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Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
ISSN: 2319-7706 Volume 3 Number 9 (2014) pp. 573-581

Original Research Article
Studies on the comparison of phytochemical constituents and
antimicrobial activity of Curcuma longa varieties
S.Shanmugam* and P.Bhavani
Department of Biochemistry, Asan Memorial College, Chennai-600 100, Tamil Nadu, India *Corresponding author A B S T R A C T
Turmeric comes from the root of curcuma longa plant and has a tough brown skin and a deep orange flesh. Turmeric has long been used as a powerful anti-inflammatory in both the Chinese and Indian systems of medicine. Turmeric was traditionally Called Indian saffron because of its deep yellow-orange color and has been used throughout history and condiment, healing remedy and textile dye. Turmeric is rich in curcuminoids. Curcuminoids vary in chemical structures, physiochemical characteristics. The present work reports on extraction method using Soxhlet extractor, characterization and separation of phytochemicals using Curcuma longa, TLC and FT-IR methodology. The present study aimed at comparing the in vitro antimicrobial activity of two varieties (cosmetics and food) of turmeric and to screen in bacterial (E.coli, Staphylococcus aureus) and fungal (Aspergillus niger, Aspergillus fumigatus) species. Introduction
Medicinal Plant:
India has a rich culture of medicinal herbs and spices, which includes about more than Organization, most populations still rely on 2000 species and has a vast geographical traditional medicines for their psychological area with high potential abilities for and physical health requirements (Rabe and Van Stoden, 2000), since they cannot afford medicines but only very few have been the products of Western pharmaceutical studied chemically and pharmacologically industries (Salie et al., 1996), together with for their potential medicinal value (Gupta et their side effects and lack of healthcare al., 2005; Sandhu et al., 2005). Human facilities (Griggs et al., 2001). beings have used plants for the treatment of diverse ailments for thousands years Rural areas of many developing countries (Sofowara, 1982; Hill et al., 1989). still rely on traditional medicine for their Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
primary health care needs and have found a yield of essential oil in various parts is 1.3% place in day-to-day life. in leaf, 0.3% in flower, 4.3% in root and 3.8% in rhizome. Curcuma longa:
Minimum inhibitory concentration:
Turmeric is the rhizome or underground stem of ginger like plant. The plant is an In microbiology, Minimum
Inhibitory
herbaceous perrineal, 60 90 cm high with a Concentration (MIC)
short stem tufted leaf. Its flowers are yellow, lowest concentration of an antimicrobial that between 10 15 cm in length and they group visible growth of together in dense spikes, which appear from a microorganism after overnight incubation. the end of spring until the middle session. Minimum inhibitory concentrations are important in diagnostic laboratories to No fruits are known for this plant. The confirm resistance of microorganisms to an whole turmeric rhizome, with a rough, antimicrobial agent and also to monitor the segmented skin. The rhizome is yellowish- activity of new antimicrobial agents. A MIC brown with a dull orange interior that looks is generally regarded as the most basic bright yellow when powdered. laboratory measurement of the activity of an antimicrobial agent against an organism. Rhizome measures 2.5 7.0 cm (in length), and 2.5 cm (in diameter) with small tuber Antimicrobial activity:
branching off. Turmeric held a place of honor in Indian traditional ayurvedic An antimicrobial or antibiotic is an agent
medicine (Fig.1). that kills microorganisms or inhibits their growth. Antimicrobial medicines can be Molecular constituents in turmeric:
grouped according to the microorganisms they act primarily against. For example, Turmeric has hundreds of molecular antibacterial are used against bacteria and constituents, each with a variety of antifungal are used against fungi. They can biological activities. For instance, there are also be classed according to their function. at least 20 molecules that are antibiotic, 14 Antimicrobials that kill microbes are are known cancer preventives, 12 that are called microbicidal; those that merely inhibit anti-tumor, 12 are anti-inflammatory and their growth are called micro biostatic. there are at least 10 different anti-oxidants. Infect, 326 biological activities of turmeric are known. This is also testimony to the use infectious agents are becoming more of whole herbs and not just isolated compounds (Hancock et al., 2012). Speaking of molecules by far the most The necessity to develop new drugs requires researcher in turmeric are the three gold- varied strategies, among them, the bio Demethoxycuccumin produced by medicinal plants (Dionisi et Bisdemethoxycurcumin. al., 2012; Benko-Iseppon et al., 2010). research done is with 95% curcuminoids extract of turmeric, through in its raw state turmeric is only 3 5% curcuminoids. The Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
Materials and Methods
dispensed into the wells of inoculated plates. Collection & extraction:
Sterilized distilled water and ethanol were The two varieties (cosmetic used turmeric used as a control which were introduced into and food used turmeric) plant material of the well instead of plant extract. The plates Curcuma longa or turmeric rhizomes is used thus prepared were refrigerated for 60 in this study was collected from provision minutes allowing the diffusion of the extract into the agar. After incubation for 24 hr at 37°C, the plates were observed. Fresh rhizomes were cleaned washed with deionized water. Sliced and dried in the If antimicrobial activity was present on the sun/shade for one week and again dried at plates, it was indicated by an inhibition zone 50°C in a hot air oven for 6 hours. Dried surrounding the well containing the extract. rhizomes were cut in small pieces, and The zone of inhibition was measured and ground it into a powder. expressed in millimeters (mm). Approximately 10g of sample were taken Separation by TLC:
into a thimble and placed in a Soxhlet Apparatus. 150ml of solvent was added and Slurry of silica gel is prepared by dissolving extracted according to their boiling point for 20 g of gel and 50 mg of calcium sulphate in 6 hours. The solvents used were aqueous about 50 ml of distilled water. This is coated 70 to 90°C) and ethanol uniformly over a glass plate and the plate is 40 to 50°C) for the actively by heating at 100°C. Standards and unknown solution are spotted at the base of completion of extraction the dark brown the plate. Then the plate is then developed in extract was stored in refrigerated condition a chamber saturated with the solvent. The solvent is marked and the plate is dried in air. The lipids are located by keeping the Antimicrobial activity:
plate in an iodine chamber and keeping the plate in a hot air oven at 100°C for 15 Method performed by a sterile cotton swab minutes. The sample is the unknown was dipped into the respective microbial solutions are identified by comparing their suspension and surplus removed by rotation Rf value with those of the standard. of the swab against the sides of the tube above the fluid level. The agar media plates Characterization by FT-IR:
organisms by even streaking of the swab Accurately weighed quantity of plant extract over the entire surface of the plate three about 10 g was dissolved in ethanol by using times, rotating the plate approximately 60 volumetric flask. To this 1.5ml of each degrees after each application to ensure an sample (Cosmetic used turmeric and Food even distribution of the inoculums. Finally, it was swabbed all around the edge of the transmittance of composition present in agar surface. Wells of 7 mm size were made clove and it performed under Fourier with sterile borer into agar plates containing the bacterial and fungal inoculums. 0.1 Instrument name is CPU ml/100µl volume of each of the plant extract Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
Different wavelength shows presence of the Second method to determination of TLC particular functional group of compounds. in Cosmetic used and Food used Curcuma From above data one can confirm the longa varieties. phytochemical analysis and chemical test As a result of Rf values for Petroleum ether: which is further reconfirmed by maximum Ethyl ether: Glacial acetic acid solvent is wavelength, Refractive Index (Rf) value and about 0.3 and 0.7 for cosmetic used extract such as aqueous and ethanol respectively. And also 0.2 and 0.5 for food used extracts components was confirmed by comparison such as aqueous and ethanol respectively. of wavelength and authentic standards. The results were shown in (Table.2 and Fig. 3 & 4). Results and Discussion
Better resolution of Rf value showed that inhibition
Petroleum ether, Ethyl ether and glacial antimicrobial activity
acetic acid can be suitable solvent for the separation using TLC method in this study. Among all the zone of inhibition for extracts of plant part, Food used turmeric ethanol different varieties of turmeric:
bacterial and also fungal microbes. Other extracts were also found effective against In Fourier Transform-Infrared Spectroscopy microbes except Staphylococcus aureus due Analysis the Cosmetic and Food used to its inhibition zone formation (Table. 1 and ethanolic extract of Curcuma longa analyzed trough FT-IR Spectrophotometer where it showed 10 wavelengths in cm-1with their TLC method using two different mobile
respective transmittance at %T in Cosmetic used turmeric extracted (ethanol extract) sample and also 12 wavelengths in cm-1 Different compositions of mobile phase were their respective transmittance at %T in Food tested in TLC (Thin Layer Chromatography) used turmeric extracted (ethanol extract) for the separation and its Rf values were determined. Chloroform: Methanol in 95:5 ratios is a Mobile Phase for the First method Graph showed many peaks for different to determination of TLC in Cosmetic used functional groups present in plant extracts and Food used Curcuma longa varieties. As a result of Rf values for Chloroform: Methanol solvent is about 0.2 and 0.6 for Fourier Transform-Infrared Spectroscopy cosmetic used extract such as aqueous and analysis showed different functional group ethanol respectively. wavelength in graph. Modern instrumental And also 0.5 and 0.6 for food used extracts technique play an indispensable role in the such as aqueous and ethanol respectively. Petroleum ether: Ethyl ether: Glacial acetic Spectroscopy is an invaluable tool for acid in 90:10:1 ratio is a Mobile Phase for determining the presence or absence of Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
certain functional group or hydroxyl group Previous estimation of Curcuma longa done such as carbon-carbon multiple bonds, by FT-IR spectrum of eugenol showed the aromatic rings, carbonyl group or hydroxyl presence of alcoholic group (Reddy et al., group in a molecule (Berger and Sicker, Table.1 Maximum Zone of inhibition of all Microbes
ZONE OF INHIBITION (mm) FOOD USED TURMERIC Staphylococcus Aspergillus niger Aspergillus fumigatus Table.2 TLC Method in two different mobile phases
TLC Mobile Phase Chloroform: Methanol Petroleum ether: Ethyl Glacial acetic acid Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
Fig. 1 Medicinal properties of Curcumin
Fig. 2 Maximum Zone of Inhibition







Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
Fig. 3 Mobile Phase Chloroform: Methanol in 95:5 Ratios
Fig. 4 Mobile Phase Petroleum ether: Ethyl ether: Glacial acetic acid in 90:10:1 Ratio
Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
Fig. 5 Characterization of compounds in cosmetic used turmeric extract
Fig. 6 Characterization of compounds in food used turmeric extract
Int.J.Curr.Microbiol.App.Sci (2014) 3(9) 573-581
Concluded from the present study is Chemistry, Technology and Quality. ethanolic extract of food used turmeric have CRC Food Sci. Nutr., 12: 199 301. the most potential antimicrobial activity Gupta, A., Gupta, M., Sushil Kumar, 2005. when compare to other extracts. However, ethanolic and aqueous extract was found to Curcuminoids in curcuma sample using be inhibiting fungi (Aspergillus niger), but HPLC. J. Liq.Chrome. Rel. Technol., (Staphylococcus aureus). Comparison with Hill, Hidaka, K., Masuda, T., Yamaguchi, H., Gentamycin and Fluconazole showed 18 1989. Chemical studies on antioxidant mm which is nearer inhibition zone of food mechanism of curcumin analysis of used turmeric extract against Aspergillus oxidative coupling products from niger and Staphylococcus aureus. And also curcumin and linoleate. J. Agri. Food Chem., 49: 2539 2547. characterization using TLC and FT-IR the Honcock, F., Greger, H., 2012. Testing of food used turmeric is rich in phytochemical components when compare to cosmetic used methodology, comparability of result turmeric. The finding of the present study and assay choice. Phytochem. Anal., clearly indicates that the extract of food used rhizome which has proved evidence for its Rahe, Van Stoden, Benny Antory, 2000. antimicrobial potential. Isolation purification and identification of chromatography. J. Exp. Sci., 2: 21 25. I express my deep indebtedness to my Reddy, C.S., Ravindran, P., Babu, K.N., supervisor/guide Mr. S. Shanmugam, M.Sc., Sivaraman, K., 2009. In: Turmeric: the M.Phil., Assistant Professor, Department of genus curcuma, Boca. p.150 155. Biochemistry, Asan Memorial College of Salie, Simon, A., 1996. Inhibitory effect of Arts and Science for his unfailing guidance, curcuminoids on MCF valuable suggestion and constant help structure-activity throughout the period of study and to my relationship. 129: 111 116. sincere thanks to Dr. S.T. Asheeba, M.Sc., Sandhu, Heinrich, Sogi, S.D., 2005. Effect of M.Phil., Ph.D., Head of the Department of extraction parameters on curcumin Biochemistry, Asan Memorial College for yield from turmeric. J. Food Sci. her encouragement and support in caring out Technol., 47(3): 300 304. the project work. Sofowara Schieffer, G.W., 2002. Pressurized liquid extraction of curcuminoids and References
curcuminoid degradation product from turmeric subsequent HPLC assay. J. liq. Benko-Iseppon, Crovella, Bansod, S., Rai, Chromatogr. Related Technol. 25: M., 2010. World J. Med. Sci., 3(2): 81. Berger, S., Sicker, D., 2009. In: Classic in Spectroscopy, isolation and structure elucidation of natural products, Wiley VCH, Weinheim, Germany. Dionisi, Davis, 2012. J. Sci., 264: 375 382. Griggs, Govendarajan, 2001. Turmeric -

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