Journal of Chinese Pharmaceutical Sciences

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2005, 14(2): 1-01.

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Chemical Constituents of Hedysarum gmelinii

LIU Yi, MA Xiao-xia, CHEN Hu-biao, TU Guang-zhong, HE Jiu-ming, ZHAO Yu-ying^*

2005, 14(2): 75-78.

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Aim To study the chemical constituents of Hedysarum gmelinii. Methods The constituents were separated and purified by different methods of chromatography, and their structures were elucidated by IR, MS and NMR. Results Eight compounds were isolated from Hedysarum gmelinii, including three triterpenoids, two flavonoids and two other compounds. Their structures were identified as squasapogenol (1), soyasapogenol (2), lupeol (3), 3, 9-dihydroxy coumestan (4), 3-hydroxy-9-methoxy pterocarpan (5), β-sitosterol (6), palmatic acid (7), and hexadecanoic acid 2, 3-dihydroxypropyl ester (8). Conclusion All the compounds have been isolated from this plant for the first time. Compounds 1-4 and 8 were obtained from this genus for the first time. The NMR data of 1 are reported for the first time.

Optimization for Purification and Characterization of Recombinant Hirudin III from E. coli

WEI Li-jun, LIU Jun, WU Bin, LI Xue-feng, YE Shuang-ning, ZHANG Liang, WU Wu-tong^*

2005, 14(2): 79-85.

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Aim To optimize purification conditions of recombinant hirudin 3 in the fermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated and purified from the fermentation broth by three column chromatography steps with macroporous resin, DEAE cellulose DE52 and preparative RP-HPLC, respectively, and the optimal conditions were obtained. Purity of the product was determined by SDS-PAGE and analytical RP-HPLC. The molecular weight was determined by mass spectrometry. The structure of the product was analyzed by peptide map. Results The product with purity of 95.4786% was obtained after three purification steps in the optimum conditions with a total yield of 39%. The molecular weight of the product was 6 913.32 ± 6.55 Da, coincident to the theoretical molecular weight of r-hirudin 3. The structure of the product was coincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfully employed for purification of r-hirudin 3 from E. coli using batch-type approaches. The product obtained with high purity was confirmed to be r-hirudin 3.

Chemical Constituents from Potentilla multifida L.

XUE Pei-feng, LIANG Hong, WANG Bin, ZHAO Yu-ying^*

2005, 14(2): 86-88.

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Aim To investigate the chemical constituents from Potentilla multifida L.. Methods Chromatographic technique was employed for the isolation and purification of the constituents, and the structures were identified by spectral evidence. Results Four compounds were isolated involving adenosine (1), apigenin-6-C-arabinopyranosyl-8-C-glucopyranoside (2), apigenin-7-O-β-D-glucuronide (3) and luteolin-7-O-β-D-glucuronide (4). Conclusion The four compounds were obtained from the genus Potentilla for the first time.

A 3D-QSAR Study on a Novel Chromanol Class of I_Ks Potassium Channel Blockers

DU Lü-pei, LI Min-yong, XIA Lin^*, YOU Qi-dong

2005, 14(2): 89-94.

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Aim and Method A novel three-dimensional quantitative structure-activity relationship (3D-QSAR) method, self-organizing molecular field analysis (SOMFA), was used to investigate the correlation between the molecular properties and a class of chromanol analogs as IKs blockers. Results The cross-validated correlation coefficient q² values (0.698) and non cross-validated correlation coefficient r² values (0.701) proved a good conventional statistical correlation. Conclusion The final SOMFA model has therefore good predictive activity for the further molecular design of chromanol IKs potassium channel blockers.

Preparation, Characterization and in vitro Release of Ciprofloxacin Polylactic Acid Microspheres

YANG Fan^*, LIANG Ren, PAN Yu-fang, ZHAO Yao-ming, WANG Zhao-yang, XU An-long

2005, 14(2): 95-99.

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Aim Ciprofloxacin polylactic acid microspheres (CFX-PLA-MS) were prepared using solvent evaporation method from a solid-in-oil-in-water emulsion system. Methods Orthogonal experiment was used to optimize the method of CFX-PLA-MS preparation. Microspheres were characterized in terms of morphology, size, encapsulation efficiency, drug loading and in vitro drug release. Results The physical state of CFX-PLA-MS was determined by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). Microspheres formed were spherical with smooth surfaces. Drug was enveloped in microspheres without mixing physically with PLA. The average particle size was 280.80±0.15 μm, with over 90% of microspheres falling in the range of 250-390 μm. The encapsulation efficiency was 65.8%±0.58% and the drug loading was 34.1%±0.51%. In vitro release study revealed a profile of sustained release of ciprofloxacin from CFX-PLA-MS. The accumulated release percentage and half-life (T1/2) of ciprofloxacin microspheres were 84.0% in 53.2 h, and 31.9 h, respectively. Higuchi equation was Q = -0.0043 + 0.0039 t1/2, r = 09941. Conclusion Ciprofloxacin microspheres have been successfully prepared and sustained release of CFX from microspheres is achieved.

Formulation Study for Rotundine Rapidly Disintegrating Tablet

WANG Xiao-qiong, KE Xue, PING Qi-neng^*, XU Bo-hui

2005, 14(2): 100-104.

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Aim The aim of the present study was to prepare tablets which can rapidly disintegrate in saliva, containing active ingredient in high dose (37.5 % W/W). Methods Rapidly disintegrating tablets containing rotundine were prepared by direct compression, wet granulation and moulding, respectively. Different disintegrants and excipients were decided by single factor test. The typical disintegration time measurement and a new method of wetting time measuring were introduced for assessing rapidly disintegrating tablets. Results The tablets (80 mg) prepared by direct compression have the crushing strength of 4.0 kg·mm^-2 and rapidly disintegrate within 15 s in the saliva of healthy volunteers ; the tablets prepared by wet granulation also have sufficient strength, a little longer but acceptable disintegration time (within 25 s in saliva) ; and the tablets obtained by moulding show disintegration within 40 s in saliva but low strength (2 kg·mm^-2). Disintegration time profiles of tablets are similar to those of wetting time, and the disintegration and wetting times in vitro are similar to the disintegration time in vivo, the latter having higher correlation with that in oral cavity. Conclusion The rapidly disintegrating tablets can be prepared by using these three techniques and excipients. Both in vitro disintegration time and wetting time are necessary indexes for judgment of in vitro disintegration profile.

In vitro Dissolution and Pharmacokinetics in Beagle Dogs of a Self-Emulsifying Formulation of Tretinoin

QUAN Dong-qin^*, XU Gui-xia

2005, 14(2): 105-109.

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Aim In vitro dissolution test and pharmacokinetics in beagle dogs were conducted to assess the formulation of tretinoin in self-emulsifying systems. Methods The concentrations of tretinoin were determined by HPLC. A crossover study was performed in four fasting beagle dogs with the formulation of self-emulsifying systems and commercial capsules. Results The results showed that the dissolution rate in 15 min of tretinoin in self-emulsifying systems was higher than 80% while that of the commercial capsules was lower than 5%. The area under the plasma concentration-time curve (AUC) of the self-emulsifying formulation was significantly higher and Cmax was approximately two times greater than those of commercial capsule, respectively. In addition, the time taken to reach peak was shorter (2 h to 1.25 h) for self-emulsifying formulation of tretinoin. Conclusion The self-emulsifying drug delivery systems can significantly increase tretinoin in vitro dissolution and in vivo absorption.

Simultaneous Determination of Baicalin, Berberine and Rhein by HPLC in Traditional Chinese Patent Medicine Sanhuang Tablets

LI Yi, GAO Jian-Ping, XU Xu^*

2005, 14(2): 110-114.

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Aim To establish a reversed phase liquid chromatographic method for simultaneous determination of three main medicinal constituents, baicalin, berberine and rhein, in Sanhuang tablets. Methods The separation was performed on a Kromasil C18 column with TEA-adjusted 0.02 mol·L^-1 H3PO4 (pH 6.78)-acetonitrile-methanol (40:9:7) as mobile phase at a flow-rate of 1.0 mL·min^-1, with detection at 254 nm. Considering interaction between acidic and alkaline compounds, three standard markers were added respectively and the volume of sample solution was doubled in recovery experiments. Results Three regression equations revealed excellent linear relationship between the peak areas and concentrations and the correlation coefficients all surpassed 0.999 8. The average recovery was 96.1% (RSD = 2.1%) baicalin, 98.5% (RSD = 2.4%) for berberine, and 101.5% (RSD = 1.3%) for rhein. Conclusion The method developed can be used to control the quality of Sanhuang tablets comprehensively.

Determination of Candicidin/FR-008 and Related Components in Fermentation Broth by RP-HPLC

MAO Xiang-zhao, SHEN Ya-ling^*, WEI Dong-zhi^**, CHEN Shi, Deng Zi-xin

2005, 14(2): 115-118.

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Aim A liquid chromatographic method for the determination of candicidin/FR-008 and related components in fermentation broth has been developed. Methods There were four major components in the candicidin/FR-008 complex, which were separated by HPLC under the following conditions: SB-C8 column (4.6 mm ×250 mm, 5 μm) was used, the mobile phase consisted of acetonitrileammonium acteate (20 mmol·L^-1, pH 4.0) (40:60, V/V), with a flow rate of 1.0 mL·min^-1 the UV detection wavelength was 380 nm, and the whole process was performed at 25 ºC. Results The linearity was obtained in the range of 6.25-500 μg·mL^-1 candicidin/FR-008 with the regression equation of Y = 20 461x + 30 748 and the correlation coefficient of 0.999 1. The instrument precision was 1.84% and the method precision was 3.8%. Conclusion This method is accurate, rapid and simple; it can be used for determination of candicidin/FR-008 and related components in fermentation broth.

Neuroprotective Effects of Bushen Decoction Against Glutamate-Induced Neurotoxicity in PC12 Cells

HE Wen-bin, ZHANG Jun-long, CHEN Nai-hong, ZHANG ling, ZHU Hai-bo^*

2005, 14(2): 119-124.

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Aim The enhanced effect of Bushen (Kidney-tonifying) decoction (BS) on cultured PC12 cell proliferation and its antagonistic action on neurotoxicity induced by glutamate were investigated by serum pharmacological method of the Chinese material medica (CMM) in vitro. Methods The effect of BS on cultured PC12 cell activity and its antagonistic action on neurotoxicity induced by glutamate was observed by MTT method. Flow cytometry and fluorescence microscope techniques were employed to observe the antagonistic effect of BS on early period apoptosis of PC12 cells induced by glutamate. Results The serum with BS was able to enhance activity of PC12 cells and exert antagonistic effect on glutamate-induced neurotoxicity. Meanwhile, these beneficial effects produced by BS were found to be the strongest in 20% concentration of in serum BS. Moreover, it can inhibit apoptosis of PC12 cells induced by glutamate, which occurs in the early period. Conclusion BS may exert a potential neuroprotective effect.

Anti-CD132 Monoclonal Antibodies Inducing T Cells Apoptosis after Alloantigen Stimulation and Its Possible Clinical Applications

CHEN Bi-cheng, CHANG Sheng, TANG Li, ZHANG Xin, Xiang Fu-li, GUO Hui, CHEN Zhong-hua Klaus^*

2005, 14(2): 125-130.

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Aim To investigate the mechanism of anti-CD132 monoclonal antibodies (mAbs) inhibiting T cells proliferation in vitro, and their potential values for clinical use. Methods BALB/c and C57BL/6 mice splenocytes were harvested for two-ways mixed lymphocyte culture (MLC). Anti-CD132 mAbs (final concentration 100 mg·L^-1) were added in MLC on day 0 (group 1) or day 3 (group 2). Fluorescence activated cell sorting (FACS) was used to measure the proliferation (carboxy-fluorescein dia cetate, succinimidyl ester,CFSE), apoptosis of T cells (PE-CD3, FITC-annexin-v), and cell cycle (propidium iodide stain). The expression of survivin in T cells was detected by immunochemical stai- ning. Results Multi-generation CFSE-labeled splenocytes were found dividing and their fluorescent strength decreased in MLC. There was no noticeable change in fluorescent intensity in group 1 and group 2. On day 3, apoptosis induced by anti-CD132 mAbs was detected in part of T cells, but was not detected in the former two days in group 1. In group 2, the number of cells in M phase (activated T cells) decreased and apoptotic cells increased on day 4. The phenomena were not observed in control group (P<0.01). Expression of survivin in T cells was detected in control group but not in groups 1 and 2. Conclusion Blockade of CD132 signaling pathway inhibits T cell proliferation in vitro by means of inducing activated alloreactive T cell apoptosis but not the resting T cells. Anti-CD132 mAbs may be candidates for clinical applications.

Possible Mechanism of Effects of Etimicin and Gentamicin on Intracellular Calcium Homeostasis

LI Zhong-dong^*, WANG Jian-chang, LI Pei-zhong

2005, 14(2): 131-134.

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Aim Intracellular calcium ([Ca²⁺]i) is mainly regulated by mitochondria and endoplasmic reticula. This study was carried out to ascertain whether the elementary mechanism of the effects of etimicin (EM) and gentamicin (GM) on [Ca²⁺]i is related to their effects on mitochondrion Ca²⁺-uptake and endoplasmic reticulum Ca²⁺-uptake. Methods The effects of GM and EM on [Ca²⁺]i in LLC-PK1 were determined with a fluorescent probe of Fura-2/AM. The effects of EM and GM on mitochondrion Ca²⁺-uptake and endoplasmic reticulum Ca²⁺-uptake were determined by isotope indicator (45Ca²⁺). Results EM and GM at the concentration of 1 mmol·L^-1 had no significant effect on [Ca²⁺]i i (P > 0.05) and at 10 mmol·L^-1 significantly caused [Ca²⁺]i to increase (P <0.01). EM and GM at 1 mmol·L^-1 caused mitochondrion Ca²⁺-uptake to ascend dramatically (P< 0.05) and at 10 mmol·L^-1 caused mitochondrion Ca²⁺-uptake to descend significantly. EM and GM at more than 0.34 mmol·L-1 significantly inhibited endoplasmic reticulum Ca²⁺-uptake (P <0.05 or 0.01). Conclusion No variation of [Ca²⁺]i caused by EM and GM at lower concentrations might relate to the equilibrium of their promotion of mitochondrion Ca²⁺-uptake with their inhibition of endoplasmic reticulum Ca²⁺-uptake. The elevation of [Ca²⁺]i caused by EM and GM at higher concentrations might correlate with their inhibition of mitochondrion Ca²⁺-uptake and endoplasmic reticulum Ca²⁺-uptake.

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