A method for increasing production of hispidin by liquid fermentation of wani-shan using ethephon

Abstract

The application discloses a method for promoting the growth of a Vani Resina strain by using ethephon Sanghuangporus vaninii The application discloses a method for increasing the production of Hispidin by liquid fermentation, and belongs to the field of biological medicines.The technical scheme is as follows: a mother strain of Vani Resina is inoculated on a potato glucose agar plate culture medium to be activated;an agar block with mycelium of the Vani Resina strain after activation is inoculated into a liquid culture medium and cultured in a shaking table;water solution of ethephon is added into the culture medium immediately after inoculation or after inoculation, and sodium carbonate is used to adjust the pH of the culture medium to an initial value.Compared with the culture medium without ethephon, the content of Hispidin is increased by 0.44-2.28 times, and compared with the content of Hispidin in a fruiting body of artificial cultivation of the Vani Resina, the content of Hispidin is increased by more than 1000 times.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a method for increasing Hispidin production by using ethephon to promote the liquid fermentation of Vanisin. Background Technology

[0002] Vani Sanghuang belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricales, order Phytotrichum, family Phytotrichumceae, and genus *Inonotus*. It is also known as Yangshu Sanghuang in folk medicine and is one of the most widely cultivated Sanghuang varieties. Previous experiments determined the Hispidin content in the fruiting bodies of Vani Sanghuang from 10 different cultivation conditions and origins to be 0.0070-0.0178 mg / g. Due to the low Hispidin content in artificially cultivated fruiting bodies and the relatively high price of Sanghuang products, its medicinal development resources are scarce. Organic synthesis methods, such as those described in the article "Gonindard C, Bergonzi C, Denier C, et al. Synthetichispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro.[J]. Cell biology and toxicology, 1997, 13(3).", synthesize Hispidin using 4-hydroxy-6-methyl-2-pyranone and 3,4-dihydroxybenzaldehyde as raw materials. However, organic synthesis involves many steps, is complex, and has difficult-to-remove byproducts. Compared with natural product extracts, it not only has less medicinal potential but also has a greater impact on the environment. Therefore, this invention discloses a method for increasing Hispidin yield by promoting the liquid fermentation of Vanisanghuang with ethephon, which has a short fermentation time, low environmental pollution, simple operation, readily available experimental materials, and good reproducibility. Summary of the Invention

[0003] The purpose of this invention is to address the problems in existing Hispidin production methods by disclosing a method for increasing Hispidin yield by using ethephon to promote liquid fermentation of Vanilla linteus. The steps are as follows:

[0004] Hispidin is obtained by fermenting Vanisin with ethephon added to the culture medium.

[0005] In some preferred embodiments, after the mycelium of *Vanilla sanguisorba* is inoculated into a liquid culture medium, ethephon is added for fermentation to obtain Hispidin, the structural formula of which is as follows:

[0006] .

[0007] In some embodiments, the *Vanilla sanguisorba* is a product cultured on potato dextrose agar (PDA) plates for 10-14 days, containing *Vanilla sanguisorba* mycelium. A block of PDA agar containing *Vanilla sanguisorba* mycelium is taken using a punch and inoculated into a liquid culture medium in a block-like manner. The liquid culture medium is also a commonly used medium in the art. In this embodiment, the liquid culture medium consists of 20 g / L glucose, 5 g / L yeast extract, 0.5 g / L MgSO4, 0.2 g / L (NH4)2SO4, 0.1 g / L CaCl2, 1 g / L KH2PO4, and 0.01 g / L VB1.

[0008] The ethephon was added immediately after inoculation or on days 1 to 12 after inoculation.

[0009] The concentration of ethephon in the culture medium is 0.2-20 mM, more preferably 0.4-10 mM.

[0010] After adding ethephon, the pH was adjusted with sodium carbonate to the original pH of vanilla linteum in the culture medium.

[0011] In the above method, the culture is carried out on a shaker at 28°C and 140 rpm.

[0012] This invention also discloses a method for extracting and detecting Hispidin in Vani Sanguine liquid culture medium. The extraction method is as follows:

[0013] Step 1: Add 95% ethanol to the culture medium after fermentation;

[0014] Step 2: Use an ultrasonic cleaner to ultrasonically extract the culture medium containing 95% ethanol mentioned in Step 1.

[0015] Step 3: Filter the mycelium and fermentation broth;

[0016] Step 4: After the fermentation broth is dried, it is dissolved in ethanol or methanol and then filtered to obtain the final product.

[0017] The detection method is as follows:

[0018] Step 1: Filter the solution from step 4 above using a 0.22 μm nylon filter membrane;

[0019] Step 2: Detect using high-performance liquid chromatography (HPLC) under the following conditions:

[0020] The mobile phase consisted of A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) under the following conditions: 0 min, 20% B; 5 min, 20% B; 45 min, 40% B; λ = 353 nm, column temperature 30 ℃, and injection volume 20 μL.

[0021] The Hispidin content obtained from the above-mentioned liquid fermentation of Vanisanghuang can be more than 1,000 times higher than that in artificially cultivated fruiting bodies. Attached Figure Description

[0022] Figure 1 These are high-performance liquid chromatography (HPLC) chromatograms of Hispidin standard and Vanishangu extract at 353 nm. In the figures, A is 0.01 mg / mL Hispidin standard with an average peak area of ​​1049.451; B is the extract from Vanishangu fruiting bodies (1B, planted for 4 months) with an average peak area of ​​49.383; C is the Vanishangu extract without ethephon fermentation with an average peak area of ​​3854.048; and D is the Vanishangu extract fermented with ethephon at a final concentration of 2 mM on day 0 of cultivation with an average peak area of ​​12548.876. Detailed Implementation

[0023] The present invention and its embodiments are described below. This description is not restrictive, and actual embodiments are not limited thereto. In summary, if those skilled in the art, inspired by this description, design similar experimental methods and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

[0024] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0025] Example 1: A method for increasing Hispidin yield by promoting the liquid fermentation of Vanisin using ethephon:

[0026] I. Cultivation of Vanisang Huang inoculation plates.

[0027] Step 1: Subculturing of strain: Take a certain amount of mycelium from the test tube of Vanisinus sanguinis mother culture and inoculate it into potato dextrose agar plate (PDA medium) (purchased from Haibo Biotechnology), culture at 28℃, and subculture twice to obtain a relatively stable Vanisinus sanguinis PDA mother plate, and store it at 5℃.

[0028] Step 2: Inoculation plate cultivation: Take a 5 mm PDA mother plate agar block containing Vanilla sanguinis mycelium in the exponential growth phase from Step 1 using a punch, and activate it on another PDA plate medium at 28℃ for 14 days to obtain the inoculation plate.

[0029] II. Liquid fermentation of Vanisanghuang.

[0030] Step 1: Culture of fungi: Using a punch, take 2 agar blocks of Vanilla sanguinis mycelium in the exponential growth phase from the Φ=5 mm agar blocks in Step 2 above and inoculate them into liquid culture medium (composed of glucose 20 g / L, yeast extract 5 g / L, MgSO4 0.5 g / L, (NH4)2SO4 0.2 g / L, CaCl2 0.1 g / L, KH2PO4 1 g / L, VB1 0.01 g / L).

[0031] Step 2: Add ethephon: Immediately after inoculation or on days 3, 6, 9, and 12 after inoculation, take 1 mL of ethephon aqueous solution and add it to the culture medium to make its final concentration in the culture medium system 0.2, 0.4, 2, 10, and 20 mM, respectively. Adjust the pH back to the initial value using 1 M sodium carbonate. Set up 3 replicates for each condition and incubate in a shaker at 28 ℃ and 140 rpm for 15 days.

[0032] III. Extraction of Hispidin from Vanisin Sanguine after liquid fermentation.

[0033] Step 1: Add 95% ethanol to the culture medium after fermentation to make the ethanol concentration in the system 60%;

[0034] Step 2: Use a handheld tissue grinder to break up the mycelial balls in the culture medium from Step 1, allowing the contents to flow out, and then extract by ultrasonication at 50°C for 45 min;

[0035] Step 3: Filter the mycelium and fermentation broth, and collect the fermentation broth and mycelium separately;

[0036] Step 4: Dry the fermentation liquid;

[0037] Step 5: Add an appropriate amount of ethanol or methanol to the dried fermentation broth described in Step 4 to dissolve it, and then filter to obtain the final product.

[0038] IV. Detection of Hispidin in Vanisan yellow fruiting bodies and after liquid fermentation:

[0039] Step 1: Extraction of Hispidin from Vanisang Huang fruiting bodies: After grinding 10 different cultivation conditions and origins of Vanisang Huang fruiting bodies into powder, Hispidin was extracted according to the steps above.

[0040] Step 2: HPLC conditions: The mobile phase consisted of A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid), and the following conditions were set: 0 min, 20% B; 5 min, 20% B; 45 min, 40% B; λ = 353 nm, column temperature 30 ℃, and injection volume 20 μL.

[0041] Step 3: Establishment of the standard curve for Hispidin standard: Accurately weigh the Hispidin standard (purchased from Maclean's) and prepare standard solutions with concentrations of 0.04 mg / mL, 0.02 mg / mL, 0.01 mg / mL, 0.005 mg / mL, 0.0025 mg / mL, and 0.00125 mg / mL using chromatographic methanol.

[0042] Step 4: Sample detection: Filter the Hispidin extract using a 0.22 μm nylon filter membrane and dilute it to an appropriate concentration for detection.

[0043] Step 5: Result Calculation: Substitute the measured peak area into the Hispidin peak area-concentration standard curve. The calculation results are shown in the table below.

[0044] Table 1 shows the Hispidin content (Hispidin - mycelial dry weight) of Vanilla mulberry fruiting bodies (commercially available Vanilla mulberry products) under different cultivation conditions and from different origins.

[0045]

[0046] Table 2 shows the results of the determination of the increase in Hispidin content of Vanilla mulberry fermented by the process of Example 1 as a function of ethephon concentration (Hispidin - mycelial dry weight).

[0047]

[0048] Table 3 shows the results of the measurement of Hispidin content in Vanilla mulberry fermented using the process of Example 1 as a function of ethephon addition time (Hispidin - mycelial dry weight).

[0049]

[0050] Comparative Example 1: Hispidin extraction and detection of *Vanilla sambac* fruiting bodies under 10 different cultivation conditions and origins were performed using the above method. The Hispidin content was found to be 0.0070-0.0178 mg / g. At day 0 of cultivation, after liquid fermentation, the Hispidin content in *Vanilla sambac* increased by 0.44, 0.46, 2.28, 1.16, and 1.07 times, respectively, in media with ethephon added at final concentrations of 0.2 mM, 0.4 mM, 2 mM, 10 mM, and 20 mM, compared with the media without ethephon. At days 0, 3, 6, 9, and 12 of cultivation, the Hispidin content increased by 2 times. After liquid fermentation with mM medium and medium without ethephon, the Hispidin content in Vani Sanghae increased by 2.30, 0.14, 0.34, 0.50 and 0.68 times, respectively; the Hispidin content in Vani Sanghae fermented using the process of Example 1 was more than 1000 times higher than the Hispidin content in artificially cultivated fruiting bodies.

[0051] Figure 1 These are high-performance liquid chromatography (HPLC) chromatograms of Hispidin standard and Vanishangu extract at 353 nm. In the figures, A is 0.01 mg / mL Hispidin standard with an average peak area of ​​1049.451; B is the extract from Vanishangu fruiting bodies (1B, planted for 4 months) with an average peak area of ​​49.383; C is the Vanishangu extract without ethephon fermentation with an average peak area of ​​3854.048; and D is the Vanishangu extract fermented with ethephon at a final concentration of 2 mM on day 0 of cultivation with an average peak area of ​​12548.876.

Claims

1. A method for increasing Hispidin yield by promoting the liquid fermentation of Vanisin using ethephon, characterized in that, The steps are as follows: Ethephon is added to the Vani Sanguinea culture medium and fermented to obtain Hispidin. The concentration of ethephon in the culture medium is 0.2-20 mM. The ethephon was added immediately after inoculation or on days 1 to 12 after inoculation; After adding ethephon, the pH was adjusted to the original pH value in the Vani Sanguine medium using sodium carbonate.

2. The method according to claim 1, characterized in that, Hispidin was obtained by fermenting Vani Sanguine mycelium into liquid culture medium with the addition of ethephon.

3. The method according to claim 1, characterized in that, The concentration of ethephon in the culture medium is 0.4-10 mM.

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