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.
Female ; Rats ; Animals ; Oryza ; Chromatography, Liquid ; Calcium ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; Drugs, Chinese Herbal/pharmacology* ; Osteoporosis/drug therapy* ; Rehmannia ; Adjuvants, Pharmaceutic

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.
Humans ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/chemistry* ; Plant Extracts/chemistry* ; Rehmannia/chemistry* ; Flavonoids/analysis*

The present study explored the effect and mechanism of repeatedly steamed and sundried Rehmanniae Radix Praeparata(RRP) in delaying brain aging in ovariectomized mice. After ovariectomy, the mice were randomly divided into a model group, an estradiol valerate group(0.3 mg·kg~(-1)), and low-(1.0 g·kg~(-1)), medium-(2.0 g·kg~(-1)), and high-dose(4.0 g·kg~(-1)) RRP groups, and a sham operation group was also set up, with 15 mice in each group. One week after the operation, intragastric administration was carried out for 15 consecutive weeks. The step-down test and Morris water maze test were used to detect the behavioral changes of mice. HE staining and Nissl staining were used to observe the morphological changes of mouse brain tissues. Immunohistochemistry was used to detect the expression of Aβ and ER_β in mouse brain tissues. The serum estrogen levels and cholinesterase and cholinesterase transferase levels in brain tissues of mice were detected by assay kits. The extracted hippocampal protein was detected by the Nano-ESI-LC-MS system, identified by the Protein Discovery, and analyzed quantitatively and qualitatively by the SIEVE. The PANTHER Classification System was used for GO analysis and KEGG pathway enrichment analysis of the differential proteins. Compared with the sham operation group, the model group showed decreased learning and memory ability, shortened step-down latency(P<0.05), prolonged escape latency(P<0.05), reduced platform crossings and residence time in the target quadrant, scattered nerve cells in the hippocampus with enlarged intercellular space, increased expression of Aβ-positive cells(P<0.05), declining expression of ER_β-positive cells and estrogen level(P<0.05), and weakened cholinergic function(P<0.05). Compared with the model group, the RRP groups showed improved learning and memory ability, prolonged step-down latency(P<0.05), increased estrogen level(P<0.05), neatly arranged nerve cells in the hippocampus with complete morphology, declining Aβ-positive cells, and elevated expression of ER_β-positive cells. A total of 146 differential proteins were screened out by proteomics, and KEGG pathway enrichment yielded 75 signaling pathways. The number of proteins involved in the dopaminergic synapse signaling pathway was the largest, with 13 proteins involved. In summary, RRP can delay brain aging presumedly by increasing the level of estrogen, mediating the dopaminergic synapse signaling pathway, and improving cholinergic function.
Aging ; Animals ; Female ; Hippocampus/metabolism* ; Learning ; Mice ; Plant Extracts ; Proteomics ; Rehmannia

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.
Drugs, Chinese Herbal/pharmacology* ; Humans ; Molecular Docking Simulation ; Network Pharmacology ; Neurodegenerative Diseases ; Plant Extracts ; Rehmannia

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

With the rice-steamed Rehmanniae Radix unearthed from the tomb of Haihunhou in the Western Han Dynasty as the re-ference, the present study evaluated the quality of Rehmanniae Radix and investigated the processing technology of rice-steamed Rehmanniae Radix to lay the foundation for the research on rice-steamed Rehmanniae Radix products. With catalpol and rehmannioside D as the investigation indexes, the quality and grade of Rehmanniae Radix from different producing areas were evaluated with the methods in 2020 edition of Chinese Pharmacopoeia. UPLC method was established for the determination of catalpol and rehmannioside D in the rice-steamed Rehmanniae Radix. The effects of steaming time, the amount of supplementary rice, and steaming times in the rice-steamed processing on the quality of products were investigated by L_9(3~4) orthogonal test and multi-index comprehensive balance scoring method combined with the content of catalpol and rehmannioside D and appearance characteristics. At last, the stability of the processing technology was tested. The results showed that the optimal processing technology for rice-steamed Rehmanniae Radix was as follows: Rehmanniae Radix and rice(200 g∶4 g) were steamed twice at atmospheric pressure, four hours each time. The mass fractions of catalpol and rehmannioside D were 0.184% and 0.335%, respectively, and the character score was 6.5. The processing conditions are reaso-nable, stable, and feasible. It can provide a basis for the restoration of the ancient rice-steamed processing technology and references for the development of rice-steamed Rehmanniae Radix products in the future.
Drugs, Chinese Herbal/pharmacology* ; Oryza ; Plant Extracts ; Rehmannia ; Technology

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

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.
3,4-Dihydroxyphenylacetic Acid/pharmacology* ; Animals ; Antidepressive Agents/therapeutic use* ; Chromatography, Liquid ; Depression/drug therapy* ; Disease Models, Animal ; Dopamine ; Eosine Yellowish-(YS)/pharmacology* ; Hematoxylin/pharmacology* ; Hippocampus/metabolism* ; Homovanillic Acid/pharmacology* ; Hydroxyindoleacetic Acid/metabolism* ; Methoxyhydroxyphenylglycol/pharmacology* ; Monoamine Oxidase/metabolism* ; Neurotransmitter Agents/metabolism* ; Norepinephrine/pharmacology* ; Plant Extracts ; Rats ; Rehmannia/chemistry* ; Serotonin/metabolism* ; Serotonin Plasma Membrane Transport Proteins/pharmacology* ; Stress, Psychological/metabolism* ; Tandem Mass Spectrometry ; Tryptophan Hydroxylase/metabolism*

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.
Drugs, Chinese Herbal/chemistry* ; Excipients ; Furaldehyde/analogs & derivatives* ; Iridoid Glucosides ; Plant Extracts ; Rehmannia/chemistry* ; Sugars ; Technology ; Water

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