Thiolane-type sulfides from garlic, onion, and Welsh onion

In this paper, we review our work in the last 10 years wherein we examined the sulfides in the acetone extracts of garlic (Allium sativum), onion (A. cepa), and Welsh onion (A. fistulosum), obtained and characterized the structures of new sulfides, three 3,4-dimethylthiolane-type sulfides from onion and Welsh onion, respectively, and four acyclic-type, nine 3,4-dimethyl- thiolane-type, four 2-methylthiolane (and thiane)-type, two 1,2-dithiolane-type, and two 2-oxothiolane-type sulfides, together with (E)-ajoene and one kujounin-type sulfide from garlic. During this process, structural corrections were made in onionin A group, garlicnin A, and garlicnin B group in some 3,4-dimethylthiolane-type sulfides. Next, hypothetical pathways for the production of the aforementioned sulfides were proposed. Furthermore, it was revealed that a typical 3,4-dimethylthiolane-type sulfide, onionin A1 obtained from onion, having the isomeric structure of garlicnin B1 obtained from garlic, decreased tumor proliferation and controlled tumor metastasis. These results showed that onionin A1 is an effective agent for controlling tumors, and that the antitumor effects observed in vivo are likely caused by reversing the antitumor immune system. Activation of the antitumor immune system by onionin A1 might be an effective adjuvant therapy for patients with osteosarcoma, ovarian cancer and other malignant tumors.


Introduction
Garlic (Allium sativum L.) is ranked at the top of the list of designer foods showing anti-cancer effects by the National Cancer Institute [1]. Generally, the biological activity of garlic is distinguished in two categories: cardiovascular disease prevention and cancer prevention. Activities in the former category include the inhibition of cholesterol synthesis, platelet aggregation, and arterial smooth muscle cell proliferation, as well as anti-inflammatory, antioxidant, and hydrogen sulfide-mediated vasodilatory effects. The activities in the latter category include the effects on carcinogen metabolism, i.e., enhanced cellular glutathione synthesis that induces cell cycle arrest and apoptosis, and prevention of Helicobacter pylori infection, gastric cancer, and colorectal cancer [2][3][4][5][6].
The chemistry of Allium sulfides began with the discovery of allicin and alliin in 1944 [7] and 1951 [8], respectively, in garlic. In 1971, two types of vinyldithiin derivatives [9] were identified as thermally decomposed compounds by GC analysis of allicin. In 1984, Block and Ahmad determined the structure of ajoene in ether fraction [10]. It was also found that volatile garlic oils contained many sulfur compounds, such as diallylsulfide, (Z and E)-ajoene, 1,3-vinyldithiin, and 1,2-vinyldithiin, produced by the decomposition of thiosulfinates [11]. Unexpectedly, there were few clarified sulfides from garlic; in particular, cyclic sulfides before our study. Therefore, we had started the investigation for aiming at the isolation, structural characterization, and antitumor activity of the cyclic sulfides (sulfur-containing compounds including sulfoxides) from garlic, onion (A. cepa), and Welsh onion (A. fistulosum). The present review provides a brief description of the above-mentioned study.

Extraction and separation of garlic
Acetone was selected as the extracting solvent because it was expected to prolong the lifetime of allyl (or 1-propenyl) sulfenic acid and allyl thiosulfenic acid, which are derived easily by the decomposition of allicin. The acyclic and cyclic sulfides are stabilized by the electron-inductive interaction between acetone and sulfenic acids, and between acetone and cyclic sulfide. Chinese garlic was used, which is the same as Japanese garlic, because it was readily available and the occurrence of various sulfides, due to long drying storage, was expected. Chinese garlic (1.0 kg) was chopped and blended with acetone in a mixer. The mixtures were then soaked in acetone for 3 days at room temperature. During this time, sulfenic acid analogs might undergo chemical changes, such as cyclization and artificial reactions, to produce new sulfides. In particular, we intended to obtain stable cyclic sulfides possessing antitumor activity. Next, the

Hypothetic pathways to respective sulfides
The first acyclic-type sulfides were produced by the arrangement and combination of allyl (or 1-propenyl) sulfenic acid, and allyl thiosulfenic acid derived from allicin (Fig. 4,  Fig. 5). In the case of garlicnins L-1 and L-2, vinyl (ethenyl) and methyl sulfenic acid, respectively, were used in the first step of their synthesis. Moreover, on the basis of garlicnin L-2 formation, it was hypothesized that allyl (or 1-propenyl) sulfenic acid would be involved in hydroxylation for oxidative reaction, of which example were observed in the pathway to onionin B group. In the case of garlicnin L-2 formation, 1-propenyl sulfenic acid was likely involved in the dehydroxylation for reductive reaction, for which instances were observed in the pathways to garlicnins L-3, M, I 1 , I 2 , J 2 , and onionin B group. Formation of garlicnin B group was proposed as shown in Fig. 6: allicin was firstly derived from S-allyl L-cysteine, next allicin was transformed into 1-propenyl 1-propene-thiosulfinate via double-bond rearrangement and was then converted to 2,3-dimethylbutanedithial 1-oxide via [3,3]-sigmatropic rearrangement [30]. The generated intermediate was subsequently ring-closed to form a thiolane derivative that reacted with allyl sulfenic acid to finally produce the 3,4-dimethylthiolane-type sulfides, garlicnins B 1 , B 2 , B 3 and B 4 . On the other hand, the above thiolane derivative was once hydroxylated on S in the thiolane framework to give thiolane S-oxide, and next reacted with allyl thiosulfenic acid and 1-propenyl sulfenic acid to generate the garlicnin C group, as shown in Fig. 6. The hypothetical pathway for the production of garlicnin M is shown in Fig. 7. In the production of the 2-methylthiolane(and thiane)-type sulfoxides, the combination of C-2 on allyl sulfenic acid and C-1 on 1-propenyl sulfenic was triggered in the pathways to garlicnins I 1 and I 2 as shown in Fig. 8, and the combination between the C-1 on 1-propenyl sulfenic acid and C-3 on allyl sulfenic acid occurred for the formation of garlicnin J 1 as shown in Fig. 9. In the production of 1,2-dithiolane-type sulfoxides, the first stage was initiated by the combination of C-1 on allyl sulfenic acid and C-2 on allyl thiosulfenic acid in the case of garlicnin G. To produce garlicnin P, the dehydroxylation of allyl thiosulfenic acid resulted in successive rearrangements between allyl thiosulfenic acid and 1-propenyl sulfenic acid to yield garlicnin P as shown in Fig. 10. The 2-oxothiolane-type sulfides, onionins B 1 and B 2 were produced following hydroxylation to C-2 on the thiolane framework. This method differed from garlicnins C group, in which the hydroxylation to the S atom on the thiolane framework occurred as shown in Fig. 6. Furthermore, allyl sulfenic acid preferred hydroxylation at C-2 and 1-propenyl sulfenic acid may participate in dehydroxylation at C-4 as shown in Fig. 11.

Effect of 3,4-dimethylthiolane-type sulfide [onionin A 1 (1)] on tumor progression and metastasis in tumor injected mice
3,4-Dimethylthiolane-type sulfides, such as onionins A 1 -A 3 from onion and Welsh onion, and garlicnins A, B 1 -B 4 , C 1 -C 3 , and M from garlic are common compounds among these Allium species and are regarded as major sulfides. Therefore, to examine the antitumor activity, onionin A 1 (1) [23], which is representative of the 3,4-dimethylthiolanetype sulfides, was investigated. Onionin A 1 is an isomer of garlicnin B 1 , with an allylsulfinyl group instead of a 1-propenylsulfinyl group at C-5 on the core 3,4-dimethylthiolane 2-ol framework. Therefore, if onionin A 1 is active for antitumor effects then garlicnin B 1 also expected to be active. We used onionin A 1 available at this time for antitumor examination. The effects of onionin A 1 on tumor progression and metastasis in mouse osteosarcoma and ovarian cancerbearing mouse models were investigated. Administration of onionin A 1 significantly suppressed both subcutaneous  Fig. 12). Furthermore, onionin A 1 significantly suppressed (in promotion stage) tumor progression in a mouse ovarian cancer (iMOC)bearing mouse model (B in Fig. 12), suggesting that onionin A 1 is an orally available small molecule for anti-cancer therapy [31,32]. The antitumor effects observed in vivo are likely caused by reversal of the antitumor immune system. Activation of the antitumor immune system by onionin A 1 might be an effective adjuvant therapy for patients with osteosarcoma, ovarian cancer and other malignant tumors.

Conclusion
The identification and characterization of novel sulfides isolated from garlic, onion, and Welsh onion have contributed to the identification of new chemicals and pharmaceutical compounds. Among the 3,4-dimethylthiolanetype of major sulfides, garlicnin B 1 (Table 1, Fig. 13) is expected to be developed as a novel anti-cancer agent, as it is readily isolated in high yield, representing approximately 0.05% of Chinese garlic, and is also a synthesizable target because of its structural simplicity. Based on these findings,     Fig. 11 Hypothetical pathway to 2-oxothiolane-type sulfides, onionins B 1 and B 2 pharmacological investigations will be conducted to develop healthy foods and anti-cancer agents that can prevent or combat disease.

Conflict of interest
The authors declare no conflict of interest.
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Fig. 12
Effect of 3,4-Dimethylthiolane-type sulfide (onionin A 1 : ONA) on tumor progression and metastasis in tumor injected mice Onionin A 1 (ONA) (20 mg/kg) was administered orally before and after the subcutaneous implantation of LM8 cells in the C3H mice (n = 20, each group) for 3 weeks, followed by determination of the subcutaneous tumor weight and presence of lung metastasis (A). As a murine ovarian cancer model, C57B6 mice were injected in the right ovary with iMOC cells and were administered ONA (20 mg/kg) for 3 week, followed by determination of the subcutaneous tumor weight (B) Fig. 13 Garlicnin B 1 (2)