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Cytoprotective effect of Coreopsis tinctoria extracts and flavonoids on tBHP and cytokine-induced cell injury in pancreatic MIN6 cells
Abstract
ETHOPHARMACOLOGICAL RELEVANCE: Coreopsis tinctoria flowering tops infusion is traditionally used in Portugal for treating the symptoms of diabetes. Recent studies have revealed its antihyperglycemic activity when administered for 3 weeks to a STZ-induced glucose intolerance model in the rat and glucose tolerance regain was even clearer and pancreatic function recovery was achieved when administering Coreopsis tinctoria flavonoid-rich AcOEt fraction. In this study we aimed to evaluate the protective effect of Coreopsis tinctoria flowering tops aqueous extract, AcOEt fraction and the pure compounds marein and flavanomarein, against beta-cell injury, in a mouse insulinoma cell line (MIN6) challenged with pro-oxidant tert-butyl-hydroperoxide (tBHP) or cytokines.
The protective effects of Coreopsis tinctoria flowering tops extracts and pure compounds were evaluated through pre-incubating MIN6 cells with samples followed by treatment with tBHP (400 μM for 2 h) after which viability was determined through ATP measurements. In order to assess whether plant extracts were involved in decreasing reactive oxygen species, superoxide anion production was determined through a lucigenin-enhanced chemiluminescent method. Lastly, the direct influence of Coreopsis tinctoria extracts and main compounds on cell survival/apoptosis was determined measuring caspase 3 and 7 cleavage induced by cytokines.
Coreopsis tinctoria flowering tops extracts (25-100 μg/mL) and pure compounds (200-400 μM), when pre-incubated with MIN6 cells did not present any cytotoxicity, instead they increased cell viability in a dose dependent manner when challenged with tBHP. Treatment with this pro-oxidant also showed a rise in superoxide radical anion formation in MIN6 cells. This increase was significantly reduced by treatment with superoxide dismutase enzyme (SOD) but not by pre-treatment with Coreopsis tinctoria flowering tops extracts. Caspase 3/7 activation measurements show that Coreopsis tinctoria flowering tops extracts, as well as marein and flavanomarein, significantly inhibit apoptosis.
Coreopsis tinctoria extracts and pure compounds show cytoprotection that seems to be due to inhibition of the apoptotic pathway, and not through a decrease on superoxide radical production.
Supplementary resource (1)
Data
September 2012
... Our previous study indicated that the flavonoids extracted from CT show anti-hyperlipidemic effects, particularly in lowering triglycerides, reducing lipid deposition, and protecting liver function in an animal model [9]. Some pharmacological studies have also reported that CT is rich in flavonoids and can adjust glucose metabolism, promote blood circulation, and reduce cardiovascular disease risk [10,11]. Thus, a retrospective analysis was performed in patients with hyperlipidemia in this study, and we validated a protective role of CT in patients with hyperlipidemia. ...
... ADRP is highly expressed in the early stage of adipose differentiation or lipid accumulation in hepatocytes [37], and it can induce increases in the number and size of lipid droplets in the liver, thereby leading to lipid accumulation and insulin signaling inhibition; thus, the decrease in ADRP could inhibit the development of fatty liver and decrease triglyceride levels in the liver. A recent study demonstrated that the flavonoidrich fraction of CT could promote glucose tolerance through pancreatic function recovery in streptozotocin-induced glucose-intolerant rats, possibly through a mechanism of action other than merely antioxidant mediation [11]. Meanwhile, CT extracts and pure compounds also showed cytoprotection that might be attributed to inhibition of the apoptotic pathway, and not through a decrease in superoxide radical production [11]. ...
... A recent study demonstrated that the flavonoidrich fraction of CT could promote glucose tolerance through pancreatic function recovery in streptozotocin-induced glucose-intolerant rats, possibly through a mechanism of action other than merely antioxidant mediation [11]. Meanwhile, CT extracts and pure compounds also showed cytoprotection that might be attributed to inhibition of the apoptotic pathway, and not through a decrease in superoxide radical production [11]. Our further studies of at least two differing doses of CT and longer consumption periods will verify the effect of differing doses of CT and longer consumption periods on lipid metabolism. ...
Background/aims:
The prevalence of hyperlipidemia is increasing rapidly. The role of Coreopsis tinctoria (CT) in amending lipid metabolism in hyperlipidemia patients has not been reported. This study aims to evaluate the role of CT in altering lipid metabolism in hyperlipidemia patients and to explore the possible mechanisms mediated by gut microbiota in hyperlipidemia mice models.
Methods:
A retrospective analysis in 40 hyperlipidemia patients was conducted, in which 20 patients took fenofibrate and another 20 patients normatively drank water with CT. Hyperlipidemia mice models were also established. Blood biochemical tests were performed using an automatic biochemical analyzer. Liver histopathology was observed by hematoxylin and eosin staining. Ileocecal samples were collected from mice, and bacterial DNA was extracted and sequenced by MiSeq sequencing. Bacterial composition and differences were analyzed.
Results:
In hyperlipidemia patients, CT was associated with decreased triglyceride and low-density lipoprotein (LDL) levels without liver injury. The experimental hyperlipidemia model also verified a similar result. Gut microbial richness and diversity were significantly decreased in hyperlipidemic mice, but increased after CT treatment. Bacterial communities were significantly differentiated between normal controls and hyperlipidemic mice. CT administration improved gut microbiota composition to an approximately normal status. Meanwhile, CT administration attenuated bacterial alterations at the class, order, family, and genus levels in hyperlipidemic mice. Importantly, the genera Barnesiella, Lactobacillus, and Helicobacter achieved high discriminatory power in hyperlipidemic mice relative to normal controls.
Conclusions:
CT can modulate blood lipid metabolism with improvement of liver function by decreasing LDL and improving gut microbiota compositions. These findings may provide novel therapeutic strategies for patients with hyperlipidemia.
... As a compound belonging to the chalcone subgroup of polyphenols, marein is a major active compound in Coreopsis tinctoria and has beneficial antioxidative [24], antihypertensive [25], antihyperlipidemic [26], and antidiabetic [27] effects. Few studies have focused on the biological activities of marein, but previous research has shown that marein prevents the induction of tert-butyl-hydroperoxide and cytokines in a mouse insulinoma cell line (MIN6) through inhibition of the apoptotic-signaling cascade [28]. However, there have been no reports on whether marein can affect MGinduced PC12 cell impairment. ...
... Once inside the cell, it is cleaved by endogenous esterases and can no longer pass out of the cell. DCFH becomes the fluorescent compound 2 0 ,7 0 -dichlorofluorescein (DCF) upon oxidation by ROS [28]. Briefly, 1 Â 10 4 cells/well were plated in a 96well cell culture plate. ...
... These results are in agreement with previous findings that primarily focused on mitochondria, ROS, and Ca 2þ signaling [43,44]. Our data showed that pretreatment with marein reversed the MG-induced increases in cytosolic Ca 2þ and intracellular ROS concentrations, consistent with the proposed role of Coreopsis tinctoria extracts [28]. The antioxidant and free radical scavenger activities of marein were recently established in cytokine-induced oxidative damage in pancreatic MIN6 cells [45], consistent with our current findings. ...
Diabetic encephalopathy, which is characterized by cognitive decline and dementia, commonly occurs in patients with long-standing diabetes. Previous studies have suggested that methylglyoxal (MG), an endogenous toxic compound, plays an important role in diabetic complications such as cognitive impairment. MG induces neuronal apoptosis. To clarify whether marein, a major compound from the hypoglycemic plant Coreopsis tinctoria, prevents PC12 cell damage induced by MG, we cultured PC12 cells in the presence of MG and marein. Marein attenuated MG-induced changes in the mitochondrial membrane potential (ΔΨm), mitochondrial permeability transition pores (mPTPs), intracellular Ca(2+ )levels, the production of reactive oxygen species (ROS), glutathione (GSH)/glutathione disulfide (GSSG) and adenosine triphosphate (ATP), and the increase in the percentage of apoptotic cells. Marein also increased glyoxalase I (Glo1) activity, phospho-AMPKα (Thr172) and Bcl-2 expression and diminished the activation of Bax, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD). Importantly, pretreatment of cells with marein diminished the compound C-induced inactivation of p-AMPK. Molecular docking simulation showed that marein interacted with the γ subunit of AMPK. In conclusion, we found for the first time that the neuroprotective effect of marein is due to a reduction of damage to mitochondria function and activation of the AMPK signal pathway. These results indicate that marein may be a potent compound for preventing/counteracting diabetic encephalopathy.
... (Golden tickseed) is a plant native to North America that is cultured worldwide. The flower of Coreopsis tinctoria is used as a beverage and as a nutraceutical for reducing bodyweight, high serum lipids, and blood sugar, as well as for treating cardiovascular disease, hypertension, diarrhea, and emesis in Native American and traditional Chinese and Portuguese medicine [8][9][10]. Recent studies have found that the extracts and flavonoids from Coreopsis tinctoria Nutt. ...
... flower (CTF) displayed vasorelaxant properties via inhibition of calcium movement through cell membranes in rat thoracic aortic rings [11]. The flavonoidrich fraction of the Coreopsis tinctoria flower increases glucose tolerance through pancreatic function recovery in streptozotocin-induced diabetic rats [2,8], improves highfat diet-induced hepatic insulin resistance in rats through the inhibiting of gluconeogenic pathway key proteins glucose-6-phosphatase and phosphoenolpyruvate carboxykinase [12], and protects mouse insulinoma MIN6 cells from tert-butylhydroperoxide oxidative stress and cytokine-induced injury [9]. Coreosides A, B, C, and D, C14-polyacetylene glycosides of Coreopsis tinctoria, display anti-inflammatory activity against cyclooxygenase-2 [13]. ...
... Coreopsis tinctoria flower has traditionally been used to control diabetes in Portugal [8,9]. It has been reported that Coreopsis tinctoria flower infusion reduced blood glucose levels in glucose-intolerant Wistar rats [2] and improved highfat diet-induced hepatic insulin resistance in rats through the inhibiting of gluconeogenic pathway [12] and glucose tolerance through pancreatic function recovery in streptozotocininduced diabetic rats [8]. ...
The aim of this study was to assay the effects of
Coreopsis tinctoria
Nutt. flower extracts on hyperglycemia of diet-induced obese mice and the underlying mechanisms.
Coreopsis tinctoria
flower was extracted with ethanol and water, respectively. The total phenol, flavonoid levels, and the constituents of the extracts were measured. For the animal experiments, C57BL/6 mice were fed with a chow diet, high-fat diet, or high-fat diet mixed with 0.4% (w/w) water and ethanol extracts of
Coreopsis tinctoria
flower for 8 weeks. The inhibitory effects of the extracts on
α
-glucosidase activity and the antioxidant properties were assayed
in vitro
. We found that the extracts blocked the increase of fasting blood glucose, serum triglyceride (TG), insulin, leptin, and liver lipid levels and prevented the development of glucose tolerance impairment and insulin resistance in the C57BL/6 mice induced by a high-fat diet. The extracts inhibited
α
-glycosidase activity and increased oxidant activity
in vitro
. In conclusion,
Coreopsis tinctoria
flower extracts may ameliorate high-fat diet-induced hyperglycemia and insulin resistance. The underling mechanism may be via the inhibition of
α
-glucosidase activity. Our data indicate that
Coreopsis tinctoria
flower could be used as a beverage supplement and a potential source of drugs for treatment of diabetics.
... North American Indians used dried C. tinctoria tea to treat internal organ pain and bleeding. C. tinctoria is used to treat diabetes in Portugal [10][11][12]. Pharmacological research shows that the main components of the C. tinctoria are avonoids with a variety of biological activities [13][14][15]. ...
... It has e ects on lowering blood sugar, regulating blood lipids, and anti-hypertension [3]. Experiments in vitro have proved that marein can resist oxidation [19,20], reduce triacylglycerol content, and have a certain protective e ect on pancreatic islet cells MIN6 [11]. Marein also improves glucose metabolism disorder induced by high glucose in HepG2 cells, which could signi cantly prevent insulin resistance induced by high glucose [21,22]. ...
Marein is the main active compound of Coreopsis tinctoria Nutt., and its main activities include antioxidant, hypoglycemic, and hypotensive. After oral administration of marein, the blood concentration of marein is low. The metabolites of marein have not been reported systematically. In this study, a rapid and systematic method based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) was established to detect metabolites of marein in vivo (plasma and urine) after oral administration and injection. Sixty-one metabolites were identified. The metabolites are formed through a wide range of metabolic reactions, including hydroxylation, glucuronidation, methylation, hydrolysis, and desorption of hydrogen. The liver microsome incubation was further used to investigate the metabolic rate of marein. Network pharmacology was applied to study the targets and pathways of marein and its metabolites. Marein and its metabolites act on the same targets to enhance the therapeutic effect. This research illuminates the metabolites and metabolic reaction of marein and establishes a basis for the development and rational utilization of C. tinctoria. Meanwhile, the analysis of prototype and metabolites together by network pharmacology techniques could provide a methodology for the study of component activity.
... Thus, flavanomarein may play a key role in the quality assessment of C. tinctoria. Flavanomarein significantly increased cell viability when MIN6 cells, a model of oxidative stress, were challenged with tBHP (Dias et al., 2012). Flavanomarein is rather rare in the plant kingdom, and its protective activity against oxidative stress by 6-OHDA has not been previously established. ...
... As a flavonoid, flavanomarein also prevents cell apoptosis. Dias et al. reported that flavanomarein displays cytoprotection that may be attributable to inhibition of the apoptotic pathway (Dias et al., 2012). In addition, flavanomarein inhibited the reduction in ΔΨm, which is a precursor to mitochondrial impairment. ...
Background: Flavanomarein is the main component of Coreopsis tinctoria Nutt. (C. tinctoria), which is a globally well-known flower tea that has a distinct flavor and many beneficial health effects, such as antioxidant activities. We aimed to explore the effect of flavanomarein on a 6-hydroxydopamine (6-OHDA)-lesioned cell model of oxidative stress.
Methods: In this study, we used 6-OHDA-lesioned PC12 cells and primary cortical neurons to investigate the protective effects of flavanomarein and its potential mechanism.
Results: The results indicated that pretreatment with flavanomarein (25, 50, or 100 µM for 24 h) significantly increased the cell viability, reduced the lactate dehydrogenase (LDH) release and improved the mitochondrial membrane potential (∆Ψm) and mitochondrial impairment. Additionally, flavanomarein markedly reduced the gene expression of tumor necrosis factor (TNF)-α and protein kinase C ζ (PKC-ζ), the nuclear translocation of p65, and the levels of p-AMPK-α and acetyl-p53. Flavanomarein also elevated the gene expression of P85α, PKC-β1, and Bcl-2, the protein expression of Sirt1 and ICAD, and the phosphorylation level of AKT. Conclusions: Together, these results suggest that flavanomarein protects PC12 cells and primary cortical neurons from 6-OHDA-induced neurotoxicity by upregulating the PI3K/AKT signaling pathway and attenuating the nuclear factor kappa B (NF-κB) signaling pathway. Therefore, our study provides evidence that may aid in the development of a potential compound against 6-OHDA toxicity.
Key words: Flavanomarein, 6-hydroxydopamine, Neuron, NF-κB, AKT, PC12 cells
... Coreopsis tinctoria Nutt. (Asteraceae), a traditionally used preparation for diabetes treatment in Portugal (Dias et al., 2012;Srere, 1966), is a plant native to North America that has spread worldwide. Our previous study revealed that C. tinctoria increased insulin sensitivity and regulated hepatic metabolism in rats fed with a high-fat diet (Jiang et al., 2015). ...
... Marein has many beneficial biological activities, including antihyperlipidemic (Liang SH et al., 2009), anti-oxidative (Lan et al., 2014), antidiabetic (Dias et al., 2010b), and antihypertensive effects (Ming T et al., 2012). Previous research had also found that marein prevented tert-butyl hydroperoxide and cytokine induction in a mouse insulinoma cell line (MIN6) through the inhibition of the apoptotic signaling cascade (Dias et al., 2012). In addition, marein promotes pancreatic function recovery in streptozotocin-induced glucose-intolerant rats (Dias et al., 2010a;Dias et al., 2010b). ...
Objective
To investigate the effects of the ethyl acetate extract of Coreopsis tinctoria (EAEC) on insulin resistance (IR) in rats fed a high-fat diet.
Methods
Male Sprague-Dawley (SD) rats were fed a HFD (60% fat) supplemented with EAEC for 8 weeks. The administration of EAEC to the rats with HFD-induced insulin resistance reduced hyperglycemia, plasma levels of insulin, and steatosis in the liver. Metabolomic study was used to analyse the metabolic levels of the high glucose-treated cells, control cells and marein-treated cells.
Results
High glucose and high fat conditions caused a significant increase in blood glucose, insulin, serum TC, TG and LDL-C levels, leading to abnormal IR in rats. However, treatment with EAEC protects against HFD-induced IR by improving the fasting serum glucose homeostasis and lipid homeostasis. The high glucose conditions significantly decreased glycogen synthesis and increased PEPCK, G6Pase and Krebs cycle-related enzyme protein levels, leading to an abnormal metabolic state in HepG2 cells. However, treatment with marein improved IR by increasing glucose uptake and glycogen synthesis and by downregulating PEPCK and G6Pase protein levels. The statistical analysis of the HPLC/MS data demonstrated that marein could restore the normal metabolic state.
Conclusion
The results revealed that EAEC ameliorates IR in rats, and marein has the potential to improve IR by ameliorating glucose metabolism disorders.
... hypotension. Modern pharmacological studies have not only confirmed these above effects, but also shown that it has good antioxidant activities [4][5][6]. Phytochemical studies have shown that snow chrysanthemum mainly contains flavonoids, phenolic acids, and terpenoids [7]. Flavanomarein and marein, the predominant flavonoids, were proved to have good hypoglycemic activities [3]. ...
... The whole plant has been employed in China for hundreds of years as a folk herb for heat-clearing and detoxifying , while the capitulum of C. tinctoria was taken as a drink to deter diabetes in Portugal [3]. Since the beginning of this century, it has been known that snow chrysanthemum was used by the Uyghur nationality in Xinjiang (China) to prevent cardiovascular and cerebrovascular diseases, such as hypoglycemia, hypolipidemia, and good antioxidant activities [4][5][6]. Phytochemical studies have shown that snow chrysanthemum mainly contains flavonoids, phenolic acids, and terpenoids [7]. Flavanomarein and marein, the predominant flavonoids, were proved to have good hypoglycemic activities [3]. ...
A simple, accurate and reliable high performance liquid chromatography coupled with photodiode array detection (HPLC-DAD) method was developed and then successfully applied for simultaneous quantitative analysis of eight compounds, including chlorogenic acid (1), (R/S)-flavanomarein (2), butin-7-O-β-D-glucopyranoside (3), isookanin (4), taxifolin (5), 5,7,30,50-tetrahydroxyflavanone-7-O-β-D-glucopyranoside (6), marein (7) and okanin (8), in 23 batches of snow chrysanthemum of different seed provenance and from various habitats. The results showed total contents of the eight compounds in the samples with seed provenance from Keliyang (Xinjiang, China), are higher than in samples from the other five provenances by 52.47%, 15.53%, 19.78%, 21.17% and 5.06%, respectively, which demonstrated that provenance has a great influence on the constituents in snow chrysanthemum. Meanwhile, an ultra performance liquid chromatography coupled with electrospray ionization and quadrupole time-of-flight-mass spectrometry (UPLC-ESI-QTOF-MS) was also employed to rapidly separate and identify flavonoids and phenolic acids in snow chrysanthemum from Keliyang. As a result, a total of 30 constituents, including 26 flavonoids and four phenolic acids, were identified or tentatively identified based on the exact mass information, the fragmentation characteristics, and retention times of eight reference standards. This work may provide an efficient approach to comprehensively evaluate the quality of snow chrysanthemum.
... The infusion of Coreopsis tinctoria flowering tops has been used in Portugal to control diabetes. The flowers extracts from Coreopsis tinctoria may contain the following compounds: marein, okanin, coreopsin, 3,4',5,6,7-pentahydroxyflavanone-Ohexoside, 3',5,5',7-tetrahydroxyflavanone-O-hexoside, 3,3',5,5',7-pentahydroxyflavanone-O -hexoside, flavanokanin,3,4',5,6,7-pentahydroxyflavanone, dicaffeoylquinic acid, 3',5,5',7-tetrahydroxy flavanone, maritimein, flavanomarein, luteolin, quercetin, butein, chlorogenic acid, caffeic acid, quercetagitin-7-O-glucoside, myristic acid, α-pinene, camphene, limonene, α-thujone, β-linalool, β-terpineol, verbenone, borneol, myrtenal, ciscarveol, trans-carveol, carvone, bornyl acetate, βcaryophyllene, β-caryophyllene epoxide [2][3][4][5][6][7][8][9][10][11][12][13]. ...
... -relations defining the solid / liquid ratio (N): (12) -relationship giving balance of solute (13) -relationship giving balance of the solvent from extraction liquid: (14) -relationship balance of all solute -solvent: (15) To obtain the mathematical model of unity in a series of contact stages at the relations (12) -(15) is added the equilibrium relationship for the current unit "n": (16) There is a graphical method of calculation which use the representation of equilibrium (N ~ x, y) and operating relationship. Here the operating relationship (line section) has the ability to pass through a fixed point called the pole of operation. ...
This paper presents the equilibrium data, together with their use for calculation of three separation cases, when the active principles from fruits of Coreopsis tinctoria Nutt. are extracted with n-hexane. Simple single contact extraction, simple extraction with multiple contact and countercurrent extraction stages were operating procedures that were custom equilibrium data. For simple extraction of single contact and multiple contact extraction is considered the analytical calculation, while for stage countercurrent extraction is completed by the calculating graph. Keywords: single contact extraction, simple extraction with multiple contact, countercurrent extraction Coreopsis tinctoria Nutt. (Asteraceae) is native from North America, in Romania being commonly cultivated for ornament in gardens. The plant is used to treat several disorders including diarrhoea, internal pains, bleeding, to strengthen blood and as an emetic [1]. The infusion of Coreopsis tinctoria flowering tops has been used in Portugal to control diabetes. The flowers extracts from Coreopsis tinctoria may contain the following compounds: marein, epoxide [2-13]. For elevation of this study we used the fruits of Coreopsis tinctoria Nutt., in order to extract the active principles, considering the processes of interest simple single contact extraction, simple extraction of multiple contact and countercurrent extraction stage, given that, n-hexane was the solvent used. The operating temperature is considered at 25 o C. In development of this work, we considered that between the components of dry extract from the fruits of Coreopsis tinctoria Nutt. was no big difference in physico-chemical behaviour, so they are considered species have a uniform behaviour, generically called specific extract. Also we show that the specific extract is an oil remembering that this plant is from sunflower family (Asteraceae). To determine the interphase equilibrium, characterizing the
... Local Uyghur people use it as an herb tea to treat high blood pressure and diarrhea. Modern pharmacological studies have shown that it can adjust blood sugar, blood lipid and blood pressure [5,6] . It is reported that Coreopsis tinctoria in Xinjiang is rich of flavonoids and can adjust glucose metabolism and reduce cardiovascular disease risk [5,6]. ...
... Modern pharmacological studies have shown that it can adjust blood sugar, blood lipid and blood pressure [5,6] . It is reported that Coreopsis tinctoria in Xinjiang is rich of flavonoids and can adjust glucose metabolism and reduce cardiovascular disease risk [5,6]. There has not research on lipid metabolism [7]. ...
To identify the chemical structure of Coreopsis tinctoria extracts and their effect and mechanism on reducing blood lipid in hyperlipemia mice.
The flavonoids were extracted from Coreopsis tinctoria. The chemical structure was identified by HPLC. 59 mice were divided randomly into 5 groups. (group 1: normal diet control; group 2: hyperlipemia model; group 3: hyperlipemia mice treated with Coreopsis tinctoria, low dose 100 mg/kg; group 4: hyperlipemia mice treated with Coreopsis tinctoria high dose group 200 mg/kg; group 5 hyperlipemia mice treated with Fenofibrate. After 2 week of hyperlipid diet, the treatment of Coreopsis tinctoria and Fenofibrate were given for another 6 weeks with continuous hyperlipid diet. The TC, TG, HDL, histology, adipose differentiation-related protein (ADRP) expression in different groups were compared.
Compared with normal diet group, TC, TG in hyperlipemia model group increased ( P < 0. 01). After treatment with Coreopsis tinctoria low dose group, high dose group, TC of the hyperlipemia mice decreased (P < 0. 05) without increasing AST, ALT and ALP. Fenofibrate can also decrease TC and TG but increase AST, ALT and ALP. Expression of hepatic ADRP increased in hyperlipemia mice. Coreopsis tinctoria high dose group 200 mg/kg can inhibit ADRP as Fenofibrate does.
The flavonoids from Coreopsis tinctoria extracts can reduce blood lipid without liver function damage, showing better anti- hyperlipemia effect than Fenofibrate by down-regulating ADRP.
... C. tinctoria is currently the only alpine plant comparable to Echeveria laui Moran & Meyrán. Pharmacological studies have shown that C. tinctoria possesses multiple activities, including antioxidant, antidiabetic, antihypertensive, and cytoprotective effects (Dias et al., 2012;Lan, Lin & Zheng, 2014;Jiang et al., 2016;Jiang et al., 2018). Studies have confirmed that the various effects of C. tinctoria on the human body are mainly attributed to its flavonoid-like polyphenols because this plant contains many types of flavonoids (Zhao et al., 2013;Shen et al., 2020;Zhang et al., 2022). ...
To analyze the flavonoids in Coreopsis tinctoria and compare the differences in flavonoids among C. tinctoria of different origins, the chemical composition of C. tinctoria capitulum was analyzed by ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and the flavonoid metabolites were analyzed and identified based on their retention time, mass-to-charge ratio and fragment ions in the UPLC-QTOF-MS matrix. Capitulum samples of C. tinctoria were collected from three locations in the Xinjiang region at different altitudes. A total of 204 flavonoid compounds were identified, and 31 different flavonoid metabolites were then identified from flowers of C. tinctoria of different origins. Further analysis of these 31 significantly accumulated metabolites identified seven flavonoid metabolites, namely, homoplantaginin, kaempferol, quercetin, isorhamnetin, avicularin, quercetin 3-O-(6′-galloyl)- β - D -galactopyranoside and isorhamnetin 3-O-glucoside, with high accumulation only in sample collected from Tashkurgan Tajik (TX) and low expression in sample collected from Yutian County (YT) and Shaya County (SY). Moreover, 7,4′-dihydroxyflavone and 4,4′-dimethoxychalcone showed high accumulation only in SY, and afzelin was specifically highly accumulated in YT. In addition, the identified flavonoid metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and key pathways that might regulate the biosynthesis of these flavonoid compounds were analyzed. These findings provide key information for research on flavonoids and their biosynthesis in C. tinctoria and will provide a theoretical basis for studying the herbal quality and origin of C. tinctoria .
... No flavanomarein was detected in Chrysanthemum morifolium and Florists chrysanthemum under the established chromatographic conditions. Flavanomarein was reported to have good hypoglycemic activities (26,29). Meanwhile, satisfactory antioxidant activities were reported for flavanokanin and okanin (25,30). ...
In this study, we report a simple and reliable high-performance liquid chromatography coupled with diode array detection method for simultaneous and quantitative analysis and comparison of major phenolic compounds dominant phytochemicals in Chrysanthemum morifolium, Florists chrysanthemum and snow chrysanthemum (Coreopsis tinctoria or C. tinctoria). The chromatographic separation was achieved using a reversed phase C18 column with a mobile phase of water [containing 0.1% trifluoroacetic acid (TFA)] and acetonitrile. The major phenolic compounds were completely separated within 16 min at a flow rate of 1.0 mL/min. Flavonoid and phenolic acid profiles of the ethanol extracts of the three flowers were analyzed. The results revealed that C. tinctoria possessed the highest amount of flavonoids (flavanomarein, flavanokanin, marein and okanin) and relative lower content of phenolic acid (chlorogenic acid and 3,5-dicafeoylquinic acid). The total content of the four flavonoids in C. tinctoria reached 53.99 ± 1.32 mg/g. In particular, the marein content in C. tinctoria was as high as 36.50 mg/g. Flavanomarein was only detected in C. tinctoria, whereas chlorogenic acid and 3,5-dicafeoylquinic acid were abundant in Chrysanthemum morifolium and Florists chrysanthemum. The content of marein in Chrysanthemum morifolium was slightly higher than that in Florists chrysanthemum, whereas no okanin was detected in Florists chrysanthemum under these high-performance liquid chromatography conditions. The results indicated phenolic components differ significantly depending on the cultivar, especially between C. tinctoria and common commercially available chrysanthemums. The method adopted in this study is helpful for quality control of different chrysanthemum species as well as their products, which is essential for usage and functionality clarification.
... Prolonged use of insulin is also known to activate immune response that prompts increase in the dosage and other side effects ( Paschou et al., 2018 ) ( Feingold, 2019 ) and immunosuppressive drugs like cyclosporine ( Stiller et al., 1984 ) are also being used. These synthetic remedies can help lower blood glucose but are unable to stop decrease in beta cell mass ( Dias et al., 2012 ). Majority of the recent advances are not cost effective and can pose a huge financial burden on the type 1 diabetics. ...
Background
Type 1 diabetes mellitus is characterized by loss of beta cell mass and insulin insufficiency. Beta cell apoptosis and destruction by cytotoxic T lymphocytes and pro-inflammatory cytokines like IL-1β, TNF-α and IFN-γ appears to represent a key event in pathogenesis of type 1 diabetes mellitus.
Purpose
The aim of present study was to evaluate the prophylactic effect of aqueous extract of Curcuma longa and Piper nigrum (CL-PN) in ratio 2:1 on pancreatic beta cell apoptosis and dysfunction induced by cytokine cocktail of IL-1β (50 ng/ml), TNF-α (25 ng/ml) and IFN-γ (25 ng/ml).
Methods
We used Min6 beta cell line as an in vitro model and the cells were pre-treated with herbal extract of CL-PN and then exposed to cytokine cocktail for 6 hrs to mimic beta cell destruction as in type 1 diabetes. Beta cell apoptosis and protective activity were assessed by flow cytometer based assay of annexin-V FITC and propidium iodide staining.
Results
Aqueous extract of the combination of CL-PN at 1 μg/ml concentration can effectively inhibit apoptosis in Min6 beta cell line by 1.6 fold as compared to cytokine cocktail. It also demonstrated 2.6 fold lesser production of nitric oxide, 1.4 fold reduced ROS and increasing mitochondrial membrane potential which are major markers of beta cell damage. The caspase 3 mRNA expression is also reduced by 4.7 fold despite cytokine challenge.
Conclusion
Therefore, the present study paves the way for establishing herbal oral treatment for protection of beta cells in type 1 diabetes mellitus.
... Instead, they increased cell viability in a dose-dependent manner when challenged with tBHP. Further mechanistic studies suggested the extracts and the two compounds execute cytoprotection through inhibition of the apoptotic pathway (Dias et al., 2012). The aqueous extract could promote the proliferation of islet β-cells (INS-1) in the dose range (0.1, 1, 5, and 10 μg/mL), and had a protective effect on pancreatic islet β-cells injury induced by palmitic acid (Kang et al., 2017). ...
Ethnopharmacological relevance
Coreopsis tinctoria Nutt. (family Asteraceae) is an important traditional medicine in North America, Europe, and Asia for quite a long historical period, which has received great attention due to its health-benefiting activities, inculding disinfection, treatment sexual infection, diarrhoea, acute and chronic dysentery, red-eye swelling as well as pain, heat, thirst, hypertension, palpitation, gastrointestinal discomfort, and loss of appetite.
Aim of the review
The purpose of this review is to give an overview of the current phytochemistry and pharmacological activities of C. tinctoria, and reveals the correlation among its traditional uses, phytochemistry, pharmacological profile, and potential toxicity.
Materials and methods
This review is based on published studies and books from electronic sources and library, including the online ethnobotanical database, ethnobotanical monographs, Scopus, SciFinder, Baidu Scholar, CNKI, and PubMed. These reports are related to the traditional uses, phytochemistry, pharmacology, and toxicology of C. tinctoria.
Results
Coreopsis tinctoria is traditionally used in diarrhoea, infection, and chronic metabolic diseases. From 1954 to now, more than 120 chemical constituents have been identified from C. tinctoria, such as flavonoids, polyacetylenes, polysaccharides, phenylpropanoids, and volatile oils. Flavonoids are the major bioactive components. Current research has shown thatits extracts and compounds possesses diverse biological and pharmacological activities such as antidiabetes, anti-cardiovascular diseases, antioxidant, anti-inflammatory, protective effects on organs, neuroprotective effects, antimicrobial, and antineoplastic. Studies in animal models, including acute toxicity, long-term toxicity, and genotoxicity have demenstrated that Snow Chrysanthemum is a non-toxic herb, especially for its water-soluble parts.
Conclusions
Recent findings regarding the main phytochemical and pharmacological properties of C. tinctorial have confirmed its traditional uses in anti-infection and treatment chronic metabolic disease and, more importantly, have revealed the plant as a valuable medicinal plant resource for the treatment of a wide range of diseases. The available reports indicated that most of the bioactivities in C. tinctorial could be attributed to flavonoids. However, higher animals and humans studies are requied to explore the efficacy and mechanism of action of C. tinctoria in future.
... In China, Coreopsis tinctoria Nutt.'s (C. tinctoria) capitula is consumed as herbal tea, and its extracts exhibit various biological activities including antioxidant [1][2][3][4], antihypertensive [5,6], antineuroinflammatory [7], antidiabetic activities [8][9][10][11], hepatic insulin resistance [12], and antihyperlipidemic effects [13]. Okanin refers to a major flavonoid found in C. tinctoria [14] having attracted considerable attention for its potential contribution to the pharmacological activity of C. tinctoria. ...
Okanin is a major flavonoid found in Coreopsis tinctoria Nutt., arousing huge interest recently for its considerable biological characteristics including antioxidant, antineurotoxic, and antidiabetic activities. An ultrahigh performance liquid chromatography triple-quadrupole tandem mass spectrometry (UPLC-MS) was successfully used to determine okanin in rat plasma after oral administration of okanin. Bavachalcone acted as an internal standard (IS). By gradient elution, IS and analyte were separated on a C18 column for 7 min at a flow rate of 0.25 mL/min with acetonitrile-0.1% acetic acid mobile phase. The stability, matrix effect, extraction recovery, accuracy, precision, linearity, and selectivity of the method were firstly demonstrated. The major pharmacokinetic parameters of okanin in rat plasma were then measured using the developed UPLC-MS method. An UPLC-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was finally established to obtain the specific and accurate mass of okanin in rat plasma after oral administration, and its proposed fragmentation was further elaborated.
... C. tinctoria is a tea beverage and folk medicine material with excellent pharmacological properties, such as anti-inflammation, anticancer, anti-hypertension, and anti-oxidation (Dias et al., 2012;Li et al., 2015). A large number of studies confirmed that the strong antioxidant activity of C. tinctoria were due to its flavonoids and phenolic acids Yang et al., 2014), in this study, the main components of CTBE were quantitatively identified as flavanomarein (13.644 mg/g) and marein (13.387 mg/g). ...
The study investigated the polyphenol of Coreopsis tinctoria buds extract (CTBE) and its protective effects on cognitive dysfunction and brain damage induced by d-galactose in mice. Three polyphenols and nine flavonoids were characterized by UPLC/Q-TOF-MS, with major components as flavanomarein (13.644 mg/g dry weight) and marein (13.387 mg/g dry weight). Results showed CTBE significantly improved the learning, memory, and cognitive abilities and brain index of aging mice. Besides, CTBE reduced the content of malondialdehyde while increased the activity of glutathione peroxidase and superoxide dismutase and the the total antioxidants in brain. CTBE reversed the abnormality of Ach level, AChE activity, and hippocampus changes in aging mice. Therefore, CTBE, as a novel nutraceutical, could attenuate the cognitive damage and improve parameters related to brain senescence, partly by reducing the oxidative stress and hippocampal damage.
... Its component eriodictyol 7-O-β-D glucopyranoside could ameliorate lipid disorders via suppressing lipogenesis and protecting mitochondrial function 25 . In streptozotocin-induced glucose-intolerant rats, the flavonoid-rich fraction exerted a cytoprotective effect on tBHP and cytokine-induced cell injury in MIN6 cells 26 . Extracts of Coreopsis tinctoria Nutt. ...
Flavanomarein (FM) is a major natural compound of Coreopsis tinctoria Nutt with protective effects against diabetic nephropathy (DN). In this study, we investigated the effects of FM on epithelial-mesenchymal transition (EMT) in high glucose (HG)-stimulated human proximal tubular epithelial cells (HK-2) and the underlying mechanisms, including both direct targets and downstream signal-related proteins. The influence of FM on EMT marker proteins was evaluated via western blot. Potential target proteins of FM were searched using Discovery Studio 2017 R2. Gene Ontology (GO) analysis was conducted to enrich the proteins within the protein-protein interaction (PPI) network for biological processes. Specific binding of FM to target proteins was examined via molecular dynamics and surface plasmon resonance analyses (SPR). FM promoted the proliferation of HK-2 cells stimulated with HG and inhibited EMT through the Syk/TGF-β1/Smad signaling pathway. Spleen tyrosine kinase (Syk) was predicted to be the most likely directly interacting protein with FM. Combined therapy with a Syk inhibitor and FM presents significant potential as an effective novel therapeutic strategy for DN.
... Daily intake of plants rich in flavonoids and proanthocyanidins reduces the risk of pancreatic cancer by 25% [26][27][28]. Ethyl acetate extracts of Coreopsis tinctoria rich in flavonoids such as marein and flavanomarein kill pancreatic tumor cells by inducing apoptosis [29]. Scutellaria baicalensis extracts containing baicalein, wogonin, oroxylin A, and a glucuronide effectively countered pancreatic cancer in a mouse xenograft model [30,31]. ...
2′,4′-Dihydroxy-6’-methoxy-3′,5′-dimethylchalcone (DMC), a principal natural chalcone of Cleistocalyx operculatus buds, suppresses the growth of many types of cancer cells. However, the effects of this compound on pancreatic cancer cells have not been evaluated. In our experiments, we explored the effects of this chalcone on two human pancreatic cancer cell lines. A cell proliferation assay revealed that DMC exhibited concentration-dependent cytotoxicity against PANC-1 and MIA PACA2 cells, with IC50 values of 10.5 ± 0.8 and 12.2 ± 0.9 µM, respectively. Treatment of DMC led to the apoptosis of PANC-1 by caspase-3 activation as revealed by annexin-V/propidium iodide double-staining. Western blotting indicated that DMC induced proteolytic activation of caspase-3 and -9, degradation of caspase-3 substrate proteins (including poly[ADP-ribose] polymerase [PARP]), augmented bak protein level, while attenuating the expression of bcl-2 in PANC-1 cells. Taken together, our results provide experimental evidence to support that DMC may serve as a useful chemotherapeutic agent for control of human pancreatic cancer cells.
... [169] Coreopsis tinctoria Nutt. Asteraceae Tickseed root tea for diarrhea root [15] Plant: polyacetylenes, (2S)-(3Z,11E)-decadiene-5,7,9-triyne-1,2-diol and (2R)-(3E,11Z)-decadiene-5,7,9-triyne-1,2-diol [170] Plant: seven compounds made up the major contributions of antioxidant activity in C. tinctoria, including okanin, isookanin, marein, flavanomarein, 5,7,3 ,5 -tetrahydroxyflavanone-7-O-glucoside, 3,5-dicaffeoylquinic acid, and chlorogenic acid [171] Flowers: C 14 polyacetylene glycosides coreosides A-D [172] Buds: C 14 polyacetylene glycosides coreosides E and F [173] Flowers: C 14 polyacetylene glycosides coreosides A, B, D, and E [174] Flowers: chalcone marein, flavanone flavanomarein [175] Flowers Flowers: quercetagitin-7-O-glucoside, marein (major), 1,3-dicaffeoylquinic acid, okanin, acetylmarein [180] Flowers: taxifolin-7-O-glucoside, flavanomarein, quercetagetin-7-O-glucoside, okanin 4 -O-glucoside, okanin, chlorogenic acid [181] Flowers: chlorogenic acid, (R/S)-flavanomarein, butin-7-O-β-D-glucopyranoside, isookanin, taxifolin, 5,7,3 ,5 -tetrahydroxyflavanone-7-O-β-D-glucopyranoside, marein, and okanin [182] Fruits: flavonoids (marein, flavanomarein, quercetagetin-7-O-glucoside, okanin aurone, leptosidin, luteolin, apigenin) and phenolic acids (chlorogenic acid, caffeic acid) [183] Floral EO: limonene (11.3%), α-bergamotene (7.3%) [184] Cornus florida L. Cornaceae Dogwood bark chewed for headache bark [15] bark decoction used for fevers, body aches; bark poultice used on sores/ulcers bark [22] Bark: saponins (sarsapogenin-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside and sarsapogenin-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside) [185] Datura stramonium L. Solanaceae Jimson weed leaf poultice applied to boils; leaves smoked for asthma leaves [15] Root culture: tropane alkaloid (−)-hyoscyamine [186] Root culture: tropane alkaloids (hyoscyamine and scopolamine) [187] Seeds: tropane alkaloid (−)-hyoscyamine [188] Leaves: tropane alkaloids (hyoscyamine and scopolamine) [189] Diospyros virginiana L. Ebenaceae Persimmon bark infusion for venereal diseases, sore throat and mouth; syrup for oral thrush, bloody discharge from bowels bark [17] Bark: binaphthoquinone isodiospyrin [190] Fruits: polyphenolics (methyl gallate, gallic acid, luteolin, quercetin, myricetin, yricetin 3-O-α-rhamnoside, myricetin 3-O-β-glucoside, myricetin 3-O-β-glucuronide) [191] Roots: 4-hydroxy-5,6-dimethoxynaphthalene-2-carbaldehyde, 12,13-didehydro-20,29-dihydrobetulin, 7-methyljuglone, diospyrin, isodiospyrin, shinanolone, lupeol, betulin, betulinic acid, betulinaldehyde, and ursolic acid [192] Epilobium angustifolium L. ...
Background: Native Americans have had a rich ethnobotanical heritage for treating diseases, ailments, and injuries. Cherokee traditional medicine has provided numerous aromatic and medicinal plants that not only were used by the Cherokee people, but were also adopted for use by European settlers in North America. Methods: The aim of this review was to examine the Cherokee ethnobotanical literature and the published phytochemical investigations on Cherokee medicinal plants and to correlate phytochemical constituents with traditional uses and biological activities. Results: Several Cherokee medicinal plants are still in use today as herbal medicines, including, for example, yarrow (Achillea millefolium), black cohosh (Cimicifuga racemosa), American ginseng (Panax quinquefolius), and blue skullcap (Scutellaria lateriflora). This review presents a summary of the traditional uses, phytochemical constituents, and biological activities of Cherokee aromatic and medicinal plants. Conclusions: The list is not complete, however, as there is still much work needed in phytochemical investigation and pharmacological evaluation of many traditional herbal medicines.
... The capitula of Coreopsis tinctoria, also known as snow chrysanthemum, have been used in the form of a tea-like beverage for the prevention of cardiovascular disorders, diarrhea and diabetes in traditional Chinese medicine (5). Coreopsis tinctoria has been revealed to contain high concentrations of flavonoids (6), and it has been reported to exert anti-inflammatory effects (5), to promote pancreatic cell recovery (7,8) and to regulate lipid metabolism in hyperlipidemic mice (9). However, the main active compounds of Coreopsis tinctoria, as well as their exact pharmacologic effects on hyperlipidemia, have yet to be elucidated. ...
Coreopsis tinctoria (snow chrysanthemum) has been reported to exert antihyperlipidemic effects. The present study aimed to identify the active compounds of Coreopsis tinctoria and to investigate the molecular mechanisms underlying its effects on lipid dysregulation by measuring lipid levels, reactive oxygen species, lipid peroxidation and fatty acid synthesis. The present results demonstrated that snow chrysanthemum aqueous extracts significantly reduced serum lipid levels and oxidative stress in vivo. The main compounds that were isolated were identified as flavanomarein (compound 1) and eriodictyol 7‑O‑β‑D glucopyranoside (compound 2). Compounds 1 and 2 demonstrated potent antioxidative properties, including free radical scavenging activity, inhibition of lipid peroxidation, as well as lipid‑lowering effects in human HepG2 hepatocellular carcinoma cells treated with free fatty acids (FFAs). Compound 2 was revealed to suppress the elevation of triglyceride levels and inhibit lipid peroxidation following FFA treatment. In addition, it was demonstrated to significantly reduce intracellular levels of reactive oxygen species and improve the mitochondrial membrane potential and adenosine triphosphate levels, thus protecting mitochondrial function in FFA‑treated HepG2 cells. Furthermore, compound 2 markedly suppressed the protein expression levels of disulfide‑isomerase A3 precursor and fatty acid synthase, thus suppressing FFA‑induced lipogenesis in HepG2 cells. In conclusion, the present study identified compound 2 as one of the main active compounds in Coreopsis tinctoria responsible for its lipid‑lowering effects. Compound 2 was revealed to possess antihyperlipidemic properties, exerted via reducing oxidative stress, protecting mitochondrial function and suppressing lipogenesis.
... Coreopsis tinctoria is native to North America but now distributed worldwide, and is planted and widely cultivated in plateau regions (above 3000 m) in Xinjiang Uygur Autonomous Region of China in the past decades [3]. The major active components in C. tinctoria are flavonoids, especially marein and flavanomarein, which has been reported to possess the antioxidant, cytoprotective, antihypertensive, and anti-inflammatory effects [4][5][6][7]. Although there is a latest report revealing the contents of 11 components in capitulas of C. tinctoria, the chemical characteristics of different parts of C. tinctoria remain unknown and the understanding of their differences is helpful to their proper applications [4]. ...
Coreopsis tinctoria, also called 'snow chrysanthemum' in China, is a flower tea material that has been reported to possess excellent pharmacological properties such as antioxidant and anti-diabetic activities. The chemical characteristics of different parts (flowers, buds, seeds, stems and leaves) of Coreopsis tinctoria were investigated based on microwave-assisted extraction and the simultaneous determination of 13 major active compounds by high-performance liquid chromatography, including taxifolin-7-O-glucoside, chlorogenic acid, (R/S)-flavanomarein, isocoreopsin, quercetagetin-7-O-glucoside, isookanin, 5,7,3',5'-tetrahydroxyflavanone-7-O-glucoside, marein, 3,5-dicaffeoylquinic acid, coreopsin, okanin, 5,7,3',5'-tetrahydroxyflavanone, and N(1) ,N(5) ,N(10) ,N(14) -tetra-p-coumaroylspermine. Chemometric analysis based on the contents of investigated compounds from 13 samples showed that Coreopsis tinctoria and the related flower tea materials, Chrysanthemum morifolium cv 'Hangju' and 'Gongju', were in different clusters, and different parts (flowers, buds, seeds, stems and leaves) of Coreopsis tinctoria were obviously different. This study is helpful for the quality control and pharmacological evaluation of different parts from Coreopsis tinctoria and its related products. This article is protected by copyright. All rights reserved.
... have a significant amounts of bioactive components. 6,10,11 C. tinctoria is an annual forb widespread in Canada, Northeast Mexico, much of the United States, especially the Great Plains and Southern states, and is often called "calliopsis" by the native residents. 6,10 C. tinctoria plants attain heights of 30 to 100 cm. ...
Response surface methodology (RSM) was applied to predict optimum conditions for microwave-assisted extraction of total flavonoids extraction from Coreopsis tinctoria. A central composite design was used to monitor the effect of ratio of water to material, extraction time, microwave power on yield of total flavanoid. the optimal extraction conditions were obtained as ration of water to material of 70 mL/g, extraction time of 8 min and microwave power 590 W. under this conditions, the average total flavonoids yield was of 9.95% which matched with the predicted value of 9.99%. The extraction method was applied successfully to extract total flavonoids from Coreopsis tinctoria.
... have a significant amounts of bioactive components. 6,10,11 C. tinctoria is an annual forb widespread in Canada, Northeast Mexico, much of the United States, especially the Great Plains and Southern states, and is often called "calliopsis" by the native residents. 6,10 C. tinctoria plants attain heights of 30 to 100 cm. ...
... Moreover, in diabetic mitochondria t-BOOH generates ROS, which cause activation of mitochondrial anion carrier protein, known as uncoupling protein-2 (UCP2). This protein regulates beta-cell membrane potential, which results in pancreatic beta-cell disruption [69]. Due to beta cells damage level of insulin released is very low, therefore much attention has been paid to phenolics able to elevate insulin secretion. ...
Type 2 diabetes mellitus, which is usually a result of wrong dietary habits and reduced physical activity, represents 85-95% of all diabetes cases and among other diet related diseases is the major cause of deaths. The disease is characterized mainly by hyperglycemia, which is associated with attenuated insulin sensitivity or beta cells dysfunction caused by multiple stimuli, including oxidative stress and loss of insulin secretion. Since polyphenols possess multiple biological activities and constitute an important part of the human diet, they have recently emerged as critical phytochemicals in type 2 diabetes prevention and treatment. Their hypoglycemic action results from their antioxidative effect involved in recovering of altered antioxidant defenses and restoring insulin secreting machinery in pancreatic cells, or abilities to inhibit the activity of carbohydrates hydrolyzing enzymes (α-amylase and α-glucosidase) or protein tyrosine phosphatase 1B (PTP1B), which is known as the major negative regulator in insulin signaling. This study investigates the total phenolic content (Folin-Ciocalteu and HPLC methods) and antioxidant capacity (ABTS) of 20 polyphenolic extracts obtained from selected edible plants, which were screened in terms of α-amylase, α-glucosidase and protein tyrosine phosphatase 1B inhibitors or protective agents against oxidative stress induced by tert-butylhydroperoxide (t-BOOH) in βTC3 pancreatic beta cells used as a model target for antidiabetes drugs. The study concludes that Chaenomeles japonica, Oenothera paradoxa and Viburnum opulus may be promising natural sources for active compounds with antidiabetic properties.
... Pharmacological studies have shown that CT exhibits many beneficial biological activities, including antihyperlipidemic (16), antioxidative (17), antidiabetic (18), and antihypertensive (19) effects. The effects of CT are mainly attributed to its flavonoid-like polyphenols (20); however, the effects of drinking CT tea on insulin sensitivity and hepatic glucose metabolism are still unknown. ...
An infusion of Coreopsis tinctoria (CT) flowering tops is traditionally used in Portugal to control hyperglycemia; however, the effects of CT protection against high-fat diet (HFD)-induced hepatic insulin resistance have not been systematically studied and the precise mechanism of action is not clear. The metabolomic profiles of insulin-resistant rats fed a HFD and a CT-supplemented diet (HFD supplemented with CT-drinking) for 8 weeks were investigated. Serum samples for clinical biochemistry and liver samples for histopathology and liquid chromatography-mass spectrometry (LC-MS)-based metabolomic research were collected. Western blot and quantitative real-time PCR (qPCR) analyses were further employed to measure the expression of several relevant enzymes together with perturbed metabolic pathways. Using MetaboAnalyst 3.0 ( http://www.metaboanalyst.ca ), the CT treatment was found to significantly ameliorate the disturbance in 10 metabolic pathways. Combined metabolomic, western blot and qPCR analyses revealed that CT treatment significantly improved the glucose homeostasis by, on the one hand through inhibiting the expression of gluconeogenic pathway key proteins G6Pase and PEPCK and, on the other hand via regulating the mRNA or protein levels of the Krebs cycle critical enzymes (CS, SDHA, and DLST). These results provide metabolic evidence of the complex pathogenic mechanism involved in hepatic insulin resistance and that the supplementation with CT improves insulin resistance at a global scale. LC-MS-based metabolomics approaches are helpful to further understand diabetes-related mechanisms.
... Once inside the cell, DCFH-DA is cleaved by endogenous esterases and can no longer diffuse out of the cell. DCFH converts to the fluorescent compound 2 ,7 -dichlorofluorescein (DCF) upon oxidation by ROS (Dias et al., 2012). Briefly, 1 × 10 4 cells/well were seeded in a 96-well cell culture plate. ...
Dihydromyricetin (DMY), the major bioactive flavonoid ingredient extracted from the leaves of Ampelopsis grossedentata (Hand.-Mazz) W. T. Wang, displays multiple pharmacological activities, including oxidation resistance, antitumor properties and free radical scavenging capacities. However, the role of DMY in methylglyoxal (MG)-induced diabetes-associated cognitive decline and its underlying molecular mechanisms are unclear. The aim of the present study was to evaluate the effects of DMY on oxidative stress and glucose transport activity in a MG-induced PC12 cell line and to explore the related mechanisms. The effects of DMY on cell survival and apoptosis were examined, and the dysregulation of intracellular Ca2+ was determined. Oxidative stress was evaluated by monitoring ROS production and the glutathione to glutathione disulfide ratio. The effects of DMY on glucose metabolism were investigated using a fluorescently labeled deoxyglucose analog and by measuring ATP and lactate production. Western blot analysis was performed to examine the protein levels of glyoxalase I (Glo-1), glucose transporter 4 (GLUT4), AMP-activated protein kinase (AMPKα) and phosphorylated AMPKα (p-AMPKα). The results revealed that DMY suppressed cellular oxidative stressinPC12 cells and balanced glucose metabolism. Additionally, DMY reduced GLUT4 translocation dysfunction and increased Glo-1 and p-AMPKα expression.We found that DMY protected PC12 cells against MG-induced apoptosis and glycometabolic disorders, at least in part by restraining the hyperactivation of p-AMPK activity and normalizing the translocation of GLUT4 from the intracellular compartment, resulting in a balance in glucose uptake. This result indicates that DMY may serve as a novel and effective candidate agent to treat diabetic encephalopathy by reducing the toxicity of MG.
... It was recently found that it inhibited histone deacetylase enzymes and NF-kB, a protein complex that controls the transcription of DNA, in the 100 mM range [43]. It was also observed to have antihyperglycemic activity [44]. ...
Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the "drug of last resort". Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis.
... In situ ROS production levels were investigated based on flow cytometry according to a method previously used for mouse fibroblasts [21]. BES-H 2 O 2 -Ac (measurement wavelength; k ex = 488 nm, k em = 527 nm, Wako Pure Chemical Industries Ltd.) and dihydroethidium (measurement wavelength; k ex = 488 nm, k em = 586 nm, Life Technologies Corporation, Carlsbad, CA, USA) were used as specific fluorescence probes responding to ROS produced by the UV-A As a positive control for each oxidative stress, the bacterial suspension was instead treated (30 min, 30°C, in the dark) with 0.05% (v/v) hydrogen peroxide or 400 lM tert-butyl hydroperoxide (tBHP), compounds known to induce increased levels of hydrogen peroxide or superoxide [22], respectively. The level of ROS induced by oxidative processes was expressed as arbitrary units (a.u.) normalized to the mean fluorescence values of control samples that were incubated for 30 or 60 min in the absence of both UV-A light and 3DOBP-4,10. ...
Methanol extract from the capitula of Coreopsis tinctoria Nutt. (Asteraceae), which is also known as a flowering tea or blooming tea "Snow Chrysanthemum," was found to inhibit the enzymatic activity of aromatase. A total of 24 known isolates (1-24) were identified from the extract, including three chalcones (1-3), an aurone (4), five flavanones (5-9), four flavanols (10-13), a flavonol (14), and two biflavanones (15, 16). Among them, okanin (1, Ki = 1.6 μM), (2S)-naringenin (5, 0.90 μM), isookanin (6, 0.81 μM), (2S)-7,3',5'-trihydroxyflavaone (7, 0.13 μM), and (2S)-5,7,3',5'-tetrahydroxyflavanone (8, 0.32 μM) exhibited relatively potent competitive inhibition. Specifically, the isolates 7 and 8, having a common 3',5'-resorcinol moiety at the B ring in their flavanone skeleton, exhibited potent inhibitory activities compared to those of a clinically applied aminoglutethimide (0.84 μM) and naturally occurring flavone, chrysin (0.23 μM), which is a common non-steroidal aromatase inhibitor. Importantly, the active flavonoid constituents (1 and 5-8) did not inhibit the activity of 5α-reductase enzyme, which normally reacts with the same substrate "testosterone," thus, these compounds were suggested to be specific to aromatase.
Eleven polyphenols, classified as flavonoid glycosides, flavonoid aglycones, and phenolic acids, are important bioactive components in the capitula of Coreopsis tinctoria (CCT). Nevertheless, their full pharmacokinetic profiles have not been demonstrated simultaneously. Therefore, a liquid chromatography ‐ tandem mass spectrometry (LC‐MS/MS) method was developed in the present work and used it to study the pharmacokinetics of these 11 compounds. We performed LC‐MS/MS with a gradient mobile phase composed of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid on a Proshell 120 SB C18 column (2.1 mm × 100 mm, 2.7 μm). We achieved a good chromatographic peak shape, resolution, and mass signal response, and multiple reaction monitoring facilitated the simultaneous detection of 11 analytes. In addition, we validated the selectivity, correlation coefficient, precision, extraction recovery, matrix effects, and stability of the LC‐MS/MS method to be acceptable for 11 analytes in rat plasma. Subsequently, rats were orally administered with 50% ethanol eluent of CCT (ECCT). Nine of 11 polyphenols were absorbed quickly (except for QCD and TCA), and their plasma levels peaked within 40 min. The exposure and Cmax values of flavonoid glycosides and phenolic acids were lower than those of flavonoid aglycones. This is the first report to demonstrate the pharmacokinetics of 11 polyphenols in ECCT, which may play an important role in future studies of the bioactive components of ECCT and their bioactive mechanisms.
The objectives of this study were to investigate the antithrombotic effect and physiological mechanism of okanin, a flavonoid monomer in Coreopsis tinctoria Nutt. The antithrombotic effects of okanin were determined by the anticoagulant activity test in vitro and in vivo, the venous thrombosis and arterial thrombosis test in rats. To study the antithrombotic physiological mechanisms of okanin, UV spectrophotometer and enzyme-linked immunosorbent assay (ELISA) were used to determine the effects of three concentrations of okanin on the contents of 6-keto-prostaglandin F1α (6-Keto-PGF1α), thromboxane B2 (TXB2), endothelin-1 (ET-1), antithrombin III (AT-Ⅲ), protein C (PC) and von willebrand factor (vWF) in the plasma of rats with arterial thrombosis; ELISA was used to detect the effects of okanin on the contents of plasminogen (PLG), tissue plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in the plasma of mice and Chinese white rabbits. The results showed that okanin could prolong the coagulation time in vitro and in vivo of animals (P
Coreopsis tinctoria Nutt. ( C. tinctoria ) is a special tea ingredient that adapts to certain salt stresses and shares the functions of chrysanthemum. With annual expansion of the cultivation area of C. tinctoria in Xinjiang (China), soil salinity may become a constraint for chrysanthemum cultivation. To investigate the response of C. tinctoria to salt stress, physiological and transcriptional changes in C. tinctoria in the early stages of low (50 mM NaCl) and high (200 mM NaCl) salt stress were analyzed and identified. The results showed that the contents of osmotic regulators (free proline, soluble sugar, and soluble protein) and antioxidant enzymes (catalase and peroxidase) under salt stress increased to various extents compared with those of the control (CK) within 72 h, and the increase was higher under 200 mM NaCl treatments. De novo RNA-seq was used to analyze changes in the transcripts under 50 and 200 mM NaCl treatments for up to 48 h. In total, 8,584, 3,760, 7,833, 19,341, 13,233, and 9,224 differentially expressed genes (DEGs) were detected under 12 h, 24 h, and 48 h for 50 and 200 mM NaCl treatments, respectively. Weighted correlation network analysis (WGCNA) was used to analyze the correlations between all DEGs and physiological indexes. We found that the coexpression modules blue2 and Lightskyblue4 highly correlated with osmotic regulators and CAT and identified 20 and 30 hub genes, respectively. The results provide useful data for the further study of salt tolerance in C. tinctoria .
Introduction
Development of therapy options for treatment of type 1 diabetes mellitus is hampered by non-availability of appropriate experimental models that can exactly mimic the in vivo situation. Apoptosis of beta cells by T cells and cytokine action leads to loss of beta cells. We propose a simple and elegant model using cytokine cocktail of TNF-α, IFN-γ and IL-1β, the major cytokines responsible for apoptosis in Min6 beta cell line.
Methods
A cocktail of TNF-α, IFN-γ and IL-1β was used to induce apoptosis in Min6 beta cell line. Apoptosis was assessed by flow cytometry using CytoFLEX (Beckman Coulter). The destruction of beta cells is through production of nitric oxide (NO), oxidative stress and change in mitochondrial membrane permeability. NO was measured using Griess reagent. Oxidative stress was assessed using 2,7-dichloroflurescin diacetate, a cell-permeable fluorogenic dye and mitochondrial membrane potential was determined on the basis of retention of rhodamine 123 using flow cytometer.
Results and discussion
Very low concentration of the cocktail viz. TNF-α 25 ng/ml, IFN-γ 25 ng/ml and IL-1β 50 ng/ml has demonstrated effective early and late apoptosis in as short a time period as 6 h. The experimental model used demonstrated 1.5 fold higher production of NO, 1.2 fold increased oxidative stress and lower mitochondrial membrane potential as compared to the positive control used. Hence the above model can be easily used for assessment and screening of drugs that can prevent apoptosis of beta cells and stop progression of type 1 diabetes.
Phytochemical investigation on the water-soluable portions of the EtOH extracts of the flowers of Coreopsis tinctoria Nutt. led to the isolation of six flavonoids, including a new okanin glycoside. The structures of these compounds were identified by spectroscopic analysis, chemical method, and comparison with the literature.
Introduction:Coreopsis tinctoria Nutt. is an annual plant with small flowers in yellow and claret. In dyeing process, a whole range of colours can be obtained using various methods.
Objective: The aim of the study was to present a wide range of colors of the little-known plant C. tinctoria . and its health promoting properties.
Methods: In our research, we selected 3 types of wool: Polish Merino, Żelaźnieńska, and Polish Lowland Sheep and compared the colours obtained on these wools using 6 dyeing methods.
Results: The results indicate that the basic colour of wool influences the intensity of colour after dyeing as well as the type of the used mordant, which determines the obtained colour. A whole range of very intense colours was obtained from very small flowers of C. tinctoria .
Conclusions: Flowers are a very good and efficient raw material that gives intense colors on wool. An additional advantage is the plant's health-promoting properties. The plant is still little explored in this respect.
In order to explore snow chrysanthemum polysaccharides (SCPs) as functional food ingredients and natural antioxidants for industrial applications, both microwave-assisted extraction (MAE) and ultrasonic-assisted extraction (UAE) were firstly optimized for the extraction of SCPs. Furthermore, the effects of conventional hot water extraction, UAE, and MAE on the chemical structures and antioxidant activities of SCPs were investigated. The maximum extraction yields of SCPs extracted by UAE (4.13 ± 0.24%) and MAE (4.26 ± 0.21%) were achieved at the optimized extraction parameters as follows: ultrasound amplitude (68%) and microwave power (500 W), ultrasound extraction time (21 min) and microwave extraction time (6.5 min), and ratio of liquid to raw material (42.0 mL/g for UAE and 59.0 mL/g for MAE). In addition, different extraction methods significantly affected the contents of uronic acids, the molecular weights, the molar ratio of constituent monosaccharides, and the degree of esterification of SCPs. SCPs exhibited remarkable DPPH (IC50 ≤ 1.702 mg/mL), ABTS (IC50 ≤ 1.121 mg/mL), and nitric oxide (IC50 ≤ 0.277 mg/mL) radical scavenging activities, as well as reducing power (≥ 80.17 ± 4.8 μg Trolox/mg), which suggested that SCPs might be one of the major contributors toward the antioxidant activities of snow chrysanthemum tea. The high antioxidant activities (DPPH, IC50 = 0.693 mg/mL; ABTS, IC50 = 0.299 mg/mL; nitric oxide, IC50 = 0.105 mg/mL; and reducing power, 127.79 ± 2.57 μg Trolox/mg) observed in SCP-M extracted by the MAE method might be partially attributed to its low molecular weight and high content of unmethylated galacturonic acids. Results suggested that the MAE method could be an efficient technique for the extraction of SCPs with high antioxidant activity, and SCPs could be further explored as natural antioxidants for industrial application.
Non-Camellia tea and herbal medicine help prevent the development of diabetes and other metabolic diseases. Previous studies found that Coreopsis tinctoria (CT) flower tea increases insulin sensitivity and, in some high-fat diet (HFD)-fed rats, even prevents hepatic metabolic disorders. However, the molecular mechanisms by which CT improves insulin resistance are unknown. In this study, six-week-old rats were fed a normal diet (ND), HFD or HFD supplemented with CT for 8 weeks. Serum samples were collected, and the liver was extracted for RNA-seq gene expression analysis. Real-time PCR and Western blotting further verified the RNA-seq results. In our results, dietary CT ameliorated HFD-induced hepatosteatosis, glucose intolerance, and insulin resistance. In the HFD group, 1667 differentially expressed genes (DEGs) were identified compared with the ND group. In the CT group, 327 DEGs were identified compared with the HFD group. Some of these DEGs were related to insulin signalling, hepatic lipogenesis and glucose homeostasis. This study suggested that insulin resistance with hyperinsulinaemia, not insulin insufficiency, is an early problem in HFD-fed rats, and CT downregulates insulin secretion genes (e.g., Rasd1, Stxbp1 and Sfxn1). Hepatic gene and protein expression analyses indicated that the regulatory effects of CT on glucose and lipid homeostasis are likely mediated via the Akt/FoxO1 signalling pathway and regulated by the transcription factors HES1 and SHP. Our study provides transcriptomic evidence of the complex pathogenic mechanism involved in hepatic insulin resistance and that supplementation with CT improves insulin resistance at a global scale.
Background
Coreopsis tinctoria is a traditional remedy for the management of various diseases including hepatitis. The hepatoprotective role of the plant is not scientifically explored till now. This study was designed to investigate the hepatoprotective potentials of the ethanol extract from C. tinctoria (CTEtOH) using an animal model of carbon tetrachloride (CCl4)-induced acute liver injury. MethodsCTEtOH (0.5 and 1.0 g/kg) and silymarin (200 mg/kg) were administered to the experimental mice for 7 days followed by 0.2% CCl4 (10 mL/kg of body weight (bw), ip), then all mice were sacrificed after 24 h. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured. Histological analysis of liver was performed. The tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), nitric oxide (NO), malondialdehyde (MDA), and antioxidant enzymatic activities were also measured.. ResultsThe results revealed that the serum ALT and AST levels significantly decreased after treatment with CTEtOH. Moreover, histological analyses indicated that CTEtOH (0.5 and 1.0 g/kg) and silymarin reduced the extent of CCl4-induced liver lesions. CTEtOH (0.5 and 1.0 g/kg) reduced the levels of malondialdehyde, nitric oxide, and proinflammatory cytokines (TNF-α and IL-1β). Furthermore, CTEtOH (1.0 g/kg) reduced the level of IL-6. The activities of antioxidant enzymes, namely superoxide dismutase and glutathione reductase, significantly increased after treatment with CTEtOH (0.5 and 1.0 g/kg) and that of glutathione peroxidase increased after treatment with 1.0 g/kg of CTEtOH. Conclusions
These results demonstrate the hepatoprotective effect of CTEtOH against CCl4-induced acute liver injury in mice, and the underlying hepatoprotective mechanisms are associated with antioxidant and antiproinflammatory activities.
Marein, flavanomarein and taxifolin-7-O-?-D-glucopyranoside (TDG) are functional compounds in capitula of Coreopsis tinctoria (CCT). This study demonstrated the above glycosides could be partially deglycosylated to okanin, isookanin and taxifolin, respectively, in cell free extracts from rat and human small intestine, human colon, but not from stomach and cecum. Okanin and isookanin mainly occurred as glucuronide conjugates; whereas taxifolin was in sulfate in rat plasma. The exposures of the glycosides and aglycones were positively correlated with the dosages of CCT extract in rats after oral administration. The aglycones but not glycosides were detected in the plasma of healthy volunteers after intake of CCT (2.5?g/50?kg) water solution. In conclusion, marein, flavanomarein and TDG could be deglycosylated to respective aglycones in gastrointestinal tracts, which could be absorbed and biotransformed to glucuronide or sulfate conjugates. The results will help us to better understand the function and functional components of CCT in vivo.
A simple and efficient method based on high-performance thin-layer chromatography coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH) bioautography (HPTLC-DPPH) was established for the screening and comparison of antioxidants in different parts of Coreopsis tinctoria herbal tea from different origins and other related herbal tea materials, which used Chrysanthemum morifolium cv. "Gongju" and "Hangju" in this study. Scanning densitometry after DPPH derivatization was applied for the determination of antioxidant capacities of isolated compounds in each sample. It is considered that ethanol extracts of C. tinctoria had stronger antioxidant activity and more characteristic bands than those of 2 compared samples, C. morifolium cv. "Gongju" and "Hangju." Chemometric analysis results showed that the combination of hierarchical clustering analysis and principal component analysis based on determined antioxidant capacities could be used for the discrimination of different parts of C. tinctoria and C. morifolium. Results showed that 7 compounds made up the major contributions of antioxidant activity in C. tinctoria, including okanin, isookanin, marein, flavanomarein, 5,7,3′,5′-tetrahydroxyflavanone-7-O-glucoside, 3,5-dicaffeoylquinic acid, and chlorogenic acid. Therefore, 7 compounds were identified as major antioxidant biomarkers for quality control of C. tinctoria. Results demonstrated that the established method could be applied for the identification of C. tinctoria, and were beneficial for the bioactivity-based quality control of C. tinctoria.
Ethnopharmacological relevance:
Coreopsis tinctoria Nutt mainly distributed in Hetian region of Xinjiang at an altitude of 3000 m, which is used as Uyghur traditional medicine because of its clearing heat, promoting circulation and removing toxicity and antihypertension, ect. effect.
Aim of the study:
This research was to study the four ingredients in the extracts of Coreopsis tinctoria Nutt. that are absorbed in different intestinal segments in rats to lay the foundation for further study on the effective constituents, tissue distribution, metabolism, and spectrum-effect relationships of these extracts.
Materials and methods:
High, medium, and low concentrations were prepared according to their pharmacological effects. Quantitative analysis multi-components by single marker was used to test the cumulative absorption volume Q, absorption rate constant Ka, and apparent permeability coefficient Papp of the four main ingredients in C. tinctoriaNutt. extract in different intestinal segments in rats using a Ussing chamber model and high-performance liquid chromatography.
Results:
The Papp of chorogenic acid and flavanomarein in the duodenum, jejunum, ileum, and colon were 1.0×10(-6) to 10×10(-6)cm·s(-1). Papp of marein in the duodenum and jejunum was <1.0×10(-6), and was 1.0×10(-6) to 10×10(-6)cm·s(-1) in the ileum and colon. Papp of 3,5-O-dicaffeoylquinic acid in the duodenum was <1.0×10(-6)cm·s(-1), while it was 1.0×(1)0(-6) to 10×10(-6) cm·s(-1) in the jejunum, ileum, and colon.
Conclusions:
All four chemical components of the plant extract can be absorbed by the intestinal canal of rats, which conforms to zero-order absorption; the ileum presented the best absorption.
Based on characteristic UV spectrum of the ene-diyne chromophore, one new polyacetylene glucoside and three known polyacetylene glucosides have been isolated from the EtOH extract of Coreopsis tinctoria. Their chemical structures were determined by detailed spectroscopic analysis and by comparison with literature data. Compounds 1-2 were tested for their antiadipogenic effects on 3T3-L1 adipocytes, and both of them reduced lipid accumulation dose-dependently in 3T3-L1 adipocytes.
Snow chrysanthemum (Coreopsis tinctoria Nutt.), a world-widely well-known flower tea material, has attracted more and more attention because of its beneficial health effects such as antioxidant activity and special flavor. In this study, a high performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) and 2,2'-azinobis(3-ethylbenzthiazoline-sulfonic acid)diammonium salt (ABTS) based assay was employed for comparison and identification of antioxidants in different samples of snow chrysanthemum. The results showed that snow chrysanthemum flowers possessed the highest while stems presented the lowest antioxidant capacities. Fourteen detected peaks with antioxidant activity were temporarily identified as 3,4',5,6,7-pentahydroxyflavanone-O-hexoside, chlorogenic acid, 2R-3',4',8-trihydroxyflavanone-7-O-glucoside, flavanomarein, flavanocorepsin, flavanokanin, quercetagitin-7-O-glucoside, 3',5,5',7-tetrahydroxyflavanone-O-hexoside, marein, maritimein, 1,3-dicaffeoylquinic acid, coreopsin, okanin and acetyl-marein by comparing their UV spectra, retention times and MS data with standards or literature data. Antioxidants existed in snow chrysanthemum are quite different from those reported in Chrysanthemum morifolium, a well-known traditional beverage in China, which indicated that snow chrysanthemum may be a promising herbal tea material with obvious antioxidant activity.
The aim of the present study was to determine the antioxidant activity of Coreopsis tinctoria flowering tops (CTFT). Studies were conducted to obtain suitable extraction conditions for chlorogenic acid, quercetin, luteolin, apigenin and kaempferol, which were identified and quantified by HPLC. Response surface methodology was employed to optimise the ultrasound-assisted extraction conditions including extraction time, ethanol concentration and liquid-solid ratio. The antioxidant activity of the extracts was analysed using various antioxidant models, such as DPPH, ABTS and hydroxyl radical scavenging assay. CTFT extracted for 15.0 min with ethanol at a concentration of 60.4 % and with liquid-solid ratio 27.5:1 possessed a considerable amounts of total flavonoids and polyphenols (18.9 %). This extract showed higher scavenging activity of ABTS and hydroxyl radical activity than rutin, however not in the DPPH test. We may assume that CTFT possess antioxidant and free radical scavenging potentials.
Coreopsis tinctoria Nutt. from Mt. Kunlun (located in Tibet, Xinjiang) is a new Chrysanthemum tea material, and its volatile components are reported here for the first time. C. tinctoria produces a greater quantity and better quality of volatile components compared with two other Chrysanthemum tea materials (Chrysanthemum morifolium cv. ‘Hangju’ and Chrysanthemum morifolium cv. ‘Gongju’). The volatile oil content was 0.42%, with eighty-one components. The major components included six monoterpenes (limonene, 11.308%; α-phellandrene, 3.884%; p-acetyltoluene, 2.761%; carvone, 2.666%; 1R-α-pinene, 2.656%; and α-campholenal, 2.483%), three sesquiterpenes (α-bergamotene, 7.314%; tricyclo[6.3.0.0(1,5)]undec-2-ene-4-one,2,3,5,9-tetramethyl-, 3.454%; and naphthalene, 1,2-dihydro-1,1,6-trimethyl-, 3.024%), and a fatty acid component (n-hexadecanoic acid, 2.713%). Antioxidant activity study was revealed that the volatile oil of C. tinctoria had the better activity than the two compared samples. With next further study, C. tinctoria could be a more promising medicinal raw material than other popular chrysanthemum tea materials.
The aim of this research was to investigate the chemical composition of Coreopsis tinctoria Nutt. fruits extract, to highlight the potential of ultrasound assisted extraction in the fast preparation of extracts rich in polyphenols using different solvents (55%, 78% and 96% hydrous ethanol) and to evaluate the antioxidant potential of formulated extracts. LC-MS/MS was used to characterize the ethanolic extract from Coreopsis tinctoria Nutt. dried fruits. The extract contains different flavonoids (marein, flavanomarein, quercetagetin-7-O-glucoside, okanin aurone, leptosidin, luteolin, apigenin) and phenolic acids (chlorogenic acid, caffeic acid). Several parameters that could affect extraction efficiency were evaluated. Finally, this study focused on determination of plant extracts total phenolic content and their antioxidant capacity. The experimental results allowed the selection of the optimum operating parameters leading to the highest total polyphenolic content, expressed as gallic acid equivalents, and avoiding the degradation of phenolic compounds (ethanol 55%; extraction temperature 323.15 K, extraction time 30 min, liquid/solid ratio 20/1). A good relationship between total phenolic content and antioxidant capacity was obtained.
Insulin and glucose inhibited apoptosis in the MIN6 insulin-secreting cell line. The protective effect of 25mM glucose was prevented by an anti-insulin antibody and this antibody-induced increase in apoptosis was reversed by the presence of excess insulin. Glucose stimulated MIN6 cell proliferation and this was inhibited by blockade of insulin secretion, by an anti-insulin antibody and by phosphatidylinositol-3 kinase (PI-3K) inhibition. Furthermore, MIN6 cell proliferation was stimulated by depolarising concentrations of KCl and by insulin itself. These data indicate that insulin secreted by β-cells in response to elevated glucose exerts autocrine effects to protect against apoptosis and stimulate proliferation, and suggest that the insulin signalling cascade, through the PI-3K pathway, may be an effective means of maintaining β-cell mass in diabetes.
Cytokines such as IL-1alpha, IL-1beta, and IFN-gamma have long been implicated in the pathogenesis of autoimmune diabetes, but the mechanisms through which they promote diabetogenesis remain unclear. Here we show that CD4(+) T lymphocytes propagated from transgenic nonobese diabetic (NOD) mice expressing the highly diabetogenic, beta cell-specific 4.1-T-cell receptor (4.1-TCR) can kill IL-1alpha-, IL-1beta-, and IFN-gamma-treated beta cells from NOD mice. Untreated NOD beta cells and cytokine-treated beta cells from Fas-deficient NOD.lpr mice are not targeted by these T cells. Killing of islet cells in vitro was associated with cytokine-induced upregulation of Fas on islet cells and was independent of MHC class II expression. Abrogation of Fas expression in 4.1-TCR-transgenic NOD mice afforded nearly complete protection from diabetes and did not interfere with the development of the transgenic CD4(+) T cells or with their ability to cause insulitis. In contrast, abrogation of perforin expression did not affect beta cell-specific cytotoxicity or the diabetogenic potential of these T cells. These data demonstrate a novel mechanism of action of IL-1alpha, IL-1beta, and IFN-gamma in autoimmune diabetes, whereby these cytokines mark beta cells for Fas-dependent lysis by autoreactive CD4(+) T cells.
The current model of apoptosis holds that upstream signals lead to activation of downstream effector caspases. We generated mice deficient in the two effectors, caspase 3 and caspase 7, which died immediately after birth with defects in cardiac development. Fibroblasts lacking both enzymes were highly resistant to both mitochondrial and death receptor-mediated apoptosis, displayed preservation of mitochondrial membrane potential, and had defective nuclear translocation of apoptosis-inducing factor (AIF). Furthermore, the early apoptotic events of Bax translocation and cytochrome c release were also delayed. We conclude that caspases 3 and 7 are critical mediators of mitochondrial events of apoptosis.
Oxidative stress and/or low cellular glutathione (GSH) levels are associated with the development and progression of numerous pathological conditions. Cells possess various antioxidant protection mechanisms, including GSH and phase II detoxifying enzymes. N-acetylcysteine (NAC) supplies cells with cysteine to increase GSH level but its efficacy is relatively low because of its limited tissue penetration. Allicin (diallyl thiosulfinate), a reactive sulfaorganic compound, increases cellular GSH and phase II detoxifying enzymes in vascular endothelial cells (EC). A novel compound was designed: S-allylmercapto-N-acetylcysteine (ASSNAC), a conjugate of S-allyl mercaptan (a component of allicin) and NAC. Both ASSNAC and NAC increased cellular GSH of ECs, reaching a maximum of up to four- and threefold increase after exposure for 24 or 6 h at a concentration of 0.2 or 1 mM, respectively. ASSNAC induced nuclear translocation of the activated transcription factor Nrf2 and expression of phase II detoxifying enzymes. EC exposure to tBuOOH resulted in 75% cytotoxicity, and pretreatment of cultures with 0.2 mM ASSNAC or 2mM NAC reduced cytotoxicity to 20 and 42%, respectively. In conclusion, ASSNAC is superior to NAC in protecting cells from oxidative stress because of its ability to up-regulate both GSH and the expression of phase II detoxifying enzymes.
NF-kappaB activation has been implicated as a key signaling mechanism for pancreatic beta-cell damage. Sulfuretin is one of the main flavonoids produced by Rhus verniciflua, which is reported to inhibit the inflammatory response by suppressing the NF-kappaB pathway. Therefore, we isolated sulfuretin from Rhus verniciflua and evaluated if sulfuretin could inhibit cytokine- or streptozotocin-induced beta-cell damage. Rat insulinoma RINm5F cells and isolated rat islets were treated with IL-1 beta and IFN-gamma to induce cytotoxicity. Incubation of cells and islets with sulfuretin resulted in a significant reduction of cytokine-induced NF-gamma B activation and its downstream events, iNOS expression, and nitric oxide production. The cytotoxic effects of cytokines were completely abolished when cells or islets were pretreated with sulfuretin. The protective effect of sulfuretin was further demonstrated by normal insulin secretion of cytokine-treated islets in response to glucose. Treatment of mice with streptozotocin resulted in hyperglycemia and hypoinsulinemia, which was further evidenced by immunohistochemical staining of islets. However, the diabetogenic effects of streptozotocin were completely prevented when mice were pretreated with sulfuretin. The anti-diabetogenic effects of sulfuretin were also mediated by suppression of NF-kappaB activation. Collectively, these results indicate that sulfuretin may have therapeutic value in preventing beta-cell damage.
A surplus of food supply has evoked a worldwide increase in incidence of type 2 diabetes. This trend will have a significant impact on the life span of people living in modern societies. In contrast, reduced calorie intake has significant impact on preventing type 2 diabetes and increasing longevity. Increased production of reactive oxygen species (ROS), resulting in oxidative stress, has long been proposed as a unifying mechanism linking nutrient excess and diabetes. This review describes the updated mechanism by which oxidative stress provoked by nutrient excess contributes to the development of insulin resistance and pancreatic betacell failure. However, despite the promising results in cellular and animal models, major clinical trials have failed to demonstrate beneficial effect of antioxidants on the prevention of type 2 diabetes or the degree of glycemic control in individuals with diabetes. Emerging evidence shows that ROS also function as an insulin-signaling molecule in normal physiology and casts doubt on the potential beneficial effect of antioxidants. The gap between basic research and clinical outcomes heightens the importance for elucidating the precise molecular mechanisms by which cellular redox status affects insulin signaling.
Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. However, studies on whether genistein has an effect on pancreatic beta-cell function are very limited. In the present study, we investigated the effect of genistein on beta-cell proliferation and cellular signaling related to this effect and further determined its antidiabetic potential in insulin-deficient diabetic mice. Genistein induced both INS1 and human islet beta-cell proliferation after 24 h of incubation, with 5 mum genistein inducing a maximal 27% increase. The effect of genistein on beta-cell proliferation was neither dependent on estrogen receptors nor shared by 17beta-estradiol or a host of structurally related flavonoid compounds. Pharmacological or molecular intervention of protein kinase A (PKA) or ERK1/2 completely abolished genistein-stimulated beta-cell proliferation, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein induced cAMP/PKA signaling and subsequent phosphorylation of ERK1/2 in both INS1 cells and human islets. Furthermore, genistein induced protein expression of cyclin D1, a major cell-cycle regulator essential for beta-cell growth. Dietary intake of genistein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in streptozotocin-induced diabetic mice, concomitant with improved islet beta-cell proliferation, survival, and mass. These results demonstrate that genistein may be a natural antidiabetic agent by directly modulating pancreatic beta-cell function via activation of the cAMP/PKA-dependent ERK1/2 signaling pathway.
R-spondin-1 (Rspo1) is an intestinal growth factor known to exert its effects through activation of the canonical Wnt (cWnt) signaling pathway and subsequent expression of cWnt target genes. We have detected Rspo1 mRNA in murine islets and the murine MIN6 and betaTC beta-cell lines, and Rspo1 protein in MIN6 beta-cells. Rspo1 activated cWnt signaling in MIN6 beta-cells by increasing nuclear beta-catenin and c-myc, a cWnt target gene. Rspo1 also induced insulin mRNA expression in MIN6 cells. Analysis of MIN6 and mouse beta-cell proliferation by [(3)H]thymidine and BrdU incorporation, respectively, revealed that Rspo1 stimulated cell growth. Incubation of MIN6 and mouse beta-cells with cytokines (IL1beta/TNFalpha/interferon-gamma) significantly increased cellular apoptosis; this increase was abolished by pretreatment with Rspo1. Rspo1 also stimulated insulin secretion in a glucose-independent fashion. We further demonstrated that the glucagon-like peptide-1 receptor agonist, exendin4 (EX4), stimulated Rspo1 mRNA transcript levels in MIN6 cells in a glucose-, time-, dose-, and PI3-kinase-dependent fashion. This effect was not limited to this beta-cell line, as similar time-dependent increases in Rspo1 were also observed in the betaTC beta-cell line and mouse islets in response to EX4 treatment. Together, these studies demonstrate that Rspo1 is a novel beta-cell growth factor and insulin secretagogue that is regulated by EX4. These findings suggest that Rspo1 and the cWnt signaling pathway may serve as a novel target to enhance beta-cell growth and function in patients with type 2 diabetes.
Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells.
Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis factor-alpha). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively.
We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.
Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.
Oxidative cell injury could be induced by different reactive oxygen species (ROS) operating in multiple pathways. The present work is focused on three different models of oxidative stress: the xanthine/xanthine oxidase system (XXO), an extracellular superoxide anion generator; tert-butylhydroperoxide (TBHP), an analogue of lipid hydroperoxides; and doxorubicin (Dox), an anticancer drug. Superoxide and peroxyl radicals, among other ROS, could be effectively scavenged by MnTM-4-PyP, a polyfunctional catalytic antioxidant. In this report, we have addressed the role of MnTM-4-PyP on the protection against the cytotoxicity induced by the three aforementioned oxidants. The effect of MnTM-4-PyP (0.1-100 microM) was evaluated in V79 fibroblasts using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction and the crystal violet assays, as well as the mitotic index. Also, the generation of intracellular ROS was studied by the fluorescent probe dihydroethidium. MnTM-4-PyP has shown significant protective effects against the cytotoxicity of XXO and TBHP, increasing the cell viability in approximately 40% and reducing the intracellular level of ROS. However, no considerable protection occurred against Dox. The three oxidants caused a mitotic index reduction that was not altered by MnTM-4-PyP. In summary, MnTM-4-PyP appears to be a promising agent for the protection against oxidative injury. However, it has shown differential responses, reinforcing the need to study different experimental models for the adequate evaluation of its potentialities as a catalytic antioxidant.
In the present work, we showed that a chalcone-enriched fraction (CEF) isolated from the stem bark of a Brazilian medicinal plant, Myracrodruon
urundeuva, presents neuroprotective actions on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death, in rat mesencephalic cells. In the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assay, which is an index of cell viability, CEF (1–100 μg/ml) reversed in a concentration-dependent manner the 6-OHDA-induced cell death. While cells exposed to 6-OHDA (40 μM) showed an increased concentration of thiobarbituric acid reactive substances (TBARS), the pretreatment with CEF (10–100 μg/ml) significantly decreased the 6-OHDA-induced TBARS formation, indicative of a neuroprotection against lipoperoxidation. Furthermore, the drastic increase of nitrite levels induced by 6-OHDA, indicative of nitric oxide formation and free radicals production, was prevented by CEF. Double staining with acridine orange/ethidium bromide showed that cultures exposed to 6-OHDA (40 and 200 μM) presented an increase of apoptotic and necrotic cell numbers in a concentration-dependent manner. CEF (100 μg/ml) protected cells from apoptosis and necrosis and increased number of cells presenting a normal morphology. The immunohistochemical analysis for tyrosine hydroxylase (TH) positive neurons indicated that 6-OHDA (40 and 200 μM) caused a concentration-dependent loss of TH+ and TH− neurons. CEF protected both cells types from 6-OHDA-induced cell death. All together, our results demonstrated neuroprotective effects of chalcones, which are able to reduce oxidative stress and apoptotic injury caused by 6-OHDA. Our findings suggest that chalcones could provide benefits, along with other therapies, in neurodegenerative injuries, such as Parkinson’s disease.
Nitric oxide has recently been implicated as the effector molecule that mediates IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and beta-cell specific destruction. The pancreatic islet represents a heterogeneous cell population containing both endocrine cells (beta-[insulin], alpha-]glucagon], gamma[somatostatin], and PP-[polypeptide] secreting cells) and non-endocrine cells (fibroblast, macrophage, endothelial, and dendritic cells). The purpose of this investigation was to determine if the beta-cell, which is selectively destroyed during insulin-dependent diabetes mellitus, is both a source of IL-1 beta-induced nitric oxide production and also a site of action of this free radical. Pretreatment of beta-cells, purified by FACS with IL-1 beta results in a 40% inhibition of glucose-stimulated insulin secretion that is prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (NMMA). IL-1 beta induces the formation of nitric oxide by purified beta-cells as evidenced by the accumulation of cGMP, which is blocked by NMMA. IL-1 beta also induces the accumulation of cGMP by the insulinoma cell line Rin-m5F, and both NMMA as well as the protein synthesis inhibitor cycloheximide prevent this cGMP accumulation. Iron-sulfur proteins appear to be intracellular targets of nitric oxide. IL-1 beta induces the formation of an iron-dinitrosyl complex by Rin-m5F cells indicating that nitric oxide mediates the destruction of iron-sulfur clusters of iron containing enzymes. This is further demonstrated by IL-1 beta-induced inhibition of glucose oxidation by purified beta-cells, mitochondrial aconitase activity of dispersed islet cells, and mitochondrial aconitase activity of Rin-m5F cells, all of which are prevented by NMMA. IL-1 beta does not appear to affect FACS-purified alpha-cell metabolic activity or intracellular cGMP levels, suggesting that IL-1 beta does not exert any effect on alpha-cells. These results demonstrate that the islet beta-cell is a source of IL-1 beta-induced nitric oxide production, and that beta-cell mitochondrial iron-sulfur containing enzymes are one site of action of nitric oxide.
The mechanisms of cytokine-induced beta-cell death are poorly characterised. In rat insulin-producing RINm5F cells, the combination of interleukin-1beta, interferon-gamma and tumour necrosis factor-alpha presently induced disruption of the mitochondrial membrane potential (Deltapsi(m)) as demonstrated by reduced JC-1 fluorescence. The reduction of Deltapsi(m) was maximal after 8 h and was preceded by increased formation of reactive oxygen species (ROS), as assessed by dichlorofluorescein-diacetate (DCFH-DA) fluorescence. A nitric oxide synthase-, but not a ROS-inhibitor, prevented cytokine-induced loss of Deltapsi(m). Overexpression of the anti-apoptotic protein Bcl-2 increased both JC-1 and DCFH-DA fluorescence, which was paralleled by protection against cytokine-induced apoptosis and necrosis. It is concluded that cytokines induce a nitric oxide-dependent disruption of Deltapsi(m) and that this may be a necessary event for both beta-cell apoptosis and necrosis. Bcl-2 may prevent beta-cell death by counteracting mitochondrial permeability transition.
In rat pancreatic islets chronically exposed to high glucose or high free fatty acid (FFA) levels, glucose-induced insulin release and mitochondrial glucose oxidation are impaired. These abnormalities are associated with high basal ATP levels but a decreased glucose-induced ATP production (Delta of increment over baseline 0.7 +/- 0.5 or 0.5 +/- 0.3 pmol/islet in islets exposed to glucose or FFA vs. 12.0 +/- 0.6 in control islets, n = 3; P < 0.01) and, as a consequence, with an altered ATP/ADP ratio. To investigate further the mechanism of the impaired ATP formation, we measured in rat pancreatic islets glucose-stimulated pyruvate dehydrogenase (PDH) activity, a key enzyme for pyruvate metabolism and for the subsequent glucose oxidation through the Krebs cycle, and also the uncoupling protein-2 (UCP-2) content by Western blot. In islets exposed to high glucose or FFA, glucose-stimulated PDH activity was impaired and UCP-2 was overexpressed. Because UCP-2 expression is modulated by a peroxisome proliferator- activated receptor (PPAR)-dependent pathway, we measured PPAR-gamma contents by Western blot and the effects of a PPAR-gamma antagonist. PPAR-gamma levels were overexpressed in islets cultured with high FFA levels but unaffected in islets exposed to high glucose. In islets exposed to high FFA concentration, a PPAR-gamma antagonist was able to prevent UCP-2 overexpression and to restore insulin secretion and the ATP/ADP ratio. These data indicate that in rat pancreatic islets chronically exposed to high glucose or FFA, glucose-induced impairment of insulin secretion is associated with (and might be due to) altered mitochondrial function, which results in impaired glucose oxidation, overexpression of the UCP-2 protein, and a consequent decrease of ATP production. This alteration in FFA cultured islets is mediated by the PPAR-gamma pathway.
In both type 1 and type 2 diabetes, the late diabetic complications in nerve, vascular endothelium, and kidney arise from chronic elevations of glucose and possibly other metabolites including free fatty acids (FFA). Recent evidence suggests that common stress-activated signaling pathways such as nuclear factor-kappaB, p38 MAPK, and NH2-terminal Jun kinases/stress-activated protein kinases underlie the development of these late diabetic complications. In addition, in type 2 diabetes, there is evidence that the activation of these same stress pathways by glucose and possibly FFA leads to both insulin resistance and impaired insulin secretion. Thus, we propose a unifying hypothesis whereby hyperglycemia and FFA-induced activation of the nuclear factor-kappaB, p38 MAPK, and NH2-terminal Jun kinases/stress-activated protein kinases stress pathways, along with the activation of the advanced glycosylation end-products/receptor for advanced glycosylation end-products, protein kinase C, and sorbitol stress pathways, plays a key role in causing late complications in type 1 and type 2 diabetes, along with insulin resistance and impaired insulin secretion in type 2 diabetes. Studies with antioxidants such as vitamin E, alpha-lipoic acid, and N-acetylcysteine suggest that new strategies may become available to treat these conditions.
Oxidative injury induces cellular and nuclear damage that leads to apoptotic cell death. Agents or antioxidants that can inhibit production of reactive oxygen species can prevent apoptosis. We tested the hypothesis that flavonoids can inhibit H(2)O(2)-induced apoptosis in human umbilical vein endothelial cells. A 30-min pulse treatment with 0.25 mmol/L H(2)O(2) decreased endothelial cell viability within 24 h by approximately 40% (P < 0.05) with distinct nuclear condensation and DNA fragmentation. In the H(2)O(2) apoptosis model, the addition of 50 micro mol/L of the flavanol (-)epigallocatechin gallate and the flavonol quercetin, which have in vitro radical scavenging activity, partially (P < 0.05) restored cell viability with a reduction in H(2)O(2)-induced apoptotic DNA damage. In contrast, the flavones, luteolin and apigenin, at the nontoxic dose of 50 micro mol/L, intensified cell loss (P < 0.05) after exposure to H(2)O(2) and did not protect cells from oxidant-induced apoptosis. The flavanones, hesperidin and naringin, did not have cytoprotective effects. The antioxidants, (-)epigallocatechin gallate and quercetin, inhibited endothelial apoptosis, enhanced the expression of bcl-2 protein and inhibited the expression of bax protein and the cleavage and activation of caspase-3. Therefore, flavanols and flavonols, in particular (-)epigallocatechin gallate and quercetin, qualify as potent antioxidants and are effective in preventing endothelial apoptosis caused by oxidants, suggesting that flavonoids have differential antiapoptotic efficacies. The antiapoptotic activity of flavonoids appears to be mediated at the mitochondrial bcl-2 and bax gene level.
Cytokines produced by immune cells infiltrating pancreatic islets have been implicated as one of the important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the protective effects of epigallocatechin gallate (EGCG) on cytokine-induced beta-cell destruction were investigated. EGCG effectively protected IL-1beta and IFN-gamma-mediated cytotoxicity in insulinoma cell line (RINm5F). EGCG induced a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production and reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein levels on RINm5F cells. The molecular mechanism by which EGCG inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. These findings revealed EGCG as a possible therapeutic agent for the prevention of diabetes mellitus progression.
Failure to secrete adequate amounts of insulin in response to increasing concentrations of glucose is an important feature of type 2 diabetes. The mechanism for loss of glucose responsiveness is unknown. Uncoupling protein 2 (UCP2), by virtue of its mitochondrial proton leak activity and consequent negative effect on ATP production, impairs glucose-stimulated insulin secretion. Of interest, it has recently been shown that superoxide, when added to isolated mitochondria, activates UCP2-mediated proton leak. Since obesity and chronic hyperglycemia increase mitochondrial superoxide production, as well as UCP2 expression in pancreatic beta cells, a superoxide-UCP2 pathway could contribute importantly to obesity- and hyperglycemia-induced beta cell dysfunction. This study demonstrates that endogenously produced mitochondrial superoxide activates UCP2-mediated proton leak, thus lowering ATP levels and impairing glucose-stimulated insulin secretion. Furthermore, hyperglycemia- and obesity-induced loss of glucose responsiveness is prevented by reduction of mitochondrial superoxide production or gene knockout of UCP2. Importantly, reduction of superoxide has no beneficial effect in the absence of UCP2, and superoxide levels are increased further in the absence of UCP2, demonstrating that the adverse effects of superoxide on beta cell glucose sensing are caused by activation of UCP2. Therefore, superoxide-mediated activation of UCP2 could play an important role in the pathogenesis of beta cell dysfunction and type 2 diabetes.
Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes-such as MCLA and luminol-that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.
Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed
peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by
t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino
acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate
1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial
depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion
in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation
or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies
the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated
with oxidative stress.
Apoptosis is probably the main form of β-cell death in both type 1 diabetes mellitus (T1DM) and T2DM. In T1DM, cytokines contribute to β-cell destruction through nuclear factor-κB (NF-κB) activation. Previous studies suggested that in T2DM high glucose and free fatty acids (FFAs) are β-cell toxic also via NF-κB activation. The aims of this study were to clarify whether common mechanisms are involved in FFA- and cytokine-induced β-cell apoptosis and determine whether TNFα, an adipocyte-derived cytokine, potentiates FFA toxicity through enhanced NF-κB activation. Apoptosis was induced in insulinoma (INS)-1E cells, rat islets, and fluorescence-activated cell sorting-purified β-cells by oleate, palmitate, and/or cytokines (IL-1β, interferon-γ, TNFα). Palmitate and IL-1β induced a similar percentage of apoptosis in INS-1E cells, whereas oleate was less toxic. TNFα did not potentiate FFA toxicity in primary β-cells. The NF-κB-dependent genes inducible nitric oxide synthase and monocyte chemoattractant protein-1 were induced by IL-1β but not by FFAs. Cytokines activated NF-κB in INS-1E and β-cells, but FFAs did not. Moreover, FFAs did not enhance NF-κB activation by TNFα. Palmitate and oleate induced C/EBP homologous protein, activating transcription factor-4, and immunoglobulin heavy chain binding protein mRNAs, X-box binding protein-1 alternative splicing, and activation of the activating transcription factor-6 promoter in INS-1E cells, suggesting that FFAs trigger an endoplasmic reticulum (ER) stress response. We conclude that apoptosis is the main mode of FFA-and cytokine-induced β-cell death but the mechanisms involved are different. Whereas cytokines induce NF-κB activation and ER stress (secondary to nitric oxide formation), FFAs activate an ER stress response via an NF-κB- and nitric oxide-independent mechanism. Our results argue against a unifying hypothesis for the mechanisms of β-cell death in T1DM and T2DM.
Stimulation of macrophages with lipopolysaccharide (LPS) leads to the production of cytokines that elicit massive liver apoptosis. We investigated the in vivo role of stress-responsive transcription factors (SRTFs) in this process focusing on the precipitating events that are sensitive to a cell-permeant peptide inhibitor of SRTF nuclear import (cSN50). In the absence of cSN50, mice challenged with LPS displayed very early bursts of inflammatory cytokines/chemokines, tumor necrosis factor alpha (1 h), interleukin 6 (2 h), interleukin 1 beta (2 h), and monocyte chemoattractant protein 1 (2 h). Activation of both initiator caspases 8 and 9 and effector caspase 3 was noted 4 h later when full-blown DNA fragmentation and chromatin condensation were first observed (6 h). At this time an increase of pro-apoptotic Bax gene expression was observed. It was preceded by a decrease of anti-apoptotic Bcl2 and BclX(L) gene transcripts. Massive apoptosis was accompanied by microvascular injury manifested by hemorrhagic necrosis and a precipitous drop in blood platelets observed at 6 h. An increase in fibrinogen/fibrin degradation products and a rise in plasminogen activator inhibitor 1 occurred between 4 and 6 h. Inhibition of SRTFs nuclear import with the cSN50 peptide abrogated all these changes and increased survival from 7 to 71%. Thus, the nuclear import of SRTFs induced by LPS is a prerequisite for activation of the genetic program that governs cytokines/chemokines production, liver apoptosis, microvascular injury, and death. These results should facilitate the rational design of drugs that protect the liver from inflammation-driven apoptosis.
Dietary polyphenols like quercetin and rutin are considered beneficial because of their potential protective role in the pathogenesis of multiple diseases associated to oxidative stress such as cancer, coronary heart disease and atherosclerosis. However, many of these effects may depend on the concentration of the polyphenol utilized since high doses of some phenolic compounds may be prooxidant and negatively affect cell growth and viability.
To test the potential chemoprotective effects of quercetin and rutin, two flavonols with high antioxidant capacity, on cell growth, viability and the response of the antioxidant defense system of a human hepatoma cell line (HepG2).
Cell growth was measured by diaminobenzoic acid and bromodeoxyuridine assays, cell toxicity by lactate dehydrogenase leakage assay, reduced glutathione was quantified by a fluorimetric assay, cellular malondialdehyde was analyzed by high-performance liquid chromatography, reactive oxygen species were quantified by the dichlorofluorescein assay, antioxidant enzyme activities were determined by spectrophotometric analysis and their gene expression by northern blot.
Short-term exposure (4 h) to these flavonols had no antiproliferative nor cytotoxic effect. High doses of quercetin (50-100 microM) increased glutathione concentration and gene expression of Cu/Zn superoxide dismutase and catalase inhibiting the activity of the latter enzyme, whereas lower doses (0.1-1 microM) decreased gene expression of Cu/Zn superoxide dismutase and increased that of glutathione peroxidase. All doses of quercetin and rutin diminished reactive oxygen species and high doses (10-100 microM) decreased malondialdehyde concentration.
The results indicate that both natural antioxidants induce favorable changes in the antioxidant defense system of cultured HepG2 that prevent or delay conditions which favor cellular oxidative stress.
Cytokines produced by immune cells infiltrating pancreatic islets have been implicated as one of the important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. In this study, the protective effects of epigallocatechin gallate (EGCG) on cytokine-induced P-cell destruction were investigated. EGCG effectively protected IL-1beta and IFN-gamma-mediated cytotoxicity in insulinoma cell line (RINm5F). EGCG induced a significant reduction in IL-1beta and IFN- gamma-induced nitric oxide (NO) production and reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein levels on RINm5F cells. The molecular mechanism by which EGCG inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. These findings revealed EGCG as a possible therapeutic agent for the prevention of diabetes mellitus progression.
Antioxidant enzyme expression was determined in rat pancreatic islets and RINm5F insulin-producing cells on the level of mRNA, protein, and enzyme activity in comparison with 11 other rat tissues. Although superoxide dismutase expression was in the range of 30% of the liver values, the expression of the hydrogen peroxide-inactivating enzymes catalase and glutathione per-oxidase was extremely low, in the range of 5% of the liver. Pancreatic islets but not RINm5F cells expressed an additional phospholipid hydroperoxide glutathione peroxidase that exerted protective effects against lipid peroxidation of the plasma membrane. Regression analysis for mRNA and protein expression and enzyme activities from 12 rat tissues revealed that the mRNA levels determine the enzyme activities of the tissues. The induction of cellular stress by high glucose, high oxygen, and heat shock treatment did not affect antioxidant enzyme expression in rat pancreatic islets or in RINm5F cells. Thus insulin-producing cells cannot adapt the low antioxidant enzyme activity levels to typical situations of cellular stress by an upregulation of gene expression. Through stable transfection, however, we were able to increase catalase and glutathione peroxidase gene expression in RINm5F cells, resulting in enzyme activities more than 100-fold higher than in nontransfected controls. Catalase-transfected RINm5F cells showed a 10-fold greater resistance toward hydrogen peroxide toxicity, whereas glutathione peroxidase overexpression was much less effective. Thus inactiva-tion of hydrogen peroxide through catalase seems to be a step of critical importance for the removal of reactive oxygen species in insulin-producing cells. Overexpression of catalase may therefore be an effective means of preventing the toxic action of reactive oxygen species.
In this communication we shall endeavor to present a description of some of the methods that we have used in establishing strains of human normal and tumor cells, and an account of some of our results. We believe that the value of this report lies not in the presentation of new and unique methods for maintaining tissue cultures under continuous cultivation, but in presenting evidence that very different methods and media can be used to obtain approximately the same end-results. This holds true, however, only when the highly important factor of experience in the management of tissue cultures is used to best advantage.
The opinion of many workers that human tissue cultures are difficult to maintain is not justified. Just as much time and energy are involved in the maintenance of a rapidly growing rat sarcoma as in the maintenance of a similar human tumor. In this laboratory, by the large lying-drop slide method, we have maintained the Walker rat sarcoma 338 for a period of over four years and a half, and a human chondromyxosarcoma (Table IV) for approximately the same length of time. Strains of cells from these tumors are still under cultivation.
The advantage of knowing that many different types of human tumor cells can be maintained under continuous cultivation for long periods of time may not on first mention become apparent to many. When, however, one considers their use and their importance in collecting radiosensitivity data for specific cell types, and in the study of their cytological, cultural, and metabolic activities, the value of such knowledge is readily appreciated. The concept that pure cultures of human tumor cells can be used as sources of living antigen for immunological and therapeutic purposes must not be overlooked. We are inclined to consider this aim as of greater significance than any of the others.
Infusions of Coreopsis tinctoria Nutt. flowering tops have been used traditionally in Portugal to control hyperglycaemia and a previous study revealed that daily administration of the infusion during a 3-week period promoted the recovery of glucose tolerance by a mechanism different from inhibition of glucose absorption and direct promotion of insulin secretion. We know report the study of the ethyl acetate fraction of Coreopsis tinctoria flowers infusion aiming to confirm flavonoids as bioactive metabolites. To give one step forward into the antihyperglycaemic mechanism of action of this traditionally used plant we also studied the activity of Coreopsis tinctoria flavonoids on the pancreatic function of glucose-intolerant rats. A standard antioxidant, Trolox, was also studied for comparative purposes as the antioxidant mechanism has been frequently purposed as one of the mechanisms mediating antihyperglycaemic effects of flavonoid-rich extracts.
Thirteen compounds, mainly of flavanone and chalcone flavonoidal type, have been identified in this fraction by HPLC-DAD-ESI-MS/MS, and the major one (marein) quantified by HPLC-UV. The fraction (125 mg containing 20 mg of marein/kg b.w.) and Trolox (50 mg/kg b.w.) were administered daily by oral gavage to normal and STZ (40 mg/kg b.w.)-induced glucose-intolerant Wistar rats for 3 weeks. Blood glucose levels were measured weekly by Oral Glucose Tolerance Test. Pancreatic function was evaluated by plasma lipase of treated and non-treated glucose-tolerant and- intolerant rats after the 3-week treatment period.
After 2 weeks oral treatment with Coreopsis tinctoria AcOEt fraction the animals were no longer glucose-intolerant, an effect maintained over the remaining experimental period. Additionally, plasma lipase values of glucose-intolerant animals treated with the AcOEt fraction (13.5 ± 0.84 U/L) showed a clear reduction when compared with the glucose-intolerant group (34.60 ± 1.76 U/L; P<0.001) and normoglycaemic control (8.35 ± 0.69 U/L) demonstrating recovery of pancreatic function. On the other hand, treatment with standard antioxidant Trolox had no effect on glucose homeostasis of glucose-intolerant rats. The oral treatment with Coreopsis tinctoria fraction caused no hepatotoxicity, as determined by blood alanine and aspartate transaminases, and had also no effect on glucose homeostasis and pancreatic function of normal rats.
AcOEt fraction, containing the same amount of marein as the infusion, promoted glucose tolerance regain in the rats more quickly, which means that the bioactivity is probably due to the several flavonoids present in Coreopsis tinctoria extracts and not to marein alone. The results also strongly suggest that these compounds act by promoting pancreatic cell function recovery from STZ-induced injury, possibly through a mechanism of action other than merely antioxidant mediated.
The antioxidant effects of chestnut inner shell extract (CISE) were investigated in a tert-butylhydroperoxide (t-BHP)-treated HepG2 cells, and in mice that were administered carbon tetrachloride (CCl(4)) and fed a high-fat diet (HFD). Pre-incubation with CISE significantly blocked the oxidative stress induced by t-BHP treatment in HepG2 cells (P<0.05) and preserved the activities of catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase compared to group treated with t-BHP only. Similarly, the CCl(4)- and HFD-induced reduction of antioxidant enzymes activities in liver was prevented by CISE treatment compared to control groups. Furthermore, hepatic lipid peroxidation were remarkably lower (P<0.05) in the CISE-treated groups with t-BHP or HFD. To determine the active compound of CISE, the fractionation of CISE has been conducted and scoparone and scopoletin were identified as main compounds. These compounds were also shown to inhibit the t-BHP-induced ROS generation and reduction in antioxidant enzyme activity in an in vitro model system. From these results, it was demonstrated that CISE has the ability to protect against damage from oxidative stressors such as t-BHP, CCl(4) and HFD in in vitro and in vivo models. The CISE might be useful for the prevention of oxidative damage in liver cells and tissues.