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IIIIntroductionntroductionntroductionntroduction

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Chapter 1 Introduction

Progress being made in all spheres of life due to technological development, still people

choose to depend on natural products as far as their health are concerned (Menghani et al. 2011).

As a result there has been a renewed interest in plant based products (Samuelsson 2004). There are

evidences supporting the Rigveda (4500-1600 B.C) that in India plant products were used in

various system of treatment such as Unani, Siddha and Ayurveda (Akerele 1993). Also the

Neanderthals living nearly 60,000 years ago in, Iraq, used plants such as hollyhock which are still

being used as therapeutics around the world (Thomson 1978; Stockwell 1988). Other than

therapeutics plant products were used for various other purposes since long (Ahmad et al. 2006).

According to World Health Organization (WHO) about three-quarters of world’s

population currently use plant products and other forms of traditional medicine to treat diseases

(Trivedi 2004). It is estimated that there are 250,000 to 500,000 species of plants on Earth but a

small part maximum upto 10 % are used as foods by both humans and animal species and the rest

remains to be explored (Borris 1996). Therefore scientists have laid emphasis on extensive

research on the use of plant and plant products for the benefits of mankind (Iwu et al. 1999).

The major area for research in plants are usually the leaves, roots, barks, flowers, stems

etc., because biologically active phytochemicals are present in these areas but the plant parts such

as rind of the fruit, seed and fruit shell etc, which are considered as waste part (due to insufficient

knowledge) may contain some biologically active phytochemicals, having therapeutic properties

(Sivakumar and Venkataraman 2010). Nowadays many diseases are cured using plant products eg.

HIV (de Clercq 1995). Also the use of plant products as therapeutics would be economical in

underdeveloped countries, where there is very little access to modern medicines (Madikizela

2013).

It has only been in the past two decades or so that interest in higher plant as therapeutic

agents has been reawakened and the literature in this area is becoming substantial (Mistscher and

Rao 1984). The new chemical entities introduced as drugs during 1981-2002, were developed

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

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

designed from natural products (23%) (Newman et al. 2003; Butler 2004).

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Importance of plants as therapeutics:

There is evidence that plant based indigenous knowledge was passed down from generation

to generation almost in the entire world throughout history and has contributed to the development of

different traditional systems of medicines as stated above. The use of plants as medicine has

involved the isolation of morphine from opium in the early 19th century (Kinghorn 2001) and

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

which some are still in use (Newman et al. 2003; Butler 2004). The isoquinoline alkaloid emetine

obtained from the underground part of Cephaelis ipecacuanha, has been used as an amoebicidal

agent and also for the treatment of abscesses due to the spread of Escherichia histolytica infections.

Quinine an alkaloid which occurs naturally in the bark of Cinchona tree useful in the treatment of

malaria can also be used to relieve nocturnal leg cramps. Another drug Arteether isolated from

Artemisia annu, used traditionally in China as medicine is also used for the treatment of malaria (van

Agtmael et al. 1999; Graul 2001).

Higher plants have made important contributions in the areas beyond antiinfectives, such as

cancer therapies. Early examples include the antileukaemic alkaloids, vinblastine and vincristine

which are obtained from the Madagascan periwinkle (Samuelsson 2004). Other cancer therapeutic

agents include taxol, homoharringtonine and several derivatives of camptothein. Exatecan isolated

from Camptotheca acuminate, vinflunine a modified form of vinblastine are used as an anticancer

agent with improved efficacy (Okouneva et al. 2003; Butler 2004; Cragg and Newman 2004). A well

known benzylisoquinoline alkaloid, papaverine has been shown to have potent inhibitory effect on

the replication of several viruses including cytomegalovirus, measles and HIV (Turano et al. 1989).

Galanthamine a product isolated from Galanthus woronowii is used for the treatment of Alzheimer’s

disease (Heinrich and Teoh 2004; Pritilla et al. 2004). Tiotropium isolated from Atropa belladonna

is used for the treatment of chronic obstructive pulmonary disease (Dewick 2002; Mundy and

Kirkpatrick 2004; Frantz 2005). Morphine-6-glucuronide a metabolite of morphine from Papaver

somniferum is reported as an alternative to traditionally used morphine for relieving pain with fewer

side effects (Lotsch and Geisslinger 2001).

In India, the drugs like flavopiridol (Geysen et al. 2003) and forskolin (Lombardino and

Lowe III 2004) have found worldwide recognition. Flavopiridol, an analogue of natural product,

rohitukine, isolated from Dysoxylum binectariferum is responsible for anti-inflammatory and

immunomodulatory activity and inhibitory activity against a series of lung carcinoma cell lines

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(Sausville 1999). It also showed broad-spectrum in vivo activity against human tumour xenografts

in mice (Cragg and Newman 2005). Forskolin, a labdane diterpenoid isolated from the Indian herb,

Coleus forskohlii is an agent used for the treatment of congestive cardiomyopathy, glaucoma and

asthma (Singh et al. 1994; Urizar et al. 2002).

A very important plant Piper betel having therapeutic importance is widely used in India

and other parts of the world. Betel leaves contain phytochemicals such as betel phenol and

chavicol, tannin, sugar, vitamin-C, starch and diastase (Majumdar et al. 2002). Betel leaves also

possess activities like antidiabetic, antiulcer, antiplatelet aggregation, antifertility, cardiotonic,

antitumour and antimutagenic and wound healing property (Andhikari et al. 1990). Piper betel is

also used to treat alcoholism, bronchitis, asthma (Varier 1997) leprosy and dyspepsia,

antihistaminic (Schlebusch and Kern 1972). It has also been reported to have antimicrobial

activity, antioxidant property, anti-inflammatory (Ramji et al. 2002) radioprotective and

immunomodulatory property (Ambarta 1986). Considereing all these factors scientists all over the

world are doing extensive research on Piper betel plant for the isolation, extraction of the

phytoconstituents having therapeutic potential.

Use of plants as antimicrobials:

The utilization of natural products as antimicrobials arise from three organic sources-

microbes, plants and animals. It was the discovery of penicillin that led to later discoveries of

antibiotics such as streptomycin, aureomycin and chloromycetin. Though most of the clinically

used antibiotics are produced by soil microorganisms or fungi, higher plants become a source of

untrapped therapeutics which demands further exploration of plant products due to its beneficial

effect (Murray 1995). Examples of these are the bacteriostatic and fugicidals such as Lichens,

allinine in Allium sativum, berberines in Hydrastis canadensis (Trease 1972).

As found in herbal and folklore literature for medicine of India many ayurvedic plants

have been reported to have antimicrobial activity (Mohamed et al. 2010). The species tested were:

Andrographis paniculata (leaves), Eugenia jambolana (kernel), Cassia auriculata (flowers),

Urraya koenigii (leaves), Salvadora persica (stem) and Ipomoea batatas (leaves) against five

bacteria; two gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis) and

three gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas

aeruginosa) and also against two fungi (Candida albicans and Aspergillus niger).

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According to Joshi et al. (2011) aqueous ethanolic extract of four medicinal plants -

Ocimum sanctum, Eugenia caryophyllata, Achyranthes bidentata and Azadirachta indica showed

in vitro antibacterial activity against human pathogens such as Escherichia coli, Salmonella typhi,

Salmonella paratyphi, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa.

Other types of plant such as Aegle marmelos (fresh fruit pulp), Salmalia malabarica

(bark), Punica granatum (dried peel), Myristica fragrans (fruit), Holarrhena antidysenterica

(seed), Terminalia arjuna (bark), Picorhiza kurroa (leaves), Acacia catechu (bark), Acacia

nilotica (bark), Cichorium intybus (leaves), Embelia ribes (fruit), Solanum nigrum (seed), Carum

copticum (seed), Apium graveolens (leaves), Ocimum sanctum (seed), Peucedanum graveolens

(seed) and Butea monosperma (leaves), Zingiber officinale (fruit) have been used to treat

gastrointestinal diseases (Yamahara 1990; Wood 1998; Rani and Khullar 2004). A list of plants

with antimicrobial property is presented in Table 1.

Plants Compound Class

Allspice Eugenol Essential oil

Bael tree Essential oil Terpenoid

Barberry Berberine Alkaloid

Betel pepper Catechols, Eugenol Eeesntial oil

Caraway Tannins Polyphenols

Cashew Salicylic acids Polyphenols

Chili peppers Capsaicin Terpenoid

Coca Cocaine Alkaloid

Eucalyptus Tannins Polyphenols

Garlic Allicin Sulfated terpenoids

Grapefruit peel Asiatocside Terpenoid

Green tea Catechin Flavanoid

Henna Gallic acid Phenolic acid

Legume Isoflavone Flavone

Lemon balm Tannins Polyphenos

Licorice Glabarol Phenolic alcohol

Olive oil Hexanal Aldehyde

Papaya Latex Mixture

Peppermint Menthol Terpenoid

Poppy Opium Alkaloid

Rauvolfia Resperine Alkaloid

Apple Phloretin Flavanoid derivative

Turmeric Curcumin Terpenoids

Table 1: List of plants with different antimicrobials (Cowan 1999)

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Antimicrobial compounds and plant defense in response to microbial attack:

The antimicrobial properties of plants are due to the presence of certain secondary

metabolites. They are broadly classified into two categories: phytoantipicins and phytoalexins

(Mansfield 1983). Phytoanticipins are described as low molecular weight, antimicrobial

compounds that are present in plants before challenge by micro-organisms or are produced after

infection solely, from pre-existing precursors.

Whereas phytoalexins are antimicrobial compounds that are both synthesized and

accumulated in plants after exposure to microorganisms or abiotic agents (Paxton 1980; van Etten

et al. 1994). Phytoalexins represents induced defense mechanisms used by plants including lytic

enzymes such as chitinases and glucanases, oxidizing agents, cell wall lignifications and a number

of pathogenesis-related (PR) proteins and transcripts of unknown functions (Lamb et al. 1989;

Dixon and Lamb 1990). It is important to recognise that phytoalexin accumulation may be part of a

co-ordinated defense strategy (Mansfield 1983). Fig 1 represents the pathway of phytoalexin

biosynthesis.

Avirulent isolate Virulent isolate

Attempted growth on plant surface

Attempted penetration

Entry into plant

Recognition process eg interaction gene-for-gene interaction

Recognition No recognition

HR or Lesion formation No HR

Exposure to phytoanticipins No exposureSuppression of response

Phytoalexin biosynthesis Phytoalexin biosynthesis

Phytoalexin accumulation Degradation of inhibition Phytoalexin accumulation

Fig 1. Activity of antimicrobial from different compounds at different stages of the plant-pathogen interaction. Thedashed lines refer to the routes taken by virulent pathogens to avoid plant defense (Adopted from Mansfield 1983)

Restricted growth Degradation of inhibition Colonisation of challenged plant

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Plants have an almost limitless ability to synthesize aromatic substances, most of which

are phenols or their oxygen-substituted derivatives (Geissman 1963). Most are secondary

metabolites, of which at least 12,000 have been reported, a number estimated to be less than 10%

of the total (Schultes 1978). In many cases, these substances serve as plant defense mechanisms

against predation by microorganisms, insects and others. Useful antimicrobial phytochemicals can

be divided into several categories as summarized in Table 2.

Class Subclass Examples Mechanisms References

Phenolics Simple phenols Catechols Substrate deprivation Peres et al.1997

Epicatechin Membrane disruption Toda et al.1992

Phenolic acids Cinnamic acid Do Fernandez et al.1996

Quinones Hypercin Bind to adhesions Duke 1985

Flavanoids Chrysin Complex with cell wall Perret et al. 1995

Flavones Abyssinone Inactive enzymes Brinkworth et al. 1992

Flavonols Totarol Bind to proteins Kubo et al. 1993

Tannins Elligatnnin Metal ion

complexation

Scalbert 1991

Coumarins Perret et al. 1995

Terpenoids Capsaicin Membrane disruption Cichewicz and Thorpe

1996

Alkaloids Berberine Intercalate into cell

wall

Atta-ur-Rahman and

Chowdhury1995

Lectins, polypeptides Fabatin Blockviral fusion or

adsorption

Estevez-Braun et al.1994

Phenolics and polyphenols:

Certain simplest bioactive phytochemicals consist of a single substituted phenolic ring.

Cinnamic and caffeic acids are common representatives of a wide group of phenyl propane-derived

compounds which are in the highest oxidation state. The common herbs tarragon and thyme both

contain caffeic acid, which is effective against fungi (Duke 1985), viruses (Wild 1994) and also

bacteria (Brantner et al. 1996). Catechol and pyrogallol both are hydroxylated phenols, shown to

be toxic to microorganisms. Catechol has two 2OH groups and pyrogallol has three. The site(s)

and number of hydroxyl groups on the phenol group are thought to be related to their relative

toxicity to microorganisms, the increased hydroxylation results in increased toxicity (Geissman

1963). In addition, some authors have found that more highly oxidized phenols are more

Major groups of antimicrobials from plants:

Table 2. Major classes of antimicrobials from plants

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inhibitory. The mechanisms thought to be responsible for phenolic toxicity to microorganism

(Scalbert 1991).

Quinones:

Quinones are having aromatic rings with 2 ketone substitutions. They are ubiquitous in

nature and are highly reactive (Schmidt 1988). In addition of providing a source of stable free

radicals, quinones are known to form complex irreversibly with nucleophilic amino acids in

proteins often leading to inactivation of the protein and loss of function for which quinone is

known as a potential antimicrobial agent (Stern et al. 1996). Quinone commonly targets surface-

exposed adhesions of microbial cells, cell wall polypeptides and membrane-bound enzymes.

Quinones also render substrates unavailable to the microorganism. Kazmi et al. (1994) described

an anthraquinone from Cassia italica, which was bacteriostatic for Bacillus anthracis,

Corynebacterium pseudodiphthericum and Pseudomonas aeruginosa and bactericidal for

Pseudomonas pseudomalliae. Hypericin, an anthraquinone from Hypericum perforatum is reported

to have antimicrobial activity (Duke 1985).

Flavones, flavonoids and flavonols:

Flavones are phenolic structures containing one carbonyl group (as opposed to the two

carbonyls in quinones). Flavonoids are also hydroxylated phenolic substances but occur as a C6-

C3 unit linked to an aromatic ring (Fessenden and Fessenden 1982). They are synthesized by

plants in response to microbial infection (Dixon et al. 1983). The activity based on their ability to

complex with extracellular and soluble proteins and to act with bacterial cell walls, as of quinones.

More lipophilic flavonoids may also disrupt microbial membranes (Tsuchiya et al. 1994).

Catechins, the most reduced form of the C3 unit in flavonoid compounds found in green tea

inhibits Vibrio cholerae, Streptococcus mutans, Shigella dysenteriae, Streptococcus mutans

(Batista et al. 1994; Vijaya et al. 1995; Borris 1996). Flavonoid compounds such as glycyrrhizin

(from licorice) and chrysin exhibit inhibitory effects against HIV (Pengsuparp et al. 1995;

Critchfield et al. 1996 ).

.

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Tannins:

Tannin is a group of polymeric phenolic substances capable to tann leather or precipitating

gelatin from solution, a property known as astringency. They are found in almost every part of

higher plant taxa such as bark, wood, leaves, fruits and roots (Scalbert 1991). They are divided into

two groups hydrolyzable and condensed tannins (Geissman 1963). Tannins are involved in many

human physiological activities such as stimulation of phagocytic cells, host-mediated tumor

activity and anti-infective actions (Serafini et al. 1994; Stern et al. 1996). Their mode of

antimicrobial action is similar to that of quinones. Tannins directly inactivate microorganisms: low

tannin concentrations modify the morphology of germ tubes of Crinipellis perniciosa. Tannins

have been shown to have inhibitory effect against viral reverse transcriptases (Nonaka et al. 1990).

Condensed tannins find to bind cell walls of ruminal bacteria, preventing growth and protease

activity (Taylor et al. 1996).

Coumarins:

Coumarins are phenolic substances made of benzene rings (O’Kennedy and Thornes 1997).

They are responsible for the characteristic odor of hay. They are useful in antithrombotic (Thastrup

et al. 1985) anti-inflammatory (Piller 1975) and vasodilatory (Namba et al. 1988) activities. In

addition it shows antimicrobial properties against Candida albicans (Thornes 1997). Coumarins

have been found to stimulate macrophages, which could have an indirect negative effect on

infections (Casley-Smith and Casley-Smith 1997). They have been used to prevent recurrences of

cold sores caused by HSV-1 in humans (Berkada 1978). Hydroxycinnamic acids a coumarin are

inhibitory to gram-positive bacteria (Fernandez 1996).

Terpenoids and Essential Oils:

Terpenoids are compounds based on an isoprene structure. Examples of common

terpenoids are methanol and camphor (monoterpenes) and farnesol and artemisin (Amaral et al.

1998). The triterpenoid betulinic acid has been shown to inhibit HIV (Barre 1997). The mechanism

of action of terpenes is membrane disruption by the lipophilic compounds. Food scientists have

found that the terpenoids present in essential oils of plants to be useful in the control of Listeria

monocytogenes (Aureli 1992). A terpenoid constituent, capsaicin is bactericidal

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to Helicobacter pylori (Jones 1997). Another diterpene, aframodial, from a Cameroonian spice, has

a broad-spectrum antifungal activity (Ayafor 1994).

Alkaloids

Heterocyclic nitrogen compounds are called alkaloids. The first medically useful example

of an alkaloid was morphine, isolated in 1805 from Papaver somniferum (Fessenden and

Fessenden 1982). Diterpenoid alkaloids, commonly isolated from the plants of buttercup family,

are commonly found to have antimicrobial properties (Omulokoli et al. 1997). Berberine is an

important representative of the alkaloid group. It is potentially effective against trypanosomes and

plasmodia (Freiburghaus et al. 1996). Solamargine, a glycoalkaloid from the berries of Solanum

khasianum and other alkaloids may be useful against HIV infection as well as intestinal infections

associated with AIDS (McDevitt et al. 1996). The mechanism of action alkaloids such as berberine

and harmane is attributed to their ability to intercalate with DNA (Phillipson and O’Neill 1987)

Lectins and Polypeptides

Peptides are often positively charged ions and contain disulfide bonds, which are inhibitory

to microorganisms (Balls et al. 1982). Their mechanism of action is due to the formation of ion

channels in the microbial membrane or competitive inhibition of adhesion of microbial proteins to

host polysaccharide receptors (Terras et al. 1993). They also have inhibitory activity against HIV.

Thionins are toxic to yeasts and gram-negative and gram-positive bacteria (Fernandes de Caleya et

al. 1972). Thionins AX1 and AX2 from sugar beet are active against fungi (Kragh et al. 1995).

The larger lectin molecules, which include mannose-specific lectins from several plants, MAP30

from bitter melon, GAP31 from Gelonium multiflorum are inhibitory to viral proliferation (HIV,

cytomegalovirus) probably by inhibiting viral interaction with critical host cell components

(Favero et al. 1993).

Mixtures

Papaya yields a milky sap, often called latex, which is a complex mixture of chemicals.

Chief among them is papain, a well-known proteolytic enzyme. An alkaloid, carpaine, is also

present. Terpenoids are also present and may contribute to its antimicrobial properties (Thomson

1978). It was found that the latex is bacteriostatic against Bacillus subtilis, Enterobacter cloacae,

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Escherichia coli, Salmonella typhi, Staphylococcus aureus and Proteus vulgaris (Osato et al.

1993). Propolis a crude extract of the balsam tree, contains terpenoids, flavonoids, benzoic acids

esters, substituted phenolic acids and esters are active against adenovirus and poliovirus (Amoros

et al. 1992). Fig 2. Shows some of the basic structures of antimicrobials from plants.

Fig 2: Basic structures of some antimicrobials (Cowan 1999)

Simple phenols and phenolic acids

OHHO

COOH

3-(3,4-Dihydroxy-phenyl)-acrylic acid(caffeic acid)

HOOH

Benzene-1,2-diol(catechol)

OHOCH3

4-Allyl-2-methoxy-phenol(eugenol)

Quinones

O

O[1,4]Benzoquinone

O

OHOH

H3CH3C

OH OHO

OH OH

1,3,4,6,8,13-Hexahydroxy-10,11-dimethyl-phenanthro[1,10,9,8-opqra]perylene-7,14-dione( hypericin)

Simple phenols and phenolic acids

Quinones

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Flavones and flavonoids

O

O2-Phenyl-chromen-4-one

(flavone)

O

OH

OH

OH

HO

OH

2-(3,4-Dihydroxy-phenyl)-chroman-3,5,7-triol(catechin)

OOH

HO O

5,7-Dihydroxy-2-phenyl-chromen-4-one(chrysin)

Coumarins

O O

Chromen-2-one(coumarin)

O OHO

7-Hydroxy-chromen-2-one(7-hydroxy coumarin)

O

OH

O

O

4-Hydroxy-3-(3-oxo-1-phenyl-pentyl)-chromen-2-one

(warfarin)

Flavones and Flavonoids

Coumarins

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Terpenoids

CH3

OH

H3C CH3

2-Isopropyl-5-methyl-cyclohexanol(Menthol)

OHH

CH3H

CH3

O

O

10-Hydroxy-1,8a-dimethyl-1,2,4,4a,8a,9,10,10a-octahydro-phenanthrene-3,6-dione

(artemisin)

NH

OCH3

CH3

8-Methyl-non-6-enoic acidbenzylamide(capsaicin)

Alkaloids

N

OO

H3COOCH3

9,10-Dimethoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquino[3,2-a]isoquinolin-7-ylium

(berberine)

HN N

H3C

1-Methyl-9H--carboline(harmane)

Terpenoids

Alkaloids

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Scientific approach for the development of phytomedicine:

The first generation of plant drugs were usually simple botanicals employed in more or less

in crude forms such as tinctures, teas, powders and other herbal formulations which now serve as

the basis of novel therapeutic agent from plants (Samuelsson 2004). Following the industrial

revolution, a second generation of plant based drugs emerged based on scientific processing of the

plant extracts to isolate their active constituents (Iwu et al. 1999).

The process of using plants as a therapeutic agent is multi and interdisciplinary. At present,

scientists are investigating ways to standardize methods of extraction and in vitro antimicrobial

efficacy testing so that the search for new biologically active plant products could be more

systematic and interpretation of results would be facilitated (Schultes 1978).

Efficient collaborations with pharmacologists and medical doctors, plant pathologists and

microbiologists are required for the development of a lead compound into an exploitable product

(Baker et al. 1995; Das et al. 2010).

Experimental approach

Extraction methods:

Analysis of plant components follows stepwise process. Plants are collected randomly from

the geographical areas where the plants are found (Martin 1995). Initial screenings of plants for

possible antimicrobial activities involves aqueous or alcohol extractions followed by various

organic extraction methods (Green 2004).

Extraction with methanol or ethanol is more preferred for the identified aromatic or

saturated organic compounds from plants. For alcoholic extractions, plant parts are dried, ground

to a fine texture and then soaked in methanol or ethanol for extended periods. The slurry is then

filtered, after which it is dried under reduced pressure and redissolved in alcohol to a determined

concentration (Cichewicz and Thorpe 1996).

Occasionally tannins and terpenoids are found in the aqueous phase, but they are more

often obtained by treatment with less polar solvents (Salie et al. 1996). Table 3 includes a list of

solvents used for extraction of metabolites.

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Water Ethanol Methanol Chloroform Dichloromethanol Ether Acetone

Tannins Polyphenols Terpenoids Terpenoids Terpenoids Alkaloids Flavanols

Terpenoids Alkaloids Polyphenols Flavanols Terpenoids

Tannins Polyphenols

Crude products can then be used in disc diffusion and broth dilution assays to test for

antifungal and antibacterial properties and in a variety of assays to screen for antiviral activity

discussed, as follows:

In vitro antimicrobial susceptibility testing:

Initial screening of potential antibacterial and antifungal compounds from plants can be

performed with pure substances (Afolayan and Meyer1997) or crude extracts (Freiburghaus et al.

1996). The methods used for the two types of organisms are similar. The two most commonly used

screens to determine antimicrobial susceptibility are the broth dilution assay (Ayafor et al. 1994)

and the disc or agar well diffusion assay (Navarro et al. 1996). Agar overlay method (Mayr-

Harting et al. 1972) can also be used. In some cases, the inoculated plates or tubes are exposed to

UV light (Taylor et al. 1996) to screen for the presence of light sensitizing photochemicals.

In addition to these assays, antifungal phytochemicals can be analyzed by a spore

germination assay. Samples of plant extracts or pure compounds can be added to fungal spores

collected from solid cultures, placed on glass slides and incubated at an appropriate temperature

(usually 25°C) for 24 h. Slides are then fixed in lactophenol-cotton blue and observed

microscopically for spore germination (Rana et al. 1997).

After initial screening of phytochemicals, the activity may be compared with that of a

standard antibiotic. At this stage, more specific media can be used and MICs can be effectively

compared to those of a wider range of currently used antibiotics. The investigation of plant extracts

effective against methicillin-resistant Staphylococcus aureus provides an example of prospecting

for new compounds which may be particularly effective against infections that are currently

difficult to treat (Sato et al.1997).

Natural-products chemists use different purification techniques for active chemicals from

crude extracts by a variety of methods. For example, Petalostemumol, a flavanol from purple

prairie clover, was obtained from the ethanol extract by partitioning between ethyl acetate and

Table 3: List of solvents used for extraction of plant metabolites (Dai and Mumper 2010)

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water, followed by partitioning between n-hexane and 10% methanol. The methanol fraction was

chromatographed and eluted with toluene (Hunter and Hull 1993). Terpenoid lactones have been

obtained by successive extractions of dried bark with hexane, chloroform and methanol (Rao et al.

1993). The chemical structures of the purified material can then be analyzed. Techniques for

further chemical analysis include chromatography, high-performance liquid chromatography,

bioautography, radioimmunoassay and newer tools such as fast atom bombardment mass

spectrometry, tandem mass spectroscopy (Rinehart et al. 1990), capillary zone electrophoresis,

nuclear magnetic resonance spectroscopy, and X-ray cystallography (Borris et al. 1996).

Modern course of action:

As evident from the above discussion, nature is the best combinatorial chemist and possibly

has answers to all problems of mankind. Till now, natural products compounds discovered from

medicinal plants have provided numerous clinically useful antimicrobials as therapeutics. The

diverse genetic resources enriched with traditional knowledge available form the strong basis for

bioprospecting. Proper utilization of these resources and tools in bioprospecting will certainly help

in discovering novel lead molecules from plants by employing modern drug discovery techniques

and the coordinated efforts of various disciplines. Scientists from divergent fields are investigating

plants anew with an eye to their antimicrobial usefulness. A sense of urgency accompanies the

search as the pace of species extinction continues. Laboratories of the world have found literally

thousands of phytochemicals which have in vitro inhibitory effects on all types of microorganisms.

More of these compounds should be subjected to animal and human studies to determine their

effectiveness. It would be advantageous to standardize methods of extraction and in vitro testing so

that the search could be more systematic and interpretation of results would be facilitated. Also,

alternative mechanisms of infection prevention and treatment should be included in initial activity

screenings. Attention to these issues could give rise to a new era of chemotherapeutic treatment of

infection by using plant-derived principals.

To comply with the above, the present investigation was primarily undertaken with an

objective for searching of novel antimicrobials from plant wastes having broad antimicrobial

activity. An outline of the approach for investigating the problem were:

• Screening of plant residue for antimicrobial activity.

• Extraction, isolation and purification of the compound.

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• Screening for phytochemical.

• Physical and chemical characterization of the principal compound.

• Identification of the principal compound.

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Afolayan A J, Meyer J J M (1997). The antimicrobial activity of 3,5,7-trihydroxyflavone isolatedfrom the shoots of Helichrysum aureonitens. Journal of Ethnopharmacology. 57: 177–181.

Ahmad I, Aqil F, Owais M (2006). Modern Phytomedicine. WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim.

Akerele O (1993). Summary of WHO guidelines for the assessment of herbal medicines. HerbalGram. 28: 13-19.

Amaral J A, Ekins A, Richards S R, Knowles R (1998). Effect of selected monoterpenes onmethane oxidation, denitrification and aerobic metabolism by bacteria in pure culture.Applied and Environmental Microbiology. 64: 520– 525.

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