Prenylated xanthones from mangosteen as promising cholinesterase inhibitors and their molecular docking studies

Garcinia mangostana is a well-known tropical plant found mostly in South East Asia. The present study investigated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of G. mangostana extract and its chemical constituents using Ellman's colorimetric method. Cholinesterase inhibitory-guided approach led to identification of six bioactive prenylated xanthones showing moderate to potent cholinesterases inhibition with IC₅₀ values of lower than 20.5 μM. The most potent inhibitor of AChE was garcinone C while γ-mangostin was the most potent inhibitor of BChE with IC₅₀ values of 1.24 and 1.78 μM, respectively. Among the xanthones, mangostanol, 3-isomangostin, garcinone C and α-mangostin are AChE selective inhibitors, 8-deoxygartanin is a BChE selective inhibitor while γ-mangostin is a dual inhibitor. Preliminary structure-activity relationship suggests the importance of the C-8 prenyl and C-7 hydroxy groups for good AChE and BChE inhibitory activities. The enzyme kinetic studies indicate that both α-mangostin and garcinone C are mixed-mode inhibitors, while γ-mangostin is a non-competitive inhibitor of AChE. In contrast, both γ-mangostin and garcinone C are uncompetitive inhibitors, while α-mangostin is a mixed-mode inhibitor of BChE. Molecular docking studies revealed that α-mangostin, γ-mangostin and garcinone C interacts differently with the five important regions of AChE and BChE. The nature of protein–ligand interactions is mainly hydrophobic and hydrogen bonding. These bioactive prenylated xanthones are worthy for further investigations.     

Application of microwave‐assisted extraction coupled with high‐speed counter‐current chromatography for separation and purification of Dehydrocavidine from Corydalis saxicola Bunting

Introduction – Dehydrocavidine is a major component of Corydalis saxicola Bunting with sedative, analgesic, anticonvulsive and antibacterial activities. Conventional methods have disadvantages in extracting, separating and purifying dehydrocavidine from C. saxicola. Hence, an efficient method should be established. Objective – To develop a suitable preparative method in order to isolate dehydrocavidine from a complex C. saxicola extract by preparative HSCCC. Methodology – The methanol extract of C. saxicola was prepared by optimised microwave‐assisted extraction (MAE). The analytical HSCCC was used for the exploration of suitable solvent systems and the preparative HSCCC was used for larger scale separation and purification. Dehydrocavidine was analysed by high‐performance liquid chromatography (HPLC) and further identified by ESI‐MS and 1H NMR. Results – The optimised MAE experimental conditions were as follows: extraction temperature, 60°C; ratio of liquid to solid, 20; extraction time, 15 min; and microwave power, 700 W. In less than 4 h, 42.1 mg of dehydrocavidine (98.9% purity) was obtained from 900 mg crude extract in a one‐step separation, using a two‐phase solvent system composed of chloroform–methanol–0.3 m hydrochloric acid (4 : 0.5 : 2, v/v/v). Conclusion – Microwave‐assisted extraction coupled with high‐speed counter‐current chromatography is a powerful tool for extraction, separation and purification of dehydrocavidine from C. saxicola. Copyright © 2009 John Wiley & Sons, Ltd.     

Accelerated solvent extraction of monacolin K from red yeast rice and purification by high-speed counter-current chromatography

Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L₉ (3)³. The optimum extraction conditions were determined as follows: extraction temperature 120 °C, static extraction time 7 min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26 mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300 mg of crude extract, 51.2 mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and ¹H NMR.     

山竺果壳的化学成分

从山竺（Garcinia mangostana）果壳中分离得到6个化合物,通过MS,1D NMR以及与文献对照鉴定它们为4个[口山]酮类化合物：α-rnangostin（1）,β-mangostin（2）,γ-mangostin（3）,5,9-dihydroxy-8-methoxy-2,2-dimethyl-7-（3-methylbut-2-envl）-2H,6H-pyrano-[3,2-b]-xanthen-6-one（4）,以及表儿茶素（epicatechin,5）和一个双苄类化合物egonol（6）。其中化合物5和化合物6为首次从该植物中分离得到。对化合物1～5进行抗HIV-1 RT活性筛选结果表明,化合物2和化合物5在浓度200μg/ml的条件下,其对HIV-1 RT抑制率分别为41．97％和47．72％;同一实验结果显示化合物1,3和4没有抑制HIV-1 RT作用。     

Isolation and purification of seven lignans from Magnolia sprengeri by high-speed counter-current chromatography

Seven lignans including (-)-maglifloenone, futoenone, magnoline, cylohexadienone, fargesone C, fargesone A and fargesone B were isolated and purified from Magnolia sprengeri Pamp. using high-speed counter-current chromatography (HSCCC) with two-step separation. In the first step, a stepwise elution mode with the two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (1:0.8:0.6:1.2, 1:0.8:0.8:1, v/v) was used and 15.6 mg of (-)-maglifloenone, 19.2 mg of futoenone, 10.8 mg of magnoline, 14.7 mg of cylohexadienone and 217 mg residues were obtained from 370 mg crude extract. In the second step, the residues were successfully separated by HSCCC with the solvent system composed of petroleum ether-ethyl acetate-methanol-water (1:0.8:1.2:0.6, v/v), yielding 33.2 mg of fargesone C, 47.5 mg of fargesone A and 17.7 mg of fargesone B. The purities of the separated compounds were all over 95% determined by HPLC. The chemical structures of these compounds were confirmed by (1)H NMR, (13)C NMR and ESI-MS.     

On-line coupling of dynamic microwave-assisted extraction with high-speed counter-current chromatography for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim

An on-line method based upon dynamic microwave-assisted extraction (DMAE) coupled with high-speed counter-current chromatography (HSCCC) was developed for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim. The DMAE parameters were optimized by means of the Box-Behnken design. The maximum extraction yield was achieved using 30:1 ml/g of liquid-solid ratio, 10 ml/min of solvent flow rate and 200 W of microwave power. The crude extracts were then separated by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (7:3:5:5, v/v/v/v). 13.0mg of nevadensin was isolated from 15.0 g original sample by HSCCC with five times sample injection in 12h, and the isolation yield of nevadensin was 0.87 mg/g. The average purity of nevadensin was higher than 98.0%. The chemical structure of collected fraction was identified by HPLC, ESI-MS and (1)H NMR. The results indicated that this on-line method was effective and fast for high-throughput isolation of nevadensin from L. pauciflorus Maxim.     

α-Mangostin induces apoptosis and suppresses differentiation of 3T3-L1 cells via inhibiting fatty acid synthase

α-Mangostin, isolated from the hulls of Garcinia mangostana L., was found to have in vitro cytotoxicity against 3T3-L1 cells as well as inhibiting fatty acid synthase (FAS, EC 2.3.1.85). Our studies showed that the cytotoxicity of α-mangostin with IC(50) value of 20 μM was incomplicated in apoptotic events including increase of cell membrane permeability, nuclear chromatin condensation and mitochondrial membrane potential (ΔΨm) loss. This cytotoxicity was accompanied by the reduction of FAS activity in cells and could be rescued by 50 μM or 100 μM exogenous palmitic acids, which suggested that the apoptosis of 3T3-L1 preadipocytes induced by α-mangostin was via inhibition of FAS. Futhermore, α-mangostin could suppress intracellular lipid accumulation in the differentiating adipocytes and stimulated lipolysis in mature adipocytes, which was also related to its inhibition of FAS. In addition, 3T3-L1 preadipocytes were more susceptible to the cytotoxic effect of α-mangostin than mature adipocytes. Further studies showed that α-mangostin inhibited FAS probably by stronger action on the ketoacyl synthase domain and weaker action on the acetyl/malonyl transferase domain. These findings suggested that α-mangostin might be useful for preventing or treating obesity.     

Microwave-assisted extraction of embelin from <Emphasis Type="Italic">Embelia ribes</Emphasis>

A rapid and efficient microwave-assisted extraction (MAE) process for the selective extraction of embelin from Embelia ribes was developed. Solvent selection, microwave energy input and solid loading were optimized. The rate of extraction and purity of embelin depended upon the solvent used and exposure time to microwaves. Maximum MAE was achieved in acetone with total yield of 92% (w/w) embelin with 90% (w/w) purity with 1% (w/v) raw material loading at 150 W power level in 80 s. Non-polar solvents, such as hexane and dichloromethane, were not effective for the selective extraction of embelin.     

Optimization of micellar extraction for the pre-purification of paclitaxel from<Emphasis Type="Italic">Taxus chinensis</Emphasis>

Currently, there are many reports in the literature regarding technological methods for paclitaxel purification. However, there have been few reports on the purification of paclitaxel using a micellar system. This method is based on the transfer of paclitaxel within the crude extract to an aqueous surfactant solution as a micelle, allowing the use of organic solvents to be used for the removal of lipids and non-polar impurities. In this study, we optimized the important process parameters of micellar extraction to obtain a high purity and yield of paclitaxel in a pre-purification step. The optimal surfactant (N-cetylpyridinium chloride, CPC) concentration, initial crude paclitaxel concentration, organic solvents (methylt-butyl ether/hexane) ratio, extraction temperature, and extraction time were 7.5% (w/v), 16.4 mg/mL, 1.5/1 (v/v), 25°C, and 30 min, respectively. The crude extracts from the liquid-liquid extracts were efficiently pre-purified by micellar extraction, increasing in purity from 6% to over 21%, with a yield of 92%. Overall, the use of micellar extraction in the pre-purification process allowed for rapid and efficient separation of paclitaxel from interfering compounds, and dramatically increased the yield and purity of the crucle paclitael for subsequent purification steps.     

Predictable and linear scale-up of four phenolic alkaloids separation from the roots of Menispermum dauricum using high-performance counter-current chromatography

This paper describes how distribution ratios were used for prediction of peak elution in analytical high-performance counter-current chromatography (HPCCC) to explore the method for separation and purification of bioactive compounds from the roots of Menispermum dauricum. Then important parameters related to HPCCC separations including solvent systems, sample concentration, sample loading volume and flow rate were optimized on an analytical Mini-DE HPCCC and finally linearly scaled up to a preparative Midi-DE HPCCC with nearly the same resolutions and separation time. Four phenolic alkaloids were for the first time obtained by HPCCC separation with a two-phase solvent system composed of petroleum ether–ethyl acetate–ethanol–water (1:2:1:2, v/v). This process produced 131.3 mg daurisolin, 197.1 mg dauricine, 32.4 mg daurinoline and 14.7 mg dauricicoline with the purity of 97.6%, 96.4%, 97.2% and 98.3%, respectively from 500 mg crude extract of the roots of M. dauricum in a one-step separation. The purities of compounds were determined by high-performance liquid chromatography (HPLC). Their structures were identified by electrospray ionization mass spectrometer (ESI-MS) and nuclear magnetic resonance (NMR).     

Antimicrobial actions of α-mangostin against oral streptococci

The increasing prevalence of dental caries is making it more of a major world health problem. Caries is the direct result of acid production by cariogenic oral bacteria, especially Streptococcus mutans. New and better antimicrobial agents active against cariogenic bacteria are badly needed, especially natural agents derived directly from plants. We have evaluated the inhibitory actions of α-mangostin, a xanthone purified from ethanolic extracts of the tropical plant Garcinia mangostana L., by repeated silica gel chromatography. α-Mangostin was found to be a potent inhibitor of acid production by S. mutans UA159, active against membrane enzymes, including the F(H+)-ATPase and the phosphoenolpyruvate - sugar phosphotransferase system. α-Mangostin also inhibited the glycolytic enzymes aldolase, glyceraldehyde-3-phosphate dehydrogenase, and lactic dehydrogenase. Glycolysis by intact cells in suspensions or biofilms was inhibited by α-mangostin at concentrations of 12 and 120 μmol·L?1, respectively, in a pH-dependent manner, with greater potency at lower pH values. Other targets for inhibition by α-mangostin included (i) malolactic fermentation, involved in alkali production from malate, and (ii) NADH oxidase, the major respiratory enzyme for S. mutans. The overall conclusion is that α-mangostin is a multitarget inhibitor of mutans streptococci and may be useful as an anticaries agent.     

Preparative isolation of alkaloids from Dactylicapnos scandens using pH-zone-refining counter-current chromatography by changing the length of the separation column

pH-Zone-refining counter-current chromatography was successfully applied for the preparative separation of alkaloids from Dactylicapnos scandens. The two-phase solvent system was composed of petroleum ether-ethyl acetate-methanol-water (3:7:1:9, v/v), where 20 mM of triethylamine (TEA) was added to the upper phase as a retainer and 5 mM of hydrochloric acid (HCl) to the aqueous phase as an eluter. In this experiment, the apparatus with an adjustable length of the separation column was used for the separation of alkaloids from D. scandens and the resolution of the compounds can be remarkably improved by increasing the length of the separation column. As a result, 70 mg protopin, 30 mg (+) corydine, 120 mg (+) isocorydine and 40 mg (+) glaucine were obtained from 1.0 g of the crude extracts and each with 99.2%, 96.5%, 99.3%, 99.5% purity as determined by HPLC. The chemical structures of these compounds were confirmed by positive ESI-MS and (1)H NMR.     

Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining counter-current chromatography

Two modes of high-speed counter-current chromatography (HSCCC) were successfully applied to the separation of alkaloids from crude extract of Nelumbo nucifera leaves. The conventional HSCCC separations were performed with a two-phase solvent system composed of tetrachloromethane–CHCl₃–methanol–0.1 M HCl at a volume ratio of 1:3:3:2 (v/v/v/v), and 120 mg crude extract could be successfully separated. pH-Zone-refining CCC was performed with a two-phase solvent system composed of petroleum ether (60–90 °C)–ethyl acetate–methanol–water (5:5:2:8, v/v/v/v) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluent. From 4.0 g of the crude extract, 120 mg N-nornuciferine, 1020 mg nuciferine and 96 mg roemerine were obtained in a single run each with a purity of over 98% as determined by HPLC. The structures of the isolated compounds were identified by ESI-MS, ¹H NMR and ¹³C NMR.     

Extraction and preparative purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography

A method for extraction and preparative separation of tanshinones from Salvia miltiorrhiza Bunge was successfully established in this paper. Tanshinones from Salvia miltiorrhiza Bunge were extracted using ethyl acetate as the extractant under reflux. The extracts were then purified by high speed counter-current chromatography (HSCCC) with light petroleum-ethyl acetate-methanol-water (6:4:6.5:3.5, v/v) as the two phase solvent system. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 8.2mg of dihydrotanshinone I, 5.8 mg of 1,2,15,16-tetrahydrotanshiquinone, 26.3mg of cryptotanshinone, 16.2mg of tanshinone I, 25.6 mg of neo-przewaquinone A, 68.8 mg of tanshinone IIA and 9.3mg of miltirone were obtained from 400mg of extracts from Salvia miltiorrhiza Bunge in one-step HSCCC separation, with the purity of 97. 6%, 95.1%, 99.0%, 99.1%, 93.2%, 99.3% and 98.7%, respectively, as determined by HPLC area normalization method. Their chemical structures were identified by 1H NMR.     

Preparative separation of helvolic acid from the endophytic fungus <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> Ppf9 by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane-ethyl acetate-methanol-water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min(-1) and 25°C, respectively. About 6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, (1)H-NMR and (13)C-NMR spectra.     

Microwave-assisted extraction of cyclotides from Viola ignobilis

Cyclotides are an interesting family of circular plant peptides. Their unique three-dimensional structure, comprising a head-to-tail circular backbone chain and three disulfide bonds, confers them stability against thermal, chemical, and enzymatic degradation. Their unique stability under extreme conditions creates an idea about the possibility of using harsh extraction methods such as microwave-assisted extraction (MAE) without affecting their structures. MAE has been introduced as a potent extraction method for extraction of natural compounds, but it is seldom used for peptide and protein extraction. In this work, microwave irradiation was applied to the extraction of cyclotides. The procedure was performed in various steps using a microwave instrument under different conditions. High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) results show stability of cyclotide structures on microwave radiation. The influential parameters, including time, temperature, and the ratio of solvents that are affecting the MAE potency, were optimized. Optimal conditions were obtained at 20 min of irradiation time, 1200 W of system power in 60 °C, and methanol/water at the ratio of 90:10 (v/v) as solvent. The comparison of MAE results with maceration extraction shows that there are similarities between cyclotide sequences and extraction yields.     

Microwave-assisted extraction of glycyrrhizic acid from licorice root

In the present study, a microwave-assisted extraction (MAE) technique has been developed for the extraction of glycyrrhizic acid (GA) from licorice root. Various experimental conditions, such as extraction time, different ethanol and ammonia concentration, liquid/solid ratios, pre-leaching time before MAE and material size for the MAE procedure were investigated to optimize the efficiency of the extraction. Under appropriate MAE conditions, such as extraction times of 4-5min, ethanol concentrations of 50-60% (v/v), ammonia concentrations of 1-2% (v/v) and liquid/solid ratios of 10:1(ml/g), the recovery of GA from licorice root with MAE was equivalent with conventional extraction methods. Those methods include extraction at room temperature (ERT), the traditional Soxhlet extraction, heat reflux extraction and ultrasonic extraction. Due to the considerable savings in time and solvent, MAE was more effective than the conventional methods. This novel method is suitable for fast extraction of GA from licorice root.     

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR.     

微生物发酵产辅酶Q10的高速逆流色谱法分离纯化

本文首次将高速逆流色谱法应用于微生物发酵液提取物中辅酶Q10的分离纯化,建立了一套可用于其制备分离的逆流色谱溶剂体系正庚烷－乙睛－二氯甲烷(12：7：3.5, v/v/v)。500mg发酵液粗提物经一步制备分离,可得到绝对纯度在98％以上辅酶Q10130mg。比较表明,该方法较传统的硅胶柱层析和结晶相结合的纯化方法在产物纯度、回收率及产率等方面都有一定的优势。