a spectrophotometric method for assay

Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences

Pol. J. Food Nutr. Sci., 2012, Vol. 62, No. 1, pp. 23-29DOI: 10.2478/v10222-011-0042-4

http://journal.pan.olsztyn.plOriginal PaperSection: Food Chemistry

INTRODUCTION

Garlic (Alliumsativum) isaplant known for its medicinal properties. Also, its cloves are used for consumption invari-ous regions oftheworld, either raw, cooked or fried. Garlic isapplied to spice dishes due to its specifi c pungent fl avour andused to preserve meat andvegetables because ofits an-timicrobial properties. Intact garlic bulbs contain organosul-fur compounds, particularly -glutamylcysteines and cyste-ine sulfoxides. Within thegarlic bulb, during storage, apart ofthe-glutamylcysteines convert into thecysteine sulfoxides which are precursors of thiosulfi nates (TS). Thiosulfi nates are formed during an enzymatic reaction catalysed by al-liinase (alliin lyase, EC4.4.1.4):

R-S(O)-CH2-CH(NH)2-COOH + H2O R-SOH + CH3-C(O)-COOH + NH3R-SOH + R1-SOH R-S(O)-S-R1 + H2O (1)

At fi rst pyruvic acid, ammonia andalk(en)yl sulfenic acid are formed. Afterwards rapid condensation ofsulfenic acids occurs and thiosulfi nates R-S(O)-S-R1are formed, where R andR1represent methyl, 1-propenyl or 2-propenyl group.

Thereaction occurs during garlic cloves crushing. There are eight known dialk(en)yl thiosulfi nates incrushed garlic:

* Corresponding author: Tel.: +48126646927; Fax: +48126340515E-mail : zofi [email protected] (dr Zofi a Olech)

allyl-2-propenethiosulfi nate (allicin) is the most abundant (5090%mol). Theothers are: allylmethaneTS (320%mol), trans-1-propenyl-2-propeneTS (518%mol), methyl-2-pro-peneTS (1.58%mol), allyltrans-1-propeneTS (1.52%mol), methylmethane-TS (12%mol), trans-1-propenylmethaneTS (12%mol), methyl-trans-1-propeneTS (0.5%mol) [Block, 1992; Kyung & Lee, 2001; Lawson & Gardner, 2005].

Thecharacteristic odour ofgarlic andmany ofits health benefi cial properties: antimicrobial, antithrombotic andan-tiatherosclerotic, are attributed to thiosulfi nates strong re-activity to free -SH groups and its antioxidant properties [Qi & Wang, 2003; Juszkiewicz et al., 2004; Hiyasat et al., 2009]. Although allicin, the enzyme alliinase and the reac-tion ofTS formation have been known andexamined since 1944 [Cavallito & Bailey, 1944], asimple andsensitive meth-od ofquantitative TS determination is still being searched. GCandHPLC methods are used for quantitative determina-tion ofTS [Rosen et al., 2001; Arnault et al., 2003] but for GC analysis allicin andother thiosulfi nates must beconverted to more stable compounds, not so sensitive to high temperature as TS.Theproblem does not occur when HPLC isused be-cause analyses are taken at room temperature. However, low resolution andthenecessity ofusing external standards limit theapplicability ofGC andHPLC methods. Enzymatic bio-sensors method may also beused for TS determination [Krest & Keusgen, 2002; Keusgen et al., 2003]. This method allows not only to determine TS concentration but also to monitor the course of the enzymatic reaction. Theanalysis isquick

ASpectrophotometric Assay for Total Garlic Thiosulfi nates Content.Kinetic Aspects ofReaction with Chromogenic Thiols

Zofi a Olech*, Wiesawa Zaborska

Faculty ofChemistry, Jagiellonian University, ul.Gronostajowa 3, 30387Krakw, Poland

Key words: Allium sativum, garlic, kinetics, alk(en)yl thiosulfi nates, spectrophotometric method

Garlic (Allium sativum) isaplant known for its antimicrobial, antithrombotic andantiatherosclerotic properties. Those features are attributed to thiosulfi nates, compounds formed inan enzymatic reaction occurring during garlic cloves crashing.

Aspectrophotometric method for thedetermination ofthiosulfi nate concentration ingarlic extracts andsupplements was proposed. Itisbased on reactions between thiosulfi nates andselected chromogenic thiol compounds: 2-mercaptopyridine (2-MP), 4-mercaptopyridine (4-MP), 1-oxide-2-mer-captopyridine (MPO), 2-mercaptopyrimidine (MPM) and2-nitro-5-thiobenzoic acid (NTB). Then kinetics ofthereactions was studied. Rate con-stants andorders were determined. All reactions are fi rst order with respect to thethiol andfi rst order with respect to TS so thereactions ofall tested thiols with garlic TS run inaccordance with second order kinetics. Reaction rates vary among thethiol compounds: theconstant rates differ from 6.7(mol/L)-1.min-1for 2-MP to 1129(mol/L)-1.min-1for NTB.

Knowledge of thekinetic equation for thereactions andmeasurements of the initial rates allowed determining thiosulfi nate contents ingarlic extracts andsupplements.

Unauthenticated | 210.56.31.9Download Date | 11/4/13 11:52 AM

24 Z. Olech & W. Zaborska

and precise and does not require sophisticated equipment, but itisdiffi cult to obtain theenzyme.

For measurement ofTS concentration spectrophotometric methods were also tested. Two kinds ofcompounds may beap-plied: directly or indirectly allowing to determine TS content inaUV-Vis range. Thefi rst group includes thiols (compounds with sulphydryl groups -SH), e.g. NTB [Miron et al., 1998] and4-MP [Miron et al., 2002]. These compounds react with adisulfi de bond ofthiosulfi nates. Thecourse ofthereaction isobserved bymonitoring thiols concentration. For indirect spectrophotometric determination disulfi de acid compound 5,5-dithiobis(2-nitrobenzoicacid) (DTNB) was used byHan et al. [1995]. This assay isbased on thereaction ofTS with ex-cess ofcysteine. Theresidue ofcysteine reacts with thedisulfi de acid. As aresult ofthereaction, acoloured compound absorb-ing intheUV-Vis range isformed. Another way ofthequan-titative TS determination isto monitor contents ofammonia andpyruvic acid, theother products formed during the en-zymatic reaction (Equation 1). However, both of them are present ingarlic cloves before crushing [Yoo&Pike,2001], hence itisnecessary to take into consideration thebackground ofammonia andpyruvic acid concentrations intheplant.

Inview oftheabove, we propose anew spectrophotomet-ric method for the determination of thiosulfi nates content, based on their reaction with chromogenic thiols. Itrequires knowledge ofthereaction kinetics anddetermination ofrate constants. Themethod allows determining thetotal concen-tration ofthiosulfi nates ingarlic andits supplements. Itisim-portant to know whether thesupplements contain any thio-sulfi nates andwhat istheir content because thecompounds benefi cial for health might bedestroyed during production.

MATERIALS ANDMETHODS

Chemicals2-mercaptopyridine (2-MP) and 4-mercaptopyridine

(4-MP) were purchased from Aldrich, whereas 1-oxide-2-mer-captopyridine (MPO) and 2-mercaptopyrimidine (MPM) were purchased from Fluka Chemika. 2-nitro-5-thiobenzoic acid (NTB) was prepared according to Degani & Patchornik [1971], whereas L-cysteine, 5,5-dithiobis(2-nitrobenzo-icacid)(DTNB) and2,2-dithiodipyridine (DTDP) were pur-chased from Sigma. Thechemicals were ofanalytical grade. Structural formulas ofthiol compounds used inthestudy are presented inFigure1.

Preparation ofplant materialThegarlic used was from China. Its cloves were ground

with amortar andmixed with water: 10mL per 1g ofgarlic [Lawson & Gardner, 2005]. Next, thesuspension was shaken for 30min andfi ltered through gauze. Then undissolved ma-terial was removed bycentrifugation at 300g for 4min.

Preparation ofgarlic supplementsGarlic supplements: Biogarlic (Pharma Nord, Danmark),

Garlic Extract (Unipharm, USA), andPulverised Garlic (Sol-gar, USA) all available inPolish pharmacies, were tested. Aqueous extracts ofgarlic supplements were prepared accord-ing to Lawson & Wang procedure [2001]: weighted tablets

were ground with amortar andmixed with water: 50mL per 1g oftablet. Then thesuspensions were shaken for 30min, fi ltered through gauze andcentrifuged at 300g for 4min.

UV-Vis spectra Absorption spectra oftested thiols andtheir changes dur-

ing thereaction with thiosulfi nates were recorded using Marcel Media (Marcel, Zielonka, Poland) UV-Vis spectrophotometer in a 330-550 nm range for about 100 mol/L thiols solu-tions in50mmol/L phosphate buffer, pH=7.0and1mmol/L EDTA.

Quantitative determination ofTS concentration Theaqueous garlic extracts were initially standardised for

their thiosulfi nate concentration using thespectrophotomet-ric method ofHan et al. [1995]. This method is based on the fact that one molecule of thiosulfi nate reacts with two molecules of cysteine to form two molecules of S-alkenyl or S-alkylmercaptocysteine. The drop of cysteine content ismeasured spectrophotometrically at 412nm in the form of 2-nitro-5-thiobenzoic acid obtained in a reaction with DTNB. Residual concentration of cysteine was determined also by the reaction with DTDP, followed by themeasure-ment of2-mercaptopyridine concentration at 343nm.

Kinetics examination of the reactions between chromogenic thiols andthiosulfi nates

Absorbance measurements were carried out in a 1 cm cuvette using the Marcel Media UV-Vis spectrophotometer (330800 nm range). In the kinetic measurements, 1 mL oftheTS solution was added andmixed with 1mL ofthethi-ol solution and then absorbance changes were recorded ev-ery10 or every 2minutes. Incalculations, stirring time was taken into account. Reactions were carried out inaphosphate buffer, pH=7.0 and 1 mmol/L solution of EDTA at room temperature. Absorbance was measured at max for each thiol that results inminimisation ofabsorbance measurement er-ror. For MPO andMPM molar absorbance coeffi cients are low, therefore there were used more concentrated solutions: [MPO]=250mol/L and[MPM]=510mol/L.For theother thiol solutions theconcentration was about 100mol/L.Inall cases, concentrations refer to solutions after mixing.

FIGURE 1. Chromogenic thiols used inthestudy.


Spectrophotometric Assay for Garlic Thiosulfinates Content 25

RESULTS ANDDISCUSSION

Total thiosulfi nates concentration ingarlic extracts Thiosulfi nates content ingarlic cloves depends on condi-

tions ofplant cultivation: insolation, temperature, humidity ofsoil, andalso depends on storage conditions ofgarlic bulbs [Ichikawa et al., 2006]. For this reason, the concentration ofthiosulfi nates inthetested garlic extracts was examined us-ing theHan et al. [1995] method before each kinetic measure-ment. Unreacted cysteine was determined inareaction with DTNB andwith theother disulfi de compound: 2,2-dithiodi-pyridine (DTDP) which has not been applied before. Mea-surements were repeated 10-fold. TS concentration was cal-culated as an arithmetical mean ofboth methods.

Thiosulfi nates content oftheexamined garlic extracts was inaconcentration interval from 3.1to 3.6mmol/L with preci-sion ofconcentration determination equal to 3%. Themodi-fi ed cysteine method, using DTDP in its second step, gave results which were comparable to the classic Han meth-od results. Itmeans the results ofboth methods are equal within the range of one standard deviation. An advantage ofthemodifi ed method isingreater stability ofthereaction mixture that allows measurements inawider pH range, i.e. from 2.0to 8.0, while theuse ofDTNB allows measurements only ina7.5to 8.5pH range [Egwim & Gruber, 2001; Riener et al., 2002]. Themethod using 2,2-DTDP may be useful inanalyses under simulated gastrointestinal conditions.

UV-Vis absorption spectra ofchromogenic thiolsUV-Vis absorption spectra were recorded for solutions

ofthechromogenic thiols. Themolar absorbance coeffi cients determined from the spectra and known from literature as well as max for each thiol were collected inTable1. Absorption spectra oftwo tested thiols, NTB and2-MP, recorded during their reaction with garlic thiosulfi nates are shown inFigure2. Only for NTB aband derived from thereaction product was observed intheexamined range.

Reaction of thiosulfi nates from garlic extracts with thechromogenic thiols

On thebasis ofMiron et al. [1998, 2002] studies itisknown that thiols: 4-MP andNTB react with allicine (stoichiometric ratio 2:1) by-SH group forming adisulfi de bond. Inthefi rst case 4-S-allylmercaptothiopyridine, wheras inthesecond case S-allylmercapto-5-thio-2-nitrobenzoic acid isformed. Taking

into account theprevious studies, thefollowing reaction may beproposed for thestudied chromogenic thiols andgarlic TS:

2P-SH + R1-S(O)-S-R2 P-S-S-R1 + P-S-S-R2 + H2O (2)

where: P-SH athiol reagent; R1, R2 alk(en)yl groups: methyl, 1-propenyl or 2-propenyl; andP-S-S-R1, P-S-S-R2 alk(en)yl disulfi des.

TABLE 1. Spectral characteristics of the chromogenic thiols and rate constants for their reactions with garlic thiosulfi nates.

Thiol max / nm /

((mol/L)-1.cm-1)

Rate constantk / ((mol/L)-1.

min-1)

2-mercaptopyridine 343 6680 6.70.4

4-mercaptopyridine 324 19800 * 693

1-oxide-2-mercaptopyridine 330 3860 73.20.3

2-mercaptopyrimidine 336 1796 1041

2-nitro-5-thiobenzoic acid 412 14150 ** 112913

* [Miron et al., 1998]; ** [Lawson & Gardner, 2005]

FIGURE 2. UV-Vis absorption spectra of 2-mercaptopyridine (A) and2-nitro-5-thiobenzoic acid (B) during thereaction with thiosulfi nates recorded at different time:

A 1 before thereaction, B 1 before thereaction, 2 after 40minutes 2 after 1minute ofthereaction, ofthereaction 3 120min, 3 6min 4 230min, 4 10min 5 420min 5 23min

Theinitial concentrations ofthesolutions were: A [TS] = 750mol/L , [2-MP] = 105mol/L B [TS] = 110mol/L , [NTB] = 80mol/L



For all tested chromogenic thiols courses oftheir reactions with thiosulfi nates from garlic extracts were recorded. Fig-ures 3and4depict thecourses ofselected thiol compounds. Thereaction of2-MP with TS was theslowest andtherefore itwas recorded for more than 4h, thereaction ofNTB with TS was thefastest anditwas registered for 30min. Figure3A shows thecourse ofreaction between thiosulfi nates ofgar-lic extract solutions of different concentrations and 2-MP

solution of constant concentration. Figure 3B shows thesituation when TS solution concentration was constant andTS reacted with 2-MP solutions having different con-centrations. Similar kinetic curves were recorded for MPM andNTB (Figures3CD,4A). TS with NTB reaction was also monitored by absorbance measurement of the product at =330nm. Theabsorption band oftheproduct can beob-served inthespectrum in Figure2B. Theshape ofsubstrate

FIGURE 3. Kinetics ofthereactions ofgarlic thiosulfi nates with 2-mercaptopyridine (A,B) and2-mercaptopyrimidine(C,D) . A [2-MP] = 105mol/L , TS concentrations given inthefi gure B [TS] = 750mol/L, 2-MP concentrations given inthefi gure C [MPM] = 510mol/L, TS concentrations given inthefi gure D [TS] = 750mol/L, MPM concentrations given inthefi gure



andproduct kinetic curves (Figure4AB) confi rms thesup-posed reaction stoichiometry.

Inan independent experiment initial rates 0oftheabove--mentioned reactions were determined. Concentration changes of the tested thiols were measured every 10 s for 10minutes. Intheperiod oftime when dependences ofab-sorbance upon time were linear, the initial rates are equal slopes oflines A(t).

Reaction order with respect to thiols andto thiosulfi natesReactions orders were determined using the isolation

method: initial rate 0ofTSthiol reactions were obtained applying an excess ofthiol inone experiment andthen ap-plying TS excess inthesecond one. From thelinear depen-dence of log0upon log[TS], reaction order with respect to TS was calculated as a slope. From the linear depen-dence of log 0upon log [P-SH], reaction order with re-spect to the thiol was calculated in the same way. An ex-ample oforders ofdetermination for TSNTB reaction is shown inFigure5. Theorders of the reaction between all thetested thiols andgarlic TS were determined inacor-responding way. All reactions are fi rst order with respect to thethiol andfi rst order with respect to TS so thetotal order ofthereaction istwo.

Rate constantsRate constant ofthereaction was calculated using an ex-

pression for second order reaction:

k = 0 / ( [P-SH]1[TS]1) (1F)

For theinitial linear fragment ofeach kinetic curve there-action initial rate i was determined as theslope ofA(t) line. Table2shows an example ofdetermination ofrate constant for MPMTS reaction. Rate constants for all tested chro-mogenic thiols were collected inTable 1. Theconstant rate ofthereaction was calculated as an arithmetical mean ofrate constants obtained for different TS or thiol concentrations. Uncertainties of rate constants were calculated as standard deviations of the means.

Thechromogenic thiols methodAnalysis ofthekinetic curves allowed to determine theini-

tial rates andrate constants of thereaction. Thepartial or-ders were also estimated with respect to thethiols andthio-sulfi nates, indicating that thekinetic equation for all tested reactions isas follows:

= k[P-SH]1[TS]1 (2F)

Reactions ofall tested thiols with garlic TS ran inaccor-dance with second order kinetics, but their rates were differ-

FIGURE 4. Kinetics ofthereaction between 102mol/L solution of2-ni-tro-5-thiobenzoic acid and garlic thiosulfi nates. Concentrations ofTS are given in thefi gures. The reaction course monitored at 412nm (A) and336nm (B).

FIGURE 5. Determination oftheorder ofreaction between NTB andgar-lic thiosulfi nates using theinitial rate method. A order with respect to NTB B order with respect to TS



ent (Table1). From thereaction kinetics, therate constant and the kinetic equations, TS concentration can be ob-tained bymeasuring absorbance decrease, Ap in thefi rst phase ofthereaction:

Ap = A0 A(tp) (3F)

where tp isthereaction time so short that theA(t) curve can beapproximated to aline. Then:

[TS]0 = 0 / ( k [P-SH]0 ), (4F)

where 0 = Ap / ( 2 l P-SH tp ) (5F)

Theproposed method ofTS concentration determina-tion is based on the measurement of chromogenic thiols absorbance decrease. Because of that, the determination is the more precise the greater is the measured decrease in Ap. If thedetermination ismade using the initial rates method they cannot be too large because it isdiffi cult to measure them. But, on theother hand, they cannot betoo small because abig error ismade or ittakes too long time to measure 0. It isobvious that rates of tested reactions depend not only on therate constants but also on theTS concentration. So, when alow concentration isdetermined it isbetter to use athiol reagent which rapidly reacts with thiosulfi nates, andinthecase ofhigh expected TS concen-tration aslow-reacting thiol.

Using the proposed thiol method the TS content was determined for several garlic supplements purchased from Polish pharmacies. Uncertainties ofTS concentration deter-mination for 2-MP and4-MP were about 5%. They mainly resulted from uncertainties of rate constants determination.

For theother thiols used, theuncertainty ofTS concentration determination isabout 3% anditiscaused ofinitial rates un-certainty. To minimise them thiol andthiosulfi nates solutions should bediluted insuch away that thedecrease ofabsorp-tion Ap equals at least 0.3.

Acomparison of the results andTS content indicated bytheproducers for thetested garlic supplements isshown in Table 3. Only for a few supplements the comparison was possible because others, available inPolish pharma-cies, were not standardised in terms of the content ofal-licin nor ofother thiosulfi nates. Sometimes only themass ofgarlic used for one tablet preparation isindicated. How-ever, it isnot reliable because of thementioned above de-pendence ofTS content ingarlic on cultivation and stor-age conditions. Theproposed chromogenic thiols method may beuseful for standardisation, because it is important to know theactual contents ofactive compounds inapplied garlic supplements.

CONCLUSION

On thebasis of the research we postulate that almost every chromogenic thiol is applicable for quantitative TS determination in fresh garlic extracts and supplements. It is only necessary to record the thiol UV-Vis spectrum. Knowing its max it ispossible to monitor thecourse of its reaction with TS and to determine the rate constant ofthereaction. Theproposed method enables determining the total concentration of thiosulfi nates, not only of alli-cin. This ismore accurate inthecase offresh garlic andits supplements because allicin content isfl exible andmutual proportions ofall TS are variable anddepend on cultiva-tion andstorage conditions [Ichikawa et al., 2006]. For this reason, it is diffi cult to conclude on TS content only on thebasis ofallicin content. Moreover, itisknown that not only allicin, but also all thiosulfi nates have medicinal prop-erties. Because ofthis, theproposed method isbest suited for thedetermination ofmedicinal properties ofgiven garlic supplement.

REFERENCES

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TABLE 2. Initial rates 0i and rate constants for the reaction between garlic thiosulfi nates and2-mercaptopyrimidine.

[TS]0 / mol/L[MPM]0 / mol/L

Initial rate (Ai/tp) / (min-1)

Initial rate0i / 106(mol/L).

min-1

Constant ratek / ((mol/L)-1..

min-1)

28.8 500 0.00543 1.15 105

33.4 500 0.00654 1.82 109

38.4 500 0.00695 1.93 101

57.7 500 0.01137 3.17 110

72.2 500 0.01457 4.06 112

87.5 500 0.01627 4.53 104

116 500 0.01933 5.38 93

144 500 0.02783 7.75 107

175 500 0.03383 9.42 108

292 500 0.05127 14.3 98

578 500 0.09728 27.1 94

875 500 0.15355 2.7 98

350 62.5 0.00866 2.41 110

350 125 0.01653 4.60 105

350 250 0.03214 8.95 102

350 330 0.04363 12.1 104

350 500 0.06567 18.3 104

TABLE3. Thecomparison ofthiosulfi nates content intested garlic sup-plements.

Content ofTSGarlic Extract

(Pharma Nord)

Biogarlic(Unipharm)

Pulverized garlic

(Solgar)

TS content(mol per 1g ofsupplement)

7.20.3 17.90.9 38.01.3

TS content(mg allicin per tablet) 0.8350.034 2.090.08 2.220.08

Producer-indicated TS content (mg allicin per tablet)

1.0 2.1 1.5



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Received March 2011. Revision received August 2011andaccept-ed September 2011. Published on-line on the 18th of January 2012.


a spectrophotometric method for assay

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