Cytotoxicity effects of various Juglans regia (walnut) leaf extracts in human cancer cell lines

Figures & data

Table 1.  Contents of total phenolics, flavonoids and condensed tannins in J. regia leaf extracts.

Sample	Content (mg/g of dry leaf extract)
	Total Phenolics^a	Flavonoids^b	Condensed Tannins^c
Yong leaf total extract	267.30 ± 2.19	149.00 ± 2.55	950.56 ± 4.50
Mature leaf total extract	182.70 ± 2.60	96.89 ± 2.44	237.42 ± 2.20
Hexane fraction	27.39 ± 1.75	26.73 ± 1.72	37.07 ± 2.56
Chloroform fraction	40.25 ± 0.83	43.82 ± 1.35	52.24 ± 1.97
Ethyl acetate fraction	93.08 ± 2.47	53.51 ± 1.67	183.66 ± 3.18
Methanol fraction	120.28 ± 2.32	59.44 ± 0.87	227.00 ± 4.91

^aUnits: mg of gallic acid equiv/g of dry leaf extract.

^bUnits: mg of quercetin equiv/g of dry leaf extract.

^cUnits: mg of leukocyanidin equiv/g of dry leaf extract.

Figure 1.  Contents of total phenolics, flavonoids and condensed tannins in (A) young and mature leaf total extracts, and (B) hexane, chloroform, ethyl acetate and methanol fractions. Significant differences are indicated by *p< 0.05 in each group when total mature leaf extract was compared with young extract and various fractions were compared with methanol fraction. Values are presented as mean ± SE of three independent experiments.

Table 2A.  IC₅₀ values (mg of total extracts/mL) for antiproliferative activity of young and mature leaf extracts towards BHY, MCF7, and HT-29 cells. Values are presented as mean ± SE of three independent experiments, performed in triplicate.

Mature leaf total extract	Yong leaf total extract	Time (h)	Cells
>1.5	>1.5	24	BHY
1.23 ± 0.14	0.8 ± 0.02	48
>1.5	>1.5	24	MCF7
0.8 ± 0.01	0.83 ± 0.02	48
>1.5	>1.5	24	HT-29
>1.5	>1.5	48

BHY: breast adenocarcinoma; HT-29: colon adenocarcinoma; MCF7: human oral cancer.

Table 2B.  IC₅₀ values (mg of fractions/mL) for antiproliferative activity of different fractions towards BHY, MCF7, and HT-29 cells. Values are presented as mean ± SE of three independent experiments, performed in triplicate.

Cells	Time (h)	Methanol fraction	Ethylacetate fraction	Chloroform fraction	Hexane fraction
BHY	24	>1.5	1.41 ± 0.1	0.59 ± 0.02	>1.5
MCF7	48	>1.5	0.82 ± 0.07	0.45 ± 0.01	>1.5
	24	>1.5	1.06 ± 0.05	0.5 ± 0.03	>1.5
HT-29	48	>1.5	0.52 ± 0.02	0.36 ± 0.00	>1.5
	24	>1.5	1.49 ± 0.06	0.81 ± 0.01	>1.5
	48	>1.5	1.06 ± 0.06	0.53 ± 0.01	>1.5

BHY: breast adenocarcinoma; HT-29: colon adenocarcinoma; MCF7: human oral cancer.

Figure 2.  Evaluation of time effect on IC₅₀ values of methanol, ethyl acetate and chloroform fractions in (A) human oral cancer, (B) breast adenocarcinoma, and (C) colon adenocarcinoma cells. Antiproliferative activity of fractions was evaluated after 24 and 48 h treatments. Values are presented as mean ± SE of three independent experiments, performed in triplicate. Significant differences are indicated by *p< 0.05 in each group relative to 24 h samples.

Figure 3.  Concentration effectiveness of chloroform fraction on proliferation of mouse Swiss embryo fibroblast cells. Five different concentrations of this fraction (0.25–1.5 mg/mL) were applied for 48 h. Values are presented as mean ± SE of three independent experiments, performed in triplicate. Bars marked with * are significantly different compared with the control. (p < 0.05).

Table 3.  Effect of chloroform fraction on cell cycle progression with respect to control.a

Treatment	MCF7				BHY
	Sub-G1(mean±SE)	Go/G1(mean±SE)	S(mean±SE)	G2/M(mean±SE)	Sub-G1(mean±SE)	Go/G1(mean±SE)	S(mean±SE)	G2/M(mean±SE)
Control	8.25 ± 2.06	60.14 ± 1.87	24.93 ± 2.13	10.67 ± 1.08	11.9 ± 3.85	56.68 ± 2.95	27.33 ± 0.07	9.47 ± 1.41
0.25 mg/mL	32.97 ± 5.37	50.20 ± 1.02	15.56 ± 2.9	3.87 ± 0.93	31.95 ± 1.51	50.88 ± 1.44	17.25 ± 0.51	6.31 ± 0.68
0.5 mg/mL	53.81 ± 6.86*	32.69 ± 4.85*	12.83 ± 2.3	3.11 ± 0.93	47.35 ± 2.56*	39.64 ± 1.46*	12.01 ± 2.71	4.76 ± 0.74

^aAt the indicated cell, distribution of the cells in sub-G1, G_o/G1, S, and G2/M phase was analyzed by flow cytometry as described in “Materials and Methods”. Results are expressed as total cells. Data represent means of triplicate experiment.^*Significant (p < 0.05) relative to 0.25mg/mL.