International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in ORGANOSULFUR COMPOUNDS OF ALLIUM TUNCELIANUM (KOLLMANN) OZHATAY, B.MATHEW & ŞIRANECI EXTRACTS BY SPME/GC-MS AND DETERMINING THEIR CYTOTOXIC EFFECT ON HELA CELLS 1YUSUF CAN GERCEK, 2GIZEM AKMAN, 3HANDE MORGIL, 4MAHMUT CALISKAN, 5GUL CEVAHIR OZ 1,3,4,5Istanbul University Biology Department, Botany Division, Istanbul, Turkey 2Istanbul University Biology Department, Zoology Division, Istanbul, Turkey E-mail: 1 [email protected], 2 [email protected], 3 [email protected], 4 [email protected] Abstract- Allium tuncelianum (Kollmann) Özhatay, B.Mathew & Şiraneci, known as Tunceli garlic, is an endemic species native to Turkey and distributed only in and around Tunceli district. Tunceli garlic was identified as a subspecies of A. macrochaetum Boiss. et Hausskn., however, following studies identified it as a different species, and it was named as A. tuncelianum. Because its chemical structure is similar to cultivated garlic (A. sativum), it is collected from the mountains, and sold/ used as a commercial product with the name “Rock Garlic” by local people. The plant is important in terms of being endemic and placed in the group of Vulnerable Plants in Red Data Book of Turkish Plants. In the present study, organosulfur compounds, categorized in volatile fatty acids, of the plant, Allium tuncelianum were extracted with solid phase micro extraction (SPME/PDMS) method and analyzed with Gas Chromatography-Mass Spectroscopy device. As a result of the analysis, diallyl disulphide compound was determined to be major compound at the rate of %50.51. Later on, cytotoxic activity of plant extracts was determined on HeLa cells originating from human cervical carcinoma and it was found that 72 h, 0,5 mg/ml concentration led 30 percent of the cells to apoptosis. It is clearly advantageous to rehabilitate Allium tuncelianum plant by cultivating it, increase its production, and making the plant consumed on a global basis rather than its limited consumption by local people, as an alternative to Allium sativum (cultivated garlic) which has been consumed traditionally over centuries. Keywords- Allium tuncelianum, Solid Phase Micro Extraction (SPME), GC-MS, Cytotoxic Activity, HeLa Cells I. INTRODUCTION cloves in its bulb. On the other hand, cloves are formed instead of flowers on the tip of pedicels. The flora of Turkey contains 12.054 species (Guner, Forming of cloves during blooming is pointed as the 2000; Ozhatay, 1994, 1999, 2006, 2009, 2008). reason why garlic is sterile (Koul, 1970). Therefore, Turkey shows a continent diversity in Many endemic plants are gathered from their natural terms of plant variety. Also 32.56% (3.925) of these habitat excessively. Due to senseless and excessive taxa are endemic to Turkey. The greatness of this gathering most of these plants are facing extinction. value can be seen clearer considering even the richest Allium tuncelianum was listed among the endangered country in Europe in terms of endemic plants, Greece, plants in Red Book of Plants of Turkey (Ekim, 2000). has between 800-1000 endemic plant species (Ekim, Taste of fresh garlic’s characteristics are associated 2000). Having such high ratio of endemism in Turkey with thiosulfinates and compounds that form leads to an increased interest from scientists who enzymatically from odorless substances (Cavallito, study endemic plants. 1944; Stoll, 2006; Lancaster, 1988; Iberl, 1990; Han, Allium L. genus, contains 700 biennial and perennial 1995; Ferary, 1996; Calvey, 1997; Keusgen, 2002). species which live in temperate zones of the northern Primary substance responsible for garlic’s taste is hemisphere (Konemann, 1999). There are 164 Allium allicin (2-Propene-1-sulfinothioic acid S-2-propenyl taxa in Turkey, of which 65 are indigenous to Turkey, ester). The enzyme alliinase converts alliin ((S)-3-(2- thus are endemic (Davis, 1984, 1998; Guner, 2000). Propenylsulfinyl)-L-alanine) to allicin. Allicin is an A. tuncelianum is originally named as A. unstable compound and breaks down to sulfites macrochaetum Boiss. and Haussk. subsp. which are secondary substances that give garlic it’s tuncelianum Kollman (Etoh, 2002). Although it is characteristic taste and odor (Stoll, 1951; Lancaster, indigenous to Tunceli district and especially the 1988; Iberl, 1990; Ferary, 1996; Keusgen, 2002). plateaus of Munzur Mountains in Ovacik, A. Primary substances in garlic are sulfur compounds. tuncelianum naturally exists in a limited area between These are allicin, thiosulfinates, gamma-glutamyl Sivas and Erzurum districts (Baktir, 2005). Around cysteine peptides and some other sulfur compounds. these regions, it is named “Garlic of Tunceli” or Some of these aren’t found in garlic naturally. They “Garlic of Ovacik” because of its similarity to form while crushing, cutting and chopping garlic. It is common garlic. A. tuncelianum has single clove in thought that 82% of sulfur compounds in garlic are contrast to the regular garlic which has numerous derived from alliin and gamma-glutamylcystein Organosulfur Compounds of Allium Tuncelianum (Kollmann) Ozhatay, B.Mathew & Şiraneci Extracts by Spme/Gc-Ms and Determining their Cytotoxic Effect on Hela Cells 52 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in peptides. Alliin is naturally found in garlic. When has antioxidant features (Gazzani, 1998; Yin, 1998). garlic is physically processed alliinase enzyme which It is thought that this effect is primarily a result of is stored in vacuoles is released and converts alliin to sulfur compounds (Kim, 1997; Lampe, 1999). allicin. Allicin is responsible for many biological Garlic’s benefits to human health makes it important activities of garlic. Garlic loses its allicin content to study. Garlic’s components are pharmaceutically completely when boiled. It has been proposed that active and have antibiotic, lipid-lowering, antioxidant this happens because allicin and alliin are converted and anti-tumor properties and prevents platelets from to volatile fatty acids such as diallyl-sulfides. Active accumulating together essentially preventing blood substance in garlic are allicin, ajoene and diallyl- clots from forming (Agarval, 1996; Dorant, 1996; sulfides. Best way to benefit from garlic is using Ferary, 1996; Calvey, 1997; Ankiri, 1999; Yoshida, when these three substances are together. Sulfurous 1999; Teyysier, 2001; Keusgen, 2002; Mondy, 2002). volatile fatty acids give the characteristic smell of garlic (Cavallito, 1944a, b; Barone, 1977; Hughes, II. MATERIAL AND METHOD 1991; Abu-Lhafi, 2004; Kimbaris, 2006;). Garlic (Allium sativum L.) is grown since ancient 2.1. Plant Material ages. No progenitor species are defined although Allium tuncelianum was obtained from a local market some species were suggested as such. Based on in Ovacik, Tunceli. Plants were kept in a freezer at - taxonomic studies (Fritsch and Friesen 2002), garlic 20 oC. may have a wild ancestor living in the region between Mediterranean and Central Asia. It has been stated 2.2. Solid-phase Micro Extraction of Samples that Allium longicuspis which grows in Central Asia, 2 grams of Allium tuncelianum was cut into 0.5 cm could be a progenitor of garlic (Vvedensky, 1944). pieces and a closed system was set up. In order to However latest research based on DNA markers have clean the fiber, it was kept at 250oC for 30 minutes shown that A. longicuspis is not a distant relative of before the extraction. Extraction was made using common garlic (Maass and Klaas, 1995; Al-Zahim et SPME/PDMS fiber at 40oC for 45 minutes. Extract al., 1997; Ipek et al., 2003). Mathew (1996), was manually injected to the inlet part of GC-MS. suggested that Allium tuncelianum which is used instead of garlic in eastern Turkey, could be a distant 2.3. GC-MS Analysis ancestor of garlic. In order to evaluate genetic and Samples were analyzed using GC Agilent 7890A, phylogenetic connection between A. tuncelianum and Headspace Agilent 7694E, Agilent 5975C MSD a few other Allium species, a research was done by (Mass Selective Detector), HP-5MS nonpolar colon Ipek and colleagues (2008), using amplified fragment (0.25 mm x 30.0 cm x 0.25 µm) with GC 5% phenyl length polymorphism (AFLP) and internal transcribed methyl siloxane composition. GC was kept at 40oC spacer (ITS). AFLP analysis revealed that A. for 5 minutes, heated to 110oC with 3oC/min tuncelianum and garlic are genetically distant. increments, further heated to 150ºC with 4oC/min Phylogenetic analysis done by using ITS showed that increments and programmed to stay at 210oC for 12 A. tuncelianum and garlic are distant relatives and minutes. Injector temperature was kept at 250oC placed A. tuncelianum, Allium sativum, and Allium (Kiralan et al., 2013). Helium was used as a carrier scorodoprasum on the same branch of dendrogram gas. MS detector was operated at 194oC and and parsimony analysis put them on the same branch ionization energy was adjusted to 70 eV. Scanning in a consensus tree. In addition to this, in terms of range was set to 0.9s-1, between 30 to 650 m/z and phylogenetics A. tuncelianum is more distant to adjustments were made using Wiley and NIST Allium sativum, than to A. ampeloprasum and A. Library Scanning Software. scorodoprasum. This makes scientists think that A. tuncelianum is not a direct ancestor of Allium 2.4. Preparing The Cell Culture sativum. Trying to make hybrids from A. Minimum Essential Medium (MEM, Sigma) with tuncelianum and Allium sativum can reveal more Earle's salts and L-glutamine and without sodium information on homology and genetic connections bicarbonate was put in distilled water. 100 µg/ml of between these species. streptomycin and 100 IU/ml penicillin (Multicell, Garlic has been used for various medicinal purposes Wisent Products) was added to the solution and then for many centuries. The most significant of all is its filtered through 0,22 µm filter (Millipore).