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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.
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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.
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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.
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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.
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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.
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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.
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Objective: In order to investigate the functional genes involved in terpenoids biosynthesis pathway of Rehmannia glutinosa, a new geranylgeranyl pyrophosphate synthase gene (RgGGPPS2) was isolated from R. glutinosa. Meanwhile, the bioinformatic analysis, prokaryotic expression, purification, and expression patterns of RgGGPPS2 gene were carried out. Methods: Based on the transcriptome data of R. glutinosa, specific primers of RgGGPPS2 gene were designed, and an open reading frame (ORF) of RgGGPPS2 gene was isolated from R. glutinosa. By constructing the prokaryotic expression vector pET32a-RgGGPPS2, the recombinant RgGGPPS2 protein was expressed in Escherichia coli BL21 (DE3) cells under IPTG induction. The expression pattern of RgGGPPS2 in different tissues was detected by real-time PCR. Results: RgGGPPS2 had an ORF of 867 bp, which encoded a protein of 289 amino acid residues. Bioinformatic analysis indicated that RgGGPPS2 protein contains the two conserved motifs FARM (first aspartate-rich motif, DDxxxxD) and SARM (second aspartate-rich motif, DDxxD). Phylogenetic analysis revealed that RgGGPPS2 protein showed the highest homology with GGPPS protein from Sesamum indicum, Catharanthus roseus and other dicots. Through the construction of prokaryotic expression vector pET-32a-RgGGPPS2, the recombinant RgGGPPS2 protein was successfully expressed in E. coli BL21 (DE3) cells and the recombinant RgGGPPS2 protein was purified by Ni2+ affinity chromatography. Real-time PCR analysis indicated that RgGGPPS2 was expressed in high transcript level in roots, lower level in leaves and the lowest level in stems. Conclusion: The RgGGPPS2 gene was isolated from R. glutinosa and the recombinant RgGGPPS2 protein was obtained. The results of this study provide a foundation for functional characterization of RgGGPPS2 gene involved in terpenoids biosynthesis pathway of R. glutinosa.
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The aim of this study was to investigate the regulatory effect of the total glycoside extracted from leaves of Rehmannia (TLR) and Dihuangye total glycoside capsule (DTG) on intestinal microflora in diabetic nephropathy rats. Forty-eight rats were randomly divided into the control group (C), model group (M), Huangkui capsule group (0.75 g·kg-1·d-1, HK), irbesartan group (27 mg·kg-1·d-1, YX), TLR low dose group (4.3 g·kg-1·d-1, DHYL), TLR high dose group (7.2 g·kg-1·d-1, DHYH), DTG low dose group (216 mg·kg-1·d-1, JNL), DTG high dose group (360 mg·kg-1·d-1, JNH). Rat model of diabetic nephropathy was induced by intraperitoneal injection of small dose of streptozotocin (45 mg·kg-1, STZ) and feeding high-fat diet and 5% glucose drinking water. After oral administration for two weeks, the 16S rDNA sequencing method was used to study the effects of the TLR and DTG on intestinal flora in diabetic nephropathy rats. The results showed that compared with the control group, the intestinal flora of diabetic nephropathy rats had changed from phylum units to the genus units. Moreover, the proportion of lactobacilli in the intestinal bacteria of the model group was significantly decreased, and the proportion of lactobacilli in the administration group was increased, especially the YX group, TLR low dose group and DTG low dose group. The data suggest that the total glycosides of Rehmannia glutinosa improved the disorder of intestinal flora in STZ-induced diabetic nephropathy rats.
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OBJECTIVE: To screen the estrogenic effects of fresh Radix Rehmanniae, dried Radix Rehmanniae, Radix Rehmanniae Praeparata and the leaves of Rehmannia glutinosa Libosch. METHODS: Mouse uterine weight test and MCF-7 cell proliferation assay were used to evaluate the estrogenic effects of the four kinds of Chinese traditional herbs. ICI182, 780 antagonnist assay and reporter gene assay were adopted to explore the mechanism of action of fresh and dried Radix Rehmanniae. In reporter gene assay, HEK293 cells were cotranfected with pERE-TAL-luc, pβgal-Control, pCXN2-hERα or pCXN2-hERβ, and the expression of reportr gene luc was controlled by ERE. RESULTS: Mouse uterine weight test showed that compared with the control group, fresh and dried Radix Rehmanniae could increase the uterus index of premature female mice, and both of them could promote the proliferation of MCF-7 cells. Co-incubation of MCF-7 cells with estrogen receptor blocker ICI182, 780 abolished the inductive effect of the proliferation. The reporter gene controlled by ERE technology showed that when mediated by ERβ, the normalized luciferase activity of the two groups were significantly higher than the activity of the control group. CONCLUSION: Fresh and dried Radix Rehmanniae have estrogenic activities which are mainly mediated by ERβ. Fresh Radix Rehmanniae has higher estrogenic activity than dried Radix Rehmanniae. Radix Rehmanniae Praeparata does not have estrogenic activity. The estrogenic activity may change during the processing process of Rehmannia glutinosa Libosch.
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OBJECTIVE: To investigate the antidepressant-like effect of extracts from dried root of Rehmannia glutinosa Libosch. (Dihuang) (RG).
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OBJECTIVE: To study the chemical constituents in the leaves of Rehmannia glutinosa Libosch. METHODS: The compounds were isolated and purified by Diaion HP-20, Toyopearl HW40, Sephadex LH-20, MCI Gel CHP-20, and silica gel column chromatography, and the structures were elucidated on the basis of spectral data and physiochemical properties. RESULTS: Twenty-seven compounds were obtained from the 50% acetone extract, and their structures were identified as p-hydroxybenzoic acid(1), gentisic acid(2), 6, 7-dihydroxycoumarin(3), p-hydroxyphenylethyl alcohol(4), 3, 4-dihydroxyphe-nylethyl alcohol(5), protocatechuic acid(6), 1, 2, 4-trihydroxybenzene(7), diosmetin(8), luteolin-7-O-β-D-glucuronide(9), apigenin(10), 2β, 3β, 19α-trihydroxyolean-12-ene-13, 28-dioic acid(11), 2α, 3β-dihydroxyolean-12-ene-28-oic acid(12), glutinolic acid(13), β1-hydroxyacteoside(14), echinacoside(15), jionoside B,(16), acteoside(17), isoacteoside(18), ajugol(19), catalpol(20), 8-epiloganic acid(21), luteolin(22), oleanolic acid(23), ursolic acid(24), oleanonic acid(25), β-sitosterol(26) and daucosterol(27). CONCLUSION: Compounds 1-14 and 23-25 are isolated from this plant for the first time.
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Objective: In order to establish the material basis for future studies on the biological function of miRNAs, we aimed to predict novel miRNAs from EST sequences of Rehmannia glutinosa by using bioinformatic strategies. Methods: Since most of the plant miRNAs were conserved in plant species, all plant miRNAs deposited in miRNase were aligned to the 93 172 EST sequences generated by next generation high-throughput RNA sequencing technology and the putative miRNA precursors were screened according to serious criteria. Results: Eight novel rehmannia miRNAs were identified which belonged to eight different families that were further validated by real-time PCR analysis. Then the eight rehmannia miRNAs were subjected to target prediction analysis, and the results showed that the target genes encoded the proteins related to root growth, metabolism, stress responses and other processes. Conclusion: The miRNAs and their target genes identified in this study will provide clues to their biological functions in R. glutinosa.
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Objective: To study the effect of different storage methods, processing methods, temperatures, pH values, and enzymes on the content of catalpol in Rehmannia glutinosa. Methods: To investigate the content change of catalpol in different processed R. glutinosa under the different storage conditions by HPLC. And the content of catalpol was determined after being stored under different temperatures, pH values, and enzymes. Results: The content of catalpol in fresh R. glutinosa was significantly higher than that in the processed ones. The content of catalpol in frozen under vacuum was almost unchanged. The content of catalpol was obviously influenced by pH value, especially in strong acid and alkali, and the change was enhanced at higher temperature. Meanwhile, the content of catalpol was easily influenced by β-glucosidenzyme. Conclusion: Catalpol is stable to high temperature, while unstable to acid, alkali or β-glucosidenzyme.
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Objective To investigate the cause of continuous cropping obstacle of Rehmannia glutinosa and screen beneficial bacteria.Methods In vitro cultured plantlets and potted plants were inoculated with different isolated soil bacteria. The plants were harvested and weighted in 30 and 60 d,respectively.Results In the in vitro culture experiment,11 out of 48 strains displayed promoting action on the growth of plantlets,and 11 other strains showed inhibitory or lethal action.In the potted test,16 strains showed promoting action and 13 strains showed suppressing or lethal action.Conclusion Soil bacteria influence the growth of R.glutinosa significantly.The flora of rhizosphere bacteria may be disturbed by the cultivation of R.glutinosa and inoculation of beneficial bacteria might be effective on the resolution of continuous cropping obstacle of R.glutinosa.
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Objective Oligosaccharides of Rehmannia glutinosa were separated and purified by different molecular weight cut-off ultrafiltration(UF) membranes,and then ultrafiltrate was concentrated and purified by nanofiltration(NF) membrane.Methods Different molecular weight cut-off UF unit first and then NF unit were used in process.Results Oligosaccharides were separated by twice UF,the optimum separation conditions:the concentration of feed solution was 13—132 mg/mL,the operation pressure was 0.25—0.275 MPa,and the temperature was 20—40 ℃.Then ultrafiltrate was concentrated and purified by NF membrane,the optimum separation conditions:the operation pressure was 0.59—0.79 MPa,the temperature was 20—40 ℃,and the concentration multiplegot to three.Under this condition,the total extraction rate of oligosaccharide products was 46.63% and the purity was above 93.3%,the molecular weights of oligosaccharide products determined by gel filtration were less than 6 000.Conclusion The technology is not only simple and feasible but also easy to separate and purify the oligosaccharides of R.glutinosa effectively.
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Objective To testify the screening function of stachyose on soil bacteria by investigating the bacterial culture in ammonium stachyose medium. Methods The turbidimetry was used to determine the absorbance of microbial suspension at 600 nm per 2 h under the same initial concentration of the microbial suspension and to draw their growth curves. Results Most of soil bacteria utilized stachyose ineffectively, while only a few of them grown well in ammonium stachyose medium. Conclusion Since the major soil bacteria can not take stachyose fully as their energy resources, the species and quantity of rhizobacteria may decrease largely and only a few that utilized stachyose better can develop vigorously. Those rhizobateria with better utilization of stachyose may multiply so rapidly as potential ones in the rhizosphere of Rehmannia glutinosa that the disequilibrium of soil microorganism appears.
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The effect of Liu Wei Di Huang Wan(LWDHW)and Rehmannia glutinosa Libosch(RGL)on the immune function of normal and immune-suppressed mice were observed.The results showed that the two medicinal herbs given orally can correct the suppres-sive effect of cyclophosphamide on spleen and thymus weight,serum specific antibodylevel and lymphocyte transformation.They can also improve the phagocytic activity ofperitoneal macrophages and enhance peripheral blood ANAE~+ lymphocyte ratio of mi-ce.Under the same conditions,no obvious effect of LWDHW and RGL on the normalmice was found except that RGL can increase the antibody level of normal mice.Theresults implicate that LWDHW and RGL may have some immunomodulatary effect.