In vitro and in vivo bioactivity assays
Mouse Islet Isolation
Islet isolation was as per the methods of  and . Briefly, Splenic lobe of pancreas was removed under sterile conditions from groups of three BALB/c mice killed by cervical dislocation without ductal injection and distention. Briefly, the pancreas was cut into small pieces/chopped finely ~1 mm2 and was subjected to enzymatic digestion for 10-12 min by vigorous mechanical shaking in a water bath maintained at 37°C. The dissociation medium consisted of Dulbecco's Modified Minimum Essential Medium (DMEM) supplemented with Collagenase type V (1 mg/mL; Sigma, St. Louis, MO), and 2% BSA fraction V (Hi-Media Labs, Mumbai). The digested tissue was then centrifuged at 1500 g for 10 min, washed twice in PBS (pH: 7.4) and seeded in culture flasks (25 cm2; Nunc, Denmark) containing RPMI-1640 (Hyclone, USA) supplemented with 10% (v/v) FBS (Hi-Media, India), 100 U/mL penicillin and 100 U/mL streptomycin under 95% O2 and 5% CO2 atmosphere at 37°C (Thermo, USA) in air. Under these culture conditions, most of the acinar cells degenerate within 48 hrs leaving behind islets. After 48 hrs of incubation, islets handpicked from exocrine pancreas using a binocular stereomicroscope were quantified using automated Microsoft Excel sheet and number of islet equivalents recorded based on their size. For further purification, islets (freed of acinar cells) were isolated using density gradient Ficoll reagent- Type 400 (Hi-Media Labs, Mumbai) present in 20-11% interface. The islets specificity was assessed using dithizone staining (Hi-Media, Mumbai, Maharashtra, India) while islet viability was performed using Trypan blue staining.
RINm5F and C2C12 cell lines procured from National Centre for Cell Sciences, Pune, India served as the experimental cell lines. The RINm5F cells were maintained in RPMI 1640 supplemented with 10% FCS, 100 IU penicillin/mL and 100 μg streptomycin/mL and incubated at 37°C under a humidified 5% CO2 atmosphere. The cells allowed to grow in 25 cm2 tissue culture flasks until confluence were then sub-cultured for experimentation. RINm5F cells were subjected to glucose stimulated insulin secretion and cultured in either basal (4.5 mM) or stimulated (16.7 mM) glucose concentrations. Insulin secretion assays were quantified using Mouse/Rat Insulin ELISA kit (Mercodia, Sweden).
Glucose induced insulin secretion assay (GSIS)
Isolated islets were cultured at 37°C in a humidified atmosphere of 5% CO2 in air in RPMI-1640 medium containing 10% FBS and antibiotics. Islets were seeded at a concentration of 100 or 50 islets per well in 24-well plates (Falcon, NJ) and allowed to attach overnight prior to acute tests. Wells were washed three times with Krebs-Ringer bicarbonate buffer (KRB; 115 mM NaCl, 4.7 mM KCl; 1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24 mM NaHCO3, 10 mM HEPES, 1 g/L BSA, 1.1 mM glucose; pH 7.4) and pre-incubated for 1 hr at 37°C. Unless otherwise stated, wells were then incubated for 1 h in 1 mL KRB with 4.5 mM or 16.7 mM glucose and OI extract/FRF (10, 50, 100 or 250 μg/mL). Aliquots were removed from each well, centrifuged (2000 rpm for 5 min, at 4°C), and assayed for insulin with mouse insulin ELISA kit and protein concentration was determined.
2NBDG glucose uptake
Glucose uptake studies was carried out using fluorescent probe 2-[N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]- 2-deoxy-d-glucose (2NBDG) . Briefly, Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% Fetal Calf Serum and antibiotics (Penicillin 100 IU/mL and Streptomycin 100 μg/mL) in 5% CO2 at 37°C served as the medium for maintenance of C2C12 skeletal muscle cell line. After attainment of ~70% confluency, cells switched to 2% horse serum for 3 days served the purpose of differentiation. The differentiated myotubes were seeded in 96 well fluorescence plates with BSA (1 mg/mL) and 80 μM fluorescent analogue, 2NBDG [Invitrogen, Carlsbad, CA] in presence of different concentrations of various fractions or FRF for 60 min. For stimulation experiments, 100 nM insulin was added along with fractions or FRF. The cells were washed three times to remove free 2NBDG and, plates were read at excitation wavelength of 485 nm and an emission wavelength of 535 nm using Fluorescent micro plate readers (Molecular Devices, Sunnyvale, USA).
Dose optimization (islet studies)
Optimization studies for different concentrations/doses of STZ (1 mM, 2 mM and 5 mM) and for duration of STZ exposure (2 hr, 6 hr, 8 hr and 12 hr) were carried out. Different concentrations of FRF (10, 50, 100 and 250 μg/mL) were tested for STZ insult/stress experiments. STZ (2 mM) with an exposure time of 8 hrs was characterized as the best-optimized schedule for experimental studies. Experimental groups consisted of control and STZ treated islets, pre-treated with FRF for a period of 24 hrs.
Intracellular Calcium levels
The intracellular calcium concentration, [Ca2+]i, was measured using fura-2AM (Molecular Probes, Invitrogen, USA). Fura-2AM crosses cell membranes and once inside the cell, the acetoxymethyl groups removed by cellular esterases generate fura-2, the fluorescent calcium indicator. Islets were incubated in calcium free HBSS with 5 μM fura-2AM at 37°C for 30 min in shaking water bath. After washing (2×) with calcium free HBSS, islets were suspended in complete HBSS and treated with various concentrations of FRF for 60 min in a shaking water bath. Fluorescence was measured in a spectrofluorimeter (Hitachi 7000, Japan) at an emission wavelength of 500 nm for dual excitation wavelength at 340 and 380 nm. The [Ca2+]i was expressed as nmole/50 islet equivalents.
For cAMP measurements, islets cultured in 24-well plates were exposed to 11.1 mM glucose for 60 min in presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1 mM) [IBMX] and various concentrations of FRF. At the end of incubation, islets were lysed following the manufacturer's instructions, and cAMP quantification was performed using a cAMP direct immunoassay kit (Abcam, USA) and normalized to protein concentrations. Results were expressed as fmol/μg protein.
The viability of cultured islets was determined by assaying the reduction of 3-(4, 5-dimethylthiazol-2-yl) - 2, 5-diphenyltetrazolium bromide (MTT) to formazan. Briefly, islets were seeded in 24-well microtiter plates (50 islets per well) and left overnight to adhere before being exposed to different concentrations of STZ and FRF. In each experiment, different concentrations of FRF (10, 50, 100, 250 μg/mL) along with 2 mM STZ were tested in three separate wells and the cytotoxicity assessed from at least three different experiments. After exposure to STZ and FRF, incubation of the wells was carried out in dark at 37°C for 4 h after addition of 50 μl of 5 mM MTT solution. Subsequently, formazan crystals were dissolved in 200 μl of DMSO after the removal of MTT and the absorbance measured at 570 nm using a Microplate Reader (Biotek Instruments, USA).
The intracellular formation of reactive oxygen species (ROS) was measured using 2', 7'-dichlorodihydrofluorescin diacetate (DCFH-DA) (Fluka Chemicals, USA). The non-fluorescent compound DCFH-DA penetrates into the cell and is cleaved by intracellular esterases, resulting in the formation of 2', 7'-dichlorodihydrofluorescin (DCFH), the oxidation of which (due to oxidative stress) generates the fluorescent compound dichlorofluorescein. Thus, the DCF fluorescence represents the rate and quantity of ROS produced. Fifty islets from all treatment groups, incubated in fresh media with 10 μM DCFH-DA at 37°C for 30 min, was washed twice with PBS and the fluorescence measured using a fluorimeter (Hitachi 7000, Japan) with excitation at 495 nm and emission at 538 nm. All values were corrected by subtracting auto fluorescence for respective wells and the results expressed as relative fluorescence units/50 islet equivalents
Concentration of peroxynitrite from respective treatment groups was estimated by incubating 50 islets with 10 μM dihydrorhodamine 123 (DHR123) for 30 min. Fluorescence was measured using excitation at 500 nm and emission at 530 nm (Hitachi 7000 Spectrofluorimeter). Auto-fluorescence deducted from total fluorescent count yielded corrected fluorescent intensity, expressed as relative fluorescence units/50 islets equivalents.
Nitric oxide estimation
For estimation of the concentration of Nitric oxide from respective treatment groups, 50 islets were incubated with the fluorescent probe DAF-FM (1 μM) for 30 min and then washed with PBS. Fluorescence was measured using excitation at 485 nm and emission at 520 nm (Hitachi 7000 Spectrofluorimeter). Auto-fluorescence was deducted from total fluorescent count for corrected fluorescent intensity and was expressed as relative fluorescence units/50 islets equivalents.
Evaluation of Mitochondrial Membrane Potential (Δψm)
Mitochondria are vulnerable targets for various toxicants because of their important role in maintaining cellular integrity and functions. Functional alterations occur in mitochondria due to changes in mitochondrial membrane potential. Disruption of the mitochondrial membrane potential (i.e. depolarization) is one of the earliest indicators of cellular disturbance. Fifty islets were loaded with a cationic fluorescent dye Rh-123. From the respective treatment groups, islets changed to serum free media containing Rh-123 (10 μM) were incubated for 30 min at 37°C. Results were expressed as relative fluorescent units/50 islet equivalents.
Lipid peroxidation was monitored in terms of malonaldehydes formed . Briefly, islets were suspended in 200 μl of 1.15% KCl. To this suspension were added 0.8% TBA, 1% SDS and 20% acetic acid. The reaction mixture was kept at 90°C for 45 min and immediately cooled on ice. The pink colour representative of thiobarbituric acid reactive substances was measured at 532 nm and protein concentration determined. The values were expressed as nmoles of MDA formed/mg protein.
Multiple dose Streptozotocin model
For induction of diabetes, BALB/c mice (~30 g) were fasted overnight and injected with 50 mg/kg body weight of streptozotocin dissolved in sodium citrate buffer (pH 4.5) intraperitoneally for five consecutive days to delete β-cells. Mice were provided 10% sucrose solution to prevent sudden hypoglycemia. Mice that exhibited frank diabetes (blood glucose levels > 220 mg/dl after 2 weeks) were considered for the experimental studies. Mice were injected flavonoid rich fraction (FRF) for a period of 28 days at a dose 100, 250 and 500 mg/kg of body weight once daily at 1000 hrs. Mice were fasted overnight prior sacrifice. All experiments were carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, India and approved by the Animal Ethical Committee of Department of Zoology, The M.S. University of Baroda, Vadodara (Approval No.827/ac/04/CPCSEA).
Glucose and insulin
Blood glucose was estimated using Glucometer (Roche Diagnostics, USA) and insulin levels were estimated using mouse insulin ELISA (Mercodia, Sweden) with an intra-assay of coefficient variance of > 5%.
TUNEL/Insulin immunostaining & confocal microscopy
The Terminal dUTP transferase mediated Nick End Labeling (TUNEL) assay was performed to assess the in situ DNA fragmentation in paraffin embedded pancreas sections according to the manufacturer's instructions using APO-BrdU™ TUNEL Assay Kit (Invitrogen, USA). Briefly, animals were sacrificed after treatment period and the excised pancreas preserved in 4% paraformaldehyde. Sections were deparaffinised in xylene, downgraded in alcohol grades (100, 95, 85, 70 and 50%), washed with PBS and fixed in fresh 4% paraformaldehyde (PFD) followed by Proteinase-K treatment. Slides were again fixed in 4% PFD and DNA labeling solution added. Guinea pig anti-insulin (1:100, Abcam, USA) was used to probe insulin. Nuclear staining was achieved by adding DAPI/Rnase-A. Anti-Fade solution (Vectamount, Vector Labs, USA) was used as mountant. The slides were visualized by Laser Scanning Confocal Microscope (LSM 510META, ZEISS, Germany). Apoptotic cells exhibited strong nuclear green fluorescence. Optical slices were taken at ~ 0.8 μm. Laser gains, pinhole setting, and magnification was set identical across samples.
Statistical evaluation of the data was done by one way ANOVA followed by Bonferroni's Multiple comparison test. The results are expressed as mean ± S.E using Graph Pad Prism version 5.0 for Windows, Graph Pad Software, San Diego, California USA.