Atypical Cyclic Sulfides, Garlicnins G, I, and J, Extracted from Allium sativum

Abstract

Newly characterized, atypical sulfides, garlicnins G (1), I (2), and J (3), were isolated from the acetone extracts of garlic bulbs, Allium sativum. Their production pathways are regarded as different from those of cyclic sulfoxides, 3,4-dimethyltetrahydrothiophene-S-oxide derivatives such as onionins A₁–A₃, garlicnins B₁–B₄ and C₁–C₃.

Garlic (Allium sativum L.) is ranked at the top of the list of designer foods with anticancer functions recommended by the National Cancer Institute.¹⁾ In general, the various biological activities of onion and garlic can be classified into two categories: cardiovascular disease prevention and cancer prevention.^2–5)

The acetone extracts of garlic has the potential of inhibiting the polarization of M2 activated macrophages, resulting in the suppression of tumor-cell proliferation. Therefore, it can be assumed that the ingredients of garlic may exhibit suppress tumor cell proliferation.^6–13) In previous studies, the acetone extract of garlic was separated using silica gel chromatography to obtain linear acyclic sulfide types L-1–L-4, E, and F,¹⁴⁾ and cyclic sulfoxides, garlicnins A,¹⁵⁾ B₁–B₄,^13,16) C₁–C₃,^13,16) D,¹³⁾ K_1,2, and H₁,¹⁷⁾ and their chemical structures were characterized. Furthermore, the activity of cyclic sulfoxide, onionin A₁ isolated from onion (Allium cepa L.),¹⁸⁾ corresponding to the isomer of garlicnin B series, was examined for its ability to inhibit tumor progression in both osteosarcoma and ovarian cancer mouse model. The results showed that onionin A₁ was an effective agent^19,20) against osteosarcoma and ovarian cancer in both in vitro and in vivo models, and the antitumor effects observed in vivo are likely caused by regulating the antitumor immune system. Activation of the antitumor immune system by onionin A₁ might be an effective adjuvant therapy for patients with osteosarcoma and other malignant tumors. Therefore, the isolated sulfides were regarded to be highly suitable for developing natural and healthy foods that can prevent and combat a variety of diseases, particularly cancers.

In this study, the structures of three atypical sulfide type garlicnins G (1), I (2), and J (3), isolated from the acetone-soluble extract of garlic bulbs, were characterized using various spectroscopic techniques.

Results and Discussion

Garlicnin G₁ (1) was obtained as a syrup showing [α]_D +0.7° (CHCl₃). High-resolution (HR)-FAB-mass spectroscopy (MS) gave a quasi-molecular ion peak in the spectrum of 1 corresponding to [M+H]⁺ at m/z 235.0287 (C₉H₁₅OS₃), in the positive mode. In the ¹H-NMR spectrum, sequential correlations of the signals for the methylene protons at δ 3.09 (1H, dd, J=5.2, 10.9 Hz) and 3.45 (1H, dd, J=5.8, 10.9 Hz); methine proton at δ 3.40 (1H, m); and allyl group at δ 2.34 (1H, ddd, J=7.2, 7.5, 13.8 Hz), 2.41 (1H, ddd, J=6.1, 7.2, 13.8 Hz), 5.80 (1H, dddd, J=7.2, 7.5, 10.3, 16.6 Hz), 5.14 (1H, d, J=10.3 Hz), and 5.16 (1H, d, J=16.6 Hz) were observed. These assignments were in turn confirmed by ¹H–¹H NMR correlation spectroscopy (COSY; Fig. 1). Moreover, the methine proton at δ 3.40 (1H, m) coupled with the signal for another methine proton at δ 4.23 (1H, d, J=5.2 Hz). Except the above, a second allyl group at δ 3.49 (1H, dd, J=8.6, 12.9 Hz), 3.79 (1H, dd, J=6.6, 12.9 Hz), 5.99 (1H, dddd, J=5.6, 8.6, 10.4, 17.2 Hz), 5.40 (1H, d, J=17.2 Hz), and 5.51 (1H, d, J=10.4 Hz) was observed (Fig. 1). Correlation between the proton signal at δ 4.23 and the carbon signals at δ 37.6 (where the methylene protons at δ 2.34 and 2.41 are attached), 44.0 (where the methylene protons at δ 3.09 and 3.45 are attached), and 54.5 (where the methylene protons at δ 3.49 and 3.79 are attached) was observed using heteronuclear multiple bond correlation (HMBC; Fig. 1) spectroscopy. Taking into consideration the above mentioned ¹H–¹H COSY and HMBC evidences, the chemical shifts of the respective proton signals, the molecular weight obtained by HR-FAB-MS and the fact that other garlicnins consist of a combination of an allyl sulfenic acid and an allyl thiosulfenic acid derived from allicin,^13–18) 1 was deduced to be composed of one mole each of the allyl sulfenic acid, allyl group, and allyl thiosulfenic acid. It was also deduced that the allyl sulfenic acid was situated adjacent to the carbon at δ 73.4 and that the thiosulfenic acid would form a 1,2-dithiolane ring. The nuclear Overhauser effect spectroscopy (NOESY) spectrum did not show any correlation between the proton at δ 3.40 and the proton at δ 4.23, therefore, the structure of garlicnin G₁ (1) was proposed 4-(prop-2-en-1-yl)-3-(prop-2-ene-1-sulfinyl)-1,2-dithiolane (Fig. 1).

Fig. 1. Structure of Garlicnin G (1) and Its ¹H–¹H COSY and Key HMBC

The production of 1 was hypothesized as illustrated in Chart 1. This shows the combination of the carbon at C-3 on the allyl sulfenic acid and the carbon at C-2 on the allyl thiosulfenic acid in the first stage. This combination mode is different from those observed thus far for onionin A and garlicnins B and C series.

Chart 1. Hypothetical Pathway to Garlicnin G (1)

Garlicnin I (2) was obtained as a syrup showing [α]_D −2.0° (CHCl₃). The HR-FAB-MS spectrum of 2 (recorded in the positive mode) showed a quasi-molecular ion peak in its spectrum corresponding to [M+H]⁺ at m/z 341.0194 (C₁₂H₂₁OS₅). The ¹H-NMR spectrum (in CDCl₃) and ¹H–¹H COSY spectrum (Fig. 2) of 2 showed signals for the two allyl groups at δ 3.36 (2H, d, J=7.5 Hz), 5.85 (1H, overlapped), 5.17 (1H, dd, J=1.2, 9.7 Hz), 5.22 (1H, dd, J=1.2, 16.0 Hz); 3.54 (2H, d, J=7.5 Hz), 5.85 (1H, overlapped), 5.19 (1H, dd, J=1.2, 9.8 Hz), and 5.30 (1H, dd, J=1.2, 16.6 Hz). A sequential line of proton signals for the methyl group at δ 1.41 (3H, d, J=6.9 Hz); methine proton at δ 3.10 (1H, m); methylene protons at δ 2.19 (1H, ddd, J=5.7, 8.0, 13.8 Hz) and 2.36 (1H, ddd, J=7.8, 9.8, 13.8 Hz); methine proton at δ 2.98 (1H, m), which further correlated to the methylene protons at δ 2.79 (1H, dd, J=7.5, 13.2 Hz) and 3.13 (1H, dd, J=4.0, 13.2 Hz); and methine proton at δ 3.94 (1H, d, J=10.3 Hz) were observed. The proton signal at δ 3.94 correlated to the carbon signals at δ 36.6 (where the methylene protons at δ 2.19 and 2.36 are attached), 42.4 (where the methylene protons at δ 2.79 and 3.13 are attached), 42.5 (where the methine proton at δ 2.98 is found), and 56.3 (where the proton at δ 3.10 is attached) in the HMBC spectrum (Fig. 2). This evidence suggested that 2 is a thiolane-S-oxide derivative, carrying two moles of prop-2-en-1-yl-disulfanyl groups. A correlation between the proton signal at δ 2.98 and the proton signal at δ 3.94 was observed in the NOESY spectrum; therefore, the structure with the relative configuration of garlicnin I (2) was deduced to be 5-methyl-2-[(prop-2-en-1-yldisulfanyl)-3-[(prop-2-en-1-yldisufanyl)methyl]-thiolan-1-ium-1-olate] as shown in Fig. 2. The configuration of the methyl group still remains to be confirmed. The proposed hypothetical pathway to garlicnin I (2) was deduced as outlined in Chart 2. The trigger stage was deduced to be the combination of the carbon at C-3 on the allyl sulfenic acid and the carbon at C-2 on the 2-propenic sulfenic acid.

Fig. 2. Structure of Garlicnin I (2) and Its ¹H–¹H COSY and Key HMBC

Chart 2. Hypothetical Pathway to Garlicnin I (2)

It is known that the inhibition of M2-macrophage polarization can suppress tumor cell proliferation. In the human monocyte-derived macrophages (HMDMs), interleukin 10 (IL-10) induces CD163 expression, a M2 macrophage marker, for 2 d. In the present study, we measured the effects of garlicnin I (2) on IL10-induced CD163 expression in HMDMs. As a result, garlicnin I (2) inhibited the expression of CD163 (Fig. 3), suggesting its capabilities for suppressing M2 macrophage activation. This result suggests that garlicnin I (2) may demonstrate good potential to suppress tumor-cell proliferation by inhibiting the polarization of M2 alternatively activated macrophages.

Fig. 3. The Effect of Garlicnin I (2) on CD163 Expression in HMDMs

HMDMs were incubated with garlicnin I (100 µM) during incubation with IL-10 (20 ng/mL) for 2 d, followed by determination of the CD163 expression using Cell-ELISA, as described in Experimental.

Garlicnin J (3) was obtained as a syrup showing [α]_D −8.5° (CHCl₃). The HR-FAB-MS spectrum of 3, recorded in the positive mode, showed a quasi-molecular ion peak, corresponding to [M+H]⁺ at m/z 341.0195 (C₁₂H₂₁OS₅). The ¹H-NMR spectrum (in CDCl₃) and ¹H–¹H-COSY NMR spectrum of 3 showed signals for two allyl groups at δ 3.34 (2H, d, J=7.4 Hz), 5.85 (1H, overlapped), 5.16 (1H, d, J=10.3 Hz), 5.22 (1H, d, J=16.1 Hz); 3.46 (2H, d, J=7.5 Hz), 5.85 (1H, overlapped), 5.21 (1H, d, J=10.9 Hz), and 5.25 (1H, d, J=14.9 Hz). A sequential line of proton signals for the methyl group at δ 1.43 (3H, d, J=6.3 Hz); methine proton at δ 3.05 (1H, m); methylene protons at δ 2.19 (1H, dddd, J=2.3, 8.0, 9.0, 11.0 Hz) and 2.49 (1H, overlapped); methylene protons at δ 2.85 (1H, dd, J=8.0, 13.5 Hz) and 3.02 (1H, dd, J=5.8, 13.5 Hz); methine proton at δ 2.49 (1H, m, overlapped); and methine proton at δ 3.93 (1H, d, J=5.8 Hz) were observed in the ¹H–¹H COSY spectrum (Fig. 4). The proton signal at δ 3.93 correlated with the carbon signals at δ 43.0 (where the methylene protons at δ 2.85 and 3.02 are attached), 46.3 (where the proton at δ 2.49 is found), and 56.2 (where the methyl group is attached) in the HMBC NMR spectrum (Fig. 4). This evidence suggested 3 to be a derivative of thiane-S-oxide, carrying two moles of prop-2-en-1-yl-disulfanyl groups. The NOESY spectrum suggested no correlation between the proton at δ 3.93 and the proton at δ 2.49, therefore, the propsed relative configuration for the structure of garlicnin J (3) was characterized as 6-methyl-2,3-bis(prop-2-en-1-yldisulfanyl)thian-1-ium-1-olate as shown in Fig. 4. The configuration of the methyl group is still to be elucidated. The proposed hypothetical production pathway of garlicnin J (3) is shown in Chart 3. The trigger stage was deduced to be the combination of the carbon at C-3 on the 2-propenic acid and the carbon at C-1 on the allyl sulfenic acid.

Fig. 4. Structure of Garlicnin J (3) and Its ¹H–¹H COSY and Key HMBC

Chart 3. Hypothetical Pathway to Garlicnin J (3)

Garlicnins G (1), I (2), and J (3) were produced by different pathways from those cyclic sulfoxides obtained so far, such as garlicnins B₁–B₄ and C₁–C₃ obtained from garlic and onionins A₁–A₃ obtained from onion (Chart 4). Garlicnins G, I, and J could thus be classified as atypical sulfides.

Chart 4. Combination Modes of Sulfenic Acid Derivatives at Trigger Stages

Experimental

General Experimental Procedures

Optical rotation was measured using a JASCO P-1020 (l=0.5) automatic digital polarimeter. The IR spectrum was measured using a Fourier transform (FT)/IR-4200 spectrometer (JASCO, Tokyo, Japan). The IR spectrum was measured with a FT/IR-4200 spectrometer (JASCO). The ¹H- and ¹³C-NMR spectra were measured in CDCl₃ using a JEOL alpha 500 spectrometer at 500 and 125 MHz, respectively, and the chemical shifts were found to be on the δ (ppm) scale. The HR-FAB-MS were measured using a JEOL JMS-DX303HF mass spectrometer and taken in a glycerol matrix containing NaI. Column chromatography was carried out on silica gel 60 (230–400 mesh; Merck, Germany). TLC was performed on silica gel plates (Kieselgel 60 F254; Merck). TLC spots were visualized under UV light (254/366 nm), sprayed with 10% H₂SO₄, and then heated. HPLC was conducted with the following instruments and conditions: pump, LC-10AT (Shimadzu); detecter, RID-10A (Shimadzu, Kyoto, Japan), column, Cosmosil 5C18-AR-H (10 mm i.d.×250 mm); solvent, 85% MeOH; flow rate, 2.0 mL/min.

Plant Material

The garlic bulbs (A. sativum L. family Liliaceae) imported from China were identified by Prof. Kotaro Murakami. A voucher specimen (SBGH 4-06-15-302) was deposited in the Herbarium of the Botanical Garden at Sojo University, Kumamoto, Japan.

Extraction and Isolation

Chinese garlic (886.1 g) was roughly chopped and blended with acetone in a mixer. Subsequently, the mixture was soaked in additional acetone for 3 d at room temperature. The filtrate was evaporated at 40°C in vacuo to obtain a residue, which was then partitioned between AcOEt and water. The organic layer was taken up and evaporated to give the residue (4.57 g), which was repeatedly chromatographed on silica gel with CHCl₃–methanol (200 : 1) and n-hexane–aceton (6 : 1), and subjected to HPLC to obtain three new sulfides, named garlicnins G (1, 28.1 mg), I (2, 35.2 mg) and J (3, 7.2 mg).

Garlicnin G (1) was obtained as a syrup showing [α]_D +0.7° (c=0.33, CHCl₃).

IR ν_max (KBr) 1025 cm⁻¹ (sulfoxide).

HR-FAB-MS (m/z): 235.0287 (Calcd for C₉H₁₅OS₃: 235.0285).

¹H-NMR (CDCl₃) δ: 2.34 (1H, ddd, J=7.2, 7.5, 13.8 Hz, H-1″a), 2.41 (1H, ddd, J=6.1, 7.2, 13.8 Hz, H-1″b), 3.09 (1H, dd, J=5.2, 10.9 Hz, H-5a), 3.40 (1H, m, H-4), 3.45 (1H, dd, J=5.8, 10.9 Hz, H-5b), 3.49 (1H, dd, J=8.6, 12.9 Hz, H-1′a), 3.79 (1H, dd, J=6.6, 12.9 Hz, H-1′b), 4.23 (1H, d, J=5.2, H-3), 5.14 (1H, d, J=10.3 Hz, H-3″a), 5.16 (1H, d, J=16.6 Hz, H-3″b), 5.40 (1H, d, J=17.2 Hz, H-3′a), 5.51 (1H, d, J=10.4 Hz, H-3′b), 5.80 (1H, dddd, J=7.2, 7.5, 10.3, 16.6 Hz, H-2″), 5.99 (1H, dddd, J=6.6, 8.6, 10.4, 17.2 Hz, H-2′).

¹³C-NMR (CDCl₃) δ: 37.6 (C-1″), 44.0 (C-5), 47.1 (C-4), 54.5 (C-1′), 73.4 (C-3), 118.6 (C-3″), 124.2 (C-3′), 125.6 (C-2′), 134.7 (C-2″).

Garlicnin I (2) was obtained as a syrup showing [α]_D −2.0° (c=0.41, CHCl₃).

IR ν_max (KBr) 1023 cm⁻¹ (sulfoxide).

HR-FAB-MS (m/z): 341.0194 (Calcd for C₁₂H₂₁OS₅: 341.0196).

¹H-NMR (CDCl₃) δ: 1.41 (3H, d, J=6.9 Hz, CH₃ at C-5), 2.19 (1H, ddd, J=5.7, 8.0, 13.8 Hz, H-4a), 2.36 (1H, ddd, J=7.8, 9.8, 13.8 Hz, H-4b), 2.79 (1H, dd, J=7.5, 13.2 Hz, H-6a), 2.98 (1H, m, H-3), 3.10 (1H, m, H-5), 3.13 (1H, dd, J=4.0, 13.2 Hz, H-6b), 3.36 (2H, d, J=7.5 Hz, H₂-1″), 3.54 (2H, d, J=7.5 Hz, H₂-1′), 3.94 (1H, d, J=10.3 Hz, H-2), 5.17 (1H, dd, J=1.2, 9.7 Hz, H-3″a), 5.19 (1H, dd, J=1.2, 9.8 Hz, H-3′), 5.22 (1H, dd, J=1.2, 16.0 Hz, H-3″b), 5.30 (1H, dd, J=1.2, 16.6 Hz, H-3′b), 5.85 (2H, overlapped, H-2′, H-2″).

¹³C-NMR (CDCl₃) δ: 12.9 (CH₃ at C-2), 36.6 (C-4), 42.4 (C-6, C-1′), 42.5 (C-3), 42.9 (C-1″), 56.3 (C-5), 78.6 (C-2), 119.1 (C-3″), 119.6 (C-3′), 132.9 (C-2″), 133.3 (C-2′).

Garlicnin J (3) was obtained as a syrup showing [α]_D −8.5° (c=0.06, CHCl₃).

IR ν_max (KBr) 1024 cm⁻¹ (sulfoxide).

HR-FAB-MS (m/z): 341.0195 (Calcd for C₁₂H₂₁OS₅: 341.0196).

¹H-NMR (CDCl₃) δ: 1.43 (3H, d, J=6.3 Hz, CH₃ at C-6), 2.19 (1H, dddd, J=2.3, 8.0, 9.0, 11.0 Hz, H-5a), 2.49 (2H, m: overlapped, H-3, H-5b), 2.85 (1H, dd, J=8.0, 13.5, H-4a), 3.02 (1H, dd, J=5.8, 13.5 Hz, H-4b), 3.05 (1H, m, H-6), 3.34 (2H, d, J=7.4 Hz, H₂-1″), 3.46 (2H, d, J=7.5 Hz, H₂-1′), 3.93 (1H, d, J=5.8 Hz, H-2), 5.16 (1H, d, J=10.3 Hz, H-3″a), 5.21 (1H, d, J=10.9 Hz, H-3′a), 5.22 (1H, d, J=16.1 Hz, H-3″b), 5.25 (1H, d, J=14.9 Hz, H-3′b), 5.85 (2H, overlapped, H-2′, H-2″).

¹³C-NMR (CDCl₃) δ: 11.4 (CH₃ at C-6), 38.7 (C-5), 42.1 (C-1″), 42.7 (C-1′), 43.0 (C-4), 46.3 (C-3), 56.2 (C-6), 81.4 (C-2), 119.0 (C-3″), 119.8 (C-3′), 132.5 (C-2″), 133.4 (C-2′).

Cells and Cell Culture Conditions

Peripheral blood mononuclear cells were acquired from healthy volunteer donors; written informed consent was obtained from all healthy donors. CD14⁺ monocytes were purified from peripheral blood mononuclear cells via positive selection using magnetic-activated cell sorting technology (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured with granular macrophage-colony stimulating factor (GM-CSF) (10 ng/mL, Wako Pure Chemical Industries, Ltd., Osaka, Japan) or macrophage-colony stimulating factor (M-CSF) (50 ng/mL, Wako Pure Chemical Industries, Ltd.) for 7 d in order to differentiate them into macrophages. The differentiated macrophages were then used as human monocyte-derived macrophages (HMDMs) in the present study.

Determination of the Inhibitory Effects of Garlicnin I on the CD163 Expression

HMDMs (1×10⁴ cells per well of a 96-well plate) were incubated with or without crude extracts and natural compounds for 24 h after treatment with IL-10 (30 nM) or tumor culture supernatant (TCS) for 2 d, followed by determination of the CD163 expression using a Cell Enzyme-linked Immunosorbent Assay (Cell-ELISA), as previously described.^19,20) Briefly, the cells in each well of a 96-well plate were blocked with Block Ace (DS Pharma Biomedical, Osaka, Japan) and washed three times with phosphate buffered saline (PBS) containing 0.05% Tween 20 (washing buffer). The cells were subsequently incubated with an anti-CD163 antibody, AM-3K (2 µg/mL), dissolved in washing buffer for 1 h and then washed with washing buffer three times and reacted with a horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) antibody, followed by a reaction with ULTRASENSITIVE TMB (Moss INC., Pasadena, MD, U.S.A.).

Conflict of Interest

The authors declare no conflict of interest.

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