Chemicals and reagents

Dimethyl sulfoxide (DMSO), Bovine serum albumin (BSA), Dexamethasone (DEX), Insulin, Glutaraldehyde, Ethylacetate (EA), Methanol (MeOH), Ethanol, +/+ PBS (containing Ca++ and Mg++), Phosphate buffered saline (PBS), İsobutylmethylxanthine (IBMX), Formalin, Isopropanol, 2-propanol, 4’6-diamidino-2-phenylindole (DAPI), Cloroform TRIzol, DEPC water, Lipid (Oil Red O) Staining Kit, Glycine, Sodium dodesylsulfate, Tris, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Mercaptoethanol, RIPA buffer, Protease inhibitor cocktail, Skim milk powder, Folin-Ciocalteu, Sodium carbonate, Gallic acid, Triton X-100, Tween 20, Paraformaldehyde, Hexamethyldisilazane (HMDS), Giemsa were optained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), L-Glutamine, Fetal bovine serum (FBS), Penicillin, Streptomycin were obtained from Hyclone (GE Healthcare, USA). Quick Start Bradford Protein Assay Kit 1, Immun-Blot PVDF membrane, 2x Laemmli sample buffer, 10x Tris/Glycine/SDS buffer, ECL reagent were purchased from Bio-Rad (Hercules, USA). Calcein-Acetomethoxy (C-AM) (ab141420), Ethidium homodimer-1 (Eth-1) (ab145323), anti-Glut-4 (ab35826 and ab654) anti-β-Actin (ab8226), Goat Anti-Mouse IgG secondary-antibody (ab205719), Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113) Phalloidin-iFluor 488 Reagent (ab176753) and BrightMount/Plus were purchased from Abcam (Cambrige, UK).

Preparation of B. spergulifolius extracts

For B. spergulifolius MeOH extraction; 5 g of dried plant samples was extracted with 100 mL MeOH for 1 h in an ultrasonic bath (Ultracleaner, DH-WUC-06H, Daihan, USA) at 15°C. These poses were replicated until the solvent turned colorless. After the plant extract was filtered using Whatman No. 1. Then the MeOH was removed from the filtrate of extract by a rotary evaporator (Buchi Rotavapour R-200, Marshall Scientific, Hampton, USA) at 37°C. Then, the filtrate was stored at -80°C.

For B. spergulifolius EA extraction; 5 g of dried plant samples was extracted with 100 mL EA and for 1 h in an ultrasonic bath (Ultracleaner, DH-WUC-06H, Daihan, USA) at 15°C (10 min intervals in three-times). After the filtration of extracts were performed using Whatman No. 1. The filtrate of extract was concentrated by a rotary evaporator (Buchi Rotavapour R-200, Marshall Scientific, Hampton, USA) at 37°C. Then, the filtrate was stored at -80°C.

For B. spergulifolius Aqueous extraction; 5 g of dried plant samples was extracted with 100 mL water for 1 h in an ultrasonic bath (Ultracleaner, DH-WUC-06H, Daihan, USA) at room temperature (10 min intervals in triplicates) and filtered (Whatman No.1) under vacuum. The filtrate of Aquaeous was frozen at -20°C and then dried with freeze dryer (BK-FD10P, Biobase, China).

The extracts of plant samples were weighed (TP-214 Timberline, Denver Instrument Company, UK) and the yield of all of the extraction was calculated in terms of (w/w) % per g of crude extract. The extracts were reconstituted in DMSO at a final-concentration and stored at -20°C under shade until further studies.

Folin-Ciocalteu

Total phenolic content (TPC) was evaluated by the Folin-Ciocalteu method [18]. 100 μL 10% (v/v) of Folin-Ciocalteu reagent was added to 20 μL samples of each plant extract for 5 min at room temperature. Finally, 80 μL 2% (w/v) of sodium carbonate were added and incubated in the dark for 30 min at 40°C. Then the absorbance was measured at wavelength of 765 nm with a spectrophotometer (Uvmini-1240, Shimadzu, Kyoto, Japan). According to the standard calibration curve, value of TPC for each extract was presented as gallic acid equivalents (mg GAE/g of dry weight).

Cell culture and differentiation of 3T3-L1 into mature adipocytes

3T3-L1 (CRL-173) preadipocytes were optained from the American Type Culture Collection (ATCC, Manassas, VA). Preadipocyte expansion medium was prepared 90% DMEM supplemented with 10% FBS, 1% L-Glutamine and 1% Penicillin+streptomycin antibiotics. The cells were cultured in 25 cm² flask containing a preadipocyte expansion medium. The cells were grown in an incubator (Nuaire, NU-5800, USA) with a humidified air at 37°C containing 5% CO². The culture medium was altered every two-three day in cabinet (LabGard NU-540, Nuaire, US) and cells observed using an inverted microscope (Leica, Dmil Led Fluo, Thermo Fisher, Germany).

The differentiation of 3T3-L1 preadipocytes into mature adipocytes were cultured and maintained [19]. Differentiation medium was prepared 90% DMEM supplemented with 10% FBS, 1% L-Glutamine, 1% Penicillin+streptomycin antibiotics-containing DEX (1μM), Insulin (1 μg/mL), and IBMX (0.5 mM). First, 3T3-L1 cells were seeded preadipocyte expansion medium at 8x10⁴ in 6-well plate containing 1 mL medium until reached confluence. After 48 h for the induction of differentiation 3T3-L1 preadipocyte were cultured with differentiation medium. After 72 h the induction of differentiation, the differentiation medium was removed and added by adipocyte maintenance DMEM supplemented with 10% FBS and insulin (1μg/mL) for another 48 h. After 5 days the induction of differentiation, the fresh adipocyte maintenance medium was replaced every two days until 14 days. The visible of lipid accumulation was monitored with an inverted microscope (Leica, Dmil Led Fluo, Thermo Fisher, Germany) [20, 21]. The mature 3T3-L1 adipocytes were used for further analysis.

Oil Red O staining assay

To visualize and measure of the lipid accumulation in preadipocytes, mature 3T3-L1 adipocytes and the effects of B. spergulifolius extracts on adipogenesis in 3T3-L1 adipocytes, was performed Oil Red O Staining Kit following the manufacturer’s instructions [20, 21]. The 3T3-L1 preadipocytes and adipocytes were cultured into a 24-well plate (2x10⁴ cells/well). After confluence, the medium was removed and cells were washed twice with PBS and fixed with 100 μL 10% (v/v) of formalin for 60 min at room temperature. The fixed cells were washed twice with PBS and then added with 100 μL 60% (v/v) of isopropanol for 5 min at room temperature. Oil Red O stock solution was prepared with 100% (v/v) isopropanol and was kept in 56°C water bath for 1 h. The Oil Red O working solution was prepared with 3-unit of Oil Red O stock solution to 2-unit water, and shook (Everlast Rocker 247, Benchmark, USA) for 10 min and filtered using Whatman no:1. The cells were incubated for 20 min at room temperature with working solution 100 μL. Then, the working solution was removed and wells were washed three times with water. The wells were added Hematoxylin for 1 min and then washed three times with water. Finally, cells were coated with water and images of them were taken under a microscope. Also, lipid accumulation in cells was quantified by eluting Oil Red O stain using isopropanol and optical density (OD) was measured at a wavelength of 490 nm using a spectrophotometer (Nanoprop 2000c, Thermo Scientific, USA) [22].

Thiazolyl blue tetrazolium bromide (MTT) assay

MTT was used to determine the effect of B. spergulifolius extracts on 3T3-L1 cell viability and the dosage of the extracts for further studies [23]. 3T3-L1 adipocytes were cultured in a 96-well plate (2x10³ cells/well) and incubated overnight at 37°C under 5% CO². After 24 h, the medium was removed and 3T3-L1 adipocytes were treated with fresh medium containing different concentrations of plant extracts (for MeOH extract, 0–1000 μg/mL; for EA extract; 0–2000 μg/mL; for Aqueous extract, 0–1000 μg/mL) or 0.1% (v/v) DMSO for 24 h at 37°C. Then, the medium was removed and cells were washed twice with PBS. MTT solution (0.5 g/mL in PBS) was supplemented in each well at 100 μL and the cells were incubated for 4 h at 37°C. After MTT solution was suspended and 100 μL of DMSO was supplemented to each well. 96-well plates wrapped with aluminum foil and placed in a shaker (Everlast Rocker 247, Benchmark, USA) for about 1 h. The color change observed in the wells and the relative cell viability were calculated by determining with a spectrophotometer (Uvmini-1240, Shimadzu, Kyoto, Japan) absorbance at 570 nm. The results of cytotoxicity were presented as the relative reduction of the OD₅₇₀, which correlated with the number of viable cells in relation to the percentage of control cells (as taken 100%) using the following formula. The cells half-proliferation inhibitory concentration (IC₅₀) for MeOH, EA, Aqueous extracts of B. spergulifolius were calculated from the linear regression equation. The relative cell-viability and IC₅₀ were plotted in graphs.

(1)

Live-dead staining assay

Live-dead staining was conducted to evaluate the effect of B. spergulifolius extracts on 3T3-L1 cell viability [24]. C-AM and Eth-1 dyes were used after 24 h treated with plant extracts. First, cells incubated in 24 well-plate (2x10⁴ cells/well) were removed medium and washed with 250 μL +/+ PBS twice. 1 μM C-AM and 1 μM Eth-1 dissolved in 250 μL +/+ PBS was added to each well and cells were incubated for 30 min in the dark at room temperature. After that, cells were removed the solution, and washed with 250 μL +/+ PBS. The cells were visualized at 10X under confocal microscope confocal microscope (Carl-Zeiss LSM 710 NLO, GmbH, Germany) to evaluate the spatial dispersion of the living (green/C-AM) and dead (red/Eth-1) cells. The ImageJ software (imagej.nih.gov) was used for quantification of relative cell-viability in each field [24]. The viable cells (%) were calculated: (2)

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

To investigate the effect of B. spergulifolius on apoptosis and insulin resistance, respectively the expression of Bcl-2-associated X (Bax), B-cell lymphoma-2 (Bcl-2) and Glucose transporter-4 (Glut-4) genes at the mRNA level in 3T3-L1 adipocytes following treatment of extracts was evaluated using quantitative qRT-PCR [25, 26]. The 3T3-L1 adipocytes were seeded in 6-well plates at an initial density of 3x10⁵ cells/well. Total RNA was isolated from the cells using TRIzol reagent 24 h of treatment with plant extracts according to the product manufacturer’s protocol. The RNA was dissolved in DEPC water and stored at -80°C until it was used. The concentration of RNA samples was measured using a spectrophotometer (Nanoprop 2000c, Thermo Scientific, USA). The 1 μg of the total RNA with 260/280 nm ratio: 1.8–2.1 were used for complementary DNA (cDNA) synthesis. ProtoScript II first Strand cDNA Synthesis Kit (NEB, New England Biolabs Inc., USA) was used to convert the RNA to cDNA following instructions of the manufacturer. The amplification of cDNA was prepared using the One Taq 2X Master Mix with the Standard Buffer (New England Bio labs Inc., USA) in a final volume of 25 μL using thermal cycler (SureCycler 8800, Agilent Technologies, USA) using primers synthesized by Sentebiolab, Turkey (Table 1). The qRT-PCR was performed using the GoTaq® 2-Step RT-qPCR System (Promega, ABD) on the Qiagen Rotor-Geneq (Qiagen, Hilden, Germany) by following PCR steps summarized in Table 2. The expression of target genes was analysed with the comparative threshold cycle C_T method (^ΔΔC_T) method, by which the results of each gene expression were normalized with the expression of Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh) gene [27]. The results of qRT-PCR were analysed with Rotor Gene Q Software 1.2 program (Qiagen, Germay).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Genes and sets of the primers used in qRT-PCR.

https://doi.org/10.1371/journal.pone.0252707.t001

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. The protocol PCR.

https://doi.org/10.1371/journal.pone.0252707.t002

Western-blot

The method was performed to evaluate the effect of B. spergulifolius extracts on level of Glut-4 protein in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were cultured in 6-well plate (3x10⁵ cells/well). After treatment of cell with extract for 24 h, the medium was removed and each well was washed with 1 mL cold PBS twice. Then 225 μL RIPA buffer containing 10% Protease inhibitor cocktail was added to each well on cold block and the plate was rocked orbital shaker (Everlast Rocker 247, Benchmark, USA) for 15 min at +4°C. The cells were scrapped, exposed with (Bioruptor sonicator, Belgium) and centrifuged at 15.000 rpm for 10 min. The protein pellet concentration was measured using Quick Start Bradford Protein Assay Kit 1 (Bio-Rad, Hercules, USA).

Protein samples (20 μg) were treated with Mercaptoethanol containing 5% 2x Laemmli sample buffer and loaded to 10% SDS-PAGE gel. Each sample was ran with 10x Tris/Glycine/SDS buffer for 1 h at 100 V using Mini Pretean Tetra Cell Module (Bio-Rad, Hercules, USA) and transferred onto Immun-Blot PVDF membrane using Mini Trans Blot Cell Module (Bio-Rad, Hercules, USA). The membranes were blocked in 5% skim milk powder and incubated with anti-Glut-4 and anti-β-Actin as an internal control [for two, 1:100 (v/v) in 5% skim milk powder] at overnight at 4°C. Membranes were then treated with Goat Anti-mouse IgG secondary antibody [1:1000 (v/v) 5% skim milk powder] for 2 h at room temperature and washed. Protein band was visualized with 1mL ECL reagent for 5 min in dark using VersaDoc (4000MP, Bio-Rad, Hercules, USA) and ImageLab software (Bio-Rad, Hercules, USA) was used for protein quantification [28].

Immunofluorescence staining

Immunofluorescence staining was performed to determine the effect of B. spergulifolius extracts on intracellular level of Glut-4 in 3T3-L1 adipocytes. The cells were cultured on coverslips (Nunc Thermanox Coverslips, Thermo Scientific, USA) into a 6-well plate (3x10⁵ cells/well) and then confluent cells treated with plant extracts. After 24 h, coverslips were transferred into 24-well plate and cells were washed twice with PBS. Then cells were fixed with 250 μL 4%lük (w/v) of paraformaldehyde in PBS for 10 min at room temperature. Fixed cells were wash with PBS and permeablized with 250 μL 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature. After permeabilization, cells were added to block nonspecific binding 250 μL 1% (w/v) of BSA in [PBST (PBS + %0.1 Tween 20)] for 10 min at room temperature. Blocked cells were incubated with 250 μL 1:100 (v/v) of anti-Glut4 and anti-Beta Actin in 1% (w/v) of PBST overnight at 4°C in a humidified room. Then cells were incubated with 250 μL 1:1000 (v/v) of Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) as secondary antibody for 60 min at room temperature in the dark. The nucleus was stained with 250 μL 1/1000 (v/v) of DAPI (1 μg/mL) in BSA for 5 min at room temperature. Finally, coverslips were coated with 3 μL BrightMount/Plus. The staining was imaged at 10X using a fluorescence microscope (Leica, Dmil Led Fluo, Thermo Fisher, Germany) and fluorescein signals were analysed ImageJ software (imagej.nih.gov) [29, 30].

Also, intra-ctyoplasmic staining was performed to examine the effect of each extract on cytoskeleton of 3T3-L1 adipocytes. The medium was discarded and washed with PBS. Then cells were fixed with 250 μL 4%lük (w/v) of paraformaldehyde, permeablized with 250 μL 0.1% (v/v) of Triton X-100 in PBS, and 250 μL 1% (w/v) of BSA in PBST at room temperature (10 min intervals). Then, Phalloidin-iFluor 488 reagent as primary antibody for intracytoplasmic dying performed for staining cytoskeleton of 3T3-L1 adipocytes was prepared in 1:100 (v/v) in 1% (w/v) PBST for 30 min in dark. The After all, of the intracytoplasmic stained cells were observed and imaged at 5X under confocal microscope (Carl-Zeiss LSM 710 NLO, GmbH, Germany).

Scratch assay

Scratch assay was performed to evaluate the effect of B. spergulifolius extracts on the cell migration ability of 3T3-L1 adipocytes [31]. The 3T3-L1 adipocytes were seeded in 6-well plates (3x10⁵ cells/well). When the cells approximately reached confluence, the scratch-wound in the middle of each well was created with a 200 μL sterile micropipette tip. Then, wells were removed and washed with DPBS. Medium having IC₅₀ dose of B. spergulifolius extracts was added to each well. Preadipocytes and adipocytes as controls received, respectively, only fresh medium and adipocyte maintenance medium. All cells were incubated under standard conditions. Images of the scratch-wound were taken at 0. h, 6. h, 24. h, 30. h, 48. h using a microscope (4X magnification). Also, the scratch-wound area was measured from the digital images and calculated using ImageJ software (imagej.nih.gov) [32]. The scratch closure rate (%) was calculated: (3)

Giemsa staining

Giemsa staining was conducted on 3T3-L1 cells to assess the effect of B. spergulifolius extracts on morphology. The cells were seeded into a 6-well plate (3x10⁵ cells/well) and then confluent cells were treated with plant extracts. After medium was removed and wells were washed with 750 μL PBS twice. To fix cells were incubated 750 μL 60% (v/v) of cold MeOH in PBS for 60 min. MeOH was discarded and 750 μL 100% of MeOH for 10 min at room temperature was added to each well. Then after cells were washed with 750 μL PBS twice. A Giemsa stain stock solution of 0.1 g was prepared by dissolving in 10 mL MeOH for 30 min in heat bath and filtered with Millex Milipore 0.42 μm (Millex-GP, Sigma Aldrich, USA). The fixed cells were stained with from stock solution 1 mL Giemsa from stock solution for 20 min at room temperature, washed with distile water. Then, air-dried cells were observed and imaged at 10X, under an inverted microscope (Leica, Dmil Led Fluo, Thermo Fisher, Germany) [33]. Also, The ImageJ software (imagej.nih.gov) was used for the measurement of the average cell diameter in each three fields in photographs for three wells.

SEM

Scanning electron microscope (SEM) was used to evaluate the effect of B. spergulifolius extracts on the physical morphology of 3T3-L1 adipocytes. The cells were cultured on coverslips (Nunc Thermanox Coverslips, Thermo Scientific, USA) into a 6-well plate (3x10⁵ cells/well) and then confluent cells treated with plant extracts. After 24 h, coverslips were transferred into petri dish and cells were fixed with 2.5% (v/v) glutaraldehyde in PBS for 45 min at room temperature. After that, the cells were washed with PBS twice and dehydrated in a graded series of ethanol (respectively, from 30, 50, 70, 80, 90, and 96%) for 15 min at room temperature. Then, cells were incubated with HMDS for 5 min and air-dried samples were mounted with a thin layer of gold and imaged at 500X using SEM (Supra 40 VP, Carl Zeiss, Oberkochen, Germany).

Statistical analysis

Statistical Package for Social Science (SPSS) v24.0 version (Inc., Chicago, IL, USA) package program was used for statistical analysis of the data. All experiments and measurements were performed in triplicate. All the continuous variables are expressed as the mean ± standard deviation (SD). The graphics were prepared with the Graph Pad Prism software 8.4.3 version (Graphpad Software Inc., USA). IC₅₀ is calculated by Graph Pad Prism software 8.4.3 version. It was checked whether the data that obtained in our experiments are normally distributed or not, and Mann-Whitney U or Student’s t-test (two-tailed) was used to compare groups. For a comparison of data between more than two independent groups, One-ANOVA tests followed by Tukey’s test were used. P values < 0.05 were considered statistically significant.

Total phenolic contents (TPC) of B. spergulifolius’s extracts

The extraction yield and TPC of B. spergulifolius crude extracts are summarized in Table 3. The extraction efficiency of B. spergulifolius Aqueous extract (49.3 ± 1.05%) was the highest, followed by the extraction efficiency of MeOH extract and EA extract (46.91 ± 1.60%, 23.2 ± 1.88%, respectively) lower.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 3. Yield of extraction (%) and TPCs of Bolanthus spergulifolius extracts from different solvents.

https://doi.org/10.1371/journal.pone.0252707.t003

Total phenolic compounds chelate with metal ions that can free radicals and form lipid peroxidation and contribute to antioxidant activity by scavenging free radicals [34, 35]. In our study, the TPC of B. spergulifolius extracts was evaluated as an indicator of anti-oxidant activity. TPC in B. spergulifolius extracts was calculated using Folin-Ciocalteu reagent and gallic acid equivalent to a standard curve equation (R² = 0.9970). The TPC was highest in the B. spergulifolius EA extract (52.81 ± 1.57 mg/g dry weight). Subsequently, the total phenolic content was lower in B. spergulifolius MeOH extract (48.22 ± 1.98 mg/g dry weight) and lowest in B. spergulifolius Aqueous extract (30.34 ± 1.36 mg/g dry weight) (Table 3). Each experiment was performed at least 3 times and data were given as the mean ± SD. When the extracts were compared with each other in terms of TPC content, there was a statistically significant difference (p< 0.05).

The cytotoxic effect of B. spergulifolius extracts on 3T3-L1 adipocytes

It is a critical step in evaluating the possible cytotoxicity of plant extracts and their suitability for further applications [21, 23]. The cytotoxic effect of B. spergulifolius extracts of MeOH (0–1000 μg/mL), EA (0–2000 μg/mL) and Aqueous (0–1000 μg/mL) on mature 3T3-L1 adipocyte cell line was investigated with using MTT assay. After 24 h of incubation with B. spergulifolius extracts, the cell viability results are presented in Fig 1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. MTT assay showing the effect IC₅₀ value of B. spergulifolius extracts on 3T3-L1 cell viability.

A. Effect of B. spergulifolius extracts on 3T3-L1 cell viability. The mature 3T3-L1 adipocytes were treated for 24 h with increased concentration of Methanol (MeOH), Ethylacetate (EA), and Aqueous extracts of B. spergulifolius. Also, cell treated without crude plant extract was taken as a control. B-D. The half-maximal (50%) inhibitory concentration (IC₅₀) value of the B. spergulifolius extracts. The IC₅₀ value is calculated by non-linear regression model. Data expressed as the means ± SD from three independent experiments. To analyse statistically was used one-way ANOVA test. *p = 0.033, ^#p = 0.001 and **p< 0.001, significantly different from control.

https://doi.org/10.1371/journal.pone.0252707.g001

Cell viability decreased as the extract concentration increased in all extracts. Cell viability began to decrease significantly at concentrations of B. spergulifolius MeOH extract higher than 200 μ/mL compared to control (p = 0.033 for 200 μg/mL and p< 0.001 for higher doses). Cell viability began to decrease significantly at concentrations of B. spergulifolius EA extract higher than 500 μg/mL compared to control (p< 0.001 for 500 μg/mL and higher doses). Cell viability started to decrease significantly at concentrations of B. spergulifolius Aqueous extract higher than 100 μg/mL compared to control (p = 0.001 for 100 μg/mL and p< 0.001 for higher doses) (Fig 1A). However, more than 90% cell viability was preserved in cells exposed to concentrations of less than 10 μg/mL, 350 μg/mL and 25 μg/mL for B. spergulifolius MeOH, EA and Aqueous extract, respectively. Also, final concentrations of 0.1% of DMSO, 0.1% of MeOH and 0.1% EA were not cytotoxic to 3T3-L1 cells.

The extract concentration (IC₅₀) that causes 50% decrease in the viability of the cells was observed as 305.7 ± 5.583 μg/mL, 567.4 ± 3.008 μg/mL and 418.3 ± 4.390 μg/mL for MeoH, EA and Aqueous extract, respectively (Fig 1B–1D). Based on these data, the IC₅₀ dose of B. spergulifolius MeOH, EA and Aqueous extracts was chosen to be applied to the cells in subsequent experiments.

The effect of B. spergulifolius extracts on cell viability in 3T3-L1 adipocytes

A live/dead assay was performed to verify the cytotoxic effect of B. spergulifolius extracts on the mature 3T3-L1 adipocyte cell line. Cells were exposed to IC₅₀ doses of MeOH, EA and Aqueous of B. spergulifolius extracts for 24 h. Then, double staining was performed using C-AM and Eth-1 to distinguish between live (green) and dead (red) cells.

Microscopic images of living and dead cells in preadipocytes, adipocytes and mature 3T3-L1 adipocytes treated for 24 h in staining performed using the live-dead assay were presented in Fig 2A. Under an inverted microscope, intensive green (C-AM) fluorescence was observed in preadipocytes and untreated adipocytes. Red (Eth-1) fluorescence with reduced green (C-AM) fluorescence intensity was observed in mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA and Aqueous extracts compared to untreated adipocytes.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Live-dead staining showing the effect of B. spergulifolius extracts on cell viability in 3T3-L1 adipocytes.

Live-dead staining assay was performed preadipocytes, mature adipocytes without and mature adipocytes with treated with IC₅₀ dose of MeOH, EA and Aqueous of B. spergulifolius extracts for 24 h A. Representative microscopic images at 10X magnification showed 3T3-L1 cells without and treated with B. spergulifolius extracts stained with Calcein-Acetomethoxy (C-AM) and Ethidium homodimer-1 (Eth-1). C-AM (green fluorescence) and Eth-1 (red fluorescence) staining showed respectively live and dead 3T3-L1 cells (Scale bar: 100 μm) B. Quantitative data of live-dead staining assay. Three coverslips for each cell group were evaluated. Six distinct-randomly chosen regions in each coverslip were visualized and analyzed using microscope and ImageJ software. Data are expressed as a total percentage of viable and dead cells and presented as the means ± SD. To analyze statistical was used Student’s t-test (two-tailed). *p< 0.05, significantly different from mature 3T3-L1 Adipocytes. **p< 0.05, significantly different from Aqueous.

https://doi.org/10.1371/journal.pone.0252707.g002

The percentage of viable cells after 24 h in preadipocyte and untreated adipocytes was 94.25 ± 2.98% and 97.5 ± 1.29% (p = 0.1836). However, compared to mature 3T3-L1 adipocytes, after 24 h treatment of B. spergulifolius MeOH, EA and Aqueous extracts, the percentage of viable cells was 69.75 ± 1.70%, 61.75 ± 1.70%, and 70 ± 4.24% decreased (p< 0.0001, p< 0.0001, and p = 0.0014). Also, compared to Aqueous extract, B. spergulifolius MeOH and EA extract treatments resulted in a 0.35% and 11.78% reduction in the percentage of viable cells, respectively (EA-MeOH, EA-Aqueous, and MeOH-Aqueous respectively, p = 0.0122, p = 0.0154, and p = 0.9157) (Fig 2B).

3T3-L1 preadipocyte cells differentiated into mature adipocytes and effect of B. spergulifolius extracts on lipid accumulation in 3T3-L1 adipocytes

Oil Red O staining was used to confirm the adipogenic phenotype in the differentiation of preadipocytes into mature 3T3-L1 adipocyte cells and to evaluate the effect of B. spergulifolius crude extracts on the lipid accumulation ability of mature 3T3-L1 adipocytes [20, 21].

Staining was performed to distinguish lipid droplets in preadipocytes, adipocytes and adipocytes treated with IC₅₀ doses of MeOH, EA and Aqueous of B. spergulifolius extracts for 24 h with the Oil Red O kit. Under an inverted microscope, more lipid droplets were observed in untreated adipocytes compared to preadipocytes. Lipid accumulation decreased in mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA and Aqueous extracts compared to untreated adipocytes (Fig 3A).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Oil Red O staining showing 3T3-L1 preadipocyte cells differentiated into mature adipocytes and effects B. spergulifolius extracts on lipid accumulation in 3T3-L1 cells.

A. The microscopic images at 10X magnification of lipid droplet of preadipocytes, mature adipocytes without B. spergulifolius extracts and adipocytes treated with IC₅₀ dose of MeOH, EA and Aqueous of B. spergulifolius extracts stained with Oil Red O (Scale bar: 100 μm) B. The relative lipid quantization of preadipocytes, mature adipocytes treated without B. spergulifolius extracts and with MeOH, EA, Aqueous of B. spergulifolius extracts. OD₄₉₀ of Oil Red O staining from three independent experiments was measured. Data are expressed as the relative change of the OD₄₉₀ and presented as the means ± SD. To analyze statistical was used One-way ANOVA test. *p< 0.0001 and **p< 0.001, significantly different from mature 3T3-L1 Adipocytes. ***p< 0.001, significantly different from Aqueous.

https://doi.org/10.1371/journal.pone.0252707.g003

After Oil Red O staining performing, levels of lipid accumulation were measured using isopropanol. The OD₄₉₀ value increased by 89.23 ± 6.46% in untreated adipocytes compared to preadipocytes (p< 0.0001). As shown in Fig 3B, compared to untreated mature 3T3-L1 adipocytes, 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA, and Aqueous extract decreased in OD₄₉₀ by respectively, 20.2 ± 2.56%, 23.9 ± 3.16% and 10.3 ± 2.81% (p< 0.0001, p< 0.0001, and p< 0.0016). In addition, OD₄₉₀ values decreased by 11.09% and 15.20%, respectively, in MeOH and EA extract treatment group adipocytes compared to B. spergulifolius Aqueous extract (EA-MeOH, EA-Aqueous, and MeOH-Aqueous respectively, p< 0.0572, p< 0.0001, and p< 0.0001) (Fig 3B).

The effect of B. spergulifolius extracts on apoptosis regulated by the Bax and Bcl-2 in 3T3-L1 adipocytes

qRT-PCR was used to evaluate the effect of B. spergulifolius extracts on Bax and Bcl-2 gene at mRNA levels associated with apoptosis in mature 3T3-L1 adipocytes [25]. Adipocytes were treated with IC₅₀ doses of MeOH, EA and Aqueous B. spergulifolius extracts for 24 h. Compared to preadipocytes, Bax and Bcl-2 genes at mRNA level expression were decreased by 23.22 ± 0.04% and 24.46 ± 1.70% but Bax/Bcl-2 ratio increased by 1.654 ± 0.01%, respectively in untreated adipocytes (p = 0.0015, p< 0.0001, and p = 0.3451). Pro-apoptotic Bax gene at mRNA level expression increased by, respectively, 264.05 ± 0.9%, 347.90 ± 0.57% and 161.64 ± 0.30% in mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA and Aqueous extracts compared to untreated adipocytes, while the anti-apoptotic Bcl-2 gene at mRNA level decreased by 15.71 ± 0.14%, 49.01 ± 0.57%, and 18.13 ± 0.15%, respectively (for Bax, p = 0.0020, p = 0.0002, p = 0.0003, and for Bcl-2 p = 0.0343, p< 0.0001, P = 0.0423, respectively). However, compared to B. spergulifolius Aqueous extract, an increase in the expression level of the Bax gene was 36.14% and 71.18%, respectively, in MeOH and EA extract treatment groups, while the expression level of the Bcl-2 gene increased by 2.96% and 37.71%, respectively (MeOH-EA, EA-Aqueous, MeOH-Aqueous, for Bax p = 0.0919, p = 0.0039, p = 0.0131 and for Bcl-2 p = 0.0025, p = 0.0083, p = 0.6994, respectively). Similarly, compared to untreated adipocytes, B. spergulifolius MeOH, EA and Aqueous treatment groups mature 3T3-L1 adipocytes showed an increase of 330.80%, 780.70%, and 22.18%, respectively in Bax/Bcl-2 (p = 0.0057, p = 0.0002 and p = 0.0134, respectively). In addition, compared to Aqueous extract, B. spergulifolius MeOH and EA extract treatments increased 33.71% and 173.35% respectively in Bax/Bcl-2 (EA-MeOH, EA-Aqueous, and MeOH-Aqueous p = 0.0062, p = 0.0025, and p = 0.0936, respectively) (Fig 4).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. qRT-PCR data showing the effect of B. spergulifolius extracts on apoptosis regulated by the Bax and Bcl-2 in 3T3-L1 adipocytes.

The expression at the mRNA level of Bcl-2-associated X (Bax) and B-cell lymphoma-2 (Bcl-2) genes and Bax/Bcl-2 ratio was detected by qRT-PCR in preadipocytes, mature adipocytes without B. spergulifolius extracts and adipocytes treated with IC₅₀ dose of MeOH, EA and Aqueous of B. spergulifolius extracts for 24 h. The Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene was used for normalization. qRT-PCR was analysed using the comparative threshold cycle C_T method (^ΔΔC_T) from three independent experiments. Data are expressed as the relative change of the ^ΔΔC_T and the means ± SD. To analyse statistical was used Student’s t-test (two-tailed). *p< 0.05, ^#p< 0.05, and ^✦p< 0.05, significantly different from mature 3T3-L1 Adipocytes. **p< 0.05, ^##p< 0.05, and ^✦✦p< 0.05, significantly different from Aqueous.

https://doi.org/10.1371/journal.pone.0252707.g004

The effect of B. spergulifolius extracts on expression of Glut-4 in 3T3-L1 adipocytes

qRT-PCR, Western-blot, and immunofluorescence staining were used to evaluate the effect of MeOH, EA, Aqueous of B. spergulifolius extracts on Glut-4 expression in mature 3T3-L1 adipocytes associated with insulin resistance and decreased glucose uptake [26]. Glut-4 expression was evaluated in untreated preadipocytes, adipocytes and adipocytes treated for 24 h with IC₅₀ doses of MeOH, EA and Aqueous of B. spergulifolius extracts by qRT-PCR, Western-blot and immunofluorescence staining (Fig 5).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. qRT-PCR, Western-blot, and immunofluorescence staining showing the effect of B. spergulifolius extracts on translocation and expression of Glut-4 in 3T3-L1 adipocytes.

The expression of Glucose transporter-4 (Glut-4) gene, protein and intra-cellular was quantified by qRT-PCR, Western-blot and immunofluorescence staining in preadipocytes, mature adipocytes without B. spergulifolius extracts and adipocytes treated with IC₅₀ doses of MeOH, EA and Aqueous of B. spergulifolius extracts for 24 h. A. Glut-4 gene expression was detected using qRT-PCR at the mRNA level. Gapdh gene was used for normalization. The relative fold change analysis was conducted using the ^ΔΔC_T method. qRT-PCR was analysed using ^ΔΔC_T from three independent experiments. Data are expressed as the relative changing of the ^ΔΔC_T and presented as the means ± SD. B. Representative image showing Glut-4 protein (55 kDa) levels in 3T3-L1 cells by Western-blot (20 μg/lane). C. The relative intensities of Glut-4 protein band were measured as densitometrically. Beta-Actin (β-Actin) protein was used for normalization. Data were analysed using the ImageLab software. Densitometric analysis data was presented as the relative changing of the band intensities and the means ± SD from three independent experiments. D. The microscopic image at 10X magnification showing Glut-4 expression levels and nuclear staining in 3T3-L1 cells by immunofluorescence staining. Merged images obtained using anti-Glut-4 antibody and diamido-2-phenylindole dihydrochloride (DAPI) (Scale bar: 100 μm) E. Three coverslips for each cell group were evaluated. Six distinct-randomly chosen regions in each coverslip were pohotographed using a microscope and relative intensities of immunofluorescence staining with anti-Glut4 antibody were quantified using ImageJ software. The immunofluorescence staining analysis data are presented as the relative changing of the fluorescein signal intensities and the means ± SD. To analyze statistical was used Student’s t-test (two-tailed). *p< 0.05, **p< 0.0001, and ***p< 0.0001, significantly different from Preadipocytes. ^#p< 0.05, ^##p< 0.05 and ^###p< 0.05, significantly different from mature 3T3-L1 Adipocytes. ^✦p< 0.05, ^✦✦p< 0.05, and ^✦✦✦p< 0.05, significantly different from treatment groups.

https://doi.org/10.1371/journal.pone.0252707.g005

Expression in Glut-4 mRNA decreased by 389.57 ± 1.60% in untreated adipocytes compared to preadipocytes (p = 0.0004). The mRNA expression level of the Glut-4 gene increased by 98.84 ± 1.39%, 236.80 ± 0.53%, and 46.18 ± 0.62%, respectively, in mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA and Aqueous extracts compared to untreated adipocytes (p = 0.0073, p = 0.0012, p = 0.0458, respectively). However, among the B. spergulifolius extract treatment groups, an increase of 36.02% and 130.40% was observed in the mRNA expression level of the Glut-4 gene in MeOH and EA extract treatment groups, respectively, compared to Aqueous extract (EA-MeOH, EA-Aqueous, and MeOH-Aqueous, for mRNA expression, p = 0.0021, p = 0.0013, p = 0.0106) (Fig 5A).

Western-blot showed a change in the level of Glut-4 protein expression between preadipocyte, adipocyte and treated adipocyte groups (Fig 5B). The relative intensities of Glut-4 protein band in membrane were measured as densitometrically. According to densitometric analysis data expression of Glut-4 protein decreased by 68.93 ± 9.60% in untreated adipocytes compared to preadipocytes (p< 0.0001). Expression of Glut-4 protein levels increased by 26.11 ± 11.65%, 138.66 ± 9.80% and 20.17 ± 7.92%, respectively, in mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA and Aqueous extracts compared to untreated adipocytes (p = 0.0291, p< 0.0001, p = 0.004, respectively). However, compared to Aqueous extract, Glut-4 protein expression levels increased by respectively, 4.93% and 98.59% in the MeOH and EA extract treatment groups (EA-MeOH, EA-Aqueous and MeOH-Aqueous respectively, p< 0.0001, p< 0.0001 and p = 0.3523) (Fig 5C).

Under an inverted microscope, anti-Glut-4 antibody fluorescence was weak in untreated adipocytes compared to preadipocytes. However, anti-Glut-4 antibody fluorescence density increased in mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA and Aqueous extracts compared to untreated adipocytes (Fig 5D). According to immunofluorescence staining analysis data, anti-Glut-4 antibody fluorescence intensity decreased 59.87 ± 13.35% in untreated adipocytes compared to preadipocytes (p< 0.0001). Compared to untreated adipocytes, anti-Glut-4 antibody fluorescence density increased by 33.28 ± 8.68%, 58.73 ± 13.46%, and 14.09 ± 6.96%, respectively, in mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA and Aqueous extracts (p = 0.0079, p = 0.0021, p = 0.071, respectively). However, anti-Glut-4 antibody fluorescence density in adipocytes increased by respectively, 16.813% and 39.12%, in the MeOH and EA extract treatment groups compared to Aqueous extract (EA-MeOH, EA-Aqueous, and MeOH-Aqueous p = 0.0028, p = 0.0007 and p = 0.0152) (Fig 5E).

The effect of B. spergulifolius extracts on the cell migration ability of 3T3-L1 adipocytes

In vitro scratch assay was used to evaluate the effects of MeOH, EA, Aqueous of B. spergulifolius extracts on the migration of mature 3T3-L1 adipocytes [31, 32]. Cell migration and wound closure in untreated preadipocytes, adipocytes and adipocytes treated with IC₅₀ doses of MeOH, EA and Aqueous of B. spergulifolius extracts for 24 h were evaluated with the Scratch assay. The scratch wound was viewed under the microscope at the 0^th, 6^th, 24^th, 30^th and 48^th hour (Fig 6).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Scratch assay showing the effect of B. spergulifolius extracts on the cell migration ability of 3T3-L1 adipocytes.

The cell migration ability was determined by Scratch assay in preadipocytes, mature adipocytes without B. spergulifolius extracts and adipocytes treated with IC₅₀ dose of MeOH, EA and Aqueous of B. spergulifolius extracts. Cells were cultured to confluent cell monolayers were scratched using the micropipette tip incubated for 0–48 h. A. Representative microscopic images (at 10X magnification) captured at 0. h, 6. h, 24. h, 30. h and 48. h showing in vitro scratch assay wound healing nature of B. spergulifolius extracts. The boundaries of the wounds in cells were presented by the red (initial scratch wound) and black (final scratch wound) colored lines (Scale bar: 100 μm) B. The scratch closure rate was quantified using ImageJ software. Data are presented as a percentage of the closure of scratch-wound area at time 0. h and the means ± SD from three independent experiments. To analyze statistical was used one-ANOVA tests followed by Tukey’s test were used. *p< 0.05, significantly different from Preadipocytes. ^✧p< 0.05, ^✦p< 0.05, and ^#p< 0.05, significantly different from mature 3T3-L1 Adipocytes.

https://doi.org/10.1371/journal.pone.0252707.g006

Under the inverted microscope, a large wound area was observed in untreated adipocytes compared to preadipocytes. Compared to untreated adipocytes, the treatment group mature 3T3-L1 adipocytes treated with the IC₅₀ dose of B. spergulifolius MeOH, EA and Aqueous extracts significantly inhibited wound healing (Fig 6A).

Scratch-wound area to preadipocytes at 0^th, 6^th, 24^th, 30^th and 48^th hour decreased by 77.52 ± 2.40%, 100 ± 4.58%, 100 ± 2.00%, and 100 ± 1%, respectively (Comparing to previous point of time p = 0.0003, p = 0.0234, p = 0.4380, p = 0.6220). Scratch-wound area of adipocytes decreased at 0^th, 6^th, 24^th, 30^th and 48^th hour 38.07 ± 2.17%, 48.11 ± 2.03%, 99 ± 2.00%, and 100 ± 0.05%, respectively (Comparing to previous point of time, p = 0.0011, p = 0.0002, p = 0.0002, p = 0.1181). Scratch-wound area of MeOH decreased at 0^th, 6^th, 24^th, 30^th and 48^th hour 33.76 ± 1.63%, 34.83 ± 1.69%, 40.24 ± 1.89%, and 51.18 ± 2.65% respectively (Comparing to a previous point of time, p = 0.0008, p = 0.4590, p = 0.0448, p = 0.0129). Scratch-wound area of EA decreased at 6^th, 24^th, 30^th and 48^th hour 26.27 ± 1.59%, 26.25 ± 3.55%, 9.50 ± 1.83%, and 7.33 ± 2.63% respectively (Comparing to a previous point of time, p = 0.0012, p = 0.9934, p = 0.0035, p = 0.4523). Scratch-wound area of Aqueous decreased at 6^th, 24^th, 30^th and 48^th hour 33.96 ± 2.10%, 43.29 ± 2.96%, 43.97 ± 3.28% and 55.95 ± 2.25%, respectively (Comparing to a previous point of time, p = 0.0013, p = 0.0029, p = 0.3035, p = 0.0209) (Fig 6B).

The percentage of the scratch wound closure was calculated using ImageJ software as the ratio between the scratch-wound area at the initial and at each time point of the assay [32]. According to data analysis of the percentage of the scratch wound closure, scratch-wound area of untreated adipocytes compared to pre-adipocytes decreased at 6^th, 24^th, 30^th and 48^th hour 50.88 ± 2.17%, 51.88 ± 2.03%, 2.94 ± 2.00%, and 0.33 ± 2.65% respectively (p = 0.0015, p = 0.0053, p = 0.3235, p = 0.4226). Compared to untreated adipocytes, in the treatment group mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA, and Aqueous extracts, at 6^th hour scratch-wound area decreased by 11.33 ± 1.63%, 30.99 ± 1.59%, and 10 ± 2.10%, respectively (p = 0.0052, p = 0.0009, p = 0.1061, respectively). In the treatment group 24^th hour scratch-wound area decreased by 27.60 ± 1.69%, 45.43 ± 3.55%, and 10.01 ± 2.96% respectively (p = 0.0108, p = 0.0130, p = 0.1167, respectively). 30^th hour migration area decreased by 59.35 ± 1.89%, 90.40 ± 1.83%, and 55.58 ± 3.28% respectively (p = 0.0002, p = 0.0002, p = 0.0025, respectively). Also, 48^th hour scratch-wound area decreased by 49.49 ± 2.65%, 92.75 ± 2.63%, and 44.78 ± 2.25% respectively (p = 0.0013, p = 0.0003, p = 0.0009, respectively) (Fig 6B).

In addition, scratch-wound area at 6^th hour decreased by 0.60% and 22.63% respectively, in the MeOH and EA extract treatment groups compared to Aqueous extract (EA-MeOH, EA-Aqueous, and MeOH-Aqueous respectively, p = 0.0002, p = 0.0236 and p = 0.8878). Scratch-wound area at 24^th hour decreased by 19.54% and 39.36% in the MeOH and EA extract treatment groups compared to Aqueous extract, respectively (EA-MeOH, EA-Aqueous, and MeOH-Aqueous respectively, p = 0.1055, p = 0.0454, and p = 0.0076). Scratch-wound area at 30^th hour decreased by 8.48% and 78.39% respectively, in the MeOH and EA extract treatment groups compared to Aqueous extract (EA-MeOH, EA-Aqueous, and MeOH-Aqueous respectively, p = 0.0032, p = 0.0072, and p = 0.2028). Scratch-wound area at 48^th hour decreased by 8.51% and 86.88% respectively, in the MeOH and EA extract treatment groups compared to Aqueous extract (EA-MeOH, EA-Aqueous, and MeOH-Aqueous respectively, p = 0.0004, p< 0.0001 and p = 0.0384) (Fig 6B).

The effect of B. spergulifolius extracts on the cell morphology and diameter of 3T3-L1 adipocytes

Microscopic examination and SEM, Immunofluorescent and Giemsa staining were used to evaluate the effect of MeOH, EA, Aqueous of B. spergulifolius extracts on morphology in mature 3T3-L1 adipocytes. Cells in untreated adipocytes showed spherical morphology compared to preadipocytes with phase-contrast microscopy and SEM, immunofluorescent staining and Giemsa staining. No significant morphologic difference was observed in the treatment group mature 3T3-L1 adipocytes treated with IC₅₀ doses of B. spergulifolius MeOH, EA and Aqueous extracts compared to untreated adipocytes (Fig 7A). However, cells were observed in less rounded morphology in the B. spergulifolius EA extract treatment group compared to the other treatment groups.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. The phase-contrast microscopy, SEM, Immunofluorescence staining and Giemsa staining showing the effect of B. spergulifolius extracts on the cell morphology and diameter of 3T3-L1 adipocytes.

A. Cell morphology was evaluated by phase-contrast microscopy, scanning electron microscopy (SEM), immunofluorescence staining and Giemsa staining in preadipocytes, mature adipocytes without B. spergulifolius extracts and adipocytes treated with IC₅₀ doses of MeOH, EA and Aqueous of B. spergulifolius extracts. Representative images showing changes of cell-morphology after treatment with an IC₅₀ dose of MeOH, EA and Aqueous of B. spergulifolius extracts for 24 h. The cells were imaged under the phase-contrast microscope at 10X magnification (Scale bar: 100 μm). The cells were mounted with a thin layer of gold and imaged at 500X using scanning electron microscope (SEM) (Scale bar: 200 μm). By immunofluorescence with Phalloidin (Alexa Fluor-488), F-Actin was stained and the cells were viewed under confocal microscopy at 5X magnification (Scale bar: 100 μm). By Giemsa, the cells were stained and viewed under a phase-contrast microscope at 10X magnification (Scale bar: 100 μm). B. Measurement of cell diameter in images stained with Giemsa. Three wells for each cell group were evaluated. Three distinct-randomly chosen regions in each well were imaged under a microscope and measurement of the average cell-diameter were quantified using the ImageJ software. Data presented as the average change of the cell diameter and the means ± SD. To analyze statistical was used one-ANOVA tests followed by Tukey’s test were used. *p< 0.0001, significantly different from Preadipocytes. **p< 0.0001, significantly different from mature 3T3-L1 Adipocytes. ^#p = 0.0152 and ^##p< 0.0001, significantly different from MeOH and Aqueous.

https://doi.org/10.1371/journal.pone.0252707.g007

The ImageJ software was used for the measurement of the average cell diameter in images stained by Giemsa. The cell diameter increased by 139.66 ± 12.94% in untreated 3T3-L1 adipocytes compared to preadipocytes (p< 0.0001). Cell diameter decreased by 41.10 ± 4.8%, 51.74 ± 6.37%, and 30.65 ± 7.60%, respectively, in the treatment group mature 3T3-L1 adipocytes treated with B. spergulifolius MeOH, EA and Aqueous extracts compared to untreated adipocytes (p< 0.0001, p< 0.0001, and p< 0.0001, respectively). In addition, cell diameter decreased 15.06% and 30.40%, respectively, in MEOH and EA extracts compared to Aqueous extract (EA-MeOH, EA-Aqueous, and MeOH-Aqueous respectively, p = 0.0152, p< 0.0001, and p = 0.0695) (Fig 7B).