In our studies the correlation between the biological effects and variation in garlic components thiosulfinates and ajoenes were analysed. Two different garlic powders were investigated. Reversed-phase HPLC/UV and HPLC/DAD were used to separate thiosulfinates and quantitate allicin and ajoenes prepared by 24 hour incubation of aqueous and ethanol extracts of garlic. Pure allicin was isolated by TLC. There is a great importance of analysing products before using for clinical investigations, because there was a great difference in content in garlic powders.

Most of the medicinal effects of garlic are referable to sulphur compounds thiosulphinates and sulphides, products of conversion of allin from garlic by enzyme allinase. Allicin (diallyl thiosulphinate), the main thiosulphinate from garlic (Allium sativum L., Alliaceae), has been shown to be responsible for the antimicrobial, antiprotozoal, antimutagenic, antiplatelet and antihyperlipidemic effects of garlic. Diallyl sulphide, one of the degradation compounds of allicin, was identified as a suppressing agent in the dimethylhydrazine – induced colon cancer developed in rats. In addition, immunomodulatory properties of the garlic have been attributed to this sulphide component.

Graph 1. Quantity of allicin released from garlic samples obtained
from various regions of the world (Schulz, 1998)

The amount of sulfur-containing constituents in fresh garlic is highly variable (Graph.1); hence it is important to standardize garlic-powder preparates to a specified concentration range of alliin.

Analysis of allicin has been difficult because of its instability. Direct GC determination has not been achieved because allicin undergoes rapid decomposition in the oven of a GC, even at moderate temperatures. The most promising method for analysis of allicin and degradation products (ajoenes) is reversed-phase HPLC (Lawson, 1991; Lawson, 1991; Blania, 1990).

In our studies the correlation between the biological effects and variation in garlic components thiosulfinates and ajoenes were analysed. Two different garlic powders were investigated. Garlic powders were blended in Ultrashear homogenizer for one minute using 10 ml of water per gram of garlic. The homogenate was allowed to stand at room temperature for five minutes (maximum production of all thiosulfinates was achieved within two minutes), filtered and injected directly into the HPLC. Reversed-phase HPLC/UV and HPLC/DAD were used to separate thiosulfinates and quantitate allicin (methanol/water/formic acid = 40:60:0.1 and methanol/water = 1:1, 254 nm) (Lawson, 1991).

Ajoenes were prepared by 24 hour incubation of aqueous and ethanol extracts of garlic (acetonitrile/water/tetrahydrofuran = 70:27:3, 240 nm) (Lawson, 1991).

The instability of allicin precludes its commercial availability. Therefore a pure allicin was isolated by TLC (Kiesel-gel 60 F 254, hexane/ethyl acetate = 60:40) after extraction from an aqueous solution of commercial garlic powder. The extract was rotary evaporated at room temperature , weighted and redissolved in water at 1 to 2 mg/ml. Allicin content in water was stable at room temperature for 24 to 36 h and at 4°C for two months (Lawson, 1991; Blania, 1991).

Identification of the thiosulfinates compounds in aqueous garlic extracts

Aqueous garlic extracts were analysed within five minutes and thiosulfinate compounds were isolated and allicin was identified. A comparison of two powders revealed the differences in contents of allicine (Table 1, Figure 1.)

Extract B (Fig.1. - upper chromatogram), compared to extract A (Fig.1. - lower chromatogram) is 40 times richer in allicin. Extract A is richer in other thiosulfinates (allyl methyl and methyl allil thiosulfinate, methyl propenyl and propenyl methyl thiosulfinate).

Figure 1. C18-HPLC of aqueous homogenate: extract B - upper chromatogram,
extract A - lower chromatogram

Preparation of pure allicin – was achieved by TLC. After UV visualization (Rf of allicin = 0.60), extraction and preparation of 1 mg/mL it' s UV spectrum was recorded (Fig. 3.-b). Quantification was done by HPLC/UV and HPLC/DAD.
Extract A: content of allicin was 0.08% (g/g)
Extract B: content of allicin was 0.36% (g/g)
Identification of sulfides and dialk(en)yl thiosulfinates in aqueous and ethanol extracts (Tab. 2 and 3, Fig. 2, Fig. 3.-a).

Aqueous extract B (Fig.2. GAE - chromatogram b) is richer in ajoens than aqueous extract A about 50 times (Fig.2. GAE - chromatogram (a) and Tab. 2). Ethanol extracts (Fig.2. GEE - chromatograms (a) and (b) have less ajoens than aqueous (Tab. 3). Extract A is richer in vinildithiins and dialkenyl sulfides.

Figure 2. C18-HPLC of garlic aqueous extracts A and B: GAE – chromatogram (a) and (b) garlic
ethanol extracts A and B: GEE – chromatogram (a) and (b)


Figure 3. UV spectra of (a) ajoene and (b) allicin

CONCLUSION

1. Reversed-phase HPLC was a method of choice for detection and quantitation of allicin and ajoene in garlic homogenates.

2. A great difference in content of allicin and other thiosulfinates between two analysed commercial garlic powders was found. Therefore it is important to analyze products before their use in clinical investigations.

3. Garlic powder B is rich in allicin (0.36%) compared to varieties from various regions of the world (Graph.1).

1. Schulz V., Hansel R., Tyler V.E. (1998): Rational Phytotherapy, London, 108-125.

2. Lawson L., Wood S., Hughes B. (1991): HPLC analysis of allicin and other thiosulfinates in garlic clove homogenates. Planta Med. 57 (4), 263-270.

3. Lawson L.D., Wang Z.J., Hughes B.G. (1991): Identification and HPLC quantitation of the sulfides and dialk(en)yl thiosufinates in commercial garlic products. Planta Med. 57 (4), 363-70.

4. Blania G., Spangenberg B. (1991): Allicin-freisetzung aus getocknetem knoblauch (Allium sativum): Eine einfach durchzuführende HPTLC-simultanbestimmung von allicin und ajoen in knoblauchpulver und daraus hergestellen fertigarzneimitteln. Planta Med. 57 (4), 371-375.