Soubhagya K B

TRENDS IN ADVANCED BIOLOGY Trends in Advanced Biology It encompasses 50 research articles presented as part of an International Conference on Advanced Biology organized by iCEIB, University of Kerala. It mainly includes innovative research articles at the intersection of life sciences where integrative and emerging fields of investigations go hand in hand. The articles mainly focus on the themes Stress Physiology, Disease Biology. Environmental Biology and Biodiversity Conservation and envisage the role of advanced techniques such as genomics and proteomics in disease biology, preservation of the diversity of species as well as sustainable utilization of species and ecosystem - Published by iCEIB, University of Kerala, Kariavattom, Thiruvananthapuram. TRENDS IN ADVANCED BIOLOGY Prof. Suhara Beevy S. Dr. Mariamma Cherian Dr. Darsan B. Menon Dr. Anil Kumar T. R. Dr. Mini V.S. Editors iCEIB Published by iCEIB University of Kerala, Kariavattom Thiruvananthapuram Prof. Suhara Beevy S. Dr. Mariamma Cherian Dr. Darsan B. Menon Dr. Anil Kumar T. R. Dr. Mini V.S. TRENDS IN ADVANCED BIOLOGY Editors Published by iCEIB University of Kerala, Kariavattom Thiruvananthapuram Prof. Suhara Beevy S. Dr. Mariamma Cherian Dr. Darsan B. Menon Dr. Anil Kumar T. R. Dr. Mini V.S. Editors Prof. Suhara Beevy S Dr. Mariamma Cherian Dr. Darsan B. Menon Dr. Anil Kumar T.R. Dr. Mini V. S. Cover page design and Layout Dr. Anil Kumar T. R. Copyright @ 2022 University of Kerala. This work is subject to copyright. All rights are reserved. No part of the publication may be reproduced, distributed or transmitted in any form or by any means, including photocopying, recording or mechanical methods without the prior written permission of the publisher, except in case of brief quotations embodied in critical reviews and certain other non-commercial uses permitted by copyright law. For permission requests write to the author, addressed “Attention: Permission” at s.- Published by iCEIB University of Kerala, Kariavattom Thiruvananthapuram - ISBN- Ordering information:- Contents Title Page No. Endospore forming Bacillus spp. as growth promoter in black pepper cuttings A.B. Anju, K. N. Anith, N. Chitra, S. Anu Rajan and V. I. Soumya..................................1 Antioxidant activity of three marine microalgae Nostoc sp., Chaetoceros muelleri and Nannochloropsis oculata M. Sakthipriya, P. Priyadharshini , S. Jeyanthi, P. Santhanam and M. Divya.....................7 Analysis of the lignin removal efficiency of hydrogen peroxide as a pre-treating agent on the wheat straw M.Iyyadurai, P. Merlin sobia, K. Paritosh, V. Vivekanand, M. Krishnaveni, S. Venkatesh..................................................................................................................12 Biodegradation of polycyclic aromatic hydrocarbon by bacteria from marine ecosystem S. Vineetha, Darsan B. Menon, V. S. Mini, M. C. Subhash Peter, S. Suhara Beevy and T. R Anilkumar ..........................................................................21 Golden Oak Mushroom (Shiitake) –A new member in mushroom flora of Kerala C. V. Deepa Rani, Lulu Das, N.P. Lishma...........................................................................36 Virtual screening of plant derived compounds against angiotensin converting enzyme related carboxypeptidase (ACE2) of SARS-COV-2 using molecular docking R. Rathika, S. Venkatesh, M. Krishnaveni.........................................................................................40 Documentation and macronutrients analysis of homestead-based fodder crops in Alappuzha District, Kerala S.R. Dhanya and V. Rajani..................................................................................................50 Impact of drought stress on morpho-physiological traits and membrane damage in the cultivars of sesame (Sesamum indicum L.) M. L. Anchu, S. Jeyaraj and S. Suhara Beevy...............................................................55 In vitro and in silico cytotoxicity evaluation of leaf extracts from Naregamia alata Wight &Arn. K. B Soubhagya, Sruthy Elsa Madhu and BenojMathew...............................................67 Phytoplankton diversity of Vairamkonamchira, a freshwater body of Anchal Panchayat. F. Jensy Roshan and Aparna T. Nair.............................................................................79 Short- and long-term effect of light exposure on photosynthetic and antioxidant machinery of Vigna unguiculata L. Riya Johnson and Jos T Puthur....................................................................................84 Etiology of fungi causing leaf rot disease of coconut in Kerala Susha S. Thara; Deepthi S. Nair; Divya S. and Aashitha Joy.........................................95 An induction of antioxidant defense and protease inhibitor activity in Solanum lycopersicum Linn. Pretreated with laminarin against Alternaria solani infection S. P Sowmiyarithika. and N Radhakrishnan...............................................................106 Bioaccumulation of E. coli in Villorita cyprinoides of Ashtamudi Estuary, Ramsar Site -1204 in Kerala, South west coast of India Letty Titus and B.T. Sulekha....................................................................................120 Studies on the endemic and threatened flowering plants of Vagamon hills, Western Ghats, India Anoop P. Balan and A. J. Robi..................................................................................128 Validation of anti-diabetic activity in Plectranthus vettiveroides and identification of lead molecules through in silico method R. P. Remya, S.Sreekumar and C. K.Biju...................................................................136 Role of Bioinformatics in target identification and differentiation of papillary thyroid carcinoma: A study based on GEO dataset Febby Payva and K. S. Santhy..................................................................................148 Evaluation of heavy metal pollution in water, sediment and Meretrix casta, an edible bivalve species of Asramam in Ashtamudi wetland, Kerala, India N. Parvathy, B.T. Sulekha and S. Sheeba...................................................................157 Status and distribution of medicinal plants in the Malamel rock exposure of Edamulakkal Panchayat, Kollam District Anila George, Morvin Mathew Kizhakekara, Nilja Arjun............................................165 Diversity of exopolysaccharide producing bacteria from soil based insect nesting structures N. Sruthi Suresh, N. Chitra, S. Anu Rajan, V. I. Soumya and K. N.Anith......................175 Pro-adipogenic PU.1 AS long noncoding RNA expression changes during adipogenesis B.Surumi. R. Rajalakshmi, G. M. Nair, A. Jayakumaran nairA. Gangaprasad, C. Prabhakumari................................................................................................................183 Study on butterfly diversity in a sacred groovelocated in Alapad village. C. J. Vishnupriya and Praseeja cheruparambath..........................................................194 Establishment of axenic culture of Hyophila involuta (Hook.) A. Jaeger Megha Santhosh, Meenu Mathew, Abraham Mathew.................................................201 Comparative pharmacological study of Piper nigrum L., Piper longum L. and Piper betle L. Sruthy Elsa Madhu, K. B Soubhagya. and Benoj Mathew...........................................207 (2012). In vitro micropropagation of rare and endangered moss Entosthodon hungaricus (Funariaceae). Bioscience Journal, 28(4), 632–640. Sabovljević, A., Vujičić, M., Skorić, M., Bajić-Ljubičić, J. A. S. N. A., & Sabovljević, M. A. R. K. O. (2012). Axenically culturing the bryophytes: establishment and propagation of the pleurocarpous moss Thamnobryum alopecurum Nieuwland ex Gangulee (Bryophyta, Neckeraceae) in in vitro conditions. Pakistan Journal of Botany, 44(1), 339-344. Vujičić, M., Sabovljević, A., Šinžar-Sekulić, J., Skorić, M., & Sabovljević, M. (2012). In vitro development of the rare and endangered moss Molendoa hornschuchiana (Hook.) Lindb. ex Limpr.(Pottiaceae, Bryophyta). HortScience, 47(1), 84-87. Vujičić, M., Sabovljević, A., & Sabovljević, M. (2010). Axenically culturing the bryophytes: a case study of the moss Herzogiella seligeri (Brid.) Z. Iwats. (Plagiotheciaceae). Biologica Nyssana 1, 77-82. Comparative pharmacological study of Piper nigrum L., Piper longum L. and Piper betle L. 1 * Sruthy Elsa Madhu, 1 K. B Soubhagya. & 2 Benoj Mathew Research Scholar, Rubber Research Institute of India, Kottayam 1 Research Scholar, PG & Research Department of Botany, St. Peter’s College, Kolenchery 2 Associate Professor, PG & Research Department of Botany, St. Peter’s College, Kolenchery-Abstract 1 Plants are used in traditional systems of medicine from prehistoric times due to their diverse pharmacological potentials and lesser side effects in biological systems. Piper spp. are reported to have great therapeutic value in Indian medicine. The current study was therefore carried out to evaluate the anatomical characteristics, phytochemical constituents, and antibacterial potential of the three Piper spp. namely Piper betle L., Piper longum L. and Piper nigrum L. The present study showed that the stem and leaf of the three species possess more or less comparable anatomical features with a few differences; whereas, the microscopic powder analysis in the fruits of the three species are distinctive. Antibacterial potential of ethanol and aqueous extracts of stem, leaf and fruit of the three Piper spp. were examined separately against pathogenic bacteria viz. Staphylococcus aureus and Escherichia coli using agar well diffusion method. The zone of inhibition was measured to analyse the effect of the extracts. All the plants showed significant activity against the pathogens but the two extracts of different plant parts 207 showed variation in their activity. The study will provide referential information for the correct identification of the crude drugs. Also, idea about the efficacy of different Piper spp. in counteracting the pathogens like E. coli and S. aureus can be obtained. Keywords: Piper spp., pharmacology, antibacterial potential, zone of inhibition 1. Introduction Phytochemicals are defined as bioactive nutrient chemical compounds produced by plants, which provide health benefits beyond basic nutrition and reduces the risk of many chronic diseases (Liu, 2004). They are produced in plants during bacterial, fungal, or viral infections which indicates their potential antibacterial, antifungal and antiviral activities. Due to their health benefits, they are being used in pharmaceutical industries. Polyphenols, flavonoids, isoflavonoids, terpenoids carotenoids, limonoids, glucosinolates etc. are some of the common phytochemicals present in plants. Plants are being used for medicinal purposes from prehistoric times. Pharmacologists, botanists and microbiologists are looking for new bioactive compounds which can be developed into drugs for treating infections and to use against drug resistant microbes. (Tanaka, 2006). Many plant- secondary metabolites are found as active compounds in treatment of a wide range of disorders. Piper spp. are important medicinal plants distributed widely in tropical and subtropical areas, central Asia and particularly in India. (Oyemitan, 2017). They are used in traditional medicines. (Bahare salehi piper sps.). Piper longum have been patented by US scientists for obtaining a therapeutic agent for diabetes mellitus. Piper nigrum is used for itching, constipation, nervous disorders and flatulence (Khan et al., 2007). Leaf extract of P. betle is found to have antiseptic antibacterial and antiviral properties (Ermawati et al., 2021). All the three species are therapeutically important, however, a comparative study of different parts of the plants against different bacteria (gram positive and gram negative), their anatomy and phytochemicals has not yet been studied. This study aims at a comparative study of the three spp. of Piper (P. nigrum L., P. longum L. and P. betle L.) against the two bacterial spp. Escherichia. coli and Staphylococcus. aureus. A comparative analysis on the anatomy, and phytochemical constituents is also done. 208 2. Materials and Methods 2.1. Plant collection and authentication Fresh plant parts of P. nigrum, P. longum and P. betle were collected from the premises of St. Peters College, Kolencherry. The collected plants were authenticated and kept in herbarium. The plants were washed under running tap water, air dried and finely powdered. They were stored in air tight bottles at 4oC. 2.2. Microscopic analysis 2.2.1. Powder microscopy Fine fruit powder of P. betle, P. longum and P. nigrum stained with safranin were observed under digital binocular motic microscope, photomicrographs taken and characteristics noted. 2.2.2. Anatomical characterisation of stem and petiole Anatomical features of stem and petiole of the three Piper species were studied by free hand sectioning. Safranin was used for staining. Photomicrographs taken under digital binocular motic microscope. Computer images were captured using motic image software. 2.3. Preparation of crude extracts of the samples Aqueous extracts of stem, leaves, and fruits used for evaluation were prepared using distilled water and ethanol. Five g. each of the powdered samples were dissolved separately in 50 ml ethanol, covered air tight and kept for 2 days undisturbed. The concentrated extracts filtered, stored and properly labelled. The aqueous extracts were prepared by dissolving the powdered plant parts separately in 100 ml of distilled water, kept in water bath until the final volume becomes 50 ml and filtered. All the samples were stored at 4oC. 2.4. Phytochemical analysis (a)Test for flavonoids- Shinoda test Mg/HCl Dissolved small amount of leaf and stem extracts separately in methanol or ethanol, a few magnesium turnings and a few drops of 5M HCl were also added to each of them. Development of deep red or magenta colour indicated the presence of flavonoids. (b)Test for coumarins Little amounts of extracts of leaves and stem were dissolved separately in methanol and ethanol and 3-4ml alcoholic KOH or NaOH was added to each of them. Formation of a yellow colour which disappeared on adding conc. HCl indicated the presence of coumarins. 209 (c)Test for tannins- Ferric chloride test A few drops of ferric chloride were added to the extracts of leaves and stem taken in two separate test tubes. The development of green colour reveals the presence of tannin. (d)Test for alkaloids- (1) Mayer’s test One or two drops of Mayer’s reagent was added to the acidified leaf and stem extracts taken in two separate test tubes. A white precipitate indicated the presences of the alkaloids. Mayer’s reagent- HgCl2 (1.32 g) was dissolved in 60 ml distilled water and mixed with a solution of 5mg of KI in 10 ml of water. As this reagent reacts only with the salts of alkaloids, the solution made distinctly acidic with HCl or H2SO4. (2) Wagner’s Test Alkaloids give brown flocculent precipitate with Wagner’s reagent. Wagner’s reagent: 1.27 gm of iodine and 2 g of KI were dissolved in 5ml of distilled water and solution was made up to 100ml distilled water. (e) Detection of steroids/terpenoids- Salkowski-test A few drops of concentrated sulphuric acid were added to a small quantity of extracts taken separately and was shaken for a few min. The development of red or brown colour indicated the presence of sterols. (f)Test for saponins Shake an aqueous/alcoholic leaf and stem extracts in two separate test tubes and a persistent foam indicate the presence of saponins. (g)Test for quinines To test the samples, a few drops of sodium hydroxide were added to them. Formation of blue’ green or red colour indicates the presence of quinines. (h)Test for anthraquinones- Borntrager’s test The extracts of leaves and stem were shaken with Aqueous Ammonia or caustic soda. Formation of pink, red or violet colour in the aqueous layer indicates the presence of Anthra quinines. (i)Test for phenols A few drops of alcoholic Ferric chloride solution were added to the samples dissolved in alcohol/water. Formation of violet, bluish green or bluish black colour indicates the presence of phenols. 210 (j)Test for resin A small quantity of extracts was dissolved in 5ml of alcohol separately and added a few ml of distilled water and petroleum ether respectively. The development of white turbidity indicated the presence of resin. (k)Test for detection of reducing sugar/glycoside- Benedict’s test The extracts were mixed separately and added Benedict’s reagents in equal amounts and the mixtures were heated for 2min. The appearance of brown to red colour indicated the presence of glycosides. (l) Test for protein- Xanthoprotein test A small amount extracts of leaf and stem were added with 0.5ml concentrated HNO3, appearance of white or yellow precipitate revealed the presence of protein. (m) Test for carbohydrates- Molisch’s test To a small quantity of leaf and stem extract added two drops of 1% alcoholic solution of alpha naphthol and1ml of conc. H2SO4 along the sides of the test tube. A deep violet ring formed at the junction of the two liquids indicated the presence of carbohydrates. 3. Results and Discussion 3.1. Powder microscopy 3.1.1. Powder microscopy in P. betle L. The powder is dark brown in colour and heating in taste. The microscopical study of powder stained with safranin showed fragments of parenchyma, beaker shaped stone cells from endocarp, tracheids and starch grains. 3.1.2. Powder microscopy in P. longum L. The powder of fruit is dark brown in colour pungent and heating in taste. The microscopical study of powder shows fragments of parenchyma, masses of minute compound and single, oval to round, starch grains, lignified fibres and spiral vessels. 3.1.3. Powder microscopy in P. nigrum L. Powder is dark brown to blackish in colour with pungent odour and bitter acrid taste. Powder stained with safranin shows, starch grains, fibres, oil globules and tracheids. 3.2. Anatomical characterisation of stem and petiole Anatomical characterization of stem shows many similarities and differences whereas P. longum and P. nigrum showed similarities in trichomes, cortex and endodermis. When vascular 211 bundles canals are and mucilage considered, they showed variation. The anatomical features of petioles of P. longum and P. nigrum showed many similarities when compared to P. betle. However, hydathodes are present in all the three species. The results are given in Tables 1 and 2. 3. 3. Preliminary phytochemical screening A variety of constituents belonging to different chemical classes such as terpenes, steroids, tannins, phenols, alkaloids, flavonoids, proteins etc. were screened in the leaf, fruit and stem. Saponins were absent in where all the other compounds were detected in either of the two solvents in all the three species. But presence of the chemicals varied according to the plant part used for study. The results are recorded in Tables 3-5. 3.4. Antimicrobial activity In the present study the inhibitory effect of different extracts (ethanol and distilled water) of stem and leaves of P. nigrum, P. longum and P. betle were carried out. The antimicrobial activity was determined using agar well diffusion method. The results were summarized in Tables 6-8 and Figs 1-6. The activity was measured quantitatively by measuring the inhibition zone. The antimicrobial potential of all the three experimental plants were evaluated according to their zone of inhibition against the pathogens and the results (zone of inhibition) were compared with the activity of the standards, viz., ciprofloxacin (1.0 mg/disc). The results revealed that all the samples were potent antimicrobials against all the microorganisms studied. Among the different solvents extracts studied it was observed that ethanolic extract of Piper nigrum stem was the most effective against S. aureus. Both ethanolic and water extracts showed good activity but ethanolic extract had higher activity against S. aureus (Gram positive) P. betle stem also had inhibition zone for S. aureus in both ethanolic and water extracts which was better than P. longum stem extract which had comparatively smaller inhibitory zone for ethanolic extract an no zone for water extract against S. aureus. In case of E. coli also P. longum stem had zone in ethanolic extract alone and no zone obtained for water extract whereas for P. nigrum stem no zone was obtained against E. coli in both ethanolic and water extracts. However, P. betle stem exhibited activity against E. coli in both ethanol and water extracts which was similar to its zones obtained in case of S. aureus. P. nigrum fruit also had good activity against S. aureus in its ethanolic extract however, no zone was obtained for water extract. Ethanolic extracts of P. nigrum fruit had zone similar to 212 that of P. betle leaf ethanolic extract. But water extract of P. longum leaf had no zone against S. aureus whereas P. betle exhibited a small zone against S. aureus. Both ethanol and water extract of P. nigrum fruit showed activity against E coli. Similarly, P. longum fruit also had zones for both extracts however the water extracts of P. longum leaf performed better than P. nigrum against E. coli. P. betle leaf had only activity in ethanolic extract against E. coli. P. nigrum leaf established zones against both E. coli and staphylococcus aureus against in both ethanolic and water extracts. The activity was moderate but much better than P. longum and P. betle where the latter had a better performance against E. coli compared to P. longum and P. longum had better zone against S. aureus in ethanolic extract compared to P. betle but both didn’t have zones in water extract against S. aureus. The effect of P. nigrum against E. coli and S. aureus substantiate the findings of Pundir, and Jain (2010), who reported that the ethanolic extract of P. nigrum shows good activity against E. coli and S. aureus. The finding that anti bacterial activity of P. longum fruits against S. aureus was more than that of E. coli was similar to the results of Singh et al. (2011). Though most parts of the plants showed antibacterial activity some failed to exhibit potent antibacterial effect and this can be because of the plant part used, type of extraction, the time of collection and climate can affect the amount of active constituents in plant material. (Arora et al., 1999; Parekh et al., 2007). The results revealed that there are many similarities and differences in the anatomical features of the three spp. But the phytochemical constituents of all the three species were similar. Though all the three species were potent therapeutics against both S. aureus (gram positive) and E. coli (gram negative), P. nigrum is comparatively more potent than other two. But performance varied according to the plant part and the solvent used. These plants can be further investigated for the different chemical compounds in these three species that can be used for drug development against the different bacterial spp. and the various diseases caused by them. Table 1. Anatomical characterisation of stem Features Piper betle Piper longum Piper nigrum Nonglandular Bicellular trichomes Multicellular trichomes multicellular Collenchyma 5˗6˗layered 2 layers of collenchyma 2 layers of collenchyma, interrupted, cells with slightly and one layer of Multi layers of trichomes Cortex 213 Endodermis and thickened walls and wide lumina parenchyma parenchymatous cells Indistinct absent absent In addition to the central large absent present In two concentric rings; one of Collateral, arranged in 2 Seen as concentric rings the bundles in the inner ring or 3 concentric rings, pericycle Mucilage canal mucilage canal, a ring of smaller canals seen between two rings of vascular bundles Vascular bundles 0 twisted through 90 , about 25 about 15 bundles bundles Table 2. Anatomical characterisation of petiole Nonglandular Piper betle Piper longum Piper nigrum Bicellular trichomes Multicellular trichomes multicellular Collenchyma 5˗6˗layered 2 layers of collenchyma 2 layers of interrupted, cells with slightly and one layer of collenchyma, Multi thickened walls and wide parenchyma layers of trichomes Cortex lumina; Endodermis and parenchymatous cells Indistinct absent absent In addition to the central large absent present In two concentric rings; one of Collateral, arranged in 2 Seen as concentric rings the bundles in the inner ring or 3 concentric rings, twisted through 900, about 25 about 15 bundles pericycle Mucilage canal mucilage canal, a ring of smaller canals seen between two rings of vascular bundles Vascular bundles bundles 214 Table 3. Phytochemical screening of Piper nigrum Phytochemicals P. nigrum stem Ethanol Water P. nigrum leaf Ethanol Water P. nigrum fruit Ethanol Water Alkaloids _ + _ _ _ _ Carbohydrates + + + + + + Proteins + + + + + + Tannins _ _ _ _ + + Flavonoids _ _ _ _ _ _ Cardiac glycosides + _ + + + + Terpenoids + _ _ _ + _ Steroids _ _ + + + _ Phenols + + + _ + + Coumarins + + _ + _ _ Saponins _ _ _ _ _ _ Table 4. Phytochemical screening of Piper longum Phytochemicals P. longum stem P. longum leaf P. longum fruit Ethanol Water Ethanol Water Ethanol Water Alkaloids + _ + _ + + Carbohydrates + + + + + _ Proteins + + + _ + + Tannins + _ + + _ _ Flavanoids _ _ _ _ _ _ Cardiac glycosides + _ + _ + + Terpenoids _ _ + _ + + Steroids + + _ _ _ _ Phenols + _ + + _ _ Coumarins + + + _ _ _ saponins _ _ _ _ _ _ 215 Table 5. Phytochemical screening of Piper betle Phytochemicals P. betle stem P. betle leaf P. betle fruit Ethanol Water Ethanol Water Ethanol Water Alkaloids _ + _ _ _ _ Carbohydrates + + + + + + Proteins + + + + + + Tannins _ _ _ _ + + Flavonoids _ _ _ _ _ _ Cardiac glycosides + _ + _ + _ Terpenoids + _ _ _ + _ Steroids _ _ + + + _ Phenols + _ + _ + + Coumarins + + _ + _ _ Saponins _ _ _ _ _ _ Table 6. Antimicrobial activity of P. nigrum S.no Plant Extract 1 Piper nigrum leaf Ethanol 2 1.7 water 1.5 1 Ethanol 0 3.2 water 0 2.6 Ethanol 2.2 2.4 water 0.5 0 2 3 Piper nigrum stem Piper nigrum fruit Zone of inhibition(cm) E. coli S. aureus Control E. coli S. aureus 3.4 2.2 216 Fig. 1. Zone of inhibition of Piper nigrum extract against E. coli zone of inhibition(cm) Fig. 2. Zone of inhibition of Piper nigrum extract against S. aureus 4 Zone of inhibition of piper longum extracts aganist E.coli 2 ethanol water 0 control Stem extracts Fruit Leaf control Fig. 3. Zone of inhibition of Piper longum extract against E. coli 217 Table 7. Antimicrobial activity of Piper longum S.no Plant Extract Zone of inhibition(cm) E. coli 1 2 3 Control S. aureus Piper longum Ethanol 2 1.7 leaf Water 1.5 1 Piper longum Ethanol 0 3.2 stem Water 0 2.6 Piper longum Ethanol 2.2 2.4 fruit Water 0.5 0 E. S. coli aureus 3.4 2.2 zone of inhibition (cm) Zone of inhibition of Piper longum L. extracts aganist S.aureus 3 2 ethanol 1 water 0 control Stem Fruit control Leaf Extracts Fig. 4. Zone of inhibition of Piper longum extract against S. aureus Table 8. Antimicrobial activity of P. betle S.no 1 Plant Piper betle leaf Zone of inhibition(cm) Extract Control E. coli S. aureus E. coli S. aureus Ethanol 2.2 0 3.4 2.2 water 0.1 0 218 2 Piper betle stem 3 Piper betle fruit Ethanol 2.1 2 water 1.2 1.2 Ethanol 1.3 1.5 water 0 0.5 zone of inhibition (cm) Zone of inhibition of piper betel extracts aganist E.coli 4 3 2 ethanol 1 water 0 control control Stem Fruit Leaf extracts Fig. 5. Zone of inhibition of Piper betle extract against E. coli zone of inhibition(cm) Zone of inhibition of piper betel extracts aganist S.aureus 2.5 2 ethanol 1.5 1 water 0.5 0 control Stem Fruit Leaf extracts Fig.6. Zone of inhibition of Piper betle extract against S. aureus References Arora, D.S., & Kaur, J. (1999). Antimicrobial activity of spices. International Journal of Antimicrobial Agents, 12(3),- Singh, C., Singh, S.K., Nath, G., & Rai, N.P. (2011). 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