Identification of Silymarin Constituents: An Improved HPLC–MS Method

A high-performance liquid chromatographic/tandem mass spectrometric method was developed for the determination of the major bioactive flavonolignans in silymarin, a herbal remedy extracted from the milk thistle Silybum marianum. In this study, eight active components of silymarin with the same elemental composition, including silychristins A and B, silydianin, silybin A and B, isosilybin A and B and an unknown compound were completely separated. Furthermore, three additional components were detected and partly separated; presumably two silybin stereoisomers and one isosilybin stereoisomer. The collision-induced dissociation (CID) MS/MS spectra of these silymarin constituents were studied: the spectral similarity values of the component pairs were determined, and simple criteria were found for distinguishing the components.

     

固相萃取法纯化制备水飞蓟宾和异水飞蓟宾

水飞蓟宾和异水飞蓟宾是水飞蓟素中的主要有效成分,其纯化制备主要借助柱色谱法,制备量大,纯化效果好,但过程非常费时。该研究的主要目的是利用更为快速高效的固相萃取(SPE)法从水飞蓟粗提物中分离纯化水飞蓟宾和异水飞蓟宾。建立了用于分析水飞蓟宾和异水飞蓟宾的高相液相色谱法,通过优化洗脱梯度,实现了水飞蓟宾、异水飞蓟宾与其他组分的分离。试用了3种保留机理不同的SPE填料,包括亲水亲脂(HLB)填料、亲水色谱(HILIC)填料及反相C18硅胶填料。通过对比发现C18硅胶对目标化合物的选择性最佳。进一步控制SPE的淋洗及洗脱条件,收集相应的洗脱液,经氮吹干燥后得到纯化的样品。提纯后的水飞蓟宾和异水飞蓟宾混合物的纯度可达94%以上。水飞蓟宾和异水飞蓟宾的平均回收率分别为70.5%~81.7%和66.7%~81.8%,相对标准偏差分别为2.7%~9.4%和1.5%~6.1%。该方法简单、高效,免去传统分离纯化过程中长时间的柱色谱分离过程,适合制备纯度较高的水飞蓟宾和异水飞蓟宾的二元混合标准样品。     

Separation and characterization of silybin, isosilybin, silydianin and silychristin in milk thistle extract by liquid chromatography-electrospray tandem mass spectrometry

A selective and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the characterization of silymarin in commercially available milk thistle extract. In this study, six main active constituents, including silydianin, silychristin, diastereomers of silybin (silybin A and B) and diastereomers of isosilybin (isosilybin A and B) in silymarin, were completely separated on a YMC ODS-AQ HPLC column using a gradient mobile phase system comprised of ammonium acetate and methanol/water/formic acid. Identification and characterization of the major constituents were based not only on the product ion scan, which provided unique fragmentation information of a selected molecular ion, but also on the specific fragmentation of multiple reaction monitoring (MRM) data, which confirmed the retention times of LC chromatographic peaks. The method was applied in the analysis of human plasma samples in the presence of silymarin and appeared to be suitable for the pharmacokinetic studies in which the discrimination of silymarin constituents is essential.     

Development of a capillary electrophoresis method for the separation of flavonolignans in silymarin complex

CE method for the baseline separation of structurally similar flavonolignans silybin A, silybin B, isosilybin A, isosilybin B, silychristin, silydianin, and their precursor taxifolin in silymarin complex has been developed and validated. The optimized background electrolyte was 100 mmol/L boric acid (pH 9.0) containing 5 mmol/L heptakis(2,3,6-tri-O-methyl)-β-CD and 10% (v/v) of methanol. The separation was carried out in an 80.5/72 cm (50 μm id) fused silica capillary at +25 kV with UV detection at 200 nm. Genistein (10 μg/mL) was used as internal standard. The resolution between the diastereomers of silybin and isosilybin was 1.73 and 2.59, respectively. The method was validated for each analyte in a concentration range of 2.5–50 μg/mL. The calibration curves were rectilinear with correlation coefficients ≥0.9972. The method was applied to determine flavonolignans in two dietary supplements containing Silybum marianum extract. The accuracy was evaluated by comparing the results of the CE analyses of the dietary supplements with those of the reference United States Pharmacopeial HPLC method. The unpaired t-test did not show a statistically significant difference between the results of both the proposed CE and the reference method (p > 0.05, n = 3).     

Validated Optimized Method for Simultaneous Analysis of Active Silymarin Components and Dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene Dioxybiphenyl-2,2′-dicarboxylate in a Pharmaceutical Preparation by Use of a Monolithic Silica C18 Column

A simple, rapid, and sensitive isocratic reversed-phase LC method using a monolithic column has been developed and validated for simultaneous analysis of the active components of silymarin [taxifolin, silydianin, silychristin, diastereomers of silybin (silybin A and B), and diastereomers of isosilybin (isosilybin A and B)] and dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxybiphenyl-2,2′-dicarboxylate in a commercial formulation. The mobile phase was a 45:55 (v/v) mixture of methanol and 5 mM NaH₂PO₄ (adjusted to pH 2.75 with phosphoric acid) at a flow rate of 1.5 mL min^?1. UV detection was performed at 288 nm and quantification was based on peak area. The method was validated for linearity, accuracy, precision, selectivity, and robustness.     

Preparative isolation of flavonoid glycosides from Sphaerophysa salsula using hydrophilic interaction solid‐phase extraction coupled with two‐dimensional preparative liquid chromatography

An offline preparative two‐dimensional reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography coupled with hydrophilic interaction solid‐phase extraction method was developed for the preparative isolation of flavonoid glycosides from a crude sample of Sphaerophysa salsula . First, the non‐flavonoids were removed using an XAmide solid‐phase extraction cartridge. Based on the separation results of three different chromatographic stationary phases, the first‐dimensional preparation was performed on an XAqua C18 prep column, and 15 fractions were obtained from the 5.2 g target sample. Then, three representative fractions were selected for additional purification on an XAmide preparative column to further isolate the flavonoid glycosides. In all, eight flavonoid glycosides were isolated in purities over 97%. The results demonstrated that the two‐dimensional liquid chromatography method used in this study was effective for the preparative separation of flavonoid glycosides from Sphaerophysa salsula . Additionally, this method showed great potential for the separation of flavonoid glycosides from other plant materials.     

水飞蓟素中活性黄酮的测定

采用高效液相色谱梯度淋洗法,使水飞蓟素中的７种主要成分得到完全分离。方法简便、快速,能单独测得水飞滨的含量,与紫外分光光度法相比,具有较好的重复性,变异系数在２％以下。     

Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum)

Complete separation, isolation, and structural characterization of four diastereoisomeric flavonolignans, silybins A (1) and B (2), and isosilybins A (3) and B (4) from the seeds of milk thistle (Silybum marianum) were achieved for the first time using a preparative reversed-phase HPLC method. In addition, three other flavonolignans, silychristin (5) isosilychristin (6) and silydianin (7), and a flavonoid, taxifolin (8) were isolated. Structures, including absolute stereochemistries of 1-4, were confirmed using 2D NMR and CD spectroscopy.     

Combinative application of pH‐zone‐refining and conventional high‐speed counter‐current chromatography for preparative separation of caged polyprenylated xanthones from gamboge

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography for the first time. pH‐zone‐refining counter‐current chromatography was performed with the solvent system composed of n‐hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high‐speed counter‐current chromatography with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n‐hexane/methyl tert‐butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β‐morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high‐performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.     

Phosphodiester Silybin Dimers Powerful Radical Scavengers: A Antiproliferative Activity on Different Cancer Cell Lines

Silibinin is the main biologically active component of silymarin extract and consists of a mixture 1:1 of two diastereoisomeric flavonolignans, namely silybin A (1a) and silybin B (1b), which we call here silybins. Despite the high interest in the activity of this flavonolignan, there are still few studies that give due attention to the role of its stereochemistry and, there is still today a strong need to investigate in this area. In this regard, here we report a study concerning the radical scavenger ability and the antiproliferative activity on different cell lines, both of silybins and phosphodiester-linked silybin dimers. An efficient synthetic strategy to obtain silybin dimers in an optical pure form (6aa, 6ab and 6bb) starting from a suitable building block of silybin A and silybin B, obtained by us from natural extract silibinin, was proposed. New dimers show strong antioxidant properties, determined through hydroxyl radical (HO^●) scavenging ability, comparable to the value reported for known potent antioxidants such as quercetin. A preliminary screening was performed by treating cells with 10 and 50 μM concentrations for 48 h to identify the most sensitive cell lines. The results show that silibinin compounds were active on Jurkat, A375, WM266, and HeLa, but at the tested concentrations, they did not interfere with the growth of PANC, MCF-7, HDF or U87. In particular, both monomers (1a and 1b) and dimers (6aa, 6ab and 6bb) present selective anti-proliferative activity towards leukemia cells in the mid-micromolar range and are poorly active on normal cells. They exhibit different mechanisms of action in fact all the cells treated with the 1a and 1b go completely into apoptosis, whereas only part of the cells treated with 6aa and 6ab were found to be in apoptosis.     

Purification of flavonoids from licorice using an off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method

An orthogonal (71.9%) off‐line preparative two‐dimensional normal‐phase liquid chromatography/reversed‐phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self‐made Click TE‐Cys (60 μm) solid‐phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE‐Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co‐eluted in the first dimension were selected for further purification using reversed‐phase liquid chromatography. Multiple compounds could be isolated from one normal‐phase fraction and some compounds with bad resolution in one‐dimensional liquid chromatography could be prepared in this two‐dimensional system owing to the orthogonal separation. Moreover, this two‐dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off‐line two‐dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.     

Separation of four flavonol glycosides from Solanum rostratum Dunal using aqueous two‐phase flotation followed by preparative high‐performance liquid chromatography

Aqueous two‐phase flotation followed by preparative high‐performance liquid chromatography was used to separate four flavonol glycosides from Solanum rostratum Dunal. In the aqueous two‐phase flotation section, the effects of sublation solvent, solution pH, (NH₄)₂SO₄ concentration in aqueous solution, cosolvent, N₂ flow rate, flotation time, and volumes of the polyethylene glycol phase on the recovery were investigated in detail, and the optimal conditions were selected: 50 wt% polyethylene glycol 1000 ethanol solvent as the flotation solvent, pH 4, 350 g/L of (NH₄)₂SO₄ concentration in aqueous phase, 40 mL/min of N₂ flow rate, 30 min of flotation time, 10.0 mL of flotation solvent volume, and two times. After aqueous two‐phase flotation concentration, the flotation products were purified by preparative high‐performance liquid chromatography. The purities of the final products A and B were 98.1 and 99.0%. Product B was the mixture of three compounds based on the analysis of high‐performance liquid chromatography at the temperature of 10°C, while product A was hyperoside after the identification by nuclear magnetic resonance. Astragalin, 3’‐O‐methylquercetin 3‐O‐β‐d ‐galactopyranoside, and 3’‐O‐methylquercetin 3‐O‐β‐d ‐glucopyranoside were obtained with the purity of 93.8, 97.1, and 99.2%, respectively, after the further separation of product B using preparative high‐performance liquid chromatography.     

Combined chromatographic strategy based on macroporous resin,high‐speed counter‐current chromatography and preparative HPLC for systematic separation of seven antioxidants from the fruit of Terminalia billerica

In the present study, combined chromatographic strategy based on macroporous resin, high‐speed counter‐current chromatography and preparative high‐performance liquid chromatography for systematic separation of antioxidants from crude samples guided by high‐performance liquid chromatography with 1,1‐diphenyl‐2‐picrylhydrazyl has been successfully established. Based on this strategy, seven antioxidants including isorugosin A, _β‐1,2,3,6‐tetragalloyl‐D ‐glucose, chebulinic acid, 1,2,3,4,6‐penta‐O‐galloyl‐_β‐D‐glucose, chebulagic acid, ethyl gallate, and gallic acid were obtained from the fruit of Terminalia billerica. First, high‐performance liquid chromatography with 1,1‐diphenyl‐2‐picrylhydrazyl experiment showed the presence of seven main antioxidants in the crude extract of the fruit of Terminalia billerica. Then, a macroporous resin column chromatography method was developed for the enrichment of these seven antioxidants. Finally, an efficient method based on high‐speed counter‐current chromatography and preparative high‐performance liquid chromatography was developed for the separation of these antioxidants. In the selection of solvent systems, it was found that acetic acid could be a good regulator for modifying the partition coefficient values of tannins. The present study provides a reference for systematic separation of antioxidants from crude samples. Considering the general existence of antioxidants in crude samples, this combined chromatographic strategy might lead to broader application prospects.     

Simultaneous quantification of five proteins and seven additives in dairy products with a heart‐cutting two‐dimensional liquid chromatography method

A heart‐cutting two‐dimensional high‐performance liquid chromatography method was developed to simultaneously quantify five major proteins and seven food additives (maltol, ethyl maltol, vanillin, ethyl vanillin, benzoic acid, sorbic acid, and saccharin sodium) in milk and milk powders. In this two‐dimensional system, a Venusil XBP‐C4 column was selected in the first dimension for protein separation, and a Hypersil ODS‐2 C18 column was employed in the second dimension for additive separation; a two‐position, six‐port switching valve was used to transfer the targets (additives) from the first dimension to the second dimension. Method validation consisted of selectivity, response function, linearity, precision, sensitivity, and recovery. In addition, a conventional one‐dimensional high‐performance liquid chromatography method was also tested for comparison. The two‐dimensional method resulted in significantly improved recovery of the food additives compared to the conventional method (90.6–105.4% and 65.5–86.5%, respectively). Furthermore, this novel method has a simple one‐step sample preparation procedure, which shortens the analysis time, resulting in more efficient analysis and less solvent usage.     

Application of high‐speed counter‐current chromatography and HPLC to separate and purify of three polyacetylenes from Platycodon grandiflorum

Three polyacetylenes were isolated and purified from Platycodon grandiflorum A. DC for the first time by high‐speed counter‐current chromatography using a two‐phase solvent system composed of hexane/ethyl acetate/methanol/water (1:31:1:31, v/v/v/v) and high‐performance liquid chromatography with an Agilent ZORBAX® SB‐C18 column (4.6 mm × 150 mm, 5 μm). After separation by high‐speed counter‐current chromatography and high‐performance liquid chromatography, we obtained 3.5 mg of platetyolin A, 4.1 mg of platetyolin B, and 18.1 mg of lobetyolin with purities of 97.2, 96.7, and 96.9%, respectively. The purity of each compound was assessed by high‐performance liquid chromatography and the chemical structures were evaluated by high‐resolution electrospray ionization time‐of‐flight mass spectrometry and one‐ and two‐dimensional NMR spectroscopy. Among the isolated compounds, platetyolin A and platetyolin B are newly reported compounds.     

Two‐dimensional hydrophilic interaction chromatography × reversed‐phase liquid chromatography for the preparative isolation of potential anti‐hepatitis phenylpropanoids from Salvia prattii

Traditional Tibetan medicine is important for discovery of drug precursors. However, knowledge of the chemical composition of traditional Tibetan medicines is very limited due to the lack of appropriate chromatographic purification methods. In the present work, Salvia prattii was taken as an example, and an off‐line hydrophilic interaction liquid chromatography/reversed‐phase liquid chromatography preparative method was developed for the purification of phenylpropanoids with high purity from a crude sample of Salvia prattii. Based on the separation results of four different chromatographic stationary phases, the first‐dimensional preparation was performed on an XAmide preparative column with the crude sample concentration of 62.0 mg/mL, and five main fractions were obtained from the 12.4 g crude sample with a recovery of 54.8%. An XCharge C₁₈ preparative column was applied in the second‐dimensional preparation to further isolate the phenylpropanoids from the redissolved first‐dimensional fractions with concentration of approximately 50.0 mg/mL. The purities of the phenylpropanoids isolated from the crude sample of Salvia prattii were higher than 98%, indicating that the method was efficient for the purification of phenylpropanoids with high purity from Salvia prattii. Additionally, this method showed great potential in the preparation of phenylpropanoids and can serve as a good example for the purification of phenylpropanoids from other plant materials.     

Analysis of the active components of silymarin

Silymarin is an antihepatotoxic substance isolated from fruits of Silybum marianum. Possibly due to their antioxidant and membrane stabilizing properties, the compounds have been shown to protect different organs and cells against a number of insults. The content and composition of main silymarin components (silybin, isosilybin, silydianin and silychristin) in various pharmaceuticals were analysed using HPLC and newly developed capillary zone electrophoresis method. Antioxidant properties expressed as total antioxidant status (TAS) of silymarin components were studied. Results of TAS were correlated with electropherograms and chromatograms.     

Electrochemical investigation of flavonolignans and study of their interactions with DNA in the presence of Cu(II)

Flavonolignans, silybin and its derivatives (2,3-dehydrosilybin, 7-O-methylsilybin, 20-O-methylsilybin) and isosilybin were studied using ex situ (adsorptive transfer, AdT) cyclic and square wave voltammetry (SWV). The two oxidation steps were described for flavonolignans at potentials E_p1 + 0.5 V and E_p2 + 0.85 V depending on experimental conditions. An additional oxidation peak at E_p3 + 0.35 V was observed only for 2,3-dehydrosilybin. The anodic currents of flavonolignans are related to their electron transfer processes (oxidation of hydroxyl groups), which was supported by density functional theory (DFT) and B3P86 theory level. Our electrochemical results confirmed that 2,3-dehydrosilybin is a relatively strong antioxidant, which is strictly associated with oxidation at E_p3. The oxidation processes and antioxidant parameters of flavonolignans can be affected by transition metal complexation via hydroxyl groups. We found that silybin and 2,3-dehydrosilybin are able to chelate transition metals, especially Cu²⁺. The formation of silybin/Cu complexes was studied by AdT SWV and the observation was also confirmed using fluorescence spectroscopy. The electrochemical investigation of DNA interactions and damage caused in the presence of silybin/Cu complex and hydrogen peroxide is described. We present evidence that flavonolignans are involved not only in antioxidant abilities but also in the prooxidation effects under in vitro conditions.     

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using middle chromatogram isolated gel column coupled with positively charged reversed‐phase liquid chromatography

Positively charged reversed‐phase liquid chromatography was employed for the efficient preparative separation of isoquinoline alkaloids from Corydalis impatiens. Ten commercially available columns were compared for isoquinoline alkaloids analysis. While tailing, overloading, lower resolution, and buffer salts limited the application in purification of isoquinoline compounds of many of these columns, one positively charged reversed‐phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading isoquinoline compounds on a larger, preparative scale. The general separation process is as follows. First, isoquinoline alkaloids are enriched with Corydalis impatiens extract via a middle chromatogram isolated gel column. After column selection, separation is performed on an XCharge C18 analytical column, from which two evident chromatographic peaks are readily obtained. Finally, two isoquinoline alkaloids (protopine and corydamine) are selectively purified on the XCharge C18 preparative column. These results demonstrate that a middle chromatogram isolated gel column coupled with positively charged reversed‐phase liquid chromatography is effective for the preparative separation of isoquinoline alkaloids from Corydalis impatiens.     

A practicable strategy for enrichment and separation of four minor flavonoids including two isomers from barley seedlings by macroporous resin column chromatography,medium‐pressure LC,and high‐speed countercurrent chromatography

Separation of minor compounds especially with similar polarities and structures from complex samples is a challenging work. In the present study, an efficient method was successfully established by macroporous resin column chromatography, medium‐pressure liquid chromatography, and high‐speed countercurrent chromatography for separation of four minor flavonoids from barley seedlings. Macroporous resin column chromatography and medium‐pressure liquid chromatography were used for enrichment of these four flavonoids. High‐pressure liquid chromatography analysis showed the total content of these four flavonoids increased from 2.2% in the crude extract to 95.3% in the medium‐pressure liquid chromatography fraction. It was indicated that the combination of macroporous resin column chromatography and medium‐pressure liquid chromatography could be a practicable strategy for enrichment of minor compounds from complex sample. Then, high‐speed countercurrent chromatography was employed for separation of these four flavonoids using ethyl acetate/n‐butanol/water (0.1% glacial acetic acid) (4:1:5, v/v/v) as solvent system. As a result, four flavonoids including two isomers with purities higher than 98% were obtained. Interestingly, two flavonoids existing in one high‐pressure liquid chromatography peak were also successfully separated. All these indicated high‐speed countercurrent chromatography had great potential for separation of compounds with similar structures and polarities. This study provides a reference for efficient enrichment and separation of minor compounds from complex sample.