SUMMER PROJECT REPORT

ON

“A study of antimicrobial activity of medicinal plants against various multiple drug resistant

pathogens”.

2010-11

SUBMITTED BY: HEMANT

JUNEJA ROLL NO : BTB/06/301

SECTION : N INSTITUTE :-

AIB YEAR OF

SUBMISION :- 2010

ACKNOWLEDGEMENT

Encouragement motivates a person towards ones aim while guidance helps one to achieve it. Both encouragement and guidance takes one towards success in his/her work.

I wish to express my deepest and sincere gratitude to Mr. Chaitanya Kumar ( Associate Project Director) Clonegen Biotechnology Pvt. Ltd. For their support and guidance.

I wish to express my sincere thanks to Miss Shruti Sharma for her keen interest, valuable guidance and for giving number of small but valuable tips about experiments and scientific milieu during the project work is unforgettable. While thanking it would be a bit unfair not to mention my parents for their love and support during the project.

( Hemant juneja)

CONTENTS

ABSTRACT

INTRODUCTION

REVIEW OF LITERATURE

MATERIALS AND METHODS

WET LAB WORK

PLANT SAMPLES & TEST MICROORGANISMS

PLANT EXTRACTION

ANTIMICRBIAL ASSAY

AGAR-WELL DIFFUSION METHOD

DETERMINATION OF MIC

PHYTOCHEMICAL SCREENING

RESULTS AND DISCUSSION

CONCLUSION

REFERENCES

ABSTRACT

Countries like India have been using crude plants as medicine since Vedic period. A major part of the total

population in developing countries still uses traditional folk medicine obtained from plant resources

(Farnsworth 1994). With an estimation of WHO that as many as 80% of worlds population living in rural

areas rely on herbal traditional medicines as their primary health care, the study on properties and uses of

medicinal plants are getting growing interests. In recent years this interest to evaluate plants possessing

antibacterial activity for various diseases is growing (Clark and Hufford, 1993). Based on local use of

common diseases and Ethnobotanical knowledge, an attempt has been made to assess the antibacterial

properties of selected medicinal plants.\Eukalyptus, marigold, dhatura, guava, licorice for potential

antibacterial activity against medically important bacterial strains, namely Bacillus thuringiensis,

Staphylococcus aureus, Escherichia coli, Pseudomonas aeriginosa The antibacterial activity of methanolic

extracts was determined by agar well diffusion method. The plant extracts were more active against Gram-

positive bacteria than against Gram-negative bacteria. Human pathogenic strain was also isolated from soil

and identified as Micrococcus spp. From the screening experiment each of the plant material showed the

antibacterial activity; hence this plant can be further subjected to isolation of the therapeutic antimicrobials

and pharmacological evaluation

.Key words: Antibacterial property, Gram positive bacteria, Gram negative bacteria, zone of

inhibition, Phytochemical Screening,

INTRODUCTION

The use of plants and plant products as medicines could be traced as far back as the beginning of human

civilization. The earliest mention of medicinal use of plants in Hindu culture is founds in “Rigveda”, which

is said to have been written between 4500-1600 B.C. and is supposed to be the oldest repository of human

knowledge. It is Ayurveda, the foundation of medicinal science of Hindu culture, in its eight division deals

with specific properties of drugs and various aspects of science of life and the art of healing. (Rastogi and

Mehrotra, 2002)

From prehistoric days, plants are used for shelter, food and medicine. The use of plants for medicinal

purposes is as old as our civilization. The first known written record of curative plants was of Sumerian

herbal of 2200 BC. In the 5th century BC, The Greek doctor Hippocrates list out some 400 herbs in common

use. Dioscorides, in the Ist century AD, wrote a herbal by using 600 plants which ultimately became the base

for many later works. Herbs have been used for uncounted time for various purposes like healing the sick

and infirm. Most of the people still continue to use herbs to benefit their bodies. People thought that herbs

keep the body in tune with nature as nature intended and maintain proper balance. Many scientific studies

are still continued with modern research following the lead of old folklore and herbal uses to help finding

new western medicine. Man has also been aware of the effects of Herbs on the body, mind and emotion. For

example - Flowers were utilized to attract love, food and protection. Fragrant plants were worn to heal the

body and give a sense of well being. The most costly flowers are offered to gods and goddesses and the use

of aromatic incense is recorded from the earliest of times.

Medicinal plants are a source of great economic value all over the world. Nature has bestowed on us a very

rich botanical wealth and a large number of diverse types of plants grow in different parts of the country.

Nepal is rich in all the 3 levels of biodiversity, namely species diversity, genetic diversity and habitat

diversity. In Nepal thousands of species are known to have medicinal value and the use of different parts of

several medicinal plants to cure specific ailments has been in vogue since ancient times. Herbal medicine is

still the mainstay of about 75-80% of the whole population, and the major part of traditional therapy

involves the use of plant extract and their active constituents (Akerele, 1993). Following the advent of

modern medicine, herbal medicine suffered a set back, but during last two or three decades advances in

phytochemistry and in identification of plant compounds effective against certain diseases have renewed the

interest in herbal medicines (FAO 1990).

Nowadays multiple drug resistance has developed due to the indiscriminate use of commercial antimicrobial

drugs commonly used in the treatment of infectious disease. In addition to this problem, antibiotics are

sometimes associated with adverse effects on the host including hypersensitivity, immune-suppression and

allergic reactions. This situation forced scientists to search for new antimicrobial substances. Given the

alarming incidence of antibiotic resistance in bacteria of medical importance, there is a constant need for

new and effective therapeutic agents. Therefore, there is a need to develop alternative antimicrobial drugs

for the treatment of infectious diseases from medicinal plants (Agarwal et al., 1996)

Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of

infectious diseases while simultaneously mitigating many of the side effects that are often associated with

synthetic antimicrobials. The beneficial medicinal effects of plant materials typically result from the

combinations of secondary products present in the plant. In plants, these compounds are mostly secondary

metabolites such as alkaloids, steroids, tannins, and phenol compounds, flavonoids , steroids, resins fatty

acids gums which are capable of producing definite physiological action on body. Compounds extracted

from different parts of the plants can be used to cure diarrhea, dysentery, cough, cold, cholera,

fever bronchitis etc.

Medicinal plants, herbs, spices and herbal remedies are known to Ayurveda in India since long times. There

are many medicinal herbs and spices, which find place in day-to-day uses, many of these, are used as herbal

remedies. They have always been considered a healthy source of life for all people. Therapeutical properties

of medical plants are very useful in healing various diseases and the advantage of these medicinal plants is

being 100% natural. In the absence of modern medicinal remedies people relied on herbal remedies derived

from herbs and spices. Action of herbal remedies may vary from human to human.

The medicinal properties of drugs are due to the presence of substances such as alkaloids, glycosides, resins,

volatile oils, gums, tannins, etc. Some of these are powerful poisons if administered indiscriminately, while

others are dangerously habit-forming. Even the most dangerous drugs can be of value to human beings, if

judiciously employed. In some plants, Drugs are obtained from Underground Parts, in some it may be form

Bark, Stems/Wood, Leaves, Flowers, Fruits, Seeds or All Parts of Plants.

India, being the treasure-house of biodiversity with its diverse ecological conditions, rich ethnic diversity

and a strong traditional knowledge base, accounts for 45,000 plant species, out of which more than 8,000

species are used in some 10,000 herbal drug formulations. Ninety percent of the herbal industry's

requirement is taken out from the forests, resulting into their destruction. Due to this reason many species

have become endangered. There is urgent need to conserve our precious bio-wealth for sustainable

utilization. Our knowledge about the aforesaid is still inadequate and needs further extensive and intensive

investigations, so as to have full spectrum at hand. For this more elaborate and research-oriented information

are aptly required.

There are a number of well established indigenous or traditional systems like Ayurveda, Unani, Siddha,

Homeopathy, Tibetan and Naturopathy that are practiced along with the modern medicine to provide the

total healthcare system. In all these systems, a large number of plant drugs are used. In India, medicinal

plants are an essential part of traditional healthcare systems as these are accessible and affordable. Medicinal

plants are among the most important 'Non-Timber Forest Produce' in India. As per the WHO, 80%

population in developing countries relies on traditional natural medicines, and almost 80% of the traditional

medicines involve the use of plant extracts.

The value of medicinal plants, herbs and spices as herbal remedies is being lost due to lack of awareness,

and deforestation. The result is many valuable medicinal herbs are becoming rare and precious information

is lost. Less pollution we make, more ecological balance we maintain, will add to happiness of humankind

and care should be observed in using it. Nowadays people are being bombarded with thousand of unhealthy

products, the level of sensibility in front of diseases is very high and that's why the use of medicinal plants

can represent the best solution.

The Antimicrobial activity of the plant is due to a substance that kills or inhibits the growth of

microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes

(microbicidal) or prevent the growth of microbes (microbistatic).Disinfectants are antimicrobial substances

used on non-living objects. Antibiotics are generally used to treat bacterial infections. The toxicity to

humans and other animals from antibiotics is generally considered to be low. However, prolonged use of

certain antibiotics can decrease the number of gut flora, which can have a negative impact on health. Some

recommend that, during or after prolonged antibiotic use, one should consume probiotics and eat reasonably

to replace destroyed gut flora.

An antifungal drug is medication used to treat fungal infections such as athlete's foot, ringworm, candidiasis

(thrush), serious systemic infections such as cryptococcal meningitis, and others. Antifungals work by

exploiting differences between mammalian and fungal cells to kill off the fungal organism without

dangerous effects on the host. Unlike bacteria, both fungi and humans are eukaryotes. Thus, fungal and

human cells are similar at the molecular level, making it more difficult to find a target for an antifungal drug

to attack that does not also exist in the infected organism. Consequently, there are often side effects to some

of these drugs. Some of these side effects can be life-threatening if the drug is not used properly.

However, the future effectiveness of antimicrobial therapy is somewhat in doubt. Microorganisms,

especially bacteria, are becoming resistant to more and more antimicrobial agents. Bacteria found in

hospitals appear to be especially resilient, and are causing increasing difficulty for the sickest patients–those

in the hospital. Currently, bacterial resistance is combated by the discovery of new drugs. However,

microorganisms are becoming resistant more quickly than new drugs are being made available; thus, future

research in antimicrobial therapy may focus on finding how to overcome resistance to antimicrobials, or

how to treat infections with alternative means, such as species-specific phages.

The microorganisms are ubiquitous in nature. They are found in probably every environmental condition

present on earth. They play an important role in the proper functioning of the environmental machinery by

their positive and negative impacts. The positive impacts are the role in the recycling of the important

minerals and the components of the life cycle by degrading the dead matters and when present inside the

other organism like humans and other animals help in proper absorption of the important vitamins for

example the E. Coli present in the intestine help in the absorption of the vitamin B-12. Among the negative

impacts include the disease causing bacteria, fungus and viruses. These microbes deplete the health

condition of the individual and may lead to death if not treated on time. The most common among these

pathogens are the disease causing bacteria, they cause disease in both plants and animals.

To combat these pathogens antibiotics have for long formed the backbone of the disease management

system. The antibiotics are

Historical timeline of antibiotics

Antibiotics have proven to be a major asset in the fight against infectious bacteria.

Penicillin was the first wide-scale major breakthrough in antibiotic treatment.

Louis Pasteur unknowingly described the first antibiotic in 1877 when he observed that certain

bacteria release substances that kill other bacteria.

In 1909, Paul Ehrlich discovered arsphenamine (Salvarsan), an arsenic compound that kills

Treponema palladium, the bacterium causing the sexually transmitted disease, syphilis.

In 1928 Alexander Fleming discovered that a mold inhibited the growth of staphylococcal bacteria

and named the substance it produced “penicillin” (possibly Pasteur’s unknown substance).

It was not until 1940 that Howard Florey and Ernst Chain isolated the active ingredient in Fleming’s

mold.

With wide-scale production of penicillin, the use of antibiotics increased, leading to an average

eight-year increase in human life span between 1944 and 1972. Unfortunately, many bacterial

species continued to survive penicillin treatment due to their resistance mechanisms.

The repeated emergence of antibiotic-resistant bacterial strains is a problem that has long plagued public

health. Bacteria have always possessed the ability to protect themselves from naturally occurring antibiotics

by acquiring resistance through the exchange of genetic material with other bacteria. In the last two decades,

however, the problem has escalated as the prevalence of antibiotic-resistant bacteria has increased and multi-

drug-resistant strains have emerged in many species that cause disease in humans.

There are no treatments available for infections caused by many of the antibiotic-resistant bacteria, and

resistance to commonly used antibiotics is steadily increasing. In fact, no class of drugs with a novel mode

of action has been developed since the introduction of nalidixic acid in 1962. Alternative methods to combat

antibiotic-resistant bacteria are needed and scientists have begun to search for antimicrobial drugs in

vertebrates, invertebrates, and even bacteria and fungi in Earth’s most extreme environments.

Antibiotic Resistance in Bacteria: Origins and Emergence:

Most clinically useful antibiotics exhibit their selective toxicity by specifically blocking one or another type

of bacterial macromolecular synthesis (e.g.protein, nucleic acid or cell wall synthesis) -- acting on targets

that are not present or accessible in animal/human cells. Since the 1940s, when drugs such as penicillin,

streptomycin, and chloramphenicol were introduced widely as “miraculous” agents for treating bacterial

infections, the emergence of strains resistant to these and subsequently-developed drugs has represented a

continuing clinical challenge. The eventual appearance of strains simultaneously resistant to multiple

antibiotics significantly worsened the problem. The latter was found to involve different resistance genes

linked to each other on segments of DNA able to move efficiently from one bacterial cell to another by

phenomena known as horizontal gene transfer (HGT).

 

HGT can occur by three basic mechanisms: transformation (the release of free DNA from one bacterial cell

to be actively taken up by another); transduction (transfer of DNA via the use of bacterial viruses [phages]

as “vectors”); and conjugation (the movement of DNA from one cell to another via cell-to-cell contact).

Conjugation phenomena frequently involve mobile plasmids or conjugative transposons, which encode their

ability to move copies of themselves from one bacterial cell to another and are widespread in the bacterial

world (Clewell and Francia, 2004; Lawley et al., 2004). Such transferable elements have been the subject of

much investigation (Clewell, 1993; Funnell and Phillips,2004). Genome analyses of numerous bacterial

species have revealed the great extent to which HGT has occurred over time and has, in some cases, even

brought into question what actually constitutes a bacterial species. On consideration of such data, including a

report showing that bacteria such as Enterococcus faecalis can have more than 25% of its genome appearing

to represent mobile or foreign DNA (Paulsen et al, 2003), it should not be surprising, in retrospect, that

multiple resistance genes have appeared rapidly among bacteria able to cause disease in humans – and

additionally in numerous nonpathogens that reside normally in the gut. Elevated concern has related to the

recent emergence of high-level vancomycin resistance in Staphylococcus aureas (Weigel et al., 2003) and

the growing presence of multiple drug-resistant strains of Mycobacterium tuberculosis (Wright et al, 2006).

 

Most antibiotics used clinically are produced by, and isolated from, microorganisms found normally in the

soil, and the genus Streptomyces represents one of the most common of these sources. Molds such as

Penicillium, the original source of penicillin, are also well known producers. Antibiotics produced by

bacteria represent members of a more general class of extracellular (secreted) compounds known as

bacteriocins, substances that kill other bacteria and facilitate competition in the bacterial world. Many of

these compounds represent proteins with a large variety of different inhibitory activities, although a number

of those that serve as clinically useful antibiotics represent small non-protein molecules. Inasmuch as a gram

of soil may contain up to 5 thousand or more different species (Schloss and Handelsman, 2005), there are

obvious advantages to producing such agents. Production, however, also requires a means by which

organisms can protect themselves from their own “weapons”; and this is done by conferring a self

“immunity” or “resistance”. Furthermore, soil bacteria that do not necessarily produce bacteriocins often

carry multiple “resistance” determinants. This has recently been shown to be the case for many strains of

streptomyces (D’Costa et al., 2006). Thus the soil is loaded with genes encoding antibiotics and related

resistance, and this has doubtless been the case for many millions of years. When one considers the fact that

a single human being carries trillions of bacteria in and on the body, exceeding the number of somatic cells

by as much as 10 fold and corresponding to as many as 1000 different species, it is highly likely that

interaction of at least some of these organisms with resistant strains from the environment is a common

occurrence. Therapeutic exposure to an antibiotic would easily select for increased numbers of such strains.

 

Mechanisms of resistance vary greatly and include processes involving modification of the drug or its target

site, or efflux (export) of the drug. Much is known about many of these mechanisms (Walsh, 2003), and

there can be more than one way to be resistant to a given antimicrobial agent. In some cases during clinical

therapy a given bacterium may have acquired a mutation in a gene encoding the drug target, whereas in

other cases (e.g. inactivation of the drug or its target) resistance relates to highly evolved processes

involving genes that have been around (e.g. in soil organisms) for millennia. The latter genes are frequently

located on mobile genetic elements (transposons) that find their ways onto transferable elements such a

plasmids or conjugative integrative elements (e.g. conjugative transposons), as these vehicles allow for rapid

amplification via spread through bacterial populations. Today it is common for patients to pick up

nosocomial (hospital-acquired) infections of multiply resistance strains such as Staphylococcus aureus,

Escherichia coli and species of Enterococcus and Pseudomonas while being hospitalized initially for

unrelated illnesses. The high usage of antibiotics in hospital environments and the potential for transfer of

resistant organisms between patients (e.g. via hospital personnel) is an ever-present issue.

The “golden age” of antibiotics has been over for a number of years. As new drugs are introduced, it is

usually only a short time before resistance emerges. Efforts to reduce selection through less “abuse”

(overuse) of antibiotics both clinically and as “growth promoters” in animal feeds may be helpful; however,

they are not likely, at this stage, to have a major effect. Identification of new drugs to keep a step ahead of

the resistance problem is ongoing, although a number of large pharmaceutical companies are getting out of

the antibiotic business because of the major costs incurred in bringing to market new agents likely to be

ineffective within a few years. It makes more sense for smaller biotech companies to do much of the

exploration and discovery, with subsequent involvement of the larger companies when agents with high

potential are ready for clinical trials. In the past, firms have tended to focus on developing antibiotics with a

broad antimicrobial spectrum, since this would mean bigger sales; the accompanying resistance problem,

however, raises the question of whether drugs with a more narrow-spectrum might be more strategic. Such

drugs would be needed less frequently and provide less overall selective pressure on bacterial populations;

but rapid diagnosis of the target organism would be important. New advances in biotechnology related to the

search for new agents and targets, as well as exploration of efforts to modulate the human immune

system,will hopefully facilitate the search for new drugs and targets.

Causes of antibiotic resistance

For many years it was believed that antibiotic resistance was only caused by the failure of prescribed drug

regimens. It is now accepted that human errors also contribute to the development of antibiotic-resistant

bacteria.

Misuse of antibiotics occurs in medicine, agriculture, and household products. Common examples

include erroneous antibiotic prescriptions for nonbacterial infections and the addition of antibiotics

to livestock feed and cleaning agents, which have helped create a reservoir of antibiotic-resistant

bacteria.

Anomalous combinations have perpetuated drug-resistant microbes. For example, one study on

Rhesus monkeys reports that mercury in dental amalgam fillings fostered a 61% increase in

antibiotic-resistant bacteria. Upon removal of the amalgam fillings, drug-resistant bacteria dropped

58%. In another example, S. aureus was shown to acquire vancomycin resistance genes through

cohabitation with the vancomycin-resistant bacteria, Enterococcus faecalis, in the wound of a

hospitalized patient. Through mechanisms of genetic exchange between bacterial species, the mere

coexistence of these two particular bacteria helped to bring about drug resistance in S. aureus.

Enhanced transmission of resistance factors, or the increased efficiency with which resistance

genes are exchanged, is another important way that antibiotic resistance is perpetuated. Factors that

contribute to enhanced transmission include the survival of patients with chronic disease, an

increased number of immunosuppressed individuals, substandard hospital hygiene, more

international travel, and budget cuts in health care administration.

The reservoir hypothesis suggests that antibiotic-resistant bacteria have evolved because of the

selective pressures applied by antibiotic drugs; moreover, the hypothesis states that each antibiotic

has a threshold level that is required to induce and maintain antibiotic resistance. After a decline in

the populations of susceptible bacteria from antibiotic treatment, naturally resistant bacteria begin to

thrive, creating a reservoir of antibiotic-resistant bacteria.

Antibiotic resistance is a continually evolving and dangerous problem that requires:

future research and planning to impede a global health crisis

consideration of methods other than current antibiotic therapies

active participation by physicians and patients in curbing antibiotic misuse

ANTIMICROBIAL ASSAY

Resistance to antimicrobial agents (AMR) has resulted in morbidity and mortality from treatment failures

and increased health care costs. With the introduction of a variety of antimicrobials it became necessary to

perform the antimicrobial susceptibility test as a routine. For this, the antimicrobial contained in a reservoir

was allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organisms.

The disc diffusion method of AST is the most practical method and is still the method of choice for the

average laboratory. Methods of Antimicrobial Susceptibility Testing. Antimicrobial susceptibility testing

methods are divided into types based on the principle applied in each system. They include:

Diffusion Dilution Diffusion & Dilution

Stokes method Minimum Inhibitory Concentration E-Test method Kirby-Bauer method i) Broth dilution

ii)Agar Dilution

Agar-well diffusion method -Agar diffusion refers to the movement of molecules through the matrix

that is formed by the gelling of agar. This phenomenon forms the basis of the agar diffusion assay that is

used to determine the susceptibility or resistance of a bacterial strain to an antibacterial agent, (e.g.,

including antibiotics). When the seaweed extract known as agar is allowed to harden, the resulting material

is not impermeable. Rather, there are spaces present between the myriad of strands of agar that comprise the

hardened polymer. Small molecules such as antibiotics are able to diffuse through the agar. Typically, an

antibiotic is applied to a well that is cut into the agar. Thus, the antibiotic will tend to move from this region

of high concentration to the surrounding regions of lower antibiotic concentration. If more material is

present in the well, then the zone of diffusion can be larger. Following a time to allow for growth of

the bacteria then agar is examined. If bacterial growth is right up to the antibiotic containing well, then the

bacterial strain is deemed to be resistant to the antibiotic. If there is a clearing around the antibiotic well,

then the bacteria have been adversely affected by the antibiotic. The size of the inhibition zone can be

measured and related to standards, in order to determine whether the bacterial strain is sensitive to the

antibiotic. This technique can also be done by placing disks of an absorbent material that have been soaked

with the antibiotic of interest directly onto the agar surface. The antibiotic will subsequently diffuse out of

the disk into the agar. This version of agar diffusion is known as the Kirby-Bauer disk-diffusion assay.

Minimum inhibitory concentrations (MICs) are defined as the lowest concentration 

of an antimicrobial that will inhibit the  visible growth of a microorganism after overnight incubation, and

minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial that will prevent

the growth of an organism after subculture on to antibiotic-free media. MICs are used by diagnostic

laboratories mainly to confirm resistance, but most often as a research tool to determine the in vitro activity

of new antimicrobials, and data from such studies have  been used to determine MIC breakpoints. MBC

determinations are undertaken less frequently and their major use has been reserved  for isolates from the

blood of patients with endocarditis.

An MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism. MICs can be determined by agar or broth dilution methods

PHYTOCHEMICALS

Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties.

There are more than thousand known phytochemicals. It is well-known that plant produce these chemicals to

protect itself but recent research demonstrate that they can protect humans against diseases. Some of the

well-known phytochemicals are lycopene in tomatoes, isoflavones in soy and flavanoids in fruits. They are

not essential nutrients and are not required by the human body for sustaining life. 

There are many phytochemicals and each works differently. These are some possible actions:

Antioxidant - Most phytochemicals have antioxidant activity and protect our cells against oxidative damage

and reduce the risk of developing certain types of cancer. Phytochemicals with antioxidant activity: allyl

sulfides (onions, leeks, garlic), carotenoids (fruits, carrots), flavonoids (fruits, vegetables), polyphenols (tea,

grapes). 

Hormonal action - Isoflavones, found in soy, imitate human estrogens and help to reduce menopausal

symptoms and osteoporosis.

Stimulation of enzymes - Indoles, which are found in cabbages, stimulate enzymes that make the estrogen

less effective and could reduce the risk for breast cancer. Other phytochemicals, which interfere with

enzymes, are protease inhibitors (soy and beans), terpenes (citrus fruits and cherries).

Interference with DNA replication - Saponins found in beans interfere with the replication of cell DNA,

thereby preventing the multiplication of cancer cells. Capsaicin, found in hot peppers, protects DNA from

carcinogens.

Anti-bacterial effect - The phytochemical allicin from garlic has anti-bacterial properties.

Physical action - Some phytochemicals bind physically to cell walls thereby preventing the adhesion of

pathogens to human cell walls. Proanthocyanidins are responsible for the anti-adhesion properties

of cranberry. Consumption of cranberries will reduce the risk of urinary tract infections and will improve

dental health.

Foods containing phytochemicals are already part of our daily diet. In fact, most foods contain

phytochemicals except for some refined foods such as sugar or alcohol. Some foods, such as whole grains,

vegetables, beans, fruits and herbs, contain many phytochemicals. The easiest way to get more

phytochemicals is to eat more fruit and vegetables

List of few phytochemicals-

1. Alkaloids-Caffeine, Theobromine, Theophylline

2. Anthocyanins- Cyanidin, Malvidin

3. Carotenoids-Beta-Carotene, Lutein, Lycopene

4. Flavonoids- Epicatechin, Hesperidin, Isorhamnetin, Kaempferol, Myricetin,

Naringin

5. Monoterpenes- Geraniol, Limonene

6. Phenolic Acids- Capsaicin, Ellagic Acid, Gallic acid, Rosmarinic acid, Tannic

Acid

7. Phytosterols- Beta-Sitosterol

8. Saponins

9. Triterpenoids- Ursolic acid

10. Xanthophylls- Astaxanthin

11. Isoflavons – Genistein, Diadzein

12. Melatonin

Eucalyptus

Eucalyptus is a diverse genus of flowering trees (and a few shrubs) in the myrtle family, Myrtaceae.

Members of the genus dominate the tree flora of Australia. There are more than 700 species of Eucalyptus,

mostly native to Australia, and a very small number are found in adjacent parts of New Guinea and

Indonesia and one as far north as the Philippine archipelago. Only 15 species occur outside Australia, and

only 9 do not occur in Australia. Species of Eucalyptus are cultivated throughout the tropics and subtropics

including the Americas, Europe, Africa, the Mediterranean Basin, the Middle East, China and the Indian

Subcontinent.

Eucalyptus is one of three similar genera that are commonly referred to as "eucalypts," the others being

Corymbia and Angophora. Many, but far from all, are known as gum trees because many species exude

copious sap from any break in the bark (e.g. Scribbly Gum). The generic name is derived from the Greek

words ευ (eu), meaning "well," and καλυπτος (kalyptos), meaning well "covered," which refers to the

operculum on the calyx that initially conceals the flower.

Eucalyptus has attracted attention from global development researchers and environmentalists. It is a fast-

growing source of wood, its oil can be used for cleaning and functions as a natural insecticide, and it is

sometimes used to drain swamps and thereby reduce the risk of malaria. Outside their natural ranges,

eucalypts are both lauded for their beneficial economic impact on poor populations and derided for being

invasive water-suckers, leading to controversy over their total impact.

Size and habitat

A mature Eucalyptus may take the form of a low shrub or a very large tree. There are three main habit and

four size categories that species can be divided into.

"Mallees" are multi-stemmed from ground level, usually less than 10 m (33 ft) in height, often with the

crown predominantly at the ends of the branchlets and individual plants may combine to form either an open

or closed formation. Many mallee trees may be so low-growing as to be considered a shrub

Tree sizes follow the convention of:

Small — to 10 m (33 ft) in height

Medium-sized — 10–30 m (33–98 ft)

Tall — 30–60 m (98–200 ft)

Very tall — over 60 m (200 ft)

Leaves

Nearly all Eucalyptus are evergreen but some tropical species lose their leaves at the end of the dry season.

As in other members of the myrtle family, Eucalyptus leaves are covered with oil glands. The copious oils

produced are an important feature of the genus. Although mature Eucalyptus trees are usually towering and

fully leafed, their shade is characteristically patchy because the leaves usually hang downwards.

The leaves on a mature Eucalyptus plant are commonly lanceolate , petiolate,

apparently alternate and waxy or glossy green. In contrast, the leaves of

seedlings are often opposite, sessile and glaucous. But there are many

exceptions to this pattern. Many species such as E. melanophloia and E.

setosa retain the juvenile leaf form even when the plant is reproductively

mature.

BarkThe appearance of Eucalyptus bark varies with the age of the plant, the manner of bark shed, the length of

the bark fibres, the degree of furrowing, the thickness, the hardness and the colour. All mature eucalypts put

on an annual layer of bark, which contributes to the increasing diameter of the stems. In some species, the

outermost layer dies and is annually deciduous, either in long strips (as in Eucalyptus sheathiana) or in

variably sized flakes (E. diversicolor, E. cosmophylla or E. cladocalyx). These

are the gums or smooth-barked species. The gum bark may be dull, shiny or

satiny (as in E. ornata) or matte (E. cosmophylla). In many species, the dead

bark is retained. Its outermost layer gradually fragments with weathering and

sheds without altering the essentially rough-barked nature of the trunks or stems

— for example E. marginata, E. jacksonii, E. obliqua and E. porosa. The

extraordinary coloured bark of Eucalyptus deglupta native to South East Asia.

Bark characteristicsStringybark — consists of long fibres and can be pulled off in long pieces. It is usually thick with a spongy

texture.

Ironbark — is hard, rough and deeply furrowed. It is impregnated with dried kino (a sap exuded by the tree)

which gives a dark red or even black colour.

Tessellated — bark is broken up into many distinct flakes. They are corkish and can flake off.

Box — has short fibres. Some also show tessellation.

Ribbon — this has the bark coming off in long thin pieces but still loosely attached in some places. They can

be long ribbons, firmer strips or twisted curls.

Use in medicine

Eucalyptus is a traditional Aboriginal remedy for a variety of ailments.  Today, it is used worldwide in pills,

liquids, inhalers, salves, and ointments for many common problems.

Internally, Eucalyptus appears to help relieve symptoms of colds, flu, chest congestion, sore throat,

bronchitis, pneumonia, and respiratory infections. 

For internal use, Eucalyptus can be made into a tea or tincture.  See How to Make Herbal Teas for complete

instructions on teas.  Teas can also be used as a gargle for relief of sore throat. 

Liquorice

.

Liquorice (pronounced /ˈlɪkərɪʃ/ LIK-ə-rish), also licorice, is the root of Glycyrrhiza glabra from which a

sweet flavour can be extracted. The liquorice plant is a legume (related to beans and peas), native to

southern Europe and parts of Asia. It is not related to Anise, Star Anise or Fennel, which are the source of

superficially similar flavouring compounds. It is an herbaceous perennial, growing to 1 m in height, with

pinnate leaves about 7–15 centimetres (3–6 in) long, with 9–17 leaflets. The flowers are 0.8–1.2 cm (½–

⅓ in) long, purple to pale whitish blue, produced in a loose inflorescence. The fruit is an oblong pod, 2–3

centimetres (1 in) long, containing several seeds The flavor of liquorice comes mainly from a sweet-tasting

compound called anethole ("trans"-1-methoxy-4-(prop-1-enyl)benzene), an aromatic, unsaturated ether

compound also found in anise, fennel, and other herbs. Additional sweetness in liquorice comes from

glycyrrhizic acid, an anti-viral compound sweeter than sugar.

Cultivation and uses

Liquorice grows best in deep, fertile, well-drained soils, with full sun, and is harvested in the autumn, two to

three years after planting.

Today, liquorice extract is produced by boiling liquorice root and subsequently evaporating most of the

water. In fact, the name 'liquorice'/'licorice' is derived (via the Old French licoresse), from the Ancient Greek

glukurrhiza, meaning 'sweet root'. Liquorice extract is traded both in solid and syrup form. Its active

principle is glycyrrhizin, a sweetener more than 50 times as sweet as sucrose which also has pharmaceutical

effects.

Liquorice flavour is found in a wide variety of liquorice candies. The most popular in the United Kingdom

are liquorice allsorts. In continental Europe, however, far stronger, saltier candies are preferred. It should be

noted, though, that in most of these candies the taste is reinforced by aniseed oil, and the actual content of

liquorice is very low.

In the Netherlands, where liquorice candy ("drop") is one of the most popular forms of sweet, only a few of

the many forms that are sold contain aniseed (although mixing it with mint, menthol or with laurel is

popular, and mixing it with ammonium chloride creates the very popular salty liquorice known in Dutch as

zoute drop.)

Use in medicine

Liquorice may be useful in conventional and naturopathic medicine for both mouth ulcers and peptic ulcers.

Non-prescription aphthous ulcer treatment CankerMelts incorporates glycyrrhiza in a dissolving adherent

troche. Liquorice is also a mild laxative and may be used as a topical antiviral agent for shingles,

ophthalmic, oral or genital herpes. The compound glycyrrhizic acid, found in liquorice, is now routinely

used throughout Japan for the treatment and control of chronic viral hepatitis, and its transaminase-lowering

effect is clinically well recognized. Hepatoprotective mechanisms have been demonstrated in mice. Recent

studies indicate that glycyrrhizic acid disrupts latent Kaposi sarcoma (as also demonstrated with other

herpesvirus infections in the active stage), exhibiting a strong anti-viral effect.

Tagetes

Tagetes (pronounced /təˈdʒiːtiːz/)[1] is a genus of 51 species of annual and perennial herbaceous plants in the daisy family (Asteraceae or Compositae). They are native to the area stretching from the southwestern United States into Mexico and south throughout South America. The different species vary in size from 0.05-2.2 m tall. They have pinnate green leaves, and white, golden, orange, yellow, to an almost red floral heads typically (0.1-) to 4-6 cm diameter, generally with both ray florets and disc florets.

Naming Tagetes (possibly from the name of an Etruscan god[2]) are known almost universally in North America as marigold, or variously as Mexican marigolds (or cempasúchil), African marigolds (usually referring to cultivars and hybrids of T. erecta, although this species is not native to Africa), or French marigolds (usually referring to hybrids and cultivars of T. patula, many of which were developed in France although the species is not native to that country). At least one species is a naturalized weed in Africa, Hawaii, and Australia.

"Marigold" is derived from "Mary's Gold", and the plant is associated with the Virgin Mary in Christian stories. Tagetes is not to be confused with the genus Calendula, which goes by "marigold" in some areas. See "marigold" for this and other species commonly called marigold.UsesDepending on the species, marigold foliage has a musky, pungent scent, though some varieties have been bred to be scentless. It is said to deter some common insect pests (although it is recorded as a food plant for some Lepidoptera larvae including Dot Moth), as well as nematodes. Tagetes are hence often used in companion planting for tomato, eggplant, pepper, tobacco and potato. T. minuta (Khakibush or Huacatay), originally from South America, has been used as a source of essential oil for the perfume industry known as tagette, and as a flavourant in the food and tobacco industries in South Africa, where the species is also a useful pioneer plant in the reclamation of disturbed land. Some of the perennial species are deer-, rabbit-, rodent- and javalina-resistant. The petals of Tagetes are rich in the orange-yellow carotenoid lutein and as such extracts of T. erecta are used as a food colour (INS-Number E161b).

Marigold

Marigold is a hairy annual herb with large, solitary and stout flower heads; flat and spreading flowers of the outer whorl, with color varying from light yellow to deep orange. The lower leaves surround the stem. The fruits are curved, with the lower part having sharp point and upper parts crested and slightly beaked. The plant grows upto about 50cm in height. The main constituents of the herb are carotenoids, resin, essential oil, flavonoids, sterol, saponins and mucilage.

Marigold is antiseptic and anti-bacterial and has excellent wound healing properties. A compress or poultice of the flowers forms an excellent first aid for burns, scalds, stings and impetigo, a highly contagious bacterial skin infection. The juice of the leaves can be applied beneficially over warts. The sap from the stem isuseful for warts, corns and callouses. The flowers are valuable remedy for inflamed or ulcerated conditions of the skin when used externally, as in varicose ulcers. A cold infusion of marigold, used as eyewash, gives relief in conjunctivitis. A lotion of the flowers is also a useful wash for inflamed and sore eyes.

The leaves of marigold are beneficial in treating scrofula or tuberculosis of the lymphatic glands in children, especially in the neck. As a remedy the leaves should be taken as a vegetable.

The herb is useful in the treatment of certain circulatory disorders. A compress of the herb can be applied beneficially in the treatment of varicose veins and chilblains, which is an inflamed condition of the skin of the hands, feet and sometimes ears and nose caused by poor circulation and cold weather.

Datura

.

Datura is a genus of nine species of vespertine flowering plants belonging to the family Solanaceae. Its

precise and natural distribution is uncertain, owing to its extensive cultivation and naturalization throughout

the temperate and tropical regions of the globe. Its distribution within the Americas, however, is most likely

restricted to the United States and Mexico, where the highest species diversity occurs.

Some South American plants formerly thought of as Daturas are now treated as belonging to the distinct

genus Brugmansia [1] (Brugmansia differs from Datura in that it is woody, making shrubs or small trees, and

in that it has pendulous flowers, rather than erect ones). Other related genera include Hyoscyamus and

Atropa

Cultivation

Datura are usually planted annually from the seed produced in the spiny pods, but with care, plants can be

overwintered. Most species are suited to being planted outside or in containers. As a rule, they need warm,

sunny places and soil that will keep their roots dry. When grown outdoors in good locations, the plants tend

to reseed themselves and may become invasive. In containers, they should have porous, aerated potting soil

with adequate drainage. The plants are susceptible to fungi in the root area, so organic enrichers such as

compost and manure should be avoided.

Medicinal uses

Datura is a bushy plant with large egg-shaped leaves, very large white flowers and egg-shaped fruits,

covered with prickles. It grows up to one meter in height. The dried leaves, top portion of the flower and

seeds of the plant constitute the drug. Datura counteracts spasmodic disorders and induces deep sleep.

The leaves of datura contain alkaloids. The cultivated plants produce higher percentage of alkaloids. Seeds

yield diploid I and tetraploid II besides alkaloids. Some amounts of alkaloids are three in dried seeds and

fruits.

Datura is very effective in asthma, when the smoke from the burning leaves is inhaled. The leaves rolled into

cigarettes can be smoked to relieve asthmatic attacks. It is also a useful remedy for heart disorders. It

relieves cardiac pains, distress, palpitation and aortic disorders. The fruit of datura is a specific remedy for

phlegmatic and bilious types of malarial fever. A desired quantity of the fruit should be placed in an earthen

pot and covered with a plaster composed of cloth and clay. It should be parched in 10 to 12 grams of cow

dung cakes. When the fire gets extinguished and the pot is in a phial for use in malarial fever.

Datura leaves are useful in the treatment of earache. About 125 grams each of the juice of datura leaves and

sesame oil should be boiled in a tin vessel on gentle fire. When half of the juice has evaporated, seven leaves

of gigantic swallow wort (akh) should be put in it after smearing them with oil and sprinkling with powdered

salt. The leaves should be boiled till they begin to char. This oil is then filtered through a coarse cloth and

preserved in a bottle. A few drops of this oil in the ear cure earache and suppurative conditions of the ear.

Datura is very useful in checking secretion of breast milk. In case of an unfortunate death of a newly born

baby, accumulation of milk in the breasts of the mother poses a problem, causing severe pain. In such a case,

warmed leaves of the datura tied on the breasts will help dry the milk without any pain and difficulty. Just 2

or 3 applications will bring the desired results.

Guava

Guavas are plants in the myrtle family (Myrtaceae) genus Psidium (meaning "pomegranate" in Latin),

which contains about 100 species of tropical shrubs and small trees. They are native to Mexico, Central

America, and northern South America. Most likely naturally spreading (by means of ocean drifting) to parts

of Southeast Asia, the Caribbean and some parts of Africa, guavas are now cultivated and naturalized

throughout the tropics, and due to growing demand they are also grown in some subtropical regions.

Medical uses

Since the 1950s, guavas – particularly the leaves – have been a subject for diverse research in chemical

identity of their constituents, pharmacological properties and history in folk medicine; most research has

been restricted to the Apple Guava (P. guajava) however, and any additional beneficial properties of other

species remain essentially unstudied. From preliminary medical research in laboratory models, extracts from

Apple Guava leaves or bark are implicated in therapeutic mechanisms against cancer, bacterial infections,

inflammation and pain. Essential oils from guava leaves have shown strong anti-cancer activity in vitro.

Guava leaves are used in folk medicine as a remedy for diarrhea and, as well as the bark, for their supposed

antimicrobial properties and as an astringent. Guava leaves or bark are used in traditional treatments against

diabetes. In Trinidad a tea made from the young leaves is used for diarrhoea, dysentery and fever.

BACTERIAL CULTURES

1. Escherichia coli

Escherichia coli is a Gram-negative, facultative anaerobic and non-sporulating. Cells are typically rod-

shaped and are about 2 micrometres (μm) long and 0.5 μm in diameter, with a cell volume of 0.6 - 0.7 μm 3.

It can live on a wide variety of substrates. E. coli uses mixed-acid fermentation in anaerobic conditions,

producing lactate, succinate, ethanol, acetate and carbon dioxide. that is commonly found in the lower

intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some, such as

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

product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by

producing vitamin K2, and by preventing the establishment of pathogenic bacteria within the intestine.

Laboratory diagnosis- On MacConkey agar, deep red colonies are produced as the organism is lactose

positive, and fermentation of this sugar will cause the medium's pH to drop, leading to darkening of the

medium. Growth on Levine EMB agar produces black colonies with greenish-black metallic sheen. This is

diagnosic of E. coli. The organism is also lysine positive, and grows on TSI slant with a (A/A/g+/H2S-)

profile. Also, IMViC is ++-- for E. coli; as it's indol positive (red ring) and methyl red positive (bright red),

but VP negative (no change-colorless) and citrate negative (no change-green color). Tests for toxin

production can use mammalian cells in tissue culture, which are rapidly killed by shiga toxin.

Role in Disease - Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal

meningitis. In rarer cases, virulent strains are also responsible for hæmolytic-uremic syndrome (HUS),

peritonitis, mastitis, septicemia and Gram-negative pneumonia.Certain strains of E. coli, such as O157:H7,

O121 and O104:H21, produce potentially-lethal toxins. Food poisoning caused by E. coli is usually caused

by eating unwashed vegetables or undercooked meat. O157:H7 is also notorious for causing serious and

even life-threatening complications like hemolytic-uremic syndrome (HUS). Severity of the illness varies

considerably; it can be fatal, particularly to young children, the elderly or the immunocompromised, but is

more often mild. If E. coli bacteria escape the intestinal tract through a perforation (for example from an

ulcer, a ruptured appendix, or a surgical error) and enter the abdomen, they usually cause peritonitis that can

be fatal without prompt treatment.

E. coli can harbor both heat-stable and heat-labile enterotoxins. The latter, termed LT, contains one "A"

subunit and five "B" subunits arranged into one holotoxin, and is highly similar in structure and function to

Cholera toxins. The B subunits assist in adherence and entry of the toxin into host intestinal cells, while the

A subunit is cleaved and prevents cells from absorbing water, causing diarrhea. LT is secreted by the Type 2

secretion pathway.

Antibiotic therapy and resistance- The antibiotic sensitivities of different strains of E. coli vary widely. As

Gram-negative organisms, E. coli are resistant to many antibiotics that are effective against Gram-positive

organisms. Antibiotics which may be used to treat E. coli infection include amoxicillin as well as other semi-

synthetic penicillins, many cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole,

ciprofloxacin, nitrofurantoin and the aminoglycosides. E. coli are extremely sensitive to such antibiotics as

streptomycin or gentamicin.

2. Staphylococcus aureus

Staphylococcus aureus literally the "golden cluster seed" or "the seed gold" and also known as golden

staph) is the most common cause of staph infections. It is a facultatively anaerobic, 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 on blood agar plates. It is a spherical bacterium, frequently part

of the skin flora found in the nose and on skin. About 20% of the population are long-term carriers of S.

aureus.

Laboratory Diagnosis - S. aureus is catalase positive (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 enterococci and streptococci. A small percentage of S. aureus can be

differentiated from most other staphylococci by the coagulase test: S. aureus is primarily coagulase-positive

(meaning that it can produce "coagulase", a protein product, which is an enzyme) that causes clot formation

while most other Staphylococcus species are coagulase-negative. However, while the majority of S. aureus

are coagulase-positive, some may be atypical in that they do not produce coagulase.

Strains are responsible for food poisoning through the production of an enterotoxin and pathogenicity is also

associated with coagulase positivity.

Diseases caused- Staphylococcus aureus is the most common cause of skin infections. It is frequently found

in the nose and skin. About 20% of the population is long-term carriers of S. aureus. S. aureus can cause a

range of illnesses from minor skin infections, such as pimples, impetigo (may also be caused by

Streptococcus pyogenes), boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome and

abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic

shock syndrome (TSS), bacteremia and sepsis. Its incidence is from skin, soft tissue, respiratory, bone, joint,

endovascular to wound infections. It is still one of the five most common causes of nosocomial infections,

often causing post-surgical wound infections. S. aureus may occur as a commensal on human skin; it also

occurs in the nose frequently (in about a third of the population) and throat less commonly. The occurrence

of S. aureus under these circumstances does not always indicate infection and therefore does not always

require treatment (indeed, treatment may be ineffective and re-colonisation may occur). It can survive on

domesticated animals such as dogs, cats and horses, and can cause bumblefoot in chickens. It can survive for

some hours on dry environmental surfaces, but the importance of the environment in spread of S. aureus is

currently debated. It can host phages, such as the Panton-Valentine leukocidin, that increase its virulence.In

infants S. aureus infection can cause a severe disease Staphylococcal scalded skin syndrome (SSSS).

S. aureus infections can be spread through contact with pus from an infected wound, skin-to-skin contact

with an infected person by producing hyaluronidase that destroy tissues, and contact with objects such as

towels, sheets, clothing, or athletic equipment used by an infected person. Deeply penetrating S. aureus

infections can be severe. Prosthetic joints put a person at particular risk for septic arthritis, and

staphylococcal endocarditis (infection of the heart valves) and pneumonia, which may be rapidly spread.

Treatment and antibiotic resistance - The treatment of choice for S. aureus infection is penicillin; but in

most countries, penicillin-resistance is extremely common and first-line therapy is most commonly a

penicillinase-resistant penicillin (for example, oxacillin or flucloxacillin). Combination therapy with

gentamicin may be used to treat serious infections like endocarditis, but its use is controversial because of

the high risk of damage to the kidneys. The duration of treatment depends on the site of infection and on

severity.

3.

Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility. It is a

common bacterium which can cause disease in animals and humans. It is found in soil, water, skin flora and

most man-made environments throughout the world. It thrives not only in normal atmospheres, but also with

little oxygen, and has thus colonised many natural and artificial environments.

Laboratory Diagnosis- Depending on the nature of infection, an appropriate specimen is collected and sent

to a bacteriology laboratory for identification. First, a Gram stain is performed, which should show Gram

negative rods with no particular arrangement. Then, if the specimen is pure, the organism is grown on

MacConkey agar plate to produce colorless colonies (as it does not ferment lactose); but, if the specimen is

not pure, then the use of a selective plate is essential. Cetrimide agar has been traditionally used for this

purpose. When grown on it, P. aeruginosa may express the exopigment pyocyanin, which is blue-green in

color, and the colonies will appear flat, large, and oval. It also has a characteristic fruity smell. P. aeruginosa

is catalase+, oxidase+, nitrase+, and lipase+.

Role in diseaese - An opportunistic, nosocomial pathogen of immunocompromised individuals, P.

aeruginosa typically infects the pulmonary tract, urinary tract, burns, wounds, and also causes other blood

infections.The most common cause of burn infections is P. aeruginosa. Pseudomonas is also a common

cause of post-operative infection in radial keratotomy surgery patients. The organism is also associated with

the skin lesion ecthyma gangrenosum. Pseudomonas aeruginosa is frequently associated with osteomyelitis

involving puncture wounds of the foot, believed to result from direct inoculation with P. aeruginosa via the

foam padding found in tennis shoes. It uses a wide range of organic material for food; in animals, the

versatility enables the organism to infect damaged tissues or people with reduced immunity. The symptoms

of such infections are generalised inflammation and sepsis. If such colonisations occur in critical body

organs such as the lungs, the urinary tract, and kidneys, the results can be fatal. Because it thrives on most

surfaces, this bacterium is also found on and in medical equipment including catheters, causing cross

infections in hospitals and clinics. It is implicated in hot-tub rash.

Antibiotics that have activity against P. aeruginosa include:

aminoglycosides (gentamicin, amikacin, tobramycin); quinolones (ciprofloxacin, levofloxacin, and

moxifloxacin), cephalosporins (ceftazidime, cefepime, cefoperazone, cefpirome, but not cefuroxime,

ceftriaxone, cefotaxime), antipseudomonal penicillins: ureidopenicillins and carboxypenicillins (piperacillin,

ticarcillin: P. aeruginosa is intrinsically resistant to all other penicillins), carbapenems (meropenem,

imipenem, doripenem, but not ertapenem), polymyxins (polymyxin B and colistin), monobactams

(aztreonam).

REVIEW OF LITERATURE

K Sudharameshwari and J Radhika(2007) conducted Antibacterial Screening of eukalyptus against three

gram positive and three gram negative bacteria using four different solvents (petroleum ether, chloroform,

ethanol, aqueous) and it was concluded that petroleum ether exhibited best antibacterial activity. Among the

three plants used in the study eucalyptus was considered as the most effective. Because eukalyptus exhibited

maximum zone of inhibition against all pathogens compared to other plants, may be due to the presence of

alkaloids, anthocyanin, phenols, xanthoproteins, flavanoids, carboxylic acids, coumarins and sterols.

Agrawal P, Rai V, Singh R, B.1996. Randomized, placebo-controlled, single-blind trial of holy basil leaves

in patients with noninsulin-dependent diabetes mellitus. Giving details in possible use of basil as a

therapeutic drug implying plant materials use in medicines

The polymerase chain reaction (PCR) is currently used for culture-independent isolation of antibiotic

resistance genes from environmental samples (Waters and Davies, 1997; Smalla et al ., 2000; Aminov et al .,

2001;Frana et al ., 2001; Stokes et al ., 2001), but only accesses genes that are similar to known sequences

and often does not recover complete genes. Because of the possibility of undiscovered gene families and

undiscovered genetic diversity within known gene families, we circumvented the limitations of both

culturing and PCRbased methods by extracting and cloning DNA directly from soil samples, thus

constructing libraries that include the genes of uncultured soil bacteria. Such libraries containing

environmental DNA (Stein et al.,1996), or ‘metagenomic libraries’ (Rondon et al ., 2000), have been used

previously to identify clones expressing various enzymes (Henne et al ., 2000; Rondon et al ., 2000;

Knietsch et al ., 2003) and antimicrobial activities (Brady and Clardy, 2000; Brady et al ., 2001; Gillespie et

al ., 2002; Courtois et al ., 2003). Diaz-Torres and colleagues (2003) recently constructed metagenomic

libraries from the human oral cavity and identified a novel tetracycline resistance gene.

Propionibacterium acnes and Staphylococcus epidermidis have been recognized as pus-forming bacteria

triggering an inflammation in acne. The present study was conducted to evaluate antimicrobial activities of

Thai medicinal plants against these etiologic agents of acne vulgaris. Crude extracts were tested for

antimicrobial activities by disc diffusion and broth dilution methods. The results from the disc diffusion

method showed that 13 medicinal plants could inhibit the growth of Propionibacterium acnes. Among those,

Senna alata, Eupatorium odoratum, Garcinia mangostana, and Barleria lupulina had strong inhibitory effects.

Based on a broth dilution method, the Garcinia mangostana extract had the greatest antimicrobial effect. The

MIC values were the same (0.039 mg/ml) for both bacterial species and the MBC values were 0.039 and

0.156 mg/ml against Propionibacterium acnes and Staphylococcus epidermidis, respectively. In

bioautography assay, the Garcinia mangostana extract produced strong inhibition zones against

Propionibacterium acnes. Antimicrobial activity from fractions of column chromatography revealed one of

the active compounds in Garcinia mangostana could be mangostin, a xanthone derivative. Taken together,

our data indicated that Garcinia mangostana had a strong inhibitory effect on Propionibacterium acnes and

Staphylococcus epidermidis. Therefore, this plant would be an interesting topic for further study and

possibly for an alternative treatment for acne (Mullika et al, 2005).

Workers throughout the world have found literally thousands of phytochemicals which have inhibitory

effects on all types of microorganisms in vitro. More of these compounds should be subjected to animal and

human studies to determine their effectiveness in whole-organism systems, including in particular toxicity

studies as well as an examination of their effects on beneficial normal microbiota. It would be advantageous

to standardize methods of extraction and in vitro testing so that the search could be more systematic and

interpretation of results would be facilitated. Also, alternative mechanisms of infection prevention and

treatment should be included in initial activity screenings. Disruption of adhesion is one example of an anti-

infection activity not commonly screened for currently. Attention to these issues could usher in a badly

needed new era of chemotherapeutic treatment of infection by using plant-derived principles (MARJORIE et

al, 1999).

The antibacterial activities of hot water, methanol and ethanol extracts of 5 plant extracts utilized in

Palestine in popular medicine were studied. The dried extracts of Syzyium aromaticum (Myrtaceae) (seed),

Cinnamomum cassia (Lauraceae) (cassia bark, Chinese cinnamon) (bark), Salvia officinalis (Lamiaceaea)

(leaf), Thymus vulgaris (Lamiaceaea) (leaf) and Rosmarinus officinalis (Labiatae) (leaf) were tested in vitro

against 4 bacterial species by disk diffusion and micro-dilution. 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 multidrug resistant Pseudomonas aeruginosa and enterohemorrhagic

Escherichia coli O157 EHEC. The combinations of ethanolic extracts of S. officinalis with R. officinalis and

of R. officinalis with T. vulgaris on 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 (Bassam et al 2004).

Citrullus colocynthis (Linn.) Schrad. is an important medicinal plant belonging to family Cucurbitaceae.

Flavonoid "quercetin" was isolated from in vivo (leaf, stem, fruit and root) and in vitro callus of the species.

The dried samples were separately soxhlet extracted in 80% methanol and then reextracted with petroleum

ether, diethyl ether and ethyl acetate. The fraction was concentrated and subjected to TLC. The Rf value of

isolated quercetin and standard quercetin was calculated. The purified material was subjected to its IR

spectra, HPLC and identified as "quercetin". This study is also of practical importance because quercetin is

an important ingredient of Citrullus colocynthis(Meena et al, 2008).

The petroleum ether, chloroform, methanol and aqueous extracts of Acacia catechu L. f. (bark), Acacia

nilotica L. (bark), Aegle marmelos L. Correa. (fruit) Azadirachta indica L. (leaves), Annona squamosa L.

(leaves), Trachyspermum ammi L. (seeds), Holarrhena antidysenterica L. (bark) and Ocimum basilicum L.

(leaves) were tested against Enteropathogenic Escherichia coli (EPEC). These are the plants traditionally

used by rural populace of semi arid regions of India for the treatment of diarrhoea. The

ethnopharmacological information on the plants was collected by interviewing the traditional healers,

community leaders and rural people of Gujarat State. The agar-well diffusion assay method was used to

access the activities of plant extracts against the test organism. The results obtained show the strong activity

of petroleum ether extract of A.marmelos, A. indica, T. ammi and H. antidysenterica; chloroform extract of

A. catechu, A. indica and T. ammi; and methanol extract of A. catechu, A. nilotica, A. marmelos and T.

ammi (MIC, d” 50 ì g/ml) followed by petroleum ether extract of O.

basilicum and chloroform extract of A. nilotica, A. marmelos and H. antidysenterica (MIC, 50-100 ì g/ml).

These preliminary results will be helpful in rationalizing the use of plants based traditional medicines in

modern systems of health care (Jayshree et al, 2008).

In a study to evaluate the antimicrobial activity of Thonningia sanguinea against two sensitive and two

multi-drug resistant (ESBL) Enterobacteria strains namely Escherichia coli and Klebsiella pneumoniae.

The confirmation of the ESBL producing strains was done by the double-disc ynergy tests and the broth

dilution method was used for the determination of the antimicrobial parameters (MIC and MBC) on these

sensitive and ESBL producing strains. The two sensitive strains had the same MIC and MBC values

respectively 3.125 mg /ml and 12.50 mg/ml. The ESBL producing strains also had the same MIC of 6.25 mg

/ml and MBC values of 25 mg/ml. The extract was bactericidal for all tested strains. The results suggest that

the flowers of T. sanguinea can be used in association with antibiotics for alternative therapy of diseases

caused by ESBL producing E .coli, K.pneumoniae (Guessan et al, 2007).

Four antibacterial flavonoids (morin-3-O-lyxoside, morin-3-O-arabinoside, quercetin, and quercetin-3-

Oarabinoside) were isolated from fresh and dried Psidium guajava leaves, and their concentrations were

determined. Among them, quercetin and morin-3-O-arabinoside were the most and the least abundant,

respectively. Studies on inhibitory effects of the flavonoids on spoilage and foodborne pathogenic bacteria

revealed that they had bacteriostatic mode of action against all tested spoilage and foodborne pathogenic

bacteria including Bacillus stearothermophilus, Brochothrix thermosphacta, Escherichia coli O157:H7,

Listeria monocytogenes, Pseudomonas fluorescens, Salmonella enterica,

Staphylococcus aureus and Vibrio cholerae (Pongsak et al, 2010).

Gmelina arborea is commonly known as Gamhar, a fast growing tree throughout India. In Ayurveda it has

been observed that root bark and fruits are useful in treatment of leprosy, vaginal discharge and also

anthelmenthic. Five principal bioactive compounds such as Alkaloids, Saponins, Steroids,

Flavonoids and Glycosides were investigated in Methanol and Chloroform extracts of Gmelina arborea.

Three active compounds: alkaloids saponins and steroids are positive for both the extracts. Flavonoids and

Glycosides are detected only in Methanol extract and are absent in Chloroform extract. The antimicrobial

and antioxidative activities of Methanol and Chloroform extracts of Gmeline arborea were investigated. The

extracts (ME and CE) are subjected to screening for their possible antioxidative activities by using 2, 2

diphenyl picryl - 1- hydrazyl (DPPH) and β-carotene bleaching methods. Methanol and Chloroform extracts

were found to be better when compared to synthetic antioxidant Butylated Hydroxyl Toluene (BHT) in their

antioxidant activity with Ic 50 value of 15.4 and 18.6 μg / ml respectively. Methanol extract seemed to

inhibit the oxidation of linoleic acid with an 87% and chloroform extract with 30%. The results revealed that

the Methanol extracts of G. arborea has more antioxidative activity due to the presence of Flavonoids and

Saponins. Both ME and CE have shown great antibacterial activity against five test microorganisms

(Amrutha et al, 2010).

The selected essential oils were screened against four gram-negative bacteria (Escherichia coli, Klebsiella

pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris) and two grampositive bacteria Bacillus subtilis and

Staphylococcus aureus at four different concentrations (1:1, 1:5, 1:10 and 1:20) using disc diffusion method.

The MIC of the active essential oils were tested using two fold agar dilution method at concentrations

ranging from 0.2 to 25.6 mg/ml. Out of 21 essential oils tested, 19 oils showed antibacterial activity against

one or more strains. Cinnamon, clove, geranium, lemon, lime, orange and rosemary oils exhibited significant

inhibitory effect. Cinnamon oil showed promising inhibitory activity even at low concentration, whereas

aniseed, eucalyptus and camphor oils were least active against the tested bacteria. In general, B. subtilis was

the most susceptible. On the other hand, K. pneumoniae exhibited low degree of sensitivity. Majority of the

oils showed antibacterial activity against the tested strains. However Cinnamon, clove and lime oils were

found to be inhibiting both gram-positive and gram-negative bacteria. Cinnamon oil can be a good source of

antibacterial agents (Seenivasan et al, 2006).

Licorice (or 'liquorice') is a plant of ancient origin and steeped in history. Licorice extracts and its principle

component, glycyrrhizin, have extensive use in foods, tobacco and in both traditional and herbal medicine.

As a result, there is a high level of use of licorice and glycyrrhizin in the US with an estimated consumption

of 0.027-3.6 mg glycyrrhizin/kg/day. Both products have been approved for use in foods by most national

and supranational regulatory agencies. Biochemical studies indicate that glycyrrhizinates inhibit 11beta-

hydroxysteroid dehydrogenase, the enzyme responsible for inactivating cortisol. As a result, the continuous,

high level exposure to glycyrrhizin compounds can produce hypermineralocorticoid-like effects in both

animals and humans. These effects are reversible upon withdrawal of licorice or glycyrrhizin. Other in vivo

and clinical studies have reported beneficial effects of both licorice and glycyrrhizin consumption including

anti-ulcer, anti-viral, and hepatoprotective responses. Various genotoxic studies have indicated that

glycyrrhizin is neither teratogenic nor mutagenic, and may possess anti-genotoxic properties under certain

conditions. The pharmacokinetics of glycyrrhizin have been described and show that its bioavailability is

reduced when consumed as licorice; this has hampered attempts to establish clear dose-effect levels in

animals and humans (Isbrucker et al, 2006).

OBJECTIVE

“A study of antimicrobial activity of medicinal plants against various multiple drug resistant pathogens”.

MATERIALS AND METHODS

I. PLANT SAMPLES

PLANT SAMPLES USED ARE AS FOLLOWS:

S.NO SCIENTIFIC NAME COMMON NAME PART OF THE PLANT USED

1. Eukalyptus Eukalyptus Bark 2. Eukalyptus Eukalyptus Leaves 3. Tagetes Marigold Leaves 4. Datura Dhatura Leaves 5. Psidium Guajava Guava Leaves 6. Tagetes Marigold Red flowers7. Tagetes Marigold Yellow flowers8. Glycyrrhiza glabra Licorice Root

MATERIALS

1.L.B AGAR pH 6.8(for 250 ml)

COMPONENTS AMOUNTYeast extract 0.5gmTyptonetone 1gmSodium chloride 1gmAgar 1.5gmDistilled water 100 ml

2. NUTRIENT AGAR pH 6.8(for 250 ml)

COMPONENTS AMOUNTBeef extract 0.75 gmPeptone 1.25 gmSodium chloride 1.25gmAgar 3.75 gmDistilled water 250 ml

3. POTATO DEXTROSE AGAR (for 200ml)

COMPONENTS AMOUNTPotato (boiled) 60 gmDextrose 4 gmAgar agar 4 gmDistilled water 200 ml

4. CHEMICALS FOR PHYTOCHEMICAL SCREENING

Conc. Sulfuric acidSodium hydroxideFerric chloridePotassium hydroxideDilute hydrochloric acidDistilled waterChloroform

4. GLASSWARES REQUIRED

PETRIPLATESCONICAL FLASKSBEAKERSTEST TUBES

SERIAL DILUTION

Requirements30 ml normal saline (NS), 6 test tubes, 0.5 gm soil sample

Protocol1. Label six test tubes.2. Weigh out 0.5 g of moist soil (record the exact weight) and put into a test tube containing 5ml NS.

Cap and shake well (about 10 minutes).3. Transfer 0.5ml of this solution into a new test tube containing 5ml of NS (10-1).4. Repeat step3 six times to obtain dilution till 10-6.

ANTIBIOTIC RESISTANCE TEST

RequirementNutrient agar media (150ml), ampicillin (10mg/ml), tetracycline (10mg/ml), chloramphenicol (10mg/ml), oxalgin (10mg/ml), ciprofloxacin (10mg/ml), oofloxacin (10mg/ml), 8 petriplates, flask (250ml), micropipette (50µl), well borer

DescriptionMultiple drug resistance or Multidrug resistance is a condition enabling a disease-causing organism to resist distinct drugs or chemicals of a wide variety of structure and function targeted at eradicating the organism. Organisms that display multidrug resistance can be pathologic cells, including bacterial and neoplastic (tumor) cells.

Different chemical compounds are mainly responsible for the antimicrobial activity. These compounds are diffused through the agar medium and depending on their concentration so the zone of inhibition (ring) inhibiting the growth of sensitive micro organism. This zone of inhibition for specific microorganism is directly proportional to antimicrobial activity and can be known by measuring the diameter of inhibition ring (in mm).

Protocol1. Nutrient Agar media was prepared and autoclaved.2. Autoclaved media was then poured in autoclaved petriplates, then it was left for 15-20 minutes to

solidify.3. 50µl of culture (Bacillus, Pseudomonas, E. coli, Staphylococcus) were spread it into nutrient agar

plates respectively.4. Wells were prepared on the solidified media.5. 50µl of these drug’s are poured into the wells of Bacillus, Staphylococcus, E. coli and Pseudomonas

plates respectively.6. All the petriplates were kept in an incubator at 37˚C for 24 hrs (not in an inverted position).7. After proper time of incubation growth of microbes was checked in all the petriplates.

Broth and Media

NORMAL SALINE (NS) Sodium chloride 0.24gm Distill Water 30ml

NUTRIENT AGAR (NA) MEDIA Beef Extract 3gm Peptone 5gm Sodium chloride 5gm Distill water 1000ml Agar 15gm pH 6.8-7.2 ± 0.2 at 25°C

MANNITOL SALT AGAR MEDIA (MSA) Enzymatic digest of casein 5gm Enzymatic digest of animal tissue 5gm Beef extract 1gm D-mannitol 10gm Sodium chloride 75gm Phenol red 0.025gm Agar 15gm Distil water 1000ml pH 7.4 ± 0.2 at 25°C

MAC CONKEY AGAR MEDIA Peptone 20gm Trehalose 10gm Bile salt (Difco) 1.5gm Sodium Chloride 5gm Neutral Red 0.05gm Crystal Violet 0.001gm Distil water 1000ml Agar 15gm pH 7.4 ± 0.2 at 25°C

CETRIMIDE AGAR MEDIA Gelatine peptone 20gm Magnesium chloride 1.4gm Potassium sulfate 10gm Glycerol 10ml Cetrimide 0.3gm Agar 13gm Distil water 1000ml pH 7.2 ± 0.2 at 25°C

SIMMON’S CITRATE AGAR MEDIA Ammonium dihydrogen phosphate 1gm Dipotassium phosphate 1gm Sodium chloride 5gm Sodium citrate 2gm Magnesium sulfate 0.2gm Distil water 1000ml Agar 15gm Bromophenol blue (pH indicator) 0.08gm pH below 6.9 at 25°C

NUTRIENT BROTH (1% STARCH + 1% GLYCINE) Beef Extract 3gm Peptone 5gm Sodium chloride 5gm Starch 10gm Glycine 10gm Distill water 1000ml pH 6.8-7.2 ± 0.2 at 25°C

MR-VP BROTH Glucose 5gm Peptone 7gm Phosphate buffer 5ml Distil water 1000ml pH 6.9 at 37°C

Reagents

KOVAC’S REAGENT p-Dimethyl aminobenzaldehyde 10gm Amyl alcohol 150ml Concentrated HCl 50ml

VOGES-PROSKAEUR REAGENT Baritt’s A (α-Napthol) 3ml Baritt’s B (Potassium hydroxide) 1ml

II. METHODS

A) PLANT EXTRACTION

Plant samples were collected from the garden, washed and dried.They were kept in hot air oven maintained at 40-500 C for 24-48 hours till the plant sample dried completelyThe dried samples were powdered with the help of grinder.5-10 grams of the dried plant samples were mixed with 50-60 ml of methanol in a conical flask. It was cotton plugged and kept in rotary shaker (80-110 rpm) for 2 days.The solution was filtered with the help of filter paper, transferred to a beaker and kept in the hot air oven maintained at 600 C for one or two days till the entire ethanol was evaporated.The dried plant extract was scrapped off from the beaker, dissolved in methanol or in DMSO (Dimethyl sulfoxide, C2H6OS used as a cryoprotectant, added to cell media to prevent cell death during the freezing process. Approximately 10% may be used with a slow-freeze method, and the cells may be frozen at -80°C or stored in liquid nitrogen safely)

B) AGAR-WELL DIFFUSION METHOD

Bacterial cultures were inoculated in the Nutrient broth and kept overnight in the incubator and fungal strains were inoculated in the Potato-Dextrose broth and kept at room temperature for 3-5 days.Nutrient Agar and Potato-Dextrose Agar were prepared and autoclaved with the required petriplates for 15-20 min at 15lbs.

The agar was poured in sterile petriplates and kept for cooling. The bacterial or the fungal cultures (around 50 microlitre ) were spread in the respective agar media with the help of a spreader (L-rod).Wells (with a size of 9mm approximately) were made in the agar and 100 microlitre of the plant extract was poured in the wells.The extract was allowed to diffuse in the well for some time and then the petriplates were kept overnight in the incubator for bacterial cultures and for 3 to 5 days at room temperature for fungal cultures.Diameter of zone of inhibition(if formed)were measured in mm.

DETERMNATION OF MINIMAL INHIBITORY CONCENTRATION

Nutrient agar was prepared and kept for sterilization (autoclaved) along with the petriplates as before.Agar was poured into the plates inside laminar hood and allowed to set.Bacterial or fungal cultures were spread over the solidified agar and four wells were made.Four different concentrations of the plant samples were made starting from and poured into the wells.The plates were kept aside for sometime for the samples to diffuse into the agar.They were then kept overnight inside the incubator for bacterial cultures and in case of fungal cultures the plates were kept in the room temperature for three to four days.The concentrations of the plant samples were reduced further till no zone of inhibition was visible.

D) PHYTOCHEMICAL SCREENING

1. Test For Flavonoids (Sodium Hydroxide Test): 2 ml of 10% sodium hydroxide solution was added to 1ml of the plant extract to produce yellow colouration. A change in colour from yellow to colourless on addition of dilute hydrochloric acid was an indication for the presence of flavonoids. (Trease and Evans, 2002)

2. Test for Saponins: To 1 ml of the extract, about 3 ml of distilled water was added and shaken vigorously for about 2 minutes. Frothing which persisted on warming was taken as an evidence for the presence of saponins. (Sofowora,1993).

3. Test for Tannins: Few drops of 1% ferric chloride solution were added to 2 ml of the plant extract occurrence of a blue-black, green or blue-green precipitate indicates the presence of tannins (Trease and Evans, 2002).

4. Test for Steroids: The plant extract (1ml) was mixed with 2-3ml of chloroform and equal volume of concentrated sulfuric acid was carefully poured along the sides of the test tube to form two different layer. If the upper layer turns red and sulphuric acid layer showed yellow with green fluorescence then this indicated the presence of steroids.

5. Test for Terpenoids:

The plant extract(1ml) was mixed with 2ml of chloroform and two to three ml of concentrated sulfuric acid was carefully poured along the sides of the test tube to form two different layer. Formation of reddish- violet colour indicates the presence of terpenoids

6. Test for Naphthoquinone (Dam-Karrer test): To the extract, few drops of 10% potassium hydroxide was added.formation of blue-black colour indicates the presence of naphthoquinone.

RESULTS

1. Concentration Of The Plant Samples

S.NO SCIENTIFIC NAME CONCENTRATION (mg/ml)

1. Eukalyptus 100-3502. Eukalyptus 100-3503. Tagetes 100-3504. Datura 100-3505. Psidium Guajava 100-3506. Tagetes 100-3507. Tagetes 100-3508. Glycyrrhiza glabra 100-350

2. Antimicrobial Assay

I. Diameter of zone of inhibition (in mm) of plant extracts against few microbial cultures of 300mg/ml

PLANT

SAMPLES

CULTURES

Eukalyptus Psidium

Guajava

Marigold

LEAVES BARK LEAVES LEAVES

P.aeruginosa 20-21 23 20 20

S.aureus 20 25 21 21

E.coli 20 26 22 21

II. Diameter of zone of inhibition (in mm) of plant extracts against cultures of 300mg/ml

PLANT

SAMPLES

CULTURES

Marigold

flowers

Dhatura licorice

RED YELLOW LEAVES ROOTS

P.aeruginosa 20-21 23 20 20

S.aureus 20 25 21 21

E.coli 20 26 22 21

Key: - means no zone of inhibition formed

Observation table of 200mg/ml concentarion of plant extracts on microbes in cm

Key words:- E.L – eucalyptus leaves, MR- marigold red flowers, L- licorice, G- guava, DH- dhatura,ML- marigold leaves, MY- marigold yellow, EB- eucalyptus bark.

Observation table of 200mg/ml concentarion of plant extracts on isolates from soil in cm

Plant extracts Orange isolate Yellow isolate Pale isolateE.L 1.7 - -MR 1.5 1.2 -L 2.2 - 2G 2.5 - 2.5DH 1.5 - 3M.L 1.3 1 3M.Y 1.5 1 3E.B 2 - 2

I MAGES:

Results of all plant samples on microbial cultures

Activity of licorice, eucalyptus bark &

Plant extracts E.coli P.aeriginosa S.aureusE.L - - 2MR 1.5 - 1.5L 2 0.7 1.7G - 2 2DH 0.8 1 -M.L 1.8 1.2 1.1M.Y - 1.4 2.3E.B 1.6 - 1.8

guava against Pseudomonas aeruginosa

Activity of dhatuta, eucalyptus leaves &

Marigold leaves against Pseudomonas aeruginosa

Activity of licorice, eucalyptus bark & Activity of dhatuta, eucalyptus leaves & guava against E.coli

Marigold leaves against staphylococcus aureus

Activity of licorice, eucalyptus bark & Activity of licorice, eucalyptus bark &

guava against E.coli guava against orange isolate

Activity of licorice, eucalyptus bark &

guava against yellow isolate

Activity of licorice, eucalyptus bark &

guava against pale yellow isolate

Result of phytochemical analysis for

proteins become positive

DISCUSSION

The widespread use of antibiotics both inside and outside of medicine is playing a significant role in the

emergence of resistant bacteria. They are often used in animals but also in other industries which at least in

the case of agricultural use lead to the spread of resistant strains to human populations. A

number of antibiotics have lost their effectiveness due to the development of resistant strains of bacteria,

which has primarily occurred through the expression of resistance genes. In addition to inducing resistance,

antibiotics are sometimes associated with opposing effects such as hypersensitivity, immune-suppression

and allergic reactions. Resistance to antimicrobial agents (AMR) has resulted in morbidity and mortality

from treatment failures and increased health care costs. Although defining the precise public health risk and

estimating the increase in costs is not a simple undertaking, there is little doubt that emergent antibiotic

resistance is a serious global problem. Appropriate antimicrobial drug use has unquestionable benefit, but

physicians and the public frequently use these agents inappropriately. Inappropriate use results from

physicians providing antimicrobial drugs to treat viral infections, using inadequate criteria for diagnosis of

infections that potentially have a bacterial aetiology, unnecessarily prescribing expensive, broad-spectrum

agents, and not following established recommendations for using chemo prophylaxis. The availability of

antibiotics over the counter, despite regulations to the contrary, also fuel inappropriate usage of

antimicrobial drugs in India. The easy availability of antimicrobial drugs leads to their incorporation into

herbal or "folk" remedies, which also increases inappropriate use of these agents. Widespread antibiotic

usage exerts a selective pressure that acts as a driving force in the development of antibiotic resistance. The

association between increased rates of antimicrobial use and resistance has been documented for nosocomial

infections as well as for resistant community acquired infections. As resistance develops to "first-line"

antibiotics, therapy with new, broader spectrum, more expensive antibiotics increases, but is followed by

development of resistance to the new class of drugs. Therefore, there is a need to develop

alternative antimicrobial drugs for the treatment of infectious diseases which may not show any side-effects.

There are various medicinal plants showing good antimicrobial activity and further investigation of such

plants has to be done in order to ascertain the antibacterial effect of this herb extract.

The antibacterial activity of a plant may be indicative of the presence of some metabolic toxins or broad-

spectrum antibiotic compounds. Our work is indicative of the potential use of the plants like eucalyptus,

datura, guava, marigold and licorice as possible sources of antimicrobial compounds and their utility as the

antimicrobial agents in the treatment of the multiple drug resistant strains. We found that the methanolic

extract of the guava has shown best results among all the known MDRs and also against the Soil Isolates.

The methanolic extract of the Licorice showing the second best result having maximum antimicrobial

activity against orange isolate showing 2.2cm zone of inhibition and against E. coli having 2 cm zone of

inhibition.

Preliminary phytochemical analysis during the present study also ascertains the presence of some

potential group of bioactive substances, but the nature of active phytochemical responsible for antibacterial

activity cannot be ascertained. In order to investigate components present in the plant samples, HPLC

analysis has to be performed.

Therefore all these plant materials can be further used for antimicrobial purposes.

CONCLUSION

This study revealed that plant materials show potential antibacterial activity and further studies on such

medicinal plants might warrant fruitful results. Some potential phytochemicals present in these plants, that

might be responsible for their activity against micro organisms, were also detected..Further studies on such

plants might warrant fruitful results.

Antibiotic resistance is a continually evolving and dangerous problem that requires immediate attention as

well as future planning to impede a global health crisis. Is it not time too seriously consider other methods

for which current antibiotic therapies are ineffective and therefore prolong sickness, treatment, an even

sometimes result in mortality? Since the efficacy of current therapies is waning and conventional antibiotics

are a temporary fix to bacterial multi-drug resistance, society must look elsewhere. If the reservoir

hypothesis is true, as most scientists agree, then curbing drug usage to prevent resistant bacteria should be

key. Although this viewpoint is highly debated, it holds some merit. Bacteria thrive on mutations and

removal of selective pressures should slow mutational rates. Indeed, the alternative methods mentioned have

begun to target the pathogen and not the organism.

Our work has put forward the possible potential of plants, Licorice, Eukalyptus, Marigold, Guava and

Datura as the sources of anti-microbially active compounds. Thus with further work done on them with use

of latest analytical techniques can result in isolation of many of the required compounds.

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