Selective depletion of glycyrrhizin from Si-Ni-San, a traditional Chinese prescription, blocks its effect on contact sensitivity in mice and recovers adhesion and metalloproteinases production of T lymphocytes

Abstract

In the present study, we performed to selectively deplete glycyrrhizin from Si-Ni-San, a traditional Chinese prescription that consists of 4 Chinese herbs including Radix Glycyrrhizae Uralensis, and examined its influence on the suppressing activity of Si-Ni-San against contact sensitivity in mice. An immunoaffinity column was made by covalently coupling the polyclonal antibody, obtained by the immunization with glycyrrhizin-BSA conjugate, to CNBr-activated Sepharose 4B. By using this column, glycyrrhizin in Si-Ni-San was selectively and almost completely depleted from the whole extract, which was confirmed by high-performance liquid chromatography (HPLC). Both 200 mg/kg of Si-Ni-San and 10 mg/kg of glycyrrhizin, the dose corresponding to its proportion contained in Si-Ni-San, significantly reduced the ear swelling of picryl chloride (PCl)-induced ear contact sensitivity in mice and the inhibition by Si-Ni-San was stronger than that by glycyrrhizin. The adhesion activity to type IV collagen of the isolated spleen cells from PCl-sensitized mice was significantly decreased by both Si-Ni-San and glycyrrhizin. However, the glycyrrhizin-depleted sample of Si-Ni-San (Si-Ni-San(GL-)) only showed a slight inhibition on the cell adhesion. Furthermore, the spleen cells from PCl-sensitized mice produced more matrix metalloproteinase (MMP)-2 and -9 than naive spleen cells did, and both Si-Ni-San and glycyrrhizin remarkably reduced MMP-2 and MMP-9 production. In contrast, Si-Ni-San(GL-) only showed a slight inhibition. These results suggest that glycyrrhizin may act as one of the active constituents of Si-Ni-San in inhibiting delayed-type hypersensitivity reaction via down-regulating the MMP production and the cell adhesion to extracellular matrix. The present study also provides a new approach to recognize and validate an active constituent in traditional prescription through a selective depletion.

Fig. 1

Synthesis of glycyrrhizin–BSA and glycyrrhizin–OVA conjugates.

Fig. 2

Characterization of anti-glycyrrhizin antiserum with ELISA. To examine the reactivity of the antiserum, gradiently diluted antiserum were added to each well of a 96-well immunoplate coated with 500 μg/mL glycyrrhizin–OVA.

Fig. 3

SDS-PAGE of purified IgG from antiserum. The purified IgG was separated by 7% SDS–polyacrylammide. The protein bands of the gel were stained with Commassie brilliant blue R-250. Left lane: molecular weight marker; right lane: purified IgG.

Fig. 4

HPLC chromatogram of glycyrrhizin before and after Si-Ni-San was loaded onto the immunoaffinity column. A, HPLC chromatogram of standard glycyrrhizin. B, HPLC chromatogram of Si-Ni-San before it was loaded onto the column. C, HPLC chromatogram of Si-Ni-San after it was passed through the column. Before loaded onto the column, 0.5 mg Si-Ni-San in 2 mL pH 6.4 PBS solution was filtered through a 0.22 mm polysulfone filter. Then it was eluted from the column at 0.5 M NaCl. The fractions were collected and subjected to HPLC analysis. HPLC conditions: YMC ODS-C₁₈ column (150 × 4.6 mm, i.d., 5 μm); column temperature, 30 °C; mobile phase, methanol–water–acetic acid (70 : 25 : 5 (v / v)); flow rate, 1.0 mL/min; UV detection at 254 nm; injection volume, 20 μL.

Fig. 5

Effect of Si-Ni-San, glycyrrhizin and the glycyrrhizin-depleted sample of Si-Ni-San (Si-Ni-San^GL−) on the adhesion of splenocytes from PCl-sensitized mice to type IV collagen. Mice were sensitized by PCl as described in Materials and methods. Five days after the sensitization, the spleen cells were isolated and incubated with Si-Ni-San, glycyrrhizin and Si-Ni-San^GL− all at 10^− 4 g/mL at 37 °C in 5% CO₂ for 2 h. Then the cells were washed and used for adhesion assay. Each column represents the mean ± SD of three independent experiments and each experiment includes triplicate sets. Spon: cell alone; Cont: cells + PMA; *P < 0.05, **P < 0.01 vs Cont (Dunnett's test); ^##P < 0.01 vs Si-Ni-San (Student's two-tailed t test).

Fig. 6

Effects of Si-Ni-San, glycyrrhizin, and the glycyrrhizin-depleted sample of Si-Ni-San (Si-Ni-San^GL−) on the productions of MMP-2 and MMP-9 in splenocytes isolated from PCl-sensitized mice. A and B: MMP-2. C and D: MMP-9. Five days after PCl sensitization, the isolated spleen cells were incubated with Si-Ni-San, glycyrrhizin and Si-Ni-San^GL− all at 10^− 4 g/mL at 37 °C for 24 h. After the incubation, the supernatants were aspirated and used for zymography assay. The figure shown here is the representative of three different experiments.