Elicitation of S-allyl-cysteine production in Callus of Garlic (Allium sativum L.)

Abstract

S-allyl-cysteine (SAC) is a non-volatile organosulfur compound commonly present in garlic (Allium sativum L.) with promising medicinal properties such as cholesterol-lowering, hepatoprotective and neuroprotective effects as well as antidiabetic, anticancer, antioxidant and antiinflammatory activities. In an attempt to monitor the production of this compound in garlic callus, a protocol was developed for its quantitative analysis. Standard S-allyl-cysteine was derivatized with dansy1 chloride to increase sensitivity and stability of its amino group prior to detection via high performance liquid chromatography (HPLC) on a LiChrospher C-18 column using sodium acetate buffer : methanol as mobile phase. Molar ratios of S-allyl-cysteine standard to dansyl chloride were used at 1:1, 1:5, 1:10, 1:20, 1:30 and 1:40. The molar ratio 1:20 presented the highest S-allyl-cysteine contents of 0.8110 ± 0.0005 µg. Based on the previous report that fresh garlic contained S-allyl-cysteine at approximately 20 µg/g, molar ratios of 1:20, 1:40, 1:60, 1:80, and 1:100 were subsequently employed to optimize the derivatization step. S-allyl-cysteine content in fresh garlic determined with derivatization at molar ratio 1:20 was 0.1285-0.0002 ug g which was almost the same as S-allyl-cysteine contents in other molar ratios. S-ally1 cysteine production was also investigated in garlic callus cultured on Murashige and Skoog solid medium (MS) with 2,4-dichloropheoxyacetic acid (2,4-D) at concentration of 0.05 mg/L for 8 weeks. S-allyl-cysteine was extracted from the tissue with methanol and derivatized with dansyl chloride following those molar ratios performed on fresh garlic revealing 0.2331 ± 0.0008 µg/g at the molar ratio 1:20. In the case of biological samples like those from fresh garlic and garlic callus, the molar ratio of 1:20 of S-allyl-cysteine to dansyl chloride is proposed for feasible detection and quantitative analysis of S-allyl-cysteine. In this study, callus culture samples from garlic were placed in MS medium with only 2.4-D at different concentrations of 0.1, 0.3 and 0.5 mg/L. Another set sub-culturing of garlic callus was carried out using the same medium in presence of kinetin at concentration of 0.5 mg/L combined with 2.4-D at different concentrations of 0.1, 0.3 and 0.5 mg/L for 4 weeks. The obtained callus culture had compact characteristic with yellowish color. S-allyl-cysteine contents in callus grown onto MS medium with different plant growth regulator were analyzed via HPLC and comparison with S-allyl- cysteine standard. The average S-allyl-cysteine contents in callus cultured on MS medium supplemented with 0.1 mg/L of 2.4-D offered the highest S-allyl-cysteine content at 0.3924 ± 0.0027 µg/g fresh weight. S-allyl-cysteine in callus culture from induction with only 2,4-D at concentration of 0.1 mg/L was separated via column chromatography, monitored with thin layer chromatography (TLC) and identified with nuclear magnetic resonance (NMR) spectroscopy, respectively. These techniques confirmed the presence of S-allyl-cysteine in callus extract. Moreover, with the conditions preliminarily used in this study garlic callus has shown S-allyl-cysteine content of 3-fold of that found in the fresh garlic at 0.1285 ± 0.0002 µg/g counterpart suggesting that tissue culture is an alternative approach for its manufacture of S-allyl- cysteine.