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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).
Chapter – I Introduction
2Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
3Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
4Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
5Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
6Studies on development of medicinally important plants and their antimicrobial properties
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.
Chapter – I Introduction
7Studies on development of medicinally important plants and their antimicrobial properties
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.
Chapter – I Introduction
8Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
9Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
10Studies on development of medicinally important plants and their antimicrobial properties
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.
Chapter – I Introduction
11Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
12Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
13Studies on development of medicinally important plants and their antimicrobial properties
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-
Chapter – I Introduction
14Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
15Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
16Studies on development of medicinally important plants and their antimicrobial properties
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:
Chapter – I Introduction
17Studies on development of medicinally important plants and their antimicrobial properties
(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
Chapter – I Introduction
18Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
19Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
20Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
21Studies on development of medicinally important plants and their antimicrobial properties
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.)
Chapter – I Introduction
22Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
23Studies on development of medicinally important plants and their antimicrobial properties
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,
Chapter – I Introduction
24Studies on development of medicinally important plants and their antimicrobial properties
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-
Chapter – I Introduction
25Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
26Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
27Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
28Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
29Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
30Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
31Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
32Studies on development of medicinally important plants and their antimicrobial properties
(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).
Chapter – I Introduction
33Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
34Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
35Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
36Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
37Studies on development of medicinally important plants and their antimicrobial properties
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).
Chapter – I Introduction
38Studies on development of medicinally important plants and their antimicrobial properties
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
Chapter – I Introduction
39Studies on development of medicinally important plants and their antimicrobial properties
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.
Chapter – I Introduction
40Studies on development of medicinally important plants and their antimicrobial properties
(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