Discovery of human pancreatic lipase inhibitors from root of Rhodiola crenulata via integrating bioactivity-guided fractionation,chemical profiling and biochemical assay / 药物分析学报

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(Ki)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.

Rhodiola crenulata extract decreases fatty acid oxidation and autophagy to ameliorate pulmonary arterial hypertension by targeting inhibiton of acylcarnitine in rats / 中国天然药物

Pulmonary arterial hypertension (PAH) is a devastating pulmonary circulation disease lacking high-efficiency therapeutics. The present study aims to decipher the therapeutic mechanism of Rhodiola crenulata, a well-known traditional chinese medicine with cardiopulmonary protection capacity, on PAH by exploiting functional lipidomics. The rat model with PAH was successfully established for first, following Rhodiola crenulata water extract (RCE) treatment, then analysis of chemical constituents of RCE was performed, additional morphologic, hemodynamic, echocardiographic measurements were examined, further targeted lipidomics assay was performed to identify differential lipidomes, at last accordingly mechanism assay was done by combining qRT-PCR, Western blot and ELISA. Differential lipidomes were identified and characterized to differentiate the rats with PAH from healthy controls, mostly assigned to acylcarnitines, phosphatidylcholines, sphingomyelin associated with the PAH development. Excitingly, RCE administration reversed high level of decadienyl-L-carnitine by the modulation of metabolic enzyme CPT1A in mRNA and protein level in serum and lung in the rats with PAH. Furthermore, RCE was observed to reduce autophagy, confirmed by significantly inhibited PPARγ, LC3B, ATG7 and upregulated p62, and inactivated LKB1-AMPK signal pathway. Notably, we accurately identified the constituents in RCE, and delineated the therapeutic mechansim that RCE ameliorated PAH through inhibition of fatty acid oxidation and autophagy. Altogether, RCE might be a potential therapeutic medicine with multi-targets characteristics to prevent the progression of PAH. This novel findings pave a critical foundation for the use of RCE in the treatment of PAH.

Identification of Rhodiola quadrifida and Rhodiola crenulata based on NMR fingerprint and chemical pattern recognition method / 中草药

Objective A method for classification and identification of Rhodiola quadrifida and Rhodiola crenulata was established based on nuclear magnetic resonance 1H-NMR fingerprints-chemical pattern recognition technique. Methods Using high resolution (600 MHz) NMR fingerprints pattern technique, the total component information 1H-NMR fingerprint of R. quadrifida and R. crenulata was determinated, combined with the similarity analysis, hierarchical cluster analysis, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) methods for chemical pattern recognition analysis. Results 1H-NMR fingerprints techniques combined with chemical pattern recognition analysis was an effective method to discriminate and identify R. quadrifida and R. crenulata. The difference of the 1H-NMR fingerprint of R. quadrifida and R. crenulata was obvious, which truly and comprehensively reflected the characteristic components and internal qualities of Rhodiola. The main different components of R. quadrifida and R. crenulata were terpenoids and flavonoids, in particular, crenulatin of the terpenoid was a characteristic ingredient in the identification of R. quadrifida and R. crenulata, which can be used as the identification and classification index of R. quadrifida and R. crenulata. Conclusion 1H-NMR fingerprints techniques combined with chemical pattern recognition analysis method is an effective method for classification and identification of Rhodiola, which lays the foundation for variety identification and quality evaluation of medicinal plants of Rhodiola.

Molecular mechanism of Tibetan medicine Rhodiola crenulata prevents oxidative stress through network pharmacology / 中国中药杂志

In this study, a computer-based network pharmacology approach was applied to investigate the potential mechanism and important components of Rhodiola crenulata in the protection of H9c2 cells against hydrogen peroxide (H₂O₂)-induced oxidative stress. The intestinal absorption liquid of R. crenulata enhanced the cell viability, maintained cell morphology and inhibited cell apoptosis in the H₂O₂-induced oxidative stress in H9c2. Then, computer-based network pharmacology was used to analyze the relevant mechanism. A total of 133 oxidative stress-related compounds were screened out; and 26 of them occupied the top 20%, and all of the compounds enriched in 43 oxidative stress-related key targets. Finally, a "compound-target-pathway-function" network was constructed. Based on the analysis of the network pharmacology, R. crenulata protected H9c2 cells against H₂O₂-induced oxidative stress probably by affecting apoptosis-related processes, such as cell death, nitric oxide metabolism, oxidative stress, mitochondrial mechanism, redox process, redox-related enzyme activty and other oxidative stress-related process. And salidroside, ethyl gallate and catechins, which were the main components of R. crenulata, played an important role in this process. Therefore, the potential mechanism and important components of R. crenulata revealed the protective effect on oxidative stress. This study shows a multi-component, multi-target and overall regulation effect of R. crenulata on the oxidative stress, and provides a reliable reference for subsequent systematic experimental studies for the pharmacodynamic material foundation and mechanism of action R. crenulata.

Effect of fertilization combinations of nitrogen, phosphorus, and potassium on four phenolic compounds of cultivated Rhodiola crenulata / 中国中药杂志

This study is aimed to explore the effect of nitrogen, phosphorus and potassium combined application on the active components of Rhodiola crenulata. R. crenulata was used as the research object, "3414" fertilization experiment were conducted with regular fertilization of NPK(N 60 kg·hm⁻², P₂O₅ 100 kg·hm⁻²,KCl 160 kg·hm⁻²) to study the effect of different rates of NPK fertilization on the total amount of 4 phenolic constituents of gallic acid, salidroside, tyrol and ethyl gallate through field test. The results show that the content of salidroside was higher in the treatment of N₁P₂K₁ and N₁P₂K₂, andthe total amount of four phenols was higher in the treatment of N₁P₂K₂ and N₂P₂K₂. The suitable level of nitrogen, phosphorus and potassium promoted the accumulation of the 4 kinds of phenols.The amount of fertilizer recommended by the three factor fertilizer effect equation,(N 0 kg·hm⁻²,P₂O₅ 150 kg·hm⁻²,KCl 31.71 kg·hm⁻²) obtained the highest content of salidroside, and it was 1.54%.（N 35.54 kg·hm⁻²,P₂O₅ 150 kg·hm⁻²,KCl 237.73 kg·hm⁻²）obtained the highest content of 4 kinds of phenolic compounds, and it was 1.93%. This study provides a reference for the standardization of artificial planting of endangered Tibetan medicine.

Discrimination of Rhodiola crenulata by HPLC fingerprinting and similarity analysis / 中国药学杂志

OBJECTIVE: To establish the HPLC fingerprint of Rhodiola crenulata herbs from Sichuan plateau, and compare them with commercially available samples. METHODS: RP-HPLC analysis was applied using Agilent Zorbax C18 chromatographic column (4.6 mm×250 mm, 5 μm). The mobile phase A was 0.1% formic acid aqueous solution and mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was 1 mL·min-1 and the column temperature was maintained at 35℃. The detection wavelength was set at 245 nm and injection of sample was 20 μL. The traditional Chinese medicine fingerprint chromatogram similarity evaluation system (Version 2004A), principal component analysis, and cluster analysis were used to compare 30 Rhodiola crenulata samples from various locations based on their HPLC chromatograms. RESULTS: The established HPLC fingerprint of Rhodiola crenulata was able to analyze Rhodiola crenulata from different sources. CONCLUSION: The method has good repeatability and stability, and can be used for the quality management standard of Rhodiola crenulata.

Effect of endophytic fungus on expression of key enzyme genes in pathway of salidroside biosynthesis in Rhodiola crenulata / 药学学报

One strain of endophytic fungus ZPRa-R-1 was obtained for the capacity of promoting production of salidroside in Rhodiola crenulata. To explain the mechanism of salidroside biosynthesis in host plant, eight housekeeping genes were evaluated, and the evaluation method was created for the expression activities of four key enzyme genes PAL (phenylalanine ammonia-lyase), TyDC (tyrosine decarboxylase), TAT (tyrosine transaminase), UDPGT (UDP-glucosyltransferase) referenced double reference genes in biosynthesis pathway of salidroside in R. crenulata. Stabilities of housekeeping genes were confirmed by real-time fluorescent quantitative PCR technology and three softwares including geNorm, NormFinder and BestKeeper, then relative expressions of key enzyme genes were analysized by the 2-ΔΔCt method. The results showed that the most stable gene was GAPDH, followed by PCS, and the most appropriate reference of internal genes were combination with two genes in R. crenulata inoculated with endophytic fungus ZPRa-R-1. Under symbiosis conditions, regularity changes of key enzyme genes affected by endophytic fungus ZPRa-R-1 were as follows:the relative expression activity of PAL attached to peak value, which was 4.9 times as that of control group when inoculated ten days. The relative expression of TyDC reached the maximum value, which was 2.8 times of that control after inoculating 12 days. The relative expression of UDPGT actually reach 17.1 times than that of control after inoculating 8 days. However, the relative expression of TAT was not affected by this fungus. The changes of four key enzyme genes are positively correlated with the changes of salidroside content in R. crenulata.

Flavonoids from roots and rhizomes of Rhodiola crenulata / 中草药

Objective: To study the chemical constituents from the roots and rhizomes of Rhodiola crenulata. Methods: The chemical constituents were isolated by repeated silica gel chromatography and medium pressure column chromatography. Their structures were identified by various spectroscopic data including ESI-MS, 1H-NMR, and 13C-NMR data. Results: Fourteen compounds were isolated from the ethyl acetate fractions of R. crenulata including 3,5-dihydroxy-3',4',7-trimethoxyflavone (1), 3,5,7,3'-tetrahydroxyflavone (2), 5,4'-dihydroxy-7,3'-dimethoxyflavone (3), kaemnpferol (4), kaemnpferol-3-O-β-D-glucopyranoside (5), kaemnpferol-3-O-α-L- rhamnopyranoside (6), tricin (7), tricin-7-O-β-D-glucopyranoside (8), quercetin (9), quercetin-3-O-β-D-glucopyranoside (10), quercetin-3-O-α-L-rhamnopyranoside (11), herbacetin-3-O-β-D-glucopyranoside (12), herbacetin-7-O-β-D-glucopyranoside (13), and herbacetin-7-O-α-L-rhamnoside (14). Conclusion: Compounds 1-3 are isolated from the plants in Rhodiola L. for the first time, compounds 5-6, 8, 10-13 are obtained from this plant for the first time.

Simultaneous determination of five active compounds in wild and culture materials of Rhodiola crenulata by RP-HPLC / 中国药学杂志

OBJECTIVE: To establish a method for simultaneous determination of five components in wild and tissue-culture materials of Rhodiola crenulata. METHODS: The determination was performed on a Thermo-C18 column (4.6 mm×250 mm, 5 μm) with mobile phase composed of methanol-water (32:68) at a flow rate of 0.8 mL·min-1. The injection volume was 10 μL, column temperature was set at 30℃, and the detection wavelength was set at 277 nm at 0-13 min and 250 nm at 13-60 min. RESULTS: There were linear relationships between the peak areas and contents of salidroside, p-tyrosol, rosarin, rosavin and rosin in the ranges of 2.80-280.00 (r=0.9998), 2.80-280.00 (r=0.9997), 1.20-120.00 (r=0.9996), 1.60-160.00 (r=0.9997) and 1.20-120.00 μg·mL-1 (r=0.9997), respectively. The extraction recoveries varied from 99. 32% to 100.45%. CONCLUSION: The established method is simple and accurate for quality control of wild and tissue-culture materials of Rhodiola.

Potential distribution of Rhodiola crenulata in Tibetan Plateau based on Maxent model / 中国中药杂志

Wildlife tending and artificial cultivation is an important way to protect the wild resources of Rhodiola crenulata. It is a study hotspot at present. The distribution information of R. crenulata was collected by query data and field survey, the ecological suitability regionalization was conducted based on maximum entropy model combine with ecological factors, including climate, soil and altitude. To provide the reference for production layout, suitable planting area and the selection of artificial planting base by studying the ecological suitability regionalization of R. crenulata. The potential distribution areas mainly concentrated in the easen Tibet, western Sichuan, southern Qinghai, and Gansu Gannan Tibetan Autonomous Prefecture, Yunnan Diqing Tibetan Autonomous Prefecture. There were 5 major environmental factors to have obvious influence on ecology suitability distributions of R. crenulata, including altitude (contribution rate of 61.8%), precipitation of warmest quarter (contribution rate of 19%), the coefficient of variation of precipitation seasonality (contribution rate of 4.7%), the SD of temperature seasonality (contribution rate of 4%), mean temperature of driest quarter (contribution rate of 2.5%). The AUCs of ROC curve were both above 0.9, indicating that the predictive results with the Maxent model were highly precise. The study of the ecological suitability regionalization of R. crenulata based on Maxent can provide a scientific basis for the selection of artificial planting base.

HPLC method for simultaneous quantitative determination of six active ingredients in Rhodiola crenulata from different origins of Qinghai-Tibet Plateau / 中草药

Objective: To analyze the six active ingredients in Rhodiola crenulata from the different origins of Qinghai-Tibet Plateau, to establish quantitative analysis methods, and to provide a basis for further use. Methods: Samples were extracted with 30% ethanol; In Diamonsil C18 column (250 mm × 4.6 mm,5 μm), gradient elution was carried out with acetonitrile -0.3% phosphoric acid solution as the mobile phase, detection wavelength 275 nm, and column temperature 25 ℃. Results: A good linear relationships of gallic acid, salidroside, tyrosol, catechin, ethyl gallate, and coumaric acid were at 38.2-382.0 (r = 0.9998), 301.0-3010.0 (r = 0.9999), 19.8 - 198.0 (r = 0.9996), 17.1-171.0 (r = 0.9996), 4.5-45.0 (r = 0.9998), and 6.38-63.80 ng/mL (r = 0.9994), respectively. Conclusion: The method is simple, rapid, accurate, and can be used simultaneously to determine the content of the six active ingredients in R. crenulata from the different origins of Qinghai-Tibet Plateau.

Effects of rhodiola crenulata compound on serum corticosterone and myocardial glucocorticoid receptor in rats exposed high sustained positive acceleration / 中国比较医学杂志

Objective To study the effect of rhodiola crenulata compound on serum corticosterone and myocardial glucocorticoid receptor ( GR) in rats exposed high sustained +Gz.Methods Seventy-two healthy SD rats were randomly divided into six groups: blank control group, stress control group, +10 Gz stress group and low, medium, high dose drug group, with twelve rats in each.20 mL/kg menstruum was fed to each rat once per day for 14 days.Low, medium and high dose drug group were fed with rhodiola crenulata compound at doses of 0.75 g/kg, 1.5 g/kg and 3.0 g/kg respectively, the other three groups were fed with equal volume saline.Rats were exposed to high +Gz in 15th day.The concentration of corticosterone in the serum of each group rats was detected with ELISA. Western blot was used to detect the expressions of GR in the myocardium of each group rats .Results The content of corticosterone was significantly elevated in +10Gz stress group, while the expression of GR in the myocardium was markedly declined (P <0.01,P <0.05).However, compound preconditioning could decrease concentration of corticosterone in the serum and enhance the expression of GR in the myocardium from rats after +10 Gz exposure. The corticosterone concentration of medium and high dose drug groups was significantly lower and the level of GR expression in the myocardium was significantly higher than that of +10 Gz stress group ( P <0.05 , P <0.01 ) . Conclusion Rhodiola crenulata compound preconditioning could regulate the concentration of corticosterone in the serum and the level of GR expression in the myocardium of the rats exposed +10 Gz, which may be related with its protective effect on high sustained +Gz-induced injury of myocardium.

GC-MS Comparative Analysis of Volatile Compounds Extracted from Rhodiola Crenulata Respectively by HS-SPME and SD / 中国药师

Objective: To compare the volatile compounds extracted from Rhodiola renulata respectively by HS-SPME and SD. Methods:The volatile constituents from Rhodiola crenulata were extracted respectively by HS-SPME and SD, and then the contents and the names were confirmed by GC-MS. Results:Totally 39 compounds were identified from Rhodiola crenulata by HS-SPME while 16 ones were identified by SD. Among them, 4 common compounds were detected. Conclusion: There are some differences between the two methods. Compared with SD, HS-SPME is obviously better because more volatile constituents can be extracted from the herb, furthermore, HS-SPME has notable advantages of higher retrieval matching and sensitivity.

Optimization of regeneration system and resistance screening of Rhodiola crenulata / 中草药

Objective: To optimize the regeneration system of Rhodiola crenulata, to establish the optimal conditions for resistance screening, and to lay the foundation for the establishment of the efficient genetic transformation system of R. crenulata. Methods: The leaves of Rhodiola crenulata were used as explants. The influences of different ratios of 6-BA, NAA, and IAA in the medium on callus induction and growth conditions were observed, and the implant resistance of Kanamycin (Kan) and hygromycin (Hyg) was screened by gradient method. Results: MS + 3.0 mg/L 6-BA + 1.0 mg/L NAA + 700 mg/L L-Pro was the optimal medium for the differentiation of leaf adventitious bud and the differentiation ratio of adventitious bud reached 92%; MS + 700 mg/L L-Pro was the medium for adventitious roots; The best selection of genetic transformation system for R. crenulata was 200 mg/L Kan and 10 mg/L Hyg; Adding 10 mg/L Vc could effectively inhibit the phenolic substances secretion. Conclusion: The regeneration system of R. crenulata, is optimized, and the pressure of Kan and Hyg for genetic transformation system of R. crenulata is screened.

Chemical constituents of roots Rhodiola crenulata / 中国药学杂志

OBJECTIVE: To study the chemical constituents of the roots of Rhodiola crenulata. METHODS: The roots of R. crenulata were extracted with 80% ethanol, and the extract was partitioned with ethyl acetate. The chemical components were isolated by various chromatographic methods. Their structures were elucidated by spectral analysis. RESULTS: Eleven compounds were isolated and identified as isolariciresinol-9, 9′-acetonide (1), p-coumaric acid (2), p-coumaric acid-4-O-β-D-glucopyranoside (3), tachio-side (4), dihydroconiferin (5), 2-phenylethyl β-glucopyranoside (6), 2-phenylethyl β-vicianoside (7), 2-phenylethyl-6-0-α -L-arabi-nofuranosyl-β-D-glucopyranoside(8), 2-phenylethyl β-primeveroside (9), benzyl β-D-glucopyranoside (10), and rhodiocyanoside D (11). CONCLUSION: All of the compounds were isolated from Rhodiola crenulata for the first time, and compounds 1, 3, 4, 5, 7, 9 were obtained from the genus of Rhodiola for the first time.

Genomic DNA cloning and analysis of uridine diphosphate-glucosyltransferase gene from Rhodiola crenulata / 中草药

Objective: To obtain the genomic DNA sequence of uridine diphosphate (UDP)-glucosyltransferase (GT) gene from Rhodiola crenulata and to analyze its DNA sequence at the level of bioinformatics. Methods: The optimized CTAB method was used to extract the genomic DNA from R. crenulata, and after three times genomic walking, the genomic DNA sequence of UDPGT in R. crenulata (RcUDPGT) was obtained by hiTAIR-PCR. The DNA sequence was analyzed by bioinformatics method. Results: The 2 977 bp DNA sequence of RcUDPGT was obtained, which contained the 1 499 bp gene sequence (including the 82 bp intron sequence) and 1 500 bp 5' upstream flanking sequence of coding region (including promoter sequence). The bioinformatic analysis showed that the RcUDPGT was hydrophilic, located in the cytoplasm, and had high homology with UDPGTs of other plants. The tertiary structure of RcUDPGT indicated that protein had UDPGT functional sites. Promoter analysis showed that it contained cis-acting elements responding to light, heat, pressure, temperature, anaerobic, anthocyanins biosynthesis sequences, etc. Conclusion The structure of RcUDPGT gene is integral and has functional sites. The research provides the reference for the molecular biology and metabolic engineering of R. crenulata.

RAPD analysis of common medicinal plants of Rhodiola L. in Yunnan Province / 中草药

Objective To analyze the hereditary background of common species from Rhodiola L. in Yunnan Province. Methods Fifty-nine samples of six populations in three natural medicinal species from Rhodiola L. were studied by RAPD method. Results The percentage of polymorphic bands (PPB) of three populations from R. crenulata were 37.97%, 46.15%, and 39.45% respectively; the Nei's gene diversities (h) were 0.110 3, 0.143 7, and 0.137 1, respectively; the Shannon's information indes (I) were 0.173 6, 0.222 1, and 0.206 7, respectively; at the special level, the PPB was 70.47%, I was (0.258 3); the genetic differentiation (G_(st)) was 0.263 0. The PPB of two populations from R. fastigiata were 43.67% and 44.42%, I were 0.215 3 and 0.217 4, respectively; at the special level, the PPB was 65.51%, G_(st) and I were 0.313 2 and 0.313 9, respectively; the PPB of R. yunnanensis was 36.97%, I was 0.188 1. Conclusion RAPD molecular marker could be used to molecular authentication and genetic diversity analysis of medicinal species from Rhodiola L.

Characterization of Rhodiola crenulata/?-cyclodextrin complex / 中成药

AIM:To characterize the inclusion complex of volatile oil of Rhodiola crenulata/?-cyclodextrin. METHODS: Some analytical methods,such as DTA,IR,GC,TLC were apllied to the investigation before and after the inclusion. RESULTS: The difference between the inclusion and physical mixture in differential thermal(analysis,) infrared spectra,gas chromatogram and thin layer chromatogram. CONCLUSION: The inclusion of Rhodiola crenulata volatile oil has the characteristic of intramolecular inclusion.