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.

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.

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.

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.

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.

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.

Complete Genome Sequencing and Analysis of Rehmannia Mosaic Virus Isolate from Shanxi Province

Using double-stranded RNA(dsRNA)technology and sequence-independent amplification(SIA),the molecular identification on infected Rehmannia glutinosa in the field with mosaic symptoms was performed and the whole-genome of the Rehmannia mosaic virus(ReMV)Shanxi isolate(ReMV-SX)was sequenced.Sequencing analysis showed that the virus that infected Rehmannia glutinosa was Rehmannia mosaic virus(ReMV).The full-length of the obtained ReMV-SX sequence(GenBank accession no.JX575184)was 6395 nt,containing four open reading frames(ORFs).The sequence homology analysis of the complete nucleotide sequence showed that ReMV-SX was 93.8%-97.0%homologous to ReMV in Tobamovirus subgroup Ⅰ,while only 49.8%-58.9%homologous to the isolates in subgroups Ⅱ and Ⅲ of the same genus.Phylogenetic analysis showed that ReMV-SX and ReMV-Henan formed a separate branch and had the closest genetic relationship.The results laid the foundation for ongoing researches in the taxonomic status and evolution of ReMV and for further investigating the pathogenic mechanism of ReMV infecting Rehmannia glutinosa.

地黄寡糖对3T3-L1脂肪细胞增殖及胰岛素抵抗的作用

目的:探讨地黄寡糖对脂肪细胞增殖和胰岛素抵抗的影响。方法:培养3T3-L1前脂肪细胞,用四甲基偶氮唑盐(MTT)方法检测3T3-L1前脂肪细胞及脂肪细胞的增殖情况,同时采用地塞米松诱导3T3-L1脂肪细胞建立胰岛素抵抗模型,检测地黄寡糖对细胞培养基中葡萄糖浓度的影响。结果:在DMEM高糖培养基中,地黄寡糖可促进3T3-L1前脂肪细胞增殖,抑制3T3-L1脂肪细胞增殖,作用呈明显量效关系;使3T3-L1前脂肪细胞及3T3-L1脂肪细胞葡萄糖消耗量增加,呈明显量效关系;地黄寡糖能明显增加胰岛素抵抗3T3-L1脂肪细胞培养基中的葡萄糖消耗量,增强对胰岛素的敏感性。结论:地黄寡糖可以促进前脂肪细胞的增殖,抑制脂肪细胞的增殖,地黄寡糖对地塞米松诱导的3T3-L1脂肪细胞胰岛素抵抗具有明显的改善作用。

不同方法加工生地黄的2D-IR和HPLC检测结果相关性比较

目的:建立简单、有效的评价不同方法加工的生地黄的质量的方法。方法:利用二维相关红外光谱技术(Twodimensionalinfraredcorrelationspectroscopy,2DIR)结合HPLC分别从宏观和微观角度对多种不同方法产地加工的生地黄进行质量评价。结果:表明二者具有良好相关统一性。结论:可以利用2DIR结合HPLC分别对不同方法产地加工的生地黄进行质量评价。

生地黄免疫抑制活性部位HPLC指纹图谱研究

目的:研究河南省道地药材生地黄免疫抑制活性部位HPLC指纹图谱。方法:采用高效液相色谱法,迪马Platisil-C18柱(250 mm×4.6 mm,5μm),乙腈-0.1%磷酸梯度洗脱,检测波长为210 nm,流速为1.0 mL/min,柱温为30℃。结果:对10批生地黄免疫抑制活性部位的指纹图谱进行了相似度比较,标定共有峰18个,其中5个色谱峰成分已指认,相似度在0.9以上。结论:建立了生地黄免疫抑制活性部位HPLC指纹图谱,为地黄进一步的药效学研究奠定了基础。

黄芪和地黄对食管癌TIL增殖及体外抗肿瘤作用的研究

目的 :探索黄芪、地黄在促进食管癌TIL增殖和特异性杀伤活性中的作用和食管癌TIL免疫治疗的可行性。方法 :采用3H TdR掺入法。应用黄芪和地黄联合多种细胞因子对 9例食管癌TIL的增殖和特异性杀伤活性进行研究。结果 :在促进TIL增殖、杀伤活性中 ,黄芪联合IL 2、IL 2 +IL 4、IL 2 +TNF α均较单用这些细胞因子有明显增强作用 (P <0 .0 5或 0 .0 1)。结论

UPLC法测定地黄叶及块根中的梓醇和毛蕊花糖苷

目的比较地黄叶及块根中梓醇和毛蕊花糖苷的含有量。方法地黄叶及块根甲醇提取液的分析采用ACQUITY UPLC BEH Shield RP18色谱柱(2.1 mm×100 mm,1.7μm);柱温35℃;流动相为乙腈-0.1%磷酸水溶液,梯度洗脱;体积流量0.2 m L/min;检测波长210 nm。结果梓醇含有量由高到低,依次为地黄嫩叶(149.22 mg/g)>老叶(49.29 mg/g)>块根(33.33 mg/g)>干叶(15.53 mg/g);毛蕊花糖苷含有量依次为地黄老叶(10.27 mg/g)>干叶(10.03 mg/g)>嫩叶(9.56 mg/g)>块根(0.28 mg/g)。结论地黄嫩叶中梓醇的含有量约为老叶的3倍,但毛蕊花糖苷的含有量略低于老叶。地黄叶存放一年后,毛蕊花糖苷的减少量比梓醇小,表明前者稳定性高于后者。而且,这两种成分在叶中的含有量均比在块根中更高。

地黄炮制前后化学成分的UHPLC-Q-TOF/MS比较研究

目的比较地黄生品、酒品、蒸制品各项质控指标及化学成分的差异。方法按照中国药典2010版标准,测定地黄不同炮制品的水分、总灰分、醇溶性浸出物,并建立HPLC方法比较地黄不同炮制品主要化学成分的差异,考察方法的线性、精密度、稳定性、重复性及加样回收率,同时使用超高效液相色谱-四极杆飞行时间串联质谱法(UHPLC-Q-TOF/MS)鉴定地黄不同炮制品的主要差异化学成分。结果地黄酒品和蒸品的水分分别为6.06%和10.02%,总灰分分别为6.70%和6.47%,酸不溶性灰分分别为2.63%和2.39%,水溶性浸出物分别为72.62%和77.81%,测定结果均符合中国药典2010版的要求。运用高分辨质谱鉴定了8个主要差异化学成分,即连翘酯苷E、洋地黄叶苷B、焦地黄苯乙醇苷A1、毛蕊花糖苷、异麦角甾苷、连翘酯苷、焦地黄苯乙醇苷D和地黄苷。结论地黄经炮制后梓醇含量显著上升,而其他主要化学成分的含量显著降低,这可能是不同炮制品临床功效存在差异的主要原因。

地黄水提液对2型糖尿病大鼠proinsulin基因表达的影响

目的:探讨地黄水提液治疗2型糖尿病(2-DM)的分子生物学机制。方法:以半定量RT-PCR法和SDS-聚丙烯酰胺凝胶电泳法分别检测胰岛素原(proinsulin)基因转录及翻译水平的表达。同时观察空腹血糖(FPG)、空腹胰岛素(FINS)的含量,计算胰岛素分泌指数(IS)、β细胞功能指数(HβCI)的变化,来研究不同剂量地黄水提液对上述各指标的影响。结果:与2-DM模型相比,地黄治疗组大鼠proinsulin基因mRNA和蛋白表达显著增高(P<0.01),FPG显著降低,FINS、IS、HβCI升高,差异均有显著性。结论:地黄水提液通过上调2-DM大鼠proinsulin基因mRNA和蛋白表达,可改善胰岛β细胞功能,降低2-DM大鼠血糖。

低分子质量地黄多糖体外对Lewis肺癌细胞p53基因表达的影响

目的研究低分子质量地黄多糖（ＬＲＰＳ）体外给药对Ｌｅｗｉｓ肺癌细胞ｐ５３基因表达的影响。方法利用竞争性逆转录聚合酶链反应（ＲＴ－ＰＣＲ）的方法检测ｐ５３基因表达的水平。结果ＬＲＰＳ在剂量０．１和０．５ｇ·Ｌ－１条件下，体外给药１２ｈ，Ｌｅｗｉｓ肺癌细胞内ｐ５３基因的表达水平为０．７９０和０．７１８，对照组为０．４８３，体外给药２４ｈ，ｐ５３基因的表达水平为０．９５７和０．５９０，对照组为０．２２６。结果表明，ＬＲＰＳ能使Ｌｅｗｉｓ肺癌细胞内的ｐ５３基因表达明显增加。结论ＬＲＰＳ可能通过调控ｐ５３基因的表达而影响肿瘤细胞的增殖、分化和凋亡。

一株克服地黄连作障碍有益菌的鉴定及其LuxAB基因标记

[目的]研究对克服地黄(Rehmannia glutinosa)连作障碍有益的43号菌的性质及其对地黄连作障碍的作用效果。[方法]利用细菌16S rDNA的保守序列进行比对,结合形态学观察、革兰氏染色和生理生化等特征对筛选得到的能够克服地黄连作障碍的43号菌进行鉴定;利用LuxAB发光酶基因对其进行标记,经抗性平板筛选,滴加葵醛在暗室进行检测,看是否得到了重组菌株;比较标记前后菌株对培养基中加入生地提取液的组培苗的作用效果。[结果]鉴定结果表明,43号菌为恶臭假单胞菌(Pseudomonas putida)。检测结果表明,得到重组菌株,命名为C-43。经过比较,标记前后菌株的生理生化特性基本没有变化,确定C-43对克服地黄连作障碍仍然有益。[结论]经过标记后的恶臭假单胞菌C-43号菌可用于后续的大田研究。

地黄和小肽对鲤鱼生长性能及IL-8表达的调节

研究了地黄和深海鱼源活性小肽对鲤鱼生长和免疫功能的调节作用,探讨其作为鱼类饲料添加剂的可行性。试验一选取540尾平均体质量为(7.46±1.06)g的鲤鱼,随机分为9组饲喂基础日粮及添加了2%生地黄粉,4%生地黄粉,2%熟地黄粉,4%熟地黄粉,0.5%生地黄水提物,1%生地黄水提物,0.5%熟地黄水提物,1%熟地黄水提物的饲料。试验二选取360尾平均体质量为(5.47±0.3)g的鲤鱼,随机分为4组饲喂基础日粮和添加5%、10%和15%小肽的饲料。饲养80d后,测定鲤鱼的生长指标和抗病性能,并检测鲤鱼脾脏IL-8的mRNA表达情况。结果显示,各种地黄制剂和不同剂量的小肽均可提高鲤鱼脾脏IL-8的表达,其中试验一的2%熟地黄粉、4%熟地黄粉、1%生地黄水提物、0.5%熟地黄水提物组和试验二的5%、10%小肽组与对照组均有显著性差异。地黄还可显著促进鲤的生长,提高其抗病力。因此,地黄和深海鱼源活性小肽均有望成为具有免疫调节作用的鱼类饲料添加剂。

地黄煎剂消退L-甲状腺素诱发的大鼠心肌肥厚并抑制其升高的心、脑线粒体Ca^(2+),Mg^(2+)-ATP酶活力

Ｌ甲状腺素４ｍｇ／ｋｇ·ｄ连续ｐｏ７ｄ诱发大鼠心肌肥厚和心、脑线粒体Ｃａ２＋，Ｍｇ２＋ＡＴＰ酶活力显著升高。经地黄４ｇ／ｋｇ·ｄｐｏ治疗３ｄ后，心肌肥厚及其心、脑升高的Ｃａ２＋，Ｍｇ２＋ＡＴＰ酶活力显著降低，但未恢复至正常。结论：地黄消退Ｌ甲状腺素诱发的大鼠缺血性心肌肥厚并抑制升高的心脑线粒体Ｃａ２＋，Ｍｇ２＋ＡＴＰ酶活力，保护心脑组织避免ＡＴＰ耗竭和缺血损伤。