Phytochemical Characterization and In Vitro Anti-Inflammatory, Antioxidant and Antimicrobial Activity of Combretum collinum Fresen Leaves Extracts from Benin

Abstract

Leaves from Combretum collinum Fresen (Combretaceae) are commonly used for the treatment of inflammatory conditions, wound healing and bacterial infections in traditional West African medicine. This research focuses on the characterization of the phenolic profile and lipophilic compounds of leaves extracts of C. collinum. Studies of the in vitro anti-inflammatory activity were performed in TNFα stimulated HaCaT cells and antibacterial activity was evaluated with agar well diffusion and microdilution assays. Antioxidant activity was determined by DPPH and ABTS assays and compared to standards. The phytochemical studies confirmed myricetin-3-O-rhamnoside and myricetin-3-O-glucoside as major components of the leaves extracts, each contributing significantly to the antioxidant activity of the hydrophilic extracts. GC-MS analysis identified 19 substances that were confirmed by comparison with spectral library data and authentic standards. Combretum collinum aqueous leaves extract decreased pro-inflammatory mediators in TNFα stimulated HaCaT cells. Further investigations showed that myricetin-3-O-rhamnoside has an anti-inflammatory effect on IL-8 secretion. In the antimicrobial screening, the largest inhibition zones were found against S. epidermidis, MRSA and S. aureus. MIC values resulted in 275.0 µg/mL for S. epidermidis and 385.5 µg/mL for MRSA. The in vitro anti-inflammatory, antibacterial and antioxidant activity supports topical use of C. collinum leaves extracts in traditional West African medicine.

Keywords: Combretum collinum; HaCaT; anti-inflammatory; antimicrobial; keratinocytes.

Figure 1

Representative HPLC chromatogram of the EtOH 50% extract of C. collinum (5 mg/mL extract dissolved in ACN 5%, injection volume 20 µL, detection wavelength = 320 nm). The main compounds could be confirmed by LC-MS experiments, ¹H-NMR and spiking experiments.

Figure 2

GC-MS chromatogram of the n-hexane extract of the leaves of C. collinum after silylation with BSTFA. Peaks were identified with the NIST08 database.

Figure 3

Antioxidant activity of 50% ethanolic extract of C. collinum extract, measured by ABTS (A) and DPPH assays (B). CCL EtOH 50% showed significant concentration-dependent antioxidant activity compared to control (pure ABTS or DPPH solution incubated with a blank). Trolox 6.25 µg/mL was used as a positive control. The two diagrams C and D show that the two main compounds of the extract had significantly higher antioxidant activity than CCL EtOH 50% in both the ABTS (C) and the DPPH assays (D) and thus most likely significantly contributed to the overall antioxidant activity of the extract; values in (C) and (D) are given in Trolox Equivalent Antioxidative Capacity (TEAC). n = 3 replicates, data presented as mean ± SEM, significant for * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.001 compared to the control in ordinary one-way ANOVA (analysis of variance).

Figure 3

Antioxidant activity of 50% ethanolic extract of C. collinum extract, measured by ABTS (A) and DPPH assays (B). CCL EtOH 50% showed significant concentration-dependent antioxidant activity compared to control (pure ABTS or DPPH solution incubated with a blank). Trolox 6.25 µg/mL was used as a positive control. The two diagrams C and D show that the two main compounds of the extract had significantly higher antioxidant activity than CCL EtOH 50% in both the ABTS (C) and the DPPH assays (D) and thus most likely significantly contributed to the overall antioxidant activity of the extract; values in (C) and (D) are given in Trolox Equivalent Antioxidative Capacity (TEAC). n = 3 replicates, data presented as mean ± SEM, significant for * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.001 compared to the control in ordinary one-way ANOVA (analysis of variance).

Figure 4

Influence of C. collinum aqueous leaves extract on IL-8- and IL-6-levels released by HaCaT cells after stimulation with TNFα (20 ng/mL): An aqueous leaves extract of C. collinum (CCL) concentration-dependently reduced the levels of IL-8 (A) and IL-6 (B) at nontoxic concentrations. CCL extract was tested in concentrations ranging from 1–200 µg/mL, resulting in an IC₅₀ value of 142.5 µg/mL for IL-8 (A) and a decrease of IL-6 release by 29.3% for the highest extract concentration of 200 µg/mL respectively (B). 10 µM budesonide (Bud 10) was used as a positive control, UC = Untreated control, n = 4–6 replicates, data presented as mean ± SEM, significant for * p ≤ 0.05 compared to TNFα control in ordinary one-way ANOVA.

Figure 5

Influence of myricetin-3-O-rhamnoside and myricetin on IL-8 released by HaCaT cells after stimulation with TNFα (20 ng/mL): The main phenolic compound of CCL, myricetin-3-O-rhamnoside (A) and its aglycone myricetin (B) concentration-dependently reduced the levels of IL-8 at nontoxic concentrations. The compounds were tested in concentrations ranging from 0.1–100 µM, resulting in IC₅₀ values for IL-8 of 121.9 µM for myricetin-3-O-rhamnoside (A) and 90.69 µM for myricetin (B), respectively. 10 µM budesonide (Bud 10) was used as a positive control, UC = Untreated control, n = 4–6 replicates, data presented as mean ± SEM, significant for * p ≤ 0.05 compared to TNFα control in ordinary one-way ANOVA.

Figure 6

The growth profile of MRSA (ATCC 43300) treated with increasing concentrations of C. collinum ethanolic leaves extract (CCL EtOH 50%). The results are plotted as means of three experiments ± SEM (dotted line), the control was the solvent 0.5% Tween 80.