CONTENTS |. AIM AND OBJECTIVES 12 . INTRODUCTION 3-6 3. LITERATURE REVIEW 712 |. PLAN OF WORK 13-15 . BIBLIOGRAPHY 16-19CHAPTER-1 AIM & OBJECTIVESCHAPTED. 1 AIM & OBJECTIVES AIM: Antimicrobial resistance is a growing problem due to overuse of antibiotics in humans. Many of the currently used antibacterial are associated with adverse effects such as toxicity, hypersensitivity, immune suppression, and tissue residues posing public health hazard. As the global scenario is now changing towards the use of non-toxic and eco-friendly products, development of modern drugs from traditional medicinal plants should be emphasized for the control of various human and animal diseases. In the present study, the selection of this plant for evaluation was based on its traditional usages. So further new alternative remedies for treatment of bacterial diseases are usually required. Although very few works have been done on the antimicrobial activity of his medicinal plant, it needs further study for verification of its activity against disease causing microorganisms. Thus the aim of this project work is the evaluation of the antimicrobial potency of Leucas plukenerii (F-Laminaceae) 1.2. OBJECTIVES: The present objective of the study is- > Extraction of active constituents from the dried leaves of Leucas plukenetii > Preliminary Phytochemical screening of the extract of leaves of Leucas plukeneti > Antimicrobial acti ity of the extract in comparison to suitable standard, GIPS Page 2CHAPTER-2 INTRODUCTIONCHAPTER 2 INTRODUCTION 2.1, INTRODUCTION: ‘The medicinal use of plants is very old. The writings indicate that therapeutic use of plants, is as old as 4000-5000 B.C. and Chinese used first the natural herbal preparations as medicines. In India, however, earliest references of use of plants as medicine appear in Rigveda, which is said to be written between 1600-3500 B.C. Later the properties and therapeutic uses of medicinal plants were studied in detail and recorded empirically by the ancient physicians in ‘Ayurveda’ which is a basic foundation of ancient medical science in India [1]. The Hindu Physician, Susruta enlisted seven hundred and sixty medicinal plant in Ayurveda in the 5 century AD. [2] Antibiotic resistance is a growing problem. The widespread and indiscriminate use of antibacterial agents resulted in development of drug resistance among many virulently pathogenic bacteria species. Many of the currently used antibacterial are associated with adverse effects such as toxicity, hypersensitivity, immune suppression, and tissue residues posing public health hazard. So, further new alternative remedies for treatment of bacterial diseases are usually required. As the global scenario is now changing towards the use of non- toxic and eco- friendly products, development of modern drugs from traditional medicinal plants should be emphasized for the control of various human and animal diseases. (3] GIPS Page 4CHAPTER 2 INTRODUCTION 2.2, NATURAL SOURCES OF ANTOMICROBIAL AGENTS(AMA). SI Plant ‘Scientific Name Major components No 1 Allspice: Pimenta dioica Eugenol, methyl ether cineol 2 Basil Ocimum asilicum | d-linalool, methyl chavicol, reraniol 3 | Black Pepper | __ Piper nigrum Monoterpenes, Sesquiterpenes a | Caraway Carum canvi Carvone, limonene Seed 3_| Celery Seed | Apium graveolens Himonene 6 | Cinamon ‘Cinamonum Ginnamie aldehyde, Finalool, zeylanicum p-cymene, eugenol 7 Glove | Syzygium aromaticum Eugenol, cariofilene 8 Coriander Coriandum sativum d-linalool, d-pinene, 9 Cumin ‘Cuminum cyminum ‘Cumin aldehyde, p-cymene 10 Fennel Foeniculum vulgare Anethole i Garlic Allium sativum Iyl disulphide, diethyl sulphide, allicin 12_| Lemongrass | Cymbopogon citratus Citral, geraniol 13. | Marjoram | Origanwn majorana Linalool, cineol, methyl chavicol, eugenol, terpininecol 14 Mustard Brassica hirta Ally! thiocynanate 15 ‘Onion Allium cepa ‘&-n-propyl disulphide, methyl-n- propyl disulphide 16 ‘Oregano Origanum vulgare ‘Thymol, carvacrol, p-cymene 17 | Parsley Pertroselinum Pinene, fenol-eter-apiol crispum 18 | Rosemary | Rosmarinus officinalis Borneol, cineol, borneol, thymol, eugenol 19 Sage Salvia officinalis Thujone, cineol, borneol, thymol, eugenol 20 | Tarragon “Artemisia Methyl chavicol, anethode dracunculus 21 Thyme Thymus Vulgaris | ‘Thymol, earvacol, geraniol, p- cymene 22 Vanilla Vanilla planifolia Vanillin, Vanillic, p- hydroxybenzoie, p-cumaric acids GIPS Fig-1: Sources of Natural AMA [4] Page 5CHAPTER 2 INTRODUCTION 2.3.PLANT PROFILE 2.31. BIOLOGICAL SOURCE: It consisted of the dried whole herb of Leucas Plukenetii belonging to the family Lamiaceace. 2.3.2 PLANT DESCRIPTION- © Habit: An annual herb with square stem. Leaves : Linear-oblong or oblong lanceolate. Flowers : Sessile, ciliate with long hairs. Fruit: Oblong, inner face sharply angular © Flowering and Fruiting Time : July-October Plant Form : Herb [5] 2.3.3 VERNICULAR NAME- Doron Bon [6] 2.3.4 SYNONYM- Leucas aspera (7] Fig-2. Leucas plukeneti 2.3.5 ETHNOMEDICINAL USES OF LEUCAS PLUKENETH- Y The leaves and flowers are used by the Ka (8) Y Itis applied on inflamed parts to relieve pain and inflammation. [9] ses to treat tooth infections and mucus. < Leave paste made with lime juice applied externally in the treatment of Headache. Y Flowers were macerated and extract put dropwise into opposite side of nostril to reduce migraine, Y Leaves with pepper and garlic chewed and spilt into the nostril with force in case of SNAKE BITE. Y Whole plant dugout early in the morning and made into paste with water to treat Wounds and worms. [10] GIPS Page 6CHAPTER-3 LITERATURE REVIEWCHAPTER 3 LITEARATURE REVIEW 3.1, PHYTO-CHEMICAL EVALUATIO! 3.1. Sadhu et al. (2003) isolated eight lignans namely nectandrin B , (-)-chicanine , -(4-allyl-2,6- (4-hydroxy-3-methoxy phenyl) propan-1-ol, Machilin C, (7R,8R)- meso-dihydroguaiaretic acid, macelignan , myristargenol B , erythro dimethoxyphenoxy)- and (78,88)-licarin from the methanol extract of the whole plant. 3.1.2. Manivannana and Sukumar (2007) reported free flavonoid ‘baicalein’ was reported in the ethereal fraction of hydro methanolic extract of flower. 3.1.3. Gerige et al. 2007 & Mangathayaru et al. (2006) found to contain high amount of a- farnesene, a-thujene and menthol 3.1.4. A new type of diterpenes, leucasperones A and reported by Sadhu et al. (2006). ; leucasperols A and B, have been 3.1.5. Linoleic acid were found which was contingent upon crop variation by Chen et al, (1979). 3.1.6. Rajyalakshmi et al., 2001) found Leucas aspera and Leucas cephalotes contain significant amounts of total carotenoid and -carotene. [11] 3.2, PHARMACOLOGICAL ACTIVITY: 3.2.1 Antimicrobial activity: Mangathayaru et al., (2005) reported significant antimicrobial activity was reported for the alkaloidal fraction and the total methanol extract the flowers [12] Satyal et al., (2013) reveals that the essential oil of the plant showed no activity against E. coli, P. aeruginosa, and C. albicans (MIC > 1250y1g/mL). The oil did exhibit good ity against S. aureus (MIC = 625 pg/mL), B. cereus (MIC = 313ug/mL), and A. niger GIPS Page 8 aCHAPTER 3 LITEARATURE REVIEW (Mic 13 j1g/mL), most likely attributable to the sesquiterpenes present in the oil. Both (®)-caryophyllene and a-humulene have shown antibacterial activity against B. cereus and S. aureus, and a-humulene was antifungal to A. niger [13] Tango et al., (2008) evaluated and found that Ethyl acetate extract (EAE) of whole plant exhibited moderate to significant and concentration dependent antibacterial activity against all the tested microorganisms at the concentrations of 50, 100, 200, 300 and 400 higidise and comparable to the various antibiotics used for individual microorganism. This study also reveals that EAE was found to be highly active against Staphylococcus epidermidis and Klebsiella pneumoniae [14] Chew et al. (2012) evaluated the anti obial activity of crude extracts of root, flower, leaf having notable antibacterial activity. The root extract showed the highest mean zone of inhibition ranging from 9.0-1 1.0 mm at a concentration of 100 mg/mL.[15] Antony et al. (2013) describes green synthesis of silver nanoparticles (AgNPS) utilizing the plant. Antimicrobial activity of the AgNPs was tested against Aeromonas hydrophila. Catla catla, the model organism used for the experiment was divided into six groups with 15 animals in each group. In vivo analysis of biochemical parameters and histological architecture provided evidence for the antibacterial effect of AgNPs in the fish model.{16] 3.2.2 Central nervous system activity: Rahman et al. (2007) found that ethanolic extract of root showed significant peripheral antinociceptive activity at a dose of 400 mgykg . [17] 3.2.3 Antioxidant activity: Rahman et al. (2007) reported significant activity was found in the ethanolic extract of root (IC5Q = 7.5 we/ml) -[17] Chew et al. (2012) evaluated antioxidant activity methanol extract of root and it possessed antioxidant activity near the range of vitamin E and thus could be a potential rich. source of natural antioxidant.[15] GIPS Page 9CHAPTER 3 LITEARATURE REVIEW 3.2.4 Hepatoprotective activity: According to Thenmozhi et al (2013) the Hydroalcoholic leaf extract has found to possess hepatoprotective property by suppressing the abnormal elevation of liver enzymes induced by lead acetate. [18] Mangathayaru et al.,(2005) showed The cold methanolic extract of the whole plant of was found to exhibit significant hepato protection in CCI4 induced liver damage.{19] Banu et al. (2012) found that d-galactosamin administration induced hepatotoxicity in rats which was manifested by increased levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total cholesterol, triglycerides, total bilirubin and oxidative stress. Pre-treatment with LA extract significantly protected the liver in d- galactosamin administered rats. LA extract significantly elevated antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and decreased lipid peroxidation levels in liver. The total phenolic and flavonoid content in LA aqueous extract was found to be 28.33 + 0.19 gallic acid equivalents mg/g of extract and 3.96 + 0.57 rutin equivalent mg/g of extract, respectively. LA extract (200 and 400 mg/Kg) treatment with CCM decreased the hexobarbitone-induced sleeping time in mice by 56,67 and 71.30%, respectively, which indicated the protective effect of LA on hepatic MDMESs. Histological studies showed that LA at 400 mg/kg attenuated the hepatocellular necrosis in d-galactosamin intoxicated rats.[20] 3.2.5 Ant {inflammatory activity: Reddy et al. (1986) reported that whole plant extract have anti-inflammatory activity and caused degranulation of mast cells [17] Reddy et al. (1986) reported anti-inflammatory activity of the yellow colored chromatographic fraction of extract was observed in the chronic and acute models of inflammation. It was observed that, the activity was due to the inhibition of histamine and serotonin [21]. GIPS Page 10CHAPTER 3 LITEARATURE REVIEW Similar observations were made by Saundane et al., (2000) and concluded that, on preliminary screening of ethanol and di led water extracts exhibited significant anti- inflammatory activity, whereas only ethanol (95%) extract produced long term analgesia in the experimental animals. [22] Srinivas et al. (2000) reported that the dried leaves of for the alcoholic and aqueous extracts of the plant possessed significant anti-inflammatory activity against carrageenan- induced paw oedema and cotton pellet induced granuloma. (23] Goudgaon et al. (2003) concluded that the alkaloid fraction of the crude ethanolic extract is accountable for the ant flammatory activity. (24] 3.2.6 Cytotoxicity: Krishnaraju et al. (2005) proves that the hydro alcoholic extract of whole plant exhibited cytotoxicity (LCs) = 1,900 j1g/ml)[16] and this activity was more in the root extract (LCsp = 52.8p.g/ml) [25]. Chew AL et al, (2012) performed. brine shrimp lethality bioassay , it was evident that the methanol root extract did not show significant toxicity. The LCso value for 12 h and 24h observation was 2.890 mg/mL and 1.417 mg/mL, respectively. [15] 3.2.7 Larvicidal Activity: Maheswaram et al. (2008) The findings of the present investigation revealed that L. aspera has good larvicidal activity of the plant activity against Culex quinquefasciatus Say. and Aedes aegypti [26] 3.2.8 Antihyperglycemic Activity: Mannan et al. (2010) performed an experiment upon antihyperglycemic activity of plant extract at mice, The methanol extract of leaves, when administered to glucose-challenged mice demonstrated dose-dependent and significant antihyperglycemic activity The stem extract also demonstrated significant and dose- dependent antihyperglycemic activity in glucose tolerance tests in mice. [27] GIPS Page 11CHAPTER 3 LITEARATURE REVIEW However, dose for the antihyperglycemic activity demonstrated by stem extract was lower than that of leaf extract. At the highest dose of 400 mg extract per kg body weight, serum glucose levels fell by 28.39% versus 34.01% obtained with the same dose of leaf extract. To summarize, antihyperglycemic activity at a statistically significant level was observed with leaf extract, when the extract was administered at doses of 200 mg per kg body weight to glucose-loaded mice. [27] GIPS Page 12CHAPTER-4 PLAN OF WORK Page 13CHAPTER 4 PLAN OF WORK 4. PLAN OF WORK Collection of Plant Material. Washing of plan material. Authentication of Plant Material by GU. Drying and Grinding of plant material. Extraction of Plant Material (Maceration Process). Preliminary phytochemical screening of extract. Determination of Antimicrobial Activity. Fig 3-Plan of work GIPS Page 14CHAPTER 4 PLAN OF WORK 4.1, Collection of Plant Material: The fresh whole aerial parts of the plant Lewcas plukentii was collected from various parts of the District Lakhimpur, Assam. 4.2, Washing of Plant Material: The collected plant material was washed using water and then it is sundried to remove moisture. 4.3, Authentication of Plant Material: Herbarium sheet was prepared using the cleaned plant material. Then it was authenticating at Department of Botany, Gauhati University. 4.4. Drying and grinding of plant material: The cleaned plant material will be dried at 60° using Tray dryer and then it will be grinded to form powder. 4.5. Extraction of Plant Material: The sieved powder drug will be extracted by maceration process using ethanol as solvent. 4.6. Preliminary phytochemical screening of extract: The Preliminary phytochemical screening of extract will be done by tested the extract for glycosides, alkaloids ete, 4,7. Determination of Antimicrobial Activity: The potency of extract will be determined by agar disc diffusion method against a standard. GIPS Page 15CHAPTER-5 BIBLIOGRAPHY Page 16CHAPTER 5 BIBLIOGRAPHY BIBLIOGRAPHY 1. Prakash P and Gupta N. Therapeutic uses of Ocimum sanctum linn (tulsi) with a note on eugenol and its pharmacological actions; a short review. Indian Journal of Physiology and Pharmacology 2005; 49: 125-131. 2. Guthrie D.A. History of Medicine. London, Thomas Nelson and Sons Ltd: 1945; 3. Das A. A study of antibacterial activity of ethanolic extracts and aqueous extracts of Leucas longifolia (doron) leaves against Eschreiashia coli. Intemational Research Journal of Pharmacy 2012; 3: 130-132. 4. Michael P, Sofos J, and Branen A . Antimicrobials in Food, Tailor & Francis group; ‘Third Edition : 2005; p- 429-507. Http://www efloraofgandhinagar.in/herb/leucas-aspera [Accessed 21 October 2016] 6. Hitp:/hvww.flowersofindia.net/catalog/slides/Common%20Leucas.html [Accessed 02 November 2016] 7. htip://www.theplantlistorg/tpl1.I/record/kew-111857 [Accessed 02 November 2016) 8. Mohammed R, Das AK, Ariful Md., Haque M, Rownak J, Khan M, Rahman T, and Chowdhury MH. An Ethnomedicinal Survey of Dhamrai Sub-district in Dhaka District, Bangladesh. American-Eurasian Journal of Sustainable Agriculture 200% 881-888, 9, Rahmatullah Q, Bhatti R, and Rabia A. Ethnomedi part of nara desert,Pakistan. Pakistan Journal of Botany 2010; 42(2): 839-851. 10, Parinitha M, Harish GU, Vivek NC, Mahesh T, and Shivanna MB. Ethnobotanical al uses of herbs from northern wealth of Bhadra Wild life sanctuary of Karnataka. Indian Journal of Traditional knowledge 2004; 3(1): 37-50. 11, Singh SK, and Chowhan HS. A review of plants of genus Leucas. Journal of Pharmacognosy and Phytotherapy 2011; 3: 13-23. 12. Mangathayaru K, Lakshmikant J, Sundar NS, Swapna R, Grace XF, and Vasantha J. Antimicrobial activity of Leucas aspera flowers. Fitoterapia 2005; 76: 752-754, GIPS Page 17CHAPTER 5 BIBLIOGRAPHY 13, Satyal P, Paudel P, Poudel A, and Setzer WN. Microbiological activities of volatile constituents of Leucas aspera (Willd). Nepal Journal of Natural Pharmaceuticals 2012 ; 3: 118-119. 14, Hangos k. Ramya s and Gopinath G. Antibacterial activity of Leucas aspera spreng. International Journal of Chemical Sciences 2008; 6 :526-530. 15. Chew AL , Jessica JJA and Sasidharan S. Antioxidant and antibacterial activity of different parts of Leucas aspera. Asian Pacific Journal of Tropical Biomedicine, 2012; 2: 176-180. 16. Antonya JJ, Nivedheethaa M, Sivaa D, Pradeephaa G, Kokilavania P, Kalaiselvia S, Sankarganesha A, Balasundaramb A, Masilamanib V, and Achiramana S. Antimicrobial activity of Leucas aspera engineered silver nanoparticles against Aeromonas hydrophila in infected Catla catla. Colloids and Surfaces B: Biointerfaces 2013; 109: 20-24. 17. Rahman MS, Sadhu SK, and Hasan CM. Preliminary antinociceptive, antioxidant and cytotoxic activities of Leucas aspera root. Fitoterapia 2007; 78: 552-55: M, Dhanalakshmi M, Devi KM, Sushila K, and Thenmozhi S. Evaluation 18. Thenmoz! of hepatoprotective activity of Leucas aspera hydroalcoholic leaf extract during exposure to lead acetate in male albino wistar rats. Asian Journal of Pharmaceutical and Clinical Research 2013; 6 : 78-81 19, Mangathayaru K, Fatima GX, Bhavani M, Meignanam E, Karna SLR and Kumar DP. Effect of Leucas aspera on hepatotoxicity in rats. Indian Journal of Pharmacology 2005 ; 37 : 329-330. 20. http://www. tandfonline.com/doi/abs/10.3109/13880209.2012.685130 21, Redy MK, Viswanathan $, Sambantham PT, Ramachandran $, and Kameswaran L. Effects of Leucas aspera on experimental inflammation and mast cell degranulation. Ancient Science of Life 1986; 5: 168-171. 22, Saha K, Mukherjee PK, Mandal SC, Saha BP, and Pal M. Antiinflammatory evaluation of Leucas lavandulaefolia Rees Extract, Natural Product Sciences 1997; 2: 119-122. GIPS Page 18CHAPTER 5 BIBLIOGRAPHY 23. Srinivas K, Rao MEB, and Rao SS. Anti-inflammatory activity of Heliotropium indicum Linn, And Leucas aspera Spreng. in albino Rats. Indian Journal of Pharmacology 2000; 32: 37-38. 24, Goudgaon NM, Basavaraj NR, and Vijayalaxmi A. Anti flammatory activity of different fractions of Leucas aspera spreng. Indian Journal of Pharmacology 2003; 35: 397-398. 25. Krishnaraju AV, Rao TVN, Sundararaju D, Vanisree M, Tsay HS, and Subbaraju GV. Assessment of bioactivity of Indian medicinal plants using brine shrimp (Artemia salina) lethality assay. International Journal of Applied Science and Engineering 2005; 3: 125-134. 26. Maheswaran R, Sathish S, and $ Ignacimuthu S. Larvicidal activity of Leucas aspera (Willd) against the larvae of Culex quinquefasciatus Say. and Aedes aegypti L. International Journal of Integrative Biology 2008; 2 : 214-217. 27.Mannan A, Das S, Rahman M, Jesmin J, Siddika A, Rahman M, Rahman S, Chowdhury M and Rahmatullah M. Antihyperglycemic Activity Evaluation of Leucas Aspera (Willd.) Link Leaf and Stem and Lannea Coromandelica (Houtt.) Merr. Bark Extract in Mice. Advances in Natural and Applied Sciences 2010; 4(3): 385-388. GIPS Page 19