Skullcap (Scutellaria Baicalensis) Hexane Fraction Inhibits the Permeation of Ovalbumin and Regulates Th1/2 Immune Responses

Abstract

Skullcap (Scutellaria baicalensis) is well known for its anti-inflammatory and anti-allergic effects. In our previous study, we found that skullcap could inhibit allergen permeation and regulate Th1/2 immune balance. To reveal the key fractions and components of skullcap, we fractionated skullcap extract into five fractions: hexane, chloroform, ethyl acetate, butanol, and water fraction. Among these fractions, the hexane fraction significantly suppressed the production of Th2-mediated cytokines (Interleukin (IL)-4, 5, 10 and 13) and increased Th1-mediated cytokines (Interferon (IFN)-γ and IL-12). Furthermore, the hexane fraction inhibited the permeation of ovalbumin (OVA), used as an allergen, across the intestinal epithelial cell monolayer. To confirm the active compounds in the hexane fraction, fatty acids were analyzed. Linoleic acid (LA, C18:2 (>59.7%)) was identified as the most important fatty acid in the skullcap hexane fraction. LA significantly suppressed IL-4 production and increased IFN-γ secretion, as well as inhibiting OVA permeation. Thus, LA significantly diminished the permeation of allergen by enhancing intestinal barrier function and regulated allergic responses to maintain Th1/Th2 immune balance.

Keywords: Scutellaria baicalensis; Th1/2 immune response; Th1/Th2 balance; linoleic acid; ovalbumin permeation; skullcap.

Figure 1

Solvent fractions of skullcap with ethanol, hexane, ethyl acetate, chloroform, butanol, and water.

Figure 2

Effects of skullcap extract (ethanol, hexane, chloroform, ethyl acetate, butanol, and water) on ex vivo splenocytes from ovalbumin (OVA)-sensitized BALB/c mice. (A) Interleukin (IL)-4 and (B) Interferon (IFN)-γ were quantified by enzyme-linked immunosorbent assay (ELISA) using splenocytes; (C) Cell viability and cytotoxicity were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; (D) Inhibition rate (%). Values are presented as mean ± standard deviation (SD) (n = 4 in each group). Data were analyzed by analysis of variance (ANOVA), followed by Duncan’s multiple-range test. Data are statistically different (p < 0.05) among those columns with different symbols. The order of value is termed as order of alphabet.

Figure 3

Effects of skullcap hexane fraction (50 µg/L on OVA-induced Th2 immune response ex vivo. (A) Th2-associated cytokines such as IL-4, IL-5, IL-10 and IL-13; (B) Th1-associated cytokines, such as IFN-γ and IL-12, were quantified by enzyme-linked immunosorbent assay (ELISA) using splenocytes. Values are presented as mean ± SD (n = 4 in each group). Data were analyzed by ANOVA, followed by Duncan’s multiple-range test. * p < 0.05, ** p < 0.01, significantly different from the OVA value.

Figure 4

Effects of linoleic acid in skullcap hexane fraction on OVA-induced Th2 immune response ex vivo. (A) LNA, PA, and LA were evaluated the capacities for IL-4 inhibition rate (%); (B) IL-4 and (C) IFN-γ were quantified by ELISA using splenocytes. Values are presented as mean ± SD (n = 4 in each group). Data were analyzed by ANOVA, followed by Duncan’s multiple-range test. (A) * p < 0.05, ** p < 0.01, significantly different from the OVA value. (B,C) Data are statistically different (p < 0.05) among those columns with different symbols. The order of value is designated as alphabetical order.

Figure 5

Effects of skullcap hexane fraction and key compound (linoleic acid) on TEER in Caco-2 cell monolayers and ovalbumin (OVA) flux in Caco-2 cell monolayers. (A,B) Effects of skullcap hexane fraction (100 µg/mL) on TEER value and OVA influx. (C,D) Effects of linoleic acid, a key compound in the hexane fraction, on TEER and OVA flux. Values are presented as mean ± SD (n = 4 in each group). Data were analyzed by ANOVA, followed by Duncan’s multiple-range test. (A,B) * p < 0.05, ** p < 0.01, significantly different from the control value. (C,D) Data are statistically different (p < 0.05) among those columns with different symbols. The order of value is designated as alphabetical order.