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Preparative isolation and purification of two benzoxazinoid glucosides from Acanthus ilicifolius L. by high-speed counter-current chromatography
Journal of Chromatography A, 1205 (2008) 177–181
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Preparative isolation and purification of two benzoxazinoid glucosides from Acanthus ilicifolius L. by high-speed counter-current chromatography Hao Yin a,b , Si Zhang a,b,∗ , Xiongming Luo a , Yonghong Liu a a Guangdong Key Laboratory of Marine Material Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, Guangdong, China b Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Science, 164 West Xingang Road, Guangzhou 510301, Guangdong, China
a r t i c l e
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Article history: Received 6 June 2008 Received in revised form 29 July 2008 Accepted 4 August 2008 Available online 9 August 2008 Keywords: Benzoxazinoids (2R)-2-O--d-glucopyranosyl-2H-1,4benzoxazin-3(4H)-one (HBOA-Glc) (2R)-2-O--d-glucopyranosyl-4-hydroxy2H-1,4-benzoxazin-3(4H)-one (DIBOA-Glc) High-speed counter-current chromatography (HSCCC) Acanthus ilicifolius L
a b s t r a c t The first preparative separation of two benzoxazinoids, (2R)-2-O--d-glucopyranosyl-2H-1,4benzoxazin-3(4H)-one (HBOA-Glc) and (2R)-2-O--d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin3(4H)-one (DIBOA-Glc), by means of high-speed counter-current chromatography (HSCCC) from the n-butanol extract of Acanthus ilicifolius L. is presented. The two-phase solvent system containing ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v, system B) was selected for the one-step HSCCC separation of HBOA-Glc and DIBOA-Glc according to the partition coefficient values (K) for target compounds and the separation factor (˛) between the two target compounds. In the one-step HSCCC separation using solvent B, from 100 mg n-butanol extract of A. ilicifolius, 6.3 mg HBOA-Glc and 6.8 mg DIBOA-Glc were isolated with purities of 90.3% and 80.2%, respectively. In order to obtain the two target compounds with higher purity, a second separation process was developed comprising two steps. In the two-step separation, the sample was first pre-purified by HSCCC using ethyl acetate–n-butanol–water (2:3:5, v/v/v, system A) solvent system and then purified using solvent system B. A 100-mg amount of the n-butanol extracts of A. ilicifolius was separated to yield 5.8 mg of HBOA-Glc and 4.8 mg of DIBOA-Glc with purities of 97.1% and 94.8%, respectively, which were directly used for NMR analyses. © 2008 Elsevier B.V. All rights reserved.
1. Introduction High-speed counter-current chromatography (HSCCC), first invented by Ito [1], is widely used in the preparative separation of natural products. HSCCC does not employ a solid-phase support and thus there is no irreversible adsorption associated with the solid supports, and allows the direct application of crude extracts and an excellent recovery of the analytes [2]. In this study, a HSCCC separation and purification of the two main benzoxazinoids, (2R)-2O--d-glucopyranosyl-2H-1,4-benzoxazin-3(4H)-one (HBOA-Glc) and (2R)-2-O--d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin3(4H)-one (DIBOA-Glc), from the n-butanol extract of Acanthus ilicifolius L. are presented (Fig. 1). In Chinese folk medicine, all parts of A. ilicifolius have been used as crude drug for the treatment of lymphadenectasis, hepatitises, stomach aches, coughs, and asthmas [3]. Pharmacological
∗ Corresponding author at: Guangdong Key Laboratory of Marine Material Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, Guangdong, China. Tel.: +86 2089023105; fax: +86 2089023105. E-mail address: zhs [email protected] (S. Zhang). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.08.010
research had shown that the extracts from A. ilicifolius in particular had many bioactivities, including antivirus, anticarcinogeni, antioxidative, hepatoprotecti, antimutagenieit, and antiinflammatory activities [3–5]. In previous phytochemical investigations of this plant, six benzoxazinoids had been reported [3,6–8]. Benzoxazinoids are a unique class of natural products abundant in family of Gramineae, such as maize, wheat, rye, and wild grasses. Also they are found in different species of Acanthaceae, Ranunculaceae, Scrophulariaceae, and others [9]. In host plant, benzoxazinoids serve as important factors of resistance against microbial diseases and insects, allelochemics used in competition with other plants, and endogenous ligands. These metabolites also were proposed as natural pesticides for agricultural use by interdisciplinary investigations by biologists, biochemists, and chemists [10]. Therefore, the analyses and separation of benzoxazinoids are of great interest [6,10–12]. Being a support free liquid–liquid chromatography technology with no solid support matrix, HSCCC method for separation and purification of benzoxazinioids could be more efficient than conventional methods which are tedious and requires repeated chromatographic steps [6,8,11]. However, no report has been published on the use of HSCCC for separation and purification of this class of compounds.
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2.3. Preparation of sample
Fig. 1. Chemical structures of HBOA-Glc and DIMBOA-Glc.
The main purpose of this contribution was to study the isolation and purification possibility of the two main benzoxazinoids, HBOAGlc (1) and DIBOA-Glc (2), on a preparative scale from A. ilicifolius by HSCCC. As far as we know, this is the first report on successful isolation and purification of benzoxaziniods from a natural source using HSCCC. 2. Experimental 2.1. Apparatus HSCCC was carried out using a Model TBE-300A high-speed counter-current chromatography (Tauto Biotech, Shanghai, China) containing a self-balancing three-coil centrifuge rotor equipped three preparative multilayer coils with a total capacity of 260 mL, the internal diameter of PTFE tubing was 2.0 mm. The revolution radius or the distance between the holder axis and central axis of the centrifuge (R) was 9.5 cm. The ˇ value varied from 0.46 at the internal terminal to 0.73 at the external terminal (ˇ = r/R, where r is the distance from the coil to the holder shaft). The HSCCC system was equipped with a Model TBP-50 constant flow pump (Tauto Biotech, Shanghai, China), and a LC757 UV detector with preparative flow cell (Shanghai Huixing Instrument, China), a Model HX-1050 constant-temperature controller (Beijing Detianyou Technology, China), and a N2010 chromatogram workstation (Zhejiang University, Hangzhou, China). A Luna 5 m C18 100A ODS column (250 mm × 4.6 mm; Phenomenex, Torrance, CA, USA) was used for analytical HPLC, along with 600E multisolvent delivery system and a Waters 996 Photodiode Array Detector (Waters, Milford, MA, USA). NMR spectra were obtained on an Avance 500 magnetic resonance spectrometer (500 MHz for 1 H NMR, and 125 MHz for 13 C NMR; Bruker, Karlsruhe, Germany). UV spectra were measured with a coulter DU 640 nucleic acid and protein analyzer (Beckman, Fullerton, CA, USA). 2.2. Reagents All organic solvents used for HSCCC were of analytical grade and purchased from Baishi (Tianjing, China). Methanol and acetonitrile used for HPLC were obtained from Merck (Darmstadt, Germany). Water was purified by Milli-Q system (Millipore, Bedford, MA, USA), and ammonium hydroxide (25–28% NH3 stock solution) were from Guangzhou Chemical Reagent Factory (Guangzhou, Guangdong, China). HBOA-Glc and DIBOA-Glc standards (purity >95%, determined by HPLC) were obtained in our laboratory by column chromatography, identity of the isolated compounds were confirmed by comparing their spectroscopic properties (1 H and 13 C NMR) with those reported in the literature [8,13,14].
A. ilicifolius was collected in April 2006 from Hainan Province, Southern of China. The identification of the plant was confirmed by Professor Zhang Si, director of Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences. A voucher sample (GKLMMM-001A) is kept in the Herbarium of the South China Sea Institute of Oceanology). The dry powdered aerial part (1 kg) of A. ilicifolius was extracted with hot EtOH (95%) three times. After removal of the solvent by evaporation, the residue (135 g) was suspended in water and then successively extracted with petroleum ether, EtOAc and n-butanol. The n-butanol extract (40 g) was stored at 4 ◦ C before HSCCC separation. 2.4. Determination of partition coefficient Experimental procedure: approximately 1 mg of target compound was weighed in a 10-mL test tube into which 1 mL of each phase of the preequilibrated two-phase solvent system was added. The test tube was capped and shaken vigorously for 1 min, and allowed to stand until it separated completely. An aliquot of 100 L of each layer was taken out and evaporated separately to dryness in vacuo. The residue was dissolved in 100 L 10% acetonitrile and analyzed by HPLC for determining the partition coefficient (K) of compounds 1 and 2. The K value was expressed as the peak area of target compound in the upper phase divided by that in the lower phase. 2.5. Preparation of two-phase solvent system and sample solution Several ethyl acetate–n-butanol–water solvent systems with different volume ratios and their modified form (Table 1) were prepared. All these two-phase solvent systems were thoroughly equilibrated in a separation funnel by repeatedly vigorously shaking at room temperature, and then separated shortly before use. The following two two-phase solvent systems were selected for the HSCCC separation of HBOA-Glc and DIBOA-Glc from A. ilicifolius: • Solvent system A: ethyl acetate–n-butanol–water (2:3:5, v/v/v). • Solvent system B: ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v). The samples were dissolved in 4 mL of each phase of selected solvent systems for preparative HSCCC separation.
Table 1 The K (partition coefficient) values of compounds 1 and 2 in different solvent systemsa Solvent system (volume ratio)
HBOA-Glc
DIBOA-Glc
Ethyl acetate–n-BuOH–water (1:0:1) Ethyl acetate–n-BuOH–water (4:1:5) Ethyl acetate–n-BuOH–water (3:2:5) Ethyl acetate–n-BuOH–water (2:3:5), A Ethyl acetate–n-BuOH–1% acetic acid (2:3:5) Ethyl acetate–n-BuOH–1%NH4 OH (2:3:5) Ethyl acetate–n-BuOH–0.5%NH4 OH (2:3:5), B
0.01 0.13 0.52 0.67 0.65 0.57 0.72
0.01 0.12 0.49 0.62 0.63 0.01 0.03
a Experimental procedure: approximately 1 mg of target compound was weighed in a 10-mL test tube into which 1 mL of each phase of the preequlibrated solvent system was added. The test tube was capped and shaked vigorously for 1 min, and allowed to stand until it separated completely. An aliquot of 100 L of each layer was taken out and evaporated separately to dryness in vacuo. The residue was dissolved in 100 L 10% acetonitrile and analyzed by HPLC for determining the partition coefficient (K) of compounds 1 and 2. The K value was expressed as the peak area of target compound in the upper phase divided by that in lower phase.
H. Yin et al. / J. Chromatogr. A 1205 (2008) 177–181
2.6. HSCCC separation procedure Two protocols for preparation of HBOA-Glc and DIBOA-Glc from n-butanol A. ilicifolius, one-step and two-step HSCCC separation, were used in present study. With the exception of the two-phase solvent system and sample, all HSCCC separation steps were identical in operation, and were performed as following: the multilayer coiled column was first entirely filled with the upper organic phase of solvent system. The lower phase was then pumped into the head end of the column at a flow rate of 2.0 mL/min, while the apparatus was run at a revolution speed of 850 rpm. After hydrodynamic equilibrium was established, as indicated by a clear mobile phase eluting at the tail outlet, sample solution was injected through the sample port. The effluent from the tail end of the column was continuously monitored with a UV detector at 254 nm. Each peak fraction was collected according to the chromatogram, respectively. Analyses of fractions were performed by HPLC-diode array detection (DAD). The peak fractions of desired compounds were evaporated to dryness in vacuo for NMR analysis. In the one-step separation, 100 mg n-butanol extract dissolved in 4 mL of each phase of solvent system B was subjected to HSCCC using solvent system B directly (CCC 1) for preparation of target compounds. In the two-step separation, 100 mg n-butanol extract dissolved in 4 mL of each phase of solvent A was subjected to HSCCC using solvent system A for a pre-purification (CCC 2). The peak fraction containing target compounds confirmed by HPLC analysis was evaporated to dryness in vacuo, and subsequently was dissolved in 4 mL of each phase of solvent system B. The pre-purified sample solution was re-subjected to the HSCCC using solvent system B (CCC 3) for separation of compounds 1 and 2. 2.7. Analytical HPLC For the separation of analytes, the following gradient system was used: acetonitrile (A) and water (B); gradient program: 90% B, 0–15 min; 90% B to 30% B, 15–35 min; 30% B to 90% B, 35–40 min. The injection volume was 10 L. The mobile phase was eluted at a flow rate of 1.0 mL/min and the effluent was monitored by a UV detector at 254 nm. 2.8. NMR spectroscopy 1H
and 13 C NMR spectra of the isolated HBOA-Glc and DIBOAGlc were recorded in [2 H4 ]MeOH with TMS as an internal reference. Chemical shifts (ı) are reported in ppm and coupling constants (J) in Hertz (Hz). 2.9. Preparation of HBOA-Glc and DIBOA-Glc standards The n-butanol extract of A. ilicifolius (1 g) was subjected to Diaion HP-20, and eluted with H2 O, 10% MeOH, 30% MeOH, 60% MeOH and MeOH, successively. The fraction eluted with 10% MeOH was subjected to successive silica column chromatography (ethyl acetate-methanol-formic acid, 5:1:0.1, v/v/v) and repeated Sephadex LH-20 column chromatography (10% MeOH), to afford HBOA-Glc (27 mg) and DIBOA-Glc (24 mg). 3. Results and discussion 3.1. HSCCC solvent systems The selection of suitable solvent system is the first and critical step in performing preparative HSCCC. Two “golden rules for
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HSCCC” mentioned in the literature [2] were (1) “find systems with K (partition coefficient) values of the target compounds in a proper range: the suitable K values for HSCCC are 0.5 ≤ K ≤ 1.0” and (2) for HSCCC separation of two similar compounds, “the ratio of the two K values or the separation factor (˛ = K1 /K2 , where K1 > K2 ) ought to be greater than 1.5 in the semipreparative multilayer separation column of a commercial HSCCC unit”. In present study, the suitable solvent systems for HSCCC separation of HBOA-Glc and DIBOA-Glc were developed according these rules. The K values of target compounds in several classical solvent systems previously used for CCC separation of alkaloids, CHCl3 –MeOH–water, CHCl3 –ethanol–water, and nHex–EtOAc–MeOH–water [15–20], were tested, but none of them were successful because the target compounds were mostly distributed in the aqueous phase in these systems. Therefore, the search was directed to more polar solvent systems. Several ethyl acetate–n-butanol–water solvent systems with different volume ratios and their modified form were tested, and the measured K values of target compounds were summarized in Table 1. Among them, solvent system A, ethyl acetate–n-butanol–water (2:3:5, v/v/v), provided K values at 0.67 and 0.62 for compounds 1 and 2, respectively, which met the rule (1) mentioned above, but the ˛ value that approximated to 1 did not meet rule (2), and this system provide a poor separation between target compounds (CCC 2). Improving the separation factor value between the two target compounds in solvent system A was necessary. Modifying volume ratio and adding acetic acid were tried to adjust the ˛ value but failed, due to the high similarity between the structures of target compounds. Significant increase of ˛ value was made in adding NH4 OH, which made compound 2 almost entirely distributed in the aqueous phase, and had little influence on the partition of compound 1 in the two-phase solvent system. In the ethyl acetate-n-butanol 0.5%NH4 OH (2:3:5, v/v/v, solvent system B) solvent system, the K value of compound 1 (K1 ) and ˛ value between compounds 1 and 2 were suitable. Though this solvent system produce too small a K value for compound 2, the one-step HSCCC using solvent system B (CCC 1) resulted in a relatively successful separation of both compounds 1 and 2 (Fig. 2B), because of the small amounts of highly polar impurities existing in nbutanol extract of A. ilicifoulius (Fig. 2A), which were co-eluted with compound 2 in this HSCCC. Further more, for preparation of both compounds 1 and 2 with higher purity, two solvent systems, solvent systems A and B, were used in a two-step HSCCC isolation protocol: the n-butanol extract was subject to the HSCCC using solvent system A (CCC 2) for removing highly polar impurities, and then both of the target compounds with higher purity were successfully separated from the pre-purified analyte by the HSCCC using solvent system B (CCC 3). For the specific action of NH4 OH to DIBOA-Glc, the proposed mechanism is as follows: adding NH4 OH to the solvent system gives higher pH. Under this condition, DIBOA-Glc, which contains a hydroxamic acid group, is deprotonated [21], become more polar, and favor partition to the aqueous phase. 3.2. HSCCC separation of HBOA-Glc and DIBOA-Glc from A. ilicifolius The typical HSCCC chromatograms are shown in Fig. 2. The nbutanol extract of A. ilicifolius and the peak fractions separated by HSCCC were analyzed by HPLC to assess purity of target compounds (Fig. 2). The result of CCC 1 is given in Fig. 2B. 6.3 mg compound 1 (fraction collected during 125–145 min) and 6.8 mg compound 2 (fraction collected during 100–105 min) were obtained from
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H. Yin et al. / J. Chromatogr. A 1205 (2008) 177–181
Fig. 2. HSCCC chromatograms of the n-butanol extract of aerial part of A. ilicifolius, along with the HPLC chromatograms of the crude n-butanol extract and the fractions containing HBOA-Glc (1) and DIBOA-Glc (2) from HSCCC. (A) HPLC profile of the crude n-butanol extract of A. ilicifolius; (B) CCC 1, solvent system: ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v); sample, 100 mg n-butanol extract dissolved in 8 mL of the mixture of ethyl acetate–n-butanol–0.5% NH4 OH (2:3:5, v/v/v); (C) CCC 2, solvent system, ethyl acetate–n-butanol–water (2:3:5, v/v/v); sample, 100 mg extract dissolved in 8 mL of the mixture of ethyl acetate–n-butanol–water (2:3:5, v/v/v); (D) CCC 3, solvent system: ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v); sample: peak fraction of compounds 1 and 2 in (C) was evaporated to dryness in vacuo, and dissolved in 8 mL of the mixture ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v). HPLC experimental conditions: column, Phenomenex Luna 5 m C18 100A ODS column (250 mm × 4.6 mm) and a C18 guard column; mobile phase, acetonitrile (A) and water (B); gradient program: 90% B, 0–15 min; 90% B to 30% B, 15–35 min; 30% B to 90% B, 35–40 min; flow rate, 1.0 mL/min. The sample injection volume was 10 L. HSCCC experimental conditions: coil volume, 260 mL; rotation speed, 850 rpm; flow rate, 2.0 mL/min; retention of stationary phase in (B), (C), and (D) were about 40%, 42%, and 36%, respectively.
100 mg n-butanol extract. The purity of the two compounds was 90.3% and 80.2% (relative area percentage). Fig. 2C shows the CCC 2 separation of a sample containing 100 mg n-butanol extract. The peak fraction of compounds 1 and 2 (fraction collected during 115–130 min) was confirmed by HPLC to contain only compounds 1 and 2. This fraction was evaporated to dryness in vacuo and re-subjected to CCC 3. As shown in Fig. 2D, CCC 3 provide a baseline separation for target compounds, and yield 5.8 mg compound 1 (fraction collected during 130–145 min) and 4.8 mg compound 2 (fraction collected during 103–112 min) with purity of 97.1% and 94.8%, respectively, which were directly used for NMR analyses. 3.3. Identification of the compounds The structures of all substances were confirmed by UV, 1 H and spectra. Spectroscopic data of HBOA-Glc and DIBOA-Glc yielded by HSCCC separation were in agreement with published data [8,13,14]. HBOA-Glc (1): UV (MeOH) max 251.1 nm; 1 H NMR (500 MHz, MeOH-d4 ): ı 5.77 (s, H-2), 6.95 (m, H-5), 7.05 (m, H-6, 7), 7.11 (m, H-8), 3.21 (dd, J = 8.2, 8.3 Hz, H-2 ), 3.32–3.40 (m, H-3 ,4 ,5 ), 3.87 (dd, J = 12.0, 2.5 Hz, H-6 ), 3.70 (dd, J = 11.9, 4.7 Hz, H-6 ); 13 C NMR (125 MHz, MeOH-d ): ı 96.5 (C-2), 163.2 (C-3), 116.9 4 (C-5), 125.1 (C-6), 124.2 (C-7), 119.1 (C-8), 142.2 (C-9), 127.2 (C10), 104.0 (C-1 ), 74.9 (C-2 ), 78.5 (C-3 ), 71.2 (C-4 ), 78.0 (C-5 ), 62.4 (C-6 ). 13 CNMR
DIBOA-Glc (2): UV (MeOH) max 254.6 nm; 1 H NMR (500 MHz, MeOH-d4 ): ı 5.96 (s, H-2), 7.38 (m, H-5), 709–7.16 (m, H-6, 7, 8), 3.20 (dd, J = 8.2, 8.2 Hz, H-2 ), 3.32–3.40 (m, H-3 ,4 ,5 ), 3.87 (dd, J = 12.0, 2.5 Hz, H-6 ), 3.69 (dd, J = 11.9, 4.7 Hz, H-6 ); 13 C NMR (125 MHz, MeOH-d4 ): ı 97.8 (C-2), 158.3 (C-3), 114.3 (C-5), 125.8 (C-6), 124.3 (C-7), 118.7 (C-8), 142.5 (C-9), 129.3 (C-10), 103.8 (C-1 ), 74.9 (C-2 ), 78.6 (C-3 ), 71.1 (C-4 ), 78.0 (C-5 ), 62.6 (C-6 ). 4. Conclusion Efficient one-step and two-step HSCCC methods were developed for the isolation and purification of HBOA-Glc and DIBOA-Glc from A. ilicifoulius. The one-step separation was more rapid and less solvents consumption, while the two-step method gave higher purity. Conventional methods of CC for preparative separation of benzoxazinoids from plant extracts are rather time-consuming. For instance, by this way, the total time of the preparation of the two standards used in this study was more than 60 h. Using the HSCCC methods described here, the two target compounds can be isolated from the n-butanol extract with higher purity and less working time (8 h). Furthermore, with the HSCCC methods, the two main benzoxazinoids in A. ilicifolius can be isolated on a preparative scale which may then be used for reference substances for chromatographic purposes, bioavailability and bioactivity studies. The present study proved that HSCCC is a powerful technique for the isolation of HBOA-Glc and DIBOA-Glc from plant crude extracts.
H. Yin et al. / J. Chromatogr. A 1205 (2008) 177–181
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Preparative isolation and purification of two benzoxazinoid glucosides from Acanthus ilicifolius L. by high-speed counter-current chromatography Hao Yin a,b , Si Zhang a,b,∗ , Xiongming Luo a , Yonghong Liu a a Guangdong Key Laboratory of Marine Material Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, Guangdong, China b Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Science, 164 West Xingang Road, Guangzhou 510301, Guangdong, China
a r t i c l e
i n f o
Article history: Received 6 June 2008 Received in revised form 29 July 2008 Accepted 4 August 2008 Available online 9 August 2008 Keywords: Benzoxazinoids (2R)-2-O--d-glucopyranosyl-2H-1,4benzoxazin-3(4H)-one (HBOA-Glc) (2R)-2-O--d-glucopyranosyl-4-hydroxy2H-1,4-benzoxazin-3(4H)-one (DIBOA-Glc) High-speed counter-current chromatography (HSCCC) Acanthus ilicifolius L
a b s t r a c t The first preparative separation of two benzoxazinoids, (2R)-2-O--d-glucopyranosyl-2H-1,4benzoxazin-3(4H)-one (HBOA-Glc) and (2R)-2-O--d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin3(4H)-one (DIBOA-Glc), by means of high-speed counter-current chromatography (HSCCC) from the n-butanol extract of Acanthus ilicifolius L. is presented. The two-phase solvent system containing ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v, system B) was selected for the one-step HSCCC separation of HBOA-Glc and DIBOA-Glc according to the partition coefficient values (K) for target compounds and the separation factor (˛) between the two target compounds. In the one-step HSCCC separation using solvent B, from 100 mg n-butanol extract of A. ilicifolius, 6.3 mg HBOA-Glc and 6.8 mg DIBOA-Glc were isolated with purities of 90.3% and 80.2%, respectively. In order to obtain the two target compounds with higher purity, a second separation process was developed comprising two steps. In the two-step separation, the sample was first pre-purified by HSCCC using ethyl acetate–n-butanol–water (2:3:5, v/v/v, system A) solvent system and then purified using solvent system B. A 100-mg amount of the n-butanol extracts of A. ilicifolius was separated to yield 5.8 mg of HBOA-Glc and 4.8 mg of DIBOA-Glc with purities of 97.1% and 94.8%, respectively, which were directly used for NMR analyses. © 2008 Elsevier B.V. All rights reserved.
1. Introduction High-speed counter-current chromatography (HSCCC), first invented by Ito [1], is widely used in the preparative separation of natural products. HSCCC does not employ a solid-phase support and thus there is no irreversible adsorption associated with the solid supports, and allows the direct application of crude extracts and an excellent recovery of the analytes [2]. In this study, a HSCCC separation and purification of the two main benzoxazinoids, (2R)-2O--d-glucopyranosyl-2H-1,4-benzoxazin-3(4H)-one (HBOA-Glc) and (2R)-2-O--d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin3(4H)-one (DIBOA-Glc), from the n-butanol extract of Acanthus ilicifolius L. are presented (Fig. 1). In Chinese folk medicine, all parts of A. ilicifolius have been used as crude drug for the treatment of lymphadenectasis, hepatitises, stomach aches, coughs, and asthmas [3]. Pharmacological
∗ Corresponding author at: Guangdong Key Laboratory of Marine Material Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, Guangdong, China. Tel.: +86 2089023105; fax: +86 2089023105. E-mail address: zhs [email protected] (S. Zhang). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.08.010
research had shown that the extracts from A. ilicifolius in particular had many bioactivities, including antivirus, anticarcinogeni, antioxidative, hepatoprotecti, antimutagenieit, and antiinflammatory activities [3–5]. In previous phytochemical investigations of this plant, six benzoxazinoids had been reported [3,6–8]. Benzoxazinoids are a unique class of natural products abundant in family of Gramineae, such as maize, wheat, rye, and wild grasses. Also they are found in different species of Acanthaceae, Ranunculaceae, Scrophulariaceae, and others [9]. In host plant, benzoxazinoids serve as important factors of resistance against microbial diseases and insects, allelochemics used in competition with other plants, and endogenous ligands. These metabolites also were proposed as natural pesticides for agricultural use by interdisciplinary investigations by biologists, biochemists, and chemists [10]. Therefore, the analyses and separation of benzoxazinoids are of great interest [6,10–12]. Being a support free liquid–liquid chromatography technology with no solid support matrix, HSCCC method for separation and purification of benzoxazinioids could be more efficient than conventional methods which are tedious and requires repeated chromatographic steps [6,8,11]. However, no report has been published on the use of HSCCC for separation and purification of this class of compounds.
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2.3. Preparation of sample
Fig. 1. Chemical structures of HBOA-Glc and DIMBOA-Glc.
The main purpose of this contribution was to study the isolation and purification possibility of the two main benzoxazinoids, HBOAGlc (1) and DIBOA-Glc (2), on a preparative scale from A. ilicifolius by HSCCC. As far as we know, this is the first report on successful isolation and purification of benzoxaziniods from a natural source using HSCCC. 2. Experimental 2.1. Apparatus HSCCC was carried out using a Model TBE-300A high-speed counter-current chromatography (Tauto Biotech, Shanghai, China) containing a self-balancing three-coil centrifuge rotor equipped three preparative multilayer coils with a total capacity of 260 mL, the internal diameter of PTFE tubing was 2.0 mm. The revolution radius or the distance between the holder axis and central axis of the centrifuge (R) was 9.5 cm. The ˇ value varied from 0.46 at the internal terminal to 0.73 at the external terminal (ˇ = r/R, where r is the distance from the coil to the holder shaft). The HSCCC system was equipped with a Model TBP-50 constant flow pump (Tauto Biotech, Shanghai, China), and a LC757 UV detector with preparative flow cell (Shanghai Huixing Instrument, China), a Model HX-1050 constant-temperature controller (Beijing Detianyou Technology, China), and a N2010 chromatogram workstation (Zhejiang University, Hangzhou, China). A Luna 5 m C18 100A ODS column (250 mm × 4.6 mm; Phenomenex, Torrance, CA, USA) was used for analytical HPLC, along with 600E multisolvent delivery system and a Waters 996 Photodiode Array Detector (Waters, Milford, MA, USA). NMR spectra were obtained on an Avance 500 magnetic resonance spectrometer (500 MHz for 1 H NMR, and 125 MHz for 13 C NMR; Bruker, Karlsruhe, Germany). UV spectra were measured with a coulter DU 640 nucleic acid and protein analyzer (Beckman, Fullerton, CA, USA). 2.2. Reagents All organic solvents used for HSCCC were of analytical grade and purchased from Baishi (Tianjing, China). Methanol and acetonitrile used for HPLC were obtained from Merck (Darmstadt, Germany). Water was purified by Milli-Q system (Millipore, Bedford, MA, USA), and ammonium hydroxide (25–28% NH3 stock solution) were from Guangzhou Chemical Reagent Factory (Guangzhou, Guangdong, China). HBOA-Glc and DIBOA-Glc standards (purity >95%, determined by HPLC) were obtained in our laboratory by column chromatography, identity of the isolated compounds were confirmed by comparing their spectroscopic properties (1 H and 13 C NMR) with those reported in the literature [8,13,14].
A. ilicifolius was collected in April 2006 from Hainan Province, Southern of China. The identification of the plant was confirmed by Professor Zhang Si, director of Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences. A voucher sample (GKLMMM-001A) is kept in the Herbarium of the South China Sea Institute of Oceanology). The dry powdered aerial part (1 kg) of A. ilicifolius was extracted with hot EtOH (95%) three times. After removal of the solvent by evaporation, the residue (135 g) was suspended in water and then successively extracted with petroleum ether, EtOAc and n-butanol. The n-butanol extract (40 g) was stored at 4 ◦ C before HSCCC separation. 2.4. Determination of partition coefficient Experimental procedure: approximately 1 mg of target compound was weighed in a 10-mL test tube into which 1 mL of each phase of the preequilibrated two-phase solvent system was added. The test tube was capped and shaken vigorously for 1 min, and allowed to stand until it separated completely. An aliquot of 100 L of each layer was taken out and evaporated separately to dryness in vacuo. The residue was dissolved in 100 L 10% acetonitrile and analyzed by HPLC for determining the partition coefficient (K) of compounds 1 and 2. The K value was expressed as the peak area of target compound in the upper phase divided by that in the lower phase. 2.5. Preparation of two-phase solvent system and sample solution Several ethyl acetate–n-butanol–water solvent systems with different volume ratios and their modified form (Table 1) were prepared. All these two-phase solvent systems were thoroughly equilibrated in a separation funnel by repeatedly vigorously shaking at room temperature, and then separated shortly before use. The following two two-phase solvent systems were selected for the HSCCC separation of HBOA-Glc and DIBOA-Glc from A. ilicifolius: • Solvent system A: ethyl acetate–n-butanol–water (2:3:5, v/v/v). • Solvent system B: ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v). The samples were dissolved in 4 mL of each phase of selected solvent systems for preparative HSCCC separation.
Table 1 The K (partition coefficient) values of compounds 1 and 2 in different solvent systemsa Solvent system (volume ratio)
HBOA-Glc
DIBOA-Glc
Ethyl acetate–n-BuOH–water (1:0:1) Ethyl acetate–n-BuOH–water (4:1:5) Ethyl acetate–n-BuOH–water (3:2:5) Ethyl acetate–n-BuOH–water (2:3:5), A Ethyl acetate–n-BuOH–1% acetic acid (2:3:5) Ethyl acetate–n-BuOH–1%NH4 OH (2:3:5) Ethyl acetate–n-BuOH–0.5%NH4 OH (2:3:5), B
0.01 0.13 0.52 0.67 0.65 0.57 0.72
0.01 0.12 0.49 0.62 0.63 0.01 0.03
a Experimental procedure: approximately 1 mg of target compound was weighed in a 10-mL test tube into which 1 mL of each phase of the preequlibrated solvent system was added. The test tube was capped and shaked vigorously for 1 min, and allowed to stand until it separated completely. An aliquot of 100 L of each layer was taken out and evaporated separately to dryness in vacuo. The residue was dissolved in 100 L 10% acetonitrile and analyzed by HPLC for determining the partition coefficient (K) of compounds 1 and 2. The K value was expressed as the peak area of target compound in the upper phase divided by that in lower phase.
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2.6. HSCCC separation procedure Two protocols for preparation of HBOA-Glc and DIBOA-Glc from n-butanol A. ilicifolius, one-step and two-step HSCCC separation, were used in present study. With the exception of the two-phase solvent system and sample, all HSCCC separation steps were identical in operation, and were performed as following: the multilayer coiled column was first entirely filled with the upper organic phase of solvent system. The lower phase was then pumped into the head end of the column at a flow rate of 2.0 mL/min, while the apparatus was run at a revolution speed of 850 rpm. After hydrodynamic equilibrium was established, as indicated by a clear mobile phase eluting at the tail outlet, sample solution was injected through the sample port. The effluent from the tail end of the column was continuously monitored with a UV detector at 254 nm. Each peak fraction was collected according to the chromatogram, respectively. Analyses of fractions were performed by HPLC-diode array detection (DAD). The peak fractions of desired compounds were evaporated to dryness in vacuo for NMR analysis. In the one-step separation, 100 mg n-butanol extract dissolved in 4 mL of each phase of solvent system B was subjected to HSCCC using solvent system B directly (CCC 1) for preparation of target compounds. In the two-step separation, 100 mg n-butanol extract dissolved in 4 mL of each phase of solvent A was subjected to HSCCC using solvent system A for a pre-purification (CCC 2). The peak fraction containing target compounds confirmed by HPLC analysis was evaporated to dryness in vacuo, and subsequently was dissolved in 4 mL of each phase of solvent system B. The pre-purified sample solution was re-subjected to the HSCCC using solvent system B (CCC 3) for separation of compounds 1 and 2. 2.7. Analytical HPLC For the separation of analytes, the following gradient system was used: acetonitrile (A) and water (B); gradient program: 90% B, 0–15 min; 90% B to 30% B, 15–35 min; 30% B to 90% B, 35–40 min. The injection volume was 10 L. The mobile phase was eluted at a flow rate of 1.0 mL/min and the effluent was monitored by a UV detector at 254 nm. 2.8. NMR spectroscopy 1H
and 13 C NMR spectra of the isolated HBOA-Glc and DIBOAGlc were recorded in [2 H4 ]MeOH with TMS as an internal reference. Chemical shifts (ı) are reported in ppm and coupling constants (J) in Hertz (Hz). 2.9. Preparation of HBOA-Glc and DIBOA-Glc standards The n-butanol extract of A. ilicifolius (1 g) was subjected to Diaion HP-20, and eluted with H2 O, 10% MeOH, 30% MeOH, 60% MeOH and MeOH, successively. The fraction eluted with 10% MeOH was subjected to successive silica column chromatography (ethyl acetate-methanol-formic acid, 5:1:0.1, v/v/v) and repeated Sephadex LH-20 column chromatography (10% MeOH), to afford HBOA-Glc (27 mg) and DIBOA-Glc (24 mg). 3. Results and discussion 3.1. HSCCC solvent systems The selection of suitable solvent system is the first and critical step in performing preparative HSCCC. Two “golden rules for
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HSCCC” mentioned in the literature [2] were (1) “find systems with K (partition coefficient) values of the target compounds in a proper range: the suitable K values for HSCCC are 0.5 ≤ K ≤ 1.0” and (2) for HSCCC separation of two similar compounds, “the ratio of the two K values or the separation factor (˛ = K1 /K2 , where K1 > K2 ) ought to be greater than 1.5 in the semipreparative multilayer separation column of a commercial HSCCC unit”. In present study, the suitable solvent systems for HSCCC separation of HBOA-Glc and DIBOA-Glc were developed according these rules. The K values of target compounds in several classical solvent systems previously used for CCC separation of alkaloids, CHCl3 –MeOH–water, CHCl3 –ethanol–water, and nHex–EtOAc–MeOH–water [15–20], were tested, but none of them were successful because the target compounds were mostly distributed in the aqueous phase in these systems. Therefore, the search was directed to more polar solvent systems. Several ethyl acetate–n-butanol–water solvent systems with different volume ratios and their modified form were tested, and the measured K values of target compounds were summarized in Table 1. Among them, solvent system A, ethyl acetate–n-butanol–water (2:3:5, v/v/v), provided K values at 0.67 and 0.62 for compounds 1 and 2, respectively, which met the rule (1) mentioned above, but the ˛ value that approximated to 1 did not meet rule (2), and this system provide a poor separation between target compounds (CCC 2). Improving the separation factor value between the two target compounds in solvent system A was necessary. Modifying volume ratio and adding acetic acid were tried to adjust the ˛ value but failed, due to the high similarity between the structures of target compounds. Significant increase of ˛ value was made in adding NH4 OH, which made compound 2 almost entirely distributed in the aqueous phase, and had little influence on the partition of compound 1 in the two-phase solvent system. In the ethyl acetate-n-butanol 0.5%NH4 OH (2:3:5, v/v/v, solvent system B) solvent system, the K value of compound 1 (K1 ) and ˛ value between compounds 1 and 2 were suitable. Though this solvent system produce too small a K value for compound 2, the one-step HSCCC using solvent system B (CCC 1) resulted in a relatively successful separation of both compounds 1 and 2 (Fig. 2B), because of the small amounts of highly polar impurities existing in nbutanol extract of A. ilicifoulius (Fig. 2A), which were co-eluted with compound 2 in this HSCCC. Further more, for preparation of both compounds 1 and 2 with higher purity, two solvent systems, solvent systems A and B, were used in a two-step HSCCC isolation protocol: the n-butanol extract was subject to the HSCCC using solvent system A (CCC 2) for removing highly polar impurities, and then both of the target compounds with higher purity were successfully separated from the pre-purified analyte by the HSCCC using solvent system B (CCC 3). For the specific action of NH4 OH to DIBOA-Glc, the proposed mechanism is as follows: adding NH4 OH to the solvent system gives higher pH. Under this condition, DIBOA-Glc, which contains a hydroxamic acid group, is deprotonated [21], become more polar, and favor partition to the aqueous phase. 3.2. HSCCC separation of HBOA-Glc and DIBOA-Glc from A. ilicifolius The typical HSCCC chromatograms are shown in Fig. 2. The nbutanol extract of A. ilicifolius and the peak fractions separated by HSCCC were analyzed by HPLC to assess purity of target compounds (Fig. 2). The result of CCC 1 is given in Fig. 2B. 6.3 mg compound 1 (fraction collected during 125–145 min) and 6.8 mg compound 2 (fraction collected during 100–105 min) were obtained from
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Fig. 2. HSCCC chromatograms of the n-butanol extract of aerial part of A. ilicifolius, along with the HPLC chromatograms of the crude n-butanol extract and the fractions containing HBOA-Glc (1) and DIBOA-Glc (2) from HSCCC. (A) HPLC profile of the crude n-butanol extract of A. ilicifolius; (B) CCC 1, solvent system: ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v); sample, 100 mg n-butanol extract dissolved in 8 mL of the mixture of ethyl acetate–n-butanol–0.5% NH4 OH (2:3:5, v/v/v); (C) CCC 2, solvent system, ethyl acetate–n-butanol–water (2:3:5, v/v/v); sample, 100 mg extract dissolved in 8 mL of the mixture of ethyl acetate–n-butanol–water (2:3:5, v/v/v); (D) CCC 3, solvent system: ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v); sample: peak fraction of compounds 1 and 2 in (C) was evaporated to dryness in vacuo, and dissolved in 8 mL of the mixture ethyl acetate–n-butanol–0.5%NH4 OH (2:3:5, v/v/v). HPLC experimental conditions: column, Phenomenex Luna 5 m C18 100A ODS column (250 mm × 4.6 mm) and a C18 guard column; mobile phase, acetonitrile (A) and water (B); gradient program: 90% B, 0–15 min; 90% B to 30% B, 15–35 min; 30% B to 90% B, 35–40 min; flow rate, 1.0 mL/min. The sample injection volume was 10 L. HSCCC experimental conditions: coil volume, 260 mL; rotation speed, 850 rpm; flow rate, 2.0 mL/min; retention of stationary phase in (B), (C), and (D) were about 40%, 42%, and 36%, respectively.
100 mg n-butanol extract. The purity of the two compounds was 90.3% and 80.2% (relative area percentage). Fig. 2C shows the CCC 2 separation of a sample containing 100 mg n-butanol extract. The peak fraction of compounds 1 and 2 (fraction collected during 115–130 min) was confirmed by HPLC to contain only compounds 1 and 2. This fraction was evaporated to dryness in vacuo and re-subjected to CCC 3. As shown in Fig. 2D, CCC 3 provide a baseline separation for target compounds, and yield 5.8 mg compound 1 (fraction collected during 130–145 min) and 4.8 mg compound 2 (fraction collected during 103–112 min) with purity of 97.1% and 94.8%, respectively, which were directly used for NMR analyses. 3.3. Identification of the compounds The structures of all substances were confirmed by UV, 1 H and spectra. Spectroscopic data of HBOA-Glc and DIBOA-Glc yielded by HSCCC separation were in agreement with published data [8,13,14]. HBOA-Glc (1): UV (MeOH) max 251.1 nm; 1 H NMR (500 MHz, MeOH-d4 ): ı 5.77 (s, H-2), 6.95 (m, H-5), 7.05 (m, H-6, 7), 7.11 (m, H-8), 3.21 (dd, J = 8.2, 8.3 Hz, H-2 ), 3.32–3.40 (m, H-3 ,4 ,5 ), 3.87 (dd, J = 12.0, 2.5 Hz, H-6 ), 3.70 (dd, J = 11.9, 4.7 Hz, H-6 ); 13 C NMR (125 MHz, MeOH-d ): ı 96.5 (C-2), 163.2 (C-3), 116.9 4 (C-5), 125.1 (C-6), 124.2 (C-7), 119.1 (C-8), 142.2 (C-9), 127.2 (C10), 104.0 (C-1 ), 74.9 (C-2 ), 78.5 (C-3 ), 71.2 (C-4 ), 78.0 (C-5 ), 62.4 (C-6 ). 13 CNMR
DIBOA-Glc (2): UV (MeOH) max 254.6 nm; 1 H NMR (500 MHz, MeOH-d4 ): ı 5.96 (s, H-2), 7.38 (m, H-5), 709–7.16 (m, H-6, 7, 8), 3.20 (dd, J = 8.2, 8.2 Hz, H-2 ), 3.32–3.40 (m, H-3 ,4 ,5 ), 3.87 (dd, J = 12.0, 2.5 Hz, H-6 ), 3.69 (dd, J = 11.9, 4.7 Hz, H-6 ); 13 C NMR (125 MHz, MeOH-d4 ): ı 97.8 (C-2), 158.3 (C-3), 114.3 (C-5), 125.8 (C-6), 124.3 (C-7), 118.7 (C-8), 142.5 (C-9), 129.3 (C-10), 103.8 (C-1 ), 74.9 (C-2 ), 78.6 (C-3 ), 71.1 (C-4 ), 78.0 (C-5 ), 62.6 (C-6 ). 4. Conclusion Efficient one-step and two-step HSCCC methods were developed for the isolation and purification of HBOA-Glc and DIBOA-Glc from A. ilicifoulius. The one-step separation was more rapid and less solvents consumption, while the two-step method gave higher purity. Conventional methods of CC for preparative separation of benzoxazinoids from plant extracts are rather time-consuming. For instance, by this way, the total time of the preparation of the two standards used in this study was more than 60 h. Using the HSCCC methods described here, the two target compounds can be isolated from the n-butanol extract with higher purity and less working time (8 h). Furthermore, with the HSCCC methods, the two main benzoxazinoids in A. ilicifolius can be isolated on a preparative scale which may then be used for reference substances for chromatographic purposes, bioavailability and bioactivity studies. The present study proved that HSCCC is a powerful technique for the isolation of HBOA-Glc and DIBOA-Glc from plant crude extracts.
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