Multiphase extraction system (M.E.S.) for extracting biologically active compounds from plant substrates using combined extraction techniques and vehicles containing same

Abstract

The present invention relates to the technical field related to extracts of natural substances, in particular plant products, where such extracts are biologically active compounds or agents, which can be intelligently delivered for transport and release, such as in nutritional and pharmaceutical applications. In particular, the present invention relates to methods for extracting bioactive compounds from a plant matrix and for preparing micro-and / or nano-particles containing them. The method according to the invention advantageously represents a novel integrated device for producing micro-and / or nano-particles functionalized for specific targets, delivering active ingredients extracted from plant substrates. Furthermore, since such microparticles and / or nanoparticles may comprise bioactive compounds or ingredients, the present invention relates to methods of producing microparticles and / or nanoparticles, as well as such microparticles and / or nanoparticles comprising bioactive compounds or ingredients. Thus, the extracted and obtained bioactive compounds or ingredients can be advantageously and effectively incorporated into vehicles such as nanoemulsions, alginate-based microspheres or clays, which allow for the transport or delivery of high biological value substances extracted / recovered from various plant substrates using the methods of the invention. Practical applications can advantageously be found in the delivery of active ingredients ("drug delivery"). Advantages of using this structural form are multiple, including gastric protection of biologically active compounds or ingredients, formation of special delayed forms, and the likelihood of multiple response of micro-nanoparticles to specific stimuli.

Description

Technical Field

[0001] This invention relates to the technical field of natural substances, particularly extracts of plant products, wherein such extracts consist of bioactive compounds or agents, and delivery systems for their transport and release, for example in nutritional and pharmaceutical applications.

[0002] Specifically, the present invention relates to a method for extracting bioactive compounds from plant matrices and using them to produce micron and / or nanoparticles containing them. Furthermore, the present invention relates to a method for preparing micron and / or nanoparticles, and such micron and / or nanoparticles containing bioactive compounds or components. Background Technology

[0003] As is well known, so-called "bioactive" compounds, agents, or ingredients are bioactive compounds that can impart beneficial health effects to organisms (including humans, in particular). Polyphenolic compounds and carotenoids can, of course, be mentioned as non-limiting examples.

[0004] The use of natural products for the prevention and treatment of various diseases is expanding worldwide; this is especially true for polyphenols, which represent a large number of molecules with enormous potential for beneficial health effects. These substances are widely found in several key foods of the Mediterranean diet. Numerous studies have highlighted several pharmacological properties of these substances in the cardiovascular system. In particular, the Mediterranean diet, which includes a daily intake of 25-50 ml of extra virgin olive oil, is associated with reduced cardiovascular risk, neurodegenerative diseases, and some forms of cancer, especially colon cancer. Typically, the daily intake in the Mediterranean diet is estimated at about 1 g / day, and recent studies have also shown that chocolate, especially dark chocolate, is rich in these molecules.

[0005] Currently, the potential effects of polyphenols on the gastrointestinal tract are of particular interest. Specifically, a low incidence of colon cancer has been demonstrated in animal and human models. This effect appears to be related to how they interfere with various intracellular signaling mechanisms (NF-κB, MARK, etc.), regulating gene expression responsible for cell proliferation, apoptosis, detoxification enzyme expression, and the immune system. These effects influence numerous mechanisms of inflammatory processes, which are often the cause of tumor manifestations. The beneficial effects are attributed not only to this pharmacological action but also to their significant antioxidant capacity. Numerous studies have shown an imbalance between oxidants and antioxidants on the basis of the development of several gastrointestinal diseases characterized by ischemia, inflammation, or colon cancer. Vulnerable regions of these molecules manifest as poor absorption and unclear metabolism; on the other hand, the gastrointestinal tract is exposed to relatively high levels of this substance, thus the “local” beneficial effects on this apparatus can overcome absorption problems, with concentrations reaching very high levels (hundreds of μM). It is further known that synergistic effects may exist between the various components, and the potency of activity is reduced due to the use of a single component compared to polyphenol extracts.

[0006] Generally, daily and regular consumption of fruits and vegetables is known to have beneficial effects on, for example, combating the risk of developing chronic noncommunicable diseases (NCDs), and more generally, improving health and quality of life.

[0007] As widely reported and described in scientific literature, herbal extracts with high levels of phenolic and carotenoid compounds have high antioxidant, anti-inflammatory, and anti-proliferative activities.

[0008] In fact, fruits and vegetables, which are believed to typically contain significant amounts of antioxidant compounds, can have beneficial health effects, such as reducing the likelihood of triggering many chronic disease events by counteracting oxidative stress. These antioxidants are primarily found in the form of phenolic compounds, such as flavonoids, phenolic acids, stilbene, tocopherols, tocotrienols, ascorbic acid, carotene, and lutein. Their beneficial effects are attributed to their ability to donate electrons, scavenge free radicals, and exert their reducing power. However, they also possess anti-protein denaturation activity, which leads to damage to biological structures, and they also act on enzymes, particularly those inactivated by metabolic alterations. Furthermore, natural antioxidants have the ability to improve food quality and stability, and in nutritional supplements, they can act as compounds that interrupt free radical chain reactions in biological systems, thus providing additional benefits to human health.

[0009] Most important fruits and vegetables, such as tomatoes, goji berries, watermelon, papaya, mango, carrots, spinach, marigolds, sweet potatoes, and squash, are sources of carotenoids. Carotenoids are a group of molecules based on C-40 isoprene-like compounds, with over 600 representatives in nature, of which about 30 are important in our daily diet. This class of phytochemicals has recently attracted considerable attention due to the potential health benefits associated with carotenoid consumption. Several research groups have shown that the cis isomer of lycopene is absorbed better than the all-trans form due to its shorter length, higher solubility in mixed micelles, and / or lower tendency to aggregate. Carotenoid pigments are ubiquitous in nature, where they play a variety of roles. Synthesized de novo only in plants, algae, bacteria, and fungi, they are essential for photosynthesis, act as antioxidants, and are involved in the coloration of many animals and plants. Carotenoids cannot be synthesized by humans or animals, therefore their absorption depends on diet. Carotenoids and their derivatives have many functions essential for development, immune responses, and the visual cycle. Indeed, in animals, including humans, the conversion of pro-vitamin A carotenoids into the retina is essential for health, and hydroxycarotenoids (lutein, zeaxanthin, and meso-zeaxanthin) have the function of protecting the central retina from photo-oxidative damage. Numerous scientific evidences support their preventative role in a range of chronic or age-related diseases, partly but not solely related to their antioxidant properties. For example, carotenoids have been proposed to prevent the onset and proliferation of several types of cancer, including lung, prostate, and breast cancer.

[0010] New evidence from epidemiological studies, cell cultures, and animal studies suggests that certain carotenoids, such as lycopene, when consumed in the form of fruits and vegetables or processed foods, may help prevent cancer and cardiovascular disease, particularly stroke, high blood pressure, and prostate cancer, and may also have beneficial effects on health and bone density. There is considerable scientific evidence that edible plant species are of exotic origin. For example, traditional Chinese medicine has used compounds found in wolfberry fruit to prevent the onset and progression of cancer. Wolfberry also exhibits immunostimulatory activity. Compounds found in wolfberry have been confirmed in numerous studies to have pro-apoptotic and anti-proliferative activities against cancer cells.

[0011] Therefore, the aforementioned beneficial substances can be obtained / derived from what nature provides. Furthermore, zero-cost processing waste from the food industry (tomatoes, grapes, plant water from olive oil production, pomace) or agriculture (olive leaves, various pruning processes) can be used as a starting substrate. Considering the circular economy, ecological sustainability, and / or ecocompatibility, this possibility represents a significant “green” investment opportunity. For example, residual biomass from wine and tomato processing represents a very rich and varied source of bioactive compounds such as resveratrol, catechins, epicatechin, gallic acid, tartaric acid, proanthocyanidins, lycopene, lutein, zeaxanthin, and tocopherols, which have potential applications in various fields, including pharmaceuticals and nutrition.

[0012] Of particular interest is the growing interest, especially in recent years, in specific compounds that can be obtained from byproducts of the food industry, such as tomatoes and wine. The reasons can be found in two fundamental aspects: firstly, production companies can thus reduce the amount of waste generated, leading to lower disposal costs; and secondly, a reduction in environmental impact, the latter now being a growing concern for consumers.

[0013] Therefore, the possibility of having available methods that allow for the efficient and convenient recovery of processing by-products rich in high-value-added substances is increasingly strategic and relevant. Thus, it is important to develop methods that allow for the recovery of these bioactive compounds from processing by-products of relevant crops using innovative industrial approaches.

[0014] Against this backdrop, from both a scientific and production perspective, the stage of extracting active ingredients from plant sources presents a real challenge for companies in this field.

[0015] Traditionally, solid-liquid phase extraction (impregnation, percolation, perfusion, etc.) is used to obtain target compounds from various wastes.

[0016] Typically, this type of extraction technology requires large amounts of organic solvents and is very energy-intensive due to its long processing times and frequent high temperatures. On the other hand, supranational agencies (EFSA, FDA, EMA) do not permit the indiscriminate use of the organic solvents almost always required for this technology; in fact, many organic solvents are incompatible with food applications because they have negative impacts on human health and the environment.

[0017] Specifically concerning the extraction of active ingredients from plant sources, techniques exist to date for the extraction of bioactive compounds from nature. Some conventional techniques, such as impregnation, remain widely used due to their inexpensiveness and ease of application. However, these techniques have several drawbacks, including long extraction times that can vary from hours to days, even tens of days. These times are not always compatible with the production needs of the companies involved. Furthermore, this technique has another limitation: it is impossible to standardize the method and control all stages of extraction kinetics because a series of equilibrium, reaction, physical, chemical, and biochemical methods are established over the required long time, which are not always predictable and often introduce other problems particularly relevant to the final quality of the extract.

[0018] Typically, extraction processes are based on physical components such as permeation and diffusion, which ensure that the solvent penetrates into the plant matrix while simultaneously extracting bioactive compounds. Therefore, conventional extraction techniques such as impregnation and percolation, relying solely on diffusion and permeation phenomena, significantly slow down the extraction of substances contained within the plant matrix. In fact, they require considerable time to ensure complete extraction of the matrix and sometimes necessitate high temperatures, thus promoting oxidation / isomerization of the plant matrix, largely due to the failure to eliminate key issues such as contact with atmospheric oxygen, temperature control, and light.

[0019] This technique (although still in use) is typically and continuously being replaced at the industrial level by more modern extraction methods, which are essentially based on different physical, chemical, and mechanical components, such as the use of ultrasound, microwaves, supercritical fluids, and / or high pressure.

[0020] In all cases, the common goal of all extraction techniques is to ensure maximum extraction yield. To this end, the key and limiting step is represented by cell disruption. Indeed, bioactive compounds are separated within vacuolar structures such as chloroplasts and chromoplasts, exhibiting considerable resistance to physical and mechanical stress. Therefore, it is important to perform appropriate manipulations to disrupt the membranes and walls of plant cells, with the aim of optimizing extraction yield and ensuring excellent extraction from both quantitative and qualitative perspectives.

[0021] Among the active ingredient extraction technologies used in recent years to improve extraction yield, ultrasound-assisted extraction technology is certainly worth mentioning, as it precisely utilizes the mechanical energy of ultrasound. This relatively new technology can be defined as an unconventional extraction technique.

[0022] Ultrasonic-assisted extraction (ALE) technology is based on the use of ultrasonic, mechanical waves, which belong to a broad spectrum and conventionally begin at 20 kHz. This technique is based on the fact that when ultrasound is applied to a liquid, the waves propagate within it, creating a continuous alternation of compression and decompression. The very rapid sequence of compression and decompression cycles generates millions of microbubbles, called cavities, whose volume increases with each cycle. This phenomenon is known as…cavitation (cavitation), and has been shown to be used to induce the rupture of biofilms, thereby facilitating the extraction of bioactive components from plant cells.

[0023] To date, ultrasound has been used to accelerate and make extraction processes more efficient. The increased efficiency is primarily due to the following reasons: - Mass transfer enhancement: Ultrasound promotes the formation of microparticles, emulsions and solvent exchange around the plant material wall; - Cell rupture: Ultrasound can disrupt cell walls, promote the extraction of contents, and break down plant materials, increasing the contact surface with solvents; -Increased solvent permeation: When the cavity collapses, the resulting ultrasonic solvent jet forces the solvent into the cell, promoting its passage through the cell membrane; - Capillary action of ultrasound: Ultrasound improves capillary diffusion of solutes, especially polar and ionic solutes, through complex mechanisms, which are still under investigation and appear to involve the development of electric fields. - Reduced extraction of heavy components; - Significantly reduced bacterial load in the extract.

[0024] Microwaves are non-ionizing waves with electromagnetic properties, ranging from 300 MHz to 300 GHz. These waves lie in the electromagnetic spectrum between infrared and X-rays. Their direct effect on matter is the conversion of 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 the sample heating process. Furthermore, it is governed by two phenomena: dipole rotation and ion conduction. Dipole rotation refers to the realignment of molecular dipoles with a rapid change in the electric field; this allows dielectric materials and solvents with persistent dipoles to be heated by microwave action. Conversely, ion conduction refers to the transfer of ions caused by changes in the electric field. Thus, migration generates friction responsible for heating the solution due to the resistance provided by the solution. Extraction can be performed on both wet and dry matrices, utilizing trace amounts of moisture present in the sample being heated (e.g., water contained in biomass plant cells). The resulting evaporation creates extremely high pressure within the cells, ultimately leading to cell wall rupture and increasing the recovery yield of plant condensates in the culture medium. The disadvantages of this technology are plant cost, plant size, its management, extract quality, and the addition of (albeit in small amounts) solvents (which are often necessary to promote extraction).

[0025] The critical point of a fluid defines the end of its vapor-liquid coexistence curve. Regardless of the applied pressure, a fluid cannot pass through the liquid phase at temperatures above its critical temperature. Therefore, a fluid at any pressure and temperature above its critical point is called "supercritical." Supercritical liquids possess unique characteristics, exhibiting physical and chemical properties intermediate between those of gases and liquids. Due to its properties, CO2 is one of the most widely used supercritical fluids. It is an inexpensive, green solvent, eco-compatible, non-toxic, non-carcinogenic, and non-flammable, with a relatively easily achievable critical pressure (74 bar) and temperature (31 °C). Supercritical CO2 allows for selective extraction of target compounds by varying the temperature and pressure. CO2-assisted supercritical fluid extraction (SFE) has demonstrated high-quality extracts, with yields comparable to those recovered from organic solvents; furthermore, the addition of modifiers such as ethanol to CO2 increases its polarity and efficiency, particularly in the extraction of polar molecules such as phenolic compounds. However, this technique has several drawbacks, including high plant costs, its management, hazards, and low flexibility in defining different molecular targets for extraction (a co-solvent must be used to modify the polarity). Therefore, based on the above, a solution is needed that can overcome the aforementioned technical problems and related shortcomings. Invention Overview The method according to the appended claims, which involves extracting bioactive compounds from a plant matrix and using them to produce micron and / or nanoparticles containing them, fulfills this need.

[0027] In fact, the applicant has discovered a method that allows the extraction of bioactive compounds from plant matrices, as well as the production of micron and / or nanoparticles containing them.

[0028] According to a first aspect, the present invention relates to the method as defined in claim 1.

[0029] In particular, one aspect of the present invention is a method for extracting bioactive compounds from plant matrices and producing micron and / or nanoparticles containing them.

[0030] Therefore, according to one aspect, the present invention relates to a method for extracting bioactive compounds from plant matrices and using them to produce micron and / or nanoparticles containing them, said method comprising the following steps: A-1) Plant material is pulverized with water using high-speed rotating blades through a first turbo emulsifier, preferably under vacuum; A-2) Intra-line homogenization is used for further pulverization; A-3) Solid-liquid separation is performed using a sedimentation centrifuge; A-4) Using a second turbine emulsifier, extract polar and / or moderately polar substances with an alcohol solvent, preferably ethanol, or a water-alcohol solvent or an alcohol / water mixture; A-5) Solid-liquid separation was performed using a second sedimentation centrifuge; A-6) Using a turboemulsifier to extract non-polar substances from edible oils; A-7) Solid-liquid separation is performed using a sedimentation centrifuge to obtain the extract; A-8) Ozonolysis extract; A-9) Micro / nanostructure the extract obtained in step A-8 using an alginate microencapsulation generator, a clay microencapsulation system, or a nanoemulsion generator.

[0031] These steps, from A-1 to A-9, can be identified as those steps that form the so-called extraction stage.

[0032] Regarding step A-1, pulverization is preferably carried out using a turbine emulsifier equipped with blades, and more preferably using a turbine emulsifier equipped with blades having high rotational capacity. Water is preferably used as the first extraction solvent.

[0033] Regarding step A-2, homogenization is inline, i.e., performed via an inline homogenizer, preferably at a speed of 300-3000 rpm. The purpose of this step is to complete, thereby ending, the rupture phase of the cell membranes and walls of the starting plant material. Preferably, speed management within the aforementioned range is achieved by using an inverter. According to a preferred aspect, the use of an inverter is included in step A2.

[0034] Regarding step A-3, which involves solid-liquid separation using a sedimentation centrifuge, solvent recovery may also be included in such a step. This recovery can be performed by incorporating a suitable solvent recovery system.

[0035] Regarding step A-4, in this step, a turboemulsifier is used with an alcohol solvent, preferably ethanol or a water-alcohol solvent, to extract polar and / or moderately polar substances. The turboemulsifier is preferably the type defined in step A-1. Preferably, the solvent mentioned in step A-4 is ethanol, more preferably undenatured ethanol with a minimum of 96°, and even more preferably a type compatible with food or with alcohol / water mixtures, preferably water / ethanol. Depending on the quality and physicochemical characteristics of the bioactive compound to be recovered, the solvent system may also consist of ethanol / water mixtures with different v / v volume ratios, preferably 90 / 10; 80 / 20; 0 / 30; 50 / 50; 25 / 75. Preferably, in step A-4, the alcohol / water mixture, preferably ethanol / water ratio, is from 90:10 to 25:75. According to a preferred aspect, the alcohol / water mixture is an ethanol / water mixture.

[0036] Regarding step A-5, solid-liquid separation occurs in this step, preferably via a second centrifugal settler. Furthermore, a further solvent recovery step may be included.

[0037] Regarding step A-6, in this step, a turboemulsifier is used for the extraction of non-polar substances, preferably using oil, more preferably edible oil, and even more preferably olive oil. The turboemulsifier is preferably the type defined in step A-1. According to a particularly preferred aspect, the edible oil is extra virgin olive oil, EVOO oil, or other similar oils.

[0038] Regarding step A-8, in such a step, ozonation of the oil extract preferably occurs. This step advantageously allows for the yield of ozonated oil / stable ozonides. An example of such a step is the system developed by Erbagil for producing precisely ozonated oil / stable ozonides.

[0039] Regarding step A-9, in such a step, the extract is micro / nanostructured using an alginate microencapsulation generator, a clay microencapsulation system, or a nanoemulsion generator.

[0040] According to a preferred aspect, one or more solid extracts are obtained after step A-9. Such a step can be represented as step A-10.

[0041] According to a preferred aspect, solvent recovery includes at least one step from A-1 to A-9. Preferably, solvent recovery includes at least steps A-3 and / or A-5.

[0042] In addition to the above, according to further preferred embodiments, the following preferred aspects may be considered, wherein the preferred aspects may be included in / used in the extraction method of the present invention.

[0043] Preferably, a spray dryer can be used to dry the nanoemulsion to obtain a solid substance intended for packaging in capsules (pharmaceutical form). According to another preferred aspect, a capsule packaging line (mixer, granulator, capsule filler, block counter, foaming machine, cartoner) can be provided. Preferably, a line is provided for rod packaging of the extract in a stable emulsion form. A fluidized bed drying system is preferably included for dehydrating the fibrous material obtained from the second extraction stage after the removal of ethanol (or an alcohol-water mixture).

[0044] As described above, a preliminary extraction preparation stage may precede the extraction stage. According to a preferred aspect, this preliminary stage includes the following steps: • Select and wash plant materials; • Dry the cleaned plant material, preferably through a ventilated dryer at a programmed temperature; • Store by vacuum packing and sealing in a suitable bag, preferably made of an opaque material that is opaque and / or airtight.

[0045] The selection and washing steps advantageously allow for the removal of foreign materials in order to obtain food-compatible materials (i.e., clean materials free of various foreign residues).

[0046] The method according to the invention advantageously includes the use of innovative integrated equipment to produce micro / nanoparticles that respond to various physical and chemical stimuli (temperature, pH) in order to deliver active ingredients extracted from the plant matrix of consideration. Furthermore, the method according to the invention can be considered an integrated extraction system with solvent recovery pathways (particularly water and ethanol), ensuring the eco-compatibility and eco-sustainability of the entire method carried out through the system itself.

[0047] Another advantage of the present invention is that the latter involves a combination of different techniques organized in sequence.

[0048] This section specifically refers to drug delivery systems that deliver drugs or bioactive components in the body in a targeted and controlled manner. These systems are particularly suitable for lipophilic components (vegetable oils, tocopherols, carotenoids, and Ozoile® as an ozonated oil), which are substances characterized by low solubility in water but greater affinity for lipids and adipose tissue. Delivery of these components requires specific solutions to ensure their effectiveness, stability, and controlled release.

[0049] Extracts obtained according to existing extraction methods can be advantageously and effectively incorporated into media such as alginate-based microspheres, microcapsules obtained by clay encapsulation, or nanoemulsions, which allow for the transport or delivery of substances of high biological value extracted / recovered from a variety of plant matrices considered by the methods of this invention. Therefore, by developing innovative... Drug delivery Practical applications can be found in the delivery of active ingredients. In this context, the advantages of using microspheres or micro and / or nanoparticles for the delivery of active ingredients are multiple, including gastric protection of the compound, formation of specific delayed forms, and the possibility of enabling microparticles to respond multiple times to specific stimuli.

[0050] Other aspects, implementation schemes, features, and advantages will be derived from the implementation scheme described below.

[0051] Furthermore, the invention is also described in the accompanying drawings and claims, the definitions of which form an integral part of this specification. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of a system according to the method of the present invention, which allows for the acquisition of a dry extract. Specifically, the system in this diagram particularly includes first, second, and third turboemulsifiers with rotating blades, followed by first, second, and third settling tanks, respectively.

[0053] Figure 2A representative diagram of an electrospray technique that begins with a stable microemulsion obtained using high-intensity ultrasound is shown, which allows for various types of microspheres.

[0054] Figure 3 Microscopic images of microspheres obtained using the optimized method of the present invention are shown.

[0055] Figure 4 The reactor type used for preparing microspheres is shown. Detailed Implementation

[0056] For the purposes of this invention, definitions of certain terms used in this specification and the appended claims are given below.

[0057] In this document, the term "bioactive" refers to a compound or ingredient that is capable of conferring beneficial health effects on organisms, including humans. Alternatively, such a term may be referred to as "bioactive agent" or simply "compound," "agent," or "active ingredient."

[0058] according to Figure 1 The embodiments shown in the invention, particularly the system according to the method, include a rotary high-capacity turboemulsifier, an inline homogenizer with an inverter for speed management of 300 to 3000 rpm, a first sedimentation centrifuge for solid-liquid separation, a second turboemulsifier for solvent extraction, preferably with a green solvent, more preferably with pure ethanol or a solvent-water mixture, preferably water / water, a second centrifugal sedimentation unit, a turboemulsion extractor, an alginate microencapsulation generator, a clay microencapsulation system, and a high-pressure homogenizer for generating nanoemulsions. Furthermore, a spray dryer tool is used for the solid reduction of the aforementioned nanoemulsions to promote the formation of capsule-like drug forms.

[0059] The first step of the method of the present invention involves pulverizing plant material. This is done using a turboemulsifier with blades having a high rotational capacity, which allows for the initial and crucial cell rupture. The high rotational capacity is associated with greater efficiency in the pulverizing operation. For example, cell membranes can be disrupted by shear stress applied to the liquid. As understood herein, the blade rotational speed refers to a speed preferably from 1500 to 2200 revolutions per minute (rpm), more preferably from 1800 to 2100 rpm.

[0060] According to a preferred aspect, the matrix / solvent ratio can vary from 1:10 to 1:20 during both the initial and subsequent extractions. Therefore, as a non-limiting example, depending on the characteristics of the starting matrix (humidity, plant part, hardness), 10 to 20 liters of solvent are added to 1 kg of matrix.

[0061] Following a first pulverization step using a turboemulsifier preferably employing water as the extraction solvent, a second pulverization / extraction process using water is performed via an inline homogenizer with an inverter (for speed management from 300 to 3000 rpm), which advantageously allows the plant tissue to be completely pulverized, ensuring cell rupture and preparing a plant matrix for subsequent extraction stages.

[0062] A solvent recovery system can also be used in the method according to the invention. Furthermore, in the method according to the invention, the solvent used for extraction via the second turboemulator is ethanol or an ethanol / water mixture. Depending on the mass and physicochemical characteristics of the bioactive compound to be recovered, the solvent system can also consist of ethanol / water mixtures with different volume ratios v / v (90 / 10; 80 / 20; 70 / 30; 50 / 50; 25 / 75).

[0063] As described above, one aspect of the present invention relates to a method for extracting bioactive compounds from plant matrices and using them to produce micron and / or nanoparticles containing them.

[0064] Regarding step A-1 of pulverizing / extracting plant material, at this point in the extraction procedure according to the method of the invention, a rotating blade turbine emulsifier and an aqueous solvent are used to dissolve minerals, simple carbohydrates, proteins, amino acids, some fibrous materials, organic acids, and water-soluble vitamins. Preferably, this solvent is softened water. At the end of this first step, depending on the consistency of the original matrix (leaves, flowers, inflorescences, twigs, stems, roots, bark, rhizomes), the suspension is transferred via a dedicated connection to an inline homogenizer (with an inverter for speed management, preferably 300 to 3000 rpm) at a high blade rotation speed, preferably 1800-2200 rpm. This completes the disintegration stage, producing a highly homogeneous product consisting of particles with an average size of 10-100 micrometers.

[0065] Specifically, regarding the inline homogenization step A-2 using an aqueous solvent, its use can be 1-10m 3 The machine operates at a speed of up to 23 mt / sec using specialized machinery.

[0066] Step A-3 begins at the end of step A-2. In particular, the highly homogeneous suspension can be conveyed to a sedimentation centrifuge to remove water containing up to 15% moisture (sent to a collection container), while the residue is transferred to the next mixer.

[0067] The aqueous liquid, rich in minerals, simple carbohydrates, proteins, amino acids, some fibrous material, organic acids, and water-soluble vitamins, is partially removed and collected at a high flow rate in a steel container. From this container, it is pushed into a filtration / reverse osmosis system for water purification, thus recovering at least 80% and allowing it to be reused in subsequent extraction cycles.

[0068] Then, the residue, which contains most of the precious phenolic substances and all fat-soluble bioactive substances such as carotenoids and tocopherols, is removed and proceeded to the next extraction stage.

[0069] Regarding step A-4, extraction is performed using an alcohol or aqueous alcohol 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, the mixture has a v / v ratio of 90 / 10 to 25 / 75, more preferably 90 / 10; 80 / 20; 70 / 30; 50 / 50 or 25 / 75.

[0070] Specifically, the residue from the settling tank contains mostly bioactive substances with moderately polar characteristics (mainly polyphenols) and non-polar characteristics (mainly carotenoids and tocopherols). Based on the homogenization of the residue, the mixer used in this step can be the same as that in step A-1. Extraction can be extended for 2-10 minutes, preferably 3-7 minutes. Preferably, the speed of the rotating blades is set to the maximum speed, more preferably 1800 to 2200 rpm.

[0071] The resulting suspension can then be transferred to a subsequent sedimentation centrifuge capable of separating the solvent phase from the fibrous residue. The solvent phase can be collected in a high-capacity steel container containing most phenolic substances with high biological activity and moderately polar compounds, such as xanthophyll and chlorophyll. This solution can be concentrated and then lyophilized to form a mixture of substances with high biological and nutritional value. The composition of this dry extract will depend on the initial matrix. For example, if the starting matrix is ​​a leafy plant matrix such as olive leaves, a dry extract rich in polyphenols and chlorophyll will be present in this step, while if the starting matrix is ​​tomato or tomato derivatives or their processing byproducts (peel, concentrate, etc.), an extract rich in phenolic substances and xanthophyll (primarily xanthophyll) will be present. Preferably, the alcohol phase or aqueous alcohol phase is distilled in a dedicated turret for alcohol recovery. Thus, the recovered alcohol can be advantageously reused in subsequent extractions.

[0072] Depending on the starting matrix, the bulk residue at this level will have a different composition. For example, if it originates from leaves, or from tomatoes and their byproducts, or from other berries or red fruits (e.g., goji berries, watermelons), it will consist of dietary fiber (which can be used in nutrition or for animal feed), and the residue will consist of lycopene-rich fiber, which can be used, for example, in nutrition or further extracted for the recovery of highly nonpolar carotenoids. If it is desired to form a product rich in dietary fiber (especially insoluble fiber) but still containing a wide range of bioactive compounds (e.g., polyphenols, carotenoids), then at this point in the procedure, a fluidized bed drying system can be used, which is capable of dehydrating the above materials into powder and automatically distilling the amount of alcohol present in the matrix to recover valuable solvents for subsequent extraction cycles.

[0073] Preferably, for step A-6, which involves the extraction of non-polar substances, extraction is performed using edible oil (preferably extra virgin olive oil + Oil® from Erbagil Tenuta).

[0074] When processing plant matrices rich in nonpolar substances such as carotenoids and / or tocopherols (etc.), such as tomatoes, goji berries, and watermelons, a final extraction is necessary to recover them. In this sense, the previous extraction is used to prepare the matrix for the final extraction, simplifying the initial matrix and concentrating it into compounds of nonpolar nature (carotenoids and tocopherols). For this purpose, the current procedure can advantageously include the following steps: using the same turboemulsifier as the two previously used, and extraction with EVO + Oil (or alternatively, other edible oils such as sunflower oil, corn oil, coconut oil, etc.), for a duration varying between 2 and 10 minutes under cold vacuum.

[0075] Step A-7. The suspension of oil and fiber can then be transferred to another centrifugal settling tank, preferably of the same type as those previously used, for separating the carotenoid-rich oily liquid component from the solid component of the fiber source: the more carotenoids enriched in the matrix, the redder the oil collected in the high-capacity steel container will acquire, and a functional ingredient will be formed for the formulation of high-value-added nutritional products. The final fibrous material can be used to form feed with high nutritional value. An advantage immediately inferred from this procedure is the acquisition of a solvent-free extract rich in carotenoids and nonpolar substances, eliminating any upstream food safety concerns that could arise from the use of harmful solvents, even if effective for the extraction purpose.

[0076] For the ozonation step A-8 of the extract obtained after bulk extraction, it is preferably carried out by oil, more preferably by Evo+Oil®, thereby obtaining an oil rich in nonpolar substances, particularly carotenoids and tocopherols, which produces a strong red-orange color to the oily matrix. Such an oil can undergo an ozonation process to further functionalize such a matrix: in fact, the oily matrix rich in carotenoid components provides not only oil (preferably EVO oil) but also C 40 The classic structure of carotenoids (up to 11 conjugated double bonds, compared to 13 in lycopene) ensures a large number of double bonds, providing considerable potential for ozone addition. This allows for the formation of significant amounts of ozonides, advantageously ensuring a matrix with superior oxygen-carrying capacity. This could be advantageously used in modern and innovative medical devices and other specific fields, such as pharmaceuticals, nutritional supplements, and cosmeceuticals.

[0077] Step A-9, concerning the micro / nanostructuring of the extract, aims to obtain an efficient method for delivering the active ingredient extracted according to the current procedure.

[0078] According to a preferred aspect, micron and / or nanoparticles obtained by the method of the present invention are used for the delivery of active ingredients. Preferably, delivery forms considered in the present procedure are nanoemulsions, alginate-based microspheres, and microcapsules obtained by clay encapsulation. Such delivery forms are advantageous for transporting or delivering substances of high biological value recovered from various plant matrices considered by the method of the present invention. Therefore, all three possible media are suitable for the active ingredient as… Drug delivery Applications. As anticipated above, this delivery method offers several advantages: the importance of gastric protection of the compound, ensuring its immutability at the gastric level and / or chemical degradation by acid attack; the promotion of the formation of specific delayed forms of the bioactive ingredient with time-delayed and prolonged release; and the possibility of allowing micro / nanoparticles to respond multiple times to specific stimuli (e.g., pH and temperature). Thus, at the end of the extraction process, three instruments can be placed side-by-side for the production of: - Nanoemulsion; - Alginate-based microspheres; - Microencapsulation with clay.

[0079] According to a preferred aspect, the nanoemulsion is obtained through homogenization. Specifically, such a nanoemulsion can be prepared using a homogenization technique capable of forming an oil-in-water emulsion at the nanoscale, in which the extracted substances are encapsulated. Through this method, nonpolar substances acquire more polar properties, becoming water-soluble, meaning they can translocate into the bloodstream and reach various cell populations in the body, promoting beneficial health effects. Advantageously, the preparation method intended to form the nanoemulsion is expected to allow for the acquisition of very stable products starting from naturally derived materials of plant origin. Another advantage is that stability lasts for more than one year (2-5 years) due to specific low-temperature methods and chitosan coating. Furthermore, due to chitosan coating, the product is gastric-tolerant, ensuring adequate release in the intestine, resulting in increased absorption of the active ingredients. Another particularly relevant advantage is the significantly improved bioavailability of the bioactive ingredients, which can be administered at lower doses due to a significant increase in absorption and metabolic rates. Therefore, nanoemulsion-based food supplements are more effective, and from a commercial point of view, this means that production is also cheaper due to the lower amount of active ingredients required.

[0080] Alginates are a family of polysaccharides derived from various brown algae or phyllophytes, characterized by a wide range of chemical compositions, molecular weights, and functional properties. Chemically, alginates are unbranched linear copolymers of β-D-mannuronic acid and its C-5 epimer α-L-guluronic acid. They form effective supports for the formation of microspheres in which extracted plant material is microdispersed. According to a preferred aspect, microspheres are obtained by microdispersing the extracted plant material. Alginates are commonly used for delivering natural active ingredients from various sources and pharmaceutical molecules from synthetic processes. Again, in this case, delivery ensures improved bioavailability of the bioactive ingredients with the aforementioned advantages.

[0081] According to a preferred aspect of the invention, the medium is obtained by microencapsulating a bioactive compound or component with clay. Preferably, the clay is halloysite. An advantageous feature of this medium is its high biocompatibility. Its advantages are the same as those described for the other two delivery methods.

[0082] Regarding the polymers used in the present invention, details of some preferred polymers are given below, with natural polymers being preferred, which can be used in the design of microspheres.

[0083] 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 biocompatibility and biodegradability 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, particularly 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 various drug forms.

[0084] In particular, pH-responsive natural and biodegradable polymers, such as alginate and chitosan, are widely used in pharmaceutical applications, especially in the form of biocompatible microspheres. Furthermore, thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) can be added to biomedical delivery systems due to its lower critical solution temperature, which is close to physiological conditions.

[0085] Advantageously, the method of the present invention can yield “smart” hydrogel microspheres that are responsive to physiological stimuli such as changes in temperature and pH, for therapeutic release of active ingredients in specific areas of the human body.

[0086] In particular, hydrogels can be used because they offer several advantages: - Formulation versatility: Hydrogels can be designed and formulated using a wide range of synthetic and natural polymers, allowing for specific customization for the type of drug or bioactive component to be delivered. This provides flexibility in tuning gel properties, such as crosslinking density, release rate, and loading capacity for lipophilic drugs; - Biocompatibility and biodegradability: The body tolerates the hydrogel well and it does not cause significant adverse reactions. Furthermore, many hydrogels can be designed to be biodegradable, reducing the risk of long-term side effects; - Controlled Release: Hydrogels allow for the controlled and gradual release of lipophilic components over time. Their ability to trap active ingredients within their gel matrix prevents sudden release, ensuring greater stability and a predictable release profile; - Drug protection: Hydrogels can provide physical and chemical protection for carried drugs. In particular, they can protect lipophilic components susceptible to enzymatic degradation or oxidation from the surrounding environment until release at the site of action; - Specific targeting: Hydrogels can be functionalized with specific targeting molecules such as antibodies or ligands, which direct drugs to the desired site of action. This targeting capability increases therapeutic efficacy and reduces the risk of undesirable side effects; -Possibility of non-invasive application: Hydrogels can be applied in various forms, including pre-formed gels, creams, ointments, and hydrogels, which can simplify the non-invasive application of drugs. This is particularly advantageous for treating skin or eye diseases, where direct application to the affected site is preferred; - Reduced dosing frequency: Due to the controlled and prolonged release of the drug, hydrogels allow for a reduction in the frequency of administration, improving patient adherence to treatment and increasing overall therapeutic efficacy.

[0087] In particular, according to a preferred aspect, two controlled-release systems for lipid substances can be developed by the method of the present invention: a first system is a pH-responsive system based on sodium alginate; and a second system is a thermo-pH-responsive system based on sodium alginate and PNIPAM (poly(N-isopropylacrylamide)).

[0088] For both types of microspheres, stable microemulsions obtained from high-intensity ultrasound were first used using electrojet technology. Figure 2 ).

[0089] In particular, pH-responsive microspheres can be obtained, and process parameters (collection distance, CaCl2 concentration, flow rate, applied voltage, needle type) can be optimized.

[0090] Advantageously, micro / nano encapsulation techniques with clay minerals can be used.

[0091] Clay minerals are natural materials that are inexpensive, readily available in large quantities, and in some cases, highly pure (haloysite and sepiolite). In fact, clays ensure the controlled and sustained release of loaded active ingredients over time. Furthermore, they possess the ability to penetrate cell membranes, thus making them suitable for delivering hydrophobic molecules in biofluids.

[0092] Micro / nano encapsulation of clay minerals onto plant substrates and various organic molecules can be carried out using a method that includes the following steps: - Disperse the clay in an aqueous environment and insert a concentrated solution of the plant matrix into its soluble solvent (e.g., on a laboratory scale, place 100 mg of clay in 10 mL of water and add the plant matrix at a variable matrix / clay ratio, e.g., 1:1, 2.5:1, 5:1, 7.5:1, 10:1). - Stir the resulting dispersion at room temperature overnight; - Centrifuge and wash the resulting solid with water to remove any physically adsorbed components (or, a filtration and filter washing system may be used). -The solid product obtained by freeze drying.

[0093] The amount of plant matrix adsorbed on the clay was determined by thermogravimetric analysis and qualitatively evaluated by FT-IR to ensure that the results met expectations.

[0094] The loading depends on the properties and morphology of the clay used (haloysite, lithium montmorillonite, palygorskite, sepiolite, and bentonite). Haloysite is preferred to allow for a maximum organic loading of 10% w / w, but lithium montmorillonite and bentonite with loadings of about 50% w / w and palygorskite and sepiolite with loadings of about 70% w / w are also allowed.

[0095] A significant advantage of this encapsulation technology is that the non-interacting plant matrix can be easily recovered and reused for further adsorption cycles, while also taking into account the concept of sustainability. Industrial scale-up is advantageously simple, as it is sufficient to use the initiator in proportion to the amount of product to be prepared.

[0096] The stages of dispersion, stirring, centrifugation, washing, and freeze drying can be easily achieved using reaction vessels, mechanical stirrers, industrial centrifuges, and large-volume freeze drying.

[0097] According to a preferred aspect, the method for preparing nanoemulsions includes using plant materials in the process to form an oil-in-water emulsion. Such emulsions are particularly stable (lasting 3-5 years) because a polydisperse is applied around the oil nanodroplets (edible oil used as a solvent in a previous stage) and thickening is ultimately promoted using a polymer coating (in this case, chitosan). All of this occurs within a high-pressure homogenizer. The result is the formation of particularly stable nanoemulsions with an average diameter of 50-200 nm. This process respects the structure and chemophysical characteristics of the treated molecules because it does not require high temperatures (below 40°C) and can be used to encapsulate lipophilic molecules of various properties and origins, such as lipids, carotenoids, luteinoids, tocopherols, sterols, fat-soluble vitamins, etc. One advantage of this process is that, in addition to producing extremely stable nanoemulsions, the resulting product has gastric tolerance properties due to its chitosan coating. Furthermore, chitosan naturally has the ability to be incorporated into liquids through swelling, which results in increased intestinal retention and consequently increased absorption capacity.

[0098] According to a preferred aspect, regarding the method for preparing the solid extract, this is carried out after the formation of a nanoemulsion derived from a final oily extract using a high-pressure homogenizer, where an appropriate amount of water for preparing the primary emulsion must be added to the final oily extract. This nanostructure, including natural materials such as chitosan 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.

[0099] The main advantage of this technology is that solids can be easily administered to prepare simple and universal drug forms such as capsules.

[0100] Another significant advantage is the ability to associate fat-soluble active ingredients with those that are water-soluble; in fact, the aqueous phase required to form the primary emulsion can be pre-enriched with water-soluble components, while the fat-soluble components can be dissolved in the oil phase. After dehydration and subsequent collapse of the oil phase on a solid support, the oil phase itself will be incorporated into the solid, ultimately forming the solid material used to prepare the capsules.

[0101] The following examples are intended to further illustrate certain embodiments of the present invention and should not be construed as limiting it.

[0102] Example Shredded plant material Using a turboemulator with rotating blades and 10 liters of softened water as solvent, 1 kg of double-strength tomato concentrate was treated for 5 minutes at a blade rotation speed of 2200 g / min to dissolve minerals, simple carbohydrates, proteins, amino acids, some fibrous material, organic acids, and water-soluble vitamins. The method was carried out under vacuum using a liquid-loop vacuum pump equipped with a water supply on / off solenoid valve and a one-way safety valve on the suction device. The vacuum was programmed to a value up to 700 mm / Hg. During the extraction phase, the temperature was maintained below 25°C, ensured by the cooling jacket of the lined extractor, and continuously monitored using a PT100 probe.

[0103] Process with an inline homogenizer The resulting suspension is then transferred to an inline homogenizer via a dedicated tube located at the bottom of the extractor. A tube connected to a suction pump ensures the removal of the suspension from the first extractor and delivery to a second extractor, which is tasked with completing the cell lysis phase and preparing the matrix for subsequent extraction stages. The suspension is then delivered from the inline homogenizer to a sedimentation centrifuge using the instrument's centrifugal force to remove water, stabilizing the moisture content at 10-20%, preferably 15%, while transferring the residue to the next mixer. Water (rich in the polar substances listed above), which can be calculated as 80-90% of the initial volume, is recovered and collected at high flow rates in a steel container and pushed to a filtration / reverse osmosis system for purification before being used in the next extraction cycle.

[0104] Extracted with ethanol The bulk residue from the settling tank, containing mostly bioactive substances with moderate polarity (primarily polyphenols) and non-polarity (primarily carotenoids and tocopherols), is treated with the same type of mixer used in the first step. Based on the homogenization of the residue, ethanol is used as the solvent, or alternatively with a mixture of ethanol and water (90 / 10; 80 / 20; 70 / 30; 50 / 50; 25 / 75 v / v) to promptly extract bioactive substances with different polarities. Extraction is performed for 5 minutes at the maximum speed of the rotating blades (2200 g / min), after which the suspension is transferred to a subsequent settling centrifuge capable of separating the ethanol (or ethanol / water) phase from the fibrous residue. The ethanol (or ethanol / water) phase, containing mostly highly bioactive phenols and moderately polar compounds (i.e., xanthophylls and chlorophylls), is collected in a high-capacity steel container. The solution is concentrated by distilling alcohol in a specially designed distillation column to recover the solvent, and then freeze-dried to form a mixture of substances with high biological and nutritional value (lutein and other lutein compounds).

[0105] Extraction of nonpolar substances Using the same turboemulators as those previously used, extraction was performed for 10 minutes under cold vacuum with Evo+Oil®. The oil and fiber suspensions were then transferred to a further sedimentation centrifuge for oil / fiber separation.

[0106] Ozone processing Following extensive extraction of EVO oil, an oil rich in nonpolar substances (carotenoids and tocopherols) is obtained, all of which is also evidenced by the intense red-orange color imparted to the oily matrix. Ozonation processing of the oil further functionalizes the matrix, resulting in the formation of ozonides. To obtain an ozonation level corresponding to 6% of the total double bonds of the unsaturated fatty acids (primarily oleic acid) in the triacylglycerols that form the oil, ozonation processing is applied (Erbagil srl patent). Indeed, such an amount offers the possibility of not altering the fundamental quality parameters of EVO and allows for the retention of all its significant properties. For all these crucial properties, those derived from ozonation processing are added, namely those attributed to ozonides, representing the internal peroxide structure of the main reaction products between unsaturated fatty acids and ozone. Another advantage of ozonation processing is the microbial stability derived from this method, a sterilization technique known for its application in food.

[0107] Micro / nanostructuring of extracts Nanoemulsions were prepared using homogenization techniques to form nanoscale oil-in-water emulsions, in which bioactive compounds previously extracted from chitosan coatings were encapsulated.

[0108] Methods and related systems for extracting bioactive compounds or components from plant matrices, and media containing them, have been described.

[0109] Controlled release systems for lipid substances: - A pH-responsive system based on sodium alginate; - A thermo-pH-responsive system based on sodium alginate and PNIPAM.

[0110] For both types of microspheres, stable microemulsions obtained from high-intensity ultrasound were first used using electrojet technology. Figure 2 ).

[0111] pH-responsive microspheres were obtained by optimizing process parameters (collection distance, CaCl2 concentration, flow rate, applied voltage, and needle type). Following this setup, lipophilic components, oil types, ozonated oils, various nonpolar substances (10-50 wt%), Tween 80 (5 wt%), and xanthan gum (0.25 wt%) were added to a sodium alginate solution (1.8 wt%), and a stable emulsion was obtained using an ultrasonic homogenizer. Microspheres were then prepared on a laboratory scale via electrospray irradiation: the emulsion was loaded into a syringe from a collection distance of 10 cm at a constant extrusion rate of 20 mL / min. A voltage of 30 kV was applied between the needle (24 G) and the CaCl2 gel bath (20% w / v). The crosslinking time was set to 30 min, followed by three rinses of the MS in deionized water. Chitosan coating was applied by vortexing the microspheres in a 0.5 wt% chitosan solution at 700 rpm for 30 min. The analysis results indicate that 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 wt% of nonpolar active ingredient, and a coating process at 700 rpm. Indeed, the high rpm leads to greater penetration of chitosan into the polymer network, resulting in partial collapse of the complex. The chitosan-coated microspheres exhibit a high water content, which can be attributed to the hydrophilicity of the polymer. Degradation of the polymer network leads to the release of nonpolar oily substances, occurring at pH > 7.4.

[0112] In addition, thermo-pH-responsive microspheres were obtained. Sodium alginate and PNIPAM were mixed in deionized water at a 1:1 ratio (w / w) in a cold water bath for 30 minutes. Then, ozonated or unozonated oily substances (10-50% by weight), nonionic surfactant Tween 80 (5% w / v), and emulsifier xanthan gum (0.25% w / v) were added, and a stable emulsion was obtained by high-intensity sonication (HIU). This method was performed using an ultrasonic homogenizer at 100% power for 2.5 minutes. The microemulsion was placed in a syringe and injected into a stainless steel needle (0.311 mm inner diameter) at a constant rate of 20 mL / min using an infusion pump. The spinneret was connected to a 30 kV high-voltage power supply. Droplets formed at the tip of the spinneret separated and fell into the crosslinking bath below. The collection distance was set to 10 cm. All experiments were conducted at a temperature of 25 °C and a humidity not exceeding 50%.

[0113] Results obtained from the laboratory, particularly from the morphological analysis of the samples, revealed that all types of microspheres had an average diameter of less than 450 μm. For this reason, they can be considered microspheres suitable for all purposes. Figure 2 The thermal response properties of PNIPAM were confirmed by the evaluation of the volume transition peak. Based on the results obtained, from both morphological and thermal response perspectives, the optimal microspheres are those with an oil concentration of 30% by weight. Therefore, it can be said that this type of microsphere is capable of delivering and releasing the incorporated active ingredient at a temperature of 37°C and a pH > 6. Laboratory-scale studies enabled the setting of optimal process parameters to obtain multi-responsive microspheres (Table 1), and this method was transferred to the use of... Figure 4 The reactor shown is used for large-scale production of microspheres with the aforementioned characteristics.

[0114] Table 1. Optimization of microsphere formation parameters

[0115] The foregoing should be understood as a non-limiting example. Furthermore, those skilled in the art will understand that changes can be made without departing from the scope of the invention. Claims (as amended under Article 19 of the Treaty) 1. A method for extracting bioactive compounds from plant matrices and using them to produce micron and / or nanoparticles containing them, comprising the following steps: A-1) Plant material is pulverized with water by rotating blades at a speed of 1500 to 2200 rpm, preferably 1800 to 2100 rpm, through a first turbo emulsifier, preferably under vacuum. A-2) In-line homogenization via an in-line homogenizer is used for further pulverization; A-3) Solid-liquid separation is performed using a sedimentation centrifuge; A-4) Using a second turbo emulsifier, polar and / or moderately polar substances are extracted from the remaining bulk residue after A-3 using an alcohol solvent, preferably ethanol, or a water-alcohol solvent or an alcohol / water mixture. A-5) Solid-liquid separation was performed using a second sedimentation centrifuge; A-6) Using a third turbo emulsifier, non-polar substances are extracted from the remaining residue after A-5 using edible oil; A-7) Solid-liquid separation is performed using a third centrifugal sedimentation unit to obtain the extract; A-8) A nonpolar extract obtained by ozonation of A-7; A-9) Micro / nanostructure the extract obtained in step A-8 using an alginate microencapsulation generator, a clay microencapsulation system, or a nanoemulsion generator. 2. The method according to claim 1, wherein the first turbo emulsifier provided with blades is a vacuum turbo emulsifier provided with blades. 3. The method according to any one of the preceding claims, wherein the inline homogenization step A-2 for further pulverization is carried out at a speed of 300-3000 rpm. 4. The method of claim 3, wherein step A2 includes using an inverter. 5. The method according to any one of the preceding claims, wherein the alcohol solvent in step A-4 is ethanol. 6. The method according to any one of claims 1-4, wherein the ratio of alcohol / water mixture in step A-4 is from 90:10 to 25:75. 7. The method of claim 6, wherein the alcohol / water mixture is an ethanol / water mixture. 8. The method according to any one of the preceding claims, wherein at least one step of steps A-1 to A-9 includes solvent recovery. 9. The method of claim 8, wherein solvent recovery occurs in steps A-3 and / or A-5. 10. The method according to any one of the preceding claims, wherein one or more solid extracts are obtained in steps A-9.

Claims

1. A method for extracting bioactive compounds from plant matrices and using them to produce micron and / or nanoparticles containing them, comprising the following steps: A-1) Plant material is pulverized with water using high-speed rotating blades through a first turbo emulsifier, preferably under vacuum; A-2) Intra-line homogenization is used for further pulverization; A-3) Solid-liquid separation is performed using a sedimentation centrifuge; A-4) Using a second turbine emulsifier, extract polar and / or moderately polar substances with an alcohol solvent, preferably ethanol, or a water-alcohol solvent or an alcohol / water mixture; A-5) Solid-liquid separation was performed using a second sedimentation centrifuge; A-6) Using a turboemulsifier to extract non-polar substances from edible oils; A-7) Solid-liquid separation is performed using a centrifugal sedimentation device to obtain the extract; A-8) Ozonolysis extract; A-9) Micro / nanostructure the extract obtained in step A-8 using an alginate microencapsulation generator, a clay microencapsulation system, or a nanoemulsion generator.

2. The method according to claim 1, wherein the first turbo emulsifier provided with blades is a vacuum turbo emulsifier provided with blades.

3. The method according to any one of the preceding claims, wherein the inline homogenization step A-2 for further pulverization is carried out at a speed of 300-3000 rpm.

4. The method according to any one of the preceding claims, wherein step A2 includes the use of an inverter.

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

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

75.

7. The method of claim 6, wherein the alcohol / water mixture is an ethanol / water mixture.

8. The method according to any one of the preceding claims, wherein at least one step of steps A-1 to A-9 includes solvent recovery.

9. The method of claim 8, wherein solvent recovery occurs in steps A-3 and / or A-5.

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

11. The method according to any one of the preceding claims, wherein the obtained micron and / or nanoparticles are used to deliver the active ingredient.

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