A method for producing glabriden using tissue culture technique

Abstract

The application provides a method for producing glabridin by using tissue culture technology, and mainly comprises the following steps: (1) selecting full-granular Russian licorice seeds, sterilizing the seeds, and then culturing licorice aseptic seedlings; (2) selecting leaves of the licorice aseptic seedlings as explants to induce callus; (3) selecting callus with vigorous growth and fast division speed, subculturing the callus, and then placing the callus into a liquid culture medium to perform suspension culture; and (4) extracting glabridin from the licorice suspension cells cultured in step (3). The glabridin obtained by the cell suspension culture has the advantages of short production cycle, no limitation of region, season and time, and strong antioxidant property, and is widely applied to high-end cosmetics and the pharmaceutical industry, and has a wide development prospect in the future.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for producing glycyrrhizin using tissue culture technology. Background Technology

[0002] Licorice is a perennial herb belonging to the genus *Glycyrrhiza* of the legume family. Its medicinal parts are mainly the roots and rhizomes, and it is a tonic traditional Chinese medicine. Glycyrrhizin, on the other hand, is a natural whitening agent, a brownish-yellow or pale yellow fine powder. It can inhibit the activity of tyrosinase, dopachrome tautomerase, and DHICA oxidase. It is a promising fast-acting, highly effective, and green whitening and spot-removing cosmetic additive with great development potential. It has a similar ability to scavenge oxygen free radicals as SOD (superoxide dismutase) and an antioxidant capacity similar to vitamin E. It is currently widely used in the cosmetic industry, such as in lotions, creams, and ointments.

[0003] In today's environmentally conscious world, plant-based active ingredients are highly effective and have few side effects, aligning with the trend of "embracing green and returning to nature." Glycyrrhizin, often referred to as "whitening gold," is a well-known cosmetic additive. However, glycyrrhizin only accounts for about 0.1% of finished licorice products, and its cultivation is easily limited by natural conditions, generally requiring 3-4 years before it can be used medicinally. Currently, scholars both domestically and internationally have conducted extensive research and development on the active ingredients of some traditional Chinese medicinal plants, achieving practical results in whitening and removing blemishes. However, methods for producing glycyrrhizin using plant tissue culture technology have not yet been reported.

[0004] The low content of glycyrrhizin, an active ingredient in licorice, and its long natural production cycle have limited its industrial development. In recent years, the over-harvesting of wild licorice resources and the sharp increase in demand have created a supply shortage. This invention aims to establish a cell suspension culture method for glycyrrhizin based on plant tissue culture technology. Specifically, it utilizes tissue culture technology to overcome the key technical bottleneck of large-scale enrichment of glycyrrhizin, a natural active ingredient in licorice. This will provide a new plant source for cosmetic raw material development and provide technical support for the industrial application of plant-derived active ingredients in cosmetics. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing glycyrrhizin using tissue culture technology, which solves the problem of scarce licorice resources.

[0006] This invention is achieved through the following techniques: A method for producing glycyrrhizin using tissue culture technology includes the following steps: (1) Cultivation of Russian licorice sterile seedlings: Select plump Russian licorice seeds, sterilize them with alcohol and mercuric chloride in sequence, rinse them with sterile water, and inoculate them into basic MS (sugar-free and agar-free) medium, with 5 g / L agar and 20-30 g / L sucrose added. After the Russian licorice seeds germinate, they are transferred to light culture at a light intensity of 2000-2400 lx for 10 h / d. They can be used after 1 week of culture. (2) Induction of Russian licorice callus and subculture: Select sterile seedlings with good growth from (1) as explants to induce callus. After 26 days of culture, select vigorous callus for subculture. Optimize 3 to 5 times to obtain yellow-green embryogenic callus. (3) Cell suspension culture: Select yellow-green, vigorously dividing callus tissue from (2) and inoculate it into liquid culture medium. After three subcultures, a stable licorice cell suspension system is established. (4) Extraction and detection of glycyrrhizin: glycyrrhizin obtained in step (3) was extracted by ultrasonic extraction and the content of glycyrrhizin was detected by high performance liquid chromatography.

[0007] The specific method for inducing sterile licorice seedlings in step (1) is as follows: (1) Cultivating sterile Russian licorice seedlings: Select plump Russian licorice seeds, sterilize them with 75% alcohol for 30 seconds, rinse them with sterile water 4-5 times, sterilize them with 0.1% mercuric chloride for 6-8 minutes, rinse them with sterile water, and inoculate them into basic MS (sugar-free and agar-free) medium, with 5g / L agar and 20-30g / L sucrose added. After the Russian licorice seeds germinate, they are transferred to light culture with a light intensity of 2000-2400 lx, 10h / d. They can be used after 1 week of culture. Step (2) Induction of Russian licorice callus and subculture: Select well-growing sterile seedlings and leaves from (1) as explants to induce callus, and add NAA (0.5~2.0mg / L), 2,4-D (0.5~2.1 mg / L), 6-BA (0.5~2.0mg / L), sucrose 25~30g / L, agar 4~6g / L, pH 5.6~6.2 before sterilization, light 1500~2000lx, light 10h per day, temperature (23~27℃), after 26 days of culture, further select vigorous callus for subculture, and optimize continuously 3~5 times to obtain yellow-green embryogenic callus; Step (3) Cell suspension culture: Select yellow-green, vigorously dividing callus tissue from (2) and inoculate it into MS liquid culture medium. Add hormones NAA (0.5~2.0 mg / L), 2,4-D (0.5~2.1 mg / L), 6-BA (0.5~2.0 mg / L), sucrose 25~30 g / L, pH 5.6~6.2 before sterilization, temperature (23~27℃), rotation speed 110~120 r / min. After 3 subcultures, establish a stable licorice cell suspension system. Step (4) Extraction and detection of glycyrrhizin: The glycyrrhizin obtained in step (3) was extracted by ultrasonic extraction, and the content of glycyrrhizin in licorice cells was detected by high performance liquid chromatography.

[0008] This invention provides a method for rapidly producing glycyrrhizin using tissue culture technology. This method is not limited by natural conditions, has high economic benefits, and has broad development prospects.

Claims

1. A method for obtaining glycyrrhizin using cell suspension culture technology, characterized in that... The following steps are adopted: (1) Cultivation of Russian licorice sterile seedlings: Select plump Russian licorice seeds, sterilize them with alcohol and mercuric chloride in sequence, rinse them with sterile water, and inoculate them into basic MS (sugar-free and agar-free) medium, with 5 g / L agar and 20-30 g / L sucrose added. After the Russian licorice seeds germinate, they are transferred to light culture at a light intensity of 2000-2400 lx for 10 h / d. They can be used after 1 week of culture. (2) Induction of Russian licorice callus and subculture: Select sterile seedlings with good growth from (1) as explants to induce callus. After 26 days of culture, select vigorous callus for subculture. Optimize 3 to 5 times to obtain yellow-green embryogenic callus. (3) Cell suspension culture: Select yellow-green, vigorously dividing callus tissue from (2) and inoculate it into liquid culture medium. After three subcultures, a stable licorice cell suspension system is established. (4) Extraction and detection of glycyrrhizin: glycyrrhizin obtained in step (3) was extracted by ultrasonic extraction and the content of glycyrrhizin was detected by high performance liquid chromatography.

2. The method for producing glycyrrhizin according to claim 1, characterized in that, The specific method for inducing sterile licorice seedlings in step (1) is as follows: (1) Cultivating sterile Russian licorice seedlings: Select plump Russian licorice seeds, sterilize them with 75% alcohol for 30 seconds, rinse them with sterile water 4-5 times, sterilize them with 0.1% mercuric chloride for 6-8 minutes, rinse them with sterile water, and inoculate them into basic MS (sugar-free and agar-free) medium, with 5g / L agar and 20-30g / L sucrose added. After the Russian licorice seeds germinate, they are transferred to light culture with a light intensity of 2000-2400 lx for 10h / d. They can be used after 1 week of culture.

3. The method for producing glycyrrhizin according to claim 1, characterized in that, Step (2) Induction of Russian licorice callus and subculture: Select well-growing sterile seedlings and leaves from (1) as explants to induce callus. The culture medium required for callus induction and subculture is MS medium, with the addition of naphthaleneacetic acid (NAA 0.5~2.0 mg / L), 2,4-dichlorophenoxyacetic acid (2,4-D 0.5~2.1 mg / L), 6-benzyladenine (6-BA 0.5~2.0 mg / L), sucrose 25~30 g / L, agar 4~6 g / L, pH 5.6~6.2 before sterilization, light intensity 1500~2000 lx, light intensity 10 h per day, temperature (23~27℃). After culturing for 26~28 days, further select vigorous callus for subculture. Optimize continuously for 3~5 times to obtain yellow-green embryogenic callus.

4. The method for producing glycyrrhizin according to claim 1, characterized in that, Step (3) Cell suspension culture: Select yellow-green, vigorously dividing callus tissue from (2) and inoculate it into MS liquid medium. Add NAA (0.5~2.0 mg / L), 2,4-D (0.5~2.1 mg / L), 6-BA (0.5~2.0 mg / L), sucrose 25~30 g / L, pH 5.6~6.2 before sterilization, temperature (23~29℃), rotation speed 110~120 r / min, and establish a stable licorice cell suspension system after 3 subcultures.

5. The method for producing glycyrrhizin according to claim 1, characterized in that, Step (4) Extraction and detection of glycyrrhizin: The glycyrrhizin obtained in step (3) was extracted by ultrasonic extraction, and the content of glycyrrhizin in licorice cells was detected by high performance liquid chromatography.