Cannabinoid concentrate, method of obtaining the same and use thereof

Abstract

The invention relates to a pesticide-free, liquid cannabinoid concentrate, method of obtaining the same and use thereof.

Description

[0001] Title:

[0002] “CANNABINOID CONCENTRATE, METHOD OF OBTAINING THE SAME AND USE THEREOF”

[0003] ★★★ ★★★ ★★★

[0004] DESCRIPTION

[0005] FIELD OF THE INVENTION

[0006] The invention relates to a pesticide-free, liquid cannabinoid concentrate, method of obtaining the same and use thereof.

[0007] BACKGROUND

[0008] Cannabis sativa L. is a prolific, but not exclusive, producer of a diverse group of isoprenylated resorcinyl polyketides collectively known as cannabinoids (Hanus et al. 2016). Cannabinoids are the lipid based exogenous compounds interacting with the endocannabinoid system. In the last few 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, fungus, or bacteria.

[0009] The endocannabinoid system consists of the endogenous cannabinoids (endocannabinoids), cannabinoid receptors and the enzymes that synthesize and degrade endocannabinoids. Many of the effects of cannabinoids and endocannabinoids are mediated by two G protein-coupled receptors (GPCRs), CB1 and CB2, although additional receptors may be involved. CB1 receptors are present in very high levels in several brain regions and in lower amounts in a more widespread fashion. These receptors mediate many of the psychoactive effects of cannabinoids. CB2 receptors have a more restricted distribution, being found in a number of immune cells and in a few neurons. Both CB1 and CB2 couple primarily to inhibitory G proteins and are subject to the same pharmacological influences as other GPCRs. Thus, partial agonism, functional selectivity and inverse agonism all play important roles in determining the cellular response to specific cannabinoid receptor ligands.

[0010] By interacting with the endocannabinoid system, exogenous cannabinoids or terpenoids, such ones from Cannabis, are used to reduce nausea and vomiting during chemotherapy, to improve appetite in people with HIV / AIDS, and to treat chronic pain and muscle spasms. Cannabis, its constituent cannabinoids, and terpenes are used to treat diseases or improve symptoms. Cannabinoids are synthesized in plants in their carboxylic acid forms. Cannabinoid acids, such as CBGA, THCA, CBDA, CBCA, and CBDVA short for cannabigerolic acid, tetrahydrocannabinolic acid, cannabidiolic acid, cannabichromenic acid and cannabidivarinic acid respectively, are precursors to their metabolites, the neutral forms CBG (cannabigerol), THC (tetrahydrocannabinol), the primary psychotropic cannabinoid found in cannabis, CBD (cannabidiol), CBC (cannabichromene) and CBDV (cannabidivarin). Neutral forms are obtained from acidic forms through decarboxylation.

[0011] The most popular route of cannabinoid administration is inhalation through the form of smoking, vaporization, or “dabbing”. During Cannabis inflorescences burning, carcinogenic compounds are formed such as acetaldehyde, formaldehyde, ammonia, polycyclic aromatic hydrocarbons, phenols, nitrosamines, and many others.

[0012] Vaporization, which relies on heating Cannabis inflorescences or liquid extracts and releasing active forms of cannabinoids at temperatures below the burning point, reduces the number of toxic compounds and consequently the irritation of the respiratory system. It should be noted that changing the inhalation method from smoking to vaporization improved lung function.

[0013] Liquid extracts are products obtained by extracting and concentrating cannabinoids from Cannabis. Vaporization of liquid extracts is becoming a popular route to inhale cannabinoids. However, presence of contaminants in liquid extracts is a growing concern. Cannabis plant is well known for efficiently adsorbing contaminants, including pesticides and heavy metals. Cannabis is able to remove pesticides and heavy metals from substrate soils and deposit these in its tissues, by virtue of its bioaccumulative capacity. Additionally, cross-contamination may occur during processing (e.g., during drying). Additionally, the plant provides host to a variety of organisms and its complex microbiome continues to be deciphered. Grey and academic literature highlight the presence of pathogenic microbial contaminants, particularly bacteria and fungi (mold), within cannabis preparations. Most of the microbial contamination occurs during the improper preparation and storage of cannabis products. For example, harvesting whilst wet, drying and storage under wet, humid conditions can lead to fungal infections such as powdery mildew and botrytis, and budworm or mite infestations. As mandated by the Medicinal and Adult-Use Cannabis Regulation and Safety Act, all cannabis and cannabis products in the legal market of California, the largest in the US, are required to be tested for 68 pesticides, 4 inorganics, 20 solvents, 6 microbes, and 5 mycotoxins. The cannabis manufacturers must submit their products - including cannabis flowers and cannabis products, such as edibles, concentrates, and other consumables - to a state-licensed cannabis testing laboratory. Limits for the concentration of residual solvents and / or pesticides for inhalable products are typically lower than for products such as edibles, as low as Limit of Detection (LCD). For pesticides listed in Category 1 the limit is 0.1 ppm. Inhalable products failing on pesticides content can be obtained from inflorescences that test negative for pesticides due to the concentration action of the extraction method. Therefore, the failure rate on pesticide testing for extracts is often higher than that of flowers. Products that fail the state’s regulatory levels in the compliance testing are subject to recalls. However, it is possible that some of these products end in the black market.

[0014] A wide study of California extracts market, analyzing 5,654 cured cannabis flower samples and 3,760 concentrate samples (including oil extracts and vape cartridges) provided by -300 cannabis producers and manufacturers in various regions in California for compliance testing between June 2020 and October 2021 , found a failure rate of 2.3% and 9.2% for flowers and extracts respectively, against the regulatory limits for pesticides and solvents (Jameson, 2022, Comparison of State-Level Regulations for Cannabis Contaminants and Implications for Public Health). Insecticides represented the largest subcategory, which was followed by herbicides, fungicides, and miticides. The most common pesticides found in the cannabis extracts were boscalid (60 samples), chlorfenapyr (55 samples), chlorpyrifos (50 samples), bifenazate (44 samples), and myclobutanil (41 samples), which was similar to the flower data set analyzed above.

[0015] Chlorfenapyr is a pesticide, and specifically a pro-insecticide, derived from a class of microbially produced compounds known as halogenated pyrroles. The United States Environmental Protection Agency initially denied registration in 2000 for use on cotton primarily because of concerns that the insecticide was toxic to birds and because effective alternatives were available. However, it was registered by the EPA in January 2001 for use on non-food crops in greenhouses. Chlorfenapyr has a solubility in water of about 0.14 mg / L and Log P (Kow) of 4.83. Trifloxystrobin is a systemic broad-spectrum foliar strobilurin fungicides, active against a wide range of fungal plant pathogens, that enters the aquatic environment during agricultural application. Trifloxystrobin has a solubility in water of about 0.16 mg / L and Log P (Kow) of 4.5.

[0016] The presence of these contaminants presents a potential health hazard not only to regular cannabis users and the general public but also to people with specific health conditions that make them susceptible to harmful contaminants. Immunocompromised patients with cancer and the human immunodeficiency virus (HIV), women of reproductive age, and patients with seizures and epilepsy are among those who are more susceptible to the health hazards of pesticide and microbial contaminants.

[0017] All pesticides found in extracts by Jameson et al. are lipophilic compounds that are therefore efficiently extracted by organic solvents, es. butane, or by supercritical CO2.

[0018] Cannabinoid liquid extracts can be produced through several techniques. However, none of them makes no use of organic solvents and eliminates pesticides.

[0019] Cannabinoid concentrates can be obtained from biomass that has been previously dried by means of supercritical fluid extraction (SFE), as with supercritical CO2, followed by a winterization step to remove chlorophyll and waxes. Winterization encompasses the use of ethanol or butane at low temperatures (US 9186386 B2, US 6403126 B1 ). Such process presents several drawbacks such as the high investment required, the need for highly skilled technicians to utilize complex equipment, the use of flammable and harmful organic solvents to winterize the crude extract, the high energy consumption. It is challenging to completely remove organic solvents used in combination with CO2 during the extraction step or to remove chlorophyll in the winterization step. The technical challenge to overcome has led policymakers to set content limits for organic solvents, some of which are known cancerogenic compounds, as high as 5.000 ppm (source Health Canada). Additionally, supercritical CO2 has high selectivity for a wide range of toxic components present in pesticides, therefore a risk associated to their presence in concentrated form in the final product is present. The use of chromatography can be considered to remove pesticides, however that results in further costs and complexities. All these aspects make the whole process not an ideal option to extract and concentrate cannabinoids, especially for inhalable products. A more recent alternative technique to obtain cannabinoid concentrates is represented by cryogenic-ethanol, a process in which a biomass that has been previously dried is extracted at very low temperatures (-40°C) to avoid extraction of chlorophyll and waxes into the solvent. The cannabinoids-enriched ethanol solution is then evaporated to recover the solvent. Such activity is energy intensive and it can be very time consuming, considering the large volumes of solvents to be evaporated (up to 20 times biomass weight). The use of organic solvents inherently results in safety, health and environmental issues. Additionally, as per SFE, eventual pesticides present in the biomass are typically co-extracted along with cannabinoids and concentrated in the extract, requiring the use of post-processing techniques such as expensive chromatography for pesticides removal.

[0020] Another technique for extracting cannabinoids is represented by ice water extraction. The method consists in washing Cannabis with ice water to make trichomes containing cannabinoids brittle, mixing mechanically for breaking trichomes, sieving of water for recovering trichomes mixed with plant material, and pressing the trichomes mixed with plant material in hot press for obtaining a concentrate. The method is being progressively used because is solventless. However, despite making no use of organic solvents, such technique does not discern cannabinoids from contaminants, such as pesticides or heavy metal, which can be concentrated in the final product.

[0021] WO2022049232A1 describes a method to obtain a THC-free concentrate from a lipid extract, wherein the concentrate presents a decreased THCA to other cannabinoids ratio as compared to the ratio THCA to other cannabinoids of the lipid extract. The lipid extract is mixed with an alkaline solution and sodium hydrochloride to form a mixture having a pH of at least 12. An aqueous phase containing cannabinoid salts is then separated from the lipid carrier that still contain THCA and THC. Finally, a THC- and THCA-free concentrate is obtained from the aqueous phase. The Applicant notes that the method does not teach how to obtain a concentrate rich in THC or THCA or a concentrate having a THC content higher than the THC content of the initial lipid extract or of the starting biomass, and / or THC content higher than 70% in weight.

[0022] US20210053900A1 describes a method to recover acidic cannabinoids microcrystalline powder by leaching Butane Hash Oil (BHO) after ethanol winterization and addition of sodium chloride. The Applicant notes that the method comprises dissolving winterized BHO in alcohol, dispersing the BHO / solution into a large volume (400 times) of alkaline solution containing 1 % sodium hydrochloride, forming an emulsion, mixing, letting leached particles to settle, clearing the leachate by filtration or centrifugation, acidifying the leachate to precipitate a microcrystalline powder rich in cannabinoid acids recovered through filtration or centrifugation. The Applicant notes that the solubility of THCA is assessed in the range 20-25 °C. The Applicant further notes that the method does not teach how to obtain a concentrate that is rich in THCA or THC from a lipid extract. Additionally, the Applicant notes that the method does not teach how to obtain a THCA or THC concentrate that is liquid. Furthermore, the Applicant notes that the method requires the use of butane to obtain the initial extract, ethanol for winterizing the initial extract as well as sodium chloride as emulsifier. Finally, the Applicant notes that to provide with an initial extract having a high content of THCA to be leached by the alkaline solution, butane extraction is the only viable option, as CO2 extract and distillates are typically characterized by a high degree of decarboxylation of cannabinoids.

[0023] A method to consistently obtain THCA or THC extracts that are liquid, free of pesticides, making no use of organic solvents, would therefore be desirable.

[0024] There is an increasingly felt need for an improved method for obtaining a pesticide- free cannabinoid-rich concentrate having a high THC content, that is more costefficient and avoids the use of harmful solvents.

[0025] Object of the present invention is the development of a more efficient and cost-effective process for obtaining pesticide-free, THC-rich liquid concentrates that have the advantage of avoiding the use of toxic, environment-threatening chemical substances, such as organic solvents.

[0026] SUMMARY OF INVENTION

[0027] The Applicant noted that, even if methods for obtaining cannabinoids concentrates are known, they result in very long and expensive operations that present several limits and need still to be improved, in particular in terms of cannabinoid concentration, efficiency, cost-effectiveness, environmental impact, utilization of toxic or harmful solvents, presence of residual organic solvents and pesticides in the products.

[0028] For example, the Applicant noted that, even if WO 2018 / 130682 provides a novel and environmentally friendly method of enzyme-assisted lipid-based extraction showing a remarkable efficiency in extracting and stabilizing cannabinoids, even in their original acidic forms, such method presents some limitations in obtaining cannabinoid concentrates having a THC content higher than 40% in weight. Furthermore, the presence of a vegetable oil carrier in the extract does not allow to obtain inhalable products. Finally, such method does not allow to obtain a pesticide-free cannabinoid concentrate.

[0029] The Applicant further noted that, although WO2022 / 049232 provides a method for preparing a THC-free cannabinoid concentrate from lipid extract which avoids the use of organic solvents, the process does not allow to attain a concentrate which is both rich in THC and pesticide-free.

[0030] The Applicant also noted that purification techniques commonly used to purify cannabinoid concentrates and eliminate pesticides typically apply techniques that result in the use of organic solvents.

[0031] Hence, the Applicant felt that a simpler way to obtain THCA and THC solventless liquid concentrates would therefore be desirable and that a process that could efficiently generate such liquid concentrates, preserving a high level of cannabinoids, without making use of any organic solvent or costly techniques, such as chromatography, to purify THC and eliminate pesticides would represent a healthier and safer process for workers and consumers as well as a more environmentally friendly and convenient solution.

[0032] An object of the present invention is therefore the provision of method for preparing a cannabinoid liquid concentrate making no use of organic solvents, wherein the content of pesticide is significantly reduced or brought to undetectable level, capable of attaining a high concentration of cannabinoids, that is efficient, environmentally friendly and cost-effective.

[0033] Therefore, the present invention relates, in a first aspect, to a method for preparing a pesticide-free, liquid cannabinoid concentrate comprising more than 70% by dry weight, with respect to the total dry weight of the concentrate, of Tetrahydrocannabinol, comprising the steps of: a) providing a lipid extract comprising at least 1 % by dry weight of Tetrahydrocannabinolic acid; b) mixing said lipid extract with an alkaline aqueous solution to form Tetrahydrocannabinolic acid salt; c) heating the mixture of step b) to at least 40 °C. d) separating from the mixture of step c) an aqueous phase containing cannabinoid acid salts; e) obtaining from the aqueous phase said pesticide-free, liquid cannabinoid concentrate, wherein the pesticide content is below 0.1 ppm.

[0034] Surprisingly, the Applicant has found out that by applying the described method it’s possible to obtain cannabinoid products not only i) liquid, but also having ii) a higher content of Tetrahydrocannabinol, and also iii) a lower pesticide content, as compared to the starting lipid extract as well as to the starting biomass, making no use of organic solvents at any step.

[0035] The advantages of these cannabinoid concentrates according to the present invention have been disclosed in relation to the method according to the first aspect of the present invention and are not herewith repeated.

[0036] Thanks to its compositional and purity properties, said cannabinoid concentrate may be advantageously used for preparing pharmaceutical or nutraceutical products, cosmetics, food or feed products, antimicrobial, antibacterial, insecticidal or biopesticides containing Tetrahydrocannabinol.

[0037] In a further aspect, therefore, the present invention relates to a method for preparing a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide, comprising the step of:

[0038] - providing a cannabinoid concentrate according to the present invention and / or preparing a cannabinoid concentrate according to the present invention; and

[0039] - obtaining a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide comprising Tetrahydrocannabinol. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention relates, in a first aspect, to a method for preparing a cannabinoid concentrate comprising more than 70% by dry weight, with respect to the total dry weight of the concentrate, of Tetrahydrocannabinol, comprising the steps of: a) providing a lipid extract comprising at least 1 % by dry weight of Tetrahydrocannabinolic acid; b) mixing said lipid extract with an alkaline aqueous solution to form Tetrahydrocannabinolic acid salt; c) heating the mixture of step b) to at least 40 °C. d) separating from the mixture of step c) an aqueous phase containing cannabinoid acid salts; e) obtaining from the aqueous phase said pesticide-free, liquid cannabinoid concentrate, wherein the pesticide content is below 0.1 ppm.

[0041] Surprisingly, the Applicant has found out that by applying the described method it’s possible to obtain cannabinoid products having not only i) a liquid consistency, but also ii) a higher content of Tetrahydrocannabinol, and also iii) a pesticide content lower as compared to the starting lipid extract, making no use of organic solvents at any step.

[0042] Furthermore, the Applicant has found out that by applying the described method it’s possible to obtain cannabinoid concentrates having more than 70% by dry weight, with respect to the total dry weight of the concentrate.

[0043] Preferably, said cannabinoid concentrate comprising more than 70% by dry weight, with respect to the total dry weight of the concentrate, of Tetrahydrocannabinol, more preferably more than 75% by dry weight, even more preferably more than 80% by dry weight, even more preferably more than 85% by dry weight.

[0044] Within the framework of the present description and in the subsequent claims, except where otherwise indicated, all the numerical entities expressing amounts, parameters, percentages, and so forth, are to be understood as being preceded in all instances by the term "about". Also, all ranges of numerical entities include all the possible combinations of the maximum and minimum values and include all the possible intermediate ranges, in addition to those specifically indicated herein below. Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0045] Unless defined otherwise, 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 belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0046] As used herein, the articles “a” and “an” refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0047] As used herein, the term “cannabinoid” includes, but is not limited to, cannabinol (CBN), cannabinolic acid (CBNA), A(9)-tetrahydrocannabinol (A(9)-THC), A(9)- tetrahydrocannabinolic acid (A(9)-THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), A(8)-tetrahydrocannabinol (A(8)-THC), A(8)-tetrahydrocannabinolic acid (A(8)-THCA), cannabivarin (CBV), cannabivarinic acid, cannabigerol (CBG), cannabigerolic acid (CBGA), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabicyclol (CBL), cannabicyclolic acid (CBLA), Cannabidivarin (CBDV) and cannabidivarin acid (CBDVA).

[0048] As used herein, with the expression “THC” is meant tetrahydrocannabinol, encompassing its isomeric forms A(9)-tetrahydrocannabinol (A(9)-THC) and A(8)- tetrahydrocannabinol (A(8)-THC).

[0049] As used herein, with the expression “CBD” is meant cannabidiol.

[0050] As used herein, with the expression “THCA” is meant tetrahydrocannabinolic acid, encompassing its isomeric forms A(9)-tetrahydrocannabinolic acid (A(9)-THCA) and A(8)-tetrahydrocannabinolic acid (A(8)-THCA).

[0051] As used herein, with the expression “CBDA” is meant cannabidiolic acid.

[0052] As used herein, the term “cannabinoid acids” includes, but is not limited to, cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), cannabicyclolic acid (CBLA), cannabidivarinic acid (CBDVA), cannabigerovarinic acid (CBGVA), tetrahydrocanabivarinic acid (THCVA), cannabichromevarinic acid (CBCVA), cannabidiphorol acid (CBDPA) and A9- tetrahydrocannabiphorol acid (THCPA).

[0053] As used herein, the term “total THC” is equal to the sum of THCA content multiplied by the molecular weight ratio 0.877 plus THC content, as per the formula: total THC = THCA x 0.877 + THC.

[0054] As used herein, the term “terpenes” includes, but is not limited to, pinene, limonene, a-terpinene, terpinen-4-ol, carvacrol, carvone, 1 ,8-cineole, p-cymene, fenchone, [3- myrcene, cannaflavin A, cannaflavin B, nerolidol, phytol and squalene.

[0055] As used herein, the term “lipids” includes, but is not limited to, olive oil, coconut oil, vegetable oil, milk, butter, liposomes, glycerine, polyethylene glycol, ethyl acetate, d- limonene, liquid paraffin, butylene glycol, propylene glycol, ethylhexyl palmitate.

[0056] As used herein, the term “pesticide” includes, but is not limited to, a compound having insecticidal or fungicidal activity, for example a member of the class of pyrroles such as Chlorofenapyr, or a strobilurine compound such as Tryfloxystrobin, Azoxystrobin, Bifenazate, Boscalid, Carbaryl, Chlorantraniliprole, Chlorpyrifos, Dimethomorph, Ethoprophos, Fludioxonil, Imidacloprid, Malathion, Methiocarb, Pentachloronitrobenzene, Pyrethrins, Spinosad.

[0057] As used herein, the term “pesticide-free” includes, but is not limited to, a product wherein the pesticide content is below 0.1 ppm. It is known in the art that during the preparation of an extract from plant material, extract concentration may bring to concentrating the initial pesticide which was present on the plant material, with the consequential product having high pesticide levels.

[0058] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1 %, and ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. The present invention may present in one or more of the above aspects one or more of the characteristics disclosed hereinafter.

[0059] Further features and advantages of the invention will appear more clearly from the following description of some preferred embodiments thereof, made hereinafter by way of a non-limiting example with reference to the following exemplary examples.

[0060] In a preferred embodiment, the alkaline aqueous solution of step b) comprises at least one hydroxide of at least one metal selected from the group consisting of: alkali metal, and alkaline earth metal.

[0061] Preferably, the alkaline aqueous solution of step b) comprises NaOH, KOH or a mixture thereof.

[0062] Preferably, the alkaline aqueous solution of step b) is 0.5 M NaOH or KOH.

[0063] More preferably, the alkaline aqueous solution of step b) is 0.1 M NaOH or KOH.

[0064] Preferably, in step b) the alkaline aqueous solution is added to the lipid extract in a weight ratio (alkaline aqueous solution):(lipid extract) of at least 1 :1 , more preferably of at least 3:1 , even more preferably of at least 4:1. The method according to the present invention comprises the step b) of adding an alkaline aqueous solution to the lipid extract in a weight ratio (alkaline aqueous solution) :(lipid extract) of at least 4:1 or 5:1 .

[0065] Preferably, in step b) the alkaline aqueous solution has a molarity of NaOH calculated on the base of the total acidity of the lipid extract expressed as moles of KOH required for acidic titration of the lipid extract, so that the ratio NaOH mol I KOH mol is in the range of 2-20.

[0066] Preferably, said aqueous alkaline solution of step b) has a molarity of NaOH or KOH of at least 0.05.

[0067] Preferably, said mixture of step b) has a pH value ranging from 12.6 to 13.5.

[0068] Preferably, said mixture of step b) is stirred for a time in the range from at least 5 seconds to less than 24 hours, more preferably from 5 to 20 minutes, even more preferably from 5 to 10 min. In case the mixing is performed in continuous, the contact time between the lipid extract and the alkaline solution is preferably less than 5 minutes. Preferably, said mixture of step c) is heated to a temperature to at least 40 °C, more preferably to at least 50 °C, even more preferably to at least 60 °C.

[0069] In a further preferred aspect, a filtration step is added after step b) or step c).

[0070] In an embodiment, before step d) at least one salt and / or at least one sugar is added to the mixture of step c), so that an increase of the density of the aqueous phase is obtained, thus facilitating its separation in step d).

[0071] Preferably, said at least one salt is selected from the group consisting of: sodium chloride (NaCI), and calcium chloride (CaCh).

[0072] Preferably said at least one sugar is selected from the group consisting of: glucose and fructose.

[0073] In a preferred embodiment, said at least one salt is selected from the group consisting of: sodium chloride, potassium chloride and calcium chloride.

[0074] Advantageously, when said at least one salt is calcium chloride, said salt may be subsequently removed after separation of the aqueous phase in step c).

[0075] Preferably, wherein said step d) of separating comprises separating from the mixture of step c):

[0076] - a lighter oily phase containing lipophilic pesticides,

[0077] - a heavier aqueous phase, wherein the heavier aqueous phase is the aqueous phase containing cannabinoid acid salts rich in THCA salt.

[0078] In a preferred aspect, an additional step comprising the addition of concentrated soda to hydrolyze residual triglycerides is added after step d).

[0079] In a still preferred aspect, in the method of the present invention step e) comprises: i) acidifying said aqueous phase of step d) to a pH lower than 4 to obtain: - a lighter liquid lipid phase containing cannabinoid acids, and - a heavier aqueous phase, wherein the lighter lipid phase is said liquid cannabinoid concentrate; ii) separating said lighter lipid phase from the remaining aqueous phase; iii) heating the lipid phase of step ii) to at least 70 °C for at least 30 min to decarboxylate Tetrahydrocannabinolic acid, thus forming a lipid phase containing THC and fatty acids; iv) mixing said lipid phase containing THC and fatty acids of step iii) with an alkaline solution to form fatty acid salts; v) separating the mixture of step iv) to obtain: - a lighter phase containing THC, and - a heavier aqueous phase containing fatty acid salts; wherein the lighter phase is said pesticide-free, liquid cannabinoid concentrate.

[0080] Preferably, step e) comprises: i) acidifying said aqueous phase of step d) to a pH lower than 4 to obtain:

[0081] - a lighter liquid lipid phase containing cannabinoid acids, and

[0082] - a heavier aqueous phase, wherein the lighter lipid phase is said pesticide-free, liquid cannabinoid concentrate; ii) separating said lighter lipid phase from the remaining aqueous phase; iii) distilling the lighter lipid phase of step ii) to at least 130 °C and a vacuum of at least 0.1 mbar, to obtain a distillate, wherein the distillate is said pesticide-free, liquid cannabinoid concentrate.

[0083] In a further preferred aspect, step e) comprises: i) acidifying said aqueous phase of step d) to a pH lower than 4 to obtain: - a lighter liquid lipid phase containing cannabinoid acids, and - a heavier aqueous phase, wherein the lighter lipid phase is said pesticide-free, liquid cannabinoid concentrate; ii) separating said lighter lipid phase from the remaining aqueous phase; iii) heating the lipid phase of step ii) to at least 70 °C for at least 30 min to decarboxylate Tetrahydrocannabinolic acid, thus forming a lipid phase containing THC; iv) distilling the lipid phase of step iii) to at least 130 °C and a vacuum of at least 0.1 mbar, to obtain a distillate, wherein the distillate is said pesticide-free, liquid cannabinoid concentrate.

[0084] Preferably, in the method of the present invention step v) comprises washing the lighter phase with water to remove residual fatty acid salts.

[0085] Preferably, step e) comprises heating to at least 80 °C said aqueous phase of step d) to obtain - a lighter phase of decarboxylated Tetrahydrocannabinol, free of fatty acids and pesticides, and

[0086] - a heavier phase containing fatty acid salts, wherein the lighter phase is said pesticide-free, liquid cannabinoid concentrate.

[0087] Preferably, said step e) comprises filtering said aqueous phase of said step d).

[0088] Preferably, said filtering is carried out with a fiberglass filter or a paper filter.

[0089] Preferably, said filtering is carried out with a filter having pores diameter of less than 2.5 microns, more preferably of less than 2 microns, even more in the range from 0.5 to 1 .8 microns, even more preferably of about 1 micron.

[0090] Preferably, said step e) comprises: i. acidifying the aqueous phase of said step d) to a pH of less than 4, thus forming a precipitate comprising cannabinoid acids; even more preferably to a pH from 1 to 2. ii. separating the precipitate of step i. from the remaining aqueous phase, wherein said precipitate is said pesticide-free liquid cannabinoid concentrate.

[0091] Preferably said step c) comprises: i. modifying the pH of the mixture of said step c) to a value ranging from 7 to 1 1 ; ii. separating a first aqueous from the remaining mixture, wherein said first aqueous phase contains impurities, such as free fatty acids; iii. adding a new alkaline solution to the remaining mixture of step i. to reach a pH higher than 12.5; iv. separating a second aqueous phase from the mixture, wherein said second aqueous phase is said pesticide-free, liquid cannabinoid concentrate.

[0092] Preferably said step e) comprises the step of re-crystallizing THCA with a suitable solvent.

[0093] Preferably, said cannabinoid concentrate comprises at least 75% by weight, with respect to the total dry weight of the concentrate, of THCA.

[0094] Preferably, said step e) comprises drying, decarboxylating, distilling or re-crystallizing, said cannabinoid concentrate.

[0095] Preferably, said liquid cannabinoid concentrate of step e) has a total THC content higher than 80%. Preferably, said liquid cannabinoid concentrate of step e) has a total THC content higher than 85%, even more preferably more than 90% by dry weight, even more preferably more than 95% by dry weight.

[0096] In the method of the invention, the content of fatty acids in the pesticide-free, liquid cannabinoid concentrate is preferably of less than 5% by weight.

[0097] Preferably the lipid extract of step a) contains pesticide above a level of quantification of 0.01 ppm and the concentrate of step e) contains no pesticides or contains pesticides below 0.01 ppm.

[0098] Preferably, the lipid of said lipid extract comprising at least 1 % by dry weight of THCA of step a) is selected from the group consisting of: vegetable oil, milk, butter, liposomes, ethyl acetate, glycerine, d-limonene, caryophyllene, liquid paraffin, mineral oil, paraffin wax, microcrystalline wax, mineral wax, ozokerite, polyethylene, polyoxyethylene and hydrocarbon waxes derived from carbon monoxide and hydrogen, cerosin; cetyl esters; hydrogenated joioba oil, butylene glycol, propylene glycol, polyethylene glycol, liposomes, lecithin, ethylhexyl palmitate, or mixtures thereof.

[0099] In another embodiment, the lipid is a vegetable oil.

[0100] Preferably, said vegetable oil is selected from the group consisting of: olive oil, coconut oil, sunflower oil, sesame oil, hemp seed oil, Cannabis seeds oil or a mixture thereof.

[0101] In an embodiment, the lipid is olive oil. In another embodiment, the lipid is coconut oil. In yet another embodiment, the lipid is sunflower oil. In yet another embodiment, the lipid is sesame oil. In another embodiment, the lipid is Cannabis seeds oil. In an embodiment, the lipid is milk. In a further embodiment, the lipid is butter. In yet another embodiment, the lipid is a liquid paraffin.

[0102] Preferably, the lipid is a vegetable oil having a free-fatty acids (FFA) content below 1 %, preferably below 0.5%, even more preferably below 0.1 %.

[0103] In an embodiment the lipid extract of step a) is winterized before mixing with alkaline solution of step b).

[0104] In a preferred embodiment, the lipid extract of step a) is winterized by stirring at a temperature below 6 °C for at least 3 hours.

[0105] In a preferred embodiment of the method, the lipid extract of step a) is vacuum distilled first to recover terpenes. In a preferred embodiment of the method according to the present invention, the lipid extract of step a) comprising at least 1 % by dry weight of THCA is obtained from a biological material containing cannabinoids, preferably chosen from the group consisting of a plant, an alga, a bacterium, a yeast, a fungus, a genetically engineered micro-organism, or a mixture thereof.

[0106] That is, the method according to the invention preferably comprises a step of obtaining a lipid extract containing cannabinoids from a biological material containing cannabinoids, preferably chosen from the group consisting of a plant, an alga, a bacterium, a yeast, a fungus, a genetically engineered micro-organism, or a mixture thereof.

[0107] In an even more preferable embodiment, the lipid extract of step a) containing cannabinoids is obtained by putting in contact a biological material containing cannabinoids with a vegetable oil.

[0108] In an embodiment the initial lipid extract is obtained by mixing a lipid with a cannabinoid extract obtained by means of supercritical CO2 extraction or by means of organic solvents.

[0109] Preferably, the lipid extract of step a) is obtained from a biological material containing cannabinoids by means of the steps of:

[0110] I. comminuting a biological material containing cannabinoids;

[0111] II. mixing the comminuted biological material with lipids to obtain a mixture;

[0112] III. stirring the mixture at a temperature range of 1 to 80 °C; and

[0113] IV Obtaining a lipid extract from the mixture of step III.

[0114] Preferably, said biological material containing cannabinoids is selected from a plant, an alga, a bacterium, a yeast, a fungus, a genetically engineered micro-organism, or a mixture thereof.

[0115] In said step L, the biological material is comminuted to increase the surface contact. Then water and / or lipids are added to the plant material to form a homogeneous mixture or slurry; the mixture may be agitated through stirring or other agitation methods preferably for at least 30 min. Ultrasound / sonication or microwaves or steam explosion may advantageously be used before or after adding lipids to the mixture to reduce the time necessary to achieve biological material dissolution and high cannabinoids lipid-extraction yield.

[0116] The mixture obtained is then separated for example via density separation (i.e. centrifugation) or pressing (French press) and / or filtration to recover a lipid fraction highly enriched with cannabinoids and waxes free.

[0117] In said preferred embodiment, steps I. and II. may be also inverted.

[0118] Preferably, said biological material containing cannabinoids is selected from the Cannabis genus of plants, wherein said biological material is pure, a hybrid or genetically modified variant thereof. Preferably, said biological material containing cannabinoids selected from the Cannabis genus of plants, belongs to the species C. sativa (hemp), C. indica and C. ruderalis.

[0119] Preferably, the biological material containing cannabinoids has a moisture content of at least 20% of the biological material weight.

[0120] Preferably, said biological material containing cannabinoids is newly harvested and has a moisture content of at least 30%, preferably at least 40%.

[0121] Preferably, said biological material can be used in said step I. of the method according to the invention either fresh or dried. In an embodiment, the biological material is newly harvested and contain high level of moisture; in such a case addition of extra water to the biological material is unnecessary.

[0122] Preferably, the biological material containing cannabinoids has a total cannabinoid content of at least 0.1% by weight, more preferably of at least 0.2 % by weight, even more preferably of at least 1 % by weight, even more preferably of at least 2% by weight.

[0123] Preferably, said biological material contains at least 0.5% cannabinoids in weight.

[0124] In a preferred aspect, said biological material is chosen from the group consisting of buds, flowers, leaves, stalks, stems, roots and seeds or a mixture thereof. In an embodiment, the biological material includes seeds. In another embodiment, when the biological material includes seeds, no lipid is added. In a further embodiment, when the biological material includes seeds, a lipid is added. Biological material including seeds may be rich in lipids, and thus may not need the further addition of lipids. Preferably, in step II. the lipids have been neutralized prior being added to the mixture.

[0125] Preferably, enzymes are added to the mixture of said step II.

[0126] Preferably, said enzymes are one or more independently selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases, cellulase, hemicellulase, xylanase, glucanase, beta-glucanase, pectinase, glucuronyltransferase, lipase, amylase, alpha-amylase, beta-amylase, phospholipase, arabanase, galacto-, beta-mannanase, protease, esterase, phytase, cannabinoid synthase, THCA synthase, CBCA synthase.

[0127] In an embodiment, said enzyme is cellulase. In another embodiment, said enzyme is beta-glucosidase. In another embodiment, said enzyme is hemicellulase. In another embodiment, said enzyme is xylanase. In yet another embodiment, said enzyme is glucanase. In yet another embodiment, said enzyme is pectinase. In still another embodiment, said enzyme is amylase. In yet another embodiment, said enzyme is lipase or phospholipase. In said another embodiment, said enzyme is glucuronosyltransferase or alcohol dehydrogenase. In yet another embodiment, said enzyme is arabinanase. In still another embodiment, said enzyme is phytase. In a further embodiment, said enzyme is protease. In yet another embodiment, said enzymes is esterase. In yet another embodiment, said enzymes is a cannabinoid synthase. In still another embodiment, said enzyme is THCA synthase. In yet another embodiment said embodiment is CBDA synthase. In another embodiment, said enzyme is CBCA synthase.

[0128] Preferably, in step II. the temperature varies in the range from 40 to 70 °C.

[0129] Preferably, in step II. the pH varies in the range from 4 to 6.

[0130] Preferably, the mixture of said step III. is degummed with phosphoric acid or citric acid.

[0131] Preferably, said step IV comprises separating the mixture into a lipid phase, an aqueous phase, and a solid phase; wherein the lipid phase comprises the lipid extract.

[0132] In an embodiment, in step IV. the mixture is separated by density. In a further embodiment, in step IV. the mixture is separated by pressing and / or filtering.

[0133] In a further embodiment, in step IV. the mixture is separated into a lipid-soluble phase and a wet solid phase. In an embodiment, the lipid-soluble extract is recirculated any number of times to achieve higher cannabinoid or terpene content.

[0134] In a further embodiment, at least 50%, preferably 70% of the terpenoids, at least 70% of the diterpenoids and at least 50%, preferably 70% of monoterpenes contained in the plant material are extracted into the lipid-soluble extract.

[0135] In a still further embodiment at least 70% of the sesquiterpenes and at least 50% of the mono-terpenes contained in the plant material are extracted into the lipid-soluble extract.

[0136] In an embodiment, the lipid-soluble extract has a total cannabinoid content of at least 2% by weight. In a further embodiment, the lipid-soluble extract has a total cannabinoid content of at least 3% by weight. In yet another embodiment, the lipid-soluble extract has a total cannabinoid content of at least 5% by weight.

[0137] In an embodiment, the two main cannabinoids in the lipid-soluble extract are preferably CBGA and THCA.

[0138] Preferably, less than 10%, preferably less than 5%, more preferably less than 2%, of cannabinoids are decarboxylated during said steps I. -IV. of obtaining the lipid extract containing cannabinoids from a biological material containing cannabinoids.

[0139] In a preferred embodiment of the method according to the present invention, the aqueous phase resulting from said 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 in the production of nutraceutical, antimicrobial, antibacterial products or biopesticides.

[0140] In a further aspect, the present invention relates to a pesticide-free liquid cannabinoid concentrate obtainable by means of the process according to the first aspect of the invention, said liquid cannabinoid concentrate:

[0141] - comprising more than 70% by weight, with respect to the total dry weight of the concentrate, of Tetrahydrocannabinol;

[0142] - comprising not more than 0.01 ppm of a pesticide chosen from the group consisting of Acephate, Dimethomorph, Methomyl, Azoxystrobin, Ethoprop(hos), Myclobutanil, Fludioxonil, Propiconazole, Carbaryl, Imidacloprid, Tebuconazole, Chlorantraniliprole, Malathion, Thiamethoxam, Diazinon, Metalaxyl, Dimethoate, Methiocarb, Chlorphenapyr, Tryfloxystrobin, Boscalid, Bifenazate, Pentachloronitrobenzene, Pyrethrins, Spinosad and Chlorpyrifos:

[0143] - being obtained without making use of an organic solvent, and

[0144] - being obtained from plant material containing one of more of said pesticides in an amount above 0.01 ppm.

[0145] Thanks to the specific conditions of the method according to the invention, a cannabinoid concentrate is indeed obtained, showing an unexpectedly high level of Tetrahydrocannabinol.

[0146] In a still further aspect, the present invention relates also to a cannabinoid concentrate being obtained without making use of an organic solvent, wherein said organic solvent is selected from a group consisting of acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, propane, toluene, m-xylene, o- xylene, and p-xyleneheptane.

[0147] In a still further aspect, the present invention relates also to a pesticide-free liquid cannabinoid concentrate:

[0148] - comprising more than 70% by weight, with respect to the total dry weight of the concentrate, of Tetrahydrocannabinol;

[0149] - comprising not more than 0.1 ppm of a pesticide chosen from the group consisting of Azoxystrobin, Bifenazate, Boscalid, Carbaryl, Chlorantraniliprole, Chlorphenapyr, Chlorpyrifos, Dimethomorph, Ethoprophos, Fludioxonil, Imidacloprid, Malathion, Methiocarb, Pentachloronitrobenzene, Pyrethrins, Spinosad, Tryfloxystrobin.

[0150] - being obtained without making use of an organic solvent, and

[0151] - being obtained from plant material containing one of more of said pesticides in an amount above 0.01 ppm.

[0152] In a still further aspect, the present invention relates also to a cannabinoid concentrate comprising more than 70% by weight, with respect to the total dry weight of the concentrate, of cannabinoids and not more than 0.0001 % per cent by weight of one or more organic solvents selected from a group consisting of acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, propane, toluene, m- xylene, o-xylene, and p-xyleneheptane.

[0153] The Applicant has noted that the combination of a high content of THC and / or THCA and the absence of pesticides and organic solvents content is particularly surprising compared to the prior art concentrates, in which a high cannabinoid acids content is usually achieved by means of concentration or purification treatments that lead to an increase of pesticides or that involve the use of organic solvents, the elimination of which may result troublesome, expensive and not in compliance with regulatory requirements.

[0154] The other advantages of the cannabinoid concentrate according to the present invention have been disclosed in relation to the method according to the first aspect of the present invention and are not herewith repeated.

[0155] Preferably, said cannabinoid concentrate comprises more than 70% by weight, even more preferably more than 85% by weight, with respect to the total dry weight of the concentrate, of cannabinoid acids.

[0156] Preferably, in the cannabinoid concentrate according to the invention the cannabinoid acids are selected from the group consisting of: cannabinolic acid (CBNA), cannabigerolic acid (CBGA), cannabichromenic acid (CBCA), cannabicyclolic acid (CBLA), cannabidivarinic acid (CBDVA), cannabigerovarinic acid (CBGVA), tetrahydrocanabivarinic acid (THCVA), cannabichromevarinic acid (CBCVA), cannabidiphorol acid (CBDPA) and A9-tetrahydrocannabiphorol acid (THCPA).

[0157] Preferably, in the cannabinoid concentrate according to the invention the cannabinoid acids is CBGA.

[0158] According to the present invention, a cannabinoid concentrate is therefore provided.

[0159] Thanks to its compositional and purity properties, said cannabinoid concentrate may be advantageously used for preparing pharmaceutical or nutraceutical products, cosmetics, food or feed products, antimicrobial, antibacterial, insecticidal or biopesticides containing one or more cannabinoids.

[0160] In a further aspect, therefore, the present invention relates to a method for preparing a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide, comprising the step of:

[0161] - providing a cannabinoid concentrate according to the present invention and / or preparing a cannabinoid concentrate according to the present invention; and

[0162] - obtaining a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide comprising Tetrahydrocannabinol.

[0163] Further features and advantages of the invention will appear more clearly from the following description of some preferred embodiments thereof, made hereinafter by way of a non-limiting example with reference to the following exemplary examples.

[0164] EXPERIMENTAL PART

[0165] Example 1

[0166] A refined sunflower oil based soluble extract (“Lipid extract”) obtained according to Example 1 of WO 2018 / 130682 and artificially added with pesticides Trifloxystrobin and Chlorfenapyr. The lipid extract had the composition reported in Table 1 .

[0167] Table 1

[0168] The quantitative determination of cannabinoids (THCA, THC) was performed by HPLC-DAD Shimadzu Nexera XR, equipped with a reverse phase C18 column NexLeaf CBX for Potency 150 x 4.6 mm, 2.7 pm.

[0169] Chromatographic conditions: Solvent A: water + 0.085% phosphoric acid (v / v); Solvent B: acetonitrile + 0.085% phosphoric acid (v / v). Flow: 1 .6 ml / min. Oven temperature: 35 °C. Manual injection: loop 20 pL. Detection wavelength: 228 nm for THCA and THC; Gradient elution: 70% of B up to 3 min, 85% of B to 7 min, 95% of B to 7.01 up to 8.00 min, and 70% of B up to 10 min.

[0170] Retention times: THC about 6.5 minutes; THCA about 7.5 minutes. The quantification of said cannabinoids was performed by an external standard method through the preparation of a calibration curve, using pure standard of cannabinoids (THCA) in the range 2.5 - 250 ppm.

[0171] The THC quantification was done on the base of CBD calibration curve, using relative response factor (RRF) reported in the analytical monograph of the pharmacopeia (OMC I Farmalyse BV Version 7.1 I November 28, 2014).

[0172] The instrumental LOD was 0.5 ppm and the instrumental LOQ was 2.5 ppm.

[0173] LOD and LOQ values for the analytical method for THC and THCA are reported, for each matrix, in Table 2 below: Table 2

[0174] Pesticides content was determined by an accredited laboratory in San Diego, CA. Pesticides analysis has been performed utilizing: LC-MS-MS & GC-MS-MS; samples analyzed according to SOPs PESTMYCQ-LC-INST-004 and PEST-GC-INST-003. LOD and LOQ values for the analytical method for pesticides are described in Table 3:

[0175] Table 3 150 g of said lipid extract were obtained according to Example 1 of WO2022049232A1 . Said lipid extract was mixed at room temperature with 450 g of an aqueous solution 0.3 M NaOH in a kitchen robot Moulinex Companion, so to reach a pH of the mixture of about 13. The mixture was kept under stirring for about 15 min. About 22.5 g NaCI were added to the mixture and mixed for 5 min, so to completely dissolve it before centrifugation. After mixture centrifugation (4.500 rpm for 10 min), 148 g of an oily lighter phase and 468 ml of a heavier aqueous phase were recovered.

[0176] The heavier aqueous phase was filtered, utilizing a lab vacuum filter with a fiberglass filter having a pores diameter of about 1.6 micron. About 461 ml of filtered aqueous solution were recovered. A sample was taken and analyzed for cannabinoids content.

[0177] The filtered aqueous phase was added with sodium chloride so to reach a final concentration of 5% NaCI, and then acidified utilizing a solution of H3PO4 at 85% concentration to reach a pH of about 1 .5-2. No appreciable quantity of precipitate was formed or recovered. THCA content of the oily lighter phase was 6.2%, while the THCA content of the filtered aqueous phase was 0.018%. It is evident that THCA and THCA salt remained unextracted in the oily lighter phase.

[0178] Example 2

[0179] The same test as per example 1 was executed, with the only difference of not adding sodium chloride to the mixture between the lipid extract and the aqueous alkaline solution. No appreciable quantity of precipitate was formed or recovered. THCA content of the oily lighter phase was 6.1 %, while the THCA content of the filtered aqueous phase was 0.031 %. THCA and THCA salt remained unextracted in the oily lighter phase.

[0180] Example 3

[0181] The same test as per example 1 was executed, with the only difference of increasing the temperature of the mixture to 60 °C before centrifugation. THCA content of the oily lighter phase was still 5.1 %, while the THCA content of the filtered aqueous phase was 0.19%. After acidification, about 1 .5 g of a liquid precipitate was formed having a Total THC content of about 65%. So THCA and THCA salt still remained largely unextracted in the oily lighter phase. Example 4

[0182] The same test as per example 3 was executed, with the only difference of not adding sodium chloride to the mixture between the lipid extract and the aqueous alkaline solution. THCA content of the oily lighter phase was 0.8%, while the THCA content of the filtered aqueous phase was 1 .7%.

[0183] After acidification of the aqueous phase, about 9.5 g of a liquid precipitate as a top layer were collected having a Total THC content of 75%. The pesticides content in the in the lighter oily phase were 0.27 and 0.10 ppm for Trifloxystrobin and Chlorfenapyr respectively. Therefore, pesticides Trifloxystrobin and Chlorfenapyr were not detected in the liquid precipitate with a limit of detection (LCD) of 0.003 ppm and 0.025 ppm respectively.

[0184] It is evident from the examples that i) addition of sodium chloride in the mixture of lipid extract and aqueous alkaline solution negatively affects the recovery of THCA, and ii) increasing the temperature of the mixture before mixture centrifugation is fundamental to extract THCA salt and THC in the heavier aqueous phase.

[0185] Surprisingly, while THC was recovered with an efficiency of about 80%, pesticides Trifloxystrobin and Chlorfenapyr were not detected in the final liquid concentrate obtained after acidifying the filtered aqueous phase.

[0186] The cannabinoid content of the precipitate was also remarkably high, considering no organic solvents were utilized.

[0187] Example 5

[0188] 150 g of said lipid extract were processed according to Example 1 of WO2022049232A1 . Then 150 g of said extract were mixed at room temperature with 225 g of an aqueous solution 0.27 M NaOH in a kitchen robot Moulinex Companion, so to reach a pH of the mixture of about 13. The mixture was kept under stirring for about 15 min. About 22.5 g NaCI were added to the mixture and mixed for 5 min, so to completely dissolve it before centrifugation. After mixture centrifugation (4.500 rpm for 10 min), 148 g of an oily lighter phase and 222 ml of a heavier aqueous phase were recovered. The heavier aqueous phase was discarded.

[0189] The oily lighter phase was mixed with 225 g of a second aqueous solution 0.27 M NaOH to form a new mixture. No sodium chloride was added to the new mixture. Such a mixture was then heated to reach 60 °C and then centrifuged. After centrifugation, 140 g of an oily lighter phase and 223 g of a heavier aqueous phase were recovered. The heavier aqueous phase was filtered, utilizing a lab vacuum filter with a fiberglass filter having a pores diameter of about 1.6 micron. About 219 ml of filtered aqueous solution were recovered. The filtered aqueous phase was added with sodium chloride so to reach a final concentration of 5% NaCI and then acidified utilizing a solution of H3PO4 at 85% concentration to reach a pH of about 1 .5-2.

[0190] After acidification of the aqueous phase, about 9.4 g of a liquid precipitate as a top layer were collected having a Total THC content of 81 %. The pesticides content in the in the lighter oily phase were 0.27 and 0.10 ppm for Trifloxystrobin and Chlorfenapyr respectively. Therefore, pesticides Trifloxystrobin and Chlorfenapyr were not detected in the liquid precipitate with a limit of detection (LCD) of 0.003 ppm and 0.025 ppm respectively.

[0191] The liquid concentrate has been decarboxylated and vacuum distilled. A final concentration of 95% of THC in the liquid distillate has been achieved.

[0192] Example 6

[0193] A butane lipophilic extract containing cannabinoids (“butane extract”) and contaminated with pesticides was provided by a licensed California cannabis manufacturer (Table 4).

[0194] Table 4

[0195] The butane extract (100 g) was then mixed with 500 g of an aqueous solution 0.5 M NaOH containing 1 % sodium chloride at room temperature in a kitchen robot Moulinex Companion, so to reach a pH of about 13. Without heating the mixture was very difficult to homogenize. The mixture was centrifuged and an aqueous phase was recovered. The aqueous phase was acidified utilizing a solution of H3PO4 at 85% concentration to reach a pH of about 1 .5-2. No precipitate was recovered. The experiment was repeated with the difference that the mixture was heated to reach 60 °C and kept under stirring for 15 min. The heated mixture was then centrifuged. 440 g of aqueous phase were recovered and filtered, utilizing a lab vacuum filter with a fiberglass filter having a pores diameter of about 1.6 micron. About 430 g of filtered aqueous solution were recovered. The filtered aqueous phase was acidified utilizing a solution of H3PO4 at 85% concentration to reach a pH of about 1.5-2. About 16 g of precipitate were recovered. The composition of the precipitate was as described in Table 5.

[0196] Table 5

[0197] As it can be noticed, applying the method according to the invention to a butane lipophilic extract was not effective in reducing the pesticides content below the LOD in the final concentrate.

[0198] This confirmed the effectiveness of the method according to the invention for recovering a pesticide-free THC liquid concentrate from a lipid extract, wherein the pesticide content in the concentrate is below the limit of quantification, preferably below the limit of detection.

[0199] Table 6 sums up the conditions used in Examples 1 - 6 as far as temperature and addition of NaCI are concerned and the resulting THCA content resulting in the aqueous phase, showing that addition of sodium chloride in the mixture of lipid extract and aqueous alkaline solution negatively affects the recovery of THCA, while increasing the temperature of the mixture before mixture centrifugation is fundamental to extract THCA salt and THC in the heavier aqueous phase. Table 6

Claims

CLAIMS1 . A method for preparing a pesticide-free, liquid cannabinoid concentrate comprising more than 70% by dry weight, with respect to the total dry weight of the concentrate, of Tetrahydrocannabinol, comprising the steps of: a) providing a lipid extract comprising at least 1 % by dry weight of Tetrahydrocannabinolic acid; b) mixing said lipid extract with an alkaline aqueous solution to form Tetrahydrocannabinolic acid salt; c) heating the mixture of step b) to at least 40 °C. d) separating from the mixture of step c) an aqueous phase containing cannabinoid acid salts; e) obtaining from the aqueous phase said pesticide-free, liquid cannabinoid concentrate, wherein the pesticide content is below 0.1 ppm.

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 the group consisting of: alkali metal and alkaline earth metal.

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

4. The method according to any one of claims 1 -3, wherein in step b) the alkaline aqueous solution has a molarity of NaOH calculated on the base of the total acidity of the lipid extract expressed as moles of KOH required for acidic titration of the lipid extract, so that the ratio NaOH mol I KOH mol is in the range of 2-20.

5. The method according to any one of claims 1 -4, wherein the quantity of NaOH or KOH is added at least in stoichiometric ratio with Tetrahydrocannabinolic acid.

6. The method according to any one of claims 1 -5, wherein the mixture of step b) is stirred for a time in the range from at least 5 seconds to less than 24 hours.

7. The method according to any one of claims 1 -6, wherein the mixture of step b) has a pH value ranging from 12.6 to 13.5.

8. The method according to any one of claims 1 -7, wherein the mixing of step b) is at a temperature of at least 60 °C.

9. The method according to any one of claims 1 -8, wherein a filtration step is added after step b) or step c).

10. The method according to any one of claims 1 -9, wherein before step c) at least one sugar is added to the mixture of step b).1 1 . The method according to any one of claims 1 -10, wherein said step d) of separating comprises separating from the mixture of step c):- a lighter oily phase containing lipophilic pesticides,- a heavier aqueous phase, wherein the heavier aqueous phase is the aqueous phase containing both cannabinoid acid salts rich in THCA salt.

12. The method according to any one of claims 1 -1 1 , wherein an additional step comprising the addition of concentrated soda to hydrolyze residual triglycerides is added after step d).

13. The method according to any one of claims 1 -12 wherein step e) comprises: i) acidifying said aqueous phase of step d) to a pH lower than 4 to obtain: - a lighter liquid lipid phase containing cannabinoid acids, and - a heavier aqueous phase, wherein the lighter lipid phase is said liquid cannabinoid concentrate; ii) separating said lighter lipid phase from the remaining aqueous phase; iii) heating the lipid phase of step ii) to at least 70 °C for at least 30 min to decarboxylate Tetrahydrocannabinolic acid, thus forming a lipid phase containing THC and fatty acids; iv) mixing said lipid phase containing THC and fatty acids of step iii) with an alkaline solution to form fatty acid salts; v) separating the mixture of step iv) to obtain: - a lighter phase containing THC, and - a heavier aqueous phase containing fatty acid salts; wherein the lighter phase is said pesticide-free, liquid cannabinoid concentrate.

14. The method according to any one of claims 1 -12 wherein step e) comprises:i) acidifying said aqueous phase of step d) to a pH lower than 4 to obtain: - a lighter liquid lipid phase containing cannabinoid acids, and - a heavier aqueous phase, wherein the lighter lipid phase is said pesticide-free, liquid cannabinoid concentrate; ii) separating said lighter lipid phase from the remaining aqueous phase; iii) distilling the lighter phase of step ii) to at least 130 °C and a vacuum of at least 0.1 mbar, to obtain a distillate, wherein the distillate is said pesticide-free, liquid cannabinoid concentrate.

15. The method according to any one of claims 1 -12 wherein step e) comprises: i) acidifying said aqueous phase of step d) to a pH lower than 4 to obtain: - a lighter liquid lipid phase containing cannabinoid acids, and - a heavier aqueous phase, wherein the lighter lipid phase is said pesticide-free, liquid cannabinoid concentrate; ii) separating said lighter lipid phase from the remaining aqueous phase; iii) heating the lipid phase of step ii) to at least 70 °C for at least 30 min to decarboxylate Tetrahydrocannabinolic acid, thus forming a lipid phase containing THC; iv) distilling the lipid phase of step iii) to at least 130 °C and a vacuum of at least 0.1 mbar, to obtain a distillate, wherein the distillate is said pesticide-free, liquid cannabinoid concentrate.

16. The method according to claim 13, wherein the step v. comprises washing with water the lighter phase to remove residual fatty acid salts.

17. The method according to any one of claims 1 -15 wherein the step e) comprises heating to at least 80 °C said aqueous phase of step c) to obtain:- a lighter phase of decarboxylated Tetrahydrocannabinol, free of fatty acids and pesticides, and- a heavier phase containing fatty acid salts, wherein the lighter phase is said pesticide- free, liquid cannabinoid concentrate.

18. The method according to any one of claims 1 -16, wherein said liquid cannabinoid concentrate of step e) has a total THC content higher than 80%.

19. The method according to any one of claims 1 -17, wherein the content of fatty acids in the pesticide-free, liquid cannabinoid concentrate is of less than 5% by weight.

20. The method according to any one of claims 1 -19 wherein the lipid extract of step a) contains pesticide above 0.01 ppm and the concentrate of step e) contains no pesticides or contains pesticides below 0.01 ppm.21 . A pesticide-free liquid cannabinoid concentrate obtainable from the method of any one of claims 1 to 20, said liquid cannabinoid concentrate:- comprising more than 70% by weight, with respect to the total dry weight of the concentrate, of Tetrahydrocannabinol;- comprising not more than 0.01 ppm of a pesticide chosen from the group consisting of: Azoxystrobin, Bifenazate, Boscalid, Carbaryl, Chlorantraniliprole, Chlorphenapyr, Chlorpyrifos, Dimethomorph, Ethoprophos, Fludioxonil, Imidacloprid, Malathion, Methiocarb, Pentachloronitrobenzene, Pyrethrins, Spinosad, Tryfloxystrobin:- being obtained without making use of an organic solvent, and- being obtained from plant material containing one of more of said pesticides in an amount above 0.01 ppm.

22. The pesticide-free liquid cannabinoid concentrate according to claim 21 , wherein said organic solvent is chosen from the group consisting of: 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.

23. The cannabinoid concentrate according to any of claims 20 or 21 comprising less than 0.0001 % per cent by weight, with respect to the total dry weight of the concentrate, of one or more organic solvents selected from a group consisting of acetone, benzene, butane, chloroform, cyclohexane, dichloromethane, ethanol, ethyl acetate, ethylbenzene, heptane, hexane, isobutane, isopropanol, methanol, pentane, propane, toluene, m-xylene, o-xylene, and p-xyleneheptane.

24. A method for preparing a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide, comprising the step of: providing a cannabinoid concentrate according to any one of claims 20 to 22 and / or preparing a cannabinoid concentrate according to any one of claims 1 - 19; andobtaining a pharmaceutical product, a nutraceutical product, a cosmetic product, a food product, a feed product, an antimicrobial, an antibacterial, an insecticide, a biopesticide comprising Tetrahydrocannabinol.

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