Ultrasound-Assisted Ionic Liquid Solid–Liquid Extraction Coupled with Aqueous Two-Phase Extraction of Naphthoquinone Pigments in Arnebia euchroma (Royle) Johnst.

  • Qianhui Sun
  • Baiquan Du
  • Chenzhao Wang
  • Weili Xu
  • Zhuang Fu
  • Yu Yan
  • Shouzhi Li
  • Zhibing WangEmail author
  • Hanqi Zhang


A simple, rapid and green ultrasound-assisted ionic liquid solid–liquid extraction coupled with aqueous two-phase extraction method was developed and applied to the extraction of shikonin, acetylshikonin and β,β′-dimethylacrylshikonin in Arnebia euchroma (Royle) Johnst. The separation and determination of the analytes was performed by high-performance liquid chromatography coupled with diode array detection. In the study, 1-butyl-3-methylimidazolium tetrafluoroborate ([C4MIM][BF4]) was used as extraction solvent. Ionic liquid-based solid–liquid extraction and aqueous two-phase extraction were performed simultaneously in one tube under the assistance of ultrasound. The liquid–liquid–solid three-phase system, namely surfactant-rich phase, water-rich phase and sample-rich phase, was formed. The target analytes were enriched in the surfactant-rich phase. Under the optimal experimental conditions, the calibration curves of target analytes showed good linear relationship (r > 0.9997). The inter-day and intra-day precision were in the range of 3.42–8.53 and 1.98–3.14%, respectively. The LOD and LOQ for the target analytes were in the range of 5.0–19.2 and 16.7–64.2 ng mL−1, respectively. The recoveries of naphthoquinone pigments ranged from 90.00 to 97.73% and relative standard deviations were less than 4.50%. Compared with heat reflux extraction and ultrasonic-assisted extraction, the proposed method is simpler, faster and more effective, because the mass transfer process for the analytes was always performed in one centrifugal tube and the steps such as filtration, concentration and redissolution were avoided. The present method was demonstrated to be efficient and satisfactory for the extraction of naphthoquinone pigments in medicinal plants.

Graphic Abstract


High-performance liquid chromatography Aqueous two-phase extraction Ionic liquid Naphthoquinone pigments Ultrasound-assisted extraction Arnebia euchroma (Royle) Johnst 



This study was financially supported by the Education Department of Jilin Province (Grant no. JJKH20181014KJ) and the Department of Science and Technology of Jilin Province (Grant no. 20190304011YY).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. 1.
    Lu HT, Jiang Y, Chen F (2004) Preparative high-speed counter-current chromatography for purification of shikonin from the Chinese medicinal plant Lithospermum erythrorhizon. J Chromatogr A 1023:159–163CrossRefGoogle Scholar
  2. 2.
    Hu YN, Jiang ZH, Leung KSY, Zhao ZZ (2006) Simultaneous determination of naphthoquinone derivatives in Boraginaceous herbs by high-performance liquid chromatography. Anal Chim Acta 577:26–31CrossRefGoogle Scholar
  3. 3.
    Xiao Y, Wang Y, Gao SQ, Zhang R, Ren RB, Li N, Zhang HQ (2011) Determination of the active constituents in Arnebia euchroma (Royle) Johnst. by IL-based ultrasonic-assisted extraction high-performance liquid chromatography. J Chromatogr B 879:1833–1838CrossRefGoogle Scholar
  4. 4.
    Kim JY, Jeong HJ, Park JY, Kim YM, Park SJ, Cho JK, Park KH, Ryu YB, Lee WS (2012) Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases. Bioorg Med Chem 20:1740–1748CrossRefGoogle Scholar
  5. 5.
    Yang RF, Huang PP, Qiu TQ (2013) Ultrasound-enhanced subcritical water extraction of naphthoquinone pigments from purple Gromwell (Lithospermum erythrorhizon) to higher yield and bioactivity. Food Sci Biotechnol 22:671–676CrossRefGoogle Scholar
  6. 6.
    Bozan B, Baser KHC, Kara S (1999) Quantitative determination of naphthoquinones of Arnebia densiflora by TLC-densitometry. Fitoterapia 70:402–406CrossRefGoogle Scholar
  7. 7.
    Dresler S, Kubrak T, Kocka AB, Szymczak G (2015) Determination of shikonin and rosmarinic acid in Echium vulgare L. and Echium russicum J.F. Gmel. by capillary electrophoresis. J Liq Chromatogr Relat Technol 38:698–701CrossRefGoogle Scholar
  8. 8.
    Glazunov VP, Tchizhova A, Pokhilo ND, Anufriev V, Elyakov GB (2002) First direct observation of tautomerism of monohydroxynaphthazarins by IR-spectroscopy. Tetrahedron 58:1751–1757CrossRefGoogle Scholar
  9. 9.
    Ozgen U, Miloglu FD, Bulut G (2011) Quantitative determination of shikonin derivatives with UV-Vis spectrophotometric methods in the roots of Onosma nigricaule. Rev Anal Chem 30:59–63CrossRefGoogle Scholar
  10. 10.
    Noula E, Samanidou VF, Assimopoulou AN, Papageorgiou VP, Papadoyannis IN (2010) Solid-phase extraction for purification of alkannin/shikonin samples and isolation of monomeric and dimeric fractions. Anal Bioanal Chem 397:2221–2232CrossRefGoogle Scholar
  11. 11.
    Sharma N, Sharma UK, Gupta AP, Devla Sinha AK, Lal B, Ahuja PS (2009) Simultaneous densitometric determination of shikonin, acetylshikonin, and β-acetoxyisovaleryl-shikonin in ultrasonic-assisted extracts of four Arnebia species using reversed-phase thin layer chromatography. J Sep Sci 32:3239–3245CrossRefGoogle Scholar
  12. 12.
    Gao SQ, You JY, Wang Y, Zhang R, Zhang HQ (2012) On-line continuous sampling dynamic microwave-assisted extraction coupled with high performance liquid chromatographic separation for the determination of lignans in Wuweizi and naphthoquinones in Zicao. J Chromatogr B 887–888:35–42CrossRefGoogle Scholar
  13. 13.
    Zhang Q, Cai DF, Wang L, Yang XT, Fan SJ, Zhang KY (2018) Rapid and sensitive determination of shikonin and its derivatives in the roots of Arnebia euchroma (Royle) Johnst using matrix solid-phase dispersion extraction and ultrahigh-performance liquid chromatography with photodiode array detector. J Liq Chromatogr Relat Technol 41:489–497CrossRefGoogle Scholar
  14. 14.
    Feng L, Ji HW, Gu HX, Wang DC, Cui JC, Liu RM, Zhai J (2009) An efficient method for extraction, separation and purification of naphthoquinone pigments from Lithospermum erythrorhizon Sieb. et Zucc. by SFE and HSCCC. Chromatographia 70:1197–1200CrossRefGoogle Scholar
  15. 15.
    Flieger J, Czajkowska AZ (2008) Ionic liquids in separation techniques. J Chromatogr A 1184:6–18CrossRefGoogle Scholar
  16. 16.
    Wei ZF, Zu YG, Fu YJ, Wang W, Luo M, Zhao CJ, Pan YZ (2013) Ionic liquids-based microwave-assisted extraction of active components from pigeon pea leaves for quantitative analysis. Sep Purif Technol 102:75–81CrossRefGoogle Scholar
  17. 17.
    Ma WY, Lu YB, Hu RL, Chen JH, Zhang ZZ, Pan YJ (2010) Application of ionic liquids based microwave-assisted extraction of three alkaloids N-nornuciferine, O-nornuciferine, and nuciferine from lotus leaf. Talanta 80:1292–1297CrossRefGoogle Scholar
  18. 18.
    Ma CH, Liu TT, Yang L, Zua YG, Chen XQ, Zhang L, Zhang Y, Zhao CJ (2011) Ionic liquid-based microwave-assisted extraction of essential oil and biphenyl cyclooctene lignans from Schisandra chinensis Baill fruits. J Chromatogr A 1218:8573–8580CrossRefGoogle Scholar
  19. 19.
    Bhan M, Satija S, Garg C, Dureja H, Garg M (2017) Optimization of ionic liquid-based microwave assisted extraction of a diterpenoid lactone-andrographolide from Andrographis paniculata by response surface methodology. J Mol Liq 229:161–166CrossRefGoogle Scholar
  20. 20.
    Hou KX, Chen FL, Zu YG, Yang L (2016) Ionic liquids-lithium salts pretreatment followed by ultrasound-assisted extraction of vitexin-4″-O-glucoside, vitexin-2″-O-rhamnoside and vitexin from Phyllostachys edulis leaves. J Chromatogr A 1431:17–26CrossRefGoogle Scholar
  21. 21.
    Wang WC, Li QY, Liu YH, Chen BB (2015) Ionic liquid-aqueous solution ultrasonic-assisted extraction of three kinds of alkaloids from Phellodendron amurense Rupr and optimize conditions use response surface. Ultrason Sonochem 24:13–18CrossRefGoogle Scholar
  22. 22.
    Zhang H, Chen XQ, Jiang XY (2011) Determination of phthalate esters in water samples by ionic liquid cold-induced aggregation dispersive liquid-liquid microextraction coupled with high-performance liquid chromatography. Anal Chim Acta 689:110–111CrossRefGoogle Scholar
  23. 23.
    Lin HM, Zhang YG, Han M, Yang LM (2013) Aqueous ionic liquid based ultrasonic assisted extraction of eight ginsenosides from ginseng root. Ultrason Sonochem 20:680–684CrossRefGoogle Scholar
  24. 24.
    Vallecillos L, Borrull F, Pocurull E (2012) Determination of musk fragrances in sewage sludge by pressurized liquid extraction coupled to automated ionic liquid-based headspace single-drop microextraction followed by GC-MS/MS. J Sep Sci 35:2735–2742CrossRefGoogle Scholar
  25. 25.
    Wu HW, Chen ML, Fan YC, Elsebaei F, Zhu Y (2012) Determination of rutin and quercetin in Chinese herbal medicine by ionic liquid-based pressurized liquid extraction–liquid chromatography–chemiluminescence detection. Talanta 88:222–229CrossRefGoogle Scholar
  26. 26.
    Liu F, Wang D, Liu W, Wang X, Bai AY, Huang LQ (2013) Ionic liquid-based ultrahigh pressure extraction of five tanshinones from Salvia miltiorrhiza Bunge. Sep Purif Technol 110:86–92CrossRefGoogle Scholar
  27. 27.
    Wu H, Zhang LB, Du LM (2011) Ionic liquid sensitized fluorescence determination of four isoquinoline alkaloids. Talanta 85:787–793CrossRefGoogle Scholar
  28. 28.
    Zhang HF, Shi YP (2010) Temperature-assisted ionic liquid dispersive liquid-liquid microextraction combined with high performance liquid chromatography for the determination of anthraquinones in Radix et Rhizoma Rhei samples. Talanta 82:1010–1016CrossRefGoogle Scholar
  29. 29.
    Zhang LS, Hu S, Che X, Bai XH, Li QS (2013) A new ionic liquid-water-organic solvent three phase microextraction for simultaneous preconcentration flavonoids and anthraquinones from traditional Chinese prescription. J Pharm Biomed Anal 86:36–39CrossRefGoogle Scholar
  30. 30.
    Sharma N, Sharma UK, Malik S, Bhushan S, Kumar V, Verma SC, Sharma N, Sharma M, Sinha AK (2008) Isolation and purification of acetylshikonin and β-acetoxyisovalerylshikonin from cell suspension cultures of Arnebia euchroma (Royle) Johnston using rapid preparative HPLC. J Sep Sci 31:629–635CrossRefGoogle Scholar
  31. 31.
    Gutowski KE, Broker GA, Willauer HD, Huddleston JG, Swatloski RP, Holbrey JD, Rogers RD (2003) Controlling the aqueous miscibility of ionic liquids: aqueous biphasic systems of water-miscible ionic liquids and water-structuring salts for recycle, metathesis, and separations. J Am Chem Soc 125:6632–6633CrossRefGoogle Scholar
  32. 32.
    Kaler EW, Murthy AK, Rodriguez BE, Zasadzinski JAN (1989) Spontaneous vesicle formation in aqueous mixtures of single-tailed surfactants. Science 245:1371–1374CrossRefGoogle Scholar
  33. 33.
    Tan ZJ, Li FF, Xu XL (2012) Isolation and purification of aloe anthraquinones based on an ionic liquid/salt aqueous two-phase system. Sep Purif Technol 98:150–157CrossRefGoogle Scholar
  34. 34.
    Wu B, Zhang YM, Wang HP (2008) Aqueous biphasic systems of hydrophilic ionic liquids + sucrose for separation. J Chem Eng Data 53:983–985CrossRefGoogle Scholar
  35. 35.
    Shukla SK, Pandey S, Pandey S (2018) Applications of ionic liquids in biphasic separation: aqueous biphasic systems and liquid-liquid equilibria. J Chromatogr A 1559:44–61CrossRefGoogle Scholar
  36. 36.
    Liu Q, Chen XX, Guo Y, Han CH, Li J, Jia LH, Liu J, Wei XL (2019) Thermodynamic study of the aqueous two-phase systems of 1-butyl-3-methylimidazolium tetrafluoroborate and sodium dodecylbenzenesulfonate. J Mol Liq 279:18–22CrossRefGoogle Scholar
  37. 37.
    Nan YQ, Liu HL, Hu Y (2006) Interfacial tension in phase-separated aqueous cationic/anionic surfactant mixtures. J Colloid Interface Sci 293:464–474CrossRefGoogle Scholar
  38. 38.
    Yu W, Liu ZL, Li Q, Zhang HQ, Yu Y (2015) Determination of sudan I-IV in candy using ionic liquid/anionic surfactant aqueous two-phase extraction coupled with high-performance liquid chromatography. Food Chem 173:815–820CrossRefGoogle Scholar
  39. 39.
    Yang X, Zhang SH, Yu W, Liu ZL, Lei L, Li N, Zhang HQ, Yu Y (2014) Ionic liquid-anionic surfactant based aqueous two-phase extraction for determination of antibiotics in honey by high-performance liquid chromatography. Talanta 124:1–6CrossRefGoogle Scholar
  40. 40.
    Li FF, Liu Y, Lin WP (2018) Phase equilibrium and protein partitioning in aqueous two-phase systems containing imidazolium ionic liquids and surfactant at low voltage levels. J Mol Liq 256:372–379CrossRefGoogle Scholar
  41. 41.
    Vicente FA, Lario LD, Pessoa A Jr, Ventura SPM (2016) Recovery of bromelain from pineapple stem residues using aqueous micellar two-phase systems with ionic liquids as co-surfactants. Process Biochem 51:528–534CrossRefGoogle Scholar
  42. 42.
    Pharmacopoeia Committee of China (2015) Chinese pharmacopoeia. Chemical Industrial Press, BeijingGoogle Scholar
  43. 43.
    Martins MAR, Neves CMSS, Kurnia KA, Carvalho PJ, Rocha MAA, Santo LMNBF, Pinho SP, Freire MG (2016) Densities, viscosities and derived thermophysical properties of water-saturated imidazolium-based ionic liquids. Fluid Phase Equilib 407:188–196CrossRefGoogle Scholar
  44. 44.
    Shang Y, Liu H, Hu Y, Prausnitz JM (2007) Effect of salts on the aqueous two-phase system in mixed solutions of Gemini (12-3-12,2Br) and sodium dodecyl sulfate. Colloids Surf A 302:58–66CrossRefGoogle Scholar
  45. 45.
    Yang X, Yu R, Zhang SH, Cao BC, Liu ZL, Lei L, Li N, Wang ZB, Zhang LY, Zhang HQ, Chen YH (2014) Aqueous two-phase extraction for determination of triazine herbicides in milk by high-performance liquid chromatography. J Chromatogr B 972:111–116CrossRefGoogle Scholar
  46. 46.
    Yu W, Liu ZL, Gao SQ, Cui SS, Yang X, Qiu W, Zhang HQ, Yu AM, Huan YF (2013) Determination of sulfonamides in blood using acetonitrile-salt aqueous two-phase extraction coupled with high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Anal Methods 5:5983–5989CrossRefGoogle Scholar
  47. 47.
    Guan LY, Luo Q, Shi JY, Yu W (2018) Application of ionic-liquid-magnetized stirring bar liquid-phase microextraction coupled with HPLC for the determination of naphthoquinones in Zicao. J Sep Sci 4:868–876CrossRefGoogle Scholar
  48. 48.
    Guo YX, Han J, Zhang DY, Wang LH, Zhou LL (2012) An ammonium sulfate/ethanol aqueous two-phase system combined with ultrasonication for the separation and purification of lithospermic acid B from Salvia miltiorrhiza Bunge. Ultrason Sonochem 19:719–724CrossRefGoogle Scholar
  49. 49.
    Yu W, Li K, Liu ZL, Zhang HQ, Jin XQ (2018) Novelty aqueous two-phase extraction system based on ionic liquid for determination of sulfonamides in blood coupled with high-performance liquid chromatography. Microchem J 136:263–269CrossRefGoogle Scholar
  50. 50.
    Woo YA, Kim HJ, Cho JH, Chung H (1999) Discrimination of herbal medicines according to geographical origin with near infrared reflectance spectroscopy and pattern recognition techniques. J Pharm Biomed Anal 21:407–413CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Qianhui Sun
    • 1
  • Baiquan Du
    • 1
  • Chenzhao Wang
    • 1
  • Weili Xu
    • 1
  • Zhuang Fu
    • 1
  • Yu Yan
    • 1
  • Shouzhi Li
    • 1
  • Zhibing Wang
    • 1
    • 2
    Email author
  • Hanqi Zhang
    • 2
  1. 1.College of Chemistry and Life ScienceChangchun University of TechnologyChangchunPeople’s Republic of China
  2. 2.College of ChemistryJilin UniversityChangchunPeople’s Republic of China

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