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Published in final edited form as: J Liq Chromatogr Relat Technol. 2013 March 1; 36(8): 983–999.

Preparative isolation and purification of iridoid glycosides from Fructus Corni by high-speed countercurrent chromatography Jinru Liang1, Jiao He1, Sha Zhu1, Wenna Zhao1, Yongmin Zhang2, Yoichiro Ito3, and Wenji Sun1,* 1Biomedicine

Key Laboratory of shaanxi province, Northwest University, Xi’an 710069, China

2Institut

Parisien de Chimie Moléculaire, Université Pierre et Marie Curie-Paris 6 (UMR CNRS 7201), 4 place Jussieu, 75005 Paris, France

3Bioseparation

Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

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Abstract Using a two-phase solvent system composed of dichloromethane–methanol–n-butanol–water– acetic acid (5:5:3:4:0.1, v/v/v/v/v), high-speed countercurrent chromatography was successfully performed for isolation and purification of three iridoid glycosides from Fructus Corni for the first time. From 100 mg of a crude extract of Fructus Corni 7.9 mg of sweroside, 13.1 mg of morroniside, and 10.2 mg of loganin were obtained in less than 3 h with purities of 92.3, 96.3 and 94.2%, respectively. These target compounds were identified by ESI-MS, 1H NMR and 13C NMR.

Keywords high-speed countercurrent chromatography; Fructus Corni; iridoid glycosides

INTRODUCTION NIH-PA Author Manuscript

Fructus Corni, which is called Shanzhuyu in Chinese, is dry ripe sarcocarp of the cornaceae plant Cornus officinalis Sieb. et Zucc. and is considered as a precious herb and food material in China. Both crude and precessed Fructus Corni are frequently used as an important traditional Chinese medicine (TCM) clinically used for the treatment of various health problems such as spontaneous perspiration, night sweating, spermatorrhea, enuresis, vertigo, tinnitus, impotence, metrorrhagia and pain in waist and knees[1]. Phytochemistry research during the past several decades showed that iridoid glycosides including morroniside, sweroside and loganin are the main active components in Fructus Corni[2]. Pharmacological studies on these components showed that they all have good biological activities. Morroniside showed the effect of neuroprotection[3,4], neural stem cell and mesangial cell proliferation[5,6], inhibiting abnormal lipid metabolism and inflammation due to reactive oxygen species in the kidneys in type 2 diabetes[7]. Sweroside showed antipyretic and

*

Corresponding author: Wenji Sun. Biomedicine Key Laboratory of Shaanxi Province, Northwest University, No. 229 Taibai North Road, Xi’an 710069, People’s Republic of China [email protected].

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antishock effect [8] and could resist D-aminogalactose hepatic injury [9]. Loganin exhibits Neuroprotection[10], anti-amnesic activity[11,12], protective effect against neurodegenerative diseases[13], hepatic injury and other diabetic complications[14]. These three components could be considered as biologically active components representative in Fuctus Corni. Considering such good biological activities of three iridoid glycosides, it is important to develop an efficient method to isolate and purify each of them with high purity for quality control and pharmacological research. At present, the conventional methods including column chromatography and preparative HPLC were used for the separation and purification of iridoid glycosides from Fuctus Corni, which require several steps and result in unsatisfactory sample recovery. Moreover, repeated column chromatography always consumes large amounts of organic solvents. Therefore, a green and preparative separation method is of great interest in recent years.

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High-speed counter-current chromatography (HSCCC) is a support-free liquid–liquid partition chromatography, which has an excellent sample recovery, shorter isolation time, wider range of selection of two-phase solvent systems, compared with the conventional column method [15–17]. It eliminates the risk of irreversible adsorption of sample components that is often caused by solid supports used in conventional column chromatography. In HSCCC the separation process is entirely based on the composition of the two-phase solvent system, which provides an ideal partition coefficient of the target compound between the mobile and stationary phases. HSCCC has been widely used for separation and purification from various natural products for years [18–23]. However, to our knowledge, no report was focused on the isolation and purification of iridoid glycosides from Fuctus Corni by HSCCC. In this paper, we would like to report on an efficient method for separation and purification of three iridoid glycosides including sweroside, morroniside and loganin. (Fig. 1) from Traditional Chinese medicine Fuctus Corni.

EXPERIMENTAL Apparatus

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The preparative HSCCC instrument employed in the present study was a TBE-300A highspeed countercurrent chromatograph (Tauto Biotech Co., Shanghai, China) with three multilayer coil separation columns connected in series (I. D. of the tubing = 1.5 mm, total volume = 280 mL) and a 20 mL sample loop. The β values of the multilayer coil varied from 0.5 at the internal terminal to 0.8 at the external terminal (b = r/R, where r is the distance from the coil to the holder shaft and R is the revolution radius or the distance between the holder axis and central axis of the centrifuge). The rotation speed of the apparatus could be ranged from 0 to 1000 rpm, and 850 rpm was used in the present study. An HX-1050 constant-temperature circulating implement was used to control the separation temperature. The solvent was pumped into the column with a model TBP5002 constant flow pump (Tauto Biotech Co. Ltd, Shanghai, China). Continuous monitoring of the effluent was achieved with a Model 500A-UV Monitor (Tauto Biotech Co. Ltd, Shanghai, China) at 240 nm. The data were collected with the Model N2000 chromatography workstation (Zhejiang University, Hangzhou, China).

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The high-performance liquid chromatography (HPLC) analysis was performed using a Waters Alliance 2695 system (Waters, Milford, MA, USA) equipped with a vacuum degasser, a low pressure quaternary pump, an autosampler and a dual-k absorbance detector, controlled by “Empower” software. A Welchrom C18 column (250 mm×4.6 mm, i.d., 5 μm) was used to separate iridoid glycosides. The nuclear magnetic resonance (NMR) spectrometer was a Bruker DRX-500 spectrometer (Bruker BioSpin, Rheinstetten, Germany). Reagents and Plant Materials

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Dichloromethane, methanol and n-butanol for preparation of crude samples and HSCCC separation were all of analytical grades and were purchased from Tianjin Chemical Factory (Tianjin, China). Methanol used for HPLC analyses was of chromatographic grade and purchased from Supervision of Kermel Chemical Reagents Development Center (Tianjin, China). Phosphoric acid used for HPLC analysis was of analytical grade and purchased from Beijing Chemical Factory (Beijing, China). Water was purified in a Milli-Q plus system (Millipore, Madrid, Spain). Macroporous resin were purchased from Xi’an LanXiao technology Co., LTD ( Xi’an, China). Fructus Corni was purchased from Xi’an Wanshou Road Chinese crude drug market (Shaanxi, China). Preparation of Crude Extract Powdered Fructus Corni (2.0 kg) were first extracted by refluxing in 18 L of 75% ethanol for three times. Then the ethanol extracts were pooled and concentrated at 80°C under reduced pressure. The obtained crude extracts (212.5 g) were dissolved in water (500 mL) and passed through a glass column (5 cm × 40 cm, containing 600 g macroporous resin), using water as elution untill the effluents became colourless. Then, the column was eluted with 30% (v/v) ethanol (7-fold of column volume). The ethanol effluents were then combined and evaporated under reduced pressure to give 97.5 g of crude sample which was submitted to HSCCC separation. Selection of Two-Phase Solvent System

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The two-phase solvent system was selected according to the partition coefficient (K) of each target component. The K values were determined by HPLC analysis as follows: a suitable amount of crude sample was added into a 10 mL test tube to which about 2 mL of each phase of the pre-equilibrated two-phase solvent system were added. The capped tube was shaken vigorously for 1 min to thoroughly equilibrate the sample between the two phases. Then, an equal volume of the upper and lower phases were transferred and evaporated separately. The residue of each phase was dissolved in an equal volume of methanol and analyzed by HPLC determine K value of each component. The peak area of the upper phase was recorded as AU (area of upper phase) and that of the lower phase was recorded as AL (area of lower phase). The K value was calculated according to the following equation: K = AU/AL.

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Preparation of Two-Phase Solvent System and Sample Solution

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The two-phase solvent system composed of dichloromethane–methanol–n-butanol–0water– acetic acid (5:5:3:4:0.1, v/v/v/v/v) was mixed and equilibrated thoroughly in a separatory funnel at room temperature and left for one night. Then the two phases were separated and degassed by sonication for 30 min prior to use. The sample solution for HSCCC separation was prepared by dissolving 100 mg of crude extract in the mixture of 5 mL of each phase used for separation. HSCCC Separation Procedure

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The preparative separation was performed with the Model TBE300A HSCCC apparatus with the selected solvent system composed of dichloromethane–methanol–n-butanol–water– acetic acid (5:5:3:4:0.1, v/v/v/v/v). The multilayer coil column was first entirely filled with the upper stationary phase. Then the HSCCC apparatus was rotated at 850 rpm. Meanwhile, the lower mobile phase was pumped into the head inlet of the coiled column at a flow rate of 1.5 mL min−1. After a clear mobile phase eluting at the tail outlet indicatng that hydrodynamic equilibrium was reached, the sample solution (100 mg of the crude extract in 5 mL of each phases was injected through the injection valve. The effluent from the tail end of the column was continuously monitored with a UV detector at 240 nm and the chromatogram was recorded. Each peak fraction was collected into the test tubes with a fraction detector set at 5 min for each tube. Peak fractions were collected and analyzed by HPLC, ESI-MS and NMR. HPLC Analysis and Identification of HSCCC Peak Fractions HPLC analyses of the crude sample and HSCCC peak fractions were performed with a Welchrom C18 column (250 mm×4.6 mm, i.d., 5 μm) at 35 °C. The mobile phase was methanol–water (containing 0.1% phosphoric acid) in the gradient mode as follows: 28% methanol for 0–8 min and 35% methanol for 8–15 min. The flow rate was kept at 1.0 mL min-1 and UV detection was set at 240 nm. The injection volume was 10 mL. All solvents were filtered through a 0.45 μm filter before use. Each peak fraction from HSCCC separation was collected according to the obtained chromatogram and evaporated under reduced pressure and then dissolved in methanol for HPLC analysis. The area normalization method was used to determine the purity of each component by HPLC.

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To identify HSCCC peak fractions, ESI-MS experiment was carried out using a Thermo Scientific LTQ XL ion trap mass spectrometer (Themo Finnigan, San Jose, CA, USA) equipped with an electrospray ionization source, and NMR spectra were recorded with an INOVA spectrometer (Varian Co. Ltd, America).

RESULTS AND DISCUSSION Selection of Two-Phase Solvent System and Other Conditions of HSCCC In order to achieve an ideal separation result, the selection of a two-phase solvent system is the most significant step in HSCCC separation. The partition coefficient (K) is the ratio of solute distributed between the mutually equilibrated two solvent phase, the suitable K values for HSCCC are between 0.5 and 1.0. If the K value is smaller, the compounds would be

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eluted near the solvent front without sufficient peak resolution, while a larger K value tends to give better resolution but broader, more dilute peaks due to a longer elution time [15]. In order to achieve an ideal separation of target compounds, a series of two-phase solvent systems have been carefully screened to optimize the solvent system for HSCCC separation and their K values of the target compounds were measured. The results indicated that the two-phase solvent system of dichloromethane–methanol–water (5:4:3, v/v/v) and ethyl acetate–n-butanol–water (4:3:2, 4:4:2, v/v/v) gave too large K values to all target compounds, while dichloromethane–methanol–n-butanol–water system (5:5:2:4, v/v/v/v) were found to have suitable K values (sweroside, K = 1.41; morroniside, K = 1.21; loganin, K = 1.53), but the retention of the stationary phase was too low to give a peak resolution. Dichloromethane–methanol–n-butanol–water at a volume ratio of 5:5:3:4 gave suitable K values with a satisfactory level of stationary phase retention.. Acetic acid was added to this system as a modifier for better separation. Among all two-phase solvent systems examined, dichloromethane–methanol–n-butanol–water–acetic acid (5:5:3:4:0.1 v/v/v/v/v) was found to be satisfactory for separation of target compounds from the crude sample (sweroside, K = 1.81; morroniside, K = 1.70; loganin, K = 1.93).

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We have also investigated other factors such as the flow rate of the mobile phase, the temperature and the revolution speed of the separation on HSCCC. To shorten the separation time while still maintaining an adequate resolution, the flow rate of mobile phase was selected at 1.5 mL min−1. The high revolution speed at 900 rpm resulted in loss of peak resolution. Meanwhile, we found that increasing temperature could improve the HSCCC separation, but a high temperature may run the risk of stripping away the stationary phase due to the emulsification of the solvent system. So the optimum separation condition was determined as follows: the flow rate was 1.5 mL/min, the revolution speed was 850 rpm and the separation temperature was set at 25 °C. Under this experimental condition the retention of the stationary phase was 44%. HSCCC Separation

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Figure 2 shows the HSCCC chromatogram of the preparative separation of 100 mg crude sample using the above selected two-phase solvent system composed of dichloromethane– methanol–n-butanol–water–acetic acid (5:5:3:4:0.1, v/v). The retention of the stationary phase was 44%, and the total separation time was 180 min. This separation yielded 7.9 mg of compound 1 (fraction collected during 115–130 min), 13.1 mg of compound 2 (fraction collected during 135–155 min) and 10.2 mg of compound 3 (fraction collected during 160– 175 min), with 7.9, 13.1 and 10.2%, respectively. The purities of compounds 1, 2 and 3 were 92.3, 96.3 and 94.2% yields, respectively, that were directly determined by NMR analyses. Figure 3 shows the HPLC chromatogram of the crude sample and in the combined peak fractions of each iridoid glycoside. Structural Identification Compounds 1, 2 and 3 were identified as sweroside, morroniside and loganin, respectively, by comparison of ESI-MS, 1H NMR and 13C NMR data with those in references [24–29].

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CONCLUSION NIH-PA Author Manuscript

The results of our studies show that in a one-step operation, HSCCC can provide a highly efficient preparative separation of sweroside, morroniside and loganin, respectively. This is the first example to use HSCCC for the separation and purification of the iridoid glycosides from Fructus Corni, which could be used for further chemical research and pharmacological studies or as reference substances.

Acknowledgments The authors thank Prof. Zhongfu Wang of the Key Laboratory of Resource Biology and Biotechnology in western China for assistance in ESI-MS experiments.

References

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1. Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China [M]. Vol. 1. China Medical Science and Technology Publishing Co; Beijing: 2010. p. 25-26. 2. Cao G, Zhang Y, Cong XD, Cai H, Cai BC. J Chin Pharmaceu Sci. 2009; 18:208–213. 3. Wang W, Sun FL, An Y, Ai HX, Zhang L, Huang WT, Li L. Eur J Pharmacol. 2009; 613:19–23. [PubMed: 19379729] 4. Wang W, Xu JD, Li L, Wang PC, Ji XM, Ai HX, Zhang L, Li L. Brain Res Bull. 2010; 83:196–201. [PubMed: 20637265] 5. Wang W, Huang WT, Li L, Ai HX, Sun FL. Cell Mol Neurobiol. 2008; 28:293–305. [PubMed: 17647102] 6. Xu HQ, Shen J, Liu H, Shi Y, Li L, Wei M. Can J Physiol Pharmacol. 2006; 4:1267–1273. [PubMed: 17487235] 7. Park CH, Noh JS, Tanaka T, Yokozawa T. J Pharm and Pharmacol. 2010; 62:374–380. [PubMed: 20487222] 8. Song WZ. Chin J Chin Mater Med. 1986; 11:643–647. 9. Hu RQ, Rao XY. Chin J Clin Hepatol. 1988; 4:41–43. 10. Kwon SH, Kim JA, Hong SI, Jung YH, Kim HC, Lee SY, Jang CG. Neurochem Int. 2011; 58:533– 541. [PubMed: 21241762] 11. Lee SJ, Shin EJ, Son KH, Son KH, Chang HW, Kang SS, Kim HP. Arch Pharma Res. 1995; 18:133–135. 12. Lee KY, Sung SH, Kim SH, Jang YP, Oh TH. Arch Pharmacal Res. 2009; 32:677–683. 13. Kwon SH, Kim HC, Lee SY, Jang CG. Eur J Pharmacol. 2009; 619:44–49. [PubMed: 19666019] 14. Yamabe N, Noh JS, Park CH, Kang KS, Shibahara N, Tanaka T, Yokozawa T. Eur J Pharmacol. 2010; 648:179–187. [PubMed: 20826139] 15. Ito Y. J Chromatogr A. 2005; 1065:145–168. [PubMed: 15782961] 16. Wood P, Ignatova S, Janaway L, Keay D, Hawes D, Garrard I, Sutherland IA. Chromatogr A. 2007; 1151:25–30. 17. Huang XY, Fu JF, Di DL. Sep Purif Technol. 2010; 71:220–224. 18. Ou-Yang XK, Jin MC, He CH. Sep Purif Technol. 2007; 56:319–324. 19. Sun QH, Sun AL, Liu RM. J Chromatogr A. 2006; 1104:69–74. [PubMed: 16364341] 20. Yin H, Zhang S, Luo XM, Liu YH. J Chromatogr A. 2009; 1205:177–181. [PubMed: 18723179] 21. Yang Y, Huang Y, Gu DY, Yili A, Sabir G, Aisa HA. Chromatographia. 2009; 69:963–967. 22. Xiao GD, Li GW, Chen L, Zhang ZJ, Yin JJ, Wu T, Cheng ZH, Wei XH, Wang ZT. J Chromatogr A. 2010; 1217:5470–5476. [PubMed: 20663508] 23. Wei Y, Xie QQ, Fisher D, Sutherland IA. Chromatographia. 2009; 70:1185–1189. 24. Zhao SP, Xue Z. Acta Pharm Sin. 1992; 27:845–848. 25. Yang J, Chen SQ, Ji CR, Liu YZ. Chin Trad and Herb Drugs. 2005; 36:1780–1782.

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Figure 1.

Chemical structures of three iridoid glycosides

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NIH-PA Author Manuscript Figure 2.

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HSCCC separation chromatogram of the crude extract from Fructus Corni. Solvent system: dichloromethane–methanol–n-butanol–water (5:5:3:4, v/v); stationary phase: upper phase; flow rate of the mobile phase: 1.5 mL min−1; revolution speed: 850 rpm; column temperature: 25 °C; sample: 100 mg of crude extract dissolved in 10 mL two-phase solvent system; UV detector:set at 240 nm. (peak 1: sweroside); (peak 2: morroniside); (peak 3: loganin)

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NIH-PA Author Manuscript Figure 3.

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HPLC chromatograms of crude extracts and HSCCC peak fractions. Conditions: column, Welchrom C18 column (250 × 4.6 mm, 5 μm); mobile phase: methanol–water (containing 0.1% phosphoric acid) in gradient mode as follows: acetonitrile: 0–8 min, 28%; 8–15 min, 35%; flow rate: 1.0 mL/min; detection wavelength: 254 nm; column temperature 30 °C; injection volume, 20 μL. (a) Crude sample; (b) combined fractions, peak 1; (c) combined fractions, peak 2; (d) combined fractions, peak 3

NIH-PA Author Manuscript J Liq Chromatogr Relat Technol. Author manuscript; available in PMC 2014 June 02.

Preparative isolation and purification of iridoid glycosides from Fructus Corni by high-speed countercurrent chromatography.

Using a two-phase solvent system composed of dichloromethane-methanol-n-butanol-water-acetic acid (5:5:3:4:0.1, v/v/v/v/v), high-speed countercurrent ...
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