RESULTS: A lactic acid bacteria strain S165 with glycosidase-producing activity was isolated from traditional Chinese fermented foods, which was identified and grouped according to API 50 CHL kit and 16S rDNA sequence analysis. Subsequently, notoginsenoside R1 underwent a 30-day fermentation period by the strain S165, and the resulting products were analyzed using High-performance liquid chromatography (HPLC), Ultra-performance liquid chromatography (UPLC)-mass spectrometry (MS)/MS, and 13C-Nuclear magnetic resonance (NMR) techniques. Employing a model of Lipopolysaccharide (LPS)-induced damage to Caco-2 cells, the damage of Caco-2 cells was detected by Hoechst 33 258 staining, and the activity of notoginsenoside R1 biotransformation product was investigated by CCK-8 and western blotting assay. The strain S165 was identified as Lactiplantibacillus plantarum and was used to biotransform notoginsenoside R1. Through a 30-day biotransformation, L. plantarum S165 predominantly converts notoginsenoside R1 into 3β,12β-dihydroxydammar-(E)-20(22),24-diene-6-O-β-D-xylopyranosyl-(1→2)-β-D-glucopyranoside, temporarily named notoginsenoside T6 (NGT6) according to HPLC, UPLC-MS/MS, and 13C-NMR analysis. Results from CCK-8 and Hoechst 33258 staining indicated that the ability notoginsenoside T6 to alleviate the intestinal injury induced by LPS in the Caco-2 cell was stronger than that of notoginsenoside R1. In addition, Western blotting result showed that notoginsenoside T6 could prevent intestinal injury by protecting tight junction proteins (Claudin-1, Occludin, and ZO-1).
CONCLUSIONS: Notoginsenoside R1 was biotransformed into the notoginsenoside T6 by L. plantarum S165, and the biotransformed product showed an enhanced intestinal protective effect in vitro.
方法:从中国传统发酵食品中分离出一株具有产糖苷酶活性的乳酸菌S165,根据API50CHL试剂盒和16SrDNA序列分析进行鉴定和分组。随后,三七皂苷R1经过菌株S165的30天发酵期,UPLC-MS/MS,和13C-NMR技术。采用LPS诱导的Caco-2细胞损伤模型,通过Hoechst33.258染色检测到caco-2细胞的损伤,用CCK-8和免疫印迹法检测三七皂苷R1生物转化产物的活性。
结果:菌株S165被鉴定为植物乳杆菌,并用于生物转化三七皂苷R1。通过30天的生物转化,植物乳杆菌S165主要将三七皂苷R1转化为3β,12β-二羟基多巴胺-(E)-20(22),24-二烯-6-O-β-D-吡喃木糖基-(1→2)-β-D-吡喃葡萄糖苷,根据HPLC暂时命名为三七皂苷T6(NGT6),UPLC-MS/MS和13C-NMR分析。CCK-8和Hoechst33.258染色结果表明,三七皂苷T6减轻LPS诱导的Caco-2细胞肠损伤的能力强于三七皂苷R1。此外,Westernblotting结果显示三七皂苷可通过保护紧密连接蛋白(Claudin-1,Occludin,ZO-1).
结论:三七皂苷R1经植物乳杆菌S165生物转化为三七皂苷T6,其生物转化产物在体外具有增强的肠道保护作用。