Boiling enriches the linear polysulfides and the hydrogen sulfide-releasing activity of garlic

Accepted Manuscript Boiling enr iches the linear polysulfides and the hydr ogen sulfide-r eleasing activity of gar lic Restituto Tocmo, Yuchen Wu, Dong Liang, Vincenzo Fogliano, Dejian Huang PII: DOI: Reference:

S0308-8146(16)31724-1 http://dx.doi.org/10.1016/j.foodchem.2016.10.076 FOCH 20064

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

6 July 2016 6 October 2016 18 October 2016

Please cite this article as: Tocmo, R., Wu, Y., Liang, D., Fogliano, V., Huang, D., Boiling enr iches the linear polysulfides and the hydr ogen sulfide-r eleasing activity of gar lic, Food Chemistry (2016), doi: http://dx.doi.org/ 10.1016/j.foodchem.2016.10.076

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Boiling enriches the linear polysulfides and the hydrogen sulfide-releasing activity of

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garlic

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Restituto Tocmoa, Yuchen Wub, Dong Lianga, Vincenzo Foglianob, Dejian Huang*,a,c

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a) Food Science and Technology Programme, Department of Chemistry, National University of

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Singapore, 117543 Singapore

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b) Food Quality Design Group, Wageningen University, PO Box 8129, Wageningen,

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Netherlands

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c) National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou

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Industrial Park, Jiangsu 215123, China

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Running title: Boiling enriches garlic polysulfides

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*Corresponding author: Dejian Huang, e-mail: [email protected], Tel: (65)6516-8821.

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Tel: 65- 65168821, Fax (65)-67757895

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Abstract

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Garlic is rich in polysulfides, and some of them can be a dietary source of H2S donors. This

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study was conducted to explore the effect of cooking on garlic’s organopolysulfides and

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H2S-releasing activity. Garlic bulbs were crushed and boiled for a period ranging from 3 to

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30 minutes and the solvent extracts were analyzed by GC-MS/FID and HPLC. A cell-based

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assay was used to measure the H2S-releasing activity of the extracts. Results showed that

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the amounts of allyl polysulfides increased in crushed garlic boiled for 6 to 10 min;

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however, prolonging the thermal treatment to 20 or 30 min decreased their

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concentrations. Data of the H2S-releasing activity, expressed as diallyl trisulfide equivalents

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(DATS-E), parallel this trend, being significantly higher at 6 and 10 min boiling. Our results

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showed enhancement of H2S-releasing activity upon moderate boiling, suggesting that

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moderately cooked garlic may maximize its health benefits as a dietary source of natural

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H2S donors.

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Keywords: garlic, hydrogen sulfide-releasing capacity, boiling, diallyl trisulfide

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1. Introduction

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Garlic (Allium sativum) is one of the most popular condiments and seasonings

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worldwide. It is the second most widely cultivated Allium species, after onion (Kamenetsky

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et al., 2005). Native to Central Asia, garlic is now widely cultivated in the rest of Asia, Africa

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and Europe. The medicinal functions of garlic date back 4000 years ago when it was

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traditionally used as an antiseptic lotion by Indians, as a stimulant by Greek athletes, and as

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a tea for fever and headache by the Chinese (Block, 1985). This traditional wisdom has

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been translated into evidence-based knowledge with advancement of research. Garlic is

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well-known for its broad spectrum of biological activities, including antioxidant (Argüello-

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García, Medina-Campos, Pérez-Hernández, Pedraza-Chaverrí, & Ortega-Pierres, 2010; Ho,

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Cheng, Chau, & Yen, 2012; Yin, Hwang, & Chan, 2002), anticancer (Milner & Schaffer, 1998;

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Tang, Chiang, & Pai, 2010), and cardioprotective effects (Ou et al., 2010; Vazquez-Prieto,

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González, Renna, Galmarini, & Miatello, 2010; Guo, Cheng, & Zhu, 2013). Organosulfide

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compounds are the main bioactive constituents responsible for these effects, although the

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exact mechanisms and the structure‒activity relationships are still not clear (Gardner et al.,

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2007). During processing and digestion, garlic organosulfides undergo complex inter-

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conversion chemical reactions. The rupture of cell structure caused by garlic cutting and

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crushing initiates the reaction between the enzyme alliinase (EC 4.4.1.4) and the substrate

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alliin (S-allyl-L-cysteine-sulfoxides) leading to the formation of allicin (allyl 2-

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propenethiosulfinate) in variable amounts (16 to 130 g/kg dry weight) (Baghalian, Ziai,

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Naghavi, Badi, & Khalighi, 2005). However, allicin is a very unstable compound (Fujisawa,

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Suma, Origuchi, Kumagai, Seki, & Ariga, 2008). It rearranges and transforms into various

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oil-soluble sulfur-containing organosulfides, particularly at high temperatures (Block,

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1985). These organosulfides include mainly diallyl disulfide (DADS), diallyl trisulfide

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(DATS), methyl allyl trisulfide (MATS), and diallyl tetrasulfide (DATTS) (Amagase, Petesch,

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Matsuura, Kasuga, & Itakura, 2001). Under certain conditions (i.e. nonpolar solvents and

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oil), other oil-soluble organosulfides, collectively called allicin transformation products,

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which include ajoenes ((E)- and (Z)-4,5,9-trithiadodeca-1,6,11-triene-9-oxides) and vinyl

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dithiins are generated (Freeman & Kodera, 1995). Due to their different chemical

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structures, these compounds may have different bioactivities.

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Recently, the ability of organopolysulfides to act as "H2S donors" under

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physiological conditions has gained much attention. H2S is now considered the third

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endogenous gasotransmitter along with NO and CO, and is associated in regulation of key

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biological functions (Szabo, 2016). Intensive studies have been carried out to discover

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synthetic (Bełtowski, 2015; Song et al., 2014; Zhao, Biggs, & Xian, 2014) and natural H2S

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donors (Pluth, Bailey, Hammers, Hartle, Henthorn, & Steiger, 2015; Song, et al., 2014).

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Polysulfides from garlic, mainly DADS and DATS, exhibited H2S-releasing capacity in both in

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vitro and animal-based studies (Benavides et al., 2007; Predmore et al., 2012). The H2S-

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releasing capacity of garlic polysulfides is the main mediator of the vasoactivity of garlic

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(Benavides, et al., 2007). Since the H2S donating activity of garlic is highly dependent on the

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concentration and on the structure of organosulfides, it is imperative to investigate how

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changes in the organosulfide profiles in various garlic preparations affect garlic’s H2S-

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releasing potential.

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Worldwide, garlic products are prepared from a variety of culinary practices and

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different processing methods. Differences in cultural and customary practices have

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resulted in various garlic products, such as garlic essential oil, garlic macerate, and garlic

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powder (Corzo-Martínez, Corzo, & Villamiel, 2007). Unique garlic preparations such as

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aged garlic extracts (Amagase, 2006) and pickled-fermented (De Castro, Montaño, Sánchez,

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& Rejano, 1998) garlic have also gained international fame. Domestically, garlic is typically

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cooked before consumption. Most household cooking methods involve varying amounts of

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heat intensity, boiling being one of the most common. A recent study looked into the effect

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of simmering and boiling (6 and 15 min) on garlic organosulfides (Locatelli, Altamirano,

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González, & Camargo, 2015). Boiling of garlic for 6 or 15 min may not result in a complete

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conversion of allicin to other organosulfides. In fact, it has been suggested that allicin and

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other thiosulfinates (TS) are more tolerant to heat than alliinase (e.g., half-life of TS at 80 °C

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and pH 4.5 ranged from 50 min to 10 h, depending on the TS) (Shen, Xiao, & Parkin, 2002).

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This suggests a variable effect of boiling on the degradation of allicin into other

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polysulfides. Therefore, boiled garlic may or may not contain allicin depending on the

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length of boiling. Previous studies showed that allicin is completely transformed into stable

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polysulfides after distillation (Locatelli, Altamirano, Luco, Norlin, & Camargo, 2014).

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Distilled garlic oil is analyzed by gas chromatography (GC). However, GC analysis of garlic

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extracts containing allicin (i.e. minimally boiled garlic) may not provide faithful results of

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organosulfide profile in the sample due to the thermolabile characteristic of allicin. At the

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high temperature employed for the GC analysis (typically 250 °C), allicin can be destroyed

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leading to artifact formation (Block, 1993). Hence, it has been recommended that

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organosulfur compounds (OSCs) should be analyzed using techniques that avoid artifact

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formation, such as HPLC (Locatelli, Altamirano, Luco, Norlin, & Camargo, 2014).

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To our knowledge, there is a dearth of literature that systematically tracks

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degradation of allicin and the changes in its transformation products in boiled garlic. In this

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study, crushed garlic was cooked for different times, covering typical household boiling

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conditions. We analyzed the extracts by GC and HPLC and compared the organosulfides

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profile obtained from both methods. The changes in the organosulfides profile induced by

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boiling were correlated with the variation of garlic’s H2S-releasing capacity.

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2. Materials and methods

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2.1. Materials

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White garlic from China was purchased from a local supermarket in Singapore and

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stored at 4 °C until further use. DADS (80% purity) and analytical grade anhydrous sodium

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sulfate (Na2SO4) were purchased from Sigma-Aldrich (Singapore). DATS was purified by

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distillation of the 80% DATS and DADS mixture. Analytical grade hexane, dichloromethane,

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diethyl ether, acetonitrile, and methanol were purchased from Merck Pte Ltd (Singapore).

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Reagent grade glacial acetic acid, 30% hydrogen peroxide, potassium hydroxide, and litmus

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paper were bought from local chemical companies in Singapore. Fetal bovine serum (FBS)

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was purchased from Hyclone Ltd (Cramlington, UK). 1,2-Dioleoyl-3-trimethylammonium-

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propane (DOTAP) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Human

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breast cancer (MCF-7) cells were purchased from American Type Culture Collection

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(ATCC), grown in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and

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100 µg/mL streptomycin, and maintained at 37 °C and 5% CO2. Penicillin and streptomycin

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were purchased from PAN Biotech (Adenbach, Germany).

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2.2. General description of methods

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We first conducted experiments to evaluate the effect of boiling on garlic

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organosulfides. This was followed by solvent extractions to obtain garlic oils. A scaled-up

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extraction was conducted to isolate allicin and allicin transformation products. In addition

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to scaled-up extractions, allicin was prepared using a previously reported method to obtain

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gram-scale amounts of crude extract of allicin. Crude extracts were then subjected to semi-

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preparative HPLC isolations to obtain pure compounds. Boiled garlic extracts and

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individual compounds were analyzed by LC-MS and GC–MS to confirm identities and

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chemical structures. Standard curves of pure compounds were prepared to quantify

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individual organosulfides. Finally, the oil samples were tested for their H2S-releasing

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capacity using a cell-based assay.

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2.3. Effect of boiling on garlic organosulfides

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Approximately 10 g of garlic cloves were peeled and crushed using a commercial

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garlic press. Crushed garlic was placed in aluminum foil and incubated for 30 min at room

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temperature to allow alliinase-mediated reaction to occur. Deionized water was brought to

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boiling in an aluminum pan. As soon as the water started boiling, garlic homogenates were

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immersed for 3, 6, 10, 20 and 30 min. The heated sample was immediately transferred to a

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50-mL beaker containing cold water, to cool the sample to room temperature. The resulting

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garlic homogenates were transferred to 50-mL blue cap tubes, and extracted with hexane

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(10 mL) with shaking (150 rpm) at room temperature. After 2 hours, the organic layer was

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collected and the extraction was repeated two more times. Subsequently, 5 g of anhydrous

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Na2SO4 were added to the combined extracts to absorb residual water. The organic layer

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was then stored at ‒20 °C overnight to separate traces of water by freezing. The solvent of

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the extract was evaporated using a rotary evaporator at 40 °C to yield yellowish and

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pungent oil. Crushed and boiled samples were assigned as C + 0 for control and C + 3, C + 6,

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C + 10, C + 20, C + 30 for extracts obtained after 3, 6, 10, 20, and 30 min boiling,

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respectively. All the samples were run in triplicate.

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2.4. GC-MS analysis of garlic extract

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GC-MS analysis of the extracts was performed with an Agilent 7890A gas

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chromatography system equipped with a fused silica DB-5MS (Santa Clara, CA) (30 m ×

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0.25 mm i.d., 0.25 μm) column and an autosampler (Agilent 7683B). For GC-MS detection,

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the GC system was interfaced with an Agilent 5975C MS unit. Oil samples (100 μL) were

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diluted with dichloromethane to 1.0 mL and transferred into a GC vial. For GC-MS

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detection, the GC system was operated in EI mode (70 eV) with an ion source temperature

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of 200 °C and a mass range of 40‒500. Carrier gas was helium (99.99%) and flow rate was

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1.0 mL/min. Injector and MS transfer line temperatures were set at 250 and 270 °C,

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respectively. The initial temperature was 50 °C (5 min), and then ramped to 250 °C (5 min)

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at a rate of 4 °C/min. Diluted sample (1.0 μL) was injected using an autosampler in splitless

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mode. Identifications of the organosulfur compounds were based on comparison of their

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mass spectra with those of the National Institute of Standards and Technology (NIST 0.5a)

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database library of the GC-MS system and those of the literature.

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2.5. Isolation of allicin and its transformation products

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Allicin was prepared according to a reported method (Nikolic, Stankovic, Nikolic, &

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Cvetkovic, 2004). In order to obtain pure compounds for HPLC quantification, semi-

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preparative HPLC isolation was carried out. Garlic (1 kg) was crushed using a food

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homogenizer and incubated for 30 min at room temperature. The homogenate was

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extracted 3 times with hexane and the organic layer was concentrated using a rotary

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evaporator. Both fresh garlic extracts and crude allicin extracts were subjected to semi-

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preparative HPLC isolations. Isolations were performed on a Waters HPLC system

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equipped with 717 plus autosampler, a Waters 515 HPLC pump, and a Waters 2996 PDA.

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The column used was a YMC (Kyoto, Japan) C-18 semi-preparative HPLC column (250 mm

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× 10 mm i.d., 5 μm). Mobile phase was acetonitrile: water: methanol (50:41:9, v/v). Flow

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rate was 3.0 mL/min and the column temperature was 25 °C. The injection volume was 80

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μL. Collections of fractions were repeated multiple times to obtain enough material. Each

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fraction obtained was combined and extracted with hexane (150 mL) 3 times. The organic

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extract was evaporated and the oil obtained was mixed with 1 mL acetonitrile and

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transferred to an HPLC vial.

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2.6. HPLC analysis and identity confirmation by LC-MS and GC-MS HPLC analysis of the extracts was performed on a Waters HPLC system (Milford, MA)

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equipped with a Waters 2996 photodiode array detector (PAD) and a Waters 2695

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separation module (Waters Corporation, Milford, MA). The column was a Phenomenex

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Luna C18 (250 mm × 4.60 mm i.d., 5 μm; Torrance, CA). The elution conditions for the HPLC

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analysis of the collected fractions were as follows: flow rate, 1.0 mL/min; column

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temperature, 25 °C; injection volume, 10 μL; detector wavelength, 254 nm; mobile phase,

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acetonitrile: water: methanol (50:41:9, v/v). Compounds were subjected to LC-APCI-MS2

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and GC-MS analyses for structural elucidation and identity confirmation. LC-MS spectra

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were acquired using a Finnigan/MAT LCQ ion trap mass spectrometer (San Jose, CA)

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equipped with an atmospheric pressure chemical ionization (APCI) source. The GC-MS

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conditions were similar to those described above. Quantification of individual compounds

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was carried out using standard curves of the pure compounds isolated (r2 = 0.99).

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2.7. H2S-releasing activity measurement

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A cell-based (MCF-7) assay previously developed in our laboratory was used to

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measure the H2S-releasing capacity of boiled garlic extracts and those of the individual

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compounds (Liang, Wang, Tocmo, Wu, Deng, & Huang, 2015; Wu et al., 2014). Garlic extract

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(2.5 mg/mL) dissolved in DMSO was used as stock mixture. The highest concentration of

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garlic extract added per well after a series of dilutions was 0.25 mg/mL. A Synergy HT

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microplate reader (Biotek, Winooski, VT) was used to measure fluorescence intensity for

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the H2S-releasing activity assay.

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2.8. Statistical analysis

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Data were expressed as means ± standard deviations (SD) of three separate

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replicates. The effect of boiling on the concentrations of organosulfides and H2S-releasing

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capacity was analyzed by ANOVA (p < 0.05) using OriginPro 8 (OriginLab, Northampton,

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MA). Post-hoc analysis using Tukey’s test was carried out to determine significant

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differences among the means (p < 0.05).

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3. Results and discussion

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3.1. Organosulfide profiles of garlic

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It is known that garlic extract, particularly its main thiosulfinate content, allicin, is

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thermally sensitive under GC analysis conditions (Block, 1992). In order to confirm the

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thermolabile characteristic of allicin, we synthesized and purified this compound by semi-

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preparative HPLC and monitored its transformation in GC. Figure 1 shows the HPLC (a)

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and GC (b) traces of pure allicin. By semi-preparative HPLC isolations, allicin was obtained

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at around 72% purity with a maximum absorbance at 205.5 nm. It was observed that, in

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GC, allicin is transformed into other organosulfides, mainly 2-vinyl-[4H]-1,3-dithiin (2-

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VDT), 3-vinyl-[4H]-1,2-dithiin (3-VDT), DADS, and DATS. LC-ESI-MS analysis of allicin did

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not result in the same degradation products. Our results were in agreement with those of

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Ilic et al. (2012), who also found VDTs and DADS as the major transformation products

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after analyzing pure allicin by GC. In this study, 2-VDT (57.03%) was the most abundant

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allicin transformation product followed by 3-VDT (23.56%), DADS (5.88%), and DATS

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(1.21%).

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Heat-treated garlic homogenates were extracted with hexane to yield yellowish

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garlic oil with pungent odor, which is indicative of the presence of allicin (Iberl, Winkler, &

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Knobloch, 1990). There was no observable trend in oil yields (1.85 to 2.32 g oil/kg raw

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material) as an effect of boiling time. Based on GC-MS data, eight major sulfur-containing

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compounds were identified. These organosulfur compounds can be further classified,

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depending on their R-substituents, into diallyl sulfides, methyl allyl sulfides, cyclic vinyl

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dithiins and vinyl dithianes. A representative GC chromatogram of garlic extracts is shown

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in the Data in Brief associated with this article (see Figure 1 in Tocmo, Wu, Dong, Fogliano,

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& Huang, 2016). The dominant peaks numbered 1 to 10 were identified by comparing their

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mass spectra to those of the NIST library of the GC system. The identified compounds along

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with their chemical structures, mass spectral characteristics, and GC retention times are

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listed in Table 1 (Tocmo, Wu, Dong, Fogliano, & Huang, 2016). The organosulfides

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identified included linear polysulfides (DADS and MADS, DATS and MATS) and cyclic

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organosulfides (3-vinyl-1,2-dithiane (3-VIN), 2-vinyl-1,3-dithiane (2-VIN), 2-VDT and 3-

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VDT). These are the same organopolysulfides typically detected in garlic, as reported by

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previous studies (Huang, Teng, Liou, Ho, Liu, & Chuang, 2010; Kimbaris, Siatis, Daferera,

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Tarantilis, Pappas, & Polissiou, 2006; Rose, Whiteman, Moore, & Yi, 2005; Sowbhagya,

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Purnima, Florence, Appu Rao, & Srinivas, 2009; Yabuki et al., 2010). The cyclic compounds

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all contain two sulfur atoms, the formation of which was proposed to be via Diels-Alder

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reaction (Rose, Whiteman, Moore, & Yi, 2005). Peak 2, identified as N,N′-dimethylthiourea

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had a good match with the NIST library mass spectral data. This compound has also been

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detected in garlic oil (Huang et al., 2010). Peak 8, is an unknown compound, with an M+ ion

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of 146. Its mass fragmentation patterns did not indicate that this chemical species is an

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organosulfur compound.

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Results from the GC analysis indicate that DATS, DADS, and VDTs are the major

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organosulfides in garlic extracts. These are the same major organosulfides detected when

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pure allicin was analyzed by GC. This observation suggests that the main organosulfides,

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including DATS, DADS, and VDTs detected in garlic extracts may be artifacts generated

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from allicin under the high temperature conditions of GC. Therefore, GC is not a suitable

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method for the analysis of garlic extracts that contain high amounts of allicin. Furthermore,

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it should be noted that when extraction is carried out using non-polar organic solvents,

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some of the allicin in the extracts might readily transform into 2-VDT, 3-VDT, and ajoene

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(Iberl et al., 1990; Ilić, et al., 2012). Therefore, garlic oil extracted by less polar solvents

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may already contain certain amounts of cyclic organosulfides prior to GC analysis. This

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makes interpretation of GC results even more complicated. However, it is remarkable that

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thermal treatment of allicin at high temperature may be a good way to obtain VDTs. VDTs

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are known for their bioactivity, particularly as antithrombotic and blood pressure-lowering

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agents (Ilić, et al., 2012).

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To clarify the actual organosulfide profiles of garlic, the extracts were analyzed by

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HPLC (Figure 2 in Tocmo, Wu, Dong, Fogliano, & Huang, 2016). Only six major

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organosulfide peaks were identified by HPLC as compared to eight by GC. DADS and DATS,

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which were major peaks by GC, were detected as very small peaks by HPLC. On the

286

contrary, allicin and ajoene, which were not detected by GC, eluted as major peaks by HPLC.

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It has been reported that ajoene was undetectable at high temperature (220 °C) during GC

288

analysis (Iberl et al., 1990). Separation of the E- and Z- isomers of ajoene was not achieved

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in the present study. Previously, cis- and trans- isomers of ajoenes were only separated by

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normal-phase HPLC (Iberl et al., 1990). Hence, we tentatively identified Peak 3 as E/Z-

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ajoene. It is also notable that VDTs are major organosulfides in solvent extracts. This result

292

confirms previous reports that allicin readily transforms to VDTs when garlic is extracted

293

using non-polar solvents (Ilić, et al., 2012). As shown in Figure 2 (Tocmo, Wu, Dong,

294

Fogliano, & Huang, 2016), allicin is the main organosulfur compound in fresh garlic extracts.

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Hence, since allicin generates artifacts under GC conditions, it is recommended that fresh

296

garlic extracts should be analyzed using analytical methods that do not expose the extracts

297

to high temperature.

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3.2. Effect of boiling on the organosulfide concentrations

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A comparison of the HPLC chromatograms obtained at different boiling times is shown

301

the Data in Brief associated with this article (see Figure 3 in Tocmo, Wu, Dong, Fogliano, &

302

Huang, 2016). Variations in the peak areas of the individual compounds were observed, which

303

suggest obvious changes in the concentrations of the polysulfides. In order to quantify the

304

individual organosulfide, semi-preparative HPLC was used to separate and purify each

305

compound from a scaled-up extraction. Individual organosulfides were identified by LC-APCI-

306

MS2 or EI-MS. The results are summarized in Table 1, along with the retention times, chemical

307

structures, and MS data for each compound. A standard curve for each compound was obtained

308

from regression lines, which were generated and used for quantification. Figure 2 shows the

309

HPLC chromatograms and UV traces of the individual compounds. All isolated standards

310

reached greater than 80% purity except allicin (72%), as it undergoes spontaneous

311

transformation even under refrigerated storage (Block, Ahmad, & Jain, 1984).

312

The quantitative profiles of organosulfides in boiled garlic extracts analyzed by

313

HPLC are presented in Figure 3. In raw garlic, allicin (5874 mg/kg) and ajoene (9116

314

mg/kg) predominate, whereas DADS (230 mg/kg) and DATS (19 mg/kg) had the lowest

315

concentrations. GC analysis, as previously discussed, revealed 2-VDT, DADS, and DATS as

316

the major organosulfur compounds in fresh garlic extracts. The large peak areas of 2-VDT

317

and DADS observed by GC were apparently a result of allicin disintegration at high

318

temperature while DADS and DATS concentrations were strongly influenced by boiling

319

time. DATS concentration significantly (p < 0.05) increased as boiling was increased from 3

320

to 20 minutes, as compared to control and 30 min. The highest concentration (584 mg/kg)

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321

was observed at 10 min, although this was not significantly different (p < 0.05) from those

322

of 3, 6 and 20 min. After 30 min of boiling, DATS concentration, although higher than in the

323

control, significantly decreased (p < 0.05) as compared to 3, 6, 10 and 20 min. DADS

324

concentration slightly increased at 6 min boiling but this change was not significant (p <

325

0.05) compared to control, 3, 6 and 20 min. It is also notable that MATS (Peak 8) was

326

present only in boiled samples. Boiling for 30 min resulted in significantly lower

327

concentration of DADS as compared to the rest of the treatments. For 2-VDT and 3-VDT, the

328

concentrations significantly decreased with prolonged boiling time, with 30 min showing

329

lowest values. Our results show that shorter boiling time, 3 to 10 min, enriches the two

330

major H2S-releasing polysulfides, DATS and DADS. The observed increase was apparently

331

an effect of boiling on allicin, favoring the formation of allyl polysulfides. It is known that

332

formation of linear polysulfides from allicin is favored at high temperatures (Block, 1985).

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Figure 4 shows a scatter plot of (a) DATS and (b) DADS as an effect of boiling time. Fitting a

334

polynomial regression showed a curvilinear relationship between boiling time and allyl

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polysulfides. Boiling time significantly accounts for the variability in DATS (r2 = 0.91; p =

336

0.028) and DADS (r2 = 0.98; p = 0.002). Since allicin is favorably transformed to DATS and

337

DADS at higher temperature, it is reasonable to expect that 2-VDT, 3-VDT, and ajoene

338

concentrations decrease.

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Taken together, results suggest that to obtain optimal concentrations of allyl

340

polysulfides, garlic should be subjected to shorter boiling (3 to 10 min). Longer boiling

341

times may result in the evaporation of these volatiles; hence, the observed reductions at

342

C+20 and C+30. In general, we conclude that boiling influences the amounts of

343

organosulfur compounds in garlic and that the effect is rather different between cyclic and

15

344

linear polysulfides. Intermediate boiling times (6 to 10 min) increase the concentrations of

345

some of the organosulfides, such as DADS and DATS, while cyclic organosulfides, mainly, 2-

346

VDT and 3-VDT, significantly decreased even after a few minutes of boiling.

347 348

3.3. H2S-releasing activity of boiled garlic extracts

349

Table 2 summarizes the H2S-releasing capacity of garlic extracts as influenced by

350

boiling. Both C+6 min (5.74 mmol DATS/g oil) and C+10 min (4.23 mmol DATS/g oil) were

351

significantly higher than control (2.39 mmol DATS/g oil). Boiling for 6 minutes resulted in

352

the highest DATS-E value compared to the rest of the treatments. This means that the H2S

353

released from a gram of oil extracted from fresh garlic boiled for 6 min is equal to the

354

amount of H2S released from 5.74 mmol (or 1.02 g) of DATS. Boiling for 10 min also

355

apparently showed higher H2S-releasing activity, but there was no statistical difference

356

compared to that of 20 min and 30 min. Control, C+3 min, C+20 min, and C+30 min showed

357

no significant (p < 0.05) differences in their DATS-E values. A curvilinear relationship was

358

observed between boiling time and DATS-E. Boiling time significantly accounts for the

359

changes in the DATS-E values (r2 = 0.64; p = 0.009). However, no significant relationship

360

was observed when a regression model was fitted between DATS or DADS concentrations

361

and DATS-E values (results not shown). This can be explained by the fact that, although

362

boiling increased the major H2S donors DATS and DADS, especially at 6 and 10 min, other

363

organosulfide components that have H2S-releasing activity (discussed in detail in the next

364

section) decreased as boiling time was prolonged. Similar trends were observed when the

365

oil yields were factored in, to obtain DATS-E values per kg of raw material (Table 2).

16

366

H2S release from garlic polysulfides is concentration-dependent and highly

367

correlated to polysulfides with allyl substituents and to the number of tethering sulfur

368

atoms in the chemical structure (Benavides, et al., 2007). This is apparently the reason why

369

DATS was found to be a good H2S donor. This suggests that the major changes in the DATS-

370

E values of boiled garlic extracts may have been brought about by the increase in DATS

371

concentration. However, despite the very low concentration of DATS and DADS in the

372

control sample, its DATS-E value was comparable to those of C+20 and C+30 min, which

373

have higher DATS content. This suggests a potential H2S-releasing activity of VDTs, ajoene,

374

and allicin, which were significantly higher in the control than in C+20 and C+30 samples.

375 376

3.4. H2S-releasing activity of allicin transformation products

377

Dose‒response curves of the H2S-releasing capacity of (a) 2-VDT, (b) 3-VDT, and (c)

378

ajoene are presented in the Data in Brief associated with this article (see Figure 4 in

379

Tocmo, Wu, Dong, Fogliano, & Huang, 2016). Between the isomeric forms of vinyl dithiins,

380

3-VDT showed a much higher DATS-E value (0.41) as opposed to a negligible value for 2-

381

VDT (0.008). This result suggests that structural differences even for isomeric

382

organosulfides significantly affect an individual compound’s H2S-releasing activity.

383

Moreover, this result shows that, relative to the allyl polysulfide, DATS, cyclic

384

organosulfides are less potent H2S-donors. Ajoene showed a much lower DATS-E value

385

(0.16) than 3-VDT but this value is comparably higher than that of 2-VDT. Therefore, in

386

terms of their potency as in vitro H2S donors, allicin transformation products can be ranked

387

in the following order: 3-VDT > ajoene > 2-VDT. Fresh garlic (control) extracts, despite

388

their very low concentration of DATS and DADS, still showed a considerable H2S-releasing

17

389

activity compared to samples subjected to prolonged boiling, which owe their higher DATS-

390

E values to much higher allyl polysulfide contents. It can, therefore, be deduced that the

391

H2S-releasing activity of the control sample is mainly due to the bioconversion of 3-VDT by

392

MCF-7 cells, generating H2S.

393

In summary, we systematically studied the effect of boiling using two analytical

394

techniques to evaluate effects on polysulfide profile and measured H2S-releasing capacity

395

of garlic. We have shown that shorter boiling time enriches garlic’s linear polysulfides,

396

especially allyl disulfides and trisulfides, which translated into an increase in its H2S-

397

releasing capacity in vitro. Moreover, we have shown, for the first time, the H2S donating

398

capacity of major cyclic organosulfides in garlic. Results of this study augment existing

399

literature, particularly that of Benavides et al. (2007), in further elucidating the structure

400

and activity relationship of individual organosulfides in garlic in terms of their H2S-

401

releasing activity. We found that between the two major allicin transformation products, 3-

402

VDT showed better H2S-releasing activity compared to 2-VDT. 3-VDT approximately has

403

half the H2S-releasing capacity of DATS. Prolonging boiling time up to 30 min decreased the

404

concentrations of vinyl dithiins and ajoenes but did not totally convert allicin into oil-

405

soluble polysulfides. Our results support literature recommendations on using analytical

406

techniques that do not expose organosulfides to high temperatures. This study gives

407

important insights on the effect of thermal processing on garlic as a potential source of

408

bioactive H2S-releasing polysulfides.

409 410 411

Acknowledgments Authors thank the Agency of Science, Technology and Research (A*Star) of

18

412

Singapore for financial support (grant number: 112 177 0036) and support of a Jiangsu

413

Province Grant for Food Science and Technology (Platform 2).

19

414

References

415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457

Amagase, H. (2006). Clarifying the real bioactive constituents of garlic. Journal of Nutrition, 136(3), 716S-725S. Amagase, H., Petesch, B. L., Matsuura, H., Kasuga, S., & Itakura, Y. (2001). Intake of garlic and its bioactive components. Journal of Nutrition, 131(3 SUPPL.), 955S-962S. Argüello-García, R., Medina-Campos, O. N., Pérez-Hernández, N., Pedraza-Chaverrí, J., & Ortega-Pierres, G. (2010). Hypochlorous acid scavenging activities of thioallyl compounds from garlic. Journal of Agricultural and Food Chemistry, 58(21), 11226-11233. Baghalian, K., Ziai, S. A., Naghavi, M. R., Badi, H. N., & Khalighi, A. (2005). Evaluation of allicin content and botanical traits in Iranian garlic (Allium sativum L.) ecotypes. Scientia Horticulturae, 103(2), 155-166. Bełtowski, J. (2015). Hydrogen sulfide in pharmacology and medicine - An update. Pharmacological Reports, 67(3), 647-658. Benavides, G. A., Squadrito, G. L., Mills, R. W., Patel, H. D., Isbell, T. S., Patel, R. P., Darley-Usmar, V. M., Doeller, J. E., & Kraus, D. W. (2007). Hydrogen sulfide mediates the vasoactivity of garlic. Proceedings of the National Academy of Sciences of the United States of America, 104(46), 17977-17982. Block, E. (1985). The chemistry of garlic and onions. Scientific American, 252(3), 114-119. Block, E. (1992). Allium chemistry: GC-MS analysis of thiosulfinates and related compounds from onion, leek, scallion, shallot, chive, and chinese chive. Journal of Agricultural and Food Chemistry, 40(12), 2431-2438. Block, E. (1993). Flavor artifacts. Journal of Agricultural and Food Chemistry, 41(4), 692. Block, E., Ahmad, S., & Jain, M. K. (1984). (E,Z)-Ajoene: A potent antithrombotic agent from garlic. Journal of the American Chemical Society, 106(26), 82958296. Corzo-Martínez, M., Corzo, N., & Villamiel, M. (2007). Biological properties of onions and garlic. Trends in Food Science and Technology, 18(12), 609-625. De Castro, A., Montaño, A., Sánchez, A. H., & Rejano, L. (1998). Lactic acid fermentation and storage of blanched garlic. International Journal of Food Microbiology, 39(3), 205-211. Freeman, F., & Kodera, Y. (1995). Garlic chemistry: Stability of S-(2-propenyl) 2propene-1-sulfinothioate (allicin) in blood, solvents, and simulated physiological fluids. Journal of Agricultural and Food Chemistry, 43(9), 23322338. Fujisawa, H., Suma, K., Origuchi, K., Kumagai, H., Seki, T., & Ariga, T. (2008). Biological and chemical stability of garlic-derived allicin. Journal of Agricultural and Food Chemistry, 56(11), 4229-4235. Guo, W., Cheng, Z-Y., Zhu, Y-Z. (2013). Hydrogen sulfide and translational medicine. Acta Pharmacologica Sinica 34, 1284–1291.

20

458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503

Gardner, C. D., Lawson, L. D., Block, E., Chatterjee, L. M., Kiazand, A., Balise, R. R., & Kraemer, H. C. (2007). Effect of raw garlic vs commercial garlic supplements on plasma lipid concentrations in adults with moderate hypercholesterolemia: A randomized clinical trial. Archives of Internal Medicine, 167(4), 346-353. Ho, C. Y., Cheng, Y. T., Chau, C. F., & Yen, G. C. (2012). Effect of diallyl sulfide on in vitro and in vivo Nrf2-mediated pulmonic antioxidant enzyme expression via activation ERK/p38 signaling pathway. Journal of Agricultural and Food Chemistry, 60(1), 100-107. Huang, T. C., Teng, K. C., Liou, Y. Y., Ho, C. T., Liu, H. J., & Chuang, K. P. (2010). Diallyl disulphide, but not diallyl sulphide, increases leucocyte function-associated antigen-1 expression and cellular adhesion in monocytes. Food Chemistry, 120(1), 113-120. Iberl, B., Winkler, G., & Knobloch, K. (1990). Products of allicin transformation: Ajoenes and dithiins, characterization and their determination by HPLC. Planta Medica, 56(2), 202-211. Ilić, D., Nikolić, V., Stanković, M., Nikolić, L., Stanojević, L., Mladenović-Ranisavljević, I., & Šmelcerović, A. (2012). Transformation of synthetic allicin: The influence of ultrasound, microwaves, different solvents and temperatures, and the products isolation. The Scientific World Journal, 2012. http://dx.doi.org/10.1100/2012/561823. Kamenetsky, R., London Shafir, I., Khassanov, F., Kik, C., Van Heusden, A. W., Vrielink-Van Ginkel, M., Burger-Meijer, K., Auger, J., Arnault, I., & Rabinowitch, H. D. (2005). Diversity in fertility potential and organo-sulphur compounds among garlics from Central Asia. Biodiversity and Conservation, 14(2), 281-295. Kimbaris, A. C., Siatis, N. G., Daferera, D. J., Tarantilis, P. A., Pappas, C. S., & Polissiou, M. G. (2006). Comparison of distillation and ultrasound-assisted extraction methods for the isolation of sensitive aroma compounds from garlic (Allium sativum). Ultrasonics Sonochemistry, 13(1), 54-60. Liang, D., Wang, C., Tocmo, R., Wu, H., Deng, L. W., & Huang, D. (2015). Hydrogen sulphide (H2S)-releasing capacity of essential oils isolated from organosulphur-rich fruits and vegetables. Journal of Functional Foods, 14, 634-640. Locatelli, D. A., Altamirano, J. C., González, R. E., & Camargo, A. B. (2015). Homecooked garlic remains a healthy food. Journal of Functional Foods, 16, 1-8. Locatelli, D. A., Altamirano, J. C., Luco, J. M., Norlin, R., & Camargo, A. B. (2014). Solid phase microextraction coupled to liquid chromatography. Analysis of organosulphur compounds avoiding artifacts formation. Food Chemistry, 157, 199-204. Milner, J. A., & Schaffer, E. M. (1998). Garlic and associated allyl sulfur constituents depress chemical carcinogenesis. In ACS Symposium Series, vol. 702 (pp. 144152). Nikolic, V., Stankovic, M., Nikolic, L., & Cvetkovic, D. (2004). Mechanism and kinetics of synthesis of allicin. Die Pharmazie-An International Journal of Pharmaceutical Sciences, 59(1), 10-14. 21

504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548

Ou, H. C., Tzang, B. S., Chang, M. H., Liu, C. T., Liu, H. W., Lii, C. K., Bau, D. T., Chao, P. M., & Kuo, W. W. (2010). Cardiac contractile dysfunction and apoptosis in streptozotocin-induced diabetic rats are ameliorated by garlic oil supplementation. Journal of Agricultural and Food Chemistry, 58(19), 1034710355. Pluth, M. D., Bailey, T. S., Hammers, M. D., Hartle, M. D., Henthorn, H. A., & Steiger, A. K. (2015). Natural products containing hydrogen sulfide releasing moieties. Synlett, 26(19), 2633-2643. Predmore, B. L., Kondo, K., Bhushan, S., Zlatopolsky, M. A., King, A. L., Aragon, J. P., Bennett Grinsfelder, D., Condit, M. E., & Lefer, D. J. (2012). The polysulfide diallyl trisulfide protects the ischemic myocardium by preservation of endogenous hydrogen sulfide and increasing nitric oxide bioavailability. American Journal of Physiology - Heart and Circulatory Physiology, 302(11), H2410-H2418. Rose, P., Whiteman, M., Moore, P. K., & Yi, Z. Z. (2005). Bioactive S-alk(en)yl cysteine sulfoxide metabolites in the genus Allium: The chemistry of potential therapeutic agents. Natural Product Reports, 22(3), 351-368. Shen, C., Xiao, H., & Parkin, K. L. (2002). In vitro stability and chemical reactivity of thiosulfinates. Journal of Agricultural and Food Chemistry, 50(9), 2644-2651. Song, Z. J., Ng, M. Y., Lee, Z. W., Dai, W., Hagen, T., Moore, P. K., Huang, D., Deng, L. W., & Tan, C. H. (2014). Hydrogen sulfide donors in research and drug development. Medicinal Chemical Communications, 5(5), 557-570. Sowbhagya, H. B., Purnima, K. T., Florence, S. P., Appu Rao, A. G., & Srinivas, P. (2009). Evaluation of enzyme-assisted extraction on quality of garlic volatile oil. Food Chemistry, 113(4), 1234-1238. Szabo, C. (2016). Gasotransmitters in cancer: From pathophysiology to experimental therapy. Nature Reviews Drug Discovery, 15(3), 185-203. Tang, F. Y., Chiang, E. P., & Pai, M. H. (2010). Consumption of S-allylcysteine inhibits the growth of human non-small-cell lung carcinoma in a mouse xenograft model. Journal of Agricultural and Food Chemistry, 58(20), 11156-11164. Tocmo, R., Wu, Y., Liang, D., Fogliano, V., & Huang, D. (2016). Data on the effect of boiling on the organosulfides and the hydrogen sulfide-releasing activity of garlic. Data in Brief. Submitted. Vazquez-Prieto, M. A., González, R. E., Renna, N. F., Galmarini, C. R., & Miatello, R. M. (2010). Aqueous garlic extracts prevent oxidative stress and vascular remodeling in an experimental model of metabolic syndrome. Journal of Agricultural and Food Chemistry, 58(11), 6630-6635. Wu, H., Krishnakumar, S., Yu, J., Liang, D., Qi, H., Lee, Z. W., Deng, L. W., & Huang, D. (2014). Highly selective and sensitive near-infrared-fluorescent probes for the detection of cellular hydrogen sulfide and the imaging of H2S in mice. Chemistry - An Asian Journal, 9(12), 3604-3611. Yabuki, Y., Mukaida, Y., Saito, Y., Oshima, K., Takahashi, T., Muroi, E., Hashimoto, K., & Uda, Y. (2010). Characterisation of volatile sulphur-containing compounds generated in crushed leaves of Chinese chive (Allium tuberosum Rottler). Food Chemistry, 120(2), 343-348.

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Yin, M. C., Hwang, S. W., & Chan, K. C. (2002). Nonenzymatic antioxidant activity of four organosulfur compounds derived from garlic. Journal of Agricultural and Food Chemistry, 50(21), 6143-6147. Zhao, Y., Biggs, T. D., & Xian, M. (2014). Hydrogen sulfide (H2S)-releasing agents: chemistry and biological applications. Chemical communications (Cambridge, England), 50(80), 11788-11805.

23

595 596 597 598 599

600 601 602 603 604

Table 1. Identity, chemical structures, and LC-MS2/EI-MS data of sulfur-containing compounds in garlic. Peak No.a

Retention time (min)

Compound

Structure

1

4.51

Allicin

162, 121, 87, 73

3

5.88

(E/Z)-Ajoene

234, 145,111, 103, 73

5

9.14

2-Vinyl-[4H]1,3-dithiin

144,111, 103

6

13.37

3-Vinyl-[4H]1,2-dithiin

144,111, 103,

7

17.80

Diallyl disulfide

146,113, 105, 81, 79, 73, 45, 41*

8

29.80

Diallyl trisulfide

178, 114, 113, 73, 72, 71, 45, 41*

MS2 [M+H]+ or EI-MS* fragmentations

a

Refers to peaks in Figure 2 (Data in Brief; Tocmo, R., Wu, Y., Liang, D., Fogliano, V., & Huang, D. (2016). * Determined with Agilent 7890A quadruple mass spectrometer and DB-5MS (30 m × 0.25 mm i.d., 0.25 μm) column.

605 606 607 608

24

609 610 611 Table 2. H2S-releasing capacity of boiled garlic extracts.

612

613 614 615

Treatments Oil yields (g of oil/kg raw material) 0 min 2.00 ± 0.43

DATS-E of oil (mmol DATS/g of oil)a 2.39 ± 0.02a

DATS-E (mmol DATS/kg of a garlic) 4.76 ± 1.20a

3 min

2.22 ± 0.61

2.46 ± 0.49ab

4.94 ± 0.97ab

6 min

1.85 ± 0.63

5.74 ± 1.31c

10.64 ± 2.90c

10 min

1.89 ± 0.24

4.23 ± 0.14b

7.99 ± 1.15b

20 min

2.23 ± 0.30

2.36 ± 0.95ab

5.24 ± 0.26ab

30 min

2.32 ± 0.06

2.16 ± 0.21ab

5.01 ± 0.41ab

aData

are expressed as mean ± SD. Difference among treatments was analyzed by Tukey’s test at a level of significance p < 0.05. Values in the same column not sharing a common letter differ significantly.

25

Figure Captions

616 617

Figure 1.

The peak at 23.5 min is an unknown compound.

618 619

HPLC chromatogram of (a) allicin and its degradation products in (b) GC.

Figure 2.

HPLC chromatograms, standard curves, and UV traces of pure compounds

620

isolated by semi-preparative HPLC. Flow rate during HPLC analysis was

621

varied from 0.8 to 1.2 mL/min to achieve better separations.

622

Figure 3.

determined by HPLC.

623 624

Effect of boiling on the concentrations of individual organosulfides

Figure 4.

Scatter plots of (a) DATS and (b) DADS as an effect of boiling time.

625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641

26

642 643 644 645 646 Allicin

A

0.60 0.50

AU

0.40 0.30 0.20 0.10 0.00 0.0

Peak Area

5.0e+06

5.0

10. 0

15. 0

20. 0

25. 0

30.0

35. 0

40.0

45.0

B 2-VDT

4.0e+06 3.0e+06

3-VDT

2.0e+06

DADS

1.0e+06

DATS 14.0

647

16.0

18.0

20.0

22.0

24.0

26.0

28.0

Retention time (min)

648 649

Figure 1

650 651 652 653 654 655 656 657

27

658 659 660 661 662 AU

Allicin

0.40 0.20

5.00

0.80

10.00

0.60

20.00

15.00

0.20

25.00

30.00

35.00

2-VDT

5.00

10.00

15.00

20.00

25.00

30.00

35.00

0.80 0.40

0.60

3-VDT

2.50 200.8 2.00

0.80

AU

1.20

0.00 0.00

1.00

2.00 198.5 1.60 225.5 1.20 0.80 0.40 0.00 200.0 250.0 300.0 350.0 Wavelength (nm)

1.60

1.50 1.00 0.50 0.00

0.40 0.20

317.6 200.0 250.0 300.0 350.0 Wavelength (nm)

0.00 10.00

15.00

DADS

25.00

30.00

35.00

2.00

2.0 198.5 1.6 1.2 0.8

0.60 0.50

AU

0.30

20.00

0.20

0.4 0.0

0.10

5.00

10.00

0.40

AU

5.00

0.40

0.00 0.00

Ajoene

0.40

200.0 250.0 300.0 350.0 Wavelength (nm)

2.00

0.00 0.00

2.50 2.00 205.5 1.50 1.00 0.50 0.00 200.0 250.0 300.0 350.0 Wavelength (nm)

AU

0.60

0.00 0.00

1.00

210.2

AU

0.80

2.50 2.00 1.50 1.00 0.50 0.00

AU

1.00

0.30 0.20

200.0 250.0 300.0 350.0 Wavelength (nm)

15.00 20.00 25.00 30.00 Retention Time (min)

35.00

40.00

0.10

45.00

0.00 0.00

6.00

10.00

14.00

2.0 198.5 1.6 1.2 0.8 0.4 0.0 200.0 250.0 300.0 350.0 Wavelength (nm) 5.00

10.00

18.00

22.00

26.00

30.00

DATS

15.00 20.00 25.00 30.00 Retention Time (min)

35.00

40.00

45.00

663 664

Figure 2

665 666 667 668 669 670 671 28

672 673 674 675

Concentration (mg/kg raw material)

3000

a

3-VDT

2500

2-VDT

2000 1500

b

1000

bc

a

500

bc

b

3 min

6 min

cd cd

d d

d d

20 min

30 min

0

0 min

Concentration (mg/kg raw material)

10000

10 min

a Allicin

8000 6000

Ajoene a b

4000

b

b

c

c bc

2000

cd cd

d d

0

0 min

3 min

6 min

10 min

20 min

Concentration (mg/kg raw material)

700

a ab

400

a

a

a

a

200 100

DATS

a

500

300

DADS

a

600

30 min

a

bc a

c

0

0 min

676 677

3 min

6 min

10 min

20 min

30 min

Boiling time (min)

Figure 3

678 679 680 681 682

29

683 684 685 686 700

A

DATS (mg/kg)

600 500 400 300

y = -2.240x2 + 69.20x + 106.9 R² = 0.907

200 100 0 0

5

10

15

20

25

30

35

25

30

35

300

B DADS (mg/kg)

250 200 150

y = -0.382x2 + 5.889x + 229.1 R² = 0.984

100 50 0 0

687 688

5

10

15

20

Boiling time (min)

Figure 4

689 690 691 692 693 694 695 30

696 697 698 699 700 701 702 703 704 705 706

Highlights: Allyl polysulfide concentrations increased in moderately boiled (6 to 10 minutes) garlic. Enhancement in H2S-releasing activity parallels the increase in allyl polysulfides. Major allicin transformation products were tested for their H2S-releasing activity in vitro.

707 708

31