Results
In the DPPH photometric analyses, the methanolic extract of A.bracteolata showed a similar antioxidant activity with that of ascorbic acid as standard at varying concentration tested (5, 10, 25, 50,125, 250, 500 μg/mL). Fig.-1 shows that extract reduced DPPH radicals significantly as compared to the control (p<0.05). As shown in Fig. 1 EC50 value of A. bracteolata was found to be 43.88 µg/mL compared with ascorbic acid which showed the EC50 value of 6.03 μg/mL. There was a dose-dependent increase in the percentage antioxidant activity for all concentrations tested (Fig.1). A. bracteolata at a concentration of 1 µg/mL showed a percentage inhibition of 16.65±0.64, and for 100 µg/ml, it showed 93.29±0.64, respectively. Ascorbic acid was used as the reference drug. The range of ascorbic acid varied from 1 to100 μg/mL. Ascorbic acid at a concentration of 1µg/mL exhibited a percentage inhibition of 24.80±0.98 and for 100 µg/ml 77.74±1.16 (Fig.1). Table 1 displayed the DPPH assay results.
HPTLC analysis:
Quantification of quercetin from A. bracteolata extracts by HPTLC
The Methanolic extracts of A. bracteolata leaves were prepared, and HPTLC quantified the amount of quercetin present in these extracts. As shown in Fig.2A, spots of quercetin seen at UV-254 nm in the TLC chromatogram. The bluish fluorescent spots of quercetin, however, were more easily detected at UV-365 nm in the chromatogram of both the extract. The data of peak areas were plotted against the corresponding concentrations as shown in Fig.2B.The amount of quercetin was found to be 1.28w/w The identity of the quercetin bands in sample chromatograms was further confirmed by comparing retention factors of quercetin from the sample and standard solutions.
Cytotoxicity effect on HDF and HaCaT cells:
The MTT assay determined the cytotoxicity effect of A. bracteolata on HDF and HaCaT cell lines. As shown in Fig. 3A and 3B the plant extract significantly increased the number of cells in a dose-dependent manner. IC50 values were 87.60±1.67 µg/mL for A. bracteolata for HDF and 85.50±1.65 µg/mL for HaCaT after 24h treatment analyzed by Graph Pad Prism 5.0 software, respectively. At low concentrations (3.125 µg/mL), the cell proliferation rates of HDF and HaCaT increased in a dose-dependent manner and peaked at 88.03% ±1.78 for HDF and 89.15% ±0.31 for HaCaT respectively (Fig. 3A and 3B). There was a significant increase in cell number when compared to the control (p<0.05). At higher concentrations (250 µg/mL), the plant extract was cytotoxic to cells, and the cell survival rates dropped to 20–40% in all cells (Fig. 3C and 3D). The vehicle (DMSO) did not affect basal cell viability. Studies with different concentrations of DMSO (0.2–1.5%) showed toxic effects at concentrations above 1%, whereas lower concentrations (<0.5%) of DMSO exhibited negligible effects 9. Therefore, only DMSO concentrations less than 0.5% were used in further experiments.
Thymidine incorporation assay:
To confirm the ability of A. bracteolata to stimulate cell proliferation 3[H] Thymidine incorporation assay was performed in both HDF and HaCaT cell lines. Thymidine is incorporated into newly synthesized DNA during the proliferative period of the cells. As shown in Fig. 4, 2 fold increase in thymidine incorporation was observed with the treatment of A.bracteolata at a concentration of 25 µg/mL in HDF compared with the control (p>0.05) within 24h of treatment. Similarly, in HaCaT cell lines, a 2.5 fold increase was observed in the 50 µg/mL of A. bracteolata compared with the control (p<0.05), (fig 4). The results indicate that significantly higher levels of thymidine incorporation were observed in cells treated with A. bracteolata in both HDF and HaCaT cell lines (p<0.05). Accordingly, based on the thymidine proliferation results in a dose of 25µg/mL concentration of A.bracteolata for HDF and 50 µg/mL of extracts in HaCaT for treatment selected for all our further studies.
Wound migration assay:
The effect of two plant extracts on proliferation and migration of HDF cells was tested in an in vitro (cell culture) wound healing model, in which scrape wounds were generated in confluent cell cultures. Cells with or without treatment of plant extracts were allowed to migrate into the denuded area for 24–48 h at 37°C and wound migration was determined by using image J software. We first investigated the effect of A. bracteolata on human dermal fibroblast cell migration and proliferation with a Standard in vitro model of wounding based on a scratch wound assay. The examined methanolic extract of A. bracteolata demonstrated a migration and proliferation activity with values of 50.38%±1.39 and 69.81%±1.89, respectively. Cells with A. bracteolata treatment were less mobile, as indicated by fewer cells in the denuded area at 24 and 48 h after scratching (Fig. 5A-C) at a concentration of 25 μg/mL, respectively (p<0.05).
In vitro anti-inflammatory study:
Effect of A. bracteolata on cytotoxicity and NO production in RAW 264.7 cells
The cytotoxicity of plant extracts was evaluated using MTT assay, A. bracteolata was found not to affect RAW264.7 cell viability at concentrations of up to 100 μg/mL. However, the cytotoxicity of A. bracteolata was evaluated at a high concentration of 250 μg/mL (Fig. 6A). The viability of RAW264.7 was found to be more than 80% (Fig. 6B) at a concentration between 3–25 μg/mL, and there is no effect on vehicle level.
NO production
To investigate the effect of plant extracts on NO production, we measured the accumulation of nitrite, a stable oxidized product of NO, in culture media. NO production was examined in RAW 264.7 cells stimulated with LPS in the presence or absence of plant extracts for 24 h. LPS (200 ng/mL) stimulated cells had significantly increased nitrite levels compared with control. This stimulation was inhibited by A.bracteolata treatment in a concentration-dependent manner (Fig. 6C). A. bracteolata caused 67% (p<0.05) inhibition of NO production at a concentration of 100 μg/mL that showed statistical significance with the baseline control (100%).
Anti-inflammatory activities of A. bracteolata on RT-PCR analysis:
iNOS, IL-1β, and TNF-α are differentially expressed in response to various inflammatory stimuli such as lipopolysaccharide (LPS). The RT-PCR analysis was done to determine whether the inhibitory effects of A. bracteolata on the proinflammatory mediators NO is related to the modulation of the expression of iNOS. In unstimulated RAW 264.7 cells which were exposed to LPS, iNOS was strongly expressed, and A. bracteolata significantly inhibited iNOS expression in a dose-dependent manner (Fig. 6D). We further investigated the effect of plant extracts on LPS-induced TNF-α and IL-1β release by RT-PCR. The LPS-activated RAW 264.7 cells produced inflammatory cytokines at significantly higher levels than plant extracts (p<0.01). The TNF-α and IL-1β levels were augmented in the treatment of LPS-stimulated RAW 264.7 cells, and this increase in levels was significantly decreased in a concentration-dependent manner by treatment with A. bracteolata (Fig. 6D).
RT-PCR analysis
Real-Time PCR analysis was performed to investigate the relative mRNA expression levels of selected remodeling enzymes, extracellular matrix components which are associated with wound repair, in HDF fibroblast culture. RT-PCR was performed on Type I and IV collagen and MMP-2. Figure 7 represents a significant upregulation of the extracellular matrix components such as Collagen Type I, Collagen Type IV in the treatment group compared with control (p<0.05) while that of GAPDH remained unaffected. The mRNA levels of MMP-2 were further elucidated by real-time PCR after HDF cells were treated with A.bracteolata, As shown in Fig. 7, MMP2 showing a significantly higher expression in dermal fibroblasts compared with control (P <0.01). These data suggest that A.bracteolata in fibroblasts can directly induce the expression of ECM components.