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.
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 45
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