Journal of Chinese Pharmaceutical Sciences

Evaluation of the intracellular trafficking of siRNAs in A375 cells by confocal laser scanning microscopy

Yiping Diao, Jing Sun, Mengyi Yang, Bo Xu, Lihe Zhang, Zhenjun Yang

2016, 25(12): 859-868. DOI: 10.5246/jcps.2016.12.096

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Investigation intracellular trafficking of siRNAs following their delivery to cells is of great interest to elucidate dynamics of siRNA in cytoplasm. In this study, we present a novel confocal laser scanning microscopy (CLSM) method to evaluate a novel delivery system of 3′-peptide-siRNA therapeutic, which was named 3′-pAs-siRNA/CLD. This method could not only calculate the content of the intracellular 3′-peptide-siRNA, but also quantify its co-localization with cellular substructure. We observed that 3′-pAs-siRNA/CLD, which provided the better antitumor capability, also had a better cell uptake, endosome escape and a longer retention time in A375. This novel strategy was proved to be efficient, quantified and visualized, thus making the dynamics research of siRNA in cytoplasm clear and simplified.

A comparative study of the methods in estimating pharmacokinetic parameters with single-observation-per-animal type data

Tingjie Guo, Anyue Yin, Tianyan Zhou, Wei Lu

2016, 25(12): 869-875. DOI: 10.5246/jcps.2016.12.097

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During pre-clinical pharmacokinetic research, it is not easy to gather complete pharmacokinetic data in each animal. In some cases, an animal can only provide a single observation. Under this circumstance, it is not clear how to utilize this data to estimate the pharmacokinetic parameters effectively. This study was aimed at comparing a new method to handle such single-observation-per-animal type data with the conventional method in estimating pharmacokinetic parameters. We assumed there were 15 animals within the study receiving a single dose by intravenous injection. Each animal provided one observation point. There were five time points in total, and each time point contained three measurements. The data were simulated with a one-compartment model with first-order elimination. The inter-individual variabilities (IIV) were set to 10%, 30% and 50% for both clearance (CL) and apparent volume of distribution (V). A proportional model was used to describe the residual error, which was also set to 10%, 30% and 50%. Two methods (conventional method and the finite resampling method) to handle with the simulated single-observation-per-animal type data in estimating pharmacokinetic parameters were compared. The conventional method (M1) estimated pharmacokinetic parameters directly with original data, i.e., single-observation-per-animal type data. The finite resampling method (M2) was to expand original data to a new dataset by resampling original data with all kinds of combinations by time. After resampling, each individual in the new dataset contained complete pharmacokinetic data, i.e., in this study, there were 243 () kinds of possible combinations and each of them was a virtual animal. The study was simulated 100 times by the NONMEM software. According to the results, parameter estimates of CL and V by M2 based on the simulated dataset were closer to their true values, though there was a small difference among different combinations of IIVs and the residual errors. In general, M2 was less advantageous over M1 when the residual error increased. It was also influenced by the levels of IIV as higher levels of IIV could lead to a decrease in the advantage of M2. However, M2 had no ability to estimate the IIV of parameters, nor did M1. The finite resampling method could provide more reliable results compared to the conventional method in estimating pharmacokinetic parameters with single-observation-per-animal type data. Compared to the inter-individual variability, the results of estimation were mainly influenced by the residual error.

Spectrophotometric and spectrofluorimetric methods for the determination of ropinirole content in pharmaceutical dosage forms

Effat Souri, Milad Torabadi, Maliheh Barazandeh Tehrani

2016, 25(12): 876-881. DOI: 10.5246/jcps.2016.12.098

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Two simple and rapid spectrofluorimetric and spectrophotometric methods were described for the determination of ropinirole hydrochloride content in pharmaceutical dosage forms. Both methods are based on the reaction of ropinirole hydrochloride and eosin Y in aqueous medium at pH 4.0. Quenching of the fluorescence intensity of eosin Y at 540 nm upon excitation at 350 nm was used for the determination of ropinirole hydrochloride levels after ion-pair complex formation. Also, the absorbance increase of eosin Y at 546 nm after ion-pair complex formation was used for spectrophotometric measurements. Both methods showed linear relationships between the fluorescence quenching or absorbance increase and ropinirole concentration in the range of 6–150 µg/mL and 50–500 µg/mL for spectrofluorimetric and spectrophotometric methods, respectively. As no organic solvents were used in these two methods, they could be categorized as green analytical methods. Both methods were accurate (Error<1.2%) and precise (CV<1.9%), as shown by statistical analysis results. Both methods were used for determination of ropinirole hydrochloride content in pharmaceutical dosage forms without any significant interference from associated impurities.

Protective effects of orally administered honokiol on cerebral ischemia reperfusion in rats and on stroke in SHRsp

Xiaoyan Liu, Zhenyu Hu, Shizhong Chen, Jiancheng Wang, Yinye Wang

2016, 25(12): 882-891. DOI: 10.5246/jcps.2016.12.099

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Honokiol is a protective agent for cerebral ischemia injury when administered intravenously. In the present study, we aimed to investigate the oral effect of honokiol microemulsion on cerebral ischemia-reperfusion (I-R) injury in rats and stroke in SHRsp. Both tMCAO and SHRsp models in rats were used to evaluate the efficacy of the microemulsion. Rat aortic segment contraction test, primary rat aortic endothelial cells and primary brain microvascular endothelial cells (BMECs) injured by OGD-R were used to explore its potential action mechanism. Oral honokiol microemulsion significantly reduced infarct volume, neurological score and brain water content in tMCAO model, and it evidently reduced neurological score and increased the survival rate of SHRsp. Moreover, honokiol significantly inhibited aortic contraction induced by KCl and phenylephrine, and L-NAME suppressed these inhibitory effects. On the other side, honokiol increased NO and p-eNOS levels in rat endothelial cells. In addition, it also protects BMECs against OGD-R injury and increased eNOS expression in BMECs. In conclusion, oral honokiol administration has protective effects in tMCAO and in SHRsp rats, and its action mechanism is likely to be associated with its vasodilative effect produced by eNOS activation and with its protective effect on BMECs.

Activity of tamarindus indica pulp water extract in high carbohydrate diet rats as a treatment for obesity and insulin resistance

Finna Setiawan, Elin Yulinah Sukandar, Sukrasno, Ketut Adnyana I

2016, 25(12): 892-897. DOI: 10.5246/jcps.2016.12.100

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Obesity is often identified as a nutritional disorder, and this condition continues to be one of the most important yet preventable health hazards. It may be a complex group of diseases and should be characterized as a syndrome that results from an imbalance between energy intake and expenditure. Furthermore, obesity is associated with several diseases such as diabetes mellitus, atherosclerosis, cardiovascular diseases, osteoarthritis, and hypertension. In previous studies, we found that in Asia, especially in the Asean region, carbohydrates are heavily represented in the diet. Therefore, we used a high carbohydrate diet to induce obesity in rats. The aim of this study was to evaluate the potency of Tamarindus indica pulp as an anti-obesity agent in a high carbohydrate diet animal model. The antiobesity effect was evaluated in this in vivo model at doses of 75 mg/kg·bw and 225 mg/kg·bw. Treatment was started in week 3 and week 9, and biochemical characteristics were measured every 3 weeks. In the end of the study period, insulin resistance and adiponectin levels were measured. TIWE (Tamarindus indica water extract) at a dose of 75 mg/kg·bw showed better efficacy than at a dose of 225 mg/kg·bw, and treatment started on week 3 was better than treatment started on week 9. TIWE has an anti-obesity effect associated insulin resistance by increasing adiponectin levels and insulin sensitivity, which can impact body weight, triglyceride levels, blood glucose levels, and body fat.

Chemical constituents from Portulaca oleracea and their bioactivities

Tianyun Jin, Tao Shen, Mingxing Zhou, Ailing Li, Da Feng, Bolun Zheng, Jie Gong, Jiawei Sun, Lingyu Li, Lan Xiang

2016, 25(12): 898-905. DOI: 10.5246/jcps.2016.12.101

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In the present study, we aimed to intensively study the chemical constituents, especially organic acids from a medicinal plant Portulaca oleracea L., and screen their anti-inflammatory and quinone reductase (QR, a phase II detoxyfication enzyme) inductive activity. A total of 20 compounds were isolated and identified based on spectroscopic methods, as succinic acid (1), mono-methyl succinate (2), L-malic acid (3), L-1-methyl malate (4), L-4-methyl malate (5), L-dimethyl malate (6), L-6-ethyl citrate (7), L-1-methyl citrate (8), L-1,5-dimethyl citrate (9), 4-hydroxy-5-methylfuran-3-carboxylic acid (10), 5-hydroxymethyl-furoic acid (11), stearic acid (12), L-pyroglutamic acid (13), cyclo-(tyrosine-leucine) (14), L-isoleucine (15), (–)-dehydrovomifoliol (16), (–)-epiloliolide (17), 3,4-dihydroxyphenylethanol (18), succinimide (19), and uracil (20). Among them, 14 compounds (2, 4–8, 10, 11, 13–18) were isolated from P. oleracea for the first time. Compound 18 (12.5 μM) exhibited potent anti-inflammatory effect in lipopolysaccharide (LPS)-induced macrophage cells (RAW264.7) by reducing NO production, and it also increased QR activity in Hepa lclc7 cells. Compound 16 (50 μM) showed weak QR inductive activity. None of other compounds showed anti-inflammatory or QR inductive activities.

Simultaneous determination of 6 organic acids, 3 nucleosides, and ephedrine in Pinellia ternata by HPLC

Qiongfang Wen, Yanfang Zhang, Jingqing Zhang, Hua Zhao

2016, 25(12): 906-913. DOI: 10.5246/jcps.2016.12.102

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Pinellia ternata has a long history of medicinal use in China, Japan, and Korea. Herein, we present a sensitive, selective, and robust HPLC method for the simultaneous determination of 6 organic acids (oxalic acid, malic acid, citric acid, formic acid, acetic acid, and succinic acid), 3 nucleosides (uridine, guanosine hydrate, and adenosine), and ephedrine in P. ternata. The Rhizoma pinelliae samples were collected from different parts of Southwest China, and the 10 constituents were detected at a wavelength of 210 nm. Chromatographic separation was achieved on a Boston Green ODS reversed-phase column along with a gradient mobile phase of methanol and phosphate buffer (pH 2.3, 10 mmol/L, v/v) at a ﬂow rate of 1.0 mL/min. The range of recovery was between 90.8% and 101.9%, and the calibration curves showed high linearity. The relative standard deviations of the new method were less than 3.0% for intra- and inter-day analyses. All samples were stable for at least 48 h. The abundance of the 10 compounds were dependent on their specific locations. The new HPLC method is a powerful technique for the quality control of P. Ternata.