In Vitro Metabolism Of Kaempferol And Isorharmnetin And Drug-Drug Interaction

Ginkgo biloba was the rarity tree only grown in china. Several pharmacological activities attributed to ginkgo biloba leaves extract was as follows: 1) improving cardiovascular circulation. 2) reducing serum cholesterin. 3) antibacterial and antiinflammatory activity. 4) antiviral acitvity 5) scavenging free radical and antioxidant activity.Recently, the preparation of ginkgo biloba leaves was firstly chosen to cure hypertension, arteriosclerosis, diabetes, stroke, cancer and Alzheimer as one of main herbal medicine.Some studies in vivo or in vitro indicated that ginkgo biloba flavonoids were metabolized in intestine and liver after absorbed in intestine. The main metabolicroute was glucuronidation. For the complexity environment in body, the metabolites of ginkgo biloba in vivo were different.Quercetin and kaempferol could decrease the protein activity of CYP1A1, 1A2, 3A4, 2B based on the literatures. The co-administration of ginkgo biloba leaves preparation with other drugs are conmon because ginkgo biloba leaves preparation are taken to treat the cardiovascular disease for long periods. Drug-drug interactions in metabolism occurred in two drugs taken at the same time or fore-and-aft, having an action of enhancing the curative effect or toxic effect. Recent years scholars have attached more importance to the drug-drug interactions about herbal medicine, but there are few literatures were reported in china.Our study focused on the metabolic route and drug-drug interaction of the flavonoids aglycone kaempfeol, isorhamnetin and quercetin. The paper was divided to three chapters: 1) Metabolism of kaempferol and isorhamnetin in vitro. 2) In vitro inhibitive effect of kaempferol on cytochrome P450. 3 ) The metabolic drug interaction between kaempferol and other two ginkgo biloba flavonoids or other drugs.1. Metabolism of kaempferol in ginkgo biloba flavonoid in vitroAIM Study the metabolism of flavonoid kaempferol and isorhamnetin in different source of hepatic microsomes (rat, human or dog) and different treated microsomes (induced or uninduced) in order to obtain the information for metabolic route and enzymes participated. METHODS 1) Phase I metabolism of kaempferol and isorhamnetin. The substrates were incubated with different rat microsomes (lmg protein-mL"1, 37°C) pretreated by PB, DEX, BNF and control for different time. The remaining substrate concentration was determined by HPLC. 2) Glucuronidation of kaempferol and isorhamnetin in rat hepatic microsome was investigated in five kinds of microsomal incubates pretreated by phenobarbital(PB), DEXamethasone(DEX), fi -naphthoflavone(BNF), diphenytriazol (DIPH), and control. The substrate wasincubated with rat hepatic microsome (0.2mg protein-mL"1) at 25 °C and the remaining concentration was determined by HPLC. The catalyse activity of different rat microsome was compared with control. LC/MS was used to analyse the structure of metabolites. 3) Metabolism of kaempferol and isorhamnetin in human hepatic microsome. The substrate was incubated with human normal hepatal microsome(lmg proteinmL"')at 37 °C and the remaining concentrarion was determined by HPLC. Metabolism in dog normal hepatic microsome was the same as in rat. RESULTS 1) Kaempferol metabolic rate was 18.26% in microsome pretreated by BNF after 60min incubate and the main metabolite was quercetin. The metabolic rate was only 3-7% in other microsomes. The metabolism was hardly observed for isorhamnetin in different microsomes ( 2-8% of metabolic rate ) . 2) The glucuronidation of kaempferol and isorhamnetin was extensive in different microsomes. The metabolic rate of kaempferol was 62.9% in the microsome induced by DIPH, 40.1% by BNF, 21.1 % by PB, 23.7% by DEX and 18.0% by control, respectively, after 45 min incubation. The Ajm of kaempferol in control microsome and the microsomes induced by BNF or by DIPH was (1.85 ±1.05), (9.41+2.45 ) and ( 72.4 + 3.08) umol-L'1 respectively;vmaxwas (2.45 + 0.63), (7.55 + 1.40) and (25.2 + 1.08) umol-g'1 min'1 respectively. The metabolic rate of isorhamnetin, after 60 min incubation, was 70.9% in the microsome induced by DIPH, 43.7% by BNF, 26.3 % by PB, 26.8% by DEX and 19.0% by control, respectively. The Km of Isorhamnetin in control microsomes and the microsomes induced by BNF or by DIPH was (1.95±0.54), (7.13 + 0.73 )and( 61.6+6.50) nmol-L"1 respectively;Vmaxwas (1.15±0.17),(3.05+ 0.26) and(22.7+2.13) nmolg'Vin'1 respectively. Two glucuronides of kaempferol and four glucuronides of isorhamnetin were detected. The results by HPLC-MS analysis indicated that four metabolites of isorhamnetin in rat microsome were all monomolecular of glucuronic acid combination. 3) The metabolisms of kaempferol and isorhametin in human and dog hepatal microsome were weak. After 60min incubate, the metabolic rate was 16.04%for kaempferol and 13.36% for isorhamnetin in human hepatal microsome, and 19.55% for kaempferol and 21.12% for isorhamnetin in dog hepatal microsome. CONCLUSION The results showed that glucuronidation was the main metabolic route for kaempferol and isorhamnetin, and the microsomes induced by DIPH and BNF played major role.2. In vitro inhibitive effect of kaempferol on cytochrome P450. AIM: To study the inhibitive effect of kaempferol toward cytochrome P450 enzymes in rat liver microsomes in vitro. METHODS: The inhibitive effect of kaempferol on CYP was investigated by coincubating kaempferol with the specific substrates of CYP1A—ethoxyresorufin in the microsome induced by BNF, with the specific substrates of CYP2B—pentoxyresorufin in the microsome induced by PB, with the specific substrates of CYP3A—diazepam, testosterone and nifedipine in the microsome induced by DEX. The inhibitive effect of kaempferol on ethoxyresorufin -O-deethylase (EROD) or pentoxyresorufin -O-dealkylase (PROD) activity was determined by measuring the product resorufin formation with fluorescence spectrophotometer directly, while the inhibitive effect on the metabolism of other substrates was determined by monitoring the substrate loss through determining the remaining concentration by RP-HPLC. Results: 1) Kaempferol inhibited the EROD activity significantly. The inhibition constant (Ki) of kaempferol was 5.86 + 0.84 umol-L"1, the result was compared with fluvoxamine, the specific inhibitor of CYP1A. The inhibition constant (Ki) of the latter was 11.03 + 0.27 umol-L"1. Kaempferol inhibitive effect on PROD was also significantly. The inhibition constant (K{) of kaempferol was 7.78+1.36 umol-L"1, the specific inhibitor of CYP2B, Sodium secobarbitate was 6.65 ±2.41 umol-L"1. And kaempferol inhibited the metabolism of diazepam, testosterone and nifedipine in different degree, but the inhibition potency was relatively weaker comparing with the typical inhibitor ketoconazole. The Ki of kaempferol on the diazepam, testosterone and nifedipine was 182.6,158.3, 86.8 umol-L"1 and ketoconazole was 2.14, 1.89,2.89 umol-L"1.CONCLUSION: kaempferol inhibited CYP1A and CYP2B enzyme significantly and partly inhibited C YP3 A enzyme.3. The metabolism interaction of kaempferol and other two ginkgo biloba flavonoids or some commonly used drugs.AIM To obtain the information about the interaction between kaempferol and other two ginkgo biloba flavonoids or some commonly used drugs. METHODS 1 )Two of three flavonoids aglycone were coincubated in rat microsomes pretreated by BNF at 25 °C. The remaining substrate concemtration was determined by HPLC and the inhibition constant Ki was calculated. 2) Some UGT substrates (paracetamol, propofol, 4-nitrophenol, dobutamine hydrochloride) were selected to coincubated with kaempferol at 25 "C. The inhibition of these drugs on glucuronidation of kaempferol was investigated by monitoring the loss of kaempferol. 3) Propranolol hydrochloride was coincubated with kaempferol in microsome pretreated with BNF, and the remaining concentration of propranolol was determined by RP-HPLC. RESULTS 1) The Ki of kaempferol on quercetin and isorhamnetin was 4.64 ± B^S^mol-L"1 and 18.42+3.87|xmol-L'';quercetin on kaempferol and isorhamnetin was lLlO + OJOixmol-L"1 and 2.26 + 0.99 umol-L"1;isorhamnetin on kaempferol and quercetin was 24.99 ± 2.87jimol-L"' and 5.27 + 2.40 umolL'1. 2) Among UGT substrates tested , only 4-nitrophenol had weak inhibition to kaempferol glucuronidation, and the Ki was 204.0±8.66^unol-L"1. 3) It was observed that inhibiton of kaempferol on propranolol metabolism mediated by CYP with the Ki 59.54+1.36jimol-L"'. CONCLUSION There were significant interactions among two of three ginkgo biloba flavonoids. Kaempferol had inhibiton on propranolol hydrochloride metabolism.