Indian Journal of Chemical Technology Vol. 8, March 2001, pp. 107-111

Conversion of alliin to allicin in garlic -A kinetic study

Rashmi Mishraa , S K Upadhyay*a & P N Maheshwarib

aDepartment of Chemistry,

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

Received 14 May 1999; accepted 18 September 2000

A method for estimation of rate of conversion of alliin to allicin based on quantitation of pyruvic acid has been developed using sodium pyruvate and the kinetics of conversion of alliin to allicin have been studied. The effect of acetic acid (5-30% strength), ethanol (5-30% strength), buffers (pH 4.0-6.0) and temperature (30-50°C) on the rate of conversion of alli in to allicin is reported which elucidate that the rate of conversion decreases by increasi ng concentration of acetic acid or ethanol (rectified spirit) and increases with an increase in pH towards neutrality, the optimum temperature for thi s conversion being about 35°C.

Garlic is valued for its sulphur containing compounds. It has been widely used as flavouring agent. It finds pharmacological uses 1-6 as potent antibacterial, antimicrobia l, antifungal and antiplatelet agent. It reduces levels of serum triglycerides and cholesterol, increases insulin level, treats leprosy, cures certain breathing problems, lowers ocular pressure and reduces gastric cancer. Ayurvedic and Unani systems of medicine consider it as a stimulant that aids in digestion and absorption of food . As a result, it finds its importance in serving not only as condiment but also as a popular remedy for various ailments and physiological disorders .

0

Allicin [CH2= CH-CH2- ~ -S-CH2-CH = CH2], a thiosulfinate ester, is sulphenyl derivative formed by enzymatic cleavage of sulfoxide called alliin,

0

[CH2=CH-CH2- ~-CH2-CH=COOH], found m

I NH2

garlic as its major flavour precursor7. Alliin, S-allyi­

L-cysteine sulfoxide, is crystalline colourless solid. It decomposes at 164-166°C. It has a specific rotation [a] 0

21 of +62.8°C. Isolation and identification of S­allyi-L-cysteine sulfoxide, purified and quantitated by HPLC8

·9 has been made by IR, NMR and MS. Alliin is

enzymatically cleaved by alliin lyase, a phosphopyridoxal enzyme, commonly known as alliinase [EC.4.4.1.4] 10 to form diallyl thiosulfinate

*For correspondence.

(commonly called allicin), ammonia and pyruvic acid as follows 1 1,

2RSOCH2CH(NH2)COOH + H20 (Alliin)

2NH3+ 2CH3COCOOH + RSSOR (Pyruvic acid) (Allicin)

(AIIiinase)

where R represents allyl group (CH2 = CH-CH2-). The allicin resulting from cleavage of alliin by

alliinase is major and pharmaceutically active constituent of garlic extract. It is unstable and further undergoes non-enzymatic decomposition to either disulphides and thiosulphonate or disulphide, monosulphide and S02 as,

RSSR + RSS0 2R

2RSSOR -c Disulphide Thiosulphonates RSSR + RSR + S0 2 Disulphide Monosulphides

Disulphide so formed, decomposes and rearranges to trisulphide and monosulphide as,

2RSSR -t RSSSR + RSR

Finally a complex mixture of mono-,di-, tri- and poly-sulphides is formed.

The rapid loss of activity of allicin on heating and in presence of alkali had been reported long back. It is most stable in garlic juice than in extracted state. Alliin has been reported to undergo cyclization during isolation from garlic. Cycloalliin is not attacked by

108 INDIAN J. CHEM. TECHNOL., MARCH 2001

. ~ e ~ <(

,,, 0.30

025

020

0.11

0.10

o.li;

0.00

.. o 42o 4l0 .uo aso '55 460 470 qo 490 soo Wavelength

Fig. !-Plot of Absorbance versus wavelength [Sodium Pyruvate] = 10.0 X 10-5 g/L, NaOH = 15 mL, Salicylaldehyde = 0.2 mL Total volume of reaction-mix. = 50 mL kept fo r 2 h.

0.7,----------------~

Concentration X 1 o·3 (giL)

Fig. 2-Standard curve for estimation of sodium pyruvate/pyruvic acid.

alliinase. It is unstable to heat under cooking conditions. Thus, it invites special attention of chemists to stabilize alliin and allicin, which have great therapeutic importance.

Further studies on the stability of alliin and allicin and on the rate of conversion of alliin to allicin as function of pH, temperature and solvent will be helpful in understanding chemical nature and activity of alliin and allicin.

The rate of conversion of alliin to allicin has been studied spectrophotometrically by estimating pyruvic acid, as one of the product formed during enzymatic cleavage of alliin, at different time intervals. Sodium pyruvate has been used in place of pyruvic acid and accordingly, a method for quantitation of pyruvic acid has been developed and tested. The effect of pH, solvent and temperature has been studied on the rate of conversion of alliin to allicin and the results are reported in present communication.

Experimental Procedure The chemicals used were of AR grade (CDH, Loba,

Ranbaxy, Reidel, Sarabhai-M, BDH). Glacial acetic

Table !-Effect of NaOH and sal icy laldehyde on the determination of sodium pyruvate at 30°C.

Total volume of reaction-mix= 50 mL, Sodium Pyruvate= 5.0 X 10-5 giL

NaOH (25%) Salicylaldehyde* Absorbance at mL mL 455 nm

5.0 0.20 0.75 10.0 0.20 0.80 15.0 0.20 0.90 20.0 0.20 0.85 15.0 0.05 0.20 15.0 0.10 0.38 15.0 0.30 1.05 15.0 0.50 1.15 15.0 0.70 1.25

I. Density = 1.164g/mL Refractive Index = 1.573 at 20°C

Table 2-Effect of temperature on the determination of sodium pyruvate Sodium Pyruvate = 5.0 X 10-5 giL, NaOH (25%) = 15 mL and Salicylaldehyde = 0.2 mL, Total volume of reaction-mix =50 mL.

Temperature (°C)

20 30 40 50 75 100

Absorbance of 455 nm

0.80 0.90 0.85 0.70 0.56 0.50

acid (GR) and rectified spirit (95% ethanol) were used for investigating their effects on the rate of conversion of alliin to allicin.

Standard aqueous solution of AR sodium pyruvate (lxl0-3N) was prepared. Sodium hydroxide (M/5) was prepared and standardised with oxalic acid. The buffer solutions for the experimental work were prepared by mixing definite proportions of solution 12

of potassium hydrogen phthalate - hydrochloric acid (pH 4.0 buffer), potassium hydrogen phthalate­sodium hydroxide (pH 4.5, 5.0 and 5.5 buffers) and sodium hydroxide and potassium dihydrogen phosphate (pH 6.0 buffer). The pH of buffer solutions were checked with the help of pH-meter.

Determination of pyruvic acid Pyruvic acid is one of the products formed during

enzymic cleavage of alliin. Therefore, the rate of conversion of alliin to allicin has been studied spectrophotometrically by estimating pyruvic acid formed at different time intervals.

The spectrophotometric determination of pyruvic acid by salicylaldehyde method 13 has been reported in

MISHRA et al.: CONVERSION OF ALLIIN TO ALLICIN IN GARLIC 109

1.40

,,0

~ ~ 0.80 .. ~ 0.00

~ ~ 0.40 ..

0.20

0.00 0.00 0.10 0.20 0.30 0.40 0.50 0.00 0.70

Salicylaldehyde (ml)

Fig. 3-Effect of salicylaldehyde on absorbance i.e. plot of (Absorbance at 455 nm) versus [Salicylaldehyde]; other conditions same as in Fig. I.

25,---------- ---------,

20 0 ln2mlgra!lcextrou:l

6 lnJmlgr•Jicedrecl

- - -rr - - - - _4 - - - - - - - - - -

- - -.[)--- - ._[) - - - - -- - ---

~ 15 . ~ -~ 10

!

lncubalion time (min)

Fig. 4-Plots of [Pyruvic Acid] versus incubation time for garlic extract prepared in di stilled water and incubated 30°C for upto 120 min.

literature. It is reported that the complex of pyruvic acid and salicylaldehyde absorbs maximum at 456 nm. Since pyruvic acid is a costly chemical, sodium pyruvate has been used and accordingly, a method for quantitation of pyruvic acid has been developed during this work . The spectrophotometric studies were made on Spectronic-20 spectrophotometer (Milton & Roy Co., USA) .

The absorbances versus wavelengths at different concentrations of sodium pyruvate were obtained. The curves had a maxima at 455 nm independent of sodium pyruvate concentration; one of them is shown in Fig. 1. Thus, it is deduced that sodium pyruvate in presence of NaOH and salicylaldehyde absorbs maximum (i.e. Amax) at 455 nm. Since the reagent blank had a considerable light absorption at 455 nm, the readings were made against reagent blank.

A series of solutions were prepared in 50 mL volumetric flasks, cons isting 5 mL of NaOH (25% w/v) and a known quantity of sodium pyruvate. The solutions were slightly diluted and 0.2 mL salicylaldehyde was added to every reaction-mix.

Table 3-The values of rate constants (Kobs) at various initial concentrations of acetic acid.

Experimental Kobs 104 giL min- I garlic extract Acetic 10% 20% 30%

mL acid= 5%

I. 1.06 1.00 0.78 0.57 2. 1.90 1.78 1.28 1.07 3. 2.74 2.69 2.18 1.53

Table 4-The values of rate constants (Kobs) at various intitial concentration of rectified spirit/ethanol.

Experimental Kobs 104 g/Lmin- 1

garlic extract Acetic 10% 20% 30% mL acid =5 %

I. 1.98 1.86 1.39 1.06 2. 3.00 2.66 2.48 1.83 3. 3.60 3.20 3.11 2.64

Table 5-The values of rate constants (Kobs) at different initial pH of buffers .

Experimental Kobs 104 giL min- I garlic extract pH=4.0 4.5 5.0 5.5 6.0 (mL)

I. 0.79 1.25 1.27 1.72 2.02 2. 1.46 2.16 2.21 2.63 3.14 3. 2.08 2.61 2.71 3.08 3.74

After shaking the contents, 10 mL of 25% NaOH was added to each flask and volume was madeup to the mark (50 mL). The intensity of the colour of mixtures i.e. absorbances were measured after 2 h (required for maximum colour development) at 455 nm. The absorbances of the solutions were plotted against concentrations of sodium pyruvate (Fig. 2). This plot was linear and used as standard curve for determination of sodium pyruvate or pyruvic acid in sample considering that one mole of pyruvic acid corresponds to one mole of pyruvic acid.

Estimation of pyruvic acid in garlic Depending upon the experimental set, a sample of

10 g deskinned garlic cloves was macerated with either distilled water or 5-30% acetic acid (v/v) or 5.30% rectified spirit (vlv) or buffers (pH 4.0- 6.0) at about 10°C in Bajaj Super Mixer at medium speed. The slurry so formed was made to 100 mL with medium and filtered using Whatman filter paper No. 42. The filtered garlic extract taken in 1, 2 and 3 mL quantities was incubated at specified fixed temperature in range 30-50°C for definite time periods of upto 2 h. After the incubation, the reaction

110 INDIAN J. CHEM. TECHNOL., MARCH 2001

•.oo,------"""7"""~-----------,

J.SO Q l"lml .. >II<O><VO<"~~ 6 In 2ml gartlc extnw:t 1 J.OO e In 3ml g.~rlic uttacl

§ 2.50 .

~ 2.00 . . 1.50 .Q

>2 1.00

0.50

0.00

20 25 30 " •• 45 50 55

Incubation temperature (oC)

Fig. 5-Piots of (Kobs) versus incubation temperature for different aliquots of garlic extract.

35.,.------- - ----------,

30 o In I ml garlic n tr.cl

n ln2mlgarhcertrltl

~ 25

6 lnlmlgarllc ertract

e 20 . '2 ~ 15

Incubation time (min)

Fig. 6-Piots of [Pyruvic Acidl versus incubation time for garlic extract prepared in 10% acetic acid and incubated at 35°C for upto 120 min.

filtrates were diluted, then treated with salicylaldehyde (0.2 mL) and NaOH (15 mL of 25%) and made to a total volume of 50 mL with distilled water. The absorbances of reaction-mixes, so formed, were measured at 455 nm and corresponding amounts of pyruvic acid were calculated with help of standard curve (Fig. 2).

Results and Discussion The effect of variation of concentration of reagent

employed viz. sodium hydroxide, salicylaldehyde and temperature in this method has been studied against distilled water to achieve the optimum conditions for colour development and its spectroscopic measurements. The results are given in Tables 1-2. It was observed that in a total 50 mL reaction-mix, an aliquot of 15 mL of 25% NaOH was required for getting the maxium absorbance at 455 nm. It was also observed that an increasing amount of salicylaldehyde from 0.05 to 0.70 mL in reaction-mix increases its absorbance; however, the Beer's law was observed to be valid in this analytical procedure for upto about 0.3 mL of salicylaldehyde (Fig. 3).

Therefore, the pyruvic acid in the experimental sample was measured using 15 mL of 25% NaOH and 0.2 mL of salicylaldehyde throughout this wmk. The absorbance of reaction-mix was also found to be temperature dependent and maximum absorbance of reaction-mix was observed at about 30°C (Table 2) .

The effect of incubation temperature, acetic acid, rectified spirit (ethanol) and pH has been studied on the decomposition or enzymic conversion of alliin to allicin. The [Pyruvic acid] versus time plots were straight line in initial stages of the reaction (Fig. 4) and, therefore, the rate constants for the formation of pyruvic acid or decomposition of alliin (Kobs) have been evaluated from the slopes of straight line portion of these plots.

The amounts of pyruvic acid formed in garlic macerates obtained by macerating deskinned! garlic cloves with distilled water incubated at different temperatures from 30-50°C as a function of time were determined by using 1 mL, 2 mL and 3 mL filtered extracts and the rate constants (Kobs) for the formation of pyruvic acid i.e. rate of decomposition of alliin to allicin have been evaluated from the slopes of the plots of [Pyruvic acid] versus incubation time at different incubation temperatures. It was observed that the rate constant for conversion of alliin to allicin were temperature dependent. The plots of (Kobs) versus incubation temperature for different aliquots of garlic extract (Fig. 5) suggested the optimum temperature for conversion of alliin to allicin of about 35°C which is in good agreement with the reported 14

•15 optimum temperature of 37°C for this

enzymatic conversion. The rate constant observed with distilled water at 35°C incubation temperature shall serve as reference point for assessment of effect of acetic acid, retified spirit and buffer/pH as macerating medium.

In order to observe the effect of acetic acid, rectified spirit and pH on the rate of formation of pyruvic acid i.e. conversion of all iin to allicin, the amounts of pyruvic acid formed in garlic macerates, obtained by macerating deskinned garlic cloves with solutions of acetic acid of 5-30% strength or 5-30% aqueous solutions of rectified spirit/ethanol or buffers of pH 4.0 - 6.0, as function of time were determined by using 1 mL, 2 mL and 3 mL filtered extracts and corresponding rate constants (Kobs) calculated from the slopes of [Pyruvic acid] versus time plots are given in Tables 3, 4 and 5, respectively.

MISHRA eta/. : CONVERSION OF ALLIIN TO ALLICIN IN GARLIC 111

The rate of conversion of alliin to allicin decreased with increasing strength of acetic acid (Table 3) or rectified spirit (Table 4) in macerating medium.

On increasing the pH of macerating medium in limited range (pH 4.0 - 6.0) the rate of conversion of alliin to allicin increased (Table 5).

It was also observed that plots of [Pyruvic acid] versus incubation time in presence of different initial concentrations of rectified spirit/ethanol or at different pH or at different incubation temperatures were linear for incubation periods of 60 min, beyond which these tended to flatten out. Therefore, it was apparent that most of alliin was converted to allicin in about 60-75 min under the aforesaid conditions. However, the plots of [Pyruvic acid] versus incubation time in presence of various initial acetic acid concentrations were linear upto 2 h (Fig. 6) suggesting relatively slow rates of conversion of alliin to allicin in presence of acetic acid. The slow rate of conversion of alliin to allicin were also confirmed by low values of K obs in presence of acetic acid.

Thus, on the basis of experimental results the favourable conditions for conversion of alliin to allicin are deduced to,

(i) Enzymatic action temperatures of about 35°C, (ii) pH of the reaction towards neutrality of medium

as the acidic conditions of reaction mixture decreases the rate of decomposition of alliin.

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