Study On Therapeutic Basis And Pharmacokinetics Of Saposhnikovia Divaricata

Saposhnikovia divaricata,usually called Fangfeng,is one of the important member of traditional Chinese medicines（TCM）.In this paper,anti-inflammatory activity,quality control method, therapeutic basis and pharmacokintics on the active constituents of Fangfeng were studied.The acute and chronic inflammation model were established to systematically investigated the anti-inflammatory activity of Saposhnikovia divaricata.The acute inflammation model was established by subcutaneous injection of 0.1%carrageenin solution into the right foot of rats.The inhibition ratio of swelling was used as index.The results of the study show that the low,middle and high dose of Saposhnikovia divaricata could significantly inhibit the swelling caused by carrageenin.The chronic inflammation model was established by subcutaneous injection of 0.1 mL FCA（Freund’s complete adjuvant） into the right foot of rats.The swelling of the right foot,which was injected,was used as index to observe the effect of Saposhnikovia divaricata on primary affection of adjuvant arthritis（AA）.The results of the study show that the low,middle and high dose of Saposhnikovia divaricata could significantly inhibit the swelling（P<0.01） and prevent the primary affection of AA.Nine days after injection,Saposhnikovia divaricata was successively given for 18 days by oral administrtion and two times each day.The swelling of the left foot was usea as the index to observe the Saposhnikovia divaricata on secondary affection of AA.The results of the study show that the high dose of Saposhnikovia divaricata could significantly inhibit the swelling（P<0.01）,but the inhibition was not significant by given the low and middle dose.The preventive and therapeutic effect on the secondary affection of the high dose of Saposhnikovia divaricata could be confirmed.The active ingredients with anti-inflammatory effect was isolated from Saposhnikovia divaricata by various modern isolation and purification techniques.The secondary swelling of AA was used as index to investigate the anti-inflammatory activity of alcohol extract,chloroform extract, acetoacetate extract and n-butanol extract.The results of the study show that the acetoacetate extract and n-butanol extract possess the better anti-inflammatory activity.Eight constituents were isolated from the acetoacetate extract and n-butanol extract by silica column chromatogrphy,gel column chromatography and preparation HPLC techniques.They were identified to be sec-o-glucosylhamaudol,hamaudol,prim-o-glucosylcimifugin,cimifugin, 4’-O-D-glucosyl-5-O-methylvisamminol,5-O-methylvisamminol,bergapten and sucrose.The standards could guarantee the further study.HPLC fingerprints of nineteen batches Saposhnikovia divaricata from different origin was developed on Jiangshen BDS C₁₈ column,with methanol-water system as mobile phase. 4’-O-D-glucosyl-5-O-methylvisamminol was used as the reference.Sixteen common peaks of the fingerprints were marked,and six of them were identified to be sec-o-glucosylhamaudol,hamaudol, 4’-O-D-glucosyl-5-O-methylvisamminol,5-0-methylvisamminol,prim-o-glucosylcimifugin and cimifugin.The quality evaluation standard was established by determination the cosine between the standard chromatogram and samplesHPLC fingerprints of the plasma samples and urine samples of normal rats and AA rats was firstly developed after oral administratin of Saposhnikovia divaricata.With the same chromatographic condition,twenty samples of rats containing plasma samples obtained at 0,0.5,2 h after oral administratin of Saposhnikovia divaricata and urine samples obtained 0-12 h after oral administratin of Saposhnikovia divaricata were analyzed.Nineteen chromatographic peaks were detected from the plasma samples of the normal rats.Compared with the fingerprints in vitro,six peaks were identified to be from Saposhnikovia divaricata.And five peaks were chromones,which were sec-o-glucosylhamaudol,5-O-methylvisamminol,4’-O-D-glucosyl-5-O-methylvisamminol, cimifugin and prim-o-glucosylcimifugin.The HPLC fingerprints of normal rats and AA rats were compared,the results show that the constituents from Saposhnikovia divaricata of the model plasma samples are less than the normal plasma samples,but the model urine samples were complex than the normal urine samples.The results suggest that the inflammatory condition could influence the metabolic process in rats.The total componet analysis of the plasma samples was carried out by fingerprint method.The chemical data and the pharmacologic data was combined to confirmed the therapeutic basis of anti-inflammatory action of Saposhnikovia divaricata by methematical statistics method.The plasma fingerprints of eighteen AA rats were analyzed to obtained the chemical data.Eighteen chromatographic peaks were marked and calculated,and 18*18 matrix was used as the chemical information.The pharmacologic data were obtained by determination of the swelling after oral administration of low,middle and high dose of Saposhnikovia divaricata.The chemical data of plasma and the pharmacologic data was combined by the software SPSS 13.0.Through the analysis by correlation and regression,prim-o-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol were validated to be the therapeutic basis of anti-inflammatory action of Saposhnikovia divaricata.A accurate and sensitive HPLC-MS method was firstly developed for simultaneous determination of prim-o-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol in biological samples.And the pharmacokinetics of the two active constituents was systematically investigated.The ESI source and the selected ion monitoring with positive ion mode was chosed. The plasma or urine samples were prepared by protein precipitation.Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C₁₈ column with a mobile phase consisted of methanol,water and 0.1%formic acid.Puerarin was added as the internal standard.The method was validated with the concentration range 1.0-100 ng/mL in rat plasma for prim-O-glucosylcimifugin,1.5-150 ng/mL in plasma for 4’-O-D-glucosyl-5-O-methylvisamminol.The lower limit of quantitation（LLOQ） of prim-O-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL, respectively.The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0%as terms of relative standard deviation（R.S.D.）.Accuracy determined at three quality control concentrations（2.0,25 and 75 ng/mL for prim-O-glucosylcimifugin;3.0,37.5 and 112.5 ng/mL for 4’-O-D-glucosyl-5-O-methylvisamminol） ranged from -1.9 to 3.9%as terms of relative error（R.E.）.The LC-ESI-MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Saposhnikovia divaricata to rats.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol were as follow: t_1/2 wasl.31h and1.96 h,C_max was39.92 and 41.53 ng/mL,t_max was 0.54 and 0.56 h,AUCo-t was 66.77 and 65.65 ng/L*h,respectively.The differences of pharmacokinetics between normal rats and AA rats were firstly studied after multidose oral administraion of Saposhnikovia divaricata.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol were as follow:in normal rats t_1/2 was1.68 and 1.97 h,C_max was 58.53 and 55.01 ng/mL,t_max was 0.47 and 0.5h,AUC_SS was 111.0 and 108.4 ng/L*h,respectively;in the AA rats,t_1/2 was 3.35 and 2.99 h,C_max was 60.23 and 61.34 ng/mL,t_max was 0.33 and 0.20 h,AUC_SS was 71.79 and 153.8 ng/L*h.Compared with the normal rats group,t_max becomes faster,t_1/2 bocames longer in the AA rats group,which suggest that the inflammatory condition could influence the absorption and metabolic process in ratsA selective,sensitive and rapid UPLC/MS/MS method was firstly developed and validated for determination of prim-O-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol in rat plasma.With puerarin as internal standard,the plasma samples were precipitated by methanol adding with 0.1%HC1.The separation was performed on an ACQUITY UPLC^TM BEH C₁₈ column （50 mm×2.1 mm,i.d.,1.7μm） with the mobile phase consisting of methanol-water-0.1%formic acid at a flow rate of 0.25 mL/min.The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring（MRM）. Linear calibration curves were obtained in the concentration range of 0.5 - 100 ng/mL,with the lower limit of quantification of 0.5 ng/mL.The intra- and inter-day precision（R.S.D.） values were below 15%and accuracy（R.E.） was from -2.6%to 0.84%at all QC levels（2.0,25 and 75 ng/mL）.The new method was fully validated and successfully applied to a pharmacokinetic study of prim-O-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol in 12 rats by oral administration and intravenous injection.The pharmacokinetic parameters of prim-o-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol were as follow:after oral administration and intravenous injection of prim-O-glucosylcimifugin,t_1/2 was 1.65 h and 1.44 h, C_max was 39.14 and 93.28 ng/mL,t_max was 0.54 and 0.05 h,AUC_0-t was 68.22 and 92.00 ng/L*h, respectively;after oral administration and intravenous injection of 4’-O-D-glucosyl-5-O-methylvisamminol,t_1/2 was 1.91 and 1.73 h,C_max was 48.41 and 89.85 ng/mL, t_max was 0.30h and 0.05 h,AUC_0-t was 23.28 and 45.77 ng/L*h. Compared the AUC of prim-O-glucosylcimifiigin and 4’-O-D-glucosyl-5-O-methylvisamminol afer administration the monomer by two ways,the results show that the absolute bioavailability of prim-O-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol was no more than 1%, which suggests that the absorption should be improved when given the monomer for clinical cure. Compared the pharmacokinetic differences of prim-O-glucosylcimifugin and 4’-O-D-glucosyl-5-O-methylvisamminol between oral administration of the monomer and the herb, the results show that the pharmacokinetics of prim-O-glucosylcimifugin was similar between oral administration of the monomer and the herb,but the pharmacokinetics of prim-O-glucosylcimifugin was some changed,which shows that after oral administration of the monomer t_max became shorter, absorption dose became less,but t_1/2 was the same.The results suggest that some ingredients of Saposhnikovia divaricata could promote the absorption of 4’-O-D-glucosyl-5-O-methylvisamminol.