Elsevier

Journal of Chromatography B

Volume 842, Issue 1, 14 September 2006, Pages 28-35
Journal of Chromatography B

Using of liquid chromatography coupled with diode array detector for determination of naphthoquinones in plants and for investigation of influence of pH of cultivation medium on content of plumbagin in Dionaea muscipula

https://doi.org/10.1016/j.jchromb.2006.05.009Get rights and content

Abstract

The interest of many investigators in naphthoquinones is due to their broad-range of biological actions from phytotoxic to fungicidal. The main aim of this work was to investigate the influence of different pH values of cultivation medium on naphthoquinone content in Dionaea muscipula. For this purpose, we optimized the simultaneous analysis of the most commonly occurring naphthoquinones (1,4-naphthoquinone, lawsone, juglone and plumbagin) by high performance liquid chromatography coupled with diode array detector (HPLC-DAD). The most suitable chromatographic conditions were as follows: mobile phase: 0.1 mol l−1 acetic acid:methanol in ratio of 33:67 (%, v/v), flow rate: 0.75 ml min−1 and temperature: 42 °C. Moreover, we looked for the most suitable technique for preparation of plant samples (D. muscipula, Juglans regia, Paulownia tomentosa, Impatience glandulifera, Impatience parviflora, Drosera rotundifolia, Drosera spathulata and Drosera capensis) due to their consequent analysis by HPLC-DAD. It clearly follows from the results obtained that sonication were the most suitable technique for preparation of J. regia plants. We also checked the recoveries of the determined naphthoquinones, which were from 96 to 104%. Finally, we investigated the changes in content of plumbagin in D. muscipula plants according to different pH of cultivation medium. The content increased with increasing pH up to 5 and, then, changed gradually. The lower content of plumbagin at lower pH values was of interest to us. Therefore, we determined the content of this naphthoquinone in the cultivation medium, what has not been studied before. We discovered that the lower tissue content of plumbagin was due to secretion of this naphthoquinone into the cultivation medium.

Introduction

Interaction of species in an ecosystem is known to be determined by many factors, including the ability of microorganisms or plants to excrete substances that affect the development of other species. Such substances called allelopathic metabolites cover a broad-range of metabolites including both simple (organic acids, sugars, etc.) and complex (hormones, secondary metabolites, etc.) compounds inhibiting and/or stimulating metabolic processes [1].

Naphthoquinones are one of the secondary metabolic groups widespread in nature, where they mostly appear as chromatic pigments. They have been found in higher plants such as Plumbaginaceae, Juglandaceae, etc. [2], [3], [4], [5], fungi (Marasmius gramium and Verticillium dahliae) [1] and microorganisms (Streptomyces and Fusarium) [6]. Plant extracts containing naphthoquinones have been used for a long time in traditional medicines of number of nations for cancer and rheumatoid arthritis treatment, particularly extracts from Plumbago europaea for mitigation of toothache, extracts from Plumbago zeylanica for treatment of diarrhoea, skin diseases and digestion malfunction [7], [8].

The interest of many investigators in this class of compounds is due to their broad-range of biological activities: phytotoxic [9], [10], [11], insecticidal [1], antibacterial [12], [13], [14] and fungicidal [13], [14]. Besides that they have also cytostatic [13] and anticarcinogenic [1] properties. Their cytostatic and antimicrobial activities emerge due to their ability to act as potent inhibitors of electron transport [15], as uncouplers of oxidative phosphorylation [16], as intercalating agents in the DNA double helix, as bioreductive alkylating agents of biomolecules, and as producers of reactive oxygen radicals by redox cycling under aerobic conditions [16], [17]. An anticancer effect of naphthoquinones stimulates an interest in determination and characterization of single derivatives of 1,2- and 1,4-quinones in biological samples. High performance liquid chromatography with UV detection and electrochemical techniques are the most commonly used methods for these purposes [18], [19], [20], [21]. In addition de Paiva et al. [22] used mass spectrometry as a tool for the study of plumbagin. The main aim of this work was to investigate the influence of different pH values of cultivation medium on naphthoquinone content in Venus's flytrap (D. muscipula). For these purposes it was necessary to optimize the simultaneous analysis of the most commonly occurring naphthoquinones (1,4-naphthoquinone, lawsone, juglone and plumbagin; Fig. 1) by high performance liquid chromatography coupled with a diode array detector (HPLC-DAD). The optimized technique was consequently used for detection and quantification of naphthoquinones in plant materials (D. muscipula, Juglans regia, Paulownia tomentosa, Impatience glandulifera, Impatience parviflora, Drosera rotundifolia, Drosera spathulata and Drosera capensis).

Section snippets

Chemicals

Naphthoquinones (1,4-naphthoquinone, lawsone, juglone and plumbagin) were purchased from Sigma Aldrich Chemical Corp. (St. Louis, USA). Methanol for HPLC and other analytical reagents of ACS purity were also purchased from Sigma Aldrich. Solutions were prepared using deionised ACS water (Sigma). The stock standard solutions of naphthoquinones at (100 μg ml−1) were prepared in ACS methanol and stored in the dark at 4 °C. The working standard solutions were prepared daily by dilution of the stock

Results and discussion

The development of new analytical techniques used for a determination of biologically active compounds is one of the most important tasks of analytical chemistry, biochemistry and pharmacy [24], [27], [28], [29], [30], [31], [32], [33], [34], [35]. Here we wished to study the content of naphthoquinones (1,4-naphthoquinone, lawsone, juglone and plumbagin; Fig. 1) in different plant species by HPLC-DAD. Therefore, primarily it was necessary to optimize their simultaneous analysis. The HPLC-DAD

Conclusions

Secondary metabolites level monitoring of naphthoquinones is helpful to obtain quantitative information about the distribution and expression of these compounds in plants during cultivation at different conditions. Here, we optimized and used HPLC-DAD to simultaneously determine of naphthoquinones in different plant species. Plumbagin was determined in almost all of the analyzed samples except J. regia, where we quantified milligrams of juglone per gram. Moreover, we studied the changes in the

Acknowledgements

The authors wish to thanks to Karl J. Kramer for correction and peer review of the manuscript. This work was supported by grants: IGA FaF VFU No. IG342012 and No. VV352003/2005, IGA MZLU No. 250061/2005 and GACR No. 525/04/P132.

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