Cloning the full-length CDNA of actin gene and analysing alliinase gene expression in tillering onion

Tillering onion is a herbaceous plant belonging to the Liliaceae family. We cloned the cDNAs of the actin gene (AcACT, GenBank: MF919598) of tillering onion using rapid amplification of the cDNA ends. The full-length cDNA of AcACT was 1,357 bp long with an open reading frame of 1,131 bp encoding 376 amino acids. The amino acid sequence of AcACT shared > 96% similarity with the amino acid sequences of other ACTs and was found (by means of phylogenetic tree analysis) to be closely related to those of Ananas comosus and Papaver somniferum. AcACT expressions showed no significant differences (p > 0.01) in two cultivars L-SH and L-SY over three growth periods and under suitable conditions, low temperature, and short-day conditions. In addition, AcACT was used as an internal reference gene to analyse the expression of the alliinase gene (AcALL). AcALL expression trends in the roots, stems and leaves were consistent with those of diallyl disulphide and diallyl trisulphide. Thus, AcACT is highly conserved and can be used as a suitable internal reference gene when analysing gene expression in tillering onion.

Preparation and in vivo evaluation of a novel allicin pro-drug tablet / 中国药科大学学报

@#A novel allicin pro-drug tablet containing antacid pellets was developed to realize pH-regulated allicin release and to guarantee allicin yield in stomach environment.Firstly, allicin precursor pellets containing antacid pellet were prepared and artificial gastric juice was used as the medium to determine the yield of the allicin.Then, the total lipid cholesterol (TC), triglyceride cholesterol (TG), high density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) were used as indicators to study the hypolipidemic effect of allicin precursor pellets in rats.The dissolution test showed that in artificial gastric juice, the yield of allicin-containing antacid pellets exceeded 90%.In pharmacodynamic studies, it was found that antacid pellets showed the expected hypolipidemic effect on hyperlipidemia rats compared without antacid pellets.There was a very significant difference in blood lipid levels between the two test groups (P < 0.05).The allicin pro-drug tablets containing antacid pellets can effectively lower blood lipids.

Synthesis of folic acid-alliinase conjugate and its in situ inhibition of tumor cell proliferation by combination of alliin / 中国药科大学学报

@#To synthesize the folic acid-alliinase conjugate(FA-Alliinase), and to study its targeting and antitumor activity on cervical cancer HeLa cells. FA-Alliinase I and FA-Alliinase II were synthesized by two methods. The couping ratios of two conjugates measured were 12 and 31, respectively. The FA-Alliinase II with high coupling ratio was selected and its structure was characterized preliminarily. The activity of alliinase retained about 50% in FA-Alliinase II determined by HPLC. The specific effect of FA-Alliinase II on HeLa cells was observed by confocal laser and flow cytometry. The antitumor activity of conjugate combined with alliin was determined by MTT, and IC50 of alliin was(127. 6±2. 3)μmol/L. This study provides a direct evaluation method for the synthesis and optimization of FA-Alliinase.

Anti-cancerous/Anti-bacterial Activities of Allicin Generated In situ from Diastereo Pure Alliins by Alliinase.

Aims: To facilitate allicin generation in-situ from pure diastereomers of alliin by enzymatic reaction of alliinase and assess its anti-cancerous/anti-bacterial activities. Study Design: Chemical synthesis and in-vitro assay of anti-cancerous/anti-bacterial activities. Place and Duration of Study: Protein Research Laboratory, Research Resources Center, University of Illinois at Chicago, between February 2014 and February 2015. Methodology: Cancer cell viability assay MTT assay, bacterial plate-diffusion growth inhibition assay, and flow cytometry cell cycle analysis have been used to demonstrate the anticancerous/ anti-pathogen activities of the in-situ allicin. Diastereomers of alliin are produced by H2O2 oxidation of deoxyalliin, which is prepared by mixing L-cysteine and allyl bromide. Deoxyalliin and diastereomers of alliin are purified to high purity with repeated fractional crystallization. In addition, fluorenylmethyloxycarbonyl (Fmoc) protected alliin and alliin methyl ester are synthesized and purified with RP-HPLC to test the importance of amino and carboxyl groups of alliin in alliinase enzymatic reaction. Alliinase is produced by a simple and effective method from an aqueous garlic extract Results: Results from spectrophotometric alliinase activity assay indicate that (+)-L-alliin is more reactive toward alliinase than (-)-L-alliin, and both amino and carboxyl groups of the cysteine portion of alliin are critical in alliinase enzymatic reaction. Results from cancer cell viability assay MTT assay, bacterial plate-diffusion growth inhibition, and flow cytometry cell cycle analysis confirm that the in-situ allicin is as active as allicin purified from aqueous garlic extract or allicin synthesized chemically in a dose-dependent manner. Conclusion: We describe here facile pathways to synthesize diastereomerically pure alliins and isolate allinase. The in-situ allicin conversed from alliin by allinase is very active. The data obtained here provide useful information on the design of the in-situ allicin strategy.

Cloning of allanase gene from garlic bulb and its expression in Pichia pastoris / 中草药

Objective To clone the alliinase gene from the garlic bulb and construct the eukaryote expression plasmid for expressing the recombinant alliinase in Pichia pastoris system and analyzing its bioactivity. Metheds The alliinase gene was cloned from the Zhejiang garlic bulb by RT-PCR and the eukaryote expression plasmid of alliinase was constructed with the pPIczαC vector. The recombinant plasmid was transformed into Pichia pastoris X-33 by eletroporation. The positive clones were screened and were induced by methanol. Supernatants after induction were analyzed by SDS-PAGE and Western blotting. The activities of the recombinant protein and the extracted alliinase were detected by the pyruvic acid method and compared by specific activity. The contents of the two kinds of alliinase were detected by Lowry method. Results The alliinase gene was successfully cloned from the garlic bulb, the length of alliinase gene was 1 500 bp, the molecule of the recombinant alliinase was about 5.5 × 104, existed in the supernatant of Pichia pastoris. The specific activity of the recombinant protein was (82.09 ± 3.89) U/mg and the nature alliinase was (176.49 ± 5.06) U/mg. Conlusion The alliinase gene is successfully expressed in Pichia pastoris system. The recombinant alliinase has the activity of enzyme, but is lower than that of the extracted alliinase.

Research on Alliinase Immobilization and Its Properties / 中国生物工程杂志

Alliinase is immobilized on pre-activated chitosan microspheres by glutaraldehyde.The optimum immobilization conditions are as follows: glutaraldehyde concentration is 4%,alliinase concentration 20.2U,immobilization time 2h.For immobilized alliinase,the highest activity was allowed at pH 7.0 and temperature 35℃,and the Michaelis constant(Km)was disclosed to be 7.9mmol/L by Lineweaver-Burk plot,and after 10 times reuses the immobilized alliinase lost no more than 10% activity.

Isolation of alliin from Allium slltivllm L. in pilot scale

S-allylcysteine sulfoxide (alliin), a colouless and odorless solid, existed in intact garlic (Allium sativum L.) cloves. Alliin itself possessed no antibacterial activity, but it quickly converts into allicin, an antibacterial component by the enzyme alliinase, which naturally occurs in garlic. This paper reported an isolation method for alliin from garlic that was in pilot scale