Food Analytical Methods

, Volume 11, Issue 4, pp 980–991 | Cite as

Partition Behaviors of Different Polar Anthocyanins in Aqueous Two-Phase Systems and Extraction of Anthocyanins from Nitraria tangutorun Bobr. and Lycium ruthenicum Murr.

  • Jun Sang
  • Kai-kai Dang
  • Qun Ma
  • Bing Li
  • Ya-ya Huang
  • Cui-qin Li


This study aimed to investigate the partition behaviors of various polar anthocyanins in NaH2PO4/(NH4)2SO4-ethanol aqueous two-phase systems (ATPS) and to extract anthocyanins from Nitraria tangutorun Bobr. and Lycium ruthenicum Murr. Anthocyanins in Hibiscus sabdariffa L., Morus atropurpurea Roxb., N. tangutorun, and L. ruthenicum were profiled using HPLC-ESI-MS/MS and HPLC-DAD, and the partition behaviors of total anthocyanins and main anthocyanins were studied. The partition coefficient of anthocyanins increased with increased hydrophobicity, and low-polarity anthocyanins exhibited a higher preference for the top phase in NaH2PO4/(NH4)2SO4-ethanol ATPS. Additionally, the NaH2PO4-ethanol ATPS gave higher selectivity and total anthocyanin yield than the (NH4)2SO4-ethanol system. Extraction at 65 °C for 45 min and at 45.5 °C for 45 min using 28% NaH2PO4 and 26% ethanol (w/w) led to the recovery of 98.91 ± 0.03% of N. tangutorun anthocyanins (3.62 ± 0.05 mg/g) and 99.84 ± 0.01% of L. ruthenicum anthocyanins (13.16 ± 0.29 mg/g) from raw material; more than 70% of total sugars were removed in a single step. NaH2PO4-ethanol aqueous two-phase extraction is a promising method for extracting anthocyanins from N. tangutorun and L. ruthenicum.


Anthocyanins Aqueous two-phase extraction HPLC-ESI-MS/MS Response surface methodology Natural pigments Food analysis Food composition 



We thank Prof. Zhe-zhi Wang for his generosity in sharing his lab facilities.


This work was supported by the Fundamental Research Funds for the Central Universities [grant number GK201303009] and Science and Technology Cooperation Project between Shaanxi Province and Qinghai Province [grant number 2014SJ-07].

Compliance with Ethical Standards

Conflict of Interest

Jun Sang declares that he has no conflict of interest. Kai-kai Dang declares that he has no conflict of interest. Qun Ma declares that she has no conflict of interest. Bing Li declares that she has no conflict of interest. Ya-ya Huang declares that she has no conflict of interest. Cui-qin Li declares that she has no conflict of interest.

Ethical Approval

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

Informed Consent

Not applicable.

Supplementary material

12161_2017_1071_MOESM1_ESM.docx (2.7 mb)
ESM 1 (DOCX 2716 kb).


  1. Barnes JS, Schug KA (2011) Structural characterization of cyanidin-3,5-diglucoside and pelargonidin-3,5-diglucoside anthocyanins: multi-dimensional fragmentation pathways using high performance liquid chromatography-electrospray ionization-ion trap-time of flight mass spectrometry. Int J Mass Spectrom 308:71–80CrossRefGoogle Scholar
  2. Barnes JS, Nguyen HP, Shen S, Schug KA (2009) General method for extraction of blueberry anthocyanins and identification using high performance liquid chromatography-electrospray ionization-ion trap-time of flight-mass spectrometry. J Chromatogr A 1216:4728–4735CrossRefGoogle Scholar
  3. Bensch M, Selbach B, Hubbuch J (2007) High throughput screening techniques in downstream processing: preparation, characterization and optimization of aqueous two-phase systems. Chem Eng Sci 62:2011–2021CrossRefGoogle Scholar
  4. Chen S, Zeng Z, Hu N, Bai B, Wang H, Suo Y (2018) Simultaneous optimization of the ultrasound-assisted extraction for phenolic compounds content and antioxidant activity of Lycium ruthenicum Murr. Fruit using response surface methodology. Food Chem 242:1–8CrossRefGoogle Scholar
  5. Da-Costa-Rocha I, Bonnlaender B, Sievers H, Pischel I, Heinrich M (2014) Hibiscus Sabdariffa L. – a phytochemical and pharmacological review. Food Chem 165:423–443CrossRefGoogle Scholar
  6. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith P (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  7. Espitia-Saloma E, Vázquez-Villegas P, Aguilar O, Rito-Palomares M (2014) Continuous aqueous two-phase systems devices for the recovery of biological products. Food Bioprod Process 92:101–112CrossRefGoogle Scholar
  8. Fattahi M, Rahimi R (2016) Optimization of extraction parameters of phenolic antioxidants from leaves of Capparis Spinosa using response surface methodology. Food Anal Methods 9:2321–2334CrossRefGoogle Scholar
  9. Giusti MM, Wrolstad RE (2003) Acylated anthocyanins from edible sources and their applications in food systems. Biochem. Biochem Eng J 14:217–225CrossRefGoogle Scholar
  10. 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
  11. Jampani C, Raghavarao K (2015) Process integration for purification and concentration of red cabbage (Brassica Oleracea L.) anthocyanins. Sep Purif Technol 141:10–16CrossRefGoogle Scholar
  12. Jin H, Liu Y, Yang F, Wang J, Fu D, Zhang X, Peng X, Liang X (2015) Characterization of anthocyanins in wild Lycium ruthenicum Murray by HPLC-DAD/QTOF-MS/MS. Anal Methods 7:4947–4956CrossRefGoogle Scholar
  13. Khazaei KM, Jafari SM, Ghorbani M, Kakhki AH, Sarfarazi M (2016) Optimization of anthocyanin extraction from saffron petals with response surface methodology. Food Anal Methods 8:1993–2001CrossRefGoogle Scholar
  14. Kong J, Chia L, Goh N, Chia T, Brouillard R (2003) Analysis and biological activities of anthocyanins. Phytochemistry 64:923–933CrossRefGoogle Scholar
  15. Liang L, Wu X, Zhao T, Zhao J, Li F, Zou Y, Mao G, Yang L (2012) In vitro bioaccessibility and antioxidant activity of anthocyanins from mulberry (Morus Atropurpurea Roxb.) following simulated gastro-intestinal digestion. Food Res Int 46:76–82CrossRefGoogle Scholar
  16. Liu Y, Han J, Wang Y, Lu Y, Zhang G, Sheng C, Yan Y (2013a) Selective separation of flavones and sugars from honeysuckle by alcohol/salt aqueous two-phase system and optimization of extraction process. Sep Purif Technol 118:776–783CrossRefGoogle Scholar
  17. Liu X, Mu T, Sun H, Zhang M, Chen J (2013b) Optimisation of aqueous two-phase extraction of anthocyanins from purple sweet potatoes by response surface methodology. Food Chem 141:3034–3041CrossRefGoogle Scholar
  18. Liu Z, Dang J, Wang Q, Yu M, Jiang L, Mei L, Shao Y, Tao Y (2013c) Optimization of polysaccharides from Lycium ruthenicum fruit using RSM and its anti-oxidant activity. Int J Biol Macromol 61:127–134CrossRefGoogle Scholar
  19. Lu M, Li Z, Liang H, Shi M, Zhao L, Li W, Chen Y, Wu J, Wang S, Chen X, Yuan Q, Li Y (2015) Controlled release of anthocyanins from oxidized konjac glucomannan microspheres stabilized by chitosan oligosaccharides. Food Hydrocoll 51:476–485CrossRefGoogle Scholar
  20. Ma F, Gu C, Li C, Luo M, Wang W, Zu Y, Li J, Fu Y (2013) Microwave-assisted aqueous two-phase extraction of isoflavonoids from Dalbergia Odorifera T. Chen leaves. Sep Purif Technol 115:136–144CrossRefGoogle Scholar
  21. Ni W, Gao T, Wang H, Du Y, Li J, Li C, Wei L, Bi H (2013) Anti-fatigue activity of polysaccharides from the fruits of four Tibetan plateau indigenous medicinal plants. J Ethnopharmacol 150:529–535CrossRefGoogle Scholar
  22. Raghavarao KSMS, Ranganathan TV, Srinivas ND, Barhate RS (2003) Aqueous two phase extraction–an environmentally benign technique. Clean Technol Environ 5:136–141CrossRefGoogle Scholar
  23. Rodríguez-Medina IC, Beltrán-Debón R, Molina VM, Alonso-Villaverde C, Joven J, Menéndez JA, Segura-Carretero A, Fernández-Gutiérrez A (2009) Direct characterization of aqueous extract of Hibiscus Sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS. J Sep Sci 32:3441–3448CrossRefGoogle Scholar
  24. Rogers RD, Willauer HD, Griffin ST, Huddleston JG (1998) Partitioning of small organic molecules in aqueous biphasic systems. J Chromatogr B 711:255–263CrossRefGoogle Scholar
  25. Sang J, Sang J, Ma Q, Hou X, Li C (2017a) Extraction optimization and identification of anthocyanins from Nitraria tangutorun Bobr. Seed meal and establishment of a green analytical method of anthocyanins. Food Chem 218:386–395CrossRefGoogle Scholar
  26. Sang J, Ma Q, Li B, Li C (2017b) An approach for extraction, purification, characterization and quantitation of acylated-anthocyanins from Nitraria tangutorun Bobr. Fruit Food Measure.
  27. Tan Z, Wang C, Yi Y, Wang H, Li M, Zhou W, Tan S, Li F (2014) Extraction and purification of chlorogenic acid from ramie (Boehmeria Nivea L. gaud) leaf using an ethanol/salt aqueous two-phase system. Sep Purif Technol 132:396–400CrossRefGoogle Scholar
  28. Wang L, Ding C, Wang H, Che G, Suo Y (2008) Extraction process of polysaccharide from Nitraria tangutorum Bobr. Food Sci 29:233–236Google Scholar
  29. Wang Y, Han J, Xu X, Hu S, Yan Y (2010) Partition behavior and partition mechanism of antibiotics in ethanol/2-propanol-ammonium sulfate aqueous two-phase systems. Sep Purif Technol 75:352–357CrossRefGoogle Scholar
  30. Wu X, Liang L, Zou Y, Zhao T, Zhao J, Li F, Yang L (2011) Aqueous two-phase extraction, identification and antioxidant activity of anthocyanins from mulberry (Morus Atropurpurea Roxb.) Food Chem 129:443–453CrossRefGoogle Scholar
  31. Wu Y, Wang Y, Zhang W, Han J, Liu Y, Hu Y, Ni L (2014) Extraction and preliminary purification of anthocyanins from grape juice in aqueous two-phase system. Sep Purif Technol 124:170–178CrossRefGoogle Scholar
  32. Wu T, Lv H, Wang F, Wang Y (2016) Characterization of polyphenols from Lycium ruthenicum fruit by UPLC-QTOF/MSE and their antioxidant activity in Caco-2 cells. J Agric Food Chem 64:2280–2288CrossRefGoogle Scholar
  33. Zheng J, Li H, Ding C, Suo Y, Wang L, Wang H (2011a) Anthocyanins composition and antioxidant activity of two major wild Nitraria tangutorun Bobr. Variations from Qinghai-Tibet plateau. Food Res Int 44:2041–2046CrossRefGoogle Scholar
  34. Zheng J, Ding C, Wang L, Li G, Shi J, Li H, Wang H, Suo Y (2011b) Anthocyanins composition and antioxidant activity of wild Lycium ruthenicum Murr. From Qinghai-Tibet plateau. Food Chem 126:859–865CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Jun Sang
    • 1
  • Kai-kai Dang
    • 1
  • Qun Ma
    • 1
  • Bing Li
    • 1
  • Ya-ya Huang
    • 1
  • Cui-qin Li
    • 1
  1. 1.Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China, College of Life SciencesShaanxi Normal UniversityXi’anChina

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