Eng Shi Ong

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.

Simultaneous analysis of different classes of phytohormones in coconut (Cocos nucifera L.) water using high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry after solid-phase extraction

Coconut (Cocos nucifera L.) water, which contains many uncharacterized phytohormones is extensively used as a growth promoting supplement in plant tissue culture. In this paper, a high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of various classes phytohormones, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellic acid (GA), zeatin (Z), N(6)-benzyladenine (BA), alpha-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in young coconut water (CW). The analysis was carried out using a reverse-phase HPLC gradient elution, with an aqueous mobile phase (containing 0.1% formic acid, pH adjusted to 3.2 with triethylamine (TEA)) modified by methanol, and solute detection made at 265 nm wavelength. The method was validated for specificity, quantification, accuracy and precision. After preconcentration of putative endogenous phytohormones in CW using C(18) solid-phase extraction (SPE) cartridges, the HPLC method was able to screen for putative endogenous phytohormones present in CW. Finally, the identities of the putative phytohormones present in CW were further confirmed using independent liquid chromatography-tandem mass spectrometry (LC-MS/MS) equipped with an electrospray ionization (ESI) interface.

Validation of green-solvent extraction combined with chromatographic chemical fingerprint to evaluate quality of Stevia rebaudiana Bertoni.

An approach that combined green-solvent methods of extraction with chromatographic chemical fingerprint and pattern recognition tools such as principal component analysis (PCA) was used to evaluate the quality of medicinal plants. Pressurized hot water extraction (PHWE) and microwave-assisted extraction (MAE) were used and their extraction efficiencies to extract two bioactive compounds, namely stevioside (SV) and rebaudioside A (RA), from Stevia rebaudiana Bertoni (SB) under different cultivation conditions were compared. The proposed methods showed that SV and RA could be extracted from SB using pure water under optimized conditions. The extraction efficiency of the methods was observed to be higher or comparable to heating under reflux with water. The method precision (RSD, n = 6) was found to vary from 1.91 to 2.86% for the two different methods on different days. Compared to PHWE, MAE has higher extraction efficiency with shorter extraction time. MAE was also found to extract more chemical constituents and provide distinctive chemical fingerprints for quality control purposes. Thus, a combination of MAE with chromatographic chemical fingerprints and PCA provided a simple and rapid approach for the comparison and classification of medicinal plants from different growth conditions. Hence, the current work highlighted the importance of extraction method in chemical fingerprinting for the classification of medicinal plants from different cultivation conditions with the aid of pattern recognition tools used.

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.

Inhibitory effects of a chemically standardized extract from Scutellaria barbata in human colon cancer cell lines, LoVo.

Scutellaria barbata (SB) is a medicinal plant that contains flavonone compounds such as scutellarein, scutellarin, carthamidin, isocarthamidin, and wogonin. A functional proteomic approach was used to study the inhibitory effects of a chemically standardized extract from SB in human colon adrencarcinoma, LoVo. In this work, a stable isotope was not used in the proposed method developed. The whole cell lysates from the control and treated cells were digested with trypsin, and the peptides were separated by two-dimensional (cation-exchange and reversed-phase) liquid chromatography and tandem mass spectrometry. The differentially expressed proteins identified using the current approach supported the data obtained from cell-cycle analysis with flow cytometry. With flow-cytometry analysis, a significant increase in the sub G1 phase was observed with a higher dose of extract from SB. Our results suggest that the chemically standardized extract from SB can induce cell death in the human colon cancer cell line. Our current work showed that the proposed platform provided a rapid approach to study the molecular mechanism because of the inhibitory effects of different doses of the botanical extracts on LoVo cell lines. This included a network of proteins involved in metabolism, regulation of the cell cycle, and transcription-factor activity.

Determination of aristolochic acids in medicinal plants (Chinese) prepared medicine using capillary zone electrophoresis.

Aristolochic acids (I and II) are commonly found in medicinal plants such as Radix aristolochiae and have been reported to cause acute hepatitis and end‐stage renal failure. The aim of this work was to develop a method for the analysis of aristolochic acids in medicinal plant/Chinese prepared medicine (CPM) using (CZE). The buffer used was 30 mM sodium tetraborate at pH 9.5, detection was at 254 nm, applied voltage at 18 kV and the temperature was set at 25°C. The effect of ionic strength, pH, and applied voltage on the separation was investigated. The precision values (relative standard deviation, RSD, %) for the relative migration time and peak area or peak height for aristolochic acids I and II were found to be less than 0.3% and between 2.6 to 4.0%, respectively. The limit of detection for aristolochic acids I and II was found to be 1.2 and 0.9 mg/L, respectively. The proposed method using pressurized liquid extraction (PLE) with CZE was used to determine the amount of aristolochic acids in medicinal plants or CPM samples with complex matrix and the results were compared with high‐performance liquid chromatography (HPLC). Method precision (RSD, n = 6) was found to be less than 4% when those from applied to medicinal plants and CPM samples.

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:H(2)O (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 microg/g to 1.04 micro/g and 1.32 micro/g to 5.29 microg/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.

Metabonomics investigation of human urine after ingestion of green tea with gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry and 1H NMR spectroscopy

A method using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and (1)H NMR with pattern recognition tools such as principle components analysis (PCA) was used to study the human urinary metabolic profiles after the intake of green tea. From the normalized peak areas obtained from GC/MS and LC/MS and peak heights from (1)H NMR, statistical analyses were used in the identification of potential biomarkers. Metabolic profiling by GC/MS provided a different set of quantitative signatures of metabolites that can be used to characterize the molecular changes in human urine samples. A comparison of normalized metabonomics data for selected metabolites in human urine samples in the presence of potential overlapping peaks after tea ingestion from LC/MS and (1)H NMR showed the reliability of the current approach and method of normalization. The close agreements of LC/MS with (1)H NMR data showed that the effects of ion suppression in LC/MS for early eluting metabolites were not significant. Concurrently, the specificity of detecting the stated metabolites by (1)H NMR and LC/MS was demonstrated. Our data showed that a number of metabolites involved in glucose metabolism, citric acid cycle and amino acid metabolism were affected immediately after the intake of green tea. The proposed approach provided a more comprehensive picture of the metabolic changes after intake of green tea in human urine. The multiple analytical approach together with pattern recognition tools is a useful platform to study metabolic profiles after ingestion of botanicals and medicinal plants.

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: H(2)O (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.

A green and effective approach for characterisation and quality control of Chrysanthemum by pressurized hot water extraction in combination with HPLC with UV absorbance detection

Chrysanthemum is a ubiquitous plant with many species and wide uses, and it is usually consumed as functional food. The main aim of this paper is to demonstrate that chromatographic fingerprints obtained from the HPLC/UV analysis of the pressurized hot water extraction (PHWE) extracts together with the aid of principal component analysis (PCA), allowed for the clustering of various chrysanthemums of different species and provenance. In addition, a parallel study of pressurized fluid extraction (PFE) with methanol was carried out for comparison. From the results, a clearer separation and clustering was obtained with the environmentally-benign water extracts compared with methanol extracts. This study shows that PHWE in combination with HPLC/UV and PCA can be used successfully as a green and effective approach for characterisation and quality control of ubiquitous functional food such as chrysanthemum.