Effects of Jiaohong Pills and Its Prescription on Scopolamine-induced Alzheimer's Disease Mice / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Jiaohong pills （JHP） and its prescription， Pericarpium Zanthoxyli （PZ） and Rehmanniae Radix （RR） cognitive dysfunction in scopolamine-induced Alzheimer's disease （AD） mice and its mechanism through pharmacodynamic and metabolomics study. MethodThe animal model of AD induced by scopolamine was established and treated with PZ， RG and JHP， respectively. The effects of JHP and its formulations were investigated by open field test， water maze test， object recognition test， avoidance test， cholinergic system and oxidative stress related biochemical test. Untargeted metabolomics analysis of cerebral cortex was performed by ultra-performance liquid chromatography-Quadrupole/Orbitrap high resolution mass spectrometry （UPLC Q-Exactive Orbitrap MS）. ResultThe behavioral data showed that， compared with the model group， the discrimination indexes of the high dose of JHP， PZ and RR groups was significantly increased （P<0.05）. The staging rate of Morris water maze test in the PZ， RR， high and low dose groups of JHP was significantly increased （P<0.05， P<0.01）， the crossing numbers in the PZ， JHP high and low dose groups were significantly increased （P<0.05， P<0.01）； the number of errors in the avoidance test were significantly reduced in the PZ and high-dose JHP groups （P<0.01）， and the error latencies were significantly increased in the JHP and its prescription drug groups （P<0.01）. Compared with the model group， the activities of acetylcholinesterase in the cerebral cortex of the two doses of JHP group and the PZ group were significantly increased （P<0.05， P<0.01）， and the activity of acetylcholinesterase in the high-dose JHP group was significantly decreased （P<0.05）， and the level of acetylcholine was significantly increased （P<0.01）. At the same time， the contents of malondialdehyde in the serum of the two dose groups of JHP decreased significantly （P<0.05， P<0.01）. The results of metabolomics study of cerebral cortex showed that 149 differential metabolites were identified between the JHP group and the model group， which were involved in neurotransmitter metabolism， energy metabolism， oxidative stress and amino acid metabolism. ConclusionJHP and its prescription can antagonize scopolamine-induced cognitive dysfunction， regulate cholinergic system， and reduce oxidative stress damage. The mechanism of its therapeutic effect on AD is related to the regulation of neurotransmitter， energy， amino acid metabolism， and improvement of oxidative stress.

Rehmanniae Radix Praeparata Improves Neurological Function of Ischemic Stroke Rats by Inhibiting Autophagy and Ferroptosis / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Rehmanniae Radix Praeparata on neurological function injury in ischemic stroke rats and explore its mechanism. MethodMale SD rats were randomized into sham operation， model， low- and high -dose （3.5 g·kg-1 and 7 g·kg-1） Rehmannia Radix Praeparata， and nimodipine （0.01 g·kg-1） groups. The rat model of middle cerebral artery occlusion （MCAO） was established with the modified suture occlusion method. Zea-Longa 5-point scoring was employed to evaluate the neurological function of rats. The cerebral infarction volume was detected by 2，3，5-triphenyltetrazolium chloride （TTC） staining. Hematoxylin-eosin staining and Nissl staining were employed to observe the morphology and damage of the brain tissue. Meanwhile， the serum levels of lactate dehydrogenase （LDH）， oxidative stress-related indicators superoxide dismutase （SOD）， glutathione peroxidase 4 （GPX4）， and malondialdehyde （MDA）， and the iron （Fe） content in the brain tissue were determined. To explore the mechanism of Rehmanniae Radix Preparata in mitigating the neurological damage in ischemic stroke rats， Western blotting was employed to determine the expression levels of proteins in the ischemic brain tissue. The autophagy-associated proteins included autophagy effector （beclin-1）， microtubule-associated protein light chain 3 （LC3B）， and ubiquitin-binding protein p62 （p62）. The ferroptosis-associated proteins included transferrin （TF）， transferrin receptor 1 （TFR1）， ferritin heavy chain 1 （FTH1）， and ferropotin （FPN1）. The neurological function injury-associated proteins included brain-derived neurotrophic factor （BDNF） and tyrosine kinase receptor B （TrkB）. ResultCompared with the sham operation group， the model group showed increased neurological function score， cerebral infarction volume， and appearance of nuclear pyknosis and vacuole of cells in the cerebral cortex. In addition， the model group presented elevated levels of LDH， MDA， and Fe （P<0.01） and lowered levels of SOD and GPX4 （P<0.01）. Compared with the model group， Rehmanniae Radix Praeparata decreased the content of LDH， MDA， and Fe （P<0.05， P<0.01） and elevated the levels of SOD and GPX4 （P<0.05， P<0.01）. Compared with the sham operation group， the modeling promoted the expression of beclin-1，LC3B Ⅱ/Ⅰ， TF， and TFR1 and inhibited the expression of p62， FTH1， FPN1， BDNF， and TrkB （P<0.01）. The expression levels of these proteins were recovered after the treatment with Rehmanniae Radix Praeparata. ConclusionRehmanniae Radix Praeparata may inhibit ferroptosis and improve the neurological function in ischemic stroke rats by down-regulating the autophagy level in the brain tissue.

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.

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 / 中国中药杂志

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.

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.

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.

Targeted Metabonomics Combined with 16SrRNA Gene Sequencing to Analyze the Effect of Rehmanniae Radix on Intestinal Flora in Rats with Kidney Yin Deficiency Syndrome Before and After Processing / 中药新药与临床药理

Objective To investigate the effects of Rehmanniae Radix before and after processing on the intestinal flora of rats with kidney yin deficiency syndrome.Methods SD rats were randomly divided into blank group,model group,probiotic group(0.35 g·kg-1),high-/medium-/low-dose groups of Rehmanniae Radix Praeparata(3.5,1.75,0.875 g·kg-1),and high-/medium-/low-dose groups of Rehmanniae Radix(3.5,1.75,0.875 g·kg-1),with 9 rats in each group.Except for the blank group,rats in each group were injected intramuscularly with Dexamethasone Sodium Phosphate Injection(0.35 mg·kg-1)once a day for 21 days.The drug was administered by gavage once a day on the seventh day of modelling for 14 days.The adrenal histopathological changes were observed by using HE staining;the levels of serum cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP),corticotropin-releasing hormone(CRH),adrenocorticotropic hormone(ACTH),and corticosterone(CORT)were detected by ELISA;and the levels of short-chain fatty acids in the feces and changes in the diversity of intestinal flora were detected by a targeted metabolomic approach in conjunction with 16SrRNA sequencing in the rats in each group.Results(1)Compared with the blank group,the body mass of rats in the model group was significantly decreased on days 7,14 and 21(P＜0.05,P＜0.01);serum levels of cAMP,CRH,ACTH,CORT and the cAMP/cGMP ratio were all significantly increased(P＜0.01),and the cGMP content was significantly decreased(P＜0.01);and the adrenal cortex was thinned,with the boundaries of various layers of the cortex unclear.Compared with the model group,the body mass of rats in the Rehmanniae Radix Praeparata administration group on day 21 were all significantly increased(P＜0.05,P＜0.01),and the serum content of cAMP,CRH,ACTH,CORT and the cAMP/cGMP ratio were all significantly decreased(P＜0.05,P＜0.01),and the content of cGMP was significantly increased(P＜0.05,P＜0.01)in the rats;body mass of rats in the Rehmanniae Radix administration group did not change significantly(P＞0.05),CRH and CORT contents in serum of rats in the high-dose group of Rehmanniae Radix were significantly reduced(P＜0.01),and ACTH contents in serum of rats in the medium-dose group of Rehmanniae Radix were significantly reduced(P＜0.05);the adrenal cortex of rats in all the administration groups were improved,in particular,the thickening of the adrenal cortex layers was obvious in the Rehmanniae Radix Praeparata group,and the improvement effect was superior to that in the Rehmanniae Radix group.(2)Compared with the blank group,the difference of Coverage index was not statistically significant(P＞0.05),and the coverage of each group was good;the abundance index(Sobs,Ace,Chao)and diversity index(Shannon)of the model group were significantly increased(P＜0.01),and the Simpson index was significantly decreased(P＜0.01).Compared with the model group,Sobs index was significantly decreased in the medium-and high-dose groups of Rehmanniae Radix Praeparata(P＜0.05),Chao index was significantly decreased in the administered groups of Rehmanniae Radix Praeparata and high-dose group of Rehmanniae Radix(P＜0.05,P＜0.01),and Simpson index was significantly increased in the high-dose group of Rehmanniae Radix Praeparata(P＜0.05).The changes of Rehmanniae Radix on the richness and diversity of intestinal microbial community in kidney yin-deficient rats were small,while Rehmanniae Radix Praeparata could better maintain the stability of the richness and diversity of intestinal microbial community in kidney yin-deficient rats.(3)Compared with the blank group,the abundance of phylum firmicutes in the feces of the model group was significantly decreased,while the abundance of bacteroides and actinomycetes was significantly increased.The abundance of Lactobacillus was significantly decreased(P＜0.01),while the abundance of norank_f__Muribaculaceae and Bifidobacterium was significantly increased(P＜0.01).Compared with the model group,the trend of recovery of bacterial abundance in the probiotic group and the high-dose group of Rehmanniae Radix Praeparata was more similar to that of the blank group,which showed that it had the best regulating effect on the ratio of bacterial flora;the abundance of lactobacillus in all administered groups was increased,with that of the probiotic group was significantly increased(P＜0.01);the abundance of norank__f__Muribaculaceae and Bifidobacterium were all decreased,among which the probiotic group and the medium-and high-dose groups of Rehmanniae Radix Praeparata were significantly decreased(P＜0.01),and the effect was significantly superior to that of Rehmanniae Radix.The COG functions of the samples in each group were mainly focused on amino acid transport and metabolism,carbohydrate transport and metabolism,translation,ribosomal structure and biogenesis,replication,recombination and repair,but the abundance information of each function was different between groups,which may be due to the differences caused by dysbiosis of intestinal flora.(4)Compared with the blank group,the levels of acetic acid,butyric acid and propionic acid in the faeces of rats in the model group were significantly decreased(P＜0.05,P＜0.01),and the level of isobutyric acid was significantly increased(P＜0.01).Compared with the model group,the levels of acetic acid,butyric acid and propionic acid in the faeces of rats in the probiotic group and the low-,medium-and high-dose groups of Rehmanniae Radix Praeparata were significantly increased(P＜0.05,P＜0.01)and the levels of isobutyric acid were significantly decreased(P＜0.05,P＜0.01);although the above indexes in the Rehmanniae Radix group were improved,the difference was not statistically significant(P＞0.05).Conclusion The enhanced therapeutic effect of Rehmanniae Radix after processing on rats with kidney yin deficiency syndrome may be related to its adjusting effect on intestinal flora.

Gender differences in antidepressant effect of raw Rehmanniae Radix / 中国中药杂志

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.

Effect of Rehmanniae Radix combined with Scrophulariae Radix on renal microinflammation in diabetic nephropathy rats based on NF-κB pathway / 国际中医中药杂志

Objective:To explore the effect of Rehmanniae Radix combined with Scrophulariae Radix on renal microinflammation in diabetic nephropathy (DN) rats. Methods:50 Sprague Dawley (SD) rats were adaptively fed for 1 week, and then 10 rats were randomly selected as the blank control group, and the rest were treated with STZ intraperitoneal injection combined with high-fat diet to induce DN model. After 4 weeks, the successful modeled rats were randomly divided into model group, Rehmannia glutinosa Scrophularia group (5.25 g/kg) and metformin group (200 mg/kg), with 10 rats in each group. After 8 weeks of administration, fasting blood glucose was measured by blood glucose meter; microalbuminuria was measured by benzalkonium chloride turbidimetry; serum cystatin, TNF-α, IL-6 and hs-CRP levels were measured by ELISA kit; renal pathological changes were detected by HE staining, Masson staining and PAS staining; the expression of MCP-1, NF-κB (total) and p-NF-κB protein in renal tissue was detected by Western blot.Results:Compared with the model group, the body weight of rats in DHXS group was significantly decreased ( P<0.05). The content of fasting blood glucose[(18.06 ± 5.69) mmol/L vs. (29.42 ± 0.63)mmol/L], 24-hour urine protein [(11.02 ± 1.77)mg/d vs. (31.61 ± 0.65)mg/d], serum cystatin [(208.16 ± 12.07)ng/ml vs. (278.05 ± 19.33)ng/ml], TNF-α [(9.13 ± 1.46)pg/ml vs. (73.16 ± 8.30)pg/ml], IL-6[(4.27 ± 1.07)pg/ml], hs-CRP[(219.36 ± 22.02)ng/ml vs. (266.97 ± 15.80)ng/ml] in DHXS group were significantly decreased ( P<0.05), and the expression level of p-NF-κB (0.49 ± 0.07 vs. 0.84 ± 0.12) and MCP-1 (0.44 ± 0.02 vs. 0.64 ± 0.11) in renal tissue of rats in DHXS group were significantly reduced ( P<0.05). Conclusion:Rehmanniae Radix combined with Scrophulariae Radix can protect kidney by inhibiting the over activation of NF-κB, and reducing the expression of MCP-1 related protein to reduce renal micro inflammation.

Study on the molecular mechanism of Cinnamomi Cortex-Rehmanniae Radix in preventing and treating osteoporosis based on multi-directional regulation of inflammatory factors, estrogen and bone homeostasis / 药学学报

In this study, the molecular mechanism of Cinnamomi Cortex-Rehmanniae Radix (CR) in the prevention and treatment of osteoporosis (OP) was investigated by integrating compatibility analysis of compound, bioinformatics and metabolomics. The rat OP models were established, and the Micro-CT indexes and pathological sections were comprehensively evaluated. The results showed that compared with the model group, the indexes such as bone mineral density (BMD) and bone volume/tissue volume (BV/TV) were significantly increased after CR treatment (P < 0.05), and the bone trabeculae were arranged into mesh. The results of UHPLC-Q-TOF/MS mainly involved amino acid metabolism, lipid metabolism and estrogen metabolism pathways. Integrating bioinformatics and metabolomics analysis, it was finally found that: ① cinnamic acid and ethylcinnamate inhibit inflammatory factors such as TNF, IL-1β, and IL-13, thereby preventing and treating OP; ② multiple active ingredients of CR target ESR2, PPARG, and CYP19A1, GABRA1 and other targets, regulate cAMP synthesis, AMPK signaling pathway and lipid metabolism, thereby regulating estrogen levels to prevent and treat OP; ③ oleic acid, arachic acid, etc. act on AR, VDR and other targets, and regulate HIF-1 signaling pathway and AGE-RAGE signaling pathway, thereby regulating osteoblasts and osteoclasts, and affecting calcium and phosphorus absorption to maintain bone homeostasis. This study clarified the molecular mechanism of CR in preventing and treating OP from the perspective of multi-directional regulation of inflammatory factors, estrogen and bone homeostasis, and provided theoretical basis for the clinical application of CR and the development of compound. This experiment complied with the ethical standards of animal experiments and was approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine (No. SUCMDL20210309002).

Preparation of Rehmanniae Radix Juice for Baihe Dihuangtang Based on UPLC-Q-TOF-MS / 中国实验方剂学杂志

ObjectiveTo compare the four preparation methods of Rehmanniae Radix juice described in ancient literature and find the method that is most suitable for the preparation of Rehmanniae Radix juice used in Baihe Dihuangtang. MethodThe ancient medical books record four methods for preparing Rehmanniae Radix juice： crushing fresh Rehmanniae Radix for juice， steaming fresh Rehmanniae Radix for juice， boiling fresh Rehmanniae Radix for juice， and boiling dry Rehmanniae Radix for juice. Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS） was employed to detect the compounds in the four juice samples， followed by principal component analysis （PCA）. Result① Totally 27 compounds were identified in the juice samples， including 10 iridoid glycosides， 14 phenylethanoid glycosides， 2 phenolic acids， and 1 irisone. Among them， 15 common compounds were shared by the four juice samples， including 7 iridoid glycosides， 7 phenylethanoid glycosides， and 1 phenolic acid. ② Five common compounds in the four juice samples can be matched with the reference standards， which were catalpol， aucubin， rehmannioside D， ajugol， and purpureaside C. ③ Verbascoside and isoacteoside were not detected in the juice prepared by crushing fresh Rehmanniae Radix， while it was detected in the other three juice samples， which indicated that the two components were produced after heating rather than being the original components in fresh Rehmanniae Radix. ④ The comparison of the ion fragments demonstrated that verbascoside was produced from purpureaside C after the cleavage of the glycosidic bond and removal of a molecule of mannose. ⑤ Isoacteoside could be isomerized from verbascoside， and its relative content increased with the extension of heating time. However， the relative content of verbascoside and purpureaside C did not decrease significantly. Therefore， it was hypothesized that purpureaside C was produced from its upstream component. ConclusionThe juice prepared by crushing fresh Rehmanniae Radix has the chemical composition significantly different from the juice samples prepared with the other 3 methods， while the latter 3 juice samples had similar chemical composition. Although all the four methods can be used， it is more suitable to prepare Rehmanniae Radix juice by steaming fresh Rehmanniae Radix， boiling fresh Rehmanniae Radix， and boiling dry Rehmanniae Radix.

Quality Evaluation in Processing of Rehmanniae Radix Praeparata Processed with Amomi Fructus and Citri Reticulatae Pericarpium Based on "Color as Paint，Sweet as Maltose" / 中国实验方剂学杂志

ObjectiveTo study the correlation between the apparent color， comprehensive sweetness and the content of main components in the preparation of Rehmanniae Radix Praeparata processed with Amomi Fructus and Citri Reticulatae Pericarpium， so as to lay a foundation for revealing the processing principle of Rehmanniae Radix. MethodThe color of Rehmanniae Radix Praeparata sample powder was measured by automatic colorimeter， the contents of 14 active components in samples with different heating time points were determined by high performance liquid chromatography， including 7 glycosides of catalpol， rehmannia glycoside D， leonurine glycoside， 5-hydroxymethyl furfural， verbascoside， isoacteoside and hesperidin， and 7 carbohydrates of D-fructose， glucose， sucrose， melibiose， raffinose， manninotriose and stachyose）， and the mobile phase was acetonitrile-water for gradient elution. The comprehensive sweetness difference of sample was calculated by the sweetness of saccharides， SPSS 21.0 was used to analyze the relationship between the color， comprehensive sweetness and the main component contents in the processing of Rehmanniae Radix Praeparata processed with Amomi Fructus and Citri Reticulatae Pericarpium， the quality comprehensive evaluation index of Rehmanniae Radix Praeparata by triangular area method was established. ResultDuring the processing， the color value of the powder increased， and the apparent color of the sample became darker. the content determination results showed that the content of glycosides decreased， monosaccharides and comprehensive sweetness increased with the increase of heating time. The results of correlation analysis showed that chromaticity value， comprehensive sweetness were significant negatively correlated with the content of iridoid glycosides （P<0.01）， the chromaticity value was significant positively correlated with the contents of furaldehyde derivatives， phenylethanoid glycosides， flavonoids and comprehensive sweetness was significant positively correlated with the contents of furaldehyde derivatives， phenylethanoid glycosides （P<0.01）， and the comprehensive sweetness was positively correlated with the content of flavonoids （P<0.05）. After 52 h of processing， the comprehensive evaluation index of samples reached 0.99. ConclusionThe overall trend of cluster analysis of powder chromaticity value of Rehmanniae Radix Praeparata is basically consistent with that of naked eyes， the comprehensive quality evaluation of Rehmanniae Radix Praeparata processed with Amomi Fructus and Citri Reticulatae Pericarpium can be carried out by combining the three indexes of powder chromaticity value， comprehensive sweetness and glycosides content.

Historical Evolution and Modern Research Progress of Rehmanniae Radix / 中国实验方剂学杂志

Rehmanniae Radix is a common medicine of traditional Chinese medicine， which has the function of nourishing Yin and tonifying the kidney， and has a long application history of processing. This medicine was first recorded in Synopsis of Golden Chamber （《金匮要略》）， which was mainly produced by steaming and boiling. Its processing materials were diverse. In addition to rice wine， honey， Amomi Fructus， milk， Aquilariae Lignum Resinatum， and Carthami Flos were also recorded in ancient books， but with the evolution of time， the characteristic excipients gradually disappeared. Based on this， starting with different excipients， the author consulted the classics of materia medica and processing specifications in various regions， sorted out the historical evolution of Rehmanniae Radix processing， and explored new methods and new ideas to exert the maximum efficacy on this basis. At the same time， the effects of different processing excipients on the chemical components and pharmacodynamic effects of Rehmanniae Radix were analyzed. After literature review， it was found that Rehmanniae Radix mainly had the effects of clearing heat and cooling blood， nourishing Yin and generating fluid. Its traditional processing excipients generally used rice wine， Carthami Flos and others. After processing with different excipients， there was different effects on the chemical components and pharmacological effects of Rehmanniae Radix. In summary， this paper can provide useful reference for standardized research on different processed products of Rehmanniae Radix.

Mechanism analysis of repeatedly steamed and sundried Rehmanniae Radix Praeparata in delaying brain aging in ovariectomized mice based on proteomics / 中国中药杂志

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.

Anti-depressant components and mechanism of Rehmanniae Radix based on UPLC-Q-Orbitrap HRMS and network pharmacology / 中国中药杂志

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.

Processing technology of rice-steamed Rehmanniae Radix unearthed from tomb of Haihunhou in the Western Han Dynasty / 中国中药杂志

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.

Mechanisms of catapol inhibiting differentiation and glycolysis of Thl7 cells through MiR-143-3p / 中国药理学通报

Aim To investigate the mechanism of eata- pol (CAT) inhibiting differentiation and glyeolysis of Thl7 eel Is through miR-143-3p.Method The peripheral hloorl CD4 ∗ T eells of HA patients were obtained to deteet the expression of miR-143-3p and the mRNA levels of key glycolytic enzymes, ineluding glucose transporter 1 ( Glutl ) , hexokinase 2 ( HK2 ) , pyruvate kinase 2 (PKM2) , laetate dehydrogenase A ( LDHA).The differentiation of Thl7 eells was induced in vitro, and the ShRNA/lentivirus was applied to achieve the overexpression or knockdown of miR- 143-3 p.Un-transfected eells were divided into control group and CAT group (20, 40, 80 mg • L 1 ) , and transfected eells were divided into four groups: negative control group, miR-143-3p inhibitor group, miR- 143-3p mimies group, miR-143-3p inhibitor + CAT group.The percentage of Thl7 eells was deteeted by flow cytometry, and the level of IL-17A was detected by EL1SA.Quantitative real-time PCR was used to detect the mRNA expression of miR-143-3p and key glycolytic enzymes, and the levels of pyruvate and lactate were also detected.Results The mRNA expression of miR-143-3p in RA peripheral blood CD4 ∗ T cells was negatively correlated with disease severity ( DAS28 ) , transcription factor ROR-yt, and the key glycolytic enzymes Glutl/HK2/LDHA.Compared with negative control group, the down-expression of miR-143-3p markedly elevated the mRNA expression of ROR-yt, Glutl, HK2, LDHA, and the levels of IL-17A, pyruvate, lactate.Catalpol groups significantly up-regula- ted the expression of miR-143-3p, decreased the mRNA expression of HK2/LDHA and the levels of pvru- vate/lactate, and inhibited Thl7 cells differentiation.Compared with miR - 1 4 3 - 3 p inhibitor group , catapol could significantly inhibit the abnormal up-regulated of HK2/LDHA mRNA relative expression, pyruvate/lactate levels and the abnormal differentiation of Thl7 eells.Conclusion MiR-143-3p inhibits the differentiation and glycolysis of Thl7 cells.Catalpol could sup-press the glycolysis and differentiation of Thl7 eells by regulating mill-143-3p.

A combined quality evaluation method that integrates chemical constituents, appearance traits and origins of raw Rehmanniae Radix pieces / 中国天然药物

The quality control of Chinese herbal medicine is a current challenge for the internationalization of traditional Chinese medicine. Traditional quality evaluation methods lack quantitative analysis, while modern quality evaluation methods ignore the origins and appearance traits. Therefore, an integrated quality evaluation method is urgent in need. Raw Rehmanniae Radix (RRR) is commonly used in Chinese herbal medicine. At present, much attention has been drwan towards its quality control, which however is limited by the existing quality evaluation methods. The present study was designed to establish a comprehensive and practical method for the quality evaluation and control of RRR pieces based on its chemical constituents, appearance traits and origins. Thirty-three batches of RRR pieces were collected from six provinces, while high-performance liquid chromatography (HPLC) was applied to determine the following five constituents, including catalpol, rehmannioside A, rehmannioside D, leonuride and verbascoside in RRR pieces. Their appearance traits were quantitatively observed. Furthermore, correlation analysis, principal components analysis (PCA), cluster analysis and t-test were performed to evaluate the qualities of RRR pieces. These batches of RRR pieces were divided into three categories: samples from Henan province, samples from Shandong and Shanxi provinces, and those from other provinces. Furthermore, the chemical constituents and appearance traits of RRR pieces were significantly different from diverse origins. The combined method of chemical contituents, appearance traits and origins can distinguish RRR pieces with different qualities, which provides basic reference for the quality control of Chinese herbal medicine.

Rehmanniae Radix and Rehmanniae Radix Praeparata improve diabetes induced by high-fat diet coupled with streptozotocin in mice through AMPK-mediated NF-κB/NLRP3 signaling pathway / 中国中药杂志

This study investigated the differential mechanisms of Rehmanniae Radix and Rehmanniae Radix Praeparata in improving diabetes in mice through AMPK-mediated NF-κB/NLRP3 signaling pathway. The diabetic mouse model was established with high-fat diet coupled with streptozotocin(STZ, intraperitoneal injection, 100 mg·kg~(-1), once a day for three consecutive days), after which the mice were randomly divided into model group, low-dose(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix groups, low-dose(5 g·kg~(-1)) and high-dose(15 g·kg~(-1)) Rehmanniae Radix Praeparata groups, catalpol group(250 mg·kg~(-1)), 5-hydroxymethylfurfural(5-HMF) group(250 mg·kg~(-1)), metformin group(250 mg·kg~(-1)), with the normal group also set. The organ indexes of heart,liver, spleen, lung, kidney and pancreas were calculated after four weeks of administration. The pathological changes and fibrosis of pancreas, kidney and liver in mice were observed by hematoxylin-eosin(HE) staining and Masson staining. Western blot was used to determine the expression levels of Toll-like receptor-4(TLR4), nuclear factor-κB(NF-κB), Nod-like receptor protein 3(NLRP3),interleukin-1β(IL-1β), adenosine monophosphate-activated protein kinase(AMPK), phosphorylated AMPK(p-AMPK) in the pancreas, kidney and liver of mice. Compared with the model group, the administration groups witnessed significant decrease in the liver,spleen, kidney, pancreas and fat indexes of diabetic mice, and there was no significant difference in heart and lung indexes. The pathological states and fibrosis of pancreatic, kidney and liver tissues were significantly improved after administration. Additionally, the expression levels of TLR4, NF-κB and NLRP3 in pancreas, kidney and liver of diabetic mice were significantly lowered. The expression levels of p-AMPK/AMPK were enhanced significantly in kidney and liver of mice in Rehmanniae Radix group while in pancreas, kidney and liver in Rehmanniae Radix Praeparata group. This suggests that Rehmanniae Radix and Rehmanniae Radix Praeparata differ in the mechanism of regulating energy metabolism of multiple organs and thereby exerting anti-inflammatory effects to alleviate symptoms of diabetic mice.

Establishment of Thin Layer Chromatography Identification Method for Different Processed Products of Rehmanniae Radix / 中国实验方剂学杂志

Objective:To establish a thin layer chromatography （TLC） identification method for different processed products of Rehmanniae Radix. Method:Catalpol， D-fructose， sucrose， raffinose， stachyose， melibiose and manninotriose were used as control substances， and the effects of extraction solvents （water， 20% methanol， 50% methanol， 80% methanol）， developing solvents （n-butanol-methanol-chloroform-glacial acetic acid-water， ethyl acetate-pyridine-glacial acetic acid-water， n-butanol-glacial acetic acid-water）， color reagents （aniline-diphenylamine-phosphoric acid solution， ninhydrin solution）， sampling volumes （2， 4， 6 μL） and inspection conditions （sunlight， bottom lamp of sunlight， 365 nm， 254 nm） on TLC were investigated to determine the preparation method of sample solution of Rehmanniae Radix and Rehmanniae Radix Praeparata and the optimal TLC conditions. Result:High performance silica gel G plate was used for TLC， n-butanol-methanol-chloroform-glacial acetic acid-water （13∶5∶5∶1∶2） was used as developing agent， aniline-diphenylamine-phosphoric acid solution was sprayed and heated at 110 ℃ for color development， and then inspected under the bottom lamp of sunlight. The separation and color development of different processed products of Rehmanniae Radix were good with clear spots and good characteristics. Conclusion:The established TLC is simple and easy to operate with obvious qualitative characteristics and intuitive results. It can effectively identify different processed products of Rehmanniae Radix and provide experimental basis for determining the end point of processing of Rehmanniae Radix Praeparata.