Essential oil from fruit of Xylopia langsdorffiana: antitumour activity and toxicity

Figures & data

Table 1. Chemical composition of EOX.

Peak	Compound	(%) GC-MS	(%) GC-FID	RI exp.^a	RI lit.^b
1	α-Pinene	34.57	37.73	931	932
2	Camphene	11.50	12.82	947	946
3	β-Pinene	4.04	4.12	975	980
4	D-Limonene	31.75	31.42	1027	1024
5	1,8-Cineol	1.15	0.91	1031	1041
6	α-Terpineol	1.08	0.95	1194	1186
7	Spathulenol	1.74	1.59	1575	1577
8	Caryophyllene oxide	3.79	3.20	1580	1582
9	Sclarene	10.38	7.72	1967	1967
Total			100

^a RI exp, Retention Index Experimental: components identified based on IR and GC-MS and listed according to the elution order in DB-5 column (30 m).

^b RI lit, Retention Index Literature: components identified by the Kovats index in the literature.

Figure 1. Percentage of haemolysis in red blood cells of Swiss mice after treatment with EOX (μg/ml). Each dot represents the average ± SEM of three experiments with three replicates, with 95% CI.

Table 2. Antiproliferative activity of EOX and doxorubicin against human cancer cell lines^a.

	TGI (μg/ml)^b
Cell lines	DOX	EOX
Glioma (U251)	0.06	118.2
Breast (MCF-7)	0.20	102.1
Ovary multidrug resistance phenotype (NCI-ADR/RES)	1.30	45.4
Kidney (786-O)	0.04	107.3
Lung (NCI-H460)	0.01	191.9
Prostate (PC-3)	0.30	78.4
Ovary (OVCAR)	0.30	>250
Colon (HT-29)	0.20	118.8
Leukaemia (K562)	0.40	1.8
Skin (line of non-tumour cells) (HaCat)	0.20	74.5

^a Assessed by the SRB assay.

^b TGI values represent the necessary concentration (μg/ml) for total inhibition of cancer cells proliferation. Values were determined through non-linear regression analysis using the ORIGIN 8.0^® (OriginLab Corporation). Dose range tested: 0.25–250 μg/ml to EOX and 0.025–25 μg/ml to doxorubicin.

Table 3. Behavioural effects of acute intraperitoneal treatment of EOX in mice.

Dose (mg/kg)	Gender	D/T^a	Symptoms
–	Male	0/6	None
	Female	0/6	None
250	Male	0/6	Aggressivity
	Female	0/6	Hyperactivity, decreased touch response
375	Male	3/6	Hyperactivity, loss of corneal reflex, loss of ear reflex, decreased ambulation, catatonia
	Female	4/6	Hyperactivity, decreased touch response, catatonia, loss of corneal reflex, loss of ear reflex
500	Male	5/6	Hyperactivity, aggressivity, loss of corneal reflex, loss of ear reflex, sedation, increased urination, forced breathing
	Female	6/6	Hyperactivity, ptosis, sedation, anaesthesia, analgesia, loss of corneal reflex, loss of ear reflex

^a D/T, the number of mice dead/number of mice treated.

Table 4. Effect of intraperitoneal administration of EOX (250, 375 e 500 mg/kg) on feed and water consumption and body weight gain in mice (14 d).

Group	Gender	Dose (mg/kg)	Water consumption (ml)	Feed consumption (g)	Body weight gain (g)
Control	Male	–	55.3 ± 1.8	35.96 ± 0.7	9.70 ± 0.5
	Female	–	57.3 ± 1.2	38.29 ± 0.5	11.50 ± 1.1
EOX	Male	250	59.2 ± 2.0	38.38 ± 1.8	6.81 ± 1.1
	Female		58.0 ± 2.5	34.92 ± 1.2	2.98 ± 0.8^a
EOX	Male	375	33.75 ± 1.6^a	18.19 ± 0.8^a	4.73 ± 0.9^a
	Female		24.1 ± 0.8^a	13.03 ± 0.8^a	2.55 ± 0.7^a

Data presented as mean ± SEM of six animals analyzed by ANOVA followed by Tukey test.

^a p <.05 compared to tumour control.

Table 5. Number of micronucleated erythrocytes in peripheral blood of mice treated with single doses of EOX and cyclophosphamide (n = 6).

Group	Dose (mg/kg)	Number of micronucleated cells
Control	–	4.3 ± 0.6
Cyclophosphamide	50	26.0 ± 5.0^a
EOX	88	2.8 ± 0.3
EOX	176	4.3 ± 0.2
EOX	281	3.5 ± 0.4

Data presented as SEM of the mean of six animals analyzed by ANOVA followed by Tukey test.

^a p < .05 compared to tumour control.

Table 6. Feed and water consumption and body weight gain of animals (n = 6) subjected to different treatments (7 d).

Group	Dose (mg/kg)	Water consumption (ml)	Feed consumption (g)	Body weight gain (g)
Healthy animals	–	35.0 ± 1.0	31.7 ± 0.8	3.4 ± 0.5
Tumour control	–	35.3 ± 1.6	26.7 ± 0.6^c	8.9 ± 1.0^c
5-FU	25	43.8 ± 3.5	26.9 ± 1.3^c	0.03 ± 1.1^a
EOX	50	38.9 ± 3.2	23.4 ± 1.1^c	6.7 ± 0.7^b^,^c
EOX	100	30.7 ± 2.7^b	19.9 ± 1.7^a^,^b^,^c	1.9 ± 0.5^a

Data presented as mean ± SEM of six animals analyzed by ANOVA followed by Tukey test.

^a p < .05 compared to tumour control.

^b p < .05 compared to the 5-FU group.

^c p < .05 compared to healthy animals.

Table 7. Effects of 5-FU and EOX on the mice organ indices (n = 6) subjected to different treatments (7 d).

Group	Dose (mg/kg)	Heart index (mg/g)	Liver index (mg/g)	Kidney index (mg/g)	Thymus index (mg/g)	Spleen index (mg/g)
Healthy animals	–	3.6 ± 0.09	56.3 ± 0.4	10.6 ± 0.1	1.9 ± 0.1	5.2 ± 0.2
Tumour control	–	3.8 ± 0.06	53.7 ± 1.9	10.1 ± 0.4	1.9 ± 0.1	6.8 ± 0.3
5-FU	25	3.2 ± 0.1	49.2 ± 1.8	10.3 ± 0.2	1.08 ± 0.1^a^,^c	3.3 ± 0.2^a
EOX	50	3.7 ± 0.2	69.2 ± 1.7^a^,^b^,^c	11.5 ± 0.2	2.3 ± 0.2^b	9.7 ± 0.7^a^,^b^,^c
EOX	100	3.8 ± 0.2	51.2 ± 3.8	11.3 ± 0.9	2.10 ± 0.2^b	7.6 ± 0.5^b^,^c

Data presented as mean ± SEM of six animals analyzed by ANOVA followed by Tukey test.

^a p < .05 compared to tumour control.

^b p < .05 compared to the 5-FU group.

^c p < .05 compared to healthy animals.

Figure 2. Effect of EOX and 5-FU on tumour weight and inhibition of tumour growth in mice transplanted with sarcoma 180. Data are expressed as mean ± SEM of six animals and were analyzed by ANOVA followed by Tukey’s test. ^ap < .05 compared to tumour control. ^bp < .05 compared to EOX (50 mg/kg). ^cp < .05 compared to EOX (100 mg/kg).

Figure 3. Histopathology of tumours of different experimental groups. (A) Tumour control: asymmetric mitoses. (B) 5-FU (25 mg/kg): coagulative necrosis areas. (C) EOX (50 mg/kg): invasion of adipose tissue (arrows). (D) EOX (50 mg/kg): invasion of skeletal muscle (arrows). (E) EOX (100 mg/kg): tumour growth advancing neural filament, without nerve infiltration (arrows). (F) EOX (100 mg/kg): coagulative necrosis areas (arrows). Haematoxylin-eosin (A, C, D, F) and Masson’s trichrome (B, E).

Table 8. Effects of 5-FU and EOX on biochemical and haematological parameters of peripheral blood of mice (n = 6) subjected to different treatments (7 d).

				EOX
Parameter	Healthy animals	Tumour control	5-FU 25 mg/kg	50 mg/kg	100 mg/kg
AST (U/L)	264.0 ± 22.9	336.7 ± 30.4	237.3 ± 23.3	382.4 ± 30.7^b	324.2 ± 32.8
ALT (U/L)	63.4 ± 2.7	62.0 ± 5.2	44.0 ± 12.0	70.0 ± 6.0	74.8 ± 4.2^b
Urea (mg/dl)	41.2 ± 2.2	43.6 ± 4.5	36.5 ± 1.6	39.7 ± 2.7	36.0 ± 3.3
Creatinine (mg/dl)	0.3 ± 0.04	0.4 ± 0.05	0.4 ± 0.03	0.4 ± 0.04	0.6 ± 0.05
Red blood cells (10^6/mm^3)	9.9 ± 0.2^a	6.8 ± 1.4^c	12.5 ± 0.4^a	7.0 ± 0.7^b^,^c	10.9 ± 0.2^a
Haemoglobin (g/dl)	14.5 ± 0.3^b	13.2 ± 0.4^b	15.6 ± 0.4^a^,^c	13.3 ± 0.3^b	13.9 ± 0.1^b
Haematocrit (%)	42.9 ± 0.6^b	45.4 ± 1.2^b	52.7 ± 1.5^a^,^c	48.3 ± 1.6^b^,^c	46.6 ± 0.5^b
MCV (fm^3)	44.0 ± 0.9	44.0 ± 0.6	42.5 ± 0.6	61.7 ± 2.0^a^,^b^,^c	42.8 ± 0.5
MCH (pg)	14.7 ± 0.3	15.6 ± 1.1	12.6 ± 0.2^a	17.5 ± 0.2^b^,^c	12.7 ± 0.2^a
MCHC (g/dl)	29.4 ± 0.1	29.5 ± 0.7	29.6 ± 0.2	28.5 ± 1.0	29.9 ± 0.3
Total leukocytes (10^3/mm^3)	8.0 ± 0.7^a^,^b	14.5 ± 1.1^b^,^c	2.0 ± 0.4^a^,^c	9.5 ± 1.9^a^,^b	15.8 ± 0.4^b^,^c
Lymphocytes	76.0 ± 1.5^a^,^b	40.6 ± 2.4^b^,^c	92.7 ± 1.5^a^,^c	49.4 ± 2.9^a^,^b^,^c	33.6 ± 2.0^b^,^c
Segmented neutrophils	27.8 ± 1.7^a^,^b	45.5 ± 4.5^b^,^c	6.2 ± 1.6^a^,^c	49.8 ± 3.3^b^,^c	64.8 ± 3.2^a^,^b^,^c
Monocytes	1.3 ± 0.2^a	5.0 ± 0.7^c	1.8 ± 0.3^a	4.2 ± 1.0^c	4.3 ± 0.8^c
Eosinophils	0.0 ± 0.0	1.0 ± 0.0	0.0 ± 0.0	1.0 ± 0.0	0.0 ± 0.0

Data presented as mean ± SEM of six animals analyzed by ANOVA followed by Tukey test.

^a p < .05 compared to tumour control.

^b p < .05 compared to the 5-FU group.

^c p < .05 compared to healthy animals.

Figure 4. Histopathology of livers of different experimental groups. (A) Tumour control: normal portal triad, liver beams radially distributed from vein centre lobular, and presence of hepatic sinusoids. (B) 5-FU (25 mg/kg): microvesicular steatosis (arrows). (C) EOX (50 mg/kg): lobular architecture preserved, especially the zonal distribution of hepatocytes (zones I, II and III). Presence of mild steatosis. (D) EOX (50 mg/kg): Kupffer cell intrasinusoidal hypertrophy. (E) EOX (50 mg/kg): discreet inflammatory infiltrate on space port. (F) EOX (100 mg/kg): macrovesicular steatosis (arrow), the presence of THV-terminal hepatic vein. (G) EOX (100 mg/kg): necroinflammatory focus (short arrow) bordering dividing hepatocyte (thick arrow). (H) EOX (100 mg/kg): portal inflammatory infiltrate. PVB, portal vein branch; THV, terminal hepatic vein; BD, bile duct. Haematoxylin-eosin (A, B, C, E, F) and Masson’s trichrome (D, G, H).