Anti-inflammatory effects of the extracts from Hylocereus polyrhizus fruit peel on primary peritoneal cells from BABL/c mice

Fang-Yu Tu 1, Hui-Hsiang Chang 2, Ting-Hsi Chang1, Po-Chuen Shieh 1*

1. Department of Pharmacy and Graduate Institute of PharmaceuticalTechnology, Tajen University, Taiwan

2. Department of Biotechnology, Tajen University, Taiwan 

Pitaya ( （ Hylocereus undatus (Haw.) Britton et Rose ） ) or dragon fruit is

native to Central and South America and has been grown in Taiwan for at least 20

years. A member of the Cactaceae, it has trailing cladode stems modified to act as

leaves and bears spectacular ovoid fruit year-round which has a bright red color

when mature, and contains white, crimson, or pale yellow flesh, depending on the

cultivar, interspersed with small black seeds. Hylocereus polyrhizus, a red pitaya

with red flesh, is widely grown in Taiwan. Pitaya fruit has been reported as a

source of beta-carotene, lycopene and vitamin E. The recent studies showed that

betacyanin, a major component of betalains, which rich in Hylocereus polyrhizus

fruit peels exhibited higher antioxidant activities. The aims of the present study

were to investigate the anti-inflammatory compounds and mechanisms of

Hylocereus polyrhizus fruit peels (HPFP) which extracted by water, 50% or 95%

ethanol solution on primary peritoneal cells from BABL/c mice.

1. Materials and experimental animals a. Samples

The fresh Hylocereus polyrhizus fruit peels (HPFP) were respectively

extracted by water, 50% or 95% ethanol solution, and dried by freeze dryer.

b. TCM medium

The medium contained 20% of TCM Serum Replacement and 0.5% of

Penicillin-Streptomycin Amphotericin B Solution in RPMI 1640 medium.

c. LPS (lipolpolysaccharid)

d. Animals

Female BALB/c ByJNarl mice (7 weeks old) were obtained from the National

Laboratory Animal Center, National Applied Research Laboratories, National

Science Council in Taipei. The animal room was maintained a 12-h light

and 12-h dark cycle. Constant temperature (25 ± 2oC) and humidity were

maintained. The mice were housed and fed with a chow diet to acclimatize for

one week before experimental beginning. Mice were anesthetized with diethyl

ether, exsanguinated using retro-orbital venous plexus puncture and

immediately euthanized with CO2 inhalation.

2. Cell cultures

After the mice were euthanized, peritoneal cells were collected and suspended

in TCM medium. Total cells were counted with a hemocytometer using the

trypan blue dye exclusion method and the cell density was adjusted to 2× 106

cells/ml in TCM medium. The cell suspensions were plated into the wells of

48-well plates with sample solution (from 31.25 to 2000 μg/ml) and incubated

without or with LPS, respectively. The final concentration of LPS presented in

wells was 2.5 μg/ml. The plates were incubated at 37 in a humidified ℃ incubator with 5% CO2 and 95% air for 48 h. The supernatants of cell cultures

were collected and stored at -80 ℃ for cytokine assays.

3. Measurement of cytokine levels in supernatants of cell cultures by an ELISA

The levels of pro-inflammatory cytokine (interleukin (IL)-6) and anti-

inflammatory cytokine (IL-10) were respectively determined according to the

cytokine ELISA protocol of manufacturer’s instructions. The sensitivity of these

cytokine assays was < 15.6 pg/ml.

4. Statistical analysis

Data were expressed as mean ± SD. Data were analyzed using analysis of

variance (ANOVA), followed by Student’s t-test. Differences among the

experimental groups were considered statistically significant if P < 0.05.

. .

Introduction

Materials and Methods

Results

Conclusions

Table 1 Effects of Hylocereus polyrhizus fruit peel extracts on the pro-inflammatory cytokine IL-6 secretion by peritoneal cell from BALB/c mice

IL-6 (ng/ml)Sample(μg/ml)

31.25 62.5 125 250 500 1000 2000

spontaneous

WEHPFP 1.20 ± 0.36 1.31 ± 0.41 1.45 ± 0.49 1.91 ± 0.57 2.45 ± 0.49* 3.86 ± 1.43* 6.13 ± 1.67*

50%EHPFP 1.29 ± 0.51 1.16 ± 0.45 1.16 ± 0.41 1.31 ± 0.52 1.42 ± 0.34 1.60 ± 0.18 1.75 ± 0.45

95%EHPFP 1.13 ± 0.31 1.05 ± 0.31 1.13 ± 0.38 1.23 ± 0.41 1.85 ± 0.71 1.53 ± 0.39 2.02 ± 0.43

Control 1.36 ± 0.33

LPS-stimulation

WEHPFP 8.30 ± 1.46 7.41 ± 1.53 7.86 ± 1.39 8.11 ± 1.00 8.64 ± 1.10 8.98 ± 1.13 11.50 ± 1.11*

50%EHPFP 8.53 ± 0.86 7.84 ± 0.39 8.06 ± 0.92 7.98 ± 1.10 8.34 ± 1.17 8.63 ± 0.90 12.02 ± 0.76*

95%EHPFP 7.66 ± 1.40 7.32 ± 0.74 7.62 ± 0.82 7.58 ± 0.22 7.78 ± 0.35 8.39 ± 0.82 10.49 ± 1.12

Control 8.75 ± 1.18

Data are presented as mean ± SD (n = 3). Data within the same column are analyzed using analysis of variance (ANOVA), followed by Student’s t-test. Asterisk ( ＊ ) means significantly (p ＜ 0.05) different from the control group in same treatment. The cell density was 1 × 106 cells/ml.. WEHPFP, 50% EHPFP and 95% EHPFP are represented the extracts of Hylocereus polyrhizus fruit peel by water, 50% and 95% ethanol solution, respectively.

Spontaneous

IL-6

/IL

-10 (

pg/p

g)

Sample concentration (g/ml)

31.25 62.5 125 250 500 1000 2000 control

0

2

4

6

8

10

12

14

WEPSPL 50%AEPSPL 95%AEPSPL control

*

*

*

*

LPS - stimulation

IL-6

/IL

-10 (

pg/p

g)

Sample concentration (g/ml)

31.25 62.5 125 250 500 1000 2000 control

0

1

2

3

4

5WEPSPL 50%AEPSPL 95%AEPSPL control

Table 2 Effects of Hylocereus polyrhizus fruit peel extracts on the anti-inflammatory cytokine IL-10 secretion by peritoneal cell from BALB/c mice

IL-10 (pg/ml)Sample(μg/ml)

31.25 62.5 125 250 500 1000 2000

spontaneous

WEHPFP 343 ± 173 361 ± 193 371 ± 195 479 ± 260 493 ± 256 587 ± 285 850 ± 342

50%EHPFP 307 ± 102 249 ± 111 290 ± 123 330 ± 148 470 ± 279 422 ± 180 571 ± 194

95%EHPFP 271 ± 98 292 ± 125 372 ± 187 369 ± 174 512 ± 233 543 ± 225 804 ± 244 *

Control 311 ± 135

LPS-stimulation

WEHPFP 3706 ± 683 3673 ± 1170 3614 ± 945 3812 ± 1220 3816 ± 861 4214 ± 1052 4279 ± 1182

50%EHPFP 3562 ± 899 3508 ± 1038 3766 ± 966 3835 ± 965 4360 ± 1392 4358 ± 1327 3900 ± 1157

95%EHPFP 3323 ± 990 3187 ± 1082 3606 ± 1300 3833 ± 963 4104 ± 1483 4220 ± 1227 4233 ± 881

Control 3545 ± 668

Data are presented as mean ± SD (n = 3). Data within the same column are analyzed using analysis of variance (ANOVA), followed by Student’s t-test. Asterisk ( ＊ ) means significantly (p ＜ 0.05) different from the control group in same treatment. The cell density was 1× 106 cells/ml. WEHPFP, 50% EHPFP and 95%EHPFP are represented the extracts of Hylocereus polyrhizus fruit peel by water, 50% and 95% ethanol solution, respectively.

Fig.1 Effects of Hylocereus polyrhizus fruit peel extracts on the pro-/anti-inflammatory cytokine secretion ratios (IL-6/IL-10) by peritoneal cells from BALB/c mice. Data are presented as mean ± SD (n = 3) and analyzed using analysis of variance (ANOVA), followed by Student’s t-test. Asterisk ( ＊ ) means significantly (P ＜ 0.05) different from the control group. The cell density was 2 × 106 cells/ml. WEHPFP, 50% EHPFP and 95% EHPFPare represented the extracts of Hylocereus polyrhizus fruit peel by water, 50% and 95% ethanol solution, respectively.

The results showed that the water extract ( ＞ 500 μg/ml) significantly increased the pro-inflammatory cytokine (IL-6) level and 95% ethanol extract (2000 μg/ml) markedlyincreased the anti-inflammatory cytokine (IL-10) level in the spontaneous peritoneal cellcultures. Furthermore, the water and 50% ethanol extract (2000 μg/ml) also significantlyincreased the IL-6 level in the lipopolysaccharide (LPS)-stimulated cultures, the peelextracts did not significantly affect the cytokine ratios of IL-10/IL-6 secreted by peritoneal cells in vitro. It is suggested that the Hylocereus polyrhizus fruit peel extracts could not play an anti- inflammatory role.