Efficiency of Recovery of the Bioactive Principles of Plants by Comparison between Solid–Liquid Extraction in Mixture and Single-Vegetable Matrices via Maceration and RSLDE

The term “officinal” derives from the Latin and includes all medicinal, aromatic and perfume plant species, which have long been a subject of interest for multiple purposes: health, food, pharmacological, cosmetic and so on. In this work, a study on six different species of medicinal plants, particularly characterized by digestive, choleretic and diuretic properties, was carried out: rosemary (Rosmarinus officinalis), sage (Salvia officinalis), laurel (Laurus nobilis), gentian (Gentiana lutea), dandelion (Taraxacum officinale) and rhubarb (Rheum palmatum). The roots and aerial parts of plants were separately extracted with two different techniques—maceration and rapid solid–liquid dynamic extraction (RSLDE)—and the quali/quantitative analysis of active ingredients have been determined by applying dry residue, Folin–Ciocalteu and DPPH assays. Data obtained have provided useful answers regarding the efficiency of the extraction carried out on a mixture or on single plants, allowing us to evaluate the best choice according to the cases and the final uses.


Introduction
The use of medicinal plants is very ancient and, in fact, for many centuries, they were used as the only available medical remedies [1,2]. Fortunately, many plants can supply active ingredients widely used by the pharmaceutical industry (cardiotonics, salicylic acid, anticancer drugs, etc.), contributing to human wellbeing and health. In fact, medicinal plants are characterized by the presence of certain classes of active ingredients, such as polyphenols, glycosides and tannins, that could be a natural aid to illnesses. For example, based on the chemical structure, polyphenols are divided into ten or more different groups, but the main classes are stilbenes, lignans, flavonoids and phenolic acids [3]. Moreover, the flavonoids can then be divided into further classes, among which it is possible to find isoflavones and anthocyanins. These substances have various properties: first of all, their chemical structure allows them to function as antioxidant molecules and therefore to protect the plant from oxidative stress and free radicals. However, their intake also determines numerous beneficial effects on human health, as demonstrated by numerous studies [4][5][6][7]. The chemical compounds contained in plants, and which positively act on the human (or animal) organism, are called active principles, responsible for some specific biological activity. They are very varied, including polyphenols, alkaloids, flavonoids, glycosides, Once this equilibrium is reached, the solution rich in extracted compounds is separated by physical processes of filtration and centrifugation. Many different parameters can influence the extraction yield, such as the size and physical state of the matrix particles, the temperature, the solvent/matrix ratio and the relative contact times [21,22]. In this work, a comparison between two solid-liquid extraction techniques-conventional maceration and the innovative RSLDE-was carried out in order to evaluate the differences in the extraction efficiency of the bioactive compounds obtained from the plants extracted in mixture or individually and, consequently, to establish the extraction methods according to the different fields of application of the extracts obtained.

Maceration vs. RSLDE
The natural products of medicinal plants, both as pure compounds and as standardized extracts, due to their chemical diversity, offer numerous opportunities for use in various fields of human and animal applications. Thus, interest in edible plants in particular has grown worldwide due to the presence of plant extracts of various types of bioactive compounds and their proven beneficial effects on health [23][24][25]. However, since extraction is the most important step in the analysis of the constituents present in plant matrices, the strengths and weaknesses of two extraction techniques, such as maceration and RSLDE, have been discussed in this work, but above all, for the first time, the effects of the extraction carried out on a mixture of plants were evaluated compared with a mixture made up of single extracts from the same plants.
Maceration is one of the oldest and simplest techniques. The extraction process is generally characterized by a long period of extraction: two or three weeks to exhaust the plant. The Official Pharmacopoeia-that is, the reference text for the preparations in herbalists' sector-specifies twenty-one days to obtain the major parts of extracts from medicinal plants, with occasional mixing of the maceration batch. The diffusion and osmosis processes used in this extraction are speeded up through the use of ultrasound or microwaves or through an increase in temperature so as to act on the kinetic energy of the molecules of the solid [26]. Furthermore, to ensure the diffusion of the extracted substances throughout the mass of the extracting liquid, it is necessary to agitate the system and remove the microequilibrium established near the solid matrix, in this way avoiding the premature stopping of extraction phenomenon. Among the disadvantages of this technique, in addition to the long times required and it not always being compatible with the properties of the matrix, there is the incomplete extraction of the matrix itself and the non-reproducibility of the extract content (standardized extract) because this type of solid-liquid extraction can be defined as passive extraction. Furthermore, the vegetable matrices cannot be macerated in water as they undergo degradation processes. However, it is still a valid and in-use technique; in fact, in a recent work, an innovative method of successive macerations was proposed using a mixture of solvents with the aim of simultaneously improving the yield, the distribution of the compounds between the different phases and reducing the volume of extraction solvents [27]. On the other hand, the most recent extraction techniques try to balance a series of factors, such as the quality of the product obtained, the efficiency of the process, the production costs and a low environmental impact [28][29][30]. From this point of view, RSLDE combines all these factors, in fact, it is an innovative technique which allows for extraction at room temperature in a short time with a reduced environmental impact (green) [31]; this kind of solid-liquid extraction can be defined as an active process because compounds are forced to exit from the inner of vegetable thanks to difference of pressure. The Naviglio extractor works by alternating a static phase, in which the pistons present in it push simultaneously on the liquid causing an increase in pressure, with a dynamic phase, during which the pistons are moved from their equilibrium position, and there is an alternation of thrusts between the two pistons themselves, with a reduction in pressure and the generation of a mixture of the liquid throughout the system to diffuse substances and thus to reduce the concentration around the solid matrix. It is at this moment that the extraction of the solid matrix takes place, made possible by a difference in pressure between the inside and the outside of the sample. The extractable substances, not chemically bonded to the principal structure of the solid matrix, at each extraction cycle, are dragged out by a mechanical effect. The dynamic phase also allows for the rapid and complete mixing of the solid matrix and the instantaneous diffusion of the extracted substances throughout the mass of the liquid, avoiding supersaturation phenomena around the solid that could stop the extractive process. This system makes this technique effective both in terms of extraction time and recovery efficiency and the quality of extract of the active ingredients contained in the plant matrix, as demonstrated by its use in various fields of application, e.g., herbal, pharmaceutical, cosmetic and food [32][33][34][35][36][37].  themselves, with a reduction in pressure and the generation of a mixture of the liquid throughout the system to diffuse substances and thus to reduce the concentration around the solid matrix. It is at this moment that the extraction of the solid matrix takes place, made possible by a difference in pressure between the inside and the outside of the sample. The extractable substances, not chemically bonded to the principal structure of the solid matrix, at each extraction cycle, are dragged out by a mechanical effect. The dynamic phase also allows for the rapid and complete mixing of the solid matrix and the instantaneous diffusion of the extracted substances throughout the mass of the liquid, avoiding supersaturation phenomena around the solid that could stop the extractive process. This system makes this technique effective both in terms of extraction time and recovery efficiency and the quality of extract of the active ingredients contained in the plant matrix, as demonstrated by its use in various fields of application, e.g., herbal, pharmaceutical, cosmetic and food [32][33][34][35][36][37].         As can be seen from Figures 1-4, the comparison of the data shows that the dry residue, expressed in g/L, increases with the passing of the days as regards the extraction during maceration, while for the extracts using RSLDE, the increase is a matter of hours.  As can be seen from Figures 1-4 the comparison of the data shows that the dry residue, expressed in g/L, increases with the passing of the days as regards the extraction during maceration, while for the extracts using RSLDE, the increase is a matter of hours. due, expressed in g/L, increases with the passing of the days as regards the extraction during maceration, while for the extracts using RSLDE, the increase is a matter of hours. Figures 5-7 show the comparison of the data obtained from the determination of the dry residue, the concentration of polyphenols and the antioxidant activity via extraction via maceration from the mixture composed of a triad of rosemary, sage and laurel and from the mixture of the individual macerates of the same plants in the same proportions in the two different solvents.          It is possible to highlight from Figures 5-10 that the mixture of this first triad of plants in both solvents shows a higher concentration in terms of dry residue, polyphenol concentration and antioxidant activity, i.e., 10-15% higher than the mixture prepared by mixing the single extracts via maceration and the single extracts via RSLDE in the same proportions.

Root Extraction
The same determinations reported previously were carried out on the second set of gentian, dandelion and rhubarb plants, whose matrix extracted in these cases is represented by the roots. Figures 11-13 show the comparison of the data obtained via the de- It is possible to highlight from Figures 5-10 that the mixture of this first triad of plants in both solvents shows a higher concentration in terms of dry residue, polyphenol concentration and antioxidant activity, i.e., 10-15% higher than the mixture prepared by mixing the single extracts via maceration and the single extracts via RSLDE in the same proportions.

Root Extraction
The same determinations reported previously were carried out on the second set of gentian, dandelion and rhubarb plants, whose matrix extracted in these cases is represented by the roots. Figures 11-13 show the comparison of the data obtained via the determination of the dry residue, the concentration of polyphenols and the antioxidant activity via extraction via maceration from the mixture composed of the triad of gentian, dandelion and rhubarb and from the mixture composed of individual macerates of the same plants in the same proportions in the two different solvents: alcoholic solution (96% vol.) and hydroalcoholic solution (40% vol.).  As can be seen from  in this case also, the comparison of the data shows that the dry residue, expressed in g/L, increases with the passing of the days as regards the extraction during maceration, while for the extracts using RSLDE, the increase a matter of hours.
Furthermore, in the extraction via maceration in a hydroalcoholic solution (40% vol.), there is a decrease in the value of the dry residue as regards dandelion and rhubarb, probably due to a degradation of the matrix after a maceration period of more than 10-15 days.    As can be seen from Figures 11-14, in this case also, the comparison of the data shows that the dry residue, expressed in g/L, increases with the passing of the days as regards the extraction during maceration, while for the extracts using RSLDE, the increase a matter of hours. out on the roots of gentian, dandelion and rhubarb individually and in mixtures.
As can be seen from Figures 11-14, in this case also, the comparison of the data shows that the dry residue, expressed in g/L, increases with the passing of the days as regards the extraction during maceration, while for the extracts using RSLDE, the increase a matter of hours.   Furthermore, in the extraction via maceration in a hydroalcoholic solution (40% vol.), there is a decrease in the value of the dry residue as regards dandelion and rhubarb, probably due to a degradation of the matrix after a maceration period of more than 10-15 days.       As previously reported, the same comparison was made for plants extracted via RSLDE. Figures 18-20 show the comparison of the data obtained via the determination of the dry residue, the concentration of polyphenols and the antioxidant activity by means of RSLDE from the mixture composed of the triad of gentian, dandelion and rhubarb and from the mixture of the individual macerates of the same plants in the same proportions in the two different solvents.     The comparison of the results obtained shows that also for this second triad of plants, the mixed extraction of the three plants turns out to be better performing, with a variation of the order of 10-15% more than the mixture prepared by mixing the individual extracts in the same proportions of the mixture and carrying out the appropriate determinations. In order to obtain a more correct evaluation of the antioxidant activity of the obtained extracts, a comparison was made with another method, i.e., the FRAP assay, which is based on the ferrous-reducing activity of the antioxidant compounds. The results obtained showed that the values for the FRAP ranged from 13.65 to 79.89 mg TE/g. Moreover, in this case (in both series of the three plants), the results of the antioxidant activity of the The comparison of the results obtained shows that also for this second triad of plants, the mixed extraction of the three plants turns out to be better performing, with a variation of the order of 10-15% more than the mixture prepared by mixing the individual extracts in the same proportions of the mixture and carrying out the appropriate determinations. In order to obtain a more correct evaluation of the antioxidant activity of the obtained extracts, a comparison was made with another method, i.e., the FRAP assay, which is based on the ferrous-reducing activity of the antioxidant compounds. The results obtained showed that the values for the FRAP ranged from 13.65 to 79.89 mg TE/g. Moreover, in this case (in both series of the three plants), the results of the antioxidant activity of the three plants in the mixture are higher than in the preparation obtained by mixing the single extracts in the same proportions of the mixture.
In summary, although further studies are underway to identify and quantify the bioactive compounds present in the various extracts obtained, two extraction methods were compared in this study to obtain bioactive compounds from two different plant parts, i.e., the leaves and roots. The experimentation conducted in this work is part of a larger project which includes instrumental analytical determinations for deep chemical characterization and biological assays on cell lines to evaluate their antioxidant and antiinflammatory properties. In this first phase, attention was focused on the effectiveness of the extraction method and on the innovation of the extraction. The results obtained will enable the use of the innovative extraction method with proven efficacy in order to guarantee the best quality of the extract. On the other hand, the choice of the matrix to be extracted depends on both the type of plant and on the part of the plant with the higher content of bioactive compounds or the greater interest [38]. Furthermore, to the best of our knowledge, this is the first study in which the extraction efficiency of plants taken individually was compared with those mixed of other plants to evaluate the differences. Therefore, a conventional extraction method, i.e., maceration, was used and compared with an innovative one, i.e., RSLDE, which allows a faster, more efficient, but above all, greener extraction, while also allowing for the recovery of the solvent used. On the other hand, RSLDE can be considered, to all intents and purposes, a "green technique" as it operates at room temperature and with minimal use of solvent and minimal energy. Among other things, the extraction solvent can be recovered and reused in line with the current principle of the circular economy. In fact, several applications of RSLDE in various sectors are reported in the literature with respect to both conventional techniques and innovative ones, in which its efficiency is highlighted [39].
In the mixed extractions (1:1:1), the matrix used in the first triad were as follows: rosemary, sage and laurel plants were represented by the dried and chopped leaves; while in the case of the second triad-made up of gentian, dandelion and rhubarb-by the dried and shredded roots. The solvents used to extract the active ingredients were ethyl alcohol (96% v/v) and a hydroalcoholic solution (40% v/v).
The macerates of the 6 single plants in the 2 different solvents were prepared by weighing 50 g of each single plant, while 51 g (17 g of rosemary, 17 g of sage and 17 g of laurel) were used for the mixtures (part used: leaves), and the same were used for the mixture prepared with gentian, dandelion and rhubarb (part used: roots).
Maceration: The plants were placed in a closed glass container with 500 mL of solvent and kept in the dark for 21 days (Official Farmacopoeia), occasionally shaking. Therefore, at intervals of 2, 4 and 24 h, 15 mL of extract were taken, which was then filtered on filter paper and used to determine the dry residue, the yield and the concentration of polyphenols in g/L by assay with Folin-Ciocalteu reagent and antioxidant activity by DPPH assay. Lastly, 15 mL of fresh solvent were added.
RSLDE: The extractions of the individual plants and their respective mixtures were extracted through the use of the Naviglio extractor, which allows for faster and more efficient extraction. The vegetable matrices and solvents were used in the same quantities and volumes of the extraction by maceration (50 or 51 g in 500 mL). Therefore, at intervals of 2, 4 and 24 h, 15 mL of extract were taken, which was then filtered on filter paper and used to calculate the dry residue, the yield and the concentration of polyphenols in g/L via assay with Folin-Ciocalteu reagent and antioxidant activity via DPPH assay. Lastly, 15 mL of fresh solvent were added.
The dry residue was obtained by drying exactly 10 mL of the sample in an oven at 105 • C for 12 h. The percentage yield was then obtained from the latter.

Analysis of Total Phenols by Folin-Ciocalteu Reagent
The Folin-Ciocalteu reagent is used for the colorimetric determination of phenols and polyphenols. This reagent is a mixture of sodium phosphomolybdate Na 3 PMo 12 O 40 and sodium phosphotungstate Na 3 PW 12 O 40 . The method is based on a redox reaction which leads to the formation of a blue chromophore, whose maximum absorption depends on the concentration of the phenolic compounds. It is detectable with a spectrophotometer in the range between 690 and 710 nm; in this research, a wavelength of 710 nm was used. A standard gallic acid (GA) was used to obtain the calibration curve, and the results for polyphenol content were expressed as mg/mL of gallic acid (GAE) [40].

DPPH Assay
The DPPH assay allows us to determine the antioxidant power by reacting the sample to be analyzed with a solution of DPPH [2,2-diphenyl-1-picrylhydrazyl] and analyzing the decrease in the radical peak under the visible wavelength at 517 nm. Antioxidant compounds (AOH), which are capable of transferring a hydrogen atom to the radical, cause a discoloration of the solution. The decrease in the peak at 517 nm of the radical (DPPH) after a pre-established incubation time (blank) is then analyzed under visible wavelength. This decrease (discoloration) is proportional to the antioxidant load present in the sample by applying the formula in [41].

FRAP Assay
Ferric reducing-antioxidant power (FRAP) assay represents another method by which to evaluate the antioxidant power. In particular, 0.1 mL of extract was added to 2 mL of reagent in acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric chloride (20 mM) in a final ratio of 10:1:1 (v/v/v). After 30 min of incubation at room temperature, the absorbance was read at 593 nm. Similarly, a blank sample was prepared (prepared in the same way but without the extract). The unit of measure was the milligram equivalent of trolox per gram of dry extract (TEs/g of extract) [42].

Statistical Analysis
The results of each experiment were performed in triplicate and are presented as mean ± standard deviations (SD). Data were analyzed using multifactorial analysis of variance (ANOVA).

Conclusions
The study carried out on the selected plants had the aim of comparing the extract obtained from a mixture prepared with three plants in equal proportions (1:1:1) with that obtained by mixing the extracts of single plants in the same proportions to verify the efficiency of the two procedures.
To this end, two solid-liquid extraction techniques were used, namely, conventional maceration and the innovative RSLDE extraction technology.
The analyses carried out for the determination of the dry residue-the tests by Folin-Ciocalteu and DPPH-have enabled us to highlight that the extraction of the ternary mixture of plants is "richer" in bioactive substances compared to the mixture obtained by mixing the pure extracts in the same amounts. The probable explanation is that the higher values obtained via extraction in the mixture can be attributed to a "synergistic" effect due to the presence of other plants.
In the future, further studies will be carried out on the identification and quantification of the bioactive compounds present in the various types of extracts obtained.
Furthermore, from the comparison of the two extraction techniques used, it can be deduced that RSLDE, thanks to faster extraction kinetics and better efficiency in recovering the extract, could certainly replace the techniques of maceration, infusion and percolation.
Finally, it must be noted that the extraction of single plants, although less efficient, has its advantages; it allows us to obtain individual extracts to be used for the formulation of infinite blends, allowing the creation of different products in the food sector such as bitters and various drinks, as well as various types of supplements, and also in the pharmacological sector. Funding: This research received no external funding. The authors wish to thank the Department of Chemical Sciences for contributing to the publication of this manuscript.

Conflicts of Interest:
The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.