Plant materials and chemiclas
The dried aerial parts of Saussurea grandifolia were extracted with methanol under reflux. 1,4-Di-O-caffeoylquinic acid (1,4-DCQA) and 1,5-di-O-caffeoylquinic acid (1,5-DCQA) was isolated from S. grandifolia. Dicaffeoylquinic acid derivatives such as 3,4-di-O-caffeoylquinic acid (3,4-DCQA), 4,5-di-O-caffeoylquinic acid (4,5-DCQA), and 3,5-di-O-caffeoylquinic acid (3,5-DCQA) were isolated from Acanthopanax henryi and obtained Natural Product Institute of Science and Technology (www.nist.re.kr, Anseong, Korea).
NMR data of dicaffeoylquinic acid derivatives
1,4-DCQA (purity: 99.7%): 1H-NMR (DMSO-d6, 500 MHz) δ: 7.51 (2H, d, J = 15.5 Hz, H-7ʹ, 7ʹʹ), 7.02 (2H, br s, H-2ʹ, 2ʹʹ), 6.98 (2H, d, H-6ʹ, 6ʹʹ), 6.76 (2H, dd, H-5ʹ, H-5ʹʹ), 6.24 (2H, d, J = 15.5 Hz, H-8ʹ, 8ʹʹ), 5.05 (1H, br s, H-3), 4.75 (1H, br s, H-4), 4.16 (1H, br s, H-5), 2.20 (3H, m, H-6a, 6b, 2a), 1.80 (1H, br s, H-2b).
1,5-DCQA (purity: 99.7%): 1H-NMR (DMSO-d6, 500 MHz) δ: 7.40 (2H, t, J = 16.5 Hz, H-7ʹ,7ʹʹ), 7.00 (2H, br s, H-2ʹ, 2ʹʹ), 6.88 (2H, dd, J = 8.0 Hz, H-6ʹ, 6ʹʹ), 6.66 (2H, d, J = 8.5 Hz, H-5ʹ, H-5ʹʹ), 6.21 (1H, d, J = 16.0 Hz, H-8ʹʹ), 6.06 (1H, d, J = 16.0 Hz, H-8ʹ), 5.28 (1H, dd, J = 7.5 Hz, H-5), 3.99 (1H, br s, H-3), 3.49 (1H, br s, H-4), 1.71–2.51 (4H, m, H2-2, H2-6).
3,4-DCQA (purity: 98.4%): 1H-NMR (DMSO-d6, 500 MHz) δ: 7.45 (2H, m, H-7’, 7’’), 7.03 (2H, dd, J = 10.0 Hz H-2ʹ, 2ʹʹ), 6.95 (2H, m, H-6ʹ, 6ʹʹ), 6.73 (2H, d, J = 8.5 Hz, H-5ʹ, H-5ʹʹ), 6.20 (1H, m, H-8ʹ, H-8ʹʹ), 5.42 (1H, br s, H-3), 4.94 (1H, br s, H-4), 4.05 (1H, br s, H-5), 1.91–2.11 (4H, m, H2-2, H2-6).
3,5-DCQA (purity: 98.7%): 1H-NMR (DMSO-d6, 500 MHz) δ: 7.47 (2H, t, J = 16.5 Hz, H-7ʹ, H-7ʹʹ), 7.05 (2H, dd, J = 8.5 Hz, H-2ʹ, H-2ʹʹ), 6.99 (2H, m, H-6ʹ, 6ʹʹ), 6.77 (2H, dd, J = 8.0 Hz, H-5ʹ, H-5ʹʹ), 6.25 (1H, d, J = 16.0 Hz, H-8ʹʹ), 6.16 (1H, d, J = 15.5 Hz, H- 8ʹ), 5.20 (1H, m, H-3), 5.11 (1H, br s, H-5), 3.84 (1H, br s, H-4), 1.91–2.17 (4H, m, H2-2, H2-6).
4,5-DCQA (purity: 99.9%): 1H-NMR (DMSO-d6, 500 MHz) δ: 7.49 (1H, d, J = 16.0 Hz, H-7ʹʹ), 7.42 (1H, d, J = 16.0 Hz, H-7ʹ), 7.02 (2H, dd, J = 4.5 Hz, H-2ʹ, 2ʹʹ), 6.97 (2H, m, H-6ʹ, 6ʹʹ), 6.74 (2H, dd, J = 8.0 Hz, H-5ʹ, H-5ʹʹ), 6.24 (1H, d, J = 16.0 Hz, H-8ʹʹ), 6.14 (1H, d, J = 16.0 Hz, H-8ʹ), 5.35 (1H, br s, H-5), 4.96 (1H, dd, J = 7.5 Hz, H-4), 4.17 (1H, br s, H-3), 1.87–2.18 (4H, m, H2-2, H2-6).
α-Glucosidase-inhibitory activity assay
Dicaffeoylquinic acid derivatives were assessed for α-glucosidase-inhibitory activity as described previously, with slight modifications [21, 22]. In brief, acarbose and dicaffeoylquinic acid derivatives (80 μL) at varying concentrations (12.5 to 100 μM) in 120 μL of 0.1 M phosphate buffer (pH 6.8) were incubated with 100 μL of 0.5 U/mL α-glucosidase at 37 °C. Enzyme activity was calculated as: α-glucosidase-inhibitory activity (%) = [(Ablank-Asample)/Ablank] × 100.
Cell culture and determination of cell viability
Rat pancreatic INS-1 line (Biohermes, Shanghai, China) was maintained routinely in the Roswell Park Memorial Institute (RPMI) 1640 medium (Cellgro, Manassas, VA, USA) supplemented with 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 10 mM HEPES, 11 mM D-glucose, 2 mM L-glutamine, and 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Invitrogen Co., Grand Island, NY, USA) under 5% CO2 and 95% humidity at 37 °C. To determine the non-toxic dose ranges of dicaffeoylquinic acid derivatives, INS-1 cells were seeded at 104 cell per well in 96-well plates. After 24 h of incubation, cells were treated with gliclazide and dicaffeoylquinic acid derivatives (100 μL) at varying concentrations (2.5 to 10 μM) for 24 h. The cells were then incubated for 2 h with 10 μL of Ez-Cytox reagent (Daeil Lab Service Co., Seoul, Korea) as described in published methods [23].
GSIS assay
INS-1 cells plated on 12-well plates for 24 h were used to measure the effects of dicaffeoylquinic acid derivatives on GSIS. To this end, INS-1 cells were kept in Krebs–Ringer bicarbonate HEPES buffer (KRBB) supplemented with 2.8 mM glucose for 2 h. Thereafter the INS-1 cells were incubated for 1 h in the fresh KRBB with the denoted glucose concentrations (2.8 or 16.7 mM glucose) and test agents (gliclazide and dicaffeoylquinic acid derivatives). Glucose stimulated index (GSI) was calculated by dividing the insulin concentration that had accumulated during exposure to 16.7 mM glucose by the insulin accumulated during exposure to 2.8 mM glucose. After incubation a cell culture supernatant was analyzed using a rat insulin ELISA kit (Gentaur, Shibayagi Co. Ltd., Shibukawa, Gunma, Japan) as recommended by the producer to measure the GSIS.
Western blot analysis
In the Western blot analysis, INS-1 cells plated on 12-well plates for 24 h were used to measure the effect of 4,5-DCQA on protein expression changes of PI3K, Akt, P-IRS-2 (Ser731), IRS-2, P-ERK, ERK, P-PI3K, P-Akt (Ser473), and PDX-1. To this end, the cells were treated with 4,5-DCQA for 24 h. The cells were lysed on ice for 20 min in radioimmunoprecipitation assay buffer (Cell Signaling, Danvers, MA, USA) with protease inhibitor. The concentration of protein in the lysates was determined using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Samples containing 20 μg concentration of protein were subsequently transferred onto polyvinylidene difluoride membranes. The membranes were incubated treated with first and second antibodies against PI3K, Akt, P-IRS-2 (Ser731), IRS-2, P-ERK, ERK, P-PI3K, P-Akt (Ser473), PDX-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Statistical analysis
All analyses were conducted using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA). Nonparametric comparisons of samples were conducted with the Kruskal–Wallis test to analyze the results. Statistical significance was set at p < 0.05.