| BackgroundAllergic rhinitis (AR) is an allergic disease which is normal. Many factors can lead to AR, such as heredity, circumstance and exposing to allergen. The incidence of AR becomes higher and higher with the change of the circumstance.In order to relief the pain of the people who is suffering AR, many researcher is looking for the best therapeutic regimen. The methods of treating AR are far away allergen, drug therapy, immunotherapy and so on. Except being far away allergen, drug therapy is in common use. Drug therapy including western medicine therapy and traditional medicine therapy. Drug therapy includes two western medicine and Chinese medicine. Western medicine therapy quick and effective, but it has more adverse reactions, these drugs are generally only control the symptoms of AR, can not achieve the purpose of cure. The adverse reactions such as nasal corticosteroids can cause nasal dryness, nasal bleeding and perforation of the nasal septum, and some even cause systemic side effects, such as the hypothalamus-pituitary-adrenal axis suppression, inhibiting the growth of children, causing cataracts and glaucoma and other eye complications. Side effects of antihistamines are more obvious, such as the patient can cause drowsiness, headache, dizziness, decreased ability to sense and manipulate the system and cause side effects effort. Due to the relative small side effects of Chinese medicine with western medicine, the effects are more durable, and therefore get the majority of people of all ages. In recent years, many researchers have begun to study the traditional Chinese medicine for the treatment on AR.Mahuang-Fuzi-Xixin decoction is a classic decoction in Shang Han Lun written by Zhang Zhongjing. It includes three herbs, there are Mahuang, Fuzi and Xixin. Because of the different drug properties of the three herbs, their effects on disease are complementary. Previous studies showed that Mahuang-Fuzi-Xixin decoction has a good effect in the treatment of AR. Drug-containing serum sampled at different time points after administrated Mahuangfuzixixin decoction can reduce the total re-lease of histamine and β-hexosaminidase from RBL-2H3cells induced by antigen, and inhibit proliferation of splenocytes induced by LPS.ObjectiveThis study intends to study anti-allergic rhinitis and anti-inflammatory mecha-nisms of verification of the active parts of Mahuang-Fuzi-Xixin decoction (Mahuang alkaloids extract, Fuzi alkaloids extract, volatile oil and decoction of Xixin) and part of the active ingredients(ephedrine, methylephedrine, pseudoephedrine, hypaconitine, aconitine, benzoylmesaconine, benzoylhypacoitine, benzoylaconine, mesaconitine, norpseudoephedrine and norephedrine). According to previous studies, in order to investigate the active parts and active components of Mahuang-Fuzi-Xxixin decoc-tion, we will intend to use macrophages and mast cells, which participate in the pathogenesis of allergic rhinitis, as the research objectcs to study the mechanism of the the active parts and components of Mahuang-Fuzi-Xixin Decoction on treating AR. Methods1. Preparation and pilot study of the active parts of Mahuang-Fuzi-Xixin De-coction: Mahuang alkaloids extract and Fuzi alkaloids extract were obtained by re-flux extraction, concentrating and so on. The volatile oil of Xixin was obtained as the method in Chinese Pharmacopeia, the2010Edition. The relative amounts of the components of the two alkaloids were rough determination by HPLC.2. To research the effect of the active parts and components of Ma-huang-Fuzi-Xixin Decoction on LPS-induced RAW264.7cells. The LPS-induced RAW264.7was interfered by active parts and components. Different concentrations of the active parts and components of Mahuang-Fuzi-Xixin Decoction were selected to be done, the change of viability of the cells was observed by MTT method.3. To research the effect of the active parts and components of Ma-huang-Fuzi-Xixin Decoction on the RBL-2H3induced by antigen. In the test, the induced RBL-2H3was interfered by active parts and components. Enzyme-linked immunosorbent assay was used to detect the release of cytokine IL-4, TNF-α and β-hexosaminidase.4. In the test, the induced RBL-2H3was interfered by active parts and compo-nents. Gene expression of cytokine IL-4and TNF-α were used by Real-Time PCR.Results1. The preparation and detection of active parts:6.77g total alkaloids of Ma-hang,13.32g total alkaloids of Fuzi and7mL volatile oil of Xixin were obtained. Detecting by HPLC, there were1.00percent of norpseudoephedrine,0.99percent of norephedrine,23.06percent of ephedrine,6.54percent of pseudoephedrine and1.97percent of methylephedrine in the total alkaloids of Mahuang,1.13percent of Ben-zoylmesaconine,0.08percent of benzoylaconine,0.07Benzoylhypacoitine and0.02percent of mesaconitine. 2. The effect of the active parts and components of Mahuang-Fuzi-Xixin De-coction on the viability of RAW264.7induced by lipopolysaccharide:after24hours later, the LPS group, compared to the normal group, the viability of RAW264.7in-creased significantly (p<0.05). The total alkaloids of Mahuang (100μg/L), volatile oil of Xixin (2.2μl/L) and ephedrine, methylephedrine, pseudoephedrine, benzo-ylhypacoitine, benzoylaconine at the concentration of10μg/L to100μg/L could in-hibited the proliferation of RAW264.7induced by lipopolysaccharide, compared with controlled group, the differences was siginificant (P<0.05). The others had no this effect.3. The effects of the active parts and components of Mahuang-Fuzi-Xixin De-coction on the release of cytokine IL-4, TNF-a and β-hexosaminidase:Mahuang al-kaloids extract, Fuzi alkaloids extract, volatile oil of Xixin, ephedrine, methylephed-rine, pseudoephedrine, hypaconitine, aconitine, benzoylmesaconine, benzoylhypa-coitine, benzoylaconine, mesaconitine, norpseudoephedrine and norephedrine can inhibit the release of β-hexosaminidase significantly (P<0.05), volatile oil of Xixin only the low dose can inhibit it. Except the volatile oil of Xixin and hypaconitine, all above can inhibit the release of the cytokine IL-4significantly (P<0.05). Total alka-loids of Fuzi, pseudoephedrine, norpseudoephedrine, ephedrine and hypaconitine can inhibit the release of the cytokine TNF-a and histamine significantly (P<0.05).4. The effect of active parts and components of Mahuang-Fuzi-Xixin Decoc-tion on mRNA expression of cytokine IL-4mRNA and TNF-a mRNA:compared to normal group, the mRNA expression of cytokine IL-4and TNF-a levels of con-trolled group induced by IgE were significantly increased (P<0.05). The mRNA ex-pression of cytokine TNF-a levels were increased significantly (P<0.05) after appli-cation of Mahuang alkaloids extract, Fuzi alkaloids extract, volatile oil of Xixin. The mRNA expression of cytokine IL-4levels were decreased significantly (P<0.05) af- ter application of benzoylhypacoitine, benzoylaconine, ephedrine, methylephedrine, aconitine and pseudoephedrine. The mRNA expression of cytokine TNF-α levels were decreased significantly (P<0.05) after application of ephedrine, methylephed-rine, pseudoephedrine, norpseudoephedrine, norephedrine, hypaconitine, benzo-ylmesaconine, benzoylhypacoitine and benzoylaconine.Conclusion1. Compared the effects of the active parts and components of Ma-huang-Fuzi-Xixin decoction and Mahuang alkaloids extract and the volatile oil of Xixin can inhibit the viability of LPS-induced RAW264.7. The Fuzi alkaloids extract without this inhibition. The groups including benzoylhypacoitine group, ben-zoylaconine group, ephedrine group, methylephedrine group, and pseudoephedrine group can also inhibit the viability of LPS-induced RAW264.7. The groups of ben-zoylmesaconine, aconitine, hypacoitine, mesaconine and norephedrine without inhi-bition on the LPS-induced RAW264.7. Effects of Mahuang alkaloids exact may due to one or more of its components. The low contents of its compents can’t lead the inhibition of Fuzi alkaloids exact2. The results of enzyme-linked immunosorbent assay can explain that the ef-fects of the active parts and components of Mahuang-Fuzi-Xixin Decoction on the release of cytokine IL-4, TNF-a and β-hexosaminidase on the antigen-induced RBL-2H3cells are not in full uniform. All parts and components can inhibite the re-lease of β-hexosaminidase, but only ephedrine, norpseudoephedrine, methylephed-rine and hypaconitine can inhibit the release of histamine. It may be due to the his-tamine is part of a secretory granules, and these components may only act on the ly-sosomes. Mahuang alkaloids extract, Fuzi alkaloids extract, decoction of Xixin, ephedrine, pseudoephedrine, methylephedrine, mesaconitine and aconitine with sim-ultaneous inhibition of IL-4, TNF-a and β-hexosaminidase release effect, suggest- ing that these components may the effective substance of Mahuang-Fuzi-Xixin de-coction for allergic rhinitis. Also verify the preliminary experiment Pseudoephedrine is presumed Mahuang-Fuzi-Xixin decoction.3. The results of the active parts and components of Mahuang-Fuzi-Xixin de-coction on the expression of IL-4mRNA, TNF-α mRNA level of antigen-induced RBL-2H3cells showed that:decoction of Xixin, ephedrine, pseudoephedrine, ben-zoyl hypaconitine, benzoylaconine and aconine can inhibit the mRNA expression level of both IL-4and TNF-α. These parts and components may exert their an-ti-inflammatory gene expression by inhibiting the action of both. Fuzi total alkaloids extract, benzoylmesaconine, norpseudoephedrine, methylephedrine, hypacoitine and mesaconitine only inhibit the mRNA expression level of TNF-α, they may play an anti-inflammatory effect through inhibition the mRNA expression level of TNF-α. Other parts and components include Mahuang alkaloids extract, volatile oil of Xixin and high-dose of norephedrine group increase the mRNA expression level of TNF-α, indicating that the anti-inflammatory effect may played by other aspects. |
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