Phytochemical studies on Cichorium intybus L. (chicory) from Kashmir Himalaya using GC-MS

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1 Research Article ISSN: Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), Available online through Phytochemical studies on Cichorium intybus L. (chicory) from Kashmir Himalaya using GC-MS Bisma Malik 1, Tanveer Bilal Pirzadah 1, Inayatullah Tahir 2, Malik Zainul Abdin 3, Reiaz Ul Rehman 1* 1 Department of Bioresources, University of Kashmir, Srinagar, Jammu and Kashmir , India 2 Department of Botany, University of Kashmir, Srinagar, Jammu and Kashmir ,India. 3 Department of Biotechnology, Jamia Hamdard, New Delhi , India. *Corresponding author. Dr. Reiaz Ul Rehman Assistant Professor Department of Bioresources, University of Kashmir, Srinagar, Jammu and Kashmir , India Received on: ; Revised on: ; Accepted on: ABSTRACT Background:Cichorium intybus commonly called as chicory, coffee weed and blue sailor s succory is traditionally used for the treatment of almost every disease particularly diseases related to heart, kidney and liver. Method: 0.2 g of the powdered seed, roots, stem and flower samples of Cichorium intybus from Kashmir Himalaya was equilibrated with 200 d/m of Cichorium intybusmethanol for 24 h, separately. Supernatant was later reduced by heating to 2 d/m. The concentrated methanolic extracts were further subjected to GC-MS analysis. Results: The GC-MS analysis determined the presence of 109 compounds from different parts of Cichorium intybus L. (chicory) in which 24 compounds were identified from seed, 22 compounds from root, 28 compounds from stem and 35 compounds from flower. The major metabolites from seed were 9,12-Octadecadienoic acid (Z,Z)- (35.03) and n-hexadecanoic acid (34), from root were n-hexadecanoic acid (24.37), 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (1.6), from stem were n-hexadecanoic acid (35.14), 9,12-octadecadienoic acid (16.01) and from flower were 5-(hydroxymethyl)-2-furaldehyde (33.26), 9,12-Octadecadienoic acid (Z,Z)- (26.86) in terms of their percent area. Conclusion: This study helps to predict the formula and structures of metabolites which can be further used in drug formulations and chicory from Kashmir Himalaya can be considered as potent candidate for pharmaceutical sector. KEYWORDS:Cichorium intybus, GC-MS, ethenobotanical, phytochemical 1. INTRODUCTION From the last few decades, medicinal plants with intensive pharmacological studies forms the backbone of the traditional system of medicines. Medicinal plants are regarded as possible sources of novel compounds with potent therapeutic properties as well as sources of new compounds which can be utilized in the development of new drug formulations. It was estimated that 80-90% of the world s population still relies on traditional herbal medicines according to the World Health Organization [1,2]. Therefore the need of the hour is to screen medicinal plants for various phytochemical constituents who form the basis for further pharmacological studies [3]. Medicinal plants are known rich sources of phytochemical constituents called secondary metabolites commonly with simple or complex structures. In general, these secondary metabolites are considered as powerful sources with a wide range of structures and properties [4]. Several phytochemical constituents have been found to exhibit important biological activities and finds application in pharmaceutical, neutraceutical, natural dyes, cosmetics and insecticides [5,6]. Currently microbial resources have been considered as the primary source of natural products (particularly antibiotics), but with the increasing recognition of herbal medicines as an alternative form of health care system, the isolation, identification and characterization of medicinal plants for secondary metabolites has become very important [7]. Asteraceae family consists of various important medicinal plants with wide range of biological properties and interesting phytochemical constituents. Cichorium intybus L. commonly called as chicory, coffee weed and blue sailor s succory a perennial herb belonging to the family Asteraceae. Traditionally, Cichorium intybus L. is used as food and medicinal crop in temperate parts of Europe and Asia and finds its application in food and pharmaceutical industries [8,9]. Chicory is found to be effective in the treatment of jaundice, asthma, gout, rheumatic complaints [10] as well as against cardiac ailments [11]. Chicory contains polyphenol compounds like phenol, flavonoid, coumarins which are considered as a source of natural pro and antioxidants [12]. Roasted chicory roots are being used as coffee substitute, as it neutralizes caffeine and help in digestion and also enhance the flavor [13]. Inulin from chicory roots is being currently used as a substrate of fibre in health and functional foods [14] and also boosts the immune system particularly in elderly people [15]. Currently chicory has gained world-wide attention due to its highly nutritive health promoting benefits and its demand for the industrial feedstock [16] and nowadays in Europe it has been ranked as a functional vegetable

2 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), of the 21 st century [17]. Cichorium intybus is a plant used in Ayurvedic medicines however there are no reports on the thorough phytochemical analysis of this plant from Kashmir Himalaya. As far as phytochemical analysis was concerned in the last few years, GC- MS technique has become firmly established technique for secondary metabolite profiling in both plant and non-plant species [18,19]. Therefore, the present study was aimed to explore the phytochemical constituents of Cichorium intybus L. (chicory) from Kashmir Himalaya using GC-MS technique. 2. MATERIALS AND METHODS volatile matter, long chain, branched chain hydrocarbons, alcohols, esters, ketones, terpenoids, steroids etc. The results pertaining to GC-MS analysis leads to the identification of number of compounds from the GC fractions of methanolic extract of wild chicory seed, root, stem and flower. These compounds were identified through mass spectrometry attached with GC. In the present study, the GC- MS analysis revealed the presence of 109 compounds in different parts of Cichorium intybus L. in which the flower comprises the highest number of compounds followed by stem, root and seed (Figure 1). The GC-MS analysis of methanolic extract of seed revealed 2.1 Chemical reagents All solvents were of analytical grade purchased from Merck (Germany) and all the standard chemicals were purchased from Sigma Aldrich (USA). All other reagents and unlabelled chemicals were of analytical grade and used without further purification. 2.2 Plant material and collection Healthy, fresh and disease free plants of Cichorium intybus L. (chicory) were collected from the natural habitats of Bandipora district of Srinagar Jammu and Kashmir, India. The samples were washed gently with distilled water (without squeezing) to remove debris and dust particles. The plant material is shade-dried at room temperature for 15 days and pulverized into a uniform material using a surface sterilized mortar and pestle. The powdered samples were stored in air tight polythene bags until use. 2.3 Plant sample extraction The 0.2g of dried extract powder of Cichorium intybus L. (Chicory) samples (root, seed, stem and flower) was dissolved in 10ml of methanol solvent properly mixed and kept for 72 hours, then filtered through 0.45μm syringe filter (Millipore Corp., Bedford, MA, USA). 1μl aliquot of the sample was then injected into the GC-MS port for the metabolite analysis (Shimadzu QP-2010 Plus with Thermal Desorption System TD 20). GC-MS analysis of methanolic leaf extract of wild chicory was determined according to the method [20]. Identification of the mass spectrum GC-MS was done by using NIST (National Institute Standard and Technology), WILEY8 and FAME libraries having more than patterns. By using these libraries, the spectrum of the unknown compound was compared to the spectrum of known compound stored in these libraries. The name of the compound, molecular weight, chemical formula and structure of the test sample were ascertained. 3. RESULTS GC-MS is one of the best technique to identify the constituents of Figure. 1. Metabolites present in different parts of Cichorium intybus L. (chicory) analyzed by GC-MS technique the presence of twenty-four (24) individual compounds (phytochemicals) that could contribute the medicinal quality of the plant. The identification of the phytochemical compounds was confirmed based on the retention time, molecular formula, molecular weight and peak area percent. The phytochemicals identified through GC-MS analysis showed many activities/uses relevant to this study are listed in table 1. The compounds identified by using NIST library database and the activities listed are based on Dr. Duke s phytochemical and ethenobotanical database created by Dr. Jim Duke s of the Agricultural Research Service/USDA. The Total Ion Chromatogram (TIC) of the GC-MS analysis of chicory seed extract is given in Figure 2A. The peaks in the chromatogram were integrated and were compared with the database of spectrum of known compounds stored in the GC-MS library. The major metabolites (>1%) identified from the seed of Cichorium intybus L. in terms of percent area are: 9,12-Octadecadienoic acid (Z,Z)- (35.03), n- Hexadecanoic acid (34), 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- (6.89), Lupeol (4.29), 5H-3,5a-Epoxynaphth[2,1-c]oxepin, dodecahydro- 3,8,8,11a-tetramethyl-,[3S-(3.alpha.,5a.alpha.,7a.alpha., 11a.beta., 11b.alpha.)] (3.51),4,4,6a,6b,8a,11,11,14 boctamethyl 1,4,4a,5,6,6a, 6b,7,8,8a,9,10,11,12,12a,14,14a,14b-octadecahydro-2H-picen-3-one (2.64), 4-Methyl-5-(phenylmethyl)-2,3-dihydrothiophene 1,1-dioxide (1.94). The major compounds present in the seed extract were in the order of fatty acid à ester à terpenoid à hydrocarbon à steroid à ketone (Figure 2B).

3 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), Table. 1. Important bioactive compounds identified in the methanolic seed extract of Cichorium intybus L. (chicory) by GC-MS and their uses S. No R.Time Compound name Mol. Formula Mol.Wt Area % Major group Uses Tetradecanoic acid C 14 H Fatty acid Antioxidant, cancer preventive, nematicide, hypercholesterolemic, lubricant Pentadecanoic acid C Fatty acid Flavoring agent, antioxidant ,2-Benzenedicarboxylic acid, C Ester Used in cosmetics bis(2-methylpropyl) ester Hexadecanoic acid, methyl ester C Ester Antioxidant, Flavor, Hypocholesterolemic Pesticide, 5-Alpha reductase inhibitor n-hexadecanoic acid C Fatty acid Antioxidant, hypocholesterolemic, nematicide, hemolytic, 5-alpha reductase inhibitor ,4,4a,5-Tetrahydrobenzo[g] C 13 H 13 NO Ketone NA isoquinolin-10(2h)-one Octadecenoic Acid (Z)- C Fatty acid Insect pheromone, pharmaceuticals, emulsifying agent, emollient, ,12-Octadecadienoic acid, C Ester Antiinflammatory, Nematicide, Insectifuge, methyl ester Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic, ,12-Octadecadienoic acid C Fatty acid Antiinflammatory, Nematicide, Insectifuge, (Z,Z)- Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic ,12,15-Octadecatrienoic acid, C Fatty acid Antiinflammatory, Hypocholesterolemic, Cancer (Z,Z,Z)- preventive, Nematicide, Hepatoprotective, Insectifuge, Antihistaminic, Antieczemic, Antiacne, 5-Alpha reductase inhibitor, Antiandrogenic, Antiarthritic, Anticoronary, Octadecanoic acid C 18 H Fatty acid Dietry supplements, softening agent, surfactant Verrucarol C Terpenoid Antitumor activity ,8,11-Heptadecatriynoic acid, C 18 H Ester Dyestuffs, feed additives, pharmaceuticals, methyl ester pigment coatings Methyl-5-(phenylmethyl)-2,3- C 12 H 14 S Hydrocarbon Lubricants dihydrothiophene 1,1-dioxide Di-n-octyl phthalate C 24 H Ester cosmetics, pesticides, plasticizer, Docosane C 22 H Hydrocarbon Used in organic synthesis, calibration, and temperature sensing equipment Octacosane C 28 H Hydrocarbon Pheromones, lubricants Stigmasterol C Steroid Anti cancer, antioxidant beta.-sitosterol C Steroid Cholesterol lowering and symptom improvement in mild to moderate benign prostatic hypertrophy. Possible role in the control of chronic inflammatory conditions R,4S,7S,11R-2,2,4,8- C 15 H Terpenoid Nutrient, stabilizers, surfactants and emulsifiers Tetramethyltricyclo[ (4,11)]undec-8-ene ,4,6A,6B,8A,11,11,14B- C Terpenoid NA octamethyl- 1,4,4A,5,6, 6A,6B,7,8,8A,9,10,11,12,12A, 14,14A,14B-octadecahydro- 2H-Picen-3-one H-3,5a-Epoxynaphth C Hydrocarbon Fragrance agents [2,1-c]oxepin, dodecahydro- 3,8,8,11a-tetramethyl-, [3S (3.alpha.,5a.alpha., 7a.alpha., 11a.beta.,11b.alpha.)] ,19-Cyclolanost-23-ene-3,25- C 32 H Terpenoid Flavoring agent diol, 3-acetate, (3.beta.,23E) Lupeol C Terpenoid Antiprotozoal, antimicrobial, antiinflammatory, antitumor and chemopreventive properties (A) (B) Figure. 2 (A) Shows the Total Ion Chromatogram and (B) Major groups present in the seed extract of Cichorium intybus L. (chicory) analyzed by GC-MS. A92

4 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), GC-MS chromatogram of methanolic root extract of Cichorium intybus L. as per the aforementioned experinmental procedures exhibits several peaks indicating the presence of different phytochemicals in the extract (Figure 3A). The GC-MS analysis revealed that the methanolic root extract of Cichorium intybus L. possesses twenty-two compounds that are later identified and characterized using NIST library database. The various compounds with their different retention time, molecular formula, molecular weight and peak area percent and their activities were tabulated in table 2. Table. 2. Important bioactive compounds identified in the methanolic root extract of Cichorium intybus L. (chicory) by GC-MS and their uses S. No. R.Time Compound name Mol. Formula Mol. Wt Area % Major groups Uses Decenal, (Z)- C 10 H 18 O Aldehyde Food flavoring agent ,4-Decadienal, (E,E)- C 10 H 16 O Aldehyde Flavoring agent ,4-Decadienal, (E,E)- C 10 H 16 O Aldehyde Flavoring agent Octadecenoic Acid C Fatty acid Reducing blood pressure (Hypotensive effect) Tetradecanoic Acid C 14 H Fatty acid Used in cosmetics and in topical medicinal preparation Ethylhexyl salicylate C Ester Used in cosmetics ,2-Benzenedicarboxylic acid, bis(2- C Ester Used in cosmetics methylpropyl) ester Hexadecanoic acid, methyl ester C Ester Antioxidant, Flavor, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5- Alpha reductase inhibitor cis-9-hexadecenoic acid C Fatty acid increase insulin sensitivity by suppressing inflammation, as well as inhibit the destruction of insulin-secreting pancreatic beta cells n-hexadecanoic acid C Fatty acid Antioxidant, Flavor, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5- Alpha reductase inhibitor ,12-Octadecadienoic acid, methyl ester C Ester Antiinflammatory, Nematicide, Insectifuge, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic ,11,14-Eicosatrienoic acid, (Z,Z,Z)- C Fatty acid Anti-inflamatory, antithrombotic effect Eicosanoic acid C Fatty acid used in the manufacture of Pharmaceuticals,soaps, cosmetics, and food packaging ,2-Benzenedicarboxylic acid, mono(2- C Ester Anti fouling, Antimicrobial ethylhexyl) ester Hexadecane C Hydrocarbon Used as a substrate for the production of biosurfactants Cyclopropane, 1,1-dichloro-2,2,3,3-tetramethyl- C 7 H 12 C Hydrocarbon Used in pharmaceuticals, agrochemicals, food additive ,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1 C 21 H Ester Co-solvents, oil carrier, antioxidant, -(hydroxymethyl)ethyl ester antiacne Docosane C 22 H Hydrocarbon Used in organic synthesis, calibration, and temperature sensing equipment Stigmasta-5,22-dien-3-ol C Steroid used as a biomarker for the presence of. (marine) algal matter in the environment beta.-sitosterol C Steroid Cholesterol lowering and symptom improvement in mild to moderate benign prostatic hypertrophy. Possible role in the control of chronic inflammatory conditions ,4,6a,6b,8a,11,11,14b-Octamethyl- C Terpenoid No activity reported 1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a, 14,14a,14b-octadecahydro-2H-picen-3-one Lupeol C Terpenoid antiprotozoal, antimicrobial, antiinflammatory, antitumor and chemopreventive properties (A) (B) Figure. 3 (A) Total ion chromatogram (B) major groups present in a root extract of Cichorium intybus L. (chicory) using GC-MS analysis technigue.

5 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), The activities listed are based on Dr. Dukes database (Duke, 2012). The major phytochemicals identified (>1%) in the root extract in terms of percent area were found to be n-hexadecanoic acid (24.37), 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester (1.6), 2,4- decadienal, (E,E)- (1.04) and 9,12-Octadecadienoic acid, methyl ester (0.78). The major compounds present in the root extract were in the order of fatty acid > ester > hydrocarbon > terpenoid > aldehyde (Figure 3B). The present study also revealed the presence of twenty-eight (28) compounds from the GC-MS analysis of the methanolic stem extract of Cichorium intybus L. Various compounds with their retention time, molecular formula, molecular weight and peak area percent and their various activities were given in table 3. The Total Ion Chromatogram (TIC) of the GC-MS analysis confirms the presence of several compounds and their different retention time is given in Table. 3. Important bioactive compounds identified in the methanolic stem extract of Cichorium intybus L. (chicory) by GC-MS and their uses S. No. R. Time Compound name Mol. Formula Mol. Wt Area% Major groups Uses Furancarboxaldehyde, 5-(hydroxymethyl)- C 6 H Aldehyde Food flavoring agent, antimicrobial, preservative beta.-d-glucopyranose, 1,6-anhydro- C 6 H 10 O Sugar Biomarker Benzeneacetic acid, 3-hydroxy- C 8 H Phenol NA Tetradecanoic acid C 14 H Fatty acid Antioxidant, cancer preventive, nematicide, hypercholesterolemic, lubricant ,6,10-Trimethyl,14-ethylene-14-pentadecne C 20 H Hydrocarbon Antiproliferative Undecanone, 6,10-dimethyl- C 13 H 26 O Ketone NA Hexadecen-1-ol, 3,7,11,15-tetramethyl-, C Terpenoid Antimicrobial, Anticancer, Diuretic, [R-[R*,R*-(E)]]- (Phytol) Antiinflammatory Pentadecanoic acid C Fatty acid Flavoring agent, antioxidant ,7,11,15-Tetramethyl-2-hexadecen-1-ol C Terpenoid Antimicrobial, Anticancer, Diuretic, Antiinflammatory Pentadecanone, 6,10,14-trimethyl- C 18 H Hydrocarbon Flavor and fragrance agent Pentadecanoic acid, 14-methyl-, methyl ester C Ester Antioxidant n-hexadecanoic acid C Fatty acid Antioxidant, Flavor, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5- Alpha reductase inhibitor Heptadecanoic acid C Fatty acid Dairy products Palmitaldehyde, diallyl acetal C 22 H Aldehyde NA ,12-Octadecadienoic acid C Fatty acid Antiinflammatory, Nematicide, Insectifuge, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic Octadecanoic acid C 18 H Fatty acid Dietry supplements, softening agent, surfactant Eicosanoic acid C Fatty acid Detergents, photographic materials and lubricants Naphtho[1,2-b]furan-2,8(3h,4h)-dione, C 15 H Terpenoid Antiparasitic,anti-inflammatory, 3a,5,5a,9b-tetrahydro-4-hydroxy-3,5a,9- antipyretic and analgesic trimethyl-, [3s-(3.alpha.,3a.alpha., Eicosene C Hydrocarbon Pharmaceticals Di-n-octyl phthalate C 24 H Ester cosmetics, pesticides, plasticizer, beta.-Acetoxystigmasta-4,6,22-triene C Steroid NA Cholesta-4,6-dien-3-ol, (3.beta.)- C 27 H Steroid Adhesive Stigmasterol C29H Steroid Anti cancer, antioxidant gamma.-sitosterol C Steroid Anti-diabetic, Anti-angeogenic, Anticancer, antimicrobial, anti-inflammatory, antidiarrhoeal and antiviral ,4,6a,6b,8a,11,11,14b-Octamethyl- C Terpenoid NA 1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a, 14b-octadecahydro-2H-picen-3-one Lupeol C Terpenoid Antiprotozoal, antimicrobial, antiinflammatory, antitumor and chemopreventive properties ,19-Cycloergost-24(28)-en-3-ol, 4,14- C 32 H Terpenoid Flavoring agent dimethyl-, acetate, (3.beta.,4.alpha.,5.alpha.) Lup-20(29)-en-3-yl acetate C 32 H Terpenoid Antimicrobial and antiinflammatory

6 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), (A) (B) Figure. 4. (A) Total ion chromatogram (B) major groups present in a stem extract of Cichorium intybus L. (chicory) using GC-MS technique Figure 4A. Different compounds were identified and characterized by using NIST library database. The phytochemicals identified during GC-MS analysis and the activities listed are based on Dr. Duke s database (Duke, 2012). The major phytochemical ( >1%) identified from the stem of Cichorium intybus L. in terms of percent area are: n- Hexadecanoic acid (35.14), 9,12-octadecadienoic acid (16.01), 2- Furancarboxaldehyde, 5-(hydroxymethyl)- (9.57), Lupeol (7.68), lup- 20(29)-en-3-yl acetate (4.2), 2,6,10-trimethyl,14-ethylene-14- pentadecne (2.69) beta.-d-glucopyranose, 1,6-anhydro- (2.61) 4,4,6a,6b,8a,11,11,14b-Octamethyl-1,4,4a,5,6,6a,6b,7,8,8a,9, 10,11,12,12a,14,14a,14b-octadecahydro-2H-picen-3-one (2.5), 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (1.61) and Cholesta-4,6-dien- 3-ol, (3.beta.)- (1.17). The major compounds present in the stem extract of wild chicory were in the order of fatty acid > terpenoid > steroid > hydrocarbon > ester > aldehyde > sugar > ketone (Figure 4B). Lastly, the GC-MS analysis also revealed the presence of thirty-five (35) different compounds from the methanolic flower extract of Cichorium intybus L. The various compounds with their different retention time, molecular formula, molecular weight and percent area confirmed the presence of different compounds in the extract. The Total Ion Chromatogram (TIC) of methanolic flower extract of Cichorium intybus L. exhibits different peaks indicating the presence of different phytochemicals in the extract (Figure 5A). The compounds identification and characterization were done by using NIST library. Some of the phytochemicals identified from GC-MS analysis of flower extract exhibits potent biological activities whose details were given in (table 4) which are based on Dr. Duke s phytochemical and ethenobotanical database. The major phytochemicals (>1%) identified from the methanolic flower extract of Cichorium intybus L. in terms of percent area are: 5-(hydroxymethyl)-2-furaldehyde (33.26), 9,12-Octadecadienoic acid (Z,Z)- (26.86), n-hexadecanoic acid (13.77), 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- (6.44), beta.- D-Glucopyranose, 1,6-anhydro- (3.66), Octadecanoic acid (2.97),2- propenyl nonanoate (1.67) and Cyclohexanol, 5-methyl-2-(1- methylethyl)-, acetate (1.48). The major compounds present in the flower extract of Cichorium intybus L. were in the order of fatty acid > ketone > hydrocarbon > ester > steroid > terpenoid > aldehyde > alcohol > phenol > sugar (Figure 5B). The structures of the major compounds identified from the chemical profile of whole chicory plant (seed, root, stem, leaf and flower) using GC-MS analysis technique were given in Figure 6. Hence, GC-MS analysis indicates the presence of several compounds in different parts of wild chicory in which some of the phytochemicals possesses potent biological activities which in turn supports the ethnomedicinal usage of this plant.

7 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), Table. 4. Important bioactive compounds identified in the methanolic flower extract of Cichorium intybus L. (chicory) by GC-MS and their uses S.No. R.Time Compound name Mol. Mol. Area Major Uses Formula Wt % groups H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C 6 H Ketone Antioxidant (hydroxymethyl)-2-furaldehyde C 6 H Aldehyde Antioxidant Cis-dimethyl morpholine C 6 H 13 NO Hydrocarbon Fungicide beta.-d-glucopyranose, 1,6-anhydro- C 6 H 10 O Sugar Biomarker (1-Hydroperoxy-2,2-dimethyl-6-methylene- C Ketone NA cyclohexyl)-pent-3-en-2-one Tetradecanoic acid C 14 H Fatty acid Antioxidant, cancer preventive, nematicide, hypercholesterolemic, lubricant Dodecanone C 12 H 18 O Ketone Flavor and fragrance agent ,4-Methanoazulene, decahydro-4,8,8-trimethyl-9- C 15 H Hydrocarbon Flavor and fragrance agent, methylene-, [1s-(1.alpha.,3a.beta.,4.alpha.,8a.beta.)]- cosmetics Pentadecanone, 6,10,14-trimethyl- C 18 H Hydrocarbon Flavor and fragrance agent Pentadecanoic acid C Fatty acid Flavoring agent, antioxidant Tetradecane C Hydrocarbon NA Heptadecanone C Ketone Flavoring agent n-hexadecanoic acid C Fatty acid Antioxidant, Flavor, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5- Alpha reductase inhibitor Heptadecanoic acid C Fatty acid Dairy products Tetradecanol C Alcohol Surfactant ,12-Octadecadienoic acid (Z,Z)- C Fatty acid Antiinflammatory, Nematicide, Insectifuge, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic Octadecanoic acid C 18 H Fatty acid Dietry supplements, softening agent, surfactant Cycloheptanone, 2-(3-buten-1-yl)- C 11 H 18 O Ketone Pharmaceuticals, fragrance agent Nonadecanoic acid C 19 H Fatty acid Insect pheromones Gamolenic Acid C Fatty acid Pharmaceuticals, Functional foods H-Pyran, 2-(2-heptadecynyloxy)tetrahydro- C Phenol Antimicrobial, Anti-inflammatory, Antioxidant Eicosanoic acid C Fatty acid Detergents, photographic materials and lubricants Cyclohexanol, 5-methyl-2-(1-methylethyl)-, acetate C Ester Flavor and fragrance agent Hexadecanoic acid, 2-oxo-, methyl ester C Ester NA Eicosene, (E)- C Fatty acid Antiviral, antiaging, anticancer, antioxidant Di-n-octyl phthalate C 24 H Ester cosmetics, pesticides, plasticizer, Propenyl nonanoate C Ester Flavor and fragrance agent Palmitic acid vinyl ester C Ester Emulsifying agents for flavors used in fruit flavoured beverages Octadecanoic acid, 2-propenyl ester C Ester Dietary supplements, surfactants, softening agent, cosmetics, detergents and soaps ,16-Hentriacontanedione C 31 H Ketone NA Cholest-24-en-16-one, (5.alpha.,20.xi.)- C 27 H Steroid NA Stigmasterol C Steroid Anti cancer, antioxidant beta.-sitosterol C Steroid Cholesterol lowering and symptom improvement in mild to moderate benign prostatic hypertrophy. Possible role in the control of chronic inflammatory conditions (1,5-Dimethyl-4-hexenyl)-3a,6,6,12a- C 32 H Terpenoid NA tetramethyltetradecahydro-1hcyclopenta[a]cyclopropa[e]phenanthr H-3,5a-Epoxynaphth[2,1-c]oxepin, dodecahydro- C Hydrocarbon Fragrance agents 3,8,8,11a-tetramethyl-, [3S -(3.alpha.,5a.alpha.,7a.alpha.,11a.beta.,11b.alpha.)]-

8 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), (A) (B) Figure. 5. (A) GC-MS chromatogram (B) major group present in the methanolic flower extract of Cichorium intybus L. (chicory) by GC-MS technigue. n-hexadecanoic acid 9,12-Octadecadienoic acid (Z,Z) Lupeol 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 5H-3,5a-Epoxynaphth[2,1 c]oxepin, dodecahydro-3,8,8,11atetramethyl-, [3S (3.alpha.,5a.alpha.,7a.alpha.,11a.beta.,11b.alpha.)] 4,4,6a,6b,8a,11,11,14boctamethyl1,4,4a,5,6,6a,6b,7,8,8a, 9,10,11,12,12a,14,14a,14b-octadecahydro-2H-picen-3-one

9 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), 2,4-decadienal 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester 4-Methyl-5-(phenylmethyl)-2,3- dihydrothiophene 1,1-dioxide 2-Furancarboxaldehyde, 5 (hydroxymethyl)- 9,12-Octadecadienoic acid, methyl ester 2,6,10-trimethyl,14-ethylene-14-pentadecne 3,7,11,15-Tetramethyl-2-hexadecen-1-ol lup-20(29)-en-3-yl acetate beta.-d-glucopyranose, 1,6-anhydro- Octadecanoic acid 4H-Pyran-4-one, 2,3-dihydro- 3,5-dihydroxy-6-methyl- Cholesta-4,6-dien-3-ol, (3.beta.)- 2-propenyl nonanoate Cyclohexanol, 5-methyl-2-(1-methylethyl)-, acetate Figure. 6. Structures of the major compounds identified from Cichorium intybus L. (chicory).

10 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), 4. DISCUSSION The more precise information in qualitative analysis can be obtained by gas chromatography coupled with mass spectrometry (GC-MS) [21] For quantitative determination, gas chromatography with flame ionization detector (GC-FID) and GCMS are preferred [22-24]. In the present study, we characterized the whole plant chemical profile of wild Cichorium intybus L. (Chicory) using GC-MS technique. The results revealed the presence of 113 compounds in different parts of chicory (seed, root, stem and flower). The identified compounds exhibit many biological properties. The prediction of the biological activities by applying the duke s databases was confirmed with previous observations and supplemented the traditional usage of chicory [25-27]. By interpreting these compounds, it is found that chicory possess various therapeutic applications. The gas chromatogram shows the relative concentration of various compounds getting eluted as a function of retention time. The height of the peaks indicates the relative concentration of the compounds present in chicory. The mass spectrometer analyzes the compounds eluted at different times to identify the nature and structure of the compounds. The large compound fragments into small compounds giving rise to appearance of peaks at different m/z ratio. These mass spectra are fingerprints of that compound which can be identified from the data library. In the present study, it has been found that the result of GC- MS analysis revealed the presence of 24 compounds in the seed of chicory which possesses many biological activities. For instance, 9,12-octadecadienoic acid and 9,12,15-octadecatrienoic acid (fatty acids) are major metabolites present in terms of percent area (35.03 and 6.89 respectively) having anti-inflammatory, anticancer, hypocholesterolemic, nematicide, hepatoprotective, anti histaminic, antiacne, antiezemic, antiarthritic properties. n-hexadecanoic acid- a fatty acid having percent area of 34.0 possess antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant activities as well as hemolytic and 5-alpha reductase inhibitor activity. Another metabolite called Lupeol- a terpenoid having percent area of 4.29 have been reported to have antiprotozoal, antimicrobial, antiinflammatory, antitumor and chemopreventive properties [5,6,28]. It was also revealed in the present study that 28 different compounds were indentified from the GC-MS analysis of Cichorium intybus L. roots which exhibits potent biological activities. The major metabolite present in chicory roots in terms of percent area is 1,2- benzenedicarboxylic acid mono (2-ethylhexyl) ester (1.6) possesses antifouling and antimicrobial properties. The GC-MS analysis also revealed the presence of 28 compounds from the stem and 36 different compounds from the flower of Cichorium intybus L. respectively, in which the major metabolites identified from stem in terms of percent area are: n-hexadecanoic acid (35.14), 9,12-octadecadienoic acid (16.01) and 2-furancarboxyaldehyde,5-hydroxymethyl (9.57). The first two compounds are fatty acids possessing several therapeutic applications as mentioned above while as 2-furancarboxyaldehyde,5- hydroxymethyl is an aldehyde which was found to possess antimicrobial and food preservative properties. The major metabolite identified from the flower of chicory in terms of percent area is 5- hydroxymethyl, 2-furaldehyde (33.26) an aldehyde compound that acts as a strong antioxidant. In addition to this, the results of the GC-MS profile can be used as pharmacognostical tool for the identification of the plant. The result of the present study supported and supplemented the previous observations [29,30]. GC-MS analysis showed the existence of various compounds with different chemical structures. The presence of various bioactive compounds confirms the application of Cichorium intybus L. (chicory)for various ailments by traditional practitioners. However, isolation of individual phytochemical constituents may proceed to find a novel drug. Thus, chicory can be utilized as an important source of bioactive compounds for various pharmaceutical applications. 5. CONCLUSION In the present study, we report the presence of some of the important compounds resolved by GC-MS analysis and their biological activities. The present study also helps to predict the formula and structure of biomolecules which can be used as drugs. This also enhances the traditional usage of Cichorium intybus which possess many known and unknown compounds. However isolation of individual compounds and their biological activities needs to be uncovered further to enhance its pharmacological importance and open new avenues in research. It could be concluded that Cichorium intybus from Kashmir Himalaya is a reservoir of bioactive constituents which could be used in various diseases in future. Hence, Cichorium intybus from Kashmir Himalaya may be recommended as a plant of phytopharmaceutical importance. Competing interest The authors declare that they have no competing interests. Acknowledgement RUR is thankful to University of Kashmir for the financial support. REFERENCES 1. WHO Report, World Health Organization Geneva, WHO/ EDM/ TRM/, 2002, 21: Vijayan A, Liju VB, John JV, Reena B, Parthipan C, Renuka,Indian Journal of Traditional Knowledge, 6(4): 2007,

11 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), 3. Hasan P, Yasa N, Ghanbari SV, Mohammadirad A, Dehghan G, Abdollahi M,In vitro antioxidant potential of Teucriumrolium, as compared to a-tocopherol. Acta Pharmaceutica, 57: 2007, de-fatima A, Modolo LV, Conegero LS, Pilli RA, Ferreira CV, Kohn LK, de-carvalho JE,Lactones and their derivatives: biological activities, mechanisms of action and potential leads for drug design. Current Medicinal Chemistry, 13: 2006, Selvamangai G, Anusha B, GC-MS Analysis of phytocomponents in the methanolic extract of Eupatorium triplinerve. Asian Pacific Journal of Tropical Medicines, 2: 2012, S Burns TA, Kadegowda AKG, Duckett SK, Pratt SL, Jenkins TC, Palmitoleicm (16:1 cis-9) and cis-vaccenic (18:1 cis-11) Acid Alter Lipogenesis in Bovine Adipocyte Cultures. Lipids., 47: 2012, Bursal E, Koksal E, Gulcin I, Bilsel G, Goren AC, Antioxidant activity and polyphenol content of cherry stem (Cerasusavium L.) determined by LCMS/MS. Food Research International, 51(1): 2013, Scholz E, Monographien: Cichorium, Cichorium intybus L., Cichorii folia et radix, Cichorii radix. In HagersHandbuch der Drogen und Arzneistoffe, Hager ROM, Eds W., E., Blaschek, S., Ebel, E., Hackental U., Holzgrabe, K., Keller, J., Reichling, and J., Schulz. Springer MedizinVerlag, Heidelberg, Blumenthal M (1998). The complete German Commission E monographs. American Botanical Council, Austin, TX, Poletti AH, Schilcher, Müller A, In HeilkraftigePflanzen, Walter HadeckeVerlag, 1989, Vilkhu KR, Mawson L, Simons Bates D, Applications and opportunities for ultrasound assisted extraction in the food industry- A review. Innovative Food Science and Emerging Technology, 9: 2008, Wootton-Beard PC, Moran A, Ryan L, Stability of the total antioxidant capacity and total polyphenol content of 23 commercially available vegetable juices before and after<i> in vitro</i> digestion measured by FRAP, DPPH, ABTS and Folin Ciocalteu methods. Food Research International, 44(1): 2011, Bais HP, Ravishankar GA, Cichorium intybus L.-cultivation, processing, utility, value addition and biotechnology, with an emphasis on current status and future prospects. Journal offood Science and Agriculture, 81: 2001, Sun LY, Touraud G, Charbonnier C, Tepfer D,Modification of phenotype in Belgian endive (Cichorium intybus) through genetic transformation by Agrobacterium rhizogenes: conversion from biennial to annual flowering. Transgenic Research, 1: 1991, Demigne C, Jacobs H, Moundras C, Davicco MJ, Horcajada MN, Bernalier A, Coxam V, Comparison of native or reformulated chicory fructans, or non-purified chicory, on rat cecal fermentation and mineral metabolism. European Journal of Nutrition, 47: 2008, Znidarcic D, Ban DA, Helena S, Carotenoid and chlorophyll composition of commonly consumed leafy vegetables in Mediterranean countries. Food Chemistry, 129(3): 2011, Sinkovic L, Hribar J, Vidrih R, Influence of cultivar and storage of chicory (Cichorium intybus L.) plants on polyphenol composition and antioxidative potential. Czech Journal of Food Science, 32: 2014, Fernie AR, Trethewey RN, Krotzky AJ, Willmitzer L,Innovation- Metabolite profiling: from diagnostics to systems biology. Nature ReviewsMolecular Cell Biology, 5: 2004, Kell DB, Brown M, Davey HM, Dunn WB, Spasic I, Oliver SG,Metabolicfootprinting and systems biology: The medium is the message. Nature Reviews of Microbiology, 3: 2005, Roessner U, Wagner C, Kopka J, Trethewey RN, Willmitzer L,Simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. Plant Journal, 23: 2000, Cong Z, Meiling Q, Qinglong S, Shan Z and Ruonong F,Analysis of the volatile compounds in Ligusticum chuanxiong Hort. using HS-SPMEGC-MS. Journal of Pharmaceutical and Biomedical Analysis., 44 (2): 2007, Lee SJ, Umano K, Shibamoto T, Lee KG, Identification of volatile components in basil (Ocimumbasilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chemistry, 91: 2005, Lampronti I, Saab AM, Gambari R,Antiproliferative activity of essential oils derived from plants belonging to the Magnoliophyta division. International Journal of Oncology, 29(4): 2006, Haznagy-Radnal E, Czigle S, Mathe I,TLC and GC analysis of the essential oils of Stachys species. Journal of Planar Chromatography, 20: 2007,

12 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), 25. Purabi R, Sarika A, Kumar A, Singh V, Preliminary study of the antioxidant properties of flowers and roots of Pyrostegiavenusta(Ker Gawl) Miers. BMC Complementary and Alternate Medicines, 11: 2011, Hema R, Kumaravel S, Alagusundaram K,GC/MS determinationof bioactive components of Murrayakoenigii. Journal of American Science,7(1): 2011, Ezhilan B, Neelamegam R, GC-MS Determination of bioactive compounds of Polygonumglabrum(Wild). Journal of Phytology, 3(9): 2011, Dantas da Silva LL, Nascimento M, Siqueira Silva DH, Furlan M, da Silva Bolzani V,Antibacterial activity of a stearic acid derivative from Stemodiafoliosa. Planta Medica, 68: 2002, Wu L, Gao H, Wang X, Ye J, Lu J, Liang Y,Analysis of chemical composition of Chrysanthemum indicumflowers by GC/MS and HPTLC. Journal of Medicinal Plants and Research, 4(5): 2010, Vohra A, Kaur H, (2011). Chemical investigation of medicinal plant Ajugabracteosa. Journal of Natural Product and Plant Resources, (1): 2011, Source of support: University of Kashmir, India, Conflict of interest: None Declared