Anti-aging activities of Rehmannia glutinosa Libosch. crude polysaccharide in Caenorhabditis elegans based on gut microbiota and metabonomic analysis.

Aging is a degenerative progress, accompanied by oxidative damage, metabolic disorders and intestinal flora imbalance. Natural macromolecular polysaccharides have shown excellent anti-aging and antioxidant properties, while maintaining metabolic and intestinal homeostasis. The molecular weight, monosaccharide composition, infrared spectrum and other chemical structure information of four Rehmannia glutinosa polysaccharides (RG50, RG70, RG90, RGB) were determined, and their free radical scavenging ability was assessed. Molecular weight and monosaccharide composition analysis exhibited that RG50 (2-72 kDa), RG70 (3.2-37 kDa), RG70 (3-42 kDa), and RGB (3.1-180 kDa) were heteropolysaccharide with significant different monosaccharide species and molar ratios. We found that RG70 had the best antioxidant activity in vitro and RG70 could enhance the antioxidant enzyme system of Caenorhabditis elegans, diminished lipofuscin and reactive oxygen species levels, up-regulate the expression of daf-16, skn-1 and their downstream genes, and down-regulate the expression of age-1. Metabolomics results showed that RG70 mainly influenced glycine, serine and threonine metabolism and citric acid cycle. 16S rRNA sequencing showed that RG70 significantly up-regulated the abundance of Lachnospiraceae_NK4B4_group, which were positively correlated with amino acid metabolism and energy cycling. These results suggest that RG70 may delay aging by enhancing antioxidant effects, affecting probiotics and regulating key metabolic pathways.

Structural characterization and anti-inflammatory effects of an arabinan isolated from Rehmannia glutinosa Libosch.

Considering that natural polysaccharides are potential anti-inflammatory agents, in this study, an arabinan (RGP70-2) was isolated and purified from Rehmannia glutinosa Libosch. (R. glutinosa) and its structure was characterized. RGP70-2 was a homogeneous polysaccharide with a molecular weight of 6.7 kDa, with the main backbone comprising →5)-α-L-Araf-(1→, →3)-α-L-Araf-(1→, →2,3,5)-α-L-Araf-(1→, and →2,5)-α-L-Araf-(1 â†’ linkages and the side chain comprising an α-L-Araf-(1 â†’ linkage. In vivo experiments showed that RGP70-2 inhibited ROS production and downregulated the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6). In vitro experiments showed that RGP70-2 decreased levels of pro-inflammatory cytokines, inhibited ROS production, and attenuated NF-κB-p65 translocation from the cytoplasm to the nucleus. Our results showed that RGP70-2 may delay inflammation by regulating the ROS-NF-κB pathway. Thus, RGP70-2 has potential applications as an anti-inflammatory agent in the biopharmaceutical industry.

Preventive effects of the Rehmannia glutinosa Libosch and Cornus officinalis Sieb herb couple on chronic kidney disease rats via modulating the intestinal microbiota and enhancing the intestinal barrier.

CKD is a clinical syndrome with slow development and gradual deterioration of renal function. At present, modern medicine still lacks an ideal treatment method for this disease, while TCM has accumulated rich clinical experience in the treatment of CKD, which can effectively improve renal function and delay renal failure, and has unique advantages. RC is widely used in clinical practice to treat CKD, especially the "Kidney-Yin" deficiency syndrome. However, the compatibility mechanisms responsible for its effects in experimental studies, including preclinical and clinical research studies, are still not fully understood. Adenine-induced CKD rats were used to investigate the preventive effect of RC on CKD rats. Based on the high-throughput 16S rRNA gene sequencing results from Illumina, we discussed the intestinal flora abundance in rats in different treatment groups. According to a PCA and a PCoA based on a distance matrix, there was a clear separation of gut microbiome profiles between normal rats and model rats in terms of beta diversity. The abundance of Firmicutes in CKD rats was relatively increased, while that of Bacteroidetes was decreased. It is clear that the plot for the RC group was closer to that of the normal group, suggesting that the RC group had higher similarities among bacterial members with N rats. Ussing chamber, Western blot, and PCR assays were used to investigate the effects of RC on intestinal barrier function and its molecular mechanism in model animals. The results indicated that the protein expressions of ZO-1, claudin-1, and occludin-1 were decreased significantly in chronic kidney disease rats with the induction of adenine. With the treatment of RG, CO, and RC, the intestinal barrier was repaired due to the upregulated expressions of the aforementioned proteins in CKD rats. Based on our findings, RC appears to strengthen the intestinal barrier and modulate gut microbiota in adenine-induced CKD rats. This project revealed the compatibility mechanism of RC in regulating the intestinal microecology and barrier function to intervene in CKD and provided the basis and ideas for the clinical application of RC and the development of innovative drugs for CKD.

The Effects of NAA on the Tuberous Root Yield and Quality of Rehmannia glutinosa and Its Regulatory Mechanism by Transcriptome and Metabolome Profiling.

Naphthylacetic acid (NAA) was used to increase the tuberous root yield of Rehmannia glutinosa, but the differences between its NAA-treated and control tuberous roots (NT and CG) and the regulatory mechanism of NAA effect remain unclear. In order to investigate them, NTs and CGs were used as materials, and both yield-related indices were measured; the metabolomics and transcriptomics were used to capture differentially accumulated metabolites (DAM) and to validate them via mining differentially expressed genes (DEGs), respectively. The effects of NAA treatment: increased NT mass per plant by 21.14%, through increasing the number of roots and increasing the mean root diameter; increased catalpol content by 1.2234% (p < 0.05); up-regulated 11DAMs and 596DEGs; and down-regulated 18 DAMs and 517DEGs. In particular, we discovered that NAA regulated its DAMs and biomass via 10 common metabolic pathways, and that the number of NAA-down-regulated DAMs was more than that of NAA-up-regulated DAMs in its tuberous root. Furthermore, HPLC validated the changes of several DAMs and 15 DEGs (4CL, ARF, CCoAOMT, ARGOS, etc.) associated with the yield increase and DAMs were verified by RT-qPCR. This study provided some valuable resources, such as tuberous root indices, key genes, and DAMs of Rehmannia glutinosa in response to NAA for distinguishing the CGs from NTs, and novel insights into the regulatory mechanism of NAA effects on both at the transcriptomic and metabolomic levels, so it will lay a theoretical foundation for NAA-regulated plant yield and quality, and provide references for prohibiting the uses of NAA as a swelling agent in medicinal tuber plants in China.

Genome-wide identification and characterization of the HD-Zip gene family and expression analysis in response to stress in Rehmannia glutinosa Libosch.

The HD-Zip family of transcription factors is unique to the plant kingdom, and play roles in modulation of plant growth and response to environmental stresses. R. glutinosa is an important Chinese medicinal material. Its yield and quality are susceptible to various stresses. The HD-Zip transcription factors is unique to the plant, and roles in modulation of plant growth and response to environmental stresses. However, there is no relevant research on the HD-ZIP of R. glutinosa. In this study, 92 HD-Zip transcription factors were identified in R. glutinosa, and denominated as RgHDZ1-RgHDZ92. Members of RgHDZ were classified into four groups (HD-ZipI-IV) based on the phylogenetic relationship of Arabidopsis HD-Zip proteins, and each group contains 38, 18, 17, and 19 members, respectively. Expression analyses of RgHDZ genes based on transcriptome data showed that the expression of these genes could be induced by the endophytic fungus of R. glutinosa. Additionally, we showed that RgHDZ genes were differentially expressed in response to drought, waterlogging, temperature, and salinity treatments. This study provides important information for different expression patterns of stress-responsive HD-Zip and may contribute to the better understanding of the different responses of plants to biotic and abiotic stresses, and provide a molecular basis for the cultivation of resistant varieties of R. glutinosa.

Isolation of endophytic bacteria from Rehmannia glutinosa Libosch and their potential to promote plant growth.

In order to study the growth promoting potential of endophytic bacteria from Rehmannia glutinosa Libosch, a total of 25 different bacteria belonging to 7 genera were identified by 16S rRNA gene sequencing, including Bacillus, Micrococcus, Lysinibacillus, Brevibacterium, Halomonas, Kocuria and Terribacillus. In this study, thirteen bacterial strains were found to solubilize inorganic phosphate, with the isolate Kocuria rosea (EH15) having the highest phosphorus dissolution activity (3.70 µg/mL). Twelve isolates were positive for nitrogen fixation abilities. Twenty-two strains produced indole-3-acetic acid (IAA) in the presence of L-tryptophan, and eleven of the twenty-two isolates synthesized IAA in the absence of L-tryptophan. The strain K. rosea (EH15) was capable of producing the highest IAA amount (15.36 and 7.98 mg/L) in Luria Bertani (LB) broth containing 0.2% L-tryptophan and lacking L-tryptophan, respectively. Ten isolates had siderophore production abilities with Bacillus amyloliquefacieus EH10 (0.26) and Brevibacterium frigoritolerans EH13 (0.32) showing high siderophore production characteristics. Five bacteria endogenous were selected to evaluate the growth parameters of Brassica napus L. and all isolates exhibited a significantly greater increase in seedling height, root length, fresh weight and dry weight, than the control plants. The greatest improvement appeared in the case of co-inoculation of EH10 and EH15, except in dry weight, and the biggest enhancement in dry weight occurred in the strain EH15. In general, these endophytic bacteria indicate a potential as microbial fertilizers to promote the growth of R. glutinosa Libosch.

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.

Rehmannia glutinosa Libosch Extracts Prevent Bone Loss and Architectural Deterioration and Enhance Osteoblastic Bone Formation by Regulating the IGF-1/PI3K/mTOR Pathway in Streptozotocin-Induced Diabetic Rats.

Rehmanniae Radix Praeparata (RR, named as Shudihuang in traditional Chinese medicine), the steamed roots of Rehmannia glutinosa Libosch (Scrophulariaceae), has been demonstrated to have anti-diabetic and anti-osteoporotic activities. This study aimed to explore the protective effect and underlying mechanism of RR on diabetes-induced bone loss. It was found that RR regulated the alkaline phosphatase activity and osteocalcin level, enhanced bone mineral density, and improved the bone microarchitecture in diabetic rats. The catalpol (CAT), acteoside (ACT), and echinacoside (ECH) from RR increased the proliferation and differentiation of osteoblastic MC3T3-E1 cells injured by high glucose and promoted the production of IGF-1 and expression of related proteins in BMP and IGF-1/PI3K/mammalian target of rapamycin complex 1 (mTOR) signaling pathways. The verifying tests of inhibitors of BMP pathway (noggin) and IGF-1/PI3K/mTOR pathway (picropodophyllin) and molecular docking of IGF-1R further indicated that CAT, ACT, and ECH extracted from RR enhanced bone formation by regulating IGF-1/PI3K/mTOR signaling pathways. These findings suggest that RR may prove to be a promising candidate drug for the prevention and treatment of diabetes-induced osteoporosis.

Xijiao Dihuang Decoction () and Rehmannia glutinosa Libosch. protect mice against lipopolysaccharide and tumor necrosis factor alpha-induced acute liver failure.

OBJECTIVE: To investigate the hepatoprotective effect of Xijiao Dihuang Decoction (, 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.

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.

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.

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.

Alleviatory effect of spent Pleurotus eryngii Quel substrate on replant problem of Rehmannia glutinosa Libosch.

Rehmannia glutinosa Libosch. is a medicinal plant cultivated at a commercial scale in China. However, replanting problems result in a severe decline in both the biomass and quality of its roots, which are of greatest medicinal value. This study attempted to remediate the replant soil using spent Pleurotus eryngii Quel substrate for alleviating this issue, and to investigate the underlying mechanisms. Results showed that R. glutinosa grew successfully in fresh soil and remedial replant soil, while no roots were harvested in the unremedied replant soil. Overall, the nutritional status in the remedial soil was higher than that of the unremedied and fresh soil, while the concentration of allelopathic phenolic acids was lower. When planted in unremedied soil, the growth of five plant pathogens was induced and one beneficial fungus was suppressed. When planted in remedied soil, four out of the five pathogens were suppressed, while two beneficial fungi were identified in the remedial soil. This study suggests that the spent P. eryngii substrate significantly alleviates the replant problem of R. glutinosa, and that the alleviatory function reflects a synergetic effect, including the supplementation of soil nutrition, the degradation of allelochemicals, and the remediation of unbalanced microbial community.

Cloning, bioinformatic analysis and expression analysis of RgGGPPS2 gene from Rehmannia glutinosa / 中草药

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.

Regulatory effect of the leaves of Rehmannia glutinosa Libosch on intestinal microflora in diabetic nephropathy rats / 药学学报

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.

Two new ionone glycosides from the roots of Rehmannia glutinosa Libosch.

Two new ionone glycosides, named frehmaglutoside G (1) and frehmaglutoside H (2), together with six known compounds, rehmapicroside (3), sec-hydroxyaeginetic acid (4), dihydroxy-ß-ionone (5), trihydroxy-ß-ionone (6), rehmaionoside A (7) and rehmaionoside C (8), were isolated from the 95% EtOH extract of the dried roots of Rehmannia glutinosa Libosch. Their structures were determined on the basis of extensive spectroscopic analyses, including HR-ESI-MS, UV, IR, 1D and 2D NMR ((1)H-(1)H COSY, HSQC, HMBC and NOESY) methods. The absolute configurations were confirmed via the circular dichroism spectra.

Evaluation of the estrogenic effects of Rehmannia glutinosa Libosch / 中国药学杂志

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.

Antidepressant-like effect of extracts from dried root of Rehmannia glutinosa (Dihuang) / 中国药学杂志

OBJECTIVE: To investigate the antidepressant-like effect of extracts from dried root of Rehmannia glutinosa Libosch. (Dihuang) (RG).