key: cord-0793974-obd48wwg authors: Chen, Zhongjian; Kong, Sisi; Song, Feifeng; Li, Liping; Jiang, Huidi title: Pharmacokinetic study of luteolin, apigenin, chrysoeriol and diosmetin after oral administration of Flos Chrysanthemi extract in rats date: 2012-09-19 journal: Fitoterapia DOI: 10.1016/j.fitote.2012.09.011 sha: eb123ebe33919de898e3d9b8f22dc3fc913281c1 doc_id: 793974 cord_uid: obd48wwg Flos Chrysanthemi (the flower of Chrysanthemum morifolium Ramat.) is widely used in China as a food and traditional Chinese medicine for many diseases. Luteolin and apigenin are two main bioactive components in Flos Chrysanthemi, and chrysoeriol and diosmetin are two methylated metabolites of luteolin in vivo by cathechol-O-methyltransferase (COMT). However, there was lack of pharmacokinetic information of chrysoeriol and diosmetin after oral administration of Flos Chrysanthemi extract (FCE). The present study aimed to develop an HPLC-UV method for simultaneous determination of rat plasma concentration of luteolin, apigenin, chrysoeriol and diosmetin and utilize it in pharmacokinetic study of the four compounds after orally giving FCE to rats. The method was successfully validated and applied to the pharmacokinetic study when oral administration of FCE to rats with or without co-giving a COMT inhibitor, entacapone. Chrysoeriol and diosmetin were detected in rat plasma after oral administration of FCE and their concentrations were significantly decreased after co-giving entacapone. Furthermore, AUC of luteolin was significantly increased by entacapone, while that of chrysoeriol was decreased by entacapone, which revealed COMT might play an important role in the disposition of luteolin in rats after dosing of FCE. In conclusion, a sensitive, accurate and reproducible HPLC-UV method for simultaneous determination of luteolin, apigenin, chrysoeriol and diosmetin in rat plasma were developed, pharmacokinetics of chrysoeriol and diosmetin combined with luteolin and apigenin were characterized after oral administration of FCE to rats, which gave us more information on pharmacokinetics and potential pharmacological effects of FCE in vivo. The flower of Chrysanthemum morifolium Ramat (Flos Chrysanthemi) is a traditional Chinese medicine, and has been widely used as a healthy food for thousands of years. Many studies reported its healthy benefits, such as antioxidation [1] , cardiovascular protection [2] , hepato-protective effect [3] , antiarrhythmic effect [4] and anticomplementary activity in severe acute respiratory syndrome [5] . A new drug candidate of Flos Chrysanthemi extract (FCE) for treatment of cardiovascular disease had been in clinical trial in China [6] . Luteolin (3′, 4′, 5, 7-tetrahydroxyflavone) and apigenin (4′, 5, 7trihydroxyflavone) (Fig. 1 ) are two main bioactive ingredients FCE. These two typical flavonoids also widely distribute in fruits and vegetables, which exhibit as anticancer [7] [8] [9] [10] or cancer preventive [11] , anti-inflammatory [12, 13] , neuroprotective [14, 15] agents in the past studies. Luteolin and apigenin mainly underwent conjugation metabolisms by uridine diphospho-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) in vivo. Recently, luteolin had been identified to be a good substrate of catechol-Omethyltransferase (COMT), and two methylated metabolites of luteolin, chrysoeriol (4′, 5, 7-trihydroxy-3′-methoxyflavone) and diosmetin (3′, 5, 7-trihydroxy-4′-methoxyflavone), were identified in the rat plasma after hydrolylsis treatment when intravenously given luteolin to rats [16] . Since these flavonoids occurred mainly as glucuronidated and sulfated forms in the blood circulation, and there was lack of standard compounds of these glucuronidated and sulfated flavonoids, the total forms (free, glucuronidated and sulfated) flavonoids were analyzed after a hydrolysis treatment in our previous study [21] . Chrysoeriol and diosmetin not only possessed many biological effects similar to luteolin, such as antioxidant, antiinflammatory, but also exhibited their own effects such as osteoporosis [17, 18] . Furthermore, chrysoeriol and diosmetin were natural prodrugs in cancer prevention which could be converted to luteolin by CYPs [19, 20] . Therefore it was crucial to study the pharmacokinetics of chrysoeriol and diosmetin when FCE were orally taken. On the other hand, luteolin exhibited a much faster elimination rate than apigenin when FCE was orally administrated to rats, COMT was probably an important mechanism causing luteolin a lower exposure [16, 21, 22] . However it was needed to confirm the role of COMT in the disposition of luteolin when orally dosing FCE to rats. Therefore, the present study aimed to develop an improved quantitative method to determine luteolin, apigenin, chrysoeriol and diosmetin in rat plasma using HPLC-UV and utilize it to pharmacokinetic study of the four flavonoids in rats after oral administration of FCE with or without a COMT inhibitor, entacapone. The pharmacokinetic data could give us more information on pharmacokinetics and potential pharmacological effects of FCE in vivo 2. Materials and methods Luteolin and apigenin were purchased from Hangzhou Skyherb technologies Co., Ltd., Hangzhou, China (purity> 99%). Diosmetin was obtained from Shaanxi Huangteng Biotechnology Co., Ltd., Sian, China (purity>95%). Chrysoeriol was synthesized by Department of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University (purity >99%). FCE was provided by the Institute of Material Medica, Zhejiang University, China, containing 6.5% (w/w) luteolin and 5.4% (w/w) apigenin determined by HPLC after hydrolysis with hydrochloric acid. Entacapone was purchased from Jinan Wedo Industrial Co., Ltd., Jinan, China (purity>99%). Methanol, acetonitrile, formic acid, and water were HPLC-grade. All other chemicals were from standard commercial sources and were of the highest quality available. Analysis was performed using high-performance liquid chromatographic system HP1100 (Agilent Technologies, USA) equipped with an on-line degasser, an auto-sampler, a VWD detector, and a thermostated column compartment. Male Sprague Dawley rats weighing 200 to 250 g were obtained from Experimental Animal Center of Zhejiang Academy of Medical Sciences. All procedures were according to an approved animal use protocol of Zhejiang University. They were housed in cages at 21 ± 1°C and exposed to a 12:12 h light-dark cycle, free access to food and water. Animals were fasted but free access to water for 12 h before experiment and 2 h after drug administration. The HPLC analysis was performed on an Agilent Zorbax SB-C 18 column (250 mm×4.6 mm, 5 μm) with a mobile phase consisting of 0.1% formic acid, acetonitrile, and methanol at 30± 1°C with a constant rate of 1 ml/min. The injection volume was 50 μl, and the wavelength was set at 350 nm for quantitative analysis, the proportion of components in the mobile phase was optimized to obtain a well separation of luteolin, apigenin, chrysoeriol, diosmetin and internal standard (quercetin). Stock standard solution of luteolin, apigenin, chrysoeriol, diosmetin (approximately 0.5 mg/ml) and the internal standard, quercetin (approximately 1.0 mg/ml), were prepared by dissolving appropriate amounts of these references in methanol, respectively. A series of working solutions containing luteolin, apigenin, chrysoeriol and diosmetin were prepared by subsequent dilution of the above stock solution with methanol to reach a concentration range of 0.313-125 μg/ml, and the working solution of quercetin (100 μg/ml) was also prepared. Rat plasma samples were prepared according to the method developed in our laboratory [21] , and with some modifications. In brief, to detect the total form (free, glucuronidated, sulfated) of flavonoids, a 100 μl plasma was hydrolyzed by hydrochloric acid (2.0 M) at 80°C for 1.5 h in a 1.5 ml tube, and then 1.0 ml ethyl acetate (containing 0.05 μg/ml quercetin) was added to the mixture. After vortexing for 3 min, the tube was centrifugated for 10 min at 3500 rpm. Then 0.8 ml of the supernatant organic phase was carefully transferred to another 1.5 ml tube and evaporated to dryness under the vacuum desiccator at room temperature. The residue was reconstituted in 100 μl mobile phase, and the concentrations of luteolin, apigenin, and chrysoeriol and diosmetin were determined by the HPLC methods described above. The method was validated according to the "Guidance for preclinical pharmacokinetic study of chemical drug" recommended by State Food and Drug Administration (SFDA) of China. Blank plasma from at least six rats, blank plasma spiked with four analytes and sample obtained from rat after oral administration of FCE were processed and assayed as described in 2.6. Interference from endogenous or exogenous materials should not occur at the retention time of luteolin, apigenin, chrysoeriol, diosmetin or quercetin. The calibration curves ranging from 0.0250 to 10.0 μg/ml were assessed by weighted (1/x) least squares liner regression based on analyte/quercetin peak area ratio prepared in triplicate. The LLOQ was defined as the lowest concentration that could be quantitated with precision and accuracy b 20%. The standard samples spiked with luteolin, apigenin, chrysoeriol and diosmetin at low, medium and high concentrations (0.05, 1, and 8 μg/ml for all the analytes above) were used for accuracy and precision studies. Five replicates for each concentration were processed and analyzed as described above in 2.6. for accuracy study. The assay recovery and extract recovery were calculated. The intra-and inter-day precisions (relative standard deviations, RSD) were evaluated by analyzing homogeneous samples in five replicates, in 1 or consecutive 3 days. Intra-and inter-day precisions (RSD) were required to be b15%. Freeze-thaw (− 80°C), short-term (25°C for 4 h), longterm (− 80°C for 3 or 7 days), stock solution (25°C for 24 h or 4°C for 7 days) and post-preparative stability (25°C in auto-sampler for 24 h) of luteolin, apignin, chrysoeriol, and diosmetin were evaluated according to FDA guidance (http:// www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf). Low, medium and high concentrations (0.05, 1, and 5 μg/ml for all the analytes above) with triplicates were used in all the stability study except stocking solution stability (only 0.5 mg/ml analytes was used). Data was expressed as remaining(%)of initial determined. 10 male Sprague-Dawley rats were randomly allocated into FCE and (entacapone + FCE) groups with five rats each. Rats were fasted 12 h with free access to water, then a dose of 100 mg/kg FCE combined with or without 20 mg/kg entacapone [dissolved in 20% hydroxypropyl-β-cyclodextrin (w/v)] were orally administered to (entacapone + FCE) and FCE groups, respectively. The blood samples were collected from the orbital venous sinus to heparinized tubes at predose and post-dose at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 h, and then centrifuged at 2000 g for 10 min at 4°C, the plasma were collected and stored at −80°C until analysis. Pharmacokinetic parameters were calculated with pharmacokinetic software DAS 2.0 (China) and differences between the FCE and (entacapone + FCE) groups were compared with 2-sample t-test (two tails) using Excel 2003 (Microsoft). Chrysoeriol and diosmetin possessed an identical LogP value due to their similar structures (Fig. 1) , and they could not be separated well using a mixture of 0.1% formic acid and methanol (or 0.1% formic acid and acetonitrile). Using 0.1% formic acid, acetonitrile and methanol could significantly improved the separation, and after optimization of their combinations, an mobile phase of 0.1% formic acid, acetonitrile and methanol (60:16:24, v/v/v) was obtained to successfully separate analytes (luteolin, apigenin, chrysoeriol and diosmetin) and internal standard (quercetin) (Fig. 2A) . Quercetin was selected as an internal standard due to its similar structure with the four analytes, which would exhibit behavior similar to the analytes during the sample extraction and chromatographic elution. The specificity of this method to plasma matrix was evaluated with plasma from six rats. The typical chromatograms of (A) a blank plasma sample, (B) a blank plasma sample spiked with 0.05 μg/ml luteolin, apigenin, chrysoeriol and diosmetin and 0.5 μg/ml quercetin (C) a plasma sample from a rat at 30 min after oral administration of 100 mg/kg FCE are shown in Fig. 2 . No interferences from endogenous substances in rat plasma was observed at the retention times of interest(All samples were treated with hydrolysis preparation). The peak area ratios of luteolin, apigenin, chrysoeriol and diosmetin to quercetin all displayed a good linear relationship over the range of 0.025-10 μg/ml. The typical regression equations were as follows: Y = 3.191X − 0.01150, R 2 = 0.9969, n = 8 (luteolin); Y = 2.766X + 0.01260, R 2 = 0.9919, n = 8 (apigenin); Y = 3.195X + 0.01178, R 2 = 0.9932, n = 8 (chrysoeriol); Y = 2.820X + 0.0003733, R 2 = 0.9947, n = 8 (diosmetin). The LLOQ of all analytes was 0.025 μg/ml. Recovery, precision and accuracy data are presented in Table 1 . The extract recoveries for luteolin, apigenin, chrysoeriol, diosmetin were 81.8%-93.3%, 76.94%-96.2%, 84.5%-96.7% and 79.08%-94.1%, respectively, while the assay recoveries of luteolin, apigenin, chrysoeriol, diosmetin were 96.7%-103.4%, 94.5%-104.4%, 93.2%-99.3% and 98.1%-98.3%, respectively. The intra-day precisions (RSDs) for luteolin, apigenin, chrysoeriol, diosmetin were b 4.9%, b5.4%, b7.0%, b6.5%, respectively, and the inter-day precisions (RSDs) were b3.5%, b6.4%, b 4.2%, b3.2%, respectively. The mean extract recovery of quercetin in samples was 89.5% (detail data not shown). The freeze-thaw, short-term and long-term stability results of luteolin, apigenin, chrysoeriol, diosmetin in rat plasma are shown in Table 2 , which revealed the four analytes were relatively stable. In addition, the four analytes in stocking solution at 25°C for 24 h or at 4°C for 7 days were stable (remaining of initial determined were >99.1%), and the four analytes and the internal standard (quercetin) during analyzing process in the HPLC auto-sampler (25°C for 24 h) were stable, too (remaining of initial determined were >96.9%). The method was successfully applied to analysis of plasma obtained from rats following a single oral dose of 100 mg/kg FCE with or without 20 mg/kg entacapone. Concentration-time profiles for luteolin, apigenin, chrysoeriol, diosmetin after giving FCE with or without 20 mg/kg entacapone were shown in Fig. 3 . The plasma concentrations of luteolin in (entacapone +FCE) group were significantly higher than that of the FCE group, and the AUC 0-t and C max of luteolin were significantly increased when entacapone was co-administered with FCE (Table 3) , whereas, the plasma concentrations of chrysoeriol diosmetin and the AUC 0−t of chrysoeriol were significantly decreased. (3), chrysoeriol (4), diosmetin (5) and 0.500 μg/ml quercetin (1); C: plasma sample 30 min after oral administration of 100 mg/kg FCE to rats. All samples above were treated with hydrolysis. In our previous pharmacokinetic studies of FCE in rat, dog and human, we mainly focused on luteolin, apigenin and their glucuronidated and sulfated forms, but methylated metabolites of luteolin, chrysoeriol and diosmetin were never mentioned. Since chrysoeriol and diosmetin were with similar structures, close LogP values and shared the same mass spectrum [16] , an optimized chromatographic condition was very important in the present study. In our study, an Agilent Zorbax SB-C 18 column (250 mm× 4.6 mm, 5 μm) was applied to separate these flavonoids. To get a suitable mobile phase condition, the mixtures of 0.1% formic acid and acetonitrile, or 0.1% formic acid and methanol, and the three above were compared, the combination of the three components in the mobile phase was found to make a well separation. After optimizing the proportion of acetonitrile and methanol, a mobile phase of 0.1% formic acid, acetonitrile and methanol (60: 16:24 , v/v/v) was determined. In addition, chrysoeriol and diosmetin still had free hydroxyl, which could be further conjugated by UGTs or SULTs, therefore a hydrolysis treatment of sample was very important to assay the methylated metabolites of luteolin in vivo. The method was successfully utilized in plasma pharmacokinetics of luteolin, apigenin, chrysoeriol and diosmetin after oral administration of 100 mg/kg FCE co-administrated with or without 20 mg/kg entacapone to rats. Plasma concentrationtime profiles of chrysoeriol and diosmetin were obtained, which indicated the two flavonoids would exhibit their biological effects in vivo when FCE was taken. And the methylated luteolin were more permeable in vivo due to their relatively higher hydrophobicity than luteolin, so they might be more easily distributed into tissues. In addition, these two methylated luteolin could be demethylated by some CYPs [19, 20] , so they also could play the biological effects of luteolin in vivo. On the other hand, compared with the control group, the concentrations of luteolin in (entacapone + FCE) group were significantly increased while the methylated metabolites were obviously decreased, which indicated that COMT might play a role in the disposition of luteolin in vivo. Because luteolin could be methylated by COMT but not apigenin, so it was speculated that COMT might be a mechanism responsible Fig. 3 . Mean-plasma concentration-time profiles of luteolin, apigenin, chrysoeriol and diosmetin after oral administration of 100 mg/kg FCE with or without co-giving 20 mg/kg entacapone to rats. Data were expressed as mean ± S.E.M, n = 5. Compared with the entacapone + CME group,*: P b 0.05, **: P b 0.01. for the pharmacokinetics differences between luteolin and apigenin when oral FCE to rats [22] . The present study directly evaluated the role of COMT in vivo in disposition of luteolin after oral dose of FCE to rats. However, the increase in concentrations of luteolin by COMT inhibition was limited, which could not reverse the pharmacokinetics differences between luteolin and apigenin. It was possible that the inhibition of COMT by entacapone was limited due to the very wide tissue distribution of COMT in vivo, and/or other metabolism enzymes, such as UGTs and SULTs, especially in the small intestinal could compete with COMT to decrease the contribution of COMT to disposition of luteolin. The concentrations of apigenin were also increased by entacapone, but the mechanism for this was unclear and needs further investigation. In conclusion, the present study provided a method for simultaneous determination of luteolin, apigenin, chrysoeriol and diosmetin in rat plasma, which had been successfully utilized in pharmacokinetics study of FCE in rats, and also could be applied in future clinical study of FCE. The pharmacokinetics of the two methylated luteolin in vivo could enlarge our knowledge about the disposition profile of the luteolin and pharmacological effects of FCE in vivo when oral administration of FCE. Evaluation of antioxidant activity and inhibitory effect on nitric oxide production of some common vegetables Endothelium-dependent and direct relaxation induced by ethyl acetate extract from Flos Chrysanthemi in rat thoracic aorta The hepatoprotective action of ten herbal extracts in CCl4 intoxicated liver Antiarrhythmic effect of ethyl acetate extract from Chrysanthemum morifolium Ramat on rats Anticomplementary principles of a Chinese multiherb remedy for the treatment and prevention of SARS Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC Luteolin suppresses growth and migration of human lung cancer cells Luteolin induces apoptosis in multidrug resistant cancer cells without affecting the drug transporter function: involvement of cell line-specific apoptotic mechanisms Apigenin induces apoptosis via extrinsic pathway, inducing p53 and inhibiting STAT3 and NFkappaB signaling in HER2-overexpressing breast cancer cells Apigenin induces apoptosis and blocks growth of medroxyprogesterone acetate-dependent BT-474 xenograft tumors Antigenotoxicity, cytotoxicity, and apoptosis induction by apigenin, bisabolol, and protocatechuic acid Luteolin inhibits microglial inflammation and improves neuron survival against inflammation Anti-inflammatory activity of structurally related flavonoids, apigenin, luteolin and fisetin Neuroprotective effect of luteolin on amyloid beta protein (25-35)-induced toxicity in cultured rat cortical neurons The flavonoid apigenin protects brain neurovascular coupling against amyloid-beta )-induced toxicity in mice Role of catechol-Omethyltransferase in the disposition of luteolin in rats Chrysoeriol isolated from the leaves of Eurya ciliata stimulates proliferation and differentiation of osteoblastic MC3T3-E1 cells Main pharmacokinetic parameters of luteolin, apigenin, chrysoeriol and diosmetin in rats after oral administration of 100 mg/kg FCE with or without co-administration of entacapone Diosmetin induces human osteoblastic differentiation through the protein kinase C/p38 and extracellular signal-regulated kinase 1/2 pathway Bioactivation of the phytoestrogen diosmetin by CYP1 cytochromes P450 Phytoestrogens as natural prodrugs in cancer prevention: dietary flavonoids Absorption and excretion of luteolin and apigenin in rats after oral administration of Chrysanthemum morifolium extract The exposure of luteolin is much lower than that of apigenin in oral administration of Flos Chrysanthemi extract to rats Apigenin Data are expressed as mean ± SD, n= 5; *: Pb 0.05,**: P b 0.01 compared with the FCE group.