| As one of the four major Huaiyao, rehmannia is generally used in traditional Chinese medicine as nourishing medicine. Its main active ingredient, Catalpol, has a variety of biological activities, such as prevention of dementia, treating for cerebral ischemia, diuresis and lowering of serum glucose, etc., which indicates the Catalpol is of great medicinal potential to explore. Current researches showed that different ways of Catalpol administration would express significant difference on effects or dosages, however, with unclear reasons. This paper was aiming to use ALX-induced diabetes model to study whether it wouldl appear dosage variance when treating diabetes with Catalpol by different administration ways. Besides, the following items were studied to explore the potential mechanism of this phenomenon for a preliminary basis of administration ways’ and dosage forms’ choosing:(1) the influence of gastrointestinal physicochemical environment on the stability of Catalpol;(2) the impact of hepatic microsomal enzyme on the metabolism of Catalpol;(3) the variance in Catalpol’s pharmacokinetics dynamics by different administration ways.Part1:Therapeutic effects of Catalpol by different administration ways on ALX-induced diabetic ratsThe purpose was to investigate the variance of therapeutic effects of Catalpol on diabetic rats by different administration ways. The8rats in normal group was randomly selected and normally fed, without any treatment. While the model rats were prepared as follows:healthy rats were fed for12h under the condition of abrosia but no lack of water. Then they were administrated with Catalpol by two steps:the first step was to give freshly prepared1%alloxan by intraperitoneal injection to prepare diabetic rats models. A single intraperitoneal injection of120mg/kg of1%alloxan was given on the first day, and a supplemental100mg/kg was given on the second day. After that, the rats were obtained72h of free food and drink. Then abrosia for12h, they were taken blood from fossa orbitalis in order to determine the serum glucose by glucose meter. The rats whose serum glucose values were over16.7mmol/L were selected as diabetic rats models.500mg/kg of Metformin was used as a positive control drug. Experimental groups were divided into two groups:intragastric administration group and intraperitoneal injection group. The rats of intragastric administration group were then divided into three subsets, each was intragastricly administrated with50,100, and200mg/kg of Catalpol, respectively. The rats of intravenous injection group were also divided into three subsets, each was intravenouseally injected with5,10, and20mg/kg of Catalpol, respectively. The administration was began after the successful preparation of diabetic rats models. The normal and model control groups were given relative volume of saline. All of the administrations were conducted once a day, lasting for two weeks. Finally, the influences of Catalpol on serum glucose, blood fat and carbohydrate tolerance were determined.The results showed that different ways of administration of Catalpol had significant variance in dosages for lowering serum glucose and blood fat. When intragastricly administrated, the effective dosage was200mg/kg. When intravenouseally injected, all of the5,10, and20mg/kg dosages of Catalpol have significant effects on lowering serum glucose and blood fat, and improving the glucose tolerance, which presented as an obvious dose-dependent manner.Part2:Exploring for the reason of dosage variance when administrating with Catalpol by different waysIn order to investigate the reason of dosage variance when administrating with Catalpol by different ways, we studied the stability of Catalpol under different pH conditions, the metabolism of hepatic microsomal, and the pharmacokinetic dynamics of Catalpol by different administration ways.(1) The influence of gastrointestinal physicochemical environment on the stability of CatalpolCatalpols were set in solutions of different pH value (1,2,3,5,7.4,9and12, respectively) at37℃.14h later, samples were taken to determine the concentration changes of Catalpols. SD rats were fasted for36h, then they were anesthetized by3.5%chloral hydrate. The stomach was exposed by surgery. The cardia was ligated with cannula, and the same with pylorus. Gastric lavage was conducted for twice with5mL protease-free artificial gastric juice. After that,5mL artificial gastric juice was injected and maintained for2h, then they were used to determine the concentration changes of Catalpols; Otherwise, SD rats were fasted for36h, then they were anesthetized by3.5%chloral hydrate. The intestinal tract was exposed by surgery. Ligation was made at10cm from the ligament of Treitz,10cm from the back portion of the blind intubation and ligation.20mL of artificial intestinal fluid was injected for washing, and the washing was conducted for twice. Then10mL of artificial intestinal fluid was injected mad maintained for2h, then they were used to determine the concentration changes of Catalpols. The results showed that Catalpol was instability when pH was at1,2, and3. The concentration of Catalpol linearly declined along with the change of time, and the higher the acidity, the sooner the declination. When pH>5, the concentration changes were little along with the time. When pH>7, Catalpol concentrations did not change. It indicated that Catalpol was easily damaged in an acidic environment, and relatively stable in an alkaline environment. In the intragastric administration experiment, the concentration of Catalpol was obviously decreased with the time, presenting as a linear declination trend. In the intraperitoneal injection experiment, the concentration of Catalpol was also obviously decreased with the time, presenting as a linear declination trend, but the declination speed was slower than that in the intragastric administration experiment.(2) The metabolism of Catalpol in hepatic microsomal enzyme systemThe metabolism of Catalpol in hepatic microsomal enzyme system was measured to acquire the influence of hepatic microsomal enzyme on the stability of Catalpol. The whole volume of hepatic microsomal incubation system was0.5mL, among which,0.1mol/L Tris-HCl buffer solution (pH=7.4) was used as balanced solution,10mg/L of Catalpol,1g/L of hepatic microsomal protein,10mmol/L of MgCl2,10mmol/L of KC1were contained in the system. After the system was set at37℃for5min,1mmol.L-1NADPH was added in and for a30min of water bath. Finally, the reaction was stopped at a ice bath. A screening was made for the reaction time, protein concentration, and catalpol concentration, and the optimal reaction condition was found out. Then the enzyme kinetic parameters Km, Vmax, and CLint were measured by Substrate Elimination Method. And the inhibitory effect of CYP450enzyme specific inhibitors on the metabolism of Catalpol was measured so that the subtypes of CYP450metabolic enzyme which participated in the metabolism of Catalpol could be identified out. When incubated for60min, the Catalpol’s linearity was good in the period of0～30min. When incubated in solutions whose protein concentration was0.2～2.0g/L, Catalpol could obtain increasing protein concentration in the range of0.2～1.0g/L. Therefore, the incubation protein concentration of Catalpol in the microsomal system was selected as1.0g/L. Different concentrations of Catalpol (2～100m g/L) were incubated in microsomal system with1.0g/L of protein concentration for30min. The metabolic speed began to slow down when the substrate concentration was over10m g/L. When the substrate concentration was over50m g/L, the declination trend was more obvious. Therefore, the optimal incubation concentration of Catalpol in the microsomal system was selected as10m g/L. As a result, the obtained enzyme kinetic parameters Km, Vmax, and CLint were0.44mg.L-1,3.24mg.L-1.min-1.mg-1, and7.3mL.min-1.mg-1, respectively. The results indicated that the specific inhibitor of CYP2C19-Ticlopidine was effective on the metabolism of Catalpol at a low concentration (1.25μmol/L). When the concentration of Ticlopidine was up to40μmol.L-1, the inhibitory effect was over50%. Besides, the specific inhibitor of CYP3A4-Ketoconazole was effective on the metabolism of Catalpol at a concentration of0.1μmol.L-1. When the concentration of Ketoconazole was at4μmol/L, the inhibitory effect was over50%. Moreover, the specific inhibitor of CYP2D6-Quinidine also expressed inhibitory effect on the metabolism of Catalpol at a concentration of1μmol/L. When the concentration of Quinidine was at20μmol/L, the inhibitory effect was between20%and50%. While other drugs had no obvious influence on the metabolism of Catalpol. It was namely that the metabolism of Catalpol was mainly related to CYP2C19, CYP3A4, and CYP2D6.(3) The pharmacokinetic variances among different administration ways (intramuscular injection, orally, intravenously)This part was aimed at investigating the difference of catalpol in pharmacokinetics by different administrations including column of Koelliker, intragastric administration and intravenous injection.18SD rats (SPF) were randomly divided into3groups which were administrated through column of Koelliker, intragastric administration and intravenous injection respectively. The dose of catalpol in all groups was50mg/kg. And all rats were inhibited to access to food for12h before entering the experiment. The samples of blood were collected via orbital artery at specific points of time, including0min,2min,2min,5min,15min,30min,60min,120min,180min,300min,480min and600min after administration. The prepared and processed samples were analyzed by HPLC. The date of catalpol concentration in blood at different times were analyzed and calculated by DAS3.5. The results showed that the ti/2of catalpol via intragastric administration (tl/2z=1.39±0.22h) was much longer than via column of Koelliker (tl/2z=0.84±0.41h) and intravenous injection (tl/2z=0.68±0.24h). Analogously, the MRT of catalpol via intragastric administration (1.62±0.20h) was also much longer than via column of Koelliker (1.62±0.20h) and intravenous injection (1.71±0.29h)(P<0.01). The Tmax of column of Koelliker (0.25±0.1h) was much shorter than intragastric administration (1.66±0.58h). Generally speaking, while the Vz/F of rat is under5L suggests that drug mainly distributes in plasma.Therefore, the distribution of catalpol was mainly in plasma depending on its Vz/F date in different ways (Vz/F of intragastric administration:1.17±0.16L/kg; Vz/F of column of Koelliker:0.40±0.20L/kg; Vz/F of intravenous injection:0.57±0.19L/kg). The intramuscular bioavailability was71.63%, which was greater than oral bioavailability (49.38%). Catalpol was instable in the gastrointestinal tract, but the oral bioavailability was close to50%. This result was nearly10-fold different from the efficacy of Catalpol on diabetes by oral or by intraperitoneal injection. It might be related to the metabolic product of Catalpol in vivo. |
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