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Sandra Bowles 1,*, Elizabeth Joubert 2,3, Dalene de Beer 2,3, Johan Louw 1,4, ... peak areas at 288 nm, while the flavones (isoorientin and orientin) were .... from Sample 3 (Aspalathin MLM T60_1) of MLM neg.wiff (Turbo Spray), Centroide.

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Intestinal Transport Characteristics and Metabolism of C-Glucosyl Dihydrochalcone, Aspalathin Sandra Bowles 1,*, Elizabeth Joubert 2,3, Dalene de Beer 2,3, Johan Louw 1,4, Christel Brunschwig 5,6,7, Mathew Njoroge 5,6,7, Nina Lawrence 6, Lubbe Wiesner 7, Kelly Chibale 5,6,8,9, and Christo Muller 1,4,10 Biomedical Research and Innovation Platform, South African Medical Research Council, Tygerberg, Cape Town, 7130, South Africa; [email protected] (J.L.); [email protected] (C.M.) 2 Plant Bioactives Group, Post-Harvest and Wine Technology Division, Agricultural Research Council, Infruitec-Nietvoorbij, Stellenbosch 7600, South Africa; [email protected] (E.J.); [email protected] (D.d.B.) 3 Department of Food Science, Stellenbosch University, Stellenbosch 7600, South Africa 4 Department of Biochemistry and Microbiology, University of Zululand, Kwa-Dlangezwa 3886, South Africa 5 Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa; [email protected] (C.B.); [email protected] (M.N.); [email protected] (K.C.); 6 Drug Discovery and Development Centre (H3D), University of Cape Town, Rondebosch 7701, South Africa; [email protected] 7 Division of Clinical Pharmacology, University of Cape Town, Observatory, Cape Town 7925, South Africa; [email protected] 8 Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory 7925, Cape Town, South Africa 9 South African Medical Research Council Drug, Discovery and Development Research Unit, University of Cape Town, Rondebosch 7701, South Africa 10 Department of Medical Physiology, Stellenbosch University, Tygerberg 7507, South Africa * Correspondence: [email protected]; Tel./Fax: +27-219-380-479 1

1.

Caco-2 Transport Experiments

1.1. Cytotoxicity

Figure S1: The cytotoxic effects of aspalathin at various concentrations in HBSS buffer (pH 6.0) on Caco-2 cells was assessed by the VialightTM plus kit. Caco-2 cells were seeded at a density of 4 x 104 cells/cm2 into white clear bottomed 96-well plates and cytotoxicity was assessed according to the manufacturer’s recommendation after 12–13 days.

1.2. Analytical Conditions of High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) with Example Chromatograms.

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HPLC-DAD analysis was performed on an Agilent 1200 system (Agilent Technologies, Inc., Santa Clara, CA) equipped with an in-line degasser, quaternary pump, autosampler, column thermostat, and DAD, controlled by Chemstation software (Agilent Technologies, Waldbronn, Germany). The chromatographic conditions were as follows: Separation was performed at 30 °C on a Poroshell SB-C18 column (50 × 4.6 mm, 2.7 μm particle size; Agilent Technologies, Inc.) protected by an Acquity UPLC in-line filter (Waters; 0.2 μm) and an Acquity UPLC VanGuard pre-column (Waters; stationary phase: BEH C18 1.7 μm). The flow rate was 1.0 mL/min, and a multilinear gradient was performed as follows: 0.0–10.0 min, 12.4–16.6%B; 10.0–10.5 min, 16.6–80.0% B; 10.5–11.5 min, 80.0% B; 11.5–12.0 min, 80.0–12.4% B; 12.0–16.0 min, 12.4% B, with solvents A and B being acetonitrile and 0.1% aqueous formic acid, respectively. UV spectra were recorded between 220 and 450 nm. Stock solutions of the phenolic standards were prepared in dimethylsulfoxide (DMSO) at concentrations of approximately 1 mg/mL and diluted with water according to experimental requirements. All diluted solutions were filtered through 0.22 µ m polyvinylidene difluoride (PVDF) filters (Merck Millipore) prior to use. Six-point calibration curves were set up for all standards. The calibration mixtures were injected at different injection volumes, giving on-column levels of 0.008–1.7 µ g for aspalathin, 0.04–0.8 µ g for caffeine, and 0.01–0.2 µ g for both isoorientin and orientin. Nothofagin was quantified using a previously determined response factor applied to the aspalathin calibration curve. Linear regression, using the least-squares method (Microsoft Excel 2013, Microsoft Corporation, Redmond, WA), was performed on the calibration curve data for each compound to determine the slope and y-intercept. Dihydrochalcones (aspalathin and nothofagin) and caffeine were quantified using their respective peak areas at 288 nm, while the flavones (isoorientin and orientin) were quantified at 350 nm.

(a)

(b)

Figure S2: Example HPLC-DAD chromatograms of (a) apical (time = 0 h) and (b) basolateral (time = 2 h) samples taken from Caco-2 monolayers after incubation with aspalathin.

1.3. Protein Detection

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Protein was isolated from differentiated Caco-2 monolayer cell lysates. The total protein content was determined and analysed by SDS-PAGE. 50 ug of total protein was denatured and loaded onto a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane, using a wet-blot system. Nonspecific binding was blocked, using 5% w/v low-fat milk in Tris-buffered saline with Tween-20 (10 mmol/L Tris-HCl, pH 7.5, 200 mmol/L NaCl, 0.05% Tween-20) at room temperature for 2 h. Subsequently, the membrane was incubated overnight at 4°C in the presence of the following primary antibodies: anti- SGLT1 ab14686 (1:1000 dilution), anti-GLUT2 ab54460 (1:1000), anti-MDR sc55510 (1:1000), and carnitine palmitoyltransferase (CPT1; 1:1000) with the relevant horseradish peroxidase conjugated secondary antibodies applied the following day for 90 min at room temperature. β-Actin (1:4000) antibody was added as a loading control. Proteins were detected and quantified using a Chemidoc-XRS imager and Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Figure S3: Western Blot analysis of (a) SGLT1 (72 kDa), (b) GLUT2 (60 kDa), and (c) MDR-1 (170 kDa).

1.4. Stability of Aspalathin. In order to determine the stability of aspalathin in the buffer employed for the Caco-2 assay, the aspalathin-enriched extract (SB1) were incubated in sealed vials with deionised water, HEPES buffer pH 6 and HEPES buffer pH 7.4, respectively. The aspalathin content were determined for these samples prior to incubation and after 2 h incubation. The percentage aspalathin degradation obtained in deionised water, HEPES buffer at pH 6 and HEPES buffer at pH 7.4 were 1, 10, and 11%, respectively.

2.

In Vivo Metabolism of Aspalathin 2.1. Fragmentation of Aspalathin in Negative Mode

Figure S4: Diagnostic ions for the negative product ion spectra fragmentation of aspalathin and its metabolites

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2.2. In Vitro Incubation of Aspalathin in Mouse Liver Microsomes XIC of -EMS: Exp 1, 451 to 452 Da from Sample 2 (Aspalathin MLM T0_2) of MLM neg.wiff (Turbo Spray)

Max. 5.3e7 cps.

XIC of -EMS: Exp 1, 451 to 452 Da from Sample 3 (Aspalathin MLM T60_1) of MLM neg.wiff (Turbo Spray)

Max. 4.5e7 cps.

(b)

4.21 5.0e7

5.2e7 5.0e7

4.8e7

4.8e7

4.6e7

4.6e7

4.4e7

4.4e7

4.2e7

4.2e7

4.0e7

4.0e7

3.8e7

3.8e7

3.6e7

3.6e7

3.4e7

3.4e7

3.2e7

3.2e7

In te n s ity , c p s

In te n s ity , c p s

(a)

Aspalathin

5.2e7

3.0e7 2.8e7 2.6e7 2.4e7

3.0e7 2.8e7 2.6e7 2.4e7

2.2e7

2.2e7

2.0e7

2.0e7

1.8e7

1.8e7

1.6e7

1.6e7

1.4e7

1.4e7

1.2e7

1.2e7

1.0e7

M1

1.0e7 3.88

8.0e6

8.0e6

6.0e6

6.0e6

4.0e6

4.0e6

2.0e6

2.0e6

0.0

0.0

1.5

2.0

2.5

3.0

3.5

Aspalathin

4.23

4.0

4.5 Time, min

5.0

5.5

6.0

6.5

7.0

7.5

8.0

3.88

1.5

2.0

2.5

3.0

3.5

4.0

4.5 Time, min

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Figure S5: Extracted ion chromatograms of aspalathin and its products formed in (a) no incubation control and (b) mouse liver microsome incubation. XIC of -EMS: Exp 1, 451 to 452 Da from Sample 17 (Aspalathin PB T0) of blood_PB neg.wiff (Turbo Spray)

5.0e6

4.8e6

4.8e6

4.6e6

4.6e6

4.4e6

4.4e6

4.2e6

4.2e6

4.0e6

4.0e6

3.8e6

3.8e6

3.6e6

3.6e6

3.4e6

3.4e6

3.2e6

3.2e6

3.0e6 2.8e6 2.6e6 2.4e6

3.0e6 2.8e6 2.6e6 2.4e6

2.2e6

2.2e6

2.0e6

2.0e6

1.8e6

1.8e6

1.6e6

1.6e6

1.4e6

1.4e6

1.2e6

1.2e6

1.0e6

1.0e6

8.0e5

8.0e5

6.0e5

6.0e5

4.0e5

4.0e5

2.0e5

2.0e5

0.0

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5 Time, min

5.0

5.5

6.0

6.5

7.0

7.5

Max. 7.0e6 cps.

(b)

5.2e6

In te n s ity , c p s

In te n s ity , c p s

(a)

Aspalathin

5.0e6

XIC of -EMS: Exp 1, 451 to 452 Da from Sample 18 (Aspalathin PB T120) of blood_PB neg.wiff (Turbo Spray)

Max. 3.0e7 cps.

4.20 5.2e6

0.0

8.0

1.05 4.22

M1 Aspalathin 4.70 1.24

1.0

1.73

1.5

5.25 5.89

4.96

2.0

2.5

3.0

3.5

4.0

4.5 Time, min

5.0

6.00

5.5

6.0

6.23

6.58

7.21

6.5

7.0

7.41 7.75

7.85

7.5

8.0

Figure S6: Extracted ion chromatograms of aspalathin and its products formed in the (a) no incubation control and (b) phosphate buffer incubation. -EPI (451.13) Charge (+0) FT (64.637): Exp 2, 4.260 min from Sample 5 (Aspalathin MLMT0_1NL120 IDA) of Optimisation.wiff (Turbo Sp...

Max. 2.7e6 cps.

-EPI (449.12) Charge (+1) FT (250): Exp 2, 4.002 min from Sample 3 (Aspalathin MLMT60_1) of MLMneg.wiff (Turbo Spray), Centroide...

(a)

331.1 2.6e6

Max. 2.1e5 cps.

449.0

2.1e5

(b)

2.0e5 2.4e6

1.8e5

2.2e6

1.6e5

1.8e6

In t e n s it y , c p s

In t e n s it y , c p s

2.0e6

451.1

1.6e6 1.4e6 1.2e6 1.0e6

1.2e5

209.1

6.0e4

6.0e5

361.1

4.0e4

4.0e5 2.0e5

149.4

0.0 140

160

179.0 180

328.9

1.0e5 8.0e4

167.1

8.0e5

1.4e5

239.3

312.8

196.9 220.9 200

220

289.0 240

260

280

300

193.1 223.0

2.0e4

343.1

320 340 m/z, Da

397.0 360

380

400

359.2

432.9 420

440

460

480

500

0.0 160

180

200

220

240

260

280

300

320 340 m/z, Da

360

380

400

420

440

460

480

500

Figure S7. Product ion spectrum of the [M–H]- ion of (a) aspalathin and (b) M1 (identified as C-glycosyl eriodictyol).