PROCESS FOR EXTRACTING BIOACTIVE COMPOUNDS FROM PLANT SUBSTRATES AND PRODUCING MICROPARTICLES AND / OR NANOPARTICLES CONTAINING THESE COMPOUNDS

Abstract

The invention relates to a technical field concerning extracts from natural substances, specifically plant products, wherein such extracts are biologically active compounds or agents capable of intelligent delivery for transport and release, e.g., in food and pharmaceutical applications. Specifically, the invention relates to a process for extracting biologically active compounds from plant substrates and producing microparticles and / or nanoparticles containing such compounds. The process under the invention represents in a beneficial way an innovative integrated device for the production of microparticles and / or nanoparticles functionalized toward specific targets, delivering the extracted active substances from plant substrates. Furthermore, since such microparticles and / or nanoparticles may contain biologically active compounds or components, the invention relates to a process for producing microparticles and / or nanoparticles, as well as such microparticles and / or nanoparticles containing biologically active compounds or components.The bioactive compounds or components thus extracted and obtained can be conveniently and efficiently incorporated into carriers such as nanoemulsions, alginate-based or clay-based microspheres, enabling the transport or distribution of highly bioactive substances extracted / recovered from various plant substrates using the process of the invention. Practical applications can be found favorably in the delivery of active substances (“drug delivery”). The advantages of using such structural forms are numerous, including gastrointestinal protection of the bioactive compounds or components, the formation of special slow-release formulations, and the ability to make the nanoparticles multi-responsive to specific stimuli.

Description

[0001] MULTI-PHASE EXTRACTION SYSTEM (M.E.S.) WITH COMBINED EXTRACTION TECHNIQUES OF BIOACTIVE COMPOUNDS FROM PLANT MATRICES AND VEHICLES CONTAINING THEM

[0002] The invention relates to the technical field related to extracts of natural substances, in particular of plant products, where such extracts consist of bioactive compounds or agents, as well as delivery systems thereof for their transport and release, for example in nutraceutical and pharmaceutical applications.

[0003] Specifically, the invention relates to a process of extracting bioactive compounds from plant matrices and for producing micro- and / or nanoparticles containing them. Moreover, the invention relates to a process of producing micro- and / or nanoparticles, as well as such micro- and / or nanoparticles containing bioactive compounds or ingredients.

[0004] As is known, a so-called "bioactive" compound or agent or ingredient is a biologically active compound, which is capable of imparting a beneficial effect on the health of organisms or living beings, clearly including humans. By way of non-limiting example, polyphenolic substances and carotenoids can certainly be mentioned.

[0005] The use of natural products for the prevention and treatment of various diseases is constantly expanding throughout the world; this is particularly true for polyphenols, which represent a large number of molecules with great potential in terms of beneficial effects on health. These substances are widely present in several key foods of the Mediterranean diet. Many studies have highlighted the several pharmacological properties that these substances have in the cardiovascular system. In particular, the Mediterranean diet includes a daily intake of 25-50 ml of extra virgin olive oil and is associated with a reduction in cardiovascular risks, neurodegenerative diseases, and some forms of cancer, in particular colon cancer. In general, the daily intake through the Mediterranean diet is estimated at around Ig / day and recent studies also show that chocolate, in particular dark chocolate, is also rich in these molecules.

[0006] Nowadays, special attention is paid to the potential effect that polyphenols could carry out on the GI tract. In particular, a low incidence of colon cancer has been demonstrated in both animal and human models. Such an effect seems to be linked to how they interfere with different intracellular signaling mechanisms (NFkB, MARKs, etc.), regulating gene expressions responsible for cell proliferation, apoptosis, the expression of detoxifying enzymes, and the immune system. Such effects have repercussions on a large number of mechanisms underlying the inflammatory process, which is often the cause of tumor manifestations. The beneficial effects are not only attributable to such pharmacological actions, but also to the significant antioxidant power thereof. Many studies have shown that at the base of the development of several diseases of the gastro-intestinal tract (characterized by ischemia, inflammation or cancer of the intestine), there is an imbalance between oxidizing agents and antioxidants. An area of vulnerability of these molecules is represented by the poor absorption thereof and a still unclear metabolism; on the other hand, the gastro-intestinal tract is exposed to relatively high levels of such substances, therefore the "local" beneficial effect on such an apparatus could overcome the problem of absorption, where the concentration could reach very high levels (several hundred pM). It is further known that the potency of activity is reduced as a result of the use of the single component when compared to the polyphenolic extract assuming a possible synergism between the various components.

[0007] In general, it is known that the daily and regular consumption of fruits and vegetables has beneficial implications, for example, in combating the risk of the onset of chronic non- communicable diseases (NCDs) and more generally in improving well-being and quality of life.

[0008] As widely reported and described in the scientific literature, herbal extracts, with high levels of phenolic and carotenoid compounds, have high antioxidant, anti-inflammatory and antiproliferative activities.

[0009] In fact, it is believed that fruits and vegetables, generally containing significant amounts of antioxidant compounds, can have beneficial effects on health, for example by counteracting the phenomenon of oxidative stress, and thereby reducing the likelihood of triggering events of many chronic diseases. These antioxidants are mainly found in the form of phenolic compounds such as flavonoids, phenolic acids, stilbenes, tocopherols, tocotrienols, ascorbic acid, carotenes and xanthophylls. The beneficial effects are attributed to the ability thereof to donate electrons, the scavenging of free radicals and the reducing power thereof. But they are also active against protein denaturation, which causes damage to biological structures, and further act on enzymes, inactivating in particular those to which metabolic alterations are attributed. Moreover, natural antioxidants have the ability to improve food quality and stability and, in nutraceuticals, can also act as compounds which interrupt free radical chain reactions in biological systems and can therefore provide additional benefits to human health.

[0010] The most important fruits and vegetables, such as tomatoes, goji berries, watermelon, papaya, mango, carrot, spinach, calendula, sweet potato and pumpkin are all sources of carotenoids. Carotenoids are a group of C-40 isoprenoid-based molecules with >600 representatives in nature, of which about 30 are important in our daily diet. This class of phytochemicals has recently attracted much attention due to the potential health benefits associated with carotenoid consumption. Several research groups have suggested that the cis isomers of lycopene are better absorbed than the aZZ-trans form due to the shorter length of the cis isomer, the higher solubility of the cis isomers in mixed micelles, and / or as a result of the lower tendency of the cis isomers to aggregate. Carotenoid pigments are ubiquitous in nature, where they carry out a variety of roles. Synthesized de novo only in plants, algae, bacteria, and fungi, they are essential for photosynthesis, function as antioxidants, and are involved in the coloration of many animals and plants. Carotenoids cannot be synthesized by humans or animals, so the absorption thereof depends on diet. Carotenoids and derivatives thereof have many functions essential for development, immune response and the visual cycle. In fact, for animals, including humans, the conversion of provitamin A carotenoids into retinal is essential for health, and the hydroxy carotenoids (lutein, zeaxanthin and meso-zeaxanthin) have the function of protecting the central retina from photo-oxidative damage. There is much scientific evidence of the preventive role thereof in a range of chronic or age-related diseases, partly but not solely related to the antioxidant properties thereof. For example, carotenoids have been proposed to prevent the onset and proliferation of several types of cancer, including lung, prostate, and breast cancer.

[0011] Emerging evidence from epidemiological studies, as well as on cell cultures and animals, indicates that certain carotenoids, such as lycopene, when consumed in the form of fruits and vegetables or also as processed foods, can be protective against the risk of cancer and cardiovascular disease, in particular stroke, hypertension and prostate cancer, and can carry out a beneficial role for health and bone density. There is much scientific evidence of edible botanical species of exotic origin. For example, traditional Chinese medicine has used the compounds present in goji fruits to prevent the onset and progression of cancer. Goji berries also exhibit immuno stimulatory activity. It has been confirmed in many tests that the compounds present in goji berries have pro-apoptotic and antiproliferative activity against cancer cells.

[0012] Starting from what nature provides us, it is therefore possible to obtain / derive the aforesaid beneficial substances. Moreover, processing waste products from the food industry (tomatoes, grapes, plant waters from the production of olive oil, spent pomace), or from agriculture (olive leaves, various pruning) which have zero cost can be used as starting matrices. Such a possibility, with a view to circular economy, eco-sustainability and / or eco-compatibility, can represent a great "green" investment opportunity. For example, the residual biomass of wine processes and that of tomato processing represent very rich and varied sources of bioactive compounds such as resveratrol, catechins, epicatechins, gallic acid, tartaric acid, proanthocyanidins, lycopene, lutein, zeaxanthin, tocopherols, with potential applications in various fields, including medicine and nutraceuticals.

[0013] It is particularly useful to note that, especially in recent years, interest has increasingly shifted towards specific compounds which can be obtained from by-products of the food industry, such as that of tomato and wine. The reason can be found in two fundamental aspects: on the one hand, production companies can thus reduce the volume of waste generated, resulting in a reduction of the related disposal costs, on the other hand, the reduction of environmental impact, where the latter is now an aspect to which consumers increasingly pay enormous attention.

[0014] Therefore, the possibility of having processes available which allow efficient and convenient recovery and thus enhancement of processing by-products rich in substances with high added value is increasingly strategic and relevant. It is therefore important to develop methods which allow recovering, with innovative industrial methods, these bioactive compounds from processing by-products of the relevant crops.

[0015] In this context, the stage of extraction of active ingredients from plant sources forms the real challenge for companies in the field, from both a scientific and productive point of view.

[0016] Solid- liquid phase extraction (maceration, percolation, infusion...) is traditionally used to obtain target compounds from different waste materials.

[0017] In general, this type of extraction technique requires organic solvents in high amounts and is very energy-intensive since it requires long treatment times and often reaching high temperatures. On the other hand, supranational bodies (EFSA, FDA, EMA) do not allow an indiscriminate use of organic solvents that such a technique almost always requires; in fact, many organic solvents are not compatible with food use due to the negative effects the latter have on human health and the environment.

[0018] With particular reference to the extraction of active ingredients from plant sources, to date there are known technologies in the field of the extraction of bioactive compounds from nature. Some conventional techniques are still widely used since they are inexpensive and simple to apply, such as maceration. However, there are many disadvantages to using this technique, including long extraction times, which can vary from a few hours, to a few days, up to several tens of days. Such times are not always compatible with the production needs of the companies involved. Moreover, such techniques have another limit, namely the impossibility of standardizing the process, controlling all the stages of the extraction dynamics, since in the long times required a series of equilibria, reactions, physical, chemical and biochemical processes are established which are not always predictable and which often introduce other problems related above all to the final quality of the extract.

[0019] In general, the extraction process is based on physical ingredients such as osmosis and diffusion which ensure solvent penetration in the plant matrix with the relative extraction of the bioactive compounds. Therefore, in summary, the conventional extraction techniques such as maceration, percolation, are based exclusively on the phenomena of diffusion and osmosis, considerably slowing down the extraction process of the substances contained in plant matrices. In fact, they require long times to ensure exhaustive extractions of the matrix, and sometimes require high temperatures, thereby promoting oxidation / isomerization phenomena of plant matrices, also because major critical issues such as contact with atmospheric oxygen, temperature control and light are not eliminated.

[0020] Such techniques, although still used, are generally and continuously supplanted at an industrial level by more modern extraction methodologies, substantially based on different physical, chemical and mechanical ingredients such as the use of ultrasound, microwaves, supercritical fluids and / or high pressures.

[0021] In any case, the common goal shared by all extraction techniques is to ensure maximum extraction yield. To this end, the critical and limiting step is represented by cell disruption. In fact, bioactive compounds are compartmentalized within vacuolar structures such as chloroplasts and chromoplasts with considerable resistance to physical and mechanical stresses. It is therefore essential to carry out the appropriate operations to break the membranes and walls of the plant cells for the purpose of optimizing the extraction yield and ensuring an excellent extraction from both a quantitative and qualitative point of view.

[0022] Among the extraction techniques of active ingredients used in recent times to improve the extraction yield, the ultrasound-assisted extraction technique is certainly worth mentioning, which precisely uses the mechanical energy of ultrasound. Such a technique, which is relatively new, can be defined as an unconventional extraction technique.

[0023] The ultrasound-assisted extraction technique is based on the use of ultrasound, mechanical waves which belong to a wide spectrum of frequencies, which begins by convention at 20KHz. This technique is based on the ingredient whereby, when ultrasound is applied to a liquid, waves propagate therein, generating a continuous succession of compressions and decompressions. The very fast sequence of compression and decompression cycles generates millions of micro-bubbles, referred to as cavities, which grow in volume with each cycle. The phenomenon is known as cavitation and has proved useful in causing the rupture of biological membranes, thereby facilitating the extraction of bioactive ingredients from plant cells.

[0024] To date, sonication is used to speed up and make the extraction process more efficient. The increase in efficiency is mainly due to the following reasons:

[0025] - intensification of mass transfer: ultrasound facilitates the formation of microparticles, emulsion and solvent exchange around the walls of the plant material;

[0026] - cell disruption: ultrasound can disrupt the cell wall, facilitating the extraction of contents, and disintegrate the plant material, increasing the contact surface with the solvent;

[0027] - increased solvent penetration: when the cavities collapse, the ultrasonic solvent jets which are generated force the solvent into the cell, facilitating the passage thereof through the cell membrane;

[0028] - capillarity effect of ultrasound: ultrasound improves the diffusion by capillarity of solutes, in particular polar and ionic ones, through a complex mechanism, still under study, which would seem to involve the development of electric fields;

[0029] - reduced extraction of heavy components;

[0030] - significant reduction of the bacterial load of the extract.

[0031] Microwaves are non-ionizing waves of an electromagnetic nature with a frequency between 300 MHz and 300 GHz. These waves are positioned in the electromagnetic spectrum between infrared rays and X-rays. The direct action of these waves on the substance is to transform electromagnetic energy into thermal energy. Microwaves consist of two oscillating perpendicular fields: magnetic and electric, responsible for heating. Microwave assisted extraction (MAE) depends on the solvent and sample heating process. Moreover, it is governed by two phenomena: dipole rotation and ionic conduction. Dipole rotation refers to the realignment of the molecule's dipoles with the rapid change in electric field; this causes both dielectric material and solvents with persistent dipoles to be heated by the action of microwaves. Ionic conduction instead refers to the transfer of ions caused by the change in the electric field. The migration thus generates, due to the resistance offered by the solution, friction responsible for heating the solution. The extraction can be carried out on both wet and dry matrices, exploiting the traces of moisture present in the sample which are heated (e.g., water contained in biomass plant cells). The resulting evaporation creates extremely high pressures within the cells which ultimately cause the cell wall to rupture, increasing the recovery yields of phytocondensates in the medium. The disadvantages of the technique are in the plant costs, plant sizing, the management thereof, the extract quality, and in the addition, albeit in low amounts, of solvents, often necessary to facilitate the extraction.

[0032] The critical point of a fluid defines the end of the vapor-liquid coexistence curve. Regardless of the pressure applied, a fluid cannot pass to the liquid phase at temperatures above the critical temperature. A fluid is therefore referred to as “supercritical” at any pressure and temperature above the pressure and temperature of the critical point. Supercritical liquids have unique features with physical and chemical properties which are intermediate between those of gases and liquids. CO2 is one of the most widely used supercritical fluids due to the properties thereof. It is an inexpensive green solvent which is eco-compatible, non-toxic, non-carcinogenic and non-flammable, with a critical pressure (74 bar) and temperature (31 °C) which are relatively easy to reach. Supercritical CO2 allows a selectivity of the extraction process towards the target compounds by varying temperature and pressure. CCh-assisted SFE has shown high quality extracts with yields comparable to those recovered from organic solvents; moreover, the addition of modifiers, such as ethanol to CO2 increases the polarity and efficiency thereof, in particular in terms of extraction of polar molecules such as phenolic compounds. However, such a technique has several disadvantages including the high plant cost, the management thereof, the danger, the low ductility of use in defining different molecular targets to be extracted (co-solvents must be used to vary the polarity). Therefore, in light of the above, the need is felt for a solution which allows the aforesaid technical problems and related disadvantages to be overcome.

[0033] The need is met by a process of extracting bioactive compounds from plant matrices and for producing micro- and / or nanoparticles containing them by a process in accordance with the appended claims.

[0034] In fact, the Applicant has found a process which allows extracting bioactive compounds from plant matrices, as well as producing micro- and / or nanoparticles containing them.

[0035] In accordance with a first aspect, the invention relates to a process as defined in claim 1.

[0036] In particular, an aspect of the invention is a process of extracting bioactive compounds from plant matrices and for producing micro- and / or nanoparticles containing them.

[0037] Therefore, according to an aspect, the present invention relates to a process of extracting bioactive compounds from plant matrices and for producing micro- and / or nanoparticles containing them comprising the steps of:

[0038] A-l) crushing the plant material with water by means of a first turboemulsifier, preferably under vacuum, with high-speed rotating blades;

[0039] A-2) in-line homogenization for further crushing; A-3) solid-liquid separation with a decanter centrifuge;

[0040] A-4) extraction of polar and / or moderately polar substances with alcoholic solvent, preferably ethanol, or hydroalcoholic solvent or an alcohol / water mixture, using a second turboemulsifier;

[0041] A-5) solid-liquid separation with a second decanter centrifuge;

[0042] A-6) extraction of apolar substances with edible oil using a turboemulsifier;

[0043] A-7) solid-liquid separation by means of a decanter centrifuge to obtain extracts;

[0044] A-8) extracts ozonization;

[0045] A-9) micro / nano structuring of the extracts obtained from step A-8 by means of an alginate micro-encapsulation generator, a clay micro-encapsulation system, or a nano-emulsion generator.

[0046] Such steps from A-l to A-9 can be identified as those steps forming the so-called the extraction stage.

[0047] With reference to step A-l, the crushing preferably occurs by means of a turboemulsifier provided with blades, more preferably a turboemulsifier provided with high rotating capacity blades. Preferably water is used as the first extraction solvent.

[0048] With reference to step A-2, the homogenization is in-line, i.e., performed by an in-line homogenizer, preferably at a speed between 300 and 3000 rpm. Such a step has the purpose of completing, thus finalizing, the rupture stage of the cell membranes and walls of the starting plant material. Preferably, the management of the speed within the aforesaid range occurs by including the use of an inverter. According to a preferred aspect, the use of an inverter is included in step A2.

[0049] With reference to step A-3 of solid-liquid separation with a decanter centrifuge for solidliquid separations, the solvent recovery can also be included in such a step. Such a recovery can occur by including suitable solvent recovery systems.

[0050] With reference to step A-4, in this step the extraction of the polar and / or moderately polar substances with alcoholic solvent, preferably ethanol or hydroalcoholic solvent, occurs using a turboemulsifier. Said thermoemulsifier preferably being a thermoemulsifier of the type as defined with reference to step A-l. Preferably the solvent referred to in step A-4 is ethanol, more preferably undenatured ethyl alcohol min 96°, even more preferably of the type compatible with food, or with an alcohol / water mixture, preferably water / ethanol. Depending on the quality and physical-chemical features of the bioactive compounds to be recovered, the solvent system can also consist of ethanol / water mixtures in different v / v volumetric ratio, preferably 90 / 10; 80 / 20; 70 / 30; 50 / 50; 25 / 75. Preferably, in step A-4 the alcohol / water mixture, preferably ethanol / water, is at a ratio from 90:10 to 25:75. According to a preferred aspect, the alcohol / water mixture is the ethanol / water mixture.

[0051] With reference to step A-5, the solid-liquid separation occurs in such a step, preferably by means of a second centrifugal decanter. Moreover, a further solvent recovery step can be included.

[0052] With reference to step A-6, in such a step the extraction of apolar substances occurs, preferably with oil, more preferably edible oil, even more preferably olive oil, using a turboemulsifier. Said thermoemulsifier preferably being a thermoemulsifier of the type as defined in step A-l. According to a particularly preferred aspect, the edible oil is extra virgin olive oil or EVOO oil or other similar oil.

[0053] With reference to step A-8, in such a step the ozonation of oily extracts preferably occurs. This step advantageously allows obtaining ozonated oil / stable ozonides. An example of such a step is the system developed by Erbagil for producing precisely ozonated oil / stable ozonides.

[0054] With reference to step A-9, in such a step the micro / nano structuring of the extracts occurs by means of an alginate micro-encapsulation generator, a clay micro-encapsulation system or a nano-emulsion generator.

[0055] According to a preferred aspect, one or more solid extracts are obtained following step A- 9. Such a step can be indicated as step A- 10.

[0056] According to a preferred aspect, the recovery of solvent is included in at least one step between steps A-l and A-9. Preferably the solvent recovery is included at least in steps A- 3 and / or A-5.

[0057] In addition to the above, the following preferred aspects can be considered, in accordance with further preferred embodiments, in which said preferred aspects can be included / used in said extraction process of the invention.

[0058] Preferably, a spray dryer can be used to dry the nano-emulsions to obtain a solid substance intended to be packaged in capsules (pharmaceutical form). According to another preferred aspect, a capsule packaging line (mixer, granulator, capsule filler, piece counter, blistering machine, cartoner) can be provided. Preferably, a line for the stick-pack packaging of the extracts in the form of stable emulsions is provided. Preferably a fluid bed drying system is included for dehydrating the fibrous material resulting from the second extraction stage after the removal of ethanol (or hydroalcoholic mixtures). The extraction stage, as reported above, can be preceded by a preliminary extraction preparation stage. According to a preferred aspect, such a preliminary stage comprises the following steps:

[0059] • selecting and washing plant material;

[0060] • drying the cleaned plant material, preferably by means of a ventilated dryer at a programmed temperature;

[0061] • storing by means of vacuum bagging and sealing in appropriate bags, preferably of opaque material, not passable by light, and / or non-breathable.

[0062] The selecting and washing step advantageously allows to remove foreign material, so as to obtain a compatible material from a food perspective (i.e., a clean material without foreign residues of various kinds).

[0063] The process according to the present invention advantageously includes the use of innovative integrated equipment for producing micro / nanoparticles multi-responsive to different physical and chemical stimuli (temperature, pH) in order to deliver the active ingredients extracted from the plant matrices considered. Moreover, the process according to the invention can be considered an integrated extraction system with solvent recovery routes, in particular water and ethanol, ensuring the eco-compatibility and eco-sustainability of the entire process carried out through the system itself.

[0064] Another advantage of the invention is that the latter refers to the combination of different technologies organized in sequence.

[0065] With particular reference to drug delivery systems, such systems deliver drugs or bioactive components in a targeted and controlled manner within the body. These systems are particularly useful for lipophilic components (vegetable oils, tocopherols, carotenoids, Ozoile® as ozonized oil), which are substances characterized by a low solubility in water but a greater affinity for lipids and fatty tissues. The delivery of such components requires specific solutions to ensure the effectiveness, stability and controlled release thereof.

[0066] The extracts obtained according to the extraction process in hand can advantageously and effectively be incorporated into vehicles such as alginate-based microspheres, microcapsules obtained by encapsulation with clays or nano-emulsions, which allow transporting or delivering the substances of high biological value extracted / recovered from the various plant matrices considered through the process of the invention. Practical applications can, therefore, be found in the delivery of active ingredients by developing innovative drug deliveries. In such a context, the advantages of using microspheres or micro- and / or nanoparticles for the delivery of the active ingredients are multiple, including gastroprotection of the compounds, the formation of special retard forms and the possibility of making the micro-nanoparticles multi-responsive to specific stimuli.

[0067] Further aspects, embodiments, features and advantages will result from the embodiments set forth in the following description.

[0068] Moreover, the invention is also described in the figures and claims, the definitions of which form an integral part of the present description.

[0069] Figure 1 is a schematic view of the system according to the process of the invention which allows obtaining a dry extract. Specifically, the system of the figure comprises in particular a first, a second and a third turboemulsifier with rotating blades, followed by a first, a second and a third decanter, respectively.

[0070] Figure 2 shows a representative diagram of the electrospray technique starting from stable microemulsions obtained with high intensity ultrasound, which allows various types of micro spheres.

[0071] Figure 3 shows a microscope image of microspheres obtained with the optimized process of the invention.

[0072] Figure 4 shows the type of reactor for preparing microspheres.

[0073] For the purposes of the invention, definitions of certain terms used in the present description and the appended claims are given below.

[0074] In the present text, the term "bioactive" compound or ingredient is a biologically active compound, which is capable of imparting a beneficial effect on the health of organisms or living beings, including humans. In the present text, such a term can alternatively be referred to as "bioactive agent" or simply "compound" or "agent" or "active ingredient".

[0075] A system according to the process of the present invention shown, according to an embodiment in figure 1, comprises, inter alia, a rotating high capacity turboemulsifier, an in-line homogenizer with inverter for speed management between 300 and 3000 rpm, a first decanter centrifuge for solid-liquid separations, a second turboemulsifier for solvent extraction, preferably green solvent, more preferably pure ethanol or solvent water mixtures, preferably water / water, a second centrifugal decanter, a turboemulsion extractor, alginate microencapsulation generator, clay microencapsulation system, high pressure homogenizer for generating nano-emulsions. Moreover, a spry-dryer tool will be used for the solid reduction of the aforesaid nano-emulsions, to facilitate the formation of capsule-like pharmaceutical forms.

[0076] A first step of the process of the invention relates to crushing the plant material. This is carried out using a turboemulsifier with high rotating capacity blades, which allows a first and important cell rupture. The feature of high rotating capacity is linked to a greater efficiency of the crushing operation. For example, the cell membranes can be disrupted by shear stress applied to the liquid. As understood in the present text, blade rotation speed means a speed preferably between 1500 and 2200 rotations per minute (rpm), more preferably between 1800 and 2100 rpm.

[0077] According to a preferred aspect, in the initial extraction, as well as for the subsequent ones, the matrix / solvent ratio is variable within the range between 1:10 and 1:20. Therefore, by way of non-limiting example, 10 to 20 liters of solvent will be added to 1 kg of matrix depending on the features of the starting matrix (degree of moisture, part of the plant, hardness).

[0078] Following the first crushing step carried out with a turboemulsifier preferably using water as the extraction solvent, a second crushing / extraction treatment with water is included, by means of an in-line homogenizer with inverter (for speed management between 300 and 3000 rpm), which will advantageously allow the plant tissue to be completely crushed, ensuring cell rupture and preparing the plant matrix for the subsequent extraction stages.

[0079] Solvent recovery systems can also be used in the process according to the invention. Moreover, in the process according to the invention, the solvent used for the extraction carried out by means of the second turboemulsifier is ethanol or ethanol / water mixtures. Depending on the quality and physical-chemical features of the bioactive compounds to be recovered, the solvent system can also consist of ethanol / water mixtures at a different volume ratio v / v (90 / 10; 80 / 20; 70 / 30; 50 / 50; 25 / 75).

[0080] As reported above, an aspect of the present invention relates to a process of extracting bioactive compounds from plant matrices and for producing production of micro- and / or nanoparticles containing them.

[0081] As for step A-l of crushing / extracting the plant material, at this point of the extraction procedure according to the process of the invention, using a rotating blade turboemulsifier and an aqueous solvent, the mineral, simple carbohydrate, protein, amino acid substances, part of the fibrous substances and organic acids and water-soluble vitamins are solubilized. Preferably, such a solvent is demineralized water. At the end of this first step, which will have a variable duration from 2 to 10 minutes, depending on the consistency of the original matrix (leaves, flowers, inflorescences, twigs, stems, roots, bark, rhizomes), at high blade rotation speed, preferably 1800-2200 rpm, the suspension will be transferred, by means of a special connection, to an inline homogenizer (with inverter for speed management preferably between 300 and 3000 rpm) which will complete the disintegration stage, generating a highly homogeneous product with a particulate consisting of particles with an average size of 10-100 microns.

[0082] Specifically, as for this in-line homogenization step A-2 with aqueous solvent, it is carried out with special machinery capable of working with 1-10 m3 / h, with turbine speed up to 23 mt / sec.

[0083] Step A- 3 begins at the end of step A-2, in particular, the highly homogeneous suspension can be conveyed to a decanter centrifuge to remove the water (sent to a collection vessel) containing moisture within 15%, while the residue will be transferred to the next mixer.

[0084] The aqueous liquid part rich in mineral, simple carbohydrate, protein, amino acid substances, part of the fibrous ones and organic acids and water-soluble vitamins, is removed and collected in a steel vessel, with a high flow rate, from which it will be pushed towards a filtration / reverse osmosis system for water purification, which will thus be recovered for at least 80% and will therefore be reusable for a subsequent extraction cycle.

[0085] The residual bulk, deprived of all substances with a strong polar character, but still containing most of the precious phenolic substances and all of the fat-soluble bioactive substances, such as carotenoids and tocopherols, will then be subjected to the next extraction stage.

[0086] As for step A-4, the extraction occurs with alcoholic or hydroalcoholic solvent phase or solvent. Preferably the solvent is a water-soluble polar solvent, more preferably ethanol or a mixture of ethanol and water. Preferably, when the solvent is a mixture of ethanol and water, said mixture has a v / v ratio from 90 / 10 to 25 / 75, more preferably 90 / 10; 80 / 20; 70 / 30, 50 / 50 or 25 / 75.

[0087] In particular, the residual bulk from the decanter contains most of the bioactive substances with a moderately polar character, mainly polyphenols, and apolar character, mainly carotenoids and tocopherols. The mixer which can be used in this step can be the same as in step A-l, based on the homogenization of the residue. The extraction can be prolonged for a time from 2 to 10, preferably between 3 and 7 minutes. Preferably, the speed of the rotating blades is set to a maximum speed, more preferably from 1800 to 2200 rpm.

[0088] The suspension obtained can then be transferred to a subsequent decanter centrifuge capable of separating the solvent phase from the fibrous residue. The solvent phase can be collected in a high capacity steel vessel, containing most of the phenolic substances and moderately polar compounds with high bioactivity, for example, xanthophylls and chlorophylls. Such a solution can be concentrated and then dried by lyophilization, forming a mixture of substances of high biological and nutraceutical value. The composition of such a dry extract will depend on the initial matrix. For example, if starting from a leafy plant matrix such as olive leaves or the like, in this step there will be dry extract rich in polyphenols and chlorophylls, while if the starting matrix is tomato or tomato derivatives or processing by-products thereof (skins, concentrates, etc.), there will be an extract rich in phenolic and xanthophyll substances (mainly lutein). Preferably, the alcoholic or hydroalcoholic phase is subjected to a distillation process in a special turret for the recovery of the alcohol. The recovered alcohol can thus be advantageously reused in subsequent extractions.

[0089] Depending on the starting matrix, the residual bulk at this level will have a different composition, for example it will consist of dietary fiber (usable in the nutraceutical field or for feed) if starting from leaves, or if starting from tomato and by-products thereof or from other berries or red fruits (e.g., goji, watermelon), the residue will consist of lycopene-rich fiber, usable as such as a nutraceutical or further extractable for the recovery of highly apolar carotenoids. If the desire is the formation of a product rich in dietary fibers (especially insoluble) still containing a wide range of bioactive compounds (e.g., polyphenols, carotenoids), at this point of the procedure a fluid bed drying system can be used, which is capable of dehydrating the aforesaid material into powder and automatically distilling the amount of alcohol present in the matrix, in order to recover the precious solvent for the subsequent extraction cycles.

[0090] Preferably, as for step A-6 of extracting apolar substances, the extraction is carried out by using edible oil (preferably extra virgin olive oil +Oil® from Erbagil Tenuta).

[0091] When treating plant matrices such as tomatoes, goji berries, watermelon, rich in apolar substances such as carotenoids and / or tocopherols (etc.), a final extraction must be carried out for the recovery thereof. In such a sense, the previous extractions will serve to prepare the matrix for the final extraction, simplifying the initial matrix and concentrating it in compounds of an apolar nature (carotenoids and tocopherols). To this end, the procedure in hand can advantageously include the following procedure: use of a turboemulsifier identical to the first two already used previously and the extraction occurs with EVO +Oil (or alternatively other edible oils such as sunflower oil, corn oil, coconut oil, etc.), for a time varying between 2 and 10 minutes under cold vacuum.

[0092] Step A-7. The suspension consisting of oil and fibers can then be transferred to a further centrifugal decanter, preferably of the same type as those previously used, for the separation of the oily liquid component rich in carotenoids from the solid one of fibrous origin: the oil, collected in a high capacity steel container, will acquire a much redder color the more the matrix is rich in carotenoids and will form a functional ingredient for the formulation of high added value nutraceuticals. The final fibrous substance can be used to form feeds of high nutraceutical value. The advantage immediately inferable from this procedure is that a solvent free extract rich in carotenoids and apolar substances will be obtained, eliminating upstream any problem related to food safety which can derive from the use of solvents which are dangerous to health, although effective for extraction purposes.

[0093] As for the ozonation step A-8 of the extracts obtained following the bulk extraction, preferably by means of oil, more preferably Evo +Oil®, an oil rich in apolar substances, in particular carotenoids and tocopherols, is thus obtained, which give an intense reddish-orange color to the oily matrix. Such oil can be subjected to an ozonation process to further functionalize such a matrix: in fact, the oily matrix enriched with carotenoid ingredients offers a considerable possibility of ozone addition by providing an exceptional amount of double bonds ensured not only by the oil, preferably EVO oil, but also by the classic structure of C40 carotenoids (up to 11 conjugated double bonds and 13 total for lycopene). This will allow the formation of significant amounts of ozonides, advantageously ensuring a matrix with exceptional oxygen-carrying capacity. This can be advantageously exploited and used both for modern and innovative medical devices and other specific fields, such as the pharmaceutical, nutraceutical and cosmeceutical sectors.

[0094] As for step A-9 of micro - / nano structuring the extracts, the purpose is to obtain an effective method of delivering the active ingredients extracted according to the procedure in hand.

[0095] According to a preferred aspect, the micro- and / or nanoparticles obtained by the process of the invention are used in the delivery of active ingredients. Preferably, the forms of delivery considered in the procedure in hand are nano-emulsions, alginate-based microspheres and microcapsules obtained by encapsulation with clays. Such forms of delivery are advantageously obtained so as to transport or deliver substances of high biological value recovered from the various plant matrices considered through the process of the invention. Therefore, the three possible vehicles are all suitable for the application of active ingredients as a drug delivery. As anticipated above, such a delivery has several advantages: the importance of the gastroprotection of the compounds, which ensures non-alterability and / or chemical degradation by acid attack at the stomach level, the formation of special retard forms which promote a delayed and prolonged release of the bioactive ingredients over time, the possibility of making the micro-nanoparticles multi-responsive to specific stimuli, such as pH and temperature. Whereby, at the end of the extraction process, three instruments can be placed side by side for producing:

[0096] - nano-emulsions;

[0097] - alginate-based micro spheres;

[0098] - microencapsulation with clays.

[0099] According to a preferred aspect, the nano-emulsions are obtained by homogenization. In particular, such nano-emulsions can be produced by homogenization technique capable of forming an oil-in-water emulsion of nanometric dimensions in which the extracted substances will be encapsulated. By virtue of this process, the apolar substances will acquire a more polar character, becoming soluble in water, which means that they can be transferred into the bloodstream and reach the various cell populations of the human body, promoting beneficial health effects. Advantageously, the preparation process contemplated for the formation of nanoemulsions allows obtaining very stable products starting from naturally derived materials of plant origin. A further advantage is that, by virtue of the particular low-temperature process and the coating with chitosan, the stability lasts for more than one year (2-5 years). Moreover, by virtue of the coating with chitosan, the product is gastro-resistant, ensuring an adequate release in the intestine resulting in an increase in the absorption capacity of the active ingredients. A further particularly relevant advantage is that by significantly improving the bio availability of the bioactive ingredients, they can be administered at a lower dosage, since the rate of absorption and metabolization is significantly increased. Therefore, food supplements based on nanoemulsions are much more effective and this, from a commercial point of view, means that production also becomes cheaper by virtue of the lower amount of active ingredients needed.

[0100] Alginates are a family of polysaccharides derived from various species of brown algae or Phaeophyceae, characterized by a wide range of chemical composition, molecular weight and functional properties. Chemically, alginates are unbranched linear copolymers of -D- mannuronic acid and the C-5 epimer a-L-guluronic acid thereof. They form a valid support for the formation of microspheres in which the extracted plant material is micro-dispersed. According to a preferred aspect, the microspheres are obtained by micro-dispersion of the extracted plant material. Alginates are commonly used in the delivery of natural active ingredients of different origin and pharmaceutical molecules derived from synthetic processes. Also in this case, the delivery ensures an improvement in the bioavailability of the biologically active ingredients with the above advantages. According to a preferred aspect of the invention, the vehicle is obtained by microencapsulation of the bioactive compound or ingredient with clays. Preferably, the clay is halloysite. Such a vehicle is advantageously characterized by high biocompatibility. The advantages are the same as those described for the other two forms of delivery.

[0101] As for the polymers used in the context of the present invention, a detail is given below on some preferred polymers, preferably natural polymers, which can be used for the design of micro spheres.

[0102] According to a preferred aspect, the polymer is a natural polymer. Preferably the natural polymer is at least one selected from alginate or chitosan. Alginate is a natural polymer extracted from seaweed. It can form hydrogels with biocompatible and biodegradable properties, useful for the delivery of lipophilic components. Chitosan is a natural polymer derived from the deacetylation of chitin, which can be used for the delivery of lipophilic components, in particular for mucosal or topical drug delivery. Gelatin can also be used. Gelatin is a natural protein derived from collagen, used for the delivery of lipophilic drugs in different pharmaceutical forms.

[0103] With particular reference to pH-responsive natural and biodegradable polymers, such as alginate and chitosan, they are widely used for drug administration applications, especially in the form of biocompatible microspheres. Moreover, thermo-responsive poly (N-isopropyl- acrylamide) (PNIPAM) can be added to biomedical administration systems by virtue of the lower critical solution temperature thereof, which is close to physiological conditions.

[0104] Advantageously, by virtue of the process of the invention it will be possible to obtain "smart" hydrogel microspheres, which are capable of responding to physiological stimuli such as temperature and pH variations, for the therapeutic release of active ingredients in specific districts of the human body.

[0105] In particular, hydrogels can be used, as they offer multiple advantages:

[0106] - formulation versatility: hydrogels can be designed and formulated with a wide range of synthetic and natural polymers, allowing a specific customization for the type of drug or bioactive component to be delivered. This offers flexibility in modulating the properties of the gel, such as cross-linking density, release rate and loading capacity of lipophilic drugs;

[0107] - biocompatibility and biodegradability: hydrogels are well tolerated by the body without causing significant adverse reactions. Moreover, many hydrogels can be designed to be biodegradable, reducing the risk of long-term side effects; - controlled release: hydrogels allow a controlled and gradual release of lipophilic components over time. The ability thereof to trap the active ingredients within the gelatinous matrix thereof prevents sudden releases, ensuring greater stability and a predictable release profile;

[0108] - drug protection: hydrogels can offer physical and chemical protection to the drugs carried. In particular, lipophilic components, which can be susceptible to enzymatic degradation or oxidation, can be protected from the surrounding environment until release at the site of action;

[0109] - specific targeting: hydrogels can be functionalized with specific targeting molecules, such as antibodies or ligands, which direct the drug to the desired site of action. This targeted targeting ability increases treatment effectiveness and reduces the risk of undesired side effects;

[0110] - possibility of non-invasive administration: hydrogels can be administered in different forms, including preformed gels, creams, ointments and hydrogels, which can simplify the non-invasive administration of drugs. This is particularly advantageous for the treatment of skin or eye diseases, where a direct administration to the affected site can be preferable;

[0111] - reduced dosing frequency: by virtue of the controlled and prolonged release of drugs, hydrogels can allow a reduction in the administration frequency, improving the patient's adherence to treatment and increasing overall therapeutic efficacy.

[0112] In particular, according to a preferred aspect, through the process of the invention, it is possible to develop two controlled release systems for lipid substances: a first system is the "pH- responsive" system based on sodium alginate; a second system is the "thermo-pH-responsive" system based on sodium alginate and PNIPAM (Poly(N-isopropylacrylamide).

[0113] For both types of microspheres, the electrospray technique is used starting from stable microemulsions obtained with high intensity ultrasound (Figure 2).

[0114] In particular, pH-responsive micro spheres can be obtained, optimizing the process parameters (collection distance, CaCh concentration, flow rate, applied voltage, needle type).

[0115] Advantageously, micro / nano-encapsulation techniques with clay minerals can be used.

[0116] Clay minerals are natural materials, of low cost and easily available in large amounts and in some cases with a high degree of purity (halloysite and sepiolite). In fact, the clays ensure a controlled and sustained release of the loaded active ingredients over time. Moreover, they have the ability to penetrate cell membranes and are therefore useful for the delivery of hydrophobic molecules in biological fluids.

[0117] Micro / nano-encapsulation with clay minerals on a plant matrix, as well as various kinds of organic molecules, can be carried out according to a process which includes the following steps:

[0118] - dispersion of the clays in an aqueous environment and insertion of a concentrated solution of the plant matrix in the solvent in which it is soluble (e.g., on a laboratory scale, 100 mg of clay are placed in 10 mL of water to which the plant matrix is added in a variable matrix / clay ratio, e.g., 1:1, 2.5:1, 5:1, 7.5:1, 10:1);

[0119] - stirring the dispersion obtained at room temperature for one night;

[0120] - centrifugation and washing the solid obtained with water to remove any physically absorbed components (alternatively, a filtration and filter washing system can be used);

[0121] - lyophilization of the solid product obtained.

[0122] The amount of plant matrix adsorbed on the clays is determined by thermogravimetric analysis and qualitatively evaluated by FT-IR, which ensures compliance with the expected result.

[0123] The loading is dependent on the nature and morphology of the clay used (halloysite, hectorite, palygorskite, sepiolite and bentonite), preferably halloysite which advantageously allows obtaining nanomaterials with an organic portion loading maximum of 10% w / w, but also hectorite and bentonite which allow a loading of approximately 50%, and palygorskite and sepiolite which allow loadings of approximately 70% w / w.

[0124] One of the great advantages of this encapsulation technique is that the non-interacting plant matrix can be easily recovered and reused for further adsorption cycles, while respecting the concept of sustainability. The possibility of industrial scale-up is advantageously simple since it is enough to use the starting reagents proportionally to the amount of product to be prepared.

[0125] The stages of dispersion, stirring, centrifugation, washing, lyophilization are easily obtainable by means of reaction tanks, mechanical stirrers, industrial centrifuges, and lyophilization for large volumes.

[0126] According to a preferred aspect, with reference to the process of preparing the nanoemulsions, this includes formations of oil-in-water emulsions using, in such a procedure, plant material. Such emulsions are particularly stable (duration 3-5 years) since a multi-dispersion is exerted and finally a thickening is promoted using a polymeric coating, in this case chitosan, around the oil nano-droplets (the edible oils used in the previous stages as solvents). All this occurs inside a high-pressure homogenizer. The result is the formation of particularly stable nano-emulsions with an average diameter of 50-200 nm. The procedure respects the structure and chemical-physical features of the treated molecules since it does not require high temperatures (within 40°C) and is usable to encapsulate lipophilic molecules of various nature and origin such as lipids, carotenoids, xanthophylls, tocopherols, sterols, fat-soluble vitamins, etc. One of the advantages of this procedure is that, in addition to producing extremely stable nano-emulsions, since the resulting product boasts a chitosan coating, it has gastro-resistant properties. Moreover, chitosan naturally has the ability to incorporate liquids by swelling, this produces an increase in retention in the intestine resulting in an increase in absorption capacity.

[0127] According to a preferred aspect, with reference to the production process of solid extracts, this is carried out after the formation of nano-emulsions, derived from high pressure homogenizer applications on the final oily extract to which the appropriate amount of water for the preparation of the preliminary emulsions must be added. Such a nano structured form, including natural material such as chitosan or the like as a solid support, can be dehydrated and reduced to a solid with a high percentage (30-50%) of oil, by using a spray-dryer.

[0128] The main advantage of this technique is that the solid can be easily dosed for the preparation of simple and versatile pharmaceutical forms such as capsules.

[0129] Another notable advantage is to be able to associate fat-soluble active ingredients with those of a water-soluble nature; in fact, the aqueous phase necessary to form the preliminary emulsions can be previously enriched with water-soluble ingredients, while the fat-soluble ingredients can be dissolved in the oily phase. After dehydration and the consequent collapse of the oily phase on the solid support, the oily phase itself will be incorporated into the solid with the final formation of the solid material to be used for preparing the capsules.

[0130] The following examples are intended to further illustrate certain embodiments of the present invention and are therefore not to be construed as limiting thereof.

[0131] EXAMPLES

[0132] Crushing plant material

[0133] Using a turboemulsifier with rotating blades and 10 liters of demineralized water as solvent, 1 kg of double tomato concentrate was treated for 5 minutes, at a blade rotation speed of 2200 g / min so as to solubilize the mineral, simple carbohydrate, protein, amino acid substances, part of the fibrous ones, organic acids and water-soluble vitamins. The process is carried out under vacuum by means of a liquid loop vacuum pump with water supply opening / c losing solenoid valve and provided with a one-way safety valve on the suction. The vacuum was programmed up to reaching the value of 700 mm / Hg. During the extraction stage, the temperature was maintained within 25 °C, ensured by a cooling jacket lining the extractor and continuously detected by a PT100 probe.

[0134] Treatment with in-line homogenizer

[0135] The suspension thus obtained was then transferred, by means of a special tube placed on the bottom of the extractor, to an in-line homogenizer. The tube being connected to a suction pump ensures the removal of the suspension from the first extractor and the delivery to the second extractor which has the task of completing the cell rupture stage and preparing the matrix for the subsequent extraction stages. From the in-line homogenizer, the suspension was then delivered to a decanter centrifuge for the removal of water, using the centrifugal force of the instrument so as to settle the moisture content between 10 and 20%, preferably 15%, while the residue was transferred to the next mixer. The water (rich in the polar substances listed above), which can be calculated in volume between 80 and 90% of the initial volume, is recovered and collected in a steel vessel, at a high flow rate, and pushed towards a filtration / reverse osmosis system to be purified and then reused for the next extraction cycle.

[0136] Extraction with Ethanol

[0137] The residual bulk from the decanter containing most of the bioactive substances with a moderately polar character (mainly polyphenols) and apolar character (mainly carotenoids and tocopherols) was treated with the same type of mixer used in the first step and based on the homogenization of the residue, using ethanol as a solvent or alternatively with mixtures of ethanol and water (90 / 10; 80 / 20; 70 / 30; 50 / 50; 25 / 75 v / v) in order to extract bioactive substances with different degrees of polarity in a timely manner. The extraction was carried out for 5 minutes with the maximum speed of the rotating blades (2200 g / min), after which the suspension was transferred to a subsequent decanter centrifuge capable of separating the ethanol (or ethanol / aqueous) phase from the fibrous residue. The ethanol (or ethanol / aqueous) phase was collected in a high capacity steel vessel, said phase containing most of the phenolic substances and moderately polar compounds with high bioactivity (i.e., xanthophylls and chlorophylls). Such a solution was concentrated, with recovery of the solvent by distillation of the alcohol on an ad hoc distillation turret, and then dried by lyophilization, forming a mixture of substances of high biological and nutraceutical value (luteins and other xanthophylls).

[0138] Extraction of apolar substances Using a turboemulsifier identical to those already used previously, the extraction occurred with Evo +Oil® for 10 minutes under cold vacuum. The oil and fiber suspension was then transferred to a further decanter centrifuge for oil / fiber separation.

[0139] Ozonation process

[0140] Following the bulk extraction by means of EVO oil, an oil rich in apolar substances (carotenoids and tocopherols) was obtained, all also confirmed by the intense red-orange color given to the oily matrix. The oil was subjected to an ozonation process to further functionalize the matrix, resulting in the formation of ozonides. The ozonation process (Erbagil s.r.l. patent) was applied in order to obtain a degree of ozonation corresponding to the addition of ozone to 6% of the total double bonds of the unsaturated fatty acids (mainly oleic acid) forming the triacylglycerols of the oil. In fact, such an amount gives the possibility of not altering the fundamental quality parameters of the EVO and allows preserving all the remarkable properties thereof. To all such very important properties, those deriving from the ozonation process will be added, i.e., those ascribed to ozonides, endoperoxide structures that represent the main reaction products between unsaturated fatty acids and ozone. A further advantage of the ozonation process is given by the microbiological stability deriving from such a process, already known as sterilization technique applied to food.

[0141] Micro / nano-structuring of extracts

[0142] Nano-emulsions were produced by homogenization technique, forming a nanometricsized oil-in-water emulsion in which the bioactive compounds previously extracted in chitosan coatings were encapsulated.

[0143] A process and related system of extracting bioactive compounds or ingredients from plant matrices and vehicles containing them has been described.

[0144] Controlled release systems of lipid substances:

[0145] - pH-responsive system based on sodium alginate;

[0146] - thermo-pH-responsive system based on sodium alginate and PNIPAM.

[0147] For both types of microspheres, the electrospray technique is used starting from stable microemulsions obtained with high intensity ultrasound (Figure 2). pH-responsive microspheres were obtained by optimizing the process parameters (collection distance, CaCh concentration, flow-rate, applied voltage, needle type). After such a setting, the lipophilic ingredients, oil type, ozonated oil, different apolar substances (10-50% by weight), Tween 80 (5% by weight) and xanthan gum (0.25% by weight) were added to a sodium alginate solution (1.8% wt%), obtaining a stable emulsion using an ultrasonic homogenizer. On a laboratory scale, the microspheres were then prepared by electrospray: the emulsion was loaded into a syringe with a constant extrusion rate of 20 mL / min from a collection distance of 10 cm. A voltage of 30 kV was applied between the needle (24 G) and the CaC12 gelling bath (20% w / v). The cross-linking time was set to 30 minutes, followed by three 3 rinses of the MS in deionized water. The chitosan coating was applied by vortexing the microspheres at 700 rpm in a 0.5% by weight chitosan solution for 30 minutes. As a result of the analysis carried out, the particularly preferred parameters for producing microspheres are: a voltage of 30 kV, a flow rate of 20 mL / h, a 24 G needle, a concentration of 20% by weight of apolar active ingredients, and a coating obtained at 700 rpm. In fact, high rpm leads to a greater penetration of chitosan in the polymer mesh resulting in the partial collapse of the complex. Chitosan-coated microspheres have a higher water content, and this can be attributed to the hydrophilic nature of the polymer. The degradation of the polymer mesh, resulting in the release of apolar oily substances, occurs at pH values > 7.4.

[0148] Moreover, thermo-pH-responsive microspheres were obtained. Sodium alginate and PNIPAM were mixed in deionized water for 30 minutes in a cold water bath in a 1:1 ratio (w / w). The oily substances, ozonated or not (10-50% by weight), the non- ionic surfactant Tween 80 (5% w / v) and the emulsifier xanthan gum (0.25% w / v) were then added to obtain a stable emulsion with the high intensity ultrasound (HIU) method. The process was carried out using the ultrasonic homogenizer for 2.5 minutes at 100% power. The microemulsion is placed in a syringe and pushed into a stainless steel needle (inner diameter 0.311 mm) using an infusion pump with a constant rate of 20 mL / min. The spinneret is connected to a 30 kV high voltage power supply. The drops formed on the tip of the spinneret detach and fall into the cross-linking bath below. The collection distance was set to 10 cm. All the experiments were conducted at a temperature of 25°C and a humidity of not more than 50%.

[0149] From the results obtained in the laboratory, and in particular from the morphological analysis of the samples, it emerged that all types of microspheres have an average diameter of less than 450 pm, for such a reason they can be considered for all purposes as microspheres (Figure 2). From the evaluation of the volumetric transition peaks, the thermo-responsive nature of PNIPAM was confirmed. Based on the results obtained, the best microspheres from both a morphological and thermal response perspective are those with an oil concentration of 30% by weight. Therefore, on this type of microspheres it can be said that they are capable of delivering and releasing the incorporated active ingredient for pH values > 6 at a temperature of 37°C. The laboratory- scale study made it possible to set the best process parameters to obtain multi- responsive microspheres (Table 1), and such a process was transferred to large-scale production using a reactor such as the one shown in Figure 4, for producing microspheres with the aforesaid features. Table 1. Optimization of parameters for micro sphere formation

[0150] The above is to be understood by way of non-limiting example. Moreover, those skilled in the art will be able to understand that changes may be made without departing from the scope of the present invention.

Claims

AMENDED CLAIMS received by the International Bureau on 14 March 2025 (14.03.2025)1. A process of extracting bioactive compounds from plant matrices and for producing micro- and / or nanoparticles containing them, comprising the steps of:A- 1) crushing the plant material with water by means of a first turboemulsifier, preferably under5 vacuum, with rotating blades at a speed between 1500 and 2200 rpm, preferably between 1800 and 2100 rpm;A-2) in-line homogenization by means of an in-line homogenizer for further crushing;A-3) solid-liquid separation with a decanter centrifuge;A-4) extraction of the polar and / or moderately polar substances from the residual bulk10 remaining after A-3 with alcoholic solvent, preferably ethanol, or hydroalcoholic solvent or an alcohol / water mixture, using a second turboemulsifier;A-5) solid-liquid separation with a second decanter centrifuge;A-6) extraction of apolar substances from the residual bulk remaining after A-5 with edible oil using a third turboemulsifier;15 A-7) solid-liquid separation by third centrifugal decanter to obtain extracts;A-8) ozonation of the apolar extracts obtained in A-7;A-9) micro / nano structuring the extracts obtained from step A-8 by means of an alginate microencapsulation generator, a clay micro-encapsulation system, or a nano-emulsion generator.

2. A process according to claim 1, wherein said first turboemulsifier provided with blades is a20 vacuum turboemulsifier provided with blades.

3. A process according to any one of the preceding claims, wherein the in-line homogenization step A-2 for further crushing occurs at a speed between 300 and 3000 rpm.

4. A process according claim 3, wherein in step A2 the use of an inverter is included.

5. A process according to any one of the preceding claims, wherein in step A-4 the alcoholic25 solvent is ethanol.

6. A process according to any one of claims 1-4, wherein in step A-4 the alcohol / water mixture is at a volumetric ratio from 90: 10 to 25:75.

7. A process according to claim 6, wherein the alcohol / water mixture is the ethanol / water mixture.

8. A process according to any one of the preceding claims, wherein at least one step between steps30 A- 1 and A-9 includes the recovery of solvent.

9. A process according to claim 8, wherein the solvent recovery occurs in steps A-3 and / or A-5.

10. A process according to any one of the preceding claims, wherein in step A-9 one or more solid extracts are obtained.

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