In vitro antioxidant capacities and fatty acid compositions of three Centaurea species collected from Central Anatolia region of Turkey

Antioxidant capacities of methanolic extract and fatty acid composition of three Centaurea species were investigated. The antioxidant capacity of the extracts was evaluated by different assays, including total phenolic content, phosphomolybdenum assay, free radical scavenging activity (DPPH assay), β-carotene/linoleic acid bleaching assay, iron (III) and cupric reduction assay. The findings showed that the methanolic extract of Centaurea pulchella has the strongest antioxidant capacity compared to other two Centaurea species. The order of the antioxidant properties of Centaurea species were C. pulchella > C. patula > C. tchihatcheffii. Thirty fatty acids were identified in the oils of three Centaurea species. The major fatty acids of these species were found to be linoleic acid from C. pulchella and C. tchihatcheffii, and α-linolenic acid from C. patula. The study concluded that the Centaurea species can be used as a source of natural antioxidants and essential fatty acids.     

密蒙花与其替代品结香总提物体外抗氧化活性作用的比较研究

目的通过体外抗氧化活性检测,比较密蒙花与其替代品结香总提物体外抗氧化能力的差异。方法采用福林酚法测定密蒙花、结香总提物中总酚,并采用DPPH自由基清除能力法、ABTS清除能力法、Fe~(3+)还原能力法和Fe~(2+)螯合能力法分别测定两者体外抗氧化能力,选取维生素C和EDTA-Na_2作为阳性对照。结果密蒙花中总酚为结香的3~5倍;除亚铁离子螯合试验外,密蒙花的抗氧化作用均明显优于结香,这与总酚的量呈正相关性。结论两者总提取物中总酚和抗氧化能力差异显著,密蒙花明显优于结香。     

In vitro and in vivo antioxidant properties of different fractions of Moringa oleifera leaves

The antioxidant potency of different fractions of Moringa oleifera leaves were investigated by employing various established in vitro systems, such as β-Carotene bleaching, reducing power, DPPH/superoxide/hydroxyl radical scavenging, ferrous ion chelation and lipid peroxidation. On the basis of in vitro antioxidant properties polyphenolic fraction of M. oleifera leaves (MOEF) was chosen as the potent fraction and used for the DNA nicking and in vivo antioxidant properties. MOEF shows concentration dependent protection of oxidative DNA damage induced by HO and also found to inhibit the toxicity produced by CCl₄ administration as seen from the decreased lipid peroxides (LPO) and increased glutathione (GSH) levels. Among the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) levels were restored to almost normal levels compared to CCl₄ intoxicated rats. The HPLC analysis indicated the presence of phenolic acids (gallic, chlorogenic, ellagic and ferulic acid) and flavonoids (kaempferol, quercetin and rutin). Thus, it may be concluded that the MOEF possess high phenolic content and potent antioxidant properties, which may be mediated through direct trapping of the free radicals and also through metal chelation.     

HPLC-ELSD同时测定铁破锣中3种三萜皂苷含量及体外抗氧化性研究

目的 建立同时测定铁破锣中3种三萜皂苷BeesiosideⅠ、Ⅱ、Ⅲ含量的方法,并研究铁破锣中三萜皂苷的体外抗氧化性。方法 运用反相高效液相色谱-蒸发光散射检测法（HPLC-ELSD）对铁破锣中三萜皂苷进行含量测定,并对所用含量测定方法进行方法学考察;同时,对铁破锣中三萜皂苷清除自由基的能力和总的抗氧化能力进行体外试验。结果 HPLC-ELSD方法可同时测定BeesiosideⅠ、Ⅱ、Ⅲ的含量,且该方法线性关系良好、精密度、重复性、稳定性均好,回收率高;铁破锣中三萜皂苷能够有效清除ABTS⁺、DPPH·和羟基自由基,并且具有很好的抗氧化能力。结论 HPLC-ELSD方法简便、快速、准确、重现性好,可作为铁破锣中三萜皂苷（BeesiosideⅠ、Ⅱ、Ⅲ）的含量测定及质量控制标准方法,体外试验表明铁破锣中三萜皂苷具有良好的自由基清除和抗氧化能力。     

泻白散及其主药体外抗氧化活性研究

目的 建立泻白散和方中3味主药甘草、地骨皮、桑白皮的体外抗氧化活性测定方法,并对31批药材和10批泻白散煎液的抗氧化活性进行测定。方法 采用紫外可见分光光度法检测一定浓度的药材提取液引起DPPH溶液吸光度（A）值降低,考察波长为517 nm,分别探索3味药材抗氧化活性成分的提取条件;并进行不同溶剂的吸收考察、专属性考察、DPPH线性考察、药材提取液线性考察、精密度试验、重复性试验、耐用性考察等方法学验证;以清除DPPH自由基的半抑制浓度（IC₅₀）作为评价指标,对泻白散和方中3味药材的体外抗氧化活性进行考察。结果 地骨皮、甘草、桑白皮和泻白散提取液的IC₅₀均值为0.31、1.24、1.49和0.91 g/L,泻白散提取工艺对方中药物抗氧化活性的保留均值为56%。结论 建立的抗氧化活性测定方法可用于泻白散及方中主药的抗氧化活性测定,为多维度评价中药和中药材质量提供新思路。     

槲皮素纳米混悬剂的制备及其性能研究

目的制备高载药量、小粒径的槲皮素纳米混悬剂,并进行性质考察。方法采用纳米沉淀–高压均质法制备,以粒径分布、药载比、稳定性为指标对处方和工艺进行优化,并对最优处方进行体外性质考察。结果以超声注入联合高压均质法制备,最佳处方为TPGS为稳定剂、DMF为有机试剂,药载比为5∶1;最佳工艺条件为低温10℃超声、200 MPa、循环5次。制备得到的槲皮素纳米混悬剂粒径为(173.21±0.90)nm,电位为(-19.02±0.15)m V,载药量为(80.40±1.44)%,包封率为(96.41±1.72)%。药物在纳米粒中以结晶状态存在,能够体外缓释36 h。结论利用TPGS可制备高载药量、小粒径的槲皮素纳米混悬剂,其体外具有明显缓释作用,解决了槲皮素水溶性差的难题,具有较好的应用前景。     

Studies on the antioxidant activity of essential oil and different solvent extracts of Vitex agnus castus L. fruits from Turkey

This study is designed to examine the chemical composition and antioxidant activity of the essential oil and different solvent extracts of Vitex agnus castus. GC and GC–MS analysis was resulted in the detection of 27 components, representing 94.5% of the oil. Major components of the oil were 1,8-cineole (24.98%), sabinene (13.45%), α-pinene (10.60%), α-terpinyl acetate (6.66%), and (Z)-β-farnesene (5.40%). Antioxidant activities of the samples were determined by three different test systems, DPPH, β-carotene/linoleic acid and reducing power assays. In all systems, water extract exhibited excellent activity potential than those of other extracts (hexane, dichloromethane, ethyl acetate and methanol) and the oil. As expected, amount of total phenolics was very high in this extract (112.46 ± 1.22 μg GAEs/mg extract). Dichloromethane extract has been found to be rich in flavonoids. A positive correlation was observed between the antioxidant activity potential and total phenolic and flavonoid levels of the extracts.     

金花茶花的指纹图谱建立、成分鉴定及体外抗氧化活性研究

目的 建立金花茶花的高效液相（HPLC）指纹图谱,对其化学成分进行鉴定,并考察其体外抗氧化能力。方法 70%乙醇溶液回流法提取10批次金花茶花,采用Agilent Eclipse Plus C₁₈（250 mm×4.6 mm,5 μm）色谱柱,梯度洗脱,建立金花茶花指纹图谱,应用HPLC-MS法分析其化学成分,并用1,1-二苯基-2-苦基肼自由基（DPPH）和2,2-联氮-二（3-乙基-苯并噻唑-6-磺酸）二铵盐（ABTS）法评价金花茶花的抗氧化能力。结果 建立金花茶花共有HPLC色谱图,标定共有峰11个,经过中药色谱指纹图谱相似度评价软件（2012版）计算,相似度均在0.98以上。HPLC-MS分析结果显示,金花茶花中成分多为黄酮类化合物。抗氧化能力检测结果显示,金花茶花对DPPH自由基和ABTS自由基均具有较好的清除能力,并呈浓度相关性。结论 建立了金花茶花药材的指纹图谱,其含有丰富的黄酮类化合物,具有良好的体外抗氧化作用。     

Potential in vitro antioxidant and protective effects of Gymnema montanum H. on alloxan-induced oxidative damage in pancreatic β-cells, HIT-T15

The present study describes the antioxidant activities of ethanol extract from Gymnema montanum (GLEt) which is an endemic plant of India. Antioxidant activity of the GLEt was studied in vitro based on scavenging of hydroxyl radicals, superoxide anions, nitric oxide, hydrogen peroxide, peroxynitrite, reducing power and inhibition of lipid peroxidation estimated in terms of thiobarbituric acid reactive substances (TBARS). Further, we examined its protective effect against alloxan-induced oxidative stress in pancreatic β-cells, HIT-T15 by measuring the free radical generation, malonaldehyde formation and antioxidant levels such as CAT, GPx and GSH. Results showed that G. montanum leaves exhibited significant antioxidant activities measured by various in vitro model systems. The HIT-T15 cell line studies showed the tendency of GLEt to increase antioxidant levels meanwhile decrease the free radical formation and inhibit the lipid peroxidation. The antioxidant activity was found to be well correlated with the phenolic phytochemicals present in the extract. GC–MS analyses revealed the presence of few phenolic compounds in the extract. As this plant has already been demonstrated for a variety of medicinal properties from our laboratory, results of this study suggest that G. montanum is an interesting source for antioxidant compounds and useful for various therapeutic applications.     

In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert.

The aim of this study was to assess the in vitro potential of ethanolic leaf extract of Stevia rebaudiana as a natural antioxidant. The DPPH activity of the extract (20, 40, 50, 100 and 200 μg/ml) was increased in a dose dependent manner, which was found in the range of 36.93–68.76% as compared to ascorbic acid 64.26–82.58%. The IC₅₀ values of ethanolic extract and ascorbic acid in DPPH radical scavenging assay were obtained to be 93.46 and 26.75 μg/ml, respectively. The ethanolic extract was also found to scavenge the superoxide generated by EDTA/NBT system. Measurement of total phenolic content of the ethanolic extract of S. rebaudiana was achieved using Folin–Ciocalteau reagent containing 61.50 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The ethanolic extract also inhibited the hydroxyl radical, nitric oxide, superoxide anions with IC₅₀ values of 93.46, 132.05 and 81.08 μg/ml, respectively. However, the IC₅₀ values for the standard ascorbic acid were noted to be 26.75, 66.01 and 71.41 μg/ml respectively. The results obtained in this study clearly indicate that S. rebaudiana has a significant potential to use as a natural antioxidant agent.     

Antrodia camphorata inhibits proliferation of human breast cancer cells in vitro and in vivo

Antrodia camphorata (A. camphorata) has been shown to induce apoptosis in cultured human breast cancer cells (MDA-MB-231). In this study, we report the effectiveness of the fermented culture broth of A. camphorata in terms of tumor regression as determined using both in vitro cell culture and in vivo athymic nude mice models of breast cancer. We found that the A. camphorata treatment decreased the proliferation of MDA-MB-231 cells by arresting progression through the G1 phase of the cell cycle. This cell cycle blockade was associated with reductions in cyclin D1, cyclin E, CDK4, cyclin A, and proliferating cell nuclear antigen (PCNA), and increased CDK inhibitor p27/KIP and p21/WAF1 in a dose and time-dependent manner. Furthermore, the A. camphorata treatment was effective in delaying tumor incidence in the nude mice inoculated with MDA-MB-231 cells as well as reducing the tumor burden when compared to controls. A. camphorata treatment also inhibited proliferation (cyclin D1 and PCNA) and induced apoptosis (Bcl-2 and TUNEL) when the tumor tissue sections were examined histologically and immunohistochemically. These results suggest that the A. camphorata treatment induced cell cycle arrest and apoptosis of human breast cancer cells both in vitro and in vivo.     

不同产地五倍子体外抗菌作用研究

目的 考察五倍子不同产地（河北曲阳、浙江嘉兴、湖南桑植、陕西西乡、云南凤县）的体外抗菌作用。方法 五倍子醇提浸膏用倍半稀释法稀释成不同浓度药液,采用管碟法测定各产地五倍子的最小抑菌浓度（MIC）。结果 河北、浙江、湖南、陕西、云南所产五倍子醇提浸膏对金黄色葡萄球菌的MIC依次为6.25、1.562 5、1.562 5、3.125、0.781 25 mg/L;对大肠杆菌的MIC依次为3.125、12.5、6.25、6.25、3.125 mg/L;对白色念珠菌的MIC依次为6.25、1.562 5、3.125、1.562 5、3.125 mg/L;对绿脓杆菌的MIC依次为12.5、6.25、6.25、6.25、3.125 mg/L。结论 河北、浙江、陕西、云南、湖南所产五倍子均有抑菌作用,其中云南产五倍子醇提物对金黄色葡萄球菌、大肠杆菌、绿脓杆菌抑菌作用最强,陕西、浙江产五倍子醇提物对白色念球菌的抑菌作用强。     

注射用丹参多酚酸体外抗氧化研究

目的 建立注射用丹参多酚酸（SAFI）体外抗氧化活性测定方法,并对29批样品体进行测定。方法 一定浓度的SAFI溶液与等体积的DPPH溶液混合均匀并放置一定时间后,采用紫外可见分光光度法,测定其吸光度（A）值相对于DPPH控制液（由DPPH溶液与等体积溶剂混合）的变化情况（DPPH自由基清除情况）,考察波长为517 nm,并进行空白吸收考察、专属性考察、反应平衡时间的确定、DPPH线性考察、YQFM线性考察、精密度试验、重复性试验等方法学验证,以V_C作为阳性对照,以SAFI清除DPPH自由基的半抑制浓度（IC₅₀）和相对抗氧化活性作为衡量其抗氧化活性指标。结果 方法学验证结果表明,建立的体外抗氧化研究方法符合要求;29批样品IC₅₀均值为25.26 μg/mL,相对于Vc抗氧化活性为61.8%。结论 SAFI具有较强的抗氧化活性,所建方法适用于SAFI体外抗氧化活性测定,为多维度评价中药注射剂质量提出新思路。     

Leaves of Cassia tora as a novel cancer therapeutic – An in vitro study

Cassia tora Linn (Leguminacea) is a medicinal plant traditionally used as laxative, for the treatment of leprosy and various skin disorders. Preliminary phytochemical analysis of leaf showed the presence of polyphenols (3.7 mg gallic acid equivalent per gram dried leaves). The presence of phenolic compound prompted us to evaluate its antioxidant and antiproliferative potential. In the present study C. tora methanolic leaf extract (CTME) was evaluated for its nitric oxide scavenging activity and reducing power assays using Rutin and BHT as standards. The extract was studied for its lipid peroxidation inhibition assay using rat liver and brain. In all assays, a correlation existed between concentration of extract and percentage inhibition of free radical, reducing power and inhibition of lipid peroxidation. The antiproliferative activity of CTME with Cisplatin, anticancer drug was studied using human cervical cancer cells (HeLa). Proliferation of HeLa was measured by MTT assay, cell DNA content by modified diphenylamine method and apoptosis by Caspase 3 activity. The plant extract induced a marked concentration dependent inhibition on proliferation, reduced DNA content and apoptosis in HeLa. These results clearly indicate that C. tora is effective against free radical mediated diseases.     

去甲泽拉木醛的体外抗真菌作用研究

目的 研究去甲泽拉木醛的体外抗真菌作用。方法 采用微量液基稀释法测定去甲泽拉木醛与氟康唑单独应用于23株真菌的最低抑菌浓度（MIC）,以棋盘式微量液基稀释法测定两药联合抗耐药白念珠菌的协同指数（FICI）,判断两药联合抗菌效果;并通过纸片扩散实验直观验证两药联合的协同作用。最后通过CCK-8法测定去甲泽拉木醛的细胞毒性。结果 去甲泽拉木醛单用时呈现广谱的抗真菌作用,MIC范围为4~32 g/L。两药联用时,可将氟康唑的有效浓度从大于64 g/L降至0.25 g/L,FICI值介于0.129~0.254之间,两药表现出协同抗耐药白念珠菌作用。CCK-8结果显示,去甲泽拉木醛在高于MIC值4倍浓度下才展示出细胞毒性。结论 去甲泽拉木醛表现出较好的抗真菌作用,与氟康唑联合时有很好的协同效果,且毒性较低。     

注射用益气复脉（冻干）体外抗氧化研究

目的 建立注射用益气复脉（冻干）（YQFM）体外抗氧化活性测定方法,并对30批样品体外抗氧化活性进行测定。方法 一定浓度的YQFM溶液与等体积的DPPH溶液混合均匀并放置一定时间后,采用紫外可见分光光度法,测定其吸收值相对于DPPH控制液（由DPPH溶液与等体积溶剂混合）的变化情况（DPPH自由基清除情况）,考察波长为517 nm,并进行空白吸收考察、专属性考察、反应平衡时间的确定、DPPH线性考察、YQFM线性考察、精密度试验、重复性试验、耐用性试验等方法学验证;以YQFM随行样品作为对照,以YQFM清除DPPH自由基的半数抑制浓度（IC₅₀）和相对抗氧化强度作为衡量其抗氧化活性的指标。结果 方法学验证结果表明,建立的体外抗氧化研究方法符合要求;30批YQFM样品IC₅₀均值为4.07 mg/mL。结论 建立的方法适用于YQFM体外抗氧化活性测定,YQFM具有较强的抗氧化活性。     

GLP体系下体外替代技术的质量保证要点

随着动物福利指导原则的提出, 大量替代方法逐步应用于药物非临床安全性评价领域。然而, 我国在药物非临床研究质量管理规范(GLP)中, 体外替代技术的发展仍处于起步阶段。从人员、实验设施、标准操作规程、质量保证体系、电子数据管理等方面, 阐述在GLP体系下如何规范替代方法, 严格控制可能影响实验结果准确性的各种主客观因素, 降低实验误差, 确保实验结果的真实性, 以推动替代技术在药物非临床安全性评价中的应用。     

HPLC法检测瑞巴派特片体外溶出度

目的 建立瑞巴派特片体外溶出度HPLC检测方法及方法学验证。方法 溶出度试验采用桨法,转速50 r/min;以水、pH 1.2盐酸（含氯化钠）、pH6.0枸橼酸-磷酸二氢钠缓冲液和pH6.8磷酸缓冲液为溶出介质,HPLC法测定溶出量。结果 瑞巴派特在5.533~221.334 μg/mL内线性关系良好（r=0.999 9）,专属性、精密度、准确度、溶液稳定性及耐用性等均良好。结论 本方法简单方便,提高了瑞巴派特溶出度测定的专属性和准确性,可用于该制剂的质量控制。     

京尼平苷体内外微透析回收率的研究

目的 探究京尼平苷在自制线性微透析探针上的体内外回收率与灌流速度、药物浓度、透析膜长之间的关系。方法 采用HPLC测定微透析样品浓度,考察不同灌流速度、不同药物浓度和不同透析膜长对体外正向和体外反向回收率、体内反向回收率的影响。结果 自制探针性质稳定;探针回收率与京尼平苷的药物浓度无关,与灌流速度成反比,随透析膜长增加而增加;体外的正向与反向探针回收率有显著性差异（P<0.01）,而体内反向回收率低于体外反向回收率,与体外正向回收率无显著性差异;新探针体内回收率大于使用过1次的探针体内回收率（P<0.05）。结论 在京尼平苷的皮肤药动学研究中,宜采用体内反向回收率或体外正向回收率作为药物的探针回收率来校正。     

Water extracts of Brassica oleracea var. costata potentiate paraquat toxicity to rat hepatocytes in vitro

Tronchuda cabbage extracts have been proven to have antioxidant potential against various oxidative species in cell free systems, though its antioxidant potential in cellular models remained to be demonstrated. In the present study, we used primary cultures of rat hepatocytes for the cellular assay system and paraquat PQ exposure as a pro-oxidant model agent, to test whether tronchuda cabbage hydrolysed water extracts provide protective or aggravating effects towards PQ-induced oxidative stress and cell death. For this purpose cellular parameters related to oxidative stress were measured, namely the generation of superoxide anion, glutathione oxidation, lipid peroxidation, intracellular ATP levels, activation of nuclear factor-κB (NF-κB), activity of antioxidant enzymes, and cell death.The obtained results demonstrated that the studied hydrolysed water extracts of tronchuda cabbage, especially rich in kaempferol (84%) and other polyphenols, namely hydroxycinnamic acids and traces of quercetin, can potentiate the toxicity of PQ in primary cultures of rat hepatocytes. These results highlight that prospective antioxidant effects of plant extracts, observed in vitro, using non-cellular systems, are not always confirmed in cellular models, in which the concentrations required to scavenge pro-oxidant species may be highly detrimental to the cells.