ABSTRACT
OBJECTIVE To establish the fingerprint of Zhuang medicine Jinmu granules and carry out chemometric analysis ， and determine the contents of three components . METHODS High performance liquid chromatography （HPLC） method was adopted. Using rutin as the reference ，HPLC fingerprints of 10 batches of Jinmu granules were drawn and similarity evaluation was performed by Similarity Evaluation of Chromatographic Fingerprint of Traditional Chinese Medicine （2012 edition）；the common peak was determined by comparing with mixed control ；SPSS 21.0 software was used for cluster analysis ，and SIMCA 14.1 software was used for principal component analysis and orthogonal partial least squares-discriminant analysis. The differential components affecting the quality of Jinmu granules were screened by taking the variable importance in projection （VIP）value ＞1 as the standard ；the HPLC method was used to determine the contents of astilbin ，polydatin and berberine hydrochloride in Jinmu granules. RESULTS There were 22 common peaks in 10 batches of Jinmu granules ，and the similarities were 0.962-0.997；five common peaks were identified ，namely gallic acid （peak 2），polydatin（peak 9），rutin（peak 11），astilbin（peak 13）and kaempferol （peak 20）. The results of cluster analysis showed that 10 batches of Jinmu granules could be clustered into 3 categories：S1 and S 3-S4 were grouped into one category ；S5-S6 and S 9 were grouped into one category ；S2，S7-S8 and S 10 were grouped into one category. The results of principal component analysis showed that the parameter of model interpretation was 0.951 and that of prediction ability was 0.723. The classification results were basically consistent with cluster analysis. The classifica tion results of orthogonal partial least squares- com discriminant analysis were also ba sically consistent with clus- ter analysis. The common peaks with VIP value ＞1 in the order were peak 7＞peak 11（rutin）＞peak 17＞peak 13（astilbin）＞ peak 3＞peak 8＞peak 6＞peak 16 respectively. The linear ranges of astilbin ，polydatin and berberine hydrochloride were 0.012 6- 1.225 0，0.010 8-1.052 5 and 0.020 0-1.562 5 mg/mL，respectively（all R 2＝0.999 9）. RSDs of precision ，stability（24 h）and repeatability tests were all less than 3％. The average recoveries were 99.48％（RSD＝2.67％，n＝9），98.57％（RSD＝1.77％，n＝ 9）and 100.84％（RSD＝2.49％，n＝9）. The contents were 1.221 0-7.011 6，2.251 1-4.462 9，1.252 4-3.328 7 mg/g，respectively. CONCLUSIONS Established fingerprint and the method of content determination are accurate ，stable and simple. Combined with chemometric analysis ，it can be used for the quality control and evaluation of Zhuang medicine Jinmu granules.
ABSTRACT
OBJECTIVE：To optimize the extraction technology of Zhideke granules. ME THODS：The extraction technology （water extraction ，alcohol extraction ，water extraction and ethanol precipitation ）of Zhideke granules was initially screened by ammonia-induced cough experiment and xylene-induced ear swelling experiment in mice. Based on its preparation route ，the immersion time of medicinal materials containing volatile oil was investigated with water absorption as index firstly. The single factor test was adopted to investigate the amount of water added and the extraction time taking the volatile oil yield as index to optimize the extraction technology of medicinal materials containing volatile oil. Taking the contents of irisflorentin and total flavonoids as indicators ，on the basis of single factor investigation ，orthogonal test was adopted to examine the influence of three factors including the amount of water added ，extraction time and extraction frequency ，so as to optimize the water extraction technology of Zhideke granules and the validation tests were conducted. RESULTS ：The results of pharmacodynamics experiment showed that the cough latency of mice in water extract low-dose and high-dose groups （6.34，12.68 g/kg，by crude drug ）and water-extraction alcohol-precipitation extract high-dose group （12.68 g/kg，by crude drug ）were significantly longer than those inmodel group ，and the number of cough within 2 minutes was significantly reduced （P＜0.05 or P＜0.01）. Compared with model group ， the ear swelling of mice in water extract low-dose and high-dose groups （6.34，12.68 g/kg，by crude drug），ethanol extract high-dose group （12.68 g/kg，by crude drug） and water-extraction alcohol-precipitation extract hig dose group （12.68 g/kg，by crude drug ） were decreased significantly （P＜0.05 or P＜0.01）. The swelling inhibition rates were 42.26％，55.08％，33.49％，51.56％，39.57％ and 44.36％ in low-dose and high-dose groups of water extract ，alcohol extract ， water-extraction and alcohol-precipitation extract respectively ，indicating that the water extract had better antitussive and anti-inflammatory effects. The optimal extraction technology of volatile oil was adding 5-fold water ，soaking for 30 minutes，and extracting for 3 hours. The optimal water extraction technology was adding 12-fold water ，extracting for 3 times after soaked for 50 min，lasting for 1 h each time. Results of 3 times of validation tests showed that average content of irisflorentin in the extract obtained by optimal technology was 76.47 μg/g（RSD＝ 2.15％，n＝3）and the average content of total flavonoids was 92.45 mg/g（RSD＝0.48％，n＝3）. CONCLUSIONS ：The optimal extraction technology of Zhideke granules is stable and feasible.
ABSTRACT
OBJECTIVE：To study the effects of chitosan graphene oxide car rier（CS-GO）loaded with oridonin （CS-GO- oridonin）on the proliferation and apoptosis of human lung cancer A 549 cells. METHODS ：Taking A 549 cells as objects ，the survival rate of cells were detected by CCK- 8 method after treated with different concentrations of CS-GO （3，6，12，24，48 μg/mL）and CS-GO-oridonin loaded with same mass of oridonin （3，6，12，24，48 μg/mL，by the weight of oridonin ，the same below）. IC 50 of CS-GO-oridonin was calculated. The cell morphology were observed by microscope after treated with CS-GO and CS-GO-oridonin（both 32 μg/mL）for 2，4，10，24 h. The uptake of CS-GO ，oridonin，CS-GO-orionin（all 32 μg/mL）by cells was observed with fluorescence labeling method. The apoptosis of cells and the content of ROS were observed by flow cytometry after treated with different concentrations of CS-GO （16，32，64 μg/mL）and CS-GO-oridonin （16，32，64 μg/mL）. The expression of anti-apoptosis related proteins （Mcl-1，Bax and Bak ）were detected by Western blot. RESULTS ：After treated with different concentrations of CS-GO ，the survival rate of cells was still above 90％ ；after treated with different concentrations of CS-GO-oridonin，the survival rate of cells showed a downward trend ，and was significantly lower than that of CS-GO group （P＜ 0.01）；IC50 of CS-GO-oridonin was 32.61 μg/mL. After CS-GO treatment，the cell morphology did not change significantly ；after CS-GO-oridonin treatment ，the cells shrunk and fell off in clusters ，and the suspended matter increased ；the fluorescence of oridonin and CS-GO-orionin taken up by cells was enhanced than CS-GO. Compared with blank group ，there was no significant change in the apoptosis rate ，the content of ROS and the expression of apoptosis-related protein in 16，32，64 μg/mL CS-GO groups（P＞0.05）；apoptosis rate ，the content of ROS ，the protein expression of Bax and Bak in 16，32，64 μg/mL CS- GO-oridonin groups were increased significantly ，while the protein expression of Mcl- 1 were decreased significantly. Above indexes were significantly high er or lower than the same concentration CS-GO group （P＜0.05）. CONCL USIONS：CS-GO dose not affect the proliferation and apoptosis of A 549 cells；CS-GO-oridonin has obvious inhibition and apoptosis promoting effect on cells ，which may be related to increasing ROS production and regulating the expression of apoptosis related proteins.