| BackgroundMa Huang Tang is a traditional Chinese herbal preparation composed of two herbs, Herb Ephedra (Ma Huang in Chinese) and Ramulus Cinnamomi (Gui Zhi in Chinese), which mutually potentiate their activities. These herbs, when combined at a crude weight ratio of 3:2, are used to treat asthma, nose and lung congestion, and fever with anhidrosis. These effects have also been confirmed experimentally.Ma Huang is derived from Ephedra sinica Stapf, Ephedra intermedia Schrenk et C. M. Mey., and Ephedra equisetina Bge., and contains 0.5-2.5% (by weight) total alkaloids, of which ephedrine accounts for between 30% and 90%. Ma Huang has been used to treat various respiratory conditions in China for many years, with only three related poisoning cases due to overdose reported in the literature. Further, the Chinese Pharmacopoeia reports that Ma Huang is non-toxic suggesting that it is safe for use when applied correctly. However, several cases of Ma Huang poisoning have been reported, due to overdose and/or long-term consumption of the herb, or by using preparations containing ephedrine without the supervision of qualified healthcare professionals.Ephedrine is an amphetamine with a pharmacological profile similar to d-amphetamine and methamphetamine. It functions as a central nervous system (CNS) stimulant; thus, ephedrine can induce insomnia, nervousness, tremors, and anxiety. Long-term therapy with high doses of ephedrine may cause psychotic episodes such as paranoia, hallucinations, and other mental disturbances. Death has been reported following chronic use of Ma Huang extract. In our previous study, we found that chronic administration of Ma Huang induced obvious neurodegeneration in rat brains, with the prefrontal cortex showing the greatest effect.Gui Zhi is derived from the young twigs of Cinnamomum cassia Presl. Its main component and major active ingredient is cinnamaldehyde, which is known for its sedative, antioxidant, and anti-neuroinflammation activity. Several other active components in Gui Zhi extract also have been reported to have the neuroprotective effect. As the compounds in Gui Zhi extract shows a sedative and anti-inflammatory effect, we hypothesized that Gui Zhi may exhibit a similar neuroprotective effect against Ma Huang-induced hyperactivity and CNS oxidative damage in the rat prefrontal cortex when used together.Mu1ti-herb therapy is an essential component of traditional Chinese medicine. Herb pairs, a unique combination of two herbs in the clinic, is the most fundamental and the simplest form of mu1ti-herb therapy used to achieve a specific efficacy. Many herb pairs were recorded in the Treatise on Febrile and Miscellaneous Diseases and in the Synopsis of the Golden Chamber. There are several methods to determine herb compatibility, including singular application, mutual promotion, mutual assistance, mutual restraint, mutual detoxification, mutual inhibition, and mutual intoxication, which are called the "seven relations of Chinese Medicine." However, herb pairs used for mutual potentiation are the most common according to Chinese records and classic Chinese books about herbs. Mutual potentiation, also called as mutual promotion, significantly improved pharmacological efficacy compared to individual herbs. The Ma Huang-Gui Zhi herb pair is a classic herb pair used for mutual potentiation, and most previous studies have focused on mutual promotion. Due to the adverse effects of Ma Huang, its medicinal use has been restricted. Further, it can only be prescribed by registered traditional Chinese medicine practitioners. However, the potential mutual detoxification effect of the herb pair has not been thoroughly considered, and may be the reason why Ma Huang is a relatively safe medicinal herb in clinical practice.Objectives"Seven relations of Chinese Medicine" is the basic theory of the compatibility of TCM. Most of them means there are potential synergy effect or restrictive effect. The herb compatibility is divided into mutual promotion, mutual assistance, mutual restraint and mutual detoxification, while mutual inhibition and mutual intoxication should avoid. Herb compatibility is affect by flavor and meridian tropism and dose and ratio are the key influence factors. Under the guidance of TCM theory, our group takes ephedra as the research object, dose a lot of experimental research work of ephedra, herb pairs and compounds of ephedra for a long time. Some laws of the relevant toxicology, pharmacology, pharmacokinetics and pharmacokinetic have been studyed. Ephedra is a central nervous system (CNS) stimulant. The role of ephedra on the central nervous system is associated with amino acid neurotransmitter release. Ephedrine is the main ingredient of ephedra and it could raised the rease of excitatory amino acid neurotransmitter while reduce the proportion of inhibitory neurotransmitter release. Gui Zhi could adersely affect excitement caused by Ma Huang when treated with Ma Huang-Gui Zhi herb pair, and adjust the release of amino acid neurotransmitter. The aim of present study is to reveal the advantage of the two herbs used in combination. It may be the reason why Ma Huang is relative save for use in traditional Chinese medicine.MethodsThe effective dose of Ma Huang on SD rats is 6 g/kg, and the sensitive dose is 15g/kg in the previous study. Therefor, the present study take 7.5、15、30g/kg as the low and high groups of Ma Huang. The Ma Huang-Gui Zhi herb pair was prepared with crude weight ratios of 3:1,3:2, and 3:4, and the low and high doses of Gui Zhi are according to the ratios.1 Ephedrine in the crude extracts of Ma Huang and Ma Huang-Gui Zhi and dose formulationDevelop and validate the high-performance liquid chromatography method to determin the content of ephedrine. Ephedrine in the crude extracts of Ma Huang and Ma Huang-Gui Zhi herpair (3:1,3:2,3:4) were determined. The Ma Huang and Ma Huang-Gui Zhi preparations were adjusted based on their ephedrine content, such that the levels of Ma Huang in each extract were normalized by the ephedrine content.2 Behavioral assessment of Ma Huang and Ma Huang-Gui Zhi herb pairsThe animals were tested for 5min before and after administration a single oral gavage dose of the stock solutions at 1、2、4、6、8h. The alterations in general behavior and autonomic activities of the rat were recorded.The changes in hypnotic action indices (sleep latency, sleep-lasting time) for the rat placed on pentobarbital at dose 40mg/kg in advance were observed. After 20 min, observe the reflex of touch about eyelashes and stimulate leg muscles of experimental animal with a needle.On the first day, the animals were conducted to the EPM test that consists of a plus-shaped wooden maze with two opposite open arms (50×10 cm) and two enclosed arms (50x10x40 cm), spreading out from a central platform (10x10 cm), elevated to a height of 50 cm from the floor. According tothe animals were individually placed in the center of the EPM, facing one of the enclosed arms, and were allowed to explore the apparatus for 5 min. The following factors were measured:(a) frequency of open arms entries (OAE); (b) frequency of enclosed arms entries (EAE); (c) open arms time (OAT); and (d) enclosed arms time (EAT). The % OAE was calculated according to the formula:open/tota1×100. An entry was counted whenever the animal placed four paws on each arm.On each of the 7 days, animals were transported to the testing room and placed into the acrylic cage. After 20 min, two animals were tested simultaneously and administered a single oral gavage dose of the stock solutions. One hour after administration, the session was started by placing the rats in the central area of the open field. Each session lasted 5 min. The movements of the rats were tracked with two digital cameras, and the movies were analyzed by the computer software Smart (RWD Life Science, China). The number of total and central squares crossed, and the frequencies of rearing were measured on the first day [1]. The area surrounding the open field was defined as zone 1, while the sub-central area and central area were defined as zone 2 and zone 3, respectively. The placement of only one, two, or three paws in a square followed by a return to the previous square was not considered as a crossing.3 Biochemical assessment and HE stain of prefrontal cortexBiochemical tests and HE stain of prefrontal cortex were conducted after performing the last behavioral task. The animals were sacrificed by decapitation. The brains were removed and rinsed with ice cold isotonic saline. The prefrontal cortex was separated from the brain on a stainless steel plate on ice, and the samples were homogenized with ice-cold 0.1 mmol/L phosphate buffer (pH 7.4). The homogenates (10% w/v) were centrifuged at 10,000× g for 15 min, and the supernatants were used for the biochemical analysis. The total activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and the malonic dialdehyde (MDA) and nitric oxide (NO) content in the supernatants were measured using commercial kits on a microplate reader.4 Ephedrine content and amino acid neurotransmitter release in the prefrontal cortexDevelop and validate the ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to determin the content of ephedrine and release of amino acid neurotransmitter in the prefrontal cortex. On each of the 7 days, the content of ephedrine and release of amino acid neurotransmitter in the prefrontal cortex were determined.5 Study of PK-PD modelingDevelop and validate the ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/NS) method to determin the content of ephedrine in hemodialysis solutions and brain dialysate and amino acid neurotransmitter in brain dialysat. In the microdialysis, take the change of ephedrine concentration in hemodialysis solutions and brain dialysate as pharmacokineties (PK) index, while the change of amino acid neurotransmitter concentration in brain dialysate as pharmacodynamics (PD) index. 6 Statistial analysisThe differences of samples in different groups were conducted with univariate statistical analysis using software of SPSS 13.0. The measurement data was presented as mean ± SD. Multiple groups differences were compared with One-way ANOVA. The analysis of repeated measures data used the variance of repeated measures data. The reciprocal action between Ma Huang and Gui Zhi used analysis of variance of factorial design. A value of P<0.05 was considered statistically significant. Study of PK-PD modeling using software of Winnonlin4.0.1.Results1 Ephedrine in the crude extracts of Ma Huang and Ma Huang-Gui Zhi and dose formulationThe HPLC instrument used was an Agilent HPLC Model 1100 (Palo Alto, CA, USA) equipped with an Agilent 1100 vacuum degasser, quaternary pump, autosampler, column compartment, and diode-array detector. Separation was performed on an Alltima Phenyl Column (250 × 4.6 mm i.d.,5-μm particle size). The HPLC column was equilibrated with a mobile phase containing acetonitrile-water-potassium dihydrogen phosphate-triethylamine (40 ml:960 ml: 2.72 g:2.8 ml), at pH 3.43. The wavelength of the UV detector was set at 210 nm. After filtering with 0.45 μm nylon filters,10 μl of each extract was injected into the HPLC system. The column temperature was held at 25℃ and the isocratic flow rate was maintained at 0.6 ml/min.The content of ephedrine in Ma Huang was the highest among the four preparations. Increasing the proportion of Gui Zhi in the Ma Huang-Gui Zhi herb pair significantly decreased the ephedrine content. These results indicated that the dissolution of ephedrine was affected when Ma Hung and Gui Zhi were prepared together.2 Behavioral assessment of Ma Huang and Ma Huang-Gui Zhi herb pairsIn the behavioral tests, following the administration of Ma huang at various dose levels (3.000、1.500、0.750g·mL-1) and schedules, the alterations in general behavior and autonomic activities of the rat were recorded, the changes in hypnotic action indices (sleep latency, sleep-lasting time) for the rat placed on pentobarbital in advance were also observed. Ma huang administrated ig significantly increased the autonomic activities and inhibited the hypnotic action induced by previously given pentobarbital. The different doses of Ma huang decocotions decreased the time spent in the open arm of the elevated plus-maze. Similar findings were observed also in the central area of open field, however this result was influenced by inhibition of motor activity of the rats in 3.000 g·mL-1 group. Also, Ma huang decocotions could inhibited the hypnotic action induced by pentobarbital sodium and these results were statistically significant.Gui Zhi decocotions were found to exert anxiolytic activity in elevated plus maze with no affection of locomotor activity. The different doses of Gui Zhi decocotions increased the time spent in the open arm of the elevated plus-maze. Similar findings were observed also in the central area of open field. With the increase of the dose, the number and time of entries into the open arm of elevated plus maze significantly increased. Number of entries into the closed arms decreased significantly. Compared with the control group,12.33,30.09 g/kg dose group was statistically significant difference (. The residence time and the proportion in the central area of the open field of 12.33,30.09g/kg dose group was significantly increased. The number of rats crossing and grooming increased significantly. Compared with the control group, the difference was statistically significant. The total distance traveled in Gui Zhi 5.27,12.33,30.09g/kg dose group were significantly lower Compared with the control group. Also, Gui Zhi decocotions could increase the hypnotic action induced by pentobarbital sodium, although these results were not statistically significant.All of the rats administered Ma Huang or the Ma Huang-Gui Zhi herb pair exhibited higher total locomotor activity than the saline treated rats. However, the hyperactivity induced by Ma Huang was inhibited by Gui Zhi in a dose-dependent manner. Examination of anxiety parameters showed that Ma Huang decreased central locomotion. However, HP3:2 and HP3:4 increased the time spent in the central area compared to rats treated with Ma Huag. Vertical exploration was reduced in Ma Huag-, HP3:1-, and HP3:2-treated rats, compared to the saline group. All rats treated with W1, W2, and W3 exhibited an increased rearing frequency compared to E-treated rats.3 Biochemical assessment and HE stain of prefrontal cortexOxidative damage in prefrontal cortex of rats as indicated by a rise in MDA and NO concentration and depleted reduced SOD, CAT and GSH-Px activity compared to control group were observed in Ma Huang and Ma Huang-Gui Zhi herb pair. Concentrations of MDA and NO in Ma Huang extract normalized by its ephedrine contents was significantly higher than pure ephedrine, indicating the presence of toxins other than ephedrine in the extracts.Chronic administration of Ma Huang significantly increased the levels of MDA and NO in the prefrontal cortex compared to the saline group. HP3:1, HP3:2, and HP3:4 treatment reduced MDA and NO levels as compared to Ma Huang-treated rats. This finding suggested that Gui Zhi decreased MDA and NO production induced by Ma Huang, therefore indicating that Gui Zhi reduces lipid peroxidation caused by Ma Huang.The total SOD, CAT, and GSH-Px activity in the prefrontal cortex of E-treated rats was significantly lower than that of the saline group. However, Gui Zhi counteracted the effects of Ma Huang on SOD, CAT, and GSH-Px in the HP3:1, HP3:2, and HP3:4-treated groups. In rats treated with HP3:4, SOD and CAT activities remained at normal levels.4 Ephedrine content and amino acid neurotransmitter release in the prefrontal cortexThe ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to determin the content of ephedrine in and amino acid neurotransmitter in prefrontal cortex have been developed and validated. The absorption and distribution of ephedrine in prefrontal cortex of rats were different among the groups. The absorption and distribution were significantly accelerated with increase of the proportion of Guizhi compatibility. The metabolic process of ephedrine did not change with the number of days change. Ephedrine increased the contents of excitatory amino acids and inhibitory amino acids. Glu and GABA were more sensitive compared to Gly and Asp. Guizhi inhibited ephedrine-induced excitotoxicity.5 Study of PK-PD modelingThe ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/NS) method to determin the content of ephedrine in hemodialysis solutions and brain dialysate and amino acid neurotransmitter in brain dialysat have been developed and validated. In the microdialysis, there were no different in AUC, Tmax and Cmax among groups, while T1/2, MRT and Vz/F of HP3:2 were significantly different from E and MM in hemodialysis solutions. There were no different in AUC and Cmax among groups, while T1/2, Tmax, MRT and Vz/F of HP3:2 were significantly different from E and MM in brain dialysate. The best PK-PD modeling is Sigmoid-Emax when take the change of ephedrine concentration in hemodialysis solutions and brain dialysate as pharmacokineties (PK) index, while the change of amino acid neurotransmitter concentration in brain dialysate as pharmacodynamics (PD) index. Each group of Glu E-C curve showed a clockwise loop, and E-C curve of GAB A of each group showed a counterclockwise loop.Conclutions1 The dissolution of ephedrine was affected when Ma Hung and Gui Zhi were prepared together. It may be the reason why Ma Huang is relative safe when treated with Ma Huang-Gui Zhi pair.2 The effect of Ma Huang is similar to ephedrine. Acute administration of Ma Huang produced hyperactivity, and with repeated drug administration, sensitization developed. Gui Zhi inhibited Ma Huang-induced hyperactivity in the CNS. Gui Zhi also altered Ma Huang-induced hyperactivity after acute administration of the Ma Huang-Gui Zhi herb pair, and prevented the development of locomotor sensitization to Ma Huang.3 Chronic administration of Ma Huang induced neuronal cell death and increased the levels of oxidative biomarkers, which was demonstrated by elevated levels of MDA and NO and concomitant reductions in SOD, CAT, and GSH-Px activity in the prefrontal cortex. Conversely, Gui Zhi effectively protected against CNS-OT and reversed the changes in the above measures.4 Ephedrine could increase excitatory amino acid neurotransmitters in the prefrontal cortex. Conversely, Gui Zhi effectively decreased excitatory amino acid neurotransmitters and increased inhibitory amino acid neurotransmitters.5 The best PK-PD modeling of ephedrine is Sigmoid-Emax. Ephedrine has tachyphylaxis and excitatory changes induced by ephedrine could be inhibited by Gui Zhi. |
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