One-step separation and purification of 3,4-dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography

Three kinds of polyphenols of Salvia miltiorrhiza Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde, were separated and purified in one step with solvent system n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1.5:8) by high-speed counter-current chromatography. Acetic acid was successfully used to increase the partition of high polar target compounds in organic phase to modify partition coefficient value. 3,4-Dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde were purified from 100mg water extracted crude sample of Salvia miltiorrhiza Bunge at purity of 97.6%, 94.2% and 98.2% and at yield of 98.6%, 73.6% and 90.2%. High-speed counter-current chromatography together with organic/aqueous solvent system supplied an efficient method to purify water-soluble compounds directly from crude samples of traditional Chinese medicines.     

One-step separation and purification of hydrolysable tannins from Geranium wilfordii Maxim by adsorption chromatography on cross-linked 12% agarose gel

The hydrolysable tannins corilagin and geraniin, the major active components of the traditional Chinese medicine Geranium wilfordii Maxim, have been separated and purified from crude extracts in one step by adsorption chromatography on cross‐linked 12% agarose gel (Superose 12 10/300 GL). The separation was achieved by gradient elution using mobile phase A composed of 5% ethanol and 5% acetic acid and mobile phase B composed of 30% ethanol and 30% acetic acid. The gradients were composed as follows: 0–240 mL, 0–25% B; 240–480 mL, 25–40% B; after 480 mL, 100% B. The purities of the collected corilagin and geraniin were 92.4 and 87.2%, and the corresponding yields were 88.0 and 76.8%, respectively.     

Purification of the isoflavonoid puerarin by adsorption chromatography on cross-linked 12% agarose

The isoflavonoid puerarin in extracts of the well-known traditional Chinese drug Radix puerariae (root of the plant Pueraria lobata) can be separated from other isoflavonoids by adsorption chromatography on the cross-linked 12% agarose gel Superose 12 equilibrated in distilled water. The adsorption is totally quenched by the addition of 50% acetic acid. The separation of the isoflavonoids is tentatively ascribed to interaction with the residues of the cross-linking reagents used in the manufacturing process of Superose 12. Thus, no useful separation can be achieved with non-cross-linked 12% agarose gel media. Symmetric elution profiles at high sample loadings (16 mg on a 24 ml column) suggest linear adsorption isotherms for the isoflavonoids.     

Preparative isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography

High-speed counter-current chromatography was applied to the isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude salvianolic acid B was obtained by extraction with ethanol-water from S. miltiorrhiza Bunge. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (3:7:1:9, v/v) was successfully performed yielding 342 mg salvianolic acid B at 98% purity from 500 mg of the crude extract in a one-step separation.     

Separation of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of tanshinones from the roots of Salvia miltiorrhiza Bunge by stepwise elution. A set of three solvent systems and other experimental conditions were determined by analytical HSCCC. Using the optimized conditions, the preparative HSCCC separation was performed on 50 mg of crude light petroleum extract yielding pure tanshinones of tanshinone HA (7 mg), tanshinone I (3 mg) and cryptotanshinone (4 mg) all at purities of over 95% in a single run.     

One-step purification of epigallocatechin gallate from crude green tea extracts by mixed-mode adsorption chromatography on highly cross-linked agarose media

(-)-Epigallocatechin gallate (EGCG) was purified in one step from a green tea polyphenol (GTP) crude extract by adsorption chromatography on a Superose 12 HR 10/30 column. The mobile phase used was a mixture of acetonitrile and water with an optimum mobile phase compositions regarding purity, recovery and separation time of 78/22 (v/v). Maximum practical sample loading was 100 mg GTP per run (corresponding to 4.2 mg/ml Superose). An EGCG purity of 99% with recoveries in the range 60-65% was achieved in one step directly from the crude GTP extract. Full column regeneration was obtained using solvents in the following order: 0.5 M NaOH, distilled water and 30% acetic acid.     

Large-scale separation of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by pH-zone-refining countercurrent chromatography

pH-Zone-refining countercurrent chromatography was successfully applied to the separation of salvianolic acid B from the Chinese medicinal plant, Salvia miltiorrhiza Bunge, using a multilayer coil planet centrifuge. A 2.0 g quantity of sample was separated using the following two-phase solvent system: methyl tert-butyl ether (MtBE)-water, 10 mM TFA in organic stationary phase and 10 mM ammonia in aqueous mobile phase. The obtained fractions were analyzed by HPLC and ESI-MS. The separation yielded 572 mg of the main component of salvianolic acid B with a purity of 94.1%.     

Separation and identification of water-soluble salvianolic acids from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography and ESI-MS analysis

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been applied to separate and purify salvianolic acids from the water extract of Danshen, Salvia miltiorrhiza Bunge. High efficiency HSCCC separation was achieved on a two-phase solvent system composed of a mixture of n-hexane-ethyl acetate-water-methanol (1.5:5:5:1.5, v/v) by eluting the lower mobile phase at a flow-rate of 1.7 ml/min and a revolution of 850 rpm. A total of five well separated peaks were obtained in the HSCCC chromatogram, and their purities determined by HPLC-UV absorption. These peaks were characterized by UV-vis spectra and ESI-MS, and the data compared with the reference standards. Salvianolic acid B was positively identified as one of the major peaks. Three of the remaining four peaks were also tentatively identified as rosmarinic acid, lithospermic acid, and salvianolic acid E, an isomer of salvianolic acid B, all are members of the salvianolic acids group. In a typical run, tens of milligrams of samples can be separated with high efficiency to yield tens of milligrams of purified materials with over 98% purity. HSCCC thus provides a cost-effective alternative to preparative scale HPLC for the semi-preparative scale separation and purification of salvianolic acids in Danshen. With appropriate modifications, the technique should also be applicable to other herbs in general.     

Rapid and high-throughput purification of salvianolic acid B from Salvia miltiorrhiza Bunge by high-performance counter-current chromatography

A large-scale purification of salvianolic acid B from Salvia miltiorrhiza Bunge is presented. The method development began with selection of the solvent system, then optimization of the operating parameters and ended up with linear scale-up from an analytical to a preparative instrument. Three factors were used for method optimization and scale-up estimation: purity, process throughput and process efficiency. Preparation was achieved using a two-phase solvent system comprising hexane–ethyl acetate–methanol–acetic acid–water (1:5:1.5:0.00596:5, v/v). This preparation yielded 475 mg of salvianolic acid B with a purity of 96.1% from 1.5 g of crude extract. The process throughput of crude was 2.23 g/h while process efficiency per gram of target compound was 0.769 g/h. Two factors—process environmental risk factor and process evaluation factor were used for evaluation of the separation process.     

Isolation and purification of flavonoid and isoflavonoid compounds from the pericarp of Sophora japonica L. by adsorption chromatography on 12% cross-linked agarose gel media

A method for isolation and purification of flavonoid and isoflavonoid compounds in extracts of the pericarp of Sophora japonica L. was established by adsorption chromatography on the 12% cross-linked agarose gel Superose 12. The crude extracts were pre-separated to two parts, sample A and sample B, on a D-101 macroporous resin column by elution with 20% ethanol and 40% ethanol, respectively. Samples A and B were then separated by adsorption chromatography on Superose 12 with 40% methanol as the mobile phase. Eight compounds including four kinds of flavonoids and four kinds of isoflavonoids were obtained by the proposed method. The adsorption mechanisms of flavonoids and isoflavonoids on Superose 12 were also discussed.     

Isolation and purification of chemical constituents from the pericarp of Sophora japonica L. by chromatography on a 12% cross-linked agarose gel

A chromatographic method using 12% cross-linked agarose gel Superose 12 as the separation medium was developed for isolation and purification of the chemical constituents from the pericarp of Sophora japonica L. The mobile phase used for the separation was 2% acetic acid and 7% acetic acid in gradient elution. As a result, eight compounds including four kinds of flavonoids and four kinds of isoflavonoids were obtained in a one-step separation. A straightforward explanation of the separation mechanism of flavonoids and isoflavonoids on Superose 12 is also given. The flavonoids and isoflavonoids are retained on Superose 12 by a combination of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of aglycone and the residues of the cross-linking reagents used in the manufacture of Superose 12.     

Purification of salvianolic acid B from the crude extract of Salvia miltiorrhiza with hydrophilic organic/salt-containing aqueous two-phase system by counter-current chromatography

Establishment of hydrophilic organic/salt-containing aqueous two-phase system and purification of salvianolic acid B from crude extract of S. miltiorrhiza by counter-current chromatography with said system were studied. Ethanol and n-propanol were selected to constitute biphasic systems with ammonia sulphate, sodium chloride and phosphate separately, and related system characteristics including phase diagrams, phase ratio, separation time were tested. The partition coefficient of crude salvianolic acid B was also tested in above systems and further finely adjusted by altering the constitution of phosphate in n-propanol/phosphate system. Salvianolic acid B was purified to 95.5% purity by counter-current chromatography in 36% (w/w) n-propanol/8% (w/w) phosphate system with the ratio between dipotassium hydrogen phosphate and sodium dihydrogen phosphate of 94:6. One hundred and eight milligrams of salvianolic acid B was purified from 285 mg crude extract with the recovery of 89%.     

Minor compounds of the high purity salvianolic acid B freeze-dried powder from Salvia miltiorrhiza and antibacterial activity assessment

The study explored the isolation and characterisation of three compounds of high purity salvianolic acid B freeze-dried powder extracted from Salvia miltiorrhiza Bunge. A new salvianolic acid, salvianolic acid V (2) together with two known compounds (3–4) was identified. The antibacterial activity tests showed that compound 2 combined with clinical antibiotics such as Levofloxacin or Colistin sulphate together exhibited potent effects against MRSA or Acinetobacter baumanii. This report has considerably extended our knowledge about the diversity and bioactivity of caffeic acid derivatives from S. miltiorrhiza.     

Preparative separation of lithospermic acid B from Salvia miltiorrhiza by polyamide resin and preparative high-performance liquid chromatography

Adsorption on polyamide resin was investigated as a means of separating lithospermic acid B (LAB) from a crude extract of the roots of the traditional Chinese medicine Salvia miltiorrhiza Bunge ("Danshen"). Variables affecting adsorption capacity (solution pH, contact time on resin, initial LAB concentration) were studied. Adsorption was strongly dependent upon the initial concentration of LAB and pH. In all conditions, the polyamide resin gave optimal adsorption of LAB at an initial concentration of 2.66 mg/mL and pH <3.0. The adsorption isotherm correlated well with the Langmuir-type adsorption isotherm. Maximal adsorption capacity was calculated to be 380 mg/g at pH 2.0 and 25°C. LAB purity of 85.30% could be obtained by polyamide resin adsorption followed by elution with 70% ethanol solution, and the recovery was 87.1%. After preparative HPLC, the maximum HPLC purity obtained was 99.28% with a recovery of 75.2%. This method provides an efficient and low-cost method for LAB purification for industrial applications.     

Extraction and isolation of lithospermic acid B from Salvia miltiorrhiza Bunge using aqueous two‐phase extraction followed by high‐performance liquid chromatography

A rapid and effective method integrating separation and purification of lithospermic acid B from Salvia miltiorrhiza Bunge was developed by combining an aqueous two‐phase system extraction with preparative chromatography. An aqueous two‐phase system of n‐butyl alcohol/KH₂PO₄ was chosen from seven systems. The influence of parameters including concentration of KH₂PO₄, n‐butyl alcohol concentration, pH, and the ratio of an aqueous two‐phase system to crude extract were investigated using a single factor design. Response surface methodology was subsequently used to find the optimal compositions of an aqueous two‐phase system. Keeping a solvent‐to‐solid ratio of 10, the final optimized composition of an aqueous two‐phase system was 39.1% w/w n‐butyl alcohol and 22.6% w/w KH₂PO₄. Under these conditions a recovery yield of 99.8% and a high partition coefficient of 310.4 were obtained. In a pilot‐scale experiment using optimized conditions, 18.79 g of lithospermic acid B with a purity of 70.5% and in a yield of 99.8% was separated from 0.5 kg of crude extract. Subsequently, 9.94 g lithospermic acid B with a purity of 99.3% and recovery yield of 70.3% was obtained with a preparative chromatographic process, and the two‐step total recovery was 70.1%.     

HPLC method for comparative study on tissue distribution in rat after oral administration of salvianolic acid B and phenolic acids from Salvia miltiorrhiza

A sensitive and selective high-performance liquid chromatography method was developed and validated to determine the prototype of salvianolic acid B and the metabolites of phenolic acids (protocatechuic acid, vanillic acid and ferulic acid) in rat tissues after oral administration of total phenolic acids and salvianolic acid B extracted from the roots of Salvia miltiorrhiza, respectively. The tissue samples were treated with a simple liquid-liquid extraction prior to HPLC. Analysis of the extract was performed on a reverse-phase C(18) column with a mobile phase consisting of acetonitrile and 0.05% trifluoracetic acid. The calibration curves for the four phenolic acids were linear in the given concentration ranges. The intra-day and inter-day relative standard deviations in the measurement of quality control samples were less than 10% and the accuracies were in the range of 88-115%. The average recoveries of all the tissues ranged from 78.0 to 111.8%. This method was successfully applied to evaluate the distribution of the four phenolic acids in rat tissues after oral administration of total phenolic acids of Salvia miltiorrhiza or salvianolic acid B and the possible metabolic pathway was illustrated.     

Preparative isolation and purification of six diterpenoids from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of six diterpenoids. dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude diterpenoids were obtained by extraction with ethanol-n-hexane (1:1, v/v) from S. miltiorrhiza Bunge. Preparative HSCCC with the two-phase solvent systems A composed of n-hexane-ethanol-water (10:5.5:4.5, v/v) and B composed of n-hexane-ethanol-water (10:7:3, v/v) was successfully performed in a stepwise elution yielding six relatively pure diterpenoids from 300 mg of the crude extract in a single run. The purities of dihydrotanshinone I, cryptotanshinone, methylenetanshiquinone, tanshinone I, tanshinone IIA and danshenxinkun B were 88.1, 98.8, 97.6, 93.5, 96.8 and 94.3%, respectively.     

Direct purification of tanshinones from Salvia miltiorrhiza Bunge by high‐speed counter‐current chromatography without presaturation of the two‐phase solvent mixture

Tanshen, the rhizome of Salvia miltiorrhiza Bunge, is a famous Traditional Chinese Medicine for multiple therapeutic remedies. This work presents the isolation and purification of tanshinone I and tanshinone IIA from the extract of the rhizome of S. miltiorrhiza by using high‐speed counter‐current chromatography (CCC) without presaturation of the two‐phase solvent mixture. The CCC method combines the results of CCC solvent system selection and components analyses of solvent mixture by GC, and thus it is possible to add accurately each individual solvent to prepare single saturated solvent phase without presaturation. The optimum CCC solvent system is a system of hexane–ethyl acetate–ethanol–water (8:2:7:3, v/v), which has been determined by usual solvent system selection and CCC runs. As a result, over 98% pure tanshinone IIA and over 94% pure tanshinone I have been obtained by using less solvent volume. Their structures have been identified by ESI‐MS, NMR spectra.     

Rapid and simultaneous determination of salvianolic acid A and salvianolic acid B by complex chromatography and study on related mechanism of coordination

Salvianolic acid A (Sal A) and salvianolic acid B (Sal B) are water-soluble phenolic acids in Danshen extract and they have high medicinal value. A rapid and novel complex high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of Sal A and Sal B within 10 min. The determination of Sal A and Sal B was carried out on a Waters Symmetry Shield RP18 column (5 μm, 3.9 mm × 150 mm), using methanol-deionized water (55:45, v/v, with 5 mmol L⁻¹ CaCl₂ and 1% acetic acid) as the mobile phase at the flow rate of 0.8 mL min⁻¹ within 10 min. The wavelength was set at 280 nm. It changed the peak sequence of Sal A and Sal B and improved their effect of determination by adding CaCl₂ in the mobile phase because Sal A and Sal B coordinated with Ca²⁺. The mechanism of coordination between Sal A, Sal B and Ca²⁺ has been studied by way of Fourier transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS) and computer model. The possible structures of the complex and complex ratio are provided in this article. The experiments have facilitated the study of Sal A-Ca²⁺ complex, Sal B-Ca²⁺ complex and provide a theoretical basis for industrialized extraction of Sal A and Sal B in the future.     

Interactions of acetylcholinesterase with salvianolic acid B and rosmarinic acid from Salvia miltiorhiza water extract investigated by NMR relaxation rate

In order to understand whether the ameliorating effect on old ages memory disorder by the root of Salvia miltiorhiza is related to the acetylcholinesterase （ACHE） inhibition, two main ingredients, salvianolic acid B （1） and rosmarinic acid （2）, which were isolated from S. miltiorhiza water extract, were investigated in vitro by NMR relaxation rate in this work. The results showed that the proton selective relaxation rates and the molecular rotational correlation time of proton pairs for compounds 1 and 2 increased significantly by adding of AChE in mixing solution. The study reveals that the two compounds might bind to the enzyme and have ACHE inhibitory effect, which could contribute to the ameliorating effect at some extent on old ages memory disorder.