Research On The Ovarian Protective Components And Protective Mechanisms Of Pine Pollen

Pine pollen is an important kind of pure natural pollen that can be used for both medicine and food.It is not only widely used in traditional Chinese medicine prescriptions,but also in the research and development of health food in recent years.It has been proved to possess antiinflammation,antioxidant and other functions.The ovary is an important organ of women,and the preservation of ovarian function has received extensive attention in recent years.A variety of plant polysaccharides containing uronic acid and phenolic substances with antioxidant active groups have been proved to possess ovarian protective functions.The phenolic substances contained in pine pollen are similar in structure to those reported to possess ovarian protective functions.Pine pollen also contains polysaccharides and other components that may possess ovarian protective functions.However,the protective effects of pine pollen and its extracts on ovarian function are rarely reported,so do its specific mechanisms.In order to broaden the application directions of pine pollen,this paper established a cyclophosphamide-induced premature ovarian failure model and a natural aging model in rats,and analyzed the protective effects of pine pollen on ovaries and functional properties for delaying ovarian aging from four mechanisms: hormone regulation,antioxidation,anti-apoptosis and anti-inflammatory.On the basis of verifying the protective function on ovary,a rat model of premature ovarian failure was selected to compare and analyze the differences in ovarian protective effect of pine pollen extracts containing different components obtained by water extraction,ethanol extraction and ultrafiltration.The above four mechanisms were synchronously analyzed to clarify the effect and mechanism of different functional components in pine pollen on ovarian protection,and it can provide theoretical and technical basis for the development of pine pollen ovarian protective functional products in the future.The main research contents and results are as follows:First,the cyclophosphamide(60 mg/kg BW 1 d+10)-induced premature ovarian failure model rats were gavaged with pine pollen of high(1.5 g/kg BW),medium(1.0 g/kg BW)and low(0.5 g/kg BW)doses for 4 weeks.After the experiment,the rat ovaries and serum were collected for the detection of morphology,hormones,antioxidant capacity,anti-apoptosis capacity and anti-inflammatory capacity.The results showed that all three doses of pine pollen could improve the ovarian index of rats with premature ovarian failure,promote the normal development of follicles,regulate the levels of serum sex hormones and gonadotropins,improve the ovarian reserve,reduce ovarian oxidative damage and the degree of apoptosis of granulosa cells,increase the expression of ovarian anti-apoptotic proteins,reduce the expression of ovarian pro-apoptotic proteins,and reduce the level of serum inflammatory factors.The above results indicated that pine pollen can improve ovarian function in rats with premature ovarian failure,and there was a dose-effect relationship.Secondly,the natural aging model rats(12 months old,with abnormal estrous cycle)were gavaged with pine pollen of high(1.5 g/kg BW),medium(1.0 g/kg BW)and low(0.5 g/kg BW)doses for 4 weeks.After the experiment,the ovaries and serum of the rats were collected for the detection of morphology,hormones,antioxidant capacity,anti-apoptosis capacity and antiinflammatory capacity.The results showed that both medium and high dose of pine pollen can improve the ovarian index of naturally aging rats,reduce follicular atresia,delay follicle consumption,regulate serum sex hormone and gonadotropin levels,improve ovarian reserve to a certain extent,improve the activity of ovarian antioxidant enzymes,attenuate ovarian oxidative damage,reduce the degree of granulosa cell apoptosis,increase the expression of ovarian anti-apoptotic proteins,reduce the expression of ovarian pro-apoptotic proteins,reduce the level of ovarian pro-inflammatory factors and increases the level of anti-inflammatory factors.This showed that pine pollen had the effect of delaying the process of ovarian aging in naturally aging rats,but the effect was limited,and there was still a large gap between the ovarian status of them and young rats.Finally,on the basis of confirming the above functions,the pine pollen was extracted with water and 70% ethanol,and the macromolecular substances(molecular weight greater than 5k Da)in the aqueous extract and the small molecular substances(molecular weight less than 5k Da)in the ethanol extract were obtained by ultrafiltration in order to explore the material basis of pine pollen on ovary protection.Aqueous extract,ethanol extract,macromolecular and micromolecular were obtained after extraction.The components and contents of carbohydrates and phenolic compounds in the four extracts was determined,and their differences in ovarian protection on rats with premature ovarian failure were compared.The results showed that the active substances in the aqueous extract were mainly polysaccharides and phenolic acids(gallic acid and digallic acid).The active substances in the macromolecular substances were mainly polysaccharides,and the content was significantly higher than that in the aqueous extract,and its monosaccharide composition is close to the aqueous extract.The active substances in the ethanol extract were mainly phenolic acids(coumaroylquinic acid,caffeic acid,p-coumaric acid)and flavonoids(taxifolin,naringenin),and contained very few polysaccharides.The contents of various phenolic compounds in micromolecules were significantly higher than those in the ethanol extract,and the protocatechuic acid,which was not detected in the ethanol extract,was identified,and its polysaccharide content was less.In the experiment to explore the ovarian protective function of the active substances in different extracts,the premature ovarian failure model with better protective effect of pine pollen was selected from the two models as the administration object.The four extracts were used as gavage drug,and the same indicators as the previous animal experiments were determined.The results showed that all four kinds of extracts could improve the function of the ovary to different degrees,reduce the apoptosis and atresia of the follicles,and promote the normal development of the follicles.Pine pollen phenols in ethanol extracts and micromolecules had strong ovarian protective functions.The protective effect of the aqueous extract was in the middle,and the protective effect of macromolecules was the worst,but the anti-inflammatory ability was the same as the other three extracts.From the above research results combined with the molecular mechanism of the active ingredients in the extracts,it can be deduced that the phenolic substances in pine pollen and polysaccharides both had the effect of ovarian protection,and phenolic substances are the main components of ovarian protection.