Preparation of penta-O-galloyl-β-D-glucose from tannic acid and plasma pharmacokinetic analyses by liquid-liquid extraction and reverse-phase HPLC

Abstract

The gallotannin penta-O-galloyl-beta-D-glucose (PGG) has many biological activities including in vivo anti-cancer efficacy. We present in this paper a scaled-up protocol for its preparation in high purity from tannic acid by acidic methanolysis with typical yield of 15%. We also describe a method for the analysis of PGG in mouse plasma by HPLC and its application in preliminary pharmacokinetic studies. A liquid-liquid extraction (LLE) protocol was optimized for the extraction of PGG from mouse plasma. The extraction efficiency for PGG at 1 μg/mL in mouse plasma was 70.0±1.3% (n=5). The limit of detection (LOD) for PGG was approximately 0.2 μg/mL. Preliminary pharmacokinetic parameters of PGG following a single i.p. injection with 5% ethanol/saline vehicle in mice were established. The peak plasma PGG concentrations (C(max)) were approximately 3-4 μM at a dose of 0.5 mg per mouse (∼20 mg/kg) at 2 h post-injection (T(max)).

Figure 1

(A) Methanolysis of tannic acid to yield PGG under weak acidic condition; (B) Structure of PGG and EGCG.

Figure 2

Representative chromatograms: (A) EGCG (IS) and PGG standard; (B) Commercial C57BL/6 mouse plasma spiked with EGCG (IS) at 25 μg/mL and PGG at 5 μg/mL; (C) Commercial C57BL/6 mouse plasma spiked with EGCG (IS) at 25 μg/mL; (D) Typical chromatogram from C57BL/6 mouse plasma after i.p. injection of 0.5 mg PGG (~20mg/kg). Before extraction, EGCG was spiked at 25 μg/mL as IS.

Figure 3

Impact of acidification solutions on extraction efficiency for PGG and EGCG from spiked C57BL/6 mouse plasma. PO4: 0.1 M Na₂HPO₄ buffer, pH 3.2; AA: 2% acetic acid solution; none: nothing added.

Figure 4

Plasma pharmacokinetic characteristics of injected PGG (0.5 mg per mouse, i.p.) in male (squares) and female (triangles) C57BL/6 mice. Each data point represents mean ± SE, n = 4 mice.

Figure 5

Lack of effect of glucuronidase and sulfatase on the extractable level of PGG in plasma. Plasma samples were from C57BL/6 mice subjected to one dose of 2 mg PGG by i.p. injection.