Chemical analyses and in vitro and in vivo toxicity of fruit methanol extract of Sechium edule var. nigrum spinosum

Figures & data

Figure 1. In vitro effects of the S. edule var. nigrum spinosum extract on the proliferation of the P388 leukemia cell line and mononuclear bone marrow cells of healthy mice (MNCBMs). The cells were exposed to the S. edule var. nigrum spinosum extract, and the cellular proliferation was evaluated using a crystal violet assay. The data are presented as the means ± SDs (n = 4) and are representative of four independent experiments. The significance of the differences were determined by Tukey’s test (p ≤ 0.05).

Table 1. Percentage of viability and apoptosis of P388 and bone marrow mononuclear cells from mice.

	Viability (%)		Apoptosis (%)
	P388	MNCBM	P388	MNCBM
CONTROL	98.3 ± 5.8	95.9 ± 4.9	0.3 ± 0.2	40.4 ± 10.7
S.e. nigrum spinosum	39.2 ± 11.3*	88.5 ± 14.0	95.5 ± 4.2*	47.7 ± 8.7
ARA-C	24.6 ± 9.5*	36.1 ± 11.4*	99.1 ± 8.4*	77.0 ± 7.9*

The cells were treated in vitro with the IC₅₀ value of the S. edule var. nigrum spinosum extract. The cell viability was determined using the Trypan blue exclusion assay in relation to the untreated population. The degree of apoptosis was estimated through an Annexin-V assay, and the cells were analyzed with a FACS Aria II flow cytometer. The data are presented as the means ± SDs (n = 4) and are representative of four independent experiments.

* Indicates a significant difference (Tukey’s test, p ≤ 0.05).

Table 2. Spleen, liver and thymic indexes of healthy mice treated i.p. with the S.e. nigrum spinosum extract at a dose of 800 mg/kg.

Index	Splenic	Hepatic	Thymic
CONTROL	0.0048 ± 0.0004	0.0672 ± 0.0083	0.0013 ± 0.0002
S.e. nigrum spinosum	0.0059 ± 0.0011	0.0689 ± 0.0079	0.0012 ± 0.0002
ARA-C	0.0039 ± 0.0004*	0.0629 ± 0.0056	0.0009 ± 0.0002*

The organ indexes were determined as the ratio of the organ weights to the body weight. The data are represented as the means ± SDs and are representative of 20 mice per treatment.

* Indicates a significant difference (Tukey’s test, p ≤ 0.05).

Table 3. Blood biochemistry of healthy mice treated with the S.e. nigrum spinosum extract at a dose of 800 mg/kg.

	Control	S.e. nigrum spinosum	Ara-C
Glucose (mg/dL)	115.72 ± 16.6	72.27 ± 3.8*	106.33 ± 25.8
Cholesterol (mg/dL)	69.73 ± 13.4	66.07 ± 13.6	68.91 ± 10.9
Triglycerides (mg/dL)	79.27 ± 16.2	88.9 ± 13.6	86.6 ± 22.9
Uric acid (mg/dL)	1.8 ± 0.9	2.1 ± 0.7	3.7 ± 0.6*
Urea (mg/dL)	47.66 ± 6.7	45.81 ± 5.9	44.80 ± 2.0
Creatinine (mg/dL)	0.50 ± 0.1	0.48 ± 0.1	0.56 ± 0.2*
AST (U/L)	120.67 ± 18.6	128.33 ± 16.7	248.00 ± 30.8*
ALT (U/L)	59.00 ± 8.7	59.67 ± 13.5	58.00 ± 3.0
Total bilirubin (mg/dL)	0.35 ± 0.1	0.39 ± 0.1	0.23 ± 0.6*

Serum was used to determine the biochemical parameters of the blood using a clinical automatic biochemical analyzer. The aminotransaminase (AST) and alanine aminotransaminase (ALT) levels were determined. The data are presented as the means ± SDs and representative of 20 mice per treatment.

* Indicates a significant difference (Tukey’s test, p ≤ 0.05).

Table 4. Blood count of healthy mice treated with the S.e. nigrum spinosum extract at a dose of 800 mg/kg.

	CONTROL	S.e. nigrum spinosum	ARA-C
WBC (10^3/mm^3)	6.40 ± 1.40	10.53 ± 2.83*	6.96 ± 2.23
RBC (10^6/mm^3)	9.37 ± 0.83	9.82 ± 1.37	9.57 ± 1.28
Hb (g/dL)	13.87 ± 1.37	14.88 ± 1.02	16.05 ± 0.79
Hct (%)	47.79 ± 5.43	49.06 ± 4.55	50.08 ± 6.15
HCMC (g/dL)	31.43 ± 1.78	32.70 ± 2.17	29.35 ± 2.89
VCM (mm^3)	51.38 ± 3.17	51.15 ± 3.33	53.56 ± 3.46
HCM (pg)	16.34 ± 1.18	16.10 ± 1.44	15.84 ± 0.41
PLT (10^6/mm^3)	80.54 ± 19.54	89.93 ± 21.82	147.15 ± 35.71*

Plasma was used to analyze the total leukocytes (WCBs), total red blood cells (RCBs), hemoglobin (Hb), hematocrit (Hct), mean corpuscular hemoglobin (HCMC), mean corpuscular volume (MCV), platelets (PLTs) and differential counts of lymphocytes, granulocytes and monocytes with an automated hematology MICROS 60 instrument. The data are presented as means ± SDs and are representative of 20 mice per treatment.

* Indicates a significant difference (Tukey’s test, p ≤ 0.05).

Figure 2. Differential peripheral blood counts of healthy mice treated with the S. edule var. nigrum spinosum extract at a dose of 800 mg/kg. n = 20 mice per treatment. The data are presented as the means ± SDs; *indicates a significant difference (Tukey’s test, p ≤ 0.05).

Figure 3. Mitotic index (MI) of bone marrow cells from healthy mice treated with the S. edule var. nigrum spinosum extract at a dose of 800 mg/kg. n = 20 mice per treatment. The data are presented as the means ± SDs; *indicates a significant difference (Tukey’s test, p ≤ 0.05).

Figure 4. Representative high-performance liquid chromatography (HPLC) chromatogram of the S. edule var. nigrum spinosum extract: (A) phenolic acids, (B) cucurbitacins C and D, and (C) flavonoids.