Hot water-extracted Lycium barbarum and Rehmannia glutinosa inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells

AIM： To investigate the effect of hot water-extracted Lydurn barbarum （LBE） and Rehrnannia glutinosa （RGE） on cell proliferation and apoptosis in rat and/or human hepatocellular carcinoma （HCC） cells. METHODS： Rat （H-4-Ⅱ-E） and human HCC （HA22T/ VGH） cell lines were incubated with various concentrations （0-10 g/L） of hot water-extracted LBE and RGE. After 6-24 h incubation, cell proliferation （n = 6） was measured by a colorimetric method. The apoptotic cells （n = 6） were detected by flow cytometry. The expression of p53 protein （n = 3） was determined by SDS-PAGE and Western blotting. RESULTS： Crude LBE （2-5 g/L） and RGE （2-10 g/L） dose-dependently inhibited proliferation of H-4-Ⅱ-E cells by 11% （P 〈 0.05） to 85% （P 〈 0.01） after 6-24 h treatment. Crude LBE at a dose of 5 g/L suppressed cell proliferation of H-4-Ⅱ-E cells more effectively than crude RGE after 6-24 h incubation （P 〈 0.01）. Crude LBE （2-10 g/L） and RGE （2-5 g/L） also dose-dependently inhibited proliferation of HA22T/VGH cells by 14%-43% （P 〈 0.01） after 24 h. Crude LBE at a dose of 10 g/L inhibited the proliferation of HA22T/VGH cells more effectively than crude RGE （56.8% ＋ 1.6% vs 70.3% ＋ 3.1% of control, P = 0.0003 〈 0.01）. The apoptotic cells significantly increased in H-4-Ⅱ-E cells after 24 h treatment with higher doses of crude LBE （2-5 g/L） and RGE （5-10 g/L） （P 〈 0.01）. The expression of p53 protein in H-4-Ⅱ-E cells was 119% and 143% of the control group compared with the LBE-treated （2, 5 g/L） groups, and 110% and 132% of the control group compared with the RGE -treated （5, 10 g/L） groups after 24 h. CONCLUSION： Hot water-extracted crude LBE （2-5 g/L） and RGE （5-10 g/L） inhibit proliferation and stimulate p53-mediated apoptosis in HCC cells.

Study on Trace Elements in Rehmannia glutinosa Libosch. by Principal Component Analysis and Clustering Analysis

[Objective] This study aimed to investigate the trace elements in Rehman- nia glutinosa Libosch. by using principal component analysis and clustering analysis. [Method] Principal component analysis and clustering analysis of R. glutinosa medicinal materials from different sources were conducted with contents of six trace elements as indices. [Result] The principal component analysis could comprehen- sively evaluate the quality of R. glutinosa samples with objective results which was consistent with the results of clustering analysis. [Conclusion] Principal component analysis and clustering analysis methods can be used for the quality evaluation of Chinese medicinal materials with multiple indices.

Screening and Verification of Genes Specifically Responding to Continuous Cropping Obstacle in Rehmannia glutinosa L.

Rehmannia glutinosa L.is one of the important medicinal crops in China.Continuous cropping obstacle severely restricts the yield and quality of R.glutinosa,but its molecular mechanism is still unclear.In this study,with widely-planted "Wen 85-5" as an experiment material,based on the digital gene expression profiling (DGE) data of previous five stress treatments (continuous cropping,phenolic acid,salt,drought and waterlogging) and the first cropping and continuous cropping treatments of R.glutinosa in five different periods (seedling period,elongation period,early expanding period,middle expanding period and later expanding period),80 candidate genes (|log 2 ratio|≥1,FDR <0.001) specifically responding to continuous cropping obstacle in R.glutinosa were screened.Functional analysis revealed that the differentially expressed genes were involved in the secretion and endocytosis of root cells,which may suggest that the recognition and absorption of allelopathic autotoxins by the roots of R.glutinosa is an important factor that restricts the development of roots in continuous cropping of R.glutinosa.In order to accurately lock genes specifically responding to continuous cropping obstacle in R.glutinosa,continuous cropping soil extract and ferulic acid and p-hydroxybenzonic acid were used to treat aseptic plantlets of R.glutinosa,respectively,and it was confirmed through qRT-PCR that the expression levels of some genes under phenolic acid treatment changed more severely than that under the continuous cropping soil extract treatment,and four key genes involved in the response of R.glutinosa to continuous cropping were finally locked.This study lays a foundation for further exploration of the molecular mechanism of continuous cropping obstacle.

Effect of Hoechunyangkyeok-San Extract on Melanogenesis

Forsythia fructus has been shown to have antioxidative, anti-inflammatory, antibacterial, anti-aging and whitening effects. Hoechunyangkyeok-san (Forsythia viridissima-prescription) is a traditional herbal medicine, which has been clinically used for treating febrile and inflammatory disorders. This work was carried out to investigate the skin whitening effects of Forsythia viridissima-prescription extract (a hydrolyzed extract of Hoechunyangkyeok-san: SID White HYC) on skin. The effects of SID White HYC were assessed the melanin contents in B161 melanoma cells and the pigmented equivalent with HMB45 and Fontana Masson staining in 3D skin model. Then, we examined the expression of major pigment enzymes regulating melanin synthesis and melanosome transport related proteins in B16F1 cells. SID White HYC significantly inhibited the melanin synthesis (56.7% and 30.6% inhibition at 100 μg/mL, intracellular and secreted, respectively) in B16F1 cells and 3D skin model. In addition, western blotting analysis showed that SID White HYC reduced the expression of melanin synthesis and melanosome transport related proteins in B16F1 cells. In clinical trials, the cream containing 0.05% SID White HYC showed skin depigmentation effect without any irritation. These results suggest that SID White HYC may be useful inhibition of melanogenesis and melanosome transport. Therefore, SID White HYC may have potential as a skin-whitening ingredient in cosmetics.

MOFs对毛蕊花糖苷的吸附解吸性能研究

为了探究MOFs材料对地黄叶提取物中毛蕊花糖苷的吸附解吸性能,首先制备MOFs材料,随后以吸附量、解吸量、解吸率为指标筛选最适宜分离毛蕊花糖苷的MOFs和大孔树脂,以单因素试验对ZIF-8吸附、解吸毛蕊花糖苷的能力进行优化,并在优化所得条件下比较ZIF-8和D-101对毛蕊花糖苷的吸附、解吸能力。结果表明:ZIF-8对毛蕊花糖苷的吸附量和解吸量分别为40.78和25.42 mg·g^(-1),其分别是D-101大孔树脂对毛蕊花糖苷吸附量和解吸量的1.85倍和1.83倍。因此,ZIF-8对毛蕊花糖苷的吸附、解吸性能优于D-101大孔树脂。

地黄对斜纹夜蛾生长发育的影响

【目的】斜纹夜蛾是危害棉田的重要害虫,地黄是农田常见杂草,探索并利用地黄防治斜纹夜蛾对于棉花可持续生产具有重要意义。【方法】分别将地黄根或叶干粉混拌于斜纹夜蛾人工饲料(地黄干粉与饲料的质量比分别为1∶3、1∶6、1∶9和1∶18),初步明确地黄对斜纹夜蛾幼虫的死亡率、发育历期和体重的影响;进一步采用药膜法,研究地黄根或叶干粉的95%乙醇提取液(地黄干粉与提取溶剂的料液比分别为1∶50、1∶30和1∶10)对1~6龄斜纹夜蛾幼虫死亡率的影响。【结果】取食混有地黄根或叶干粉饲料的斜纹夜蛾幼虫生长发育受到一定的影响,随着饲料中地黄根或叶干粉含量的增大,斜纹夜蛾幼虫死亡率升高、发育历期延长、体重降低。地黄根或叶干粉与饲料的质量比为1∶3时,其对斜纹夜蛾幼虫的抑制效果最好。地黄根或叶提取液对低龄斜纹夜蛾幼虫有一定抑制作用,对高龄幼虫的毒杀作用较差,同一测定时间随地黄提取液浓度增大,斜纹夜蛾低龄幼虫的死亡率升高。地黄根或叶干粉与提取溶剂的料液比为1∶10时,对斜纹夜蛾1~6龄幼虫的毒杀作用效果最好。【结论】地黄对斜纹夜蛾有一定的抑制作用,在一定范围内,用量越大其抑制作用越强,研究结果可为有效利用农田杂草资源开发植物源杀虫剂奠定理论基础。

河南温县蚕豆萎蔫病毒2的鉴定与多样性分析

蚕豆萎蔫病毒2(broad bean wilt virus 2,BBWV2)属豇豆花叶病毒科蚕豆病毒属。2019年7月从河南温县采集20株具有褪绿和皱缩等典型病毒病症状的地黄,采用PCR技术从cDNA中扩增目的片段,有13株样品扩增出与预期大小相符的目的条带,检出率为65%;并通过克隆测序获得BBWV2 CP序列,将其与GenBank中其他48个BBWV2分离物进行序列分析,结果显示有75.4%~82.95%的一致率。系统发育分析表明,BBWV2可分为3个组,其中河南温县BBWV2地黄分离物(No.2279841-China-R.glutinosa-11)位于第Ⅲ组。

生地百合西洋参胶囊对小鼠睡眠的促进作用及其安全性研究

为了研究生地百合西洋参胶囊对小鼠睡眠的作用并对其毒理安全性进行评价,本试验以低[200 mg/(kg·bw)]、中[400 mg/(kg·bw)]、高[800 mg/(kg·bw)]剂量的生地百合西洋参胶囊连续给小鼠灌胃30 d,通过直接诱导睡眠试验、延长戊巴比妥钠睡眠时间试验、戊巴比妥钠阈下剂量催眠试验,以及小鼠脑组织中5羟色胺(5-HT)、γ-氨基丁酸(GABA)和多巴胺(DA)含量的检测,分析其改善睡眠的作用;以最大给药剂量试验(MTD)进行生地百合西洋参胶囊的小鼠急性毒性试验;以低[1 g/(kg·bw)]、中[2 g/(kg·bw)]、高[4 g/(kg·bw)]剂量的生地百合西洋参胶囊给大鼠连续灌胃30 d,评价其对大鼠的毒理安全性。结果显示,与对照组相比,生地百合西洋参胶囊对小鼠无直接诱导睡眠作用,不同剂量的生地百合西洋参胶囊均能显著延长小鼠的戊巴比妥钠睡眠时间(P<0.05或P<0.01),中、高剂量组小鼠的催眠入睡率显著提高(P<0.05或P<0.01),小鼠脑组织中5-HT和GABA的含量显著增加(P<0.05或P<0.01),高剂量组小鼠脑组织中DA的含量显著降低(P<0.05);以15 g/(kg·bw)剂量给小鼠灌胃生地百合西洋参胶囊14 d,小鼠均未出现死亡和中毒现象。大鼠在30 d喂养试验期间生长状况良好,各剂量组大鼠的体重增长量、进食量、食物利用率、血常规、血液生化和脏器系数等指标均与对照组无显著性差异(P>0.05)。结果表明,生地百合西洋参胶囊具有改善小鼠睡眠的作用,其作用机制与对小鼠脑组织中5-HT、GABA和DA含量的调节有关,本试验所用剂量范围对大鼠和小鼠均无明显毒性,安全性良好。本试验结果可为生地百合西洋参胶囊的临床应用提供参考依据。

Xijiao Dihuang Decoction(犀角地黄汤)and Rehmannia glutinosa Libosch.Protect Mice against Lipopolysaccharide and Tumor Necrosis Factor Alpha-Induced Acute Liver Failure

Objective: To investigate the hepatoprotective effect of Xijiao Dihuang Decoction(犀角地黄汤,XJDHD) on lipopolysaccharide(LPS)-and tumor necrosis factor alpha(TNF-α)-induced acute liver failure(ALF)as well as the underlying mechanism of action, and to clarify the key herbs and components of XJDHD. Methods:LPS/D-galactosamine(D-GalN) or TNF-α/D-GalN were intraperitoneally injected into C57BL/6J mice to induce ALF. Simultaneously, XJDHD or its individual herbs and components were orally administered. Survival rates, transaminase levels in serum, and hepatic histology were examined to evaluate the effects of XJDHD.The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay and real-time polymerase chain reaction were additionally performed to expound the mechanism underlying the anti-apoptotic activity of XJDHD. Results: Oral administration of XJDHD protected mice from lethal liver failure induced by LPS and TNF-α, with notable amelioration of liver injury in histology and a significant decrease in transaminase levels in serum. XJDHD signi?cantly inhibited apoptosis of hepatocytes and enhanced expression of the antiapoptosis genes, c-Flip, Iap1, Gadd45 b and A20(all P<0.05). In addition, Rehmannia glutinosa Libosch. was identi?ed as the key herb of XJDHD and galactose as the effective component of Rehmannia glutinosa Libosch.that protects against ALF. Conclusions: XJDHD inhibits TNF-α-induced apoptosis of hepatocytes by promoting the expression of nuclear factor κB-regulated anti-apoptotic genes. Rehmannia glutinosa Libosch. may be the most effective herb of XJDHD and galactose is an active component in this protection.

基于化学模式识别结合灰色关联度法的鲜地黄药材质量评价

目的:构建基于化学模式识别和灰色关联度法的鲜地黄药材多指标综合评价模型,为鲜地黄药材整体质量评价提供参考。方法:收集不同产地的32批鲜地黄药材样品,测定各批样品中总灰分,酸不溶性灰分,浸出物,梓醇、地黄苷D、铅、镉、砷、汞、铜含量与指纹图谱,采用聚类分析(HCA)、主成分分析(PCA)、正交偏最小二乘法-判别分析和灰色关联度法对各指标数据进行分析。结果:HCA和PCA均可将32批鲜地黄药材分为4类,但无明显产地聚集现象;指纹图谱中峰1~3、9、10的峰面积及总灰分、浸出物、梓醇含量是体现各产地鲜地黄药材质量差异的主要指标;32批鲜地黄药材灰色关联度为0.036~0.042,灰色关联度差异为0~13.89%,综合质量差异不大。结论:化学模式识别结合灰色关联度法构建的多指标综合评价模型分析结果客观、科学、准确,可用于鲜地黄药材质量的综合评价。

不同产地的地黄中梓醇含量比较

目的 采用薄层扫描法测定了不同产地的地黄中梓醇的含量。方法 以氯仿 -甲醇 -水 ( 7∶ 4∶ 0 .5 )为展开剂 ,10 %硫酸 -乙醇为显示剂 ,4 13 nm为扫描波长 ,反射法扫描。结果 河南温县产地黄中梓醇含量较高 ,山东嘉祥产地黄中梓醇含量较低。

地黄栽培历史及其品种考证

目的 考证地黄的栽培历史以及品种记载。方法 查阅古今文献。结果 除部分本草书籍对地黄的记载与目前不一致外 ,多数记载应为玄参科植物地黄。地黄的栽培历史可追溯到 10 0 0多年以前 ,当时就有块根膨大的类型 ,并开始了人工栽培。由于长期的栽培 ,选育了许多优良品种 ,有文献记载的品种多达 5 0余个 ,但对品种的描述过于简单 ,严重影响了品种的识别和应用。结论 地黄栽培历史悠久 ,种质资源丰富 ,可能是造成有关中药质量不稳定的原因之一。因此急需对其进行系统地收集、整理和保存研究 ,为成分分析、活性筛选和临床应用奠定基础。同时建议在进行相关研究时 ,应高度重视取样的一致性 ;在进行 GAP基地建设时 。

熟地水煎剂及其提取物对小鼠外周血象影响的比较研究

目的 :观察熟地水煎剂 (RGD)、熟地多糖 (RGP)、熟地非多糖 (RGNP)对小鼠外周血象的影响。方法 :实验各组连续灌胃 9天 ,断尾取血测定。各组小鼠于给药第 5天环磷酰胺造模后测定。各组小鼠造模后于 2d、4d、6d、8d分别取血测定。结果 :实验各组对正常小鼠外周血象无组间差异 ,对血虚模型小鼠各给药组与模型组比较 ,RGP与RGD有显著意义 ,RGNP仅对WBC有升高意义。RGP与RGNP均可对抗环磷酰胺所致小鼠WBC下降。但RGP作用优于RGNP。结论 :熟地补血功能具有双向调节作用 ,初步认为 。

高效液相色谱指纹图谱应用于地黄的研究

利用高效液相色谱建立了地黄的色谱指纹图谱。采用反相C1 8柱 (5 μm ,1 5 0mm× 4.6mm) ,流动相甲醇∶水 (V V =5∶95 ) ,检测波长为 2 80nm ,流速 1 .0mL min进行实验。根据相对保留值和相对面积值对色谱指纹图谱进行分析对比研究 ,建立了控制地黄药材质量的新方法。该法为中药样品的鉴定提供了较全面的信息 ,并为HPLC在复杂组分的样品分析领域开拓了新的应用途径。

不同产地的地黄中梓醇含量比较

目的 :测定不同产地地黄中梓醇含量。方法 :薄层扫描法 ,展开剂为氯仿 甲醇 水 (7∶4∶0 .5 ) ,显色剂为 10 %硫酸 乙醇 ,扫描波长为 4 13nm。结果 :回归方程 :Y =6 2 1.76X +118.6 9,r =0 .996 6 ,平均回收率为 99.10 % ,RSD =2 .5 0 %。结论 :河南温县产地黄中梓醇含量较高 。

地黄根系分泌物化感效应与酚酸类物质的关系研究

目的：通过研究不同生育时期地黄根系分泌物化感潜力和酚酸类物质含量动态变化,揭示酚酸类物质与地黄化感效应之间的关系。方法：利用新型根系分泌物收集装置,收集地黄不同生育时期根系分泌物,采用HPLC分析5种酚酸（香豆酸、对羟基苯甲酸、香草酸、丁香酸、阿魏酸）含量变化并通过生物测试（萝卜种子发芽试验）评价其化感潜力,分析根系分泌物化感效应与5种酚酸含量的相关性。结果：地黄不同生育时期根系分泌物浸种后萝卜种子发芽率为97.89%、92.38%、89.52%、85.71%、85.71%、84.76%和83.81%,随生育期推进呈逐渐降低趋势,其中从块根膨大前期开始与对照表现出显著差异;芽长为5.68、5.76、5.91、5.65、5.41、5.28和5.11 cm,呈先升高后降低的趋势,同样从块根膨大前期开始与对照相比差异达到显著水平。HPLC检测地黄不同生育时期根系分泌物均发现了5种酚酸,但其含量未呈现与根系分泌物化感作用相似的变化规律,对两者相关性分析发现,除丁香酸与发芽长度呈显著负相关外（r=-0.833,P〈0.05）,其他酚酸含量均未与萝卜种子发芽率和芽长表现出显著相关性（r=-0.666-0.590）。结论：地黄于块根膨大前期开始表现出显著化感作用,且化感作用随生育期的推进逐渐增强。丁香酸可能是引起地黄化感效应的主导物质。

地黄优良品种“85-5”脱毒苗的快速繁殖研究

目的 探索“85 - 5”脱毒苗的快速繁殖技术。方法 将脱毒苗接种在不同培养基 (单位 :m g/ L) (1.MS+BA0 .5 ;2 .MS+BA 0 .5 +NAA 0 .0 2 ;3.MS+BA 0 .5 +NAA 0 .0 2 +GA30 .1;4 .1/ 2 MS+BA 0 .1;5 .1/ 2 MS+BA0 .1+GA30 .1;6 .1/ 2 MS+GA30 .1)上 ,30 d后统计新生叶片数、根数以及苗高等。然后选用 2号培养基 ,在不同的培养条件下培养 ,同上记录有关数据。结果 在 1,2和 3号培养基上 ,具茎尖苗主茎发育良好 ,基部具 1～ 3个腋芽 ;无茎尖苗腋芽发育 ,主芽长 0 .5～ 1.5 cm。 4 ,5和 6号培养基上脱毒苗细弱 ,叶少根多。在相同光照条件下 ,2 8℃时 ,各项测量值均显著高于 2 3℃时的值 ;在相同温度下 ,低光照培养 ,叶小苗高 ,强光则相反。此外 ,还发现 BA和 GA3之间存在拮抗作用。结论 在 2 8℃ ,10 0 0～ 2 0 0 0 lx培养条件下 ,采用 2号培养基适于“85 - 5”脱毒苗的快速繁殖。

生地黄提取物的抗氧化活性研究

【目的】通过体外抗氧化试验,研究生地黄块根不同溶剂提取物的抗氧化活性,为生地黄的进一步开发利用提供理论依据。【方法】用体积分数70%的乙醇回流提取生地黄根粉末,减压浓缩得到乙醇浸膏,加蒸馏水制成悬浮液,依次用等体积的石油醚、氯仿、乙酸乙酯、正丁醇萃取,分别得到石油醚、氯仿、乙酸乙酯、正丁醇提取物;以2,6-二叔丁基对甲酚(BHT)为对照,分别采用DPPH法、Feton法、FRAP法和还原能力测定法,研究4种不同熔剂提取物的抗氧化活性。【结果】生地黄4种不同熔剂提取物均具有不同程度的抗氧化活性,且抗氧化活性与提取物质量浓度呈量效关系。生地黄乙酸乙酯提取物的1,1-二苯基-2-苦苯肼自由基(DPPH.)清除活性(IC50值为0.178mg/mL)、还原能力(IC50值为0.341 mg/mL)和总抗氧化活性均最高,其中乙酸乙酯提取物的DPPH.清除活性高于对照BHT(IC50值为0.456 mg/mL)。生地黄氯仿提取物的羟自由基(OH.)清除活性最强,IC50值为1.397 mg/mL。【结论】生地黄乙酸乙酯提取物具有较强的抗氧化活性,可作为一种新的自由基清除剂和天然抗氧化剂。

生、熟地黄缓解肾间质纤维化的作用及其机制研究

目的:探讨生地黄和熟地黄对单侧输尿管梗阻模型(UUO)大鼠肾间质纤维化的作用及其机制。方法:雄性SD大鼠随机分为生地黄水煎液组、熟地黄水煎液组、模型对照组、假手术组,每组5只。生地黄水煎液组、熟地黄水煎液组分别按生地黄、熟地黄剂量1.6 g/(kg·d)灌胃给药14 d,测定血清肌酐(Scr)、尿素氮(BUN)。HE染色、Masson染色观察肾脏组织病理学变化,免疫印迹检测TGF-β1、α-SMA和Collagen-Ⅰ的表达。结果:与模型组比较,各给药组大鼠Scr、BUN值显著降低(P<0.05);肾小球玻璃样病变减轻,肾小管囊性扩张和空泡变性程度缓解,胶原沉积减少,肾间质纤维化程度明显改善;TGF-β1、α-SMA和Collagen-Ⅰ表达显著降低(P<0.05)。与熟地黄水煎液组比较,生地黄水煎液组的Scr、BUN值较低,胶原沉积较少,肾间质纤维化改善较明显,α-SMA表达显著减少(P<0.05)。结论:生地黄和熟地黄均能缓解肾间质纤维化,以生地黄作用较强,其作用机制可能与下调TGF-β1、α-SMA和Collagen-Ⅰ的表达有关。

怀地黄连作对土壤微生物区系的影响

运用传统平板培养方法及变性梯度凝胶电泳(DGGE)分析法对地黄的连作问题进行了研究.结果表明,地黄连作引起土壤根际、根外细菌数量减少;根际放线菌数量增加,根外数量变化不大;真菌种类和数量都有较大变化.DGGE分析表明,头茬和重茬地黄生长过程中根际土壤微生物区系发生了明显变化,细菌数量减幅较大,有些条带消失,说明种群数量减少;放线菌种群没有大的变化,数量有所上升;真菌数量有所增加,种群发生了明显改变,表现为条带的缺失和增加.地黄连作后土壤微生物多样性发生了较大变化,细菌数量减少,木霉和黄曲霉数量增加,土壤生态系统已开始失调,这可能是地黄连作障碍产生的原因.