Content determination of seven active components of Eucommiae Cortex in aortic vascular endothelial cells of spontaneously hypertensive rats by UPLC-MS/MS / 中国中药杂志

In the present study, the contents of seven active components [genipinic acid(GA), protocatechuic acid(PCA), neochlorogenic acid(NCA), chlorogenic acid(CA), cryptochlorogenic acid(CCA),(+)-pinoresinol di-O-β-D-glucopyranosid(PDG), and(+)-pinoresinol 4'-O-β-D-glucopyranoside(PG)] of Eucommiae Cortex in aortic vascular endothelial cells of spontaneously hypertensive rats(SHR) were simultaneously determined by ultra-high liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The qualified SHR models were selected. The primary aortic endothelial cells(VECs) of rats were separated and cultured by ligation and adherence, followed by subculture. After successful identification, an UPLC-MS/MS method for simultaneously determining the contents of GA, PCA, NCA, CA, CCA, PDG, PG in seven components of Eucommiae Cortex in VECs was established, including specificity, linearity, matrix effect, recovery, accuracy, precision and stability. The established method had the lo-west limit of quantification of 0.97-4.95 μg·L~(-1), accuracy of 87.26%-109.6%, extraction recovery of 89.23%-105.3%, matrix effect of 85.86%-106.2%, and stability of 86.00%-112.5%. Therefore, the established accurate UPLC-MS/MS method could rapidly and simultaneously determine the contents of the seven active components of Eucommiae Cortex in VECs of SHRs, which provided a refe-rence for the study of cellular pharmacokinetics of active components of Eucommiae Cortex extract.

Herbal Textual Research on Eucommiae Cortex in Famous Classical Formulas / 中国实验方剂学杂志

In order to provide the basis for the development of famous classical formulas， the name， origin， quality evaluation， harvesting and processing of Eucommiae Cortex were systematically researched by consulting the ancient herbal and medical books， combining with the modern literature. According to the textual research， materia medica in the past dynasties used Eucommiae Cortex as the correct name. Combined with characteristics， origin and efficacy， Eucommiae Cortex in ancient times to the present is the dry bark of Eucommia ulmoides from family Eucommiaceae. The earliest producing areas of Eucommiae Cortex are Henan， Shanxi， Shaanxi and Sichuan. Since the Ming dynasty， the producing areas have expanded to most of the regions in the country， and Sichuan， Shaanxi， Chongqing， Guizhou and Hubei are regarded as the authentic producing areas. It has been concluded that the quality of Eucommiae Cortex is best if the bark has thick body， large block， scraped rough skin， multi silk section and dark purple internal surface. In ancient times， the processing methods of Eucommiae Cortex were mainly included removing rough bark and cutting for raw use， processing with auxiliary materials such as honey， ginger juice， salt water， wine， and so on. While in modern times， the processing methods have become increasingly simplified which are mainly cutting raw materials after cleansing and salt processing. It is need to excavate the connotation of different processed products and restore the traditional main processing methods through standards. Based on the requirement of Eucommiae Cortex in Sanbitang， it is suggested to use ginger-processed products according to the research results， which is used ginger juice as auxiliary material and processed with stir frying method according to the 2020 edition of Chinese Pharmacopoeia.

Mechanism of Eucommiae Cortex and Its Active Components in Treatment of Knee Osteoarthritis： A Review / 中国实验方剂学杂志

Knee osteoarthritis （KOA） is a common degenerative joint disease in the middle-aged and elderly. The incidence of KOA is rising as the population aging aggravates and the obese population grows. KOA seriously affects the health and daily life of the patients. The commonly used drugs for the symptomatic treatment of KOA include non-steroidal anti-inflammatory drugs， cartilage protective drugs， and opioid analgesics， which have limited therapeutic effects and induce obvious adverse drug reactions. Eucommiae Cortex is one of the commonly used Chinese herbal medicines for the treatment of KOA， while its pharmacological material basis and mechanism remain unclear， which limits its clinical application. The active ingredients of Eucommiae Cortex for treating KOA mainly include iridoids （geniposide， aucubin）， lignans （pinoresinol diglucoside）， flavonoids （quercetin， astragaloside， baicalein， hyperoside， and kaempferol）， phenylpropanoids （chlorogenic acid）， and polysaccharides. These compounds regulate the levels of inflammatory cytokines， inhibit oxidative stress， protect chondrocytes， balance the synthesis and degradation of extracellular matrix， and control the progression of KOA via the mitogen-activated protein kinase， nuclear factor-κB， phosphatidylinositol-3-kinase/protein kinase B， and Janus kinase 1/signal transducer and activator of transcription 3 signaling pathways. This paper introduces the mechanisms of Eucommiae Cortex and its active components in the treatment of KOA， aiming to provide a theoretical basis for the development of new drugs for KOA.

Pharmacokinetic investigation of principal active constituents in renal fibrotic rats after oral administration of crude and salt-processed eucommiae cortex extracts / 药学学报

A quantitative analysis method for six principal active constituents (acubin, geniposidic acid, chlorogenic acid, pinoresinol di-O-glucopyranoside, geniposide, and pinoresinol 4-O-glucopyranoside) of crude Eucommiae Cortex (EC) and its salt-processed product extracts was developed to investigate and compare their pharmacokinetic behaviors in adenine-induced renal fibrotic rats in vivo. UHPLC-QqQ-MS/MS technology was employed. Scan was conducted in negative ion mode and quantitative determination was carried out by MRM paired ion. The established method was fully validated by specificity, linearity, precision, accuracy, stability, recovery, and matrix effect, and the results of methodological investigation met the requirements of biological sample analysis. Then, a quick, sensitive, and accurate method was successfully established, which could simultaneously measure the contents of six active constituents of crude and salt-processed EC extracts in rat plasma. After a single administration to renal fibrotic rats of crude EC and its salt-processed product extracts, the plasma concentration of each constituent at different time points was measured, the pharmacokinetic parameters were calculated and the concentration time curves were structured. The experiment was approved by the experimental animal ethics committee from Nanjing University of Chinese Medicine (No. 202103A008). The results showed that compared to the crude Eucommiae Cortex group, the tmax of aucubin, pinoresinol di-O-glucopyranoside, geniposide, and pinoresinol 4-O-glucopyranoside in the salt-processed Eucommiae Cortex group rat plasma were significantly lower than those in the crude group (P < 0.05, P < 0.01); the Cmax and AUC0-48 h of chlorogenic acid, the Cmax, AUC0-48 h and AUC0-∞ of pinoresinol di-O-glucopyranoside, and the Cmax of geniposide and pinoresinol 4-O-glucopyranoside were significantly higher than those in the crude group (P < 0.05, P < 0.01). Our investigation found that compared to crude Eucommiae Cortex, a variety of active ingredients could play a role of quick effect with higher peak blood concentration and bioavailability after oral administration of salt-processed Eucommiae Cortex, which were consistent with the traditional Chinese medicine theory of "salt-processing enhancing drug into kidney meridian", providing an experimental basis for the selection of quality control indexes and the in-depth study of processing mechanisms and metabolic rules in vivo of Eucommiae Cortex and its salt-processed product.

Efficacy of the herbal pair, Radix Achyranthis Bidentatae and Eucommiae Cortex, in preventing glucocorticoid-induced osteoporosis in the zebrafish model / 中西医结合学报

OBJECTIVE@#In traditional Chinese medicine, the herbal pair, Radix Achyranthis Bidentatae (RAB) and Eucommiae Cortex (EC), is widely used to treat osteoporosis. Herein, we determined whether this herbal pair can be used to ameliorate glucocorticoid (GC)-induced osteoporosis (GIOP) and find its optimal dosage in zebrafish.@*METHODS@#The characteristics of the aqueous extract of RAB and EC were separately characterized using high-performance liquid chromatography. Osteoporosis was induced in 5-day post-fertilization zebrafish larvae by exposing them to 10 μmol/L dexamethasone (Dex) for 96 h. Seven combinations of different ratios of RAB and EC were co-administered. Treatment efficacy was determined by calculating zebrafish vertebral area and sum brightness, via alizarin red staining, and by detecting alkaline phosphatase (ALP) activity. Multiple regression analysis was conducted to test the optimal dosage ratio.@*RESULTS@#According to the Chinese Pharmacopoeia (2015), β-ecdysone (β-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of β-Ecd and PDG content values aligned with the Chinese Pharmacopoeia standards. Treatment with 10 μmol/L Dex reduced zebrafish vertebral area, sum brightness, and ALP activity, but RAB and EC attenuated these effects. Combining 50 µg/mL RAB and 50 µg/mL EC was optimal for preventing GIOP in zebrafish. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the mRNA expression of osteogenesis-related genes. A treatment of 10 μmol/L Dex decreased runt-related transcription factor 2 (Runx2), osteogenic protein-1 (OP-1), bone γ-carboxyglutamic acid-containing protein (BGLAP), and β-catenin levels. This effect was counteracted by RAB and EC co-treatment (P < 0.05). Additionally, the effect of using the two herbal extracts together was better than single-herb treatments separately. These results demonstrated that RAB and EC preserve osteoblast function in the presence of GC. The best mass ratio was 1:1.@*CONCLUSION@#RAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.

Overview of quality standards of Eucommiae Cortex in China and overseas / 中国中药杂志

Eucommiae Cortex is an authentic medicinal material with broad growing areas( such as Hunan and Sichuan provinces in China. It is well-known for its efficacy in tonifying liver and kidney,strengthening muscles and bones,and stabilizing fetus. It has also been proven in pharmacology to possess the functions such as lowering blood pressure and lipids. Hence,Eucommiae Cortex has attracted increasing attention. The current quality standards of Eucommiae Cortex vary in different countries or regions. The quality of Eucommiae Cortex products on the market is affected by mix-ups of non-medicinal parts and insufficient growth years. In view of these problems,this paper summarizes the current quality standards and research progress of Eucommiae Cortex in China and overseas,aiming to provide a reference for the establishment of the quality standards of Eucommiae Cortex.

Study on anti-osteoporosis effect of Eucommiae Cortex based on GC-MS metabonomics / 中国中药杂志

Based on GC-MS metabolomics and biochemical index analysis, the mechanism of bone mass loss in osteoporosis and the evaluation of anti-osteoporosis in Eucommiae Cortex were studied. The OVX rats model was established by bilateral ovariectomized. The routine indexes such as BMC, BMD, BGP and TRAP5 b were determined. The GC-MS technique was used to analyze the metabolism profile of serum samples between the control group, model group and medicine group, and multiple statistical analysis methods including principal component analysis(PCA), partial least squares-linear discriminant analysis(PLS-LDA) and subwindow rearrangement analysis(SPA) were used to screen and identify biomarkers. Five metabolites were selected as potential biomarkers, glycine, lysine, tryptophan, docosahexaenoic acid and glucose. Except for the significant increase of tryptophan in serum of OVX rats, the other four metabolites were significantly decreased. Moreover, the five biomarkers of the medicine group had a trend of returning to rats in control group. The significantly altered metabolite levels indicated that Eucommiae Cortex may relieve the symptoms of osteoporosis by regulating amino acid metabolism and oxidative stress.

Discovery and study on potential effect of herbal pair of Uncariae Ramulus cum Uncis-Eucommiae Cortex on pregnancy hypertension based on network pharmacology and molecular docking / 中国中药杂志

This study aimed to explore the optimal indications and mechanism of Uncariae Ramulus cum Uncis(UR)-Eucommiae Cortex(EC) in lowering blood pressure based on network pharmacology and molecular docking. Chemical constituents were collected and screened by TCMSP database. Swiss Target Prediction platform was used to predict the related targets of the drug. OMIM, TCMIP and GeneCards databases were used to collect hypertension-related genes, and the intersections were taken to obtain potential targets for anti-hypertensive treatment of UR-EC. FunRich software was used to enrich the clinical phenotype and expression site of potential target of lowering blood pressure to analyze and predict the optimal indications of UR-EC. STRING database was used for KEGG pathway enrichment analysis, and Cytoscape 3.7.2 was used to construct the network of "composition-target-pathway". The key targets and their corresponding components in the network were analyzed and obtained, and then molecular docking was applied for preliminary verification. Twenty potential active components of UR and 24 potential active components of EC were respectively collected, and 92 anti-hypertensive potential targets of UR-EC were obtained. According to FunRich enrichment results, the optimal indication of UR-EC was pregnancy hypertension, which involved calcium signaling pathway, HIF-1 signaling pathway, neuroactive ligand receptor interaction, renin vascular tightening, VEGF signaling pathway, etc. In addition, AKT1, NOS2, ADRB2, F2, NOS3, SCN5 A, HTR2 A and JAK2 were considered as the key targets in the network. The molecular docking results showed that the screened potential active components had high binding activity with the key targets. This study preliminarily revealed that UR-EC may have therapeutic effects on pregnancy hypertension in terms of sedation, anti-hypertension, anti-inflammatory, anti-oxidation, improvement of vascular endothelial function and so on.

Protective effect of aucubinon on the eucommia ulmoides on skin photoaging induced by UVB / 国际中医中药杂志

Objective To explore the protective effect of aucubin on the photoageing of the skin that induced by ultraviolet (UVB) radiation. Methods Human skin fibroblasts were divided into normal group, model group and high, medium and low dose groups of apocynin by random method. Except the normal group, the other groups of cells were subjected to UVB radiation with a radiation wavelength of 311 nm, irradiation intensity of 9.00 mw/cm2, radiation time of 2.8 s, radiation dose of 25 mJ/cm2. The cells in the high, medium and low doses of aglycone were cultured for 24 h at an equal volume of 400, 200, 100 μg/ml, and then subjected to UVB irradiation, and then cultured for 24 h. The normal group and the model group were cultured for 48 h in a common medium. The apoptosis rate of each group was detected by flow cytometry. The levels of IL-1, IL-6 and TNF-α in each group were detected by ELISA. The expressions of Bax and Bcl-2 mRNA in each group were detected by RT-PCR. Results Compared with the model group, the apoptosis rate (4.84% ± 0.24%, 12.32% ± 0.67% vs. 35.63% ± 2.77%) in the medium and high dose group significantly decreased (P<0.05), the content of IL-1 (21.14 ± 1.94 ng/ml, 16.17 ± 0.74 ng/ml vs. 22.55 ± 1.02 ng/ml), IL-6 (17.46 ± 0.93 ng/ml, 14.51 ± 0.79 ng/ml vs. 18.39 ± 2.05 ng/ml), TNF-α (44.21 ± 1.16 ng/ml, 35.94 ± 3.08 ng/ml vs. 45.67 ± 2.28 ng/ml) significantly increased (P<0.05). Compared with the model group, the expression of Bax mRNA (0.29 ± 0.05, 0.42 ± 0.05, 0.51 ± 0.04 vs. 0.59 ± 0.05) significantly decreased, and the expression of Bcl-2 mRNA (0.52 ± 0.04, 0.44 ± 0.03, 0.35 ± 0.03 vs. 0.26 ± 0.04) significantly increased (P<0.05). Conclusions The aucubin has protective function to the damage of skin fibroblasts which is induced by UVB radiation, indicating the protective function of decreasing Bax mRNA expression, increasing Bcl-2 mRNA expression, reducing the expression of inflammatory factor IL-1, IL-6, TNF-α, holding-up the cell apoptosis which is induced by UVB radiation, and inhibiting apoptosis of cell that caused by UVB radiation, playing an role of protective fibroblasts for delay of the photoageing.

Optimization of extraction process of Wuzi Yanzong plus and minus Recipe based on pharmacodynamic screening and orthogonal experiment / 中草药

Objective: To screen the extraction process of Wuzi Yanzong plus and minus Recipe (WYR) by pharmacodynamics and long-term toxicity test, and optimize it by orthogonal experiment to determine the best extraction process of WYR. Methods Long-term toxicity experiments were carried out on different processed WYR samples. The penile erection latency and sex hormone levels in rats with kidney-yang deficiency were used as pharmacodynamic indicators. The results of long-term toxicity and efficacy experiments were combined to screen out the optimal process plan. Using the content of ingredients and the rate of ointment as indicators, the orthogonal test was used to screen the optimal level of different extraction processes and verified. Results Long-term toxicity test results showed that all mice survived healthily and no obvious toxicity was observed. The results of pharmacodynamic experiments showed that the extracts of Morindae Officinalis Radix, Eucommiae Cortex, Taxilli Herba, Dipsaci Radix, and Glycyrrhizae Radix et Rhizoma Praeparata cum Melle were refluxed with 70% ethanol, and the remaining medicinal materials were extracted with 70% ethanol as the solvent to obtain the best extraction method of WYR. The suitable extraction process was as follows: Morindae Officinalis Radix, Eucommiae Cortex, Taxilli Herba, Dipsaci Radix, and Glycyrrhizae Radix et Rhizoma Praeparata cum Melle were extracted three times with 10 times 70% ethanol for 2.0 h each time; The remaining medicinal materials were percolated with 10 times 70% ethanol at 1.5 mL/min. The medicinal herbs were firstly soaked for 16 h before percolation. Conclusion WYR can significantly improve the penile erection latency and sex hormone levels in rats with kidney yang deficiency. The optimal process conditions are reasonable and feasible, which provides a basis for the follow-up development of WYR.

Difference of chemical constituents in Eucommiae Cortex from different habitats by LC-QTOF MS/MS / 中国中药杂志

In order to study the influence of ecological environment regarding the synthesis and accumulation of metabolites in Eucommiae Cortex, LC-QTOF MS/MS method combined with multivariate statistical analysis was used to analyze the differences of chemical constituents in Eucommiae Cortex from different habitats. Through the analysis of the multistage tandem mass spectrometry, the characteristic peaks were extracted with mass spectrometry data peak matching, peak alignment, and noise filtering. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for data processing. The chemical constituents were identified or tentative presumed according to MS accurate mass and MS/MS spectrometry fragmentation information, combined with the software of database search, comparison with reference standards and literature. The results show the differences among samples of Eucommiae Cortex from different habitats are distinguishable. A total of 23 chemical constituents in Eucommiae Cortex were identified or tentative presumed. Among of them, 14 kinds of common differential chemical constituents (aucubin, geniposidic acid, neochlorogenic acid, syringin, olivil-4',4'-di-O-β-D-glucopyranoside, chlorogenic acid, cryptochlorogenic acid, 1-hydroxypinoresinol- 4',4'-di-O-β-D-glucopyranoside, caffeic acid, pinoresinol-di-O-β-D-glucopyranoside, syringaresionl-di-O-β-D-glucopyranoside, pinoresinol-4'-O-β-D-glucopyranoside, eucommiol, isochlorogenic acid C and asiatic acid) presented different changing laws. This study provides basic information for revealing the influence law of ecological environment on the biosynthesis of metabolites in Eucommiae Cortex.

Research Progress in Differentiation of Osteoblasts by BMSCs after Intervention of Eucommiae Cortex / 中国中医药信息杂志

Eucommiae Cortex is one of the most common and effective traditional Chinese medicine to treat osteoporosis, whose mechanism of action on BMSCs has gradually been confirmed. BMSCs are pluripotent precursor cells of the osteoblast and the osteoclast. In addition to a multi-directional differentiation potential, BMSCs are also characterized of immune suppressive regulating function. This article is a review about the research on differentiation of osteoblasts by BMSCs, which provides a new thinking for further research.

Study on quality of Eucommiae Cortex using flow injection-mass spectrometric fingerprinting method combined with chemical pattern recognition approaches / 中草药

Objective: To study the quality of Eucommiae Cortex from different resources using flow injection mass spectrometric fingerprinting (FIMS) method combined with chemical pattern recognition approaches. Methods: Different Eucommiae Cortex samples were extracted in a simple way and were directly injected into the mass spectrometer for the analyses with no column used. Based on the intensities of the ions at m/z 100-1000, principal components analysis and partial least square discrimination analysis were performed using Simca-P software. Results: Eucommiae Cortex samples from different resources were classified into four clusters in scores plot, and the biomarkers which played the most important roles in classification were screened out. Conclusion: As a novel and characteristic fingerprinting method, FIMS can be widely and efficiently used in true or false identification, identification of origins, and rapid discrimination of herbs from different regions.

Simultaneous determination of five index components in Eucommiae Cortex by HPCE / 中国药学杂志

OBJECTIVE: To establish a high performance capillary electrophoresis (HPCE) method for simultaneous determination of aucubin, geniposide, pinoresinol diglucoside, harpagide, and chlorogenic acid in Eucommiae Cortex. METHODS: 60 mmol·L-1 sodium borate-20 mmol·L-1 sodium dihydrogen phosphate-10% methanol was used as buffer solution (pH 10.0), un-coated fused silica capillary (64.5 cm×75 μm id, 56 cm of effective length) was used, separation voltage was 20 kV, detection wavelength was 210 nm, column temperature was maintained at 25°C, and sample was injected at 5 kPa×6 s. RESULTS: Five index components showed good linearity (r>0.9973) in the range of the tested concentration. The average recoveries of the method were between 96.63%-103.73%. CONCLUSION: The method is simple, accurate and reproducible, and can be used for quality control of Eucommiae Cortex. Copyright 2012 by the Chinese Pharmaceutical Association.