A method for producing eleutheroside E and oleanolic acid by microbial fermentation

Abstract

A method for producing acantho-side E and oleanolic acid by microbial fermentation, relates to the field of microbial fermentation technology, and aims to solve the problem that the existing method for obtaining acantho-side E and oleanolic acid can destroy plant resources and cause environmental pollution. Method: I. Take the activated HASX4 after the formation of the seed liquid, expand the culture, and collect the fermentation broth; II. Take the fermentation broth and elute it through a macroporous resin column, collect the eluate, separate it through a silica gel column chromatography, collect the eluate, collect the components containing the same secondary metabolites, combine them, and treat them through recrystallization to obtain acantho-side E; III. Take the fermentation broth, concentrate it under reduced pressure, extract it, combine the extract, separate it through a silica gel column chromatography, collect the eluate, collect the components containing the same secondary metabolites, obtain the crude product, separate it through a silica gel column chromatography, collect the eluate, combine the same components, and treat them through recrystallization for multiple times to obtain oleanolic acid. The application is used for producing acantho-side E and oleanolic acid by microbial fermentation.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a method for producing eleutheroside E and oleanolic acid by microbial fermentation. Background Technology

[0002] Eleutherococcus senticosus (Rupr. et Maxim.) Harms, a plant belonging to the Araliaceae family, is a plant with dried roots, rhizomes, or stems. It has the effects of invigorating qi and strengthening the spleen, tonifying the kidneys and calming the mind. It can be used to treat symptoms such as deficiency of both lung and kidney, insomnia, and excessive dreaming. It is mainly used in medicine, food, health products, and daily necessities.

[0003] Studies have found that Acanthopanax senticosus possesses various pharmacological effects, including anti-tumor, anti-inflammatory, antibacterial, antidepressant, sedative, cardiovascular protective, neuroprotective, and immune-enhancing properties. It is rich in secondary metabolites such as syringin, eleutheroside E, isozymidine, chlorogenic acid, oleanolic acid, and hyperoside. Among these, eleutheroside E exhibits sedative, anti-inflammatory, antioxidant, anti-cervical cancer, antihypertensive, and memory-enhancing effects, and shows good efficacy in treating Parkinson's disease and insomnia. Oleanolic acid possesses hepatoprotective, anti-tumor, antibacterial, neuroprotective, and antioxidant effects. Furthermore, oleanolic acid effectively promotes skeletal muscle wound healing and is an important pharmaceutical raw material.

[0004] Currently, the production of eleutheroside E mainly involves extraction from plant materials. Oleanolic acid can be extracted from plant materials or prepared through chemical synthesis. Plant extraction methods damage plant resources, while chemical synthesis methods cause environmental pollution. Summary of the Invention

[0005] The present invention aims to address the problem that existing methods for obtaining eleutheroside E and oleanolic acid damage plant resources and pollute the environment, and provides a method for producing eleutheroside E and oleanolic acid by microbial fermentation.

[0006] This invention relates to a method for producing eleutheroside E and oleanolic acid using microbial fermentation, comprising the following steps:

[0007] 1. Take the activated Pantoea agglomerans HASX4 and culture it to obtain seed culture. Continue to expand the seed culture and collect the fermentation broth.

[0008] 2. Take the fermentation broth from step one and elute it with a macroporous resin column using 30% ethanol as the eluent. Collect the eluent. Then, separate it using silica gel column chromatography with chloroform-methanol as the eluent. Collect the eluent again and combine it with TLC detection. Collect the components containing the same secondary metabolites, combine them, and recrystallize them to obtain eleutheroside E.

[0009] 3. The fermentation broth from step 1 was concentrated under reduced pressure and extracted with ethyl acetate. The extracts were combined and concentrated to dryness. The mixture was then subjected to silica gel column chromatography with petroleum ether-ethyl acetate as the eluent. The eluent was collected, and fractions containing the same secondary metabolites were collected by TLC detection. These fractions were then combined to obtain the crude product. The crude product was then subjected to silica gel column chromatography again with 70% ethanol as the eluent. The eluent was collected, and fractions containing the same secondary metabolites were combined by TLC detection. After multiple recrystallization processes, oleanolic acid was finally obtained.

[0010] Furthermore, the Pantoea agglomerans HASX4 described in step one has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, on May 16, 2024, with accession number CCTCC NO: M 2024971.

[0011] Furthermore, the activation method of the clump-forming pantothenic acid HASX4 in step one is as follows: take the clump-forming pantothenic acid HASX4, inoculate it onto the surface of NA medium under aseptic conditions, and incubate at a constant temperature of 34~35℃ for 23~25h for later use.

[0012] Furthermore, the seed culture method in step one is as follows: Activated, clustered *Pantotheca HASX4* is inoculated into NB medium, shaken, and cultured to prepare 1×10⁻⁶ seed culture solution. 7 ~1×10 8 Seed culture of CFU / mL.

[0013] The shaking culture conditions are: 33~35℃, 120~130 r / min, shaking culture for 3~4 days.

[0014] Furthermore, in step one, the seed culture expansion involves inoculating the seed culture onto NB medium and culturing it at an inoculation volume of 6% to 7% (v / v).

[0015] Furthermore, the conditions for expanding the culture in step one are: 33~35℃, 120~130 r / min, and shaking culture for 5~6 days.

[0016] Furthermore, in step two, the macroporous resin column is D. 101 Macroporous resin column.

[0017] Furthermore, in step two, the volume ratio of chloroform to methanol in the eluent chloroform-methanol is 10:1.

[0018] Furthermore, in step three, the volume ratio of petroleum ether to ethyl acetate in the eluent petroleum ether-ethyl acetate is 1:2.

[0019] The beneficial effects of this invention are:

[0020] This invention employs a microbial fermentation method to ferment Pantoea agglomerans HASX4, enabling the simultaneous production of eleutheroside E and oleanolic acid. This invention discovers a new use for Pantoea agglomerans HASX4.

[0021] This invention obtains Acanthopanax senticosin E and oleanolic acid through microbial fermentation. The process is simple, does not pollute the environment, and is suitable for industrial production.

[0022] The method of this invention not only saves plant resources but also opens up a new way for the production of Acanthopanax senticosin E and oleanolic acid raw materials. Attached Figure Description

[0023] Figure 1 The results of high-performance liquid chromatography analysis of compound A in Example 1 are shown.

[0024] Figure 2 This is the mass spectrum of compound A in Example 1;

[0025] Figure 3 The results of high performance liquid chromatography analysis of compound B in Example 1;

[0026] Figure 4 This is the mass spectrum of compound B from Example 1. Detailed Implementation

[0027] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0028] Specific Implementation Method 1: This implementation method utilizes microbial fermentation to produce eleutheroside E and oleanolic acid, including the following steps:

[0029] 1. Take the activated Pantoea agglomerans HASX4 and culture it to obtain seed culture. Continue to expand the seed culture and collect the fermentation broth.

[0030] 2. Take the fermentation broth from step one and elute it with a macroporous resin column using 30% ethanol as the eluent. Collect the eluent. Then, separate it using silica gel column chromatography with chloroform-methanol as the eluent. Collect the eluent again and combine it with TLC detection. Collect the components containing the same secondary metabolites, combine them, and recrystallize them to obtain eleutheroside E.

[0031] 3. The fermentation broth from step 1 was concentrated under reduced pressure and extracted with ethyl acetate. The extracts were combined and concentrated to dryness. The mixture was then subjected to silica gel column chromatography with petroleum ether-ethyl acetate as the eluent. The eluent was collected, and fractions containing the same secondary metabolites were collected by TLC detection. These fractions were then combined to obtain the crude product. The crude product was then subjected to silica gel column chromatography again with 70% ethanol as the eluent. The eluent was collected, and fractions containing the same secondary metabolites were combined by TLC detection. After multiple recrystallization processes, oleanolic acid was finally obtained.

[0032] Specific Implementation Method Two: The Pantoea agglomerans HASX4 described in Step One of this implementation method has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan City, on May 16, 2024, with accession number CCTCC NO: M 2024971. Other steps and parameters are the same as in Specific Implementation Method One.

[0033] The Pantoea agglomerans HASX4 has been disclosed in patent CN118956664A.

[0034] Specific Implementation Method 3: The activation method for the clump-forming pantothenic acid HASX4 in step one of this implementation method is as follows: Take the clump-forming pantothenic acid HASX4, inoculate it onto the surface of NA medium under aseptic conditions, and incubate at a constant temperature of 34~35℃ for 23~25h for later use. Other steps and parameters are the same as in Specific Implementation Method 1 or 2.

[0035] The preferred culture conditions for this embodiment are: constant temperature culture at 34℃ for 24 hours.

[0036] The method for preparing NA medium (beef extract peptone solid medium) in this embodiment is as follows: 20 g of agar is slowly added to NB medium in small amounts several times, followed by 5 g of NaCl. The pH is adjusted to 7, and the medium is dispensed and sterilized in an autoclave for 30 min for later use.

[0037] Specific Implementation Method Four: The seed culture method in step one of this implementation method is as follows: Activated, clumped pantothenic acid HASX4 is inoculated into NB medium, shaken, and cultured to prepare 1×10⁻⁶ saturates. 7 ~1×10 8 Seed culture at CFU / mL. Other steps and parameters are the same as in any of the specific embodiments one to three.

[0038] The preparation method of NB medium (beef extract peptone liquid medium) described in this embodiment is as follows: First, add 3 g of beef extract to 1 L of boiling water, stir quickly to dissolve, then add 10 g of peptone, and finally add 5 g of NaCl. After it is completely dissolved, adjust the pH of the medium to 7, bottle it, and sterilize it in an autoclave for 30 min for later use.

[0039] Specific Implementation Method 5: The shaking culture conditions described in this implementation method are: 33~35℃, 120~130 r / min, shaking culture for 3~4 days. Other steps and parameters are the same as in Specific Implementation Methods 1 to 4.

[0040] The preferred shaking culture conditions for this embodiment are: 34℃, 130 r / min, shaking culture for 3 days.

[0041] Specific Implementation Method Six: In step one of this implementation method, the seed culture is expanded by inoculating the seed culture into NB medium, with an inoculation volume of 6%~7% (v / v). Other steps and parameters are the same as in any of Specific Implementation Methods One to Five.

[0042] Specific Implementation Method Seven: The conditions for scaling up the culture in step one of this implementation method are: 33~35℃, 120~130 r / min, and shaking culture for 5~6 days. Other steps and parameters are the same as in any of Specific Implementation Methods One to Six.

[0043] Specific Implementation Method Eight: In step two of this implementation method, the macroporous resin column is D. 101 Macroporous resin column. Other steps and parameters are the same as in any of the specific embodiments one to seven.

[0044] Specific Implementation Method Nine: In step two of this implementation method, the volume ratio of chloroform to methanol in the eluent chloroform-methanol is 10:1. Other steps and parameters are the same as in Specific Implementation Methods One through Eight.

[0045] Specific Implementation Method 10: In step three of this implementation method, the volume ratio of petroleum ether to ethyl acetate in the eluent is 1:2. Other steps and parameters are the same as in any of Specific Implementation Methods 1 to 9.

[0046] 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.

[0047] Example 1:

[0048] This embodiment describes a method for producing eleutheroside E and oleanolic acid using microbial fermentation, including the following steps:

[0049] I. Preparation of Culture Medium

[0050] Beef extract peptone liquid medium (NB medium): Add 3 g of beef extract to 1 L of boiling water and stir quickly to dissolve. Then add 10 g of peptone and finally 5 g of NaCl. After it is completely dissolved, adjust the pH of the medium to 7, bottle it, and autoclave it for 30 min.

[0051] Beef extract peptone solid medium (NA medium): In NB medium, slowly add 20 g of agar in small amounts several times, then add 5 g of NaCl, adjust the pH to 7, dispense, autoclave and sterilize for 30 min, and set aside.

[0052] II. Preparation of Fermentation Broth

[0053] Activated Pantoea agglomerans HASX4 was inoculated into sterile NB medium (40 mL volume) and cultured at 34℃ and 130 r / min with shaking for 3 days to prepare 1×10⁻⁶ cells / mL. 7 CFU / mL seed culture; at an inoculation rate of 6% (v / v), the seed culture was transferred to 500 mL NB medium (300 mL volume), and cultured continuously at 34℃ and 130 r / min for 5 days. After filtration, the fermentation broth was collected.

[0054] III. Extraction and separation of Acanthopanax senticosin E and oleanolic acid

[0055] Take 15L of the fermentation broth of Pantotheca HASX4 that has formed clumps, and slowly pass it through D. 101 Macroporous resin column, eluted with 30% ethanol, the eluent was collected, the solvent was recovered to dryness, and separation was performed by silica gel column chromatography with chloroform-methanol (10:1) as the eluent. The eluent (10 mL / bottle) was collected, and combined with TLC detection, fractions 354-409 (i.e. fractions containing the same secondary metabolites) were collected. Compound A was obtained by recrystallization.

[0056] 15 L of fermentation broth from *Pantotheca HASX4* was concentrated to 300 mL under reduced pressure and extracted three times with ethyl acetate (3 × 100 mL each). The extracts were combined and concentrated to dryness at 50 °C. Then, silica gel column chromatography was performed using petroleum ether-ethyl acetate (1:2) as the eluent. 10 mL of the eluent was collected per vial. The separation was examined using TLC. Fractions 458–493 (i.e., fractions containing the same secondary metabolites) were combined to obtain the crude product. The crude product was further purified using silica gel column chromatography, eluting with 70% ethanol. 10 mL of the eluent was collected per vial. Based on the TLC results, fractions 87–98 (identical fractions) were finally combined. After multiple recrystallizations, compound B was finally obtained.

[0057] The compounds A and B prepared in this embodiment were identified as follows:

[0058] Compound A: 10.622 mg, yield 0.7081 mg / L, white powder, readily soluble in organic solvents such as methanol and chloroform.

[0059] High-performance liquid chromatography (HPLC) was used. The mobile phase was acetonitrile-0.1% glacial acetic acid (20:80); the detection wavelength was 210 nm; and the flow rate was 1 mL / min. -1 Column temperature: 25℃; Injection volume: 10 μL. Eleutherococcus senticosin E was used as a control. Results are shown below. Figure 1 , Figure 1 In the table, A represents compound A + Acanthopanax senticosin E as a control, B represents compound A, and C represents Acanthopanax senticosin E.

[0060] Depend on Figure 1 It was found that both compound A and eleutheroside E reference standard showed a characteristic peak at 4.316 min. When the two were mixed in equal proportions and injected, a single chromatographic peak appeared. HPLC analysis was performed using three different mobile phases, and compound A still showed the same chromatographic peak at the same retention time as eleutheroside E reference standard, indicating that compound A is eleutheroside E. Quantitative analysis showed that the purity of this compound reached 98.36%. To fully confirm its structure, high-performance liquid chromatography-mass spectrometry (HPLC-MS) was used for analysis, and the results are shown below. Figure 2 .

[0061] Depend on Figure 2 The ESI-MS (m / z) of compound A is 743.75 [M+H]+, suggesting a molecular weight of 742. The loss of a glucose molecule at 581.41 [M-162]+ is likely due to glycosidic bond cleavage. The base peak at m / z 418 [M-324]+ in the mass spectrum may also originate from the loss of glucose molecules on either side. These data are consistent with reported data for eleutheroside E in the literature. Based on the combined results of HPLC and HPLC-MS analysis, compound A is identified as eleutheroside E, with a relative molecular mass of 742 and a molecular formula of C2. 34 H 46 O 18 The structural formula is as follows.

[0062]

[0063] Compound B: 8.989 mg, yield 0.5993 mg / L, is a pale yellow needle-like crystal, readily soluble in methanol, and soluble in organic solvents such as ether, acetone, and chloroform.

[0064] HPLC method was used. Mobile phase: methanol-0.2% acetic acid solution; detection wavelength: 210 nm; flow rate: 1 mL / min.-1 Column temperature: 25℃; Injection volume: 10 μL. Oleanolic acid was used as a control. The analytical results are shown below. Figure 3 , Figure 3 In the table, A is compound B + oleanolic acid control, B is compound B, and C is oleanolic acid control.

[0065] Depend on Figure 3 It was found that both compound B and oleanolic acid reference standard showed characteristic peaks at 28.453 min. After mixing the two in equal proportions and injecting the mixture, a single chromatographic peak was obtained, suggesting that compound B is oleanolic acid. Analysis showed that the purity of this compound reached 99.57%. To fully confirm its structure, subsequent HPLC-MS analysis was performed, and the results are shown below. Figure 4 .

[0066] Depend on Figure 3 It was found that both compound B and oleanolic acid reference standard showed characteristic peaks at 28.453 min. After mixing the two in equal proportions and injecting the mixture, a single chromatographic peak was obtained. HPLC analysis was performed using three different mobile phases. Compound B showed the same chromatographic peak at the same retention time as the oleanolic acid reference standard, thus suggesting that compound B is oleanolic acid. The purity of this compound reached 99.57%. To fully confirm its structure, subsequent HPLC-MS analysis was performed, and the results are shown below. Figure 4 .

[0067] Depend on Figure 4 The m / z of compound B is 457.24 [M+H]+, suggesting a molecular weight of 456. The 438.79 [M-H2O]+ peak, formed by the loss of an H2O group, is inferred to originate from an OH group. The base peak at m / z 346 may be due to the loss of the side chain (C7H7) at the D / E ring junction of oleanolic acid, forming a stable tetracyclic skeleton ion. These data are consistent with literature reports on oleanolic acid. Based on the combined HPLC-HPLC-MS analysis results, compound B is identified as oleanolic acid with a relative molecular mass of 456 and a molecular formula of C... 30 H 48 O3. The structural formula is as follows.

[0068]

Claims

1. A method for producing eleutheroside E and oleanolic acid using microbial fermentation, characterized in that, The method includes the following steps:

1. Take the activated clumps of Pantothenia gravis ( Pantoea agglomerans HASX4 culture yields seed culture, which is then further cultured and fermentation broth is collected.

2. Take the fermentation broth from step one and elute it with a macroporous resin column using 30% ethanol as the eluent. Collect the eluent. Then, separate it using silica gel column chromatography with chloroform-methanol as the eluent. Collect the eluent again and combine it with TLC detection. Collect the components containing the same secondary metabolites, combine them, and recrystallize them to obtain eleutheroside E.

3. The fermentation broth from step 1 was concentrated under reduced pressure and extracted with ethyl acetate. The extracts were combined and concentrated to dryness. The mixture was then subjected to silica gel column chromatography with petroleum ether-ethyl acetate as the eluent. The eluent was collected, and fractions containing the same secondary metabolites were collected by TLC analysis. These fractions were combined to obtain the crude product. The crude product was then subjected to silica gel column chromatography with 70% ethanol as the eluent. The eluent was collected, and fractions containing the same secondary metabolites were combined by TLC analysis. After multiple recrystallization processes, oleanolic acid was finally obtained. Step 1 describes the formation of pantothenic bacteria ( Pantoea agglomerans HASX4 has been deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, on May 16, 2024, with accession number CCTCC NO: M2024971.

2. The method for producing eleutheroside E and oleanolic acid by microbial fermentation according to claim 1, characterized in that, The activation method of Pantothenia glutinosa HASX4 in step one is as follows: Take Pantothenia glutinosa HASX4, inoculate it onto the surface of NA medium under aseptic conditions, and incubate at 34~35℃ for 23~25h for later use.

3. The method for producing eleutheroside E and oleanolic acid by microbial fermentation according to claim 2, characterized in that, The seed culture method in step one is as follows: Activated, clumped pantothenic acid HASX4 is inoculated into NB medium, shaken, and cultured to prepare 1×10⁻⁶ seed culture solution. 7 ~1×10 8 Seed culture of CFU / mL.

4. The method for producing eleutheroside E and oleanolic acid by microbial fermentation according to claim 3, characterized in that, The shaking culture conditions are: 33~35℃, 120~130 r / min, shaking culture for 3~4 days.

5. The method for producing eleutheroside E and oleanolic acid by microbial fermentation according to claim 1, characterized in that, In step one, the seed culture expansion involves inoculating the seed culture onto NB medium and culturing it at a concentration of 6% to 7% (v / v).

6. The method for producing eleutheroside E and oleanolic acid by microbial fermentation according to claim 5, characterized in that, The conditions for scaling up the culture in step one are: 33~35℃, 120~130 r / min, and shaking culture for 5~6 days.

7. The method for producing eleutheroside E and oleanolic acid by microbial fermentation according to claim 1, characterized in that, In step two, the macroporous resin column is D. 101 Macroporous resin column.

8. The method for producing eleutheroside E and oleanolic acid by microbial fermentation according to claim 1, characterized in that, In step two, the volume ratio of chloroform to methanol in the eluent chloroform-methanol is 10:

1.

9. The method for producing eleutheroside E and oleanolic acid by microbial fermentation according to claim 1, characterized in that, In step three, the volume ratio of petroleum ether to ethyl acetate in the eluent petroleum ether-ethyl acetate is 1:2.

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