Separation, Lipid-lowering And Anti-obesity Activity Of Anthocyanins From Purple Potato

Purple potato (Solanum tuberosum L.), which derives in the Andes Mountains of South America, due to the presence of rich anthocyanins in the tubers, more and more attentions are focused on their contributions to industry as natural colorants and potential health benefits for humans. In this study, High-speed Counter-current Chromatography (HSCCC) and Preparative High Performce Liquid Chromatography (Pre-HPLC) were used to separate the purple potato anthocyanin extract, and the UV-vis spectroscopy and MS were applied to identify the chemical structures of two kinds of athocyanin monomers. Then the effects of purple potato anthcyanin extracts on the proliferation and differentiation of3T3-L1preadipocytes were investigated. Last, SD rats were fed with high fat diet which was supplemented with purple potato anthocyanin extracts to assess its anti-obesity effects. This study could help to provide theoretical supports for the isolation and identification of natural pigments and the developments of health care products from purple potatoes. The main conclusions were as follows:(1)Two anthocyanins were isolated from anthocyanin extracts of purple potato flesh (AEPPF) by the combination of High-speed Counter-current Chromatography (HSCCC) and Preparative HPLC (Pre-HPLC). The biphasic solvent system of HSCCC was composed of ethyl acetate-n-butanol-water-trifluoroacetic acid (1:3:5:0.1%, v/v). The upper organic phase was used as stationary phase and the lower aqueous phase as mobile phase. The flow rate of mobile phase was2mL/min and100mg of AEPPF was injected for a single run. The HSCCC separation yielded40mg of anthocyanin mixture A (purity56.4%) and llmg of B (purity45.6%). To get high-purity anthocyanins, mixture A and B were applied to the preparative HPLC subsequently. At last,10mg of anthocyanin A (purity97.8%) and3mg of anthocyanin B (purity97.7%) were gained after the separation. The chemical structures of the isolated compounds were predicted by combination of UV-Vis spectroscopy and ESI-MS/MS, which were petunidin-3-p-coumaroylrutinoside-5-glucoside and peonidin-3-p-coumaroylrutinoside-5-glucoside respectively.(2)The effects of purple potato anthocyanin extracts on the proliferation and differentiation of3T3-L1preadipocytes were investigated. MTT assay was used for measuring the effects of purple potato anthocyanin extracts on the proliferation of3T3-L1and the50%growth inhibitory concentrations (IC50). Cell cycle and apoptosis were analyzed by flow cytometry. Then the differentiation of3T3-L1preadipocytes was induced and oil red O staining and spectrometry was used to analyze the effect of purple potato anthocyanin extracts on the differentiation of3T3-L1preadipocytes. The results showed that purple potato anthocyanin extracts had an inhibition effect on the proliferation of3T3-L1preadipocytes, in a dose-dependent way. The50%growth inhibitory concentrations (IC50) of AEPPF and AEPPP on3T3-L1preadipocytes after treatment for24h were92.0μg/mL and26.9μg/mL respectively. Treatment of cells with the anthocyanin extracts resulted in induction of apoptosis and G2/M phase cell cycle arrest. The differentiation of preadipocytes into adipocytes was suppressed with the treatment with anthocyanin extracts and the accumulation of fat in adipocytes was reduced.20μg/mL,40μg/mL and80μg/mL of AEPPF lowered the lipid content in adipocytes by16.1%,26.7%and29.6%respectively, and20μg/mL of AEPPP decreased the lipid content by39.5%, compared with the control (p<0.01).(3)To further study the anti-obesity effects of purple potato anthocyanin extracts on rats with high fat diets, the rats were randomly divided into6groups to receive4-week diets as follows:regular diet (control); high fat diet (HF); high fat diet supplemented with1g of AEPPF/kg (1‰AEPPF),5g of AEPPF/kg (5%oAEPPF),0.5g of AEPPP/kg (0.5%oAEPPP),1g of AEPPP/kg (1%oAEPPP) diets respectively. The daily diet intake and body weight of rats were recorded. After4weeks of feeding, results showed that the body weights of1‰AEPPF and5%oAEPPF groups significantly lowered compared to that of high fat diet (HF) group (p<0.05) and Lee’s index was significantly lower in the anthoyanin groups than HF group. The total weights of epididymal and perirenal fat of1%oAEPPF group were significantly less than that of HF group(p<0.05).The dietary purple popato anthocyanin extract increased the activity of SOD and had a positive effect on serum level of total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C). Purple potato anthocyanin extracts could have potential effects on improving the antioxidant status in vivo, lowering body weight and modulating lipid metabolism.