lee MS (2009). Antioxidative and antimutagenic effects of Agaricus bisporus ethanol extracts. J. Kor. Soc. Food Sci. Nutr. 38(1):19-24. Zhang X, Geoffroy P, Miesch M, Julien-David D, Raul F, Aoude-werner D, Marchioni E (2005). Gram-scale chromatographic purification of bsitosterol synthesis and characeterization of b-sitosterol oxides. Steroides 70: 886-895. Zia-Ul-Haq M, Iqbal S, Ahmad S, Bhanger MI, Wiczkowski W, Amarowicz R (2008). Antioxidant Potential of desi chickpea varieties commonly consumed in Pakistan. J. Food Lipid 15: 26-342. Zia-Ul-Haq M, Ćavar S, Qayum M, Imran I, Feo V (2011a). Compositional studies: antioxidant and antidiabetic activities of Capparis decidua (Forsk.) Edgew. Int. J. Mol. Sci. 12(12): 8846-8861. Zia-Ul-Haq M, Ahmad S, Iqbal S, Luthria DL, Amarowicz R (2011b). Antioxidant potential of lentil cultivars. Oxid. Comm. 34:819-831. Zia-Ul-Haq M, Shahid SA, Ahmad S, Qayum M, khan I (2012). Antioxidant potential of various parts of Ferula assafoetida L. J. Med. Plants Res. 6:3254-3258.

Journal of Medicinal Plants Research Vol. 6(312), pp. 4625-4632, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1301 ISSN 1996-0875©2012 Academic Journals

Full Length Research Paper

Survey of herbal remedies used by Fulani herdsmen in the management of animal diarrhoea in Plateau State, Nigeria Nkechi Veronica Offiah1,2, Christiana Joshua Dawurung1, Olusola Olalekan Oladipo1, Micah Shehu Makoshi1, Sunday Makama1, Ishaku Leo Elisha1*, Jurbe Gofwan Gotep1, Ann Lohlum Samuel1 and David Shamaki1 1

Biochemistry Division, National Veterinary Research Institute, P. M. B. 01, Vom, Plateau State, Nigeria. School of Veterinary Medicine, Faculty of Medical Sciences, the University of the West Indies, St. Augustine, Trinidad and Tobago.

2

Accepted 26 October, 2011

The Fulani herdsmen of Nigeria are known to use herbs for the treatment and control of different human and livestock diseases. This study was designed to identify and document the medicinal plants used by the Fulani herdsmen in Plateau State, Nigeria, in the management of animal diarrhoea, and to harness such plants for the purpose of drug development. Open-ended questionnaires and guided dialogue techniques were used to interview the Fulani pastoralists in nine Local Government Areas (LGAs) spread across the three senatorial districts of Plateau State. Seventy-nine plants were mentioned as being used for treatment and control of diarrhoea in animals. Fabaceae was the most common family mentioned followed by Combretaceae, Moraceae and Verbanaceae. The leaves were mentioned as the most common plant part used. Most anti-diarrhoeal preparations are administered by drenching while a few others are mixed with feed, salt or potash to improve palatability. The Fulani herdsmen have appreciable understanding of medicinal plants and could constitute a relevant source of information about herbal remedies. Plateau State has a large reserve of medicinal plants used for the management of diarrhoea in livestock; such plants are potential sources of novel anti-diarrhoeal medicaments. Key word: Animal diarrhoea, Fulani, herbs, Plateau State, Nigeria, survey.

INTRODUCTION The Fulani tribe found mainly in Central, Western and Northern Africa hold a large number of livestock population. In Nigeria and most parts of Africa, mobile pastoralism is the dominant system of livestock management practiced by pastoralists. This involves the movement of herdsmen, their families, and herds from one grazing area to another with availability of fodder, water and animal health as determining factors (Adekunle

*Corresponding author. E-mail: [email protected]. Tel: +2348035956638. Abbreviations: LGA, Local Government Areas; WHO, World Health Organization; ABU, Ahmadu Bello University.

et al., 2002). The economic burden of diseases worldwide (Bennett et al., 1999) and the declining provision of animal health services in developing

countries have undermined the efficiency of livestock production by Fulani nomads in Nigeria (Ilemobade, 2009). It is generally believed that Fulani herdsmen have good knowledge of medicinal plants because as they move from one place to another they depend on these plants to tackle their health challenges as well as those of their animals. More recently veterinarians and other scientists in recognition of the fact that livestock owners posses considerable understanding of herbal remedies and their application in disease management (Adekunle et al., 2002) have intensified their efforts towards harnessing

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this knowledge in dealing with livestock diseases and other problems (Adekunle et al., 2002). Most livestock diseases present diarrhoea as a symptom with adverse effects reported to include anorexia, weight loss, general malaise and death (Gattuso and Kamm, 1994). Current management of diarrhoea is achieved using drugs such as antibiotics, atropine sulphate, loperamide, kaolin, anthelminthics, fluid and electrolyte replacement therapy (Hall, 2011; Sur and Bhattacharya, 2006). Despite the availability of a vast spectrum of approaches for diarrhoeal management, many people in developing countries still rely on herbal drugs for the management of diarrhoea. World Health Organization (WHO) has encouraged studies for the treatment and prevention of diarrhoeal disease using traditional medical practices (Atta and Mouneir, 2004). The use of herbal medicines is common among peasant farmers and pastoralist because orthodox medicines have been found to be either not available or too expensive as a result; the Fulanis have resorted to the use of indigenous plants as remedy for animal diseases (Abubakar et al., 2007; Ibrahim, 1984). Furthermore, several investigators have contributed to reports which establish the use of plants in the treatment of diarrhoea in South Africa (De Wet et al., 2010; Appidi et al., 2008; Mathabe et al., 2006), Mozambique (Ribeiro et al., 2010), India (Tetali et al., 2009) and Sokoto State, Nigeria (Etuk et al., 2009). A review of available literature shows that such survey has not been conducted in Plateau State, Nigeria. The state has a large population of Fulani nomads probably due to the favourable climate all year round (FAO, 2009). The need to preserve and transfer indigenous knowledge from one generation to another is imperative in order to prevent the rapid depletion of such knowledge (Prance, 1991; Cox, 1990) This study is therefore intended to record the medicinal plants used for the treatment of diarrhoea by Fulani herdsmen in Plateau State, and to evaluate such plants for the purpose of developing new drugs. MATERIALS AND METHODS The data was collected through oral interview of Fulani herdsmen from 9 selected local government areas of Plateau state Nigeria (Figure 1) which spread across the 3 senatorial zones of the state during the months of October to December 2010. Letters were written seeking for assistance and cooperation of the Local Government Agricultural Departments in mobilizing community leaders and Fulani herdsmen. The Local Government Areas (LGAs) surveyed include; Bassa, Jos East, Jos South and Barkin-Ladi in the Northern zone; Bokkos and Pankshin in the Central Zone; Langtang North, Shendam, Qua‟an-Pan and Wase in the Southern Zone. The selected LGAs are known to have high population of cattle and favourable environment for livestock production (Bertu et al., 2010). The Fulani pastoralists were interviewed using a well structured, open-ended questionnaire and guided dialogue techniques (Jacob et al., 2004). The questionnaire was designed by the team based on the needed information and validated by the epidemiology and

extension units of the Institute. The team was made up of five veterinarians, one pharmacist, one pharmacologist and two trained veterinary extension officers. Members of the team were randomly divided into two on a rotational basis, with one extension officer in each group at any given time. Fulfulde and Hausa languages were used to conduct the interview. Active participation in the survey was gained by giving out some incentive to stimulate cooperation. These included free consultancy services by the veterinarians, remuneration in some instances for the field staff and the promise to organize seminar for the communities visited after the conclusion of the research. Those who consented to participate in the survey were asked to share their knowledge and experiences on the medicinal plants used in their communities to manage diarrhoea. Information was received on part(s) of the plant used, methods of herbal preparation, mode of administration, dosage estimation, the effectiveness of the herbal remedy and adverse effects observed. The conversation was built on trust, with the clear understanding of the aim of the survey (Okoli et al., 2002). Plants claimed to be beneficial in the treatment of diarrhoea were collected based on the guided field-walk method (Rashid et al., 2010). The plant specimens collected were pressed, labelled with their local names where available and sent to the herbarium of the Department of Biological Sciences, Ahmadu Bello University (ABU), Zaria, and Identified, authenticated and voucher number assigned by Mallam U.S Gallah.

RESULTS One hundred and five questionnaires were administered directly during the survey. A total of 87 (82.86%) respondents admitted having used antidiarrhoeal medicinal plants or were still using them to treat their animals. Eighteen (17.14%) had no knowledge of herbs or medicinal plants used for the treatment of diarrhoea in animals. Most of the respondents were able to give adequate description of the nature of the diarrhoea often seen in their animals. Data generated from the survey indicated seventy-nine (79) medicinal plants as remedies in use for diarrhoea management out of which twenty-eight (28) were properly identified by their scientific nomenclature and local names (Table 1). The 28 plants scientifically identified represents 23 genera distributed among 17 families (Table 1), with the families Fabaceae (21.43%) having the highest frequency of occurrence followed by Combretaceae (17.86%). Moraceae and Verbanaceae had 2 (7.14%) members each while all other families were mentioned once (3.57%). Khaya senegalensis 26 (24.76%) was the most common plant mentioned followed by Adansonia digitata 10 (9.52%). Vitex doniana was mentioned 9 (8.57%) times while Combretum glutinosum, Terminalia avicennioiodes and Terminalia macroptera were mentioned 7(6.67%) times each. Various parts of these plants in use were also indicated (Figure 2), with the leaves being the most commonly mentioned (42.86%). Plant parts to be used are usually prepared by soaking the fresh or dried plant parts in water and the extract administered by drenching. In some cases, the plant materials are mixed with feed and/or potash to improve palatability.

Offiah et al.

4627

-LGAs visited

Figure 1. Map of Plateau State, Nigeria showing LGAs visited. Bassa (1), Jos East (2), Barkin-Ladi (3), Bokkos (4), Pankshin (5), Qu‟an Pan (6), Shendam (7), Langtang North (8) and Wase (9) LGAs. Pink – North, Green – Central and Blue – Southern geopolitical zones.

DISCUSSION Cattle-rearing is the main occupation of Fulani herdsmen in Nigeria while other ethnic groups usually engage in livestock farming as a secondary occupation (Abdu et al., 2000). Out of the 105 Fulani herdsmen interviewed in this survey, 87 (82.86%) indicated that they use herbal remedies to manage animal diarrhoea, while 18 (17.14%) stated that they rely on orthodox veterinary preparations. This agrees with earlier reports on the relevance of different traditional healing practices in Nigeria as well as other parts of the world (Abdu et al., 2000; Mathias, 1994; McCorkle, 1986). The reliance of pastoralists on herbal remedies for both prophylactic and therapeutic purposes in Nigeria has been reported (Abdu et al., 2000; Kudi and Myint, 1999). The Fulani herdsmen exhibited good knowledge of the pathology of various animal diseases and the corresponding plant(s) used in the treatment. Most of them were able to clearly describe the type of diarrhoea passed by their animals which they called “saaroo or dauda”. Others could identify and name disease conditions responsible for diarrhoea such as: helminthosis (goli), white scour in calves (shanin-koje or gortoyel), fascioliasis (hanta) and rinderpest (bushiya). Their understanding of animal diseases is partly due to experiences gathered during grazing and interaction with butchers when they take sick animals for slaughter (Ibrahim, 1984).

From Table 1, K. senegalensis 26 (24.76%) and A. digitata 10 (9.52%) are the plants commonly used by Fulani herdsmen in the management of diarrhoea in livestock. Another survey of ethnoveterinary practices of agropastoralist in eleven selected states of Nigeria also reported that K. senegalensis and A. digitata as the most common plants used as remedies for various livestock diseases (Abdu et al., 2000). Other plants mentioned were V. doniana, 9 (8.57%), T. avicennioiodes and T. macroptera 7 (6.67%) times each. These medicinal plants are either used singly or in combination with other plants. A similar checklist of the plants listed in Table 1 has been reported in a survey of ethnoveterinary plants useful in the treatment of poultry diseases in Ekiti State, Nigeria (Kayode et al., 2009). Thus, agreeing with reports that medicinal plants have a wide range of application in the treatment of different animal species (Eisenberg et al., 1998). A. digitata (baobab) is commonly found in the northern part of Nigeria. Earlier works have reported its use in the management of diarrhoea, malaria and cough (De Caluwe et al., 2009; Woolfe et al., 1977) .The anthelminthic effect of K. senegalensis (mahogany) has been reported (Ndjonka et al., 2010). Apart from eliminating matured adult worms, the plant has also been shown to have ovicidal activity (Chiezey et al., 2000). These may justify its use in diarrhoea management. Fabaceae is the most common plant family reported in this study, having 6 genera (21.43%), followed by

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Table 1. Medicinal plants used in the management of diarrhoea by Fulani herdsmen in Plateau State.

S/N

Botanical/Scientific name

Family

Common Name (Eng)

1

Acacia albida

2

Adansonia digitata,

Fabaceae (Mimosaceae) Bombacaceae

3 4

Aloe buettneri Anogeissus leiocarpus

Liliaceae Combretaceae

Apple-ring Acacia, Winter Thorn Baobab Tree, Judas Fruit West African aloe African Birch

5

Bauhinia rufescens

Fabaceae

Bauhinia

6

Boswellia dalzielii Hutch

Burseraceae

Frankincense tree

7 8 9

Carica papaya Combretum glutinosum Combretum lamprocarpum

Caricaceae Combretaceae Combretaceae

Paw-paw -

10

Erythrophloem africanum

Fabaceae

African blackwood

11

Ficus ingens

Moraceae

Red-leaved fig

12

Ficus platyphylla

Moraceae

13 14

Khaya senegalensis Kigelia africana

Meliaceae Bignoniaceae

15 16 17

Mitragyna inermis Moringa oleifera Parkia biglobosa

18

Piliostigma reticulatum

19

Piliostigma thonningii

Rubiaceae Asclepiadaceae Fabaceae (Mimosaceae) Leguminosae Caesalpiniaceae Fabaceae

20

Prosopis africana

Fabaceae

Flake/Red Kano rubber tree African Mahogany Cucumber or Sausage tree False abura Drumstick Tree African locust bean; Monkey cutlass tree English: camel’s foot (Etkin). camel's foot, monkey bread, Rhodesian bauhinia Iron wood; Axlewood

21

Psidium guajava

Myrtaceae

Guava

Nigeria language name (H; Y; I; F) H: Gawo

Folkloric Evidence of Use

Leaves

Stem bark

Roots

Fruits

Seeds

Flower

Whole

1 (0.95%)

+

+

-

-

-

-

-

10 (9.52%)

+

-

-

-

-

-

-

H: Zabuwa; F: Zabuwahi H: Marke; Y: Pako dudu, ayin H: Matsagi, Kalgon Allah; F: Nammare H: Ararabi; Hano; F: Mangalede H: Gwanda H: kantakara, Baushe H: Bauli; F: Buski daneehi; Zindi; Y: ajantiro H:Goska; F: Naretibahi F: Nunahi; Bakurahi; H: Kawuri H: Gamji; F: Dundehi

2 (1.90%)

+

-

-

-

-

-

-

2 (1.90%)

+

+

-

-

+

-

-

1 (0.95%)

+

-

-

-

-

-

-

1 (0.95%)

-

-

-

-

-

-

-

2 (1.90%) 7 (6.67%)

+

+ -

+

-

+ -

-

-

2 (1.90%)

+

-

-

-

-

-

-

1 (0.95%)

-

-

-

-

-

-

-

1 (0.95%)

+

-

-

-

-

-

-

2 (1.90%)

-

+

-

-

-

-

-

H: Madaci H: Nonon giwa; F: Jillarehi

26 (24.76%)

-

+

-

-

-

-

-

2 (1.90%)

-

+

-

-

-

-

-

1 (0.95%) 1 (0.95%)

+

+

-

-

+

-

-

3 (2.86%)

-

+

-

-

-

-

-

1 (0.95%)

+

-

-

-

-

-

-

1 (0.95%)

+

-

-

-

-

-

-

H: Kuka

H: Giyayya; F: Koli H: Zogale H: Doruwa H: ‘kalgo; F: Batehi; Y: abafin; I: okpo atu Kalgo / Kargo (Hausa)

H: Kirya; F: Kwahi

3 (2.86%)

-

+

-

-

-

-

-

H: Gwaiva

1 (0.95%)

+

-

-

-

-

-

-

Offiah et al.

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Table 1. Contd.

22 23

Solanum dasyphyllum Starchytarpheta angustifolia

Solanaceae Verbanaceae

Devil's coach whip

24

Scrophulariaceae

25

Striga hermonthica (Del.) Benth Terminalia avicennioides

Combretaceae

Witchweed; purple witchweed -

26

Terminalia macroptera

Combretaceae

-

27

Vitellaria paradoxa Gaertn. (Butyrospermum paradoxum) Vitex doniana

Sapotaceae

Sheabutter tree

28

Verbanaceae

black plum

H: Gautan Kaji; H: Tsarkiyar kuusuu, Wutsiyan kadangare H: Wuta-wuta; F: Turguel H: Baushe; Y: Igiodan; I: Edo H: Baushe; F: Bodi H: Kadanya; I: okwuma; Y: akú malapa H: Dinya; F: Bodilohi (Munjiriya); I: Utakiri; Y: Ori-nla

1 (0.95%)

-

-

-

+

-

-

-

2 (1.90%)

+

-

+

-

-

-

-

1 (0.95%)

-

-

-

-

-

-

+

7 (6.67%)

-

-

-

-

-

-

-

7 (6.67%)

+

-

+

-

-

-

-

1 (0.95%)

-

+

-

-

-

-

-

9 (8.57%)

+

+

-

+

-

-

-

* H, Y, I, F: Hausa, Yoruba, Igbo, Fulfulde. +, plant part in use; -, no information on use.

Combretaceae which has 5 (17.86%). A similar observation suggesting that the Fabaceae family may be more likely to have antidiarrhoeal effect than plants from other families has been made (Appidi et al., 2008). The Fabaceae family contains many genera that have been shown to be useful in the treatment of many other ailments besides diarrhoea (Joudi and Ghasem, 2010; Appidi et al., 2008). In contrast, an ethnoveterinary plant survey in Ethiopia reported Asteraceae as the highest, followed by Solanaceae, with Fabaceae and Lamiaceae being third (Yinegar et al., 2007). This difference may be due to the fact that their survey was not specific on diarrhoea but on medicinal plants used in all animal diseases. It was also observed that the leaves (42.88%) constitute the most frequently used plant part, followed by the stem bark (31.43%) as shown in Figure 2. A similar survey of plant parts used in Dheera town Arsi zone in Ethiopia also reported

that leaves are the most frequently used plant part in herbal preparations followed by the roots (Wondimu et al., 2007). Communities using herbal medicaments have indicated preference for the use of leaves because it is more convenient collecting leaves than root parts, flowers and fruits (Giday et al., 2009). However, some authors have reported that roots are more commonly collected plant parts in ethnoveterinary practice (Yinegar et al., 2007; Hunde et al., 2004; Tibuti et al., 2003). The use of leaves in combination with other plant parts has also been reported (Ayyanar and Ignacimuthu, 2011). It is known that leaves are actively involved in photosynthesis and the production of metabolites (Ghorbani, 2005). Thus, the numerous constituents found in leaves could explain their efficacy in the treatment of various ailments in both humans and animals. Collection of leaves for herbal preparations ensures sustainability as long

as some leaves are left on the parent plant (Yinegar et al., 2007). This is opposed to the collection of roots which could be a severe threat for rare and slowly producing plants. The herbal remedies were often prepared by pounding either the fresh or dried parts of the plants followed by either soaking or boiling them in water, and the infusions or decoctions administered by drenching. These practices have also been reported by other researchers (Ermias et al., 2008; Abdu et al., 2000). Sometimes, the plant portions are mixed with bran or grain and fed to the animals or mixed with potash (kanwa) or salt and given to the animals to lick, an observation corroborated by Abdu et al. (2000). The dosages often administered varied with the parts of the plant used and the mode of preparation. However most Fulani herdsmen administer the preparations once or twice a day for 3 to 5 days, or keep treating until the animal recovers. Full recovery is confirmed when the

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Figure 2. Percentage distribution of medicinal plant parts used in the management of diarrhea by Fulani herdsmen.

animals resume feeding and activities. Similarly, administration of herbal medicines to sick animals by pastoralists for 3 to 7 days once or twice daily or until there is visible improvement of condition has been reported (Abdu et al., 2000). Most respondents claimed that their herbal remedies were efficacious and healing was achieved without visible adverse effects. A study carried out in Kerela, South India indicated that majority of farmers used traditional medicine because it had no side effects (Padmakumar, 1998). This may be due to their holistic properties (Majumdar, 1989). Some of the plants listed in Table 1 have been investigated in some other parts of Nigeria and the world for their antidiarrhoeal properties using castor oil in rats or mice model as well as the antimicrobial activity of the extracts (Ahmadu et al., 2007; Agunu et al., 2005; Abdullahi et al., 2001; Mujumdar et al., 2000). During this survey, the researchers experienced unwillingness to part with indigenous knowledge and the problem of inconsistent dosage regimen in the administration of the herbal preparations. This is not uncommon with researches on ethnobotanical surveys (Souto et al., 2011; Bisi-Johnson et al., 2010). The guardians of indigenous knowledge of herbal remedies do not usually document their practices; hence transfer of knowledge to subsequent generations becomes difficult following their demise. This type of survey serves to fill that important gap. To the best of our knowledge, this is the first report of herbal remedies used in the management of diarrhoea by the Fulani herdsmen in livestock in Plateau State. Plants identified from this study will be evaluated to determine their phytochemical constituents and biological activities in order to validate the claims.

The Fulani herdsmen are a relevant source of information on medicinal plants used for the management of diarrhea in livestock owing to their nomadic nature. Such plants could be harnessed and used as potential drug sources for the production of anti-diarrhoeals that could be used for the treatment and control of diarrhea in livestock. It is therefore, strongly recommended that further studies be carried out on all the above listed plants that were collected during the survey to validate their efficacy in the treatment of diarrhea in animals for the purpose of drug development. ACKNOWLEDGEMENT The authors are grateful to the management of the National Veterinary Research Institute Vom, for funding the project; the University of the West Indies, St. Augustine, Trinidad and Tobago for releasing Dr. Offiah for this project, Mr Jamo Aliyu and Simon Emmanuel of the Extension services Department who served as linkmen with the farmers, Mr. U. S. Gallah of Biological Sciences Department, ABU, Zaria for identifying the plants. We are grateful also to all Chairmen of LGAs visited and their extension Staff, the Miyetti Allah Cattle Rearers Association, all village heads and their subjects for their cooperation and assistance. REFERENCES Abdu PA, Jagun AG, Gefu JO, Mohammed AK, Alawa CB, Omokanye AK (2000). A survey of ethnoveterinary practices of agropastoralist in Nigeria. In Gefu JO, Abdu PA, Alawa CB (eds) Ethnoveterinary Practices, Research and Development. Proceedings of the International Workshop on ethnoveterinary practices held in Kaduna, Nigeria, pp. 25-37.

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Abdullahi AL, Agho MO, Amos S, Gamaniel KS, Wambebe C (2001). Antidiarrhoeal Activity of the Aqueous Extract of Terminalia avicennoides Roots. Phytother. Res. 15:431-434. Abubakar MS, Musa AM, Ahmed A, Hussaini IM (2007). The perception and practice of traditional medicine in the treatment of cancers and inflammations by the Hausa and Fulani tribes of Northern Nigeria. J. Ethnopharmacol. 111(3):625-629. Adekunle OA, Oladele OI, Olukaiyeja TD (2002). Indigenous control methods for pest and diseases of cattle in Northern Nigeria. Livestock Res. Rural Dev. 14(2): http://www.cipav.org.co/lrrd/lrrd14/2/adek142.htm Accessed10.09.2011. Agunu A, Yusuf S, Andrew GO, Zezi AU, Abdulrahman EM (2005). Evaluation of five medicinal plants used in diarrhoea treatment in Nigeria. J. Ethnopharmacol. 101:27-30. Ahmadu AA, Zezi AU, Yaro AH (2007). Antidiarrhoeal activity of the leaf extracts of Daniellia oliveri Hutch and Dalz (Fabaceae) and Ficus sycomorus Miq (Moraceae). Afr. J. Trad. CAM 4(4):524528. Appidi JR, Grierson DS, Afolayan AJ (2008). Ethnobotanical study of plants used for the treatment of diarrhoea in the Eastern Cape, South Africa. Pak. J. Biol. Sci. 11(15):1961-1963. Atta AH, Mouneir SM (2004). Antidiarrhoeal activity of some Egyptian medicinal plant extracts. J. Ethnopharmacol. 92:303309. Ayyanar M, Ignacimuthu S (2011). Ethnobotanical survey of medicinal plants commonly used by the Kani tribals in Tirunelveli hills of Western Ghats, India. J. Ethnopharmacol. 134(3):851864. Bennet R, Christiansen K, Clifton-Hadley R (1999). Estimating the costs associated with endemic diseases of dairy cattle. J. Dairy. Res. 66:455-459. Bertu WJ, Ajogi I, Bale JOO, Kwaga JKP, Ocholi RA (2010). Seroepidemiology of brucellosis in small ruminants in Plateau State, Nigeria. Afr. J. Microbiol. Res. 4(19):1935-1938. Bisi-Johnson MA, Obi CL, Kambizi L, Nkomo M (2010). A survey of indigenous herbal diarrhoeal remedies of O.R. Tambo district, Eastern Cape Province, South Africa. Afr. J. Biotechnol. 9(8):1245-1254. Chiezey NP, Gefu JO, Jagun AG, Abdu PA, Alawa CBI, Magaji SO, Adeyinka, JA, Eduvie LO (2000). Evaluation of some Nigerian plants for anthelminthic activity in young cattle. In Gefu JO, Abdu PA, Alawa CB (eds) Ethnoveterinary Practices, Research and Development. Proceedings of the International Workshop on ethnoveterinary practices held in Kaduna, Nigeria, pp. 38-48. Cox PA (1990). Ethnopharmacology and the search for new drugs. Ciba Found. Symp. 154:40-47. De Caluwe E, Halamova K, Van Damme P (2009). Adansonia digitata L. A review of traditional uses, phytochemistry and pharmacology. In: Juliani HR, Simon JE, Ho CT (eds) African natural plant products: discoveries and challenges in quality control. Am. Chem. Soc. Symp. Ser. 1021:51-84. De Wet H, Nkwanyana MN, van Vuuren SF (2010). Medicinal plants used for the treatment of diarrhoea in Northern Maputaland, KwaZulu-Natal Province, South Africa. J. Ethnopharmacol. 130(2):284-289. Eisenberg D, Davis R, Ettner S (1998). Trends in alternative medicine use in the United States 1990-1997; results of a follow up survey. J. Am. Med. Assoc. 280:1569-1575. Ermias L, Ensermu K, Tamrat B, Haile Y (2008). An ethnobotanical study of medicinal plants in Mana Angetu District, southeastern Ethiopia. J. Ethnobiol. Ethnomed. 4:10 doi:10.1186/1746-4269-410. Etuk EU, Ugwah MO, Ajagbonna OP, Onyeyili PA (2009). Ethnobotanical survey and preliminary evaluation of medicinal plants with antidiarrhoea properties in Sokoto state, Nigeria. J. Med. Plants. Res. 3(10):763-766. FAO (2009). Country pasture/forage resource profiles - Nigeria.

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http://www.fao.org/ag/AGP/AGPC/doc/counprof/nigeria/nigeria.ht m. Gattuso JM, Kamm MA (1994). Adverse effects of drugs used in the management of constipation and diarrhoea. Drug Saf. 10(1):4765. Ghorbani A (2005): Studies on pharmaceutical ethnobotany in the region of Turkmen Sahra, north of Iran (Part 1): general results. J. Ethnopharmacol. 102:58-68. Giday M, Asfaw Z, Woldu Z (2009). Medicinal plants of the Meinit ethnic group of Ethiopia: An ethnobotanical study. J. Ethnopharmacol. 124:513–521. Hall EJ (2011). Antibiotic-responsive-diarrhoea in small animals. Vet. Clin. North. Am. Small Anim. Pract. 41(2):273-286. Hunde D, Asfaw Z, Kelbessa E (2004). Use and management of ethnoveterinary medicinal plants by indigenous people in „Boosat‟, Welenchetti area, Ethiopia. J. Biol. Sci. 3:113-132. Ibrahim MA (1984). Veterinary traditional practices in Nigeria. Proceedings of the second ILCA/NAPRI symposium, held in Kaduna State, Nigeria. Ilemobade AA (2009). Tsetse and trypanosomosis in Africa: the challenges, the opportunities. Onderst. J. Vet. Res. 76(1):35-40. Jacob MO, Farah KO, Ekaya WN (2004). Indigenous knowledge: The basis for the Maasai ethnoveterinary diagnostic skills. J. Hum. Ecol. 16(1):43-48. Joudi L, Ghasem HB (2010). Exploration of medicinal species of Fabaceae, Lamiaceae and Asteraceae families in Ilkhji region, Eastern Azerbaijan Province (Northwestern Iran). J. Med. Plants Res. 4(11):1081-1084. Kayode J, Olanipekun MK, Tedela PO (2009). Medicobotanical studies in relation to veterinary medicine in Ekiti State, Nigeria: (1) checklist of botanicals used for the treatment of poultry diseases. Ethnobotan. Leaf. 13:40-46. Kudi AC, Myint SJ (1999). Antiviral activity of some Nigerian medicinal plant extracts. J. Ethnopharmacol. 68:289-294. Majumdar AK (1989). Ayuverda and Modern Medicine. Ancient Sci. Life 8:117-190. Mathabe MC, Nikolova RV, Lall N, Nyazema NZ (2006). Antibacterial activities of medicinal plants used for the treatment of diarrhoea in Limpopo Province, South Africa. J. Ethnopharmacol. 105(1-2):286-293. Mathias ME (1994). Magic, myth and medicine. Econ. Botan. 48(1):3-7. McCorkle CM (1986). An introduction to ethnoveterinary research and development. J. Ethnobiol. 129:129-140. Mujumdar AM, Upadhye AS, Misar AV (2000). Studies on antidiarrhoeal activity of Jatropha curcas root extract in albino mice. J. Ethnopharmacol. 70:183-187. Ndjonka D, Agyare C, Luersen K, Djafsia B, Achukwi D, Nukenine EN, Hensel A, Liebau E (2010). In vitro activity of Cameroonian and Ghanian medicinal plants on parasitic (Onchocerca ochengi) and free-living (Caenorhabditis elegans) nematodes. J. Helminthol. 24:1-9. Okoli IC, Okoli CG, Ebere CS (2002). Indigenous livestock production paradigm: survey of plants of Ethnoveterinary importance in southeastern Nigeria. Trop. Ecol. 43(2):257-263. Padmakumar V (1998). Farmers‟ reliance on ethnoveterinary practices to cope with common cattle ailments. Indigen. Knowl. Dev. Monit. 6(2):14-15. Prance DT (1991). What is ethnobotany today? J. Ethnopharmacol. 32(1-3):209-216. Rashid MH, Tanzin R, Ghosh KC, Jahan R, Khatun MA, Rahmatullah M (2010). An ethnoveterinary survey of medicinal plants used to treat cattle diseases in Birishiri area, Netrakona district, Bangladesh. Adv. Nat. Appl. Sci. 4(1):10-13. Ribeiro A, Romeiras MM, Tavares J, Faria MT (2010). Ethnobotanical survey in Canbane village, district of Massingir, Mozambique: medicinal plants and traditional knowledge. J. Ethnobiol. Ethnomed. 3:6-33.

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Souto WMS, Mourao JS, Barboza RRD, Alves RRN (2011). Parallels between zootherapeutic practices in ethnoveterinary and human complementary medicine in northeastern Brazil. J. Ethnopharmacol. 134(3):753-767. Sur D, Bhattacharya SK (2006). Acute diarrhoeal diseases – an approach to management. J. Ind. Med. Assoc. 104(5):220-223. Tetali P, Wagchaure C, Daswani PG, Antia NH, Birdi TJ (2009). Ethnobotanical survey of antidiarrhoeal plants of Parinche valley, Pune district, Maharashtra. Ind. J. Ethnopharmacol. 123(2):229236. Tibuti JR, Dhillion SS, Lye KA (2003). Ethnoveterinary medicines for cattle (Bos indicus) in Bulamogi county Uganda: plant species and mode of use. J. Ethnopharmacol. 88:279-286.

Wondimu T, Asfaw Z, Kelbessa E (2007). Ethnobotanical study of medicinal plants around Dheera town, Arsi zone, Ethiopia. J. Ethnopharmacol. 112:152-161. Woolfe ML, Martin FC, Otchere G (1977). Studies on the mucilages extracted from okra fruits (Hibiscus esculentus L.) and baobab leaves (Adansonia digitata L.). J. Sci. Food Agric. 28:519-529. Yinegar H, Kelbessa E, Bekele T, Lulekal E (2007). Ethnoveterinary medicinal plants in Bale Mountains National Park, Ethiopia. J. Ethnopharmacol. 112:55-70.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4633-4639, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1573 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

In vitro anti-angiogenic activity fractions from hydroalcoholic extract of Elaeagnus angustifolia L. flower and Nepeta crispa L. arial part Badrhadad A.1, Piri Kh1* and Mansouri K.2 1

Department of Biotechnology, Faculty of Agriculture of Bu-Ali Sina University, Hamadan, Iran. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.

2

Accepted 22 December, 2011

Angiogenesis is an essential event in the tumor growth and Metastasis. The aim of our research is to study the effect of Elaeagnus angustifolia and Nepeta crispa extracts on anti-angiogenic activities in human umbilical endothelial cells (HUVEC). Hydroalcoholic extract and its successive hexane, ethyl acetate, chloroform and aqueous fractions were used in different concentration by three dimensional cytodex-collagen model. Hydroalcoholic extracts of E. angustifolia flower in 200 µg/ml and N. crispa aerial part in 400 µg/ml potentialy inhibited angiogenesis activity of HUVEC and 10 µg/ml both of ethyl acetate and chloroform fractions exerted prevention of this activity. Therefore, E. angustifolia flower and N. crispa aerial part could be candidate for therapeutic or preventive activity against angiogenesis related disorders. Key words: Anti-angiogenesis, Elaeagnus angustifolia, Nepeta crispa, human umbilical endothelial cells.

INTRODUCTION The formation of Neovascularization from an existing capillaries network, angiogenesis, is a process involving the proliferation, extracellular matrix degradation, survival, migration, and anastomosis of endothelial cells (ECs). It is associated with a number of physiologic and pathologic conditions including malignancies, diabetic retinopathy, rheumatoid arthritis and skin diseases, particularly psoriasis (Creamer et al., 2002). Angiogenesis, tightly modulated through a balance of positive and negative regulatory factors, is to operate by pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epithelial growth factor (EGF) (Hanahan and Folkman, 1996), which in turn induce activation of their respective receptors on the surface of endothelial cells, resulting in angiogenesis (Hynu-JooJung et al., 2006) Identification of endostatin as an inhibitor of angiogenesis (Folkman,

*Corresponding author. E-mail: [email protected]. 00980198130783. Fax: 0098811 4224012.

Tel:

2006), a variety of anti-angiogenic compounds, such as soybean trypsin inhibitor (Shakiba et al., 2007), withaferin A from withania somniferous (Mohan et al., 2004), a peptide from shark cartilage (Hassan et al., 2005) and green tea catechin (Tang et al., 2007) have been isolated from natural products. (Keshavarz et al., 2010). Therefore, identification of new agents that inhibit growth in endothelial cells could have potential to inhibit tumor angiogenesis and subsequently repress tumor growth. No doubts, plants are the source of many bioactive compounds and a lot of them may possess significant biological activity. However, besides enthusiasm which many people uncritically express towards natural products, there are several problems which should be discussed (Dulkan, 2005). The genus Elaeagnus and Nepeta respectively belongs to the family Elaeagnaceae and Lamiaceae, which comprises some important species that growing in Iran, with the common local name Senjed and Mofarrah (because of its sweet odor) has been of great interest to Iranian traditional medicine, especially in Hamedan province (Mozaffarian, 1996) .The major compound of E. angustifolia flower show ethyl cinnamate, 2-phenyl-ethyl

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benzoate, 2-phenyl-ethyl isovalerate, nerolidole, squalene and acetophenone (Bucur et al., 2007) and the main constituents in N. crispa aerial part indicate 1,8cineol (47.9%) and 4aα,7α,7aβ-nepetalactone (20.3%) (Sefidkon and Jamzad, 2006). There are various reports showing beneficial effects of E. angustifolia and N. crispa such as antioxidant, antiinflammatory, antifungal, antibacterial, antinociceptive activities, sedative, relaxant, carminative, restorative tonic for nervous, respiratory disorders and prevention of heart diseases (Mozaffarian, 1996; Ahmadiani et al., 2000; Sonboli and Salehi, 2004; Bucur et al., 2007). This work evaluates the in vitro antiangiogenic activity of extracts and fractions of E. angustifolia and N. crispa.

MATERIALS AND METHODS Rat tail collagen (Sigma Chemical Co.), Dulbecco’s modified minimum essential medium (DMEM), RPMI 1640, fetal bovine serum (FBS) (Gibco, New York, USA), dextran-coated cytodex 3 microcarriers (Amersham Pharmasia Biotech) and Human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection, lactate dehydrogenase (LDH) cytotoxicity assay kit (Roch Chemical Co.).

microcarriers at a ratio of 30 cells per bead in 1 ml of DMEM/F12 medium (Auerbach et al., 2003). Beads with cells were shaken gently every 20 min for 4 h at 37ºC and 5% CO2. The mixture were transferred to a 24-well tissue culture plate and left for 12 to 16 h in 1 ml of DMEM/F12 at 37ºC and 5% CO2. The following day, beads with cells re-suspended in type 1 collagen gel, and 50 μl of collagen/bead mixture was added to each well of a 96-well tissue culture plate and allowed to clot for 20 min at 37ºC, 5% CO 2. Then, 250 μl of DMEM/F12 medium was added to each well and after 8 to 12 h different concentrations of the extracts were added. After 3 to 5 days of treatment, anti-angiogenic effects of the extracts were monitored microscopically (Keshavarz et al., 2010).

Cytotoxicity assay Cytotoxic concentrations were determined by growth of HUVECs in medium containing different concentrations of fractions (10, 20, 40, 80, 160 μg/ml). Cell viability was determined after 48 h of incubation, by LDH assays compared with controls. The absorbance of converted dye in LDH assay was measured at wave length of 490 nm with background subtraction at 630 nm (Decker and Lohmann-Matthes, 1988).

Aint -proliferative assay

Flowers and aerial parts of respectively E. angustifolia and N. crispa were collected in July from Hamedan province and then identified by the Agricultural College of Bu-Ali sina University The plants were cleaned, and dried at 25°C at room condition.

Anti-proliferative assay was performed (achived) on HUVECs because they are representative of microvascular endothelial cells. The cells were seeded on to a 24-wells culture plate at a density of 2×104 cells/well in DMEM/F12 supplemented with 10% FBS. After 24 h incubation at 37ºC and 5% CO2 (10, 20, 40, 80 and 160 µg/ml) of EA and NC fractions were added to the wells, and the cells were cultured for additional three days, then trypsinized and counted with cell counter (KX-21 SYSMEX Co.) against control wells.

Preparation of hydroalcohic extract and its fractions

Statistical analysis

The powder of plants was extracted with 70% (v/v) hydroalcohic ethanol for 48 h. The extracts were filtered through filter paper Whatman No. 1 and were then concentrated with a rotary evaporator (40°C) to simplify its further process. The hydroalcohic extract was successively fractionated in to n-hexane (5.9%), ethyl acetate (5.9%), chloroform (16.7%) and aqueous (72.2%) fractions. The cell cultures have been treated with extracts at the concentrations ranging from 10 to 1000 µg/ml of hydroalcohic extract and 10 to 160 µg/ml of fractions. Maximal concentration of Demethyl sulfoxide (DMSO) added to cells was 0.1%, and the solvent was always used as control.

The mean values were calculated for each group of concentrations and control. For the determination of the significance among the means, One way ANOVA test was applied (p< 0.05).

Plant material

Cell line Human umbilical vein endothelial cells (HUVEC) were purchased from Pasture Institute of Iran and grown in DMEM/F12 culture medium was supplemented with 10% of fetal calf serum, 100 IU ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycint, then incubation at 37°C in a 5% CO2.

Human umbilical vein endothelial cells (HUVEC) capillary tube formation in three-dimensional collagen matrix HUVECS were grown in DMEM/F12 supplemented with 10% FBS at 37ºC and 5% CO2. The cells were mixed with cytodex 3

RESULTS Angiogenesis, tightly modulated through a balance of positive and negative regulatory factors, is to operate by pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF), which in turn induce activation of their respective receptors on the surface of endothelial cells, resulting in angiogenesis (HynuJooJung et al., 2006). Therefore, identification of new agents that inhibit growth in endothelial cells could have potential to inhibit tumor angiogenesis and subsequently repress tumor growth. Three-dimensional culture of HUVECs is an in vitro model to screen the inhibitory activity of E. angustifolia and N. crispa extracts and its fractions on vascular development. After 3 to 5 days of treatment, untreated control wells gave branching pattern of tube like capillaries. In contrast, capillary tube formation was strongly suppressed in wells which treated with E. angustifolia (200 to 1000 µg/ml) and N. crispa

Badrhadad et al.

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Figure 1. Effect of E. angustifolia and N. crispa hydroalcoholic extracts on angiogenesis inhibition of HUVEC. A: Control group: Formation blood vessel on human umbilica endothelial cells. B: inhibition of angiogenesis on 200 µg/ml E. angustifolia extract. C: inhibition of angiogenesis on 400 µg/ml N. crispa extract.

(400 to 1000 µg/ml) (Figure 1). E. angustifolia flower and N. crispa aerial part were successively fractionated using hexane, ethyl acetate and chloroform to basically figure out the chemical characters of active principle(s) present in E. angustifolia and N. crispa . Among the obtained fractions, the ethyl acetate and chloroform fractions of both plant showed highest inhibitory activity at 10 µg/ml concentration (minimum concentration) on three-dimensional culture of HUVEC (Figures 2 and 3). E. angustifolia flower fractions in the range of 10 to 80 µg/ml concentration had no significant effect on the proliferation of HUVECs, but at 160 µg/ml and higher, a significant inhibition has been observed in cells proliferation (Figures 4 and 5). Among the used fractions, most reduction on cell proliferation was observed in N. crispa chloroform fraction in the range of 40 to 160 µg/ml. Aqueous fractions have no anti proliferation effects on HUVEC. The E. angustifolia and N. crispa fractions could inhibit endothelial cell growth in a dose dependent manner (Figure 6 and 7) so their hexane, ethyl acetate and chloroform fractions in 80 and 160 µg/ml concentrations were significantly reduced survival cells. Furthermore, in these concentrations, inhibitory effect did not result from cytotoxic effect, as assessed by LDH cytotoxicity assays, compared with controls. Based on these criteria, many natural or synthetic chemicals were found to inhibit tumor angiogenesis (Singh and Agarwa, 2003).

controlling primary growth and development of tumors as well as secondary metastatic tumors. Various strategies have been tested to inhibit endothelial cell proliferation and their survival (Agarwa and Singh, 2004). Over the recent years, more attention has been focused on the anti-angiogenic and antineoplastic effects of non toxic compounds from natural products. Several antiangiogenic drugs are at present in different phases of clinical trials (Kerbel, 2000). The taken together E. angustifolia and N. crispa chloroform and ethyl acetate fractions at 200 and 400 µg/ml concentrations respectively, indiquant significative inhibitory effects on endotelial cell angiogenesis. Among the obtained fractions, the ethyl acetate and chloroform fractions of both plant showed highest inhibitory activity at 10 µg/ml concentration on three-dimensional culture of HUVEC. Fractions of E. angustifolia and N. crispa in these concentrations have not attribute toxicity and inhibition of human umbilica endothelial cell on endolelial cell, angiogenesis may contain major active antiangiogenic compound(s), as flavonoid responsible for anti-angiogenic properties of E. angustifolia and N. crispa. However, based on these findings, further investigations are required to evaluate the in vivo antiangiogenic potential of EA and NC, especially in tumors for its possible usefulness in the prevention of growth and metastasis of tumors.

Conclusions DISCUSSION Tumorigenesis is a multi-step process where angiogenesis plays an important role in growth, progression and metastasis of all solid tumors. Therefore, the agents that inhibit angiogenesis could be effective in

In conclusion, the present study demonstrated that E. angustifolia flower and N. crispa aerial part extracts at 200 and 400 µg/ml concentrations respectively could inhibit angiogenesis in HUVEC. Our results also showed that, the ethyl acetate and chloroform fractions of both

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Figure 2. Effect of E. angustifolia fractions on angiogenesis inhibition of HUVEC. A: Hexane on 20 µg/ml, B: Ethyl acetate on10 µg/ml, C: Chloroform on 10 µg/ml and D: Aqueous on 40 µg/m.

Figure 3. Effect of N. crispa fractions on angiogenesis inhibition of HUVEC. A: Hexane fraction on 40 µg/ml, B: Ethyl acetate fraction on10 µg/ml, C: chloroform on 10 µg/ml) and D: Aqueous fraction on 160 µg/ml.

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Cells proliferation (%)

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Fractions concentrations (µg/ml) Figure 4. Effect of different concentration of E. angustifolia fractions on human umbilica endothelial cells proliferation.

Figure 4 . Effect of different concentration of E. angustifolia fractions on human umbilica

Cells proliferation (%)

endothelial cells Proliferation.

Fractions concentrations (µg/ml) Figure 5. Effect of Nepeta crispa fractions on HUVEC proliferation.

E. angustifolia and N. crispa at 10 µg/ml concentration contains strong anti-angiogenic activity in vitro condition. It has been suggested that the use of quantitative angiogenesis assay in clinical trials may be helpful in the early detection of the disease and monitoring the efficacy

of the agents under test (Bostwick and Iczkowski, 1998). These findings provide additional pharmacological information of the therapeutic efficacy of E. angustifolia and N. crispa, and it would be considered as a novel starting point for the development of a new anti-

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Toxicity (%)

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Fractions concentrations (µg/ml)

Toxicity (%)

Figure 6. Toxicity effect of N. crispa fractions on HUVEC.

Fractions concentrations (µg/ml) Figure 7. Toxicity effect of E. angustifolia fractions in high concentration in HUVEC.

angiogenic drugs. REFERENCES Ahmadiani A, Hosseiny J, Semnanian S, Javan M, Saeedi F, Kamalinejad M, Saremi S (2000). Antinociceptive and antiinflammatory effects of Elaeagnus angustifolia fruit extract. J. Ethnopharmacol. 72:287-292.

Agarwa Ch, Singh RP (2004). Anti-angiogenic efficacy of grape seed extract in endothelial cells. Oncol. Rep. 11:681-685. Auerbach R, Lewis R, Shinners B, Ubai L, Akhtar N (2003). Angiogenesis assays. A critical overview. Clin. Chem. 49(1):32-40. Bostwick DG, Iczkowski KA (1998). Microvessel density in prostate cancer: prognostic and therapeutic utility. Semin. Urol. Oncol. 16:118123. Bucur L, Stanciu G, Istudor V (2007). The GC-MS Analysis of Elaeagnus Angustifolia L. Flowers. Essential Oil Rev. Chim. 58(11): 1027-1029.

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Creamer D, Sullivan D, Bicknell R (2002). Angiogenesis in psoriasis. Angiogenesis 5:231-236. Decker T, Lohmann-Matthes ML (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Method. 115:61–69. Hanahan D, Folkman J (1996). Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353–364. Hynu-Joo J, Hye-Jin J (2006). Anti-angiogenic activity of the methanol extract and its fractions of Ulmus davidiana var. japonica. J. Ethnopharmacol. 112(2):406-409. Keshavarz M, Mostafaie A , Mansouri K (2010). In vitro and ex vivo antiangiogenic activity of Salvia officinalis. Phytother. Res. 24(10):15261531. Kerbel RS (2000). Tumor angiogenesis: past, present and the near future. Carcinogenesis 21:505-515. Mozaffarian V (1996). A Dictionary of Iranian Plant, Names. Farhang Moaser, Tehran, Iran.

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Sefidkon F, Jamzad Z (2006). Chemical composition of the essential oil of five Iranian Nepeta species (N. crispa, N. mahanensis, N. ispahanica, N. eremophila and N. rivularis. Flavour Fragr. J. l:764767. Shakiba Y, Mansouri K, Mostafaie A (2007). Anti-angiogenic effect of soybean kunitz trypsin inhibitor on human umbilical vein endothelial cells. Fitotherapia, 78(7-8):587-589. Singh RP, Agarwal R (2003). Tumor angiogenesis: a potential target in cancer control by phytochemicals. Curr. Cancer Drug Targets 3:205217. Sonboli A, Salehi P (2004). Antimicrobial Activity and Chemical Composition of the Essential Oil of Nepeta crispa Willd. from Iran. Z. Naturforsch, 59:653-656.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4640-4646, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1651 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Glycyrrhizin and isoliquiritigenin production by hairy root culture of Glycyrrhiza glabra Zahra Shirazi1, Khosro Piri1*, Asghar Mirzaie Asl1 and Tahereh Hasanloo2 1

Department of Biotechnology, Bu Ali Sina University, Hamedan, Iran. Department of Molecular Physiology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran.

2

Accepted 12 March, 2012

Glycyrrhizin and isoliquiritigenin production by hairy root culture of Licorice (Glycyrrhiza glabra) was investigated using Agrobacterium rhizogenes strain AR15834. Hairy roots were induced by inoculation of the leaf and stem explants with A. rhizogenes. Presence of the rolB gene in the hairy root lines was performed by polymerase chain reaction (PCR) and using rolB gene specific primers. The amount of glycyrrhizin and isoliquiritigenin in roots was measured by high-performance liquid chromatography (HPLC). The highest transformation frequency was obtained (50%) in leaf explants. The obtained results on hairy roots of G. glabra showed two distinguishable phenotypes, typical hairy roots and callus-like roots. In contrast to the previous studies, hairy root cultures of G. glabra synthesized glycyrrhizin in this research. In all of the hairy root lines, biomass and glycyrrhizin production was more than normal roots. The highest amount of glycyrrhizin and isoliquiritigenin was produced in callus-like roots. Key words: Agrobacterium rhizogenes, Glycyrrhiza glabra, glycyrrhizin, hairy root, isoliquiritigenin.

INTRODUCTION Licorice roots and stolons are commercially desired parts of Glycyrrhiza glabra that contain a number of important chemical compounds (Mousa et al., 2006). Licorice, which is one of the most popular medicinal plants in the world, is widely used in many fields such as flavoring agent, medicament, and commodity (Hanrahan, 2001). The wide usage of G. glabra is due to two kinds of main constituents, the saponin and flavonoids (Nomura and Fukai, 1998). Glycyrrhizin is the most sweet-tasting triterpene saponin in roots and stolons of Glycyrrhiza plant, and its sweetness is measured about 200 times as much as that of the sucrose, and is a conjugate of two molecules of glucuronic acid and glycyrrhetinic acid, and oleanane-type triterpene (Hayashi, 2009). Various pharmacological activities of glycyrrhizin, including antiinflammatory, immunomodulatory, antiulcer, and antiallergy activities has been reported. It has also antiviral agent, various DNA and RNA viruses including HIV and severe acute respiratory syndrome (SARS)-

*Corresponding author. E-mail: [email protected]. 009801988130783. Fax: 0098811 4224012.

Tel:

associated with coronavirus (Ito et al., 1987; Baba et al., 1988; Cintal et al., 2003). Isoliquiritigenin is a simple chalcone-type flavonoid that has been evaluated in the terms of its antioxidative, anti-inflammatory, antispasmodic, and estrogenic properties (Han et al., 2010). Isoliquiritigenin has strong inhibitory effect on tyrosinase activity, which is known to be a key enzyme in melanin biosynthesis, involved in determining the color of mammalian skin and hair (Cao et al., 2004). Agrobacterium rhizogenes, the causative agent of hairy root syndrome, is a common soil organism capable of entering in a plant through a wound (Wordragen et al., 1992). The hairy roots are well established as experimental systems and most importantly, they have been characterized by a high growth rate and are able to synthesize root derived secondary metabolites (Giri and Narasu, 2000). This bacterium transfers a DNA segment (T-DNA) from its large root-inducing (Ri) plasmid into the genome of the infected plant (Guillon et al., 2006). Four loci involved in root formation have been identified in the T-DNA of the Ri plasmid and designated root loci (rol) A, B, C and D (Ayala-Silva et al., 2007). Callus and cell suspension cultures were established from various organs of G. glabra, but they failed to produce detectable

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amounts of glycyrrhizin (Hayashi et al., 1988). The hairy root system is stable and high productive under hormone free culture condition (Hu and Du, 2006). The greatest advantage of hairy root is the same or greater biosynthesis capacity for secondary metabolite production compared to their mother plants (Kim et al., 2002).

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ATGGATCCCAAATTGCTATTCCCCACGA-3' and reverse primer 5'-TTAGGCTTCTTTCATTCGGTTTACTGCAGC-3' were used according to the DNA sequence of the rolB gene described by Rahnama et al. (2008). PCR was carried out by amplification under following conditions: Denaturation at 94°C for 1 min, primer annealing at 58°C for 1 min, and extension at 72°C for 1 min for 37 cycles. The amplified product was observed by 1/2% agarose gel.

Preparation of hairy root extracts MATERIALS AND METHODS

A. rhizogenes strain AR15834 (a gift from Dr. T. Hasanloo, Agricultural Biotechnology Research Institute Karaj, Iran). Prior to inoculation, mono clone of AR15834 was grown for 24 h in liquid Luria-Bertani (LB) medium with rifampicin 0.50 g/L antibiotic (Sigma Chemical Co.) at 28°C on a shaker at 110 rpm.

The growth of G. glabra was slow. In some studies, the Glycyrrhizin production and growth rate of G. glabra have been measured in about two month ages culturing (Shabani et al., 2009). In this research, two-month hairy roots were harvested from the liquid medium and washed twice using doubled distilled water, then were blotted filter paper to remove excess water. Dry weight was measured by drying the fresh hairy roots in room temperature for 48 h. Statistical significance for dry weight (three replications for each line) was calculated using the Duncan test for unpaired data (α≤0.01) and the analysis of variance (ANOVA) method was used for comparisons of the means. To measure, Glycyrrhizin and Isoliquiritigenin, 40 mg powdered dry hairy root was extracted with 1 ml 80% (v/v) methanol at 60°C for 6 h. Extractions were centrifuged at 4000 rpm for 15 min at room temperature. The supernatant was transferred to a new tube (Hayashi et al., 1998), and then was evaporated to dryness by blowing nitrogen. The residue extracts were used for high-performance liquid chromatography (HPLC). The amount of Glycyrrhizin and Isoliquiritigenin were calculated by the average of two experimental replications for each line.

Induction of hairy root cultures

High-performance liquid chromatography (HPLC) analysis

Leaf and stem explants obtained from different age (2, 3, 4 and 8 week older) in vitro grown plants were used for inoculation with A. rhizogenes. The explants were pricked with sterile scalpel and then immersed in an overnight culture of bacteria suspension (Optical density at 600 nm OD600=0.6) for 20 min and then dried on a sterile filter paper. Explants were pricked with sterile scalpel dipped in sterile distilled water served as control, and incubated in the same medium. To eliminate bacteria, the explants were transferred on fresh MS medium supplemented with 0.5 g/L cefotaxime antibiotic (Sigma Chemical Co.) after 48 h. This activity was repeated 4 to 5 times and during sub-culturing the antibiotic level gradually reduced. Determination of transformation frequency response of the leaf and stem explants to bacterial infection in terms of the hairy root emergence was observed. The transformation frequency was determined as follows.

Standard of glycyrrhizin (glycyrrhizic acid ammonium salt) was purchased from Fluka and isoliqiritigenin from Indofine company. Before HPLC analysis, each sample residual extract was dissolved in water and methanol for glycyrrhizin and isoliquiritigenin receptivity and filtered through 0.4 µm filter. A 20 µl aliquot of each sample extract was analyzed by HPLC at 25°C. The HPLC system consisted of water HPLC 510 pump, a waters 2478 detector. The separation for glycyrrhizin was performed according to the method reported by Hurst et al. (1983) with an isocratic elution using methanol-water-acetic acid (60: 34:6) at a flow rate of 1 ml/min with UV absorption detection at 254 nm, RP column (3.9×150 mm). The isoliquiritigenin separation was performed according to the Liu et al. (2005) method. The mobile phase consisted of the solvents: acetonitrile: acetic acid (1%) (45:55) at a flow rate of 1 ml/min with UV absorption detection at 350 nm, RP column (4.6×250 mm).

Plant material Seeds of G. glabra were provided by Pakan- Bazer Seed production Company (Isfahan, Iran). The seeds were disinfected with H2SO4 (98%) for 40 min and washed with sterile water. Then, they were cultured on semi solid hormone-free MS (Murashige and Skoog, 1962) medium and incubated at 25±2°C for a photoperiod of 16 h light.

Bacterial strain

RESULTS Hairy root lines were cultured by the transfer of 3 to 4 cm long root pieces to hormone-free Ms liquid medium with 3% sucrose at 25±2°C on a rotary shaker (110 rpm) in 16 h photoperiod and subcultured every 1 week.

DNA isolation and polymerase chain reaction (PCR) analysis Total DNA was isolated from the original hairy root clones derived from the strain AR15834 and from untransformed roots (negative control) by the cetyl trimethylammonium bromide (CTAB) method (Cai et al., 1997). Plasmid DNA from AR15834 was isolated by Sambrook et al. (1989) method used as a positive control. Isolated DNA was analyzed by polymerase chain reaction (PCR) for the presence of the rolB gene in the T-DNA. Forward primer 5'-

Establishment of hairy root cultures Hairy root formation was observed directly 3 to 4 weeks after inoculation on the wounded sites of the leaf and stem explants of G. glabra without callus formation (Figure 1). Young leaf and the stem explants showed higher transformation frequency which decreased with an increase in explants age. Wounded leaves of G. glabra were more susceptible to inoculation with A. rhizogenes. The highest transformation frequency (50%) was observed in the leaf disc explants at the 3 weeks age (Table 1). These roots exhibited characteristics typical of

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Table 1. Influence of the source (explant) and age (time) of explants in frequency of the transformation.

Time 2 week 3 week 4 week 8 week

Transformation frequency (%) Leaf (Explant) Stem (Explant) 43 21 50 33 28 16 19 9

Figure 1. Hairy roots were emerged from wounded sites of the leaf explants of G. glabra via A. rhizogenes.

Figure 2. PCR analysis of hairy roots was performed using primers of the rooting locus gene TL-rolB. 1: Molecular weight marker, 2 and 9 Negative control (normal roots), 7: positive control (Plasmid DNA from AR15834), 3-6 and 8, 10, 11 hairy roots lines.

transformed roots that had rapid growth, extensive lateral branch and a lack of geotropism (Chang et al., 2005). DNA of hairy root was extracted and analyzed for the presence of rolB gene. DNA was Isolated from the roots of normal G. glabra seedling and used as negative control. After PCR analysis, the products were subjected

to electrophoresis in 1.2% agarose gel and stained in ethidium bromide. PCR amplified a 780 base pair (bp) fragment and the presence of rolB gene in hairy roots DNA was confirmed (Figure 2). The obtained hairy roots showed two morphological characters: callus-like roots (CR) and typical hairy roots (HR) (Figure 3).

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Figure 3. Callus-like roots (CR) and typical hairy roots (HR) in G. glabra hairy root lines.

Figure 4. The normal (right) and transformed roots (left) in MS liquid medium.

Table 2. Dry weight (D.W( produced glycyrrhizin and isoliquiritigenin contents )µg/gDW( by non-hairy root and hairy root lines of G. glabra after 2 month sub-culturing in MS liquid medium.

DW )mg(

Line

b

A (HR) B (CR) C (CR) D (HR) E (HR) Non-HR

473.43 c 357 a 602.73 e 252.66 d 304.36 f 111.13

Isoliquiritigenin )µg/g)

Glycyrrhizin )µg/g(

51.7 52.2 157.5 107 39.5 54

43.75 259.8 57.42 68.45 60.65 25.62

Data of three replications per line followed by the different letters in the DW column, are significantly different (α≤0.01) using the Duncan test. Amount of glycyrrhizin and isoliquiritigenin (with HPLC) were average of two replications for each line.

Biomass and production isoliquiritigenin in the hairy roots

of

glycyrrhizin,

Measuring dried biomass of the normal and transformed

roots after 2 months of sub-culturing under the same condition showed that the transformed samples produced more biomass in comparison with the normal samples (Figure 4 and Table 2). The C line that was a sample of

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Figure 5. The HPLC chromatograms of the glycyrrhizic acid ammonium salt standard (a) and one of the hairy root line extraction of G. glabra (b) from only one of experiments.

callus-like roots, had faster growth capacity (high biomass). The result of HPLC analysis showed different glycyrrhizin and isoliquiritigenin production in hairy root lines (Table 2). In this case, glycyrrhizin also accumulated in the hairy root cultures (Figure 5) and the glycyrrhizin content was higher in all the hairy root lines than normal root cultures. The highest amount of glycyrrhizin and isoliquiritigenin was produced in B and C lines respectively that both of them were in callus-like roots.

DISCUSSION Establishment of hairy root cultures A number of previous studies showed that, type and age of explants had a great influence on hairy root induction, since the age of explants is a major factor that alters the physiological properties of the cell (Dupre et al., 2000) and the juvenile explant is optimal for hairy roots induction (Hu and Du, 2006). Mehrotra et al. (2008) reported the

same results of maximum transformation frequency in the leaf explants of G. glabra at the 3 weeks age. The callus-like phenotype has been observed in transformed root lines of other plant species, including Datura metel, Dubosia hybrid, Nicotiana tabacum (Martin-Tanguy et al., 1990; Van Larebeke et al., 1974) and Withania sominifera (Bandyopadhyay et al., 2007; Mirjalili et al., 2009). In the agropine-type A. rhizogenes as AR15834, two sets of pRi genes are involved in the root induction process: aux and ages genes

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located in the TR region and the rol (root loci) gene of the TL region. The ages and aux genes responsible for providing transformed cells with an additional source of auxin (Chiriqui et al., 1996; Morris and Genes, 1986). Auxin excess could induce disorganization of hairy roots (Robins et al., 1991; Moyano et al., 2007), Several loci on the TL-DNA of Ri plasmids have been shown to be essential for hairy root induction but the TR-DNA are not essential for production of hairy roots (Veena and Taylor, 2007). Thus, probably produced callus–like roots morphology was due to the presence of TR-DNA in genome of some hairy root lines.

Biomass and production isoliquiritigenin in the hairy roots

of

glycyrrhizin,

Normally, root cultures need an exogenous phytohormone supply and grow very slowly, resulting in the poor or negligible synthesis of secondary metabolites (Hu and Du, 2006). Owing to the site of T-DNA integration into the host plant genome, the hairy roots derived often show different accumulation pattern of secondary metabolites (Mano et al., 1986). In spite of the fact that, Hayashi et al. (1988, 2003) reported that there were no detectable glycyrrhizin in control of cell suspension cultures of G. glabra, and MeJa-treated cultured cells by HPLC analysis and also the obtained results by Toivonen and Rosequist (1995), Li et al. (2000), Kovalenko et al. (2003), showed no detectable glycyrrhizin in hairy roots of G. glabra. In our study, the hairy root cultures of G. glabra with the ability of producing glycyrrhizin can be a promising source for continuous and standardized production of glycyrrhizin and isoliquiritigenin under controlled conditions and hormone free medium. It is also necessary to evaluate and screen the effects of various elicitors with different mechanisms on the production of glycyrrhizin and isoliquiritigenin. Elicitors have been considered as effective strategies to enhance the production of secondary metabolites. Conclusions In the present study, the establishment of hairy root cultures of G. glabra realized leading to the production of bioactive compound as glycyyrhizin and isoliquiritigenin. The transformed roots with A. rhizogenes have an altered phenotype such as lateral growth, lack of geotropism and fast growth in the culture. This system provides an alternative way to produce the pharmaceutical glycyyrhizin and isoliquiritigenin, and makes it possible for metabolic engineering of these metabolites in G. glabra. The most importantly point in this research was an increased ability to synthesize useful metabolites that could not be produced by unorganized cells even higher than plant roots. These roots can also synthesize more

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than a single metabolite and therefore prove economical for commercial production. REFERENCES Ayala-Silva T, Bey CA, Dortch G (2007). Agrobacterium rhizogenes mediated transformation of Asimina triloba L. Cuttings. Pak. J. Biol. Sci. 10:132-136. Baba M, De Cleroq S, Nakashima H, Yamamoto N (1988). Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV). Antiviral Res. 10:289-298. Bandyopadhyay M, Jha S, Tepfer D (2007). Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera. Plant Cell Rep. 26:599–609. Cai D, Kleine M, Kifle S, Horloff HJ, Sandal NN, Marcker KA, Lankhorst RMK, Salentijn EMJ, Lange W, Stiekema WJ, Wyss V, Grundler FMW, Jung C (1997). Positional cloning of a gene for nematode resistance in sugarbeet. Science 275:832-834. Cao Y, Wang Y, Ji C, Ye J (2004). Determination of liquiritigenin and isoliquiritigenin in Glycyrrhiza uralensis and its medicinal preparations by capillary electrophoresis with electrochemical detection. J. Chromatogr. A. 1042:203–209. Chang CK, Chang KS, Lin YC, Liu SY, Chen CY (2005). Hairy root cultures of Gynostemma pentaphyllum (Thunb) Makino: a promising approach for the production of gypenosides as an alternative of ginseng saponins. Biotechnol. Lett. 27:1165–1169. Chiriqui D, Guivarch A , Dewitte W, Prinsen E, Onkelen H (1996). Rol genes and root initiation and development. Plant Soil 187:47–55. Cintal J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr HW (2003). Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 361:2045-2046. Dupre P, Lacoux J, Neutelings G, Mattar-Laurain D, Fliniaux MA, David A, Jacquin-Dubreuil A (2000). Genetic transformation of Ginkgo biloba by A. tumefaciens. Physiol. Plant 108(4):413-419. Giri A, Narasu LM (2000). Transgenic hairy roots: Recent trends and applications. Biotechnol. Adv. 18(1):1-22. Guillon S, Tremouillaux-Guillen J, Pati PK, Rideau M, Gantet P (2006). Harnessing the potential of hairy roots. Trends Biotechnol. 24:403409. Han B, Zheng Q, Wang J, Chen W, Tang H, Wang Q, Wang X, Li J (2010). Isoliquiritinenin extractied from licorice Glycyrrhiza uralensis roots by A facile conversion technique. Chem. Nat. Compd. p. 46. Hanrahan C (2001). Gale encyclopedia of alternative medicine, licorice (book on CD-ROM). Farmington Hills, MI: Thomson Gale. Hayashi H (2009). Molecular Biology of Secondary Metabolism: Case Study for Glycyrrhiza Plants. Recent Adv. Plant Biotechnol. 1:89-103. Hayashi H, Fukui H, Tabata M (1988). Examination of triterpenoids produced by callus and cell suspension culutres of Glycyrrhiza glabra. Plant. Cell. Rep. 7:508-511. Hayashi H, Hiraoka N, Ikeshiro Y, Yamamoto H, Yoshikawa T (1998). Seasonal variation of glycyrrhizin and isoliquiritigenin in the root of Glycyrrhiza glabra. Biol. Pharm. Bull. 21(9):987-989. Hayashi H, Hung P, Inoue K (2003). Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell. Physiol. 44:404-411. Hu ZB, Du M (2006). Hairy root and its application in plant genetic engineeering. J. Integr. Plant. Biol. 48(2):121-127. Hurst WJ, Mckim JM, Martin RA (1983). High-performance liquid chromatographic determination of glycrrhizin in licorice products. J. Agric. Food Chem. 31:387-389. Ito M, Nakashima H, Baba M, Pauwels R, De Clercq E, Shigeta S, Yamamoto N (1987). Inhibitory effect of glycyrrhizin on the in vitro infectivily and cytopathic activity of the human immunodeficiency virus (HIV). Antiviral. Res. 7:127-137. Kim YJ, Wyslouzil BE, Weathers PJ (2002). Secondary metabolism of hairy root cultures in bioreactors. In vitro Cell. Dev. Biol. Plant 38:110. Kovalenko PG, Antonjuk VP, Maliuta SS (2003). Secondary metabolites production from transformation cells of Glycyrrhiza glabra and potentilla alba as productions of radioprotective compounds. Uk.

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Bioorgan. Acta, 1:21-32. Li W, Asad Y, Yoshikawa T (2000). Flavonoid constituents from Glycyrrhiza glabra hairy root cultures. Phytochemistry 55:447-456. Liu XR, Li L, Wang Q, Wang W, Bi SK, Guo DA (2005). Simultaneous determination of nine flavonoids in dalbergia odorifera by LC. Chromatographia 61:409–413. Mano H, Nabeshima S, Matsui C, Ohkawa H (1986). Production of Tropane Alkaloids by Hairy Root Cultures of Scopolia japonica. Agric. Biol. Chem. 50:2715-2722. Martin-Tanguy J, Tepfer D, Paynot M, Burtin D, Heisler L, Martin C (1990). Inverse relationship between polyamine levels and the degree of phenotypic alteration induced by the root- inducing, lefthand transferred DNA from Agrobacterium rhizogenes. Plant. Physiol. 92:912–918. Mehrotra S, Kukreja AK, Khanuja SPS, Mishra BN (2008). Genetictransformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor. Electron. J. Biotechnol. 11(2):1-7. Mirjalili HM, Fakhrtabatabaei SM, Bonfill M, Alizadeh H, Gusido RM, Ghassempour A, Palazon J (2009). Morphology and withanolide production of Withania coagulans hairy root cultures. Eng. Life Sci. 9(3):97–204. Morris R, Genes O (1986). Specifying auxin and cytokinin biosynthesis in phytopathogens. Annu. Rev. Plant. Physiol. 37:509–538. Mousa N, Siaguru P, Wiryowidagdo S, Wagih ME (2006). Rapid clonal propagation of Licorice (Glycyrrhiza Glabra) by in vitro. Sugar Technol. 8(4):292-298. Moyano E, Palazon J, Bonfill M, Osuna L, Cusido RM, OksmanCaldentey KM , Pinol MT (2007). Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures. J. Plant. Physiol. 164:521–524. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15:473-497.

Nomura T, Fukai T (1998). Phenolic constituents of licorice (Glycyrrhiza species). In: Herz W, Kirby GW, Moore RE, Steglich W, Tamm C. (Eds). Fortschritte der Chemie Organischer Naturstoffe.SpringerVerlag, New York. 73:1-40. Rahnama H, Hasanloo T, Shams MR, Sepehrifar R (2008). Silymarin production in hairy root culture of Silybum marianum (L.) Gaertn. Iran. J. Biotechnol. 6:113-118. Robins RJ, Bent FG, Rhodes MJC (1991). Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures. Part 3: the relationship between morphological integrity and alkaloid biosynthesis. Planta, 185:385–390. Sambrook J, Fritrsch EF, Maniatis T (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Laboratory Press. Cold Spring. Harbor. NY. Shabani L, Ehsanpour AA, Asgari G, Emami J (2009). Glycyrrhizin production by in vitro cultured Glycyrrhiza glabra elicited by methyl Jasmonate and salicylic acid. Russ. J. Plant. Physiol. 56(5):621–626. Toivonen L, Rosequist H (1995). Establishment and growth characteristics of Glycyrrhiza glabra hairy root cultures. Plant. cell. Tissue. Organ. Cult. 11:243-258. Van Larebeke N, Engler G, Holsters M, Van den Esacker S, Schilperoot RA, Schell J (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature 252:169–170. Veena V, Taylor CG (2007). Agrobacterium rhizogenes: recent developments and promising applications. In vitro Cellular and Development. Biol. Plant 43:383–403. Wordragen MF, Ouwerkerk PBF, Dons HJM (1992). A. rhizogenes mediated induction of apparently untransformed roots and callus in Crysanthemum. Plant. Cell. Tissue. Organ Cult. 30:149-157.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4647-4652, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11. 1724 ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Effects of irrigation and nitrogen (N) fertilization levels on yield, morphological traits and water use efficiency of chicory (Cichorium intybus L.) Seyyed Gholam Reza Moosavi Islamic Azad University, Birjand Branch, Birjand, Iran. E-mail: [email protected]. Tel: 00989155615768. Fax: 00985614342171. Accepted 16 January, 2012

In order to evaluate the different irrigation and nitrogen levels on yield, morphological traits and water usage efficiency of Cichorium intybus L., an experiment was conducted in the Agricultural Research Station of Islamic Azad University, Birjand Branch, in 2009. The experimental design was split plots based on randomized complete block design in three replications. The main plots were three irrigation levels (irrigation after 60, 120 and 180 mm evaporation from pan class A), and the sub-plots were four nitrogen rates (0, 60, 120, and 180 kg N/ha). The results showed that irrigation after 180 mm evaporation from pan treatment in comparison with irrigation after 60 mm evaporation from pan treatment decreased number of leaves per plant, root diameter, the number of root branches, leaf, root, and biological dry yields by 51.1, 28.2, 46.3, 58.5, 57, and 58.3%, respectively, while root: leaf dry weight ratio and water use efficiency (WUE) for root production were 8.6 and 30.4% higher under severe water deficit stress than under moderate stress and no-stress treatments, respectively. Also, as nitrogen (N) -1 rate was increased from 0 to 180 kg.ha , the number of leaves per plant, root diameter and the number of root branches, leaf, root and total dry yields were increased by 77.1, 25, 50, 125.8, 69.1, and 118.9%, respectively, while root: leaf dry weight ratio was decreased by 25.7%. Also, increasing the applied nitrogen rate caused an improvement in leaf, root, and total dry matter WUE. Overall, the results showed that irrigation after 60 mm accumulative evaporation from evaporation pan and the application -1 of 180 kg N.ha can be recommended for the cultivation of chicory in Birjand, Iran, because of its higher economical yield in spite of the resulting partial loss of WUE. Key words: Cichorium intybus, water stress, nitrogen, yield, water use efficiency (WUE), morphology.

INTRODUCTION Chicory (Cichorium intybus L.) belongs to the family of Asteraceae. It is a short-living, long-day biennial herbaceous plant and native of Europe, North Africa and hot and temperate regions of Asia with broad leaves, and a life cycle of 2 to 5 years. At the first year, chicory only complements its vegetative growth and then starts its flowering and completes its life cycle during the second year (Moore et al., 2006; Nandagopal and Ranjithakumari, 2006). The roots of chicory cleanse the alimentary tract and blood. Its leaves contain potash whose tea is used in purifying the blood, belly and organs. All parts of chicory, particularly its roots and leaves, are gall stimulant. Additionally, it is useful in the treatment of gastroenteritis, kidney disorders and

hepatitis, and it is easily digested (Nandagopal and Ranjithakumari, 2006). Water is an important environmental factor affecting the growth of the crops particularly in arid and semi-arid regions like Iran; so, its optimum use for the production of the crops is vital (Mirzaei et al., 2005). On the other hand, the increase in the costs of water supply and the decrease in the amount of available water has brought the application of water deficit stress into focus in these regions (Winter, 1990). Low irrigation, during which water deficit stress is applied either at a certain growth stage or during the whole growing season, is a technique for maximizing water usage efficiency (WUE), and increasing the yield per unit of applied water (Kirda, 2002). In

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addition, the availability of nutrients like nitrogen (N) plays a key role in the growth of the crops, which can in turn be influenced by the availability of water to the roots. Water deficit stress which often occurs in summer can greatly affect the yield of chicory (Danuso, 2001; Schittenhelm, 1999). In a study on the application of irrigation after 50, 100 and 150 mm evaporation from evaporation pan, Taheri et al. (2009) showed that the impact of water deficit stress was significant on the number of leaves, leaf dry weight and root length of chicory, but it did not significantly affect root diameter. Moreover, water deficit stress significantly declined biological yield of chicory. They obtained the highest number of leaves, root diameter and length, biological yield and leaf dry weight from the treatment of irrigation after 50 mm evaporation. Aliabadi et al. (2008) studied the effect of two irrigation levels on coriander (Coriandrum sativum L.), including the irrigation after 30 and 60 mm evaporation from pan and concluded that low irrigation increased WUE and the highest WUE (on the -3 average, 0.45 kg.m ) was obtained under water deficit stress. Also, Asad et al (2000) reported 96.6% increase in WUE for sugar beet root production with the increase in irrigation interval from 7 to 14 days, though root yield decreased by 8%. Furthermore, in a study on the effects of P and water deficit stress on coriander, Sani and Aliabadi (2010) concluded that irrigation after 60 mm accumulative evaporation significantly decreased root yield by 43.2% as compared with irrigation after 30 mm accumulative evaporation treatment. Johnson (1995) applied water deficit stress to Spanish thyme and observed that the plant dry and fresh weights were decreased as the stress was increased. In a study on the effect of two N levels on growth and partitioning of assimilates in Veronica herbaceae, Cuzzuol et al. (2005) reported that with the -1 increase in N application rate from 1.3 to 10.7 mmol.L , the number of leaves and leaf area were increased 4.3 and 2.8 times, respectively. Also, in a study on different NPK rates, Custic et al. (2003) recommended the -1 application of 50 to 75 kg N.ha for the production of chicory. The increase in root: shoot ratio of chicory under N deficiency conditions has been reported, too (Ameziane et al., 1997; Schittenhelm, 1999). Moreover, Jelaini et al. (2008) found that although WUE of sugar beet was improved with the increase in N application, but statistically significant difference was not observed in WUE among N fertilization rates. The current study was carried out in order to examine the effect of irrigation and N fertilization levels on morphological traits, yield and WUE of chicory in Birjand, Iran. MATERIALS AND METHODS This study was carried out in the Research Station of Islamic Azad University, Birjand Branch, Iran (Longitude 59°13′ E., Latitude 32°52′ N, altitude 1400 m above sea level) in 2009. The soil was loam-sandy with pH of 8.1, EC of 4.49 µmho.cm-1, organic carbon

content of 0.32% and total nitrogen content of 0.018% at the depth of 0 to 30 cm. The average long-time minimum and maximum temperature is 4.6 and 27.5°C, with average annual precipitation of 169 mm, and average minimum and maximum relative humidity of 23.5 and 59.6%, respectively. The regional climate is hot and arid. Experimental design was split plots based on randomized complete block design in three replications. The main plots were three irrigation levels (irrigation after 60, 120 and 180 mm evaporation from pan class A) and the sub-plots were four nitrogen rates (0, 60, 120 and 180 kg N/ha). Moreover, the field had been left fallow in the previous year. It was prepared in early-April by plowing and twice vertical disking. Given the soil analysis, 150 kg.ha-1 super phosphates triple was added to the soil before final disk. The seeds were sown at the depth of about 2 cm on May 17 in furrows. They had been disinfected with the fungicide, carboxin thiram (2:1000). Each experimental plot included six 6 m long sowing rows with inter-row spacing of 50 cm. A 2 m space was left between adjacent plots as well as between replication to prevent the water exchange between them. The plots were irrigated by pressurized system using hose and contour. Nitrogen fertilizer from urea source was applied at two phases (half after thinning and another half at mid-growing period). In order to measure morphological traits including the number of leaves per plant, root length, root diameter and the number of root branches, 10 plants were randomly selected from each plot and these traits were measured. For the determination of root and leaf dry yield and total dry matter, the plants of two middle rows with an area of 2 m 2 were harvested. Their leaves and roots were separated and oven-dried at 65°C for 48 and 72 h, respectively and then weighed by 0.1precision digital scale. Root, leaf and biomass WUEs were obtained by dividing their yields by the amount of applied water. Finally, the data were analyzed by statistical software MSTAT-C and the means were compared by Duncan’s multiple range test at 5% level.

RESULTS Morphological traits and root: leaf dry weight ratio The results of analysis of variance for the effect of irrigation and N levels on morphological traits and root: leaf dry weight ratio indicated that irrigation levels affected the number of leaves per plant at 1% level and root length, root diameter, the number of root branches and root: leaf ratio at 5% level. Also, N fertilization rate significantly impacted on all these traits, except root length at 1% level. In addition, the interaction between irrigation and N was significant on the number of leaves per plant at 5% level, but it did not affect other traits (Table 1). As shown by means comparison, severe water deficit stress resulted in significantly lower number of leaves per plant, root diameter and the number of root branches, so that they were decreased by 51.1, 28.2 and 46.3%, respectively as compared with no-stress treatment, but root: leaf dry weight ratio was 15.6 and 8.6% higher under severe water deficit stress than under moderate stress and nostress treatments, respectively (Table 2). Also, as N -1 fertilization rate was increased from 0 to 180 kg.ha , the number of leaves per plant, root diameter and the number of root branches were increased by 77.1, 25 and 50%, respectively; while, root: leaf dry weight ratio was

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Table 1. Mean square of morphological traits and root: leaf dry weight ratio of chicory as affected by irrigation and nitrogen levels.

Sources of variation Replication irrigation (A) Error a Nitrogen rate (B) A×B Error b C.V. (%)

Number of leaves per plant ns 2591.42 24508.38** 553/7 9933.83** 1337.6* 435.3 15.52

df 2 2 4 3 6 18 -

Root length ns 13.89 47.51* 6.92 ns 1.28 ns 3.72 3.38 11.93

Number of root branches ns 1.57 145.724* 9.96 43.83** ns 9.01 8.202 25.6

Root diameter ns

16.88 52.25* 3.39 14.84** ns 1.86 2.13 11.45

Root: leaf dry weight ratio ns

0.0001 0.001* 0.0001 0.002** ns 0.001 0.0001 14.88

ns, Non Significant at 0.05 probability level; * and ** significant at 0.05 and 0.01 probability levels, respectively.

Table 2. Effect of irrigation levels on morphological traits and root: leaf dry weight ratio of chicory.

Irrigation after evaporation from pan (mm)

Number of leaves per plant a

60 120 180

174.06 b 143.92 c 85.2

Root length (cm) a 17.48 ab 15.22 b 13.51

Root diameter (mm) a 14.7 a 12.99 b 10.55

Number of root branches a

13.31 a 13.04 b 7.15

Root: leaf dry weight ratio b

0.116 b 0.109 a 0.126

Means followed by the same letters in each column-according to Duncan’s multiple range test are not significantly (P