| Background and SignificanceHerbal couples, also called herbal pairs, which as the basic composition units of Chinese herbal formulas have special clinical significance in traditional Chinese medicine (TCM), and are much simpler than other complex formulas without altering their basic therapeutic features. Herbal couples is the bridge of the single Chinese herb and TCM formula, is the essence and core of the prescription compatibility, have the basic function of the attending of prescription, and also reflects the overall efficacy of the TCM formula.The clinical efficacy of Chinese herbs largely depends on the compatibility of medicine. The herbal couples, as the compatibility unit between single herb and Chinese herbal formulas, and have the inseparable connection of Chinese herbal formulas, which composed according to the principle of "monarch, minister, assistant and guide". The herbal couples have less herbal medicines, and chemical composition is relatively simple, which not only have the features of formula, but also easy to carry out the characteristics of the modern medical science research. So research on the herbal couples, as a starting point, may be beneficial to elucidate its mechanism of actions of formulas, seek the best the ratio of compatibility, and explore the basic effects, and function expansion. Carrying out research on the compatibility lawof herbal couples, is of great significance to reveal the scientific contents of formulas compatibility, mine the theory of TCM compatibility, and expand the clinical ideas of the treatment, as well as provide clues to the basis for in-depth study of herbal compatibility law, and also the basis for the formation of the new formulas. Especially with the increasing of compatibility using active ingredients or effective parts, applying the traditional compatibility theory to guide the new form of compatibility, is of practical significance to promote the new products of traditional Chinese medicine for research and innovation.In recent years, foreign agents use preparation which containing ephedra as food supplements of exercise and weight-loss, which lead to a series of adverse events, especially the side effects of the cardiovascular system, the United States has introduced legislation to disabled the preparation which containing ephedra. Epidemiological studies have proven this associated with long-term use of ephedra, and the side effects of ephedra and its preparations mainly associated with the ephedra alkaloids. Containing ephedra prescriptions of TCM have been used for2000years in clinical, the curative effects of that is distinct, and fewer side effects have reported in domestic. The dosage of foreign preparation which containing ephedra are far below the TCM prescriptions, but the side effects of the event are far less than the former. Investigate its reason, in addition to the differences in clinical use, the essence of compatibility of TCM cannot be ignored.This study is the part of basic research on the composition of the law of Herba Ephedrae herbal couples, funded by the National Science Foundation. Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb couples (also called Mahuang-Gancao and Mahuang-Guizhi herb-couple in Chinese) are commonly used in TCM clinical, which used in many classical prescriptions, such as Mahuang decoction and Daqinglong decoction. The combination of Mahuang and Gancao can moderated the drying chamber of Mahuang, and strengthen the sweating aproll, cough and asthma of Mahuang. While the combination of Mahuang and Guizhi can sweating nourish, and have good treatment on chill cold, bad cold fever, headache and body pain. In the actual clinical application, using the different ratio of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb couples for different syndromes, is a question worth considering concering the connotation of compatibility.ObjectivesTo improve effect and decrease toxicity is the main purpose of the compatibility of TCM, study on prescription-formulating principle of herb-couples from enhancing pharmacological efficacy and reduce side effects are two feasible platforms. We think no matter synergy or attenuated is the result of interaction between components, the comprehensive effect which is the effect of a dynamic process in vivo of the composition influence each other. The object of this topic is Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb couples, using the modern technology, have a more comprehensive and systematic study on the blood drug concentration time curve, pharmacokinetic parameters, tissue distribution and excretion of these two herbal couples in different proportion and compatibility of their bioactive compounds. By explore the changes in the pharmacokinetic parameters, tissue distribution and excretion, it will not only further to validate the compatibility-relationship between mahuang and gancao (also mahuang and guizhi) and also play a role in finding the best compatibility proportion. Furthermore, by finding out the relationship of dose-efficiency, time-efficiency and toxic-efficiency, this will to bring into further validation and depth of the compatibility relationship of "mutual-assistance","mutual promotion", to reveal the intracorporal process of material basis and action mechanism of improve effect and decrease toxicity of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb couples. That can provide the experimental basis for the clinical treatment of TCM, development of new drug-couples and drugs.ContentsTo clarify the compatibility rules of Traditional Chinese herb-couple Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi with different compatibility ratio, we compared the changes of active ingredients in plasma pharmacokinetics, tissue distribution and excretion. Mainly containing:1. Study on the plasma pharmacokinetics of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herbal couples.Develop and validate the ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to determination the content of norpseudoephedrine (NMP), norephedrine (NME), ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) in rat plasma, which are the major active ingredients of Mahuang; the content of glycyrrhizic acid (GLY), glycyrrhetinic acid (GA) and liquiritin (LQ) in rat plasma, which are the major active ingredients of Gancao; the content of coumarin (CN), cinnamic alcohol (CAL) and cinnamic acid (CA) in rat plasma, which are the major active ingredients of Guizhi. Using the developed method to determined the major active ingredients content of Mahuang (also Gancao and Guizhi) in rat plasma after orally administered Mahung, Mahuang-Gancao herb-couple (ratios12:3,12:6,12:12,w/w) and Mahuang, Mahuang-Guizhi herb-couple (atios3:1, 3:2,3:4,w/w) extracts (also after orally administered the Gancao (3), Gancao (6), Gancao (12) and Guizhi (2) extracts). Compare the changes of blood drug concentration-time curve and the main pharmacokinetic parameters of the main bioactive ingredients of Mahuang (also Gancao and Guizhi). Discuss the influence of compatibility for the plasma pharmacokinetic behavior of Mahuang, Gancao and Guizhi, analysis the drug interaction of Mahuang and Gancao (also Mahuang and Guizhi).2. Study on the tissue distribution of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herbal couplesDevelop and validate the ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to determinate the content of norpseudoephedrine (NMP), norephedrine (NME), ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) in rat tissues, which are the major active ingredients of Mahuang. Using the developed method to determined the major active ingredients content of mahuang in rat tissues after orally administered Mahung, Mahuang-Gancao herb-couple (ratio12:6,w/w) and Mahuang, Mahuang-Guizhi herb-couple (atio3:2,w/w) extracts. Compare the changes of tissue distribution, and discussed the accumulation of the ephedrine alkaloids in each tissue.3. Study on the excretion from urine and feces of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herbal couplesDevelop and validate the ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to determinate the content of norpseudoephedrine (NMP), norephedrine (NME), ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) in rat urine and feces, which are the major active ingredients of Mhuang. Using the developed method to determined the major active ingredients content of mahuang in rat urine and feces samples after orally administered Mahuang, Mahuang-Gancao herb-couple (ratio12:6, w/w) and Mahuang, Mahuang-Guizhi herb-couple (ratio3:2, w/w) extracts. Compared and analysis the changes of its excretion.4. Statistial analysisThe pharmacokinetic parameters of the major active compound of mahuang (also gancao and guizhi) were calculated by the non-compartmental analysis of plasma concentration vs. time data using Drug and Statistics (DAS)3.0software (BioGuider Co., Shanghai, China). The differences of samples in different groups were conducted with univariate statistical analysis using software of SPSS16.0. The measurement data was presented as mean±SD (x±s); multiple groups means were compared with One-way ANOVA, among them, using the Dunnett method to compared the three compatibility groups and Mahuang group; two groups means were compared with indepent-samples T tese; the analysis of tissue distribution using the variance of repeated measurement data. A value of p<0.05was considered statistically significant.Results1. Study on pharmacokinetics of the major component in rat plasma after oral Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herbal couples in different proportionWe have established UPLC-MS/MS method to determine the norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, methyleephedrine; glycyrrhizic acid, glycyrrhetinic acid, liquiritin; and cinnamic acid, cinnamic alcohol, coumarin in rat plasma, which pass the methodological study with high accuracy, good reproducibility and stability, extraction recovery and matrix effects in biological samples meet the testing requirements. Under these UPLC method, separation of each analyte is good, there have no endogenous interfering substances and possible metabolites, so the method is rapid, sensitive and accurate.The pharmacokinetic parameters of ephedra alkaloids and the active ingredients of Gancao were different after oral administration of Mahuang, Gancao and Mahuang-Gancao herb-couple. The AUC0-t, MRT0-t, Tmax, t1/2z, CLz/F of NME; the AUCo-t, MRTo-t, Tmax, ti/2z, Vz/F, CLz/F of NMP; the AUC0-t, MRT0-t, CLz/F of E; the AUCo-t, MRTo-t, ti/2z, Vz/F, CLz/F of PE; and the AUC0-t, CLz/F of ME in Mahung group, Mahuang-Gancao (12:3) group, Mahuang-Gancao (12:6) group and Mahuang-Gancao (12:12) group have significant difference(p<0.05). Compared with the Mahuang group, Mahuang-Gancao (12:3) group reduced the MRT0-t of NME and NMP, reduced the t1/2z and Vz/F of PE (p<0.05); Mahuang-Gancao (12:6) group increased the AUC0-t of NME, E and PE(all p<0.05), reduced the CLz/F of E and PE(all p<0.05), reduced the Vz/F of PE(p<0.05); Mahuang-Gancao (12:12) group also increased the MRT0-t of E and PE, the CLz/F of ME(all p<0.05).Compared with Gancao (3) group, Mahuang-Gancao (12:3) reduced the AUC0-t and Cmax of LQ, but increased its Vz/F and CLz/F value (all p<0.05); It can reduced the AUC0-t, MRT0-t and Tmax of GLY, but increased its Vz/F value (all p<0.05); reduced the AUC0-t and Cmax of GA, increased its MRT0-t, Tmax, tmz, Vz/F and CLz/F value (all p<0.05).Compared with Gancao (6) group, Mahuang-Gancao(12:6) group reduced the AUC0-t and Cmax of LQ, but increased its MRT0-t, t1/2z, Vz/F and CLz/F value (all p<0.05); reduced the AUC0-t, Tmax and Cmax of GLY, increased its MRT0-t, tmz, Vz/F and CLz/F value(all p<0.05); reduced the AUC0-t and Cmax of GA, but increased its MRT0-t, Tmax, t1/2z and Vz/F value(all p<0.05).Compared with Gancao (12) group, Mahuang-Gancao(12:12) group reduced the AUC0-t and Cmax of LQ, but increased its MRT0-t, Vz/F and CLz/F value(all p<0.05); increased the AUC0-t and Cmax of GLY, and reduced its CLz/F value(all p<0.05); reduced the CLz/F of GA, but increased its MRT0.t, Tmax and t1/2z value(all p<0.05).The phanrmacokinetic parameters of ephedra alkaloids and the active ingredient of Guizhi (CA, CN and CAL) were different after oral administration of Mahuang, Guizhi and Mahuang-Guizhi herb-couple. The MRT0-t, Cmax, t1/2z, Vz/F of NME, the AUC0-t, MRT0-t, Cmax, t1/2z Vz/F, CLz/F of NMP, the AUC0-t, MRT0-t, Tmax, Cmax, t1/2z, Vz/F, CLz/F of E, the MRT0-t, Tmax, Cmax, t1/2z, Vz/F of PE, and the MRT0-t, Cmax, t1/2Z, Vz/F of ME in Mahung group, Mahuang-Guizhi (3:1) group, Mahuang-Guizhi (3:2) group and Mahuang-Guizhi (3:4) group have significant difference(p<0.05). Compared with the Mahuang group, Mahuang-Guizhi (3:1) group reduced the t1/2z, MRT0-t and Vz/F of NME, E, PE and ME(all p<0.05), increased the Tmax of E (p=0.001); Mahuang-Guizhi (3:2) group reduced the MRT0-t of NME, E, PE and ME(all p<0.05), the t1/2z and Vz/F of NME, E and ME(all p<0.05), the CLz/F of NMP (p<0.05), but increased the Cmax of the five ephedra alkaloids, the AUC0-t of NMP(p<0.05); Mahuang-Guizhi(3:4) group also reduced the t1/2Z, MRT0-t and Vz/F of the five ephedra alkaloids(all p<0.05), the CLz/F of NMP(p<0.05); but increased the Cmax of five ephedra alkaloids(all p<0.05), the AUC0-t of NMP, the Tmax of E and PE (all p<0.05).Compared with the Guizhi (2) group, Mahuang-Guizhi(3:2) group increased the AUC0-t and MRT0-t of CN, but reduced its t1/2z, Vz/F and CLz/F value(all p<0.05); increased the AUC0-t and MRT0-t of CAL, but reduced its Vz/F and CLz/F value(all p<0.05); also increased the AUC0-t and MRT0-t of CA, reduced the t1/2Z, Vz/F and CLz/F of CA(all p<0.05).2. Study on tissue distribution of the major component of mahuang in rat after oral Ephedra sinica-Glycyrrhiza uralensis and Ephedra sinica-Ramulus Cinnamomi herbal-couple decoction.Compared with the Mahuang decoction, after compatibility of Mahuang and Gancao(12:6), the concentration of NME in heart, brain, liver, spleen, lung and kidney are changed along with time(The p of group interaction with time0.05), at0.25h after oral administration, the concentration of NME in six tissue were all increased (p<0.05), at1.5h the concentration of NME in heart is still higher than Mahuang group, at3h the concentration of NME in brain、heart、liver、lung and kidney are reduced(p<0.05); after compatibility of Mahuang and Gancao, the concentration of NMP in brain and heart are changed along with time, at0.25h after oral administration the concentration of NMP are reduced, at1.5and3h the concentration of NMP in brain and heart are reduced (all p<0.05); after compatibility of Mahuang and Gancao, the concentration of E in heart, brain, liver, spleen, lung and kidney are changed along with time(from increase to reduce), at0.25and1.5h after oral administration, the concentration of E in tissues are increased(all p<0.05), and at3h, the concentration of E in brain, heart and spleen are reduced(all p<0.05); after compatibility of Mahuang and Gancao, the concentration of PE in heart, brain, spleen and kidney are changed along with time, at0.25,1.5and3h, the concentration of PE in heart, lung, liver, spleen and kidney are increased(all p<0.05), the concentration of PE in brain changes from increased (0.25and1.5h, p<0.05) to reduced (3h, p<0.05); after compatibility of Mahuang and Gancao, the concentration of ME in heart, lung, liver and kidney are changed along with time, at0.25and1.5h after oral administration, the concentration of ME in heart are reduced(p<0.05), the concentration of ME in brain, spleen and kidney are reduced at3h, the concentration of ME in lung are increased at0.25and1.5h, and reduce at3h(all p<0.05).Compared with the Mahuang decoction, after compatibility of Mahuang and Guizhi(3:2), the concentration of NME in heart, lung, liver, spleen and kidney are changed along with time(The p of group interaction with time<0.05), the concentration of NME in brain and liver are reduced at0.25and1.5h after oral administration,the concentration of NME in brain are increased at3h, the concentration of NME in heart are reduced at3h, the concentration of NME in spleen are reduced at0.25h, the concentration of NME in kidney are increased at1.5and3h(all p<0.05); after compatibility of Mahuang and Guizhi, the concentration of NMP in brain, heart, lung, liver, spleen and kidney are changed along with time, the concentration of NMP in lung are increased at0.25and3h, the concentration of NMP in brain are also increased at1.5and3h, the concentration of NMP in spleen are increased at three time, the concentration of NMP in heart, liver and kidney are increased at1.5h(all p<0.05); after compatibility of Mahuang and Guizhi, the concentration of E in brain, lung, spleen and kidney are changed along with time, the concentration of E in heart are reduced at0.25and3h, the concentration of E are changed from reduced(1.5h) to increase(3h), the concentration of E are from reduced(0.25h) to increase(1.5h), and reduced at3h, the concentration of E in spleen(0.25h) and kidney(3h) are increased; after compatibility of Mahuang and Guizhi, the concentration of PE in tissues are similar with E; after compatibility of Mahuang and Guizhi, the concentration of ME in heart, lung and kidney are changed along with time, the concentration of ME in brain are increased at3h, and the concentration of ME in heart are changed from increased (1.5h) to reduced (3h), the concentration of ME in lung and kidney(0.25h) are incresed(all p<0.05).3. Study on the urine and feces excretion of the bioactive ingredients of mahuang in rat after oral Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb-couple decoction.Compared with the Mahuang decoction, Mahuang-Gancao herb-couple can reduced the cumulative feces excretion of E (t=3.430, p=0.009), reduced the cumulative urinary excretion of ME (t=3.955, p=0.004). Five ephedra alkaloids in feces excretion were more moderate than urine. In Mahuang decoction, the urine and fecal excretion of NME, NMP, E, PE and ME accounted for a total dose was12.69%,13.56%,3.48%,3.65%and3.26%, respectively; while it was12.28%,14.52%,3.29%,3.21%and2.27%of NME, NMP, E, PE and ME in Mahuang-Gancao(12:6) group.Compared with the Mahuang decoction, Mahuang-Guizhi herb-couple increased the cumulative urinary excretion of NME (t=2.462, p=0.039), NMP (t=3.187, p=0.013) and E (t=2.703, p=0.027), reduced the cumulative feces excretion of E (t=2.905, p=0.020); In Mahuang decoction, the urine and fecal excretion of NME, NMP, E, PE and ME accounted for a total dose was11.34%,14.76%,6.35%,4.54%and8.08%, respectively; while it was15.01%,18.07%,5.89%,4.35%and7.35%of NME, NMP, E, PE and ME in Mahuang-Guizhi(3:2) group.Conclusion1. The results of the plasma pharmacokinetic showed that:After oral administration of Mahuang, Gancao and Mahuang-Gancao herb-couple decoction in rat, the pharmacokinetic parameters of ephedra alkaloids and the active ingredients of Gancao were different. Mahuang-Gancao (12:6) group can improve the degree of bioavailability of ephedra alkaloids, reaching synergistic effect. Therefore, Gancao can strengthen the pharmacological effects of Mahuang. The ratio of this is consistent with "GanMahuang-Tang", and this is also verified the science thought of the compatibility of "mutual-assistance" of Mahuang and Gancao. Also compared the the active ingredient of Gancao between Gancao and Mahuang-Gancao herb-couple decoction, with the increasing amount of Gancao, the bioavailability and existence time in vivo of glycyrrhizic acid are changed from inhibiting to promoting; the bioavailability of liquiritin and glycyrrhetinic acid in all Mahuang-Gancao groups are also reduced, compared with all Gancao group, the average residence time of them are extended.Study on the plasma pharmacokinetic of Mahuang, Guizhi and Mahuang-Guizhi herb-couple in rat, the pharmacokinetic parameters of ephedra alkaloids and the active ingredients of Guizhi are different. After the combined use of them, the bioavailability of the each ingredient has been improved, and the accumulation of ephedra alkaloids in rat was reduced. When Mahuang combination with Guizhi, the pharmacological effects of them all enhanced, and there are have synergism between them; Guizhi also can reduced the accumulation of ephedra alkaloids in vivo, can reduced the side effects. The results of this confirmed the scientific connotation of "mutual promotion" of Mahuang-Guizhi.2. Studies on the tissue distribution of ephedra alkaloids before and after drug compatibility. Compared with Mahuang group, Mahuang-Gancao (12:6) group can promoted the absorption and distribution of NME, E and PE in heart, brain, lung, liver, spleen and kidney, make them play a role of pharmacological quickly. Accelerated the elimination of NME, E, PE and ME from brain, reduced their accumulation in brain. Inhibited the absorption and distribution of NMP in the heart and brain, accelerated its elimination from the heart and brain, and reach the role of attenuated. After combined the Mauang with Gancao, it can accelerated the elimination of NME from heart, liver, spleen and kidney, accelerated the elimination of E from heart and spleen, and accelerated the elimination of ME from lung, liver, spleen and kidney, reduced the accumulation of them in the tissues, also have the effect of attenuated.Compared with Mahuang group, Mahuang-Guizhi group can inhibited the absorption of E and PE in heart, lung and spleen, inhibited the absorption of NME and NMP in spleen, and promoted the absorption of NMP and ME in lung; increased the distribution of E and PE in lung, increased the distribution of NME in lung and kidney, increased the distribution of NMP in brain, heart, liver, spleen and kidney, and reduced the distribution of NME, E and PE in brain; delayed the elimination of five ephedra alkaloids from brain, at a certain extent, have an effect of increasing pharmacological; Mahuang-Guizhi group can accelerated the elimination of E and PE from heart and lung, also accelerated the elimination of NME and ME from heart, reduced the accumulation of them in heart.3. The excretion study of ephedra alkaloids before and after two drugs matched in the urine and feces. After rat oral administration of Mahuang, Mahuang-Gancao (12:6) and Mahuang-Guizhi (3:2) decoction, the proportion of cumulative excretion in urine and feces of ephedra alkaloids from the original drug was very low. It showed ephedra alkaloids might exist some biotransformation in the body. Whether Mahuang and Gancao matched, or Mahuang and Guizhi matched, it had litter significant impact on cumulative urinary and feces excretion rate of ephedra alkaloids. |
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