Fig 1.
Effects of HWHF on insulin secretion from (A & B) BRIN-BD11 cells and (C) pancreatic islets, (D) protein glycation, (E) insulin release in the presence of established stimulators or inhibitors and (F) absence of extracellular calcium.
Values are Mean±SEM for n = 4–8 for insulin secretion and n = 3 for protein glycation. *p<0.05, **p<0.01 and ***p<0.001 compared to control (5.6/16.7 mM glucose and 220 mM glucose + insulin (1 mg/ml)). ϕp<0.05, ϕϕp<0.01 and ϕϕϕp<0.001 compared to 5.6 mM glucose in the presence of HWHF. Δp<0.05, ΔΔp<0.01 and ΔΔΔp<0.001 compared to incubations without HWHF.
Fig 2.
Effects of HWHF on (A) membrane potential and (B) cytoplasmic calcium in BRIN BD11 cells, (C, D, E, F & G) glucose uptake by differentiated 3T3L1 adipocytes, (H) starch digestion and (I) in vitro glucose diffusion. Fluorescence intensity was monitored in cells incubated with HWHF without (E) or with (F) 100 nM insulin.
Images are taken at X10 magnification. Values are Mean±SEM for n = 6 for membrane potential and cytoplasmic calcium, n = 4 for glucose uptake, starch digestion and glucose diffusion. *p<0.05, **p<0.01 and ***p<0.001 compared to control.
Fig 3.
Acute effects of HWHF on (A) DPP-IV enzyme activity in vitro, (B) glucose tolerance, (C) plasma insulin and (D) plasma DPP-IV in high fat fed rats. Parameters were assessed before and after oral administration of 18 mmol glucose/kg, body weight (control) with or without HWHF (250 mg/5ml/kg, b.w.).
Established DPP-IV inhibitors: sitagliptin and vildagliptin, were used as positive controls. Values are Mean±SEM, n = 4 for DPP-IV enzyme activity in vitro and n = 6 for glucose tolerance, plasma insulin and DPP-IV in vivo. *p<0.05, **p<0.01 and ***p<0.001, compared to normal control and Δp<0.05, ΔΔp<0.01 and ΔΔΔp<0.001 compared to control.
Fig 4.
Effects of HWHF on (A-F) sucrose content in the gut after oral sucrose loading in high fat fed rats. Rats were fasted for 24 h before the oral administration of sucrose solution (2.5 g/kg b.w.) with or without HWHF (250 mg/5ml/kg, b.w.).
Values are Mean ± SEM, n = 6. *p<0.05 and **p<0.01, compared to control.
Fig 5.
Effects of HWHF on (A & B) gut perfusion, (C) intestinal disaccharidase enzyme activity and, (D) GI motility in high fat fed rats.
Rats were fasted for 36 h, and intestinal perfusion was performed with glucose (54 g/l) with or without HWHF (250 mg/5ml/kg, b.w.). BaSO4 was administered at 1 h following the initial oral dosing. Acarbose (200 mg/5ml/kg, b.w.) and Bisacodyl (1 mg/5ml/kg, b.w.) were used as positive controls for disaccharidase enzyme activity and GI motility, respectively. Values are Mean ± SEM, n = 8. *p<0.05, **p<0.01 and ***p<0.001, compared to control.
Fig 6.
Representative HPLC profile of HWHF.
Crude HWHF was chromatographed with flow rate of 1.0 ml/min on a (10 x 250 mm) semi-preparative 5μm C-18 column (Phenomenex, UK). The concentration of the eluting solvent was raised using linear gradients of acetonitrile (0–20% from 0 to 10 min, 20–70% from 10 to 40 min and 70–20% from 40 to 60 min). Compounds were detected by measurement of absorbance at 254-360nm.
Fig 7.
Molecular mass of peak samples of HWHF by LC-MS analysis.
Peak fractions were separated on a Spectra System LC using a Kinetex 5μm F5 LC column (150 x 4.6 mm) (Phenomenex). The mass-to-charge ratio (m/z) versus peak intensity was determined. Samples of “peaks (P) 1 to 4” with retention times of 19, 20, 23 and 28 min were used to determine the molecular weights of unknown compounds with m/z 449.9, 447.9, 431.9 and 594.0 Da, respectively.
Fig 8.
(A) 1H-NMR, (B) C13-NMR spectrum and isolated compounds (C) Quercitrin of P-2 sample obtained from RP-HPLC of HWHF.
Proton-decoupled natural abundance 1H- NMR and C13- NMR spectrum of peak-2 sample of HWHF (obtained from chromatograph over the period of 70% acetonitrile from 10 to 40 min with retention time of 20 min) at 40 °C. The spectrum was obtained at 600 MHz after 119044 transients (14 h) by the pulsed Fourier transform method on a Varian XL-100 A spectrometer. Representative structure of flavonoids, corresponding to the molecular formula of quercitrin is C21H20O11.
Fig 9.
Insulin releasing effects of (A & B) Peak-1 & 2, (C) Quercitrin from BRIN-BD11 cells and, (D & E) glucose tolerance and plasma insulin in mice.
Fasted (12 h) mice were administered glucose (18 mmol /5ml/kg, body weight) orally with or without (D & E) quercitrin (30 mg/5ml/kg b.w.). Values are Mean±SEM for n = 8 for insulin release and n = 7 for glucose tolerance and plasma insulin. *p<0.05, **p<0.01 and ***p<0.001 compared to control.