Gas chromatography‑mass spectrometer (GC–MS) analysis of fig latex.
The GC-MS analysis of fresh fig crude latex showed the presence of twenty-four bioactive compounds with concentrations more than 0.1% of the total latex contents. The most abundant with the highest percentage of the compounds was the Taraxasterol-acetate that represented the highest concentration (more than 50% of the total latex content). Moreover, the olean-12-en-3-ol, acetate, (3.beta.)- was detected, and represented 32% of the total latex contents. Finally, 9,19-Cyclolanost-24-en-3-ol, (3.beta.)- (3.999%), and Lanosta-8,24-dien-3-ol, acetate, (3.beta.)- (3.346%). All these four major chemical constituents represented 90% of the latex. Other active compounds with their concentration (peak area %), and retention time (RT) were presented with the chromatogram in Fig. 1 and in details in Table 1 for the chemical components obtained from the GC-MS analysis.
Fig latex exerts antibacterial activity using well diffusion method.
After isolation of the bacteria using a swab method, they were screened for the bacterial community. We got a lot of pure strains using (streak plate method) technique spreading on a different culture media like MSA for gram-positive bacteria and Macconkey for gram-negative bacteria. The Antibacterial activity of fresh fig latex was tested against five DFUs biofilm-forming bacterial isolates “Staphylococcus haemolyticus AUMC B-331, two Gram negative Pseudomonas sp., Bacillus sp., and Paenibacillus sp.”, to determine the efficacy of the latex as an antibacterial drug. The diameter of the inhibition zone is clarified in Fig. 2. The highest clear zones formed by fresh fig latex were 32, 30, and 28 mm, against St. haemolyticus AUMC B-331, Bacillus sp., and Paenibacillus sp., respectively, whereas the lowest inhibition zone observed was 18 mm against Pseudomonas sp. MS2, and there was no significant effect with no clear zone nor growth inhibition against Pseudomonas sp. MS4. The stored latex showed less activity than fresh crude latex as presented in Table 2.
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).
The MIC value was found to be 250 mg/mL demonstrating a great promising effect of the fresh fig latex as a natural antibacterial agent. According to broth macrodilution results, MIC was 250 mg/ml for St. haemolyticus AUMC B-331, Bacillus sp., Pseudomonas sp. MS2, and Paenibacillus sp. and the MBC was 500 mg/mL, and Pseudomonas sp. MS4 not reported MIC with fig latex as demonstrated in Fig. 3.
Fig latex reduces bacterial biofilm formation.
The resulting plate shown in Fig. 4A was quantified using a microplate reader at 600 nm, revealing that there were five isolates-forming biofilm Staphylococcus haemolyticus UMC B-331, Pseudomonas sp. MS2, Pseudomonas sp. MS4, Bacillus sp., and Paenibacillus sp. The results of OD 600nm were interpreted as low, moderate, and high bacterial biofilm-former when OD600 nm was < 1; 1–2.9 and > 2.9 respectively. Accordingly, Pseudomonas sp. MS4 was a high biofilm-former while, Pseudomonas sp. MS2 and St. haemolyticus had moderate ability. On the other hand, Bacillus sp., and Paenibacillus sp., were low biofilm-formers bacteria. In Fig. 4B, the ability of fig latex to reduce the bacterial biofilm for these isolates was illustrated. Notably, it was found that the MIC value of fresh fig latex had the ability to significantly reduce the biofilm of four isolates out of the five tested, Staphylococcus haemolyticus UMC B-331, Pseudomonas sp. MS2, Bacillus sp., and Paenibacillus sp. but, it had no significant activity on Pseudomonas sp. The percentages of the biofilm inhibition post treatment with fig latex were shown in Fig. 4C. Additionally, after treatment with fig latex, the biofilm formed by Staphylococcus haemolyticus AUMC B-331 was decreased 73% with the percentage of inhibition, Pseudomonas sp. MS2 inhibited with 37%, Paenibacillus sp. inhibited with 39% and Bacillus sp. inhibited with 67%. Additionally, fig latex inhibited the biofilm formed by Pseudomonas sp. MS4 with only 11%, so there is no significant effect of latex on Pseudomonas sp. MS4.
Survival curve of DFUs pathogenic bacteria in the presence of fig latex.
The survival curve test of isolated bacterial demonstrated slightly similar increase in the growth along 24 hours. While the growth curves were decreased in case of treatment of the bacterial culture with the MIC value of fresh fig latex nearly after 8 hours with a significant growth inhibition started at 16 hours of the bacteria cultured within the fig latex as demonstrated in Fig. 5 (A, B, C, E). In contrast, the growth curve of Pseudomonas sp. MS4 did not show any significant decrease after treatment with fig latex, suggesting a resistance stain to fig latex Fig. 5D.
Fig latex enhances the healing process of diabetic wounds.
The in vivo experiment on the mice model, in Fig. 6A monitored the morphological changes occurring in the three animal groups with each time point from day 0 to day 15-post wound-injury. It was noted that in the diabetic animals the healing process of wounds was delayed, and the abscess was found in the diabetic wounds with no healing as compared to the healing process of wounds in the control group. Most importantly, topical application of fig latex on the diabetic wounds twice daily improved and accelerated the wound closure nearly to those found in the control group. Figure 6B showed the wound size starting from 8mm, in the three animal groups at day 0 post-wounding. The diabetic group showed a significance increase in the wound diameter (4mm) (impaired healing) as compared to the wound diameters (0mm) in the control group at day 15 post-wounding. Interestingly, when the wounds were topically treated with fresh fig latex, the wound diameters (0mm) were similar to those found in the control group. The percentage of wound closure was calculated from accumulated data (Fig. 6C). The diabetic animals exhibited a significance decrease in the wound closure as compared to the control non-diabetic group. Amazingly, when the diabetic wounds were topically treated with fresh fig latex, the wound closure was significantly enhanced nearly to those found in the control group.
Fig latex restores collagen formation in the wounded skin tissues of diabetic mice.
Figure 7A-E demonstrated representative pictures of wounded skin tissues stained with sirrus red to monitor the collagen deposition. In row A, at day3 the three animal groups showed similar collagen deposition. However, from row B to E it was found that from day 6 the expression of collagen began to obviously increase in the control group with days until reaching the maximal expression at day15 post-wounding. However, the diabetic animals showed an obvious decrease in the expression of collagen as compared to the control. Most importantly, treatment of diabetic mice with fig latex restored the collagen expression nearly to those found in the control group. Using Image J software, the collagen expression in three animals per each group is quantified, and accumulated data are represented in (Fig. 7F). From day 6 to day 15 post-wounding both control and fig latex-treated diabetic animals demonstrated a similar expression of the collagen in the wounded skin tissues. Nevertheless, the diabetic animals exhibited a significant decrease in the expression of collagen.
Fig latex enhances vascularization in diabetic wounded skin tissues.
Figure 8A-E illustrated representative pictures for Immunohistochemical (IHC) expression of platelet endothelial cell adhesion molecule (PECAM-1) as a marker of vascularization in the wounded skin tissues. The diabetic animal group showed a decrease in the expression of PECAM-1 from day 3 to 15 post-wounding with mild positive reaction as compared to those found in the control animals which exhibited strong positive reaction of PECAM-1 expression. Amazingly, the wounded tissues of diabetic skin mice treated with fig latex exhibited a moderate reaction as compared to the control group. Using Image J software, accumulated data for PECAM-1 expression was quantified and presented in Fig. 8F. The diabetic mice showed a significant decrease in the expression levels of PECAM-1 from day 3 to day 15 post-wounding. On contrast to that was found in the diabetic group, both control and fig latex-treated mice exhibited a significant increase in the expression of PECAM-1 from day 3 to day 15 post-wounding.
Fig latex decreased the expression level of inflammatory CCL2 in the diabetic wounded skin tissues.
Figure 9A-E showed one representative pictures for the expression of CCL2 (inflammatory chemokine). Predominantly, it was noticed that there was an increased CCL2 expression in diabetic wounds from day 3 to 15 after wounding compared to the CCL2 expression in the wounds of control group, suggesting severe complications and protein expression dysregulation that associated with diabetes. Topical treatment of diabetic mice with fig latex downregulated the CCL2 expression nearly to those found in the control group. The accumulated data from three animals per group (Fig. 9F) was quantified using Image J software. The data demonstrated gradual significant elevation in the CCL2 expression from day 3 to 15 post-wounding. Nevertheless, the expression levels of CCL2 were significantly decreased in the wounded tissues of the control and fig latex-treated groups with a notable mild reaction.
Fig latex accelerates the healing process of diabetic wounds via restoration in the expression levels of β-defensin-1 and ZO-1.
It was concluded that fig latex inhibits pathogenic growth in vitro studies, hence, the expression of antibacterial protein (β-defensin) and tight junction protein Zonula occludens (ZO-1) were monitored using ELISA and are shown in Fig. 10 (A and B) respectively.
Our results showed decreased levels of β-defensin-1 and elevated level of ZO-1 in the diabetic mice compared to control animals. Interestingly, fig latex treated diabetic mice exhibited significantly restoration in the levels of β-defensin-1 and ZO-1 compared to the vehicle-treated diabetic animals.