Critical approach to PLE technique application in the analysis of secondary metabolites in plants

Accepted Manuscript Critical approach to PLE technique application in the analysis of secondary metabolites in plants Dorota Wianowska, Marta Gil PII:

S0165-9936(19)30086-X

DOI:

https://doi.org/10.1016/j.trac.2019.03.018

Reference:

TRAC 15454

To appear in:

Trends in Analytical Chemistry

Received Date: 21 February 2019 Revised Date:

19 March 2019

Accepted Date: 20 March 2019

Please cite this article as: D. Wianowska, M. Gil, Critical approach to PLE technique application in the analysis of secondary metabolites in plants, Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2019.03.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Critical approach to PLE technique application in the analysis of secondary metabolites in plants

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Dorota Wianowska* & Marta Gil

Department of Chromatographic Methods, Faculty of Chemistry, Maria Curie-Skłodowska University.

Tel: +48 81 537 55 01

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Fax: +48 81 533 33 48

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Pl. Maria Curie-Skłodowska 3, 20-031 Lublin, Poland

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*Corresponding author E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract The aim of this paper is to present in the most comprehensive way the facts about the application of PLE in the analysis of secondary metabolites in plants. It is true that due to its specific extraction conditions, PLE is one of the most effective and versatile techniques of plant constituents isolation. It is also true that it has popularized the use of aqueous organic mixtures, and even water alone, for the extraction of nonpolar compounds. Yet, the same

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specific extraction conditions can lead to erroneous results of plant constituent content analysis. Disclosure of new facts about the application of PLE in the analysis of secondary plant metabolites provides an insight into an

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often overlooked aspect of extraction of active compounds from plants.

Keywords:

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pressurized liquid extraction; pressurized hot water extraction; PHWE; enhanced extraction techniques; sample preparation; chromatographic analysis; extraction of bioactives; extraction of phenolics.

Abbreviation:

EOs – essential oils

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ASE – accelerated solvent extraction

MSPD – matrix solid-phase dispersion

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PFE – pressurized fluid extraction

PHWE – pressurized hot water extraction

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PLE – pressurized liquid extraction

PSE – pressurized solvent extraction SFE – supercritical fluid extraction SPME – solid phase microextraction SWE – superheated water extraction or subcritical water extraction

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ACCEPTED MANUSCRIPT 1. Introduction Secondary plant metabolites belong to a vast and diverse assortment of organic compounds synthesized by plants. They are not directly involved in the normal growth and development of a plant. Their functions, though many of which remain unknown, are being elucidated with increasing frequency. It is already known that these compounds are extremely sensitive to a change in the environmental conditions and that they mediate many

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aspects of plants interactions with their environment. For this reason they are referred to as signal substances playing an important role in a plant defence and their adaptation to their environmental changes. They can be synthetized by a plant as a result of biotic factors, such as e.g. excitation by insects and, as a result of abiotic

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factors, such as UV radiation, environment pollutants or mechanical breakage of a plant. Some of them as hormones are the main tool for regulation and coordination of all physiological processes of a plant organism. Interestingly, these compounds were initially rejected as anti-nutritional plant substances in terms of human

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nutrition. However, over the last twenty years the importance of secondary plant metabolites for human nutrition has been reconsidered due to the discovery of their protective potential and health benefits. Many of them stimulate the human immune system, exhibit antioxidant activity and are characterized by anti-inflammatory, antibacterial, antifungal and antiviral actions [1]. Among them, there are also compounds which exhibit antitumor activity, or can relieve the side effects of chemotherapy. For these reasons secondary plant metabolites

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are currently an important source of pharmaceuticals. At present, however, not only the pharmaceutical industry is interested in their application. The consequence of their many valued properties is that they are the desired natural product used by many industries, for example, as flavour and flavour additives for food and cosmetics,

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dyes, polymers, fibers, adhesives, oils, waxes, flavours, perfumes. Hence it is not surprising that the analysis of secondary metabolites in plants is a very important area in the field of analytical chemistry.

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Most of the procedures for determination of organic compounds involve chromatographic techniques. This is because chromatography, owing to high separation power of the chromatographic systems and the application of very sensitive detectors, provides capability of analyzing even the smallest amount of a single compound in a complex sample. However, considering the samples with which we have to do in in everyday laboratory practice, the capabilities of chromatographic systems are not sufficient to ensure proper results of the analysis. In fact, the matrix of the sample may not be suitable for direct introduction onto the analytical system, or is characterized by the presence of a great number of substances that are not to be analyzed. Finally, the analyte cannot meet the basic requirements of the target analytical technique or, the analyte is present in the untreated sample in the smaller amount than defined by the limit of quantification of a given method. Therefore

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ACCEPTED MANUSCRIPT sample preparation is a process required for its transformation into a form which is suitable for performing the analysis or, which improves the analysis. For almost two decades a sample preparation step for chromatographic analysis has attracted interest of both researchers, analysts and companies producing analytical equipment and that supporting the analysis step. This interest reflects the awareness of the significance of sample preparation impact on the quality of analytical

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results. Moreover, this is a result of a general trend towards the use of faster and more efficient methods which should be also more environmentally friendly [2,3]. The problem of proper preparation of a sample for analysis is particularly important in the case of complex natural materials, especially such as plant material. The

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distinguishing feature of plant material, besides its diversity and characteristic tissue architecture, is abundance of various compounds, which are additionally present in plant material at different concentrations. Therefore a good method of plant sample preparation should be independent of the location of a compound in the matrix and

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the matrix type (hard, elastic or soft). Additionally, the results obtained by this method should not be dependent on the presence of other components (e.g. lipids) in a matrix. The method should also extract a desired compound in an easy and quantitative way, independent of the compound physicochemical properties, i.e. whether a compound is volatile or non-volatile, polar or non-polar, sensitive or resistant to the impact of high temperatures. Taking into account the application area of raw or pre-processed plant material and the importance

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of plants analysis, the lack of influence of the applied method on the change of the qualitative and quantitative compositions of the investigated material should be particularly strongly emphasized, considering the characteristics of a good method of plant sample preparation.

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Extraction methods are most frequently applied to prepare plant material for chromatographic analysis. Among them the assisted extraction techniques deserve a special attention. One of them is the Pressurized Liquid

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Extraction (PLE) technique. Due to its specific extraction conditions it overcomes most of the drawbacks of traditional techniques used for plant sample preparation providing better opportunity for the extraction process selectivity control. At present the choice of extraction solvent type is not the sole key to the success in the extraction process. In PLE not only temperatures above the boiling points of the solvents are commonly applied but temperature can be also changed. Pressure conditions and duration time of the process can be varied too. Moreover, PLE has introduced water as the extraction medium of nonpolar compounds and popularized the use of multicomponent extractants. Based on these facts, growing interest in the use of PLE is not surprising. In recent years, several articles have been published summarizing the use of PLE. In most of them, however, the PLE technique is presented as one of many techniques available for extracting compounds from solid samples

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ACCEPTED MANUSCRIPT [4]. Among those devoted exclusively to PLE, the majority focuses on the use of PLE for environmental analysis [5–7]. Some of them show the suitability of PLE to isolate specific groups of compounds from plants [8]. Several shows the use of the green approach of PLE, i.e. PHWE, for this purpose [9–11]. To our knowledge, however, there is no review of the use of PLE for the analysis of secondary metabolites in plants, in which not only the beneficial but also unfavourable features of its use are discussed. The advantages of PLE and the

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growing commercial demand for secondary metabolites make the use of PLE in routine and research laboratories steadily increasing. However, increasing the extraction capacity of the PLE technique is accompanied by the concern about the quality of obtained extracts and the veracity of provided results of the analysis of plants

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composition. Hence, this paper discusses various aspects of PLE application for isolation of secondary metabolites from plants and their analysis. The overall aim of this review is to share knowledge and long-term experience of using PLE in the analysis of plant components in order to present in the most comprehensive way

General information about PLE

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all the facts about the use of PLE in this specific field of analytical chemistry.

The basic principle in Pressurized Liquid Extraction is the use of elevated pressure to maintain the liquid state of solvents applied at temperatures above their boiling points. Therefore this technique is an alternative to a

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classical version of a solvent extraction of solids that is accomplished in the Soxhlet apparatus under atmospheric pressure. Initially, it was known as Accelerated Solvent Extraction (ASE). The term was derived from the trade name of the first commercially available system made by Dionex, i.e. Accelerated Solvent

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Extractor (ASE®100). Currently this technique has received different names, such as Pressurized Solvent Extraction (PSE) and Pressurized Fluid Extraction (PFE). When water is employed as the extraction solvent, to

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highlight the use of this environmentally friendly solvent, the terms such as Pressurized Hot Water Extraction (PHWE), Superheated Water Extraction or Subcritical Water Extraction (SWE) can be found in the literature. As follows from Fig. 1, however, the term Pressurized Liquid Extraction (PLE) is the most widely used. For clarification, the data presented in Fig. 1 refer to the papers, in which full names of techniques (not abbreviations) appear in the paper title or are indicated by the author/s as a keyword (according to ScienceDirect, January 10th, 2019). The first presentation of PLE was in 1995 at the Pittcon Conference (The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy). The technique was designed for environmental applications, therefore its use was focused on extraction of environmental pollutants present in soil matrices, sediments,

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ACCEPTED MANUSCRIPT sewage sludge and fly ash. Soon, however, the field of the PLE use was extended to other types of solid matrices, including polymers and plastics, pharmaceuticals, food, animal and plant tissues. Fig. 2 shows the progress in the PLE application area presented within the years as the change of the number of papers published in a given area by the Elsevier and Springer journals. A more detailed scrutiny of publication texts devoted to the PLE applications is presented in Fig. 3. These data show that in addition to the environmental applications

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constituting 30 % of all applications, PLE is widely used for preparation of whole herbs, leaves, flowers, roots, needles and bark (almost 30 % of applications). In addition, this method is also frequently used for analysis of food (including meat, bread, cereals, honey and eggs), seafood (fish, mollusk, shrimp, bivalve and oysters); as

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well as for fruits and vegetables analysis (altogether almost 27 % of applications). One of the newer and more interesting uses of PLE is for preparation of samples of beverages, cosmetics and even solid and liquid human samples such as hair, placental, urine and breast milk.

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The expansion of the use of PLE from the area focusing on monitoring environmental pollutants onto other areas of analytical chemistry, results mainly from the fact that this technique is fully automated, radically shortens the time needed for sample extraction and reduces the consumption of organic solvents. Shortening the extraction process duration is particularly advantageous in the isolation of compounds for which exposure time at high temperatures is important. The fact that in PLE a sample is extracted in an inert atmosphere protected

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from light increases additionally the attractiveness of its application. The principle of PLE is simple and involves three consecutive steps carried out one after another (see Fig. 4). First, a sample loaded to the extraction vessel is extracted by a solvent under pre-selected temperature

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and pressure conditions for a specific period of time (this step is commonly referred to as static extraction). Second, a solvent is pumped through the vessel to remove the obtained extract and to flush the sample with a

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fresh portion of the solvent (this step is called a stage of flushing). Finally, the vessel is purged by inert gas to ensure complete expulsion of the solvent from the vessel and tubes of the PLE system. This stage is commonly referred to as purge time. A more detailed description of the influence of different parameters that affect the PLE performance is presented further. Commercially available PLE systems allow to use a wide range of extraction temperatures. It usually ranges from room temperature to 200 oC and the pressure range is 35 - 200 bar. A static extraction time in the process can be varied from 1 to 99 minutes. However, the time required for solids extraction is most often equal to a few minutes (usually 10 - 15 min.). The standard PLE conditions (commonly referred to as the default conditions), recommended by Dionex, are as follows: the temperature 100 oC and the pressure 60 bar. In

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ACCEPTED MANUSCRIPT addition, a wide range of different solvents can be used in PLE. Highly corrosive liquids as well as those characterized by a low self-ignition temperature (e.g. dioxane, diethyl ether) are not advisable. In the PLE system the quaternary gradient pump, which simplifies the creation process of mixed extractants greatly, is applied. However, these are usually two-component extraction mixtures. Generally, the PLE procedure is performed under static conditions in which the compounds released

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from the solid matrix are transferred to a solvent which is immobile. The amount of the used solvent, depends on the volume of the extraction vessel, the amount of a sample and the degree of vessel packing with a sample. In the static set-up, the extraction process consists of one or several extraction cycles with replacement of the

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solvent between them. The above described three-stage procedure refers to the static PLE conditions. Alternatively, the process can be conducted at a constant and defined flow of a solvent through the extraction

available on market.

Versatility of PLE applications

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vessel, i.e. under dynamic conditions. As a matter of fact, there are no commercial dynamic PLE systems

Plants synthetize a great number of different secondary metabolites. It is estimated that more than 50,000 of

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them are known and described in literature. In general, they can be divided into volatile and non-volatile groups of compounds. The first one is mainly represented by terpenes. The second, apart from terpenes, includes a number of different groups of compounds, ubiquities and the most numerous of which are phenolics and

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alkaloids. Only the former are grouped in at least 10 classes of compounds, ranging from simple, low molecularweight, single aromatic-ringed compounds to large and complex tannins and derived polyphenols. In addition,

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phenolics are commonly found connected to sugars and organic acids [1]. Contrary to free phenolics (aglycones), these conjugated ones form phenolic glycosides, mainly monoglycosides. The glycone moiety of the phenolic glycoside can be D-glucose, D-galactose, D-fructose, L-rhamnose, D-xylose, L-arabinose or D-glucuronic acid. D-Glucose is the most common in the natural glycosides, whereas D-fructose and D-glucuronic acid are rare. Many of phenolic compounds display positive biological activity in the human organism. Some of them e.g. furanocoumarins are considered undesirable in human food due to their ‘toxic’ effects. Yet, a low daily intake of these ‘toxins’ may be an important factor in the search for an explanation of the beneficial effects of fruit and vegetables on human health. Alkaloids, the second of the aforementioned groups, are indicated as those among the metabolites that are most commonly exploited as pharmaceuticals, stimulants, narcotics and poisons (over

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ACCEPTED MANUSCRIPT 12,000 different alkaloids). In contrast, terpenes are mainly identified as flavour and fragrance compounds, preservatives, plant hormones and insect attractants. Although there are also anti-cancer compounds (toxoids) among them. Volatile secondary plant metabolites compose mixtures of essential oils. Though they constitute a very small part of a plant composition, these compounds are valuable and sought-after for many industries. For

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centuries essential oils have been considered to possess the most therapeutic and rejuvenating properties of all botanical extracts. Modern research has shown that these compounds are also active antioxidants. Putting them into the composition of many groups of products can reduce or eliminate the use of artificial preservatives.

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Moreover, essential oils have an ability to interact with insect receptors. Thus, they are used in repellents in order to scare insects and/or to mask the odour of human blood and sweat. For the same reason they are used as environmentally friendly pesticides. In addition, essential oils are applied as natural plant growth factors. It

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should be also remembered that essential oils like colours on a painter's palette, in the hands of an experienced chef can change mediocre food into a culinary masterpiece. Yet, the contrast of physicochemical properties of essential oils against the aforementioned groups of compounds, manifested also in sample preparation, imposes the need for a separate discussion of the PLE effectiveness for the essential oil analysis. The review of representative applications of PLE in the extraction and analysis of volatile and non-volatile secondary plant

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metabolites is presented in Table 1.

Extraction of non-volatile compounds

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At present there are many studies demonstrating the usefulness of the PLE technique for isolation of various non-volatile secondary metabolites from plants, including anthocyanins [12,13], chlorogenic acids [14–16],

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curcuminoids [17], furanocoumarins [18,19], flavanols [20], flavonols [21,22], flavones [23], flavonolignans [24], hydroxyanthraquinones [25], alkaloids [20,26], taxoids [27], phenolic acids [22,28] and many others. In most cases, these compounds are isolated in a single extraction cycle lasting several-minutes in the temperature range from 40 to 100 oC using acidified (or not) aqueous mixtures of alcohols for the isolation of more polar compounds, and solvents without admixed water for extraction of less polar compounds. In rare cases, the PLE extraction must be longer or carried out in several cycles repeated using the same portion of biological material. In [27] it was proved that in the case of toxoids extraction (10-deacetylbaccatin III, cephalomannine and paclitaxel) from common yew (Taxus baccata L.) for their quantitative isolation four consecutive extraction cycles of the same sample under the optimal PLE conditions are needed. Comparison of

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ACCEPTED MANUSCRIPT the PLE results with those obtained using the method recommended by Pharmacopeia (the 21-hour Soxhlet procedure) revealed that the PLE yield is considerably greater. Taxoids are thermolabile compounds shortening the duration time of their exposure to high temperatures in the PLE process favours their higher yields. It is worth noticing that in the case of taxoids isolation the PLE effectiveness is also higher in comparison to the other techniques of assisted extraction, such as Microwave Assisted Solvent Extraction (MASE) and Ultrasound

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Assisted Solvent Extraction (UASE). The analogous result was also obtained during the use of PLE for the isolation of furanocoumarins (umbelliferone, xanthotoxin, isopimpinellin, bergapten, pimpinellin, imperatorin, phellopterin and umbelliprenin) from garden angelica (Archangelica officinalis Hoffm.) [18] and parsnip

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(Pastinaca sativa L.) fruits [19].

Pereira et al. [12] applied the sequential PLE process to extract bioactive compounds from grape marc and proved that this is a viable strategy to obtain two extract fractions with specific compositions. In the first one

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monomeric anthocyanins were isolated using the ethanol-water mixture of pH 2.0 (50% w/w) at 40 °C. In the second, phenolic compounds were recovered using the solvent ethanol-water (50% w/w) at 100 °C. The results of the research presented in [13] discussing the application of sequential PLE process to extract phenolics and anthocyanins from industrial residue of jucara (Euterpe edulis Mart.) lead to similar conclusions. In the paper, the PLE capability was evaluated at 10 MPa and 40, 60 and 80 °C using ethanol, water, acidified (pH 2.0)

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mixture of ethanol/water (50 % v/v) and acidified (pH 2.0) water as solvents. It was proved that the best PLE conditions for anthocyanins and phenolics are not the same. The highest concentration of phenolics was obtained with the acidified mixture of ethanol/water at 80 °C and the highest anthocyanin content was achieved with

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acidified water at 40 °C. Thus, combining the optimal conditions for both groups of compounds in a sequential extraction process allows to take full advantage of the technique possibilities. Another paper showing the great

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potential of PLE to obtain bioactive compounds from agro-industrial residues is by Okiyama et al. [20]. It presents the PLE process with absolute ethanol for flavanols (catechin, epicatechin and procyanidin B2) and alkaloids (caffeine and theobromine) extraction from cocoa shells which is a co-product of the cocoa industry mainly used as fuel for boilers but with the additional applications as a fertilizer and animal feed. Vigano et al. [23] developed the ethanolic-water PLE to obtain flavones (isoorientin, vicenin, orientin, vitexin, and isovitexin) from the passion fruit (Passiflora edulis Sims) industry by-products (rinds). Ethanol, water, and their mixtures were used as solvents. The PLE conditions were: temperature 30 – 60 °C, ethanol concentration 70 – 100 % (v/v), pressure 10 MPa. With regard to the results, it was found that the increase of temperature and decrease of ethanol percentage favor the recovery of phenolic compounds.

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ACCEPTED MANUSCRIPT The vast majority of PLE applications, including those presented above, relate to the isolation of secondary metabolites abundantly present in a given plant matrix. Nevertheless, the results presented in [18,19,27] prove the great potential of PLE to recover bioactive compounds existing in plants at the trace level of concentrations from plant matrices rich in ballast substances, e.g. lipids. In addition to the effective furanocoumarins and toxoids extraction, presented in [18,19] and [27] respectively, the results contained in [24]

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also prove the usefulness of PLE for the extraction of flavonolignans (silychristin, silydianin, silybin A, silybin B, isosilybin A, and isosilybin B) from milk thistle (Silybium marianum L.) fruits in which the lipid fraction represents almost 30 % of the fruits weight. It was proved that, for each compound belonging to the same group

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of substances, the optimal conditions of its pressurized extraction vary a little. The latter result is worth stressing because identical extraction conditions are usually assumed for the compounds belonging to the same group. These data revealed the cognitive value of PLE and, that they encouraged investigations of the possibility of PLE

Extraction of volatile compounds

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application for extraction of other secondary metabolites from plants.

Essential oils (EOs) are characterized by a complex composition of substances of various physicochemical nature. Two fractions of compounds can be distinguished in the EO mixture: a volatile fraction that constitutes

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approximately 90 - 95 % of the total oil amount, and a non-volatile fraction that contains fatty acids, sterols, carotenoids, flavonoids, etc. The volatile fraction contains monoterpenes and sesquiterpene hydrocarbons and their oxygenated derivatives. The quantities of the individual components of essential oils are various. Apart

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from single components constituting even 60 % of the total oil amount, in the EOs composition a large group of such components the content of which is very small is present. These less-represented compounds are very often

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responsible for the characteristic smell of the plant. Among them there are unsaturated compounds that are susceptible to transformation leading to a change in the volatile composition. Thus, the challenge for the analyst is preparation of plant samples in such a way that they do not change the essential oil composition characteristic of a given plant material.

Steam distillation and/or hydrodistillation are the routine methods recommended by pharmacopoeias for analysis and controlling the quality of plant materials as essential oil sources [29]. These standard methods, however, in the EO analysis are very time consuming and therefore not efficient enough for screening numerous plant samples for their aroma composition. Furthermore, during these processes the EO components are subjected to various chemical transformations, leading to enrichment of EO in the compounds that are not

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ACCEPTED MANUSCRIPT characteristic of a given plant (e.g. chamazulene in the chamomile oil) or to depletion as a result of degradation of individual oil components. Matrix Solid-Phase Dispersion (MSPD) is one of the newer and uncommonly applied processes of EO isolation that can be accomplished even in the solventless mode [30,31]. Another approach is characteristic of the modern solvent free extraction techniques, such as Solid Phase Microextraction (SPME) and Supercritical Fluid Extraction (SFE) [32,33]. However, the techniques are not free from drawbacks.

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In SPME, for example, the analysis of compounds existing in a sample on a trace level is difficult. Moreover, for aromatic compounds with higher boiling points longer equilibrium times are needed. In addition, SPME is not recommended for quantitative analysis of the individual components of essential oils [34]. In SFE, in turn,

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because in most applications carbon dioxide acts as a supercritical fluid, the usage of SFE is mainly limited to the extraction of non-polar and medium polar substances of high volatility [33]. As follows from these facts, the technique which allows to optimize the extraction conditions for all EO representatives while reducing the

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organic solvents use and shortening the duration of sample preparation is PLE.

The possibility of the PLE technique application for the essential oil components analysis in plants is discussed in detail in [35]. In the paper the qualitative and quantitative compositions of extracts obtained by PLE from Thymus vulgaris L. were compared with the analogous data obtained using steam distillation, SPME and SFE. Additionally, the results were compared with those obtained by the Soxhlet apparatus using n-hexane,

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which is most often employed as an extracting solvent in the liquid extraction processes of essential oil components from herbs. From the obtained data, it was concluded that among the tested methods the PLE technique reveals the essential oil composition analogous to that obtained by steam distillation. Hence, PLE is

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the best alternative to the method of classical steam distillation of essential oils. Additionally, it was shown that under the PLE conditions ethyl acetate exhibits the highest extraction power towards volatile constituents of

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plants (the extraction power of n-hexane is slightly lower). Comparison of the results obtained during searching for the PLE conditions (temperature, pressure and time) under which, for a given solvent (n-hexane and/or ethyl acetate), the highest EO yield is observed, led the authors of the paper to another interesting conclusion. Regardless of the extractant type, the highest PLE yield of EO components is observed for very similar extraction conditions and, the conditions are indicated by Dionex as default extraction ones. Thus, the PLE optimization procedure can be avoided which is an extra advantage of the method application for the analysis of essential oil compositions in plants. In [36] the results of n-hexane extraction of thyme essential oils were compared with those obtained using superheated water under the static and dynamic PLE conditions. The obtained results proved that the

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ACCEPTED MANUSCRIPT dynamic superheated water extraction of essential oil constituents is characterized by the efficiency similar to that of the n-hexane extraction. Summing up, the PLE technique can be used to extract essential oils from plants. Moreover, it does not require an optimization procedure and allows complete elimination of organic solvents, making the process more environmentally friendly and the obtained extracts healthier.

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Usefulness of PLE applications

The versatility of PLE applications for the isolation of various groups of secondary metabolites currently makes it one of the more commonly used sophisticated extraction techniques. This is also due to the fact that it is a fast

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and effective technique. Due to the limitation and even complete elimination of the use of organic solvents, it is not only more environmentally friendly but also allows to obtain healthier extracts with high antioxidant activity. These are beneficial features of this technique. They also include the fact that the technique is useful in

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extremely subtle research of plant stress, and in the assessment of the actual content of the compound in the plant material. These areas of the use of PLE for secondary metabolites analysis are briefly presented below in the form of separate subsections.

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Enhancing the antioxidant power of obtained extracts

Oxygen determines life on the Earth, however, it can be toxic too. The negative effect of oxygen results from its ability to form reactive species belonging to free radicals. Their excess is indicated as the cause of many adverse

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processes, starting with the diseases, through the premature aging of organisms, finishing with food spoilage. Natural and synthetic antioxidants are used in the fight against free radicals and the effects of their unfavourable

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action. As synthetic antioxidants are suspected of toxicity and mutagenicity, therefore, more and more attention is paid to the antioxidants of plants origin. The antioxidant properties of the individual secondary metabolites as well as their mixtures are determined. In these attempts the ways to enhance the antioxidant power of extracts are also searched for.

In [37] it was tested whether greater extraction efficiency of PLE affects the increase in antioxidant

properties of the obtained extracts. Phenolic compounds, especially flavonoids, are known to be involved in oxidation-reduction processes, occurring both inside and outside cells. In the cited paper owing to the comparison of the contents of the most representative flavonols (rutin, isoquercitrin, astragaline) and anthocyanins (cyanidin-3-sambubioside, cyanidin-3-glucoside) in the alcoholic extracts of Sambucus nigra L. with the antioxidant power of these extracts, it was found out that the pressurized extraction conditions are

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ACCEPTED MANUSCRIPT conducive to obtaining extracts with higher antioxidant properties. The same conclusion was drawn by the other researchers [16]. The properties of the obtained extracts depend essentially on temperature and flavonoids contents in the extracts. The higher concentrations of flavonoids in the extract, the higher its antioxidative ability. However, direct correlation between the flavonoids contents and the antioxidant properties does not exist. This is a consequence of the fact that determination of antioxidant properties depends on many factors

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(extraction solvent type, pH, water content in a sample, metal concentration and/or metal type). These are the factors responsible for the lack of correlation between the antioxidant properties of extracts and phenolics

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contents.

Enabling the study of changes on the trace levels of metabolites in plants response to stress As it was stated, the synthesis of secondary metabolites is a form of a plant defence against adverse changes in

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its environment. Plants response to the so-called environmental stress is fast and lead to changes in their physiological state. These changes are at the earliest visible on young plants, initially as rust stains on the leaves and, eventually they lead to a marked drop in the crop. The analytical challenge is determination of the change in the concentration of compounds involved in plants defence against the stress. Isoflavonoids are most often

stress.

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reported in the literature about the studies on the changes in physiological status of the plant under heavy metal

In [38] due to development of the PLE procedure suitable for determining low concentrations of flavonols (quercetin-3-O-D-rhamnoside), kaempferol-3-O-D-rhamnoside, quercetin-3-O-D-glucuronide, and

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kaempferol-3-O-D-glucuronide) in young and older primary leaves of runner bean (Phaseolus coccineus L.) just after their harvesting, it was shown that flavonols, besides isoflavonoids, are also involved in the response. More

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important, however, is the fact that by estimating their amounts in the leaves of control plants, where their level was much lower than in the plants grown under heavy metal stress, for the first time it was shown that the change in their concentration is a symptom of antioxidant defence under heavy metal stress. The process is more evident in the younger plants, where the level of flavonols increased from 200 % to 490 % of the control, depending on the metal (Cd2+, Cu2+) and its concentration as well as on the flavonol type. Jasmonic acid is a compound very sensitive to any biotic and abiotic factors, and that is why it is one of the major compounds signalling the changes in plant cells. As a phytohormone it is present in a plant at a very low concentration. In addition, its amount varies depending on the plant type, its parts, and even on the type of leaves within the same plant. Analysis of jasmonic acid is, in fact, an example of the analysis that is

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ACCEPTED MANUSCRIPT characterized by many levels of difficulty. Limiting the experimental research material to the primary leaves of the Arabidopsis thaliana and P. coccineus plants providing a small amount of sample, is the first of them. The analysis must be carried out on a plant material that has not been subjected to the pre-drying and grinding procedure since a change in the osmotic pressure or damage to the plant may lead to a change in its content. Finally, the initiation of plant response to stress is short-lived and the metabolic cycle of the cells occurs even

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after harvesting that is why it is necessary to provide conditions that allow rapid analysis.

In [39] due to the choice of optimal PLE conditions allowing to develop a short and efficient extraction procedure and the idea of the initial cryogenic treatment of leaves guaranteeing a minimal impact of other factors

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on the jasmonic acid amount change, the correctness of hypothesis about induction of jasmonic acid synthesis in plants as a result of toxic action of heavy metals was verified. For the first time, the authors showed that a heavy metal stress causes induction of jasmonic acid accumulation in intact plants. They proved that this phenomenon

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occurs in plants of a different reaction to exogenous jasmonic acid, and possesses variable dynamics depending on the group of the studied plants and their growth stage. The inductive influence of heavy metals is of biphasic character. In the first phase, after 7 h. (A. thaliana) or 14 h (P. coccineus) of exposure to Cu2+ or Cd2+, a rapid increase of jasmonic acid level takes place. In the second one, a slow increase of its value is observed after much longer time. It is worth noticing that the quality of the results presented in [39] was quickly recognized by the

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world scientific community as evidenced by the number of citations of the paper.

Allowing determination of the authentic content of compounds in the plant material

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Analysis of secondary metabolites in a plant material is a complex and difficult issue. The quality of the results depends on both sample preparation and final analysis step. Nevertheless, in the case of the analysis of natural

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samples constituents a reliable result depends mainly on the first step of the analytical procedure, more specifically on the extraction efficiency. Efficiency is influenced by a number of variable factors. The most important of them, discussed in more

detail below, are as follows: location of metabolites in the plant (characteristic tissue structure); presence of metabolites in several forms (glycosides, aglycones, esters, etc.); type of interactions between the metabolite molecule and the other compounds present in the cell/tissue as well as type and amount of interfering substances. It should be remembered that the extraction efficiency is also influenced by the type of extractant and conditions of the extraction. Obviously, such solvents and extraction conditions are applied that provide the greatest extraction yield of desired compounds. (The conditions which yield the largest amounts of compounds are often

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ACCEPTED MANUSCRIPT referred to as the optimal extraction conditions.) In the light of so many factors influencing the efficiency, it is not surprising that examining the recovery of analytes is necessary. For this purpose a known amount of a foreign substance (internal standard) is added to a sample. The added compound should have physicochemical properties similar to those of the designated substance. However, taking into account the cellular architecture of the plant tissue and the force with which different metabolites are retained within the cell and tissue, the

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extraction efficiency of the internal standard (substance that is artificially introduced to the sample) will never be equal to the recovery of natural substances amount. Therefore a more popular way is to compare the yields obtained for the same aliquot of plants using two different extraction techniques.

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In the studies focusing on the use of new extraction techniques the result obtained by the tested method under the optimal conditions is compared with that obtained using one of the classical and/or recommended extraction techniques. In this approach the higher one is very often assumed as a reliable analysis result. Though

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this approach is quite commonly used it is not entirely correct. The results presented in [25] demonstrate that the transformation of other compounds (e.g. glycoside forms of polyphenols) to the analysed form of the compound (i.e. the aglycone form) may be the cause of greater analyte yields.

In another approach to verify the effectiveness of the extraction method, the same sample is subjected to the cyclic extraction until the amount of the compound in the extract reaches a detection level of

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chromatographic analysis. This approach is called multiple or exhaustive extraction, repeated using a new portion of a fresh solvent each time. Each extraction cycle is another equilibrium state and the extraction effectiveness depends on their number. In this approach experiments do not need to be carried out under optimal

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conditions which is its advantage. The method was tested in [40] and as it turned out it is suitable for subtle plant matrices (flowers, leaves) for which high pressure in the extraction vessel hinders analytes diffusion into the

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volume of a solvent. However, in the case of other matrices the total recovery of the analytes may require up to several extraction cycles of the same plant sample [26]. This leads to considerable elongation of the analytical process duration. Moreover, the overall time of analysis is dependent on the time of multiple extraction. Keeping this in mind, in [40] another manner for evaluation of the extraction efficiency is proposed. The method presented in [40] consists in extraction accomplished in a single step (so-called one-cyclic extraction) of the samples with different mass. According to this approach, to determine the efficiency of extraction in the liquid-solid system the dependence well known in the liquid-liquid system is used. The correctness of this hypothesis was verified experimentally with reference to the analysis of various compounds in different plant matrices. The data obtained by the authors show that the reciprocal value of the analyte amount

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ACCEPTED MANUSCRIPT extracted in the single-step PLE from a plant matrix is a linear function of the ratio of the plant material mass to the extractant volume. Thus the dependence permits to estimate the total amount of the analyte (its actual concentration value) in plant matrices. Correctness of the presented way of determining analyte concentration in plants was verified by the authors comparing the results obtained by the proposed method and by the multiple PLE. Based on the obtained data, only a few experimental points (at least two) are necessary to calculate the total

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amount of the analyte. Hence the multi-step PLE can be successfully replaced by the single-step PLE performed

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at different ratios of the plant mass to the solvent volume to save time and volume of the used extractant.

Factors differentiating the PLE efficiency

As it has been already mentioned, the PLE technique distinguishes the application of high pressures and high

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temperatures. Elevated pressure allows to maintain a liquid state of the extraction medium, although the boiling point at atmospheric pressure of the applied solvent has been exceeded. The elevated pressure also increases the contact area between the matrix and the solvent, entering the solvent into such regions of the solid matrix which are not accessible to the solvent under normal pressure. Elevated temperatures of extraction facilitate the desorption of an analyte from solid matrices, its diffusion into a solvent and furthermore, it allows to achieve

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higher values of partition coefficients. As a result, in the PLE process the increased ability of an extractant to dissolve a larger amount of an analyte is observed. Furthermore, by using multicomponent extractants PLE allows for a larger change of the extractant polarity, and thereby better control of the extraction process

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selectivity. These are generally known facts about pressurized liquid extraction. However, the literature rarely (if at all) reports that the increase in the extraction temperature contributes to a decrease in the extraction selectivity

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(1); the increase of pressure in the extraction vessel can lead to a decrease in the extraction performance (2); the use of multiple-component extractants can lead to a transformation of complex forms of compounds (3), and further that the purge stage in the PLE procedure can lead to changes in the amount of volatile components of extracts (4). These problems relate to the use of the PLE technique for analysis of the secondary metabolites in plants and they are discussed below in separate subsections.

Elevated temperature as a factor reducing the process selectivity PLE enables the use of extractants at elevated pressure and, hence at temperatures above their boiling point. Higher temperature assists in breaking down the analyte – matrix interactions caused by the van der Waals

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ACCEPTED MANUSCRIPT forces, hydrogen bonding as well as dipole attractions of the analyte molecules and the matrix, encourages analyte diffusion to the liquid solvent. The use of higher temperatures implies also reduction in solvent viscosity, thereby increasing the solvent ability to wet the matrix and to solubilize the target analytes. In consequence, the contact of analytes with the solvent is improved and the extraction efficiency is evidently enhanced. Moreover, owing to the application of elevated temperatures in the PLE process, the amount of needed solvents is

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considerably decreased and the time needed for quantitative extraction is reduced to a few minutes. These are the facts on which researchers frequently rely, discussing the effectiveness of the technique. Yet few people reported that the increase in the extraction temperature often causes that the basic rule “like dissolves like”, which

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indicates the use of polar solvent for extraction of polar analytes and likewise nonpolar analytes are dissolved in nonpolar solvents, is not always true under the PLE conditions. As a consequence, deterioration of extraction selectivity can be observed. The results presented in [24,27] demonstrate the problem of the analytes loss as the

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effect of lower extraction selectivity at higher temperatures of the PLE process.

In [27] concerning the search for the most effective technique for taxoids extraction from yew twigs (Taxus baccata L.) it was shown, that under the PLE conditions the preliminary extraction of ballast substances leads to loss of analytes. The results presented in the cited paper proved that solvents not extracting taxoids in the traditional extraction techniques (n-hexane, toluene, chloroform) do exhibit some extraction ability under the

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PLE conditions. Hence the removal of ballast substances from yew twigs by preliminary PLE extraction using nhexane or toluene could lead to unreliable analytical results. Similar conclusions can be drawn from the results of experiments on the search of the best PLE conditions for the extraction of silymarin from the milk thistle fruit

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(Silybum marianum L. Gaertner). These studies are discussed in detail in [24] and based on them, it can be concluded that the preliminary PLE extraction of lipids ballast material using n-hexane leads to loss of

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flavonolignans valuable for human health. It should be stressed that these compounds are not present in the nhexane extracts obtained by the Soxhlet extraction.

Elevated pressure as a factor hindering diffusion of an analyte from a plant matrix to a solvent The main advantage of applying elevated pressure during the PLE procedure is that a temperature above the boiling point can be used while a solvent maintains its liquid state. In practice, the effect of pressure on the recovery of substances from different matrices is usually negligible. Hence lower pressures (in the range 40 - 60 bar) are commonly applied. However, owing to the introduction of a solvent under pressure to the extraction vessel the solvent can be squeezed into such areas of the porous matrices that would not normally be contacted

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ACCEPTED MANUSCRIPT by the solvent under atmospheric conditions, e.g. due to the presence of air bubble or water in small pores of the matrix. The use of elevated pressure will force the solvent into the pore to contact analytes. As a result, a contact area between the matrix and the solvent is increased. This argument is most often used by researchers explaining higher efficiency of PLE in comparison to that of conventional solvent extraction techniques [41]. However, in

process can lead to reduced efficiency of the extraction.

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[26] it was clearly shown that in the case of some plant matrices the application of elevated pressure in the PLE

Dawidowicz et al. [26] used PLE to isolate caffeine from powdered green tea leaves and coffee beans and noticed that the separation of caffeine from the coffee is easy whilst from green tea leaves is difficult and

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pressure-dependent. The higher pressure applied in the PLE process, the lower the caffeine yield from the leaves. As some amount of caffeine still remained in the tea matrix after the PLE procedure, he deduced and proved that the elevated pressure squeezes the soft tea matrix making the diffusion of caffeine from the inside to the outside

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of the matrix difficult and/or it hinders with the penetration of the inner part of the matrix by the extractant. Obviously, the optimization of PLE conditions allows to increase the effectiveness of the one-cycle PLE. Nevertheless, the obtained results are still far away from the actual caffeine content in tea. The only way to resolve this problem is the right packaging of the extraction vessel. The way consists in dispersing the ground

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plant material in a solid inert material such as sand.

Effect of multi-component extractant mixtures

PLE is one of very few techniques of solids extraction which allows to enhance easily the extraction selectivity

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using multi-component extractants. Additionally, in PLE aqueous-organic mixtures are commonly applied in order to reduce the consumption of organic solvents. Generally optimisation of the PLE process begins with the

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choice of an appropriate extraction solvent, giving the highest yield of the analyte. This course of investigations may, however, be unreliable in the study of the chemical composition of plants. Application of mixtures containing water for the isolation of glycoside/aglycone forms of polyphenols is extremely doubtful. Glycosides can be easily transformed into aglycones under such conditions. This transformation can lead to false conclusions about the chemical composition of plants. The problem of stability of glycosides during their extraction process, especially in short-lasting PLE, however, is rarely taken into consideration during plants analysis. Dawidowicz's team of researchers was one of the first who spotted the problem of instability of secondary metabolites during their extraction from plants under the PLE conditions [15,25,42–44].

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ACCEPTED MANUSCRIPT One of the first papers dealing with the transformation of secondary metabolites during PLE is the paper by Wianowska [25]. In the paper the effect of extraction conditions on the yield of hydroxyanthraquinones from the Rumex crispus L. root using the methanol/water mixtures as an extractant is presented. It was found that the extraction efficiency of emodin, chrysophanol and physcion is better in more polar methanol/water mixtures than in less polar methanol while their corresponding glucopyranoside glycosides are better extracted by

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methanol and methanol/water mixtures containing a small amount of water. The results were surprising as the opposite extraction ability of methanol and methanol/water mixtures for aglycones and their glycosides is expected. Aglycones, less polar than their glycosides, are better soluble in methanol and water mixtures

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containing its significant concentration. Methanol/water mixtures rich in water, in turn, are recommended for the extraction of glycosides. Detailed analysis of the extracts showed that chemical composition of the extracts changes with the increase of water content in the extractant mixture suggesting transformation of glycosides to

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the corresponding aglycones. To verify the hypothesis the experiments were repeated using the standard of one of the tested glycosides instead of plant material. The experiments were accomplished using extractants differing in the water content in the extraction mixture. They proved that the increase of water concentration in the extractant mixture increases the degree of glycoside forms transformation to the corresponding aglycones increasing their concentration. The factors affecting the transformation degree are temperature and the time of

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static extraction. The extraction temperature change alters the quantitative relationships between the glycoside and aglycone forms of hydroxyanthraquinones. The extension of the extraction time also intensifies the hydrolytical degradation of the glycoside forms of hydroxyanthraquinone. Yet not only hydroxyanthraquinone

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glycosides under the PLE conditions are sensitive to the temperature and time of the process. It has been shown that this is also the case for quercetin glycosides [43,44] and depside forms of phenolic [15], especially when

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water is used as a component of the extractant mixture.

Effect of purge times on the yields of volatile compounds The impact of the third step of the PLE procedure (purge stage) on the technique effectiveness has not been taken into consideration during the PLE process optimization so far. Yet the results published in [36] show that the purge step can lead to loss of plants volatile components when water is applied as an extractant. In [45] it was demonstrated that in the case of D-limonene the application of organic solvents does not eliminate the loss of this volatile plant constituent. The extensive research on the loss of essential oil components during different times of the sample purge by nitrogen (0, 30 and 60 s) at three different extraction temperatures (50, 100 and

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ACCEPTED MANUSCRIPT 150 oC) and in the solvents with various extraction abilities (n-hexane, ethyl acetate and methanol) using herbs with the main compounds of essential oils differing markedly in the boiling point [rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.) and chamomile (Chamomilla recutita L.)] are discussed in [46]. Their results prove that the estimated yield of essential oil components extracted from plants in the PLE process is influenced by purge time. Generally, loss of easily volatile components during the purge process is observed. For

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less volatile components the increase of purge time leads to the increase of the concentration of extracted components in the receptacle due to solvent evaporation. In other words, the effect of purge time on the PLE yield of essential oil from plants depends on the evaporation rate of individual constituents and the applied

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extractants in the nitrogen stream. The magnitude of the EO component loss is also influenced by the strength of the solvent-analyte interactions (which corresponds to the solvent extraction ability). Thus the EO components undergo the co-evaporation process together with solvent molecules in a manner similar to co-distillation. If the

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loss of an individual EO component - as a result of purging of the sample by nitrogen - is comparable to the

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evaporation rate of the applied solvent, the effect of purge time on the yield of this compound is negligible.

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ACCEPTED MANUSCRIPT Conclusions Analysis of secondary metabolites in plant materials constitutes a highly complex research issue. The quality of the obtained results is largely dependent on the stage of plant sample preparation for chromatographic analysis. This step is most often realized by means of extraction. The information collected herein demonstrates that the PLE technique can replace the traditionally used techniques for isolation of compounds from solids (extraction in

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the Soxhlet apparatus, maceration, and distillation) in the analysis of secondary metabolites in plants. This replacement results in the fulfilment of the demands made before the modern analytics in the context of reducing its costs, increasing the number of analyses performed per unit of time and reducing/eliminating the use of

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organic solvents. It is also important, in view of the constantly growing demand for herbal products and/or extracts for wider and safer applications that the PLE technique enables obtaining in a short time high quality extracts with low processing costs and higher process efficiency that meet the requirements of many industries.

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These facts are the positive aspects of the use of pressurized liquid extraction (its advantages) in the analysis of secondary metabolites in plants. The additional advantages of the PLE use are as follows: -

the technique is extremely useful for the isolation of compounds, existing in the plant material at a trace level, from plant matrices rich in lipids;

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PLE increases the extraction efficiency of thermally labile compounds by shortening their exposure

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times to the impact of high temperatures;

it promotes the release of compounds from hard and mechanically resistant plant tissues and, those compounds that form strong bonds with lignin;

the extraction conditions of volatile plant components do not need to be optimized;

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PLE facilitates research on metabolism of plants just after their harvesting, and

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it can be used as a quick reference method.

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The diversity of physicochemical structures and properties of compounds belonging to secondary metabolites, and the diversity of plant matrices make, however, that there is no such extraction technique that would have only advantages without disadvantages. The use of PLE for the isolation and analysis of plant metabolites reveals the technique effectiveness and usefulness which is an advantage of PLE. However, it also points out to the disadvantages of this technique. These shortcomings of PLE should be treated as new directions for further research aimed at making an excellent method out of the good method.

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ACCEPTED MANUSCRIPT References W. Steglich, Plant Secondary Metabolites. Occurrence, Structure and Role in the Human Diet. Edited by Alan Crozier, M. N. Clifford and H. Ashihara., 2007. doi:10.1002/anie.200685491.

[2]

D. Wianowska, M. Gil, Recent advances in extraction and analysis procedures of natural chlorogenic acids, Phytochem. Rev. (2018). doi:10.1007/s11101-018-9592-y.

[3]

M. Plaza, C. Turner, Pressurized hot water extraction of bioactives, TrAC Trends Anal. Chem. 71 (2015) 39–54. doi:10.1016/J.TRAC.2015.02.022.

[4]

T. Belwal, S.M. Ezzat, L. Rastrelli, I.D. Bhatt, M. Daglia, A. Baldi, H.P. Devkota, I.E. Orhan, J.K. Patra, G. Das, C. Anandharamakrishnan, L. Gomez-Gomez, S.F. Nabavi, S.M. Nabavi, A.G. Atanasov, A critical analysis of extraction techniques used for botanicals: Trends, priorities, industrial uses and optimization strategies, TrAC Trends Anal. Chem. 100 (2018) 82–102. doi:10.1016/J.TRAC.2017.12.018.

[5]

L. Martín-Pozo, B. de Alarcón-Gómez, R. Rodríguez-Gómez, M.T. García-Córcoles, M. Çipa, A. ZafraGómez, Analytical methods for the determination of emerging contaminants in sewage sludge samples. A review, Talanta. 192 (2019) 508–533. doi:10.1016/J.TALANTA.2018.09.056.

[6]

R.B. Hoff, T.M. Pizzolato, Combining extraction and purification steps in sample preparation for environmental matrices: A review of matrix solid phase dispersion (MSPD) and pressurized liquid extraction (PLE) applications, TrAC Trends Anal. Chem. 109 (2018) 83–96. doi:10.1016/J.TRAC.2018.10.002.

[7]

M. Herrero, A. del P. Sánchez-Camargo, Plants, seaweeds, microalgae and food by-products as natural sources of functional ingredients obtained using pressurized liquid extraction and supercritical fluid extraction, TrAC Trends Anal. Chem. 71 (2015) 26–38. doi:10.1016/J.TRAC.2015.01.018.

[8]

D. Bursać Kovačević, M. Maras, F.J. Barba, D. Granato, S. Roohinejad, K. Mallikarjunan, D. Montesano, J.M. Lorenzo, P. Putnik, Innovative technologies for the recovery of phytochemicals from Stevia rebaudiana Bertoni leaves: A review, Food Chem. 268 (2018) 513–521. doi:10.1016/J.FOODCHEM.2018.06.091.

[9]

P. Panja, Green extraction methods of food polyphenols from vegetable materials, Curr. Opin. Food Sci. 23 (2018) 173–182. doi:10.1016/J.COFS.2017.11.012.

[10]

J. Giacometti, D. Bursać Kovačević, P. Putnik, D. Gabrić, T. Bilušić, G. Krešić, V. Stulić, F.J. Barba, F. Chemat, G. Barbosa-Cánovas, A. Režek Jambrak, Extraction of bioactive compounds and essential oils from mediterranean herbs by conventional and green innovative techniques: A review, Food Res. Int. 113 (2018) 245–262. doi:10.1016/J.FOODRES.2018.06.036.

[11]

K. Ameer, H.M. Shahbaz, J.-H. Kwon, Green Extraction Methods for Polyphenols from Plant Matrices and Their Byproducts: A Review, Compr. Rev. Food Sci. Food Saf. 16 (2017) 295–315. doi:10.1111/1541-4337.12253.

[12]

D.T.V. Pereira, A.G. Tarone, C.B.B. Cazarin, G.F. Barbero, J. Martínez, Pressurized liquid extraction of bioactive compounds from grape marc, J. Food Eng. 240 (2019) 105–113. doi:10.1016/J.JFOODENG.2018.07.019.

[14]

SC

M AN U

TE D

EP

AC C

[13]

RI PT

[1]

M. del P. Garcia-Mendoza, F.A. Espinosa-Pardo, A.M. Baseggio, G.F. Barbero, M.R. Maróstica Junior, M.A. Rostagno, J. Martínez, Extraction of phenolic compounds and anthocyanins from juçara (Euterpe edulis Mart.) residues using pressurized liquids and supercritical fluids, J. Supercrit. Fluids. 119 (2017) 9–16. doi:10.1016/J.SUPFLU.2016.08.014.

D. Wianowska, R. Typek, A.L. Dawidowicz, How to eliminate the formation of chlorogenic acids artefacts during plants analysis? Sea sand disruption method (SSDM) in the HPLC analysis of chlorogenic acids and their native derivatives in plants, Phytochemistry. 117 (2015) 489–499. doi:10.1016/j.phytochem.2015.07.006.

[15]

D. Wianowska, R. Typek, A.L. Dawidowicz, Chlorogenic acid stability in pressurized liquid extraction conditions, J. AOAC Int. 98 (2015) 415–421. doi:10.5740/jaoacint.14-200.

[16]

I. Pagano, A.L. Piccinelli, R. Celano, L. Campone, P. Gazzerro, M. Russo, L. Rastrelli, Pressurized hot water extraction of bioactive compounds from artichoke by-products, Electrophoresis. 39 (2018) 1899– 1907. doi:10.1002/elps.201800063.

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ACCEPTED MANUSCRIPT I.-C. Chao, C.-M. Wang, S.-P. Li, L.-G. Lin, W.-C. Ye, Q.-W. Zhang, Simultaneous Quantification of Three Curcuminoids and Three Volatile Components of Curcuma longa Using Pressurized Liquid Extraction and High-Performance Liquid Chromatography., Molecules. 23 (2018). doi:10.3390/molecules23071568.

[18]

M. Waksmundzka-Hajnos, A. Petruczynik, A. Dragan, D. Wianowska, A.L. Dawidowicz, Effect of extraction method on the yield of furanocoumarins from fruits of Archangelica officinalis Hoffm, Phytochem. Anal. 15 (2004). doi:10.1002/pca.784.

[19]

M. Waksmundzka-Hajnos, A. Petruczynik, A. Dragan, D. Wianowska, A.L. Dawidowicz, I. Sowa, Influence of the extraction mode on the yield of some furanocoumarins from Pastinaca sativa fruits, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 800 (2004). doi:10.1016/j.jchromb.2003.07.006.

[20]

D.C.G. Okiyama, I.D. Soares, M.S. Cuevas, E.J. Crevelin, L.A.B. Moraes, M.P. Melo, A.L. Oliveira, C.E.C. Rodrigues, Pressurized liquid extraction of flavanols and alkaloids from cocoa bean shell using ethanol as solvent, Food Res. Int. 114 (2018) 20–29. doi:10.1016/J.FOODRES.2018.07.055.

[21]

G. Tripodo, E. Ibáñez, A. Cifuentes, B. Gilbert-López, C. Fanali, Optimization of pressurized liquid extraction by response surface methodology of Goji berry ( Lycium barbarum L .) phenolic bioactive compounds, Electrophoresis. 39 (2018) 1673–1682. doi:10.1002/elps.201700448.

[22]

M. Waksmundzka-Hajnos, D. Wianowska, A. Oniszczuk, A.L. Dawidowicz, Effect of samplepreparation methods on the quantification of selected flavonoids in plant materials by high performance liquid chromatography, Acta Chromatogr. 20 (2008). doi:10.1556/AChrom.20.2008.3.13.

[23]

J. Viganó, I.Z. Brumer, P.A. de C. Braga, J.K. da Silva, M.R. Maróstica Júnior, F.G. Reyes Reyes, J. Martínez, Pressurized liquids extraction as an alternative process to readily obtain bioactive compounds from passion fruit rinds, Food Bioprod. Process. 100 (2016) 382–390. doi:10.1016/J.FBP.2016.08.011.

[24]

D. Wianowska, M. Wiśniewski, Simplified procedure of silymarin extraction from Silybum marianum L. Gaertner, J. Chromatogr. Sci. 53 (2014). doi:10.1093/chromsci/bmu049.

[25]

D. Wianowska, Hydrolytical instability of hydroxyanthraquinone glycosides in pressurized liquid extraction, Anal. Bioanal. Chem. 406 (2014) 3219–3227. doi:10.1007/s00216-014-7744-5.

[26]

A.L. Dawidowicz, D. Wianowska, PLE in the analysis of plant compounds: Part I. The application of PLE for HPLC analysis of caffeine in green tea leaves, J. Pharm. Biomed. Anal. 37 (2005). doi:10.1016/j.jpba.2004.10.038.

[27]

D. Wianowska, M.Ł. Hajnos, A.L. Dawidowicz, A. Oniszczuk, M. Waksmundzka-Hajnos, K. Głowniak, Extraction methods of 10-deacetylbaccatin III, paclitaxel, and cephalomannine from Taxus baccata L. twigs: A comparison, J. Liq. Chromatogr. Relat. Technol. 32 (2009). doi:10.1080/10826070802671622.

[28]

M. Waksmundzka–Hajnos, A. Oniszczuk, K. Szewczyk, D. Wianowska, Effect of sample-preparation methods on the HPLC quantification of some phenolic acids in plant materials, Acta Chromatogr. 19 (2007) 227–237.

[29]

R. Carabias-Martínez, E. Rodríguez-Gonzalo, P. Revilla-Ruiz, J. Hernández-Méndez, Pressurized liquid extraction in the analysis of food and biological samples, J. Chromatogr. A. 1089 (2005) 1–17. doi:10.1016/j.chroma.2005.06.072.

[31]

SC

M AN U

TE D

EP

AC C

[30]

RI PT

[17]

A.L. Dawidowicz, N.B. Czapczyńska, D. Wianowska, Relevance of the sea sand disruption method (SSDM) for the biometrical differentiation of the essential-oil composition from conifers, Chem. Biodivers. 10 (2013). doi:10.1002/cbdv.201200001.

D. Wianowska, M. Gil, New insights into the application of MSPD in various fields of analytical chemistry, Trends Anal. Chem. 112 (2019) 29–51. doi:10.1016/j.trac.2018.12.028.

[32]

J. Richter, I. Schellenberg, Comparison of different extraction methods for the determination of essential oils and related compounds from aromatic plants and optimization of solid-phase microextraction/gas chromatography, Anal. Bioanal. Chem. 387 (2007) 2207–2217. doi:10.1007/s00216-006-1045-6.

[33]

S.M. Pourmortazavi, S.S. Hajimirsadeghi, Supercritical fluid extraction in plant essential and volatile oil analysis, J. Chromatogr. A. 1163 (2007) 2–24. doi:10.1016/J.CHROMA.2007.06.021.

[34]

M. Mardarowicz, D. Wianowska, A.L. Dawidowicz, R. Sawicki, Comparison of terpene composition in Engelmann spruce (Picea engelmannii) using hydrodistillation, SPME and PLE, Zeitschrift Fur Naturforsch. - Sect. C J. Biosci. 59 (2004).

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ACCEPTED MANUSCRIPT A.L. Dawidowicz, E. Rado, D. Wianowska, M. Mardarowicz, J. Gawdzik, Application of PLE for the determination of essential oil components from Thymus vulgaris L., Talanta. 76 (2008). doi:10.1016/j.talanta.2008.04.050.

[36]

A.L. Dawidowicz, E. Rado, D. Wianowska, Static and dynamic superheated water extraction of essential oil components from Thymus vulgaris L., J. Sep. Sci. 32 (2009). doi:10.1002/jssc.200900214.

[37]

A.L. Dawidowicz, D. Wianowska, B. Baraniak, The antioxidant properties of alcoholic extracts from Sambucus nigra L. (antioxidant properties of extracts), LWT - Food Sci. Technol. 39 (2006). doi:10.1016/j.lwt.2005.01.005.

[38]

E. Skórzyńska-Polit, M. Dra̧ zkiewicz, D. Wianowska, W. Maksymiec, A.L. Dawidowicz, A. Tukiendorf, The influence of heavy metal stress on the level of some flavonols in the primary leaves of Phaseolus coccineus, Acta Physiol. Plant. 26 (2004).

[39]

W. Maksymiec, D. Wianowska, A.L. Dawidowicz, S. Radkiewicz, M. Mardarowicz, Z. Krupa, The level of jasmonic acid in Arabidopsis thaliana and Phaseolus coccineus plants under heavy metal stress, J. Plant Physiol. 162 (2005). doi:10.1016/j.jplph.2005.01.013.

[40]

A.L. Dawidowicz, D. Wianowska, PLE in the analysis of plant compounds - Part II: One-cycle PLE in determining total amount of analyte in plant material, J. Pharm. Biomed. Anal. 37 (2005). doi:10.1016/j.jpba.2004.10.025.

[41]

M.A. Rostagno, A. Villares, E. Guillamón, A. García-Lafuente, J.A. Martínez, Sample preparation for the analysis of isoflavones from soybeans and soy foods, J. Chromatogr. A. 1216 (2009) 2–29. doi:10.1016/J.CHROMA.2008.11.035.

[42]

R. Typek, A.L. Dawidowicz, D. Wianowska, K. Bernacik, M. Stankevič, M. Gil, Formation of aqueous and alcoholic adducts of curcumin during its extraction, Food Chem. (2019). doi:10.1016/j.foodchem.2018.10.006.

[43]

D. Wianowska, A.L. Dawidowicz, K. Bernacik, R. Typek, Determining the true content of quercetin and its derivatives in plants employing SSDM and LC–MS analysis, Eur. Food Res. Technol. 243 (2017). doi:10.1007/s00217-016-2719-8.

[44]

D. Wianowska, A.L. Dawidowicz, Effect of Water Content in Extraction Mixture on the Pressurized Liquid Extraction Efficiency—Stability of Quercetin 4′-Glucoside During Extraction from Onions, J. AOAC Int. 99 (2016) 744–749. doi:10.5740/jaoacint.16-0019.

[45]

A.L. Dawidowicz, N.B. Czapczyńska, D. Wianowska, The loss of essential oil components induced by the Purge Time in the Pressurized Liquid Extraction (PLE) procedure of Cupressus sempervirens, Talanta. 94 (2012) 140–145. doi:10.1016/j.talanta.2012.03.008.

[46]

D. Wianowska, The Influence of Purge Times on the Yields of Essential Oil Components Extracted from Plants by Pressurized Liquid Extraction, J. AOAC Int. 97 (2014) 1310–1316. doi:10.5740/jaoacint.13318.

[47]

R. Typek, A.L. Dawidowicz, D. Wianowska, K. Bernacik, M. Stankevič, M. Gil, Formation of aqueous and alcoholic adducts of curcumin during its extraction, Food Chem. 276 (2019) 101–109. doi:10.1016/J.FOODCHEM.2018.10.006.

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[35]

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ACCEPTED MANUSCRIPT Fig. 1. Scopus search results depicting the number of papers which were published over 1995-2019

PHWE PFE ASE

SWE PSE PLE

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550 500 450 400 350 300 250 200 150 100 50 0

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Number of papers

related to the use of PLE in chemical analysis

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1995-2000 2001-2005 2006-2010 2011-2015 2016-2019 (January)

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ACCEPTED MANUSCRIPT Fig. 2. Scopus search results depicting the number of papers which were published over 1995-2019 related to the use of PLE in different areas of analytical chemistry. The presented data refer to the publications in which the full names of the techniques (not abbreviations) appear in the publication

RI PT

Other samples Plant samples Environment samples

SC

240 220 200 180 160 140 120 100 80 60 40 20 0

2001-2005 2006-2010

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1995-2000

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Number of papers

title or are indicated by the author/s as a keyword (according to ScienceDirect, January 10th, 2019)

26

2011-2015 2016-2019 (January)

ACCEPTED MANUSCRIPT Fig. 3. Number of papers in the Elsevier and Springer journals devoted to the use of PLE for the preparation of different matrices for its analysis. The presented data refer to the publications in which the full names of the techniques (not abbreviations) appear in the publication title or are

RI PT

indicated by the author/s as a keyword (according to ScienceDirect, January 10th, 2019)

Environmental, 30.1% Herbs, 28.8%

Fruits, vegetables and seeds, 11.1%

SC

Food, 10.9%

Biomass, 5.7% Seafood, 4.8%

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Aquatic products, 2.8% Beverage, 2.6% Human samples, 1.9% Cosmetics, 1.0%

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Dietary suplements, 0.4%

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ACCEPTED MANUSCRIPT

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Fig. 4. Stages of the PLE process

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ACCEPTED MANUSCRIPT

Table 1 Selected representative applications of PLE in the extraction and analysis of secondary plant metabolites

phenolic compounds; anthocyanins caffeoylquinic acids; flavone glycosides

Fruits of parsnip (Pastinaca sativa L.) Cocoa beans

Goji berry (Lycium barbarum L.)

Flower of black elder (Sambucus nigra L.); herb of common knotgrass (Polygonum aviculare L.) Passion fruit rinds (Passiflora edulis Sims.) milk thistle fruit

UHPLC-UV

[13]

UHPLC-UV

[16]

HPLC-UV

[17]

Methanol; temperature 100 – 130 °C; time 10 min; pressure 60 bar; flush 60%; purge time 120 s; static extraction

HPLC-UV

[18]

HPLC-UV

[19]

UPLC-MS/MS

[20]

HPLC-DADMS/MS

[21]

HPLC-UV

[22]

UPLC-MS/MS

[23]

HPLC-UV

[24]

TE D

Fruits of garden angelica (Archangelica officinalis Hoffm.)

curcuminoids: curcumin, bisdemethoxycurcumin, demethoxycurcumin; volatiles: α-turmerone, βturmerone, ar-turmerone furanocoumarins: umbelliferone, xanthotoxin, bergapten, isopimpinellin, phellopterin imperatorin furanocoumarins: xanthotoxin, bergapten, isopimpinellin, phellopterin, imperatorin flavonols: catechin, epicatechin, procyanidin B2; alkaloids: theobromine; caffeine phenolic compounds

Reference [12]

Methanol or petrol; temperature 100 °C; time 10 min; pressure 60 bar; flush 60%; purge time 120 s; static extraction Ethanol; temperatures 60, 75 and 90 °C; pressure 103.5 bar; time ranging from 5 to 50 min; static extraction

EP

Rhizome of turmeric (Curcuma longa L.)

AC C

Artichoke by-products

Analysis UHPLC-UV UPLC-QTOFMS

RI PT

Jucara (Euterpe edulis Mart.)

PLE conditions 5 g of sample; pressure 100 bar; extraction of MACs: ethanol-water pH 2.0 (50:50 v/v), 100 °C, 40 min; extraction PCs: ethanol-water (50:50 v/v), 100 °C, 4 h; dynamic extraction 2,5 g of sample; pressure 100 bar; ethanol-water (50:50 v/v); 80 °C; dynamic extraction 1 g of sample with 1 g of diatomaceous earth; modifier, ethanol 10% v/v; temperature 93 °C; pressure 103 bar; time 5 min; flush 150%, purge time 100 s; static extraction Ethanol; temperature 100 °C, time 5 min, pressure 103 bar, flush volume 60%, static extraction

SC

Analyte monomeric anthocyanins (MACs); phenolic compounds (PCs)

M AN U

Matrix type Grape marc

rutin, isoquercetrin

phenolic compounds: isoorientin, vicenin, vitexin, orientin, isovitexin flavonolignans: silychristin,

1 g of sample mixed with 3 g of sea sand; etanol-water (86:14 v/v); temperature 180 °C; time 20 min; pressure 100 bar; flush volume 60%; purge time 60 s; static extraction 1 g of sample mixed with neutral glass; flowers of S. nigra L. were extracted with methanol-water mixture (80:20v/v) and P. aviculare herb was extracted with methanol; time 10 min; pressure 60 bar; temperature 100 °C; static extraction 3 g of sample; methanol-water (70:30 v/v); temperature 60 °C; pressure 100 bar; time 30 min; dynamic extraction 0.5 g of sample mixed with neutral material; acetone; time

29

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Flower of black elder (Sambucus nigra L.); herb of common knotgrass (Polygonum aviculare L.) Marjoram (Origanum majorana L.) Caraway (Carum carvi L.) Sage (Salvia officinalis L.) Thyme (Thymus vulgaris L.) Engelmann Spruce (Picea engelmannii)

RI PT

Twigs of Taxus baccata L.

SC

Green tea leaves

10 min; temperature 125 °C; pressure 60 bar; flush 60%; purge time 60 s; static extraction 0.5 g of sample mixed with neutral glass; methanol-water (40:60 v/v); time 10 min; temperature 100 °C; pressure 60 bar; flush 60%; purge time 60 s; static extraction 0.5 g of sample mixed with neutral glass; water; time 10 min; pressure 60 bar; temperature 100 °C; one-cycle PLE and multiple PLE of the same sample; static extraction toxoids: 10-deacetylbaccatin III, Portion of plant mixed with neutral glass; methanol; paclitaxel, cephalomannine temperature 115 °C; time 15 min; pressure 60 bar; flush 60%; purge time 120 s; one-cycle PLE and multiple PLE of the same sample; static extraction phenolic acids: protocatechuic 1 g of sample; flowers of S. nigra L. were extracted with acid, gallic acid, ferulic acid, p- methanol-water mixture (80:20v/v) and P. aviculare herb hydroxybenzoic acid was extracted with methanol; time 10 min; pressure 60 bar; temperature 100 °C; static extraction Essential oil components 10 g sample of fresh or 5 g of freeze-dried plant material was mixed with 5 g diatomite; solvent n-hexane; time 10 min; pressure 140 bar; temperature 100 °C; static extraction

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Root of Rumex crispus L.

silydianin, silybin A, silybin B, isosilybin A, isosilybin B hydroxyanthraquinones: emodin, chrysophanol, physcion and their glycosides caffeine

Terpens

Portion of plant mixed with neutral glass; solvent hexane; temperature 100 °C; time 10 min; pressure 60 bar; static extraction 0.5 g of sample; solvent n-hexane; temperature 100 °C; time 10 min; pressure 60 bar; flush 100%; purge time 60 s; static extraction 0.5 g of sample; solvent n-hexane; temperature 100 °C; time 10 min; pressure 40 bar; static extraction 0.5 g of sample; solvent water; temperature 100 °C; time 10 min; pressure 40 bar; static and dynamic extraction 1 g of sample mixed with neutral glass; solvent ethanolwater (80:20 v/v); temperature from 20 to 200 °C; time 10 min; pressure 60 bar; flush 60%; purge time 120 s; static extraction 3 g of sample; solvent methanol; ambient temperature; time 10 min; pressure 60 bar; flush 60%; purge time 120 s; static extraction

TE D

(Silybum marianum L. Gaertner)

Essential oil components

Thyme (Thymus vulgaris L.)

Essential oil components

Leaves, berries and flowers of Sambucus nigra L.

rutin, isoquercitrin, astragaline, cyanidin-3-sambubioside, cyanidin-3-glucoside

leaves of runner beans (Phaseolus coccineus L.)

quercetin-3-O-D-rhamnoside; kaempferol-3-O-D-rhamnoside; quercetin-3-O-D-glucuronide; kaempferol-3-O-D-glucuronide

AC C

EP

Thyme (Thymus vulgaris L.)

30

HPLC-UV HPLC/ESI/MS

[25]

HPLC-PDA HPLC-UV

[26]

HPLC-DAD

[27]

HPLC-UV

[28]

GC-FID GC-MS

[32]

GC-MS

[34]

GC-MS

[35]

GC-MS

[36]

HPLC-PDA HPLC-UV

[37]

HPLC-PDA HPLC-UV

[38]

ACCEPTED MANUSCRIPT

jasmonic acid

3 g of sample; solvent methanol; ambient temperature; time 10 min; pressure 60 bar; flush 60%; purge time 120 s; static extraction

GC-MS

[39]

rutin, caffeine

HPLC-PDA HPLC-UV

[40]

Rhizome of turmeric (Curcuma longa L.)

curcumin

LC-MS

[47]

White and red onion (Alium cepa L.)

quercetin 4′-glucoside; quercetin

HPLC-PDA HPLC-UV

[44]

Rosemary (Rosmarinus officinalis L.), Thyme (Thymus vulgaris L.) Chamomile (Chamomilla recutita L.)

rosemary: α-pinene; 1,8cineole, camphor; thyme: thymol, βcaryophyllene; chamomile: β-farnesene; αbisabolol oxide B; α-bisabolol oxide A

For S. nigra L. flowers: methanol-water (80:20v/v), temperature 100 °C, time 10 min, pressure 100 bar; static extraction For tea and coffee: water; temperature 100 °C, time 10 min, pressure 60 bar; static extraction 200 mg of sample; methanol-phosphoric buffer (pH 3 or 6.5 or 9) mixtures (75/25 v/v); temperature 150 °C; pressure 40 bar; time 20 min; flush 60%; purge time 60 s; static extraction 0.3 g of plant material; solvent methanol and methanolwater mixture; pressure 60 bar; time 10 min; temperature 75 and 125 °C; flush 60%; purge time 60 s; static extraction 500 mL of sample; solvent n-hexane, ethyl acetate and/or methanol; temperature 50, 100 or 150 °C; time 5 min; pressure 40 bar; flush 60%; purge time 30 or 60 s; static extraction

GC-MS

[45]

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thale cress herb Arabidopsis thaliana (L.) Heynh. runner bean herb (Phaseolus coccineus L.) Flowers of Sambucus nigra L. Tea leaves and coffee beans

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ACCEPTED MANUSCRIPT Highlights: 1. This review updates knowledge about the applicability of PLE. 2. It discusses the effectiveness and usefulness of PLE in the context of its achievements. 3. It discloses the limitations of PLE in the plant constituents content analysis

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4. Knowing all these facts can lead to the creation of a perfect method with a good method.