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CCCChapterhapterhapterhapter 1111
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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Chapter 1 Introduction
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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Chapter 1 Introduction
(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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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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Chapter 1 Introduction
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)
15 |
Chapter 1 Introduction
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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Chapter 1 Introduction
• Screening for phytochemical.
• Physical and chemical characterization of the principal compound.
• Identification of the principal compound.
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Chapter 1 Introduction
References:Adhikari P, Banerji J, Choundhuri D, Das A K, Deb C C, Mukherjee P, Chatterjee S R
(1990). Effect of oral administration of stalk of leaves of Piper betel Linn. on ostrous cycleand its antifertility activity in rats. Indian Journal of Physiology and Allied Science. 44: 116-123.
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.
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