Isolation And Purification Of Hydroxysafflor Yellow A And Its Mechanism To Improve Exercise Endurance

Hydroxysafflor yellow A(HSYA)is the main active substance of safflower.Although HSYA has a variety of physiological functions,its effect on exercise endurance and the potential molecular mechanisms remain clear.Exercise endurance is one of the important indicators of physical health.Improving exercise endurance not only helps to improve metabolic health but also reduces the risk of getting cardiovascular or other diseases.Accordingly,in this paper,HSYA was isolated and purified from safflower aqueous extract by macroporous adsorbent resin-preparative-grade high-performance liquid chromatography(PHPLC)coupling technique.Furthermore,the effect of HSYA intervention on exercise endurance and its underlying mechanism were investigated in depth.The main results of this paper are as follows.Firstly,the optimized macroporous adsorption resin for the purification of HSYA was screened using static adsorption-desorption experiments.Subsequently,the content of HSYA was used as an indicator to optimize the purification process of the macroporous adsorption resin.The crude HSYA was further purified by PHPLC,and the obtained products were compared with the standards for identification.The results showed that the optimal process of purification of HSYA by macroporous sorbent resin was as follows: AB-8 macroporous sorbent resin,sample solution concentration 5 mg/m L,loading volume 48 m L sample solution/g wet resin,loading flow rate 3 m L/min,elution flow rate 2 m L/min,and eluent 70% ethanol solution.The purity of HSYA could be improved from 29.22% to 59.67% under the optimal extraction conditions.After further purification by PHPLC,HSYA with a purity of 93.94% was obtained.Secondly,mice were gavaged with 50 mg/kg HSYA daily for 3 weeks,and the food intake,body weight,and skeletal muscle weight of the mice were measured.In addition,the exercise capacity of the mice was examined using a rotating bar fatigue meter and a platform treadmill.Results showed that HSYA had no significant influence on the feeding and body weight of mice while reducing the weight of gastrocnemius,tibialis anterior,and soleus muscles of mice.In addition,HSYA intervention significantly increased the exercise endurance of mice.Thirdly,the effect of HSYA on muscle fiber-type transition was further investigated.The C2C12 myotubes were stimulated with HSYA in a concentration and time gradient.Then,q RTPCR,Western Blot,and immunofluorescence were used to detect the effect of HSYA on myofiber type conversion in C2C12 myotubes,which was further verified in animal experiments.The results revealed that HSYA intervention could increase the expression of slow muscle-related gene expression,aerobic enzyme activity,and mitochondrial biosynthesis in C2C12 myotubes.In contrast,HSYA intervention decreased the expression of fast musclerelated gene expression.The results of in vivo experiments showed a consistent trend.Furthermore,the possible mechanism underlying the role of HSYA in regulating myofiber type switching was further explored using molecular docking and small interfering RNA techniques,and it was found that the effect of HSYA on myofiber remodeling in myotubes was mediated by forkhead transcription factor O1(Fox O1)signal pathway.Finally,the effect of HSYA on the fuel selection of myotubes was further investigated.The glucose uptake and the expression of genes related to glucose metabolism and lipid metabolism in C2C12 myotubes were detected after HSYA treatment.Then,the effect of HSYA intervention on fuel selection in skeletal muscle of mice was examined by the comprehensive monitoring system,oil red staining,q RT-PCR,and Western Blot.In addition,the molecular mechanism underlying the effect of HSYA on fuel selection of myotubes was further explored by using small interfering RNA.The results showed that HSYA promoted the shift of substrate utilization from glucose to fat in C2C12 myotubes.Similarly,HSYA intervention significantly reduced the respiratory exchange rate and triggered a shift in fuel selection from glucose to fat in the skeletal muscle of mice.Silencing the expression of peroxisome proliferator-activated receptor δ(PPARδ)reversed this effect of HSYA on fuel selection in myotubes,suggesting that the PPARδsignaling pathway is required for HSYA on fuel selection in myotubes.Taken together,HSYA prolonged endurance performance by promoting the conversion of muscle fiber from fast twitch to slow-twitch in myotubes,which in turn regulated the shift of fuel selection from glucose toward fat.These findings suggested the potential use of HSYA-based health food products for improving exercise endurance and provided a new strategy for the development of novel endurance sports nutrition foods.