Gc-Ms Analysis of Ethanolic Stem Extract of Clausena anisata (Willd.) Hook F Ex Benth

Objective: In the present study, the bioactive components present in the ethanol stem extract of Clausena anisata was analyzed by using Gas Chromatography Mass Spectrometry analysis technique (GC-MS). Clausena anisata, a medicinal plant belonging to the family Rutaceae, is represented by 20 species available in India and used traditionally for the treatment of several ailments but there is a requirement to identify its phytoconstituents, its target, mode of action and treatment using plant products either alone or in combination with synthetic drugs. Methods: Clausena anisata stem was procured from Manamettupatti, a village of Pudukottai District, Tamil Nadu. The shade dried stem was powdered and extracted using ethanol by maceration method. One microlitre of the extract was subjected to GC-MS analysis to detect the presence of bioactive compounds present in the stem of C. anisata. Results:The results showed that the ethanol stem extract of C. anisata contained nine bioactive compounds, of which the major one is n-hexadecanoic acid (78.54%), followed by 8-octadecenoic acid, methyl ester, [E](6.638%). The total number of compounds obtained was compared with National Institutes of Standard and Technology (NIST) library that contains more than 62,000 known compounds based on retention time and molecular mass. Conclusion: In this study, nearly nine compounds have been identified from the ethanolic stem extract of C. anisata using GC-MS analysis which was mainly composed of fatty acids and sterols. The GC-MS analysis is used to understand the nature of active principles present in this plant revealed that the plant can be used as a potential source of new useful drugs.


INTRODUCTION
Clausenaanisataof family Rutaceae is locally known as "Kattukarveppillai", is a deciduous shrubs or small treethat grows up to 10m, found in India, Tropical and South East Asia,which is indigenous in South Africa.Leaves are pinnately compound with 10 to 17 alternate leaflets and a terminal leaflet.The leaves are densely dotted with pellucid glands and have a strong scent when bruised.Flowers are small and attractive, white with orange-yellow stamens and havinggynophores.In Mozambique, it is locally known as "Horse wood". 1 In Clausena anisata is the iron content was found to be >1000 µg/day and this may be useful for the persons with micronutrient deficiency. 2 The dry mass per unit area of C. anisatais 3.24, nitrogen concentration per unit dry mass is 22.3 and water concentration per unit dry mass is 2.5. 3 They have the ability to withstand heavy pruning, easy to grow and free of pests and diseases.Nearly about 9 out of 23 known species of Clausena genus were explored and identified for chemical and biological studies.

Plant collection:
The fresh stems of C. anisata were procured during the months of October to November,2013 from Manamettupatti, Viralimalai Taluk (Pudukottai District, Tamilnadu).The botanical identity of the plant specimen was identified by Dr. S. John Britto, Director, The Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph's College (Campus) Tiruchirappalli-620002 and autheniticated as Clausena anisata (Willd.)Hook f. ex.Benth.The stem parts were washed with tap water followed by distilled water and shade dried in a well ventilated room.The dried stem materials were powdered, stored in an air tight container and used for further solvent extraction.Preparation of the ethanol extract: The powdered stem was extracted using ethanol in the ratio of 1:10 by maceration method.After 48 hrs, the extracts were filtered through Whatmans No: 1filter paper.The stem material was then macerated again with ethanol and the combined filtrate obtained from the first and the second maceration was then concentrated to dryness under controlled temperature 40-50°C using Rota evaporator (Yamato, Japan (Model-RE801)).The extract was preserved for further use.GC-MS analysis: GC-MS analysis was carried out on a JEOL GCMATE II and gas chromatograph interfaced to a mass spectrometer (GC -MS) instrument.High resolution mass spectra were acquired at a resolving power of 5000 (20% height definition) and scanning the magnet from m/z 65 to m/z 350 at 1 second per scan.The column (HP5) was fused silica 50 m x 0.25 mm.Analysis conditions were 20 min at 100°C, 3 min at 235°C for column temperature, 240°C for injector temperature, helium was the carrier gas and split ratio was 5:4.The sample (1 µl) was evaporated in a split less injector at 300°C.Run time was 30 min.

Identification of compounds:
The compounds were identified by gas chromatography coupled with mass spectrometry.Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns.The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library.The name, molecular weight and structure of the components were ascertained.

DISCUSSION
The phytoconstituents are important compounds that are responsible for the medicinal properties and act as a potential source for neutraceuticals.In the present study, GC-MS analysis of ethanol stem extract of C. anisata showed the presence of nine compounds.The compounds were identified based on the retention time and molecular formula.Among the identified phytochemicals, n-hexadecanoic acid(78.54%)and 8-octadecenoic acid, methyl ester, [E]-(6.638%)was found to be predominant in the extract and was corroborated with 12 in the ethanolic leaf extracts of Indigofera suffruticosa.This compound was reported to have hypercholesterolemic, anti-inflammatory, antioxidant 13 and antimicrobial activity. 14Pentadecanoic acid, 13-methyl ester-, methyl ester(1.876%) was reported to possess antioxidant activity, 14 Octadec -9-enoic acid(2.67%)activity was found to have cancer preventive and insectifuge.Dipyrimadole (0.686%) is reported to prevent blot clots, to dilate blood vessels, 15 it inhibits the replication of mengovirus RNA. 16The bioactive compounds isolated from crude culture extracts produced by C. albicans isolated from the coastal mangrove ecosystem 17 also reported the presence of is oxazole, 5-(3,3-dicyano-1-cyclohexylidene-2-morpholino-prop-2-enyl)-3-p-methoxy phenyl-, that was in accordance with this study.Thus this type of GC-MS analysis is used to understand the nature of active principles in C. anisata and that may be helpful for further detailed studies.

CONCLUSION
GC-MS method is a direct and fast analytical approach for identification of bioactive compounds and only limited amount of plant material is required.The present study, which reveals the presence of bioactive compounds in ethanolic stem extract of C. anisata possess antioxidant, anti-inflammatory, antimicrobial, in sectifuge and cancer preventive that suggests to evaluate the contribution of these bioactive compounds in pharmacology.However, further studies are needed to ascertain its bioactivity.