TER -1

INTRODUCTION
 INTRODUCTION

1. INTRODUCTION
Plants are the most beautiful creation of nature which fornis the basis of almost all life on
the Earth, providing protection and substance for organisms ranging from bacteria to
large mammals. They are the main source of carbohydrate, proteins, fats and other useful
materials like wood, cellulose, fuel, gum, rubber and many others. Infect, plants are the
self governed factories producing a vast- array of compounds from simple components
Hke CO2, H2O and in ions. Besides this, they also have other important roles making them
an attractive thing to be explored by the researcher. They grab the attention of the
researchers by showing some medicinal properties. India has been a heartland for
traditional medicinal knowledge and practices since ancient times. Out of approximately
3200 taxa in India so far known having medicinal value, a total of about 343 species
under 298 genera and 98 families are known to be used as medicines. There is an old
saying "for every disease that arises on this planet, plants or herbs gives a cure".
These medicinal plants have been used for centuries as remedies and the richest bio
resources of drugs of traditional medicinal systems in pharmaceuticals, folk medicines,
nutraceuticals and synthetics drugs. According to the World Health Organization (WHO,
1977) "a medicinal plant" is any plant, which in one or more of its organ contains
substances that can be used for the therapeutic purposes or which, are precursors for the
synthesis of useful drugs. This definition distinguishes those plants whose therapeutic
properties and constituents have been established scientifically and plants that are
regarded as medicinal but which have not yet been subjected to thorough investigation.
Medicinal plants or herbal remedies are valuable in the treatment of various health
problems (Das et ah, 2010). Furthermore, WHO (2001) defines medicinal plant as herbal
preparations produced by subjecting plant materials to extraction, fractionation,
purification, concentration or other physical or biological processes which may be
produced for immediate consumption or as a basis for herbal products. The medicinal
properties of plants could be based on the antioxidant, antimicrobial antipyretic effects of
the phytochemicals in them (Cowman, 1999; Adesokan et al, 2008).
A large number of these medicinal plants are used in several foraiulations for the
treatment of various diseases caused by microbes. According to World Health
Organization, medicinal plants would be the source of obtaining a variety of drugs.

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 INTRODUCTION

Various societies across the world have shown great interest in curing diseases using
plants or plant based drugs. Microbes are closely associated with the health and welfare
of human beings. Some are beneficial and some are detrimental. As preventive and
curative measures, plants and their products are used in the treatment of infections for
many centuries ago. WHO estimated that 80% of the people worldwide rely on plant
based medicines for their primary healthcare (Famsworth, 1985) and India happens to be
the largest user of traditional medical cure.
Traditions of collecting, processing and applying plants and plant-based medication have
been handed down from generation to generation (Fyhrquist et al, 2002). It appears that
science is becoming a full circle. In the beginning, all drugs were natural, since
everything we used to treat our illness, cure our discomforts, and protect us came from
the world around us, which is from plants, herbs, and in some cases, the animal world
(Katz, 2002). Plants have served as a source of new phannaceutical products and
inexpensive starting materials for the synthesis of many known drugs.
Medicinal plants produce bioactive compounds used mainly for medicinal purposes.
These compounds either act on different systems of animals including man, and/or act
through interfering in the metabolism of microbes infecting them. The microbes may be
pathogenic or symbiotic. In either way the bioactive compounds from medicinal plants
play a determining role in regulating host-microbe interaction in favour of the host. So
the identification of bioactive compound in plants, their isolation, purification and
characterization of active ingredients in crude extracts by various analytical methods is
important. These bioactive constituents and their derivatives represent more than 50% of
the drugs in clinical use in the world (Cowan, 1999, Sofowora, 1982) .Although the first
chemical substance to be isolated from plants was benzoic acid in 1560, the search for
useful drugs of known structures did not begin until 1804 when morphine was separated
from Papaver somniferum. Since then many drugs from higher plants have been
discovered, but less than 100 with defined structures are in common use. Less than half
of these are accepted as usefiil drugs in industrialized countries (Famsworth, 1985).
Considering the great number of chemicals that have been derived from plants as
medicine, scientific evaluation of plants used traditionally for the treatment of bacterial

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 INTRODUCTION

infection seems to be a logical step of exploiting the antimicrobial compounds, which

may be present in plants.
Bioactive compounds in plants
As mentioned earlier, plants are able to synthesize many aromatic secondary metabolites
or commonly known as bioactive compound in examples phenols or its oxygen-substitute
derivatives. There are several major groups of secondary compounds exists in the
medicinal plants includes alkaloids, phenolic compounds and terpenoids, hi phenolic
compounds, phytochemicals such as tannins, flavonoids and coumarins amongst the well-
known among the researchers. These bioactive compounds are abundance in parts of
plants such as roots, stems, flowers, barks, leaves and seeds. Most of them have been
fully studied by many researchers for their ability to treat infectious diseases.
Phenolic compounds
Flavonoids
One of the examples of bioactive compound is phenolic compounds including their
subcategory which is flavonoid. This compound are usually present in all plants and
mostly being discovered in soybeans, nuts, vegetables, fruits, tea and olive oils (Kris-
Etherton et al, 2002). Flavonoid is a plant pigment mostly derived from benzo-y-pyrone
similar to chromone that gives color to the flower petals. This colored functions to attract
the pollinator animals for pollination pmposes (Cushnie et al, 2005 and Havsteen, 2002).
It has a molecular structure consists of two aromatic rings, A and B linked by three
carbon bridges. Groups of studies proves that the structures of flavonoid have certain
effects toward antimicrobial, antiviral and antifungal activity (Cushnie et al, 2005).
Flavonoids can be divided into six subclasses comprises of flavones, flavanones,
flavanols, flavonols, isoflavones, and anthocyanidin.
Flavonoids are commonly synthesis by plant in response to microbial infection. The
antimicrobial activity is probably due to their ability to form a complex with extracellular
and soluble proteins and to complex with bacterial cell wall. Lipophilic flavonoids may
interrupt the microbial membranes (Cowan, 1999). In addition, in another study, they
have found that it can inhibit the DNA synthesis. The B ring of robinetin, myricetin and
(-)-epigallocathecin can intercalate or fonn hydrogen bond with the stacking of nucleic

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 INTRODUCTION

acid bases thus explaining the inhibitory action of DNA and RNA synthesis (Cushnie and
Lamb, 2005).

Tannins
Tannins are one of the phenolic compounds that are naturally occurring in the plants. It is
a polymeric phenolic substance with astringent properties. These properties give tannins
ability to dissolves in water, alcohol and acetone thus precipitating gelatin from solution
(Das et. al., 2010). It combines with protein and other polymer to fonn a stable complex
(Lim et. al., 2006). They are found in most parts of the plants example bark, wood, leaves
fruits and roots. Tannins can be divided into two groups, hydrolyzable and condensed
tannins. Hydrolyzable tannin is basically gallic acid whereas condensed tannin comes
from flavonoid monomers. Formation of tannins might be from the condensation of
flavan derivatives transported to woody tissues of plants (Cowan, 1999).
Tannins have been reported in the treatment of various diseases in humans such as
diarrhea, gastric ulcers, snake bites and wounds (Lim et. al., 2006). Moreover, some also
consider tannin to have antiviral properties. Despite less reports of any involvement of
tannins on antimicrobial activity, Dass et. al, 2010 have showed some positive results on
the antibacterial activity of tannins towards Staphylococcus aureus. Streptococcus
pyrogens, Slamonella typhi, Pseudomonas aeruginosa, Proteus vulgaris and Escherichia
coli.

Coumarins
Coumarins are also parts of the phenolic groups along with flavonoids and tannins. They
are made of fused benzene and a-pyrone rings and responsible for the odour release by
hay (Cowan, 1999). It can be divided into two benzo-a-pyrones for coumarin and benzo-
y-pyrones where flavonoids one of its member. Coumarin exists in most of the plant
kingdom such as fruits, green tea, and chicory.
Coumarins are phytoalexins and synthesized de novo by the plant following an infection
by a bacterium or fungus. These phytoalexins are antimicrobial for example scopoletin
which is synthesized by the potato (Solanum tuberosum) followed by infection of fungal.
Coumarin has been known for its antithrombotic, anti-inflammatory, vasodilatory, anti-
tumour and antimicrobial activities (Lacy and O' Kennedy, 2004, Cowan, 1999). There

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 INTRODUCTION

are many studies related to the role of coumarin in the treatment of diseases. Warfarin is
one of the coumarin have been used as an oral anticoagulant and rodenticide. Moreover,
there has been evidence of the antimicrobial properties of coumarin where it can inhibit
Candida albicans in vitro thus can be used as an agent for treatment of vagina
candidiasis. Derivatives of coumarin like phytoalexins are produce by carrot in response
towards fungal infection (Cowan, 1999). Besides the antimicrobial activities, the
bacteriostatic and anti-tumor of coumarin offers great interest to researchers to use them
as a therapeutic agent. Coumarin and its derivatives 7-hydroxycoumarin have anti-tumor
activity towards several tumor cell lines (Lacy and O' Kennedy, 2004).

Terpenoids
Nomially we can smell some fragrance release by plants due to the content of essential
oils inside them. These essential oils are secondary metabolites highly enriched with
isoprene based compound. They are called terpenes with general chemical structure
C10H16 and occur as diterpenes, triteipenes, tetraterpenes, hemiterpenes and
sesquiterpenes. If the terpenes contain additional elements such as oxygen, they are called
terpenoids. Terpenoids share the same origin as fatty acids because they are both
synthesized from acetate units. The only difference between those two is that they have
widespread branching and cyclized.
Terpenoids have been reported active against bacteria in many researchs such as by
Okwu and Ohenhen, 2009; Edeoga et ai, 2005; Enwuru et ah, 2008. Essential oils also
posses' strong antimicrobial properties. 60% of the essential oil derivatives inhibit the
growth of fungi while another 30% goes to bacteria inhibition. Terpenoids presences in
the essential oils of plants are found to be useful in the control oiListeria monocytogenes.
Alkaloids
Another natural compounds usually found in the plants is alkaloid. It is defined as
heterocyclic nitrogen compounds. However, alkaloid is not uniquely from plants because
they also have been isolated from various animal sources. Morphine is an alkaloid
isolated in 1805 from the opium poppy Papaver somniferum. Plant with alkaloid such as
Ranunculaceae family is found to have antimicrobial properties. As the time passes,
alkaloid draws attention of the researcher because of their physiological activities in
humans and animals. Taxol is the most known form of alkaloid isolated from Taxus

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 INTRODUCTION

brevivolis useful for anticancer treatment. Many other plants in example Cephalotaxus
harringtona plant containing homoharringtonine can cure leukemias, while Tapia can
modulate inflammatory disorders (Lopes et. al., 2009).
Moreover, antimicrobial activities of alkaloids are explained by its effects on Giardia and
Entamoeba species due to their localization in small intestine. One of the alkaloid,
berberin is found to be effective against trypanosomes and plasmodial. Its reaction is
based on its ability to intercalate with DNA similar to harmane.

Plant-based antimicrobial represent a vast untapped source of medicines with enonnous

therapeutic potential (Cowan, 1999). They are supposedly effective in treatment of
infectious diseases while simultaneously minimizing many of the side effects that are
often associated with synthetic antimicrobials.

1.1 Antibiotic resistance

Antibiotic is a substance of biological, semisynthetic or synthetic origin that shows

selective activity against bacteria and have potential to use in treatment of infections.
Antibiotic agent generally cures disease by killing the causative agent through the
inhibition of a unique and vital function in the pathogen. (Baquero and Blazquez, 1997).
Microbial resistance to antibiotics is a widespread phenomenon that is blamed on a
combination of the following factors. The first major factor to blame is the huge
consumption and indiscriminate use of commercial antimicrobial drugs commonly used
in the treatment of infectious diseases (Baquero and Blazquez, 1997; Guillemot, 1999).
The incorrect use (for too short a time period, at too low a dose, at inadequate potency or
for the wrong reasons), as well as the under-use (through a lack of access, inadequate
dosing, poor adherence and substandard antimicrobials), of antimicrobials, has
substantially contributed to the emergence of resistance (WHO, 2004).
The combined effects of fast growing rates, high concentrations of cells, genetic
processes of mutation and selection and the ability to exchange genes, account for the
extraordinary rates of adaptation and evolution that can be observed in bacterial
resistance to antibiotics. A bacterial strain is either inherently resistant to an antibiotic
agent because they lack the specific target site for the drug, or due to acquired resistance
to an antibiotic.

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Almost since the beginning of the antibiotic era, bacterial resistance has been seen as the
major obstacle to successful treatment (Iwu et al, 1999). Microbial resistances to
antibiotics in the clinic emerged soon after their first use in the treatment of infectious
disease, and continue to pose a significant challenge for the health care sector. Resistance
has now firaily emerged as a problem in the wider community. At the end of the 1960s
the Surgeon General of the United States stated that: "we could close the book on
infectious diseases." At the time he uttered these words the emergence of resistance did
not seem to affect therapeutic options. Although S. aureus had become resistant to
benzylpenicillin and showing resistance to thethincillin, it remained sensitive to
gentamicin and infections could therefore still be treated. At the start of the next century,
things looked very different. Already at least three bacterial species, capable of causing
life-threatening illness (Enterococcus faecalis, Mycobacterium tuberculosis and
Pseudomonas aeruginosa), had become resistant to every one of the 100 antibiotics,
available except for vancomycin (Iwu, 1999). Vancomycin is the antibiotic of last resort
for treatment of resistant infections and within the past year scientists have found strains
of Streptococcus pneumoniae and S. aureus to be resistant to this antibiotic. This is
attested by the spread, with associated deaths, of infection by methicillin-resistant
Staphylococcus aureus and the increased prevalence of drug-resistant S. pneumoniae in
patients suffering from pneumonia. Anti-microbial resistance is driven by inescapable
evolutionary pressures and is therefore predictable and inevitable. The emergence in the
past year of vancomycin-resistant S. aureus an event that has been anticipated for the past
decade with great dread, punctuates this assertion.

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 INTRODUCTION

Table 1.1 The discovery of antibiotics and the date when the resistance was reported
(Guilfoile and Calamo, 2006).

Antibiotics Introduced Resistance Occurred

Sulfonamides 1930s 1940s
Penicillin 1943 1946
Streptomycin 1943 1948
Bacitracin 1945 1953
Chloramphenicol 1947 1959
Cephalosporin 1960s late 1960s
Neomycin 1949 1950
Tetracycline 1948 1953
Erythromycin 1952 1988
Vancomycin 1956 1988
Kanamycin 1957 1966
Methicillin 1960 1961
Ampicillin 1961 1973
Gentamicin 1963 1969
Carbenicillin 1964 1974
Clindamycin 1969 1970
Amoxicillin 1972 1975
Piperacillin 1980 1981
Augmentin 1984 1984
Aztreonam 1984 1985
Imipenem 1985 1985
Ciprofloxacin 1987 1987
Quinupristin-Dalfopr istin 1999 2000
LinezoHd 2000 2002

Due to emergence of drug resistant bacteria, the search for new antibacterial compounds
with improved activity is necessary (Harold and Heath 1992).

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1.2 Need for new, novel antimicrobial agents

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
gives the alarming incidence of antibiotic resistance in bacteria of medical importance;
hence, 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 ah, 1996)
The increasing failure of chemotherapies and antibiotic resistance exhibited by
pathogenic microbial infections agents have led to the screening of several medicinal
plants for their potential antimicrobial activity (Ritch-Rrc et al., 1996; Martins et ah,
2001). Antibacterial properties of various plants parts, such as leaves, seeds and fruits
have been well documented for some of the medicinal plants for the past two decades.

1.3 Search for alternative antimicrobial agents; Plants as a potential source of

antibiotics
The search for antimicrobial agents has continued to be concentrated on higher plants.
The use of medicinal plants and preparations taken from them for the treatment of
infections diseases caused by microbial pathogens has a long history in almost all
cultures (Mitcher et al., 1987). Emetine, quinine, berberine and artemesinin are well
known examples of plant-derived antimicrobial compounds that remain highly effective
instruments in the fight against microbial infections (Schulz et al, 2001). However,
plants have been used as drugs for over millennia by human beings.
Plants historically have served as models in drug development for some major reasons:
The first being that each plant is a unique chemical factory capable of synthesizing large
numbers of highly complex and unusual chemical substances. It has also been estimated
by the World Health Organization (WHO) that about 80% of the population of the
developing countries rely exclusively on plants to meet their health care needs
(Famsworthe/a/., 1985).

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The second reason involves biologically active substances derived from plants have
served as templates for synthesis of phannaceuticals. Such compounds may have poor
phannacological and toxicological profiles. While the reason concerns the fact that highly
active secondary plant constituents have been instrumental as phannacological tools to
evaluate physiological processes (Famsworth, 1984). There are numerous illustrations of
plant-derived drugs.
Despite the expense involved in the development of a drug today, at least US$230
million and a time span between 1 0 - 2 0 years (Famworth, 1984), nature remains the
most reliable and most important source of novel drug molecules. Nature provides 80%
of all pharmacological and therapeutic lead compounds and the NCI estimates that over
60% of the compounds currently in pre-clinical and clinical development in its
laboratories are of natural origin. Thus higher plants remain an important and reliable
source of potentially useful chemical compounds not only for direct use drugs, but also as
unique prototypes for synthetic analogues and as tools that can be used for a better
understanding of biological processes (Famsworth, 1984).
There are numerous illustrations of plant-derived drugs. Some selected examples are
presented in Table 1.2.

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 INTRODUCTION

Tabic -1.2 List of selected drugs originally derived from plants (Taylor, 2000 & Safowora,
1982)

Plant Drug Use

Aloe vera Resin Purgative
Cinchona succirubra Quinine Bitter tonic
Claviceps purpurea Ergometrine Uterine stimulant
Digitalis purpurea Digoxin Myocardial stimulant
Hammamelis virginiana Gallic acid Astringent
Papaver somniferum Morphine Analgesic
Penicillium Penicillin Antibiotic
Rauwolfia serpentina Reserpine Antihypertensive
Slix alba Salicin Analgesic
Cassia species Sennoside A, B Laxative
Berberis vulgaris Berbine Baccillary dysentery
Ephedra sinica Ephedrine Sympatho mimetic
Vasicinece cerebral Vinca minor Cerebral stimulant
Chondodendron tomentosum Tubocuranine Skeletal muscle relaxant

From the numerous published research articles and similar studies it is evident that
antimicrobial agents from higher plants are plentifiil and that there are a large amount of
unexploited natural sources of antimicrobial compounds in higher plants. A wide variety
of higher plants have been investigated for the presence of antibacterial (Rabe and Van
Staden, 1997; Eloff, 2001, Kotze and Eloff, 2002), antifungal (Afolayan et al, 2002;
Motsei et al, 2003) antimycobacterial (Seidel and Taylor, 2004) and antiplasmodial
(Clarkson et al., 2004) activities. These studies have resulted in the isolation and
characterization of a number of compounds with activity against a range of microbial
pathogens, many of which are important human pathogens.
In India, the ayurvedic system has described a large number of such medicines based on
plants or plant product and the determination of their morphological and pharmacological

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or pharmacognostical characters can provide a better understanding of their active

principles and mode of action. However a large number of tropical plants have not been
studied in detail for their chemical constituents, phannacological properties of the
extracts and their phannacognostical characterization including DNA sequencing etc.
Today, there are 121 pure chemical substances extracted from about 130 species of
higher plants used in the modem pharmacopoeias throughout the world. Out of these, 89
plant derived drugs, currently used in modem medicine, were originally discovered
through the study of traditional cures and folk knowledge of indigenous people (Bhattarai
and Karki, 2004). Some of the studied antimicrobial substances, obtained from the
plants are still required to further study of their extraction, fractionation, purification,
concentration etc. to obtain a strong antimicrobial nontoxic fraction, so that in future it
would be possible to develop an antimicrobial dmgs from plants which may replace the
over use and misuse of antibiotic. Considering the rich diversity of Indian medicinal
plants we selected the three plants commonly used in our daily life and one wild plant for
the research work.

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 INTRODUCTION

Clove

1|F . ^^

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 INTRODUCTION

1.4 CLOVE:

Kingdom: Plantae
<
Division: Magnoliophyta
Class: Magnoliopsida
Order: Myrtales
Family: Myrtaceae
Genus: Syzygium L.
Species: S. aromaticiim L.
Popular Name(s): Laung
Parts Used: Floral buds (http://'en.wikipedia.org/wiki/clove)
Clove is the dried unopened flower bud obtained from a handsome, middle-sized,
evergreen tree. The tree has a straight trunk and grows up to a height of 10 to 12 meters.
Cloves come under the group of spices and condiments. It is commercially in demand
today as a food item, and as a medicine ingredient. Clove is also extensively used in
soaps, lotions, and toothpastes, and other topical applications. It is also used in
perfumery, pharmaceutical and flavoring industries.
y An analysis of clove shows it to consist of carbohydrates moisture, protein, volatile oil,
non-volatile ether extract (fat), and crude fiber besides mineral matter, ash insoluble in
hydrochloric acid, calcium, phosphorus, iron, sodium, potassium, thiamine, riboflavin,
niacin, vitamins C and A. Its calorific value is 430.
Plant constituents
The compound eugenol is responsible for most of the characteristic aroma of cloves.
Eugenol comprises 72-90% of the essential oil extracted from cloves, and is the
compound most responsible for the cloves' aroma. Other important essential oil
constituents of clove oil include acetyl eugenol, beta-caryophyllene and vanillin;
crategolic acid; tannins, gallotannic acid, methyl salicylate (painkiller); the flavonoids
eugenin, kaempferol, rhamnetin, and eugenitin; triterpenoids like oleanolic acid,
stigmasterol and campesterol; and several sesquiterpenes.

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 [NTRODUCTION

Uses & Benefits of cloves

• Healing Power and Curative Properties of Clove
Cloves have many medicinal virtues. They are stimulant. They are useful in counteracting
spasmodic disorders and in relieving flatulence. They help stimulate sluggish circulation
and thereby promote digestion and metabolism. In the Indian system of medicine, cloves
are used in various conditions either in the form of a powder or a decoction made from
them. Clove oil contains ingredients that help stabilize blood circulation and regulate
body temperature. Clove oil, applied outwardly, has stimulating effects on the skin,
producing heat and redness.
• Digestive Disorders
Cloves promote enzymatic flow and boost digestive functioning. They are used in various
fonns of gastric irritability and dyspepsia. Licking the powder of fried cloves mixed with
honey is effective in controlling vomiting. The anesthetic action of clove numbs the-
guUet and stomach and stops vomiting.
• Cholera
Cloves are very useful for treating cholera. About 4 grams of cloves are boiled in 3 litres
of water until half the water has evaporated. This water, taken in draughts, will check
severe symptoms of the disease.
• Respiratory disorders
Clove oil has a cooling and anti inflammatory effect, and thereby clears the nasal
passage. This expectorant is useful in various respiratory disorders including coughs,
colds, bronchitis, asthma, sinusitis, and tuberculosis. Chewing a clove bud eases sore
throats. Clove is an effective remedy for asthma. A teaspoon of decoction prepared by
boiling 6 cloves in 30 ml of water can be taken with honey thrice daily as an expectorant.
• Teeth Disorders
The most prominent use of clove oil is in dental care. The germicidal properties of the oil
make it very effective for relieving dental pain, tooth ache, sore gums and mouth ulcers.
Clove oil contains the compound eugenol, which has been used in dentistry since
numerous years. Gargles with diluted clove oil help in easing the throat. The
characteristic smell of clove oil helps in removing bad breath. Clove is also effective
against cavities. Traditionally, in India, clove oil was added to a small cotton ball and put

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 INTRODUCTION

at the end of the tooth which has cavity daily before sleeping. The cavity would vanish in
a few days. As a result, clove oil is added to numerous dental products and medications,
including, mouth wash and tooth pastes.

• Earache
A clove sauted in a teaspoon of sesame ( til) oil and 3 to 5 drops of this (warm) oil put
into the ear can cure earache.
• Headaches
A paste of clove and salt crystals in milk is a common household remedy for headaches.
Salt, as a hygroscopic agent, absorbs fluid and decreases tension.
• Infections:
Due to its antiseptic properties, clove oil is useful for wound, cuts, scabies, athlete's foot,
fungal infections, bruises, prickly heat, scabies, etc. It can also be used for treating insect
bites and stings. Clove oil is very strong in nature and hence should be used in diluted
forni. Further, it should not be used on sensitive skin.
• Skin Care: Clove oil is often recommended for skin care, especially to acne patients.
• Stress: Clove oil is aphrodisiac in nature and hence serves as an excellent stress reliever.
It has a stimulating effect on the mind and removes mental exhaustion and fatigue. When
taken internally, in appropriate amounts, it refreshes the mind. Clove oil also induces
sleep and is helpful to insomnia patients. It is useful for treating mental problems such as
loss of memory, depression and anxiety.
• Nausea: Clove oil is helpftil in case of nausea and vomiting and is often used for
pregnancy related vomiting.
• Blood Circulation: Clove oil is increases your body metabolism by increasing blood
circulation and reducing body temperature.
• Blood Purification: Clove oil also helps in purifying the blood.
• Diabetes: Along with blood purification, clove oil also helps in controlling the blood
sugar levels and hence is useful to diabetics.
• Immune System: Both clove and clove oil are useful for boosting the immune system.
Its antiviral properties and ability to purify blood increases your resistance to diseases.

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 INTRODUCTION

Premature Ejaculation: Research has indicated that clove can be useful for treating
premature ejaculation. Further research needs to be carried out to confirm these results.
Sty: Clove and clove oil is a very effective home remedy for treating sty. Sty is an
inflammation on the eyelash and is a very imtating condition. Sty is not only painful, but
also causes difficulty in the proper functioning of the eye.

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 INTRODUCTION

-<,

Cinnamon

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 INTRODUCTION

1.5 CINNAMON:

Kingdom : Plantae
Division : Magnoliophyta
Class : Magnoliopsida
Order : Laurales
Family : Lauraceae
Genus : Cinnamomum
Popular Name(s): Tvak, Dalchini, Daruchini Kaniva, Vazhana, Tamalapatra.
Parts Used: Bark, (http://en.wikipedia.org/wiki/cinnamon)

It's one of the oldest known spices and it's the bark of the cinnamon tree that is dried and
rolled into sticks, which is also called quills. The characteristic flavor and aroma of
cinnamon comes from a compound in the essential oil of the bark called
cinnamonaldehyde.
Plant Constituents:
{+)- cinnamaldehyde (65-80%), lesser percentages of other phenols and terpenes,
eugenol, trans-cinnamic acid, hydroxycinnamaldehyde, o-methoxycinnamaldehyde,
cinnamyl alcohol and its acetate, limonene, alpha-terpineol, tannins, mucilage, oligomeric
procyanidins, gum, mannitol, trace amounts of coumarin.

Uses & Benefits of Cinnamon

• Anti-Microbial Activity
Cinnamon's essential oils act as an "anti-microbial agent" which is used to stop the
growth of bacteria as well as fungi. Essential oil of Cinnamon used against the Gram-
positive bacteria Enterococcus faecalis. Bacillus cereus, and also against the gram
negative bacteria like E. coli, Aspergillus niger etc. Ground cinnamon is used in diarrhea
and dysentery; for cramps of the stomach, gastric irritation; for checking nausea and
vomiting; used externally in toothache, neuralgia and rheumatism.
• Antidiabetic Activity
In Ayurvedic and in folklore medicine, cinnamon is used for the treatment of diabetes.
Researcher demonstrates that cinnamon reported to reduce the blood glucose level in

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 INTRODUCTION

non-insulin dependent diabetes. Consummg half teaspoon of the spice each day helps in
reducing blood sugar, cholesterol and triglyceride levels by as much as 20%.
• Anti-oxidant property
Due to having anti-oxidant property cinnamon & its essential oil have been used as
preservative in food.
• Anti-Clotting Actions
Cinnamaldehyde (also called cinnamic aldehyde) has been well-researched for its effects
on blood platelets. Platelets are constituents of blood that are meant to clump together
under emergency circumstances (like physical injury) as a way to stop bleeding, but
under normal circumstances, they can make blood flow inadequate if they clump together
too much. The cinnaldehyde in cinnamon-spice helps prevent unwanted clumping of
blood platelets.
• Calcium and Fiber Improve Colon Health and Protect Against Heart Disease
In addition to its unique essential oils, cinnamon is an excellent source of the trace
mineral manganese and a very good source of dietary fiber, iron and calcium. The
combination of calcium and fiber in cinnamon is important and can be helpful for the
prevention of several different conditions. Both calcium and fiber can bind to bile salts
and help remove them from the body. By removing bile, fiber helps to prevent the
damage that certain bile salts can cause to colon cells, thereby reducing the risk of colon
cancer. In addition, when bile is removed by fiber, the body must break down cholesterol
in order to make new bile. This process can help to lower high cholesterol levels, which
can be helpful in preventing atherosclerosis and heart disease.
• Local Actions:
It is blood alterative, stimulant and analgesic. Mouth refresher and gives strength to
gums. Hence, a piece of its bark should be chewed to stop vomiting and nausea. A swab
made in its oil is held between teeth for dental caries. A thin paste should be applied to
the skin in skin diseases. Applicafion of oil cleanses and heals a tubercular ulcer.
• Other applications:
l.It is widely used in cakes and other baked recipe, along with milk and rice puddings,
chocolate dishes and fmit desserts, especially apples and pears.
2. The spice is used in Indian curries and forms a part of the garam masala.

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 INTRODUCTION

3. Cinnamon is also used to spice mulled wines, creams and syrups.

4. It is used to treat nausea, tlatulence and diaiThea.
5. Chewing and swallowing a small pinch of powdered cinnamon is helpful in treating
cough accompanied by spitting of whitish phlegm. The remedy is also helpful to people
having cold feet and hands at night.
6. The spice is significant in treating loss of appetite and indigestion.
7. It is used in flatulent dyspepsia, dyspepsia with nausea, intestinal colic and digestive
atony associated with cold and debilitated conditions.
8. Cinnamon helps in relieving vomiting, due to its mild astringency.
9. The spice has the ability to stop medication-resistant yeast infections.
10. It reduces the proliferation of leukemia and lymphoma cancer cells.
11. Cinnamon has an anti-clotting effect on blood.
12. Smelling cinnamon boosts cognitive function and memory.
13. People suffering from arthritis should be given half a teaspoon of cinnamon powder,
mixed with one tablespoon of honey every morning, before breakfast. It relieves the pain
and the patient becomes capable of walking without pain within one month.
14. The spice is commonly used in Middle Eastern and North African dishes for flavoring
Iamb tagines or stuffed aubergines.

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 INTRODUCTION

Datura

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 INTRODUCTION

1.6 DATURA:

Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Solanales
Family: Salanaceae
Genus: Datura L.
Species: Datura stramonium L.
Popular Name(s): Jimson Weed, Stink weed, Mad Apple, Thorn Apple Stramonium,
Dhatura Tatula, Datura Seeds
Parts Used: Seeds(http://en.\vikipedia.org/wiki/datura)
Datura is a bushy plant growing up to one meter in height. It has large egg shaped leaves,
very large white flowers and egg shaped fruits, covered with prickles. The dried leaves,
top portion of the flower and seeds of the plant constitute the dmg. Datura is referred to
in ancient Indian literature as shivashekhra because the flowers are believed to be
associated with Lord Shiva. It was known to the ancient Hindu physicians. They regarded
the drag as an intoxicant, with emetic, digestive and healing powers.
PlantConstituents-
Datura leaves contain a far larger proportion of alkaloids. The alkaloid consists chiefly of
hyoscyamine, associated with atropme and hyoscine (scopolamine). Malic acid is also
present.
Uses & Benefits of Datura
• Respiratory disorders
Datura is very useful 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 used m
the treatment of Parkinsonism and Hemorrhoids. Its leaves, applied after roasting, are
useful in relieving pain.
• Malaria
Datura fruit 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

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 INTRODUCTION

composed of cloth and clay. It should be parched in to 12 grams of cow dung cakes.
When the fire gets extinguished and the pot is cold, the burnt fruit should remove,
powdered and kept safely in a phial for use in malarial fever.
• Heart Disorders
The herb is valuable remedy for heart disorders. It relieves cardiac pains, distress,
palpitation and aortic disorders.
• Impotency
Datura is useful in impotency. The seeds of 15 ripe fruits should be extracted and boiled
in eight kilograms of cow's milk on gentle fire. This milk should then be made into curd
in the usual way and churned the next morning to extract butter which is stored m a
broad-mouthed bottle. This butter, massaged every morning and evening on the penis and
the spine, will provide the desired effect. It is also used as an oral medicine. Four grains
with betel leaf can be taken. The use of this butter both externally and internally in this
way will gradually promote health and vigor and restore absolute fitness in the body.
• Baldness
A preparation made from datura seeds with other ingredients is useful in patchy baldness.
A paste made with datura seeds, liquorice, saffron and milk cream is heated in coconut
oil thoroughly fill the solid mass converts to a charred powder. The oil when applied on
bald patches has the power to sfimulate hair growth. As datura is poisonous, hands should
be washed thoroughly after handling the oil. The mixture should not be used on the scalp
of children.
• Problem of Breast Milk Secretion
The herb is highly beneficial in checking secretion of breast milk. In case of an
unfortunate death of a newly bom baby, accumulation of milk in the breasts of the mother
poses a problem, causing severe pain. In such a case, wanned 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.
• Other applications
• The bitter narcotic plant relieves pain and encourages the healing process. The plant has a
very long history of being used as herbal medicine.

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 INTRODUCTION

Its leaves, flowering tops and seeds have anodyne, antiasthmatic, antispasmodic,
hallucinogenic, hypnotic, mydriatic and narcotic properties. The seeds of the plant are the
most active medicinally.
Datura is internally used for treating giddiness, dry mouth, hallucinations and coma.
It's leaves, containing hyoscyamine and atropine, can be used as an immensely powerful
mind-altering drug.
The seeds of datura are analgesic, anthelmintic and anti-inflammatory and as such, they
are used in the treatment of stomach and intestinal pain that results from womi
infestation, toothache, and fever from inflammations.
The juice of its fruit is applied to the scalp, to treat dandmff and falling hair.
The growing plant works as an insect repellant, which protects neighboring plants from
insects.

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 INTRODUCTION

Tulsi

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 INTROnUCTlON

Kun'/Jun^i : Plamae

Class : N'];ig!i.iiiO[)iiUia
Order : Lanuaies
Ftiin:'•: •Lai!V.icei;c
Genus ; Qjunum (http://en.wikipedia.org/wiki/tulsi)

Apart from its religious significance it is of great medicinal significance, and is a prime
herb in Ayurvedic treatment. Tulsi has been widely known for its health promoting and
medicinal value for thousands of years. Commonly called sacred or holy basil, it is a
principal herb of Ayurveda, the ancient traditional holistic health system of India, Tulsi is
known as "The Incomparable One", "The Mother Medicine of Nature", and "The Queen
of Herbs". Vagbhata, Nighantu Adarsha, Agnipurana, Vishnupurana, Padmapurana,
Garudapurana and Tulsi Kavacham, written between 500 BC and 1200 AD, the plant
Tulsi is continuously mentioned as one of the main pillars of herbal medicine.
Chemical Constituents:
A variety of biologically active compounds have been isolated from the leaves including
ursolic acid, apigenin and luteolin. Essential oil of Tulsi has antibacterial, antifungal and
antiviral properties and possesses 100% larvicidal activity against the Culex
mosquitoes. Tulsi contains keenol, ascorbic acid, carotene and alkalide.
Uses & Benefits of Tulsi
Tulsi has been widely known for its health promoting and medicinal value for thousands
of years. In Ayurvedic medicines it is used as a whole plant, the leaves, root, stem and
the essential oil. Tulsi used in medicines as-
Pharmacological Effects - In traditional Ayurvedic system of medicine, several
medicinal properties have been attributed to this plant. Recent phannacological studies
have established the anabolic, hypoglycemic, smooth muscle relaxant, cardiac depressant,
antifertility, adaptogenic and immunomodulator properties of this plant.

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 INTRODUCTION

Antimicrobial effects - Essential oil of Tuisi have antibacterial, antifungal and antiviral
properties. It inhibits the growth of E. coli. B.anthracis, M.liiherciilosis etc. It's
antitubercular activity is one-tenth the potency of streptomycin and one-fourth that of
isoniazid. Preperations containing Tulsi extract significantly shorten the course of illness,
clinical symptoms and the biochemical parameters in patients with viral hepatitis and
viral ejicephalitis.
Antimalarial effects Essential oil
of Tulsi has been reported to possess 100% larvicidal activity against the Culex
mosquitoes. Trials have shown excellent antimalarial activity of Tulsi. It's extracts have
marked msecticidal activity against mosquitoes. It's repellant action lasts for about two
hours
Anti allergic and Immunomodulator effects - Essential oil of Tulsi was found to have
anti-allergic properties. When administered to laboratory animals, the compound was
found to inhibit mast cell degranulation and histamine release in the presence of allergen.
These studies reveal the potential role of Ocimum sanctimi extracts in the iTianagement of
immunological disorders including allergies and asthma.
Antistress/Adaptogenic effects - Extracts from the plant have been found to reduce
stress.
Antifertility effect - One of the major constituents of the leaves, ursolic acid has been
reported to possess antifertility activity in rats and mice, this effect has been attributed to
its antiestrogenic effect which may be responsible for arrest of spennatogenesis in males
and inhibitory effect on implantation of ovum in females. This constituent may prove to
be a promising antifertility agent devoid of side effects.
Anti diabetic effect - A randomized, placebo-controlled cross-over single blind trial on
40 human volunteers suffering from Type II diabetes was perfonned. During the four
week trial, subjects alternately received a daily dose of 2.5 g of Tulsi leaves powder or a
placebo for two week periods. The results showed 17.6 % reduction in fasting blood
glucose and 7.3% decline in postprandial blood glucose on treatment v/ith Tulsi as
compared to the blood glucose levels during treatment with placebo.
For Heart ailments - As 'Tulsi' (basil) has a positive effect over blood pressure and also
a detoxicant, its regular use prevents heart attacks. A tonic may be prepared by mixing 1

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 INTRODUCTION

gm of dry 'Tulsi' leaves with a spoonful of butter and some candy sugar or honey. Take
twice a day; ilrst thing m the morning and before going to bed at night. The drini<:ing of
Tulsi-leaf tea keeps the blood pressure even
In S>vine Flu: Tulsi can not only keep the dreaded Swine flu or HlNl flu at bay but also
help in fast recovery of an afflicted person, Ayurvedic practitioners claim. "The anti-flu
property of Tulsi has been discovered by medical experts across the world quite recently.
Tulsi improves the body's overall defence mechanism including its ability to fight viral
diseases. Tulsi can control swine flu and it should be taken in fresh fomi. Juice or paste
of at least 20-25 medium sized leaves should be consumed twice a day on an empty
stomach.
Other effects - The leaves in the fonn of a paste are used in parasitical diseases of the
skin and also applied to the finger and toe nails during fever when the limbs are cold. The
juice of the leaves is given in catairh and bronchitis in children. The plant is said to have
canninafive, diaphorefic and stimulant properties. A decoction of the plant is used for
cough and also as mouth washes for relieving tooth ache. It is good for headache,
convulsions, cramps, fever and cholera.
The drinking of Tulsi tea keeps one free from cough and colds and other ailments
associated with 'cough ' in the body. This Tulsi tea is an instant pick-me-up (energy
drink).

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 INIRODUCTION

1.8 Microorganisms used for study:

Staphylococcus aureus meaning the "golden grape-cluster berry," is a facultative
anaerobic Gram-positive coccal bacterium. It is frequently part of the skin flora found in
the nose and on skin, and in this manner about 20% of the human population is long-term
carriers of 5. aureus.
S. aureus is the most common species of staphylococci to cause Staph mfections. One of
the reasons for this is a carotenoid pigment staphyloxanthin that is responsible for the
characteristic golden colour of 5". aureus colonies. This pigment acts as a virulence factor,
with an antioxidant action that helps the microbe evade death by reactive oxygen species
used by the host immune system.
S. aureus can cause a range of illnesses from minor skin infections, such as pimples,
impetigo, 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, respiratoiy, bone, joint, endovascular to wound infections. It is still one
of the five most common causes of nosocomial infections, often causing postsurgical
wound infections. Each year, some 500,000 patients in American hospitals contract a
staphylococcal infection.
Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose
fennenting, facultative anaerobic, rod shaped bacterium found in the nonnal flora of the
mouth, skin, and intestines. It is clinically the most important member of the Klebsiella
genus of Enterobacteriaceae.
In recent years, Klebsiella have become important pathogens in nosocomial infections.
Klebsiella can cause different types of infections, including urinary tract infections,
pneumonia, bloodstream, infections, wound or surgical site infections and meningitis.
Klebsiella is a significant cause of healthcare associated infections (HAIs). Increasingly,
Klebsiella bacteria have developed antimicrobial resistance, most recently to the class of
antibiotics known as carbapenems. Klebsiella bacteria are normally found in the human
intestines (where they do not cause disease) and are also found in human stool.
Escherichia coli (commonly abbreviated E. coli) is a Gram-negative, rod-shaped
bacterium that is commonly found in the lower intestine of wanii-blooded organisms.

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 INTRODUCTION

Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in
humans, and are occasionally responsible for product recalls. The hannless 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.
People can contract an E. coli infection by drinking contaminated water, eating fruit or
vegetables that have been watered with contaminated water, drinking unpasteurized milk,
or eating undercooked ground meat. The E. coli infection can also be caught by coming
into contact with others who are infected or by working in environments where one might
come into contact with human or animal feces, such as fanns, day care centers, nursing
homes, or hospitals. The most common way to contract an E. coli infection is by eating
hamburgers that are not fully cooked. The symptoms of E. coli infection are primarily
acute dian-hea that may or may not be bloody, severe stomach cramps, bloating, and gas.
While these are the most prevalent symptoms, many people infected with E. coli might
also experience continuous abdominal pain, loss of appetite, fever, and in rare cases,
vomiting.
There are many types of E. coli, and most of them are harmless. But some can cause
bloody diaixhea. These are called enterohemon'hagic E. coli (EHEC). One common type
is called E. coli 0157:H7. In some people, this type of £". coli may also cause severe
anemia or kidney failure, which can lead to death. Other strains of E. coli can cause
urinary tract infections or other infections.
Proteus vulgaris is a rod-shaped. Gram negative bacterium that inhibits the intestinal
tracts of humans and animals. It can be found in soil, water and fecal matter. It is grouped
with the enterobacteriaceae and is an opportunistic pathogen of humans. It is known to
cause urinary tract infections and wound infections. P. vulgaris occurs naturally in the
intestines of humans and a wide variety of animals; also manure soil and polluted waters.
Proteus can cause urinary tract infections and hospital-acquired infections. Proteus is
unique, however, because it is highly motile and does not forai regular colonies. Instead,
Proteus fonns what are known as "swarming colonies" when plated on non-inhibitory
media. The most important member of this genus is considered to be P.mirabilis, a cause
of wound and urinary tract infections. Fortunately, most strains of P. mirabilis are
sensitive to ampicillin and cephalosporins. Unlike its relative, P. vulgaris is not sensitive

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 INTFWDL'CTION

to these antibiotics. However, this organism is isolated less often in the laboratory and
usually only targets immunosuppressed individuals. P. mirahilis and P. vulgaris can be
differentiated by an indole test for which only P. vulgaris tests positive.

The objective of the present research work was to find a scientific rationale for the use of
traditional medicine, against some pathogenic microorganisms, by investigating the
plants for their antimicrobial activity, which the traditional healers claim they have.
Furthennore mixing of the isolated compounds in different ratio and analyzing the
additive antimicrobial activity of isolated compounds and then Preparation of herbal
fomiulation by mixing of isolated component from different plant extracts.

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