Journal of Applied Botany and Food Quality 89, 235 - 242 (2016), DOI:10.5073/JABFQ.2016.089.030 1Chiang Mai University, Department of Plant Science and Soil Science, Faculty of Agriculture, Chiang Mai, Thailand 2 Queen Sirikit Botanic Garden, Chiang Mai, Thailand Chemical composition and comparison of genetic variation of commonly available Thai garlic used as food supplement S. Sommano1*, N. Saratan2, R. Suksathan2, T. Pusadee1 (Received April 2, 2016) * Corresponding author Summary In order to classify true garlic cultivars, comparisons of oil composi- tion and genetic of three garlic cultivars (Allium sativum L.) com- monly used for essential oil production in the northern Thai market [viz., Thai (TH), Chinese (CH) and Pingpong (PP) cultivars] were carried out. Garlic essential oils were obtained by hydrodistilla- tion and microwave hydrodistillation which were then analysed for chemical components by gas chromatography-mass spectrometry. The RAPD data suggests similarity (>95 %) of the three cultivars in chemical compositions, and the major compounds are trisulphide, di-2-propenyl, the disulphide, di-2-propenyl, and the trisulphide, methyl 2-propenyl. Sulphur-containing compounds (Rf = 0.18-0.2) were detected by thin-layer chromatography (TLC) with ninhydrin staining reagent. The essential oil of CH from hydrodistillation and microwave hydrodistillation showed the highest alliin content. The RAPD analysis of the three garlic cultivars presents 45 fragments. A dendrogram shows genetic similarity between the garlic cultivars. The TH and the CH showed similarity value as 0.93, while the PP was classified as a different cluster. Though there was considerable similarity between the chemical and the genetic profiles of the TH and the CH, the CH demonstrated high potential as an ingredient in food supplement products due to its high alliin content. Abbreviations: CAS, chemical abstracts service; CH, garlic cv. Chinese; GC-MS, gas chromatography-mass spectrometry; MOG, garlic essential oil from microwave hydrodistillation; OG, garlic es- sential oil from hydrodistillation; PP, garlic cv. Pingpong; TH, garlic cv. Thai; TLC, thin layer chromatography. Introduction Besides being an important spice in many cultures, garlic (Allium sativum L.) is also well known for its excellent medicinal properties (VELÍŠEK et al., 1997). The major components found in garlic cloves are sulphur-containing compounds such as allicin, alliin, ajoene, diallyl disulphide, dithiin and S-allylcysteine. These compounds are responsible for the pungent smell and taste of garlic (GAFAR et al., 2012). Among those, allicin is the most abundant (70 % of the overall thiosulphates) present in fresh garlic which can be activated upon mechanical crushing (MIRON et al., 2004). This compound possesses powerful antibiotic, antimicrobial, anticancer and other properties. Essential oil of garlic also contains a number of sulphides such as diallyl disulphide and dilly trisulphide; however, allicin can be com- pletely eliminated during thermal or chemical extraction processes (BLOCK, 1985). Food supplement products made from garlic are available in plen- ty, including, for example, garlic essential oil capsules, dehydrated garlic powder, garlic essential oil macerate and aged garlic extract (AMAGASE et al., 2001; GAFAR et al., 2012). The challenge for food processors is to control the products in terms of quality, and, thus, at the starting point, searching for a suitable raw material is vitally important (VELÍŠEK et al., 1997). By dealing with major fresh gar- lic suppliers in Thailand, we identified that there was a minimum of three garlic cultivars in the market. These types include locally grown cultivars, such as ‘Thai’ and ‘Pingpong’. The latter is named after its colour and shape. The ‘Chinese’ cultivar is said to be im- ported through the Thai-Burmese border; however, its real origin is unknown. Classification of ‘true’ variety of garlic can be done by using different techniques including morphological comparison and chemical analyses such as volatile components, DNA finger- prints and contents of bioactive compounds (TRIRONGJITMOAH et al., 2015). In this study, we compared three garlic specimens generally avail- able in Thai markets based on their morphology, bioactive compo- nents, volatile profiles of essential oils and variation in their DNA fingerprints. The aim was to classify the sources of raw materials and to control for quality the final products produced by Thai food producers. Materials and methods Plant material Garlic bulbs were supplied by Chiang Mai Harvest Co., Ltd., the major garlic supplier from Chiang Mai, Thailand. Three garlic cul- tivars, commonly known as (1) garlic cv. Thai -(TH), (2) garlic cv. Chinese -(CH) and (3) garlic cv. Pingpong -(PP) were examined for morphology and stored in commercial warehouse conditions until use. Extraction of garlic essential oil Essential oil of garlic was extracted by hydrodistillation from fresh garlic cloves (200 g) with 1 L of water for 3 h. The essential oil was collected from a Clevenger trap and dried over anhydrous sodium sulphate. The yield of the oil obtained (OG) was calculated as a per- centage (v/w) (SOWBHAGYA et al., 2009). The same amount of gar- lic was also used for garlic essential oil extraction using microwave hydrodistillation and gravity unit at 700 W without the addition of water (SOMMANO et al., 2015). The yield of the extract (MGO) was also accessed. Analysis of chemical composition of garlic essential oil by gas chromatography-mass spectrometry (GC-MS) The oil was diluted in hexane (0.01%) prior to injection onto a GC- MS system (BRUKER, SCION SQ 436-GC). The gas chromato- graphic column was RESTEK (Rxi®-5sil MS, 30 m, 0.25 mmID, 0.25 um). The column was maintained at 45 °C for 1 min, and then heated to 245 °C at 4 °C min-1 using helium as the carrier gas (2 cm3/min) (WEINBERG et al., 1993). The mass spectrometer was run in electron ionization mode (EI 70 eV); source temperature, 100-325 °C; quadrupole temperature, 90 °C; mass scan range, m/z 1-1200 Da; and scan rate, up to 14,000 Da s-1, in a complete scan 236 S. Sommano, N. Saratan, R. Suksathan, T. Pusadee acquisition mode. The comparison of MS fragmentation patterns with those from the National Institute of Standards and Technology (NIST) MS 98 library was performed. The relative peak areas (RAs) of a single compound were expressed in relation to the total peak area of the identified compounds. Cluster analysis was performed based on 75 chemical compositions of garlic essential oil using the statistical package XLSTAT version 2015.5.01.23234 software. The coefficients of genetic similarity for all pair-wise comparisons were computed using Jaccard’s coefficient of similarity and then the dis- tance matrix was subjected to cluster analysis using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) to produce a dendrogram. Identification of sulphur-containing compounds by Thin Layer Chromatography Separation of chemical compositions from essential oil (5-10 μl) was achieved by Thin Layer Chromatography (TLC silica gel 60 F254, Merck) with glacial acetic acid:propanol:water:ethanol (20:20:20:20) as the mobile phase. Sulphur-containing compounds were visible after spraying the TLC plates with ninhydrin reagent (BELEMKAR et al., 2013; KEUSGEN, 1997). The retention factor (Rf) is the result of the distance travelled by the compound divided by the distance travelled by the mobile phase. Analysis of alliin content by spectrophotometry The alliin content was analysed using the spectrophotometry method with some adaptations (MIRON et al., 2002). Briefly, garlic essential oil (4 μl) was incubated with 1.1 × 10-4 M of mercaptopyridine (4- MP) in 50 mM sodium phosphate buffer, pH 7.2, containing alliinase. The decrease in the optical density (OD) at 324 nm was determined after 30 min of incubation at room temperature. The alliin concentra- tion was calculated using the following equation: [alliin] = ΔA324 × dilution × [εM]-1, where ΔA324 = [OD without extract] − [OD with extract] εM = Molar extinction coefficient at 324 (4MP) = 19,800 DNA analysis Total genomic DNA was extracted using the CTAB (hexadecyltrim- ethylammonium bromide) method with some modification (DOYLE and DOYLE, 1987). Into the ground sample, 1000 μl of the extrac- tion buffer [100 mM Tris–HCl pH 8.0, 20 mM EDTA (ethylenedi- aminetetracetate) pH 8.0, 1.4 mM NaCl and 4 % (w/v) CTAB] was added and the sample was incubated at 65 °C for 1 h. Thereafter, the sample was further extracted with 600 μl chloroform:isoamyl alco- hol [24:1 (v/v)] and centrifuged at 13,000 rpm for 10 min. The re- sultant supernatants were transferred to a new microcentrifuge tube. The air-dried pellet was re-suspended in 50 μl TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA and pH 8.0). The DNA samples were stored at -20 °C prior to RAPD analysis. DNA quantification DNA was quantified by using nano-drop spectrophotometer (ND- 1000, spectrophotometer). For re-quantification, the extracted DNA was run on 1 % agarose gel electrophoresis using 1× TBE buffer at 5-8 V/ml for 30 min and visualised under BLook LED transillumi- nator (Genedirex, Taiwan) by staining with MaestroSafeTM (Mae- strogen, USA). The DNA solution was diluted with sterile distilled water to a concentration of 50 ng/μl for PCR analysis and kept in -20 °C until use. RAPD-PCR protocols For RAPD analysis of the genomic DNA, 10-base primers from Operon Technologies (Alameda, USA) and UBC (University of Brit- ish Columbia, Canada) were chosen (Tab. 1). A total of 10 primers were screened. The polymerase chain reaction (PCR) was adjusted to 10 μl containing 8 μl of OnePCRTM Plus (Genedirex, Taiwan), 1 μl of 1 μM RAPD primer and 1 μl of 10 ng genomic DNA. All the reactions were carried out on a Flexcycler2 thermal cycler (Analytik Jena, Germany) using the following profile: 1 cycle, 94 °C, 4 min; 40 cycles, 94 °C, 30 s; 37 °C, 30 s; 72 °C, 60 s; 1 cycle, 72 °C, 10 min. The sample was separated in a 1.5 % agarose gel in 1× TBE buffer. The samples were run at 70 V for 90 min. The gels were then visualised using the BLooK LED transilluminator (Genedirex, Taiwan). Tab. 1: Sequences of oligonucleotide primers used for RAPD analysis Primer Sequence Number of Number of Percent name score bands polymorphic poly- bands morphism OPA08 GTGACGTAGG 8 7 87.5 UBC106 AGGAGTCGGA 6 4 66.6 UBC120 AGACCCTTGG 7 7 100 UBC155 CTGGCGGCTG 8 3 37.5 UBC184 CAAACGGCAC 6 5 83.3 UBC215 TCACACGTGC 5 2 40 UBC237 CGACCAGAGC 5 0 0 UBC275 CCGGGCAAGC 5 4 80 Statistical analysis The extractions were performed in triplicate. The data were analysed by using the IBM SPSS statistical software version 22. The means were subjected to comparison by using the one-way analysis of vari- ance (ANOVA) and the Ducan’s post hoc multiple comparisons at the 99 % confidence level. The banding pattern for each primer was scored as diallelic (1 = band present, 0 = band absent), and stored in an Excel (Microsoft) spreadsheet file in the form of a binary matrix. In order to assess the genetic differentiation between the three garlic accessions, eight RAPD markers were analysed using the statisti- cal package XLSTAT version 2015.5.01.23234 software. The coef- ficients of genetic similarity for all the pair-wise comparisons were computed using the Jaccard’s coefficient of similarity, and then the distance matrix was subjected to cluster analysis by using the Un- weighted Pair Group Method with Arithmetic Mean (UPGMA) to produce a dendrogram. Results and discussion Garlic bulb is solitary with globose to applanate-globose shapes de- pending upon the variety. In general, garlic is 2-6 cm in diameter, consisting of several bulblets. The bulblets are covered with white to purple tunic and underneath the tunic are layers of scales which are held together by the basal plate. The basal plate is located at the bottom of the bulb (WU et al., 1998). Morphological differences of the Thai garlic bulbs used in this experiment are shown in Tab. 2 and Fig. 1. The major chemical components of garlic are sulphur-containing compounds which are measured in the forms of cysteine sulfoxides [i.e., alliin (1)] and non-volatile γ-glutamylcysteine peptides (>82 %) (Sendl, 1995). The representatives of this group are as shown in Fig. 2: thiosulphinates [i.e., allicin (2)], ajoenes [i.e., E-ajoene (3) Chemical and genetic variations of Thai garlic bulbs 237 Fig. 1: Garlic bulb and bulblets of A. sativum L. cv. Thai (A-B), cv. Chinese (C-D) and cv. Pingpong (E-F). Scale bar = 5 cm. Tab. 2: The morphological descriptions of three garlic cultivars Cultivars Bulb Weight Shape Colour of No. of Clove size Weight Garlic flavour (cm) (g) tunic bulblets (cm) (g) TH 3.7–4.3 14.78–20.85 Round Pinkish white 21–23 1×2–2.3 0.79–1.33 ++ CH 1.8–2.5 7.91–12.87 Oval Opaque 4–11 0.9-2.1×1.9-3.0 0.68–2.35 +++ white–purple PP 5–5.5 32.23–51.33 Round Pinkish 12–14 1.3-1.7×3.2-3.5 2.28–5.97 ++ white–purple and Z-ajoene (4)], vinyldithiins [i.e., 2-vinyl-(4H)-1,3-dithiin (5) and 3-vinyl-(4H)-1,2-dithiin (6)] and sulphides [i.e., diallyl disul- phide (7) and diallyl trisulphide (8)]. Among those, diallyl sulphides (57 %), allyl methyl (37 %) and dimethyl mono- to hexasulphides (6 %) are most commonly found in garlic essential oil (CERELLA et al., 2011). Essential oil from garlic specimens of the TH, CH and PP was ana- lysed for the chemical profiles by GC-MS. All the tested samples consisted of similar major chemical components, which were trisul- phide, di-2-propenyl (40.97- 45.17 %), disulphide, di-2-propenyl (CAS) (16.95-25.59 %) and trisulphide, methyl 2-propenyl (CAS) (13.41-14.43 %) (Tab. 3). These results are in line with the findings of the work reported by LALLA (2013). The garlic essential oil from A. sativum for. pekinen- se Makino had disulphide, di-2-propenyl, 32.82 %, and trisulphide, di-2-propenyl, 29.12 %, and essential oil of A. sativum var. Jam Nagar gave trisulphide, di-2-propenyl, as high as 60 %, and disul- phide, di-2-propenyl, 13.07 % (PYUN and SHIN, 2006; SOWBHAGYA Fig. 2: The chemical structures of sulphur-containing compounds found in garlic. 238 S. Sommano, N. Saratan, R. Suksathan, T. Pusadee Ta b. 3 : C he m ic al p ro fil es o f g ar lic e ss en tia l o il an al ys ed b y G C -M S N o. C om po un ds % o f t ot al c om po un ds o f g ar lic e ss en ti al o il T H 1 C H 1 P P 1 M or oc co C hi ne se E gy pt ia n M ex ic an fo r. p e- Tu ni si an va r. va r. or ig in 2 or ig in 3 or ig in 3 or ig in 3 ki ne ns e or ig in 5 Ja m N ag ar 6 sa ti vu m 7 M ak in o4 1 1, 2- D itr ia cy cl o- pe nt an e 0. 83 ±0 .0 5a 0. 85 ±0 .1 1a 0. 81 ±0 .1 4a - - - - - - - - 2 1- Pr op en e, 3, 3- th io bi s (C A S) 0. 74 ±0 .0 6a 0. 45 ±0 .0 6a 1. 39 ±0 .1 2b 3. 93 - - - - - - - 3 D is ul ph id e, m et hy l 2 – p ro pe ny l ( C A S) 2. 07 ±0 .2 0a 1. 63 ±0 .1 0a 4. 12 ±0 .1 4b 1. 71 - - - - - - - 4 3- C hl or oc ro to na l- de hy de 1. 28 ±0 .4 6a 0. 97 ±0 .2 2a 0. 99 ±0 .3 8a - - - - - - - - 5 E th ox yc yc lo he xy ld i- m et hy ls ila ne - - - - - - - - - - - 6 A lly l t hi ol - - - - 0. 15 0. 21 0. 14 - - - - 7 M et hy l a lly l s ul ph id e - - - - 0. 64 1. 21 3. 15 - - - - 8 D im et hy l d is ul ph id e - - - - 0. 23 0. 41 0. 88 - - - 1 9 D ia lly l s ul ph id e - - - - 4. 25 3. 69 6. 96 - 2. 30 - 1. 20 10 M et hy l a lly l d is ul ph id e - - - - 2. 23 5. 37 15 .2 5 - 1. 70 - - 11 A lly l m et hy l s ul ph id e - - - - - - - - - - - 12 D im et hy l d is ul ph id e - - - - - - - - - - - 13 D ia lly l s ul ph id e - - - - - - - - - - - 14 A lly l m et hy l d is ul ph id e - - - - - - - - - - - 15 3, 3- T hi o bi s- 1- pr op en e - - - - - - - 0. 87 - - - 16 2, 4- D im et hy l t hi op he ne - - - - - - - 0. 03 - - - 17 2, 5- D im et hy l t hi op he ne - - - - - - - 0. 06 - - - 18 M et hy l c is -p ro pe ny l d is ul ph id e - - - - - - - 0. 13 - - - 19 M et hy l t ra ns -p ro pe ny l d is ul ph id e - - - - - - - 0. 24 - - - 20 N ,N ’- D im et hy l t hi ou re a - - - - - - - 1. 46 - - - 21 Tr is ul ph id e, d im et hy l ( C A S) 0. 80 ±0 .1 7a 0. 58 ±0 .1 1a 0. 85 ±0 .1 4a - 0. 18 1. 3 1. 44 0. 51 - - - 22 D is ul ph id e, d i- 2- pr op en yl (C A S) 14 .7 4± 3. 72 a 16 .0 0± 0. 66 a 18 .0 6± 4. 74 a 14 .3 0 - - - 32 .8 2 - 13 .0 7 - 23 D ia lly l d is ul ph id e 2. 19 ±0 .9 0a 0. 92 ±0 .1 0a 1. 60 ±0 .4 4a 0. 46 - - - - - - - 24 D ia lly l d is ul ph id e 1. 64 ±0 .1 1a 1. 25 ±0 .1 1a 3. 19 ±0 .4 0b - - - - - - - - 25 M et hy l i so pe nt yl d is ul ph id e - - - - - - - - - - 19 .0 0 26 M et hy l a lly l d is ul ph id e - - - - - - - - - - - 27 D ip ro py l d is ul ph id e - - - - - - - 0. 11 - - - 28 D ia lly l d is ul ph id e - - - - - - - - - - - 29 Tr an s- pr op en yl p ro py l d is ul ph id e - - - - - - - 0. 30 - - - 30 Tr is ul ph id e, m et hy l 2 -p ro pe ny l ( C A S) 11 .3 4± 1. 37 a 13 .1 4± 0. 17 a 11 .1 2± 1. 54 a 10 .8 8 - - - 7. 40 - - - 31 A lly 1 m et hy l t ri su lp hi de - - - - - - - - - - - 32 M et hy l p ro py l t ri su lp hi de - - - - - - - 0. 15 - - - 33 1, 3, 5- Tr ith ia ne - - - - - - - 0. 26 - - - 34 3- M et hy l t hi o 1- pr op en e - - - - - - - 0. 03 - - - 35 4, 5- D im et hy l t hi az ol e - - - - - - - 0. 02 - - - 36 1- M et hy l t hi o- 1- pr op en e - - - - - - - 0. 12 - - - 37 3- V in yl -1 ,2 -d ith ia ne - - - - - - - - - 1. 46 - 1 D at a ar e ex pr es se d as m ea n ± SD o f t ri pl ic at e hy dr od is til la tio n (O G ) e xt ra ct s. M ea ns in th e sa m e ro w w ith d iff er en t l et te rs (a –b ) a re s ig ni fic an tly d iff er en t ( P <0 .0 1) , A N O VA , D uc an , I B M S PS S St at is tic s. 2 G ar lic es se nt ia l o il of M or oc co o ri gi n (D O U IR I e t a l., 2 01 4) 3 G ar lic e ss en tia l o il of C hi ne se , E gy pt ia n an d M ex ic an o ri gi n (S H A A T H a nd F L O R E S, 1 99 5) 4 G ar lic e ss en tia l o il of f or . p ek in en se M ak in o of S eo ul ( PY U N a nd SH IN , 2 00 6) 5 G ar lic e ss en tia l o il of T un is ia n or ig in (D Z IR I e t a l., 2 01 4) 6 G ar lic e ss en tia l o il of J am N ag ar v ar ie ty o f I nd ia (S O W B H A G Y A e t a l., 2 00 9) 7 G ar lic e ss en tia l o il of s at iv um v ar ie ty o f I ra n (K H A R A Z I, 20 05 ). D at a w as c om pa re d w ith ou t c on si de ra tio n of d iff er en t G C M S co nd iti on s fr om d iff er en t r ep or ts . Chemical and genetic variations of Thai garlic bulbs 239 38 2- V in yl -1 ,3 -d ith ia ne - - - - - - - - - 3. 42 - 39 3- V in yl -[ 4H ]- 1, 2- di th iin 2. 00 ±0 .4 3a 1. 83 ±0 .0 9a 1. 43 ±0 .2 2a 2. 76 - - - 1. 99 - - - 40 D ia lly l t ri su lp hi de - - - - - - - - - - - 41 2- V in yl -[ 4H ]- 1, 3- di th iin - - - 1. 64 - - - - - 1. 85 - 42 2- V in yl +H )- 1 ,3 -d ith iin - - - - - - - 5. 87 - - - 43 1, 2, 3- T hi ad ia zo le ,5 -m et hy l- (C A S) 2. 12 ±0 .1 0a 2. 42 ±0 .3 0a 1. 71 ±0 .2 3a - - - - - - - - 44 3- V in yl -1 ,2 -d ith ia cy cl oh ex -5 -e ne 4. 60 ±0 .5 4a 4. 61 ±0 .2 4a 3. 44 ±0 .1 3a - - - - - - - - 45 2, 5- D im et hy lth ia zo le - - - - - - - 0. 01 - - - 46 Tr is ul ph id e, d i- 2- pr op en yl 21 .5 9± 11 .0 5a 42 .5 7± 2. 60 a 14 .0 3± 8. 44 a 46 .5 2 - - - 29 .1 2 - 60 .0 0 - 47 Is ob ut yl is ot hi oc ya na te 1. 57 ±0 .3 0a 1. 28 ±0 .2 0a 1. 73 ±0 .1 6a - - - - - - - - 48 3, 5- D ie th yl -1 ,2 ,4 -T ri th io la ne - - - - - - - - - 1. 00 - 49 5- M et hy l- 1, 2, 3, 4- te tr at hi a- cy cl oh ex an e - 0. 72 ±0 .0 9b 0. 45 ±0 .1 1b - - - - - - - - 50 D is ul ph id e, m et hy l 2 -p ro pe ny l ( C A S) - 0. 90 ±0 .2 1b 0. 64 ±0 .1 5b - - - - - - - - 51 1, 2- D ih yd ro cy cl ob ut ab en ze ne 0. 64 ±0 .2 1a 1. 00 ±0 .2 7a 0. 50 ±0 .1 1a - - - - - - - - 52 D ia lly l d is ul ph id e 4. 34 ±0 .8 1a 4. 74 ±1 .1 0a 4. 01 ±1 .0 2a - 28 .6 27 .4 5 42 .4 6 - - - - 53 tr an s- Pr op en yl m et hy l d is ul ph id e - - - - - - - - 0. 40 - - 54 SU L PH U R ,M O L .(S 8) 1. 15 ±0 .1 8a 1. 28 ±0 .1 3a 0. 87 ±0 .1 6a - - - - - - - - 55 A lly l p ro py l d is ul ph id e - - - - 0. 57 0. 74 0. 28 - - - - 56 C 6H 10 S 2 (T en t I D ) - - - - 0. 91 1. 92 0. 03 - - - - 57 M et hy l a lly l t ri su lp hi de - - - - 6. 77 16 .8 2 10 .3 6 - - - - 58 D im et hy l t ri su lp hi de - - - - - - - - 0. 20 - - 59 D ia lly l d is ul ph id e - - - - - - - - 29 .1 0 - - 60 M et hy l a lly l t ri su lp hi de - - - - - - - - 10 .4 0 - - 61 2- V in yl -1 .3 -d ith ia ne - - - - - - - - 3. 90 - - 62 1. 4- D im et hy l t et ra su lp hi de - - - - - - - - 0. 40 - - 63 D ia lly l t ri su lp hi de - - - - 50 .9 2 35 .3 0 12 .5 2 - 37 .3 0 - 3. 20 64 E ug en ol - - - - - - - - 0. 40 - - 65 α -C ar yo ph yl le ne - - - - - - - - 0. 30 - - 66 α -G ua ie ne - - - - - - - - 1. 00 - - 67 A ro m ad en dr en e - - - - - - - - 1. 70 - - 68 α -B is ab ol en e - - - - - - - - 2. 10 - - 69 γ- C ad in en e - - - - - - - - 4. 30 - - 70 D i- 2- pr op en yl te tr as ul ph id e - - - - - - - - - 5. 01 - 71 D ia lly l t et ra su lp hi de (T en t I D ) - - - - 0. 74 0. 70 1. 09 6. 35 3. 00 - - 72 1, 4- D im et hy l t et ra su lp hi de - - - - - - - 0. 28 - - - 73 1, 2- D ith ia ne -4 -o ne - - - - - - - 0. 05 - - - Ta b. 3 : C he m ic al p ro fil es o f g ar lic e ss en tia l o il an al ys ed b y G C -M S (c on tin ue d) N o. C om po un ds % o f t ot al c om po un ds o f g ar lic e ss en ti al o il T H 1 C H 1 P P 1 M or oc co C hi ne se E gy pt ia n M ex ic an fo r. p e- Tu ni si an va r. va r. or ig in 2 or ig in 3 or ig in 3 or ig in 3 ki ne ns e or ig in 5 Ja m N ag ar 6 sa ti vu m 7 M ak in o4 1 D at a ar e ex pr es se d as m ea n ± SD o f t ri pl ic at e hy dr od is til la tio n (O G ) e xt ra ct s. M ea ns in th e sa m e ro w w ith d iff er en t l et te rs (a –b ) a re s ig ni fic an tly d iff er en t ( P <0 .0 1) , A N O VA , D uc an , I B M S PS S St at is tic s. 2 G ar lic es se nt ia l o il of M or oc co o ri gi n (D O U IR I e t a l., 2 01 4) 3 G ar lic e ss en tia l o il of C hi ne se , E gy pt ia n an d M ex ic an o ri gi n (S H A A T H a nd F L O R E S, 1 99 5) 4 G ar lic e ss en tia l o il of f or . p ek in en se M ak in o of S eo ul ( PY U N a nd SH IN , 2 00 6) 5 G ar lic e ss en tia l o il of T un is ia n or ig in (D Z IR I e t a l., 2 01 4) 6 G ar lic e ss en tia l o il of J am N ag ar v ar ie ty o f I nd ia (S O W B H A G Y A e t a l., 2 00 9) 7 G ar lic e ss en tia l o il of s at iv um v ar ie ty o f I ra n (K H A R A Z I, 20 05 ). D at a w as c om pa re d w ith ou t c on si de ra tio n of d iff er en t G C M S co nd iti on s fr om d iff er en t r ep or ts . 240 S. Sommano, N. Saratan, R. Suksathan, T. Pusadee Fig. 3: The dendrogram of the chemical composition of garlic oil obtained from A. sativum L. cv.Thai (TH), cv. Chinese (CH) and cv. Ping- pong (PP), of Morocco origin (DOUIRI et al., 2014), var. Jam Nagar (SOWBHAGYA et al., 2009), for. pekinense Makino (PYUN and SHIN, 2006), var. sativum (KHARAZI, 2005), Egyptian origin, Chinese origin, Mexican origin (SHAATH and FLORES, 1995) and Tunisian origin (DZIRI et al., 2014) derived by UPGMA from the similarity matrix of 75 chemical compositions. Fig. 4: The dendrogram of three garlic cultivars, cv. Thai (TH), cv. Chinese (CH) and cv. Pingpong (PP), derived by UPGMA from the similarity matrix of 75 chemical compositions. et al., 2009). Garlic essential oils of different origins (viz., Chinese, Egyptian, Mexican and Tunisian) also contained two major com- pounds, diallyl trisulphide (12.52-50.92 %) and diallyl disulphide (28.6-42.46 %) (SHAATH and FLORES, 1995; DZIRI et al., 2014). Cluster analysis of 75 chemical compositions of garlic essential oil from the three cultivars and eight references divided garlic into three groups on a dendrogram (Fig. 3). Group 1 includes garlic cv. CH, cv. TH, of Morocco origin (DOUIRI et al., 2013), cv. PP, var. Jam Nagar (SOWBHAGYA et al., 2009), and for. pekinense Makino (PYUN and SHIN, 2006). Group 2 includes garlic var. sativum (KHARAZI, 2005) and group 3 includes garlic of Egyptian, Chinese, Mexican (SHAATH and FLORES, 1995) and Tunisian origin (DZIRI et al., 2014) (Fig. 3). The TH, CH and PP were classified into the same group where the CH and the TH showed 0.997, while the PP gave 0.981 similarity values (Fig. 4). Identification of sulphur-containing compounds from the three cultivars was achieved on TLC. Staining these compounds with nin- hydrin revealed the compounds with Rf to be in the range of 0.18- 0.2 in all the tested cultivars and extraction methods (Tab. 4). This result is in agreement with the report by BELEMKAR (2013), which suggested the Rf of sulphur-containing compounds as being about 0.2, and those compounds were identified as diallyl disulphide, dial- lyl sulphide, alliin and thiosulphinate. However, the oil extracted by the hydrodistillation of the PP showed no evidence of the presence of such compounds. The chemical component of garlic essential oil varied, depending largely upon the method of extraction: for exam- ple, allylsulphides were apparent in hydrodistillated oil, while sol- vent extraction using ethanol at the ambient temperature gave thio- sulphinates, and temperatures below 0 °C were suitable for alliin and amino acid (LI et al., 2010; SENDL, 1995). As one of the heat stable compounds found in garlic, the alliin content was analysed using spectrophotometry (MIRON et al., 2002). The alliin content of the oil extract by hydrodistillation of the three cultivars was in the range of 40.0-90.0 mg/100 ml, while the content was less in the oil extracted using microwave-assisted apparatus (15.0-60.0 mg/100 ml) (Tab. 4). In any case, the CH gave the highest alliin content among the tested cultivars. Variations in the alliin content among the cultivars and be- cause of the types of processing as well as products have also been observed in previous studies (BHANDARI et al., 2014; VELÍŠEK et al., 1997). Alliin is a bioactive compound that shows high anti-human colon cancer anti-stomach cancer activities and stimulates peripheral blood cell immune functions (KHANUM et al., 2004; SALMAN et al., 1999). In addition, several reports have illustrated its health benefits inclu- ding biological activities such as anti-platelet aggregatory effect, in addition to decreasing of systolic blood pressure, lipid peroxides, HL-60 human leukaemia uric acid, blood glucose, total lipid, tri- glyceride and cholesterol, glycemic controlling, antioxidant system modulation of red blood cell and anti-atherogenic effect (YUN et al., 2014; ANWAR and MEKI, 2003; LIU et al., 2005; WU et al., 2001; JAIN and KONAR, 1978). In this study, RAPD-PCR fingerprints were generated from three garlic cultivars (cv. TH, cv. CH and cv. PP). Ten randomly designed 10-mer oligonucleotide primers were ini- tially used for screening the DNA samples to obtain reproducible RAPD fingerprints. Out of the ten primers tested, only eight primers provided consistent well-resolved and reproducible band patterns, and were therefore selected for further analysis.The total number of fragments observed among the three garlic cultivars based on the RAPD analysis with eight polymorphic primers was 45 fragments (Fig. 5). A dendrogram based on the similarity matrix generated with the RAPD primers is presented in Fig. 6. The dendrograms at a simi- larity value of 0.93 grouped the TH and the CH together, while the PP was separated into different clusters with 0.31 genetic similarity. Tab. 4: Quantitative and qualitative comparisons of sulphur-containing compounds in garlic bulbs Thai, Chinese and Pingpong cultivars Garlic Time of % Yield Sulphur-containing compounds Alliin content essential extraction retention factor (Rf) (mg 100/ml) oil (min) TH CH PP TH CH PP TH CH PP OG 120 0.36 ± 0.01b 0.39 ± 0.02b 0.45 ± 0.02a 0.20 0.19 - 88.70 ± 0.20b 91.53 ± 0.11a 40.76 ± 0.20c MOG 45 0.22 ± 0.02a 0.21 ± 0.01a 0.16 ± 0.01b 0.19 0.20 0.18 15.04 ± 0.29c 60.43 ± 0.18a 22.91 ± 0.09b All data are expressed as mean±SD of triplicate measurements. Means in the same row with different letters (a–c) are significantly different (P<0.01), ANOVA, Ducan, IBM SPSS Statistics. Chemical and genetic variations of Thai garlic bulbs 241 It is noteworthy that the alliin content of the garlic essential oil from the CH was higher than that of the TH from both hydrodistillation and microwave hydrodistillation. 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