Study On The Safety Evaluation Of Apigenin And Its Effect On Decreasing Blood Pressure As Well As Its Possible Mechanisms

BackgroundApigenin is one of natural flavonoids that are almost ubiquitously present in many fruits,vegetables and beans,with the highest in celery.There has been a long history for people ingesting flavonoids.However,high doses of flavonoids may act as pro-oxidant,form lipid peroxidate,induce genetic mutation,cause DNA damage and breakage.The potential toxicological properties of apigenin remain largely unkown.Hypertension is one of the cardiovascular diseases which threatens the health of human being.It is an important risk factor of many diseases such as coronary heart disease,cerebrovascular and renal disease.Prevention and treatment of hypertension and its complications have an important clinical significance.The effects of long over-loading pressure on the vascular endothelium and myocardium in spontaneously hypertension rats(SHR) are similar to those in patients with essential hypertension. Apigenin has a varity of bioactivities,such as antioxidantive,anti-inflammatory, antiallergic effects.However,the mechanisms of hypotensive effect of apigenin remain largely unkown.Therefore,the studies on effects and mechanisms of apigenin on blood pressure of SHR will contribute to supply useful experimental evidence for the reasonable use of apigenin.ObjectivesTo evaluate the safety of high ingestion of apigenin,dose-response relationship as well as possible mechanisms of apigenin in lowering blood pressure,so as to provide scientifc support for the reasonable use and development of apigenin.Methods1.Safety evaluation of apigeninAccording to the procedure and methods of food toxicological safety evaluation,a series of toxicity studies were conducted on the ingestion of apigenin(including acute toxicity,genetic toxicity as well as sub-chronic toxicity experiments). 2.Study on the effects of apigenin on lowering blood pressure and its possible mechanisms2.1 Effects of apigenin on lowering blood pressure of SHR and its possible mechanismsAccording to body weight and blood pressure,male SHR rats aged 12 weeks were randomly divided into 6 groups,10 rats of each group,with different levels of 0,0.007, 0.026,0.104,0.417g/kg bw of apigenin as well as 12mL/kg bw captopril positive control group.Systolic blood pressure(SBP) and heart rate were measured once a week,body weight was measured twice a week.At the end of the above animal study,all rats were anesthetized with 1%sodium pentobarbital(i.p.),and 6mL arterial blood was drawn for the determination of blood plasm renin-angiotensin-aldosterone system(RAAS),possible sensitive indexes of blood pressure,including uric acid(UA),C-reactive protein(CRP),interleukin-6(IL-6), oxidative stress level,the effect of apigenin on SHR left ventricular hypertrophy index(LVHI),as well as the effect of apigenin on SHR kidney RAAS by real-time PCR and immunohistochemistry methods.2.2 Study on possible mechanisms of apigenin relaxing rat aortic rings effectsRats were anesthetized with 1%sodium pentobarbital(i.p.) and killed by exsanguination.The thoracic aorta was immediately isolated and put in oxygenated Kreb solution at 4℃.The adherent connective tissue was cleaned and the vessel was cut into 3-4 mm rings with special care not to damage the endothelium.In some preparations,endothelium was mechanically removed by gently rubbing the lumen with a wet cotton tip.Vascular rings were suspended in an organ bath filled with 20 mL Kreb solution which was maintained at 37℃and ventilated continuously with 95%O₂ and 5%CO₂. The contraction was measured with a multichannel acquisition and analysis system (BIO-PAC MP150,America).Related studies were conducted on the effect of apigenin on the contraction induced by PE and role of endothelium,K⁺ channel as well as Ca²⁺ channel in PE-induced relaxation. Results1.Safety evaluation of apigenin1) For the acute toxicity,the MTD was higher than 8g/kg bw.2) No apigenin-related genetic toxicity was found.3)Except for GST in 8g/kg bw dose of serum and 2g/kg bw dose of liver homogenate of male rats was significantly lower than control group(P＜0.05 or 0.01);4g/kg bw dose of GSH-P_X in serum of both sex as well as 2,4g/kg bw dose of SOD and GSH-Px of male rats liver homogenate were significantly lower than control groups((P＜0.05), there were no apigenin-related adverse effects on the body weight,food intake,food consumption,hematology,serum biochemistry,urine routine as well as histopathology.2.Study on the effects of apigenin on lowering blood pressure and its possible mechanisms2.1 Effects of apigenin on lowering blood pressure of SHR and its possible mechanisms1) After apigenin administration for 2 weeks,SBP of captopril positive group and 0.417g/kg bw group was significantly lower than control group(P＜0.05).From the 3th of experiment,SBP of all groups except 0.007g/kg bw was significantly lower than the control group(P＜0.05).There was no significant difference between body weight and heart rate throughout the experiment(P＞0.05).2) Effect of apigenin on RAAS of SHR blood plasmAngâ…¡and ACE in blood plasm of captopril positive control group and 0.417g/kg bw group was significantly lower than control group(P＜0.05).Ald of captopril positive control group was also significantly lower than control group(P＜0.05).3) Effect of apigenin on ACE2,ACE,eNOS and ET-1 of SHR kidneyAfter apigenin administration for 4 weeks,transcription level of ACE2 mRNA and eNOS mRNA of captopril positive control group and 0.417g/kg bw group was significantly higher than control group(P＜0.05).Transcription level of ACE and ET-1 mRNA was significantly lower than control group(P＜0.05).The immunohistochemistry results were consistent with the above gene expression results. 4) Effect of apigenin on vasorelaxation and vasoconstriction factors of SHRCalcitonin gene related protein(CGRP),eNOS and NO of 0.417g/kg bw group were significantly higher than control group(P＜0.05).cGMP and NO/ET of 0.026,0.104,0.417g/kg bw were also significantly higher than control group(P＜0.05).NO/Angâ…¡of captopril positive control group and 0.007,0.104,0.417g/kg bw groups were also significantly higher than control group(P＜0.05).There was no significant difference between ET-1 and PGI₂ in all groups(P＞0.05).5) Effect of apigenin on oxidative stress of SHRAfter apigenin administration for 4 weeks,O₂ level was greatly lower than control group(P＜0.05),while SOD activity was greatly higher(P＜0.05) than control group(P＜0.05).6) Effect of apigenin on LVHI of SHRAfter apigenin administration for 4 weeks,although there were descending tendency among LVHI and heart index,yet no significant difference was found(P＞0.05).7) Effect of apigenin on UA,CRP and IL-6 of SHR blood plasmAfter apigenin administration for 4 weeks,there was no obvious difference among UA,CRP as well as IL-6 in all groups(P＞0.05).2.2 Study on possible mechanisms of apigenin relaxing rat aortic rings effects1) Apigenin had no effect on the basal tension in rat aortic rings.2) Apigenin can relax PE pre-contracted rings in both endothelium-intact aortic and endothelium-denuded aortic in a dose-dependent manner,with the effect of endothelium-intact aortic significantly stronger than that of endothelium-denuded aortic(P＜0.05)(E_max=76%,EC₅₀=4.55×10^-6M with endothelium-intact aortic,while for endothelium-denuded aortic,E_max=44%,EC₅₀=4.84×10^-5M).3) Pre-incubation of endothelium-intact rings with L-NAME,an inhibitor of NOS, and methylene blue,an inhibitor of soluble guanylyl cyclase,significantly reduced apigenin-induced relaxation(P＜0.05).However,indomethacin,a cyclooxygenase blocker,did not significantly affect the apigenin-induced relaxation in endothelium-intact rings(P＞0.05).4) Voltage-dependent K⁺ channel blocker 4-AP,mito-ATP-sensitive K⁺ channel blocker 5-HD,Ca²⁺-activated K⁺ channel blocker TEA as well as inward rectifying K⁺ channel blocker BaCl₂ significantly inhibited apigenin-induced relaxation in endothelium-denuded rings pre-contracted by PE(P＜0.05).However,unspecific ATP-sensitive K⁺ channel blocker glibenclamide had no significant effect(P＞0.05).5) In the K⁺-free solution,apigenin can significantly inhibit PE pre-contracted aortic rings(P＜0.05).Apigenin can strongly inhibit 60 mM KCl induced aorta rings contraction(P＜0.05).In the Ca²⁺-free solution plus PE,cumulative addition of CaCl₂ induced a stepwise tension increase of aortic rings.Pretreated with apigenin for 20 min significantly attenuated CaCl₂ induced contraction.Conclusions1) The NOAEL of apigenin was 2g/kg bw.Apigenin belongs to actually non-toxic substance and it had no genetic-related effects.2) The lowest effective dose of apigenin in lowering blood pressure of SHR was 0.026g/kg bw.3) Possible mechanisms of apigenin in lowering blood pressure●Up-regulation of the expression of ACE2 and down-regulation of the expression of ACE gene in kidney,thereby improving RAAS of SHR.●Up-regulation of the expression of eNOS and down-regulation of the expression of ET-1 gene in kidney,decrease of the Angâ…¡content in blodd plasm,increase the content of blood plasm NO,eNOS,CGRP,cGMP,up-regulation the ratio of NO/ET-1 and NO/Angâ…¡,increase the activity of blood plasm SOD,lowering the content of blood plasm O₂,thereby,improving endothelium fuction.●NO-cGMP pathway was involved in the relaxation of apigein in endothelium-intact aorta,whereas production of PGI₂ by the endothelial cells was not attributed to the effect.●The vasorelaxant effect of apigenin was partially mediated by the opening of the K⁺ channels in vascular smooth muscle cells,including Kca,Kv,K_IR as well as mito-K_ATP,and by inhibiting of receptor-operated Ca²⁺ channel,voltage-dependent Ca²⁺ channel as well as extracellular Ca²⁺ influx.