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INTRODUCTION

Chapter – I Introduction

1Studies on development of medicinally important plants and their antimicrobial properties

Chapter I

INTRODUCTION

Traditional medicine has been practiced for many centuries by a substantial

proportion of the population for the treatment of various human ailments. Over the

period the interest in the study of medicinal plants as a source of

pharmacologically active compounds has increased worldwide.

It is recognized that in some developing countries, plants are the main medicinal

source to treat infectious diseases. Plant extracts represent a continuous effort to

find new compounds with the potential to act against multi-resistant bacteria

(Cowan, 1999). Approximately 20% of the plants found in the world have been

submitted to pharmacological or biological test, and a substantial number of new

antibiotics introduced in the market are obtained from natural or semi-synthetic

resources (Marino et al., 1999; Paulo et al., 2010).

Antibiotics are an essential part for combating harmful bacterial infections in vivo.

During the last decade infectious diseases have played a significant role in the

death of millions around the world, especially in developing countries like India.

Because of the mutagenic nature of bacterial DNA, the rapid multiplication of

bacterial cells, and the constant transformation of bacterial cells due to plasmid

exchange and uptake, pathogenic bacteria continue to develop antimicrobial

resistance and thus rendering certain antibiotics useless. An increased number of

pathogens have also developed resistance to multiple antibiotics (Multiple Drug

Resistance), threatening to develop complete immunity against all antimicrobial

agents and, therefore, be untreatable (Cohen, 1992; Gold and Moellering, 1996).

Thus, the search for novel antimicrobial agents is of the utmost importance. The

indiscriminate use has led to an alarming increase in antibiotic resistance among

microorganisms thus necessitating the need for development of novel

antimicrobials (Hart and Karriuri, 1998; Burt, 2004).



Plants have been used for centuries as remedy for human diseases because they

contain components of therapeutic values. They are natural sources of

antimicrobial agents primarily because of the large biodiversity of such organisms

and the relatively large quantity of metabolites that can be extracted from them

(Copp, 2003; Yu R et al., 2011). The acceptance of these traditional medicines as

an alternative form of health care has led researchers to investigate the

antimicrobial activity of medicinal plants (Zaika, 1998; Singh et al., 2005).

Medicinal plants are relied upon by 80% of the world's population, and in India,

the use of plants as therapeutic agents remains an important component of the

traditional medicinal system (Zafar, 1994; Zavala et al., 1997). A lot of work has

been done which aims at knowing the different antimicrobial and phytochemical

constituents of medicinal plants and using them for the treatment of microbial

infections as possible alternatives to chemically synthetic drugs to which many

infectious microorganisms have become resistant (Dorman and Deans 2000;

Scazzocchio et al., 2001).

In recent times, the search for potent antibacterial agents has shifted to plants.

However, the major part of the search has focused mainly on higher plants

(Mothana and Lindequist, 2005). Most plants are medicinally useful in treating

diseases in the body and in most cases; the antimicrobial efficacy value attributed

to some plants is beyond belief. Conservative estimates suggest that about 10% of

all flowering plants on earth have at one time, been used by local communities

through out the world but only 1% have gained recognition by modern scientists

(Yu X et al., 2011).

A number of plants have been documented for their biological and antimicrobial

properties (Aswal et al., 1984; Ahmad et al., 1998; Ahmed and Beg, 2001; Arora

and Kaur, 2007). In an effort to expand the spectrum of antimicrobial agents from

natural resources, 3 medicinal plants have been selected based on their traditional

usage in India to access their antibacterial and antifungal potential. These Plants

are Acacia catechu, Cassia fistula and Bauhinia purpurea. Their Classification



and medicinal uses are as follows:

Acacia catechu (L.) Willd. Var., Oliv. lies in the family Fabaceae: Mimosoideae

and is commonly known as catechu, cachou and black cutch. It is a deciduous,

thorny tree which grows up to 15 m in height and is found in Asia, China, India

and the Indian Ocean area. A. catechu is a medicinal plant used for varied

purposes (Yadav, 2001). The extract of this plant is used to treat sore throats and

diarrhoea. The bark of this plant is strong antioxidant, astringent, anti-

inflammatory, anti-bacterial and antifungal in nature. It is useful in passive

diarrhoea, high blood pressure, dysentery, colitis, gastric problems, bronchial

asthma, cough, leucorrhoea and leprosy (Seigler, 2003). It is used as mouthwash

for mouth, gum, sore throat, gingivitis, dental and oral infections. The heartwood

is used to yield concentrated aqueous extract i.e. cutch which is astringent, cooling

and digestive. It is useful in cough, ulcers, boils and eruptions of the skin.

Decoction of the bark is given internally in case of leprosy. Acacia spp. produces

gum exudates, traditionally called gum Arabic or gum Acacia, which are widely

used in the food industry such as emulsifiers, adhesives, and stabilizers

(Kishimoto et al., 2006). There is also use of Acacia gum in the chronic renal

failure. Acacia gum is a bifidogenic dietary fibre with high digestive tolerance in

healthy humans and believed to benefit intestinal health (Garcia et al., 2006). The

effects of increasing doses of GUM were compared to those (SUC) on stool

output, concentration of the main bacterial populations in stools, and occurrence

and severity of intestinal symptoms (flatulence, bloating, abdominal cramps and

diarrhoea). Ingestion of GUM 10 and 15 gm/day for 10 days increased total lactic

acid producing bacteria and bifidobacteriae counts in stools without affecting total

anaerobic and aerobic counts. The magnitude of this selective effect was greater in

subjects with a low initial faecal concentration of bifidobacteria. Faecal

digestibility 95% and its caloric value were around estimated to range between 5.5

and 7.7 KJ/gm. In addition, stool weight increased 30% because of greater faecal

water content (Singhal and Joshi, 1984; Khan et al., 2006).



Cassia fistula (L.) lies in the family Fabaceae: Caesalpinioideae and is

commonly known as Golden shower tree or Golden shower Cassia. Other names

also include Indian laburnum, golden shower and drumstick tree. It is a medium-

sized tree growing to 10-20 m tall with fast growth. The leaves are deciduous or

semi evergreen, 15-60 cm long, the fruit is a legume, is 30-60 cm long and

containing several seeds. It is found throughout India, Ceylon, Malaya, China,

South Africa and the West Indies.

C. fistula is a medicinal plant used for varied purposes. This plant is also used in

haematemesis, pruritus, leucoderma, diabetes and many other ailments (Pari and

Latha, 2002; Wang et al., 2007). The antifungal and antibacterial activities of this

plant have already been reported (Hemlata and Kalidhar, 1994; Chow et al., 1998;

Sharma et al., 2000). In Aurvedic medicine, Golden shower tree is known as

‘ARAGVADHA (disease killer). It contains elevated quantities of anthraquinones

and is consequently mainly useful against gastrointestinal conditions (e.g.

constipation or acid reflux). The seeds are used in the treatment of biliousness and

to improve the appetite. Its root is useful in the treatment of skin diseases, leprosy,

tuberculosis, throat troubles, liver complaints, rheumatism and asthma

(Hostettmann and Marston, 2002). A new biologically active flavone glycoside

was isolated from the acetone soluble fraction of the defatted seeds of C. fistula

(Linn). It was characterized as a new bioactive flavone glycoside 5,3’, 4’-

trihydroxy-6-methoxy-7-0-α-L-rhamnopyranosyl-(1→2)-0-ß-D-galactopyranoside

by several colour reactions, spectral analysis and chemical degradations. It also

showed antimicrobial activity (Yadava and Verma, 2003). The leaves of C. fistula

are laxative, antiperiodic; heal ulcers, used in rheumatism and cure cough. The leaf

juice of this plant is used in folklore to treat cough in Tripura, India. Studies were

undertaken to evaluate the anticough effect of the leaf extract against sulphur

dioxide induced cough reflex in mice. The antitussive activity of the methanol

extract of C. fistula extract was comparable to that of codeine phosphate, a

prototypes antitussive agent. The C. fistula extract (400, 600 mg/kg, p.o.) showed

maximum inhibition of cough by 44.44% and 51.85% with respect to control

group (Chopra et al., 1997; Bhakta et al., 1998; Mahida and Mohan, 2006).



According to the Ayurvedic and Unani system of medicine various parts of C.

fistula are highly useful in curing various diseases and as an antifertility agent. The

pulp from the pod is of great therapeutic value. It is rich in proteins and

carbohydrates. Besides eleven essential aminoacids, the pulp contains a large

amount of aspartic and glutamic acid. It is also a source of Fe and Mn. It could be

a good source of some important nutrients and energy. Fistulic acid was isolated

from the pods and Kaempferol & leucopelargonidin tetramer was isolated from the

flowers of C. fistula (Bhawasar et al., 1965). Ethanolic extract of fruits of this

plant has been reported to possess anti-implantation and estrogenic effects in rats.

The antifertility effect of aqueous extract of seeds of C. fistula was reported in

female rats (Yadava and Jain, 2000). Oral administration of aqueous extract of

seeds of C. fistula to mated female rats from day 1-5 of pregnancy at the doses of

100 and 200 mg/kg body weight resulted in 57.14% and 71.43% prevention of

pregnancy, respectively, whereas 100% pregnancy inhibition was noted at 500

mg/kg bw (Hamburger and Hostettmann, 1991).

Bauhinia purpurea (L.) lies in the family Fabaceae: Caesalpinioideae and is

commonly known as purple orchid tree. It is a small to medium sized deciduous

tree growing to 17 m tall. The leaves are 10-20 cm long and broad rounded and

biolobed at the base of apex. B. purpurea is also a medicinal plant and has been

used medicinally.

Many species of Bauhinia are used traditionally in Nepal to treat virus caused

diseases (Pokhrel et al., 2002). The leaves of B. purpurea contain a mixture of

phytol fatty esters, lutein and ß-sitosterol. The structure was elucidated by NMR

spectroscopy, while the chain lengths of the esterified fatty acids were determined

by mass spectroscopy (Albert et al., 2004). Antimicrobial tests indicated that

phytol fatty esters has low activity against the fungi, Aspergillus niger and

Candida albicans and inactive against the bacteria, Pseudomonas aueruginosa,

Staphylococcus aureus, Bacillus subtilis, Escherichia coli and the fungus,

Trichophyton mentagrophytes (Ragasa et al., 2004). A novel flavone glycoside, 5,

6-dihydroxy-7-methoxyflaone-6-ß-d-xylopyranoside was isolated from the



chloroform soluble fraction of the ethanolic extract of Bauhinia purpurea stems

(Yadava and Tripathi, 2000). B. purpurea now used as a new paraffin section

marker for Reed-Sternberg cells of Hodgkin’s disease in comparison with Leu-M1

(CD 15), LN 2 (CD 74), peanut agglutinin and Ber-H2 (CD 30). Thirty-three cases

of Hodgkin’s disease were studied with Bauhinia purpurea agglutinin (BPA),

peanut agglutinin (PNA), anti-Leu-M 1, LN 2, and Ber-H 2 by the avidinbiotin-

peroxidase complex (APC) method in paraffin sections. Reed-Sternberg (RS) cells

and variants were stained positively with one or more of the reagents in all cases

but in all BPA can be accepted as a useful marker due to its high detection rate,

reproducible staining pattern and resistance to fixatives (Pinto-da-Silva et al.,

2002). BPA is a Galß1-3GalNAc (T) specific leguminous lectin that has been

widely used in multifarious cytochemical and immunological studies of cells and

tissues under pathological or malignant conditions (Sarkar et al., 1993). BPA also

used as a marker in hyperblastic human tonsil and peripheral blood mononuclear

cells by immunohistological, immunoelectron microscopic, and flow cytometric

techniques (Sarkar et al., 1992). Bauhinia purpurea lectin (BPA) was purified from

seeds of B. purpurea alba. The purified lectin was digested with an

endoproteinase, Asp-N, or trypsin and then the amino acid sequenences of the

resultant fragments were analyzed. Ac-DNA library was constructed using RNA

isolated from germinated B. purpurea seeds (Kusui et al., 1991).

B. purpurea lectin facilated the purification of Leishmania braziliensis metacyclic

promastigotes, which is the causative agent of mucocutaneous leishmaniasis from

stationary phase culture by negative selection. The ultrasound analysis showed

that B. purpurea non agglutinated promastigotes have a dense and thicker

glycocalyx. They are resistant to complement lysis, and highly infective for

macrophase in vitro and hamsters in vivo. These results suggest that the B.

purpurea non-agglutinated promastigotes are the metacyclic forms of Leishmania

braziliensis (Brown and Thorpe, 1995).

Rapid and progressive deforestation is endangering several plant species.

Micropropagation systems have the potential for rapidly multiplying economically

important genotypes for reforestation, which help to increase forest productivity.



Conventional propagation methods i.e. through coppicing are not successful.

Therefore, for the mass multiplication of the important tree species, tissue culture

techniques are applied (Debnath et al., 2006).

Plant tissue culture is not a separate branch of plant science like taxonomy, plant

physiology etc. rather it is collection of experimental methods of growing large

number of isolated cell or tissue under sterile and controlled conditions (Lowe et

al., 1996; Govil and Gupta, 1997).

Tissue culture has widened the aspect of in vitro techniques since embryo or plant

parts grown ascetically and subjected to stipulated conditions can show the desired

effects on metabolism. Thus, tissue culture has been used as a tool for the large

scale propagation of genetically manipulated superior clones and for ex-situ

conservation of valuable germplasm. It is a relatively novel method that has been

widely used for genetic improvement of cultivars; rescue of rare and threatened

species, reduction of growth time and overcoming barriers to progress in

multiplication (Collin, 2001; Herman, 2004).

Plant hormone referred to as ‘phytohormone’ is an organic substance other than

nutrient, active in very minute amounts and evokes biochemical, physiological and

morphological responses. In most plants growth is proportional to the logarithm of

the concentration of the applied hormone (Khanam et al., 2000).

Auxins, which constitute a small group of plant hormone, were identified by their

promoting effect on tropism. These were the first growth hormone to be

discovered in plants. Indole acetic acid (IAA) was the first natural auxin to be

identified (Bandureki et al., 1995). Auxins play a central role in the regulation of

plant growth and development including cell elongation, differentiation, tropism

(phototropic and gravitropic), apical dominance, fruit ripening, rooting etc. In

tissue culture, auxins have been used for cell division and root differentiation.



The term cytokinin was suggested by Letham, 1963. Cytokinins play an important

role in cell division by activating DNA synthesis, orderly development of embryo,

expansion of cell and breaking of dormancy of seeds (Kaminek et al., 1997).

Auxins and cytokinins are essential for success of the culture and the level of

hormones and their ratio in the medium affects the differentiation of cultured cells

(Bhojwani and Razdan, 1983). There are several effects of cytokinin/auxin

combinations on organogenesis, shoot regeneration and tropane alkaloid

production (Pacheco et al., 2007). The whole plant can also be regenerated in large

number from callus tissue through manipulations of nutrient and hormonal

constituent in culture medium.

Callus tissue means an unorganized proliferate mass of cells without any

differentiation. Callus tissue is important to plant tissue culture and is produced

experimentally from an excised portion called explants of any living healthy plant.

The most important characteristic feature of callus is that it has the potential to

develop into normal roots, shoots and embryoids that can form complete plant and

in addition be used to initiate a suspension culture (Razdan, 1993).

Organogenesis through callus depends on source and origin of callus, genotype,

age, endogenous hormonal levels and physical factors. The general growth

characteristics of a callus involve a complex relationship between a plant material

used to induce callus and the composition of the medium and the environment

conditions during the incubation period (Murashige, 1974). Moreover, plant

growth regulators especially the auxins and cytokinins play an important role in

organ redifferentiation from the dedifferentiated callus tissue (Singh et al., 2002).

Establishment of callus from explants can be divided roughly into three stages-

induction, cell division and dedifferentiation. During the initial induction phase

metabolism is stimulated prior to mitotic activity. The length of this phase depends

on physiological status of the explant cells as well as culture conditions and

subsequently there is a phase of active cell division as explant cell reverts to

meristematic state; third phase involves the appearance of cellular differentiation



and the expression of certain metabolic pathways that lead to the formation of

secondary products. Secondary product biosynthesis in relation to callus

differentiation has been reviewed (Yang et al., 2006).

Exogenous application of hormones is one of the most common methods for

research into the mode of action of such substances. The rate of metabolism of the

applied hormone depends on its rate of penetration into cells and tissues. There are

several influences of exogenous hormone on the growth and secondary metabolite

formation (Rhodes et al., 1994).

It is also known fact that the regeneration rate of leguminous trees in natural

habitats is quite low. Therefore, improved methods of in vitro propagation need

to be explored. There are difficulties in propagation of woody species, through

conventional techniques such as cutting and layering, owing to the seasonal effects

and poor rooting. Micropropagation, which is often used successfully for the

multiplication of several woody plants, represents an interesting alternative for

these species. Acacia species, in general are recalcitrant to regenerate and they

pose various problems during conditions (Mittal et al., 1989; Ide et al., 1994).

Although regeneration of plants from hypocotyls derived callus of Acacia sinuata

has been achieved, the technique has not yet been fully developed. Somatic

embryogenesis and plant regeneration from callus culture of A. catechu has been

achieved (Rout et al., 1995; Sultana et al., 2007).

The availability of an efficient propagation technique would allow large scale

multiplication for commercial exploitation. Vegetative propagation is worth

special attention, in particular cloning by tissue culture. Tissue culture cloning

may overcome the limited success of more conventional techniques, especially as

far as the capacity to produce adventitious roots is concerned (Bon et al., 1998;

Monteuuis and Bon, 2000; Paek et al., 2005).



Here we are highlighting the Review of literature of tissue culture of Acacia

catechu, Cassia fistula and Bauhinia purpurea.

Plant hormones called phytohormones are defined as organic substance active in

very minute amounts and evoke specific biochemical, morphological and

physiological response. Various research strategies are used to study the

correlation between hormone metabolism and functions of hormones in plant

development (Moore, 1994).

Auxins

Auxins are a class of phytohormones, playing a central role in the regulation of

plant growth and development including cell elongation and differentiation,

tropism (phototropic and gravitropic), apical dominance and fruit ripening.

Emergence of Concept of Auxins

Auxins were the first type of plant hormones to be discovered. The term ‘auxin’ is

derived from the Greek ‘auxein’ which means ‘to grow’ a particular substance that

had the property of promoting curvature in Avena coleoptile curvature test. A

committee of plant physiologists in 1954 defined ‘auxin’ as a generic term for

compounds characterized by their capacity to induce elongation in shoot cells.

Types of Auxins

There are two types of Auxins:-[A] Natural Auxin [B] Synthetic Auxins

[A] Natural Auxins

Indole 3-acetic acid is the most abundant, physiologically relevant and only native

indole auxin in higher plants. It was the first auxin isolated and later several other

auxins were discovered in higher plants.



Structure of indole-3-acetic acid

IAA is universally distributed throughout the plant kingdom including non seed

plants e.g. bacteria, fungi and algae (Epstein and Ludwig, 1993).

One nonindolic compound that is widely recognized as a naturally occurring auxin

is phenyl acetic acid (PAA) which was studied extensively by Frank Wightman

and his associates (Wightman and Lighty, 1982). PAA occurs with IAA as native

auxin in tomato and sunflower shoots.

[B] Synthetic Auxins

These are similar in structure and physiological action with IAA and are

synthesized and tested for biological activity. They may be categorized into 5

groups.

1. Indole acid e.g. Indole butyric acetic acid (IBA).

2. Napthalene acid e.g. Napthaline acetic acid (NAA).

3. Chlorophenoxy acetic acid e.g. 2, 4-dichlorophenoxy acetic acid.

4. Benzoic acid e.g., 2, 3, 6 and 2, 4, 6 trichlorobenzoic acid (TBA).

5. Piconilic acid e.g., Picloram or tordon (4-amino 3, 5, 6-tricholropiconilic acid,

TPA) (Moore, 1994).

Auxin Biosynthesis

The control of concentration of auxin in the various parts of the plant is complex,

involving multiple processes. The amino acid tryptophan is commonly regarded as

the precursor for the biosynthesis of auxin (IAA) in plants. Several other indolic

compounds also function as precursors of IAA in higher plant systems e.g.

cucumber seedlings contain Indole-3-ethanol. Members of cruciferacae contain

Indole-3-acetonitrile and a nitralase that converts the nitrile directly to IAA

Auxin Transport

Polarity of auxin transport was recognized by the classical experiments of Went



with avena coleoptiles sections (Went, 1932). Apical dominance and other growth

relations (e.g. cambial activation) in the plants are co-related with polar movement

of endogenous auxin. Auxin is the only plant growth hormone that is transported

polarly and it contributes to the formation of an auxin gradient from the shoot to

root. It affects various developmental processes including stem elongation, apical

dominance, and wound healing and leaf senescence. The basis for the directional

nature of auxin transport is thought to be the basal localization of the efflux

carrier. Thus auxin enters cells from all direction but is pumped out basally. In

coleoptiles polar transport of auxin appears to occur in non vascular tissues

whereas in intact dicot stems it is restricted to the parenchyma associated with the

vascular tissue (Murashige, 1974).

Auxin transport in roots is less completely understood than movement in shoots.

Evidences from experiments in which movement of 14 CIAA in root segments

was studied, indicates that movement is acropetal i.e. from the base of the stem

towards the root tip. The polarity of movement in roots is an order of magnitude

lower than that in shoots. Besides IAA, polarity of movement has been

demonstrated for several synthetic auxin including IBA, NAA and 2, 4-D.

Hormone Conjugation

The term ‘Conjugated plant hormones’ is given to those plant hormones which are

metabolically bound to other low molecular weight compounds by covalent

binding. Conjugation may be involved in regulation of the level of active

hormones by alteration of their physical, chemical and biological properties.

Auxin Conjugation

Conjugation of auxin (IAA) is important because content of IAA conjugates is

much higher than that of the amount of the free IAA . Most of the IAA in plants is

conjugated via ester linkages to sugar or myoinositol or via amide linkages to

amino acids, peptides or proteins (Bandureki et al., 1995). IAA conjugate



formation and hydrolysis activities are tissue specific and developmentally

regulated and conjugated. IAA has several fates: storage, transport, protection

from peroxidation and catabolism (Kleczkowski and Schell, 1995).

Conjugation is a means of regulating free IAA level. Conjugated IAA is a storage

form of IAA that is slowly hydrolysed to yield free IAA and the observed

biological effects are due to the degree to which free IAA level are regulated by

hydrolysis of the conjugates. The first structurally identified IAA conjugate; the

Indole 3-acetyl 2-aspartate (IAA) was isolated from Pisum sativum seedlings after

application of IAA. Exogenous IAA is biotransformed to its aspartate conjugate by

roots of lycopersicon esculentum (Aniket et al., 2008).

Besides IAA, some synthetic auxins also form bound auxins e.g. IBA, Benzoic

acid and 2, 4-D which are reported to conjugate with aspartic acid in pea epicotyls

sections (Moore, 1994). IBA has also been shown to exist in free and conjugated

form. The greater stability of IBA and IBA conjugates is sufficient to explain its

efficiency in rooting. NAA has been reported to form glycosyl ester in plant of

several families as well as to form a peptide with aspartate (Kato et al., 1996).

Cytokinins

Cytokinins were discovered in 1955 when Miller et al., working in the laboratories

at the University of Wisconsin, isolated a substance called ‘KINETIN’ (6-

furfurylaminopurine) from an autoclaved sample of herring sperm DNA and

demonstrated it to be very active in promoting mitosis and cell division in tobacco

callus tissue in vitro (Agarwal et al., 1997).

Natural Cytokinins

Natural cytokinins were isolated in crystalline form from immature corn (Zea

mays) kernels, called as zeatin. The most probable structure of zeatin is 6-(4-



hydroxy 3-methyltrans-2-butenyl amino) purine (Letham, 1974). Several other

cytokinins were also isolated from various sources and characterized chemically.

All the naturally occurring cytokinins are considered to be isopentyl adenine

deviates. N6-isopentyl) adenosine (I6A) occurs in all t-RNA preparations and i6A is

the only cytokinin that has been found in t-RNA from animals (Liu, 1995).

Occurrence of Cytokinins

Cytokinin containing extracts was prepared from approximately 40 species of

higher plants. The cytokinins are ubiquitous among seed plants and through out

the plant kingdom. They are reported to occur specifically in t-RNA of numerous

animals and microorganisms, indicating high probability of occurrence in all

organisms (Tripathi and Tripathi, 2003).

Biosynthesis and Metabolism of Cytokinins

Cytokinin biosynthesis in seed plants occurs generally in tissues and loci that are

meristematic and have growth potential. Cytokinins are synthesized in roots and

translocated acropetally in shoots. This acropetal transport of cytokinins in the

vascular tissue is involved in co-relative growth phenomena such as apical

dominance. There are investigations of biosynthesis of cytokinins in pea (Pisum

sativum L.) plant organs and carrot (Daucus carota L.) root tissues (Miller, 1961).

Enzyme called A2-isopentyl pyrophosphate: AMP transferase or cytokinin

synthease synthesizes cytokinins has been found in plants.

Transport of Cytokinins

In whole plants root apical meristem are major sites of synthesis of the free

cytokinins in whole plants. The cytokinins synthesized in roots appear to move

through the xylem into the shoots, along with the water and minerals taken up by

the roots. The cytokinins in the xylem exudates are mainly in the form of zeatin

ribosides, once reacted the some portion of this nucleoside is converted to the free

bases or to glucosides (Torrey, 1975).



Ratio of Auxin to Cytokinin

High level of auxins relative to cytokinins is known to stimulate the formation of

roots where as high levels of cytokinins relative to auxin lead to the formation of

shoots. At intermediate levels, the tissue grows as an undifferentiated callus

(Skoog and Miller, 1957). To investigate the significance of the auxin/cytokinins

ratio in regulating morphogenesis in crown gall tissues, mutated T-DNA of

Agrobacterium Ti-plasmid was used.

Cytokinin Conjugation

There are several cytokinins conjugates which have chemical, biochemical and

physiological properties. After application of zeatin to Alnus glutinosa conjugated

cytokinins were formed (Meyer et al., 1982). Application of dihydrozeatin to the

leaves led to the formation of diHZ-OZ and Z-OG. In Raphanus sativus, seedlings,

zeatin is converted to ZR, its phosphate and Z-7G. In leaves of Phaseoulus

vulgaris exogenous zeatin is converted to urea, ureides and unknown basic

compounds. Callus cultures of Vinca rosea a crown gall tissue formed after

adenine application rapidly form zeatin and zeatin glucosides (Millvi, 1965).

Exogenous Application of Hormones

Exogenous application of auxins and cytokinins is one of the most common

methods, owing to their mode of action. The rationale behind such approaches is

based mainly on the idea of replacement of endogenous naturally occurring

hormone, the level of which may be controlled and its effects can be monitored.

Exogenous hormones affect endogenous levels of other hormones in vitro (Miller

et al., 1955).



Effect of Exogenous Auxins

Exogenous auxin has two major effects in the initiation and enhancement of root

organogenesis and inhibition of cytokinins induced shoot initiation. Auxin

application is also important in rooting shoots obtained through micropropagation.

NAA promoted root formation and IAA alone or in high ratio to kinetin lead to

adventitious root formation (Skoog and Tsui, 1951). NAA or IBA are generally

more effective than the phenoxyacetic acids, 2, 4-D or 2, 4, 5-T which induce

callusing even at very low concentrations likewise 2, 4-D is more effective

inhibitor of shoot initiation than IAA, IBA or NAA. Auxin prevents shoot

initiation and their action is modified by other hormones, nutritional and

environmental factors (Martin, 1980).

Effect of Exogenous Cytokinins

Early work with 6-amino purine (adenine) (Skoog and Tsui, 1951; Miller, 1961)

and later kinetin established that application of exogenous cytokinins induced

vegetative bud organogenesis in tobacco tissue cultures. Cytokinin alone or high

ratio of exogenous cytokinins to exogenous auxin will induce adventitious shoot

formation. The use of cytokinins to break apical dominance and promote axillary

bud growth is the most widely used technique of micropropagation (Tiwari et al.,

2000). Lower concentrations of cytokinins tend to be ineffective while high

concentrations are inhibitory during induction. Cytokinin action is also modified

in the presence of other exogenous hormones and by light (Niederwieser and

Staden, 1990).

Organogenesis

Organogenesis is usually induced by manipulation of exogenous growth regulator

levels and occurs either directly from the explant tissues or indirectly from callus

that forms on the explants. Organogenesis can be grouped into the following

categories:



(a) direct shoot regeneration

(b) regeneration via callus

Direct Shoot Regeneration

Extensive in vitro research has been conducted on Acacia species with varying

degree of success. Clonal propagation of A. catechu Willd. by shoot tip culture has

been achieved (Kaur and Kant, 2000). Tissue culture of A. auriculiformis using the

aseptically germinated seedlings has achieved and in vitro development of

plantlets from axillary buds of A. auriculiformis has also been achieved. In vitro

micropropagation of A. tortilis, a legume adapted to acrid lands has also been

achieved (Badji, 1993). Multiple shoots were produced from nodal explants, of 30

days old in vitro grown seedling and pretreated 3 and 9 months old greenhouse

grown A. mearnsii plants, respectively (Nandwani, 1995). Plants were

acclimatized in transparent plastic containers under greenhouse conditions with a

90% success rate. It is evident that there is a strong and intricate interaction

between the explant, plant growth regulators, culture conditions and genotype. The

meristem culture of A. mearnsii is employed for the elimination of virus. Plants

are obtained from the khair tree, A. catechu using mature nodal segments

(Bhattacharya and Bhattacharya, 1997; Beck et al., 1998, 2000). Maximum shoot

bud development (8-10) from a single explant was achieved on Murshiege and

Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) 4.0 mg/l and

α-napthaleneacetic acid (NAA) 0.5 mg/l. Addition of adenine sulphate (25.0 mg/l),

ascorbic acid (20.0 mg/l) and glutamine (150.0 mg/l) to the medium was found

beneficial for maximum shoot bud induction. In vitro micropropagation through

shoot apices of A. catechu was obtained (Kaur et al., 1998). Explants were excised

from 15-day old in vitro grown seedlings raised from superior seed stocks. A

maximum of 12 shoots were obtained on MS medium supplemented with 1.5 mg/l

BAP and 1.5 mg/l kinetin. The influence of different macronutrient solutions and

growth regulators on micropropagation of juvenile A. mangium explants has been

reported (Kaur and Kant, 2000). The influence of auxins and darkness on in vitro

rooting of micropropagated shoots from mature and juvenile A. mangium has been



reported by (Azad et al., 2005). Shoot regeneration from seedling explants of A.

mangium Willd has been reported (Douglas and Mcnamara, 2000).

Cassia family are known for their recalcitrant nature, yet some successful attempts

have been made on in vitro organogenesis of Cassia fistula, Cassia siamea and

Cassia alata. Regeneration via calli has been the potent source of producing

somaclonal variants in plants and thus the improvement of the species. The high

efficiency shoot regeneration was achieved through leaflet and cotyledon derived

calli in C. augustifolia-an important medicinal plant. Dark brown callus was

induced at the cut ends of the explants on Murashiege and Skoog’s medium

augmented with 1 μΜ N6-BAP + 1 Μm 2, 4-D (Ammirato, 1983; Agarwal and

Sardar, 2006).

In vitro propagation protocol has been developed from mature lianas of Bauhinia

vahlii (Dhar and Upreti, 1999). Browning was the major obstacle in the

establishment of cultures. For B. vahlii an improved regeneration protocol was

developed using seedling explants (Bhatt and Dhar, 2000). A combination of

thidiazuron and kinetin (1.0 μM each) increased the number of shoots significantly

up to four successive subculture cycles. In vitro propagation protocol was

established for B. variegata and in this case axillary shoot proliferation was

achieved (Mathur and Mukunthakumar, 1992). Micropropagation of B. purpurea,

a mature leguminous tree was also achieved (Kumar, 1992).

Regeneration via Callus

Somatic embryogenesis in leguminous plants has been achieved. Regeneration of

Acacia mangium through somatic embryogenesis and whole plant regeneration

were achieved in callus cultures derived from immature zygotic embryos of A.

mangium. Embryogenic callus was induced on MS medium containing

combinations of TDZ (1-2 mg/L), IAA (0.25-2 mg/L) and a mixture of amino

acids (Xie and Hong, 2001; Nanda and Rout, 2003).



In vitro somatic embryogenesis and subsequent plant regeneration was achieved in

callus culture derived from immature zygotic embryos of A. arabica on semi-solid

Murashige and Skoog (MS) basal salts and vitamins supplemented with 8.88 μΜ

BA, 6.78 μΜ 2, 4-D and 30 g/L (w/v) sucrose.

In vitro morphogenesis via organogenesis was achieved by callus cultures derived

from hypocotyls explants of A. sinuata on MS medium. Calli were induced from

hypocotyls explants excised from 7-day-old seedlings on MS medium containing

3% sucrose, 0.8% agar, 6.78 μΜ 2, 4-D and 2.22 μΜ 6-BAP (Guohua, 1998;

Vengadesan et al., 2002). Somatic embryogenesis and plant regeneration from

callus culture of A. catechu has been achieved (Anjaneyulu et al., 2004).

Plant regeneration from phyllode explants of A. crassicarpa via organogenesis

was achieved by phyllode (leaf) explants excised from 60-day-old in vitro

seedling. It was used for green compact nodule induction and tested MS media

supplemented with various concentrations of TDZ and NAA (Arnold et al., 2002).

In vitro regeneration through somatic embryogenesis as well as organogenesis was

reported in C. angustifolia using cotyledons. The cotyledons dissected from semi-

mature seeds inoculated on MS medium supplemented with auxin alone or in

combination with cytokinin produced direct and indirect somatic embryos

(Agarwal and Sardar, 2007).

Plants have been used for centuries as remedy for human diseases because they

contain components of therapeutic values (Kaur et al., 2002). They are natural

sources of antimicrobial agents primarily because of the large biodiversity of such

organisms and the relatively large quantity of metabolites that can be extracted

from them. The acceptance of these traditional medicines as an alternative form of

health care has led researchers to investigate the antimicrobial activity of

medicinal plants (Nakatani, 1994). Plants exudes these therapeutic chemicals

through various organs e.g. fungitoxic exudates on the leaves to inhibit fungal

spores. Phenolic compounds, tannins and some fatty acid like compounds in cells



of young fruit, leaves or seeds, have been responsible for the resistance in young

tissues to pathogenic microorganisms such as Bortrytis sp. As young tissues grow

older, their inhibitor content and their resistance to infection decrease steadily.

Several other preformed compounds like saponins (glycosylated steroidal),

tomatine in tomato and avinacin in oats have antifungal membranolytic activity.

Plants produce these substances before or after the infection. These chemicals act

alone or in concert to provide resistance to the plants against insects/pathogens

(Dorman and Deans, 2000; Elizabeth, 2005; Eldeen and Staden, 2007).

The development of bacterial resistance to presently available antibiotics has

necessitated the search for new antibacterial agents (Hancock, 1997). The gram

positive bacterium such as Staphylococcus aureus is mainly responsible for post

operative wound infections, toxic shock syndrome, endocarditis, osteomyelitis and

food poisoning (Elizabeth, 2001). Bacillus subtilis are rod shaped aerobic bacteria

and are reported to have some pathogenic role (Gorden et al., 1973). The gram-

negative bacterium such as Escherichia coli is present in human intestine and

causes lower urinary tract infection, coleocystis or septicemia (Levine, 1987;

Eftekhar et al., 2005). Pseudomonas is an aerobic, non fermentative, oxidase

positive bacillus which mainly causes urinary tract infection, wound or burn

infection, chronic otitis media, septicemia etc. in human (Body, 1983; Fuchs et al.,

2011) and also causes several diseases in fishes (Bullock et al., 1965; Livermore,

2002).

Modern science has identified several secondary metabolites of various plant

species that contain antimicrobial properties. A lot of work has been done which

aims at knowing the different antimicrobial and phytochemical constituents of

medicinal plants and using them for the treatment of microbial infections as

possible alternatives to chemically synthetic drugs to which many infectious

microorganisms have become resistant (Kaushik and Dhiman, 2000; Robledo et

al., 2005). The effects of herbal and phytochemical compounds on pathogenic and

economically important bacteria have been well studied (Sato et al., 1996; Binto,

1997; Sampietro et al., 1997; Kim et al., 2008).



The antimicrobial activity of the plant extracts can be determined by various

methods such as disc diffusion, agar well diffusion and two fold serial dilution

techniques (Kaushik, 1985). For screening of the antimicrobial activity of

medicinal plants, the agar well diffusion technique is easy and popular. Pepeljnjak

et al. used this method for the evaluation of the antimicrobial activity of the

ethanol extract of Saturja montana ssp. montana (Pepeljnjak et al., 1999; Kharwar

et al., 2010). Determination of the minimum bactericidal concentration (MBC)

value of a particular plant extract is also essential during evaluation of

antimicrobial activity (Al-Bayati and Al-Mola, 2008; Amor et al., 2008).

Here we are highlighting the review of literature of Microbiological work of

Acacia catechu, Cassia fistula and Bauhinia purpurea.

Microbiological Work of Acacia Species

Acacia is an important medicinal plant and economically important forest tree. It

has great ecological value because it helps in controlling erosion and improving

soil fertility. The genus Acacia comprises many species which are important for

firewood, fodder, shelterbelts and soil improvements.

A. farnesiana ethanolic extract inhibited growth, enterotoxin production and

adhesion of Vibrio cholerae strains 01 and 0139 (Garcia et al., 2006). The effect of

this plant extract on enterotoxin production and adhesion of V. cholerae to Chinese

hamster ovary (CHO) cells were determined. The minimal bactericidal

concentration (MBC) for growth was 4.0-7.0 mg/ml.

Acacia nilotica L. bark has the potential to remove toxic elements from solution by

corroboration from toxicity bioassay using Salix viminalis L. in hydroponic system

(Prasad et al., 2001). This work showed that the A. nilotica bark could be used to

prevent metal mobilization and metal uptake by plants due to its adsorbtion

capacity. Toxicity bioassay with S. viminalis cuttings indicated that the presence of

A. nilotica bark powder did not affect the growth of the plant. A correalitive study

on antimutagenic and chemopreventive activity of A. auriculiformis (A. Cunn.)



and A. nilotica (L.) willd. have been carried out. Antimutagenic and chemo

preventive activity of bark of A. auriculiformis and A. nilotica has been reported

(Kunii et al., 1996). A. auriculiformis is reported to act as a central nervous

depressant, have spermicidal and filaricidal activities, and rich in triterpenoid

Saponin and A. nilotica has been used for colds, bronchitis, diarrhoea, dysentery,

biliousness, bleeding piles and leucoderma. And it also serves as a source of

polyphenols (Fatimi et al., 2007). Acacia gum is a bifidogenic dietary fibre with

high digestive tolerance in healthy humans and believed to benefit intestinal health

(Jeppesen et al., 2000). The effects of increasing doses of GUM were compared to

those of sucrose (SUC) on stool output, concentration of the main bacterial

populations and occurrence and severity of intestinal symptoms (flatulence,

bloating, abdominal cramps and diarrhoea). Ingestion of GUM 10-15 g for 10 days

increased total lactic acid producing bacteria and bifidobacteria counts in stools,

without affecting total anaerobic and aerobe counts (Khan, 1997). A new

triterpenoidal saponin was isolated from A. auriculiformis (Uniyal et al., 1992).

And a novel flavone glycoside was isolated from the stem of A. catechu (Zhou et

al., 2009). The antimicrobial activity of saponins from A. auriculiformis has been

determined (Mandal et al., 2005).

Microbiological Work of Cassia Species

Chemical investigations on plants belonging to leguminous have indicated that

flavonoids are their important components (Dornenburg and Knorr, 1996). Plants

of Genus cassia have been used as remedies in traditional systems of medicines.

Cassia angustifolia Vahl. is employed in various indigenous systems of medicine

against several diseases. The seeds are used as an anthelmintic, digestive and to

treat piles, skin diseases and abdominal troubles. The pharmacognostic

investigations on the seeds of C. angustifolia have been reported.The study

includes macro and microscopically details, SEM studies and HPTLC

fingerprinting (Silva et al., 2008).



Cassia nodosa is used as a folk medicine for the treatment of cheloid tumours,

ring- worm, insect-bite and rheumatism. Earlier work includes the isolation and

characterization of 2, 3-dihydrokaempferol-3-0-rhamnoside, quercetin-3-0-

rhamnoside, kaempferol-3-0-rhamnoside, nodosin, 8-C-flucosyl genistein and

dalpaitin from the flowers of C. nodosa. The isolation and characterization of a

new chromon (5, 4’-dihydroxy-7-methyl 3-benzyl chromone) along with three

known flavonoid compounds, the unsubstituted flavone, Kaempferol-3-0-

rhamnoside and quercetin-3-0-arabinoside from the leaves of C. nodosa have been

reported (Kumar et al., 2006). C. fistula is a medicinal plant used for varied

purposes. This plant is also used in haematemesis, pruritus, leucoderma, diabetes

and many other ailments (Govindarajan et al., 2008). The antifungal and

antibacterial activities of this plant have already been reported (Chukwujekwu et

al., 2006). And emodin, an antibacterial anthraquinones were isolated from the

roots of C. occidentalis (Abbas et al., 2008). In Aurvedic medicine Golden shower

tree is known as ‘ARAGVADHA (disease killer). It contains elevated quantities of

anthraquinones and consequently is mainly useful against gastrointestinal

conditions (e.g. constipation or acid reflux). The seeds are used in the treatment of

biliousness and to improve the appetite. Its root is useful in the treatment of skin

diseases, leprosy, tuberculosis, throat troubles, liver complaints, rheumatism and

asthma. A new biologically active flavone glycoside was isolated from the acetone

soluble fraction of the defatted seeds of C. fistula (Linn). It was characterized as a

new bioactive flavone glycoside 5,3’, 4’-trihydroxy-6-methoxy-7-0-α-L-

rhamnopyranosyl-(1→2)-0-ß-D-galactopyranoside by several colour reactions,

spectral analysis and chemical degradations. It also showed antimicrobial activity

(Samappito et al., 2002). The leaves of C. fistula are laxative, antiperiodic; heal

ulcers, used in rheumatism and cure cough. The leaf juice of this plant is used in

folklore to treat cough in Tripura, India. Studies were undertaken to evaluate the

anticough effect of the leaf extract against sulphur dioxide induced cough reflex in

mice (Arora and Kaur, 1999). The antitussive activity of the methanol extract of C.

fistula extract was comparable to that of codeine phosphate, a prototypes

antitussive agent. The C. fistula extract (400, 600 mg/kg, p.o.) showed maximum

inhibition of cough by 44.44% and 51.85% with respect to control group (Erturk,



2006). According to the Ayurvedic and Unani system of medicines various parts of

C. fistula are highly useful in curing various diseases and as an antifertility agent.

The pulp from the pod is of a great therapeutic value. It is rich in proteins and

carbohydrates. Besides eleven essential amino acids, the pulp contains a large

amount of aspartic and glutamic acid. It is also a source of Fe and Mn. It could be

a good source of some important nutrients and energy. Fistulic acid was isolated

from the pods and kaempferol and leucopelargonidin tetramer from the flowers of

C. fistula. Ethanolic extract of fruits of this plant has been reported to possess anti-

implantation and estrogenic effects in rats (Hanif et al., 2007). The antifertility

effect of aqueous extract of seeds of C. fistula was reported in female rats (Khalid

et al., 1996). Oral administration of aqueous extract of seeds of C. fistula to mated

female rats from day 1-5 of pregnancy at the doses of 100 and 200 mg/kg body

weight resulted in 57.14% and 71.43% prevention of pregnancy, respectively,

whereas 100% pregnancy inhibition was noted at 500 mg/kg bw.

Cassia occidentalis has potent antimutagenic and anticarcinogenic activities

against mutagens requiring metabolic activation. The aqueous extract of C.

occidentalis Linn. was screened for effectiveness in inhibiting mutagenecity of

alfatoxin B1 (AFB1) and benzo[a]-pyrene (B [a] P) in the Ames test.

Antimutagenicity was evaluated using Salmonella typhimurium strains TA 98 and

TA 100. In vivo antimalarial activity of C.occidentalis were also evaluated in mice

against Plasmodium berghei ANKA by taking ethanolic, dichloromethane and

lyophilized extracts of root bark. At doses of 200 mg/kg, all the ethanolic and

dicholoromethane extracts produced significant chemosuppressions of

parasitaemia when administered orally (Nostro et al., 2000).

Cassia auriculata seeds, flower and leaves decoction mediate an anti-diabetic

effect. Its flower extract has hypoglycaemic action. Oral administration of 0.45

gm/kg body weight of the aqueous extract of the flower for 30 days resulted in a

significant reduction in blood glucose and increase in plasma insulin in

streptozotocin diabetes rats (Denyer and Stewart, 1998). In the leaves and flowers

of C. siamea Lam. Barakol (3a, 4-dihydro-3a, 8-dihydroxy-2, 5-dimethyl-1, 4-



dioxaphenaline) is found. It has been reported that intravenous injections of

barakol (0.5-15 mg/kg) show hypertensive effects in rats and cats (Chen et al.,

1999). The methanolic extract of C. nigricans leaves was investigated for its

contraceptive activity in mice and rats. Its anticonceptive effect may be due to in

parts to its anti-implantation, estrogenic and direct effect on the uterus (Nwafor

and Okwuasaba, 2001). C. nigricans methanol extract was also investigated for its

antiplasmodial activity against chloroquine resistant strains of Plasmodium

falciparum. The in vitro activity against P. falciparum strain K1 was assessed

using the parasite lactate dehydrogenase assay method. The main antiplasmodial

principle 1, 3, 8-trihydroxy-6-methyl-9, 10-anthracenedione has been isolated

from C. nigricans. This was found to have in vitro activity against P. falciparum

(Obiageri et al., 2005).

Extract of Cassia tora and Cassia sophera completely inhibited conidial

germination of Phyllactinia corylea (Yen and Chung, 1999). Studies were carried

out on the antifungal properties and preservative effect of ground Cassia on the

self life of bread. At 2% concentration, cassia delayed mold growth in bread for

9.5 days. The anti-cryptococcus activity of combination of ethanolic extracts of

leaves of Cassia alata and Ocimum sanctum was reported. The activity of

combination of the extracts was heat-stable and worked at acidic pH (Bridges,

1987; Ranganathan and Balajee, 2000). Methanolic extracts of 22 Indian plants

belonging to 12 families were studied for their antibacterial activity. All the

examined plant extracts were effective against more than one organism and the

results are comparable with antibiotics. Out of the tested plants, Cassia alata L.,

Cassia biflora L., Cassia fistula L. extracts were found to be more effective

against both Gram-positive and Gram-negative bacteria (Liu, 2005; Samie et al.,

2005). A new chromone from Cassia nodusa has been isolated. And assessment of

antimutagenic and genotoxic potential of senna (C. angustifolia Vahl.) from

aqueous extract using in vitro assays has been determined (Grover et al., 2002;

Landa et al., 2006).



Microbiological Work of Bauhinia Species

Bauhinia variegata alcoholic extract was found to have antimicrobial activity

against Bacillus subtilis (ATCC 6635), Pseudomonas aeruginosa (ATCC 27853),

Salmonella typhi, Shigella dysenteriae, Staphylococcus aureus (ATCC 292213)

and Vibrio cholerae. The largest zone of inhibition (18 mm) was found to be

exhibited against B. subtilis. For this organism the minimum bactericidal

concentration (MBC) of the crude extract was 0.39 mg/ml. The extract was found

to be more effective against gram-positive than gram-negative bacteria. The

antimicrobial activity was found to be decreased during purification (Zaka et al.,

2006; Pitta-Alvarez et al., 2008). From the roots of B. variegata a novel flavonol

glycoside 5,7,3’4’-tetrahydroxy-3-methoxy-7-0-α-L-rhamnopyranosyl (1→3)-0-ß-

D-galactopyranoside was isolated. This novel compound showed anti-

inflammatory activity. A new phenanthraquinone, named bauhinione has been

isolated from B. variegata L. stem extracts and its structure has been elucidated as

2, 7-dimethoxy-3-methyl-9, 10-dihydrophenanthrene-1, 4-dione on the basis of

spectroscopic analysis. It showed antitumor activity on human myeloid leukaemia

K562 cells (Gupta et al., 1980; Yadav and Reddy, 2002; Neto et al., 2008). B.

purpurea leaves contain mixture of phytol fatty esters, lutein and ß-sitosterol. The

structure was elucidated by NMR spectroscopy, while the chain lengths of the

esterified fatty acids were determined by mass spectroscopy. Antimicrobial tests

indicated that phytol fatty esters has low activity against the fungi, A. niger and C.

albicans, and inactive against the bacteria, P. aeruginosa, S. aureus, B. subtilis, E.

coli and the fungus, T. Mentagrophytes (Kumar et al., 2005). Bauhinia racemosa

L. stem bark has antioxidant and antimicrobial activities. The ethanolic extract of

leaves of B. racemosa shows analgesic, antipyretic, anti-inflammatory and

antispasmodic and antimicrobial activity (Gupta et al., 2004a). The cytotoxicity

against CA-9 kb in cell culture, hypotensive and hypothermic activities were

reported from the hydroalcohalic extract of B. racemosa. Several phytochemical

constituents of this plant have been isolated and it chefiely include flavonoids

(kaempferol & quercetin), coumarins (scopoletin & scopolin), triterpenoids (ß-

amyrin), steroids (ß-sitosterol), and stibenes (resveratrol). The methanol extract of



B. racemosa Lam. Stem bark was investigated for the antioxidant and

hepatroprotective effects in wistar albino rats (Gupta et al., 2004b). Secondary

metabolites and a novel flavone glycoside were isolated from the stem of Bauhinia

purpurea (Gupta et al., 1979). And antitumour activity of Bauhinia variegata

against Ehrlich ascites carcinoma induced mice has also been determined

(Rajkapoor et al., 2003).

The use of medicinal herbs in the treatment of infection is an age old practice and

several natural products are used as phytotherapy for treatment of many diseases.

Human infections constitute a serious problem and most frequent pathogens are

microorganisms such as bacteria and fungi. Therefore, the search for discovery of

new antimicrobial agents is necessary and stimulates the research of new

chemotherapeutic agents in the medicinal plants (Kianbakht and Jahaniani, 2003).

Terpenes are naturally occurring substances produced by a variety of plants and by

some animals. They are abundantly found in fruits, vegetables, and flowers

(Dudareva et al., 2005). Their concentration is generally high in plant reproductive

structures and foliage during and immediately following flowering (Thongson et

al., 2004).Terpenes are also major components of plant resins. In plants they

function as infochemicals, attractants or repellents, as they are responsible for the

typical fragrance of many plants. On the other hand, high concentration of terpens

can be toxic and are thus an important weapon against herbivores and pathogens

(Crowell, 1997; Dikusar et al., 2001; Thesis and Lerdau, 2003).

Animal cholesterol and steroids and the higher triterpenes and phytosterols of

plants are directly involved in the stabilization of cell membranes and are

regulators of permeability and enzymatic reactions (Carvalho and Fonseca, 2006).

Terpenes are biosynthetically derived from isoprene units with the molecular

formula C5H8. The basic formula of all terpens is (C5H8) n, where n is the number

of linked isoprene units (Gao and Singh, 1998). Isoprene units may be linked

“heads to tail” to form linear chains or they may be arranged to form rings.

Terpenes can exist as hydrocarbons or have oxygen-containing compounds such as



hydroxyl, carbonyl, ketone, or aldehydes groups. After chemical modification of

terpens, the resulting compounds are referred to as terpenoids.

The terpenes are classified in order of size into hemiterpenes, monoterpenes,

sesquiterpenes, diterpenes, triterpenes, tetraterpenes, and polyterpenes. Plant

triterpenes and their derivates are a large group of biologically active substances.

Tetraterpenes contain eight isoprene units (C40). Biologically important

tetraterpenes include lycopene (tomatoes) and carotenes. Polyterpenes consist of a

long chain of many isoprene units. Natural rubber is a polyisoprene with several

cis double bonds. The diverse array of terpenoid structures and functions has

provoked increased interest in their commercial use. Terpenoids have been found

to be useful in the prevention and therapy of several diseases, including cancer,

and also to have antimicrobial, antifungal, antiparasitic, antiviral, anti-allergenic,

antispasmodic, antihyperglycemic, anti-inflammatory, and immunomodulatory

properties (Niedermeyer et al., 2005). They also act as natural insecticides and can

be of use as protective substances in storing agricultural products (Monteriro et al.,

2004; Sottomayor et al., 2004).

Here we are giving the Review of literature of Terpenes

Terpenes in Acacia and Other Species

Acacia belongs to the leguminosae family which has been a rich source of

secondary metabolites. Triterpene esters, hydroxycinnamoyl esters of the amyrin

and lupeol were isolated from the leaves and twigs of Acacia linarioides and

Acacia trineura (Wang et al., 1999).

A. catechu predominant catechins, catechin, epicatechin, epicatechin-3-0-gallate,

andepigallocatechin-3-0-gallate and other major secondary products including

caffeine, flavonol dimmers, and flavonol glycosides were identified by their

molecular ion peaks and fragmentation peaks using LC-MS and LC-MS/ MS (Ali

et al., 1997).



The methanol extract of Acacia pennatula contain catechin, epigallocatechin,

eriodictyol, ß – sitosteryl – ß – D - glucopyranoside, and stigmasteryl – ß – D -

glucopyranoside (Shen et al., 2006). A new triterpenoid saponin has been isolated

from an aqueous EtOH extract of the legumes of Acacia auriculiformis (Rios,

2005). The antimicrobial efficacy of four major monoterpenes contained in

essential oils (thymol, carvacrol, p-cymene, and γ-terpinene) against the Gram-

positive bacterium S. aureus and the Gram-negative bacterium E. coli has been

reported (Vardar-Unlu et al., 2008). Antimicrobial terpenes were obtained from

oleoresin of Pinus ponderosa. These monoterpenes were active primarily against

fungi and also against Gram-positive bacteria (Cristani et al., 2007). From the

aerial parts of Saussurea pulchella Fisch (Compositae) seven terpenes and eight

phenolics were isolated. The isolated compounds were examined for cytotoxic

activity against four human cancer cell lines in vitro using the sulforhodamin bio

assay method (Himejima et al., 2007). Cytotoxic terpenes hydroperoxides were

also isolated from the aerial parts of Aster spathulifolius. These compounds

showed moderate cytotoxicity against human cancer cells with ED50 values

ranging from 0.24 to 13.27 μg/ml (Lee et al., 2006).

Different Activities of Terpenes

Antimicrobial and Antifungal Activities

Many terpenes have been found to be active against a variety of microorganisms.

Test has been performed on Gram-positive and Gram-negative bacteria and also

on fungi (Kaminska et al., 2004). In general, Gram-positive bacteria are more

sensitive to terpenes than Gram-negative (Trombetta et al., 2005). This is mainly

determined by differences in the permeability, composition, and charge of the

outer structures of the microorganisms. The mechanism of antimicrobial action of

terpenes is closely associated with their lipophilic character. Monoterpenes

preferentially influence membrane structures which increase membrane fluidity

and permeability, changing the topology of membrane proteins and inducing

disturbances in the respiration chain (Kedzia et al., 2000). The rank order of the



antibacterial activities of terpenes against S. aureus was: farsenol > (+)-nerolidol >

plunotol > monoterpenes such as (-) -citronellol, geraniol, nerol, and linalool.

Thymol and (+) -menthol (monoterpenes) also expressed high toxicity when

analyzed with S. aureus and additionally (+) -menthol was toxic against

Escherichia coli (Trombetta et al., 2002; Halda et al., 2003). Recently it was

proposed that the antibacterial activity against S. aureus depends on the length of

the aliphatic chains of terpene alcohols and the presence of double bonds

(Habtemariam, 2000). In turn, the sesquiterpenoids nerolidol, farnesol, bisabolol,

and apritone enhanced bacterial permeability and susceptibility to antibiotics

(Inoue et al., 2004). The most important terpene that can be used in antimicrobial

therapy is (4R) - (+) -carvone (monoterpene), which was effective against Listeria

monocytogenes and showed activity towards Enterococcus faecium and

Escherichia coli (Brehm-Stecher and Johnson, 2003). Terpenoid derivatives have

been reported to possess antimycobacterial activity, as well. Several metabolites

have been tested, where the most active was ferruginol (diterpene), which

exhibited inhibitory activity towards Mycobacterium smegmatis, M. intracellulare,

and M. chelonei. On the other hand, benzoxazole-containing diterpene,

tetracycline diterpene elisapteroosin B, and triterpenoid zeorin were shown to

reduce growth of M. tuberculosis (Breitmaier, 2007).

Terpenes also displayed antifungal activity. Carvone and perillaldehyde inhibited

the transformation of Candida albicans from the coccal to the filamentous form,

which is responsible for the pathogenicity of the fungus (Cappuccino and

Sherman, 1999). The development of C. albicans, C. krusei, and C. tropicalis was

also limited by a composition of monoterpenes, which included terpinen -4 -ol, α-

pinene, ß-pinene, 1,8-cineole, linalool, and α-terpineol. They inhibited the

development of dermatophytes such as Trichophyton mentagrophytes, T. rubrum,

and Microsporum gypseum, as well as α-terpinene also exhibited antifungal

activity similar to that of commonly used antifungal drugs (Bauer et al., 1996;

Hammer et al., 2003).



Antiviral Activities

Terpenoids are an interesting group of natural agents with both specific and wide-

ranging antiviral activities that could be used to improve the therapeutic efficacy

of standard antiviral therapy.

The terpenoid constituents of Ganoderma pfeifferi oil lucialdehyde D and

ganoderon A and C were also potent inhibitors of herpes simplex virus (HSV)

(Parveen et al., 2004). Terpene compounds moronic and betulinic acids have been

found to possess anti-HSV-1 activity. Moreover, betulin and betulinic acid, as well

as several of their derivatives have been described as very active anti-HIV agents

affecting virus-cell fusion, reverse transcriptase activity, and virion assembly and

budding. Betulin, betulinic acid, and oleanolic acid have also been seen to inhibit

the replication of vesicular stomatitis virus and encepphalomyocarditis virus

(Mooney and Emboden, 1968).

Anticancer Activity

Epidemiological studies suggest that dietary monoterpenes may be helpful in the

prevention and therapy of cancers (Huijbregts et al., 2000; Bartzatt et al., 2007).

Among dietary monoterpenes, D-limonene and perillyl alcohol have been shown

to posses chemopreventive and therapeutic properties against many cancers

(Reddy et al., 1997). Several plant terpenes exhibited in vitro antitumor activity.

Betulinic acid has been shown to induce apoptosis of several human tumour cells.

It was also shown that betulinic acid is an inhibitor of topoisomerase I, a nuclear

enzyme that catalyses changes in DNA topology (Chowdhury et al., 2002; Kris-

Etherton et al., 2002). Other plant triterpenes, such as ursolic acid and oleanolic

acid, found in natural wax on apples and other fruits, reduced leukemia cell

growth (Fulda et al., 1998) and inhibited the proliferation of several transplantable

tumours in animals by inducing nitric oxide (NO) and tumour necrosis factor

(TNF) production (Cipak et al., 2006). These triterpenoids and their derivatives act

at various stages of tumour development, inhibiting initiation and promotion as

well as inducing tumour cell differentiation and apoptosis. Moreover, they are

effective inhibitors of angiogenesis, invasion, and metastasis of tumour cells



(Patocka, 2003).

Antihyperglycemic Activity

Diterpene, stevioside is a steviol glycoside extracted from leaves of the plant

Stevia rebaudiana which possesses insulinotropic, glucagonostatic, and

antihyperglycemic effects (Loza-Tavera, 1999).

Anti-Inflammatory Activity

Monoterpene, eucalyptol isolated from eucalyptus oil was found to be especially

useful in curing chronic ailments such as Bronchitis sinusitis and steroid-

dependent asthma or as a preventive agent in returning respiratory infections

(Hanari et al., 2002; Peana et al., 2003). Terpinen-4-ol, the main component of the

oil of Melaleuca alternifolia, Tea tree oil and cineole derivatives present in many

plant oils also suppressed the production of several proinflammatory substances

such as PGE2, TNF-α, and interleukin 1ß (Hammer et al., 1999; Phillips and

Croteau, 1999; Santos and Rao, 2000).

Antiparasitic Activity

The antiparasitic activity of terpenoids is explained by their interaction with heme

and Fe (II) groups where free radicals are released to kill parasites. In a group of

monoterpenes, espintanol and piquerol A have been found to have some

antiprotozon parasite activity. Thymol and its structural derivatives also possess an

anti-leishmanial potential (Hart et al., 2000).

Fine Chemicals from Terpenes

Terpenes constitute a class of natural products that can be transformed into novel

and valuable compounds commercially important for the industrial production of

fragrances, perfumes, flavours, and pharmaceuticals as well as useful synthetic

intermediates and chiral building blocks (Sa Roberto et al., 2007).



Terpenoids as Skin Penetration Enhancers

Terpenes are used in numerous areas of medicine. There are many reports

indicating that terpenes are skin penetration-enhancing agents. Currently, terpenes

are being analyzed as supplementary agents in tropical dermal preparation,

cosmetics, and toiletries (Kohlert et al., 2000).

Terpenes as Natural Substrates

Plant terpenes and lignin act as natural co substrates in inducing the

biodegradative pathways of PCBs (Polyclorinated Biphenyls) and PAHs

(Polycyclic Aromatic Hydrocarbons) (Lerdau et al., 1994; Koh et al., 2000;

Predieri and Rapparini, 2007).

Isolation of pure and pharmacologically active constituents from plants remains a

long and tedious process. For this reason, it is necessary to have methods available

which eliminate unnecessary separation procedures. Biological screening includes

agar diffusion, direct TLC bio autographic detection and agar-overlay. The number

of available targets for biological screening is limited. Furthermore, bioassays are

often not reliable for clinical efficiency. For these reasons, it is extremely helpful

to have chemical screening techniques available as complementary approach for

the discovery of new molecules which might serve as lead compounds. Chemical

screening is thus performed to allow localization and targeted isolation of new and

useful types of constituents with potential activities. The procedure enables

recognition of known metabolites in extracts or at the earliest stages of separation

and is thus economically very important.

TLC is the simplest and cheapest method of detecting plant constituents because

the method is easy to run, reproducible and requires little equipment (Golkiewiez

and Gadzikowska, 1999). However for efficient separation of metabolites, good

selectivity and sensitivity of detection, together with the capability of providing

on-line structural information, high performance liquid chromatographic (HPLC),

liquid chromatographic/ mass spectroscopic (LC-MS), liquid chromatographic/

nuclear magnetic resonance (LC-NMR) and gas chromatography/ mass



spectroscopic (GC-MS) techniques are preferred (Marston et al., 1997;

Hostettmann et al., 1997). They play an important role as an analytical support in

the work of phytochemists for the efficient localization and rapid characterization

of natural products.

Plants contain thousand of constituents and are a valuable source of new and

biologically active molecules. For their investigation it is important to have the

necessary tools at hand. These include suitable biological assays and chemical

screening methods.

Chromatography

The Russian botanist Mikhail Tswett is credited with the original development of a

separation technique that we now recognize as a form of chromatography. In 1903

he reported the successful separation of a mixture of plant pigments using a

column of calcium carbonate (Strain and Sherma, 1967).

The basis of all forms of chromatography is the partition or distribution coefficient

(Kd), which describes the way in which a compound distributes itself between

two immiscible phases. For the two immiscible phases A and B, the value for this

coefficient is a constant at a given temperature and is given by the expression:

The term effective distribution coefficient is defined as the total amount, as

distinct from the concentration, of substance present in one phase divided by the

total amount present in the other phase. It is in fact the distribution coefficient

multiplied by the ratio of the volumes of the two phases present. Basically, all

chromatographic systems consist of the stationary phase, which may be solid, gel,

liquid or a solid/liquid mixture that is immobilized, and the mobile phase, which

may be liquid or gaseous and which flows over or through the stationary phase.

The choice of stationary and mobile phases is made so that the compounds to be

Kd =Concentration in phase A

Concentration in phase B



separated have different distribution coefficients. From that time there are several

advancements in this field which are reviewed below:

Thin layer chromatography has been used to screen plant samples for ecdysteroids

(Bathori et al., 1988). Normal phase silica and actadecyl silica plates were used to

differentiate a polar and polar ecdysteroids respectively. The major advantages of

thin-layer chromatography are multiple detections which enables specific

detection of the ecdysteroids. Thin layer chromatography, has also been used to

screen plant samples of Salix psammophila and separated by column

chromatography, and its structure was identified by nuclear magnetic resonance

(NMR) (Guan-hui and Jin-tian, 2008).

Two-dimensional Thin-layer chromatography of plant ecdysteroids has also done

(Bathori et al., 2000). Separation of palm carotene from crude palm oil by

adsorption chromatography with a synthetic polymer adsorbent has been achieved

(Andronikashvili et al., 1999). A new approach for the determination of relative

retention in thin-layer liquid chromatography has been achieved (Berezkin, 2007).

Spot and mobile-phase front anomalies in planer (thin-layer) chromatography have

been done (Kalasz, 2005). Thin-layer Chromatography of neutral sugars as

influenced by the nature of the cation of impregnating salt has been achieved

(Kalinina and Litvinova, 2001). Characterization of Barium sulphate as a TLC

material for the separation of plant carboxylic acids has been reported (Rathore

and Khan, 1987). Low temperature liquid chromatographic separation of

oxygenated terpenes from terpene hydrocarbons has also achieved (Yamasaki et

al., 1986).

Gas Chromatography-Mass Spectrometry (GC-MS)

It is a method that combines the features of gas liquid chromatography and mass

spectrometry to identify different substances with in a test sample. The use of

mass spectrometer as the detector in gas chromatography was developed by

Roland Gohlke and Fred McLafferty (Gohlke and McLafferty, 1993).



The GC-MS is composed of two major building blocks: the gas chromatograph

and the mass spectrometer. The gas chromatograph utilizes a capillary column

which depends on the column’s dimensions (length, diameter, film thickness) as

well as the phase properties (e.g. 5% phenyl polysiloxane). The difference in the

chemical properties between different molecules in a mixture will separate the

molecules as the sample travels the length of the column. The molecules take

different amount of time (called retention time) to come out of (elute from) the gas

chromatograph, and this allows the mass spectrometer downstream to capture,

ionize, accelerate, deflect and detect the ionized molecules separately. The mass

spectrometer does this by breaking each molecule into ionized fragments and

detecting these fragments using their mass to charge ratio. These two components,

used together, allow a much finer degree of substance identification than either

unit used separately. It is not possible to make an accurate identification of a

particular molecule by gas chromatography or mass spectrometry alone. The mass

spectrometry process normally requires a very pure sample while gas

chromatography using a traditional detector (e.g. Flame Ionization Detector)

detects multiple molecules that happen to take the same amount of time to travel

through the column (i.e. have the same retention time) which results in two ormore

molecules to co-elute. Sometimes two different molecules can also have a similar

pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining

the two processes makes it extremely unlikely that two different molecules will

behave in the same way in both a gas chromatograph and a mass spectrometer.

Therefore, when an identifying mass spectrum appears at a characteristic retention

time in a GC-MS analysis, it typically lends to increase certainty that the analyte

of interest is in the sample. GC-MS is increasingly being used for the detection

and identification of plant compounds these days because it is cheap and less time

consuming as compared to liquid chromatography. It is also useful for the

identification and separation of components from closely related compounds from

liquid or gaseous mixture.

The analysis of phenolic and other aromatic compounds in honeys by solid-phase

microextraction followed by gas chromatography- mass spectrometry has been



achieved (Daher and Gulagar, 2008). GC-MS analysis of essential oils from Greek

aromatic plants and their fungitoxicity on Penicillium digitatum has been reported

(Daferera et al., 2000). Selectivity of several liquid phases for the separation of

pine terpenes by gas chromatography has been done (Diaz et al., 2004). Analysis

of phenolics of bud exudate of Populous angustifolia by GC-MS. Phytochemistry

has been reported (Greenaway and Whatley, 1990). The effect of the nature of the

carrier gas on the character of the gas chromatographic separation of isomeric

mixtures of benzene derivatives has been reviewed (Baharin et al., 1998).

In Acacia species 4-hydroxypipecolic acid and pipecolic acid has been reported

and their determination by high performance liquid chromatography, its

application to leguminous plants, and configuration of 4-hydroxypipecolic acid has

been reported. Determination of the predominant catechins in Acacia catechu by

liquid chromatography/electrospray ionization- mass spectrometry has also been

achieved. Analysis of chemical composition of catechu, quantitative determination

of catechin and epicatechin by RP-HPLC has also achieved (Henon et al., 2001;

Shen et al., 2006).



1.1 AIMS AND OBJECTIVES

The search for biologically active compounds from natural sources has always

been of great interest to scientists looking for new sources of drugs useful in

infectious diseases. In recent years a number of studies have been reported,

dealing with antimicrobial screening of extracts of medicinal plant for chemical

composition, biological and therapeutic activities.

Screening programmes for biologically active natural products require the proper

bioassay. Detection of compounds with the desired activity in complex plant

extracts depends on the reliability and sensitivity of the test system used.

Bioassays are also essential for monitoring the required effects throughout

activity-guided fractionation: all fractions are tested and those continuing to

exhibit activity are carried through isolation and purification until the active

monosubstances are obtained. Bioassay must be simple, inexpensive and rapid in

order to cope with the large numbers of samples. They must also be sensitive

enough to detect active principles which are generally present only in small

concentrations in crude extracts. The amount of active components in plant

extracts differ considerably depending on several factors like the plant tissue used

and the season during which the plant is harvested. The development of methods

for detection and quantitation of an active substance is fundamental for quality

control of medicinal pants. In view of the large number of plant species potentially

available for study, it is essential to have efficient systems available for the rapid

biological and chemical screening of the plant extracts selected for investigation.

The development of bacterial resistance to presently available antibiotics has

necessitated the search for new antimicrobial agents. At the same time, there is

continuing interest in the discovery of antibacterial and antifungal agents which

are effective against pathogenic bacteria and fungi. Since the plant kingdom

provides a useful source of lead compounds of novel structure, a wide scale

investigation of three medicinal plant species has been undertaken. The present

study was concentrated on following areas: Tissue Culture Work, Microbiological



Work and Bioassay with the following objectives:

(1) To study the response of selected medicinal plant species for in vitro

regeneration. To achieve the above objective, studies were conducted for in

vitro establishment and multiplication of Acacia catechu, Cassia fistula and

Bauhinia purpurea.

(a) Response of different explants to different concentrations of cytokinins

(b) Response of different growth hormones on nodal proliferation and mean

length of shoot established from nodal segments.

(c) Response of different growth hormones on callus induction

(2) To study the effect of different antimicrobial agents on the sensitivity of

microorganisms for therapeutic purposes. To achieve the above objective,

studies were conducted for in vitro screening of A. catechu, C. fistula and

B. purpurea for potential antimicrobials.

(a) Response of aqueous plant extracts on the range of microorganisms

(b) Response of leaves extracts prepared in different organic solvents on the

range of microorganisms

(c) Response of callus extracts prepared in different organic solvents on the

range of microorganisms

(d) Determinations of minimum inhibition concentration (MIC) of active plant

extracts

(3) To study the different separation techniques available for identification of

active plant constituents. To achieve the above objective, studies were

conducted on bioassay guided fractionation, purification and isolation of

active plant components of A. catechu, C. fistula and B. purpurea.



(a) Chemical screening of plant extracts by chromatographic methods

(b) Purification of plant extracts by gel column methods

(c) Structure identification of the isolated compounds by GC-MS analysis

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