Preparation And Properties Of Disulfide Conjugates Between Allicin And Soy Glycinin/peptides

The biological activities of Allium vegetables are mainly attributed to allicin(TS). TS has excellent anti-tumor, immune regulation, anti-inflammatory and antioxidant activities. However, the poor stability of TS limits its commercial exploitations. It is generally supposed that the disulfide conjugates formed by TS and free thiols of peptides or proteins in vivo may contribute to the physiological activities of TS. Soy glycinin(11S) is rich of cysteine residues, and the obtained thiol-containing peptides from 11 S show excellent activities like antioxidant, heavy metal detoxification et al. But the sulfhydryls are easy to be oxidized, thus the application of those peptides are greatly limited. For those reasons, the conjugates(RSSA) were prepared to testified whether they still own the benefits of TS and thiols, which would gain theoretical and practical values. In this research, TS as well as soybean glycinin and thiol-containing peptides under various processings were prepared, and the storage stability of TS and enriched thiol-containing peptide(EP) were explored individually. Then, the disulfide conjugates between TS and soybean glycinin or peptides were prepared, and the conjugated reaction conditions were optimized. Furthermore, the stability and bioactivities of the disulfide conjugates(RSSA) were investigated, aimed to prepare RSSA with good stability and bioactivities.Firstly, alliin was synthesized and catalyzed by nature alliinase extracted from raw garlic to prepare TS. The structure and purity were analyzed, and the purity was proved to be more than 95%. The stability of TS was investigated, and it revealed that the half-life of pure TS was only 1 d, and the water solution of TS was sensitive to high temperature and pH with the halflife of 18 d at 20°C, pH 7.0. Soy proteins/peptides with different content of sulfhydryl were prepared with soy glycinin(11S). The sulfhydryl peptides were enriched and the storage stability was further investigated. It turned out that the sulfhydryl peptides were more stable at low pH, while they were quite unstable under high temperature and alkaline conditions with the half-life of 1.87 d at 20°C, pH 7.0.The optimal conjugated reaction conditions of TS and thiols from protein/peptides were further determined as follows: 24°C, pH 7.0, molar ratio of [TS] and [R-SH] is 5:4, and reaction time of 1 h. A series of RSSA were prepared under optimal conditions, all of them owned the completion ratio more than 94%. RP-HPLC results showed that compared with that of thiolcontaining peptides, the retention time of disulfide conjugated increased, which meant the hydrophobicity increased. As the MS revealed, the relative molecular weight of RSSA was 72 Da higher than that of RSH. The UV spectra showed that the specific absorbance of thiosulfinate group(196 nm) was disappeared after the formation of RSSA. All the results indicated that the thiosulfinate group of TS will be broken and react with thiols to form a new disulfide bond, thus RSSA formed. The disulfide conjugates have better storage stabilities with the half-life of 231 d and 2 years at 20°C, pH 7.0 for EPSSA, SP1SSA/SP2 SSA, respectively.The bioactivities of the disulfide conjugates, including the induction of quinone reductase(QR), the inhibition of lipopolysaccharide-stimulated nitric oxide(NO) production, antioxidant acitivity in vitro, were further evaluated. The results showed that all the RSSA could induce QR and inhibit NO production with varied concentration from 12.5 μg/mL to 4 mg/mL. In addition, they all showed dose-concentration relationships, and had no toxic effect on cell proliferation. Of them, the conjugate(EPSSA) from TS and EP showed better induction of QR and inhibition of NO procuction with CD value and IC50 value of 0.29 mg/mL and 0.062 mg/mL respectively. The results here showed that RSSA had favourable bioactivities of anti-cancer and antiinflammatory, which might also imply that TS may exert its bioactivities by conjugating with thiols to form RSSA in vivo. However, RSSA showed a humble activity of antioxidant in vitro. Finally, the interaction between thiol-containing peptides and RSSA were further discussed with GSH or EP. The results showed that RSSA could react with thiols by thiol-disulfide exchange under neutral conditions, with the formation of new disulfide compounds and release of thiol-containing peptides. This indicated that thiol balance in vitro might be affected like that, some pathways like Nrf2-ARE would be activated as well, which, may be part of the mechanism of RSSA or even TS exerting their activities.