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SUMMARY: Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) can protect diabetic kidneys, but the mechanisms are unclear. This work is to investigate the potential mechanisms of C. tinctoria Nutt. in the treatment of diabetic nephropathy based on network pharmacology analysis of its active ingredients. Twelve small molecular compounds of C. tinctoria Nutt. and targets related to diabetic nephropathy were docked by Discovery Studio 3.0. DAVID database was used for GO enrichment and KEGG pathway analysis. Cytoscape 3.6.1 was used to construct active ingredient-target network. Cell viability was detected with MTT. Glucose consumption was analyzed with glucose oxidase method. Protein expression was measured with Western blot and immunofluorescence. Electron microscopy observed autophagosomes. The core active ingredients of C. tinctoria Nutt. included heriguard, flavanomarein, maritimein, and marein. Twenty-one core targets of the 43 potential targets were PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK, and ABL2. These 21 core targets were significantly enriched in 50 signaling pathways. Thirty- four signaling pathways were closely related to diabetic nephropathy, of which the top pathways were PI3K/AKT, insulin, and mTOR, and insulin resistance. The enriched GO terms included biological processes of protein phosphorylation, and the positive regulation of PI3K signaling and cytokine secretion; cellular components of cytosol, extracellular region, and extracellular space; and molecular function of protein kinase activity, ATP binding, and non-membrane spanning protein tyrosine kinase activity. In vitro experiments found that marein increased the expression of phosphorylated AKT/AKT in human renal glomerular endothelial cells of an insulin resistance model induced by high glucose, as well as increased and decreased, respectively, the levels of the microtubule-associated proteins, LC3 and P62. C. tinctoria Nutt. has many active ingredients, with main ingredients of heriguard, flavanomarein, maritimein, and marein, and may exert anti-diabetic nephropathy effect through various signaling pathways and targets.
RESUMEN: Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) puede proteger riñones diabéticos, sin embargo los mecanismos son desconocidos. Este trabajo se realizó para investigar los potenciales mecanismos de C. tinctoria Nutt. en el tratamiento de la nefropatía diabética basado en el análisis de farmacología en red de sus principios activos. Doce compuestos moleculares pequeños de C. tinctoria Nutt. y los objetivos relacionados con la nefropatía diabética fueron acoplados por Discovery Studio 3.0. La base de datos DAVID se utilizó para el enriquecimiento GO y el análisis de la vía KEGG. Se usó Cytoscape 3.6.1 para construir una red de ingrediente-objetivo activa. La viabili- dad celular se detectó mediante MTT. El consumo de glucosa se analizó con el método de glucosa oxidasa. La expresión proteica fue determinada mediante Western blot e inmunofluorescencia. En la microscopía electrónica se observó autofagosomas. Los principales ingredientes activos de C. tinctoria Nutt. incluyeron heriguard, flavanomarein, maritimin y marein. Veintiún de los 43 objetivos potenciales fueron PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK y ABL2. Estos 21 objetivos principales se enriquecieron significativamente en 50 vías de señalización. Treinta y cuatro vías de señalización estuvieron estrechamente relacionadas con la nefropatía diabética, de las cuales las principales vías fueron PI3K/ AKT, insulina y mTOR, y resistencia a la insulina. Los términos GO enriquecidos incluyeron procesos biológicos de fosforilación proteica, la regulación positiva de la señalización de PI3K y la secreción de citoquinas; componentes celulares del citosol, región extracelular y espacio extracelular; y la función molecular de la actividad de la proteína quinasa, la unión de ATP y la actividad de la proteína tirosina quinasa que no se extiende por la membrana. Los experimentos in vitro encontraron que la mareína aumentaba la expresión de AKT/AKT fosforilada en células endoteliales glomerulares renales humanas en un modelo de resistencia a la insulina inducida por niveles elevados de glucosa, así como aumentaron y disminuyeron respectivamente, los niveles de las proteínas asociadas a los microtúbulos, LC3 y P62. C. tinctoria Nutt. tiene muchos principios activos, con ingredientes principales de heriguard, flavanomarein, maritimain y marein, y puede ejercer un efecto de nefropatía antidiabética a través de distintass vías de señalización y objetivos.
Subject(s)
Coreopsis/chemistry , Diabetic Nephropathies , Network Pharmacology , Microscopy, Electron , Blotting, Western , Fluorescent Antibody Technique , ChalconesABSTRACT
Objective To study the chemical constituents of the water extract of capitula of Coreopsis tinctoria. Methods The chemical constituents from water extract were isolated and prepared by silica gel column, reversed-phase ODS, and pre-HPLC, and their structures were identified according to the physical and chemical properties and spectral data. Results A total of 13 compounds were isolated and identified as 8,3’,4’-trihydroxyflavone-7-O-β-D-glucopyranoside (1), 6-Hydroxyluteolin-7-O-β-D-glucoside (2), kaempferol (3), quercetin-7-O-β-D-glucopyranoside (4), (2S)-3’,4’,5,8-tetrahydroxyflavanone-7-O-β-D-glucoside (5), (2S)- eriodictyol-5-O-β-D-glucoside (6), butin-7-O-β-D-glucopyranoside (7), plathymenin (8), (Z)-6-O-β-D-glucopyranosyl-6,3’,4’- trihydroxyaurone (9), 5,6,3’,4’-tetrahydroxyaurone (10), 6,3’,4’-trihydroxyaurone (11), okanin-5’-O-β-D-glucopyranoside (12), and 4’-O-β-D- glucopyranosyl-3,4,2’,4’,5’-pentahydroxychalcon (13). Conclusion Thirteen compounds are isolated from C. tinctoria for the first time.
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Aim To compare the inhibition of lipid peroxidation of ethyl acetate extract( EAE) and n-buta-nol( BE) extract from Coreopsis tinctoria Nutt. in vitro. To investigate the parameters such as body weight, bio-chemical indexes in plasma, and viscera indexes on type 2 diabetes mice by intraperitoneal injection of streptozotocin ( STZ ) . Methods The extracts were prepared by response surface methodology. The ex-tracts were suspended in distilled water and defatted with petroleum ether. The aqueous layer was succes-sively extracted with ethyl acetate and n-butanol. The inhibition of lipid peroxidation activity was determined by thiobarbituric acid method. The effects of extract BE on diabetic mice were observed at the dosage of 0. 2,0. 4,0. 8 g·kg-1 ( ig) for 4 weeks. The parame-ters were observed such as weight of body changes, or-gan coefficients of liver, pancreas and kidney, bio-chemical indexes in plasma and viscera pathological sections. Results In the linoleic acid reaction system, the SC50 value of the EAE and BE was ( 443. 96 ± 11. 24) mg·L-1, (840. 29 ± 16. 38) mg·L-1, re-spectively, and that in rat liver homogenate was (23. 59 ± 3. 67 ) mg · L-1 , ( 60. 37 ± 4. 27 ) mg · L-1 , respectively. Compared with diabetic model group, BE could significantly improve the trend of weight loss, and increase viscera indexes. The patho-logical sections showed that BE had the recovery and improvement effects on the damage of liver, pancreas and kidney. Conclusions The extracts of C. tinctoria have a certain anti-lipid peroxidation activity in vitro. And BE has a certain capacity to improve and repair damaged organs for DM mice.
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Objective Response surface methodology ( RSM ) was applied to optimize the ultrasonic extraction conditions for flavonoids from Coreopsis tinctoria Nutt. Methods The influence factors of ultrasonic extraction were evaluated using the Box-Behnken central component experiments and analyzed by RSM. Results The optimum extraction conditions were confirmed as follows:extraction time 30. 0 min, ratio of liquid to solid 21∶1, concentration of ethanol 60%. The yield of flavonoids under this condition was (4.65±0.036)% (n=3). Conclusion The flavonoids could be extracted with stability and higher yield from Coreopsis tinctoria Nutt under optimized conditions.
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Objective: By comparing the acute toxicity of different extracts from Coreopsis tinctoria on mice, combined with the HPLC fingerprint and multiple linear regression to analyze the element which plays the most important role in causing the death of mice, and to provide the safety data for improving the extraction technology. Methods: To measure the maximum dose and maximal tolerance dose (MTD) of all the extracts, to measure the median lethal dose (LD50) by Bliss, and to record the death and weight changes; To measure the fingerprints of the extracts by HPLC, and to determine the element which mostly induced the death of mice by analyzing the absorption peak of the extracts by HPLC fingerprint with multiple linear regression. Results: The extracts include aqueous extract by spray drying (SD), aqueous extract by vacuum drying (VD) process, ethanol extract (ETE), ethyl acetate extracted component (AC), and the ethyl acetate extracted residuum (AR). Among those extracts, the maximum dose of SD and AR is 36 g/kg, the MTD of the VD is 26 g/kg, the LD50 (95% confidence limits) of ETE and AC are 19.565 (17.558-21.734) g/kg and 16.414 (13.987-34.725) g/kg, respectively; Under the high dose situation, 3,5-dicaffeoylquinic acid properly is the component which mostly contributes to the death of mice. Conclusion: Under the high dose situation, the ETE and AC will lead the death, and 3,5-dicaffeoylquinic acid properly is the component which mostly contributes to the death of mice.
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Objective: The high performance liquid chromatography diode array detection method was used to establish the determination methods for the contents of chlorogenic acid, rutin, quercetin, and luteolin in Kunlun snow daisy (Coreopsis tinctoria, Coreopsis Tinctoriae Folium). Methods: Agilent HC-C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was CH3OH (A)-0.01% H3PO4 (B) for the gradient elution with flow rate of 1.0 mL/min, the detection wavelength was 315 nm and the column temperature was 30 ℃. Results: There was a linear correlation between the concentration of chlorogenic acid 2.0-60 μg/mL (r = 0.999 8), rutin 5.0-100 μg/mL (r = 0.999 9), quercetin 1.0-40 μg/mL (r = 0.999 9), and luteolin 0.5-40 μg/mL (r = 0.999 9). The average recovery rates of chlorogenic acid, rutin, quercetin, and luteolin were 102.4% (RSD = 1.05%, n = 5), 98.6% (RSD = 1.28%, n = 5), 103.6% (RSD = 0.95%, n = 5), and 101.2% (RSD = 1.12%, n = 5), respectively. Conclusion: The analysis method is simple, rapid, reproducible, accurate, and reliable. It could be used to identify and evaluate the quantitative determination of chlorogenic acid, rutin, quercetin, and luteolin contents in Kunlun snow daisy.
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Objective: To study the chemical constituents in the capitulum of Coreopsis tinctoria. Methods: The constituents were separated and purified by AB-8 macroporous resin, silica gel, Sephadex LH-20, ODS column chromatography, and preparative HPLC. The structures were elucidated on the basis of chemical evidences, spectroscopic methods, and optical rotation data. The new compound was evaluated for its inhibitory activity against LPS-activated NO production in BV-2 microglial cells using the Griess assay. Results: Five polyacetylenes were obtained from the fraction of 50% ethanol extract of C. tinctoria identified as (3S)-(6E,12E)-tetradecadiene-8,10-diyne-1-ol-3-O-β-D-glucopyranoside (1),(2S)-(3Z,11E)-decadiene-5,7,9-triyne-1,2-diol (2),(2R)-(3E,5E,11E)-triene-7,9-diyne-1,2-diol (3),(E)-7-phenyl-2-heptene-4,6-diyn-1-ol (4), and (Z)-7-phenyl-2-heptene-4,6-diyn-1-ol (5). Conclusion: Compound 1 is a new polyacetylene glycoside named coreoside E and shows weak anti-inflammatory activity. Compounds 3 and 5 are isolated from the plants of C. tinctoria for the first time.
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The 3T3-L1 preadipocytes were used as carriers in the investigation of total extract, n-butanol extract, CB-1 and CB-2 of Coreopsis tinctoria Nutt. on cell proliferation and differentiation. Three groups at different doses were set for each of the four extract regions of C. tinctoria Nutt., respectively. MTT assay was used to detect 3T3-L1cell proliferation by four extract regions of C. tinctoria Nutt. Oil Red O staining was used to analyze the formation and accumulation of cytoplasmic lipid during cell differentiation. The results showed that compared with the control group, there were significant inhibition on cell proliferation when thetotal extract of C. tinctoriaNutt. at 100 μg·mL-1, n-butanol extract at 0.5, 5, and 50 μg·mL-1, CB-1 and CB-2 at 50 μg·mL-1 (P< 0.01). N-butanol extract showed certain dose-dependent manner (r = -0.903). Oil Red O staining showed that compared with the control group, thetotal extract of C. tinctoria Nutt. at 1, 10, 100 μg·mL-1 can obviously inhibit cell differentiation, reduce the formation of cytoplasmic lipid (P< 0.01). N-butanol extract can inhibit cell differentiation in a dose-dependent manner (r= -0.779). CB-1 and CB-2 obviously inhibited cell differentiation at the concentration of 50 μg·mL-1 (P < 0.01). It was concluded that thetotal extract, n-butanol extract, CB-1 and CB-2 of C. tinctoria Nutt. can inhibit the proliferation and differentiation of 3T3-L1 preadipocytes and reduce the formation of cytoplasmic lipid.
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Aim To investigate the effects of the alco-hol extract from Coreopsis tinctoria Nutt. on urinary metabolomics of spontaneous hypertension rats ( SHR) . Methods SHR were fed with normal diet for 1 week and then, they were randomly divided into six groups:untreated control, the high, middle, low dos-age group of the alcohol extract(3.2 g·kg-1 ,1.6 g· kg-1,0.8 g·kg-1) , captopril group(4 mg·kg-1) and Duzhong tablet group(187.5 mg·kg-1). The u-rine of normal rats and SHR hypertension model rats was collected on 1,2,3,4 weeks. The metabolic pro-files were analyzed using 1 H-NMR. PLS-DA methods were used to discriminate the difference and the bio-markers. Results Compared with model group, the blood pressure of all groups was significantly lowered after 4 weeks ( P <0.01 ) . The alcohol extract from Coreopsis tinctoria Nutt. could significantIy reduce blood pressure, and the urinary metabolic profiles trea-ted with Coreopsis tinctoria Nutt. were changed signifi-cantly such as IIL,creatine,β-glucose,etc. Conclusion The alcohol extract from Coreopsis tinctoria Nutt. could significantIy reduce blood pressure and change the urinary metabolomics profiles of SHR rats.