Jean Wan Hong Yong

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.

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.

Analyses of gibberellins in coconut (Cocos nucifera L.) water by partial filling‐micellar electrokinetic chromatography‐mass spectrometry with reversal of electroosmotic flow

In this paper, we present the results of simultaneous screening of eight gibberellins (GAs) in coconut (Cocos nucifera L.) water by MEKC directly coupled to ESI‐MS detection. During the development of MEKC‐MS, partial filling (PF) was used to prevent the micelles from reaching the mass spectrometer as this is detrimental to the MS signal, and a cationic surfactant, cetyltrimethylammonium hydroxide, was added to the electrolyte to reverse the EOF. On the basis of the resolution of the neighboring peaks, different parameters (i.e., the pH and concentration of buffer, surfactant concentrations, length of the injected micellar plug, organic modifier, and applied separation voltage) were optimized to achieve a satisfactory PF‐MEKC separation of eight GA standards. Under optimum conditions, a baseline separation of GA standards, including GA1, GA3, GA5, GA6, GA7, GA9, GA12, and GA13, was accomplished within 25 min. Satisfactory results were obtained in terms of precision (RSD of migration time below 0.9%), sensitivity (LODs in the range of 0.8–1.9 μM) and linearity (R2 between 0.981 and 0.997). MS/MS with multiple reaction monitoring (MRM) detection was carried out to obtain sufficient selectivity. PF‐MEKC‐MS/MS allowed the direct identification and confirmation of the GAs presented in coconut water (CW) sample after SPE, while, the quantitative analysis of GAs was performed by PF‐MEKC‐MS approach. GA1 and GA3 were successfully detected and quantified in CW. It is anticipated that the current PF‐MEKC‐MS method can be applicable to analyze GAs in a wide range of biological samples.