Effect of kumquat (Fortunella crassifolia) pericarp on natural killer cell activity in vitro and in vivo

Figures & data

Fig. 1. Fractionation of KP.

Fig. 2. NK cell activation effects of KP fractions in KHYG-1 cells.

Notes: Cells were treated in the presence or absence of KP fractions (50 μg/mL) at 24 h and IFN-γ production in culture supernatant was measured by ELISA. The relative IFN-γ production is presented as the mean ± standard error (n = 6). The data obtained were statistically analyzed using one way ANOVA followed by Dunnett’s test for individual comparison of groups with control. *p < 0.05 and ***p < 0.001.

Fig. 3. NK cell activation effects of KP-AF in KHYG-1 cells.

Notes: Cells were treated in the presence or absence of unheated and heated KP-AF (50 μg/mL) at 24 h. (a) Relative IFN-γ production in culture supernatant. (b) NK cytotoxic activity. The relative IFN-γ production and specific lysis ratio are presented as the mean ± standard error (n = 5 or 6). Multiple comparisons were performed using one-way ANOVA followed by Tukey–Kramer as a post hoc test. *p < 0.05 and ***p < 0.001.

Fig. 4. Effect of IFN-γ production enhancement by carotenoids and other constituents on KHYG-1 cells.

Notes: Cells were treated in the presence or absence of chrysin (50 μg/mL), auraptene (50 μg/mL), β-cryptoxanthin (25 μg/mL), and d-limonene (1 and 10 μg/mL) at 24 h. (a) Relative IFN-γ production in culture supernatant by chrysin, auraptene, β-cryptoxanthin, and (b) d-limonene. The relative IFN-γ productions are presented as the mean ± standard error (n = 6). The data obtained were statistically analyzed using one way ANOVA followed by Dunnett’s test for individual comparison of groups with control. *p < 0.05 and ***p < 0.001.

Fig. 5. NK cell activation effects of carotenoids in KHYG-1 cells.

Notes: Cells were treated in the presence or absence of β-cryptoxanthin, β-carotene, lutein, and zeaxanthin at 24 h. (a) Relative IFN-γ production in culture supernatant. (b) NK cytotoxic activity by β-cryptoxanthin (25 μg/mL). The relative IFN-γ production and specific lysis ratio are presented as the mean ± standard error (n = 5 or 6). Statistically significant differences were determined by one way ANOVA followed by Dunnett’s test for individual comparison of groups with control (a) or Student’s t-test. (b) ***p < 0.001.

Table 1. Carotenoids contents in unheated and heated KP-AF.

	Carotenoids contents (μg/g^a)^b
	Unheated KP-AF	Heated KP-AF
β-carotene	56.5 ± 10.1	55.1 ± 34.8
β-cryptoxanthin	98.2 ± 7.1	91.5 ± 62.5
Lutein	19.8 ± 14.1	12.1 ± 2.4
Zeaxanthin	10.1 ± 7.2	6.1 ± 2.0
Total carotenoids^c	184.6 ± 24.7	164.8 ± 101.0
	Percent of carotenoids per total carotenoids (%)
	Unheated KP-AF	Heated KP-AF
β-carotene	30.7 ± 4.6	33.4 ± 1.2
β-cryptoxanthin	53.9 ± 5.6	50.1 ± 11.2
Lutein	10.2 ± 6.1	11.3 ± 7.7
Zeaxanthin	5.2 ± 3.1	5.2 ± 2.9

^a Expressed as microgram per gram of KP-AF.

^b Data are presented as mean ± standard deviation (n = 3).

^c A total of four carotenoids.

Table 2. Effect of KP-AF on spleen weight, spleen index, and spleen lymphocyte number in restraint-stressed mice.

Group	Spleen weight (mg)	Spleen index (%)	Spleen lymphocyte number (10^7)
Normal (n = 10)	73.67 ± 4.58^a	0.30 ± 0.02^a	8.4 ± 0.7^a
Model (Restraint) (n = 15)	50.73 ± 2.06^b	0.25 ± 0.01^b	3.3 ± 0.2^b
Restraint + KP-AF (0.6 mg/kg) (n = 10)	46.91 ± 1.86^b	0.24 ± 0.01^b	3.6 ± 0.3^b

Notes:

The results represent mean ± standard error. Different alphabet letters in same row indicate the statistical significance at p < 0.01 (ANOVA followed by Tukey–Kramer’s test).

Fig. 6. Effect of KP-AF on plasma corticosterone levels, IFN-γ level and NK cell cytotoxicity of restraint-stressed mice.

Notes: (a) Plasma corticosterone levels were determined by a quantitative competitive ELISA. (b) Plasma IFN-γ levels were determined using an ELISA. (c) and (d) In the determination of NK cell cytotoxic activity, calcein AM-labeled YAC-1 (target) cells were mixed with spleen lymphocytes and incubated for 4 h. After incubation, NK cell activity was determined using an calcein-AM-release assay. (c) NK cytotoxic activity per splenocyte (LU₁₀/10⁶ cells). (d) NK cytotoxic activity per spleen (LU₁₀/spleen). The results data are presented as the mean ± standard error (n = 10 or 15). Multiple comparisons were performed using one-way ANOVA followed by Tukey–Kramer as a post hoc test. ***p < 0.001.