Antimicrobial Study

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ANTIMICROBIAL STUDIES IN

ANDROGRAPHIS PANICULATA

PROJECT REPORT

submitted in partial fulfillment of the requirements

for the award of the degree of

BACHELOR OF TECHNOLOGY

inBIOTECHNOLOGY

by

PREETY PRIYA (10904215)

&

K.S.PRASHANSA (10904202)

under the guidance of

Mrs. S. Rupachandra, M.Sc., M.Phil.,(Lecturer, Department of Biotechnology,School of Bio-engineering, SRM University)

DEPARTMENT OF BIOTECHNOLOGY

SCHOOL OF BIOENGINEERING

FACULTY OF ENGINEERING AND TECHNOLOGY

SRM UNIVERSITY

KATTANKULATHUR 603 203

April 2008

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CERTIFICATE

Certified that the project report entitled ANTIMICROBIAL STUDIES IN

ANDROGRAPHIS PANICULATA submitted by PREETY PRIYA

(10904215) is a record of project work done by her under my supervision. This

project has not formed the basis for the award of any degree, diploma,

associateship or fellowship.

INTERNAL GUIDE HEAD OF THE DEPARTMENT

For the purpose of viva voce

1.

2.

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CERTIFICATE

Certified that the project report entitled ANTIMICROBIAL STUDIES IN

ANDROGRAPHIS PANICULATA submitted by K.S.PRASHANSA

(10904202) is a record of project work done by her under my supervision. This

project has not formed the basis for the award of any degree, diploma,

associateship or fellowship.

INTERNAL GUIDE HEAD OF THE DEPARTMENT

For the purpose of viva voce

1.

2.

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DECLARATION

I do hereby declare that the project report entitled ANTIMICROBIAL

STUDIES INANDROGRAPHIS PANICULATA is a record of original

work carried out by me under the supervision of Mrs. Rupachandra, Lecturer,

Department of Biotechnology,School of Bio-engineering, SRM University.This

project has not been submitted earlier in part or full for the award of any

degree, diploma, associateship or fellowship.

Kattankulathur PREETY PRIYA

Date

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DECLARATION

I do hereby declare that the project report entitled ANTIMICROBIAL

STUDIES INANDROGRAPHIS PANICULATA is a record of original

work carried out by me under the supervision of Mrs. Rupachandra, Lecturer,

Department of Biotechnology,School of Bio-engineering, SRM University.This

project has not been submitted earlier in part or full for the award of any

degree, diploma, associateship or fellowship.

Kattankulathur K.S.PRASHANSA

Date

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ACKNOWLEDGEMENTS

It gives us great pleasure to humbly place on record my heartfelt thanks and gratitude

to Dr. K. Ramasamy, Dean, School of Bioengineering, SRM University.

Our sincere gratitude and thanks to Dr. D. Kantha Arunachalam, Head ofDepartment of Biotechnology.

We would also like to sincerely thank Dr. David Paul Raj, Assistant professor andHead of department of Bio process engineering, SRM University, for his guidanceand support.

Our special thanks are to our internal guide Mrs. S. Rupa Chandra, Lecturer,Department of Biotechnology, School of Bio-engineering, SRM University, for herconstant support and help in the presentation and editing of the thesis.

We would also like to thank Dr. Lakshmi Narasu, Head of the Department, Centrefor Biotechnology, Institute of Science and Technology, JNT University, for theample facilities provided in completing the project work.

We take this opportunity to respectfully thank our guide Dr. Archana Giri, Assistantprofessor, Centre for Biotechnology, for her encouragement and her keen interest tocarry out this work.

We also wish to acknowledge and express our sincere thanks to senior researchscholars at JNT University, Ms. Bhuwaneshwari.C.Hand Ms. Kiranmayee Rao,fortheir valuable assistance and encouragement.

We would like to extend our appreciation and thanks to the supporting staff members,Ms. Shabanaand Ms. Bharatiand other lab assistants for their timely help, extendedthroughout the course of the project.

Last but not the least, we would like to thank one and all who have been a part of

making this project work a success.

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LIST OF TABLES

Table

No.

Title

1.0 Antimicrobial activity ofAndrographis paniculataMethanol extract.

2.0 Antimicrobial activity ofAndrographis paniculataEthyl acetate extract

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LIST OF FIGURES

Fig.No. Title

1.0 Structure of Andrographolide

2.0 Zone of Inhibition shown byAndrographis paniculataMethanol extract

againstBacillus subtilis.


againstEnterococcous faecalis.


against Salmonella typhimurium

5.0Zone of Inhibition shown byAndrographis paniculataEthyl acetate extract

against Bacillus subtilis.


against Staphylococcous epidermis.



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Fig.No. Title

8.0 Chromatogram of the Methanol extract

9.0 Chromatogram of the Ethyl acetate extract

10.0 Chromatogram of the authenticAndrographis paniculatasample

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CONTENTS

S.No. CHAPTER PAGE No.

1.0 ABSTRACT 01

2.0 INTRODUCTION 02

3.0 REVIEW OF LITERATURE 10

4.0 MATERIALS AND METHODS 19

5.0 RESULTS 28

6.0 DISCUSSION 30

7.0 SUMMARY 32

8.0 CONCLUSION 33

REFERENCES I-IV

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1.0 ABSTRACT

Andrographis paniculata, a well known medicinal plant has been used since ancient

times in Traditional Chinese medicine and Indian Ayurvedic medicine. It has been

used as bitter tonic to treat digestive problems, snake bite, and infections ranging from

Malaria to Dysentery. Recent research has thrown light on the anti-microbial activity

of the plant. The most significant pharmacological activities of the plant that have

been discovered are anti-allergic, anti-cancer and anti HIV effect.

In the present study, the antimicrobial activity of the Andrographis paniculataplant

extract has been tested against nine common human-affecting bacterial pathogens.

Two different solvents - Methanol and Ethyl acetate have been used to prepare the

Andrographis paniculata plant extract and the antimicrobial activity of the two

extracts has been tested separately.

The effectiveness of the extract against the pathogen is taken as the measure of the

diameter of zones of inhibition, formed on the bacterial culture plates after a 24 hour

Incubation period. The growth medium used is Mueller Hinton Agar.

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2.0 INTRODUCTION

Many higher plants are sources of natural products used as pharmaceuticals,

agrochemicals, flavor and fragrance ingredients, food additives, and pesticides(Balandrin and Klocke, 1988).The search for new plant derived chemicals should

thus be a priority in current and future efforts towards sustainable conservation and

rational utilization of biodiversity (Phillipson, 1990). In search for alternatives to

production of desirable medicinal compounds from plants, biotechnological

approaches, specifically, plant tissue cultures, are found to have potential as a

supplement to traditional agriculture in the industrial production of bioactive plant

metabolites (Ramachandra Rao and Ravishankar, 2002).

Plants are a tremendous source for the discovery of new products of medicinal value

for drug development. Today, several distinct chemicals derived from plants are

important drugs currently used in many countries in the world. The evolving

commercial importance of secondary metabolites has in recent years resulted in a

great interest in secondary metabolism, particularly in the possibility of altering the

production of bioactive plant metabolites by means of tissue culture technology.

Medicinal plants are the most important source of life saving drugs for the majority of

the worlds population. Plants have been an important source of medicine for

thousands of years. Even today, the World Health Organization (WHO) estimates that

up to 80% of people still rely mainly on traditional remedies such as herbs for their

medicines. It is estimated that approximately one quarter of prescribed drugs contain

plant extracts or an active ingredient obtained from or modeled on plant substances.

Throughout the history, secondary metabolites of plants have been utilized by

humanity. There are approximately four major classes of secondary compounds that

are significant to humans, viz. Alkaloids, Phenyl propaniods, Flavonoids and

Terpenoids (Edwards and Gatehouse, 1999).

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Various medical plants have been used for years in daily life to treat disease all over

the world. According to a study performed by the WHO based on publications on

pharmacopoeias and medical plants in 91 countries, the number of medicinal plants is

nearly 20,000. The characteristics of the plants that inhibit microorganisms and are

important for human health have been researched in laboratories since 1926. Many

efforts have been made to discover new antimicrobial compounds from various kinds

of sources such as soil, microorganisms, animals and plants. One such resource is folk

medicine and systematic screening of these many results in the discovery of novel

effective compounds (Janovska et al, 2003).

2.1. ABOUT THE PLANTANDROGRAPHIS PANICULATA

Andrographis paniculata is traditionally known as Kalmegh. The plant belongs to

family Acanthaceae and is widely used in Traditional Chinese, Ayurvedic and

Homeopathic systems of medicine. The plant grows in waste grounds and prefers

moist habitat. The herb is bitter in taste and has weak odour. The whole plant is used

in medicine, with leaves and roots being the mostly used parts. It is widely cultivated

in southern Asia, where it is used to treat infections and some diseases.

2.1.1. SYSTEMIC POSITION

Division: Angiosperms

Class: Dicotyledonae

Subclass: Gamopetalae

Series: Bicarpellatae

Order: Personales

Tribe: Justicieae

Family: Acanthaceae

Genus: Andrographis

2.1.2. DESCRIPTION

It grows erect to a height of 30-110 cm in moist, shady places with glabrousleaves

and white flowers with rose-purple spots on the petals. Stem dark green, 0.3 - 1.0 m in

height, 2 - 6 mm in diameter, quadrangular with longitudinal furrows and wings on

the angles of the younger parts, slightly enlarged at the nodes; leaves glabrous, up to

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8.0 cm long and 2.5 cm broad, lanceolate, pinnate; flowers small, in lax spreading

axillary and terminal racemes or panicles; capsules linear-oblong, acute at both ends,

1.9 cm x 0.3 cm; seeds numerous, sub quadrate, yellowish brown.

2.1.3. CHARACTERISTICS

Since ancient times,A. paniculata is used as a wonderdrugin traditional Siddha and

Ayurvedic systems of medicine as well as in tribal medicine in India and some other

countries for multiple clinical applications. The therapeutic value of Kalmegh is due

to its mechanism of action which is perhaps by enzyme induction. The plant extract

exhibits antityphoid and antifungal activities. Kalmegh is also reported to possess

antihepatotoxic, antibiotic, antimalarial, antihepatitic, antithrombogenic,

antiinflammatory, antisnakevenom, and antipyretic properties to mention a few,

besides its general use as an immunostimulant agent. A recent study conducted at

Bastyr University, confirms anti-HIV activity of andrographolide.

2.1.4. DISTRIBUTION

Andographis paniculata is distributed in tropical Asian countries, often in isolated

patches. It can be found in a variety of habitats ie. plains, hill slopes, waste lands,

farms, dry or wet lands, sea shore and even road sides. Native populations of A.

paniculataare spread throughout south India and Sri Lanka which perhaps represent

the centre of origin and diversity of the species. The herb is also available in northern

stations of India, Java, Malaysia, Indonesia, West Indies and elsewhere in Americas

where it is probably introduced. The species is also available in Hong Kong, Penang,

Malacca, Pangkor Island (south of Penang), Malaya, Thailand, West Java, Borneo,

Celebes, Brunei, West Indies, Jamaica, Barbados, Bahamas etc. However, precise

data are lacking on the introduction and naturalization of the species in these

countries.

Unlike other species of the genus, A. paniculatais of common occurrence in most of

the places in our country including the plains and hilly areas up to 500 m, which

accounts for its wide use. Since time immemorial, village and ethnic communities in

India have been using this herb for treating a variety of ailments.

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2.1.5. CULTIVATION

It prefers a sunny situation. The seeds are sown during May-June. The seedlings are

transplanted at a distance of 60 cm x 30 cm.

2.1.6. PHARMACOLOGY

Andrographis paniculata plant extract is known to possess a variety of

pharmacological activities. Andrographolide (Fig.1.0), the major constituent of the

extract is implicated towards its pharmacological activity. A study has been conducted

on the cellular processes and targets modulated by andrographolide treatment in

human cancer and immune cells. Andrographolide treatment inhibited the in vitro

proliferation of different tumor cell lines, representing various types of cancers.

The compound exerts direct anticancer activity on cancer cells by cell cycle arrest at

G0/G1 phase through induction of cell cycle inhibitory protein p27 and decreased

expression of cyclin dependent kinase 4 (CDK4). Immunostimulatory activity of

andrographolide is evidenced by increased proliferation of lymphocytes and

production of interleukin 2. Andrographolide also enhanced the tumor necrosis factor

production and CD marker expression, resulting in increased cytotoxic activity of

lymphocytes against cancer cells, which may contribute for its indirect anticancer

activity. The in vivo anticancer activity of the compound is further substantiated

against B16F0 melanoma syngenic and HT 29 xenograft models. These results

suggest that andrographolide is an interesting pharmacophore with anticancer and

immunomodulatory activities and hence has the potential for being developed as a

cancer therapeutic agent (Rajagopal et al, 2003).

2.1.7. PHYTOCHEMISTRY

Andrographolideis the major constituent extracted from the leaves of the plant which

is a bicyclic diterpenoid lactone. This bitter principle was isolated in pure form by

Gorter (1911). Andrographolide is also attributed with other such activities like liver

protection under various experimental conditions of treatment with galactosamine,

paracetamol etc. (Saraswat et al, 1996). Systematic studies on chemistry of A.

paniculatahad been carried out by various researchers during various times.

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http://en.wikipedia.org/wiki/Andrographolidehttp://en.wikipedia.org/wiki/Immune_cellhttp://en.wikipedia.org/wiki/In_vitrohttp://en.wikipedia.org/wiki/Cyclinhttp://en.wikipedia.org/wiki/CDK4http://en.wikipedia.org/wiki/Lymphocytehttp://en.wikipedia.org/wiki/Interleukin_2http://en.wikipedia.org/wiki/Tumor_necrosis_factorhttp://en.wikipedia.org/wiki/In_vivohttp://en.wikipedia.org/wiki/Melanomahttp://en.wikipedia.org/wiki/Xenografthttp://en.wikipedia.org/wiki/Pharmacophorehttp://en.wikipedia.org/wiki/Immunomodulatorhttp://en.wikipedia.org/wiki/Andrographolidehttp://en.wikipedia.org/wiki/Lactonehttp://en.wikipedia.org/wiki/Galactosaminehttp://en.wikipedia.org/wiki/Paracetamolhttp://en.wikipedia.org/wiki/Paracetamolhttp://en.wikipedia.org/wiki/Galactosaminehttp://en.wikipedia.org/wiki/Lactonehttp://en.wikipedia.org/wiki/Andrographolidehttp://en.wikipedia.org/wiki/Immunomodulatorhttp://en.wikipedia.org/wiki/Pharmacophorehttp://en.wikipedia.org/wiki/Xenografthttp://en.wikipedia.org/wiki/Melanomahttp://en.wikipedia.org/wiki/In_vivohttp://en.wikipedia.org/wiki/Tumor_necrosis_factorhttp://en.wikipedia.org/wiki/Interleukin_2http://en.wikipedia.org/wiki/Lymphocytehttp://en.wikipedia.org/wiki/CDK4http://en.wikipedia.org/wiki/Cyclinhttp://en.wikipedia.org/wiki/In_vitrohttp://en.wikipedia.org/wiki/Immune_cellhttp://en.wikipedia.org/wiki/Andrographolide


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Fig. 1.0Structure of Andrographolide

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2.2. CONSTITUENTS OF ANDROGRAPHIS PANICULATA

Some known constituents are:

14-Deoxy-11-dehydroandrographolide, Plant

14-Deoxy-11-oxoandrographolide, Plant

5-Hydroxy-7,8,2',3'-Tetramethoxyflavone, Plant

5-Hydroxy-7,8,2'-Trimethoxyflavone, Tissue Culture

Andrographine, Root

Andrographolide, Plant

Neoandrographolide, Plant

Panicoline, Root

Paniculide-A, Plant

Paniculide-B, Plant

Paniculide-C, Plant

2.3. SECONDARY METABOLITES FROM ANDROGRAPHISPANICULATA

The secondary metabolites obtained fromAndrographis paniculataare two new

flavonoid glycosides, 5-hydroxy-7,8-dimethoxy (2R)-flavanone-5-O--D

glucopyranoside and 5-hydroxy-7,8,2,5-tetramethoxy-flavone-5-O--D-

glucopyranoside and a new diterpenoid, andrographic acid along with

andrographidine A. Their structures were determined on the basis of physicochemical

and spectroscopic analysis.

2.4. ABOUT THE PATHOGENS

2.4.1.Bacillus subtilis

Bacillus subtilis is a Gram-positive, catalase-positive bacterium commonly found in

soil. A member of the genus Bacillus, B. subtilis has the ability to form a tough,

protective endospore, allowing the organism to tolerate extreme

environmentalconditions. B. subtilis has proven highly amenable to genetic

manipulation, and has therefore become widely adopted as a model organism for

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laboratory studies, especially of sporulation, which is a simplified example of cellular

differentiation. It is also heavily flagellated, which givesB.subtilisthe ability to move

quite quickly.

2.4.2.Enterobacter cloacae

Enterobacter cloacae is a clinically significant Gram-negative, facultatively-

anaerobic, rod-shapedbacterium. The optimum growth temperature for this bacterium

is 30C, Appropriate growth media is nutrient agar and nutrient broth. The Genomic

sources for the restriction enzymes are Ecl136II, EclHKI, EclXI. The bacteria is Gram

Negative and the mode of respiration is Facultatively anaerobic. Motility is by

Peritrichous flagella.

2.4.3. Staphylococcus epidermidis

Staphylococcus epidermidis is a member of the bacterial genus Staphylococcus,

consisting of Gram-positive cocci arranged in clusters. It is catalase-positive and

coagulase-negative and occurs frequently on the skin of humans and animals and in

mucous membranes.

2.4.4.Enterococcus faecalis

Enterococcus faecalis is a Gram-positive commensal bacterium inhabiting the

gastrointestinal tractsof humans and other mammals. Like other species in the genus

Enterococcus, E. faecalis can cause life-threatening infections in humans and

monkeys, especially in the nosocomial(hospital) environment.

2.4.5. Salmonella typhimurium

S. enterica has an extraordinarily large number of serovars or strainsup to 2000

have been described. Salmonella enterica Serovar Typhi (historically elevated to

speciesstatus as S. typhi) is the disease agentin typhoid fever. Other serovars such as

Typhimurium (also known as S. typhimurium) can lead to a form of human

gastroenteritis sometimes referred to as salmonellosis. Salmonella Typhi possesses

three main antigenic factors: the O, or somatic antigen; the Vi, or encapsulation

antigen; and theH, or flagellar antigen.

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2.4.6. Staphylococcus aureus

S. aureus is a Gram-positive coccus, which appears as grape-like clusters when

viewed through a microscope and has large, round, golden-yellow colonies, often with

hemolysis, when grown onblood agar plates. S. aureusis catalasepositive (meaning

that it can produce the enzyme "catalase") and able to convert hydrogen peroxide

(H2O2) to water and oxygen, which makes the catalase test useful to distinguish

staphylococci from enterococciand streptococci.

2.4.7. Escherichia coli

Escherichia coli is a bacterium that is commonly found in the lower intestine of

warm-blooded animals. MostE. colistrainsare harmless, but some, such as serotype

O157:H7, can cause serious food poisoning in humans, and are occasionally

responsible for costlyproduct recalls.E. coliis Gram-negative, facultative anaerobic

and non-sporulating. It can live on a wide variety of substrates. E. coliuses mixed-

acid fermentation in anaerobic conditions, producing lactate, succinate, ethanol,

acetateand carbon dioxide.

2.4.8.Klebsiella pneumoniae

Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose

fermenting, facultative anaerobic, rod shapedbacteriumfound in the normal flora of

the mouth, skin, and intestines. It is clinically the most important member of the

Klebsiellagenusof Enterobacteriaceae; it is closely related to K. oxytocafrom which

it is distinguished by being indole-negative and by its ability to grow on both

melezitoseand 3-hydroxybutyrate. It naturally occurs in the soil and about 30% of

strains can fix nitrogenin anaerobic condition.

2.4.9.Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with

unipolar motility. An opportunistic human pathogen, P. aeruginosa is also an

opportunistic pathogen of plants. P. aeruginosa is the type species of the genus

Pseudomonas

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2.5. OBJECTIVES

The present study involves the test of the antimicrobial activity of Andrographis

paniculata plant extract against nine common human-affecting bacterial pathogens

with the following objectives:

1. To check the antimicrobial activity of the plant extract against humanpathogens

2. To evaluate the activity of the plant extract in 2 different solvents - Methanoland Ethyl acetate being the solvents used respectively.

3. To perform HPLC of the extract and compare it with the authenticAndrographolide sample

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3.0. REVIEW OF LITERATURE

The use of alternative medical therapy has increased the interest of pharmacologists

and herbalists over the past decade. Historically, plants have provided a source of

inspiration for novel drug compounds, as plant derived medicines have made large

contributions to human health and well being. On the other hand, there is an increase

in use of herbal products all over the world; in USA, it reached 380% between 1990

and 1997.

The success story of chemotherapy lies in the continuous search for new drugs to

counter the challenge posed by resistant strains of microorganisms. The investigation

of certain indigenous plants for their antimicrobial properties may yield useful results.

A large number of plants indeed were used to combat different diseases and known to

possess antimicrobial activity.

The antibacterial activities of hot water, methanol and ethanol extracts of five plant

extracts utilized in Palestine in popular medicine were studied. The dried extracts of

Sygyium aromaticum (Myrtaceae) (seed), Cinnamomum cassia (Lauraceae) (cassia

bark, Chinese cinnamon) (bark), Salvia officinalis (Lamiaceaea) (leaf), Thymus

vulgaris(Lamiaceaea) (leaf) andRosmarinus officinalis( Labiatae) (leaf) were tested

in vitro against four bacterial species by disc diffusion and micro-dilution (Bassam

Abu-Shanab et al, 2004). The patterns of inhibition varied with the plant extract, the

solvent used for extraction, and the organism tested. Methicillin-resistant

Staphylococcus aureus (MRSA) and Bacillus subtilis ATCC 6633 were the most

inhibited microorganisms S. aromaticum extract was the most active against multi

drug resistant Pseudomonas aeruginosaand enterohaemorrahagicE.coliO157 EHEC.

The combinations of ethanolic extracts of S.officinalis with R. officinalis and of R.

officinaliswith T. vulgarison bacterial species tested exhibited a higher effect than

that of any individual extract. Results of this kind herald the interesting promise of

designing a potentially active antibacterial synergized agent of plant origin.

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Voravuthikunchai et al., (2006), prepared ethanolic extracts of eight Thai medicinal

plants (representing five families) that are used as traditional remedies for treating

diarrhea were examined with a salt aggregation test for their ability to modulate cell

surface hydrophobicity of enterohemorrhagic Escherichia coli strains, including E.

coli O157:H7. Four of these medicinal plants, Acacia catechu, Peltophorum

pterocarpum, Punica granatum,and Quercus infectoria,have high bacteriostatic and

bactericidal activities. The ethanolic extract of Q. infectoriawas the most effective

against all strains ofE. coli,with MICs of 0.12 to 0.98 mg/ml and MBCs of 0.98 to

3.91 mg/ml. The ethanolic extract of P. granatumhad MICs of 0.49 to 1.95 mg/ml

and MBCs of 1.95 to 3.91 mg/ml. Ethanolic extracts of Q. infectoria, P. pterocarpum,

and P. granatumwere among the most effective extracts against the two strains ofE.

coli O157:H7. The other four plants, Andrographis paniculata, Pluchia indica,

Tamarindus indica, and Walsura robusta, did not have high bacteriostatic and

bactericidal activities but were able to affect hydrophobicity characteristics on their

outermost surface. All plants except Q. infectoria had some ability to increase cell

surface hydrophobicity. There appears to be no correlation between antibacterial

activity and cell aggregative properties.

Poolsup. N et al., (2004) assessed the efficacy of Andrographis paniculata in the

symptomatic treatment of uncomplicated upper respiratory tract infection. Methods:

Systematic review of the literature and meta-analysis of randomized controlled trials.

Mean difference in the reduction in symptom severity scores between treatment and

control groups was calculated to obtain an overall estimate of effect. Four studies

met our inclusion criteria and were reviewed. A total of 433 patients reported in three

trials were included in the statistical analysis. Andrographis paniculata fixed

combination withAcanthopanax senticosuswas more effective than placebo.

. Current evidence suggests thatA. paniculataextract alone or in combination withA.

senticosus extract may be more effective than placebo and may be an appropriate

alternative treatment of uncomplicated acute upper respiratory tract infection.

Jada, et al, (2006), identified a new diterpenoid lactone of the plant Andrographis

paniculata , known to possess antitumour activity in in vitro and in vivo breast cancer

models was subjected to semisynthesis leading to the preparation of a number of

novel compounds.

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Zhang. et al.,(2006) investigate diterpenoids from the aerial parts of Andrographis

paniculata (Burm. f.) Nees, three new ent-labdane diterpenoids, namely 19-

norandrographolides A-C (compounds 1-3), were isolated from the ethanolic extract

of A. paniculata.Their structures were established by HRESIMS and NMR spectral

data in combination with X-ray crystallographic analysis.

Sinha. J, et al., (2000), despite the rapid development in medicinal and

pharmaceutical technology, the targeting of drugs to phagocytic cells in macrophage-

related diseases still remains a major unsolved problem. By using the mannosyl-

fucosyl receptors on macrophages, attempts were made to target antileishmanial drugs

encapsulated in mannosylated or fucosylated liposomes to treat experimental

leishmaniasis in the hamster model. Mannosylated liposomes were found to be more

potent in delivering antileishmanial drugs to phagocytic cells. Liposomes loaded with

an indigenous drug, andrographolide, a labdane diterpenoid isolated from Indian

medicinal plant Andrographis paniculata, were prepared and tested against

experimental leishmaniasis in a hamster model. Mannosylated liposomes loaded with

the drug were found to be most potent in reducing the parasitic burden in the spleen as

well as in reducing the hepatic and renal toxicity. In addition, mannosylated drug-loaded liposome-treated animals showed a normal blood picture and splenic tissue

histoarchitecture when compared with those treated with free drug or regular

liposomal drug. Such a drug-vehicle formulation may be considered for clinical trials.

Tipakorn . N et al., (2004), conducted studies to determine the antibacterial activity

of A. paniculata (AP) leaf extracts (at 1:10, 1:100 and 1:1000) diluted with three

solvents (distilled water, 70 and 85% alcohol). The extracts were tested against

Salmonella typhimurium, Salmonella spp. (from Lampang and Phitsanulok

provinces),Escherichia coli(from chicken, pig, deer and duck, and ATCC 25922 as

standard), and Pasteurella multocida (from buffalo tissue, buffalo liver, beef tissue

and beef heart). Streptomycin 2 mg/ml was used as the control. The concentration

1:10 of 70 and 85% alcoholic extract showed moderate to intermediate activity

against S. typhimurium, with inhibition zones of 12 and 10 mm, respectively. The

minimum inhibitory concentration of 70 and 85% AP alcohol extracts AP was 1:10.

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Likewise, the 70 and 85% AP alcohol extracts showed antibacterial activity towards

P. multocidaisolated from buffalo tissue, buffalo liver and beef tissue. E. colistrain

ATCC 25922 as well as those isolated from chicken, pig and duck, Salmonella sp.

from Phitsanulok, and P. multocidaisolated from buffalo liver and beef tissue showed

resistance to Streptomycin. Thus, the use of AP leaves as antibacterial agents against

bacteria causing diarrhoea is promising.

Youhong Xu, et al., (2006), prepared aqueous and two ethanolic extracts of

Andrographis paniculata, used in traditional Chinese, Thai and Indian medicine and

andrographolide, an active principle of Andrographis paniculata, were investigated

for their antimicrobial activity against nine bacterial species including Salmonella

typhimurium,Escherichia coli, Shigella sonnei,Staphylococcus aureus,Pseudomonas

aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes, Legionella

pneumophila andBordetella pertussis, using the disc diffusion method. Of all tested

concentrations, direct antimicrobial activity of the two ethanolic Andrographis

paniculata extracts was observed for only two human pathogens, Legionella

pneumophila andBordetella pertussis.

P Borgna, et al., (1996), N-Hydroxyalkyl derivatives of 1,2-benzisothiazol-3(2H)-

one and 1,2-benzisothiazol-3(2H)-thione have been prepared and their antifungal and

antibacterial activity evaluated. Several compounds were active against selected fungi

and Gram-positive microorganisms. Interesting activity was observed against the

anaerobic strain Clostridium perfringens. Generally the more active compounds

belong to the class of 1,2-benzisothiazol-3(2H)-ones. The retardation matches RMof

the compounds was also evaluated but the results obtained show that lipophilicity has

only a minor effect on the antimicrobial activity.

G Daidone, et al., (1996), prepared number of derivatives of new 4-diazopyrazole

reaction of 1-R-3-methyl-5(R1-substituted)benzamidopyrazoles with a sevenfold

excess of nitrous acid in acetic medium. The compounds were tested for activity

against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus,

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Staphylococcus epidermidis, Streptococcus faecalis, Listeria monocytogenes, Candida

albicans, Candida tropicalis and Paecilomyces varioti. The highest microbial

susceptibility was shown by Gram-positive bacteria, with minimum inhibitory

concentrations (MIC) in the range 0.512.5 g/mL. For S aureus the R1substituents

were screened utilizing the Topliss operational scheme. The 4-nitro group was found

to be the best substituent. We also tested the compounds 41,o,p, found to be the most

active in the test against S aureus ATCC 25923, on ten clinical S aureus strains, five

of which were sensitive and five resistant to methicillin. The above compounds were

active in the range 28 g/mL against methicillin-resistant S aureus strains. An X-ray

analysis of compounds 4i and 4q is reported.

zlem Temiz, , et al., (1998), synthesis of a new series of 5- or 6-methyl-2-(2,4-disubstituted phenyl)benzoxazoles (4, 5) is disubstituted phenyl)benzoxazoles (4, 5) is

described in order to determine their antimicrobial activities and feasible structure-

activity relationships. The synthesized compounds were tested in vitro against three

Gram-positive bacteria, three Gram-negative bacteria and the yeast Candida albicans,

in comparison with several control drugs.

OM Walsh, et al., (1996),synthesized a series of 3-acetoxyazetidin-2-ones 3an and

3-hydroxyazetidin-2-ones 6aj is reported together with the antibacterial andantifungal evaluation of these compounds. An additional series of 3-acetoxyazetidin-

2-ones 11ah which possess a free carboxylic acid group on the N-1 aryl ring were

obtained by treatment of suitably substituted Schiff bases 10ah with acetoxyacetyl

chloride. The novel bicyclic structures 7-acetoxy-6-phenyl-5-thia-1-

azabicyclo[4.2.0]octan-8-one 13 and 7-hydroxy-6-phenyl-5-thia-1-

azabicyclo[4.2.0]octan-8-one 14 were also obtained. Many of the compounds

displayed antifungal activity in vitro when evaluated against the pathogenic fungiCryptococcus neoformans, Candida albicans, Candida tropicalis, Candida

parapsilosis, Candidaglabrata, and Trichosporon cutaneum, while 3-

acetoxyazetidin-2-ones 11ah containing a free carboxylic acid group on the N-1 aryl

ring displayed antibacterial activity against Staphylococcus aureus,Proteus vulgaris,

Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella aerogenes and Escherischia

coli

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Prajjal K. Singha, (2003),studied the antimicrobial activity ofaqueous extract,

andrographolides and arabinogalactanproteins fromAndrographis paniculata were

evaluated. The aqueous extract showed significant antimicrobial activity, which may

be due to the combined effect of the isolated arabinogalactanproteins and

andrographolides.

Essawi and M. Srour. Of the 15 plants tested, eight showed antibacterial activity.

Each plant species has unique against different bacteria. The most active antibacterial

plants against both gram-positive and gram-negative bacteria were Thymus vulgaris

and Thymus origanium. The organic and aqueous extract from the same plants

showed different fecalis. Of the 15 plants tested, eight showed antibacterial activity.

Each plant species has unique against different bacteria. The most active antibacterialplants against both gram-positive and gram-negative bacteria were Thymus vulgaris

and Thymus origanium. Finally, the holeplate diffusion method showed larger

activity than the disc diffusion method.

Jonathan E et al., (2000), prepared aqueous, methanolic and ethyl acetate extracts of

14 plants used in traditional Zulu medicine for treatment of ailments of an infectious

nature were screened for antibacterial activity. Most of the activity detected was

against Gram-positive bacteria. Tuber bark extracts of Dioscorea sylvatica hadactivity against Gram-negative Escherichia coli and extracts of Dioscorea dregeana,

Cheilanthes viridis and Vernonia colorata were active against Pseudomonas

aeruginosa. The highest antibacterial activity was found in extracts of C. viridis, D.

dregeana, D. silvatica, Melianthus comosus and V. colorata. In general, methanolic

extracts exhibited higher activity than aqueous and ethyl acetate extracts.

Owais .M, et al, (2005), evaluated the antibacterial activity of ashwagandha

(Withania somnifera L. Dunal (Solanaceae; root and leaves), an Indian traditional

medicinal plant against pathogenic bacteria. Both aqueous as well as alcoholic

extracts of the plant (root as well as leaves) were found to possess strong antibacterial

activity against a range of bacteria, as revealed by in vitro Agar Well Diffusion

Method. The methanolic extract was further sub-fractionated using various solvents

and the butanolic sub-fraction was found to possess maximum inhibitory activity

against a spectrum of bacteria including Salmonella typhimurium. Moreover, in

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contrast to the synthetic antibiotic (viz. chloramphenicol), these extracts did not

induce lysis on incubation with human erythrocytes, advocating their safety to the

living cells. Finally, the antibacterial efficacy of the extracts isolated from plant (both

root and leaves) was determined against experimental salmonellosis in Balb/C mice.

Oral administration of the aqueous extracts successfully obliterated salmonella

infection in Balb/C mice as revealed by increased survival rate as well as less

bacterial load in various vital organs of the treated animals.

Mice studies have shown that Andrographis paniculata is a potent stimulator of the

immune system in two ways:

1. Antigen-specific response: antibodies are made to counteract invadingmicrobes, and

2. Nonspecific immune response: macrophage cells scavenge and destroyinvaders.

A. paniculata activated both responses-making it effective against a variety of

infectious and oncogenic (cancer-causing) agents. AP has a record of effective

treatment rooted in its mechanism of immune boosting. Cancer results when cells do

not respond to signals that are intended to limit growth. If a cancer cell can be made to

mature (or differentiate), it will not have the ability to grow out of control. AP was

chosen because it contained substances (Terpenes) which were known to cause

differentiation of cancer cells. AP extracts from the leaves of the plant are also

cytotoxic (Cell killing) against cancer cells. This cancer cell-killing ability was

demonstrated against human epidermoid carcinoma (Squamous cell carcinoma) of the

skin lining of the Nasopharynx and against lymphocytic leukemia cells. It was the

andrographolide component that was found to have the cancer cell-killing ability. It

was recommended by National Cancer Institute as a cytotoxic substance. Japanese

researchers have reported that AP stopped stomach cancer cells from multiplying.

After 3 days, there were less than 8 cancer cells growing in the presence of AP, while

the untreated cancer cells numbered 120.

Another group of Japanese researchers tested AP on sarcoma cells. These very

malignant cancers affect muscles, connective tissue and bones. When tumor cells

were examined under the microscope, AP was found to inhibit the growth of tumors.

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Laboratory tests conducted in Buffalo, New York, demonstrated that AP inhibited the

growth of human breast cancer cells. Extracts of AP are much less toxic than most

chemotherapeutic agents used to fight cancer. In 1977, a human study was conducted

using AP in 60 cancer patients, including 41 with confirmed metastasis (the cancer

was spreading). As reported in theJournal of Chinese Medicine, 12 patients given AP

and its compounds alone, recovered. All other patients were given AP along with

standard drugs; there was no tumor regrowth in 47 of these patients. In 1996, early

trials showed that the extract safely and effectively blocked growth of both prostate

and breast cancer cells, grown in laboratory. Researchers believed that AP probably

inhibits synthesis of cancer cell DNA.

Immune deficiency is at the root of susceptibility to a variety of infections, and its is

the basis of the Acquired Immune Deficiency Syndrome (AIDS). HIV, like all virus,

cannot reproduce by itself or even live, without using the resources of other cells.

When HIV virus finds a suitable cell, it attaches to the cell, using proteins on its cell

surface. In the case of human cells, the HIV virus enters the cells by binding to

molecules on the cell surface. The first of these to be identified was CD4; other more

recently identified molecules are CCR5 and CXCR4. The HIV virus actually subverts

the cells messengers, tricking them into producing more viral particles. Using signal

transduction technology (methods to investigate the cell message systems), scientists

found that AP contains substances that destroyed the virus communications

mechanism. One component of the herd Andrographolide prevented transmission

of the virus to other cells and stopped the progress of the disease by modifying

cellular signal transduction. Andrographolide probably does this by inhibiting

enzymes that facilitate the transfer of phosphates. AP can thus interfere with key

enzymes that result in viral reproduction.

HIV alters the action of central information -processing enzyme, Cyclin dependent

Kinase (CDK), particularly CDK1, that coordinates all events relating to cell division.

Agents that can prevent this phosphorylation can lessen the severity of AIDS. The

new class of anti viral compounds with this ability is called Tyrosin Kinase inhibitors

which includes Andrographolides. An extract of AP can, in fact, inhibit CDK1 that

has been altered by HIV. Cooperative research at National Cancer Institute has shownthat Andrographolide can also inhibit HIVs toxic effect on cells. Testing of AP done

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at Frederick Research Centre, demonstrated that extracts of AP increased AZTs

ability to inhibit replication of HIV. An added benefit is that lower doses of AZT

could be used.

Some researchers believe that AP extracts may also be useful in combating other

viruses, including the Ebola virus and the viruses associated with Herpes, Hepatitis,

and Influenza.

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4.0. MATERIALS AND METHODS

4.1. Materials

1. Conical flasks2. Petri plates3. Test tubes4. Micropipettes5. Mueller Hinton Broth6. Bacteriological Agar7.

Plant extract

8. Methanol9. Ethyl acetate.10.Distilled water11.Cotton12.Ethanol13.Para film14.Glass markers15.Rubber bands16.Aluminum foil

4.1.1. Equipment

1. Laminar Air Flow

2. Decontaminator

3. Autoclave

4. Ultra sonicator

5. High Pressure/Performance Liquid Chromatography (HPLC) column and

detector

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4.2. Methods

4.2.1. Sterilization of culture vessels and instruments

Culture vessels and instruments were sterilized by exposure to hot dry air (160 C) for

2-4 hr in a hot air oven. All the vessels were properly sealed with aluminum foil

before sterilization.

4.2.2. Preparation of the Media

21gms of the Mueller Hinton broth was taken and added to 1000ml of distilled water.

The pH of the media was then adjusted to 7, and then 7 gms of agar was added. The

media was then sterilized in an autoclave at 121 C, at 15 lbs pressure for 15 minutes.

4.2.3. Media composition

Beef Infusion - 300 g/l

(from Caesin Hydrolysate- 17.50 g/l and Starch 1.50 g/l)

Final pH at 25 C is 7.4 (plus or minus) 0.2

4.2.4. Sterilization of Mueller Hinton Agar Media

The media used in the present investigation was sterilized in an autoclave at 121 C

under 15 lbs for 15-20 minutes. Distilled water, micro-nutrients, macro-nutrients and

other stable mixtures were autoclaved. Culture media in glass containers were sealed

with cotton plugs and autoclaved at 14 lbs at 121 C for 15-20 minutes.

4.2.5. Plant Material

In the present study, the plant material used isAndrographis paniculata,belonging to

the family Acanthaceae. Extracts were prepared using two different solvents

Methanol and Ethyl acetate, using leaves of the plant.

4.2.6. Preparation of the Plant extract

In the present study, the Methanol and Ethyl acetate extracts of leaves of

Andrographis paniculata were used for evaluation of their antimicrobial activity

against human pathogens.

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4.2.6.1. Methanol extract:

250 gms of powdered AP plant material was carefully weighed and it was carefully

transferred into a round bottomed flask of soxhlet extractor. Then 2 liters of Methanol

was added and the plant material was soaked in Methanol for 24 hours at room

temperature. Then the methanol extract of the plant was extracted using the soxhlet

extractor. The methanol present in the methanol extract was evaporated under reduced

pressure to yield the residue.

4.2.6.2. Ethyl acetate extract:

250 gms. of powdered AP plant material was carefully weighed and it was carefully

transferred into a round bottomed flask of soxhlet extractor. Then 2 liters of Ethyl

acetate was added and the plant material was soaked in Ethyl acetate for 24 hours at

room temperature. Then the Ethyl acetate extract of the plant was extracted using the

soxhlet extractor. The Ethyl acetate present in the Ethyl acetate extract was

evaporated under reduced pressure to yield the residue.

4.2.7. Preparation of pure cultures

To prepare pure cultures, Mueller Hinton Agar was prepared. Then, a loopful of

culture was taken from stock culture and it was inoculated in the test tubes containing

about 25 ml of the medium. These tubes were incubated at 37 C for 24 hours and

used for further experimental procedure.

The following bacterial strains for the antimicrobial assay have been collected from

microbial type culture collection (MTCC) of IMTECH, Chandogarh. The

microorganisms that were used in the antimicrobial assay were:

1. Bacillus subtilis2. Enterobacter cloacae3. Staphylococcous epidermis4. Enterococcus faecalis5. Salmonella typhimurium

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6. Staphylococcous aureus7. Escherichia coli8. Klebsiella pneumoniae9. Pseudomonas aeroginosa

4.2.8. Procedure

The following steps were performed under sterile conditions in the laminar air flow:

1. 25ml of hot Mueller Hinton Agar was poured into 9 test tubes respectivelynumbered 1-9.

2. These tubes were then placed in a beaker containing hot water, to prevent the agarfrom solidifying.

3. In each of the test tube, the respective pathogen was added. (10 micro liter vol. forBacillus subtilis, and 20 micro liters for each of the other pathogen), and mixed

properly.

4. The culture was then poured into the respective petri plates (numbered 1-9) andkept aside for about 20 mins, for solidification.

5. Four wells were then punched, using the blue micropipette tip, in the fourquadrants of every petri plate.

6. Next, increasing concentrations of the extract was poured into each of the fourwells.

7. In the case of the Methanol extract, the concentrations used were 25, 50, 75 and100 micrograms of the extract.

8. In the case of the Ethyl acetate extract, the concentrations used were 20, 30, 40,50 micrograms of the extract.

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9. The volume in each well was made up using the respective solvent (Methanol(Table 1.0, Fig 2.0, 3.0, 4.0) and Ethyl acetate (Table 2, Fig 5.0, 6.0, 7.0)).

10.The plates were then sealed with parafilm, and placed in the freezer for 5-10minutes.

11.The plates are then removed from the freezer and placed in the bacterial incubatorat 37 C, for 24 hours.

12.The zone of inhibition for each well is checked and its diameter is measured. Incase of negative activity of the extract against the pathogen, there is no zone of

inhibition formed.

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Table 1.0 - Antimicrobial activity ofAndrographis paniculataMethanol extract

S.No. Name of theorganism Diameter ofzone in 25

micro gm of

extract(cm)

Diameter ofzone in 50

micro gm of

extract(cm)


micro gm of

extract(cm)


micro gm of

extract(cm)

1.0 Bacillus subtilis0.4/0.8 0.6/0.8 0.6/0.8 0.2/0.8

2.0 Enterobacter

cloacae1.4/0.8 1.4/0.8 1.6/0.8 1.6/0.8

3.0 Staphylococcus

epidermis

- - - -

4.0 Enterococcus

faecalis1.6/0.8 1.6/0.8 1.8/0.8 1.8/0.8

5.0 Salmonella

typhimurium1.2/0.8 1.6/0.8 1.2/0.8 1.6/0.8

6.0 Staphylococcus

aureus

- - - -

7.0 Escherichia coli - - - -

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Fig.2.0. Zone of Inhibition shown byAndrographis paniculataMethanol extract

againstBacillus subtilis.

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againstEnterococcous faecalis.

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Table 2.0.Antimicrobial Activity ofAndrographispaniculata -Ethyl acetateextract

S.No. Name of the

organism

Diameter of

zone in 20

micro gm of

extract

Diameter of

zone in 30

micro gm of

extract

Diameter of

zone in 40

micro gm of

extract

Diameter of

zone in 50

micro gm of

extract

1.0 Bacillus subtilis 2.0/0.8 2.0/0.8 2.0/0.8 1.8/0.8

2.0 Enterobacter

cloacae

- - - -

3.0 Staphylococcus

epidermis1.4/0.8 1.4/0.8 1.6/0.8 1.8/0.8

4.0 Enterococcus

aecalis1.4/0.8 1.4/0.8 1.5/0.8 1.5/0.8

5.0 Salmonella

typhimurium1.2/0.8 1.4/0.8 1.2/0.8 1.4/0.8

6.0 Staphylococcus

aureus

2.0/0.8 2.0/0.8 2.2/0.8 2.2/0.8

7.0 Escherichia coli 1.6/0.8 1.3/0.8 1.6/0.8 1.4/0.8

8.0 Klebsiella

pneumoniae

- - - -

9.0 Pseudomonas

aeroginosa

1.2/0.8 1.3/0.8 1.2/0.8 1.3/0.8

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Fig.5.0. Zone of Inhibition shown byAndrographis paniculataEthyl acetate extract against

Bacillus subtilis.

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Fig.6.0. Zone of Inhibition shown byAndrographis paniculataEthyl acetate extract against

Staphylococcous epidermis.

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Fig.7.0. Zone of Inhibition shown byAndrographis paniculata Ethyl acetateextract against Salmonella typhimurium

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4.2.9. High Performance Liquid Chromatography (HPLC):

High-performance liquid chromatography (or High pressure liquid

chromatography, HPLC) is a form of column chromatographyused frequently in

biochemistry and analytical chemistry. HPLC is used to separate components of a

mixture by using a variety of chemical interactions between the substance being

analyzed (analyte) and the chromatography column.

4.2.9.1 Operation

The sample to be analyzed is introduced in small volume to the stream of mobile

phase and is retarded by specific chemical or physical interactions with the stationary

phase as it traverses the length of the column. The amount of retardation depends on

the nature of the analyte, stationary phase and mobile phase composition. The time at

which a specific analyte elutes (comes out of the end of the column) is called the

retention time and is considered a reasonably unique identifying characteristic of a

given analyte. The use of pressure increases the linear velocity (speed) giving the

components less time to diffusewithin the column, leading to improved resolution in

the resulting chromatogram. Common solvents used include any miscible

combinations of wateror various organic liquids (the most common are methanoland

acetonitrile). Water may contain buffers or salts to assist in the separation of the

analyte components, or compounds such as Trifluoroacetic acidwhich acts as an ion

pairing agent.

4.2.9.2 Reversed phase chromatography

Reversed phase HPLC (RP-HPLC or RPC) consists of a non-polar stationary phase

and an aqueous, moderately polar mobile phase. One common stationary phase is a

silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group

such as C18H37or C8H17. The retention time is therefore longer for molecules which

are more non-polar in nature, allowing polar molecules to elute more readily.

45
http://en.wikipedia.org/wiki/Column_chromatographyhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Analytical_chemistryhttp://en.wikipedia.org/wiki/Analytehttp://en.wikipedia.org/wiki/Velocityhttp://en.wikipedia.org/wiki/Diffusionhttp://en.wikipedia.org/wiki/Chromatographyhttp://en.wikipedia.org/wiki/Solventhttp://en.wikipedia.org/wiki/Water_%28molecule%29http://en.wikipedia.org/wiki/Methanolhttp://en.wikipedia.org/wiki/Acetonitrilehttp://en.wikipedia.org/wiki/Trifluoroacetic_acidhttp://en.wikipedia.org/wiki/Trifluoroacetic_acidhttp://en.wikipedia.org/wiki/Acetonitrilehttp://en.wikipedia.org/wiki/Methanolhttp://en.wikipedia.org/wiki/Water_%28molecule%29http://en.wikipedia.org/wiki/Solventhttp://en.wikipedia.org/wiki/Chromatographyhttp://en.wikipedia.org/wiki/Diffusionhttp://en.wikipedia.org/wiki/Velocityhttp://en.wikipedia.org/wiki/Analytehttp://en.wikipedia.org/wiki/Analytical_chemistryhttp://en.wikipedia.org/wiki/Biochemistryhttp://en.wikipedia.org/wiki/Column_chromatography


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Retention Time (RT) is increased by the addition of polar solvent to the mobile phase

and decreased by the addition of more hydrophobic solvent.

RPC operates on the principle of hydrophobic interactions, which result from

repulsive forces between a polar eluent, the relatively non-polar analyte, and the non-

polar stationary phase. The binding of the analyte to the stationary phase is

proportional to the contact surface area around the non-polar segment of the analyte

molecule upon association with the ligand in the aqueous eluent. This solvophobic

effect is dominated by the force of water for "cavity-reduction" around the analyte and

the C18-chain versus the complex of both. The energy released in this process isproportional to the surface tension of the eluent (water: 73 erg/cm, methanol: 22

erg/cm) and to the hydrophobic surface of the analyte and the ligand respectively.

The retention can be decreased by adding less-polar solvent (MeOH, ACN) into the

mobile phase to reduce the surface tension of water.

Structural properties of the analyte molecule play an important role in its retentioncharacteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-

C, and generally non-polar atomic bonds, such as S-S and others) results in a longer

retention time because it increases the molecule's non-polar surface area, which is

non-interacting with the water structure. On the other hand, polar groups, such as -

OH, -NH2, COO- or -NH3

+ reduce retention as they are well integrated into water.

Very large molecules, however, can result in an incomplete interaction between the

large analyte surface and the ligands alkyl chains and can have problems entering thepores of the stationary phase.

RT increases with hydrophobic - non-polar - surface area. Branched chain compounds

elute more rapidly than their corresponding linear isomers because the overall surface

area is decreased. Similarly organic compounds with single C-C-bonds elute later than

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the ones with a C=C or C-C-triple bond, as the double or triple bond is shorter than a

single C-C-bond.

Aside from mobile phase surface tension (organizational strength in eluent structure),

other mobile phase modifiers can affect analyte retention. For example, the addition

of inorganic salts causes a moderate linear increase in the surface tension of aqueous

solutions (ca. 1.5 erg/cm pro Mol for NaCl, 2.5 erg/cm pro Mol for (NH4)2SO4), and

because the entropyof the analyte-solvent interface is controlled by surface tension,

the addition of salts tend to increase the retention time. This technique is used for mild

separation and recovery of proteins and protection of their biological activity inprotein analysis (hydrophobic interaction chromatography, HIC).

Another important component is the influence of the pH since this can change the

hydrophobicity of the analyte. For this reason most methods use a buffering agent,

such as sodium phosphate, to control the pH. A volatile organic acid such as formic

acidor most commonly trifluoroacetic acidis often added to the mobile phase, if massspectrometry is applied to the eluent fractions. The buffers serve multiple purposes:

they control pH, neutralize the charge on any residual exposed silica on the stationary

phase and act as ion pairing agents to neutralize charge on the analyte. The effect

varies depending on use but generally improve the chromatography.

4.2.9.3 HPLC Technique

1. The HPLC technique (using Reverse phase adsorptive C18 column) wasperformed for the Andrographis paniculata aunthentic sample and for both

the extracts of theAndrographis paniculataplant.

2. HPLC was performed on Shimadzu LC 10 AT VP series using a supelcocolumn (250 x 46 mm, C18, ODS with particle size of 5 um).

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3. The sample was injected into the sample injection port at regular timeintervals.

4. The mobile phase used was the mixture of water: acetonitrile: Methanol-55:30:15/100ml at a flow rate of 1 ml /min.

5. Andrographis paniculataauthentic sample and the Methanol and Ethyl acetateextracts were monitored at 223 nm with an UV Vis detector (shumadzu uv

visible SPD LC 10 AVP Series) (Tikhomiroff and Jolicoeur 2002)

6. The chromatograms of both the extracts were compared respectively to thechromatogram of the authentic sample.

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Fig.8.0 Chromatogram of the Methanol extract

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Fig. 9.0 Chromatogram of the Ethyl acetate extract

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Fig.10.0 Chromatogram of the AuthenticAndrographolide paniculata Sample

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5.0. RESULTS

5.1. Antimicrobial activity of Andrographis plant

5.1.1. Methanol extract

The methanol extract of the plant showed antimicrobial activity against 4 out of the 9

pathogens tested (Table 1).

Varying concentrations of 25, 50, 75 and 100 microgram of the Methanol extract was

used in each of the four wells of the petri plate cultures.

The zones of inhibition of growth were formed in all the 4 wells containing the varying

concentrations of the Methanol extract.

The 4 microorganisms, against which the Methanol extract was effective were Bacillus

subtilis (Fig.2.0), Enterobacter cloacae, Enterococcous faecalis(Fig.3.0) and

Salmonella typhimurium(Fig.4.0).

5.1.2. Ethyl acetate extract

The ethyl acetate extract of the plant showed antimicrobial activity against 7 out of the

total 9 pathogens tested (Table 2).

Varying concentrations of 20,40,60 and 80 microgram of the Ethyl acetate extract was

used in each of the four wells of the petri plate cultures.

The zones of inhibition of growth were formed in all the 4 wells containing the varying

concentrations of the Methanol extract.

The 7 microorganisms, against which the Ethyl acetate extract was effective were

Bacillus subtilis (Fig.5.0), Staphylococcous epidermis (Fig.6.0), Enterococcous

faecalis, Salmonella typhimurium (Fig.7.0), Staphylococcous aureus, Escherichia coli,

Pseudomonas aerogenus.

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5.2. HPLC

TheAndrographis paniculataauthentic sample and both the extracts were monitored at

223 nm with an UV Vis detector (shumadzu uv visible SPD LC 10 AVP

Series) (Tikhomiroff and Jolicoeur 2002).

5.2.1. Chromatogram of Methanol extract (Fig.8.0)

The active constituent Andrographolide is the second compound to elute out in the

methanol extract at a retention time of 3.460 minutes.

The first compound eluted out at a retention time of 1.380 minutes and the third

compound eluted out at a retention time of 4.493 minutes.

5.2.2. Chromatogram of the Ethyl acetate extract (Fig.9.0)

The active constituent Andrographolide is the second compound to elute out in the

Ethyl acetate extract at a retention time of 3.460 minutes.

The first compound eluted out at a retention time of 2.870 minutes and the third, fourth,

fifth and sixth compound eluted out at retention times of 3.997, 4.207, 4.497 and 4.753

minutes respectively.

5.2.3. Chromatogram of the AuthenticAndrographis paniculatasample (Fig.10.0)

The Andrographolide is the first compound to elute out in the authentic sample at a

retention time of 3.460 minutes, and the second compound eluted out at a retention time

of 4.490 minutes.

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6.0. DISCUSSION

6.1. Antimicrobial activity of Andrographis paniculata

In the present study, varying concentrations of 25, 50, 75 and 100 microgram of the

Methanol extract was used in each of the four wells of the nine petri plate cultures.

The zones of inhibition of growth were formed in all the four wells containing the

varying concentrations of the Methanol extract.

The methanol extract of the plant showed antimicrobial activity against four out of thenine pathogens tested.

Bacillus subtilis, Enterobacter cloacae, Enterococcous faecalis and Salmonella

typhimurium were the microorganisms against which the antimicrobial activity of the

Methanol extract was detected.

For the Ethy acetate extract, varying concentrations of 20, 30, 40 and 50 micrograms

was used in each of the four wells of the nine petri plate cultures.

The zones of inhibition of growth were formed in all the four wells containing the

varying concentrations of the Methanol extract.

The Ethyl acetate extract of the plant showed antimicrobial activity against seven out of

the nine pathogens tested.

Bacillus subtilis, Staphylococcous epidermis, Enterococcous faecalis, Salmonella

typhimurium, Staphylococcous aureus, Escherichia coli, Pseudomonas aerogenus were

the microorganisms against which the antimicrobial activity of the Ethyl acetate extract

was detected.

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The Ethyl acetate extract is inferred to have higher antimicrobial activity when

compared to the extract prepared using Methanol as the solvent.

This is inferred from the fact that the Ethyl acetate extract is effective against seven of

the nine pathogens, when compared to the effectiveness of the Methanol extract, which

was effective against only four of the nine pathogens tested.

The reason for the higher effectiveness of the Ethyl acetate can be due to a higher

content of active Andrographolide being present in the Ethyl acetate extract, in

comparison with that present in the Methanol extract.

Andrographolide, the active compound confers the antimicrobial property to the

Andrographis paniculata plant and hence the higher content of the Andrographolide

present in the Ethyl acetate extract, makes it more effective against the bacterial

pathogens.

6.2. HPLC

HPLC technique (using Reverse phase adsorptive C18 column) was performed for the

Andrographis paniculata aunthentic sample and for both the extracts of the

Andrographis paniculataplant. The chromatograms of both the extracts were compared

respectively to the chromatogram of the authentic sample.

It is inferred that the peak of the active constituent, Andrographolide present in both

Methanol and Ethyl acetate extracts respectively was obtained at a Retention time of

3.460 minutes, which was same as the retention time of the Andrographolide compound

present in the authentic sample ofAndrographis paniculata.

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7.0 SUMMARY

In the present study, the antimicrobial activity ofAndrographis paniculataplant extract

was tested against nine common human-affecting bacterial pathogens. The evaluationwas carried out using two different solvents - Methanol and Ethyl acetate. Pure stock

cultures of the nine bacterial pathogens were prepared and maintained throughout the

study. 10 micro liter volume forBacillus subtilis, and 20 micro liters for each of the other

pathogens, were taken from the pure stock culture and were then added to 25 ml of the

Mueller Hinton Agar present in nine test tubes numbered 1-9. This mixture was then

poured into the Petri plates and left for solidification. Next, four wells were punched into

each Petri plate and the extract was added in increasing concentrations in each of the fourwells. The plates were sealed and then kept for Incubation in a bacterial incubator for 24

hours at 37 C. The same procedure is performed for both the extracts.

After the 24 hour incubation period, each of the nine plates was checked for the zones of

inhibition of growth. The diameter of the zones were measured and noted down. For

microorganisms that showed zones of inhibition of growth, it is inferred that the plant

extract is effective against the respective microorganisms. The evaluation of both the

extracts is done based on its effectiveness against higher number of microorganisms.

Hence, it is concluded that Ethyl acetate is more effective against the bacterial pathogens,

in comparison to the Methanol extract.

The HPLC of the authentic Andrographis paniculata sample and both the extracts was

performed and their respective chromatograms were compared. In this, it is found that the

active compound Andrographolide, is present in all three samples and therefore has the

same retention time of 3.460 minutes. It is further noted that the antimicrobial property of

the Andrographis paniculata plant is conferred by its active component,

Andrographolide.

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8.0. CONCLUSION

Based on the results of the present study, it can be concluded that Andrographis

paniculata plant extract shows significant antibacterial activity against most of the

human- affecting pathogens. Extracts made from solvents that are more effective against

the pathogens can be used to develop drugs that can be taken along with other

Antibiotics. It is of common knowledge that the Andrographis paniculataplant can be

used to treat a wide range of ailments. Hence, the advantage of such a combinatorial drug

therapy would be the absence of side-effects and also increased effectiveness, in

comparison with the present day anti-biotic based drug therapy.

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