Optimization of Preparation of Oregano Oil Microspheres by Box-Behnken Response Surface Methodology

[Objectives]To optimize the formulation and preparation of oregano oil microspheres by Box-Behnken response surface methodology.[Methods]Chitosan was used as the carrier material to prepare oregano oil microspheres by emulsion crosslinking method.The encapsulation efficiency,drug loading and ID 50 were used as the evaluation indicators,and the comprehensive score(OD)obtained by"coefficient of variation-AHP comprehensive weighting method"was used as the final evaluation indicator.The formulation design and preparation process were optimized by single factor experiment and Box-Behnken response surface methodology,and the optimal process parameters were determined.[Results]The optimal formulation and preparation process parameters of oregano oil microspheres were as follows:the ratio of oregano oil to chitosan was 2∶1,the emulsifying speed of double emulsion was 200 r/min,the amount of emulsifier in the colostrum was 4%,and the volume of curing agent was 1.0 mL.The average encapsulation efficiency was 45.33%±1.32%,the average drug loading was 30.59%±2.45%,and the median diameter(ID 50)was 52.596μm±0.023%.[Conclusions]The encapsulation efficiency,drug loading and ID 50 of oregano oil chitosan microspheres prepared by emulsion crosslinking method met the requirements.The drug-loaded microsphere not only can be used as a preparation finished product for direct application,but also be used as a product intermediate to lay a foundation for the research and development of subsequent dosage forms.

Erzhi pills ameliorates cognitive dysfunction and alter proteomic profiles of hippocampus in ovariectomized Alzheimer disease model rats induced by D-galactose and Aβ1-40 injection

OBJECTIVE Erzhi pills is a clas⁃sic prescription of Chinese medicine originated from Fu Shou Jing Fang in Ming dynasty,with the effects of nourishing kidney-Yin and hemosta⁃sis,black hair,strengthening muscles and bones.As a classical prescription for nourishing kidney-Yin,Erzhi pills has been used to treat senile dementia in China for many years.Herein,our study aimed to investigate the protective effects of Erzhi pills in rat models of Alzheimer disease(AD)induced by ovariectomy as well as D-galactose and Aβ1-40 injection and to explore its potential mechanism.METHODS The model of AD rats was established by ovariectomy com⁃bined with D-galactose and Aβ1-40 injection.Ovariectomized rats were randomly divided into four groups:model group,estradiol valerate(0.80 mg·kg-1)group,Erzhi pills high(1.50 mg·kg-1)and low(0.75 mg·kg-1)doses group.In addition,rats of sham operation were selected as the sham operated group.Except for the sham oper⁃ated group,rats were injected intraperitoneally with D-galactose(100 mg·kg-1 per day,for 49 d)on the 8th day,then they were given intracerebro⁃ventricular injection of Aβ1-40(10μg per rat,1 g·L-1)on the 36th day,while the corresponding drugs were given by gastrointestinal administra⁃tion on the 22nd day.In our study,Morris water maze test was used to evaluate the learning and memory abilities,while ELISA kit was used to an⁃alyze serum estrogen level.The morphology of hippocampal neuron cells was observed by HE staining,and Nissl staining was utilized to ob⁃serve the Nissl body in cytoplasm.Then,the ex⁃pression of ERβpositive cells and hippocampal Aβ1-40 and p-Tau404 proteins was determined by immunohistochemistry.In order to further explore the molecular mechanism of Erzhi pills preven⁃tion and treatment of AD,proteomics was used to find potential targets and related pathways,Western blotting was used to verify the expres⁃sion of candidate differential protein.According to the results of proteomics in our experiment,Western blotting was used to detect the protein expression of PI3K,Akt,Bcl-2,Bcl-xl,Bad,14-3-3 and GSK3β.RESULTS The escape latency was significantly shortened,the number of crossing platform was increased,the neuron arrange⁃ment was more orderly and with less nuclear pycnosis in rats of Erzhi pills groups compared to the model group.In rats treated with Erzhi pills,the number of neurons,Nissl bodies,the estro⁃gen levels and ERβpositive cells were increased,while the number of Aβ1-40 and p-Tau404 positive cells was significantly decreased.Proteomics found that there were more than one hundred differentially expressed proteins of rats treated with Erzhi pills,which were involved in 48 signal⁃ing pathways.Among these proteins,five of them were involved in the PI3K/Akt signal path⁃way.As the down-stream protein of PI3K/Akt sig⁃naling pathway,the content of 14-3-3 protein was significantly increased.Western blotting analysis showed that the expression of p-GSK3β/GSK3βand Bad was decreased,while that of p-Akt/Akt,p-PI3K/PI3K,14-3-3,Bcl-xl and Bcl-2 was up-regulated in rats from the Erzhi pills groups compared with the model group.CON⁃CLUSION Erzhi pills can improve estrogen levels,alter proteomics expression of the hippocampus and activate PI3K/Akt pathway in AD rats,reduce Aβaggregation,inhibit the hyperphos⁃phorylation of Tau protein,maintain the morphol⁃ogy of hippocampal neurons and decrease the apoptosis of hippocampal neurons,thereby improving the learning and memory abilities of ovariectomized AD rats induced by D-galactose and Aβ1-40 injection.This study may provide an experimental basis for the clinical treatment of Erzhi pills.