Phenolic Elderberry Extracts, Anthocyanins, Procyanidins, and

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Phenolic Elderberry Extracts, Anthocyanins, Procyanidins and Metabolites Influence Glucose and Fatty Acid Uptake in Human Skeletal Muscle Cells Giang Thanh Thi Ho, Eili Tranheim Kase, Helle WANGENSTEEN, and Hilde Barsett J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 17, 2017

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Journal of Agricultural and Food Chemistry

Phenolic Elderberry Extracts, Anthocyanins, Procyanidins and Metabolites Influence Glucose and Fatty Acid Uptake in Human Skeletal Muscle Cells Giang Thanh Thi Ho1,*, Eili Tranheim Kase2, Helle Wangensteen1 and Hilde Barsett1 1

Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O.

Box 1068 Blindern, 0316 Oslo, Norway 2

Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, P.O.

Box 1068, Blindern, 0316 Oslo, Norway

*Corresponding author: Tel:+47 22856015; Fax: +47 22854402; E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT 1

The uptake of glucose and fatty acids in skeletal muscle is of interest for type 2 diabetes

2

treatment. The aim was to study glucose and fatty acid uptake in skeletal muscle cells,

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antioxidant effects and inhibition of carbohydrate-hydrolyzing enzymes by elderberries.

4

Enhanced glucose and oleic acid uptake in human skeletal muscle cells were observed after

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treatment with phenolic elderberry extracts, anthocyanins, procyanidins and their metabolites.

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The 96% EtOH and the acidified MeOH extracts were highly active. Of the isolated

7

substances, cyanidin-3-glucoside and cyanidin-3-sambubioside showed highest stimulation of

8

uptake. Phloroglucinol aldehyde was most active among the metabolites. Isolated

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anthocyanins and procyanidins are strong radical scavengers and are good inhibitors of 15-

10

lipoxygenase and moderate inhibitors of xanthine oxidase. As α-amylase and α-glucosidase

11

inhibitors, they are considerably better than the positive control acarbose. The antidiabetic

12

property of elderberry phenolics increases the nutritional value of this plant and indicates a

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potential as functional food against diabetes.

14 15 16 17 18 19 20 21 22

KEYWORDS: Elderberry; Sambucus nigra; type 2 diabetes; human skeletal muscle cells;

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metabolic disorder; glucose uptake; oleic acid uptake; anthocyanin; procyanidins; metabolites 2 ACS Paragon Plus Environment

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INTRODUCTION Type 2 diabetes (T2D) is a metabolic disorder characterized by chronic hyperglycemia

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caused by insulin resistance, pancreatic β-cell failure and enhanced gluconeogenesis in the

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liver. 1 The World Health Organization (WHO) estimates that 422 million adults live with

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diabetes worldwide and that 90% suffer from T2D.1 The prevalence of T2D is rising

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exponentially, and is estimated to reach 366 million cases by year 2030.2 Supplementary

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nutraceuticals and plant-based medicines might be alternative approaches for treatment of

31

T2D. Polyphenol-rich foods have been linked to a reduced risk of cardiovascular diseases,

32

and might be useful in the prevention and control of T2D and diabetes associated

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complications as well.3

34

The fruit of Sambucus nigra, the elderberries, have been used as dietary supplements

35

and in ethnomedicine throughout centuries in the treatment of diabetes. 4 Elderberries contain

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high amounts of flavonoids, such as flavonols, anthocyanins, proanthocyanidins and phenolic

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acids. 4-6 Studies have shown that elderberry extracts help prevent high blood glucoseby

38

improving insulin activity and possibly by preventing β-cell failure in diabetic rats. It may

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also increase the activity of antioxidant enzymes in serum and reduce lipid oxidation.4, 7 In a

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recent study, elderberry extracts were shown to enhance insulin resistance in mice induced

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with high-fat diet.8

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The skeletal muscle plays an important role in blood glucose control, storage and

43

utilization of glucose. 9 Increased levels of plasma free fatty acids (FFA) are associated with

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cardiovascular diseases. Thus, substances that stimulate glucose and fatty acid uptake in the

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skeletal muscles play an important role in the blood glucose homeostatis. Another therapeutic

46

goal for treating diabetes is the regulation of postprandial hyperglycemia. This can be done by

47

inhibiting carbohydrate hydrolyzing enzymes such as α-amylase and α-glucosidase, thereby

48

delaying carbohydrate digestion and glucose absorption from the instestine.10 Oxidative stress



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and increased production of free radicals have been implicated as contributing factors in the

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development and progression of diabetes and its complications. 11 Sources of reactive species

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(ROS) such as 15-lipoxygenase (15-LO) and xanthine oxidase (XO) play an important role in

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production of reactive oxygen species in diabetes.12 Since oxidative stress is implicated in the

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development of diabetes complications, inhibition of ROS production, utilizing substances

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that act as antioxidants or radical scavengers, might have a positive effect on cardiovascular

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function. 11

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Anthocyanins are extensively metabolized in vivo, suggesting that the accumulation of

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multiple phenolic metabolites may ultimately be responsible for the reported bioactivity of

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anthocyanins. 18 However, anthocyanins may also be detected unmetabolized (as glycosides)

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in plasma. Human intervention studies feeding elderberry extracts and anthocyanins have

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indentified many metabolites in urine, serum and feces. The metabolites that are included in

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our study are hippuric acid, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, protocatechuic

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acid (PCA), caffeic acid, p-coumaric acid, ferulic acid, homovanillic acid, vanillic acid and

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phloroglucinol aldehyde (PGA). These metabolites were selected due to their high

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concentration in urine and plasma after elderberry intake. 13-15 The objective of this study was

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to investigate elderberry extracts and its selected polyphenols and metabolites for uptake of

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glucose- and oleic acid in human skeletal muscles. Furthermore, inhibition of the enzymes α-

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amylase, α-glucosidase, 15-LO and XO, and the radical scavenging activity of the 1,1-

68

diphenyl-2-picrylhydrazyl (DPPH) radical were tested.

69 70 71

MATERIALS AND METHODS Plant Material. Elderberries and pressed juice were gifts from cultivator Rune Hatleli,

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Fresvik in Sogn, Norway. The elderberries (Sambucus nigra ‘Sampo’, a Danish cultivar) were

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harvested in October 2014 and stored at -20 °C until extraction. A voucher specimen (No. 4 ACS Paragon Plus Environment

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EB1010) is deposited in the Pharmacognosy section, School of Pharmacy, University of Oslo,

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Norway.

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Chemicals. 4-Hydroxybenzaldehyde, 4-hydroxybenzoic acid, acarbose, caffeic acid,

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homovanillic acid, vanillic acid, protocatechuic acid (PCA), phloroglucinol aldehyde (PGA),

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hippuric acid, ferulic acid, p-coumaric acid, quercetin, linoleic acid, 15-lipoxygenase (15-LO)

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from soybeans, CD3OD, 4-nitrophenyl α-D-glucopyranoside (PNP-G), 2-chloro-4-

80

nitrophenyl-α-D-maltotrioside (CNPG3), xanthine oxidase from bovine milk, hypoxanthine,

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diphenylpicrylhydrazyl (DPPH) radical, acarbose, 22-S-hydroxycholesterol (22-SHC) and

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bovine serum albumin (BSA) (essentially fatty acid-free) were purchased from Sigma-Aldrich

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(St. Louis, MO, USA). Proanthocyandins B2, B5 and C1 were obtained from Plantchem

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(Klepp, Norway). T0901317 was obtained from Cayman Chemicals (Ann Arbor, Michigan,

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USA). Dulbecco‘s modified Eagle's medium (DMEM-Glutamax™, 5.5 mM), DMEM, fetal

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bovine serum, Ultroser G, penicillin–streptomycin–amphotericin B and trypsin-EDTA were

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obtained from Gibco, Life Technologies (Paisley, UK). D-[14C(U)]glucose (1 µCi/ml, 100 µM)

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and [14C]oleic acid (37 kBq, 100 µM) were purchased from ARC (American Radiolabeled

89

Chemicals) (St. Louis, MO, USA). Corning CellBIND tissue culture plates were obtained

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from Corning Life-Sciences (Schiphol-Rijk, The Netherlands). The protein assay reagent was

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obtained from BioRad (Copenhagen, Denmark). All other reagents were of the highest purity

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available.

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Extraction. Frozen berries were crushed and lyophilized, then grinded to a powder

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and extracted on an accelerated solvent extraction system, ASE 350 (Dionex, Sunnyvale, CA,

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USA). For extraction 30 gram freeze-dried berries were mixed with diatomaceous earth

96

(Dionex, Sunnyvale, USA) (4:1) and loaded in a 100 mL steel cartridge.The elderberries were

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then extracted successively with dichloromethane (DCM) (40 °C) followed by 96% EtOH

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(70 °C), 50% EtOH (70 °C), water at 50 °C and 100 °C. The extractions were performed at 5 ACS Paragon Plus Environment


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1500 psi, with 5 minutes heating, 5 minutes static time, and a 60 seconds purge for a total of

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three cycles. Cold-pressed elderberries were filtered to remove the peels and seeds to obtain

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the raw pressed juice and heated at 68 °C. Extracts and pressed juice were taken to dryness by

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rotary evaporation (IKA ® HB 10 basic, IKA-Werke Gmbh & Co. KG, Staufen) or by

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lyophilization (Alpha 1-4 LD plus, Christ, Germany).

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Total Phenolic Content. Test substances were dissolved in DMSO, and the assay was

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carried out as previously described. 16 A linear calibration curve of gallic acid was obtained in

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the range 3.1-25 µg/ml to calculate the amount of gallic acid equivalents in each sample.

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Isolation of Anthocyanins. The anthocyanins from elderberries were extracted in

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accordance with Bräunlich et al. 17 with minor modifications. Freeze-dried elderberries (1 kg

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dry weight) were extracted by maceration with 2 × 3 L MeOH (0.5% TFA v/v) for 24 h. The

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extract (0.8 kg) was applied on an Amberlite XAD-7HP column (5 × 50 cm) with water as

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mobile phase, followed by 1 L MeOH (0.5% TFA) to obtain the anthocyanins. The

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anthocyanin enriched fraction was then fractionated on a Sephadex LH-20 column (5 ×100

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cm) by use of a step gradient of 3L 15% MeOH (0.1% TFA), 5 L 30% MeOH (0.1% TFA),

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3L 50% MeOH (0.1% TFA) and 2L 100% MeOH. The anthocyanins were eluted with 50%

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EtOH, and this fraction was purified by preparative HPLC using a ProStar Polaris system

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(Varian, Palo Alto, California, USA) equipped with a Microsorb 60-8 C18 (250 × 21.4 mm)

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column (particle size 8 µm) (Varian, Palo Alto, California, USA). The fractions were

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evaporated to dryness in a Genevac vacuum centrifuge (40 °C) (Genevac Ltd, Ipswich, UK).

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Mobile phase A (0.5% TFA in water) and mobile phase B (0.5% TFA in acetonitrile), were

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mixed with the following gradient: 10% B, 0-2 min; 10-20% B, 2-4 min; 20-85% B, 4-24 min,

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the flow rate was 20 ml/min.

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HPLC Analysis of Anthocyanins. HPLC analysis was performed on a LaChrom Elite HPLC system (Hitachi, Tokyo, Japan) with a Chromolith Performance RP18e 100 x 4.6 mm 6 ACS Paragon Plus Environment

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column (Merck, Darmastadt, Germany) as previously described.18 Elution was performed

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using a gradient of mobile phase A (0.5% TFA in water) and mobile phase B (0.5% TFA in

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acetonitrile) with the following time schedule: 10 % B, 0-1 min; 10-20 % B, 1-3 min; 20-85 %

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B, 3-4 min; 85-10%, 4-5 min; 10% B, 5-6 min.

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NMR Spectroscopy. 1H and 13C nuclear magnetic resonance (NMR) were performed

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on a Bruker DPX 300 or a Bruker AVII 400 instrument (Bruker, Rheinstetten, Germany) as

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previously described. 19 CD3OD or CD3OD:TFA (95:5) were used as solvents and

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tetramethylsilane (TMS) as reference.

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Culturing of Human Myotubes. Satellite cells were isolated from the Musculus

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obliquus internus abdominis or musculus vastus lateralis of four healthy donors, age 34.8 (±

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19) years, body mass index 22.9 (± 2.7) kg/m2, fasting glucose 4.9 (± 0.6) mM, insulin,

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plasma lipids and blood pressure within normal range and no family history of diabetes as

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previously described by Ho et al. 19

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Glucose- and Oleic Acid Uptake. Uptake of glucose and fatty acids was performed 19, 20

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by using the substrate oxidation assay as previously described.

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test samples (0.1, 1, 10 and 100 µg/mL (extracts) or 0.1, 1, 10 and 100 µM (pure compounds)

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in final concentrations) solved in 0.1% DMSO that were added directly to the medium (pH

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7.4) for 2 days.

142 143

144 145 146 147

Cells were exposed to

Inhibition of α-Glucosidase Activity. Assay for inhibition of α-glucosidase was done as described previously.19 Inhibition of α-Amylase Activity. Assay for inhibition of α-amylase was done as described previously.19 Scavenging of DPPH Radicals. DPPH scavenging was measured as described previously. 16 7 ACS Paragon Plus Environment


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150 151

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Xanthine Oxidase Inhibition. Assay for inhibitory activity towards XO with hypoxanthine as substrate was performed as described previously. 17 15-Lipoxygenase Inhibition. Inhibition of 15-lipoxygenase (15-LO) from soybeans was measured as described previously. 17

Statistical Analysis. Data and figures for glucose- and oleic acid uptake are given as

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mean (±SEM) from n = number of separate experiments. At least 3 parallels were included in

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each experiment. Comparisons of different treatments were evaluated by two-tailed

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Student's t-test, and p< 0.05 was considered significant. Samples for α-glucosidase, α-amylase,

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DPPH, 15-LO and XO assays were analyzed in triplicate and results are given as averages ±

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SD. Student’s t test was used for statistical evaluation and p< 0.05 was considered statistically

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significant.

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RESULTS AND DISCUSSION

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Extraction and Chemical Characterization. Freeze-dried elderberries were

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successively extracted with dichloromethane, 96% EtOH, 50% EtOH and water (50 °C and

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100 °C). The phenol-sulfuric acid method 21 and 1H NMR analyses revealed that

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carbohydrates were present in the 50% EtOH, 50 °C and 100 °C water extracts, whereas 1H

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and 13C NMR analyses showed signals from aromatic protons and carbons, organic acids and

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carbohydrates in the 96% EtOH crude extract. Due to the risk of anthocyanin degradation at

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high temperatures, 22 the elderberries were also extracted with acidic methanol at room

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temperature. A study by Sadilova et al.23 shows that the elderberry anthocyanin content was

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reduced by approximately 16% after heating for 1 hour at 95 °C at pH 1. Thus with our ASE

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extraction at 70 °C we could anticipate some degradation for the anthocyanin in the ethanol

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extracts. The pressed juice (69.1 ± 1.1 gallic acid equivalents (GAE)/g extract), the 96%

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EtOH (55.3 ± 3.0 GAE/g extract) and the acidic MeOH (49.4 ± 2.0 GAE/g extract) extracts


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were found to have the highest content of phenolics. Anthocyanins from elderberries were

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extracted with 0.5% TFA in MeOH and purified by column chromatography (Amberlite

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XAD-7HP and Sephadex LH-20). Cyanidin-3-glucoside (340 mg) and cyanidin-3-

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sambubioside (250 mg), the major anthocyanins in elderberry (Figure 1), were isolated, in

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addition to the aglycone cyanidin (270 mg). The substances were identified by NMR

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spectroscopy (1H, 13C, COSY, APT, HSQC and HMBC) and the spectroscopic data were in

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accordance with those reported in the literature.24, 25 Chemical structures of anthocyanins,

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procyanidins and metabolites from elderberries employed in this study are shown in Figure 1

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and Figure 2.

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Uptake of Glucose and Oleic Acid in Human Skeletal Muscle Cells. Elderberry

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extracts, anthocyanins, procyanidins and metabolites were screened for their glucose

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stimulation (Figure 3A, B and C) and oleic acid uptake (Figure 4A, B and C) in the human

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skeletal muscle cells. In this study, differentiated human myotubes were treated with test

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compounds for up to 48 h, and then the glucose uptake was measured as the sum of glucose

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oxidation and cell-associated glucose. In line with this, cellular uptake of oleic acid, assessed

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as the sum of cell-associated and CO2-trapped radioactivity, was measured. The 96% EtOH

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and the acidic MeOH extracts showed an increased uptake of glucose at the highest

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concentration tested (50 µg/mL) by 37.4 ± 4.8% and 42.6 ± 6.7%, respectively, compared to

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DMSO control (Figure 3A). The effects of water extracts were less prominent than of the

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phenolic rich extracts, where only the highest concentration of water extracts (50 µg/mL)

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showed a significant increase of glucose uptake. The anthocyanins and proanthocyanidins

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exhibited significant stimulation of uptake at all concentrations tested (0.1-10 µM), where

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cyanidin-3-glucoside and cyanidin-3-sambubioside showed the highest maximal efficacy on

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glucose uptake at 10 µM (38.0 ± 2.0 % and 44.0 ± 3.7%, respectively) (Figure 3B). Since

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proanthocyanidin B2 (dimer) and C1 (trimer) appear to have almost the same activity at the 9 ACS Paragon Plus Environment


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same molar concentration, no clear correlation between polymerization and glucose uptake is

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evident. The anthocyanins and other phenolics such as rutin are present in high amounts in

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elderberries and could therefore be important contributors to the increased glucose uptake.

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Rutin is reported to stimulate glucose- and oleic acid uptake in the same cell model.19 The

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molecular interaction of cyanidin and its glycosides on specific binding sites on human

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skeletal muscles remains unclear. Metabolites that are detected in urine, feces and plasma

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after consumption of elderberry were also investigated for their possible biological effect.

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The effect of the metabolites on glucose uptake in skeletal muscle cells are illustrated in

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Figure 3C. Caffeic acid, a metabolite detected in urine and feces after intake of elderberry

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extract, caused a significant increase in glucose uptake with respect to the control at all tested

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concentrations. Caffeic acid, p-coumaric acid and ferulic acid have been reported previously

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to increase glucose uptake in porcine myotubes.26 Our results showed somewhat higher

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glucose uptake at 10 µM for caffeic acid, p-coumaric acid and ferulic acid. The different cell

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lineages and experimental setups used might be the explanation for the inconsistency in the

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effects of these compounds. The highest increase in glucose uptake was observed at the

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concentration of 10 µM for PGA and PCA (26.7 ± 5.7% and 28.6 ± 3.5%, respectively). PCA

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and PGA are degradation products of cyanidin-3-glucoside which have been detected in urine,

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plasma and feces. 14, 15 Vanillic acid, a methylated degradation product of PCA, which has

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been detected in urine, serum and feces showed a significant increase by 8.7 ± 7.9% at 10 µM

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compared to DMSO control. 4-Hydroxybenzaldehyde, one of the abundant metabolites in

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plasma, showed an increase in glucose uptake at all tested concentrations. 4-

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Hydroxybenzaldehyde has shown to enhance insulin resistance by decreasing body fat in rats

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with obesity. 27

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There is a positive correlation between the intake of unsaturated fatty acids and the

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prevention of T2D. 28 Oleic acid is a common monounsaturated fatty acid in human diet, it


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has a beneficial effect towards glucose transport in adipocytes, and it prevents T2D.29 The

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uptake of oleic acid in human myotubes after exposure to crude extracts, anthocyanins,

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procyanidins and metabolites are shown in Figure 4 A, B and C. Among the crude extracts the

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acidic MeOH and the 96% EtOH extracts showed highest increase in oleic acid uptake in a

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dose-dependent manner. For the acidic MeOH extract, the increase in oleic acid uptake was

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statistically consistent within the tested concentration range of 12.5-50 µg/mL, with a

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numerical maximum increase of 34.0 ± 4.6% at 50 µg/mL compared to DMSO control. The

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high oleic acid uptake of the acidic MeOH and EtOH extracts may be ascribed to the high

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content of polyphenols. Cyanidin-3-glucoside and cyanidin-3-sambubioside increased the

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oleic acid uptake at 10 µM by 26.5 ± 2.2% and 29.3 ± 5.1%, respectively. Small differences

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were observed between the anthocyanins and the procyanidins. Vanillic acid and ferulic acid,

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which have been detected in serum, showed a significant increase in oleic acid uptake in all

234

concentrations. p-Coumaric acid and homovanillic acid, metabolites detected in urine, showed

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a significant increase only at 10 µM (12.3 ± 4.2% and 14.0 ± 7.3%, respectively). 4-

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Hydroxybenzoic acid, that is both urine and feces metabolite, showed a small but significant

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increase in oleic acid uptake at 1 and 10 µM. PCA and PGA, the major metabolites from

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cyanidin-3-glucoside, showed the highest increase of oleic acid uptake at 10 µM (30.8 ± 8.4%

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and 31.3 ± 6.2, respectively).

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Several plant extracts and dietary polyphenols have been reported to enhance glucose-

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and fatty acid uptake in vitro.30 However, their mechanism of action and structural elements

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important for the enhanced uptake is still rather scarce. In human skeletal muscle cells, energy

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substrates are generally believed to be transported into the cells. Basal glucose uptake is

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mediated by glucose transporter (GLUT) 1, while insulin-stimulated glucose transport is

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mediated by GLUT 4. 31 It has been reported that rosemary extract increases the glucose

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upake in the muscle cell by a mechanism that involves AMP-activated protein kinase



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(AMPK) pathway. Activation of AMPK might leads to increased plasma GLUT 1 and GLUT

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4 activity. 31 and has been considered as a new treatment for T2D.32, 33 It has also been

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suggested that polyphenols affect phosphatidylinositide 3-kinase (PI3k) as a key signaling

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pathway for up-regulation of glucose- and fatty acid uptake. 34, 35 The fatty acid translocase

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CD36 (FAT/CD36) is a membrane protein implicated in fatty acid uptake and its upregulation

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is associated with insulin resistance. 35 It has been reported that quercetin and other

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polyphenols down-regulate FAT/CD36. 35 Different plant extracts such as elderflower, anise

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and thyme have been reported to have an effect on the PPARs. The PPARs are transcription

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factors within the large family of nuclear receptors that are crucially involved in the

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regulation of carbohydrate and lipid metabolism. 36 The findings in this paper, that elderberry

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extracts, anthocyanins, procyanidins and metabolites, influence glucose- and fatty acid uptake

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in human skeletal muscle cells are only observational and the mechanisms are unknown.

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The bioavailability of anthocyanins is reported to be low, less than 0.1% of the

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ingested dose is commonly detected. Due to poor absorption and extensive metabolism by gut

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microbiota the effects of the anthocyanins in vivo can be very limited. 37 However,

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unmetabolized anthocyanins (with the glycosidic part of the molecule intact) have been

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detected in plasma. There are big differences in the number of recovered metabolites in blood

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and urine and the amounts of recovered parent anthocyanins in the human studies. The major

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finding in bioavailability studies in humans with elderberry anthocyanins was the

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determination of 0.1-0.4 µM in blood and urine after intake of elderberry extract (500-700 mg

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anthocyanins). 14, 15, 38 The bioavailability of the anthocyanin metabolites was reported be 60-

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and 45-fold higher than their parent compounds in urine and plasma, respectively. 14, 15

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Knowledge about how anthocyanins and proanthocyanidins are fully absorbed is still missing.

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Williamson and Manach 37 reported that physiologic concentrations of polyphenols do not

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exceed 10 µM and in vitro concentrations above 10 µM are generally not valid. Thus, the


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concentrations used in this study might have a clinical relevance, especially for the

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metabolites. The activity of the metabolites might be physiologically more relevant compared

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to the anthocyanins as they are better absorbed and will therefore easier reach the systemic

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tissues.

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Inhibition of α-Amylase and α-Glucosidase. One therapeutic approach for treating

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T2D is to control postprandial hyperglycemia. This can be done through inhibition of the

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carbohydrate-hydrolyzing enzymes α-amylase and α-glucosidase, and thus delay the digestion

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of carbohydrates and the absorption of glucose.10 The inhibitory activity of the water and

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alcoholic extracts, the pressed juice, anthocyanins, procyanidins and the metabolites towards

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α-glucosidase and α-amylase were much stronger than the positive control acarbose, an anti-

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diabetic drug (Table 1 and Table 2). The 96% EtOH extract (IC50 7.2 ± 1.3 µg/mL for α-

283

glucosidase, IC50 6.8 ± 2.1 µg/mL for α-amylase) and the acidic MeOH extracts (IC50 4.6 ±

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1.9 µg/mL for α-glucosidase, IC50 3.9 ± 1.3 µg/mL for α-amylase) were strong α-glucosidase

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and α-amylase inhibitors, and may be ascribed to their high content of polyphenols. The

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potential of the polysaccharides to inhibit α-glucosidase and α-amylase, and act as a

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hypoglycemic agent have previously been reported. 39 However, the mechanism of action is

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unclear. The 50 °C water and 100 °C water extracts from elderberries showed higher α-

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glucosidase and α-amylase inhibition compared to acarbose which could be due to the

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polysaccharides present in these extracts. 40 Cyanidin-3-glucoside and cyanidin-3-

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sambubioside are the predominant anthocyanins in elderberry and both showed strong

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inhibition of α-glucosidase and α-amylase. These results indicate that the presence of 3-O-

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glucoside and 3-O-sambubioside is important for inducing inhibition of α-amylase and α-

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glucosidase. Previous reports have shown that cyanidin-3-glucoside and cyanidin-3-

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sambubioside possessed α-amylase and α-glucosidase inhibitory activities, and the

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anthocyanins have been shown to reduce blood glucose concentration and enhance insulin 13 ACS Paragon Plus Environment


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sensitivity in type 2 diabetic mice. 17, 41, 42 The inhibitory activity of anthocyanins might

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depend on hydrogen bonds between the hydroxyl groups of the polyphenol ligands and

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certain sugar position that serve as the region for binding to the active site of α-amylase and

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α-glucosidase. 41 Proanthocyanidins that are present in a small amount in elderberries

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possessed also strong inhibitory activity on the carbohydrate-hydrolyzing enzymes. Trimeric

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procyanidin C1 were stronger α-glucosidase and α-amylase inhibitors compared to the

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dimeric procyanidins B2 and B5. 43 It appeared that the activity increased as the molecular

304

weight increased, that is in good accordance with previously reported results.43 α-Glucosidase

305

is a membrane-bound enzyme, which means that ingested products do not need to be

306

absorbed from the gastrointestinal tract to have α-glucosidase inhibitory activity in vivo.

307

Anthocyanins as well as oligomeric proanthocyanidins could therefore have local effects as α-

308

glucosidase inhibitors in the GI tract. 44 The metabolites and degradation products were all

309

much more active than the positive control, where PCA was the most active one. In our

310

experiments, PCA is considerably more active than previously reported.45, 46 PCA, however,

311

was reported in a study to be inactive as α-glucosidase inhibitor.47 PCA appears to have not

312

been tested for inhibition of α-amylase previously. The inhibitory activity varies depending

313

on the structural aspects of the compounds, differences in the experimental setup or to

314

different enzyme sources. Antioxidant Activities. It has been suggested that an imbalance between generation

315 316

and scavenging of free radicals, as well as lipid peroxidation, may be involved in the

317

development of diabetes and it is also known that T2D patients are under oxidative stress. 11,

318

12

319

progression of diabetes-associated complications.45 The activity of crude extracts, isolated

320

compounds and the metabolites from elderberries as DPPH scavengers, and as 15-LO and XO

321

inhibitors are shown in Table 1 and Table 2. The DPPH, 15-LO and XO assays cover

Substances that can contribute to a reduction of ROS might have a positive effect on the


Page 15 of 33


322

different aspects of the antioxidant action and the results derived from these tests give a

323

broader view on the antioxidant activity of elderberry substances. In previous studies

324

elderberry extracts have been reported to have high antioxidant activity in DPPH assay,

325

ABTS scavenging assay and ORAC assay. 48-51 In this study, the acidic MeOH extract (IC50

326

95.9 ± 3.9 µg/mL) showed the highest radical scavenging activity followed by pressed juice

327

and the 96% EtOH extract (IC50 128.3 ± 4.3 µg/mL). The DCM extract and the water extracts

328

were inactive in all assays. Among the anthocyanin-type compounds isolated, the aglycone

329

cyanidin was the strongest scavenger of the DPPH radical, with an IC50 value of 22.1 ± 1.1

330

µM. Cyanidin-3-glucoside (IC50 33.6 ± 1.9 µM) and cyanidin-3-sambubioside (IC50 29.2 ±

331

1.4 µM) were slightly less active. Glycosylated anthocyanins decreased antioxidant capacity

332

compared to the corresponding aglycone. Thus, glycosylation of the anthocyanidin and

333

increased molecular weight may seem to have an effect on the antioxidant activities. Radical

334

scavenger activity and antioxidant effect of cyanidin and cyanidin-3-glucoside have been

335

reported earlier. 17, 52 The trimeric procyanidin C1 (IC50 3.2 ± 0.4 µM) was a more efficient

336

scavenger of the DPPH radical than the dimeric ones, procyanidin B2 (IC50 7.4 ± 0.3 µM) and

337

B5 (IC50 8.5 ± 1.1 µM). The radical scavenging ability of the procyanidins is in good

338

accordance with the literature. 53 The number and position of hydroxyl groups (OH) are

339

important factors for scavenging of free radicals and an increase in molecular weight enhance

340

the activity.54 Procyanidins have high hydrogen donating capacity, and the number of OH

341

groups is correlated to the molecular size of procyanidins, that are important for the reaction

342

with DPPH. PCA (IC50 66.9 ± 3.2 µM) and PGA (IC50 75.9 ± 3.5 µM) possessed weak radical

343

scavenging activity when compared to quercetin (positive control; IC50 9.3 ± 1.5 µM).

344

Anthocyanins and procyanidins possessed moderate 15-LO and weak XO inhibitory ability

345

compared to quercetin. Among the procyanidins, procyanidin C1 showed the highest

346

inhibitory activity, and cyanidin was the best inhibitor among the anthocyanins. It has



Page 16 of 33

347

previously been suggested that aglycones are better XO inhibitors than glycosides, and that

348

glycosyl groups decrease inhibitor activity.55 The 15-LO inhibitory activity of anthocyanins

349

and procyanidins are in fair accordance with previous investigations.17 The metabolites and

350

degradation products showed moderate 15-LO inhibitory activities with PCA and PGA as the

351

most active metabolites (IC50 120.8 ± 6.7 µM and 124.2 ± 2.2 µM, respectively). The

352

metabolites were inactive as XO inhibitors, except for PCA, PGA and caffeic acid that

353

showed moderate activity. The acidic MeOH extract was, in particular, the most active of all

354

the extracts, whereas the DCM and aqueous residues were the least active. This might suggest

355

that acidic MeOH extract is more efficient in extraction of phenolics and substances with

356

antioxidant activity.

357

In conclusion, anthocyanins and procyanidins from elderberries and some of their

358

metabolites show ability to increase the glucose and oleic acid uptake in human skeletal

359

muscle cells. The bioavailability of polyphenols is complex, and from the present data it is

360

difficult to point out which substances are the most important ones after human consumption

361

of elderberries. The anthocyanins are poorly absorbed, and therefore the clinical effect can be

362

limited. The metabolites however are better absorbed, and PGA, ferulic acid, PCA and caffeic

363

acid showed pronounced stimulation of uptake of both glucose and oleic acid in human

364

skeletal muscle cells. It is also noteworthy that cyanidin, its glycosides and metabolites have a

365

strong potential in the control of postprandial hyperglycemia by inhibiting intestinal α-

366

glucosidase and pancreatic α-amylase. To our knowledge, this is the first report giving an

367

overview of phenolic elderberry extracts, anthocyanins, procyanidins and metabolites with

368

antidiabetic properties. These properties may increase the nutritional value of elderberries and

369

qualify the use as functional food against diabetes. These results were derived from in vitro

370

experiments and follow-up testing in vivo is necessary to validate these findings.

371


Page 17 of 33


372

ORCID ID: Giang Thanh Thi Ho: 0000-0001-9237-0356

373

Funding

374

No funding

375

Notes

376

The authors declare no competing financial interest.

377

ABBREVIATIONS USED. 15-LO, 15-lipoxygenase; 22-S-hydroxycholesterol, 22-SHC;

378

DCM, dichloromethane; DMSO, dimethyl sulfoxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl;

379

EtOH, ethanol; MeOH, methanol; NMR, nuclear magnetic resonance; PCA, protocatechuic

380

acid; PGA; phloroglucinol aldehyde; ROS, reactive oxygen species; T2D, type 2 diabetes;

381

TFA, trifluoroacetic acid; TMS, tetramethylsilane; XO, xanthine oxidase.

382

ACKNOWLEDGEMENTS. The authors are grateful to Rune Hatleli for the supply of

383

elderberries and pressed juice. The University of Oslo NMR Center is acknowledged for the

384

spectrometer facilities. We are grateful to Karl Egil Malterud for valuable assistance with

385

NMR and enzymatic assays.

386 387

REFERENCES

388

1.

389

http://who.int/mediacentre/factsheets/fs312/en/ accessed 01.10.2016

390

2.

391

estimates for the year 2000 and projections for 2030. Diabetes Care 2004, 27, 1047-1053.

392

3.

393

and disease. Oxid. Med. Cell. Longev. 2009, 2, 270-278.

394

4.

395

benefit of elderberry (Sambucus nigra) in food - A review. J. Funct. Foods 2014, 18B, 941-

396

958.

World Health Organization. Diabetes fact sheets. WHO, Geneva, Switzerland, 2016.

Wild, S.; Roglic, G.; Green, A.; Sicree, R.; King, H. Global prevalence of diabetes

Pandey, K. B.; Rizvi, S. I. Plant polyphenols as dietary antioxidants in human health

Sidor, A.; Gramza-Michałowska, A. Advanced research on the antioxidant and health



Page 18 of 33

397

5.

Mikulic-Petkovsek, M.; Schmitzer, V.; Slatnar, A.; Todorovic, B.; Veberic, R.;

398

Stampar, F.; Ivancic, A. Investigation of anthocyanin profile of four elderberry species and

399

interspecific hybrids. J. Agric. Food Chem. 2014, 62, 5573-5580.

400

6.

401

phenolic composition of different elderberry species and hybrids. J. Food Sci. 2015, 80, 2180-

402

2190.

403

7.

404

fructus effect and efficacy profiles. Phytother. Res. 2010, 24, 1-8.

405

8.

406

improves inflammatory markers and insulin resistance in a high-fat diet-induced obesity

407

model of mice. FASEB J. 2015, 29, 402.

408

9.

409

peroxisome proliferator-activated receptor δ. Pharmacol. Rev. 2009, 61, 373-393.

410

10.

411

Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus. J.

412

Ethnopharmacol. 2006, 107, 449-455.

413

11.

414

in diabetes: implications for vascular and other complications. Int. J. Mol. Sci. 2013, 14,

415

21525-21550.

416

12.

417

Suzuki, H.; Toyama, K.; Spin, J. M.; Tsao, P. S. Diabetic cardiovascular disease induced by

418

oxidative stress. Int. J. Mol. Sci. 2015, 16, 25234-25263.

419

13.

420

degradation products by the intestinal microflora. Bioorg. Med. Chem. 2005, 13, 5195-5205.

Mikulic-Petkovsek, M.; Ivancic, A.; Todorovic, B.; Veberic, R.; Stampar, F. Fruit

Vlachojannis, J.; Cameron, M.; Chrubasik, S. A systematic review on the Sambuci

Farrell, N.; Norris, G.; Ryan, J.; Blesso, C. Anthocyanin-rich black elderberry extract

Ehrenborg, E.; Krook, A. Regulation of skeletal muscle physiology and metabolism by

Ali, H.; Houghton, P.; Soumyanath, A. α-Amylase inhibitory activity of some

Pitocco, D.; Tesauro, M.; Alessandro, R.; Ghirlanda, G.; Cardillo, C. Oxidative stress

Kayama, Y.; Raaz, U.; Jagger, A.; Adam, M.; Schellinger, I. N.; Sakamoto, M.;

Keppler, K.; Humpf, H.-U. Metabolism of anthocyanins and their phenolic


Page 19 of 33


421

14.

Ferrars, R. M.; Cassidy, A.; Curtis, P.; Kay, C. D. Phenolic metabolites of

422

anthocyanins following a dietary intervention study in post-menopausal women. Mol. Nutr.

423

Food Res. 2014, 58, 490-502.

424

15.

425

pharmacokinetics of anthocyanins and their metabolites in humans. Br. J. Pharmacol. 2014,

426

171, 3268-3282.

427

16.

428

from coriander. Food Chem. 2004, 88, 293-297.

429

17.

430

Extracts, anthocyanins and procyanidins from Aronia melanocarpa as radical scavengers and

431

enzyme inhibitors. Nutrients 2013, 5, 663-678.

432

18.

433

H. Anthocyanins, proanthocyanidins and total phenolics in four cultivars of aronia:

434

Antioxidant and enzyme inhibitory effects. J. Funct. Foods 2014, 7, 746-752.

435

19.

436

from elderflowers on glucose- and fatty acid uptake in human myotubes and HepG2-cells.

437

Molecules 2017, 22, 90.

438

20.

439

Cell-based multiwell assays for the detection of substrate accumulation and oxidation. J. Lipid

440

Res. 2007, 48, 961-967.

441

21.

442

for determination of sugars and related substances. Anal. Chem. 1956, 28, 350-356.

443

22.

444

anthocyanin stability in foods; mechanisms and kinetics of degradation. Trends Food Sci.

445

Technol. 2010, 21, 3-11.

Ferrars, R.; Czank, C.; Zhang, Q.; Botting, N.; Kroon, P.; Cassidy, A.; Kay, C. The

Wangensteen, H.; Samuelsen, A. B.; Malterud, K. E. Antioxidant activity in extracts

Bräunlich, M.; Slimestad, R.; Wangensteen, H.; Brede, C.; Malterud, K. E.; Barsett, H.

Wangensteen, H.; Bräunlich, M.; Nikolic, V.; Malterud, K. E.; Slimestad, R.; Barsett,

Ho, G. T. T.; Kase, E. T.; Wangensteen, H.; Barsett, H. Effect of phenolic compounds

Wensaas, A.; Rustan, A.; Lövstedt, K.; Kull, B.; Wikström, S.; Drevon, C.; Hallen, S.

Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P.; Smith, F. Colorimetric method

Patras, A.; Brunton, N. P.; O'Donnell, C.; Tiwari, B. Effect of thermal processing on



Page 20 of 33

446

23.

Sadilova, E.; Stintzing, F.; Carle, R. Thermal degradation of acylated and nonacylated

447

anthocyanins. J. Food Sci. 2006, 71, C504-C512.

448

24.

449

cyanidin-3-sambubioside and assignments of the 1H and 13C NMR resonances through two-

450

dimensional shift-correlated NMR techniques. Phytochem. Anal. 1991, 2, 175-183.

451

25.

452

coumaroyl)-2-(xylosyl)-glucoside]-5-glucoside and other anthocyanins from fruits of

453

Sambucus canadensis. Phytochemistry 1991, 30, 4137-4141.

454

26.

455

Færgeman, N. J.; Kristiansen, K.; Young, J. F.; Oksbjerg, N. Bioactive components from

456

flowers of Sambucus nigra L. increase glucose uptake in primary porcine myotube cultures

457

and reduce fat accumulation in Caenorhabditis elegans. J. Agric. Food Chem. 2013, 61,

458

11033-11040.

459

27.

460

resistance by decreasing body fat in diet-induced obese rats: vanillin and 4-

461

hydroxybenzaldehyde are the bioactive candidates. Eur. J. Nutr. 2011, 50, 107-118.

462

28.

463

Prog. Lipid Res. 2009, 48, 44-51.

464

29.

465

and the Mediterranean diet: a beneficial effect of oleic acid on insulin sensitivity, adipocyte

466

glucose transport and endothelium-dependent vasoreactivity. QJM-Int. J. Med. 2000, 93, 85-

467

91.

468

30.

469

hypoglycemic drugs on glucose uptake in myotubes. Phytomedicine 2009, 16, 1119-1126.

Andersen, Ø. M.; Aksnes, D. W.; Nerdal, W.; Johansen, O. P. Structure elucidation of

Johansen, O.-P.; Andersen, Ø. M.; Nerdal, W.; Aksnes, D. W. Cyanidin 3-[6-(p-

Bhattacharya, S.; Christensen, K. B.; Olsen, L. C.; Christensen, L. P.; Grevsen, K.;

Park, S.; Kim, D. S.; Kang, S. Gastrodia elata blume water extracts improve insulin

Risérus, U.; Willett, W. C.; Hu, F. B. Dietary fats and prevention of type 2 diabetes.

Ryan, M.; McInerney, D.; Owens, D.; Collins, P.; Johnson, A.; Tomkin, G. Diabetes

Prabhakar, P. K.; Doble, M. Synergistic effect of phytochemicals in combination with


Page 21 of 33


470

31.

Naimi, M.; Tsakiridis, T.; Stamatatos, T. C.; Alexandropoulos, D. I.; Tsiani, E.

471

Increased skeletal muscle glucose uptake by rosemary extract through AMPK activation. Appl.

472

Physiol. Nutr. Metab. 2014, 40, 407-413.

473

32.

474

insulin signaling. Circ. Res. 2007, 100, 328-341.

475

33.

476

Wierzbicki, M.; Verbeuren, T. J.; Cohen, R. A. Polyphenols stimulate AMP-activated protein

477

kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor–deficient

478

mice. Diabetes 2006, 55, 2180-2191.

479

34.

480

potentiate glucose uptake and up-regulate Glut-4, PPARγ and PI3K. J. Ethnopharmacol. 2009,

481

126, 533-537.

482

35.

483

Flórez, S.; Fernandez, A.; Benet, M.; Olcoz, J. L.; Jover, R.; Gonzalez-Gallego, J.; Sanchez-

484

Campos, S. Quercetin ameliorates dysregulation of lipid metabolism genes via the PI3K/AKT

485

pathway in a diet‐induced mouse model of nonalcoholic fatty liver disease. Mol. Nutr. Food

486

Res. 2015, 59, 879-893.

487

36.

488

Rimbach, G.; Wein, S.; Wolffram, S.; Kristiansen, K. Identification of plant extracts with

489

potential antidiabetic properties: effect on human peroxisome proliferator-activated receptor

490

(PPAR), adipocyte differentiation and insulin-stimulated glucose uptake. Phytother. Res.

491

2009, 23, 1316-1325.

492

37.

493

bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am. J. Clin.

494

Nutr. 2005, 81, 230S-242S.

Towler, M. C.; Hardie, D. G. AMP-activated protein kinase in metabolic control and

Zang, M.; Xu, S.; Maitland-Toolan, K. A.; Zuccollo, A.; Hou, X.; Jiang, B.;

Kumar, R.; Balaji, S.; Uma, T.; Sehgal, P. Fruit extracts of Momordica charantia

Pisonero-Vaquero, S.; Martínez-Ferreras, Á.; García-Mediavilla, M. V.; Martínez-

Christensen, K. B.; Minet, A.; Svenstrup, H.; Grevsen, K.; Zhang, H.; Schrader, E.;

Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Rémésy, C. Bioavailability and



Page 22 of 33

495

38.

Czank, C.; Cassidy, A.; Zhang, Q.; Morrison, D. J.; Preston, T.; Kroon, P. A.; Botting,

496

N. P.; Kay, C. D. Human metabolism and elimination of the anthocyanin, cyanidin-3-

497

glucoside: a 13C-tracer study. Am. J. Clin. Nutr. 2013, 97, 995-1003.

498

39.

499

polysaccharides in the treatment of type 2 diabetes: A review. Carbohydr. Polym. 2016, 144,

500

474-494.

501

40.

502

Structure–activity relationship of immunomodulating pectins from elderberries. Carbohydr.

503

Polym. 2015, 125, 314-322.

504

41.

505

Inhibitory activities of cyanidin and its glycosides and synergistic effect with acarbose against

506

intestinal α-glucosidase and pancreatic α-amylase. Int. J. Mol. Sci. 2010, 11, 3387-3396.

507

42.

508

antihyperglycemic effects of polyphenols in the fruit of Viburnum dilatatum Thunb. J. Agric.

509

Food Chem. 2006, 54, 4588-4592.

510

43.

511

oxidative and α-glucosidase inhibitory activities of Cynomorium songaricum. Food Chem.

512

2010, 118, 116-119.

513

44.

514

Antioxidants 2016, 5, 13.

515

45.

516

complications. Diabetes Care 1996, 19, 257-267.

517

46.

518

activity of bioactive compounds from maize (Zea mays L.). Chem. Biol. Drug Des. 2014, 83,

519

119-125.

Wu, J.; Shi, S.; Wang, H.; Wang, S. Mechanisms underlying the effect of

Ho, G. T. T.; Ahmed, A.; Zou, Y.-F.; Aslaksen, T.; Wangensteen, H.; Barsett, H.

Akkarachiyasit, S.; Charoenlertkul, P.; Yibchok-Anun, S.; Adisakwattana, S.

Iwai, K.; Kim, M.-Y.; Onodera, A.; Matsue, H. α-Glucosidase inhibitory and

Ma, C.-M.; Sato, N.; Li, X.-Y.; Nakamura, N.; Hattori, M. Flavan-3-ol contents, anti-

Tsuda, T. Recent progress in anti-obesity and anti-diabetes effect of berries.

Giugliano, D.; Ceriello, A.; Paolisso, G. Oxidative stress and diabetic vascular

Nile, S. H.; Park, S. W. Antioxidant, α−glucosidase and xanthine oxidase inhibitory


Page 23 of 33


520

47.

Schäfer, A.; Högger, P. Oligomeric procyanidins of French maritime pine bark extract

521

(Pycnogenol®) effectively inhibit α-glucosidase. Diabetes Res. Clin. Pract. 2007, 77, 41-46.

522

48.

523

alcoholic extracts from Sambucus nigra L. (antioxidant properties of extracts). LWT-Food Sci.

524

Technol. 2006, 39, 308-315.

525

49.

526

and antioxidant activity of small fruits in Lithuania. J. Food. Agric. Environ. 2010, 8, 259-263.

527

50.

528

proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant

529

capacity. J. Agric. Food Chem. 2004, 52, 7846-7856.

530

51.

531

Comparison of major taste compounds and antioxidative properties of fruits and flowers of

532

different Sambucus species and interspecific hybrids. Food Chem. 2016, 200, 134-140.

533

52.

534

DeWitt, D. L. Antioxidant and antiinflammatory activities of anthocyanins and their aglycon,

535

cyanidin, from tart cherries. J. Nat. Prod. 1999, 62, 294-296.

536

53.

537

Radical scavenging ability of polyphenolic compounds towards DPPH free radical. Talanta

538

2007, 71, 230-235.

539

54.

540

the inhibitory effect of tannins and flavonoids against the 1, 1-diphenyl-2-picrylhydrazyl

541

radical. Biochem. Pharmacol. 1998, 56, 213-222.

542

55.

543

flavonoids that inhibits xanthine oxidase. Biochem. Biophys. Res. Commun. 2002, 294, 167-

544

172.

Dawidowicz, A. L.; Wianowska, D.; Baraniak, B. The antioxidant properties of

Viskelis, P.; Rubinskienė, M.; Bobinaitė, R.; Dambrauskienė, E. Bioactive compounds

Wu, X.; Gu, L.; Prior, R. L.; McKay, S. Characterization of anthocyanins and

Mikulic-Petkovsek, M.; Ivancic, A.; Schmitzer, V.; Veberic, R.; Stampar, F.

Wang, H.; Nair, M. G.; Strasburg, G. M.; Chang, Y.-C.; Booren, A. M.; Gray, J. I.;

Villano, D.; Fernández-Pachón, M.; Moyá, M.; Troncoso, A.; García-Parrilla, M.

Yokozawa, T.; Chen, C. P.; Dong, E.; Tanaka, T.; Nonaka, G.-I.; Nishioka, I. Study on

Lin, C.-M.; Chen, C.-S.; Chen, C.-T.; Liang, Y.-C.; Lin, J.-K. Molecular modeling of

545 23 ACS Paragon Plus Environment


Page 24 of 33

FIGURE CAPTIONS Figure 1. Anthocyanins and procyanidins from elderberries Figure 2. Chemical structures of elderberry metabolites. Figure 3. Effects of elderberry extracts, anthocyanins, procyanidins and metabolites on glucose uptake in human myotubes. Myotubes were treated with A) 12.5, 25, and 50 µg/mL of different crude extracts, B) 0.1, 1 and 10 µM of anthocyanins and proanthocyandins and C) 0.1, 1 and 10 µM of selected metabolites for 2 days. Thereafter, the cells were exposed to D[14C(U)]glucose (1 µCi/ml, 100 µM) for 4 h as described in Materials and Methods. 22SHC (10 µM) was used as positive control. The figures show D-[14C(U)]glucose uptake given as means ± SEM (n=3) from separate experiments. *p < 0.05 vs. control (0.1% DMSO). Figure 4. Effects of elderberry extracts, anthocyanins, procyanidins and metabolites on oleic acid uptake in human myotubes. Myotubes were treated with A) 12.5, 25, and 50 µg/mL of different crude extracts, B) 0.1, 1 and 10 µM of anthocyanins and proanthocyandins and C) 0.1, 1 and 10 µM of selected metabolites for 2 days. Thereafter, the cells were exposed to [114

C]oleic acid (37 kBq, 100 µM) for 4 h as described in Materials and Methods. T0901317

(10 µM) was used as positive control. The figures show [1-14C]oleic acid uptake given as means ± SEM (n=3) from separate experiments. *p < 0.05 vs. control (0.1% DMSO).


Page 25 of 33


Table 1. Scavenging of DPPH radical, 15-LO, XO, α-Glucosidase and α-Amylase Inhibitory Activity of Elderberry Crude Extracts. IC50 Values ± SD are shown. Elderberry extract

DPPH

15-LO

XO

α-glucosidase

α-amylase

(µg/mL)

(µg/mL)

(µg/mL)

(µg/mL)

(µg/mL)

DCM

>167

>167

>167

123 ± 5.3

115 ± 4.9

96% EtOH

138.8 ± 5.1

153.9 ± 3.6

>167

7.2 ± 1.3

6.8 ± 2.1

50% EtOH

158.9 ± 5.2

>167

135.9 ± 4.2

13.9 ± 2.1

15.6 ± 2.3

50 °C Water

>167

>167

>167

63.1 ± 3.9

56.8 ± 4.1

100 °C Water

>167

>167

>167

45.8 ± 4.6

32.2 ± 3.6

0.5% TFA in MeOH

95.9 ± 3.9

108.3 ± 4.5

89.8 ± 3.9

4.6 ± 1.9

3.9 ± 1.3

Pressed juice

128.3 ± 4.3

143.5 ± 3.9

145.9 ± 4.4

10.8 ± 3.1

8.9 ± 2.6

Quercetin (control)

2.8 ± 0.3

29.3 ± 1.9

0.7 ± 0.2

nt

nta

Acarbose (control)

nta

nta

nta

84.7 ± 3.8

73.3 ± 4.3

a

a

Not tested



Page 26 of 33

Table 2. Scavenging of DPPH Radical, 15-LO, XO, α-Glucosidase and α-Amylase Inhibitory Activity of Phenolic Compounds and Metabolites from Elderberry. IC50 Values ± SD are shown. Test compound

DPPH

15-LO

XO

α-glucosidase

α-amylase

(µM)

(µM)

(µM)

(µM)

(µM)

Cyanidin

22.1 ± 1.1

102.6 ± 4.4

109.8 ± 2.9

18.4 ± 1.3

16.2 ± 2.7

Cyanidin-3-glucoside

33.6 ± 1.9

132.8 ± 4.8

129.0 ± 2.9

5.0 ± 0.2

3.7 ± 0.8

Cyandin-3-sambubioside

29.2 ± 1.4

123.4 ± 3.2

126.6 ± 2.8

2.8 ± 0.9

2.3 ± 0.5

Procyanidin B2

7.4 ± 0.3

113.6 ± 5.3

110.4 ± 4.7

9.0 ± 0.9

6.6 ± 2.7

Procyanidin B5

8.5 ± 1.1

120.7 ± 3.8

115.6 ± 3.9

11.9 ± 4.7

7.0 ± 5.5

Procyanidin C1

3.2 ± 0.4

104.3 ± 4.5

108.7 ± 5.8

5.2 ± 0.5

2.6 ± 0.9

p-Coumaric acid

>167

129.9 ± 3.2

>167

29.3 ± 4.4

25.9 ± 4.4

Homovanillic acid

>167

153.9 ± 4.2

>167

35.7 ± 3.1

26.3 ± 2.5

Phloroglucinol aldehyde (PGA)

75.9 ± 3.5

124.2 ± 2.2

114.4 ± 4.5

16.0 ± 1.7

12.5 ± 0.9

4-Hydroxybenzoic acid

>167

155.4 ± 5.8

>167

43.5 ± 2.8

35.4 ± 2.7

Hippuric acid

>167

128.6 ± 4.8

>167

71.2± 3.6

62.8 ± 4.6

Ferulic acid

125.8 ± 5.7

135.4 ± 4.2

>167

19.3 ± 3.4

15.9 ± 4.1

4-Hydroxybenzaldehyde

>167

145.1 ± 5.3

>167

21.3 ± 3.8

14.8 ± 2.8

Protocatechuic acid (PCA)

66.9 ± 3.2

120.8 ± 6.7

103.3 ± 2.8

13.3 ± 0.8

10.4 ± 0.6

Caffeic acid

90.3 ± 4.3

125.9 ± 4.7

107.3 ± 3.2

18.5 ± 0.9

13.9 ± 0.7

Vanillic acid

>167

129.7 ± 3.3

>167

25.6 ± 3.2

19.5 ± 4.2

Quercetin (control)

9.3 ± 1.5

96.9 ± 1.3

2.3 ± 0.3

nta

nta

Acarbose (control)

nta

nta

nta

131.2 ± 19

113.5 ±16

Phenolic compounds

Metabolites

Positive control

a

Not tested


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Cyanidin

Cyanidin-3-glucoside

Procyanidin B2

Cyanidin-3-sambubioside

Procyanidin B5

Procyanidin C1

Figure 1.



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Chemical compound

R1

R2

R3

R4

R5

R6

p-Coumaric acid Homovanillic acid Phloroglucinol aldehyde 4-Hydroxybenzoic acid Hippuric acid Ferulic acid 4-Hydroxybenzaldehyde Protocatechuic acid Caffeic acid Vanillic acid

CH=CH-COOH CH2-COOH CHO COOH CO-NH-CH2-COOH CH=CH-COOH CHO COOH CH=CH-COOH COOH

H H OH H H H H H H H

H H H H H OCH3 H H H H

OH OH OH OH H OH OH OH OH OH

H OCH3 H H H H H OH OH OCH3

H H OH H H H H H H H

Figure 2.


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A

B



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C

Figure 3.


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A

B



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C

Figure 4.


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TABLE OF CONTENTS GRAPHICS


Phenolic Elderberry Extracts, Anthocyanins, Procyanidins, and

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