Screening the effective components in treating dampness stagnancy due to spleen deficiency syndrome and elucidating the potential mechanism of Poria water extract / 中国天然药物

Poria is an important medicine for inducing diuresis to drain dampness from the middle energizer. However, the specific effective components and the potential mechanism of Poria remain largely unknown. To identify the effective components and the mechanism of Poria water extract (PWE) to treat dampness stagnancy due to spleen deficiency syndrome (DSSD), a rat model of DSSD was established through weight-loaded forced swimming, intragastric ice-water stimulation, humid living environment, and alternate-day fasting for 21 days. After 14 days of treatment with PWE, the results indicated that PWE increased fecal moisture percentage, urine output, D-xylose level and weight; amylase, albumin, and total protein levels; and the swimming time of rats with DSSD to different extents. Eleven highly related components were screened out using the spectrum-effect relationship and LC-MS. Mechanistic studies revealed that PWE significantly increased the expression of serum motilin (MTL), gastrin (GAS), ADCY5/6, p-PKAα/β/γ cat, and phosphorylated cAMP-response element binding protein in the stomach, and AQP3 expression in the colon. Moreover, it decreased the levels of serum ADH, the expression of AQP3 and AQP4 in the stomach, AQP1 and AQP3 in the duodenum, and AQP4 in the colon. PWE induced diuresis to drain dampness in rats with DSSD. Eleven main effective components were identified in PWE. They exerted therapeutic effect by regulating the AC-cAMP-AQP signaling pathway in the stomach, MTL and GAS levels in the serum, AQP1 and AQP3 expression in the duodenum, and AQP3 and AQP4 expression in the colon.

Study on the mechanism of Poria cocos polysaccharides on the regulation of gluconeogenesis in liver of type 2 diabetic mellitus model rats / 中国药房

OBJECTIVE To investigate the effect and mechanism of Poria cocos polysaccharides on the regulation of blood glucose in type 2 diabetes mellitus （T2DM）model rats by phosphatidylinositol 3-kinase（PI3K）/protein kinase B （Akt）/forked box transcription factor O 1（FoxO1）pathway. METHODS SD rats were randomly divided into blank control group （no modeling ，no administration），model group （modeling，no administration ），metformin group （modeling，200 mg/kg）and P. cocos polysaccharide low-dose，medium-dose and high-dose groups （modeling，100，200，400 mg/kg），8 in each group. Except for blank control group ， other groups were given high fat diet combined with streptozotocin to construct the model of T 2DM rats. At the same time ， administration groups were given relevant dose of medicine intragastrically ，and blank control group and model group were given constant volume of water intragastrically ，once a day ，for consecutive 42 days. During the experiment ，general condition and bodyweight of rats were observed every day ；fasting blood glucose （FBG）of rats were collected ，and oral glucose tolerance test were conducted and area under curve （AUC）was calculated the day before last administration. After last medication ，the heart ，liver， kidney organ index were calculated ；the levels of HbA 1c，TC，TG，MDA，SOD，GSH-Px and hepatic glycogen content were detected. HE staining was used to observe the pathological changes of liver and pancreatic tissue ，and the pathological grade score was calculated. Western blot assay was used to detect the protein expressions of p-PI 3K，p-Akt，p-FoxO1， PEPCK and G 6Pase in liver tissues. RESULTS Compared with blank control group ，the rats of model group suffered cc1965@163.com from polydipsia ，polyphagia and polyuria ；the body weight ， the levels of SOD and GSH-Px ，the protein expressions of p-PI 3K，p-Akt and p-FoxO 1 were significantly decreased （P＜0.05）；liver and kidney organ index ，blood glucose level at 0，0.5 and 2 hours after intragastric administration of glucose solution ，AUC， FBG，HbA1c，serum levels of MDA ，TC，TG and hepatic glycogen content ，liver and pancreatic pathological grade score ，the protein expressions of PEPCK and G 6Pase were all increased significantly （P＜0.05）. Compared with model group ，the general condition of rats in P. cocos polysaccharide groups were all improved ，and all of above indicators had been reversed to varying degrees. CONCLUSIONS P. cocos polysaccharide can downregulate protein expressions of PEPCK and G 6Pase which are key enzymes of gluconeogenesis ，inhibit hepatic gluconeogenesis ，effectively decrease blood glucose levels and regulate glucolipid metabolism in T 2DM model rats by weakening oxidative stress and upregulating PI 3K/Akt/FoxO1 pathway.

Study on Repair ，Anti-inflammation and Analgesia Effects of Compound Crocodile Oil Burn Ointment on Super- ficial Second-degree Burned Skin / 中国药房

OBJECTIVE：To study the repa ir，anti-inflammatory and analgesic effects of Compound crocodile oil burn ointment on superficial second-degree burned skin. METHODS ：The heated weight was attached to the right depilated skin of guinea pigs for 4 s to induce the model of superficial second-degree burn. After modeling ，guinea pigs were randomly divided into model group ， Jingwanhong ointment group （positive control ），formula Ⅰ，Ⅱ and Ⅲ groups of Compound crocodile oil burn ointment （volume fraction 1.5％，3％，4.5％，hereinafter），with 8 guinea pigs in each group. Except for model group ，other groups were smeared with 0.7 g/guinea pigs twice a day for 14 consecutive days. The wound healing was recorded every day ，the healing rate of wound was calculated. HE staining was used to observe the histopathological changes of the wound. The serum levels of EGF ，VEGF， SOD，MDA，TNF-α and IL-1 were detected by ELISA. Eighty Kunming mice were divided into 2 groups，and then sub-grouped into model group ，Jingwanhong ointment group （positive control ），formula Ⅰ and Ⅲ groups of Compound crocodile oil burn ointment，with 10 mice in each group. Then xylene auricle swelling method and acetic acid writhing method were used to investigate the anti-inflammatory and analgesic effects of Compound crocodile oil burn ointment. RESULTS ：In the burn repair experiment，after intervention of Compound crocodile oil burn ointment ，the wound area of guinea pigs gradually decreased ，and on the 14th day ，the wound had healed greatly ，and the wound healing rate increased significantly （P＜0.01）；serum levels of EGF and SOD were increased significantly （P＜0.01），while the levels of VEGF ，MDA，TNF-α and IL-1 were decreased significantly（P＜0.05 or P＜0.01）. The thick new epidermal layer was found in wound tissue ，and the connective tissue and neovascularization in the dermis increased significantly. In the anti-inflammatory and analgesic experiment ，after intervention of Compound crocodile oil burn ointment ，the degree of ear swelling and the times of writhing decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：Compound crocodile oil burn ointment shows good skin repair ，anti-inflammatory and analgesic efficacy；the mechanism may be associated with increasing the serum levels of EGF and SOD and reducing the levels of VEGF ， MDA，TNF-α，IL-1.

Establishment of UPLC Fingerprint of Poria cocos Aqueous Extract and Study on Its Spectrum-effect Relationship with Sedative and Hypnotic Effect / 中国药房

OBJECTIVE：To establis h the UPLC fingerprint of Poria co cos aqueous extract ，and to investigate its relationship with sedative and hypnotic effect. METHODS ：Ten batches of P. cocos from different areas were extracted with water to obtain the aqueous extract. UPLC method was adopted. The determination was performed on Waters HSS-C 18 column with mobile phase consisted of 0.1％ phosphoric acid solution-acetonitrile-methanol （gradient elution ） at the flow rate of 0.4-0.2 mL/min. The detection wavelengths were set at 210 and 242 nm. The column temperature was 40 ℃，and sample size was 2 μL. The fingerprints of 10 batches of P. cocos aqueous extracts were established by using the Similarity Evaluation System of TCM Fingerprint （2012A version），and the common peaks were identified. The sedative and hypnotic effects of 10 batches of P. cocos aqueous extracts from different areas under the synergistic action of pentobarbital sodium were investigated by taking the sleeping rate ，sleep latency and sleep duration of mice as the single efficacy index. After data transformation of single efficacy index and total efficacy （single indexes calculated by analytic hierarchy process ），grey correlation analysis was used to analyze the correlation between the common peaks in fingerprint of P. cocos aqueous extract and the single efficacy index and total efficacy. RESULTS ：There were 24 common peaks in 10 batches of aqueous extract of P. cocos ， and 11 components were identified ， i.e. 16 α-hydroxydehydrotrametenolic acid （peak 6），16α-hydroxytrametendic acid （peak 7），poricoic acid B （peak 9），dehydrotumulosic acid（peak 10），poricoic acid A （peak 12），polyporenic acid C （peak 15），3-O-acetyl-16α-hydroxydehydrotrametenolic acid （peak 17），dehydropachymic acid （peak 20），pachymic acid （peak 21），dehydrotrametenolic acid （peak 22），dehydroeburicoic acid （peak 24）. Grey correlation analysis showed ，the correlation between 24 peaks and sleep duration was greater than 0.6（0.611 5- 0.811 8）；the correlation between 24 peaks and sleep latency was greater than 0.6（0.605 9-0.790 4），except for peaks 14，24 and 2；the correlation of 24 peaks between sleeping rate was greater than 0.6（0.606 4-0.721 6），except for peaks 23，19，17 and 5； the correlation of 24 peaks between total efficacy was greater than 0.6（0.619 0-0.781 2），except for peaks 2，5，19. The top 10 chromatographic peaks related to the total efficacy were peak 15（polyporenic acid C ），peak 16，peak 8，peak 11，peak 12 （poricoic acid A ）， peak 1， peak 7 （16 α-hydroxytrametendicacid）， peak 3， peak 9 （poricoic acid B ） and peak 20 （dehydropachymic acid ）. CONCLUSIONS ：UPLC fingerprint of P. cocos aqueous extract was established and 11 components were identified. Ten components such as polyporus acid C are closely related to the total efficacy of sedation and hypnosis ，which preliminarily reveal the material basis of the sedative and hypnotic effect of P. cocos .

Content Determination and Cluster Analysis of 7 Kinds of Triterpenes in Poria cocos from Different Habitats / 中国药房

OBJECTIVE：To establish a method for th e content determination of 7 kinds of triterpenoids in Poria cocos ，and to compare the differences of the above components in P. cocos from different habitats ，so as to provide reference for the quality control of P. cocos . METHODS ：Using 36 batches of P. cocos from different habitats as samples ，HPLC method was used for content determination of dehydrotomorphic acid ， polyporhinic acid C ， 3-epidehydrotomorphic acid ， 3-O-acetyl-16 α-hydroxy-hydrogenolysaccharic acid ，dehydrotomorphic acid ，pachymic acid and dehydrotrametenolic acid. The column was performed on Thermo Acclaim 120 C18 with mobile phase consisted of acetonitrile-phosphoric acid water （gradient elution ）at the flow rate of 1 mL/min. The detection wavelength was set at 210 nm. The column temperature was 30 ℃，and the injection volume was 20 μL. SPSS 21.0 statistical software was used for cluster analysis of 36 batches of P. cocos from different habitats. RESULTS ： The linearity of 7 triterpenoids was good in the range of their mass concentration （all r≥0.999 0）；average recoveries were 96.74％-104.04％（RSD＝0.54％-1.55％，n＝6）. RSDs of precision ，and reproducibility stability （24 h）tests were all lower than 3.0％（n＝6）. RSD of durability test was lower than 5.0％（n＝2）. There were some differences in the single content of 7 indicator components among samples from different habitats ，but the total content difference was not obvious （the total content of most samples was in the range of 1.3-1.9 mg/g）. After cluster analysis ，36 batches of sample were clustered into 5 categories，i.e. S 27 was clustered into class Ⅰ；S30 and S 34 were clustered into class Ⅱ；S2，S8 and S 9 were clustered into class Ⅲ；S10，S11，S12 and S 14 were clustered into class Ⅳ；and the remaining 26 batches of samples were clustered into class Ⅴ. CONCLUSIONS ：The method is simple ，and has good precision ，repeatability and durability. It can be used for the simultaneous determination of above 7 components in P. cocos . There has no significant difference in the quality of P. cocos from different habitats. The content of P. cocos in most producing areas is uniform in content and stable in quality ，only a few of them are different. Δ 基金项目 ：国家重点研发计划中医药现代化研究重点专项

Study on “Qi-invigorating”Effect and Its Mechanism of Total Saponins of Astragalus membranaceus on Rats with Qi-deficiency / 中国药房

OBJECTIVE：To study “Qi-invigorating”effect and its possible mechanism of total saponins of Astragalus membranaceus on rats with Qi-deficiency ，and to provide reference for elucidating the material basis of “Qi-invigorating”effect of A. membranaceus . METHODS ：Forty male Wistar rats were randomly divided into normal group ，model group ，positive control group [Buzhong yiqi pills ，4.5 g/（kg·d）]，A. membranaceus total saponins high-dose and low-dose groups [ 252，28 g/（kg·d），by the amount of total saponins] according to body weight ，with 8 rats in each group. Except for normal group ，the model of Qi-deficiency was made in other groups by the method of “diet disorder+fatigue ”. At the same time ，administration groups were given relevant medicine intragastrically ，and normal group and model group were given constant volume of water，once a day ，for consecutive 21 days. After last administration ，the general situation of rats was observed ；the body weight ，spleen index and thymus index of rats were detected ；weight-bearing swimming time was recorded ；the levels of spleen T lymphocyte subsets CD 3 and CD 4，the levels of ATP and ADP in liver tissue ，serum levels of ALB ，RBC and HBG in blood as well as the serum levels of SOD，MDA，lactate，LDH，CK，IL-2，IL-12 and TNF-α were all detected. RESULTS：Compared with normal group ，body weight，thymus index ，spleen index ，weight-bearing swimming time ，the level of spleen T lymphocyte subsets CD 3，ATP，ADP， ALB，IL-2 and IL- 12 were decreased or shortened significantly in model group （P＜0.05 or P＜0.01）. The levels of MDA ， lactate，CK and TNF-α were increased significantly （P＜ 0.05）. Compared with model group ，body weight ，spleen index，weight-bearing swimming time ，the level of spleen T lymphocyte subsets CD 3 and the levels of ATP ，ADP，ALB， RBC and IL- 2 were increased significantly or prolonged（P＜0.05）；while the levels of MDA ，lactate，CK and TNF-α were decreased significantly in A. membranaceus total saponins high-dose group（P＜0.05 or P＜0.01）.Weight-bearing swimming time ，the levels of ATP ，ADP and IL- 2 in A. membranaceus total saponins low-dose group were increased significantly or prolonged （P＜0.05 or P＜0.01），while the levels of MDA ，lactate，CK and TNF-α were decreased significantly （P＜0.05 or P＜0.01）. Compared with positive control group ，spleen index ，spleen T lymphocyte subsets CD 3，weight-bearing swimming time and ATP level of A. membranaceus total saponins high-dose group were increased significantly or prolonged （P＜0.05 or P＜0.01），while MDA levels of A. membranaceus total saponins high-dose and low-dose groups were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：A. membranaceus total saponins can reduce the body ’s accumulation of blood lactic acid ，the activity of CK ，the level of lipid peroxide and regulate immunity to tonify Qi ，delay fatigue and improve exercise ability.

Comparison of Total Protein Content Difference of Poria cocos from Different Producing Areas / 中国药房

OBJECTIVE：Compare the difference of total protein content of Poria cocos from different producing areas. METHODs：Using bovine serum albumin （BSA） as control ，0.4 mol/L sodium hydroxide solution as exctraction solution ， Coomassie brilliant blue G- 250 as chromogenic reagent ，visible spectrophotometry at 595 nm was used to determine the contents of total protein of P. cocos ；cluster analysis was used to classify 34 batches（S1-S34）of P. cocos from different producing areas. RESULTS：The linear range of BSA was 1.45-17.40 μ g/mL （r＝0.999 6）. RSDs of precision ，stability （20 min） and reproducibility tests were all lower than 3％；recoveries were 100.14％-104.26％（RSD＝1.43％，n＝9）. The contents of total protein in 34 batches of P. cocos from different producing areas were 0.388 4％-1.129 7％. The results of cluster analysis showed that among 34 batches of P. cocos ，the total protein content of P. cocos produced in Yingshan county of Hubei province （S2，S3） was higher than 1％，clustered into one category ；the total protein contents of P. cocos produced in Hubei ，Yunnan，Anhui and Hunan（S1，S5-S10，S12，S13，S16，S17，S19-S21，S23-S25，S28，S30，S31）were 0.653 5％-0.946 1％，clustered into one categony，and the remaining batch content were 0.388 4％-0.601 2％，clustered into one category. CONCLUSIONS ：Established method is suitable for the content determination of total protein content of P. cocos . The protein content of P. cocos from Yingshan county of Hubei province is the highest ，followed by Yunnan and Anhui in 34 batches of P. cocos from different producing areas.

Effect on Blood Lipid and Hepatoprotective Efficacy of Hemp Seed Oil in Hyperlipidemic Rats / 中国药师

Objective:To observe the effect of hemp seed oil on the blood lipid and its hepatoprotective efficacy in hyperlipidemic rats. Methods:High fat diet was used to make the hyperlipidemia model in SD rats,and the rats were randomly divided into 6 groups:the normal control group,high fat model group,high dose group(1. 5 g·kg-1 ),medium dose group(1. 0 g·kg-1 ),low dose group (0. 5 g·kg-1 )and Xuezhikang group(0. 5 g·kg-1 ). After 35-day treatment,serum TC,TG,HDL-C,LDL-C,SOD,MDA,ALT, AST and liver SOD were detected. Results:Compared with the model group,hemp seed oil at different dosages could significantly de-crease the contents of serum TC,TG,ALT and the atherogenic index(AI)( P<0. 05 or P<0. 01),and significantly increase the SOD activity in serum and liver(P<0. 01). The contents of serum MDA and AST in the medium and high dose groups were signifi-cantly decreased(P<0. 01),and the content of LDL-C in serum and the content of MDA in liver in the high dose group were signifi-cantly reduced(P<0. 01). Conclusion:Hemp seed oil shows promising blood lipid reducing ability and liver protective effect in hy-perlipidemic rats,and the mechanism may be related with liver lipid metabolism improvement and antioxidant function enhancement.

Quality standard of Compound Shouwu Granule / 中成药

AIM:To establish the quality standard of Compound Shouwu Granule (Radix Polygoni multiflori,Radix et Rhizoma Thalictri Baicalensis,Herba Rabdosiae Rubescentis,etc.) METHODS:TLC was performed to identify Radix Polygoni Multiflori,Radix et Rhizoma Thalictri Baicalensis,Herba Rabdosiae Rubescentis.HPLC was used to determine 2,3,5,4'-tetrahydroxystilbene-2-0-?-D-glucoside. RESULTS:The study on the quality control showed that the characteristic of identification by TLC was distinct and highly specific.The quantitative evaluation had the linear range of 0.04-0.72 ?g.The average recovery was 99.99% and RSD was 1.3%. CONCLUSION: The method for identification and quantitation was simple,accurate,realizable and reproducible.It can be used effectively for the quality control of Compound Shouwu Granule.

Quality standard for Tongduhuoxue Tablets / 中成药

Objective:To establish the quality standard for Tongduhuoxue Tablets.Methods: Radix Salvia miltiorrhiza and Rhizoma Corydalis in this prescription were identified by TLC. Tanshinone Ⅱ A was determined by HPLC and astragaloside was determined by dual wavelength TLC Scanning.Results:Radix Salvia miltiorrhiza and Rhizoma Corydalis could be detected by TLC. Determination method of tanshinone Ⅱ A and astragaloside was linear at the range of 0.04 ～ 0.8 ?g( r= 0.99998 ,n=7 ) and 0.3 ～ 6.0 ?g( r= 0.9991 ,n=7 ),average recovery was 98.6% ( RSD= 1.1% ,n=5 ) and 97.7% ( RSD= 1.8% ,n=5 ),respectively. Conclusions: The established method is accurate and reproducible. This study provides a method for the quality control of Tongduhuoxue Tablets.