Djoko Agus , Purwanto

M. Rasjad, Indra

Susilowati, Susilowati

Sutini, -

English

UPN Jatim Repository

THE EXTRACTION PROCESS OF TRIMETHYL XANTHINA IN VITRO CULTURE OF CALLUS CAMELLIA SINENSIS  WITH ETHYL ACETATE SOLVENT  Sutini 1, Susilowati 2, Djoko Agus Purwanto 3, M. Rasjad Indra4 1Agrotechnology Department of Agriculture Faculty  UPN ”Veteran” Jawa Timur, Indonesia 2Chemical Engineering Department of  Industrial Technology Faculty UPN ”Veteran” Jawa Timur, Indonesia 3Department of  Pharmaceutical , Faculty of Pharmacy, Airlangga  University, Jawa Timur,  Indonesia 4Department of the school physiology  Faculty of  Medical Brawijaya  University, Malang, Indonesia E-mail: tien_basuki@yahoo.com  ABSTRACT Trimethyl xanthina is one of the compounds contained bioactive culture in vitro Cammelia sinensis callus which is widely used in the field of food, beverage, agriculture and health industries. The presence of trimethyl   xanthina on food, beverages and health is needed in a certain amount depending on the use which is achieved by the user. To get a certain amount of trimethyl   xanthina from callus culture of Cammelia sinensis, the extraction process is performed on the water solvent, as well as non-solvent water / organic solvent such as ethyl acetate. The purpose of this study was to obtain profile of trimethyl xanthina in the extraction of Cammelia sinensis callus. The experimental methods used consisted of dissolution, filtration, extraction with water solvent and ethyl acetate, then followed by identification of trimethyl   xanthina using HPLC. The results shows the profile form of trimethyl xanthina of Cammelia sinensis callus have similarities with the standard form of trimethyl xanthina.  Keywords: cultured in vitro, Cammelia sinensis callus, trimethyl   xanthina.   INTRODUCTION Trimethyl xanthina is one of the bioactive compounds found in vitro culture of callus Cammelia sinensis (Shervington et.al., 1998). Trimethyl xanthine, according to a study of Wei et.al. (2008), could be raised in production by culture in vitro by addition of precursors of purine. Research conducted by Li et.al.., (2008) shows that gene trimethyl xanthina could expressed on new Cammelia sinensis leaves. According to Shane et.al. (2013) on new Cammelia sinensis leaves, trimethyl xanthina is synthesized in the chloroplasts of cell during photosynthesis than subsequently transported to vascular tissue for plant defense of against pathogens and predators  (Ministry for primary Industries Manato Ahu matua New Zealand Government.  2012). Trimethyl   xanthina is widely used in many fields of food and beverage industry, agriculture, and health.  For the health, trimethyl xanthina plays a role in physiological effect on increasing the freshness of the human body (Gramza-Michałowska 2014). According to Rebecca et.al. (2013), trimethyl xanthina is classified as food and medicine with a particular dose. Toxic dose is 100 mg for an average adult per day. But according to Krueger and Howard (2011), dose of trimethyl xanthina most likely to be effective without causing undesirable side effects is between 100-600 mg. In the field of food-beverage industry, bioactive Trimethyl xanthina increase levels of dopamine, which will be activated the body's metabolism (Susita 2014). In the beverage industry, trimethyl xanthina of Cammelia sinensis leaves presence is expected and loved by the British people (Kato 1989).  In the agricultural industry, trimethyl xanthina is used to eradicate the beetle (Hewavitharanage et.al.., 1999). Almost all varieties of tea plants containing  trimethyl xanthina, but the content of this  trimethyl  xanthina varies depending on the age of the plant, harvesting  techniques, the particle size of the harvested leaves, (Hyong et.al.., 2007) and the equipment used in the manufacture of liquid extraction (Astill et.al.., 2001). According Komes et.al.., (2009) the content of trimethyl xanthina has been associated with the origin of plants and plant growth conditions. The use of liquid in  liquid-liquid extraction method includes the separation of trimethyl xanthina compound that soluble in chloroform solvent, is partially soluble in the solvent ethyl acetate, but in this study in addition to extraction using chloroform also using ethyl acetate solvent. The use of another solvent is ethanol at the optimum temperature (temperature of 60° C) extraction time of 240 minutes, resulting in the highest trimethyl xanthina  biomass. While the extraction temperature of 40 ° C, 15 min extraction time decreased biomass (Setyo Pratomo 2014). The purpose of this study was to obtain profile of trimethyl xanthina found in in vitro callus cultures of Camellia sinensis with the method of callus extraction using ethyl acetate solvent and using HPLC method for the analysis.  METHODOLOGY  Callus Cammelia sinensis was made from leaf explants of tea plucked from PT Perkebunan  Nusantara (PTPN) XII Lawang, Malang, East Java. Distilled water, chloroform, ethyl acetate, and trimethyl xanthina raw material supplies from specialized sales agents from Sigma. The experimental method was performed   as follows: (1) in vitro callus induction by growing tea leaf explants on media with growth regulating substances and the maintenance (2) harvesting callus followed by weighing callus and callus morphological observation. (3) Isolation and trimethyl xanthina bioactive extraction (4) qualitative identification of trimethyl xanthina of tea callus by HPLC.      , Web of Conferences DOI: 10.1051/  conf/2016MATEC 58 matec 501026 (2016) 801026BISSTECH 2015       © The Authors,  published  by EDP Sciences.  This  is  an  open  access  article  distributed  under  the  terms  of  the Creative  Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Tools   for analysis included High-performance liquid chromatography (HPLC) Agilent 1100   with the specification: detector spectrophotometer UV-ST diode array, with the column RP 18 Waters μ Bondapak 10 μm, 3.3 x 300mm. (2) filter  "Nylon  membrane f ilter "0.2 μm (3) analytical balance (Shimadzu) with 0.001 mg sensitivity (3) pumpkin separating funnel, 5 ml volumetric flask (4) rotavapour.Callus induction in vitro The results of in vitro callus induction was obtained callus growth starts from explant   changes from inflated shape of tea leaf tip, morphological changes such as the formation of callus from the cut edge of the wound leaves until the entire surface filled with callus.Harvesting callus Formed callus was harvested then weighed and the callus morphology is observed in micro and macroscopicly (Figure 1). Isolation and extraction Isolation and extraction of trimethyl xanthinawas done by carrying out the extraction of tea callus by smoothing callus   then  carefully weighed with ± 415 mg (predetermined water content by gravimetric method) and adding hot distilled water with temperature of 70-80 º C 25.0 m L, settling for ± 30 minutes (Shirai et.al.., 1994). After that filtered, and put into a 50.0 mL volumetric flask. The dregs of tea callus was rinsed with 10 mL of hot water 2 times, settling ± 30 minutes and then filtered. The second and the third extract results were subsequenlycollected in the same flask, then add distilled water up to 50.0 m L. Put 25.0 mL Tea extract solution was then gently shaken with 25 mL of chloroform in a separating funnel, it will form two layers, the bottom and the top. Bottom layer is the chloroform phase while the upper layer is the water phase. Mixing with chloroform was repeated twice. From obtained water phase, take 25.0 mL then extract with 25 mL of ethyl acetate 3 times to form two layers. Bottom layer is the water phase and the upper layer is the ethyl acetate phase. Ethyl acetate phase is accommodated and rotavapored to obtain a dry extract. Identification by HPLC The obtained dry extract was dissolved in methanol and then injected into the HPLC (Karlina 2006), then it will obtain the form of trimethyl xanthina profile with retention time (RT) which is approximately equal to the standard   (Figure 2).Figure-1. The shape of  macroscopis callus (A). Microscopic callus with microscope triokuler with 400x magnification (B). (Bars = 1cm)BA   , Web of Conferences DOI: 10.1051/  conf/2016MATEC 58 matec 501026 (2016) 801026BISSTECH 2015    2RESULTS AND DISCUSSIONSGreenish white callus induction, along with greenish white growth on the wound surface is response to damage. According to Sutini (2010) when the tea leaf is cut, there will be thickening wound called callus. According to research by Borzabad et.al. (2010) on new leaves of the Artemisia vulgaris L plant could induce callus and regenerated with MS medium with growth regulators containing optimum concentrations of 1.0 mg-1 BAP and 3.0 mg-1 NAA.Morphology of callus is microscopically obtained irregular shapes since the cells have not differentiated.Final extraction uses ethyl acetate to get trimethyl   xanthina in a certain amount as research need. Results of research conducted by Amra et.al.(2006) says that the solvent extraction using water, acetone, ethanol, methanol, acetonitrile at a temperature of 60 to 100 C for 240 minutes produces 36 grams of trimethyl xanthina / caffeine / kg dry component.Identification using HPLC obtained chromatogram with the standard retention time is the same with sample retention time. This study using in vitro culture techniques which is relevant to Maria (2013) that in vitro culture can be as an alternative for the production of secondary metabolites Camellia sinensis.CONCLUSIONSTrimethyl xanthine compound is secondary metabolite compounds that are bioactive which can be produced through in vitro culture.ACKNOWLEDGEMENTS The author would like thank Directorate General of Higher Education and National Department of Education Indonesian, which has funded the research Competence Grants 2013-2015.REFERENCESAmra P. U., Mojca S K., Knez M. (2006).  Extraction of active ingredients from green tea (Camellia sinensis): Extraction eﬃciency of major catechins and caﬀeine. J. Food Chemistry  96,  597–605. Figure-2. Chromatogram trimethyl xanthina standard (A) Chromatogram tri trimethyl xanthina methyl   xanthina   callus   (B) with   R is approximately equal. AB   , Web of Conferences DOI: 10.1051/  conf/2016MATEC 58 matec 501026 (2016) 801026BISSTECH 2015    3Astill C., Mark R. B., Dacombe C.( 2001). Factors Affecting the Caffeine and Polyphenol Contents of Black and Green Tea Infusions. J. Agric. Food Chem,49, 5340-5347Borzabad, Karami R,  Sudarshana. 2010. In vitro Plant Regeneration from Leaf Explants of Artemisia vulgaris L. - A Medicinal Herb. J. Modern Applied Science  ( 4): 130Gramza-Michałowska A (2014). Caffeine in tea Camellia sinensis — Content,absorption, benefits and risks of consumption. Abstract.  Journal of nutrition, health & aging (18): 143-149 Hewavitharanage P., Karunaratne S., Kumar S., 1999.  Efect of ca}eine on shot!hole borer beetle "Xyleborus fornicatus of tea "Camellia sinensis. J. Phytochemistry (40): 24-30Hyong S.K., a, Hyung K. C., Sung J.L. (2007). Effect of mass transfer on the removal of caffeine from green tea by supercritical carbon dioxide. J. of Supercritical Fluids ( 42): 205–211. www.elsevier.com/locate/foodchemKato (1989). Camellia sinensis L. (Tea): In Vitro Regeneration. Abstract.  J. Biotechnology in Agriculture and Forestry (7): 82-98Karlina 2006. Determination of Levels Epigallocatechin gallate (EGCG) in tea leaves with HPLC method, Thesis Faculty of Pharmacy Universitas Airlangga  Surabaya.Komes D., HoržIc D., BelšcAK, A. 2009. Determination of Caffeine Content in Tea and Maté Tea by using Different Methods. Czech J. Food Sci. 27:213-216Krueger G., and Howard M.L. 2011. Effects ofPsychoactive Chemicalson Commercial Driver Health and Performance: Stimulants, Hypnotics,Nutritional, and Other Supplements A Synthesis of Safety Practice.www.national-academies.org. Diakses  22-3-2015.Li Y, ogita S, Charman A K, (2008). Expression of caffeine biosynthesis gene in tea (Camellia sinensis). J. Springer Verlag. 267-270Wei W D, Yeyun Li Y,  Ogita S, Ashihara H. (2008). 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The Extraction Process Of Trimethyl Xanthina In Vitro Culture Of Callus Camellia Sinensis With Ethyl Acetate Solvent

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The Extraction Process Of Trimethyl Xanthina In Vitro Culture Of Callus Camellia Sinensis With Ethyl Acetate Solvent

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