A method for biosynthesis of glycyrrhizinic acid

Abstract

The application provides a glycyrrhizic acid biosynthesis method, characterized in that cell groups of Glycyrrhiza inflate are inoculated in MS culture medium added with 6-benzylaminopurine, naphthaleneacetic acid and water-soluble squalene, after a period of culture, gibberellic acid and helminthosporium are added into the MS culture medium in time sequence, after 15 days of culture, glycyrrhiza culture is obtained through filtration, and finally, the glycyrrhiza culture is subjected to low-pressure treatment, thereby obtaining glycyrrhiza callus for extracting glycyrrhizic acid. The application innovatively proposes to use glycyrrhiza cells as material to culture and produce glycyrrhizic acid, and through the method of multiple induction of helminthosporium and water-soluble squalene, the ability of the Glycyrrhiza inflate cell groups to synthesize glycyrrhizic acid is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for the biosynthesis of glycyrrhizic acid. Background Technology

[0002] Glycyrrhizic acid, the most representative pentacyclic triterpenoid active ingredient in licorice, is not only a core substance in traditional Chinese medicine for clearing heat and detoxifying, anti-inflammatory and liver-protecting, and harmonizing other medicines, but also an important raw material in modern medicine, food, and cosmetics. Clinically, glycyrrhizic acid and its derivatives are widely used in the treatment of chronic hepatitis, liver damage, inflammation, and viral infections, possessing extremely high medicinal and economic value. For a long time, glycyrrhizic acid has mainly relied on extraction from wild or cultivated licorice plants. This method is not only limited by production location, season, and growth cycle, but also faces problems such as resource shortages, uneven quality, and ecological damage caused by over-harvesting, making it difficult to meet continuous and stable market demand. Therefore, developing efficient and green industrial production methods is of great significance.

[0003] Plant cell culture is a crucial method for the large-scale production of glycyrrhizic acid, primarily encompassing callus culture, suspension cell culture, and hairy root culture systems. Stable culture systems obtained through explant induction, combined with culture medium optimization, hormone regulation, and treatment with inducers such as methyl jasmonate, can significantly enhance cell biomass and the accumulation of secondary metabolites. Compared to traditional extraction methods, cell culture offers advantages such as shorter production cycles, controllable conditions, no land occupation, and sustainable production. A cell culture-based biosynthetic strategy is a key pathway to alleviating glycyrrhizic acid resource dependence and achieving green pharmaceutical manufacturing, possessing broad research and application prospects. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for the biosynthesis of glycyrrhizic acid, which can achieve the goal of large-scale biosynthesis of glycyrrhizic acid. To solve the above technical problem, the technical solution adopted by this invention is: a method for the biosynthesis of glycyrrhizic acid, characterized in that cell clusters of *Glycyrrhiza uralensis* are used as material, inoculated into MS medium supplemented with 6-benzylaminopurine, naphthaleneacetic acid, and water-soluble squalene, and cultured. After a period of culture, gibberellic acid and *Helicobacter pylori* are added to the MS medium sequentially at different time intervals. After culturing for 15 days, the licorice culture is obtained by filtration, and finally, the licorice culture is subjected to low-pressure treatment to obtain licorice callus tissue for extracting glycyrrhizic acid.

[0005] Preferably, the addition standards for 6-benzylaminopurine, naphthaleneacetic acid, and water-soluble squalene are: 0.5 mg 6-BA, 1.5 mg NAA, and 0.1-0.5 g water-soluble squalene per 1 L MS medium.

[0006] Preferably, on the 8th day of culture, gibberellic acid (GA3) is added to the MS medium, wherein the addition standard of gibberellic acid (GA3) is 5-20 mg per 1L of MS medium.

[0007] Preferably, gibberellic acid (GA3) is added to the MS medium on the 8th day of culture, wherein the addition standard of gibberellic acid (GA3) is 10-15 mg of gibberellic acid per 1L of MS medium.

[0008] Preferably, on days 8-12 of cultivation, fermentation broth containing Helicobacter pylori is added to the MS medium, wherein the addition standard for the fermentation broth containing Helicobacter pylori is: 5-20 mL of fermentation broth containing Helicobacter pylori per 1 L of MS medium.

[0009] Preferably, on days 9-12 of cultivation, fermentation broth containing Helicobacter pylori is added to the MS medium, wherein the addition standard for the fermentation broth containing Helicobacter pylori is: 8-15 mL of fermentation broth containing Helicobacter pylori per 1 L of MS medium.

[0010] Preferably, the cell clusters of Glycyrrhiza inflata are cell aggregates produced by culturing the leaves or stem segments of Glycyrrhiza inflata.

[0011] Preferably, the specific conditions for low-pressure treatment of the licorice culture are: treatment at 30 kPa for 12-48 hours.

[0012] Preferably, the culture conditions are: shaking culture in a shaker at 25°C, 120 rpm, and 4000 Lux of light.

[0013] The spirulina used in this invention is *Hymenocortis chinensis*, which is disclosed in the invention patent CN 111944697 A (publication number) filed on June 24, 2020.

[0014] Compared with the prior art, the present invention has the following advantages: 1. This invention is the first to promote the biosynthesis of glycyrrhizic acid in the cells of Glycyrrhiza uralensis by inducing the biosynthesis of glycyrrhizic acid through the induction of Helicobacter pylori isolated from the same plant, Oxycodon grandiflorus.

[0015] 2. This invention provides a novel method for the biosynthesis of glycyrrhizic acid. This method combines and optimizes a series of steps, including the cultivation of licorice cells, the addition of Helicobacterium spp., the addition of gibberellic acid, and low-pressure induction in the later stage of cultivation, to achieve the synthesis of high-content (up to 19.3 mg / g) glycyrrhizic acid. The glycyrrhizic acid content is 3.9 times higher than that of conventional licorice cell culture (without the addition of Helicobacterium spp. fermentation broth and without low-pressure treatment). This synthesis process provides an effective means for the industrialization of glycyrrhizic acid.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is the high-performance liquid chromatogram of glycyrrhizic acid standard.

[0018] Figure 2 This is a high-performance liquid chromatogram of the sample powder containing glycyrrhizic acid prepared in Example 1 of the present invention. Detailed Implementation

[0019] Example 1

[0020] This embodiment discloses a method for the biosynthesis of glycyrrhizic acid, the method being as follows: S1. Obtaining cell clusters from inflated licorice fruits: The seeds of *Glycyrrhiza uralensis* were sterilized and then placed in MS medium. After germination and the emergence of the second true leaf, the leaf was cut into 0.3 cm pieces. 2 The small pieces were then placed in MS medium supplemented with 6-BA (6-benzylaminopurine) and NAA (NAA: naphthaleneacetic acid). After culturing for 30 days, licorice cell clusters grew on the leaf surface. The amount of 6-BA added was 0.5 mg per 1 L of MS medium, and the amount of NAA added was 1.5 mg per 1 L of MS medium. S2, Helicobacter: The *Hylocereus* species mentioned is *Oxylocereus*, disclosed in invention patent CN 111944697 A (publication number), filed on June 24, 2020. The specific methods for obtaining, culturing, and preparing the fermentation broth are as follows: First, randomly select disease-free and plump seeds from air-dried Oxytropis ochrocephala seeds and place them in sterile EP tubes. Immerse the tubes in a 75% ethanol solution for 2 min, then wash three times with sterile dH2O for 2 min each time. Next, treat with a 2% NaClO solution for 5 min, followed by three to four washes with sterile dH2O. Then, cut the seeds in half with a sterile blade and place them on PDA medium containing 100 μg / mL chloramphenicol. Seal with parafilm and incubate in the dark at 20±2℃ for 4 days. Finally, obtain Helicobacter sp. on the seed surface. Identify the fungal ITS fragment using molecular biology methods; the product is 583 bp. NCBI homology comparison shows a close relationship ≥ 1 / 2 with Undifilum sp. 99% of the Undifilum sp. is deposited at the China Microbial Culture Collection Center (CGMCC) on November 19, 2018, with accession number CGMCC NO.16997. The rDNA gene sequence of the Undifilum sp. is shown in SEQ ID NO:1.

[0021] Helicobacter pylori was inoculated into PDA medium and cultured for 5 days at 28°C and 120 rpm. Then, the mycelium was filtered out to obtain the filtrate, which is the Helicobacter pylori fermentation broth, for later use.

[0022] S3. Liquid culture of licorice cell clusters and induction with glycyrrhizic acid: S301. Licorice cell clusters were inoculated into MS medium containing 6-BA, NAA and water-soluble squalene (CAS: 7683-64-9) and then placed in a shaker at 25°C, 120 rpm and 4000 Lux of light for incubation.

[0023] The addition amounts of 6-BA, NAA, and water-soluble squalene described in S301 are as follows: 0.5 mg 6-BA, 1.5 mg NAA, and 0.3 g water-soluble squalene per 1 L MS medium. S302. On the 8th day of culture, gibberellic acid (GA3) is added to MS medium, and on the 10th day of culture, Helicobacter pylori fermentation broth is added; the addition standard of gibberellic acid (GA3) is: 5 mg gibberellic acid per 1L MS medium; the addition standard of Helicobacter pylori fermentation broth is: 10 mL Helicobacter pylori fermentation broth per 1L MS medium. S303. On the 15th day of culture, the culture medium was filtered out to obtain licorice culture, which was then placed under a low pressure of 30 kPa for 24 hours to obtain licorice callus. S4, Extraction and Detection of Glycyrrhizic Acid S401, Extraction of glycyrrhizic acid The licorice callus obtained from S303 was dried in a 60℃ oven to constant weight. The dried sample was then removed, thoroughly ground into powder, and passed through a 60-mesh sieve to obtain sample powder. 0.2 g of sample powder was weighed, 5 mL of methanol was added, and the mixture was placed in an ultrasonic cleaner for ultrasonic extraction for 60 min. After ultrasonic extraction, the mixture was centrifuged, and the licorice extract was collected.

[0024] Extraction and detection of S402 and glycyrrhizic acid Glycyrrhizic acid was separated from the licorice extract collected in S4 using HPLC. The mobile phase was acetonitrile-0.2% formic acid aqueous solution; the detection wavelength was 254 nm; the flow rate was 1.0 mL / min; the temperature was 30 ℃; the injection volume was 10 μL; and the gradient elution program was shown in Table 1. Table 1

[0025] like Figure 2 As shown, the retention time (peak elution time) of glycyrrhizic acid in the licorice extract prepared in this embodiment is 34.5 min; compared with... Figure 1 The peak elution times of the glycyrrhizic acid standard were consistent. Based on the comparison with the peak area of ​​the standard, the content of glycyrrhizic acid in the licorice extract was calculated. The calculated content of glycyrrhizic acid in the licorice cell clusters (i.e., sample powder) was 19.3 mg / g (dry weight).

[0026] In this embodiment, the MS medium is a commercially available liquid MS medium with the following specific formulation: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate 370 mg / L, calcium chloride 440 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, disodium EDTA 37.25 mg / L, ferrous sulfate 27.85 mg / L, inositol 100 mg / L, glycine 2 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 0.5 mg / L, and sucrose 30 g / L.

[0027] In this embodiment, the preparation process of the PDA culture medium is as follows: Weigh 200 g of peeled potatoes, shred them into thin strips using a potato shredder, place them in a 2000 mL beaker, add 900 mL of single-distilled water, boil on an electric stove for 20 min, filter through 4 layers of gauze and collect the filtrate, add 2.0% Sucrose and 1.5% Agar powder, and finally add sterile dH2O to make up to 1000 mL, dispense into tissue culture flasks, and sterilize at 121℃ for 20 min to obtain the PDA culture medium.

[0028] Example 2

[0029] This embodiment discloses a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: S301. Licorice cell clusters were inoculated into MS medium containing 6-BA, NAA and water-soluble squalene (CAS: 7683-64-9) and then placed in a shaker at 25°C, 120 rpm and 4000 Lux of light for incubation.

[0030] The addition amounts of 6-BA, NAA, and water-soluble squalene described in S301 are as follows: 0.5 mg 6-BA, 1.5 mg NAA, and 0.2 g water-soluble squalene per 1 L MS medium. S302. On the 8th day of culture, GA3 is added to MS medium, and on the 10th day of culture, Helicobacter pylori fermentation broth is added; the addition standard of GA3 is: 10 mg of GA3 per 1L of MS medium; the addition standard of Helicobacter pylori fermentation broth is: 5 mL of Helicobacter pylori fermentation broth per 1L of MS medium. S303. On the 15th day of culture, the culture medium was filtered out to obtain licorice culture, which was then placed under a low pressure of 30 kPa for 12 hours to obtain licorice callus.

[0031] Experiments showed that the glycyrrhizic acid content in the licorice cell clusters prepared in this embodiment was 15.8 mg / g (dry weight).

[0032] Example 3

[0033] This embodiment discloses a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: This embodiment discloses a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: S301. Licorice cell clusters were inoculated into MS medium containing 6-BA, NAA and water-soluble squalene (CAS: 7683-64-9) and then placed in a shaker at 25°C, 120 rpm and 4000 Lux of light for incubation.

[0034] The addition amounts of 6-BA, NAA, and water-soluble squalene described in S301 are: 0.5 mg 6-BA, 1.5 mg NAA, and 0.5 g water-soluble squalene per 1 L MS medium. S302. On the 8th day of culture, GA3 is added to the MS medium, and on the 10th day of culture, Helicobacter pylori fermentation broth is added. The standard for adding GA3 is 20 mg of GA3 per 1 L of MS medium. The standard for adding Helicobacter pylori fermentation broth is 20 mL of Helicobacter pylori fermentation broth per 1 L of MS medium. S303. On the 15th day of culture, the culture medium was filtered out to obtain licorice culture, which was then placed under a low pressure of 30 kPa for 48 hours to obtain licorice callus.

[0035] Experiments showed that the glycyrrhizic acid content in the licorice cell clusters prepared in this embodiment was 12.6 mg / g (dry weight).

[0036] Example 4

[0037] This embodiment discloses a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: This embodiment discloses a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: S301. Licorice cell clusters were inoculated into MS medium containing 6-BA, NAA and water-soluble squalene (CAS: 7683-64-9) and then placed in a shaker at 25°C, 120 rpm and 4000 Lux of light for incubation.

[0038] The addition amounts of 6-BA, NAA, and water-soluble squalene described in S301 are: 0.5 mg 6-BA, 1.5 mg NAA, and 0.1 g water-soluble squalene per 1 L MS medium. S302. On the 8th day of culture, GA3 is added to the MS medium, and on the 10th day of culture, Helicobacter pylori fermentation broth is added. The standard for adding GA3 is: 10 mg of GA3 per 1 L of MS medium; the standard for adding Helicobacter pylori fermentation broth is: 15 mL of Helicobacter pylori fermentation broth per 1 L of MS medium.

[0039] S303. On the 15th day of culture, the culture medium was filtered out to obtain licorice culture, which was then placed under a low pressure of 30 kPa for 12 hours to obtain licorice callus.

[0040] Experiments showed that the glycyrrhizic acid content in the licorice cell clusters prepared in this embodiment was 12.6 mg / g (dry weight).

[0041] Comparative Example 1 This comparative example provides a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: step S2, which prepares the fermentation broth of *Helicobacter pylori*, is not performed; and steps S302 and S303 are not performed in S3, which is to culture the bacteria in a shaker at 25°C, 120 rpm, and 4000 Lux of light for 15 days as described in S301.

[0042] In this comparative example, the glycyrrhizic acid content in the licorice cell clusters was measured to be 2.8 mg / g (dry weight).

[0043] Compared with Example 1, without the addition of GA3 and Helicobacter pylori fermentation broth, and without low-pressure treatment, the glycyrrhizic acid content decreased by 16.5 mg / g.

[0044] Comparative Example 2 (Original Example 6) This comparative example provides a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: step S2, which prepares the fermentation broth of *Helicobacter pylori*, is not performed; steps S302 and S303 are not performed in S3; and 0.2 g of water-soluble squalene is added to every 1 L of MS medium in S301. The culture process is carried out in a shaker at 25°C, 120 rpm, and 4000 Lux of light for 15 days as described in S301.

[0045] In this comparative example, the glycyrrhizic acid content in the licorice cell clusters was measured to be 4.4 mg / g (dry weight).

[0046] Compared with Comparative Example 1, no GA3 and Helicobacter pylori fermentation broth were added, no low-pressure treatment was performed, and the amount of water-soluble squalene added was reduced from 0.3 g to 0.2 g. Compared with Comparative Example 2, the amount of water-soluble squalene added was reduced from 0.3 g to 0.2 g. The result was that the glycyrrhizic acid content increased by 1.6 mg / g. As an inducer, water-soluble squalene can stimulate the activity of glycyrrhizic acid biosynthetic enzyme, thereby increasing the amount of glycyrrhizic acid biosynthesis. However, the amount of biosynthesis is dose-dependent. Too low or too high a dose will not achieve the desired effect. Excessive doses of water-soluble squalene may even inhibit the amount of glycyrrhizic acid biosynthesis.

[0047] Comparative Example 3 This comparative example provides a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: S302. On the 8th day of cultivation, add Helicobacter pylori fermentation broth to MS medium; the addition standard for Helicobacter pylori fermentation broth is: 10 mL of Helicobacter pylori fermentation broth per 1 L MS medium.

[0048] In this comparative example, the glycyrrhizic acid content in the licorice cell clusters was measured to be 8.2 mg / g (dry weight).

[0049] Compared with Example 1, no GA3 was added and no low-pressure treatment was performed. Compared with Comparative Example 1, Helicobacter spp. fermentation broth was added. The results showed that the glycyrrhizic acid content decreased from 19.3 mg / g in Example 1 to 8.2 mg / g, a difference of 11.1 mg / g, and increased from 2.8 mg / g in Comparative Example 1 to 8.2 mg / g. This indicates that although adding Helicobacter spp. alone can increase the glycyrrhizic acid content of the product, the potential for improvement is limited.

[0050] Comparative Example 4 This comparative example provides a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: Operation S2, namely the preparation of Helicobacter pylori fermentation broth, was not performed; S302. On the 8th day of culture, add GA3 to MS medium; the standard for adding GA3 is: 15 mL of GA3 per 1 L of MS medium.

[0051] In this comparative example, the glycyrrhizic acid content in the licorice cell clusters was measured to be 3.9 mg / g (dry weight).

[0052] Compared with Example 1, no Helicobacter spp. fermentation broth was added, and no low-pressure treatment was performed. Compared with Comparative Example 1, GA3 was added. The results showed that the glycyrrhizic acid content decreased from 19.3 mg / g in Example 1 to 3.9 mg / g, a difference of 15.4 mg / g, and increased from 2.8 mg / g in Comparative Example 1 to 3.9 mg / g. This indicates that although adding GA3 alone can increase the glycyrrhizic acid content of the product, the potential for improvement is limited.

[0053] Comparative Example 5 This comparative example provides a method for the biosynthesis of glycyrrhizic acid, which is the same as the method in Example 1, except that: The S2 operation was not performed, and the fermentation broth of *Helicobacter pylori* was prepared. Step S302 was not executed in S3; The treatment time under low and medium pressure in S303 has been reduced from 24 hours to 12 hours; In this comparative example, the glycyrrhizic acid content in the licorice cell clusters was measured to be 4.1 mg / g (dry weight).

[0054] Compared to Example 1, no GA3 and Helicobacter spp. fermentation broth were added, and the low-pressure treatment time was changed to 12 hours. Compared to Comparative Example 1, low-pressure treatment was added. The results showed that the glycyrrhizic acid content decreased from 19.3 mg / g in Example 1 to 4.1 mg / g, a difference of 15.2 mg / g, and increased from 2.8 mg / g in Comparative Example 1 to 4.1 mg / g. This indicates that low-pressure treatment helps to increase the glycyrrhizic acid content, but the increase is limited.

[0055] By comparing Example 1 and Comparative Examples 1-5, it can be clearly seen that the addition of GA3 and Helicobacter pylori fermentation broth during the middle stage of cultivation, and the low-pressure treatment in the later stage of cultivation, together induce the biosynthesis of glycyrrhizic acid, resulting in a significant increase in the content of glycyrrhizic acid.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for the biosynthesis of glycyrrhizic acid, characterized in that, Cell clusters of Glycyrrhiza inflata were inoculated into MS medium supplemented with 6-benzylaminopurine, naphthaleneacetic acid and water-soluble squalene and cultured. After a period of culture, gibberellic acid and Helicobacter spp. were added to the MS medium in sequence at different time intervals. After 15 days of culture, the licorice culture was obtained by filtration. Finally, the licorice culture was subjected to low pressure treatment to obtain licorice callus tissue for the extraction of glycyrrhizic acid.

2. The method for biosynthesizing glycyrrhizic acid according to claim 1, characterized in that, The addition standards for 6-benzylaminopurine, naphthaleneacetic acid, and water-soluble squalene are as follows: 0.5 mg 6-BA, 1.5 mg NAA, and 0.1-0.5 g water-soluble squalene per 1 L MS medium.

3. The method for biosynthesizing glycyrrhizic acid according to claim 2, characterized in that, On day 8 of culture, gibberellic acid (GA3) was added to the MS medium. The addition standard for gibberellic acid (GA3) was 5-20 mg per 1 L of MS medium.

4. The method for biosynthesizing glycyrrhizic acid according to claim 3, characterized in that, On day 8 of culture, gibberellic acid (GA3) was added to MS medium at a standard of 10-15 mg per 1 L of MS medium.

5. The method for biosynthesizing glycyrrhizic acid according to claim 3, characterized in that, On days 8-12 of culture, fermentation broth containing Helicobacter pylori was added to MS medium. The addition standard for the fermentation broth containing Helicobacter pylori was 5-20 mL per 1 L of MS medium.

6. The method for biosynthesizing glycyrrhizic acid according to claim 5, characterized in that, On days 9-12 of culture, fermentation broth containing Helicobacter pylori was added to MS medium. The addition standard for the fermentation broth containing Helicobacter pylori was 8-15 mL per 1 L of MS medium.

7. The method for biosynthesizing glycyrrhizic acid according to claim 1, characterized in that, Cell clusters of Glycyrrhiza inflata are cell aggregates produced by culturing the leaves or stem segments of Glycyrrhiza inflata.

8. The method for biosynthesizing glycyrrhizic acid according to claim 1, characterized in that, The specific conditions for low-pressure treatment of the licorice culture are: treatment at 30 kPa for 12-48 hours.

9. The method for biosynthesizing glycyrrhizic acid according to claim 1, characterized in that, The cultivation conditions were as follows: shaking culture in a shaker at 25°C, 120 rpm, and 4000 Lux of light.

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