Chemicals
3T3-L1 mouse embryonic fibroblasts and bovine calf serum were purchased from the American Type Culture Collection (ATCC, CL-173, USA). Insulin solution from bovine, methyl isobutyl xanthine (IBMX), lipase from porcine pancreas type 2, dimethyl sulphoxide (DMSO), 4-Nitrophenyl dodecanoate (pNP), simvastatin and orlistat were purchased from Sigma-Aldrich (St. Louis, USA). Dulbecco’s modified Eagle’s medium with high glucose (DMEM), penicillin, streptomycin, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from GIBCO Invitrogen (Grand Island, NY). Dexamethasone was acquired from G Bioscience (St. Louis, USA). Oil Red O was purchased from amresco (USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah).
Preparation of O. indicum extract
O. indicum (fruit pods) fresh samples were purchased from the local market at Wang Nam Khiao district, Nakhon Ratchasima province, Thailand. The voucher specimens (SOI0808U) were deposited at the flora of Suranaree University of Technology (SUT) Herbarium and authenticated by Dr.Santi Wattatana, a lecturer and a plant biologist at Institute of Science, SUT, Thailand. Fresh pods were washed thoroughly with tap water, cut into small pieces and then dried in the oven at 40 °C for 2 days. The dried pieces were pulverised using a mechanical grinder, and the resulting coarse powder was preserved from moisture. The O. indicum dry powder (500 g) was extracted with 95% ethanol by a soxhlation for 8 h. The extract was filtered through Whatman filter paper and concentrated using a rotary evaporator at 50 °C under vacuum to remove the ethanol. The remaining extract was stored at − 80 °C until required. Subsequently, the sample was lyophilized in a freeze dryer (LABCONCO), automatic mode, vacuum 240 × 10− 3 mBar, and collector − 55 °C. The extracted powder was stored at − 20 °C until required. The lyophilized O. indicum extract was used within 3 months of preparation. The extract was resuspended in the differentiation medium containing 0.1 v/v DMSO (vehicle) and added to the cells at concentrations ranging from 0 to 1500 μg/mL.
Phytochemical screening
The stock concentration of the lyophilized extract (10 mg/mL) was prepared and tested for the presence of bioactive phytochemical compounds, including total phenolics, flavonoids, alkaloids, steroids, glycosides, tannin, and saponins.
Test for flavonoids
Briefly, 1 mL of the OIE was mixed with 2 mL of 2% w/v of NaOH. A 2 mL aliquot of 10% w/v lead acetate solution was added to 1 mL of the alkaline extract. The formation of a yellow colour indicated the presence of flavonoids [17, 18].
Test for alkaloids
The Mayer’s and Wagner’s test was performed. Briefly, 1 mL of the OIE was added to 2 mL of 1% v/v HCl and heated. A few drops of Mayer’s and Wagner’s reagents was added to the mixture, the presence of a precipitate suggested the presence of alkaloids in the sample [17, 18].
Test for steroids
The steroids containing in OIE were investigated. In short, 1 mL of the OIE was mixed with 2 mL each of chloroform and concentrated H2SO4. A red colour in the lower layer indicated the presence of steroids [17, 18].
Test for glycoside
The detection of glycosides was performed using Salkowski’s test. In brief, 2 mg of the OIE was mixed with 2 mL of chloroform and a few drops of concentrated H2SO4. A reddish brown colour was associated with the presence of glycosides [17, 18].
Test for tannin
The Gelatin test was performed to identify tannins in the extract. To summarise, 1 mL of the OIE was added to 2 mL of a 1% w/v gelatin solution, the formation of a white precipitate indicated the presence of tannins [17, 18].
Test for saponin
The foam test was performed. Briefly, a 5 mL aliquot of the OIE was shaken for 5 min. If saponins were present, they formed a stable foam in the test tube [17, 18].
Total phenolic content determination (TPC)
The total phenolic content was measured following the method outlined by Kohoude with slight modifications [19]. In brief, 20 μL of extract (0.625 mg/mL) or a standard solution of gallic acid (0–7.5 μg/mL) were added into 100 μL of Folin-Ciocalteu reagent. The mixture was incubated at room temperature for 6 min, prior to the addition of 7.5% w/v of Na2CO3. After a 1 h incubation at room temperature, the absorbance was recorded at 760 nm against a DMSO blank. TPC of the sample was expressed as mg of gallic acid equivalents (GAE) per g of dry weight.
Total flavonoid content determination (TFC)
The total flavonoid content was measured according to a previously published method with slight modifications [20]. To summarise, a 25 μL of extract (3 mg/mL) or a standard solution of catechin (0–200 μg/mL) were added to 125 μL of deionised water, followed by the addition of 10 μL of 5% w/v NaNO2. This mixture was incubated at room temperature, and after 6 min, 15 μL of 10% AlCl3 was added. Upon mixing the reaction was allowed to proceed for 5 min, then 50 μL of 1 M NaOH was added. The absorbance of the mixture was determined at 595 nm versus prepared DMSO blank. Total flavonoid of the sample was expressed as mg of catechin equivalent (CE) per g of dry weight.
Cell culture and differentiation procedures
The differentiation procedures of 3T3-L1 preadipocyte were performed as following the ATCC recommended protocol. Briefly, adherent 3T3-L1 cells were cultured in DMEM containing a high glucose concentration, supplemented with 10% of bovine calf serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin, until they reached 70–80% confluently. Two days after confluence (day 0), the cells were stimulated to differentiate with differentiation medium containing 10% FBS, 1.0 μM dexamethasone, 0.5 mM of IBMX, and 1.0 μg/mL of insulin in DMEM. On day 2, the differentiation medium was changed to maintain a medium consisting of 10% of FBS and 1.0 μg/mL of insulin in DMEM. The maintenance medium was replaced every 48 h for the next 8 days. On day 10, the differentiation of 3T3-L1 pre-adipocytes into adipocytes was observed. The cells were maintained in 5% CO2 incubator and at 37 °C throughout the whole process. The required doses of OIE were added to the 3T3-L1 cell culture during the differentiation (at day 0, 2, 4, 6, and day 8).
Cytotoxicity assay
The cytotoxic effects of the OIE on the proliferation of preadipocytes and adipocytes were determined by MTT assay largely following the method of Dunkhunthod et al. and Denizot [21, 22]. In brief, the 3T3-L1 cells were seeded in 96-well plates at a density of 5 × 103 cells/well. Two days after reaching confluence, the dividing cells were treated with the OIE at concentrations ranging from 0 to 1500 μg/mL. Both treated and control cells were incubated for a further 48 h. At the end of the treatment period, the cell viability was assessed by using the MTT assay. The culture medium was removed, and 100 μL of MTT solution (0.5 mg/mL in phosphate buffer saline) was added, then incubated at 37 °C for 4 h. After incubation, 150 μL of DMSO was added to dissolve formazan crystal. The absorbance of the intracellular formazan is proportional to the number of viable cells present was determined at 540 nm against a blank medium (Benchmark Plus, Bio-Rad, Japan). The percentage of formazan product was calculated to determine cytotoxicity [23]. OIE at concentrations of 0–200 μg/mL were used to assess any cytotoxic effects of mature adipocytes and Oil Red O assay for lipid accumulation. The IC50 of the extract was also calculated from a dose-response curve using linear regression analysis.
Oil red O staining
The intracellular triglyceride content was determined using an Oil Red O staining method as previously described [21, 24]. Briefly, on day 10, cells were washed with PBS and fixed with 1 mL/well of 10% (v/v) formalin for 1 h at room temperature. After fixation, the cells were washed, and 500 μL of 0.5% of the Oil Red O solution was added. The cells were incubated for 30 min at room temperature. The Oil Red O solution was removed by gentle aspiration, and the cells were washed with PBS. The nucleus was then stained with 0.10% (w/v) haematoxylin. Fat droplets were observed under an inverted microscope at an appropriate magnification. To determine the percent of lipid accumulation, the cells were extracted with 250 μL of isopropanol and 200 μL of the eluted solution was transferred to a new 96 well plate. The absorbance was measured at 490 nm with a microplate spectrophotometer. The simvastatin at 1.67 μg/mL was used as a positive control. The 3T3-L1 cells, treated with 200 μg/mL OIE were selected for FTIR studies.
Fourier-transform infrared spectroscopy
The effect of OIE on 3T3-L1 adipocyte cells using FTIR measurement was performed following the method of Dunkhunthod et al. [21]. Briefly, on day 10, cells were collected and centrifuged at 4000×g for 5 min, the medium was removed by gentle aspiration, and the cells were agitated and washed with 0.85% w/v NaCl. The cell suspensions were centrifuged at 4000×g for 5 min. The acquired cell pellets were dropped onto a window slide (MirrIR, Kevley Technologies) and dried for 30 min in a desiccator to eliminate the excess water. The dried cells were stored in a desiccator prior to FTIR analysis.
FTIR spectra were obtained at the Synchrotron Light Research Institute (Public Organization), Thailand. FTIR spectra were acquired with a Bruker Vertex 70 spectrometer coupled with a Bruker Hyperion 2000 microscope (Bruker Optics Inc., Ettlin-Gen, Germany) equipped with nitrogen cooled MCT (HgCdTe) detector with a 36 x IR. The spectra were obtained in the reflection mode with the wavenumber range of 4000–600 cm− 1, using an aperture size of 50 μm × 50 μm, with a resolution of 6 cm− 1. Each spectrum was produced following 64 scans. OPUS 7.2 software (Bruker Optics Ltd., Ettlingen, Germany) was used to acquire FTIR spectral data and control instrument system.
The spectral ranges of biochemical interest were identified using Principal Component Analysis (PCA) as being between 3000 and 2800 cm− 1 and 1800–850 cm− 1. The preprocessing of the spectra was performed by second derivative transformations using the Savitzky-Golay algorithm (nine smoothing points) and normalised with extended multiplicative signal correction (EMSC). Score plots (3D) and loading plots were used to represent the different classes of data and relations among variables of the data set, respectively.
The FTIR spectra datasets were submitted for Unsupervised Hierarchical Cluster Analysis (UHCA), to collect similar spectra in groups or clusters, using the OPUS 7.2 software (Bruker). Cluster analysis was performed on the second derivatives, and vector normalises spectra using Ward’s algorithm.
Lipase activity
Measurement of lipase activity was performed as previously described by Guo et al. and Dunkhunthod et al. [21, 25]. In brief, lipase of porcine pancreas type 2 was dissolved in distilled water at 5 mg/mL, the solution was centrifuged at 10,000 xg for 5 min, and the supernatant was used for the assay. A 0.1% w/v solution of pNP laurate was prepared in 5 mM of sodium acetate (pH 5.0) containing 1% v/v Triton X-100. The solution was heated to 80 °C and cooled to room temperature prior to use. A 30 μL volume of the lipase was added to a 96 well plate, followed by 40 μL of reaction buffer (100 mM of Tris buffer pH 8.2). Either 20 μL of OIE or 50% v/v DMSO was added prior to the addition 30 μL of the substrate solution. The mixtures were incubated at 37 °C for 6 h and measured at 409 nm using a microplate spectrophotometer. Orlistat at 12.5 to 100 μg/mL was used as a positive control. The inhibition rate (%) was calculated using the following equation. [((OD control – blank control) – (OD sample – blank sample)) /OD control] × 100 [26].
Statistical analysis
All the data were expressed as a mean ± standard error of the mean (SEM). The statistical significances difference between treatment and control groups of cell viability, the amount of lipid accumulation, biomolecular changes, and lipase activity were analysed by One-way analysis of variance (ANOVA) with a Turkey’s HSD post-hoc test (SPSS v 23). Values were considered statistically significant when p < 0.05 and data were representative of at least three independent experiments (n ≥ 3). Most experiments were performed in triplicate.