Full-length transcriptome sequence and identification of genes involved in phenylethanol glycoside biosynthesis in Rehmannia glutinosa / 药学学报

italic>Rehmannia glutinosa belongs to the Scrophulariaceae family with important medicinal value. In order to effectively explore the transcriptome information of R. glutinosa and identify the genes encoding enzymes involved in phenylethanol glycoside (PhGs) biosynthesis, the leaves, stems and tuberous roots of R. glutinosa were used for transcriptome sequencing using Pacific Biosiences RS II platform. A total of 27 773 transcripts were generated with an average length of 2 380 bp, and 27 236 coding sequences (CDS) were predicted. Using BLAST software, non-redundant transcript sequences were annotated with NR, NT, GO, COG, KEGG, SwissProt and Interpro databases and a total of 27 399 annotated genes were obtained. Among them, the number of genes related to Sesamum indicum in the NR database was the highest (81.44%), which is consistent with their evolutionary relationship. Enzymes likely involved in the biosynthesis of isoacteoside, echinacoside, cistanosides A, cistanosides F, 2′-acetylacteoside and leonoside F were identified, and 143 genes were identified in R. glutinosa full-length transcriptome. The expression levels of 19 genes correlated with acteoside content in twelve tissues of R. glutinosa, and most showed higher expression levels in leaf tissues and floral organs. This study provides more reliable transcriptome data for screening R. glutinosa for functional genes and provides a foundation for the study of the molecular mechanisms of PhGs biosynthesis.

Identification and biological characteristics of a pathogen causing leaf blight of Rehmannia glutinosa / 中国中药杂志

Leaf blight outbroke in Rehmannia glutinosa plantation in Wenxian county, Henan province in 2019. R. glutinosa plants with diseased leaves were collected from the plantation, and three strains were isolated from the diseased leaf samples. Pathogenicity test, morphological observation, and phylogenetic analysis of ITS, EF1-α, and Tub suggested that they were respectively Fusarium proliferatum, F. oxysporum, and F.acuminatum. Among them, F. acuminatum, as a pathogen of R. glutinosa leaf disease, had never been reported. To clarify the biological characteristics of F. acuminatum, this study tested the influence of light, pH, temperature, medium, carbon source, and nitrogen source on the mycelial growth rate of the pathogen during a 5-day culture period, and explored the lethal temperature. The results showed that the mycelia grew well under the photoperiod of 12 h light/12 h darkness, at 5-40 ℃(optimal temperature: 25 ℃), at pH 4-11(optimal pH: 7.0), on a variety of media(optimal medium: oatmeal agar), and in the presence of diverse carbon and nitrogen sources(optimal carbon source: soluble starch; optimal nitrogen source: sodium nitrate). The lethal temperature was verified to be 51 ℃(10 min). The conclusion is expected to lay a scientific basis for diagnosis and control of R. glutinosa leaf diseases caused by F. acuminatum.

Acetylation of Rehmannia glutinosa polysaccharides and antioxidant activity of acetylated derivatives / 中国中药杂志

This study aims to acetylate Rehmannia glutinosa polysaccharides by acetic anhydride method, optimize process parameters and evaluate their antioxidant activity. With the degree of substitution(D_s) as a criterion, the effects of reaction time, acetic anhydride-to-polysaccharides ratio and temperature were investigated. Process parameters were optimized by single-factor experiment and response surface methodology. The infrared spectroscopy(IR) and scanning electron microscopy(SEM) proved the successful acetylation and were employed to preliminarily analyze the structural characteristics of acetylated derivatives. The results showed that the D_s was 0.327 under the optimal technological conditions, including m(acetic anhydride):m(R. glutinosa polysaccharides)=2.70, reaction time 3.0 h and temperature 48 ℃. Further, the antioxidant properties of acetylated derivatives were investigated in vitro and acetylation was found effective to improve the antioxidant activity of R. glutinosa polysaccharides. This study provides a reference for the further development and application of R. glutinosa polysaccharides.

Evaluation of the quality of different germplasms of Rehmannia glutinosa based on iridoid glycosides / 中国药房

OBJECTIVE To evaluate the quality of different germplasms of Rehmannia glutinosa based on iridoid glycosides. METHODS The contents of total iridoid glycosides ，catalpol，rehmaionoside D ，rehmaionoside A ，and leonuride in 18 batches of R. glutinosa from 6 germplasms（85-5，JinJiu，BX，BJ-1，Shandong，QH-1）were determined by ultraviolet spectrophotometry and high performance liquid chromatography. After normalization of the above content determination results ，the quality of different germplasm of R. glutinosa were evaluated by multiple statistical methods such as cluster analysis ，factor comprehensive analysis and partial least squares discriminant analysis （PLS-DA）. RESULTS Among 6 germplasms of R. glutinosa ，the content of total iridoid glycosides in R. glutinosa 85-5 was the highest ，and the content of catalpol in R. glutinosa BX was the highest ；the contents of rehmannioside D and rehmannioside A in R. glutinosa JinJiu were the highest ，and the content of leonuride in R. glutinosa BX was the highest. Cluster analysis showed that R. glutinosa JinJiu were clustered into one category ，R. glutinosa BX clustered into one category ，R. glutinosa Shandong and R. glutinosa BJ-1 were clustered into one category ，and R. glutinosa QH-1 and 85-5 were clustered into one category. Through factor comprehensive analysis ，there were differences in the quality of different germplasms of R. glutinosa . The comprehensive score of R. glutinosa BX，Shandong，85-5，BJ-1，QH-1，JinJiu were 2.283 9，1.689 1，1.664 8， 1.503 3，1.469 0，1.214 6，respectively. PLS-DA showed that variable importance projection value of total iridoid glycosides ， catalpol and leonuride were all higher than 1. CONCLUSIONS The quality difference of R. glutinosa from different germplasms may be caused by total iridoid glycosides ，catalpol and leonuride.

Analysis of Volatile Organic Compounds in Rhizosphere Soil of Rehmannia glutinosa Under Intercropping Zea mays Based on GC-MS Metabonomics / 中国实验方剂学杂志

Objective:To explore the composition characteristics of rhizosphere soil under <italic>Rehmannia glutinosa-Zea mays</italic> intercropping model，and screen out special signal substances in rhizosphere soil of <italic>R. glutinosa</italic> under intercropping <italic>Z. mays</italic>， so as to provide the basis for the study of allelopathic substances in continuous cropping obstacle of <italic>R. glutinosa</italic>. Method:In this experiment，rhizosphere soils of <italic>R. glutinosa</italic> under <italic>Z. mays </italic>intercropping and <italic>R. glutinosa </italic>single cropping models in July，August，September and October were taken as the research objects， and the volatile organic compounds in ethyl acetate fraction were analyzed by gas chromatography-mass spectrometry （GC-MS）. Principal component analysis （PCA）， hierachical cluster analysis （HCA） and orthogonal partial least squares discriminant analysis （OPLS-DA） analysis were performed on the data by SIMCA 14.1 to screen out potential differences in volatile organic compounds between the two models. Result:The types of volatile organic compounds in intercropping and single cropping models were mainly hydrocarbons， alcohols， esters， ketones， amides， acids and other substances. Specifically， the average relative contents of hydrocarbons，esters and amides in intercropping model were 58.46%，32.15% and 5.42% respectively，while the relative contents of hydrocarbons，esters and amides in single cropping model were 37.27%，36.11% and 21.13%. The results of PCA and HCA showed that the characteristics of volatile organic compounds in the ethyl acetate fraction of rhizosphere soil under intercropping and single cropping models could be clearly divided into two categories，the screening results of potential differential components based on OPLS-DA analysis indicated that various components， such as dibutyl phthalate，（<italic>Z</italic>）-9-oleamide，<italic>β</italic>-caryophyllene，dioctyl iso-phthalate， phthalate （2-propylamyl） diester， <italic>n</italic>-hexadecane，octodecane， <italic>n</italic>-heneicosane， were screened from rhizosphere soil under the two models. Conclusion:The <italic>R. glutinosa-Z. mays</italic> intercropping model has certain effects on the volatile organic compounds in the rhizosphere soil of <italic>R. glutinosa</italic>，and the effect of the selected components on the growth and quality characteristics of <italic>R. glutinosa</italic> still need to be further studied.

Effects of density on growth and gene transcription characteristics of Rehmannia glutinosa / 中国中药杂志

The present study analyzed the effects of planting density on the development, quality, and gene transcription characte-ristics of Rehmannia glutinosa using 85-5 and J9 as materials with three planting densities of 5 000, 25 000, and 50 000 plants/Mu(1 Mu≈667 m~2). The agronomic characteristics of leaves and tuberous roots, the content of catalpol and acteoside, and the changes of gene expression were determined. The results showed that the leaf size, the diameter of tuberous root, leaf biomass, tuberous root number, and tuberous root biomass per plant at low density were significantly higher than those of medium and high densities. The content of catalpol and acteoside in leaves was higher at high density. The content of catalpol in tuberous roots was higher at low density, and the change trend was similar to that in leaves, while the content of acteoside in tuberous roots was higher at high density. Transcriptome analysis found that about 1/2 of the expansin genes could change regularly in response to density treatment, which was rela-ted to the development of tuberous roots. The change trend of the gene expression of multiple catalytic enzymes involved in the biosynthesis of catalpol and acteoside was consistent with that of their content, which was presumedly involved in the accumulation and regulation of density-responsive medicinal components. Based on the analysis of the development, medicinal components, and gene expression characteristics of R. glutinosa at different densities, this study is expected to provide an important basis for regulating the quality and yield of medicinal materials of R. glutinosa by managing the planting density.

Identification and expression analysis of NRT1 family genes in Rehmannia glutinosa / 中国中药杂志

NRT1 family proteins play an important roles for absorbing and transporting of nitrate in different plants. In order to identify the NRT1 family genes of Rehmannia glutinosa, this study used 11 NRT1 homologous proteins of Arabidopsis as probe sequences and aligned with the transcriptome data of R. glutinosa by using NCBI BLASTN software. Resulting there were 18 NRT1 proteins were identified in R. glutinosa. On basis of this, a series of the molecular characteristics of R. glutinosa NRT1 proteins including the conserved domains, the transmembrane structure, the subcellular location and phylogenetic features were in detail analyzed. At same time, it were systematically analyzed that the temporal and spatial expression patterns and characteristics of R. glutinosa NRT1 family genes in response to different stress factors. The results indicated that 18 R. glutinosa NRT1 family genes with the length of coding region from 1 260 bp to 1 806 bp, encoded proteins ranging from 419 to 601 amino acids, and all of they owned the domains of typical peptide transporter with 7 to 12 transmembrane domains. These R. glutinosa NRT1 family proteins mostly were found to locate on cellular plasma membrane, and belonged to the hydrophobic proteins. Furthermore, the evolutionary analysis found that the 18 R. glutinosa NRT1 protein family could be divided into two subfamilies, of which 14 NRT1 family genes might occur the positive selection, and 4 genes occur the passivation selection during the evolution process of R. glutinosa. In addition the expression analysis showed that 18 R. glutinosa NRT1 family genes have the distinct expression patterns in different tissues of R. glutinosa, and their expression levels were also obvious difference in response to various stress. These findings infield that 18 R. glutinosa NRT1 family proteins might have obviously different functional roles in nitrate transport of R. glutinosa. In conclusion, this study lays a solid theoretical foundation for clarifying the absorption and transport molecular mechanism of N element during R. glutinosa growth and development, and at same time for deeply studying the molecular function of R. glutinosa NRT1 proteins in absorption and transport of nitrate.

Isolation and identification of a new phytopathogen causing root rot of Rehmannia glutinosa / 中国中药杂志

Root rot was occurred widely in the production area of Rehmannia glutinosa, and which result in serious influence on the yield and quality of R. glutinosa. In the present work, a new phytopathogen was isolated from roots with root rot symptom in the production area of R. glutinosa. The colony of the pathogen growing on PDA medium was gray-black, the structure of hyphae was compact, the aerial hyphae was less developed, and the back of the colony was black. The hyphae of the pathogen were uneven in size, about 2 to 3 μm in diameter and twined with each other, the conidia of the pathogen were small, nearly round and about 1 μm in diameter. The healthy roots of R. glutinosa were inoculated with the pathogen in vitro, black-brown rot was observed at the inoculate sites after a few days' incubation. The rhizosphere soil of healthy R. glutinosa seedlings were inoculated in vivo, the leaves were wilted and the roots were black-brown rotted after several days' normal culture, the symptoms were consistent with those observed in the field. The genomic DNA of the pathogen was amplified by fungus rDNA-ITS universal primer ITS1/ITS4 and homologous analyzed, the pathogen was in a branch with Heterophoma sp., Phoma sp., P. novae-verbascicola and P. herbarum with the nuclear acid homology of 99.21% to 99.43%. The pathogen shown 97.00% to 98.02% nuclear acid homology with H. verbascicola, H. novae-verbascicola, H. poolensis, P. herbarum, H. sylvatica, H. verbascicola and H. verbasci-densiflori when amplified by the tub2 gene special primer Btub2 fd/Btub4 rd, and H. novae-verbascicola was the highest. The pathogen was in a branch with H. novae-verbascicola when amplified by the lsu gene special primer LR0 R/LR7. Based on the morphological characteristics, nucleotide sequence analysis and Koch's test results, the isolated pathogen causing root rot of R. glutinosa was identified as H. novae-verbascicola. This study is of great significance for the further theoretical research on root rot of R. glutinosa and root rot control in field.

Effect of catalpol from the root of rehmannia glutinosa on the expression of inflammatory proteins in synovium of the knee joint in rats with early osteoarthritis / 中国组织工程研究

BACKGROUND: Inflammatory transmitters secreted from the synovium may be one of the important factors inducing the onset of knee osteoarthritis, and further exacerbate knee osteoarthritis. OBJECTIVE: To observe the effect of different concentrations of catalpol from the root of Rehmannia glutinosa on the expression of interleukin-1β, galectin 3 and S100A12 in the synovium of the knee joint in rats with early knee osteoarthritis, and to explore the mechanism of catalpol from the root of Rehmannia glutinosa in the treatment of early knee osteoarthritis. METHODS: The 14 of 48 adult male Wistar rats were randomly selected as a normal control group, and the remaining 34 rats were injected with 4% papain and 0.03 mol/L cysteine solution (0.2 mL) into the right knee joint cavity at 1, 4, 7 days after the initialization of the experiment to duplicate the model of early knee osteoarthritis. At 10 days after the initialization of the experiment, 4 rats in each group were randomly selected for histological observation using hematoxylin-eosin staining and validation of the model using Mankin and OARSI grading evaluation. After the model was successfully verified, the remaining 30 rats in the model group were randomly divided into model control group, low-dose group and high-dose group. At 2 days after successful modeling, each treatment group was infused with corresponding drugs at a dose of 0.2 mL/kg, and normal control group and model control group were infused with normal saline, once a day for 28 consecutive days. The synovium of the right knee joint was then taken from rats in each group, and the expression of interleukin-1β, galectin 3 and S100A12 in the rat synovium was detected by ELISA. RESULTS AND CONCLUSION: Mankin score and OARSI grading were significantly higher in the model control group than the normal control group (P < 0.05). The levels of S100A12, interleukin-1β and galectin 3 in the synovium were significantly higher in the model control group than the normal control group (P < 0.01). Compared with the model control group, the levels of S100A12, interleukin-1β and galectin 3 in the synovium was decreased somewhat in the low-dose group (P < 0.05), but significantly decreased in the high-dose group (P < 0.01). Therefore, catalpol from the root of Rehmannia glutinosa can delay the progression of knee osteoarthritis by reducing the level of inflammatory factors in the synovium.

Protection of inflammatory osteoblasts in neonatal rats using catalpol from the root of rehmannia glutinosa / 中国组织工程研究

BACKGROUND: Studies have shown that abnormal osteoblast metabolism may cause abnormal subchondral bone mineralization in knee osteoarthritis. OBJECTIVE: To verify the effects of catalpol from the root of Rehmannia glutinosa on osteoblast proliferation and drug toxicity, and the anti-inflammatory protective effect on inflammatory osteoblasts induced by lipopolysaccharide. METHODS: (1) Osteoblasts from newborn Sprague-Dawley rats were primarily extracted, cultured, and passaged. The activity and proliferation of osteoblasts were determined by observing the morphology of osteoblasts alkaline phosphatase staining and staining of mineralized nodules. (2) Cell counting kit-8 (CCK-8) method was used to observe the effect of different concentrations of catalpol from the root of Rehmannia glutinosa on osteoblast activity. (3) Different concentrations of lipopolysaccharide (0, 10, 20, 40, 80, 160 mg/L) were used to induce inflammatory osteoblasts in fetal rats. CCK-8 method was used to verify the best concentration. (4) The experiment was divided into three groups: blank group, model group, low-, medium- and high-concentration catalpol groups. Inflammatory osteoblasts were induced by lipopolysaccharide at 80 mg/L, and then treated for 8 hours. CCK-8 method was used to observe the effect of different concentrations of catalpol on inflammatory osteoblasts. The study protocol was approved by the Medical Ethics Committee of Liuhe District People’s Hospital in Nanjing. RESULTS AND CONCLUSION: When osteoblasts were cultured to the fourth generation, followed by alkaline phosphatase staining, black grey granules were found in the cytoplasm of the positive cells, and the morphology of the cells was irregular. When the concentration of catalpol was lower than 1 mg/L, it had no significant effect on osteoblasts; when it was higher than 10 mg/L, it had a significant effect on the proliferation of osteoblasts but no drug toxicity (P < 0.05). When the concentration of lipopolysaccharide was higher than 80 mg/L, there was a significant trend of inflammatory damage to osteoblasts (P < 0.01). Therefore, 80 mg/L was selected as the best injury concentration in this experiment. When the concentration of catalpol from the root of Rehmannia glutinosa was lower than 1 mg/L, it had no significant protective effect on the inflammatory response of osteoblasts (P < 0.05); when it was higher than 10 mg/L, it had significant protective effect on the inflammatory response of osteoblasts (P < 0.05). Therefore, when the concentration of catalpol from the root of Rehmannia glutinosa reaches a certain level, it has no toxic effect on osteoblasts, promotes proliferation, and has anti-inflammatory protective effect on inflammatory osteoblasts induced by lipopolysaccharide.

Cloning, subcellular location and expression analysis of an acteoside synthase gene from Rehmannia glutinosa / 中草药

Objective: To clone the acteoside synthase gene (RgAcS1) from Rehmannia glutinosa, and analyze its subcellular localization and expression pattern. Methods: The cDNA sequence of RgAcS1 was identified based on the annotation of the transcriptome data of R. glutinosa, and the RgAcS1 gene was cloned by polymerase chain reaction (PCR). Constructing the GFP fusion expression vector and observing the subcellular localization of RgAcS1 mediated by Agrobacterium tumefaciens. The expression pattern of RgAcS1 in different parts of tuberous root of R. glutinosa was detected by real-time fluorescence quantitative PCR (qRT-PCR). Results: A full-length coding sequence of a shikimate-O-hydroxy cinnamoyl transferase from R. glutinosa was obtained and named RgAcS1. The length of the RgAcS1 cDNA was 1659 bp, including an open reading frame (ORF) of 1 296 bp, encoding 431 amino acid residues, the molecular weight of the protein was 475 900, and it has a typical domain of shikimic acid-O-hydroxy cinnamoyl transferase. The result of subcellular localization showed that RgAcS1 was mainly distributed in cytoplasm and also in nucleus. The qRT-PCR analysis showed that the expression levels of RgAcS1 were higher in the periderm and root hair of R. glutinosa tuberous root, but lower in the xylem and phloem. The expression levels of RgAcS1 were higher in non-radial striation than that in radial striation of BJ1, QH1 and 85-5. Conclusion: In this study, we obtained the cDNA sequence of RgAcS1, and analyzed the subcellular location and expression patterns of RgAcS1, which will lay foundations for further study on roles of RgAcS1 gene in the synthesis of acteoside in R. glutinosa.

Genetic diversity analysis on cultivar and wild population of Rehmannia glutinosa based on ISSR markers / 中草药

Objective: To study the genetic diversity and genetic relationship of cultivar and wild population of Rehmannia glutinosa and its relative species by ISSR molecular marker technique, and provide the reference for R. glutinosa germplasm protection and breeding. Methods: 106 samples including cultivar and wild population of R. glutinosa and its relative species were studied by ISSR-PCR markers. Nei's genetic diversity index (H) and other parameters of genetic information were calculated by POPGEN 32, and a cluster dendrogram of different samples was established based on the unweighted pair-group method with arithmetic mean (UPGMA) by NTSYS-pc software. Results: Seven ISSR primers generated 85 loci of which 83 loci were polymorphic. The percentage of polymorphie bands (PPB) of all samples was 97.65%. Nei's genetic diversity index (H) and Shannon's information index (I) were 0.2659 and 0.4125. The percentage of polymorphie bands (PPB) of cultivar of R. glutinosa was 30.59%. The percentage of polymorphie bands (PPB) of wild population of R.glutinosa in Henan province was 83.53%. In the cluster dendrogram, all samples were clustered into seven groups at the level of Genetic similarity coefficient (GS) 0.67. Conclusion: The results of ISSR analysis revealed that the level of genetic diversity between wild populations of R. glutinosa was higher than that within cultivar populations of R. glutinosa. The genetic diversity among wild populations of R. glutinosa in Henan province was higher than other region, which was consistent with authentic producing areas of R. glutinosa in this area. The relationships of wild population of R. glutinosa had no obvious correlation with their geographical distribution pattern.

Comparison of Antioxidant Effect of Rehmannia glutinosa Decoction Pieces and Its Polysaccharides on Aging Model Rats before and after Processing Based on the Characteristic Ancient Method / 中国药房

OBJECTIVE：To compare the difference in the antioxid ant effect of fresh Rehmannia glutinosa ，dried R. glutinosa ， R. glutinosa preparata during ancient characteristic processing and its polysaccharides before and after processing on aging model rats，and to provide reference for the processing of R. glutinosa . METHODS ：The sample of R. glutinosa preparata was prepared according to ancient characteristic method. During the processing ，the fresh and dried R. glutinosa samples were retained. Then crude polysaccharide were extracted from fresh R. glutinosa and Rehmanniae radix preparata by water extraction and alcohol precipitation. Totally 96 rats were divided into blank group （water），model group （water），positive control group [vitamine C ，100 mg/（kg·d）]，fresh R. glutinosa group [ 700 mg/（kg·d）]，dried R. glutinosa group [ 135 mg/（kg·d）] ，Rehmanniae radix preparata group [ 135 mg/（kg·d）]，fresh R. glutinosa polysaccharide group [ 1 400 mg/（kg·d），by the weight of fresh R. glutinosa ] and Rehmanniae radix preparata polysaccharide group mg/（kg·d），by the weight of Rehmanniae radix preparata] ， with 12 rats in each group. Except for blank group ，other 制。E-mail：zhouyan1221@163.com groups were given D-galactose [ 125 mg/（kg·d）] on neck and back to induce sub-acute aging model. At the same time ，they were given relevant medicine in tragastrically，once a day ，for consecutive 56 days. After last admin istration，the liver ，brain，kidney，spleen，heart and thymus indexes were determined. The total antioxidant capacity （T-AOC），superoxide dismutase （SOD）activity，catalase（CAT）activity and MDA content in serum ， liver，brain and kidney were determined. RESULTS ：Compared with blank group ，organ indexes of rats in the model group were decreased significantly （P＜0.05 or P＜0.01）；T-AOC，SOD activity and CAT activity in serum ，brain，liver and kidney tissue were decreased significantly （P＜0.01），while MDA content increased significantly （P＜0.01）. Compared with model group ，the organ indexes of brain ，liver and kidney ，SOD activity in serum and kidney of fresh R. glutinosa group were not significantly increased （P＞0.05）；kidney index ，T-AOC in serum and brain ，SOD activity in serum ，liver and kidney tissue were not significantly increased in the dried R. glutinosa group（P＞0.05）；kidney index ，T-AOC in serum and cerebral tissue ，SOD activity in serum were not significantly increased in fresh R. glutinosa group（P＞0.05）；other organ indexes ，T-AOC，SOD activity and CAT activity in serum and tissues were increased significantly in other groups （P＜0.05 or P＜0.01），while MDA content in serum and tissues were decreased significantly in all administration groups （P＜0.05 or P＜0.01）. Compared with fresh R. glutinosa group，T-AOC in serum was decreased significantly in dried R. glutinosa group（P＜0.01），and there was no significant difference in other indexes （P＞0.05）；kidney and spleen indexes of rats in Rehmanniae radix preparata group were increased significantly （P＜0.05），T-AOC in renal tissue ，SOD activity in serum ，cerebral tissue and renal tissue ，CAT activity in cerebral and liver tissue were increased significantly （P＜0.05 or P＜0.01），while MDA in cerebral and liver tissue were significantly decreased （P＜ 0.01）. CAT in cerebral tissue and liver tissue of rats in Rehmanniae radix preparata group were significantly higher than those in positive control group （P＜0.01）. Compared with fresh R. glutinosa polysaccharide group ，spleen and renal indexes of rats in Rehmanniae radix preparata group were increased significantly （P＜0.05 or P＜0.01），T-AOC，SOD activity and CAT activity in serum and cerebral ，liver，renal tissues were increased significantly （P＜0.05 or P＜0.01）. T-AOC and CAT activity of cerebral ， liver and renal tissues in Rehmanniae radix preparata group were all significantly higher than those in positive control group （P＜ 0.05 or P＜0.01）. CONCLUSIONS ：In the aspect of increasing organ index and improving the activity of antioxidant enzymes in serum，cerebral，liver and

Xijiao Dihuang Decoction () and Rehmannia glutinosa Libosch. protect mice against lipopolysaccharide and tumor necrosis factor alpha-induced acute liver failure / 中国结合医学杂志

OBJECTIVE@#To investigate the hepatoprotective effect of Xijiao Dihuang Decoction (, 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 significantly inhibited apoptosis of hepatocytes and enhanced expression of the antiapoptosis genes, c-Flip, Iap1, Gadd45b and A20. In addition, Rehmannia glutinosa Libosch. was identified 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 κ B-regulated anti-apoptotic genes. Rehmannia glutinosa Libosch. is the effective herb of XJDHD and galactose is an active component in this protection.

Molecular cloning and expression analysis of iridoid synthase genes from Rehmannia glutinosa / 中国中药杂志

Iridoid synthase( IS),the key enzyme in the natural biosynthesis of vegetal iridoids,catalyzes the irreversible cyclization of 10-oxogeranial to epi-iridodial. In this study,we screened the Rehmannia glutinosa transcriptome data by BLASTn with Catharanthus roseus CrIS cDNA,and found four c DNA fragments with length of 1 527,1 743,1 425,1 718 bp,named RgIS1,RgIS2,RgIS3 and RgIS4,respectively. Bioinformatics analysis revealed that the four iridoid synthase genes encoding proteins with 389-392 amino acid residues,protein molecular weights were between 44. 30-44. 74 k Da,and theoretical isoelectric points were between 5. 30 and 5. 87. Subcellular localization predictions showed that the four iridoid synthase were distributed in the cytoplasm. Structure analysis revealed that R. glutinosa iridoid synthases contain six conserved short-chain dehydrogenase/reductase( SDR) motifs,and their 3 D models were composed typical dinucleotide-binding " Rossmann" folds covered by helical C-terminal extensions. Using the amino acid sequences of four R. glutinosa iridoid synthases,phylogenetic analysis was performed,the result indicated that RgIS3,CrIS and Olea europaea OeIS were grouped together,the other R. glutinosa iridoid synthases and fifteen proteins in other plants had close relationship. Real-time fluorescent quantitative PCR revealed that RgIS1 and RgIS3 highly expressed in unfold leaves,however,RgIS2 and RgIS4 highly expressed in stems and tuberous roots,respectively. RgIS3 showed higher expression levels in non-radial striations( nRS) of the two cultivars,and RgIS1 and RgIS2 had higher expression levels in nRS of QH,while RgIS4 had less expression levels in nRS of QH1. RgIS1,RgIS2 and RgIS3 were up-regulated by Me JA treatment,although the time and degree of response differed. Our findings are helpful to reveal molecular function of R. glutinosa iridoid synthases and provide a clue for studing the molecular mechanism of iridoid biosynthesis.

Cloning and expression analysis of LncRNA-RgAGT2 responding to consecutive monoculture in Rehmannia glutinosa / 中国中药杂志

The consecutive monoculture obstacle is a major problem in the field of Rehmannia glutinosa( R. glutinosa),has severely declined the yield and quality of R. glutinosa. Here,using hi TAIL-PCR and RACE techniques,we have cloned the full-length transcript( 1 573 bp) of Unigene 29334＿All screened by DGE as a consecutive monoculture obstacle response gene of R. glutinosa. Based on ORF Finder prediction,all ORFs detected in the full-length transcript were less than 300 nt,which suggested that the above transcript was confirmed to be a long non-coding RNA( LncRNA). With alignment in R. glutinosa transcriptome,this LncRNA was partially homologous to alanine glyoxylate transaminase 2 gene( Rg AGT2),which was named LncRNA-RgATG2. To further explore the function of LncRNA-RgAGT2,we have examined expression patterns of LncRNA-RgAGT2 and Rg AGT2 at five critical development stages( seedling,elongation,pre-expanding,mid-expanding,late-expanding) in the first and second year replanting of R. glutinosa,respectively. The results indicated that LncRNA-RgAGT2,as a potential regulator,is possible to play a vital role in Rg AGT2 expression regulation. Meanwhile,LncRNA-RgAGT2 has presented significant variation in all development stages of R. glutinosa,which could be used as a " diagnostic label" to assess consecutive monoculture obstacle. This study,for the first time,showed that LncRNA was responsible for the response and regulation of consecutive monoculture obstacle,which would be a powerful supplement to reveal the molecular mechanisms of consecutive monoculture obstacle of R. glutinosa.

Effects of shading on tuberous root traits, photosynthetic characteristics and gene transcription of Rehmannia glutinosa / 中草药

To reveal the effects of shading on tuberous root and photosynthetic characteristics of Rehmannia glutinosa, and analyze the molecular mechanism of shading affecting the expansion of R. glutinosa tuberous root by transcriptome sequencing. Methods: R. glutinosa plants were treated with full light, 60% shading and 90% shading. High-throughput sequencing was used to sequence the transcriptome of the R. glutinosa tuberous roots treated with full-light and 90% shading, and the differentially expressed genes were screened out. The expression characteristics of some genes in tuberous roots and leaves were analyzed by real-time fluorescence quantitative PCR. Results: After shading, the number of parenchyma cell layers in the tuberous roots was decreased, but the proportion of ducts was increased, the length, diameter and fresh weight of tuberous roots were decreased significantly, and the tuberous roots barely expanded under 90% shading treatment. The number of parenchyma cell was decreased and the proportion of duct was increased in root tubers of R. glutinosa. With the increase of shading degree, the content of chlorophyll a and b and total chlorophyll content were gradually decreased, and photosynthetic capacity was decreased. A total of 3 348 differentially expressed genes were obtained by transcriptome analysis, of which 1 396 were down-regulated and 1 952 were up-regulated. Through enrichment analysis of KEGG metabolic pathway, 1 668 differentially expressed genes (53.4%) were enriched into 117 metabolic pathways, and 17 of them were significantly enriched pathways. The plant hormone signaling pathway was enriched firstly, followed by the plant pathogen interaction pathway, the phenylethanoid glycoside biosynthesis pathway, starch and sucrose metabolic pathways were also enriched significantly. In the hormone signaling pathway, most of different expressed genes were up-regulated. Eleven expansin genes were down-regulated under 90% shading, only five expansin genes were up-regulated. Two of beta-amylase genes (Bmy) related to starch degradation were up-regulated when shading treated, while the sucrose phosphate synthase genes (SPS) were down-regulated. Most of the genes involved in lignin synthesis were down-regulated and most of the genes involved in cellulose synthesis were up-regulated. Conclusion: The photosynthetic capacity of R. glutinosa was decreased under shading conditions, and led to the accumulation of photosynthate decreased in its leaf and tuberous root. R. glutinosa plant responded to shading by regulating the differential expression of a series of hormone pathway genes, which prevent the expansion of tuberous roots.

HPLC fingerprint and catalpol content determination of Rehmannia glutinosa / 中草药

Objective: The fingerprint of Rehmannia glutinosa was established by HPLC to provide scientific basis for improving the quality of R. glutinosa. Methods: The characteristic silicagel C18 column-Atlantis T3 was used, and the mobile phase was 0.1% phosphate water and acetonitrile by gradient elution. Absorption wavelength was 203 nm, and flow rate was 1.0 mL/mL with column temperature of 35 ℃. Setting catalpol as the control peak, 20 batches of R. glutinosa fingerprint was established. Fingerprint similarity evaluation software was used for data evaluation and determinating the catalpol content of 20 batches of R. glutinosa. Results: The standard contrast chromatographic fingerprint similarity of 20 batches R. glutinosa reached above 0.917. The 20 batches catalpol content was above 0.2%. Conclusion: The established fingerprint chromatography was proved that it had good precision, stability, and repeatability. The catalpol content determination met requirement which can avoid interference of saccharide compared with China Pharmacopoeia method and provide scientific reference for improving the quality of R. glutinosa.

Advances in molecular identification and functional genes of Rehmannia glutinosa / 中草药

Rehmannia glutinosa is one of the most common bulk medicinal materials in China and it has a long history of medicinal use. In recent years, with the development of molecular biology, especially for the emergence of high-throughput sequencing technology, it not only provides new ways to identify R. glutinosa quickly, and reveal the genetic diversity and relationship of R. glutinosa, but also lays the vital foundation for explaining the mechanism on metabolism, root tuber growth, stress response and continuous cropping obstacles of R. glutinosa. The present paper reviews the recent study progress in molecular biology research of R. glutinosa from molecular systematics, molecular identification and functional genes, and puts forward three research prospects in order to provide a reference for further study on molecular biology of R. glutinosa.

Synergistic interactions of catalpol and stachyose in STZ-HFD induced diabetic mice: Synergism in regulation of blood glucose, lipids, and hepatic and renal function / 中草药·英文版

Objective: Rehmanniae Radix has been traditionally used to treat diabetes. Catalpol (CAT) and stachyose (STA) are two of the main bioactive compounds in Rehmannia Radix and found to have similar therapeutic effects on diabetes and its complications. In this paper, we aimed to investigate whether there were synergistic therapeutic effects of CAT and STA on diabetes. Methods: Streptozotocin (STZ) with the feeding of high-sugar-high-fat diet (HFD) was applied to induce diabetic C57BL/6 mice. STZ-HFD induced diabetic mice were then divided into model and six medical-treated groups: metformin (MET), STA, CAT, and three combinations of CAT:STA (1:1, 1:2, 2:1). Blood, liver, and kidney samples were isolated after six-week oral administration for biochemical assays of serum lipids, the indicators of kidney and liver functions and HE staining for liver tissues. Results: It turned out that CAT, STA and their three combinations (1:1, 1:2, 2:1) could effectively control body weight, blood glucose, kidney weight and liver weight index, and well regulate levels of TC, HDL-c, TG, ALT, and TBA. In addition, CAT and its combination with STA at the ratio of 2:1 could significantly improve albumin content, compared to that in model group. STA and CAT and their combinations showed the improvements on kidney function in terms of urinary creatinine (Ucr). However, there were no such consistent observations on serum creatinine (Scr) and creatinine clearance rate (Ccr). The combination of CAT and STA at the ratio of 1:1 exhibited the better adjusting effects on kidney weight and liver weight indexes and the levels of ALT, Ucr, Scr, and Ccr. Our results demonstrated that the combinations of CAT and STA especially 1:1 showed similar or better improvements on diabetes-associated complications, compared to the sole CAT or STA treatment. Conclusion: Thus, we concluded that there were synergistic therapeutic effects between CAT and STA on STZ/HFD-induced type 2 diabetes. This project provided insights and technical supports for the innovation of discovering bioactive constituents in Rehmannia Radix and studying its integrative mechanism in curing diabetes.