Study On Increasing The Active Ingredients And Lipid-lowering Effects Of Pine Pollen By Fermentation

Pine pollen is a kind of medicinal and edible material that is rich in bioactive ingredients.It has multiple physiological effects,such as antioxidation,liver protection,anti-inflammatory,etc.In recent years,it has been widely used in the development of functional foods.Studies have shown that pine pollen also has weight loss effect.It can significantly control the weight gain of mice and maintain the balance of lipid metabolism,and the phenolic components may be the effective ingredients for controlling obesity.However,previous research in our laboratory found that the phenolic ingredients in pine pollen mostly exist in a bound state,which prevents them from fully exerting their effects.How to improve the conversion of phenolic substances from the bound state to the free state,and verify the activity and study the mechanism have become the key scientific issues to realize the high-quality utilization of pine pollen.In this study,we fermented pine pollen and optimized the fermentation conditions to increase the content of active substances such as phenolics and flavonoids.Thus,we constructed a 3T3-L1 cell differentiation model and oleic acid-induced lipid accumulation Hep G2 cell model to investigate the effects of NFPP and FPP on lipid deposition in these two in vitro models,as well as the expression of genes and proteins related to lipid metabolism.We explored the mechanism and possible reasons for the improvement of lipid metabolism by FPP,so as to provide theoretical basis and guidance for the development of pine pollen functional products with good lipid-lowering effects.The main research contents and results are as follows.Firstly,the fermentation of pine pollen was evaluated using total phenolics content,total flavonoids content,ABTS,and FRAP as indicators.Four types of edible fungi,Rhizopus oryzae,Mucor racemosus,Cunninghamella echinulata,and Monascus purpureus were screened.The results have demonstrated that Rhizopus oryzae can increase the total phenolics content,total flavonoids content,ABTS,and FRAP ability the most.Clean and dry pine pollen was used as the sole source of nutrients（substrate）for liquid medium（solid-liquid-ratio 1:20）and was fermented by Rhizopus oryzae in flask with 5%（m/v）inoculum（containing 10⁶ spores/m L）.The fermentation conditions for Rhizopus oryzae were explored,and the optimal fermentation conditions were determined as:fermentation duration of 4 days,fermentation temperature of30℃,and inoculum amount of 10%（v/v）.Under these fermentation conditions,the total phenolics and total flavonoids content of the pine pollen significantly increased by 7.89%,87.71%and 88.89%,respectively.Furthermore,the results of HPLC showed that compared with NFPP,the content of catechin,p-coumaric acid,pinocembrin and naringenin were significantly increased to 448.66μg/g,179.23μg/g,246.99μg/g and 2027.30μg/g,as 2.76,1.10,1.88 and4.27 times of NFPP,among which naringenin increased most.The effects of NFPP and FPP on lipid accumulation were studied with the 3T3-L1 cell differentiation model.The results showed that NFPP and FPP at concentrations of 0-400μg/m L were generally non-toxic to 3T3-L1 cells.Within this concentration range,the intracellular lipid content decreased as the dose of both NFPP and FPP,FPP could significantly reduce the lipid accumulation in 3T3-L1 cells,which was better than NFPP under the same concentration.In addition,compared to the NFPP group,FPP（200,400μg/m L）can activate adenosine monophosphate-activated protein kinase（AMPK）,reduce the expression of PPARγ,C/EBPα,FAS and increase the expression of PPARα,ATGL,and HSL.This study preliminarily explored the possible reasons for the reduction of lipid deposition in 3T3-L1 cells by FPP.Treat 3T3-L1cells with 1.5μmol/L and 3.0μmol/L naringenin（corresponding to the naringenin content of the FPP group at concentrations of 200μg/m L and 400μg/m L,respectively）,the results showed that 1.5μmol/L naringenin inhibited the expression of C/EBPαand PPARγproteins,promoted the expression of PPARαand ATGL,while PPARγwas effective at a concentration of 3.0μmol/L.The results suggest that FPP may reduce the accumulation of intracellular lipids by activating the AMPK signaling pathway,inhibiting the expression of genes and proteins related to fat production,and promoting the expression of genes and proteins related to fatty acid oxidation and lipid decomposition.Compared to NFPP,the reason why FPP has a better lipid-lowering effect may be due to the increase in phenolic substances,including the significant increase in naringenin content after fermentation.Finally,using a Hep G2 oleic acid-induced lipid accumulation cell model,we investigated the effects of NFPP and FPP on hepatic lipid metabolism.The results showed that at concentrations of 0-400μg/m L,the survival rate of Hep G2 cells was 100%.We selected 200μg/m L and 400μg/m L for further experiments.The results showed that the TG and TC contents in the FPP treatment group were significantly lower than those in the NFPP treatment group.Compared to NFPP group,the FPP group could increase the enzyme activity of SOD and GSH-Px,and decrease the content of MDA.In addition,the FPP group could down-regulate the expression of SREBP-1,FAS,and ACC,and up-regulate the expression of PPARαand ATGL.Further preliminary exploration revealed that FPP may have a good potential for inhibiting lipid deposition in Hep G2 cells,as treatment with naringenin at 1.5μmol/L and 3.0μmol/L could inhibit the expression of SREBP-1,FAS,and ACC,and promote the expression of PPARαand ATGL genes and proteins in a dose-dependent manner.It is speculated that the FPP group may affect hepatic lipid metabolism through inhibiting lipid synthesis,promoting fatty acid oxidation into H₂O and CO₂,promoting the decomposition of triglycerides,and bioactive substances from pinepollen including naringenin possibly playing an important role in lipid lowering.