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A sensitive liquid chromatography–electrospray tandem mass spectrometric method for lancemaside A and its metabolites in plasma and a pharmacokinetic study in mice

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Abstract

A high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method employing electrospray ionization (ESI) has been developed for simultaneous determination of lancemaside A (3-O-β-d-glucuronopyranosyl-3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester) and its metabolites in mouse plasma. When lancemaside A (60 mg/kg) was orally administered to mice, echinocystic acid was detected in the blood. Tmax and Cmax of the echinocystic acid were 6.5 ± 1.9 h and 56.7 ± 29.1 ppb. Orally administered lancemaside A was metabolized to lancemaside X (3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester) by intestinal microflora in mice, which was metabolized to echinocystic acid by intestinal microflora and/or intestinal tissues. Human intestinal microflora also metabolized lancemaside A to echinocystic acid via lancemaside X. These results suggest that the metabolism by intestinal microflora may play an important role in pharmacological effects of orally administered lancemaside A.

Introduction

Lancemaside A, 3-O-β-d-glucuronopyranosyl-3β, 16α-dihydroxyolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester, is a triterpenoid saponin isolated from BuOH extract of the rhizome of Codonopsis lanceolata (family Campanulaceae) [1]. Triterpenoid saponins of the rhizome of C. lanceolata, which contain lancemaside A as a major compound, are identified by centrifugal partition chromatography and liquid chromatography–mass spectrometry [2], [3], [4]. The rhizome of C. lanceolata is been used in herbal medicines for inflammatory diseases such as bronchitis and cough in Asian countries [5], [6], [7]. Their saponins exhibit anti-inflammatory and anti-tumor effects [7], [8]. We also reported that lancemaside A isolated from its BuOH fraction potently inhibited colitis via TLR-linked NF-κB activation in mice [9]. Similar to other saponins, such as ginsenoside Rb1, the absorption of orally administered lancemaside A from intestine into the blood may be difficult due to its hydrophilicity [10], [11], [12], [13]. Thus, orally administered lancemaside A comes into contact with intestinal microflora in intestine and is metabolized to hydrophobic compounds and its metabolites may be absorbed into the blood.

Therefore, to understand its bioactive form, we performed a pharmacokinetic study of lancemaside A in mice.

Section snippets

Chemicals, materials and reagents

Lancemaside A was isolated from C. lanceolata (CL) as previously reported by Joh et al. [9]. Compound K was isolated using the previously published method of Bae et al. [14]. β-d-Glucuronidase was purchased from Sigma (St Louis, MO, USA). Acetonitrile, methanol and formic acid (HPLC grade) were purchased from Samchun Chemicals (Pyeongtaek, Gyeonggi, Korea). All other reagents were of analytical grade.

Animals

Male ICR mice (24–28 g) were supplied from Orient animal breeding center (Seoul, Korea). All

Method validation

Lancemaside A, lancemaside X and echinocystic acid 3-O-β-d-glucuronopyranoside gave a fairly strong mass response in positive ESI mode, while echinocystic acid gave a fairly strong mass response in negative ESI mode. The ion peaks of lancemaside A and its metabolites were difficult to find. Overall, lancemaside A, lancemaside X, EAG and echinocystic acid gave a strong mass response in negative electrospray ionization (ESI) mode. By negative ESI, lancemaside A, lancemaside X, EAG, echinocystic

Conclusion

In this study, we developed a rapid, sensitive and selective LC–MS/MS method and validated its use for the determination of lancemaside A, lancemaside X, EAG, and echinocystic acid in mouse plasma. When lancemaside A (60 mg/kg) was orally administered to mice, echinocystic acid was detected in the blood, but lancemaside A, lancemaside X and EAG were not detected. Tmax and Cmax of echinocystic acid were 6.5 ± 1.9 h and 56.7 ± 29.1 ppb, respectively. Orally administered lancemaside A was metabolized to

Acknowledgement

This research was supported by a grant (09172 KFDA 996) from Korean Food and Drug Administration in 2009.

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