Anti-pruritic effect of baicalin and its metabolites, baicalein and oroxylin A, in mice

Abstract

Aim: To explore whether intestinal microflora plays a role in anti-pruritic activity of baicalin, a main constituent of the rhizome of Scutellaria baicalensis (SB).

Methods: Baicalin was anaerobically incubated with human fecal microflora, and its metabolites, baicalein and oroxylin A, were isolated. The inhibitory effect of baicalin and its metabolites was accessed in histamine- or compound 48/80-induced scratching behavior in mice.

Results: Baicalin was metabolized to baicalein and oroxylin A, with metabolic activities of 40.2+/-26.2 and 1.2+/-1.1 nmol.h(-1).mg(-1) wet weight of human fecal microflora, respectively. Baicalin (20, 50 mg/kg) showed more potent inhibitory effect on histamine-induced scratching behavior when orally administered than intraperitoneally. In contrast, baicalein and oroxylin A had more potent inhibitory effect when the intraperitoneally administered. The anti-scratching behavior activity of oral baicalin and its metabolites was in proportion to their inhibition on histamine-induced increase of vascular permeability with oroxylin A more potent than baicalein and baicalin. In Magnus test using guinea pig ileum, oroxylin A is more potent than baicalein and baicalin in inhibition of histamine-induced contraction. The anti-scratching behavioral effect of oral baicalin was significantly reduced when oral antibiotics were simultaneously administered, whereas the effect of baicalein and oroxylin A were not affected.

Conclusion: Oral baicalin may be metabolized by intestinal microflora into baicalein and oroxylin A, which ameliorate pruritic reactions through anti-histamine action.

Figure 1

Metabolism of baicalin by human fecal microflora. (A) Identification of baicalin isolated (a) and baicalin metabolites with LC-MS/MS; (i) baicalin, (ii) baicalein, and (iii) oroxylin A. (B) Activity of baicalin metabolism by human fecal microflora. (n=5). (C) Proposed pathway of baicalin metabolism by human intestinal microflora. →, main pathway; , minor pathway.

Figure 2

Inhibitory effects of orally administered Scutellariae rhizome extract (SB ex), baicalin, its metabolites, and azelastine, on scratching behavior induced by histamine or compound 48/80 in mice. The scratching behavior was induced by histamine and compound 48/80 in ICR and BALB/c mice, respectively. (A) Effect of SB ex. Treatment with SB ex (100 mg/kg) or azelastine (10 mg/kg) orally 5 h before the intradermal injection of 300 μg/50 μL of histamine or 50 μg/50 μL of compound (Cpd) 48/80. (B) Effect of baicalin, baicalein, oroxylin A and azelastine in histamine-induced scratching behavior in ICR mice. (C) Effect of baicalin, baicalein, oroxylin A and azelastine on compound 48/80-induced scratching behavior in BALB/c mice. These agents [10 (gray bar), 20 (white bar) and 50 mg/kg (black bar)] were administered orally 1 or 5 h before the intradermal injection of histamine or compound 48/80. The frequency of scratching behavior in the normal group treated with saline alone for 1 h were 2±1, respectively, and the frequency of scratching behavior in the control group treated with histamine in ICR mice and compound 48/80 in BALB/c mice was 89±5 and 168±18, respectively. The values indicate the mean±SD (n=6). ^bP<0.05.

Figure 3

Inhibitory effects of baicalin, its metabolites, and azelastine administered intraperitonelly on histamine-induced scratching behavior in mice. The scratching behavior was induced by histamine in ICR mice. Mice were treated with or without intraperioneal administration of the test agents [5 (gray bar), 10 (white bar) and 20 mg/kg (black bar)] 1 h before the intradermal injection of 300 μg/50 μL of histamine into the skin on the backs of mice. The frequency of scratching behavior in the normal (treated with saline alone) and control groups (treated with histamine) for 1 h was 2±1 and 85±4, respectively. The values indicate the mean±SD. (n=6). ^bP<0.05.

Figure 4

Inhibitory effects of baicalin, baicalein, and azelastine administered orally on histamine-induced scratching behavior in mice treated with or without antibiotics. The scratching behavior was induced by histamine in ICR mice treated with or without antibiotics. The antibiotics were administered orally 1 d before histamine treatment. Oral administration of test agents [10 (gray bar), 20 (white bar), and/or 50 mg/kg (black bar)] was given 1 h before the intradermal injection of 300 μg/50 μL of histamine into the skin on the backs of mice. The frequency of scratching behavior in the normal group treated with saline alone in normal and antibiotics-treated mice for 1 h were 2±2 and 2±1, respectively, and the frequency in the control group treated with histamine in normal and antibiotics-treated mice was 84±4 and 87±5, respectively. Ba, baicalin; ABa, baicalin with antibiotics; Be, baicalein; ABe, baicalein with antibiotics; Oa, oroxylin A; AOa, oroxylin A with antibiotics; A, azelastine; AA azelastine with antibiotics. The values indicate the mean±SD. n=6. ^bP<0.05.

Figure 5

Inhibitory effects of baicalin, its metabolites, and azelastine administered orally on vascular permeability increased by histamine in mice. (A) Effect in vascular permeability. The vascular permeability was increased by histamine in ICR mice. Mice were treated with or without the oral administration of test agents [10 (gray bar), 20 (white bar), and/or 50 mg/kg (black bar)] 1 h before the intradermal injection of 300 μg/50 μL of histamine into the skin on the backs of mice. In the vascular permeability assay, the amount of Evan blue extravasated from the dorsal skin (1 cm×1 cm) of the control group stimulated with histamine and the vehicle-treated group was 12±3 μg and 4±2 μg, respectively. The values indicate the mean±SD. (n=6). ^bP<0.05 vs baicalin (50 mg/kg) treated group. (B) Histologic photograph. The skin tissues were stained with hematoxylin-eosin and assessed by light microscopy.

Figure 6

Anti-contraction effect of baicalin, its metabolites, and azelastine in Magnus test using guinea pig ileum. The ileal strip was set in a 10 mL Magnus tube (32 °C, 95% O₂+5% CO₂) containing Tyrode's solution. Each test agent [0.1 mmol/L (gray bar) and 0.4 mmol/L (black bar), dissolved in 2% trition X-100] was added to the preparation 30 s before treatment with histamine (1×10⁻⁶mol/L). The values indicate mean±SD. (n=3). ^bP<0.05 vs 0.1 mmol/L baicalin treated group. ^eP<0.05 vs 0.4 mmol/L baicalin treated group.