H2O-Induced Hydrophobic Interactions in MS-Guided Counter-Current Chromatography Separation of Anti-Cancer Mollugin from Rubia cordifolia.

Counter-current chromatography (CCC) is a unique liquid-liquid partition chromatography and largely relies on the partition interactions of solutes and solvents in two-phase solvents. Usually, the two-phase solvents used in CCC include a lipophilic organic phase and a hydrophilic aqueous phase. Although a large number of partition interactions have been found and used in the CCC separations, there are few studies that address the role of water on solvents and solutes in the two-phase partition. In this study, we presented a new insight that H2O (water) might be an efficient and sensible hydrophobic agent in the n-hexane-methanol-based two-phase partition and CCC separation of lipophilic compounds, i.e., anti-cancer component mollugin from Rubia cordifolia. Although the n-hexane-methanol-based four components solvent systems of n-hexane-ethyl acetate-methanol-water (HEMWat) is one of the most popular CCC solvent systems and widely used for natural products isolation, this is an interesting trial to investigate the water roles in the two-phase solutions. In addition, as an example, the bioactive component mollugin was targeted, separated, and purified by MS-guided CCC with hexane-methanol and minor water as a hydrophobic agent. It might be useful for isolation and purification of lipophilic mollugin and other bioactive compounds complex natural products and traditional Chinese medicines.

Modeling gradient elution in countercurrent chromatography: efficient separation of tanshinones from Salvia miltiorrhiza Bunge.

Countercurrent chromatography (CCC) is a support-free liquid-liquid chromatography using centrifugal fields to hold the liquid stationary phase. CCC has been widely applied in the separation of various natural and synthetic components using a variety of biphasic liquid systems. The related hexane or heptane/ethyl acetate/methanol or ethanol/water biphasic liquid systems demonstrated their significance in CCC. Gradient is difficult in CCC since any composition change in one phase induces a composition change of the other phase to maintain phase equilibrium. This work provides a new insight into linear gradient elution in CCC that is feasible with some biphasic liquid systems such as selected compositions of the hexane/ethyl acetate/ethanol/water systems. The equations modeling solute motion inside the CCC column are proposed. Particular compositions of the liquid system, namely the hexane/ethyl acetate/ethanol/water 8:2:E:W compositions with E + W = 10, were studied from W = 1 to 9. They showed moderate changes in the upper organic phase compositions. The model is tested with the separation of tanshinones from the rhizome of Salvia miltiorrhiza Bunge. Different linear solvent gradient profiles were experimentally performed between 8:2:5:5 and 8:2:3:7 compositions and the results were evaluated using the proposed model. Five tanshinones including dihydrotanshinone I, cryptotanshinone, tanshinone I, 1,2-dihydrotanshinquinone, and tanshinone IIA have been successfully separated (>95% purities) using a gradient profile optimized by the developed model. The gradient model can be used only with biphasic liquid systems in which one phase shows minimum composition changes when the other phase composition changes notably. This case is not the general case for biphasic liquid systems but can be applied with specific compositions of the quaternary hexane or heptane/ethyl acetate/methanol or ethanol/water most useful CCC liquid systems.

A novel protocol for the preparation of sodium tanshinone sulphonates by direct ultrasound-assisted sulphonation of the crude extract of the roots of Salvia miltiorrhiza Bunge and following counter-current chromatography purification.

INTRODUCTION: Sodium tanshinone sulphonates are water-soluble derivatives of tanshinones originated from Tanshen (or Danshen, Salvia miltiorrhiza Bunge), a famous Traditional Chinese Medicine, which have potent biological activities, especially in the treatment of cardiovascular disorders. However, the classical preparation processes of sodium tanshinone sulphonates often involve multiple time- and solvent-consuming steps after purification of tanshinones, resulting in relatively low yields. OBJECTIVE: To develop a simple protocol for direct preparation of sodium tanshinone sulphonates from the complex crude extract of the roots of S. miltiorrhiza without pre-purification of tanshinones. METHODOLOGY: The 100 mg crude tanshinone extract of S. miltiorrhiza was first sulphonated in a ultrasound bath with glacial acetic acid, acetic anhydride and concentrated sulphuric acid for 20 min, and then subjected to counter-current chromatography (CCC) separation using a optimum two-phase solvent system composed of n-hexane:ethylacetate:ethanol:5% sodium chloride aqueous solution (1:8:4:10, v/v). Based on the UV detection and HPLC analyses, the sulphonated fractions were collected. RESULT: Sodium tanshinone IIA sulphonate (7.1 mg) and sodium tanshinone I sulphonate (2.8 mg) with over 95% purity were obtained successfully for the first time by ultrasound-assisted sulphonation and following CCC purification. CONCLUSION: The study has shown that the method combining ultrasound-assisted sulphonation and CCC purification is an efficient way to prepare tanshinone sulphonates without pre-purification of tanshinones from the complex extracts of Tanshen, and can be explored as a new protocol for wide natural product modification directly from a crude complex extracts without pre-purification.

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.

Methanol linear gradient counter-current chromatography for the separation of natural products: Sinopodophyllum hexandrum as samples.

Counter-current chromatography (CCC) is a unique, liquid-liquid partition chromatography process. Both the mobile and stationary phases are liquids, so no solid support matrix is used. CCC has gained wide acceptance as a preparative technique in a variety of fields. Because the mobile and stationary phases are both liquids, gradient elution is difficult to perform with CCC. Phase equilibrium must be maintained, so any change in the composition of one phase may induce a compositional change in the other. In this work, a new linear gradient elution method was developed for CCC. Biphasic solvent systems containing heptane, ethyl acetate, methanol, and water (HepEMWat) in various ratios were prepared and used to optimize both isocratic and linear gradient CCC separation with methanol. We first separated a test mixture of four standard compounds with partition coefficients ranging from 0.8 to 7.8. The separation resembled a reversed-phase process, and elution was performed while progressively decreasing the polarity of the mobile phase. Target molecules with small partition coefficients eluted first in the lower phase of the optimized HepEMWat solvent system. Elution of constituents with large partition coefficients was quite slow under isocratic conditions. Separation time was significantly reduced when elution was performed with a linear gradient using methanol and the optimal HepEMWat system. Elution with a 3:7:4:6 (v/v/v/v) HepEMWat system took approximately 200 min. This included an 80-min isocratic step, followed by gradient elution with methanol from 0% to 30%. The optimized methanol linear gradient CCC method was then used to separate a complex mixture of natural products isolated from Sinopodophyllum hexandrum (Royle) Ying roots. Twelve compounds with a wide range of polarities were well-resolved in a single separation. We have developed a convenient and cost-effective strategy for the separation of complex mixtures. No tedious mobile phase preparation step is required. The volume of unused mobile phase is minimal, so little solvent is wasted. The method is an important advance for the separation of mixtures that contain many compounds with a large range of polarities and partition coefficients, which are common features of natural products.

A novel 9 × 9 map-based solvent selection strategy for targeted counter-current chromatography isolation of natural products.

Counter-current chromatography (CCC) is an efficient liquid-liquid chromatography technique for separation and purification of complex mixtures like natural products extracts and synthetic chemicals. However, CCC is still a challenging process requiring some special technical knowledge especially in the selection of appropriated solvent systems. In this work, we introduced a new 9 × 9 map-based solvent selection strategy for CCC isolation of targets, which permit more than 60 hexane-ethyl acetate-methanol-water (HEMWat) solvent systems as the start candidates for the selection of solvent systems. Among these solvent systems, there are clear linear correlations between partition coefficient (K) and the system numbers. Thus, an appropriate CCC solvent system (i.e., sweet spot for K = 1) may be hit by measurement of k values of the target only in two random solvent systems. Besides this, surprisingly, we found that through two sweet spots, we could get a line ("Sweet line") where there are infinite sweet solvent systems being suitable for CCC separation. In these sweet solvent systems, the target has the same partition coefficient (K) but different solubilities. Thus, the better sweet solvent system with higher sample solubility can be obtained for high capacity CCC preparation. Furthermore, we found that there is a zone ("Sweet zone") where all solvent systems have their own sweet partition coefficients values for the target in range of 0.4 < K< 2.5 or extended range of 0.25 < K < 16. All results were validated by using 14 pure GUESSmix mimic natural products as standards and further confirmed by isolation of several targets including honokiol and magnolol from the extracts of Magnolia officinalis Rehd. Et Wils and tanshinone IIA from Salvia miltiorrhiza Bunge. In practice, it is much easier to get a suitable solvent system only by making a simple screening two to four HEMWat two-phase solvent systems to obtain the sweet line or sweet zone without special knowledge or comprehensive standards as references. This is an important advancement for solvent system selection and also will be very useful for isolation of current natural products including Traditional Chinese Medicines.

Overlapping elution-extrusion counter-current chromatography: a novel method for efficient purification of natural cytotoxic andrographolides from Andrographis paniculata.

Counter-current chromatography (CCC) is extremely useful for the separation, purification, and isolation of natural products. Recently, Berthod et al. established an elution-extrusion CCC method in metabolic analysis by combining regular chromatographic elution with stationary-phase extrusion, which extends the hydrophobicity window of a counter-current separation. In this study, a novel overlapping elution-extrusion CCC method was developed and applied to the preparation of natural cytotoxic andrographolides from the aerial parts of Andrographis paniculata, a well-known Traditional Chinese Medicine (TCM) with potent anti-inflammatory effect and anti-cancer activity. Its theory was first developed, and then a series of CCC experiments were performed to investigate the efficiency of the method in the separation of the ethanol extracts from A. paniculata. Results show that overlapping elution-extrusion CCC is an efficient method to prepare a cytotoxic natural diterpenoid combination of 14-deoxy-andrographolide and 14-deoxy-11,12-didehydroandrographolide with the molar ratio of 1:2 as well as andrographolide using an optimized solvent system composed of hexane-ethyl acetate-ethanol-water (5:5:4:6, v/v) with an on-demand solvent preparation mode. All components obtained showed potent cytotoxic activity against human hepatocellular liver carcinoma cells HepG2 and doxorubicin-resistant R-HepG2 cells. Molecular structures have been identified by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), one- and two-dimensional nuclear magnetic resonance (1D- and 2D-NMR). The method appears to be very useful for the high-throughput purification of natural products.

Andrographolide enhances 5-fluorouracil-induced apoptosis via caspase-8-dependent mitochondrial pathway involving p53 participation in hepatocellular carcinoma (SMMC-7721) cells.

Despite recent significant advances in the treatment of human carcinoma (HCC), the results of chemotherapy to date remain unsatisfactory. 5-Fluorouracil (5-FU) still represents the cornerstone of treatment of carcinoma, and resistance to the actions of 5-FU is a major obstacle to successful chemotherapy. More effective treatment strategies may involve combinations of agents with activity against HCC. Andrographolide (ANDRO), a natural bicyclic diterpenoid lactone isolated from Andrographis paniculata, has been shown to suppress the growth of HCC cells and trigger apoptosis in vitro. To assess the suitability of ANDRO as a chemotherapeutic agent in HCC, its cytotoxic effects have been evaluated both as a single agent and in combination with 5-FU. ANDRO potentiates the cytotoxic effect of 5-FU in HCC cell line SMMC-7721 through apoptosis. ANDRO alone induces SMMC-7721 apoptosis with p53 expression, Bax conformation and caspase-3,8,9 activation. Surprisingly, the addition of ANDRO to 5-FU induces synergistic apoptosis, which could be corroborated to the increased caspase-8, p53 activity and the significant changes of Bax conformation in these cells, resulting in increased losses of mitochondrial membrane potential, increased release of cytochrome c, and activation of caspase-9 and caspase-3. Suppression of caspase-8 with the specific inhibitor z-IETD-fmk abrogates largely ANDRO/5-FU biological activity by preventing mitochondrial membrane potential disappearance, caspase-3,9 activation and subsequent apoptosis. The results suggest that ANDRO may be effective in combination with 5-FU for the treatment of HCC cells SMMC-7721.