Investigation On Quality Equivalence Of Zanthoxyli Pericarpium From Two Species

Zanthoxyli Pericarpium came from dry ripe pericarp of Zanthoxylum schinifolium Sieb. et Zucc. or Zanthoxylum bungeanum Maxim.which belonged to Rutaceae. Zanthoxyli Pericarpium was the variety which had two species. Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc.had been used as sources of Zanthoxyli Pericarpium together since 1985 version of Chinese Pharmacopoeia. But it must be studied that if the two species could be used as the same medical material and if the two species had same chemical constituents, drug actions and toxicity. The comparative studies on herbal records, genetic diversity, chemical compositions, main drug actions, acute toxicity and pharmacokinetics characteristic between Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc. had been reported less. The article therefore first compared deeply and systematically these several aspects between Z. bungeanum Maxim and Z.schinifolium Sieb. et Zucc. which were the two species of Zanthoxyli Pericarpium in Chinese Pharmacopoeia. The study provided scientific basis if the two species could be used as the same medical material.It established foundation for revising standard of medical materials in Chinese Pharmacopoeia. It also provided an example of quality evaluation for another medical materials which had many species. The content and results of study were as follows:1 Herbal textualThe article researched herbal records of Z. bungeanum Maxim.and Z. schinifolium Sieb.et Zucc..Z. bungeanum Maxim.was wrote down in Shi Jing for the first time. It was called Hui in ancient times. According to its habitat, it was divided into two kinds, namely Qing Jiao and Shu Jiao in herbals. From the delineations of their appearances and the figures attached in herbals, they were both Z. bungeanum Maxim.of Zanthoxylum of Rutaceae. There was not any record about Z. schinifolium Sieb. et Zucc. in herbals. As sibling kindred plant of Z. bungeanum Maxim., Z. schinifolium Sieb. et Zucc. was used as one of the soures of Zanthoxyli Pericarpium in "the example of traditional Chinese medicine in China Republic". Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc. were clearly listed together below the item of Zanthoxyli Pericarpium in 1985 version of Chinese Pharmacopoeia.2 Comparison of genetic diversityZ. bungeanum Maxim.and Z.schinifolium Sieb.et Zucc.respectively belonged to Zanthoxyli Pericarpium subgenus and Radix Zanthoxyli subgenus of Zanthoxylum of Rutaceae. There were differences in quality between them and medical materials from different habitats.There had not been any correlated report about if the differences were induced by genetic variation at present. The article thus first studied genetic diversity of Z.bungeanum Maxim.and Z. schinifolium Sieb.et Zucc.to approach genetics basis of the differences in quality of them. The study provided scientific basis for consummating quality evaluation method of Zanthoxyli Pericarpium. Analytical technique of random amplified polymorphism DNA (RAPD), which was based on polymerase chain reaction (PCR), was used to analyze genetic diversity of Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc.,and then cluster analysis was done. The polymorphism ratio of Z. bungeanum Maxim.and Z. schinifolium Sieb.et Zucc. were respectively 78.3% and 67.5%.They both had ditissimus genetic diversity. The polymorphism of Z. bungeanum Maxim.was larger than Z. schinifolium Sieb. et Zucc.. The genetic distance of Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc. was between 0.6406 and 0.7368.Their genetic relationship was relatively far and they were clustered into two groups in cluster analysis, which coincided with that they respectively belonged to different subgenus in plant taxonomy.It was thus evident that Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc. had same genetic background. But there ware also some differences between them.3 Comparison of chemical compositionsThe topic group had once compared essential oils and flavonoids compositions of Z. bungeanum Maxim.and Z. schinifolium Sieb.et Zucc.. Gas chromatograph mass spectrometer, high performance liquid chromatography and ultraviolet spectrophotometry were respectively used to determine. The result was that the content and composition of essential oils and the content of Rutosids and total flavonoids all had significant differences. The contents of essential oils,Rutosids and total flavonoids of Z. bungeanum Maxim. were all higher than those of Z. schinifolium Sieb.et Zucc.. Mode of much index, which used finger printing determination plus quantitative determination of effect compositions, was used to compare the differences of chemical compositions between Z. bungeanum Maxim. and Z. schinifolium Sieb.et Zucc..3.1 Comparison of finger printThere were abundant effective components in Z. bungeanum Maxim. and Z. schinifolium Sieb.et Zucc., such as essential oils, amides, flavones, alkaloids and so on. The differences of their categories and contents were showed by finger prints. But only GC finger prints of essential oils in Z bungeanum Maxim. and Z schinifolium Sieb.et Zucc. had been reported at present. There had not been any study reports about HPLC finger prints of another compositions. For this reason, the article first set up and compared HPLC finger prints of Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc. to consider the differences of their main chemical compositions. The study provided scientific basis for consummating quality evaluation method of Zanthoxyli Pericarpium. The main chemical compositions were extracted by 50% methanol. The finger prints were determined by HPLC.Then the study compared the differences in HPLC finger prints of Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc.. Similarity of Z. bungeanum Maxim. was 0.909-0.992.There were twenty-seven common peaks in HPLC finger prints of Z. bungeanum Maxim. Similarity of Z. schinifolium Sieb. et Zucc.was 0.930-0.999.There were twenty-four common peaks in HPLC finger prints of Z. schinifolium Sieb.et Zucc.. There were differences in relative peak areas of common peaks in their HPLC finger prints. The results above-mentioned explained that there were obvious differences in their HPLC finger prints, and in the constructions and relative contents of their chemical compositions. Amides compositions were most abundant. The finger prints that this article set up could obviously distinguish Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc.. For this reason, it was suggested that finger prints and the contents of main effective compositions should be used together to control the quality of Zanthoxyli Pericarpium. 3.2 Content comparison of amides compositionsAmides compositions were the main substance which tasted numb in Z. bungeanum Maxim.and Z. schinifolium Sieb.et Zucc.. They had many physiologic functions such as anesthesia, excitement, bacteriostasis, expelling wind-evil and removing wetness, anthelminthic, analgesia, etc.From their HPLC finger prints, amides compositions were the most abundant compositions.But there had been few study reports about comparison of their contents in Z. bungeanum Maxim. and Z. schinifolium Sieb.et Zucc.at present. The article accordingly first compared the content of amides compositions which were the typical numbing substances in ten batch Z. bungeanum Maxim.and ten batch Z. schinifolium Sieb.et Zucc.The study provided scientific basis for revising standard of medical materials in Chinese Pharmacopoeia. Amides compositions were extracted with ethyl ethe by Soxhlet extraction and their contents were determined by HPLC. Then the study compared the differences in amides compositions'contents of Z. bungeanum Maxim.and Z. schinifolium Sieb.et Zucc.. The contents of amides compositions in ten batch Z. bungeanum Maxim.were between 1.038% and 1.660%.The average content was 1.276%. The contents of amides compositions in ten batch Z. schinifolium Sieb. et Zucc. were between 1.997% and 2.777%. The average content was 2.322%. The contents had obvious differences, which indicated that their main effects also had differences. The contents of amides compositions in Z. schinifolium Sieb. et Zucc. were double that in Z. bungeanum Maxim.. Amides compositions in Z. schinifolium Sieb. et Zucc. were accordingly more abundant than that in Z. bungeanum Maxim.. It was thus evident that the contents of amides compositions in them were not controlled by same content limit. It was suggested that the content limits of Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc. should be set separately in medical material standards.The contents of amides compositions in Z. bungeanum Maxim. were not below 1.0%.The contents of amides compositions in Z. schinifolium Sieb. et Zucc. were not below1.9%.In a word, it had some limitations that medical material quality was only control by the content of essential oil in Zanthoxyli Pericarpium item of Chinese Pharmacopoeia.4 Comparison of main drug actionsAccording to the traditional effects of Zanthoxyli Pericarpium, which was wanning middle energizer to relieve pain and killing parasites to relieve itching, the topic group had once compared gastrointestinal tract effects of Z. bungeanum Maxim. and Z. schinifolium Sieb.et Zucc.. The result indicated that there were significant differences in gastrointestinal tract effects of them. The anti-diarrhoea effect and inhibiting effect of facilitating movement of small intestine of normal mice of Z. bungeanum Maxim.were both better than those of Z. schinifolium Sieb.et Zucc..4.1 Comparison of local anesthetic effectThe effects of Zanthoxyli Pericarpium were warming middle energizer to relieve pain and killing parasites to relieve itching. Local anesthetic effect was one of its main effects and of connotations of warming middle energizer to relieve pain. Local anesthetic effects of Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc. mainly came from their amides compositions. The experiments verified that the contents of their amides compositions had obvious differences. So it should be studied if their local anesthetic effects also had differences. But the reports about comparison in local anesthetic effects of Z. bungeanum Maxim. and Z schinifolium Sieb. et Zucc. were few. So the article first compared local anesthetic effect which was their main effect. The study provided scientific basis for clinical application of Zanthoxyli Pericarpium. Methods of administration on frog's skin, frog's leg and mouse's foot were used to compare the differences in surface anesthesia effects, conduction anesthesia effects and infiltration anesthesia effects of Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc.. The result was that both Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc. had local anesthetic effects, such as surface anesthesia effects,conduction anesthesia effects and infiltration anesthesia effects. But there were significant differences in their local anesthetic effects. Local anesthetic effect of Z. schinifolium Sieb. et Zucc. was better than that of Z. bungeanum Maxim., which coincided with the conclusion that the contents of amides compositions in Z. schinifolium Sieb. et Zucc., which was the main compositions that could cause local anesthetic effects, were higher than that in Z. bungeanum Maxim..4.2 Comparison of analgesic effectThe effects of Zanthoxyli Pericarpium were warming middle energizer to relieve pain and killing parasites to relieve itching. Analgesic effect was one of its main effects. Local anesthetic effect was the connotation of analgesic effect of Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc.. The experiments verified that their local anesthetic effect had obvious differences. So it should be studied if their analgesic effects also had differences. The article thus compared analgesic effect which was their another main effect, and first determined half effective dose of analgesic effect of Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc.. The study provided scientific basis for clinical application of Zanthoxyli Pericarpium. Half effective dose of analgesic effect was determined with pain model induced by hot plate in mice. Then the study compared the differences in analgesic effects of Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc.. The result was that both Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc.had analgesic effects.Their half effective doses of analgesic effect were respectively 18.43±5.31 g·kg-1 and 11.18±2.28 g·kg-1.There were significant differences in their analgesic effects. Analgesic effect of Z. schinifolium Sieb.et Zucc. was better than that of Z. bungeanum Maxim., which coincided with the conclusion that local anesthetic effect of Z. schinifolium Sieb. et Zucc., which was one of the connotations of analgesic effect, was better than that of Z. bungeanum Maxim..5 Comparison of acute toxicityHerbals recorded that Zanthoxyli Pericarpium was slightly poisonous. So the article compared their acute toxicities. The study provided scientific basis for clinical application of Zanthoxyli Pericarpium. The differences in acute toxicities of Z. bungeanum Maxim. and Z. schinifolium Sieb.et Zucc.were compared by determining their half lethal doses. The result was that both Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc. had acute toxicities. Their half lethal doses were respectively 51.14±4.47 g·kg-1 and 78.02±6.86 g·kg-1.There were significant differences in their acute toxicities. Acute toxicitiy of Z. bungeanum Maxim.was larger than that of Z. schinifolium Sieb. et Zucc..6 Comparison of pharmacokinetics characteristicAnalgesic effect and acute toxicity of Z. bungeanum Maxim. and Z.schinifolium Sieb. et Zucc. had significant differences. There had not been any related study report about that whether the differences concerned with the different internal pharmacokinetics process of drugs or not. So the article first studied the internal pharmacokinetics characteristic of Z. bungeanum Maxim. and Z.schinifolium Sieb. et Zucc. to approach the internal reasons that analgesic effect and acute toxicity of them had differences. The study provided scientific basis for instituting reasonable clinical application project of of Zanthoxyli Pericarpium. After water-extracted solutions of Z. bungeanum Maxim. and Z. schinifolium Sieb. et Zucc. intragastricly administered in mice, apparent pharmacokinetics parameters of them were determine with pharmacodynamics effect method which took analgesic effect as index and drug cumulative method which took acute toxicity as index. Then the study compared the differences in pharmacokinetics characteristic between Z bungeanum Maxim. and Z. schinifolium Sieb.et Zucc.. Main apparent pharmacokinetics parameters of Z. bungeanum Maxim. with pharmacodynamics effect method were that t1\2α=0.421 h, Ka=3.519 h-1, AUC(0-t)= 8.365 g·h·kg-1, Tmax=4.000 h, Cmax=5.480 g·kg-1.Main apparent pharmacokinetics parameters of Z. schinifolium Sieb. et Zucc. with pharmacodynamics effect method were that t1/2=0.676 h,Ka=1.523 h-1,AUC( =17.111 g·h·kg-1,Tmax=5.000 h,Cmax=6.710 g·kg-1.Main apparent pharmacokinetics parameters of Z. bungeanum Maxim.with drug cumulative method were that t1/2=1.321 h,AUC(0-t)=102.015 g·h·kg-1,Tmax=0.500 h,Cmax=46.720 g·kg-1.Main apparent pharmacokinetics parameters of Z. schinifolium Sieb.et Zucc. with drug cumulative method were that t1/2=0.607 h,AUC(0-t)=83.561 g·h·kg-1,Tmax =0.250 h,Cmax=75.050 g·kg-1.Apparent pharmacokinetics parameters of them respectively with pharmacodynamics effect method which took analgesic effect as index and drug cumulative method which took acute toxicity as index both had differences, which coincided with the conclusion that there were differences in their analgesic effects and acute toxicities. To sum up, there were many differences between Z. bungeanum Maxim.and Z. schinifolium Sieb. et Zucc. in herbal records,genetic diversity, chemical compositions, main drug actions, acute toxicity and pharmacokinetics characteristic. So it was sugfested that Z. Bungeanum Maxim. and Z. Schinifolium Sieb. et Zucc. should be used separately in the new version of Chinese Pharmacopoeia. In addition, Z. Bungeanum Maxim.was used more than Z Schinifolium Sieb.et Zucc.in ancient herbals and modern clinical application. But the experiments verified that local anesthetic effect and analgesic effect of Z. Schinifolium Sieb. et Zucc. were better than that of Z. Bungeanum Maxim., and that acute toxicity of Z Schinifolium Sieb.et Zucc. was less than that of Z. Bungeanum Maxim.. So the apply of Z. Schinifolium Sieb. et Zucc. should be increased in clinical application. Especially in local anesthetic and analgesic application, Z. Schinifolium Sieb. et Zucc. could took the place of Z. Bungeanum Maxim..