Article pubs.acs.org/JAFC
Potential of Polygonum cuspidatum Root as an Antidiabetic Food: Dual High-Resolution α‑Glucosidase and PTP1B Inhibition Profiling Combined with HPLC-HRMS and NMR for Identification of Antidiabetic Constituents Yong Zhao, Martin Xiaoyong Chen, Kenneth Thermann Kongstad, Anna Katharina Jag̈ er, and Dan Staerk* Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark S Supporting Information *
ABSTRACT: The worldwide increasing incidence of type 2 diabetes has fueled an intensified search for food and herbal remedies with preventive and/or therapeutic properties. Polygonum cuspidatum Siebold & Zucc. (Polygonaceae) is used as a functional food in Japan and South Korea, and it is also a well-known traditional antidiabetic herb used in China. In this study, dual high-resolution α-glucosidase and protein-tyrosine phosphatase 1B (PTP1B) inhibition profiling was used for the identification of individual antidiabetic constituents directly from the crude ethyl acetate extract and fractions of P. cuspidatum. Subsequent preparative-scale HPLC was used to isolate a series of α-glucosidase inhibitors, which after HPLC-HRMS and NMR analysis were identified as procyanidin B2 3,3″-O-digallate (3) and (−)-epicatechin gallate (5) with IC50 values of 0.42 ± 0.02 and 0.48 ± 0.0004 μM, respectively, as well as a series of stilbene analogues with IC50 value in the range from 6.05 ± 0.05 to 116.10 ± 2.04 μM. In addition, (trans)-emodin-physcion bianthrone (15b) and (cis)-emodin-physcion bianthrone (15c) were identified as potent PTP1B inhibitors with IC50 values of 2.77 ± 1.23 and 7.29 ± 2.32 μM, respectively. These findings show that P. cuspidatum is a potential functional food for management of type 2 diabetes. KEYWORDS: α-glucosidase, PTP1B, HPLC-HRMS, NMR, Polygonum cuspidatum
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INTRODUCTION
as PTP1B are important therapeutic targets for managing blood glucose.4−6 T2D is a multifactorial disease that calls for a multifactorial response, and bioactive natural products from functional foods and medicinal herbs have been successfully applied for the treatment and prevention of diseases such as diabetes and obesity.7 Recent results have shown that many bioactive constituents in functional foods and medicinal herbs inhibit multiple T2D target enzymes in vitro or that functional foods and medicinal herbs contain different classes of constituents that inhibit different target enzymes in vitro.8,9 The stem and root of Polygonum cuspidatum Siebold & Zucc. (Polygonaceae) are used as functional foods in East Asian countries such as Japan and South Korea.10,11 It is also a well-known traditional Chinese medicine used for the treatment of diabetes, hyperlipemia, infection, and inflammation as documented in the Chinese Pharmacopoeia.12 Compounds such as anthraquinones, flavonoids, and stilbenes have been identified as the pharmacologically active constituents.11,13,14 Previous studies revealed that tannins, which are widely distributed in this plant, contribute to its α-glucosidase inhibitory activity,15 and several stilbene glycosides have also been identified as α-glucosidase
Globally, it is estimated that 380 million people suffer from diabetes, with type 2 diabetes (T2D) accounting for up to 90% of all diabetes cases. The main risk factors for the development of T2D are excess weight and lack of exercise, and it is expected that a rapidly increasing proportion of the population will develop T2D due to the global obesity epidemic.1 It has been shown that pharmacological intervention with, for example, metformin without lifestyle modifications decreases the progression of T2D by 30%,2 but may cause different side effects such as vitamin B12 deficiency.3 Lifestyle changes with increased physical exercise as well as intake of weight-reducing and/or functional foods (i.e., foods with health-promoting effects beyond their nutritional value) are more effective methods for controlling blood glucose levels.2 The major source of blood glucose is derived from dietary polysaccharides such as starch. α-Glucosidase and α-amylase are two important digestive enzymes that break down polysaccharides into monosaccharides, which are subsequently absorbed into the bloodstream via the small intestine.4,5 Protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of the insulin and leptin signaling pathways, through dephosphorylation of the activated insulin receptor as well as the insulin receptor substrate. PTP1B inhibitors can therefore improve and/or prolong the action of insulin.6 Previous studies have reported that the digestive enzymes α-glucosidase and α-amylase as well © 2017 American Chemical Society
Received: Revised: Accepted: Published: 4421
March 24, 2017 May 11, 2017 May 12, 2017 May 12, 2017 DOI: 10.1021/acs.jafc.7b01353 J. Agric. Food Chem. 2017, 65, 4421−4427
Article
Journal of Agricultural and Food Chemistry
α-Amylase Inhibition Assay. The α-amylase inhibition assay was performed in 96-well microplates according to a previously reported procedure.20 In brief, in each well was added sample dissolved in 10 μL of DMSO and 90 μL of phosphate buffer (0.1 M, pH 6, 0.02% NaN3). Then 80 μL of α-amylase solution (2.0 U/mL) was added and incubated for 10 min at 37 °C. The reaction was started by adding 20 μL of substrate solution (CNP-G3, 10 mM in phosphate buffer). The absorbance was measured at 405 nm every third minute for 30 min. The percentage inhibition was calculated using the same formula as described for the α-glucosidase inhibition assay. PTP1B Assay. The PTP1B inhibition assay was performed in 96well microplates according to a previously reported procedure.24 In brief, the assay was performed at 25 °C in a final volume of 180 μL, using a buffer containing 50 mM Tris, 50 mM Bis-Tris, and 0.1 M NaCl (pH adjusted to 7.0 with citric acid). Samples were dissolved in 18 μL of DMSO followed by the addition of 52 μL of EDTA solution (3.46 mM in the above buffer) and 60 μL of substrate solution consisting of 1.5 mM p-NPP and 6 mM DTT. After incubation at 25 °C for 10 min, 50 μL of 0.001 μg/μL PTP1B stock solution was added into each well to start the reaction. The absorbance was measured at 405 nm every 30 s for 10 min, and the percentage inhibition was calculated using the same formula as described for the α-glucosidase inhibition assay. Analytical-Scale HPLC Separations and High-Resolution αGlucosidase and PTP1B Inhibition Profiles. Analytical-scale HPLC separations were performed with an Agilent 1200 system (Santa Clara, CA, USA) consisting of a G1367C high-performance autosampler, a G1311A quaternary pump, a G1322A degasser, a G1316A thermostated column compartment, a G1315C photodiode array detector, and a G1364C fraction collector, all controlled by Agilent ChemStation version B.03.02 software. Separations were performed at 40 °C on a Phenomenex Luna C18(2) reversed-phase column (150 × 4.6 mm i.d., 3 μm particle size, 100 Å pore size; Phenomenex, Torrance, CA, USA), using a flow rate of 0.5 mL/min with a combination of solvent A (H2O/MeCN 95:5 with 0.1% (v/v) formic acid) and solvent B (H2O/MeCN 5:95 with 0.1% (v/v) formic acid). For the high-resolution α-glucosidase and PTP1B inhibition profiling of crude EtOAc extract, 10-μL injections (20 mg/mL in MeOH) were separated using the following elution gradient profile: 0 min, 0% B; 30 min, 100% B; 40 min, 100% B. The eluate from 10 to 35 min was fractionated into 88 wells of a 96-well microplate, yielding a resolution of 3.52 data points per minute. For the active fraction, either 3-μL (for α-glucosidase) or 10-μL (for PTP1B) injections (20 mg/mL in MeOH) were separated using the following elution gradient profile: 0 min, 10% B; 30 min, 40% B. The eluate from 10 to 30 min was fractionated into 88 wells of a 96-well microplate, yielding a resolution of 4.4 points per minute. Subsequently, the microplates were evaporated to dryness using an SPD121P Savant SpeedVac concentrator equipped with an OFP400 oil Free Pump and an RVT400 Refrigerated Vapor Trap (Thermo Scientific, Waltham, MA, USA). α-Glucosidase and/or PTP1B inhibition assays were performed as described above, and the result from each well was plotted at its respective chromatographic retention time to yield high-resolution αglucosidase and/or PTP1B inhibition profiles (biochromatograms). Preparative-Scale Isolation and Purification. The crude extract of P. cuspidatum (1.4 g) was dissolved in 14 mL of MeOH and centrifuged for 5 min. The supernatant (0.9 mL per injection) was separated using an Agilent 1100 series instrument consisting of two G1361A preparative-scale pumps, a G1365B multiple-wavelength detector, a G2260A preparative autosampler, and a G1364B preparative-scale fraction collector, all controlled by Agilent ChemStation ver. B.01.01 software and equipped with a reversed-phase Phenomenex Luna C18 (2) column (250 × 21.2 mm, 5 μm, 100 Å) operated at room temperature. The flow rate was maintained at 20 mL/min, using the following elution gradient profile: 0 min, 0% B; 30 min, 100% B; 40 min, 100% B. The active fraction and peaks 13, 14, and 15 were collected manually at retention times of 10−16, 20.6, 24.9, and 30 min, respectively. Three peaks (15a, 15b, and 15c) were further isolated from peak 15 (∼5 mg) by analytical-scale HPLC isocratically eluted with 70% B for 25 min, which afforded compounds
inhibitors.16,17 Furthermore, Sohn and co-workers showed that the extract of P. cuspidatum reduces diabetes-induced mesangial cell dysfunction (via inhibition of platelet-derived growth factor BB),18 and attenuates diabetes-induced retinopathy (via inhibition of the high-mobility group box 1 signaling pathway).19 However, a systematic investigation of antidiabetic constituents in P. cuspidatum, and especially investigation of antidiabetic constituents beyond α-glucosidase inhibitors, has still not been performed. Hyphenation of high-performance liquid chromatography with various bioassays (HPLC-bioassay) has proven to be an effective tool for pinpointing the bioactive constituents directly from crude plant extracts. High-resolution bioassays combined with other analytical techniques such as HPLC-HRMS and NMR allow structural identification of these bioactive metabolites. In our previous papers, this platform has been successfully applied for the identification of antidiabetic constituents from foods such as cinnamon,20 apple peel,21 and edible seaweeds,22 as well as from medicinal herbs.8,23 In our continued search for functional foods with antidiabetic properties and/or antidiabetic drug leads, P. cuspidatum extract was disclosed as a potent inhibitor of α-glucosidase and PTP1B. Dual high-resolution α-glucosidase and PTP1B inhibition profiling combined with HPLC-HRMS and NMR was used to identify the bioactive metabolites.
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EXPERIMENTAL PROCEDURES
Chemicals. α-Glucosidase type I (EC 3.2.20, from Saccharomyces cerevisiae, lyophilized powder), p-nitrophenyl α-D-glucopyranoside (pNPG), α-amylase type VI-B (EC 3.2.1.1, from porcine pancreas, lyophilized powder), 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3), p-nitrophenyl phosphate (p-NPP), dimethyl sulfoxide (DMSO), NaH 2 PO 4 , Na 2 HPO 4 , NaN 3 , NaCl, tris(hydroxymethyl)aminomethane (Tris), bis(2-hydroxyethyl)iminotris(hydroxymethylmethane) (Bis-Tris), dithiothreitol (DTT), N,N,N′,N′-ethylenediaminetetraacetate (EDTA), and HPLC grade MeCN were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human protein tyrosine phosphatase 1B (PTP1B) (BML-SE332-0050, EC 3.1.3.48) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Formic acid and calcium acetate were purchased from Merck (Darmstadt, Germany). Water was purified by deionization and 0.22 μM membrane filtration (Millipore, Billerica, MA, USA). Plant Material and Extraction. Roots of P. cuspidatum Siebold & Zucc. were collected in the Sichuan province of China. A voucher specimen (voucher specimen no. zyc087) has been deposited at Southwest Treasure Herbs (Chendu, China). Approximately 70 g of powered plant material was extracted with 700 mL of EtOAc and sonicated for 2 h at room temperature. The mixture was filtered and subsequently evaporated to dryness by rotary evaporation (100 mbar, 35 °C), which yielded 1.4 g of dry EtOAc extract. α-Glucosidase Inhibition Assay. The α-glucosidase inhibition assay was performed at 28 °C in 96-well microplates as previously reported.21 In short, samples were dissolved in 10 μL of DMSO, and to each well was added 90 μL of phosphate buffer (0.1 M, pH 7.5, 0.02% NaN3); the plates were then shaken for 10 min. To each well was then added 80 μL of α-glucosidase solution in the same phosphate buffer (2.0 U/mL), and incubation was continued for an additional 10 min. The enzyme reaction was hereafter started by adding 20 μL of pNPG solution (10 mM in phosphate buffer). The absorbance of the cleavage product (the strongly chromogenic p-nitrophenolate ion) was measured at 405 nm with a Thermo Scientific Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA) coupled to SkanIt ver. 2.5.1 software. Measurements were performed every 30 s for 35 min to yield the cleavage rate (kinetic measurements) as ΔAU/s. The inhibition of the enzyme was calculated as follows: percentage inhibition = [(slopeblank − slopesample)/slopeblank] × 100%. 4422
DOI: 10.1021/acs.jafc.7b01353 J. Agric. Food Chem. 2017, 65, 4421−4427
Article
Journal of Agricultural and Food Chemistry 15a (3 mg), 15b (0.8 mg), and 15c (0.7 mg) at retention times of 18.1, 19.9, and 21.8 min, respectively. The active fraction (approximately 0.5 g) was dissolved in 5 mL of MeOH and subsequently separated on preparative-scale HPLC (injection volume = 500 μL for each separation) with a gradient elution similar to the one used for high-resolution screening (0 min, 10% B; 30 min, 40% B). The targeted peaks (1, 2, 3, 6, 7, 8, 9, 10, 11, and 12) were collected manually at retention times of 11.2, 11.8, 12.7, 16.0, 17.9, 18.5, 20.1, 21.9, 25.3, and 27.2 min, respectively. Compounds 3, 7 and 9 were further purified on Sephadex LH-20 (MeOH) due to the low purity detected by analytical-scale HPLC. Peaks 4 and 5 were collected together due to insufficient separation with preparative-scale HPLC and further separated by analytical-scale HPLC with isocratic elution at 14% B for 25 min, which afforded the pure compound 4 and a mixture of compound 4 and 5 (approximately 2:1). HPLC-HRMS and NMR Experiments. HPLC-HRMS analyses were performed on an Agilent 1260 HPLC system consisting of a G1311B quaternary pump with a built-in degasser, a G1329B autosampler, a G1316A thermostated column compartment, and a G1315D photodiode array detector, coupled with a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonik, Bremen, Germany). Mass spectra were acquired in both positive and negative modes, and a solution of sodium formate clusters was automatically injected to enable internal mass calibration. Separations were performed with the same column, temperature, and solvent systems as described above. For the crude ethyl acetate extract, 2-μL injections of a 20 mg/mL solution in MeOH were separated using the following elution gradient profile: 0 min, 0% B; 30 min, 100% B; 40 min, 100% B. For the active fraction, 2-μL injections of a 20 mg/mL solution in MeOH were separated using elution the following elution gradient profile: 0 min, 10% B; 30 min, 40% B. For the mixture containing 15a, 15b, and 15c, 2-μL injections of a 5 mg/mL solution in MeOH were separated using isocratic elution at 70% B. 1D 1H NMR and 2D homo- and heteronuclear NMR experiments were recorded on a 600 MHz Bruker Avance III instrument (operating frequency of 600.13 MHz) equipped with a cryogenically cooled 1.7mm TCI probe head and a Bruker SampleJet sample changer (Bruker Biospin, Karlsruhe, Germany). Compounds 1−7 and 9−14 were dissolved in CD3OD, whereas compounds 15a, 15b, and 15c were dissolved in CDCl3. All experiments were acquired in automation (temperature equilibration to 300 K, optimization of lock parameters, gradient shimming, and setting of receiver gain). 1H and 13C NMR spectra were acquired with 30° pulses and 64K data points. IconNMR ver. 4.2 (Bruker Biospin, Karlsruhe, Germany) was used for controlling automated sample change and acquisition of NMR data, whereas Topspin ver. 3.5 (Bruker Biospin) was used for acquisition and processing of NMR data. Chemical shifts of 1H and 13C were calibrated on the basis of the residual solvent signal (δ 3.31 and 49.00 for compounds 1−7 and 9−14 and δ 7.26 and 77.16 for compounds 15a, 15b, and 15c).
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determined from the inhibition curves shown in the Supporting Information (Figure S1: 8.33 ± 1.42 μg/mL for α-glucosidase and 16.21 ± 0.38 μg/mL for PTP1B). Dual High-Resolution α-Glucosidase and PTP1B Inhibition Profiling of the Crude Extract. To identify the constituents responsible for the observed inhibition, the extract was separated using reversed-phase analytical-scale HPLC, and two successive separations were fractionated into two 96-well microplates. The α-glucosidase and PTP1B inhibition was subsequently assessed for each well, and the inhibition values were plotted at their respective retention times to provide the dual high-resolution α-glucosidase and PTP1B inhibition profile. This is shown in Figure 1, where the HPLC chromatogram of a 10-μL injection (20 mg/mL of the crude extract) is shown as the black line, the high-resolution αglucosidase inhibition profile as the red line, and the highresolution PTP1B inhibition profile as the blue line. All of the active inhibitors for α-glucosidase were eluted from 11 to 18 min, with broadening of the inhibition peaks due to insufficient separation of the HPLC peaks and strong α-glucosidase inhibition. Although not very clear, inhibition of PTP1B seemed to occur in the same region as a broad flat hump. Additionally, peak 15 showed >70% inhibition of PTP1B, whereas peaks 13 and 14 showed weak PTP1B inhibition. To identify the active constituents, the active fraction from 11 to 18 min as well as the three active peaks (13, 14, and 15) were separated using preparative-scale HPLC. Dual High-Resolution α-Glucosidase and PTP1B Inhibition Profiling of the Active Fraction. With the aim of obtaining a biochromatogram with higher resolution than shown in Figure 1, two successive separations of the active fraction collected by preparative-scale HPLC (vide supra) were fractionated into two 96-well microplates after optimization of the HPLC method. The α-glucosidase and PTP1B inhibition was measured and plotted as high-resolution inhibition profiles as shown in Figure 2. Peaks 3 and 5 showed strong αglucosidase inhibition peaks (100%), whereas peaks 1, 2, 6, 7, 8, and 10 exhibited weak inhibition peaks with an injection volume of 3 μL (20 mg/mL). However, no strong PTP1B inhibition was observed, even though the injection volume was increased to 10 μL. The most intense PTP1B inhibition peak was peak 12, with an inhibitory effect of 26%. Identification of α-Glucosidase Inhibitors. To determine the structures of the active peaks and the possible structure−activity relationships (SARs) of the potential inhibitors, 12 peaks from the active fraction and 3 peaks from the crude extract were subjected to HPLC-HRMS analysis. A total of 16 metabolites were further purified (Figure 3) for NMR experiments and IC50 determination. The obtained HRMS and NMR data are presented in the Supporting Information (Table S1), and comparison of these data with data reported in the literature allowed the identification of resveratrol-4′-O-β-D-glucoside (1),10 3-O-feruloylquinic acid (2),25 procyanidin B2 3,3″-O-digallate (3),26,27 resveratrol-3O-β-D-glucoside (4),10 pieceid-6″-O-gallate (6),28 pieceid-2″-Ogallate (7),29 emodin-1-O-β-D-glucoside (9),13 resveratrol (10),13 torachrysone-8-O-β-D-glucoside (11),30 and emodin-8O-β-D-glucoside (12),30 respectively. In an attempt to purify peak 5, a mixture of compounds 4 and 5 in the ratio of 2:1 was obtained, as the limited amount did not allow further separation. However, by subtracting the 1 H NMR signals of the known compound 4, peak 5 was identified as (−)-epicatechin gallate (5).31 An authentic sample
RESULTS AND DISCUSSION
The α-glucosidase, α-amylase, and PTP1B inhibitory activities of the crude ethyl acetate extract of P. cuspidatum were assessed in vitro (Table 1). This showed that the crude extract inhibited α-glucosidase and PTP1B by >50% at a concentration of 50 μg/mL, and the IC50 values for the two enzymes were Table 1. Inhibitory Activity at 50 μg/mL and IC50 Values of Crude P. cuspidatum Extract against α-Glucosidase, αAmylase, and PTP1B
a
enzyme
% inhibition at 50 μg/mL
IC50a (μM)
α-glucosidase α-amylase PTP1B
99.27 5.34 99.23
8.33 ± 1.42 16.21 ± 0.38
Values are expressed as the mean ± SD (n = 3, p < 0.05). 4423
DOI: 10.1021/acs.jafc.7b01353 J. Agric. Food Chem. 2017, 65, 4421−4427
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Journal of Agricultural and Food Chemistry
Figure 1. HPLC chromatogram of crude P. cuspidatum extract monitored at 280 nm (black), high-resolution α-glucosidase inhibition profile (red), and high-resolution PTP1B inhibition profile (blue) (10 μL injection, 20 mg/mL).
Figure 2. HPLC chromatogram of the active fraction monitored at 280 nm (black), high-resolution α-glucosidase inhibition profile (red) (3 μL injection, 20 mg/mL), and high-resolution PTP1B inhibition profile (blue) (10 μL injection, 20 mg/mL).
its inhibitory effect on α-glucosidase, and this reduction was more pronounced for glucosylation at C-3 (4) than at C-4′ (1). For compounds 6 and 7, the gallate moieties connected to the glucose at C-2′ or C-6′, respectively, improved the αglucosidase inhibitory effect compared to 4. Compounds 3 and 5, both containing a gallate group, also exhibited strong αglucosidase inhibition, which suggests that the gallate moiety could be an important determinant contributing to the observed inhibition. Identification of PTP1B Inhibitors. Peaks 13 and 14 showed only weak PTP1B inhibition (Figure 1), whereas peak 15 showed >70% inhibition. Peaks 13 and 14 were purified and identified as 2-methoxystypandrone (13)33 and emodin (14)34 on the basis of comparison of HRMS and 1H NMR data with the literature. During the purification of the material eluted as peak 15, we were excited to find this peak in fact consisted of three compounds (15a, 15b, and 15c) when the analytical-scale HPLC method was optimized (Figure 4). The three compounds were subsequently separated by analytical-scale HPLC and identified as parietin (15a),35 (trans)-emodinphyscion bianthrone (15b),36 and (cis)-emodin-physcion bianthrone (15c)36 by comparison of HRMS and 1H NMR data with those reported in the literature.
of 5 was purchased from Cayman Europe (Tallinn, Estonia) and used for spiking experiments as well as for IC 50 determination. Peak 8 showed a [M + H]+ ion with m/z 419.1625 and a [M − H]− ion with m/z 417.1183, which suggested a molecular formula of C22H26O8. However, this compound was rapidly degraded into compound 4 during purification, indicating that the structure of compound 8 was an ethyl analogue of resveratrol-3-O-β-D-glucoside. It is suggested that the ethyl group might come from the EtOAc used during extraction, because it was observed that peak 8 was absent in a methanol extract using the same HPLC conditions. The α-glucosidase inhibitory activity of the purified metabolites 1−4, 6, 7, and 10 together with the commercial sample of 5 was assessed. The IC50 values are presented in Table 2, and the IC50 curves are shown in the Supporting Information (Figure S2). The IC50 value of 5 was 0.48 ± 0.00 μM, which is in agreement with a previous study.32 Compound 3, which was a dimer of 5, also showed strong inhibition with an IC50 value of 0.42 ± 0.02 μM. A series of stilbene analogues (1, 4, 6, 7, and 10) showed inhibition with IC50 values in the range of 6.05−116.10 μM. Interestingly, it was observed that the glycosylation of resveratrol (10) could significantly reduce 4424
DOI: 10.1021/acs.jafc.7b01353 J. Agric. Food Chem. 2017, 65, 4421−4427
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Journal of Agricultural and Food Chemistry
Figure 3. Structures of compounds 1−15c identified in P. cuspidatum.
Table 2. IC50 Values of 1−7 and 10 in α-Glucosidase Inhibition Assay
a
compd
structure
1 2 3 4 5 6 7 10
resveratrol-4′-O-β-D-glucoside 3-O-feruloylquinic acid procyanidin B2 3,3″-O-digallate resveratrol-3-O-β-D-glucoside (−)-epicatechin gallate pieceid-6″-O-gallate pieceid-2″-O-gallate resveratrol
IC50a (μM) 59.76 57.12 0.42 116.10 0.48 6.05 8.20 10.35
± ± ± ± ± ± ± ±
7.00 2.52 0.02 2.04 0.00 0.05 0.60 1.32
Figure 4. Analytical-scale HPLC separation of preparative-scale HPLC peak 15.
Values are expressed as the mean ± SD (n = 3, p < 0.05).
Table 3. IC50 Values of PTP1B Inhibitory Activity by Compounds 12−15c
PTP1B inhibition by respectively compounds 12, 13, 14, 15a, 15b, and 15c was assessed. IC50 values are shown in Table 3, and IC50 curves are shown in the Supporting Information (Figure S3). It was intriguing to find that the dominating compound (15a) in peak 15 did not show any inhibition of PTP1B at a concentration up to 500 μM, whereas the minor compounds 15b and 15c, a pair of bianthrones, displayed strong inhibition. The IC50 value of the trans form was 2.77 ± 1.23 μM, and the IC50 value of the cis form was 7.29 ± 2.32 μM. Compounds 13 and 14 showed lower inhibitory activity with IC50 values of 83.87 ± 7.45 and 171.88 ± 35.89 μM,
a
4425
IC50a (μM)
compd
structure
12 13 14 15a 15b 15c
emodin-8-O-β-D-glucoside 2-methoxystypandrone emodin parietin (trans)-emodin-physcion bianthrone (cis)-emodin-physcion bianthrone
>500 83.87 171.88 >500 2.77 7.29
± 7.45 ± 35.89 ± 1.23 ± 2.32
Values are expressed as the mean ± SD (n = 3, p < 0.05). DOI: 10.1021/acs.jafc.7b01353 J. Agric. Food Chem. 2017, 65, 4421−4427
Journal of Agricultural and Food Chemistry
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ACKNOWLEDGMENTS We acknowledge Arife Ö nder and Katrine Krydsfeldt for technical assistance
respectively, which was in accordance with the weak inhibition observed in the biochromatogram shown in Figure 1. For compound 14, either glucosylation at C-8 (compound 12) or methylation at 6-OH (compound 15a) causes a decrease in the PTP1B inhibitory activity. Collectively, dual high-resolution α-glucosidase and PTP1B inhibition profiling of both the crude extract and the active fraction of P. cuspidatum allowed identification of the active peaks directly from these complex mixtures. HPLC-HRMS and NMR analyses of material isolated from these peaks resulted in the identification of 16 compounds. Tannins have previously been reported to be abundant in the stem and root of P. cuspidatum and could potentially also be responsible for the α-glucosidase inhibition observed for the crude extract in this study. However, in our research only two tannins (3 and 5) were found to exhibit strong α-glucosidase inhibition with IC50 values of 0.42 and 0.48 μM, respectively. In addition, a series of stilbene analogues showed inhibition with IC50 in the range of 6.05−116.10 μM. A pair of emodin-physcion bianthrones was identified in this plant for the first time, and they displayed strong PTP1B inhibitory activity with IC50 values of 2.77 and 7.29 μM. The results show that P. cuspidatum is a source of both α-glucosidase and PTP1B inhibitors and, therefore, a possible candidate as a future functional food for management of diabetes and obesity. However, more detailed safety studies, in vivo studies, and human intervention studies are needed before P. cuspidatum root can be considered a safe evidencebased functional food.
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REFERENCES
(1) King, H.; Aubert, R. E.; Herman, W. H. Global burden of diabetes, 1995−2025: prevalence, numerical estimates, and projections. Diabetes Care 1998, 21, 1414−1431. (2) Knowler, W. C.; Barrett-Connor, E.; Fowler, S. E.; Hamman, R. F.; Lachin, J. M.; Walker, E. A.; Nathan, D. M. Diabetes Prevention Program Research Group, reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N. Engl. J. Med. 2002, 346, 393−403. (3) Liu, Q.; Li, S.; Li, J. Vitamin B12 status in metformin treated patients: systematic review. PLoS One 2014, 9, e100379. (4) He, L. α-Glucosidase inhibitors as agents in the treatment of diabetes. Diabetes Rev. 1998, 6, 132−145. (5) Whitcomb, D. C.; Lowe, M. E. Human pancreatic digestive enzymes. Dig. Dis. Sci. 2007, 52, 1−17. (6) Johnson, T. O.; Ermolieff, J.; Jirousek, M. R. Protein tyrosine phosphatase 1B inhibitors for diabetes. Nat. Rev. Drug Discovery 2002, 1, 696−709. (7) Abuajah, C. I.; Ogbonna, A. C.; Osuji, C. M. Functional components and medicinal properties of food: a review. J. Food Sci. Technol. 2015, 52, 2522−2529. (8) Tahtah, Y.; Kongstad, K. T.; Wubshet, S. G.; Nyberg, N. T.; Joensson, L. H.; Jager, A. K.; Sun, Q.; Staerk, D. Triple aldose reductase/α-glucosidase/radical scavenging high-resolution profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy for identification of antidiabetic constituents in crude extract of Radix Scutellariae. J. Chromatogr., A 2015, 1408, 125−132. (9) Wubshet, S. G.; Tahtah, Y.; Heskes, A. M.; Kongstad, K. T.; Pateraki, I.; Hamberger, B.; Moeller, B. L.; Staerk, D. Identification of PTP1B and α-glucosidase inhibitory serrulatanes from Eremophila spp. by combined use of dual high-resolution PTP1B and α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR. J. Nat. Prod. 2016, 79, 1063−1072. (10) Kirino, A.; Takasuka, Y.; Nishi, A.; Kawabe, S.; Yamashita, H.; Kimoto, M.; Ito, H.; Tsuji, H. Analysis and functionality of major polyphenolic components of Polygonum cuspidatum (Itadori). J. Nutr. Sci. Vitaminol. 2012, 58, 278−286. (11) Peng, W.; Qin, R.; Li, X.; Zhou, H. Botany, phytochemistry, pharmacology, and potential application of Polygonum cuspidatum Sieb. et Zucc.: a review. J. Ethnopharmacol. 2013, 148, 729−745. (12) Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China.; China Medical Science Press: Beijing, China, 2010; pp 194−195. (13) Uddin, Z.; Song, Y. H.; Curtis-Long, M. J.; Kim, J. Y.; Yuk, H. J.; Park, K. H. Potent bacterial neuraminidase inhibitors, anthraquinone glucosides from Polygonum cuspidatum and their inhibitory mechanism. J. Ethnopharmacol. 2016, 193, 283−292. (14) Zhang, H.; Li, C.; Kwok, S. T.; Zhang, Q.-W.; Chan, S.-W. A review of the pharmacological effects of the dried root of Polygonum cuspidatum (Hu Zhang) and its constituents. Evidence-Based Complement. Altern. Med. 2013, 2013, 208349. (15) Tang, W.; Shen, Z.; Yin, J. Inhibitory activity to glycosidase of tannins from Polygonum cuspidatum. Nat. Prod. Res. Dev. 2006, 18, 266−268. (16) Li, F.; Zhan, Z.; Liu, F.; Yang, Y.; Li, L.; Feng, Z.; Jiang, J.; Zhang, P. Polyflavanostilbene A, a new flavanol-fused stilbene glycoside from Polygonum cuspidatum. Org. Lett. 2013, 15, 674−677. (17) Yang, Y. N.; Li, F. S.; Liu, F.; Feng, Z. M.; Jiang, J. S.; Zhang, P. C. A novel adduct of ECG fused to piceid and four new dimeric stilbene glycosides from Polygonum cuspidatum. RSC Adv. 2016, 6, 60741−60748.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01353. Table S1, HRMS and NMR data of compounds isolated from P. cuspidatum; Figure S1, α-glucosidase and PTP1B IC50 curves of the crude EtOAc extract of P. cuspidatum; Figure S2, α-glucosidase IC50 curves for compounds 1−7 and 10; Figure S3, PTP1B IC50 curves for compounds 12−15c (PDF)
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Article
AUTHOR INFORMATION
Corresponding Author
*(D.S.) Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Phone: +45 35336177. Fax: +45 35336001. E-mail:
[email protected]. ORCID
Dan Staerk: 0000-0003-0074-298X Funding
HPLC equipment used for high-resolution bioassay profiles was obtained via a grant from The Carlsberg Foundation. The HPLC-HRMS and NMR system used in this work was acquired through a grant from “Apotekerfonden af 1991”, The Carlsberg Foundation, and the Danish Agency for Science, Technology and Innovation via the National Research Infrastructure funds. Y.Z. acknowledges the Chinese Scholarship Council for a Ph.D. scholarship (CSC Grant 201508530222). Notes
The authors declare no competing financial interest. 4426
DOI: 10.1021/acs.jafc.7b01353 J. Agric. Food Chem. 2017, 65, 4421−4427
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Journal of Agricultural and Food Chemistry (18) Sohn, E.; Kim, J.; Kim, C.-S.; Jo, K.; Lee, Y. M.; Kim, J. S. Root of Polygonum cuspidatum extract reduces progression of diabetesinduced mesangial cell dysfunction via inhibition of platelet-derived growth factor-BB (PDGF-BB) and interaction with its receptor in streptozotocin-induced diabetic rats. BMC Complementary Altern. Med. 2014, 14, 477. (19) Sohn, E.; Kim, J.; Kim, C.-S.; Lee, Y. M.; Kim, J. S. Extract of Polygonum cuspidatum attenuates diabetic retinopathy by inhibiting the high-mobility group box-1 (HMBG1) signalling pathway in streptozotocin-induced diabetic rats. Nutrients 2016, 8, 140. (20) Okutan, L.; Kongstad, K. T.; Jager, A. K.; Staerk, D. Highresolution α-amylase assay combined with high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy for expedited identification of α-amylase inhibitors: proof of concept and α-amylase inhibitor in cinnamon. J. Agric. Food Chem. 2014, 62, 11465−11471. (21) Schmidt, J. S.; Lauridsen, M. B.; Dragsted, L. O.; Nielsen, J.; Staerk, D. Development of a bioassay-coupled HPLC-SPE-ttNMR platform for identification of α-glucosidase inhibitors in apple peel (Malus × domestica Borkh.). Food Chem. 2012, 135, 1692−1699. (22) Liu, B.; Kongstad, K. T.; Wiese, S.; Jager, A. K.; Staerk, D. Edible seaweed as future functional food: identification of α-glucosidase inhibitors by combined use of high-resolution α-glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR. Food Chem. 2016, 203, 16− 22. (23) Liu, B.; Kongstad, K. T.; Sun, Q.; Nyberg, N. T.; Jager, A. K.; Staerk, D. Dual high-resolution α-glucosidase and radical scavenging profiling combined with HPLC-HRMS-SPE-NMR for identification of minor and major constituents directly from the crude extract of Pueraria lobata. J. Nat. Prod. 2015, 78, 294−300. (24) Tahtah, Y.; Wubshet, S. G.; Kongstad, K. T.; Heskes, A. M.; Pateraki, I.; Moeller, B. L.; Jager, A. K.; Staerk, D. High-resolution PTP1B inhibition profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy: proof-of-concept and antidiabetic constituents in crude extract of Eremophila lucida. Fitoterapia 2016, 110, 52−58. (25) Dokli, I.; Navarini, L.; Hameršak, Z. Syntheses of 3-, 4-, and 5O-feruloylquinic acids. Tetrahedron: Asymmetry 2013, 24, 785−790. (26) Suda, M.; Katoh, M.; Toda, K.; Matsumoto, K.; Kawaguchi, K.; Kawahara, S.-i.; Hattori, Y.; Fujii, H.; Makabe, H. Syntheses of procyanidin B2 and B3 gallate derivatives using equimolar condensation mediated by Yb (OTf) 3 and their antitumor activities. Bioorg. Med. Chem. Lett. 2013, 23, 4935−4939. (27) Genichiro, N.; Itsuo, N.; Tetsuro, N.; Hikokichi, O. Tannins and related compounds. I. Rhubarb (1). Chem. Pharm. Bull. 1981, 29, 2862−2870. (28) Okasaka, M.; Takaishi, Y.; Kogure, K.; Fukuzawa, K.; Shibata, H.; Higuti, T.; Honda, G.; Ito, M.; Kodzhimatov, O. K.; Ashurmetov, O. New stilbene derivatives from Calligonum leucocladum. J. Nat. Prod. 2004, 67, 1044−1046. (29) Lee, J. P.; Min, B. S.; An, R. B.; Na, M. K.; Lee, S. M.; Lee, H. K.; Kim, J. G.; Bae, K. H.; Kang, S. S. Stilbenes from the roots of Pleuropterus ciliinervis and their antioxidant activities. Phytochemistry 2003, 64, 759−763. (30) Liu, Y.; Nielsen, M.; Staerk, D.; Jäger, A. K. High-resolution bacterial growth inhibition profiling combined with HPLC−HRMS− SPE−NMR for identification of antibacterial constituents in Chinese plants used to treat snakebites. J. Ethnopharmacol. 2014, 155, 1276− 1283. (31) Kim, H. J.; Lee, J. Y.; Kim, S. M.; Park, D. A.; Jin, C.; Hong, S. P.; Lee, Y. S. A new epicatechin gallate and calpain inhibitory activity from Orostachys japonicus. Fitoterapia 2009, 80, 73−76. (32) Deng, S.; Xia, L.; Xiao, H. Screening of α-glucosidase inhibitors from green tea extracts using immobilized enzymes affinity capture combined with UHPLC-QTOF MS analysis. Chem. Commun. 2014, 50, 2582−2584.
(33) Alemayehu, G.; Abegaz, B.; Snatzke, G.; Duddeck, H. Bianthrones from Senna longiracemosa. Phytochemistry 1993, 32, 1273−1277. (34) Wei, X. H.; Yang, S. J.; Liang, N.; Hu, D. Y.; Jin, L. H.; Xue, W.; Yang, S. Chemical constituents of Caesalpinia decapetala (Roth) alston. Molecules 2013, 18, 1325−1336. (35) Goncalves, M. L. S.; Mors, W. B. Vismiaquinone, a Δ1isopentenyl substituted anthraquinone from Vismia reichardtiana. Phytochemistry 1981, 20, 1947−1950. (36) Du, L.; Zhu, T.; Liu, H.; Fang, Y.; Zhu, W.; Gu, Q. Cytotoxic polyketides from a marine-derived fungus Aspergillus glaucus. J. Nat. Prod. 2008, 71, 1837−1842.
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