[Role of mitochondrial autophagy and the curative effect of rehmannia glutinosa leaves total glycoside capsules on nucleos(t)ide drug-induced renal injury].

Objective: To study the curative effect of rehmannia glutinosa leaves total glycoside capsules and the role of mitochondrial autophagy on nucleos(t)ide drug-induced renal injury. Methods: Adefovir dipivoxil (ADV) was used to construct a hepatitis B virus (HBV) transgenic mouse model for renal injury. Renal function was measured in each group at one and two weeks of modeling. Mitochondrial autophagy indicators were measured at two weeks of modeling in renal tissue. Transmission electron microscopy was used to detect mitochondrial autophagy phenomena in renal tissue. The model was established for two weeks. Mouse with renal injury were treated with rehmannia glutinosa leaves total glycoside capsules or isotonic saline for eight weeks by intragastric administration. Renal function was measured. Renal tissue morphology was observed. Mitochondrial autophagy indicators were detected in renal tissue. The protective effect of different concentrations of verbascoside (the main active ingredient of rehmannia glutinosa capsule) was observed on HK-2 cell damage induced by ADV. HK-2 cells were divided into control, ADV, and ADV plus verbascoside groups. The effects of verbascoside at different times and concentrations were observed on the HK-2 mitochondrial autophagy indicators. Fifty patients with chronic hepatitis B were collected who presented with renal injury after treatment with nucleos(t)ide analogs. The random number method was used to divide 29 cases into a control group that received conventional treatment. The treatment group of 21 cases was treated with rehmannia glutinosa leaves total glycoside capsules on the basis of the control group. Serum creatinine (Scr) and urinary protein were detected at eight weeks.The χ(2) test or t-test was used for statistical analysis. Results: Compared with the control group, two weeks of modeling in the ADV group induced renal function injury in HBV mice. The expression of autophagy indicators was higher in the renal tissue of the ADV group than that of the control group. Transmission electron microscopy had revealed mitochondrial autophagy in the renal tissue of the ADV group. Compared with the control group, the renal function of HBV mice treated with rehmannia glutinosa leaves total glycoside capsules improved for two months, and the expressions of autophagy indicators were down-regulated.Verbascoside promoted proliferation in ADV-damaged HK-2 cells, and the expression of autophagy indicators was down-regulated compared with the ADV alone group. In 50 patients with renal function injury, the urinary protein improvement was significantly superior in the treatment group than that in the control group, with eighteen and three cases being effective and ineffective in the treatment group and 12 and 17 cases being effective and ineffective in the control group, with a statistically significant difference (χ(2) = 9.975 0, P = 0.001 6). Serum creatinine was decreased in the treatment group compared with the control group, with 11 and 10 cases being effective and ineffective in the treatment group and 12 and 17 cases being effective and ineffective in the control group, with no statistically significant difference (χ(2) = 0.593 5, P = 0.441 1). Conclusion: Rehmannia glutinosa leaves total glycoside capsule can improve the nucleos(t)ide drug-induced renal function injury in chronic hepatitis B, possibly playing a role via inhibiting PINK1/Parkin-mediated mitochondrial autophagy.

Transcriptome Analysis Provides Insights into Catalpol Biosynthesis in the Medicinal Plant Rehmannia glutinosa and the Functional Characterization of RgGES Genes.

Rehmannia glutinosa, a member of the Scrophulariaceae family, has been widely used in traditional Chinese medicine since ancient times. The main bioactive component of R. glutinosa is catalpol. However, the biogenesis of catalpol, especially its downstream pathway, remains unclear. To identify candidate genes involved in the biosynthesis of catalpol, transcriptomes were constructed from R. glutinosa using the young leaves of three cultivars, Beijing No. 3, Huaifeng, and Jin No. 9, as well as the tuberous roots and adventitious roots of the Jin No. 9 cultivar. As a result, 71,142 unigenes with functional annotations were generated. A comparative analysis of the R. glutinosa transcriptomes identified over 200 unigenes of 13 enzymes potentially involved in the downstream steps of catalpol formation, including 9 genes encoding UGTs, 13 for aldehyde dehydrogenases, 70 for oxidoreductases, 44 for CYP450s, 22 for dehydratases, 30 for decarboxylases, 19 for hydroxylases, and 10 for epoxidases. Moreover, two novel genes encoding geraniol synthase (RgGES), which is the first committed enzyme in catalpol production, were cloned from R. glutinosa. The purified recombinant proteins of RgGESs effectively converted GPP to geraniol. This study is the first to discover putative genes coding the tailoring enzymes mentioned above in catalpol biosynthesis, and functionally characterize the enzyme-coding gene in this pathway in R. glutinosa. The results enrich genetic resources for engineering the biosynthetic pathway of catalpol and iridoids.

Exploration of the Dynamic Variations of the Characteristic Constituents and the Degradation Products of Catalpol during the Process of Radix Rehmanniae.

Radix Rehmanniae (RR), a famous traditional Chinese medicine (TCM) widely employed in nourishing Yin and invigorating the kidney, has three common processing forms in clinical practice, including fresh Radix Rehmanniae (FRR), raw Radix Rehmanniae (RRR), and processed Radix Rehmanniae (PRR). However, until now, there has been less exploration of the dynamic variations in the characteristic constituents and degradation products of catalpol as a representative iridoid glycoside with the highest content in RR during the process from FRR to PRR. In this study, an ultra-performance liquid chromatography coupled with photodiode array detector (UPLC-PDA) method was successfully established for the simultaneous determination of ten characteristic components to explore their dynamic variations in different processed products of RR. Among them, iridoid glycosides, especially catalpol, exhibited a sharp decrease from RRR to PRR. Then, three degradation products of catalpol were detected under simulated processing conditions (100 °C, pH 4.8 acetate buffer solution), which were isolated and identified as jiofuraldehyde, cataldehyde, and norviburtinal, respectively. Cataldehyde was first reported as a new compound. Moreover, the specificity of norviburtinal in self-made PRR samples was discovered and validated, which was further confirmed by testing in commercially available PRR samples. In conclusion, our study revealed the decrease in iridoid glycosides and the production of new degradation substances during the process from FRR to PRR, which is critical for unveiling the processing mechanism of RR.

Comprehensive Analysis of the Complete Mitochondrial Genome of Rehmannia chingii: An Autotrophic Species in the Orobanchaceae Family.

Rehmannia chingii is an important medicinal plant with immense value in scientific research. However, its mitochondrial genome (mitogenome) has not yet been characterized. Herein, based on whole-genome Illumina short reads and PacBio HiFi reads, we obtained the complete mitogenome of R. chingii through a de novo assembly strategy. We carried out comparative genomic analyses and found that, in comparison with the plastid genome (plastome) showing a high degree of structural conservation, the R. chingii mitogenome structure is relatively complex, showing an intricate ring structure with 16 connections, owing to five repetitive sequences. The R. chingii mitogenome was 783,161 bp with a GC content of 44.8% and contained 77 genes, comprising 47 protein-coding genes (CDS), 27 tRNA genes, and 3 rRNA genes. We counted 579 RNA editing events in 47 CDS and 12,828 codons in all CDSs of the R. chingii mitogenome. Furthermore, 24 unique sequence transfer fragments were found between the mitogenome and plastome, comprising 8 mitogenome CDS genes and 16 plastome CDS genes, corresponding to 2.39% of the R. chingii mitogenome. Mitogenomes had shorter but more collinear regions, evidenced by a comparison of the organelles of non-parasitic R. chingii, hemiparasitic Pedicularis chinensis, and holoparasitic Aeginetia indica in the Orobanchaceae family. Moreover, from non-parasitic to holoparasitic species, the genome size in the mitogenomes of Orobanchaceae species did not decrease gradually. Instead, the smallest mitogenome was found in the hemiparasitic species P. chinensis, with a size of 225,612 bp. The findings fill the gap in the mitogenome research of the medicinal plant R. chingii, promote the progress of the organelle genome research of the Orobanchaceae family, and provide clues for molecular breeding.

Neuroprotective effects of Rehmannia glutinosa polysaccharide on chronic constant light (CCL)-induced oxidative stress and autophagic cell death via the AKT/mTOR pathway in mouse hippocampus and HT-22 cells.

Rehmannia glutinosa polysaccharide (RGP) has been reported to exhibit anti-anxiety effects, yet the underlying mechanism remains unclear. Chronic constant light (CCL) induced cognitive dysfunction associated with oxidative stress in mice has been reported. Here, the neuroprotective effect of RGP on hippocampal neuron damage in CCL-treated mice was investigated. In vivo study, mice were subjected to CCL for 4 weeks and/or oral administration of 100, 200 and 400 mg/kg RGP every other day. In vitro experiment, hippocampal neuron cells (HT-22) was exposed to LED light and/or supplemented with 62.5, 125 and 250 µg/mL RGP. Mice exposed to CCL showed impaired cognitive and depressive-like behavior in the hippocampus, which were reversed by RGP. Meanwhile, RGP reversed light-induced oxidative stress and autophagy both in mice and hippocampal neuron cells (HT-22). Furthermore, compared with Light-exposed group, RGP treatment activated the AKT/mTOR pathway. Importantly, the AKT inhibitor Perifosine significantly weakened the neuroprotective of RGP on Light-induced oxidative stress and autophagy in HT-22 cells by inhibiting AKT/mTOR pathway and increasing the content of autophagy-related protein. Our data demonstrated, for the first time, that oxidative stress and the AKT/mTOR pathway plays a critical role in Light-induced apoptosis and autophagic cell death in mice and HT-22 cells.

Effects of Catalpol from Rehmannia glutinosa Extract on Skin Flaps.

BACKGROUND: Flaps are commonly used for repairing tissues and wounds in surgery. However, various factors can cause postoperative necrosis in these flaps. Catalpol is a bioactive component in extracts from Rehmannia glutinosa , which has pharmacologic characteristics that may improve flap survival. METHODS: The experiments were performed in 36 male Sprague-Dawley rats divided into three groups: control, low-dose catalpol, and high-dose catalpol. The flap survival rate, neutrophil density, microvessel density, superoxide dismutase, and malondialdehyde levels were measured; histopathologic analysis was performed 7 days after surgery. Blood flow was measured by laser Doppler flowmetry and lead oxide-gelatin angiography. The levels of vascular endothelial growth factor, toll-like receptor 4, nuclear factor-kappa B, tumor necrosis factor-α, interleukin (IL)-6, nod-like receptor 3, cysteinyl aspartate specific proteinase-1 (caspase-1), IL-1ß, and IL-18 were determined by immunohistochemistry. RESULTS: Catalpol treatment increased flap survival, reduced neutrophil recruitment and release, decreased malondialdehyde levels, and increased superoxide dismutase levels; thus, it effectively reduced oxidative stress, up-regulated the expression of vascular endothelial growth factor, and increased microvessel density. Laser Doppler flowmetry and lead oxide-gelatin angiography showed that catalpol treatment improved angiogenesis. Immunohistochemical analyses showed that catalpol inhibited the production of inflammatory factors, such as tumor necrosis factor-α and IL-6, by down-regulating toll-like receptor 4 and nuclear factor-κB. Furthermore, catalpol reduced cell pyroptosis by inhibiting the production of nod-like receptor 3 inflammasomes, thereby down-regulating the release of IL-1ß and IL-18. CONCLUSION: Catalpol can improve the rate of flap survival. CLINICAL RELEVANCE STATEMENT: The research verified that the Rehmannia extract catalpol, through angiogenesis, inflammatory response, ischemia-reperfusion injury, and pyroptosis-related pathways, effectively improved the flap survival rate, which will provide new ideas for clinical medication.

Progress of research into the pharmacological effect and clinical application of the traditional Chinese medicine Rehmanniae Radix.

The traditional Chinese medicine (TCM) Rehmanniae Radix (RR) refers to the fresh or dried root tuber of the plant Rehmannia glutinosa Libosch of the family Scrophulariaceae. As a traditional Chinese herbal medicine (CHM), it possesses multiple effects, including analgesia, sedation, anti-inflammation, antioxidation, anti-tumor, immunomodulation, cardiovascular and cerebrovascular regulation, and nerve damage repair, and it has been widely used in clinical practice. In recent years, scientists have extensively studied the active components and pharmacological effects of RR. Active ingredients mainly include iridoid glycosides (such as catalpol and aucuboside), phenylpropanoid glycosides (such as acteoside), other saccharides, and unsaturated fatty acids. In addition, the Chinese patent medicine (CPM) and Chinese decoction related to RR have also become major research subjects for TCM practitioners; one example is the Bolus of Six Drugs, which includes Rehmannia, Lily Bulb and Rehmannia Decoction, and Siwu Decoction. This article reviews recent literature on RR; summarizes the studies on its chemical constituents, pharmacological effects, and clinical applications; and analyzes the progress and limitations of current investigations to provide reference for further exploration and development of RR.

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.

Omics analyses of Rehmannia glutinosa dedifferentiated and cambial meristematic cells reveal mechanisms of catalpol and indole alkaloid biosynthesis.

BACKGROUND: Rehmannia glutinosa is a rich source of terpenoids with a high medicinal reputation. The present study compared dedifferentiated cells (DDCs) and cambial meristematic cells (CMCs) cell cultures of R. glutinosa for terpenoid (catalpol) and indole alkaloid (IA) biosynthesis. In this regard, we used widely targeted metabolomics and transcriptome sequencing approaches together with the comparison of cell morphology, cell death (%), and catalpol production at different time points. RESULTS: We were able to identify CMCs based on their morphology and hypersensitivity to zeocin. CMCs showed higher dry weight content and better catalpol production compared to DDCs. The metabolome analysis revealed higher concentrations of IA, terpenoids, and catalpol in CMCs compared to DDCs. The transcriptome sequencing analysis showed that a total of 27,201 genes enriched in 139 pathways were differentially expressed. The higher catalpol concentration in CMCs is related to the expression changes in genes involved in acetyl-CoA and geranyl-PP biosynthesis, which are precursors for monoterpenoid biosynthesis. Moreover, the expressions of the four primary genes involved in monoterpenoid biosynthesis (NMD, CYP76A26, UGT6, and CYP76F14), along with a squalene monooxygenase, exhibit a strong association with the distinct catalpol biosynthesis. Contrarily, expression changes in AADC, STR, and RBG genes were consistent with the IA biosynthesis. Finally, we discussed the phytohormone signaling and transcription factors in relation to observed changes in metabolome. CONCLUSIONS: Overall, our study provides novel data for improving the catalpol and IA biosynthesis in R. glutinosa.

Identification of raffinose family oligosaccharides in processed Rehmannia glutinosa Libosch using matrix-assisted laser desorption/ionization mass spectrometry image combined with machine learning.

RATIONALE: Currently, research on oligosaccharides primarily focuses on the physiological activity and function, with a few studies elaborating on the spatial distribution characterization and variation in the processing of Rehmannia glutinosa Libosch. Thus, imaging the spatial distributions and dynamic changes in oligosaccharides during the steaming process is significant for characterizing the metabolic networks of R. glutinosa. It will be beneficial to characterize the impact of steaming on the active ingredients and distribution patterns in different parts of the plant. METHODS: A highly sensitive matrix-assisted laser desorption/ionization mass spectrometry image (MALDI-MSI) method was used to visualize the spatial distribution of oligosaccharides in processed R. glutinosa. Furthermore, machine learning was used to distinguish the processed R. glutinosa samples obtained under different steaming conditions. RESULTS: Imaging results showed that the oligosaccharides in the fresh R. glutinosa were mainly distributed in the cortex and xylem. As steaming progressed, the tetra- and pentasaccharides were hydrolyzed and diffused gradually into the tissue section. MALDI-MS profiling combined with machine learning was used to identify the processed R. glutinosa samples accurately at different steaming intervals. Eight algorithms were used to build classification machine learning models, which were evaluated for accuracy, precision, recall, and F1 score. The linear discriminant analysis and random forest models performed the best, with prediction accuracies of 0.98 and 0.97, respectively, and thus can be considered for identifying the steaming durations of R. glutinosa. CONCLUSIONS: MALDI-MSI combined with machine learning can be used to visualize the distribution of oligosaccharides and identify the processed samples after steaming for different durations. This can enhance our understanding of the metabolic changes that occur during the steaming process of R. glutinosa; meanwhile, it is expected to provide a theoretical reference for the standardization and modernization of processing in the field of medicinal plants.

Rehmannia Glutinosa Polysaccharide Regulates Bone Marrow Microenvironment via HIF-1α/NF-κB Signaling Pathway in Aplastic Anemia Mice.

Aplastic anemia (AA), a rare disorder, is associated with bone marrow microenvironment (BMM). Presently, AA treatment is of great difficulty. This study aimed to explore the mechanism of action of Rehmannia glutinosa polysaccharide (RGP) in AA. Busulfan was used to induce AA in BALB/c mice; blood cell count and Ray's Giemsa staining were used to assess the severity of hematopoietic failure; HE was performed to assess the pathological state of the marrow cavity; ELISA was performed to assess IL-4, IL-10, IL-6, IL-12, IL-1ß, TNF-α, MCP-1, VEGF, and EPO; and WB was performed to evaluate the effects of RGP on the HIF-1α/NF-κB signaling. Significant downregulation of hemocyte levels in the blood and nucleated cells in the bone marrow was reversed by RGP and Cyclosporine A (CA). Compared with the AA group, dilating blood sinusoids, inflammation, hematopoiesis, decreased bone marrow cells and megakaryocytes were alleviated by RGP and CA, and the HIF-1α/NF-κB signaling was inhibited too. Notably, RGP was more effective when used in combination with CA. In this study, we established a relationship between BMM and the HIF-1α/NF-κB signaling pathway and found that RGP regulates BMM by suppressing the activation of the HIF-1α/NF-κB signaling. Thus, RGP exerts a pharmacological effect on AA.

Angiotensin I-Converting Enzyme-Inhibitory Activity and Phytochemical Profile of Constituents of the Leaves of Rehmannia glutinosa f. hueichingensis.

The root of Rehmannia glutinosa Liboschitz forma hueichingensis HSIAO has been used as a tonic and treatment for urinary and skin disorders in Japanese Kampo medicine. Phytochemical investigation of the root has been well reported, but that of the leaves is limited. To explore the potential value of R. glutinosa leaves, we focused on the angiotensin I-converting enzyme (ACE)-inhibitory activity. The leaf extract exhibited ACE-inhibitory activity, and the inhibitory potency of leaves was stronger than that of roots. Using this activity as an indicator, we isolated linaride (1), 6-O-hydroxybenzoyl ajugol (2), acteoside (3), leucosceptoside A (4), martynoside (5), luteolin (6), apigenin (7), and chrysoeriol (8) by separating and purifying the extract. We then examined the ACE-inhibitory activities of 1-8, catalpol (9), aucubin (10), ajugol (11), and echinacoside (12). Among them, 3, 6, and 12 displayed the most potent inhibitory activity. A simultaneous analytical method was also developed using compounds contained in R. glutinosa leaves and roots, and their contents were compared. The method consisted of extraction with 50% aqueous methanol under sonication for 60 min and LC/MS measurement. R. glutinosa leaves tended to have higher levels of majority of the analytes than the roots, including 3 and 6, which had higher ACE-inhibitory activity. These results suggest that 3 and 6 contribute to the ACE-inhibitory activity of R. glutinosa leaves, which may represent a useful medicinal resource for hypertension.

Magnetic molecularly-imprinted microspheres with a core-shell structure for the extraction of catalpol from both Rehmannia glutinosa Libosch and biological samples.

We report on the successful development of a magnetic molecularly imprinted polymer (MMIP) for the separation and detection of catalpol in Rehmannia glutinosa extract and biological samples. Catalpol was used as the template molecule for the MMIPs, which were synthesized using the reverse prediction approach and differential UV-visible spectra to optimize the synthesis conditions. The resulting MMIPs were characterized using techniques including scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and vibrating sample magnetometry, which confirmed their excellent nuclear shell construction and simple rapid magnetic separation. Static and dynamic adsorption experiments were performed to assess the sensitivity, repeatability, and adsorption capacity of the MMIPs. Adsorption capacity was found to be high (59.09 µg/mg). The MMIPs also exhibited strong identification specificity for catalpol analogs (IF = 2.41). Magnetic solid phase extraction (MSPE) was optimized to establish an MSPE-HPLC approach based on the MMIPs. Under ideal conditions, the new method showed excellent determination coefficient (R2 > 0.99) in the ranges of 0.1-1.0 mg/mL and 0.03-0.18 mg/mL for R. glutinosa extract and biological material, respectively, with limits of detection of 5.4 ng/mL and 1.7 ng/mL. The novelty of this research lies in the preparation of magnetic surface MMIPs using a magnetic carrier. These MMIPs exhibit excellent magnetic properties and possess specific adsorption capability for catalpol, enabling rapid separation. They offer advantages such as low cost, high specificity, chemical stability, physical robustness, and recyclability (97.09%-106.53%, 91.32%-100.99%). The application of these polymers in the pretreatment method for determining the catalpol content in R. glutinosa extract and biological samples enhances the extraction efficiency and detection accuracy for catalpol. It eliminates interference and influences from other components in catalpol determination while exhibiting high sensitivity, good enrichment effects, excellent reusability, and a high recovery rate.

A Rehmannia glutinosa caffeic acid O-methyltransferase functional identification: Reconstitution of the ferulic acid biosynthetic pathway in Saccharomyces cerevisiae using Rehmannia glutinosa enzymes.

Rehmannia glutinosa produces many pharmacological natural components, including ferulic acid (FA) which is also an important precursor of some medicinal ingredients, so it is very significant to explore FA biosynthesis for enhancing the production of FA and its derivations. This study aimed to determine and reconstitute the R. glutinosa FA biosynthetic pathway from phenylalanine (Phe) metabolism in Saccharomyces cerevisiae as a safe host for the biosynthesis of plant-derived products. Although plant caffeic acid O-methyltransferases (COMTs) are thought to be a vital catalytic enzyme in FA biosynthesis pathways, to date, none of the RgCOMTs in R. glutinosa has been characterized. This study identified an RgCOMT and revealed its protein enzymatic activity for FA production in vitro. The RgCOMT overexpression in R. glutinosa significantly increased FA yield, suggesting that its molecular function is involved in FA biosynthesis. Heterologous expression of the RgCOMT and reported R. glutinosa genes, RgPAL2 (encoding phenylalanine ammonia-lyase [PAL] protein), RgC4H (cinnamate 4-hydroxylase [C4H]), and RgC3H (p-coumarate-3-hydroxylase [C3H]), in S. cerevisiae confirmed their catalytic abilities in the reaction steps for the FA biosynthesis. Importantly, in this study, these genes were introduced into S. cerevisiae and coexpressed to reconstitute the R. glutinosa FA biosynthetic pathway from Phe metabolism, thus obtaining an engineered strain that produced an FA titer of 148.34 mg L-1 . This study identified the functional activity of RgCOMT and clarified the R. glutinosa FA biosynthesis pathway in S. cerevisiae, paving the way for the efficient production of FA and its derivatives.

Add-on Rehmannia-6-Based Chinese Medicine in Type 2 Diabetes and CKD: A Multicenter Randomized Controlled Trial.

BACKGROUND: Diabetes is the leading cause of CKD and kidney failure. We assessed the real-world effectiveness of Rehmannia-6-based Chinese medicine treatment, the most used Chinese medicine formulation, on the change in eGFR and albuminuria in patients with diabetes and CKD with severely increased albuminuria. METHODS: In this randomized, assessor-blind, standard care-controlled, parallel, multicenter trial, 148 adult patients from outpatient clinics with type 2 diabetes, an eGFR of 30-90 ml/min per 1.73 m 2 , and a urine albumin-to-creatinine ratio (UACR) of 300-5000 mg/g were randomized 1:1 to a 48-week add-on protocolized Chinese medicine treatment program (using Rehmannia-6-based formulations in the granule form taken orally) or standard care alone. Primary outcomes were the slope of change in eGFR and UACR between baseline and end point (48 weeks after randomization) in the intention-to-treat population. Secondary outcomes included safety and the change in biochemistry, biomarkers, and concomitant drug use. RESULTS: The mean age, eGFR, and UACR were 65 years, 56.7 ml/min per 1.73 m 2 , and 753 mg/g, respectively. Ninety-five percent ( n =141) of end point primary outcome measures were retrievable. For eGFR, the estimated slope of change was -2.0 (95% confidence interval [CI], -0.1 to -3.9) and -4.7 (95% CI, -2.9 to -6.5) ml/min per 1.73 m 2 in participants treated with add-on Chinese medicine or standard care alone, resulting in a 2.7 ml/min per 1.73 m 2 per year (95% CI, 0.1 to 5.3; P = 0.04) less decline with Chinese medicine. For UACR, the estimated proportion in the slope of change was 0.88 (95% CI, 0.75 to 1.02) and 0.99 (95% CI, 0.85 to 1.14) in participants treated with add-on Chinese medicine or standard care alone, respectively. The intergroup proportional difference (0.89, 11% slower increment in add-on Chinese medicine, 95% CI, 0.72 to 1.10; P = 0.28) did not reach statistical significance. Eighty-five adverse events were recorded from 50 participants (add-on Chinese medicine versus control: 22 [31%] versus 28 [36%]). CONCLUSIONS: Rehmannia-6-based Chinese medicine treatment stabilized eGFR on top of standard care alone after 48 weeks in patients with type 2 diabetes, stage 2-3 CKD, and severely increased albuminuria. CLINICAL TRIAL REGISTRY: Semi-individualized Chinese Medicine Treatment as an Adjuvant Management for Diabetic Nephropathy (SCHEMATIC), NCT02488252 .

[Adjuvant rice optimization of rice-steamed Rehmanniae Radix and its anti-osteoporosis effect].

The present study aimed to investigate the effect of various adjuvant rice on the quality of rice-steamed Rehmanniae Radix(RSRR) with Japonica rice, millet, yellow rice, black rice, and glutinous rice as raw materials, and analyze the anti-osteoporosis effect of RSRR by the optimal adjuvant rice. On the basis of the established UPLC-MS/MS method for the determination of the content of catalpol and rehmannioside D, comprehensive weighted scoring method was employed to evaluate the effect of various auxiliary rice on the quality of RSRR with the content of catalpol and rehmannioside D, character score, and taste score as indicators to optimize adjuvant rice. The osteoporosis model was induced by ovariectomy in rats. SD rats were randomly divided into a sham operation group, a model group, a positive control group, and low-dose and high-dose groups of Rehmanniae Radix, RSRR, steamed Rehmanniae Radix, and Epimedii Folium-RSRR. After treatment for 12 weeks, body weight, bone calcium content, and bone mineral density were mea-sured. The results showed that Japonica rice was selected as the optimal adjuvant due to the highest comprehensive score of RSRR steamed by Japonica rice. Rehmanniae Radix, RSRR, steamed Rehmanniae Radix, as well as Epimedii Folium-RSRR, could improve osteoporosis by increasing bone calcium content and bone mineral density. RSRR was superior to Rehmanniae Radix in improving osteo-porosis. However, there was no significant difference between RSRR and steamed Rehmanniae Radix. This study confirmed that Japo-nica rice was the optimal adjuvant rice of RSRR and verified the anti-osteoporosis effect of RSRR, which laid a foundation for further research on the pharmacological action and mechanism of RSRR.

Purification and structural analysis of a novel polysaccharide from Rehmannia Radix Praeparata.

In this paper, the purification, structure, and antioxidant activity of Rehmannia Radix Praeparata polysaccharide (RRPP) were studied. The RRPP was separated using DEAE-52 cellulose and Sephadex G-100. The RRPP consisted of xylose, glucose, rhamnose, galactose, and mannose in ratios of 10.64:5.58:3.52:1.39:1.0. No protein was detected in the RRPP fraction, and the molecular weight of RRPP was about 1.75 × 106 Da. The basic skeleton information was obtained using periodic acid oxidation-Smith degradation, and RRPP contained 1→, 1 → 2, 1 → 3, 1 → 4, 1 → 2,6, 1 → 4,6 or 1 → 6, 1 → 2,3, 1 → 2,3,4, and other glycosidic bonds. Fourier transform infrared spectroscopy also showed that RRPP has both α- and ß-glycosidic bonds. The in vitro antioxidant activity test showed that RRPP could potentialize scavenging effect on ABTS+· and its scavenging rate was 91.3%.

Combination of Internal Extractive Electrospray Ionization Mass Spectrometry and Statistical Analysis for High-Throughput Molecular Differentiation of Rehmannia glutinosa Samples.

Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey. is a perennial herb of the Scrophulariaceae family, which has long enjoyed a good reputation in China, and has a wide range of pharmacological effects and clinical applications. The place of origin is an important factor affecting the chemical composition of R. glutinosa, resulting in different pharmacological effects. Herein, internal extractive electrospray ionization mass spectrometry (iEESI-MS) combined with statistical techniques was established for high-throughput molecular differentiation of different R. glutinosa samples. Dried and processed R. glutinosa samples from four different places of origin were analyzed by iEESI-MS with high throughput (>200 peaks) and rapidness (<2 min/sample) without sample pretreatment. Clear separation models created by OPLS-DA were then established for distinguishing the places of origin of dried and processed R. glutinosa by using the obtained MS data. In addition, the molecular differences between the pharmacological effects of dried and processed R. glutinosa were also investigated by OPLS-DA, and 31 different components were screened out. This work provides a promising method for evaluating the quality of traditional Chinese medicines and studying the biochemical mechanism of processing.

Dried root of Rehmannia glutinosa extracts prevents steroid-induced avascular necrosis of femoral head by activating the wingless-type (Wnt)/ß-catenin signal pathway.

Steroid-induced avascular necrosis of femoral head (SANFH) is one of the most common complications caused by long-term or excessive clinical use of glucocorticoids. This study aimed to investigate the effects of dried root of Rehmannia glutinosa extracts (DRGE) in SANFH. First, SANFH rat model was established by dexamethasone (Dex). Tissue change and proportion of empty lacunae were detected by hematoxylin and eosin staining. Protein levels were detected by western bloting analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to assess apoptosis of femoral head tissue. Cell viability and apoptosis of MC3T3-E1 cells were assessed by Cell Counting Kit-8 assay and flow cytometry. ALP activity and cell mineralization were detected by ALP staining assay and Alizarin red staining. The findings showed that DRGE improved tissue damage, inhibited apoptosis, and promoted osteogenesis in SANFH rats. In vitro, DRGE increased cell viability, inhibited cell apoptosis, promoted osteoblast differentiation, reduced the levels of p-GSK-3ß/GSK-3ß, but increased the levels of ß-catenin in cells treated with Dex. Furthermore, DKK-1, an inhibitor of the wingless-type (Wnt)/ß-catenin signaling pathway, reversed the effect of DRGE on cell apoptosis and ALP activity in cells treated with Dex. In conclusion, DRGE prevents SANFH by activating the Wnt/ß-catenin signaling pathway, indicating that DRGE may be a hopeful choice drug to prevent and treat patients with SANFH.

Purple Rehmannia: Investigation of the activation of R2R3-MYB transcription factors involved in anthocyanin biosynthesis.

Engineering anthocyanin biosynthesis in herbs could provide health-promoting foods for improving human health. Rehmannia glutinosa is a popular medicinal herb in Asia, and was a health food for the emperors of the Han Dynasty (59 B.C.). In this study, we revealed the differences in anthocyanin composition and content between three Rehmannia species. On the 250, 235 and 206 identified MYBs in the respective species, six could regulate anthocyanin biosynthesis by activating the ANTHOCYANIDIN SYNTHASE (ANS) gene expression. Permanent overexpression of the Rehmannia MYB genes in tobacco strongly promoted anthocyanin content and expression levels of NtANS and other genes. A red appearance of leaves and tuberous/roots was observed, and the total anthocyanin content and the cyanidin-3-O-glucoside content were significantly higher in the lines overexpressing RgMYB41, RgMYB42, and RgMYB43 from R. glutinosa, as well as RcMYB1 and RcMYB3 in R. chingii and RhMYB1 from R. henryi plants. Knocking out of RcMYB3 by CRISPR/Cas9 gene editing resulted in the discoloration of the R. chingii corolla lobes, and decreased the content of anthocyanin. R. glutinosa overexpressing RcMYB3 displayed a distinct purple color in the whole plants, and the antioxidant activity of the transgenic plants was significantly enhanced compared to WT. These results indicate that Rehmannia MYBs can be used to engineer anthocyanin biosynthesis in herbs to improve their additional value, such as increased antioxidant contents.