Determination of Biologically Actie Constituents of the Fruits of Cucumis Sativus Linn Using GC-MS Analysis

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  523 Gopalakrishnan S and Kalaiarasi T. et al  . / International Journal of Biological & Pharmaceutical Research. 2013; 4(7): 523-527.   e- ISSN 0976 - 3651 Print ISSN 2229 - 7480 International Journal of Biological & Pharmaceutical Research Journal homepage:  DETERMINATION OF BIOLOGICALLY ACTIVE CONSTITUENTS OF THE FRUITS OF CUCUMIS SATIVUS   LINN. USING GC- MS ANALYSIS Gopalakrishnan S 1,*  and Kalaiarasi T 2   1* Department of Chemistry, Noorul Islam University, Kumaracoil-629180, K.K.District, Tamil Nadu, India. 2 Department of Pharmaceutical Chemistry, Manonmaniam Sundaranar University, Tirunelveli-627012, Tamil Nadu, India.   ABSTRACT The plant Cucumis sativus   (Fam.Cucurbitaceae) commonly known as “Mullu vellari” in Tamil, “Sakusa” in Sanskrit, “Kheera” in Hindi and   “Cucumber” in English is reported to possess a number of medicinal values. The present investigation was carried out to determine the possible bioactive components of the ethanolic extract of the fruits of Cucumis sativus  using GC-MS analysis. Seven compounds were identified by GC-MS analysis. The major phytoconstituents were 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (10.86 %), 5-(Hydroxyl methyl)-2-furancarboxaldehyde (64.69 %), 4-Hydroxy-3-methyl-2-butenyl-acetate (3.08 %), 2-(2-Methylcyclohexylidene)-hydrazinecarboxamide (11.54 %),n-Hexadecanoic acid (2.95 %), 1,2-Benzenedicarboxylic acid-diisooctyl ester (6.88 %) which are possessing many biological activities. Hence this study creates a  platform to screen many bioactive components to treat liver disorder. Key Words : Cucumis sativus , GC-MS analysis, Bioactive components, Liver disorder. INTRODUCTION Herbal medicines are safe than synthetic medicines because the phytochemicals in the plant extract target the biochemical pathway (Zaidan et al  ., 2005). The herbs are constantly being screened for their biological and  pharmacological activities such as anti-diabetic, anti-oxidant, anti-microbial, laxative, and anti-cancer activities (Evans and Trease, 1997; Anbuselvi et al  ., 2012; Makky et al  ., 2012; Kavitha et al  ., 2011; Meurer-Grimes et al  ., 1996; Koduru et al  ., 2006). The herbs are having numerous  bioactive components which are identified (at less than 1ng) by using GC or LC-MS. The cucumber plant is an annual climber which grows to a height of 15-30 cm and has large leaves that form a canopy over the fruit. The cucumber is the edible Corresponding Author S. Gopalakrishnan Email: fruit of the cucumber plant Cucumis sativus , which belongs to the gourd family Cucurbitaceae. C ucumis sativus  fruit is shown to possess various activities such as anti-hyperglycemic activity (Roman-Ramos et al  ., 1995), inhibitory effects on protein kinase C (PKC) activity Sudheesh et al  ., 2007), anti-oxidant activity (Stratil et al  ., 2006; Chu YF et al  ., 2002; Pellegrini et al  ., 2003), amylolytic activity (Repka and Fischerova, 1999), anti-cancer activity (Villasenor et al  ., 2002), anti-clastogenic activity (Edenharder et al  ., 1998), and anti-mutagenicity activity (Edenharder et al  ., 1994; Zhao et al  ., 1992). The  juice is used in many beauty products (Katsambas and Lotti 2003). C. sativus  is amongst the constituents of cosmetics marketed as treatments for skin inflammations and other skin disorders, and as skin protectants (Aburjai and Natsheh, 2003). Taking into consideration of the medicinal importance of this plant, the ethanol extract of fruits of C ucumis sativus  was analyzed for the first time using GC-MS. This work will be helpful to identify the compounds of therapeutic value. GC-MS is IJBPR  524 Gopalakrishnan S and Kalaiarasi T. et al  . / International Journal of Biological & Pharmaceutical Research. 2013; 4(7): 523-527.   one of the best techniques to identify the bioactive constituents of long chain branched chain hydrocarbons, alcohols, acids, ester etc. MATERIALS AND METHODS Collection of plant materials The fruits of Cucumis sativus was collected in the month of July from Alangulam, Tirunelveli District, Tamil  Nadu and identified by Prof. P. Jayaraman, Plant Anatomy Research Centre, West Thambaram, Chennai- 600 045, Tamil Nadu, India. A voucher specimen (MSU/PHAR/HER   –  141) has  been preserved in the Herbarium of the Department of Pharmaceutical Chemistry, Manonmaniam Sundaranar University, Tirunelveli -627 012, Tamil Nadu, India. Preparation of extracts The collected fruits were cut into pieces, shade-dried at room temperature and powdered. The dried fruit  powder (500 g) was successively extracted using  petroleum ether (40°- 60°C), benzene, chloroform, ethanol and water by using Soxhlet apparatus. The last trace of solvent was removed under reduced pressure distillation and then vacuum dried. The dried crude ethanolic extract of the fruits has been used for the GC-MS analysis. GC-MS ANALYSIS Preparation of extract 2 μl of the ethanolic extract of the fruits of Cucumis sativus was employed for GC-MS analysis (Merlin et al  ., 2009). Instruments and chromatographic conditions GC-MS analysis of the ethanolic extract was carried out on a GC-MS Clarus 500 Perkin Elmer system comprising a AOC-20i autosampler and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the following conditions: column Elite-1 fused silica capillary column (30 mm x 0.2 5mm ID x 1μMdf, composed of 100 % Dimethyl poly si loxane), operating in electron impact mode at 70 eV; helium (99. 999 %) was used as carrier gas at a constant flow of 1ml/min and an injection volume of 0.5 μl was employed (split ratio of 10:1); injector temperature 250°C. The oven temperature was programmed from 110°C (isothermal for 2 min), with an increase of 10°C/min, to 200°C, then 5°C / min to 280°C, ending with a 9 min isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of 0.5 seconds and fragments from 40 to 550 Da. Identification of phytochemical constituents Interpretation on mass spectra of GC-MS was conducted using the database of National Institute of Standards and Technology (NIST). The mass spectra of the unknown components were compared with those of the known components stored in the NIST library. The name, molecular weight and structure of the phytochemical constituents of the ethanolic extract were ascertained (Nezhadali et al  .,   2010; Sathyaprabha et al  ., 2011). RESULTS AND DISCUSSION  The biologically active constituents present in the ethanolic extract of the fruits of Cucumis sativus were identified by GC-MS analysis (Fig. 1) and the mass spectra of the six major constituents were obtained (Fig. 2 to Fig. 7). The mass spectra of the constituents were compared with those of the known spectra which are stored in the  NIST library. The retention time (RT), molecular formula, molecular weight and peak area (%) are presented in Table 1. Table 1. Phytoconstituents identified in the ethanolic extract of the fruits of Cucumis sativus by GC-MS analysis. No. RT Name of the compound Molecular formula Molecular weight Peak Area % Compound Nature **Activity 1 3.61 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one C 6 H 8 O 4  144 10.86 Flavonoid fraction Antimicrobial Anti-inflammatory 2 4.59 5-(Hydroxymethyl)- 2-furancarboxaldehyde C 6 H 6 O 3  126 64.69 Aldehyde compound Antimicrobial Preservative 3 7.50 4-Hydroxy-3-methyl-2- butenyl- acetate C 7 H 12 O 3  144 3.08 Acetate compound Antimicrobial 4 10.60 2-(2-Methylcyclohexylidene)- hydrazinecarboxamide C 8 H 15  N 3 O 169 11.54 Amino compound Antimicrobial 5 13.32 n-Hexadecanoic acid C 16 H 32 O 2  256 2.95 Palmitic acid Antioxidant, Hypo cholesterolemic  Nematicide, Pesticide, Lubricant, Antiandrogenic, Flavor, Hemolytic 5-Alpha reductase inhibitor 6 24.24 1,2-Benzenedicarboxylic acid-diisooctyl ester C 24 H 38 O 4  390 6.88 Plasticizer compound Antimicrobial Anti-fouling **Source: Dr.Duke's phytochemical and ethnobotanical databases [Online database].  525 Gopalakrishnan S and Kalaiarasi T. et al  . / International Journal of Biological & Pharmaceutical Research. 2013; 4(7): 523-527.   Fig 1. GC-MS Chromatogram of ethanol extract of the fruits of Cucumis sativus Fig 2. Mass spectrum of 2, 3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (RT: 3.61) Fig 3. Mass spectrum of 5-(Hydroxymethyl)-2-furancarboxaldehyde (RT: 4.59) Fig 4. Mass spectrum of 4-Hydroxy-3-methyl-2-butenyl-acetate (RT: 7.50) 20 30 40 50 60 70 80 90 100 110 120 130 140 150   0   50   100273141435561 7184126OHO O    526 Gopalakrishnan S and Kalaiarasi T. et al  . / International Journal of Biological & Pharmaceutical Research. 2013; 4(7): 523-527.   Fig 5. Mass spectrum of 2-(2-Methylcyclohexylidene)-hydrazinecarboxamide (RT: 10.60) 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180   0   50   100264155 61 6781 9598110126141 152169NNHH2N O   Fig 6. Mass spectrum of n-Hexadecanoic acid (RT: 13.32) Fig 7. Mass spectrum of 1, 2-Benzenedicarboxylic acid, diisooctyl ester (RT: 24.24)  In the present study, six phytochemical constituents have been identified from the ethanol extract of the fruits of Cucumis sativus  by Gas Chromatography-Mass spectrometry (GC-MS) analysis. All the identified  phytoconstituents, 2,3-Dihydro-3,5-dihydroxy-6-methyl- 4H-pyran-4-one, 5-(Hydroxylmethyl)-2-furan carboxal dehyde, 4-Hydroxy-3-methyl-2-butenyl-acetate, 2-(2-Methylcyclohexylidene)-hydrazinecarboxamide, 1,2-Benzene dicarboxylic acid-diisooctyl ester are reported to  possess antimicrobial activity.n-Hexadecanoic acid has antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant, antiandrogenic, flavor, hemolytic 5- α reductase inhibitor activities. Out of these six bioactive constituents 2-Furancarboxaldehyde-5-(hydroxyl methyl) (64.69 %) is  present in high quantity and n-Hexadecanoic acid (2.95 %) is present in less quantity, and the other constituents are  present in considerable amounts. Thus, this type of GC-MS analysis is the first step towards understanding the nature of active principles in this medicinal plant and this type of study will be helpful for further detailed phytochemical study. The investigation concluded that the stronger extraction capacity of ethanol could have produced number of active constituents responsible for many biological activities. CONCLUSION The six phytochemical constituents identified by GC-MS of the ethanolic extract of Cucumis sativus  are medicinally valuable and possess various pharmaceutical applications. GC-MS analysis of the ethanolic extract showed the presence of Hemolytic 5- α reductase inhibitor like n-Hexadecanoic acid which may be responsible for the liver disorder curing effect. The identified  phytoconstituents needs further research on toxicological aspects to develop safe drug.

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