An endophytic fungus strain HASZ2 from Acanthopanax senticosus and its application

Abstract

A strain of Acanthopanax senticosus endophytic fungus HASZ2 producing isofraquinone and protocatechuic acid and its application, relating to the field of microorganisms, the Acanthopanax senticosus endophytic fungus HASZ2 is Rhodotorula glutinosus HASZ2, which has been preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, the preservation date is May 16, 2024, and the preservation number is CCTCC NO: M2024973. The strain has good antioxidant activity. After secondary metabolite analysis of its fermentation broth, it was found that the strain can produce isofraquinone and protocatechuic acid. The present invention is used for anti-oxidation and production of Acanthopanax senticosus secondary metabolites.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to an endophytic fungus HASZ2 of Acanthopanax senticosus producing isoflurane and protocatechuic acid and an application thereof. Background Art

[0002] Acanthopanax belongs to the genus Acanthopanax of the family Acanthopanax and is a traditional medicinal plant. The effective ingredients of Acanthopanax include protocatechuic acid, eleutheroside B, eleutheroside E, chlorogenic acid, rutin, hyperoside, syringin, saponin, polysaccharides, etc. The glycoside compounds in the rhizome of Acanthopanax can improve the phenomenon of myocardial ischemia in the human body and the local damage caused by it. The glycosides and flavonoids rich in Acanthopanax have a wide range of effects on the central nervous system. Acanthopanax can regulate the excitation and inhibition processes of the central nervous system, improve the blood supply to the brain, and promote brain cell metabolism and repair. Acanthopanax glycoside E, eleuthero polysaccharides, flavonoids, terpenoids and coumarins in Acanthopanax are all natural anti-ultraviolet protective agents. The glycosides and polysaccharides in Acanthopanax also have anti-tumor effects.

[0003] At present, the separation and extraction of Acanthopanax senticosus secondary metabolites from Acanthopanax senticosus plant tissues requires a large amount of Acanthopanax senticosus raw materials, resulting in high costs and is not conducive to industrial production. Summary of the invention

[0004] The invention aims to solve the problem that the existing method for extracting secondary metabolites of Acanthopanax senticosus has high cost and is not conducive to industrial production, and provides an Acanthopanax senticosus endophytic fungus HASZ2 producing isoflurane and protocatechuic acid and its application.

[0005] The present invention provides an endophytic fungus HASZ2 of Acanthopanax senticosus that produces isoflurane and protocatechuic acid, which is a member of the group consisting of Rhodotorula glutinosae ( Rhodotorula mucilaginosa )HASZ2 has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is May 16, 2024, and the deposit number is CCTCC NO: M2024973.

[0006] The colony of the Acanthopanax senticosus endophytic fungus HASZ2 of the present invention is orange-red, the hyphae are sticky, and the matrix is ​​light pink.

[0007] The ITS rDNA sequence of the Acanthopanax senticosus endophytic fungus HASZ2 of the present invention was submitted to the NCBI database, and BLAST analysis and comparison were performed. The strain sequences with high homology of the Acanthopanax senticosus endophytic fungus HASZ2 sequence were analyzed using MEGA7 software. Rhodotorula mucilaginosa (MT378424.1) reached 100%. The endophytic fungus HASZ2 of Acanthopanax senticosus was identified as Rhodotorula glutinosus ( Rhodotorula mucilaginosa ).

[0008] The invention also provides application of Acanthopanax senticosus endophytic fungus HASZ2 in fermentation to produce isoflurane and protocatechuic acid.

[0009] The present invention also provides the use of Acanthopanax senticosus endophytic fungus HASZ2 in in vitro anti-oxidation.

[0010] Beneficial effects of the present invention:

[0011] The present invention isolates an endophytic fungus HASZ2 from Acanthopanax senticosus, which is identified as Rhodotorula glutinosus. The strain has good antioxidant activity. After the secondary metabolites of the fermentation broth are analyzed, it is found that the strain can produce isofraquinone and protocatechuic acid. Among them, isofraquinone is one of the coumarin components contained in Acanthopanax senticosus, and is also an indicator component of Acanthopanax senticosus. Isofraquinone has the effects of anti-liver cancer, inhibiting the proliferation of human colorectal cancer cells, and treating neurodegenerative diseases, and has a protective effect on lipid metabolism disorders and non-alcoholic fatty liver induced by a high-fat diet, and is widely used in the fields of medicine, food, and health products. Protocatechuic acid, also known as 3,4-dihydroxybenzoic acid, is a phenolic acid substance contained in Acanthopanax senticosus, and has antibacterial, anti-inflammatory, antioxidant, anti-tumor and other active effects. Microbial fermentation produces Acanthopanax secondary metabolites. Since the microorganisms reproduce rapidly and the product extraction is simple, the cost is low and it is suitable for industrial production. Since Acanthopanax senticosus is a Class III endangered plant under national protection, the secondary metabolites of Acanthopanax senticosus from microbial sources are of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the colony morphology of the endophytic fungus HASZ2 in Acanthopanax senticosus;

[0013] Figure 2 This is the hyphae morphology of the endophytic fungus HASZ2 of Acanthopanax senticosus;

[0014] Figure 3 This is the phylogenetic tree of the endophytic fungus HASZ2 from Acanthopanax senticosus;

[0015] Figure 4 The scavenging ability of the fermentation liquid of Acanthopanax senticosus endophytic fungus HASZ2 on DPPH free radicals;

[0016] Figure 5 The HPLC analysis results of isoflurane in the fermentation broth of endophytic fungus HASZ2;

[0017] Figure 6 The results of HPLC analysis of protocatechuic acid in the fermentation broth of endophytic fungus HASZ2;

[0018] Figure 7 For crystal A 1 H NMR spectroscopy;

[0019] Figure 8 For crystal B1 H NMR spectrum. DETAILED DESCRIPTION

[0020] 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.

[0021] Example

[0022] The endophytic fungus HASZ2 of Acanthopanax senticosus in this example is Rhodotorula glutinosus ( Rhodotorula mucilaginosa )HASZ2 has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is May 16, 2024, and the deposit number is CCTCC NO: M2024973.

[0023] The method for obtaining the Acanthopanax senticosus endophytic fungus HASZ2 in this embodiment is as follows:

[0024] Acanthopanax senticosus Acanthopanax seuticosus (Rupr. et Maxim.) Harms stems (this Acanthopanax senticosus was collected from Mishan City, Heilongjiang Province), surface disinfection, disinfection procedure: soak the seeds in 75% alcohol for 30 seconds, then wash with sterile water, rinse in sodium hypochlorite for 10 minutes, wash with sterile water, then soak in 75% alcohol for 30 seconds, and finally wash with sterile water three times.

[0025] Use a sterile scalpel to remove the outer skin of the disinfected stem and cut it in the middle. Place the cut surface in PDA culture medium and culture it at a constant temperature of 28℃ for 5~7 days.

[0026] The last sterile water wash of the sterilization procedure was removed and inoculated into the PDA medium. The sterilized experimental materials without cutting were placed on the PDA medium, rolled once, and then taken out and cultured together with the endophytic fungus isolation medium as a control.

[0027] Preparation of PDA culture medium: Peel 200g of potatoes, cut into small pieces, add into boiling water and boil for 30min, filter (8 layers of gauze), add 20g agar and 20g glucose in small amounts several times, stir, and adjust the volume to 1000mL after dissolution.

[0028] The endophytic fungus HASZ2 was isolated from the stem of Acanthopanax senticosus. No bacteria grew in the negative control plate and the negative control liquid culture medium, and this was repeated many times, thus proving that the isolated bacteria were endophytic fungi of Acanthopanax senticosus.

[0029] Example 2: Identification of the endophytic fungus HASZ2 in Acanthopanax senticosus

[0030] The strains were identified by morphological observation and ITS rDNA sequence analysis.

[0031] Colony morphology of the endophytic fungus HASZ2 in Acanthopanax senticosus Figure 1 As shown in the figure, the mycelial morphology is as follows Figure 2 As shown in the figure, the colony of the endophytic fungus HASZ2 of Acanthopanax senticosus is orange-red, the hyphae are sticky, and the matrix is ​​light pink.

[0032] The ITS rDNA sequence (591 bp) of the Acanthopanax senticosus endophytic fungus HASZ2 of the present invention was submitted to the NCBI database, and BLAST analysis and comparison were performed. The phylogenetic tree of the endophytic fungus HASZ2 was constructed using the MEGA7 software. Figure 3 Endophytic fungus HASZ2 and Acanthopanax senticosus Rhodotorula mucilaginosa (MT378424.1) reached 100%. The endophytic fungus HASZ2 of Acanthopanax senticosus was identified as Rhodotorula glutinosus ( Rhodotorula mucilaginosa ).

[0033] The ITS rDNA sequence of the endophytic fungus HASZ2 from Acanthopanax senticosus is as follows:

[0034] .

[0035] Example 3: Analysis of Antioxidant Activity of Acanthopanax Senticosus Endophytic Fungus HASZ2

[0036] Activation of endophytic fungus HASZ2: Take endophytic fungus HASZ2, inoculate it on the surface of PDA culture medium under sterile conditions, and culture it at 28°C for 3 days for later use.

[0037] Preparation of fermentation broth of endophytic fungus HASZ2: Take the activated endophytic fungus HASZ2, pick the hyphae under sterile conditions and inoculate them into 50 mL PDB medium, 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 inoculation rate of 20% (v / v), and cultured in a shaking incubator at 28°C and 120r / min for 14d, the fermentation broth was collected, concentrated under reduced pressure, extracted with ethyl acetate (3×100mL), and concentrated at low temperature to an extract, and dried to obtain the fermentation broth powder of the endophytic fungus HASZ2 for later use.

[0038] 1. Determination of DPPH free radical scavenging ability

[0039] Dissolve 5.9 mg DPPH in methanol and dilute to 100 mL to prepare a solution with a mass concentration of 0.15 mmol•L -1 DPPH solution; 2 mg of Vc was dissolved in methanol and the volume was adjusted to 10 mL to obtain 200 μg•mL -1 of reference solution;

[0040] The fermentation broth powder of Acanthopanax senticosus endophytic fungus HASZ2 was dissolved in an appropriate amount of methanol to prepare a 200 μg•mL -1 of the test sample solution.

[0041] 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:

[0042]

[0043] 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.

[0044] The scavenging ability of the fermentation liquid of Acanthopanax senticosus endophytic fungus HASZ2 on DPPH free radicals is shown in Figure 4 .

[0045] 2. Determination of OH radical scavenging ability

[0046] 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 -1The 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:

[0047]

[0048] 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.

[0049] from Figure 4 It can be seen that the scavenging rates of DPPH free radicals and •OH free radicals are related to the concentration of the fermentation liquid, and the concentration of the fermentation liquid of the endophytic fungus HASZ2 from Acanthopanax senticosus is positively correlated with the antioxidant capacity. The experimental results show that the fermentation liquid of the endophytic fungus HASZ2 from Acanthopanax senticosus has good antioxidant activity.

[0050] When the concentration of the fermentation broth of Acanthopanax senticosus endophytic fungus HASZ2 was 100 μg mL -1 When the scavenging rate of DPPH free radicals was 66.24%, the half clearance rate (IC 50 value) reached 40.62μg·mL -1 ; The scavenging rate of •OH free radicals reached 70.16%, and the half scavenging rate (IC 50 value) reached 37.46μg·mL -1 .

[0051] Example 4: HPLC analysis of fermentation broth of Acanthopanax senticosus endophytic fungus HASZ2

[0052] Activation of endophytic fungus HASZ2: Take endophytic fungus HASZ2, inoculate it on the surface of PDA culture medium under sterile conditions, and culture it at 28 ℃ for 3 days for later use.

[0053] Preparation of fermentation broth of endophytic fungus HASZ2: Take the activated endophytic fungus HASZ2, 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, collect the fermentation liquid and set aside.

[0054] (1) Preparation of test solution

[0055] Take 300 mL of the fermentation broth of the endophytic fungus HASZ2 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.

[0056] (2) Preparation of reference solution

[0057] Accurately weigh 1 mg each of isoflurane and protocatechuic acid reference substances, place them in a 1 mL centrifuge tube, add 1 mL of chromatographic methanol to dissolve, and prepare reference substance solutions for later use.

[0058] 10 g of Acanthopanax senticosus stem powder was dissolved in 100 mL of ethyl acetate solution, extracted by ultrasonic for one hour, filtered, and the solution was concentrated to 10 mL of Acanthopanax senticosus solution under low temperature environment, which was used as a reference substance.

[0059] 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 for 3 times (3×100 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.

[0060] (3) HPLC conditions

[0061] Chromatographic column: Venusil XBP-C 18 Column (4.6 mm × 250 mm, 5 μm, USA) Mobile phase: methanol: water (30:70) 0.2% phosphoric acid; Flow rate: 1 mL•min -1 ; Column temperature: 25℃; Detection wavelength: 254nm; Injection volume: 10μL.

[0062] The fermentation broth of endophytic fungus HASZ2 was analyzed by HPLC. Figure 5 , 6 shown.

[0063] Depend on Figure 5 It can be seen that the fermentation broth of the endophytic fungus HASZ2 from Acanthopanax senticosus has the same chromatographic peak as the isofraquinone reference substance at the same retention time, and the blank control (PDB) has no interference, indicating that the fermentation broth of the fungus may contain isofraquinone. In order to further confirm that the secondary metabolite contained in it is isofraquinone, three different HPLC mobile phase systems were used to analyze it, and the chromatographic peak was also well confirmed by the standard addition method. The results showed that the fermentation broth of the endophytic fungus HASZ2 from Acanthopanax senticosus contains isofraquinone.

[0064] Depend on Figure 6It can be seen that the fermentation broth of the endophytic fungus HASZ2 from Acanthopanax senticosus has the same chromatographic peak as the protocatechuic acid reference substance at the same retention time, and the blank control (PDB) has no interference, indicating that the fermentation broth of the fungus may contain protocatechuic acid. In order to further confirm that the secondary metabolite contained in it is protocatechuic acid, three different HPLC mobile phase systems were used to analyze it, and the chromatographic peak was also well confirmed by the standard addition method. The results showed that protocatechuic acid was contained in the fermentation broth of the endophytic fungus HASZ2 from Acanthopanax senticosus.

[0065] Example 5: Separation of isoflurane and protocatechuic acid from the fermentation broth of Acanthopanax senticosus endophytic fungus HASZ2

[0066] According to the HPLC analysis results of the fermentation broth of the endophytic fungus HASZ2 from Acanthopanax senticosus, a separation process was designed for isofraxidin and chlorogenic acid contained therein.

[0067] Take 10L of HASZ2 strain fermentation broth, concentrate it to 200mL under reduced pressure at 50℃, extract it with ether 3 times (3×100mL), and combine the extracts. Concentrate the extract to dryness under reduced pressure at 50℃ to obtain the ether extraction part. Separate the ether extraction part by silica gel column, elute with petroleum ether-ethyl acetate (5:1) and petroleum ether-ethyl acetate (1:1), collect the eluent (10mL / bottle), combine the 1-36 components of the petroleum ether-ethyl acetate (5:1) eluent, and obtain crude product A; separate crude product A by silica gel column again, elute with chloroform-methanol (2:1), collect the eluent, recover the solvent, and obtain crude crystal A. Refrigerate the crude crystal A at 4℃ for 24h to precipitate crystal A; combine the 52-88 components of the petroleum ether-ethyl acetate (1:1) eluent, recrystallize with ethanol, and obtain crystal B.

[0068] Crystal A: Yellow oily crystalline substance, easily soluble in methanol, soluble in acetone, ethanol, chloroform, methanol and other organic solvents. 1 H-NMR (400MHz, MeOD) δppm: 7.43 (1H, s, H-2), 7.41 (1H, d, J = 2.1Hz, H-6), 6.80 (2H, dd, J = 7.8Hz, H-5), there are three substituents on the benzene ring, the results are as follows Figure 7 shown.

[0069] After HPLC and 1 After H-NMR identification, it was found that the chemical formula of crystal A was C7H6O4, and based on its specific physical and chemical properties and reference to literature, it was finally determined that crystal A was protocatechuic acid.

[0070] Crystal B: Pale yellow needle crystal, easily soluble in methanol, ethanol, chloroform, soluble in organic solvents such as ether. Crystal B 1H-NMR (400MHz, MeOD) δppm: 7.26 (d, J=9.5Hz, 1H, H-4), 6.33 (s, J=9Hz, 1H, H-5), 4.30 (s, 6H), 3.36 (s, 3H, C8-OCH3), 3.31 (s, 3H, C6-OCH3), the results are as follows Figure 8 shown.

[0071] After HPLC and 1 After H-NMR identification, it was found that the chemical formula of crystal B is C 11 H 10 O2, and based on its specific physical and chemical properties and reference to literature, it was finally determined that crystal B was isoflurane.

Claims

1. An endophytic fungus of Acanthopanax senticosus HASZ2, characterized in that: The Acanthopanax endophytic fungus HASZ2 is Rhodotorula mucilaginosa HASZ2, which has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan City, the deposit date is May 16, 2024, and the deposit number is CCTCC NO: M 2024973.

2. Use of the Acanthopanax senticosus endophytic fungus HASZ2 as claimed in claim 1 in in vitro antioxidant activity.

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