Investigation of Wound Healing and Anti-Inflammatory Activities of Solvent Fractions of 80% Methanol Leaf Extract of Achyranthes aspera L. (Amaranthaceae) in Rats

Figures & data

Figure 1 Achyranthes aspera from site of collection (Captured on 20/04/2019).

Table 1 Master and Reduced Formula Used to Formulate Simple Ointment

Ingredients	Master Formula	Reduced Formula
Wool fat	50 g	2.5 g
Hared paraffin	50 g	2.5 g
Cetostearyl alcohol	50 g	2.5 g
White soft paraffin	850 g	42.5 g
	1000 g	50 g

Table 2 Effect of Solvent Fractions of 80% Methanol Leaf Extract of A. aspera on Percentage Wound Contraction in Rats

Days of Wound Area Measurement in mm^2 (Mean±SEM) and %			Contraction
Treatment group	Day 2	Day 4	Day 6	Day 8	Day 10
SO	307.97±2.5[1.92]	297.85±2.91[5.14]	282.13±1.14[11.30]	247.82±2.56[21.08]	221.44±3.59[29.48]
NF 0.2%	297.85±2.91[4.3]	284.10±1.33[9.24]*a	240.60±7.52[31.15]*a	164.37±6.55[47.59] *aef	131.39±6.63[58.16]*a
5% w/w AF	303.93±1.79[3.2]	290.82±1.11[7.38]	247.82±2.56[26.77]	229.03±7.23[27.06]	131.75±15.89[58.04]*a
10% w/w AF	305.48±1.56[2.71]	286.05±1.34[8.9]	227.20±7.71[39.01]*a	186.14±10.55[40.72]*af	102.88±10.36[67.24]*a
5% w/w BF	308.22±2.1[1.83]	289.49±1.88[7.81]	252.85±7.70[24.74]	229.66±7.72[26.86]	157.84±6.81[49.73]*a
10% w/w BF	302.52±1.5[3.66]	284.130±1.27[9.51]	198.53±11.91[36.77]*a	170.09±5.36[45.83]*af	113.49±5.10[63.85]*ad
5% w/w CF	303.48±1.72[3.35]	290.67±2.83[7.43]	264.00±3.45[19.04]	201.29±7.26[35.89]*a	149.73±3.62[52.32]*a
10% w/w CF	298.06±0.89[5.08]	284.5±0.76	199.49±10.07[39.30]*abcd	172.82±8.51[44.96]*adf	127.80±4.29[59.30]*a
Day 12	Day 14		Day 16	Day 18	Day 20
183.5±2.78[41.56]	95.50±6.31[69.59]		59.80±3.05[80.95]	38.99±4.02[87.58]	35.33±3.61[88.75]
75.88±6.59[75.83]*a	30.24±2.10[90.37]*ae		5.76±0.83[98.17]*aef	1.39±0.41[99.56]*adef	0.15±0.13[99.95] *af
107.64±13.01[65.72]*a	54.95±9.84[82.62]*a		30.23±4.21 [90.37]*a	26.36±3.52 [91.60]	15.99±2.15[94.0]*a
83.81±8.17[73.31]*a	53.56±0.75[82.81]*a		10.50±1.18[96.66]*af	7.44±0.59[97.63]*af	5.33±0.77[98.30]*af
114.21±8.4[63.63]*a	62.70±1.27[80.03]*a		21.23±4.29[92.51]*a	14.79±3.74[95.29]*a	8.87±2.14[97.18]*a
73.58±7.04[76.57]*ad	41.07±2.81[86.92]*a		20.61±2.44[93.44]*a	15.19±1.65[95.31]*a	8.94±0.98[97.19]*a
102.85±2.5[67.25]*a	43.00±2.94[86.31]*a		9.16±1.64[97.08]*af	5.69±0.67[98.19]*af	0.58±0.0999.82]*af
67.52±4.75[78.50]*af	23.38±4.2[92.55]*adefg		6.74±1.31[97.85]*af	1.97±0.53[99.37]*aef	0.27±0.17[99.92]*af

Notes: Values are expressed as mean ± SEM (n = 6 rats in each group) and analyzed by one-way ANOVA followed by post hoc Tuckey test; ^acompared with simple ointment; ^bcompared with NF 0.2%; ^ccompared with 5% chloroform fraction; ^dcompared with 5% n-butanol fraction; ^ecompared with 10% n-butanol fraction; ^fcompared with 5% aqueous fraction; ^gcompared with 10% aqueous fraction; w/w = weight by weight; SO = Simple ointment; * = p ˂0.01; NF 0.2% = nitrofurazone 0.2% ointment; AF, BF and CF are aqueous, n-butanol and chloroform fractions of 80% methanol leaf extract of A. aspera (Amaranthaceae), respectively. Initial wound area was 314 mm². Values in the bracket are percentages of contractions.

Table 3 Effect of Topical Application of Solvent Fractions of 80% Methanol Leaf Extract of A. aspera on Period of Epithelialization (No. of Days) Post Wound Creation in Rats

Treatment Group	Period of Epithelialization in Days (Mean ± SEM)
SO	19.33±0.21
NF 0.2%	14.17±0.31*abc
5% w/w AF	16.67±0.21*a
10% w/w AF	15.50±0.22*a
5% w/w BF	16.17±0.21*a
10% w/w BF	14.83±0.21*a
5% w/w CF	15.33±0.33*a
10% w/w CF	14.00±0.26*abcd

Notes: Values are expressed as mean ± SEM (n = 6 rats in each group) and analyzed by one-way ANOVA followed by post hoc Tuckey test; ^acompared with simple ointment, ^bcompared with 5% n-butanol fraction, ^ccompared with 5% aqueous fraction, ^dcompared with 10% aqueous fraction; w/w = weight by weight; SO = Simple; * = p ˂0.01; NF 0.2% = nitrofurazone 0.2% ointment; AF, BF and CF are aqueous, n-butanol and chloroform fractions of 80% methanol leaf extract of A. aspera (Amaranthaceae), respectively.

Table 4 Effect of the Solvent Fraction Ointments of 80% Methanol Leaf Extract of A. aspera on Tensile Strength in Rats

Treatment Group	Tensile Strength (g)	Tensile Strength (%)
LU	445.82 ± 42.21	_
SO	484.37 ± 73.22	8.74
NF 0.2%	876.15 ± 42.32*abcde	80.89
5% w/w AF	698.10 ± 65.61*a	43.09
10% w/w AF	779.14 ± 65.89*ae	60.89
5% w/w BF	736.12 ± 51.66*ae	51.97
10% w/w BF	835.58 ± 21.68*ade	72.51
5% w/w CF	739.31 ± 51.19*ae	52.63
10% w/w CF	873.15 ± 69.38*abcde	80.26

Notes: Values are expressed as mean ± SEM (n = 6 rats in each group) and analyzed by one-way ANOVA followed by post hoc Tuckey test; ^acompared with simple ointment, ^bcompared with 5% chloroform fraction, ^ccompared with 5% n-butanol fraction, ^dcompared with 5% aqueous fraction, ^ecompared with untreated group; LU = left untreated; w/w = weight by weight; SO = Simple ointment; * = p ˂0.01; NF 0.2% = nitrofurazone 0.2% ointment; AF, BF and CF are aqueous, n-butanol and chloroform fractions of 80% methanol leaf extract of A. aspera (Amaranthaceae), respectively.

Figure 2 Excision wound on rats treated with solvent fraction ointments of leaves of A. aspera in rats.

Table 5 Anti-Inflammatory Effect of the Solvent Fractions of 80% Methanol Leave Extract of A. aspera on Carrageenan-Induced Paw Edema in Rats

Treatment Group	Basal Volume (mL)	Change in Paw Volume (Mean ± SEM) (mL) [Percentage Inhibition (%)]
		1 Hour	2 Hours	3 Hours	4 Hours
2% Tween 80,10mL/kg	0.22±0.01	0.42 ± 01	0.43 ± 0.01	0.41 ± 0.01	0.40 ± 0.01
Indo-10mg/kg	0.22±0.1	0.29±0.01(24.79)*^ab	0.26±0.20(39.99) *^ab	0.22±0.01(47.18)*^ab	0.18±0.01(53.33)*^abc
100mg/kg AF	0.18± 0.02	0.38 ± 0.15(3.85)	0.37 ± 0.02(14.67)	0.35 ± 0.01(14.92)	0.31 ± 0.03(22.50)*^a
200mg/kg AF	0.22± 0.01	0.36± 0.01(8.55)	0.31± 0.02(27.41)*^a	0.24±0.01(37.09)*^ab	0.22± 0.01(45.00)*^ab
400mg/kg AF	0.23± 0.00	0.31±0.02(20.94)*^a	0.34±0.029(29.34)*^a	0.28±0.03(42.33)*^ab	0.24±0.01(45.83)*^ab
100mg/kg BF	0.22± 0.01	0.35 ±0.02(11.11)	0.34 ±0.02(21.24)*^a	0.28±0.03(31.85)*^a	0.24 ±0.01(39.16)*^a
200mg/kg BF	0.23± 0.00	0.32±0.02(16.23)*^a	0.33 ±0.00(22.78)*^a	0.26±0,01(37.10)*^ab	0.23 ±0.00(42.5)*^ab
400mg/kg BF	0.22± 0.01	0.30±0.00(24.36)*^ab	0.27±0.01(36.67)*^ab	0.24±0.00(42.74)*^ab	0.22 ±0.01(45.0)*^ab
100mg/kgCF	0.21 ±0.02	0.33±0.02(13.68)*^a	0.33±0.01(22.78)*^a	0.28±0.01(31.85)*^a	0.27 ±0.02(31.25)*^a
200mg/kgCF	0.15± 0.02	0.30±0.02(24.36)*^ab	0.26 ± 0.02(37.12)*^ab	0.23±0.02(42.75)*^ab	0.12± 0.03(51.25)*^abc
400mg/kgCF	0.20 ±0.01	0.30±0.02(23.93) *^ab	0.28 ± 0.02(35.91)*^ab	0.22±0.01(44.76)*^ab	0.19±0090(52.50)*^abc

Notes: Values are expressed as mean ± SEM (n = 6 rats in each group) and analyzed by one-way ANOVA followed by post hoc Tuckey test; ^acompared with 2% Tween 80, ^bcompared with 100 mg/kg aqueous fraction, ^ccompared with 100 mg/kg chloroform fraction; * = p ˂0.01; Indo = Indomethacin; AF, BF and CF are aqueous, n-butanol and chloroform fractions of 80% methanol leaf extract of A. aspera (Amaranthaceae), respectively.

Table 6 Effects of Solvent Fractions of 80% Methanol Leave Extract of A. aspera on Cotton Pellet-Induced Granuloma in Rats

Treatment Group	Mean Weight of Exudates in mg (Mean ± SEM)	% Inhibition of Exudation	Mean Weight of Granuloma in (mg) (Mean ± SEM)	% Inhibition of Granulation
2% Tween 80,10mL/kg	123.33 ± 0.54	–	143.70 ±0.77	–
Indo 10mg/kg	74.85 ± 2.01^*ab1b2c1d1	39.31	67.39± 0.83^*a b1b2c1d1	53.10
AF 100mg/kg	121.83 ± 0.97	1.22	132.54 ± 2.45^*a	7.76
AF 200mg/kg	95.33 ± 2.64^*ab1	22.71	98.15 ± 3.17^*a b1	31.69
AF 400mg/kg	84.56 ± 0.72^*ab1c1d1	31.44	75.42 ± 0.55^*ab1b2	47.51
BF 100mg/kg	100.85 ± 0.61^* ab1	18.47	80.04 ± 1.37^*a b1	44.30
BF 200mg/kg	91.37 ± 2.27^*ab1	25.92	76.50 ±0.93^*ab1b2	46.75
BF 400mg/kg	85.92 ± 1.94^*ab1c1d1	30.33	69.36 ± 1.06^*ab1b2c1d1	51.73
CF 100mg/kg	98.54 ± 1.26^*ab1b2	20.10	79.71± 1.4^*ab1b2	44.53
CF 200mg/kg	88.32 ± 1.81^*ab1b2	28.39	74.87 ± 0.87^*ab1b2	47.90
CF 400mg/kg	77.06 ± 2.77^*ab1b2c1c2d1	37.52	67.81 ± 0.67^*ab1b2c1c2d1	52.81

Notes: Values are expressed as mean ± SEM (n = 6 rats in each group) and analyzed by one-way ANOVA followed by post hoc Tuckey test; ^acompared with 2% Tween 80, ^b1compared with 100 mg/kg aqueous fraction, ^b2compared with 200 mg/kg aqueous fraction, ^c1100 mg/kg n-butanol fraction, ^c2200 mg/kg n-butanol fraction, ^d1compared with 100 mg/kg chloroform fraction; * = p ˂0.01; Indo= indomethacin; AF, BF and CF are aqueous, n-butanol and chloroform fractions of 80% methanol leaf extract of A. aspera (Amaranthaceae), respectively.

Table 7 Preliminary Phytochemical Screening of Solvent Fractions of 80% Methanol Leaf Extract of A. aspera

Secondary Metabolite	Aqueous Fraction	n-Butanol Fraction	Chloroform Fraction
Saponin	+	+	-
Terpinoids	+	+	+
Tanins	-	-	+
Flavonoid	+	+	+
Glycosides	+	+	+
Steroids	-	+	+
Alkaloids	+	+	+

Notes: (+, Present); (-, Absent).