Inducer, reagent combination and application of inducer and reagent combination in preparation of anisodamine

Abstract

The invention relates to the technical field of biology, in particular to an inducer, a reagent combination and application of the inducer in preparation of anisodamine. The inducing agent comprises silver nitrate, acetylsalicylic acid and methyl jasmonate, the components of the inducing agent cooperate with one another, compatibility is high, synergistic interaction can be achieved, and the efficiency of the inducing agent for rapidly converting hyoscyamine into anisodamine is further improved. According to the invention, a suspension cell culture technology is adopted, and an optimized callus culture medium, a precursor substance hyoscyamine and an inducer are combined, so that compared with an initial material anisodamine tanguticum used by an existing anisodamine hydrobromide raw material medicine, the anisodamine hydrobromide raw material medicine has the advantages of short growth cycle, stable content and components, environmental protection and no damage to natural resources; the invention provides a new approach for obtaining a starting material for the production of an anisodamine hydrobromide bulk drug, and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an inducer, a reagent combination and their application in the preparation of anisodamine. Background Art

[0002] Anisodus tanguticus is an important medicinal plant, and its main active ingredient is anisodamine. Anisodamine has various pharmacological effects, such as anticholinergic, spasmolytic, analgesic, etc., and is widely used in clinical treatment. The traditional production of anisodamine relies on the excavation of Anisodus tanguticus plants. Starting from Scopolia tangutica Maxim plants, the raw material of anisodamine hydrobromide is prepared through purification. Since the content of anisodamine in Anisodus tanguticus plants is low and the growth is slow, it takes more than 3 years to grow before it can be used for production. The natural resources are limited and the excavation causes damage to the ecological environment. The content of anisodamine and the impurity level fluctuate greatly due to the growth environment, resulting in large fluctuations in the production process of anisodamine hydrobromide raw materials and poor quality controllability. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide an inducer, a reagent combination and their application in the preparation of anisodamine. The present invention provides a technical solution for producing anisodamine using suspension cells, and the inducer provided can rapidly convert hyoscyamine into anisodamine.

[0004] The present invention provides an inducer, which comprises silver nitrate, acetylsalicylic acid and methyl jasmonate in a mass ratio of (4.5~13.5):(6.5~20.4):(28~84).

[0005] Compared with the prior art, the components of silver nitrate, acetylsalicylic acid and methyl jasmonate provided by the present invention cooperate with each other, have strong compatibility, can synergistically enhance the effect, and further improve the efficiency of rapidly converting hyoscyamine into anisodamine, thereby achieving a more accurate technical effect.

[0006] In some embodiments, it comprises silver nitrate, acetylsalicylic acid and methyl jasmonate in a mass ratio of 8.5:13.6:56.

[0007] Through experimental verification, at the above mass ratio, the cooperation degree among the components of silver nitrate, acetylsalicylic acid and methyl jasmonate is the highest, the compatibility is the strongest, they can synergistically enhance the effect, and further improve the efficiency of rapidly converting hyoscyamine into anisodamine, thereby achieving a more accurate technical effect.

[0008] The present invention provides the application of the above-mentioned inducer in the preparation of anisodamine.

[0009] The present invention provides a reagent combination for preparing anisodamine, which comprises the above-mentioned inducer and a callus medium.

[0010] In some embodiments, the callus medium comprises N6 medium, 3 - 8 mg / L calcium pantothenate, 30 - 80 mg / L arginine, 10 - 30 mg / L α - ketoglutaric acid, 10 - 30 mg / L vitamin C, and 0.1 - 0.5 mg / L naphthylacetic acid

[0011] In some specific embodiments, the callus medium comprises N6 medium, 5 mg / L calcium pantothenate, 50 mg / L arginine, 20 mg / L α - ketoglutaric acid, 20 mg / L vitamin C, and 0.2 mg / L naphthylacetic acid.

[0012] The present invention provides a method for preparing anisodamine, comprising the following steps:

[0013] Step 1: Inoculate the anisodus explant into the callus medium in the reagent combination, and obtain a callus tissue filtrate after dark culture, light culture, and filtration;

[0014] Step 2: Inoculate the callus tissue filtrate obtained in Step 1 into the callus medium, and obtain a suspension cell culture solution after dark culture and light culture;

[0015] Step 3: Add scopolamine and the inducer in the reagent combination to the suspension cell culture solution obtained in Step 2, and obtain a culture containing the anisodamine after light culture;

[0016] Step 4: Take the anisodamine culture obtained in Step 3, adjust the pH to alkaline, extract with chloroform, concentrate under reduced pressure, dissolve in acetone, adjust the pH to acidic with hydrobromic acid, crystallize, and dry to obtain the anisodamine.

[0017] In some embodiments, the anisodus explant is selected from at least one of the cotyledon tip, root, stem, and cotyledon base of the anisodus plant;

[0018] The time of dark culture is 2 - 8 days, the time of light culture is 15 - 25 days, the light intensity of light culture is 1500 - 2000 lx, the temperature of dark culture and light culture is 25 ± 1 °C, and the oscillation speed of dark culture and light culture is 50 - 200 r / min.

[0019] In some specific embodiments, the anisodus explant is the root of the anisodus plant;

[0020] The time of dark culture is 5 days, the time of light culture is 25 days, the light intensity of light culture is 1500 - 2000 lx, the temperature of dark culture and light culture is 25 ± 1 °C, and the oscillation speed of dark culture and light culture is 50 - 200 r / min.

[0021] In some embodiments, in step 2,

[0022] the volume ratio of the callus filtrate to the callus culture medium is 1:(5 - 15);

[0023] the time of dark culture is 2 - 8 days, the time of light culture is 15 - 25 days, the light intensity of light culture is 1500 - 2000 lx, the temperatures of dark culture and light culture are 25 ± 1°C, and the oscillation rotation speed of dark culture and light culture is 50 - 200 r / min.

[0024] In some specific embodiments, in step 2,

[0025] the volume ratio of the callus filtrate to the callus culture medium is 1:10;

[0026] the time of dark culture is 5 days, the time of light culture is 20 days, the light intensity of light culture is 1500 - 2000 lx, the temperatures of dark culture and light culture are 25 ± 1°C, and the oscillation rotation speed of dark culture and light culture is 100 r / min.

[0027] In some embodiments, in step 3, the working concentration of hyoscyamine is 0.1 - 0.5 g / L, and the time of light culture is 2 - 10 days.

[0028] In some specific embodiments, in step 3, the working concentration of hyoscyamine is 0.15 g / 558 mL, and the time of light culture is 5 days.

[0029] In some embodiments, in step 4, adjusting the pH to alkaline includes adjusting the pH to above 10.0 using ammonia water, the volume ratio of chloroform to the culture is 1:(0.5 - 3), the number of chloroform extractions is 2 - 6 times, the volume ratio of the solution after reduced pressure concentration to acetone is 1:(3 - 8), and adjusting the pH to acidic using hydrobromic acid is to adjust the pH to 2 - 5.

[0030] In some specific embodiments, in step 4, the volume ratio of chloroform to the culture is 1:1, the number of chloroform extractions is 4 times, and the volume ratio of the solution after reduced pressure concentration to acetone is 1:5.

[0031] The present invention provides the anisodamine prepared by the described preparation method.

[0032] Compared with the prior art, the beneficial effects of the present invention include:

[0033] 1. The present invention provides an inducer comprising silver nitrate, acetylsalicylic acid and methyl jasmonate. The components of the inducer cooperate with each other, have strong compatibility, can synergistically enhance the effect, and further improve the efficiency of rapidly converting scopolamine into anisodamine.

[0034] 2. The present invention adopts the suspension cell culture technology, combined with an optimized callus culture medium, a precursor substance scopolamine and an inducer. Compared with the starting material Anisodus tanguticus plants used in the current raw material drug of anisodamine hydrobromide, it has the advantages of a short growth cycle, stable content components, environmental protection and no damage to natural resources, providing a new way to obtain the starting material for the production of anisodamine hydrobromide raw material drug, and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 shows the technical route of the present invention;

[0036] Figure 2 shows the HPLC detection result diagram of the mixed control sample in Example 1;

[0037] Figure 3 shows the HPLC detection result diagram of the culture solution after induction and transformation in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention provides an inducer, a reagent combination and their application in the preparation of anisodamine. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0039] The suspension culture technology can overcome the current disadvantages of scopolamine production. By using the suspension culture of Anisodus roots, the growth is not restricted by the natural environment, the culture period is short, only 2-3 months are required, the content and components are relatively stable, and it has little impact on the production process of anisodamine hydrobromide raw material drug. The suspension has a high content, low cost, and is environmentally friendly, solving the problem of a large amount of Anisodus plants required for the production of anisodamine hydrobromide raw material drug.

[0040] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market. The present invention will be further described below in conjunction with the examples.

[0041] Example 1

[0042] The technical route of the present invention is as Figure 1 shown.

[0043] (1)Selection of callus

[0044] Select Anisodus plants from different parts. First, wash them with dishwashing liquid for 3 - 5 minutes, then rinse with tap water for 5 - 8 minutes. Transfer them to the ultra - clean workbench, disinfect with 75% alcohol for 30 seconds, wash twice with sterile water, then soak and shake in 0.1% mercuric chloride solution for 15 minutes, and wash 4 - 5 times with sterile water. After blotting dry with sterile paper towels, set aside. On the ultra - clean workbench, cut the root epidermis into small pieces with a disinfected scalpel and inoculate it into the optimized N6 medium. Cover the bottle cap, first culture in the dark for 5 days and then in the light for 25 days. The culture temperature is 25 ± 1 °C, and the shaker speed is 100 r / min. The shaker speed is 100 r / min, and the light intensity for light culture is 1500 - 2000 lx. Observe the growth status and the content of anisodamine. The results are shown in Table 1 below:

[0045] Table 1

[0046]

[0047] The components of the optimized N6 medium are shown in Table 2 below.

[0048] Table 2

[0049]

[0050] Finally, select the root as the explant for callus.

[0051] (2)Suspension cell culture

[0052] Filter the well - cultured callus, take 50 mL of the filtrate and inoculate it into 500 mL of the optimized N6 medium treated aseptically. First, culture in the dark for 5 days (temperature 25 ± 1 °C, shaker speed 100 r / min), and then culture in the light for 20 days (temperature 25 ± 1 °C, shaker speed 100 r / min, light 1500 - 2000 lx).

[0053] (3)Induction of transformation

[0054] Add 5 mL of atropine sterile solution (containing 0.15 g of atropine), 1 mL of silver nitrate sterile solution (containing 8.5 mg of silver nitrate), 1 mL of acetylsalicylic acid sterile solution (containing 13.6 mg of acetylsalicylic acid), and 1 mL of methyl jasmonate sterile solution (containing 56.0 mg of methyl jasmonate) to the culture solution, and continue light culture for 5 days.

[0055] (4)Sample detection

[0056] Take out the culture solution and detect the alkaloid content by HPLC. The detection method is as follows:

[0057] Chromatographic conditions: Use octadecylsilyl silica gel bonded with water as the filler, with acetonitrile - 30 mmol / L potassium dihydrogen phosphate (containing 0.08% triethylamine, adjusted to pH 6.0 with phosphoric acid) (13:87) as the mobile phase, the detection wavelength is 210 nm, and the column temperature is 35°C. The number of theoretical plates calculated based on the anisodine peak shall not be less than 3000.

[0058] Preparation of test solution: Filter this product and use it as the test solution.

[0059] Preparation of reference solution: Accurately weigh about 10 mg of anisodamine hydrobromide reference substance, place it in a 50 ml volumetric flask, dissolve it with the mobile phase and dilute to the mark.

[0060] Assay method: Accurately measure 20 μL of each of the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the content according to the external standard method based on the peak area.

[0061] The results are shown in Table 3 as follows:

[0062] Table 3

[0063]

[0064] (5) Purification

[0065] Adjust the pH of the culture solution to above 10.0 with ammonia water, extract it 4 times with chloroform in a 1:1 ratio, combine the extraction solutions, concentrate under reduced pressure to remove chloroform, add 5 times the amount of acetone to dissolve, dropwise add hydrobromic acid to adjust the pH to 2 - 5, stir to crystallize, and dry to obtain crude anisodamine hydrobromide with a purity of 99.2%.

[0066] Comparative Example 1

[0067] Under the same conditions, perform the same operation using N6 medium. Compared with the composition in Table 1, the N6 medium does not contain calcium pantothenate, arginine, α - ketoglutaric acid, vitamin C, and NAA (naphthaleneacetic acid) components. The alkaloid conversion rate is shown in Table 4 as follows:

[0068] Table 4

[0069]

[0070] The results show that: Using the conventional N6 medium without components such as calcium pantothenate, arginine, α - ketoglutaric acid, vitamin C, and NAA (naphthaleneacetic acid), the conversion rate of anisodamine is relatively low.

[0071] Comparative Example 2

[0072] Use the optimized N6 medium, add the same amount of scopolamine, without adding an inducer, and the alkaloid conversion is shown in Table 5 as follows:

[0073] Table 5

[0074]

[0075] The results showed that without the inducer, the content and conversion rate of anisodamine were low.

[0076] Comparative Example 3

[0077] Without methyl jasmonate inducer, the method was the same as before, and the alkaloid conversion rates were as shown in Table 6 below:

[0078] Table 6

[0079]

[0080] Compared with Example 1, the conversion rate decreased slightly.

[0081] Comparative Example 4

[0082] Without acetylsalicylic acid inducer, the method was the same as before, and the alkaloid conversion rates were as shown in Table 7 below:

[0083] Table 7

[0084]

[0085] Compared with Example 1, the conversion rate decreased.

[0086] Comparative Example 5

[0087] Without silver nitrate inducer, the method was the same as before, and the alkaloid conversion rates were as shown in Table 8 below:

[0088] Table 8

[0089]

[0090] Compared with Example 1, the conversion rate decreased slightly.

[0091] Comparative Example 5

[0092] The inducer remained unchanged, and the concentration was reduced: The method was the same as before, 1 mL of silver nitrate sterile solution (containing 4.5 mg of silver nitrate), 1 mL of acetylsalicylic acid sterile solution (containing 6.8 mg of acetylsalicylic acid), and 1 mL of methyl jasmonate sterile solution (containing 28.0 mg of methyl jasmonate). The alkaloid conversion rates were as shown in Table 9 below:

[0093] Table 9

[0094]

[0095] Compared with Example 1, the conversion rate decreased.

[0096] Comparative Example 6

[0097] The inducer remains unchanged while the concentration is increased: The method is the same as before. 1 mL of a sterile silver nitrate solution (containing 13.5 mg of silver nitrate), 1 mL of a sterile acetylsalicylic acid solution (containing 20.4 mg of acetylsalicylic acid), and 1 mL of a sterile methyl jasmonate solution (containing 84.0 mg of methyl jasmonate). The alkaloid conversion rates are shown in Table 10 below:

[0098] Table 10

[0099]

[0100] Compared with Example 1, the conversion rate decreased.

[0101] Comparative Example 7

[0102] The inducer is reduced to 1 type while the concentration remains unchanged: The method is the same as before. Add 5 mL of a sterile hyoscyamine solution (containing 0.15 g of hyoscyamine) and 1 mL of a sterile acetylsalicylic acid solution (containing 13.6 mg of acetylsalicylic acid) to the culture medium, and continue light cultivation for 5 days. The alkaloid conversion rates are shown in Table 11 below:

[0103] Table 11

[0104]

[0105] Comparative Example 8

[0106] The inducer is reduced to 2 types while the concentration remains unchanged: The method is the same as before. Add 5 mL of a sterile hyoscyamine solution (containing 0.15 g of hyoscyamine), 1 mL of a sterile acetylsalicylic acid solution (containing 13.6 mg of acetylsalicylic acid), and 1 mL of a sterile methyl jasmonate solution (containing 56.0 mg of methyl jasmonate) to the culture medium, and continue light cultivation for 5 days. The alkaloid conversion rates are shown in Table 12 below:

[0107] Table 12

[0108]

[0109] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An inducer, characterized in that, It comprises silver nitrate, acetylsalicylic acid and methyl jasmonate with a mass ratio of (4.5~13.5):(6.5~20.4):(28~84).

2. The inducer according to claim 1, wherein It comprises silver nitrate, acetylsalicylic acid and methyl jasmonate with a mass ratio of 8.5:13.6:

56.

3. Use of the inducer according to claim 1 or 2 in the preparation of anisodamine.

4. A reagent combination for preparing anisodamine, characterized in that, It comprises the inducer according to claim 1 or 2 and a callus medium.

5. The reagent combination according to claim 4, wherein The callus medium comprises N6 medium, 3~8 mg / L calcium pantothenate, 30~80 mg / L arginine, 10~30 mg / L α-ketoglutaric acid, 10~30 mg / L vitamin C and 0.1~0.5 mg / L naphthylacetic acid.

6. Preparation method of anisodamine, characterized in that, It comprises the following steps: Step 1: Take the anisodus explant and inoculate it into the callus medium in the reagent combination according to claim 4 or 5, and obtain a callus filtrate after dark culture, light culture and filtration; Step 2: Take the callus filtrate obtained in Step 1 and inoculate it into the callus medium, and obtain a suspension cell culture solution after dark culture and light culture; Step 3: Add scopolamine and the inducer in the reagent combination according to claim 4 or 5 to the suspension cell culture solution obtained in Step 2, and obtain a culture containing the anisodamine after light culture; Step 4: Take the culture of anisodamine obtained in Step 3, adjust the pH to alkaline, extract with chloroform, concentrate under reduced pressure, dissolve with acetone, adjust the pH to acidic with hydrobromic acid, crystallize, and dry to obtain the anisodamine.

7. The preparation method according to claim 6, characterized in that In the said Step 1, The anisodus explant is selected from at least one of cotyledon tip, root, stem and cotyledon base of anisodus plants; The time of the dark culture is 2~8 days, the time of the light culture is 15~25 days, the light intensity of the light culture is 1500~2000 lx, the temperature of the dark culture and the light culture is 25±1 °C, and the oscillation speed of the dark culture and the light culture is 50~200 r / min.

8. The preparation method according to claim 6 or 7, characterized in that, In the said Step 2, The volume ratio of the callus filtrate to the callus medium is 1:(5~15); The time of the dark culture is 2~8 days, the time of the light culture is 15~25 days, the light intensity of the light culture is 1500~2000 lx, the temperature of the dark culture and the light culture is 25±1 °C, and the oscillation speed of the dark culture and the light culture is 50~200 r / min.

9. The preparation method according to any one of claims 6 to 8, characterized in that, In the said Step 3, the working concentration of the scopolamine is 0.1~0.5 g / L, and the time of the light culture is 2~10 days; In the said Step 4, adjusting the pH to alkaline includes adjusting the pH to above 10.0 with ammonia water, the volume ratio of the chloroform to the culture is 1:(0.5~3), the number of times of chloroform extraction is 2~6 times, the volume ratio of the solution concentrated under reduced pressure to the acetone is 1:(3~8), and adjusting the pH to acidic with hydrobromic acid is adjusting the pH to 2~5.

10. The anisodamine prepared by the preparation method according to any one of claims 6~9.

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