| Ginsenoside Rg3(3β,12β,20(R)-dihydroxydammar-24-ene-O-[β-D-glucopyranosyl (1→2)-β-D-glucopyranoside]),a minor ginsenoside from the Panax ginseng,has been shown multiple pharmacological activities,including anti-fatigue,anti-tumor, platelet aggregation inhibition enhance immunity and memory improvement and so on. However,the plasma concentration of Rg3 after oral administration in human is very low.Previous researches obtained some human pharmacokinetic(PK) parameters, including Cmax of(16.00±6.00) ng/ml and tmax of(0.66±0.10) h at 3.2 mg/kg from the oral experiments.It is widely accepted that the short half-life and low plasma concentration of drugs had many reasons,firstly,the drug absorbed by gastrointestinal was low;secondly,the drug is metabolized quickly by the enzymes and bacterias in the gastrointestinal tract;thirdly,the drug can be absorbed by the gastrointestinal mocosal,but is metabolized by the enzymes in intestinal or liver,which is called first-pass effect.Recently,the interest arises from the different possible advantages presented by the nasal cavity,such as:the direct transport of absorbed drugs into the systemic circulation thereby avoiding the first-pass effect in peroral administration,the lower enzymatic activity compared with the gastrointestinal tract and the liver,the convenience for administration when there is no water.For all these reasons the nasal route can be considered as an useful alternative both to parenteral and oral routes.In this study,we used chitosan as the carrier material to prepare ginsenoside Rg3 microspheres,which was considered to have a bioadhesive effect and also have an absorption enhance effect as the carrier material.Firstly,in order to provide an animal experimental evidence of Rg3 intranasal administrated preparation,in this study,we investigated the anti-fatigue effect of Rg3 after intranasal administration.In the present study,we investigated the effects of Rg3 on performance of the weight-loaded swimming test and on biochemical parameters related to fatigue:serum urea nitrogen(SUN),lactic dehydrogenase(LDH),superoxide dismutase(SOD),malondialdehyde(MDA),blood lactic acid(LA) and hepatic glycogen.Ginsenoside Rg3 was administrated intranasally to mice for two weeks at three different doses.Compared with the negative control group,the intermediate-dose and the high-dose groups significantly prolonged the weight-loaded swimming time(p＜0.05;p＜0.01);and the hepatic glycogen levels were significantly increased(p＜0.05) in these two groups;SUN levels were significantly decreased in three Rg3-treated groups(p＜0.01);in addition,the low-dose group obviously decreased the content of blood LA(p＜0.05).However,the levels of LDH,SOD and MDA did not show a significant change.The results predict a benefit of Rg3 as an anti-fatigue treatment by intranasal administration.The mechanism is related to increase the storage of hepatic glycogen,and decrease the accumulation of metabolite such as lactic acid and SUN.Secondly,we investigated the physical and chemical properties of Rg3 before prescription.Then we used rat in vivo in-situ method as an experimental model to investigate the regulation of nasal absorption,The effects of drug volume and concentration on nasal absorption of Rg3 were studied.Then we studied the effects of three different absorption enhancers such as:chitosan,β-cyclodextrin and bomeol through rats in-situ nasal cavity stagnation method,combined with the results of ciliotoxity experiments,we finally chose chitosan as a carrier material because of its good promotion effect and low ciliotoxity.Thirdly,a detailed investigation of the preparation of chitosan microspheres was carried out.It was found that the loading efficiency of multiple emulsion method was higher than emulsion method,so the multiple emulsion method was selected to prepare microspheres.The effects of processing parameters,including concentration of chitosan,emulsifying agents and speed of emulsifying and so on were also investigated according to the Single-factor Experimental Design.In order to optimize the formulation,a Central Composite Design(CCD) was employed for three independent variables,the ratio of drug to chitosan,the ratio of organic phase to water phase and ratio of first emulsion to oil phase.The dependent response variables were measured as particle size,drug loading and loading efficiency.Consequently,when the drug ratio was 0.45,the ratio of organic phase to water phase was 0.5 and the ratio of first emulsion to oil phase was 0.15,microspheres with a good shape were prepared. The average diameter was(44.99±12.59)μm,the drug loading capacity was (10.25±0.08)%,the encapsulation efficiency was(30.61±1.46)%.At the same time, we established the method to determine the in-vitro release of Rg3 chitosan microspheres and approached the release mechanism,then investigated the influencing factors related to drug release.The test was carried out under 37℃and constantly stirred at the rate of 100 rounds per minute.The media for release was saline(pH=6.5) contained 3%SDS.Then,we studied the ciliotoxicity and mucoadhesion of Rg3 chitosan microspheres.Rats were used for irritation tests.No obvious flare on the nasal region was found in three dosage levels after successive intranasal administered for 2 weeks.To evaluate the ciliotoxicity of Rg3 chitosan microspheres,the lasting time of ciliary movement was recorded with in situ toad palate model.The degree of mucoadhesion was also investigated by determining the mucociliary transport rate(MTR)of the microspheres.The results showed that Rg3 chitosan microspheres could effectively reduce ciliotoxicity and had a good mucoadhesive property.Finally,we evaluated the anti-fatigue effect of Rg3 chitosan microspheres.The test animals were divided into three groups:negative control group,positive control group and Rg3-treated group.We investigated the effects of Rg3 on performance of the weight-loaded swimming test and on biochemical parameters related to fatigue: serum urea nitrogen(SUN),lactic dehydrogenase(LDH),superoxide dismutase(SOD), malondialdehyde(MDA),blood lactic acid(LA) and hepatic glycogen.The results suggested that the Rg3 chitosan microsphere had an anti-fatigue effect.
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