Active Component And Its Mechanism Of Urtica Fissa In Diabetic Hypogonadism

Diabetes is one of the most common metabolic disorders nowadays,and it is also the third non communicable disease after cardiovascular disease and malignant tumor.Persistent hyperglycemia in diabetic patients can cause organ damage,dysfunction and failure.Man patients with hypogonadism are characterized by decreased sexual desire and erectile dysfunction.Urtica fissa E.Pritz is widely distributed in Shaanxi,Sichuan,Guizhou,Hubei and other places in China.It is an ethnic medicine and folk medicine with a long history of application for Han,Uygur,Tibetan and other ethnic groups.U.L.shows the effects of menstruation,diuresis,breast enhancement and aphrodisiac in Uygur Medicine,but there is no systematic study on the material basis of efficacy.In this paper,U.fissa,which is rich in resources in the Qinling Mountains,was taken as the research object to study its active components and the mechanism of its aphrodisiac effect.The details were as follows.1.Extraction and separation of effective components from U.fissa and the antioxidant and hypoglycemic activities in vitro.The roots of U.fissa(21.8 kg)were systematically isolated,and the ethanol extract(2.8kg)was obtained by 95%ethanol extraction.After suspension in water,it was extracted with different polar-solvents to obtain petroleum ether phase(433.4 g),ethyl acetate phase(480.5 g),n-butanol phase(1058.3 g)and raffinate water phase(378.5 g).MLTC-1 cell activity assay showed that the level of total testosterone in ethyl acetate phase was the highest.Silica gel column chromatography was chosed to separate ethyl acetate phase,which eluted with petroleum ether/ethyl acetate in different polarity gradients to obtain Fr.1～Fr.7 components.The results showed that the total testosterone level in Fr.3 component was the highest.Fr.3(15.3 g)was eluted with ethyl acetate/methanol by silica gel column chromatography.Six compounds were separated:p-methoxybenzaldehyde(1),quercetin(2),kaempferol-3,7-rhamnoside(3),3,4-divanillyltetrahydrofuran(4),emodin(5)and p-hydroxycinnamic acid(6).Compound(4),3,4-divanillyltetrahydrofuran(DVTF),showed the highest level of total testosterone in MLTC-1.The maximum scavenging rates of DPPH free radical and hydroxyl free radical by DVTF were 84.12±2.05%and 62.49±2.63%,and IC50 were 9.30 μg/mL and 139.90 μg/mL,respectively,indicating that DVTF effected the antioxidant activity in vitro.The maximum inhibition rates of a-glucosidase and α-amylase by DVTF were 59.09±1.61%and 76.84±2.38%,and IC50 were 7.53 mg/mL and 6.96 mg/mL,respectively,indicating that DVTF effected the hypoglycemic activity in vitro.2.Effect of DVTF on gonadal function in normal mice and preliminary evaluation in safety.The three administrations of DVTF in mice were compared:subcutaneous injection,intraperitoneal injection and intragastric administration,and the intragastric administration was determined.The acute toxicity test of DVTF on mice were studied for 14 days.The results showed that DVTF had no obvious toxic reaction in mice.Healthy ICR male mice were divided into control group,positive control group(sildenafil)and DVTF with low,medium,high dose groups(25,50 and 100 mg/kg)for 4 weeks.The physiological indexes and mating behavior in mice were observed,and the levels of serum total testosterone and free testosterone were measured,and the histomorphology of main organs were observed.Compared with the control group,there were no significant difference in food intake,water intake and body weight of mice in groups of DVTF.The low,medium and high dose groups of DVTF could significantly shorten mount latency,increase mount number and intromission number of the mice.Mount latency of mice in the high dose group was shortened by 46.32%(p<0.01),and mount number increased by 14.67%(p<0.01),and intromission number increased by 32.00%(p<0.01).Serum total testosterone increased by 8.21%,17.31%and 43.98%in low,medium and high dose groups of DVTF,and free testosterone increased by 1.02%,12.17%and 14.98%,respectively(p<0.05,p<0.01).The liver,kidney,heart,testis,epididymis and pancreas of mice showed no abnormalities.These results showed that DVTF increased the levels of serum total testosterone and free testosterone in mice,promoted sexual ability,enhanced mating behavior,and DVTF was safe and non-toxic to the mice.3.Protective effects of DVTF on gonadal function in diabetic mice.Type 2 diabetes mellitus were induced by high fat and high glucose combined with streptozotocin in ICR male mice.They were divided into control group,model group,positive control group(metformin)and DVTF low,medium and high dose groups(25,50 and 100 mg/kg)and continuously administered for 4 weeks.Compared with the model group,DVTF had reduced the food and water intake,and increased their weight in the diabetic mice.The food intake decreased by 11.82%,and the water intake decreased by 31.45%,and the body weight increased by 24.80%(p<0.01)in DVTF high dose group.The fasting blood glucose of mice decreased by 22.28%,31.58%and 43.33%respectively in DVTF low,medium and high dose groups(p<0.01).DVTF increased fasting insulin level and insulin sensitivity index(IS)in diabetic mice,and lowered the homeostatic model assessments for insulin resistance(HOMA IR).However,HOMA IR only in high dose group had statistical significance(p<0.05),indicating that DVTF can improve insulin resistance in diabetic mice.In DVTF low,medium and high dose groups,mount latency in diabetic mice was shortened by 22.86%,32.00%and 34.85%(p<0.01),and mount number increased by 10.20,10.40 and 11.60 times(p<0.01),and intromission number increased by 18.00,21.50 and 22.00 times(p<0.01),indicating that DVTF could improve sexual arousal and enhance mating behavior in diabetic mice.In addition,the total testosterone levels in DVTF medium and high dose groups increased by 45.36%and 87.29%,and the level of free testosterone increased by 51.56%and 62.88%respectively(p<0.05,p<0.01).In DVTF high dose group,the number of spermatogenic cells and spermatocytes increased,and the gap decreased and the number of spermatozoa increased.The structure of testicular spermatogenic cells was basically complete,no obvious cell gap was found,the cell membrane was complete,the nucleus was clear,the mitochondrial structure was basically norrmal,and the expansion of endoplasmic reticulum was reduced.The mitochondria of leydig cells increased and the expansion of smooth endoplasmic reticulum decreased.Thus,DVTF restored the gonadal function of diabetic mice and relieved testicular tissue and cell injury.DVTF had protective effects on gonadal function in diabetic mice.4.Protective mechanism of DVTF on gonadal function in diabetic mice.The metabolites in the serum of diabetic mice were analyzed and detected with UPLC-Q/TOF-MS.The metabolite data were analyzed by XCMS online database,SIMCA-P software,HMBD database and MetaboAnalyst database.34 differential metabolites and 12 metabolic pathways were found,in which the steroid metabolic pathway was obviously changed,involving two biomarkers:22β-hydroxycholesterol and 20 α-hydroxycholesterol.Western blotting was used to study the expression of proteins affecting testosterone synthesis in testis of diabetic mice.It was found that the expression of transcription factor Nur77,steroidogenic acute regulatory(StAR)and cytochrome P450 family 11 subfamily A member 1(P450scc)in testis of diabetic mice were downregulated.After DVTF intervention,the expression of Nur77,StAR and P450scc in diabetic mice was up-regulated,indicating that DVTF can regulate the testosterone synthesis-related proteins in diabetic mice.The binding of the ligand DVTF to the receptor protein Nur77 was simulated by molecular docking calculation.It was found that DVTF could form intermolecular hydrogen bonds at GLY45,TYR122,LYS125 and LEU 166 and hydrophobic interactions with ARG123 of Nur77.In conclusion,DVTF has a binding force with Nur77,a transcriptional regulator upstream of testosterone synthesis protein,which promotes testosterone synthesis.Nur77 is a key target of DVTF against diabetic hypogonadism.In summary,DVTF is the one of the material basis for the anti-diabetic hypogonadism of U.fissa.DVTF effects the antioxidant and hypoglycemic activity in vitro.DVTF can improve insulin resistance in diabetic mice,and promote the gonadal function of normal and diabetic male mice,and does not produce obvious toxic effects.The mechanism of DVTF improving the gonadal function of diabetic mice is related to the regulation of Nur77.