Study On The Effect Of Radices Paeoniae Alba Eye Drops On Dry Eye Model Based On The Yin Deficiency Theory

Objective:To extract,isolate and purify paeoniflorin and polysaccharides from Radices Paeoniae Alba(RPA),and prepare them to eye drops.The experimental dry eye mouse model was prepared by combining the application of scopolamine with dry environment and was used to investigate the effect of those eye drops on dry eye disease and its mechanismMethod:The paeoniflorin was extracted by alcohol extraction and the slag was used to extract the polysaccharide from aqueous extraction.Chitosan flocculation experiment was operated to deproteinization,and the optimal macroporous resin or cation exchange resin for separation and purification was test by static adsorption method.I Used silica gel column to purify paeoniflorin,and used cellulose DE 52 gel column to purify the polysaccharides Component.After desalting and lyophilization,the power dissolve in suitable solvent for eye drops prepared.The dry eye model mouse was prepared by both subcutaneous injection of scopolamine hydrobromide and low-humidity environment.After successfully set up the model,those 42 mouse were randomly divided into model group,vehicle group,sodium hyaluronate(SH)group,paeoniflorin group,RPA polysaccharide-1(BS-P1)group and RPA polysaccharide-2(BS-P2)group,and were treated with the corresponding drug eye drops for 10 days.(The SH group was used 1%sodium hyaluronate eye drops——an ordinary clinical eye drops for dry eye patient,and the vehicle group using the blank eye drops.)Another 7 normal mouse that were not modeled were used as the normal group,receiving no eye drops or any special treatmeant.After the treatmeant,Symptoms of dry eye were assess by tear secretion,fluorescein sodium corneal staining and corneal irregular test;HE stai.ning of corneal,conjunctival,periocular gland and conjunctival electron microscope scan(SEM)were use for morphological observation;molecular level indicators:The protein levels of tumor necrosis factor-?(TNF-?)?interleukin-1?(IL-1?)and mucin 1(MUC1)on ocular surface were detected by Western Blot.In a word,explore the different effects of othse eye drop on experimental dry eye mouse.Results:The aqueous extract of total glucosides of RPA is preferably separated and purified by using D101 and polyamide resin to obtain a crude paeoniflorin having a purity of 67.04%,and the yield is 2.62%.It was further purified by silica gel column to obtain paeoniflorin with a purity of 94.53%.The best effect of removing protein from RPA aqueous extraction is adding 3%chitosan mother liquor(1%(v/v))to the solution(0.1g·crude drug·mL-1),and the rate of deproteinization is 64.90%±0.33%.The next,choosed Type 732-H+ cation exchange resin,the protein removal rate can up to 85.67%,and the polysaccharide retention rate to 94.96%,and 2.6 g crude polysaccharide,whose yield is 1.3%was received.Finally,the crude polysaccharide was eluted by DE 52 cellulose column,fractionated and desalted to obtain power:BS-P1(pure white),BS-P2(pure white),BS-P3(light yellow),BS-P4 and BS-5 components.GPC showed the weight average molecular weight of BS-P1 and BS-P2 is 19,712 and 178,291,732.In animal experiments,compared with the normal group,the tear secretion was significantly reduced and corneal flouresein or irregularityscores were both significantly increasing in the model group(P<0.05),and morphology indicated a more common of cornea epithelialahy thinness and ocular surface defection,and the number of goblet cells in the conjunctiva was decreased.SEM show that the goblet cells in the model group are obviously atrophied,and a large number of abnormally thick secretions are covered in the conjunctival sac.Compared with the model group,only BS-P1 group could significantly increase the amount of tears(P<0.05).Conversely,except for the BS-P1 group,each drug can significantly reduce both the corneal staining scores and irregularity scores(P<0.05),indicating that sodium hyaluronate,paeoniflorin and BS-P2 do some protecting to the cornea.The pathological changes of dry eye have a certain improvement effect in all four drug groups.Western blot found that each drug can significantly correct the abnormally expression of IL-1 and MUC 1.But for the abnormal expression of TNF-?,only the SH and paeoniflorin have significant effects.Athough there is a trend to improve the abnormal expression of TNF-? in BS-P1 and BS-P2 group,the difference is not statistically significant.Conclusion:Using high concentration ethanol and water as extraction solvent,paeoniflorin and Paeonia lactiflora polysaccharide were extracted by microwave,which could greatly improve the utilization rate of Paeonia lactiflora,reduce the production cost and promote the reuse of resources.High-purity paeoniflorin can be obtained by using D101 macroporous resin combined and silica gel column chromatography.By type 732-Na+ cationic exchange resin,the purification of polysaccharide of RPA could reached at a higher protein clearance rate and polysaccharide retention rate.And after DE 52 column polysaccharide elution,two main homogeneous components,BS-P1 and BS-P2,were obtained.The former was neutral polysaccharide and the latter was acidic polysaccharide.Both paeoniflorin and polysaccharide component(BS-P2)in the extract of RPA showed a corneal protection in experimental dry eye mice.Those were probably occurred by inhibiting the inflammatory pathway.The polysaccharide component BS-P1 has a good effect on increasing the amount of tears.All is suggested that most components are involved in the different ways to play the role of "nourishing yin" in the treatment of dry eye model.