Assessment of Wound-Healing Properties of Medicinal Plants: The Case of Phyllanthus muellerianus

Abstract

Phyllanthus muellerianus (Family Euphorbiaceae) is a shrub, which is widely distributed in West Africa and employed traditionally as a wound-healing agent especially in Ghana. The aim of the study was to determine the in vivo wound-healing activity of aqueous aerial part extract of P. muellerianus (PLE) and its major isolate, geraniin. Excision and incision wound models were used to determine the wound-healing activity. Wounds were treated with PLE (0.25, 0.5, and 1% w/w) and geraniin (0.1, 0.2, and 0.4% w/w) aqueous creams. PLE and geraniin significantly (p < 0.001) increased wound contraction rate and hydroxyproline production compared to untreated wounds. Histological studies of wound tissues showed high levels of fibroblasts and increased collagen content and cross-linking in PLE and geraniin-treated wound tissues. Immuno-histochemical investigations revealed high levels of TGF-β₁ in PLE and geraniin-treated wound tissues compared to the untreated wound tissues. Tensile strength of incised wounds was significantly (p < 0.05) high in PLE and geraniin-treated wounds. PLE (0.1-100 μg/mL) significantly (p < 0.001) reduce LDH release from HaCaT-keratinocytes compared to the untreated cells. PLE and geraniin possess wound healing and cytoprotective effect.

Keywords: Phyllanthus muellerianus; TGF-β1; collagen; cytotoxicity; excision model; geraniin; hydroxyproline; incision model.

FIGURE 1

Influence of PLE on rate of wound closure. Full thickness wounds of approximately 20 mm in diameter were excised on the dorsal region of rats. The excised wounds were topically treated with 0.25, 0.5, and 1% w/w PLE formulated as aqueous cream. Wound diameter was measured on days 1, 3, 5, 7, 9, 11, and 13 post injuries. (A) Time-course curve, (B) area under curve (AUC) of time course curve. PLE: aqueous extract of the aerial parts of P. muellerianus. Vehicle group was treated with aqueous cream base. SSPD: 1% w/w silver sulphadiazine-treated wounds (positive control). Values are mean ± SEM, n = 5. ^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05 compared to untreated group (two-way ANOVA followed by Bonferroni’s post hoc test).

FIGURE 2

Influence of geraniin on rate of wound closure. Full thickness wounds of approximately 20 mm in diameter were excised on the dorsal region of rats. The excised wounds were topically treated with geraniin (0.1, 0.2, and 0.4% w/w) formulated as aqueous cream. Wound diameter was measured on days 1, 3, 5, 7, 9, 11, and 13 post injuries. (A) Time-course curve, (B) AUC of time course curve. Excised wounds were treated with 0.1, 0.2, and 0.4% w/w geraniin. Vehicle group was treated with aqueous cream base. SSPD: 1% w/w silver sulphadiazine-treated wounds. Values are mean ± SEM, n = 5. ^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05 compared to untreated group (two-way ANOVA followed by Bonferroni’s post hoc test).

FIGURE 3

Histological images (×400) representing the influence of PLE and geraniin in excised wound tissues from both treated and untreated rat wounds. Full thickness wounds were created on the dorsal region and topically treated with PLE, geraniin, silver sulphadiazine, vehicle (cream only). Wound tissues were excised 13 days post wounding and sectioned. Serial sections were stained with hematoxylin and eosin and evaluated microscopically for fibroblast proliferation, neovascularization, epithelial regeneration, and collagen deposition (H&E stain ×400). (A) Untreated wound tissues, (B) vehicle-treated (aqueous cream only) wound tissues, (C) 1% w/w silver sulphadiazine-treated wound tissues, (D) 0.25% w/w PLE-treated wound tissues, (E) 0.5% w/w PLE-treated wound tissues, (F) 1% w/w PLE-treated wound tissues, (G) 0.1% w/w geraniin-treated wounds tissues, (H) 0.2% w/w geraniin-treated wound tissues, and (I) 0.4% w/w geraniin-treated wound tissues. CO, collagen strands; MA, macrophages and monocytes; BV, blood vessels; FB, fibroblast.

FIGURE 4

Influence of PLE (A) and geraniin (B) treatment on hydroxyproline production in excised wound tissues. Hydroxyproline content was determined using acid hydrolysate of excised wound tissue on day 7 post injury. Excised wounds were treated with PLE (0.25, 0.5, and 1% w/w), geraniin (0.1, 0.2, and 0.4% w/w), silver sulphadiazine, and cream only. PLE, aqueous extract of the aerial parts of P. muellerianus. Vehicle group was treated with aqueous cream only. SSPD, 1% w/w silver sulphadiazine-treated wounds. Values are mean ± SEM, n = 5. ^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05 compared to untreated wound tissues (One-way ANOVA followed by Dunnet’s post hoc test).

FIGURE 5

Histological images showing influence of PLE and geraniin on collagen production in both PLE and geraniin-treated and untreated wound tissues. Full thickness wounds were created on the dorsal region and topically treated with PLE, geraniin, silver sulphadiazine, vehicle (cream only). Wound tissues were excised 13 days post wounding and sectioned. Serial sections were stained with Verhoeff–Van Gieson stain and evaluated microscopically for degree collagen deposition (Van Gieson stain ×400). Wound tissues were blindly examined by a pathologist. (A) Untreated wound tissues, (B) vehicle-treated (aqueous cream only) wound tissues, (C) 1% w/w silver sulphadiazine-treated wound tissues, (D) 0.25% w/w PLE-treated wound tissues, (E) 0.5% w/w PLE-treated wound tissues, (F) 1% w/w PLE-treated wound tissues, (G) 0.1% w/w geraniin-treated wound tissues, (H) 0.2% w/w geraniin-treated wound tissues, and (I) 0.4% w/w geraniin-treated wound tissues. CO, collagen fibers; MF, muscle fibers.

FIGURE 6

Histology of wound tissues showing influence of PLE and geraniin on TGF-β₁ levels in excised wound tissues. Full thickness wounds were created on the dorsal region and topically treated with PLE, geraniin, silver sulphadiazine, vehicle (cream only). Wound tissues were excised on day 7 post wounding and sectioned. Serial sections were incubated in the primary antibody overnight. Sectioned tissues were then incubated in the biotinylated secondary antibody, which was further conjugated with HRP to form a complex. The addition of DAB to the complex produced a brownish chromophore and counter stained with Mayers haematoxylin. The slides were blindly examined under the microscope. Region of positive immunoreactivity (brownish chromophore) is indicated by the blue line. Representative images of the treatment groups revealed the following morphological features: (A) Untreated-wound tissues, (B) vehicle-treated (aqueous cream only) wound tissues, (C) 1% w/w silver sulphadiazine-treated wound tissues, (D) 0.25% w/w PLE-treated wound tissues, (E) 0.5% w/w PLE-treated wound tissues, (F) 1% w/w PLE-treated wound tissues, (G) 0.1% w/w geraniin-treated wound tissues, (H) –0.2% w/w geraniin-treated wound tissues, and (I) 0.4% w/w geraniin-treated wound tissues.

FIGURE 7

Influence of PLE (A) and geraniin (B) on tensile strength of incised wound tissue. Paravertebral incisions were made through the full thickness of the skin on each side of the vertebral column and sutured. The sutured wounds were treated with PLE (0.25, 0.5, and 1% w/w) and geraniin (0.1, 0.2, and 0.4% w/w) and tensile strength determined on 10 days post incision. PLE, aqueous extract of the aerial parts of P. muellerianus; SSPD, 1% silver sulphadiazine-treated wound tissues. Vehicle group was treated with aqueous cream base only. Values are mean ± SEM, n = 5. ^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05 compared to untreated group (one-way ANOVA followed by Dunnett’s post hoc test).

FIGURE 8

Influence of PLE (A) and geraniin (B) on LDH release from HaCaT-keratinocytes. HaCaT-keratinocytes were seeded into 96-well plates and incubated to achieve 90% confluency. Cells were then treated with PLE (0.1 to 100 μg/mL) and geraniin (0.1 to 100 μM). Fetal calf serum (1% FCS) was used as positive control. The absorbance was measured at 490 against 690 nm. PLE, aqueous extract of the aerial parts of P. muellerianus. Values are mean ± SEM, n = 3. ^∗∗∗p < 0.001 compared to untreated cells (one-way ANOVA followed by Dunnett’s post hoc test).