Separation And Identification Of Litchi Pericarp Procyanidins And Their Action Mechanism In Attenuating Atherosclerosis

Litchi(Litchi chinensis Sonn.)is a typical fruit of subtropical areas.Its planting area and production in China ranks first in the world.It was found that the by-products of litchi such as pericarp and seed,are rich in phenolics.Studies have shown that litchi pericarp was rich in A-type procyanidins,which are relatively rare phenolics in nature when compared to B-type procyanidins.The chemical composition of procyanidins in litchi pericarp has not been clearly clarified up till now,due to their complex structures and the existence of many isomers.The biological activity of procyanidins are closely related with their chemical structures,and many studies have confirmed that B-type procyanidins from grape seed exhibited a variety of biological activities including anti-atherosclerosis.However,the biological activity and action mechanism of A-type procyanidins have not been systematically investigated.Dysregulation of lipid metabolism and inflammation were the main mechanism in promoting the development of atherosclerosis.It has been demonstrated that the anti-atherosclerotic activity of B-type procyanidins was related to their regulatory effect on lipid metabolism.It has been reported that procyanidins have significant anti-inflammatory activity in animals suffering from cancers or acute inflammation induced by LPS.However,it is unknown whether anti-inflammatory effect of procyanidins was related with their anti-atherosclerotic activity.Due to the differences in structures between A-type and B-type procyanidins,the biological activity of A-type procyanidins is waiting to be investigated.Therefore,the main work done in the present study was as follows.Litchi pericarp procyanidins oligomers(LPPO)was purified after optimizing ultra-high-pressure-assisted extraction of procyanidins from litchi pericarp,and a qualitative and quantitative analysis method of procyanidin compounds by HPLC-QqQ-MS was establish.The HPLC-QqQ-MS method was then used to compare the procyanidin profiles of litchi pericarp from different cultivars cultivated in Southern China and to analyze the changing tendency of procyanidins in pericarp samples stored under different temperature.Finally,the anti-atherosclerotic effect of LPPO on apoE knockout mice fed with high fat diet was investigate to explore the molecular mechanism of LPPO from regulating lipid metabolism in liver and intestine and inhibiting inflammation in aorta and the whole body.The main results were presented as follows1.Extraction,isolation and characterization of procyanidins from litchi pericarpA response surface analysis method was adopted to establish the optimum ultra-high-pressure-assisted extraction of procyanidins from litchi pericarp with pressure,pressure holding time,ethanol concentration and ratio of solid to liquid as the analysis factors.The optimized conditions are as follows:295 MPa pressure,13 min pressure holding time,70%ethanol concentration and 16:1 liquid to solid ratio,under which the yield of procyanidins from litchi pericarp was 2.4%.Compomared to conventional ethanol extraction and ultrasonic-assisted extraction methods,UHP process significantly increased the yields of the total procyanidins and the antioxidant activity of the extracts.Purification through macroporous resin adsorption and ethyl acetate partitation significantly increased the total procyanidins content and ORAC and CAA activity of LPPO.Several phenolic compounds were identified in litchi pericarp extracts,including 8 flavonoid compounds such as epicatechin,kaempferol-3-rutinoside,narcissin,rutin,quercetin-3-rutinose-7-rhamnoside,quercetin,isoquercetin and typhaneoside,four procyanidin oligomers PA1,PA2,PB2 and PC1,two A-type dimer isomers,one B-type dimer isomers,six A-type trimer isomers and four B-type trimer isomers,using HPLC-QqQ-MS method.HPLC-QqQ-MS quantitative analysis showed that the composition of procyanidins in extracts before and after purification remained the same,but the total content of epicatechin,PA2 and other procyanidin oligomers increased from 16.05%to 87.3%after purification.2.Procyanidin profiles and antioxidant activities of litchi pericarp of different cultivars cultivated in South ChinaThe total phenolics,total flavonoids,total procyanidins contents and the ORAC and CAA antioxidant activities of litchi pericarp of 14 cultivars were measured.The results showed that there were significant varietal differences in the content of total procyanidins,total phenolics and total flavonoids of litchi pericarp.Phenolic compound with the highest content in litchi pericarp of different cultivars was epicatechin identified by HPLC-QqQ-MS method,followed by PA2.Significant varietal differences in the content of procyanidin compounds were found.The content of A-type procyanidin oligomers and B-type procyanidin oligomers varied from 950 to 3077 ?g/g and 94.8 to 446.4 ?g/g in litchi pericarp of 14 cultivars respectively,as well as the content of total procyanidin compounds(including epicatechin)from 1421 to 5233 ?g/g accounting for 76.9-94.6%of the total content of detected phenolic compounds,which showed that procyanidins were the main phenolic compounds in litchi pericarp.Also,there were significant varietal differences in ORAC and CAA antioxidant activity of litchi pericarp that had a significant positive correlation with total procyanidins content(P<0.01),suggesting that procyanidins may be the main active compounds of antioxidant activity from litchi pericarp.Based on the cluster analysis of procyanidins content and antioxidant activity,four cultivars including Heiye,Feizixiao,Nuomici and March Red were clustered into a group with the highest phenolic contents and antioxidant activity.3.The variation of the composition and antioxidant activities of phenolic compounds in litchi pericarp during storage periodThe results showed that the content of total procyanidins in litchi pericarp were decreased as the increase of storage time in both conditions of 4?,7 d and room temperature(26±2?),96 h,which were decreased most quickly during storage period of 1 d at 4? and 12 h at room temperature,but slowly after that.Finally,the content of total procyanidins was decreased by 24.3%after 7 d at 4? and 53.9%after 96 h at room temperature.HPLC-QqQ-MS analysis showed that the contents of 24 phenolic compounds including epicatechin and PA2 from litchi pericarp were decreased as the increase of storage time in both storage conditions.PC1,PA 1 and isoquercitrin were more degraded to reduce by 44%,63%and 86%respectively after 7 d at 4? but other phenolic compounds about 35%.At room temperature,total contents of epicatechin,kaempferol-3-rutinoside,rutin,isoquercitrin,PA1,PC1,A-type dimer isomer-1 and B-type trimer isomer-4 were decreased by more than 40%of the fresh litchi pericarp content.Therefore,the storage condition at 4? was more favorable to retard the degradation of phenolic compounds in litchi pericarp.4.Safety evaluation of LPPO and study on its anti AS effectSafety evaluation:the safety of LPPO was evaluated by acute toxicity test and 30d feeding subacute toxicity experiment,which found that an intragastric administration of 2000 mg/kg bw LPPO exhibited no toxicity in male and female KM mice.In addition,an intragastric administration of more than 2000 mg/kg·bw LPPO had no acute toxicity.Given on the food utilization,hemogram and blood biochemical indexes,continuous intragastric administration of 250,500 or 1000 mg/kg·bw LPPO in the weaning male and female SD mice also exhibited no subacute toxicity.Study on the effect of anti AS:after 16 w continuous intragastric administration of low dose(100 mg/kg bw)and high dose(200 mg/kg bw)LPPO in apoE-KO mice fed a high-fat diet,serum and liver biochemical indexes were measured to study the effects of LPPO on AS plaque area in aortic sinus,hepatic lipid accumulation,blood,liver and fecal lipid content and the body antioxidant state.The results showed that LPPO significantly reduced AS plaque area/lumen area in aorta lesions by 12.7%(P<0.05)and 25.1%(P<0.05),compared with model group,showing a significant effect against AS in apoE-KO mice.LPPO significantly reduced hepatic steatosis induced by high fat diet,serum low density lipoprotein cholesterol(LDL-c)and total cholesterol(TC)level(P<0.05)so that the fatty degeneration score in liver tissue decreased from 2.75 in HFD group to 2.10(P<0.05)in low dose of LPPO group and 1.64(P<0.05)in high dose of LPPO group.Cholesterol and triglyceride levels and total bile acid in feces in two LPPO group were significantly increased(P<0.05);furthermore,LPPO treatment significantly reduced MDA content and increased the activity of antioxidant enzymes SOD,GSH-Px and CAT in serum and liver(P<0.05),compared to that of model group.These results suggested that the anti-atherosclerotic effect of LPPO may be related to regulating cholesterol metabolism and inhibiting oxidative stress in apoE-KO mice fed a high-fat diet.5.Molecular mechanism of the anti-atherosclerotic activity of LPPOThe study confirmed the anti-atherosclerotic effect of LPPO to further elucidate the effects of LPPO on the expression of intestinal cholesterol absorption and transport related genes,the nuclear translocation of PPARa,PPARy,SREBP2 and LXRa and the expression of their downstream cholesterol metabolism related genes,as well as aorta NF-?B activation and inflammatory protein expression levels by qPCR,western blot and liquid chip.The results showed that LPPO significantly inhibited the expression of NCP1L,ACAT2 and MTP and increased ABCG5/G8 expression in intestinal mucosa in apoE-KO mice fed a high fat diet(P<0.05);LPPO promoted the expression of LDLR,CYP7A1 and ABCG5/G8 as the increased nuclear translocation of PPAR?,PPAR? and LXRa in liver(P<0.05),which indicated that LPPO improved cholesterol metabolism through inhibiting cholesterol absorption in intestinal epithelial cells,promoting LDL-c uptake in the liver and the metabolism of cholesterol to bile acids into the intestine.Additionally,LPPO reduced aorta I?B? phosphorylation level and the expression of ICAM-1,VCAM-1 and COX-2(P<0.05),while the levels of serum IL-1,IL-6,IL-12p70,TNF-?,Rantes and MIP-la were reduced(P<0.05)and anti-inflammatory cytokine IL-10 level were increased(P<0.05).These results indicated that LPPO inhibited aorta NF-?B activation,down-regulated the expression of inflammatory factors and attenuated the inflammatory reaction in the vessels.Therefore,LPPO played an anti-atherosclerotic role by improving dysfunction of cholesterol metabolism and inhibiting inflammation of blood vessels.The main innovations of our research as follows:? The anti-atherosclerotic effect of LPPO,the predominant components of which are A-type procyanidins,have been confirmed in the study.The moleculaf mechanism has been demonstrated as to improve the dysregulation of cholesterol metabolism by influencing the absorption and transportation of cholesterol in intestine and the intake and metabolism of cholesterol in liver and to alleviate the inflammatory response in aorta and the whole body by inhibiting the nuclear translocation of NF-?B and the subsequent increased expression of inflammatory factors,adhesion molecules and chemokines.?UHP-assisted extraction of procyanidins from litchi pericarp was optimized and a method for qualitative and quantitative analysis of procyanidin compounds by HPLC-QqQ-MS was established in the study.PA2,PA1,PB2,PC1 and 13 procyanidin oligomer isomers with A-type or B-type structures were identified from litchi pericarp.This method makes the accurate quantification of procyanidin isomers possible under the condition where the isomers can not be separated well by HPLC.? The varietal differences of procyanidin profiles and antioxidant activity of litchi pericarp were analyzed in this study.The changing tendency of procyanidins under different storage temperature was investigated and 4 ? storage was found to slow down the degradation of procyanidins in litchi pericarp effectively in 7 days.The significance of our study:The anti-atherosclerotic effect of litchi pericarp extract rich in A-type proycianidins was explored and their molecular action mechanism was demonstrated as improving cholesterol metabolism dysfunction in liver and small intestine and inhibiting inflammation in aorta.These findings provide a theoretical basis for the development and utilization of plant resources rich in A-type procyanidin.Efficient extraction and qualitative and quantitative analysis method were established to purify and identify procyanidins from litchi pericarp.A storage condition was found under which the degradation of procyanidins was slow.The results would provide techniques for the deep processing of litchi by-products and the development of functional food,which is of high significance for the sustainable development of the litchi industry.