Cannabinoid concentrate free of thc, method for obtaining thereof and use thereof

Abstract

The present invention relates to cannabinoid concentrates free of THC, methods for obtaining them and uses thereof. The methods for obtaining comprise an alkaline wash to purify a lipid extract.

Description

Technical Field

[0001] This invention relates to THC-free cannabinoid concentrates, methods of obtaining them, and their uses. Background Technology

[0002] Cannabis (Cannabis sativa L.) is a prolific producer of various isoprene-resorcinol polyketides (called cannabinoids), but not the only producer. (et al. 2016) Cannabinoids in cannabis are not the only lipid-based exogenous compounds that interact with the endocannabinoid system. In recent years, other plants have been found to produce cannabinoid-like compounds, and several non-traditional cannabinoid plant natural products have been reported to act as cannabinoid receptor ligands. Cannabinoids can also be produced from yeast, fungi, or bacteria.

[0003] The endocannabinoid system consists of endocannabinoids, cannabinoid receptors, and enzymes that synthesize and degrade endocannabinoids. Many of the effects of cannabinoids and endocannabinoids are mediated by two G protein-coupled receptors (GPCRs), namely CB1 and CB2, although other receptors may also be involved. CB1 receptors are present at very high levels in several regions of the brain, and at lower levels in a more generalized manner. These receptors mediate many of the psychoactive effects of cannabinoids. CB2 receptors have a more limited distribution, present in many immune cells and a few neurons. CB1 and CB2 are primarily coupled to inhibitory G proteins and are subject to the same pharmacological effects as other GPCRs. Therefore, partial agonism, functional selectivity, and anti-agonism all play important roles in determining cellular responses to specific cannabinoid receptor ligands.

[0004] Exogenous cannabinoids, or terpenoids, such as those derived from cannabis, are used to alleviate nausea and vomiting during chemotherapy, improve appetite in HIV / AIDS patients, and treat chronic pain and muscle spasms through interaction with the endocannabinoid system. Cannabinoids, along with their constituent cannabinoids and terpenes, are used to treat diseases or improve symptoms. Cannabinoids are synthesized in plants in the form of carboxylic acids. Cannabinoid acids, such as cannabigerolic acid, tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid, and cannabidivarinic acid, are abbreviated as CBGA, THCA, CBDA, CBCA, and CBDVA, respectively. The metabolites of these precursors are the neutral forms of cannabigerol (CBG), tetrahydrocannabinol (THC; the main psychoactive cannabinoid found in cannabis), cannabidiol (CBD), cannabichromen (CBC), and cannabidivarin (CBDV), respectively. The neutral forms are obtained from the acidic forms through decarboxylation.

[0005] Until recently, cannabinoid acids were considered to be unable to undergo metabolism (i.e., inhaled by the lungs or digested by the gastrointestinal tract and processed by the liver); nor were they considered to have any pharmacological activity in themselves (Jung et al 2007; Takeda et al 2008).

[0006] However, recent in vitro and animal studies using extracted CBGA, THCA, CBDA, or CBDVA have shown measurable effects on certain enzymes and receptor sites, suggesting that these cannabinoids have some potential therapeutic effects. Furthermore, there is a need to elucidate and refine specific extraction techniques to preserve these specific acidic forms of these cannabinoids, thereby providing material for further experiments and research.

[0007] Specifically, acidic forms of cannabinoids, such as CBDA or CBDVA, have been shown to provide specific biological activities useful for treating health conditions, and in some cases even superior to their respective neutral forms (WO2017025712A1 - Use of cannabinoids in the treatment of epilepsy; WO / 2019 / 012267 - Use of cannabinoids in the treatment of neurodegenerative diseases or conditions).

[0008] Cannabinoid acid (CBGA) is the acidic form of cannabinoid (CBG). It is a dihydroxybenzoic acid, specifically an oleic acid in which the hydrogen at the 3-position is replaced by a geranyl group. CBGA is a biosynthetic precursor of Δ(9)-tetrahydrocannabinol, the main psychoactive component of the cannabis plant.

[0009] CBGA can help diabetic patients combat complications and comorbidities, such as cardiovascular disease. In vitro studies have found that CBGA significantly inhibits aldose reductase, a major contributor to oxidative stress that leads to heart and other problems. As expected, CBGA test results are highly dose-dependent. Synthetic inhibitors have serious side effects in many patients, making plant-derived CBGA a promising treatment option (Smeriglio et al., 2018).

[0010] Finally, the researchers examined the cytotoxic effects of CBGA extracted from cannabis and found that CBGA not only killed colon cancer cells but also accelerated the death of early-stage cancer cells and halted the cancer cell cycle. While more research is certainly needed, the researchers are encouraged that CBGA can not only effectively target colon cancer cells but also prevent the growth and proliferation of polyps. If left untreated, these polyps can develop into cancer (Nallathambi, 2018).

[0011] Tetrahydrocannabinol acid (THCA) is a precursor to THC produced by plants, which is decarboxylated to THC by heat, light, and time (e.g., through heating, smoking, or cooking). Unlike THC, THCA is not associated with psychoactive effects in monkeys, mice, or dogs because we know these effects are due to the activation of the CB1 receptor, suggesting that THCA is not a strong activator of this receptor.

[0012] Cannabidiol (CBDA) is a plant-derived precursor of CBD, decarboxylated to CBD through heating, light exposure, and time. Research on CBDA is limited, with most studies focusing on its antinausea effects. Like CBD, CBDA inhibits nausea and vomiting in rats and shrews via the serotonin receptor (5HT1A) and reduces intestinal motility, suggesting a moderating effect on nausea, as seen in patients undergoing chemotherapy (Bolognini et al 2013). Similar to CBD, CBDA has also been shown to reduce stress in rats via the serotonin receptor. Other identified pharmacological targets of CBDA include inhibition of enzymes in the endocannabinoid system, activation of TRPV1, and inhibition of cyclooxygenase (COX). The pharmacological effects of CBDA in vivo and in vitro appear to be more similar to those of CBD (e.g., both are activated via the serotonin receptor), although CBDA is more potent than CBD in its serotonin receptor-mediated action. Furthermore, CBDA has been shown to block COX1 and COX2 to varying degrees in vitro; both enzymes are different mediators of inflammation and inflammation-related pain. Non-steroidal anti-inflammatory drugs (NSAIDs), such as acetylsalicylic acid (aspirin), ibuprofen, naproxen, indomethacin, and diclofenac, all exert their effects by inhibiting COX1 and COX2, and like CBDA, contain a carboxylic acid group in their structure, indicating that this part of the molecule is indispensable to their function.

[0013] In one study, CBDA significantly inhibited both COX 1- and COX 2-mediated oxidation activities, with CBDA exhibiting a strong preference for specifically inhibiting COX 2 (Takeda et al., 2008). A second study showed that CBDA significantly inhibited COX 1, but only THCA inhibited COX 2, with an inhibition rate only slightly above 30% (Ruhaak, L. et al., 2011). However, both studies indicated that the carboxylic acid forms of CBDA and THCA possessed stronger overall COX-inhibiting activity than their decarboxylated forms, CBD and THC.

[0014] Finally, CBDAs exhibited in vitro activity on several different cation channel receptors (collectively referred to as transient receptor potentials), which play important roles in pain and inflammatory signal transduction, such as TRPV1 and TRPV4 (“vanillic acid” type); TRPA1 (“ankylin” type); and TRPM8 (“melastatin” type). These receptors can be blocked, activated, or desensitized by another activator (Cascio and Pertwee 2014). These may be additional mechanisms by which the carboxylic acid form of cannabinoids functions independently of its decarboxylated form to alleviate central and peripheral pain and inflammation.

[0015] To facilitate the manufacture of various products that can be safely administered or used by patients and / or consumers, cannabinoids are typically extracted, concentrated, and purified from biomass to obtain various concentrates or isolates. However, typical methods for extracting neutral cannabinoids are not ideal for extracting cannabinoid acids and have some limitations.

[0016] In certain applications, cannabinoid acids exhibit superior chemophysical properties compared to their neutral forms. For example, in evaporation applications, using a concentrate with a high CBDA content (instead of CBD) may help prevent crystal formation in the evaporation chamber.

[0017] Cannabinoid concentrates can be produced using several technologies. Typically, they are obtained from biomass that has been pre-dried by supercritical fluid extraction (SFE) (e.g., using supercritical CO2), followed by a winterization step to remove chlorophyll and waxes. Winterization involves the use of ethanol or butane at low temperatures (US 9186386 B2, US 6403126 B1). These methods have several drawbacks, such as high investment costs, the need for highly skilled technicians to operate complex equipment, the use of flammable and hazardous organic solvents for winterization of the crude extract, and high energy consumption. Completely removing the organic solvents used in conjunction with CO2 during the extraction step or removing chlorophyll during the winterization step is challenging. These technical challenges have prompted policymakers to set limits on the levels of organic solvents (some of which are known carcinogens), up to 5,000 ppm (from Health Canada). Furthermore, supercritical CO2 is highly selective for potentially toxic components found in pesticides, thus posing a risk that these toxic components may be present in concentrated form in the final product. Furthermore, since drying biomass and removing solvents require heat, which is generated through the CO2 extraction step, it is difficult to preserve well the heat-sensitive acidic form that can be decarboxylated. The cannabinoid content obtained using this method is not high enough to proceed directly to the crystallization step. An intermediate distillation step is usually required. Finally, supercritical CO2 cannot extract acidic cannabinoids with the same efficiency because the acidic form has a higher molecular weight compared to the neutral form. All these factors make the method as a whole unsuitable for extracting and concentrating acidic cannabinoids.

[0018] Recent alternative technologies represent low-temperature ethanol extraction, in which pre-dried biomass is extracted at very low temperatures (-40°C) to prevent chlorophyll and waxes from being extracted into the solvent. The cannabinoid-rich ethanol solution is then evaporated to recover the solvent. Given the large amounts of solvent to be evaporated (up to 20 times the weight of the biomass), this activity is energy-intensive and very time-consuming. Avoiding the decarboxylation of cannabinoid acids in this step is challenging. Finally, the use of organic solvents inevitably leads to safety, health, and environmental issues.

[0019] WO2019057994A1 describes a method for preparing a whole-plant extract from cannabis plant material comprising at least 50 w / w% cannabidiol (CBD) and less than 0.2 w / w% tetrahydrocannabinol (THC), comprising the following steps:

[0020] a) Provide cannabis plant material including cannabinoids and their acids.

[0021] b) Incubate the plant material from step a) in an aqueous medium containing salts and / or hydroxides at 25-75°C in a medium containing 30 to 100 v / v% ethanol for 0.5 to 1 hour to convert cannabinoid acids and cannabinoids into the corresponding cannabinoid salts and / or their complexes.

[0022] c) Separate the cannabinoid salts and / or complexes from step b) from the waxes contained in the plant material to obtain aqueous cannabinoid salts or complexes on the surface of the raw material.

[0023] d) Mix the aqueous cannabinoid salt or complex from step c) with ethanol to obtain an alcohol-containing cannabinoid extract.

[0024] e) To achieve a water / alcohol ratio of (30-70):(70-30)v / v% for the alcohol-containing extract in step c), providing an aqueous extract comprising cannabinoid salts and complexes.

[0025] f) Acidification of the aqueous extract from step e) converts cannabinoid salts and complexes into their acids, producing an aqueous acidic extract.

[0026] g) Decarboxylating the cannabinoid acid from step f) with alkaline Al2O3 at a temperature of at least 20°C, such that the formation of THC preferentially takes precedence over the formation of CBD, to provide a decarboxylated mixture.

[0027] h) Optionally, the decarboxylation mixture from step g) is further incubated with alkaline Al2O3 at a temperature of at least 20°C to convert cannabinoid acids and cannabinoids into cannabinoid salts and / or complexes, respectively.

[0028] i) Add ethanol to the mixture from step h) to achieve a water / ethanol ratio of (30-70):(70-30), where THC dissolves while cannabinoid salts and the complex do not.

[0029] j) Contact the mixture from step i) with an adsorbent or absorbent to remove THC from the adsorbent or absorbent, resulting in a THC-depleted mixture.

[0030] k) Acidifying the THC-depleted mixture of step j) to convert the cannabinoid salts and complexes in the THC-depleted mixture into cannabinoid acids, and removing the mixture from the adsorbent or absorbent with ethanol.

[0031] l) Cannabinoid acids are decarboxylated to their corresponding cannabinoids at temperatures ranging from 50 to 150°C and under a vacuum ranging from 0 to 1000 mBar, to achieve the desired degree of decarboxylation within 1 to 10 hours, yielding a decarboxylated, THC-depleted mixture.

[0032] m) Recover one or more cannabinoids from the decarboxylated, THC-depleted mixture of step l).

[0033] The applicant specifically noted that the method includes using an organic solvent on plant material, decarboxylating acidic cannabinoids in the presence of Al2O3 to preferentially form THC, and filtering an ethanol solution.

[0034] US2019359550 describes a method for recovering acidic cannabinoids after winterization by leaching plant material or a lipophilic extract (e.g., butane-hash oil-BHO). The applicant notes that by employing a pH of at least 8.8, the method produces microcrystalline powders indiscriminately enriched with all cannabinoid acids present in the initial lipophilic extract, and the method does not teach how to obtain concentrates with low THCA content and high content of other cannabinoid acids (e.g., CBDA or CBGA) unless chromatography is considered a post-refining step. Specifically, the applicant notes that the method produces concentrates with high THCA content, exceeding that of the initial lipophilic extract, and the method does not teach how to obtain cannabinoid concentrates with low THCA content (where the THCA content is lower than that of the initial lipophilic extract). Furthermore, the applicant notes that the method requires the use of harmful solvents (i.e., butane or ethanol). Additionally, the applicant notes that US2019359550 specifies that the leaching step should be performed for 20 minutes. Finally, the applicant notes that the method does not allow for obtaining a concentrate rich in CBDA or CBGA without concentrating THCA at any step of the production process.

[0035] US7592468B2 describes a method for producing THC, comprising extracting THC and THC carboxylic acid from plant material using a first solvent, extracting the acid using a second solvent, wherein the solvent preferentially dissolves the acid compared to THC, converting the THC acid into a derivative, and extracting the derivative into a third solvent.

[0036] US9340475B2 teaches a method to decarboxylate CBDA in cannabis oil, followed by distillation of CBD from the decarboxylated cannabis oil, conversion of THC to CBN, winterization with isopropanol, and finally elution of the silica plug with hexane-ethyl acetate to remove impurities.

[0037] As a precursor to THC, although it lacks psychoactivity, THCA is often subject to the same content limits as THC. Therefore, further purification and concentration are usually necessary to remove or reduce THCA and THC to regulatory levels, such as 0.3% by weight.

[0038] In some cases, the concentration levels of THC and / or THCA must meet prescribed levels at any point during the production process. For example, in the United States, federal law requires manufacturers wishing to produce CBD concentrates to ensure that the total THC content does not exceed a threshold of 0.3% w / w at any step of the production process. Current extraction techniques, such as CO2 or low-temperature ethanol, yield intermediates with THC content exceeding the permissible 0.3% w / w level through chromatography or crystallization.

[0039] Chromatography can be a very time-consuming and expensive process, and there are some limitations to scale-up. In addition, chromatographic purification methods (such as rapid chromatography) can have a significant environmental impact because they often involve large quantities of harmful or toxic solvents running at high flow rates.

[0040] Crystallization also involves the use of large amounts of organic solvents (such as pentane or heptane), making the process dangerous and environmentally unfriendly. Summary of the Invention

[0041] The applicant notes that even though methods for obtaining cannabinoid acid concentrates free of THCA or THC are known, they result in very long and expensive operations and have limitations that require improvement, particularly in the following areas: retention of cannabinoid acid, cannabinoid concentration, efficiency, cost-effectiveness, environmental impact, use of toxic or harmful solvents, presence of residual organic solvents in the product, and THC and / or THCA concentration levels throughout the production process in compliance with the regulatory framework.

[0042] For example, the applicant noted that while WO 2018 / 130682 provides a novel, environmentally friendly, enzyme-assisted lipid-based extraction method that demonstrates significant efficiency in extracting and stabilizing cannabinoids (even in their original acidic form), this method has some limitations in obtaining concentrates (cannabinoid content >40%), particularly when starting with materials with low cannabinoid content (e.g., cannabis biomass). Furthermore, such methods cannot selectively separate acidic and neutral forms in lipid extracts. Finally, such methods cannot yield cannabinoid concentrates free of THCA or THC.

[0043] The applicant also noted that purification techniques commonly used to purify cannabinoid concentrates typically involve extraction, concentration, and purification processes that result in the decarboxylation of cannabinoid acids and require the use of organic solvents.

[0044] Therefore, the applicant believes there is a need for a simpler method to obtain cannabinoid concentrates, or even their acidic forms, that do not contain THCA or THC, and a method that can efficiently produce such concentrates while retaining high levels of cannabinoid acids without using any organic solvents or expensive techniques (such as chromatography) to remove THC or THCA. This would be a healthier and safer method for workers and consumers, as well as a more environmentally friendly and convenient solution.

[0045] Therefore, the object of the present invention is to provide a method for preparing cannabinoid concentrates without the use of organic solvents, wherein the contents of THC and THCA are significantly reduced or reduced to undetectable levels, enabling the acquisition of high concentrations of cannabinoids while retaining cannabinoid acids other than THC and THCA. This method is efficient, economical, and environmentally friendly, even when starting from materials with low cannabinoid content (e.g., cannabis biomass).

[0046] Therefore, in a first aspect, the present invention relates to a method for preparing a cannabinoid concentrate, the concentrate comprising more than 50% by dry weight of cannabinoids relative to the total dry weight of the concentrate, wherein the weight ratio (THCA):(other cannabinoid acids) is less than 1:50, the method comprising the following steps:

[0047] a) Provide a lipid extract, relative to the total dry weight of cannabinoids, said lipid extract comprising at least 1% by dry weight of cannabinoid acids, wherein the weight ratio (THCA):(other cannabinoid acids) is less than 1:2;

[0048] b) The lipid extract is mixed with an alkaline aqueous solution to form a mixture with a pH of at least 12;

[0049] c) Separate the aqueous phase containing cannabinoid salts from the mixture of step b); and

[0050] d) Obtain the cannabinoid concentrate.

[0051] Surprisingly, the applicant has discovered that by applying the method, cannabinoid products can be obtained, without the use of organic solvents, having not only i) a higher content of cannabinoids and ii) a higher content of cannabinoid acids, but also iii) a lower THCA and / or THC content and iv) a reduced (THCA):(other cannabinoid) ratio compared to the starting lipid extract.

[0052] Furthermore, the applicant discovered that by applying the method, it is possible to obtain cannabinoid concentrates with a THCA content not exceeding 0.3% w / w in any intermediate at any step of the production process, fully complying with regulatory requirements.

[0053] Due to the specific conditions of the method according to the invention, a cannabinoid concentrate was indeed obtained, which showed an unexpectedly high level of retention of cannabinoid acids other than THCA.

[0054] In another aspect, the present invention relates to a concentrate comprising more than 50% by weight of cannabinoid acid relative to the total dry weight of the concentrate, and comprising no more than 0.3% by weight of total THC relative to the total dry weight of the concentrate.

[0055] In another aspect, the present invention also relates to a cannabinoid concentrate comprising, relative to the total dry weight of the concentrate, greater than 50% by weight of cannabinoid acid and not more than 0.0001% by weight of one or more organic solvents selected from acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, propane, toluene, m-xylene, o-xylene, and p-xylene-heptane.

[0056] The advantages of these cannabinoid concentrates according to the invention have been disclosed in the method according to the first aspect of the invention and will not be repeated here.

[0057] Due to its composition and purity characteristics, the cannabinoid concentrate can be advantageously used in the preparation of pharmaceutical or nutritional products, cosmetics, food or feed products, antimicrobial agents, antibacterial agents, insecticides or biopesticides containing one or more cannabinoids.

[0058] Therefore, in another aspect, the present invention relates to methods for preparing pharmaceutical products, nutritional products, cosmetic products, food products, feed products, antimicrobial agents, antibacterial agents, insecticides, and biopesticides, comprising the following steps:

[0059] Providing cannabinoid concentrates according to the invention and / or preparing cannabinoid concentrates according to the invention; and

[0060] Obtain pharmaceutical products, nutritional products, cosmetic products, food products, feed products, antimicrobial agents, antibacterial agents, insecticides, and biopesticides containing one or more cannabinoids. Attached Figure Description

[0061] Figure 1 The HPLC chromatogram of the lipid extract of Example 1 is shown;

[0062] Figure 2 The HPLC chromatogram of the lighter oil phase obtained from the separation step of Example 1 is shown;

[0063] Figure 3 The HPLC chromatogram of the intermediate viscous phase obtained from the separation step of Example 1 is shown; and

[0064] Figure 4The HPLC chromatograms of the heavier aqueous phase obtained from the separation step of Example 1 and the precipitate obtained from the heavier aqueous phase are shown. Detailed Implementation

[0065] In a first aspect, the present invention relates to a method for preparing a cannabinoid concentrate, the concentrate comprising more than 50% by dry weight of cannabinoids relative to the total dry weight of the concentrate, wherein the weight ratio (THCA):(other cannabinoid acids) is less than 1:50, the method comprising the following steps:

[0066] a) Provide a lipid extract, relative to the total dry weight of cannabinoids, said lipid extract comprising at least 1% by dry weight of cannabinoid acids, wherein the weight ratio (THCA):(other cannabinoid acids) is less than 1:2;

[0067] b) The lipid extract is mixed with an alkaline aqueous solution to form a mixture with a pH of at least 12;

[0068] c) Separate the aqueous phase containing cannabinoid salts from the mixture of step b); and

[0069] d) Obtain the cannabinoid concentrate.

[0070] Surprisingly, the applicant has discovered that by applying the method, cannabinoid products can be obtained, without the use of organic solvents, having not only i) a higher content of cannabinoids and ii) a higher content of cannabinoid acids, but also iii) a lower total THC content and iv) a reduced (THCA):(other cannabinoid) ratio compared to the starting lipid extract.

[0071] Furthermore, the applicant discovered that by applying the method, it is possible to obtain cannabinoid concentrates with a THCA content not exceeding 0.3% w / w in any intermediate at any step of the production process, fully complying with regulatory requirements.

[0072] Within the framework of this specification and in the following claims, unless otherwise stated, all numerical entities representing quantities, parameters, percentages, etc., shall in all cases be understood to be preceded by the term "about". Furthermore, in addition to the ranges specifically indicated below, all ranges of numerical entities include all possible combinations of maximum and minimum values, and include all possible intermediate ranges.

[0073] The following lists the definitions of various terms used to describe the invention. These definitions apply to the terminology used throughout this specification and claims, unless otherwise limited in the specific circumstances, either individually or as part of a larger group.

[0074] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Typically, the terms used herein and laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are well-known and commonly used in the art.

[0075] As used herein, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical objects of that article. For example, “an element” means one or more elements. Furthermore, the term “including” and the use of other forms such as “include,” “includes,” and “included” are not restrictive.

[0076] As used herein, the term "cannabinoid" includes, but is not limited to, cannabinol (CBN), cannabinolic acid (CBNA), Δ(9)-tetrahydrocannabinol (Δ(9)-THC), Δ(9)-tetrahydrocannabinolic acid (Δ(9)-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), Δ(8)-tetrahydrocannabinol (Δ(8)-THC), Δ(8)-tetrahydrocannabinolic acid (Δ(8)-THCA), cannabivarin (CBV), cannabivarinic acid, cannabigerol (CBG), cannabichromene (CBGA), cannabichromene (CBC), cannabicyclophenolic acid (CBCA), cannabicyclol (CBL), and cannabicyclolic acid. Cannabidiol (CBLA), cannabidiol (CBDV), and cannabidiol acid (CBDVA).

[0077] As used herein, the term “THC” refers to tetrahydrocannabinol, including its isomers Δ(9)-tetrahydrocannabinol (Δ(9)-THC) and Δ(8)-tetrahydrocannabinol (Δ(8)-THC).

[0078] As used in this article, the term "CBD" refers to cannabidiol.

[0079] As used herein, the term “THCA” refers to tetrahydrocannabinol acid, including its isomers Δ(9)-tetrahydrocannabinol acid (Δ(9)-THCA) and Δ(8)-tetrahydrocannabinol acid (Δ(8)-THCA).

[0080] As used in this article, the term "CBDA" refers to cannabidiol.

[0081] As used herein, the term “cannabinoid acid” includes, but is not limited to, cannabidiol (CBDA), cannabinol (CBNA), cannabipolyphenolic acid (CBGA), cannabicyclic cyclopolyphenolic acid (CBCA), cannabicyclic acid (CBLA), cannabidiol (CBDVA), cannabigerovarinic acid (CBGVA), tetrahydrocannabiabivarinicacid (THCVA), cannabichromevarinic acid (CBCVA), cannabidiphorol acid (CBDPA), and Δ9-tetrahydrocannabiphorol acid (THCPA).

[0082] As used herein, the term "total THC" equals the sum of the THCA content multiplied by the molecular weight ratio 0.877 and the THC content, as shown in the following formula:

[0083] Total THC = THCA x 0.877 + THC.

[0084] As used herein, the term "terpene" includes, but is not limited to, pinene, limonene, α-terpinene, terpinene-4-ol, carvacrol, carvacrol, 1,8-cineole, p-cymene, anisone, β-myrcene, cannaflavin A, cannaflavin B, nerolidol, phytol, and squalene.

[0085] As used herein, the term "lipid" includes, but is not limited to, olive oil, coconut oil, vegetable oil, milk, butter, liposomes, glycerol, polyethylene glycol, ethyl acetate, d-limonene, liquid paraffin, butylene glycol, propylene glycol, and ethylhexyl palmitate.

[0086] As used herein, the term “about” will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. As used herein, when referring to measurable values, such as quantity, duration, etc., the term “about” means a variation covering ±20% or ±10% of a particular value, including ±5%, 1%, and ±0.1%, such variation being suitable for implementing the disclosed methods.

[0087] The present invention may exhibit one or more of the features disclosed below in one or more of the above aspects.

[0088] Other features and advantages of the invention will become clearer from the following description of some preferred embodiments thereof, which are described below by way of non-limiting examples with reference to the following exemplary embodiments.

[0089] In a preferred embodiment, the total THC content in any stream at any step of the method according to the invention does not exceed 0.3% dry weight. In other words, none of the solutions, phases, extracts or other streams used or available according to the method contain more than 0.3% dry weight of total THC.

[0090] The method according to the invention includes step a) providing a lipid extract, relative to the total dry weight of cannabinoids, said lipid extract comprising at least 1% by dry weight of cannabinoid acids, wherein the weight ratio (THCA):(other cannabinoid acids) is less than 1:2.

[0091] Preferably, the lipid extract in step a) comprises no more than 1% by dry weight of THCA.

[0092] Preferably, the lipid extract of step a) comprises at least 20% by dry weight of cannabinoid acid relative to the total cannabinoid weight.

[0093] Preferably, the lipid extract has a total cannabinoid content of at least 2% by dry weight, more preferably at least 3% by dry weight, and even more preferably at least 5% by dry weight.

[0094] Preferably, the lipid extract has a cannabinoid acid content of at least 1% dry weight, more preferably at least 2% dry weight, and even more preferably at least 3% dry weight, wherein more preferably, the cannabinoid acid is selected from cannabinoid acid (CBGA) and tetrahydrocannabinoid acid (CBDA).

[0095] Preferably, the lipids in the lipid extract of step a) are selected from: vegetable oils, milk, butter, liposomes, ethyl acetate, glycerol, d-limonene, caryophyllene, liquid paraffin, mineral oil, paraffin wax, microcrystalline wax, mineral wax, ceresin, polyethylene, polyoxyethylene, and hydrocarbon waxes derived from carbon monoxide and hydrogen, sucrose wax; cetyl ester; hydrogenated jojoba oil, butanediol, propylene glycol, polyethylene glycol, liposomes, lecithin, ethylhexyl palmitate, or mixtures thereof.

[0096] In another embodiment, the lipid is a vegetable oil.

[0097] Preferably, the vegetable oil is selected from: olive oil, coconut oil, sunflower seed oil, sesame oil, hemp seed oil, medium-chain triglyceride (MCT) oil, or mixtures thereof.

[0098] In one embodiment, the lipid is olive oil. In another embodiment, the lipid is coconut oil. In another embodiment, the lipid is sunflower seed oil. In another embodiment, the lipid is medium-chain triglyceride (MCT) oil. In one embodiment, the lipid is milk. In another embodiment, the lipid is butter. In another embodiment, the lipid is liquid paraffin.

[0099] Preferably, the lipid is a vegetable oil with a free fatty acid (FFA) content of less than 1%, more preferably less than 0.5%, and even more preferably less than 0.1%.

[0100] In a preferred embodiment of the method according to the invention, the lipid extract containing cannabinoids in step a) is obtained from cannabinoid-containing biological material, preferably selected from plants, algae, bacteria, yeast, fungi, genetically engineered microorganisms or mixtures thereof.

[0101] In other words, the method according to the invention preferably includes the step of obtaining a lipid extract containing cannabinoids from a biological material containing cannabinoids, preferably, the biological material being selected from plants, algae, bacteria, yeast, fungi, genetically engineered microorganisms or mixtures thereof.

[0102] In a more preferred embodiment, the lipid extract containing cannabinoids of step a) is obtained by contacting the biomaterial containing cannabinoids with plant oil.

[0103] In one embodiment, a starting lipid extract is obtained by mixing the lipid with a cannabinoid extract obtained by supercritical CO2 extraction or by an organic solvent.

[0104] Preferably, the lipid extract of step a) is obtained from biomaterials containing cannabinoids through the following steps:

[0105] I. Crush biological materials containing cannabinoids;

[0106] II. The pulverized biological material is mixed with lipids to obtain a mixture;

[0107] III. Stir the mixture within a temperature range of 1 to 80°C; and

[0108] IV. Obtain the lipid extract from the mixture of step III.

[0109] Preferably, the cannabinoid-containing biological material is selected from plants, algae, bacteria, yeast, fungi, genetically engineered microorganisms, or mixtures thereof.

[0110] In step I, the biomaterial is pulverized to increase surface contact. Water and lipids are then added to the plant material to form a homogeneous mixture or slurry; the mixture can be stirred by agitation or other agitation methods, preferably for at least 30 minutes. Ultrasonic / ultrasonic treatment, microwave, or steam blasting can be advantageously used before or after adding lipids to the mixture to reduce the time required to achieve dissolution of the biomaterial and high cannabinoid lipid extraction rates.

[0111] The resulting mixture is then separated, for example by density separation (i.e., centrifugation) or pressure filtration (French pressure filtration) and / or filtration, to recover the highly cannabinoid-rich and wax-free lipid fraction.

[0112] In the preferred embodiment described above, steps I and II can also be interchanged.

[0113] Preferably, the cannabinoid-containing biological material is selected from plants of the genus *Cannabis*, wherein the biological material is a pure, hybrid, or genetically modified variant. Preferably, the cannabinoid-containing biological material is selected from plants of the genus *Cannabis*, belonging to the following species: *Cannabis sativa* (hemp), *Cannabis indica*, and *Cannabis ruderalis*.

[0114] Preferably, the cannabinoid-containing biomaterial is industrial hemp of the species *C. sativa*. In the context of this invention, preferred hemp plant material is fiber hemp or industrial hemp, specifically the following varieties: Fedora 17, Felina 34, Ferimon 12, Futura 75, Carmagnola, and Santhica 70, which have a relatively high CBDA content by weight%.

[0115] Preferably, the water content of the biomaterial containing cannabinoids is at least 20% of the weight of the biomaterial.

[0116] Preferably, the cannabinoid-containing biological material is freshly harvested and has a moisture content of at least 30%, preferably at least 40%.

[0117] Preferably, the biological material can be used in step I of the method according to the invention, either fresh or dried. In one embodiment, the biological material is newly harvested and contains a high level of moisture; in such cases, it is not necessary to add additional water to the biological material.

[0118] Preferably, the biomaterial containing cannabinoids has a total cannabinoid content of at least 0.1% by weight, more preferably at least 0.2% by weight, even more preferably at least 1% by weight, and even more preferably at least 2% by weight.

[0119] Preferably, the biomaterial contains at least 0.5% by weight of cannabinoids.

[0120] Preferably, the biomaterial containing cannabinoids is industrial hemp containing less than 0.6% by weight of total THC, more preferably less than 0.2% by weight of total THC, or hemp containing more than 0.2% by weight of total THC, more preferably more than 0.6% by weight of total THC, or hybrids and genetically modified variants thereof.

[0121] In a preferred aspect, the biological material is selected from buds, flowers, leaves, stems, trunks, roots, and seeds, or mixtures thereof. In one embodiment, the biological material includes seeds. In another embodiment, when the biological material includes seeds, no lipids are added. In yet another embodiment, when the biological material includes seeds, lipids are added. Biological material including seeds may be rich in lipids, and therefore may not require further addition of lipids.

[0122] Preferably, the lipids in step II have been neutralized before being added to the mixture.

[0123] Preferably, the enzyme is added to the mixture in step II.

[0124] Preferably, the enzyme is independently selected from one or more of the group consisting of: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases, cellulases, hemicellulases, xylanases, glucans, β-glucans, pectins, glucuronyltransferases, lipases, amylases, α-amylases, β-amylases, phospholipases, arabinoseases, galactosidases, β-mannanases, proteases, phytases, cannabinoid synthases, THC synthases, CBD synthases, and CBCA synthases.

[0125] In one embodiment, the enzyme is a cellulase. In another embodiment, the enzyme is a β-glucosidase. In another embodiment, the enzyme is a hemicellulase. In another embodiment, the enzyme is a xylanase. In another embodiment, the enzyme is a glucanase. In another embodiment, the enzyme is a pectinase. In another embodiment, the enzyme is an amylase. In another embodiment, the enzyme is a lipase or phospholipase. In another embodiment, the enzyme is a glucuronyl transferase or alcohol dehydrogenase. In another embodiment, the enzyme is an arabinase. In another embodiment, the enzyme is a phytase. In another embodiment, the enzyme is a protease. In another embodiment, the enzyme is a cannabinoid synthase. In another embodiment, the enzyme is a THCA synthase. In another embodiment, the enzyme is a CBDA synthase. In another embodiment, the enzyme is a CBCA synthase.

[0126] Preferably, the temperature in step II varies within the range of 40 to 70°C. Preferably, the pH value in step II varies within the range of 4 to 6.

[0127] Preferably, the aqueous phase in step III is degummed with phosphoric acid.

[0128] Preferably, step IV includes separating the mixture into a lipid phase, an aqueous phase, and a solid phase; wherein the lipid phase includes a lipid extract.

[0129] In one embodiment, the mixture is separated by density separation in step IV. In another embodiment, the mixture is separated by pressure filtration and / or filtration in step IV.

[0130] In another embodiment, the mixture is separated into a lipophilic phase and a wet solid phase in step IV.

[0131] In one embodiment, the fat-soluble extract is recycled an arbitrary number of times to achieve a higher cannabinoid or terpene content.

[0132] In one embodiment, the fat-soluble extract is recycled an arbitrary number of times to achieve a higher cannabinoid content.

[0133] In another embodiment, at least 50%, preferably 70%, of the terpenoids, at least 70% of the diterpenoids and at least 50%, preferably 70% of the monoterpenes contained in the plant material are extracted into a fat-soluble extract.

[0134] In another embodiment, at least 70% of the sesquiterpenes and at least 50% of the monoterpenes contained in the plant material are extracted into a fat-soluble extract.

[0135] In one embodiment, the fat-soluble extract has a total cannabinoid content of at least 2% by weight. In another embodiment, the fat-soluble extract has a total cannabinoid content of at least 3% by weight. In yet another embodiment, the fat-soluble extract has a total cannabinoid content of at least 5% by weight.

[0136] In one embodiment, the two main cannabinoids in the fat-soluble extract are preferably CBGA and CBDA, or any cannabinoid other than THCA.

[0137] Preferably, during steps I to IV of obtaining a lipid extract containing cannabinoids from a biological material containing cannabinoids, less than 10%, preferably less than 5%, more preferably less than 2% of the cannabinoids are decarboxylated.

[0138] In one embodiment, the lipid extract of step a) is not winterized before being mixed with the alkaline solution of step b). In a preferred embodiment of the method according to the invention, the aqueous phase obtained from the step of separating the mixture into a lipid phase, an aqueous phase, and a solid phase (wherein the lipid phase comprises the lipid extract) can also be used to produce nutritional products, antimicrobial products, antibacterial products, or biopesticides.

[0139] The method according to the invention includes step b), mixing the lipid extract with an alkaline aqueous solution to form a mixture with a pH of at least 12.

[0140] Preferably, the alkaline aqueous solution in step b) comprises at least one hydroxide of at least one metal selected from alkali metals and alkaline earth metals.

[0141] Preferably, the alkaline aqueous solution in step b) includes NaOH, KOH, or a mixture thereof.

[0142] Preferably, the alkaline aqueous solution in step b) is 0.5M NaOH or KOH.

[0143] More preferably, the alkaline aqueous solution in step b) is 0.1M NaOH or KOH.

[0144] Preferably, in step b), the alkaline aqueous solution is added to the lipid extract at a weight ratio of at least 2:1 (alkaline aqueous solution): (lipid extract), more preferably at least 3:1, and even more preferably at least 4:1.

[0145] Preferably, in step b), the NaOH molar concentration of the alkaline aqueous solution is calculated based on the total acidity of the lipid extract, which is expressed as the number of KOH moles required for the acidic titration of the lipid extract, such that the ratio of NaOH mol / KOH mol is 5 to 12, or even more preferably 7 to 10.

[0146] Preferably, the mixture in step b) is mixed at 25°C for at least 5 seconds to less than 60 minutes, more preferably 5 to 20 minutes, and even more preferably 5 to 10 minutes. In the case of continuous mixing, the contact time between the lipid extract and the alkaline solution is preferably less than 5 minutes.

[0147] Preferably, the pH of the mixture in step b) is 12.6 to 13.5, and most preferably about 13.

[0148] The method according to the invention includes step c), separating an aqueous phase containing cannabinoid salts from the mixture of step b).

[0149] Preferably, before step c), at least one salt and / or at least one sugar are added to the mixture in step b).

[0150] By adding at least one salt and / or at least one sugar to the mixture of step b), the density of the aqueous phase is increased, thereby facilitating its separation in step c).

[0151] Preferably, the at least one salt is selected from sodium chloride (NaCl) and calcium chloride (CaCl2).

[0152] Preferably, the at least one sugar is selected from glucose and fructose.

[0153] In a preferred embodiment, the at least one salt is sodium chloride.

[0154] Preferably, the at least one salt is calcium chloride.

[0155] Advantageously, when the at least one salt is calcium chloride, the salt can then be removed after separating the aqueous phase in step c).

[0156] In one embodiment, when the method according to the invention includes the addition of at least one salt and / or at least one sugar prior to step c), the separation in step c) comprises separation from the mixture of step b):

[0157] The lighter oil phase,

[0158] intermediate viscous phase, and

[0159] The heavier aqueous phase, which is the aqueous phase containing cannabinoids.

[0160] In this way, three phases are advantageously obtained. The lighter oil phase contains neutral cannabinoids and most of THCA, but the cannabinoid acids other than THCA are depleted; the intermediate viscous phase contains impurities and the remaining amount of THCA; and the heavier aqueous phase contains cannabinoid salts, which are different from the salts of THCA, given that THCA is divided into three parts in this three-phase system.

[0161] Preferably, the lighter oil phase, after being depleted of cannabinoid acid, is distilled to recover decarboxylated cannabinoids and / or terpenes.

[0162] In this way, fractions suitable as broad-spectrum oil components can be obtained advantageously.

[0163] The method according to the invention includes step d), obtaining the cannabinoid concentrate.

[0164] Preferably, step d) includes filtering the aqueous phase from step c).

[0165] Preferably, the filtration is performed using a glass fiber filter or a paper filter.

[0166] Preferably, the filter is made of a filter with a pore size of less than 2.5 μm, more preferably less than 2 μm, even more preferably 0.5 to 1.8 μm, and even more preferably about 1 μm.

[0167] Preferably, step d) includes:

[0168] i. Acidify the aqueous phase of step c) to a pH value less than 4, thereby forming a precipitate including cannabinoid acid; even more preferably, acidify to a pH value of 1 to 2.

[0169] ii. Separate the precipitate from step i from the remaining aqueous phase, wherein the precipitate is the cannabinoid concentrate.

[0170] Preferably, step d) includes:

[0171] i. Adjust the pH of the aqueous phase in step c) to 12.5 to 9 to form a first precipitate;

[0172] ii. Separate the first precipitate from the remaining aqueous phase, wherein the first precipitate contains impurities, such as free fatty acids;

[0173] iii. Adjust the pH of the remaining aqueous phase from step ii to less than 12.5 to form a second precipitate;

[0174] iv. Separate a second precipitate from the remaining aqueous phase, wherein the second precipitate is the cannabinoid concentrate.

[0175] Preferably, step d) includes drying the aqueous phase to form a dried product, wherein the dried product is the cannabinoid concentrate.

[0176] Preferably, the cannabinoid concentrate comprises less than 0.3% by weight of total THC relative to the total dry weight of the concentrate, more preferably less than 0.1% by weight of total THC.

[0177] Preferably, the cannabinoid concentrate comprises less than 0.05% by weight of total THC relative to the total dry weight of the concentrate.

[0178] Preferably, the cannabinoid concentrate comprises at least 75% by weight of cannabinoids relative to the total dry weight of the concentrate.

[0179] Preferably, the cannabinoid concentrate comprises at least 70% by weight of cannabinoid acids other than THCA, relative to the total dry weight of the concentrate.

[0180] Preferably, the THCA content of the cannabinoid concentrate in step d) is lower than the THCA content of the lipid extract in step a).

[0181] Preferably, the total THC of the cannabinoid concentrate in step d) is lower than the total THC of the lipid extract in step a).

[0182] Preferably, step d) includes drying, decarboxylating, distilling, or crystallizing the cannabinoid concentrate.

[0183] In another aspect, the present invention relates to cannabinoid concentrates that can be obtained by the method of the first aspect of the present invention.

[0184] Due to the specific conditions of the method according to the invention, a cannabinoid concentrate was indeed obtained, which showed an unexpectedly high level of retention of cannabinoid acids other than THCA.

[0185] In another aspect, the present invention relates to cannabinoid concentrates, which comprise more than 50% by weight of cannabinoid acid relative to the total dry weight of the concentrate, and comprise no more than 0.3% by weight of total THC relative to the total dry weight of the concentrate.

[0186] In another aspect, the present invention also relates to cannabinoid concentrates, which, relative to the total dry weight of the concentrate, comprise greater than 50% by weight of cannabinoid acid and not more than 0.0001% by weight of one or more organic solvents selected from acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, propane, toluene, m-xylene, o-xylene, and p-xylene-heptane.

[0187] In another aspect, the present invention also relates to cannabinoid concentrates, which comprise more than 50% by weight of cannabinoids and less than 0.3% by weight of total THC relative to the total dry weight of the concentrate.

[0188] In another aspect, the present invention also relates to cannabinoid concentrates, which comprise more than 50% by weight of cannabinoid acid and less than 0.3% by weight of total THC relative to the total dry weight of the concentrate.

[0189] In another embodiment, the cannabinoid concentrate advantageously exhibits a lower THCA content relative to the total dry weight of the concentrate compared to the THCA content of the starting biological material.

[0190] In another embodiment, the cannabinoid concentrate advantageously exhibits a lower THCA content relative to the total dry weight of the concentrate compared to the THCA content of the lipid extract of step a).

[0191] In another embodiment, the cannabinoid concentrate advantageously exhibits a lower total THC content relative to the total dry weight of the concentrate compared to the total THC content of the starting biological material and / or lipid extract.

[0192] The applicant has noted that the combination of high cannabinoid acid content with particularly low THC and / or THCA content and the absence or at most very low levels of organic solvents is particularly surprising compared to prior art concentrates, in which high cannabinoid acid content is typically achieved through concentration or purification processes that result in an increase in THC and / or THCA, or involves the use of organic solvents whose removal can be troublesome, costly, and non-compliant with regulations.

[0193] Other advantages of the cannabinoid concentrate according to the invention have been disclosed in the method according to the first aspect of the invention and will not be repeated here.

[0194] Preferably, the cannabinoid concentrate comprises more than 70% by weight, or even more preferably more than 85% by weight, cannabinoid acid relative to the total dry weight of the concentrate.

[0195] Preferably, the cannabinoid concentrate according to the invention comprises less than 0.1% total THC relative to the total dry weight of the concentrate.

[0196] Preferably, in the cannabinoid concentrate according to the present invention, the cannabinoid acid is selected from: cannabidiol (CBDA), cannabinol (CBNA), cannabinoid acid (CBGA), cannabinoid acid (CBCA), cannabinoid acid (CBLA), hypocannabidiol (CBDVA), hypocannabinoid acid (CBGVA), tetrahydrohypocannabinol (THCVA), hypocannacroxyl (CBCVA), cannabidiphorol acid (CBDPA), and Δ9-tetrahydrocannabiphorol acid (THCPA).

[0197] Preferably, in the cannabinoid concentrate according to the invention, the cannabinoid acid is CBDA. Preferably, in the cannabinoid concentrate according to the invention, the cannabinoid acid is CBGA.

[0198] Therefore, according to the present invention, a cannabinoid concentrate is provided.

[0199] Due to its composition and purity characteristics, the cannabinoid concentrate can be advantageously used in the preparation of pharmaceutical or nutritional products, cosmetics, food or feed products, antimicrobial agents, antibacterial agents, insecticides or biopesticides containing one or more cannabinoids.

[0200] Therefore, in another aspect, the present invention relates to methods for preparing pharmaceutical products, nutritional products, cosmetic products, food products, feed products, antimicrobial agents, antibacterial agents, insecticides, and biopesticides, comprising the following steps:

[0201] Providing cannabinoid concentrates according to the invention and / or preparing cannabinoid concentrates according to the invention; and

[0202] Obtain pharmaceutical products, nutritional products, cosmetic products, food products, feed products, antimicrobial agents, antibacterial agents, insecticides, and biopesticides containing one or more cannabinoids.

[0203] Other features and advantages of the invention will become clearer from the following description of some preferred embodiments thereof, which are described below by way of non-limiting examples with reference to the following exemplary embodiments.

[0204] Experimental Section

[0205] Example 1

[0206] A refined soluble extract (“lipid extract”) based on sunflower seed oil, obtained according to Example 1 of WO 2018 / 130682, is provided, having the composition determined by HPLC analysis as reported in Table 1. Figure 1 ).

[0207] Table 1

[0208] weight% THCA THC CBDA CBD lipid extracts 0.06 <0.05 3.10 0.80

[0209] At room temperature, 100 g of the extract was mixed with 480 g of 0.355 M NaOH aqueous solution in a Mulinex Companion kitchen robot to achieve a pH of approximately 13. The mixture was stirred for approximately 15 min. Approximately 40 g of NaCl was added to the mixture, and it was mixed for 5 min, followed by centrifugation. After centrifugation (4500 rpm, 10 min), 86 g of the lighter oil phase, 9 g of the intermediate semi-solid layer, and 525 mL of the heavier aqueous phase were recovered. Samples of the lighter oil layer and the intermediate layer were analyzed for cannabinoids by HPLC (see [link to HPLC analysis]). Figure 2 and Figure 3 ).

[0210] The heavier aqueous phase was filtered using a laboratory vacuum filter with a glass fiber filter having a pore size of approximately 1.6 μm. Approximately 520 mL of the filtered aqueous solution was recovered. Samples were collected and the cannabinoid content was analyzed.

[0211] The filtered aqueous phase was acidified with an 85% H3PO4 solution to achieve a pH of approximately 1.5–2, thereby precipitating cannabinoid acid. Cannabinoid acid was recovered using a laboratory vacuum filter with a 1.6 μm pore size glass fiber filter. After air-drying at room temperature for 24 hours, 3.06 g of cannabinoid precipitate was recovered. The cannabinoid precipitate was sampled and sent for HPLC analysis. Figure 4 ).

[0212] Quantitative determination of cannabinoids (CBD, CBDA, THCA) was performed using HPLC-DAD Shimadzu Nexera XR, equipped with a reversed-phase C18 column (NexLeaf CBX) with a titer of 150 x 4.6 mm and 2.7 μm.

[0213] Chromatographic conditions: Solvent A: Water + 0.085% phosphoric acid (v / v); Solvent B: Acetonitrile + 0.085% phosphoric acid (v / v). Flow rate: 1.6 mL / min. Column oven temperature: 35℃. Manual injection: 20 μL quantitative loop. Detection wavelength: 228 nm for CBD, THCA, and THC; 307 nm for CBDA. Gradient elution: 70% B to 3 min, 85% B to 7 min, 95% B to 7.01 to 8.00 min, 70% B to 10 min.

[0214] Retention time: CBDA approximately 3.5 min; CBD approximately 4 min; CBN approximately 5.5 min; THC approximately 6.5 min; THCA approximately 7.5 min.

[0215] Figure 1 The HPLC chromatogram of the lipid extract is shown. Figure 2 The HPLC chromatogram of the lighter oil phase is shown. Figure 3 The HPLC chromatogram of the intermediate viscous phase is shown. Figure 4 The HPLC chromatograms of the heavier aqueous phase obtained therefrom and the precipitate obtained from the heavier aqueous phase are shown, indicating the assignment of the cannabinoid (CBDA, CBD, THC, THCA) chromatographic peaks.

[0216] Using pure standards of cannabinoids (CBD, CBDA, THCA) in the range of 2.5 to 250 ppm, the cannabinoids were quantified by external standard method after preparing calibration curves.

[0217] The quantification of THC was performed based on the CBD calibration curve, using the relative response factor (RRF) reported in the pharmacopoeia analysis monograph (OMC / Farmalyse BV Version 7.1 / November 28, 2014).

[0218] The instrument's limit of detection (LOD) is 0.5 ppm, and its limit of quantitation (LOQ) is 2.5 ppm.

[0219] The table below reports the LOD and LOQ values ​​for each matrix for the analytical method:

[0220] Table 2

[0221]

[0222] The following cannabinoid concentrations (% w / w) in each fraction of the obtained cannabinoids are reported:

[0223] Table 3

[0224] Dry weight % THCA THC CBDA CBD lighter oil phase 0.04 <0.05 0.06 0.78 intermediate phase 0.30 <LOD 0.57 0.21 Filtered water phase <LOD <LOD 81.34 1.98 Sediments from the aqueous phase <LOD <LOD 84.75 2.10

[0225] Considering the extraction efficiency of cannabinoids in different chemical forms, a striking difference was observed. Although the recovery efficiency of CBDA was approximately 84%, neither THCA nor THC was found in the samples obtained from the filtered aqueous phase or in the precipitate obtained after acidifying the aqueous phase. THCA and THC were mostly retained in the lighter oil phase.

[0226] Considering that no organic solvents were used, the cannabinoid content in the precipitate was also very high, approximately 85%.

[0227] This confirms the effectiveness of the method according to the invention, which recovers cannabinoids from the starting lipid extract without causing significant decarboxylation.

[0228] Example 2

[0229] The same test as in Example 1 was performed, except that 5g of calcium chloride was added to the aqueous phase and mixed for 30 minutes before filtration.

[0230] Approximately 2.66 g of precipitate was collected, with a CBDA content of 88.2%, and no detectable THCA or THC. Therefore, calcium chloride helps to achieve higher purity.

[0231] Example 3

[0232] The same tests as in Example 1 were performed, the only difference being that liquid paraffin was used to obtain the lipid extract.

[0233] Table 4

[0234] weight% THCA THC CBDA CBD Lipid extract (paraffin) 0.08 0.07 3.30 1.10

[0235] The following cannabinoid concentrations (% w / w) were obtained in each fraction of cannabinoids:

[0236] Table 5

[0237] weight% THCA THC CBDA CBD lighter oil phase <LOD 0.06 <LOD 0.93 intermediate phase 0.25 <LOD 0.42 0.11 Filtered and dried aqueous phase <LOD <LOD 78.21 1.45 Sediments from the aqueous phase 0.02 <LOD 81.37 2.01

[0238] It can be noted that cannabinoid acid was completely removed from the oil phase. On the other hand, trace amounts of THCA can still be measured in the precipitate.

[0239] Example 4

[0240] The same tests as in Example 1 were performed, the only difference being that the tests started with a lipid extract containing CBGA.

[0241] Table 6

[0242] weight% THCA THC CBGA CBG lipid extracts 0.113 <LOD 9.45 0.12

[0243] The following cannabinoid concentrations (% w / w) were obtained in each fraction of cannabinoids:

[0244] Table 7

[0245]

[0246]

[0247] It can be noted that no THCA and THC were detected in the obtained CBGA concentrate.

[0248] Example 5

[0249] The same tests as in Example 1 were performed, the only difference being that the aqueous phase was acidified in two steps. In the first step, the pH of the aqueous phase was adjusted from approximately 13 to 12 using a 1M H3PO4 solution.

[0250] The aqueous phase was then filtered using a 1.6 μm glass fiber filter to remove the first precipitate. The filtered solution was then further acidified with an 85% H₃PO₄ solution to achieve a pH of approximately 1.5–2, yielding the second precipitate. The cannabinoid content of the first and second precipitates was analyzed. The first precipitate contained 2.1% CBD and 1.07% CBDA, while the second precipitate contained 0.5% CBD and 90.5% CBDA.

[0251] Example 6 (Reference)

[0252] The following steps are followed to obtain an ethanolic lipophilic extract containing cannabinoids (“ethanolic extract”):

[0253] a. Mix 150g of Futura 75 cannabis with 750g of 99.9% pure ethanol at room temperature to form a mixture;

[0254] b. After stirring for 15 min, the mixture was separated into a lighter fraction rich in cannabinoids and an heavier fraction containing waste biomass by centrifugation (4500 rpm, 10 min).

[0255] c. The lighter ethanol fraction was kept overnight at 4°C to precipitate wax (wintering), and then separated by filtration; the winterized ethanol fraction was then sampled and the cannabinoid content was analyzed by HPLC.

[0256] Then, at room temperature, the winterized ethanol extract was mixed with 480 g of 0.355 M NaOH aqueous solution in a Mulinex Companion kitchen robot to achieve a pH of approximately 13. The mixture was stirred for approximately 15 min. Approximately 10 g of NaCl was added to the mixture and mixed for 5 min. The mixture was then filtered using a laboratory vacuum filter with a glass fiber filter having a pore size of approximately 1.6 μm. Approximately 573 g of the filtered solution was recovered. The filtered aqueous phase was acidified with an 85% H3PO4 solution to achieve a pH of approximately 1.5–2, thereby precipitating cannabinoid acid. Cannabinoid acid was recovered using a laboratory vacuum filter with a glass fiber filter having a pore size of 1.6 μm. After air-drying at room temperature for 24 hours, 2.1 g of cannabinoid precipitate was recovered. The cannabinoid precipitate was sampled and sent for HPLC analysis.

[0257] Table 8 reports the cannabinoid concentrations (% w / w) in the ethanol fraction and cannabinoid precipitate:

[0258] Table 8

[0259] Dry weight % THCA THC CBDA CBD Ethanol lipophilic extract 1.5 0.4 36,4 5.2 Sediments from the aqueous phase 2.7 0.1 71 0.4

[0260] It can be noted that when the method according to the invention is applied to ethanol lipophilic extracts, CBDA and THCA are extracted and recovered with similar efficiency, and the THCA content of the resulting concentrate is higher than that of the initial lipophilic extract. Concentrates with total THC (THC+THCA) below the detection limit cannot be obtained. This confirms the effectiveness of the method according to the invention for recovering cannabinoid concentrates from lipid extracts, wherein the total THC content of the cannabinoid concentrate is lower than the total THC content of the initial lipid extract, and preferably lower than the quantification level.

Claims

1. A method for preparing a cannabinoid concentrate, wherein the concentrate comprises more than 50% cannabinoids by dry weight relative to the total dry weight of the concentrate, wherein the weight ratio of THCA to other cannabinoid acids is less than 1:50, the method comprising the following steps: a) Provide a lipid extract, relative to the total dry weight of cannabinoids, said lipid extract comprising at least 1% by dry weight of cannabinoid acids, wherein the weight ratio of THCA: other cannabinoid acids is less than 1:2; b) The lipid extract is mixed with an alkaline aqueous solution to form a mixture with a pH of 12.6 to 13.5; c) Separate the aqueous phase containing cannabinoid salts from the mixture of step b); and d) Obtain the cannabinoid concentrate; Prior to step c), at least one salt and / or at least one sugar are added to the mixture of step b); the separation in step c) includes separation from the mixture of step b): The lighter oil phase, intermediate viscous phase, and The heavier aqueous phase, wherein the heavier aqueous phase is an aqueous phase containing cannabinoid acid salts.

2. The method according to claim 1, wherein the alkaline aqueous solution of step b) comprises at least one hydroxide of at least one metal selected from alkali metals and alkaline earth metals.

3. The method according to claim 1 or 2, wherein in step b), the alkaline aqueous solution is added to the lipid extract at a weight ratio of at least 2:1 (alkaline aqueous solution: lipid extract).

4. The method according to claim 1 or 2, wherein the mixture in step b) is mixed at 25°C for at least 5 seconds to less than 60 minutes.

5. The method according to claim 1 or 2, wherein step d) comprises: i. Acidify the aqueous phase of step c) to a pH value less than 4, thereby forming a precipitate including cannabinoid acid; ii. Separate the precipitate from step i from the remaining aqueous phase, wherein the precipitate is the cannabinoid concentrate.

6. The method according to claim 1 or 2, wherein step d) comprises: i. Adjust the pH of the aqueous phase in step c) to 12.5 to 9 to form a first precipitate; ii. Separating the first precipitate from the remaining aqueous phase, wherein the first precipitate contains impurities; iii. Adjust the pH of the remaining aqueous phase from step ii to less than 12.5 to form a second precipitate; iv. Separate a second precipitate from the remaining aqueous phase, wherein the second precipitate is the cannabinoid concentrate.

7. The method according to claim 1 or 2, wherein step d) comprises drying the aqueous phase to form a dried product, wherein the dried product is the cannabinoid concentrate.

8. The method according to claim 1 or 2, wherein the cannabinoid concentrate comprises less than 0.3% by weight of total THC relative to the total dry weight of the concentrate.

9. The method of claim 8, wherein the cannabinoid concentrate comprises less than 0.1% by weight of total THC relative to the total dry weight of the concentrate.

10. The method according to claim 1 or 2, wherein the THCA content of the cannabinoid concentrate in step d) is lower than the THCA content of the lipid extract in step a).

11. The method according to claim 1 or 2, wherein the THCA content of the cannabinoid concentrate in step d) is lower than the THCA content of the starting biological material.

12. The method according to claim 1 or 2, wherein the total THC of the cannabinoid concentrate in step d) is lower than the total THC of the lipid extract in step a).

13. A method for preparing pharmaceutical products, nutritional products, cosmetic products, food products, feed products, antimicrobial agents, insecticides, and biological pesticides, comprising the following steps: The method for preparing cannabinoid concentrates according to any one of claims 1 to 12; and Obtain pharmaceutical products, nutritional products, cosmetic products, food products, feed products, antimicrobial agents, pesticides, and biopesticides that include one or more cannabinoids.

14. The method according to claim 13, wherein, The antimicrobial agent includes an antifungal agent.

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