Study On The Antiasthmatic Effect And Mechanism Of Limax Extract And Its Quality Control

Objective: To study the effect and mechanism of Limax extract on bronchial asthma in rats,and its relieving cough and eliminating phlegm in mice,and to screen the active ingredients of Limax for relieving cough and eliminating phlegm,and establish a content determination method based on the active ingredients,which can provide experimental basis for the establishment of Limax quality control system and the comprehensive utilization of Limax extract.Methods: 1.Forty SD rats were randomly divided into six groups with 8 rats in each group: normal group,model group,dexamethasone group,high-dose group and low-dose group of Limax extract.Except for the normal group,the other groups were peritoneal injection with 10%OVA solution 1m L on the first day and the eighth day of the experiment.Add to subcutaneous injection of 10%OVA solution 0.2m L on the eighth day,to establishe the model of bronchial asthma in rats.The normal group was injected with saline of equal volume.From the 15 th day,rats were placed in an ultrasonic nebulizer.Rats was sprayed with 2%OVA solution for 30 minutes and 10 m L,once a day for 3 weeks.Normal rats were sprayed with normal saline instead of 2%OVA solution.One hour before each atomization sensitization,different groups were intervened by isovolumic gastric perfusion with corresponding drugs.Normal group and model group were given isovolumic gastric perfusion with normal saline.After the last sensitization stimulation,rats were anesthetized with 10% chloral hydrate and specimens were collected as required.To determinate of serum and BALF LT,IL-4,IFN-? in rats by enzyme-linked immunosorbent assay.Pathological sections of lung tissue were made and HE staining was used to observe the lung tissue structure and inflammatory infiltration.The immunohistochemical method was used to detecte of PTEN protein expression in lung tissue.The content of PTEN m RNA in lung tissues was detected by RT-PCR.2.Forty eight Kunming mice were divided into six groups with 8 mice in each group.They are normal control group,carbetapentane citrate group,high-dose group and low-dose group of Limax extract,and high-dose group and low-dose group of Limax polysaccharide.Each mouse was treating with medicine by intragastric administration.Carbetapentane citrate group treat with carbetapentane citrate 50mg·kg-1 by intragastric administration.High-dose group and low-dose group of Limax extract treat with Limax extract 300 mg·kg-1,100mg·kg-1 by intragastric administration.High-dose group and low-dose group of Limax polysaccharide treat with Limax polysaccharide 200mg·kg-1,50 mg·kg-1 by intragastric administration.Five day after administration,the latency and frequency of cough in mice within 3 minutes were recorded by 20% ammonia gas induced cough.The same amount of Kunming mice were divided into groups and given the same dosage.Ambroxol hydrochloride group treat with ambroxol hydrochloride 100mg·kg-1 by intragastric administration.After Five days of administration,5% phenol red was injected intraperitoneally to detect the excretion of phenol red in trachea of mice,and the active ingredients of antitussive and phlegmatic slugs were preliminarily were screened.3.Qualitative identification of Limax medicinal materials was carried out by microscopic identification and TLC.Referring to the general principles of the Chinese Pharmacopoeia(2015 edition),detections of water,total ash,acid-insoluble ash,extracts and other related items of Limax medicinal materials from different habitats were done.The content of polysaccharides in of Limax medicinal materials from different habitats was determined by ultraviolet spectrophotometer(UV).Results: 1.Compared with model group,dexamethasone group and high dose group of Limax extract could prolong the latency of asthma in rats(P < 0.05).The contents of LT and IL-4 in serum and bronchoalveolar lavage fluid(BALF)of model group rats were increased,while the contents of IFN-? were decreased(P<0.05)by treatment with Limax extract compared with the normal group.The levels of LT and IL-4 in serum of rats in dexamethasone group and high dose group of Limax extract were decreased significantly,while the levels of IFN-? was increased(P<0.05)compared with model group.The LT and IL-4 contents in BALF in dexamethasone group,and Limax extract groups were decreased significantly,while IFN-? content was increased(P<0.05)compared with model group.HE staining showed that the structure of lung tissue and bronchial epithelium in normal group was intact,there was without inflammatory exudation in the lumen,the size of alveoli was normal,without adhesion and inflammatory secretion.In model group,the bronchial epithelium was markedly exfoliated and necrotic,the bronchial wall became thicker,the inflammatory infiltration around the bronchi was serious,the lumen became smaller,the inflammatory excretion gathered in the lumen,the alveolar wall adhesion became thicker,the number of alveoli decreased,and inflammatory secretions were seen in the alveolar cavity.In dexamethasone,high dose group and low dose group of Limax extract,the bronchial wall was slightly thicker,the inflammatory substances in the lumen were reduced,the bronchial epithelium were not fall off and necrosis,and the adhesion of alveolar wall was reduced.Immunohistochemical staining of PTEN protein in bronchus of rats showed that PTEN protein was abundantly expressed around bronchus in normal group,while decreased in dexamethasone group,and high dose group of Limax extract.The PTEN protein expression was negative in model group and low dose group of Limax extract.The results of PCR showed that compared with the model group,the expression of PTEN gene in lung tissue of rats in dexamethasone group and high does group of Limax extract were increased.2.Compared with the normal control group,the cough frequency and latency of mice in the carbetapentane citrate group,high-dose group and low-dose group of Limax extract,and high-dose group and low-dose group of Limax polysaccharide were decreased significantly(P<0.05).Compared with the normal control group,the phenol red excretion of mice in the ambroxol hydrochloride group,polysaccharide high-dose group increased significantly(P<0.05).3.Qualitative identification of Limax from different habitats was carried out by microscopic identification and TLC identification.The results showed that Limax from different habitats had similar characteristics,fluorescence spots showed in the same position.The test items of Limax from different habitats conformed to the regulations.A method for determining the content of Limax polysaccharide by UV was established.The results of methodological investigation showed that the precision,stability,repeatability and recovery test of the method met the relevant standards of Chinese Pharmacopoeia(2015 edition).Conclusion: 1.The therapeutic effect of Limax extract on bronchial asthma was investigated by establishing a rat model of bronchial asthma.The results shows that the Anti-bronchial asthma effect of Limax extract effect is remarkable and its mechanism might be related to the influence of Th1/Th2 cytokine balance and the up-regulation of PTEN gene expression.2.It is shown that polysaccharide is the main active ingredient of Limax in relieving cough and resolving phlegm.3.A method for identifying Limax is established.The reference limits of Limax moisture,total ash,acid-insoluble ash and extract are established.A method for the determination of polysaccharide content in Limax is established to provide scientific basis for the development and utilization of Limax.