A method for producing polygonatum sibiricum polysaccharide by using polygonatum sibiricum tissue culture

Abstract

This invention belongs to the field of plant tissue culture technology, specifically disclosing a method for producing Polygonatum polysaccharides using Polygonatum yunnanense tissue culture, comprising the following steps: explant disinfection, callus induction, callus proliferation and induction culture: selecting vigorous callus tissue, transferring it to a culture medium: White medium + 2,4-D 1.5 mg / L + VC 20 mg / L + sucrose 40 g / L + 30 mg / L inducer + agar 6 g / L, pH 5.8, after culture, collecting the callus, drying it, and then extracting Polygonatum yunnanense polysaccharides; the inducer is Polygonatum yunnanense endophytic basidiomycetes (… Talaromyces sp. The fermentation broth extract of HJF23; the HJF23 preservation number is CCTCC NO: M2021344. This invention not only realizes the efficient biosynthesis of Polygonatum yunnanense polysaccharide, but also significantly enhances its functional activity.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for producing Polygonatum polysaccharides using Polygonatum yunnanense tissue culture. Background Technology

[0002] Polygonatum kingianum Coll. et Hemsl., a plant belonging to the genus Polygonatum in the family Liliaceae, is one of the three original sources of the traditional Chinese medicine "Huangjing" listed in the Chinese Pharmacopoeia. It is mainly distributed in southwestern my country. Its rhizome is rich in polysaccharides, saponins, flavonoids, alkaloids, and other secondary metabolites, possessing pharmacological activities such as tonifying qi and nourishing yin, strengthening the spleen and moistening the lungs, and benefiting the kidneys and relieving fatigue. In recent years, its demand has surged in the development of functional foods, new drugs, and health products.

[0003] Traditional methods of obtaining secondary metabolites mainly rely on wild or artificially cultivated rhizomes. However, wild resources of Polygonatum yunnanense are nearing depletion due to over-harvesting, and artificial cultivation faces the following bottlenecks: asexual reproduction based on tubers is the main method, which requires a large amount of seeds, is costly, and is prone to rotting; seed propagation requires a long period of dormancy, has a low germination rate, and a long growth cycle; tissue culture seedlings also have a long growth cycle and can only be harvested after 4 to 5 years of cultivation. The content of target components in rhizomes fluctuates greatly due to the influence of climate, soil, and pests and diseases, making it difficult to achieve standardized supply.

[0004] Plant tissue culture technology can achieve rapid proliferation of cells and callus tissues under controlled conditions, and obtain secondary metabolites with the same or higher content as the original medicinal materials in a short period of time by inducing and regulating metabolic pathways. Currently, both domestically and internationally, the industrial production of active ingredients such as saponins and phenolic acids from medicinal plants such as ginseng, Panax notoginseng, and Salvia miltiorrhiza has been achieved through tissue culture. However, there is no relevant research on Polygonatum yunnanense. Existing research on Polygonatum yunnanense tissue culture mainly focuses on rapid propagation of seedlings. Therefore, developing a tissue culture-based technology for the production of secondary metabolites from Polygonatum yunnanense to achieve the efficient synthesis of target products such as Polygonatum yunnanense polysaccharides and saponins is of great practical significance for alleviating resource pressure and ensuring the upgrading of the Polygonatum yunnanense industrial chain. Summary of the Invention

[0005] The main objective of this invention is to provide a method for producing Polygonatum polysaccharides using tissue culture of Polygonatum yunnanense, aiming to achieve large-scale industrial production of Polygonatum yunnanense polysaccharides. Specifically, this invention provides the following technical solution;

[0006] A method for producing Polygonatum polysaccharides using Polygonatum yunnanense tissue culture includes the following steps:

[0007] (1) Disinfection of explants: The petioles of Polygonatum yunnanense were collected as explants and disinfected.

[0008] (2) Callus induction: The disinfected petioles were inoculated into the induction medium: MS + 2,4-D 2.0 mg / L + 6-BA 0.5 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8, culture environment: temperature 25±1℃, relative humidity 50%~70%, and loose callus tissue was formed by dark culture.

[0009] (3) Callus proliferation and polysaccharide induction culture: Select vigorous callus tissue and transfer it to the culture medium: White medium + 2,4-D 1.5mg / L + VC 20mg / L + sucrose 40g / L + 30mg / L inducer + agar 6g / L, pH 5.8, culture environment: 25±1℃, light / dark 16h / 8h, light intensity 3000lx, relative humidity 50%~70%, change the culture medium every 20 days, culture for no less than 60 days, collect the callus, dry it and extract polysaccharide from Polygonatum yunnanense;

[0010] The inducer is the fermentation broth extract of *Talaromyces sp.* HJF23, an endophytic bacterium of *Polygonatum yunnanense*; the preservation number of HJF23 is CCTCC NO: M2021344.

[0011] In step (1), the explant disinfection method is as follows: rinse the petiole of Polygonatum yunnanense with running water, soak it in 75% ethanol for 30 seconds, rinse it twice with sterile water; then disinfect it with 0.1% HgCl2 solution for 8-10 minutes, and finally rinse it 5-6 times with sterile water until there is no residue.

[0012] The method for preparing the inducer includes the following steps:

[0013] (1) The HJF23 strain of *Basilaria basilicas* was activated on PDA medium;

[0014] (2) The activated strain was inoculated into ME culture medium: 20 g / L malt extract, 10 g / L peptone, 20 g / L glucose, pH 5.5; and cultured in the dark at 28℃ and 180 rpm for 7 days. After the fungus fermented fully, the mycelium was filtered to collect the fermentation broth. The collected fermentation broth was concentrated, dried and ground into fine powder. Methanol was added at a mass-volume ratio of 1:20. After ultrasonic treatment for 1 hour, the mixture was filtered and the filter residue was thoroughly washed with an appropriate amount of methanol. The filtrate was collected and evaporated by rotary evaporation until the methanol was evaporated to dryness. An appropriate amount of purified water was added to redissolve the extract to obtain a fermentation broth extract of 30 mg / mL. The extract was stored at 4℃ for later use.

[0015] The technical effects achieved by this invention are as follows:

[0016] This invention successfully established a highly efficient callus induction and polysaccharide synthesis system. Experimental results showed that, under the co-culture induction of the fermentation broth extract of the specific endophytic fungus *Talaromyces sp.* HJF23, the polysaccharide content in *Polygonatum yunnanense* callus was significantly increased to 7.16%, far exceeding the 1.12% of the untreated control group (CK) and the 2.04% of the HJF23 mycelial extract, exhibiting a strong synthesis-promoting effect. More importantly, the obtained polysaccharide showed excellent free radical scavenging ability in in vitro antioxidant tests: ABTS cationic free radical scavenging rate of 83.35%; DPPH free radical scavenging rate of 91.01%; and superoxide anion free radical scavenging rate of 68.71%. These indicators suggest that the polysaccharide not only has high yield but also significant antioxidant activity, possessing good potential for development as a natural antioxidant.

[0017] In summary, this invention not only achieves efficient biosynthesis of Polygonatum yunnanense polysaccharides but also significantly enhances their functional activity, providing high-value-added raw materials for the development of deep-processed Polygonatum yunnanense products. It also provides an innovative path for utilizing plant endophytic fungi resources to enhance the content of active ingredients in traditional Chinese medicine, and has broad prospects for industrial application. Attached Figure Description

[0018] Figure 1 Callus induction state diagram;

[0019] Figure 2 Image of callus proliferation and induced culture for 30 days after adding extract of Bacillus basiliformis HJF23 fermentation broth;

[0020] Figure 3 Images of callus proliferation and induced culture for 60 days after adding the fermentation broth extract of Bacillus simulans HJF23. Detailed Implementation

[0021] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. For the experimental methods, purchased goods, unless specific conditions are specified, shall be carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used that do not specify the manufacturer can be commercially available conventional products. Unless otherwise defined herein, the scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials described herein can also be used in the practice and testing of this invention.

[0022] The endophytic talaromyces sp. HJF23 used in this invention was deposited on April 9, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2021344. Detailed information about this strain is disclosed in patent CN202110802292.0, and the portions concerning the strain's origin and preservation in that patent are incorporated herein by reference.

[0023] Example 1

[0024] 1. Preparation of reductants

[0025] Set up different inducer treatment groups (3 replicates per group):

[0026] Control group (CK): No inducers were added;

[0027] Basilaria lobata HJF23 mycelial extract group: final concentration 30 mg / L;

[0028] Basilaria lobata HJF23 fermentation broth extract group: final concentration 30 mg / L;

[0029] (1) Activation of Basiliforme HJF23

[0030] Select the preserved basketweed strain HJF23 and inoculate it onto a PDA plate (200g peeled potato, 20g glucose, 1000mL distilled water, 20g agar), and incubate at 28℃ until the mycelium covers the plate.

[0031] (2) Inducer fermentation culture

[0032] Take a 1 / 3 section of the HJF23 basket-shaped fungus that has grown on a PDA plate and add it to 20 mL of ME culture medium (20 g malt extract, 10 g peptone, 20 g glucose, 1 L distilled water; pH 5.5). After thoroughly breaking it up, inoculate 2 mL of the mycelial mixture into 100 mL of ME culture medium and incubate in the dark at 28 °C and 180 rpm for 7 days. After the fungus has fully fermented, filter it through 4 layers of sterile gauze and collect the mycelium and fermentation broth separately.

[0033] (3) Preparation of mycelial extract of Bassicrys edulis HJF23

[0034] The collected mycelia were rinsed multiple times with purified water, freeze-dried in liquid nitrogen, and ground into a fine powder to obtain the mycelial dry powder. The prepared mycelial dry powder was added to methanol (1:20, mass-to-volume ratio), sonicated for 1 hour, filtered, and the filter residue was thoroughly washed with an appropriate amount of methanol. The filtrate was collected, and the methanol was evaporated to dryness using a rotary evaporator. The filtrate was weighed to calculate the dry weight of the mycelial extract. The mycelial extract was redissolved in an appropriate amount of purified water to obtain a 30 mg / mL mycelial extract, which was stored at 4°C for later use.

[0035] (4) Preparation of extract from fermentation broth of Bassicium argyi HJF23

[0036] The collected fermentation broth was concentrated and freeze-dried, and then processed in the same way as the mycelial extract preparation procedure to obtain a 30 mg / mL fermentation broth extract, which was stored at 4°C for later use.

[0037] 2. Induction and subculture of Polygonatum odoratum callus

[0038] (1) Disinfection of explants: Rinse the petioles of Polygonatum yunnanense with running water, soak in 75% ethanol for 30 seconds, rinse twice with sterile water; then disinfect with 0.1% HgCl2 solution for 8-10 minutes, and finally rinse with sterile water 5-6 times until there are no residues.

[0039] (2) Callus induction: After disinfection, the petioles were inoculated into the induction medium: MS + 2,4-D 2.0 mg / L + 6-BA 0.5 mg / L + sucrose 30 g / L + agar 6 g / L, pH 5.8, culture environment: temperature 25±1℃, relative humidity 50%~70%, and loose callus was induced after 10 days of dark culture.

[0040] (3) Callus proliferation and polysaccharide induction culture: Select vigorous callus tissue and transfer it to the culture medium: White medium + 2,4-D 1.5mg / L + VC 20mg / L + sucrose 40g / L + 30mg / L inducer + agar 6g / L, pH 5.8, culture environment: 25±1℃, light / dark ratio of 16h / 8h, light intensity of 3000lx, change the culture medium every 20 days, and collect the callus after 60 days of culture.

[0041] 3. Extraction and content determination of Polygonatum polysaccharides

[0042] (1) Callus pretreatment: Collect the treated callus, rinse it three times with deionized water, absorb the surface moisture, dry it at a constant temperature of 60℃, and pulverize it through a 40-mesh sieve for later use.

[0043] (2) Preparation of reference solution: Weigh 33 mg of anhydrous glucose reference standard accurately, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the mark, shake well, and you will get the solution (each 1 ml contains 0.33 mg of anhydrous glucose).

[0044] (3) Preparation of standard curve: Accurately measure 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, and 0.6 ml of the reference solution and place them into 10 ml stoppered graduated test tubes respectively. Add water to each tube to 2.0 ml, shake well, and slowly add 0.2% anthrone-sulfuric acid solution to the mark in an ice-water bath. Mix well, cool, and then incubate in a water bath for 10 minutes. Remove and immediately cool in an ice-water bath for 10 minutes. Use the corresponding reagent as a blank. Measure the absorbance at a wavelength of 582 nm using ultraviolet-visible spectrophotometry. Plot the standard curve with absorbance as the ordinate and concentration as the abscissa: Y = 0.1335X + 0.0319(R² + π / 4)². 2 =0.992).

[0045] (4) Extraction and determination process: Take about 0.25g of the fine powder of this product dried to constant weight at 60℃ (the difference between two consecutive weighings shall not exceed 5mg), accurately weigh it, put it in a round bottom flask, add 150ml of 80% ethanol, heat in a water bath and reflux for 1 hour, filter while hot, wash the residue with 10ml of 80% hot ethanol 3 times each time, put the residue and filter paper in a flask, add 150ml of water, heat in a boiling water bath and reflux for 1 hour, filter while hot, wash the residue and flask with 10ml of hot water 4 times each time, combine the filtrate and washings, cool, transfer to a 250ml volumetric flask, add water to the mark, shake well, accurately measure 1ml, put it in a 10ml stoppered dry test tube, and determine the absorbance according to the method under the preparation of the standard curve, starting from "add water to 2.0ml", read the amount of anhydrous glucose in the test solution from the standard curve, calculate, and obtain the content of Polygonatum yunnanensis polysaccharide.

[0046] This invention uses *Polygonatum yunnanense* petioles to induce callus tissue. After 60 days of culture under different induction treatments, the polysaccharide content of the *Polygonatum yunnanense* callus tissue is shown in Table 1. Significant differences in polysaccharide content were observed among the different treatment groups (p<0.01). The polysaccharide content of the HJF23 fermentation broth extract group was as high as 7.16%, which was 6.4 times higher than the control group (CK) at 1.12%, and significantly higher than the HJF23 mycelial extract group at 2.04%. The high efficiency of the HJF23 fermentation broth extract group indicates that the inducing active ingredients are mainly found in the secretory metabolites of *Polygonatum yunnanense* HJF23, rather than in mycelial structural components.

[0047] Table 1. Polysaccharide content of *Polygonatum odoratum* callus.

[0048]

[0049] 4. In vitro antioxidant activity assay

[0050] Extraction steps of Polysaccharide from Polygonatum yunnanense: Weigh the callus powder, add deionized water at a material-to-liquid ratio of 1:50 (g / mL), soak for 60 min, then microwave at 450W for 5 min; centrifuge at 8000rpm for 15 min at 4℃ to remove residue, concentrate the supernatant under reduced pressure to 1 / 5 of the original volume; add 4 times the volume of anhydrous ethanol (final concentration 80%), let stand overnight at 4℃ to precipitate polysaccharides, centrifuge at 10000rpm for 15 min to collect the precipitate, wash once each with anhydrous ethanol, acetone and diethyl ether, and freeze-dry under vacuum to obtain crude polysaccharide (PPS) for testing.

[0051] Using Trolox as a positive control, the scavenging abilities of Polygonatum yunnanense polysaccharide against ABTS cationic free radicals, DPPH free radicals, and superoxide anion free radicals were determined according to the instructions of the purchased kit.

[0052] The in vitro antioxidant activities of Polygonatum yunnanense polysaccharides under different induction treatments are shown in Table 2. The antioxidant activity of the polysaccharides from the HJF23 fermentation broth extract group comprehensively surpassed that of the CK and mycelial extract groups, with a DPPH scavenging rate as high as 91.01%, while the ABTS and superoxide anion scavenging rates reached 83.35% and 68.71%, respectively. The antioxidant activity of Polygonatum yunnanense polysaccharides was significantly enhanced, especially in terms of DPPH and ABTS free radical scavenging ability. Its excellent antioxidant performance may be closely related to the abundance of uronic acid, sulfation modification, and specific monosaccharides in its polysaccharide structure.

[0053] Table 2. In vitro antioxidant activity of Polygonatum yunnanense polysaccharides

[0054]

[0055] Those skilled in the art will understand that the described embodiments are only some embodiments of this application, and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

Claims

1. A method for producing Polygonatum polysaccharides using tissue culture of Polygonatum yunnanense, characterized in that, Includes the following steps: (1) Disinfection of explants: The petioles of Polygonatum yunnanense were collected as explants and disinfected. (2) Callus induction: The disinfected petioles were inoculated into the induction medium: MS medium supplemented with 2,4-D 2.0 mg / L, 6-BA 0.5 mg / L, sucrose 30 g / L and agar 6 g / L, pH 5.8, culture environment: temperature 25±1℃, relative humidity 50%~70%, and loose callus tissue was formed in the dark. (3) Callus proliferation and polysaccharide induction culture: Select vigorous callus tissue and transfer it to the culture medium: White medium supplemented with 2,4-D 1.5mg / L, VC 20mg / L, sucrose 40g / L, 30mg / L inducer and agar 6g / L, pH 5.8, culture environment: 25±1℃, light / dark 16h / 8h, light intensity 3000lx, relative humidity 50%~70%, change the culture medium every 20 days, culture for no less than 60 days, collect the callus, dry it and extract polysaccharide from Polygonatum yunnanense; The inducer is *Polygonatum yunnanense* endophytic *Basilella* (… Talaromyces sp . Fermentation broth extract of HJF23; the HJF23 accession number is CCTCC NO: M2021344.

2. The method according to claim 1, characterized in that, In step (1), the explant disinfection method is as follows: rinse the petiole of Polygonatum yunnanense with running water, soak it in 75% ethanol for 30 seconds, rinse it twice with sterile water; then disinfect it with 0.1% HgCl2 solution for 8-10 minutes, and finally rinse it 5-6 times with sterile water until there is no residue.

3. The method according to claim 1, characterized in that, The method for preparing the inducer includes the following steps: (1) The HJF23 strain of *Basilaria basilicas* was activated on PDA medium; (2) The activated strain was inoculated into ME culture medium: 20 g / L malt extract, 10 g / L peptone, 20 g / L glucose, pH 5.5; and cultured in the dark at 28℃ and 180 rpm for 7 days. After the fungus fermented fully, the mycelium was filtered to collect the fermentation broth. The collected fermentation broth was concentrated, dried and ground into fine powder. Methanol was added at a mass-volume ratio of 1:

20. After ultrasonic treatment for 1 hour, the mixture was filtered and the filter residue was thoroughly washed with an appropriate amount of methanol. The filtrate was collected and evaporated by rotary evaporation until the methanol was evaporated to dryness. An appropriate amount of purified water was added to redissolve the extract to obtain a fermentation broth extract of 30 mg / mL. The extract was stored at 4℃ for later use.

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