Indian Journal of Chemical Technology Vol. 8, May 2001, pp. 195-199

Stability of allicin in garlic-A kinetic study

Rashmi Mishra", S K Upadhyay"* & P N Maheshwarib

"Department of Chemistry

bDepartment of Biochemi cal Engineering & Food Technology, H B Technological Institute, Kanpur 208 002, India

Received 30 Jun e 1999: accepted 22 No vember 2000

In order to study the stability of allicin , an active constituent of garlic extract, the rate of formation of allicin from allicin and that of decompos iti on of allicin to volatile oil/sulphides has been determined by chloramine- T method. The effect of aceti c acid (5-30%), ethanol (5-30%). buffers (pH 4.0-6.0) and temperature (30-50°C) on the rate of formation of allicin and on the rate of its further decomposition to volatile oil has been studied. It has been observed that the decomposition of allicin dec reases with an increase in the strength of acetic acid and increases with an increase in the pH of macerating medium. The increasing strength of ethanol in macerating medium had a little effect of the rate of decompositi on, which was found to be maximum at an incubation temperature of 35°C.

0

Allicin , [CH2=CH-CH2-~ -S-CH2-CH=CH2], a thiosulphinate ester, is the major and pharmaceutically active constituent of garlic extract and responsible for fresh garlic flavour'. It results from enzymic cleavage of alliin, [CH2=CH-CH2-

0 II S - CH2-CH-COOH], S-allyl-L-cysteine sulph-

1

NH, oxide, as follo~s2 , 2RSOCH2CH(Nl-h)COOH+H20 ___:<_AI_li i_na-'-sel~

(Alliin) 2NH3+ 2CH3COCOOH + RSSOR

(Pyruvic acid) (Allicin) where R represents allyl group (CH2 = CH-CH2-)

Allicin in unstable and further undergoes non­enzymatic decomposition to a complex mixture of mono-, di- , tri- and polysulphides as follows,

RSSR + RSS0 2 R

2RSSOR-cDisulphide Thiosulfonate RSSR + RSR + so 2

Disulphide Monosulphide

2RSSR ~ RSSSR + RSR Disulphide Trisulphide Monosulphide

The amount of allicin in garlic has been quantitated

*For correspondence.

by estimating volatile sulphur compounds or pyruvic acid3 using gravimetric, spectrophotometric~ and chromatographic5

·6 techniques .

It has been observed that allicin possesses low stability and high reactivity associated with weak S-S bond (bond energy 46 KCal mol- 1 or less) and thus have facile pathways available for decomposition. As a result, studies on the stability of alliin and allicin have diverted the attention of many chemists. The rapid loss of activity of allicin on heating and in presence of alkali had been reported earlier in the literature.

. The rate of conversion of alliin to allicin and effect of temperature, solvent and pH on the rate of th is enzymic conversion has been studied and reported in previous communication 7. Further studies on the decomposition of allicin to volatile oil/sulphides as a function of pH, solvent and temperature could be helpful in understanding the chemical nature and stability of allicin . Therefore, the rate of formation of allicin from alliin and that of further decomposition of allicin to volatile oi l/su lphides has been studied using chloramine-T method8 and the results are reported in the present communication.

Experimental Procedure Materials

The chemicals, chloramine-T, sodium thiosul­phate, acetic acid, rectified spirit (95% ethanol) etc. used were of CDH, Loba, Ranbaxy, Reidel and of AR grade. All the solutions were prepared in doubly distilled water. Aqueous stock solution of chlor-

196 INDIAN J. CHEM. TECHNOL.. MAY 2001

Table !-Values of % volatile oil in garlic samples at various temperature maintained during enzymic re.1ction, solvent-distilled water, steam di stilled for 90 min

Incubation time (min )

30 45 60 75 Value of Kobsx !0-3 (min-1

)

(obtained from slope of plot between % volatile oil versus incubation time (at fi xed steam distillation time)

0.090 0. 116 0.145 0.173 1.853

% volatile oi l in garlic sample

0.107 0.148 0.1 89 0.230 2.733

0.079 0.112 0.142 0.165 1.920

0.123 0.052 0. 178 0.205 1.793

Table 2-Values of % volatile o il in the garli c sample prepared in distilled water, incubated at fixed period of 75 min at different temperature

Steam di stillation time (min)

10 20 30 40 50 60 70

Values of K obsX I 03/ min

obtained from slope of straight line plotted between o/o volati le oil and steam di stillati on time.

30°C

0.034 0.047 0.064 0.077 0.097 0.116 0.139 1.735

amine-T (0.2N) was prepared*. Potassium iodide (20% wlv), sodium hydroxide (25 % wlv) and freshly prepared starch solution were used. Aqueous solution of sodium thiosulphate (hypo) was prepared in double di still ed water and its strength was checked i odometri call y.

The buffer solutions for experimental work were prepared by mixing solutions9 of 0 .1 M potassium hydrogen phthalate and 0.1 M HCl (pH 4.0) ; 0.1 M potassium hydrogen phthalate and O.l M NaOH (pH 4.5, 5.0 and 5.5) and 0.1 M NaOH and 0.1 M potassium dihydrogen phosphate (pH 6.0).

Method The rate of decomposition of allicin to

sulphides/volatil e oil has been studied using chloramine-T method 8

. The detail s of the method are:

*Strength was checked by iodomctric method or wi th a standard arsenite solution.

% volatile oi l in Garlic sample 35°C 40°C 50°C

0.046 0.044 0.036 0.066 0.053 0.048 0.090 0.082 0.064 0.1 22 0. 122 0.073 0.136 0.134 0.083 0.156 0. 142 0.094 0.178 0. 167 O. lOl 2.228 2. 103 1.092

Depending upon the experimental set, a sample of 10 g de-skinned garl ic cloves was macerated with 70 mL distilled water or 5-30% acetic acid (vlv) or 5-30% rectified spirit (vlv) or buffer (pH , 4 .0-6.0) at about l0°C in Bajaj super mixer operated at medium speed. The sl urry so formed was transferred to 500 mL three-necked stoppered d istill ation flask contain ing few glass beads and incubated at specified fixed temperature in range 30-50°C for specified periods of 30-75 min for enzymic cleavage of alliin to allicin. Thereafter, the flask was connected to di stillation assembly, whose tip at the receiving end was kept dipped in a solution containing 20 mL of 0.2 N chloramine-T, 35 mL of ION sulphuric acid and 15 mL di stilled water taken in an Erlenmeyer flask . Then, the samples were steam disti lled for 90 min to collect distillate in the receiving flask.

In another set, incubation time was kept 75 min and di stillation time was vari ed from 10-70 min for determining the rate of decomposition of allicin. Excess chloramine-T in the receiv ing flask left

MISHRA eta/. : STABILITY OF ALLICIN IN GARLIC 197

0.16 ,-----

0 -14 0 10"'4 acetic acid

l:J. 20% acetic acid 0.12

0.10

0 .08

0 .06

0.04

0 .02

o.ooL--- - --'----- -'--- - ·--'-------::' 15 30 45 60 75

Incubation time (min) at JSOC

Fig. !- Plots of % volatile oil versus incubation time for garlic extract prepared in various strengths of acetic acid, for a fixed period of steam di stillation of 90 min .

0.25,---- ------------- ---, 0 5 % rectil led spiri t D 10 "'- recl ified a-piri l

0.20 6 20 % rectif ied &plril e 30 % rec:lllled aplrll

0 ~ 0.15

~ I!. 0.10

0.05

0.00 !-;-15 -------+-,oc---------:,1:-, -------+. .. ,.--------:!,.

Incubation time (min) at JSOC

Fig. 2- Plots of % volatile oil versus incubation time for gralic extract prepared in various strength of rectified spirit, for a fixed period of steam di stillation of 90 min.

unutilized after oxidation of volatile sulphur compounds was determined by adding lO mL of 20% (wl v) KI solution and titrating liberated iodine against hypo using starch as an indicator. The difference in the titre value between the blank and the sample corresponded to consumption of chloramine-T. The volatile oil at different time interval was calculated by using the equation,

V x N x iOO Volatile oil % = --'-1

---­

W x F

where, V, = Volume of hypo being titre difference between

blank and sample at any time t, N = Normality of hypo. W = Weight of sample, F = Factor of 90 being mL of IN chloramine-T for oxidation of 1 g volatile oil of garlic.

Results and Discussion The plots % volatile oil (obtained in fixed steam

distillation time of 90 min for conversion of allicin to

Table 3- The values of rate constants for the formation of allicin from alliin (Kobs) and for decomposition of allicin (Kobs) at 35°C in garlic macerates obtained by macerating de-skinned garlic cloves with different solvents

Solvent

Distilled water 5% acetic acid I 0% acetic acid 20% acetic acid 30% acetic acid 5% rectified spirit 10% rectified spirit 20% rectified spirit 30% rectified spirit Buffer of pH =4.0

4.5 5.0 5.5 6.0

Kobs X 103

(min-1) from

slope of plot of % volati le oil

versus incubation time

2.733 1.273 1.046 0.593 0.346 2.166 1.646 1.206 1.000 0.613 1.113 1.500 2.100 2.386

KobsXI03

(min.1) from slope

of plot of % volatile oil versus steam distillation time

2.228 1.471 1.175 1.028 0.717 2.182 2.117 2.046 1.900 0.566 0.700 1. 196 1.375 2.103

sulphides/volatile oil) versus incubation time were linear and, therefore, the rate constants for the formation of allicin from alliin were obtained from the slopes of straight lines plotted between % volatile oil versus incubation time. Likewise, the plots of % volatile oil (for fixed incubation period of 75 min for conversion of alliin to allicin) versus stream distillation time were also linear and, therefore, the rate constants for the decomposition of alliicin to sulphides/volatile oil have been evaluated from the slopes of straight lines plotted between % volatile oil versus steam distillation time.

The effect of incubation temperature, acetic acid, rectified spirit/ethanol and pH has been studied on the rate of formation of allicin from alliin and rate of decomposition of allicin to sulphides/volatile oil.

The amounts of volatile oil (%) obtained from allicin formed under various incubation temperatures in incubation periods of 30-75 min by steam distillation for a fixed period of 90 min are given in Table 1. Likewise, the amounts of sulphides/volatile oil (%) from allicin under different incubation temperatures in fixed incubation period of 75 min but varying steam distillation periods of 10-70 min were determined and are reported in Table 2. The corres­ponding rate constants (Kobs) obtained from the plots of (% volatile oil) versus incubation time (in fixed steam distillation time of 90 min) and (% volatile oil)

198 INDIAN J . CHEM. TECHNOL., MAY 2001

025

0 Buffets of pH 4.0

0.20

0 0.15 . a g

"" 0.10

0.05

0.00 15 30 45 60 7S

Incubation time tmin) at JS°C

Fig. 3-Pl ots o f % volatile oil versus incubatio n time for garlic ex tract prepared in buffers o f different pH, for a fixed period o f steam di stillation of 90 min .

0.16

0.14 0 S% aeetie • cld

0.12 0 10 % Bce1ic..cld

0 0.10

~ g 0.08

•• 006

0.04

0.02

000 0

Steam distillation time (min)

Fi g. 4-Piots o f % volatil e oil versus steam di stillation time for garlic extract prepared in various strength o f acetic acid, incubation at 35°C for 75 min .

versus steam distillation time (for fixed incubation period of 75 min) have been obtained and also reported in last column of Tables 1 and 2, respectively.

Alliin is enzymatically converted into allicin, which on steam distillation decomposes to sulphides i.e. volatile oil. Therefore, the rate constant for formation of volatile oil obtained from the plots of (% volati le oil) versus incubation time corresponds to the rate of formation of allicin from alliin , while the rate constant for the formation of volatile oil obtained from the plots of(% volatile oil) versus steam distillation time corresponds to the decomposition of allicin to sulphides/volatile garlic oil.

It is apparent from Table 1 that the highest rate constant (2.733 x 10-3 min- 1

) for conversion of alliin to allicin in garlic macerates prepared in distilled water in this study has been obtained at 35°C. This rate constant shall serve as a reference point for assessment of effect of acetic acid, rectified spirit, buffers of various pH used as macerating media. Similarly, the highest rate constant (2.228 x 10-3

0. 18

0.16

0.14

0 .12

=& . 0.10 . g 0.08

"" 0.06

0.04

0.02

0.00 0 10 20 30 40 " 60 70

Steam distillation l ime (min)

Fig . 5~Plots o f % volatile oil versus steam di stillation time for garlic extract prepared in various strengths of rectified spirit , incubated at 35°C for 75 min.

0.18 0 Buflers ofpHh•

0.16 0 Buf11t1 of pH 4.5

0.14 6 Butt. ,.. ol pH 5.0

0.12 8 Buffer• ol pH 5.5

~ X Butters of pH 6.0

.! 0.10

J 0.08

" 0.06

O. OA

0.02

0.00 0 10 20 30 " 60 70

Steam di5!lllalion time (min)

Fi g. 6- Plots o f % vo lati le oil versus steam d istillation time for garlic ext ract prepared in buffers o f differe nt pH incubated a t 35°C for 75 min.

min- 1) for conversion of allicin to sulphides/volatile

oil in garlic macerates prepared in distilled water in this study has also been obtained at 35°C which shall serve as reference point for assessment of the effect of acetic acid/rectified spirit/buffers of various pH.

It can be safely deduced that a proportionately higher rate constant for conversion of allicin to su lphides/volatile oil than that for conversion of alliin to allicin at incubation temperature of 40°C (refer Tables 2 and 1) is indicative of some decomposition of allicin at this temperature. Thus decomposition accordingly should have increased at incubation temperature of 50°C. However, the K obs IS

proportionately low suggesting that some loss of volatile oil at this incubation temperature plausibly occurred during incubation period of 75 min used in the experiment. It is also apparent from the results that optimum temperature for enzymic conversion of all iin to allicin is about 35°C.

MISHRA et al.: STABILITY OF ALLICIN IN GARLIC 199

In order to observe the effect of acetic acid/rectified spirit/pH, the amounts of allicin formed as a function of incubation time in garlic macerates obtained by macerating de-skinned garlic cloves with 5-30% acetic acid or 5-30% rectified spirit (ethanol) or buffers (pH-4.0 to 6.0) and decomposition of thus formed allicin to sulphides/volatile oil as a function of steam distillation time have been determined. The amount of % volatile oil have been obtained from allicin formed (in presence of various strength of acetic acid or rectified spirit or buffers of various pH) in incubation periods of 30-75 min and for a fixed steam distillation period of 90 min . The results are represented graphically in Figs 1 to 3 for effect of acetic acid, rectified spirit and buffers of various pH values, respectively.

Likewise, the amounts of % volatile oil have also been obtained from allicin formed (in presence of various strength of acetic acid, rectified spirit or buffers of various pH) in fixed incubation time of 75 min and varying steam distillation periods of 10-70 min. The results have been shown in Figs 4 to 6, respectively. The plot of % volatile oil (obtained in fixed steam distillation time of 90 min for conversion of allicin to sulphide/volatile oil) versus incubation time in each case was linear (Figs 1 to 3) and, therefore, the rate constants (Kabs) for the formation of allicin from alliin have been evaluated from the slopes of these plots. Similarly the rate constants (Kabs) for the decomposition of allicin to sulphides/volatile oil have been calculated from the slopes of the linear plots of % volatile oil versus steam distillation time (for a fixed incubation periods of 75 min, for conversion of alliin to allicin) i.e. Figs 4 to 6. The rate constants for the formation of allicin from alliin (Kabs) and that for the decomposition of allicin to volatile oil/sulphides (Kabs) have been summarised in Table 3.

It is apparent from the results that increasing the strength of acetic acid or rectified spirit in macerating medium decreased the rate constant for the formation of allicin from alliin . A decrease in the rate of decomposition of allicin to sulphides/volatile oil has also been observed with an increase in the strength of acetic acid in macerating medium. However, a little effect in the observed rate constant for decomposition of allicin to sulphides/volatile oil with an increase in the strength of rectified spirit in macerating medium has been observed. On increasing pH of macerating medium, the rate constants for formation of allicin from alliin as well as that for the decomposition of allicin increased. These results are in agreement with

earlier reports 7"10 that more allicin was formed at pH

6.5 than at pH 5.5, and that it decomposed even faster at pH 6.5 than at pH 5.5 with heat. Thus, the results of this study may be summarised as: (i) The rate of decomposition of allicin has been

found to be maximum at incubation temperature of 35°C. This is because, at 35°C the enzymatic conversion of alliin to allicin in maximum.

(ii) There are some indications that some decomposition of allicin occurs at incubation temperature exceeding 35°C.

(iii) The rate of decomposition of allicin decreases with an increase in the strength of acetic acid in macerating medium.

(iv) The increasing strength of rectified spirit/ethanol (5-30%) in macerating medium had little effect on observed rate constant for decomposition of allicin .

(v) An increase in the pH (4.0-6.0) in macerating medium resulted in an increase in observed rate constant for decomposition of allicin.

The experimental results obtained with chloramine-T method for the formation of allicin from alliin i.e. maximum rate of formation of allicin at incubation temperature of 35°C, retarding effect of acetic acid (5 to 30%) or rectified spirit (5 - 30%) on the rate of formation of allicin and an increase in the rate of formation of allicin with an increase in the pH (4.0-6.0) of the macerating medium are in agreement with our previous results reported during spectrophotometric studies7 on the conversion of allin to allicin in garlic.

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