In vivo antifungal effect of Combretum and Terminalia species extracts on cutaneous wound healing in immunosuppressed rats

Figures & data

Table 1.  Evaluation of erythema and exudate.

Score	Erythema	Exudate
0	No red color at all	No exudate
1	Light red just visible	Exudate just visible
2	Clearly red	Easily visible
3	Dark red, not whole area	Substantial quantity
4	Dark red, widespread	Large quantity

Figure 1.  The average size of lesions infected with C. albicans (A), C. neoformans (B), M. canis (C), and S. schenckii (D) and treated with four extracts, isolated compound, and amphotericin B (positive control).

Figure 2.  The influence of the crude extracts, isolated compound, and amphotericin B (positive control) on wound erythema in rats infected with C. albicans (A), C. neoformans (B), M. canis (C), and S. schenckii (D).

Figure 3.  The influence of the crude extracts, isolated compound, and amphotericin B (positive control) on exudate formed in rats infected with C. albicans (A), C. neoformans (B), M. canis (C), and S. schenckii (D).

Table 2.  Quantitative histopathological findings of wounds of rats infected with C. albicans after topical application of different creams.

Rat nr.	Treatment^a	Fibrosis	Necrosis	Hypertrophy of subcutaneous muscle fibers	Infiltration of					Angiogenesis	Epithelialization	Presence of fungal spores and hyphae
					Neuts^b	Lymphocytes/plasma cells	Eosinophils	Mast cells	Macs^b
1	A	2(++)	0	0	1&2(+++)	3(+)	3(±)	3(±)	3(++)	3(++)	2	1(±)
	B	2(++)	0	0	2(±)	3(++)	3(±)	3(±)	3(+++)	3(++)	2	1(±)
	C	2(+)	2(+)	0	1(+++);2(±)	2(+)	2(±)	2(±)	2(+++)	2(+)	2	1(±)
	D	2(++)	0	0	2(±)	2(++)	2(±)	2(±)	2(+++)	2(++)	2	1(±)
	E	2(++)	0	0	2(±)	2(+++)	2(±)	0	2(+++)	2(+)	2	1(±)
	F	2(+)	2(±)	0	2(±)	2(+)	2(++)	0	2(++)	2(+)	2	1(±)
	G	2(+)	0	0	0	2(++)	0	0	2(+++)	2(+)	2	1(±)

^aA, negative control (untreated); B, compound 1 (mixture of asiatic acid and ajurnolic acid); C, C. nelsonii crude extract; D, C. imberbe crude extract; E, C. albopunctatum crude extract; F, T. sericea crude extract; G, positive control (amphotericin B).

^bNeuts, neutrophils; Macs, macrophages.

Parameters were marked as follows:

• Severity: –, absent; ±, scant; +, mild; ++, moderate; +++, severe/marked.

• Distribution: 1, dermal; 2, dermal and subdermal (i.e. subcutaneous skeletal muscle); 3, locally extensive (dermal and subdermal).

• Epithelialization: 0, absent; 1, partial; 2, complete.

Table 3.  Quantitative histopathological findings of wounds of rats infected with C. neoformans after topical application of different creams.

Rat nr.	Treatment^a	Fibrosis	Necrosis	Hypertrophy of subcutaneous muscle fibers	Infiltration of					Angiogenesis	Epithelialization	Presence of fungal spores and hyphae
					Neuts^b	Lymphocytes/plasma cells	Eosinophils	Mast cells	Macs^b
7	A	3(++)	2(+)	0	1&2(++)	3(++)	3(±)	0	3(++)	3(++)	1	1(±)
	B	2(++)	0	0	0	2(+)	2(±)	0	2(+)	2(++)	2	1(±)
	C	2(+++)	1(++)	0	1(++);2(+)	2(+++)	2(+)	2(+)	2(+++)	2(++)	1	1(±)
	D	2(++)	2(±)	0	0	2(+++)	2(+)	2(±)	2(++)	2(+)	2	1(±)
	E	3(++)	2(±)	0	2(+)	3(++)	3(+)	3(+)	3(++)	3(++)	2	1(±)
	F	2(++)	2(±)	+	0	2(+)	3(±)	0	3(++)	2(++)	2	1(±)
	G	2(++)	2(±)	±	2(±)	2(++);3(+)	2(+)	0	2(+)	2(+)	2	1(±)

^aA, negative control (untreated); B, compound 1 (mixture of asiatic acid and ajurnolic acid); C, C. nelsonii crude extract; D, C. imberbe crude extract; E, C. albopunctatum crude extract; F, T. sericea crude extract; G, positive control (amphotericin B).

^bNeuts, neutrophils; Macs, macrophages.

Parameters were marked as follows:

• Severity: –, absent; ±, scant; +, mild; ++, moderate; +++, severe/marked.

• Distribution: 1, dermal; 2, dermal and subdermal (i.e. subcutaneous skeletal muscle); 3, locally extensive (dermal and subdermal).

• Epithelialization: 0, absent; 1, partial; 2, complete.

Table 4.  Quantitative histopathological findings of wounds of rats infected with M. canis after topical application of different creams.

Rat nr.	Treatment^a	Fibrosis	Necrosis	Hypertrophy of subcutaneous muscle fibers	Infiltration of					Angiogenesis	Epithelialization	Presence of fungal spores and hyphae
					Neuts^b	Lymphocytes/plasma cells	Eosinophils	Mast cells	Macs^b
13	A	3(++)	3(±)	0	0	3(++)	3(±)	3(±)	3(++)	3(++)	2	1(±)
	B	3(++)	0	0	3(±)	3(+)	3(±)	0	3(++)	3(++)	2	1(±)
	C	2(++)	2(±)	±	2(±)	2(+++)	2(±)	0	2(++)	2(++)	2	1(±)
	D	2(++)	2(±)	0	2(±)	2(++)	2(+)	3(+)	2(++)	2(++)	2	1(±)
	E	2(++)	2(++)	+	2(+)	2(+++)	2(±)	0	2(+++)	2(++)	2	1(±)
	F	2(++)	0	0	0	2(++)	2(+)	0	2(+)	2(++)	2	1(±)
	G	3(+++)	0	0	2(±)	3(++)	2(±)	0	3(+++)	2(++)	2	1(±)

^aA, negative control (untreated); B, compound 1 (mixture of asiatic acid and ajurnolic acid); C, C. nelsonii crude extract; D, C. imberbe crude extract; E, C. albopunctatum crude extract; F, T. sericea crude extract; G, positive control (amphotericin B).

^bNeuts, neutrophils; Macs, macrophages.

Parameters were marked as follows:

• Severity: –, absent; ±, scant; +, mild; ++, moderate; +++, severe/marked.

• Distribution: 1, dermal; 2, dermal and subdermal (i.e. subcutaneous skeletal muscle); 3, locally extensive (dermal and subdermal).

• Epithelialization: 0, absent; 1, partial; 2, complete.

Table 5.  Quantitative histopathological findings of wounds of rats infected with S. schenckii after topical application of different creams.

Rat nr.	Treatment^a	Fibrosis	Necrosis	Hypertrophy of subcutaneous muscle fibers	Infiltration of					Angiogenesis	Epithelialization	Presence of fungal spores and hyphae
					Neuts^b	Lymphocytes/plasma cells	Eosinophils	Mast cells	Macs^b
19	A	4(++)	4(±)	0	1(+++)	4(+++)	4(+)	4(+)	4(++)	3(++)	1	1(±)
	B	4(++)	1(++)	0	1(+++)	4(+++)	4(+)	4(±)	4(++)	3(++)	0	1(±)
	C	2(+++)	0	0	2(+)	2(+++)	2(+)	0	2(++)	2(+++)	1	1(±)
	D	2(+++)	2(±)	0	0	2(+++)	2(+)	0	2(++)	2(+++)	2	2(±)
	E	2(+++)	2(+)	0	2(+)	2(+++)	2(+)	0	2(+++)	2(+++)	2	1(±)
	F	2(++)	2(+)	0	0	2(+++)	2(+)	0	2(+)	2(++)	2	2(+++)
	G	4(+++)	4(++)	0	2(++)	4(+++)	4(±)	0	3(+++)	4(++)	1	4(+++)

^aA, negative control (untreated); B, compound 1 (mixture of asiatic acid and ajurnolic acid); C, C. nelsonii crude extract; D, C. imberbe crude extract; E, C. albopunctatum crude extract; F, T. sericea crude extract; G, positive control (amphotericin B).

^bNeuts, neutrophils; Macs, macrophages.

Parameters were marked as follows:

• Severity: –, absent; ±, scant; +, mild; ++, moderate; +++, severe/marked.

• Distribution: 1, dermal; 2, dermal and subdermal (i.e. subcutaneous skeletal muscle); 3, locally extensive (dermal and subdermal).

• Epithelialization: 0, absent; 1, partial; 2, complete.

Table 6.  Minimum inhibitory concentration (MIC) values of selected extracts after 24 h incubation at 37°C.

Microorganism	MIC value (mg/mL)
	C. imberbe	C. nelsonii	C. albopunctatum	T. sericea	Amphotericin B (μg/mL)
C. albicans	2.5	0.04	0.64	0.64	0.4
C. neoformans	0.16	0.04	0.08	0.16	0.3
M. canis	0.04	0.02	0.02	0.02	0.2
S. schenckii	2.5	0.02	0.02	0.08	0.4
A. fumigatus	2.5	0.16	0.64	0.16	0.2

Table 7.  LC₅₀ values from MTT cytotoxicity assay of the optimal extracts.

Plant extract	LC50 (μg/mL)
C. imberbe	168.6
C. nelsonii	75.7
C. albopunctatum	102.9
T. sericea	121.7
Berberine (+ve control)	4.347