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

Research Article ISSN: 0974-6943 Bisma Malik et al. / Journal of Pharmacy Research 2016,10(11), Available online through http://jprsolutions.info 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-190006, India 2 Department of Botany, University of Kashmir, Srinagar, Jammu and Kashmir-190006,India. 3 Department of Biotechnology, Jamia Hamdard, New Delhi-110062, India. *Corresponding author. Dr. Reiaz Ul Rehman Assistant Professor Department of Bioresources, University of Kashmir, Srinagar, Jammu and Kashmir-190006, India Received on:31-08-2016; Revised on: 27-09-2016; Accepted on: 23-11-2016 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

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 65000 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).

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 1 13.176 Tetradecanoic acid C 14 H 28 228 0.25 Fatty acid Antioxidant, cancer preventive, nematicide, hypercholesterolemic, lubricant 2 14.214 Pentadecanoic acid C 11 186 0.45 Fatty acid Flavoring agent, antioxidant 3 14.31 1,2-Benzenedicarboxylic acid, C 16 278 0.35 Ester Used in cosmetics bis(2-methylpropyl) ester 4 14.782 Hexadecanoic acid, methyl ester C 17 270 0.37 Ester Antioxidant, Flavor, Hypocholesterolemic Pesticide, 5-Alpha reductase inhibitor 5 15.272 n-hexadecanoic acid C 16 256 34 Fatty acid Antioxidant, hypocholesterolemic, nematicide, hemolytic, 5-alpha reductase inhibitor 6 15.811 3,4,4a,5-Tetrahydrobenzo[g] C 13 H 13 NO 199 1.44 Ketone NA isoquinolin-10(2h)-one 7 16.168 9-Octadecenoic Acid (Z)- C 18 282 0.46 Fatty acid Insect pheromone, pharmaceuticals, emulsifying agent, emollient, 8 16.438 9,12-Octadecadienoic acid, C 19 294 0.51 Ester Antiinflammatory, Nematicide, Insectifuge, methyl ester Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic, 9 16.915 9,12-Octadecadienoic acid C 19 294 35.03 Fatty acid Antiinflammatory, Nematicide, Insectifuge, (Z,Z)- Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic 10 16.975 9,12,15-Octadecatrienoic acid, C 18 278 6.89 Fatty acid Antiinflammatory, Hypocholesterolemic, Cancer (Z,Z,Z)- preventive, Nematicide, Hepatoprotective, Insectifuge, Antihistaminic, Antieczemic, Antiacne, 5-Alpha reductase inhibitor, Antiandrogenic, Antiarthritic, Anticoronary, 11 17.099 Octadecanoic acid C 18 H 36 284 0.82 Fatty acid Dietry supplements, softening agent, surfactant 12 17.505 Verrucarol C 15 266 0.64 Terpenoid Antitumor activity 13 18.102 5,8,11-Heptadecatriynoic acid, C 18 H 24 272 0.91 Ester Dyestuffs, feed additives, pharmaceuticals, methyl ester pigment coatings 14 19.727 4-Methyl-5-(phenylmethyl)-2,3- C 12 H 14 S 222 1.94 Hydrocarbon Lubricants dihydrothiophene 1,1-dioxide 15 21.104 Di-n-octyl phthalate C 24 H 38 390 0.31 Ester cosmetics, pesticides, plasticizer, 16 23.341 Docosane C 22 H 46 310 0.35 Hydrocarbon Used in organic synthesis, calibration, and temperature sensing equipment. 17 25.787 Octacosane C 28 H 58 394 0.2 Hydrocarbon Pheromones, lubricants 18 32.033 Stigmasterol C 29 12 0.83 Steroid Anti cancer, antioxidant 19 33.378 beta.-sitosterol C 29 14 0.45 Steroid Cholesterol lowering and symptom improvement in mild to moderate benign prostatic hypertrophy. Possible role in the control of chronic inflammatory conditions. 20 33.817 1R,4S,7S,11R-2,2,4,8- C 15 H 24 204 0.5 Terpenoid Nutrient, stabilizers, surfactants and emulsifiers Tetramethyltricyclo[5.3.1. 0(4,11)]undec-8-ene 21 34.273 4,4,6A,6B,8A,11,11,14B- C 30 24 2.64 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 22 35.507 5H-3,5a-Epoxynaphth C 18 278 3.51 Hydrocarbon Fragrance agents [2,1-c]oxepin, dodecahydro- 3,8,8,11a-tetramethyl-, [3S (3.alpha.,5a.alpha., 7a.alpha., 11a.beta.,11b.alpha.)]- 23 37.709 9,19-Cyclolanost-23-ene-3,25- C 32 H 52 484 0.92 Terpenoid Flavoring agent diol, 3-acetate, (3.beta.,23E)- 24 38.169 Lupeol C 30 26 4.29 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

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 1 6.748 2-Decenal, (Z)- C 10 H 18 O 154 0.49 Aldehyde Food flavoring agent 2 7.244 2,4-Decadienal, (E,E)- C 10 H 16 O 152 0.83 Aldehyde Flavoring agent 3 7.579 2,4-Decadienal, (E,E)- C 10 H 16 O 152 1.04 Aldehyde Flavoring agent 4 10.896 9-Octadecenoic Acid C 18 282 0.16 Fatty acid Reducing blood pressure (Hypotensive effect) 5 13.181 Tetradecanoic Acid C 14 H 28 228 0.31 Fatty acid Used in cosmetics and in topical medicinal preparation 6 13.685 2-Ethylhexyl salicylate C 15 250 0.07 Ester Used in cosmetics 7 14.304 1,2-Benzenedicarboxylic acid, bis(2- C 16 278 0.09 Ester Used in cosmetics methylpropyl) ester 8 14.776 Hexadecanoic acid, methyl ester C 17 270 0.17 Ester Antioxidant, Flavor, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5- Alpha reductase inhibitor 9 15.075 cis-9-hexadecenoic acid C 16 254 0.18 Fatty acid increase insulin sensitivity by suppressing inflammation, as well as inhibit the destruction of insulin-secreting pancreatic beta cells 10 15.504 n-hexadecanoic acid C 16 256 24.37 Fatty acid Antioxidant, Flavor, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5- Alpha reductase inhibitor 11 16.448 9,12-Octadecadienoic acid, methyl ester C 19 294 0.78 Ester Antiinflammatory, Nematicide, Insectifuge, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic. 12 18.265 8,11,14-Eicosatrienoic acid, (Z,Z,Z)- C 20 306 0.29 Fatty acid Anti-inflamatory, antithrombotic effect 13 18.996 Eicosanoic acid C 20 312 0.47 Fatty acid used in the manufacture of Pharmaceuticals,soaps, cosmetics, and food packaging. 14 21.183 1,2-Benzenedicarboxylic acid, mono(2- C 16 278 1.6 Ester Anti fouling, Antimicrobial ethylhexyl) ester 15 21.68 Hexadecane C 16 226 0.06 Hydrocarbon Used as a substrate for the production of biosurfactants 16 22.724 Cyclopropane, 1,1-dichloro-2,2,3,3-tetramethyl- C 7 H 12 C 12 166 0.06 Hydrocarbon Used in pharmaceuticals, agrochemicals, food additive 17 23.543 9,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1 C 21 H 38 354 0.46 Ester Co-solvents, oil carrier, antioxidant, -(hydroxymethyl)ethyl ester antiacne 18 24.697 Docosane C 22 H 46 310 0.04 Hydrocarbon Used in organic synthesis, calibration, and temperature sensing equipment. 19 32.059 Stigmasta-5,22-dien-3-ol C 29 12 0.05 Steroid used as a biomarker for the presence of. (marine) algal matter in the environment 20 33.392 beta.-sitosterol C 29 14 0.06 Steroid Cholesterol lowering and symptom improvement in mild to moderate benign prostatic hypertrophy. Possible role in the control of chronic inflammatory conditions. 21 34.278 4,4,6a,6b,8a,11,11,14b-Octamethyl- C 30 24 0.05 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 22 35.516 Lupeol C 30 26 0.22 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.

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 1 6.585 2-Furancarboxaldehyde, 5-(hydroxymethyl)- C 6 H 6 126 9.57 Aldehyde Food flavoring agent, antimicrobial, preservative 2 10.597 beta.-d-glucopyranose, 1,6-anhydro- C 6 H 10 O 5 162 2.61 Sugar Biomarker 3 11.543 Benzeneacetic acid, 3-hydroxy- C 8 H 8 152 0.95 Phenol NA 4 13.162 Tetradecanoic acid C 14 H 28 228 0.46 Fatty acid Antioxidant, cancer preventive, nematicide, hypercholesterolemic, lubricant 5 13.878 2,6,10-Trimethyl,14-ethylene-14-pentadecne C 20 H 38 278 2.69 Hydrocarbon Antiproliferative 6 13.968 2-Undecanone, 6,10-dimethyl- C 13 H 26 O 198 0.7 Ketone NA 7 14.134 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, C 20 96 0.58 Terpenoid Antimicrobial, Anticancer, Diuretic, [R-[R*,R*-(E)]]- (Phytol) Antiinflammatory 8 14.209 Pentadecanoic acid C 15 242 0.37 Fatty acid Flavoring agent, antioxidant 9 14.325 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C 20 96 1.61 Terpenoid Antimicrobial, Anticancer, Diuretic, Antiinflammatory 10 14.688 2-Pentadecanone, 6,10,14-trimethyl- C 18 H 36 68 0.81 Hydrocarbon Flavor and fragrance agent 11 14.771 Pentadecanoic acid, 14-methyl-, methyl ester C 17 270 0.26 Ester Antioxidant 12 15.331 n-hexadecanoic acid C 16 256 35.14 Fatty acid Antioxidant, Flavor, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5- Alpha reductase inhibitor 13 16.168 Heptadecanoic acid C 17 270 0.25 Fatty acid Dairy products 14 16.586 Palmitaldehyde, diallyl acetal C 22 H 42 338 0.85 Aldehyde NA 15 16.906 9,12-Octadecadienoic acid C 18 280 16.01 Fatty acid Antiinflammatory, Nematicide, Insectifuge, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic 16 17.114 Octadecanoic acid C 18 H 36 284 2.72 Fatty acid Dietry supplements, softening agent, surfactant 17 18.889 Eicosanoic acid C 20 312 0.47 Fatty acid Detergents, photographic materials and lubricants. 18 19.75 Naphtho[1,2-b]furan-2,8(3h,4h)-dione, C 15 H 18 262 0.37 Terpenoid Antiparasitic,anti-inflammatory, 3a,5,5a,9b-tetrahydro-4-hydroxy-3,5a,9- antipyretic and analgesic trimethyl-, [3s-(3.alpha.,3a.alpha., 19 20.256 1-Eicosene C 20 280 0.42 Hydrocarbon Pharmaceticals 20 21.109 Di-n-octyl phthalate C 24 H 38 390 0.71 Ester cosmetics, pesticides, plasticizer, 21 27.683 3.beta.-Acetoxystigmasta-4,6,22-triene C 31 452 0.42 Steroid NA 22 28.557 Cholesta-4,6-dien-3-ol, (3.beta.)- C 27 H 44 84 1.17 Steroid Adhesive 23 32.047 Stigmasterol C29H4812 0.36 Steroid Anti cancer, antioxidant 24 33.41 gamma.-sitosterol C 29 14 0.81 Steroid Anti-diabetic, Anti-angeogenic, Anticancer, antimicrobial, anti-inflammatory, antidiarrhoeal and antiviral 25 34.315 4,4,6a,6b,8a,11,11,14b-Octamethyl- C 30 24 2.5 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 26 35.598 Lupeol C 30 26 7.68 Terpenoid Antiprotozoal, antimicrobial, antiinflammatory, antitumor and chemopreventive properties 27 37.742 9,19-Cycloergost-24(28)-en-3-ol, 4,14- C 32 H 52 468 0.65 Terpenoid Flavoring agent dimethyl-, acetate, (3.beta.,4.alpha.,5.alpha.)- 28 38.575 Lup-20(29)-en-3-yl acetate C 32 H 52 468 4.2 Terpenoid Antimicrobial and antiinflammatory

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.

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 1 5.228 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C 6 H 8 144 6.44 Ketone Antioxidant 2 6.954 5-(hydroxymethyl)-2-furaldehyde C 6 H 6 126 33.26 Aldehyde Antioxidant 3 8.058 Cis-dimethyl morpholine C 6 H 13 NO 115 0.58 Hydrocarbon Fungicide 4 11.01 beta.-d-glucopyranose, 1,6-anhydro- C 6 H 10 O 5 162 3.66 Sugar Biomarker 5 12.767 4-(1-Hydroperoxy-2,2-dimethyl-6-methylene- C 14 238 0.11 Ketone NA cyclohexyl)-pent-3-en-2-one 6 13.172 Tetradecanoic acid C 14 H 28 228 0.19 Fatty acid Antioxidant, cancer preventive, nematicide, hypercholesterolemic, lubricant 7 13.51 6-Dodecanone C 12 H 18 O 178 0.12 Ketone Flavor and fragrance agent 8 13.815 1,4-Methanoazulene, decahydro-4,8,8-trimethyl-9- C 15 H 24 204 0.16 Hydrocarbon Flavor and fragrance agent, methylene-, [1s-(1.alpha.,3a.beta.,4.alpha.,8a.beta.)]- cosmetics 9 13.969 2-Pentadecanone, 6,10,14-trimethyl- C 18 H 36 68 0.18 Hydrocarbon Flavor and fragrance agent 10 14.221 Pentadecanoic acid C 15 242 0.31 Fatty acid Flavoring agent, antioxidant 11 14.477 Tetradecane C 14 198 0.15 Hydrocarbon NA 12 14.561 2-Heptadecanone C 17 54 0.21 Ketone Flavoring agent 13 15.364 n-hexadecanoic acid C 16 256 13.77 Fatty acid Antioxidant, Flavor, Hypocholesterolemic Nematicide, Pesticide, Lubricant, Antiandrogenic, Hemolytic, 5- Alpha reductase inhibitor 14 16.187 Heptadecanoic acid C 17 270 0.51 Fatty acid Dairy products 15 16.457 4-Tetradecanol C 14 14 0.8 Alcohol Surfactant 16 17.074 9,12-Octadecadienoic acid (Z,Z)- C 18 280 26.86 Fatty acid Antiinflammatory, Nematicide, Insectifuge, Hypocholesterolemic, Cancer preventive, Hepatoprotective, Antihistaminic, Antiacne, Antiarthritic, Antieczemic 17 17.222 Octadecanoic acid C 18 H 36 284 2.97 Fatty acid Dietry supplements, softening agent, surfactant 18 17.678 Cycloheptanone, 2-(3-buten-1-yl)- C 11 H 18 O 166 0.22 Ketone Pharmaceuticals, fragrance agent 19 17.996 Nonadecanoic acid C 19 H 38 298 0.45 Fatty acid Insect pheromones 20 18.164 Gamolenic Acid C 18 278 0.25 Fatty acid Pharmaceuticals, Functional foods 21 18.576 2H-Pyran, 2-(2-heptadecynyloxy)tetrahydro- C 22 336 0.79 Phenol Antimicrobial, Anti-inflammatory, Antioxidant 22 18.921 Eicosanoic acid C 20 312 0.38 Fatty acid Detergents, photographic materials and lubricants 23 19.659 Cyclohexanol, 5-methyl-2-(1-methylethyl)-, acetate C 12 198 1.48 Ester Flavor and fragrance agent 24 20.751 Hexadecanoic acid, 2-oxo-, methyl ester C 17 284 0.29 Ester NA 25 20.847 5-Eicosene, (E)- C 20 280 0.22 Fatty acid Antiviral, antiaging, anticancer, antioxidant 26 21.125 Di-n-octyl phthalate C 24 H 38 390 0.14 Ester cosmetics, pesticides, plasticizer, 27 21.518 2-Propenyl nonanoate C 12 198 1.67 Ester Flavor and fragrance agent 28 22.276 Palmitic acid vinyl ester C 18 282 0.24 Ester Emulsifying agents for flavors used in fruit flavoured beverages 29 23.154 Octadecanoic acid, 2-propenyl ester C 21 324 0.64 Ester Dietary supplements, surfactants, softening agent, cosmetics, detergents and soaps 30 24.562 14,16-Hentriacontanedione C 31 H 60 464 0.47 Ketone NA 31 27.374 Cholest-24-en-16-one, (5.alpha.,20.xi.)- C 27 H 44 84 0.08 Steroid NA 32 32.047 Stigmasterol C 29 12 0.1 Steroid Anti cancer, antioxidant 33 33.384 beta.-sitosterol C 29 14 0.12 Steroid Cholesterol lowering and symptom improvement in mild to moderate benign prostatic hypertrophy. Possible role in the control of chronic inflammatory conditions. 34 35.311 1-(1,5-Dimethyl-4-hexenyl)-3a,6,6,12a- C 32 H 52 468 0 Terpenoid NA tetramethyltetradecahydro-1hcyclopenta[a]cyclopropa[e]phenanthr 35 35.523 5H-3,5a-Epoxynaphth[2,1-c]oxepin, dodecahydro- C 18 278 0.26 Hydrocarbon Fragrance agents 3,8,8,11a-tetramethyl-, [3S -(3.alpha.,5a.alpha.,7a.alpha.,11a.beta.,11b.alpha.)]-

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

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).

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. 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