Analysis of Differences in the Chemical Composition of Glycosides and Sugars between Four Forms of Fresh Rehmanniae Radix.

Fresh Rehmanniae Radix, as well as its processed products, are widely used in the clinical practice of traditional Chinese medicine. It is mainly available in four forms: fresh Rehmanniae Radix, raw Rehmanniae Radix, prepared Rehmanniae Radix, and nine-steamed, nine-dried Rehmanniae Radix. Pharmacological studies have shown that all Rehmanniae Radix forms contain iridoid glycosides and sugar compounds with various effects, including hypoglycemic, anti-inflammatory, neuroprotective, immunological enhancement, and bone marrow hematopoiesis-promoting activities. Differences in the efficacy among these Rehmanniae Radix forms and their processed products have been attributed to variations in their chemical compositions, particularly in iridoid glycosides and sugar compounds; however, the specific compositional differences in glycosides and sugars among the four forms of Rehmanniae Radix have not been clarified. Therefore, this study aims to qualitatively characterize the iridoid glycosides and sugar compounds in fresh Rehmanniae Radix, raw Rehmanniae Radix, prepared Rehmanniae Radix, and nine-steamed, nine-dried Rehmanniae Radix.

[Gender differences in antidepressant effect of raw Rehmanniae Radix].

For the first time, this study evaluated the gender differences and mechanisms of the antidepressant effects of raw Rehmanniae Radix(RRR) based on the classic depression model with traditional Chinese medicine syndrome of Yin deficiency and internal heat. The depression model with Yin deficiency and internal heat was established by the widely recognized and applied method of thyroxine induction of the classic depression model with Yin deficiency and internal heat(chronic unpredictable mild stress). Male and female mice were simultaneously treated with RRR. The study analyzed indicators of nourishing Yin and clearing heat, conventional antidepressant efficacy test indicators, and important biomolecules reflecting the pathogenesis and prevention and treatment mechanisms of depression, and conducted a correlation analysis of antidepressant efficacy, Yin-nourishing and heat-clearing efficacy, and biological mechanism in different genders, thereby comprehensively assessing the antidepressant effects of RRR on depression of Yin deficiency and internal heat, as well as its gender differences and mechanisms. RRR exhibited antidepressant effects in both male and female mouse models, and its antidepressant efficacy showed gender differences, with a superior effect observed in females. Moreover, the effects of RRR on enhancing or improving hippocampal neuronal pathology, nucleus-positive areas, postsynaptic dense area protein 95, and synaptophysin protein expression were more significant in females than in males. In addition, RRR significantly reversed the abnormal upregulation of nuclear factor(NF)-κB/cyclooxygenase 2(COX2)/NOD-like receptor thermal protein domain associated protein 3(NLRP3) pathway proteins in the hippocampus of both male and female mouse models. The antidepressant effects of RRR were more pronounced in depression female mice with Yin deficiency and internal heat syndrome, possibly due to the improvement of neuronal damage and enhancement of neuroplasticity. The antidepressant mechanisms of RRR for depression with Yin deficiency and internal heat syndrome may be associated with the downregulation of the NF-κB/COX2/NLRP3 pathway to reduce neuronal damage and enhance neuroplasticity.

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.

Potential benefits of Rehmanniae Radix after ancient rice-steaming process in promotion of antioxidant activity in rats' health.

Rice steam processed product of Rehmanniae Radix (RSRR), one of the processed products of Rehmanniae Radix (RR), is popular as an herbal medicine and food. However, the health-promoting effects and mechanisms of RSRR are still unclear. In this study, 10-week-old Sprague-Dawley female rats were treated with different processed products of RR. No organ coefficient differences were observed between RSRR and the control group, indicating that RSRR did not cause damage to the rats. Compared with other RR products, superoxide dismutase, glutathione, and catalase levels were significantly higher and malondialdehyde levels were significantly lower in the RSRR group, indicating that RSRR exerted a better antioxidant effect. Gene expression analysis showed that hemoglobin genes (Hba-a1, Hba-a2, Hbb-bs, Hbb, Hbq1b, Hbb-b1, and LOC103694857) may be potential biomarkers to evaluate the antioxidant effect of RSRR. Antioxidation-related signaling pathways in GO annotation, including cellular oxidant detoxification, hydrogen peroxide metabolic process, hemoglobin complex, and oxygen binding signaling pathways were significantly enriched, indicating these pathways may represent the antioxidant mechanism of RSRR. To explore the main active compounds primarily responsible for the antioxidant activity of RSRR, UPLC-Q-TOF-MS was used and six components (catalpol, rehmannioside A, rehmannioside D, melittoside, ajugol, and verbascoside) were identified in rat serum. Catalpol and rehmannioside A were predicted to be the major active components by network pharmacology. These results suggested that RSRR exhibits antioxidant activity and has health-promoting properties. This study provides a scientific basis for the antioxidant mechanism and clinical use of RSRR.

[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.

[Effects of Rehmanniae Radix and Rehmanniae Radix Praeparata on proteomics and autophagy in mice with type 2 diabetes mellitus induced by high-fat diet coupled with streptozotocin].

To compare the pancreatic proteomics and autophagy between Rehmanniae Radix-and Rehmanniae Radix Praeparata-treated mice with type 2 diabetes mellitus(T2DM). The T2DM mouse model was established by high-fat diet coupled with streptozotocin(STZ, intraperitoneal injection, 100 mg·kg~(-1), once a day for three consecutive days). The mice were then randomly assigned into a control group, low-(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix groups, low-(150 mg·kg~(-1)) and high-dose(300 mg·kg~(-1)) catalpol groups, low-(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix Praeparata groups, low-(150 mg·kg~(-1)) and high-dose(300 mg·kg~(-1)) 5-hydroxymethyl furfuraldehyde(5-HMF) groups, and a metformin(250 mg·kg~(-1)) group. In addition, a normal group was also set and each group included 8 mice. The pancreas was collected after four weeks of administration and proteomics tools were employed to study the effects of Rehmanniae Radix and Rehmanniae Radix Praeparata on protein expression in the pancreas of T2DM mice. The expression levels of proteins involved in autophagy, inflammation, and oxidative stress response in the pancreatic tissues of T2DM mice were determined by western blotting, immunohistochemical assay, and transmission electron microscopy. The results showed that the differential proteins between the model group and Rehmanniae Radix/Rehmanniae Radix Prae-parata group were enriched in 7 KEGG pathways, such as autophagy-animal, which indicated that the 7 pathways may be associated with T2DM. Compared with the control group, drug administration significantly up-regulated the expression levels of beclin1 and phosphorylated mammalian target of rapamycin(p-mTOR)/mTOR and down-regulated those of the inflammation indicators, Toll-like receptor-4(TLR4) and Nod-like receptor protein 3(NLRP3), in the pancreas of T2DM mice, and Rehmanniae Radix showed better performance. In addition, the expression levels of inducible nitric oxide synthase(iNOS), nuclear factor erythroid 2-related factor 2(Nrf2), and heine oxygenase-1(HO-1) in the pancreas of T2DM mice were down-regulated after drug administration, and Rehmanniae Radix Praeparata demonstrated better performance. The results indicate that both Rehmanniae Radix and Rehmanniae Radix Praeparata can alleviate the inflammatory symptoms, reduce oxidative stress response, and increase the autophagy level in the pancreas of T2DM mice, while they exert the effect on different autophagy pathways.

Comparing steamed and wine-stewed Rehmanniae Radix in terms of Yin-nourishing effects via metabolomics and microbiome analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rehmanniae Radix Praeparata (RRP), the processed root of Rehmannia glutinosa, has been widely used to treat Yin deficiency syndrome in traditional Chinese medicine. RRP is available in two forms: processed by steaming with water (SRR) or processed by stewing with yellow rice wine (WRR). Previous work has documented chemical differences in the secondary metabolomes and glycomes of SRR and WRR. AIM OF THE STUDY: This study aimed to compare SRR and WRR in terms of Yin-nourishing effects via metabolomics and microbiome analysis. MATERIALS AND METHODS: ICR mice were orally administered with thyroxine for 14 d to induce Yin deficiency. Changes in biochemical indices and histopathology were detected. Serum metabolomics analysis and microbial 16S rRNA sequencing were performed to compare the therapeutic effects and mechanisms between SRR and WRR in treating thyroxine-induced Yin deficiency. RESULTS: Both SRR and WRR decreased serum T3, T4 and MDA levels, and increased SOD activity. SRR more effectively decreased serum Cr, and ameliorated kidney injury, while WRR showed better regulation on ratio of cAMP/cGMP and serum TSH, and relieved thyroid injury. Both SRR and WRR regulated tyrosine, glycerophospholipid, and linoleic acid metabolism and the citric acid cycle. Additionally, SRR regulated fatty acid metabolism, while WRR influenced alanine, aspartate and glutamate metabolism, and bile acid biosynthesis. SRR significantly enriched the genera Staphylococcus and Bifidobacterium in the gut microbiome, while WRR significantly enriched the genera Akkermansia, Bacteroides and Parabacteroides, and decreased the abundance of Lactobacillus. CONCLUSIONS: SRR displayed better protective effects on kidney, while WRR showed stronger effects on thyroid in thyroxine-induced Yin deficient mice. These differences might be due to different regulating effects of SRR and WRR on the metabolome and gut microbiota.

[Effect of processing method on chemical constituents of Rehmanniae Radix: based on UHPLC-LTQ-Orbitrap MS].

This study aims to explore the chemical composition of Rehmanniae Radix braised with mild fire and compare the effect of processing method on the chemical composition of Rehmanniae Radix. To be specific, ultra-high performance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) was used to screen the chemical constituents of Rehmanniae Radix. The chemical constituents were identified based on the relative molecular weight and fragment ions, literature information, and Human Metabolome Database(HMDB). The ion peak area ratio of each component before and after processing was used as the index for the variation. SIMCA was employed to establish principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) models of different processed products. According to the PCA plot, OPLS-DA plot, and VIP value, the differential components before and after the processing were screened out. The changes of the content of differential components with the processing method were analyzed. A total of 66 chemical components were identified: 57 of raw Rehmanniae Radix, 55 of steamed Rehmanniae Radix, 55 of wine-stewed Rehmanniae Radix, 51 of repeatedly steamed and sundried Rehmanniae Radix Praeparata, 62 of traditional bran-braised Rehmanniae Radix, and 63 of electric pot-braised Rehmanniae Radix. Among them, the 9 flavonoids of braised Rehmanniae Radix were from Citri Reticulatae Pericarpium. PCA suggested significant differences in the chemical composition of Rehmanniae Radix Praeparata prepared with different processing methods. OPLS-DA screened out 32 chemical components with VIP value >1 as the main differential components. Among the differential components, 9 were unique to braised Rehmanniae Radix(traditional bran-braised, electric pot-braised) and the degradation rate of the rest in braised(traditional bran-braised, electric pot-braised) or repeatedly steamed and sundried Rehmanniae Radix was higher than that in the steamed or wine-stewed products. The results indicated the chemical species and component content of Rehmanniae Radix changed significantly after the processing. The 32 components, such as rehmapicrogenin, martynoside, jionoside D, aeginetic acid, hesperidin, and naringin, were the most important compounds to distinguish different processed products of Rehmanniae Radix. The flavonoids introduced by Citri Reticulatae Pericarpium as excipient may be the important material basis for the effectiveness of braised Rehmanniae Radix compared with other processed products.

Integrated Metabonomic Study on the Relationship between Medicinal Properties and Efficacy of Rehmanniae Radix.

Each Chinese medicine has its own properties and effects. However, the close connection between the medicinal properties and the effects of the medicine remains unclear. To export the scientific connection between the medicinal properties and efficacy of Rehmanniae Radix (RR), this study established a model and evaluated the therapeutic effects of RR on cold-heat syndrome to access the properties of RR, and then established a blood-heat syndrome model through the injection of rats with dry yeast combined with anhydrous ethanol. Related biochemical indicators (coagulation factors and central pyrogenic factor) were measured to assess the efficacy of RR. Finally, metabonomic technology was used to study the blood-cooling mechanism of RR from two aspects: medicinal properties and efficacy. The comprehensive results suggest that RR can significantly reduce the rectal temperature of blood-heat syndrome model rats and increase both the expression levels of coagulation factors (TNF-[Formula: see text], IL-1[Formula: see text], and IL-6) and the central pyrogenic factors (c-AMP, PGE-2). RR also cools the blood through regulating arginine, proline, phenylalanine, taurine, hypotaurine, sulfur, glycerophospholipid, primary bile acid metabolic pathways, and the tricarboxylic acid cycle. Therefore, RR plays the role of cooling blood by virtue of its cold property. The medicinal property of RR has a guiding effect on the clinical application. Moreover, the integrated metabolomic approach is a powerful tool for studying the properties and efficacy of Chinese medicine.

Analysis of Suitable Processing Time of Rehmanniae Radix Praeparata Processed with Amomi Fructus and Citri Reticulatae Pericarpium Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

ObjectiveTo investigate the relative content changes of differential metabolites and reducing sugars during the processing process of Rehmanniae Radix Praeparata （RRP） processed with Amomi Fructus （AF） and Citri Reticulatae Pericarpium （CRP）， and to lay the foundation for revealing the processing principle of this characteristic variety. MethodThe samples of the 0-54 h processing process of RRP processed with AF and CRP were taken as the research object， and their secondary metabolites were detected by ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS）. The 0.1% formic acid aqueous solution （A）-acetonitrile （B） was used as the mobile phase for gradient elution （0-1 min， 1%-3%B； 1-10 min， 3%-9%B； 10-15 min， 9%-12%B； 15-22 min， 12%-18%B； 22-31 min， 18%-24%B； 31-35 min， 24%-100%B； 35-36 min， 100%-5%B； 36-40 min， 5%-1%B； 40-45 min， 1%B）， column temperature was 40 ℃， injection volume was 3 μL， flow rate was 0.3 mL·min-1. Electrospray ionization （ESI） was used to scan and collect MS data in the negative ion mode， the scanning range was m/z 50-1 250. Data analysis was carried out using PeakView 1.2 software， and the chemical composition of RRP processed with AF and CRP was identified by combining the literature information and chemical composition databases. The MS data were normalized by MarkerView 1.2， and then the multivariate statistical analysis was applied to screen the differential metabolites， and the changes of the relative contents of the differential metabolites with different processing times was analyzed， finally， correlation analysis was performed between the differential metabolites， the change of the reducing sugar content was combined to determine the most suitable processing time of RRP processed with AF and CRP. ResultA total of 121 compounds were identified from RRP processed with AF and CRP at different processing times， and 12 differential metabolites were screened out by multivariate statistical analysis， including catalpol， hesperidin， isoacteoside， acteoside， narirutin， echinacoside， isomartynoside， decaffeoylacteoside， 6-O-E-feruloylajugol， dihydroxy-7-O-neohesperidin， jionoside D， and rehmapicroside. With the prolongation of processing time， the relative contents of these 12 differential metabolites and reducing sugars changed slightly at 52-54 h. ConclusionUPLC-Q-TOF-MS can comprehensively and accurately identify the chemical constituents of RRP processed with AF and CRP at different processing times， and the suitable processing time of 52-54 h is determined according to the content changes of different metabolites and reducing sugars， which provides a basis for revealing the scientific connotation of the processing principle of this variety.

Effect of processing method on chemical constituents of Rehmanniae Radix: based on UHPLC-LTQ-Orbitrap MS / 中国中药杂志

This study aims to explore the chemical composition of Rehmanniae Radix braised with mild fire and compare the effect of processing method on the chemical composition of Rehmanniae Radix. To be specific, ultra-high performance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) was used to screen the chemical constituents of Rehmanniae Radix. The chemical constituents were identified based on the relative molecular weight and fragment ions, literature information, and Human Metabolome Database(HMDB). The ion peak area ratio of each component before and after processing was used as the index for the variation. SIMCA was employed to establish principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) models of different processed products. According to the PCA plot, OPLS-DA plot, and VIP value, the differential components before and after the processing were screened out. The changes of the content of differential components with the processing method were analyzed. A total of 66 chemical components were identified: 57 of raw Rehmanniae Radix, 55 of steamed Rehmanniae Radix, 55 of wine-stewed Rehmanniae Radix, 51 of repeatedly steamed and sundried Rehmanniae Radix Praeparata, 62 of traditional bran-braised Rehmanniae Radix, and 63 of electric pot-braised Rehmanniae Radix. Among them, the 9 flavonoids of braised Rehmanniae Radix were from Citri Reticulatae Pericarpium. PCA suggested significant differences in the chemical composition of Rehmanniae Radix Praeparata prepared with different processing methods. OPLS-DA screened out 32 chemical components with VIP value >1 as the main differential components. Among the differential components, 9 were unique to braised Rehmanniae Radix(traditional bran-braised, electric pot-braised) and the degradation rate of the rest in braised(traditional bran-braised, electric pot-braised) or repeatedly steamed and sundried Rehmanniae Radix was higher than that in the steamed or wine-stewed products. The results indicated the chemical species and component content of Rehmanniae Radix changed significantly after the processing. The 32 components, such as rehmapicrogenin, martynoside, jionoside D, aeginetic acid, hesperidin, and naringin, were the most important compounds to distinguish different processed products of Rehmanniae Radix. The flavonoids introduced by Citri Reticulatae Pericarpium as excipient may be the important material basis for the effectiveness of braised Rehmanniae Radix compared with other processed products.

Adjuvant rice optimization of rice-steamed Rehmanniae Radix and its anti-osteoporosis effect / 中国中药杂志

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.

[Study on mechanism of Rehmanniae Radix Praeparata for treatment of osteoarthritis based on network pharmacology and molecular docking].

The mechanism of Rehmanniae Radix Praeparata against osteoarthritis was investigated based on network pharmacology, molecular docking, and in vitro experiments in the present study. Osteoclast models were established via receptor activator of nuclear factor-κB ligand(RANKL) and macrophage colony-stimulating factor(M-CSF) inducing RAW264.7 cells. Further, the influence of Rehmanniae Radix Praeparata on the activity of tartrate-resistant acid phosphatase(TRAP) was evaluated and the efficacy of Rehmanniae Radix Praeparata in the treatment of osteoarthritis was verified. The active components of Rehmanniae Radix Praeparata were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and literature, and the potential targets of the components were collected from SwissTargetPrediction. Osteoarthritis disease targets were searched in Online Mendelian Inheritance in Man(OMIM), Therapeutic Target Database(TTD), GeneCards, and DisGeNET. The intersection targets of Rehmanniae Radix Praeparata and osteoarthritis were obtained by Venny platform. The protein-protein interaction(PPI) network was constructed by Cytoscape 3.8.2, and key targets were obtained based on topology algorithm. The Database for Annotation, Visualization and Integrated Discovery(DAVID) was used to perform Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Finally, the mRNA expression of the key targets was determined by RT-qPCR and the binding activity between the components and key targets was validated by molecular docking. The results showed that Rehmanniae Radix Prae-parata inhibited the TRAP activity, thus inhibiting bone resorption by osteoclasts and treating osteoarthritis. By network pharmacology, 14 active components of Rehmanniae Radix Praeparata and 126 intersection targets were obtained. The network pharmacology enrichment results revealed 432 biological processes and 139 signaling pathways. Key targets such as proto-oncogene tyrosine-protein kinase Src(SRC), signal transducer and activator of transcription 3(STAT3) and transcription factor p65(RELA) were obtained according to the degree in topological analysis. SRC was highly expressed in osteoclasts, which accelerated the development of osteoarthritis. Therefore, SRC was selected for subsequent verification, and Rehmanniae Radix Praeparata decreased the gene expression level of SRC. The molecular docking showed that acteoside, isoacteoside, raffinose had good bonding activity with SRC, suggesting that they might be the critical components in treating osteoarthritis. In conclusion, Rehmanniae Radix Praeparata can inhibit bone resorption by osteoclasts and balance the metabolism of articular cartilage and subchondral bone via acting on SRC, thus playing a therapeutic role in osteoarthritis. In addition, Rehmanniae Radix Praeparata may exert overall efficacy on osteoarthritis through other targets such as STAT3 and RELA, and other related pathways such as PI3 K-AKT and IL-17 signaling pathways.

[Effect of Rehmanniae Radix on depression-like behavior and hippocampal monoamine neurotransmitters of chronic unpredictable mild stress model rats].

To investigate the effect of Rehmanniae Radix on depression-like behavior and monoamine neurotransmitters of chronic unpredictable mild stress(CUMS) model rats. CUMS combined with isolated feeding was used to induce the depression model of rats. The depression-like behavior of rats was evaluated by sucrose preference test, open field test, and forced swim test. Hematoxylin-Eosin(HE) staining was used to investigate the pathological changes of neurons in the CA1 and CA3 area of hippocampus. Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS) was used to detect the contents of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), 3,4-dihydroxyphenylacetic acid(DOPAC), homovanillic acid(HVA), norepinephrine(NE), and 3-methoxy-4-hydroxyphenyl glycol(MHPG) in rats. Western blot was used to detect the protein expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), and monoamine oxidase A(MAO-A) in the hippocampus of rats. Compared with the normal group, depressive-like behavior of rats was obvious in the model group. The arrangements of neurons in the CA1 and CA3 area of hippocampus were loose and disorderly. The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in the hippocampal area were decreased(P<0.01). The protein expression of TPH2 was decreased(P<0.01), but those of SERT and MAO-A were increased(P<0.01). In the Rehmanniae Radix groups with 1.8 g·kg~(-1) and 7.2 g·kg~(-1), the depression-like behavior of CUMS rats and pathological changes of neurons in CA1, CA3 area of hippocampus were improved. The protein expression of TPH2(P<0.05, P<0.01) was increased, and those of SERT and MAO-A were down-regulated(P<0.05, P<0.01). The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in hippocampus were increased(P<0.05, P<0.01). The changes in DA, DOPAC, HVA, DA/(DOPAC +HVA), NE, DHPG, and NE/DHPG were not statistically significant. The results suggested that Rehmanniae Radix improved depression-like behavior of CUMS rats, and the mechanism might be related to the regulation of synthesis, transportation, and metabolism of 5-HT neurotransmitter in the hippocampus.

[Optimization of processing technology of braised Rehmanniae Radix based on multiple indexes and response surface technology and correlation between components and color].

This study aims to explore the key factors influencing the processing of braised Rehmanniae Radix, optimize the processing, and determine the correlation between the components in different processed products and chroma values, which is expected to add quantitative indexes for the processing of braised Rehmanniae Radix and better control the processing. The weights of the indexes catalpol, rehmannioside D, verbascoside, isoacteoside, 5-hydroxymethylfurfural, reducing sugar, and appearance were calculated based on analytic hierarchy process(AHP) in combination with coefficient of variation, and the overall desirability(OD) was obtained. Box-Behnken design was used to explore the optimal amount of water added, time for soaking with rice wine, and steaming time in the processing of braised Rehmanniae Radix. Colorimeter was employed to determine the chroma of 17 samples and raw samples, and SPSS, Prism, and other software to investigate the correlation between the components in braised Rehmanniae Radix and the chroma values. The results showed that each factor influenced the processing, and the influence followed the order of steaming time>amount of water added>time for soaking with rice wine. The optimal processing process is as below: A total of 100 g medicinal material was added with 7 times of water, followed by soaking with rice wine for 5 h and steaming in a pot for 6 h. The correlation analysis suggested the extremely significantly positive correlation between L~* and content of catalpol, between a~* and 5-hydroxymethylfurfural content, and between b~* and catalpol content, and the extremely significantly negative correlation between L~* and the content of 5-hydroxymethylfurfural and reducing sugar, and between b~* and the content of 5-hydroxymethylfural and reducing sugar. In this experiment, response surface methodology was used to optimize the processing technology of braised Rehmanniae Radix and the optimized process was rational and feasible. The content of chemical components in braised Rehmanniae Radix was significantly correlated with the chroma. This study provided a new method for the quality evaluation of braised Rehmanniae Radix.

Neuroprotective Effects of Oligosaccharides in Rehmanniae Radix on Transgenic Caenorhabditis elegans Models for Alzheimer's Disease.

Rehmanniae Radix (RR, the dried tuberous roots of Rehmannia glutinosa (Gaertn.) DC.) is an important traditional Chinese medicine distributed in Henan, Hebei, Inner Mongolia, and Northeast in China. RR is frequently used to treat diabetes mellitus, cardiovascular disease, osteoporosis and aging-related diseases in a class of prescriptions. The oligosaccharides and catalpol in RR have been confirmed to have neuroprotective effects. However, there are few studies on the anti-Alzheimer's disease (AD) effect of oligosaccharides in Rehmanniae Radix (ORR). The chemical components and pharmacological effects of dried Rehmannia Radix (DRR) and prepared Rehmannia Radix (PRR) are different because of the different processing methods. ORR has neuroprotective potential, such as improving learning and memory in rats. Therefore, this study aimed to prove the importance of oligosaccharides in DRR (ODRR) and PRR (OPRR) for AD based on the Caenorhabditis elegans (C. elegans) model and the different roles of ODRR and OPRR in the treatment of AD. In this study, we used paralysis assays, lifespan and stress resistance assays, bacterial growth curve, developmental and behavioral parameters, and ability of learning and memory to explore the effects of ODRR and OPRR on anti-AD and anti-aging. Furthermore, the accumulation of reactive oxygen species (ROS); deposition of Aß; and expression of amy-1, sir-2.1, daf-16, sod-3, skn-1, and hsp-16.2 were analyzed to confirm the efficacy of ODRR and OPRR. OPRR was more effective than ODRR in delaying the paralysis, improving learning ability, and prolonging the lifespan of C. elegans. Further mechanism studies showed that the accumulation of ROS, aggregation, and toxicity of Aß were reduced, suggesting that ORR alleviated Aß-induced toxicity, in part, through antioxidant activity and Aß aggregation inhibiting. The expression of amy-1 was downregulated, and sir-2.1, daf-16, sod-3, and hsp-16.2 were upregulated. Thus, ORR could have a possible therapeutic effect on AD by modulating the expression of amy-1, sir-2.1, daf-16, sod-3, and hsp-16.2. Furthermore, ORR promoted the nuclear localization of daf-16 and further increased the expression of sod-3 and hsp-16.2, which significantly contributed to inhibiting the Aß toxicity and enhancing oxidative stress resistance. In summary, the study provided a new idea for the development of ORR.

[Mechanism of moistening process of Rehmanniae Radix based on kinetic process].

The moistening process of Rehmanniae Radix was characterized quantitatively by moisture phase, texture properties, and component content based on water absorption kinetics and expansion kinetics. Non-linear fitting of water absorption kinetics and expansion kinetics in the moistening process of Rehmanniae Radix was carried out. Low-field nuclear magnetic resonance and imaging(LF-NMR/MRI) technology was used to investigate the phase state and distribution changes of water during the moistening process. The Texture Analyzer was used for the determination of texture properties. The correlations between water absorption rate, expansion rate, water phase state, hardness, and compression cycle work of Rehmanniae Radix at different moistening time were analyzed. The results showed that the water absorption kinetics and expansion kinetics of Rehmanniae Radix were in accordance with the first-order kinetics. Moreover, the water absorption rate and expansion rate increased with the increase in temperature but decreased with the increase in the size of the medicinal materials.In the moistening process, the moisture was transferred from the outside to the inside, and the proportion of the moisture phase changed significantly.Within 16 hours, free water increased from 0.825% to 97.7%,while bound water decreased from 99.2% to 2.33%.Within 28 hours, the texture properties, such as hardness and compression cycle work, decreased gradually with the prolongation in moistening time.At 32 hours, water was evenly distributed throughout the whole medicinal material, and the texture properties also tended to be stable.Pearson correlation bivariate analysis showed that moistening time, water absorption rate, expansion rate, the relative content of free water and bound water, hardness, and compression cycle work were significantly correlated, suggesting that water absorption kinetics and expansion kinetics, LF-NMR/MRI,and Texture Analyzer could directly and quantitatively characterize the moistening process.This study is expected to provide a scientific basis for clarifying the scientific connotation of the moistening process of Rehmanniae Radix.

[Effect of Rehmanniae Radix Praeparata on energy metabolism in prefrontal cortex of rats with attention deficit hyperactivity disorder based on "static Yin and dynamic Yang" theory].

The present study investigated the effect of Rehmanniae Radix Praeparata(RRP) on the energy metabolism of prefrontal cortex(PFC) of spontaneously hypertensive rats with attention deficit hyperactivity disorder(ADHD) based on the "static Yin and dynamic Yang" theory.Thirty spontaneously hypertensive male rats aged 3 weeks were randomly divided into a model group, a methylphenidate(MPH) group(2 mg·kg~(-1)), and an RRP group(2.4 g·kg~(-1)).Wistar-Kyoto(WKY) male rats of the same age were assigned to the normal group.Rats were treated with corresponding drugs twice per day, and those in the model group and the normal group received the same volume of 0.9% sodium carboxymethyl cellulose(CMC-Na) solution by gavage.The open-field test was performed to evaluate the spontaneous and impulsive behaviors of rats before treatment and on the 4~(th) week after treatment.Four weeks after treatment, PFC was isolated and mitochondria were prepared.The content of adenosine triphosphate(ATP), adenosine diphosphate(ADP), and adenosine monophosphate(AMP) in the PFC was determined by high-performance liquid chromatography(HPLC), and energy charge(EC) was calculated.The parameters related to mitochondrial respiratory function were measured by the Clark oxygen electrode, specifically, state 3 respiration(ST3), state 4 respiration(ST4), and respiratory control rate(RCR).Enzymatic activities of succinate dehydrogenase(SDH), cytochrome C oxidase(COX), Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase were measured by chemical colorimetry.Mitochondrial permeability transition pore(mPTP) opening was measured by spectrophotometry.Protein expression of glucose transporter 1(GLUT1) and GLUT3 in PFC was tested by Western blot.Compared with the results in the model group, RRP could significantly reduce the total distance of movement, vertical times, and distance in the central area in the open field test(P<0.05 or P<0.01), increase the content of ATP and EC, decrease the content of AMP(P<0.05), elevate ST3 and RCR(P<0.05), potentiate activities of SDH, COX, Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase(P<0.05 or P<0.01), inhibit the opening of mPTP, and increase the expression levels of GLUT1 and GLUT3 proteins(P<0.05).It was inferred that RRP could inhibit hyperacti-vity and impulsivity by improving the energy metabolism disorder in PFC of ADHD rats, and its mechanism may be related to the improvement of mitochondrial respiratory function, potentiation of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, and mitochondrial respiratory enzymes, inhibition of the opening of mPTP, and up-regulation of the expression of glucose transporter proteins.This study initially reveals the biological connotation of the "static Yin and dynamic Yang" theory in ADHD.

[Metabolomic profiling reveals blood-tonifying effect of Rehmanniae Radix Praeparata based on theory of activating spleen and generating blood].

Based on the theory of activating spleen and generating blood, this study explored the effect of Rehmanniae Radix Prae-parata on the spleen metabolome of the rat model with blood deficiency syndrome.The rat model of blood deficiency syndrome was established by combining with cyclophosphamide(CTX) and N-acetyl-phenylhydrazine(APH), and the metabolomes of the spleen samples were analyzed with ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS).Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were carried out for the metabolite profiles of spleen samples.The MEV heatmap and metabolic network were established based on the potential biomarkers.Finally, the blood routine indexes were combined with the metabolomic profile to reveal the mechanism of Rehmanniae Radix Praeparata in activating spleen and generating blood.The treatment with CTX and APH decreased the blood routine indexes such as white blood cell count(WBC), red blood cell count(RBC), platelet(PLT), and hematocrit(HCT), indicating that the rat model of blood deficiency syndrome was successfully established.The administration of Rehmanniae Radix Praeparata significantly improved the blood routine indexes, which suggested that Rehmanniae Radix Praeparata played a role in replenishing blood.In addition, the metabolomics analysis identified 41 potential biomarkers.The PCA and MEV heatmap also showed significant improvement effect of Rehmanniae Radix Praeparata on the spleen metabolic profile.These potential biomarkers were mainly involved in tricarboxylic acid cycle, niacin and nicotinamide metabolism, phenylalanine metabolism, tyrosine metabolism, taurine and hypotaurine metabolism, and sphingolipid metabolism.Therefore, we hypothesize that Rehmanniae Radix Praeparata may regulate energy metabolism, peripheral blood production, and oxidative injury of hemocytes to tonify blood.

[Anti-depressant components and mechanism of Rehmanniae Radix based on UPLC-Q-Orbitrap HRMS and network pharmacology].

This study aimed to explore the anti-depressant components of Rehmanniae Radix and its action mechanism based on network pharmacology combined with molecular docking. The main components of Rehmanniae Radix were identified by ultra-high performance liquid chromatography-quadrupole/Orbitrap high resolution mass spectrometry(UPLC-Q-Orbitrap HRMS), and the related targets were predicted using SwissTargetPrediction. Following the collection of depression-related targets from GeneCards, OMIM and TTD, a protein-protein interaction(PPI) network was constructed using STRING. GO and KEGG pathway enrichment analysis was performed by Metascape. Cytoscape 3.7.2 was used to construct the networks of "components-targets-disease" and "components-targets-pathways", based on which the key targets and their corresponding components were obtained and then preliminarily verified by molecular docking. Rehmanniae Radix contained 85 components including iridoids, ionones, and phenylethanoid glycosides. The results of network analysis showed that the main anti-depressant components of Rehmanniae Radix were catalpol, melittoside, genameside C, gardoside, 6-O-p-coumaroyl ajugol, genipin-1-gentiobioside, jiocarotenoside A1, neo-rehmannioside, rehmannioside C, jionoside C, jionoside D, verbascoside, rehmannioside, cistanoside F, and leucosceptoside A, corresponding to the following 16 core anti-depression targets: AKT1, ALB, IL6, APP, MAPK1, CXCL8, VEGFA, TNF, HSP90 AA1, SIRT1, CNR1, CTNNB1, OPRM1, DRD2, ESR1, and SLC6 A4. As revealed by molecular docking, hydrogen bonding and hydrophobicity might be the main action forms. The key anti-depression targets of Rehmanniae Radix were concentrated in 24 signaling pathways, including neuroactive ligand-receptor interaction, neurodegenerative disease-multiple diseases pathway, phosphatidylinositol 3-kinase/protein kinase B pathway, serotonergic synapse, and Alzheimer's disease.