A glycyrrhiza uralensis dry cell line and application thereof

Abstract

The present application relates to the technical field of biology, and particularly provides a licorice stem cell line and application thereof. The licorice stem cell line of the present application has been preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 46600, is named as licorice stem cell strain gc001, has a high content of secondary metabolites, and can be directly used in high-end skin care products and medical products, and expands the value of licorice resources.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a licorice root stem cell line and its applications. Background Technology

[0002] Licorice (Glycyrrhiza glabra), an important medicinal plant, contains a variety of valuable secondary metabolites in its roots and rhizomes, including glycyrrhizin, glycyrrhizic acid, and glycyrrhizin flavonoids. These compounds have significant application potential in the fields of skincare, pharmaceuticals, and cosmetics. For example, glycyrrhizin, with its powerful antioxidant and whitening effects, is widely used in high-end skincare products, effectively inhibiting tyrosinase activity and reducing melanin production; glycyrrhizic acid has anti-inflammatory and anti-allergic properties and is often used to treat skin inflammation; while glycyrrhizin flavonoids are also highly favored in anti-aging and moisturizing products. With the growth of market demand, the commercial value of these secondary metabolites is becoming increasingly prominent, and the development of efficient and sustainable production methods has become a focus of industry attention.

[0003] However, the production of licorice secondary metabolites faces significant bottlenecks in existing technologies. The content of target compounds such as glycyrrhizin and glycyrrhizic acid in natural licorice plants is generally low. Traditional extraction methods are not only inefficient but also limited by the long plant growth cycle, resource scarcity, and environmental impact. These challenges are particularly pronounced in industries such as skincare products, where low content leads to high extraction costs, increased product prices, and difficulty in meeting the demands of large-scale industrial production.

[0004] Therefore, there is an urgent need to screen for a Glycyrrhiza glabra stem cell line that has a stable expression of secondary metabolites. Summary of the Invention

[0005] The purpose of this invention is to provide a *Glycyrrhiza glabra* stem cell line and its applications. This invention relates to a *Glycyrrhiza glabra* stem cell line, which was deposited on September 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 46600. Named *Glycyrrhiza glabra* stem cell line gc001, it has a high content of secondary metabolites and can be directly used in high-end skincare and pharmaceutical products, expanding the value of licorice resources.

[0006] In a first aspect, the present invention provides a licorice root stem cell line, the preservation number of which is CGMCC No.46600.

[0007] Optionally, the licorice stem cell line is derived from the cambium of a licorice (Glycyrrhiza glabra) stem segment.

[0008] In a second aspect, the present invention provides a derivative of the licorice stem cell line described in the first aspect.

[0009] Optionally, the derivative is a culture, lysate, extract, or a combination thereof.

[0010] Thirdly, the present invention provides the application of the licorice stem cell line described in the first aspect or the derivatives described in the second aspect in the preparation of food, health products, pharmaceuticals or cosmetics.

[0011] Optionally, the application is based on the stem cell line or its derivatives containing high levels of glycyrrhizin, glycyrrhizin flavonoids, and glycyrrhizic acid.

[0012] In summary, the present invention includes at least one of the following beneficial technical effects:

[0013] 1. Achieved efficient and stable production of secondary metabolites: The Licorice glabra stem cell line with accession number CGMCC No.46600 can stably and efficiently synthesize a variety of high-value active ingredients such as glycyrrhizin, glycyrrhizin flavonoids and glycyrrhizic acid, with a content far exceeding the extraction level of traditional cultivated licorice, providing a new way to solve the problem of natural product resource shortage.

[0014] 2. Eliminates dependence on wild licorice resources: This invention utilizes plant stem cell culture technology to produce active substances on a large scale under laboratory and factory conditions. It does not depend on land cultivation and is not affected by seasons, climate, or pests and diseases. It is an environmentally friendly and sustainable production method that is conducive to the protection of wild licorice resources.

[0015] 3. Controllable and consistent product quality: Produced through cell culture, the process is easy to standardize and control, avoiding product quality fluctuations caused by different raw material origins and harvesting seasons in traditional extraction methods, and ensuring the stability of the active ingredient content and efficacy of the final product.

[0016] 4. Wide range of applications: This stem cell line and its derivatives (such as extracts) are rich in whitening ingredient glycyrrhizin, anti-inflammatory ingredient glycyrrhizic acid, and antioxidant ingredient glycyrrhizin flavonoids. They can be directly applied to high-end skin care products, pharmaceutical products, and health products, which greatly expands the commercial application value of licorice resources. Attached Figure Description

[0017] Figure 1 Microscopic image of suspension cells of the Glycyrrhiza glabra stem cell line gc001.

[0018] Figure 2 Image showing the staining of Glycyrrhiza glabra stem cell line GC001 suspension cells (neutral red + potassium iodide). Detailed Implementation

[0019] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0020] Unless otherwise specified, the experimental methods in the following examples were performed according to standard procedures, and the materials and reagents used were all commercially available.

[0021] Example 1: Stem cell line screening

[0022] 1.1 Disinfection

[0023] Licorice stem segments (from *Glycyrrhiza glabra* collected from Ningxia, a gift from the South China Botanical Garden) were placed in sterile tubes and disinfected by shaking with 75% ethanol for 1 minute. The ethanol disinfectant was poured out, and the tubes were rinsed three times with sterile water. Then, 10% sodium hypochlorite was added for disinfection for 5 minutes. The sodium hypochlorite disinfectant was poured out, and the tubes were rinsed five times with sterile water. The tubes were then dried with sterile filter paper and set aside for use.

[0024] 1.2 Stem cell induction

[0025] After disinfection, licorice stem segments were glass-crystallized in a clean bench, cut into 0.5cm pieces, and inoculated onto solid agar plates. They were then incubated statically at 25°C for 21-28 days. Solid culture medium: MS medium + plant hormones: 0.5mg / L 2,4-D, 0.5mg / L NAA, 0.5mg / L 6-BA, 5g / L plant gel, pH 5.8.

[0026] 1.3 Cell line screening

[0027] Licorice stem cell clusters on solid culture medium were crushed and inoculated into 100 mL of liquid culture medium at a 5% inoculum. The cells were then cultured in the dark on a shaker at 120 rpm at 25°C. Cell clusters with good growth were selected and inoculated into solid culture medium at a depth of 0.5 cm. The cells were crushed again and inoculated into 100 mL of liquid culture medium at a 5% inoculum. This process was repeated to select cell clusters with fast growth and good stability. Liquid culture medium: MS medium + plant hormones: 0.5 mg / L 2,4-D, 0.5 mg / L NAA, 0.5 mg / L 6-BA, pH 5.8.

[0028] 1.4 Ultraviolet mutagenesis of licorice stem cells

[0029] The licorice stem cell suspension obtained from Example 1.3, which was in the logarithmic growth phase, was passed through 100µm and 70µm cell sieves in successive stages to prepare a single-cell suspension for later use; the cell count was adjusted to approximately 10. 5Calculate the concentration of single licorice cells per mL and place 2 mL of the single-cell suspension at the bottom of a 60 mm culture dish. Place the culture dish 30 cm away from a 20 W UV lamp with a wavelength of 253.7 nm. Open the lid of the culture dish and irradiate for 2 min. Shake the culture dish during irradiation to ensure uniform cell irradiation. After irradiation, transfer the suspended cells to a solid culture medium (same as in 1.2) for semi-solid culture. Protect the cells from light with aluminum foil and incubate at 35 °C for 21-28 days. Select viable cell clusters and transfer them to a new solid culture medium. Repeat the screening process in Example 1.3 to obtain a genetically stable stem cell line, named gc001.

[0030] Microscopic examination and staining images of suspension cells from the Glycyrrhiza glabra stem cell line GC001 are shown below. Figure 1 and Figure 2 As shown.

[0031] Example 2: Detection of secondary metabolites

[0032] The glycyrrhiza glabra stem cell line gc001 obtained in Example 1 and the original cell line before mutagenesis (as a control) were cultured under the same conditions for 60 days. Cell clusters of 2 mm in size were collected, and the content of their main secondary metabolites was detected. The detection method is as follows.

[0033] 2.1 Glycyrrhizin

[0034] Sample preparation: 0.1 g of the sample material after grinding the small cell clusters was added to 2 mL of 90% ethanol (ensuring a material-to-liquid ratio of 1:20), and extracted by ultrasonication at 30-40℃ for 60 min;

[0035] After ultrasonic extraction, the sample supernatant is transferred to a centrifuge tube and centrifuged. The sample supernatant is then filtered through a filter membrane into a chromatographic injection bottle for analysis.

[0036] The glycyrrhizin in licorice was determined by HPLC under the following chromatographic conditions: column: Shim-pack GIST ODS (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A) - 0.05% phosphoric acid (B) aqueous solution; gradient elution (elution program shown in Table 1); flow rate: 0.8 mL / min; detection wavelength: 280 nm; column temperature: 30 ℃; injection volume: 10 μL.

[0037] Table 1

[0038] T / min A / % B / % 0 46 54 10 46 54 30 53 47 50 55 45 55 80 20 60 46 54

[0039] 2.2 Licorice Flavonoids

[0040] The small cell clusters were freeze-dried, ground into powder using a mortar and pestle, and then passed through a 50-mesh sieve. 0.1 g of the sieved powder was accurately weighed, and 1 mL of 90% ethanol was added. The mixture was extracted using an ultrasonic extractor at 60°C for 30 min. After ultrasonic extraction, the mixture was centrifuged at 10,000 rpm for 10 min at room temperature. The supernatant was collected and diluted to 1 mL with 90% ethanol for analysis.

[0041] Take 60 μL of the test solution (blank is 90% ethanol), add 15 μL of 5% sodium nitrite solution, let stand for 6 min, add 15 μL of 10% aluminum nitrate solution, let stand for 6 min, then add 120 μL of 1mol / L sodium hydroxide solution, and finally add 90 μL of 90% ethanol. Shake well and let stand for 15 min. Measure the absorbance at 502 nm using an ELISA reader. Calculate the sample content using a standard curve plotted with standards (T / CI 187-2023, Extraction and Content Detection of Glycyrrhiza Polysaccharide and Glycyrrhiza Flavonoids).

[0042] 2.3 Glycyrrhizic acid

[0043] Glycyrrhizic acid was extracted using the same method as glycyrrhizin. High-performance liquid chromatography (HPLC) was used for glycyrrhizic acid detection. Chromatographic conditions: Kromasil-C18 column (4.6 mm × 250 mm, 5 μm), acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, gradient elution, column temperature: 35℃, flow rate: 1.0 mL / min, detection wavelength: 237 nm, injection volume: 10 μL.

[0044] The results are shown in Table 2. Compared with the cell line before UV mutagenesis, the contents of glycyrrhizin, glycyrrhizin flavonoids, and glycyrrhizic acid in the *Glycyrrhiza glabra* stem cell line gc001 screened in this invention were significantly increased. This stem cell line was deposited on September 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46600.

[0045] Table 2

[0046] cell lines glycyrrhizin content Licorice flavonoid content glycyrrhizic acid content Premutation cell lines 326.48 μg / g 48.4 mg / g 688.01 μg / g mutagenized cell line gc001 581.46 μg / g 59.63 mg / g 901.3 μg / g

[0047] Example 3: Scale-up culture of Glycyrrhiza glabra stem cell line gc001

[0048] 30L of liquid culture medium (Example 1.3) was loaded into a 50L fermenter, and 3L (10%) of licorice stem cell line gc001 suspension cells (passaged 16 times) were inoculated. The culture was carried out at pH 5.8, 25℃, dissolved oxygen (DO) 40%, and rotation speed 50 r / min. After 60 days of culture, the contents of glycyrrhizin, glycyrrhizin flavonoids, glycyrrhizic acid and glycyrrhizic acid were tested according to the method in Example 2. The contents of glycyrrhizin flavonoids were 602.2 μg / g, glycyrrhizin flavonoids were 100.3 mg / g and glycyrrhizic acid was 1.11 mg / g.

[0049] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A licorice root stem cell line, characterized in that, The preservation number of the *Glycyrrhiza glabra* stem cell line is CGMCC No. 46600.

2. The *Glycyrrhiza glabra* stem cell line according to claim 1, characterized in that, The glabra stem cell line is derived from the cambium of glabra stem segments.

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