Determination of Naphthazarin Derivatives in Endemic Turkish Alkanna Species by Reversed Phase High Performance Liquid Chromatography

Abstract

The quantification of free and total alkannins in 13 endemic Anatolian Alkanna species is described for the first time. Extraction of the samples was performed by sonication with hexane, followed by hydrolysis in 1 N NaOH. For analysis, a new HPLC method, utilizing reversed phase material (Synergi Max RP), was developed and successfully validated. The obtained data confirmed that the assay is sensitive (LOD of 13 ng on-column for alkannin), accurate (the recovery rate was 92.3 %) and precise (RSD ≤ 6.8 % for intra-day precision; RSD ≤ 4.8 % for inter-day precision). LC-MS experiments were performed to assure peak identity and purity. Quantitative results revealed that roots of Alkanna tubulosa showed the highest amount of total alkannins (0.84 %), while Alkanna mughlae contained the highest percentage of free alkannins (0.04 %). Compared to the commonly used A. tinctoria (total alkannins: 0.11 to 0.54 %; free alkannins: 0.002 to 0.01 %) these two species could therefore serve as alternative sources for medicinally important alkannin derivatives.

Key words

Alkanna spp. - alkannin - shikonin - HPLC - LC-MS

References

• 1 Papageorgiou V P, Assimopoulou A N, Couladouros E A, Hepworth D, Nicolaou K C. The chemistry and biology of alkannin, shikonin and related naphthazarin natural products.  Angew Chem Int Ed Engl. 1999;  38 270-300.
• 2 Hazra B, Sarma M D, Sanyal U. Separation methods of quinonoid constituents of plants used in Oriental tradition medicines.  J Chromatogr B. 2004;  812 259-75.
• 3 Sasaki K, Abe H, Yoshizaki F. In vitro antifungal activity of naphthoquinone derivatives.  Biol Pharm Bull. 2002;  25 669-70.
• 4 Chen X, Yang L, Oppenheim J J, Howard O MZ. Cellular pharmacology studies of shikonin derivatives.  Phytother Res. 2002;  16 199-209.
• 5 Assimopoulou A N, Boskou D, Papageorgiou V P. Antioxidant activities of alkannin, shikonin and Alkanna tinctoria root extracts in oil substrates.  Food Chem. 2004;  87 433-8.
• 6 Assimopoulou A N, Papageorgiou V P. Radical scavenging activity of Alkanna tinctoria root extracts and their main constituents, hydroxynaphthoquinones.  Phytother Res. 2005;  19 141-7.
• 7 Yang F, Chen Y, Duan W, Zhang C, Zhu H, Ding J. SH-7, a new synthesized shikonin derivative, exerting its potent antitumor activities as a topoisomerase inhibitor.  Int J Cancer. 2006;  119 1184-93.
• 8 Assimopoulou A N, Papageorgiou V P. Study on polymerization of the pharmaceutical siubstances isohexenylnaphthazarins.  Biomed Chromatogr. 2004;  18 492-500.
• 9 Plyta Z F, Li T, Papageorgiou V P, Mellidis A S, Assimopoulou A N, Pitsinos E N. et al . Inhibition of topoisomerase I by naphthoquinone derivatives.  Bioorg Med Chem Lett. 1998;  8 3385-90.
• 10 Hu C -M, Cheng Y -W, Cheng H -W, Kang J -J. Impairment of vascular function of rat thoracic aorta in an endothelium-dependent manner by shikonin/alkannin and derivatives isolated from roots of Macrotomia euchroma .  Planta Med. 2004;  70 23-8.
• 11 Staniforth V, Shyur L F, Wang S -Y, Shyur L -F, Yang N -S. Shikonins, phytocompounds from Lithospermum erythrorhizon, inhibit the transcriptional activation of human tumor necrosis factor α promoter in vivo .  J Biol Chem. 2004;  279 5877-85.
• 12 Lu Q, Liu W, Ding J, Cai J, Duan W. Shikonin derivatives: synthesis and inhibition of human telomerase.  Bioorg Med Chem Lett. 2002;  12 1375-8.
• 13 Song G -Y, Kim Y, Zheng X -G, You Y -J, Cho H, Chung J -H. et al . Naphthazarin derivatives (IV): synthesis, inhibition of DNA topoisomerase I and cytotoxicity of 2- or 6-acyl-5,8-dimethoxy-1,4-naphthoquinones.  Eur J Med Chem. 2000;  35 291-8.
• 14 Yesilada E, Sezik E, Aslan M, Yesilada A. Quantitative analysis of the enantiomeric naphthaquinone derivatives from Boraginaceous roots by high performance liquid chromatography.  J Liq Chromatogr R T. 1996;  19 3369-81.
• 15 Assimopoulou A N, Papageorgiou V P. Study on the enantiomeric ratio of the pharmaceutical substances alkannin and shikonin.  Biomed Chromatogr. 2004;  18 791-9.
• 16 Ikeda Y, Ishida N, Fukaya C, Yokoyama K, Tabata M, Fuku H. et al . Determination of the ratio between optical isomers, shikonin and alkannin by high performance liquid chromatography analysis.  Chem Pharm Bull. 1991;  39 2351-2.
• 17 Kang J S, Ahn B Z, Blaschke G. Enantiomeric ratio of shikonin derivatives as a possible key for the determination of the origin of Lithospermi radix.  Arch Pharm Res. 1998;  21 565-9.
• 18 Spyros A, Assimopoulou A N, Papageorgiou V P. Structure determination of oligomeric alkannin and shikonin derivatives.  Biomed Chromatogr. 2005;  19 498-505.
• 19 Papageorgiou V P, Assimopoulou A N, Kyriacou G. Determination of naturally occurring hydroxynaphthoquinone polymers by size-exclusion chromatography.  Chromatographia. 2002;  55 423-30.
• 20 Yamamoto H, Yazaki K, Inoue K. Simultaneous analysis of shikimate-derived secondary metabolites in Lithospermum erythrorhizon cell suspension cultures by high-performance liquid chromatography.  J Chromatogr B. 2000;  738 3-15.
• 21 Bozan B, Baser K HC, Kara S. Quantitative determination of naphtaquinones of Arnebia densiflora (Nordm.) Ledeb. by an improved high-performance liquid chromatographic method.  J Chromatogr A. 1997;  782 33-6.
• 22 Chaisuksant R, Niopas I, Voulgaropoulos A, Mellidis A, Papageorgiou V P. Analysis of some isohexenylnaphthazarins by reversed phase HPLC.  Pharmazie. 1995;  50 363.
• 23 Assimopoulou A N, Karapanagiotis I, Vasiliou A, Kokkini S, Papageorgiou V P. Analysis of alkannin derivatives from Alkanna species by high-performance liquid chromatography/photodiode array/mass spectrometry.  Biomed Chromatogr. 2006;  20 1359-74.
• 24 Papageorgiou V P, Mellidis A S, Assimopoulou A N, Tsarbopoulos A. Use of matrix-assisted laser desorption/ionization mass spectrometry for the rapid detection of low-mass components in the Alkanna tinctoria pigments fraction.  J Mass Spectrom. 1998;  33 89-91.
• 25 Assimopoulou A N, Papageorgiou V P. Study on isohexenylnaphthazarins polymerization in alkali media.  Biomed Chromatogr. 2004;  18 508-22.
• 26 Papageorgiou V P, Assimopoulou A N. Lipids of the hexane extract from the roots of medical Boraginaceous species.  Phytochem Anal. 2003;  14 251-8.