| BackgroundHerba Ephedrae-Radix Aconiti Lateralis and Herba Ephedrae-Semen Armeniacae Amarum are classical herb pairs derived from Herba Ephedrae-Decoction and its serial formulations. The representative formulations are Mahuang-Fuzi-Xixin Decoction and San’ao Decoction, which is comprised Ephedrae Herba-Armeniacae Semen Amarum-Radix Glycyrrhizae and Herba Ephedrae-Radix Aconiti Lateralis-Asarum heterotropoides, respectively. Herba Ephedrae-Radix Aconiti Lateralis can warm (or reinforce) the lung Qi and dissipate the exterior cold. Herba Ephedrae-Semen Armeniacae Amarum exhibits the ability of rebalancing the Lung Qi movement to suppress cough and to calm panting. As herb composition art recorded in ancient medical work, the combination mechanism underlying the prescription characteristic summarized as "treatment with both elimination and reinforcement" and "couple hardness with softness" was unclear and merits investigation. Herba Ephedrae (Mahuang in Chinese), Radix Aconiti Lateralis (Fuzi in Chinese) and Semen Armeniacae Amarum (Xingren in Chinese) are all slight toxic which could result in side effects. Mahuang extracts and its preparations have been banned and removed from market by Food and Drug Administration due to obvious adverse reactions. Xingren or Fuzi intoxication and even fatal poisoning were occasionally presented. Drug concentration in plasma and target organs (or cells) is depended on drug disposition in vivo, which can induce the pharmacology efficacy. Hence, proper knowledge on pharmacokinetics, distribution and excretion is not only vital to investigate the major target sites and explain the mechanism of efficacy improvement and detoxification in vivo, but also be useful for therapeutic drug monitoring and rational application of medicinal herbs.Aim of the studyCombined with the results from a previous study on herb consitituents and pharmacology, comparative pharmacokinetics, tissue distribution and excretion profiles of the bioactive compounds in single herb and herb pair were performed to explore the relationships of "dose-efficacy" and "composition-efficacy" between herbs combination and efficacy (toxicity), and then enriched and demonstrated the mechanism of mutual assistance and potential mutual restraint effect in Mahuang-Fuzi and Mahuang-Xingren herb pair. These results could provid insights into the mechanisms of efficacy improvement and detoxification. Therefore, following research contents were conducted:(1) Changes in profiles of pharmacokinetics, tissue distribution and excretion of Mahuang-Fuzi herb pair.(2) Variation of bioactive compounds in Mahuang-Xingren herb pair underlying different combination ratios(3) Comparative analysis of acute toxicity of Mahuang-Xingren and herbs oral administered individually by median-effect analysis.(4) Changes in profiles of pharmacokinetics, tissue distribution and excretion of Mahuang-Xingren herb pair.(5) Cytotoxicity test of amygdalin in context of different configuration ratios and the primary study on in vivo metabolism of amygdalin.Methods1 Development of an analytical method for determination of the representative bioactive compounds in Mahuang-Fuzi and Mahuang-Xingren herb pairs in context of different biological samples.Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method were developed for simultaneous determination of eleven alkaloids in Mahuang-Fuzi, including norephedrine (NE), norpseudoephedrine (NPE), ephedrine (E), pseudoephedrine (PE), methylephedrine (ME), aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BAC), benzoylmesaconine (BMA), benzoylhypaconine (BHA) and NE, NPE, E, PE, ME amygdalin and prunasin in Mahuang-Xingren in rat biological matrix (plasma, tissue, urine and feces) sample. Developed method were validated according to the guidance for bioanalytical method validation.2 Comparative study on in vivo disposition of the representative bioactive compounds in Mahuang-Fuzi herb pairBased on previous results of acute toxicity and pharmacodynamics experiments, Mahuang-Fuzi 1:1 (original ratio in Mahuang-Fuzi-Xixin Decoction) was selected for continuous study of herb disposition in vivo.According to an extraction method described in the Treatise on Cold Damage and Miscellaneous Diseases (Shanghan Lun in Chinese) and dose conversion of human dose to rat, Mahuang-Fuzi extract preparation method was developed as follows:Mahuang (120 g) was immersed in water (8000 mL) for 30 min and boiled for 30 min. Fuzi (220 g) was then added and boiling continued for 70 min. Water in the extracts was evaporated under reduced pressure and the extracts were concentrated to proper concentration and then obtained the experimental herb extracts. The identification, quantification, and chromatographic separation of the alkaloids were performed using our validated analytical method.2.1 Comparative pharmacokinetics studies of the representative alkaloids in rats after administration of Mauang-Fuzi extractSprague-Dawley (SD) rats were randomly divided into three groups, i.e., a Mahuang group, a Fuzi group, and a Mahuang-Fuzi group. Rodents in these groups were administered an oral dose of Mahuang, Fuzi and Mahuang-Fuzi 1:1 extracts. Blood samples were collected from the orbital plexus at designed time points after dosing to obtain rat plasma. The plasma concentrations of alkaloids were calculated using the validated UPLC-MS/MS method. Data from these samples were used to construct pharmacokinetic profiles by plotting the drug concentration over time and then calculate the pharmacokinetic parameters.2.2 Comparative tissue distribution studies of the representative alkaloids in rats after administration of Mauang-Fuzi extractSD rats were oral administrated of Mahuang, Fuzi and Mahuang-Fuzi 1:1 extracts and then sacrificed at designed endpoints to obtain heart, liver, spleen, lung, kidney and brain. The validated UPLC-MS/MS method was used to determine the content of these compounds in organs. Tissues distribution profile and pharmacokinetics parameters were characterized and calculated respectively.2.3 Comparative excretion studies of the representative alkaloids in rats after administration of Mauang-Fuzi extractSD rats were oral administrated of Mahuang, Fuzi and Mahuang-Fuzi 1:1 extracts and then collected urine and feces samples at designed time quantums. The validated UPLC-MS/MS method was used to determine the concentration of alkaloids in these samples. Cumulative excretion-time profile and excretion kinetics were characterized and calculated respectively.3 Study on acute toxicity and herb-herb interactions of Mahuang-xingren in terms of different combination ratiosAccording to an extraction method described in the Prescriptions from the Great Peace Imperial Grace Pharmacy (TaipingHuiminHejiJu-Fang in Chinese), Mahuang (9 g) and Xingren (9 g) were immersed in 220 mL water for 30 min and decocted t for 60 min. Herb dregs was decocted by 160 mL water again for 60 min. The solution was filtered and concentrated to obtain the Mahuang-Xingren 1:1 decoction. The obtained solution was concentrated and then freeze-dried to obtain the lyophilized powder form of the extract. Amount of crude drug was remained to be consistent underlying different combination ratios of herbs. The same procedure was followed for the preparation of the single herb, Mahuang-Xingren 4:1,2:1,1:2, and 1:4 extracts. Lyophilized powder samples were reconstituted with distilled deionized water and 5 dose levels were obtained by geometric dilution. Mice were randomly assigned to 8 groups, including Mahuang, Xingren, Mahuang-Xingren 4:1, 2:1,1:1,1:2,1:4 and control groups, rodents were administered a single dose of one of the extracts via oral gavage. General behaviors, moribund condition and mortalities were used for herb combination analysis. Combination index (CI) and dose-reduction index (DRI) were calculated using CalculSyn software based on the median-effect method.4 Study on the variation of bioactive compounds in extracts in context of different combination ratios of Mahuang and XingrenSimultaneous determination of NE, NPE, E, PE, ME, L-amygdlin and D-amygdalin by high performance liquid chromatography (HPLC) was developed and validated. Lyophilized powders of extracts were dissolved separately in deionized water. Suspension was precipitation with methanol and centrifuged to obtain 3 portions of resulting solutions for HPLC analysis. Decoction efficiency of ephedra-alkaloids and amygdalin of different combination ratios groups was analyzed according to the validated method above.5 Comparative study on in vivo disposition of the representative bioactive compounds in Mahuang-Xingren herb pair5.1 Comparative pharmacokinetics, tissue distribution and excretion studies of the representative bioactive compounds in rats after administration of Mauang-Xingren extractBased on previous results of acute toxicity and pharmacodynamics experiments, Mahuang-Xingren 1:1 (original ratio in San’ao Decoction) was selected for continuous study of herb disposition in vivo. SD rats were randomly divided into three groups, i.e., a Mahuang group, a Xingren group, and a Mahuang-Xingren group. Rodents in these groups were administered an oral dose of Mahuang, Xingren and Mahuang-Xingern 1:1 extracts. Blood samples (or tissue, urine and feces) were collected at designed time points after dosing. The concentrations of these compounds were calculated using the validated UPLC-MS/MS method. Data from these samples were used to characterize pharmacokinetic (or tissue distribution and cumulative excretion) profiles by plotting the drug concentration over time and then calculate the kinetics parameters.5.2 Cytotoxicity test of amygdalin in different configuration ratiosThe existing form (epimerization) of amygdalin varied in Mahuang-Xingren combination. Moreover, stereoselective metabolism of amygdalin with different configurations was found in vivo for the first time. Therefore, amygdalin cytotoxicity in terms of different chemical configurations is a proof and further investigation compared with drug disposition in vivo.D-amygdalin stock solutions were placed in tubes in a boiling water bath for different time to obtain 100%,81.9%,68.8% and 49.9% D-amygdalin respectively (ratios calculated by D-amygdalin/L-amygdalin+D-amygdalin). Effect of amygdalin on growth inhibition carried out in the presence of β-glucosidase was estimated by the CCK-8 assays.5.3 Primary study of metabolism of amygdalin in vivoSD Rats were oral administrated of Xingren aqueous extract. Plasma and urine samples were collected and detected by UPLC-MS/MS. Possible metabolism pathway were conducted based on the primary analysis of mass information.Results1 UPLC-MS/MS method were developed and validated for bioanalysis of the representative bioactive compounds of Mahuang-Fuzi and Mahuang-Xingren herb pair in rat plasma, tissue, urine and feces samples.The validated method demonstrated adequate sensitivity, selectivity, precision, accuracy, recovery, matrix effect and processed efficiency for the quantitative analysis of complex herbal components after rat oral administrated of Mahuang-Fuzi and Mahuang-Xingren aqueous extracts.2 In vivo disposition of the representative bioactive compounds in Mahuang-fuzi herb pair(1) Peak concentrations in plasma of most alkaloid, half-life or the mean residence time has been prolonged. Half-life of HA (aconitum diester alkaloids) had been delayed in all tissues. Meanwhile, bio-clearance rate decreased significantly.(2) All alkaloids had a higher concentration in lung. Quick absorption and distribution of these alkaloids were observed after combination. Meanwhile, peak concentration and bioavailability had been reduced (Cmax/dose and AUC0-5/dose decreased). Otherwise, bio-clearance of alkaloids (CLz increased except HA) was accelerated underlying combination application.(3) Urine and fecal excretion are the main excretion pathways of ephedra-alkaloids and aconitum-alkaloids respectively. There was no significant difference in kinetics parameter of excretionbetween combination and single application. Excretion half-life of HA and monoester-diterpenoid alkaloids (BAC, BMA, BHA) decreased. Cumulative excretion ratio of HA increased while monoester-diterpenoid decreased.3 Obvious antagonistic effects were observed when the acute toxicity of the Mahuang-Xingren combinations was testedThe results indicated that Xingren was more toxic than Mahuang and demonstrated that the safety profile of the Mahuang-Xingren combination had been improved significantly in comparison with that of Xingren alone (the Mahuang-Xingren treatments had LD50 concentrations greater than that of Xingren). Using median effect analysis, obvious antagonistic effects of acute toxicity (CI>1) were observed in all Mahuang-Xingren combinations. Moreover, the results of these experiments were consistent with the herb combination ratios:higher proportions of Mahuang were associated with stronger antagonistic effects. After combined treatment in mice, the DRI50 (DRI at LD50) of Xingren was decreased 37% to 669% (except Mahuang-Xingren 1:4) and the DRI50 of Mahuang was increased just 6% to 57% when compared with the two herbs taken individually. The results of the combination analysis suggested that improvement of the Mahuang-Xingren safety profile was mainly due to Xingren detoxification.4 The D-amygdalin composition of herbal extracts varies greatly in response to combination with MahuangThe extraction efficiencies (mg/g) of NE, NPE, E, PE, ME and amygdalin in combination (except Mahuang-Xingren 1:1) were not significantly different from those of the single herbs. However, the extraction efficiency of D-amygdalin in Mahuang-Xingren was increased significantly in comparison with that of Xingren alone. The epimeric forms of amygdalin varied in response to Mahuang-Xingren combination. The Pearson correlation coefficient indicated that the ratio of D-amygdalin/L-amygdalin and CI were very strongly related. In other words, the proportion of D-amygdalin was closely correlated with the level of antagonism. 5 In vivo disposition of the representative bioactive compounds in Mahuang-Xingren herb pairExtract efficiency of D-amygdalin was improved in Mahuang-Xingren combination, but the absorption, distribution and metabolism of amygdalin was also inhibited. Meanwhile, absorption and elimination of ephedra-alkaloids in vivo were accelerated by Xingren. The pharmacokinetics characteristics can be summarize as follows:(1) Peak concentration, biodistribution and residence time(MRTo-t and/or Ua) were accelerated in vivo; (2) Absorption and bioavalability (AUC0-5/dose and Cmax/dose) of ephedra-alkaloids in heart, liver, spleen and lung by Mahuang-Xingren. Besides, combined with Xingren decreased plasma concentration of NE and promoted its elimination. (3) Distribution and metabolism of the D-amygdalin was postponed. Not only concentration of D-prunasin but also absorption in tissues decreased significantly. (4) Cumulative urinary excretion ratio of prunasin was observed underlying Mahuang-Xingren.6 Sereoselective metabolism of amygdalin may be part of the mechanism underlying detoxification upon Mahuang-Xingren combinationD-prunasin was less abundant than L-prunasin in rat plasma, although there was more D-amygdalin than L-amygdalin in the extract. Interestingly, the process was facilitated by Mahuang, which led to virtual dose reduction of the D-prunasin dose in vivo and thus reduced toxicity. Stereoselective metabolism, in which the metabolic pathway is manipulated to some extent, may be partial mechanism for detoxification of Xingren in the Mahuang-Xingren combination. This result provides a preliminary explanation for the hypothesis regarding the "mutual restraint" relationship of Mahuang and Xingren in herb pair combination.7 Primary analysis on in vivo metabolism of amygdalinStereoselective metabolism of amygdalin-based study had been proved to be correlated with detoxification of Xingren in the Mahuang-Xingren herb pair. It offers a aspiration of thinking regarding research into the metabolism underlying detoxification effects. The result indicated that amygdalin was not only hydrolyzed to prunasin by deglucosidation, but also could be metabolized into amides or carboxylic acids through cyano-hydroxylation process. Metabolism of nitrile grouping plays an important role in toxicity of amygdalin. The hypothesis that amygdalin metabolism through amide pathway broaden our horizon upon detoxification of amygdalin through metabolism in vivo.Conclusion1 Combination mechanism of Mahuang-Fuzi herb-pair:mutual restraint besides mutual assistanceMutual inhibition of extract efficiency of bioactive alkaloids were observed in Mahuang-Fuzi combination, antd thus led lower peak concentration in plasma and tissues. Clearance of these alkaloids in tissues was also accelerated. As meanwhile, cumulative excretion ratios of aconitum alkaloids were reduced. Therefore, we hypothesized that detoxification may be achieved by "mutual restraint" on extraction efficiency and absorption in vivo, and synergistic effects of analgesic anti-inflammatory effect may be related to the fact that bioavalability improvement of Mahuang alkaloids and benzoylaconine, prolonged biological half-time of these alkaloids and the lower excretion ratios of monoester-diterpenoid alkaloids which is effective with slight toxicity. Moreover, highest distribution of these alkaloids were observed in lung tissue, the orientation of the medicinal action remains unclear with its meridian/channel on therapeutic action manifested and merits further investigation. The combination art of Mahuang-Fuzi herb pair can be summarize as mutual assistance accompanied with mutual restraint. However, there is still an overlapping dose range between acute toxicity and analgesic effects. Mahuang-Fuzi herb pair can delay mean residence time of most alkaloids. Moreover, hypoaconitine elimination in plasma and tissues was also delayed. These results indicated that the potential risk of Mahuang-Fuzi combination shoule be paid more attention.2 Mutual assistance accompanied with mutual detoxification is the combination mechanism of Mahuang-Xingren herb pairLower dosage of Mahuang and Xingren were achieved underlying its synergistic anti-asthma effects. Thus, for identical doses, Mahuang-Xingren facilitated lower intake and poor absorption of ephedra-alkaloids in heart, lung, spleen and kidney without decrease in bioavailability. Besides, plasma concentration of norephedrine were also decreased. In general, results above indicate that the combination of Mahuang and Xingren had higher elimination and decreases the risk of accumulation of their active constituent alkaloids in vivo and, thus, decreases their toxicity. Bioavalability of amygdlain and prunasin was improved comparatively. Although there was more D-amygdalin than L-amygdalin in the extract, stereoselective metabolism of amygdalin facilitated by Mahuang, as a result, absorption, tissue distribution of D-prunasin were disrupted. What’more, Mahuang-Xingren acts to enhance the elimination of prunasin through urine. To some extent, hydrocyanic acid generated from prunasin could be blocked in vivo and thus reduced toxicity. These results illustrate the rationale behind the combination of Mahuang and Xingren. Not only merely mutual assistance but also mutual detoxification were achieved in Mahuang-Xingren herb pair, which enriched scientific context of combination mechanisms on its effectiveness and safety. |
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