Cloning and Sequence Analysis of Actin Gene from Rehmannia glutinosa

[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.

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.

Effects of Consecutively Monocultured Rehmannia glutinosa L.on Diversity of Fungal Community in Rhizospheric Soil

Continuous monoculture problems, or replanting diseases, are one of the key factors affecting productivity and quality of Chinese medicinal plants. The underlying mechanism is still being explored. Most of the studies on continuous monoculture ofRehmannia glutinosa L. are focused on plant nutritional physiology, root exudate, and its autotoxieity. However, the changes in the diversity of microflora in the rhizosphere mediated by the continuous monoculture pattern have been remained unknown. In this study, terminal restriction fragment length polymorphism （T-RFLP） technique was used for fingerprinting fungal diversity in the rhizosphere soil sampled from the fields ofR. glutinosa monocultured for 1 and 2 yr. The results showed that the structure of fungal community in consecutively moncultured rhizosphere soil was different from that in control soil （no cropping soil）, and varied with the consecutive monoeulture years （1 and 2 yr）. The comprehensive evaluation index （D） of fungal community estimated by principal component analysis of fragment number, peak area, Shannon-Weiner index, and Margalef index was higher in 1 yr monoculture soil than that in 2 yr monoculture soil, suggesting that consecutive monoculture of R. glutinosa could be a causative agent to decrease the diversity of fungal community in the rhizosphere soil.

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.

Advances of Allelopathic Autotoxicity in Rehmannia glutinosa L.

Allelopathic autotoxicity occurs when a plant releases toxic chemical substances into the environment which inhibits development and growth of the same plant species.Rehmannia glutinosa L.( R.glutinosa ) is one of the most common traditional Chinese medicines,whose productivity and quality,however,are seriously impacted by consecutive monoculture obstacle.Allelopathic autotoxicity is one reason for consecutive monoculture obstacle.In this paper,we reviewed the categories of allelochemicals,the methods of allelochemicals identification,and the mechanisms of allelopathic autotoxicity,which provides clues for further study of the molecular mechanisms of allelopathic autotoxicity and consecutive monoculture obstacle.

Analysis on Pigment and Photosynthetic Characteristics of Leaves of New Strain of Rehmannia glutinosa

Through the analysis on the leaf color and photosynthetic characteristics of new strains and main cultivars of Rehmannia glutinosa,it is expected to provide theoretical basis for breeding of new varieties. Chlorophyll,anthocyanin,and net photosynthetic rate（Pn）,stomatal conductance（Cond）,transpiration rate（Tr）,and intercellular CO2concentration（Ci） in 8 varieties of Rehmannia glutinosa were measured by spectrophotometer and LI-6400 XT Portable Photosynthesis System. The results showed that the chlorophyll content of Huaidijin 8（2. 84 mg/g）,Huaidi 81（2. 71 mg/g）,Huaidi 85-5（2. 69 mg/g）,Jinjiu（2. 66 mg/g） and Huaidi 83（2. 63 mg/g） was higher; the anthocyanin content of Jinjiu（0. 169） and Huaidijin 8（0. 165） was higher,while the anthocyanin content Huaidi 83（0. 060） was the lowest; Pn of Huaidi81[2. 41 μmol/（m2·s） ],Huaidi 83[2. 37 μmol/（m2·s） ]and Huaidijin 8[2. 25 μmol/（m2·s） ]was higher,and the anthocyanin content was positively correlated with Pn,while the anthocyanin content was negatively correlated with Pn; Huaidijin 8 and Huaidi 83 showed dominant advantages in single plant fresh weight,indicator component,and resistance over the main cultivars. This indicates that the new variety Huaidijin 8 and Huaidi 83 have excellent comprehensive traits and can be properly popularized.

Rehmannia glutinosa exhibits anti-aging effect through maintaining the quiescence and decreasing the senescence of hematopoietic stem cells

Background: The time-related decline in regenerative capacity and organ homeostasis is a major feature of aging. Rehmannia glutinosa and Astragalus membranaceus have been used as traditional Chinese herbal medicines for enhanced immunity and prolonged life. However, the mechanism by which this herbal medicine slows aging is unknown. In this study, we investigated the mechanism of the herbal anti-aging effect.Methods: Mice were fed diets supplemented with R. glutinosa or A. membranaceus for 10 months; the control group was fed a standard diet. The phenotypes were evaluated using a grading score system and survival analysis. The percentages of the senescence phenotypes of hematopoietic stem cells(HSCs) were determined by fluorescence-activated cell sorting analysis. The function and the mechanism of HSCs were analyzed by clonogenic assay and the real-time polymerase chain reaction.Results: The anti-aging effect of R. glutinosa is due to the enhanced function of HSCs. Mice fed with R. glutinosa displayed characteristics of a slowed aging process,including decreased senescence and increased rate of survival. Flow cytometry analysis showed decreased numbers of Lin–Sca1^+c-kit–(LSK) cells, long-term HSCs(LT-HSCs) and short-term HSCs(ST-HSCs) in the R. glutinosa group. In vitro, clonogenic assays showed increased self-renewal ability of LT-HSCs from the R. glutinosa group as well as maintaining LSK quiescence through upregulated p18 expression. The R. glutinosa group also showed decreased reactive oxygen species levels and the percentage of β-gal^+ cells through downregulation of the cellular senescence-associated protein p53 and p16.Conclusion: Rehmannia glutinosa exerts anti-aging effects by maintaining the quiescence and decreasing the senescence of HSCs.

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.

铅胁迫对地黄生长及光合特性的影响

为了阐明铅(Pb)胁迫对地黄生长量及光合特征的影响,探讨药用植物对重金属胁迫的响应机制,以‘北京3号’地黄为试验材料,采用土培盆栽试验,以自然土壤加水为对照(0 mg·kg^(-1)),设置5个外源Pb浓度(50、100、200、300和400 mg·kg^(-1))处理,分析不同Pb浓度处理对地黄不同生长时期的生长量、叶片光合色素含量、光合参数和叶绿素荧光参数的影响。结果表明,高浓度Pb处理抑制了地黄叶片数、叶长、叶宽和叶厚的增长,光合色素含量和叶绿素荧光参数在整个生育期内随Pb浓度的升高均表现为先升高后降低,当Pb浓度高于200 mg·kg^(-1)时,电子传递速率、光合量子产量、最大荧光和初始荧光均显著降低;随Pb浓度的升高,地黄叶片的净光合速率、气孔导度和胞间CO_(2)浓度在地黄膨大初期至膨大前期逐渐降低,在膨大中期至收获期则表现为先升高后降低;蒸腾速率在整个生育期内随Pb浓度的升高呈下降趋势;地黄的光合特性对Pb的耐受性总体表现为50 mg·kg^(-1)处理>100 mg·kg^(-1)处理>0 mg·kg^(-1)处理>200 mg·kg^(-1)处理>300 mg·kg^(-1)处理>400 mg·kg^(-1)处理。综上所述,当Pb浓度低于200 mg·kg^(-1)时,地黄通过吸收光能、增强光合作用对抗Pb胁迫,当Pb浓度过高时,地黄光合作用减弱,对地黄的生长产生显著的抑制作用。研究比较了不同Pb浓度处理对地黄不同生育期生长量及光合特征的影响,解析了地黄对Pb元素的抗性机制,以期为农田土壤的安全利用及地黄的种植管理提供重要技术参考。

地黄UDP-鼠李糖:鼠李糖基转移酶基因家族的生物信息学和表达分析

尿苷二磷酸鼠李糖基转移酶能将活性鼠李糖基供体转移到其糖基化受体分子上,合成鼠李糖基化的化合物.它们结构多样,活性广泛,具有药理、维持细胞结构、信号转导和化学防御及调节植物体内激素水平等重要作用.迄今,已从多种植物中分离得到这类酶,但有关其基因家族的结构特性与在植物次生代谢产物毛蕊花糖苷生物合成中的作用研究较少.以地黄尿苷二磷酸鼠李糖基转移酶基因(RgURT)家族的4个成员RgURT1~RgURT4为材料,利用生物信息学对该家族的特征进行了分析,比较了RgURT在高、低毛蕊花糖苷地黄品种块根中的表达模式.结果表明,其cDNA大小为645~1 422 bp,编码蛋白质分子量为24.05~53.24 kDa,由214~473个氨基酸残基组成,属于糖基转移酶GT-B型超家族,C端具有保守结构域盒(PSPG盒),无信号肽,亚细胞定位在细胞质中,二级结构以无规则卷曲和α-螺旋为主;RgURT4与RgURT1~RgURT3分别聚类为2个簇,RgURT4蛋白与其他3个URT蛋白序列之间的同源性低;RT-qPCR分析表明,RgURT基因在高毛蕊花糖苷地黄块根中的表达量高于低毛蕊花糖苷地黄块根中的表达量.研究首次揭示了地黄RgURT家族的特征,初步验证了RgURT与地黄毛蕊花糖苷合成相关,将为进一步研究毛蕊花糖苷合成提供了重要酶基因.

地黄梓醇调控ATDC5软骨细胞的衰老

背景:课题组前期体内、体外研究结果表明地黄梓醇能够显著降低膝骨关节炎大鼠滑膜组织中炎症指标水平,同时能够延缓膝骨关节炎进展,但是否通过影响软骨细胞衰老进而延缓膝骨关节炎的进展尚未明确。目的:探讨地黄梓醇能否调控ATDC5软骨细胞衰老及可能的机制。方法:将ATDC5软骨细胞分为空白组(0.1%牛血清白蛋白)、模型组(0.1%牛血清白蛋白+1μmol/L阿霉素)、地黄梓醇低剂量组(0.1%牛血清白蛋白+1μmol/L阿霉素+20μmol/L地黄梓醇)及地黄梓醇高剂量组(0.1%牛血清白蛋白+1μmol/L阿霉素+80μmol/L地黄梓醇)。应用阿霉素诱导构建ATDC5软骨细胞衰老模型,按上述分组予以对应的处理。CCK-8法检测地黄梓醇对ATDC5软骨细胞活力的影响,筛选地黄梓醇最佳给药浓度。相应处理后应用β-半乳糖苷酶染色法检测各组ATDC5软骨细胞衰老情况;实时荧光定量PCR法检测相关基因表达(P21、P53、Ⅱ型胶原、基质金属蛋白酶13、白细胞介素6);Western blot检测P21、P53、Ⅱ型胶原、基质金属蛋白酶13、白细胞介素6的表达水平;免疫荧光法检测P21、P53和Ⅱ型胶原表达情况;流式细胞仪检测各组细胞凋亡情况。结果与结论:(1)经鉴定成功诱导ATDC5软骨细胞并诱导衰老模型;(2)地黄梓醇浓度在0,20,40,80μmol/L时对细胞活力均无明显影响,提示地黄梓醇对细胞无毒性,可安全使用(P> 0.05);当浓度≥100μmol/L时,细胞活力降低,提示可能存在毒性,故选择80μmol/L作为高剂量进行后续实验;(3)与空白组β-半乳糖苷酶阳性细胞百分率(17.32±0.72)%比较,模型组(86.93±2.18)%显著升高(P <0.05);与模型组比较,地黄梓醇低、高剂量组(57.28±1.73)%、(27.18±0.97)%均显著降低(P <0.05);(4)与模型组比较,地黄梓醇低、高剂量组的P21、P53、基质金属蛋白酶13、白细胞介素6 mRNA和蛋白相对表达量均显著下调,而Ⅱ型胶原的mRNA和蛋白相对表达量显著上调(P <0.05),高剂量组更为明显(P <0.05);(5)与模型组比较,地黄梓醇低、高剂量组的P21、P53荧光信号均显著减弱,而Ⅱ型胶原的荧光信号显著增强(P <0.05),高剂量组更为明显(P <0.05);(6)经Annexin V/PI法检测各组细胞凋亡情况,与空白组比较,模型组的凋亡情况无明显变化(P> 0.05);与模型组比较,地黄梓醇低、高剂量组的凋亡指标均显著升高,且以高剂量组更为明显(P <0.05);(7)提示地黄梓醇能够通过促进衰老的ATDC5软骨细胞凋亡,进一步清除衰老的ATDC5软骨细胞,降低衰老相关表型进而延缓骨关节炎的进展。

地黄连作障碍研究进展

总结了连作障碍的危害、形成机制及消减策略,明确连作障碍形成的主要原因为土壤理化性质劣化、土传病虫害加剧和化感自毒作用,是由地黄-土壤-微生物3者共同作用的结果。针对连作障碍,综述了选用抗连作优质品种、强还原土壤灭菌、土壤淹水处理、添加食用菌菌渣、施加修复微生物菌剂等方法进行防控与缓解,以期为解决连作障碍问题提供理论指导。

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。

地黄氧-酰基转移酶WSD基因的鉴定与表达分析

植物蜡酯合成酶催化长链醇和长链脂肪酸合成蜡酯,对植物蜡质合成及其抗旱、抗致病菌袭击和紫外辐射、抗寒和昆虫侵害等环境胁迫具有非常重要的作用;镉是环境中含量最高的有毒重金属之一,严重威胁植物的生长发育、质量、产量和食用安全。为研究地黄蜡酯合成酶基因镉胁迫表达,该文从地黄全长转录组测序数据中鉴定其成员,并用生物信息学技术与qRT-PCR对其编码蛋白质的理化性质、系统进化和保守结构域及其组织表达与镉胁迫表达进行分析。结果表明:(1)鉴定出两个蜡酯合成酶基因RgOATWSD1与RgOATWSD2,其编码蛋白质的长度、理论等电点和相对分子量依次为463 aa与473 aa、8.86与9.34、51.31 kD与52.49 kD,均为不稳定蛋白。(2)二者均具有acyl_WS_DGAT保守域与DUF1298超家族,前者占其氨基酸序列的92.65%~94.50%。(3)二者均定位于内质网中,二级结构以无规卷曲与α螺旋为主;RgOATWSD1为跨膜蛋白,而RgOATWSD2不是。(4)二者均在地黄根、茎、叶中差异表达。(5)二者表达均受镉胁迫诱导,但其表达变化趋势不同。该研究鉴定了两个镉胁迫应答反应的蜡酯合成酶基因,为地黄RgOATWSD的镉胁迫表达及功能研究奠定了基础。

地黄的主要化学成分及药理作用研究进展

地黄为玄参科地黄属植物,是我国药食同源常用中药材之一,始载于《神农本草经》,临床应用广泛,主产于河南、河北、山东、山西等地,以河南怀庆府产最为道地,是著名的“四大怀药”之一。近年来,随着国内外学者对地黄全面深入地研究,其结构多样的化学成分、广泛的药理作用及开发应用备受关注。本文对地黄化学成分和药理活性的相关文献进行综述,旨在为相关研究提供参考。

生地低聚糖雾化吸入后小鼠体内药动学研究

目的 考察生地低聚糖雾化吸入后小鼠体内药动学。方法 66只小鼠随机分为11组,雾化吸入药液,于0、0.16、0.33、0.67、1、2、4、8、12、18、24 h采血,取出肺组织。LC-MS/MS分析采用Shodex Asahipak NH2P-50 4E色谱柱(250 mm×4.6 mm, 5μm)和Asahipak NH2P-50G 4A保护柱(10 mm×4.6 mm);流动相水-乙腈,梯度洗脱;体积流量900μL/min;柱温25℃;电喷雾离子源;负离子扫描;多反应监测模式,计算主要药动学参数。结果 生地低聚糖在50~36 000 ng/mL范围内线性关系良好(r>0.999 0),定量限为50 ng/mL。血浆、肺组织中AUC_(0~t)分别为14 726.56 ng/mL·h、18 544.11 ng/g·h,T_(max)分别为0.16、0 h,t_(1/2z)分别为0.838、3.739 h。结论 生地低聚糖可通过雾化吸入迅速分布到小鼠肺组织,并维持一段时间。

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。

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。

熟地黄多糖含量近红外模型的建立及其对肥胖小鼠肾损伤炎症的抑制作用

建立近红外光谱法快速测定熟地黄多糖(RGP)含量的方法,并探讨不同含量RGP对肥胖小鼠炎症的影响。采集熟地黄的近红外漫反射光谱,结合偏最小二乘法(PLS),采用多元散射校正法、直接差分法和一阶导数法对光谱进行处理,以4040.12~11221.66 cm^(-1)为波长区间,19为主成分数建立RGP含量的定量分析模型,其含量的预测值为34.63%~45.28%,测定值为34.49%~45.09%,绝对偏差为-0.41%~0.26%,相对偏差为0.121%~0.995%。并建立高脂饮食诱导的肥胖小鼠模型,以不同含量RGP给药14周后测定空腹血糖(FPG)、总胆固醇(TC)、甘油三酯(TG)、肌酐(Cr)和肾脏指数,观察小鼠肾脏组织病理形态。与模型组相比,RGP高含量组小鼠的体质量、TC、TG、Cr、FPG和肾脏指数分别下降11.29%、31.59%、47.63%、34.63%、28.57%、19.00%;RGP的干预可明显改善肾脏组织结构损伤,抑制炎症细胞的浸润,且作用效果与含量高低呈正相关。由此表明建立近红外光谱模型可快速准确地测定RGP含量;RGP具有调节肥胖小鼠血脂异常,抑制肾脏炎症反应,保护肾脏结构的作用,为RGP的开发利用提供实验依据。

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.