Food Sci. Biotechnol. 19(2): 497-502 (2010) DOI 10.1007/s10068-010-0069-z
RESEARCH ARTICLE
Large Scale Isolation and Purification of Salvianolic Acid B in High Purity from Roots of Dansham (Salvia miltiorrhiza Bunge) Hyoung Jae Lee, Ki Hoon Lee, Keun-Hyung Park, and Jae-Hak Moon
Received: 2 November 2009 / Revised: 29 December 2009 / Accepted: 31 December 2009 / Published Online: 30 April 2010 © KoSFoST and Springer 2010
Abstract Salvianolic acid B (Sal B) is the most abundant 80 million kilograms per year in China (5). The roots representative compound in roots of dansham (Salvia miltiorrhiza) which is beneficial in treatment of cardiovascular disease. The present study was performed to establish a large scale isolation method of Sal B in high purity from dansham roots. Water suspension of dansham roots’ (100 g d.w.) methanol extract (13.16 g) was partitioned with chloroform, and the water layer was adjusted to pH 3.0 by triflouroacetic acid, because the Sal B partition coefficient in acidic condition was predominately higher than that in neutral condition, and then continuously extracted with ethyl acetate (EtOAc). The EtOAc-soluble acidic fraction was subjected to Sephadex LH-20 column chromatography. The fractions (Ve/Vt 1.75-2.71, 1.82 g) containing Sal B were finally purified by preparative high performance liquid chromatography using an octadecylsilane column. The isolated Sal B fraction (1.08 g) represented a 75.0% recovery, with a purity exceeding 99.2%.
Keywords: salvianolic acid B, dansham (Salvia miltiorrhiza), solvent fractionation, Sephadex LH-20, preparative high performance liquid chromatography
Introduction Roots of dansham or danshen (Salvia miltiorrhiza Bunge) have been widely used in Chinese traditional medicine for the treatment of cardiovascular diseases such as coronary heart disease, hyperlipidemia, and cerebrovascular disease (1-4). The consumption of dansham roots amounts to about Hyoung Jae Lee, Ki Hoon Lee, Keun-Hyung Park, Jae-Hak Moon ( ) Department of Food Science & Technology, and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Korea Tel: +82-62-530-2141; Fax: +82-62-530-2149 E-mail:
[email protected]
contain diterpenoids, flavonoids, sterols, and water-soluble phenolics (6,7). Of these constituents, the phenolics, which are the main compounds in water-soluble exudate - in particular salvianolic acid B (Sal B, also known as lithospermic acid B; Fig. 1) - are most commonly used to treat patients in Chinese clinics. Because of its prevalence in water-soluble extracts, Sal B is used as a chemical marker of dansham roots as a phytomedicine. Sal B content ranges widely from 0.6-0.7% of the root total dried weight (8) depending of cultivation-related variation (9). The biological activities of Sal B and its magnesium salt include strong antioxidant and free radical scavenging activities (10-13), angiotensin converting enzyme (ACE) inhibition (14), inhibition of glucose-induced cell proliferation (15), hepatoprotection (16), elicitation of endotheliumdependent vasodilation (17), lowering of blood pressure in hypertension (18), and inhibition of replication of human immunodeficiency virus-1 (19). Chromatography-based isolation and purification of Sal B from dansham root extracts has recently been reported (14, 20-25). In one of these studies, a multi-step procedure involving solvent fractionation, silica gel column chromatography, Sephadex LH-20 column chromatography, and thin layer chromatography (TLC) was successfully applied (14). In another study, solvent fractionation was followed by high-speed counter-current chromatography (HSCCC) after adjustment of the pH to 2.0 using HCl (24). HSCCC was also successful when utilized after primary purification by D101 macroporous resin (25). While HSCCC is less laborious than the multi-step chromatographic regimen, access to the specialized instrumentation has limited this approach. Presently, we sought to establish a simple method for large-scale purification of Sal B from dansham roots using solvent fractionation, single-pass column chromatography, and the more readily available and
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Fig. 1. Stucture of salvianolic acid B (lithospermic acid B).
economical method of high-pressure liquid chromatography (HPLC).
Materials and Methods Material and chemicals Dansham (Salvia miltiorrhiza) roots imported from China were purchased from Jeonnam Herbal Medicine Agricultural Cooperation (Hwasun, Korea). The dried pieces were ground using a DY 8585 grinder (Dongyang PCS, Incheon, Korea). The powder was extracted just after grinding. Solvents used for analyses were of HPLC grade and purchased from Fisher Scientific Korea (Seoul, Korea). Spectrophotometric grade trifluoroacetic acid (TFA) was obtained from SigmaAldrich (St. Louis, MO, USA). Methanol (MeOH) used for extraction, and chloroform (CHCl3), ethyl acetate (EtOAc), and n-buthanol (BuOH) used for solvent fractionation were of extra pure quality and were obtained from Duksan (Ansan, Korea). Authentic Sal B was isolated from dansham roots and identified by electrospray ionization mass spectrometry (ESI-MS) and 1H-nuclear magnetic resonance (NMR) experiments in our laboratory.
partitioned with 3 times of 100 mL volumes of CHCl3. The H2O layer was extracted by the application of 3 times of 100 mL volumes of EtOAc and was then continuously extracted by H2O-saturated BuOH (100 mL, 3 times) (Method A). Each layer was evaporated in vacuo at 38oC, and the concentrated extracts were dissolved in 5 mL MeOH and filtered through a Millex-HV 13-mm, 0.45-µm pore size polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Each filtrate of the CHCl3, EtOAc, BuOH, and H2O layers was injected (10 µL, representing 2 mg d.w. eq.) into a HPLC apparatus of analysis scale (conditions of analytical HPLC-1). The contents of Sal B obtained from the HPLC chromatograms were compared with that from MeOH extract of dansham roots. As well, another solvent fractionation for purification of Sal B (Method B) was examined, in which the H2O layer obtained after partition between H2O suspension of MeOH extract (2.63 g) of dansham roots (20 g d.w. eq.) and CHCl3 (100 mL, 3 times) was adjusted to pH 3.0 by TFA, and then successively partitioned with EtOAc and H2Osaturated BuOH by the same scale as Method A.
Isolation and purification of Sal B in large scale Solvent fractionation for purification of Sal B in large scale was
Extraction procedure Dansham root powder (240 g) conducted in 10 times scale (dansham roots in 200 g d.w. was extracted with MeOH (1,200 mL) for 24 hr at room temperature. The extract was filtered under vacuum through Whatman (Maidstone, England) No. 2 filter paper. The extraction and filtering steps were repeated. The filtrates were combined and concentrated by vacuum evaporation at 38oC.
Determination of condition for solvent fractionation in small scale MeOH extracts (2.63 g) of dansham roots (20 g d.w. eq.) were suspended in 100 mL water (H2O) and
eq.) of small scale used to determine conditions for solvent fractionation. A 400 mL H2O suspension of MeOH extract of dansham root powder (200 g) was solvent fractionated with CHCl3 (400 mL, 3 times). The H2O layer adjusted to pH 3.0 by TFA was extracted by EtOAc (400 mL, 3 times). The EtOAc-soluble acidic fraction (4.2 g) was evaporated in vacuo at 38oC and half (2.1 g) of the fraction was subjected to column chromatography on a Sephadex LH20 column (2.0 × 130 cm, 25-100 mesh, Pharmacia Fine Chemicals, Uppsala, Sweden), with a total elution volume
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of 2,200 mL and 10 mL/fr. (26). The elution was performed with H2O/MeOH (8:2, v/v) and each fraction was spotted on a silica gel TLC plate (silica gel 60 F254, 0.25-mm thickness; Merck, Darmstadt, Germany). The TLC plate was developed with BuOH/acetic acid/H2O (4:1:2, v/ v/v). Then, 200 µM 1,1-diphenyl-2-picrylhydrazyl (DPPH; Wako, Osaka, Japan) in ethanol was sprayed on the dried TLC plate. Sal B-containing fractions were finally purified by HPLC of preparative scale (conditions of preparative HPLC). The purity of the isolated compound was determined by analytical scale HPLC (conditions of analytical HPLC-2). In addition, the isolated compound was subjected to ESI-MS and 1H-NMR analyses.
Conditions for instrumental analyses Analytical HPLC-1: Analytical scale HPLC-1 was carried out using a
ChromeleonTM system (Dionex, Sunnyvale, CA, USA) equipped with P580 pump, ASI-100 automated sample injector, and UVD-170S UV/VIS detector, and using a 4.6 × 150 mm octadecylsilane (ODS) 80Ts column (Tosoh, Kyoto, Japan) at room temperature. The mobile phase was composed of CH3CN/H2O/acetic acid (10:88:2, v/v/v) (A) and CH3CN/H2O (40:60, v/v) (B). The gradient program was as follows: starting with 100% A, rising to 100% B after 70 min, and maintaining 100% B for 15 min. The flow rate was 1.0 mL/min and detection was monitored at 280 nm. Analytical HPLC-2: Analytical scale HPLC-2 was carried out using a Shimadzu HPLC-PDA system (Shimadzu, Kyoto, Japan) equipped with LC-6AD pump, SPD-M20A detector, DGU-20A3 degasser, CBM-20A controller, and Shimadzu LC solution program, and using a ODS 80Ts (4.6 × 150 mm) column at room temperature. The mobile phase was composed of CH3CN/H2O (10:88, v/v) (A) adjusted to pH 2.65 by TFA and CH3CN/H2O (40:60, v/v) (B). The gradient program was as follows: starting with 100% A, rising to 100% B after 70 min, and maintaining
100% B for 15 min. The flow rate was 1.0 mL/min and detection was monitored at 280 nm. Preparative HPLC: Preparative scale HPLC was performed with the same instrumentation as that of analytical HPLC2 except for preparative mode equipped with a Shim-pack PREP-ODS(H)KIT column (20× 250 mm). The mobile phase consisted of CH3CN/H2O (10:90, v/v) (A) adjusted to pH 2.65 by TFA and CH3CN/H2O (40:60, v/v) (B). The following gradient program was used: linear change from 0% B to 50% B from 0-15 min; maintain 50% B from 1530 min, linear change from 50% B to 100% B from 30-45 min, and maintain 100% B from 45-60 min. The flow rate was 9.9 mL/min and detection was monitored at 280 nm. MS analysis: Mass spectra were acquired on a LCMS hybrid ion trap-time of flight (IT-TOF) mass spectrometer (Shimadzu) equipped with an ESI source in negative ion mode at a mass resolution of 10,000 full-width at half maximum. Each sample solution was prepared by dissolving the isolated compound in MeOH to a final concentration of 50 µg/µL. Data acquisition and analysis was performed using LC Solution 3.0 software. NMR analysis: 1H-NMR was recorded on a 500 MHz unit INOVA 500 spectrometer (Varian, Walnut Creek, CA, USA). The chemical shift was measured with tetramethylsilane serving as an internal standard. The isolated compound was dissolved in deuterium acetone (acetone-d6; Acros Organics, Geel, Begium). Results and Discussion
Determination of conditions for solvent fractionation in small scale To determine solvent fractionation conditions
for purification of Sal B, 2 different methods (A and B) were investigated. A portion (2 mg d.w. eq.) of each fraction was subjected to analytical HPLC to determine the Sal B contents in each layer (Table 1). As shown in Fig. 2,
Table 1. Contents of Sal B in MeOH extract of dansham roots (100 g d.w. eq.) and in each layer after solvent fractionation of the extract under different condition Content of salvianolic acid B g/100 g d.w. eq. % 1.44±0.21 100.00±14.39 0.02±0.00 1.42±0.03 0.99±0.01 68.35±0.66 0.25±0.06 17.40±2.01 0.07±0.01 09.75±0.17 0.02±0.00 01.31±0.00 1.48±0.01 102.84±0.380 0.03±0.00 00.06±0.00 trace 1)
MeOH extract of dansham roots CHCl layer EtOAc layer Method A BuOH layer H O layer CHCl layer EtOAc layer Method B BuOH layer H O layer 3
2
3
2
Each data represents the mean±SD (n =3).
1)
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Fig. 2. HPLC chromatograms of each layer after solvent fractionation by method A without adjustment of pH of H2O suspension of dansham roots MeOH extract. a, CHCl3 layer; b, EtOAc layer; c, BuOH layer; d, H2O layer; e, authentic compound (Sal B)
method A yielded 68.35±0.66% of the total Sal B content of the MeOH extract of dansham roots in the EtOAc fraction. Therefore, to elevate the Sal B partition coefficient, the pH of the H2O layer obtained after solvent fractionation of H2O suspension of MeOH extracted dansham root with CHCl3 was adjusted to 3.0 by TFA (Method B). TFA has a low boiling point (72oC) and so can be simply be removed by vacuum evaporation without any other elimination processes. The pH 3.0 H2O layer was continuously partitioned with EtOAc and BuOH in a similar manner as Method A. Virtually all (102.84±0.38%) of the Sal B was detected in the EtOAc-soluble acidic layer obtained after solvent fractionation by Method B (Fig. 3). The observations indicate that purification of Sal B by solvent fractionation of H2O suspension of dansham roots MeOH extract was more efficient under an acidic condition (Method B) than under a neutral condition (Method A).
Isolation and purification of Sal B in large scale
Purification by solvent fractionation in large scale was started from a 10× extract (200 g d.w. eq. dansham roots, representing 26.32 g) of the small scale preparation obtained by Method B. Half (2.1 g) of the EtOAc-soluble acidic fraction was subjected to Sephadex LH-20 column chromatography to purify Sal B. Each fraction obtained from column chromatography was analyzed by TLC (Fig 4). TLC patterns were compared with that of the authentic Sal B previously isolated from dansham roots and identified in our laboratory. The presence of Sal B was
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Fig. 3. HPLC chromatograms of each layer after solvent fractionation by method B with adjustment of pH of H2O suspension of dansham roots MeOH extract. a, CHCl3 layer; b, EtOAc layer; c, BuOH layer; d, H2O layer; e, authentic compound (Sal B)
suggested from the fractions of Ve/Vt (elution volume/total bed volume) 1.75-2.71 (fr. 64-110 of Fig. 4) by comparison with Rf value (0.72) of the authentic Sal B. These fractions (1.82 g) were comprised of 88% Sal B; they were further purified by preparative scale HPLC (Fig. 5) to highly purify Sal B. The HPLC chromatogram and UV/VIS spectrum of Sal B isolated from preparative scale HPLC are shown in Fig. 6. The isolated compound (tR 33.6 min, 1.08 g, Fig. 5) was subjected to ESI-MS and 1H-NMR analyses to identify the chemical structure.
Identification of isolated compound The ESI-MS and
H-NMR determined structure of the isolated compound was: ESI-MS (negative): m/z 717.1 [M-H]+; 1H-NMR (acetone-d6, 500 MHz): δ 6.90 (1H, d, J =8.5 Hz, H-5), 7.22 (1H, d, J =8.5 Hz, H-6), 7.57 (1H, d, J =16.0 Hz, H7), 6.25 (1H, d, J =16.0 Hz, H-8), 6.84 (1H, d, J =1.8 Hz, H-2'), 6.80 (1H, d, J =8.0 Hz, H-5'), 6.70 (1H, dd, J =1.8, 8.0 Hz, H-6'), 5.85 (1H, d, J =4.8 Hz, H-7'), 4.45 (1H, d, J =4.8 Hz, H-8"), 6.86 (1H, d, J =1.9 Hz, H-2"), 6.73 (1H, d, J =7.9 Hz, H-5"), 6.65 (1H, dd, J =1.9, 7.9 Hz, H-6"), 2.91 (1H, dd, J =8.9, 14.3 Hz, H-7"a), 3.07 (1H, dd, J =3.8, 14.3 Hz, H-7"b), 5.19 (1H, dd, J =3.8, 8.9 Hz, H-8"), 6.68 (1H, d, J =1.8 Hz, H-2"'), 6.64 (1H, d, J =8.0 Hz, H-5"'), 6.44 (1H, dd, J =1.8, 8.0 Hz, H-6"'), 3.02 (1H, dd, J =8.5, 14.3 Hz, H-7"'a), 3.10 (1H, dd, J =3.9, 14.3 Hz, H-7"'b), 5.22 (1H, dd, J =3.9, 8.5 Hz, H-8"'). These 1H-NMR data corresponded with previous reports (27-29) and to authentic Sal B (data not shown). Consequently, the isolated compound was unambiguously identified as Sal B. 1
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Fig. 4. TLC analysis of a part of fractions obtained after Sephadex LH-20 column chromatography.
Fig. 5. HPLC chromatogram in preparative scale of main fractions (Ve/Vt 1.75-2.71, fr. 64-110) containing Sal B after Sephadex LH-20 column chromatography of the EtOAc-acidic layer.
Sal B is the most abundant representative compound in roots of dansham which is beneficial in treatment of cardiovascular disease. Therefore, dansham and Sal B have recently attracted considerable attention as a potent functional food ingredient for prevention of cardiovascular diseases such as coronary heart disease. The presently described method provides a convenient and economical way to obtain Sal B with an acceptable yield and high purity, and may offer useful information for extraction, purification, analysis, and application of Sal B.
Acknowledgments ESI-MS and NMR spectral data were obtained from the Korea Basic Science Institute, Ochang and Gwangju, respectively.
References
Fig. 6. HPLC chromatogram (A) and UV/VIS spectrum (B) in HPLC-PDA system of Sal B isolated by preparative HPLC.
Recovery and purity of Sal B Sal B content in MeOH
extract (13.16 g) of dansham roots (100 g dry wt. eq.) was determined to be 1.44 g, with the final isolated amount being 1.08 g. Therefore, on the basis of Sal B content, the yield of Sal B was calculated as 75.0%. Recovery of Sal B based on the weights of MeOH extract (13.16 g) and isolated Sal B (1.08 g) was 8.2%. In addition, the finally isolated Sal B was detected as a single HPLC peak (Fig. 6) of analytical scale, whose purity was determined to be more than 99.2%.
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