10.8
CiteScore
 
5.3
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
10.8
CiteScore
5.3
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
View/Download PDF

Translate this page into:

Original article
11 (
8
); 1247-1259
doi:
10.1016/j.arabjc.2017.10.002

Identification and inhibitory activities of ellagic acid- and kaempferol-derivatives from Mongolian oak cups against α-glucosidase, α-amylase and protein glycation linked to type II diabetes and its complications and their influence on HepG2 cells’ viability

, , , , , ,
 National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Qinghuadonglu No. 35, Haidian District, Beijing 100083, China

⁎Corresponding author. yjliubio@bjfu.edu.cn (Yujun Liu)

Received: , Accepted: ,
© The Author(s) 2017
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
1 These two authors made contributions to this article equally and should be considered as co-first authors.

Abstract

This study was to characterize phenolic composition of 50% ethanol crude extract (ECE) by UPLC-QTOF-MS/MS and to investigate anti-diabetic activities of the ECE and its four fractions from Mongolian oak cups. The results show that 24 phenolics were identified from the ECE, and ellagic acid (EA)- and kaempferol-derivatives were the main phenolic components in oak cups. Dominant constituents in each of the four fractions were subsequently characterized by HPLC fingerprints. Acid hydrolysis exhibited that oak cups contained both ellagitannins and gallotannins, and ellagitannins were the dominant hydrolysable tannins. Furthermore, ECE and its four fractions exhibited much more drastic inhibitory activities against α-glucosidase than α-amylase, and formation of advanced glycation end-products was inhibited differently by ECE and Frs I-IV. Overall, EA- and kaempferol-derivatives in oak cups were the main anti-diabetic contributors, and EA-derivatives exhibited superior inhibition against α-glucosidase and glycation while kaempferol-derivatives showed stronger α-amylase inhibitory activity. In addition, Frs I-IV affected cell viability differently and kaempferol-derivatives in Fr. IV resulted in its highest anticancer activity. Aforementioned results first indicated that oak cups, being underutilized plant byproducts, should be a novel dietary phytonutrient for diabetes management with inhibitory activities against α-glucosidase, α-amylase and formation of AGEs, as well as for cancer treatment.

Keywords

Mongolian oak cup extract
Macroporous resin fractions
UPLC-QTOF-MS/MS
Ellagic acid-/kaempferol-derivatives
Anti-diabetes
HepG2 cells
1

1 Introduction

Diabetes mellitus is one of the most costly and burdensome chronic diseases in the world characterized by hyperglycaemia (Bhandari et al., 2008). It has been predicted that patient number of diabetes will increase to approximate 592 million by 2035 (Guariguata et al., 2014). In addition to risks of death, hyperglycaemia resulting from diabetes can lead to many complications, such as cardiovascular disease, nephropathy, urinary problems, and skin infections (Giovannini et al., 2016). Therefore, it is urgent to explore effective therapeutic methods for diabetes mellitus.

One promising approach for the management of diabetes is to decrease postprandial hyperglycemia, which is mainly caused by ingestion of carbohydrates. Inhibition of catalytic activities of both α-amylase and α-glucosidase could delay and prolong digestion of overall carbohydrates, leading to the retardation of glucose adsorption and consequently blunting in postprandial blood glucose level (Bhandari et al., 2008; Zhang et al., 2014). Effective inhibitors can significantly reduce fasting and postprandial hyperglycemia as well as the incidence of late diabetic complications (Zhang et al., 2016). However, artificially synthesized anti-diabetes medicines like acarbose and voglibose extensively used now, which can effectively inhibit α-glucosidase and/or α-amylase, are far from satisfying the urgency for their undesirable side effects (Kim et al., 2011). For decades, natural antidiabetic agents are receiving increasing attention due to their functionality, effectiveness, and last but not least, safety. Thus, seeking a natural and healthy antidiabetic product throughout plant kingdom is quite essential.

Another helpful strategy for preventing diabetes is to control the development of diabetic complications by inhibiting formation of advanced glycation end-products (AGEs) (Matsuda et al., 2003). AGEs are final products of the non-enzymatic reaction between reducing sugars and lipids, nucleic acids and/or free amino groups of proteins. At the first step forming AGEs, the carbonyl groups of reducing-sugars react with nucleic acids, phospholipids and/or amino groups of proteins to form fructosamines via a Schiff base by Amadori rearrangement. Next, both the Schiff base and the Amadori product further undergo a series of reactions to form the important precursors, α-dicarbonyl compounds, which are finally oxidized and crosslinked to form AGEs with various chemical structures (Matsuda et al., 2003; Reddy and Beyaz, 2006; Sang et al., 2007). AGEs’ accumulation in vivo has been implicated as a major pathogenic process in diabetic complications, such as cataract, atherosclerosis, Alzheimer and normal aging (Peng et al., 2008). Therefore, the discovery and application of inhibitors against formation of AGEs would offer a potential therapeutic approach for the prevention of diabetic or other pathogenic complications.

Mongolian oak, belonging to Fagaceae, is largely distributed in China, Russia, Mongolia, Japan and Korea and its acorns were popularly used for food. Documents have revealed that extracts of leaves, acorns or woods from oak trees possess antioxidant, antiinflammatory, anticarcinogenic, antidiabetic and antimicrobial activities (Custódio et al., 2014; Moreno-Jimenez et al., 2015; Tahmouzi, 2014), yet few investigation has been done on Mongolian oak cups, the byproduct with large biomass. It had been reported that dietary phenolic compounds were found to be effective in the control of diabetes and its complications. Phenol-rich extracts obtained from plants could modulate the activity of selected digestive enzymes, such as α-glucosidase (Toshiro et al., 2001) and α-amylase (Yang and Kong, 2016), and inhibit glycation by acting as antioxidants (Matsuda et al., 2003; Verzelloni et al., 2011). For instance, Zhang et al. (2014) reported that polyphenol extracts from burs of Castanea mollissima, also belonging to the Fagaceae, exhibited remarkable inhibition against α-glucosidase. In addition, our previous study (Yin et al., 2016) showed that the cups exhibited excellent antioxidant activities in both chemical and cellular level and possessed abundant phenolic compounds. Therefore, it is worthy to explore the anti-diabetic ability of oak cup phenolic extracts through determining the inhibitory activities against α-glucosidase, α-amylase and formation of AGEs.

Over the past few decades, ellagitannins (ETs) and ellagic acid (EA) have drawn increasing attention due to their beneficial effects to human health such as antioxidant, anti-inflammatory, anti-adipogenic and anti-carcinogenic activities (Ding et al., 2014; Naiki-Ito et al., 2015; Nunez-Sanchez et al., 2016; Okla et al., 2015; Theocharis and Andlauer, 2013). Onem et al. (2014) reported that acorn cups and beards enriched in hydrolysable tannins, especially ETs. We also found that Mongolian oak cups were rich in hydrolysable tannins as well as EA (Yin et al., 2016). Most of the EA in nature is present as ETs, which requires an acid hydrolysis step to release EA for accurately determination (da Silva Pinto et al., 2008; Espín et al., 2013). Therefore, an acid hydrolysis step was adopted to determinate the EA of oak cups in this study. Moreover, as the key component abundant in oak cups, EA’s inhibitory activities againstα-amylase, α-glucosidase and AGEs, as well as cytotoxicity on HepG2 cells were also detected separately.

In our previous study, we obtained the optimal 50% ethanol crude extract (ECE) from Mongolian oak cups and its subsequent four fractions with high antioxidant activities (Yin et al., 2016). In view of the research gaps on anti-diabetes of oak cups, the objectives of the present study were to determine the phenolic composition by UPLC-QTOF-MS/MS, and to evaluate the potentials of purified EA, ECE and fractions I-IV (Frs I-IV) as inhibitors against α-amylase, α-glucosidase and formation of AGEs for prevention and treatment of diabetes and its complications, as well as to accurately quantify EA in oak cups. These information should be able to contribute to further utilization of Mongolian oak cup as anti-diabetes resources.

2

2 Materials and methods

2.1

2.1 Chemicals and plant materials

Quercetin, rutin and ellagic acid were obtained from National Institutes for Food and Drug Control (Beijing, China). Yeast α-glucosidase from Sccharomyces cerevisiae, α-amylase, and p-nitrophenyl-α-glucopyranoside (pNPG) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Girard-T was bought from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). HepG2 cells were purchased from China Academy of Medical Sciences (Beijing, China). DMEM medium, bovine serum albumin (BSA), PBS, trypsin, fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Hyclone (Logan, Utah, USA).

Mongolian oak (Quercus mongolica Fisch. ex Ledeb) cups were collected on September 2015 from Grand Khingan in Heilongjiang province, and authenticated by Dr. Z. Liu, Beijing Forestry University, Beijing, China. The oak cups were air-dried until equilibrium humidity, ground and screened through a 1-mm mesh, then stored in darkness at room temperature for further use.

2.2

2.2 Preparation of the ethanol crude extract (ECE) and its four fractions (Frs I-IV)

The ECE and Frs I-IV were prepared according to our previous report (Yin et al., 2016). In brief, 50 g oak cup powders were extracted by heating reflux for 1 h at 80 °C with 500 mL of 50% aqueous ethanol for three times, and the combined supernatants (∼1500 mL) were rotarily evaporated till one third of the volume left, which were then diluted to 1500 mL with distilled water. Next, 300 mL of the 1500 mL solution were evaporated then dried in the water bath at 60 °C to prepare ECE, the rest 1200 mL was subjected to HPD100 macroporous resin (Cangzhou Bonchem Co., Ltd, Cangzhou, China) sequentially eluted with 2000 mL of distilled water, 2000 mL of 30% aqueous ethanol, 1500 mL of 50% aqueous ethanol, and 1500 mL of 80% aqueous ethanol, and these four collected fractions were evaporated and dried in the water bath at 60 °C to obtain dried Frs I-IV. Contents of Frs I-IV in ECE were 14.49%, 45.23%, 3.87% and 3.25%, respectively. Finally, Frs I-IV, together with ECE, were stored at −20 °C for further analyses.

2.3

2.3 UPLC-QTOF-MS/MS analyses of phenolic composition in ECE

The UPLC-QTOF-MS/MS system was comprised of an Acquity Ultra-Performance Liquid Chromatography (UPLC) system (Waters, Milford, MA, USA) and a QTOF-MS mass spectrometer (Xevo G2-XS, Waters). A Diamonsil C18 column (250 mm × 4.6 mm, 5µm) was used for separation, and the column temperature was set at 25 °C. The mobile phase was consisted of water with 0.4% formic acid (v:v) (A) and acetonitrile (B) under the following gradient program: 0–30 min, 5–30% B; 30–40 min, 24–45% B; 40–50 min, 45–63% B. The flow rate was set at 1 mL/min with an injection volume of 10 µL. Mass spectra were recorded in the range of m/z 100–1500. MS experiments were performed both in positive and negative ionization mode under the following conditions: nitrogen drying gas flow, 10.0 L/min; nebulizer pressure, 45 psi; gas drying temperature, 370 °C; capillary and fragmentor voltage, 2.500 kV; and with MS/MS collision energies set at 20 V. Peak identification was performed by comparing the mass spectra and fragmentation ions with data from reported literatures and METLIN database (https://metlin.scripps.edu).

2.4

2.4 HPLC fingerprints of ECE and Frs I-IV

HPLC analyses were performed with a Shimadzu HPLC system (Shimadzu, Japan) equipped with two LC-10AT VP pumps, a SPDM20A ultraviolet detector, and a SIL-20AC TH autosampler controlled by an analytical software (LC Solution-Release 1.23SP1). The Diamonsil C18 column (250 mm × 4.6 mm, 5 µm) was used, and the column temperature was set at 25 °C. Solvent system and gradient program same to those in the UPLC analysis described above were used for comparison. The flow rate was also set at 1 mL/min with an injection volume of 10 μL. Detection wavelength was set at 280 nm to monitor more phenolic compounds simultaneously. At the end of each running, the column was flushed with 95% acetonitrile for 10 min to remove strongly retained constituents, and then equilibrated for 10 min under initial conditions.

2.5

2.5 Acid hydrolysis of ECE and quantification of EA and GA by HPLC

The acid hydrolysis was adopted to hydrolyze ETs and gallotannins (GTs) for determining EA and gallic acid (GA) in oak cups. A 20-mg ECE was diluted in 10 mL solvent mixture comprising ethanol/HCl/water (50:10:40), and hydrolyzed at 80 °C for various durations (0, 4, 6, 8 and 10 h). Subsequently, the acid-hydrolyzed solutions were evaporated to dryness with a rotary evaporator (Heidolph Instrument, Schwabach, Germany) at 60 °C to obtain one non-hydrolyzed (i.e., ECE) and four acid hydrolyzed samples. Then, they were dissolved with methanol to a concentration of 2 mg/mL. Prior to injection into HPLC system, these solutions were filtered through a 0.22-μm nylon membrane. EA and GA determination was conducted with HPLC system described above using our previous conditions (Zhang et al., 2014).

2.6

2.6 Determinations of α-amylase and α-glucosidase inhibitory activities by EA, ECE, and Frs I-IV

α-Amylase inhibitory activity was determined as reported previously (Yang and Kong, 2016) with some modification. In brief, soluble starch solution (0.5%) was prepared by dissolving starch in phosphate buffer (pH 6.9, 20 mmol/L, containing 6.7 mM NaCl) and gelatinized at 90 °C for 20 min, sample (‘sample’ here and after referred to EA, ECE or Frs I-IV) was dissolved in DMSO at 25 mg/mL as stock solution and diluted with phosphate buffer to different concentrations (0, 100, 300 and 500 μg/mL), and 500 μL sample solution and α-amylase (3.185 unit/mL) were incubated at 37 °C for 15 min. After incubation, 500 μL soluble starch were added at 37 °C for 5 min and the mixture was stopped with 1.0 mL dinitrosalicylic acid color reagent. After that, the mixture was boiled for 10 min and cooled to room temperature. The reaction mixture was then diluted 2 times with distilled water, and absorbance was measured at 540 nm using a spectrophotometer (Shimadzu UV-1700, Japan). The readings were compared with the control, which contained phosphate buffer instead of sample solution. The inhibitory effect was calculated using following formula: Inhibitory effect (%) = (ODcontrol − ODsample) /ODcontrol × 100. For determination of IC50 for α-amylase, individual samples and acarbose were prepared at different concentrations (0, 10, 100, 300, 500, 1000 and 3000 μg/mL).

Inhibitory effect of a sample against α-glucosidase was carried out using pNPG as the substrate as described previously by (Zhang et al., 2014). Briefly, 10 μL α-glucosidase (1.0 unit/mL) were mixed with 60 μL phosphate buffer (0.1 mM, pH 6.8) and 100 μL different concentrations of a sample in corresponding well of a 96-well plate and incubated for 10 min at 37 °C. Then, 2 mM pNPG solution in 0.1 mM phosphate buffer was added quickly to initiate the enzyme reaction. The absorbance was monitored at 405 nm every 15 min for 2 h using a microplate reader (Tecan infinite 200, Swiss). The same volume (100 μL) of phosphate buffer and acarbose (200, 500, 1000 and 2000 μg/mL) were used as the negative and positive control, respectively. The enzyme inhibitory activity was determined by calculating the net area under the curve (AUC) for each sample or acarbose and comparing the net AUC with that of the negative control. The formula was as following: Inhibitory activity (%) = (An − Ai)/An × 100, where An is the AUC of negative control and Ai is the AUC of solution with inhibitors (sample or the positive control, acarbose). In addition, individual samples and acarbose were also prepared at different concentrations (0, 1, 2, 4, 10, 100 and 1000 μg/mL) for determination of IC50 for α-glucosidase.

2.7

2.7 Determinations of AGEs inhibitory activity by EA, ECE and Frs I-IV

2.7.1

2.7.1 Preparation of AGEs-BSA

Fluorescent AGEs were prepared using the method of Chompoo et al. (2011). In brief, stock solution at 60 mg/mL was prepared by dissolving a standard (quercetin or rutin) or sample (EA, ECE or each of Frs I-IV) in DMSO. Then, the stock solution was diluted to 0.1 and 0.2 mg/mL with phosphate buffer. To obtain AGEs, BSA (0.8 mg/mL) was incubated at 60 °C for 30 h with d-glucose (200 mM) in 50 mM phosphate buffer (pH 7.4) with or without 10 μL of the diluted standard or sample. Quercetin and rutin were used as the positive control. The AGEs solution was used for further as glycated material.

2.7.2

2.7.2 Fluorescence measurement of BSA glycation

BSA glycation assay was performed according to a method developed by Chompoo et al. (2011). An aliquot of glycated material obtained above (100 μL) was mixed with 10 μL of 100% (w/v) trichloroacetic acid. After centrifugation (20627g, 4 °C, 4 min), supernatant was removed, and precipitate of AGEs-BSA was dissolved with 100 μL alkaline PBS. Formation of AGEs was measured with an excitation at 530 nm and an emission at 485 nm using a multi-functional fluorescence detector (Tecan infinite 200, Swiss). Inhibition percentage of the AGEs formation was calculated using the following equation: Inhibitory activity (%) = (1 − Fi/Fo) × 100 where Fi and Fo are fluorescences with and without inhibitor, respectively.

2.7.3

2.7.3 Measurement of fructosamine adducts

Fructosamine adduct was measured using the NBT assay as described by Baker et al. (1994). An aliquot of glycated material (20 μL) was added to 160 μL NBT reagent (300 μM) dissolved in sodium carbonate buffer (100 mM, pH10.35), and reaction mixture was incubated at room temperature for 30 min. Absorbance was measured at 530 nm using the microplate reader. Inhibition percentage of fructosamine was calculated using the following equation: Inhibitory activity (%) = (1 − Ai/Ao) × 100, where Ai and Ao are absorbances with and without inhibitor, respectively.

2.7.4

2.7.4 Measurement of α-dicarbonyl compounds

Formation of α-dicarbonyl compounds was measured as reported by Chompoo et al. (2011) with modifications. Briefly, 5 μL glycated material was incubated with 10 μL Girard-T solution (500 mM) and 170 μL sodium formate (500 mM, pH2.9) for 1 hat room temperature, and absorbance was measured at 290 nm using the microplate reader. Inhibition percentage of α-dicarbonyl compounds was calculated using the same equation as that for fructosamine.

2.8

2.8 Cell viability assay

HepG2 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2 in a humidified atmosphere. Cell viabilities were assessed using the MTT assay (Khan et al., 2012). Briefly, HepG2 cells at a density of 2 × 104 cells/well were cultured in a 96-well plate for 12 h, then treated with different concentrations of EA, ECE or Frs I-IV (50, 100, 200 and 500 μg/mL) for 24 h. Subsequently, 20 μL MTT (5 mg/mL) was added into each well and incubated at 37 °C for 4 h. Finally, the medium was removed, 150 μL DMSO was added to each well, and absorbance was read at 570 nm using the microplate reader. HepG2 cells without treatment were used as control and the DMEM medium with or without corresponding sample (EA, ECE or each of Frs I-IV) was used as blank. The experiment was performed six times and the percentage of cell viability was calculated as follows: cell viability (%) = (A570sample – A570blank)/(A570control – A570blank) × 100.

2.9

2.9 Statistical analysis

Data were presented as mean ± SD of three parallel measurements. The statistical significance (t-test: two-sample equal variance, using two-tailed distribution) was determined by using Microsoft Excel statistical software (Microsoft Office Excel 2016, Microsoft Corp. Redmond, WA, USA). Values of P < .05 were set to be significant.

3

3 Results and discussion

3.1

3.1 Twenty-four phenolic compounds were identified by UPLC-QTOF-MS/MS from ECE

Fig. 1 and Table 1 show the 24 phenolic compounds identified from ECE through UPLC-QTOF-MS/MS analysis, along with their retention times (RT), experimental m/z, calculated m/z, error values (ppm), molecular formula, and MS/MS fragments listed in the table. Among them, compound 2 was identified as quinic acid based on its [M-H]− ion at m/z 191.0568, and MS/MS spectrum showing ions at 173 (M-H-H2O) and 127 (M-H-CO-2H2O) and also other fragments at 93 and 85 (Santos et al., 2013). Compound 14 was identified as catechin as its [M-H]- was observed at m/z 289.0707 and it fragmented at 245 (Yang et al., 2017). Compound 19 with a parent ion at m/z 447.0916 was identified as quercetin-3-O-rhamnoside (quercitrin) by comparison with the MS/MS data of a previous study (Dorta et al., 2014). The other 21 compounds could be classified into following two groups, namely, 5 kaempferol-derivatives and 16 EA-derivatives. Since this was the first report on its detailed phenolic composition, all phenolics other than EA were considered to be first characterized in Mongolian oak cup. Moreover, 22, 10, and 9 phenolics were identified for the first time in Mongolian oak, Quercus and Fagaceae species, respectively.

UPLC-QTOF-MS profile of ECE in negative ion mode. Each peak number was in accordance with that in Table 1. Ellagic acid-derivatives and kaempferol-derivatives were divided into two groups based on their retention times as indicated by the dashed line.
Fig. 1
UPLC-QTOF-MS profile of ECE in negative ion mode. Each peak number was in accordance with that in Table 1. Ellagic acid-derivatives and kaempferol-derivatives were divided into two groups based on their retention times as indicated by the dashed line.
Table 1 Phenolic compounds tentatively identified from the ECE by UPLC-QTOF-MS/MS analyses.

Peak No.		 RT Exp. m/z Calc. m/z Error (ppm) Formula MS/MS fragments (m/z) Proposed compound
1 2.34 631.0557 631.0571 −2.2 C27H19O18 569.0467, 467.0154 castalin
2 2.51 191.0568 191.0556 6.3 C7H11O6 85.0304, 93.0354, 127.0401, 173.0457 quinic acid
3 3.19 331.0660 331.0665 −1.5 C13H15O10 169.0170 galloyl glucose
4 3.38 481.0630 481.0618 2.5 C20H17O14 300.9980, 275.0200 HHDP-glucose
5 5.81 633.0737 633.0728 1.4 C27H21O18 301.0000, 275.0200, 169.0118 isostrictinin
6 6.24 169.0146 169.0137 5.3 C7H5O5 125.0238 gallic acid
7 7.15 1055.0718 1055.0638 7.6 C47H27O29 300.9991, 481.0215, 169.0124 castacrenin E
8 7.39 633.0674 633.0728 −8.5 C27H21O18 481.0628, 301.0038, 169.0087 strictinin
9 8.32 933.0681 933.0634 5.0 C41H25O26 915.0485, 301.0020 vescalagin
10 9.40 783.0657 783.0681 −3.1 C34H23O22 301.0038, 275.0206, 481.0611 pedunculagin β
11 10.85 933.0674 933.0634 4.3 C41H25O26 915.0789, 631.05, 300.9963 castalagin
12 12.88 783.0673 783.0681 −1.0 C34H23O22 481.0586, 300.9980, 275.0128 pedunculagin-α
13 14.83 785.0826 785.0837 −1.4 C34H25O22 300.9997, 275.0154 tellimagrandin I β
14 15.98 289.0707 289.0712 −1.7 C15H13O6 245.0669 catechin
15 18.27 785.0875 785.0837 4.8 C34H25O22 300.9993, 275.0152, 169.0114 tellimagrandin I α
16 24.82 433.0420 433.0407 3.0 C19H13O12 301.0029, 300.0003 ellagic acid-pentoside
17 26.06 1083.0547 1083.0587 −3.7 C48H27O30 301.0035, 915.1547 punicalagin
18 27.32 300.9985 300.9984 0.3 C14H5O8 284.0000, 245.0118, 229.0318, 201.0212 ellagic acid
19 31.41 447.0916 447.0927 −2.5 C21H19O11 300.0256, 301.0338, 255.0251 quercetin-3-O-rhamnoside
20 37.82 593.1297 593.1295 0.3 C30H25O13 285.0404, 447.1001, 145.0283 kaempferol-3-O-(6″-di-E-p-coumaroyl)-glucoside
21 38.36 593.1290 593.1295 −0.8 C30H25O13 285.0339, 447.0804, 145.0134 kaempferol-3-O-(6″-di-Z-p-coumaroyl)-glucoside
22 42.48 739.1643 739.1663 −2.7 C39H31O15 285.0399, 453.1180, 145.0291 kaempferol-3-O-(2″,6″-di-E-p-coumaroyl)-glucoside
23 43.00 739.1647 739.1663 −2.2 C39H31O15 593.1290, 285.0398, 453.1169, 145.0291 kaempferol-3-O-(2″-Z,6″-E-dip-coumaroyl)-glucoside
24 43.51 739.1664 739.1663 0.1 C39H31O15 593.1336, 285.0417, 453.1120, 145.0319 kaempferol-3-O-(2″,6″-di-Z-p-coumaroyl)-glucoside

As signals in a negative mode was stronger than those in a positive mode, data collected in the negative mode was thus chosen to conduct the identification.

The peak number of each compound was in accordance with that in Fig. 1.

3.1.1

3.1.1 Kaempferol-derivatives

Compounds 20 and 21 were assigned as kaempferol-3-O-coumaroyl-glucoside isomers as they both shared a [M-H]− at 593 and fragments at 447 (M-H-p-coumaroyl), 285 (kaempferol-H), and 145 (p-coumaroyl-H). Accordingly, compound 20 was identified as kaempferol-3-O-(6″-di-E-p-coumaroyl)-glucoside and compound 21 as kaempferol-3-O-(6″-di-Z-p-coumaroyl)-glucoside based on their eluting order in the C18 columns (Karioti et al., 2010). Compounds 22, 23 and 24 were assigned as kaempferol-3-O-di-coumaroyl-glucoside isomers by comparison with the MS/MS data of a previous study (Karioti et al., 2010). Likewise, they were identified as kaempferol-3-O-(2″,6″-di-E-p-coumaroyl)-glucoside, kaempferol-3-O-(2″-Z,6″-E-di-p-coumaroyl)-glucoside, and kaempferol-3-O-(2″,6″-di-Z-p-coumaroyl)-glucoside, respectively, based on their elution order in the C18 columns (Karioti et al., 2010).

3.1.2

3.1.2 EA-derivatives

Compound 1, which gave a [M-H]− ion at 631.0557, was identified as castalin (an ellagitannin) as its fragmentation pattern shows a predominant fragment ion at 613 originating from the loss of a water (M-H-H2O) and further fragments at 569 and 467 (Muccilli et al., 2017). Compound 3 showed a [M-H]− at 331.0660 and a fragment at 169 which is typical of gallic acid, and thus was identified as galloyl glucose (Dorta et al., 2014). Compound 6 gave a [M-H]− at 169.0137 and a major fragment at 125 caused by the loss of a –CO2 group, and thus was identified as gallic acid (GA), which was normally found in Quercus spp. extracts (Muccilli et al., 2017). Galloyl glucose and GA were related to the biosynthesis of EA, thus they were also classified here as EA-derivatives.

Compound 18 was identified as EA by comparing their retention time and MS/MS spectra with its standard. Moreover, the EA ion at 301 occurred not only in compounds 4 and 5, but also in compounds 7–13 and 15–17, indicating that all these compounds shared the EA moieties. Compound 4 was identified as HHDP-glucose (i.e., a glucose esterified by hexaydroxyhexadiphenic acid) and showed a [M-H]− at 481.0630 and a predominant fragment at 301 (M-H-glucose) due to the loss of a glucose. Compounds 5 and 8 shared a common parent ion at 633 and daughter ions at 301 and 169, which were assigned as EA and GA moieties, thus they were identified as isostrictinin and strictinin, respectively (Jackrel et al., 2016). Compound 7 exhibited a [M-H]− at 1055.0718 with the molecular formula C47H27O29 and was identified as castacrenin E as its MS/MS profile corresponded with that reported by Tanaka et al. (1997). Compounds 9 and 11 gave a common [M-H]− signal at 933.0681, and fragmented at 915 with a loss of H2O, at 631 with a loss of an HHDP moiety, and a further signal at 301 suggesting the loss of vescalin or castalin, thus were identified as vescalagin and castalagin, respectively (Muccilli et al., 2017). Likewise, compounds 10 and 12 with the same molecular ion at 783 were identified as pedunculagin β and pedunculagin α, respectively, as they fragmented at 481 due to the loss of an ellagic acid unit (302) and at 301 with a loss of one HHDP-glucose (482) (Jackrel et al., 2016). Compounds 13 and 15 both showing significant [M-H]− signals at 785 were determined as tellimagrandin Iβ and tellimagrandin Iα, respectively, by comparison with the MS/MS data of a previous study (Jackrel et al., 2016). Compound 16 was identified as ellagic acid-pentoside based on their characteristic [M-H]− at 433.0420 and a MS/MS fragment at 301 due to the loss of a pentose unit (Santos et al., 2013). Compound 17 yielded a [M-H]− at 1083 which corresponded with the molecular formula C48H27O30, a fragment at 301 corresponding with the radical anion of EA, and a fragment at 781, and was thus determined as punicalagin (Borges and Crozier, 2012).

The above results showed that EA- and kaempferol-derivatives were the main phenolic constituents in ECE. Furthermore, these conclusions could also be deduced from the HPLC profiles of ECE and Frs I-IV (Fig. 2) conducted with the same conditions as those in UPLC-QTOF-MS/MS analysis shown in Fig. 1. The profiles show that small portion of EA-derivatives existed in Fr. I (see the red curve), Fr. II consisted of almost entire EA-derivatives (green curve), Fr. III were the mixture of EA- and kaempferol-derivatives (yellow curve), whereas Fr. IV were composed of almost all kaempferol-derivatives (blue curve).

HPLC chromatograms of the ECE (black curve) and Frs I-IV (red, green, yellow and blue curves, respectively). All these five curves were obtained under a uniform HPLC condition, which was also the same as that described in the UPLC analysis. The range of the figure’s y-axis was adjusted to clearly display all curves. Ellagic aid- and kaempferol-derivatives can be divided into two groups based on their retention times as indicated by the dashed line.
Fig. 2
HPLC chromatograms of the ECE (black curve) and Frs I-IV (red, green, yellow and blue curves, respectively). All these five curves were obtained under a uniform HPLC condition, which was also the same as that described in the UPLC analysis. The range of the figure’s y-axis was adjusted to clearly display all curves. Ellagic aid- and kaempferol-derivatives can be divided into two groups based on their retention times as indicated by the dashed line.

3.2

3.2 ECE under acidic conditions released large amount of EA but little GA

Most of the EA is present as ETs in nature, which demanding an acid hydrolysis step to release EA for accurately determining the total EA content (da Silva Pinto et al., 2008; Espín et al., 2013). In this study, ECE was hydrolyzed with 10% HCl for the quantification of total EA.

As shown in Fig. 3A, EA was 14.30 mg/g d.w. in ECE, and significantly increased to 176.67 after acid hydrolysis for 4 h. The EA content reached to the maximum at 6 h (198.17) and showed slight declines but with no significant difference at 8 and 10 h. Thus conclusion can be drawn that the hydrolysis of ETs was completed at 6 h. GA (Fig. 3B; 7.80 mg/g d.w.) was much less than EA in ECE, and it increased also abruptly during the first 4 h then linearly with a much lower slop but all with significant differences between two acid hydrolysis treatments from 4 to 10 h. Taken together, the data indicated that oak cups are rich in EA or ETs rather than GA or GTs, and acid hydrolysis was a superior way to improve both the levels of EA and GA from ETs and GTs, respectively, for determining their total contents precisely.

Effects of acid hydrolysis on EA and GA contents in ECE determined by HPLC. (A and B) EA (A) and GA (B) after acid hydrolysis were presented as mean ± SD (n = 3). Different letters mean significant difference (P < .05). (C-E) The right side figures show standards’ profiles (C; a, GA; b, EA) and those of ECE before (D) and after (E) acid hydrolyses for 6 h. Note that y-axes of the three profiles were set at the same scale for comparison, and the left most was the solvent peak.
Fig. 3
Effects of acid hydrolysis on EA and GA contents in ECE determined by HPLC. (A and B) EA (A) and GA (B) after acid hydrolysis were presented as mean ± SD (n = 3). Different letters mean significant difference (P < .05). (C-E) The right side figures show standards’ profiles (C; a, GA; b, EA) and those of ECE before (D) and after (E) acid hydrolyses for 6 h. Note that y-axes of the three profiles were set at the same scale for comparison, and the left most was the solvent peak.

Fig. 3D and E shows HPLC profiles of ECE before (D) and after (E) acid hydrolyses for 6 h. No matter with or without the 6 h acid hydrolysis, the peaks of substances were mainly occurred between 2 and 20 min and EA was the most intense peak. After the acid hydrolysis (Fig. 3E), intensities of certain peaks showed a huge rise (marked with ∗∗ in Fig. 3E), among which EA showed the biggest increase, while several other peaks decreased or nearly disappeared (both marked with ∗ in Fig. 3D). It can be interpreted that the compounds declined or disappeared were converted or hydrolyzed to other compounds, including EA and/or GA. On the other hand, certain undetectable compounds at 280 nm might be transformed to other simple phenolics that were detected after 6 h acid hydrolysis. Furthermore, as EA and GA increased after acid hydrolysis, it can be concluded that oak cups contained both ETs and GTs. In view of the high EA and low GA contents, it can be inferred that ETs was the dominant hydrolysable tannins in oak cups, also indicating that EA-derivatives were the main phenolic constituents in ECE with numerous varieties and large amount, and thus EA was employed for further research.

3.3

3.3 EA, ECE, and Frs I-IV exhibited distinguishing inhibitory effects against α-amylase and α-glucosidase

Activity inhibitions of α-amylase and α-glucosidase are considered to be effective strategies for the control of diabetes by diminishing the absorption of glucose (Kim et al., 2011). Inhibitory effects of EA, ECE, and Frs I-IV on α-amylase and α-glucosidase were determined and acarbose was used as a positive control (Fig. 4). As shown in Fig. 4A, ECE and Frs I-IV exhibited inhibitory activities against α-amylase differently, and the inhibitory effects of Fr. IV was better than Fr. II. Considering that Fr. IV were almost all kaempferol-derivatives and Fr. II consisted of almost entire EA-derivatives, it could be concluded that kaempferol-derivatives exhibited stronger inhibitory activities against α-amylase than EA-derivatives. In addition, the inhibitory activity of Fr. III was the highest, next only to acarbose. Since Fr. III were the mixture of EA- and kaempferol-derivatives, most of the EA-derivatives in it was EA, and EA exhibited no inhibitory activity against α-amylase, it could be inferred that kaempferol-derivatives in Fr. III were much more superior than those in Fr. IV. Meanwhile, there might be synergistic effects between EA- and kaempferol-derivatives in Fr. III. Irondi et al. (2017) reported that Adansonia digitata leaves extract, with high kaempferol contents, exhibited a strong inhibitory activity against α-amylase, which also supported the superiority of kaempferol-derivatives by the current work.

Inhibitions of acarbose, EA, ECE and Frs I-IV against α-amylase and α-glucosidase. Inhibitory effects of α-amylase (A) and α-glucosidase (B) were measured with different concentrations of ECE, Frs I-IV, EA or acarbose. Inhibitory effect of EA against α-amylase was not detected within the experiment concentrations and was thus not present in (A). Data of acarbose could not be showed in (B) as its effective concentration (200, 500, 1000 and 2000 μg/mL) was far more beyond the range of this figure’s x-axis.
Fig. 4
Inhibitions of acarbose, EA, ECE and Frs I-IV against α-amylase and α-glucosidase. Inhibitory effects of α-amylase (A) and α-glucosidase (B) were measured with different concentrations of ECE, Frs I-IV, EA or acarbose. Inhibitory effect of EA against α-amylase was not detected within the experiment concentrations and was thus not present in (A). Data of acarbose could not be showed in (B) as its effective concentration (200, 500, 1000 and 2000 μg/mL) was far more beyond the range of this figure’s x-axis.

α-Glucosidase, which is readily available in a pure form and has been widely used in nutraceutical research and medical investigations as a model for screening its potential inhibitors (Zhang et al., 2014; Zhang et al., 2011), is used to detect the inhibitory activities of EA, ECE or Frs I-IV. As shown in Fig. 4B, EA, ECE, and Frs I-IV all exhibited potent inhibition against α-glucosidase and the order was as follows: Fr. II > ECE > Fr. III > EA > Fr. I > Fr. IV. When the concentration was lower than 2 μg/mL, the inhibitory effects of ECE and Fr. II were much stronger than those of Frs III, I and IV. The effects of ECE and Fr. II, Fr. III and EA, and Frs I and IV reached their highest points at 4, 6, and 10 μg/mL, respectively, with that of Fr. IV was the lowest and less than half of all the others at the highest concentration. Relating the inhibitory activities of Frs I-IV to their phenolic compositions, it can be inferred that EA-derivatives in Fr. II contributed to most α-glucosidase inhibitory activities, and could be a promising objective for a next research. According to our previous study, Fr. III contained considerable EA (Yin et al., 2016). Since the high inhibition of EA and its similar increase pattern of inhibitory effect comparing with Fr. III, the strong inhibitory activity of Fr. III against α-glucosidase might be due to its abundant EA. As for Fr. IV, the weakest inhibitory activity against α-glucosidase might be due to its enrichment in kaempferol-derivatives, but the inhibition of Fr. IV was still better than acarbose, whose inhibitory values were too low to be displayed in Fig. 4A at the same concentration ranges with other samples. In this sense, it could be concluded that EA-derivatives exhibited much better inhibitory effect against α-glucosidase than kaempferol-derivatives and they both showed stronger inhibition than that of acarbose.

Moreover, the inhibitions of ECE and EA against α-glucosidase were reversible and noncompetitive (data not shown), which was the same as that reported by Zhang et al., (2014), who reported that the burs of Castanea mollissima also exhibited noncompetitive inhibition against α-glucosidase.

IC50 values of ECE and Frs I-IV against α-amylase and α-glucosidase (Table 2) were defined as micrograms of the ECE or each of the four fractions per mL for 50% inhibition of enzymatic activities. Rank order of IC50 values against α-amylase was: acarbose < Fr. III < Fr. IV < Fr. II < ECE < Fr. I < EA (Table 2; A lower IC50 value indicated a stronger inhibitory ability to the enzyme; IC50 of EA against α-amylase was deduced as the highest in that its inhibitions was not detected within the experiment concentration range). Tan et al. (2017) reported that the crude extract and three major column fractions of black soybean showed effective inhibitory capacity against α-amylase (IC50 values: 250–2250 μg/mL), which was similar to those of ECE and Frs I-IV (119.59–2933.90). Especially, the inhibitory activities of Frs III and IV (IC50 values: 119.59 and 166.11, respectively) were much stronger than those of black soybean. Thus, the results demonstrated the potential of oak cups for use in the inhibition of α-amylase as well.

Table 2 Inhibitory effects reflected in IC50 values (μg/mL) of EA, ECE and FrsI-IV against α-glucosidase and α-amylase.
α-amylase α-glucosidase
ECE 803.38 ± 18.66a 1.63 ± 0.03a
Fr.I 2933.90 ± 166.71b 4.59 ± 0.11b
Fr.II 272.14 ± 3.91c 1.17 ± 0.01c
Fr.III 119.59 ± 0.30d 2.85 ± 0.16d
Fr.IV 166.11 ± 0.53e 13.02 ± 0.42e
EA The highest* 3.65 ± 0.09f
Acarbose 25.30 ± 0.25f 973.02 ± 19.65g

Different letters indicate significant differences (P < .01).

* For explanation, see the third paragraph of this subsection.

Rank order of IC50 values against α-glucosidase was: Fr. II < ECE < Fr. III < EA < Fr. I < Fr. IV < acarbose, with inhibitions of the lowest (Fr. IV, 13.02 μg/mL) being even 75- and the highest (Fr. II, 1.17) being 832-fold more effective than that of acarbose (973.02). And the IC50 values of ECE and Frs I-IV was similar to the report of Sheikh et al. (2015), who found that IC50 value of Quercus serrata leaves extract against α-glucosidase was 1.85 μg/mL. It can be concluded that oak cups could be, if not the most, a potent α-glucosidase inhibitor for the treatment of diabetes. Grapes, rich in phenolic compounds, particularly flavonols, anthocyanins and procyanidins, exhibited excellent antioxidant activity. Zhang et al. (2011) had reported that the IC50 of Norton grape skin extract was 384 μg/mL on inhibiting α-glucosidase, which was much higher than that of ECE (1.63 μg/mL). Moreover, Tundis et al. (2016) found the juice of trifoliate orange, which has been proven to have health-promoting properties, inhibited α-glucosidase with an IC50 value at 81.27 μg/mL, that was much higher than the IC50 values of ECE and Frs I-IV. The results above also demonstrate potent inhibitory activity of oak cups against α-glucosidase.

Furthermore, ECE and Frs I-IV had relatively lower inhibition abilities against α-amylase, while displaying superior inhibitory effect against α-glucosidase. This is in accordance with previous study reporting that natural compounds from plants have usually a stronger inhibitory activity against α-glucosidase when compared with α-amylase (Custódio et al., 2014; Tan et al., 2017). Overall, the results demonstrated the potential of oak cups extract (especially its Frs II and III) for diabetes management.

3.4

3.4 Formations of AGEs were inhibited differently by EA, ECE, and Frs I-IV

3.4.1

3.4.1 EA, ECE, and Frs II and III exhibited superior inhibitory effects on BSA glycation

Protein glycation is extremely relevant in the progression of several diabetes complications (Verzelloni et al., 2011). Inhibitory effects of EA, ECE, and Frs I-IV on formation of AGEs were thus evaluated using the BSA-glucose assay, in which BSA served as the model protein and glucose as the glycating agent. As shown in Fig. 5A, quercetin, rutin, EA, ECE, and Frs I-IV all exhibited certain inhibitory effects on BSA glycation in a more or less dose-dependent manner. Moreover, the inhibitory activities of ECE, Frs II and III, and EA were all higher than those of Frs I and IV. At 0.1 mg/mL, EA showed the strongest inhibitory effect (77.36%), followed by Fr. II (69.46%), quercetin (69.18%), ECE (54.08%), Fr. III (43.60%), rutin (34.17%), Fr. I (13.16%) and Fr. IV (6.87%). At 0.2 mg/mL, the order of the inhibitory effect on glycation of BSA was a little different from that at 0.1 mg/mL. Taken together, the inhibitory effect of EA was the highest in both concentrations, and those of ECE, Frs II and III were higher or a little lower than quercetin but all higher than rutin, while those of Frs I and IV were the lowest. The above results might also indicate that EA-derivatives including EA itself enriched in Fr. II and III were better inhibitors on BSA glycation than kaempferol-derivatives, which were enriched in Fr. IV (Fig. 5). Moreover, our previous study had demonstrated that the ECE and Frs I-IV were rich in phenolic compounds with considerable antioxidant activity and the content of phenolic compounds in ECE and Frs II-III were significantly higher than Frs I and IV (Yin et al., 2016). Thus, we predicted the abundant phenolic compounds in ECE and Frs I-IV were the important AGEs inhibitors by acting as antioxidants. The results were consistent with that of Peng et al. (2008), who reported that the phenolic compounds might be the major contributors to anti-glycation activity.

Inhibition (%) by quercetin, rutin, EA, ECE and Frs I-IV on BSA glycation (A) and formations of fructosamine adducts (B) and α-dicarbonyl compounds (C). Different lowercase or capital letters indicate significant difference (P < .05) at 0.1 and 0.2 mg/mL, respectively.
Fig. 5
Inhibition (%) by quercetin, rutin, EA, ECE and Frs I-IV on BSA glycation (A) and formations of fructosamine adducts (B) and α-dicarbonyl compounds (C). Different lowercase or capital letters indicate significant difference (P < .05) at 0.1 and 0.2 mg/mL, respectively.

3.4.2

3.4.2 EA, ECE, and Frs II and III could exert their inhibitory effects on formation of fructosamine adducts at the early stage of glycation

Fructosamine is a marker of early glycation (Joglekar et al., 2014) and it is an Amadori product formed by glycation of amino acid via Schiff’s base. In order to further verify AGEs inhibition, the amount of fructosamine formed was evaluated using a colormetric assay by measuring the reducing activity of serum in alkaline solution (Chompoo et al., 2011). As shown in Fig. 5B, EA, ECE, and Frs I-IV could inhibit the formation of fructosamine adducts in different degrees. The inhibitory effects of EA, ECE, and Frs II and III were higher than those of Frs I and IV at both experimental concentrations. At 0.1 mg/mL, quercetin and Fr. II showed the strongest inhibitory effects by 39.78% and 38.40% (with no significant difference), respectively, followed by ECE (24.85%), Fr. III (20.62%), rutin (18.09%), EA (10.62%), Fr. I (10.32%) and Fr. IV (9.54%), and there was no significant difference among EA, Fr. I and Fr. IV. At 0.2 mg/mL, the order of inhibitory rate was quercetin > rutin > Fr. III > Fr. II > ECE > EA > Fr. I > Fr. IV, and the inhibitory rates of ECE and Frs II and III were similar to that of rutin. It had been reported that most of AGEs inhibitors prevented the formation of AGEs at the late stage of glycation, but a few of them exerted their effects at its early stage (Mesías et al., 2013). As shown in Fig. 5B, fructosamine adduct formation, a marker of early glycation, could be inhibited by EA, ECE, and Frs I-IV in different extents, indicating that EA, ECE, and Frs I-IV (especially Frs II and III) could exert their effects at the early stage of glycation.

3.4.3

3.4.3 Frs I and IV contained components counteracting the superior inhibitory effects of Frs II and III in ECE on formation of α-dicarbonyl compounds

Reactive α-dicarbonyl compounds such as glyoxal and methylglyoxal are produced during oxidative degradation of Amadori products. They are highly reactive towards protein-NH2 groups and can form protein crosslinks leading to formation of AGEs, which can be measured as Protein Bound Carbonyls (PCO) using the DNPH assay and are considered as markers of oxidative protein modification (Liggins and Furth, 1996). As shown in Fig. 5C, quercetin, rutin, EA, and Frs II and III could inhibit the formation of α-dicarbonyl compounds in a concentration-dependent manner. Nevertheless, there was no inhibitory (or even little stimulatory) activities of ECE and Frs I and IV at 0.1 mg/mL, but they showed slight activities at 0.2 mg/mL. These variations may be explained by different kinds and/or different contents of active compounds in ECE and Frs I-IV. In addition, the strong inhibition of Frs II and III might be due to strong antioxidant activities resulting from EA-derivatives mentioned above several times. By the same token, weak inhibitory activities on formation of α-dicarbonyl compounds in Frs I and IV might attribute to their low levels of these phenolic compounds.

Meanwhile, although ECE contained the compounds leading to the strong inhibitory effects of Frs II and III, the inhibitory effect of ECE on formation of α-dicarbonyl compounds was still weak. This fact might be explained by a probability that in ECE either the compounds with weak inhibitory effects existing in Frs I and IV could counteract those compounds with strong inhibitory effects existing in Frs II and III, or ECE contained not only compounds with low inhibitory activities as those enriched in Frs I and IV but also other compounds counteracting or cancelling the strong effects of those compounds existing in Frs II and III.

In conclusion, EA-derivatives were superior inhibitor to prevent the formation of AGEs both at the early and late stage of glycation, and EA- and kaempferol-derivatives might have synergetic effects.

3.5

3.5 EA, ECE, and Frs I-IV affected cell viability differently and Fr. IV might contain components with high anticancer activity

To investigate viabilities of EA, ECE, and Frs I-IV against HepG2 cells, MTT assay was used and results were depicted in Fig. 6. HepG2 cells without treatment were used as control and the percent of cell viability was regarded as 100%. The viability of HepG2 cells decreased after treating with ECE and Frs I-IV and there were significant differences between control and each of the treatments at all concentrations. Nonetheless, EA could improve cell viability at experimental concentrations. When the concentration was at 50 or 100 μg/mL, all the samples could significantly improve the cell viability comparing to the control except for Fr. IV. At 200 μg/mL, EA and Frs I and III significantly increased, while ECE and Frs II and IV significantly decreased the cell viability. At 500 μg/mL, ECE and its four fractions all inhibited the growth of HepG2 cells. In general, EA exhibited the strongest effect to improve the cell viability and Fr. IV showed the strongest cytotoxicity, which might be explained by its abundant kaempferol-derivatives. The results demonstrated that oak cup extract could improve the cell viability at the low concentrations and exhibited cytotoxicity at high dose, indicating the potential anticancer activity of oak cups with kaempferol-derivatives, which are promising and require further elucidation.

Effects of EA, ECE, and FrsI-IV on HepG2 cells’ viabilities. Results were expressed as the percentage of cell growth relative to the control (0 μg/mL). The asterisks indicate significant differences with the control at *P < .05 and **P < .01.
Fig. 6
Effects of EA, ECE, and FrsI-IV on HepG2 cells’ viabilities. Results were expressed as the percentage of cell growth relative to the control (0 μg/mL). The asterisks indicate significant differences with the control at *P < .05 and **P < .01.

4

4 Conclusion

The present study first characterized phenolic composition, detected the total EA of Mongolian oak cups, and investigated the anti-diabetic effect of EA, ECE and its four fractions (Frs I-IV) by acting as inhibitors of α-glucosidase, α-amylase and AGEs. Totally, 24 phenolics were identified in ECE, EA- and kaempferol-derivatives were the main phenolic components in oak cups, and ETs were the main hydrolysable tannins in oak cups. In particular, all phenolics other than EA were considered to be first characterized in Mongolian oak cup. And 22, 10, and 9 phenolics were identified for the first time in Mongolian oak, Quercus and Fagaceae species, respectively. Furthermore, ECE and Frs I-IV exhibited splendid inhibitory capacity to diabetes and its complications, especially Frs II and III. And Fr. II exhibited superior α-glucosidase and AGEs inhibitory activity while Frs III and IV showed higher α-amylase inhibition effect, which indicated that EA- and kaempferol-derivatives contribute to the different aspects on the antidiabetic effect. Moreover, results of the MTT assay demonstrated that Fr. IV exhibited cytotoxicity at all concentrations which resulted from its abundant kaempferol-derivatives. Thus Frs II and III, which exhibited high antidiabetic capacities, might be developed for the management of the digestion of starch for improving the health of people with a diabetes problem, while Fr. IV could be used for the treatment of cancer. In conclusion, oak cups might be a source of EA and a potential natural resource as a novel dietary phytonutrients for diabetes management with inhibitory activities against α-glucosidase, α-amylase and formation of AGEs, as well as for cancer treatment.

Acknowledgement

This work was financially supported by the Fundamental Research Funds for the Central Universities (No. BLYJ201412) and Ph.D. Programs Foundation of Ministry of Education (20120014110006), China.

Author contributions

Peipei Yin, Lingguang Yang and Yujun Liu designed the study, carried out the research and drafted the manuscript. Qiang Xue, Miao Yu, Fan Yao and Liwei Sun participated in the experiments. Yujun Liu provided facilities and reviewed the manuscript. All authors read and approved the final manuscript.

Conflict of interest

The authors declared that there was no conflict of interest.

References

  1. , , , , . Chemistry of the fructosamine assay: D-glucosone is the product of oxidation of Amadori compounds. Clin. Chem.. 1994;40:1950-1955.
    [Google Scholar]
  2. , , , , . α-Glucosidase and α-amylase inhibitory activities of Nepalese medicinal herb Pakhanbhed (Bergenia ciliata, Haw.) Food Chem.. 2008;106:247-252.
    [Google Scholar]
  3. , , . HPLC-PDA-MS fingerprinting to assess the authenticity of pomegranate beverages. Food Chem.. 2012;135:1863-1867.
    [Google Scholar]
  4. , , , , , . Advanced glycation end products inhibitors from Alpinia zerumbet rhizomes. Food Chem.. 2011;129:709-715.
    [Google Scholar]
  5. , , , , , , . Phenolic composition, antioxidant potential and in vitro inhibitory activity of leaves and acorns of Quercus suber on key enzymes relevant for hyperglycemia and Alzheimer's disease. Ind. Crop. Prod.. 2014;64:45-51.
    [Google Scholar]
  6. , , , . Bioactive compounds and quantification of total ellagic acid in strawberries (Fragaria x ananassa Duch.) Food Chem.. 2008;107:1629-1635.
    [Google Scholar]
  7. , , , , , , , , , . Dietary ellagic acid improves oxidant-induced endothelial dysfunction and atherosclerosis: role of Nrf2 activation. Int. J. Cardiol.. 2014;175:508-514.
    [Google Scholar]
  8. , , , , , . Screening of phenolic compounds in by-product extracts from mangoes (Mangifera indica L.) by HPLC-ESI-QTOF-MS and multivariate analysis for use as a food ingredient. Food Res. Int.. 2014;57:51-60.
    [Google Scholar]
  9. , , , , . Biological significance of urolithins, the gut microbial ellagic acid-derived metabolites: the evidence so far. Evid. Based Complement. Alternat. Med.. 2013;2013:270418.
    [Google Scholar]
  10. , , , . Medicinal plants used in the traditional management of diabetes and its sequelae in Central America: a review. J. Ethnopharmacol.. 2016;184:58-71.
    [Google Scholar]
  11. , , , , , , . Global estimates of diabetes prevalence for 2013 and projections for 2035. Diabetes Res. Clin. Pract.. 2014;103:137-149.
    [Google Scholar]
  12. , , , , , . Blanching influences the phenolics composition, antioxidant activity, and inhibitory effect of Adansonia digitata leaves extract on alpha-amylase, alpha-glucosidase, and aldose reductase. Food Science & Nutrition. 2017;5:233-242.
    [Google Scholar]
  13. , , , . Intraspecific leaf chemistry drives locally accelerated ecosystem function in aquatic and terrestrial communities. Ecology. 2016;97:2125-2135.
    [Google Scholar]
  14. , , , . Inhibition of advanced glycation end product formation by cymene – A common food constituent. J. Funct. Foods. 2014;6:107-115.
    [Google Scholar]
  15. , , , . Quercus ilex L.: A rich source of polyacylated flavonoid glucosides. Food Chem.. 2010;123:131-142.
    [Google Scholar]
  16. , , , , , , , , , , , . Isoalantolactone induces reactive oxygen species mediated apoptosis in pancreatic carcinoma PANC-1 cells. Int. J. Biol. Sci.. 2012;8:533-547.
    [Google Scholar]
  17. , , , . The inhibitory effects of ethanol extracts from sorghum, foxtail millet and proso millet on α-glucosidase and α-amylase activities. Food Chem.. 2011;124:1647-1651.
    [Google Scholar]
  18. , , . Mixed dimers formed by crosslinking of native and glycated proteins in the absence of free sugar. Biochem. Bioph. Res. Co.. 1996;219:186-190.
    [Google Scholar]
  19. , , , , . Structural requirements of flavonoids for inhibition of protein glycation and radical scavenging activities. Bioorgan. Med. Chem.. 2003;11:5317-5323.
    [Google Scholar]
  20. , , , , . Antiglycative effect of fruit and vegetable seed extracts: inhibition of AGE formation and carbonyl-trapping abilities. J. Sci. Food Agric.. 2013;93:2037-2044.
    [Google Scholar]
  21. , , , , , , , , . Antioxidant, anti-inflammatory and anticarcinogenic activities of edible red oak (Quercus spp.) infusions in rat colon carcinogenesis induced by 1,2-dimethylhydrazine. Food and Chem. Toxicol.: Int. J. Publ. British Indus. Biol. Res. Assoc.. 2015;80:144-153.
    [Google Scholar]
  22. , , , , , . alpha-Glucosidase inhibition and antioxidant activity of an oenological commercial tannin. Extraction, fractionation and analysis by HPLC/ESI-MS/MS and (1)H NMR. Food Chem.. 2017;215:50-60.
    [Google Scholar]
  23. , , , , , , , , , . Ellagic acid, a component of pomegranate fruit juice, suppresses androgen-dependent prostate carcinogenesis via induction of apoptosis. Prostate. 2015;75:151-160.
    [Google Scholar]
  24. , , , , , , , , . In vivo relevant mixed urolithins and ellagic acid inhibit phenotypic and molecular colon cancer stem cell features: A new potentiality for ellagitannin metabolites against cancer. Food and Chem. Toxicol.: Int. J. Publ. British Indus. Biol. Res. Assoc.. 2016;92:8-16.
    [Google Scholar]
  25. , , , , , , , . Ellagic acid modulates lipid accumulation in primary human adipocytes and human hepatoma Huh7 cells via discrete mechanisms. J. Nutr. Biochem.. 2015;26:82-90.
    [Google Scholar]
  26. , , , , . Optimization of tannin isolation from acorn and application in leather processing. Ind. Crop. Prod.. 2014;53:16-22.
    [Google Scholar]
  27. , , , , , , , , . Cinnamon bark proanthocyanidins as reactive carbonyl scavengers to prevent the formation of advanced glycation endproducts.J Agric. Food Chem.. 2008;56:1907-1911.
    [Google Scholar]
  28. , , . Inhibitors of the Maillard reaction and AGE breakers as therapeutics for multiple diseases. Drug Discov. Today. 2006;11:646-654.
    [Google Scholar]
  29. , , , , , , . Tea polyphenol (-)-epigallocatechin-3-gallate: a new trapping agent of reactive dicarbonyl species. Chem. Res. Toxicol.. 2007;20:1862-1870.
    [Google Scholar]
  30. , , , , , , . Phenolic composition and antioxidant activity of industrial cork by-products. Ind. Crop. Prod.. 2013;47:262-269.
    [Google Scholar]
  31. , , , , , , , . Anti-diabetic potential of selected ethno-medicinal plants of north east India. J. Ethnopharmacol.. 2015;171:37-41.
    [Google Scholar]
  32. , . Optimization of polysaccharides from Zagros oak leaf using RSM: antioxidant and antimicrobial activities. Carbohyd. Polym.. 2014;106:238-246.
    [Google Scholar]
  33. , , , . Comparison of alpha-amylase, alpha-glucosidase and lipase inhibitory activity of the phenolic substances in two black legumes of different genera. Food Chem.. 2017;214:259-268.
    [Google Scholar]
  34. Tanaka, T., Ueda, N., Shinohara, H., Nonaka, G., Kouno, I., 1997. Four new C-glycosidic ellagitannins, castacrenins D-G, from Japanese chestnut wood (Castanea crenata SIEB. et ZUCC). Chem. Pharm. Bull. 45.
  35. , , . Innovative microwave-assisted hydrolysis of ellagitannins and quantification as ellagic acid equivalents. Food Chem.. 2013;138:2430-2434.
    [Google Scholar]
  36. , , , , , , . α-Glucosidase inhibitory action of natural acylated anthocyanins. 2. α-Glucosidase inhibition by isolated acylated anthocyanins. J. Agric. Food Chem.. 2001;49:1952-1956.
    [Google Scholar]
  37. , , , , , , , , . Poncirus trifoliata (L.) Raf.: Chemical composition, antioxidant properties and hypoglycaemic activity via the inhibition of α-amylase and α-glucosidase enzymes. J. Funct. Foods. 2016;25:477-485.
    [Google Scholar]
  38. , , , , , , , , , , , . Antiglycative and neuroprotective activity of colon-derived polyphenol catabolites. Mol. Nutr. Food Res.. 2011;55(Suppl 1):S35-43.
    [Google Scholar]
  39. , , , , , , , , . Response surface methodology optimization of ultrasonic-assisted extraction of Acer Truncatumleaves for maximal phenolic yield and atioxidant activity. Molecules 2017
    [CrossRef] [Google Scholar]
  40. , , . Effects of tea polyphenols and different teas on pancreatic α-amylase activity in vitro. LWT - Food Sci. Technol.. 2016;66:232-238.
    [Google Scholar]
  41. , , , , , , , , . Bioactive components and antioxidant activities of oak cup crude extract and its four partially purified fractions by HPD-100 macroporous resin chromatography. Arab. J. Chem. 2016
    [CrossRef] [Google Scholar]
  42. , , , , , , , , , . alpha-Glucosidase inhibitory activity of polyphenols from the burs of Castanea mollissima Blume. Molecules. 2014;19:8373-8386.
    [Google Scholar]
  43. , , , , , , , . Grape skin extract inhibits mammalian intestinal α-glucosidase activity and suppresses postprandial glycemic response in streptozocin-treated mice. Food Chem.. 2011;126:466-471.
    [Google Scholar]
  44. , , , , , , . Antioxidants and alpha-glucosidase inhibitors from Ipomoea batatas leaves identified by bioassay-guided approach and structure-activity relationships. Food Chem.. 2016;208:61-67.
    [Google Scholar]

Fulltext Views
158

PDF downloads
46
View/Download PDF
Download Citations
BibTeX
RIS
Show Sections

Suggested read for related articles:

© Copyright 2025 – Arabian Journal of Chemistry.

Published by Scientific Scholar on behalf of King Saud University together with Saudi Chemical Society, Riyadh, Saudi Arabia.

ISSN (Print): 1878-5352
ISSN (Online): 1878-5379
Scientific Scholar CrossRef Creative Commons Open Acess Portico