Anti-inflammatory Activity Screening Of Xianglian Pills Composed Of Different Parts Of Aucklandia Lappa Decene And Acute Toxicity Evaluation Of New Xianglian Pills

Objective:To screen the Xianglian pills composed of different parts of Aucklandia lappa Decne with anti-inflammatory activity,and to evaluate the acute toxicity of the most active Xianglian pill.As a result,it may provide useful information for not only formulation improvement but safe medication as well as patient compliance of the new Xianglian Pills.Methods and results1?Preparation and content determination of different parts of Aucklandia lappa DecneThe volatile oil of Aucklandia lappa Decne was extracted by steam distillation and solvent extraction respectively and the main components of the volatile oil were determined by GC-MS.At the same time,Aucklandia lappa Decne was extracted by 75%ethanol reflux firstly.Then the ethanol extract was adsorbed by D101macroporous resin and eluted by 8 BV?Column volume?of distilled water,20%,60%and 95%ethanol successively.The fractions of water,20%,60%and 95%ethanol eluent were collected separately,and concentrated under vacuum condition?ethanol was recovered?followed by freeze-drying.The different parts namely water elute?WE?,20%ethanol elut?20EE?,60%ethanol elute?60EE?and 95%ethanol elute?95EE?of Aucklandia lappa Decne were obtained.The contents of costunolide and dehydrocostus lactone in different parts of Aucklandia lappa Decne were determined by HPLC.The preparation and analysis of essential oil showed that the volatile oil yield of steam distillation was 0.69%,the relative percentage content of Dehydrocostus lactone was 4.58%,and the yield by volatile oil extracted by ethyl acetate was 5.44%,the relative percentage content of Dehydrocostus lactone was 43.89%.These data indicated that both the yield of volatile oil and the percentage of active component by ethyl acetate extraction were higher than that by steam distillation.The ethanol extract of Aucklandia lappa Decne was segmented into four parts by D101 macroporous resin,and the weight of them were188.49 g of WE?37.80%relative to crude drug yield?,7.08 g of 20 EE?1.42%?,9.58 g of60 EE?1.92%?and 21.50 g of 95 EE?4.30%?respectively.Further HPLC analysis showed that the costunolide and Dehydrocostus lactone were mainly concentrated in the95 EE,in with the content of Dehydrocostus lactone reached 36.14%,while only1.85%in the original herbs,thus,the effective component transfer rate reached 84.01%.2?Preparation and content Determination of Xianglian Pills composed of Different Parts of Aucklandia lappa DecneYuhuanglian was prepared according to the method described in the Chinese Pharmacopoeia?the 2015 edition?.Prepared Yuhuanglian was extracted by 45%ethanol percolation.The extract of Yuhuanglian were combined with WE,20EE,60EE,95EE and volatile oil separately to form a series of xianglian pills consisting of the different part of Aucklandia lappa Decne according to the prescription of xianglian pills documented in Chinese pharmacopoeia 2015 edition,and named as xianglian pill 1-5.The contents of berberine,costunolide and dehydrocostus lactone in those xianglian pills were determined by HPLC.Acetonitrile?A?and 0.05 mol/L potassium dihydrogen phosphate solution?B?were used as mobile phases.Gradient elution?0-15 min,35%A;15-40 min,70%A?was performed.The elute was monitored at 225 nm.The results showed that after percolation extraction of Yuhuanglian,86.56g of extract was obtained form 309 g raw materials,calculated yield was of 28.57%.Meanwhile,HPLC analysis showed the content of berberine in Xianglian Pills 1-5 was 51.08 mg/g,58.34 mg/g,54.12 mg/g,52.70 mg/g and 53.57 mg/g respectively,the content of dehydrocostus lactone in Xianglian Pills 4 and 5 were 21.91 mg/g and 22.61 mg/g respectively.The costunolide in those Xianglian pills were undetectable.3?Screening of Anti-inflammatory Activities of Xianglian Pills composed of Different Parts of Aucklandia lappa DecneThe anti-inflammatory activities of Xianglian pills composed of different parts of Aucklandia lappa Decne were screened by LPS induced macrophage?RAW264.7?inflammation model and xylene induced ear swelling model in mice.The results showed that the optimal concentration of LPS to induce inflammation on macrophage was 1?g/mL and the production of NO,TNF-?,IL-6 and the expression of TNF-?,IL-6,COX-2 and iNOS were significantly increased after LPS treatment.Xianglian Pills4 and 5 significantly reduced the levels of NO,TNF-?and IL-6 in the cells,Meanwhile the reduced expressions of TNF-?,IL-6,COX-2 and iNOS proteins treated by Xianglian Pill 4 and 5 were also observed.No such effects,however,were observed for Xianglian Pills 1-3.Animal test showed that Xianglian Pill 4 and Xianglian Pill 5 had obvious inhibitory effects on xylene induced mice ear swelling.The inhibitory rates of swelling degree were 55.12%and 56.24%?1285 mg/kg?respectively,while the inhibitory rates of aspirin were 47.10%?150 mg/kg?.No inhibitory effects on xylene induced mice ear swelling was observed for Xianglian pill 1-3.Considering the factors of extraction cost and efficiency,Xianglian pill with 95EE were selected and named Xinxianglian pills.4?Acute Toxicity Evaluation of New xianglian PillsThe acute toxicity of the New xianglian Pills?NXP?was evaluated in the following work.According to the"Guidelines for the Experimental Technology of Acute Toxicity of Traditional Chinese Medicines and Natural Medicines",the pre-experiments were carried out in advance.The results showed that the doses of 100%mouse death?D_m?and 0%mouse death?D_n?caused by NXP were 37.28g/kg,4.68 g/kg respectively.Therefore,NXP of different concentrations?4.68g/kg,7.86g/kg,13.21g/kg,22.19g/kg,37.28g/kg?the inter-group dose ratio was 1:1.68??were intragastrically administered to mice twice a day.6 h interval between two doses.After 14 days of observation,the survival of mice was recorded,LD₅₀ was calculated,and the dead mice corpses were examined by gross anatomy and histopathology immediately.The GSH,SOD and MDA levels in liver were determined by corresponding test kits,and the expressions of occludin,claudin-1 and ZO-1 in ileum were detected by Western blot.The results showed that the toxic reaction of NXP in mice was positively correlated with the dosages.The LD₅₀ was 11.56 g/kg,and the 95%confidence interval was 8.39g/kg-15.17 g/kg.The gross anatomy of the death mouse showed that the liver was dark and dull,the liver tissue was congested,the stomach and small intestine were flatulent,the small intestine had edema and blood stasis,and no obvious organ damage were found in other organs.Meanwhile,the surviving mouse were executed and no obvious organ damage were found.HE staining showed that the structure of liver and small intestine of poisoned mice was disordered and inflammatory cells infiltrated when compared with the normal group.At the same time,compared with the normal group,the contents of GSH and SOD in liver of poisoned mice decreased significantly?P<0.01,P<0.05?,and the content of MDA increased significantly?P<0.01?.The expression of occludin,claudin-1 and ZO-1 in ileum decreased significantly?P<0.01?in dead mice.In summary,the 95EE and the volatile oil of Aucklandia lappa Decne are the active parts to enhance the anti-inflammatory activity of Xianglian Pill.The anti-inflammatory potency may be attributed to costunolide and dehydrocostus lactone.The LD₅₀ of Xinxianglian pill,which is composed of the extract of Yuhuanglian and the95%alcohol extract of Aucklandia lappa Decne,is 11.56 g/kg.According to the acute toxicity classification standard of chemical substances issued by WHO,New xianglian pill is micro-toxicity,and its target organs may be liver and small intestine.