Five new eremophilane-type sesquiterpenes from the fresh roots of Rehmannia glutinosa.

Five undescribed eremophilane-type sesquiterpenes, remophilanetriols E-I (1-5), along with seven known compounds (6-12) were isolated from the fresh roots of Rehmannia glutinosa. Their structures were characterized by extensive spectroscopic data analysis and their absolute configurations were determined by comparing their calculated electronic circular dichroism (ECD) spectra and experimental ECD spectra. The anti-pulmonary fibrosis activities of all compounds were evaluated in vitro by MTT methods, and compounds 2, 8, 10, and 12 exhibited excellent anti-pulmonary fibrosis activities. In addition, compound 2 can reduce the levels of ROS and apoptosis in TGF-ß1-induced BEAS-2B cells.

Ten cyclopentanoid monoterpenes from the whole plant of Rehmannia piasezkii maxim.

Ten new cyclopentanoid monoterpenes (1-10) were isolated from the whole plant of Rehmannia piasezkii. The structures of these compounds were elucidated based on spectroscopic data analysis. In in-vitro assays, compounds 3, 7, and 9 exhibited weak hepatoprotective activities against APAP-induced HepG2 cell damage. Compound 9 exhibited protective effect on hapassocin carbon tetrachloride model.

Catalpol attenuates ischemic stroke by promoting neurogenesis and angiogenesis via the SDF-1α/CXCR4 pathway.

BACKGROUND: Stroke is a leading cause of disability and death worldwide. Currently, there is a lack of clinically effective treatments for the brain damage following ischemic stroke. Catalpol is a bioactive compound derived from the traditional Chinese medicine Rehmannia glutinosa and shown to be protective in various neurological diseases. However, the potential roles of catalpol against ischemic stroke are still not completely clear. PURPOSE: This study aimed to further elucidate the protective effects of catalpol against ischemic stroke. METHODS: A rat permanent middle cerebral artery occlusion (pMCAO) and oxygen-glucose deprivation (OGD) model was established to assess the effect of catalpol in vivo and in vitro, respectively. Behavioral tests were used to examine the effects of catalpol on neurological function of ischemic rats. Immunostaining was performed to evaluate the proliferation, migration and differentiation of neural stem cells (NSCs) as well as the angiogenesis in each group. The protein level of related molecules was detected by western-blot. The effects of catalpol on cultured NSCs as well as brain microvascular endothelial cells (BMECs) subjected to OGD in vitro were also examined by similar methods. RESULTS: Catalpol attenuated the neurological deficits and improved neurological function of ischemic rats. It stimulated the proliferation of NSCs in the subventricular zone (SVZ), promoted their migration to the ischemic cortex and differentiation into neurons or glial cells. At the same time, catalpol increased the cerebral vessels density and the number of proliferating cerebrovascular endothelial cells in the infracted cortex of ischemic rats. The level of SDF-1α and CXCR4 in the ischemic cortex was found to be enhanced by catalpol treatment. Catalpol was also shown to promote the proliferation and migration of cultured NSCs as well as the proliferation of BMECs subjected to OGD insult in vitro. Interestingly, the impact of catalpol on cultured cells was inhibited by CXCR4 inhibitor AMD3100. Moreover, the culture medium of BMECs containing catalpol promoted the proliferation of NSCs, which was also suppressed by AMD3100. CONCLUSION: Our data demonstrate that catalpol exerts neuroprotective effects by promoting neurogenesis and angiogenesis via the SDF-1α/CXCR4 pathway, suggesting the therapeutic potential of catalpol in treating cerebral ischemia.

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

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

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

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

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

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

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

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

[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.

Extraction, structure and bioactivities of polysaccharides from Rehmannia glutinosa: A review.

ETHNOPHARMACOLOGIC RELEVANCE: Rehmannia glutinosa (Gaertn.) DC. (RG) is a widely used herb for clearing heat and cooling the blood. Polysaccharides from Rehmannia glutinosa (Gaertn.) DC. (RGPs) have a variety of biological activities, including antioxidation, hypoglycemia, immune enhancement, hematopoiesis promotion, and antianxiety. AIM OF THE STUDY: This review provides up-to-date and comprehensive information on the extraction and separation methods, structural characteristics, and pharmacological activities of RGPs. A more in-depth study on the structure and clinical pharmacology of the RGPs was investigated. To further explore the pharmacological effects of RGPS, and lay a foundation for the safe clinical application and expansion of application scope. MATERIALS AND METHODS: Use Google Scholar, Scifinder, PubMed, Springer, Elsevier, Wiley, Web of Science and other online database search to collect the literature on extraction, separation, structural analysis and pharmacological activity of RGPs published before December 2022. The key words are "extraction", "isolation", "purification" and "pharmacological action" and "Rehmanniae polysaccharide". RESULTS: Rehmannia glutinosa has been widely used in the treatment of diabetes since ancient times, and is known as one of the "Four Sacred Medicines" for the treatment of diabetes, along with Ginseng, Psidium Guajava and Pueraria Mirifica. The active ingredients of Rehmannia glutinosa that have been studied more in the treatment of diabetes are Rehmannia glutinosa polysaccharide and Rehmannia glutinosa oligosaccharide. The content of polysaccharides varies due to different extraction methods, and separation and purification methods. RGPs have a wide range of pharmacological activities, including antitumor, immunomodulatory, neuroprotective effect, hypoglycemic activity, cardioprotective and antioxidant activities. These pharmacological properties lay a foundation for the treatment of tumors, inflammation, hyperglycemia, myocardial ischemia, oxidative stress and other diseases with RGPs. CONCLUSION: Based on its effects of promoting hematopoiesis, antitumor and enhancing immunity, RGPs have been clinically applied in the treatment of chronic aplastic anemia and esophageal cancer, but other effects of RGPs have not been reflected in the clinical practice. In the future, more in-depth research can be conducted on the molecular structure analysis, toxicity, side effects and clinical pharmacological effects of RGPs to further explore the pharmacological effects of RGPs and to lay the foundation for safe clinical application and expansion of application scope.

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.

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.

Development of Highly Sensitive Method for Sugar Determination in Herbal Medicine; Application of Monosaccharides and Oligosaccharides in Japanese Angelica Root and Rehmannia Root.

We have developed a simple and accurate method for quantifying sugars in herbal medicines, which have hitherto been difficult to quantify. Using ultra performance liquid chromatography-quadrupole-time-of-flight (UPLC-Q-TOF)-MS and two types of columns with different chemical properties, we determined the optimum conditions for separating nine sugars (fructose, galactose, glucose, mannitol, sucrose, melibiose, raffinose, manninotriose, and stachyose) commonly found in herbal medicines. Separation was completed within 10 min when an apHera NH2 HPLC column was used, although galactose and glucose could not be separated. On the other hand, the nine sugars were completely separated within 16 min when a hydrophilic interaction chromatography (HILIC)pak VG-50 2D column was used. The calibration curves obtained using those two columns gave good linearity for the sugar standards, and the coefficient of determination was 0.995 or higher. Both columns showed excellent performance with short analysis time and high sensitivity. Using our developed method, we were able to quantify sugars in galactose-free herbal medicines within 10 min and in herbal medicines containing galactose within 16 min. We revealed that our method could be used for the analysis of sugars in Angelica acutiloba and Rehmannia glutinosa roots.

[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.

[Synthesis, characterization, and immunological activity of Rehmannia glutinosa seleno-polysaccharides].

The present study explored the optimum synthesis process of Rehmannia glutinosa seleno-polysaccharides with acetic acid as a catalyst, characterized the structure of R. glutinosa seleno-polysaccharides by Fourier transform infrared spectroscopy(FT-IR), scanning electron microscopy(SEM), thermogravimetry(TG), and atomic force microscopy(AFM), and preliminarily investigated the immunological activity of R. glutinosa seleno-polysaccharides. The results showed that the optimal conditions for the synthesis of R. glutinosa seleno-polysaccharides included m(acetic acid)∶m(R. glutinosa polysaccharides)=0.80, m(Na_2SeO_3)∶m(R. glutinosa polysaccharides)=1.25, reaction temperature of 80.0 ℃, and reaction time of 7.0 h. Under these conditions, the selenium content of R. glutinosa seleno-polysaccharides was 2.239 mg·g~(-1). The acetic acid catalysis method was milder than the nitric acid method, without affecting the structure of polysaccharides. The results of IR, SEM, TG, and AFM showed that R. glutinosa seleno-polysaccharides were properly prepared. The results of immunological activity showed that compared with the control group, R. glutinosa seleno-polysaccharides could significantly promote the phagocytic capacity of mouse monocyte macrophages and improve the spleen index and thymus index of mice. In the concentration range of 15-240 µg·mL~(-1), the proliferation of spleen lymphocytes of mice was strengthened, and the IL-2 and IFN-γ secretion by Th1 cytokines was promoted. This study can provide references for the further development and application of R. glutinosa polysaccharides.

[Acetylation of Rehmannia glutinosa polysaccharides and antioxidant activity of acetylated derivatives].

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.

Rehmannioside A improves cognitive impairment and alleviates ferroptosis via activating PI3K/AKT/Nrf2 and SLC7A11/GPX4 signaling pathway after ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Rehmannioside A is derived from Rehmannia glutinosa Libosch, which is widely used as an important ingredient in diverse traditional Chinese medicines to treat diseases caused by "kidney deficiency" such as cerebral arteriosclerosis, aging-related stroke and dementia in China. Recent studies have proved that Rehmannia glutinosa Libosch and Rehmannioside A can improve memory capability and recover nerve damage. AIM OF THE STUDY: To investigate the effect of Rehmannioside A on cognitive impairment after ischemia in rats and SH-SY5Y cells, and further evaluate the anti-oxidative and anti-ferroptosis mechanisms. MATERIALS AND METHODS: Differentially expressed proteins (DEPs) in patients after cerebral ischemic stroke were revealed by a RayBio protein array. Cognitive impairment model was established by middle cerebral artery occlusion and reperfusion (MCAO) 14 days in rats. Rehmannioside A was administered intraperitoneally injection at dose of 80 mg/kg. The SH-SY5Y cells were exposed to H2O2 for 24 h and treated with Rehmannioside A (80 µM) for 24 h. The neuroprotecion of Rehmannioside A were evaluated by infarct volume (TTC), neurological defects (Garcia score) and learning memory (Morris water maze test) in vivo, and cell viability (CCK-8 or LDH) in vitro. Superoxide dismutase (SOD), malondialdehyde (MDA) and myeloperoxidase (MPO) activity of rats, glutathione (GSH), oxidized glutathione (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH) of cells were detected by biochemical assay. Intracellular reactive oxygen species (ROS) were measured by DCFH-DA assay. Myeloperoxidase (MPO), PI3 kinase (PI3K), p-PI3K, Akt, p-Akt, heme oxygenase-1 (HO-1), nuclear factor-E2-related factor 2 (Nrf2), SLC7A11, glutathione peroxidase 4 (GPX4) of the cerebral cortex in rats or SH-SY5Y cells were examined by western blotting. RESULTS: Compared with model group, the cognitive impairment and neurological deficits of Rehmannioside A group were significantly improved, and the cerebral infarction was reduced in MCAO rats. Moreover, the cell viability obviously increased and the H2O2-induced toxicity was reduced in Rehmannioside A group. Further research indicated that the expression of p-PI3K, p-Akt, nuclear Nrf2, HO-1 and SLC7A11 in Rehmannioside A group was significantly higher than model group. CONCLUSION: Rehmannioside A has neuroprotection effect and improves cognitive impairment after cerebral ischemia by inhibiting ferroptosis and activating PI3K/AKT/Nrf2 and SLC7A11/GPX4 signaling pathway. These findings provide valuable insight into the pathogenesis and therapeutic target of ischemic stroke.

Polysaccharides derived from Chinese medicinal herbs: A promising choice of vaccine adjuvants.

Adjuvants have been used in vaccines for a long time to promote the body's immune response, reducing vaccine dosage and production costs. Although many vaccine adjuvants are developed, the use in human vaccines is limited because of either limited action or side effects. Therefore, the development of new vaccine adjuvants is required. Many studies have found that natural polysaccharides derived from Traditional Chinese medicine (TCM) possess good immune promoting effects and simultaneously improve humoral, cellular and mucosal immunity. Recently polysaccharide adjuvants have attracted much attention in vaccine preparation because of their intrinsic characteristics: immunomodulation, biocompatibility, biodegradability, low toxicity and safety. This review article systematically analysed the literature on polysaccharides possessing vaccine adjuvant activity from TCM plants, such as Astragalus polysaccharide (APS), Rehmannia glutinosa polysaccharide (RGP), Isatis indigotica root polysaccharides (IRPS), etc. and their derivatives. We believe that polysaccharide adjuvants can be used to prepare the vaccines for clinical use provided their mechanisms of action are studied in detail.

Three New 2,2'-Difurylketone Derivatives and Two New Chromones from the Rehmanniae Radix Praeparata.

Rehmanniae Radix Praeparata is the processed products of the root of Rehmannia glutinosa. It has been used as a Traditional Chinese Medicine for thousands of years, and it has been found to possess widely pharmacological activities. In this study, three new 2,2'-difurylketone derivatives (rehmanniaeketone A-C) and two new chromones [3,8-dihydroxy-2-(2-hydroxyethyl)chromone and 3,8-dihydroxy-2-[(2-O-α-D-galactopyranosyloxy)ethyl]chromone] were isolated from the Rehmanniae Radix Praeparata. Furthermore all of the compounds were subjected to cytotoxic testing against the human lung carcinoma A549 cells. The cytotoxic results showed that rehmanniaeketone B and rehmanniaeketone C exhibited more stronger inhibition effects on the cell activity of A549 cells with the IC50 5.23 µM and 2.05 µM than other compounds. And 3,8-dihydroxy-2-(2-hydroxyethyl)chromone exhibited moderately inhibitory activity with the IC50 61 µM. Rehmanniaeketone A and 3,8-dihydroxy-2-[(2-O-α-D-galactopyranosyloxy]chromone showed no inhibitory effects.

Exploration of the Effect and Mechanism of Fructus Lycii, Rehmanniae Radix Praeparata, and Paeonia lactiflora in the Treatment of AMD Based on Network Pharmacology and in vitro Experimental Verification.

PURPOSE: The aim of this study was to observe the mechanism of Fructus Lycii (FL), Rehmanniae Radix Praeparata (RRP) and Paeonia lactiflora (PL) in treating age-related macular degeneration (AMD) based on network pharmacology and biological experiments. METHODS: Bioactive compounds, potential targets of FL, RRP and PL, and genes related to AMD, were acquired from public databases. Functional and pathway enrichment analyses of the core targets were conducted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, the finding was further verified with cell experiments. The MTT assay and flow cytometric analysis were used to assess cell viability and apoptosis. The production of reactive oxygen species (ROS) was analyzed by DCFH-DA staining; the activity of antioxidant enzymes was chemically measured with assay kits. The expression of key proteins was evaluated by Western blot analysis. RESULTS: Fifty-nine active compounds, 182 potential targets, and 2536 AMD-related human genes were identified. A total of 103 key targets of the three herbs on AMD were identified by protein-protein interaction (PPI) analysis. The abovementioned targets were correlated with nuclear receptor activity, oxidative stress, and apoptosis pathways according to the GO and KEGG analyses. MTT assay and flow cytometry demonstrated that pretreatment of ARPE-19 cells with the three herbs significantly increased cell viability and decreased apoptosis induced by H2O2. The three herbs might reduce the intracellular ROS levels and increase the SOD and CAT activities after H2O2. Furthermore, the three herbs significantly inhibited oxidative stress via increasing the expression of Nrf2, HO-1 and NQO1. CONCLUSION: The combined results of network pharmacology and validation experiments showed that FL, RRP and PL reduce oxidative stress and apoptosis in RPE cells to exert its effect in the treatment of AMD.