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??OBJECTIVE To evaluate the reference standard of recombinant human interleukin-15 (IL-15) for the effective quality control of IL-15 products according to the requirements of Chinese Pharmacopeia.METHODS The biological activity, concentration, purity, and isoelectric point of IL-15 were tested according to Chinese Pharmacopeia (volume ??, 2010 edition). The primary structure was confirmed by analyzing the N-terminal amino acid sequence and relative molecular mass and peptide mass mapping. RESULTS The measured biological activity of IL-15 was 1.03??10⁷ IU??mg^-1, the content was (0.879??0.065) mg??mL^-1, the purities tested by SDS-PAGE and SEC-HPLC were all 100%, and the isoelectric point was 5.2, which all met the criteria specified in the quality standard. The observed relative molecular mass, 12 900.80, was consistent with theoretical value (12 900.71). Its amino acid sequence was verified with coverage of 100%. The disulfide bonds were identified to to be Cys36-Cys86/Cys43-Cys89, which was in accordance with the published papers. CONCLUSION This reference standard, which is qualified and has correct amino acid sequence, can be used for the routine quality control of IL-15.     

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??OBJECTIVE To study the chemical constituents of the chloroform extract from the aerial parts of Artemisa sacrorum. METHODS The chemical constituents were isolated and purified by silica gel and LH-20 column chromatography and preparation HPLC. Their structures were identified by spectral analysis methods. RESULTS Thirteen compounds were obtained and identified as 5-hydroxyl-7,4??-dimethoxyflavone(1), 4-hydroxylacetophenone(2), 5,4??-dihydroxyl-7,3??-dimethoxyflavone(3), 5,7-dihydroxyl-6,4??-dimethoxyflavone(4), 5,7-dihydroxyl-4??-methoxyflavone(5), 5,4??-dihydroxyl-7-methoxyflavone(6), caffeic acid(7), 8-hydroxyl-6,7-dimethoxycoumarin(8), 3,4-dihydroxylbenzoic acid(9), acetophenone-4-O-??-D-glucoside(10), 6-methoxycoumarin-7-O-??-D-glucoside(11), 6,8-dimethoxycoumarin-7-O-??-D-glucoside(12), and 2-hydroxyl-6-methoxyacetophenone-4-O-??-D-glucoside(13). CONCLUSION Compounds 3, 4, 5, 9, 10 and 12 are isolated from this plant for the first time.
     

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??OBJECTIVE To investigate the chemical constituents of the aerial parts of Ribes diacanthum Pall. METHODS The compounds were isolated and purified by silica gel, Sephadex LH-20 colunm chromatography and HPLC. The structures were elucidated on the basis of spectral data and physiochemical properties. RESULTS Nineteen compounds were isolated from 95% ethanol extracts and identified as quercetin (1), quercetin-3-O-??-D-glucopyranoside (2), quercetin-3-O-??-L-rhamnopyranoside (3), quercetin-3-O-??-D-neohesperoside (4), mearnsetin (5), myricetin-3-O-??-L-rhamnoside (6), myricetin-3-O-??-D-glucopyranoside (7), mearnsetin 3-O-??-D-glucopyranoside (8), mearnsetin 3-O-??-L-rhamnopyranoside (9), kaempferol-3-O-??-D-glucopyranoside (10), kaempferol 3-O-??-D-(2-O-??-L-rhamnopyranosyl) glucopyranoside (11), kaempferol 3-(2??,6??-di-O-??-L-rhamnosyl)-??-D-glucoside (12), 1,2,4-trihydroxybenzene (13), vanillic acid (14), protocatechuic acid (15), 4-hydroxy benzoic acid (16), gallic acid (17), blumenol C glucoside (18), conocarpan (19). CONCLUSION All the compounds are isolated from the title plant and the NMR data for 8 is reported here for the first time.     

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??OBJECTIVE To establish the detection technology of riboflavin polymorphism, and investigate the moisture sorption properties of riboflavin. METHODS The two crystal forms of riboflavin were characterized by different analysis methods, such as powder X-ray diffraction method (p-XRD), differential scanning calorimetry method (DSC) and infrared spectrum method (IR). Dynamic vapor sorption (DVS) analysis was adopted to acquire moisture sorption dynamics of the two crystal forms. The influences of temperature, humidity and light on crystal transformation were explored. RESULTS The crystal forms of two batches of riboflavin samples were identified as anhydrate ?? and monohydrate, respectively. The monohydrate could be obtained by recrystallizing in DMSO. The monohydrate was hygroscopic, the number of crystal water may change at different relative humidities, and the moisture sorption dynamics of the monohydrate was greatly influenced by temperature. The anhydrate ??was almost non-hygroscopic, and temperature hardly affected its moisture sorption dynamics. CONCLUSION Characteristic data for riboflavin polymorphism are obtained, and the results show that anhydrate ?? is more stable than monohydrate. This study indicates that DVS could be used in studies on drug polymorphism.     

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??OBJECTIVE To isolate the chemical constituents and screen out the activity index of Dendrobium officinale, and establish a reliable method for isolation and determination. METHODS A supercritical fluid extraction (SFE) combined with simulated moving bed (SMB) chromatography method was established for the chemical composition extraction of D.officinale. RESULTS Under the optimal SFE condition 46 g crude extract and 30 mg naringenin could be obtained from 1 kg of dried D.officinale. Naringenin and peak X could be fully extracted and isolation by the optimal supercritical fluid simulated moving bed (SF-SMB) operation. The content of naringenin increased from 1 199 mg to 2 400 mg in 1 kg of crude extract and the percentage content of peak X increased from 4.99% to 20.44%. The pure peak X was isolation by SMB and identified as 2,6-dimethoxy-4-(2-propen-1-yl)-phenol using GC-MS, ¹H-NMR, and IR. CONCLUSION The technology of SFE combined with SMB is stable and excellent for extraction and isolation of the active ingredients from D. officinale, and it provides a new idea for the industrial extraction.     

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??OBJECTIVE To isolate and purify 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid from the roots of Actinidia eriantha Benth, establish its determination method, and compare the contents in different extracted parts and samples from different sources. METHODS An HPLC-PDA method was established for the content determination. The contents of 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid in the roots of Actinidia eriantha Benth from five different sources and different extracted parts were determined by standard curve method. RESULTS The content of 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid was higher in the samples from Shouning county of Fuzhou city and Yunhe county of Lishui city while lower in those from Yongjia county of Wenzhou city and Lishui city. And in different extracted parts, the content was the highest in dichloromethane part, lower in ethanol part, and the lowest in methanol part. CONCLUSION The method is stable and accurate with good reproducibility and can be used for the determination of 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid in the roots of Actinidia eriantha Benth. The content of 2??,3??,24-trihydroxy-12-alkene-28-ursolic acid is different in Actinidia eriantha Benth samples from different sources, which is the highest in dichloromethane part.     

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??The rapid development of modern analytical technology provides many methods of detecting drugs and their metabolites. LC-MS/MS technology has become one of the most commonly used methods in drug metabolites analysis with its characteristics of simpleness and high efficiency. In this paper, based on the published articles of in vivo drug metabolites study by LC-MS/MS in recent years, the processes of data acquisition and processing, metabolites detecting and identification and software-assisting were summarized. This article provides references for research on drug metabolism using LC-MS/MS.     

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??OBJECTIVE To study the chemical constituents from the leaves of Jiangxi genuine medicinal material Chimonanthus nitens Oliv. METHODS The compounds were isolated and purified by silica gel column chromatography, Sephadex LH-20, ODS column chromatography, semi-preparative HPLC, and so on. Their structures were elucidated on the basis of physiochemical properties and spectral data. RESULTS Ten compounds were isolated and elucidated as uracil (1), 6,7-dimethoxycoumarin (2), 6,7,8-trimethoxycoumarin (3), p-hydroxybenzoic acid ethyl ester (4), 1??1 mixture of two diastereomers identical with (3RS,6RS)-2, 6-dimethyl-octa-1,7-dien-3,6-diol (5 and 6), kaempferol (7), isofraxidin (8), scopoletin (9), and loliolide (10). CONCLUSION Compounds 1, 3-6 and 10 are isolated from this plant for the first time and compounds 1, 4-6 are isolated from this genus for the first time.     

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??OBJECTIVE To study the chemical constituents of Swertia binchuanensis. METHODS The constituents were isolated and purified by column chromatography of silica gel. Their structures were identified on the basis of spectral analysis and chemical properties. RESULTS Five compounds are isolated and identified as 7-O-????-L-rhamnopyranosyl-(1??2)-??-D-xylopyranosyl??-1,8-dihydroxy-3-methoxyxanthone(1), 3-O-??-D-glucopyranosyl-1,8-dihydroxyl-5-methoxyxanthone(2), 7-O-??-D- glucopyranosyl-1,8-dihydroxyl-3-methoxyxanthone(3), amarogentin(4), and amaroswerin(5). CONCLUSION All of the compounds were isolated from S.binchuanensis for the first time.     

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??OBJECTIVE To identify the metabolites of Pulsatilla oleanolic acid 3-??-L-rhamnopyranosyl (1??2) -??-L-pyran arabinoside (B7) in rat plasma, bile, urine, feces, and in vitro liver microsomes incubation system. METHODS An ultra-high performance liquid chromatography combined with electrospray ionization quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed and successfully applied to the study on the metabolites of B7 in rat plasma, bile, urine, feces, and in vitro liver microsomes incubation system after oral administration. RESULTS A total of 23 metabolites were identified, of which 12 metabolites were present in rat liver microsomes incubation system. CONCLUSION Deglycosylation, demethylation glucuronidation, and hydroxylation are the major metabolic transformation forms of B7 in rats in vivo and in vitro.     

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??OBJECTIVE To evaluate the effects of drug concentration and perfusion rate on the recoveries of self-made linear microdialysis probes for further ocular pharmacokinetic study. METHODS Brimonidine tartrate was selected as the model drug. The in vitro recovery was determined using positive dialysis and retrodialysis at different perfusion rates and drug concentrations. And the in vivo recovery was determined using retrodialysis method. RESULTS The microdialysis recoveries of brimonidine tartrate were inversely proportional to perfusion rate,while independent of drug concentration. The positive dialysis and retrodialysis recoveries in vitro were different at 1.0 ??L??min^-1, but no significant difference at 2.0 and 3.0 ??L??min^-1. The in vitro recoveries were greater than those in vivo. CONCLUSION The self-made microdialysis probe has stable recovery and can be used in ocular pharmacokinetic study of brimonidine tartrate.     

替格瑞洛的体外代谢及其与他汀类药物的相互作用研究

目的 考察替格瑞洛在大鼠肝微粒体的酶动力学及其与CYP3A的底物药物辛伐他汀、洛伐他汀及阿托伐他汀的相互作用,以期为临床上替格瑞洛与他汀类药物的合理使用提供科学依据。方法 将替格瑞洛与大鼠肝微粒体进行体外共孵育,孵育一定时间后用含有内标(地西泮,10 ng·mL^-1)的甲醇终止反应,并沉淀蛋白,14 000 r·min^-1离心10 min后取上清液进行分析,采用 LC-MS/MS测定微粒体酶孵育体系中活性代谢产物AR-C124910XX的浓度,使用 Prism 5软件计算主要的酶促动力学参数K_m,V_max与CL_int。在得到 K_m后,反应体系中底物替格瑞洛的浓度选择为1/3K_m~3K_m内的3个浓度,辛伐他汀、洛伐他汀及阿托伐他汀的浓度范围为1~100 μmol·L^-1,通过体外大鼠肝微粒体代谢实验研究其与替格瑞洛代谢性相互作用。使用 SigmaPlot 12.3 软件酶动力学模块,根据 Dixon公式计算各他汀对替格瑞洛代谢的可逆性抑制常数 K_i,并根据标准差值选择最符合的模型。结果 替格瑞洛在大鼠肝微粒体的代谢符合米氏反应动力学,其转化生成AR-C124910XX的K_m值为32.2 μmol·L^-1,V_max为149.0 pmol·min^-1·mg(pro)^-1。表观清除率CL_int为4.63 nL·min^-1·mg(protein)^-1;辛伐他汀显著抑制替格瑞洛活性代谢产物的生成,其抑制符合混合抑制模型。抑制常数K_i为0.58 μmol·L^-1,α值为7.5,β值为0.56。洛伐他汀及阿托伐他汀对替格瑞洛代谢呈现出中等强度的抑制作用,其抑制亦符合混合抑制模型,抑制常数K_i分别为2.9和7.5 μmol·L^-1,α 值分别为3和1.7,β值为0.34和0.29。 结论 替格瑞洛在大鼠肝微粒体的代谢符合米氏反应酶动力学;辛伐他汀对替格瑞洛有显著程度的代谢性抑制作用,洛伐他汀和阿托伐他汀对替格瑞洛有中等程度的抑制作用,临床上替格瑞洛与他汀类药物合用时可能需要考虑其与辛伐他汀的相互作用。     

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??OBJECTIVE To investigage the effects of celastrol-triggered HeLa cells autophagy and the molecular mechanisms in vitro and in vivo. METHODS The antiproliferative effect of celastrol was detected using MTT assay. Apoptotic rate and cell cycle were evaluated using flow cytometric analysis. Autophagy was detected using fluorescence microscope. Protein expression was evaluated using Western blotting. Tumor growth was evaluated by subcutaneous xenograft model in vivo. RESULTS Celastrol inhibited HeLa cells proliferation and induced HeLa cells autophagy and cell cycle arrest at G₀/G₁ phase, but not induced HeLa cell apoptosis in vitro. The protein expression of Beclin 1 was up-regulated and the conversion from LC3 ?? to LC3 ?? was increase in HeLa cells in vitro after treatment with celastrol. Moreover, celastrol promoted the protein expression of PTEN??p-ERK1/2??p-MEK1/2 and inhibited the phosphorylated of Akt, p70S6K and mTOR in HeLa cells. After pretreatment with 3-methyladenine (5 mmol??L^-1), the antiproliferative and induced-autophagy effects of celastrol were reversed. Furthermore, celastrol inhibited tumor growth and the protein expression of p-Akt and p-mTOR, but up-regulated the protein expression of LC3 ?? and Beclin 1 in vivo. CONCLUSION Antitumor effect of celastrol dependent on cells autophagy in HeLa cells via inhibition of Akt/mTOR signaling pathway in vitro and in vivo.     

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??OBJECTIVE To prepare the tulobuterol crystal reservoir patch, and to evaluate morphology, stability and crystallization factors of the crystal in the patch, adhesive force, dissolution, transdermal properties in vitro and the pharmacokinetics in rabbits. METHODS The transdermal patch was prepared on the basis of drug recrystallization and characterized by morphology, stability and crystallization factors using microscope and adhesive force using initial adhesion tester, adhesion tester and peel tester. The dissolution and transdermal properties were evaluated by using the dissolution tester and transdermal tester. In addition, pharmacokinetics was studied using New Zealand rabbits as experimental animals. RESULTS The drug crystals were evenly distributed in the form of filaments, which had average width of (4.4??1.8)??m and kept stable at 2-40 ??. The crystallization in patches is affected by tulobuterol supersaturation and temperature. The adhesive force of patch was suitable and its dissolution matched standard which can be fitted by the Higuchi equation. In the diffusion cell in vitro, the drug penetrated through the skin in a Zero-order kinetic equation, and the cumulative penetration percentage and skin retention concentration were 92.04% and 10.36 ??g??cm^-2 with in 24 h. The pharmaceutic parameters showed that the tulobuterol blood concentration can be maintained within 24 h, whose t_max and ??_max were (6.67??3.06)h and (3.08??1.32) ng??mL^-1, respectively. CONCLUSION The tulobuterol crystal reservoir patch can be established by control of recrystallization conditions. The patch has good adhesive properties and sustained release characteristics in vitro and in vivo, which has the practical significance for further study.     

HPLC-MS/MS测定大鼠血浆中刺五加、贯叶连翘提取物主要活性成分浓度及其药动学研究

目的 建立HPLC-MS/MS测定刺五加提取物、贯叶连翘提取物主要活性成分在大鼠血浆中药物浓度的方法,应用于刺五加提取物、贯叶连翘提取物及其联用时主要活性成分在大鼠体内的药动学研究,并使用DAS 3.2.8软件对主要活性成分的药动学参数进行非房室模型拟合。方法 以Welch Ultimate XB-C₁₈(2.1 mm ×100 mm,4.6 μm)为色谱柱,乙腈(含0.1%甲酸)-0.1%甲酸水溶液(含5 mmol·L^-1乙酸铵)为流动相,以300 μL·min^-1的流速进行梯度洗脱,采用电喷雾离子源进行正负离子同时扫描,多反应监测模式进行监测。结果 所建立的方法精密度、准确度良好,提取回收率基本符合生物样品分析方法要求。结论 药动学结果表明,相比于单独给药,联合用药后刺五加苷E t_max缩短,金丝桃素ρ_max升高、t_1/2延长。该药动学结果表明,刺五加提取物、贯叶连翘提取物联用时,很可能通过增加金丝桃素血药浓度、缩短刺五加苷E的达峰时间,从而在体内产生协同抗抑郁作用。     

巨噬细胞膜仿生白蛋白纳米体系的构建及其胶质瘤靶向递药性能评价

目的 构建包载WEE1激酶抑制剂adavosertib的巨噬细胞膜仿生白蛋白纳米粒(MM-BSA/Ada),体外评估其作为胶质瘤靶向递药体系的可行性。方法 制备MM-BSA/Ada并筛选最佳膜-核比和最佳药-载比,检测其载体安全性和对C6胶质瘤细胞抗增殖活性,考察其体外细胞摄取、跨血脑屏障转运和跨膜后摄取的能力。结果 MM-BSA/Ada具有良好的稳定性,CCK-8结果初步显示,未载药纳米粒在体外细胞实验中对脑血管内皮细胞呈低毒性;与未包膜纳米粒和游离药物相比,MM-BSA/Ada给药后的体外抗胶质瘤细胞增殖活性(P＜0.001)、胶质瘤细胞摄取量(P＜0.001)、体外血脑屏障透过量(P＜0.01)及跨膜后摄取量(P＜0.001)均显著提高。结论 MM-BSA/Ada有较好的胶质瘤靶向递药性能,有望为胶质瘤提供新的放射增敏策略。     

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??OBJECTIVE To evaluate the anti-hepatitis B virus (HBV) activity of herpetrione nanosuspension (PEDX-NS) both in vitro and in vivo. METHODS HepG2 2.2.15 cells and duck hepatitis B virus (DHBV) infected ducks as in vitro and in vivo models were used to compare the anti-HBV activity of PEDX-NS and PEDX coarse suspension (PEDX-CS). RESULTS In the HepG2 2.2.15 cell, PEDX-NS effectively suppressed the secretion of the HBV antigens (HBsAg and HBeAg) in a dose-dependent manner with significant difference from PEDX-CS (P<0.05 or P<0.01). In the in vivo evaluation, PEDX-NS with high dose (100 mg??kg^-1) and middle dose (60 mg??kg^-1) significantly reduced the serum HBV DNA level (P<0.05 or P<0.01) and the effect was better than that of PEDX-CS (P<0.05 or P<0.01). CONCLUSION The result revealed that PEDX-NS exhibits anti-HBV activity both in vitro and in vivo and its effect was superior to that of PEDX-CS. The mechanism is probably that the small particle size of PEDX-NS provides a large specific surface area that resulted in better absorption in vivo, thus enhancing its anti-HBV activity.
     

国产非那雄胺片与原研制剂体外溶出一致性评价及采用计算机模拟技术建立体内外相关性模型研究

目的 通过比较国产非那雄胺片与原研制剂的体外溶出行为,评价仿制药与参比制剂的质量一致性,利用计算机模拟技术分析体内外相关性。方法 参照《中国药典》2015年版方法(2020年版《中国药典》该项目没有变化),分别考察国产制剂与原研制剂在4种不同溶出介质(pH 1.2盐酸溶液、pH 4.5醋酸盐溶液、pH 6.8磷酸盐溶液、水)中的体外溶出行为。同时借助GastroPlus^TM软件结合体外溶出试验结果,建立非那雄胺片体内外相关性模型。结果 在选定条件下,国内15家制药公司中有3家公司产品在4种溶出介质中的溶出曲线均与原研制剂相似。软件分析结果提示,体外溶出曲线与软件模拟的体内行为不相似。结论 大部分非那雄胺片仿制制剂在体外的溶出曲线与原研制剂存在一定的差异,国产非那雄胺片工艺水平及处方有待提高,反映体内释放行为的生物体相关溶出条件有待进一步研究。     

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??OBJECTIVE To study the chemical constituents of Noni enzyme (Morinda citrifolia L.) and their antitumor activities. METHODS Compounds were isolated by various chromatographic techniques, including silica gel, TLC, sephadex LH-20, and semi-preparative HPLC, and their structures were identified by their physicochemical properties and ¹H-NMR and ¹³C-NMR data. The in vitro antitumor activities of the isolated compounds were studied by MTT method. RESULTS Sixteen compounds were isolated from Noni enzyme. They were xylogranatinin(1), pelargonic acid(2), 1,5,15-tri-O-methylmorindol(3), sesquipinsapol B(4), (+)-syringaresinol(5), pinonesinol(6), 3-methylhexahydropyrrolopyrazine-1,4-dione(7), (2S)-3??-hydroxybutan-2-yl-2-hydroxypropanoate (8), 3-(sec-butyl)-6-methylpiperazine-2,5-dione(9), cyclo-(L-Pro-L-Leu)(10), gentisic acid(11), vomifoliol(12), scopoletin(13), 3-(2-hydroxy-4,5-dimethoxyphenyl) propanoic acid(14), medioresinol(15), hydroxychavicol(16). CONCLUSION Compounds 1-10, 12 and 14-16 are isolated from Noni enzyme for the first time. Compound 10 displays the stronger cytotoxicity against HepG2 and HeLa cells with an IC₅₀ value of 23.73, 16.55 ??g??mL^-1. Compound 5 had a certain inhibitory activity against HeLa cells with an IC₅₀ value of 47.12 ??g??mL^-1.