i antioxidant activities of bioactive compounds from

i

ANTIOXIDANT ACTIVITIES OF BIOACTIVE COMPOUNDS FROM

BARRINGTON AUGUSTA KURZ. AND SYZYGIUM GRATUM

(WIGHT) S.N. MITRA VAR. GRATUM IN BAN ANG-ED

OFFICIAL COMMUNITY FOREST PROJECT

(THE CHAIPATTANA FOUNDATION)

WILAILUCK LEAMKLANG

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE MASTER DEGREE OF SCIENCE

IN BIOLOGICAL SCIENCE

FACULTY OF SCIENCE

BURAPHA UNIVERSITY

AUGUST 2018

COPYRIGHT OF BURAPHA UNIVERSITY

iii

ACKNOWLEDGMENT

In the successful of this thesis, I would like to express my sincere gratitude

and deep appreciation to my principal advisor, Dr. Chatchawin Petchlert and my co-

advisor, Assistant Professor Dr. Ekaruth Srisook for their attention, technical

assistance, helpful suggestion and comment, encouragement and support throughout

my study.

Moreover, I would like to thank Assistant Professor Dr. Orasa Suriyaphan as

an external examiner and Dr. Waeowalee Choksawangkarn as an examiner from

Faculty of Science, for all of their guidance, and valuable advice to this thesis.

Great appreciation is also given to the Biological Science Graduate Program,

Department of Biochemistry and Department of Chemistry for giving me the chemicals

and instruments to do research.

I also thank to Ban Ang-Ed Official Community Forest Project (The

Chaipattana Foundation) at Chantaburi province for supporting the indigenous plants

in this research. I would like to give special thank Dr. Benchawan Chewpreecha for

botanically classified the plants. Surely, special thanks to Burapha University for

financial support through research grant in 2014, and thanks to National Research

Council of Thailand for financial support research grants in 2014 and 2016 .

Finally, great respect is given to my family for their love, kindness and care.

Thank you Ms. Cholsittapan Asawesna, Ms. Natthasri Sirichonpakdee, Ms. Sasithorn

Uttatree, Ms. Phanutchanat Nontesa and our members in laboratory for inspiration,

encouragement and motivation to finish study. I offer special thanks to all my friends

for their friendship, suggestion, solidarity, and togetherness.

Wilailuck Leamklang

iv

56910048: MAJOR: BIOLOGICAL SCIENCE; M.Sc.

(BIOLOGIAL SCIENCE)

KEYWORDS: FREE RADICAL/ ANTIOXIDANTS/ BIOACTIVE COMPOUND/

INDIGENOUS PLANT

WILAILUCK LEAMKLANG: ANTIOXIDANT ACTIVITIES OF

BIOACTIVE COMPOUNDS FROM Barringtion augusta KURZ. AND Syzygium

gratum (WIGHT) S.N. MITRA VAR. GRATUM IN BAN ANG-ED OFFICIAL

COMMUNITY FOREST PROJECT (THE CHAIPATTANA FOUNDATION).

ADVISORY COMMITTEE: CHATCHAWIN PETCHLERT, Ph.D., EKARUTH

SRISOOK, Ph.D. 116 P. 2561.

Barrington augusta Kurz. (Jig) and Syzygium gratum (Wight) S.N. Mitra var.

gratum (Sameddang) are found in the Eastern region of Thailand. They are indigenous

plants collected from Ban Ang-Ed Official Community Forest Project, Chantaburi

province. They have been used in healthcare prevention several diseases by traditional

doctors. In addition, a previous report of both plants in hot water extract showed high

antioxidant activities, but there is still no study of the antioxidant activity of bioactive

compound from these plants. Therefore, this work aims to evaluate the bioactive

compounds by determining the total phenolic content, and the antioxidant activity from

sub-extracts and sub-fractions after purification via TLC and column chromatography

compared with the crude extract. The results showed that water extract of B. augusta

had the highest total phenolic contents of 267.589 ± 1.544 mg GAE/g extract, and it

could strongly inhibit DPPH radical (EC50 = 0.052 ± 0.005 mg/mL). On the other hand,

ethyl acetate extract expressed the greatest FRAP value of 434.340 ± 1.344 mg TE/g

extract and showed ferrous ion-chelating activity of 83.18%. From three assays, it

indicated that ethyl acetate fraction exhibited the high antioxidant activities. Therefore,

we decided to choose this extract to further isolate by column chromatography. After

isolation, the percent of DPPH radical scavenging activity of active fractions from B.

augusta (38.38%) was lower than crude ethyl acetate extract (65.67%). Moreover, both

of FRAP value and %ferrous ion-chelation were decreased to 144.287 ± 1.273 mg TE/g

extract and 36.79%, respectively. For the result of S. gratum, ethyl acetate extract had

the highest total phenolic contents with 151.458 ± 1.360 mg GAE/g extract, and it could

v

remarkably inhibit DPPH radical (EC50 = 0.071 ± 0.002 mg/mL). In addition, it was

also expressed the highest FRAP value of 890.885 ± 4.724 mg TE/g extract and showed

ferrous ion-chelating activity of 30.43%. The results suggested that the ethyl acetate

extract showed high antioxidant activity. Thus, this extract was selected to further

isolate by column chromatography. After isolation, active fractions from S. gratum had

still high DPPH radical scavenging activity (94.36%) and FRAP value of 767.373 ±

3.272 mg TE/g extract. The %ferrous ion-chelating activity of active fraction seemed

to be lower (39.11%) than its crude extract. From those results indicate that B. augusta

and S. gratum were showed remarkable antioxidant activity in ethyl acetate extract, and

active compound can provide additional information for identification and

characterization the bioactive compounds. For the future, their bioactive compounds

can be applied in nutritional and dietary supplemented applications with their

antioxidant activities and total phenolic contents.

vi

CONTENTS

Page

ABSTRACT........................................................................................................ iv

CONTENTS........................................................................................................ vi

LIST OF TABLES............................................................................................... xi

LIST OF FIGURES............................................................................................. xii

CHAPTER

1. INTRODUCTION.................................................................................... 1

1.1 Introduction................................................................................... 1

1.2 Objective……………………………..……………………….… 2

1.3 Hypothesis…………………………………..…………………... 3

1.4 Contribution to the knowledge………………………………...... 3

1.5 Scope of the study.......................................................................... 3

2. LITERATURE REVIEWS....................................................................... 4

2.1 Free radicals……........................................................................... 4

2.1.1 Formation of free radicals……….......................................... 4

2.1.2 Type of free radicals............................................................... 5

2.1.3 Oxidative stress…................................................................... 5

2.1.4 Reaction of free radicals with biomolecules........................... 7

2.1.4.1 DNA................................................................................ 7

2.1.4.2 Lipid…………………………………………………… 8

2.1.4.3 Protein……………………………………………........ 8

2.1.5 Antioxidants……………..…………………………………. 8

2.1.6 Type of antioxidant………………………………………… 9

2.1.6.1 Enzymatic antioxidant………………………………… 9

2.1.6.2 Non-enzymatic antioxidant…………………………… 9

2.2 Indigenous plants……………..…………………………………. 15

2.2.1 Barrington augusta Kurz……………………………..….… 15

2.2.2 Syzygium gratum (Wight) S.N. Mitra var. gratum……...….. 18

vii

CONTENTS (CONTINUED)

Chapter Page

2.3 Chromatographic technique……...…………………………….… 21

2.3.1 Relationship between column diameter, length and sample

size……………………...…………………………………… 22

2.3.2 Column preparation…………………………………............. 22

2.3.3 Bioassay guide-isolation…………………………...………... 23

3. RESEARCH METHODOLOGY………………………………………… 25

3.1 Overview of experiments…………………………..…………….. 25

Part I: Plants preparation and extraction

3.2 Chemicals and equipments………………………………..……...

3.2.1 Chemicals…………………………………..………….……..

3.2.2 Equiments……………………………………………...……..

27

27

27

3.3 Methods………………………………………...…………………

3.3.1 Plants preparation……...………………………………….….

28

28

3.3.2 Plants extraction……………………………………...…….... 28

3.3.3 Monitored the compounds using Nuclear Magnetic

………………….Resonance spectrometer (NMR) …………………………….

29

Part II: Sub-extract determination

3.4 Chemicals and equipments……..………………………………... 29

3.4.1 Chemicals……….....……………………...……………….… 29

3.4.2 Equipments………………………………...…….................... 30

3.5 Methods……………………………............................................... 30

3.5.1 Total phenolic content………...………….............................. 30

3.5.2 DPPH radical scavenging assay……....................................... 30

3.5.3 Ferric reducing antioxidant power (FRAP) assay.................... 31

3.5.4 Ferrous ion-chelating activity……………………...………... 32

viii


Chapter Page

Part III: Bioactive compound isolation by column chromatography

3.6 Chemicals and equipments……..………………………………... 32

3.6.1 Chemicals…………………………………………………….. 32

3.6.2 Equipments…………………………………………………… 33

3.7 Methods….……………………………………………………… 33

3.7.1 Selection of solvent system for elution……………………… 33

3.7.2 Isolation of bioactive compound from B. augusta and

………………….S. gratum by column chromatography…………..…………...

34

3.7.3 Determination of compounds using thin layer

………………….chromatography (TLC)………………………………………

34

3.7.4 Determination of the antioxidant compounds using TLC

………………….screening for DPPH radical scavengers……………………...

34

3.7.5 Elucidation of the compounds using Nuclear Magnetic

………………….Resonance spectrometer (NMR)……………………………..

34

3.7.6 Determination of the active fractions using DPPH radical

………………….scavenging assay……………………………………………..

35

3.7.7 Determination of the active fractions using ferric reducing

………………….antioxidant power assay (FRAP)…………………………….

35

3.7.8 Determination of the active fractions using ferrous ion-

………………….chelating activity……………………………………………..

35

3.8 Statistical analysis…………………………………………...…… 35

4. RESULTS…...……………………………………………………………. 36

Part I: Plants preparation and extraction of B. augusta and Syzygium

………………...gratum

ix


Chapter Page

4.1 The extraction yield…………………………………………….... 36

4.2 Barrington. augusta Kurz………………………………………... 37

Part II: Sub-extract determination of B. augusta

4.2.1 Total phenolic content……..………………………………... 37

4.2.2 DPPH radical scavenging assay………………………...…… 38

4.2.3 Ferric Reducing Antioxidant Power assay (FRAP)………..... 39

4.2.4 Ferrous ion-chelating activity assay……………………...….. 41

Part III: The 1st Column Chromatography of B. augusta

4.2.5 Antioxidant-guided isolation of the fractions from the 1st

column.chromatography…………………………………......

43

4.2.5.1 DPPH radical scavenging assay………………...…... 43

4.2.5.2 Ferric Reducing Antioxidant Power assay (FRAP).... 44

4.2.5.3 Ferrous ion-chelating activity assay……………..…. 45

Part IV: the 2nd Column Chromatography of B. augusta

4.2.6 Determination of the fractions from the 2nd column

…………………chromatography………………………………………………

47

4.2.6.1 DPPH radical scavenging assay…………………….. 48

4.2.6.2 Ferric Reducing Antioxidant Power assay (FRAP)… 48

4.2.6.3 Ferrous ion-chelating activity assay………………… 49

4.3 Syzygium gratum (Wight) S.N. Mitra var. gratum..…………..…. 52

Part II: Sub-extract determination of S. gratum

4.3.1 Total phenolic content…………………………………….… 52

4.3.2 DPPH radical scavenging assay……………………………... 53

4.3.3 Ferric Reducing Antioxidant Power assay (FRAP)………..... 54

4.3.4 Ferrous ion-chelating activity assay………………………… 55

x


Chapter Page

Part III: The 1st Column Chromatography of S. gratum

4.3.5 Antioxidant activity of the fractions from the 1st column

………………... chromatography……………………………………….…….

57

4.3.5.1 DPPH radical scavenging assay……………………...… 58

4.3.5.2 Ferric Reducing Antioxidant Power assay (FRAP)…..... 58

4.3.5.3 Ferrous ion-chelating activity assay………………….... 60

5. DISCUSSION AND CONCLUSIONS …………..……………………. 63

REFERENCES…………………………………………………………….. 71

APPENDIX………………………………………………………………… 78

Appendix A……………………………………………………..…….. 79

Appendix B…………………………...………………….……... 82

Appendix C……………………………………………….……….….. 85

Appendix D……………………………..……….….………………… 92

Appendix E………………………………………………………...…. 96

Appendix F……………………………….………………………….... 100

BIOGRAPHY……………………………………………………………… 116

xi

LIST OF TABLES

Tables Page

2-1 Different types of free radical……..……………………………………… 5

2-2 Nomenclature of reactive species………………………………………… 6

2-3 Contents of phenolic compounds from leaves, sticks and barks extract of

B. racemose……………………………………………………………….. 17

2-4 DPPH radical scavenging assay, ferric reducing antioxidant power

(FRAP) assay, total phenolic content of crude extract of S. gratum…….... 20

2-5 Guidelines for the choice of column diameter according to sample size… 22

4-1 Dry weight and percent yield of B. augusta and S. gratum………..……... 37

4-2 Total phenolic content from ethanol, hexane, ethyl acetate and water

extracts at 1 mg/mL…………………………………………………,,…… 38

4-3 The percent of DPPH radical scavenging from ethanol, hexane, ethyl

acetate and water extracts at 0.1 mg/mL and EC50 of DPPH radical

scavenging assay……………………………………………..…………… 39

4-4 FRAP value from ethanol, hexane, ethyl acetate and water extracts at 0.1

mg/mL…………………………………………………………………….. 40

4-5 The percent of ferrous ion-chelating from ethanol, hexane, ethyl acetate

and water extracts at 0.1 mg/mL………………………………………… 41

4-6 Dry weight and percent yield of each fractions from the 1st column

chromatography…………………………………..……………………… 42

4-7 The percent of DPPH radical scavenging of each fractions at 0.1 mg/mL

from the 1st column chromatography……………..………………………. 43

4-8 FRAP value of each fractions at 0.1 mg/mL from the 1st column

chromatography…………………………………………………………... 44

4-9 The percent of ferrous ion-chelating of each fractions at 0.1 mg/mL from

the 1st column chromatography…………………………………...………. 46

xii

LIST OF TABLES (CONTINUED)

Tables Page

4-10 Dried weight and percent yield of each fraction from 2nd column

chromatography…………………………………………………………… 47


from the 2nd column chromatography…..………………………………… 48

4-12 FRAP value of each fractions at 0.1 mg/mL from the 2nd column

chromatography…………………………………………………………… 49

4-13 The percent of ferrous ion-chelating at 0.1 mg/mL of each fractions from

2nd column chromatography………………………………………………. 50


extracts at 0.05 mg/mL…………………………………………………... 52

4-15 The percent of DPPH radical scavenging from ethanol, hexane, ethyl


scavenging assay………………………………………………………….. 54

4-16 FRAP value from ethanol, hexane, ethyl acetate and water extracts at

0.05 mg/mL……………………………………………………………….. 54

4-17 The percent of ferrous ion-chelating from ethanol, hexane, ethyl acetate

and water extracts at 0.05 mg/mL………………………………………... 56

4-18 Dried weight and percent yield of each fractions from the 1st column

chromatography…………………………………………………………… 57


from 1st column chromatography…………………………………………. 58


chromatography…………………………………………………………… 59

4-21 The percent of ferrous ion-chelating of each fractions at 0.05 mg/mL

from the 1st column chromatography…..…………………………………. 61

xiii

LIST OF FIGURES

Figures Page

2-1 Chemical structures of 2-butylated hydroxyanisole (BHA) and butylated

hydroxytoluene (BHT)……………………………………………………. 10

2-2 Chemical structure of Ethlenediaminetetraacetic acid (EDTA)…………... 10

2-3 Chemical structure of propyl gallate……………………………………... 11

2-4 Chemical structure of ascorbic acid………………………………………. 11

2-5 Chemical structure of α-Tocopherol……………………………………… 12

2-6 Chemical structures of phenol and flavonoid……………………………... 12

2-7 Chemical structures of anthocyanin, catechin, chalcone and flavone…….. 13

2-8 Chemical structures of β-carotene, lycopene, lutein and zeaxanthin……... 15

2-9 Barrington augusta Kurz…………………………………………………. 16

2-10 The structures of gallic acid, ferulic acid, naringin, rutin, luteolin and

kaempferol………………………………………………………………… 18

2-11 Syzygium gratum (Wight) S.N. Mitra var. gratum………………………... 19

2-12 Sample separation by column chromatography…………………………... 23

3-1 Experimental workflow……………………………….………………….. 26

3-2 Steps of extraction……………………………...…………………………. 29


extracts at 1 mg/mL……………………………………………………… 38

4-2 FRAP value from ethanol, hexane, ethyl acetate and water extracts at 0.1

mg/mL…………………………………………………………………….. 40

4-3 FRAP value of each fraction at 0.1 mg/mL from the 1st column

chromatography…………………………………………………………… 45

4-4 FRAP value of each fractions at 0.1 mg/mL from the 2nd column

chromatography…………………………………………………………… 49

4-5 Extraction diagram for the isolation of bioactive compounds from aerial

parts of B. augusta 51

xiv

LIST OF FIGURES (CONTINUED)

Figures Page


extracts at 0.05 mg/mL…………………………………………………... 53

4-7 FRAP value from ethanol, hexane, ethyl acetate and water extracts at

0.05 mg/mL……………………………………………………………….. 55


chromatography…………………………………………………………… 60

4-9 Extraction diagram for the isolation of bioactive compounds from aerial

parts of S. gratum 62

1

CHAPTER 1

INTRODUCTION

1.1 Introduction

Free radicals are atoms or groups of atom with unpaired electrons that are

generated by the interaction of oxygen and certain molecules. Once radicals are formed,

these highly reactive radicals can start a chain reaction (Clarkson, 1995). Free radicals

are also generated by different types of radiation, with X-ray-generated hydroxyl

radical and irradiation with ultraviolet light generating electronically excited states with

the subsequent radical formation. The antioxidants terminate the chain reactions by

oxidized themselves and inhibit other oxidation reactions. Thus, an antioxidant is often

reducing agents such as thiols, ascorbic acid or polyphenols (Sies, 1997).

Although oxidation reactions are important for the life, they can also damage

to plant and animal cells. Plants and animals maintain complex systems of multiple

types of antioxidants such as vitamin C, vitamin A, vitamin E, and glutathione as well

as enzymes such as catalase, superoxide dismutase, and various peroxidases. Deficient

levels of antioxidants or inhibition of the antioxidant enzymes cause oxidative stress

and can damage or kill cells. Oxidative stress causes dangerous to cells structural and

function by over-reactive oxygen molecules and chronic excessive inflammation. It

seems to role in many human diseases including cancer (Jha, Flather, Lonn,

Farkouh, & Yusuf, 1995) cardiovascular disease (Dhalla, Temsah, & Netticadan, 2000),

stroke, heart attacks, atherosclerosis, rheumatoid arthritis, neurodegeneration, diabetes

(Fang, Yang, & Wu, 2002) and Alzheimer's disease (Sayre, Smith, & Perry, 2001).

In the present, the use of antioxidants in pharmacology has been intensively

studied, particularly as treatments for stroke and neurodegenerative diseases (Jha et al.,

1995). In developing countries, traditional medicine and natural extracts began to play

a major role for healthcare (Farnsworth, Akerele, Bingel, Soejarto, & Guo, 1985) and

treatment of various diseases such as inflammation, rheumatism, circulatory problems,

asthma and bronchitis, epilepsy and immune system deficiencies (Farnsworth &

Bunyapraphatsara, 1992). Ban Ang-Ed Official Community Forest Project, it locates in

the eastern region of Thailand. There are many medicinal plants, which have been used

2

in healthcare by traditional medicine. In addition, a previous report remarkably showed

antioxidant activities of several plants from Ban Ang-Ed Official Community Forest

Project, Chantaburi province. In 2013, Thepmongkon, Rungreungburanakul, and

Petchlert determined the antioxidant activities of Barrington augusta Kurz, Syzygium

gratum (Wight) S.N. Mitra var. gratum, Lepisanthes fruticosa (Roxb.) Leenh, Sauropus

amabilis Airy Shaw, Brucea javanica (Linn.) Merr. And Lasia spinosa (L.) Thwaites

in hot water extract. The extracts of B. augusta and S. gratum exhibited better

antioxidant activities such as DPPH scavenging assay, reducing power, total phenolic

and flavonoid contents than the other plant extracts. In the same year, Petchlert,

Wongla, and Phumphinich also investigated the ferrous-ion chelation, ABTS radicals

decolorization assay and ferric ion reducing antioxidant power (FRAP) of Barrington

augusta Kurz, Syzygium gratum (Wight) S.N. Mitra var. gratum, Lepisanthes fruticosa

(Roxb.) Leenh, Sauropus amabilis Airy Shaw, Brucea javanica (Linn.) Merr. and

Cissus hastate Miq. in hot water extract. They found that B. augusta Kurz. and S.

gratum expressed high activities of ferrous-ion chelating, ABTS radicals decolorization

assay and ferric ion reducing antioxidant power than other plants extract. Those reports

indicated that B. augusta and S. gratum displayed good antioxidant activities and they

also expressed high total phenolic and total flavonoid contents. Moreover, B. augusta

is often used as a traditional remedy for several diseases like diarrhea, scurvy, laxative,

conjunctivitis, emetics, and fever (อุไร จิรมงคลการ, 2547) whereas S. gratum is often

used to protect against several diseases like sprain, expectorate, asthma, bronchitis and

anti-parasite (อุไร จิรมงคลการ, 2547). However, there is lack of the study about the

bioactive compounds and their antioxidant activities from these plants. Therefore, in

this study, the bioactive compounds of B. augusta and S. gratum extracts were isolated

and determined their antioxidant activities in order to compare to the crude extracts.

1.2 Objectives To isolate the bioactive compounds from B. augusta Kurz. and S. gratum

(Wight) S.N. Mitra var. gratum by chromatographic techniques and to determine the

antioxidant activities of B. augusta Kurz. and S. gratum (Wight) S.N. Mitra var. gratum

from sub-extracts and active fractions in order to compare to the crude extracts.

3

1.3 Hypothesis Bioactive compounds isolated from selected indigenous plants will express

antioxidant activities.

1.4 Contribution to the knowledge Bioactive compounds extracted from selected indigenous plants can be

applied to nutritional and dietary supplemented applications with their antioxidant

activities and total phenolic content. The extracts may play important roles in the

discovery, development and manufacturing of the functional foods.

1.5 Scope of study B. augusta Kurz. and S. gratum (Wight) S.N. Mitra var. gratum young leaves

obtained from Ban Ang-Ed Official Community Forest Project (The Chaipattana

Foundation) were cleaned and dried by hot air oven. They were extracted with different

solvents including ethanol, hexane, ethyl acetate, and water. The extracts were collected

from each step and they were monitored by NMR spectroscopy. Total phenolic contents

of all extracts were determined. Additionally, antioxidant activities were determined

using DPPH radical scavenging assay, ferrous ion-chelating activity assay and ferric

reducing antioxidant power assay in order to guide to the isolation of the bioactive

compounds. Sub-extract with the highest activities was isolated by column

chromatography. Each isolate was monitored by TLC; the same fraction was collected

and screened DPPH radical scavenger by TLC screening. Additionally, All fractions

were elucidated the structure using NMR spectroscopy. After that, antioxidant activities

of active fractions were evaluated again including DPPH radical scavenging, ferric

reducing antioxidant power and ferrous ion-chelating activities.

4

CHAPTER 2

LITERATURE REVIEWS

2.1 Free radicals

Free radicals are highly reactive chemicals having an unpaired electron in

outer orbital, such as hydroxyl radicals and superoxide anions. The free radicals

produced in the body by normal metabolic activities have beneficial effects in a

physiological. When they reach the pathologic level because of exogenous reasons such

as extreme sports, infection, radiation, smoking and diets rich in free radicals (Erbas &

Sekerci, 2011), after that the free radicals were formed. These highly reactive radicals

can start a chain reaction, like dominoes. Their chief danger comes from the damage

when they react with important cellular components such as DNA, or the cell

membrane. Cells may loss functional or die (Halliwell & Gutteridge, 2008).

2.1.1 Formation of free radicals

Steps involved free radical generation:

Initiation reactions are those results by heat, UV radiation or metal catalysis.

This is a homolytic cleavage at covalent bond of a normal molecule.

RH + initiator R• + H•

Propagation reactions are those reactions involving free radicals in which

the total number of free radicals remains the same.

R• + O2 ROO•

ROO• +RH ROOH + R•

Termination reactions are those reactions resulting in a net decrease in the

number of free radicals. Two free radicals combine to form a more stable species.

R• + R• RR

ROO• + ROO• ROOR + O2

light T

5

2.1.2 Type of free radicals

Most free radicals called reactive oxygen species (ROS) such as superoxide,

hydroxyl, peroxyl and alkoxyl radicals are generated from oxygen atoms. Chlorine

radical and trichloromethyl are also free radical that called reactive chlorine species

(RCS). Moreover, carbon atom (e.g. trichloromethyl) and nitrogen atom (e.g. nitrogen-

centred radical) could also be sources of free radical (Table 2-1).

Table 2-1 Different types of free radical

Name Formula

Hydrogen atom

Trichloromethyl

Superoxide

Hydroxyl

Thiyl/perthiyl

Peroxyl, alkoxyl

Nitrogen-centred radicals

Chlorine radical

H•

CCl3•

O2• ‾

OH•

RS•/RSS•

RO2•, RO•

C6H5N=N•

Cl•

(Halliwell & Gutteridge, 2008)

2.1.3 Oxidative stress

Oxygen radicals can occur as alkyl group of peroxyl radicals, e.g. in lipids.

Moreover, there are some oxide of nitrogen (e.g. nitric oxide), one of the radicals of

biological interest. Free radicals are also generated by different types of radiation, with

X-irradiation generating the hydroxyl radical. Ultraviolet light generated electronically

excited states with subsequent radical formation. Microwave radiation can also

generate reactive oxygen species. Even shear stress, e.g. homogenization, could

generate radicals (Sies, 1997).

Oxidative stress was defined as an imbalance between production and

destruction of free radicals and reactive oxygen species (ROS). This imbalance leads to

damage of biomolecules and cells with potential impact on the whole organism. ROS

and products of cellular metabolism play vital roles in the stimulation of signaling

pathway in plant and animal cells in response to changes in intracellular and

6

extracellular conditions. Most ROS were generated by the mitochondrial respiratory in

cells (Reuter, Gupta, Chaturvedi, Bharat, & Aggarwal, 2010). Indeed, there are not only

ROS that play roles in biology, but also reactive nitrogen species (RNS), reactive

chlorine species (RCS), reactive bromine species (RBS) and reactive sulphur species

(Halliwell & Gutteridge, 2008). Several of reactive species are showed in Table 2-2.

Table 2-2 Nomenclature of reactive species

Free radical Non-radicals

Reactive oxygen species (ROS)

Superoxide, O2• ‾

Hydroxyl, OH•

Hydroperoxyl, HO2•

Carbonate, CO3• ‾

Peroxyl, RO2•

Alkoxyl, RO•

Carbon dioxide, CO2• ‾

Single 1O2

Reactive chlorine species (RCS)

Atomic chlorine, Cl•

Reactive bromine species (RBS)

Atomic bromine, Br•

Hydrogen peroxide, H2O2

Hypobromous acid, HOBr

Hypochlorous acid, HOCl

Ozone, O3

Singlet oxygen, 1O2

Organic peroxides, ROOH

Peroxynitrite, ONOO‾

Peroxynitrate, O2NOO‾

Peroxynitrous acid, ONOOH

Nitrosoperoxycarbonate, ONOOCO2‾

Peroxomonocarbonate, HOOCO2‾

Hypochlorous acid, HOCl

Nitryl chloride, NO2Cl

Chloramines

Chlorine gas, Cl2

Bromine chloride, BrCl

Chlorine dioxide, ClO2

Hypobromous acid, HOBr

Bromine gas, Br2

Bromine chloride, BrCl

7

Table 2-2 (continued)

Free radicals Non-radicals

Reactive nitrogen species (RNS)

Nitric oxide, NO•

Nitrogen dioxide, NO2•

Nitrate, NO3•

Reactive nitrogen species (RNS)

Nitrous acid, HNO2

Nitrosylcation, NO+

Nitroxyl anion, NO‾

Dinitrogen tetroxide, N2O4

Dinitrogen trioxide, N2O3

Peroxynitrite, ONOO‾

Peroxynitrate, O2NOO‾

Peroxynitrous acid, ONOOH

Nitronium (nitryl) cation, NO2+

Alkyl peroxynitrites, ROONO

Alkyl peroxynitrates, RO2ONO

Nitryl chloride, NO2Cl

Peroxyacetylnitrate,CH3C(O)OONO2

(Halliwell & Gutteridge, 2008)

2.1.4 Reaction of free radicals with biomolecules

Free radicals and reactive species imbalance leads to damage of biomolecules.

In the cells, metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic,

nickel) could mediate for mation of free radicals (e.g. Fenton reaction). Biomolecules

have been damage including DNA (both mitochondrial and nucleus), lipids and proteins

(Valko, Rhodes, Moncol, Izakovic, & Mazur, 2006).

2.1.4.1 DNA

Reactive species are involved in the development of cancer, both by

direct effects on DNA and by modulating signal transduction, cell proliferation,

senescence, and cell death. Indirect damage to DNA by RS can lead to activation of

Ca2+-dependent endonucleases. Direct damage by RS can affect the purine or

pyrimidine bases. For example; if Fenton-reaction generate hydroxyl radical (OH•) in

the nucleus, hydroxyl radical oxidizes guanosine to 8-hydroxyl-2-deoxyguanosine (8-

8

OHdG) (Halliwell & Gutteridge, 2008). 8-OHdG has been noted in various tumors,

strongly implicating such damage in the etiology of cancer (Valko et al., 2006).

2.1.4.2 Lipid

Membrane lipids present in subcellular organelles contain

polyunsaturated fatty acid residues of phospholipids, which are highly sensitive to

oxidation reaction lead to damage by free radical (Marnett, 1999). The overall process

of lipid peroxidation consists of three stages: initiation, propagation and termination.

Once formed, peroxyl radicals (ROO•) can be rearranged through cyclisation reaction

to endoperoxides with the final product of lipid peroxidation reaction being

malondialdehyde (MDA). MDA is mutagenic in bacterial and mammalian cells by

reacts with the free amino group of proteins, phospholipids, and nucleic acids induce

dysfunction of immune systems (Valko et al., 2006).

2.1.4.3 Protein

Mechanisms involved in the oxidation of proteins by ROS were

elucidated by studies in which amino acids, simple peptides and proteins were exposed

to ionizing radiations under conditions where hydroxyl radicals or a mixture of

hydroxyl/superoxide radicals are formed (Stadtman, 2004; Valko et al., 2006). It can

impair the functions of receptors, antibodies, signal transduction, transport proteins and

enzyme. Protein damage can lead to secondary damage to other biomolecules, for

example; raising Ca2+ levels and activating nucleases (Halliwell & Gutteridge, 2008).

2.1.5 Antioxidants

An antioxidant is a molecule that inhibits the oxidation reaction of other

molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a

substance to an oxidizing agent. Oxidation reactions can produce free radicals. When

radicals can start chain reactions, the chain reaction occurs in a cell, it can cause damage

to the cell. Antioxidants could terminate these chain reactions by removing free radical

intermediates and inhibit other oxidation reactions (Sies, 1997). The functional of

antioxidant include radical scavenging, singlet oxygen quenching, metal chelation,

chain-breaking, synergism and enzyme inhibition. The reaction of glutathione with the

radical R• can be described:

9

GSH + R• GS• + RH

Thiyl radicals generated may dimerise to form the non-radical product,

oxidized glutathione (GSSG)

GS• + GS• GSSG

Thiol compounds are due to the sulphur atom which can easily accommodate

the loss of a single electron. Oxidized glutathione GSSG is accumulated inside the cells

and the ratio of GSH/GSSG is a good measure of oxidative stress of organisms (Valko

et al., 2006).

Furthermore, metal chelation is one important of scavenge free radicals

mechanism. In Fenton reaction, iron (II) is oxidized by hydrogen peroxide to iron (III),

forming a hydroxyl radical and a hydroxide ion in the process. Antioxidants are stop

reaction by chelate ferrous (Fenton, 1894).

Fe2+ + H2O2 Fe3+ + HO• + OH−

2.1.6 Types of antioxidant

Antioxidants can be classified to 2 major groups, which are enzymatic

antioxidants and non-enzymatic antioxidants.

2.1.6.1 Enzymatic antioxidant such as superoxide dismutase, catalase,

glutathione reductase and glutathione peroxidases are exert synergistic actions in

scavenging free radicals.

2.1.6.2 Non-enzymatic antioxidant can be divided into 2 types

1) Synthetic antioxidant

- Butylated Hydroxyanisol (BHA) and butylatedhydroxytoluene

(BHT) are the most commonly used antioxidants and present constant intractable

problems to the food industry (Figure 2-1) (Shibamoto & Bjeldanes, 2009).

10

BHA BHT

Figure 2-1 Chemical structures of 2-butylated hydroxyanisole (BHA) and butylated

hydroxytoluene (BHT)

- Ethylenediaminetetraacetic acid (EDTA) is a colorless, water-

soluble solid and widely used to dissolve timescale. EDTA has a claw like molecular

structure that binds to metal ions and other toxins in the practice of chelation therapy,

e.g., for treating mercury and lead poisoning. It is used in a similar manner to remove

excess iron from the body. This therapy is used to treat the complication of repeated

blood transfusions, as would be applied to treat thalassemia (Seely, Wu, & Mills, 2005).

The structure is shown in Figure 2-2.

Figure 2-2 Chemical structure of Ethylenediaminetetraacetic acid (EDTA)

- Propyl gallate (n-propyl-3,4,5-trihydroxybenzoate) found in

vegetable oils and butter (Figure 2-3) (Shibamoto & Bjeldanes, 2009).

11

Figure 2-3 Chemical structure of propyl gallate

2) Natural antioxidant

- L-Ascorbic acid (Vitamin C) is water-soluble antioxidant in

humans (Lee et al., 2003). Ascorbic acid has two ionize –OH groups (Figure 2-4).

Plants and most animals can synthesize ascorbate from glucose, but human, other

primate, some fish and fruit bats lost gulonolactone oxidase enzyme, so they need

ascorbate in diet food (Appenroth, Fröb, Kersten, Splinter, & Winnefeld, 1997).

Vitamin C can reduce cadmium toxicity and excess doses prolong the retention time of

an organic mercury compound in a biological system (Shibamoto & Bjeldanes, 2009).

Figure 2-4 Chemical structure of ascorbic acid

- α-Tocopherol (Vitamin E) occurs in many kind of plants such as

lettuce and alfalfa (Shibamoto & Bjeldanes, 2009). The richest natural sources of tocols

are wheat germ, soybean, corn, oils, margarines, nuts, seeds, cereal grains and green

vegetables. Vitamin E is the most important lipophilic antioxidant in human organism

(Püssa, 2008). Tocopherol and tocotrienols can inhibit lipid peroxidation. It scavenge

lipid peroxyl radicals (LO2•) much faster than other radicals can react with adjacent

fatty acid side chains or with membrane proteins (Halliwell & Gutteridge, 2008). The

structure is shown in Figure 2-5.

12

Figure 2-5 Chemical structure of α-Tocopherol

- Phenolic compounds are phytochemicals that important aromatic

secondary metabolites in plants, many of which are commonly substituted by sugar

moieties such as glucose, arabinose, xylose, rhamnose and galactose. The distribution

and composition of phenolic phytochemicals are affected by maturity, cultivars,

horticultural practices, geographic origin, growing season, postharvest storage

conditions and processing procedures (Kim, Jeong, & Lee, 2003). Such compounds are

present in the human diet, mostly through the ingestion of fruits and vegetables

(Balasundram, Sundram, & Samman, 2006; O’Shea, Arendt, & Gallagher, 2012). In

addition, many phenolic phytochemicals have anti-oxidative, anti-carcinogenic, anti-

microbial, anti-allergic, anti-mutagenic and anti-inflammatory activities (Ito et al.,

1998; Cao & Cao, 1999; Kawaii, Tomono, Katase, Ogawa, & Yano, 1999; Eberhardt,

Lee, & Liu, 2000; Kim, Choi, & Chung, 2000). Some phytochemicals including

flavonoids such as quercetin in onion, wine, teas and other plant products (Halliwell &

Gutteridge, 2008), consumed as part of our daily diet, can reduce and exhibit the risk

of cardiovascular disease (Cook & Samman, 1996).

Phenol Flavonoid

Figure 2-6 Chemical structures of phenol and flavonoid

Phenol is any compound that contains –OH group attached to a

benzene ring. Most phenols exert antioxidant effects in vitro, inhibiting lipid

13

peroxidation by acting as chain-breaking peroxyl radical scavengers. In addition,

phenols often scavenge other RS, such as OH•, NO2•, N2O3, ONOOH and HOCl

(Halliwell & Gutteridge, 2008).

Flavonoids are water soluble polyphenolic molecules containing

15 carbon atoms, and belong to the polyphenol family. It 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, thus forming a third middle ring, which can

be five or six-membered. The flavonoids consist of 6 major subgroups: chalcone,

flavone, flavonol, flavanone, anthocyanins and isoflavonoids (Figure 2-7) (Erdman

et al., 2007).

(a) (b)

(c) (d)

Figure 2-7 Chemical structures of (a) anthocyanin, (b) catechin, (c) chalcone and (d)

flavone

- Carotenoids are natural pigments found in photosynthetic

organisms, act as vitamin A precursors and efficient antioxidants (Naves & Moreno,

1998) and sensitive to oxidation and degradation response to oxygen, heat and light

(You, Jeon, Byun, Koo, & Choi, 2015). Carotenoids were known to prevent oxidative

14

damage as potent antioxidants; besides prevent the cancers, cardiovascular disease and

molecular degeneration (Edge, McGarvey, & Truscott, 1997; Castenmiller & West,

1998). Chemical structure determines its stability as well as chemical and biological

reactivity of compound. Previous studies have reported that antioxidant activity of

carotenoids was originated either from the functional groups or the conjugated polyene,

for example; the effective of anti-oxidation tends to vary depending on the polyene

length of carotenoids. Carotene without functional groups other than carbon and

hydrogen shows better antioxidant activity than xanthophyll with oxo and hydroxyl

functional groups at the terminal rings (Mortensen & Skibsted, 1997). Carotenoids are

occurring naturally such as β-carotene, lycopene, lutein and zeaxanthin (Figure 2-8).

(a)

(b)

(c)

15

(d)

Figure 2-8 Chemical structures of (a) β-carotene, (b) lycopene, (c) lutein and

(d) zeaxanthin.

The antioxidant activity of carotenoids arises primarily as a

consequence of the ability of the conjugated double-bonded structure to delocalize

unpaired electrons. This is primary responsible for the excellent ability of β-carotene to

physically quench singlet oxygen without degradation, and for the chemical reactivity

of β-carotene with free radicals such as the peroxyl (ROO•), hydroxyl (OH•), and

superoxide radicals (O2• −) (Valko et al., 2006).

2.2 Indigenous plants 2.2.1 Barrington augusta Kurz.

Barrington augusta Kurz. belongs to the family Lecythidaceae. (Figure 2-9)

Thai folk name is Jig. It is always found in the wetland of Thailand. In Thailand, it is

used by decoction of leaves against scurvy, dysentery and diarrhea. Barks are used for

the laxative. Seeds are used medicinally as antipyretics and anti-conjunctivitis (อุไร

จิรมงคลการ, 2547).

16

Figure 2-9 Barrington augusta Kurz (Courtesy by Juraiporn Hannarong)

In 2007, Ali, Muse, and Mohd reported that the antioxidant activities of

Barringtonia racemosa L. which is a member of the Lecythidaceae family using Ferric

Thiocyanate method (FTC), Thiobarbituric acid method (TBA) and DPPH radical

scavenging methods. The results showed that its chloroform extract exhibited the

highest activity by FTC method (79.13 ± 1.75%) followed by hexane extract (76.52 ±

1.64%), tocopherol (76.52 ± 1.64%) and ethanol extract (51.39 ± 0.30%). By TBA

method, it showed that chloroform extract presented the highest activity (58 ± 1.42%)

followed by hexane extract (54 ± 1.14%), tocopherol (46.4 ± 1.54%) and ethanol extract

(33 ± 0.54%). IC50 values for DPPH radical scavenging of α-tocopherol, chloroform,

hexane and ethanol extracts were found to be 32, 54, 63 and 125 µg/mL, respectively.

All two non-polar extracts (chloroform and hexane extracts) could inhibit DPPH radical

at significantly higher (P< 0.05) than polar extract (ethanol extract).

Later, Hussin et al. (2011) determined the polyphenol and flavonoid

compounds of Barrintonia racemosa L. that is in the same family with Barrington

augusta Kurz. from its leaf, stick and bark in boiling water, methanol and ethanol

extracts, respectively. The results are shown in Table 2-3, the leaves extract of B.

racemosa showed two different phenolic acids include gallic acid and ferrulic acid and

four different flavonoids include naringin, rutin, luteolin and kaempferol with values of

171.81, 65.80, 62.94, 59.10, 10.29 and 5.75 μg/g freeze-dried weight tissue,

17

respectively). In stick, gallic acid, naringin and luteolin could be detected from the

extract (103.53, 51.17 and 5.22 μg/g freeze-dried weight tissue, respectively). Further

HPLC analysis on the extracts of B. racemosa bark revealed that gallic acid, ferrulic

acid and naringin were present as the major phenolic compounds with 56.92, 25.67 and

13.76 μg/g freeze-dried weight tissue.

Table 2-3 Contents of phenolic compounds from leaves, sticks and barks extract of

B. racemosa

Sample

extract

Content (µg/g freeze-dried weight tissue)

Phenolic Flavonoid

gallic acid ferulic acid naringin rutin luteolin kaempferol

Leaf

Stick

Bark

171.81

103.53

56.92

65.80

-

25.67

62.94

51.17

56.92

59.1

-

-

10.29

5.22

-

5.75

-

-

(Hussin et al., 2011)

The structures of phenolic and flavonoid compounds found in this research

are shown in Figure 2-10.

(a) (b)

18

(c) (d)

(e) (f)

Figure 2-10 The structures of (a) gallic acid, (b) ferulic acid, (c) naringin, (d) rutin, (e)

luteolin and (f) kaempferol

2.2.2 Syzygium gratum (Wight) S.N. Mitra var. gratum

S. gratum (Wight) S.N. Mitra var. gratum belongs to the family Myrtaceae.

Thai folk name is Sameddang or Mek. It is always found in India, Myanmar, Thailand

and Malaysia. Sameddang can be found in the dry forest all over Thailand. In

Thailand, medicinal use is an essential oil from leaves to protect sprain and swell.

Decoction of leaves is medicinally used against expectorate, asthma and bronchitis

(อุไร จิรมงคลการ, 2547).

19

Figure 2-11 Syzygium gratum (Wight) S.N. Mitra var. gratum (Courtesy by Juraiporn

Hannarong)

Kukongviriyapan, Luangaram, Leekhaosoong, Kukongviriyapan, and

Preeprame (2007) studied the DPPH free radical scavenging and ferric reducing

antioxidant power assay of Syzygium gratum (synonym: Eugenia grata WIGHT) in hot

water extract. Syzygium gratum was collected between January and March from local

agricultural field in Khon Kaen province, Thailand. Researchers demonstrated that

plant extracts possessed high free radical scavenging (IC50 = 4.08 ± 0.50 µg/mL) and

total antioxidant capacity (FRAP assay) was 261 ± 25 µg ascorbic acid equivalence/mg

extract.

Senggunprai, Kukongviriyapan, Prawan, and Kukongviriyapan (2010)

determined the antioxidant activities from Syzygium gratum. leaves extracted in hot

water and 95% ethanol. They tested DPPH radical scavenging assay, ferric reducing

antioxidant power (FRAP) assay and total phenolic assay. The results are shown in

Table 2-4. Ethanolic extract of S. gratum showed strong DPPH radical scavenging

activity (IC50 values of 0.58 ± 0.18 µg/mL) when compared with water extract (IC50

values of 2.04 ± 0.14 μg/ml). From FRAP assay, water extract (295 ± 38 µg ascorbic

acid equivalence/mg extract) was also displayed the highest activity than ethanolic

extract (262 ± 48 µg ascorbic acid equivalence/mg extract). Moreover, water extract

had contain the total phenolic content (31.3 ± 1.0 g gallic acid/100 g extract) higher

than ethanolic extract (12.1 ± 0.3 g gallic acid/100 g extract). These data suggest that

S. gratum had a rich source of antioxidant compounds.

20

Table 2-4 DPPH radical scavenging assay, ferric reducing antioxidant power

(FRAP) assay, total phenolic content of crude extract of S. gratum

S. gratum extract

IC50 of DPPH

(µg/mL)

FRAP assay

(μg ascorbic acid

equivalence/mg

extract)

Total phenolic

content (g gallic

acid/100 g

extract)

Water extract

Ethanolic extract

2.04 ± 0.14

0.58 ± 0.18

295 ± 38

262 ± 48

31.3 ± 1.0

12.1 ± 0.3

(Senggunprai et al., 2010)

Chanudom and Tangpong (2011) determined the antioxidant activity (by kit

assay) and screened for total phenolic contents (by Folin-Ciocalteu method) from

ethanol and water extracts of S. gratum (Wight) S.N. Mitra var. gratum. The results

showed that total phenolic contents in water extracts and ethanol extracts were 111.583

± 0.001 and 84.917 ± 0.001 mg GAE/g dry weight, respectively. Moreover, water

extracts also showed higher antioxidant activity than ethanol extracts (38.375 ± 0.16

and 5.731 ± 0.16 mM Trolox, respectively).

Stewart, Boonsiri, Puthong, and Rojpibulstit (2013) assessed the antioxidant

activities using the ABTS system and analyzed for total phenolic content from ethyl

acetate extract of S. gratum. They suggested that S. gratum showed the highest level

of ABTS scavenging activity of 2,823.521 ± 27.521 mM TEAC/g dry weight.

Additionally, S. gratum also displayed total phenolic values at 149.789 ± 0.381 mg

GAE/g dry weight.

Later, Thepmongkon et al. (2013) also determined the antioxidant activities

of B. augusta Kurz. and S. gratum (Wight) S.N. Mitra var. gratum in hot water extract.

They showed that the high activity against DPPH radical with IC50 of B. augusta Kurz.

and S. gratum (Wight) S.N. Mitra var. gratum were 0.0419 ± 0.006 and 0.0417 ± 0.004

mg/mL, respectively. The researchers suggested that B. augusta also expressed the

great reducing power at 30.27 mg gallic acid equivalent/g extract. B. augusta Kurz. also

presented high total phenolic and flavonoid contents by 251.26 ± 0.005 mg gallic acid

equivalent/g extract and 146.74 ± 0.023 mg quercetin equivalent /g extracts,

21

respectively whereas S. gratum (Wight) S.N. Mitra var. gratum presented total phenolic

and flavonoid contents by 231.33 ± 0.026 mg gallic acid equivalent/g extract and

131.72 ± 0.006 mg quercetin equivalent /g extracts, respectively.

In addition, Petchlert et al. (2013) also investigated the ferrous-ion chelation,

ABTS radicals decolorization assay and ferric ion reducing antioxidant power (FRAP)

of B. augusta Kurz. and S. gratum (Wight) S.N. Mitra var. gratum in hot water extracts.

B. augusta Kurz. possessed the higher iron chelating effect (99.18%) than S. gratum

(Wight) S.N. Mitra var. gratum (91.85%). Furthermore, B. augusta Kurz. at 5 mg/mL

can decolorize ABTS radicals. They presented the highest TEAC value (72.41 ± 0.001

μM trolox equivalent/mg extract). S. gratum (Wight) S.N. Mitra var. gratum also

showed good FRAP value by 15.61 ± 0.026 mM FeSo4/mg extract.

In 2013, Gohar, Maatooq, Gadara, and Aboelmaaty isolated compounds from

the bark of Callistemon viminalis of Myrtaceae family (the same family with S.

gratum). Structures of these compounds were elucidated on the basis of their

spectroscopic data (NMR, MS, IR spectra and COSY and HR-MS). They found lupeol,

octacosanol, β-sitosterol, betulin, betulinic acid, ursolic acid, corosolic acid, β-

sitosterol-3-O-β-D-glucoside, methyl gallate, gallic acid, catechin, ellagic acid and 3-

O-acetylursolic acid.

Later, Zhang et al. (2015) determined total phenolic content of Callistemon

lanceolatus (the same family with S. gratum). Its ethyl acetate extract showed high total

phenolic content (68.35 ± 0.16 mg GAE/g extract) followed by methanol extract (89.26

± 0.19) and aqueous extracts (16.76 ± 0.21 mg GAE/g, respectively. They suggested

that antioxidant compounds such as methyl gallate, catechin, gallic acid, ellagic acid

and other phenolics can exhibit antioxidant and anticancer activities.

2.3 Chromatographic technique Chromatographic technique is used to isolate compounds from a mixture.

Samples were separated by column chromatography. Column chromatography is

generally used as a purification technique. The mixture analyzed by column

chromatography is administered from the top of the column. If the solvent is flown

down through the column by gravity, it is called gravity column chromatography. If the

22

solvent is forced down the column by positive air pressure, it is called flash

chromatography (Claeson, 1993).

2.3.1 Relationship between column diameter, length and sample size

The gradient elution furthermore gives a better separation on a column of a

shorter length than the originally described 15 cm. For rather nonpolar compounds we

generally use column lengths of 10-12 cm, for more polar compounds columns with a

length of 6-8 cm. Even though the largest column (diameter of 14 cm), we usually

employ lengths of 14-16 cm and 8-10 cm, respectively (Claeson, 1993). The following

Table 2-5 is an approximate guideline for the choice of column diameter according to

sample size.

Table 2-5 Guidelines for the choice of column diameter according to sample size

Sample size (g) Column diameter (cm) silica gel in use (g)

1-2

3-6

7-30

30-50

50-80

80-200

3

4

6

8

10

14

25

50

100

200

400

700-1000

(Claeson, 1993)

2.3.2 Column preparation

The methods are generally used to prepare a column including dry and wet

methods. For the dry method, the column is first filled with dry stationary phase

powder, followed by addition of mobile phase. For the wet method, a slurry is prepared

of the eluent with the stationary phase powder and then carefully poured into the

column avoid air bubbles. A solution is added on top of the stationary phase. This layer

is usually topped with a small layer of sand or cotton or glass wool to protect the shape

of the organic layer from the velocity of newly added eluent. Eluent is slowly passed

through the column (Patra, Gouda, Sahoo, & Thatoi, 2012).

23

Additionally, the polarity of solvent is important factor in chromatographic

technique. The polarity of the solvent which is passed through the column affects the

relative rates at which compounds move through the column. Polar solvents are

competitive with the polar molecules of a mixture for the polar sites on the adsorbent

surface and will also better solvent the polar constituents. Consequently, a highly polar

solvent will move even highly polar molecules rapidly through the column. If a solvent

is too polarity, movement becomes too rapid, that will result the components of a

mixture no separate or little separate. Often a series of increasingly polar solvent

systems are used to elute a column. A less-polar solvent is first used to elute a less-

polar compound. Once the less-polar compound is off the column, a more-polar solvent

is added to the column to elute the more-polar compound (Mann & Saunders, 1978).

Separation of the compounds through the column is in Figure 2-12. Thin-Layer

Chromatography (TLC) is used to determine the system for a column chromatography

separation.

Figure 2-12 Sample separation by column chromatography

(http://organicchemistrysite.blogspot.com/2013/08/column-

chromatography.html)

2.3.3 Bioassay guide-isolation

For the study of plants that used in the traditional medicine, bioassay-guided

isolation were used to preliminarily selected for the study of plants extracts in

developing countries (Pieters & Vlietinck, 2005). ประภาพร ชนยทุธ (2545) isolated the

24

chemical constituents of Gardenia Collinsae craib. G. collinsae. He used TLC

screening for DPPH radical scavenging as bioassay guide-isolation lead to isolate the

bioactive compound from plant extract. Sometimes it is called “bioassay-guided

fractionation” if the sample passes through the column chromatography.

Another previous report studied the isolation of active compounds responsible

for the antioxidant property from Lippia nodiflora L. through the bioassay-guided

fractionation using DPPH assay. L. nodiflora were extracted with 90% methanol. And

then, it was successively partitioned into ethyl acetate, n-butanol and water. All the

fractions were collected and tested for antioxidant property by DPPH assay. The ethyl

acetate fraction exhibited the highest free-radical scavenging activity. The highest

DPPH activity fraction was loaded to column chromatography. Finally, the active

compound structures were analyzed by nuclear magnetic resonance spectra (recorded

on BRUKER, AVANCE 400 MHz), and HPLC analysis was performed using a C-18

column. Moreover, antioxidant activities of pure compounds were determined

including DPPH assay, ferric reducing antioxidant power assay, superoxide radical-

scavenging assay, hydroxyl radical scavenging assay, nitric oxide radical scavenging

assay and lipid peroxidation assay (Sudha & Srinivasan, 2014).

25

CHAPTER 3

RESEARCH METHODOLOGY

3.1 Overview of experiments Young leaves of Barrington augusta Kurz. and Syzygium gratum (Wight)

S.N. Mitra var. gratum were extracted first with ethanol before partitioned with hexane,

ethyl acetate, and water. Sub-extracts were collected and then total phenolic content

was determined. Additionally, antioxidant activities were evaluated for bioassay-

guided isolation using DPPH scavenging, ferric reducing antioxidant power (FRAP)

and ferrous ion-chelating activity assays. Each extract was monitored by NMR

spectroscopy. The best activity extract was further isolated by column chromatography.

Each isolate was monitored by TLC; the same fraction was collected and screened

DPPH radical scavenger by TLC screening. Additionally, the purified fractions were

elucidated the structure using NMR spectroscopy. After that, the antioxidant activity of

such fraction was evaluated the antioxidant activities again including DPPH radical

scavenging, ferric reducing antioxidant power, ferrous ion-chelating activity. The

experimental workflow was shown in Figures 3-1.

26

Figure 3-1 Experimental workflow

Total phenolic content

DPPH radical scavenging assay

FRAP assay

Ferrous ion-chelating activity

High activity extract

Solvent system screening by TLC

Column chromatography

Fractions

Determined the compounds using TLC DPPH radical scavenger by TLC screening

Bioassay guide-isolation

All extracts (Hexane, ethyl acetate, and water extracts)

DPPH radical scavenging assay

FRAP assay

Ferrous ion-chelating activity

Fraction

Plants were prepared and extracted in ethanol

Hexane, ethyl acetate, and water extracts

Sub-extraction

NMR spectroscopy

NMR spectroscopy

27

Part I: Plants preparation and extraction

3.2 Chemicals and equipments 3.2.1 Chemicals

1. Ascorbic acid (APS, Australia)

2. Deuterated chloroform (CDCl3) (Merck, Germany)

3. Dimethyl sulfoxide (DMSO) (Merck, Germany)

4. Dimethyl sulfoxide-d6 (DMSO-d6) (Merck, Germany)

5. Distilled water

6. Ethanol (Merck, Germany)

7. Ethyl acetate (Merck, Germany)

8. Hexane (Honey Well B&J, USA)

9. Methanol (Honey Well B&J, USA)

10. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) (Sigma, Germany)

3.2.2 Equipments

1. Air oven incubator (Bindea, Germany)

2. Autopipette (Gilson, France)

3. Blender (Electrolux, China)

4. Chromatography paper number 1 (Whatman, UK)

5. Flask evaporating pear shape (Schott, Germany)

6. Freeze-dryer (GAST, USA)

7. Microplate reader (Versa max, USA)

8. Nuclear magnetic resonance spectrometer 400 MHz (NMR)

(Bruker, Germany)

9. Rotary evaporator (EYELA, Japan)

10. Separating funnel (Witeg, Germany)

11. Vacuum pump (GAST Mfg., USA)

12. 96 well microplate (Costar, USA)

28

3.3 Methods 3.3.1 Plants preparation

Young leaves of Barrington augusta Kurz. and Syzygium gratum (Wight)

S.N. Mitra var. gratum that were Thai indigenous plants were obtained from Ban Ang-

Ed Official Community Forest Project (The Chaipattana Foundation). They were

botanically classified by Dr. Benchawon Chewpreecha, Department of Biology,

Faculty of Science, Burapha University. Fresh plants were washed with tap water, cut

into tiny and small pieces and dried in hot air oven at 50oC to remove water in plant

cells. Each dried plant was blended using a blender to make a powder for extraction in

the next experiment.

3.3.2 Plants extraction

Solvents used in this study were ethanol, hexane, ethyl acetate, and water.

Briefly, dried leaves from two plants were macerated in absolute ethanol 1:10 (w/v) at

room temperature and kept out UV lights for 5 days. This step was repeated three times

prior to collect all ethanol extract together. The extract was filtered with Whatman No.

1 and evaporated using a rotary evaporator to collect crude extracts (ethanol extracts).

The ethanol extracts were suspended with water and then successively partitioned into

hexane and ethyl acetate by separating funnels. Each organic layer was evaporated by

a rotary evaporator with a vacuum pump to give hexane, ethyl acetate, and water

extracts. All extracts were weighed and kept in -20oC until use. The steps of extraction

were shown in Figure 3-2.

29

Figure 3-2 Steps of extraction. Each extract was collected for total phenolic content

and antioxidant activity determination.

3.3.3 Monitored the compounds using Nuclear Magnetic Resonance

spectrometer (NMR)

1H NMR spectra were recorded on AVANCE 400 NMR spectrometer (400

MHz) with tetramethylsilane (TMS) as an internal standard. Ethanol, hexane, ethyl

acetate and water fractions were dissolved with CDCl3 of DMSO-d6 before were then

analyzed by NMR spectroscopy.

Part II: Sub-extract determination

3.4 Chemicals and equipments

3.4.1 Chemicals

1. Acetic acid (Carlo erba, Germany)


3. Deionized water

4. Dimethyl sulfoxide (DMSO) (Fishes Scientific, UK)

5. Distilled water

6. Ethylene-dinitrilotetraacetic acid (EDTA) (Merck, Germany)

Young leaves

Ethanol

Ethanol extract

Hexane Water

Hexane extract Water extract

Ethyl acetate extract Water extract

Water Ethyl acetate

30

7. Ferrous sulphate (Lobachemie, India)

8. Ferrozine (Sigma, Germany)

9. Folin-Ciocalteu’s reagent (Carlo erba, Germany)

10. Hydrochloric acid (Merck, Germany)

11. Iron (III) Chloride hexahydrate (MERCK, Germany)


13. Silica gel size 0.040-0.063 mm. (Merck, Germany)

14. Sodium carbonate (Carlo erba, Germany)


16. 2,4,6-Tripyridyl-s-triazine (TPTZ) (Sigma, Germany)

3.4.2 Equipments




3.5 Methods

3.5.1 Total phenolic content

The total phenolic content was performed using Folin-Ciocalteu reaction with

some modification from the method of Deng et al. (2014). Briefly, 0.125 mL of extract

was mixed with 0.5 mL of distilled water and 0.125 mL of Folin-Ciocalteu’s reagent.

The mixture was incubated for 6 min. After incubation, 1.25 mL of sodium carbonate

(7%w/v) was added and distilled water was added to make volume up to 3 mL. The

reaction was incubated for 30 min at room temperature and the absorbance was

measured at 765 nm. Total phenolic contents were expressed in mg gallic acid

equivalent per g extract.

3.5.2 DPPH radical scavenging assay

Each extract was determined for DPPH radical scavenging activity with some

modification from the method of Srisook et al. (2012). Briefly, 100 µL of 0.2 mM

DPPH solution in methanol was mixed with 50 µL of various concentrations of plant

31

extract in DMSO containing the required amount of test sample in a 96 well microplate.

The solution was mixed well and incubated for 30 min at room temperature in darkness.

After 30 min, the absorbance was measured at 517 nm using a microplate reader. The

test was carried out in triplicate for each concentration at least three times. The

percentage of DPPH radical scavenging activity was calculated from the absorbance

measurements using the equation below:

% scavenging = [Acontrol - (Asample- Ablank)/Acontrol] × 100

Where, Acontrol is the absorbance of control solution containing DPPH and DMSO

after incubation.

Asample is the absorbance in the presence of sample solution in DPPH after

incubation.

Ablank is the absorbance of the sample solution without DPPH for baseline

correction arising from the unequal color of the sample solution (optical blank for

Asample)

The EC50 value (the concentration of sample required to scavenge 50% of

DPPH free radical after exposure time) was obtained from the curve between the

percentage of inhibition and sample concentrations. Ascorbic acid was used as a

positive control.

3.5.3 Ferric reducing antioxidant power (FRAP) assay

The procedure was performed according to Guo et al. (2003). The principle

of this method is to study the reduction of a ferric-tripyridyltriazine (Fe3+-TPTZ)

complex to ferrous-tripyridyltriazine (Fe2+-TPTZ), in the presence of antioxidants. In

brief, the FRAP reagent was prepared from 2.5 mL of 10 mM TPTZ (2,4,6,-tripyridyl-

s-triazine) solution in 40 mM HCl, 2.5 mL of 20 mM FeCl3 and 25 mL of 0.28 mM

acetate buffer, pH 3.6. The reagent was warmed at 37oC. However, FRAP reagent

should be prepared freshly before use. 0.3 mL of plant extract was mixed with 2.7 mL

of FRAP reagent. The solution was incubated at 37oC for 10 min. The absorbance was

then measured at 593 nm. This experiment used Trolox as a standard solution and

expressed as a FRAP value.

32

3.5.4 Ferrous ion-chelating activity

The ferrous ion-chelating activity was carried out according to the method of

Singh and Rajini (2004) with minor modifications. In this experiment, the reaction

mixture with 100 µL of various concentrations of plants extract, 5µL FeCl2 (2 mM),

and 10 µL of 5 mM ferrozine was shaken completely and incubated for 10 min at room

temperature. After that, the absorbance was measured at 562 nm. EDTA was used as a

positive control. The values were expressed as the percentage of the extract that could

chelate ferrous ion.

% ferrous ion-chelating ability = [Acontrol - (Asample - Ablank)/Acontrol] × 100

Where, Acontrol is the absorbance of the control solution containing deionized water,

ferrous chloride and ferrozine

Asample is the absorbance in the presence of sample solution, ferrous chloride

and ferrozine

Ablank is the absorbance of the sample solution, ferrous chloride and deionized

water

Part III: Bioactive compound isolation by column chromatography

3.6 Chemicals and equipments

3.6.1 Chemicals

1. Acetic acid (Carlo erba, Germany)


3. Deionized water

4. Dichloromethane (Merck, Germany)

5. Dimethyl sulfoxide (DMSO) (Fishes Scientific, UK)

6. Distilled water

7. Ethyl acetate (Merck, Germany)

8. Ethylene-dinitrilotetraacetic acid (EDTA) (Merck, Germany)

9. Ferrous sulphate (Lobachemie, India)

10. Ferrozine (Sigma, Germany)

33

11. Folin-Ciocalteu’s reagent (Carlo erba, Germany)

12. Hydrochloric acid (Merck, Germany)

13. Hexane (Honey Well B&J, USA)

14. Iron (III) Chloride hexahydrate (MERCK, Germany)


16. Silica gel size 0.040-0.063 mm. (Merck, Germany)

17. Sodium carbonate (Carlo erba, Germany)

18. TLC silica gel 60 F254 aluminium sheet 20×20 cm. (Merck,

Germany)


20. 2,4,6-Tripyridyl-s-triazine (TPTZ) (Sigma, Germany)

3.6.2 Equipments


2. Column chromatography

3. Flask evaporating pear shape (Schott, Germany)


5. Rotary evaporator (EYELA, Japan)

6. Vacuum pump (GAST Mfg., USA)


3.7 Methods 3.7.1 Selection of solvent system for the elution

The solvent mixtures were used for isocratic elution followed by Claeson

(1993). The selection of components for solvent mixture was guided by thin layer

chromatography (TLC). Solvent mixtures that commonly used for nonpolar compounds

are mixtures of hexane with ethyl acetate, chloroform, dichloromethane or acetone. On

the other hand, methanol in chloroform, dichloromethane or ethyl acetate is usually

selected for polar compounds.

34

3.7.2 Isolation of bioactive compound from B. augusta and S. gratum by

column chromatography

To isolate the highly active compound from extracts, each extract was

screened by DPPH radical scavenging assay. The active extract was selected and then

separated by column chromatography. The dry filling method was used for the packing

of silica gel columns. The silica gel was packed into the column under gravity to give

a very homogeneous packing, which should not be disturbed by stirring and was

incubated overnight. The dissolved extract was mixed with silica gel in ratio 1:1.5 (v/v

sample:silica gel). Then, the solvent was removed by a rotary evaporator with a

vacuum; the samples were loaded on the top of the column. After that, the solvent

gradient was used to elute the solution.

3.7.3 Determination of compounds using thin layer chromatography

(TLC)

TLC technique was introduced to preliminarily separate the chemical

compounds present in B. augusta Kurz. and S. gratum (Wight) S.N. Mitra var. gratum.

Fractions from column chromatography were spotted on TLC plate (silica gel 60 F254

aluminium sheet) by capillary tube and placed in the chamber with saturated solvent

until the mobile phase reached to the front line. Then the chromatogram was detected,

the same fractions were collected and concentrated with a rotary evaporator.

3.7.4 Determination of the antioxidant compounds using TLC screening

for DPPH radical scavengers

After determining the chemical compounds using TLC, each fraction was

screened the DPPH radical scavenging activity by spraying DPPH solutions on a TLC

plate before detecting under the UV light.

3.7.5 Elucidation of the compounds using Nuclear Magnetic Resonance

spectroscopy (NMR)

NMR spectra were recorded on AVANCE 400 NMR spectrometer (400 MHz)

with TMS as an internal standard. The fractions from the column were dissolved with

CDCl3 or DMSO-d6 before analyzed by NMR spectroscopy.

35

3.7.6 Determination of the active fractions using DPPH radical

scavenging assay

This experimental study with active fractions was performed instead of the

crude extract as described in 3.5.2 at the same condition. The values were expressed as

the percentage of the fraction to DPPH radical scavenging.

3.7.7 Determination of the active fractions using ferric reducing

antioxidant power assay (FRAP)

FRAP assay of active fractions was performed as described in 3.5.3.

3.7.8 Determination of the active fractions using ferrous ion-chelating

activity

The active fractions were evaluated for ferrous ion-chelating activity as

previously described in 3.5.4.

3.8 Statistical analysis

Each experiment was carried out in triplicate for each concentration at least

three times. The data were expressed as mean ± SD. The difference among groups was

analyzed by one-way ANOVA and Duncan’s multiple range tests using Minitab

program version 18. A significant difference was statistically considered at the level of

P<0.05.

36

CHAPTER 4

RESULTS

Part I: Plants preparation and extraction of B. augusta and S. gratum

4.1 The extraction yield B. augusta and S. gratum young leaves were collected from Ban Ang-Ed

official community forest project, Chanthaburi province. B. augusta powder of shade-

dried leaves (800 g) was soaked in absolute ethanol (1 grams of sample per 10 mL of

solvent). The crude extract was continuously extracted by ethanol, hexanes, ethyl

acetate and water. All sub-extracts were collected and removed the solvent using a

rotary evaporator. Then, dry weight and percent yield of each extract were measured

and shown in Table 4-1. The dry weight of each extract was 82.23 g (ethanol extract),

25.56 g (hexanes extract), 27.47 g (ethyl acetate extract), and 20.40 g (water extract),

respectively. However, when the results were expressed as percent yields, we found

that the highest yield was obtained in ethyl acetate extract (33.0%) followed by hexane

extract (30.71%), water extract (24.51%), and ethanol extract (10.40%), respectively.

Dry leaf powder (800 g) of S. gratum was primarily extracted in the absolute

ethanol (1 gram of sample per 10 mL of the solvent). The crude ethanol extract was

successively partitioned into hexane, ethyl acetate, and water. All sub-extracts were

collected and evaporated to remove the solvent. Then, dry weight and percent yield of

each extract were measured as the results in Table 4-1. The dry weight of each extract

was 118.203 g (ethanol extract), 7.45 g (hexane extract), 17.79 g (ethyl acetate extract),

and 29.78 g (water extract), respectively. When the results were expressed as percent

yields, we found the highest yield was obtained in water extract (25.63%) followed by

ethyl acetate extract (15.31%), ethanol extract (14.75%), and hexane extract (6.41%),

respectively.

37

Table 4-1 Dry weight and percent yield of B. augusta and S. gratum

Extract B. augusta S. gratum

Dry weight (g) % yield Dry weight (g) % yield

Ethanol

Hexane

Ethyl acetate

Water

83.23

25.56

27.47

20.40

10.40

30.71

33.00

24.51

118.203

7.45

17.79

29.78

14.75

6.41

15.31

25.63

4.2 B. augusta Kurz.

Part II: Sub-extract determination of B. augusta After sub-extraction in hexane, ethyl acetate, and water, all three extracts were

collected from this step. Then, the compounds from ethanol, ethyl acetate, hexane and

water extracts were characterized by NMR spectroscopy. Ethanol and ethyl acetate

extracts were dissolved in CDCl3 that mixed with DMSO-d6. Hexane extract was

dissolved in CDCl3, whereas water extract was dissolved in DMSO-d6. Then, they were

analyzed by NMR spectroscopy. 1H NMR spectra of all extracts were shown in Figures

F-1 to F-4 (Appendix F). All extracts (ethanol, hexane, ethyl acetate and water) were

evaluated the total phenolic content and determined the bioactivities by DPPH radical

scavenging assay, ferric reducing antioxidant power assay (FRAP) and ferrous ion-

chelating activity.


This experiment was performed using Folin-Ciocalteu reaction modified from

the method of Deng et al. (2014). The color of Folin-Ciocalteau reagent changes from

yellow to blue upon the detection of phenolics in the extracts because of the chemical

reduction of tungsten and molybdenum oxides mixture in the reagent (Lim et al., 2007).

Gallic acid was used as a reference for a standard curve. The selected extract was

prepared in a final concentration at 1 mg/ml (Table 4-2, and Figure 4-1). The total

phenolic content in water extract had the highest amount (267.589±1.544 mg GAE/g

extract) when compared to the other extracts (228.349 ± 0.257, 96.494±0.257 and

38

52.099±0.086 mg GAE/g extract for ethyl acetate, ethanol, and hexane extracts,

respectively.

Table 4-2 Total phenolic content from ethanol, hexane, ethyl acetate, and water

extracts at 1 mg/mL

Extract Total phenolic content

(mg GAE/g extract)

Ethanol

Hexane

Ethyl acetate

Water

96.494 ± 0.257c

52.099 ± 0.086d

228.349 ± 0.257b

267.589 ± 1.544a

The results were expressed as mean ± SD. a,b,c,d Different letters within the same column indicate statistical differences (P<0.05).

Figure 4-1 Total phenolic content from ethanol, hexane, ethyl acetate, and water

extracts at 1 mg/mL


This assay was determined with some modification from the method of

Srisook et al. (2012). The radical scavenging activity was measured with DPPH and

extract as radicals and radical-scavenger in the reaction, respectively. In the chemical

mechanism, antioxidants react with DPPH and convert it to the yellow colored 2,2-

0

50

100

150

200

250

300

Ethanol Hexane Ethyl acetate Water

mg

GA

E/g

extra

ct

a b

c

d

39

diphenyl-1-picryl hydrazine. The results were expressed with EC50 value and used

ascorbic acid was used as a positive control. Antioxidant activities of sub-extracted

were shown in Table 4-3. Among these plant extracts, all sub-extracts were shown their

DPPH radical scavenging activity when compared to an ascorbic acid (100% DPPH

radical-scavenging).

The plant extracted in water had the lowest EC50 = 0.052 ± 0.005 mg/mL

followed by ethyl acetate (0.083 ± 0.003 mg/mL), ethanol (0.179 ± 0.005 mg/mL), and

hexane (1.447 ± 0.068 mg/mL), respectively. That means water extract showed the

strongest DPPH radical scavenging activity and its EC50 value was very close to a

standard antioxidant, ascorbic acid (0.022 ± 0.001 mg/mL). In addition, the maximum

level of DPPH radical scavenging activity was detected in the presence of water fraction

of 86.69% followed by ethyl acetate, ethanol and hexane fractions (65.67%, 38.15%,

and 12.3%, respectively).

Table 4-3 The percent of DPPH radical scavenging from ethanol, hexane, ethyl acetate

and water extracts at 0.1 mg/mL and EC50 of DPPH radical scavenging

assay

Extract % DPPH radical

scavenging

EC50

(mg/mL)

Ethanol

Hexane

Ethyl acetate

Water

Ascorbic acid

38.15

12.3

65.67

86.69

100

0.179 ± 0.005b

1.447 ± 0.068a

0.083 ± 0.003c

0.052 ± 0.005c

0.022 ± 0.001c

The results were expressed as mean ± SD. a,b,c, Different letters within the same column indicate statistical differences (P<0.05).

4.2.3 Ferric Reducing Antioxidant Power assay (FRAP)

FRAP assay is commonly used to assess the antioxidant power by the

reduction of ferric-tripyridyltriazine (Fe3+-TPTZ) to ferrous-tripyridyltriazine (Fe2+-

TPTZ) in the presence of antioxidants. This method was followed to Guo et al. (2003),

and Trolox was used as a reference standard compound. Table 4-4 and Figure 4-2,

40

describes the ferric reducing activity of the extract. The results were expressed as FRAP

value at 0.1 mg/mL. This result was found that the ethyl acetate extract showed the

highest activity of ferric reducing (highest FRAP value = 434.340 ± 1.344 mg TE/g

extract) and water extract also had high FRAP value (343.610 ± 1.0607 mg TE/g

extract), whereas the hexane extract exhibited the lowest activity as presented in this

study (42.165 ± 3.415 mg TE/g extract) and ethanol extract was detected as 205.640 ±

1.089 mg TE/g extract.

Table 4-4 FRAP value from ethanol, hexane, ethyl acetate and water extracts at 0.1

mg/mL

Extract FRAP value (mg TE/g extract)

Ethanol

Hexane

Ethyl acetate

Water

205.640 ± 1.089c

42.165 ± 3.415d

434.340 ± 1.344a

343.610 ± 1.0607b


Figure 4-2 FRAP value from ethanol, hexane, ethyl acetate and water extracts at 0.1

mg/mL a,b,c,d Different letters over the bar indicate statistical differences (P<0.05).

050

100150200250300350400450500


mg

TE/g

ext

ract

a

b

c

d

41

4.2.4 Ferrous ion-chelating activity assay

The experiment prefers to study the reaction of antioxidants that can stop the

ferrous ion chelating reaction. This method was carried out according to the method of

Singh and Rajini (2004). The results were shown in Table 4-5. Ethyl acetate and water

extracts at 0.1 mg/mL showed high chelating activity, they could chelate the ferrous ion

of 83.18% and 78%, respectively. However, ethanol and hexane extracts could also

chelate ferrous ion (64.74% and 58.25%, respectively) even less than ethyl acetate and

water extracts.

Table 4-5 The percent of ferrous ion-chelating from ethanol, hexane, ethyl acetate and

water extracts at 0.1 mg/mL

Extract % ferrous ion-chelating

Ethanol

Hexane

Ethyl acetate

Water

EDTA

64.74c

58.25c

83.18b

78.00b

100a

The results were expressed as a percentage of ferrous ion-chelating activity. a,b,c,d Different letters within the same column indicate statistical differences (P<0.05).

Part III: The 1st Column Chromatography of B. augusta

From the study above, ethyl acetate extract showed the highest ferric reducing

antioxidant power and ferrous ion-chelating activity compared with hexane and water

extracts. It was further monitored by thin layer chromatography (TLC) to preliminarily

select the solvent system for column chromatography. Figure D-1 (Appendix D)

showed TLC screening the compounds of ethyl acetate extract from B. augusta. The

appropriate solvent system for separating the substance is ethyl acetate mixed with

hexane and methanol mixed with ethyl acetate. Then, the ethyl acetate extract was

isolated by column chromatography. The powder of dried extract (26.12 g) was

resuspended with methanol+dichloromethane, and it was then packed by mixing with

42

silica gel in ratio 1:1.5 (v/v) by dried packing method. Silica gel 7734 (pored size 0.063-

0.2 mm) was used as a stationary phase packed in column (diameter 9 cm). The ground

silica gel was loaded upper into the column. The solvent systems were used to elute a

compound mixture of ethyl acetate with hexane and methanol with ethyl acetate

(Claeson, 1993). The solvents from the combined extract were concentrated using a

vacuum rotary evaporator, and then they were weighed. Sub-fractions were collected

and determined by TLC (Figure D-2), the compounds were screened DPPH radical

scavenger on TLC (Figure E-1). The results of the fraction were shown in Table 4-6.

The quantity of each fraction was calculated for the dry weight and percent yield. Eluted

fractions were FBA1 to FBA8 separated as the active fractions. The result showed that

the fraction FBA7 had the highest dry weight and %yield values (Table 4-6) followed

by FBA8 and FBA6, respectively (4.8158 g and 18.437% for FBA8 and 4.0623 g and

15.552% for FBA6) – however, the others were relatively low.

Table 4-6 Dry weight and percent yield of each fraction from the 1st column

chromatography

Fraction Dry weight (g) % yield

FBA1

FBA2

FBA3

FBA4

FBA5

FBA6

FBA7

FBA8

0.1935

0.1035

0.1814

0.2581

0.4909

4.0623

6.9319

4.8158

0.741

0.396

0.694

0.988

1.879

15.552

26.539

18.437

After sub-fraction, all fractions were monitored the compound by NMR

spectroscopy. FBA1, FBA2, FBA3, FBA4, FBA5 and FBA6 fractions were dissolved

in CDCl3, whereas FBA7 and FBA8 fractions were dissolved in DMSO-d6. Then, they

were analyzed by NMR spectroscopy. 1H NMR spectra of these fractions were shown

in Figures F-5 to F-12 (Appendix F).

43

4.2.5 Antioxidant-guided isolation of the fractions from the 1st column

chromatography

Eight fractions (FBA1, FBA2, FBA3, FBA4, FBA5, FBA6, FBA7 and FBA8)

that separated from the 1st column chromatography were aliquoted to determine the

bioactivities using DPPH radical scavenging assay, ferric reducing antioxidant power

assay (FRAP) and ferrous ion-chelating activity.

4.2.5.1 DPPH radical scavenging assay

From the result, the DPPH radical scavenging activity of the FBA7

fraction showed the maximum activity compared to ascorbic acid, a positive control

(100% DPPH radical scavenging). Fractions FBA1 to FBA8 slightly showed DPPH

radical scavenging activity (Table 4-7). FBA5 and FBA6 fractions gave the inhibitory

effect around 21%.

Table 4-7 The percent of DPPH radical scavenging of each fraction at 0.1 mg/mL

from the 1st column chromatography

Fraction % DPPH radical scavenging

FBA1

FBA2

FBA3

FBA4

FBA5

FBA6

FBA7

FBA8

Ascorbic acid

8.54f

6.87g

9.82e

9.93e

21.27c

21.01c

29.63b

14.67d

100a

The results were expressed as a percentage of DPPH radical scavenging activity. a,b,c,d,e,f,g Different letters within the same column indicated statistical differences

(P<0.05).

44

4.2.5.2 Ferric Reducing Antioxidant Power assay (FRAP)

Ferric Reducing Antioxidant Power assay (FRAP) was performed the

fractions that might show as an electron donor capacity. In this study, no reducing

power was found in fraction FBA1 whereas the excellent effect was observed in

fractions FBA6, FBA7 and FBA5 (109.673 ± 3.502, 99.709 ± 2.478 and 92.775 ± 0.294

mg TE/g extract, respectively) with approximately FRAP value of 0.1 mg TE/g extract.

The activity of ferric reducing power in fractions FBA2, FBA3, FBA4, and FBA8 were

lower level (FRAP value <30 mg TE/g extract). The results were shown in Table 4-8

and Figure 4-3.

Table 4-8 FRAP value of each fraction at 0.1 mg/mL from the 1st column

chromatography

Fraction FRAP value (mg TE/g extract)

FBA1

FBA2

FBA3

FBA4

FBA5

FBA6

FBA7

FBA8

-13.909 ± 1.644g

3.3527 ± 0.046f

13.016 ± 0.360e

18.867 ± 0.525e

92.775 ± 0.294c

109.673 ± 3.502a

99.709 ± 2.478b

26.797 ± 1.700d

The results were expressed as mean ± SD. a,b,c,d,e,f,g Different letters within the same column indicate statistical differences

(P<0.05).

45

Figure 4-3 FRAP values of each fraction at 0.1 mg/mL from the 1st column

chromatography a,b,c,d,e,f,g Different letters over the bar indicate statistical differences (P<0.05).

4.2.5.3 Ferrous ion-chelating activity assay

Ferrous ion-chelating activity assay was studied to predict the

mechanism of secondary antioxidants. The results were compared to EDTA as a

positive control expressed as 100% ferrous ion-chelating (Table 4-9). All of them had

lower ferrous ion-chelating activities than EDTA at a concentration of 0.1 mg/mL

especially FBA1 had the lowest ferrous ion-chelating activity (16.16%). FBA7 fraction

had the highest activity (45.13%) followed by FBA8 (43.06%), FBA6 (40.80%), FBA2

(40.74%), FBA5 (40.17%), FBA3 (31.0%) and FBA4 fraction (23.47%), respectively.

-40

-20

0

20

40

60

80

100

120

FBA1 FBA2 FBA3 FBA4 FBA5 FBA6 FBA7 FBA8

mg

TE/g

ext

ract

a b

c

d e

e f g

46

Table 4-9 The percent of ferrous ion-chelating of each fraction at 0.1 mg/mL from the

1st column chromatography

Fraction % ferrous ion-chelating

FBA1

FBA2

FBA3

FBA4

FBA5

FBA6

FBA7

FBA8

EDTA

16.16g

40.74d

31.00e

23.47f

40.17d

40.80d

45.13b

43.06c

100a

The results were expressed as a percentage of ferrous ion-chelating activity. a,b,c,d,e,f,g Different letters within the same column indicated statistical differences

(P<0.05).

Part IV: The 2nd Column Chromatography of B. augusta

From the study above, FBA7 fraction showed the highest DPPH radical

scavenging and ferrous ion-chelating activities compared to the other fractions.

Therefore, this fraction was chosen to further isolate by column chromatography. FBA7

fraction was monitored by TLC to preliminarily select the solvent system for elution

the compounds from column chromatography. Figure D-3 (Appendix D) showed TLC

screening the compounds of FBA7. The appropriate solvent system for separating the

substance was the mixture of methanol with dichloromethane. 5 g of this fraction was

resuspended with ethanol+methanol, and it was packed with the silica gel in ratio 1:1.5

(v/v) via dried packing method. A column (diameter 5 cm) was packed with the silica

gel 9385 (pored size 0.040-0.063 mm) as stationary phase. The ground silica gel was

loaded upper into the column. The solvent systems were used to elute the compound

mix with methanol and dichloromethane. The solvents from the combined extract were

concentrated using a vacuum rotary evaporator, and then they were weighed. Sub-

47

fractions were collected and determined by TLC (Figure D-2), the compounds were

screened for DPPH radical scavenger on TLC (Figure E-2). The results were shown in

Table 4-10. The fractions showing high production were selected for the 2nd column

chromatography. The dry weight and percent yield were determined after elution. The

fractions in the present study were F2-BA1, F2-BA2, F2-BA3, F2-BA4, and F2-BA5,

respectively. F2-BA5 fraction with the highest yield was approximately 2.4415 g

(48.83% yield). Other obtained fractions were lower dry weight and %yield than 1.1664

g and 23.33% yield, respectively.

Table 4-10 Dry weight and percent yield of each fraction from the 2nd column

chromatography


F2-BA1

F2-BA2

F2-BA3

F2-BA4

F2-BA5

0.2649

0.3136

0.7844

1.1664

2.4415

5.29

6.27

15.69

23.33

48.83

After isolation, all fractions from the 2nd column chromatography were

monitored the compounds by NMR spectroscopy. F2-BA1 fraction was dissolved in

CDCl3, whereas F2-BA2, F2-BA3, F2-BA4 and F2-BA5 fraction were dissolved in

DMSO-d6. And then, they were analyzed by NMR spectroscopy. 1H NMR spectra of

five fractions showed in Figures F-13 to F-17 (Appendix F).

4.2.6 Determination of the fractions from the 2nd column

chromatography

Five fractions above (F2-BA1, F2-BA2, F2-BA3, F2-BA4 and F2-BA5) that

separated with the 2nd column chromatography were aliquoted to determine the

bioactivity by antioxidant-guided isolation again using DPPH radical scavenging assay,

ferric reducing antioxidant power assay (FRAP) and ferrous ion-chelating activity.

48


DPPH radical scavenging assay was studied under the same condition

as assay above. Antioxidant activity was considered DPPH removal activity by fraction

F2-BA1, F2-BA2, F2-BA3, F2-BA4, and F2-BA5. F2-BA5 fraction showed the highest

antioxidant activity (about 38.38 % DPPH radical scavenging). This was similar to the

results of F2-BA1, F2-BA2, F2-BA3 and F2-BA4 fractions, their antioxidant activity

still remained less than 30%. However, among of sub-fractions showed less capacity of

antioxidant when compared to a standard antioxidant; ascorbic acid at 0.1 mg/mL.


from the 2nd column chromatography


F2-BA1

F2-BA2

F2-BA3

F2-BA4

F2-BA5

Ascorbic acid

21.09d

15.90e

29.17c

16.62e

38.38b

100a

The results were expressed as a percentage of DPPH radical scavenging activity. a,b,c,d,e Different letters within the same column indicated statistical differences

(P<0.05).


From Table 4-12 and Figure 4-4, F2-BA5 fraction at 0.1 mg/mL

exhibited the greatest FRAP value of 144.287 ± 1.273 mg TE/g extract when compared

to the other fractions. F2-BA3 fraction had also high activity (127.975 ± 0.572 mg TE/g

extract), followed by F2-BA1 and F2-BA4 fraction (62.493 ± 0.830 and 34.946 ± 1.249

mg TE/g extract). While F2-BA2 fraction showed the lowest FRAP value (22.549 ±

0.554 mg TE/g extract).

49

Table 4-12 FRAP value of each fractions at 0.1 mg/mL from the 2nd column

chromatography


F2-BA1

F2-BA2

F2-BA3

F2-BA4

F2-BA5

62.493 ± 0.830c

22.549 ± 0.554e

127.975 ± 0.572b

34.946 ± 1.249d

144.287 ± 1.273a

The results were expressed as mean ± SD. a,b,c,d,e Different letters within the same column indicate statistical differences (P<0.05).

Figure 4-4 FRAP value of each fraction at 0.1 mg/mL from the 2nd column

chromatography a,b,c,d,e Different letters over the bar indicate statistical differences (P<0.05).


The experiment was done under the condition of 0.1 mg/mL ferrous ion-

chelating and the results were shown in Table 4-13 below. The table indicated that F2-

BA5 fraction was the highest level of ferrous ion-chelating activity by 36.79%. The

next two lower level were F2-BA1 and F2-BA3 and these two fractions did not show

0

20

40

60

80

100

120

140

160

F2-BA1 F2-BA2 F2-BA3 F2-BA4 F2-BA5

mg

TE/g

ext

ract

b

d

a

c

e

50

different activity at the same concentration (23.66% and 23.53%). For the others, all of

them were much lower than the standard reference (EDTA).

Table 4-13 The percent of ferrous ion-chelating at 0.1 mg/mL of each fraction from

the 2nd column chromatography


at 0.1 mg/mL

F2-BA1

F2-BA2

F2-BA3

F2-BA4

F2-BA5

EDTA

23.66c

19.14d

23.53c

16.12e

36.79b

100a

The results were expressed as a percentage of ferrous ion-chelating activity. a,b,c,d,e Different letters within the same column indicated statistical differences

(P<0.05).

51

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52

4.3 Syzygium gratum (Wight) S.N. Mitra var. gratum

Part II: Sub-extract determination of S. gratum

Hexane, ethyl acetate, and water extracts were collected from sub-extraction.

Then, ethanol, ethyl acetate, hexane and water extracts were characterized the

compound by NMR spectroscopy. Ethanol and hexane extracts were dissolved in

CDCl3. Ethyl acetate and water extracts were dissolved in DMSO-d6. Then, they were

analyzed by NMR spectroscopy. 1H NMR spectra of all extracts were shown in Figures

F-18 to F-21 (Appendix F). Then, all extracts (ethanol, hexane, ethyl acetate and water)

were evaluated the total phenolic content and determined the antioxidant activities by

DPPH radical scavenging assay, ferric reducing antioxidant power assay (FRAP) and

ferrous ion-chelating activity.


Among the collected extracts of S. gratum, the ethyl acetate extract contained

the excellent total phenolic content of 151.458 ± 1.360 mg GAE/g extract while other

extracts including ethanol, water, and hexane had total phenolic contents as 71.195 ±

0.392, 46.115 ± 1.251, 30.117 ± 0.598 mg GAE/g extract, respectively. The results were

shown in Table 4-14 and Figure 4-6. Ethyl acetate extract containing high phenolic acid

content relatively indicated a good extract for antioxidant activities.

Table 4-14 Total phenolic content from ethanol, hexane, ethyl acetate, and water

extracts at 0.05 mg/mL

Extract Total phenolic content

(mg GAE/g extract)

Ethanol

Hexane

Ethyl acetate

Water

71.195 ± 0.392b

30.117 ± 0.598d

151.458 ± 1.360a

46.115 ± 1.251c


53

Figure 4-6 Total phenolic content from ethanol, hexane, ethyl acetate and water

extracts at 0.05 mg/mL a,b,c,d Different letters over the bar indicate statistical differences (P<0.05).


The DPPH radical scavenging assay was studied, DPPH radical scavenging

activity in ethyl acetate, water, ethanol and hexane extracts were 49.62%, 24.02%,

21.1% and 11.44%, respectively. The inhibitory effect of all fractions had lower than

ascorbic acid. The results in DPPH radical scavenging assay related to EC50 values.

EC50 values in ethyl acetate, water, ethanol and hexane extracts were 0.071 ± 0.002,

0.164 ± 0.011, 0.143 ± 0.002 and 0.667 ± 0.062 mg/mL, respectively.

020406080

100120140160180


mg

GA

E/g

extra

ct

a

b c

d

54

Table 4-15 The percent of DPPH radical scavenging from ethanol, hexane, ethyl


scavenging assay

Extract % DPPH radical

scavenging

EC50

(mg/mL)

Ethanol

Hexane

Ethyl acetate

Water

Ascorbic acid

21.1

11.44

49.62

24.02

95.09

0.143 ± 0.002b,c

0.667 ± 0.062a

0.071 ± 0.002c,d

0.164 ± 0.011b

0.022 ± 0.001d

The results were presented as mean ± SD.

a,b,c,d Different letters within the same column indicate statistical differences (P<0.05).

4.3.3 Ferric Reducing Antioxidant Power assay (FRAP)

Table 4-16 and Figure 4-7 describes the ferric reducing antioxidant power of

the extracts. The results were expressed as FRAP value at 0.05 mg/mL. This result

found that ethyl acetate extract showed the highest FRAP value (890.885 ± 4.724 mg

TE/g extract) followed by ethanol and water extracts (222.414 ± 0.370 and 109.128 ±

3.433 mg TE/g extract, respectively) whereas hexane extract exhibited the lowest

activity as presented in this study (15.12 ± 3.732 mg TE/g extract).

Table 4-16 FRAP value from ethanol, hexane, ethyl acetate and water extracts at 0.05

mg/mL

Extract FRAP value (mg TE/g extract)

Ethanol

Hexane

Ethyl acetate

Water

222.414 ± 0.370b

15.12 ± 3.732d

890.885 ± 4.724a

109.128 ± 3.433c


55

Figure 4-7 FRAP value from ethanol, hexane, ethyl acetate and water extracts at

0.05 mg/mL a,b,c,d Different letters over the bar indicate statistical differences (P<0.05).

4.3.4 Ferrous ion-chelating activity assay

The results of the ferrous ion-chelating activity assay were compared to

EDTA as a positive control expressed as 99.89% ferrous ion-chelating (Table 4-17).

All of them had lower ferrous ion-chelating activity than EDTA at a concentration of

0.05 mg/mL especially ethanol extract had the lowest ferrous ion-chelating activity

(22.76%). Ethyl acetate extract had ferrous ion-chelating activity 30.43% followed by

hexane (27.49%) and water extracts (24.89%).

0100200300400500600700800900

1000


mg

TE/g

ext

ract

b

a

c d

56

Table 4-17 The percent of ferrous ion-chelating from ethanol, hexane, ethyl acetate

and water extracts at 0.05 mg/mL

Extract % ferrous ion-chelating

Ethanol

Hexane

Ethyl acetate

Water

EDTA

22.76e

27.49c

30.43b

24.89d

99.89a

The results were expressed as a percentage of ferrous ion-chelating activity. a,b,c,d,e Different letters within the same column indicated statistical differences

(P<0.05).

Part III: The 1st Column Chromatography of S. gratum

From the study above, ethyl acetate extract seemed to exhibit the best activity

by DPPH radical scavenging assay, ferric reducing antioxidant power assay and ferrous

ion-chelating activity assay compared to the other extracts. Therefore, the ethyl acetate

extract was chosen to isolate by column chromatography. Ethyl acetate extract was

monitored by TLC to preliminarily select the solvent system for elution the compounds

from column chromatography. Figure D-5 (Appendix D) showed TLC screening the

compounds of FBA7 from the 1st column chromatography. The appropriate solvent

system for separating the substance was ethyl acetate mix with hexane. 15 g of ethyl

acetate extract was mixed with silica gel in ratio 1:1.5 (v/v) via dried packing method.

A column (diameter 5 cm) was packed with silica gel 9385 (pored size 0.040-0.063

mm). The solvent systems were used to elute the compounds mixture of ethyl acetate

with hexane and methanol with ethyl acetate. Fractions were collected and determined

by TLC, the compounds were screened DPPH radical scavenger on TLC (Figure E-3).

The ground silica gel as loaded into the column. The solvent systems were used to elute

the compounds mixtures of ethyl acetate with hexane and methanol with ethyl acetate.

The solvents from the combined extract were concentrated using a vacuum rotary

evaporator, then they were weighed. Sub-fractions from the 1st column chromatography

57

were collected and determined by TLC (Figure D-2), the compounds were screened

DPPH radical scavenging activity on TLC. Dry weights and percent yields were shown

in Table 4-18. From Table 4-18, FSG6 fraction had the highest values (5.152 g and

34.347% yield) while FSG7 and FSG8 were still high in both values (3.0606 g and

20.404% yield for FSG7 and 2.052 g and 13.68% yield for FSG8, respectively);

however, the others were relatively low.

Table 4-18 Dry weight and percent yield of each fraction from the 1st column

chromatography


FSG1

FSG2

FSG3

FSG4

FSG5

FSG6

FSG7

FSG8

0.0883

0.1242

0.1018

0.239

0.175

5.152

3.0606

2.052

0.5886

0.828

0.6787

1.5933

1.1667

34.3467

20.404

13.68

After purification, all fractions from 1st column chromatography were

monitored the compounds by NMR spectroscopy. The FSG1 fraction was dissolved in

CDCl3, whereas FSG2, FSG3, FSG4, FSG5, FSG6, FSG7 and FSG8 fractions were

dissolved in DMSO-d6. And then, they were analyzed by NMR spectroscopy. 1H NMR

spectra of all fractions were shown in Figure F-22 to F-29 (Appendix F).

4.3.5 Antioxidant activity of the fractions from the 1st column

chromatography

All fractions (FSG1, FSG2, FSG3, FSG4, FSG5, FSG6, FSG7 and FSG8)

separated from the 1st column chromatography were aliquoted to determine the

antioxidant activities by DPPH radical scavenging assay, ferric reducing antioxidant

power assay (FRAP) and ferrous ion-chelating activity.

58


In Table 4-19, the results showed the percent of DPPH radical

scavenging of sub-fractions at 0.05 mg/mL. The DPPH radical scavenging activity of

FSG7 showed the maximum activity (94.36%), which is comparable to ascorbic acid

as a positive control (96.46%). FSG7 fraction could strongly inhibit DPPH radical

(94.36%) followed by FSG5 (91.68%), FSG8 (83.81%) and FSG6 (82.94%),

respectively. FSG4 fractions showed the inhibitory effect nearly 36%, while FSG1 to

FSG3 fractions had DPPH radical scavenging activity less than 30%.


From the 1st column chromatography


FSG1

FSG2

FSG3

FSG4

FSG5

FSG6

FSG7

FSG8

Ascorbic acid

4.44f

14.28e

12.94e

36.39d

91.68b

82.94c

94.36a

83.81c

96.46a

The results were expressed as a percentage of DPPH radical scavenging activity. a,b,c,d,e,f Different letters within the same column indicated statistical differences

(P<0.05).


The results expressed as the FRAP value at the concentration of 0.05

mg/mL and used Trolox as a reference for a standard curve. FSG5 and FSG6 fractions

were strongly active with 767.373 ± 3.272 and 747.173 ± 1.425 mg TE/g extract while

FSG7 and FSG4 fractions could highly reduce ferric ion in the assay by 642.604 ± 2.157

and 347.27 ± 2.192 mg TE/g extract. In contrast, FRAP value of FSG1 (35.912 ± 1.421

59

mg TE/g extract), FSG2 (57.852 ± 0.198 mg TE/g extract) and FSG3 fractions (92.859

± 0.872 mg TE/g extract) slightly displayed the reducing ability to ferric ions (Table 4-

20 and Figure 4-8).

Table 4-20 FRAP value of each fraction at 0.05 mg/mL from the 1st column

chromatography


FSG1

FSG2

FSG3

FSG4

FSG5

FSG6

FSG7

FSG8

35.912 ± 1.421h

57.852 ± 0.198g

92.859 ± 0.872f

347.27 ± 2.192d

767.373 ± 3.272a

747.173 ± 1.425b

642.604 ± 2.157c

314.020 ± 1.175e

The results were expressed as mean ± SD. a,b,c,d,e,f,g,h Different letters within the same column indicate statistical differences

(P<0.05).

60

Figure 4-8 FRAP value of each fraction at 0.05 mg/mL from the 1st column

Chromatography a,b,c,d,e,f,g,h Different letters over the bar indicate statistical differences (P<0.05).


The results suggested that all fractions had lower ferrous ion-chelating

activity than EDTA at a concentration of 0.05 mg/mL (Table 4-21). FSG6 fraction

showed the ferrous-ion chelating activity of 39.11%. FSG4 and FSG5 fractions had

slightly different to each other (31.44 and 31.25%, respectively). FSG7, FSG3, FSG8

and FSG2 fractions slightly exhibited the ferrous ion-chelating activity (<30%),

particularly the FSG1 fraction showed the lowest ferrous ion-chelating activity

(16.07%).

0

100

200

300

400

500

600

700

800

900

FSG1 FSG2 FSG3 FSG4 FSG5 FSG6 FSG7 FSG8

mg

TE/g

ext

ract

a b c

d e

f g h

61

Table 4-21 The percent of ferrous ion-chelating of each fraction at 0.05 mg/mL from the 1st column chromatography


FSG1

FSG2

FSG3

FSG4

FSG5

FSG6

FSG7

FSG8

EDTA

16.07g

17.81g

23.83e

31.44c

31.25c

39.11b

28.38d

20.66f

99.89a

The results were expressed as a percentage of ferrous ion-chelating activity. a,b,c,d,e,f,g Different letters within the same column indicated statistical differences

(P<0.05).

62

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. gra

tum

63

CHAPTER 5

DISCUSSION

Most natural antioxidants are phytochemicals like L-ascorbic acid and α-

tocopherol (Lee et al., 2003; Shibamoto & Bjeldanes, 2009). Phenolic compounds are

also phytochemicals that belong to important aromatic secondary metabolites in plants

(Kim et al., 2003). Some phenolics like flavonoids or polyphenols have antioxidative,

anticarcinogenic, antimicrobial, antiallergic, antimutagenic and anti-inflammatory

activities (Ito et al., 1998; Cao & Cao, 1999; Kawaii et al., 1999; Eberhardt et al., 2000;

Kim et al., 2000). In the present, traditional medicine and natural extracts began to play

a major role in health care and treatment for several diseases (Farnsworth et al., 1985).

In the Eastern region of Thailand, there are many medicinal plants at Ban Ang-Ed

Official Community Forest Project which have been used in healthcare by traditional

folklore. However, the study about antioxidant activity and antioxidant compounds of

medicinal plants including Barrington augusta Kurz. and Syzygium gratum (Wight)

S.N. Mitra var. gratum is still limited. B.augusta Kurz. even though they have been

used as traditional remedies for several diseases like diarrhea, scurvy, laxative,

conjunctivitis, emetics, and fever (อุไร จิรมงคลการ, 2547). S. gratum (Wight) S.N. Mitra

var. gratum is often used to prevent against several diseases like sprain, expectorate,

asthma, bronchitis and anti-parasite (อุไร จิรมงคลการ, 2547). Previous reports of both

plants in water extract showed high antioxidant activity including DPPH scavenging

assay, reducing power, total phenolic and flavonoid content (Thepmongkon et al.,

2013), ferrous-ion chelating, ABTS radicals decolorization assay and ferric ion

reducing antioxidant power (Kukongviriyapan et al., 2007; Petchlert et al., 2013).

Although leaves of B. augusta and S. gratum had been used as traditional medicine and

study about antioxidant activity; nevertheless, no research had been done on their

antioxidant activity of bioactive compound from these plants. In this study, we aimed

to evaluate their bioactive compounds by antioxidant assay-guided isolation and

compared with the crude extract from sub-extracts and sub-fractions after purification

via TLC and column chromatography.

64

B. augusta Kurz. was collected from Ban Ang-Ed Official Community Forest

Project, Chantaburi province. It was successively partitioned with ethanol, hexane,

ethyl acetate and water. All extracts were characterized the compound by NMR

spectroscopy. 1H NMR spectra of all extracts were shown in Figures E-1 to E-4

(Appendix E). Then they were determined the total phenolic content. From the result,

total phenolic content was performed using Folin-Ciocalteu reaction modified from the

method of Deng et al. (2014). Folin-Ciocalteu reagent changed the color from yellow

to blue when the phenolic structure occurred in the extracts. It was due to the chemical

reducing of tungsten and molybdenum oxides mixture in the reagent (Lim, Lim, & Tee,

2007). The total phenolic content in extracts were measured with gallic acid as a

standard (standard curve equation: y=17.03x + 0.0257, R2 = 0.9936). Water extract had

the greatest total phenolic content (267.589 ± 1.544 mg GAE/g extract) followed by

ethyl acetate, ethanol, and hexane extract (Table 4-2). This is very close to a previous

report of Thepmongkon et al. (2013), they suggested that hot water extract of B. augusta

leaves contained high total phenolic content of 251.26 ± 0.005 mg GAE/g extract.

Furthermore, the major phenolic compounds of Barringtonia racemosa, which is the

same family with B. augusta have been previously reported in methanol, ethanol and

boiling water extracts by Hussin et al. (2011). They found that gallic acid seemed to be

a major compound in leaf extracts (171.81 μg/g freeze-dried weight tissue) and also the

ferulic acid (65.80 μg/g freeze-dried weight tissue). Moreover, Rachana (2009) also

demonstrated that gallic acid, ferulic acid, and others phenolic acids, secondary

metabolites, which often presented in several plants, possessed antioxidant, antifungal,

antipyretic and anti-inflammatory properties.

In addition, all extracts were determined the antioxidant activities by DPPH

radical scavenging assay, ferric reducing antioxidant power assay (FRAP) and ferrous

ion-chelating activity assay before isolating by column chromatography. For DPPH

radical scavenging assay, ascorbic acid was used as a standard. The results were

expressed as EC50 values (the concentration required to inhibit 50% of the DPPH

radicals) and %scavenging showed the powerful antioxidant property. The color of

DPPH solution can be changed from the violet (DPPH radical) to yellow or colorless

(non-radical DPPH-H form) in the presence of antioxidant capacity in the extracts

(Srisook et al., 2012). From the results in Table 4-16, water extract could strongly

65

inhibit DPPH radical (EC50 = 0.052 ± 0.005 mg/mL) that was greater than the other

three extracts. This was in agreement with previous results from Thepmongkon et al.

(2013), they indicated that hot water extract of B. augusta leaves showed high DPPH

radical scavenging activity (0.0419 ± 0.005 mg/mL). That means some compounds in

a water extract may donate the H atom to DPPH radical efficiently.

The FRAP assay measures the antioxidant effect by the reduction of ferric-

tripyridyltriazine (Fe3+-TPTZ) complex to ferrous-tripyridyltriazine (Fe2+-TPTZ), in

the presence of antioxidants. FRAP value will express the highest electron donor

capacity in the extract (Guo et al., 2003). Ethyl acetate extract of B. augusta displayed

the greatest FRAP value of 434.340 ± 1.344 mg TE/g extract when compared to other

extracts (Table 4-4). This is an interesting point for further study which compounds in

this extract can donate an electron to the ferric ion.

In Fenton reaction, ferrous ion was oxidized by hydrogen peroxide to ferric

ion, forming a hydroxyl radical in the process. Antioxidants can stop the reaction by

chelating the ferrous ion (Decker & Welch, 1990). The ferrous ion-chelating assay was

monitored by the absorbance of the ferrous ion-ferrozine complex at 562 nm. We found

that the ethyl acetate extract presented the highest iron chelating ability of 83.18%

followed by water extract (78%). That means any compounds in this extract may

prevent the generation of Fenton reaction by Fe2+.

From those results, there was a significant positive correlation between

phenolic content and antioxidant activity by the report of Rachana (2009). We found

that ethyl acetate extract showed good activity by ferric reducing antioxidant power and

ferrous ion-chelating activity when compared with ethanol, hexane and water extracts.

Then, ethyl acetate extract was monitored by thin layer chromatography (TLC) to

preliminarily select the solvent system for column chromatography. TLC is a quick and

easy way to find compounds in the mixture. Additionally, TLC is also used to identify

the compounds in the extract when Rf of a compound is compared with Rf of a known

compound (Sasidharan, Chen, Saravanan, Sundram, & Latha, 2011). This has also been

used to confirm the purity and to identify the isolated compounds. Figure D-1 showed

TLC of ethyl acetate extract from B. augusta. The appropriate solvent system for

separating the substance mixtures are ethyl acetate with hexane and methanol with ethyl

acetate. The solvent mixtures were used for gradient elution followed by Claeson

66

(1993). After solvent flow through the column, the fractions were collected and

monitored by TLC. The fractions were pooled by the principle "The substance that

expresses at the same distance may be the same compound". The solvents from the

combined extract were concentrated using a vacuum rotary evaporator, then they were

weighed.

Fraction FBA7 showed higher dry weight (6.932 g) and % yield (26.539%)

than other fractions. All sub-fractions were aliquoted to determine the bioactivities by

antioxidant guided-isolation using DPPH radical scavenging assay, ferrous ion-

chelating activity assay and ferric reducing antioxidant power assay. In DPPH radical

scavenging assay, FBA7 fraction showed more percent DPPH radical scavenging

(29.63%) activity than other fractions when compared with ascorbic acid at 0.1 mg/mL.

Additionally, the FBA7 fraction was also expressed ferrous ion-chelating activity of

45.13%. However, the activity of ferric reducing antioxidant power assay (FRAP) is

good activity in FBA6 fraction (109.673 ± 3.502 mg TE/g extract). From the result

above, FBA7 fraction showed the highest DPPH radical scavenging and ferrous ion-

chelating activities compared to the other fractions. Therefore this fraction was chosen

to isolate by the 2nd column chromatography. The fraction was monitored by TLC to

preliminarily select the solvent system for the elution of the compounds from column

chromatography. The TLC-screening compound of FBA7 was shown in Figure D-3

(Appendix D). The appropriate solvent system to separate the substance mixtures was

methanol with dichloromethane.

After isolation, the fractions were separated into F2-BA1, F2-BA2, F2-BA3,

F2-BA4 and F2-BA5 (Table 4-10). Fraction F2-BA5 showed higher dry weight (2.4415

g) and % yield (48.83%) than other fractions. The diagram for the isolation of fractions

from aerial parts of B. augusta was shown in Figure 4-5. All fractions that collected by

the 2nd column chromatography were aliquoted to evaluate the bioactivity again using

DPPH radical scavenging assay, ferric reducing antioxidant power assay and ferrous

ion-chelating activity. For DPPH radical scavenging assay, F2-BA5 fraction showed

more percent DPPH radical scavenging (38.38%) activity than other fractions.

Apparently, the percent DPPH radical scavenging of F2-BA5 (38.38%) from the 2nd

column chromatography was more activity than FBA7 (29.63%) from the 1st column

chromatography. In addition, an F2-BA5 fraction (144.287 ± 1.273 mg TE/g extract)

67

from the 2nd column chromatography had more the FRAP value than a FBA6 fraction

(109.673 ± 3.502 mg TE/g extract) from the previous column chromatography. For the

DPPH radical scavenging and ferric reducing antioxidant power assay, sub-fraction of

B. augusta had the ability to inhibit DPPH radical by donating the H atom and reducing

Fe3+ to Fe2+ by electron donating action (Vijayalakshmi & Ruchmani, 2016). In ferrous

ion-chelating activity assay, the F2-BA5 fraction was displayed percent ion chelation

by 36.79% that was lower than a FBA7 fraction. However, the values of three

antioxidant activity assays that isolated by column chromatography had lower than

ethyl acetate extract (crude). This activity decreased may be due to the synergism

between the bioactive compound(s) in the plants, synergism is defined as the activity

of two or more substances given together that is greater than the sum of activity had the

agents have been given separately (Bennett, Dolin, & Blaser, 2014).

Syzygium gratum (Wight) S.N. Mitra var. gratum was collected from Ban

Ang-Ed Official Community Forest Project, Chantaburi province. It was first extracted

in absolute ethanol and then successively partitioned into hexane, ethyl acetate, and

water. All extracts were characterized the compound by NMR spectroscopy. 1H NMR

spectra of four fractions were shown in Figure F-18 to F-21 (Appendix F). Then they

were determined the total phenolic content. The total phenolic content in extracts were

measured with a gallic acid as a standard (standard curve equation: y=17.03x + 0.0257,

R2 = 0.9936). For the results, ethyl acetate extract had the highest total phenolic content

of 151.485 ± 1.360 mg GAE/g extract followed by ethanol, water and hexane extracts

(Table 4-15). This is similar to the previous report of Stewart et al. (2 0 1 3 ) , S. gratum

in ethyl acetate extract displayed total phenolic values of 149.789 ± 0.381 mg GAE/g

dry weight. The fractions of S. gratum in ethanol, hexane, ethyl acetate, and water

extracts were examined for antioxidant activities by bioassay-guided isolation using

DPPH radical scavenging assay, ferrous ion- chelating activity assay and ferric

reducing antioxidant power assay. Three assays that were performed based on the

ability of an antioxidant to act as reducing agents and radical scavengers. Antioxidants

activity used several mechanisms such as hydrogen/electron donors, metal ions

chelators, inducers through the activities of antioxidant enzymes. Hence, the various

methods that determined the different effects on antioxidant activity to observe the

better candidate antioxidant for the future use in various applications. The inhibitory

68

effect on DPPH radical of S. gratum, the ethyl acetate extract showed the highest

scavenging activity (EC50 = 0.071 ± 0.002 mg/mL). For ferrous ion-chelating activity

assay, the ethyl acetate extract significantly showed (P<0.05) higher ferrous ion

chelating activity (30.43%) than ethanol, hexane, and water extracts. Moreover, the

ethyl acetate extract also presented the highest FRAP value (890.885 ± 4.724 mg TE/g

extract). Globally, it indicates that ethyl acetate extract exhibits the high-antioxidant

activity. Gohar et al. (2013) who isolated Callistemon viminalis of Myrtaceae family

(the same family as S. gratum) suggested that antioxidant compounds such as methyl

gallate, catechin, gallic acid, ellagic acid and other phenolics can display the antioxidant

activity, those compounds would expect to be found in Syzygium gratum (Wight) S.N.

Mitra var. gratum as well. Therefore, ethyl acetate extract is suitable for sub-fraction

isolated by column chromatography to further evaluate the active fraction and

antioxidant activity.

In the part of column chromatography, the ethyl acetate extract exhibited the

high antioxidant activity. Therefore, this extract was chosen to further isolate by column

chromatography. Ethyl acetate extract was monitored by thin layer chromatography

(TLC) to preliminarily select the solvent system for column chromatography. Figure

D-5 showed TLC of ethyl acetate extract from S gratum. The appropriate solvent

systems for separating the mixtures were ethyl acetate with hexane and methanol with

ethyl acetate. The solvent mixture was used for gradient elution according to Claeson

(1993). After solvent flew through the column, the obtained fractions were collected

and monitored by TLC. A diagram for the isolation of fractions from aerial parts of S.

gratum was shown in Figure 4-9. Eight fractions were obtained including FSG1, FSG2,

FSG3, FSG4 FSG5, FSG6, FSG7, and FSG8 (Table 4-18). Fraction FSG6 showed more

dry weight (5.125 g) and %yield (34.35%) than the other fractions. All sub-fractions

were aliquoted to determine the antioxidant activities using DPPH radical scavenging

assay, ferrous ion-chelating activity assay and ferric reducing antioxidant power assay.

For DPPH radical scavenging assay, FSG7 fraction showed the highest percentage of

DPPH radical scavenging (94.36%) followed by FSG5 (91.68%) when compared with

ascorbic acid (100%) at concentration 0.05 mg/mL. Apparently, the ability of DPPH

radical scavenging of sub-fraction FSG7 increased from selected the ethyl acetate

extract (49.62%) as a result of isolation and purification of the crude extract.

69

Purification and isolation of bioactive compounds from plants are a technique offers

the ability to parallel the development and availability of many advanced bioassays.

The goal when searching for bioactive compounds is to find an appropriate method that

can screen the material source for bioactivity such as antioxidant, antibacterial, or

cytotoxicity, combined with simplicity, specificity, and speed (Altemimi, Lakhssassi,

Baharlouei, Watson, & Lightfoot, 2017). Likewise, the results of the ferrous ion-

chelating activity assay also showed more percentage of ferrous ion-chelating of FSG6

(39.11%) than the original ethyl acetate extract (30.43%). Whereas, FSG5 fraction

presented the FRAP value of 767.373 ± 3.272 mg TE/g extract. It slightly decreased

from the ethyl acetate fraction (890.885 ± 4.724 mg TE/g extract) at the same

concentration (0.05 mg/mL). However, the FRAP values of FSG6 (747.173 ± 1.425 mg

TE/g extract) and FSG7 (642.604 ± 2.157 mg TE/g extract) fractions showed good

activity as a result of bioactive compounds in each fraction may have a synergistic

effect of the activity. For the bioactivity of sub-fractions in three assays, it indicated

that FSG5 fraction is suitable to separate the substances and confirm antioxidant

compounds by high-performance liquid chromatography (HPLC) and nuclear magnetic

resonance (NMR) spectroscopy.

Conclusion In conclusion, Barrington augusta Kurz. and Syzygium gratum (Wight) S.N.

Mitra var. gratum that are Thai indigenous plants from Ban Ang-Ed Official

Community Forest Project (The Chaipattana Foundation) were evaluated for total

phenolic content. The ethyl acetate extract of both plants was shown remarkably

exhibited DPPH radical scavenging, ferric reducing antioxidant power and ferrous ion-

chelating activity. Therefore, it was further isolated by the column chromatography to

evaluate their bioactive compounds. The active compounds from B. augusta fractions

showed DPPH radical scavenging, ferric reducing antioxidant power and ferrous ion-

chelating lower than ethyl acetate extract (crude), whereas the active compounds from

S. gratum still presented DPPH radical scavenging activity and ferrous ion-chelating

that were higher than the ethyl acetate extract (crude). It may be due to the synergism

of the bioactive compounds in this fraction. From those studies, we suggested that the

70

active compounds in sub-fractions can be applied to nutritional and dietary

supplemented applications, with their high antioxidant activities and total phenolic

content. The extracts may play important roles in the discovery, development, and

manufacturing of the functional foods. However, active fraction should be continued to

isolate for purified compound(s) and determine the antioxidant activity of such

compound(s) again in the future.

71

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APPENDIX

79

APPENDIX A

Preparation of solutions

80

1. 7% Sodium bicarbonate solution (Na2CO3) for total phenolic contents

7% Sodium bicarbonate solution (Na2CO3) 50 ml

100 ml = 7 g

50 ml = (7 × 50) / 100

= 3.5 g

Sodium bicarbonate 3.5 g dissolved in distilled water 50 ml

2. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) for DPPH radical

scavenging assay 0.2 mM DPPH solution (M.W. = 394.33 g/mol) in methanol 10 mL

g / M.W. = CV/1000

g = [(0.2×10-3) ×10 × 349.33] / 1000

g = 0.00078

DPPH 0.00078 g dissolved in methanol 10 mL

3. FRAP reagent for Ferric Reducing Antioxidant Power assay (FRAP)

3.1 FRAP reagent

Mixed 0.28 mM Acetate buffer solution (pH 3.6) 50 mL, 0.01 M TPTZ

solution 5 mL and 0.02 M Iron (III) chloride hexahydrate solution 5 mL in the ratio

10:1:1

3.2 0.28 mM Acetate buffer solution (pH 3.6) 100 mL

Sodium acetate (CH3COONa) 2.3 g dissolved in conc. acetic acid solution

(C2H4O2) 7.6 mL. Then, the solution was adjusted pH to 3.6 by added conc. HCl and

made the volume to 100 mL by deionized water.

3.3 40 mM hydrochloric acid solution (HCl) 100 mL

Mixed conc. hydrochloric acid 0.33 ml with deionized water 100 mL

81

3.4 0.01 M TPTZ solution (2, 4, 6- tri (2- pyridyl)- s- triazine) 10 mL

TPTZ 0.0310 g dissolved in 40 mM hydrochloric acid solution 10 mL. The

solution was dissolved at 50 °C.

3.5 0.02 M Iron (III) chloride hexahydrate solution (FeCl3•6H2O) 10 mL

Iron (III) chloride hexahydrate 0.054 g dissolved in deionized water 10 mL.

4. 2 mM Iron (II) chloride (FeCl2) 10 mL for Ferrous ion-chelating

activity 2 mM FeCl2 solution (M.W. = 126.75 g/mol) in distilled water 10 mL

g / M.W. = CV/1000

g = [(2×10-3) ×10 × 126.75] / 1000

g = 0.0025

FeCl2 0.0025 g dissolved in distilled water 10 mL

5. 5 mM ferrozine for Ferrous ion-chelating activity

5 mM ferrozine solution (M.W. = 492.5 g/mol) in distilled water 10 mL

g / M.W. = CV/1000

g = [(5×10-3) × 10 × 492.5] / 1000

g = 0.0246

ferrozine 0.0246 g dissolved in distilled water 10 mL

82

APPENDIX B

Standard curve of solutions

83

DETERMINATION OF ANTIOXIDANT ACTIVITIES

1. Total phenolic content

Figure 1-1 Standard curve of gallic acid solution. At 765 nm, it had absorbance

among 0.04-0.57.

2. DPPH radical scavenging assay

Figure 2-1 Positive control curve from ascorbic acid (vitamin C). It had repeated

three times (EC50 = 0.022 ± 0.001 mg/ml).

y = 17.03x + 0.0257R² = 0.9936

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035

Abs

orba

nce

of g

allic

aci

d

concentration of gallic acid (mg/mL)

y = 1789.7x + 9.5015R² = 0.9818

0

20

40

60

80

100

120

0 0.01 0.02 0.03 0.04 0.05 0.06

% S

cave

ngin

g

concentration of ascorbic acid (mg/mL)

84

3. Ferric Reducing Antioxidant Power assay (FRAP)

Figure 3-1 Standard curve of Trolox solution. At 593 nm, it had absorbance among

0.05-0.72.

4. Ferrous ion-chelating activity assay

Figure 4-1 Positive control curve from EDTA. It had repeated three times (EC50 =

0.017 ± 0.000 mg/mL).

y = 12.772x + 0.0904R² = 0.9879

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.01 0.02 0.03 0.04 0.05 0.06

Abs

orba

nce

at 5

93 n

m

Concentration of Trolox (mg/mL)

y = 1685.6x + 22.679R² = 0.9213

0

20

40

60

80

100

120

0 0.01 0.02 0.03 0.04 0.05 0.06

% fe

rrou

s ion

-che

latin

g

concentration of EDTA (mg/mL)

85

APPENDIX C

Calculation of percent yield from the extract

86

1. Barrington augusta Kurz. 1.1 Percent yield of sub-extract

Calculate yield directly in percent yield of the extract from B. augusta using

formula:

% yield of crude extract = (A/B) × 100

Where, A = weight of crude after extraction process

B = material weight of B. augusta powder taken for extraction

Percent yield of ethanol extract

% yield of ethanol extract = (83.2323 g/800 g) × 100

= 10.40%

Percent yield of hexane extract

% yield of hexane extract = (25.56 g/83.23 g) × 100

= 30.71%

Percent yield of ethyl acetate extract

% yield of ethyl acetate extract = (27.47 g/83.23 g) × 100

= 33%

Percent yield of water extract

% yield of water extract = (20.40 g/83.23 g) × 100

= 24.51%

1.2 Percent yield of the fractions from the 1st column chromatography

Calculate yield directly in percent yield of the fractions from the 1st column

chromatography using formula:


87

Where, A = weight of fraction after the 1st purification process

B = weight of ethyl acetate extract taken for the 1st purification

Percent yield of FBA1 fraction

% yield of FBA1 fraction = (0.1935 g/26.12 g) × 100

= 0.741%



= 0.396%



= 0.694%



= 0.988%



= 1.879%



= 15.552%



= 26.539%

88



= 18.437%

1.3 Percent yield of the fractions from the 2nd column chromatography

Calculate yield directly in percent yield of the fractions from the 2nd column



Where, A = weight of fraction after the 2nd purification process

B = weight of FBA7 fraction taken for the 2nd purification

Percent yield of F2-BA1 fraction

% yield of F2-BA1 fraction = (0.2649 g/5 g) × 100

= 5.29%



= 6.27%



= 15.69%



= 23.33%

89



= 48.83%

2. Syzygium gratum (Wight) S.N. Mitra var. gratum 2.1 Percent yield of sub-extract

Calculate yield directly in percent yield of the extract from S. gratum using

formula:


Where, A = weight of crude after extraction process

B = material weight of S. gratum powder taken for extraction

Percent yield of ethanol extract

% yield of ethanol extract = (118.203 g/800 g) × 100

= 14.75%

Percent yield of hexane extract

% yield of hexane extract = (7.45 g/116.203 g) × 100

= 6.41%

Percent yield of ethyl acetate extract

% yield of ethyl acetate extract = (17.79 g/116.203 g) × 100

= 15.305%

Percent yield of water extract

% yield of water extract = (29.78 g/116.203 g) × 100

= 25.63%

90

2.2 Percent yield of the fractions from the 1st column chromatography

Calculate yield directly in percent yield of the fractions from the 1st column



Where, A = weight of fraction after the 1st purification process

B = weight of ethyl acetate extract taken for the 1st purification

Percent yield of FSG1 fraction

% yield of FSG1 fraction = (0.0883 g/15 g) × 100

= 0.5886%



= 0.828%



= 0.6787%



= 1.5933%



= 1.1667%



= 34.3467%

91



= 20.404%



= 13.68%

92

APPENDIX D

Thin layer chromatography

93

Barrington augusta Kurz.

Figure D-1 TLC of ethyl acetate fraction from B. augusta

Figure D-2 TLC of fractions from 1st column chromatography

When: FBA1 fraction was pooled by SBA1-4, FBA2 fraction was pooled

by SBA5, FBA3 fraction was pooled by SBA6-7, FBA4 fraction was

pooled by SBA8-10, FBA5 fraction was pooled by SBA11-16, FBA6

94

fraction was pooled by SBA17-20, FBA7 fraction was pooled by 21-22

and FBA8 fraction was pooled by SBA23-24.

Figure D-3 TLC of FBA7 fraction from 1st column chromatography

Figure D-4 TLC of fractions from 2nd column chromatography

Syzygium gratum (Wight) S.N. Mitra var. gratum

Figure D-5 TLC of ethyl acetate fraction from S. gratum

95

Figure D-6 TLC of fractions from 1st column chromatography

When: FSG1 fraction was pooled by lane 1-3, FSG2 fraction was pooled

by lane 4, FSG3 fraction was pooled by lane 5-6, FSG4 fraction was pooled

by 7-9, FSG5 fraction was pooled by lane 10-20, FSG6 fraction was pooled

by lane 21-91, FSG7 fraction was pooled by lane 92-196 and FSG8 fraction

was pooled by lane 197-360.

96

APPENDIX E

Example of TLC screening by DPPH radical solution

97


Figure E-1 Example of TLC screening for DPPH radical scavengers from the 1st


Spray DPPH solution

98

Figure E-2 Example of TLC screening for DPPH radical scavengers from the 2nd


Spray DPPH solution

99


Figure E-3 Example of TLC screening for DPPH radical scavengers from the 1st


Spray DPPH solution

100

APPENDIX F 1H NMR spectra of fractions

101


Figure F-1 1H NMR spectra of ethanol extract

Figure F-2 1H NMR spectra of hexane extract

102

Figure F-3 1H NMR spectra of ethyl acetate extract

Figure F-4 1H NMR spectra of water extract

103

Figure F-5 1H NMR spectra of FBA1 fraction from the 1st column chromatography


104



105



106



107

Figure F-13 1H NMR spectra of F2-BA1 fraction from the 2nd column

chromatography


chromatography

108


chromatography


chromatography

109


chromatography

110


Figure F-18 1H NMR spectra of ethanol extract

Figure F-19 1H NMR spectra of hexane extract

111

Figure F-20 1H NMR spectra of ethyl acetate extract

Figure F-21 1H NMR spectra of water extract

112

Figure F-22 1H NMR spectra of FSG1 fraction from the 1st column chromatography


113



114



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