| The dried stigma of Crocus sativus L.(saffron),an Iridaceae herb,is a world’s precious spice,and is also a traditional Chinese medicine for reducing bruises,promoting blood circulation and reducing blood sugar.The stigma takes only 7.4%(w/w)of the total weight of the flower,resulting in wasting the rest 92.6%of the flower.Studies showed that the saffron floral bio-residues were rich in flavonoids,phenolic acids,alkaloids,etc.And they were proved to have pharmacological activities like antioxidant activity,anti-inflammation and liver protection.Accordingly,the utilization of the saffron floral bio-residues not only helps discovering its new activity,but also avoids the waste of resources.Hyperuricemia,defined as an excessively high level of uric acid(UA)in the blood,is a class of diseases caused by disorders of purine and UA metabolism.The increasing incidence has made hyperuricemia become the fourth metabolic disease after hypertension,hyperlipidemia and diabetes.Three main types of drugs,namely,xanthine oxidase(XOD)inhibitor,urate anion transporter inhibitor and UA oxidase were used in clinical treatment of hyperuricemia,in which the representative drug was allopurinol.However,the side effects or adverse reactions of these UA-lowering drugs imposed a significant burden on the body.Studies showed that Chinese herbal medicines not only had a good therapeutic effect on hyperuricemia,but also had fewer side effects compared with the western medicine.Therefore,the discovery of drugs for the treatment of hyperuricemia from Chinese herbal medicines and natural products has attracted the attention of researchers.In the previous pharmacological experiments,we found that the alcohol extract of saffron floral bio-residues has a preliminary effect on reducing UA level in potassium oxonate-induced hyperuricemia mice,and the main component flavonoid may be the active compound due to its anti-XOD activity,which needs to be further studied.In this paper,a flavonoid extract from saffron floral bio-residues was prepared,and its chemical compositions were qualitatively and quantitatively analyzed.The anti-hyperuricemia activity of the flavonoid extract was investigated using three animal models of hyperuricemia,and its UA-lowing mechanism was explored through the effects on UA-related target proteins and gut microbiota.In addition,the metabolism of the compounds from the flavonoid extract in rats was studied.The aim of this paper is to provide an experimental basis for the development of saffron new medicinal parts.1 Preparation of the flavonoid extract from saffron new medicinal partsTaking the flavonoid content and antioxidant activity as indicators,the preparation method of flavonoid extract from saffron floral bio-residues was optimized,and the method of ultrasonic extraction combined with macroporous resin purification were determined.2 Chemical composition analysis of the flavonoid extractThe UPLC-Q-TOF-MS and GC-MS methods were used to qualitatively analyze the chemical constituents of the raw material of saffron floral bio-residues and the flavonoid extract.Totally 52 flavonoids,8 nitrogen-containing components,10 polyols and 42 volatile components were detected in the flavonoid extract,which were roughly the same as those in the raw material.The HPLC method was used to quantitatively analyze the two main components in the flavonoid extract,kaempferol-3-O-sophoroside(KS)and delphinidin-3,5-di-O-glucoside(DG).The contents of KS and DG were 50.19%and 2.20%,respectively,which were 10 times and 4 times respectively higher than that of the raw material.In addition,the antioxidant capacity of the flavonoid extract was also significantly improved(DPPH clearance increased by 5 times;ORAC value increased by 100 times).3 Metabolic characterization of major chemical constituents in ratsThe components detected in rat plasma after gavage of the flavonoid extract were analyzed using the UPLC-Q-TOF-MS method.Twelve components,including 11 flavonoids(3 flavonoid glycosides and 8 metabolites)and 1 alkaloid were identified or speculated in rat plasma.Furthermore,a rapid and sensitive UHPLC-MS/MS assay for the measurement of 5 target flavonoids,namely,kaempferol-3-O-sophoroside-7-O-glucoside,quercetin-3-O-sophoroside,KS,kaempferol-3-O-glucoside,kaempferol,and 3 glucuronidation metabolites,namely,kaempferol-3-O-glucuronide,kaempferol-di-O-glucuronide,kaempferol-O-sulfate-O-glucuronide in rat plasma after gavage of the flavonoid extract was performed.And pharmacokinetic comparison of the flavonoid extract and the main component KS and its aglycon kaempferol was conducted.Results showed that the four flavonoid glycosides were poorly absorbed and eliminated quickly in plasma,and most of them were converted into aglycone and phase Ⅱ metabolites.Although the content of aglycone kaempferol in the extract was extremely low,it had high plasma concentration,indicating that flavonoid glycosides could be decomposed into kaempferol in the body.The time for phase Ⅱ metabolites to reach the maximum plasma concentrations were longer than that of the prototype flavonoids,which were less than 4 hours for prototype flavonoid glycosides and more than 6 hours for phase Ⅱ metabolites.In addition,the plasma concentrations of phaseⅡ metabolites were much higher than those of the prototype flavonoid glycosides,in which the maximum plasma concentration of the metabolite kaempferol-3-O-glucuronide was about 20 times more than that of its prototype KS.Furthermore,coexisting components in the extract affected the metabolic profiles of KS and kaempferol in vivo.At the same dosage,the absorption of KS was reduced and the elimination was accelerated in the flavonoid extract as compared with those of the KS monomer.In detail,the area under the plasma concentration-time curve decreased by nearly 60%(from 441.4 to 174.4 h·μg/L),while the clearance increased by nearly 2 times(from 271.7 to 758.6 L/h/kg).Compared with the kaempferol monomer,the absorption of kaempferol was increased and the retention time was prolonged in the flavonoid extract.Specifically,the area under the plasma concentration-time curve increased by about 7 times(from 97.1 to 753.9 h·μg/L),and the average retention time increased by more than 1 time(from 4.0 to 10.7 h).The results suggested that the coexisting compounds in the extract reduced the absorption and accelerated the elimination of flavonoid glycoside compounds;in contrast,enhanced the absorption and slowed down the elimination of flavonoid aglycone compounds in vivo.4 Antihyperuricemia activity and its mechanism of the flavonoid extractThree animal models of hyperuricemia including acute hyperuricemia mice induced by potassium oxonate(preventive administration),hyperuricemia rats induced by adenine combined with ethambutol(preventive administration),and hyperuricemia rats induced by potassium oxonate(therapeutic administration)were estabolished to investigate the anti-hyperuricemia activity and its mechanism of the flavonoid extract.The modeling drugs were selected from the perspectives of inhibiting UA decomposition,supplementing exogenous UA precursors,and inhibiting UA excretion.4.1 Effects on the biochemical indexesThe flavonoid extract effectively reduced the UA levels in blood and intestine;inhibited the XOD activities in blood and liver;improved the renal function and inflammatory state to protect kidney;enhanced the antioxidant capacity and reduced the lipid levels in blood of hyperuricemic mice/rats.4.2 Effects on the UA-related target proteinsThe effects of the flavonoid extract on UA related proteins XOD,urate transporter 1(URAT1),glucose transporter 9(GLUT9),ATP-binding cassette transporter G2(ABCG2)in hyperuricemia rats induced by potassium oxonate were investigated by the Western Blot and RT-qPCR methods.Results showed that the flavonoid extract significantly reduced the expressions of XOD in liver,URAT1 and GLUT9 in kidney and ileum;and increased the expressions of ABCG2 in kidney and ileum,suggesting that these four proteins might be the key targets for the flavonoid extract to reduce UA.4.3 Effects on the endogenous metabolites in serumThe effects of the flavonoid extract on serum endogenous metabolites in hyperuricemia rats induced by adenine combined with ethambutol and induced by potassium oxonate were investigated by the method of non-targeted metabolomics technology.Results showed that hyperuricemia caused abnormal metabolism of endogenous substances,especially lipids(glycerophospholipids and fatty acids)and amino acids in rat serum.The flavonoid extract could adjust the levels of differential metabolites such as phosphatidylcholine,sphingolipids,carnitine,aspartic acid,ceramide,palmitic acid,tyrosine and citrulline,and affected metabolic pathways related to amino acid and lipid metabolism.The five metabolic pathways of alanine,aspartate and glutamate metabolism;arginine biosynthesis;glycine,serine and threonine metabolism;histidine metabolism;glyoxylate and dicarboxylate metabolism were closely related to purine/UA metabolism.4.4 Effects on the gut microbiotaThe effect of the flavonoid extract on the metabolism of gut microbiota in hyperuricemia rats induced by potassium oxonate was studied by the 16S rRNA sequencing method.Results showed that hyperuricemia caused metabolic disturbance of the gut microbiota in rats,and decreased the richness and uniformity of the microbiota.The flavonoid extract significantly increased the diversity of the gut microbiota,adjusted the abnormal levels of 30 differential bacteria such as Roseburia,Clostridium_sp.and Gastranaerophilales,thus to improved the metabolic imbalance of the gut microbiota.In addition,results of correlation analysis showed that the abundances of differential bacteria were highly correlated with the levels of serum differential metabolites,indicating that the gut microbiota was closely related to host metabolism.4.5 Mechanism of UA-lowering effect of the flavonoid extractBased on the results of the above experiments,the mechanism of anti-hyperuricemia activity of the flavonoid extract was discussed.It was proposed that the mechanism of the flavonoid extract to reduce UA was to regulate the UA-related target proteins XOD,URAT1,GLUT9 and ABCG2;and modulate the gut microbiota closely related to host lipids and amino acids metabolism.Combined with the results of pharmacodynamics and pharmacokinetics,it was preliminarily speculated that KS,kaempferol and phase Ⅱ metabolites might be the material basis for the anti-hyperuricemia effect of the flavonoid extract.In conclusion,the flavonoid extract of saffron floral bio-residues was prepared by ultrasonic extraction combined with purification with macroporous resin purification in which the content of KS reached more than 50%of the extract.The metabolism of the flavonoid extract in rat plasma was characterized,and 12 components were identified or speculated.Pharmacokinetic studies of 5 flavonoids and 3 phase Ⅱ metabolites were conducted.Results showed that the prototype glycosides were poorly absorbed and metabolized rapidly in plasma,and were mainly converted into aglycone and phase Ⅱ metabolites.Moreover,the coexisting components in the flavonoid extract had a significant effect on the metabolic parameters of the target components.The effects of the flavonoid extract on hyperuricemic mice/rats were investigated.The flavonoid extract significantly reduced the UA levels in hyperuricemic mice/rats,which may owing to the regulation of UA-related proteins XOD,URAT1,GLUT9 and ABCG2,and the modulation of gut microbiota related to host metabolism.And KS,kaempferol and phase Ⅱmetabolites might be the material basis for the anti-hyperuricemia effect of the flavonoid extract. |
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