Arctium lappa - American Chemical Society

Article pubs.acs.org/JAFC

Chemical Analysis and Antihyperglycemic Activity of an Original Extract from Burdock Root (Arctium lappa) Didier Tousch,‡,∥ Luc. P. R. Bidel,⊥ Guillaume Cazals,# Karine Ferrare,‡,∥ Jeremy Leroy,†,§ Marie Faucanié,†,§ Hugues Chevassus,†,§ Michel Tournier,†,§ Anne-Dominique Lajoix,†,§ and Jacqueline Azay-Milhau*,†,§ †

Université Montpellier I, 4 Boulevard Henri IV, Montpellier, France Université Montpellier II, Place Eugène Bataillon, 34095 Montpellier, France § EA 7288, Centre de Pharmacologie et Innovation dans le Diabète, UFR des Sciences Pharmaceutiques et Biologiques, 15 Avenue Charles Flahault, BP14491, 34093 Montpellier Cedex 5, France ∥ UMR 95 Qualisud, 15 Avenue Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, France ⊥ INRA, UMR AGAP, Centre de Recherche de Montpellier, 2 Place Pierre Viala, F-34060 Montpellier, France # Laboratoire de Mesures Physiques, Service Commun de l’Université Montpellier 2, Plateau Technique de l’Institut des Biomolécules Max Mousseron, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France ‡

ABSTRACT: In the present study, we obtained a dried burdock root extract (DBRE) rich in caffeoylquinic acids derivatives. We performed the chemical characterization of DBRE and explored its antihyperglycemic potential in both in vitro and in vivo experiments. Chemical analysis of DBRE using LC−MS and GC−MS revealed the presence of a great majority of dicaffeoylquinic acid derivatives (75.4%) of which 1,5-di-O-caffeoyl-4-O-maloylquinic acid represents 44% of the extract. In the in vitro experiments, DBRE is able to increase glucose uptake in cultured L6 myocytes and to decrease glucagon-induced glucose output from rat isolated hepatocytes together with a reduction of hepatic glucose 6-phosphatase activity. DBRE did not increase insulin secretion in the INS-1 pancreatic β-cell line. In vivo, DBRE improves glucose tolerance both after intraperitoneal and oral subchronic administration. In conclusion, our data demonstrate that DBRE constitutes an original set of caffeoylquinic acid derivatives displaying antihyperglycemic properties. KEYWORDS: dried burdock root extract, Arctium lappa L. (Asteraceae), chemical analysis, antihyperglycemic effects, in vitro investigations, in vivo effects

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INTRODUCTION Burdock (Arctium lappa L.), a perennial herb cultivated as a vegetable in several countries, is known for its antidiabetic potential. In Africa, burdock has been traditionally used for its antidiabetic property in folk medicine.1 In Europe, burdock has essentially been taken as fresh root decoctions to lower blood sugar.1,2 However, in phytopharmacology, the challenge is to identify the active substance(s) present in the plants. Many studies have been designed to find the compound(s) responsible for the antidiabetic effect. Some compounds, such as the arctiin, inulin and sitosterol β-D-glucopyranoside, have been reported to possess antidiabetic properties.3 Arctiin, a lignan present especially in the root, has been found to be effective against diabetic complications.4 Inulin, a fructan compound often considered as suitable for diabetics, improves glycemic control as a result of a dietary fiber effect.5 Among sterols, sitosterol β-D-glucopyranoside has been reported to be a potent efficient substance able to inhibit glucosidase activities involved in the catabolic process of glycoproteins and glycogen.6 The chemical compositions of burdock root have been reported in the literature.7−9 From these different studies, it appears that the root contains a large number of polyphenols, among them being arctigenin, arctiin, caffeic acid, and many © 2014 American Chemical Society

caffeoylquinic acids derivatives such as chlorogenic acid (CGA) also known for their antihyperglycemic effect.10−12 CGA decreases hepatic glucose release, an effect resulting from the inhibition of glucose 6-phosphatase activity (G6 Pase).10 We therefore focused our study on the set of caffeoylquinic acid derivatives present in burdock roots and its potential antihyperglycemic activity. For this purpose, we produced a dried burdock root extract (DBRE), containing essentially caffeoylquinic acids drivatives, devoid of fructan, terpen, and lipidic compounds and with only very few proteins. In the present work, we describe the chemical composition of DBRE and bring both in vitro and in vivo evidence that our extract exerts significant antihyperglycemic effects.

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MATERIALS AND METHODS

Plant Material: Burdock Root. Burdock (voucher specimen; No. HBH071) root has been collected from the botanic garden of Montpellier (France) and identified by plant biologists. The plant material was dried in a chamber under airflow at 50 °C. Received: Revised: Accepted: Published: 7738

February 27, 2014 June 7, 2014 June 13, 2014 June 16, 2014 dx.doi.org/10.1021/jf500926v | J. Agric. Food Chem. 2014, 62, 7738−7745

Journal of Agricultural and Food Chemistry

Article

Chemicals and Standards. HPLC-grade acetonitrile (CarloErba) and formic acid (Merck, Darmstadt, Germany) were purchased from Merck (Darmstadt, Germany). Water was permuted using a reverse osmosis Milli-Q system (Millipore, Molsheim, France). Chlorogenic acid, caffeic acid, metformin (dimethyl-biguanide), cytochalasin B, and tolbutamide were provided by Sigma-Aldrich (St. Quentin Fallavier, France). Burdock Root Extraction. The dried burdock roots were crushed into a fine powder that was placed into a cellulose cartridge suspended in a glass graduated cylinder. Then, a sequential extraction was performed by “soaking”, i.e., pouring slowly different solutions on the powder using a peristaltic pump. The pump can extend each extraction for 12 h in a closed circulation. The extraction was started with an aqueous solution (50 mM Tris-HCl at pH 8, 150 mM NaCl, and 2 mM EDTA) and continued with a 70% ethanolic solution [ethanol/ water, 70/30 (v/v)] for 48 h. The filtrate was then treated by chloroform (0.5:1; v/v) in a “bulb” glass decanter. The alcoholic phase was collected and dried in a Rotavapor apparatus before storage at −20 °C. Before use, the powder was solubilized in 20% ethanol. Sugar, protein, and phenolic compounds content was measured by the method of Dubois et al.,13 the Bradford method,14 and the method of Singh et al.,15 respectively. GC−MS and LC−MS Analysis of Compounds. GC−MS Analysis. Gas chromatography was performed using a Focus GC oven (Thermo) fitted with a mass spectrometer as detector. The column system used was a TG-5MS (30 m × 0.25 mm i.d., 0.25 μm df) supplied by Thermo. The column was connected to a split/splitless injector. The GC conditions were as follows: the oven was temperature-programmed for 70 °C, initially held for 1 min, then raised at 10 °C/min to 300 °C and held for 5 min. The column flow was 1.2 mL·min−1 helium. Injections of 2 μL were performed in splitless mode with a splitless time of 2 min. The injector temperature was 200 °C. The mass spectrometer used was a DSQII (Thermo) with EI source (70 eV). The scan range was 40−500 Da. A scan speed of 3000 amu/s was used. The source and transfer line temperature were held at 250 and 310 °C, respectively. The data were processed using the Xcalibur system. Identifications were performed using the NIST library. LC−MS Analysis. In a first step, high-resolution mass spectrometry (HRMS/MS) was performed to characterize elemental composition of parent and fragment ions. Abundance of each compound was assessed in a second step with a diode array detector (DAD) coupled with a second HPLC−ESI−MS. For the first step, chromatographic separation was carried out on Acquity H-Class ultrahigh performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA), equipped with a 100/2 Nucleoshell RP18 2.7 μm column (MachereyNagel). The mobile phase consisted of permuted water (solvent A) and acetonitrile (solvent B), both phases acidified by 0.1% (v/v) formic acid. The extract was then characterized using a Synapt G2-S high definition mass spectrometry system (Waters Corp., Milford, MA) equipped with electrospray ionization. Mass spectra were acquired in the positive and negative ionization mode with a capillary voltage of 1 kV. The TOF mass analyzer was calibrated using phosphoric acid in 1:1 (v:v) acetonitrile:H2O from 50 to 1200 m/z to obtain mass accuracy within 3 ppm. The Synapt parameters were optimized as follows: the sample cone was set at 20 V, the source and desolvation temperature were set at 120 and 600 °C, respectively. Each sample was processed with MassLynx (V4.1) software. In the second step, absorbance spectra were acquired using a Waters 996 photodiode array detector. Chromatographic separation was carried out on a XTerra MS C18 column (100 × 2.1 mm, 3.5 μm) heated at 30 °C (Gecko 2000, Cluzeau Info Labo, France). Gradient was performed at a 210 μL·min−1 flow rate using a binary HPLC pump (Waters 1525 μ, Waters, Manchester, UK); it was composed of permuted water (solvent A) and acetonitrile (solvent B), both acidified by 0.1% (v/v) formic acid. The analysis was performed on a Micromass ZQ ESCi multimode ionization mass spectrometer (Micromass Ltd, Manchester, UK) equipped with an electrospray ionization ion source. Absorbance spectra were acquired using a Waters 996 photodiode array detector. Sensitivity of the mass

spectrometer was optimized using a chlorogenic acid standard. For peak assignment, we took as references LC−MS/MS characterizations from Lin and Harnly,11 Jaiswal and Kuhnert,16 and Matura et al.7 Compounds were identified by their retention times, UV absorbance spectra, and MS fragmentation pattern and numbered in conformity with the IUPAC numbering system.17 Isomers were assigned by using the appropriate standards from Sigma-Aldrich (chlorogenic acid, caffeic acid). Since other caffeoylquinic acid derivatives were not commercially available, they were identified by comparison with chromatograms of Arnica montana L. flower extract and of the leaf extract from Coffea canephora, Ilex paraguariensis, Cynaria scolymus L. The compound concentrations were calculated by integrating chromatogram peak areas at 326 nm (maximal absorbance of CGA) and expressed as equivalent absorbance of the authentic chlorogenic acid standard (Sigma-Aldrich). Calibration curves were performed by dilution of stock solutions (10−3 M) in aqueous methanol (30/70, v/ v), stored at −4 °C during experiments. L6 Myocyte Culture and [3H]-2-Deoxyglucose Uptake. The experimental procedure used has been previously described by Tousch et al.12 On the day of the experiment, cells were first starved during 4 h in DMEM supplemented with 0.1% BSA alone and then incubated during 1 h in KRB, 0.1% BSA, 5 mM glucose supplemented with or without 100 nM insulin,18 DBRE at 50 or 100 μg·mL−1, or CGA (100 μg·mL−1) and cytochalasin B (CCB) (1 μM), used as pharmacological controls. Cells were incubated in 1 mL of KRB containing 0.5 μCi [3H]deoxyglucose per well. Uptake was stopped by three washings in cold PBS, and cells were lysed in 0.1 N NaOH. Radioactivity was measured and the total protein concentration was evaluated by a Bradford assay.14 Results are expressed in cpm·mg protein−1·min−1. Hepatocyte Culture and Glucose Output Test. Hepatocytes were isolated from male Wistar rats (180−260 g) fed ad lib using a two-step perfusion technique, as described by Seglen.19 Cells were cultivated on collagen-coated 12-well plates in basal medium (William’s E containing 11.1 mM glucose, 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin) supplemented with 6% FCS at a cell density of 830 000 cells/well. Four hours after initial plating, the medium was replaced with a basal medium supplemented with 100 nM dexamethasone, and plates were incubated for 24 h. Hepatocytes were loaded in glycogen with a 20 h incubation in loading medium: William’s E containing 11.1 mM glucose, 100 U·mL−1 penicillin, 100 μg·mL−1 streptomycin supplemented with 100 nM dexamethasone, 13.9 mM glucose, and 100 nM insulin. After the loading period, hepatocytes were washed three times with PBS and incubated for 3 h in buffer [117.6 mM NaCl, 5.4 mM KCl, 0.82 mM MgSO4, 1.5 mM KH2PO4, 20.0 mM Hepes, 9.0 mM NaHCO3, 0.1% (w/v) BSA, 2.25 mM CaCl2 (pH 7.4)]20 without or with DBRE at 50 or 100 μg·mL−1 or CGA at 50 μg·mL−1 in the presence of 100 nM glucagon (stimulating conditions). After 3 h incubation, supernatants were collected and frozen at −20 °C until glucose quantification. Glucose release (nmol/well) was measured using a glucose oxidase kit (Megazyme, Wicklow, Ireland). Results are presented as the percentage of glucagon stimulation values. In Vitro Measurement of Hepatic Glucose 6-Phosphatase Activity. Commercial liver microsomes (BD Biosciences, Le Pont de Claix, France) were used to quantify glucose 6-phosphatase activity. In the standard glucose 6-phosphatase assay, microsome suspension samples (2 μg of proteins by assay) were incubated for 30 min at 30 °C in the buffer containing 20 mM of glucose 6-phosphate.21 The reaction was stopped by the addition of 1 mL of malachite green color reagent and incubated during 5 min before measuring optical density at 660 nm with NaH2PO4 (1−20 nmol) taken as standard. The integrity of microsomes was checked with 0.1% Triton X100 to measure the activity of the catalytic subunit of glucose 6-phosphatase after membrane disruption as previously described by Murataliev and Vufson.22 The effects of DBRE (50 and 100 μg·mL−1) and CGA (50 μg·mL−1) were tested. The results are expressed as Pi (nmol·h−1). β-Cell Culture and Measurement of Insulin Release. The experimental procedure on rat insulinoma-derived INS-1 β-cells23 has been previously described by Tousch et al.12 For incubation experiments, cells were first washed twice and incubated for 60 min 7739

dx.doi.org/10.1021/jf500926v | J. Agric. Food Chem. 2014, 62, 7738−7745


Article

Table 1. LC−MS Fingerprint with Fragmentation of DBRE Compoundsa peak

tR (min)b

parent ion mol. form.c

parent ion theor mass(m/z)d

Δppme

parent ion [M − H]−

P1 P2 P3 P4 P5 P6 P7

10.7 11.6 16.2 19.2 27.0 29.5 30.4

C16H17O9 C16H17O9 C16H17O9 C16H17O9 C25H23O12 C29H27O16 C33H31O20

353.0878 353.0878 353.0878 353.0878 515.1195 631.1305 747.1414

1.6 0.5 1.3 −1.9 −1.3 25 −0.2

353.088 353.087 353.099 353.087 515.120 631.132 747.141

P8 P9 P10 P11

31.3 30.5 31.4 32.6

C25H23O12 C29H27O16 C38H33O18 C29H27O16

515.1195 631.1305 777.1672 631.1305

2.1 0.9 −0.7 2.1

515.120 631.130 777.167 631.131

P12

34.4

C33H31O20

747.1414

1.1

747.141

P13 P14

34.8 35.2

C29H27O16 C29H27O15

631.1305 615.1355

−1.0 −1.0

631.129 615.134

P15 P16 P17

35.4 35.6 36.0

C33H31O20 C25H23O12 C33H31O19

747.1414 515.1195 731.1433

1.0 1.0 1.9

747.141 515.120 731.146

P18

36.7

C33H31O19

731.1433

−1.6

731.145

P19 P20 P21 P22

36.9 37.3 37.6 38.1

C33H31O20 C33H31O19 C29H27O15 C33H31O20

747.1414 731.1433 615.1355 747.1414

−1.0 −1.2 −0.3 −2.8

747.144 731.146 615.135 747.139

P23

39.0

C28H32O19

793.4000

−1.8

793.160

P24 P25

39.2 40.3

C33H31O18 C38H33O19

715.1516 793.4000

−2.7 −1.5

715.149 793.160

P26 P27

42.2 42.7

C38H33O18 C34H29O15

777.1672 677.1512

−2.6 −2.6

777.165 677.150

fragmentation data (m/z, relative intensity) [M − H]− 191 (100),179 (67), 93 (64) 179 (75), 161 (10), 135 (19), 85 (25) 191 (100), 85 (12) 173 (100), 191 (34), 179 (75), 135 (26) 353 (100), 299 (37), 255 (28), 203 (61), 179 (3), 173 (19) 515 (85), 469 (100), 353 (95), 307 (5), 191 (10), 179 (12), 93 (20) 631 (82), 515 (32), 469 (100), 353 (98), 307 (95), 191 (70), 179 (100), 161 (95), 93 (99) 353 (100), 191 (12), 179 (9), 85 (10) 515 (38), 469 (92), 353 (100), 307 (100) 615 (100), 515 (52), 453 (92), 353 (5) 515 (100), 469 (91), 353 (100), 191 (95), 179 (15), 161 (11), 135 (8), 111 (10) 631 (100), 469 (70), 335 (13), 453 (15), 191 (27), 161 (18), 135 (3), 93 (58) 515 (12), 469 (10), 353 (100), 335 (5), 191 (30), 179 (9), 161 (11), 93 (6) 515 (15), 453 (25), 353 (100), 335 (5), 191 (25), 127 (14), 93 (12), 85 (10) 631 (15), 469 (17), 335 (19), 453 (100), 191 (62), 161 (31), 93 (52) 353 (100), 191 (10), 173 (25), 179 (10) 631 (8), 615 (32), 515 (31), 469 (45), 453 (12), 353 (100), 307 (18), 179 (41), 173 (36), 135 (8) 631 (7), 615 (21), 515 (12), 469 (100), 453 (28), 353 (100), 307 (35), 134 (38) 631 (5), 469 (100), 453 (50), 353 (20), 191 (300), 135 (170), 85 (120) 631 (7), 615 (100), 569 (31), 515 (21), 469 (18), 453 (65), 307 (35) 515 (10), 453 (54), 353 (100), 335 (28), 191 (32) 631 (2), 453 (100), 469 (25), 335 (18), 191 (120), 173 (42), 161 (61), 93 (150) 677 (100), 631 (2), 515 (55), 469 (100), 353 (2), 307 (25), 191 (85), 161 (95), 93 (99) 615 (15), 515 (60), 453 (100), 353 (2), 191 (95), 161 (80), 93 (99) 677 (1), 631 (22), 515 (23), 469 (100), 353 (12), 335 (25), 307 (6), 191 (99), 161 (95), 93 (85) 615 (11), 515 (3), 453 (45), 353 (100), 191 (67), 179 (85), 161 (99) 615 (11), 515 (3), 453 (45), 353 (100), 191 (67), 179 (85), 161 (99)

a

Peaks obtained in DBRE are listed in the order of elution. with their names, molecular formulas, retention times (tR), precursor ions, and fragmentation data of hydroxycinnamoylquinic acids. btR (min): retention time in minutes. cMol. form.: molecular formula. dTheo. mass: theoretical monoisotopic mass of precursor ion [M − H]−. eΔppm: mass tolerance expressed in parts per million. daily administration, an oral glucose tolerance test (OGTT; 3 g·kg−1) was performed. Blood samples were collected from the tail vein, at different times before and after glucose administration. After centrifugation at 4 °C, plasma glucose levels were measured by the glucose oxidase method24 and plasma insulin concentrations using a radioimmunological method.25 Data Analysis. In vitro data are expressed as means ± SD and in vivo data as means ± SEM. Multiple group comparisons were performed by analysis of variance (ANOVA) followed by Fisher’s protected least significant difference test at *p < 0.05 or **p < 0.01, using the Stat Graphics software. For in vivo experiments, plasma glucose and insulin values are expressed in mmol·L−1 and ng·mL−1, respectively. The areas under the curve (AUCs) for the first 60 min (IPGTT) or 120 min (OGTT) were also evaluated.

in glucose-free KRB, BSA (0.1%). They were then washed once again with the same medium and incubated for 60 min in KRB, BSA (0.1%) with glucose at 2.8, 5.6, and 11.2 mM. We investigated the effects of DBRE at 50 or 100 μg·mL−1, in the presence of 5.6 mM glucose. Tolbutamide was used at 200 μM as a control insulinotropic drug. Insulin released in the medium was determined by FRET technology with the HTRF Insulin-Kit (Cis-Bio International). Fluorescence levels were measured on a RUBY star instrument (BMG LABTECH). Results are expressed in ng·mL−1. Animals. Experiments were performed in male Wistar rats (280− 320 g) obtained from Charles River Laboratories (L’Arbresle, France) and maintained on a 12 h/12 h light−dark schedule. The protocols have been reviewed and approved (2009/11/03) by the Animal Care and Use Committee Languedoc-Roussillon (CEEA-LR-0920 and CEEA-LR-0921). Glucose Tolerance Test. Two experimental sets were carried out. The first four groups received, during 4 days, daily intraperitoneal (ip) administrations of DBRE at 0.75 or 3 mg·kg−1 or metformin at 100 mg·kg−1 as a pharmacological positive control or saline (controls). On the fourth day, fasted rats were submitted to an intraperitoneal glucose tolerance test (IPGTT; 1 g·kg−1). In the second set, the animals were divided into three groups; they received four daily oral administrations of either DBRE at 3 or 15 mg·kg−1 or saline (controls). On the fourth

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RESULTS

GC−MS and LC−MS Chemical Analysis. The global composition of DBRE (percent of weight) was as follows: 12% of glucidic compounds, 86% of phenolic compounds, 0.8% proteins, and 1.2% others. The total phenolic content is 150 mg/g of dry plant material. 7740



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Figure 1. Effect of DBRE (50 or 100 μg·mL−1) on glucose uptake in L6 muscle cells without or in the presence of 100 nM insulin. CGA (100 μg· mL−1) and CCB (1 μM) were used as control pharmacological compounds. Values are means (±SD) of six independent experiments (n = 24 for each condition). *p < 0.05, **p < 0.01 versus insulin (100 nM) alone.

< 0.05). CGA induced a similar increase (p < 0.05), whereas CCB provoked a drop in glucose uptake (p < 0.01). Effect of DBRE on Hepatic Glucose Output. Glucagon (100 nM)-induced glucose release reached 450 ± 30 nmol glucose/well (100%) (Figure 2A). The presence of 50 or 100 μg·mL−1 DBRE induced a significant non-concentrationdependent decrease (p < 0.01). Similar results were obtained with CGA at 50 μg·mL−1 (p < 0.05). Effect of DBRE on Hepatic Glucose 6-Phosphatase Activity. DBRE induced a significant inhibition of glucose 6phosphatase activity on hepatic microsomal fractions (Figure 2B). A similar inhibitory effect was observed in the presence of 50 and 100 μg·mL−1 DBRE or 50 μg·mL−1 CGA (p < 0.01). Effect of DBRE on INS-1 β-Cells Insulin Release. As expected, increasing glucose concentration in the medium from 2.8 to 5.6 and 11.2 mM resulted into a clear and significant glucose-dependent stimulation of insulin release (Figure 3). Tolbutamide also induced a clear insulinotropic effect. In the presence of 5.6 mM glucose, addition of DBRE at 50 μg·mL−1 did not modify insulin release. However, a slight but significant (p < 0.05) reduction in insulin release occurred for the higher 100 μg·mL−1 concentration. Glucose Tolerance Test. After four daily DBRE (0.75 or 3 mg·kg−1) or metformin (100 mg·kg−1) ip administrations, basal glucose and insulin levels were not found to be significantly modified (Table 3A). IPGTTs clearly show that DBRE at 0.75 mg·kg−1 is able to reduce the increase in plasma glucose (p < 0.05) from minute 10 to 30. A similar effect was observed in metformin-treated rats. These results are corroborated by AUCs for the first 60 min. Glucose-induced insulin response was significantly reduced (p < 0.05) by DBRE at 0.75 mg·kg−1. In contrast, in rats treated with the higher DBRE dose (3 mg· kg−1) no significant effect could be observed. After four daily oral administrations of DBRE at 3 or 15 mg· kg−1, basal glucose and insulin levels were not found to be modified (Table 3B). Oral glucose tolerance tests bring evidence that DBRE at 15 mg·kg−1 is able to significantly

GC−MS and LC−MS allowed us to assign and characterize all substances except sugars. The data obtained by LC−MS analysis are shown in Tables 1 and 2. The GC−MS chemical monitoring allowed the detection of 27 hydroxycinnamic acids, of which 19 have been previously described by Lin and Harnly11 and Jaiswal and Kuhnert;16 hence, our analysis allowed us to identify eight other additional isomers in our original extract. The chemical analysis of DBRE has been performed by LC− MS and GC−MS. LC−MS analysis revealed the presence of a large majority of dicaffeoylquinic acid derivatives (75.4%). Four maloyl-dicaffeoylquinic acid isomers were identified by their parent ion (m/z 631.129) and m/z 469.099 fragment, corresponding to [maloyl−caffeoylquinic acid−H] after the loss of one caffeoyl group. The three peaks P6, P9, and P11 have been previously observed by Jaiswal and Kuhnert.16 Among them, 1,5-di-O-caffeoyl-4-O-maloylquinic acid (peak P11) is the most abundant DBRE compound and contributes to 44% of chlorogenic acid equivalent absorbance. 1,4-O-Dicaffeoylquinic acid (peak P5) and 1,5-O-dicaffeoylquinic acid (cynarin, peak P16) represent respectively 1 and 3%. P5 and P16 correspond to the two major dicaffeoylquinic acids of Cynaria scolymus leaf extract (i.e., 1,4-di-O-caffeoylquinic acid and 1,5-di-O-caffeoylquinic acid. Likewise, peak P8 corresponds to the 3,5-dicaffeoylquinic acid peak of Coffea robusta and Ilex paraguariensis leaf extracts. DBRE is poor in monocaffeoylquinic acids (m/z 353.098 [M − H], peaks P1−P4), representing 12%, of which 7% is chlorogenic acid (5-O-caffeoylquinic acid, named CGA and used as “ authentic standard” in in vitro experiments, peak P3). Effect of DBRE on Glucose Uptake in L6 Myocytes. In the absence of insulin, DBRE has no effect on glucose uptake by L6 myocytes (Figure 1). In the presence of insulin (100 nM), glucose uptake increased from 629 to 862 cpm·mg−1· min−1 (i.e., +37%, p < 0.01). Addition of DBRE (100 μg·mL−1) significantly increased insulin-induced glucose uptake (+16%, p 7741



Article

Figure 2. Effect of DBRE at 50 or 100 μg·mL−1 on glucagon-induced glucose release from rat isolated hepatocytes (A) and on glucose 6-phosphatase activity of liver microsomes (B). CGA (50 μg·mL−1) was used as control. For panel A, results are expressed as percent of glucose release in the presence of glucagon alone. Data are means (±SD) of three independent experiments (n = 14 values for each condition). *p < 0.05 and **p < 0.01 versus glucagon alone (100% effect). For panel B, results are expressed as Pi nmol·h−1. Data are means (±SD) of three experiments (n = 9 for each condition). **p < 0.01.

are largely due to our sequential partitioned extraction procedure but also could be due at the plant growth geographic conditions and also the burdock genotypes, as was shown by Liu at al.26 None of the six lignans previously described in burdock roots were present in DBRE. Likewise, no fructan, terpen, or lipid could be detected. Our in vitro experiments bring evidence that DBRE is able to increase glucose uptake in myocytes (L6 cells) in the presence of insulin, pointing to an insulin-sensitizing effect of the extract. Upon stimulation of glucose production by glucagon in rat isolated hepatocytes, DBRE induced a strong decrease in glucose release which was paralleled by a significant decrease in glucose 6-phosphatase activity. These results are in agreement with previous studies investigating CGA effects.10,12 In contrast, in the presence of glucose (5.6 mM), DBRE (100 μg·mL−1) significantly decreased insulin release by INS-1 β-cells. This result is in discordance with the presence of CGA, which has been described as an insulinotropic compound,12 suggesting that in DBRE another compound is present that is able to counteract and reverse the CGA effect.

reduce hyperglycemia from minute 60 to 90 (p < 0.05) (Table 3B). Interestingly, a clear increase in insulin response to oral glucose preceded the reduction in hyperglycemia at 30 min (p < 0.05) and 60 min (p < 0.01). AUCs for the first 120 min confirm this observation. No significant change could be observed for DBRE at 3 mg·kg−1.

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DISCUSSION Our extraction procedure allowed us to get an original polyphenol-rich extract of dried burdock roots. The total phenolic content is 150 mg/g of dry plant material, in comparison with 28.7 mg/g of dry plant material in the hydromethanolic (70:30, v/v) burdock root extract obtained by Ferracane et al.8. The chemical analysis of DBRE by GC−MS and LC−MS led to the identification of 27 caffeoylquinic acids derivatives and notably 1,5-di-O-caffeoyl-4-O-maloylquinic (44%) and monocaffeoylquinic acid (CGA) (7%). DBRE differs clearly from the extracts obtained by Ferracane et al.,8 Lin and Harnly,11 and Jaiswal and Kuhnert.16 These differences 7742



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Table 2. Concentrations of Each Compound Were Calculated by Integrating Peak Areas of Chromatograms at 326 nm (maximal absorbance of CGA) and Are Expressed as Equivalent Absorbance of the Authentic Chlorogenic Acid Standard at 10−3 M

Figure 3. Effect of DBRE on insulin release in the INS-1 β-pancreatic cell line. DBRE (50 and 100 μg·mL−1) or tolbutamide (200 μM) as a positive control were tested in the presence of 5.6 mM glucose. Data are means (±SD) of six independent experiments; they are expressed as ng·mL−1 (n = 24) for each experimental condition. *p < 0.05, **p < 0.01 versus 5.6 mM glucose.

Our in vivo data obtained after IP subchronic treatments clearly show that DBRE (0.75 mg·kg−1) improves IP glucose tolerance, an effect that occurs together with a decrease in insulin secretion. These data confirm and extend in vivo and in vitro experiments pointing to an insulin sensitizing effect. It is noteworthy that similar results could be obtained with metformin, another well-known insulin-sensitizing agent. At the higher dose (3 mg·kg−1), DBRE was ineffective, which suggests probably an adverse effect, as already described for pro-oxidants, showing once again and emphasizing the importance of avoiding high dosages upon treatments with polyphenols.27,28 It is interesting to note that a treatment of diabetic db/db mice with a rich caffeolyquinic acid extract of Pandanus tectorius for 3 weeks induced an antihyperglycemic effect by insulin-sensitizing action.29 Concerning oral repeated administrations of DBRE, our extract, at the dose of 15 mg· kg−1, remained able to improve oral glucose tolerance. A similar beneficial effect on plasma glucose levels has been previously reported in rats with orally ingested synthetic CGA (3.5 mg· kg−1);30 these authors ascribed the reduced increase in plasma glucose to a possible reduced glucose intestinal absorption. From a physiological point of view, it is worthy to mention that a tricaffeoylquinic acid (3,4,5-tri-O-caffeoylquinic acid) present in the brazilian propolis exerts an antihyperglycemic effect through the inhibition of intestinal maltase activity.31 Anyhow, our experiments bring evidence that subchronic oral DBRE administrations result in a clear increase of glucose-induced insulin response. Previous studies have reported that heavy coffee (with CGA as the chief polyphenol present) consumption is associated with increased plasma levels of GLP-1.32 We therefore suggest that the increased insulin response and the beneficial effect on oral glucose tolerance could be due to a DBRE-induced increase in incretin activity, all the more since insulin response to glucose is not modified after repeated ip DBRE administrations, which is also in line with the absence of insulinotropic effect in vitro on INS cells. In conclusion, we realized and chemically characterized an extract of dried Burdock roots (DBRE). Our data demonstrate that the original set of caffeoylquinic acid compounds present in DBRE at 86% displays both in vitro and in vivo compelling antihyperglycemic properties with potential therapeutic relevance. However, further investigations are required to address

peak

tR (min)

name

Abs326

ref

P1 P2 P3 P4 P5 P6

10.7 11.6 16.2 19.2 27.0 29.5

0.38 4.08 7.52 0.06 0.97 4.17

11 11 11, 16 11 11, 16 16

P7 P8 P9

30.4 31.3 30.5

0.00 2.95 0.95

11, 16 16

P10 P11

31.4 32.6

P12

34.4

P13 P14

34.8 35.2

P15

35.4

P16

35.6

1-O-caffeoylquinic acid 3-O-caffeoylquinic acid 5-O-caffeoylquinic acid 4-O-caffeoylquinic acid 1,4-di-O-caffeoylquinic acid 1,5-di-O-caffeoyl-3-O-maloylquinic acid dimaloyl-dicaffeoylquinic acid isomer 1 3,5-di-O-caffeoylquinic acid 1,4-di-O-caffeoyl-3-O-maloylquinic acid succinoyl-tricaffeoylquinic acid isomer 1,5-di-O-caffeoyl-4-O-maloylquinic acid 1,3-di-O-caffeoyl-4,5-di-Omaloylquinic acid maloyl-dicaffeoylquinic acid isomer 1,5-di-O-caffeoyl-3-O-succinoylquinic acid 1,4-di-O-maloyl-3,5-di-Ocaffeoylquinic acid 1,5-di-O-caffeoylquinic acid

P17

36.0

P18

36.7

P19 P20

36.9 37.3

P21

37.6

P22 P23 P24

38.1 39.0 39.2

P25

40.3

P26

42.2

P27

42.7

dicaffeoyl-succinoyl-malonylquinic acid isomer 1 dicaffeoyl-succinoyl-malonylquinic acid isomer 2 dimaloyl-dicaffeoylquinic acid isomer 2 1,5-di-O-caffeoyl-3-O-succinoyl-4-Omaloylquinic acid 1,5-di-O-caffeoyl-4-O-succinoylquinic acid dimaloyl-dicaffeoylquinic acid isomer 3 maloyl-tricaffeoylquinic isomer 1,5-di-O-caffeoyl-3,4-di-Osuccinoylquinic acid 1,3,5-tri-O-caffeoyl-4-O-maloylquinic acid 1,3,5-tri-O-caffeoyl-4-Osuccinoylquinic acid 1,3,5-tri-O-caffeoylquinic acid

0.24 43.95

16

1.18

16

0.32 12.29 1.83 2.96

7, 11, 16 16 7, 11, 16

0.44 0.48 0.03 0.45 1.23 0.09 2.18 2.08

16 7, 11, 16

16

7.04 1.40 0.73

7, 11, 16 11

and understand the specific role of each caffeoylquinic acid compound, in the beneficial effects of DBRE.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +33 4 11 75 94 89. Fax: +33 4 11 75 95 47. E-mail: [email protected]; UFR des Sciences Pharmaceutiques et Biologiques, Centre de Pharmacologie et Innovation dans le Diabète, EA7288, 15 avenue Charles Flahault, B.P. 14491, 34093 Montpellier cedex 5, FRANCE. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Excellent technical support from Yves Baissac is acknowledged. We sincerely thank Dr. Gérard Ribes, Dr. René Gross, and Prof. Pierre Petit for consenting to give us some precious scientific advice. 7743



Article

Table 3. Effects of DBRE on Glycemia and Insulinemia during either an Intraperitoneal Glucose Tolerance Test (IPGTT) (A) or an Oral Glucose Tolerance Test (OGTT) (B) in Wistar Ratsa (A) Intraperitoneal Glucose Tolerance Testb 0 min glycemia (mmol·L−1) controls DBRE, 0.75 mg·kg−11 DBRE, 3 mg·kg−1 metformin, 100 mg·kg−1 insulinemia (ng·mL‑1) controls DBRE, 0.75 mg·kg−1 DBRE, 3 mg·kg−1 metformin, 100 mg·kg−1

glycemia (mmol·L−1) controls DBRE, 3 mg·kg−1 DBRE, 15 mg·kg−1 insulinemia (ng·mL‑1) controls DBRE, 3 mg·kg−1 DBRE, 15 mg·kg−1

10 min

5.3 5.6 5.4 5.7

± ± ± ±

0.1 0.2 0.2 0.2

10.3 6.3 10.3 7.9

0.3 0.6 0.3 0.4

± ± ± ±

0.1 0.2 0.1 0.1

3.3 1.8 2.4 2.5

± ± ± ±

20 min

0.9 0.5* 0.6 0.5*

10 5.7 8.7 7.8

± 0.6 ± 0.5* ± 0.4 ± 0.1 (B) Oral

± ± ± ±

30 min

0.8 0.4* 0.9 0.7*

9.7 7 7.7 7.1

± ± ± ±

0.5 0.3* 0.9 0.7*

1.7 ± 0.4 1.2 ± 0.4 1.1 ± 0.2 0.8 ± 0.3 1.2 ± 0.3 0.9 ± 0.3 1.9 ± 0.4 1.2 ± 0.3 Glucose Tolerance Testc

60 min

90 min

120 min

AUCs (60 min)

7.7 6.8 7.9 7.1

± ± ± ±

0.5 0.8 0.7 0.6

6.3 6.3 7.3 5.8

± ± ± ±

0.6 0.6 0.4 0.2

7.1 5.9 6.2 6.3

± ± ± ±

0.2 0.4 0.2 0.4

538 390 488 434

± ± ± ±

31 20* 29 25*

1.7 0.7 1.2 1.0

± ± ± ±

0.7 0.1 0.3 0.3

1.3 0.9 0.9 1.3

± ± ± ±

0.4 0.3 0.2 0.4

1.0 1.0 1.1 1.3

± ± ± ±

0.4 0.2 0.2 0.4

102 60 76 87

± ± ± ±

27 13* 10 15

0 min

15 min

30 min

60 min

90 min

120 min

180 min

AUCs (120 min)

5.2 ± 0.3 4.9 ± 0.3 5.7 ± 0.2

8.6 ± 0.5 8.4 ± 0.4 7.6 ± 0.6

9.5 ± 0.8 8.0 ± 0.4* 8.4 ± 0.5*

8.7 ± 0.5 8.3 ± 0.3 8.3 ± 0.2*

8.0 ± 0.3 8.0 ± 0.2 8.3 ± 0.2

8.1 ± 0.2 7.2 ± 0.3 7.7 ± 0.3

6.1 ± 0.3 5.6 ± 0.1 6.1 ± 0.2

1002 ± 50 938 ± 31 962 ± 34

0.5 ± 0.3 0.4 ± 0.1 0.4 ± 0.1

2.6 ± 0.3 2.5 ± 0.3 3.1 ± 0.2*

1.7 ± 0.6 1.5 ± 0.2 3.6 ± 0.6**

1.7 ± 0.3 1.3 ± 0.2 1.8 ± 0.3

0.7 ± 0.2 1.5 ± 0.3 1.4 ± 0.2

0.9 ± 0.3 0.8 ± 0.2 1.2 ± 0.6

0.6 ± 0.1 0.7 ± 0.2 1.1 ± 0.4

169 ± 23 170 ± 15 244 ± 5*

Values are means (±SEM) expressed in mmol·L−1. bTime courses of plasma glucose (mmol·L−1) and plasma insulin (ng·mL−1) during IPGTTs (1 g·kg−1) in rats previously treated by intraperitoneal DBRE (0.75 and 3 mg·kg−1), saline, or metformin (100 mg·kg−1) during 4 days; n = 5 for each experimental condition. AUCs were calculated for the first 60 min. *p < 0.05. cTime courses of plasma glucose (mmol·L−1) and plasma insulin (ng· mL−1) during OGTTs (3 g·kg−1) in rats treated with oral DBRE (3 and 15 mg·kg−1) or saline during 4 days; n = 5 for each experimental condition. AUCs were calculated for the first 120 min. *p < 0.05, **p < 0.01. a

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Arctium lappa - American Chemical Society

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