Determination Of The Contents Of Strychnine And Brucine With HPLC And Toxicology Study On SouFeng Pills

Objective: SouFeng Pills (SFP) is the compound praeparatum we developed, which has effects of warming meridians, dispelling cold and beriberoid diseases and activating collaterals. It is mainly applied to treat wind cold wetness evil infestating the body and chronic obstinate diseases. The main active components are strychnine and brucine. The contents of strychnine and brucine and the methodology should be determined by HPLC to control the toxical components. Meanwhile comparative study of strychnine and brucine contents from different processed products was performed. Toxic reactions of rats administered by SFP (nux vomica soaked by acetic acid) consecutively and repeatedly, first symptoms and severity, target organs and the recovery were observed to determine appropriate dosage having no toxic reactions and to provide safe dosage for human being. Comparative study of the difference between rude nux vomica and processed nux vomica in toxic reactions was performed to provide scientific base on quality control and safe clinical application of SFP.Methods:1 Determination of the Contents of Strychnine and Brucine in Compound SouFeng Pills with HPLCHPLC method was used to establish the measurement method of Strychnine and Brucine, as well as the methodology.2 Toxicology Study on SouFeng PillsEighty Wistar rats were randomly divided into four groups: control group, low dose SFP (0.7g crude drug/kg), hingh dose SFP (2.8g crude drug/kg), and NSFP group (rude nux vomica, 2.8g crude drug/kg) .Rats were raised in cage and fed freely. SFP was continuously administrated 10ml/kg for 12 weeks after one week adaptation period. Rats were weighed weekly and dosage was regulated iaccording to weight changes. Ethological changes were observed in open-field experiment and Pole-jumping test during the administration period. The organ coefficient, blood cell counting, blood biochemistry index, histological and pathological indexes were measured at the end of the experiment and after two weeks of the experiment. The latter was measured as the indexes in convalescent period.Results:1 The HPLC methods for measurement of Strychnine and Brucine in Compound SouFeng Pills and was established and comparative study of different processed products was performed Chromatographic conditions and system suitability test: 18 alkyl silane bonded silica was used as the filler and methanol-water– triethylamine (54:45.5:0.5) was used as the mobile phase, the column temperature was 30℃, the flow rate was 1ml/min, and the detection wavelength was 260nm.Theoretical plate number calculated by Strychnine should not be lower than 5000.Reference standard solution preparation: precisely take appropriate amounts of Strychnine and Brucine reference substance and put it into methanol to make it containing 40.16μg and 20.12μg, respectively, in 1ml solution.Test solution preparation: precisely weigh 5g specimen, comminute and put it into triangular flask. Precisely add Sodium hydroxide 3ml, misce bene, keep it 15 minutes and then precisely add trichloromethane 25ml. Make it airtight, weigh it and extract active components for 2h with backflow extracting method. Cool and weigh it again. Supplement the lost weight by trichloromethane and agitate it. Filter it with the filter paper containing a little Anhydrous Sodium. Precisely draw the filtrate 10ml and put it into the separatory funnel, extract it 5 times with 20ml per time with 20% sulphuric acid. Put the acid liquor together and regulate PH value to 11 by the 20% caustic soda solution. Extract it 5 times with 20ml per time by trichloromethane, Put the trichloromethane layer together, recovery and dry it, and then add the methanol to the residue, transfer it to volumetric 25ml flask, dilute it to the scale, misce bene.Determination: draw the test solution and reference substance solution 10μl, respectively, and inject them into HPLC to determine the contents.Strychnine and Brucine in nux vomica soaked by acetic acid were lower significantly in the comparative study of contents of different processed products.2 Result of the toxicology Study on SouFeng Pills: (1) Growth condition and ethological changes: rats in NSFP group were observed restless and excited, and there were no death in the rest of rats during the experiment. Compared with the control group, there was no significant change (P>0.05) in the frequency of stepping over the grid, the frequency of erecting and the detention time in the center during the open-filed experiment. During the Pole-jumping test, the low dose SFP group and the NSFP group were significantly decreased (P <0.05), but the others had no significant difference compared with the control group (P>0.05). (2) Food consumption and weight: the food consumptions of female and male rats in low dose SFP group were increased at the seventh week and the ninth week respectively (P<0.05). The weights of all other groups were not changed significantly compared with the control group (P>0.05). (3)Hematology: HB and GR in the high SFP group and the NSFP group were significantly decreased (P <0.05). LY in the high dose SFP group and the NSFP group were significantly increased (P<0.05). Neutrophilic leucocyte and lympholeukocyte in NSFP group were increased significantly (P<0.05) compared with the control group. There were not significant difference (P>0.05) in other hematological indexes compared with the control group. (4) Biochemical indicator of blood: The ALT in the high dose SFP group and the NSFP group were significantly increased compared with the control group (P<0.05) at the twelfth week, and there was no significant difference (P>0.05) in other Biochemical indicators of blood compared with the control group. (5)Organ coefficient: The coefficients of liver and kidney in high dose SEP group and NSFP group were increased significantly (P<0.05), but there was no significant difference in the other groups compared with the control group (P>0.05). (6) Pathomorphology inspection: Liver: bile duct hyperplasia and hepatic cells nephelo were observed with midrange in the high dose SFP group. The hepatic cells necrosis and phlogocyte infiltrating were observed in the NSFP group. Kidney: kidney glomerulus vacuolization and kidney tubules necrosis were observed in high dose SFP group and NSFP group. Muscles: carcoplasm disintegration and fibrous tissue hyperplasia were observed in the high dose SFP group, carcoplasm disintegration and phlogocyte infiltrating were observed in the NSFP group. No cell denaturation, necrosis and phlogocyte infiltrating were observed in the other organs of other groups. (7) Observation in convalescent period: there was no significant change of growth condition, ethological detection, weight and food consumption in all experimental groups compared with the control group (P>0.05).There was no significant difference compared with the control group (P>0.05) with the HB. GR and LY in the NSFP group were higher than the control group (P<0.05). There were no significant difference compared with the control group (P>0.05) with the ALT in the high dose SFP group. The ALT in NSFP group was still higher than that of the control group (P<0.05). The coefficients of liver and kidney were still higher than the control group (P<0.05). Tiny granuloma was observed in the high dose SFP group by microscope and necrosis was still in the NSFP group. Kidney glomerulus vacuolization and Kidney tubules necrosis were still in kidney. Muscles cell nucleus were disarranged in the high dose SFP group and carcoplasm disintegration and phlogocyte infiltrating were still in the NSFP group.Conclusions:1 The method of determining contents of Strychnine and Brucine by HPLC in Compound SouFeng Pills is simple, reliable and suitable for quality control of SouFeng Pills. The results show that the contents of strychnine and Brucine in nux vomica soaked by acetic acid are the lowest, according to the comparative study of different processed products of nux vomica.2 SouFeng Pills (2.8g/kg) could induce HB and GR to decrease significantly and LY and ALT to increase significantly; the coefficients of liver and kidney were increased.Pathomorphology observation: hepatic cells nephelo spread widely, and kidney glomerulus vacuolization was observed as well. Hepatic cells necrosis and kidney tubules necrosis were observed in liver and kidney respectively in the NSFP group .The toxicity of NSFP group is higher than that of the SPF groups.3 The rats'concordant, muscle endurance and tensity were impacted by the SFP (2.8g/kg). Pathomorphology observation: carcoplasm disintegration and fibrous tissue hyperplasia were observed in the high dose SFP group, and carcoplasm disintegration and phlogocyte infiltrating were observed in the NSFP group.