1.1. Assam and present scope of study:

1.2. Antioxidant

1.3. Antimicrobial

1.4 Phytochemistry

References

1

1. Introduction

1.1. Assam and present scope of study:

The North-Eastern India is constituted by eight states (Assam, Arunachal Pradesh,

Manipur, Meghalaya, Mizoram, Nagaland, Tripura and Sikkim) and seven states excluding

seven sisters

sq km, which is 25% of the tota

geographical area supporting 50% of the flora (8000 species), of which 31.58% (2526

species) is endemic (Yumnam 2008) and forms a significant portion of both the Himalaya

and Indo-Burma biodiversity hotspots. North-East India has a relatively complex geography

(e.g. altitude ranging from 100 to > 7000 m above sea level) and habitat diversity from

tropical to alpine (Pawar et al., 2007). The region is also the abode of approximately 225

tribes, out of 450 in India (Chatterjee et al., 2011).

Assam consists of Karbi Anglong & north cachar as hilly terrain of varying altitude and

Brahmaputra & Barak Valley plains. In Assam, there are as many as 23 tribal communities,

which constitute 12.82 per cent of the total population of the state (Ali et al., 2003). About

80% population of Assam reside in remote areas, and is totally dependent on plants for their

day-to-day life (Singh et al., 2011). These tribes have their unique knowledge of medicinal

plants to combat various diseases. Availability of diverse medicinal plants and their

knowledge of use make Assam an important study area.

Human learnt the use of herbs as medicines and for other purposes since time

immemorial. But, the depth was unknown till science tried to explore the underlying

phenomena. Early human was acquainted with the value of the herbs, but the modern science

unfurled the complex envelop and showed the humanity, the actual agent.

The discovery of novel drugs from nature is also important because many isolated

molecules are quite complex and would not be obtained by a simple synthetic approach. In

2

some cases, the isolated lead compounds may not be potent enough to be drugs in their own

right, but they can serve as pharmacophores for chemical modification and drug design.

Ethno botany provides the tentative feasible path to move forward the avenue of modern drug

discovery. Moreover, since the isolation of active compound is guided by ethnobotany i.e. the

plant source has been in use by human for long time, there is less probability for the

compound to be toxic. Assam has diverse ethnic group and hence a diversity of knowledge,

particularly the use of different plants. From the information provided by ethnobotany,

phytochemistry can make a sure shot and can lead a pathway within its possibility to utilise

the natural resources to its best for the mankind. Therefore, there is a growing interest mostly

in plant derived compounds particularly from the ethnomedicinal plants and their potential

use in food and pharmaceutical industries.

Search of antioxidant and antimicrobial is highly demaded in the present day situation.

Antioxidant phytochemical has gained enough thrust due to free radical imbalance in the

metabolic system due to different endogenous and exogenous factors appeared, leading to the

generation of disease and disease complexity. For example, physical stress results in the

generation of free radicals in the body, therefore, Indian soldiers to whom physical stress is a

routine matter and reaches its peak during operations are more likely to undergo oxidative

stress. This hinders the competence of the Indian defence forces. While, antimicrobial

phytochemicals are need of the hour, due to the emergence of drug resistance and to find a

less toxic and effective antimicrobial substitute. In both the case phytochemical study is

necessary not only to uncover a novel compound but also to standardise and monitor herbal

formulation in use, to maintain quality control. Therefore, the present study was undertaken.

3

1.2: Antioxidant

concentrations compared to that of an oxidisable substrate, significantly delays or inhibits the

et al., 1995).

Pokorny J. (2007) described types of antioxidants as follows:

1. Inhibitors of free-radical oxidation reactions called preventive antioxidants,

which inhibit the formation of free lipid radicals (Fig. 1A); the most important

substances of the type are compounds inhibiting the decomposition of lipid

hydroperoxides into free radicals (Fig. 1B), as this is the most frequent source

of free radicals in the period of initiation.

2. Inhibitors interrupting the propagation of the autoxidation chain reaction (the

proper antioxidants) are called chain-breaking antioxidants (Fig. 1C).

3. Singlet oxygen quenchers (e.g. carotenes, especially lycopene).

4. Synergists of proper antioxidants, i.e. substances not efficient as antioxidants

when applied alone, but increasing the activity of chain-breaking antioxidants

in a mixture; citric acid is the best-known example.

5. Reducing agents, such as thiols or sulfides (thioethers), which convert

hydroperoxides into stable components in a non-radical way.

6. Metal chelators, which convert metal pro-oxidants, especially iron or copper

derivatives, into stable products. If not chelated, heavy metals promote the

decomposition of lipid hydroperoxides into free radicals (Fig. 1B). Quercetin,

tannins, and phytates are good examples of efficient metal chelators.

4

7. Inhibition of pro-oxidative enzymes (especially lipoxygenases).

R-H R + H [1A]

ROOH RO + OH [1B]

R + O2 RO2

RO2 + RH RO2H + H [1C]

R , RO , RO2 stable products

Figure 1.1: Mechanism of lipid oxidation

In recent years, a substantial body of evidence has developed supporting a key role for

free radicals in many fundamental cellular reactions and suggesting that oxidative stress

might be important in the pathophysiology of common diseases including atherosclerosis,

chronic renal failure, and diabetes mellitus. Reactive oxygen species (ROS) includes

superoxide radicals, hydroxyl radicals, and hydrogen peroxide, are often generated as

byproducts of biological reactions. They are known to cause oxidative damage to living

systems, they may also cause great damage to cell membranes and DNA, inducing oxidation

that causes membrane lipid peroxidation, decreased membrane fluidity, and DNA mutations

leading to cancer, atherosclerosis, hypertension and arthritis, cirrhosis, diabetes, heart disease,

inflammatory conditions and other diseases. (Cerutti 1991; Frenkel 1992; Harman 2006;

Dreher et al., 1996; Diaz et al., 1997; Hecht 1999; Finkel et al., 2000), and neuronal

Christen 2000) Lang et al.,

1998) as well as being involved in the process of aging (Ames et al., 1993; Ashok et al.,

1999). In a review, Young (2001) described mechanisms of free radical formation in the

5

body, the consequences of free radical induced tissue damage, and the function of antioxidant

defence systems in health and disease.

Halliwell B winner of Lifetime Achievement Award, 2008 from the Society for Free

Radical Biology and Medicine (SFRBM)) mentioned in a review (2009) that, antioxidants

seem, in general, better at decreasing oxidative damage in rodent models of disease, and

simultaneously beneficially affecting the disease progression. He exemplified two research

work where it has been shown that antioxidants are beneficial in murine models of

amyotrophic lateral sclerosis, but do not seem to help human patients. He concluded with that

antioxidants could be beneficial, if we could find ones that actually work in humans, getting

to the correct sites of action and diminishing oxidative damage at those sites.

Plant-derived antioxidants are capable to delay or prevent the onset of degenerative

diseases because of their redox properties (Govindarajan et al., 2005.). Considerable attention

towards them as potentially protective factors against cancer and heart diseases is due to their

antioxidant potency and availability in a wide range of commonly consumed foods of plant

origin (Kamei et al., 1995; Rice-Evans 2001; Muselik et al., 2007).

Polyphenolic compounds exhibit profound antioxidant properties. This type of compounds is

commonly found in both edible and inedible plants, and they have been reported to have

multiple biological effects, including antioxidant activity. Wojdylo (2007) described that

crude extracts of herbs and spices, and other plant materials rich in phenolics are of

increasing interest in the food industry because they retard oxidative degradation of lipids and

thereby improve the quality and nutritional value of food.

Flavonoids are another important class of antioxidant compounds. The basic flavonoids

structure is the flavan nucleus, which consists of 15 carbon atoms arranged in three rings. The

differences in the structure and substitution influence the phenoxyl radical stability and

thereby the antioxidant properties of the flavonoids.

6

Few examples from various types of flavonoids are presented below:

1. Chemical structure of flavonols (quercetin, kaempferol, myricetin, isorhamnetin)

O

OH

HO

OH

R1

OH

R2

O

2. Chemical structure of flavones (luteolin, apigenin)

O

OH

HO

OH

R1

O

3. Chemical structure of flavanones (eriodictyol, hesperetin, naringenin)

O

OH

HO

R2

O

R1

4. Structure of flavan-3-ols (catechins, epicatechins, theaflavins, and thearubigins)

O

OH

HO

OH

OH

OH

R

Flavonol R1 R2

Quercetin OH H

Kaempferol H H

Myricetin OH OH

Isorhamnetin OMe H

Flavone R1

Apigenin H

Luteolin OH

Flavonone R1 R2

Eriodictyol OH OH

Hesperetin OH OMe

Naringenin H OH

Catechins R

(+)-Catechin H

(+)-Gallocatechin OH

7

O

OH

HO

OH

OH

OR2

R1

OH

OH

OH

O

Gallate

Epicatechins R1 R2

(-)-Epicatechin (EC) H H

(-)-Epigallocatechin (EGC) OH H

(-)-Epicatechin-3-gallate (ECG) H Gallate

(-)-Epigallocatechin-3-gallate (EGCG) OH Gallate

5. Chemical structure of anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin,

peonidin, and petunidin)

O

OH

HO

OH

R1

OH

R2

Moyer et al., (2002) analyzed fruits from 107 genotypes of Vaccinium L., Rubus L., and

Ribes L., for total anthocyanins, total phenolics and antioxidant capacities as determined by

ORAC and FRAP. They demonstrated the wide diversity of phytochemical levels and

antioxidant capacities within and across these three genera of small fruit.

Anthocyanidin R1 R2

Cyanidin OH H

Delphinidin OH OH

Malvidin OMe OMe

Pelargonidin H H

Petunidin OMe OH

Peonidin OMe H

8

Ou et al., (2002) studied a total of 927 freeze-dried vegetable samples, including 111

white cabbages, 59 carrots, 51 snap beans, 57 cauliflower, 33 white onions, 48 purple onions,

130 broccoli, 169 tomatoes, 25 beets, 88 peas, 88 spinach, 18 red peppers, and 50 green

peppers, were analyzed using the ORAC and FRAP methods. The data show that the ORAC

and FRAP values of vegetable are not only dependent on species, but also highly dependent

on geographical origin and harvest time.

The above points clearly indicate the importance of research on antioxidant

phytochemicals to cope with the imbalance between free radical generation and self-defence

system of the body.

1.3 Antimicrobial:

Microbial diseases are ubiquitous throughout the tropical and subtropical regions of the

world. They are of serious health concern, not only because they are life-threatening but also

due to the extreme discomfort, stress, pain and ugliness they cause. The incidence of such

infections rises alarmingly as reported from various parts of India as well as other countries

(Jain et al., 2008; Kannan et al., 2006; Nweze, 2010; Prasad et al., 2005; Straten et al., 2003).

The hot and humid climate of Assam is highly conducive for the growth of many pathogenic

microorganisms. Investigations on the occurrence of microbial diseases in some parts of

Northeast India revealed the prevalence of different types of pathogenic microbes, like

dermatophytes, yeasts and bacteria (Das, 2003; Devi and Zamzachin, 2006; Jaiswal, 2002;

Sharma and Borthakur, 2007). It is a serious health problem of the common people,

particularly those residing in rural area. The Indian soldiers working in hostile environment in

remote border areas, particularly during operations are more prone to such ailments,

hampering the efficiency of the defence forces.

9

Many of the synthetic drugs presently used in treatment of microbial diseases have

undesirable side effect, are ineffective against new or re-emerging microbes, or lead to the

rapid development of resistance. Increase incidence of microbial diseases and resistance to

microbial agents has important implications for morbidity, mortality and health care costs all

over the world. These adverse effects together with the emergence of multidrug-resistant

strains of various pathogenic microbes call attention to the need of development of new

generation of antimicrobial agents. Substantial attention has been focussed on developing

novel and effective antimicrobial options for the treatment of infections and to prevent the

emergence and spread of newly emerged microorganisms.

In this context, ethnopharmacolgy hold a paramount importance, to cope with various

ailments using natural resources from the traditional knowledge. Medicinal plants constitute

an excellent source of new drugs, mainly considering that the molecular diversity of plant

resources is much higher than that derived from chemical syntheses. Traditionally used

medicinal plants produce a variety of compounds of known therapeutic properties. This fact

has stimulated investigation of the antimicrobial activity of different plant extracts, fraction

and compounds in order to promote the development of new pharmaceuticals that can control

many diseases including microbial diseases. As a consequence, many research groups have

concentrated efforts on the assessment of the antimicrobial properties of plant based products,

aiming to detect new antimicrobial compounds. In recent years, antimicrobial properties of

medicinal plants are increasingly reported different parts of the country as well as overseas.

It is a well-known fact that one of the most famous natural product discoveries is that of

penicillin derived from a fungus (microorganism) Penicillium notatum discovered by A.

Fleming in 1929. A counter current extractive separation technique that produced penicillin

in high yields was required for the in vivo experimentation that ultimately saved countless

lives and won Chain and Florey (together with Fleming) the 1945 Nobel Prize in Physiology

10

and Medicine. This discovery led to the re-isolation and clinical studies by Chain, Florey and

co-workers in the early 1940s and commercialization of synthetic penicillin, which ultimately

revolutionized drug discovery research.

After publication of the first clinical data on penicillin G in between 1942 1944 there

was a worldwide endeavour to discover new antibiotics from microorganisms and bioactive

natural products. During the 1970s, advancedment in screening methods, the production of

bacterial strains supersensitive to -lactams, tests for the inhibition of -lactamases and

specificity for sulphur containing metabolites resulted in the discovery of novel antibiotic

structural classes (norcardicins, carbapenems and monobactams) including the isolation of

the antibiotics, norcardicin, imipenem and aztreonam, respectively (Dias et al., 2012).

NO N

S

O OH

H

O

OH2N

CO2HN

H

NO

O

NOH

H

OH

CO2H

H

Penicillin Norcardicin

N

OHH

COOH

SNH

NH

O

H

N

CH3

O SO3H

NH

O

NO

NS

H2N

O

HO

Imipenem Aztreonam

The use of plants as medicines involves isolation of active compounds. This began with

the isolation of morphine from opium in the early 19th century (Huxtable and Schwarz,

11

2001). Of the several hundred thousand plant species around the globe, only a small

proportion has so far been investigated from phytochemical and pharmacological point of

view. The ultimate success of an investigation leading to discovery of bioactive compounds

depends on selection of appropriate plant material, suitable biological assays and chemical

screening methods. Biological and chemical screenings are complementary approaches for

the rapid detection and isolation of interesting new bioactive plant compounds. Bioassay-

guided fractionation has been used successfully for the discovery of many bioactive

compounds like antifungal, antibacterial etc (Hostettmann, 1999). Various chromatographic,

spectroscopic and chemical methods were employed for monitoring the fractionation,

isolation and identification of bioactive components.

The majority of natural products derived from medicinal plants are secondary

metabolites viz., terpenoids, steroids, cardenolides, quinine lignans, flavonoids or alkaloids.

The active molecules isolated from traditional medicinal plants might provide valuable drugs.

Many active compounds from traditional medicine sources could serve as good scaffolds for

rational drug design. Challenges in bioassay screening and identification of active

components remain an important issue in the future of drug discovery from medicinal plants.

The emergence of resistant strains due to unjustified use of drug is another serious problem.

Hence, there is an urgent need for the development of new antimicrobial drugs, which can

take care of disease, efficacy and cost.

1.4 Phytochemistry

Since the dawn of medicine, compounds derived from animals, plants, and microbes

have been used as therapeutic agents. Carlson E. E. (2010) Indicated that, although about

200,000 natural compounds are currently known but prior to the 1800s, the active

constituents of most medicines, which were generally plant-based, were unknown. Isolation

12

of the well-known drug, morphine, from the opium poppy occurred in 1817. Although the

therapeutic value of natural products is clear, use of these compounds presents a number of

nd it

has become clear that renewed interest in natural products will be essential to the future of

both biological studies and drug development.

The biosynthesis and breakdown of proteins, fats, nucleic acids and carbohydrates,

which are essential to all living organisms, is known as primary metabolism with the

. Secondary

metabolites are generally not essential for the growth, development or reproduction of an

organism and are produced either as a result of the organism adapting to its surrounding

environment or are produced to act as a possible defence mechanism against predators to

assist in the survival of the organism. (Dias et al., 2012 and Croteau et al., 2000).

Traditional medicinal practices have formed the basis of most of the early medicines

followed by subsequent clinical, pharmacological and chemical studies. Few early examples

noted by Dias et al., (2012) are synthesis of the anti-inflammatory agent, acetylsalicyclic acid

(aspirin) derived from the natural product, salicin isolated from the bark of the willow tree

Salix alba L. Investigation of Papaver somniferum L. (opium poppy) resulted in the isolation

of several alkaloids including morphine, a commercially important drug, first reported in

1803. Digitalis purpurea L. (foxglove) had been traced back to Europe in the 10th century

but it was not until the 1700s that the active constituent digitoxin, was found to enhance

cardiac conduction. The anti-malarial drug quinine was isolated from the bark of Cinchona

succirubra Pav. Ex Klotsch, used for centuries for the treatment of malaria, fever,

indigestion, mouth and throat diseases and cancer.

13

O

HOO

OCH3

O

OHHO

HO

O

OH

HO HO

O

HO

NCH3

H

1 2 3

O O

O O

O O

O

O

OH

HO

H

OH

H H

H

OH

H

OH

H

N

HONH

H3CO

4 5

Acetylsalicyclic acid (1), Salicin (2), Morphine (3), Digitoxin (4), Quinine (5)

The most widely used breast cancer drug is paclitaxel (Taxol), isolated from the bark of

Taxus brevifolia (Pacific Yew). In 1962 the United States Department of Agriculture (USDA)

first collected the bark as part of their exploratory plant screening program at the National

Cancer Institute NCI). The bark from about three mature 100 year old trees is required to

provide 1 gram of Taxol, given that a course of treatment may need 2 grams of the drug.

Current demand for Taxol is in the region of 100 200 kg per annum (i.e., 50,000 treatments

per year) and is now produced synthetically. Taxol is present in limited quantities from

natural sources, its synthesis (though challenging and expensive) has been achieved. Baccatin

III present in much higher quantities and readily available from the needles of T. brevifolia

and associated derivatives is an example of a structural analogue that can be efficiently

transformed into Taxol.

14

OH

OOH

O

OO O

H

O

O

O

OH

NHO

O

O

OH

OOH

O

HOO O

H

O

O

O

O

Taxol Baccatin

A plant based antimalarial drug isolated from the Chinese plant Artemisia annua

(Asteracae) was used. (Klayman 1985) However, the use of Artemisinin, an endoperoxide

sesquiterpene lactone is somewhat limited because of its relatively high cost , limited

production to GMP standards and reports of toxicity. Derivatives of Artemisinin such as

artemether, arteether, artesunate, and dihydroartemisinin are prepared semi-synthetically

from purified extract of Artemisia annua. (Haynes, 2001).

O

O O

H

H

O

O

O

O O

H

H

O

OCH2CH3

Artemisinin Arteether

Grandisines A and B are two indole alkaloids which were isolated from the leaves of

the Australian rainforest tree, Elaeocarpus grandis (Figure/////). Grandisine A contains a

unique tetracyclic skeleton, while Grandisine B possesses an unusual combination of

isoquinuclidinone and indolizidine ring systems. Both Grandisines A and B are potential

leads for analgesic agents. Galantamine hydrobromide is an Amaryllidaceae alkaloid

obtained from the plant Galanthus nivalis and has been used traditionally in Turkey and

15

Apomorphine is a derivative of morphine isolated from the poppy (P. somniferum) and is

. Tubocaurarine isolated from the climbing plant,

Chondrodendron tomentosum (Menispermaceae) is one of the active constituents used as a

muscle relaxant in surgical operations, reducing the need for deep anesthesia. The limited

availability of tubocurarine has led to the development of a series of synthetic analogues

which are now preferred over the natural product.

O

O

NH

H

H

O

N

N

O

1 2

N

HO

HO H

3

NO

HCH3

H

H3CO

OH

O

Me2NOH

OCH3

H

4

Grandisine A (1), Grandisine B (2), apomorphine (3) and tubocaurarine (4).

Written records of the use of plants as medicinal agents date back thousands of years. Ancient

system of medicine like Sidhdha and Unani are based on the use of plants. The oldest records

come from Mesopotamia and date from about 2600 BC. However, it was not until the early

that among the first active principles to be isolated were strychnine, morphine, atropine, and

colchicine. In 1826, this resulted in E. Merck producing the first commercially pure natural

product, morphine.

16

O

O

CH2OH

MeN

O

OMe

OMe

MeO

MeO

H

NH

O

Atropine Colchicine

N

N

O

H

H

HH

O

H

O

HO

NMeH H

HO

Strychnine Morphine

Phillipson (2001) mentioned that 6 out of the top 20 pharmaceutical prescription drugs

dispensed in 1996 were natural products and that over 50% of the top 20 drugs could be

linked to natural product research. In recent years, the development of sensitive biological

testing systems, mainly by industry, has led to the procedure of high throughput screening.

Such screens are carried out robotically and it is possible for a pharmaceutical or

biotechnological company to run 50,000 biological tests per day. The isolation and use of

natural products such as digitoxin, morphine and quinine has resulted in replacing the plant

extracts used with single chemical entities. There is a basic supposition that any plant

possessing clinical effectiveness must contain an active principle that can completely replace

the plant extract, however Phillipson (2001) pointed out that this may not necessarily be true.

The use of plants as medicines has involved the isolation of active compounds, beginning

with the isolation of morphine from opium in the early 19th century (Kinghorn A. D. 2001)

and subsequently led to the isolation of early drugs such as cocaine, codeine, digitoxin and

17

quinine, of which some are still in use (Butler M. S. 2004, Newman et al., 2000). Isolation

and characterization of pharmacologically active compounds from medicinal plants continue

today. More recently, drug discovery techniques have been applied to the standardization of

herbal medicines, to elucidate analytical marker compounds.

According to the opinion of Newman et al., (2003), 61% of the 877 small-molecule

(New Chemical Entities) introduced as drugs worldwide during 1981 2002 was inspired by

natural products. These include, natural products (6%), natural products derivatives (27%),

synthetic compounds with natural products-derived pharmacophore (5%) and synthetic

compounds designed from natural products (natural products mimic, 23%).

Keeping a view of the above facts, the present study makes an effort towards search of

antioxidant and antimicrobial compound, with the following objectives:

1. Evaluation of antioxidant and antimicrobial activity of certain medicinal plants of

Assam.

2. Bioassay guided fractionation to find active components.

3. Isolation and identification of the active compound(s).

18

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