Antibacterial, Antibiofilm, and Wound Healing Activities of Rutin and Quercetin and Their Interaction with Gentamicin on Excision Wounds in Diabetic Mice

Abstract

Phytochemicals are effective and are gaining attention in fighting against drug-resistant bacterial strains. In the present study, rutin and quercetin were tested for antibacterial, antibiofilm, and wound healing activities on excision wounds infected with MDR-P. aeruginosa in diabetic mice. Antibacterial and antibiofilm activities were studied in vitro using broth dilution assay and crystal violet assay, respectively. These phytochemicals were tested alone for wound-healing activities at different concentrations (0.5% and 1% in ointment base) and in combination with gentamicin to evaluate any additive effects. Rutin and quercetin demonstrated effectiveness against MDR-P. aeruginosa at higher concentrations. Both phytochemicals inhibited biofilm formation in vitro and contributed to the healing of diabetic wounds by eradicating biofilm in the wounded tissue. Rutin at a low concentration (0.5%) had a lesser effect on reducing the epithelization period and regeneration of the epithelial layer compared to quercetin. When combined with gentamicin, quercetin (1%) displayed the maximum effect on epithelium regeneration, followed by rutin (1%) in combination with gentamicin. Both phytochemicals were found to be more effective in controlling biofilm and wound-healing activities when used as an additive with gentamicin. The study supports the traditional use of phytochemicals with antibacterial, antibiofilm, and wound-healing activities in managing diabetic infections.

Keywords: antibacterial compounds; antibiotic resistance; bioactive compounds; histology; quercetin; rutin; wound healing.

Figure 1

In vitro antibiofilm activity of rutin and quercetin against MDR-P. aeruginosa. Each experiment was repeated three times (n = 3) and each sample was in duplicate. Bars indicate means ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to control (0 µg/mL). Statistical analysis was completed using one-way ANOVA followed by Tukey’s test.

Figure 2

Epithelization period in biofilm-induced excision wound in mice after different treatments. The epithelization period is the day on which there was a falling of the scar, leaving no raw wound. Bars represent mean ± SEM, n = 6, *** p < 0.001 as compared to base-treated control. +++ p < 0.001 as compared to individual quercetin (1%) treatment. Statistical analysis was completed using one-way ANOVA followed by Tukey’s test.

Figure 3

Effect on wound contraction. MDR-P. aeruginosa-infected wounded tissue contracted differently after various treatments. The bars in the graph represent mean ± SEM, n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate significance compared to the base-treated control. +++ p < 0.001 indicate significance compared to the individual quercetin (1%) or rutin (1%) treatment. Statistical analysis was completed using one-way ANOVA followed by Tukey’s test.

Figure 4

Images showing macroscopic appearance and microscopic changes in the wounded tissues. The wounded tissue images on Day 0 show pus formation in all the groups. Treatment with gentamicin (0.1%), rutin (1%), quercetin (1%), and their combinations accelerated the healing of wounds to varying degrees. Wounded tissue histology at 200× from different treatment groups is shown. The regenerated epithelial tissue is shown by black arrows. The regenerated epithelial tissue height was lowest in the infected control groups. Gentamicin (0.1%) and the combination of gentamicin (0.1%) with quercetin (1%) increased the regeneration of skin epithelium as indicated by the thickness of the tissue in histological sections. Rutin (1%) produced less regenerative effect compared to other treatments. Note: the images are from groups that showed maximum treatment effect, and the epithelial tissue histology from other groups with intermediate effects has not been shown.The bacteria count in the wounded tissue 20 days after using the phytochemical ointment was significantly less compared to the control treated with the base. However, there was no significant reduction in the count of P. aeruginosa at the 0.5% concentration of rutin, while the higher concentration (1%) of rutin and both the concentration of quercetin (0.5% and 1%) significantly decreased the bacterial count. The growth and counting of other bacteria was avoided by using cetrimide agar which is selective for P. aeruginosa. Treatment with gentamicin significantly decreased the bacterial load compared to the control. A combination of quercetin (1%) + gentamicin (0.1%) was significantly more effective in reducing CFU in the tissue when compared to quercetin (1%) alone while no such action could be observed with the combination of rutin (1%) and gentamicin (0.1%) as compared to rutin (1%) alone (Table 3).