Inhibitory Effects of Bioassay-Guided Isolation of Anti-Glycation Components from Taraxacum coreanum and Simultaneous Quantification

Inhibition of the formation of advanced glycation end products (AGEs) is an attractive strategy in diabetes treatment. Taraxacum coreanum extracts were suggested to have antidiabetic effects. However, studies on the components of T. coreanum are lacking, and there is no report on the inhibitory effects of T. coreanum on the formation of AGEs. Therefore, T. coreanum extracts and fractions were tested for their inhibitory effects on α-glucosidase and AGEs formation in two systems (bovine serum albumin (BSA)–glucose and BSA–methylglyoxal (MGO)). Bioassay-guided isolation of compounds from T. coreanum led to six flavones (1–6) and four hydroxycinnamic acid derivatives (7–11). Compound 11 exhibited the highest inhibitory activity against α-glucosidase and AGEs formation and had the highest content in T. coreanum extract. All compounds except compound 9 showed a stronger inhibition than the positive control in the BSA-glucose and BSA-MGO system. In addition, T. coreanum showed a higher content of bioactive compounds and stronger inhibition of AGE formation and α-glucosidase activity than T. officinale. Our study demonstrated the preventive and therapeutic efficacy of T. coreanum and its potential use as a cost-effective phytopharmaceutical in complementary therapy against type-2 diabetes and its complications.


Introduction
Protein glycation, also called Maillard reaction, is a nonenzymatic reaction that occurs between the amino and carbonyl groups of proteins, lipids, and nucleic acids and results in the formation of a group of heterogeneous compounds called advanced glycation end products (AGEs). These compounds form covalent bonds with proteins, which results in changes in the structural and functional properties of proteins. The interaction between AGEs and their receptors (RAGE) causes oxidative stress, thrombosis, and inflammatory reactions [1,2]. The rate of AGE formation is accelerated in diabetes [3]. Unless long-term uncontrolled, it can lead to a series of complications such as cataracts, atherosclerosis, neuropathy, retinopathy, nephropathy, and delayed wound healing. The tissue concentrations of AGE were twofold higher in diabetic patients with end-stage renal disease than in diabetic patients without renal disease [4]. Additionally, diabetic patients with Alzheimer's disease had increased accumulation of AGEs and upregulated RAGE in the brains [5], and patients with heart failure-associated cardiac stiffness showed myocardial accumulation of AGEs [6]. AGEs formation can be suppressed through use of inhibitors [7]. Some synthetic compounds, such as aminoguanidine (AMG) exhibit a high AGE-inhibitory activity; however, they are associated with various adverse effects in vivo and are not Accumulation of AGEs in the body is implicated in the development of chronic degenerative diseases [21]. AGE inhibition is a therapeutic option for diabetes that is not based on controlling of postprandial blood glucose level. This approach could be useful in the prevention or reduction of diabetic complications [22]. Therefore, studies have been performed to develop AGE inhibitors. Owing to the importance of AGEs in the pathogenesis of various related diseases, AGE inhibitors have received increased attention. methylglyoxal (MGO) is a crucial intermediate for the formation of AGEs in vivo [10]. In addition, α-glucosidase inhibitors are believed to act as anti-diabetic agents by impeding sugar degradation and attenuating postprandial hyperglycemia. Thus, inhibition of α-glucosidase activity and carbohydrate hydrolysis would be beneficial for controlling blood glucose levels in diabetic patients [23]. In this study, inhibitory effects on AGE formation were determined in bovine serum albumin (BSA)-glucose and BSA-MGO systems to demonstrate the effects of T. coreanum in preventing diabetic complications. We also examined α-glucosidase inhibitory activity of T. coreanum to demonstrate its potential in preventing diabetes. The results are summarized in Table 1.

AGE Formation in BSA-glucose and BSA-MGO Systems and α-Glucosidase Inhibitory Activities of Compound 1-11 Isolated from T. coreanum
Regarding AGE formation inhibitory activity in BSA-glucose system, glycosylation at the C-7 position of flavones improved chelation effect and glycosylation at the C-4 position of flavones decreased the inhibitory capacity [36]. It was same result that the lower inhibitory activity of compound 3, which was glycosylated at C-4 , had than that of compounds 2-6 (glycosylation at the C-7 position). In addition, compound 2, which was glycosylated at the C-7 position of luteolin-type flavonols, showed the highest inhibitory activity, followed by luteolin (compound 1) with no glycosylation, and compounds 5 and 6 with glycosylation at the C-7 and C-2 position of isoetin-type flavonols. The IC 50 values of compounds 2-6 were 122.81 ± 2.02, 423.30 ± 18.04, 253.3 ± 18.04, 268.18 ± 3.41, and 238.05 ± 13.82 µM, respectively. In contrast, compound 3 (only glycosylation at C-2 position) had the lowest IC 50 values with 423.30 ± 18.04 mM. Among hydroxycinnamic acids, derivatives of both caffeic acid and tartaric acid (compound 11) showed higher inhibitory activity than derivatives of caffeic acid and quinic acid (compound 10). Compounds 8 and 9 showed mild activity, with IC 50 values of 324.21 ± 8.29 and 306.99 ± 10.16 µM. Compound 10 demonstrated slight inhibition, with IC 50 value of 704.86 ± 167.44 µM.
In BSA-MGO system, compound 1 showed the highest inhibitory activity with IC 50 value of 66.11 ± 17.06 µM, followed by compounds 2 and 3, which was glycosylated at the C-7 or C-4 position of flavones, and compounds 4-6, which had two glycosylations. The inhibitory capacity of compounds 4-6 was weaker than that of other flavone compounds, but stronger than compounds 8-9. Among hydroxycinnamic acids, compounds 9-11, which have a glycerol group, quinic acid, or caftaric acid, showed similar inhibitory potentials, with IC 50 values of 140.72 ± 67.36, 138.18 ± 1.91 µM, and 141.21 ± 8.76, respectively. Compound 8, which had an IC 50 value of 151.67 ± 65.36, exhibited weaker inhibitory activities than flavones and other compounds.

Quantitative HPLC Analysis of Six Bioactive Compounds
HPLC analysis was performed for quantitative evaluation of the active components of T. coreanum extract (Figure 2). After a preliminary screening of the collected samples, compound 11 was identified as the major component of T. coreanum extract, and compounds 9 and 10 were the second major components. The six compounds (1, 2, 7, 9-11) that showed the most potent inhibitory activity against AGE formation were examined. To establish a quality-control standard, this study developed a standard extraction method. The six major compounds were extracted using different solvent compositions and extraction times (Table 4). These six compounds were extracted with 50% ethanol (216.69 mg/g), and the content was extracted from 30 to 90 min stably. Based on the contents of the six compounds, 30-90 min extraction with 50% ethanol was the optimized solvent condition. Among T. coreanum and T. officinale, the contents of all compounds except compound 7 were higher in T. coreanum than in T. officinale, but the content of compound 7 was significantly lower than that of other compounds. In particular, the content of compound 11, which is the major bioactive compound, is about more 125.84 (mg/g) than T. officinale. (Figure 3). The quantitative study suggested that the IC 50 values of the tested samples were inversely proportional to the total content of compounds 1, 2, 7, 9-11, indicating that these six compounds could play important roles in the inhibition of AGE formation. This finding suggested that, for medicinal purposes, HPLC analysis of these six compounds cac be performed for obtaining quality-control standards of T. coreanum. Actually, the T. coreanum extraction (extracted with 50% ethanol) demonstrated better inhibition activity than the 100% methanol extraction in all assay. In addition, we collected eight samples of T. coreanum from different regions, including Gyeongsangnam-do, Gyeongsangbuk-do, Gyeonggi-do, Chungcheongnam-do, Jeollabuk-do, and Gangwon-do. The contents of compounds 1 and 11 were the highest in samples obtained from Gyeongsangnam-do, Sancheong; compound 2 was highest in samples from Gyeongsangbuk-do, Yeongcheon; compound 8 was highest in samples from Gyeongsangnam-do, Sancheong, and Chungcheongnam-do, Taean; and compound 10 was highest in samples from Gyeongsangbuk-do, Yeongcheon and Gangwon-do, Yanggu. The content of compound 9 was 5 mg/g in average, and contained almost the same amount in all samples. Considering the total content of the six major bioactive compounds of T. coreanum, the most abundant compounds were harvested from Gyeongsangnam-do, Sancheong. The average content of major components in the extract was calculated based on quantitative analysis. We suggested that T. coreanum harvested from Gyeongsangnam-do, Sancheong was the best useful natural alternative medicine for diabetic complications. The results are summarized in Table 5.    Data are mean ± SD (n = 3) in mg/g dried sample. Data are mean ± SD (n = 3) in mg/g dried sample.
A portion of the BuOH fraction was separated using a Sephadex LH-20 column chromatography with a gradient elution solvent system of 30% to 100% MeOH to give six sub-fractions. Sub-fraction 2 was applied to MCI gel chromatography using a 10% to 60% MeOH gradient elution solvent system to yield sub-fractions 2-1 to 2-12. Compound 5 was isolated from sub-fraction 2-9 using a Sephadex LH-20 with 20% MeOH. In addition, compound 6 was isolated from sub-fraction 2-10 using MCI column chromatography with 50% MeOH. Sub-fractions 2-11 was isolated by recrystallization to yield compounds 10.
Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) were used as gradient linear mobile phases (0-18 min, 0-50% B; 18-20 min, 50-100% B). All eluents were filtered with a 0.45 µm polyvinylidene fluoride (PVDF) syringe filter. The flow rate was adjusted to 0.3 mL/min. The injection volume was 5.0 µL for the standard solution that was used. The optimized conditions for the analysis were as follows: heater temperature, 300 • C; capillary temperature, 360 • C; aux gas flow rate, 10 L/h; sheath gas flow rate, 45 L/h; S-lens RF level, 50.0 V; spray capillary voltage, 3.0 kV; full MS resolution, 35,000 (FWHM @ m/z 200); full MS AGC target, 3e 6 ; and full MS maximum IT, 200 ms.

Measurement of BSA-glucose and Fructose Inhibitory Activity
Inhibition of AGE formation in BSA-glucose system was determined using the spectrophotometric method described previously [37]. All test samples were dissolved in 10% DMSO. The assay mixture was 50 mM phosphate buffer (pH 7.4) with 0.02% sodium azide, BSA (10 mg/mL), 0.4 M D-fructose and D-glucose, and sample or buffer. This mixture was incubated at 60 • C for 2 days. After incubation, the fluorescence was measured (excitation and emission wavelengths 350 and 450 nm, respectively) in a 96-black well plate. We used AG as a positive control. Three replicate samples were run for each set. The inhibitory activity on AGE formation was calculated using the following formula: {(Ac − As)/Ac} × 100, where Ac is fluorescence of the control, and As is the fluorescence of the sample.

Measurement of BSA-MGO Inhibitory Activity
This assay was performed according to a previously described method, with modifications [38]. The inhibitory effect on protein glycation induced by MGO (40% aqueous solution) and the main reactive intermediate compound formed in Maillard reaction were evaluated. All test samples were dissolved in 10% DMSO. The assay mixture was 50 mM phosphate buffer (pH 7.4) with 0.02% sodium azide, BSA (1 mg/mL), 7 mM MGO, and sample or buffer. This mixture was incubated at 60 • C for 2 days. After incubation, fluorescence was measured on (excitation and emission wavelengths of 340 and 420 nm, respectively) in a 96-black well plate. We used AG as a positive control. Three replicate samples were run for each set. The inhibitory activity was calculated using the same equation applied in BSA-glucose assay.

Measurement α-Glucosidase Inhibitory Activity
This assay was performed using a 96-well microplate reader and a spectrophotometer. The reaction mixture contained 100 mM phosphate buffer (pH 6.8), 2.5 mM p-NPG, and sample or buffer. A chromogenic substance was used for quantification purposes. After the addition of mixture solution to each well, 10 mM phosphate buffer (20 µL, pH 6.8) containing 0.2 U/mL α-glucosidase was added. This mixture was incubated at 37 • C for 15 min. The reaction was stopped by adding 80 µL of 0.2 mol/L sodium carbonate solution. The absorbance was measured at 405 nm immediately after stopping the reaction using a SUNRISE microplate reader. Acarbose dissolved in 10% DMSO was used as the positive control. Three replicate samples were run for each set. The inhibitory activity was calculated using the equation applied in BSA-glucose assay, but absorbance was measured instead of fluorescence.

Statistical Analysis
All assays were performed in triplicate. Data were presented as mean ± standard deviation (SD) and analyzed by using one-way ANOVA. The data were considered to have statistical significance at p < 0.05.

HPLC Analysis
To identify the six major compounds from T. coreanum, a Waters Kromasil column C18 (4.6 × 250 mm, 5 µm) column was used for determination major compounds in T. coreanum. Solvent A (0.2% phosphoric acid in water) and solvent B (methanol) were used as gradient linear mobile phases (0-15 min, 93-81% B; 15-25 min, 81-60% B; 25-45 min, 60-40% B) at a mobile phase rate of 1 mL/min. All eluents were filtered with a 0.45 µm PVDF syringe filter. The injection volume was 10 µL, and compounds were measured at a wavelength of 330 nm. For preparation of extract stock solutions, plant powders were sonicated with MeOH for 60 min and dried under vacuum by using a rotary evaporator at 50 • C. Then, they were dissolved in MeOH to a concentration of 10,000 ppm. Standard compound stock solutions were dissolved in MeOH. All analyzed solutions were strained using a 0.45 µm PVDF membrane filter prior to injection. The standard calibration curve was constructed using five concentrations. The linear relationship between peak area and concentration is presented in Table 6. The concentrations of the six major compounds were calculated using regression equations based on the calibration curves. To optimize the extraction conditions, a patent extraction method was performed using MeOH and EtOH with different solvent compositions (30%, 50%, 70%, and 100%) and extracted for 30, 60, 90, and 120 min, respectively.

Conclusions
After confirming that EA, BuOH, and water fraction of T. coreanum exhibited the strongest inhibitory capacities, we isolated the 11 major bioactive compounds of T. coreanum and characterized their structures. The isolated bioactive compounds inhibited AGE formation in two systems and α-glucosidase activity, which is related to diabetes and its complications. Compounds 3, 4, and 11 exhibited stronger inhibitory activity than other compounds against α-glucosidase. All compounds except compound 9 showed a stronger inhibition than the positive control and compound 11 showed the strongest activity in the BSA-glucose system. In the BSA-MGO system, all compounds showed a higher inhibition rate than the positive control. Compound 11 exhibited higher AGE-inhibitory activity than the positive control in the two systems and showed higher inhibitory potency against α-glucosidase, followed by compound 10. Compounds 1, 2, 7, 9-11 with a high inhibitory effect were investigated content of T. coreanum from variety region in Korea and T. officinale using HPLC. T. coreanum showed higher content of these bioactive compounds than T. officinale. Indeed, T. coreanum showed stronger inhibition of AGE formation and α-glucosidase activity than T. officinale. We suggested that T. coreanum was superior to T. officinale and that compounds 10 and 11 were the major components, which are responsible for the antidiabetic effects of T. coreanum. We also suggested that 30-90 min extraction with 50% ethanol was the optimized solvent condition and T. coreanum harvested from Gyeongsangnam-do, Sancheong was the most useful natural alternative medicine for diabetic complications. Based on these results, we demonstrated the preventive and therapeutic efficacy of T. coreanum and its potential use as a cost-effective phytopharmaceutical medicine in complementary therapy against type-2 diabetes and its complications. It is required to evaluate whether further research, including in vivo studies and clinical trials on the efficacy of these isolated compounds, are sufficient to the compounds be used in clinical applications. In addition, the isolated compound may have a beneficial therapeutic effect on other diseases.