Pharmaceutical compositions, formulations and uses thereof comprising cannabinoids

Abstract

The present invention relates to a pharmaceutical composition comprising cannabinoids and uses thereof. The present invention also provides a beneficial use of waste material produced during industrial production of cannabidiol. The composition according to the present invention can be used for the treatment of Helicobacter pylori associated diseases, elimination or reduction of oral malodor and treatment of periodontal diseases.

Description

Technical Field

[0001] This invention relates to a composition with medicinal uses, specifically an extract of industrial hemp. Background Technology

[0002] Cannabidiol (CBD) is one of the main components of the cannabis plant and has various pharmacological effects, such as anti-inflammatory, antioxidant, anti-anxiety, and anticonvulsant properties. CBD can reduce inflammatory responses, relieve pain and muscle spasms, and also reduce anxiety and psychological stress. It also has some therapeutic effects in treating neurological disorders such as epilepsy and Parkinson's disease.

[0003] Due to the expanding applications, the global demand for CBD is continuously increasing. The applicant company is one of the major CBD producers in China and has been committed to the research and implementation of cannabidiol (CBD) preparation processes and related technologies.

[0004] In a method for producing CBD that the applicant currently uses, which involves supercritical extraction, a large amount of residue is generated after extraction. These residues are complex in composition and have very low abundance, making them impossible to extract individually. They are usually discarded or incinerated as waste. Summary of the Invention

[0005] One of the objectives of this invention is to make full use of a waste product generated during the industrial production of cannabidiol.

[0006] Another object of the present invention is to provide a novel composition derived from industrial hemp.

[0007] A first aspect of the present invention provides a composition extracted from industrial hemp, which is prepared by the following method:

[0008] 1) Supercritical CO2 extraction of industrial hemp flowers and leaves yields hemp extract;

[0009] 2) Dissolve the cannabis extract in a non-polar solvent, and then perform a first extraction with an alcohol solution of the first concentration to obtain oil phase 1 and aqueous phase 1. The non-polar solvent is pentane, hexane, heptane, octane, methylcyclohexane or a mixture thereof, and the alcohol solution is a 60-65% ethanol solution or a 65-75% methanol solution.

[0010] 3) Extract the aqueous phase 1 with an equal volume of the nonpolar solvent 5-10 times to obtain the aqueous phase 4. Concentrate the aqueous phase 4 to remove the solvent to obtain the first composition.

[0011] In another embodiment of the invention, the composition is prepared by the following method.

[0012] 1) Supercritical CO2 extraction of industrial hemp flowers and leaves yields hemp extract;

[0013] 2) Dissolve the cannabis extract in a non-polar solvent, and then perform a first extraction with an alcohol solution of the first concentration to obtain oil phase 1 and aqueous phase 1, wherein the alcohol solution of the first concentration is a 60-65% ethanol solution or a 65-75% methanol solution.

[0014] 3) Concentrate aqueous phase 1 to obtain aqueous extract. Dissolve the aqueous extract in 8-10 times its volume of 65-70% alcohol. Filter out the insoluble matter to obtain aqueous phase 4.

[0015] 4) Extract aqueous phase 4 5-10 times with 2-3 times the volume of the above-mentioned nonpolar solvent to obtain aqueous phase 5. Concentrate aqueous phase 5 to remove the solvent and obtain the first composition.

[0016] In a preferred embodiment, the second composition is obtained by molecular distillation of the first composition.

[0017] In one specific embodiment, the nonpolar solvent is pentane, hexane, heptane, octane, methylcyclohexane or a mixture thereof, and the amount used in step 2) is 2-4 times the weight of the cannabis extract.

[0018] A second aspect of the present invention provides a pharmaceutical preparation comprising the composition described in the first aspect of the present invention, which is any one of tablets, pills, capsules, granules, powders, syrups, drops, ointments, and emulsions.

[0019] The above-described compositions and pharmaceutical preparations of the present invention can be used to treat Helicobacter pylori-related diseases, as well as to eliminate or reduce bad breath and treat periodontal diseases. Detailed Implementation

[0020] The technical concept of the present invention will be described in detail below with reference to specific embodiments. It is readily understood that the embodiments described in this disclosure are merely illustrative and not all implementations of the present invention. Those skilled in the art, upon obtaining the description of the basic concept of the present invention in this disclosure and in conjunction with the given embodiments, can obtain other ways of implementing the present invention without creative effort, and can also make further improvements based on the basic concept of the present invention. All such variations or improvements fall within the scope defined by the claims of this application.

[0021] In one of the applicant's implemented CBD production processes, industrial hemp flowers and leaves are first subjected to supercritical extraction to obtain hemp extract. This extract is then dissolved in a non-polar solvent and subjected to a first extraction with a first-concentration alcohol solution, yielding oil phase 1 and aqueous phase 1. The first-concentration alcohol solution is a 60-65% ethanol solution or a 65-75% methanol solution. Oil phase 1 is then subjected to a second extraction with a second-concentration alcohol solution, yielding oil phase 2 and aqueous phase 2. Oil phase 2 is then subjected to a third extraction with a third-concentration alcohol solution, yielding oil phase 3 and aqueous phase 3. Aqueous phases 2 and 3 are combined, concentrated, and crystallized to obtain high-purity CBD. The second-concentration alcohol solution is a 90-92% ethanol solution or an 88-95% methanol solution, and the third-concentration alcohol solution is an 80-88% ethanol solution or methanol solution. This method achieves a total CBD transfer rate of 50-60% or even higher. The term "transfer rate" here refers to the percentage of cannabidiol (CBD) transferred from cannabis extract to another physical or phase state. The term "total transfer rate" refers to the percentage of CBD crystals obtained as the final product relative to the total amount of CBD in the extract; the total transfer rate is the product yield.

[0022] In the above method, the aqueous phase 1 obtained from the first extraction still contains a considerable proportion of CBD. Approximately 30% of the CBD in the cannabis extract is transferred to this aqueous phase 1, with a transfer rate of about 30-35%. To make the most of the cannabis resources, the aqueous phase 1 is extracted multiple times with alkanes. The alkane phases are then combined to obtain the oil phase 4. The oil phase 4 undergoes tertiary molecular distillation and contains 40-60% CBD. The residue obtained after solvent recovery from the separated aqueous phase 4 typically contains less than 5% CBD, making further extraction of it less valuable. It is usually discarded or incinerated as waste.

[0023] The concept of this invention is based on a discovery made by the inventor while working at the applicant's company. During a recurrence of severe toothache, the inventor casually took a residue from the concentrated aqueous phase 4 solution in the workshop and bit it in the mouth. After several hours, the pain lessened, and after repeated attempts, the toothache gradually decreased to a slight level. Based on this discovery, in the following months, the inventor frequently bit down on the extracted residue or inserted it into his tooth cavity, and surprisingly found that his long-standing halitosis (bad breath) caused by stomach issues had essentially disappeared.

[0024] Currently, gastrointestinal causes of halitosis are believed to include Helicobacter pylori infection, reflux esophagitis, gastrointestinal dysfunction, and other diseases. The inventors therefore speculate that the extraction residue generated during the CBD production process of the company at the time of the application is likely effective against at least one of these four diseases, with Helicobacter pylori infection being the causative factor in most cases of gastric ulcers. If the extraction residue is effective against Helicobacter pylori, it means it could be used to treat Helicobacter pylori-related diseases, such as gastric ulcers. The inventors propose this invention based on this finding.

[0025] In a typical embodiment of the present invention, the composition according to the present invention is obtained by the following method:

[0026] 1) Supercritical CO2 extraction of industrial hemp flowers and leaves yields hemp extract;

[0027] 2) Dissolve the cannabis extract in a non-polar solvent, and then perform a first extraction with an alcohol solution of the first concentration to obtain oil phase 1 and aqueous phase 1. The alcohol solution of the first concentration is a 60-65% ethanol solution or a 65-75% methanol solution.

[0028] 3) Concentrate aqueous phase 1 to obtain aqueous extract, dissolve the aqueous extract in 8-10 times its volume of the alcohol solution, and filter out the insoluble matter;

[0029] 4) Extract aqueous phase 4 5-10 times with 2-3 times the volume of nonpolar solvent to obtain aqueous phase 4. Concentrate aqueous phase 4 to remove solvent to obtain the first composition.

[0030] 5) The first composition is subjected to preferred molecular distillation to obtain the second composition.

[0031] Supercritical extraction

[0032] Generally, any supercritical fluid extraction extract using carbon dioxide as a medium in existing technologies can be used in this invention. However, preferably, the extract used in this invention contains no less than 40% cannabidiol (CBD), ideally between 40% and 60%. In this invention, the industrial hemp flowers and leaves are used. The selected materials are crushed, roasted, and granulated, and then fed into a supercritical extraction device for extraction. The roasting process removes some moisture and decarboxylates CBDA, converting it into CBD.

[0033] In this invention, supercritical extraction is carried out at 30-55°C and 20-40 MPa, with a preferred method using entrainers of alcohols and alkanes. A prior publication by the applicant, CN 112279752A, describes a specific method for supercritical extraction of industrial hemp, and the extract obtained by this method can also be used in this invention. Preferably, after supercritical extraction, the extract is subjected to heat treatment again to achieve complete decarboxylation. Another prior publication by the applicant, CN 112386948A, details a method for decarboxylating cannabidiol (CBDA) from industrial hemp extract. This method involves spreading the extract obtained from supercritical extraction in a drying tray to form a thin layer with an average thickness of 1-7 mm, then placing it in a vacuum oven and heating it at 70-110°C for 2-3 hours under a vacuum of -0.06 to -0.09 MPa. This method can improve the conversion rate of CBDA in the obtained product. This invention preferentially uses the supercritical extract obtained by this method.

[0034] Nevertheless, this invention does not exclude industrial hemp extracts obtained through other means, such as products obtained by extracting industrial hemp with alkanes (typically hexane or heptane) under normal pressure, provided that their CBD content is between 40% and 60%, which may be suitable for the methods of this invention. Therefore, this disclosure refers to raw materials suitable for the methods of this invention as hemp extracts.

[0035] One extraction

[0036] In an extraction reactor, cannabis extract is contacted with a nonpolar solvent to prepare an extract solution. The nonpolar solvent is suitably selected from the group consisting of pentane, hexane, heptane, octane, methylcyclohexane, and mixtures thereof, with hexane and heptane being preferred. Then, an alcohol solution of a first concentration is added for extraction, wherein the alcohol is selected from methanol and ethanol. The purpose of this extraction is to remove as many impurities as possible from the cannabis extract with the alcohol solution, thereby increasing the abundance of CBD in the oil phase 1.

[0037] The concentration and amount of nonpolar solvents and alcohol solutions affect production efficiency. Cannabis extract contains cannabinoids, flavonoids, and terpenes. A first-concentration alcohol solution with high water content is used to extract the more water-soluble components of the cannabis extract, such as cannabinoids and flavonoids. By selecting an appropriate solvent amount, as much of these substances as possible can be extracted while retaining as much cannabidiol as possible in oil phase 1, which is then transferred to another production line for the purification of CBD.

[0038] In a preferred embodiment of the invention, heptane is used as the nonpolar solvent, and the amount of solvent used is 2-4 times the weight of the cannabis extract. The amount of alcohol solution used is 6-8 times the weight of the cannabis extract. In one preferred embodiment, the alcohol solution is an ethanol solution with a mass concentration of 60-65%. In another preferred embodiment, hexane is used as the nonpolar solvent, and the amount of solvent used is 2-4 times the weight of the cannabis extract, and the alcohol solution is a methanol solution with a mass concentration of 65-75%, preferably 68-72%, and most preferably 70%.

[0039] In this disclosure, unless otherwise specified, all references to alcohol solution concentration are made as a mass percentage. If too much heptane is used and too little alcohol solution is used, insufficient water-soluble components (such as flavonoids) will be extracted. These components are more likely to remain in aqueous phases 2 and 3 during subsequent extractions and are less likely to separate from cannabidiol (CBD). If too little heptane is used and too much alcohol solution is used, excessive CBD will enter the aqueous phase, which is undesirable for CBD-targeted production and ultimately reduces product yield. Furthermore, the concentration of the alcohol solution also affects the process effect. If the water content in the alcohol solution is too high, fewer "impurities" will be extracted; if the water content is too low, more CBD will be carried away, reducing product yield.

[0040] The first extraction process is preferably carried out at a temperature of 15-40°C, preferably at room temperature, and the stirring time can be 1-4 hours, preferably 2-4 hours. After stirring, the mixture is allowed to stand for 1-2 hours, and the lower aqueous phase 1 is separated and sent to the next stage, while the oil phase 1 is transferred to the cannabidiol production line.

[0041] Secondary extraction

[0042] Aqueous phase 1 still contains a considerable amount of cannabidiol, from which further extraction of CBD remains cost-effective. For this purpose, it is advantageous to use a small amount of nonpolar solvent for multiple extractions. In a typical embodiment of the invention, aqueous phase 1 is extracted 5-10 times, for example 6-8 times, using a nonpolar solvent of equal volume to aqueous phase 1, or 1 / 10 to 1 / 3, preferably 1 / 8 to 1 / 4, of the volume of aqueous phase 1, using a nonpolar solvent to separate aqueous phase 4 and oil phase 4. The obtained oil phase 4 is used for further CBD production, and aqueous phase 4 is concentrated to remove the solvent, yielding the first extract, i.e., the product according to the invention, which has a mass of 30-40% of that before the second extraction.

[0043] In another specific embodiment, the aqueous phase 1 is first concentrated into an aqueous extract, and then the aqueous extract is dissolved in 65-70% alcohol at 8-10 times its volume. The insoluble matter is filtered off, and then the aqueous extract is extracted multiple times with a small amount of non-polar solvent, preferably 2-3 times its volume of the above-mentioned non-polar solvent, for 5-10 extractions to obtain the aqueous phase 4. The aqueous phase 4 is concentrated to remove the solvent, and the first composition is obtained.

[0044] Molecular distillation

[0045] In this invention, preferably, the high-boiling-point components are removed by molecular distillation to obtain the second composition. The distillation pressure is 0.005-0.02 MPa, preferably not exceeding 0.015 MPa. The distillation temperature can be between 140-180°C. In embodiments of this invention, a scraped-film molecular distillation apparatus is used for molecular distillation, with a scraped-film temperature of 125-135°C, a distillation pressure less than 0.015 MPa, and a molecular distillation temperature of 150-160°C. The resulting second composition has a mass of 80-90% of the first composition and is a colorless amorphous substance after drying. Studies have found that the second composition obtained after molecular distillation has a lower minimum inhibitory concentration (MIC) for Helicobacter pylori.

[0046] Uses and dosage forms

[0047] The first and second compositions according to the invention can be used for the prevention, treatment, and relief of gastrointestinal diseases, particularly chronic gastric ulcers. In such applications, the daily dose for adults can be between 0.1 and 5.0 g, typically between 0.2 and 3.0 g, and can be taken in two or three divided doses. When using the second composition, the daily dose can be reduced to between 0.1 and 4.0 g, for example, between 0.2 and 2.0 g. The compositions of the invention are suitable for formulation into oral dosage forms such as tablets, pills, capsules, granules, powders, and syrups, and can also be formulated into sustained-release formulations.

[0048] These oral dosage forms may contain: binders such as tragacanth gum, gum arabic, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose, or flavorings such as peppermint, wintergreen oil, or cherry flavoring. When the unit dosage form is a capsule, in addition to the above types of materials, it may also contain a liquid carrier such as vegetable oil or polyethylene glycol. Other materials may also be added as coatings or to otherwise modify the shape of the unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, etc. Of course, any materials used to prepare any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in dosage.

[0049] The above dosage forms are also suitable for eliminating oral inflammation and halitosis caused by gastrointestinal diseases.

[0050] The compositions of the present invention can also be formulated into dosage forms such as drops, ointments, and emulsions, and applied directly to the oral cavity for the prevention and treatment of periodontal diseases. For example, they can be applied as ointments or emulsions to the gaps between teeth or cavities, or as drops to the cavities, several times a day.

[0051] It is also anticipated that the compositions of the present invention can be formulated into chewing gum for frequent chewing to prevent and eliminate bad breath, and also for the prevention and treatment of periodontal disease and gastrointestinal disease. For this purpose, the compositions of the present invention can be mixed with a colloidal matrix at a mass ratio of 1:3 to 2:1, and then combined with appropriate amounts of other additives such as sweeteners, emulsifiers, stabilizers, preservatives, etc.

[0052] In one embodiment of the present invention, the composition is made into capsules by simply drying the composition obtained by the above extraction method into a dry powder, and then encapsulating it, with each capsule containing 0.4g.

[0053] Example 1 Preparation of the composition

[0054] Preparation of materials:

[0055] Industrial hemp flower and leaf powder was spread evenly on a baking tray and baked at 130℃ for 3 hours. After cooling to room temperature, it was granulated. 150 kg of industrial hemp flower and leaf granules were loaded into the hopper of a supercritical extraction device. A mixed solvent of heptane and ethanol (heptane:ethanol = 1:1) of 10-15% by weight of the hemp granules was added as an entrainer. The extraction temperature was set at 45℃ and the pressure at 30 MPa. Then, carbon dioxide was introduced for supercritical extraction, yielding 13.7 kg of industrial hemp extract. The content of cannabidiol (CBD) in the extract was determined to be 42.71%.

[0056] The cannabis extract was placed in a homogenizer, and 40 kg of heptane was added and stirred to dissolve it. Then, 100 kg of 65% ethanol was added, and the mixture was stirred at room temperature for 3 hours. After standing for 2 hours, the lower aqueous phase 1 was separated. The oil phase 1 was used to prepare CBD. The aqueous phase 1 was concentrated to obtain 4.21 kg of aqueous extract.

[0057] The aqueous extract was dissolved in 32 kg of 70% ethanol and filtered to remove insoluble matter. The aqueous phase was extracted with 5 kg of heptane, and the extraction was repeated a total of 8 times. The oil phase 4 was combined and used for the other phases. The aqueous phase 4 was concentrated to obtain 2.23 kg of the first composition A1.

[0058] The first composition A1 was molecularly distilled using a scraped-film molecular distillation apparatus at a scraped-film temperature of 125-135℃ and a vacuum degree of less than or equal to -0.085 MPa. The molecular distillation temperature was set at 150-160℃ and the vacuum degree was adjusted accordingly. The resulting light components were collected to obtain 1.76 kg of the second composition B1.

[0059] Example 2 Preparation of the composition

[0060] As in Example 1, to prepare supercritical extraction cannabis extract, 1 kg of cannabis extract (containing 432 g of CBD) was placed in a homogenizer, 3 kg of heptane was added and stirred to dissolve, then 6 kg of 70% methanol was added, and the mixture was stirred and mixed at room temperature for 3 hours. After standing for 2 hours, the lower aqueous phase 1 was separated and concentrated to obtain 284 g of aqueous extract.

[0061] The aqueous extract was dissolved in 2.5 kg of 70% methanol, filtered to remove insoluble matter, and extracted with 0.6 kg of heptane. The extraction was repeated 8 times to obtain aqueous phase 4. The solvent was removed by concentration to obtain 139 g of the first composition A2.

[0062] The first composition A1 was molecularly distilled using a scraped membrane molecular distillation apparatus, and the resulting light components were collected to obtain 118g of the second composition B2.

[0063] Example 3: Determination of MIC of Helicobacter pylori using the agar double-strength method

[0064] Four strains were isolated from patients with chronic gastric ulcers at the First Affiliated Hospital of Kunming Medical University in May 2022, including two strains from male patients and two strains from female patients. Gastric mucosal specimens obtained by gastroscopy were inoculated into 4-5 ml of hydrolyzed casein (MH) broth and cultured in a tri-gas incubator for 3-5 days under microaerophilic conditions (82% N2, 7% O2, 11% CO2), 37°C, and humidity >90%. The specimens were subcultured every 2-3 days, and after two subcultures, morphology and viability were confirmed by staining. The specimens were then stored in cryopreservation buffer containing peptone and glycerol at ultra-low temperature until use.

[0065] Samples A1 and B1 obtained in Example 1 were serially diluted twofold from 512 μg / mL to 4 μg / mL, i.e., 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL. 99.0% pure CBD (self-made by the applicant) was serially diluted twofold from 128 μg / mL to 1 μg / mL, i.e., 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL. Amoxicillin was used as a positive control, with eight double-dose gradients ranging from 3.2 μg / mL to 0.025 μg / mL: 3.2 μg / mL, 1.6 μg / mL, 0.8 μg / mL, 0.4 μg / mL, 0.2 μg / mL, 0.1 μg / mL, 0.05 μg / mL, and 0.025 μg / mL. Each culture dish was inoculated with 9 mL of culture medium and 1 mL of antibiotic.

[0066] Four cryopreserved clinical strains were removed from the ultra-low temperature freezer and brought to room temperature. They were inoculated onto solid culture media and cultured at 37°C under microaerophilic conditions for 3-5 days to prepare Helicobacter pylori bacterial suspension (OD600 = 1). 100 μL of the bacterial suspension was inoculated into each culture dish and placed in an incubator under microaerophilic conditions (82% N2, 7% O2, 11% CO2), 37°C, and humidity >90%. After 48 hours, the lowest concentration of the drug in the sterile culture dish was taken as the minimum inhibitory concentration (MIC).

[0067] Table 2. MIC test results of the product in Example 1 and CBD inhibiting Helicobacter pylori.

[0068]

[0069] The experimental results showed that CBD did not exhibit inhibitory activity against any of the four strains. The first composition A1 and the second composition B1 obtained in Example 1 showed inhibitory activity against all strains, but their MIC values ​​were much higher than those of amoxicillin. This means that the inhibitory activity of the compositions of the present invention against Helicobacter pylori is unrelated to CBD; the inhibitory activity is due to other components, likely the combined effect of multiple components, which requires further investigation.

[0070] The technical solution of the present invention has been described above in conjunction with the embodiments. It should be noted that for those skilled in the art, other similar implementation methods based on the technical concept of the present disclosure without departing from the technical concept of the present disclosure are also within the protection scope of the embodiments of the present disclosure.

Claims

1. A pharmaceutical composition comprising cannabinoids, prepared by the following method: 1) Supercritical CO2 extraction of industrial hemp flowers and leaves yields hemp extract; 2) The cannabis extract was dissolved in a nonpolar solvent, and then subjected to a first extraction with an alcohol solution of the first concentration to obtain oil phase 1 and aqueous phase 1, wherein, The nonpolar solvent is pentane, hexane, heptane, octane, methylcyclohexane or a mixture thereof, and the alcohol solution is a 60-65% ethanol solution or a 65-75% methanol solution; 3) Extract the aqueous phase 1 with an equal volume of the nonpolar solvent 5-10 times to obtain the aqueous phase 4. Concentrate the aqueous phase 4 to remove the solvent to obtain the first composition, which is the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein in step 2), the amount of the nonpolar solvent is 2-4 times the weight of the cannabis extract, and the amount of the alcohol solution is 6-8 times the weight of the cannabis extract.

3. The pharmaceutical composition according to claim 1, wherein, Step 3) is replaced by the following steps: 3-1) Concentrate aqueous phase 1 to obtain aqueous extract, dissolve the aqueous extract in 8-10 times its volume of 65-70% alcohol solution, and filter out insoluble matter; 3-2) After extraction with 2-3 times the volume of the nonpolar solvent of the aqueous extract 5-10 times, the aqueous phase 4 is obtained. The aqueous phase 4 is concentrated to remove the solvent, and the first composition is obtained.

4. The pharmaceutical composition according to claim 1, wherein the second composition is obtained by molecular distillation of the first composition, wherein the distillation pressure is 0.005-0.02 MPa and the distillation temperature is between 140-180°C.

5. A formulation comprising the pharmaceutical composition of any one of claims 1-4, wherein the pharmaceutical composition is any one of tablets, pills, capsules, granules, powders, syrups, drops, ointments, and emulsions.

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