A method for isolating branched sphingosines from bacteroides fragilis, products and uses thereof

Abstract

The application provides a method for isolating branched sphingosine from Bacteroides fragilis, products and applications. The method comprises the following steps: (1) using an organic solvent as an extraction solvent to extract intracellular metabolites and obtain a total extract; (2) using an organic solvent as an extractant to extract the total extract to obtain an extract; (3) using a reverse phase chromatographic column to perform liquid chromatography separation on the extract to obtain the branched sphingosine; wherein the liquid chromatography separation conditions comprise elution with an acetonitrile / water solution containing formic acid as an elution system. The application can obtain a plurality of different high-purity active branched sphingosines from the fermentation broth of Bacteroides fragilis through simple treatment, and the method is simple to operate and does not require complex equipment and technology.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more specifically, to a method, product, and application for isolating branched-chain sphingosine from Bacteroides fragilis. Background Technology

[0002] Bacteroides fragilis is an important anaerobic gut microbiome belonging to the genus Bacteroides. It is one of the most common bacteria in the human gut, typically occupying a central position in the gut microbiota and playing a vital role in human health. Bacteroides fragilis and its components can be used to study immune regulatory mechanisms, thus holding promise as a new direction for treating inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease) and regulating intestinal immune dysregulation.

[0003] Studies have found that sphingosine produced by Bacteroides can enter the host's metabolic pathways, thereby affecting ceramide levels and host lipid metabolism. Its branched-chain sphingosine has significant biological and medical implications. Recent research indicates that immunomodulatory α-galactosphosine plays a regulatory role in intestinal immune maturation within the host-microbe environment, and the unique terminal branched structure of sphingosine is a key determinant of its specific immunomodulatory activity. These studies suggest that the development of methods for isolating active branched-chain sphingosine from Bacteroides fragilis is of great importance.

[0004] In addition, sphingosine derivatives have received widespread attention in dermatology and cosmetic research and development because they play an important role in skin physiology and have a variety of biological functions, including regulating cell proliferation, cell differentiation, programmed cell death, participating in inflammatory responses, and possessing antibacterial, anti-inflammatory, and skin barrier repair properties. They can help maintain the skin's acid-base balance (pH value), enhance the skin's natural barrier function, reduce damage to the skin from external environmental factors such as ultraviolet radiation and pollutants, and have moisturizing effects and the ability to promote skin health. This makes them useful ingredients for treating dry skin, eczema, and other skin conditions, and they are becoming increasingly common in skin care products on the market.

[0005] Given the important activity of sphingosine and its wide application in the cosmetics industry, its production currently mainly focuses on extraction and isolation from the fermentation broths of animals, plants, and various yeasts. Methods for isolating and purifying sphingosine are relatively cumbersome and require significant time and resources. For example, patent CN115372521A discloses a method for the isolation and identification of phytosphingosine and / or N-acetylphytosphingosine, providing a reliable detection and analysis method for the quality control of phytosphingosine. To date, no method has been reported for preparing large quantities of branched-chain sphingosine from Bacteroides fragilis through fermentation. Summary of the Invention

[0006] One object of the present invention is to provide a method for isolating branched-chain sphingosine from Bacteroides fragilis;

[0007] Another object of the present invention is to provide a branched sphingosine;

[0008] Another object of the present invention is to provide the use of the branched sphingosine.

[0009] To achieve the above objectives, in one aspect, the present invention provides a method for isolating branched-chain sphingosine from Bacteroides fragilis, wherein the method comprises the following steps:

[0010] (1) The steps of using organic solvents as extraction solvents to extract intracellular metabolites of bacteria and obtain total extract;

[0011] (2) The step of using an organic solvent as an extractant to extract the total extract to obtain the extract;

[0012] (3) The step of separating the extract by liquid chromatography using a reversed-phase column to obtain the branched sphingosine; wherein the liquid chromatography separation conditions include elution using an acetonitrile / water solution containing formic acid as the elution system.

[0013] According to some specific embodiments of the present invention, step (1) uses an aqueous methanol solution or an aqueous ethanol solution with a volume concentration of 70%-100% as the extraction solvent.

[0014] According to some specific embodiments of the present invention, the volume of the extraction solvent is 3-5 times the mass of the bacterial cells.

[0015] According to some specific embodiments of the present invention, the volume of the extraction solvent is 3-4.2 times the mass of the bacterial cells.

[0016] According to some specific embodiments of the present invention, step (1) includes using an organic solvent as an extraction solvent to extract intracellular metabolites of bacteria to obtain a primary extract, centrifuging the primary extract to obtain the supernatant to obtain an extract, and concentrating the extract to obtain a total extract.

[0017] According to some specific embodiments of the present invention, step (1) includes adding the bacterial cells to the extraction solvent and extracting the initial extract by shaking or ultrasonication.

[0018] Both oscillation and ultrasound can be performed using conventional oscillation and ultrasound devices in the art. According to some specific embodiments of the present invention, the oscillation frequency is 30-50 kHz and the oscillation time is 0.5-1.5 h.

[0019] According to some specific embodiments of the present invention, the oscillation frequency is 40 kHz.

[0020] According to some specific embodiments of the present invention, the oscillation time is 1 hour.

[0021] According to some specific embodiments of the present invention, the ultrasonic power is 300-500W and the ultrasonic time is 0.5-1.5h.

[0022] According to some specific embodiments of the present invention, the power of the ultrasound is 400W.

[0023] According to some specific embodiments of the present invention, the ultrasound time is 1 hour.

[0024] According to some specific embodiments of the present invention, the centrifugal speed in step (1) is 3000-4000.

[0025] According to some specific embodiments of the present invention, the centrifugal speed in step (1) is 3500-3700.

[0026] According to some specific embodiments of the present invention, the centrifugation time in step (1) is 15-30 min.

[0027] According to some specific embodiments of the present invention, the centrifugation time in step (1) is 15-20 min.

[0028] According to some specific embodiments of the present invention, in step (1), after centrifuging the initial extract to obtain the supernatant, the centrifuged precipitate is extracted again using the extraction solvent 1-3 times.

[0029] According to some specific embodiments of the present invention, the extractant in step (2) is a chloroform / methanol solution, and the volume ratio of chloroform to methanol is (2-3):1.

[0030] According to some specific embodiments of the present invention, step (2) includes dispersing the total extract in water to obtain a total suspension, then extracting the total suspension with an extractant, and concentrating and drying the extract to obtain an extract; the volume of the extractant is 2-3 times the volume of the total suspension.

[0031] According to some specific embodiments of the present invention, in step (2), after extracting the total suspension with an extractant, the organic phase is taken as the extractant.

[0032] According to some specific embodiments of the present invention, the volume of water is 8-10 times the mass of the total extract.

[0033] According to some specific embodiments of the present invention, the volume of water is 9-10 times the mass of the total extract.

[0034] According to some specific embodiments of the present invention, in step (3), the volume fraction of acetonitrile in the acetonitrile / water solution is 50%-60%; and the volume fraction of formic acid is 0.1%-0.2%.

[0035] According to some specific embodiments of the present invention, the amount of formic acid used is such that the pH of the acetonitrile / water solution is adjusted to 3.5-4.5.

[0036] According to some specific embodiments of the present invention, the amount of formic acid used is such that the pH of the acetonitrile / water solution is adjusted to 4-4.5.

[0037] According to some specific embodiments of the present invention, the reversed-phase chromatographic column in step (3) is selected from C8 or C18 chromatographic columns.

[0038] According to some specific embodiments of the present invention, the reversed-phase chromatographic column in step (3) has a length of 20cm-30cm and a diameter of 0.46cm-2.1cm.

[0039] According to some specific embodiments of the present invention, the reversed-phase chromatography column in step (3) has a length of 25cm-27cm.

[0040] According to some specific embodiments of the present invention, the liquid chromatography separation conditions in step (3) further include an injection volume of 0.1-10 ml and a flow rate of 1-25 ml / min.

[0041] According to some specific embodiments of the present invention, the liquid chromatography separation in step (3) is performed by detection at 210-220 nm using an ultraviolet detector.

[0042] According to some specific embodiments of the present invention, step (3) includes dissolving the extract with an organic solvent as the loading solvent, and then performing liquid chromatography separation using a reversed-phase column.

[0043] According to some specific embodiments of the present invention, the loading solvent is an aqueous solution of acetonitrile with a volume fraction of 40%-60%.

[0044] According to some specific embodiments of the present invention, the loading solvent is an aqueous solution of acetonitrile with a volume fraction of 50%-55%.

[0045] According to some specific embodiments of the present invention, in step (3), after dissolving the extract with an organic solvent as the loading solvent, the concentration of the extract in the solution is 40-60 mg / ml.

[0046] According to some specific embodiments of the present invention, in step (3), after dissolving the extract with an organic solvent as the loading solvent, the concentration of the extract in the solution is 50-55 mg / ml.

[0047] According to some specific embodiments of the present invention, step (3) includes dissolving the extract, filtering it through a microporous membrane, and then performing liquid chromatography separation.

[0048] According to some specific embodiments of the present invention, the pore size of the microporous membrane is in the range of 0.22-0.4 micrometers.

[0049] According to some specific embodiments of the present invention, step (3) further includes concentrating and drying the eluent obtained by liquid chromatography to obtain the branched sphingosine.

[0050] According to some specific embodiments of the present invention, the method further includes a step of culturing Bacteroides fragilis, and then using an organic solvent as an extraction solvent to extract intracellular metabolites of the bacteria. The culturing step includes culturing Bacteroides fragilis seed culture in a culture medium under anaerobic conditions at 35℃-40℃ for 24-72h, and then centrifuging the fermentation broth at high speed to obtain bacterial precipitate.

[0051] According to some specific embodiments of the present invention, the OD of the Bacteroides fragilis seed solution is 6-8.

[0052] According to some specific embodiments of the present invention, the culture medium is BHI or mGAM culture medium.

[0053] The present invention may also add other components to the BHI or mGAM medium, for example, one or more of the following components in combination may be added per 1L of medium: 3-8 mg / L heme, 0.3-0.8 mg / L vitamin, 0.7-1.5 g / L cysteine; preferably 5 mg / L heme, 0.5 mg / L vitamin, and 1 g / L cysteine.

[0054] According to some specific embodiments of the present invention, the Bacteroides fragilis seed culture is cultured in a culture medium under anaerobic conditions at 37°C-38°C for 24-72 hours.

[0055] According to some specific embodiments of the present invention, the centrifugation speed in the step of culturing Bacteroides fragilis is 3000-4000 rpm.

[0056] According to some specific embodiments of the present invention, the centrifugation speed in the step of culturing Bacteroides fragilis is 3500-3700 rpm.

[0057] According to some specific embodiments of the present invention, the centrifugation time in the step of culturing Bacteroides fragilis is 15-30 min.

[0058] According to some specific embodiments of the present invention, the centrifugation time in the step of culturing Bacteroides fragilis is 15-20 min.

[0059] On the other hand, the present invention also provides a branched sphingosine mixture obtained by the method of any one of the present invention, wherein the purity of the branched sphingosine in the mixture is greater than or equal to 90%; the structure of the branched sphingosine is shown in formula (I) below:

[0060]

[0061] n is 5-15.

[0062] According to some specific embodiments of the present invention, the purity of the branched sphingosine in the mixture is 90%-99%, preferably 90%-95%; more preferably 92%-95%; and even more preferably 93%-95%.

[0063] According to some specific embodiments of the present invention, the branched sphingosine is selected from one of the following structures:

[0064]

[0065] Furthermore, the present invention also provides the application of the branched-chain sphingosine mixture described herein in the preparation of immunomodulators or skin care products.

[0066] According to some specific embodiments of the present invention, the immunomodulator is an intestinal immunomodulator.

[0067] According to some specific embodiments of the present invention, the immunomodulator is an immunomodulator used to treat inflammatory bowel disease.

[0068] According to some specific embodiments of the present invention, the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

[0069] In summary, this invention provides a method, product, and application for isolating branched-chain sphingosine from Bacteroides fragilis. The method of this invention has the following advantages:

[0070] 1. This invention can obtain a variety of high-purity active branched-chain sphingosine from the fermentation broth of Bacteroides fragilis through simple processing. The method of this invention is simple to operate and does not require complicated equipment and technology.

[0071] 2. This method employs a three-step separation process: extraction, extraction, and separation. This significantly reduces sample loss due to additional separation steps, improves the recovery rate of the target product, and allows for the simultaneous acquisition of multiple monomeric compounds, thereby increasing the utilization rate of raw materials. It enables rapid and efficient separation and purification of branched-chain sphingosine.

[0072] 3. The scale of preparation by this invention can reach the milligram level. It adopts rapid preparative reverse high performance liquid chromatography elution separation, and the separation time for a single injection is only 10 minutes. This can save a lot of column equilibration and rinsing time, greatly improve the preparation efficiency, reduce the use of solvents, and reduce separation costs. It is suitable for the extraction of large quantities of branched sphingosine, and it is easy to realize large-scale production. Moreover, the obtained branched sphingosine has high purity and activity. Attached Figure Description

[0073] Figure 1 The chromatogram for the high-performance liquid chromatography preparation in Example 1 (UV: 210nm);

[0074] Figure 2 This is a high-resolution secondary mass spectrometry identification diagram of branched sphingosine 1 obtained in Example 1;

[0075] Figure 3 This is a high-resolution secondary mass spectrometry identification diagram of branched sphingosine 2 obtained in Example 1;

[0076] Figure 4 This is a high-resolution secondary mass spectrometry identification chromatogram of branched sphingosine 3 obtained in Example 1. Detailed Implementation

[0077] The following detailed embodiments illustrate the implementation process and beneficial effects of the present invention, aiming to help readers better understand the essence and characteristics of the present invention, and are not intended to limit the scope of implementation of this case.

[0078] Example 1

[0079] 10 mL of Bacteroides fragilis BHI overnight seed culture was inoculated into an Erlenmeyer flask containing 1 L of fresh sterile BHI medium (Oxoid, with additional 5 mg / L heme, 0.5 mg / L vitamin, and 1 g / L cysteine) and incubated at 37°C for 24 hours in an anaerobic incubator. After centrifugation at 3500 rpm for 15 min, approximately 4.5 g of cells were collected. 15 mL of anhydrous ethanol was added to the cells, and extraction was performed by shaking for 1 hour. The supernatant was collected after centrifugation at 3500 rpm for 15 min. The ethanol extraction was repeated three times, and the extracts were combined to obtain approximately 50 mL of ethanol extract. After concentration under reduced pressure to remove the ethanol solvent, approximately 0.8 g of total extract was obtained. The total extract was thoroughly dispersed in 8 mL of water, and 24 mL of a chloroform-methanol (2:1) mixture was added. After thorough shaking and extraction, the layers were allowed to separate. Approximately 18 mL of the lower chloroform fraction was obtained using a separatory funnel. The chloroform extract was concentrated under reduced pressure and dried to obtain approximately 90 mg of crude sphingosine extract. The crude extract was dissolved in 1.8 mL of 50% acetonitrile aqueous solution to obtain a 50 mg / mL sample to be separated. 0.1 mL of the sample was injected for separation. Reverse high-performance liquid chromatography (RP-HPLC) separation conditions were: C8 analytical column, diameter 0.46 cm, column length 25 cm, elution with 60% acetonitrile-water (with 0.1% formic acid added), and detection of the eluent at 210 nm using a UV detector. The chromatographic peaks corresponding to the branched-chain sphingosine monomers were collected. The eluent was concentrated and dried to obtain the target product. The separation results are as follows: Figures 1 to 4 After repeated injections and separation, branched sphingosine monomer compounds 1, 2, and 3 were obtained in amounts of approximately 0.2 mg (HPLC purity 92%), 3.5 mg (HPLC purity 90%), and 1.1 mg (HPLC purity 95%), respectively.

[0080] The HPLC conditions were as follows: a preparative liquid chromatograph (LC-2050) from Beijing Qingbohua Technology Co., Ltd., equipped with a UV2000D dual-wavelength ultraviolet detector, was used. Before injection, the column was fully equilibrated with 60% acetonitrile-water (with 0.1% formic acid added). After injection, the ultraviolet detector was automatically activated to begin data acquisition. The elution mobile phase was kept constant at 60% acetonitrile-water (with 0.1% formic acid added), and the total elution time was 10 minutes. Eluents were collected at 2.5–3.0 minutes, 4.5–4.75 minutes, and 5.6–6.0 minutes to obtain branched-chain sphingosine 1, 2, and 3.

[0081] Example 2:

[0082] 10 mL of Bacteroides fragilis mGAM overnight seed culture was inoculated into an Erlenmeyer flask containing 1 L of fresh, sterile mGAM medium (Haibo Biotechnology, with an additional 1 g / L cysteine) and incubated at 37°C for 48 hours in an anaerobic incubator. After centrifugation at 3500 rpm for 15 min, approximately 6.0 g of cells were collected. 25 mL of anhydrous ethanol was added to the cells, and the mixture was sonicated for 1 hour, followed by centrifugation at 3500 rpm for 15 min to collect the supernatant. The ethanol extraction was repeated three times, and the supernatant was collected. The extract was then combined to obtain approximately 75 mL of ethanol extract. After concentration under reduced pressure to remove the ethanol solvent, approximately 1.1 g of total extract was obtained. The total extract was thoroughly dispersed in 10 mL of water, and 30 mL of a chloroform-methanol (3:1) mixture was added. After thorough shaking and extraction, the mixture was allowed to stand and separate into layers. Approximately 25 mL of the lower chloroform fraction was obtained using a separatory funnel. The chloroform extract was concentrated under reduced pressure and dried to obtain approximately 150 mg of crude sphingosine extract. The crude extract was dissolved in 3.0 mL of 50% acetonitrile aqueous solution to obtain a sample concentration of 50 mg / mL to be separated. 3 mL of the sample was injected for separation using reversed-phase high-performance liquid chromatography (RP-HPLC) under the following conditions: a C18 preparative column with a diameter of 2.1 cm and a column length of 25 cm; elution with 50% acetonitrile-water (with 0.1% formic acid added); detection of the eluent at 220 nm using a UV detector; collection of the chromatographic peaks corresponding to the branched-chain sphingosine monomers; concentration and drying of the fraction to obtain the target product. Branched-chain sphingosine monomers 1, 2, and 3 were separated at approximately 0.3 mg (HPLC purity 92%), 5.0 mg (HPLC purity 93%), and 1.0 mg (HPLC purity 95%), respectively. Their mass spectra were essentially the same as in Example 1.

[0083] Example 3:

[0084] 40 mL of Bacteroides fragilis mGAM overnight seed culture was inoculated into an Erlenmeyer flask containing 4 L of fresh sterile mGAM medium (as above) and incubated at 37°C for 72 hours in an anaerobic incubator. After centrifugation at 3500 rpm for 30 min, approximately 25.0 g of cells were collected. 100 mL of 70% ethanol aqueous solution was added to the cells, and the mixture was extracted by sonication for 1 hour, followed by centrifugation at 3500 rpm for 20 min to collect the supernatant. This ethanol aqueous solution extraction was repeated three times, and the supernatant was collected. The extract was combined to obtain approximately 300 mL of ethanol extract. After concentration under reduced pressure to remove the ethanol solvent, approximately 4.1 g of total extract was obtained. The total extract was thoroughly dispersed in 40 mL of water, and 120 mL of a chloroform-methanol (3:1) mixed solvent was added. After thorough shaking and extraction, the mixture was allowed to stand and separate into layers. Approximately 80 mL of the lower chloroform fraction was obtained using a separatory funnel. The chloroform extract was concentrated under reduced pressure and dried to obtain approximately 550 mg of crude sphingosine extract. The crude extract was dissolved in 11 mL of 50% acetonitrile aqueous solution to obtain a sample concentration of 50 mg / mL to be separated. 10 mL of the sample was injected for separation using reversed-phase high-performance liquid chromatography (RP-HPLC) under the following conditions: a C18 preparative column with a diameter of 2.1 cm and a length of 25 cm; elution with 50% acetonitrile-water (with 0.1% formic acid added); detection of the eluent at 210 nm using a UV detector; collection of the chromatographic peaks corresponding to the branched-chain sphingosine monomers; concentration and drying of the fraction to obtain the target product. Approximately 1.2 mg (HPLC purity 91%), 15.5 mg (HPLC purity 92%), and 2.2 mg (HPLC purity 92%) of branched-chain sphingosine monomers 1, 2, and 3 were obtained. Their mass spectra were essentially the same as in Example 1.

Claims

1. A method for isolating branched-chain sphingosine from Bacteroides fragilis, wherein, The method includes the following steps: (1) The steps of using organic solvents as extraction solvents to extract intracellular metabolites of bacteria and obtain total extract; (2) The step of using an organic solvent as an extractant to extract the total extract to obtain the extract; (3) The step of separating the extract by liquid chromatography using a reversed-phase column to obtain the branched sphingosine; wherein the liquid chromatography separation conditions include elution using an acetonitrile / water solution containing formic acid as the elution system; The branched sphingosine is selected from one of the following structures: 。 2. The method according to claim 1, wherein, In step (3), the volume fraction of acetonitrile in the acetonitrile / water solution is 50%-60%; the volume fraction of formic acid is 0.1%-0.2%.

3. The method according to claim 1, wherein, The amount of formic acid used is to adjust the pH of the acetonitrile / water solution to 3.5-4.

5.

4. The method according to claim 1, wherein, The amount of formic acid used is to adjust the pH of the acetonitrile / water solution to 4.

5. The method according to claim 1, wherein, The reversed-phase column in step (3) is selected from C8 or C18 columns.

6. The method according to claim 1, wherein, The chromatographic column is 20-30 cm long and 0.46-2.1 cm in diameter.

7. The method according to claim 1, wherein, The conditions for liquid chromatography separation in step (3) also include an injection volume of 0.1-10 ml and a flow rate of 1-25 ml / min.

8. The method according to claim 1, wherein, The liquid chromatography separation in step (3) is performed by detection at 210-220 nm using an ultraviolet detector.

9. The method according to claim 1, wherein, Step (3) involves dissolving the extract using an organic solvent as the loading solvent, followed by liquid chromatography separation using a reversed-phase column.

10. The method according to claim 9, wherein, The sample loading solvent in step (3) is an acetonitrile aqueous solution with a volume fraction of 40%-60%.

11. The method according to claim 9, wherein, Step (3) involves dissolving the extract, filtering it through a microporous membrane, and then performing liquid chromatography separation.

12. The method according to claim 1, wherein, Step (1) uses a methanol aqueous solution or ethanol aqueous solution with a volume concentration of 70%-100% as the extraction solvent.

13. The method according to claim 12, wherein, The volume of extraction solvent used should be 3-5 times the mass of the bacterial cells.

14. The method according to claim 1, wherein, Step (1) includes using an organic solvent as the extraction solvent to extract intracellular metabolites of the bacteria to obtain a primary extract, centrifuging the primary extract to obtain the supernatant to obtain an extract, and concentrating the extract to obtain a total extract.

15. The method according to claim 14, wherein, The centrifugation speed is 3000-4000.

16. The method according to claim 1, wherein, The extractant in step (2) is a chloroform / methanol solution with a volume ratio of chloroform to methanol of (2-3):

1.

17. The method according to claim 1, wherein, Step (2) includes dispersing the total extract in water to obtain a total suspension, then extracting the total suspension with an extractant, concentrating and drying the extract to obtain the extract; the volume of the extractant is 2-3 times the volume of the total suspension.

18. The method according to claim 17, wherein, The volume of water is 8-10 times the total mass of the extract.

19. The method according to any one of claims 1 to 18, wherein, The method also includes a step of culturing Bacteroides fragilis, and then using an organic solvent as an extraction solvent to extract intracellular metabolites of the bacteria. The culturing step includes culturing the Bacteroides fragilis seed culture in a culture medium under anaerobic conditions at 35℃-40℃ for 24-72h, and then centrifuging the fermentation broth at high speed to obtain bacterial precipitate.

20. The method according to claim 19, wherein, The culture medium is BHI or mGAM medium.

21. The method according to claim 19, wherein, The cultivation steps include incubating the Bacteroides fragilis seed culture in a culture medium under anaerobic conditions at 37°C-38°C for 24-72 hours.

22. The method according to claim 19, wherein, The centrifugation speed of the high-speed centrifuge is 3000-4000 rpm.

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