Garlic Chemistry: Chemical and Biological Properties of Sulfur

Chapter 30

Downloaded by COLUMBIA UNIV on June 13, 2013 | http://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch030

Garlic Chemistry: Chemical and Biological Properties of Sulfur-Containing Compounds Derived from Garlic Yukihiro Kodera, Hiromichi Matsuura, Hiromichi Sumiyoshi, and Shin-ichiro Sumi Healthcare Research Institute, Wakunaga Pharmaceutical Company, Ltd., 1624, Shimokotachi, Koda-cho, Takata-gun, Hiroshima 739-1195, Japan

Garlic (Allium sativum L.) is rich in biologically active organosulfur compounds. Although intact garlic cloves contain a limited number of organosulfur compounds, such as alliin, methiin and y-glutamyl-S-alk(en)yl-cysteines, processing initiates a cascade of chemical transformation reactions. Allicin is one of the well-known transformation compound, however, it is highly unstable and reactive, and quickly decomposes into other organosulfur compounds including alk(en)ly-polysulfides, ajoenes and vinyldithiins. Thus, allicin is a transient compound and likely only its transformation compounds contribute to the health benefits of garlic. Recently, biological activities of water-soluble organosulfur compounds derived from garlic, such as S-allyl-L-cysteine and S-allylmercapto-L-cysteine, have become the center o f attention because they are stable, odorless and safe. Studies on structure-activity relationships have revealed that the S-allyl group plays an important role in the pharmacological activities of organosulfur compounds derived from garlic. Further, consumption of garlic shows feasibility to prevent cancer and affect immunomodulation or enhancement of the immune systems.

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© 2003 American Chemical Society In Food Factors in Health Promotion and Disease Prevention; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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For thousand of years, people have used garlic for medicinal purposes because they were aware of the existance of its biologically active constituents from their own experience. One may ask in what form(s) the garlic yielded such benefits. Various processing methods have been noted in the historical remedies using garlic for treatment and healing (1-4). It is generally believed that the biological activities of garlic are derived from organosulfur compounds, however, intact constituents in raw garlic do not have strong biological activities. There is a legend called "Four Thieves Vinegar" that states that the lives of four condemned criminals were saved from terrible plague in Marseilles by drinking a concoction consisting of macerated garlic in wine, and this legend is still in circulation today (5). Macerated preparation do not contain the allicin which has strong antimicrobial activity (3,4% but it might be possible that there were other beneficial compounds derived from garlic in that Vinegar. Processing activates constituents in garlic preparations through enzymatic and/or chemical reactions and composition of generated compound varies greatly depending on the processing method employed.


9

GTPase Ο

COOH

y-Glutamyl-S-alk(en)yl-L-cysteine

S-Alk(en)yl-L-cysteine

W Oxidase? HOOC S-Alk(en)ylmercapto-L-cysteine Alliinase (Rapid) Oil soluble compounds (DAS,DADS, Ajoene, Dithiins)

HOOC S-Alk(en)ly-L-cysteine sulfoxide

Alk(en)ylthiosulfmate

Figure 1. Key reaction pathway in transformation reaction of sulfur containing compound derived from garlic

In Food Factors in Health Promotion and Disease Prevention; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Additionally, processing methods greatly effect biological activities of resulting preparations. In this chapter, the effect of processing on chemical constituents and biological activities of garlic preparation will be discussed and potential benefits of various processing methods be elucidated.


Processing Intact garlic contains sulfur constituent such as S-alk(en)yl-L-cysteine sulfoxides and y-glutamyl-S-alk(en)yl-L-cysteines. These constituents in garlic dramatically change to other compounds by processing (Figure 1). Processing involves various reactions and these reactions greatly affect constituents in each preparation (Table 1) (3,4). In the garlic cells, S-alk(en)yl-L-cysteine sulfoxide (for example, alliin) stably exists as the end product which is produced from Salk(en)yl-L-cysteine and/or y-glutamyl-S-alk(en)yl-L-cysteines by hypothetical oxidase and/or transferase (Figure 1). However, once garlic is processed, C-S lyase, such as alliinase, stored in vacuoles produces alk(en)ylthiosulfinates (for example, allicin) which are highly reactive. Produced

Table I. Content of Organosulfur Containing Compounds in Intact Garlic and Garlic Preparations Raw Garlic 5.4-14.5 ND >2

;

FD* Garlic >1 ND >1

Garlic Oil ND ND ND

b)

Oi\ Macerate ND ND ND

Soaking (AGE) 0.01> ND ND

Heating*

c;

Alliin >1 Allicin ND AllicinND Release Dithiin ND ND ND ND ND 0.6-0.7 Ajoene ND ND ND 0.01 ND 0.02-0.12 AllylpolyND 0.01> ND 0.06-0.22 0.01-0.05 >1 Sulfide γ-GSAC 1.9-8.2 >1 >1 ND 0.2-1 ND SAC Trace >1 0.01> Trace ND ND SAMC ND 0.01-0.05 ND ND ND ND NOTE: a) Freeze dried garlic, b) Clear oil isolatedfrommaceration or grinding of garlic cloves in vegetable oil. c) Sliced garlic was soaked with alcohol containing water for more than 10 months at room temperature, d) Whole raw garlic was boiled for one hour, e) Amount of allicin produced after homogenating or mixing with water. γ-GSAC ; γglutamyl-S-allyl-L-cysteine, SAC ; S-allyl-L-cysteine, SAMC ; S-allylmereapto-Lcysteine, ND ; Not detected. Units are mg/g-materials. e)



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alk(en)ylthiosulfinates then transform to various compounds through nonenzymatic chemical cascade reactions. Thus, alk(en)ylthiosulfinates are important transient compounds that generate various organosulfur compounds via chemical transformations (genaration of oil soluble compounds, see Figure 1) (6). On the other hand, enzymatic reaction was related to production of Salk(en)yl-L-cysteines form y-glutamyl-S-alk(en)yl cysteines, such as production of S-allyl-L-cysteine (SAC), in processing. GTPase activity during aging of garlic, which is one of soaking in an aqueous-alcoholic solution, was examined and it was observed that its activity was retained even after 300 days with a resultant increase in S A C content (Figure 2) (7). There is more evidence that GTPase relate to S A C production. When cysteine was added to the soaking mixture, the amount of S A C produced was dramatically increased as compared with no addition of L-cysteine (Figure 3). The assumed reaction mechanism is that added cysteine became a glutamin acceptor on transformation of S A C and S A C was released by GTPase, as shown in Figure 4. Processing under around room temperature, such as soaking, generates various compounds because both chemical and enzymatic reactions are able to work at same time, however, enzymatic reactions, such as reaction of GTPase, are inhibited at high temperatures (i.e. distillation or heating). Therefore, the number of compounds in preparation produced under high temperature treatment are fewer than those produced under low temperture treatment (Table I).

0

100

200

300

Aging Period (Days)

Figure 2. The content of S-allyl-L-cysteiene and GTPase activity during aging process.


350


5

Added L-Cysteine (% against garlic)

Figure 3. Effect of L-cysteine addition for S-allyl-L-cysteine (SAC) production. The preparation was incubated at 37°Cfor 7 days after addition of L-cysteine.

Acceptor

GIu-Acceptor

Figure 4. Hypothetical mechanism of S-allyl-L-cysteine production with addition of L-cysteine.

Processing affects not only the cascade reactions of sulfur containing compounds, but also other constituents, such as saponins, amino acids and carbohydrates, among others. For example, proto-eruboside-B, one of intact saponins in raw garlic, transforms to eruboside-B during the extraction process. Eruboside-B inhibits tumor promotion activity but intact compound, protoeruboside-B, does not (8,9). Recently, several kind of strong and unique


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antioxidants, Na-(l-deoxy-D-fiiictos-l-yl)-L-arginine (10) and 1,2,3,4tetrahydro-P-carbolines, were isolated from aged garlic extract (AGE). These antioxidants did not exist in raw garlic, but increased upon soaking, and their antioxidant activity was stronger than ascoribic acid in H 0 scavenging assay system. Additionally, processing change the potency of biological activity of garlic. Two research groups investigated the improvement of peripheral blood circulation by garlic preparations, independently (11,12). They observed that the effectiveness of A G E and potency of A G E was greater than raw garlic or heattreated garlic on both rats and human. Considering the above findings, processing is obviously necessary for taking advantage of the beneficial biological activity of garlic. Downloaded by COLUMBIA UNIV on June 13, 2013 | http://pubs.acs.org Publication Date: June 19, 2003 | doi: 10.1021/bk-2003-0851.ch030

2

2

Pharmacokinetic Behavior and Metabolism To evaluate biological activities of herbs or chemicals for human health promotion, it is also important to assess chemistry, pharmacokinetic behavior and metabolism of action of these mateirals. To find interaction or metabolism of sulfur-containing compounds with biological materials, some representative compounds derived from garlic, such as allicin, diallyldisulfide (DADS), ajoene, vinyldithiins, were incubated with blood and allylmercaptan was detected (13). Additionally, D A D S , one representative compound in garlic oil, was also detected along with allylmercaptan in our follow-up reinvestigations. A perfusion experiment of D A D S in isolated rat livers yielded allylmercaptan as the metabolite (14). Pushpendran et al (15) found that sulfates were recovered as main metabolite in the mouse liver cytosol fraction in their pharmacokinetic study using radioactive D A D S . Therefore, it is suggested D A D S was first reduced to allylmercaptan which was then oxidized to sulfate by hepatic oxidases. Vinyldithiins, representative compounds in oil macerate, were quickly absorbed (Tma^lSmin, rat) and metabolic rate between 1,2-dithiin and 1,3-dithiin was very different in peroral administration. A part of both administrated vinyldithiins remained in the kidney and fat tissue even 24 hours after administration, but metabolites were not identified (16). Metabolites and metabolic pathway of vinyldithiins is still unknown. There are no clear reports which have studied the absorption and detection of allicin, but not transformation confounds, in a living body, though more than half a century has passed since allicin was discovered. When allicin was added to whole blood, allicin completely disappeared in whole blood within a few minutes and allylmercaptan and D A D S were identified (6,13,17). In addition, it has been shown that hemoglobin in the blood rapidly and irreversibly changes to methemoglobin through interaction with allicin. High reactivity is one chemical


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property of allicin, thus, detection of allicin and its mechanism of action in living body is difficult to elucidate.

Table II. L D

5 0

Values of S-AIlyl-L-cysteine in Rat and M i c e


Route Sex p.o. Male Female i.p. Male Female N O T E : a) L D values were 5 0

LD50

values(g/kg)* Rat Mice 10.94 8.89 9.50 9.39 3.34 6.91 3.34 3.65 calculated by Probit method.

On the other hand, alliin, a hydrophilic compound in the intact garlic and the precursor of allicin, was investigated along with its pharmacokinetic behavior including allicin production in in vivo (4,6,14,17). of this compound was 10 min and its bioavailability was 16.5%. In addition, no evidences was found of allicin production in living bodies. S-Allyl-L-cysteine, produced during the soaking of garlic in aqueous alcohol, was investigated in detail. This compound was quickly absorbed and excreted slowly in both experimental animals and humans. Bioavailability of S A C was very high, in mice - 100%, in dog - 87%. N-Acetyl-S-allyl-L-cysteine was identified as the metabolite of this compound in rats urine after administration of S A C (6) and also human urine after consumption of garlic (18). Toxicity of this compound was very weak and its

0

10

20

30

40

50

60

70

Incubation Time(min)

Figure 5. Biotransformation of S-allylmercapto-L-cysteine (SAMC) in rat liver homogenate. DADS, diallyldisulfide; DATS, diallyltrisulfide; DATES, diallyltetrasulfide; and DAPS, diallylpentasulfide.


353 LD o was >8.8g/kg on p.o. and >3.3g/kg on i.p. in mice and rat, respectively (Table Π ), and subacute toxicity test revealed non-toxic dose for rats with as much as 250mg/kg. L D values of this compound were almost the same as for essential amino acids such as He (6.81g/kg), Leu(6.54g/kg), Phe(5.28g/kg) and Met(4.3 lg/kg) in rat by i.p. administration (19). Another hydrophilic compound, S-allylmercapto-L-cysteine (SAMC), showed a unique metabolite profile in which allylpolysulfides (S=2~5) gradually increased after incubation of S A M C with rat liver homogenete (Figure 5) (6). Allylpolysulfides have been reported to have protective effects on lipid peroxidation in a rat liver microsome. S A M C has been shown to have antioxidative activity in vitro and hepatoprotective effect both in vitro and in vivo. Therefore, these results suggest that S A M C might be responsible for some of biological activity of garlic through biotransformation. Considering these findings, metabolic passway shown in Figure 6 suggests that the allyl-(S) - group containig compounds (n>l) might be first reduced to allylmercaptan and then either oxidized to S 0 " by hepatic oxidases or reversibley transformed to allylpolysulfides (75). These reactions may occur concurrently. 5


5 0

n

2

4

Allyl-(S) -group containing compounds (n>l) n

( S )

n

/ ^

Allylpolysulfide

SO

2-

Figure 6. Hypothetical metabolic passway of allyl - (S) - group containing compounds derived from garlic. n

Important Structure It is obvious that sulfur containing compounds derived from garlic are responsible, at least in part, for biological activity of garlic. However, there is little knowledge of specific chemical structures responsible for biological compounds containing S-allyl group were effective. In addition, they revealed the necessity of allyl group being attached to the sulfur atom for manifestation of neurotrophic activity. These findings strongly indicated that the structure of Sallyl group plays important roles in biological activity of sulfur containing compounds derived from garlic. In Food Factors in Health Promotion and Disease Prevention; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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activity although numerous garlic compounds have been investigated for biological activities. Considering the cascade reaction of sulfur containing compounds following processing and transformation in living bodies, the S-allyl group might be the key structure responsible for biological activity of garlic and for generation of new compounds. To evaluate the importance of S-allyl group, investigations were conducted to reveal the relationships between chemical structure and biological activities. Inhibition of platelet aggregation was investigated using garlic compounds containing methyl and allyl moieties, and compounds containing the allyl moiety were found to be more active than those containing the methyl moiety (20). Hatono et al. (21) investigated structure-activity relationship of garlic chemicals and chemopreventive activity using the rat aberrant crypt assay. Figure 7 shows re-evaluation of their results with regard to carbon number, sulfur number, unsaturated bonds and their position. Some relationships were observed among these factors. First, crypt number decreased by increasing of carbon numbers on thioalkyl and thioalkenyl compounds. Second, the crypt number increased by increasing of carbon numbers on alkyl disulfide compounds, and other findings indicated that S-allyl group had the highest potency for colon cancer prevention and was safer than the other groups. Moriguchi et al. (22) investigated the importance of the S-allyl group on the survival of neurons using analogues or derivatives of S-allyl-L-cysteine and y-glutamyl-S-alk(en)yl cysteines. Only

Figure 7. The relationships between carbon number, sulfur number, unsaturated bond of alk(en)y[sulfides and number of aberrant crypts. Position of each boxes with chemical structure indicates the score of aberrant crypt assay. * ; the score of S-propargyl was almost the same as for propyldisulfide.


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Potency of Cancer Prevention


National Cancer Institute (NCI) has considered numerous natural products for their cancer preventive activity; garlic was given the most crucial position in the N C I experimental foods program (23). Recently, several epidemiological or intervention studies for cancer prevention have been reported or are on-going. These reports show that garlic consumption has a tendency to prevent cancer(2428). For example consumption of 28g-garlic/week (about half to one bulb of garlic) is associated with cancer protective effects against stomach and colorectal cancers (28).

2000 ι

-i

1

2 Week after Incubation

3

Figure 8. Anti-tumor activities of aged garlic extract. Astarisks denote significant difference from control : * ; p

Garlic Chemistry: Chemical and Biological Properties of Sulfur

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