Evaluation of the extraction efficiency of thermally labile bioactive compounds in Gastrodia elata Blume by pressurized hot water extraction and microwave-assisted extraction

Our earlier work showed that the stability of the bioactive compounds gastrodin (GA) and vanillyl alcohol (VA) in Gastrodia elata Blume behaved differently with varying compositions of water-ethanol using pressurized liquid extraction (PLE) at room temperature. To have a better understanding of the extraction process of these thermally labile compounds under elevated temperature conditions, pressurized hot water extraction (PHWE) and microwave-assisted extraction (MAE) methods were proposed. PHWE and MAE showed that GA and VA could be extracted using pure water under optimized conditions of temperature and extraction time. The extraction efficiency of GA and VA by the proposed methods was found to be higher or comparable to heating under reflux using water. The marker compounds present in the plant extracts were determined by RP-HPLC. The optimized conditions were found to be different for the two proposed methods on extraction of GA and VA. The method precision (RSD, n=6) was found to vary from 0.92% to 3.36% for the two proposed methods on different days. Hence, PHWE and MAE methods were shown to be feasible alternatives for the extraction of thermally labile marker compounds present in medicinal plants.     

Pressurized hot water extraction of bioactive or marker compounds in botanicals and medicinal plant materials

To reduce the use of organic solvent, pressurized hot water extraction (PHWE) has been shown to be a feasible option for the extraction of bioactive and marker compounds in botanicals and medicinal plants. The parameters that may affect the extraction efficiencies in PHWE include temperature, extraction time and addition of small percentage of organic solvent or surfactants. Currently, applications of PHWE for the extraction of thermally labile compounds in botanicals are still rather limited. PHWE with and without the additional of a small percentage of organic solvent such as ethanol is highly suited for the chemical standardization and quality control of medicinal plants. At the same time, it can be applied at the pilot scale as a manufacturing process for medicinal plants. Surfactant assisted PHWE was found to enhance the extraction of thermally labile and more hydrophobic species in medicinal plants at a lower temperature. The addition of small amount of surfactants in PHWE is highly suited for the determination of bioactive or marker compounds in medicinal plants. With proper optimization, PHWE was observed to have good extraction efficiency and precision when compared to other reference methods of extraction.     

Evaluation of surfactant‐assisted pressurized hot water extraction for marker compounds in Radix Codonopsis pilosula using liquid chromatography and liquid chromatography/electrospray ionization mass spectrometry

In the move towards the elimination of organic solvents in the extraction process in botanicals, a new method combining surfactant and pressurized hot water extraction (PWHE) with an applied temperature below the boiling point and lower pressure from 10 to 20 bar was developed for the analysis of marker compounds that are reasonably hydrophobic such as tetradeca‐4E,12E‐diene‐8,10‐diyne‐1,6,7‐triol and tetradeca‐4E,12E‐diene‐8,10‐diyne‐1,6,7‐triol‐O‐β‐D‐glucoside in Radix Codonopsis pilosula (DangShen). Because reference substances for the proposed botanicals were not available, a method was developed to isolate the marker compounds in Radix Codonopsis pilosula. Other than surfactant‐assisted PHWE, the marker compounds present in Radix Codonopsis pilosula were extracted using pressurized liquid extraction (PLE) with methanol and PHWE with a mixture of water/ethanol (80:20). The extracts were analyzed using liquid chromatography and liquid chromatography/electrospray ionization mass spectrometry. With surfactant‐assisted PHWE, the effects of different added surfactants such as sodium dodecyl sulfate and Triton X‐100 was studied. Surfactant assisted PHWE with Triton X‐100 proved to be at least equivalent or better compared to Soxhlet extraction in terms of quantitative analysis of marker compounds in Radix Codonopsis pilosula. The method precision was less than 8% (RSD, n = 6). The presence of surfactants in PHWE was found to enhance the solubility of target compounds naturally occurring in medicinal plants.     

Determination of senkirkine and senecionine in Tussilago farfara using microwave-assisted extraction and pressurized hot water extraction with liquid chromatography tandem mass spectrometry

Tussilago farfara (Kuan Donghua) is an important Chinese herbal medicine which has been shown to contain many bioactive compounds and widely used to relieve cough and resolve phlegm. However, besides therapeutic bioactive compounds, this herb has been found to contain toxic pyrrolizidine alkaloids (PAs), mainly senkirkine and traces of senecionine. In this report, conditions for microwave-assisted extraction (MAE) and pressurized hot water extraction (PHWE) were optimized for the extraction of the PAs. The results were compared against heating under reflux. It was found that the binary mixture of MeOH:H₂O (1:1) acidified using HCl to pH 2-3 was the optimal solvent for the extraction of the PAs in the plant materials. Liquid chromatography (LC) with ultra-violet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) in the positive mode was used for the determination and quantitation of senkirkine and senecionine in the botanical extract. The proposed extraction methods with LC/MS allow for the rapid detection of the major and the minor alkaloids in T. farfara in the presence of co-eluting peaks. With LC/MS, the quantitative analysis of PAs in the extract was done using internal standard calibration and the precision was found to vary from 0.6% to 5.4% on different days. The limits of detection (LODs) and limits of quantitation (LOQs) for MAE and PHWE were found to vary from 0.26 μg/g to 1.04 μg/g and 1.32 μg/g to 5.29 μg/g, respectively. The method precision of MAE and PHWE were found to vary from 3.7% to 10.4% on different days. The results showed that major and minor alkaloids extracted using MAE and PHWE were comparable to that by heating under reflux. Our data also showed that significant ion suppression was not observed in the analysis of senkirkine and senecionine in the botanical extracts with co-eluting peaks.     

Pressurized hot water extraction (PHWE)

Pressurized hot water extraction (PHWE) has become a popular green extraction method for different classes of compounds present in numerous kinds of matrices such as environmental, food and botanical samples. PHWE is also used in sample preparation to extract organic contaminants from foodstuff for food safety analysis and soils/sediments for environmental monitoring purposes. The main parameters which influence its extraction efficiency are namely the temperature, extraction time, flow rates and addition of modifiers/additives. Among these different parameters studied, temperature is described as the most important one. It is reported that the extraction of certain compounds is rather dependent on pressurized water with different applied temperature. Thus, the stability and reduced solubilities of certain compounds at elevated temperatures are highlighted in this review. With some modifications, a scaled-up PHWE could extract a higher amount of desirable compounds from solid and powdered samples such as plant and food materials. The PHWE extracts from plants are rich in chemical compounds or metabolites which can be a potential lead for drug discovery or development of disease-resistant food crops.     

Determination of metabolites in Uncaria sinensis by HPLC and GC-MS after green solvent microwave-assisted extraction

Uncaria sinensis (Oliv.) Havil (Rubiaceae) has been used as an important Traditional Chinese Medicine (TCM) herb for the treatment of fevers and various nervous disorders. The major bioactive secondary metabolites from different classes of chemical compounds, i.e. organic acid, flavonoid and alkaloid, present in this TCM herb, namely catechin, caffeic acid, epicatechin and rhynchophylline, were extracted by microwave-assisted extraction (MAE) method with ultra-pure water as the extraction solvent. The optimal extraction conditions for this green solvent MAE method were found to be 100 °C for 20 min. The recoveries of the compounds were found to be comparable to that of heating under reflux using ultra-pure water for 60 min. The method precision (RSD, n = 6) was found to vary from 0.19% to 5.60% for the proposed method on different days for the secondary metabolites. Simultaneously, the key primary metabolites such as sucrose and phenylalanine for the biosynthesis of bioactive secondary metabolites were successfully characterized by GC-MS. Furthermore, an approach using the combination of primary and secondary metabolite profiling based on their chemical fingerprints with Principal Component Analysis (PCA) was successfully developed to evaluate the quality of U. sinensis obtained from different sources. This approach was shown to be feasible in discriminating U. sinensis from different origins and thus a potential application for the quality control of other medicinal herbs.     

Study of polyethylene glycol as a green solvent in the microwave-assisted extraction of flavone and coumarin compounds from medicinal plants

In this paper, the application of polyethylene glycol (PEG) aqueous solution as a green solvent in microwave-assisted extraction (MAE) was firstly developed for the extraction of flavone and coumarin compounds from medicinal plants. The PEG solutions were optimized by a mono-factor test, and the other conditions of MAE including the size of sample, liquid/solid ratio, extraction temperature and extraction time were optimized by means of an orthogonal design L(9) (3(4)). Subsequently, PEG-MAE, organic solvent-MAE, and conventional heating reflux extraction (HRE) were evaluated with nevadensin extraction from Lysionotus pauciflorus, aesculin and aesculetin extraction from Cortex fraxini. Furthermore, the mechanism of PEG-MAE was investigated, including microwave-absorptive property and viscosity of PEG solutions, the kinetic mechanism of PEG-MAE and different microstructures of those samples before and after extraction. Under optimized conditions, the extraction yields of nevadensin from L. pauciflorus, aesculin and aesculetin from C. fraxini were 98.7%, 97.7% and 95.9% in a one-step extraction, respectively. The recoveries of nevadensin, aesculin and aesculetin were in the range of 92.0-103% with relative standard derivation lower than 3.6% by the proposed procedure. Compared with organic solvent-MAE and conventional extraction procedures, the proposed methods were effective and alternative for the extraction of flavone and coumarin compounds from medicinal plants. On the basis of the results, PEG solution as a green solvent in the MAE of active compounds from medicinal plants showed a great promising prospect.     

Pressurized hot water extraction of berberine, baicalein and glycyrrhizin in medicinal plants

Pressurized hot water extraction (PHWE) using a laboratory made system was applied for the extraction of thermally labile and reasonably polar components such as berberine in coptidis rhizoma, glycyrrhizin in radix glycyrrhizae/liquorice and baicalein in scutellariae radix. PHWE was carried out dynamically at a flow of 1 ml/min, temperature between 95 and 140 °C, an applied pressure of 10-20 bar and extraction time of 40 min. Extraction by PHWE was found to give efficiencies comparable to Soxhlet extraction for baicalein in scutellariae radix and sonication for berberine in coptidis rhizoma, and glycyrrhizin in radix glycyrrhizae. Effects of ethanol added into the water used in PHWE were explored. Pressurized liquid extraction (PLE) with methanol as solvent was used for extraction of baicalein in scutellariae radix. The marker compounds present in the various medicinal plant extracts were determined by gradient elution HPLC.     

Application of ionic liquids in the microwave-assisted extraction of polyphenolic compounds from medicinal plants

Ionic liquids (ILs) solutions as solvents were successfully applied in the microwave-assisted extraction (MAE) of polyphenolic compounds from medicinal plants. ILs, its concentration and MAE conditions were investigated in order to extract polyphenolic compounds effectively from Psidium guajava Linn. (P. guajava) leaves and Smilax china (S. china) tubers. The results obtained indicated that the anions and cations of ILs had influences on the extraction of polyphenolic compounds as well as the ILs with electron-rich aromatic π-system enhanced extraction ability. Under the optimized conditions, the extraction yields of the polyphenolic compounds were in the range of 79.5-93.8% with one-step extraction, and meanwhile the recoveries were in the range of 85.2-103% with relative standard deviations (R.S.D.s) lower than 5.6%. Compared to conventional extraction procedures, the results suggested that the proposed method was effective and alternative for the extraction of polyphenolic compounds from medicinal plants. In addition, the extraction mechanisms and the structures of samples before and after extraction were also investigated. ILs solutions as green solvents in the MAE of polyphenolic compounds from medicinal plant samples showed a great promising prospect.     

Evaluation of pressurized liquid extraction and pressurized hot water extraction for tanshinone I and IIA in Salvia miltiorrhiza using LC and LC-ESI-MS

Pressurized liquid extraction (PLE) and pressurized hot water extraction (PHWE) using a laboratory-made system are applied for the extraction of thermally labile components such as tanshinone I and IIA in Salvia miltiorrhiza. PLE and PHWE are carried out dynamically at a flow of 1 mL/min, temperature between 95-140 degrees C, applied pressure of 10-20 bars, and extraction times of 20 and 40 min, respectively. Effects of ethanol added into the water used in PHWE are explored. PLE is found to give comparable or higher extraction efficiencies compared with PHWE with reference to Soxhlet extraction for tanshinone I and IIA in Salvia miltiorrhiza. The tanshinone I and IIA present in the various medicinal plant extracts are determined by liquid chromatography and liquid chromatography-mass spectrometry.     

Microwave‐assisted extraction with water for fast extraction and simultaneous RP‐HPLC determination of phenolic acids in Radix Salviae Miltiorrhizae

An optimized microwave‐assisted extraction method using water (MAE‐W) as the extractant and an efficient HPLC analysis method were first developed for the fast extraction and simultaneous determination of D (+)‐(3,4‐dihydroxyphenyl) lactic acid (Dla), salvianolic acid B (SaB), and lithospermic acid (La) in Radix Salviae Miltiorrhizae. The key parameters of MAE‐W were optimized. It was found that the degradation of SaB was inhibited when using the optimized MAE‐W and the stable content of Dla, La, and SaB in danshen was obtained. Furthermore, compared to the conventional extraction methods, the proposed MAE‐W is a more rapid method with higher yield and lower solvent consumption with a reproducibility (RSD <6%). In addition, using water as extractant is safe and helpful for environment protection, which could be referred to as green extraction. The separation and quantitative determination of the three compounds was carried out by a developed reverse‐phase high‐performance liquid chromatographic (RP‐HPLC) method with UV detection. Highly efficient separation was obtained using gradient solvent system. The optimized HPLC analysis method was validated to have specificity, linearity, precision, and accuracy. The results indicated that MAE‐W followed by HPLC–UV determination is an appropriate alternative to previously proposed method for quality control of Radix Salviae Miltiorrhizae.     

Determination of pyrrolizidine alkaloids in comfrey by liquid chromatography-electrospray ionization mass spectrometry

Symphytum officinale L. (comfrey) is a medicinal plant commonly used in decoctions and aliments. Besides therapeutic bioactive compounds present in the herb, it is found to contain hepatotoxic pyrrolizidine alkaloids (PAs), such as lycopsamine and others. In the present study, PAs such as lycopsamine, echimidine and lasiocarpine were determined using electrospray liquid chromatography-mass spectrometry (LC-MS) with the method precision (relative standard deviation, RSD) <10%. Detection of lycopsamine, symviridine and their N-oxides could be confirmed with a newly developed method based on HPLC ion-trap and orbitrap MS with electrospray ionization interface. With LC-MS, quantitative analysis of lycopsamine in the botanical extract was carried out. The effect of extraction solvent was optimized by sonication and methanol: H₂O (50:50) was selected. Then a rapid method based on pressurized hot water extraction (PHWE) was employed for the extraction of lycopsamine from comfrey followed by the comparison with heating under reflux with the RSD ranging from 2.49% to 19.32%. Our results showed a higher extraction efficiency for heating under reflux compared with PHWE. It was proposed that the lower extraction efficiency for PHWE was attributable to dissolved nitrogen from air which caused the reduction in the solubility of lycopsamine in the compressed hot solvent. In this study, quantitative analysis of PAs in comfrey was demonstrated. In addition, it was found that the use of subcritical water for extractions depended on the physical properties of the dissolved solutes and their tendency to degrade under the chosen extraction conditions.     

Differentiation of Cultivars of Flos Chrysanthemum with the Use of High-Performance Liquid Chromatography Fingerprints and Chemometrics

Flos Chrysanthemum is a generic name for a particular group of edible plants, which also have medicinal properties. There are, in fact, twenty to thirty different cultivars, which are commonly used in beverages and for medicinal purposes. In this work, four Flos Chrysanthemum cultivars, Hangju, Taiju, Gongju, and Boju, were collected and chromatographic fingerprints were used to distinguish and assess these cultivars for quality control purposes. Chromatography fingerprints contain chemical information but also often have baseline drifts and peak shifts, which complicate data processing, and adaptive iteratively reweighted, penalized least squares, and correlation optimized warping were applied to correct the fingerprint peaks. The adjusted data were submitted to unsupervised and supervised pattern recognition methods. Principal component analysis was used to qualitatively differentiate the Flos Chrysanthemum cultivars. Partial least squares, continuum power regression, and K-nearest neighbors were used to predict the unknown samples. Finally, the elliptic joint confidence region method was used to evaluate the prediction ability of these models. The partial least squares and continuum power regression methods were shown to best represent the experimental results.     

Comprehensive evaluation of chemical stability of Xuebijing injection based on multiwavelength chromatographic fingerprints and multivariate chemometric techniques

A strategy for monitoring and analyzing the chemical stability of Xuebijing injection (XBJ) by multiwavelength chromatographic fingerprints and multivariate classification techniques is presented in this paper. Multiwavelength chromatographic fingerprints were constructed using chromatographic data obtained at four wavelengths (260, 280, 320, and 400?nm). The raw chromatography data were preprocessed by noise reduction, baseline correction, data normalization, and interval correlation optimized shifting (icoshift). Using this method, fingerprints of 166 samples of XBJ subjected to different forced degradation conditions (irradiation, high temperature, and a range of pH values) were properly represented. Forty-one chemical components were identified using the iPeak program. In addition, the identified peak area profiling of chemical components were used for multivariate classification analysis. Principal component analysis (PCA) and Ward’s method were used to classify different XBJ degradation samples. The PCA score plot showed that XBJ degradation samples were clustered into four groups, and the results are confirmed by Ward’s method. Ten key chemical markers under different degradation conditions were found and identified by counterpropagation artificial neural networks (CP-ANN), statistical t-tests, and UPLC-Q-TOF-MS. The results suggest that the proposed strategy could be successfully applied to the comprehensive analysis of complex chemical systems.     

Extending the applicability of pressurized hot water extraction to compounds exhibiting limited water solubility by pH control: curcumin from the turmeric rhizome

Pressurized hot water extraction (PHWE, also known as subcritical water extraction) is commonly considered to be an environmentally friendly extraction technique that could potentially replace traditional methods that use organic solvents. Unfortunately, the applicability of this technique is often limited by the very low water solubility of the target compounds, even at high temperatures. In this paper, the scope for broadening the applicability of PHWE by adjusting the pH of the water used in the extraction is demonstrated in the extraction of curcumin (which exhibits very limited water solubility) from untreated turmeric (Curcuma longa L.) rhizomes. Although poor extraction yields were obtained, even at high temperatures when using degassed water or neutral phosphate buffer as the extraction medium, yields exceeding those obtained by Soxhlet extraction were achieved using highly acidic pH buffers due to curcumin protonation. The influence of the temperature, pH, and buffer concentration on the extraction yield were investigated in detail by means of a series of designed experiments. Optimized conditions for the extraction of curcumin from turmeric by PHWE were estimated at 197 °C using 62 g/L buffer concentration at pH 1.6. The relationships between these variables were subjected to statistical analysis using response surface methodology.     

Optimal Template Removal from Molecularly Imprinted Polymers by Pressurized Hot Water Extraction

An optimal extraction method for the removal of templates from molecularly imprinted polymers (MIPs) is presented. The extraction method is based on pressurized hot water extraction (PHWE). PHWE was evaluated by application to three distinctly colored MIPs for chlorophyll (green), quercetin (yellow) and phthalocynine (dark blue) with subsequent monitoring of template removal and template bleeding by an ultraviolet spectrophotometer. The templates were washed-off and the extraction efficiency (EE) was compared to that of soxhlet and ultrasonic extraction methods. PHWE employed hot water at an optimal temperature of 220 °C, pressure of 50 bars and flow rate of 2 mL min^?1 to thoroughly wash-off the respective templates from their MIPs. The EE evaluated for PHWE was over 99.6% for all the MIPs with no subsequent or minimal template bleeding (<0.01%). The washing procedure was simple and relatively fast as it was achieved in 70 min at the most. At 95% confidence level (n = 3), soxhlet and ultrasonic recorded EE that was not significantly different (<94.5% in all cases) from that of PHWE (>99.6% in all cases). Soxhlet and ultrasonic had washing procedures that were slower (over 18 h) and employed large quantities (400 mL) of organic solvents modified with acids. The percentage relative standard deviations (%RSD) for the EE and recovery results were less than 2.3% in all cases indicating the high reproducibility of the method. Overall, the three methods performed comparably in extracting templates. PHWE seems to be the method of choice as it employed water which poses no environmental threat.     

Quality evaluation of fingerprints of herbal medicine with chromatographic data

In this study, local least squares (LLS) and principal component analysis (PCA) were applied to deal with the disturbances in a data set of chromatographic fingerprints after necessary data transformations. It has been demonstrated that PCA with standard normal variate (SNV) transformation of data led to meaningful classification of 33 different Erigeron breviscapus herbal samples. The result was also corroborated by variance squares discriminant method. The quality of herbal objects was further evaluated, and the causes of this fact have been explained from a chemical point of view. At the same time, it implied an idea for qualitative evaluation of the herbal objects with a common class pattern of chromatographic fingerprints.     

Evaluation of microwave-assisted extraction for aristolochic acid from Aristolochiae Fructus by chromatographic analysis coupled with nephrotoxicity studies

In this paper, a microwave-assisted extraction (MAE) method was established for aristolochic acid-I from Aristolochiae Fructus, and the advantage of MAE was evaluated by chromatographic analysis coupled with nephrotoxicity studies. The experimental parameters of MAE for aristolochic acid-I in Aristolochiae Fructus were investigated and MAE was compared with Soxhlet extraction and ultrasound-assisted extraction in terms of extraction yields and extraction conditions. Under the optimum conditions, MAE could provide higher extraction yields of aristolochic acid-I (1.10 mg/g) than ultrasound-assisted extraction (0.82 mg/g) and Soxhlet extraction (0.95 mg/g), in addition to using less solvent and having a shorter extraction time. Furthermore, the nephrotoxicities of the extracts of Aristolochiae Fructus from different extraction procedures were investigated in Sprague-Dawley rats. The results of nephrotoxicity studies of, for example, general conditions, biochemistry parameters and histopathology examination showed no significantly differences in the nephrotoxicity levels of the extracts from MAE and that from Soxhlet extraction. These results indicated that MAE technique is a simple, rapid and effective extraction method, and the microwave irradiation during MAE procedure did not have any influence on the nephrotoxicity of Aristolochiae Fructus compared with Soxhlet extraction.     

Optimization of microwave-assisted extraction for alizarin and purpurin in Rubiaceae plants and its comparison with conventional extraction methods

A simple and rapid microwave-assisted extraction (MAE) procedure was developed and optimized for two common color pigments, alizarin and purpurin, in various samples of Rubiaceae plants. Several variables that can potentially affect the extraction efficiency, namely temperature, methanol concentration in the extractant mixture, time, and solvent volume were optimized by means of a central composite design approach. The results suggest that temperature and methanol concentration in the solvent mixture are statistically the most significant factors. The separation and quantitative determination of the pigments was carried out in less than 6 min by a developed high-performance liquid chromatographic method with UV detection at 250 nm. Under optimum operating conditions, MAE showed significantly higher recoveries than those obtained by the conventional extraction methods (ultrasonic and reflux extraction), ranging from 84 to 94%. In addition, a drastic reduction of the extraction time (20 min versus 6 h) and solvent consumption (20 versus 100 mL) was achieved with a reproducibility (RSDs < 10%) comparable with that provided by the reflux extraction as a reference method.     

Extraction of polycyclic aromatic hydrocarbons and organochlorine pesticides from soils: a comparison between Soxhlet extraction, microwave-assisted extraction and accelerated solvent extraction techniques

The methods of simultaneous extraction of polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) from soils using Soxhlet extraction, microwave-assisted extraction (MAE) and accelerated solvent extraction (ASE) were established, and the extraction efficiencies using the three methods were systemically compared from procedural blank, limits of detection and quantification, method recovery and reproducibility, method chromatogram and other factors. In addition, soils with different total organic carbon contents were used to test the extraction efficiencies of the three methods. The results showed that the values obtained in this study were comparable with the values reported by other studies. In some respects such as method recovery and reproducibility, there were no significant differences among the three methods for the extraction of PAHs and OCPs. In some respects such as procedural blank and limits of detection and quantification, there were significant differences among the three methods. Overall, ASE had the best extraction efficiency compared to MAE and Soxhlet extraction, and the extraction efficiencies of MAE and Soxhlet extraction were comparable to each other depending on the property such as TOC content of the studied soil. Considering other factors such as solvent consumption and extraction time, ASE and MAE are preferable to Soxhlet extraction.