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CHAPTER 1 INTRODUCTION: 1.1 BACKGROUND OF THE STUDY Plant mucilage is found in almost all classes of plants, usually in very small amount. Mucilage and gums are water soluble polysaccharides found in a widespread number of plants and also in some microorganisms. It has different purposes ranging from water storage and seed germination in plants to membrane thickener and food reserve to adhesive, in some cases.

Okra is one of the many plants which contain mucilage. It is widely distributed in tropical regions around the world. Its mucilaginous properties are used as thickening agent for food particularly in soups. On the other hand, some people use it for medicinal purposes. The upper part of the okra pods are commonly remove or cut and thrown prior to cooking. This part of okra pod also contains mucilage which gives its slimy characteristic. Okra pods specially when heated produce more sticky mucus. This mucilage has a good potential to be an alternative adhesive in the laboratory.

Egg white is commonly used in the histopathology section in the laboratory as adhesive for fishing out tissues from the flotation water bath. However, early studies have shown that okra mucilage solution is similar to egg white at higher concentrations forming threads and stabilizing foams. Both okra mucilage and egg white have proteins that have functional properties such as gel and foam formation (Alleoni, 2006). These

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properties are very important in the effectiveness of an adhesive. Furthermore, okra mucilage has been found to have potential uses as an extender for serum albumin and as an additive to dried egg white.

A good tissue adhesive must promote tissue attachment to the glass slides when use in histological preparation. It must retain tissue transparency and may not cause any tissue shrinkage. It must not damage or distort any cellular structure which may affect the diagnosis of certain disease. Tissue adhesives in histopathology are essential for methods that require the exposure of tissue sections it must retain its adhesiveness even after exposure to acids and alkalis during staining. Mayer’s egg albumin is the most commonly used tissue adhesive because it is easy to make, convenient, and relatively inexpensive (Gregorios, 2006). Moreover, it is composed of egg white and glycerol mixed together.

Processing of adhesives depends mainly on rheological parameters and the quality of the final product is determined by the interaction between the adhesive and adherent (Azeez, 2005). Since viable tissues are involved, certain characteristics of adhesives must be met to be considered effective. The tissue adhesives must be permeable to both fixative and stain and must not retain the stain. The purpose of this study is to prove the effectiveness of okra (Abelmoschus esculentus) as substitute adhesive for egg albumin in tissue preparation. This topic is being pursued so that the readers, students and lastly people who work at the hospitals will be able to expand their knowledge about the use of adhesive in the laboratory.

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1.2 OBJECTIVES OF THE STUDY General objective: The objective of the study is to evaluate the effectiveness of the binding capacity of the mucilage obtained from the plants of Okra (Abelmoschus esculentus) as an alternative adhesive of egg albumin in histopathology section. Specific objectives: 1.

What are the properties of Okra that can be a source of a good adhesive in

the laboratory? 2.

Does okra’s pod have potential to be permeable to fixative and stain in the

histopathology procedure? 3.

Does the lady’s finger upper portion do not retain the stain after being

applied to the tissue ribbons or specimens? 4.

How can okra be new effective and cheaper mucilage like egg albumin?

5.

How the findings in this study can make recommendations on the

improvement of adhesive in the histopathology section in the laboratory of hospitals? 1.3 SIGNIFICANCE OF THE STUDY This study will be beneficial to the scientific community because it can provide new information regarding the adhesive capabilities of the Abelmoschus esculentus. This development can be used as a basis for future study or experimentation related to the topic of mucilage. Since science always want to progress, this type of study will help the people in

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the scientific community like doctors, pathologists and medical technologies to increase their awareness that a plant specifically okra or lady’s finger can be an alternative adhesive like egg albumin that is commonly use in the histopathology procedures.

To the general public, their awareness about the study of okra as adhesive will help them to use it as source of income. Since okra usually grows in warm areas and Philippines is considered as a tropical country, letting the people know about the benefits of okra will persuade them to plant. However, some people are not aware about the good properties of the okra; this study will allow them to know the importance of okra particularly as source of mucilage.

To the medical technology field, this study will allow them to have a substitute adhesive that is cheaper and has good properties like egg albumin. This experimentation will help the medical technologists identify a new source of mucilage that can be used in the histopathology procedure. This study will inform the people in the laboratory to consider that okra has qualities of a good adhesive agent. 1.4 SCOPE AND DELIMITATIONS The coverage of the study is about the effectiveness of okra’s upper portion as an alternative adhesive. The (lady’s finger) plants that will be use in this experiment are purchased only on local market and the number of okra’s that will be used in this experiment is only 20-40 pieces or enough number to let the tissue ribbons stick to the glass slide after fishing out.

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The site of the experimentation of this topic is only inside the laboratories of University of Santo Tomas. Moreover, the tissue ribbons that will be used in testing the adhesive are from the University of Santo Tomas Hospital only. Lastly, this research is about the helpfulness of okra as adhesive in the tissue ribbon in the histopathology procedure only. This study does not cover the effectiveness of the other parts of okra as adhesive like its leaves, seeds and flowers. In addition, this study is limited in using only local plants; other okra’s from other countries are not included. Our group will avoid using okras that are too much for tissue ribbons to meet the group’s budget. Doing the experimentation outside the university is not applicable in this study. Likewise, there will be no specimens that will be used in this study from other hospital. The experimentation will not cover some external factors that may affect the result of the test like temperature and contamination. 1.5 DEFINITION OF TERMS 1. adhesive – refers to the substance used for attachment of tissues to glass slides in tissue

preparation 2. effectiveness – refers to the ability of the okra mucilage to adhere tissues to glass slides

with certain criteria met 3. egg albumin – the standard tissue adhesive used in histopathology section in the

laboratory 4. extender – refers to a substance added to a product especially in the capacity of a diluent,

adulterant, or modifier

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5. fishing out- refers to the process of getting the tissue ribbon with the use of slides in a

warm water. 6. fixative- refers to the reagent or combination of reagents used to fix the tissue. 7. foam – refers to a colloid in which particles of a gas are dispersed throughout a liquid 8. gel – refers to the cross-linking among polymeric molecules which make an

intermolecular network within a liquid medium 9. germination- refers to the beginning of the spore or seed of the plant. 10. histopathology- refers to the study of microscopic anatomical changes in diseased tissue

in the laboratory. 11. permeability- refers to the ability of substance to penetrate to fixative and stain of the

specimen. 12. mucilage – refers to a complex glutinous carbohydrate secreted by certain plants. 13. specimen- refers to a small fragment of tissue for microscopic study. 14. stain- refers to a dye or mixture of dyes used to impart color to the substance during the

procedure in the laboratory. 15. tissue ribbon – refers to thin, long slices from paraffin blocks where the specimen is

embedded.

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Chapter 2 REVIEW OF RELATED LITERATURE

This chapter will show the different journals, books and articles that will provide additional information related to the study of the researches entitled the Effectiveness of Abelmoschus esculentus (Okra) Fruit Extract as Substitute for Egg Albumin in Histopathology Procedure.

Vernacular names Okra’s

scientific

names

are Abelmoschus

esculentus

and

also Hibiscus

esculentus. It is known in different parts of the world, it is commonly called as Okra, Ochro, Okoro, Quimgombo, Quingumbo, Ladies Fingers, Gombo, Kopi Arab, Kacang Bendi, Bhindi in Malaysia and other parts of South East Asia. However, in Middle East it is known as Bamia, Bamya or Bamieh and Gumbo in Southern USA. On the other hand, in Portugal and Angola, okra is known as Quiabo , and known as Quimbombo in Cuba and in Japan it is known as Okura. Lastly, it is also found in Taiwan, where it is called Qiu Kui" pronounced Cheeoh Kway. (Seed To Supper,2000)

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There are three general types of okra: tall green, dwarf green, and ladyfinger. Each of these is again divided according to length and color of the pods. Varieties in most common use are known to the seed trade as Perkins Mammoth, Long Green, Dwarf Green, and White Velvet. Clemson Spineless is of the same type as Perkins Mammoth Podded but has spineless pods and somewhat sparse foliage, making it less troublesome to harvest than other varieties.

Structure and Physiology The okra plants are fast growing, heat loving, annual or perennial species, and can grow up to 2 meters tall, usually not branched. The leaves are coarse, palmately lobed with 5 to 7 lobes, and ranges from 10 to 20 cm long. Flowers are 4 to 8 cm in diameter, cream colored and have the shape and form of a hibiscus blossom but do not splay flat. Okra pods are capsule-like that are up to 18 cm long. Most are green, some are red pigmented. Pods also mature quickly, regardless of shape or color. Okra plants are cultivated throughout the warm and temperate regions of the world for its fibrous pods containing round, white seeds. The seeds are soaked overnight prior to planting. Germination occurs between 6 days and 3 weeks. The seed pods rapidly become fibrous and woody and must be harvested within a week of the fruit being pollinated to be edible. After that, the walls of the pod quickly lignified and become inedible. It is an easy vegetable to grow in any average garden soil. Its seeds can be saved from late season pods because it is self-pollinated (Klingaman, 2005).

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Mucilage is a thick, glutinous substance, related to natural gums. It is a water soluble polysaccharide found in a widespread number of plants (Woolfe, et al). It is secreted by the seed covers of various plants, and form slippery, aqueous colloidal dispersions. In plants, it sometimes serves to check the loss of water to aid germination, to facilitate seed dispersal, and to store food. Mucilage occurs in nearly all classes of plants in various parts of the plant, usually in moderately small percentages, and is not occasionally associated with other substances. Okra mucilage has a relatively narrow range of molecular weight distribution for both protein and carbohydrate. Electrophoresis of the mucilage indicates that it is highly negatively charged specie. Neutral sugars identified with qualitative carbohydrate analysis are rhamnose, galactose and glucose. It also contains galacturonic acid. The viscosity of okra mucilage is at maximum in the neutral pH range, similar to the other acidic polysaccharides, such as gum arabic. Also, boiling the mucilage caused a marked decrease in viscosity (Woolfe, et al,). Okra mucilage is like a thick, slime. Its consistency is good for use as thickening agents in soups, and is very popular in Middle East countries. Popular dishes around the world use okra mucilage to make their food thick. For instance, a typical Ghanaian okra soup would contain approximately 0.2-0.3 % mucilage by weight. Okra seeds should be planted in areas which are constantly exposed to sunlight. Temperatures of 60 degrees during night and 85 degrees in the morning in the soil are preferred by the seeds. Sandy loam is the best soil, but clay soil suffices if it contains organic matter and formed into high, stable rows for drainage. Okra grows best at soil pH

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levels of 6.0 to 7.0 and dislikes excessively acidic soils. Okra seeds will root in the soil and will sprout in three to four weeks (Jones, B. 2008). The seeds can tolerate poor soils with heavy clay and intermittent moisture. The seed pods rapidly become fibrous and woody and must be harvested within a week of the fruit being pollinated to be edible. The seedlings of okra possess taproots that are not easily damaged. Okra is among the most heat-tolerant vegetables in the world and it can survive severe drought conditions. Okra reaches up to 6 feet in height with proper care (Opapo, L. 2009). Mucilage is defined as a sticky substance obtained from certain plants which are used as an adhesive (American Heritage Dictionary, of the English Language, 4th Ed., 2000). According to Collins English Dictionary, it is a complex glutinous carbohydrate secreted by certain plants (2003). An example of plant which secretes mucilage is the okra fruit. The physical composition of the okra mucilage is that it mainly pertains to its viscosity. According to Woolfe, M., the okra mucilage has varied viscosity depending on different factors: concentration, pH and temperature. The relationship between the concentration and the viscosity of the okra mucilage is that when it is increased, the viscosity is also increased. In terms of pH, the viscosity of the okra mucilage is at maximum in neutral pH range. It has a slightly broad maximum (pH 6-9). Lastly, having the okra mucilage in a high temperature condition will decrease the viscosity of it. Therefore, the mucilage is not stable to heat.

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The appearance of the okra mucilage is cloudy and opaque at neutral or acid pH conditions (El-Mahdy). Okra extracts has the mucilage property that is used as a pharmaceutical adjuvant and other pharmaceutical applications such as a gelling and emulsifying

agent, furthermore, it has a bulk laxative qualities which is mainly

manifested by its ability to lubricate and adhere (Kumar, 2009). However, according to BeMiller , okra mucilages are complex macromolecules composed of acidic polysaccharides, proteins and minerals. The acidic polysaccharide of it is composed of galacturonic acid, galactose, rhamnose and glucose (1.3: 1.0: 0.1: 0.1) (Whistler and Conrad). Okra mucilage has also found to have potential uses as an extender for serum albumin and as an additive to dried egg white (Kelkar, et al.). Below is a table showing the different chemical composition of crude and purified okra mucilage (percentage of wet wt.), Woolfe, M., 1977: Table 1. The Different Chemical Composition of Crude and Purified Okra Mucilage (Percent of Wet Wt.):

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Okra Mucilage Crude

Purified

Moisture content

9.35

9.37

Protein (N x 6.25)

9.42

7.31

Ash

5.95

4.81

Magnesium

0.51

0.41

Calcium

2.02

2.83

Potassium

0.68

0.11

Phosphorus

0.18

0.14

The table showed that there is a small change of protein content in a process of purification. This could indicate a fairly strong interaction of protein with the polysaccharide. Ash content is accounted by calcium, magnesium, potassium and phosphate ions. In a purification process, there is an alteration in the distribution of ions, which showed an increase in concentration.

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Origin and Distribution Okra originated somewhere around Ethiopia, and was cultivated by the ancient Egyptians by the 12th century B.C. Its cultivation spread throughout North Africa and the Middle East. The seed pods were eaten cooked, and the seeds were toasted and ground, used as a coffee substitute (Ehler, J. 2010). Okra Abelmoschus esculentus L. (Moench), it is grown in different parts of the world, especially in tropical and sub-tropical countries. This crop can be grown on large commercial farm or could be a garden crop. It is grown commercially in India, Turkey, Iran, Western Africa, Yugoslavia, Bangladesh, Afghanistan, Pakistan, Burma, Japan, Malayasia, Brazil, Ghana, Ethiopian, Cyrpus and the Southern United States. 70% of total production came from India with 3.5 tonnes. (FAOSTAT 2008). Abelmoschus esculentus is found all around the world from Mediterranean to equatorial areas as may be seen from the geographical distribution of cultivated and wild species. There are Eight Abelmoschus species occur in India. Out of these, esculentus is the only known cultivated species. Abelmoschus moschatus is cultivated for aromatic seeds and also occur as wild species, while the rest six are truly wild types. The wild species occupy diverse habitats. The species Abelmoschus ficulneus and Abelmoschus tuberculatus is spread over the semi-arid areas in northwestern India and north India; Abelmoschus crinitus and Abelmoschus manihot (tetraphyllus and pungens types) in tarai

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range and lower Himalayas; Abelmoschus. manihot (tetraphyllus types), Abelmoschus angulosus, and Abelmoschus moschatus in western and eastern ghats; and Abelmoschus crinitus and Abelmoschus manihot (mostly pungens types) in the northeastern region depicts their broad range of distribution in different regions of the country. Existence of different Abelmoschus species in different areas of India observed in a recent survey is presented in the following: Abelmoschus angulosus can be found in Tamil Nadu and Kerala while Abelmoschus cancellatus can be found in Uttaranchal, Himachal Pradesh, Uttar Pradesh and Orissa. Abelmoschus criniturs are distributed in Uttranchal, Madhya Pradesh and Orissa. Abelmoschus ficulneus is well distributed in Jammu and Kashmir, Rajasthan, Madhya Pradesh, Chhattisgarh, Maharashtra, Tamil Nadu, Andrah Pradesh and Uttar Pradesh. Abelmoschus manihot ssp. Tetraphyllus var. tetraphyllus can be found in Uttar Pradesh, Rajasthan, Madhya Pradesh, Maharashtra, Orissa and Chhattisgarh. Abelmoschus manihot ssp. Tetraphyllus var. pungens in Uttaranchal, Himachal Pradesh, Jammu and Kashmir, Assam, Adaman and Nicrobar Islands.Abelmoschus moschatus spp. moschatus inUttranchal, Orissa, Kerala, Karnataka, Adaman and Nicobar Islands. Other species of Okra, Abelmoschus moschatus spp. tuberosus can be found in Kerala and parts of Western Ghats in Tamil Nadu. Last Specie is found in Uttar Pradesh, Rajasthan, Madhya Pradesh and Maharashtra this Specie is known as Abelmoschus tuberculatus. (Bhist and Bhat, 2006)

Properties

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The okra mucilage has a light pungent flavour and a soft to mucous texture. The mucous texture comes from over cooked okra. The mucous texture can be averse by adding lemon juice or another acidic liquid (like vinegar). Okra contains vitamins A and C and is a good source of iron and calcium. It also contains starch, fat, ash, thiamine and riboflavin (Kochar, R. 2006). The superior fiber found in okra helps stabilize blood sugar by curbing the rate at which sugar is absorbed from the intestinal tract. Okra helps lubricate the large intestines due to its bulk laxative qualities. The okra fiber absorbs water and ensures bulk in stools. This helps prevent and relieves constipation. Okra's mucilage soothes and facilitates elimination more comfortably by its slippery characteristic (Wilson J. 2006). Okra binds excess cholesterol and toxins. Okra is used for healing ulcers and to keep joints limber. It helps to neutralize acids, being very alkaline, and provides a temporary protective coating for the digestive tract. Protein and oil contained in the seeds of okra serves as the source of first-rate vegetable protein. It is enriched with amino acids on the likes of tryptophan, cysteine and other sulfur amino acids (Kochar, R 2006).

Uses

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Okra pods were commonly use as source of food. It is one of plants that is known for its mucilaginous quality. Due to its mucilage it is used as soup binder which gives soup its desired slimy consistency. It is also use as binder and adhesives (Ameena, 2010). Not only okra pods were used in an okra plant but also leaves and stem of okra were often used as source of fibers and ropes. Morever, the plant has been used medicinally in treatment of several disorders, anti-cancer, anti-microbial and hypoglycemic activities of the plant have been reported. Okra mucilage binds cholesterol and bile acid carrying toxins dumped into it by filtering liver, thus might act as a hepatoprotective agent (Ameena,2010). The ripe seeds of okra are sometimes roasted and ground as a substitute for coffee. In the 1800s, slaves from Africa used ground okra as part of their diet, and this apparently led to the use of ground okra seeds as a coffee substitute by other southerners during the American Civil War blockades of the 1860s. The Austin State Gazette noted: “An acre of okra will produce seed enough to furnish a plantation of 50 Negroes with coffee in every way equal to that imported from Rio” (Another Medicinal Vegetable, 2009). Today, okra is widely used in a thick stew made with vegetables and meat in Egypt, Greece, Iran, Iraq, Jordan, Lebanon, Turkey, Yemen and other parts of the Mediterranean. In Indian cooking, okra is sautéed or added to gravy-based preparations and is very popular in South India. In the Caribbean islands, okra is cooked up and eaten as soup, often with fish. Okra became a popular vegetable in Japanese cuisine. It is served

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with soy sauce and katsuobushi or as tempura. Breaded, deep fried okra is served in the southern United States. The immature pods may also be pickled. Okra remains an important ingredient throughout the American South, where it is blanched and sauced, made into succotash with ham hocks, cornmeal-coated and deep fried, pickled, cooked with tomatoes, lettuce, cheese, mint and corn, steamed and frittered. When it is sliced

CHAPTER 3 METHODOLOGY

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3.1 Introduction The primary objective of the study is to evaluate the effectiveness of the binding capacity of the mucilage obtained from the plants of Okra (Abelmoschus esculentus) as an alternative adhesive of egg albumin in histopathology section. This chapter will provide the description of the site where the study is being conducted, the kind of the research design, flowchart of the procedure, target of the study and the instrumentation. 3.2 Research / Study Design The Research design is experimental, so that the researchers will be able to prove the effectiveness of okra as an alternative adhesive of egg albumin in histopathology section.

3.3 Flowchart of the Procedure Schematic Diagram

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The group researchers will conduct the experiment at the fourth floor laboratory in the University of Santo Tomas. The okra pods that will be using in this study are purchased

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at the local department store. To obtain the Okra extracts, the fruit is cut into two, the lower portion will be disregarded and the upper portion is will be collected. The collected portion is then pound to extract the mucilage needed. To separate the mucilage from the pounded portion, there is a need of filter paper. The pounded portion will be discarded while the extract will be collected in a small beaker. While performing the extraction of the Okra mucilage, the flotation water bath is being prepared, and will be filled half way with water, and turned it on. After the flotation water bath is being prepared, and the okra extract is collected, the tissue ribbons from the hospital of the University of Santo Tomas are made floating on the water. The ribbons are cut into a single tissue ribbon. A small amount of okra extract will be smeared on the center of a glass slide using an applicator stick. When the okra extract is already smeared, place the tissue ribbon on the smeared part of the glass slide using the “fish out” technique. Adhering of the tissue ribbon on the okra extract smeared part of the glass slide indicates that the Okra extract has the ability to be an adhesive using its mucilage property. 3.4 INSTRUMENTS A knife will be used to cut the okra into two portions. The upper portion will be pounded with the use of mortar and pestle. Filter papers will be used for separating the extracts from the whole portions and a beaker for the storage of the extracts. The tissue ribbons will be prepared by using a microtome. Glass slides are where the extracts from

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okra will be smeared with the use of an applicator stick. The fish out technique will be used on the tissue ribbon to get it from the floatation water bath.

3.5 ETHICAL CONSIDERATIONS Most people are removing the upper portion of the fruit when cooking. In this experiment, the researchers will make use of that part as a substitute for an adhesive in histopathology. Hence, no bit of the okra pod will be put to waste. Furthermore, this experiment will encourage farmers to plant and cultivate okras because of their useful benefits. Lastly, no animals will be used or harmed at the expense of this experiment.

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BIBLIOGRAPHY JOURNAL: I-Min Liu, Shorong-Shii Liou, Ting-Wei Lan, Feng-Lin Hsu, Juei-Tang Cheng (2005). Myricetin as the active principle of Abelmoschus moschatus to lower plasma glucose in streptozotocin-induced diabetic rats. Planta Med. 71 (7), 617-621. Myricetin as the active principle of Abelmoschus moschatus to lower plasma glucose in streptozotocin-induced diabetic rats. Planta Med. 71 (7), 617-621.

Kahlon.(2007) In vitro binding of bile acids by okra, beets, asparagus, eggplant, turnips,green beans, carrots, and cauliflower. Food chemistry. 103 (2), 676-680. Lengsfeld, C., Titgemeyer, F., Faller, G., Hensel, A. (2004) . Glycosylated compounds from okra inhibit adhesion of Helicobacter pylori to human gastric mucosa. J. Agric. Food Chem. 52 (6), 1495–1503.

Odeleye, F. O., O. M. O. Odeleye, O. A. Dada and A. O. Olaleye (2005). The response of okra to varying levels of poultry manure and plant population density under sole cropping. Journal of Food, Agriculture and Environment, 3(3,4):68-74.

Olasotan, F. O. (2001). Optimum population density for okra (Abelmoschus esculentus (L) Moench) in a mixture with cassava (Manihot esculentus) and its relevance to rainy season-based cropping system in south-western Nigeria. Jr. of Agric. Sc. 136:207-214.

World Journal of Agricultural Sciences (2006). 2 (1):119-122. THESIS:

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Akanbi, W. B. (2002). Growth, nutrient uptake and yield of maize and okra as influenced by compost and nitrogen fertilizer under different cropping systems. Ph.D. Thesis, Department of Crop Protection and Environmental Biology, University of Ibadan, Nigeria. Akande MO, Oluwatoyinbo FI, Adediran JA, Buari KW, Yusuf IO (2003). Soil amendments affect the Release of P from rock phosphate and the development and Yield of Okra. J. Vegetable Crop Prod. 2: 3-9. Yasmeen Siddiqui, (2006). Bio-efficacy of compost extracts for the control of Choanephora wet rot disease of okra (Abelmoschus esculentus L.). PhD Thesis, Universiti Putra Malaysia.

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