Method for extracting benzophenone through radix cynanchi bungei endophytic fungi

Abstract

The invention discloses a method for extracting benzophenone through radix cynanchi bungei endophytic fungi, which comprises the following steps: centrifuging a fermentation supernatant of the radix cynanchi bungei endophytic fungi at 10,000 r / min for 10 minutes, pre-freezing the supernatant obtained by centrifuging at-20 DEG C for 10-12 hours, and transferring into a freeze dryer for drying for 20-22 hours to obtain dry powder; and uniformly mixing the dried powder and cyclohexane, transferring to a magnetic stirring electric heating jacket at 50 DEG C, sealing, fully stirring for 2-3 hours, transferring a sample to an environment at 4 DEG C, and standing until crystals are obtained by chromatography, thereby obtaining the bacterium source benzophenone extract. The method has the advantages of simple process, high extraction efficiency of the obtained bacterium source benzophenone, high purity and the like, and is suitable for industrial application.

Description

Technical Field

[0001] The invention belongs to the technical field of benzophenone extraction, and in particular relates to a method for extracting benzophenone through endophytic fungi of Cynanchum multiflorum. Background Art

[0002] Cynomorium benzophenone has important physiological activities such as protecting nerves, anti-oxidation, anti-aging, protecting brain cells and blackening hair. Studies have shown that Cynomorium benzophenone may be the essence of Cynomorium benzophenone blackening hair, and Cynomorium benzophenone derivatives have been used in shampoo.

[0003] Most of the existing extraction processes of benzophenone from Cynanchum multiflorum use stems and leaves of Cynanchum multiflorum as raw materials, and adopt an organic solvent extraction method or a petroleum ether extraction-organic solvent elution-silica gel column chromatography extraction method. Among them, in the organic solvent extraction method, since benzophenone from Cynanchum multiflorum is very soluble in organic solvents, ultrasonic extraction of benzophenone from Cynanchum multiflorum can be performed using methanol or ethanol. In the extraction method of petroleum ether extraction-organic solvent elution-silica gel column chromatography, a compound containing a variety of acetophenone substances is separated and purified by a petroleum ether extraction layer, and then extracted with ethanol as a solvent, and two elution systems of PE-EA and chloroform-methanol are selected for gradient elution, and finally 95% and 75% ethanol are used for percolation in turn, and the filtrate obtained by reduced pressure concentration is gradient eluted by silica gel column chromatography (chloroform-methanol system (20:1-10:1, volume ratio)) to obtain benzophenone from Cynanchum multiflorum. However, these extraction methods still have problems such as low efficiency. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a method for extracting benzophenone by using endophytic fungi of Cynanchum multiflorum.

[0005] The present invention is achieved by a method for extracting benzophenone by endophytic fungi of Radix Cynanchum multiflorum, the method comprising the following steps:

[0006] (1) centrifuging the fermentation supernatant of the endophytic fungus of Cynanchum multiflorum at 10,000 r / min for 10 min, pre-freezing the supernatant obtained by centrifugation at -20°C for 10 to 12 h, and then transferring it to a freeze dryer for drying for 20 to 22 h to obtain a dry powder;

[0007] (2) The dry powder and cyclohexane are mixed and then transferred to a 50° C. magnetic stirring electric heating mantle, sealed and stirred for 2 to 3 hours, and the sample is transferred to a 4° C. environment and allowed to stand until crystals are precipitated, which is the bacterial-derived benzophenone extract.

[0008] Preferably, in step (1), the endophytic fungus of Cynanchum multiflorum is Acremonium sp Z-13, with a preservation number of CCTCC NO: M 20241239 and a preservation address of Wuhan University Collection Center, Luojia, Wuchang District, Wuhan City, with a zip code of 430072.

[0009] Preferably, in step (1), the preparation of the fermentation supernatant comprises the following steps: inoculating the Radix Cynanchum multiflorum Z-13 at a 1% inoculation rate into a PDA liquid culture medium, activating and enriching the culture medium at 28°C and 180 r / min until the logarithmic growth phase, transferring the culture medium to the fermentation medium, culturing the culture medium at 28°C and 180 r / min for 1 day, centrifuging the culture medium for 10 minutes, and obtaining the supernatant as the fermentation supernatant.

[0010] Preferably, the PDA culture medium comprises: 12 g potato powder, 20 g glucose, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate, 2 g peptone, 1,000 mL distilled water, and a pH of 7.

[0011] Preferably, the fermentation medium comprises: 12 g potato powder, 16 g soluble starch, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate, 1 g peptone, 0.1% NaCl, 2.9 mg / L L-phenylalanine, 1,000 mL distilled water, and a pH of 7.

[0012] Preferably, in step (2), the mass volume ratio of the dry powder to cyclohexane is 1 g:5 mL.

[0013] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: the present invention freezes and dries the fermentation supernatant of the endophytic fungus of Cynanchum multiflorum, and the obtained dry powder is successively subjected to cyclohexane heat extraction and low-temperature chromatography to finally obtain the fungus-derived benzophenone. The method of the present invention has the advantages of simple process, high extraction efficiency and high purity of the obtained fungus-derived benzophenone, and is suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the response surface analysis of the fermentation medium optimization of strain Z-13; Figure 1 -a is the optimal carbon-nitrogen ratio, and the horizontal axis A is the carbon-nitrogen ratio; Figure 1 -b is the optimal NaCl concentration, and the horizontal axis B (%) is the NaCl concentration. Figure 1 -c is the optimal L-phenylalanine concentration, and the abscissa C:C (mg / mL) is the L-phenylalanine concentration;

[0015] Figure 2 is the full-band scanning result of the sample; among them, Figure 2 -a is the full-band scanning result of the standard product of Cynanchum multiflorum benzophenone, Figure 2-b is the full-band scanning result of the bacterial benzophenone extract sample;

[0016] Figure 3 This is the LC-MS analysis result of the bacterial benzophenone extract. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0018] 1. Preparation of bacterial-derived benzophenone

[0019] 1. Strain selection and cultivation

[0020] The endophytic fungus of Cynanchum multiflorum in this embodiment is selected as Acremonium sp Z-13 Z-13, which was deposited in the China Center for Type Culture Collection on June 17, 2024, with a deposit number of CCTCC NO: M20241239, and a deposit address of Wuhan University Collection Center, Luojia, Wuchang District, Wuhan City, with a postal code of 430072.

[0021] Response surface design was used to optimize the culture conditions of Z-13 and regulate the metabolic level of benzophenone. Based on the results of the single factor experiment, the carbon-nitrogen ratio was set to 6:1, 8:1, and 10:1, the NaCl concentration was 0.1%, 0.2%, and 0.3%, and the concentration of the precursor L-phenylalanine was 1 mg / L, 3 mg / L, and 5 mg / L, respectively. The fermentation medium was optimized (see Figure 1 ).

[0022] Acremonium Z-13 was inoculated into PDA liquid culture medium at a 1% inoculation rate, activated and enriched to the logarithmic growth phase at 28°C and 180r / min, and then transferred to the optimized fermentation medium for expansion culture. The culture was carried out at a constant temperature of 28°C and 180r / min for 24h. After the benzophenone content reached a peak, the culture was centrifuged for 10min and the fermentation supernatant was collected.

[0023] The PDA medium is composed of 12 g potato powder, 20 g glucose, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate, 2 g peptone, 1,000 mL distilled water, and a pH of 7. The fermentation medium is composed of 12 g potato powder, 16 g soluble starch, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate, 1 g peptone, 0.1% NaCl, 2.9 mg / L L-phenylalanine, 1,000 mL distilled water, and a pH of 7.

[0024] 2. Fermentation

[0025] The obtained fermentation supernatant was centrifuged at 10,000 r / min for 10 minutes, the supernatant obtained by centrifugation was pre-frozen at -20°C for 10 to 12 hours, and then transferred to a freeze dryer for drying for 20 to 22 hours to obtain a dry powder.

[0026] 3. Hot extraction and cold chromatography

[0027] The dry powder and cyclohexane were mixed at a mass volume ratio of 1 g:5 mL and then transferred to a 50°C magnetic stirring electric heating mantle. After sealing, the mantle was fully stirred (2 to 3 h). The sample was transferred to a 4°C environment and allowed to stand until crystals were precipitated, which was the bacterial-derived benzophenone extract.

[0028] 2. Qualitative and quantitative analysis of bacterial benzophenone extracts

[0029] 1. Add the above-mentioned bacterial-derived benzophenone extract (30 mg) into methanol (1 mL) to fully dissolve it to obtain a bacterial-derived benzophenone solution.

[0030] 2. The bacterial benzophenone solution was scanned in the whole band, and the results showed that it was consistent with the main peak information of the standard product of Cynanchum multiflorum benzophenone (see Figure 2 ).

[0031] 3. Filter the bacterial-derived benzophenone solution through a 0.2 μm filter membrane and perform LC-MS analysis (see Figure 3 ), its [MH] + The mass-to-charge ratio is 180, so the molecular weight of the compound is 182. After comparison with the spectrum, the sample was identified as the acetophenone substance Cynanchum multiflorum B.

[0032] 4. Yield and purity

[0033] The calculation formulas for the yield of bacterial-derived benzophenone in the fermentation broth and the purity of the benzophenone extract are as follows:

[0034]

[0035] The analysis showed that the yield of benzophenone from the fungus of Baishouwu Z-13 reached 77.00±1.00%, and the purity reached 86.50±1.50%.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting benzophenone by endophytic fungi of Cynanchum multiflorum, characterized in that: The method comprises the following steps: (1) centrifuging the fermentation supernatant of the endophytic fungus of Cynanchum multiflorum at 10,000 r / min for 10 min, pre-freezing the supernatant obtained by centrifugation at -20°C for 10 to 12 h, and then transferring it to a freeze dryer for drying for 20 to 22 h to obtain a dry powder; (2) The dry powder and cyclohexane are mixed and then transferred to a 50° C. magnetic stirring electric heating mantle, sealed and stirred for 2 to 3 hours, and the sample is transferred to a 4° C. environment and allowed to stand until crystals are precipitated, which is the bacterial-derived benzophenone extract.

2. The method according to claim 1, characterized in that In step (1), the endophytic fungus of Cynanchum multiflorum is Acremonium sp Z-13, with a preservation number of CCTCC NO: M 20241239 and a preservation address of Wuhan University Collection Center, Luojia, Wuchang District, Wuhan City, with a zip code of 430072.

3. The method according to claim 2, characterized in that In step (1), the preparation of the fermentation supernatant comprises the following steps: The Z-13 bacteria of Baishouwu were inoculated into PDA liquid culture medium at a 1% inoculation rate, activated and enriched at 28°C and 180 r / min until the logarithmic growth phase, then transferred to fermentation medium, cultured at 28°C and 180 r / min for 1 day, centrifuged for 10 minutes, and the obtained supernatant was the fermentation supernatant.

4. The method according to claim 3, characterized in that The PDA culture medium comprises: 12 g potato powder, 20 g glucose, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate, 2 g peptone, 1,000 mL distilled water, and a pH of 7.

5. The method according to claim 3, characterized in that The fermentation medium comprises: 12 g potato powder, 16 g soluble starch, 2 g potassium dihydrogen phosphate, 2 g magnesium sulfate, 1 g peptone, 0.1% NaCl, 2.9 mg / L L-phenylalanine, 1,000 mL distilled water, and a pH of 7.

6. The method according to claim 1, characterized in that In step (2), the mass volume ratio of the dry powder to cyclohexane is 1 g:5 mL.

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