Extraction method of cannabidiolic acid

Abstract

The present invention relates to the field of biotechnology, and in particular to a method for extracting cannabidiol acid. The present invention provides a method for extracting cannabidiol acid, comprising: sterilizing industrial hemp, mixing it with a strain with a deposit number of CGMCC: 1.3604, fermenting it, adjusting the pH value to be alkaline, ultrasonically extracting it, filtering it, collecting the extract, extracting it with an organic solvent, and purifying it to obtain the cannabidiol acid. The present invention provides a method for extracting cannabidiol acid, which fills the gaps in the prior art, and most of the current extraction methods for industrial hemp are based on chemical extraction methods. The present invention uses microorganisms to increase the selection of methods for extracting related components. By the method provided by the present invention, the cannabidiol acid extracted has a high purity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for extracting cannabidiolic acid. Background Art

[0002] Industrial hemp has gained market traction due to the medicinal properties of its cannabinoids, leading to widespread research into their extraction and purification. Since cannabidiol (CBD) has become a popular active ingredient in recent years and its efficacy has been well-established, most extraction and purification efforts focus on it. Cannabidiolic acid, a derivative of CBD, undergoes decarboxylation under various conditions, transforming it into CBD. Many studies have exploited this decarboxylation property to increase the CBD content in extracts. However, as a hydrophilic cannabinoid, CBD possesses unique biological activities, many of which cannot be achieved by CBD itself. Therefore, it is necessary to conduct research on CBD extraction and purification technologies, as well as systematic and in-depth biological activity studies. This will contribute to the healthy and diversified development of industrial hemp in the medical and health sectors.

[0003] Although cannabidiol acid and cannabidiol differ only in one carboxyl group, their solubility, physicochemical properties, and biological activities are all different. The extraction and purification technology for cannabidiol is not completely suitable for cannabidiol acid, so targeted optimization is needed. Cannabidiol acid is relatively water-soluble and has insufficient thermal stability, so special attention should be paid during the experiment. There are relatively few invention patents specifically for cannabidiol acid, and most of the previous extraction methods for industrial hemp are based on basic chemical extraction methods. Therefore, it is necessary to find a new extraction method to fill the corresponding gap. Summary of the Invention

[0004] In light of this, the present invention provides a method for extracting cannabidiol acid. This method addresses a gap in the existing technology. While most current extraction methods for industrial hemp rely on chemical extraction, the present invention utilizes microbial extraction to expand the options for extracting relevant components. This method, using the cannabidiol acid extraction method, allows for the extraction of cannabidiol acid with a high degree of purity.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for extracting cannabidiolic acid, comprising:

[0007] After sterilizing industrial hemp, the mixture is mixed with a strain with a preservation number of CGMCC: 1.3604, fermented, the pH value is adjusted to alkaline, ultrasonically extracted, filtered, the extract is collected, extracted with an organic solvent, and purified to obtain the cannabidiolic acid.

[0008] Specifically, in some embodiments of the present invention, the content of cannabidiolic acid (CBDA) in the industrial hemp in the above extraction method is 40%.

[0009] In some embodiments of the present invention, the strain is taken from the mixture in the above extraction method to prepare a bacterial solution, the culture temperature of the strain is 37°C, the time is 72 hours, and the number of viable bacteria in the bacterial solution is 6×10 8 CFU / mL.

[0010] Specifically, in some embodiments of the present invention, the culture medium for culturing the strain in the above-mentioned extraction method includes Horikoshi I medium; the Horikoshi I medium includes: 100 g / L glucose, 5.0 g / L polypeptone, 5.0 g / L yeast extract, 1.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 10.0 g / L sodium carbonate and 20.0 g / L sodium chloride; the pH of the Horikoshi I medium is 10.

[0011] More specifically, in some embodiments of the present invention, the culturing time in the above extraction method is 48 hours.

[0012] In some embodiments of the present invention, the fermentation in the above-mentioned extraction method uses a culture solution, and the solid-liquid ratio of the industrial hemp to the culture solution is 1:20 kg / L.

[0013] Specifically, in some embodiments of the present invention, the culture solution in the above extraction method includes: 12.0 g / L konjac flour, 15.0 g / L soybean cake powder, 5.0 g / L yeast extract, 40.0 g / L sodium chloride, 1.5 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate and 10.0 g / L sodium carbonate; the pH of the culture solution is 10.

[0014] In some embodiments of the present invention, the fermentation temperature in the above extraction method is 37° C., the fermentation time is 72 h, and the ventilation volume is 0.3 vvm.

[0015] In some embodiments of the present invention, the solution used in the above extraction method for adjusting the pH value to alkaline is an alkaline solution.

[0016] Specifically, in some embodiments of the present invention, the alkaline solution in the above extraction method includes 2% sodium chloride.

[0017] In some embodiments of the present invention, the pH value in the above-mentioned extraction method of adjusting the pH value is 9-12.

[0018] In some embodiments of the present invention, the ultrasonic power in the above extraction method is 400w and the time is 20 to 40 minutes.

[0019] In some embodiments of the present invention, after adjusting the pH value to alkaline in the above-mentioned extraction method, before the ultrasonic extraction, the material-liquid ratio of the industrial hemp to the total amount of the culture solution, the bacterial solution and the alkaline solution is 1:(35-45) kg / L.

[0020] Specifically, in some embodiments of the present invention, the above extraction method further includes a step of adjusting the pH to acidic after collecting the extract and before extracting with an organic solvent.

[0021] More specifically, in some embodiments of the present invention, the solution used to adjust the pH to acidic in the above extraction method is 0.2 M hydrochloric acid.

[0022] Specifically, in some embodiments of the present invention, the organic solvent in the above extraction method includes: n-hexane, n-heptane and ethyl acetate; and the extraction is performed four times.

[0023] In some embodiments of the present invention, the purification method in the above extraction method is liquid chromatography; the conditions of the liquid chromatography are: injection volume of 1 to 5 mL, flow rate of 2 to 5 mL / min; column temperature of 15 to 35°C.

[0024] Specifically, in some embodiments of the present invention, the column temperature in the above extraction method is 25°C.

[0025] Specifically, in some embodiments of the present invention, the above extraction method further comprises a freeze-drying or rotary evaporation step after the purification.

[0026] Specifically, in some embodiments of the present invention, in order to fully extract the cannabinoids in the industrial hemp, the ultrasonic extraction in the above-mentioned extraction method does not require too long after the strain is cultured, which correspondingly saves the time of using the ultrasonic extraction alone.

[0027] Specifically, in some embodiments of the present invention, the cannabidiol acid in the industrial hemp in the above-mentioned extraction method will be more easily dissolved in the organic solvent due to solubility issues, thereby purifying the cannabidiol acid from the alkaline solution.

[0028] Specifically, in some embodiments of the present invention, the liquid chromatography in the above extraction method is a common natural active ingredient purification process that can be easily scaled up, and therefore the simplest and most direct method is adopted in this embodiment.

[0029] The present invention provides a method for extracting cannabidiolic acid, comprising: sterilizing industrial hemp, mixing it with a strain with a preservation number of CGMCC:1.3604, fermenting it, adjusting the pH value to alkaline, ultrasonically extracting it, filtering it, collecting the extract, extracting it with an organic solvent, and purifying it to obtain the cannabidiolic acid.

[0030] The present invention provides a method for extracting cannabidiol acid, addressing a gap in the existing technology. While most current extraction methods for industrial hemp rely on chemical extraction, the present invention utilizes microbial extraction to expand the options for extracting related components. The method provided by the present invention allows for the extraction of cannabidiol acid with high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0032] Figure 1 Figure 4 shows the HPLC chromatogram of the organic extract, wherein the main peak is CBDA;

[0033] Figure 2 1 shows the HPLC chromatogram of the preparative liquid chromatography purified solution; wherein the main peak is CBDA;

[0034] Figure 3 Shows the liquid chromatograms of the CBDA products obtained in Examples 5 to 7;

[0035] Figure 4 1 shows the HPLC chromatogram of the alkaline extract obtained in Example 2; wherein the main peak is CBDA;

[0036] Figure 5 1 is a high performance liquid chromatogram of the alkaline extract of Comparative Example 1, wherein the main peak is CBDA. DETAILED DESCRIPTION

[0037] The invention discloses a method for extracting cannabidiolic acid.

[0038] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0039] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0040] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0041] The use of any and all examples or exemplary language, such as "such as" or "including," herein is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0042] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, all ranges, amounts, values, and percentages used in this disclosure should be understood to be modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specified value or range.

[0043] In the preparation of the bacterial solution, ultrasound-assisted extraction, organic solvent extraction, refined treatment, comparative examples and verification tests of the present invention, all raw materials and reagents used can be purchased from the market.

[0044] The present invention will be further described below in conjunction with the embodiments:

[0045] Example 1 Extraction of CBDA by alkaliphilic bacteria

[0046] Alkaliphilic Bacillus sp. was purchased from the China General Microbiological Culture Collection (CGMCC No. 1.3604). Alkaliphilic Bacillus sp. was cultured using Horikoshi I medium containing 100 g / L glucose, 5.0 g / L polypeptone, 5.0 g / L yeast extract, 1.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 10.0 g / L sodium carbonate, and 20.0 g / L sodium chloride, with a pH of 10.0. The culture temperature was 37°C.

[0047] 1) Preparation of bacterial solution: After culturing alkaliphilic Bacillus for 48 hours, when the number of viable bacteria reaches 6×10 8After the CFU / mL was reached, a 1% (v / v) inoculum (10 mL) was inoculated into 1 L of sterilized liquid culture medium. The liquid culture medium contained: 10.0 g / L peptone, 5.0 g / L yeast extract, 10.0 g / L soluble starch, 20.0 g / L sodium chloride, 1.5 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate, 10.0 g / L sodium carbonate, pH = 10.0. The culture temperature was 37°C, the shaking speed was 200 r / min, and the incubation time was 72 h.

[0048] 2) Fermentation and extraction of industrial hemp: Add 2L of fermentation medium into the fermentation tank according to the ratio, sterilize it and cool it to 37°C.

[0049] Industrial hemp was sterilized by ultraviolet irradiation for 1 hour and then added to the fermenter at a material-liquid ratio of 1:20 (100 g). The fermenter temperature was controlled at 37°C. The concentration of the bacterial solution obtained in step 1) was adjusted to 6×10 8 CFU / mL, remove 100mL of the adjusted bacterial solution and add it to the fermenter. Set the stirring speed to 30r / min, the ventilation volume to 0.3vvm, and the fermentation reaction for 72h. The industrial hemp in this example is the variety studied by this institute, and the CBDA content in the sample is 4.0%.

[0050] The fermentation medium formula is as follows: 12.0 g / L konjac flour, 15.0 g / L soybean cake powder, 5.0 g / L yeast extract, 40.0 g / L sodium chloride, 1.5 g / L potassium hydrogen phosphate, 0.3 g / L magnesium sulfate, 10.0 g / L sodium carbonate, pH = 10.0.

[0051] Example 2 Ultrasound-assisted extraction

[0052] The bacterial culture solution containing industrial hemp raw materials obtained in Example 1 was collected and a certain volume (2 L) of alkaline water with a pH value of 12 and 2% sodium chloride was added to the mixed state, and the pH was adjusted to 11. The ratio of the total volume of the mixed solution to the industrial hemp feed was controlled at 45:1. After stirring and mixing evenly, the mixed solution was placed in an ultrasonic cleaning machine (power: 400W) for auxiliary extraction. After full power extraction for 20 minutes, it was allowed to stand. The mixed solution was filtered through filter paper to obtain about 4.5 L of alkaline extract.

[0053] Example 3 Ultrasonic assisted extraction

[0054] The bacterial culture solution containing industrial hemp raw materials obtained in Example 1 was collected and a certain volume (2 L) of alkaline water with a pH value of 12 and 2% sodium chloride was added to the mixed state, and the pH was adjusted to 10. The ratio of the total volume of the mixed solution to the industrial hemp feed was controlled at 40:1. After stirring and mixing evenly, the mixed solution was placed in an ultrasonic cleaning machine (power: 400W) for auxiliary extraction. After full power extraction for 30 minutes, it was allowed to stand. The mixed solution was filtered through filter paper to obtain approximately 4 L of alkaline extract.

[0055] Example 4 Ultrasound-assisted extraction

[0056] The bacterial culture solution containing industrial hemp raw materials obtained in Example 1 was collected and a certain volume (2 L) of alkaline water with a pH value of 12 and 2% sodium chloride was added to the mixed state, and the pH was adjusted to 9. The ratio of the total volume of the mixed solution to the industrial hemp feed was controlled at 35:1. After stirring and mixing evenly, the mixed solution was placed in an ultrasonic cleaning machine (power: 400W) for auxiliary extraction. After full power extraction for 40 minutes, it was allowed to stand. The mixed solution was filtered through filter paper to obtain approximately 3.5 L of alkaline extract.

[0057] Example 5 Organic solvent extraction

[0058] A certain amount (100 mL) of 0.2 M hydrochloric acid was added to the alkaline extract obtained in Example 2 to make the solution acidic. A certain volume (1 L) of an organic solvent (n-hexane, n-heptane, ethyl acetate) was added for extraction four times, and the extracts were combined.

[0059] Example 6 Organic solvent extraction

[0060] A certain amount (100 mL) of 0.2 M hydrochloric acid was added to the alkaline extract obtained in Example 3 to make the solution acidic. A certain volume (1 L) of an organic solvent (n-hexane, n-heptane, ethyl acetate) was added to perform extraction four times, and the extracts were combined.

[0061] Example 7 Organic solvent extraction

[0062] A certain amount (100 mL) of 0.2 M hydrochloric acid was added to the alkaline extract obtained in Example 4 to make the solution acidic. A certain volume (1 L) of an organic solvent (n-hexane, n-heptane, ethyl acetate) was added for extraction four times, and the extracts were combined.

[0063] Example 8 Refining treatment

[0064] The extracts obtained in Examples 5 to 7 were subjected to rotary evaporation or vacuum drying to remove the solvent and then dissolved in a predetermined volume (20 mL) of methanol. Purification was performed by preparative liquid chromatography using a standard C18 column, with an injection volume of 1 to 5 mL, a flow rate of 2 to 5 mL / min, a column temperature of 15 to 35°C (25°C being optimal), and a detection wavelength of 220 nm. After injection, the effluent fraction corresponding to the CBDA peak time was intercepted and freeze-dried or rotary evaporated to obtain a high-purity sample powder. The preparative liquid chromatography treatment described above yielded a CBDA product with a purity exceeding 90%.

[0065] The results are as follows Figure 1 and Figure 2 As shown, after adjusting the acidity and extracting with an organic solvent, the chromatographic purity of CBDA in the organic solvent was 90.5%. After further treatment with preparative liquid chromatography, the chromatographic purity of the obtained product was 95.1%.

[0066] The liquid chromatogram of the CBDA product obtained according to the above steps is as follows: Figure 3 As shown in Figure 2, the chromatographic purities of the three products are 94.1%, 91.7%, and 90.5%, respectively. This shows that organic solvent extraction can rapidly increase the purity of CBDA, while obtaining high-purity CBDA requires preparative liquid chromatography. This allows us to obtain CBDA products of varying purities.

[0067] Comparative Example 1

[0068] In order to compare the effect of bacterial extraction assistance, ultrasonic extraction was used as a comparison. In this method, alkaline water was directly used to ultrasonically extract the sample. The specific method is as follows:

[0069] 100g of industrial hemp powder was added to 4L of alkaline water with a pH of 12 and 2% sodium chloride, maintaining a 40:1 ratio of total volume of alkaline water to industrial hemp. After stirring and mixing thoroughly, the mixture was placed in an ultrasonic cleaner (power: 400W) for assisted extraction. After 20 minutes of full power extraction, the mixture was allowed to stand. The alkaline water was filtered through filter paper to obtain approximately 4L of alkaline extract. The CBDA concentration in this extract was compared with that of the alkaline extract obtained in Example 2.

[0070] The results are as follows Figure 4 、 Figure 5As shown in Table 1, the effect of bacterial extraction combined with ultrasound is compared to ultrasound alone. Bacterial culture supplemented with ultrasound-assisted extraction achieves excellent extraction results. The extraction yield of the alkaline extract after bacterial culture and ultrasound-assisted extraction reached 90.5%, which is higher than the extraction yield of ultrasound-assisted extraction alone (58.6%), indicating that bacterial culture effectively improves the extraction efficiency.

[0071] Table 1

[0072] Processing method / sample CBDA content (g) Extraction rate (%) CBDA chromatographic purity (%) Bacteria + ultrasound 3.61±0.11 90.3±2.6 70.9±2.1 Ultrasound alone 2.28±0.07 57.1±1.8 62.0±1.5

[0073] The experiment was repeated three times, and the mean ± deviation was taken; the CBDA chromatogram of bacteria + ultrasound is shown in Figure 4 As shown; the chromatogram of ultrasonic CBDA alone is as shown Figure 5 shown.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for extracting cannabidiolic acid, comprising: After sterilization, take industrial hemp and deposit it with the accession number CGMCC: 1.3604 strains, fermented, adjusted to alkaline pH, ultrasonically extracted, filtered, collected extract, extracted with an organic solvent, and purified to obtain cannabidiolic acid; The mixing step is as follows: the strain is prepared into a bacterial solution, the culture temperature of the strain is 37°C, the culture time is 72 hours, and the number of viable bacteria in the bacterial solution is 6×10 8 CFU / mL; taking the bacterial solution and mixing it with sterilized industrial hemp for fermentation; The fermentation uses a culture solution, and the solid-liquid ratio of the industrial hemp to the culture solution is 1:20 kg / L; The fermentation temperature was 37° C., the fermentation time was 72 h, and the ventilation rate was 0.3 vvm.

2. The extraction method according to claim 1, wherein The solution used in adjusting the pH value to alkaline is an alkaline solution.

3. The extraction method according to claim 2, wherein The pH value in the pH adjustment is 9-12.

4. The extraction method according to claim 3, wherein The power of the ultrasound is 400W, and the time is 20 to 40 minutes.

5. The extraction method according to claim 4, wherein After the pH value is adjusted to alkaline, before the ultrasonic extraction, the material-liquid ratio of the industrial hemp to the total amount of the culture solution, the bacterial solution and the alkaline solution is 1:(35-45) kg / L.

6. The extraction method according to any one of claims 1 to 5, characterized in that The purification method is liquid chromatography; the conditions of the liquid chromatography are: injection volume of 1 to 5 mL, flow rate of 2 to 5 mL / min; and column temperature of 15 to 35°C.

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