Analysis of the anti-inflammatory and chemopreventive potential and description of the antimutagenic mode of action of the Annona crassiflora methanolic extract

Figures & data

Figure 1. Treatments to evaluate the mutagenicity and antimutagenicity of A. crassiflora methanolic extract (ACME). Groups: negative control = distilled water 120 h; MMS = aqueous solution of methyl methanesulfonate; pre-treatment (48 h ACME + 48 h MMS), simultaneous simple (48 h distilled water + 48 h of association of ACME and MMS), simultaneous with pre-incubation (48 h distilled water + 48 h MMS with ACME pre-incubated for 1 h at 37 °C in the oven), post-treatment (48 h MMS + 48 h ACME), continuous (48 h ACME + 48 h MMS) without washing the seeds.

Figure 2. Experimental design in vivo.

Figure 3. Effect of oral administration of ACME on carrageenan-induced paw edema in mice. Animal received ACME (30, 100, or 300 mg/kg, p.o.), dexamethasone (DEX – 1 mg/kg, s.c.) or vehicle and after 1 h, an intraplantar injection of carrageenan (300 µg/paw) was performed. In (A), the inhibition induced by 30, 100, and 300 mg/kg of ACME and DEX in paw edema (µm) at 4 h after carrageenan injection is shown. In (B), bars show the effect of different doses of 30, 100, and 300 mg/kg of ACME and DEX in increasing of myeloperoxidase (MPO) activity at 6 h after carrageenan injection is shown ACME and DEX. The bars express the mean ± SE of five animals, compared with the vehicle (V) versus the treated group. *p < 0.05, **p < 0.01, one-way ANOVA followed by the Student–Newman–Keuls test.

Figure 4. Effects of ACME on total leukocytes (A) and protein extravasation (B) induced by carrageenan in the pleural cavity of mice. Animal received the oral treatment with ACME (100 or 300 mg/kg), or vehicle, and after 1 h, they received an intrapleural injection of Cg (100 μL of a 1% solution/cavity). Control animals received only the vehicles and Naïve animal did not received carrageenan or ACME treatment. Animals were killed after Cg injection. The bars express the mean ± SEM of five animals, compared with the vehicle (V) versus the treated group. **p < 0.01, ***p < 0.001, one-way ANOVA followed by the Student–Newman–Keuls test. #p < 0.001.

Figure 5. Effect of ACME on carrageenan-induced leukocyte migration and plasma leakage into the air pouch. Mice (n = 5) were pretreated 1 h before with ACME (10–300 mg/kg p.o.), dexamethasone (1 mg/kg, s.c., diluted in saline) or vehicle. Pouches were washed with PBS-containing heparin. Cells were counted and plasma leakage was analyzed. Results are expressed as cell 10⁶/cavity. **p < 0.01, ***p < 0.001, #p < 0.001, compared with the vehicle group. Difference between groups were analyzed by analysis of variance (one-way ANOVA) followed by the Newman–Keuls test.

Table 1. Distribution of chromosomal aberrations, MI, MIDR, and DR% in Allium cepa treatments associated to A. crassiflora methanolic extract.

			Chromosomal aberrations
Treatment	DRMI%	MI	MN	BRI	BRE	LO	SPR	DLA	MA	Total	DR%
Mutagenicity
Control	–	3.70	19	17	0	0	0	4	0	40	–
MMS	3.51	3.57	204	16	2	5	2	9	0	238^a	–
A. crassiflora 5 mg/L	−1.62	3.76	13	11	0	1	0	6	0	31^a	–
A. crassiflora 10 mg/L	−11.35	4.12	14	10	0	0	0	2	1	27^a	–
A. crassiflora 15 mg/L	−29.46	4.79	24	8	1	1	0	0	0	34^a	–
Antimutagenicity
5 mg/L
Pre-treatment	−31.35	4.86	322	24	6	4	1	12	0	369^b*	−66.17
Simple simultaneous	−44.86	5.36	63	20	1	4	1	5	0	94^b*	72.72
Simultaneous with pre-incubation	−2.43	3.79	21	14	0	0	0	4	0	39^b*	100.5
Post-treatment	−58.38	5.86	37	20	0	0	0	4	0	61^b*	89.39
Continuous	−37.03	5.07	36	18	0	1	0	13	0	68^b*	86.36
10 mg/L
Pre-treatment	−64.05	6.07	52	26	0	1	1	8	0	82^b*	75.75
Simple simultaneous	−15.95	4.29	151	15	3	1	0	2	0	172^b*	33.33
Simultaneous with pre-incubation	−35.68	5.02	19	26	0	2	0	5	0	52^b*	93.93
Post-treatment	−21.35	4.49	58	14	0	1	1	6	0	80^b*	79.79
Continuous	−35.95	5.03	47	22	0	1	0	7	0	77^b*	81.31
15 mg/L
Pre-treatment	45.95	2.00	82	4	7	2	3	3	0	101 ^b*	69.19
Simple simultaneous	−23.78	4.58	148	33	2	3	2	6	0	194^b*	22.22
Simultaneous with pre-incubation	−16.49	4.31	10	22	0	0	1	2	0	35^b*	102.52
Post-treatment	−36.76	5.06	38	26	0	0	3	4	0	71^b*	84.34
Continuous	−44.59	5.35	33	15	1	4	0	0	0	53^b*	93.43

DRMI%, damage reduction percentages of the mitotic index; MI, mitotic index; DR%, damage reduction percentages; MN, micronucleus; BRI, bridge; BRE, break; LO, loss; SPR, sprout; DLA, delay; MAN, multipolar anaphase. MMS, methyl methanesulfonate. Experimental groups: control = distilled water 96 h; MMS = aqueous solution of MMS; pre-treatment (48 h A. crassiflora + 48 h MMS), simultaneous simple (48 h distilled water + 48 h of association of A. crassiflora and MMS), simultaneous with pre-incubation (48 h distilled water + 48 h MMS with A. crassiflora pre-incubated for 1 h at 37 °C in the oven), post-treatment (48 h MMS + 48 h A. crassiflora), continuous (48 h A. crassiflora + 48 h MMS without change the seeds). *Statistically significant differences (Chi-square test, p < 0.05).

^aValues compared with the control group.

^bValues compared with the MMS group.

Table 2. Initial, final, and body weight gain of the animals; absolute and relative weights of the organs of animals treated with A. crassiflora methanolic extract and/or cyclophosphamide (mean ± S.E.).

	Experimental groups
Parameters	Control	Cycplophosphamide	ACME	Pre-treatment	Simultaneous	Post-treatment
Initial (g)	32.49 ± 1.59^a	31.43 ± 2.22^a	28.91 ± 1.46^a	28.17 ± 1.59^a	29.09 ± 0.91^a	28.13 ± 1.53^a
Final (g)	32.44 ± 2.56^a	31.56 ± 1.46^a	28.79 ± 1.29^a	27.68 ± 1.57^a	28.97 ± 0.58^a	27.50 ± 1.21^a
Liquid weight gain (g)	1.60 ± 0.86^a	0.52 ± 2.15^a	0.12 ± 0.59^a	0.49 ± 2.11^a	0.11 ± 0.69^a	0.63 ± 0.40^a
Absolut weight of organs (g)
Lungs	0.1939 ± 0.0127^a	0.2175 ± 0.0284^a	0.1762 ± 0.0135^a	0.1864 ± 0.0078^a	0.1862 ± 0.0140^a	0.2057 ± 0.0098^a
Heart	0.1842 ± 0.0098^a	0.1859 ± 0.0161^a	0.1695 ± 0.0129^a	0.1426 ± 0.0093^a	0.1826 ± 0.0142^a	0.1472 ± 0.0070^a
Liver	1.6591 ± 0.1489^a	1.6818 ± 0.1410^a	1.3960 ± 0.0689^a	1.3633 ± 0.0736^a	1.6277 ± 0.0997^a	1.3961 ± 0.0586^a
Spleen	0.1644 ± 0.0290^a	0.1148 ± 0.0141^a	0.1325 ± 0.0079^a	0.1051 ± 0.0112^a	0.1697 ± 0.0629^a	0.0889 ± 0.0034^a
Kidneys	0.3827 ± 0.0150^a	0.3602 ± 0.0258^a	0.3351 ± 0.0134^a	0.3310 ± 0.0196^a	0.3307 ± 0.0148^a	0.3223 ± 0.0114^a
Relative weight of organs (g)
Lungs	0.0061 ± 0.0006^a	0.0068 ± 0.0006^a	0.0060 ± 0.0003^a	0.0068 ± 0.0003^a	0.0064 ± 0.0004^a	0.0076 ± 0.0004^a
Heart	0.0058 ± 0.0003^a	0.0058 ± 0.0003^a	0.0058 ± 0.0002^a	0.0051 ± 0.0002^a	0.0063 ± 0.0004^a	0.0054 ± 0.0002^a
Liver	0.0513 ± 0.0025^a	0.0528 ± 0.0022^a	0.0479 ± 0.0016^a	0.0493 ± 0.0008^a	0.0561 ± 0.0031^a	0.0508 ± 0.0013^a
Spleen	0.0050 ± 0.0008^a	0.0035 ± 0.0003^a	0.0045 ± 0.0002^a	0.0038 ± 0.0002^a	0.0060 ± 0.0024^a	0.0033 ± 0.0002^a
Kidneys	0.0120 ± 0.0005^a	0.0114 ± 0.0004^a	0.0114 ± 0.0004^a	0.0120 ± 0.0002^a	0.0114 ± 0.0004^a	0.0118 ± 0.0003^a

ACME Annona crassiflora methanolic extract; g, grams.

^aValues compared with the control group (Chi-square test, p < 0.05).

Table 3. Total frequency, frequency mean ± S.E., percentage of damage reduction related to tests for mutagenicity and antimutagenicity in micronucleus test in peripheral blood of Swiss female mice.

Experimentals groups	MN frequency	Mean ± SE	DR%
Mutagenicity
Control	38	7.60 ± 1.03^a	–
Cyclophosphamide	134	26.80 ± 1.83^b	–
ACME	55	11.00 ± 1.38^a	–
Antimutagenicity
Pre-treatment	62	12.40 ± 1.50^a	75.00
Simultaneous	72	14.40 ± 0.98^a	64.58
Post-treatment	178	35.60 ± 2.34^c	−45.83

MN, micronucleus. S.E., standard error of mean. DR%, percentage of damage reduction. Different letters mean significant differences (Chi-square test, p < 0.05).