Optimisation of a microwave-assisted method for extracting withaferin A from Withania somnifera Dunal. using central composite design

Introduction – Recently, there have been growing attention on the modification and optimisation of new extraction and quantification methods, caused by the lack of environmentally friendly methodologies for the extraction of phytochemicals from complex matrices. In the case of pharmaceutical compounds, not only the extraction procedure but also the analysis method should be efficient, precise, fast and easy. Objectives – The essential pharmaceutical characteristics and trace concentration of withanolides led us to modify and optimise the previously reported extraction and quantification procedure for withaferin A (WA) as a candidate for withanolides. Matrial and methods – The WA from the air‐dried aerial part of Withania somnifera Dunal. was extracted using a microwave‐assisted extraction (MAE) technique. Four variables affecting the extraction procedure were optimised using the central composite design approach. The method of high‐performance thin‐layer chromatography assay was validated and applied for the quantification of each experiment. Results – The optimum values of factors were: extraction time (150 s), extraction temperature (68°C) and 17 mL of methanol : water in the ratio 25 : 75 as extracting solvent. The solvent system consisted of ethyl acetate : toluene : formic acid : 2‐propanol (7.0 : 2.0 : 0.5 : 0.5, v/v/v/v), and densitometric scanning at 220 nm was applied for the analysis. The dynamic linear range, LOD, LOQ and recovery with the inter‐day, and intra‐day RSDs of the developed method indicated the validity of the method. Conclusion – A pressurised MAE method for extracting WA from the plant's aerial part was optimised using factorial‐based design. The net effect of time, temperature, solvent volume and its ratio suggests that the yield of WA increases until each factor reaches its optimum value, and decreases with further increase in temperature or solvent ratio. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

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.     

Microwave assisted extraction of ursolic acid and oleanolic acid from Ocimum sanctum

Present study deals with the microwave assisted extraction (MAE) of ursolic acid (UA) and oleanolic acid (OA) from Ocimum sanctum leaves. UA and OA have been reported to possess significant medicinal properties. Various experimental parameters such as selection of solvent, solvent composition, irradiation time, microwave power, solid to solvent ratio, preleaching time and number of cycles were investigated to optimize the extraction process. Under optimum conditions of irradiation time (3 min), microwave power (272 W), solid to solvent ratio (1:30), preleaching time (10 min), maximum UA and OA has been extracted in one extraction cycle with ethanol: water (80:20) as a solvent. Maximum 86.76 and 89.64% of UA and OA was extracted under above mentioned optimized experimental conditions. MAE was also compared with the batch and ultrasound assisted extraction (UAE) method. As compared to batch and UAE, higher extraction yield of these important phytochemicals have been obtained through MAE in only 3 min.     

Microwave-assisted extraction of homoharringtonine from <Emphasis Type="Italic">Cephalotaxus koreana</Emphasis>

An efficient method using microwave energy was developed to extract homoharringtonine (HHT), an alkaloid component effective in the treatment of leukemia, from Cephalotaxus koreana. The effects of major process parameters on extraction efficiency were also investigated. Using a fixed biomass-to-methanol ratio of 1:8 (w/v), an extraction temperature of 30°C, an extraction time of 20 min, and a stirrer velocity of 250 rpm, a 25% higher yield of HHT was achieved using microwave-assisted extraction (MAE) than using conventional solvent extraction. It was possible to recover more than 95% of the HHT by extracting twice using MAE. In addition, the HHT yield increased as the extraction temperature increased, but the content of plant-derived tar and waxy compounds increased as well. Removal of these impurities and of the pigments from extracts was most effectively accomplished at a mixing ratio of biomass-to-sylopute of 1:1.5 (w/w). The effects of using different organic solvents (acetone, chloroform, ethanol, or methanol) for MAE were also assessed; the highest extraction efficiency was obtained using methanol. When the agitation speed was altered, most of the HHT (> 99%) was recovered at 250 rpm. A mixing ratio of biomass-to-methanol of 1:6 (w/v) at an extraction temperature of 40°C and an extraction time of 10 min proved to be the most effective for reducing processing time and organic solvent usage while enabling nearly all of the HHT (> 99%) to be recovered.     

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.     

微波辅助提取荔枝核黄酮类化合物及其抗氧化性研究

研究微波辅助法提取荔枝核黄酮类化合物的工艺,考察了提取溶剂、微波功率、溶剂用量、辐射时间、提取次数等因素对提取的影响。通过正交实验确定最佳的提取参数为:60%乙醇作为提取溶剂,微波功率700 W,料液比1∶25,辐射时间150 s,提取一次。在此优化条件下用微波辅助,黄酮类化合物的得率为6.86%,提取物中黄酮含量达到36.7%。抗氧化性研究表明荔枝核黄酮类化合物有良好的抗氧化活性,能有效清除羟基自由基(OH.)和超氧阴离子自由基(O2-.)。     

密闭微波辅助间歇提取RP-HPLC测定三七中三种皂苷类化合物

本文建立了采用微波辅助间歇提取三七样品中的皂苷类化合物,并以反相高效液相色谱（RP—HPLC）测定其中三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1含量的实验方法。分别通过单因素实验和正交实验设计,优化了萃取溶剂浓度、溶剂用量、微波功率和微波辐射时间等提取条件。与传统的索氏提取法、冷浸法和超声波提取法比较,微波辅助萃取法具有快速、提取率高、溶剂消耗少等优点,间歇萃取较连续萃取更是大大节省了微波能耗。     

Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods

Usually marine algae are an excellent source of pigments for different commercial sectors. Freshwater macroalgae can be exploited as a good source of biologically active compounds provided an appropriate extraction method is developed. The efficiency of four methods, like microwave‐assisted (MAE), ultrasound‐assisted extraction (UAE), supercritical fluid extraction (SFE) with ethanol as a co‐solvent, as well as conventional Soxhlet extraction were studied in the same conditions (time, solvent and temperature) for the recovery of chlorophylls and carotenoids from three freshwater green algae species: Cladophora glomerata, Cladophora rivularis and Ulva flexuosa. UV‐Vis spectrophotometry was used to determine chlorophyll a, chlorophyll b and total carotenoid content in obtained extracts. The results of this study showed that the advantages of novel extraction techniques (MAE and UAE) include higher yield and, in consequence, lower costs compared to traditional solvent extraction techniques. These methods were much more efficient in freshwater green algae pigment recovery than the classic Soxhlet extraction as well as SFE.     

超临界CO_2和微波辅助萃取艾叶挥发油工艺的研究

通过超临界CO2萃取均匀设计实验和微波辅助萃取艾叶挥发油的正交实验比较,考察影响提取的主要因素,寻求最佳萃取工艺。超临界CO2萃取最佳工艺条件为:萃取压力16MP,萃取温度31℃,CO2流量20kg/h和时间80min,得率3.75%;微波萃取最佳工艺条件为:辐射功率720w,辐射时间200s,溶剂量400mL,洗涤剂量50mL,得率4.85%。水蒸馏法提取率为1.87%。结果表明超临界CO2和水蒸馏法萃取艾叶挥发油品质最好;微波萃取收率最高,但品质较差。     

Contribution of microwave accelerated distillation in the extraction of the essential oil of Zygophyllum album L.

Introduction – The aerial parts of Zygophyllum album L. are used in folk medicine as an antidiabetic agent and as a drug active against several pathologies. In this work we present the chemical composition of Algerian essential oils obtained by microwave accelerated distillation (MAD) extraction, a solventless method assisted by microwave. Objective – Under the same analytical conditions and using GC‐FID and GC‐MS, the chemical composition of the essential oil of Zygophyllum album L. extracted by MAD was compared with that achieved using hydrodistillation (HD). Methodology – The extracted compounds were hydrosoluble, and they were removed from the aqueous solution by a liquid extraction with an organic solvent. Results – Employing MAD (100°C, 30 min), the essential oil contained mainly oxygenated monoterpenes with major constituents: carvone and α‐terpineol. However, most of the compounds present in the hydrodistilled volatile fraction were not terpene species, with β‐damascenone as a major constituent. Conclusion – The MAD method appears to be more efficient than HD: after 30 min extraction time, the obtained yields (i.e. 0.002%) were comparable to those provided by HD after 3 h extraction. MAD seems to be more convenient since the volatile fraction is richer in oxygenated monoterpenes, species that are recognised for their olfactory value and their contribution to the fragrance of the essential oil. Copyright © 2010 John Wiley & Sons, Ltd.     

亚麻木酚素的微波辅助提取工艺研究

采用微波辅助提取法从脱脂亚麻籽壳中提取亚麻木酚素,以磷钼酸显色的方法定量测定亚麻木酚素,通过单因素试验、中心组合试验及响应面分析,确定微波辅助提取的最优工艺条件为:乙醇浓度40.9%(v/v)、液固比21.9:1(mL/g)、超声处理5 min进行预浸、辐照时间90.5 s、微波功率为130 W。与常规溶剂提取法和索氏提取方法相比,微波辅助提取法显著提高了亚麻木酚素的得率,大大缩短了提取时间,并节省了能耗。     

Recovery of solanesol from tobacco as a value-added byproduct for alternative applications

Solanesol in the waste streams of a bioprocess designed for alternative applications of low-alkaloid tobacco was recovered using three different extraction methods. Compared to the conventional heat-reflux extraction (HRE) and ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) using 1:3 hexane:ethanol (v/v) as the solvent after saponification treatment of tobacco biomass was found the most effective in terms of solanesol yield, processing time, and volume of solvent consumed. Quantification of solanesol was achieved by optimizing the mobile phase at 60/40 acetonitrile–isopropanol and lowering the oven temperature to 22 °C using a standard reverse-phase high performance liquid chromatography (RP-HPLC). The total solanesol recovered from tobacco biomass and chloroplast accounted for 30% (w/w) of the total solanesol in the fresh leaves. Since solanesol is the precursor of metabolically active quinones such as coenzyme Q10 and vitamin K analogues, extraction of solanesol from tobacco bioprocess waste is a feasible operation and could leverage the overall profitability of biorefining tobacco for alternative, value-added uses.     

Analysis of fatty acids in A. szechenyianum Gay. by microwave‐assisted extraction and gas chromatography‐mass spectrometry

Introduction – Aconitum szechenyianum Gay. is a traditional Chinese medicinal herb with the detumescent and styptic effects and antitumor activity. There have been only a few researches on its chemical components, but no detailed report has appeared on its fatty acids. Objective – To develop a simple and effective method for the extraction of fatty acids from A. zechenyianum Gay. and then to investigate the fatty acid components. Methodology – Microwave‐assisted extraction (MAE) was optimized with response surface methodology, and the fatty acid compositions of extract were determined by GC–MS with previous derivatisation to fatty acid methyl esters (FAMEs). The results were compared with that obtained by classical Soxhlet extraction (SE). Results – Compared with SE, MAE showed significantly higher fatty acid yields, shorter extraction time, and lower energy and solvent consumption. The major fatty acids in A. szechenyianum Gay. are linoleic acid, palmitic acid, linolenic acid, oleic acid and stearic acid, and the unsaturated fatty acids occupy 66.4% of the total fatty acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Optimization of a rapid microwave-assisted extraction method for the simultaneous determination of opiates,cocaine and their metabolites in human hair

A rapid and cleanup-free microwave-assisted extraction (MAE) method is proposed for the simultaneous extraction of six illegal drugs of abuse – cocaine, benzoylecgonine (BZE), cocaethylene (CCE), morphine, 6-monoacethylmorphine (6AM) and codeine – from human hair samples. The analytes were determined using high performance liquid chromatography (HPLC) with photodiode array UV detection. The influence of several variables on the efficiency of the MAE procedure was investigated in detail by a multi-objective optimization approach based on a hybrid experimental design (17 experiments) and desirability functions. Six drugs were successfully extracted from human hair with recoveries close to 100% and good reproducibility (<3.6% RSD) under the optimal MAE conditions: 11 mL dichloromethane (DCM) extraction solvent, 60 °C extraction temperature, 9 min extraction time and 0.5 mL of methanol (MeOH) added to 50 mg of the hair sample in the extraction vessels. Limits of quantification of 0.2 ng mg^?1 were found for the studied compounds. A comparison of sample preparation procedures, including MAE, enzymatic digestion and digestion by aqueous acids, was also conducted. The results indicated that the global behaviour of sample procedures provided similar satisfactory recoveries ranging from 86 to 100%. Indeed, the MAE procedure resulted in a reduction of extraction time by 100-fold and the elimination of cleanup steps. Slightly higher recoveries of morphine, 6AM, BZE and CCE, at 1 ng mg^?1 concentration level and cocaine at 40 ng mg^?1 concentration level, were achieved using MAE. Lastly, the proposed MAE method was applied to several human hair samples from multidrug abusers.     

A robust sampling approach for identification and quantification of methyl jasmonate in leaf tissue of oilseed rape for analysis of early signaling in sclerotinia sclerotiorum resistance

Introduction – Methyl jasmonate (MJA), which is a natrual hormonal regulator, is thought to be essential for the regulation of systemic defence responses. The information about MJA levels in plant tissues is helpful for the study of the disease resistance mechanism and genetically engineered cultivars with increased resistance. Therefore, the quantification of MJA levels in plant tissues by means of a sensitive and reliable method is of interest. Objective – Development of a film extraction method coupled with GC for determination of methyl jasmonate in leaf tisssue of oilseed rape for analysis of early signalling in sclerotinia sclerotiorum resistance. Methodology – A robust polydimethylsiloxane film was prepared and used for extraction of MJA in leaf tissues. By using in‐solution extraction mode, optimum extraction efficiency was achieved with methanol–water (1 : 5, v/v) as extraction medium at 40°C for 60 min. Results – Under the optimal conditions, a detection limit of 0.2 ng/mL was achieved. Excellent reproducibility was found over a linear range of 1–1000 ng/mL. MJA in leaves infected by sclerotinia sclerotiorum was determined, with the results showing that basal levels of MJA (15 ng/g) were present in noninfested controls, but increased to 313 ng/g 10 h after fungal attack. Conclusion – The film extraction method is a simple, rapid and inexpensive sampling technique for determination of endogenous MJA in plant tissues that can be applied to most plants. Copyright © 2009 John Wiley & Sons, Ltd.     

响应面法优化微波辅助提取发酵虫草菌丝体多糖工艺

为优化发酵虫草菌粉多糖的微波辅助提取工艺,在单因素实验基础上,以液固比、微波功率以及提取时间为自变量,多糖提取率为响应值,采用中心组合设计的方法,研究各自变量及其交互作用对多糖提取率的影响。利用SAS软件和响应面分析相结合的方法对发酵虫草菌粉多糖的微波辅助提取工艺进行优化,确定了微波辅助提取多糖的最佳条件：液固比值12．2,微波功率650．5W,提取时间11．8min,在此条件下,多糖提取率达到6．41％。采用此法提取的虫草菌丝体多糖,当质量浓度为1mg／mL时,对二苯代苦味肼基自由基（DPPH）清除率达到76％。     

正交试验优化梓醇的微波辅助提取工艺

目的:通过正交实验优选了地黄中梓醇的微波提取工艺。方法:以地黄粗提液中梓醇含量为指标,HPLC为含量测定方法,采用正交实验法,选取乙醇浓度(A)、料液比(B)、提取时间(C)和提取次数(D)4个因素,每个因素选取3个水平进行实验,确定了最佳提取工艺。结果:研究结果表明乙醇浓度为60%,料液比为4,微波提取3次,每次3 min为梓醇的最佳提取工艺。结论:微波辅助提取地黄中梓醇效率高,提取完全,方法可行。     

An efficient strategy based on MAE, HPLC-DAD-ESI-MS/MS and 2D-prep-HPLC-DAD for the rapid extraction, separation, identification and purification of five active coumarin components from Radix Angelicae Dahuricae

Introduction – Further studies of active coumarin components in Radix Angelicae Dahuricae (AE) are absolutely essential to provide data on pharmacology, toxicology and quality for innovative drug candidates. Thus, the preparation of active component standards and the administration of coumarin monomers should be carried out. The isolation of the low‐level active components from complex Traditional Chinese Medicine (TCM) samples necessitates the development of rapid, simple and economical modern extraction, separation, identification and purification methods. Objective – To develop an efficient strategy for the rapid extraction, separation, identification and purification of coumarins from AE. Methodology – First, active coumarins in AE were extracted with microwave‐assisted extraction (MAE) after the extraction conditions were optimised. Second, gradient extraction methods with MAE were used to partially purify AE. Third, a high‐performance liquid chromatography–diode array detection‐electrospray ionisation tandem mass spectrometry (HPLC‐DAD‐ESI‐MS/MS) method was applied for the preliminary on‐line identification and screening of the main coumarins in AE extract. Finally, a two‐dimensional preparative high‐performance liquid chromatography–diode array detection (2D‐prep‐HPLC‐DAD) system was developed for further preparative separation of those target components. Results – Altogether 10 coumarins have been identified and five of them including xanthotoxol, osthenol, oxypeucedanin hydrate, byakangelicin and imperatorin were deemed as target components for the preparative isolation. All of the five isolated coumarins were at high purities of over 99% and the production rate was much higher than the traditional methods. Conclusion – The present paper demonstrates that these consecutive approaches are very useful for to isolate chemical constituents from TCM.