In vitro Antioxidant and in vivo Hepatoprotective Activities of Root Bark Extract and Solvent Fractions of Croton macrostachyus Hochst. Ex Del. (Euphorbiaceae) on Paracetamol-Induced Liver Damage in Mice

Figures & data

Table 1 Phytochemical Constituents of the Root Bark Extracts of Croton Macrostachyus

Constituents	Test		Crude Extract	Aqueous Fraction	Chloroform Fraction	Indicator
Alkaloids	Mayer’s test		+	+	+	Whitish opalescence
	Wagner’s test		+	+	+	Reddish brown
Anthraquinones	Borntrager’s Test		+	–	+	Pink to red
Polyphenol	Ferric Chloride Test		+	+	+	Blue green
Flavonoids	Shinoda reduction test	Magnesium	+	+	+	Orange
		Zink	–	–	–
Saponins	Foam test		+	+	+	Foam persistence
Tannins	Gelatin test		–	–	–	Precipitate formation
Coumarins	Ammonia test		+	+	+	Intense fluorescence

Notes: +→ presence, – → absence.

Table 2 DPPH Scavenging Activities of Crude Extract and Solvent Fractions

Concentration (µg/mL)	Percentage Radical Scavenging Activity
	Ascorbic Acid	Crude Extract	Aqueous Fraction	Chloroform Fraction
12.5	40.9	17.7	6.6	5.8
25	47.5	21.8	8	6.4
50	58.7	26.5	17.5	9.7
100	70.7	40.6	34	17.1
200	86	71	57.2	26.2
IC50 (µg/µL)	30.8	128.6	168.9	406

Abbreviation: IC₅₀, 50% inhibitory concentration.

Table 3 Effects of Crude Extract and Solvent Fractions on Liver Weight Variation

Treatment Group	Dose	Liver Weight (g)
Normal	5%DMSO	1.16±0.09
Paracetamol	250mg/kg	1.47±0.07^a
Silymarin	100mg/kg	1.17±0.06*
Crude	100mg/kg	1.30±0.03
	200mg/kg	1.23±0.04
	400mg/kg	1.18±0.02*
Aqueous	100mg/kg	1.33±0.06
	200mg/kg	1.28±0.05
	400mg/kg	1.19±0.06*
Chloroform	100mg/kg	1.33±0.10
	200mg/kg	1.26±0.08
	400mg/kg	1.21±0.04*

Notes: N= 6, Results were expressed as Mean ±SEM. ^aP<0.01Vs Normal; *P<0.05 Vs paracetamol. Data were analyzed by One-Way ANOVA followed by Dunnet multiple comparison test.

Abbreviation: DMSO, dimethyl sulfoxide.

Table 4 Effects of Crude Extract and Fractions on Biochemical Parameters Against Paracetamol-Induced Hepatotoxicity in Mice

Serum Enzyme Levels
Treatment Group	AST (U/L)	ALT(U/L)	ALP (U/L)	Total Bilirubin (mg/dl)	Total Protein (mg/dl)
Normal(5% DMSO)	105.83±3.46	56.83±2.91	219.66±7.12	0.45±0.04	8.48±0.93
Paracetamol (250mg/kg)	313.50±13.75^a	164.06±6.95^a	506.72±16.38^a	1.27±0.07^a	4.68±0.48^b
Silymarin (100mg/kg)	161.50±5.60***	82.33±3.49***	268.83±6.26***	0.70±0.08***	8.00±0.57**
Crude(100mg/kg)	250.55±9.49***	122.6±5.48***	381.50±10.86***	1.07±0.05	6.67±0.45
Crude(200mg/kg)	214.46±5.91***	103.43±6.87***	325.28±11.47***	0.99±0.06*	6.91±0.37
Crude (400mg/kg)	183.42±5.86***	91.10±4.26***	303.98±8.47***	0.72±0.04***	7.90±0.55*
Aqueous(100mg/kg)	242.03±7.35***	119.67±4.66***	402.04±9.28***	1.02±0.03	6.73±0.84
Aqueous(200mg/kg)	205.87±6.05***	98.72±5.08***	360.12±8.05***	0.96±0.0.07**	7.16±0.45
Aqueous (400mg/kg)	197.65±5.68***	86.33±4.67***	317.21±10.72***	0.82±0.0.06***	7.86±0.37*
Chloroform(100mg/kg)	256.47±8.14***	121.47±6.90***	416.32±12.72***	1.08±0.03**	6.38±0.51
Chloroform(200mg/kg)	215.10±7.62***	104.57±5.48***	389.18±8.52***	0.98±0.05*	6.93±0.49
Chloroform(400mg/kg)	196.55±4.84***	96.42±4.34***	321.50±9.35***	0.86±0.04***	7.29±0.68

Notes: N= 6; Results were expressed in mean ± SEM. ^aP<0.001;^bP<0.01 Vs Normal group animals; ***P<0.001,**P<0.01, *P<0.05 vs paracetamol group animals. Data were analyzed by One-Way ANOVA followed by Tukey-Kramer multiple comparison test.

Abbreviation: DMSO, dimethyl sulfoxide.

Figure 1 Continued.

Figure 1 Histopathological changes of liver tissues (10x and 40x). (A) Normal control group (Normal hepatic cells with normal liver architecture) (B) paracetamol group (Necrotic hepatocytes, severe degeneration, hyperaemic/condensed blood vessels and cells with inflammation); Black arrow shows damaged hepatocytes. (C) Standard group (Liver architecture normal, cells with mild inflammation); Black arrow shows normal hepatocytes (D) Crude extract 100 mg/kg (Aggravated necrotic cells); Black arrow shows necrotized cells. (E) Crude extract 200 mg/kg (More necrotic hepatocytes and regenerated cells); Black arrow shows regenerated cells. (F) Crude extract 400mg/kg (Hepatocytes are normal in appearance with some mitotic figures); Black arrow shows normal hepatocytes. (G) Aqueous fraction 100mg/kg (Necrotized cells are by far more than normal cells); Black arrow shows necrotized hepatocytes. (H) Aqueous fraction 200 mg/kg (Normal hepatocytes are fewer than in Aqueous fraction 400mg/kg and haemorrhage); Black arrow shows haemorrhages. (I) Aqueous fraction 400 mg/kg (Most cells are normal with some dead cells and regenerated cells); Black arrow shows regenerated cells. (J) Chloroform fraction 100mg/kg (Shows inflammation and degeneration of hepatocytes); Black arrow shows degenerated cells. (K) Chloroform fraction 200 mg/kg (Mild inflammation and degeneration; regeneration). (L) Chloroform fraction 400mg/kg (Almost normal hepatocytes with mitotic figures); Black arrow shows normal hepatocytes.