An endophytic fungus from Schisandra chinensis WWZZ01 producing schisandrin A and its application

Abstract

A Schisandra endophytic fungus WWZZ01 producing Schisandra alcohol A and its application, relating to the field of microorganisms, the Schisandra endophytic fungus WWZZ01 is Geotrichum candidum WWZZ01, which has been preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, the preservation date is July 18, 2024, and the preservation number is CCTCC NO: M 20241605. The strain has good antioxidant activity. After the secondary metabolite analysis of its fermentation broth, it is found that the strain can ferment to produce Schisandra alcohol A. The present invention is used for in vitro antioxidant and production of Schisandra secondary metabolites.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to an endophytic Schisandrae Chinensis fungus WWZZ01 producing Schisandrin A and an application thereof. Background Art

[0002] Schisandra chinensis Schisandra chinensis The dried mature fruit of Turcz. Baill. is a commonly used Chinese herbal medicine in clinical practice. It has the effects of astringency, invigorating qi and promoting body fluid, and nourishing the kidney and calming the mind. Schisandra contains a variety of lignans, among which Schisandraein A is one of the main active ingredients in Schisandrae and is also an indicator ingredient for Schisandraein quality control included in the Chinese Pharmacopoeia. Schisandraein A has sedative, tranquilizing, hypnotic, anti-depressant, anti-inflammatory, and antioxidant effects, and can inhibit the release of transaminase, protect liver cell membrane damage, and prevent osteoporosis.

[0003] The basic skeleton structure of Schisandrin A is connected by C6-C3. It was originally extracted from Schisandrin fruit. Due to its various pharmacological activities and unique skeleton structure, the synthesis of Schisandrin A has become the most challenging focus in the field of organic synthesis. In the past, the synthesis reaction route of Schisandrin A had as many as 20 steps. Although some people summarized the experience of their predecessors and synthesized Schisandrin A with gallic acid as raw material through 9 steps of reaction, the reaction conditions were harsh, there were many by-products, and the yield was low. Therefore, plant sources are still the preferred production method of Schisandrin A, but the extraction of Schisandrin A from Schisandrin plant raw materials requires the consumption of a large amount of plant raw materials, and Schisandrin is a national third-level protected plant, which is not conducive to industrial production. Summary of the invention

[0004] The invention aims to solve the problem that the existing method for extracting schisandra alcohol A is not conducive to industrial production, and provides schisandra alcohol A-producing endophytic fungus WWZZ01 of schisandra chinensis and application thereof.

[0005] The present invention provides an endophytic fungus strain WWZZ01 of Schisandra chinensis, which is a strain of Geotrichum candidum ( Geotrichum candidum )WWZZ01, has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is July 18, 2024, and the deposit number is CCTCC NO: M 20241605.

[0006] The colony of the Schisandra endophytic fungus WWZZ01 of the present invention is white, the hyphae are sticky, and the matrix is ​​grayish white. After the hyphae mature, they break into single or chained long cylindrical arthrospores with blunt ends.

[0007] The 18S rDNA sequence of the endophytic fungus WWZZ01 of Schisandra chinensis of the present invention was submitted to GenBank for Blast homology comparison, and the strain sequence with high homology of the endophytic fungus WWZZ01 of Schisandra chinensis was analyzed using MEGA7 software. Geotrichum candidum (KJ543497.1) reached 100%. The endophytic fungus WWZZ01 of Schisandra chinensis was identified as Geotrichum candidum ( Geotrichum candidum ).

[0008] The present invention also provides application of endophytic fungus WWZZ01 of Schisandra chinensis in fermentation to produce secondary metabolites of Schisandra chinensis.

[0009] Furthermore, the secondary metabolite of Schisandra chinensis is Schisandrin A.

[0010] The present invention also provides application of endophytic fungus WWZZ01 of Schisandra chinensis in in vitro anti-oxidation.

[0011] Beneficial effects of the present invention:

[0012] The present invention isolated an endophytic fungus WWZZ01 from Schisandra chinensis, which was identified as Geotrichum candidum ( Geotrichum candidum ). The strain has good antioxidant activity. After analyzing the secondary metabolites of its fermentation broth, it was found that the secondary metabolites of the strain contained schisandrin A, which can be fermented to produce schisandrin A.

[0013] The invention can produce schisandrin A by microbial fermentation, and is suitable for industrial production because the microorganisms reproduce rapidly and the product extraction is simple, and can provide a new method for producing schisandrin A by microbial fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the colony morphology of the endophytic fungus WWZZ01 in Schisandra chinensis;

[0015] Figure 2 This is the hyphae morphology of the endophytic fungus WWZZ01 in Schisandra chinensis;

[0016] Figure 3 is the phylogenetic tree of the endophytic fungus WWZZ01 in Schisandra chinensis;

[0017] Figure 4 The scavenging ability of the fermentation liquid of Schisandra chinensis endophytic fungus WWZZ01 on DPPH free radicals;

[0018] Figure 5 The scavenging ability of the fermentation liquid of Schisandra chinensis endophytic fungus WWZZ01 on •OH free radicals;

[0019] Figure 6The results of HPLC analysis of schisandrin A in the endophytic fungus WWZZ01 of Schisandra chinensis;

[0020] Figure 7 For crystal A 1 H-NMR analysis results. DETAILED DESCRIPTION

[0021] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. Embodiment 1:

[0022] The endophytic fungus WWZZ01 of Schisandra chinensis in this example is Geotrichum candidum ( Geotrichum candidum )WWZZ01, has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is July 18, 2024, and the deposit number is CCTCC NO: M 20241605.

[0023] The isolation method of the endophytic fungus WWZZ01 of Schisandra chinensis in this embodiment is as follows:

[0024] Schisandra chinensis Acanthopanax seuticosus The seeds of (Rupr. et Maxim. )Harms (collected from Qinghe County, Heilongjiang Province) were disinfected after removing the seed coat. The disinfection procedure was as follows: the seeds were soaked in 75% alcohol for 30 s, washed with sterile water, rinsed in sodium hypochlorite for 10 min, washed with sterile water, then soaked in 75% alcohol for 30 s, and finally washed three times with sterile water.

[0025] Use a sterile scalpel to cut the sterilized seeds in the middle, place the cut surface in MS culture medium, and culture at 28°C for 7 days.

[0026] The last sterile water wash of the sterilization procedure was removed and inoculated into the MS culture medium. The sterilized uncut experimental materials were placed on the MS culture medium, rolled once, and then taken out and cultured with the endophytic fungus isolation culture medium as a control.

[0027] Preparation of MS medium: Take 4.74 g MS and add it into boiling water. After it dissolves, add 10 g agar and 30 g sucrose, stir, and adjust the volume to 1000 mL after it dissolves.

[0028] The endophytic fungus WWZZ01 was isolated from Schisandra seeds. No bacteria grew in the negative control plate and negative control liquid culture medium, and this was repeated many times, thus proving that the isolated bacteria were endophytic fungi of Schisandra.

[0029] Example 2: Identification of the endophytic fungus WWZZ01 in Schisandra chinensis

[0030] The strains were identified by colony and spore morphology observation and 18S rDNA sequence analysis.

[0031] Colony morphology of endophytic fungus WWZZ01 in Schisandra chinensis Figure 1 As shown in the figure, the mycelial morphology is as follows Figure 2 As shown. Its colony is white, the hyphae are sticky, and the matrix is ​​grayish white. When the hyphae mature, they break into single or chain-shaped arthrospores with obtuse ends.

[0032] The 18S rDNA sequence (1295 bp) of the endophytic fungus WWZZ01 of Schisandra chinensis of the present invention was submitted to GenBank for Blast homology comparison. The strain sequences with high homology to the endophytic fungus WWZZ01 of Schisandra chinensis were analyzed using MEGA7 software to construct a phylogenetic tree. Figure 3 As shown, the strain sequence of the endophytic fungus WWZZ01 in Schisandra chinensis is Geotrichum candidum (KJ543497.1) reached 100%. The endophytic fungus WWZZ01 of Schisandra chinensis was identified as Geotrichum candidum ( Geotrichum candidum ). The 18S rDNA sequence of the endophytic fungus WWZZ01 in Schisandra chinensis is shown in SEQ ID NO: 1 in the sequence listing.

[0033] Example 3: Antioxidant activity analysis of endophytic fungus WWZZ01 from Schisandra chinensis

[0034] Activation of endophytic fungus WWZZ01: Take endophytic fungus WWZZ01, inoculate it on the surface of PDA culture medium under sterile conditions, and culture it at 28 ℃ for 3 days for later use.

[0035] Preparation of fermentation broth of endophytic fungus WWZZ01: Take the activated endophytic fungus WWZZ01, pick the hyphae and inoculate them into 50 mL PDB medium under sterile conditions, and culture them in a shaking incubator at 28 °C and 120 r / min for 3 days to make 1×10 7 CFU / mL seed liquid; the seed liquid was transferred to 3L sterilized PDB medium at an inoculum size of 20% (v / v), and cultured in a shaking incubator at 28°C and 120 r / min for 14 days. The fermentation liquid was filtered and collected, and concentrated under reduced pressure, extracted with ethyl acetate (3×100mL), and concentrated at low temperature to an extract, and freeze-dried to obtain the freeze-dried powder of the fermentation liquid of the endophytic fungus WWZZ01 of Schisandra chinensis for later use.

[0036] 1. Determination of DPPH free radical scavenging ability

[0037] Dissolve an appropriate amount of DPPH in methanol and dilute to 100 mL to prepare a solution with a mass concentration of 0.15 mmol•L -1 DPPH solution; take an appropriate amount of Vc and dissolve it in methanol and make up to 10 mL, then 200 μg•mL -1 of reference solution;

[0038] The lyophilized powder of the fermentation liquid of the endophytic fungus WWZZ01 of Schisandra chinensis was dissolved in an appropriate amount of methanol to prepare a concentration of 200 μg•mL -1 of the test sample solution.

[0039] Dilute the above-mentioned reference and test solutions in sequence, and dilute each solution to a concentration gradient of 100 μg•mL -1 , 50μg•mL -1 , 25μg•mL -1 , 12.5μg•mL -1 、6.25μg•mL -1 2 mL of each solution was pipetted into a test tube, and methanol was used as a blank control group. 0.15 mmol•L -1 4 mL of DPPH solution was placed in the dark for 30 min, and the absorbance of each sample was measured at 517 nm (n=3). The DPPH free radical scavenging rate was calculated as follows:

[0040]

[0041] In the formula, A i Indicates the absorbance value when the test product and DPPH solution are added simultaneously; A j A0 represents the absorbance value when the test sample and blank control are added at the same time; A1 represents the absorbance value when the blank control and DPPH solution are added at the same time.

[0042] The scavenging ability of the fermentation liquid of Schisandra chinensis endophytic fungus WWZZ01 on DPPH free radicals is shown in Figure 4 .

[0043] 2. Determination of OH radical scavenging ability

[0044] Pipette 2 mL of each test solution of different concentrations into a test tube, and add 2 mL of 6 mmol•L -1 FeSO4, 2mL 6mmol•L -1 of H2O2, shake and mix, let stand for 10 min, then add 2mL 6mmol•L -1 The salicylic acid-ethanol solution was placed at 37°C for 30 min, and the absorbance of each sample was measured at 510 nm (n=3). The •OH radical scavenging rate was calculated as follows:

[0045]

[0046] In the formula, A1 represents the absorbance value of the added sample; A2 represents the absorbance value when distilled water replaces H2O2 to eliminate the influence of the sample's own color; A0 represents the absorbance value when anhydrous ethanol replaces the sample as a blank control.

[0047] The scavenging ability of •OH free radicals in the fermentation broth of endophytic fungus WWZZ01 from Schisandra chinensis is shown in Figure 5 .

[0048] from Figure 4 and Figure 5 It can be seen that the scavenging ability of DPPH free radicals and •OH free radicals is related to the concentration of the fermentation broth, and the concentration of the fermentation broth of the endophytic fungus WWZZ01 in Schisandra chinensis is positively correlated with the antioxidant activity. The above results indicate that the fermentation broth of the endophytic fungus WWZZ01 in Schisandra chinensis has good antioxidant activity.

[0049] Example 4: HPLC analysis of fermentation broth of endophytic fungus WWZZ01

[0050] Activation of endophytic fungus WWZZ01: Take endophytic fungus WWZZ01, inoculate it on the surface of PDA culture medium under sterile conditions, and culture it at 28 ℃ for 3 days for later use.

[0051] Preparation of fermentation broth of endophytic fungus WWZZ01: Take the activated endophytic fungus WWZZ01, pick the hyphae and inoculate them into 50 mL PDB medium under sterile conditions, and culture them in a shaking incubator at 28 °C and 120 r / min for 3 days to make 1×10 7 CFU / mL of seed liquid; transfer the seed liquid to 3L sterilized PDB medium at a 20% (v / v) inoculation rate, culture at 28°C, 120 r / min shaking for 14 days, filter and collect the fermentation liquid.

[0052] 1. Preparation of test solution

[0053] Take 300 mL of the fermentation broth of endophytic fungus WWZZ01 and concentrate it under reduced pressure at 50 °C. Extract the concentrated fermentation broth with ethyl acetate three times (3×100 mL). Combine the extracts, concentrate to dryness at 50 °C, and dissolve it in 1 mL of methanol as the test solution for later use.

[0054] 2. Preparation of reference solution

[0055] Accurately weigh 1 mg of Schisandrin A reference substance, place it in a 1 mL centrifuge tube, add 1 mL of chromatographic methanol to dissolve it, and prepare the reference substance solution for later use.

[0056] 10 g of Schisandra chinensis seed powder was added to 100 mL of ethyl acetate, extracted with ultrasonic wave for 1 h, filtered, and the solvent was recovered to 10 mL for use as a reference.

[0057] Take 300 mL of PDB culture medium and concentrate it under reduced pressure at 50°C. Extract the concentrated PDB culture medium with ethyl acetate three times (3×30 mL). Combine the extracts, concentrate to dryness at 50°C, dissolve them in 1 mL of methanol and use them as blank control solution for later use.

[0058] 3. HPLC conditions

[0059] Chromatographic column: Venusil XBP-C 18 Column (4.6 mm × 250 mm, 5 μm, USA) Mobile phase: methanol: acetonitrile: water (32:38:30); Flow rate: 1 mL•min - 1 ; Column temperature: 25℃; Detection wavelength: 250 nm; Injection volume: 10 μL.

[0060] The fermentation broth of endophytic fungus WWZZ01 of Schisandra chinensis was analyzed by HPLC. Figure 6 shown. Figure 6 A is the fermentation liquid of WWZZ01; B is schisandra alcohol A; C is the crude drug of Schisandra chinensis seeds; and D is the PDB blank control. Figure 6 It can be seen that the fermentation broth of the endophytic fungus WWZZ01 of Schisandra chinensis has the same chromatographic peak at the same retention time as the schisandrin A reference substance, and the blank control (PDB) has no interference, indicating that the fermentation broth of the fungus may contain schisandrin A.

[0061] Example 5: Extraction and separation of schisandra alcohol A from the fermentation broth of endophytic fungus WWZZ01 of Schisandra chinensis

[0062] According to the HPLC analysis results, the fermentation broth of the endophytic fungus WWZZ01 may contain schisandrin A, so a separation process was designed specifically and crystalline A was separated from its fermentation broth.

[0063] Take 5L of fermentation broth of endophytic fungus WWZZ01, concentrate it to 300mL under reduced pressure, extract it with ethyl acetate three times (3×100mL), combine the ethyl acetate extracts, recover the solvent, sample for silica gel column chromatography, and elute with petroleum ether-ethyl acetate gradient (15:1, 10:1, 4:1, 2:1), collect the petroleum ether-ethyl acetate (2:1) elution fraction (10mL / bottle), combine it, recover the solvent, separate it with AB-8 macroporous resin column, and elute with water, 10% and 20% ethanol respectively, collect the 20% ethanol eluate, recover the solvent, and recrystallize it with ether-ethyl acetate to obtain crystal A.

[0064] Crystal A: White powder, soluble in methanol, ethanol, DMSO and other organic solvents. 1 H- NMR (400MHz, MeOD) δ: 6.74 (s, 1H, PhH-4), 6.69 (s, 1H, PhH-11), 3.89 (d, J = 11.8 Hz, 6H, 2×OCH3-3, 12), 3.83 (d, J = 1.1 Hz, 6H, 2×OCH3-2, 13), 3.46 (d, J = 3.2 Hz, 6H, 2×OCH3-1,14), 2.85 (dd, J = 14.2, 2.3 Hz, 1H, CH H -9), 2.57 (d, J = 13.5 Hz, 1H, C H H-6), 2.40 (dd, J = 13.9, 7.4 Hz, 2H, C H H-9), 1.22 (s, 3H, CH3-18), 0.83 (d, J = 7.2 Hz, 3H, CH3-17). 1 The results of H-NMR analysis are as follows Figure 7 shown.

[0065] After HPLC and 1 After H-NMR analysis, it can be seen that the chemical formula of crystal A is C 24 H 32 O7, and based on its specific physical and chemical properties and reference to literature, it was finally determined that crystal A was schisandra alcohol A.

Claims

1. An endophytic fungus of Schisandra chinensis WWZZ01 producing Schisandrin A, characterized in that: The endophytic fungus WWZZ01 of Schisandra chinensis is Geotrichum candidum WWZZ01, which has been deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan. The deposit date is July 18, 2024, and the deposit number is CCTCC NO: M20241605.

2. Use of the endophytic fungus WWZZ01 of Schisandra chinensis as claimed in claim 1 in fermentation to produce Schisandra chinensis secondary metabolites; the Schisandra chinensis secondary metabolite is schisandrin A.

3. Use of the endophytic fungus WWZZ01 of Schisandra chinensis as claimed in claim 1 in in vitro antioxidant activity.

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