1. 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 Chang 1, Po-Chuen Shieh 1* 1. Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, 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 ± 2 o C) 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 CO 2 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× 10 6 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% CO 2 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) 31.2562.512525050010002000 spontaneous WEHPFP1.20±0.361.31±0.411.45±0.491.91±0.572.45±0.49*3.86±1.43*6.13±1.67* 50%EHPFP1.29±0.511.16±0.451.16±0.411.31±0.521.42±0.341.60±0.181.75±0.45 95%EHPFP1.13±0.311.05±0.311.13±0.381.23±0.411.85±0.711.53±0.392.02±0.43 Control1.36±0.33 LPS-stimulation WEHPFP8.30±1.467.41±1.537.86±1.398.11±1.008.64±1.108.98±1.1311.50±1.11* 50%EHPFP8.53±0.867.84±0.398.06±0.927.98±1.108.34±1.178.63±0.9012.02±0.76* 95%EHPFP7.66±1.407.32±0.747.62±0.827.58±0.227.78±0.358.39±0.8210.49±1.12 Control8.75±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 × 10 6 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. 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) 31.2562.512525050010002000 spontaneous WEHPFP343±173361±193371±195479±260493±256587±285850±342 50%EHPFP307±102249±111290±123330±148470±279422±180571±194 95%EHPFP271±98292±125372±187369±174512±233543±225804±244 * Control311±135 LPS-stimulation WEHPFP3706±6833673±11703614±9453812±12203816±8614214±10524279±1182 50%EHPFP3562±8993508±10383766±9663835±9654360±13924358±13273900±1157 95%EHPFP3323±9903187±10823606±13003833±9634104±14834220±12274233±881 Control3545±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× 10 6 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% EHPFP are 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) markedly increased the anti-inflammatory cytokine (IL-10) level in the spontaneous peritoneal cell cultures. Furthermore, the water and 50% ethanol extract (2000 μg/ml) also significantly increased the IL-6 level in the lipopolysaccharide (LPS)-stimulated cultures, the peel extracts 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.