| ObjectiveTo study the effects of Polyporus umbellatus on ALD, AngⅡ, ADH and ANP in normal rats with multiple-target diuretic mechanisms and to provide evidence for clinical application.To study the effects of different dosage and times of administration of Polyporus umbellatus on ACE and AngIIreceptor 1 and to disscuse the parmacological basis of its diuretic effects.To establish simutaneous determination of ergosterone and ergosterol with LC/MS and to provide the QC for Chinese herbal drug.To establish simutaneous determination of plasma concentration of ergosterone and ergosterol in rats after administration of Polyporus umbellatus with HPLC-APCI/MS/MS and to calculate the pharmacokinetic parameters with statistical momental models and compartmental models.To establish simutaneous determination of urinary concentration of ergosterone and ergosterol in rats after administration of Polyporus umbellatus with HPLC-APCI/MS/MS and to illustrate the subsequent excretion.MethodsSixty male SD rats were randomly grouped into 2 days and 8 days of administration. Each group of rats were administrered consecutively with normal saline(10mL·kg-1), furosemide(0.1 g·kg-1), low dosage Polyporus umbellatus(18.9 g·kg-1), middle dosage Polyporus umbellatus(56.7 g· kg-1), high dosage Polyporus umbellatus(94.5 g·kg-1). Immunoassasy was used to detect the plasma content of ALD, AngII, ADH and ANP.Forty-two rats with normal urinary volume, Na+, K+, and Cl- were administered consecutively for 8 days with normal saline(10 mL·kg-1), furosemide (0.1g·kg-1), captopril(0.63 g·kg-1), losartan(0.315 g·kg-1), low dosage Polyporus umbellatus(18.9 g·kg-1), middle dosage Polyporus umbellatus(56.7 g·kg-1), high dosage Polyporus umbellatus(94.5 g·kg-1) with 6 in each group. Blood was sampled 24 hours after the first, the second and the eighth administration and detected with ELISA kits.The standard solution was prepared with ultrasonic extraction method, and seperated with Zorbax Eclipse XDB-C18 (2.1 mm×150 mm,3.5 μm), the mobile phase is methanol:10 mmoL·L-1 ammonium acetate buffer (98:2, V/V), the speed is 0.4 mL·min-1. The sample was detected with HPLC-APCI/MS/MS and positive MRM.The plasma samples were extracted with acetidine. The plasma concentration of ergosterone and ergosterol was determined simutaneously with the internal standard of β-ecdysterone. After validation the PK parameters were calculated with WinNonlin.Twenty-four rats were randomly divided into 3 groups with 8 in each group and administered with low dosage Polyporus umbellatus(18.9 g·kg-1), middle dosage Polyporus umbellatus(56.7 g·kg-1), high dosage Polyporus umbellatus(94.5 g·kg-1)-After administration, the urine was collected during the time period of 0-2,2-4,4-8,8-12,12-24,24-36,36-48,48-60 hours. The urinary samples were diluted and prepared. The urine-concentration time curves and accumulative urine time curves were drew.ResultsThe 2d group and 8d group of administration of Polyporus umbel latus showed significant diuretic effects. And the high dosage of Polyporus umbellatus acted equally as furosemide. All dosages of Polyporus umbellatus may significantly decrease the content of ALD, AngⅡ, ADH, and ANP with a higher altitude of 8d than that of 2d (P< 0.05). Among them, ALD and ANP of high dosage in 8d group changed most as 56.7% and 69.2%(vs NS); ADH of middle dosage in 8d decreased as 50.8%(vs NS). However, furosemide didn’t show marked changed between 2d and 8d.Effects of Polyporus umbellatus on ACE and ATI in plasma of normal rats.Effects of Polyporus umbellatus on ACE. All dosages of Polyporus umbellatus and captopril may significantly decreased the content of ACE in rats (vs NS) with the elevated altitude as the increasing administration days. After Id admistration, all dosages of Polyporus umbellatus show effects lower than that of captopril; after 2d, high dosage equal; after 8d, all equal. The decreasing level showed certain dosage-dependency.Effects of Polyporus umbellatus on AT1. All dosages of Polyporus umbellatus and losartan may significantly decreased the content of of AT1. After Id and 2d admistration, all dosages of Polyporus umbellatus show effects lower than that of captopril; after 8d, all equal. The decreasing level showed certain dosage-dependency.The concentration of ergosterone and ergosterol between 1-3000 ng·mL showed good linearity with chromatographic peak area. The average recovery is 100.8% and 100.7%, intra- and inter-day variance (RSD) were all below 6.5%. The retention time is 4.2min and 4.6min.The plasma concentration of ergosterone and ergosterol between 1-3000 ng·mL-1 showed good linearity with chromatographic peak area. The relative recovery of control standards of ergosterone is (95.8±8.5)%, (96.0±4.4) %, (95.5±3.5)%(n=15). And the extraction recovery is (68.3±9.1)%, (91.5±6.5)%, (85.5%±4.4)%(n=5). The relative recovery of control standards of ergosterol is (97.3±7.7)%, (100.7±8.9)%, (101.4±7.5)%(n=15) And the extraction recovery is (68.7±10.5)%, (90.6±13.3)%, (90.6±8.5) %, (n=5). All the intra- and inter-day variance and stability (RSD) were below 6.5%. The PK parameters were calculated with 1st order one-compartment model and statistical momental models. After sigle i.g. administration of Polyporus umbellatus, the AUClast, Cmax and Ka of ergosterone and ergosterol increased, with the shortest Tmax and longest Half Life in the middle dosage group. But V_F of ergosterone is the largest in the high dosage group and V_F of ergosterol is the largest in the middle dosage group. MRT of ergosterone is the longest in the low dosage group and MRT of ergosterol is the longest in the middle dosage group. All the Ke and Clearance of ergosterone and ergosterol increased dependently with dosage.The urine concentration of ergosterone and ergosterol between 1-300 ng· mL-1 showed good linearity with chromatographic peak area. The relative recovery of control standards of ergosterone is (92.8±4.4)%, (98.6±7.3) %, (93.5±7.1)% (n=15). And the matix efficacy is (96.0±0.2)%, (98.9±3.0) %, (95.1±1.2)%, (n=5). The relative recovery of control standards of ergosterol is (94.8±6.2)%, (98.8±5.8)%, (s99.3±6.2)%(n=15). And the matix efficacy is (96.7±2.2)%, (99.9±4.7)%, (96.4±4.8)%, (n=5). The ergosterone and ergosterol in urine reached the highest excretion volume after 18 hours of administration. But 48 hours-60 hours after administration the urine still remained a few ergosterone and ergosterol. The whole excretion volume showed dosage-dependency.ConclusionALD, Ang Ⅱ, ADH and ANP play key roles in the modulation of body water-fluid metabolism. The diuresis of Polyporus umbellatus may be related with decreasing ALD, AngⅡ, ADH and increasing ANP. The Chinese herbal drug hereby demonstrates longer and marked efficacy to provide evidence for the multiple-target effects of inducing diuresis and excreting dampness recorded in traditional literatures.The action target of Polyporus umbellatus may be inhibition of ACE and antagony of AT1, as to lower the hormone level of RAS and exhibit strong diuresis.The HPLC-APCI-MS/MS method established in this paper was specific and economic to determine the content of ergosterone and ergosterol in the Chinese herbal drug accurately, sensitively, and quickly. The data may provide basis for the QC of Polyporus umbellatus.The simutaneous determination method of plasma concentration of ergosterone and ergosterol in rats after administration of Polyporus umbellatus with HPLC-APCI/MS/MS established in this paper provides reliable pharmacokinetic parameters with statistical momental models and compartmental models for the Chinese herbal drug, and depicts the ADME of the two active components of Polyporus umbellatus, and provide referrable optimal dosage, administration period, loaded dosage and best regimen for clinical application.The simutaneous determination method of urinary concentration of ergosterone and ergosterol in rats after administration of Polyporus umbellatus with HPLC-APCI/MS/MS established in this paper is also effective, sensitive and specific. The data showed that the active components of Polyporus umbellatus may last for a certain period in the body with slow metabolism to supplement the blank area. |
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