Effect of Aegle marmelos leaf extract on N-methyl N-nitrosourea-induced hepatocarcinogensis in Balb/c mice

Figures & data

Table 1. Set of specific genes primer.

Genes		Primer reference	Product size (bp)	Amplification conditions
IL-1β	Sense	5′-GACCTTCCAGGATGAGGACA-3′	273	94 °C for 54 s, 54 °C for 60 s, 72 °C for 60 s
	Antisense	5′-AGGCCACAGGTATTTTGTCG-3′
IL-6	Sense	5′-TCTCCAGCAACGAGGAGAAT-3′	348	94 °C for 50 s, 49 °C for 60 s, 72 °C for 60 s
	Antisense	5′-TGTGATCTGAAACCTGCTGC-3′
p53	Sense	5′-ATGACTGCCATGGAGGAGTCACAGT-3′	495	94 °C for 50 s, 54 °C for 60 s, 72 °C for 60 s
	Antisense	5′ -GTGGGGGCAGCGTCTCACGACCTCC-3′
Bcl-2	Sense	5′-CTCGTCGCTACCGTCGTGACTTCG-3′	238	94 °C for 50 s, 56 °C for 60 s, 72 °C for 60 s
	Antisense	5′-CAGATGCCGGTTCAGGTACTCAGTC-3′
IL-4	Sense	5′-TCAACCCCCAGCTAGTTGTC-3′	312	94 °C for 50 s, 51 °C for 60 s, 72 °C for 60 s
	Antisense	5′-ATCGAAAAGCCCGAAAGAGT-3′
c-jun	Sense	5′-TTTTGGAAGAGAGAACGGGA-3′	262	94 °C for 54 s, 49 °C for 60 s, 72 °C for 60 s
	Antisense	5′-ACTGAAAACACAAAACCGGC-3′
β-actin	Sense	5′-AGCCATGTACGTAGCCATCC-3′	227	94 °C for 54 s, 55 °C for 60 s, 72 °C for 60 s
	Antisense	5′-CTCTCAGCTGTGGTGGTGAA-3′

Figure 1. (A) Dose selection: The effect of varying concentrations of HEAM (50, 100, 200, 400 mg/kg body weight) and MNU on body weight of Balb/c mice were compared to control (PBS) during an 8-week period. A total of 100 mg/kg body weight dose was found suitable and was therefore retained for subsequent studies. (B) Effect of HEAM (100 mg/kg body weight) and MNU on body weight increase of balb/c mice Aegle Marmelos compared to control (PBS) for period of 28 weeks.

Figure 2. Cell viability assay (MTT) following HEAM treatment on human prostate cancer cell line (DU 145). Values are mean ± SEM. *p < 0.05 considered statistically significant.

Figure 3. (A) DNA fragmentation in the presence of MNU and HEAM (100 mg/kg b.w.) on agarose gel. (B) HEAM effect on DNA fragmentation: HEAM and MNU + Extract-treated samples show nuclear fragmentation (Lane1: N-Methyl N-nitrosourea; Lane 2: Phosphate buffer saline (PBS); Lane 3: HEAM extract; Lane 4: (MNU + HEAM).

Figure 4. (A) Effect of HEAM on the mRNA level of tumor related genes (p53, Bcl-2), inflammatory (IL-1β, IL-6 and c-jun) and anti-inflammatory (IL-4) cytokines. Representative photographs of ethidium-bromide-stained 2% agarose gels for each gene after 7 months of treatment. (B) Expression of tumor-related genes (p53, Bcl-2), inflammatory (IL-1β, IL-6 and c-jun) and anti-inflammatory (IL-4) cytokines (quantified in terms of fold change according to densitometric analysis). Each experiment was repeated three times with similar results and results are expressed as means ± SE, p < 0.05. Lane 1: MNU, Lane 2: HEAM (100 mg/kg b.w.), Lane 3: Control (PBS), Lane 4: MNU + HEAM (100 mg/kg b.w.).

Table 2. Whole blood cell counts in Group I (PBS), Group II (MNU), Group III (HEAM) and Group IV (MNU + HEAM).

Parameter	PBS (Group I)	MNU (Group II)	HEAM (Group III)	MNU + HEAM (Group IV)
Hb. (12–17) g/dl	11.72 ± 0.46^a	8.74 ± 0.47^c	11.43 ± 0.37^a	10.86 ± 0.49^b
Leucocyte (4–10) × 10^3/cmm	3.220 ± 1.87^c	6.730 ± 2.35^a	3.240 ± 1.50^c	4.010 ± 1.44^b
Platelets (1.5–4) lacs/mm	27 000 ± 2160^d	105 500 ± 6433^a	56 600 ± 1646^c	83 800 ± 2898^b

The superscripts are given to show statistical difference within the groups rather than to show level of significance.

Table 3. Leucocyte differential counts in Group I (PBS), Group II (MNU), Group III (HEAM) and Group IV (MNU + HEAM).

Parameters	PBS (Group I)	MNU (Group II)	HEAM (Group III)	MNU + HEAM (Group IV)
Lymphocytes	52.70 ± 2.21^a	30.80 ± 2.24^c	46.40 ± 2.37^b	41.20 ± 1.32^c
Neutrophils	46.4 ± 1.43^c	66.60 ± 1.43^a	46.4 ± 1.43^c	52.00 ± 1.83^b
Monocytes (%)	10	100	10	30
Eosinophils (%)	10	100	00	40

Data are presented as the mean ± SD (n = 10). Different superscripts (a, b, c, and d) with the mean values show the statistical differences between the mean values at p ≤ 0.05 level. One-way ANOVA followed by Duncan new multiple range test post-hoc comparison was performed for the studied parameters.

Figure 5. Raman spectra for all the experimental groups. Comparison of Raman laser spectra of DNA in the region 600–1600 cm⁻¹. These spectra are representative of experiments repeated eight times. Raman conditions and parameters: resolution cm⁻¹, laser power 5 mW exciting source was 514.5 nm Argon ion laser.

Table 4. Raman bands observed in the spectra of the experimental group treated DNA of liver of Balb/c mice and their tentative assignments with references of earlier reports.

	Raman spectra peak as per our experimental DNA sample and their assignments as per observations		Raman spectra peaks and their assignments as per earlier reports and references
Region of peak position (cm^−1) of Raman spectra	Peak position (cm^−1) of Raman spectra	Peak assignments for Raman spectra of liver DNA	Peak assignments of DNA Raman spectra as reported (References)	Peak position of DNA Raman spectra as reported
Region-1 (740–900)	806	O–P–O stretching	Prescott et al. (1984); Duguid et al. (1996)	807
	838	Phosphodiester stretching	Prescott et al. (1984); Duguid et al. (1996)	838
	898	Deoxyribose	Ruiz-chica et al. (2006)	895
Region-2 (900–1040)	938	Deoxyribose	Weizhong et al. (1999)	920
	970	Deoxyribose	Weizhong et al. (1999)	956
	996	Deoxyribose	Sipro (1987); Thomas & Tsuboi (1993)	1012
	1035	C=O stretching vibration	Thomas & Benevides (1985)	1032
Region-3 (1080–1190)	1152	Deoxyribose-phosphate	Weizhong et al. (1999)	1153
	1165	DNA backbone	Gao et al. (2005)	1167
Region-4 (1190–1280)	1216	O–P–O stretching	Yifu (1996)	1220
	1252	Adenine, Cytosine	Weizhong et al. (1999); Ruiz-chica et al. (2006)	1256
Region-5 (1280–1400)	1327	Adenine, Guanine	Ruiz-Chica et al. (2006)	1339
	1357	Adenine, Guanine	Ruiz-Chica et al. (2006)	1346
Region-6 (1400–1525)	1434	Deoxyribose	Ruiz-Chica et al. (2006)	1422
	1456	Deoxyribose	Weizhong et al. (1999)	1462

Figure 6. Overlay Raman spectra of DNA samples from Groups I–IV mice. Raman bands at 806, 838, 938, 970, 996, 1035, 1152, 1165, 1216, 1252, 1327, 1357, 1434 and 1456 cm⁻¹ are highlighted. The increase in intensity is observed in MNU (group II)-treated samples followed by MNU + Extract (Group IV)-treated samples while the control (PBS: group I) and Extract (group III)-treated samples had reduced peak intensities for the assigned bands. The major peak alteration is observed at 1252 cm⁻¹ and is characteristic of MNU-treated samples only.

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