Acanthopanax inborn bacteria hasx4 producing kaempferol and isofraxidin and application thereof
By isolating and identifying the endophytic bacterium HASX4 of Acanthopanax senticosus, kaempferol and isozymine were produced by microbial fermentation, which solved the problem that secondary metabolites of Acanthopanax senticosus are not conducive to industrial production, and achieved efficient production and sustainable utilization of resources.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HEILONGJIANG UNIV
- Filing Date
- 2024-08-05
- Publication Date
- 2026-08-04
AI Technical Summary
Existing methods for extracting secondary metabolites from Acanthopanax senticosus are not conducive to industrial production, and the resources of Acanthopanax senticosus are endangered and in high demand.
An endophytic bacterium, HASX4, producing kaempferol and isozymine was isolated and identified, and produced kaempferol and isozymine through microbial fermentation.
The strain has achieved efficient production of secondary metabolites of Acanthopanax senticosus, which is suitable for industrial application. The strain also has good antioxidant activity, and the products have multiple nutritional and health benefits.
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Figure CN118956664B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, specifically to an endophytic bacterium HASX4 from Acanthopanax senticosus that produces kaempferol and isozymine, and its applications. Background Technology
[0002] Eleutherococcus senticosus, the dried root, rhizome, or stem of *Acanthopanax seuticosus* (Rupr. et Maxim.) Harms, a plant in the Araliaceae family, is a key medicinal herb for treating cold-natured diseases. It is believed to invigorate qi and spleen, tonify the kidneys, and calm the mind. Eleutherococcus senticosus is rich in glycosides, flavonoids, polysaccharides, trace elements, and amino acids, among other active chemical components. It possesses anti-fatigue, anti-inflammatory, anti-tumor, antihypertensive, hepatoprotective, and hypoglycemic effects. Furthermore, it can regulate bodily dysfunction, improve cardiovascular microcirculation, improve myocardial ischemia, and enhance the body's immune function. It is now widely used in the pharmaceutical and food industries.
[0003] Acanthopanax senticosus is mainly produced in Heilongjiang Province. The cold, black soil of the region has created its unique quality, making it a leading variety of medicinal herbs from cold regions, renowned both domestically and internationally. However, due to rising demand, decreasing forest area, and indiscriminate harvesting, the resources of Acanthopanax senticosus are facing challenges. Currently, it is a Class II endangered species and a Class III key protected species in China.
[0004] Given the endangered status and quality advantages of Acanthopanax senticosus, research on various aspects of Acanthopanax senticosus has become a current hot topic. The extraction of secondary metabolites from Acanthopanax senticosus plant tissues requires a large amount of raw materials, which is not conducive to industrial production. Summary of the Invention
[0005] The present invention aims to solve the problem that existing methods for extracting secondary metabolites of Acanthopanax senticosus are not conducive to industrial production, and provides an endophytic bacterium HASX4 of Acanthopanax senticosus that produces kaempferol and isozymine, and its application.
[0006] This invention provides an endophytic bacterium, HASX4, that produces kaempferol and isozymine, specifically Pantoea agglomerans HASX4. It has been deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, on May 16, 2024, with accession number CCTCC NO: M 2024971.
[0007] The colonies of the endophytic bacteria HASX4 from Acanthopanax senticosus of this invention are light yellow and moist in texture. The bacterial cells are rod-shaped and have flagella.
[0008] The endophytic bacterium HASX4 of Acanthopanax senticosus of this invention is a facultative anaerobic bacterium. It is Gram-negative, positive for starch hydrolysis, positive for gelatin liquefaction, positive for catalase, positive for biofilm formation, and positive for VP test. Its optimal pH is 6, its optimal growth temperature is 37°C, and its optimal salt tolerance is 4%.
[0009] The endophytic bacterium HASX4 of Acanthopanax senticosus in this invention showed 100% similarity to Pantoea agglomerans (CP034469.1) through 16S rDNA sequence alignment analysis. Based on the bacterial morphology characteristics, growth conditions, and physiological and biochemical identification results, the endophytic bacterium HASX4 of this invention was determined to be Pantoea agglomerans.
[0010] This invention also provides the application of the endophytic bacterium HASX4 of Acanthopanax senticosus in the fermentation production of kaempferol and isozymine.
[0011] This invention also provides the application of the endophytic bacterium HASX4 of Acanthopanax senticosus in in vitro antioxidant activity.
[0012] The beneficial effects of this invention are:
[0013] This invention isolated an endophytic bacterium, HASX4, from Acanthopanax senticosus, which was identified as a cluster of pantothenic bacteria. This strain exhibits good antioxidant activity. Analysis of its fermentation broth revealed that the strain produces kaempferol and isozydin. Kaempferol is a flavonoid compound with antioxidant, anti-inflammatory, and anticancer properties, and is beneficial for preventing and treating diabetes, atherosclerosis, and osteoporosis, as well as protecting nerves, liver, and myocardium, and inhibiting protein kinases. Isozydin is a hydroxycoumarin monomer and one of the key components of Acanthopanax senticosus. Isozydin has anti-liver cancer, inhibits the proliferation of human colorectal cancer cells, treats neurodegenerative diseases, and protects against high-fat diet-induced lipid metabolism disorders and non-alcoholic fatty liver disease.
[0014] This invention enables the production of secondary metabolites of Acanthopanax senticosus through microbial fermentation. Due to the rapid reproduction of microorganisms and the simplicity of product extraction, it is suitable for industrial production. This provides a new method for producing the active ingredients isozymine and kaempferol from Acanthopanax senticosus using microbial fermentation. Attached Figure Description
[0015] Figure 1 A colony photograph of the endophytic bacterium HASX4 in Acanthopanax senticosus;
[0016] Figure 2 Photograph of the endophytic bacteria HASX4 from Acanthopanax senticosus;
[0017] Figure 3Phylogenetic tree of the endophytic bacterium HASX4 of Acanthopanax senticosus;
[0018] Figure 4 The ability of the fermentation broth of Acanthopanax senticosus endophytic bacteria HASX4 to scavenge DPPH free radicals;
[0019] Figure 5 The ability of the fermentation broth of Acanthopanax senticosus endophytic bacteria HASX4 to scavenge ·OH free radicals;
[0020] Figure 6 The results of HPLC analysis of isozymine in the fermentation broth of Acanthopanax senticosus endophytic bacteria HASX4;
[0021] Figure 7 HPLC analysis results of kaempferol in the fermentation broth of Acanthopanax senticosus endophytic bacteria HASX4;
[0022] Figure 8 For crystal A 1 H nuclear magnetic resonance spectrum;
[0023] Figure 9 For crystal B 1 H nuclear magnetic resonance spectrum. Detailed Implementation
[0024] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.
[0025] Example 1:
[0026] The endophytic bacterium HASX4 of Acanthopanax senticosus in this embodiment is Pantoea agglomerans HASX4, which has been deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, on May 16, 2024, with accession number CCTCC NO: M 2024971.
[0027] The method for obtaining the endophytic bacteria HASX4 from Acanthopanax senticosus in this embodiment is as follows:
[0028] Take the leaves of healthy Acanthopanax seuticosus (Rupr. et Maxim.) Harms, a plant of the Araliaceae family (this Acanthopanax was collected from Mishan City, Heilongjiang Province). Disinfect the surface by the following procedure: soak the leaves in 75% alcohol for 30 seconds, then wash with sterile water, rinse in sodium hypochlorite for 6-8 minutes, wash with sterile water, soak in 75% alcohol for 30 seconds, and finally wash three times with sterile water.
[0029] Use a sterile scalpel to cut the disinfected leaves into 1cm pieces.2 Thin slices were embedded in NA medium and incubated at 37°C for 1–3 days. The final sterile wash solution from the disinfection procedure was spread onto NA medium. Uncut, sterilized experimental materials were placed on NA medium, rolled once, and then removed and co-cultured with endophytic bacterial isolation medium as a control.
[0030] Preparation of NA culture medium: Add 6g beef extract, 20g peptone, 10g NaCl and 20g agar powder to boiling water in sequence. After the agar powder is completely dissolved, add water to make up to 1L, adjust the pH to 7.0-7.2, dispense into containers, put them in an autoclave, sterilize at 121℃ for 30min, cool to room temperature, and store for later use.
[0031] Endophytic bacteria HASX4 were isolated from Acanthopanax senticosus leaves. No bacteria grew in the negative control plates and negative control culture medium. This was repeated multiple times, thus proving that the isolated bacteria were endophytic bacteria of Acanthopanax senticosus.
[0032] Example 2: Identification of the endophytic bacterium HASX4 in Acanthopanax senticosus
[0033] 1. Morphological characteristics of the endophytic bacterium HASX4 in Acanthopanax senticosus
[0034] The colonies of the endophytic bacterium HASX4 in Acanthopanax senticosus are pale yellow and moist. The bacteria are rod-shaped and have flagella. A colony image of the endophytic bacterium HASX4 in Acanthopanax senticosus is shown below. Figure 1 As shown in the image, the bacterial cell photograph is as follows: Figure 2 As shown.
[0035] 2. Physiological and biochemical characteristics of the endophytic bacterium HASX4 in Acanthopanax senticosus
[0036] The endophytic bacteria HASX4 of Acanthopanax senticosus is a facultative anaerobic bacterium. It is Gram-negative, positive for starch hydrolysis, gelatin liquefaction, catalase, biofilm formation, and VP test. Its optimal pH is 6, optimal growth temperature is 37℃, and optimal salt tolerance is 4%.
[0037] 3. Molecular identification of the endophytic bacterium HASX4 in Acanthopanax senticosus
[0038] PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3') for the bacterial 16S rDNA gene. The product was examined by 1.0% agarose gel electrophoresis, and an amplified fragment was obtained at 1406 bp. The 16S rDNA sequence of strain HASX4, an endophytic bacterium of Acanthopanax senticosus, was sequenced by Shanghai Sangon Biotech Co., Ltd., and is shown in SEQ ID NO: 1 in the sequence listing. The sequencing results were submitted to the NCBI database, analyzed using BLAST and compared, and then a phylogenetic tree of the endophytic bacterium HASX4 was constructed using MEGA 7 software. The phylogenetic tree construction results of Acanthopanax senticosus endophytic bacterium HASX4 are shown in [link to phylogenetic tree]. Figure 3 The endophytic bacteria HASX4 of Acanthopanax senticosus showed 100% similarity to Pantoea agglomerans (CP034469.1). Based on the combination of bacterial morphological characteristics, growth conditions, and physiological and biochemical identification results, it was determined that the endophytic bacteria HASX4 of Acanthopanax senticosus is Pantoea agglomerans.
[0039] Example 3: Antioxidant Activity Analysis of the Endophytic Bacterium HASX4 of Acanthopanax senticosus
[0040] Activation of endophytic bacteria HASX4: Take endophytic bacteria HASX4 and inoculate it onto the surface of NA medium under aseptic conditions. Incubate at 37°C for 1 day for later use.
[0041] Preparation of fermentation broth for endophytic bacteria HASX4: Activated endophytic bacteria HASX4 were used. Mycelia were picked and inoculated into 50 mL of NB medium under aseptic conditions. The medium was then cultured in a shaker at 37℃ and 120 r / min for 3 days to prepare 1×10⁻⁶ fermentation broth. 7 CFU / mL seed culture; the seed culture was transferred to 3L of sterilized NB medium at an inoculation rate of 20% (v / v), and cultured on a shaker at 37℃ and 120r / min for 7 days. The fermentation broth was collected, concentrated under reduced pressure, extracted with ethyl acetate (3×100mL), and concentrated at low temperature to a paste state. After drying, the endophytic bacteria HASX4 fermentation broth powder was obtained for later use.
[0042] 1. Determination of DPPH free radical scavenging ability
[0043] Accurately weigh 5.9 mg of DPPH, dissolve it in a certain amount of methanol, and bring the volume to 100 mL. Shake well to prepare a solution with a mass concentration of 0.15 mmol·L⁻¹. -1 DPPH solution; accurately weigh 2 mg of vitamin C, dissolve it in a certain amount of methanol, and then dilute to 20 mL to obtain 100 μg / mL. -1 The reference solution;
[0044] Dissolve the above-mentioned Acanthopanax senticosus endophytic bacteria HASX4 fermentation broth powder in an appropriate amount of methanol to prepare a concentration of 200 μg·mL. -1 The test solution.
[0045] The above reference standard and test solution were diluted sequentially to a concentration gradient of 100 μg / mL. -1 50 μg·mL -1 25 μg·mL -1 12.5 μg·mL -1 6.25 μg·mL -1 Pipette 2 mL of each solution into a test tube, with methanol serving as a blank control. Add 0.15 mmol·L⁻¹ to each test tube. -1 4 mL of DPPH solution was added and placed in the dark for 30 min. The absorbance of each sample was measured at 517 nm (n=3). The formula for calculating the DPPH free radical scavenging rate is as follows:
[0046]
[0047] In the formula, A i Indicates the absorbance value when the test sample and DPPH solution are added simultaneously; A j A0 indicates the absorbance when the test sample and blank control are added simultaneously; A0 indicates the absorbance when the blank control and DPPH solution are added simultaneously.
[0048] The scavenging ability of the endophytic bacteria HASX4 fermentation broth of Acanthopanax senticosus on DPPH free radicals is as follows: Figure 4 As shown.
[0049] 2. Determination of ·OH free radical scavenging ability
[0050] Take 2 mL of each of the above-mentioned test solutions of different concentrations and place it in a test tube. Add 2 mL of 6 mmol·L⁻¹ solution to each tube. -1 FeSO4, 2 mL 6 mmol·L -1 Add H2O2, shake to mix well, let stand for 10 min, then add 2 mL of 6 mmol·L⁻¹ H₂O₂. -1 The salicylic acid-ethanol solution was allowed to stand at 37℃ for 30 min, and the absorbance of each sample was measured at 510 nm (n=3). The formula for calculating the ·OH free radical scavenging rate is as follows:
[0051]
[0052] In the formula, A1 represents the absorbance value of the added sample; A2 represents the absorbance value of distilled water instead of H2O2 to eliminate the influence of the sample's own color; and A0 represents the absorbance value of anhydrous ethanol instead of the sample as a blank control.
[0053] The scavenging ability of the endophytic bacteria HASX4 fermentation broth of Acanthopanax senticosus against ·OH free radicals is as follows: Figure 5 As shown.
[0054] from Figure 4 and Figure 5 It can be seen that the scavenging rates of DPPH and ·OH radicals are related to the concentration of the fermentation broth, and the concentration of the fermentation broth of the endophytic bacterium HASX4 from Acanthopanax senticosus is positively correlated with the antioxidant capacity. The experimental results indicate that the fermentation broth of the endophytic bacterium HASX4 from Acanthopanax senticosus possesses good antioxidant activity.
[0055] When the concentration of the endophytic bacteria HASX4 fermentation broth of Acanthopanax senticosus was 100 μg·mL -1 At that time, the scavenging rate of DPPH free radicals was 64.26%, and the half-maximal scavenging rate (IC50) was 64.26%. 50 The value reached 41.708 μg·mL -1 The scavenging rate of ·OH free radicals reached 67.95%, and the half-maximal scavenging rate (IC50) was 100%. 50 The value reached 55.28 μg·mL -1 .
[0056] Example 4: HPLC analysis of fermentation broth of Acanthopanax senticosus endophytic bacteria HASX4
[0057] Activation of endophytic bacteria HASX4: Take endophytic bacteria HASX4 and inoculate it onto the surface of NA medium under aseptic conditions. Incubate at 37°C for 1 day for later use.
[0058] Preparation of fermentation broth for endophytic bacteria HASX4: Activated endophytic bacteria HASX4 were used. Mycelia were picked and inoculated into 50 mL of NB medium under aseptic conditions. The medium was then cultured in a shaker at 37℃ and 120 r / min for 3 days to prepare 1×10⁻⁶ fermentation broth. 7 CFU / mL seed culture; transfer the seed culture to 3L sterilized NB medium at an inoculation rate of 20% (v / v), and culture at 37℃ and 120r / min on a shaker for 7 days. Collect the fermentation broth for later use.
[0059] 1. Preparation of the test solution
[0060] Take 300 mL of endophytic bacteria HASX4 fermentation broth and concentrate it under reduced pressure at 50 °C. Extract the concentrated fermentation broth three times with ethyl acetate (3 × 100 mL). Combine the extracts and concentrate them to dryness at 50 °C. Dissolve the extracts in 1 mL of methanol to obtain the test solution for later use.
[0061] 2. Preparation of reference solution
[0062] Accurately weigh 1 mg each of isozymine and kaempferol reference standards, place them in a 1 mL centrifuge tube, add 1 mL of chromatographic methanol to dissolve them, and prepare a reference solution for later use.
[0063] Dissolve 10g of Acanthopanax senticosus leaf powder in 100mL of ethyl acetate solution, extract with ultrasound for 1 hour, filter, and concentrate the solution into 10mL of Acanthopanax senticosus solution at low temperature, which is used as a reference standard.
[0064] Take 300 mL of NB culture medium and concentrate it under reduced pressure at 50 °C. Extract the concentrated NB culture medium three times with ethyl acetate (3 × 100 mL). Combine the extracts and concentrate them to dryness at 50 °C. Dissolve the solution in 1 mL of methanol and use it as a blank control solution for later use.
[0065] 3. HPLC conditions
[0066] Column: Venusil XBP-C 18 Column (4.6 mm × 250 mm, 5 μm, USA) flow 1: methanol:water (45:55) 0.2% phosphoric acid; flow rate: 1 mL·min -1 Column temperature: 25℃; Detection wavelength: 254nm; Injection volume: 10μL. Flow 2: Methanol:water (60:40) 0.2% phosphoric acid; Flow rate: 1mL·min -1 Column temperature: 25℃; Detection wavelength: 290nm; Injection volume: 10μL.
[0067] The fermentation broth of endophytic bacteria HASX4 was analyzed by HPLC, and the results are as follows: Figure 6 , 7 As shown. Figure 6 In the table, A represents HASX4 fermentation broth, B represents isopyridine, C represents Acanthopanax senticosus leaf extract, and D represents NB blank control. Figure 6 It can be seen that the test sample of the endophytic bacterium HASX4 of Acanthopanax senticosus has the same chromatographic peak as the isozinc reference standard at the same retention time, and the blank control (NB) has no interference, indicating that the fermentation broth of this bacterium may contain isozinc. Figure 7 In the table, A represents HASX4 fermentation broth, B represents kaempferol, C represents Acanthopanax senticosus leaf extract, and D represents NB blank control. Figure 7 It can be seen that the test sample of the endophytic bacterium HASX4 of Acanthopanax senticosus has the same chromatographic peak as the kaempferol reference standard at the same retention time, and the blank control (NB) has no interference, indicating that the fermentation broth of this bacterium may contain kaempferol.
[0068] Example 5: Isolation of isozymine and kaempferol from the fermentation broth of Acanthopanax senticosus endophytic bacteria HASX4
[0069] Based on the HPLC analysis results of the fermentation broth of Acanthopanax senticosus endophytic bacteria HASX4, a separation process was designed for isozymine and kaempferol contained therein.
[0070] Take 10L of endophytic bacteria HASX4 fermentation broth and slowly pass it through D 101 A macroporous resin column was used for gradient elution with 30%, 50%, 70%, and 90% ethanol. The eluates were collected, and the solvent was recovered to obtain D. 101 Elution sites of macroporous resin column.
[0071] Take 30% D 101 The elution fraction of the macroporous resin column was separated by a polyamide column and eluted with a gradient of 30%, 50%, 70%, and 90% ethanol. The eluent was collected (10 mL / bottle), and fractions 150–208 in the 70% ethanol eluent were combined to obtain crude product A.
[0072] Crude product A was separated by silica gel column chromatography using petroleum ether-ethyl acetate (1:1) as the eluent. The eluent (10 mL / bottle) was collected, and fractions 120-180 were combined and recrystallized multiple times to obtain crystalline product A.
[0073] Crystal A: Yellow crystalline powder, needle-like crystals, readily soluble in methanol, ethanol, and chloroform, and soluble in organic solvents such as diethyl ether. 1 H-NMR (400MHz, MeOD) δ: 8.1-8.05 (m, 6H), 6.94-6.87 (m, 6H), 6.40 (d, J = 2.1Hz, 3H), 6.19 (d, J = 2.1Hz, 3H). For example... Figure 8 As shown.
[0074] According to HPLC and 1 H-NMR and comparison with data in the literature, physicochemical properties of crystal A and 1 The H-NMR results are completely consistent with those of kaempferol; therefore, crystal A is kaempferol.
[0075] Take 5 L of endophytic bacterial HASX4 fermentation broth, concentrate it to 200 mL under reduced pressure at 50 °C, and then extract it three times with diethyl ether (3 × 100 mL). Combine the extracts. Concentrate the extract to dryness under reduced pressure at 50 °C to obtain the diethyl ether extract fraction. Separate the diethyl ether extract fraction by silica gel column chromatography, eluting with petroleum ether-ethyl acetate (5:1) and petroleum ether-ethyl acetate (1:1), respectively. Combine fractions 46–92 of the petroleum ether-ethyl acetate (1:1) eluent, and recrystallize from ethanol to obtain crystal B.
[0076] Crystalline B: Pale yellow, readily soluble in methanol, ethanol, acetone, and chloroform; soluble in diethyl ether. 1¹H-NMR (400MHz, MeOD) δ: 7.76 (d, J = 9.5Hz, 1H), 6.13 (d, J = 9.4Hz, 1H, vinyl proton), 6.83 (s, 1H, C5-H), 3.85 (s, 3H, C6-OH3), 3.81 (s, 3H, C8-OH3). Results are shown in […]. Figure 9 .
[0077] According to HPLC and 1 H-NMR and comparison with data in the literature, physicochemical properties of crystal B and 1 The H-NMR results were completely consistent with those of isozincide, therefore, crystal B was identified as isozincide.
Claims
1. An endophytic bacterium HASX4 producing kaempferol and isozymine, characterized in that, The endophytic bacteria HASX4 of the acanthopanax is Pantoea agglomerans Pantoea agglomerans )HASX4, has been preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on May 16, 2024, and the preservation number is CCTCC NO: M 2024971.
2. The application of the endophytic bacterium HASX4 of Acanthopanax senticosus as described in claim 1 in the fermentation production of kaempferol and isozymine.
3. The application of the endophytic bacteria HASX4 of Acanthopanax senticosus as described in claim 1 in in vitro antioxidant activity.