Analysis of Lutein Content in Microencapsulated Marigold Flower Extract (Tagetes erecta L.) Using UHPLC-Q-Orbitrap-HRMS and Its Cytotoxicity in ARPE-19 Cells.

Organic solvents are commonly used to extract lutein. However, they are toxic and are not environmental-friendly. There are only a few reports on the quantification of lutein. Therefore, this study aimed to determine a suitable extraction method by which to obtain lutein from marigold flower (Tagetes erecta L.), using coconut oil to evaluate the cytotoxicity of extract in ARPE-19 cells, to optimize the encapsulation process for the development of microencapsulated marigold flower extract, and to develop the method for analysis of lutein by using UHPLC-Q-Orbitrap-HRMS. Coconut oil was used for the extraction of marigold flowers with two different extraction methods: ultrasonication and microwave-assisted extraction. The UHPLC-Q-Orbitrap-HRMS condition for the analysis of lutein was successfully developed and validated. Marigold flower extract obtained using the microwave method had the highest lutein content of 27.22 ± 1.17 mg/g. A cytotoxicity study revealed that 16 µM of lutein from marigold extract was non-toxic to ARPE-19 cells. For the development of microencapsulated marigold extract, the ratio of oil to wall at 1:5 had the highest encapsulation efficiency and the highest lutein content. Extraction of lutein using coconut oil and the microwave method was the suitable method. The microencapsulated marigold extract can be applied for the development of functional ingredients.

Antihyperglycemic effects of Lysiphyllum strychnifolium leaf extract in vitro and in vivo.

CONTEXT: Lysiphyllum strychnifolium (Craib) A. Schmitz (LS) (Fabaceae) has traditionally been used to treat diabetes mellitus. OBJECTIVE: This study demonstrates the antidiabetic and antioxidant effects of aqueous extract of LS leaves in vivo and in vitro. MATERIALS AND METHODS: The effects of aqueous LS leaf extract on glucose uptake, sodium-dependent glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2) mRNA expression in Caco-2 cells, α-glucosidase, and lipid peroxidation were evaluated in vitro. The antidiabetic effects were evaluated using an oral glucose tolerance test (OGTT) and a 28-day consecutive administration to streptozotocin (STZ)-nicotinamide (NA)-induced type 2 diabetic mice. RESULTS: The extract significantly inhibited glucose uptake (IC50: 236.2 ± 36.05 µg/mL) and downregulated SGLT1 and GLUT2 mRNA expression by approximately 90% in Caco-2 cells. Furthermore, it non-competitively inhibited α-glucosidase in a concentration-dependent manner with the IC50 and Ki of 6.52 ± 0.42 and 1.32 µg/mL, respectively. The extract at 1000 mg/kg significantly reduced fasting blood glucose levels in both the OGTT and 28-day consecutive administration models as compared with untreated STZ-NA-induced diabetic mice (p < 0.05). Significant improvements of serum insulin, homeostasis model assessment of insulin resistance (HOMA-IR), and GLUT4 levels were observed. Furthermore, the extract markedly decreased oxidative stress markers by 37-53% reduction of superoxide dismutase 1 (SOD1) in muscle and malondialdehyde (MDA) in muscle and pancreas, which correlated with the reduction of MDA production in vitro (IC50: 24.80 ± 7.24 µg/mL). CONCLUSION: The LS extract has potent antihyperglycemic activity to be used as alternative medicine to treat diabetes mellitus.

Phytochemical Composition, Antiglycation, Antioxidant Activity and Methylglyoxal-Trapping Action of Brassica Vegetables.

Brassica vegetables are common in cuisines worldwide. The aim of this study was to investigate the antiglycation, methylglyoxal (MG)-trapping action and antioxidant activity of Brassica vegetable extract (BVE) from cabbage, cauliflower and Chinese cabbage. The results showed that cauliflower had the highest phenolic content with the strongest DPPH radical scavenging activity, ferric reducing antioxidant power and oxygen radical absorbance capacity. Seven phenolic acids and three flavonoids were identified by ESI-Q-TOF-MS analysis. The common phenolic compounds in all BVE were sinapic acid and p-hydroxybenzoic acid. The BVE (0.5 mg/mL) showed significant inhibitory activity against glucose-induced fluorescent advanced glycation end products (AGEs) formation (34 - 67%) and preserved the amount of protein thiol group (30 - 35%). In addition, all extracts (0.125 - 4 mg/mL) also had the ability to trap MG, a reactive glycating agent. Total phenolic content of BVE exhibited a positive correlation with DPPH radical scavenging activity (r = 0.524) and % inhibition of AGE formation (r = 0.570) and % MG-trapping capacity (r = 0.786). These findings suggest that the BVE possesses antioxidant and antiglycating activity that may help to protect against protein glycation and oxidation mediated by glycation reaction.

Protective role of Clitoria ternatea L. flower extract on methylglyoxal-induced protein glycation and oxidative damage to DNA.

BACKGROUND: Methylglyoxal (MG) is a highly reactive dicarbonyl precursor for the formation of advanced glycation end products (AGEs) associated with age-related diseases, including diabetes and its complications. Clitoria ternatea L. flower has been reported to possess antioxidant and antiglycating properties. Evidence indicates that the extract of Clitoria ternatea L. flower inhibits fructose-induced protein glycation and oxidative damage to bovine serum albumin (BSA). However, there is no evidence to support the inhibitory effect of CTE against MG-mediated protein glycation and oxidative damage to protein and DNA. Therefore, the aim of the present study was to investigate whether C. ternatea flower extract (CTE) prevents MG-induced protein glycation and oxidative DNA damage. METHODS: The formation of fluorescent AGEs in BSA was evaluated using spectrofluorometer. The protein carbonyl and thiol group content were used for detecting protein oxidation. DNA strand breakage in a glycation model comprising of MG, lysine and Cu2+ or a free radical generator 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) systems was investigated using gel electrophoresis. Generation of superoxide anions and hydroxyl radicals in the MG/lysine system was assessed by the cytochrome c reduction assay and thiobarbituric acid reactive substances assay, respectively. High performance liquid chromatography (HPLC) was used to measure the MG-trapping ability. RESULTS: In the BSA/MG system, CTE (0.25-1 mg/mL) significantly inhibited the formation of fluorescent AGEs and protein oxidation by reducing protein carbonyl content as well as preventing the protein thiol depletion. The concentration of CTE at 0.125-1 mg/mL prevented oxidative DNA cleavage in MG/lysine and AAPH systems associated with the inhibition of superoxide anion and hydroxyl radical formation. It also directly trapped MG in a concentration-dependent manner, ranging from 15 to 43%. CONCLUSIONS: The study findings suggest that the direct carbonyl trapping ability and the free radical scavenging activity of CTE are the underlying mechanisms responsible for the prevention of protein glycation and oxidative DNA damage.

Anthocyanin-Enriched Riceberry Rice Extract Inhibits Cell Proliferation and Adipogenesis in 3T3-L1 Preadipocytes by Downregulating Adipogenic Transcription Factors and Their Targeting Genes.

Riceberry rice (Oryza sativa L.) is a new pigmented variety of rice from Thailand. Despite its high anthocyanin content, its effect on adipogenesis and adipocyte function remains unexplored. We investigated whether Riceberry rice extract (RBE) impacted cell proliferation by examining viability and cell cycle, using preadipocyte 3T3-L1 cells. To test RBE's effect on adipocyte formation, cells were cultured in adipogenic medium supplemented with extract and adipocyte number and triglyceride levels were quantified. Furthermore, Akt1 phosphorylation along with RT-qPCR and intracellular calcium imaging were performed to obtain an insight into its mechanism of action. The effect of RBE on adipocyte function was investigated using glucose uptake and lipolysis assays. Treatment of cells with RBE decreased preadipocyte number without cytotoxicity despite inducing cell cycle arrest (p < 0.05). During adipogenic differentiation, RBE supplementation reduced adipocyte number and triglyceride accumulation by downregulating transcription factors (e.g., PPARγ, C/EBPα, and C/EBPß) and their target genes (p < 0.05). The Akt1 phosphorylation was decreased by RBE but insignificance, however, the extract failed to increase intracellular calcium signals. Finally, the treatment of adipocytes with RBE reduced glucose uptake by downregulating Glut4 mRNA expression and enhanced isoproterenol-induced lipolysis (p < 0.05). These findings suggest that RBE could potentially be used in the treatment of obesity by inhibiting adipocyte formation and proliferation.

Cyanidin-3-rutinoside acts as a natural inhibitor of intestinal lipid digestion and absorption.

BACKGROUND: Cyanidin-3-rutinoside (C3R), a naturally occurring anthocyanin, possesses anti-oxidant, anti-hyperglycemic, anti-glycation and cardioprotective properties. However, its mechanisms responsible for anti-hyperlipidemic activity have not been fully identified. The aim of the study was to investigate the lipid-lowering mechanisms of C3R through inhibition of lipid digestion and absorption in vitro. METHODS: The inhibitory activity of C3R against pancreatic lipase and cholesterol esterase was evaluated using enzymatic fluorometric and enzymatic colorimetric assays, respectively. An enzyme kinetic study using Michaelis-Menten and the derived Lineweaver-Burk plot was performed to understand the possible types of inhibition. The formation of cholesterol micelles was determined using the cholesterol assay kit. The bile acid binding was measured using the colorimetric assay. The NBD cholesterol uptake in Caco-2 cells was determined using fluorometric assay. The mRNA expression of cholesterol transporter (Niemann-Pick C1-like 1) was determined by RT-PCR. RESULTS: The results showed that C3R was a mixed-type competitive inhibitor of pancreatic lipase with the IC50 value of 59.4 ± 1.41 µM. Furthermore, C3R (0.125-1 mM) inhibited pancreatic cholesterol esterase about 5-18%. In addition, C3R inhibited the formation of cholesterol micelles and bound to primary and secondary bile acid. In Caco-2 cells, C3R (12.5-100 µM) exhibited a significant reduction in cholesterol uptake in both free cholesterol (17-41%) and mixed micelles (20-30%). Finally, C3R (100 µM) was able to suppress mRNA expression of NPC1L1 in Caco-2 cells after 24 h incubation. CONCLUSIONS: The present findings suggest that C3R acts as a lipid-lowering agent through inhibition of lipid digestion and absorption.

Acute effect of Clitoria ternatea flower beverage on glycemic response and antioxidant capacity in healthy subjects: a randomized crossover trial.

BACKGROUND: Clitoria ternatea L., a natural food-colorant containing anthocyanin, demonstrated antioxidant and antihyperglycemic activity. The aim of this study was to determine the effects of Clitoria ternatea flower extract (CTE) on postprandial plasma glycemia response and antioxidant status in healthy men. METHODS: In a randomized, crossover study, 15 healthy men (ages 22.53 ± 0.30 years; with body mass index of 21.57 ± 0.54 kg/m2) consumed five beverages: (1) 50 g sucrose in 400 mL water; (2) 1 g CTE in 400 mL of water; (3) 2 g CTE in 400 mL of water; (4) 50 g sucrose and 1 g CTE in 400 mL of water; and (5) 50 g sucrose and 2 g CTE in 400 mL of water. Incremental postprandial plasma glucose, insulin, uric acid, antioxidant capacities and lipid peroxidation were measured during 3 h of administration. RESULTS: After 30 min ingestion, the postprandial plasma glucose and insulin levels were suppressed when consuming sucrose plus 1 g and 2 g CTE. In addition, consumption of CTE alone did not alter plasma glucose and insulin concentration in the fasting state. The significant increase in plasma antioxidant capacity (ferric reducing ability of plasma (FRAP), oxygen radical absorbance capacity (ORAC), trolox equivalent antioxidant capacity (TEAC), and protein thiol) and the decrease in malondialdehyde (MDA) level were observed in the subjects who received 1 g and 2 g CTE. Furthermore, consumption of CTE protected sucrose-induced reduction in ORAC and TEAC and increase in plasma MDA. CONCLUSIONS: These findings suggest that an acute ingestion of CTE increases plasma antioxidant capacity without hypoglycemia in the fasting state. It also improves postprandial glucose, insulin and antioxidant status when consumed with sucrose. TRIAL REGISTRATION: Thai Clinical Trials Registry: TCTR20170609003 . Registered 09 September 2017. 'retrospectively registered'.

The inhibitory activity of herbal medicines on the keys enzymes and steps related to carbohydrate and lipid digestion.

BACKGROUND: Obesity and overweight are consistently associated with metabolic disorders including hyperglycemia and hyperlipidemia. Herbal medicines have been currently suggested as an alternative source of potentially useful antihyperglycemic, antihyperlipidemic, antioxidant activities. The objective of this study was to assess the in vitro inhibitory effects of eleven herbal medicines on carbohydrate and lipid digestive enzymes and the key steps of lipid digestion including the inhibition of micelle formation and the ability to bind bile acid. In addition, antioxidant activity of herbal medicines was also investigated. METHODS: α-Glucosidase, pancreatic α-amylase, pancreatic lipase, and pancreatic cholesterol esterase inhibitory activities of aqueous extract of herbal medicines were measured using the enzymatic colorimetric assay. The formation of cholesterol micelles was determined using the cholesterol assay kit. The bile acid binding was measured using the colorimetric assay. Antioxidant activities were assessed by using four methods including Trolox equivalent antioxidant capacity (TEAC), oxygen radical absorbance capacity ORAC), superoxide radical scavenging activity (SRSA), and hydroxyl radical scavenging activity (HRSA). RESULTS: The extracts (1 mg/mL) markedly inhibited intestinal maltase (5.16 - 44.33 %), sucrase (1.25-45.86 %), pancreatic α-amylase (1.75-12.53 %), pancreatic lipase (21.42-85.93 %), and pancreatic cholesterol esterase (2.92-53.35 %). The results showed that all extracts exhibited the inhibitory activity against pancreatic lipase with the IC50 values ranging from 0.015 to 4.259 mg/mL. In addition, the incorporation of cholesterol into micelles was inhibited by the extracts (6.64-33.74 %). The extracts also bound glycodeoxycholic acid (9.9-15.08 %), taurodeoxycholic acid (12.55-18.18 %), and taurocholic acid (11.91 - 18.4 %). Furthermore, the extracts possessed various antioxidant activities including the TEAC values (0.50 - 4.70 µmol trolox/mg dried extract), the ORAC values (9.14-44.41 µmol trolox/mg dried extract), the SRSA (0.31 - 18.82 mg trolox/mg dried extract), and the HRSA (0.05-2.29 mg trolox/mg dried extract). The findings indicated that Syzygium aromaticum, Phyllanthus amarus, Thunbergia laurifolia were the effective promising antihyperglycemic and antihyperlipidemic agents. Statistical analysis demonstrated strong positive significant correlations between the contents of phenolic compounds and % inhibition of pancreatic lipase (r = 0.885, p < 0.001), % inhibition of pancreatic cholesterol esterase (r = 0.761, p < 0.001), and the TEAC value (r = 0.840, p < 0.001). Furthermore, there was a strongly positive correlation between the TEAC value and % inhibition of pancreatic cholesterol esterase (r = 0.851, p < 0.001) and % inhibition of pancreatic lipase (r = 0.755, p < 0.001). CONCLUSIONS: Three herbal medicines including Syzygium aromaticum, Thunbergia laurifolia, and Phyllanthus amarus markedly inhibited the intestinal α-glucosidase, pancreatic α-amylase, pancreatic lipase, and pancreatic cholesterol esterase. They also reduced formation of cholesterol micelle and bound bile acid. The findings indicate that these herbal medicines might be a promising agent for antihyperglycemic, antihyperlipidemic, and antioxidant activities.

Cyanidin-3-rutinoside attenuates methylglyoxal-induced protein glycation and DNA damage via carbonyl trapping ability and scavenging reactive oxygen species.

BACKGROUND: Advanced glycation end-products (AGEs) play a significant role in the development and progression of vascular complication in diabetes. Anthocyanin has been recently reported to possess antiglycating activity. This study aimed to determine whether a naturally occurring anthocyanin, cyanidin-3-rutinoside (C3R) inhibits methylglyoxal (MG) induced protein glycation and oxidative protein and DNA damage. METHODS: C3R (0.125-1 mM) was incubated with bovine serum albumin and MG (1 mM) for 2 weeks. The formation of fluorescent AGEs was measured by using spectrofluorometer and thiol group content were used to detect protein oxidative damage. Gel electrophoresis was used to determine whether C3R (0.125-1 mM) reduced DNA strand breakage in a glycation model comprising lysine, MG and/or Cu(2+). The generation of superoxide anions and hydroxyl radicals were detected by the cytochrome c reduction assay and the thiobarbituric acid reactive substances assay. MG-trapping capacity was assessed by high performance liquid chromatography (HPLC). RESULTS: C3R (0.25-1 mM) reduced the formation of fluorescent AGEs and depleted protein thiol groups in bovine serum albumin mediated by MG. At 1 mM C3R inhibited oxidative DNA damage in the glycation model (p < 0.05) and at 0.5-1 mM prevented Cu(2+) induced DNA strand breakage in the presence of lysine and MG. The findings showed that C3R reduced the formation of superoxide anion and hydroxyl radicals during the glycation reaction of MG with lysine. C3R directly trapped MG in a concentration and time dependent manner (both p < 0.001). CONCLUSIONS: These findings suggest that C3R protects against MG-induced protein glycation and oxidative damage to protein and DNA by scavenging free radicals and trapping MG.

Isoferulic acid prevents methylglyoxal-induced protein glycation and DNA damage by free radical scavenging activity.

BACKGROUND: Isoferulic acid (IFA), a naturally occurring cinnamic acid derivative, is a main active ingredient of the rhizoma of Cimicifuga dahurica. It has been shown various pharmacological activities. The aim of the study was to investigate the effect of IFA against MG-induced protein glycation and oxidative DNA damage. Free radical scavenging activity and the MGO-trapping abilities of IFA were also investigated. METHODS: The fluorescent MG-derived AGEs and non-fluorescent N(ε)-(carboxymethyl) lysine (N(ε)-CML) was measured using a spectrofluorometer and an enzyme linked immunosorbant assay (ELISA). Protein carbonyl content was used to detect protein oxidation. Gel electrophoresis was used to determine DNA damage. Superoxide anion radicals and hydroxyl radicals were determined using cytochrome c reduction assay and thiobarbituric acid reactive 2-deoxy-D-ribose oxidation products, respectively. The MG-trapping capacity was performed by HPLC. RESULTS: IFA (1.25-5 mM) inhibited the formation of fluorescent MG-derived AGEs, and N(ε)-CML, and protein carbonyl in bovine serum albumin. In addition, IFA (0.1-1 mM) also prevented MG/lysine-mediated oxidative DNA damage in the presence and absence of copper ion. The protective ability of IFA was directly correlated to inhibition of hydroxyl and superoxide anion radical generation during the reaction of MG and lysine. Most notably, IFA had no the directly trapping ability to MG. CONCLUSIONS: The present results highlighted that free radical scavenging activity, but not the MG-trapping ability, is the mechanism of IFA for preventing MG-induced protein glycation and DNA damage.

Consumption of Mesona chinensis attenuates postprandial glucose and improves antioxidant status induced by a high carbohydrate meal in overweight subjects.

Edible plants constitute a potential source for controlling postprandial hyperglycemia and oxidative stress. The objective of this study was to investigate in vitro antioxidant and intestinal α-glucosidase inhibitory activities of Mesona chinensis (MC). In addition, the acute effect of MC on postprandial glucose and plasma antioxidant status after the consumption of a high carbohydrate (HC) meal by overweight subjects was also determined. The results showed that total phenolic and flavonoid contents in the extract were 212.37 ± 5.64 mg gallic acid equivalents/g dried extract and 23.44 ± 2.50 mg catechin equivalents/g dried extract, respectively. MC extract markedly inhibited the intestinal maltase and sucrose with the IC50 values of 4.66 ± 0.22 mg/mL and 1.30 ± 0.43 mg/mL, respectively. However, MC extract had no inhibitory activity against pancreatic α-amylase. In addition, MC extract had antioxidant properties including DPPH radical scavenging activity, superoxide radical scavenging activity (SRSA), hydroxyl radical scavenging activity (HRSA), trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and ferrous ion cheating activity (FICP). The significant decrease in postprandial plasma glucose, triglyceride and malondialdehyde levels, and the increase in plasma antioxidant capacity (FRAP and ORAC) were observed in overweight subjects receiving a HC meal together with MC extract (1 g). The finding supports that MC helps normalize and enhance antioxidant defense induced by a HC meal, suggesting that MC may have the potential for the prevention of chronic conditions and diseases associated with overweight and obesity.

Mesona Chinensis Benth extract prevents AGE formation and protein oxidation against fructose-induced protein glycation in vitro.

BACKGROUND: Mesona chinensis Benth (Chinese Mesona), an economically significant agricultural plant, is the most widely consumed as an herbal beverage in Southeast Asia and China. The objective of this study was to evaluate the inhibitory activity of Mesona chinensis (MC) extract on the formation of advanced glycation end products (AGEs) and protein oxidation in an in vitro model of fructose-mediated protein glycation. METHODS: The content of total polyphenolic compounds was measured by using Folin-Ciocalteu assay. Antiglycation activity was determined using the formation of AGE fluorescence intensity, Nϵ-(carboxymethyl)lysine (CML), the level of fructosamine, and the formation of amyloid cross ß-structure. The protein oxidation was examined using the level of protein carbonyl content and thiol group. RESULTS: Our results revealed that the content of total polyphenolic compound in MC extract was 212.4 ± 5.6 mg gallic acid equivalents/g dried extract. MC extract (0.25-1.00 mg/mL) significantly inhibited the formation of fluorescence AGEs in fructose-glycated bovine serum albumin (BSA) during 4 weeks of study. Furthermore, MC extract also decreased the level of Nϵ-CML, fructosamine, and amyloid cross ß-structure in fructose-glycated BSA. While the total thiol group was elevated and the protein carbonyl content was decreased in BSA incubated with fructose and MC extract. CONCLUSIONS: The extract of MC inhibits fructose-mediated protein glycation and protein oxidation. This edible plant could be a natural rich source of antiglycation agent for preventing AGE-mediated diabetic complication.