Table 1.
Genes and sets of the primers used in qRT-PCR.
Table 2.
The protocol PCR.
Table 3.
Yield of extraction (%) and TPCs of Bolanthus spergulifolius extracts from different solvents.
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.
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.
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.
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 CT method (ΔΔCT) from three independent experiments. Data are expressed as the relative change of the ΔΔCT 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.
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 ΔΔCT method. qRT-PCR was analysed using ΔΔCT from three independent experiments. Data are expressed as the relative changing of the ΔΔCT 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.
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.
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.