Liquid chromatographic separation of derivatives of diospyrin, a bioactive bisnaphthoquinonoid plant-product, and analogous naphthyl compounds

Abstract

Isocratic reversed-phase liquid chromatography (LC) method was developed using acetonitrile and water for the determination of diospyrin, a pharmacologically important bisnaphthoquinonoid plant-product. The method was validated for precision, accuracy and reproducibility, and was found to be linear over the concentration range of 1–1000 μg/ml; the limits of detection and quantitation were 8 and 20 ng, respectively. The technique was used to determine the amount of diospyrin in plant extracts from four climatic regions in India. It was also applied for differentiation and separation of 27 naphthyl compounds. While a composition of 50:50 was preferable for dimeric compounds, the composition 40:60 was a better choice for the monomers. Also, the isomeric α- and β-naphthols and their dimers could be distinguished by conversion into the respective methyl ethers.

Introduction

Quinonoid compounds are mostly present as secondary metabolites in all respiring animal and plant cells and possess a wide range of biological activities [1]. Some of the most frequently used anticancer drugs have been derived from quinononoid natural products [2]. Studies carried out during 1940s on lapachol, a plant-derived naphthoquinonoid, have led to the synthesis of a series of analogues with antiparasitic activities [3]. One such drug, atovaquone, has recently obtained clinical approval as a constituent of a novel antimalarial formulation, viz. Malarone [4]. Diospyrin (1), a bisnaphthoquinonoid isolated from the stem-bark of Diospyros montana Roxb., exhibited significant antitumour and antileishmanial activities in our laboratory [5], [6]. Hence, derivatives of 1, which were more effective than their naturally occurring precursor, were tested against human cancer cell lines, as well as some parasites and microorganisms [7], [8], [9], [10], [11], [12]. Since derivatisation of 1 would influence its polarity, and consequently, its pharmacokinetic profile, detection of such compounds might pose a challenge in studying their metabolic behaviour [13]. This consideration prompted us to look for a simple and precise analytical method for detection and estimation of 1, and its analogues.

Earlier workers pursuing different objectives have reported the separation of monomeric naphthoquinonoids [14], [15], Vitamin K analogues [16], [17], prenylquinones [18], etc. through reversed-phase liquid chromatography (LC) using widely divergent compositions of the eluent; however, no uniform solvent system for separation of naphthyl compounds, in general, and dimeric naphthoquinonoids, in particular, has been established so far. Determination of plumbagin, a plant-derived naphthoquinone, by normal-phase LC was reported by Gupta et al [19]. In the present communication, we are presenting a reversed-phase LC–UV spectrophotometric method, using a binary isocratic solvent system, which has been demonstrated to be sensitive enough for detection of some monomeric and dimeric naphthoquinonoid compounds with minor variations of the substituents (Fig. 1, Table 1). Furthermore, the scope of this study has been extended to include a few naphthyl analogues as well. Finally, the method has been validated and verified for the quantitative estimation of the dimeric naphthoquinonoid, 1, which was present in semi-purified samples of the stem-bark of D. montana Roxb. collected from four different climatic regions in eastern and north-eastern India.