| Purpose: To formulate Agei nasal spray containing essential oil of Artemisia frigida Willd and evaluate its qualitative control,antibacterial activity,anti-anflammatory,anti-alergic rhinitis.Methods: The effects of particle size of law material,soaking time,liquid to plant material ratio and extraction time on essential oil yield were investigated through a single factor and multi-factor experiments.Nasal spray was oil-in-water emulsion formulation that prepared by incorporating the oil phase into the water phase mixed together with the surfactant stirring.Quality control of Agei nasal spray carried out thin layer chromatography,gas chromatography-mass spectrometry and gas chromatography method by Chinese pharmacopoeia method.The pharmacology study of Agei nasal spray on allergic rhinitis animal model studied by ELISA,WB,q RT-PCR assay and histopathological examination.An animal model of AR in SD rats was established by sensitizing it intraperitoneally injected 1 m L of ovalbumin suspension(0.5mg/m L OVA and 30 mg/m L aluminum hydroxide)once every other day for 7 times and challenged with intranasal dropping 0.05 m L of 2% ovalbumin solution once every other day for total 5 times.The rats were grouped as normal group,model group,Agei nasal spray group,china spray group,and western spray group.Each group was consisted of 10 rats.The drugs were dropped on both sides of the nose three times for a total 11 days.The antimicrobial activities of MIC and MBC for the Agei nasal spray were studied by using micro-dilution method and agar dilution method.Results: The optimal process combination for extraction of essential oil is A1B2C3,which means 2 h of soaking time,10:1 the liquid to solid ratio,and 8 h of extraction time.The Agi nasal spray was prepared by using Mongolian medicine Agei essential oil(1%w/v),povidone K30,tween-80,benzalkonium bromide,and distilled water.The spots in TLC of Agei rhinitis spray were clear and easy to be identified.The compounds(eucalytol,camphor,and borneol)in Agei nasal spray were clearly identified by comparing their GC-MS,retention times,and MS data with those ofcorresponding reference compounds.All calibration curves showed good linearity(r2>0.9997)within the test ranges.The LOD,LOQ,specificity,precision,and accuracy for the method were validated.The average recovery rates were 97.6%(RSD= 2.5%),102.1%(RSD = 2.4%),and 101%(RSD = 3.7%)and the content of eucalytol,camphor,and borneol in different batch of Agei nasal spray were0.73?0.78mg/m L,1.28?1.4mg/m L,and 0.42?0.47 mg/m L respectively.The antibacterial effect of Agei nasal spray in vitro showed that the Agei nasal spray had obvious bacteriostatic effect on Staphylococcus aureus,Staphylococcus epidermidis,Proteus and Escherichia coli,and its concentration of MIC50 was 2.25 mg/m L,2.1mg/m L,4.31 mg/m L and 0.51 mg/m L respectively.Result of vivo anti-inflammatory showed that the swelling of ear in control group was significantly increased(P<0.01)compared with the normal group(right ear of mice).The swelling of ear in Agei nasal spray group was significantly difference(P<0.05)compared with the model group.The positive group of Chinese and Western nasal spray were significantly difference(P<0.05).Result of acute toxicity study showed that the mice in Agei nasal spray group did not die.The anatomical observation of the heart,liver,spleen,lung,kidney,stomach,and intestine showed no abnormalities.The pathological examination of nasal mucous tissue in rats was performed by HE staining.In the model group,the inflammatory cell infiltration,mucous edema,and cell degeneration of nasal tissue were changed.The Agei nasal spray group,Chinese nasal spray group,and western nasal spray group were compared with model group which the inflammatory cell infiltration,mucous edema were approaching to normal.The results of ELISA: The levels of Ig E and IL-4 in the serum of model group were significantly increased(P<0.05)compared with the normal group and the level of IFN-gamma were decreased(P<0.05).The levels of Ig E and IL-4 in the serum of Agei group were lower(P<0.01 and P<0.05)than model group.The result of WB experiment: The expression of GATA-3 protein in the nasal tissue of the model group was significantly increased compared with normal group.The expression of GATA-3 protein in Agei group was significantly decreased compared with model group.The result of q RT-PCR experiment: The expression of GATA-3 m RNA in the nasal tissue of the model groupwas significantly increased compared with normal group.The expression of GATA-3m RNA in Agei group was significantly decreased compared with model group.The expression of GATA m RNA in Chinese spray and Western spray groups were significantly decreased than model group.The expression of GATA m RNA in administration groups between no significant.Conclusion: This research carried out to prepare Agei nasal spray and its quality control,antibacterial,anti-anflammatory,anti-alergic rhinitis activities investigated that provide to development of clinical and pharmacodynamic studies. |
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