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.

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.

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.

施用生物菌肥对地黄重茬土壤的影响

以地黄(Rehmannia glutinosa)的重茬土壤为研究对象,分析生物菌肥对地黄重茬土壤的微生物群落构成及土壤内根系残留化合物的影响。通过施用不同浓度的生物菌肥,结合土壤微生物计数法和超高效液相色谱-四极杆飞行时间质谱法(UPLC-QTOF/MS)对施有生物菌肥的重茬土壤分别进行微生物种群计数和根系残留化合物分析。结果表明,不同处理下土壤微生物群落结构存在明显差异,其中,0.05%生物菌肥施用60 d后的地黄重茬土壤中细菌、放线菌之和最高,而真菌数量相对较少。筛选出9种地黄重茬土壤中特有的根系残留化合物,其中,在生物菌肥浓度为0.05%时,化合物2的含量最低;化合物5和化合物6含量在生物菌肥浓度为0.10%、施用60 d的条件下最低。可见,外施生物菌肥可有效改变地黄重茬土壤微生物数量和降解重茬土壤中根系残留的某些化合物,有效缓解地黄的连作障碍。

硫酸-香草醛法测定地黄总环烯醚萜苷含量

建立地黄(Rehmannia glutinosa)不同部位总环烯醚萜苷的含量测定方法并检测7份地黄种质的总环烯醚萜苷含量。以梓醇为对照品,采用硫酸-香草醛显色法于510 nm对地黄叶片和根中的总环烯醚萜苷含量进行测定。结果表明,该方法在0.01~0.25 mg/mL线性关系良好,回归方程A=1.1917ρ-0.0035,相关系数R2=0.9999。样品平均回收率为100.65%,RSD为1.61%。地黄叶片总环烯醚萜苷的含量普遍高于根含量。试验建立的方法简单快捷、准确,可以用于对地黄根和叶片中总环烯醚萜苷进行含量分析和质量评价。

地黄不同炮制品指纹图谱模式识别及多成分含量测定

建立地黄(Rehmannia glutinosa Libosch.)不同炮制品(鲜地黄、地黄片、生地黄、熟地黄)HPLC指纹图谱及11个指标成分含量测定方法,指认11个共有峰,结合主成分分析(PCA)法和聚类分析(HCA)法分析炮制过程中化学成分变化趋势,为地黄不同炮制品质量评价及药理药效研究奠定基础。结果表明,HPLC指纹图谱结合多指标成分含量测定的方法适用于地黄不同炮制品的HPLC指纹图谱分析,PCA和HCA结果显示,熟地黄明显区别于鲜地黄、地黄片、生地黄,鲜地黄和地黄片在PCA中分为2类,HCA中鲜地黄和地黄片聚为一类;11种指标成分线性关系良好,R^(2)≥0.9995,11种指标成分含量变化趋势各不相同。HPLC指纹图谱结合多指标成分含量测定的方法反映了地黄炮制过程中的化学成分变化,适用于不同炮制阶段地黄的质量评价,为药理药效研究及质量标记物的发现奠定研究基础。

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.

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.

A new megastigmane from fresh roots of Rehmannia glutinosa

A new megastigmane,rehmamegastigmane(1),together with eighteen known compounds lariciresinol(2),lariciresinol-4′-O-β-D-glucopyranoside(3),hierochin D(4),yemuoside YM1(5),darendoside B(6),decaffeoylacteoside(7),jionoside B1(8),catalpol(9),ajugol(10),6-O-vanilloylajugol(11),6-O-E-feruloylajugol(12),rehmapicroside(13),rehmapicrogenin(14),3-methoxy-2,6,6-trimethylcyclohexane-1-enecarboxylic acid(15),vanillic acid(16),hydroferulic acid(17),threo-1-(4-hydroxy-3-methoxyphenyl)-1,2,3-propanetriol(18),p-hydroxyphenylethyl alcohol(19)was isolated from the fresh roots of Rehmannia glutinosa.Compounds 2–6 and 16–18 were isolated from this plant for the first time.

UPLC-Q-TOF/MS-based Metabolic Profiles of Bioactive Components in Rehmannia glutinosa and Cornus officinalis Herb Pair by Rat Intestinal Bacteria

Objective To investigate the metabolic routes and metabolites of Rehmannia glutinosa and Cornus officinalis herb pair produced by gut microbiome from rats.Methods A rapid and sensitive ultra-performance liquid chromatography/quadrupole-time-offlight mass spectrometry（UPLC-Q-TOF/MS） technique combined with Metabolynx？software was established and successfully applied to identify the metabolites of the main bioactive components in the herb pair extract by rat intestinal bacteria.Results Four parent compounds（loganin,morroniside,catalpol,and acteoside） and their eight corresponding metabolites were detected and tentatively identified by the characteristics of their protonated ions.Hydrogenated and demethylated loganetin,dehydroxylated morronisid aglycone,caffeic acid,and its methylated product were the main metabolites.These metabolites suggested that the glycosides were firstly hydrolyzed to their aglycones by hydrolytic enzymes of the enteric microbial flora and subsequently to the other metabolites through hydrogenation,（de）-methylation,and de-hydroxylation.Conclusion The results may be helpful for the further investigation of the pharmacokinetic study of R.glutinosa and C.officinalis herb pair in vivo.

A NEW ALKALOID FROM THE ROOTS OF REHMANNIA GLUTINOSA

The present phytochenucal study was undertaken to investigate the chemical constituents of the 95%EtOH extract of the dried roots of Rehmannia glutinosa.The compounds were isolated and purified by Diaion HP-20,Toyopearl HW-40,silica gel column chromatography and preparative HPLC,and the structures were identified on

地黄己糖激酶RgHXK2基因的电子克隆及生物信息学分析

己糖激酶(HXK)的主要功能是在糖酵解途径中催化己糖磷酸化,是植物体内调控呼吸代谢的关键酶之一。主要催化己糖磷酸化进入糖酵解途径,可在植物体的生长发育过程中为其提供大量的能量。己糖激酶属于hexokinase-2超家族,在植物生命活动中发挥着重要的作用。为了解地黄(Rehmannia glutinosa)己糖激酶基因RgHXK2在磷酸化过程中起到的作用,该研究基于转录组测序获得的地黄己糖激酶基因ORF的部分保守序列和地黄的SRA数据库,在NCBI中进行电子克隆获得地黄RgHXK2全长cDNA序列;采用在线网上工具ProtParam、TMHMM、SOPMA、SMART、MEME、Clustal Omega以及Jalview、MegaX等软件对其进行生物信息学分析,结果显示,地黄RgHXK2基因全长1 462 bp,包含1 194 bp的开放阅读框,编码一个含有397个氨基酸、相对分子质量为43.75 kD、等电点为6.34的蛋白质。在线软件预测该蛋白含有Hexokinase-1和Hexokinase-2保守结构域,无信号肽与跨膜结构,为亲水性非分泌蛋白,主要分布于线粒体和叶绿体中,参与碳水化合物代谢、细胞葡萄糖的动态平衡以及糖酵解等三个生物反应过程。系统进化分析表明,地黄与木犀榄(Olea europaea)、一串红(Salvia splendens)、黄色猴面花(Erythranthe guttata)、紫花风铃木(Handroanthus impetiginosus)及松蒿(Phtheirospermum japonicum)、独脚金(Striga asiatica)聚为一支,其中与木犀榄HXK1蛋白的同源性最高,为90.05%。本研究为进一步揭示RgHXK2基因在地黄己糖磷酸化途径中的功能奠定基础。

核桃与地黄套种栽培技术初探

结合核桃(Juglans regia L.)前期投入大、无收益,中药材地黄(Rehmannia glutinosa Libosch.)产生效益快的特点,介绍了适宜十堰地区大面积种植的‘清香’核桃与地黄套种的实用栽培技术,以供种植户交流参考。