Zika virus (ZIKV) and Japanese encephalitis virus (JEV) can cause permanent neurological damage and death, yet no approved drugs exist for these infections. Rhodiola crenulate, an herb used in traditional Chinese medicine for its antioxidation and antifatigue properties, was studied for its antiviral activity against ZIKV and JEV in vitro. The cytotoxicity of Rhodiola crenulata extract (RCE) was evaluated using the CCK-8 reagent. Antiviral effects of RCE were assessed in ZIKV-infected or JEV-infected Vero cells via quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, fluorescent focus assay (FFA), and immunofluorescence assay (IFA). The cell-free antiviral effects of RCE were evaluated using an inactivation assay. To determine the stage of the viral life cycle affected by RCE, time-of-addition, binding, and entry assays were conducted. Three bioactive constituents of RCE (salidroside, tyrosol, and gallic acid) were tested for antiviral activity. RCE exhibited dose-dependent anti-ZIKV and anti-JEV activities at non-cytotoxic concentrations, which were likely achieved by disrupting viral binding and stability. Gallic acid exhibited antiviral activity against ZIKV and JEV. Our findings indicate that RCE disrupts viral binding and stability, presenting a potential strategy to treat ZIKV and JEV infections.

BACKGROUND: Effective control of postprandial blood glucose (PBG) level is essential for the prevention and treatment of diabetes and its complications. Several flavonoids have attracted much attention due to their significant PBG-lowering effects. However, there is still a certain gap in the in vivo hypoglycemic activity of most flavonoids compared to first-line drugs available on the market, and are still lack of the PBG-lowering effects of 8-hydroxyflavones and their structure-activity relationship.
OBJECTIVE: Evaluate hypoglycemic effects of 8-hydroxyflavones from Rhodiola crenulata in vitro and in vivo, especially comparatively analyze the relationship between hypoglycemic effects and flavonoid configuration and reveal the possible mechanism of 8-hydroxyflavones in lowering hyperglycemia.
METHODS: Starch, maltose, sucrose, and glucose tolerance tests in both diabetic and normal mice were used to evaluate and compare the hypoglycemic effects of 8-hydroxyflavones rhodiosin (RHS), rhodionin (RHN), and herbacetin (HBT). Molecular docking, enzyme kinetics, and immunofluorescence analysis were used to research the possible hypoglycemic mechanisms of 8-hydroxyflavones.
RESULTS: RHS (5 and 10 mg/kg) could efficiently decrease PBG levels in both normal and diabetes mice. Moreover, RHS, RHN, and HBT all had significant PBG-lowering effects in transgenic diabetes mice, and the effects were equivalent to or stronger than acarbose. Further mechanism research indicated that 8-hydroxyflavones achieved PBG-lowering effects by inhibiting both the activity and production of glycosidase. Notably, we have innovatively discovered that inhibiting the expression of glycosidases rather than just their activities may be a new target for hypoglycemic drugs.
CONCLUSIONS: We have firstly comprehensively and systematically clarified PBG-lowering effects of 8-hydroxyflavones from Rhodiola crenulata, and revealed their structure-activity relationships and hypoglycemic mechanisms. The study demonstrated that the substitution of 8-hydroxy groups in flavonoids could significantly enhance their hypoglycemic effects, which were equivalent to or stronger than commercially available drug acarbose. 8-Hydroxyflavones could be used as therapeutic or health drugs with significant potential to reduce postprandial hyperglycemia.

The pharmacokinetics (PK) of Rhodiola crenulata in rats were studied, and pharmacokinetic-pharmacodynamic (PK-PD) correlation analysis was performed to elucidate their time-concentration-effect relationship. The myocardial ischemia model was made with pituitrin. Rats were divided into sham operation, sham operation administration, model, and model administration groups (SG, SDG, MG, and MDG, respectively; n = 6). Blood was collected from the fundus venous plexus at different time points after oral administration. The HPLC-QQQ-MS/MS method was established for the quantification of five components of Rhodiola crenulata. CK, HBDH, SOD, LDH, and AST at different time points were detected via an automatic biochemical analyzer. DAS software was used to analyze PK parameters and PK-PD correlation. The myocardial ischemia model was established successfully. There were significant differences in the PK parameters (AUC0-t, AUC0-∞, Cmax) in MDG when compared with SDG. Two PD indicators, CK and HBDH, conforming to the sigmoid-Emax model, had high correlation with the five components, which indicated a delay in the pharmacological effect relative to the drug concentration in plasma. The difference in the PK parameters between modeled and normal rats was studied, and the time-concentration-effect of composition and effect indicators were investigated. This study can provide reference for the rational clinical application of Rhodiola crenulata and for related studies of other anti-myocardial ischemia drugs.

Rhodiola crenulata, a plant of great medicinal value found in cold high-altitude regions, has been excessively exploited due to the difficulty in cultivation. Understanding Rhodiola crenulata\'s adaptation mechanisms to cold environment can provide a theoretical basis for artificial breeding. Glutathione peroxidases (GPXs), critical enzymes found in plants, play essential roles in antioxidant defense through the ascorbate-glutathione cycle. However, it is unknown whether GPX5 contributes to Rhodiola crenulata\'s cold tolerance. In this study, we investigated the role of GPX5 in Rhodiola crenulata\'s cold tolerance mechanisms. By overexpressing Rhodiola crenulata GPX5 (RcGPX5) in yeast and Arabidopsis thaliana, we observed down-regulation of Arabidopsis thaliana GPX5 (AtGPX5) and increased cold tolerance in both organisms. Furthermore, the levels of antioxidants and enzyme activities in the ascorbate-glutathione cycle were elevated, and cold-responsive genes such as AtCBFs and AtCORs were induced. Additionally, RcGPX5 overexpressing lines showed insensitivity to exogenous abscisic acid (ABA), suggesting a negative regulation of the ABA pathway by RcGPX5. RcGPX5 also promoted the expression of several thioredoxin genes in Arabidopsis and interacted with two endogenous genes of Rhodiola crenulata, RcTrx2-3 and RcTrxo1, located in mitochondria and chloroplasts. These findings suggest a significantly different model in Rhodiola crenulata compared to Arabidopsis thaliana, highlighting a complex network involving the function of RcGPX5. Moreover, overexpressing RcGPX5 in Rhodiola crenulata hairy roots positively influenced the salidroside synthesis pathway, enhancing its pharmaceutical value for doxorubicin-induced cardiotoxicity. These results suggested that RcGPX5 might be a key component for Rhodiola crenulata to adapt to cold stress and overexpressing RcGPX5 could enhance the pharmaceutical value of the hairy roots.

OBJECTIVE: Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba (R. crenulate), a famous and characteristic Tibetan medicine, has been demonstrated to exert an outstanding brain protection role in the treatment of high-altitude hypoxia disease. However, the metabolic effects of R. crenulate on high-altitude hypoxic brain injury (HHBI) are still incompletely understood. Herein, the anti-hypoxic effect and associated mechanisms of R. crenulate were explored through both in vivo and in vitro experiments.
METHODS: The mice model of HHBI was established using an animal hypobaric and hypoxic chamber. R. crenulate extract (RCE, 0.5, 1.0 and 2.0 g/kg) and salidroside (Sal, 25, 50 and 100 mg/kg) was given by gavage for 7 days. Pathological changes and neuronal apoptosis of mice hippocampus and cortex were evaluated using H&E and TUNEL staining, respectively. The effects of RCE and Sal on the permeability of blood brain barrier (BBB) were detected by Evans blue staining and NIR-II fluorescence imaging. Meanwhile, the ultrastructural BBB and cerebrovascular damages were observed using a transmission electron microscope (TEM). The levels of tight junction proteins Claudin-1, ZO-1 and occludin were detected by immunofluorescence. Additionally, the metabolites in mice serum and brain were determined using UHPLC-MS and MALDI-MSI analysis. The cell viability of Sal on hypoxic HT22 cells induced by CoCl2 was investigated by cell counting kit-8. The contents of LDH, MDA, SOD, GSH-PX and SDH were detected by using commercial biochemical kits. Meanwhile, intracellular ROS, Ca2+ and mitochondrial membrane potential were determined by corresponding specific labeled probes. The intracellular metabolites of HT22 cells were performed by the targeted metabolomics analysis of the Q300 kit. The cell apoptosis and necrosis were examined by YO-PRO-1/PI, Annexin V/PI and TUNEL staining. In addition, mitochondrial morphology was tested by Mito-tracker red with confocal microscopy and TEM. Real-time ATP production, oxygen consumption rate, and proton efflux rate were measured using a Seahorse analyzer. Subsequently, MCU, OPA1, p-Drp1ser616, p-AMPKα, p-AMPKβ and Sirt1 were determined by immunofluorescent and western blot analyses.
RESULTS: The results demonstrated that R. crenulate and Sal exert anti-hypoxic brain protection from inhibiting neuronal apoptosis, maintaining BBB integrity, increasing tight junction protein Claudin-1, ZO-1 and occludin and improving mitochondrial morphology and function. Mechanistically, R. crenulate and Sal alleviated HHBI by enhancing the tricarboxylic acid cycle to meet the demand of energy of brain. Additionally, experiments in vitro confirmed that Sal could ameliorate the apoptosis of HT22 cells, improve mitochondrial morphology and energy metabolism by enhancing mitochondrial respiration and glycolysis. Meanwhile, Sal-mediated MCU inhibited the activation of Drp1 and enhanced the expression of OPA1 to maintain mitochondrial homeostasis, as well as activation of AMPK and Sirt1 to enhance ATP production.
CONCLUSIONS: Collectively, the findings suggested that RCE and Sal may afford a protective intervention in HHBI through maintaining BBB integrity and improving energy metabolism via balancing MCU-mediated mitochondrial homeostasis by activating the AMPK/Sirt1 signaling pathway.

Salidroside, a principal bioactive component of Rhodiola crenulata, is neuroprotective across a wide time window in stroke models. We investigated whether salidroside induced neurogenesis after cerebral ischemia and aimed to identify its primary molecular targets. Rats, subjected to transient 2 h of middle cerebral artery occlusion (MCAO), received intraperitoneal vehicle or salidroside ± intracerebroventricular HSC70 inhibitor VER155008 or TrkB inhibitor ANA-12 for up to 7 days. MRI, behavioural tests, immunofluorescent staining and western blotting measured effects of salidroside. Reverse virtual docking and enzymatic assays assessed interaction of salidroside with purified recombinant HSC70. Salidroside dose-dependently decreased cerebral infarct volumes and neurological deficits, with maximal effects by 50 mg/kg/day. This dose also improved performance in beam balance and Morris water maze tests. Salidroside significantly increased BrdU+/nestin+, BrdU+/DCX+, BrdU+/NeuN+, BrdU-/NeuN+ and BDNF+ cells in the peri-infarct cortex, with less effect in striatum and no significant effect in the subventricular zone. Salidroside was predicted to bind with HSC70. Salidroside dose-dependently increased HSC70 ATPase and HSC70-dependent luciferase activities, but it did not activate HSP70. HSC70 immunoreactivity concentrated in the peri-infarct cortex and was unchanged by salidroside. However, VER155008 prevented salidroside-dependent increases of neurogenesis, BrdU-/NeuN+ cells and BDNF+ cells in peri-infarct cortex. Salidroside also increased BDNF protein and p-TrkB/TrkB ratio in ischemic brain, changes prevented by VER155008 and ANA-12, respectively. Additionally, ANA-12 blocked salidroside-dependent neurogenesis and increased BrdU-/NeuN+ cells in the peri-infarct cortex. Salidroside directly activates HSC70, thereby stimulating neurogenesis and neuroprotection via BDNF/TrkB signalling after MCAO. Salidroside and similar activators of HSC70 might provide clinical therapies for ischemic stroke.

To fully explore the influence mechanism of interactions between different monomer units of proanthocyanidins (PAs) on biological activity, a path analysis model of the PA structure-activity relationship was proposed. This model subdivides the total correlation between each monomer unit and activity into direct and indirect effects by taking into account not only each monomer unit but also the correlation with its related monomer units. In addition, this method can determine the action mode of each monomer unit affecting the activity by comparing the direct and total indirect effects. Finally, the advantage of this model is demonstrated through an influence mechanism analysis of Rhodiola crenulata PA monomer units on antioxidant and anti-diabetes activities.

BACKGROUND: Rhodiola crenulata (Rc) is a traditional herb, used in Tibetan medicine, has shown promise efficacy in physical performance improvement, work capacity enhancement, fatigue elimination, and altitude sickness prevention. Also, Rc exhibited therapeutic effects on aging-related diseases. However, relevant researches on Rc and their bioactive components are quite few and needs further investigation.
OBJECTIVE: The objective of this study was to understand the relationship between phytochemical profiles and their activities of Rc extracts.
METHODS: Rc extracts prepared by solvents with various hydrophilicity (i.e. aqueous ethanol (70%, v/v), water, and ethyl acetate), and their chemical compositions and specific compounds were analyzed by chemical analysis method and ultra-performance liquid chromatography quadruple time-of-flight mass spectrometry (UPLC-QTOF-MS). The regulate effects of Rc extracts on senescence and antioxidant activity were evaluated using the models of LO2 cells and Caenorhabditis elegans.
RESULTS: The 70% ethanol extracts exhibited better regulating effects on senescence via the assays of senescence -associated β-galactosidase (SAβG) staining and lifespan, which was consistent with the higher antioxidant activities observed based on the results of antioxidant assays. A total of 14 phytochemicals have been identified in 70% ethanol extracts, whereas the other two extracts contained much fewer compounds in varieties. Phytochemical profile of water extract was similar to the first half (polar compounds, running time: 0-6 min) of 70% ethanol extract profile, while those of ethyl acetate extract was consistent with its second half (more nonpolar compounds, running time: 6-12 min).
CONCLUSIONS: The 14 phytochemicals in Rc might exhibit additive or synergistic effects on senescence regulating and antioxidant activities, providing theoretical basis for daily administration of Rc.

Although herbal medicines (HMs) are widely used in the prevention and treatment of obesity and obesity-associated disorders, the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood. Recently, we assessed the inhibitory potentials of several HMs against human pancreatic lipase (hPL, a key therapeutic target for human obesity), among which the root-extract of Rhodiola crenulata (ERC) showed the most potent anti-hPL activity. In this study, we adopted an integrated strategy, involving bioactivity-guided fractionation techniques, chemical profiling, and biochemical assays, to identify the key anti-hPL constituents in ERC. Nine ERC fractions (retention time = 12.5-35 min), obtained using reverse-phase liquid chromatography, showed strong anti-hPL activity, while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS/MS). Among the identified ERC constituents, 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG) and catechin gallate (CG) showed the most potent anti-hPL activity, with pIC50 values of 7.59 ± 0.03 and 7.68 ± 0.23, respectively. Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner, with inhibition constant (K i) values of 0.012 and 0.082 μM, respectively. Collectively, our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC, as well as to elucidate their anti-hPL mechanisms. These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

BACKGROUND: Rhodiola crenulata (Hook.f. & Thomson) H.Ohba has a long history of clinical application for the prevention and treatment of acute mountain sickness (AMS) in traditional Chinese medicine. However, gaps in knowledge still exist in understanding the underlying mechanisms of Rhodiola crenulata against AMS.
OBJECTIVE: To address this problem, a comprehensive method was established by combining UHPLC-Q-TOF-MS/MS analysis and network pharmacology.
METHODS: The ingredients of Rhodiola crenulata were comprehensively analyzed using UHPLC-Q-TOF-MS/MS method. On this basis, a network pharmacology method incorporated target prediction, protein-protein interaction network, gene enrichment analysis and components-targets-pathways network was performed. Finally, the possible mechanisms were verified through molecular docking, in vitro and in vivo experiments.
RESULTS: A total of 106 constituents of Rhodiola crenulata were charactered via UHPLC-Q-TOF-MS/MS. The 98 potentially active compounds out of 106 were screened and corresponded to 53 anti-AMS targets. Gene enrichment analysis revealed that hypoxia and inflammation related genes may be the central factors for Rhodiola crenulata to modulate AMS. Molecular docking revealed that TNF, VEGFA and HIF-1α had high affinities to Rhodiola crenulata compounds. Subsequently, Rhodiola crenulata extract was indicated to inhibit the protein expression level of TNF in hypoxia induced H9c2 cells. Lastly, Rhodiola crenulata extract was further verified to ameliorate heart injury and decreased the heart levels of TNF, VEGFA and HIF-1α in acute hypoxia-induced rats.
CONCLUSIONS: This study used UHPLC-Q-TOF-MS/MS analysis and a network pharmacology to provide an important reference for revealing the potential mechanism of Rhodiola crenulata in the prevention and treatment of AMS.