International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in ORGANOSULFUR COMPOUNDS OF 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 , 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 is important in terms of being endemic and placed in the group of Vulnerable 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). Prepared use as an antibiotic. It has been mixed with butter to medium was kept at +4oC. heal wounds (Koch and Lowson 1996). There are many studies on Allium species about their effect on 2.5. Passaging the Cells human health. Garlic (A. sativum) draws attention for Provided by Tokyo Institute of Technology, the its therapeutic and prophylactic features. Anecdotal tumoral cell line we used in our experiments are evidence suggests that Allium species plays an HeLa (CCL-2) cells which originate from human important role in treatment and prevention of cervical carcinoma. HeLa cells which are passaged cardiovascular diseases, tumors and pathogenic twice a week were incubated in standard tissue infections. Other studies claim that Allium species culture medium added with 10% Fetal Bovine Serum

Organosulfur Compounds of Allium Tuncelianum (Kollmann) Ozhatay, B.Mathew & Şiraneci Extracts by Spme/Gc-Ms and Determining their Cytotoxic Effect on Hela Cells

53 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in (FBS, Gibco Lab.), pH adjusted to 7,2 with 4,4% After the incubation period, medium was removed 2 NaHCO3, in 25 cm sterile culture plates, in 5% CO2 from the cells which were inoculated in 96-well and 95% humid air, at 37oC as a single-layer culture. plates. 40 µl of MTT solution was prepared in phosphate buffered saline (PBS, Multicell, Wisent 2.6. Inoculating The Cells Products) and was added on all wells and incubated at º For cytotoxicity trials, cells were inoculated in a 96- 37 C, in a dark environment with 5% CO2 content for well plates at a rate of 20,000 cells per well. After the 4 hours. After the incubation, 160 µl of dimethyl inoculation, cells were incubated in an incubator sulfoxide was added on all wells and further o which has 5% CO2 and 95% air, at 37 C, at pH 7.2 incubated for one night. for 24 hours. Experimental group’s absorbance values are measured with a spectrophotometer at 570 nm 2.7. Determining Cytotoxicity: Mitochondrial wavelength, with 690 nm reference wavelength Dehydrogenase Enzyme Activity (Kurashige et al., 1999). Cytotoxic effect on HeLa cells was determined by using MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5- III. RESULTS AND DISCUSSION diphenyltetrazolium bromide) method. The principle behind this method is based on mitochondrial Organosulfur compounds from Allium tuncelianum dehydrogenase enzymes which are found inside alive that were analyzed using the Solid-phase micro cells, reducing MTT salt which is a water soluble extraction technique are shown in Table 1. Major yellow compound, into insoluble formazan salt. compound is diallyl sulphide with 50.51 percent. Mitochondrial dehydrogenase enzyme activity Molecular structure is as shown in Figure 3. 72.14% increases as number of cells increase, thus the amount of Allium tuncelianum volatile compounds of formazan salt crystals is directly proportional to determined as organosulfur compounds. the number of cells (Mosmann, 1983).

Morphological changes of HeLa cells in control group and 0 to 72 hours cell cultures are as shown in Figure 1 and Figure 2. No deterioration observed in cell proliferation and liveliness.

Table 1. Organosulfur compounds of Allium tuncelianum found using SPME-GC-MS. Figure 1: Image of HeLa cell culture control group under phase-contrast microscope (200x) after time duration of 0 hour.

Figure 3: Molecular structure of diallyl disulphide

MTT method is used to determine the cytotoxic effects of 5 different solutions (0.001 mg/ml, 0.01 mg/ml, 0.05 mg/ml, 0.25 mg/ml, 0.5 mg/ml) which were applied on HeLa cells for 24, 48 and 72 hours durations. The average absorbance values of experiment group and control group were measured (Figure 4). After the evaluation, experiment groups were compared with the control group. The 0.5 Figure 2: Image of HeLa cell culture control group under mg/ml concentration extract of Allium tuncelianum phase-contrast microscope (200x) after time duration of 72 determined to apoptosis 30% of the cells at 72nd hours. hour.

Organosulfur Compounds of Allium Tuncelianum (Kollmann) Ozhatay, B.Mathew & Şiraneci Extracts by Spme/Gc-Ms and Determining their Cytotoxic Effect on Hela Cells

54 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in number of herbal products that are intended for medicinal use, researches on extraction of active compounds and acquiring commercial products from plants should be increased, and applicability of these in human body should be investigated thoroughly. .

CONCLUSIONS

As a result of the analyses, it has been discovered that three main components (allyl disulfide, diallyl disulphide and allyl trisulphide) of Allium tuncelianum make up of 65.70% of the extract. It is extremely important that other soluble substances in the extract are identified. Organosulfur compounds which are the major group in garlic were analyzed using SPME technique. Which substance caused the cytotoxic activity or the combined effect of these Figure 4: Percentage of live cells with extracts from Allium substances has to be tested. In order to make practical tuncelianum on 24th, 48th and 72nd hour marks. (D1:0.001 use of the plant extract, the active ingredients, after mg/ml, D2:0.01 mg/ml, D3:0.05 mg/ml, D4:0.25 mg/ml, D5:0.5 mg/ml) being obtained separately, have to be tested on HeLa cells for their cytotoxic effects, and then which active SPME is a very simple and efficient, solventless ingredients kill the cells or cause adverse effects must sample preparation method that uses a syringe-like be determined using various combinations. In mechanism. It involves the use of a fiber coated with addition to this, the afore-mentioned major an extracting phase, that can be a liquid (polymer) or substances in discussion and conclusion were found a solid (sorbent), which extracts different kinds of in different amounts than other studies. This analytes (including both volatile and non-volatile) difference in the results should be investigated by from different kinds of media, that can be in liquid or gathering more samples from different populations gas phase. Extraction is done by inserting SPME and comparing the results of the analyses all together. needle and then retracting the part that covers the It is necessary to evaluate the extraction method used fiber. Then the sample is analyzed using GC-MS. to obtain the active compounds by comparing with As a result of analyses, 3 main organosulfur other methods, to conduct experiments intended for compounds in Allium tuncelianum are identified as clinical trials and to conduct multi-disciplined allyl disulfide, diallyl disulphide and allyl trisulphide. experiments concurrently. This project is intended to By comparing the results with works of Kiralan and be a pretest and to get a better and usable results, colleagues (2013), it is found that the major extended research needs to be done. compounds have different ratios (Kiralan, 2013). Turkey has an extraordinarily rich flora. By There can be a few causes for this, one of which is increasing the amount of research on endemic species alteration of the contents according to the of Turkey, plants that could be used as medicine environmental conditions that plants grew, and the should be identified and the work on their production other is the difference in fiber structural material and and improvement should be increased. By making column. commercial products, these plants should be Extracts from Allium tuncelianum are prepared in introduced to the global market thus contributing the five different concentrations as follows: 0.001; 0.01; national economy. 0.05; 0.25 and 0.5 mg/ml. During the 24, 48 and 72 hour periods, each solution with different ACKNOWLEDGMENTS concentration applied on HeLa cultures. On 72nd hour, it is observed that 0.5 mg/ml concentration This work was supported by Scientific Research caused significantly increased rates of apoptosis. Projects Coordination Unit of Istanbul University Considering that today cancer is a rather common with project number 47349. The authors would like to disease, it should be kept in mind that the main goal thank Almila Ciftci for taking time to read the final in cancer treatment is to accelerate the cancerous manuscript. cells’ death. Therefore, knowing about cell cycle mechanisms, and molecules that take part in cell REFERENCES cycle and cell cycle checkpoints increases the [1] Abu-Lhafi, S., Dembicki, J. W., Goldshlag, P., Hanuš, L. O., efficiency of treatments. In recent years there is a & Dembitsky, V. M. (2004). The use of the tendency in using natural herbal products under the ‘Cryogenic’GC/MS and on-column injection for study of name of Alternative Medicine. High rates of failure in organosulfur compounds of the Allium sativum. Journal of other methods used in cancer treatment (radiotherapy, Food Composition and Analysis, 17(2), 235-245. chemotherapy) might have a part in this. Therefore,

Organosulfur Compounds of Allium Tuncelianum (Kollmann) Ozhatay, B.Mathew & Şiraneci Extracts by Spme/Gc-Ms and Determining their Cytotoxic Effect on Hela Cells

55 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in [2] Agarwal, K. C. (1996). Therapeutic actions of garlic Allium Crop Science: Recent Advances. CABI Publishing, constituents. Medicinal research reviews, 16(1), 111-124. New York, pp. 5–30. [3] Al-Zahim, M., Newbury, H.J., Ford-Lloyd, B.V., 1997. [22] Gazzani G, Papetti A, Daglia M, Berte F and Gregotti C, Classification of genetic variation in garlic (Allium sativum) 1998. Protective activity of water soluble components of revealed by RAPD. HortScience 32, 1102–1104. some common diet vegetables on rat liver microsome and the [4] Ankri, S., & Mirelman, D. (1999). Antimicrobial properties effect of thermal treatment. Journal of Agricultural and Food of allicin from garlic.Microbes and infection, 1(2), 125-129. Chemistry 46: 4123- 4127. [5] Baktir I, 2005. Tunceli sarimsagi’nin (Allium tuncelianum) in [23] Guner A, Ozhatay N, Ekim T and Baser KHC, 2000. Flora of vitro kosullarinda cogaltilmasi. In: Proceeding of GAP IV. Turkey and the East Aegean islands (Vol. 11) (Supplement- Tarim Kongresi. II). Edinburgh: Edinburgh University Press. [6] Barone, F. E., & Tansey, M. R. (1977). Isolation, [24] Han, J., Lawson, L., Han, G., & Han, P. (1995). purification, identification, synthesis, and kinetics of activity Spectrophotometric method for quantitative determination of of the anticandidal component of Allium sativum, and a allicin and total garlic thiosulfinates. Analytical biochemistry, hypothesis for its mode of action. Mycologia, 793-825. 225(1), 157-160. [7] Block, E., Ahmad, S., Catalfamo, J. L., Jain, M. K., & Apitz- [25] Iberl, B., Winkler, G., Müller, B., & Knobloch, K. (1990). Castro, R. (1986). The chemistry of alkyl thiosulfinate esters. Quantitative Determination of Allicin and Alliin from Garlic 9. Antithrombotic organosulfur compounds from garlic: by HPLC*. Planta Medica, 56(3), 320-326. structural, mechanistic, and synthetic studies. Journal of the [26] Ipek, M., Ipek, A., & Simon, P. W. (2008). Genetic American Chemical Society, 108(22), 7045-7055. characterization of Allium tuncelianum: An endemic edible [8] Calvey, E. M., Matusik, J. E., White, K. D., DeOrazio, R., Allium species with garlic odor. Scientia horticulturae, Sha, D., & Block, E. (1997). Allium chemistry: Supercritical 115(4), 409-415. fluid extraction and LC-APCI-MS of thiosulfinates and [27] Ipek, M., Ipek, A., Simon, P.W., 2003. Comparison of related compounds from homogenates of garlic, onion, and AFLPs, RAPD markers, and isozymes for diversity ramp. Identification in garlic and ramp and synthesis of 1- assessment of garlic and detection of putative duplicates in propanesulfinothioic acid S-allyl ester. Journal of germplasm collections. J. Am. Soc. Hort. Sci. 128, 246–252. Agricultural and Food Chemistry, 45(11), 4406-4413. [28] Keusgen, M., Schulz, H., Glodek, J., Krest, I., Krüger, H., [9] Cavallito, C. J., & Bailey, J. H. (1944). Allicin, the Herchert, N., & Keller, J. (2002). Characterization of some antibacterial principle of Allium sativum. I. Isolation, Allium hybrids by aroma precursors, aroma profiles, and physical properties and antibacterial action. Journal of the alliinase activity. Journal of agricultural and food American Chemical Society, 66(11), 1950-1951. chemistry,50(10), 2884-2890. [10] Cavallito, C. J., Buck, J. S., & Suter, C. M. (1944). Allicin, [29] Kılıç A (2008). Uçucu yağ elde etme yöntemleri. Bartın the antibacterial principle of Allium sativum. II. Orman Fakültesi Dergisi 10:13. Determination of the chemical structure. Journal of the [30] Kim SM, Kubota K and Kobayashi A, 1997. Antioxidative American Chemical Society, 66(11), 1952-1954. activity of sulfur-containing flavor compounds in garlic. [11] Chyau, C. C., & Mau, J. L. (1999). Release of volatile Bioscience, Biotechnology and Biochemistry 61: 1482-1485. compounds from microwave heating of garlic juice with 2, 4- [31] Kimbaris, A. C., Siatis, N. G., Daferera, D. J., Tarantilis, P. decadienals. Food chemistry, 64(4), 531-535. A., Pappas, C. S., & Polissiou, M. G. (2006). Comparison of [12] Cowley, J., Özhatay, N., & Mathew, B. (1994). New species distillation and ultrasound-assisted extraction methods for the of Alliaceae & Hyacinthaceae from Turkey. Kew Bulletin, isolation of sensitive aroma compounds from garlic (Allium 481-489. sativum). Ultrasonics sonochemistry, 13(1), 54-60. [13] Davis PH, 1984. Flora of Turkey and the East Aegean Islands [32] Kiralan, M., Rahimi, A., Arslan, N., & Bayrak, A. (2013). (Vol. 8). Edinburgh: Edinburgh University Press. Volatiles in an endemic Allium specie: Allium tuncelianum [14] Davis PH, 1998. (Supplement-I). Flora of Turkey and the by headspace solid phase microextraction. Journal of East Aegean islands (Vol. 10). Edinburgh: Edinburgh Essential Oil Bearing Plants, 16(3), 417-420. University Press. [33] Koch, H.P., and L.D. Lawson. 1996. Garlic. Williams and [15] Dorant, E., Van den Brandt, P. A., Goldbohm, R. A., & Wilkins, USA. Sturmans, F. E. R. D. (1996). Consumption of onions and a [34] Konemann 1999. Botanica. Hong Kong: Gordon Cheers reduced risk of stomach carcinoma. Gastroenterology, Publication, 1020p. 110(1), 12-20. [35] Koul AK and Gohil RN, 1970. Causes averting sexual [16] Ekim, T., Koyuncu, M., Vural, M., Duman, H., Aytac, Z., & reproduction in Allium sativum Linn. Cytologia 35: 197-202. Adiguezel, N. (2000). Tuerkiye bitkileri kirmizi kitabi [36] Lampe JW, 1999. Health effects of vegetables and fruit: (Egrelti ve Tohumlu Bitkiler). Assessing mechanisms of action in human experimental [17] Etoh T, Simon PW, 2002. Diversity, fertility and seed studies. American Journal of Clinical Nutrition 70: 475-490. production of garlic. In: Rabinowitch, H.D., Currah, L. [37] Lancaster, J. E. (1988). Production of flavour precursors [S- (Eds.), Allium Crop Science: Recent Advances. CABI alk (en) yl-L-cysteine sulphoxides] in photomixotrophic Publishing, New York, pp.101-117. callus of garlic. Phytochemistry,27(7), 2123-2124. [18] Etoh, T., Simon, P.W., 2002. Diversity, fertility and seed [38] Lawson, L. D., Wang, Z. Y. J., & Hughes, B. G. (1991). production of garlic. In: Rabinowitch, H.D., Currah, L. Identification and HPLC quantitation of the sulfides and dialk (Eds.), Allium Crop Science: Recent Advances. CABI (en) yl thiosulfinates in commercial garlic products. Planta Publishing, New York, pp. 101–117. medica, 57(04), 363-370. [19] Ferary, S., & Auger, J. (1996). What is the true odour of cut [39] Lucier, G., & BiingHwan, L. (2000). Garlic: flavour of the Allium? Complementarity of various hyphenated methods: ages. Agricultural Outlook, (272), 7-10. gas chromatography-mass spectrometry and high- [40] Maass, H.I., Klaas, M., 1995. Infraspecific differentiation of performance liquid chromatography-mass spectrometry with garlic (Allium sativum L.) by isozyme and RAPD markers. particle beam and atmospheric pressure ionization interfaces Theor. Appl. Genet. 91, 89–97. in sulphenic acids rearrangement components discrimination. [41] Mathew, B., 1996. A Review of Allium Section Allium. Journal of Chromatography A,750(1), 63-74. Royal Botanic Gardens, Kew Publishing, Richmond. [20] Foster, B. C., Vandenhoek, S., Hana, J., Krantis, A., Akhtar, [42] Mondy, N., Duplat, D., Christides, J. P., Arnault, I., & Auger, M. H., Bryan, M., ... & Arnason, J. T. (2003). In vitro J. (2002). Aroma analysis of fresh and preserved onions and inhibition of human cytochrome P450-mediated metabolism leek by dual solid-phase microextraction–liquid extraction of marker substrates by natural products. Phytomedicine, and gas chromatography-mass spectrometry. Journal of 10(4), 334-342. Chromatography A, 963(1), 89-93. [21] Fritsch, R.M., Friesen, N., 2002. Evolution, domestication, [43] Mosmann, T. (1983). Rapid colorimetric assay for cellular and . In: Rabinowitch, H.D., Currah, L. (Eds.), growth and survival: application to proliferation and

Organosulfur Compounds of Allium Tuncelianum (Kollmann) Ozhatay, B.Mathew & Şiraneci Extracts by Spme/Gc-Ms and Determining their Cytotoxic Effect on Hela Cells

56 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in cytotoxicity assays. Journal of immunological methods, 65(1- Enzymology and Related Areas of Molecular Biology, 2), 55-63. Volume 11, 377-400. [44] Özhatay, N., Kültür, Ş., & Aksoy, N. (1999). Check-list of [49] Teyssier, C., Amiot, M. J., Mondy, N., Auger, J., Kahane, R., additional taxa to the supplement flora of Turkey II. Turkish & Siess, M. H. (2001). Effect of onion consumption by rats Journal of Botany, 23(3), 151-170. on hepatic drug-metabolizing enzymes. Food and chemical [45] Özhatay, N., & Kültür, Ş. (2006). Check-list of additional toxicology, 39(10), 981-987. taxa to the supplement flora of Turkey III. Turkish Journal of [50] Vvedensky, A.I., 1944. The genus Allium in the USSR. Botany, 30(4), 281-316. Herbertia 11, 65–218. [46] Özhatay, N., Byfield, A., & Atay, S. (2008). Türkiye'nin 122 [51] Yin MC and Cheng WS, 1998. Antioxidant activity of several önemli bitki alanı. WWF Türkiye (Doğal Hayatı Koruma Allium members. Journal of Agricultural and Food Chemistry Vakfı). 46: 4097-4101. [47] Özhatay, N., KÜLTÜR, Ş., & Aslan, S. (2009). Check-list of [52] Yoshida, H., Katsuzaki, H., Ohta, R., Ishikawa, K., Fukuda, additional taxa to the supplement Flora of Turkey IV. Turkish H., Fujino, T., & Suzuki, A. (1999). Antimicrobial activity of Journal of Botany, 33(3), 191-226. the thiosulfinates isolated from oil-macerated garlic extract. [48] Stoll, A., & Seebeck, E. (2006). Chemical investigations on Bioscience, biotechnology, and biochemistry, 63(3), 591-594. alliin, the specific principle of garlic. Advances in

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Organosulfur Compounds of Allium Tuncelianum (Kollmann) Ozhatay, B.Mathew & Şiraneci Extracts by Spme/Gc-Ms and Determining their Cytotoxic Effect on Hela Cells

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