| Zhizhu pill (ZZP) is a traditional well-known formula for treatment of "fullness" in TCM. Since the creation by Zhang Yuan-su in Jin Dynasty, Zhizhu Pill has been used for the treatment of chronic stomach injury, chronic disease and deficient disease on clinic. Zhizhu Pill is composed by Atractylodes Rhizome and Fructus Citrus Immaturus, and the later originates from the immature fruit of both Citrus aurantium L. (CA) and Citrus sinensis Osbeck (CS). Clinical trials had confirmed its effective in the treatment of functional dyspepsia, ZZP containing of CA (CA-ZZP) showed better efficacy than the other containing of CS (CS-ZZP). The present investigation is aimed to elucidate the disparities of the two kinds of ZZP based on their chemical composition and content, on the other hand, in a pharmacodynamic point of view, to compare the gastrointestinal motility and stomach gut hormone secretion of the main bioactive components from two kinds of ZZP, with the aim of exploring the relationship between differences of ingredients in two kinds of ZZP and their related differences in efficacy. To provide the scientific basis for the development of Zhizhu quality assessment methods and their clinical uses.1 Research on extraction process of the main active ingredients in ZZP1.1 Study on optimum extraction process of ZZP volatile oilsThe yield of volatile oil was chosen as indicator, the crush degree, soak time, water volume, and extraction time were investigated as factors. The optimum extraction process for the essential oil of bran fried Atractylodes Rhizomew was determined as following:6 fold amount of water was added to herbal pieces, then the essential oil extracted by steam distillation for 10 h after soaking for 2 hours. According to our previous research on the extraction process of two kinds of Fructus Aurantii Immaturus, the method was determined as following:6 fold amount of water was added to herbal pieces, then the essential oil was extracted by steam distillation for 8 h after soaking for 2 hours.1.2 Chemical constituents and contents of ZZP volatile oil influenced by different extracted methodBasing on background of the volatile oil which is not stable, GC-MS and HPLC methods was adopted to study comparatively whether the content of volatile oil on conventional extraction method that the extractor was wrapped with black cloth to get the oil keeping out of the sun light (dark condition oil), and the other was that the extractor was not wrapped to get the oil exposed to the sun light oil (light condition oil) No significant differences of yields of volatile oil existed between the two extraction methods.The GC-MS data showed that fifty-four constitutes and sixty constitutes were identified in the two kinds of oils which were extracted in dark and light conditions, respectively. The main categories of constituents were terpenes, as well as alcohols, acids, esters and so on, but the numbers were different for each type of constitutes. Among them, forty-six constitutes were common in the two types of volatile oils, but the relative amount of each constitute was different; The most abundant constitute was limonene, and the atractylone was significantly changed; Compared with the volatile oil obtained in dark condition, eight constitutes were disappeared and 13 constituents were appeared in the volatile oil obtained in light condition. The changes of chemical constituents and contents in volatile oil of Zhizhu pills showed that compositional changes for the volatile oil of Zhizhu Pills obtained in light condition were primarily attributable to the changes in the constituents of Chao Baizhu volatile oil, including the content different of atractylone, β-eudesmol, isovelleral, and atractylenolide.Comparative analysis of contents of 4 kinds of main active components atractylone, atractylenolide-I, atractylenolide-Ⅱ, and atractylenolide-Ⅲ were performed by HPLC. The content of Atractylone in light condition oil was significantly lower than that in dark condition oil, which were 73.01mg/g and 100.3mg/g, respectively.The content of Atractylenolide-Ⅰ in light condition oil was much higher than that in dark condition oil, which were 0.7572mg/g and 0.3541mg/g, respectively. There was no significant difference in contents of Atractylenolide-Ⅱ in the two kinds of oils, which were 2.026mg/g and 2.158mg/g, respectively. The content of Atractylenolide-Ⅲ in light condition oil was obviously higher than that in dark condition oil, which were 0.4124mg/g and 0.3219mg/g, respectively (P<0.01).The results suggested that changes in the constituents caused by the experimental process should be concerned.1.3 Study on the β-cyclodextrin inclusion of ZZP volatile oil and its stabilityGiven the unstable nature of the volatile oil of ZZP, and in order to ensure the stability of the experiment samples, the essential oil of ZZP was wrapped up with β-cyclodextrin. In order to verify the stability of the inclusion complexes of β-cyclodextrin with ZZP essential oils comparing with non-inclusion of ZZP essential oils, the time-contents of four main components of Atractylodes Rhizome at different storage time, atractylone, atractylenolide-I, atractylenolide-II and atractylenolide-III were determined by HPLC.The results showed that the content of atractylone, atractylenolide-I, atractylenolide-II, and atractylenolide-Ⅲ in β-cyclodextrin inclusion of ZZP volatile oil were stability within 4 weeks, and their amplitudes was little, the content changes at the 28d were-4%,+5%,+3%and+5%, respectively; but the four component contents of non-inclusion of ZZP essential oils varied greatly, at the fourth weeks, the percentage of content changes were-71%,+19%,-4%and 9%.1.4 Research on determination of the main effective components in the bran fried Atractylodes Rhizome HPLC method was adapted to analyze the content of atractylone and atractylenolide-I,-Ⅱ,-Ⅲ. HPLC condition:Desirable chromatographic separation of samples was obtained on a Zorbax SB-C18 column (4.6mm×250mm,5μm), by use of the mobile phase A (water) and mobile phase B (acetonitrile) in a gradient elution program:0～ 22min,56%～70%B; 20-30min,70%～90%B; 30～35min,90%～100%B. The wavelength was 220nm and 275nm. The flow rate was set at 1 mL/min. The column was maintained at 25 ℃. The methodological study results show that the method is stable and reliable for the determination of content of atractylone and atractylenolide-I,-Ⅱ,-Ⅲ.1.5 Research on the optimum ethanol extract condition of the main effective components in the bran fried Atractylodes RhizomeTo further extract the effective components of the bran fried Atractylodes Rhizome, the contents of atractylone, atractylenolide-I, atractylenolide-I, and atractylenolide-III were choosen as indicates, the concentration of ethanol, the volume of water-ethanol, and extracting times were choosen as factors, orthogonal tables L9(34) was used to confirm the optimum extract condition of the active principle. The conclusion showed that the optimum extract condition of the atractylone, atractylenolide-I, atractylenolide-I, and atractylenolide-III was 8 folds amount of 95% ethanol, circumfluence distilling was 2 times and 1.5 h for every time.1.6 Research on the extraction and separation of the ethanol ingredient, flavoids and alkaloids in two kinds of fried Fructus Citrus ImmaturusIn order to determine the chemical differences source of two kinds of Zhizhu pill efficacy, we adopt our previous determinied method of separating two kinds of fried Fructus aurantii Immaturus ethanol components, flavonoids and alkaloids components. The results showed that Citrus aurantium L. ethanol fractions, flavonoids and alkaloids components’isolated rate was 53.00%,18.16% and 11.48%, respectively; Citrus sinensis Osbeck ethanol, flavonoid and alkaloids components’isolated rate was 50.70%,15.47%, and 13.61%. Citrus aurantium L. flavonoids components mainly included four kinds of flavonoids, which are narirutin, naringin, hesperidin and neohesperidin, and Citrus sinensis Osbeck mainly have 2 kinds of flavonoids, which are narirutin and hesperidin. There was no significant difference of the contents of synephrine in the two kinds of alkaloids components.2 Comparative analysis of chemical constituents and contents of two kinds of ZZP2.1 Research on the chemical constituents and contents of two kinds of ZZP volatile oilsWe utilized GC-MS to determine the differences of the chemical constituents and contents in volatile oil of the two kinds of ZZP which were extracted in dark conditions. The result shows that 57 compounds and 54 compounds were determined in Citrus aurantium L.-ZZP volatile oil and Citrus sinensis Osbeck-ZZP volatile oil, respectively, which contain 91.00% and 92.05% in total numbers of peaks and contain 96.02% and 95.30% in total volatile oil. The two different ZZP volatile oils contain fifty-one same compounds and their contents are different, five compounds are unique for Citrus aurantium L.-ZZP volatile oil, and three are unique for Citrus sinensis Osbeck-ZZP volatile oil.5 compounds are common in volatile olis of Citrus sinensis Osbeck and Atractylodes Rhizome. We transform the content of these compounds to percentage of metal, which reflects the differences in the two kinds of ZZP volatile oils directly.2.2 Eight main compounds in two kinds of ZZP determined by double-wavelength-HPLC methodHPLC was utilized to determine 8 main compounds in two kinds of ZZP. HPLC condition is as following, mobole phase is consisted of 0.01% sodium dihydrogen phosphate water solution (A)-acetonitrile (B); 0～5 min,5%B; 5～7 min,5～10% B; 7-10 min,10～16%B; 10～25 min,16～18%B; 25～42 min,18～35%B; 42～55 min, 35～58%B; 55-65 min,58%B; 65～75 min,58～100%B; 75～85 min,100% B. Temperature of column:30℃. Flow rate:lml/min. Determined wavelength:283nm (0-52min, Narirutin, Naringin, Hesperidin, Neohesperidin, Atractylenolide); 220nm (52-65 min, Atractylenolide-Ⅱ、Atractylenolide-Ⅲ、Atractylon). Both of the 8 compounds were determined in Citrus aurantium L.-ZZP, while Naringin and Hesperidin were not determined in Citrus sinensis Osbeck-ZZP.3 Effects of two kinds of Zhizhu Pills and their different compositions on gastrointestinal motility in mice3.1 Effects on gastrointestinal motility of normal miceUsing the phenol red-determination method to investigate the effects of two kinds of Zhizhu Pills and flavonoids, alkaloids and volatile oil components of two kinds of Fructus Citrus Immaturus (essential oil and ethanol extract of Atractylodes Rhizome are added) on gastric emptying and intestinal propulsion of normal mice. The results showed that there were no significant differences of gastric emptying and intestinal propulsion rates of the animals of the blank control, positive control and the Citrus aurantium L.-Zhizhu Pill (CA-ZZP) groups, but the other groups showed a certain inhibition of gastrointestinal motility in normal mice.3.2 Effects on gastrointestinal motility of atropine-induced delay model miceUsing the phenol red-determination method to investigate the effects of two kinds of Zhizhu Pills and flavonoids, alkaloids and volatile oil components of two kinds of Fructus Citrus Immaturus (essential oil and ethanol extract of Atractylodes Rhizome are added) on gastric emptying and intestinal propulsion of model mice. The results showed that the two kinds of Zhizhu Pills and flavonoids, alkaloids and volatile oil components of two kinds of Fructus Citrus Immaturus all exhibit varying degrees of improvement in gastrointestinal motility of model mice. The CA-ZZP was significantly better than the Citrus sinensis Osbeck-Zhizhu Pill (CS-ZZP) in improving the gastrointestinal motility inhibition, and there was no significant difference in the positive control and CA-ZZP groups; Compared with alkaloids of Citrus aurantium L. (CA-A) and Citrus sinensis Osbeck (CS-A), non-significant difference was observed in promoting the intestinal propulsive, but the former was significantly better than the latter in promoting gastric emptying; Both of the flavonoids (CA-F)and volatile (CA-V) components of Citrus aurantium L. were better than the flavonoids (CS-F)and volatile (CS-V) components of Citrus sinensis Osbeck in promoting the gastric emptying and intestinal propulsion, moreover, the role of promoting the gastrointestinal motility were more significant by increasing the dose of volatile oils; The improving gastrointestinal motility in mice model roles of two kinds of ZZP were better than their flavonoids, alkaloids and volatile components.Finally said, there are obvious efficacy differences between the two kinds of flavonoids and volatile oils.4 Effects of two kinds of Zhizhu Pills and flavonoids, alkaloids of Citrus aurantium L. on Functional Dyspepsia (FD) model ratsThe phenol red assay and radioimmunoassay were prepared to investigate the effects of two kinds of ZZP, CA-F and CA-A on gastrointestinal motility disorders and gastrointestinal hormones of the complex etiology of functional dyspepsia model rats. The results showed that each treatment group could increase the rate of gastric emptying and intestinal propulsion of FD rats. The CA-ZZP was significantly better than the CS-ZZP, CA-F and CA-A in improving gastrointestinal motility disorders. Each treatment group could increase levels of serum GAS and plasma MTL of rat model, and decreased levels of plasma VIP of rat model, and there was no significant difference in levels of the three kinds of gastrointestinal hormones between CA-ZZP and the blank control groups; CA-ZZP were better than CS-ZZP in improving three kinds of gastrointestinal hormone levels; In the aspect of reducing levels of plasma VIP in FD rats, no significant difference was observed in both of CA-ZZP, CA-F, CA-A and the positive control groups. In aspect of increasing serum GAS, MTL plasma levels, Ck-ZZP was significantly better than CA-F and the positive control groups; No significant differences were exist in the levels of plasma MTL between CA-ZZP and CA-A groups, but for the levels of serum GAS in CA-ZZP-treated group was obviously higher than the CA-A-treated group.5 ConclusionsThis paper studies the relationship between the different components and the corresponding efficacy, preliminary results show that chemical composition of two kinds of ZZP is markedly different, in particular, there are significant differences in the flavonoid and volatile components. In addition, the efficacy results show that there are certain levels of difference in two kinds of ZZP and corresponding components.Compared to CA-ZZP and CA-F, lacking the neohesperidin and naringin for CS-ZZP and CS-F, which may lead to gastrointestinal weaker than the former ones. All the results revealed that differences of two Zhizhu Pill pharmacodynamic may be related to differences in flavonoid components. |
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