Method for promoting synthesis of bletilla striata glycoside in bletilla striata suspension culture cells

Abstract

The invention provides a method for promoting synthesis of bletilla striata glycoside in bletilla striata suspension culture cells, and belongs to the technical field of suspension cell culture. The invention provides a method for promoting synthesis of bletilla striata glycoside in bletilla striata suspension culture cells, which comprises the following steps: adding bletilla striata calluses into a suspension culture medium, and carrying out suspension culture for 18-25 days under the condition of 130-140rpm; the suspension culture medium is prepared from the following components in concentration: 1 / 2 MS, 0.8 to 1.2 mg / L of 6-BA, 2.5 to 3.5 mg / L of 2, 4-D, 0.4 to 0.6 mg / L of NAA, 33 to 37 g / L of sucrose, 140 to 160 [mu] mol / L of NaAc and 600 to 650 mg / L of ammonium nitrate, and the pH value of the suspension culture medium is 5.85 to 6.05. The invention provides a technical support for the utilization of the traditional Chinese medicinal material rhizoma bletillae and the further research of the pharmacological activity of the bletilla striata glycoside.

Description

Technical Field

[0001] The invention relates to the technical field of suspension cell culture, in particular to a method for promoting the synthesis of bletilla striata glycosides in bletilla striata suspension culture cells. Background Art

[0002] Bletilla striata (Thunb.) Rchb.f. is a traditional and precious Chinese herbal medicine. Its dried tubers are bitter, sweet, astringent, and slightly cold; they possess astringent, hemostatic, swelling-reducing, and tissue-stimulating properties. They are effective in treating hemoptysis, hematemesis, traumatic bleeding, tuberculosis, and ulcer bleeding, serving as a hemostatic. Recent studies on its chemical composition and pharmacology have shown that various secondary metabolites in Bletilla striata, such as bibenzyls, dihydrophenanthrenes, phenanthrenes, biphenanthrene derivatives, and quinone derivatives, possess pharmacological activities such as anti-influenza virus, anti-tumor, and gastric mucosal protection.

[0003] While the successful establishment of a liquid suspension culture system for Bletilla striata cells has been reported, few studies have examined how to optimize the accumulation of militarine in these cells by regulating the suspension culture conditions. Therefore, using response surface methodology, we optimized the Bletilla striata suspension culture system to maximize militarine accumulation, which has significant practical value for the modernization of Bletilla striata, a traditional Chinese medicine. Summary of the Invention

[0004] The object of the present invention is to provide a method for promoting the synthesis of bletilla striata glycosides in bletilla striata suspension culture cells.

[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 promoting the synthesis of bletilla striata glycosides in suspension culture cells of Bletilla striata, comprising adding Bletilla striata callus to a suspension culture medium and carrying out suspension culture at 130-140 rpm for 18-25 days;

[0007] The suspension culture medium includes the following components in concentrations: 1 / 2MS+0.8-1.2 mg / L 6-BA+2.5-3.5 mg / L 2,4-D+0.4-0.6 mg / L NAA+33-37 g / L sucrose+140-160 μmol / L NaAc+600-650 mg / L ammonium nitrate, with a pH of 5.85-6.05.

[0008] Preferably, the method for preparing the Bletilla striata callus comprises the following steps:

[0009] (1) adding Bletilla striata seeds to an induction medium and culturing for 25 to 35 days to induce callus formation; the induction medium comprises the following components at the following concentrations: MS + 0.8 to 1.2 mg / L 6-BA + 1.6 to 2.4 mg / L 2,4-D + 0.4 to 0.6 mg / L NAA + 28 to 32 g / L sucrose, with a pH of 5.85 to 6.05;

[0010] (2) adding the callus obtained in step (1) to a subculture medium and culturing for 13 to 17 days to obtain Bletilla striata callus; the subculture medium comprises the following components in concentrations: 1 / 2MS+0.8 to 1.2 mg / L 6-BA+2.5 to 3.5 mg / L 2,4-D+0.4 to 0.6 mg / L NAA+28 to 32 g / L sucrose+140 to 160 μmol / L NaAc, with a pH of 5.85 to 6.05.

[0011] Preferably, in step (1), the mass volume ratio of the Bletilla striata seeds to the induction culture medium is 0.8-1.2 g: 30-40 mL.

[0012] Preferably, the callus tissue obtained in step (2) is added to the subculture culture medium at a ratio of 0.8-1.2 g: 30-40 mL.

[0013] Preferably, the cultures in step (1) and step (2) are both dark cultures, the culture temperature is 23-27° C., and the culture speed is 100-150 rpm.

[0014] Preferably, the mass volume ratio of the Bletilla striata callus to the suspension culture medium is 0.8-1.2 g:30-40 mL.

[0015] Preferably, the suspension culture is dark culture, and the temperature of the suspension culture is 23-27°C.

[0016] The invention provides an application of the method in promoting the synthesis of bletilla striata glycosides in bletilla striata suspension culture cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention optimizes the suspension culture system of Bletilla striata using response surface methodology, resulting in a method for promoting the synthesis of baiji glycosides in suspension cultured cells of Bletilla striata. The method provided by the present invention can significantly increase the accumulation of baiji glycosides in suspension cultured cells, providing technical support for further research on the utilization of Bletilla striata and the pharmacological activity of baiji glycosides, and is suitable for laboratory research and industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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 use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0020] Figure 1 The effects of different concentrations of sucrose on the callus culture state and accumulation of bletilla striata glycosides in Example 3 are shown;

[0021] Figure 2 The effects of different concentrations of ammonium nitrate on the callus culture state and accumulation of bletilla striata glycosides in Example 3 are shown;

[0022] Figure 3 The effects of different shaking speeds on the callus culture state and accumulation of bletilla striata glycosides in Example 3 are shown;

[0023] Figure 4 The growth status of the Bletilla striata suspension cells under different culture conditions in Example 4 (A) and the growth status of the Bletilla striata suspension cells cultured under the optimal conditions (B);

[0024] Figure 5 These are the response surface fitting contour map and three-dimensional surface map based on sucrose concentration (carbon source) A, ammonium nitrate concentration (nitrogen source) B and shaker speed C in Example 4. DETAILED DESCRIPTION

[0025] The present invention provides a method for promoting the synthesis of bletilla striata glycosides in suspension culture cells of Bletilla striata, comprising adding Bletilla striata callus to a suspension culture medium and carrying out suspension culture at 130-140 rpm for 18-25 days;

[0026] The suspension culture medium includes the following components in concentrations: 1 / 2MS+0.8-1.2 mg / L 6-BA+2.5-3.5 mg / L 2,4-D+0.4-0.6 mg / L NAA+33-37 g / L sucrose+140-160 μmol / L NaAc+600-650 mg / L ammonium nitrate, with a pH of 5.85-6.05.

[0027] In the present invention, the suspension culture is preferably carried out at 132-138 rpm for 19-24 days, more preferably at 134-136 rpm for 20-22 days, and even more preferably at 135 rpm for 21 days.

[0028] In the present invention, the suspension culture medium preferably includes the following components in concentrations: 1 / 2MS + 0.9-1.1 mg / L6-BA + 2.8-3.2 mg / L 2,4-D + 0.45-0.55 mg / LNAA + 34-36 g / L sucrose + 145-155 μmol / LNaAc + 620-630 mg / L ammonium nitrate, with a pH of 5.9-6.0; further preferably includes the following components in concentrations: 1 / 2MS + 1 mg / L 6-BA + 3 mg / L 2,4-D + 0.5 mg / L NAA + 35 g / L sucrose + 150 μmol / LNaAc + 625 mg / L ammonium nitrate, with a pH of 5.95.

[0029] In the present invention, the preparation method of the Bletilla striata callus comprises the following steps:

[0030] (1) adding Bletilla striata seeds to an induction medium and culturing for 25 to 35 days to induce callus formation; the induction medium comprises the following components at the following concentrations: MS + 0.8 to 1.2 mg / L 6-BA + 1.6 to 2.4 mg / L 2,4-D + 0.4 to 0.6 mg / L NAA + 28 to 32 g / L sucrose, with a pH of 5.85 to 6.05;

[0031] (2) adding the callus obtained in step (1) to a subculture medium and culturing for 13 to 17 days to obtain Bletilla striata callus; the subculture medium comprises the following components in concentrations: 1 / 2MS+0.8 to 1.2 mg / L 6-BA+2.5 to 3.5 mg / L 2,4-D+0.4 to 0.6 mg / L NAA+28 to 32 g / L sucrose+140 to 160 μmol / L NaAc, with a pH of 5.85 to 6.05.

[0032] In the present invention, preferably, Bletilla striata seeds are added to the induction medium and cultured for 27 to 33 days, more preferably 28 to 32 days, and even more preferably 30 days.

[0033] In the present invention, the induction medium preferably includes the following components in concentrations: MS + 0.9-1.1 mg / L 6-BA + 1.8-2.2 mg / L 2,4-D + 0.45-0.55 mg / L NAA + 29-31 g / L sucrose, with a pH of 5.9-6.0; further preferably includes the following components in concentrations: MS + 1 mg / L 6-BA + 2 mg / L2,4-D + 0.5 mg / L NAA + 30 g / L sucrose, with a pH of 5.95.

[0034] In the present invention, the mass volume ratio of the Bletilla striata seeds to the induction culture medium in step (1) is 0.8-1.2 g:30-40 mL, preferably 0.9-1.1 g:32-38 mL, and more preferably 1.0 g:35 mL.

[0035] In the present invention, the callus obtained in step (1) is preferably added to a subculture medium and cultured for 14 to 16 days, more preferably 15 days.

[0036] In the present invention, the subculture culture medium preferably includes the following components in concentrations: 1 / 2MS+0.9-1.1mg / L6-BA+2.8-3.2mg / L 2,4-D+0.45-0.55mg / LNAA+29-31g / L sucrose+145-155μmol / LNaAc, with a pH of 5.9-6.0; further preferably includes the following components in concentrations: 1 / 2MS+1mg / L 6-BA+3mg / L 2,4-D+0.5mg / L NAA+30g / L sucrose+150μmol / LNaAc, with a pH of 5.95.

[0037] In the present invention, the callus tissue obtained in step (2) is added to the subculture culture medium at a ratio of 0.8-1.2 g: 30-40 mL, preferably at a ratio of 0.9-1.1 g: 32-38 mL, and more preferably at a ratio of 1.0 g: 35 mL.

[0038] In the present invention, the cultures in step (1) and step (2) are both dark cultures, and the culture temperatures are both 23-27°C, preferably 24-26°C, and more preferably 25°C; the culture speeds are both 100-150 rpm, preferably 105-140 rpm, more preferably 110-130 rpm, and more preferably 120 rpm.

[0039] In the present invention, the mass volume ratio of the Bletilla striata callus to the suspension culture medium is 0.8-1.2 g:30-40 mL, preferably 0.9-1.1 g:32-38 mL, and more preferably 1.0 g:35 mL.

[0040] In the present invention, the suspension culture is dark culture, and the temperature of the suspension culture is 23-27°C, preferably 24-26°C, and more preferably 25°C.

[0041] The invention provides an application of the method in promoting the synthesis of bletilla striata glycosides in bletilla striata suspension culture cells.

[0042] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] A method for optimizing Bletilla striata suspension cell culture conditions to promote Bletilla striata glycoside synthesis, comprising the following steps:

[0045] (1) Add Bletilla striata seeds to a liquid culture medium containing MS, 1 mg / L 6-BA, 2 mg / L 2,4-D, 0.5 mg / L NAA, and 30 g / L sucrose. Add 35 mL of culture medium and 1.00 g of Bletilla striata seeds to each 100 mL culture flask. Induce callus tissue after dark culture at 25°C and 120 rpm for 30 days.

[0046] (2) 1.00 g of callus tissue obtained in step (1) was added to 35 mL of 1 / 2MS + 1 mg / L 6-BA + 3 mg / L 2,4-D + 0.5 mg / L NAA + 30 g / L sucrose + 150 μmol / L NaAc liquid culture medium for subculture. The subculture was cultured in the dark at 25° C. and 120 rpm for 15 days.

[0047] (3) 1.00 g of subcultured callus tissue was added to 35 mL of liquid culture medium containing 1 / 2 MS, 1 mg / L 6-BA, 3 mg / L 2,4-D, 0.5 mg / L NAA, 30 g / L sucrose, 150 μmol / L NaAc, and 625 mg / L ammonium nitrate. The culture was carried out in the dark at 25°C and 120 rpm for 21 days.

[0048] The above culture conditions were used as the basic culture conditions, and then cell suspension culture was performed under three different culture conditions in step (3) with different sucrose and ammonium nitrate concentrations and different shaker speeds, and the optimal condition range was determined through single-factor experiments.

[0049] Example 2

[0050] A method for extracting and determining bletilla striata glycosides in suspension culture cells of Bletilla striata comprises the following steps:

[0051] (1) Extraction of Bletilla striata glycosides from suspension culture cells

[0052] After filtration, the suspended cell pellet of Bletilla striata was dried and crushed in a mortar through a No. 4 sieve. Accurately weigh 0.20 g of the dry powder and place it in a 250 mL three-necked flask. Add 100 mL of 70% methanol-water and reflux for 2 hours. Filter through dry filter paper to obtain the extract. The extract was then decompressed and the solvent was recovered until dry. The extract was then dissolved in an appropriate amount of 70% methanol-water and transferred to a 5 mL volumetric flask, brought to volume, shaken, and filtered through a 0.22 μm filter membrane to prepare the test solution.

[0053] (2) Determination of the content of baiji glycosides in suspension culture cells of Bletilla striata

[0054] Peak areas were measured using an Agilent 1260 HPLC using a Dubhe C18 column (250 mm × 4.6 mm, 5 μm); a mobile phase consisting of methanol and water; a flow rate of 0.8 mL / min; a column temperature of 25°C; a detection wavelength of 225 nm; and injection volumes of 10 μL for the test sample and 10 μL for the reference sample. The volume fraction changes during gradient elution are shown in Table 1.

[0055] Table 1 HPLC gradient elution program

[0056] Time (min) Acetonitrile (100%) 0.1% phosphoric acid (%) 0 20 80 10 20 80 25 50 50 30 50 50 35 20 80

[0057] The formula for calculating the effective content is: ① Reference sample peak area / Test sample peak area = Reference sample content (concentration) / Test sample content (concentration); ② Active ingredient content = (Test sample concentration × volume × dilution factor) / Sample weight. SPSS 29.0 was used for correlation analysis of the experimental data.

[0058] The content of Militarine in suspension culture cells of Bletilla striata was calculated according to the regression equation of the standard.

[0059] Y=28.14+0.6136A-0.1822B-4.95C-1.06AB+0.4034AC-0.3036BC-2.33A 2 -3.66B 2 -10.4C 2

[0060] Example 3 Single factor experiment

[0061] According to the method of Example 1, a single factor experiment was conducted on the amount of sucrose and ammonium nitrate added to the culture medium in step (3) and the shaking speed under the culture conditions, and the content of militarine in the suspension culture cells of Bletilla striata was calculated according to the method described in Example 2.

[0062] 1. Amount of sucrose added to the culture medium

[0063] Keeping other components of the culture medium unchanged and the culture conditions unchanged, different concentrations of sucrose (20, 30, 40, 50 g / L) were selected to prepare the liquid culture medium. The callus tissue of Bletilla striata was inoculated into a 100 mL culture bottle filled with 35 mL of liquid culture medium to carry out the Bletilla striata liquid suspension culture cell experiment. After culturing for 21 days, the cumulative amount of secondary metabolites in the cells was determined ( Figure 1 ).

[0064] 2. Amount of ammonium nitrate added to the culture medium

[0065] Keeping other components of the culture medium unchanged and the culture conditions unchanged, different concentrations of ammonium nitrate (250, 500, 750, 1000 mg / L) were selected to prepare liquid culture medium. The callus of Bletilla striata was inoculated into a 100 mL culture bottle filled with 35 mL of liquid culture medium to carry out the Bletilla striata liquid suspension culture cell experiment. After culturing for 21 days, the cumulative amount of secondary metabolites in the cells was determined ( Figure 2 ).

[0066] 3. Shaker speed in culture conditions

[0067] Keeping other components in the culture medium unchanged and the culture conditions the same, different rotation speeds (90, 120, 150 rpm) were selected to culture for 21 days and the cumulative amount of secondary metabolites in the cells was determined ( Figure 3 ).

[0068] according to Figures 1 to 3 It can be seen that under the single-factor experimental conditions, the content of Militarine in the Bletilla striata suspension culture cells was the highest when the sucrose concentration was 40 g / L, the ammonium nitrate concentration was 750 mg / L, and the shaker speed was 120 rpm.

[0069] Example 4 Response surface experiment

[0070] According to the analysis result of the single factor experiment of embodiment 3, the response surface optimization experimental design of sucrose concentration, ammonium nitrate concentration and shaking speed of the design factor culture condition was carried out. According to the Box-Behnken experimental design scheme, software Design-Expert 13.0 was utilized to carry out the experimental design of 3 factors, 3 levels, 5 center points and 17 groups of repetitions, as shown in Table 2. Analyzed by this software, with Militarine content being the maximum as the standard, the optimization of the best combination of culture conditions of Bletilla striata liquid suspension culture cells at the non-hormone level was carried out. The best combination of conditions obtained by the response surface method was repeated 3 times in parallel to verify the reliability of the analytical method and the optimization result.

[0071] Table 2 Response surface experiment scheme and results of Bletilla striata glycoside accumulation

[0072]

[0073]

[0074] Growth status of Bletilla striata suspension cells under different culture conditions (A) and growth status of Bletilla striata suspension cells cultured under optimal conditions (B) Figure 4 The response surface fitting contour map and three-dimensional surface map based on sucrose concentration A, ammonium nitrate concentration B and shaker speed C are shown in Figure 2. Figure 5 shown.

[0075] From Table 2 and Figure 4 It can be seen that the growth state of Bletilla striata suspension cells was the best under the conditions of sucrose concentration of 35 g / L, ammonium nitrate concentration of 625 mg / L and shaker speed of 135 rpm, and the content of Militarine in Bletilla striata suspension culture cells was the highest.

[0076] The quadratic multiple linear regression equation was fitted using Design-Expert 13.0 software, and the quadratic multiple linear regression equation of the response surface model of Militarine content in Bletilla striata suspension culture cells was obtained as follows:

[0077] Y=28.14+0.6136A-0.1822B-4.95C-1.06AB+0.4034AC-0.3036BC-2.33A 2 -3.66B 2 -10.4C 2

[0078] Table 3 Analysis of variance of regression equation

[0079]

[0080]

[0081] As can be seen from Table 3, verification tests were carried out in sequence according to the optimal culture conditions obtained by the response surface methodology. When cultured under the culture conditions that meet the maximum content of Militarine, a key secondary metabolite in Bletilla striata, the average content of Militarine obtained was greater than 20 mg / g. Compared with the predicted value of the fitting equation, the average deviation was 2.15%, and the difference was not significant. The signal-to-noise ratio measured by AdeqPrecision was 10.7389 (greater than 4 is desirable), and the ratio of 10.739 indicates that the signal is sufficient. This shows that the model can predict the extraction amount within a certain range. The verification experiment showed that the accumulation of Bletilla striata glycosides extracted under the optimal culture conditions can exceed the standard specified in the pharmacopoeia (20 mg / g), indicating that the culture conditions are stable and reliable. In summary, the experimental scheme obtained by optimization through the response surface methodology is reasonable and reliable.

[0082] 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 the scope of protection of the present invention.

Claims

1. A method for promoting the synthesis of bletilla striata glycosides in suspension culture cells of Bletilla striata, characterized in that: Adding Bletilla striata callus to suspension culture medium, and culturing at 130-140 rpm for 18-25 days; The suspension culture medium includes the following components in concentrations: 1 / 2MS+0.8-1.2 mg / L 6-BA+2.5-3.5 mg / L 2,4-D+0.4-0.6 mg / L NAA+33-37 g / L sucrose+140-160 μmol / L NaAc+600-650 mg / L ammonium nitrate, with a pH of 5.85-6.

05.

2. The method according to claim 1, wherein The preparation method of the Bletilla striata callus comprises the following steps: (1) adding Bletilla striata seeds to an induction medium and culturing for 25 to 35 days to induce callus formation; the induction medium comprises the following components at the following concentrations: MS + 0.8 to 1.2 mg / L 6-BA + 1.6 to 2.4 mg / L 2,4-D + 0.4 to 0.6 mg / L NAA + 28 to 32 g / L sucrose, with a pH of 5.85 to 6.05; (2) adding the callus obtained in step (1) to a subculture medium and culturing for 13 to 17 days to obtain Bletilla striata callus; the subculture medium comprises the following components in concentrations: 1 / 2MS+0.8 to 1.2 mg / L 6-BA+2.5 to 3.5 mg / L 2,4-D+0.4 to 0.6 mg / L NAA+28 to 32 g / L sucrose+140 to 160 μmol / L NaAc, with a pH of 5.85 to 6.

05.

3. The method according to claim 2, wherein The mass volume ratio of the Bletilla striata seeds to the induction culture medium in step (1) is 0.8-1.2 g: 30-40 mL.

4. The method according to claim 2, wherein The callus tissue obtained in step (2) is added to the subculture culture medium at a ratio of 0.8-1.2 g: 30-40 mL.

5. The method according to claim 2, wherein The cultivation in step (1) and step (2) is dark cultivation, the temperature of the cultivation is 23-27° C., and the speed is 100-150 rpm.

6. The method according to claim 1, wherein The mass volume ratio of the Bletilla striata callus tissue to the suspension culture medium is 0.8-1.2 g: 30-40 mL.

7. The method according to claim 1, wherein The suspension culture is dark culture, and the temperature of the suspension culture is 23-27°C.

8. Use of the method according to any one of claims 1 to 7 in promoting the synthesis of bletilla striata glycosides in suspension culture cells of Bletilla striata.

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