Comparison of extraction efficiency and selectivity between low‐temperature pressurized microwave‐assisted extraction and prolonged maceration

Extraction is a key step in studying compounds from plants and other natural sources. The common use of high temperatures in pressurized microwave‐assisted extraction (PMAE) makes it unsuitable for the extraction of compounds with low or unknown thermal stability. This study aimed at determining the suitability of low‐temperature, short‐time PMAE in attaining yields comparable to those of prolonged maceration at room temperature. Additionally, we explored the phytochemical differences of the extracts from both techniques. Maceration at room temperature for 24 hr and PMAE at 40–45°C and 10 bar for 30 min were carried out on 18 samples from 14 plant species at a solvent‐to‐feeds ratio of 10. The PMAE yields of 16 out of 18 samples were within the proportions of 91–139.2% as compared with the respective extracts from maceration. Varying numbers of nonmatching peaks were noted in MS chromatograms of five extract pairs, indicating selective extraction of some compounds. Low‐temperature PMAE can attain reasonable extraction efficiency with the added value of sparing compounds of low thermal stability. The method can also enable the recovery of compounds distinct from those obtained by maceration.


| INTRODUCTION
The search for bioactive compounds for the treatment of diseases, among other applications, is key to ensure the continuous availability of viable treatment options. A number of approaches can be used in the discovery of new medicines. These include the screening of natural products or chemical libraries, in silico designing, and modification of existing medicines, to mention a few. Extraction is an important step toward obtaining phytochemicals of interest from plant materials. Outcomes of an extraction process are influenced by extraction duration, temperature, pressure, solvent's polarity, and acidity of the extraction medium, among other factors. [1] Different extraction methods have been reported. Maceration, percolation, infusion, decoction, and Soxhlet extraction are among the most employed techniques. This is mainly due to their less requirement for modern equipment and other infrastructure. However, other modern methods are currently in place. Most of them aim at attaining higher yields, reduced solvent use, and shorter extraction time. [1] Microwave-assisted extraction (MAE) involves the use of microwaves (300 MHz to 300 GHz) to generate thermal energy through rotation or vibration of dipoles or ionic conduction. [2] However, transfer are directed toward the solvent. [3] The rapid heating generated by MAE causes sudden evaporation of residual water or solvent molecules in plant cells. This results in a buildup of high internal pressure and rupturing of the cells. [3] These events are, thus, in favor of higher rates of desorption, diffusion, and partition of the phytochemicals from the plant matrix into the extracting solvent. [4] Attaining high recovery rates using conventional methods is a challenging task. Studies have indicated the necessity for longer extraction time and higher temperatures as possible modifications of these methods to boost their efficiency. [5][6][7] Besides prolonged exposure to atmospheric oxygen, thermal, oxidative, or enzymatic degradations, as well as crossreactions among the phytochemicals, can occur. [3,8] There are two possible equipment modes of carrying out an MAE. In the open mode, the equipment operates at an atmospheric pressure, commonly associated with a refluxing mechanism. [9][10][11] Modification of domestic microwaves to suit this mode is also a common approach. [8,12] On the contrary, the closed mode offers the choice of operating at a high pressure. The pressure is built up by the pumping of inert gas into the extraction chamber. Nevertheless, a degree of pressure may be generated by vapor pressure during heating of the extraction mixture. [9,13] The use of pressure enables the heating of the solvents above their boiling points. Depending on the phytochemicals of interest, this can result in higher yields and an overall decrease in extraction time. [13,14] Furthermore, the application of pressure is in line with the working principle of pressurized liquid extraction, whereby, besides enabling heating of the solvent above the boiling point, high pressure improves the permeation of the solvent through the plant matrices, hence favoring the desorption process. [10,15] The combination of pressure in MAE is also termed as pressurized microwave-assisted extraction (PMAE). [13][14][15][16] Current reports on the use of MAE indicate a broad use of rather high extraction temperatures, mostly in the range of 60-120°C. [4,5,[17][18][19] This approach has the benefit of achieving good yields using a few seconds to <10 min. However, it is not suitable for the extraction of heatsensitive compounds or when the compounds of interest are unknown.
In the current study, we aimed at exploring the usefulness of PMAE when conducted at low temperatures and moderate time duration. Besides evaluating the recovery efficiency of PMAE in comparison to maceration, we also wanted to determine if the obtained extracts differed in phytochemical profiles.

| RESULTS
A total of 18 plant samples were obtained from 14 plant species (Table 1). The plants were selected on the basis of a parallel study aimed at evaluating the antimicrobial activities of these plants.

| Quantitative comparison of the extract compositions
Of the 18 tested samples, 16 were found to provide MAE yields with the proportions >90% as compared with maceration ( Figure 1a). Moreover, 13 samples provided yields within 100 ± 10% of the maceration, with 79% and 139% being the lowest and highest proportions, respectively.

| Qualitative comparison of the extract compositions
The search for additional/nonmatching peaks on the UV chromatograms indicated that 4 out of the 18 chromatograms contained at least one additional peak within the extracts obtained by both methods. The additional peaks varied in intensities from small, as shown in Figure 6, to notably large, as exemplified in Figure 7a  creases MAE efficiency. [3,20] Moreover, as seen in Figure 1b impair the mass transfer of phytochemicals into the extracting solvent. [19] The need for using high temperatures in PMAE should, therefore, necessarily involve experiments to predetermine the temperature for optimal recoveries. [8,18] Low-temperature PMAE can, therefore, be considered as a very useful approach in attaining good yields of phytochemicals. This is occurring at high temperature and prolonged extraction times. [4,19] The use of PMAE can recover higher amounts of all or some of the phytochemicals from the plant matrix. Moreover, a degree of selectivity most likely based on the nature of phytochemicals and their solubility at different temperatures and time conditions is possible. [5,21] This is demonstrated by the higher intensities of UV and BPC peaks observed in chromatograms of PMAE extracts in 8 out of 14 samples that had no additional UV peaks (Figure 3a,b and Supporting Information). However, as superimposed chromatograms of other 5 out of 18 samples showed a mix in higher intensity peaks, other factors may be in play (Figure 5a,b). Other studies have also indicated differences in selectivity of extracted phytochemicals when MAE was compared with other extraction methods. [4][5][6] This can be caused by the nature of phytochemicals present in the matrix and their dependence on temperature and duration of extraction. [3,22] The magnitudes of peaks' intensities can be directly related to the quantities of respective phytochemicals. This is because, for each sample, the same sample concentration and injection volume were used during high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis.
When the phytochemicals of interest are known, the application of PMAE at low temperatures is a valid option for a selective increase of their recovery from the plant matrix. This can particularly be F I G U R E 4 (a) Prominent higher intensities of peaks in chromatograms of Zingiber officinale rhizomes extract obtained under maceration (black) as compared with pressurized microwave-assisted extraction (PMAE; pink). (b) Base peak chromatograms (BPCs) of Z. officinale rhizome extracts showing equivalent intensities of peaks A and B in both PMAE and maceration, as opposed to higher intensities for maceration observed in the UV chromatograms (a). UV peaks C and D were not found in the BPCs (x). Two additional peaks/compounds (*) are observed in the BPC from maceration (above). Green "* indicate the present peaks. Red "*" indicate missing peaks beneficial when these compounds are sensitive to high temperatures.
However, high heat stability of phytochemicals of interest warrants the use of high temperatures, with further benefits of shorter extraction times and even higher yields. [2,8,18] Differences in profiles of recovered phytochemicals may be im- Moreover, upon cross-examination of these peaks with the respective mass spectra, only one of them (peak B in Figure 7a) was confidently noted to be additional (Figure 7b,c). The compound corresponding to peak A at the RT of 6.6 min in UV chromatograms had a molecular peak at m/z 815.10 and was likely a caricaflavonol diester A. [23] However, on the basis of literature and library search, we could not ascertain the identity of the compound corresponding to an additional peak B at RT = 10.5.
Varying numbers of additional peaks/compounds were noted in five pairs of extracts from both methods when the MS BPCs were evaluated independent of the UV chromatograms ( Figure 8). This enabled us to arrive at a different conclusion in this aspect. Maceration extracts had a slightly higher mean and median numbers of additional BPCs as compared with PMAE ( Figure 8). Therefore, these findings show chances of the prospect of recovering completely different types of phytochemicals, based on the method of extraction. [14,24,25] F I G U R E 5 (a) Mixed higher peak intensities at different regions of the chromatograms of Cinnamomum verum leaves extract obtained under pressurized microwave-assisted extraction (PMAE; pink) as compared with maceration (black). (b) Base peak chromatograms for C. verum leaves extract showing higher intensities of peaks A and B and lower intensity for peak C under maceration (above) than in PMAE (below), conforming to the pattern observed in the UV chromatograms (a) Apart from the detection method, other factors such as method selectivity due to other chromatographic conditions play a role.
Through their particular influence on sensitivity and selectivity of the method, these factors are prone to affect a clear observation of additional compounds in generated chromatograms. [7] For example, this study employed a single UV detection wavelength of 254 nm; hence, compounds having chromophores with maximum absorbance at other wavelengths or compounds that lack a chromophore may be missed.
These factors may explain the non-UV detection of other compounds, which were detected on the electrospray ionization-mass spectrometry (ESI-MS) detector (Figure 3a,b).
Moreover, poor or lack of ionization of some compounds might have contributed to the observed missing MS BPCs corresponding to some peaks in UV chromatograms (Figure 4a,b). There are low chances that these additional compounds are the products of degradation or cross-reactions among phytochemicals in the crude extracts. This is based on the relatively low temperature and moderate extraction durations used in the study. [3,18] In addition to the selection of a suitable extraction method, we underline the need for using more versatile detection methods for  using the same solvent-to-matrix ratio of 10 ml/g, followed by automated stirring, in a microwave reaction system (synthWAVE; MLS GmbH, Germany). [14] Temperature, microwave energy, and pressure were set at

| Data analysis
The yields were compared by observing the percentage proportions at which the yield from PMAE differed from those of maceration.
This was carried out independently for each sample. For each sample, the UV chromatograms from both methods were superimposed and examined for differences in numbers and intensities of corresponding peaks. 18 MS BPC pairs of corresponding extracts were compared for the presence of the same peaks and the number of additional peaks.
The identity of additional/nonmatching peaks observed in UV chromatograms was cross-checked on the corresponding MS spectra.
The peak was regarded as representing an additionally extracted compound if it was not observed in the BPCs of the corresponding extract. The BPCs of each pair of extracts showing additional/nonmatching peaks were comparatively analyzed using box and whisker plots.