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ANTIMICROBIAL ACTIVITY OF SOME MEDICINAL PLANTS

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					                    INTRODUCTION
The present project was undertaken with the analysis of the

antimicrobial activity of the some aromatic plants or herbs.

          The science of ayurveda is a unique holistic system, based

on the interaction of body mind and spirit in ayurveda, the origin of all

aspects of existence is pure intellect or consciousness. The treatment

of Ayurveda is based on Indian herbs, which has a healing energy.

Ayurveda has focused on the various aspects of herbs and there

practice in our day to day life. Ayurveda is prevalent in India since

2000 B.C.and it means the science of life and drives the medicine form

nature. Ayurveda is a Sanskrit word. Ayur mean “life” and Veda mean

“knowledge”.

          Herbs are a plant or plant extract including leaves, stem,

seed, and flower, bark, which are bestowed with nourishing and

healing elements. In herbal medication herbs are used for their

therapeutic and medicinal value.

        Higher and aromatic plants have traditionally been used in folk

medicines as well as to extend the self life of foods showing inhibition

against bacteria, fungi and yeast. Most of there properties are due to

essential oils produced by their secondary metabolism. Essential oils

are   extracted   from   several   plant   species   and   able   to   control

microorganism related to skin, dental cares, and food spoilage,


                                                                            1
including Gram-negative and Gram-positive bacteria.

              Many countries have maintained research programs to

screen traditional medicines for antimicrobial activity, as is the case of

the Indian, Africa and Italy etc.

            Plants from India biomes have also been used as natural

medicines by local population in the treatment of several tropical

diseases,    including   malaria,   leishmaniasis,   fungal   and   bacterial

infections. Extracts, fractions and compounds isolated from some

aromatic plants were able to control one or more microorganisms.

Aromatic plants and spices have great importance for food, cosmetics,

and pharmaceutical industries.

             Their use has taken place since ancient time, and despite

many of them were substituted by synthetic ones, the demand for

natural products is increasing. Leaves from Mentha and Lemon Grass

have been used as spices and teas after drying. While the essential oil

is utilized in cosmetics pharmaceuticals. The essential oils contents in

different species is influenced by genetic material, culture conditions

environment and finally, by crop and post crop processes.



Objective:     The object of the present investigation was to study

invitro antimicrobial activity of the extracts from two aromatic

plants viz. Mentha and Lemon Grass.




                                                                           2
            REVIEW OF LITRATURE
                                 HERBS

An herb is a plant with no woody stem above ground distinguished
from a tree or a shrub. However, that is meaning as per botany. In
general, any part of the vegetable species that can be used for
medicine, cosmetic culinary or such purposes are known as herbs. The
roots, leaves, bark, fruits, flowers, stem or any part of the plant can
be used for such purposes.


             Also an herb is valued for flavor, scent or other qualities.
Herbs are used in cooking and for spiritual purpose. Herbs are the
highest quality food known to man containing vitamin, mineral and
trace element in natural balance and harmony (Ratnakar, 1997).



                       HISTORY OF HERBS
The use of plant for medicinal purpose is as old as our civilization. The
first known written record of creative plants was of Sumerian herbal of
2200 B.C. In the fifth century B.C, the Greek doctor Hippocrates, list
out some 400 herbs in common use. Dioscorides in the first century
A.D wrote an herbal plant by using 600 plants which ultimately
become the base for many later works.
            Herbs have been used for uncounted time for various
purposes like healing the sick. Most of the people still continue to use
herbs to benefit their bodies. 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
effect of herbs on the body; mind and emotion for e.g. flower were
utilized to attract love. Fragrant plants were work to heal the body and
give a sense of well being (Kapoor, L.D., 1990).
                                                                         3
                         PRESENT STATUS
          Herbalists today believe to help people to build their good
health with the help of natural resources. Herbs are considered to be
food rather than medicine because they are complete all natural and
pure as nature intended. When herbs are cleansed, it gets purified
itself (Bisset N.G., 1994).


For thousand of years, human have been used the herbs as following -
          In cooking for flavorings foods.
          As perfume.
          As disinfectant.
          To protect us against germs.
          As medicine to heal when we are sick.


                              INDIAN HERBS
The ancient history of Indian herbs depict that the Rig Veda, the oldest

document of human knowledge carries the evidence of the use of

medicinal plants in the treatment of various diseases. The tradition of

Ayurveda originated around 5000 years ago. The basic concept of

Ayurveda is to maintain the balance of life with the environment along

with the usage of some herbs that are widely cultivated in India for its

medicinal usages. The ancient book of Charka, which was written

around 1000 B.C., concentrates on medicine and depicts that in

ancient times the practitioners used to treat diseases with the help of

the decoctions from different herbs.

The Indian herbs are considered helpful in building good health with

the help of natural sources (Thakur and Mandal, 1992).


                                                                       4
The Indian herbs are used as culinary herbs, Aromatic herbs ,

ornamental herbs and medicinal herbs or in some cases even spiritual

usage .In medicinal and spiritual use any of    the parts of the   plant

might be considered herb including leaves, roots, flowers, seeds, resin,

root bark, inner bark, barries, and sometimes the pericarp or other

portions of the    plant . Culinary herbs are distinguished from

vegetables even spices; they are used in small amounts and provide

flavor rather than substance to food.

        The other usage of Indian herbs is as the Aromatic herbs

which are pleasant smelling flowers or foliage. Oils from these

aromatic herbs can be used to produce perfumes, toilet cologne, oils

and are some times used as essential oils (Basu, 1971).




                                                                       5
                 Types of Ayurvedic Herbs

Ayurveda is the ancient science of life, which aims to promote the
healthy lifestyle, free of diseases. The herbs have been in used
since the ancient times, when people were not familiar with allopath
and other form of treatments. The Ayurvedic herbs can be classified
into five types: according to origin, according to habitat, according
to various actions, according to actions on doshas and according to
there uses (Chunekar, 1997).


                     Classification of Herbs
According to Rastogi et al, (1995) Herbs are classified in many
ways. Some of them are: ----


   According to the usage.
   According to the active constituents.
   According to the period of life.


1) According to the Usage, the herbs are classified into four
    parts:   Medicinal   herbs,   Culinary   herbs,   Aromatic   herbs,
    Ornamental herbs.


a) Medicinal herbs:
                     Medicinal herbs have curative powers and are
used in making medicines because of their healing properties.


b) Culinary herbs:
                   Culinary herbs are probably the mostly used as
cooking herbs because of their strong flavors like mint, parsley,
basil.
                                                                      6
c) Aromatic herbs:
                    Aromatic    herbs   have     some   common      uses
because of their pleasant smelling flowers or foliage. Oils from
aromatic herbs can be used to produce perfumes, toilet water and
various scents. For e.g. mint, rosemary, basil etc.


d) Ornamental herbs:
                  Ornamental herbs are used for decoration because
they have brightly colored flowers and foliage like lavender, chives.


2) According to the active constituents present in them, the
herbs are divided into five major categories: Aromatic (volatile oils)
Astringents (tannins), Bitter (phenolic compounds, saponins, and
alkaloids),   Mucilaginous   (polysaccharides)    and   Nutritive   (food
stuffs).


a) Aromatic herbs:
                     Aromatic herbs, the name are a reflection of
the pleasant odor. They are used extensively both therapeutically
and as flavorings and perfumes. Aromatic herbs are divided into
two subcategories: stimulants and nervines.


Stimulant herbs: increase energy and activities of the body or
organs, and most often affect the respiratory, digestive, and
circulatory systems. E.g. fennel, ginger, garlic, lemon grass.


Nervine herbs: are often used to heal and soothe the nervous
system, and often affect the respiratory, digestive, and circulatory
systems. E.g. ginger, catnip.



                                                                        7
b) Astringent herbs:
                Astringent herbs have tannins, which have the
ability to precipitate proteins, and this tightens, contracts or tones
living tissue, and helps to halt discharges. They affect the digestive,
urinary, and circulatory systems, and large doses are toxic to the
liver. They are analgesic, antiseptic, antiabortive, astringent,
homostatic, and styptic. For e.g. peppermint, red raspberry.


c) Bitter herbs:
                Bitter herbs are named because of the presence of
the phenols and phenolic glycosides, alkaloids or saponins and are
divided into four subcategories: laxative herbs, diuretic herbs,
saponin-contaning herbs and aloaloid-contaning herbs.


Laxative bitter herbs: Include alterative, antipyretic, purgative,
hepatonic, vermifuge and blood purifier. For e.g. aloe, cascara,
licorice, pumpkin, senna, yellow dock, barberry, safflowers and
golden seal.


Diuretic herbs: Induce loss of fluid from the body through the
urinary system. The fluids released help cleanse the vascular
system,   kidneys,   and   liver.   They   are   alterative,   antibiotic,
antipyretic, antiseptic and blood purifier in nature. For e.g.
asparagus, blessed, burdock, butcher’s broom, corn silk, dandelion,
dog grass, grapevine and parsley.


Saponin-containing herbs: Are known for their ability to produce
frothing or foaming in solution with water. The name saponin comes
from the Latin word for soap. They emulsify fat soluble molecules in
the digestive tract, and their most important property is to enhance

                                                                        8
the body’s ability to absorb other active compounds. Saponins have
the ability to effectively dissolve the cell membranes or red blood
cells and disrupt them.


d) Mucilaginous herbs:
                      Mucilaginous herbs derive their properties from
the polysaccharides they contain, which give these herbs a slippery,
mild taste that is sweet in water. These herbs are most effective
topically as knitting agents, and are also used topically in the
digestive tract. They eliminate the toxins from the intestinal
system, help in regulating it and reduce the bowel transit time.
They are antibiotic, demulcent and detoxifier in nature. For e.g.
althea, aloe, burdock, comfrey, kelp, slippery elm, Irish moss.


e) Nutritive herbs:
                    These herbs derive both their name and their
classification from the nutritive value they provide to the diet. They
are true foods and provide some medicinal effects as fiber,
mucilage, and diuretic action. But most importantly they provide
the nutrition of protein, carbohydrates, and fats, plus the vitamins
and minerals that are necessary for adequate nutrition. For e.g.
rosehips, acerola, apple, asparagus, banana, barley grass, bee
pollen.


f) According to the period of life:
                    Herbs    also   can   be   classified   as   annuals,
biennials, and perennials. Annuals bloom one season and then die.
Biennials live for two seasons, blooming the second season only.
Ones established, perennials live over winter and each season.




                                                                        9
Annual herbs complete their life cycle in one year; start them from
seed. Annual herbs include: anise, basil, borage, calendula, and
chamomile.


Perennial herbs grow for more then one season and include sweet
marjoram, parsley, mint, thyme and chives.


Biennial herbs are plants which live two season and bloom in the
second season only. E.g. caraway, prime rose.




               HERBAL EXTRACTS AND OIL


ESSENTIAL OILS –
Essential or aromatic oil is a concentrated, hydrophobic liquid
containing odoriferous, volatile aroma compounds from plants which
are called aromatic plant. Oil is essential in the sense that it carries a
distinctive scent of the plant. It is free from enzyme and also
microbiologically stable.
        Essential    or   aromatic   oils   are   generally   extracted   by
distillation. They are used in perfumes, cosmetic and bath product for
flavoring food and drinks for medicinal purpose and household cleaning
products (Banerjee and Nigam., 1977).


         Some oils are –
                   Caraway oils
                   Eucalyptus oil
                   Cinnamon oil
                   Peppermint oil
                   Lemongrass oil

                                                                          10
ANTIMICROBIAL ACTIVITY OF SOME MEDICINAL PLANTS


Nature has been a source of medicinal agent for thousand of years and
an impressive number of modern drugs have been isolated from
natural source. Various medicinal plants have been used for years in
daily life to treat disease all over the world. Plants produce a diverse
range of bioactive molecule making them a rich source of different
types of medicine. Plants with possible antimicrobial activity should be
tested against an appropriate microbial model to confirm the activity
and to ascertain the parameter associated with it. The effect of plant
extracts on bacteria has been studied by a large number of
researchers in different part of the world (Ahmed et al., 2001).
              In the present work a few selected medicinal flora were
screened for potential antimicrobial activity.




                                                                      11
PUDINA (Mentha arvensis)
                           12
s an herbaceous perennial plant growing to 10-60cm (rarely to 100cm)
tall. The leaves are in opposite pairs, simple 2-6.5cm long and 1-2cm
broad, hairy and with a coarsely serrated margin. The flowers are pale
purple (occasionally white or pink) in clusters on the stem, each flower
is 3-4mm long (Gupta et al., 1997).


        Field mint, wild mint or corn mint is a species of mint native to
the temperate region of Europe, western and central Asia, east to the
Himalaya and eastern Siberia. It is in flower from May to October and
the seeds ripen from July to October.
Part used -   whole plant, oil


          It has characteristically pure and refreshing odor, pungent
and burning taste. Menthol and methyl acetate are responsible for the
pungent and refreshing odor; they are mostly found in older leaves
and are preferentially formed during long daily sunlight periods.


         The entire plant is antibacterial, antifebrile, it yield an
essential oil and menthol which exert through their rapid evaporation,
a slightly anesthetics and has some local effect. It is effective in
headache, rhinitis, cough and sore throat.




                                                                       13
                    TAXONOMY OF PUDINA -
                             (Mentha arvensis)


                  Kingdom             -           Plantae
                  Division            -           Angiosperm
                  Class               -           Eudicots
                  Order               -           Lamiale
                  Family              -           Lamiaceae
                  Genus               -           Mentha
                  Species             -           arvensis



           Leaves raw or cooked. A reasonably strong mint flavor with
a slight bitterness. It is used as a flavoring in salad or cooked foods. A
herb tea is made from the fresh or dried leaves. An essential oil from
the plant is used as a flavoring in sweets and beverages. The leaves
contained about 0.2% essential oil.


         Corn mint like many other members of this genus is often
used as domestic herbal remedy; it is used for antiseptic properties
and its beneficial effect on the digestion. The whole plant is
anesthetics,   antiseptic,    antiphagistic,   antispasmodic,   aromatic,
stimulant and stomachic. The tea made from the leaves has
traditionally been used in the treatment of fever, headaches, digestive
disorder and various minor ailments. The leaves are harvested as the
plant comes into flower and can be dried for later use. The plant is
used as an insect repellent. Also used to form menthol crystals, in
flavoring toothpaste, mouthwashes and pharmaceuticals.




                                                                       14
Oil is good for the nervous system acting as a regulator and sedative.
Menthol      is   well   known    as   a   cardiac   tonic   in   pharmaceutical
preparation. It is a good blood cleanser because it is antiseptic and
anti bacterial it can be used in swollen gums, mouth wash and mouth
ulcer. Philips and Foy.N.
          The essential oil of peppermint (up to 2.5% in the dried
leaves) is mostly made up from menthol (50%), menthone (10 to
30%), menthyl Easters (up to 10%) and further monoterpene
derivative        (pulegone,     piperitone,    menthofurane)        Traces   of
jasmine(0.1%) improves the oil quality remarkably(Vyas et al.,1979).


                   Antimicrobial activity of Mentha:


Several studies indicate that the essential oils of the Mentha possess
biological activity against several bacteria.
           According to Ohno et al., (1995), there are differences in the
activity of oil to other subproducts, due to the structure activity
relationship. Many of the oil components may possess the ability to
break or to penetrate the lipid structure present in the Gram-negative
bacteria walls.
          Trabulsi et al., (1992) studied that the essential oil of Mentha
showed the least susceptibility against E.coli ATCC 25922 and have the
minimum zone of inhibition.
          Regarding the strains of Staphylococcus aureus, it was
observed that all of the tested substances presented antimicrobial
activity, characterized by the presence of zone of inhibition with
diameters that varied from 9mm to 16mm for the strain S.aureus
ATCC 25923.




                                                                              15
Lemon Grass (Cymbopogon flexusosus) -




                                        16
                TAXONOMY OF LEMON GRASS-
                      Cymbopogon flexusosus


                  Kingdom          -     Plantae
                  Division         -     Angiosperm
                  Class            -     Monocots
                  Order            -     Poales
                  Genus            -     Cymbopogon
                  Species          -     flexusosus


         Lemon Grass is an aromatic, perennial, tall grass with
rhizomes and densely tufted fibrous roots. It has short underground
stems with ringed segment, coarse, green, slightly leathery leaves in
dense cluster terminating in a long bristly point. The blades of the
grass are about 90cm long and 0.5cm wide.


       Lemon grass contains an essential oil. This oil is sherry colored
with a pungent taste and lemon like odor with citral as the principal
constituent.


      Lemon grass is native to India. It is widely used as a herb in
Asian cuisine. It has a citrus flavor and can be dried and powdered or
used fresh. Cymbopogum is a genus of about 55 species of grasses
native to warm temperate and tropical region. Lemon grass is
commonly used in teas, soups and curries. It is also suitable for
poultry, fish and seafood.




Research also shows that lemongrass oil has antifungal and pesticide
properties. Lemon grass is also known as Gavati chaha. Lemon grass
                                                                      17
oil is applied on the ancient manuscript found in India in oriental
research institute Mysore for preservation, which acts as a pesticide. It
is supposed to help in relieving cough and nasal congestion.


          Lemon grass oil revitalizing the body and relieves the symptoms
of headache and helps to combat nervous exhaustion and stress
related condition. It is useful in respiratory infection such as sore
throats, laryngitis and fever and helps to prevent spreading of
infectious disease. It helps to tone the muscles and tissues, relives
muscle pain by making the muscle suppler.


          Lemon grass is beneficial in strengthening the function of
stomach and promoting its action. It is beneficial in the treatment of
indigestion. Lemon grass oil also treats spasmodic, affection of the
bowels, gastric irritability and cholera(Hugo & Russell).


      Lemon grass oil has a lemony, sweet smell and is dark yellow to
amber and reddish in color with a watery viscosity. The principle
chemical constituent are citronella, geranial and citronellal, they have
antiseptic action, hence their use in household disinfectant and soaps.
It is fresh smelling oil that can be used with success for revitalizing a
tired body and mind as well as keeping the family pet free of flies and
ticks.


     Fresh lemongrass contains an essential oil which has substantial
amount of citral. Dry herbs yield 0.4% essential oil containing 72.3%
citral.
Lemon grass is commonly used in teas, soups and curries. It is also
suitable for poultry fish and seafood (Hugo and Russell., 1995).




                                                                       18
Antimicrobial activity of Lemon Grass:


Baratta et al., (1998) studied that the zone of inhibition exhibited by
the essential oil of Lemongrass was 33mm against S.aureus MTCC
3160 whereas the zone of inhibition yielded against E.coli MTCC 1089
was 22.3mm.
Lemongrass (Cymbopogon flexosus L.) oil (ranging between 25 and
500 ppm) was tested for antifungal activity against Colletotrichum
coccodes,    Botrytis        cinerea,     Cladosporium   herbarum,     Rhizopus
stolonifer and Aspergillus niger in vitro. Oil-enrichment resulted in
significant (P < 0.05) reduction on subsequent colony development for
the examined pathogens. Fungal spore production inhibited up to 70%
at 25 ppm of lemongrass oil concentration when compared with
equivalent plates stored in ambient air. In the highest oil concentration
(500 ppm) employed, fungal sporulation was completely retarded.
Lemongrass oil reduced spore germination and germ tube length in C.
coccodes, B. cinerea, C. herbarum and R. stolonifer with the effects
dependent         on   oil     concentration.      However,     lemongrass    oil
(up to 100 ppm) accelerated spore germination for A.niger. Work is
currently focusing on the mechanisms underlying the impacts of
essential   oil    volatiles    on      disease   development   with   a   major
contribution to limiting the spread of the pathogen by lowering the
spore load in the storage/transit atmospheres as well as the use of
essential oil as an alternative food preservative.




                                                                              19
               MATERIALS AND METHODS –


MATERIAL REQUIRED FOR PREPARATION OF PLANT EXTRACT


     Plant parts (powdered form)
     Sterilized glass wares (iodine flask, test tube, funnels, test
      Tube stand, thermometer)
     Whatmans filter paper (no.1)
     Instruments      (oven,    water    bath,   chemical     balance,
       Refrigerator)
     Chemical (methanol, distilled water, hexane, RO water)




METHODS FOR PREPARATION OF PLANT EXTRACT -


    Solvent extraction -
     Hydrocarbon solvents (hexane or methanol) were generally
       used.
     Plants material was dissolved in these solvent and filtration
       was done after 24 hours.
     The filtrate was concentrated by distillation.




                                                                       20
           Protocol for solvent extraction –


             Weighed the total quantity of powdered plant parts
                using digital weight balance.
             Divided each plant powder into two equal fractions for
                dilution in – hexane and methanol.
               Equal quantities were dissolved in the above two
                solvents and kept them overnight.
             On the next day filtered out the extracts.
             The filtered extracts were kept for distillation in a water
                bath heated at 95oC for aqueous extracts and at 70oC
                for hexane and methanol extract.
             The concentrated extract were kept in separate test
                tube and covered with aluminum foils and stored in
                refrigerator for further use.




TEST ORGANISMS


The table no 1. Given below, list the range of bacterial organisms used
through   out   the   study.   These   organisms   were    used   for   the
investigation of antibacterial activity of extracts of Pudina and Lemon
grass. The bacteria were propagated using the conditions described in
table 1 according to the recommendations of the supplier.




                                                                         21
                     Bacteria used in this study
                                   Table 1


S.No. Bacterial           Liquid     Solid      Temp.(CO)      Strain
         Species          Medium     Medium                    No.


1)        S.aureus           NB         NA          37OC        MTCC – 737




2)          E.coli           NB         NA          37OC        MTCC - 452




    NA – Nutrient Agar


    NB – Nutrient Broth


    MTCC – Microbial Type Culture Collection




    METHODS FOR ANTIMICROBIAL ACTIVITY TESTING –


 Well diffusion method –
                           This method is used in case of low concentration
           extracts suspected to show some antimicrobial activity.
           Comparatively require more quantity unlike antibiotics, thus
           wells are needed to be bored in the culture plates. It is a
           qualitative.




                                                                         22
1. Media preparation:


Dissolved 18.5gms. Of Nutrient Agar in 500 ml of distilled water. The
composition of Nutrient Agar was peptone=10 gm/lt, beef extract
=10gm/lt, NaCl =5gm/lt and agar =12gm/lt.
       After dissolution the media was kept for sterilization in

autoclave at 15lb/inch pressure and 121 C temperatures for 15

minutes, ph. was checked and set at 7.



2. Culture preparation:



The surface of the laminar air flow was sterilized with UV for 15

minutes. After switching off UV. The freshly prepared media were

inoculated with 100µl of E.coli and S.aureus cultures separa .Media

was cooled to at least 45oC before inoculation with bacteria.

Fifteen ml of inoculated media was poured into each petri-plate for

each bacterial culture. One replica for each test was made for better

result. After setting of culture plates, wells were bored. The wells were

filled up with 50 µl of concentrated extract with the help of

micropipette. The petri-plates were covered and incubated for 24

hours. After 24 hours the petri-plates was examined.




                                                                      23
Methodology of testing:



      SELECTION OF PLANTS


            PLANTS


POWDERING OF THE PLANT PARTS TO
           BE USED


            BE USED


 PREPRATION OF PLANT EXTRACTS IN:
 1. HEXANE
 2. METHANOL




      FILTRATON OF EXTRACTS




         DISTILLATION OF
            EXTRACTS




    ANTIMICROBIAL ACTIVITY
        TESTING USING
    WELL- DIFFUSING METHOD




          RESULTS



                                    24
                            RESULTS

Antimicrobial activity of Mentha arvensis:


Zone of inhibition in methanol extract of Mentha arvensis against
S.aureus was 22mm as shown in Fig 1.whereas its hexane extract
yielded a zone of 15mm (Fig 2) against S.aureus.
Zone of inhibition in methanol extract of Mentha against E.coli was
12mm as shown in Fig 3 and its hexane extract yielded a zone of
13mm against E.coli (Fig 4).


Antimicrobial activity of Lemongrass:


Zone of inhibition in methanol extract of Lemon grass against S.aureus
was 23mm that shown in Fig 5 whereas its hexane extract yielded a
zone of 17mm against S.aureus.(Fig 6) .
No activity was detected in both methanol and hexane extract of
Lemon grass against E.coli as shown in Fig 7 and 8 respectively.
No activity was detected in negative control in both methanol and
hexane against E.coli that shown in Fig 9 and 10 respectively.
Zone of inhibition in case of Ciprofloxacin was 36mm against S.aureus
that shown in Fig 11.


The observations are shown in table 2. Later




                                                                    25
Fig 1:- Zone of inhibition in methanol extract of Mentha arvensis (Pudina)
                               using S.aureus.




Fig 2:-Zone of inhibition in hexane extract of Mentha arvensis (Pudina)
                             using S.aureus.

                                                                        26
   Fig 3:-Zone of inhibition in methanol extract of M.arvensis
                     (Pudina) using E.coli.




Fig4:-Zone of inhibition in hexane extract of M.arvensis (Pudina)
                           using E.coli.




                                                                 27
Fig 5:-Zone of inhibition in methanol extract of C.flexusosus
               (Lemon grass) using S.aureus




 Fig 6:-Zone of inhibition in hexane extract of C.flexusosus
               (Lemongrass) using S.aureus.



                                                                28
Fig 7:- N.A.D. in methanol extract of C.flexusosus (Lemongrass) is
                          using E.coli.




   Fig 8:-N.A.D.in hexane extract of C.flexusosus using E.coli.



                                                                  29
  .

Fig. 9:- Negative control in methanol using E.coli.




Fig. 10:- Negative control in hexane using E.coli.


                                                      30
Fig. 11:- Positive control was using S.aureus.




                                                 31
      Table 2:-Antimicrobial activity of plant extract
                                                   TEST      DIAMETER OF
S.No    PLANT USED         EXTRACT TYPE          MICROBE        ZONE OF
.                                                             INHIBITION
                                                E.coli         12mm
                                 METHANOL
                                                S.aureus       22mm

1.      M.arvenses
                                                E.coli         13mm
        (PUDINA)
                                  HEXANE
                                                S.aureus       15mm


                                                E.coli          NAD*
                                 METHANOL
                                                S.aureus         23mm
2.      C.flexusosus
        (LEMONGRASS)                            E.coli          NAD
                                  HEXANE
                                                S.aureus       17mm

                                 METHANOL       E.coli          NAD
3.      Negative control
                                  HEXANE        E.coli          NAD


                                  CIPLOX        S.aureus        36mm
4.      Positive control    (CIPLOFLOXACIN)

     No Activity Detected*


      NOTE: The diameter of well was 6mm, so the zone of inhibition has
       to be>6mm.


      Positive control showed a large diameter of 36mm while negative
       controls didn’t showed any zone of inhibition. Refer to photographs
       displayed under result.



                                                                        32
                           DISCUSSION

In this study the antimicrobial activity of Mentha and Lemongrass was
studied against E.coli and S.aureus. First we powdered the plant parts
to be used, then plant extracts were prepared in hexane and
methanol, we used the well-diffusion method for antimicrobial activity
testing.
From the observations made it is inferred that out of the two plants
selected both of them showed antimicrobial activity while Lemongrass
showed activity against S.aureus but not against E.coli in the both
methanol and hexane extract.


Moreover we can clearly observe that the maximum antimicrobial
activity was shown by methanol extract of Lemongrass followed by
methanol extract of Mentha against S.aureus.


It was also seen that these plant extracts were found to be more
effective against S.aureus which is Gram-positive bacteria.


On comparing the type of extracts, it is clear that methanol and
hexane extracts of Lemongrass show no activity against E.coli but
Mentha showed activity against both S.aureus and E.coli.


If on the other hand comparison is done on the basis of plants
selected, Mentha have good antimicrobial activity.




                                                                    33
According to Trabulsi et al.,(1995)    the Mentha shows antimicrobial
activity against both S.aureus (9-16mm) and E.coli (minimum i.e.6-
7mm), In this study, the zone of inhibition of menthe extract was
about 22mm and 15mm against S.aureus , 12mm and13mm against
E.coli in methanolic and hexane extracts respectively .
                    According to Baratta et al.,(1998), the zone of
inhibition/mm of Lemongrass against S.aureus was 33mm and against
E.coli was22.3 mm. Whereas in this study, the zone of inhibition was
23mm and 17mm against S.aureus in both methanol and hexane
extracts respectively. No activity was detected against E.coli.
      This discrepancy in the results may be due to the use of
different strains and different method for the study of antimicrobial
activity of plant extracts, as they used S.aureus ATCC 25923 and
MTCC 3160 and E.coli ATCC 25922 and MTCC1089 for Mentha and
Lemongrass respectively. Besides, they used disc-diffusion method for
their study on the other hand we used S.aureus MTCC 737 and E.coli
MTCC 452 and well diffusion method for our study.




                                                                   34
                        CONCLUSION

Plants have met various human needs from earliest times. Their
medicinal uses similarly date back to the beginning of human
civilization. The Indian systems of Medicines, Ayurveda, Siddha, Unani
and even Homeopathy are largely plant based. The emphases for
development of new biologically active molecule have been gradually
replaced by use of the plants as medicine and food supplements.
This project work involves the assessment of the antimicrobial activity
of two plants.
         The antimicrobial activity in plants may be contributed by
phytochemicals. For this two common and traditional plants were
selected processing antimicrobial properties which were Pudina and
Lemon Grass.
        For the work, the plant parts were taken in powdered form.
After the selection process, the extracts were prepared. Although there
are different methods of extract preparation but solvent extraction
method was chosen because of the simplicity and high yield, Extracts
were prepared in two solvents – Methanol and Hexane. The extracts
were prepared by soaking the plant parts in the solvents overnight
followed by filtration with Whatman paper and boiling the filtrate in
waterbath until an oily and viscous extract is obtained. After the
extract preparation, comes the antimicrobial testing process. For this
Agar well diffusion method was used. The media was inoculated with
the selected bacterial strain. After inoculation media was poured into
the petri plates and was allowed to set. After the media was solidified,
wells were bored into the plates and the extracts were poured into the
labeled wells, All this work was carried out in laminar air flow under
aseptic conditions and autoclaved glasswares. After which the petri
plates were incubated under optimum conditions in bacteriological


                                                                      35
incubator at 40oC.for 24hours. After 24 hours the plants are examined
for results i.e. Zone of inhibition. If the zones were observed their
diameter was noted. The zones indicate the extent to which the growth
of bacteria is inhibited.
      All the inoculation work was carried out with precautions like use
of shoe cover, face mask, head cap and constant sterilization with
alcohol.




 The antimicrobial activity of the selected plants can be concluded as
 On the basis of plants used : PUDINA >LEMON GRASS
 On the basis of extracts used : METHANOL > HEXANE
 Hence we can concluded on the basis of the results that these
   plants extracts which are used and known to treat fungal and
   bacterial diseases like Typhoid,Gonorrhoea ,
   Tetanus,   Syphilis,     Disentery,   Influenza   etc.have   shown   their
   medicinal importance as quoted in the ancient literature books.




                                                                          36
                 SCOPE AND APPLICATIONS

Not only in India, but in many other countries plant have played
significant and prominent role as medicine. At one time it was felt that
the chemical synthesis would completely replace the drugs of natural
origin. In spite of the emergence of many wonder drugs from the
synthetic field, the problem of senescence and so called civilization
disease e.g. immuno deficiency, arthritis and cancer. They cannot be
tackled and therefore there is a greater demand for natural medicine
and health food, today then ever before in the world.
          A large number of aromatic plants containing essential oils are
also important from the utilization point of view. Natural perfume is
one of the most remarkable phenomenons of plant utilization. The
essential oils are used in every day human life in various ways and
their consumption is rapidly increasing. A few of the common uses to
which essential oils and their derivative are put to are in the
manufacture      of   soaps,   cosmetics,   pharmaceutical   preparation,
disinfectant, detergent etc.
      There is ample scope for setting up of small scale extraction and
manufacturing units for indigenous medicine, pharmaceutical products
and the food processing unit at central and salient places. In the
medicinal plant sector, the world health organization (WHO) has
estimated that about 80% of the population of developing countries
rely on the modern medicine that also contain 25% drugs derived from
plants.


The derivatives of medicinal and aromatic plant are non-narcotic
having no side effect even if used for the prolonged time in permissible
doses. The aromatic plants provide the raw material for the production
of flavors, condiments, herbal cosmetic, perfumery, scented soaps,
and hair oils. Demand for these herbs is increasingly progressively
                                                                       37
with increase in number of star hotels and multinational establishing
consumer oriented cosmetics and pharmaceutical units.


   Why global resurgence in the use of green medicine?
         Comparatively more safe.
         Comparatively cheap.
         More eco-friendly.
         Only cure where modern drugs are either unavailable or
           unsatisfactory.
         Preventive, promotive and curative.
         Beside health benefit medicinal and aromatic plant provide
           crucial livelihood option for millions of rural people,
           particularly women, tribal and the poorest of the poor.
         In India 35 million workdays of employment per year are
           contributed by collection and processing of medicinal plant.
         95% of medicinal and aromatic plant are harvested and
           collected from the wild.
         25% modern medicine derived from the plant.




                                                                     38
                             APPENDIX:

  CLEANING & SANITATION OF MICROBIOLOGY LAB
Cleaning and Sanitation is major part of lab It is done to insure that
testing of material hading of sample instruments lab work place is
clean and sanitary conditions. Also to insure that bourdon in
microbiology lab remains under control. Housekeeper engaged in
cleaning and sanitation should be adequately trained in this programe.


In Daffohils laboratories plant cleaning program should be done
weekly.
   To clean the area suitable disinfectant with there appropriate
     dilution can be used.
   The disinfectant and there dilution should be made just before
     it’s used.
   The person who is carrying out cleaning should where proper
     clothes, apron, gloves, masks, goggles to protect his eyes and
     skin.
   They should first sweep the followed by mopping with freshly
      Prepared disinfectant solution.
   Doors windows, ventilation duct, grills and outer area of lab
     should be cleaned by same disinfectant.
   It should be keep for 10 to 15 minutes and after that it should
     be cleaned with pure milli Q water to remove the traces of
     disinfectant. Quality control supervisor of the lab shall insure
     that work place is clean and all the things are kept in proper
     order.




                                                                    39
   CLEANING AND DISINFECTION OF INSTRUMENTS
Cleaning and disinfection of instruments like oven, refrigerator,
incubator is done monthly to insure that these instruments itself does
not add any contamination to the article stored.


Incubator:-
Incubator in the microbiology lab is instruments which are operated to
allow the microbial growth on suitable medium under appropriate
temperature. It is double walled steel chamber which is adjusted to
desired temperature by using external knob controlling the thermostat
system. As the temperature influence the microbial growth therefore
instruments   is   generally    designed     that   can   allow   the   desired
microorganism      to   grow   at    particular   temperature.    Variation   in
temperature should not more than one degree.




                                    Incubator




                                                                              40
Refrigerator:-
Similarly refrigerator shall be used for storage, preservation of
microbial cultures, test samples, particular count TOC of standard.
Temperature of refrigerator near about 8 C .
     The entire article kept inside the refrigerator or incubator shall
       be properly labeled. To clean and disinfect follow the following
       steps:-
     Power supply to the refrigerator or incubator shall be switched
       off before starting the cleaning operator.
     Remove all the particles from refrigerator and incubator.
     Defrost the refrigerator to remove ice formation.
     70% of IPA, dettol or savlon use to disinfect the area outside
       and inside of refrigerator and incubator.
     Carryout the moping with the swab of disinfectant.
     After 10 minutes carryout moping with DI water in order to
       remove the residues of disinfectant.
     Microbiologist shall insure that the entire articles are put back
       in the refrigerator or incubator in orderly manner.
     All these activities control by microbiologist and quality control
       manager.




                               Refrigerator

                                                                      41
         STERILITY MONITORING OF AUTOCLAVE


Autoclave or steam sterilizer is a device like a pressure cooker in which
the killing action of heat on organism can be done by using increase in
steam. Hot saturated steam continues to enter until the chamber
reaches the desired temperature pressure (121 C and 15 pound) at
this temperature and pressure steam destroys all vegetative cells and
end spore.
         In Daffohils Laboratories, there is a modern automatically
controlled autoclave or steam sterilizer which is having different
chambers. Temperature and pressure of each point is maintained
automatically to load the material. Autoclave have one none sterile
side and to unload the contents there is a sterile door which is opened
inside the sterile lab. This autoclave operates under two cycles.


Standard Cycle


Cleaning and performance check of autoclave is done to ensure that
autoclave is functioning satisfactory and completing the sterilization
cycle the functioning of autoclave:
     Water Pressure is above 1.2 kg on gauge off water line.
     Compressed air pressure is 7.0+0.5 kg.
     Door gasket pressure on sterile and non sterile door is
       maintained at 3.0+0.5 kg.
     Door pre condition light on control panel glows during the
       process is on.
     Chamber pressure in sterilization cycle is between 1.1 to 1.2kg.
     Display on panel of recorded as well as pressure gauge on the
       panel of autoclave.
     Place one strip of autoclave places other strip along with the
       material to be sterilized inside the autoclave chamber.
                                                                      42
 At the completion of cycle check the color change if there is
  color change of strip autoclave is certified to the in proper
  order.
 All these activities are under the control of Microbiologist and
  are supervised by Quality Control Manager.




                         Autoclave




                                                                43
STERILITY MONITORING OF LAF (LAMINAR AIR FLOW)


Laminar air flow is an apparatus consist of an air blower in the
rear side of chamber which can produce air flow with the uniform
velocity along parallel flow lines. This cabinet employs a high
efficiency particles air (HEPA) filter which fro microorganism being
handled with the cabinet and prevent room contamination.


Cleaning and sterilized monitoring is done to ensure that:-
    LAF is functioning satisfactory or not.
    Air provided by filter is microbe free.
    Before using the LAF. UV light for a period of 30 min is
      switched on so to kill the germs if present.
    A suitable disinfectant is used to clean inside and outside of
      LAF.
    To ensure that there is proper cleaning sterilized SCDA
      PLATES are exposed inside the chamber and allow it for
      30-35 C for 48 hours.
    If there is no growth occurs on this plate that means it is
      properly clean.
    Any leakage is HEPA filter is checked by instrument section.




                        Laminar Air Flow Hood

                                                                  44
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Description: The present project was undertaken with the analysis of the antimicrobial activity of the some aromatic plants or herbs. The science of ayurveda is a unique holistic system, based on the interaction of body mind and spirit in ayurveda, the origin of all aspects of existence is pure intellect or consciousness. The treatment of Ayurveda is based on Indian herbs, which has a healing energy.