1. INTRODUCTION - 14.139.186.108

Phytochemical, pharmacological, in vitro propagation and molecular studies on Phyllanthus niruri L. 1

1. INTRODUCTION

India is one of the major countries, having 40 per cent of the global

biodiversity and availability of rare plant species. Medicinal and aromatic plants

constitute a major segment of the flora, which provides raw materials for use in the

pharmaceuticals and drug industries. The indigenous systems of medicines, developed

in India for centuries, make use of many medicinal herbs. These systems include

Ayurveda, Siddha, Unani and many other indigenous practices. More than 9,000

native plants have been established and recorded for their curative properties. In one

of the studies made by the World Health Organisation, it was estimated that 80 per

cent of the population of developing countries relies on traditional plant based

medicines for their health requirements (WHO, 1991). Even in many of the modern

medicines, the basic composition is derived from medicinal plants and these have

become acceptable medicines for many reasons that include easy availability, least

side effects, low prices, environmental friendliness and lasting curative property.

In India, the use of herbal medicine can be traced back from the Vedic

period and the first written reports are timed to 600 BC with Charaka Samhita. India

is a varietal emporium of medicinal plants and it is one of the richest countries in the

world as regards genetic resources of medicinal plants. The Ministry of Environment

and Forest, Government of India has identified and documented over 9,500 species of

medicinal plants that are significant for the pharmaceutical industry. Among them,

2,000 to 2,300 species are used in traditional medicines, while at least 150 species are

used commercially on a large scale (EXIM Bank, 1997). Due to this rising

international demand, many important medicinal plant species are becoming scarce

and some of them are facing the prospect of extinction. Therefore, it is important to

conserve the extensively traded medicinal plants in their natural environments or

cultivate them in favourable environments.

The importance of medicinal plants and traditional health systems in

solving the health care problems of the world is also increasing the attention of the

indigenous people. Because of this resurgence of interest, the research on plants of

medicinal importance is growing phenomenally at the international level, often to the

detriment of natural habitats and mother populations in the countries of origin. Most


of the developing countries have adopted traditional medical practice as an integral

part of their culture. Historically, all medicinal preparations were derived from plants,

whether in the simple form of raw plant materials or in the refined form of crude

extracts, mixtures, etc. Recent estimates suggest that several thousands of plants have

been known with medicinal applications in various systems (Farnsworth and Soejarto,

1991).

India is the second largest exporter of medicinal plants in the world. Instead

of exporting such a large amount of valuable resource with very low returns, we can

think about developing in our its own Research and Development capabilities and to

produce finished goods in the form of modern medicines and health care products

derived from plant origin and based on the knowledge of alternative system of

medicine (Kamboj, 2000).

Information about genetic relationships among accessions within and

between the species has several important applications in plant improvement

(Thormann et al., 1994). Therefore correct genotype identification of the plant

material remains important for protection of both the public health and industry.

Chemoprofiling and morphological evaluations are routinely used for the

identification of medicinal plants. Chemical complexity and lack of therapeutic

marker(s) are some of the limitations associated with morphological evaluation. Now

a-days genetic polymorphism in medicinal plants has been widely studied to

distinguish plants at inter- and/or intra-species level (Joshi et al., 2004).

Morphological traits are also commonly used to determine relationships but

they do not provide good estimates of genetic distance because they are influenced by

the environment and they are not variable enough to adequately characterize genetic

differences among elite genotypes (Smith and Smith, 1992). Molecular markers have

also been used to quantify genetic diversity in plants (Clegg, 1990). The advantages

of using molecular markers are to allow direct comparisons of genetic similarity to be

made at the DNA level (Newbury and Ford-Lloyd, 1993). They are not affected by

plant development and also they are not modified by the environment and they are

very abundant (Novy et al., 1994). In the last decade, molecular markers such as

RFLP, RAPD, SCAR and AFLP have been used to assess the genetic variation at the


DNA level, allowing an estimation of the degree of relatedness between individuals

without the influence of environmental factors (Miller and Tanksley, 1989; Pandian et

al., 2000). Factors such as speed, efficiency and amenability to automation make

RAPD analysis the most suitable method for effective germplasm management with

respect to estimating diversity, monitoring genetic erosion and removing duplicates

from germplasm collections (Virk et al., 1995).

Commercial venture in the herbal medicines increasing the demand for the

medicinal plants which lead to irreplaceable loss of naturally occurring plant species.

To meet the ever increasing demand for this valuable medicinal plant, it is necessary

to find out the superior varieties in terms of action against diseases and disorders and

to produce them in large scale. Culturing of callus tissue, cell suspensions, and

isolated roots are the major tissue culture technologies employed so far for the

characterization and evaluation of important secondary metabolites from plants

(Rhodes et al., 1987).

During the last two decades there has been an upsurge in the search for new

plant-derived drugs containing medicinally useful alkaloids, glycosides,

polyphenolics, steroids, and terpenoid derivatives. Farnsworth et al. (1985) identified

119 secondary metabolites, isolated from higher plants that were being used globally

as drugs.

Different environmental conditions can also affect the chemical composition

of the plants (Khan et al., 2010). The biosynthesis of secondary metabolites varies

among plants, even in different organs of plants and their biosynthesis depends on the

environmental factors in which they grow. Intra-specific variation in

phytoconstituents has been documented extensively among the plants (Chew and

Rodman, 1979; Johnson and Scriber, 1994). Differences in biosynthesis can result

from both genetic and phenotypic variations. Phenotypic variation is especially

pronounced in the physiological responses of a plant under growth conditions. Many

environmental factors like precipitation, mean temperature, soil, wind speed, low and

high temperature extremes, duration of snow-cover, length of the vegetation period

and the intensity of radiation also known to influence the biochemistry of medicinal

plants (Korner, 1999). Moreover, study on phytochemicals of wild populations of

plant at different altitudes were performed and it is not conclusive whether the


observed variations are the response of individual plants to environmental factors

related to altitude or a genetic adaptation of the populations growing at different

altitudes to their specific environment (Mc Dougal and Parks, 1984; Polle et al., 1992;

Veit et al., 1996; Ruhland and Day, 2000; Zidorn and Stuppner, 2001; Zidorn et al.,

2005). However, biochemistry of P. niruri growing in different geographical regions

of India and environmental factors is fluctuating at various altitudes. In view of the

importance of this species, its large scale multiplication and cultivation of quality

planting material (based on the content of active ingredients) is urgently required.

A wide range of plant species belonged to the genus Phyllanthus have been

phytochemically investigated. Among the studied species, P. niruri, P. urinaria, P.

emblica, P. flexuosus, P. amarus, and P. sellowianus have received the most

phytochemical and biological attention. According to the available literature, research

has either been focused on isolating all the substances in these plants, or on

determining a specific class of natural products (Calixto et al., 1998). Intensive

phytochemical examinations of this plant have been carried out by scientists belonged

to several countries. Phytochemical constituents such as alkaloids, flavonoids,

lignans, tannins, phenols and terpenes have been identified. However, the composition

of the aqueous extract, used for medicinal purposes, has not been adequately studied.

Although the specific compounds have not been precisely defined, some research

results give valid credit for the therapeutic action of urinary tract stones to the phenols

(Ishmaru et al., 1992; Calixto et al., 1998)

The aerial parts of P. niruri have been reported to contain phytochemical

compounds as mentioned in the previous paragraph. Some of these isolated

compounds have been tested for their pharmacological activities (Ishimaru et al.,

1992, Calixto et al., 1998; Huang et al., 2003; Naik and Juvekar, 2003). Lignans from

this plant have been studied most intensively and so far 17 different lignans have been

identified. Several of these lignans were tested for cytotoxicity and other biological

activities in vitro. Phyllanthin and hypophyllanthin were found to be protective

against carbon tetrachloride and galactosamine induced cytotoxicity in primary

cultured rat hepatocytes (Syamasundar et al., 1985). The major pharmacological

active compounds are gallotannins (e.g. phyllanthusiin-D, amariin, geraniin and


corilagin (Foo and Wong, 1992; Foo, 1993) and the lignans - phyllanthin and

hypophyllanthin.

Phyllanthus niruri Linn. is widespread in the tropical and temperate regions of

the world. It was named as „stone breaker‟ by the indigenous people of Africa and

used as a effective remedy to completely remove gallstone and kidney stones. It has a

wide number of traditional uses employing the whole plant for jaundice, gonorrhea,

frequent menstruation and diabetes. The plant is topically used as poultice for skin

ulcer, sores, swellings and itchiness. An aqueous extract of this plant possesses anti-

hepatitis B virus surface antigen activity in both in vivo and in vitro studies

(Thyagarajan et al., 1988; Calixto et al., 1998).

Since allopathic medicines can not give complete cure for the liver disorders

caused by hepatitis B viruses, current research has been focused on Phyllanthus niruri

as a potential plant for the treatment of this deadly dangerous diseases by suppressing

the growth and replication of the virus (Venkateswaran et al., 1987; Mehrota et al.,

1990; Thyagarajan et al., 1982; Yeh et al., 1993; Jayaram and Thyagarajan, 1996; Lee

et al., 1996; Calixto et al., 1998).

Herbal medicines for liver disease have been used in India for a long time and

have been popularized world wide by leading pharmaceuticals. Despite the significant

popularity of several herbal medicines in general, and for liver diseases in particular,

they have not become acceptable treatment modalities for liver diseases. The limiting

factors that contribute to this eventuality are (i) lack of standardization of the herbal

drugs, (ii) lack of identification of this active ingredient(s), (iii) lack of randomized

controlled clinical trials (RCTs), and (iv) lack of toxicological evaluation (Thyagrajan

et al., 2002).

Conventional propagation of this medicinally important plant is achieved

through seeds, but the viability of the seeds is limited to very few months. The

percentage of germination is drastically reduced during the storage period and is

completely lost with in a year. The embryos are also very small when present and

most of the seeds are abortive (Anon, 1990). Based on the preliminary studies

conducted on seed propagation, specific habitat conditions are required for its survival


and growth. Germination is also very slow. There is no reliable method for vegetative

propagation of this plant. All such factors, coupled with unsustainable and

indiscriminate harvesting from the wild, have posed threats to this species. Thus,

conventional propagation through seeds is not sufficiently reliable or adequate to meet

the demand for planting material. Hence, development of an in vitro propagation

method will be of great importance for the production of planting material to build up

the resource base of this particular species (Santos et al., 1994).

Based on these back ground information, the present study was justifiably

designed with the following objectives for the effective utilization of Phyllanthus

niruri as medicinally important one.

1. Collection of Phyllanthus niruri plant specimens and their seeds from the

different locations of Tamilnadu.

2. Determination of the degree of variability in plant populations using

morphometric and RAPD analysis.

3. Determination of the phytochemical compounds from all the collected plant

specimens by gas chromatography and mass spectrometry.

4. Screening of the antibacterial efficiency of the plant against some important

human pathogenic organisms.

5. Determination of the hepatoprotective potential of P. niruri by using animal

models.

6. Standardization of the in vitro culture techniques for the micropropagation of

selected superior accession of P. niruri.

The thesis encompasses six chapters including Introduction, Review of Literature,

Materials and Methods, Results, Discussions, Summary and References. The research

findings are spread over 16 plates, 28 tables and 14 figures. Results are statistically

analysed.


2. REVIEW OF LITERATURE

Phyllanthus niruri L. is an valuable medicinal plant which has been used for

centuries in ancient Hindu system of medicine i.e. „Ayurveda‟ to cure gallstones,

jaundice and diseases of urinogenital system. It is a subtropical plant of great value,

which plays an important role in health improvement around the world. Every part of

this plant has been investigated as a source of valuable compounds. The aim of the

present study was to assess the genetic diversity by RAPD, phytochemistry,

antibacterial activity, hepatoprotective property and in vitro response of this potential

medicinal herb. As this medicinal plant is widely used in several health disorders, it

has been subjected to intensive research. The earlier reports relevent to objectives of

present context are reviewed and presented here.

2.1. Biogeography and Ecology

Phyllanthus niruri is a small plant widely distributed in tropical and

subtropical regions of Central and South America, Asia including India and

Indonesia, Africa and the West Indies (Mehrota et al., 1990; Eisei, 1995; Unander,

1995a; Calixto et al., 1998). This is a common weed which can be found along the

roads, in valley, on the riverbanks and near lakes. It can grow well in moist, shady and

sunny places (Cabieses, 1993; Nanden, 1998).

2.2. Taxonomy

This plant species belongs to Euphorbiaceae, a large family of upright or

prostrate herbs or shrubs often with milky juice (Lewis, 1977). The plant genus

Phyllanthus is a large one consisting 550 to 750 species under sub genera:

Botryanthus, Cicca, Conani, Emblica, Ericocus, Gomphidium, Isocladus, Kirganelia,

Phyllanthodendron, Phyllanthus, and Xylophylla (Unander et al., 1995b; Calixto et

al., 1998).

The most common species of the genus Phyllanthus found in most of the

West African countries including Nigeria are Phyllanthus niruri and Phyllanthus

amarus. P. niruri and P. amarus are very closely related in appearance and in

phytochemical structure. The major difference between these two is that P. niruri has


larger leaves and the plants as a whole is bigger when compared to P. amarus.

Reorganization of the Phyllanthus genus has been however classified as P. amarus as

type of P. niruri (Ekwenye and Njoku, 2006). P. amarus is usually misidentified with

the closely related P. niruri in appearance, phytochemical structure and history of use

(Morton, 1981).

2.3. Herbal medicines and their pharmacological uses

Traditional systems of medicine has been in vogue for centuries in all over

the world. According to one estimate, 80 % of the world population still depends on

herbal products for their primary healthcare needs. The toxic side effect of the drugs

of modern medicine and the lack of medicines for many chronic ailments has led to

the reemergence of the herbal medicine, with possible treatments for many health

problems. Consequently, the use of plant-based medicine has been increasing in all

over the world (British Medical Association, 1993). Varieties of plants and growing

conditions according to geographical origin often play a part in determining the

quality and efficacy of these herbals (Kamboj, 2000). A rapid and accurate analytical

technique is necessary to check if these factors cause wide difference in the samples

and therefore their quality. Recently there is an increase in interest in the search of

potential drugs of plant origin that are capable of minimizing the toxicity induced by

chemotherapy to normal cells without compromising its anti-neoplastic activity.

Traditional system of Indian medicine extensively used to derive some compounds in

plants and formulations to modulate the immune system of the host. These herbal

formulations were found to be either less toxic or non-toxic (Kamboj, 2000).

Different plant parts of P. niruri were ethnobotanically reported to have

various therapeutic activities e.g. leaves as expectorant, diaphoretic and useful in

strangury and sweats; the seeds as carminative, laxative, astringent to the bowels,

tonic to the liver, diuretic, diaphoretic, useful in bronchitis, ear ache, griping,

opthalmia and ascites (Kirtikar and Basu, 2001). An aqueous infusion of the whole

plant, which is a typical preparation, is employed as a stomachic, aperitive,

antispasmodic, diuretic, against constipation, fever including malaria, dysentery,

gonorrhea, syphilis, tuberculosis, cough, diarrhea and vaginitis (Paranjape, 2001).

Fresh root is a remedy for jaundice. Leaves are stomachic. Milky juice is used as


application to offensive sore and a popular remedy against fever and infusion of

young shoots is given in dysentery. This species is also used in stomach ailments such

as dyspepsia, colic, dropsy, urinogential problems and also as external applications for

edematous swelling and inflammation (Chopra et al., 1956; Calixto et al. 1998).

Whole plants have been used in traditional medicine in Central and South America

and Asia (including India and Indonesia)for the treatment of jaundice, asthma,

hepatitis and malaria and for its diuretic, antiviral, and hypoglycemic properties

(Mehrota et al., 1990; Eisei, 1995; Calixto et al., 1998).

2.3.1. Antiviral activity

Hepatitis B is one of the major diseases inflicting human population.

Conventional treatment with interferon – alpha is very expensive and has many

serious side effects. Alternative herbal medicine using extracts of Phyllanthus niruri

and Phyllanthus urinaria has been reported to be effective against Hepatitis B and

other viral infections (Meixa et al., 1995). The genus Phyllanthus has been intensively

studied clinically for its antiviral effects. A systematic review of 22 randomized

clinical trial showed that Phyllanthus species have positive effects on antiviral activity

and on liver biochemistry in chronic hepatitis B virus infection (Calixto et al., 1998;

Liu et al., 2001). Phyllanthus amarus Schum and Thonn is an another important

medicinal plant species due to its antiviral properties and useful against hepatitis

infection (Bratati et al., 1990; Joy and Kuttan, 1998; Raphael et al., 2002).

An aqueous extract of P. niruri was found to inhibit the hepatitis B virus

(Thyagarajan et al., 1988) and also inhibits endogenous DNA polymerase of hepatitis

B virus and binds to the surface antigen of hepatitis B virus in vitro (Venkateswaran

et al., 1987). Aqueous extracts containing tannin, lignan and other isolated

compounds from Phyllanthus species have been tested for their anti- HIV activity in

vitro and in vivo. They inhibited the HIV-key enzymes like integrase, reverse

transcriptase and protease (Thyagarajan et al., 1988; Calixto et al., 1998; Shead et al.,

1992; Notka et al., 2004). P. amarus was also proved to be potential plant for the

treatment of hepatitis B by suppressing the growth and replication of the virus

(Mehrota et al., 1990, Yeh et al., 1993; Jayaram and Thyagarajan, 1998; Lee et al.,

1996). The most recent research on P. niruri reveals that its isolated molecule

niruriside‟s antiviral activity extends to human immunodeficiency virus by inhibiting


the reverse transcriptase enzyme (Qian-Cutrone, 1996). Its antiviral activity extends to

HIV-1 RT inhibition (Ogata et al., 1992, Naik and Juvekar, 2003). Nirtetralin and

niranthin were tested against human hepatitis B virus in vitro (Huang et al., 2003).

Additional studies on callus and root extracts of different species of

Phyllanthus have shown the presence of phyllemblin, a tannin which has

antimicrobial activity, and the hydrolyzable tannins inhibited DNA polymerase and

reverse transcriptase, of geraniin and its derivatives which showed high activity in the

inhibitions of HIV reverse transcriptase and angiotensin-converting enzyme involved

in diabetic complications (Ueno et al. 1988; Ogata et al. 1992; Unander, 1996).

Recently, seven ellagitannins isolated from P. urinaria showed activity against

Epstein-Barr virus DNA polymerase at a micromolar level, and the lignans

phyllmyricin B and retrojusticidin B showed strong inhibition against HIV-RT. These

results present an additional potential use of this herb against several DNA viruses

including oncogenic Epstein-Barr virus and retroviruses human (Liu et al., 1999).

2.3.2. Antiplasmodial activity

In vitro antiplasmodial activity of this plant extract has been described by

Tona et al. (2000). The in vitro and in vivo antiplasmodial activity of the ethanolic

and dichloromethane extracts as well as the toxicity of the lyophilized aqueous extract

of P. niruri have been previously reported by Tona et al. (1999; 2000).

2.3.2. Antidiabetic activity

In Brazil, infusion of leaves, stems and roots of Phyllanthus species has

used in folk medicine for treating intestinal infections, diabetes and disturbances of

the kidney (Calixto et al., 1998). An alcoholic extract of P. niruri was found to reduce

significantly the blood sugar in normal rats and in alloxan diabetes rats, and indicates

its potential antidiabetic action (Raphael et al., 2000). P. niruri extract also showed

inhibitory activities against angiotensin converting enzyme (ACE) and aldose

reductase (AR), which play a significant role in the reduction of aldose to alditol

under abnormal conditions such as diabetes (Shimizu et al., 1989). P. niruri was also

used as a hypoglycemic agent in traditional medicine to control non-insulin dependent

Diabetis mellitus (Sivarajan and Balachandran, 1994).


2.3.3. Analgesic activity / Antinociceptive effects

In South India, an infusion of the leaves is given for headache (Kirtikar and

Basu, 1987). An extract of the callus culture of P. niruri showed analgesic activity

(Santos et al., 1994). Methanol and ethanol extracts of dried callus tissue of P. niruri

administered intraperitonially (10 mg/kg) to mice and showed antinociceptive effects

on 5 different models of nociception (Olive-Bever, 1986). Main compounds identified

in the extracts of P. niruri like flavonoids, tannins, terpenes, sterols, alkaloids and

phenols were found to be responsible for the antinociceptive activity (Santos et al.,

1994; Catapan et al., 2000). Phytosterols, quercetin, gallic acid ethyl ester and

geraniin were identified in P. caroliniensis and among them quercetin, gallic acid

ethyl ester and some flavonoids were found to have antinociceptive action in mice

(Filho et al., 1996).

2.3.4. Urolithiasis

Indigenous people of Amazon are calling this herb as „stone breaker‟ and

it has been used as an effective remedy to eliminate gallstone and kidney stones by

them (Mello, 1980). P. niruri is used in Brazilian folk medicine for patients with

urolithiasis (Paulino et al., 1996). Previous clinical studies demonstrated that P. niruri

had no acute or chronic toxicity, and preliminary data suggested the effects, which

promote stone elimination in stone-forming patients, as well as the normalization of

calcium levels in hypercalciuric patients (Nishiura et al., 2004). Experimental studies

had shown that P. niruri reduced the uptake of calcium oxalate crystals by MDCK

cells, without evidence of cytotoxicity or biochemical alterations of the culture

medium (Campos and Schor, 1999). Moreover, it prevented the growth of calculi in a

model of CaOx-induced urolithiasis in rats (Freitas et al., 2002). It was also reported

that with the CaOx crystallization process in vitro by reducing crystal growth and

aggregation and favoured the formation of a less adherent dihydrate CaOx crystalline

structure (Barros et al., 2003). Its role in urolithiasis was proved to inhibit the

calcium oxalate endocytosis by renal tubular cells of experimental rats (Campos and

Schor, 1999; Freitas et al., 2002).


2.3.5. Cardioprotective

The studies of the antioxidative and cytoprotective effects using H9C2

cardiac myoblasts showed that Phyllanthus urinaria has a protective activity against

doxorubicin cardiotoxicity. This protection was mediated through multiple pathways

such as enhancement of survival factor through elevation of glutathione, activation of

catalase/superoxide dismutase activity and inhibition of lipid peroxidation. This plant

may serve as an alternative source of antioxidants for the prevention of doxorubicin

cardiotoxicity (Chularojmontri et al., 2005).

2.3.6. Lipid lowering activity

Liver damage is followed by complex disturbances in the lipolytic activity

of the vascular space which often appeared with hyperlipoproteinemia in patients

(Vadivelu and Ramakrishnan, 1986). Abnormalities with lipid metabolism have been

reported in cholesteosis (Seidel and Wall, 1983), alcoholism (Chander et al., 1988)

chemical intoxication (Dwivedi et al., 1990) and hepatitis (Dudnik et al., 2000). P.

niruri was reported to possess lipid lowering activity (Khanna, 2002). In a 2002 study,

Indian researchers reported that „chanca piedra‟ increased bile acid (Khanna, 2002)

secretion (demonstrated choleretic activity) and significantly lowered blood

cholesterol levels in rats. Administration of alcoholic extracts of P. niruri in triton

induced hyperlipidaemia rats, lowered the elevated level of low-density lipoprotein

lipids (Chandra, 2000).

2.3.7. Antitumor and anticarcinogenic activity

3, 4-methylenedioxybenzyl-3, 4-dimethoxybenzylbutyrolactone from P.

niruri has been reported to possess antitumor activity (Satyanarayana and

Venkateswarlu, 1991). Antitumor and anticarcinogenic activities of Phyllanthus

amarus have also been reported by Rajeshkumar et al. (2002). P. amarus extract

administration has been shown to inhibit the liver tumour development induced by N-

nitrosodiethylamine in rats and increased the life span of hepatocellular carcinoma

harboring animals (Joy and Kuttan, 1998; Rajeshkumar and Kuttan, 2000). Free

radicals, from both endogenous and exogenous sources, are implicated in the etiology

of several degenerative diseases, such as coronary artery diseases, stroke, rheumatoid

arthritis, diabetes and cancer (Halliwell et al., 1992). Immunomodulating effects in


treatment of cancer by influencing the function and activity of the immune system has

been reported (Ma‟at, 2002). Lignans such as phyllanthin, hypophyllanthin,

flavanoids, quercetin, astragalin, ellagitannins and hydrolysable tannins are shown to

be present in this plant. Some of these compounds have been shown to have

significant activity against experimental carcinogenesis (Calixto et al., 1998).

2.3.8. Anti-inflammatory activity

Aerial parts of P. amarus exhibited marked anti-inflammatory properties and

suggest that these lignans are the main active principles responsible for the traditional

application of this plant for the inflammatory complaints (Kassuya et al., 2005).

2.3.9. Pharmacognosy and Genetic transformation

Utility of Phyllanthus roots in traditional systems of treatment has been

studied (Anonymous, 1969). But the number of studies into the medicinal potency of

Phyllanthus roots has been limited. This may be due to constrains faced in the natural

collection of roots which are much less in quantity compared to the aerial parts. To

augment the availability of this plant organ as an alternative source of bioactive

compound, root culture or hairy root culture may be ideal. Hairy roots, produced by

genetic transformation through Agrobacterium rhizogenes, a soil bacterium, have

proved to be more potent than the roots obtained by the conventional root culture

method in respect of biomass production (Rhodes et al., 1987). Root culture, among

all the techniques, has been used more frequently because, being organized structures,

roots are more amenable to maintaining genetic stability even after a prolonged period

of culture (Arid et al., 1988). During herbal drug market survey it was observed that

P. amarus, P. fraternus and P. maderaspatensis are being sold under the trade name

„Bhuiamlki‟ in mixed form. Very little work on pharmacognostical studies on two

species of P. amarus and P. fraternus is on record (De and Datta, 1990; Bagchi et al.,

1992).

2.3.10. Antibacterial activity

The discovery, development and clinical use of antibiotics during the

nineteenth century have substantially decreased public health hazards resulting from

bacterial infections. However, there has been a parallel and alarming increase in

bacterial resistance to existing chemotherapeutic agents as a result of their injudicious


use. In addition, antibiotics are occasionally associated with adverse effects to the

host, including hypersensitivity, immune-suppression and allergic reactions (Ahmad

et al., 1998)

These developmental demands that a renewed effort to be made to seek

antibacterial agents effective against pathogenic bacteria resistant to current

antibiotics. One possible strategy is the rational localization of bioactive products

from folk medicines, with the hope that systematic screening of these will result in the

discovery of novel effective compounds with potent and useful activities against

microbes. There is an ever-increasing demand for plant-based therapeutics in both

developing and developed countries due to a growing recognition that they are natural

products, non-narcotic and no side effects in most cases and are easily available at

affordable prices (Lewis and Elvin-Lewis, 1977; Bruneton, 1999).

A number of pathogens have developed resistance (Cohen, 1992; Gold and

Moellering, 1996) to multiple antibiotics (Multiple Drug Resistance), threatening to

develop complete immunity against all antimicrobial agents and therefore be

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

importance. The increasing prevalence of multidrug resistant strains of bacteria and

the recent appearance of strains with reduced susceptibility to antibiotics raises the

specter of untreatable bacterial infections and adds urgency to the search for new

infection-fighting strategies (Sieradski et al., 1999).

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

they contain components of therapeutic values (Kaushik, 1985). 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 (Nostro et al., 2000). The systemic screening of antimicrobial plant extracts

represents a continuous effort to find new compounds with the potential to act against

multiresistant pathogenic bacteria and fungi. A special feature of higher angiospermic

plants is their capacity to produce a large number of organic chemicals of high

structural diversity. The so-called secondary metabolites (Evans et al., 1997), which

are divided into different categories based on their mechanism of function like


chemotherapeutic, bacteriostatic, bactericidal and antimicrobial (Purohit and Mathur,

1999). The accumulation of phytochemicals in the plant cell cultures had been studied

for more than thirty years and the generated knowledge had helped in realization of

using cell cultures for production of desired phytochemicals (Castello et al., 2002).

Plant-based remedies have been highlighted due to their fewer side effects in

comparison to synthetic drugs and antibiotics. Successful transformation technology

is thought to be one of the most reasonable approaches to enhance the production of

secondary metabolites through genetic manipulation of biosynthetic pathway (Mann

et al., 2000).

Several bacterial infections are associated with the risk of certain cancer, and

viruses are now recognized as the second most important cause of human cancer.

Many chemicals produced in plants are being examined for their potential to inhibit

human pathogens (Mulligen et al., 1993). In recent years there has been a resurgence

of interest in medicinal plants that are effective, safe and culturally acceptable as an

alternative treatment for many human diseases (Atmani et al., 2003).

Antifungal and antibacterial properties were recorded in P. urinaria (Cruz et

al., 1994), P. fraternus (Ramchandani and Chunganth, 1998), P. embilica (Jasril et

al., 1999), antibacterial in P. amarus (Vinayagamoorthy, 1982; Verpoorte and Dihal,

1987; Kannan and Venkatakrishnan, 2002), P. discoideus (Mensah et al., 1990;

Olukoya et al., 1993). The plant has also been reported to possess antifungal,

antibacterial and antiviral activities (Verpoorte and Dihal, 1987).

Many countries have maintained research programs to screen traditional

medicines for antimicrobial activity, as is the case of India (Ahamed et al., 1995;

Valsara et al., 1997; Perumalsamy and Ignachimuthu, 2000; Ahmad and Beg, 2001;

Kumar et al., 2006), Palestin (Ali-Shtayeh et al., 1998; Essawi and Srour, 2000),

Africa (Baba-Moussa et al., 1999), Italy (Panizzi et al., 1993), Cuba (Martinez et al.,

1996), Honduras (Lentz et al., 1998), Jordan (Mahasneh et al., 1999), Indonesia

(Goun et al., 2003), China (Janovska et al., 2003) and Brazilian south east region

(Oliveira et al., 2007).

Alcoholic extracts of various medicinal plants such as Adhatoda zeylanica

(George et al., 1947), Emblica officinalis, Terminalia chebula, T. belerica, Plumbago


zeylanica and Holarrhena anidysentrica (Ahamed et al., 1995), Thymus vulgaris and

T. origamum (Essawi and Srour, 2000), Cyperus rotundus (Puratchikody et al., 2001),

Tulbagia violacea (Invernizzi, 2002), leaf of Aloe vera (Agarry et al., 2005),

Andrographis paniculata (Xu et al., 2006) and R. communis (Al-zubaydi, 2009)

showed a prominent antibacterial activity against deadly dangerous microorganisms.

Methanol extracts of various plants such as Euphobia hirta and Camellia

sinensis (Vijaya et al., 1995), Evolvoulus alsinoides (Purohit et al., 1995); rhizome

and leaves of Aristalochia paucinercis (Gadhi et al., 1999), Terminalia catappa,

Swietenia mahagoni, Phyllanthus acuminatus, Ipomoea spp., Tylophora asthmatica

and Hyptis brevipes have the antibacterial activities (Goun et al., 2003); and aerial

parts of Anthemis tinctoria (Akgul and Saglikoglu, 2005), leaves of Cassia alata

(Owoyale et al., 2005), Toddalia asiatica, Syzygium lineare, Acalypha fruticosa and

Peltoporum pterocarpum (Duraipandiyan et al., 2006) also showed highly inhibitory

effect against several pathogenic bacteria.

The aqueous extracts of several medicinal plants such as Acalypha wilkesiana

(Alade and Irobi, 1993), Lawsonia inermis, Eclipta alba, Nyctanthes arbour-tristis,

Vinca rosea, Datura stramonium, Cleome gynandropsis and Ageratum conyzodies,

Tridax procumbens, Cleome viscose, Acalypa indica and Boerhaavia erecta

(Perumalsamy et al., 1999), Cassia accidentalis and C. auriculata (Perumalsamy and

Ignachimuthu, 2000), Azardirachta indica (leaf, stem and bark of neem) (Arora et al.,

2005), Andrographis paniculata (Xu et al., 2006) and young stem, leaf and bark of

neem (Azardirachta indica L.) (Ghangaonkar and Mukadam, 2006) were found to

possess active principles against the growth of pathogenic microbes. The acetone

plant extracts of Cyperus rotundus recorded high antibacterial activity (Puratchikody

et al., 2001). Phenolic and flavonoid extracts of several medicinal plants showed

antibacterial activity (Al-zubaydi, 2009).

2.3.11. Hepatoprotective activity

Drug-induced liver injury is a major health problem that challenges not only

health care professionals but also the pharmaceutical industry and drug regulatory

agencies. According to the United States Acute Liver Failure Study Group, drug-

induced liver injury accounts for more than 50% of acute liver failure, including


hepatotoxicity caused by overdose of acetaminophen (39%) and idiosyncratic liver

injury triggered by other drugs (13%) (Michael and Cynthia, 2005). Liver damage is

generally associated with cellular necrosis, increase in tissue lipid peroxidation and

depletion in the tissue GSH levels. In addition serum levels of many biochemical

markers like SGOT, SGPT, triglycerides, cholesterol, bilirubin, alkaline phosphatase

are elevated (Mascolo et al., 1998). Hepatotoxicity of CCl4 causes the formation of

trichloromethyl and trichloromethyl peroxyl radicals, initiating lipid peroxidation and

resulting in fibrosis and cell necrosis (Recknagel et al., 1989).

Herbal medicines have been used in the treatment of liver diseases for a

long time. In many Asian countries, the species of Phyllanthus has long been used in

folk medicine for liver protection (Gamble, 1956; Thyagarajan and Jayaram, 1992). A

number of herbal preparations are available in the market (Dhiman and Chawla,

2005). P. niruri is used as one of the components of a multiherbal preparation for the

treatment of liver ailments (Kapur et al., 1994). Among the herbals used for

hepatoprotection, Phyllanthus niruri is a well-known hepatoprotective herbal plant.

The aerial parts of P. niruri, known in Brazilian folk medicine as “quebra-pedra”

(stone breaker), was widely used as a tea in the treatment of genitourinary and liver

disorders (Venkateswaran et al., 1987; Santos, 1990). The hexane isolated fractions of

P. niruri are reported to be hepatoprotective against carbon tetrachloride and

galactosamine induced cytotoxicity in primary cultured rat hepatocytes

(Shyamasundar, 1985).

Phyllanthus amarus (Euphorbiaceae) is widely used against various liver

disorders (Bhattacharyya and Bhattacharya, 2001). It has been traditionally used in

the treatment of a variety of ailments including hepatic disorders (Nadkarni, 1976;

Kirtikar and Basu, 1993). This herb has a potent free radical scavenging activity and

could scavenge superoxides and hydroxyl radicals and can inhibit lipid peroxides (Joy

and Kuttan, 1995). Free radicals, from both endogenous and exogenous sources, are

implicated in the etiology of several degenerative diseases, such as coronary artery

diseases, stroke, rheumatoid arthritis, diabetes and cancer (Halliwell et al., 1992).


Mohammed Saleem et al. (2008) demonstrated the hepatoprotective effect of

alcoholic and water extract of Annona squamosa (custard apple) in hepatotoxic

animals with a view to explore its use for the treatment of hepatotoxicity in human. In

the isoniazid with rifampicin induced hepatotoxic animals there was a significant

decrease in total bilirubin accompanied by significant increase in the level of total

protein. ALP, AST, ALT and γ-GT levels were decreased in treatment group as

compared to the hepatotoxic group.

Hepatoprotective activity of methanol leaf extracts of Orthosiphon

stamineus against paracetamol induced hepatotoxicity in rats was investigated by

Maheshwari et al. (2008). Alteration in the levels of biochemical markers of hepatic

damage like SGOT, SGPT, ALP and lipid peroxides was tested in both paracetamol

treated and untreaed groups. Paracetamol (2 g/kg) has enhanced the SGOT, SGPT,

ALP and the lipid peroxides in liver. Treatment of methanolic extract of O. stamineus

leaves (200 mg/kg) has brought back the altered levels of biochemical markers to the

near normal levels in the dose dependent manner. The findings suggested that

O. stamineus methanol leaf extract possessed a significant hepatoprotective activity.

Hepatoprotective ayurvedic medicine - a multi herbal preparation (HPN–12)

containing Glycirrhiza glabra, Pichorhiza kurroa, Berberis aristata, Piper longum,

Phyllanthus niruri, Solanum dulcamara, Zingiber officinale, Curculigo orchioides,

Elettaria cardamomum, Tinospora cordifolia, Desmodium trifolium and Saccharum

officinarum, when orally administered to male albino rats at 1ml/100g body weight

was found to be effective against liver damage (Latha and Rajesh, 1999).

Animals with carbon tetrachloride induced hepatopathy were treated with

„catliv‟ containing extracts of Swertia chirata, Eclipta alba, Fumaria vaillanti,

Picorrhiza kurroa, Andrographis paniculata and Phyllanthus niruri at 25 ml orally,

twice daily for six days starting at 48 hours after administration of carbon

tetrachloride. On the basis of the result obtained it was concluded that the ingredients

of catliv, effectively helped in the regeneration of hepatic cells in calves (Pradhan,

2001).


Herbal preparations containing Andrographis paniculata and Phyllanthus

amarus for various liver disorders have been proved to have antihepatotoxic activity

(Ram, 2001). CCl4 induced hepatotoxicity in the liver of rats, as judged by the raised

serum enzymes, glutamate oxaloacetate transaminase and glutamate pyruvate

transaminase, was prevented by the pretreatment with the extracts of Phyllanthus

niruri, demonstrating its hepatoprotective action (Harish and Shivanandappa, 2006).

2.4. RAPD analysis

Different methods have been used to assess the diversity of plant breeding

materials. This information can be obtained by studying pedigrees and determining

the points of origin of the breeding germplasm. However, reliable and detailed

pedigree or accession records are not always available. Morphological traits are also

commonly used to determine relationships but they do not provide good estimates of

genetic distance because they are influenced by the environment and they are not

variable enough to adequately characterize genetic differences among elite genotypes

(Smith and Smith, 1992).

Correct genotype identification of the plant material, therefore, remains

important for protection of both the public health and industry. Chemoprofiling and

morphological evaluation are routinely used for identification of the plants. Chemical

complexity and lack of therapeutic marker(s) are some of the limitations associated

with chemical approach while subjective bias in morphological evaluation limits the

use.

Molecular biology offers various techniques that can be applied for plant

identification (Techen et al., 2004). Genetic polymorphism in medicinal plants has

been widely studied which helps in distinguishing plants at inter- and/or intra-species

level (Joshi et al., 2004). Among others the assessment of genetic diversity of the

germplasm has been used by plant breeders for numerous reasons like selection of

parents, germplasm management and germplasm protection (Lee, 1995).

In the last decade, molecular markers such as RFLP, RAPD, SCAR and

AFLP have been used to assess the genetic variation at the DNA level, allowing an


estimation of the degree of relatedness between individuals without the influence of

environmental factors (Miller and Tanksley, 1990; Pandian et al., 2000). Molecular

phylogenetic studies have substantially increased the understanding of the systematics

of Euphorbiaceae sensu lato (s.l.) (Samuel et al., 2005). Molecular markers have also

been used to quantify genetic diversity in plants (Clegg, 1990). The advantages of

using molecular markers are that they allow direct comparisons of genetic similarity

to be made at the DNA level (Newbury and Ford-Lloyd, 1993), they are not affected

by plant development, they are not modified by the environment and also they are

very abundant (Novy et al., 1994)

Factors such as speed, efficiency and amenability to automation which

make RAPD analysis is the most suitable method for effective germplasm

management with respect to estimating diversity, monitoring genetic erosion and

removing duplicates from germplasm collections (Virk et al., 1995). Two major

factors may be responsible for this variation are the difficulties in maintaining

homogeneity in harvesting the P. amarus population from a plethora of closely

resembled Phyllanthus species and the climatic variations resulting in biological

differences in plants occurring at various geographic regions (Lee et al., 1996).

Information about genetic relationships among accessions within and between species

has been used in several important applications and also in plant improvement

(Thormann et al., 1994).

A collection of P. amarus was made from various parts of India to

determine the extent of genetic variability using analysis at DNA level. RAPD

profiling of 33 collections from different locations, covering states of Tamil Nadu,

Karnataka, Maharashtra, Gujarat, Assam, West Bengal, Tripura, Uttar Pradesh,

Punjab and Haryana was generated (Jain et al., 2003). Analysis through UPGMA

revealed up to 65% variation among these accessions. However, intra-population

variation was found to be much larger in the accession from the southern part of the

country. Nevertheless, interpopulation variation also overlaps in the phylogenetic

clustering, which is understandable from the natural dissemination of this plant

species as a weed that has spread across the geographical boundaries.


Negi et al. (2000) used RFLP analysis to find out the relationship among

collection of W. somnifera from the mountain regions of Jammu and Kashmir

(Kashmiri type) and those from the plains of central parts of the India (Nagori type).

The cluster analysis seprated W. somnifera in to three sub classes corresponding to

Kashmir and Nagori groups and act an intermediate type. The RFLP profile of

Kashmir individuals was distinct from that of the Nagori and Kashmir individuals,

eventhough it was identified as a Kashmir morphotype. Furthermore, a low level of

variation was observed within populations, but high level of polymorphism was

observed between Nagori and Kashmiri populations.

Date palm (Phoenix dactylifera L.) varieties were fingerprinted using

fourteen random primers to detect the DNA polymorphism. Primer OPC-02 revealed

a 1400 bp fragment amplified in „Bugal White‟ (salinity tolerant) and „Khlas‟ which

is known to be drought tolerant. Primer OPD-02 distinguished „Bugal White‟, which

proved to be salinity tolerant, with a DNA fragment of about 1200 bp. Primer OPD-02

amplified a 1600 bp fragment in „Khashkar‟, „Bugal White‟, „Shaham‟ and „Khlas‟

(Kurup et al., 2009).

Inter and intra specific variation of two ginseng species Panax ginseng and P.

quinquefolius was studied by Artyukova et al. (2004) and estimated by studying 159

RAPD and 39 allozyme loci. Gene diversity in the total P. ginseng sample was

comparable with the mean expected heterozygosity of herbaceous plants. This

suggests that wild P. ginseng plants in various areas of the currently fragmented

natural habitat and cultivated plants of different origin have retained a significant

proportion of their gene pool. The mean heterozygosity calculated per polymorphic

locus for the RAPD phenotypes is similar to that of the allozyme loci and may be

helpful in estimating gene diversity in populations of rare and endangered plant

species.

The interactions between these factors can lead to complex genetic structures

within populations, which are often difficult to resolve. The use of biochemical and

molecular markers can enhance the understanding of such complexities (Dawson et

al., 1993). The assumption that forms the basis for such analysis is that genetic

structure as measured by neutral and selective genes reflects both deterministic and


stochastic evolutionary processes. Several reviews have described a detailed range of

molecular markers useful for assessing plant genetic diversity (Rafalski and Tingey,

1993; Staub et al., 1996). Molecular markers are the powerful tool for rapid and

efficient assess of genetic variability and have been used in germplasm banks and

breeding programs of various crop species (Rafalski and Tingey, 1993).

2.5. Phytochemistry

Plants produce a great number of secondary metabolites, many of them with

antibacterial and antifungal activity. Well-known examples of these compounds

include flavonoids, phenols and phenolic glycosides, unsaturated lactones, sulphur

compounds, saponins, cyanogenic glycosides and glucosinolates (Gomez et al., 1990;

Bennett and Wallsgrove, 1994; Grayer and Harborne, 1994; Osbourne, 1996). The

accumulation of phytochemicals in plant cell cultures has been studied for more than

thirty years, and the generated knowledge has helped in the realization of using cell

cultures for production of the desired phytochemicals (Castello et al., 2002). It is well

known that the plant species synthesize and accumulate various secondary metabolites

belonging to different phytochemical groups. In intact plants, the formation of these

metabolites is regulated in a coordinated fashion. Differentiation of plant cells or

tissues during development is implied in this process. On the other hand, plant cell

cultures are widely used for the comparison of biological activities of extracts,

fractions or isolated compounds from the intact plant material to that of cultured plant

material obtained in some experimental conditions (Santos et al., 1994; Sokmen et al.,

1999).

A great variety of species belonged to the genus Phyllanthus have been

phytochemically investigated and several molecules were isolated and identified.

Although most of these compounds are chemically known, their pharmacological

properties remain, in general, undetermined. Constituents such as alkaloids,

flavonoids, lignans, tannins, phenols and terpenes have been identified. However, the

composition of the aqueous extract, as used for medicinal purposes, has not been

adequately studied. Although the specific compounds have not been precisely defined,

some research results credit the therapeutic action on urinary tract stones to the

phenols (Ishmaru et al., 1992; Calixto et al., 1998). A wide range of plant species


belonged to the genus Phyllanthus has been phytochemically investigated. Among the

studied species, P. niruri, P. urinaria, P. emblica, P. flexuosus, P. amarus, and P.

sellowianus have received the most phytochemical and biological attention (Calixto et

al., 1998).

The complexity of the mixture of compounds and the presence of several

compounds in small concentrations can make the isolation and identification of these

substances present in this genus is very laborious. Different environmental conditions

can also affect the chemical constitution of the plants, and differing interpretation of

the spectral data of the complex structures has been reported to result in considerable

confusion (Khan et al., 2010). The choice of solvent in the isolation of compounds

has proved to be crucial, because the use of ethanol or methanol may lead to the

production of artefacts, e.g. ethyl gallates or methyl gallates, during the extraction

process (Calixto et al., 1998). Callus extracts of P. niruri, P. tenellus and P. urinaria

have the main compounds identified in the extracts were flavonoids, tannins and

phenols (Santos et al., 1994). The accumulation of phytochemicals in plant cell

cultures has been studied for more than thirty years, and the generated knowledge has

helped in the realization of using cell cultures for production of the desired

phytochemicals (Castello et al., 2002). Recently, seven ellagitannins isolated from P.

urinaria such as phyllmyricin-B and retrojusticidin-B etc., (Liu et al., 1999). Callus

cultures are also initiated for analytic and quantitative comparative studies of

secondary metabolites synthesis between the intact plant material and callus extracts

(Bahorun et al., 1994; El-Bahr et al., 1997; Rady and Nazif, 1997; Balz et al., 1999;

Zhentian et al., 1999).

Currently the various applications of genetic engineering are implemented in

medicinal plants to increase the production of secondary metabolites (Nisha et al.,

2003). The aerial parts of P. niruri have been reported to contain alkaloids,

flavonoids, phenols, coumarins, tannins, terpenoids and lignans. Several of these

isolated compounds have been tested for their pharmacological activities (Ishimaru et

al., 1992; Calixto et al., 1998; Huang et al., 2003; Naik and Juvekar; 2003). Lignans

from this plant have been studied most intensively; 17 different lignans have been

found so far. Several of these lignans were tested for cytotoxicity and other biological


activities in vitro. The lignans were found to enhance the cytotoxic response

mediators by vinblastine with multidrug resistant cultured cells (Somanabandhu et al.,

1993). The lignans such as niranthin, phyltetralin and nirtetralin isolated from aerial

parts of P. amarus and suggest that these lignans are the main active principles

responsible for the traditional applications (Kassuya et al., 2005). Phyllanthin and

hypophyllanthin were protective against carbon tetrachloride- and galactosamine-

induced cytotoxicity in primary cultured rat hepatocytes (Syamasundar et al., 1985).

Several compounds isolated from P. urinaria are known to have pharmacological

effects, especially rutin, β-amyrin, ellagic acid, geraniin, quercetin and β-sitosterol

(Calixto et al., 1998). Filho et al. (1996) isolated several compounds such as

alkaloids, tannins, flavonoids, lignans, phenols and terpenes and identified in various

species of Phyllanthus.

2.6. Tissue Culture

Tissue culture technology is a powerful tool for the conservation and rapid

multiplication of many threatened plant species (Fay, 1992). It has been particularly

useful for the conservation and rapid propagation of valuable, rare and endangered

medicinal species. The P. niruri is widely used in traditional medicine by simple

cultivation or collection from the wild (Unander et al., 1995a).

The conventional method of propagation of these species is through seeds.

However, poor germination potential restricts their multiplication. Micropropagation

technique offers an alternative method for cloning these plants (Unander, 1991;

Santos et al., 1994). In recent years, there has been an increased interest in in vitro

culture techniques which offer a viable tool for mass multiplication and germplasm

conservation of rare, endangered and threatened medicinal plants (Sahoo and Chand,

1998; Ajithkumar and Seeni, 1998). Commercial exploitation and elimination of

natural habitats consequent to urbanization have led to gradual extinction of several

medicinal plants. Micropropagation is an effective approach to conserve such

germplasms. Further, genetic improvement is another approach to augment drug-

yielding capacity of the plant (Tejavathi and Shailaja, 1999).

Few studies are available on the tissue culture of Phyllanthus spp. on

callus cultures of P. emblica, P. urinaria, P. amarus, P. abnormis, P. caroliniensis, P.


tenellus, and P. niruri and on transformed root cultures of P. niruri (Khanna and Nag,

1973; Unander, 1991; Ishimaru et al., 1992; Santos et al., 1994). Direct regeneration

has already been achieved (Johnson, 2006). However, the establishment of a

micropropagation protocol for P. niruri constitutes a useful tool for large scale plant

production, assuring continuous availability of plant material appropriate for the study

of factors that influence the production of the target secondary metabolites as well as

for strategies of in vitro culture to increase the yield of these active principles

accumulated in cultures of P. niruri. A way of obtaining genuine crude drug is being

limited due by large-scale destruction of natural habitat due to population pressure

and overexploitation, which have become a major threat to important bioresources of

P. niruri (Sangeeta and Buragohain, 2005). Considerable efforts have been made for

in vitro plantlet regeneration of P. amarus from shoot tips and nodal and internodal

segments (Bhattacharya and Bhattacharya, 2001; Ghanti et al., 2004).

Sivanesan (2007) compared different media (MS, SH and B5) for the shoot

multiplication from the shoot tip explants of mature plants of W. somnifera. MS

medium was found superior to SH and B5 medium. Similar observation was made in

Eclipta alba (Baskaran and Jayabalan, 2005). Liang and Keng (2006) developed a

protocol for a rapid production of P. niruri plantlets using nodal segments. Rapid and

efficient propagation of P. niruri using shoot tip culture for providing a better source

for continuous supply of plants in the manufacturing of drugs (Karthikeyan et al.,

2007). Kalidass and Mohan (2009) developed an efficient micropropagation protocol

for the medicinal plant P. urinaria Linn. using nodal segment for axillary shoot

proliferation.

The regenerated shoots were found to produce flower buds after 6 weeks of

culture in the medium supplemented with KIN (0.5 to 4 mg/l) and IAA (0.1 mg/l) in

Withania somnifera (Saritha and Naidu, 2007). Similar observation was made in

Ocimum sanctum (Karthikeyan et al., 2009). Rooting of regenerated shoots of

Physalis peruviana was occurred on hormone free MS medium (Jayasree et al.,

2005). Profuse rooting (46.8 per shoot) was induced by IBA (2.0 mg/l) with a root

length of 19.7 cm in Adhatoda vasica (Sangeetha and Buragohain, 2005). Highest

frequency of rooting (85%) was obtained in both apical and axillary bud derived

shoots of Heliotropium indicum in half strength MS medium supplemented with IBA


(0.1 mg/l) (Senthilkumar and Rao, 2007). About 90% of rooting was achieved in W.

somnifera on MS medium with IBA (3.6 µmol) (Ashutosh et al., 2004).

Micropropagated plants of Withania somnifera were hardened in half strength MS

medium and then established in sand and soil (1:1) mixture (Ujjwala Supe et al.,

2006). Rooted plants of Phyllanthus amarus were hardened on MS basal liquid

medium added sterile soil + vermiculite (1:1). The survival rate of plantlets in the

field was found to be very high (85%) (Ghanti et al., 2004). A maximum of 60%

survival rate was noticed in Macrotyloma uniflorum on a mixture of soil, sand and

manure (1:1:1 ratio) (Tejavathi et al., 2010). Rooted shoots of W. somnifera were

hardened successfully in garden soil - vermicompost (3:1 w/w) under a glass house

(Sabir et al., 2008).

Duangporn and Siripong (2009) investigated the effects of various

combinations of auxin and cytokinin on the callus growth and accumulation of

Phyllanthusol-A on P. acidus. A few reports are available for phytochemical analysis

in in vitro derived cultures. Based on this back ground information, the present study

was initiated on calli production and organogenesis in P. niruri. Therefore, the

development of an in vitro protocol is of critical importance as it will provide plants

that can be used for reintroduction in their natural habitats and for further chemical

analytical and pharmacological studies.


3. MATERIALS AND METHODS

The experimental material used was Phyllanthus niruri, popularly known as

“Stone breaker” one of the important medicinal plant often quoted in the “Siddha and

Ayurvedic” literatures. In the present study, the plants were collected from six

different locations namely Ooty, Palani, Madurai, Thanjavur, Kumbakonam, and

Nagapattinam in Tamilnadu, South India.

Classification:

Kingdom : Plantae

Division : Angiospermae

Class : Dicotyledonae

Sub class : Monochlamydeae

Series : Unisexuales

Family : Euphorbiaceae

Binomial : Phyllanthus niruri L.

Vernacular names:

Tamil : Kilanelli, Kilakkainelli

Hindi : Jamgli amli, Jaramla

Kannada : Kirunelli

Malayalam : Kilarnelli, Kilukanelli

Sanskrit : Bhumyamalaki

Bengali : Bhuiamla, Sadahazurmani

Marathi : Bhuivali

Telugu : Nela usirika

Botanical Description of Phyllanthus niruri: A herb that grows up to 20 - 60 cm

tall, erect, stem terete, younger parts rough, cataphylls 1.5-1.9 mm long, deltoid

acuminate; leaf 3.0-11.0 x 1.5-6.0 mm, elliptic oblong to obvate, obtuse or minutely

apiculate at apex, obtuse or slightly inequilateral at base; Flowers axillary, proximal

2-3 axils with unisexual 1-3 male flowers and all succeeding axils with bisexual


cymules. Male flowers -pedicel 1mm long, calyx 5, sub equal 0.7 x 0.3 mm, oblong,

elliptic, apex acute, hyaline with unbranched mid rib; disc segments 5, rounded,

stamens 3, filaments connate. Female flowers-pedicel 0.8-1.0 mm long, calyx lobes 5,

0.6 x 0.25 mm, ovate-oblong, acute at apex; disc flat deeply 5 lobed, lobes often

toothed at apex, styles 3, free, shallowly bifid at apex. Capsule 1.8 mm in diameter,

oblate and rounded, seeds about 0.9 mm long, triangular with 6-7 longitudinal ribs

and many transverse striations on the back (Gamble, 1967; Kirtikar and Basu, 1994).

Geographical distribution: Phyllanthus niruri grows wildly in all drier parts of

subtropical regions of India. It occurs in Madhya Pradesh, Andhra Pradesh, Punjab,

Tamilnadu and North Western parts of India like Gujarat and Rajasthan. P. niruri can

also be found in all the tropical regions of the world through the roads, valleys, on the

riverbanks and near lakes. It is wide spread throughout the tropics and subtropics in

sandy regions as a weed in cultivated and waste land (Ross, 1999).

Study materials and their source: Phyllanthus niruri L. belonged to Euphorbiaceae

was selected for the present study and extensive field trips were carried out to collect

the plants from the six different distinct locations such as Ooty (Centnary Botanical

garden- Hill area), Palani (Kodaikanal – Hill area), Madurai (Tamilnadu Agricultural

University campus- plain, dry habitat), Thanjavur (TNAU campus, Kattuthotam -

delta area), Kumbakonam (Govt. Arts college campus- delta area) and Nagapattinam

(sandy belt) of Tamil Nadu, South India and they were identified with the help of the

standard manuals such as „The Flora of the Presidency of Madras‟ (Gamble, 1967)

and Indian Medicinal Plants (Kiritikar and Basu, 1994). The identification was

confirmed at Rapinat Herbarium, St. Joseph‟s college (Autonomous), Tiruchirapalli,

Tamilnadu. Voucher specimen of each plant sample was dry-mounted, photographed

and preserved for future reference and deposited in the herbarium of the Post

Graduate and Research Department of Botany and Microbiology, A.V.V.M Sri

Pushpam College (Autonomous), Poondi-613502, Thanjavur district, Tamilnadu.

3.1. Analysis of physico-chemical properties of soil

The physico-chemical properties of soil samples such as pH, electrical

conductivity, nitrogen, phorphorous, potassium and micronutients, organic carbon,

zinc, copper, iron and manganese were analysed following the methods of Bernes


(1959); Muthuvel and Udhayasooriyan (1999). The soil samples were collected from

the six different localities of Tamilnadu viz, Ooty, Palani, Madurai, Thanjavur,

Kumbakonam, and Nagapattinam.

3.1.1. pH

Ten gram of air dried rhizosphere soil was taken in a beaker and 100 ml of

water was added to make a suspension of 1:10 (w/v) dilution and the pH was

determined by using digital pH meter (Systronics-335).

3.1.2. Electrical Conductivity

Ten gram of air dried rhizosphere soil was taken in a beaker and 100 ml of

water was added to make suspension of 1:10 (w/v) dilution and the electrical

conductivity was measured by using digital electrical conductivity meter (DEC-1-

USA).

3.1.3. Analysis of Soil Nutrients

The total nitrogen (N) and available phosphorus (P) were determined

respectively by micro-kjeldahl and molybdenum blue methods (Jackson, 1973).

Exchangeable K was extracted from the soil in ammonium acetate solution (pH 7) and

measured with a digital flame photometer (Jackson, 1973). The organic carbon (OC)

and organic matter (OM) present in the soil were estimated using rapid dichromate

oxidation method (Walkey and Black, 1934). The micronutrients such as Cu, Zn, Fe

and Mn were estimated by DTPA soil test described by Lindsay and Norvell (1978).

3.2. Morphometry

Morphological parameters of Phyllanthus niruri such as habit, plant height,

stem, flower, leaf size, floral structure, fruit shape were collected and determined

from the 6 different locations. In each sample, 10 replicates of the particular plant

parts from six plants each from wild populations were taken and mean length and

breadth of root were measured, calculated and tabulated. Dry weights of the plants

were obtained from each sample by oven drying at 80°C to get a constant weight.


3.3. RAPD analysis

3.3.1. Isolation of genomic DNA

Fresh leaf samples (young leaves) were collected from the field of above

study sites (1-2 month old) were used for the isolation of DNA. About 2 grams of

leaves were cut in to small bits and transferred to a prechilled mortar. The leaf tissue

was frozen in liquid nitrogen and ground in to a fine powder. The powder was

transferred to a centrifuge tube and added with 10 ml of preheated (65°C) extraction

buffer containing 1.5% (w/v) hexadecyl or cetyl trimethyl ammonium bromide

(CTAB) (Doyle and Doyle, 1987). 10 mM Tris HCL (pH 8.0), 1.4 M sodium chloride,

20 mM EDTA and 0.1% v/v 2-mercaptoethannol. The mixture was incubated in a

waterbath for 30 min at 65°C with occasional mixing. Equal volume of chloroform:

isoamyl alcohol (24:1) v/v was added, gently mixed for 15 min and centrifuged at

10,000 rpm for 20 min at room temperature (37°C).

The clear aqueous phase was transferred to a new tube and an equal volume

of isopropanol in cold ice was added and mixed gently by inversion until the DNA

was precipitated out (10-20 sec). The precipitated DNA was hooked out using a sterile

bent Pasteur pipette and air dried. The dried pellet was dissolved in 200-500 µl of TE

(10 mM Tris-pH 8.0, 1mM EDTA pH 8.0). The contaminant RNA was eliminated

from DNA by treating the DNA sample with RNase to a final concentration of 20

µg/ml. The sample was kept at room temperature for 15 minutes.

3.3.2. Purification of DNA

Genomic DNA, was purified by phenol: chloroform: isoamyl alcohol

(25:24:1) extraction mixture. An equal volume of phenol: chloroform: isoamyl

alcohol mixture was added to the DNA sample and mixed by repeated inversions. The

mixture was centrifuged at 10,000 rpm for 10 minutes at room temperature and the

aqueous phase was transferred carefully to a fresh eppendorf tube. To the aqueous

phase, an equal volume of chloroform was added and centrifuged at 10,000 rpm for 2

minutes and the aqueous phase was transferred to another tube. To the aqueous phase

1/10th

volume of 3 M sodium acetate (pH 8.0 for genomic and plastid DNA, pH 7.0

for DNA fragments) and two volumes of absolute ethanol was added and pelleted by


centrifugation at 10,000 rpm for 5 min. After discarding the supernatant the resulting

pellet was dissolved in nuclease free water and stored at -20°C.

3.3.3. Quantification of DNA

Genomic DNA and amplified products were quantified using UV

spectrophotometer. The diluted DNA samples (1:250) were read at 260 nm and

distilled water was taken as blank. The amount of DNA was calculated by using the

following formula.

Amount of DNA (µg/µl) =

3.3.4. PCR amplification

About 50 ng of DNA samples were taken in PCR tubes and mixed with 200µM

of each dNTPs, 0.5M RAPD primer (Operon Technologies, Almanda, California). 25

mM MgCl2, 1 unit of Taq polymerase and reaction buffer (Genei, Bangalore, India).

Finally the total reaction volume was made up to 25µl by nuclease free water. The

reaction tubes were placed in MJ thermal cycler using the following cycling

conditions.

Initial denaturation - 95°C for 3 min

Denaturation - 94°C for 1 min

Primer annealing - 37°C for 1 min

Extension - 72°C for 1 min and 20 sec. for 40 cycles

Final extension - 72°C for 15 min and then hold on at 4°C

3.3.5. Electrophoresis of samples

After the completion of PCR amplification, the samples were added with 2µl

of loading dye containing TBE buffer, glycerol and bromophenol blue. Agarose gel

(1.5%) was casted with 1X TBE buffer and the samples were loaded in the wells.

Electrophoresis was carried out at 60 volts for 4 hours. After electrophoresis the gels

were documented in Gel documentation system (Vilber Lourmet, France). The

amplification product‟s size was calculated by using the software photocapt MW.


3.3.6. Primer screening

Primers were selected on the basis of the number and intensity of

polymorphic amplified bands. Ten random primers from Operon Technologies

(Almanda, California) were initially screened and using in different populations of P.

niruri to determine the suitability of each primer for the study. Primers were selected

for further analysis based on their ability to detect distinct, clearly resolved and

polymorphic amplified products within the populations of P. niruri. To ensure

reproducibility, the primers generating no/weak/complex patterns were discarded. The

primers were tested on the 6 plant populations of P. niruri. From this, four primers

were selected for further studies on the basis of good DNA amplification, with at least

three sharp electrophoretic bands.

3.3.7. Agarose gel electrophoresis

Required amount of agarose was weighed out (0.75% for genomic DNA and

1.5% for amplified products) and melted in IX TBE buffer (90 mM Tris-borate and

2mM EDTA-pH 8.0)) or IX TAE buffer (40 mM tris-acetate, 1mM EDTA-pH 8.0).

After melting, the volume of the gel mixing was made up to the final volume by the

addition of water. After cooling to 50°C, ethidium bromide was added to a final

concentration of 0.5 mg/ml. The mixture was poured immediately on a preset

template with appropriate comb. After solidification, the comb and the sealing tapes

were removed and the gel was mounted in an electrophoresis tank. To the DNA

sample, required volume of sample buffer (6X sample buffer : 40% sucrose, 0.25%

bromophenol blue) was added and the samples were loaded onto the gel.

Electrophoresis was performed at 60 volts for 4 hours.

3.3.8. RAPD data analysis

PCR products from individual plants were scored as either present or

absent. Only clearly amplified fragments were analysed. Scores of 1 (present) or 0

(absent) were used to form a matrix. The genetic distance was calculated as the

precentage of total number of bands scored that were clearly different between each

pair of accessions. Each amplification fragmets was named by the source of the

primer (Operon, Advanced Biotechnologies) the kit letter or number, the primer

number and its approximate size in base pairs. Similarity indices were estimated using


the Dice coefficient of similarity (Nei and Li, 1979). Cluster analysis were carried out

on similarity estimates using UPGMA (unweighed pair-group method to produce a

dendrogram using arthimetic average) in the NTSYSpc-version 1.80 software

program (Rohlf, 1993).

3.4. Phytochemical analysis

3.4.1 Preparation of extracts (Ahmad and Beg, 2001)

The whole plants collected from different locations (one month old field

grown) were brought in to the laboratory for phytochemical studies. Phytochemical

analysis for major phytoconstituents of the plant extracts was undertaken using

standard qualitative methods as described by various authors (Vogel, 1958; Kapoor et

al., 1969; Odebiyi and Sofowora, 1990). The plant extracts were screened for the

presence of biologically active compounds such as alkaloids, carbohydrates, saponins,

phytosterols, phenolics, tannins, flavonoids, terpenoids, phlobatannins and proteins.

3.4.2 Test for alkaloids (Evans, 1997)

Mayer’s test: A drop of Mayer‟s reagent was added to a few ml of the filtrates by the

side of the test tube. The formation of a creamy or white precipitate indicated the

presence of alkaloids.

Dragandroff’s test (kraut reagent – potassium bismuth iodide): 8g of Bi(NO3)3

5H2Owas dissolved in 20ml of HNO3 and 2.72g of potassium iodide in 50 ml of water.

These were mixed and allowed to stand till KNO3 crystallised out. The supernatant

was decanted off and made up to 100 ml with distilled water. The alkaloids were

regenerated from the precipitate by treating with Na2CO3 followed by extraction of

liberated base with ether.

0.5 ml of herbal extract was added to 2 ml of HCl. To this acidic medium, 1

ml of reagent was added. An orange red precipitate was produced immediately, which

indicated the presence of alkaloids.

Wagner’s reagent (Iodine – Potassium iodide solution): 1.2 g of iodine and 2.0 g of

potassium iodide were dissolved in 5 ml of H2SO4 and the solution was diluted to 10

ml. 10 ml of herbal extracts was acidified by adding 1.5 % v/v HCl and a few drops of


Wagner‟s reagent. The formation of a yellowish brown precipitate confirmed the

presence of alkaloids.

3.4.3. Test for carbohydrates (Kokate, 1999)

Benedict’s test: 173 g of sodium citrate and 100 g of sodium carbonate were

dissolved in 500 ml of water. To this solution, 17.3 g of copper sulphate dissolved in

100 ml of water was added. To 0.5 ml of the herbal extracts, 5 ml of Benedict‟s

reagent was added and boiled for 5 minutes. The formation of a bluish green colour

showed the presence of sugar.

3.4.4. Test for saponins (Kokate, 1999)

The extract was diluted with distilled water and made up to 20 ml. The

suspension was shaken in a graduated cylinder for 15 min. The formation of 2 cm

layer of foam indicated the presence of saponins.

3.4.5. Test for phytosterols (Finar, 1986)

Libermann-Buchard’s test: The extract was mixed with 2 ml of acetic anhydride.

To this 1 or 2 drop of concentrated sulphuric acid was added slowly along the sides of

the test tubes. An array of colour change showed the presence of phytosterols.

3.4.6. Test for phenols (Mace, 1963)

Ferric chloride test: The extract was diluted to 5 ml with distilled water. To this a

few drops of neutral 5% ferric chloride solution was added. The formation a dark

green colour indicated the presence of phenolic compounds.

3.4.7. Test for tannins (Segelman et al., 1969)

About 0.5 mg of dried powdered samples was boiled in 20 ml of water in

test tubes then filtered. A few drops of 0.1 % ferric chloride was added and observed

the formation of brownish green or blue black colouration.


3.4.8. Test for flavonoids (Malick and Singh, 1980)

A portion of the aqueous extract was added to 5 ml of the dilute ammonia solution,

followed by addition of concentrated sulphuric acid. Appearance of yellow

colouration indicated the presence of flavonoids.

3.4.9. Test for terpenoids (Malick and Singh, 1980)

5 ml of the extract was mixed with 2 ml of chloroform and concentrated

sulphuric acid to form a layer. A reddish brown colouration of the interface showed

the presence of terpenoids.

3.4.10. Test for phlobatannins (Malick and Singh, 1980)

Formation of red precipitate when aqueous extract of plant sample was boiled

with 1% aqueous hydrochloric acid indicated the presence of phlobatannins.

3.4.11. Proteins and free aminoacids (Walsh and Farrel, 1961)

Biuret test: A small quantity of extract was mixed with a few ml of water and to

which biuret reagent was added. The formation of pink - purple colour indicated the

presence of proteins.

Ninhydrin test: A small quantity of extract was mixed with a few drops of ninhydrin

reagent and then heated. Formation of purple colour indicated the presence of amino

acids.

Xanthoprotien test: A small quantity of extract was mixed with a few drops of

concentrated nitric acid and then heated. Then, 40 per cent sodium hydroxide solution

was added to it. Formation of yellow colour, which turns in to orange indicated the

presence of aminoacids.

3.5. Gas chromatography and mass spectrometry (GC-MS) analysis

(Ivanova et al., 2002)

3.5.1. Sample preparation

The powdered sample (20 g) was soaked and dissolved in 75 ml of methanol

for 24 hrs. Then the filtrates were colleted and evaporated under liquid nitrogen. The


GC-MS analysis was carried out using a Clarus 500 Perkin-Elmer (Auto system XL)

Gas Chromatograph equipped and coupled to a Mass detector Turbo mass gold-

Perking Elmer Turbomas 5.1spectrometer with an Elite-1 (100% Dimethy1 ply

siloxane), 30 m x 0.25 mm ID x 1 µm df capillary column. The instrument was set to

an initial temperature of 110°C, and maintained at this temperature for 2 min. At the

end of this period, the oven temperature was raised up to 280°C, at the rate of an

increase of 5°C/min, and maintained for 9 min. Injection port temperature was

ensured as 250°C and Helium flow rate as 1 ml/min. The ionization voltage was

70eV. The samples were injected in split mode as 10:1. Mass spectral scan range was

set at 25-400 mhz. The chemical constituents were identified by GC-MS. The

fragmentation patterns of mass spectra were compared with those stored in the

spectrometer database using National Institute of Standards and Technology - Mass

Spectral database (NIST-MS). The percentage of each component was calculated

from the relative peak area of each component in the chromatogram.

3.6 Antibacterial Activity

3.6.1. Collection of culture

In the present investigation, the following pure cultures of human pathogenic

bacteria were collected from Microbial Type Culture Collection (MTCC), Institute of

Microbial Technology (IMTECH), Chandigarh, India.

3.6.2. Bacterial cultures

Staphylococcus aureus (MTCC 1144)

Klebsiella pneumoniae (MTCC 2295)

Escherichia coli (MTCC 1574)

Salmonella typhi (MTCC 0733)

Proteus mirabilis (MTCC 0425)

Streptococcus mutans (MTCC 0899)

3.6.3. Characteristics of the bacterial pathogens

Staphylococcus aureus: Gram positive cocci. Morphologically, they were spherical

and arranged characteristically in grape like clusters. They were aerobes and


facultative anaerobes. They grew on ordinary media like nutrient agar. Sheep blood

agar was recommended for isolating Staphylococcus aureus. Most of the acute

pyogenic infections were caused by these organisms. The diseases may be classified

as cutaneous, deep infection and poisioning.

Klebsiella pneumoniae: Medically important gram negative non motile capsulated

Bacillus. It grew well on ordinary media. They were widely distributed in nature,

occurring both as commensals intestine and saprophytes in soil and water. It produced

large and usually mucoid colonies on nutrient agar. The colonies on blood agar and

Mac Conkey agar were as like on nutrient agar. Most strains were lactose fermenting.

This species were found as a commensal in the mouth and upper respiratory tract, and

also in moist environment and in hospitals. This species caused chest infection, severe

pneumonia, urinary tract infection, septicemia and meningitis.

Escherichia coli: A Gram negative straight rod shaped bacterium arranged singly or

in pairs. It was motile by peritrichous flagella. Four main types of clinical syndromes

were caused by E. coli such as urinary tract infection, diarrhoea or gastroenteritis,

pyogenic infections and septicaemia. Members of the genus Escherichia were

common bacteria that colonize the human large intestine. Most were opportunistic

normal flora but some are potent pathogens. Transmission of diarrheal disease was

generally person to person, usually related to hygiene, food processing and sanitation.

Salmonella typhii: A Gram negative motile bacterium caused enteric fever and

infections of the urinary tract. Enteric or typhoid fever occured when the bacteria

leave the intestine and multiply within cells of the reticuloendothelial system. This

disease resembled other Gram-negative septicemias and was characterized by a high,

remittent fever with little gastrointestinal involvement.

Proteus mirabilis: A facultative gram negative bacteria and these organisms

fermented lactose, which was a useful characteristic for differentiating them from

other organisms. It was considered as opportunistic pathogens of humans. Proteus can

be grouped into three general categories: Nosocomial or hospital-acquired infections:

Forty percent of all nosocomial infections involve Proteus. The primary sites for


infection include the urinary tract, surgical wound, lower respiratory tract, and

primary bacteremia.

Streptococcus mutans: Streptococci were a very heterogeneous group of bacteria.

S. mutans were Gram-positive, non-motile cocci that divided in one plane, produced

chains of cells were catalase negative and may be either facultative or obligate

anaerobes. Viridans species (e.g. S. mutans) were responsible for oral caries and

subacute bacterial endocarditis following dental surgery. S. mutans was an important

contributor to dental caries.

3.6.4. Plant materials

The plant materials were collected from the six different distinct locations like

Ooty, Palani, Madurai, Thanjavur, Kumbakonam, and Nagapattinam of Tamil Nadu,

South India during the rainy season (middle of November, 2008) and shade dried at

room temperature for 10 days for further study.

3.6.5. Preparation of plant extracts

Plant extracts was prepared as described earlier (Ahmad and Beg, 2001) with

slight modification. 100 g of each sample of powdered plant material were soaked in

250 ml of ethanol, methanol and chloroform for 96 h. Each mixture was stirred every

18 h using a sterile glass rod. At the end of extraction each extract was passed through

Whatman No.1 filter paper (Whatman Ltd., England). The filtrate was concentrated

on a rotary evaporator under vacuum at 35°C and stored at 4°C for further use.

Percent yield of crude extract of each plant sample was determined in terms of mg

(dry weight)/100 g sample. Aqueous extracts was centrifuged at 5000 rpm and the

supernatant was taken and dried.

3.6.6. Culture media and inoculums

3.6.6.1. Nutrient agar medium (Difco Manual, 1953)

Nutrient agar medium is one of the most commonly used medium for several

bacteriological strains. The components are Peptone (5.0 g), Beef extract (3.0 g), Agar

(15.0 g), Sodium chloride (5.0 g), Distilled water (1000 ml) and the pH was adjusted

to 7.0.


After mixing the ingredients in to the distilled water it was melted in the water

bath and sterilized by autoclaving at 15 lbs pressure of 121°C for 15 minutes.

3.6.6.2. Inoculum preparation

The selected bacterial pathogens were inoculated into nutrient broth (liquid

medium) and incubated at 37°C for 24 hours and the suspensions were checked to

provide approximately 10-5

CFU/ml.

3.6.7. Antibacterial tests

Antibacterial activity was tested using a modification of the disc diffusion

method originally described by Bauer et al. (1966). A loop of bacteria from the agar

slant stock was cultured in nutrient broth overnight and spread with a sterile cotton

swap into petriplates containing 10 ml of Nutrient Agar. Sterile Whatman No.1 filter

paper discs were (Whatman Ltd., England) (6mm in diameter) impregnated with the

plant extract and placed on the culture plates and incubated at 25 or 37°C, depending

on the bacteria. The solvent without extracts served as negative control. After 24 h of

incubation, the diameter in mm of the inhibitory or clear zones (MIC) around the

disks was recorded. Standard antibiotic tetracycline 30 mg (Span Diagnostics Limited,

Surat, India) was used as reference or positive control.

3.7. Pharmacology - Hepatoprotective activity

3.7.1. Collection of plant material

The plant was collected from different places of Tamil Nadu, viz., Ooty, Palani,

Madurai, Thanjavur, Kumbakonam and Nagapattinam during the month of

November, 2009. The plant was dried under shade and powdered into fine powder.

The callus of the plant was used to screen for its biological activity.

3.7.2. Chemicals and biological kits

All chemicals used were of analytical grade. The biochemical kits were

procured from Nicholas Piramal India limited, Mumbai and Crest biosystems, Goa,

India.


3.7.2.1. Paracetamol (Hepatotoxins)

Paracetamol is a widely used analgesic and antipyretic drug. It is well known

that this drug exerts hepatotoxic effects in a dose-dependent manner (Cover et al.,

2006). In overdose of the drug (over 4 g/day paracetamol), centrilobular hepatic

necrosis is recognized as a dominant morphologic alteration (Knight et al., 2001).

However, pathogenesis of centrilobular hepatic necrosis is not completely understood.

Metabolic activation of paracetamol is considered to be a major mechanism of its

hepatotoxicity (Graham et al., 2005).

3.7.2.2. Silymarin (standard drug)

Silymarin is used for the treatment of numerous liver disorders characterized

by the degenerative necrosis and functional impairment. Furthermore, it is able to

antagonise the hepatotoxin and provides (hepato) protection against poisoning by

phalloidin, galactosamine, paracetamol, thioacetamide, halothane and CCl4 (Barbarino

et al., 1989).

3.7.2.3. Carboxy methyl cellulose (CMC)

CMC was used as a suspending agent, in order to get uniform dispersion.

3.7.3. Experimental animals

Sixty day-old chicks (White Leghorn cockerels) were purchased from

Namakkal district, Tamilnadu. The chicks were housed in separate cages for five days

prior to dosing for acclimatization to the laboratory conditions. During the period of

acclimatization the chicks were observed for ill health, if any. Chicks demonstrating

signs of spontaneous disease or abnormality prior to the start of the study were

eliminated from the study. The cockerels had free access to water and were fed ad

libitum with chick-mash which was later replaced with grower-mash for the later part

of the experiment. After brooding for four weeks, the birds were randomly but equally

divided into ten groups. Each group consists of six birds.


3.7.5. Paracetamol induced hepatotoxicity

3.7.5.1. Drug administration

Chicks were divided into ten groups consisting of six each. Animals of Group

I received orally 0.5% (w/v) of carboxy methyl cellulose and treated as normal

control. Group II animals received paracetamol (250 mg/kg b.wt., i.p.) suspended in

0.5% (w/v) of Carboxymethyl cellulose and treated as diseased control. Animals of

Group III were administrated with standard drug, silymarin (100 mg/kg, p.o.) and

treated as positive control. Group IV, V, VI, VII, VIII and IX animals were

administered orally with Phyllanthus niruri (250 mg/kg b.wt.), suspended in 0.5%,

w/v, Carboxy methyl cellulose. Group X animals were treated orally with callus,

raised in vitro from P. niruri (250 mg/kg, b.wt.). All the drug treatments were given

twice a day for 4 days. Paracetamol was administrated at the dose of 250 mg/kg, b.wt

by intra-peritoneal route to all treatment groups except for normal control. After 48

hours of paracetamol administration, all the birds were sacrificed and the blood was

collected by carotid cutting and the serum was separated for the assessment of

different enzyme activities. Chicken liver was carefully dissected, and extraneous

tissue was trimmed out and washed with ice cold saline to remove blood. The whole

liver was observed for gross pathology, weighed and fixed in 10 % buffered saline for

pathological examination (Bhar et al., 2005).

3.7.6. Assessment of hepatoprotective activity

The following enzymes were analyzed in order to assess the protective effect

of the P. niruri on liver. As there was a decrease in the level of these enzymes under

stress conditions, therefore, it was expected that the plant extract would be able to

revert this effect and maintain the normal level of enzymes.

Hepatic enzymes, such as AST and ALT were used as the biochemical

markers of the hepatic damage and were assayed by the method of Reitman and

Frankel (1957). Estimation of serum ALP (King and King, 1965) and serum bilirubin

(De Groot and Noll, 1986) were also carried out to assess the acute hepatic damage

caused by paracetamol.


3.7.7. Histopathology

Livers from chicks of different groups were perfused with 10% neutral

formalin solution. Paraffin sections were made and stained using hematoxylin-eosin

(H&E) stain. After staining, the sections were observed under microscope and

photographs were taken by Nickon optiphot microscope with photographic unit

(Nickon Fx camera, Japan).

3.7.7.1. Preparation of tissues for histopathological examination

The chicks were anaesthetized with ether and thereafter sacrificed by cervical

dislocation. Tissue samples were taken from the liver of the sacrificed chicken and

fixed in 10 % formalin neutral buffer solution. The trimmed tissues were first washed

with tap water followed by dehydration through a graded alcohol series and then

passed though xylol and paraffin series before finally blocked in paraffin. The

paraffin blocks were cut into 5–6 μm sections using a spencer rotary microtome

(American optical company) and stained by using hematoxylin and eosin and

examined under a light microscope.

3.7.8. Parameters studied

1. Liver function tests

a. Total bilirubin

b. SGOT (AST)

c. SGPT (ALT)

d. Alkaline phosphatase (ALP)

2. Gross examination of liver

3. Histopathology

3.7.9. Statistical analysis

The analysis of variance (ANOVA) appropriate for the design was carried out

to assess the significance of differences among the treatment means. The treatment

means were compared using Duncan‟s Multiple Range Test (DMRT) at a 5%

probability level (Gomez and Gomez, 1976).


3.8. In vitro culture technique

3.8.1. Sterilization of equipments and glasswares

All operations for in vitro culture were carried out inside a laminar air flow

cabinet under aseptic conditions using sterilized plant materials, equipments, glass

materials and chemicals. A horizontal laminar flow cabinet with HEPA (High

efficiency particulate air) filter was used. The hood surface was wiped clean with

paper towel soaked in 70 % ethanol and sterilized by germicidal ultraviolet light for at

least 10 min prior to use. All surgical instruments, glass wares and other accessories

were sterilized in autoclave at 121 ºC with 15 psi for 30 min and then dried in oven.

Surgical instruments such as scalpel, forceps, scissors etc., were sterilized by dipping

in 100 % ethyl alcohol and flaming prior to use.

3.8.2. Culture conditions

Single disinfected shoot tip segments were cultured on MS basal medium

(Murashige and Skoog, 1962) supplemented with 3% (w/v) sucrose (87.64 mM)

(Sigma, Ltd. India) and 0.8% (w/v) agar for culture initiation and these initiated

shoots were served as explant sources for subsequent experiments. The pH of the

medium (supplemented with respective growth regulators) was adjusted to 5.8 with 1

N NaOH or 1 N HCl before addition of 0.8% (w/v) agar (SLR, India). In all the

experiments, analytical grade of chemicals were used (SLR, Kelco, Merk and Sigma).

The medium was dispensed into culture vessels (Borosil, India) and

autoclaved at 105 kPa (121°C) for 15 min. The surface-disinfected explants were

implanted vertically on the culture medium [test tubes (150 x 25 mm) containing 15

ml medium] and plugged tightly with non-absorbent cotton. All the cultures were

incubated at 25±2°

C under 16 h photoperiod of 45 - 50 μmol m-2

s-1

irradiance

provided by cool white fluorescent tubes (1500 Lux, Philips, India) and with 75 - 80%

relative humidity (RH). All subsequent subcultures were performed at four weeks

intervals.

3.8.3. Preparation of culture media

MS, (Murashige and Skoog, 1962), B5 (Gamborg et al., 1968), MS-B5 (MS

nutrients with vitamins from B5), inorganic salts, organic supplements, and vitamins

were used as basal media for seed germination, callus induction, callus multiplication,


shoot and root induction and in vitro flowering. The formulation and composition of

MS, B5 and MS-B5 media were given in Table (2 and 3).

3.8.3.1. Preparation of stock solution

Stock solutions of the major components, such as macronutrients,

micronutrients, vitamins and plant growth regulators of the media were prepared and

stored in refrigerator. The macro nutrients were dissolved in 500 ml of double

distilled water and the micro nutrients are dissolved in 250 ml of double distilled

water. KI was dissolved in 250 ml of double distilled water. Minor nutrients are

dissolved in 500 ml of double distilled water. In the case of iron, the Na2 EDTA and

FeSO4 were dissolved separately in 100 ml of double distilled water. Na2 EDTA was

boiled and then slightly added to the FeSO4.7H2O gently. Then make up into 250 ml

of double distilled water. The vitamins were dissolved in 100 ml of double distilled

water. Agar, meso-inositol and sucrose were weighed and added at the time of

medium preparation. Stock solutions were stored in refrigeration.

3.8.3.2. Preparation of working media

Stock solution of macro, micro and minor elements including iron and

vitamins were prepared by dissolving adequate quantities of each element (Murashige

and Skoog, 1962; Gamborg (B5), 1968). Analytical grade of chemicals and double

distilled water were used in all the preparation. Required quantities of agar weighed

and dissolved in double distilled water with the help of water bath. Appropriate

quantities of the various stock solutions, sucrose, meso-inositol and growth regulators

were added. The final volume of the medium was made up by using double distilled

water. Before and after mixing of the agar, the pH of the medium was adjusted to 5.8

using 0.1 N NaOH and 0.1 N HCl. The medium was poured into culture tubes (about

20 ml of the medium in each culture tube) and the culture tubes were plugged with

sterile non-absorbent cotton wool which was wrapped with cheese cloth and sterilized

by autoclaving at 121ºC and 15 psi for 15 min.


3.8.4. Growth regulators

Auxin, cytokinins and gibberlins were the three major phytohormones used

in different concentrations and combinations in various media for the induction and

growth of callus, shoot, root and in vitro flowers of P. niuri.

3.8.4.1. Auxin

Powders of auxin (Sigma, India Ltd.) were dissolved in 1N NaOH and made

up the volume with sterilized distilled water and then used or stored in freezer as

stock for further use. The three auxins used in the present study were α-naphthalene

acetic acid (NAA), indole-3-butric acid (IBA) and indole-3-acetic acid (IAA).

Different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) of NAA, IBA and IAA

were tested in MS-B5 medium for callus induction, rooting and flowering.

3.8.4.2. Cytokinins

The cytokinins (Sigma, India Ltd.) were dissolved in low quantity of 1N

NaOH, mixed in distilled water made up to 100 ml, then used or stored as stock for

further use. The two cytokinins used were 6-benzyl amino purine (BAP) and Kinetin

(KIN). Six concentrations of BAP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) and KIN (0.5,

1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) were used in the medium for shoot multiplication.

3.8.4.3. Gibberllins

The gibberllic acid (GA3) (Sigma, India Ltd.) was dissolved in 1N NaOH

and made up the volume of 100 ml with sterilized distilled water and then used or

stored as stock for further use. The different concentrations of GA3 used were (0.5,

1.0 and 1.5 mg/l) for the induction of in vitro flowering.

3.8.5. Raising of seedlings

3.8.5.1. Surface sterilization of seeds

Fully matured, healthy and well dried seeds were presterilised with 70 %

ethanol for two min and washed with sterile distilled water and then surface sterilized

with 0.1% (w/v) mercuric chloride solution for 3 min. Further the seeds were


subsequently washed thoroughly (four to five times) with sterile distilled water inside

the laminar flow cabinet until the trace of mercuric chloride.

3.8.5.2. Seed germination

Six accessions were used (Ooty, Palani, Madurai, Thanjavur,

Kumbakonam and Nagapattinam) for germination. The sterilized seeds were placed in

culture tubes containing hormone free MS medium solidified with agar 0.8 % (w/v)

and the seeds placed in sterile moist cotton in a culture tubes were also used seed

germination. Individual culture tubes were wrapped with paraffin film to maintain

them free from contamination. The seed cultures were incubated under dark at 26ºC.

In vitro germinated seedlings and wildly growing plants were also used as explants

for shoot multiplication, root, callus induction and in vitro flowering.

3.8.5.3. Plant material and disinfections

Healthy young shoot tip and nodal explants with dormant axillary buds

were collected from the mature plants of P. niruri L. from Ooty grown in the

Botanical Experimental Garden of A.V.V.M. Sri Pushpam College, Poondi,

Thanjavur district. After removing leaves, the explants (1.0 - 1.5 cm) were excised

and then washed thoroughly under running tap water for 15 min. Followed by a

treatment with a aqueous solution of detergent 2% v/v Teepol (Reckitt Benckiser,

India Ltd.) for 10 min., and 70% (v/v) ethanol for 15 seconds and washed with

autoclaved sterile distilled water three to five times. The explants were then surface

disinfected with 0.1% (w/v) aqueous mercuric chloride solution for 5 - 6 min and

finally rinsed with autoclaved distilled water (five to seven changes). The shoot tip

and nodal segments were then trimmed at both ends prior to inoculation on culture

media.

3.8.5.4. Inoculation of explants for shoot multiplication

Before started inoculation all the required instruments such as media

containing culture tube, sprit lamp, sterile water, glassware etc. were transferred to

laminar air flow chamber and the platform surface of the chamber was swapped with

70 per cent alcohol. After swapping, the UV light was switched „on‟ for 30 min. After

that, the UV light became switched „off‟ and „on‟ the ordinary light. Before


inoculation, hands were rinsed with 70 per cent alcohol. Then the explants were

inoculated on the medium. The instruments used aseptic manipulation (forceps,

scalpel, needles etc.) were sterilized by dipping in 70 per cent alcohol followed by

flaming and cooling.

The inoculation was carried out in vicinity of flame. The explants were taken

out from the beaker and at the same time the cotton plug of the culture tube was

slightly opened in front of the spirit lamp flame. The explants has been put in it and

immediately covered with cotton plug. Hence, shoot tip and nodal segments were

inoculated vertically on the medium containing different combinations and

concentrations of growth regulators mentioned above.

The shoot tip and node was cut from 15 days old seedlings and placed in

shoot multiplication media. The shoot tip and nodal segment from wild populations

were also used as explants inoculated by inserting their cut-ends in the medium

supplemented with different concentrations of cytokinins to induce multiple shoots. A

single explant was placed in a single culture tube containing the medium.

3.8.5.5. Shoot multiplication and maintenance

The explants were sub cultured onto fresh media every 25 days. When the

explants started to multiply, well grown axillary shoots were separated with the help

of a sterile scalpel under the laminar air flow and put in the same media for further

multiplication. The shoot lets derived from each seed were tracked individually to

determine the total number of plants produced from single seed and their subsequent

genetic identity.

The cultures were kept under 16 h light / day (2400 Lux) photoperiod at 25

2oC. The shoot multiplication was assessed after 4 weeks in culture by counting the

proliferated shoots which attained the length of 2.0 cm and above. The subsequent sub

culture was made only on the medium which showed maximum shoot multiplication.

3.8.6. Effect of basal media

Three different media including MS (Murashige and Skoog, 1962), B5

(Gamborg et al., 1968) and MS-B5 were evaluated for their effects on in vitro growth

and development of P. niruri. All the basal media contained 3% (w/v) sucrose and


solidified with 0.8% (w/v) and different concentrations of cytokinins, including 0.5 -

3.0 mg/l of 6-benzylaminopurine (BAP) and 0.5 - 3.0 mg/l of Kinetin (KIN).

3.8.7. Effect of carbon sources

Shoot tip and nodal segments were cultured on MS agar medium

supplemented with 1.5 mg/l BAP and different types of carbon sources, including 3%

(w/v) of glucose, fructose and sucrose.

3.8.8. Effect of cytokinins

Shoot tip and nodal segments were cultured on MS medium containing 3%

(w/v) sucrose and 0.8% (w/v) agar and supplemented with different combination and

concentrations of plant growth regulators, including 1.5 mg/l BAP with (0.5 - 3.0

mg/l) KIN.

3.8.9. In vitro flowering

The in vitro raised stem nodal explants that are remained aseptic were

cultured vertically with the basal end placed in to MS-B5 supplemented with 20, 30,

50 and 70 g/l sucrose and MS medium supplemented with 0.5, 1.0, and 1.5 mg/l of

GA3

respectively. The percentage of in vitro flowering was recovered and recorded

every week for four weeks.

3.8.10. Rooting medium

Elongated shoots were excised from each culture passage and transferred to

full-strength and half-strength (1/2 MS) MS medium containing 3% (w/v) sucrose and

0.8% (w/v) agar. The medium was further supplemented with 0.5 - 3.0 mg/l indole-3-

acetic acid (IAA) or indole-3-butyric acid (IBA) or -naphthalene acetic acid (NAA)

individually.

3.8.11. Acclimatization and transfer of plantlets to soil

Plantlets with well-developed roots were removed from the culture medium

and after washing the roots gently under running tap water, plantlets were transferred

to plastic pots (7 cm diameter) containing autoclaved vermiculite. All were irrigated

with ½ strength MS basal salt solution devoid of sucrose and inositol every 4 days for

two weeks. The potted plantlets were covered with polyethylene sheets pin air holes


were made for maintaining high humidity and were maintained under the culture

room conditions. The relative humidity was reduced gradually and after 30 days the

plantlets were transplanted in to larger pot containing sterilized garden soil, farmyard

(manure) and sand (2:1:1) and then transferred to botanical experimental garden and

kept under shade in a net house for further growth and development. The morphology,

growth characteristics and floral features P. niruri were examined.

3.8.12. Induction of callus

The shoot tip and nodal explants were used for the induction of callus and

they were inoculated in MS-B5 media with different concentrations of growth

hormones. The callus induction was tested on various concentrations of BAP and KIN

(1.0 and1.5 mg/l) with NAA (0.5 to 2.5 mg/l) each. The induced callus was

subcultured at regular intervals of 30 days on the MS-B5 medium containing different

concentrations of NAA, BAP and KIN.

3.8.13. Statistical analysis

Experiments were set up in a Randomized Block Design (RBD) and each

experiment usually had 10 replicates and was repeated at least three times. Ten to

fifteen explants were used per treatment in each replication. Observations were

recorded on the frequency (number of cultures responding for axillary shoot

proliferation and root development) and the number of shoots per explants, shoot

length, roots per shoot and root length respectively. The analysis of variance

(ANOVA) appropriate for the design was carried out to assess the significance of

differences among the treatment means. The treatment means were compared using

Duncan‟s Multiple Range Test (DMRT) at a 5% probability level (Gomez and

Gomez, 1976).


4. RESULTS

4.1. The physico-chemical characteristics of soil

The physico-chemical characteristics of six different soil types are given in

the Table - 4. Among the study areas, the Ooty and Palani areas are found with peat

swamp and marshy soil. In Thanjavur and Kumbakonam the soil is sandy clay,

whereas the Madurai area has well drained sandy soil and in Nagapattinam the soil is

fine sand to sandy loam. pH was varied from 5.7 to 7.5 and the high pH (7.5) was

recorded in Nagapattinam soil, strong acidic pH was determined in Ooty and Palani

soils whereas in Madurai, Thanjavur and Kumbakonam the pH values were 6.8, 6.6

and 6.0 respectively.

Electrical conductivity was low in Ooty (0.50 dsm-1

) and Palani (0.52 dsm-

1), moderate in Madurai (0.56 dsm

-1), Thanjavur (0.54 dsm

-1) and Kumbakonam (0.55

dsm-1

) and high in Nagapattinam (0.61 dsm-1

). The organic matter content was too

high in Ooty soil when compared to other soil samples. The high amount of nitrogen

level was observed in Ooty (150.0 Kg/acre) and Palani (140.0 Kg/acre) soil samples

and low in Nagapattinam soil (94.6 Kg/acre) whereas in other soil samples it was

moderate. Phosphorous level was high in Ooty (9.6 Kg/acre) and Palani (8.8 Kg/acre)

soil samples and moderate in other study sites. The potassium level was moderate in

all the six soil types.

High level of zinc was recorded in the Ooty soil (0.18 ppm) and low level

in Nagapattinam soil sample (0.15 ppm) whereas in other soil samples it was

moderate. Iron content of the soil was high in Madurai while the other areas showed

minor variations. The level of other elements such as copper and magnesium were

observed with slight differences among the different soil samples collected.

4.2. Morphometry

Morphometric analysis of Phyllanthus niruri collected from six different

locations of Tamilnadu was recorded (Table - 5). All the plants were exhibiting their

habit as annual herb. Among the plants collected from several locations, the

Nagapattinam plant sample showed maximum mean shoot length (60±0.46 cm)

followed by the Thanjavur and Kumbakonun plant samples with similar heights


(55±0.56, 55±0.12 cm respectively). Madurai plant sample was found with a height of

48±1.2 cm followed by Palani and Ooty (40±0.8 cm and 40±0.6 cm). Stems of all the

plant samples were showed erect and their branches were alternate in position.

Leaf blades were oblong-elliptic and 4-7 pairs of lateral veins in all

populations of Phyllanthus niruri. Leaves were maximum in length in three areas

such as Thanjavur, Kumbakonam and Nagapattinam (6.0±0.2mm, 6.0±0.5mm and

6.0±0.8mm respectively) when compared to other three samples such as Ooty, Palani

and Madurai (5.0±0.4 mm, 5.2 ±0.6mm, and 5.0±0.4mm respectively). The leaf width

of all the six populations was found almost similar and showed only minor

differences. Flowers were unisexual (male and female flower seprately). Male flowers

possess of pedicel 1mm long, calyx-5, sub equal 0.7 x 0.3 mm, stamen- 3, pollen

grains were ellipsoid in shape and female flowers have pedicel 0.8-1.0 mm long,

calyx lobes 5, 0.6 x 0.25 mm were recorded in all six populations of P. niruri. Size of

the seeds was noticed as 0.9 mm long and 1.8 mm diameter width in all populations.

The root length and width were maximum in Nagapattinam plant sample

(25±0.08 cm and 6 ±0.14 mm), moderate height in Thanjavur (20 ±0.18 cm; 6 ±0.14

mm), Kumbakonam (20±0.12 cm; 6 ±0.12 mm) and Madurai (18±0.16 cm; 6±0.08

mm) and minimum in Palani (15±0.13 cm; 6±0.12 mm) and Ooty (15±0.14 cm;

4±0.08 mm). In general all the growth parameters were found to be the maximum in

the Nagapattinam sample. Hence the mean plant biomass was also high in the

Nagapattinam plant sample when compared with other five populations.

4.3. RAPD analysis

All the collected six populations from different parts of Tamilnadu were

maintained under uniform growth conditions and used for RAPD analysis. In the

present study, the RAPD technique was used to analyse the genetic variations in P.

niruri populations collected from six different locations of Tamilnadu. The RAPD

primers were listed in Table – 6 (A and B kits; 5 primers each) were procured from

Operon Technologies (USA) and after initial screening, primers OPA02, OPA03,

OPB07 and OPB17 were selected on the basis of profiles with each of the template

DNA tested. All RAPD reactions were carried out with same cycling conditions and

chemicals. Fragment sizes of the amplification products obtained using RAPD


primers were estimated from the gel by comparision with standard molecular weight

marker (DNA ladder from Genei, Bangalore).

Among the different primers screened, four primers showed good

amplification of polymeric bands (Table - 7) and three did not give any amplification

products. Three of the primers showed amplifications but the intensity of the

fragments was very low. The amplification profiles of total genomic DNA from six

different populations with four random primers produced 95 consistent RAPD

markers, ranging in size from 0.2 kb to 2.4 kb; out of which 10 were monomorphic.

Pattern of RAPD profiles produced by the primer OPB07 are shown in the figure - 1.

From the amplified products of different primers used, two different groups of unique

bands were observed. This observation clearly indicates that the populations of six

accessions can be divided in to two clusters. The cluster A includes populations of

Ooty, Palani and Madurai while cluster B comprises the populations of Thanjavur,

Kumbakonam and Nagapattinam.

The similarity indices as given in table - 8, which clearly shows P. niruri

accessions from the Tamilnadu show less varitions. Even though they may be

belonging to geographically distinct locations, they are very close nearly to 92% in

terms of similarity index. The similarity coefficient values range from 0.88 to 0.92 of

six different populations of P. niruri. These similarity coefficients were used to

generate a tree from cluster analysis using UPGMA method (Figure - 2). The cluster

analysis indicates that the six different populations of P. niruri are grouped in to two

major clusters based on similiarity indicies. One major cluster had three members in

the population i.e., Ooty, Palani and Madurai. Another major cluster includes three

populations viz., Tanjavur, Kumbakonam and Nagapattinam. Each and every

population could be identified by using four random 10-mer primers. Among the six

populations, three of each cluster showed the highest similarity index (92%). The

present study provides evidences through RAPD data to show the occurence of

genetic variations among different collections of P. niruri.

On the basis of the RAPD analysis, the chosen samples have been

grouped under two clusters and labelled A and B. The present study showed

maximum similarity indices and minor variations among the populations. All the

samples of these two clusters were selected for phytochemical studies.



In the present investigation, analysis of chemicals and bioactive compounds

were made in the plants collected from six different regions of Tamilnadu.

4.4.1. Qualitative analysis

For all the six accessions of P. niruri, a phytochemical screening was

performed to test the presence of different secondary metabolites (Table - 9). The

qualitative phytochemical analysis of methanolic and aqueous extracts revealed the

presence of alkaloids, carbohydrates, phytosterols, saponins, phenols, tannins,

flavonoids, terpenoids, phlobatannins, proteins and free aminoacids.

4.4.2. Gas chromatography – Mass spectroscopy (GC-MS) analysis

The phytochemical compounds present in the methanolic extract of P.

niruri were identified by GC-MS analysis. The active principles with their retention

time (RT), molecular formula (MF), molecular weight (MW) and concentration (%)

in the extracts of P. niruri were presented.

From Ooty sample, totally 15 compounds were identified (Table - 10). The

prevailing compounds were lauric acid (2.46%), ester compound (0.05%), alkanes

(0.05), phenolic compound (0.08%), myristic acid (2.77%), plasticizer compound

(4.15%), palmitolic acid (2.27%), palmitic acid (13.97%), diterpene (2.31%), stearic

acid (1.68%), mono unsaturated fatty (5.19%), chlorine compound (2.16%), steroid

(11.6%), alkaloid (1.78%), triterpenes (3.56%) and amino compound (39.27%).

Figure - 3 shows mass spectrum and structures of these compounds, and are suggested

to be the medicinally important compounds which can be used as antimicrobial, anti-

inflammatory, cancer preventive, antioxidant, antiviral, antifouling and

hepatoprotective agent.

Eight compounds were identified from methanolic plant extract of Palani

samples (Table - 11). The prevailing compounds in these extract were alkaloid

(28.34%), chloro compound (22.12%), nitrogen compound (18.24%), alkene

compound (12.06%) and sulphur compound (8.36%). Figure - 4 show the mass

spectrum of these compounds and are reported to have hepatoprotective, cancer

preventive, antimicrobial and anti-inflammatory properties.


Totally eight compounds were identified from the methanolic extract of the

Madurai plant samples of P. niruri and are presented in the table - 12. The plant

samples revealed the synthesis of amino acid compound (16.16%) and alkaloids

(25.16%) which are the sources of active pharmacological principles. The GC-MS

chromatogram of methanolic extracts of Madurai plant samples is shown in the

figure -5.

Totally 12 compounds were identified from the methanolic extract of

Thanjavur plant sample and presented in the table - 13. They were nitrogen (28.67%),

aromatic (14.16%), fluro (7.13%), alkaloid (8.12%), saturated hydrocarbons

(11.22%), silica (18.15%), phosphorus (11.64%), hydroxyl (16.18%) and sulphur

compounds (12.24%). These phytochemical compounds are known to have various

medicinal properties for human beings such as antimicrobial and anti-inflammatory.

The GC-MS chromatogram of Madurai plant sample is shown in the figure - 6.

The result of the GC-MS analysis of P. niruri from Kumbakonam and

Nagapattinam plant samples is presented in the table 14 and 15. The GC-MS

chromatogram of these medicinal plant extracts was shown in the figure 7 and 8.

Nearly 12 compounds were indentified in the Kumbakonam sample. They were

nitrogen (13.56%), aromatic (7.88%), fluro (28.40%), alkaloid (7.64%), silica

(5.66%), phosphorus (16.31%) and chlorine compounds (6.26%). While in the

Nagapattinam plant sample showed only 3 compounds (two alkaloids and one alkene

compound). In general the isolated compounds are reported to possess antimicrobial,

antitumor, anticarcinogenic and anti-inflammatory properties.

Observations of GC-MS analysis of in vitro grown callus tissue of P. niruri

collected from Ooty showed number of alkaloids (28.27%), ester (14.16%), amino

(11.62%), alkenes (3.56%) and nitrogen compounds (12.16%). Totally 8 compounds

were identified and they are recorded in the table - 16. All these compounds are of

pharmacological importance, as they posses the properties such as anti-inflammatory,

analgesic, anti-nociceptive, anti-diabetic, cancer preventive, anti-plasmodial and

antimicrobial. The GC-MS chromatogram of this medicinal plant extract is shown in

the figure - 9.


Among the six plant samples from different locations and in vitro callus

tissue of Ooty plant, the plant sample from Ooty was found with maximum number

and quantity of medicinally important compounds. Some of the important compounds

are depicted in the chromatogram and their molecular structures are presented in the

figure -10.

4.5. Antibacterial activity

Antibacterial activity of different solvent extracts of P. niruri was tested

against six human pathogenic Gram positive and Gram negative multidrug resistant

bacteria (Escherchia coli, Klebsiella pneumoniae, Salmonella typhii, Proteus

mirabilis, Staphylococcus aureus and Streptococcus mutans). In the present study,

extracts were derived from all the six selected populations, and their efficacy to

inhibit the growth of pathogenic bacteria was studied. The antibacterial activities of

the different solvent extracts obtained from the plants were studied by disc diffusion

method shown in the Table - 17. The obtained results showed that the methanolic

extracts inhibited significantly the growth of most of the organisms tested. It was

followed by the aqueous extract in terms of zone of inhibition. But the chloroform

showed lesser activity when compared to methanol and water. Whereas minimum

antibacterial activity was observed with the ethanolic extract. Among the different

populations of plant sample tested, accession from Ooty showed more activity than

the other plant samples. It was followed by Palani, Madurai, Thanjavur,

Kumbakonam and Nagapattinam in the order of efficiency of controlling the

pathogens (Plates, 4-9).


The effect of P. niruri collected from different populations and in vitro

grown callus from Ooty plant on serum marker enzymes are presented in table - 18.

The levels of serum total bilirubin, AST, ALT and ALP were markedly elevated in

paracetamol intoxicated animals, indicating liver damage. Administration of P. niruri

extracts at the dose of 250 mg/kg remarkably prevented paracetamol-induced

hepatotoxicity (Table – 18).


In paracetamol intoxicated chicks, a well marked increase in the content of

serum total bilirubin and enhanced SGOT (AST), SGPT (ALT) and alkaline

phosphatase (ALP) activities were observed in comparison with normal control

chicks, indicating liver damage. The treatment with P. niruri at a dose of 250 mg/kg

showed a significant decrease in total bilirubin, AST, ALT and alkaline phosphatase

in paracetamol intoxicated chicks. Standard control drug, silymarin at a dose of 100

mg/kg also prevented the elevation of above mentioned serum enzymes in the

intoxicated animals.

4.6.1. Total bilirubin

In normal control chicks, the total bilirubin showed their level as

0.2667±0.0210 (mgms %). Intoxication of paracetamol caused a significant elevation

of levels (0.3667±0.0670 U/ml) when compared to control chicks. The total bilirubin

level was restorated onormal levels on the administration of silymarin at a dose of 100

mg/kg and the total bilirubin level was 0.3000±0.0258. The total bilirubin levels in

treated birds with P. niruri of Ooty, Palani, Madurai, Thanjavur, Kumbakonam,

Nagapattinam and callus tissue were 0.3067±0.0760, 0.3126±0.0012, 0.3100±0.0365,

0.3200±0.0223, 0.3233±0.0210, 0.3300±0.0730 and 0.3100±0.0210 respectively

(Figure - 11). It was found that the maximum reduction in the level of total bilirubin

was observed in the animals administered with plant samples of Ooty and the

minimum was in the animal treated with plant samples of Nagapattinam.

4.6.2. SGOT (AST)

Aspartate aminotransferase (AST) or SGOT showed their level in control

chicks as 66.3300±4.4550 U/ml. Intoxication of paracetamol caused a significant

elevation of this enzyme level (87.0000±2.0330U/ml) when compared to control

birds. There was a significant restoration of these enzyme levels on administration of

the silymarin at a dose of 100 mg/kg and the activity of serum glutamate oxaloacetate

transaminase was 72.0000±1.3420. Serum glutamate oxaloacetate transaminase levels

of chicks administered with P. niruri of Ooty, Palani, Madurai, Thanjavur,

Kumbakonam, Nagapattinam and callus tissue treated animals were 68.0000±5.6690,

70.3300 ± 2.1080, 72.6700 ± 3.3130, 67.0000 ± 2.2360, 70.0000 ± 2.5560, 69.6700 ±

2.4860 and 68.3300 ± 1.8380 respectively (Figure - 12). Maximum reduction of


SGOT activity was observed with plant samples of Ooty and the minimum was in the

animals treated with plant samples of Madurai.

4.6.3. SGPT (ALT)

Alanine aminotransferase (ALT) or serum glutamate pyruvate

transaminase (SGPT) showed their level in control bird as 10.6700±1.1160 U/ml.

Paracetamol intoxicated birds had their SGPT level as 18.3300±2.9290. There was a

restoration in the enzyme levels on administration of the silymarin at a dose of 100

mg/kg and the serum glutamate pyruvate transaminase level was 14.1700±3.1240.

SGPT levels of P. niruri from Ooty, Palani, Madurai, Thanjavur, Kumbakonam,

Nagapattinam and callus tissue treated animals were 12.0000 ± 0.0130, 13.6670 ±

1.4760, 14.6700 ± 4.4020, 14.0000 ± 4.0250, 14.3300 ± 1.8740, 14.6700 ± 1.8740

and 15.0000 ± 0.7303 respectively (Figure - 13). Maximum reduction of Alanine

aminotransferase activity was observed with Ooty plant sample and the minimum was

in the animals treated with samples of callus tissue.

4.6.4. Alkaline phosphatase

The normal control chicks showed their enzyme level as 51.6700 ± 4.6240.

Diseased group, which received normal saline and paracetamol showed increase in the

respective liver enzyme activities with a value of 71.1700 ± 10.1600 U/ml when

compared to control birds. There was a restoration of these enzyme levels on

administration of the silymarin at a dose of 100 mg/kg and the alkaline phosphatase

level was 61.5000 ± 7.8260. The alkaline phosphatase levels of the treated birds with

P. niruri of Ooty, Palani, Madurai, Thanjavur, Kumbakonam, Nagapattinam and

callus tissue were 56.0000 ± 4.0660, 58.0000 ± 3.9500, 59.3300 ± 3.4710, 60.0000 ±

0.4472, 61.0000 ± 2.0330, 64.6700 ± 4.7660 and 51.0000 ± 5.9780 respectively

(Figure - 14). Maximum reduction of alkaline phosphatase activity was observed with

callus tissue (raised from Ooty plant population) and the minimum was in the animals

treated with plant samples of Nagapattinam.

4.6.5. Histopathological changes

Gross examination of liver was analysed in all treated and control chicks

(Plate - 2). Morphological observations clearly showed an increased size and


enlargement of the liver in paracetamol intoxicated groups. The control animals

exhibited normal liver morphology whereas the paracetamol intoxicated groups

showed increase in size and enlargement of their liver. These changes were reversed

by the treatment with silymarin and also by P. niruri plant samples collected from

different study area.

In histopathological studies (Plate - 3), paracetamol treated chick‟s liver

samples showed focal areas of necrosis with periportal chronic and centrilobular

necrosis with extensive congestion and vacuolar degenerative changes in hepatocytes.

The normal architecture of liver was completely lost in chicks treated with

paracetamol. While chicks treated with paracetamol and plant samples of different

area showed a range of restoration of damaged liver tissue. Chicks treated with

paracetamol and of P. niruri extract of Ooty sample together showed kupffer cells

hyperplasia and regeneration activities in the liver cells. Treatment with doses of

Palani plant sample of P. niruri extract showed diffused vacuolar degeneration of

hepatocytes and hyperplasia and absence of centrilobular necrosis when compared

with control. Whereas in the chicks treated with plant samples of Madurai showed

mild focal individual cell necrosis. Thanjavur, Kumbakonam and Nagapattinam plant

samples treated in chick‟s liver showed diffused vacuolar degeneration of hepatocytes

and mild periportal fibrosis, indicating its hepatoprotective efficiency in chicks.

Silymarin, callus and Ooty plant samples treated chick‟s liver sections showed

reversible regeneration with mitotic figure and most of the liver cells appeared as

normal similar to that of control. In vitro grown callus tissue and Ooty populations of

P. niruri gave the prominent effect against intoxicated liver injury.

4.7. In vitro culture technique

4.7.1. Seed germination

The percentage germination of the seeds was determined for all the

accessions collected from the different study area. Seeds (presoaked in distilled water)

were allowed to germinate under dark condition on MS basal medium (without

growth regulators) and on the sterile moist cotton in culture tubes (Table - 19).

Maximum percentage of germination (93 ± 1.7) was observed from the seeds of Ooty

accession. The percentage of germination was comparatively less and a decreased


trend was noticed in all other accessions (Palani - 88±1.3; Madurai - 86±1.4;

Thanjavur - 77±1.5; Kumbakonam - 64±2.6 and Nagapattinam - 50±3.3). Among the

two different conditions of incubation, seeds placed in sterile moist cotton under dark

gave the maximum germination percentage.

4.7.2. Effect of basal media and cytokinins on shoot regeneration

To study the influence of constituents of media on shoot regeneration,

shoot tip and nodal explants were cultured on different media containing individual

BAP or KIN (0.5-3.0 mg/l). Among the three different media along with growth

regulators tested, MS-B5 was found to be better when compared to MS and B5 media.

Among the different concentrations of BAP used, the shoot tip explants in MS-B5

medium with 1.5 mg/l BAP were healthy and grew vigorously. In MS-B5 medium,

1.5 mg/l BAP yielded 96% of shoot tip explants bearing multiple shoots after 45 days

and explants exhibited an average of 7.8 normal shoots with healthy leaves. MS and

B5 media induced vitrification with few number of shoot production. MS-B5 medium

supplemented with 2.0 mg/l KIN was more effective for the frequency of sprouting of

shoot, number of shoots and shoot length than the other concentrations of KIN (Table

- 20).

Similarly, the nodal explants produced maximum number of shoots on MS-

B5 medium supplemented with BAP 2.0 mg/l (6.3 shoots) with a height of 9.4 cm

(Table - 21). In the present study, all the media tested containing 2.0 mg/l KIN

favoured the production of maximum number of shoots and shoot length. Further

increase in the concentration of KIN (3.0 mg/l) decreased the number of shoot

production and enhanced the basal callus after 30 days of culture and the callus was

white and compact in nature. However, in each medium, increased concentrations of

KIN (2.5 mg/l) enhanced the shoot number and favoured the moderate shoot length.

The explants inoculated in to the media (MS, B5 and MS-B5) containing more

amounts of KIN (3.0 mg/l) showed decreased shoot numbers as well as shoot length.

Excised explants cultured on the MS-B5 (KIN 3.0 mg/l) medium formed white

compact callus at the proximal end of the node after 30 days of culture. Hence all

further experiments were carried out using MS-B5 medium.


4.7.3. Effect of carbon sources and shoot regeneration

The responses of in vitro cultures to different carbon sources added to the

media were also tested. Shoot tip and nodal segments were cultured on MS-B5

medium containing 3% (w/v) of glucose, fructose or sucrose. These media were

supplemented with 1.5 mg/l BAP and 0.8% (w/v) agar. Among the three carbon

sources tested sucrose proved to be better for shoot regeneration than fructose and

glucose (Table - 22). Maximum shoot proliferation was obtained from the shoot tip

and nodal explants on sucrose supplemented medium [3% (w/v)], whereas glucose

and fructose favoured less number of shoots and minimum shoot lengths.

Shoot tip explants cultured on MS-B5 medium supplemented with sucrose

produced high shoot length (11.8 cm) as well as healthy shoots. Similarly nodal

explants cultured on MS-B5 fortified with sucrose produced healthy shoots with a

moderate shoot length (10.3 cm). However, shoot elongation was less on the medium

containing fructose, followed by glucose and the induced shoots were not healthy.

Shoots of shoot tip and nodal explants on MS-B5 medium fortified with fructose

attained a length of 5.6 and 4.8 cm respectively after 45 days of culture. Among the

different carbon sources tested in the rooting medium, the isolated shoots produced

healthy normal long roots in the sucrose supplemented medium, whereas glucose and

fructose in the media induced abnormal and fewer roots which were unsuitable for

hardening and transplantation.

4.7.4. Effect of combination of cytokinins on shoot regeneration

Various concentrations of KIN (0.5-3.0 mg/l) along with constant

concentration of BAP (1.5 mg/l) were tested for shoot induction and to determine the

multiplication potential of shoot tip and nodes. Combined effect of KIN and BAP

induced the growth of axillary shoots of explants cultured (Table – 23). Multiple

shoots developed from both shoot tip and nodal explants in MS-B5 medium

containing both BAP and KIN were more when compared with the media

supplemented individually. Multiple shoots produced from shoot tip explants were

maximum (16.5 shoots) in the combination of KIN (2.0 mg//l) and BAP (1.5 mg/l)

and the shoots grew faster upto a height of 4.9 cm, while nodes with maximum


number (14.0) of shoots were observed at the level of BAP (1.5 mg/l) and KIN (2.0

mg/l) combination, with a mean shoot length of 4.5cm (Plate-10 and 11).

In the lower concentrations of KIN (0.5 mg/l) with BAP (1.5 mg/l) multiple

shoots were obtained fom the shoot tip (8.4 shoots) and node (8.3 shoots) and the

induced shoots from both explants attained a mean shoot length of 10.8 and 12.9 cm

respectively. In the combination of KIN and BAP (each 1.5 mg/l) on MS-B5 medium,

shoot tip (11.9 shoots) and nodal (9.6 shoots) explants produced multiple shoots

which were slender with a height of 8.0 and 8.3 cm respectively. Increased frequency

of shoots sprouting and maximum number of shoots were obtained when the MS-B5

medium was supplemented with BAP (1.5 mg/l) + KIN (2.0 mg/l) from shoot tip and

nodal explants (Table - 23). Thus maximum number of shoots was obtained in MS-B5

medium supplemented with BAP (1.5 mg/l) + KIN (2.0 mg/l) from both of the

explants, but when the KIN concentration was raised, the shoots became dwarf. The

shoot length was strongly affected by the higher concentrations of KIN at and above

2.5 mg/l. White-greenish compact callus developed directly from the cut ends of the

explants (shoot tip and node) on MS-B5 medium when the KIN was further increased

(KIN - 3.0 mg/l).

4.7.5. Effect of sucrose and GA3 on the in vitro flowering

MS-B5 medium supplemented with 30g L-1

sucrose promoted the growth of

healthy plantlets. Among them, 60% of the plantlets started to produce in vitro

flowers after 2 weeks of culture and from the 4th

week onwards all the in vitro grown

plantlets produced flowers. When the amount of sucrose supplemented in the MS-B5

medium was increased to 50 and 70g L-1

, the plantlets were abnormal and in vitro

flowering was inhibited. Only less than 20% of the plantlets produced flowers even

after 4 weeks of culture on MS-B5 medium supplemented with 20 g L-1

of sucrose. In

vitro plantlets when cultured on MS-B5 medium supplemented with GA3 (0.5-1.5

mg/l) started early flowering with in seven days of culture. After two weeks of

culture, 40% of the in vitro plantlets produced flowers when they were cultured on

basal MS-B5 medium without plant growth regulator. On the other hand MS-B5

medium supplemented with 1.0 or 1.5 mg/l GA3 produced 98% and 100% flowering

respectively after four weeks of culture (Table – 24; Plate -17).


4.7.6. Callus induction and organogenesis

Depending upon the concentrations and combination of plant growth

hormone, the frequency and type of callus formation varied among the two explants

(shoot tip and node) (Table - 25 & 26). The maximum percentage (80.0±1.75) of

callus formation was observed on MS-B5 medium augmented with NAA 1.5 mg/l and

1.0 mg/l of BAP. Among different types of calli obtained on different media, the

friable and creamy white callus showed high rate of proliferation (Plate - 12). At

higher concentrations of auxins, the callus was hard and dark yellowish brown in

colour. Callus formation was achieved from shoot tip and nodal explants when

cultured in a vertical position on MS-B5 medium with BAP and NAA. The mean

fresh weight of callus was increased with the increased concentrations of BAP and

NAA. Friable white bulky callus was formed in the combinations of BAP (1.0 mg/l)

and NAA (1.5 mg/l) and this combination supported the highest frequency of callus

(80%) formation from nodal explants (Table – 26; Plate - 13). In shoot tip explants,

maximum percentage of callus was observed in the combinations of NAA 1.5 mg/l

and BAP 1.0 mg/l on MS-B5 medium and the nature of the callus was friable and

yellowish in colour. The combination of NAA (2.0 mg/l) and KIN (1.0 mg/l) showed

the higher callus biomass (48.4%) from shoot tip explants when compared to other

combinations of hormones. Media containing either BAP or KIN combined with

NAA, showed superior results in callus induction and growth.

The semi-friable callus obtained from shoot tip and nodal explants were

transferred to MS-B5 medium augmented with constant concentration of BAP (1.5

mg/l) with different concentrations of KIN (0.5-3.0 mg/l) in combinations for the

purpose of organogenesis. The callus derived from shoot tip and nodal explants was

found to be organogenic. Transfer of this callus to the medium BAP and KIN each 1.5

mg/l showed maximum response and induced the maximum number of adventitious

bud differentiation after two passages of subcultures. Minimum response was noticed

in most of the treatments and lowest number of shoot buds was observed only in BAP

(1.5 mg/l) and KIN (1.0 mg/l) containing MS-B5 medium. KIN above the level of 2.0

mg/l with BAP (1.5 mg/l) did not accomplish shoot organogenesis (Plate - 14). In the

first sub culture, the amount of callus increased slightly and proliferation of cells

could be noticed on the surface of the callus. From these newly proliferated cells,

adventitious buds were initiated after one week in the second subculture. For the


complete development and growth of buds another two weeks of culture was needed

(Data not shown).

4.7.7. Effect of auxins on rooting of shoots

Excised shoots were rooted on half-strength or full-strength MS-B5

medium with different types of auxins. It was found that in P. niruri, reducing MS-B5

salt strength to ½, enhanced the rooting frequency but reduced the formation of callus.

Half strength and full strength MS-B5 medium supplemented with all concentrations

of auxins induced roots from shoots within 30 days of culture. Among the three

auxins tested, the number of roots and root length varied in both medium (Table - 27).

Full strength MS-B5 medium fortified with 2.0 mg/l IBA showed better root

formation when compared to half strength MS-B5 medium with 2.0 mg/l IBA. Full

strength MS-B5 medium significantly promoted lengthy roots and strengthened root

induction within twenty days of culture. In half strength MS-B5 medium, IBA was

found to be more effective for root induction than IAA and NAA. Full strength MS-

B5 medium supplemented with IBA (2.0 mg/l) was more effective for root induction

than IAA and NAA (Table - 27; Plate - 15). However, IAA and NAA induced the

formation of slender roots in both media. Less amount of callus formation occurred in

all the types of auxins in full strength MS-B5 media. In general IBA was found to be

more effective for root induction in both types of media than IAA or NAA.

4.7.8. Hardening of regenerated plants and examination of morphological

characteristics

Plantlets were successfully acclimatized without growth chamber facility.

100% plantlet survival was observed after hardening on garden soil, farmyard

(manure) and sand (2:1:1) mixture for three weeks under shade incubation (Plate -16).

The percentage of survival was decreased to 96.0 and 74.6%, respectively after four

and ten weeks of acclimatization (Table - 28). The initial growth rates of plant height

were 9.3 ± 0.27 cm after first two weeks of acclimatization. On the other hand, in the

following three to ten weeks, substantial increase of plant height was observed.

Initially shoot produced, two to three healthy branches each bearing an

average of two to three leaves developed adjacent to the main shoot. Thereafter, the


number of branches per plant increased to 5.4 ± 0.12 and 14.03 ± 0.23, after four and

ten weeks of acclimatization respectively. Flowering occurred at the apical portion of

the main shoot initially but after six weeks each branch also developed flowering at its

terminal region. The number of flowers per plant increased to 3.9 ± 0.21 and 40.3.8 ±

0.28, after six and ten weeks of acclimatization.


5. DISCUSSION

Soil nutrients directly or indirectly influence the presence of such elements

or components in plants (Brady and Weil, 1999). Plant communities are directly

related to geology and soil types that may occur in a specific area (Van Rooyen and

Theron, 1996). Morpholgy, phenology and biochemical constiuents of the plants also

vary in response to physico-chemical characteristics of the soil (Sumathi et al., 2008).

Phyllanthus niruri is an important medicinal plant, which possesses active principles

such as alkaloids, flavonoids, phenols, coumarins, tannins, terpenoids and lignans

which form the part of the medicinal preparations in the pharmaceutical industries.

They generally possess antiviral, antidiabetic, antiplasmodial, antinociceptive,

antitumor, anticarcinogenic, hyperlipidemic, anti-inflammatory, antitumour and

antioxidant properties.

In the present investigation, different populations of P. niruri showed a

variations in their morphology, phenology and biochemistry in relation to the physico-

chemical properties of the soil where they grow. Charcteristation of the

phytochemical variations is an essential first step towards executing any plant

conservation and improvement programme. Environmental factors have important

role in the physiology and morphology of plants. In the dynamic environment plants

can respond to the changing conditions through altered production of chemical

compounds and morphometric triats.

The soil parameters of different study areas, morphology and genetic

diversity of P. niruri collected from different areas, quantitative and qualitative

phytochemical analysis and pharmacology with reference to antihepatotoxic property,

and antibacterial activities of the plant extract obtained with different solvents; in

vitro culture and micropropagation were made in the present study.

The morphometric traits varied under different environmental conditions. At

relatively high altitudes (Ooty and Palani) the plant height was low whereas at low

altitudes (Nagapattinam) it was high. Similarly all other morphometric parameters

such as root length, shoot length, leaf area index and biomass were also comparatively

high. The reason behind wide variations in morphometric triats could be due to soil


and other environmental factors including precipitation and temperature (Korner,

1999). Similarly it has been reported that most of the morphological data in sago palm

were highly variable in relation to the environmental conditions (Kjaer et al., 2004).

Variations in morphometric triats in relation to geographical region have also been

reported in P. amarus by Khan et al. (2010).

5.1. RAPD analysis

Molecular markers have been used to study the genetic diversity of various

plants species (Rafalski and Tingey, 1993, Staub et al., 1996). RAPD-PCR

(Polymerase chain reaction) has the advantage of being quick easy, high precision and

require little plant material (Steinger et al.,1996; Gugerli et al., 1999). Molecular

markers such as RFLP, RAPD, SCAR and AFLP have been used to determine the

genetic variation at the DNA level and to estimate the degree of relatedness between

individuals without the interference of environmental factors (Miller and Tanksley,

1990; Pandian et al., 2000). Genetic polymorphism in medicinal plants has been

widely studied which helps in distinguishing plants at inter- and/or intra-species level

(Joshi et al., 2004).

The similarity indices obtained in the present investigation clearly indicate

that P. niruri accessions from the different regions of Tamilnadu show less variations.

The collections shows close releation 92% level in terms of similarity index, inspite of

the fact that they belonged to geographically distinct locations. The subclusters in the

dendrograms could be to some extent correlated to the geographical distribution.

Group A of the first major cluster represents all the accessions from hill regions (Ooty

and Palani), except one which was a collection from dry areas of Madurai. In other

subcluster (B), all the accessions were from the Cauvery delta (Thanjavur and

Kumbakonam) and the remaining one was collected from the coastal belt

(Nagapattinam).

In the present study, similarity coefficient values obtained by RAPD profile

from the six different accessions were in the range of 0.88 - 0.92. These similarity

coefficient values were used to generate a dendrogram using UPGMA method. The

cluster analysis indicates that the six different populations of P.niruri grouped in to

two major clusters based on similiarity coefficient values. One major cluster had three


members in the population i.e., Ooty, Palani and Madurai. Another major cluster

includes three populations viz., Tanjavur, Kumbakonam and Nagapattinam. Each and

every population could be identified by using four random 10-mer primers. Among

the six populations, three of each clusters showed the highest similarity index (92%).

The present study provides evidence through RAPD data for the occurrence

of genetic variations among different collections of P. niruri. These results are in

agreement with previous reports in molecular polymorphism among populations of

Frankliniella intonsa in Hungary reported by Gyulai et al., (2001). In the present

attempt, the analysis of molecular variance indicated pronounced genetic differences

among populations of P.niruri. Observed genetic differentiation among the

populations is in accordance with the geographic isolation. The geographical isolation

is the major cause of genetic variation due to the decreased gene flow among the

individuals in the populations. Accordingly, prononunced genetic transformation

among the geographically isolated populations has been reported for a number of rare

species such as Astragalus cremnophylax and Gentianella germanica (Travis, 1996;

Fischer and Matthies, 1998).

On the contrary, the observations made by Jain et al. (2003) showed that

geographical grouping did not coincide with clustering by using RAPD banding

pattern of intra specific population. Interestingly, the accesions collected from

Tamilnadu grouped in two subclusters, indicating the differences in genotypes with

wide habitat diversity. Similarly, population in varied habitat with high degree of

genetic similarity (92%) was also observed in this study. High genetic similarity is

expected among P. niruri accessions in the southern part of the country, due to the

same geographical location. But, they showed broad genetic base indicating earlier

introduction of this species from costal to plain, and subsequently leading to

accumulation of variation. This situation could arise in natural populations when there

is a possibility of free/random pollen flow and fertilization,as is the case in most of

the cross pollinated species. On the other hand, further amplifications of such cross-

hybridized seeds through dissemination by natural modes like wind is possible. This

is probably the reason that accessions like Ooty, Palani and Madurai (cluster-A) with

Thanjavur, Kumbakonam and Nagapattinam (cluster-B), appeared closely related to


the genetic level, although geographically they are from different zones of highly

distinct locations of the Tamilnadu.

In a nutshell, the RAPD method used in this study displayed appreciable intra

population variation or molecular polymorphism, which pre-existed in different

collections. In spite of their morphological identity, substantial polymorphism was

observed among the accessions under study. Despite the consideration that the per

cent genome surveyed by different primers remains extremely less, the extent of

polymorphisms was found to be high. The present study clearly reveales that though

the decamer primers are small in comparison to the large genome of P. niruri, they

produced appreciable amplicons, sufficient to demarcate in all accessions collected

from the 6 locations.

Kanawapee et al. (2007) assessed intra-specific variations using RAPD

banding pattern showed similarity coefficients of 0.67, 0.56 and 0.60 among different

populations of P. amarus, P. urinaria and P. debilis respectively. Jain et al. (2003)

also obtained similar value of similarity index (0.65) among 33 accessions of P.

amarus collected from different locations in India.

The dendrogram also established a genetic relatedness among different

accessions and quantum of changes that occurred in the genome during the course of

evolution. The study confirms the suitability of RAPD as a reliable, simple, easy to

handle and elegant tool in molecular diagnosis of different accessions of an important

medicinal plant species available in a particular area. Thus RAPD proved to be useful

in molecular profiling of different accessions of P. niruri collected from diverse

places in Tamilnadu. Concurrently, it is also proved that the entries found to be

similar in taxonomical classification based on morphological characters do have

divergence at DNA level. Two major factors may be responsible for this variation i.e.

the difficulties in maintaining homogeneity in harvesting the P. niruri population

from a plethora of closely resembled Phyllanthus species and the climatic variations

resulting in biological differences in plants occurring at various geographic regions

(Lee et al., 1996).


On the basis of the RAPD analysis, the chosen samples have been grouped

under two clusters and labeled A and B. The present study, also indicate that

similarity indices were in the range of 0.86 to 0.92 and the variations among the

populations were low. Hence it is concluded that, RAPD fingerprints can be used as

an additional tool to assist in identification of morphologically similar and closely

related species of Phyllanthus. However, using RAPD fingerprint pattern for species

identification, although simple and quick, has certain limitations due to low

reproducibility of RAPD technique when using different PCR conditions and DNA

samples. Further works are in progress to develop more specific sequence

characterized amplified region markers for identification of this group of plants.


Exploring the healing power of plants is an ancient concept. For many

centuries people have been trying to alleviate and treat diseases with different plant

extracts and formulations (Cowan, 1999). Further it would be worthwhile to isolate

and chacterized the bioactive principles which are responsible for these activities.

A several bioactive molecules, such as lignans, phyllanthin, hypophyllanthin,

flavonoids, glycosides and tannins, have been indentified in the extracts of P. niruri

(Rajeshkumar et al., 2002). Presence of secondary metabolites and carbohydrates in

P. niruri suggests that this plant is one of the potential sources of drugs, which could

be used for the preparation, formulations and delivery of medicines of various cures,

and thus it forms one of the important medicinal plants as suggested by Kapoor

(2001), Igwe et al. (2007), Gupta and Rana (2007). Similarly several compounds such

as alkaloids, tannins, flavonoids, lignans, phenols and terpenes have also been isolated

and identified in various other species of Phyllanthus, and have been shown to

possess antinociceptive action in mice and other therapeutic activities (Filho et al.,

1996).

Qualitative phytochemical screening of all the six selected accessions of P.

niruri showed the presence of alkaloids, carbohydrates, phytosterols, saponins,

phenols, tannins, flavonoids, terpenoids, phlobatannins, proteins and free aminoacid

in the present study. All these compounds were identified from the methanolic and

aqueous extracts of all the selected accessions of P. niruri. Hence an attempt has been


made to quantitatively estimate the chemical constituents present in this medicinally

important plant.

The complexity of the mixture of compounds and the presence of several

compounds in small concentrations can make the isolation and identification of the

substances present in this genus very laborious. It has been established that the choice

of solvent in the isolation of compounds is very crucial (Calixto et al., 1998). It has

been proved that different environmental conditions can affect the chemical

constituents of the plants, both qualitatively and quantitatively and differing

interpretation of the spectral data of the complex structures has been reported (Khan

et al., 2010). Observation and interpretations made on the spectral data of the

complex structures of the constituents of the plants, without giving the consideration

to these facts, have led to confusion. Hence, it becomes essential to analyse the plant

constituents of pharmaceutical importance in relation to growth parameter of the plant

and the environmental conditions. Thus varieties of plants and growing conditions

according to geographical origin often play a significant part in determining the

quality and efficacy of these herbals. So, it is emphasized that a rapid and accurate

analytical technique is necessary to check if these factors cause wide difference in the

samples and also in their quality (Melendez and Capriles, 2006).

The quantitative GC-MS phytochemical analysis of the six populations from

different sites showed the presence of compounds belonged to alkaloids, flavonoides,

carbohydrates, saponins, tannins, lignans, phenol and terpenes as reported by Mishra

et al. (2000). Methanol has been prevalently used for phytochemical screening of

several medicinal plants (Merlin et al., 2008). Methanolic extraction protocol has

been frequently used, and the compounds were isolated and identified different

species of Phyllanthus by Filho et al. (1996). In the present study also, methanol was

found to be ideal for the extraction and fractionations of different constituents of P.

niruri using GC-MS.

Among the six populations of P. niruri, the plants from Ooty yielded a

maximum of 15 compounds. Those compounds are coming under alkaloids, phenolic

compounds, terpenes, fatty acids, etc., and are considered as medicinally valuable.


Accession from Nagapattinam showed lesser (3) number of compounds. Biosynthesis

of compounds among plants depends on the environmental factors in which they grow

(Khan et al., 2010). Intra specific variation in phytochemical constituents has been

documented extensively among the plants (Johnson and Scriber, 1994; Italer et al.,

2008). In P. amarus, phyllanthin biosynthesis was highly influenced by

environmental factors prevailing in different geographical locations (Khan et al.,

2010). The biosynthesis of biochemicals in plants was positively correlated to higher

altitudes of their occurrence (Ganzera et al., 2008; Khan et al., 2010). Similarly in the

present study also, presence of high amount of secondary metabolites in the Ooty

plants corroborates with earlier observations.

In the present investigation, in vitro callus culture was also subjected to

find out the qualitative and quantitative phytochemical constituents analysis using

GC-MS techniques. Totally eight compounds were identified from the callus extracts

of P. niruri. The accumulation of phytochemicals in plant cell cultures has been

studied for more than thirty years (Santos et al., 1994; Sokmen et al., 1999), and the

generated knowledge has helped in the understanding of using cell cultures for the

production of the desired phytochemicals (Castello et al., 2002). Callus cultures are

also used for analysis, quantitative estimation and comparison of secondary

metabolites synthesis between the intact plant and callus extracts (Bahorun et al.,

1994; El-Bahr et al., 1997; Rady and Nazif, 1997; Balz et al., 1999; Zhentian et al.,

1999). In the callus extracts of P. niruri, P. tenellus and P. urinaria the main

compounds identified were flavonoids, tannins and phenols (Santos et al., 1994).

5.3. Antibacterial activity

The quest for plants with medicinal properties continues to receive the

attention of the scientists to survey plants, particularly of ethnobotanical significance,

for a complete range of biological activities, which range from antibiotic to antitumor.

Thus plants have provided active principles even for western medicine prescribed for

a variety of health problems (Lewis and Elvin-Lewis, 1977; Bruneton, 1999).

Development of multi-drug resistance in pathogenic microbes and parasites and non-

availability of safe antifungal drugs for systemic mycoses necessitates a search for


new antimicrobial substances from other sources, including plants (Chopra et al.,

1992; Bruneton, 1995).

Plant - derived medicines have been part of traditional health care in most

parts of the world for thousands of years and nowadays there is an increasing interest

in plants as sources of agents to fight microbial diseases (Portillo et al., 2001;

Natarajan et al., 2003). Plants are the important source of potentially useful chemical

constituents and active principles for the development of new chemotherapeutic

agents. The first step towards this goal is the in vitro antibacterial activity assay (Tona

et al., 1998). Many reports are available regarding the antiviral, antibacterial,

antifungal, anthelmintic, antimolluscal and anti-inflammatory properties of plants

(Samy and Ignacimuthu, 2000). Some of these observations have helped in

identifying the active principle which is responsible for such activities and in the

developing drugs for the therapeutic use in human beings. However, many reports are

not available on the exploitation of antifungal or antibacterial property of plants for

developing commercial formulations for applications in crop protection.

The present study revealed that the P. niruri also possesses antimicrobial

properties. The overall results showed that the different concentrations of methanol

extracts of the plants had antimicrobial activity against many pathogenic organisms

such as Salmonella typhii, Klebseilla pneumoniae, Proteus mirabilis, Styphylococcus

auerus, Streptococcus mutans and Escherichia coli. The antimicrobial activity of the

plant extracts have been documented with several plants such as Cassia auriculata,

Calotropis gigantea, Clerodenrum infortunatum, Lantana camara, and Morinda

tinctoria against Gram-negative bacterium, E. coli, P. aeruginosa (Valsaraj et al.,

1997; Sami and Ignacimuthu, 2000; Srinivasan, 2001).

In the present study, it was found that the pathogenic bacteria such as

Salmonella typhii, Klebseilla pneumoniae, Proteus mirabilis, Styphylococcus auerus,

Streptococcus mutans and Escherichia coli were the most sensitive organisms to the

herbal extracts. The degree of sensitivity of the test organisms may be due to the

intrinsic tolerance of microorganisms and the nature and combinations of

phytochemical compounds present in the herbal crude extracts. Some of the common


phytoconstituents such as alkaloids, tannins, phenols, glycosides and flavonoids were

identified in the extracts. These major phytochemical compounds are known to have

antimicrobial activity (Bruneton, 1995).

Thus the methanolic extracts of P. niruri displayed a broad spectrum

antibacterial activity against Gram negative and Gram positive bacteria, while the

other solvent extracts were less inhibitory than the methanol. Among the different

plant populations tested, Ooty accession exhibited high degree of inhibiton against all

the six tested microorganisms when compared to other plant accessions. The

antibacterial principles were either polar or non-polar and were extracted only through

the organic solvent medium (John Britto, 2001). Plants produce a great deal of

secondary metabolites, many of them possess antibacterial activity. Earlier reports

clearly indicated that the antibacterial activity was due to different chemical

constituents including flavonoids, terpenoids, phenols and phenolic glycosides,

unsaturated lactones, sulphur compounds, saponins, cyanogenic glycosides and

glucosinolates which were classified as active antimicrobial compounds (Gomez et

al., 1990; Rojas et al., 1992; Bennett and Wallsgrove, 1994; Grayer and Harborne,

1994; Osbourne, 1996).

In the previous attempts also methanolic extracts of P. niruri were found to

have potent antibacterial activity against Salmonella typhii, Klebseilla pneumoniae,

Proteus mirabilis, Staphylococcus auerus, Streptococcus mutans and Escherichia coli

(Karthikeyan et al., 2008). The present experiment confirmed the previous studies

which reported that methanol was a better solvent for more consistent extraction of

antimicrobial substances from medicinal plants when compared to other solvents,

such as water, ethanol and chloroform (Ahmad et al., 1998; Eloff, 1998; Lin et al.,

1999).

On the basis of the present investigations it is highlighted that the methanolic

plant extracts show promising antibacterial activities and could be exploited in herbal

preparations against bacterial infections at least for external uses. Alternatively, the

active principles of these plant extracts may be characterized and tested for their

safety and efficacy to uncover their therapeutic potential in modern and traditional

medicine against infectious diseases. The results of present investigation clearly


indicate that the antibacterial activity varies with species and the characteristics

features of the locality. Thus, the present study ascertains the potential medicinal

value of this herbal plant used in the traditional medicines, and highlights the

significance of physicochemical properties of the habitat, and extraction protocol for

the formulations and development of delivery systemsfor the newly designd drugs.


The liver is an organ of paramount importance, which plays an essential role

in the metabolism of foreign compounds entering the body. Human beings are

exposed to these compounds through environmental exposure, consumption of

contaminated food and due to deleterious chemical substances in the occupational

environment. In addition, human beings consume a lot of synthetic drugs during

diseased conditions which are alien to body organs and their constituents. All these

compounds produce a variety of toxic manifestations. Conventional drugs used in the

treatment of liver diseases are often inadequate. It is therefore necessary to search for

alternative drugs for the treatment of liver disease to replace the currently used drugs

of doubtful efficacy and safety (Rajesh, 2001).

In the ayurvedhic system of medicine, herbal extracts but not in purified forms

have been used from many centuries, because many constituents with more than one

mechanism of action are considered to be beneficial. Attempts are being made to

develop new drugs from traditional medicines for different liver diseases such as

hepatitis, jaundice etc., (Liu et al., 2001). In the present study, an attempt has been

made to establish the hepatoprotective effect of P. niruri, in liver damage of

experimental chicken caused by paracetamol.

Chicken is one of the main meat sources consumed by the human beings for

their healthy life. In poultry, chicken often met with several diseases related to liver

disorder, which resulted in their weight loss. Increasing evidence suggests that liver

injury could be induced by the exposure of the body to various pollutants, toxicants,

hazardous chemicals and also to number of drugs. This major organ, responsible for

the metabolism of drugs and toxic chemicals, is also the primary target for many toxic

materials causing hepatic disorder (Skrivan et al., 2000; Jaeschke et al., 2002). In


general mammals and the chicks show high similarity in their physiology of liver. So

the chicks were used to explore the use of P. niruri in the treatment of hepatotoxicity.

Paracetamol is a well known antipyretic and an analgesic which produces

hepatic necrosis in high doses (Maheswari et al., 2008). Paracetamol is normally

eliminated mainly as sulfate and glucuronide. By the administration of toxic doses of

paracetamol, the sulfation and glucuronidation routes become saturated and hence,

high percentage of paracetamol molecules are oxidized to highly reactive N-acetyl-p-

benzoquinemine by cytochrome-450 enzymes. Semiquinone radicals, obtained by one

electron reduction of N-acetyl-p-benzoquineimine, can covalently bind to

macromolecules of cellular membrane and increase the lipid peroxidation resulting in

the tissue damage. High doses of paracetamol and N-acetyl-p-benzoquineimine can

alkylate and oxidise intracellular GSH, which results in the depletion of liver GSH

pool subsequently leads to increased lipid peroxidation and liver damage (Cover et

al., 2006).

In the present study, silymarin (100 mg/kg b.wt) treated animals were used

as positive control. The silymarin is a flavanolignan that has been introduced fairly as

a hepatoprotective agent (Valeozvela et al., 1994). It is one of the most well known

compounds of the flavonoids. It is extracted from the seeds and fruit of Silybum

marianum (Compositae) (Desplaces et al., 1975). The seeds of S. marianum have

been used for almost 2,000 years as a natural medicament for the liver and biliary

duct. The silymarin, a pharmacologically effective substance, contains four main

constituents namely silybin (50 - 60%), isosilybin (5%), silychristin (20%) and

silydianin (10%). It is used in the treatment of numerous liver disorders characterized

by the degenerative necrosis and functional impairment. Furthermore, it is able to

antagonise the hepatotoxin and provides (hepato) protection against poisoning by

phalloidin, galactosamine, paracetamol, thioacetamide, halothane and CCl4 (Barbarino

et al., 1989).

Available literature sources state that silymarin acts in four different ways

(i) as an antioxidant, absorber and regulator of the intracellular glutathione, (ii) as a

stabiliser and regulator of cell membrane permeability that prevents the entering of


hepatotoxic substances into hepatocytes, (iii) as the ribosomal RNA synthesis

promoter simulating regeneration of the liver; and (iv) as an inhibitor of the

transformation of liver stellate cells into myofibroblasts i.e., the process is responsible

for deposition of collagen fibres in liver. Furthermore, absorption of free radicals by

silymarin is considered to be one of the key mechanisms securing liver protection

(Fraschini et al., 2002).

In the present investigation, it was observed that in the paracetamol

intoxicated group, there was an elevation in the levels of various markers of hepatic

damage such as total bilirubin, SGOT, SGPT and alkaline phosphatase (ALP) which

could be due to the toxic property of paracetamol. Carbon tetrachloride/ paracetamol

induced hepatic injuries are commonly used models for the screening of

hepatoprotective drugs and the extent of hepatic damage is assessed by the level of

released cytoplasmic alkaline phosphatase and transaminases in circulation. It is well

documented that CCl4/PCM are biotransformed under the action of microsomal

cytochrome P-450 of liver to reactive metabolites (Raucy et al., 1993). Cytochrome p-

450 exhibits a key function in the biotransformation of xenobiotics, catalyzes the

reductive transformation of foreign compounds and displays an oxidase activity

resulting in reactive oxygen species (ROS) formation. The activation of molecular

oxygen by cytochrome p-450 requires electrons from the donor NADPH -cytochrome

p-450 reductase (NADH-cytochrome b5 reductase) (Czinner et al., 2001).

Treatment with the extract of P. niruri has decreased the levels of the

biochemical markers of the hepatic damage and stabilished them to the normal levels.

Literature review shows that the P. niruri contains phenolic compound and flavonoids

and possibility of possessing hepatoprotective activity. The phytochemicals present in

P. amarus, responsible for these activities have been established as lignans

(phyllanthin and hypophyllanthin) (Khatoon et al., 2006). Further studies are needed

to elaborate wheather some other compounds present in the extracts are also

responsible for hepatoprotection in paracetamol induced liver damage and the

molecular basis of their mode of action.


Preliminary phytochemical screening of the methanol and aqueous extracts

of P. niruri revealed the presence of alkaloids, flavonoids, saponins, cardiac,

glycosides, triterpenoids, phenolic compounds and tannins. In the present state of

knowledge of the chemical constituents of the extract of this plant, it is not possible to

attribute with certainty the hepatoprotective effect to one or several active principles

among those detected in the methanol and aqueous extracts of P. niruri. However,

flavonoids (Paya et al., 1993), titerpenoids (Gao et al., 2004), saponins (Tran et al.,

2001) and alkaloids (Vijayan et al., 2003) are known to possess a cumulative

hepatoprotective activity in animals.

5.4.1. Histopathology

It is well known that toxicants like CCl4 and PCM produce sufficient injury

to hepatic parenchyma cells to cause elevation in serum bilirubin, and in contrast

decrease the level of total plasma protein content (Plaa and Hewitt, 1982).

Hepatocellular necrosis or memberane damage leads to very high levels of SGOT and

SGPT released from liver to circulation. Among these two, SGPT is a better index of

liver injury, since SGPT catalyses the conversion of alanine to pyruvate and

gultamate, and is released in a similar manner, thus liver SGPT represents 90% of

total enzyme present in the body (Achliya et al., 2003).

Histopathological analyses of liver samples showed that the focal areas of

necrosis with periportal chronic necrosis in paracetamol treated liver of chick, while

chick simultaneously treated with paracetamol and P. niruri extracts showed kupffer

cells hyperplasia and regeneration activities in cells lead to heal injury. The present

observation clearly demonstrates the potential hepatoprotective activity of P. niruri

extract.

The hepatoprotective activity of P. niruri was observed in the present study

is due to its stimulatory effect on both enzymatic and non-enzymatic antioxidant

systems in the experimental chicks. Consequently, the hepatotoxicity and damage

induced by paracetamol in the liver of chicks is suppressed with the administration of

the extract of P. niruri, which was due to the reduction in the level of reactive oxygen


species (ROS) as indicated by the reduction in the level of total bilirubin and the

induction of recovery and repair process in the liver of chicks.

Similar observations have also been reported by several others against various

chemical liver toxins CCl4, PCM and galactosamine (Liu et al., 2001) could be due to

the presence of lignans, i.e., phyllanthin and hypophyllanthin (Xin-Hua et al., 2001).

These phytochemicals present in P. niruri are also reported to act as hepatoprotective

agents and protect hepatocytes against carbon tetrachloride (CCl4) and galactosamine

induced cytotoxicity in rats (Khatoon et al., 2006).

It is of interest to note that the aqueous extract showed remarkable activity at a

concentration as low as 6.25 mg/kg. The potentiality of this plant extract is superior to

that of silymarin, a commonly used hepatoprotective herbal drug. It is likely that an

active fraction from the extract could be effective at a very low concentration. This

extract is an attractive material for drug development. Hence, it is concluded, that the

aqueous extract of P. niruri produce considerable effect of alleviation for the liver

damage from the hepatotoxic action of paracetamol in the chicks, which is in line with

the findings made by Meixa et al. (1995) and Latha and Rajesh (1999).

5.5. In vitro culture – Micropropagation

Due to large-scale, unrestricted exploitation of this natural resource to meet

the ever increasing demand of the Indian pharmaceutical industry coupled with

limited cultivation and insufficient attempts for its replenishment, this medicinally

important plant species is markedly depleted (Pandey et al., 1993). Moreover,

availability of this plant is subjected to seasonal variations leading to uncertainty in

constant supply throughout the year. Hence development of viable micropropagation

protocol becomes neessary important for the ex situ conservation and sustainable

utilization of this species. So establishment of a micropropagation protocol for P.

niruri, ensures large scale production and supply, assuring continuous availability of

plant material, and also serves as a strategy of in vitro culture to increase the yield of

active principles accumulated in cultures of P. niruri.


5.5.1. Seed germination

Treatment with sulphuric acid (2% and 5%) acts as surface sterilizing

chemical used to improve the seed germination of Phoenix dactylifera (Date palm)

(Khan et al., 1985; Ikuma and Thimann, 1960). But in the present investigation, 0.1%

mercuric chloride solution was used as surface sterilizing agent for the germinating

seeds of P. niruri. The percentage of seed germination was determined and recorded

in two different conditions (MS basal medium without growth regulators and sterile

moist cotton). The germination percentage was more in sterile moist cotton (Ooty

accessions) than the MS basal medium without growth regulators. The percentage of

seed germination was also high in Ooty accession when compared to remaining

accessions. This difference in the germination percentage of seeds might be due to

altered physiology of embryos and liberating enzymes due to the impact of different

conditions (Kattimani et al., 1999). According to Zhang and Maun (1990) size of the

seed also influences the germination percentage in the case of Agropyron

psammophilum. Thus the present study suggests that the seed germination in sterile

moist cotton under dark conditions was more economic for developing mass planting

stocks at low cost.

5.5.2. Effect of basal media and BAP/KIN on shoot regeneration

To know the effect of different media and cytokinins on shoot regeneration

of P. niruri, shoot tip and nodal explants were cultured on three types of media

supplemented with various concentrations of BAP or KIN. The degree of growth and

differentiation varied considerably with the medium constituents (Shekhawat et al.,

1993; Das et al., 1996). As nitrogen is to be a constituent of plant cell components, its

deficiency inhibits plant growth. In addition to this, total nitrogen content and the

ratio of nitrate to ammonium (NH4

-

) are very important aspect in nitrogen nutrition

(Ramage and Williams, 2002).

This is because of the fact that this ratio strongly influences the pH of the

medium, which in turn determines the absorption of other nutrients (Tefera and

Wannakrairoj, 2004). Thus, as in most of the plant species, the relatively higher

supply of nitrate-nitrogen within the MS medium could have exerted the profound


effect on shoot growth of this plant species. MS-B5 medium was also found to be

more effective than other media (Karthikeyan, 2004). In contrast to the report of

Baskaran and Jayabalan (2005) who found MS was the best basal medium for E. alba,

MS-B5 medium produced the higher number of shoots in this study. A similar

phenomenon has already been observed in the in vitro culture of Withania somnifera

(Chandran et al., 2007).

Comparing the effect of cytokinins type (BAP and KIN) on shoot production,

the best response was achieved by BAP as in Centella asiatica (Karthikeyan et al.,

2009). BAP was far more effective than KIN for inducing proliferation of axillary

buds in Cichorium intybus (Velayutham et al., 2006). BAP and KIN in MS-B5

medium showed varied responses and BAP was more effective than KIN in inducing

multiple shoots in P. niruri (Karthikeyan et al., 2009). MS-B5 medium with different

concentrations of KIN did not show any improvement for the increased production of

shoots in Eclipta alba (Baskaran and Jayabalan, 2005).

In general and also found in the present study, higher concentrations of

cytokinins (above 2.5 mg/l) reduced the shoot number as well as shoot length (Table

1). This finding is also in line with the finding of Hu and Wang (1998) who reported

that higher concentrations of cytokinin reduced the number of micropropagated

shoots. A similar response was also observed in Mentha piperita (Kiran Ghanthi et

al., 2004). In contrast, KIN was the effective cytokinin resulting in multiple shoot

induction rather than shoot elongation but BAP showed better response resulting in

multiple shoot induction as well as shoot elongation in Artemisia pallens (Usha and

Swamy, 1994).

The explants in the media (MS, B5 and MS-B5) containing more KIN

showed decreased shoot numbers as well as shoot length. Excised explants cultured

on MS-B5 medium with KIN (2.5-3.0 mg/l) formed white compact callus at their

proximal ends. Similar results were also observed in Peganum harmala (Saini and

Jaiwal, 2000) and Holostemma adakodien (Martin, 2000). The callus formation might

be due to the accumulated auxin at the basal cut ends, which stimulates cell

proliferation especially in the presence of cytokinins (Marks and Simpson, 1994). The


callus formation at the basal cut ends of nodal explants on cytokinin enriched medium

is frequent in Silver maple with strong apical dominance (Preece et al., 1991).

The MS-B5 medium supplemented with low concentrations of BAP (0.5-

1.0 mg/l) promoted only root formation rather than KIN. Similar result was observed

in Eclipta alba (Franca et al., 1995). It was difficult to isolate rooted shoots from the

culture without the damage to the roots. In each media, when the cultures were

maintained for a long time (after 4 weeks), there was gradual browning and

defoliation of leaves. A similar phenomenon was noticed in E. alba and Eupatorium

adenophorum by Borthakur et al. (2000). These observations indicate that the MS-B5

medium with specific concentrations of BAP (1.5 mg/l) favoured for promoting shoot

proliferation in P. niruri as recorded in the present study.

5.5.3. Carbon source and shoot regeneration

The responses of in vitro cultures to different carbon sources added to the

medium were also determined. Although carbohydrates are of prime importance for in

vitro organogenesis, carbon metabolism in vitro is still not clearly understood (Kozai,

1991). It is well established that carbohydrate requirements depends upon the stage of

culture and may show differences according to the species (Thompson and Thorpe,

1987).

Among the different sources of carbohydrates tested in the present research

work sucrose was more effective than the others. Similar results were already

obtained in micropropagation of cork oak (Romano et al., 1995) and Kaempferia

(Fatima et al., 2000). Sucrose has been commonly used as a carbon source in tissue

culture media (Fuentes et al., 2000). This is due to its efficient uptake across the

plasma membranes of plant tissues (Borkowska and Szezebra 1991). However,

glucose was most effective for shoot proliferation in Prunus (Hisashi and Yasuhiro,

1996). Sucrose and glucose gave a similar rate of proliferation in sour cherry

(Borkowska and Szezebra, 1991). However, sucrose and glucose induced highest

frequency of organogenesis in Bixa orrellana (De Paiva Neto et al., 2003).

In Malus robusta, fructose gave the lowest number of shoots (Pua and

Chong, 1984). However, the same authors showed that in shoot cultures of the apple


scion cultivar Macspur there was no differences in shoot multiplication between

sucrose, fructose and glucose. However, the shoot elongation was moderate on

fructose, followed by glucose but the shoots were not healthy. This difference could

not be directly linked to the carbohydrate nutritional aspects, but with carbohydrate

osmotic contribution. Pritchard et al. (1991) reported that the carbohydrates control

morphogenesis by acting as energy source and also by altering the osmotic potential

of the culture medium. But in the present investigation, sucrose gave more number of

shoots of P. niruri than the other carbohydrates tested.

5.5.4. Effect of combinations of cytokinins on shoot regeneration

Combinations of various concentrations of KIN with BAP were tested

for shoot induction and shoot multiplication potential of shoot tip and nodal explants.

Combined effect of KIN and BAP, increased the axillary shoots. Axillary shoots were

induced in BAP alone or in combination with other cytokinins in Macrotyloma

uniflorum (Varisai et al., 1999). On the other hand similar results were obtained in

Dioscorea composita shoot cultures, where the combination of NAA, IAA and IBA

stimulated significantly the more number of nodes per plantlet in comparison to

cytokinins (Alizadeh et al., 1998).

Relatively more morphologically uniform multiple shoots were developed

from the both (shoot tip and nodal) of the explants in MS-B5 medium containing BAP

combined with KN. A similar response was observed in Gloriosa (Sivakumar and

Krishnamurthy, 2000). The combined effect of cytokinins (BAP and KIN) enhanced

multiple shoot bud regeneration in Arachis hypogaea (Venkatachalam and Jayabalan,

1997). On the contrary, the combination of cytokinins (BA and KIN) failed to

improve shoot multiplication in Sterculia urens (Purohit and Ashish, 1984).

The results of the present study suggested that the shoot tip explants were

better and more suitable for the micropropagation of P. niruri than the nodal explant.

The shoot tip explants have been suggested as the best source of multiple shoot

induction in other medicinal plants also, such as Adhatoda vasica (Sangeetha and

Buagohain, 2005) and Decalepis hamiltonii (Giridhar et al., 2005). Cytokinins,

especially BAP were reported to overcome apical dominance, release lateral buds


from dormancy and promote shoot formation (George, 1993). The reason for

effectiveness of the BAP may lie in its ability to stimulate the plant tissue to

metabolize the natural endogenous hormones or could induce the production of

natural hormone system for the induction of shoot organogenesis (Ghanti et al.,

2004). The increased activity of BAP compared to kinetin was also reported by earlier

workers (Chirangini et al., 2005).

5.5.5. Effect of sucrose / GA3 on the in vitro flowering

The in vitro raised shoot tip and nodal explants that are remained aseptic

were cultured vertically with the basal end placed in to MS-B5 supplemented with

0.5, 1.0, and 1.5 mg/l of GA3

respectively. High concentration of GA3 (1.5 mg/l)

favoured maximum (100%) response of flower induction in vitro after four weeks of

culture. This study showed that GA3 could shorten the period of flowering. In contrast

Liang and Keng (2006) observed in vitro flowering of P. niruri on MS basal medium

without growth regulators. Similarly flowering of Murraya paniculata was observed

in vitro on MS basal medium without growth regulator, whereas the flowering was

inhibited or reduced when supplemented with BAP or NAA respectively (Taha,

1997).

The in vitro raised shoot tip and nodal explants that are remained aseptic were

cultured vertically with the basal end placed in to MS-B5 (GA3 1.5 mg/l)

supplemented with 20, 30, 50 or 70 g/l sucrose. Optimal concentration (30 g/l) of

sucrose favoured maximum (100%) response of flower induction in vitro after four

weeks of culture. According to Haicour et al. (1994) flowering in vitro could

overcome the reproductive barriers observed when studying the experimental

crossings within Phyllanthus subsect. Odontadenii, which shows different level of

reproductive incompatibility between taxa. Observations under light microscope

showed that the flower morphology of the in vitro plantlet was similar except the

flower size when compared to mother plant, where the flowers were bigger.

Rajasubramaniam and Saradhi (1997) reported that in vitro plantlets of P. frartenus

only flowered after ex vitro transfer. Catapan et al., (2000) also reported the

occurance of same phenomena in P. caroliniensis.


5.5.6. Effect of BAP/KIN with NAA on callus induction and organogenesis

The frequency of callus formation varied based on the concentration of the

plant growth hormone supplemented in the medium. Different types of calli were

obtained of which, the friable, semi-friable and creamy white coloured showed high

proliferation rates. The semi-friable callus was transferred to MS-B5 medium

augmented with different combinations and concentrations of BAP, KIN, and NAA

either alone or in combinations for the purpose of organogenesis. Among different

combinations tested, both BAP and KIN (each 1.5 mg/l) favoured shoot regeneration.

Orientation of explants in the culture medium plays a vital role in callus induction.

In the present study, callus formation was achieved in shoot tip and nodal

explants cultured in a vertical position on MS medium with BAP and NAA. These

results were similar to P. stipulatus and contrast to those obtained in P. caroliniensis

under the same culture conditions (Catapan et al., 2001). The results of the present

work are also in line with the observations made in P. emblica and herbaceous

members of Phyllanthus (Unander, 1991) when the combinations of cytokinins and

auxins were used for the production of organogenic callus.

For callus mediated regeneration in Phyllanthus niruri, explants were cultured

on the medium containing strong auxins and cytokinin for the production of callus.

These calli were transferred to medium supplemented with cytokinin and a weak

auxin for shoot regeneration (Karthikeyan et al., 2007). In the present study, the

semi-friable calli obtained from shoot tip and nodal explants were transferred in to

MS-B5 medium augmented with constant concentration of BAP (1.5 mg/l) with

different concentrations of KIN (0.5-3.0 mg/l) in combinations for the purpose of

organogenesis. Transfer of this organogenic callus to the medium containing BAP and

KIN each 1.5 mg/l showed maximum response and induced the maximum number of

adventitious bud differentiation after two passages of subcultures.

5.5.7. Effect of auxins on rooting of shoots

The regenerated shoots were excised and transferred on rooting medium (full

and half strength) supplemented with different concentrations of IBA or IAA or NAA

(0.5 to 2.5 mg/l). The present observations clearly indicate that auxins were found to


induce and enhance rooting from the basal cut ends of the shoots. Roots were not

induced during culture initiation, shoot formation and shoot multiplication in

the cytokinin regime. Excised shoots were rooted on half-strength or full-strength

MS-B5 medium with different types of auxins. Half strength and full strength MS

medium supplemented with all concentrations of auxins induced roots from shoots

within 30 days of culture. Addition of auxins IBA and NAA to MS-B5 medium

enchanced the rate of rhizogenesis. Highest rate of frequency of rooting was observed

in the full strength MS-B5 medium containing 2.0 mg/l (94.3%), NAA at 2 mg/l

(88%) of IBA followed by IAA at 2.0 mg/l (67.4%). The results on rooting

experiments confirms that NAA and IAA were less effective compared to IBA. The

slow movement towards growing shoot and slow degradation of IBA in the medium

facilitates its function better in inducing roots. Similar responses were also reported in

different plant species such as Vitex negundo (Sahoo and Chand, 1998), Gymnema

sylvestre (Komalavalli and Rao, 2000), Gloriosa superba (Sivakumar and

Krishnamurthy, 2000).

5.5.8. Hardening of regenerated plants and examination of morphological

characteristics

Plantlets were successfully acclimatized without growth chamber facility.

100% plantlet survival was seen after hardening on garden soil, farmyard (manure)

and sand (2:1:1). Sterilized garden soil minimized the cost of transplantation as

documented by several authors (Agretious et al., 1996; Anand et al., 1997). In the

present experiment, there was no detectable variation among the acclimatized plants

with respect to morphological growth characteristics and floral features. All the

micropropagated plants were free from external defects. In contrast to the present

reports, earlier studies of in vitro cultures of many medicinal plants, due to the

delicate nature of in vitro regnerated plantlets, required special arrangements such as

controlled green house conditions, use of soil free potting mix like perlite,

vermiculite, peat plugs and application of fungicides were needed for easy and

successful acclimatization of plantlets (Baskaran and Jayabalan, 2005). Thus the

present study provides a protocol for regeneration of complete plantlets has been

established. The results obtained in the present investigation assume significance as it

is a pioneering study on tissue culture of this medicinal plant. The protocol described


in the present study is reproducible and highly useful for large scale production of this

important medicinal plant through micropropagation. Thus this thesis concludes on

the basis of the findings of the present investigation as follows

1. RAPD analysis favoured for the division of six populations in to two cluster of

three each.

2. The Ooty plant sample contains 15 different types of compounds with

pharmacological importance.

3. P. niruri offers hepatoprotective effect against paracetamol induced

hepatotoxicity by means reducing the levels of serum markers enzymes in the

tested animals.

4. The methanolic extract of this plant possess more antimicrobial compounds

against human pathogenic bacteria.

5. Sterile moist cotton under dark condition could be used for maximum seed

germination of this plant.

6. Both shoot tip and nodal parts of the plant P. niruri could be used for

regeneration and micropropagation and

7. Acclimatization and hardening of the in vitro plantlets could be successfully

achieved using mixture of garden soil, FYM and sand and as a substrate under

open shade incubations.


6. SUMMARY

The thesis entitled “Phytochemical, pharmacological, in vitro propagation and

molecular studies on Phyllanthus niruri L.” deals with the analysis of soil

characteristics of different study area; the morphometric characteristics; biochemical

constituents including alkaloid, carbohydrates, phytosterols, saponins, phenols,

tannins, flavonoids, terpenoids, phlobatannins, protein and free aminoacids;

antibacterial activity; pharmacology and in vitro propagation of P.niruri.

The physico-chemical characteristics of the soils studied from the six

different locations showed wide variations in their soil texture, pH, organic and

inorganic nutrients contents.

The plant samples were collected from six different districts of Tamilnadu,

South India. Morphometric and phytochemical studies revealed variations in the

morphology and biochemical constituents.

Fresh leaf samples (young leaves) collected from the field (one month old)

were used for DNA isolation. The cluster analysis using RAPD data indicates that the

six different populations of P.niruri grouped in to two clusters based on similiarity

indices. One cluster has three population belonged to Ooty, Palani and Madurai.

Another cluster includes three populations viz., Tanjavur, Kumbakonam and

Nagapattinam.

Among the six populations, three of each cluster showed the highest

similarity indices (92%). The present study provides evidences for the occurance of

less genetic variations among the different collections of P. niruri.

The qualitative and quantitative phytochemical analysis confirmed the

occurrence of alkaloid, carbohydrates, phytosterols, saponins, phenols, tannins,

flavonoids, terpenoids, phlobatannins, protein and free aminoacids. GC-MS analysis

revealed that a maximum of 15 compounds were recorded from the methanolic extract

of the plant samples from Ooty. As per the report of earlier literature, these

phytochemical compounds are known to have various medicinal curative properties.


Methanolic extract of P. niruri showed maximum zone of inhibition against

multidrug resistant strains of six common human pathogenic bacteria viz., Escherchia

coli, Klebsiella pneumoniae, Salmonella typhii, Proteus mirabilis, Staphylococcus

aureus and Streptococcus mutans.

In the pharmacological study, it was observed that the paracetamol induced

hepatotoxicity was effectively controlled by P. niruri in the experimental animals.

The paracetamol intoxication enhanced serum markers such as serum total bilirubin,

SGOT (AST), SGPT (ALT) and alkaline Phosphatase (ALP). On the other hand, the

level of these enzymes were reduced and stabilized to their normal level when the

animals were treated with P. niruri.

Histopathological observations of the hepatotoxic liver treated with the

aqueous extract of samples of P. niruri showed kupffer cells hyperplasia, regeneration

activities in the liver cells and absence of centrilobular necrosis in hepatocytes which

were later recovered and became normal. Silymarin, callus and Ooty plant samples

treated chick‟s liver sections showed reversible regeneration with mitotic figure and

most of the liver cells were appeared normal, similar to that of control chicks.

Maximum of 93% seed germination was achieved when the seeds were placed

in sterile moist cotton under dark conditions.

The type of medium, carbon sources and plant growth regulators markedly

influenced the in vitro propagation of P. niruri. The in vitro plantlet production

system was investigated on Murashige and Skoog (MS-B5) medium with the

combination of BAP (1.5 mg/l) and KIN (2.0 mg/l) and 3% sucrose which induced

maximum number of shoots as well as beneficial for shoot length. Subculturing of

shoot tip and nodal segments on similar medium enabled continuous production of

healthy shoots with similar frequency.

Maximum percentage (80.0 ± 1.75) of callus (Friable white bulky) production

was achieved from the nodal explants on MS-B5 medium fortified with NAA 1.5 mg/l

and 1.0 mg/l of BAP. Callus mediated regeneration was achieved from shoot tip and

nodal explants tested. Among the two explants investigated for regeneration,

maximum percentage of regeneration and number of shoots per explant were obtained

from the shoot tip on MS-B5 medium supplemented with BAP (1.5 mg/l) and KIN


(2.0 mg/l). It was followed by nodal explants. These calli produced maximum number

of shoots when sub cultured on the MS-B5 medium containing BAP and KIN (each

1.5 mg/l).

The regenerated shoots from both of the explants were responded well for

rooting on MS-B5 medium supplemented with IBA or IAA (2 mg/l). Rooting was

highest (94.3%) on full strength MS medium containing 2.0 mg/l IBA.

Micropropagated plants established in a mixture of garden soil, farmyard (manure)

and sand (2:1:1) were uniform and identical to the donor plant with respect to growth

characteristics as well as floral features. These plants grew normally without showing

any morphological variation.

Thus the findings of the present investigation helped to find out the accession

with medicinal qualities and to use for the mass propagation of this medicinally

important herbal plant through in vitro regeneration and subsequently to offer

protection against hepatotoxicity induced by the indiscriminate use of various drugs in

human beings.


List of Publications

1. Chandran, C., Karthikeyan, K. and Kulothungan, S., 2007. In vitro propagation

of Withania somnifera (L.) Dunal from shoot tip and nodal explants. Journal of

Scientific Transactions in Environment and Technovation, 1(1): 15-18.

2. Karthikeyan, K., Chandran, C. and Kulothungan, S., 2007. Rapid regeneration

of Phyllanthus niruri L. from shoot tip and nodal explants. Indian Journal of

Applied and Pure Biology, 22(2): 337-342.

3. Karthikeyan, K., Chandran, C. and Kulothungan, S., 2008. In vitro propagation

of Phyllanthus niruri L. – A medicinal plant, Journal of Scientific Transactions

in Environment and Technovation. 1(3): 131-133.

4. Kulothungan, S., Karthikeyan, K., Chandran, C. and Ganapathi, A., 2008.

Morphogenetic responses from in vitro cultured shoot tip and nodal explants of

cowpea [Vigna unguiculata (L.) Walp]. Indian Journal of Applied and Pure

Biology, 23(2): 319-327.

5. Karthikeyan, K., Chandran, C. and Kulothungan, S. 2008. Antibacterial

activity of Phyllanthus niruri L., Indian Journal of Applied and Pure Biology.

23(2): 295-297.

6. Karthikeyan, K., Chandran, C. and Kulothungan, S., 2009. Rapid clonal

multiplication through in vitro axillary shoots proliferation of Centella asiatica

L. (Vallarai) – A rare medicinal plant. Indian Journal of Biotechnology, 8: 232-

235.

7. Karthikeyan, K., Chandran, C. and Kulothungan, S., 2009. In vitro flowering

and rapid regeneration of Ocimum sanctum – A valuable medicinal herb. Asian

Journal of Microbiology, Biotechnology and Environmental Science, 1(1): 37-

40.

Documents

1. INTRODUCTION - 14.139.186.108