Phytochemical Screening and Proximate Analysis of Castanopsis Indica

PHYTOCHEMICAL SCREENING AND PROXIMATE ANALYSIS OF CASTANOPSIS INDICA

A DISSERTATION SUBMITTED FOR THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE MASTERS DEGREE OF SCIENCE IN CHEMISTRY

BY Prakash Shrestha Exam Symbol No.: Chem.462/072 T.U. Regd. No.: 5-2-0022-0174-2011

CENTRAL DEPARTMENT OF CHEMISTRY INSTITUTE OF SCIENCE AND TECHNOLOGY TRIBHUVAN UNIVERSITY, KIRTIPUR KIRTIPUR, KATHMANDU NEPAL JUNE, 2019

BOARD OF EXAMINER AND CERTIFICATE OF APPROVAL

This dissertation entitled “PHYTOCHEMICAL SCREENING AND PROXIMATE ANALYSIS OF Castanopsis indica” by Prakash Shrestha, under the supervision of Prof. Dr. Ram Chandra Basnyat Central Department of Chemistry, Tribhuvan University, Nepal, is hereby submitted for the partial fulfillment of the Master of Science (M.Sc.) Degree in Chemistry. This dissertation has not been submitted in any other university or institution previously for the award of a degree.

Supervisior Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry Tribhuvan University Kritipur, Kathmandu, Nepal

Internal Examiner External Examiner Asst. Prof. Surya Kant Kalauni, PhD Prof. Daman Raj Gautam, PhD Central Department of Chemistry Amrit Science College Tribhuvan University Tribhuvan University Kritipur, Kathmandu, Nepal Lainchaur, Kathmandu

Head of the Department Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry Tribhuvan University Kritipur, Kathmandu, Nepal

RECOMMENDATION LETTER

This is to certify that the dissertation work entitled “PHYTOCHEMICAL SCREENING AND PROXIMATE ANALYSIS OF Castanopsis indica” has been carried out by Prakash Shrestha as a partial fulfillment for the requirement of Master Degree in Chemistry under my Supervision. To the best of my knowledge, this work has not been submitted to any other degree in this institute.

______Supervisor Prof. Ram Chandra Basnyat, PhD Central Department of Chemistry Tribhuvan, University Kirtipur, Kathmandu, Nepal

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DECLARATION

I, Prakash Shrestha, hereby declare that the work presented herein is genuine work done originally by me and has not been published or submitted elsewhere for the requirement of a degree program. Any literature, data or works done by others, presented in this dissertation are cited, has been given due acknowledgement and listed in the reference section.

______Prakash Shrestha Central Department of Chemistry Tribhuvan University Kirtipur, Kathmandu, Nepal

ACKNOWLEDGEMENTS

The successful completion of this study was possible with the support, assistance, and advice of various people. I would like to express my gratitude and appreciation to the following:

I would like to express my deepest gratitude and sincere appreciation to my supervisor and Head of the Central Department of Chemistry Prof. Ram Chandra Basnyat, PhD, Central Department of Chemistry, for his constant support, inspirable guidance, invaluable suggestions, and regular feedback at all stages of my dissertation work.

My deepest appreciation goes to all my respected teachers of CDC. I would also like to convey my thanks to all the administration staff as well as laboratory staffs of the department for their continuous assistance throughout the dissertation work.

I would especially like to thank National Herbarium and Plant Laboratories (KATH), for the identification of plants. I am thankful to the authors of books and articles which I have used as reference in this work.

I would also like to thank Department of Food Technology and Quality Control for determination of proximate analysis. I am thankful to Pushpa Bhattarai for her cooperation.

My special thanks go to all my colleagues whom I have been surrounded and enjoyed, especially for their co-operation, support and encouragement during the work. I would also like to convey my thanks to my seniors and my juniors for their help and suggestion during this work.

Last but not least, my sincere obligation goes to my dearest family. Thank you so much your love, encouragement, inspiration and cooperation that have made this dissertation possible.

Thank you all. Prakash Shrestha Email: [email protected]

LIST OF TABLES

Table 1: Chemical composition of sea water ...... 24

Table 2: Sample collection and identification ...... 33

Table 3: Table showing % yield of methanol and hexane extract ...... 33

Table 4: Phytochemical analysis of leaves and bark of Castanopsis indica ...... 34

Table 5: Total phenolic content of Castanopsis indica extracts ...... 38

Table 6: Total Flavonoid content of Castanopsis extracts ...... 39

Table 7: Antimicrobial activity of methanol extract of bark and leaves ...... 40 Table 8: Antifungal activity of methanol extract of Castanopsis indica ...... 41

Table 9: Calculation of LC50 value of methanol extract of leaves and bark of Castanopsis indica ...... 42 Table 10: Percentage composition of nutritional parameters in sample of Castanopsis indica in nut ...... 43 Table 11: Antioxidant activity of Ascorbic acid ...... 55 Table 12: Antioxidant activity of methanol extract of bark ...... 55 Table 13: Antioxidant activity of methanol extract of leaf ...... 55 Table 14: Antioxidant activity of hexane extract of bark ...... 56 Table 15: Antioxidant activity of hexane extract of leaf ...... 56 Table 16: Total phenolic content in methanol extract of bark ...... 56 Table 17: Total phenolic content in methanol extract of leaf ...... 57 Table 18: Total phenolic content in hexane extract of bark ...... 57 Table 19: Total phenolic content in hexane extract of leaf ...... 57 Table 20: Total flavonoid content in methanol extract of bark ...... 58 Table 21: Total flavonoid content in hexane extract of leaf ...... 58 Table 22: Total flavonoid content in methanol extract of leaf ...... 58 Table 23: Total flavonoid content in hexane extract of lark ...... 59 Table 24: Determination of moisture ...... 59 Table 25: Determination of total ash ...... 59 Table 26: Determination of total nitrogen ...... 59 Table 27: Determination of crude fiber ...... 60 Table 28: Determination of crude fat ...... 60

LIST OF FIGURES

Figure 1: Castanopsis indica plant ...... 3 Figure 2: Castanopsis indica nuts ...... 3

Figure 3: Mechanism of DPPH free radical scavenging ...... 6

Figure 4: Mechanism of DPPH free radical scavenging by ascorbic acid ...... 6 Figure 5: Structure of compound found in Castanopsis indica ...... 15

Figure 6: Scheme for extraction, fractionation and analysis of bark and ……………leaves of Castanopsis indica ...... 17

Figure 7: Antioxidant activity of ascorbic acid ...... 35 Figure 8: Antioxidant activity of methanol extract of Castanopsis indica leaf ...... 35 Figure 9: Antioxidant activity of methanol extract of Castanopsis indica bark ..... 36 Figure 10: Antioxidant activity of hexane extract of Castanopsis indica leaf ...... 36 Figure 11: Antioxidant activity of hexane extract of Castanopsis indica bark ...... 37 Figure 12: Absorbance vs concentration curve for gallic acid ...... 38 Figure 13: Absorbance vs concentration curve for Quercetin ...... 39 Figure 14: Percentage composition of nutritional parameters in nuts of Castanopsis indica...... 44

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LIST OF ACRONYMS AND ABBREVIATIONS C. indica : Castanopsis indica DMSO : Dimethyl Sulphoxide DPPH : 1, 1-diphenyl-2-picryl hydrazyl FCR : Folin-Ciocalteau Reagent GAE : Gallic Acid Equivalent

IC50 : Inhibitory Concentration for 50% Inhibition IR : Infra-Red

LC50 : Lethal Concentration for 50% Mortality MHA : Mueller Hinton Agar MHB : Mueller Hinton Broth ppm : Part per million QE : Quercetin Equivalent

ROS : Reactive Oxygen Species TFC : Total Flavonoid Content TPC : Total Phenolic Content UV : Ultraviolet ZOI : Zone of Inhibition

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ABSTRACT Castanopsis indica also known as Katus was selected as the plant for the dissertation work. Methanol and hexane extracts of leaves and bark of C. indica was prepared by Soxhlet extraction method. Phytochemical screening results showed the presence of glycosides, polyphenols, and tannins in all these extracts. The total phenolic and total flavonoid contents of methanol extracts of leaves of C. indica were estimated as 153.58 mg GAE/gm and 138.46 mg QE/gm respectively. The Zone of inhibition shown by methanol extracts of leaves and bark of C. indica for S. aureus were measured 18 mm and 15 mm respectively. Gram negative bacteria didn’t show any antimicrobial activity in both of the extracts. The antifungal assay showed that there is no any inhibitory activity against the growth of the fungi used under study among the methanol leaves and methanol stem fractions. In DPPH free radical scavenging test for antioxidant activity, the IC50 values of methanol and hexane extracts of leaves of C. indica were

64.94 and 114.62 μg/ml. Similarly, IC50 values of methanol and hexane extracts of bark of C. indica were found as 74.98 and 124.41 μg/mL respectively in the antioxidant assay. In brine shrimp bioassay, the LC50 for methanolic extract of leaves and bark of C. indica were found to be 758.57 and 281.83 μg/mL respectively. The percentage composition of different nutrition parameters (moisture, total ash, crude fat, protein, crude fiber, carbohydrate) were determined. This study hopes to provide valuable information for different research.

Keywords: Castanopsis indica, Methanol extract, Hexane extract, Antimicrobial and Antioxidant, Proximate analysis

TABLE OF CONTENTS

BOARD OF EXAMINER AND CERTIFICATE OF APPROVAL ...... ii RECOMMENDATION LETTER ...... iii DECLARATION ...... iv ACKNOWLEGEMENTS ...... v LIST OF TABLES ...... vi LIST OF FIGURES ...... vii LIST OF ACRONYMS AND ABBREVIATIONS ...... viii ABSTRACT ...... ix TABLE OF CONTENTS ...... x-xiii

CHAPTER I: INTRODUCTION ...... 1-12 1.1 Background ...... 1 1.1.1 Natural Product Chemistry ...... 1 1.1.2 Biodiversity of Nepal ...... 2 1.2 Introduction to the Selected plant ...... 2 1.2.1 Introduction to Castanopsis indica (Katus) ...... 2 1.2.2 Classification...... 3 1.3 Introduction to Testing Parameters ...... 3 1.3.1 Phenolic and Flavonoids ...... 3 1.3.2 Antioxidant Activity ...... 4 1.3.2.1 Mechanism of DPPH Assay ...... 5 1.3.3 Antimicrobial Activity ...... 7 1.3.4 Brine Shrimp Assay ...... 7 1.3.5 Nutritional Constituents and Composition...... 8 1.3.5.1 Moisture ...... 8 1.3.5.2 Fats ...... 8 1.3.5.3. Proteins ...... 9 1.3.5.4 Carbohydrate ...... 9 1.3.5.5 Vitamins ...... 9 1.3.5.6 Fiber ...... 10 1.3.5.7 Ash ...... 10 1.4 Objectives ...... 10 1.4.1 General Objectives ...... 10 1.4.2 Specific Objectives ...... 11

CHAPTER II: LITERATURE REVIEW ...... 12-15

CHAPTER III: MATERIALS AND METHODS ...... 16-32 3.1 Plant Extracts ...... 16 3.1.1 Collection and Identification of Plant Samples ...... 16 3.1.2 Sample Preparation ...... 16 3.1.3 Extraction Process ...... 16 3.1.4 Phytochemical Analysis ...... 17 3.1.5 Antioxidant Activity ...... 18 3.1.5.1 Preparation of 0.2 mm DPPH solution ...... 18 3.1.5.2 Preparation of ascorbic acid solution (Standard) ...... 18 3.1.5.3 Preparation of sample solutions ...... 18 3.1.5.4 Measurement of DPPH radical scavenging activity ...... 19 3.1.6 Total Phenolic Content ...... 19 3.1.6.1 Preparation of the standard gallic acid solution ...... 19 3.1.6.2 Construction of the calibration curve ...... 19 3.1.6.3 Preparation of the sample solution ...... 20 3.1.6.4 Calculation of the total phenolic content (TPC) ...... 20 3.1.6.5 Statistical analysis ...... 20 3.1.7 Total Flavonoid Content (TFC) ...... 21 3.1.7.1 Preparation of the standard quercetin stock solution ...... 21 3.1.7.2 Preparation of the sample solutions ...... 21 3.1.7.3 Calculation of the total flavonoid content ...... 22 3.1.7.4 Statistical analysis ...... 22 3.1.8 Bioassay ...... 22 3.1.8.1 Brine shrimp bioassay ...... 23 3.1.8.2 Required Materials ...... 23 3.1.8.3 General Procedure of Brine Shrimp Bioassay ...... 23 3.1.8.3.1 Sterilization of the apparatus ...... 23 3.1.8.3.2 Preparation of the artificial sea water ...... 23 3.1.8.3.3 Hatching of the brine shrimp eggs ...... 24 3.1.8.3.4 Preparation of samples ...... 24 3.1.8.3.5 Procedure for bioassay ...... 24 3.1.8.3.6 Data analysis ...... 25 3.2 Antimicrobial Assay ...... 25 xi

3.2.1 Collection of Test Organisms ...... 26 3.2.2 Preparation of Solution of Extracts ...... 26 3.2.3 Preparation of Muller Hilton Agar (MHA) Plates ...... 26 3.2.4 Nutrient Broth (NB) Solution ...... 26 3.2.5 Preparation of Standard Culture Inoculums ...... 26 3.2.6 Transfer of Bacteria in Petri Plates ...... 27 3.2.7 Screening and Evaluation of Antibacterial Activity ...... 27 3.2.8 Screening and evaluation of the Antifungal activity ...... 27 3.3 Proximate Nutritional Analysis of Castanopsis indica nut ...... 28 3.3.1 Determination of Moisture ...... 28 3.3.2 Determination of total Ash ...... 28 3.3.3 Determination of Crude Fat ...... 29 3.3.4 Determination of Protein ...... 29 3.3.4.1 Total Nitrogen ...... 30 3.3.5 Determination of Crude Fiber ...... 30 3.3.6 Estimation of Carbohydrates ...... 31 3.3.7 Estimation of Vitamin C ...... 31 3.3.7.1 Preparation of Dye ...... 31 3.3.7.2 Standardization of Dye ...... 32 3.3.7.3 Preparation of Sample ...... 32

CHAPTER IV: RESULTS AND DISCUSSION ...... 33 4.1 Identification of Selected Plant ...... 33 4.2 Percentage Yield ...... 33 4.3 Qualitative Analysis of Phytochemicals ...... 34 4.4 Antioxidant Activity ...... 34 4.5 Total Phenolic Content and Flavonoid Content ...... 38 4.6 Antimicrobial Activity ...... 40 4.7 Antifungal Activity ...... 41 4.8 Brine Shrimp Bioassay ...... 41 4.9 Proximate Analysis of Castanopsis indica ...... 42

CHAPTER V: CONCLUSIONS AND RECOMMENDATION ...... 45-46 5.1 Conclusions ...... 45

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5.2 Suggestion for further work ...... 46

REFERENCES ...... 47-50

APPENDICES ...... 51-61

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CHAPTER I

INTRODUCTION

1.1 Background

1.1.1 Natural Product Chemistry

Plants produce a huge and different variety of organic compounds, the great majority of which do not appear to participate directly in growth and development. These substances traditionally referred to as secondary metabolites. The primary metabolites, in contrast, such as phytosterols, acyl lipids, nucleotides, amino acids, and organic acids, are found in all plants and perform metabolic roles that are essential and usually evident. Natural products have been widely recognized as biologically insignificant and have received little attention from most plant biologists. Organic chemists, however, have long been interested in these novel phytochemicals and have investigated their chemical properties extensively since the 1850s. Based on their biosynthetic origins, plant natural products can be divided into three major groups: the terpenoids, the alkaloids, and the phenylpropanoids and allied phenolic compounds. All terpenoids, including both primary metabolites and more than 25,000 secondary compounds, are derived from the five-carbon precursor isopentenyl diphosphate (IPP). The 12,000 or so known alkaloids, which contain one or more nitrogen atoms, are biosynthesized principally from amino acids. The 8000 or so phenolic compounds are formed by way of either the shikimic acid pathway or the malonate/ acetate pathway.1

Throughout the ages, humans relied on natural products. Natural products have earliest records from 2900-2600 BC documenting the uses of approximately 1000 plants derived substances such as the oil of Cedrus species (cedar), Commiphora myrrha (myrrh), Cupressus sempervirens (cypress), Glycyrrhiza glabra (liquorice) and Papaver somniferum (poppy).2 Before the advent of high throughput screening (HTS) and post-genomic era, a huge amount of drug substance was a purely natural product or was inspired by the molecule derived from natural sources. An analysis into the sources of a new drug from 1981 to 2007 reveals that almost half of the drug approved since 1994 were based on natural product.2,3,4

Natural products have always played a major role in human therapy and represent a huge reservoir of bioactive chemical diversity and help to understand the cellular pathways that are an essential component of the drug discovery process.5 NPs have a large quantity of lead-like molecules, which could be used as scaffolds to expand the chemical library.6

1.1.2 Biodiversity of Nepal

Nepal is landlocked country situated in the central part of the Himalayas between 26022’ and 30027’ N latitudes and 80004’ and 88012’ E longitudes, covering an area of 147,181 square kilometers with the geographical condition. The elevation ranges from around 70 meters above sea level in the eastern alluvial plains to 8,848 meters at the Mount Everest has made Nepal a rich country in plant biodiversity.7

Biodiversity is closely linked to the livelihoods and economic well-being of most Nepalese people. At least 1,463 species of herbal medicinal plants are used by the rural people in Nepal. Biodiversity is the second largest natural source of our country after fresh water. Nepal has 118 types of the ecosystem of which highlands region contains 38, Mid-Hills 13, Siwalik Hills 13, terai 10 and other ecosystems. Out of around 7000 plants species, 1800 species are currently in use for folk medicine.8,9

1.2 Introduction to the Selected Plant

1.2.1 Introduction to Castsnopsis indica (Katus)

Castanopsis indica is a tallish tree, growing up around 8–14 m (26–46 ft) in height with a dense, full crown. The leaves are thick and leathery with a serrated edge. They are oblong and elliptical, with an acute tip, are nearly evergreen and have a short petiole. The bark of the tree is rough and grey. The fruit is reddish- brown and round, found in small clusters, and is covered with long, thin spines. The fruit is often fed upon by squirrels. Castanopsis indica (Roxb. Ex Lindl.) is commonly known as ‘Dhalne katus’ and ‘Indian chestnut tree’ found throughout the Himalayan region of North East India, Bangladesh, Laos, Myanmar, Nepal, Sikkim, Bhutan, Thailand, Vietnam.10

1.2.2 Classification

Kingdom: Plantae Division: Angiosperms Class: Eudicots Order: Fagales Family: Fagaceae Genus: Castanopsis Species: indica

Figure 1: Castanopsis indica plant Figure 2: Castanopsis indica nuts

1.3 Introduction to the Testing Parameters

1.3.1 Phenolic and Flavonoids

Phenolics are universal secondary metabolites in plants. They comprise a large group of biologically active ingredients from simple phenol molecules to polymeric structures with molecular mass above 30000 Da. On the basis of the number of phenol subunits, there are two basic groups of phenolics - simple phenols and polyphenols. Simple phenols contain phenolic acids or phenols with carboxyl group whereas polyphenols contain at least two phenol rings flavonoids belong to this group.11 Polyphenolic compounds are an essential group of plant metabolites which are essential for human diet and overall health. Also, the polyphenolics compounds display a broad range of biological activities.12,13

In nature, flavonoid compounds are products extracted from plants and they are found in several parts of the plant. Flavonoids are used by vegetables for their growth and defense against plaques.14 Flavonoids have several subgroups, which include chalcones, flavones, flavonols, and isoflavones. These subgroups have unique major sources.15 Flavonoids play a variety of biological activities in plants, animals, and bacteria. In plants, flavonoids have long been known to be synthesized in particular sites and are responsible for the color and aroma of flowers, and in fruits to attract pollinators and consequently fruit dispersion to help in seed and spore germination, and the growth and development of seedlings.16 Flavonoids protect plants from different biotic and abiotic stresses and act as unique UV filters, function as signal molecules, allopathic compounds, phytoalexins, detoxifying agents and antimicrobial defensive compounds.17

1.3.2 Antioxidant Activity

Oxidation is the transfer of electrons from one atom to another and represents an essential part of aerobic life and our metabolism, since oxygen is the ultimate electron acceptor in the electron flow system that produces energy in the form of ATP.18 Problems may arise when the electron flow becomes uncoupled (transfer of unpaired single electrons), generating free radicals. Free radicals are two types: known as “reactive oxygen species” (ROS) and “reactive nitrogen 19 - species” (RNS). They content superoxide (O2 ), peroxyl (ROO), alkoxyl (RO), hydroxyl (HO), and nitric oxide (NO). The hydroxyl (half-life of 10 -9 sec) and the alkoxyl free radicals are very reactive and rapidly attack the molecules in nearby cells.20 ROS may be very damaging, since they can attack lipids in cell membranes, proteins in tissues or enzymes, carbohydrates and DNA, to induce oxidations, which may cause cancer, atherosclerosis, aging, immunosuppressant, inflammation, ischemic heart disease, diabetes, hair loss and neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease.21,22

The mammalian cell possesses a defense mechanism for radical detoxification. The key metabolic steps are superoxide dismutase (SOD) catalysis of the dismutation of superoxide to hydrogen peroxide and oxygen, the conversion of

H2O2 into water and oxygen by catalase (CAT) and glutathione peroxidase (GPX) which destroys toxic peroxides.23

Antioxidants are molecules which can delay or inhibit cellular damages from free radicals existing in biological systems. Plants are an excellent source of bioactive compounds with high antioxidant activity such as phenolic acids, flavonoids, coumarins, stilbenes, vitamins, hydrolyzable and condensed tannins24 approximately 4,000 to 6,000 antioxidant compounds synthesized by plants as secondary products have been detected.25 There are two basic categories of antioxidants, namely, synthetic and natural. In general, synthetic antioxidants are compounds with phenolic structures of various degrees of alkyl substitution, whereas natural antioxidants can be phenolic compounds (flavonoids, and phenolic acids), nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids, and amines), or carotenoids as well as ascorbic acid. Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been suspected to be carcinogenic. Thus, the interest in natural antioxidants has increased.10

1.3.2.1 Mechanism of DPPH Assay

The DPPH (1,1-diphenyl-2-picrylhydrazyl) having molecular weight 394.32 g is considered as a stable radical because of the para magnetism conferred by its odd electron. Its solution in methanol appears as a deep violet colored solution and shows a strong absorption band at 517 nm. DPPH radical can accept hydrogen to become a stable diamagnetic molecule and has a pale violet or yellow color. If the testing solution gives rise to pale violet or yellow color, it suggests that the extract has an anti-oxidant effect by the mechanism of free radical scavenging activity.

Some compound showing anti-oxidant property are α-tocopherol, ascorbic acid, carotenoids, polyphenols, flavonoids, glutathione, etc. oxidative enzymes are superoxide dismutase (SOD) and catalase (CAT).26

Antioxidants

RH R⁰

DPPH DPPH free radical (Yellow) (Purple)

Figure 3: Mechanism of DPPH free radical scavenging

Ascorbic acid was used as a standard in the DPPH radical scavenging method.

It is commonly known as vitamin C (molecular formula C6H8O6 and the molecular weight 176.12 g). It is a natural antioxidant.

Ascorbic acid (vitamin C)

DPPH free ₊ DPPH ₊ radical

Ascorbic acid (vitamin C) Free radical of ascorbic acid

₊ H⁰

Hydrogen free Free radical of ascorbic acid Keto form of ascorbic acid radical

Figure 4: Mechanism of DPPH free radical scavenging by ascorbic acid

1.3.3 Antimicrobial Activity

In the last few years, there has been great interest in chemical and pharmacological investigations of the biological properties of medicinal plants. The use of extracts as antimicrobial agents shows a low risk of increasing resistance to their action because they are complex mixtures, making microbial adaptability very difficult and it is also reported to have minimum side effects.27

Medicinal plants are an abundant source of antimicrobial molecules. A wide range of medicinal plants extracts are used to treat several infections as they have potential antimicrobial activity.28 Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial, anti-viral, anti-parasites and anti-cancer properties.29 The ability to use most of the medicinal plants for the treatments for various diseases may lie in the antioxidant and antimicrobial effect of phytochemicals.30 Antimicrobial activity refers to the process of killing or inhibiting the disease-causing microbes. Antimicrobial may be anti-bacterial, anti-fungal, anti-viral or anti-parasitics. They all have a different mode of action. Therapies that kill microorganisms are called microbiocidal therapies and therapies that only inhibits the growth of microorganism is called microbiostatic therapies.31

1.3.4 Brine Shrimp Assay

To study the toxicity of the medicinal plants we performed brine shrimp lethality bioassay which based on the ability to kill laboratory cultured brine shrimp (Artemia nauplii). The brine shrimp assay was proposed by Michael et al. in 1956 and later developed by others. The assay is considered a useful tool for preliminary assessment of toxicity and it has been used for the detection of fungal toxins, plant extract toxicity.32 The brine shrimp assay is a very useful tool for the isolation of bioactive compounds from plant extracts. The method is attractive because it is very simple, inexpensive and low toxin amounts are sufficient to perform the test on the microwell scale.33 Minimum toxicity to no toxicity is essential for the successful development of pharmaceutical or cosmetic preparation and in this regard, cellular toxicity studies play a crucial

7 role.34 For the bioactive compound of either synthetic or natural, this test is a rapid and comprehensive test.

1.3.5 Nutritional Constituents and Composition

All human beings require sufficient food for growth, development and to live an active and healthy life and it depends upon the quality of foodstuffs. The quality of food depends upon the presence of relative concentrations of various nutrients such as proteins, fat, carbohydrates, vitamins and minerals. Carbohydrates, fat, proteins are sometimes referred to as proximate principles and form the major portion of the diet, while minerals play an important role in the regulation of the metabolic activity in the body.35

1.3.5.1 Moisture Water is a major constituent of most food products. Water content in relation to food material is normally termed “moisture content”. The approximate, expected moisture content of a food can affect the choice of the method of measurement. The ease of water removal from foods depends on how it exists in the food product.36 The three states of water in food products are: a. Free water: Free water retains its physical properties and acts as a dispersing agent for colloids and the solvent for salts. b. Adsorbed water: This water is held tightly or is occluded in cell walls or protoplasm and is held tightly to proteins. c. The water of hydration: This water is bound chemically, for example, lactose monohydrate.

1.3.5.2 Fats

Lipids are a group of substances that, in general, are soluble in ether, chloroform, or other organic solvents but are sparingly soluble in water. Some lipids, such as triacylglycerols, are very hydrophobic. Other lipids, such as di- and monoacylglycerols, have both hydrophobic and hydrophilic moieties in their molecules and are soluble in relatively polar solvents. Short-chain fatty acids such as C1–C4 are completely miscible in water and insoluble in nonpolar solvents. Fats generally refer to those lipids that are solid at room temperature and oils generally refer to those lipids that are liquid at room temperature.

Natural fats and oils are predominantly glyceryl esters of fatty acids. Crude fat obtained by normal extraction process contains not only true fat but also other minor constituents such as waxes, phospholipids, fat-soluble vitamins, sterols, pigments, and antioxidants.36

1.3.5.3 Proteins

Proteins are an essential nutrient for the human body. They are one of the building blocks of body tissue and can also serve as a fuel source. Protein can be found in a wide range of food which is very complex. They are composed of elements including hydrogen, carbon, nitrogen, oxygen, and sulfur. Twenty α- amino acids are the building blocks of proteins; the amino acid residues in a protein are linked by peptide bonds and nitrogen is the most distinguishing element present in proteins. Proteins can be classified by their composition, structure, biological function, or solubility properties. For example, simple proteins contain only amino acids upon hydrolysis, but conjugated proteins also contain non-amino acid components.36

1.3.5.4 Carbohydrate

Carbohydrates are important in foods as a major source of energy and as dietary fiber which influences physiological processes. Digestible carbohydrates are converted into monosaccharides, which are absorbed and provide metabolic energy. Carbohydrates account for more than 70℅ of the caloric value of the human diet. Monosaccharides are the only carbohydrates that can be absorbed from the small intestine. Higher saccharides (oligo- and polysaccharides) must first be digested (i.e., hydrolyzed to monosaccharides) before absorption and utilization can occur). Humans can digest only sucrose, lactose, maltooligosaccharides/maltodextrins, and starch. All are digested with enzymes found in the small intestine.36

1.3.5.5 Vitamins

Vitamins are defined as relatively low-molecular-weight compounds. With few exceptions, humans cannot synthesize most vitamins and therefore need to obtain them from food and supplements. Insufficient intake of vitamins results

9 in deficiency diseases. For example: scurvy and pellagra, which are due to the lack of ascorbic acid (vitamin C) and niacin, respectively.36

1.3.5.6 Fiber

Dietary fiber is the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. Dietary fiber includes polysaccharides, oligosaccharides, lignin, and associated plant substances. Dietary fibers promote beneficial physiological effects including laxation, and or blood cholesterol attenuation, and/or blood glucose attenuation. 36,37

1.3.5.7 Ash

Ash refers to the inorganic residue remaining after either ignition or complete oxidation of organic matter in a foodstuff. Two major types of ashing are used: dry ashing, primarily for proximate composition and for some types of specific mineral analyses; wet ashing (oxidation), as a preparation for the analysis of certain minerals. Dry ashing refers to the use of a muffle furnace capable of maintaining temperatures of 500–600 ⁰C. Water and volatiles are vaporized, and organic substances are burned in the presence of oxygen in the air to CO2 and oxides of N2. Wet ashing is a procedure for oxidizing organic substances by using acids and oxidizing agents or their combinations. Minerals are solubilized without volatilization.36

1.4 Objectives

1.4.1 General Objectives

The general objective of the study is:  To study the phytochemical and biological activities of leaves and bark of C. indica and to evaluate the proximate nutritional value of nut of C. indica.

1.4.2 Specific Objectives

Based on the general objective mentioned above, the following specific objectives are pointed out:  To analyze different classes of phytochemicals, present in the hexane and methanol extracts of C. indica leaves and barks.  To evaluate the total phenolic and total flavonoid content of hexane and methanol extracts of C. indica leaves and barks.  To study the antimicrobial activity of methanol extract of leaves and bark of C. indica.  To estimate the antioxidant activity of hexane and methanol extract of leaves and bark of C. indica.  To identify the proximate nutritional value of nuts of C. indica.

CHAPTER II

LITERATURE REVIEW

The literature survey on Castanopsis indica is done by collecting different papers published in different journals via google scholar, science direct and some are depicted below:

Castanopsis indica (C. indica), family Fagaceae is known as ‘Dhalne katus’ and ‘Indian Chestnut’ found throughout the Himalayan region of North East India, Bangladesh, Laos, Myanmar, Nepal, Sikkim, Bhutan, Thailand, Vietnam. A decoction of the leaves applied to treat stomach disorder and skin diseases.10 The leaf extract possesses a pronounced in vitro antibacterial effect.21

A decoction of the leaves applied to treat stomach disorder and skin diseases. Powdered leaves are given to cure indigestion. A plant resin is given to treat diarrhea. A paste of leaves is applied for headache. Bark paste is used to control chest pain.38

The history of chemical analysis of this plant goes back to almost four decades back. The researcher reported two coumarins and five tri-terpenoids from C. indica. Two coumarins were psoralen and angelicin whereas five triterpenoids were erythrodiol, ursolic acid, castanopsin, castanopsone and castanopsol. Amongst the triterpenoids, three were found to be new products and the structure of two of these, namely castanopsone and castanopsol were elucidated. The structure of two of the three new triterpenoids has been elucidated by chemical reaction and physical methods.39

On 1978 the researcher elucidated the structure of Castanopsin with the modern spectroscopy techniques IR, NMR, UV adsorption. The structure was supported by IR spectrum exhibited absorptions for OH (3575, 3290), gem dimethyl (1375, 1365) and a conjugated trisubstituted double bond (1630, 835 cm-1) and NMR spectrum showed the signals for eight quaternary methyls in the region δ 0.83-1.2 ppm, two carbinolic protons at 3.78(q) and 4.14 ppm respectively and two protons on a cis-conjugated double bond as a pair of doublets at 5.53 and

5.71 (J = 6 Hz). The latter was supported by UV absorption (286 nm log ɛ 3.9) due to the homoannular diene system in the molecule.40

The antioxidant properties of different parts of chestnut (flower, leaves, skins, and fruits) extracts were evaluated through several biochemical assays: DPPH

(1, 1-diphenyl-2-picrylhydrazyl) radical-scavenging activity. EC50 values were also calculated for all the methods in order to evaluate the antioxidant efficiency of each extract from which skin of chestnut revealed the best properties. Phenol and flavonoid contents were also obtained.25

C. indica showed anti-viral activity in Central Drug Research Institute Lucknow (CDRI)ʼs biological screening program.41

The methanol extract of C. indica leaves was studied on the Central Nervous System (CNS) using a number of neuropharmacological experiment models in Swiss albino mice. MECI in a dose-dependent manner significantly reduced the immobility time of mice in both forced swim test (FST) and tail suspension test (TST) and suggested that the MECI has an analgesic effect, behave as a potential anticonvulsant and CNS depressant action.42

It was reported that methanol extract of C. indica leaves also showed direct cytotoxicity of Ehrlich ascites carcinoma (EAC) cell in a dose-dependent manner and there was significant decrease in the tumor volume, viable cell count, tumor weight and elevated the life span of EAC tumor-bearing mice.43

The phytochemical analysis revealed that methnolic extract of leaves of C. indica contained flavonoids, saponin, tannins, steroids, and glycosides. Antioxidant and hepatoprotective properties of methanol extract of C. indica was evaluated. The extract showed significant activities in all antioxidant assays compared to the reference ascorbic acid in a dose-dependent manner. In carbon tetrachloride intoxicated rat, C. indica is a potential hepatoprotective agent via its free radical scavenging.10

Research on the effects of β-sitosterol glucoside from C. indica leaves showed β-sitosterol glucoside (BSSG) is a natural biologically active substance isolated from the C. indica leaves. This study explored the apoptogenic mechanism studies of BSSG against Ehrlichʼs ascites carcinoma (EAC) treated mice

13 through morphological study, comet assay, flow cytometry (FACS) and Western blotting assay method.44

The antibacterial activity of methanol extract showed resistance at the lowest concentration (1 µl) to all isolate except Methicillin-resistant S. aureus (MRSA) 6 i.e., 10 mm inhibition zone. At highest concentration (100 µl) the highest antibacterial activity was detected against MRSA 6 (25 mm inhibition zone) and its lowest antibacterial activity was detected against MRSA 5 (resistant).45

A recent study on the plant regarding phytochemical screening, antioxidant activity, total phenolic, and total flavonoid content was published which showed C. indica has potent antioxidant activity with higher total phenolic and flavonoid content in both ethanol and an aqueous solvent.46

The structure of a few of them are given in figure no. 5.39,40

S.No. Compounds Structure

1 β -sitosterol

2 Psoralen

3 Castanopsin

4 Ursolic acid

Figure 5: Structure of compound found in Castanopsis indica

CHAPTER III

MATERIALS AND METHODS

3.1 Plant Extracts 3.1.1 Collection and Identification of Plant Samples The fresh leaves and bark of C. indica were collected from Arghakhanchi District. The taxonomic identification of the plants was done at National herbarium and plant laboratories (KATH).

3.1.2 Sample Preparation The collected fresh leaves and stem of C. indica were washed with tap water to remove the contaminants. Then the leaves were shade-dried. The shade dried leaves were grounded into powder form in an electric grinder and stored in a clean plastic bag until the further use.

3.1.3 Extraction Process

The phytochemicals present in the powdered leaves and bark were extracted by Soxhlet extraction process using methanol and hexane as solvent.

Soxhlet Extraction Process: Powdered leaves and bark (60 grams) of Castanopsis indica were wrapped in a filter paper and kept in clean and dry soxhlet. The soxhlet was fitted to the condenser from upside and to the downside round bottom flak with solvent (300 ml). The instrumentation was set up and the heat was given to the RB flask with the help of heating mantle maintaining the temperature at 40 ⁰C. The condenser was fitted to the regular water supply through the tap. After half an hour the solvent starts to evaporate and the sample absorbs the solvent. And then after half an hour, the solvent from the soxhlet runs down to the RB flask and the process is continued till 6 hours. The process is continued until the hexane and methanol solvent running down to the RB flask is almost colorless. The concentrated filtrates were kept in a beaker wrapping with aluminum foil containing small pores to facilitate the evaporation of the solvent. Then next day it was concentrated more in rota

16 evaporator and finally kept in hot water bath for half day to obtain the semi- solid methanol and hexane extracts.

The percentage yields of the extracts were calculated using the following formula

Wt. of the extract (in g) % Yield of Extract = × 100% ... (1) Wt. of the powdered leaves (in g)

Plant material Proximate analysis

Hexane extract Marc

Marc Methanol extract

1. Phytochemical analysis 1. Phytochemical analysis 2. Total phenolic content 2. Total phenolic content

3. Total flavonoid content 3. Total flavonoid content

4. Antioxidant assay 4. Antibacterial assay 5. Antioxidant assay

6. Brine shrimp bioassay Figure 6: Scheme for extraction, fractionation, and analysis of bark and leaves of C. indica

3.1.4 Phytochemical Analysis

The method employed for phytochemical screening was based on the procedure put forward by Prof. I. Ciulei.47

3.1.5 Antioxidant Activity

Antioxidant activity of different plant extracts was done by DPPH radical scavenging method as described by Blois (1958).48

The percentage of the DPPH free radical scavenging activity was calculated by using the following equation:

Radical scavenging (%) = [(Ao-As)/Ao] × 100 ………. (2)

Where A0 = Absorbance of the control (DPPH solution + methanol)

As = Absorbance of the test sample

The IC50 value (50% inhibitory concentration) is the effective concentration of the sample that is required to scavenge 50% of the DPPH free radicals present in the solution. IC50 values were calculated using the inhibition curve by plotting extract concentration versus the corresponding % of free radical scavenging activity.

3.1.5.1 Preparation of 0.2 mm DPPH solution

0.0078 g DPPH was weighed out and dissolved by methanol in 100 mL volumetric flask and final volume was made up to the mark to make 0.2 mM DPPH solution. Then it was stored in dark until further use.

3.1.5.2 Preparation of ascorbic acid solution (Standard)

10 mg ascorbic acid was weighed out and dissolved in 10 mL methanol to make the stock solution of 1000 µg/ml (ppm). Then by serial dilution, ascorbic acid solutions having concentration 20 ppm, 40 ppm, 60 ppm, 80 ppm, and 100 ppm were prepared.

3.1.5.3 Preparation of sample solutions

Firstly 15 mg extracts (methanol and hexane) were weighed out and dissolved in 15 mL methanol to make the stock solution of 1000 µg/ml (ppm). Then by

18 serial dilution, extract solutions having concentration 20 ppm, 40 ppm, 60 ppm, 80 ppm, and 100 ppm were prepared.

3.1.5.4 Measurement of DPPH radical scavenging activity

2 mL ascorbic acid solution from each concentration was pipetted out and mixed with 2 mL 0.2 mm DPPH solution in triplicate and kept in dark for 30 minutes. 2 mL methanol was mixed with 2 mL 0.2 mm DPPH solution and kept in dark also. Then their absorbance value was measured at 517 nm by using spectrophotometer against methanol and DPPH as a blank.

Similarly, absorbance value for extract and DPPH solution was measured following the same procedure as ascorbic acid.

Finally, the calibration curve was drawn taking sample concentration as X-axis and % radical scavenging activity as Y-axis for both ascorbic acid and sample solution and IC50 values were calculated.

3.1.6 Total Phenolic Content

The amount of total phenolics in different extracts was determined according to the Folin-ciocalteu procedure using gallic acid as a standard.49

3.1.6.1 Preparation of the standard gallic acid solution

Firstly, 1000 µg/mL stock solution of gallic acid was prepared by dissolving 10 mg gallic acid in 10 mL methanol. Various concentrations of gallic acid such as 20, 40, 60, 80 and 100 µg/mL were prepared by serial dilution of the stock solution.

3.1.6.2 Construction of the calibration curve

Gallic acid solution (1 mL) from each concentration was poured into test tubes. Then, 5 mL of 10 % Folin-Ciocalteu reagent (FCR) and 4 mL of 7 % sodium carbonate solution (Na2CO3) were added to these test tubes to get a total volume of 10 mL. The blue colored mixture was shaken well and incubated for 30 minutes at 40 ºC in a water bath. Finally, the absorbance of the solution was measured at 760 nm wavelength using spectrophotometer against a blank solution containing all reagents except gallic acid.

All the experiments were carried out in triplicate. The average absorbance values obtained at different concentrations of gallic acid were used to plot the calibration curve.

3.1.6.3 Preparation of the sample solution

Stock solution (10000 µg/mL) of the extract was prepared by dissolving 50 mg extract in 5 mL methanol. Then triplicate of concentrations of the extract 1000 µg/mL was prepared by serial dilution and their absorbance values were measured following the same procedure as explained above for gallic acid.

3.1.6.4. Calculation of the total phenolic content (TPC)

The total phenolic content was calculated using the following relation,

cV C = ……….. (3) m

Where, C = Total content of the phenolic compounds

(mg/g) in gallic acid equivalent (GAE)

c = Concentration of gallic acid established from calibration curve

3.1.6.5 Statistical analysis

Data were recorded as a mean of three determinations of absorbance for each concentration, from which the linear correlation coefficient (R2) value was calculated. The regression equation is given as,

Y = mx + c ……… (4) Where, y = Absorbance of the extract

m = Slope from the calibration curve

x = Concentration of the extract

c = Intercept

Using this linear equation, the concentration of the extract was calculated. Thus with the calculated value of the concentration of the extract, the total phenolic content was calculated from the equation (3).

3.1.7 Total Flavonoid Content (TFC)

The total flavonoid content of the plant extract was determined by aluminum chloride colorimetric assay using Quercetin is as a standard.50

3.1.7.1 Preparation of the standard quercetin stock solution

Quercetin stock solution of concentration 1000 µg/mL (ppm) was prepared by dissolving 20 mg of quercetin in 20 mL of methanol. Then various concentrations of quercetin such as 20, 40, 60, 80 and 100 µg/mL were prepared by serial dilution of the stock solution. An aliquot of 1 mL quercetin of each concentration in methanol was poured into 20 mL test tube containing 4 mL distilled water. Then, at the zero time, 0.3 mL 5% NaNO2 was added to the test tube. After 5 minutes, 0.3 mL of 10% AlCl3 and after 6 minutes 2 mL of 1 M NaOH were added to the mixture. Immediately the total volume of the mixture was made up to 10 mL by the addition of 2.4 mL distilled water and mixed thoroughly. Finally, the absorbance of the pink color mixture was measured at 510 nm wavelength using spectrophotometer against a blank solution containing all the reagents except quercetin. The average absorbance values obtained for different concentrations of quercetin were used to plot the calibration curve.

3.1.7.2 Preparation of the sample solutions

Stock solution (10000 µg/mL) of the extract was prepared by dissolving 50 mg extract in 5 mL methanol. Then triplicate of concentrations of the extract 1000 µg/mL was prepared and their absorbance values were measured following the same procedure as explained above for quercetin.

3.1.7.3 Calculation of the total flavonoid content

The following formula was used to calculate the total flavonoid content of the extract, cV C = …………….… (5) m

Where, C = Total Flavonoid Content (in mg/g)

in Quercetin Equivalent (QE)

c = Concentration of quercetin established

from calibration curve in mg/mL

V = Volume of the extract (in mL)

m = Weight of the plant extract (in g)

3.1.7.4 Statistical analysis

Data were recorded as a mean of three determinations of absorbance for each concentration, from which the Linear Correlation Coefficient (R2) value was calculated. The regression equation is given as,

Y = mx + c …… (6)

Where, y = Absorbance of the extract

m = Slope from the calibration curve

x = Concentration of the extract, c = Intercept

Using this linear equation, the concentration of the extract was calculated. Thus with the calculated value of the concentration of extract, the flavonoid content was calculated by equation (5).

3.1.8 Bioassay

Biological screening is the study of the effect of the crude plant extracts or fractions or isolated compounds at arbitrarily fixed dose levels in a species of an organism and predicting its effect over the entire dose range. In this

22 dissertation work, Castanopsis indica is used as the general bioassay tool to perform brine shrimp bioassay as given by Meyer et al.51

3.1.8.1 Brine Shrimp Bioassay

Brine shrimp, Artemia salina is a tiny crustacean. The eggs of brine shrimp are readily available at low cost and they remain viable for years in the dry state. Upon being placed in a brine solution, the eggs hatch within 48 hours providing a large number of larvae (nauplii). These larvae are used in this assay for biological screening. This method is the rapid, inexpensive, simple and in-house approach for screening and monitoring physiologically active plant extracts.

It determines the LC50 values (µg/mL) for the crude extracts. Compounds having LC50 values less than 1000 ppm (µg/mL) are considered as potentially pharmacologically active.

3.1.8.2 Required Materials

i. Eggs of brine shrimp ii. Artificial seawater iii. Beakers for hatching iv. Table lamp v. Disposable pipette vi. Micropipette vii. Test tubes and test tube stand

3.1.8.3 General Procedure of Brine Shrimp Bioassay

3.1.8.3.1 Sterilization of the apparatus

All the apparatus used in the experiment were sterilized before their use.

3.1.8.3.2 Preparation of the artificial sea water

Sea water was prepared by dissolving following chemicals in 1 Liter of distilled water.

Table: 1 Chemical composition of sea water

S. N. Composition Amount (g/L) 1. NaCl 23.50

2. Na2SO4 4.00 3. KCl 0.68

4. H3BO3 0.027

5. MgCl2.2H2O 10.68

6. CaCl2.2H2O 1.48

7. NaHCO3 0.197

8. Na2EDTA 0.0003

3.1.8.3.3 Hatching of the brine shrimp eggs

About 50 mg eggs of brine shrimp were sprinkled on the artificial seawater taken in the beaker and the beaker was covered with aluminum foil. Several small pores were made to facilitate the passage of heat and light. Then the beaker was kept for 48 hours illuminating with the bulb (60 Watt) at room temperature.

3.1.8.3.4 Preparation of samples

Extract (20 mg) was weighed out and dissolved in 2 mL methanol to make a stock solution of concentration 10,000 ppm (µg/mL). From that stock solution, solutions of concentration 1000 µg/mL, 100 µg/mL, and 10 µg/mL were prepared by serial dilution method. 2 mL solutions from each solution (1000 ppm, 100 ppm, and 10 ppm) were transferred to nine different test tubes, three for each concentration. Similarly, 2 mL methanol was taken in three test tubes (as a blank). After labeling these test tubes, they were kept for 24 hours to evaporate the solvent (methanol).

3.1.8.3.5 Procedure for bioassay

After the complete evaporation of the solvent, 5 mL artificial sea water was added and the solution was mixed thoroughly to suspend the residue. Then ten mature brine shrimp nauplii were transferred into all twelve test tubes. After 24 hours, the numbers of the survivors were counted with the help of the disposable pipettes.

3.1.8.3.6 Data analysis

LC50 value is the lethal concentration dose required to kill 50 % of organisms used in bioassay. It can be determined as follows,

If ‘n’ is the number of replicates (here three), ‘x’ is the log of the concentration of the solution in µg/mL (log10, log100 and log1000 in this experiment) and ‘y’ is the Probit for average survivors for all replicates, Then we have,

[Σy -βΣx] α = ………………..… (7) n Now, From Probit regression,

Σx Σy Σx - y n β = ……..…….…… (8) (Σx)2 Σx2 - n

Now, from probit regression,

Y = α + βx ……………………...……….……. (9)

X = (Y-α)/β……………………………..…..… (10)

Where Y is a constant having value 5 for calculating LC50 value.

Hence,

LC50 = Antilog (X) …………………………… (11)

3.2 Antimicrobial Assay

Inhibition of bacterial growth was tested by using agar well diffusion plate method and measured in the form of the zone of inhibition (ZOI).52 Antibacterial assay was performed at NIST college.

3.2.1 Collection of Test Organisms

The bacterial strains on which antibacterial assay was carried out on three bacteria Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus. They were available in NIST College, Kathmandu.

3.2.2 Preparation of Solution of Extracts

The methanol extract of C. indica (50 mg) was dissolved in 50% dimethyl sulphoxide (DMSO) in water. Tubes were capped and stored in the refrigerator at 4 oC until further use.

3.2.3 Preparation of Muller Hilton Agar (MHA) Plates

The 9.5 g MHA media was dissolved in 250 mL distilled water in a conical flask warming on a hot plate. The conical flask was cotton-plugged, sealed with the aluminum foil and sterilized in an auto Clave at 121 ºC for 15 minutes. After cooling the media to room temperature, it was poured into the clean, dry and autoclaved Petri dishes (20 mL per plate) and allowed to set at room temperature. When the agar solidified, the plates were sealed with parafilm and stored in the refrigerator until further use.

3.2.4 Nutrient Broth (NB) Solution

The 0.65 g NB powder was weighed out and completely dissolved in 50 mL distilled water (13 g/L) in a conical flask warming on the hot plate. The conical flask was cotton-plugged, sealed with aluminum foil and sterilized in an autoclave. The solution was then allowed to cool down at room temperature and stored in the refrigerator until further use.

3.2.5 Preparation of Standard Culture Inoculums

The test organisms to be tested were aseptically touched with the help of the inoculating loop from the primary culture plate and transferred to a test tube containing 10 mL of sterile nutrient broth solution. The tube was then incubated for 24 hours at 37 oC.

3.2.6 Transfer of Bacteria in Petri Plates

The MHA plates for the assay were labeled with the name of the bacteria and the number of plates. The inoculums of bacteria were transferred into a petri dish containing solid nutrient media of agar using sterile swab. One swab was used for one species of bacteria only. The sterile cotton swab was dipped into a well- mixed saline test culture and removed excess inoculums by pressing the saturated swab against the inner wall of the culture tube. The swab was used to spread the bacteria on the media in a confluent lawn. Then, the Petri dishes were rotated at an angle of 90º to spread bacteria. The culture plates were allowed to dry for 5 minutes.

3.2.7 Screening and Evaluation of Antibacterial Activity

The wells were made in the incubated Petri plates with the help of sterile cork borer of the diameter of 6 mm and were labeled properly. Then, 15 µl of the working solution of the plant extracts were loaded into the respective wells with the help of micropipette. The plates were then left for half an hour covered with a lid so that extracts diffuse into the media. Then Petri plates were incubated overnight at 37°C.

After 24 hours, Petri plates were then observed for the zone of inhibition (ZOI) produced by the antibacterial activity of plant extracts. The inhibition zones were measured by the use of a scale. The positive control was taken as Neomycin and DMSO as that of the negative control.

3.2.8 Screening and Evaluation of the Antifungal Activity

The wells were made in the incubated media plates with the help of sterile cork borer of the diameter of 4mm and labeled properly. Then, 15 µl of the working solution of the plant extracts were loaded into the respective wells with the help of micropipette. The plates were then left for half an hour with the lid closed so that extracts diffuse into the media. The plates were incubated overnight for 24 hrs at 37°C. The plates were then observed for the zone of inhibition (ZOI) produced by the antifungal activity of plant extracts and the inhibition zones were measured by the use of a scale.

3.3 Proximate Nutritional Analysis of Castanopsis indica Nut

Proximate analysis is done to determine the amount of exact nutrients present in a particular food. The major nutrients such as carbohydrates, proteins, fats, crude fibres, minerals and vitamins helps in growth and proper functioning of a body and knowing their actual amount in any food helps in making a balanced diet. Also, precise proximate analysis helps in puissant documentation of the food which consequently helps upgrade the market of the local food in regional, national or international levels. Proximate nutritional analysis was performed according to AOAC Official Method of Analysis.53

3.3.1 Determination of Moisture 5 g of the fresh sample was weighed and kept in a tared shallow porcelain dish and evenly distributed by shaking the content. Then the sample was dried for 12 -18 hours at 100 ± 5 ºC in a hot air oven. The content was cooled in the desiccator and weighed, then the content was reheated and weighed repeatedly until the difference in weights was 1 mg. Finally, moisture content was calculated by the formula,

Finally, moisture content was calculated by the formula, W – W % Moisture = 1 2 × 100...... (12) W1 -W Where,

W1 = weight in g of the dish with the material before drying

W2 = weight in g of the dish with the material after drying

W = weight in g of the empty dish after heating and cooling

3.3.2 Determination of Total Ash 5 g of the dry sample was taken in a tared crucible and the content was charred for two hours at 100 ± 5 ºC in a hot air oven. Then moisture was incinerated in a muffle furnace at 600 ± 20 ºC till the content became white. The crucible was cooled partly on the asbestos sheet and then in a desiccator and the weights were taken. The process of reheating, cooling and weighing was repeated until the difference in weights was 1 mg.

Finally, total ash content was calculated by the formula,

W2 – W % Total Ash = × 100...... (13) W1

Where, W2 = weight in g of the crucible with ash

W1 = weight in g of the sample taken

W = weight in g of the empty crucible

3.3.3 Determination of Crude Fat 2 g of the dry sample was taken in the thimble of Whatman no. 1 and the open end was plugged with cotton to prevent sample loss. Then the sample in the thimble was subjected to fat extraction in the Soxhlet extractor using petroleum ether as a solvent. The weight of the collected fat in the beaker was weighed after drying and cooling in the desiccator. The process of reheating, cooling and weighing was repeated until the difference in weights was 1 mg.

Finally, crude fat content was calculated by the formula,

W1 – W2 % Crude Fat = × 100...... (14) W Where,

W1 = weight in g of the extraction flask with dried extract

W2 = weight of the extraction flask

W = weight in g of the material take for extraction

3.3.4 Determination of Protein The protein content of the sample was estimated using the Kjeldahl Method. Reagents Used,

a. the mixture, CuSO4.5H2O, and K2SO4 in the ratio 1:5

b. Conc. H2SO4 (sp. gr. 1.84) c. 40% NaOH

d. 4% Boric Acid (20 g acid in 500 ml water and 5 drops of methyl red) e. 0.1% Methyl red (0.1 g methyl red in 100 ml spirit) f. 0.1% Bromocresol green indicator

g. Standard H2SO4 (0.05 N)

Magnesium oxide – carbonate free

3.3.4.1 Total Nitrogen

0.5 g of accurately weighed sample was transferred to Kjeldahl flask and 2 g of digestion mixture and 10 ml of conc. sulphuric acid was added to it. The flask was then adjusted to the hot plate, in the exhaust tube and heated first at a low temperature until the frothing ceased then heated to boil the acid vigorously and digested for 3 hours. After the mixture was clear, the content of the flask was cooled, the content was diluted with 10 ml distilled water and transferred in 100 ml volumetric flask. The digestion flask was rinsed several times and transferred to the volumetric flask and the solution was made up to the mark. Then blank digestion was carried out without the sample.

Micro-Kjeldahl distillation unit was set up and the standard run to clear out impurity was carried out. Then the run with the sample was carried out for distillation whose content finally received in the conical flask was titrated with the standard sulphuric acid (0.1 N) that changed the color of the solution to pink. The blank distillation was also carried out.

Finally, total nitrogen content was calculated by the formulae

Titre × normality × 1.4 × vol. made

% Total Nitrogen by weight = Aliquot of digest ×wt. of sample ×100 × 100 ... (15)

Protein (%) = Total N2, percent by weight × 6.25 …………………………………..…..(16)

3.3.5 Determination of Crude Fiber

2 g of defatted sample was taken in a 750 ml Erlenmeyer flask with 200 ml of

1.25% H2SO4 (0.255 N) and boiled and refluxed for 30 minutes. The solution

30 was boiled and washed to make acid free. The residue was transferred to the flask and boiled and refluxed with 200 ml of 1.25% NaOH (0.3134 N) and filtered and washed. Then the residue was transferred to gooch crucible and washed with 15 ml of 95% alcohol and then dried in a hot air oven at 100 ± 1 ºC. The content was finally cooled and weighed. Thus, the content was transferred to muffle furnace at 600 ± 20 ºC for 3 – 4 hours for ashing.

Finally, the crude fiber was calculated by the formula,

W1 – W2 % Crude Fibre = × 100 ...... (17) W Where,

W1 = weight of gooch crucible with content before ashing

W2 = weight of gooch crucible with containing asbestos and ash

W = weight in g of the material taken for a test

3.3.6 Estimation of Carbohydrate

The proximate analysis of the nutritional content of the food material followed the direct estimation of the carbohydrate content of the material by subtracting the major nutritional content from 100 (percent) without following other particular protocol to estimate the quantity of carbohydrate.

3.3.7 Estimation of Vitamin C

The estimation of vitamin C in the fresh sample of fruit was carried out by 2,6–dichlorophenol-Indophenol visual titration method.

3.3.7.1 Preparation of Dye

50 g sod. salt of 2, 6 – dichlorophenol-indophenol was mixed in 150 ml hot distilled water containing 42 g NaHCO3. Then the solution was cooled and diluted with 200 ml water and stored in the refrigerator. The dye was standardized every day before use.

3.3.7.2 Standardization of Dye

5 mL of standard ascorbic acid along with 5 mL HPO3 was taken in a conical flask. The micro burette was filled with the dye and the titration was carried out till the pink color persisted for 15 s. Then the dye factor, mg of ascorbic acid per ml of dye, was determined using formula,

0.5 Dye Factor = ……………………. (18) Titre

3.3.7.3 Preparation of Sample

10 – 20 ml of fresh plant juice was made up to 100 ml in volumetric flask by adding 3% HPO3. Then the solution was filtered or centrifuged which was titrated. For the assay of extract, 2 – 10 ml of an aliquot of 3% HPO3 of the sample was taken and titrated with the standard dye to a pink endpoint.

Elimination of Interference due to SO2

SO2 if present reduces the activity of dye and interferes with ascorbic acid analysis. So SO2 elimination by formaldehyde condensation was carried out by mixing 10 ml of filtrate, 1 ml 40% HCHO, 0.1 ml HCl kept for 10 minutes before titration was carried out as before.

Finally, Vitamin C was calculated by using formula,

Dye factor × V2 × V mg of ascorbic acid per 100 g or ml = × 100 ...... (19) V1 × W

Where V2 = volume of dye required

V1 = volume of sample extract taken for dye titration, V = volume of the sample made up with HPO3 solution, W = weight of sample taken for extraction with

HPO3

CHAPTER IV

RESULTS AND DISCUSSION

4.1 Identification of Selected Plant

The plant was collected from Arghakhanchi District of Nepal and the selected plant was identified by Ganga Dutta Bhatta (National Herbarium and Plant laboratories) and taxonomically acknowledged as C. indica (Roxb. Ex Lindl.) A. DC.

Table 2: Sample collection and identification

Identified plant Common name Family Place of collection Castanopsis indica Katus Fagaceae Arghakhanchi District

4.2 Percentage Yield

Table 3: Table showing % yield of methanol and hexane extract

A specific part of the plant Methanol Extract Hexane Extract Leaves 22.58 5.07 Bark 26.32 2.81

The percentage yield of methanol and hexane extracts of Castanopsis indica leaves and bark were found to be 22.58%, 5.07%, 26.32%, 2.81% respectively.

4.3 Qualitative Analysis of Phytochemicals

The micro-chemical analysis of a crude extract of Castanopsis indica leaves and bark in methanol and hexane extract depicted the presence of a class of phytochemicals as shown in the below table.

Table 4: Phytochemical analysis of leaves and bark of C. indica S.N. Classes of Hexane Methanol Hexane Methanol phytochemicals extract of extract of extract of extract of leaves leaves bark bark 1 Phenolic + + + + compounds 2 Alkaloids - - - - 3 Terpenoids + + + + 4 Steroids + + + + 5 Saponins + + + - 6 Tannins + + + + 7 flavonoids + + + + 8 Glycosides - + - + Where ʽ+ʼ means presence and ʽ-ʼ means absent

4.4 Antioxidant Activity The antioxidant activity of different extracts of Castanopsis indica was obtained by plotting % of free radical scavenging vs concentration and IC50 values of respective extracts were calculated.

The DPPH radical assay was performed for each plant extract by using Ascorbic acid as standard according to the standard procedure. In this assay, the mixture of DPPH with different concentrations of extract solution and Ascorbic acid were separately incubated at room temperature and absorbance was recorded at 517 nm by a spectrophotometer. The observed absorbance with the different concentration of Ascorbic acid was plotted in the graph as shown in figure no. 7-11.

100 90 80

70 y = 0.8569x + 1.3452

60 R² = 0.9954

50 % scv Ascorbic acid 517nm 40

30 Linear (% scv

% OF FREE RADICAL SCAVENGING RADICAL FREE OF % Ascorbic acid) 20 10 0 0 20 40 60 80 100 120 CONCENTRATION

Figure 7: Antioxidant activity of Ascorbic acid

100 90 80 70 60 50

40 % scv of ascorbic acid 30 % scv of methanol leaf 20

% OF FREE RADICAL SCAVENGING RADICAL FREE OF % 10 0 0 20 40 60 80 100 120 CONCENTRATION

Figure 8: Antioxidant activity of methanol extract of Castanopsis indica leaf

The IC50 value of methanolic extract of C. indica leaf was 64.95 µg/ml as compared to that of standard IC50 value of ascorbic acid 56.78 µg/ml.

100 90 80 70 60 50 40 % scv of ascorbic acid 30 % scv of methanol bark 20

% OF FREE RADICAL SCAVENGING RADICAL FREE OF % 10 0 0 20 40 60 80 100 120 CONCENTRATION

Figure 9: Antioxidant activity of methanol extract of Castanopsis indica bark

The IC50 value of methanolic extract of C. indica bark was 74.98 µg/ml as compared to that of standard IC50 value of ascorbic acid 56.78 µg/ml.

100 90 80 70 60 50 40 30 % scv of ascorbic acid

% OF FREE RADICAL SCAVENGING RADICAL FREE OF % 20 % scv of hexane leaf 10 0 0 20 40 60 80 100 120 CONCENTRATION

Figure 10: Antioxidant activity of hexane extract of Castanopsis indica leaf

The IC50 value of hexane extract of C. indica leaf was 114.62 µg/ml as compared to that of standard IC50 value of ascorbic acid 56.78 µg/ml.

100 90 80 70 60 50 % scv of ascorbic acid 40 % scv of hexane bark 30 20

% OF FREE RADICAL SCAVENGING RADICAL FREE OF % 10 0 0 20 40 60 80 100 120 CONCENTRATION

Figure 11: Antioxidant activity of hexane extract of Castanopsis indica bark

The IC50 value of hexane extract of C. indica bark was 124.41 µg/ml as compared to that of standard IC50 value of ascorbic acid 56.78 µg/ml.

From the study of the antioxidant property, IC50 value of different extracts were calculated and compared with IC50 value of ascorbic acid. Antioxidant activity is inversely proportional to the IC50 values i.e. extract or fraction or compounds having small IC50 values are more potent antioxidant than those having larger

IC50 values. Among these four extracts, methanol extract of leaves of C. indica showed the highest antioxidant activity. The methanol extract of bark showed the antioxidant value of 74.98 µg/ml and that of the leaf was found out to be 64.95 µg/ml showing leaf to be more potent antioxidant than bark. The hexane extract of bark and leaves, however, showed very high IC50 value indicating that the extract does not show significant antioxidant activity.

The high content of phenolic compounds is known to have direct antioxidant property due to the presence of hydroxyl groups which can function as hydrogen donor. The scavenging ability of the phenolics is mainly due to the presence of hydroxyl groups. The result reflects that the plant studied above can act as a very good option in the field of medicine based on the antioxidant property of natural product chemistry.

4.5 Total Phenolic Content and Flavonoid Content

In this study, total phenolic content is presented in gallic acid equivalents while total flavonoid content is in quercetin equivalents. The phenolic content is most widely present phytochemicals in plants which acts as an antioxidant and thus reduce the risk of oxidative stress-induced diseases.

0.35

0.3

0.25

0.2 y = 0.003x - 0.0101 0.15 R² = 0.9909

ABSORBANCE 0.1 Absorbance 0.05 Linear (Absorbance)

0 0 20 40 60 80 100 120 -0.05 CONCENTRATION(PPM)

Figure 12: Absorbance vs concentration curve for gallic acid

Using calibration curve and absorbance values (triplicate of 1000 µg/ml) total phenolic content of methanol and hexane extracts of leaves and bark of C. indica was calculated. Total phenolic content of methanol extract of leaves and bark was calculated as 153.58 and 139.47 mg per gram Gallic acid equivalent (mg GAE/gm) respectively. Similarly, the total phenolic content of hexane extract of leaves and bark was calculated as 35.7 and 30.14 mg per gram Gallic acid equivalent (mg GAE/gm) respectively. It has been reported that the extract containing a large amount of polyphenol content possess a greater antioxidant activity.

Table 5: Total phenolic content of Castanopsis indica extracts

S.N. TPC Methanol extract Hexane extract (mg GAE/gm) (mg GAE/gm) 1 Leaves 153.58 35.7 2 Bark 139.47 30.14

Total phenolic content was found varied in between methanol and hexane extract of plant samples. Total phenolic content of methanolic extracts were found higher than hexane extract. The result indicating that phenolic compounds are generally more soluble in polar organic solvent than in non polar hexane solvent.

Flavonoids are known to have a wide range of biological and pharmacological activities. Its content is calculated by taking quercetin as standard.

0.08

0.07

0.06

0.05

0.04 y = 0.0007x + 0.0004 0.03 ABSORBANCE R² = 0.991 0.02 absorbance

0.01 Linear (absorbance)

0 0 20 40 60 80 100 120 CONCENTRATION(PPM)

Figure 13: Absorbance vs concentration curve for Quercetin

Using calibration curve and absorbance values (triplicate of 1000 µg/ml) total flavonoid content of methanol and hexane extracts of leaves and bark of C. indica was calculated. Total flavonoid content of methanol extract of leaves and bark was calculated as 138.46 and 124.18 mg per gram quercetin equivalent (mg QE/gm) respectively. Similarly, the total flavonoid content of hexane extract of leaves and bark was calculated as 34.66 and 24.59 mg per gram quercetin equivalent (mg QE/gm) respectively.

Table 6: Total Flavonoid content of Castanopsis indica extracts

S.N. TFC Methanol extract Hexane extract (mg QE/gm) (mg QE/gm) 1 Leaves 138.46 34.66 2 Bark 124.18 24.59

From the data obtained for TFC, flavonoid content of methanol extract of leaves was highest followed by that of methanol extract of bark whereas the rest of the extract had low values. Both the leaves and bark methanol extracts contained significant flavonoid and phenolic content than the extract obtained from hexane fraction attributing to the higher activity of methanol extract.

4.6 Antimicrobial Activity

The diameter of zone of inhibition (ZOI) produced by plant extract on particular bacteria was measured for the estimation of their antimicrobial activity. The methanol extract of C. indica was found to be resistant against gram-positive bacterial strain. The result obtained from the antibacterial screening of the extract is tabulated in table no. 7.

Table 7: Antimicrobial activity of methanol extract of bark and leaves

S.N. Bacteria Reference ZOI Value culture (mm) Positive Negative Methanol Methanol control Control Bark Leaves Neomycin DMSO 1 Escherichia ATCC 24 0 0 0 coli 25922

2 Staphylococcus ATCC 19 0 15 18 aureus 25923

3 Salmonella ATCC 21 0 0 0 typhimurium 14028 ZOI = zone of inhibition

The zone of inhibition for E. coli, S. aureus, S. typhimurium were found to be 0 mm, 15 mm and 0 mm respectively for methanol extract of bark of C. indica. This showed that the plant did not show any antimicrobial activity for E. coli, S. typhimurium whereas it was found to be potent against gram-positive S. aureus. Similarly, the zone of inhibition for E. coli, S. aureus, S. typhimurium were found to be 0 mm, 18 mm and 0 mm respectively for methanol extract of leaf of C. indica which showed similar activity as of bark i.e. both extracts were active against S. aureus.

4.7 Antifungal Activity

The table summarizes the results obtained from the determination of antifungal activity of the methanol fractions of Castanopsis indica stem and leaves at fixed concentration (50 mg/ml) on the tested organisms according to the procedure described in section and the results are expressed in diameter of zone of inhibition (mm) including the diameter of well (6 mm).

Table 8: Antifungal activity of the methanol extract of the Castanopsis indica

S.N. Fungal strain Positive control Zone of Inhibition (mm)

(Itraconazole) mm Methanol bark Methanol leaves

1 Candida albicans 17 - -

Both methanol extract of bark and leaves did not show any antifungal activity against Candida albicans.

4.8 Brine Shrimp Bioassay

Brine shrimp assay of methanol extract of leaves and bark of C. indica were done to assess the toxicity of these extract. The newly hatched brine shrimp nauplii were exposed to different concentrations of extracts (10 µg/mL, 100 µg/mL and 1000 µg/mL) and their toxicity towards nauplii was evaluated by calculating LC50 (µg/mL) value. LC50 is defined as the concentration that kills

50% of test organisms exposed to it. Extracts having LC50 values less than1000 µg/mL are supposed to be pharmacologically active or toxic. The calculation of the LC50 value of methanol extract of leaves and bark of C. indica was given in the following table.

Table 9: Calculation of LC50 value of methanol extract of leaves and bark of Castanopsis indica

2 Name Conc in x = No. of No. of xy x β α X LC50 = of µg/mL alive Replicate Anti (Z) log Z larvae the (y) (n) log X

Extract MeOH 10 1 8 8 1 Extract 100 2 6 3 12 4 of C. 1000 3 5 15 9 -1.5 9.33 2.88 758.57 indica leaf

Σx = 6 Σy= 19 Σxy=35 Σx2 = 14 MeOH 10 1 7 7 1 extract 100 2 6 12 4 of C. 1000 3 4 12 9 indica bark 3 Σx = 6 Σy= 17 Σxy=31 Σx2 = 14 -1.5 8.67 2.45 281.83

From the above table, LC50 value of methanol extract of C. indica leaves was calculated to be 758.57 ppm. The LC50 value is less than 1000ppm, the leaf extract was toxic towards Artemia salina larvae.

Similarly, the LC50 value of the methanol extract of C. indica bark was found to be 281.83 ppm which shows that bark extract is also toxic towards Artemia salina. As both values are less than 1000 µg/ml both extracts might be used in pharmaceutical synthesis.

4.9 Proximate Analysis of Castanopsis indica

Table 10: Percentage composition of nutritional parameters in a sample of Castanopsis indica in nut

S.N. Parameters % in nuts 1 Moisture 18.90 2 Total ash 1.26 3 Crude fat 0.30 4 Protein 10.83 5 Crude fiber 3.72 6 Carbohydrate 64.99 7 Vitamin C 4.84 mg/100g sample 8 Nutritive value 305.98 cal/100 g

The edible part of plant materials C. indica (nuts) collected from Arghakhanchi district has a relatively high carbohydrate content when compared to ash, protein, fat, fiber and moisture content.

From the above table, it can be inferred that the percentage composition of different nutrition parameters (moisture, total ash, crude fat, protein, crude fiber, carbohydrate) was found to be 18.90, 1.26, 0.30, 10.83, 3.72, 64.99 % in nuts sample respectively. Vitamin C was found to be 4.84 mg/100g. The nutritive value of nuts of C. indica was calculated to be 305.98 calories in 100 gram. The value showed a promising amount of nutrition is available on this plant. The nuts can be consumed directly. These quantitative values are represented in a pie-chart in figure no. 14.

Percentage composition in nuts

Moisture 19%

Moisture Total ash 1% Total ash Crude fat Crude fat 0.3% Protein Protein Crude fiber Carbohydrate 11% Carbohydrate 65% Crude fiber 4%

Figure 14: Percentage composition of nutritional parameters in nuts of Castanopsis indica

Thus, it can be ascertained that C. indica nuts are very rich in nutritional value and the result showed a lower percentage of fats and a higher percentage of protein depicting it to be very healthy if consumed properly.

CHAPTER V

CONCLUSIONS AND RECOMMENDATION

5.1 Conclusions

Phytochemical screening of methanol extracts of bark of C. indica showed the presence of polyphenols, terpenoids, glycosides, tannins, flavonoids, and saponins and that of leaves showed the presence of flavonoids, terpenoids, polyphenols, and saponins.

The IC50 values of different plant extracts exhibited that the methanol extract of C. indica leaves was the most potent natural antioxidant among all extracts which was confirmed by its comparable IC50 value to the standard. The methanol extract of C. indica bark also showed considerable antioxidant property. However, hexane extract of C. indica leaves and bark because of very high IC50 values are termed as a poor antioxidant.

Both methanol extract of leaves and bark contained a higher amount of phenolic content as compared to the corresponding hexane extracts. This might be one of the major factors for methanol extracts of C. indica leaves and bark exhibiting strong antioxidant activity. Among leaves and bark, TPC was found to be higher in leaves extracts.

Both methanol extract of leaves and bark contained a higher amount of flavonoid content as compared to the corresponding hexane extracts. Among leaves and bark, TFC was found to be higher in leaves extracts.

The percentage composition of different nutrition parameters (moisture, total ash, crude fat, protein, crude fiber, carbohydrate) was found to be 18.90, 1.26, 0.30, 10.83, 3.72, 64.99% in nuts sample respectively. Vitamin C was found to be 4.84 mg/100g. The nutritive value of nuts of C. indica was calculated to be 305.98 calories in 100 gram.

Among three microbes E. coli and S. typhimurium were gram-negative and S. aureus was gram-positive. The significant antimicrobial activity was shown by methanolic extract of leaf and bark in S. aureus whereas both extracts did not show antimicrobial activity against gram-negative bacteria.

The antifungal assay showed that there is no any inhibitory activity against the growth of the fungi used under study among the methanol leaves and methanol bark fraction.

From brine shrimp bioassay, it can be inferred that the bark and leaves of

Castanopsis indica have the LC50 values are less than 1000 so it’s pharmacologically active.

5.2 Suggestion for further work

This dissertation work has aimed at addressing the nutritional values of Castanopsis indica nuts. The phytochemicals are known to show a wide range of biological and pharmacological activity, it is desired to prepare the plant extracts in another major solvent apart from discussed here. So that wide varieties of phytochemical in higher amount can be extracted. Moreover, the plant extract can be subjected to other medicinal activity parameters such as anticancer test, analgesic, antiviral test. In vivo test can be done to further galvanized the result obtained here, as only in vitro test were monitored due to lack of budget.

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APPENDICES

A. Phytochemical Screening Protocol

1) Test for Volatile Oils To about 500 mg extract, 0.5 mL methanol was added, shaken vigorously and filtered. Few drops of the filtrate were put on a filter paper by means of a capillary tube. Yellow spot persistent even after evaporation of the solvent indicates the presence of volatile oils.

2) Test for Alkaloids About 500 mg extract was dissolved in 3 mL of 2 % (v/v) HCl. The solution was equally divided into two test tubes and the following tests were performed, i. Meyer’s Test Few drops of Meyer’s reagent were added to the first part. Formation of a pale yellow precipitate indicates the presence of alkaloids. ii. Dragendorff’s Test Few drops of Dragendorff’s reagent were added to the second part. Formation of an orange-red precipitate indicates the presence of alkaloids.

3) Test for Terpenoids

To about 200 mg extract, 2 mL of chloroform (CHCl3) and then 3 mL

concentrated sulphuric acid (H2SO4) was added carefully. Formation of reddish-brown coloration at the interface indicates the presence of terpenoids. 4) Test for Coumarins To about 1 mL of extract, 1 mL of 10 % sodium hydroxide (NaOH) solution was added. Formation of yellow color indicates the presence of coumarins.

5) Test for Flavonoids/Shinoda’s Test About 200 mg extract was dissolved in 2 mL methanol. To this solution, a small piece of magnesium and 4-5 drops of concentrated hydrochloric acid (HCl) were added. Formation of orange color indicates the presence of flavonoids.

6) Test for Quinones

To about 2 mL extract, 1 mL freshly prepared ferrous sulfate (FeSO4)

solution and few crystals of ammonium thiocyanate (NH4SCN) were

added and the solution was treated with conc. sulphuric acid (H2SO4) drop by drop. The appearance of persistent deep red coloration indicates the presence of quinones.

7) Test for Polyphenols/ FeCl3 Test To about 1 mL extract, 1 mL distilled water was added followed by the

addition of a few drops of 10 % (w/v) ferric chloride (FeCl3) solution. The appearance of greenish blue coloration indicates the presence of polyphenols.

8) Test for Glycosides About 500 mg extract was dissolved in 2 mL methanol and divided into two parts and the following tests were performed, i. Molisch’s Test The first part was treated with 5 mL of Molisch’s reagent and conc.

H2SO4 was added drop by drop from the side of the test tube without disturbing the solution. The appearance of a violet ring at the junction of two liquids which on shaking turns the solution into violet color indicates the presence of glycosides.

ii. To the second part 2 mL of 25 % (v/v) NH4OH solution was added and shaken vigorously. The appearance of the cherry red color indicates the presence of glycosides. 9) Test for Reducing Sugars To about 1 mL extract, 1mL distilled water was added followed by addition of 1 mL Fehling’s reagent (1,1 mixture of Fehling’s solution

A and B). Then the mixture was warmed over a water bath for 30 minutes. The appearance of a brick red precipitate indicates the presence of reducing sugars.

10) Test for Saponins About 500 mg extract was treated with hot water followed by shaking for 30 seconds. Formation of thick forth indicates the presence of saponins.

11) Test for Tannins About 200 mg extract was boiled adding 10 mL distilled water. The

mixture was cooled, filtered and few drops of FeCl3 solution were added to the filtrate. The appearance of blue-black precipitate indicates the presence of tannins.

B. Preparation of Reagents

1. Meyer’s Reagent

Mercuric chloride, HgCl2 (0.679 g) was weighed in a 50 mL volumetric flask and dissolved in distilled water. To this solution, 2.5 g potassium iodide (KI) was added. The scarlet red precipitate was dissolved by shaking and volume was made up to the mark by adding distilled water.

2. Dragendorff’s Reagent

Bismuth nitrate, Bi(NO3)3 (4.000 g) was dissolved in 5 N nitric acid (10 mL) to make solution A. Next, potassium iodide, KI (13.5 g) was dissolved in distilled water (20 mL) to make solution B. These two solutions were mixed together in a 50 mL volumetric flask.

Picric acid (0.25 g) was dissolved in 50 mL distilled water to make an aqueous picric acid solution. The solution was neutralized with sodium bicarbonate

(NaHCO3). A strip of Whatman no. 1 filter paper was dipped in the prepared solution. The paper was dried completely and protected from external

53 contamination. Thus prepared sodium picrate paper was used for Cyanogenic Glycoside detection.

3. Molisch’s Reagent

α -Naphthol (5.000 g) was dissolved in 50 mL methanol to prepare Molisch’s reagent.

4. Neutral Ferric Chloride (FeCl3) Solution,

Ferric chloride crystals (1.000 g) were dissolved in 100 mL distilled water. To this solution, sodium carbonate crystals were added little by little with stirring until the slight turbidity was persistent. Finally, the mixture was filtered and the colorless filtrate was used as neutral ferric chloride solution.

C. Antioxidant Activity

Table 11: Antioxidant activity of Ascorbic acid

S.N. Concentration Absorbance mean % of (ppm) scavenging 1 20 0.400 0.402 0.403 0.401 18.57 2 40 0.298 0.299 0.298 0.298 39.44 3 60 0.238 0.240 0.242 0.242 51.23 4 80 0.158 0.158 0.159 0.158 67.89 5 100 0.058 0.057 0.061 0.059 88.02 IC50 56.40 Blank 0.492

Table 12: Antioxidant activity of methanol extract of bark

S.N. Concentration Absorbance mean % of (ppm) scavenging

1 20 0.414 0.415 0.414 0.414 15.37 2 40 0.332 0.336 0.334 0.334 31.72 3 60 0.298 0.294 0.300 0.297 39.32 4 80 0.262 0.262 0.263 0.262 44.62 5 100 0.147 0.145 0.148 0.146 70.16 IC50 74.98 Blank 0.498

Table 13: Antioxidant activity of methanol extract of leaf

S.N. Concentration Absorbance mean % of (ppm) scavenging

1 20 0.302 0.300 0.304 0.302 39.43 2 40 0.276 0.277 0.276 0.276 44.58 3 60 0.253 0.252 0.248 0.251 49.63 4 80 0.229 0.225 0.230 0.228 54.27 5 100 0.177 0.178 0.176 0.177 64.46 IC50 64.94 Blank 0.498

Table 14: Antioxidant activity of hexane extract of bark

S. N. Concentration Absorbance mean % of (ppm) scavenging 1 20 1.145 1.145 1.147 1.146 6.07 2 40 0.985 0.989 0.987 0.987 19.05 3 60 0.920 0.926 0.926 0.926 24.08 4 80 0.800 0.796 0.800 0.799 34.53 5 100 0.755 0.759 0.760 0.758 37.86

IC50 124.41 Blank 1.220

Table 15: Antioxidant activity of hexane extract of leaf

S. N. Concentration Absorbance mean % of (ppm) scavenging 1 20 1.162 1.166 1.164 1.164 7.82 2 40 1.106 1.106 1.106 1.106 12.42 3 60 0.980 0.979 0.979 0.979 22.41 4 80 0.802 0.799 0.805 0.802 36.46 5 100 0.701 0.702 0.699 0.700 44.51

IC50 114.62 Blank

1.262

D. Total Phenolic Content

Table 16: Total phenolic content in methanol extract of bark

S.N. Sample Wt. of dry Absorbance GAE GAE TPC as Mean solution extract per conc. conc. GAE= (µg/ml) mL C C c × V m (g) (µg/ml) (mg/ml) m

1 1000 0.001 0.407 139.03 0.139 139.03

2 1000 0.001 0.410 140.03 0.140 140.03 139.47

3 1000 0.001 0.408 139.36 0.139 139.36

Table 17: Total phenolic content in methanol extract of leaf

S.N. Sample Wt. of dry Absorbance GAE GAE TPC as Mean solution extract per conc. conc. GAE (µg/ml) mL C C = c × V m (g) (µg/ml) (mg/ml) m

1 1000 0.001 0.451 153.7 0.153 153.7

2 1000 0.001 0.448 152.7 0.152 155.7 153.58

3 1000 0.001 0.453 154.36 0.154 154.36

Table 18: Total phenolic content in hexane extract of bark

S.N. Sample Wt. of dry Absorbance GAE GAE TPC as Mean solution extract per conc. conc. GAE (µg/ml) mL C C = c × V m (g) (µg/ml) (mg/ml) m

1 1000 0.001 0.082 30.7 0.03 30.7

2 1000 0.001 0.079 29.7 0.029 29.7 30.14

3 1000 0.001 0.08 30.03 0.03 30.03

Table 19: Total phenolic content in hexane extract of leaf

S.N. Sample Wt. of dry Absorbance GAE GAE TPC as Mean solution extract per conc. conc. GAE (µg/ml) mL C C = c × V m (g) (µg/ml) (mg/ml) m

1 1000 0.001 0.098 36.03 0.036 36.03

2 1000 0.001 0.094 34.7 0.034 34.7 35.7

3 1000 0.001 0.099 36.37 0.036 36.37

E. Total Flavonoid Content

Table 20: Total flavonoid content in methanol extract of bark

S.N. Sample Wt. of dry Absorbance QE conc. QE TFC as Mean solution extract per C conc. QE (µg/ml) mL (µg/ml) C = c × V m (g) (mg/ml) m

1 1000 0.001 0.087 123.71 0.123 123.71

2 1000 0.001 0.089 126.57 0.126 126.57 124.18

3 1000 0.001 0.086 122.28 0.122 122.28

Table 21: Total flavonoid content in hexane extract of leaf

S.N. Sample Wt. of dry Absorbance QE conc. QE conc. TFC as QE Mean solution extract per C C c × V (µg/ml) mL (µg/ml) (mg/ml) m m (g) 1 1000 0.001 0.023 32.28 0.32 32.28

2 1000 0.001 0.025 35.14 0.35 35.14 34.66

3 1000 0.001 0.026 36.57 0.36 36.57

Table 22: Total flavonoid content in methanol extract of leaf

S.N. Sample Wt. of dry Absorbance QE conc. QE TFC as Mean solution extract per C conc. QE (µg/ml) mL (µg/ml) C = c × V m (g) (mg/ml) m

1 1000 0.001 0.098 139.4 0.139 139.4

2 1000 0.001 0.096 136.57 0.136 136.57 138.46

3 1000 0.001 0.098 139.4 0.139 139.4

Table 23: Total flavonoid content in hexane extract of bark

S.N. Sample Wt. of dry Absorbance QE conc. QE conc. TFC as QE Mean solution extract per C C = c × V (µg/ml) mL (µg/ml) (mg/ml) m m (g) 1 1000 0.001 0.018 26.35 0.026 26.35

2 1000 0.001 0.017 23.71 0.023 23.71 24.59

3 1000 0.001 0.017 23.71 0.023 23.71

F. Proximate Analysis

Table 24: Determination of moisture

S.N. W1 W2 W % moisture = 100 (W1-W2) Mean % (g) (g) (g) moisture W1-W 1. 40.6234 39.6718 35.5784 18.86 18.90

2. 38.1408 37.1360 32.8365 18.94

Table 25: Determination of total ash

S.N. W W W Mean % 1 2 100 (W -W) (g) (g) (g) 2 total ash Fat = W1 1. 35.5784 5.0450 35.6499 1.27 1.26

2. 32.6365 5.3045 32.9028 1.24

Table 26: Determination of total nitrogen

S.N. W Titre Aliquot V N factor N2 % = Mean (g) of (ml) Titre×N×1.4×V digest ×100 Aliquot ×W×100

1. 0.5726 1.3 10 100 0.05 1.4 1.59 1.73

2. 0.5230 1.4 10 100 0.05 1.4 1.87

% crude protein = 6.25 × % of N2

= 6.25 × 1.73

= 10.83%

Table 27: Determination of crude fiber

S.N. W (g) W1 W2 % Crude Fiber = W1-W2 Mean % (g) (g) ×100 crude fiber W

1. 2.0780 27.7260 27.6486 3.72 3.72

2. 2.0676 28.7842 28.4762 3.73

Table 28: Determination of crude fat

S.N. W W1 W2 % crude fat= 100(W1-W2)/W Mean % (g) (g) (g) crude fat 1 2.1965 20.3709 20.3631 0.36 0.30

2 2.0862 22.1114 22.1062 0.23

G. Photographs

ZOI for GNB ZOI for GPB

Soxhlet Extractor Rota Evaporater