A spectrophotometric assay for allicin, alliin, and alliinase(alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine

with thiosulnates

Talia Miron, Irina Shin, Guy Feigenblat, Lev Weiner, David Mirelman,Meir Wilchek, and Aharon Rabinkov*

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

Received 5 November 2001

Abstract

Allicin (diallylthiosulnate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein

amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for

the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This

reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and

allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is

based on the reaction of 4-MP kmax 324nm with the activated disulde bond of thiosulnates S(O)S, forming the mixeddisulde, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was

conrmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form

as well as of alliin and total thiosulnates concentrations in crude garlic preparations and garlic-derived products, at micromolar

concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly ecient throughput assay of allicin, alliin, and

alliinase in garlic preparations. 2002 Elsevier Science (USA). All rights reserved.

Keywords: Garlic; Allicin; Alliin; Alliinase; 4-Mercaptopyridine; 2-Nitro-5-thiobenzoic acid

Allicin (diallylthiosulnate) is the best known activecompound of garlic, and it represents about 70% of theoverall thiosulnates present or formed upon crushingthe cloves [14]. It is produced by the interaction of thenonprotein amino acid alliin, abundant in garlic cloves,with the enzyme alliinase (alliin lyase, EC 4.4.1.4.), aspresented in Scheme 1.

Allicin is known to possess a vast variety of biologicaleects. Its antimicrobial, anti-inammatory, antithrom-botic, anticancer, and antiatherosclerotic activities, aswell as the capacity to lower serum lipid levels and ocularpressure, were reported [1,3,4]. Most of these eects canbe related to its strong SH-modifying and antioxidantproperties [58]. Garlic extracts and the majority ofcommercial garlic food supplements, such as tablets andcapsules containing garlic powder, are based on either

allicin content or on the potential to produce allicin [9].Various methods have been developed to quantify allicinsince its discovery [5]. Most of these approaches includegas chromatography and HPLC procedures [1014].These procedures are time consuming and expensive. Inthis work our aim was to develop a simple, sensitive, andhighly throughput assay with a low cost for allicin, alliin,and alliinase.

Allicin reacts very rapidly with free thiol groups, via athiol-disulde exchange reaction. Therefore, a thiol-con-taining chromophore whose optical absorbance is shiftedupon the thiol-disulde exchange reaction can be used forthe direct determination of allicin. Previously, we des-cribed an assay based on a single-step reaction of allicinwith 2-nitro-5-thiobenzoate (NTB)1 [15]. This reaction isassessed by measuring the decrease of the optical density

Analytical Biochemistry 307 (2002) 7683

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ANALYTICALBIOCHEMISTRY

* Corresponding author. Fax: +972-8-946-8256.

E-mail address: bfrabin@wicc.weizmann.ac.il (A. Rabinkov).

1 Abbreviations used: 4-MP, 4-mercaptopyridine; NTB, 2-nitro-5-

thiobenzoic acid; DTNB, 5,50-dithio-bis (2-nitrobenzoic acid).

0003-2697/02/$ - see front matter 2002 Elsevier Science (USA). All rights reserved.PII: S0003 -2697 (02 )00010-6

at 412 nm, due to the consumption of NTB by allicin andthe formation of S-allylmercapto-NTB. In this paper asimilar approach was used with another, thiol-containingchromophore, 4-mercaptopyridine (4-MP; kmax 324nm), a commercially available compound, which changesits absorbance spectrum upon reaction with allicin. Thereaction product is a mixed disulde, 4-allylmercapto-thiopyridine, characterized by a shift in the optical ab-sorbance compared to 4-MP. In this assay wemonitor thedecrease in the optical absorbance of 4-MP at 324 nm.This method was also applied to monitor the kinetics ofalliinase activity by measuring the formation of allicin.Alternatively, alliin could also be quantied bymeasuringthe amount of allicin formed in the presence of an excessof alliinase.

Materials and methods

L-Cysteine, hydroxylamine, and DTNB were pur-chased from Sigma Chemical (St. Louis, MO). 4-Mer-captopyridine and allyl bromide were purchased fromAldrich Chemical. S-Allylcysteine and alliin (+) weresynthesized as described [16]. 2-Nitro 5-thiobenzoic acidwas prepared according to [15]. Alliinase was puried byusing ConA-agarose chromatography as describedpreviously [17]. The preparation contained traces ofgarlic-mannose-specic lectin. The specic activity ofthe various preparations was 170220 units/mg protein.Allicin was prepared by applying synthetic alliin onto animmobilized alliinase column [18]. The purity andconcentration of allicin were determined by HPLC (seebelow). NTB determination of allicin content and alli-inase activity was described previously [15]. Kineticconstants for alliinase reaction using 4-MP were deter-mined in the same manner as that described for NTB[15]. NTB concentration was calculated by using eM14; 150M1 cm1 at 412 nm according to Riddles et al.(1979) [19]. The above eM was also used to determinefree SH content with DTNB. Allicin concentration wascalculated by using the NTB assay with eM28; 300M1 cm1 at 412 nm [15]. 4-MP assay was cal-culated using eM 19; 800M

1 cm1 at 324 nm [20].

Quantitative determination of allicin and alliin by HPLC

Quantitative assessment of alliin and allicin wasobtained by using an LKB HPLC system equipped

with a chromatography station for windows (Data-Apex, The Czech Republic). The separation wasachieved on a LiChrosorb RP-18 (7 lm) column byusing an isocratic system:methanol (60%) in watercontaining 0.05% triuoroacetic acid. The observedretention time, at room temperature with a ow rateof 0.55ml/min, is 4.6min for alliin and 8.1min forallicin.

Quantitative determination of allicin for calibration curves

Allicin is not stable in solutions. Therefore, in orderto obtain pure allicin as a standard for the quantitativeassay we use a prepurication step on HPLC as de-scribed above. Allicin (13 lg) was loaded onto a RP-18HPLC column, and the peak was collected. Its concen-tration was determined by the NTB assay [15]. Thisprovided us with standards for the evaluation of HPLC-separated allicin concentrations and for the calibrationcurve of allicin in the 4-MP method.

Assay of allicin concentration with 4-MP

Allicin (0.13 lg) was incubated at room tempera-ture in 1.0ml of 4-MP (104M) in 50mM Na-phos-phate, 2mM EDTA, pH 7.2 (buer A). The decreasein optical density (OD) at 324 nm was determinedafter 30- or 60-min incubation periods, at roomtemperature. A calibration curve for allicin was rununder the same conditions. eM 39; 600M

1 cm1 at324 nm was used for the calculation of allicin con-centration.

Determination of the rate constant of the reaction betweenallicin and 4-MP

The rate constant for the reaction of allicin (0.10.5mM) with 4-MP (0.1mM) was determined in buerA at room temperature. The initial rate of the disap-pearance of 4-MP was monitored spectrophotometri-cally. The following equation was used to calculate thebimolecular rate constant:

d4-MP=dt kallicin4-MP

v d4-MP=dt

k vallicin14-MP1

Scheme 1.

T. Miron et al. / Analytical Biochemistry 307 (2002) 7683 77

Synthesis of 4-allylmercaptothiopyridine

Allicin, 0.2ml (70mg/ml in ethanol), was added to 4-MP, 4.0ml (20mg dissolved in 2ml ethanol, and furtherdiluted with water to 4ml). The reaction mixture wasstirred for 30min at room temperature. The product wasextracted with diethylether and evaporated to dryness.The oily product was identied by mass specrometry as4-allylmercaptothiopyridine.

Determination of alliinase activity by using 4-MP

One enzyme unit is dened as the amount of enzymeconverting alliin into pyruvic acid at a rate of 1 lmol/min.The present assay of alliinase activity is based on the as-sessment of the production rate of allicin, using 4-MP.The molar consumption of 4-MP in its reaction with al-licin is equivalent to the production of pyruvic acid. Thereaction mixture (1.0ml) contained 4-MP (104M) in50mMNa-phosphate, 2mM EDTA, 0.02mM pyridoxal50-phosphate, pH 7.2 (buer B), and alliin (10mM). En-zymatic activity started upon addition of an aliquot ofalliinase (2:510 103 activity units) at 23 C.The initialrate of the decrease in absorbance at 324 nm was mea-sured and the enzymatic activity was calculated usinge324 19; 800M1 cm1. Endogenous allicin had to bedepleted from crude extracts by a pre-incubation with 4-MP. Therefore, crude extracts of alliinase were pretreatedwith 103 M 4-MP for 30min in an ice bath and were di-luted prior to the assay of enzyme activity.

Assay of alliin concentration with NTB and 4-MP

Alliin concentration (synthetic or in garlic prepara-tions) was assayed with either NTB or 4-MP in the pres-ence of alliinase.NTB (1:2 104M)or 4-MP (104M) inbuer A, containing alliinase (12 units/ml), was added totubes containing alliin (1mg/ml, 010 ll). The reaction(1.0ml) proceeded at room temperature and incubationperiods were 1530min or 3060min for NTB and 4-MP,respectively. The amount of alliin, which is in directproportion to the decrease in the absorbance at either412 nm (NTB) or 324 nm (4-MP), after subtraction fromthe absorbance without alliin, was calculated by using eM

14; 150M1 cm1 at 412 nm (NTB) or eM 19; 800M1

cm1 at 324 nm (4-MP). A calibration of allicin was runbecause the reaction is not complete under these condi-tions at the above-noted times.

Results and discussion

The reaction of allicin with 4-MP and analysis of itsproduct

The reaction between 4-MP and allicin was analyzedand its product was characterized. Allicin (0.2ml, 75mg/ml ethanol) was added to a solution of 4-MP (20mg in4ml of 50% ethanol) in buer A. The decrease in theabsorbance at 324 nm was monitored at a 1:400 dilutionand the reaction was stopped after 30min at roomtemperature. The product was extracted in ether and theorganic phase was evaporated to dryness, yielding anoily fraction. Upon reacting this product with cysteineor 2-ME, the typical absorption spectra of 4-MP reap-peared with a maximum at 324 nm, characteristic of 4-MP [20]. These data indicated that the reaction productof 4-MP with allicin might be a mixed disulde. HPLCseparation, under conditions that were used for the de-tection of allicin and alliin, showed that the retentiontime of the product on HPLC chromatogram could notbe discriminated from that of allicin.

Mass spectrometry data indicated that the reactionproduct of the interaction of 4-MP with allicin is 4-al-lylmercaptothiopyridine (Scheme 2). The decrease of 4-MP concentration and the appearance of 4-allylmerca-ptothiopyridine resulted from the reaction of 4-MP withallicin, as described in Scheme 2.

Determination of allicin content in solution

A static method for the assay of low allicin concen-trations. The kinetic behavior of the reaction betweenallicin and 4-MP was characterized. Dierent concen-trations of allicin (2 1062 105 M; 0.33 lg/ml)were added to 1ml 4-MP (1:0 104M) in buer A andthe decrease in the absorbance at 324 nm was monitoredat various time intervals up to 7 h (Fig. 1). The reaction

Scheme 2.

78 T. Miron et al. / Analytical Biochemistry 307 (2002) 7683

of allicin with 4-MP at a ratio of 1:20 or higher iscompleted within 90min (Figs. 1 and 2), and there is alinear relationship between the amounts of allicin andthe decrease in the optical density of 4-MP at 324 nm at0.21.5 lg/ml (Fig. 2, inset). Therefore at low concen-trations of allicin, the decrease in the absorbance at324 nm after 30min can be used directly to assay theamount of allicin based on e324 39; 600M1 cm1.

The molar concentration of allicin was calculatedaccording the equation:

allicin DA324 dilution=39; 600

DA324 A3244-MP without allicin A3244-MP with allicin:

The range of concentration for reliable allicin determi-nation by this assay is between 106 and 105M (0.11.5 lg).

A kinetic method for the assay of high allicin concen-trations. In the static method an excess of 4-MP is used;however, due to the high molar extinction value of 4-MPit cannot be used in the assay at high allicin concen-trations. In order to assay allicin at high concentrations(0.0120.75mM; 2120 lg/ml), a kinetic approach wasused [21]. The initial rate of the decrease of 4-MP atdierent allicin concentrations was followed at roomtemperature. From these data the bimolecular rateconstant was calculated (see Table 1) The observed rateconstant (k) at 24 C, pH 7.2, is 82M1 min1.

The amount of allicin in a sample can be determinedfrom the initial rate of the reaction of 4-MP(1:0 104M) with allicin by using the value of k Fig. 3:

v k4-MPallicin

v DA324 min 1=3:96 104M1

4-MP 104M

k 82M1 min 1:allicin v=k4-MP:

Fig. 1. Time course of the reaction between allicin and 4-MP. The

reaction was followed by monitoring the decrease at 324 nm after

adding allicin to 4-MP (1:2 104M) in buer A, at the followingconcentrations: 0.36lg/ml (), 0.72lg/ml (), 1.80lg/ml (), 3.60lg/ml (j). The curves were obtained by interpolation.

Fig. 2. The decrease in the absorbance of 4-MP at 324 nm, at various

allicin concentrations. Allicin (70.7lg/ml, 0100ll) was added to 4-MP (1:1 104M) in buer A (nal volume 1.0ml), and the decreasein the absorbance at 324 nm was recorded after 30 (), 60 (), and 100(x) min at room temperature. Inset: Calibration curve for allicin at low

concentrations with 4-MP. Reaction was done in 1.0ml nal volume.

Various concentration of allicin (0.11.5lg in a nal volume of 20 ll)were added to 4-MP (1:1 104M) in buer A (0.98ml) and vortexedfor 2 s. The OD at 324 nm was measured after 30min. The curve-tting

equation; Y 1:40 103 0:17X ; correlation coecient; R2 0:997.

Table 1

Initial rate of the decrease of 4-MP at dierent allicin concentrations

Allicin

(103M)Initial rate

DA324(min1)

Initial rate

(107M min1)

K (RT, pH 7.2)

(M1 min1)

0.214 0.065 16.414 76.7

0.285 0.090 22.727 79.7

0.356 0.122 30.808 86.5

0.428 0.142 35.859 83.8

0.499 0.166 41.919 84.0

0.570 0.190 47.980 84.2

0.712 0.226 57.071 80.2

4-MP concentration is 104M.

T. Miron et al. / Analytical Biochemistry 307 (2002) 7683 79

allicin DA324 min 1=3:96 104M1

82M1 min 1 104M

allicinmolar concentration 3:08 103 M DA324allicin mg=ml 0:499 DA324:

Application of NTB and 4-MP for the assay of allicin byHPLC analysis

HPLC analysis is the most common technique for thedetermination of allicin and alliin content in dierentgarlic preparations, due to their good separation pat-tern. The main drawback of this quantitative estimationis the lack of standards for calibration. Both alliin andallicin are compounds devoid of specic spectral prop-erties and therefore calibration is usually done bygravimetric analysis of the pure compounds. By usingassays based on chromogenic thiols (NTB and 4-MPin our case), which are reliably characterized by theextinction coecients e412 14150 (NTB) or e32419; 800M1 cm1 (4-MP), the calibration of standardsbecomes simple and easy. The sensitivity of these assays(0.110 lg) is adequate for the amounts loaded ontoanalytical HPLC columns.

Determination of alliinase activity with 4-MP

Most of the assays used to determine alliinase activityare based on monitoring the production of pyruvic acid.

The assay described here is based on allicin production(Scheme 1), and its reactivity with 4-MP (Scheme 2).Using a partially puried alliinase and comparing the4-MP enzymatic assay with the one previously describedwith NTB [15] showed no signicant dierences betweenKM and Vmax values (Table 2).

Since the reaction rate of allicin with 4-MP is higherthan the Vmax value of the alliinase enzymatic reaction(Figs. 4A and B), one may conclude that the enzymaticproduction of allicin is the rate-limiting step in the 4-MPmethod. The close values of the kinetic constants of thealliinase reaction, obtained by the two independentmethods, strongly support the reliability of the 4-MPassay. Both methods gave similar enzyme activity unitsper gram of garlic: 24:3 2:19 (4-MP method) and25:5 2:3 (NTB method) upon assessing alliinase in acrude garlic extract.

Inhibition of alliinase activity assayed with 4-MP

We wished to show that inhibition of alliinase can bemonitored through inhibition of allicin production, byusing the 4-MP method. S-allylcysteine (a competitiveinhibitor of alliinase) and hydroxylamine (an irreversibleinhibitor of the enzyme) were examined. Using dierentconcentrations of alliin in the presence of S-allylcysteineand performing an analysis according to the Linewe-averBurk method, we conrmed that S-allylcysteine isa competitive inhibitior of alliinase (Ki 103M, Fig.5A). Hydroxylamine is a common inhibitor of pyridoxal50-phosphate-dependent enzymes. Since alliinase is apyridoxal 50-phosphate-dependent enzyme, we deter-mined the inhibitory eect of hydroxylamine on theenzyme activity. As can be seen from Fig. 5B, a 10-minpreincubation of hydroxylamine with alliinase led to aconcentration-dependent inhibition of the enzymaticactivity (I50 0:51 103M).

Determination of alliin by using alliinase with either4-MP or NTB

Alliin (S-allylcysteine sulfoxide) is a nonproteinamino acid abundant in garlic cloves and dry garlicpowder. It is the natural substrate of alliinase (Scheme6). Alliin has no specic properties which enable its di-rect assay. Therefore its content in the pure form is

Fig. 3. Calibration curve for allicin at high concentrations with 4-MP

(kinetic method). The initial reaction rate between 4-MP (0.1mM) and

allicin (0.20.7mM, 20120lg/ml) was recorded at 324 nm. Reactionwas done at room temperature in phosphate buer, pH 7.2. The curve-

tting equation, Y 2:73 105 2:02 103X ; correlation coe-cient, R2 0:993. For calculations, the bi-molecular rate constant of82M1 min1 was used.

Table 2

Kinetic parameters of alliinase activity according to the Lineweaver

Burk methoda

Assay KM0 103(M)

Vmax 105M min1

NTB 1:90 0:80 1:27 0:204-MP 1:93 1:10 1:55 0:10aMean SD is given, n 3.

80 T. Miron et al. / Analytical Biochemistry 307 (2002) 7683

commonly analyzed by HPLC according to its retentiontime and absorption at 210 nm. Here we describe thequantitation of alliin in natural products and garlicpreparations by using alliinase as a reagent in thechromogenic thiol assay. Pure alliin, or alliin in garlicpreparation was assayed with the chromogenic thiols,4-MP or NTB. The assay was performed in 1.0-ml nalvolume. A solution of chromogenic thiol (11:3104M) containing alliinase (1 unit/ml; 0.98ml) wasadded to various samples (020 ll) of alliin (about 1mg/ml). The absorbance was determined after 30 or 90minat room temperature at 412 or 324 nm, according to thereagent used.

The molar concentration of alliin was calculated fromthe dierence in absorbance according to the followingequations:

alliin OD without alliin OD with alliin dilution eM1:

In the presence of 4-MP:

alliin DA324 dilution=19; 800:

Fig. 4. Kinetic analysis of alliinase reaction monitored by 4-MP.

Conditions of the reaction are described under Materials

and methods. Alliinase was used at 2:5 103 units/ml, at 23 C.(A) MichaelisMenten plot; (B) LineweaverBurk plot. The curve-

tting equation; Y 0:662 0:434X ; correlation coecient,R2 0:972.

Fig. 5. Inhibition of alliinase monitored by the 4-MP method. (A).

Partially puried alliinase (5 103 units/ml) was assayed eitherwithout inhibitor () or in the presence of S-allylcysteine[1:0 103M] (). Alliin (0:153:0 103M) was added to start theenzymatic reaction. The decrease in absorbance at 324 nm was re-

corded. The assay was performed at 23 C in buer B, at a 4-MPconcentration of 1:2 104M. The curve-tting equation without in-hibitor; Y 34:34 21:51X ; correlation coecient, R2 0:999. Thecurve-tting equation with inhibitor, Y 34:28 41:91X ; correlationcoecient, R2 0:997. (B). Alliinase was preincubated for 10min withhydroxylamine (010 103 M). The residual activity was measuredupon adding alliin to the reaction mixture. The curve tting equation,

Y 96:1188:24X ; correlation coecient, R2 0:987.

T. Miron et al. / Analytical Biochemistry 307 (2002) 7683 81

In the presence of NTB:

alliin DA412 dilution=14; 150:Using these assays we found that the amount of alliinin local garlic is 3:4 1:0mg/g in nonpeeled garlic(1317mg alliin/garlic clove).

Conclusions

During recent years a number of garlic-containingfood supplements have appeared on the market. Themajority of the manufacturers of these products claimthat allicin is their most active ingredient. Therefore it isnecessary to have simple and reliable methods for theassay of allicin. In a previous study [15], we developed asingle-step, direct spectrophotometric method for thedetermination of allicin and other thiosulnates, using anoncommercial NTB, which is the product of the thiol-disulde exchange reaction of the Ellman reagent withfree SH groups. In this study we use 4-MP, anotherchromogenic thiol similar to NTB, which is commer-cially available. This compound is as stable and inert asNTB and does not interact with oxidized glutathioneand cysteine. Being an activated disulde, allicin reactsvery rapidly with 4-MP (Scheme 2), as it does with otherthiols.

Due to its higher extinction coecient (e324 19; 600M1 cm1), 4-MP is more sensitive than NTB (e412 14; 150M1 cm1). Moreover, it can be used in asignicantly broader (3.48.1) pH range [20]. Theconcentration range of these assays is 106105M ofallicin concentrations. The kinetic method for allicindetermination is not dependent on allicin concentra-tion, but depends on temperature and pH. Therefore,determination of the rate constant of the 4-MP reac-tion with allicin under certain pH and temperatureconditions is a prerequisite. The advantage of thestatic assay is in its simplicity. On the other hand,the static assay of allicin must be carried out in thepresence of an excess of the reagent, limiting the rangethat can be measured. We suggest that this easy andconvenient determination of allicin with chromogenicthiols could serve as a potential tool for quality con-trol analyses to facilitate garlic product description,including the contents of alliin and allicin as well asalliinase activity.

Acknowledgments

This research was supported by a grant from theLevine Center and from Yeda Co. at the WeizmannInstitute of Science.

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