Chemical Constituents and Biological Activities from Crude Hexane Extract of Schleichera Oleosa Fruits

CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES FROM CRUDE HEXANE EXTRACT OF SCHLEICHERA OLEOSA FRUITS

XAYPHONE THATAVONG

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE MASTER DEGREE OF SCIENCE IN CHEMICAL EDUCATION THE FACULTY OF SCIENCE BURAPHA UNIVERSITY JUNE 2015 COPYRIGHT OF BURAPHA UNIVERSITY

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ACKNOWLEDGMENTS

I would like to express my sincere gratitude and deep appreciation to my principal advisor, Dr. Anan Athipornchai, for his valuable instruction, advice, guidance, understanding and intensive supervision throughout this study. His kindness and helpfulness will always be remembered. I would like to thank my master committee members, Dr Sureeporn Homvisasevongsa, Dr. Prapapan Techasauvapak and Assistant Professor Dr. Rungnapha Saeeng for your help and valuable advice. I would like to thank Dr. Akapong Suwattanamala and Dr. Prapapan Techasauvapak for giving the ways to learn and the experience in this thesis. I am also grateful to Thailand International Development Cooperation Agency (TICA) for gave me the opportunity to study and financial supporting. I would like to thank Department of Chemistry, Faculty of Science Burapha University, Thailand, where I performed my research and study. Finally, I would like to express my deepest gratitude for everyone in my family and all benefactors for everlasting love, understanding and unlimited supports.

Xayphone Thatavong

56920797: MAJOR: CHEMICAL EDUCATION; M. Sc. (CHEMICAL EDUCATION) KEYWORDS: SCHLEICHERA OLEOSA/ SAPINDACEAE/ ANTIOXIDANT ACTIVITY/ PHYTOCHEMICAL SCREENING XAYPHONE THATAVONG: CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES FROM CRUDE HEXANE EXTRACT OF Schleichera oleosa FRUITS. ADVISORY COMMITTEE: ANAN ATHIPORNCHAI, Ph.D. 101 P. 2015.

Schleichera oleosa (Lour.) Oken (Ceylon oak) is classified in Sapindaceae family, commonly called as Ta-khro in Thailand. Its fruits are consumed by the traditional people and taste sour. This plant also has many medicinal uses and is used in traditional medicine for several indications such as skin inflammations and skin infections. The present study was performed to evaluate the phytochemical screening of S. oleosa fruits and in vitro antioxidant activity of the methanolic extract and was further fractionated in solvents of different polarity. Phytochemical studies of the methanolic extracts from S. oleosa fruits revealed the presence of secondary metabolites of terpenoids, flavonoids, tannins and steroids. The extracts displayed total phenolic content of 0.38±0.05 to 2.56±0.08 mg GAE/g crude extract, total flavonoids content of 0.78±0.02 to 5.23±0.04 mg QE/g crude extract and total flavonols content of 0.81±0.04 to 2.90±0.12 mg QE/g crude extract. The highest antioxidant activity using DPPH free radical method was demonstrated by hexane fraction (60.91%) followed by EtOAc fraction (43.53%) and water fraction (34.94%), respectively. The results obtained indicate that although S. oleosa has been widely used for centuries as an astringent and against skin inflammations, ulcers, itching, acne and other skin infections, the fruits extract of the same also has possibility as an antioxidant and free radical scavenging agent. These results suggest the potential of S. oleosa fruits as a medicine or cosmetic agents against free-radical-associated oxidative damage. Investigation of the chemical constituents of the hexane fraction from S. oleosa fruits gave unsaturated fatty acid methyl esters (AA-NMR3 and AA- NMR6), unsaturated fatty acid (AA-NMR18, 24, 25, 35–37), a mixture -sitosterone v and stigmasterone (AA-NMR5), a mixture of -sitosterol and stigmasterol (AA- NMR15) and -amyrin (AA-NMR11–14). The biological of the AA-NMR3, AA-NMR6 (unsaturated fatty acid methyl esters) and AA-NMR11–14 (-amyrin) were evaluated for their antioxidant activity. The antioxidant activity of these isolated compounds was performed by DPPH free radical scavenging assay. Ascorbic acid, quercetin and gallic acid were used as reference antioxidant standards. The results showed that the unsaturated fatty acid methyl esters showed weaker antioxidant inhibitory activity than -amyrin. However, -amyrin also showed weaker antioxidant inhibitory activity than all positive controls.

CONTENTS

Page ABSTRACT...... iv CONTENTS...... vi LIST OF TABLES...... ix LIST OF FIGURES...... x LIST OF SCHEMES ...... xiii ABBREVIATION AND SYMBOLS...... xiv CHAPTER 1 INTRODUCTION...... 1 1.1 Objectives...... 4 1.2 Contribution to knowledge...... 4 1.3 Scope of the study...... 4 2 LITERATURE REVIEWS...... 5 2.1 Extraction of plant...... 5 2.2 Preliminary phytochemical...... 7 2.2.1 Steroids...... 7 2.2.2 Flavonoids...... 8 2.2.3 Alkaloids...... 9 2.2.4 Tannin...... 9 2.2.5 Terpenoids...... 10 2.2.6 Anthraquinones...... 10 2.2.7 Saponins...... 11 2.2.8 Cardiac glycosides...... 12 2.2.9 Phlobatanins...... 13 2.2.10 Coumarins...... 13 2.3 Antioxidants...... 14 2.4 Review of literatures...... 16 2.4.1 Selected examples of the biological activities of the crude extract from Schleichera oleosa (Lour.) Oken...... 16

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CONTENTS (CONTINUED)

Chapter Page 2.4.2 Selected examples of chemical constituents of Schleicher oleosa (Lour.) Oken...... 20 3 RESEARCH METHODOLOGY...... 25 3.1 Instruments and chemicals...... 25 3.2 Plant material...... 25 3.3 Extraction of S. oleosa fruits…...... 25 3.4 Phytochemical screening of S. oleosa fruits extracts…...... 26 3.4.1 Detection of alkaloids...... 27 3.4.2 Detection of anthaquinones...... 27 3.4.3 Detection of flavonoids...... 27 3.4.4 Detection of terpenoids...... 27 3.4.5 Detection of saponins...... 27 3.4.6 Detection of tannins...... 27 3.4.7 Detection of steroids...... 28 3.4.8 Detection of cardiac glycoside...... 28 3.4.9 Detection of phlobatannins...... 28 3.4.10 Detection of coumarins...... 28 3.5 Quantitative phytochemical contents of S. oleosa fruits extracts….. 28 3.5.1 Determination of total phenolic content...... 28 3.5.2 Determination of total flavonoids content...... 29 3.5.3 Determination of total flavonols content...... 30 3.6 DPPH free radical scavenging activity of S. oleosa fruits ...... 30 3.7 Chemical investigation of hexane fraction of S. oleosa fruits ...... 31 4 RESULTS AND DISCUSSION ……………...... 35 4.1 Phytochemical screening of S. oleosa fruits extracts...... 35 4.2 Quantitative phytochemical contents of S. oleosa fruits extracts...... 36

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CONTENTS (CONTINUED)

Chapter Page 4.3 DPPH radical scavenging activity of S. oleosa pulp fruits extracts... 37 4.4 Isolation and purification of hexane fraction from S. oleosa fruits... 38 4.5 Characterization of isolation compounds from hexane fraction of S. oleosa pulp fruits...... 42 4.6 DPPH radical scavenging activity of isolated compounds...... 47 5 CONCLUSION...... 49 REFERENCES...... 50 APPENDIX...... 56 BIOGRAPHY...... 101

LIST OF TABLES

Table Page 2-1 Antiulcer activity of ethanol extract from the stem bark of S. oleosa...... 17

2-2 IC50 values of the different fractions S. oleosa bark in different free radical scavenging tests...... The18 glucosidase inhibitory activity of analogues of 14-deoxy-11,12- 2-3 Antioxidant activity of ethanolic extract from the stem bark of S. oleosa by DPPH method...... 19 2-4 Antioxidant activity of ethanolic extract from the stem bark of S. oleosa by Hydrogen peroxide method...... Inhibitory19 effects of compounds (2) and(16-22) on NO production 2-5 Antioxidant activity of ethanolic extract from the stem bark of S. oleosa by Nitric oxide method...... Cytotoxic20 activities of compound (23) and (24) against tumor cell

2-6 Cancer cell growth inhibitory evaluation (GI50) of the Schleicherastatins 1-7...... 21

2-7 Cancer cell growth inhibitory evaluation (GI50) of the Shleicheols 1and2 21 2-8 Fatty acid composition in S. oleosa seed oil...... 23 4-1 Phytochemical profiles of the methanolic extract of S. oleosa pulp fruits.... The35 synthesis of C-19 derivatives of 14-deoxy-11,12-didehydro- 4-2 Total phenolic, total flavonoids and flavonols content of methanolic extract and soluble fractions of Schleichera oleosa pulp fruits...... The36 synthesis of compound (4a-4d) by acetylation...... 4-3 Fractions obtained from the hexane fraction of S. oleosa fruits...... 38 4-4 Fractions obtained from the A1...... Acetylation39 of C-19 substituted-14-deoxy-11,12-didehydroandro- 4-5 Fractions obtained from the B2...... 39 4-6 Fractions obtained from the C3...... The39 synthesis of compound (2) with benzoyl derivatives...... 4-7 Fractions obtained from the A2...... 40 4-8 Fractions obtained from the B2...... 40 4-9 Fractions obtained from the A5...... The41 synthesis of compound (2) with benzoyl derivatives...... 4-10 Fractions obtained from the G6...... 41Synthesis of analogues(12) by acetylation at C-3 position of 4-11 Fractions obtained from the A7…………………...... 42

LIST OF FIGURES

Figure Page 1-1 Schleichera oleosa (Lour.) Oken...... 3 2-1 Extraction of plant...... 6 2-2 Principle of DPPH radical scavenging capacity assay...... 15 2-3 Chemical constituents of the methanol extract from bark and stem of S. oleosa...... 21 2-4 Chemical constituents of methanol extract from the outer bark from S. oleosa...... 22 2-5 Fatty acid composition in S. oleosa seed oil...... 24 4-1 Free radical scavenging activity of methanolic extract and soluble fractions of Schleichera oleosa pulp fruits extracts...... 37 4-2 The 1H-NMR spectra of AA-NMR3...... 43 4-3 The 1H-NMR spectra of AA-NMR6...... 43 4-4 The 1H-NMR spectra of AA-NMR11...... 44 4-5 The 1H-NMR spectra of AA-NMR15...... 45 4-6 The 1H-NMR spectra of AA-NMR5...... 45 4-7 The 1H-NMR spectra of AA-NMR18...... 46 4-8 Free radical scavenging activity of AA-NMR3, AA-NMR6 and AA-NMR11...... 48 6-1 The 1H-NMR spectra of AA-NMR1...... 57 6-2 The 1H-NMR spectra of AA-NMR2...... 58 6-3 The 1H-NMR spectra of AA-NMR3...... 59 6-4 The 1H-NMR spectra of AA-NMR4...... 60 6-5 The 1H-NMR spectra of AA-NMR5...... 61 6-6 The 1H-NMR spectra of AA-NMR6...... 62 6-7 The 1H-NMR spectra of AA-NMR7...... 63 6-8 The 1H-NMR spectra of AA-NMR8...... 64 6-9 The 1H-NMR spectra of AA-NMR9...... 65 6-10 The 1H-NMR spectra of AA-NMR10...... 66 xi

LIST OF FIGURES (CONTINUED) Figure Page 6-11 The 1H-NMR spectra of AA-NMR11...... 67 6-12 The 1H-NMR spectra of AA-NMR12...... 68 6-13 The 1H-NMR spectra of AA-NMR13...... 69 6-14 The 1H-NMR spectra of AA-NMR14...... 70 6-15 The 1H-NMR spectra of AA-NMR15...... 71 6-16 The 1H-NMR spectra of AA-NMR16...... 72 6-17 The 1H-NMR spectra of AA-NMR17...... 73 6-18 The 1H-NMR spectra of AA-NMR18...... 74 6-19 The 1H-NMR spectra of AA-NMR19...... 75 6-20 The 1H-NMR spectra of AA-NMR20...... 76 6-21 The 1H-NMR spectra of AA-NMR21...... 77 6-22 The 1H-NMR spectra of AA-NMR22...... 78 6-23 The 1H-NMR spectra of AA-NMR23...... 79 6-24 The 1H-NMR spectra of AA-NMR24...... 80 6-25 The 1H-NMR spectra of AA-NMR25...... 81 6-26 The 1H-NMR spectra of AA-NMR26...... 82 6-27 The 1H-NMR spectra of AA-NMR27...... 83 6-28 The 1H-NMR spectra of AA-NMR28...... 84 6-29 The 1H-NMR spectra of AA-NMR29...... 85 6-30 The 1H-NMR spectra of AA-NMR30...... 86 6-31 The 1H-NMR spectra of AA-NMR31...... 87 6-32 The 1H-NMR spectra of AA-NMR32...... 88 6-33 The 1H-NMR spectra of AA-NMR33...... 89 6-34 The 1H-NMR spectra of AA-NMR34...... 90 6-35 The 1H-NMR spectra of AA-NMR35...... 91 6-36 The 1H-NMR spectra of AA-NMR36...... 92 6-37 The 1H-NMR spectra of AA-NMR37...... 93 6-38 The 1H-NMR spectra of AA-NMR38...... 94 6-39 The 1H-NMR spectra of AA-NMR39...... 95 xii

LIST OF FIGURES (CONTINUED) Figure Page

6-40 The 1H-NMR spectra of AA-NMR40...... 96 6-41 The 1H-NMR spectra of AA-NMR41...... 97 6-42 The 1H-NMR spectra of AA-NMR42...... 98 6-43 The 1H-NMR spectra of AA-NMR43...... 99 6-44 The 1H-NMR spectra of AA-NMR44...... 100

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LIST OF SCHEMES

Schemes Page 3-1 Extraction of Schleichera oleosa pulp fruits...... 26 3-2 Fractionation of the hexane extract from S. oleosa fruits...... 31 3-3 Fractionation A1 of the hexane extract from S. oleosa fruits...... 32 3-4 Fractionation A2 of the hexane extract from S. oleosa fruits...... 32 3-5 Fractionation A3 and A4 of the hexane extract from S. oleosa fruits...... 33 3-6 Fractionation A5 of the hexane extract of S. oleosa fruits...... 33 3-7 Fractionation A7 of the hexane extract of S. oleosa fruits...... 34

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ABBREVIATION AND SYMBOLS

Abs Absorbance

Me2CO Acetone 13C NMR Carbon Nuclear Magnetic Resonance cm centimeter

CDCl3 Chloroform-D CC Column Chromatography c Concentration COSY Correlated spectroscopy J Coupling constant  Chemical Shift Relative to TMS DCM Dichloromethane DPPH 1,1-Diphenyl-2-picrylhydrazyl

EC50 50% Effective concentration

ED50 50% Effective dose EtOAc Ethyl acetate eq. Equivalent

GI50 50%Growth inhibition g gram GAE Gallic acid equivalents Hz Hertz h Hour

IC50 50% Inhibitory concentration IR Infrared spectroscopy Kg Kilogram P-388 Lymphocytic leukemia MHz Megahertz m.p. Melting point MeOH Methanol µg Microgram xv

ABBREVIATION AND SYMBOLS (CONTINUED)

µL Microliter µ Micromolar mg milligram mmol Millimole mL Milliter m Multiplet (spectra) MIC minimum Inhibition Concentrations min minute NMR Nuclear magnetic resonance NaOAc sodium acetate s Singlet (spectra) ppm Part per Million % Percentage PLC preparative Thin-layer Chromatography 1H NMR Proton Nuclear magnetic resonance QE Quercetin equivalents cm-1 Reciprocal Centimeter (wave number)

Rf Retardation factor TLC Thin-layer chromatography t Triplet (spectra)

CCl4 Tetrachloromethane UV Ultraviolet-visible VLC vacuum liquid chromatography

CHAPTER 1 INTRODUCTION

At present, there are many researchs in natural products which were interested. Plant is important and necessary to dive into life, due to it is a source of food, traditional until modern medicine. Plants are natural source of producing wide number of bioactive chemical constituents in most efficient way and precise selectivity. Consumption of various types of fruit provides excellent health benefits because they are a good source of phytochemicals that are good for preventing diseases (Hu, 2003; Ikram et al., 2009). Fruits contain many different kinds of antioxidant compounds, including flavonoids, phenolics, carotenoids and vitamins, which are all considered beneficial to human health, for decreasing the risk of degenerative diseases by reduction of oxidative stress, and for the inhibition of macromolecular oxidation (Heber, 2004; Prior et al., 2003; Rangkadilok et al., 2007). Thailand is a tropical country with a large diversity of fruits including durian, papaya, lychee, mangosteen and star fruits. These have been shown to be good sources of antioxidants. Besides the commonly consumed local fruits, some under-utilized species are important in the diets of rural communities. Some wild-fruits of Thailand are rarely eaten, and are unknown or at least unfamiliar, especially in urban communities. These wild-fruit species that are unique to the distinct climate in Thailand may offer potential benefits to human health (Charoensiri et al., 2009; Kubola et al., 2011). Schleichera oleosa (Lour.) Oken is a plant in the family Sapindaceae, vernacularly known as Lac tree or kusum is a large deciduous forest tree species. S. oleosa is an evergreen tree with the height up to 30 m and the girth up to 3 m. The leaves are par pinnate, 20-40 cm long. The leaflets are 2 to 4 pairs, elliptic or elliptic- oblong, coriaceous, margins entire and apex rounded. The flowers are minute, yellowish green, either male or bisexual, fascicled in spike like axillaries racemes 7.5 to 12.5 cm long. The fruits are berry, globose or ovoid, and hard skinned. The seeds are brown, irregularly elliptic, slightly compressed, oily, enclosed in a succulent aril (Palanuvej & Vipunngeun, 2008). S. oleosa is widely in the sub-Himalayan region, 2

throughout central and southern India, Nepal, Sri Lanka, Thailand, Indonesia and Malaysia. The tree is popularly exploited in India. In Thailand, S. oleosa named Ta-Khro is found in the Northern, North- eastern, South-eastern, South-western and Central regions. It is used in the wood industry. The wood is suitable for fuel wood and charcoal, the bark is used as dye and the young leaves are eaten as vegetable. Different parts of S. oleosa, such as fruits, leaves, bark and seeds are used as tribal food, animal feed, seed-oil and timber. The tree also serves as important source for traditional medicines for curing pruritus, malaria, inflammation and ulcers (Bhatia et al., 2013). Most exotics sesame past research has studied the properties of S. oleosa extracts from the bark and stems of the S. oleosa and it was found to reduce the free radicals that cause the death of cancer cells (Pettit et al., 2000; Thind et al., 2010). Antimicrobial activities were also performed against some fungal and bacterial species (Ghosh et al., 2011). Oil extraction (Kusum oil or Macassar oil) from seed Ta-khro can be used for the cure of itch, acne, skin burns as a massage oil for rheumatic pains solution (Palanuvej & Vipunngeun, 2008). The water extract of the bark of S. oleosa was used to treat menstrual pain as well (Mahaptma & Sahoo, 2008). However, few researchs of the chemical constituents and antioxidant activities of S. oleosa were reported previously. Therefore, we were interested to evaluate the phytochemical screening of S. oleosa fruits and in vitro antioxidant activity of the methanolic extract and were further fractionated in solvents of different polarity.

(https://www.google.co.th/search?q=%E0%B8%95%E0%B8%B0%E0%B8%84%E0%B8%A3%E0%B9%89%E0%B8% AD&source=lnms&tbm=isch&sa=X&ei=AnodU_eNOovIrQfr-4D4BA&sqi=2&ved=0CAcQ_AUoAQ&biw=1366&bih=562)

Figure 1-1 Schleichera oleosa (Lour.) Oken.

1.1 Objectives 1. To elucidate the phytochemical screening of S. oleosa fruits. 2. To isolate, purify, and identify the chemical constituents of S. oleosa fruits. 3. To study the antioxidant activity of the methanolic extract and was further fractionated in solvents of different polarity of S. oleosa fruits.

1.2 Contribution to knowledge 1. To obtain the phytochemical screening of S. oleosa fruits. 2. To obtain the chemical constituents from S. oleosa fruits. 3. To obtain the antioxidant activity of the methanolic extract and was further fractionated in solvents of different polarity of S. oleosa fruits.

1.3 Scope of study 1. Elucidation the phytochemical screening of S. oleosa fruits. It was further fractionated in solvents of different polarity. 2. Extraction, isolation, and purification of bioactive compounds from S. oleosa using chromatographic methods. 3. Structure elucidation of bioactive compounds from S. oleosa fruits using spectroscopic analysis. 4. Evaluations of the antioxidant activity of the methanolic extract and were further fractionated in solvents of different polarity of S. oleosa fruits.

CHAPTER 2 LITERATURE REVIEWS

2.1 Extraction of plant 2.1.1 Maceration In this process, the whole or coarsely powdered crude drug is placed in a stoppered container with the solvent and allowed to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter has dissolved. The mixture then is strained, the marc (the damp solid material) is pressed, and the combined liquids are clarified by filtration or decantation after standing (Handa, Khanuja, Longo, & Rakesh, 2008).

2.1.2 Percolation This is the procedure used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is generally used. The solid ingredients are moistened with an appropriate amount of the specified solvent and allowed to stand for approximately 4 hour in a well closed container, after which the mass is packed and the top of the percolator is closed. Additional solvent is added to form a shallow layer above the mass, and the mixture is allowed to macerate in the closed percolator for 24 hour. The outlet of the percolator then is opened and the liquid contained therein is allowed to drip slowly. Additional solvent is added as required, until the percolate measures about three-quarters of the required volume of the finished product. The marc is then pressed and the expressed liquid is added to the percolate. Sufficient solvent is added to produce the required volume, and the mixed liquid is clarified by filtration or by standing followed by decanting (Handa, Khanuja, Longo, & Rakesh, 2008).

2.1.3 Hot Continuous Extraction (Soxhlet) The main advantage of Soxhlet extraction is that it is a continuous process. In this method, the finely ground crude drug is placed in a porous bag made of strong filter paper, which is placed in chamber of the Soxhlet apparatus. The extracting solvent in flask is heated, and its vapors condense in condenser. The condensed extractant drips into the thimble containing the crude drug, and extracts it by contact. When the level of liquid in chamber rises to the top of siphon tube, the liquid contents of chamber E siphon into flask. This process is continuous and is carried out until a drop of solvent from the siphon tube does not leave residue when evaporated. The advantage of this method, compared to previously described methods, is that large amounts of drug can be extracted with a much smaller quantity of solvent. This effects tremendous economy in terms of time, energy and consequently financial inputs. At small scale, it is employed as a batch process only, but it becomes much more economical and viable when converted into a continuous extraction procedure on medium or large scale (Handa, Khanuja, Longo, & Rakesh, 2008).

http://slideplayer.in.th/slide/2107753/ http://slideplayer.in.th/slide/2107753/ http://slideplayer.in.th/slide/2107753/ Maceration Extraction Percolation Extraction Soxhlet Extraction

Figure 2-1 Extraction of plant.

2.2 Preliminary phytochemical Natural products chemistry has originated from mankind’s curiosity about colour, taste, odour, and cures for human, animal and plant diseases. The term natural product is applied to materials derived from plants, microorganisms, invertebrates and vertebrates, which are fine biochemical factories for the biosynthesis of both primary and secondary metabolites. The secondary metabolites play ecologically significant roles in how the living organisms deal with their surrounding and therefore are important for their ultimate survival. They include steroids, flavonoids, coumarins, alkaloids, tannins, terpenoids, anthraquinones, saponins, cardiac glycosides and phlobatanins (Bhat, Nagasampagi, & Meenakshi, 2009).

2.2.1 Steroids Steroids are a group of cyclical organic compounds whose basis is a characteristic arrangement of seventeen carbon atoms in a four-ring structure linked together from three 6-carbon rings followed by a 5-carbon ring and an eight-carbon side chain on carbon 17. They include a wide range of naturally occurring compounds like the sterols, the bile acids (cholic acid), the adrenocortical hormones (aldosterone), the cardiac glycosides, the sapogenins (yamogenin) and some alkaloids (solasodine). The sterols, the bile acids and the adrenocortical hormones have a number of functions in human physiology and are of immense biological importance. The most important compound in this class is cholesterol. It is the most abundant steroid present in humans and the most important one as well all the steroids are derived from it (Bhat, Nagasampagi, & Meenakshi, 2009).

2.2.2 Flavonoids Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms. Flavanoids can be visualized as two benzene rings which are joined together with a short three carbon chain. One of the carbons of the short chain is always connected to a carbon of one of the benzene rings, either directly or through an oxygen bridge, thereby forming a third middle ring, which can be five or six-membered. Flavonoids protect the plant from UV-damaging effects and play a role in pollination by attracting animals by their colors. Recently, flavonoids have attracted interest due to the discovery of their pharmacological activities as anti-inflammatory, analgesic, antitumor and antioxidant. Biologically active flavonoids include hesperidin and rutin for decreasing capillary fragility and quercetin for its anti-diarrheal activity (Galeotti, Barile, Curir, Dolci, & Lanzotti, 2008). OH HO HO OH O H3C OH HO O O OCH OH 3 OH O HO HO O O OH HO O O OH OH O H3C O HO OH O HO OH OH hesperidin rutin OH

HO O

OH OH O quercetin

2.2.3 Alkaloids Alkaloids are a chemically heterogeneous group of basic nitrogen containing substance found predominantly in higher plants. However, such basic substance also occur in lower plants, animals, microorganisms and marine organisms. Alkaloids usually contain one or two nitrogen atoms although some like ergotamine may contain up to five nitrogen atoms (Bhat, Nagasampagi, & Meenakshi, 2009).

2.2.4 Tannins Tannins are a complex group of polyphenolic compounds found in a wide range of plant species commonly consumed by ruminants. They are conventionally classified into two major groups, the hydrolysable and the condensed tannins. Tannins are used against diarrhea and as an antidote in poisoning by heavy metals. Their use declined after the discovery of hepatotoxic effects of absorbed tannic acids (Frutos, Hervás, Giráldez, & Mantecón, 2004).

2.2.5 Terpenoids Terpenoids are divided from the isoprene unit (C5), which is biosynthesized from acetate by mevalonic acid. Terpinoids can be classified like monoterpenoids, sesquiterpenoinds, diterpenoids, sesterterpenoids, triterpenoids, carotenoids and polyterpenoids. Plant terpenoids are used extensively for their aromatic qualities and played a role in tradition herbal remedies for antibacterial, antineoplastic and antioxidant such as thymol, geranial and retinol (Bhat, Nagasampagi, & Meenakshi, 2009).

2.2.6 Anthraquinones Anthraquinones, called as anthracenedione or dioxoanthracene are an aromatic (a hydrocarbon characterized by general alternating double and single bonds between carbons) organic compound. Anthraquinone is an important member of the quinone family. Quinone is a class of organic compounds that are formally derived from aromatic compounds. The term is also used in the more general sense of any compound that can be viewed as an anthraquinone with some hydrogen atoms replaced by other atoms or functional groups. These derivatives include many substances that are technically useful or play important roles in living beings. Anthraquinones are natural product present in fungi, higher plants and lichens such as emodin and 1,7- dihydroxy-6-methyl-anthraquinone (Kaur & Arora, 2010).

2.2.7 Saponins Saponins are glycosylated compounds that are widely distributed in the plant kingdom and can be divided into three major groups including triterpenoid (astragalside), steroid (protodioscin), or steroidal glycoalkoloid (solanine) (Figen, 2006).

2.2.8 Cardiac glycosides Cardiac glycosides are an important class of naturally occurring drugs which actions include both beneficial and toxic effects on the heartare organic compounds containing a sugar (glycoside) and the non-sugar (aglycon) moieties. Several bioactive cardiac glycoside were reported for anti-cancer activity such as digitoxin, digoxin and ouabain (Singh & Rastogi, 1970).

2.2.9 Phlobatannins Phlobatanins are phlobaphens formed with under action of acids or heating of condensed tannin or of the fraction of tannins such as fisetinidol-(4a,10)-tetrahydro pyrido-[2,3-f]chromene (Steenkamp et al., 1985).

2.2.10 Coumarins Coumarin is a fragrant organic chemical compound in the benzopyrone chemical class, which is a colorless crystalline substance in its standard state. It is a natural substance found in many plants. Several bioactive coumarin were reported for antifungal, antioxidant, anticancer such as Osthole, Esculetin, Chartreusin (Venugopala, Rashmi, & Odhav, 2013).

2.3 Antioxidants Free radicals are atoms or groups of atoms with unpaired number of electrons and can be formed when oxygen interacts with certain molecules. Once formed these highly reactive radicals can start a chain reaction, like dominoes. Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs. To prevent free radical damage the body has a defense system of antioxidants. Antioxidants are molecules which can safely interact with free radicals and terminate the chain reaction before vital molecules are damaged. Although there are several enzyme systems within the body that scavenge free radicals, the principle micronutrient (vitamin) antioxidants are vitamin E, beta-carotene, and vitamin C. Additionally, selenium, a trace metal that is required for proper function of one of the body's antioxidant enzyme systems, is sometimes included in this category. The body cannot manufacture these micronutrients so they must be supplied in the diet. Antioxidant compounds in food play an important role as a health protecting factor. Scientific evidence suggests that antioxidants reduce the risk for chronic diseases including cancer and heart disease. Primary sources of naturally occurring antioxidants are whole grains, fruits and vegetables. Plant sourced food antioxidants like vitamin C, vitamin E, carotenes, phenolic acids, phytate and phytoestrogens have been recognized as having the potential to reduce disease risk. Most of the antioxidant compounds in a typical diet are derived from plant sources and belong to various classes of compounds with a wide variety of physical and chemical properties. Some compounds, such as gallates, have strong antioxidant activity, while others, such as the mono-phenols are weak antioxidants (Abheri, Anisur, & Ghosh, 2010). Various antioxidant activity methods have been used to monitor and compare the antioxidant activity of foods. In recent years, oxygen radical absorbance capacity assays and enhanced chemiluminescence assays have been used to evaluate antioxidant activity of foods, serum and other biological fluids. These methods require special equipment and technical skills for the analysis. The different types of methods published in the literature for the determinations of antioxidant activity of 15

foods involve electron spin resonance (ESR) and chemiluminescence methods. These analytical methods measure the radicalscavenging activity of antioxidants against free radicals like the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, the superoxide anion radical (O2), the hydroxyl radical (OH), or the peroxyl radical (ROO). The various methods used to measure antioxidant activity of food products can give varying results depending on the specific free radical being used as a reactant. The DPPH method was developed by Blois (1958) with the viewpoint to determine the antioxidant activity in a like manner by using a stable free radical α,α- diphenyl-β-picrylhydrazyl (DPPH; C18H12N5O6, M = 394.33). DPPH is characterized as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole (Fig. 2-2), so that the molecules do not dimerise, like most other free radicals. The assay is based on the measurement of the scavenging capacity of antioxidants towards it. The odd electron of nitrogen atom in DPPH is reduced by receiving a hydrogen atom from antioxidants to the corresponding hydrazine which shown in Figure 2-2 (Contreras-Guzman & Srong, 1982).

Figure 2-2 Principle of DPPH radical scavenging capacity assay.

2.4 Review of literatures 2.4.1 Selected examples of the biological activities of the crude extract from Schleichera oleosa (Lour.) Oken

Moon, Khan, and Wadher (2009) studied the aerial parts of S. oleosa for screening of antibacterial property. Effects of methanolic extracts of S. oleosa were investigated against drug resistant strains of Escherichia coli, Staphylococcus aureus, Klebsiella pnueumoniae, and Salmonella typhii. The antibiotic sensitivity pattern for all the clinical isolates was studied by Bauer-Kirby method. All the clinical isolates obtained were resistant to one or more than one antibiotic. This plant showed great potential as antibacterial agent.

Thind, Rampal, Agrawal, Saxena, and Arora (2010, 2011) studied the effect of extracts of S. oleosa for its cytotoxic and hydroxyl radical-scavenging activities. The barks and roots of the tree were used to prepare extracts with different solvents such as hexane, chloroform, ethyl acetate, methanol, and water. The extracts were initially assessed for their in vitro cytotoxicity potential in the sulforhodamine B dye assay against cancer cell lines, such as 502731 (colon), SW-620 (colon), HTC-15 (colon), A-549 (lung), HEP-2 (liver), SK-NS-H (central nervous system), and IMR-32 (neuroblastoma). It was observed that the water extract was effective against all the three colon cancer cell lines, while only methanol and water extracts were effective against A-549 (lung), and HEP-2 (liver) cell lines. As DNA damage is one of the hallmarks of cell death, so the extracts were assessed for their ability to scavenge hydroxyl radicals, in the deoxyribose degradation assay (site- and nonsite specific) as well as their protective effect against the hydroxyl radical induced DNA damage in the plasmid nicking assay, used pBR322. It was observed that all the extracts, except chloroform and hexane extracts, exhibited relatively greater antioxidant activity in the nonsite-specific than in the site-specific assay. Similarly, the extracts were also found to be effective in inhibiting the hydroxyl radical induced unwinding of super coiled DNA, which further confirmed the hydroxyl radical scavenging ability of the extracts in the deoxyribose degradation method.

Srinivas and Celestin Baboo (2011) studied the ethanolic extract from the stem bark of S. oleosa and evaluated for the antiulcer activity by aspirin induced and pylorus ligation of rats. The extract significantly decreased the gastric secretion, free acidity as well as gastric ulcer in the aspirin induced and pylorus ligated rat and the effects were compared with omeprazole as shown in Table 2-1.

Table 2-1 Antiulcer activity of ethanol extract from the stem bark of S. oleosa.

Total volume of Dose Total acidity Group Treatment gastric secretion (mg/kg) (meq/L/100g) (mL/100gm) Normal I 1 mL of 1% cmc 3.9±0.55 428.44±20.40 control Ulcer II 200mg/kg ASA 5.1±0.76*a 490.43± 22.40*a control Standard 2mg/kg III 2.6±0.25 332.91±21.12 control Omeprazole 200mg/kg of IV EESO 3.2±0.34*b 382.4± 30.45*b EESO

* Values are expressed as Mean ± SEM *a Values are significantly different from Normal control group at p<0.01 *b Values are significantly different from ulcer control group at p<0.01

Table 2-1 (continued)

Dose % Group Treatment pH Ulcer score mg/kg protection Normal I 1ml of 1% cmc 2.2±0.25 0.3±0.01 0.000 control Ulcer II 200mg/kg ASA 1.6± 0.14*a 2.0± 0.16*a 0.000 control Standard 2mg/kg III 4.0±0.52 0.5±0.10 75.00 control Omeprazole 200mg/kg of IV EESO 3.0± 0.36*b 0.8± 0.15*b 60.00 EESO

* Values are expressed as Mean ± SEM *a Values are significantly different from Normal control group at p<0.01 *b Values are significantly different from ulcer control group at p<0.01

Thind, Singh, Kaur, Rampal, and Arora (2011) studied the free radical scavenging activity of methanolic extract and the fractions from the bark of S. oleosa by employing various well-established in vitro systems such as DPPH, deoxyribose degradation (non-site-specific and site-specific), reducing power, chelating power and plasmid nicking assays. Total phenol content of the extracts was determined by the assay based on Folin-Ciocalteu’s method. In all assays, it was observed that the residue fraction, left after the precipitation, was more effective in scavenging the free radicals than the aqueous extract and precipitates. The higher activity of residue fraction may be attributed to the greater amount of phenolic content present in it (942.4 mg/g GAE) as compared to precipitates and aqueous extract. The extract and fractions were found to possess potent antiradical properties, which may be due to either direct scavenging of free radicals or through metal chelation as shown in Table 2-2.

Table 2-2 IC50 values of the different fractions S. oleosa bark in different free radical scavenging tests

Standards Extracts Assays Standard IC50 (µg/ml) Extract IC50 (µg/ml) Aqueous extract 30 DPPH assay Gallic acid 24 Precipitates 119.5 Residue fraction 29 Non-site-specific Aqueous extract 22.5 Deoxyribose Gallic acid 12 Precipitates 62 Degradation assay Residue fraction 31 Site-specific Aqueous extract 55 Deoxyribose Gallic acid 46 Precipitates 53.5 Degradation assay Residue fraction 64 Aqueous extract 52 Reducing assay Gallic acid 55 Precipitates 137 Residue fraction 54 Aqueous extract 81 Chelating assay EDTA 60 Precipitates 100 Residue fraction 108 19

Srinivas and Celestin Baboo (2013) reported the screening of the ethanolic extract from the stem bark of S. oleosa for in vitro antioxidant activity by DPPH, hydrogen peroxide and nitric oxide methods. Ascorbic acid was used as reference standard. The extract showed significant antioxidant activity with all the selected methods as compared to standard ascorbic acid as shown in Tables 2-3 to 2-5.

Table 2-3 Antioxidant activity of ethanolic extract from the stem bark of S. oleosa by DPPH method Concentration Schleichera oleosa Standard (Ascorbic acid) No (µg/ml) % Inhibition IC50 (µg/ml) % Inhibition IC50 (µg/ml) 1 10 40.13±0.47 60.41±0.10 2 20 43.07±0.59 71.36±0.05 3 30 49.22±0.29 81.47±0.46 151.59 48.06 4 40 54.60±0.28 85.93±0.04 5 50 62.43±0.24 91.23±0.03 6 100 66.07±1.58 96.46±0.08

Table 2-4 Antioxidant activity of ethanolic extract from the stem bark of S. oleosa by Hydrogen peroxide method Concentration Schleichera oleosa Standard (Ascorbic acid) No (µg/ml) % Inhibition IC50 (µg/ml) % Inhibition IC50 (µg/ml) 1 10 24.71±1.78 49.11±0.33 2 20 34.53±1.11 60.35±0.13 3 30 51.57±1.34 71.62±0.03 145.54 42.18 4 40 66.02±1.58 79.26±0.23 5 50 80.59±1.19 86.25±0.02 6 100 91.71±0.32 93.61±0.08

Table 2-5 Antioxidant activity of ethanolic extract from the stem bark of S. oleosa by Nitric oxide method Concentration Schleichera oleosa Standard (Ascorbic acid) No (µg/ml) % Inhibition IC50 (µg/ml) % Inhibition IC50 (µg/ml) 1 10 22.25±0.16 43.86±0.92 2 20 31.09±0.28 59.64±0.94 3 30 45.17±0.06 68.46±0.98 174.9 67.73 4 40 54.41±0.01 73.62±0.91 5 50 69.99±0.18 88.84±0.89 6 100 77.31±0.01 96.42±0.73

2.4.2 Selected examples of chemical constituents of Schleichera oleosa (Lour.) Oken

The first isolation of S. oleosa was reported by Pettit and co-worker in 2000. The bioassay-guided separation for evaluation of P-388 lymphocytic leukemia cell lines of the methanol extract from the bark and stem of Schleichera oleosa led to the isolation of seven cancer cell growth inhibitory hydroxylated sterols designated schleicherastatins 1-7 (1-7) and two related sterols, shleicheols 1 and 2 (8 and 9) along with the 7-oxo-β-sitosterol (10) and methyl protocatechuate (11), as shown in Figure 2-3 (Pettit et al., 2000). The cancer cell growth inhibitory properties of sterols 1-10 and phenol 11 were examined using the murine P-388 lymphocytic leukemia cell line and a section of human cancer cell lines. Schleicherastatins 1-7 (Table 2-6) and phenol 11 (P-388

ED50 1.4 µg/mL) exhibited significant inhibitory activity against the murine P-388 lymphocytic leukemia, and Shleicheols 1 and 2 (Table 2-7) showed marginal activity against a mini-panel of human tumor cell lines (Pettit et al., 2000). 21

R3 R 5 R4 CO2CH3

R1 OH HO R2 11

1: R1= OCH3, R2= H, R3= OH, R4= CH2CH3, R5= H 2: R1= H, R2= OCH3, R3= OH, R4= CH2CH3, R5= H 3: R1= OCH3, R2= H, R3= OH, R4= CH3, R5= H 4: R1= OCH3, R2= H, R3= OH, R4= H, R5= CH3 5: R1= R2= O, R3= OH, R4= CH2CH3, R5= H 6: R1= R2= O, R3= OH, R4= CH3, R5= H 7: R1= R2= O, R3= OH, R4= H, R5= CH3 8: R1= OCH3, R2= H, R3= H, R4= CH2CH3, R5= H 9: R1= H, R2= OCH3, R3= H, R4= CH2CH3, R5= H 10: R = R = O, R = H, R = CH CH , R = H 1 2 3 4 2 3 5

Figure 2-3 Chemical constituents of the methanol extract from bark and stem of S. oleosa

Table 2-6 Cancer cell growth inhibitory evaluation (GI50) of the Schleicherastatins 1-7

GI50 (µg/mL): Schleicherastatins 1-7 Cancer cell line 1 2 3 4 5 6 7 Leukemia P-388 0.19 0.72 1.20 1.20 0.34 0.78 0.78

Table 2-7 Cancer cell growth inhibitory evaluation (GI50) of the Shleicheols 1 and 2

GI50 (µg/mL): Shleicheols 1-2 Cancer cell line Compound 8 Compound 9 Leukemia P-388 14.0 15 CNS SF-295 3.2 1.9 Colon KM 20L2 2.5 1.2 Lung NCI-H460 1.8 2.2 Ovary OVCAR-3 2.1 2.5 Pancreas BXPC-3 3.3 1.4 Prostate 1.8 1.6

In 2011, Ghosh and co-worker reported the isolation of two triterpenoids, taraxerone (12) and tricadenic acid A (13) from the methanol extract of the outer bark of Schleichera oleosa available in Darjeeling foothills. Both the compounds showed prominent antimicrobial activities against the tested fungal and bacterial pathogens. Compound 12 showed better activity against all the microorganisms than compound 13 and its activity is comparable to that of Ampicilin against E. coli and Enterobactor. The activity of compound 12 is nearly comparable to that of Bavistan, when it was tested against Colletotrichum gloeosporioides and Colletotrichum camelliae (Ghosh et al., 2011).

Figure 2-4 Chemical constituents of methanol extract from the outer bark from S. oleosa

Gandhi, Ramu, and Raj (2011) studied the seeds of S. oleosa which were blended and macerated with hexane. The fatty acid composition was investigated by GC/MS after methylation. Fatty acid profile showed 14 components. Linolelaidic acid, the trans-form of linoleic acid, was found as dominant fatty acid (49.7%). The next below were eicosenoic acid or gondoic acid (29.5%), palmitic acid (7.6%), linoleic acid (5.6%) and oleic acid (2.8%) (Table 2-11).

Table 2-8 Fatty acid composition in S. oleosa seed oil

compounds Fatty acid %

14 C14:0 Myristic acid 0.01 15 C16:0 Palmitic acid 7.59 16 C16:1 Palmitoleic acid 1.80 17 C18:1 Oleic acid 2.83 18 C18:2 Linolelaidic acid 49.69 19 C18:2 Linoleic acid 5.56 20 C18:3 Alpha-linolenic acid 0.26 21 C20:1 Eicosenoic acid 29.54 22 C20:2 Eicosadienoic acid 0.24 23 C21:0 Heneicosanoic acid 0.04 24 C22:0 Behenic acid 1.14 25 C22:1 Erucic acid 1.22 26 C24:0 Lignoceric acid 0.03 27 C22:6 Docosahexaenoic aci 0.02

14 n = 5 ; Myristic acid

O 15 n = 6 ; Palmitic acid HO n 24 n = 9 ; Behenic acid

26 n = 10 ; Lignoceric acid OH O

O OH

16 Palmitoleic acid 17 cis-Oleic acid

OH O OH O

18 trans-Linolelaidic acid

19 cis-Linoleic acid

O OH O OH

20 alpha-Linolenic acid 21 Eicosenoic acid

O OH O OH

22 Eicosadienoic acid 23 Heneicosanoic acid

O OH O OH

25 Erucic acid 27 Docosahexaenoic acid

Figure 2-5 Fatty acid composition in S. oleosa seed oil

CHAPTER 3 RESERCH METHODOLOGY

3.1 Instruments and chemicals All chemical reagents were obtained from chemical companies and used 1 13 further purification. H and C NMR spectra were recorded in CDCl3 solutions on a Bruker AVANCE 400 (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometer. Chemical shifts are in  (ppm) with tetramethylsilane (TMS) as an internal standard. Vacuum liquid chromatography (VLC) and column chromatography (CC) were carried out using Merck silica gel 60 (0.06-0.23 mm). Gel filtration (size exclusion chromatography) was carried out using Sephadex LH-20 (Merck). Preparative thin layer chromatography (PLC) was carried out on glass plates using silica gel 60 F254 (20 x 20 cm, layer thickness 0.25, 0.5 and 1.0 mm, Merck).

Pre-coated thin layer chromatography (TLC) aluminum sheets of silica gel 60 F254 (20 x 20 cm, layer thickness 0.2 mm, Merck) were used for analytical purposes and the compounds were visualized under ultraviolet light or sprayed with anisaldehyde-

H2SO4 reagent followed by heating.

3.2 Plant material The fresh fruits of Schleichera oleosa (Lour.) Oken were collected at Sisaket province, Thailand, during May and July 2013. The samples were washed three times in tap water to remove any attached salt, epiphytes, and sand.

3.3 Extraction of S. oleosa fruits The fresh pulp fruits of Schleichera oleosa (2.0 kg) were ground and immersed in 80% methanol at ambient temperature for 7 days and concentrated using a rotary evaporator under reduce pressure at 50 °C to yield the methanolic extract (195.5 g). The residue (182.0 g) was suspended in water and methanol (1:4, 100 ml) and partitioned successively with hexane and ethyl acetate to give hexane (2.52 g), ethyl acetate (25.50 g) and residual aqueous (118.23 g) fractions, respectively. 26

Schleichera oleosa fruits (2.0 kg) + MeOH

Methanol extract (195.5 g)

Methanol extract (182.0 g)

+ MeOH:H2O (1:4) + Hexane

Hexane fraction Aqueous layer (2.52 g) + EtOAc

Ethyl acetate fraction Residual aqueous fraction (25.50 g) (118.23 g)

Scheme 3-1 Extraction of Schleichera oleosa pulp fruits

3.4 Phytochemical screening of S. oleosa fruits extracts The different qualitative chemical tests were performed for establishing the profile of given extract for its chemical composition. The following tests were performed using standard protocols on the extracts to detect various phytochemical constituents present in them as described in pervious publications (Sazada et al., 2009; Shyam-Krishnan et al., 2013).

3.4.1 Detection of alkaloids

About 0.2 g of the methanolic extract was warm with 2% H2SO4 for two minutes. The reaction were filter and added a few drops of Dragendroff's reagent. Orange red precipitate indicates the presence of alkaloids.

3.4.2 Detection of anthaquinones

About 0.2 g of the methanolic extract was warm with 10% H2SO4 for five minutes. The reaction were filter and extracted with chloroform for two times. The chloroform fraction was also added a few drops of 10% NH3. A pink red solution indicates the presence of anthaquinones.

3.4.3 Detection of flavonoids About 0.2 g of the methanolic extract was dissolved in 50% ethanol. The reaction were filter and added a small pieces of Mg(s) after that was boiled for two minutes. The mixture was also added a few drops of conc. HCl. A yellow or orange red solution indicates the presence of flavonoids.

3.4.4 Detection of terpenoids About 0.2 g of the methanolic extract was mixed with 2.0 ml of chloroform and concentrated H2SO4 was carefully added to form a layer. The formation of a reddish brown coloration at the interface indicates positive results for presence of terpenoids.

3.4.5 Detection of saponins About 0.2 g of the methanolic extract was shaken with 5.0 ml of distilled water and heated to boiling. Frothning (appearance of creamy miss of small bubbles) shows the presence of saponins.

3.4.6 Detection of tannins About 0.2 g of the methanolic extract was mixed with distilled water and heated on water bath and filtered. A few drops of ferric chloride (FeCl3) were added to the filtrate. A dark green solution indicates the presence of tannins.

3.4.7 Detection of steroids About 0.2 g of the methanolic extract was mixed with 1.0 ml glacial acetic acid and filtered. Then 2.0 ml of concentrated H2SO4 were added into the solution mixture. Presence of greenish blue color solution indicates the presence of steroids.

3.4.8 Detection of cardiac glycoside About 0.2 g of the methanolic extract was mixed with 1.0 ml glacial acetic acid and 5% ferric chloride (FeCl3) solution. Then few drops of concentrated H2SO4 were added to form a layer. The presence of brown ring of the interface indicates deoxy sugar characteristic of cardiac glycoside.

3.4.9 Detection of phlobatannins About 0.2 g of the methanolic extract was warm with distilled water and filtered. The filtrate was boiled in 10% HCl solution. Red precipitate indicates the presence of phlobatannins.

3.4.10 Detection of coumarins About 0.2 g of the methanolic extract was dissolved in 50% ethanol (1 ml), and 6.0 M NaOH was added. A dark yellow solution indicates the presence of coumarins.

3.5 Quantitative phytochemical contents of S. oleosa fruits extracts 3.5.1 Determination of total phenolic content Total phenolic content of the extracts were measured by employing the methods given in the literature involving Folin-Ciocalteu reagent as oxidizing agent and gallic acid was used as standard (Demiray et al., 2009; Majhenic et al., 2007). Different gallic acid solutions were prepared having a concentration ranging from 25 μg/ml to 0.1 μg/ml. In brief, 0.5 ml of Folin-Ciocalteau reagent (diluted 10 times with water) was added to 0.5 ml of gallic acid solution. The reaction mixture was pre- incubated for 5 min and then 1.0 ml of Na2CO3 (7.5 % w/v) solution was added. The mixture was incubated for 2 hour at room temperature. After incubation, the absorbance was measured at 760 nm. After plotting the absorbance in ordinate against 29

the concentration in abscissa a linear relationship was obtained which was used as a standard curve for the determination of the total phenolic content of the test samples. In addition, 0.5 ml of Folin-Ciocalteau reagent (diluted 10 times with water) was added to 0.5 ml of extract solution (conc. 4 mg/ml). The reaction mixture was pre- incubated for 5 min and then 1.0 ml of Na2CO3 (7.5 % w/v) solution was added. The mixture was incubated for 2 hour at room temperature. After incubation, the absorbance was measured at 760 nm by UV-Vis spectrophotometer and using the standard curve prepared from gallic acid solution with different concentration, the total phenolic content of the sample was measured. The total phenolic contents of the sample were expressed as mg of GAE (gallic acid equivalent) /gm of the extractive.

3.5.2 Determination of total flavonoids content The total flavonoids content of the extracts were measured by employing the methods given in the literature involving aluminium trichloride (AlCl3) reagent and quercetin was used as standard (Arvouet-Grand, Vennat, Pourrat, & Legret, 1994). Different quercetin solutions were prepared having a concentration ranging from 25

μg/ml to 0.01 μg/ml. In brief, 1.5 ml of 2% aluminium trichloride (AlCl3) methanolic solution was mixed with 0.5 ml of quercetin solution. The mixture was incubated for 10 minutes at room temperature. After 10 minutes the absorbance was measured at 415 nm. After plotting the absorbance in ordinate against the concentration in abscissa a linear relationship was obtained which was used as a standard curve for the determination of the total flavonoids content of the test samples. In addition, 1.5 ml of

2% aluminium trichloride (AlCl3) methanolic solution was mixed with 0.5 ml of extract solution (conc. 4 mg/ml). The mixture was incubated for 10 minutes at room temperature. After 10 minutes the absorbance was measured at 415 nm by UV-Vis spectrophotometer and using the standard curve prepared from quercetin solution with different concentration, the total flavonoids content of the sample was measured. The total flavonoids contents of the sample were expressed as mg of QE (quercetin equivalent)/gm of the extractive.

3.5.3 Determination of total flavonols content The total flavonols content of the extracts were estimated using the method reported by Adedapo et al. (2008) and quercetin was used as standard (Adedapo, Jimoh, Afolayan, & Masika, 2008). Different quercetin solutions were prepared having a concentration ranging from 25 μg/ml to 0.01 μg/ml. In brief, 0.5 ml of 2% aluminum trichloride (AlCl3) methanolic solution was mixed with 0.5 ml of quercetin solution and 1.0 ml sodium acetate (NaOAc) solution (50 g/l). The mixture was shaken and incubated for 2 hours at room temperature. After incubation the absorbance was measured at 440 nm. After plotting the absorbance in ordinate against the concentration in abscissa a linear relationship was obtained which was used as a standard curve for the determination of the total flavonols content of the test samples.

In addition, 0.5 ml of 2% aluminum trichloride (AlCl3) methanolic solution was mixed with 0.5 ml of extract solution (conc. 4 mg/ml) and 1.0 ml sodium acetate (NaOAc) solution (50 g/l). The mixture was shaken and incubated for 2 hours at room temperature. After incubation the absorbance was measured at 440 nm by UV-Vis spectrophotometer and using the standard curve prepared from quercetin solution with different concentration, the total flavonols content of the sample was measured. The total flavonols contents of the sample were expressed as mg of QE (quercetin equivalent)/gm of the extractive.

3.6 DPPH free radical scavenging activity of S. oleosa fruits The free radical scavenging activity of the extract, based on the scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, was determined by the method described by Braca et al. (2001). Briefly, 1.0 ml of a methanolic solution of the extract, sample or standard at different final concentration was mixed with 1.0 ml of a DPPH methanolic solution (20 µg/ml, 0.05 mM). After 30 minutes reaction period at room temperature in dark place, the absorbance was measured against at 517 nm against methanol as blank by UV-Vis spectrophotometer.

The percentage inhibition activity was calculated from [(A0–A1)/A0]×100; where A0 is the absorbance of the control, and A1 is the absorbance of the extract or standard. Gallic acid was used as positive control. 31

3.7 Chemical investigation of hexane fraction of S. oleosa fruits Separation of the hexane extract (1.0 g) was carried out by VLC using hexane and EtOAc in a polarity gradient manner. On the basis of their TLC characteristics, similar fractions were combined to give 12 fractions (A1-A12, Scheme 3-2 and Table 4-3).

Hexane Extract (1.0 g)

CC (gradient, Hexane:EtOAC)

A1 A4 A9 A12 (324.1 mg) (83.2 mg) (13.0 mg) (33.9 mg)

A2 A5 A8 A11 (114.3 mg) (122.3 mg) (22.5 mg) (143.2 mg)

A3 A6 A7 A10 (56.2 mg) (52.3 mg) (123.9 mg) (4.8 mg)

Scheme 3-2 Fractionation of the hexane extract from S. oleosa fruits

Fraction A1 (324.1 mg) was further purified by column chromatography using isocratic condition of hexane and ethyl acetate (5:0.5) to give 2 fractions (B1-B2, Table 4-4). Fraction B2 (278.4 mg) was further purified by column chromatography using isocratic condition of hexane and dichloromethane (5:0.5) to give 3 fractions (C1-C3, Table 4-5). Fraction C3 (265.6 mg) was further purified by silica gel column chromatography using isocratic condition of hexane and dichloromethane (5:1) to give 7 fractions (D1-D7, Scheme 3-3 and Table 4-6).

A1 (324.1 mg)

CC (Hexane:EtOAc; 5:0.5)

B1 (39.0 mg) B2 (278.4 mg)

CC (Hexane:DCM; 5:0.5)

C1 (1.8 mg) C2 (2.2 mg) C3 (265.6 mg)

CC (Hexane:DCM; 5:1)

D1 (3.1 mg) D3 (19.4 mg) D5 (65.0 mg) D7 (18.6 mg) D2 (14.2 mg) D4 (52.7 mg) D6 (9.1 mg)

Scheme 3-3 Fractionation A1 of the hexane extract from S. oleosa fruits

Fraction A2 (114.3 mg) was further purified by column chromatography using isocratic condition of hexane and dichloromethane (1:1) to give 10 fractions (E1-E10, Scheme 3-4 and Table 4-7).

A2 (114.30 mg) CC (Hexane: DCM; 1:1)

E1 E3 E5 E7 E9 (1.9 mg) (1.9 mg) (1.5 mg) (0.2 mg) (1.1 mg) E2 E4 E6 E8 E10 (1.6 mg) (2.0 mg) (1.3 mg) (1.2 mg) (4.8 mg)

Scheme 3-4 Fractionation A2 of the hexane extract from S. oleosa fruits

The combined A3 and A4 (139.4 mg) was further purified by column chromatography using isocratic condition of 100% dichloromethane to give 8 fraction (F1-F8, Scheme 3-5 and Table 4-8).

A3+A4 (139.4 mg) CC (DCM; 100%)

F1 (27.3 mg) F3 (6.3 mg) F5 (6.8 mg) F7 (5.1 mg)

F2 (4.4 mg) F4 (5.0 mg) F6 (6.4 mg) F8 (34.8 mg)

Scheme 3-5 Fractionation A3 and A4 of the hexane extract from S. oleosa fruits

Fraction A5 (112.3 mg) was further purified by column chromatography using isocratic condition of 100% dichloromethane to give 6 fractions (G1-G6, Table 4-9). Fraction G6 (78.8 mg) was further purified by column chromatography using isocratic condition of hexane and ethyl acetate (3:1) to give 5 fractions (H1-H5, Scheme 3-6 and Table 4-10).

A5 (112.3 mg) CC DCM (100%)

G1 (12.0 mg) G3 (3.0 mg) G5 (3.0 mg) G2 (3.0 mg) G4 (5.2 mg) G6 (78.8 mg) CC Hexane:EtOAc (3:1)

H1 (17.4 mg) H2 (9.6 mg) H3 (17.6 mg) H4 (15.8 mg) H5 (18.4 mg)

Scheme 3-6 Fractionation A5 of the hexane extract of S. oleosa fruits 34

Fraction A7 (123.9 mg) was further purified by column chromatography using isocratic condition of hexane and ethyl acetate (3:1) to give 9 fractions (I1-I9, Scheme 3-7 and Table 4-11).

A7 (123.9 mg) CC Hexane:DCM (1:1)

I1 (7.5 mg) I3 (4.2 mg) I5 (2.2 mg) I7 (3.4 mg) I9 (32.2 mg)

I2 (10.3 mg) I4 (3.0 mg) I6 (2.3 mg) I8 (1.8 mg)

Scheme 3-7 Fractionation A7 of the hexane extract of S. oleosa fruits

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Phytochemical screening of S. oleosa fruits extracts The weight percentage yield of the crude extract and further fractionation in solvents of different polarity from the fresh fruits of Schleichera oleosa are given in methanolic extract (9.10%), hexane fraction (1.38%), ethyl acetate fraction (14.01%) and residual aqueous fraction (64.96%), respectively. The preliminary phytochemistry screening of the methanolic extract from S. oleosa pulp fruits revealed the presence of secondary metabolites of terpenoids, steroids, flavonoids and tannins (Table 4-1).

Table 4-1 Phytochemical profiles of the methanolic extract of S. oleosa pulp fruits

Phytochemical Compound Resulta

Alkaloids -

Anthaquinones -

Flavonoids +

Terpenoids +++

Steroids ++

Cardiac glycosides -

Saponins -

Tannins +

Phlobatannins -

Coumarins -

a (-): Negative test, (+): Weak positive test, (++): Positive test, (+++): Test strongly positive.

4.2 Quantitative phytochemical contents of S. oleosa fruits extracts Previously, it is mentioned that the phytochemical screening of the extractives revealed the presence of terpenoids, steroids, flavonoids and tannins. Polyphenolic compounds, like flavonoids, tannins and phenolic acids, usually found in plants have been reported to have multiple biological effects, including antioxidant activity. Flavonoids and tannins present in the plant extract, as evident from phytochemical screening, may be responsible for the antioxidant action in the tested models (Hasanuzzaman et al., 2014). The result of total phenolic, total flavonoids and total flavonols contents of this plant extractives is presented in Tables 4-2. The results are expressed as the number of gallic acid or quercetin equivalents per gram of the plant extractives. Different extractive displayed total phenolic content ranging from 0.38±0.05 to 2.56±0.08 mg GAE/g of extractive, total flavonoids content ranging from 0.78±0.02 to 5.23±0.04 mg QE/g of extractive and total flavonols content ranging from 0.81±0.04 to 2.90±0.12 mg QE/g of extractive.

Table 4-2 Total phenolic, total flavonoids and flavonols content of methanolic extract and soluble fractions of Schleichera oleosa pulp fruits

Total Phenolic Total Flavonoids Total Flavonols Plant extract (mg GAE/g) (mg QE/g) (mg QE/g)

Methanol extract 2.56 ± 0.08 5.23 ± 0.04 2.90 ± 0.12

Hexane fraction 0.38 ± 0.05 2.61 ± 0.01 0.81 ± 0.04

Ethyl acetate fraction 1.88 ± 0.06 2.78 ± 0.02 1.51 ± 0.07

Residual aqueous 1.03 ± 0.16 0.78 ± 0.02 1.62 ± 0.03 fraction

4.3 DPPH radical scavenging activity of S. oleosa pulp fruits extracts The methanolic extract and further fractionation in solvents of different polarity from the fresh fruits of Schleichera oleosa were evaluated for their antioxidant potential against selected which showed good activity. The antioxidant activity was performed by DPPH radical scavenging assay. Different fractions showed activity at different level (Figure 4-1). From the results showed the highest antioxidant activity by hexane fraction (60.91%) followed by ethyl acetate fraction (43.53%) and residual aqueous fraction (34.94%), respectively.

Figure 4-1 Free radical scavenging activity of methanolic extract and soluble fractions of Schleichera oleosa pulp fruits extracts

4.4 Isolation and purification of hexane fraction from S. oleosa fruits The primary screening of antioxidant activity of variously crude extracts of Schleichera oleosa pulp fruits were evaluated. It was found that the hexane fraction showed the highest antioxidant activity using DPPH radical scavenging assay. From these results, the hexane fraction was selected for further investigation of the isolation and identification of the chemical constituents. The isolation results of the hexane fraction were summarized in Tables 4-3 to 4-11.

Table 4-3 Fractions obtained from the hexane fraction of S. oleosa fruits Fraction Weight (mg) Physical characteristic

A1 324.1 yellow oil

A2 114.3 yellow oil

A3 56.2 deep yellow solid

A4 83.2 yellow solid

A5 122.3 yellow solid

A6 52.3 yellow oil (AA-NMR 1)

A7 123.9 yellow oil

A8 22.5 yellow oil (AA-NMR 2)

A9 13.0 yellow oil

A10 4.8 yellow oil

A11 143.2 deep yellow solid

A12 33.9 deep yellow solid

Table 4-4 Fractions obtained from the A1. Fraction Weight (mg) Physical characteristic B1 39.0 yellow oil (AA-NMR 3)

B2 278.4 yellow oil

Table 4-5 Fractions obtained from the B2. Fraction Weight (mg) Physical characteristic

C1 1.8 colorless oil

C2 2.2 colorless oil (AA-NMR 4)

C3 265.6 yellow oil

Table 4-6 Fractions obtained from the C3. Fraction Weight (mg) Physical characteristic

D1 3.1 colorless oil

D2 14.2 colorless oil (AA-NMR 5)

D3 19.4 colorless oil

D4 52.7 colorless oil

D5 65.0 yellow oil (AA-NMR 6)

D6 9.1 yellow oil

D7 18.6 yellow oil (AA-NMR 7)

Table 4-7 Fractions obtained from the A2. Fraction Weight (mg) Physical characteristic

E1 27.9 yellow oil (AA-NMR 21)

E2 1.6 yellow oil (AA-NMR 22)

E3 1.9 yellow oil (AA-NMR 23)

E4 2.0 colorless oil (AA-NMR 24)

E5 1.5 colorless oil (AA-NMR 25)

E6 1.3 colorless oil (AA-NMR 26)

E7 0.2 yellow oil (AA-NMR 27)

E8 1.2 yellow oil (AA-NMR 28)

E9 1.1 yellow oil (AA-NMR 29)

E10 4.8 yellow oil (AA-NMR 30)

Table 4-8 Fractions obtained from the combined A3 and A4. Fraction Weight (mg) Physical characteristic

F1 27.3 yellow oil (AA-NMR 8)

F2 4.4 yellow oil (AA-NMR 9)

F3 6.3 white solid (AA-NMR 10)

F4 5.0 white solid (AA-NMR 11)

F5 6.8 white solid (AA-NMR 12)

Table 4-8 (continued) Fraction Weight (mg) Physical characteristic F6 6.4 white solid (AA-NMR 13)

F7 5.1 white solid (AA-NMR 14)

F8 34.8 white solid (AA-NMR 15)

Table 4-9 Fractions obtained from the A5. Fraction Weight (mg) Physical characteristic

G1 12 yellow oil (AA-NMR 16)

G2 3.0 yellow oil (AA-NMR 17)

G3 3.0 colorless oil (AA-NMR 18)

G4 5.2 colorless oil (AA-NMR 19)

G5 3.0 colorless oil (AA-NMR 20)

G6 78.8 yellow oil

Table 4-10 Fractions obtained from the G6. Fraction Weight (mg) Physical characteristic

H1 17.4 yellow oil (AA-NMR 40)

H2 9.6 yellow oil (AA-NMR 41)

H3 17.6 yellow oil (AA-NMR 42)

H4 15.8 yellow oil (AA-NMR 43)

H5 18.4 yellow oil (AA-NMR 44)

Table 4-11 Fractions obtained from the A7. Fraction Weight (mg) Physical characteristic

I1 7.5 yellow oil (AA-NMR 31)

I2 10.3 yellow oil (AA-NMR 32)

I3 4.2 yellow oil (AA-NMR 33)

I4 3.0 colorless oil (AA-NMR 34)

I5 2.2 colorless oil (AA-NMR 35)

I6 2.3 colorless oil (AA-NMR 36)

I7 3.4 yellow oil (AA-NMR 37)

I8 1.8 yellow oil (AA-NMR 38)

I9 32.2 yellow oil (AA-NMR 39)

4.5 Characterization of isolated compounds from hexane fraction of S. oleosa pulp fruits

From the isolated results, the pure fractions were identified by TLC and characterized by 1H-NMR and 13C-NMR spectroscopic techniques to give NMR spectral data (AA-NMR1–AA-NMR44).

The AA-NMR3 [Rf = 0.17 (hexane:DCM, 5:0.5)] was obtained as pale yellow oil. The 1H-NMR spectra (Figure 4-2) of this compound showed long-chain free fatty acid signals pattern at  0.95 (3H, t, J = 7.6 Hz), 1.30 (m), 1.61 (t, J = 6.8 Hz), 2.05 (m), 2.30 (t, J = 7.2 Hz), and 2.80 (m), the olefinic proton signal at  5.35 (m) and a methyl ester signal at  3.66 (3H, s). The structure of AA-NMR3 was identified as unsaturated fatty acid methyl ester. In addition, the 1H-NMR spectra

(Figure 4-3) of AA-NMR6 [Rf = 0.23 (Hexane:DCM, 5:1)] was similar to that of 43

AA-NMR3 except the signal at  2.80 (m) was missing. The structure of AA-NMR6 was thus identified as unsaturated fatty acid methyl ester.

Figure 4-2 The 1H-NMR spectra of AA-NMR3

Figure 4-3 The 1H-NMR spectra of AA-NMR6 44

The AA-NMR11–AA-NMR14 [Rf = 0.43 (DCM, 100%)] were obtained as white solid. The 1H-NMR spectra (Figures 4-4) of these compounds showed the resonances of an oxymethine proton at  3.22 (1H, dd, J = 3.6, 10.8 Hz) and an olefinic proton at  5.18 (1H, br s). The eight methyl signals were characterized as triterpenoid pattern signals at  0.81 (3H, s), 0.84 (3H, s), 0.90 (6H, s), 0.96 (3H, s), 1.00 (3H, s), 1.02 (3H, s) and 1.15 (3H, s) were observed. Comparison of the 1H- NMR data with -amyrin from literature, the structure of AA-NMR11–AA-NMR14 were identified as -amyrin (Sunil et al., 2014).

Figure 4-4 The 1H-NMR spectra of AA-NMR11

The AA-NMR15 [Rf = 0.29 (DCM, 100%)] was obtained as white solid. The 1H-NMR spectra (Figure 4-5) of this compound showed the resonances of an oxymethine proton at  3.52 (m), an olefinic proton at  5.35 (br d, J = 4.8 Hz) and trans-olefinic protons at  5.02 and 5.13 (dd, J = 8.4, 14.8 Hz). The six methyl proton signals which were characterized of steroid pattern signals at  0.68 (s), 0.81 (d, J = 6.6 Hz), 0.84 (d, J = 6.3 Hz), 0.85 (t, J = 6.3 Hz), 0.92 (d, J = 6.6 Hz) and 1.03 (s) were observed. Comparison of the1H-NMR data with those of -sitosterol and stigmasterol, the structure of AA-NMR15 was identified as a mixture of -sitosterol and stigmasterol (Moghaddam et al., 2007). In addition, the 1H-NMR spectra (Figure

4-6) of AA-NMR5 [Rf = 0.35 (Hexane:DCM, 5:1)] was similar to that of AA-NMR15 45

except the oxymethine proton signal at  3.52 (m) was missing. The structure of AA- NMR5 was thus identified as a mixture of -sitosterone and stigmasterone.

Figure 4-5 The 1H-NMR spectra of AA-NMR15

Figure 4-6 The 1H-NMR spectra of AA-NMR5 46

All of them, AA-NMR18, 24, 25, 35–37 [Rf = 0.44 (DCM, 100%)] were obtained as white amorphous solid. The 1H-NMR spectra (Figure 4-7) of these compounds showed long-chain fatty acid signals pattern at  0.88 (3H, t, J = 6.8 Hz), 1.25 (m), 1.41 (m), 2.01 (m) and 2.31 (t, J = 7.6 Hz). The olefinic protons at  4.15 (m), 4.19 (m) and 5.34 (m) were observed. The structures of AA-NMR18, 24, 25, 35– 37 were thus identified as unsaturated fatty acid compounds.

Figure 4-7 The 1H-NMR spectra of AA-NMR18

O AA-NMR3 and AA-NMR6; R = OCH3 R n AA-NMR18, 24, 25, 35-37; R = OH

H H

H HO HO

-Sitosterol Stigmasterol H HO -Amyrin

H H

O O -Sitosterone Stigmasterone

4.6 DPPH radical scavenging activity of isolated compounds.

The AA-NMR3, AA-NMR6 (unsaturated fatty acid methyl esters) and AA- NMR11–14 (-amyrin) which were isolated the large amounts from hexane fraction of S. oleosa pulp fruits were evaluated for their antioxidant activity. The antioxidant activity of these isolated compounds was performed by DPPH free radical scavenging assay. Ascorbic acid, quercetin and gallic acid were used as reference antioxidant standard. From the results found that the unsaturated fatty acid methyl esters showed weaker antioxidant inhibitory activity than -amyrin. However, -amyrin also showed weaker antioxidant inhibitory activity than all positive controls as shown in Figure 4-2. In addition, Sunil and co-worker (2014) reported the in vitro and in vivo antioxidant and free radical scavenging activities of -amyrin which showed very good scavenging effects on DPPH (IC50 89.63 ± 1.31 g/mL), hydroxyl (IC50 76.41 ±

1.65 g/mL), nitric oxide (IC50 87.03 ± 0.85 g/mL) and superoxide (IC50 81.28 ± 48

1.79 g/mL) radicals as well as high reducing power and strong suppressive effect on lipid peroxidation. In in vivo study, pre-treatment of rats with -amyrin at 50 mg/kg for 7 days showed significant reduction in the levels of SGOT, SGPT and LDH compared to CCl4 treated rats. The SOD, CAT, GSH and GPx levels were increased significantly after treatment with -amyrin. These results clearly demonstrated that

-amyrin possessed marked antioxidant activity (Sunil et al., 2014).

*a: is a compounds (0.1 mg/ml) *b: is a standards (0.001 mg/ml)

Figure 4-8 Free radical scavenging activity of AA-NMR3, AA-NMR6 and AA-NMR11

CHAPTER 5 CONCLUSION

The preliminary phytochemical screening of the methanolic extract from Schleichera oleosa fruits revealed the presence of terpenoids, flavonoids, tannins and steroids. Flavonoids, tannins and terpenoids present in this plant extract, as evident from phytochemical screening, may be responsible for the antioxidant action in the tested models. These results suggest the potential of S. oleosa fruits as a medicine or cosmetic agents against free-radical-associated oxidative damage. Investigation of the chemical constituents of the hexane fraction from S. oleosa fruits gave unsaturated fatty acid methyl esters (AA-NMR3 and AA- NMR6), unsaturated fatty acid (AA-NMR18, 24, 25, 35–37), a mixture -sitosterone and stigmasterone (AA-NMR5), a mixture of -sitosterol and stigmasterol (AA- NMR15) and -amyrin (AA-NMR11–14). The biological of the AA-NMR3, AA-NMR6 (unsaturated fatty acid methyl esters) and AA-NMR11–14 (-amyrin) were evaluated for their antioxidant activity. The antioxidant activity of these isolated compounds was performed by DPPH free radical scavenging assay. Ascorbic acid, quercetin and gallic acid were used as reference antioxidant standards. The results showed that the unsaturated fatty acid methyl esters showed weaker antioxidant inhibitory activity than -amyrin. However, -amyrin also showed weaker antioxidant inhibitory activity than all positive controls.

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APPENDIX

AA-NMR1 (52.3 mg , CDCl3)

57 1 Figure 6-1 The H-NMR spectra of AA-NMR1

Figure 6-2 The 1H-NMR spectra of AA-NMR2 58

59 1 Figure 6-3 The H-NMR spectra of AA-NMR3

60 1 Figure 6-4 The H-NMR spectra of AA-NMR4

61 1 Figure 6-5 The H-NMR spectra of AA-NMR5

62 1

Figure 6-6 The H-NMR spectra of AA-NMR6

1 63

Figure 6-7 The H-NMR spectra of AA-NMR7

64 1 Figure 6-8 The H-NMR spectra of AA-NMR8

65 1 Figure 6-9 The H-NMR spectra of AA-NMR9

66 1 Figure 6-10 The H-NMR spectra of AA-NMR10

67 1 Figure 6-11 The H-NMR spectra of AA-NMR11

Figure 6-12 The 1H-NMR spectra of AA-NMR12 68

1 69

Figure 6-13 The H-NMR spectra of AA-NMR13

1 70

Figure 6-14 The H-NMR spectra of AA-NMR14

71 1

Figure 6-15 The H-NMR spectra of AA-NMR15

1 72

Figure 6-16 The H-NMR spectra of AA-NMR16

1 73

Figure 6-17 The H-NMR spectra of AA-NMR17

74 1

Figure 6-18 The H-NMR spectra of AA-NMR18

1 75

Figure 6-19 The H-NMR spectra of AA-NMR19

76 1 Figure 6-20 The H-NMR spectra of AA-NMR20

1 77

Figure 6-21 The H-NMR spectra of AA-NMR21

1 78

Figure 6-22 The H-NMR spectra of AA-NMR22

79 1 Figure 6-23 The H-NMR spectra of AA-NMR23

80 1 Figure 6-24 The H-NMR spectra of AA-NMR24

1 81

Figure 6-25 The H-NMR spectra of AA-NMR25

82 1 Figure 6-26 The H-NMR spectra of AA-NMR26

83 1 Figure 6-27 The H-NMR spectra of AA-NMR27

84 1 Figure 6-28 The H-NMR spectra of AA-NMR28

85 1 Figure 6-28 The H-NMR spectra of AA-NMR29

86 1 Figure 6-30 The H-NMR spectra of AA-NMR30

87 1 Figure 6-31 The H-NMR spectra of AA-NMR31

1 88

Figure 6-32 The H-NMR spectra of AA-NMR32

1 89

Figure 6-33 The H-NMR spectra of AA-NMR33

Figure 6-34 The 1H-NMR spectra of AA-NMR34 90

91 1 Figure 6-35 The H-NMR spectra of AA-NMR35

92 1 Figure 6-36 The H-NMR spectra of AA-NMR36

93 1 Figure 6-37 The H-NMR spectra of AA-NMR37

Figure 6-38 The 1H-NMR spectra of AA-NMR38 94

1 95

Figure 6-39 The H-NMR spectra of AA-NMR39

1 96

Figure 6-40 The H-NMR spectra of AA-NMR40

97 1

Figure 6-41 The H-NMR spectra of AA-NMR41

98 1

Figure 6-42 The H-NMR spectra of AA-NMR42

99 1 Figure 6-43 The H-NMR spectra of AA-NMR43

100 1

Figure 6-44 The H-NMR spectra of AA-NMR44

101

BIOGRAPHY

Name Mr. Xayphone Thatavong Date of birth June 17, 1987 Place of birth Xiengkhouang province, Lao PDR Present address Phonkham village Xiengkhouang province Pek district Foreign 135/A

Education 2004-2009 Bachelor of Education (B.Ed), Faculty of Education, National University of Laos 2013-2015 Master of Science (M.Sc.), Faculty of Science, Burapha University, Chonburi, Thailand

Publication

Thatavong, X., & Athipornchai, A. (2015). Phytochemical Analysis and Antioxidant Evaluation from Ceylon oak (Schleichera oleosa) Fruits. In Pure and Applied Chemistry International Conference: Innovative Chemistry for Sustainability of the AEC and Beyond. 21st- 23rd (pp. 497-400).