PERPUSTAKAAN UMP

I llllIffi Iffi Ill Iffi IU Villil llVIllllII 0000073686

ANTIOXIDANT ACTIVITY OF DIFFERENT FRACTIONS FROM TINOSPORA C'RISPA STEMS EXTRACTS

VIDHYAA AlP PAROOINDRAN

Report submitted in partial fulfillment of the requirements for the award of the degree of Bachelor of Applied Science (Honor) Industrial Chemistry

Faculty of Industrial Sciences & Technology UNIVERSITI MALAYSIA PAl-lANG

2012

V

ABSTRACT

"Patawali (Malay) or Tinospora crispa is a plant that has many traditional uses for skin treatment. Tinospora crispa is a woody and glabrous plant in the family MenispermaCeae. In cosmetics, Tinospora crispa can be used externally for its strong antioxidant activity and' free radical scavenging properties. Therefore, the objective of the study was to fractionated all the five fractions from the Tinospora crispa stems extract, to test antioxidant activity of the extracted fractions using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay and finally to evaluate total content of phenolics in the fractions extracted from Tinospora crispa stems. The methodology of the study involves defatting of the plant material, separation and extraction of the moderately polar extract (organic extract) along with the aqueous extract. The fractions are then tested using the DPPH assay for free radical scavenging property to prove its antioxidant activity. The antioxidant activity of the plant fractions are compared with positive control ascorbic acid and butylated hydroxytoulene (BUT). On the other hand, total content of phenolics in fractions extract from the stem was determined using Folin-Ciocalteu method. Moreover, thin layer chromatography method was used to identify phenolics in fraction F3 and fraction F4 while alkaloids were also identified in fraction F4. The research carried out proved that the fractions extracted had significant antioxidant activity especially in fraction F3 and F4, 88.40 ± 0.37 % and 64.91 ± 0.58 % of inhibition repectively which was realatively comparable with the positive control ascorbic acid and butylated hydroxytoluene (BUT). Besides, the total phenolic content in F3 was reported the highest followed by F4, 73.54 ± 2.22 .tg GAE per mg of sample and 62.10 ± 1.42 tg GAB per mg of sample respectively. As an overall conclusion, the research clearly supports that the Tinospora crispa stems extract have significant antioxidant activity due to the presence of phenolics in the fractions that can be further studied. The research too emphasizes the extract from the plant stems to be a good source of herbal medication that can be produced in industries to compliment the society need for curing diseases such as cancer and rapid aging.

vi

I

"Patawali atau Tinospora crispa merupakan tumbuhan yang mempunyai banyak kegunaafl tradisional untuk rawatan kulit. Tinospora crispa berkayu dan glabrous dalam keluarga Menispermaceae. Dalam kosmetik, Tinospora crispa boleh digunakan secara luaran untuk aktiviti antioksidatifhya yang kuat kerana sifatnya yang boleh memerangkap radikal bebas. Oleh itu, objektif kajian adalah untuk mengasingkan dan mengumpul kesemua lima pecahan ekstrak daripada tumbuhan ini. Kemudian untuk menguji aktiviti ant ioksidatif pecahan yang diekstrak menggunakan "2,2-diphenyl- 1 -picrylhydrazyl" (DPPH) dan akhirnya untuk menilai kandungan jumlah fenolik yang diekstrak daripada batang Tinospora crispa di dalam setiap ekstrak. Metodologi kajian mi melibatkan pengasingan wax daripada batang tumbuhan dan pengeluaran ekstrak sederhana organik bersama-sama dengan ekstrak akueus. Pecahan kemudiannya diuji dengan menggunakan DPPH membuktikan aktiviti antioksidatiffiya serta membandingkannya dengan asid askorbik kawalan positif dan "butylated hydroxytoulene" (BHT). Sebaliknya, kandungan jumlah phenolics di dalam pecahan ekstrak daripada batang ditentukan dengan menggunakan kaedah "Folin-Ciocalteu". Selain itu, kaedah kromatografi digunakan untuk mengenal pasti phenolics dalam pecahan F3 dan F4 manakala alkaloid yang juga dikenal pasti dalam F4 pecahan. Penyelidikan yang dijalankan membuktikan bahawa pecahan yang diekstrak mempunyai aktiviti antioksidan yang ketara terutama dalam pecahan F3 dan F4, 88.40 ± 0.37 % dan 64.91 ± 0.58 % perencatan masing-masing yang setanding dengan kawalan positif asid askorbik dan "butylated hydroxytoluene" (BHT). Selain itu, jumlah kandungan fenolik dalam F3 dilaporkan tertinggi diikuti oleh F4 sebanyak 73.54 ± 2.22 j.tg GAE/ mg sampel dan 62.10 ± 1.42 .ig GAE/ mg sampel masing-masing. Kesimpulannya, penyelidikan mi dengan jelas menyokong bahawa ekstrak batang Tinospora crispa mempunyai aktiviti antioksidan yang ketara disebabkan oleh kandungan fenolik di dalamnya. Penyelidikan mi juga menekankan ekstrak daripada batang tumbuhan mi boleh menjadi sumber yang baik untul membuat ubat-ubatan herba di dalam indutri yang akan selaras dengan keperluan masyarakat bagi mengubati penyakit-penyakit seperti kanser dan penuaan yang cepat.

TABLE OF CONTENTS

Page

SUPERVISOR'S DECLARATION

STUDENT'S DECLARATION

ACKNOWLEDGEMENT iv

ABSTRACT

ABSTRAK

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES xi

LIST OF SYMBOLS

LIST OF ABBREVATION xiv

LIST OF APPENDICES xv

CHAPTER 1 INTRODUCTION

1.1 Background of the study I

1.2 Problem statement 3

1.3 Objective 3

1.4 Scope of research 4

1.5 Significance of the study 4

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction 5

2.2 Tinospora crispa plant material 5

2.3 Antioxidants 7 2.4 Types of antioxidants 8

vii

viii

2.4.1 Phenolic 8 2.4.2 Ascorbic acid 15 2.4.3 Butylated hydroxytoulene 16

2.5 2, 2-Dipheñyl-1-picrylhydrazyl (DPPH) 17

CIIMTER 3 MATERIALS AND METHODS

3.1 Introduction 19

3.2 Materials 19

3.3 Apparatus 20

3.4 Instruments 20

3.5 Methodology

3.5.1 Extraction of Tinospora crispa stem extracts 20 3.5.2 2,2-Diphenyl-1-picrylhydrazyl DPPH Assay 23 3.5.3 Folin-Ciocalteu reagent method 25 3.5.4 Identification of alkaloid compounds using

Thin Layer Chromatography (TLC) 27 3.5.5 Identification of phenolic compounds using

Thin Layer Chromatography (TLC) 28 3.5.6 Statistical analysis 29

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Introduction 31

4.2 Yield of dried fractions 31

4.3 Antioxidant activity of Tinospora Crispa stems fractions,

positive controls BHT and ascorbic acid 32

4.4 Total content of phenolics in tinospora crispa stems fractions 46

4.5 Pre-Identification of alkaloid compounds 48

4.6 Pre-Identification of phenolic compounds 49

ix

CHAPTER 5 CONCLUSION AND RECOMENDATIONS

5.1 Introduction

52

5.2 Conclusion

52

5.3 Recommendations

53

REFERENCES

54

APPENDICES

61

LIST OF TABLES

Table No. Page

4.1 Table of yield of fractions extracted 31

4.2 Table of physical properties of the fractions extracted 32

4.3 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 0 min 33

4.4 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 15 min 33

4.5 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 30 min 34

4.6 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 45 min 34

4.7 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 1 h 35

4.8 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 1 h 15 min 35

4.9 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 1 h 30 min 36

4.10 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 1 h 45 min 36

4.11 Percentage (%) of inhibition of fractions, BHT and ascorbic acid at 2 h 37

4.12 Concentration and absorbance of gallic acid equivalent 46

4.13 Concentration of fraction Fl, F2, F3, F4 and F5 from gallic acid equivalent standard curve 47

4.14 Total content of phenolic compounds in fraction Fl, F2, F3, F4 and F5

x

LIST OF FIGURES

Figure No. Page

2.1 Tinospora crispa plant material 6

2.2 Schematic biochemical pathway of the synthesis of flavanone, resveratrol, and furanocoumarin. 9

2.3 Basic plant phenolic structures with examples 11

2.4 Ascorbic acid 15

2.5 Butylated hydroxytoluene. 16

2.6 2,2- Diphenyl-1- picryihydrazyl (DPPH) 18

3.1 Flow chart of extraction of fraction 1,2,3,4 and 5 from Tinospora Crispa stems. 22

3.2 Flow chart of DPPH Assay 24

3.3 Flow chart of Folin-Ciocalteu reagent method 26

3.4 Flow Chart of Research Methodology 30

4.1 Graph of antioxidant activity of fraction (Fl, F2, F3, F4 and F5), BHT and ascorbic acid 38

4.2 Proposed reaction mechanism of ascorbic acid with DPPH. (radical) 41

4.3 Proposed reaction mechanism for BHT and DPPH • (radical) 42

4.4 Proposed reaction mechanism of phenolic with DPPH • (radical) 45

4.5 Graph of Gallic Acid Equivalent (GAE) 46

4.5 Identification of alkaloid using TLC Visualizer at Xnlax 254nm and Xax 366nm for fraction F4 48

4.6 Pre-Identification of phenolic using TLC Visualizer at Xmax 2 54n and Xmax366nm for fraction F3. 49

xi

47 Pre-Identification of phenolic using TLC Visualizer at X.max 254nm and Xmax 366nm for fraction F4. 50

xii

LIST OF SYMBOLS

XIII

Wavelength

Percentage

Uncertainty

Greater than

cm Centimeter

h Hour

Microliter

g Gram

Kg Kilogram

L Liter

min Minutes

mg Milligram

ML Milliliter

tg Microgram

nm Nanometer

Rf Retention factor

LIST OF ABBREVATIONS

BUT Butylated hydroxytoluene

DPPH 2,2-Diphenyl- 1 -picrylhydrazyl

DMSO Dimethyl Sulfoxide

EtOH Ethanol

MeOH Methanol

TLC Thin Layer Chromatography

UV-Vis Ultraviolet-Visible

xiv

LIST OF APPENDICES

Appendix No. Page

A Antioxidant activity of Tinospora crispa 61

Al DPPH Assay absorbance at 0 min 61

Percentage (%) of inhibition at 0 min 61

DPPH Assay absorbance at 15 min 62

A4 Percentage (%) of inhibition at 15 min 62

AS DPPH Assay absorbance at 30 min 63

A6 Percentage (%) of inhibition at 30 min 63

A7 DPPH Assay absorbance at 45 min 64

A8 Percentage (%) of inhibition at 45 min 64

A9 DPPH Assay absorbance at 1 h 65

AlO Percentage (%) of inhibition at 1 h 65

All DPPH Assay absorbance at 1 h 15 min 66

Al2 Percentage (%) of inhibition at 1 h 15 min 66

A13 DPPH Assay absorbance at 1 h 30 min 67

A14 Percentage (%) of inhibition at 1 h 30 nun 67

A15 DPPH Assay absorbance at 1 h 45 min 68

A16 Percentage (%) of inhibition at 1 h 45 min 68

A17 DPPH Assay absorbance at 2 h 69

A18 Percentage (%) of inhibition at 2 h 69

B Total content of phenolics in Tinospora crispa stems fractions 70

xv

xvi

BI Concentration of fraction Fl, F2, F3, F4 and F5 from gallic acid equivalent standard curve 70

B2 Total content of phenolic compounds in fraction Fl, F2, F3, F4 and F5 70

B3 Absorbance of Fl, F2, P3, F4, F5 and control DMSO at 750 nm 70

CHAPTER 1

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Tinospora crispa is a woody and glabrous plant in the family of

Menispermaceae. It is found in primary rainforests or mixed deciduous forests

throughout a large part of Asia and Africa including all parts of Thailand, Malaysia and

Indonesia (Pathak et al., 1995).

Free radicals contribute to more than one hundred disorders in humans including

atherosclerosis, arthritis, ischemia and repercussion injury of many tissues, a central

nervous system injury, gastritis and cancer (Pourmorad et al., 2006). Due to

environmental pollutants, radiation, chemicals, toxins, deep fries and spicy foods as well

as physical stress, free radicals cause depletion of the immune system antioxidants, the

change in gene expression and induce abnormal proteins. The oxidation process is one

Of the most important routs for producing free radicals in food, drugs, and even living

systems (Dullard and German, 2000 and Turkoglu et al., 2007).

Antioxidant means "against oxidation". An antioxidant is any substance that

retards or prevents deterioration, damage or destruction by oxidation. In definition of the

term by Halliwell and Gutteridge (1999) stated that an antioxidant is 'any substance that,

when present at low concentrations compared with that of an oxidizable substrate, significantly delays or inhibits oxidation of that substrate'. On the other hand, a free

radical is a compound with one or more unpaired electrons in its outer orbital. Such

2

unpaired electron(s) make these species very unstable and therefore quite reactive with

other molecules due to the presence of unpaired electron(s) and they try to pair their

electron(s) and generate a more stable compound. Antioxidants can be classified taking

into account their mechanism of action, although there are other possible classifications.

Bearing in mind the mechanism of action, they can be divided into primary antioxidants,

synergistic and secondary antioxidants (Rajalakhmi et al., 1996). Some substances

considered as antioxidants are ascorbate, tocopherols, some enzymes, carotenoids and

bioactive plant phenols. The health benefits of fruits and vegetables are largely due to

the antioxidant vitamin content supported by a large number of phytochemicals, some

with greater antioxidant properties. Sources of tocopherols, carotenoids and ascorbic

acid are well recognized antioxidants.

Antioxidants are important species which possess the ability of protecting

organisms from damage caused by free radical-induced oxidative stress. The antioxidant

activity of phenolics is mainly due to their redox properties, which allow them to act as

reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelators

(Canadanovic-Brunet et al., 2005). A number of synthetic antioxidants such as butylated

hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been extensively

added to foodstuffs, although their use has begun to be questioned because of their

toxicity (Ito et al., 1985), so there is considerable interest in preventive medicine and in

the food industry in the development of natural antioxidants obtained from botanical

sources, especially herbal plants (Djilas et al., 2003). Medicinal plants have been used

for centuries as remedies for human diseases because they contain components of

therapeutic value. Moreover, the increasing use of plant extracts in the food, cosmetic and pharmaceutical industries suggests that, in order to find active compounds, a

systematic study of medicinal plants is very important (Nostro et al., 2000).

22-Diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical which has an

Unpaired valence electron at one atom of nitrogen bridge (Eklund et al., 2005). DPPH is

one of the free radicals widely used for testing preliminary radical scavenging activity of a compound or plant extract. It is stable and it contains an odd electron in its structure

that is usually utilized for the detection of the radical scavenging activity in chemical

analysis.

1.2 PROBLEM STATEMENT

Antioxidant in general is important for the anti-ageing study. Besides, Tinospora

Crispa is reported to contain different phenolic compounds with probable free radical

scavenging property (Amom et al., 2009). The antioxidant activities may vary depending

on the structural factors, such as number of phenolic hydroxyl or methoxyl group,

flavone hydroxyl, keto groups, free carboxylic groups and other structural features.

Thus, this study is simply based on further study of on Tinospora Crispa fractions

extracted to know their variation in antioxidant properties and their total content of

phenolics. This will be done because the role of free radicals in many disease conditions

has been well established (Pathak et al., 1995; Quisumbing et al., 1978 and Rahman et

al., 1999). Several biochemical reactions in our human body can damage crucial bio-

molecules due to presence of free radical and increase the rate of ageing in human while

cause severe health risks. Hence, this study is done to extract different fractions from

Tinospora crispa stems and then to determine the antioxidant activity of the different

fractions extracted with other positive controls such as ascorbic acid and butylated

hydroxytoluene using DPPH Assay. Finally, the total content for phenolics in the

respective fractions extracted from Tinospora crispa stems is determined.

1.3 OBJECTIVES

i. To extract different fractions from Tinospora crispa stems extracts.

ii. To determine antioxidant activity of the different fractions extracted from the

Tinospora crispa stems using DPPH Assay.

lii. To determine total content of phenolics in the respective fractions extracted from

Tinospora crispa stems.

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1.4 SCOPE OF RESEARCH

As a way to accomplish the objective of this study, the scope of this research

focuses on extraction of different fractions from Tinospora CriSpa stems and the

comparison of the antioxidant activity of the different fractions extracted with other

established antioxidants such as ascorbic acid and butylated hydroxytoluene (BHT)

using DPPH Assay. Besides, it also focuses on determination of the total content of

phenolics in the respective fractions extracted from Tinospora crispa stems. Finally it

involves identification of the phenolics and alkaloids present in some selected fractions

of extract.

1.4.1 SIGNIFICANCE OF THE STUDY

The extracted fractions from Tinospora crispa stems are believed to have anti-

oxidative properties which can help in anti-ageing of cells. Ageing of cells can cause

severe health risks to humans. Therefore, through this test of free radical scavenger

using 2,2-diphenyl-1-picrylhydrazyl DPPH of the fractions extracted from Tinospora

Crispa, it is trusted to help reduce ageing of cells that may eventually decrease health

risks. If this study proves that the fractions extracted from Tinospora Crispa has anti-

oxidative properties, it may be of great use to the medicinal industry and help treat

various deceases such as cancer, atherosclerosis, senility, aging, and many more. Thus,

the fractions are further studied to determine the responsible antioxidants which can take

Part in maintenance of good health as do vitamins, minerals and enzymes. Antioxidants

can be added to foodstuffs to prevent free radical damage as they scavenge the free

radical (Botsoglou et al., 2002 and Shetty, 1997). Besides, the ready availability of the

plant source widely distributed in Asia is a major factor that influences the plant

selection in this study. Other than that, the plant source is also cheap and is viable for industrial applications.

CHAPTER 2

LITERATURE REVIEW

2.1 INTRODUCTION

In this chapter, the detailed facts on the plant material Tinospora crispa,

antioxidants, the various type of antioxidants, and 2,2-diphenyl- 1 -picryihydrazyl

(DPPH) reagent functionality were discussed.

2.2 TINOSPORA CRISPA PLANT MATERIAL

Tinospora crispa as in Figure 2.1 is an indigenous climber plant that commonly

grows wild in Asean countries including Malaysia. Known by various local names like

'akar patawali' and 'akar seruntum' (Noor and Ashcroft, 1989). Most species of the

genus Tinospora are among the most widely employed medicinal plants throughout a

large part of Asia and Africa (Pathak et al., 1995). In general folklore, the stem

decoction is considered antipyretic, useful as an antimalarial and a wash for skin ulcers.

Traditionally an infusion is used to treat fever due to malaria and also in cases of

jaundice and for use against intestinal worms. The antimalarial effect was confirmed in a

study (Ra1man et al., 1999).

An infusion of the stem is drunk as a vermiflige while a decoction of the stem is

used for washing sore eyes and syphilitic sores. Its stem has been used by traditional

folklore for various therapeutic purposes such as treatment for diabetes, hypertension, stimulation of appetite and protection from mosquito bites. Based on research done it

was also used as hypoglycemic drug an antipyretic agent in both man and domestic

animals (KongSaktrak00I et al., 1994; Noor et al., 1989 and Pathak et al., 1995). This

bitter tasting plant locally known as Makabuhai in Philippines is used for the treatment

of stomach troubles,, ulcers and fevers, as a tonic and a febrifuge for malaria and

smallpox, as a vulnerary for itches and wounds, and many other purposes (Quisumbing,

1978).

Figure 2.1: Tinospora crispa plant material

Source: Noor et al. (1989)

2.3 ANTIOXIDANTS

Antioxidant compounds play an important role as a health-protecting factor.

Antioxidants are substance that significantly prevents or delays the oxidation of other

molecules by inhibiting the initiation or propagation of oxidizing chain reactions

(HalliWell and Gutteridge, 1990). Oxygen-centered free radicals and other reactive

oxygen species (ROS) have been associated with the beginning of many diseases and

degenerative processes in ageing (Halliwell, 1994). Since oxidative stress is common in

chronic degenerative diseases, dietary antioxidants in plant sources may provide a

beneficial effect. Antioxidants are known to protect cells against the damaging effects of

reactive oxygen species (ROS) such as singlet oxygen, superoxide and hydroxyl

radicals, among others (Halvorsen et al., 2002 and Wu et al., 2004).

Almost all organisms are well protected against free radical damage by

oxidative enzymes such as superoxide dismutase and catalase or chemical compounds

such as a-tocopherol, vitamin C (ascorbic acid), carotenoids, polyphenol compounds and

glutathione (Betancur-Ancona et al., 2004). However, these systems are frequently

insufficient to totally prevent the damage, especially under the conditions of severe

oxidative stress, resulting in diseases and accelerated ageing (Ames et al., 1993). The

balance between antioxidation and oxidation is believed to be a critical concept for

maintaining a healthy biological system. Hyper physiological burden of free radicals

causes imbalance in homeostatic phenomena (mechanisms) between oxidants and

antioxidants in the body. This imbalance leads to oxidative stress involved in aging and

various human diseases like atherosclerosis, stroke, diabetes, cancer and

neurodegeneratjve diseases such as Alzheimer's disease and Parkinsonism (Halliwell,

1994).

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2.4 TYPES OF ANTIOXIDANTS.

2.4.1 Phenolics

Phenolic compounds are secondary metabolites which are synthesized in

plants. They possess biological properties such as antioxidant, anti-apoptotic, anti-aging,

anti-carcinogenic, anti-inflammatory, anti-atherosclerotic, cardiovascular protection,

improvement of the endothelial function, as well as inhibition of angiogenesis and cell

proliferation activity. Most of these biological actions have been attributed to their

intrinsic reducing capabilities (Han et al., 2007). Phenolic compounds are one of the

most important, numerous and ubiquitous groups of compounds in the vegetable

kingdom, being synthesised by plants during normal development and in response to

different situations such as stress and ultraviolet radiation (Naczk and Shahidi, 2004).

These substances are obtained from phenyipropanoid metabolism and there are more

than 8000 different known structures (Herrero et al., 2005). However, these large ranges

of structures possess a common structural feature: an aromatic ring with one or more

hydroxy substituents. The way to classify these components it is not clearly established;

a possible classification can be based on the number of constitutive carbon atoms in

conjunction with the structure of the basic phenolic skeleton, including for instance,

simple phenols, phenolic acids, coumarins, flavonoids and stilbenes (Antolovich et al.,

2000).

Plant polyphenols are aromatic hydroxylated compounds, commonly found in vegetables, fruits and many food sources that form a significant portion of our diet, and

which are among the most potent and therapeutically useful bioactive substances.

Phenolic derivatives represent the largest group known as 'secondary plant products'

synthesized by higher plants, probably as a result of antioxidative strategies adapted in evolution by respirative organisms starting from precursors of cyanobacteria. Many of

these phenolic compounds are essential to plant life, e.g., by providing defense against microbial attacks and by making food unpalatable to herbivorous predators (Bennick, 2002).

Although a precise chemical definition may be given for plant phenolics, it

would inevitably include other structurally similar compounds such as the terpenoid sex

hormones. Therefore, an operational definition of metabolic origin is preferable, and

thus the plant phenols being regarded as those substances derived from the shikimate

pathway and phenyipropanoid metabolism, following the phosphoenolpyruvate

phenylalaflhlle -p cmnamate - 4-coumarate course, leading to chalcone, flavanone,

dihydrOflavono l, and anthocyanin as in Figure 2.2 (Robards and Antolovich, 1997).

0 II CH,—CHNH1---C—OH Pnjatn*e mmoia4yese

NH3 CHCH—Lo.

Cinnamic. acid

Jr

HOQ

C14=CH—C—OH

CH=CH—C—DH

!IC

acid CInnamate-4-hYdroxrIasaj( .1

+3 HOOC—CH3CO—SCaA

Strlfran8 sthas9

HO •CH=CHO1°H2O

sprMase 0Rasvratrc,

OR

OH

HO

GH

_JJOr

o 7-Hydroxycoumann chatcope

1J3kQfl £Om$e

H0(01.JO1Cr

wi o Ftiranocouniaijn Ravanon.

Figure 2.2: Schematic biochemical pathway of the synthesis of flavanone, resveratrol, and furanocoumarin.

Source: Robards and Antolovich (1997)

10

Significant antioxidant, antitumor, antiviral and antibiotic activities are

frequently reported for plant phenols. They have often been identified as active

principles of numerous folk herbal medicines. In recent years, the regular intake of fruits

and vegetables has been highly recommended, because the plant phenols and

polvphenolS they contain are thought to play important roles in long term health and

reduction in the risk of chronic and degenerative diseases. Recognition of the benefits

brought by these natural products to human health has encouraged the inclusion in

everyday diets of some typical plant-derived food and beverages, among the most

preferred examples being olive and vegetable oils, citrus and other fruit juices,

chocolate, tea, coffee and wine.

Over eight thousand naturally occurring phenolic compounds are known

(Balasundram et al., 2006). These substances contain at least one aromatic ring with one

or more attached —OH groups, in addition to other substituents (Bennick, 2002), and can

be divided into 15 major structural classes (Harborne and Simmonds, 1964). Major

classes of plant phenolics with 'the type of carbon skeleton, class name (example)'

format include: C6, simple phenols (resorcinol); C6-C1, phenolic acids (p-

hydroxybenzoic acid); C6-C2, acetophenones and phenylacetic acids; C6-C3,

hydroxycinnamic acids (caffeic acid); C6-C4, hydroxyanthraquinones (physcion); C6-

C2-C6, stilbenes (resveratrol); C6-C3-C6, flavonoids (quercetin); (C6-C3) 2, lignans

(matairesjno 1); (C6-C3 -C6) 2, biflavonoids (agathisfiavone); (C6-C3), lignins; (C6-C3-

C6), and condensed tannins (procyanidin) (Balasundram et al., 2006).

Tannins are considered to be polyphenolic metabolites of plants with a molecular

weight larger than 500 and with the ability to precipitate gelatin and other proteins from solution (Mehansho et al., 1987) and to give typical phenol reactions such as forming a

blue colour with FeCl3 (Khanbabaee and Van Ree, 2001). Tannins may be subdivided into hydrolyzable and condensed tannins' ; the former are esters of gallic acid (gallo - and ellag i .. taflhljfl5) while the latter are polyhydroxyflavan-3-ols, also known as proanthocyaflidins (Porter et al., 1989). (Bernick, 2002) defines hydrolyzable tannins

consisting of a polyhydric alcohol, such as glucose, to which gallic acid or its dimer

I

he.xaliydrodiphenic acid is linked in ester linkages, whereas the condensed tannins

contain the monomeric unit of a flavan-3-ol such as catechin or epicatechin that is linked

through C-C bonds. Basic plant phenolic structures with examples are shown in Figure

2.3.

HO OH

HO OH

Simple phenols (e.g., resorcinol) Hydroxybenzoic acids (e.g., gallic acid)

OH

OH

a0H

OH

HO 0

Flavanols (flavan-3 -ols) (e.g., (+)-

catechin)

Hydroxyciimamjc acids (e.g., p-Coumaric acid)

Figure 2.3: Basic plant phenolic structures with examples

Source: Bennick (2002)