Efficacy of Rosmarinus officinalis leaves extract against cyclophosphamide-induced hepatotoxicity

Figures & data

Figure 1. Diagram represents the design of the experiment. Group 1 (G1): control mice which were injected with saline, group 2 (G2): mice that were injected with 100 mg/kg b.w. MEROL, group 3 (G3): mice that were injected with 200 mg/kg b.w. MEROL, group 4 (G4): mice which were injected with saline followed with a single dose of 200 mg/kg b.w. CTX, group 5 (G4): mice that were injected with 100 mg/kg b.w. MEROL followed by a single dose of 200 mg/kg CTX, group 6 (G6): mice that were injected with 200 mg/kg b.w. MEROL followed by a single dose of 200 mg/kg CTX. *(Gs 1a–6a): groups of mice that were sacrificed at day (d22) from the starting of the experiment at (d1). **(Gs 1b–6b): groups of mice that were sacrificed at day (d37) from the starting of the experiment at (d1).

Table 1. Phytochemical analysis, DPPH radical scavenging activities, IC₅₀ value, and total antioxidant capacity (TAC) of R. officinalis leaves.

R. officinalis leaves extract (MEROL)
Phenolics (mg gallic acid equivalent/g extract)	121 ± 3.86
Flavonoids (mg quercetin equivalent/g extract)	14 ± 0.085
Saponin (mg saponin equivalent/g extract)	53 ± 2.68
Anthocyanins (μmole/g extract)	0.1 ± 0.007
% DPPH at 100 μg/ml	91.0 ± 0.72
IC50 (μg/ml)	55
TAC (ascorbic acid equivalent/g extract)	568.5 ± 84.14

Table 2. Correlation coefficient (r) between total antioxidant capacity (TAC) and DPPH radical scavenging activity and the determined metabolites by spectrophotometer.

	TAC	Phenolics	Flavonoids	Saponins	Anthocyanins
DPPH	64.4	Ns	202.5	368.85	310.25
TAC		38.81	86.92	74.43	91.26

Table 3. Determination of LD₅₀ of MEROL on albino Swiss mice.

MEROL (g/kg b.w.)	No. of dead in the first exp.	No. of death in the second exp.	Mean death	Dose difference	Mean death ×  dose diff.
1	0	0	0	0	0
2	0	1	0.5	1	0.5
3	1	1	1	1	1
4	2	1	1.5	1	1.5
5	2	2	2	1	2
6	3	3	3	1	3
7	4	3	3.5	1	3.5
Sum (∑)					11.5

The LD₅₀ was calculated using the formula: LD₅₀ = LDy−∑ (Dd × md)/N, where LDy is the highest dose (LD₁₀₀), N is the number of animals per group, Dd is the dose difference, Md, mean death.

Table 4. Effect of MEROL treatment prior CTX injection on the total body and relative liver weights.

Groups/time	G1a	G2a	G3a	G4a	G5a	G6a
At day 22 (5 d post-CTX treatment)
T B W (g)	36.2 ± 6.0	42.0 ± 2.7	40.2 ± 3.2	39.1 ± 2.7	41.0 ± 1.8	40.8 ± 2.7
L R W (%)	4.4 ± 0.05	4.6 ± 0.08	4.6 ± 0.05	3.24 ± 0.19	4.8 ± 0.05	4.4 ± 0.05
Groups/time	G1b	G2b	G3b	G4b	G5b	G6b
At day 37 (20 d post-CTX treatment)
T B W (g)	37.2 ± 6.0	43.0 ± 2.0	41.6 ± 3.0	31.6 ± 2.1*	42.2 ± 4.7	38.2 ± 4.8
L R W (%)	4.4 ± 0.05	5 ± 0.006	4.7 ± 0.05	5.5 ± 0.08	4.3 ± 0.03	5.4 ± 0.05

Total body weight (TBW) and relative liver weight (RBW). Data are expressed as means ± SE with n = 6.

* p ≤ 0.05. G1: control mice, G2 & 3, mice that were injected with 100 or 200 mg/kg b.w. MEROL, respectively G4: mice which were injected with 200 mg/kg b.w. CTX, G5 & 6: mice that were injected with 100 or 200 mg/kg b.w. MEROL followed by 200 mg/kg CTX, respectively, (Gs 1a to 6a): groups of mice that were sacrificed at day 22, (Gs 1b to 6b): groups of mice that were sacrificed at day 37.

Table 5. Effect of MEROL treatment prior CTX injection on the liver function.

Groups/time	G1a	G2a	G3a	G4a	G5a	G6a
At day 22 (5 d post-CTX treatment)
AST (U/L)	18.0 ± 2.1	16.5 ± 1.7	19.5 ± 1.7	32.0 ± 0.8*	15.0 ± 1.4	19.2 ± 2.5
ALT (U/L)	10.5 ± 1.2	13.2 ± 2.0	18.0 ± 3.7*	27.5 ± 4.50*	25.0 ± 2.1*	24.0 ± 1.1*
Cholesterol	38.5 ± 3.1	40.2 ± 8.2	56.2 ± 4.7*	101.5 ± 5.9*	48.5 ± 5.8*	59.7 ± 3.3*
Triglycerides	56.0 ± 4.7	55.0 ± 2.8	134 ± 1.7*	110.2 ± 6*	62.0 ± 2.08	82.5 ± 4.4*
	G1b	G2b	G3b	G4b	G5b	G6b
At day 37 (20 d post-CTX treatment)
AST (U/L)	17.2 ± 0.9	18 ± 1.15	20.7 ± 2.0*	26.0 ± 1.7*	18.7 ± 0.9	20.5 ± 2.6*
ALT (U/L)	12.0 ± 0.8	15.2 ± 2.5	22.0 ± 3.7*	24.2 ± 1.5*	13.0 ± 0.8*	17.5 ± 1.5*
Cholesterol	32.0 ± 3.1	38.5 ± 4.3	33.0 ± 3.4	58.7 ± 2.9*	38.0 ± 3.9	49.2 ± 7.0*
Triglycerides	50.7 ± 3.9	54.7 ± 7.8	74.0 ± 2.8*	96.5 ± 9.9*	52.0 ± 1.3	62.7 ± 2.2*

Cholesterol expressed by mg/100 mL, triglycerides expressed by mg/100 mL, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Data are expressed as means ± SD with n = 6.

* p ≤ 0.05. G1: control mice, G2 & 3: mice that were injected with 100 or 200 mg/kg b.w. MEROL, respectively, G4: mice which were injected with 200 mg/kg b.w. CTX, G5 & 6: mice that were injected with 100 or 200 mg/kg b.w. MEROL followed by 200 mg/kg CTX, respectively, (Gs 1a–6a): groups of mice that were sacrificed at day 22, (Gs 1b to 6b): groups of mice that were sacrificed at day 37.

Figure 2. Haematoxylin and eosin-stained liver sections: (a) and (b) control mice at days 22 and 37, respectively, showing normal hepatic architecture; (c) and (d) mice that were injected with 200 mg/kg CTX at days 22 and 37, respectively, showing vacuolar-degenerated hepatocytes with piknotic nuclei (arrows). Also, notice the coagulative necrosis of many hepatocyte (arrowheads); (e) and (f) mice that were pretreated with 100 mg/kg MEROL before CTX injection at days 22 and 37, respectively, showing that hepatocytes has partial improvement at day 22 and regained its normal architecture at day 37; (g) and (h) mice that were pretreated with 200 mg/kg MEROL before CTX injection at days 22 and 37, respectively, showing partial improvement in the hepatic architecture at 22 d while showing a hepatic degeneration at day 37 representing in the detection sever centrilobular pattern of degeneration and necrosis (arrowheads) with wide vacuolar degeneration (arrows) (haematoxylin- and eosin-stained paraffin sections; H& E ×400).