A strain of Streptomyces and a fermentation method for producing pieridin glycoside and its application

Abstract

The present invention discloses a culture medium, a fermentation method and an application of Streptomyces for producing pieridian glycosides. Through this fermentation method, the Streptomyces can produce abundant pieridian glycosides, and its effective part contains more than 8 types of pieridian glycosides at the same time, and the content of the four types of pieridian glycosides, namely compound 4, compound 5, compound 6 and compound 8, is greater than 5%. The extract obtained by the strain through this fermentation method and its effective part can be used to prepare drug lead compounds for treating renal cancer, organ fibrosis, acute kidney injury and chronic kidney disease, and can provide drug source guarantee for China's marine drug research and development.

Description

Technical field:

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Streptomyces, a fermentation method for producing pieridium glycoside and applications thereof. Background technology:

[0002] Piericidins are α-pyridone antibiotics derived from microorganisms. Approximately 80 piericidin derivatives have been discovered, all produced by terrestrial and marine microorganisms. Piericidin compounds have been reported to exhibit insecticidal and antibacterial activities, as well as inhibitory activity against some tumor cells. Their pharmaceutical applications await further exploration (Journal of Antibiotics, 2016, 69:582). Piericidin glycosides are glycosyl-containing compounds of the piericidin family, and 23 have been reported. Previous research by the inventors has shown that piericidin glycosides have significant potential in treating renal cancer, inhibiting organ fibrosis, and treating acute kidney injury and chronic kidney disease. For example, several piericidin glycoside compounds have shown significant anti-renal cancer potential in ACHN renal cancer cells and mouse animal models (such as glucopiericidinA, GPA) (Journal of Medicinal Chemistry, 2019, 62, 7058), and the glycoside GPA is significantly less toxic than the piericidin aglycone piericidin A (PA) (Journal of Medicinal Chemistry. 2021, 64, 9943); the piericidin glycoside 13-hydroxyglucopiericidinA (13OH-GPA) has significant anti-acute kidney injury efficacy (Acta Pharmaceutica Sinica B, 2024.doi:10.1016 / j.apsb.2024.03.005); and significant anti-renal fibrosis activity and potential for treating chronic kidney disease (Theranostics.2022;12:7158); as a liver kinase B1 (LKB1) activator, 13OH-GPA also has potential therapeutic effects on fibrosis of organs such as the liver and lungs. The present invention further confirmed that multiple pierisin glycoside compounds have anti-acute kidney injury activity through the human proximal tubular epithelial cell hypoxia / reoxygenation injury model. However, the content of pierisin glycoside in microorganisms is relatively low, and there is no report on the chemical synthesis of pierisin glycoside, which limits its in-depth pharmaceutical research and development. Summary of the invention:

[0003] The present invention provides a culture medium, a fermentation method and an application for producing pieridian glycoside by Streptomyces psammoticus SCSIO NS126 (deposit number: GDMCC No: 64524). By improving the fermentation method, a large yield of pieridian glycoside is obtained, thereby solving the problem of insufficient drug supply in the research and development of drugs for treating renal cancer, organ fibrosis, acute kidney injury and chronic kidney disease.

[0004] The present invention is achieved through the following technical solutions:

[0005] The invention provides an H9 culture medium for fermenting Streptomyces psammoticus SCSIO NS126 to produce pieridium glycoside compounds. The H9 culture medium is characterized in that the H9 culture medium comprises soluble starch, cottonseed powder, yeast extract powder, maltodextrin, malt extract powder, anhydrous magnesium sulfate, sodium chloride, calcium carbonate, and the solvent is water.

[0006] Preferably, the formula of the H9 culture medium is: 20 g / L soluble starch, 10 g / L cottonseed powder, 5 g / L yeast extract powder, 20 g / L maltodextrin, 5 g / L malt extract powder, 2 g / L anhydrous magnesium sulfate, 2 g / L sodium chloride, 2 g / L calcium carbonate, and the solvent is water.

[0007] The present invention also provides a fermentation method for producing pieridium glycoside compounds, which comprises inoculating Streptomyces psammoticus SCSIO NS126 into the H9 culture medium according to claim 1 or 2 for fermentation and culture.

[0008] Preferably, the pieridin glycoside compound comprises:

[0009] 13-hydroxypiericidin A 10-O-α-D-glucose(1→6)-β-D-glucoside、4'-O-β-D-glucose piericid in A 10-O-α-D-glucose(1→6)-β-D-glucoside、piericidin A 10-O-β-D-glucoside(1→6)-α-D-gala ctose, 13-hydroxyglucopiericidin A, 4'-O-β-glucoseglucopiericidin A, 7-demethyl-13-hydroxyglucopiericidin A and glucopiericidin A.

[0010] Preferably, the fermentation culture is cultured under the conditions of shaking at 28° C. and 180 rpm.

[0011] Preferably, Streptomyces psammoticus SCSIO NS126 is cultured in a seed liquid medium at 28°C with shaking at 180 rpm for 48 hours; then, amplified fermentation is carried out in H9 medium at 28°C with shaking at 180 rpm for 7 days. The seed liquid medium comprises 20 g / L D-mannitol, 10 g / L soy peptone, 2.5 g / L soybean oil, and 0.35 g / L dipotassium hydrogen phosphate, and the solvent is water.

[0012] Preferably, the method comprises the following steps:

[0013] (1) Streptomyces psammoticus SCSIO NS126 was cultured in a seed liquid medium for 48 hours at 28°C with shaking at 180 rpm; then, the fermentation was scaled up in H9 medium, with each liter of seed liquid scaled up to a fermentation volume of 30 liters at 28°C with shaking at 180 rpm; after 7 days of culture, the fermentation liquid was extracted with ethyl acetate, and the extract was concentrated to obtain an extract;

[0014] (2) The extract obtained in step (1) was subjected to ODS reverse phase column chromatography to obtain 8 fractions SP1-SP8, under the following conditions: methanol / water, with a volume ratio of 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 9:1, 10:0 gradient elution, and SP1-8 fractions were obtained in sequence; the three fractions SP5-7 were combined and concentrated to obtain the effective part;

[0015] (3) The effective fraction was separated and purified by semi-preparative HPLC with a mobile phase of acetonitrile / water in a volume ratio of 60:40 and a flow rate of 3 mL / min to obtain compound 1 with a retention time of 22 min, compound 2 with a retention time of 23 min, compound 4 with a retention time of 26.4 min, compound 5 with a retention time of 27.5 min, compound 6 with a retention time of 28.9 min, compound 7 with a retention time of 32.5 min, and compound 8 with a retention time of 34.8 min.

[0016] The present invention also provides the use of 13-hydroxypiericidin A 10-O-α-D-glucose (1→6)-β-D-glucoside, piericidi n A 10-O-β-D-glucoside (1→6)-α-D-galactose, 13-hydroxyglucopiericidin A, 4'-O-β-glucose gl ucopiericidin A (4'Glc GPA) or glucopiericidin A in the preparation of drugs for treating renal cancer, organ fibrosis, acute kidney injury and chronic kidney disease.

[0017] Preferably, the drug further contains acceptable excipients as a pharmaceutical composition.

[0018] The structural formula of compound 8 (glucopiericidinA) is shown in formula (I):

[0019]

[0020] The structural formula of the compound 5 (13-hydroxyglucopiericidinA) is shown in formula (II):

[0021]

[0022] The structural formula of the compound 6 (4'-O-β-glucose glucopiericidinA) is shown in formula (III):

[0023]

[0024] The structural formula of the compound 4 (piericidinA 10-O-β-D-glucoside (1→6)-α-D-galactose) is shown in formula (IV):

[0025]

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides a culture medium, a fermentation method and an application for producing pieridian glycoside by Streptomyces psammoticus SCSIO NS126 (deposit number: GDMCC No: 64524). By improving the fermentation method, a large yield of pieridian glycoside is obtained, which solves the problem of insufficient drug sources in the research and development of drugs for treating renal cancer, organ fibrosis, acute kidney injury and chronic kidney disease, and is of great significance to the development of China's marine drug resources.

[0028] The Streptomyces psammoticus SCSIO NS126 described in the present invention is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, the deposit date is April 19, 2024, and the deposit number is GDMCC No: 64524. Description of the drawings:

[0029] Figure 1 This is the LCMS analysis chart of the effective portion of the pieritin glycoside prepared in Example 1;

[0030] Figure 2 This is the H NMR spectrum (DMSO-d6, 700 MHz) of compound 1 (6″Glc-13OH-GPA) isolated and purified in Example 2;

[0031] Figure 3 This is the C NMR spectrum (DMSO-d6, 175 MHz) of compound 1 (6″Glc-13OH-GPA) isolated and purified in Example 2;

[0032] Figure 4 This is the H NMR spectrum (CD3OD, 700 MHz) of compound 2 (6"Gal-4'Glc GPA) isolated and purified in Example 2;

[0033] Figure 5 This is the C NMR spectrum (CD3OD, 175 MHz) of compound 2 (6"Gal-4'Glc GPA) isolated and purified in Example 2;

[0034] Figure 6 This is the H NMR spectrum (DMSO-d6, 700 MHz) of compound 4 (6″Gal-GPA) isolated and purified in Example 2;

[0035] Figure 7 This is the C NMR spectrum (DMSO-d6, 175 MHz) of compound 4 (6″Gal-GPA) isolated and purified in Example 2;

[0036] Figure 8 This is the H NMR spectrum (DMSO-d6, 700 MHz) of compound 5 (13OH-GPA) isolated and purified in Example 2;

[0037] Figure 9 This is the C NMR spectrum (DMSO-d6, 175 MHz) of compound 5 (13OH-GPA) isolated and purified in Example 2;

[0038] Figure 10 The H NMR spectrum (in DMSO-d6, 700 MHz) of compound 6 (4'Glc GPA) isolated and purified in Example 2;

[0039] Figure 11 The C NMR spectrum (DMSO-d6, 175 MHz) of compound 6 (4'Glc GPA) isolated and purified in Example 2;

[0040] Figure 12 This is the H NMR spectrum (CD3OD, 700 MHz) of compound 7 (7DeMe-GPA) isolated and purified in Example 2;

[0041] Figure 13 This is the C NMR spectrum (CD3OD, 175 MHz) of compound 7 (7DeMe-GPA) isolated and purified in Example 2;

[0042] Figure 14 This is the H NMR spectrum (DMSO-d6, 700 MHz) of compound 8 (GPA) isolated and purified in Example 2;

[0043] Figure 15 This is the C NMR spectrum (DMSO-d6, 175 MHz) of compound 8 (GPA) isolated and purified in Example 2;

[0044] Figure 16 This is the anti-acute kidney injury activity of compounds 1, 4, 5, 6, and 8 in Example 3. Specific implementation method:

[0045] It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention. However, many changes can be made in the disclosed specific embodiments and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0046] Example 1 Fermentation Method of Streptomyces psammoticus SCSIO NS126 and Preparation of Effective Fraction

[0047] Streptomyces psammoticus SCSIO NS126 was cultured in a seed medium containing 20 g of D-mannitol, 10 g of soy peptone, 2.5 g of soybean oil, 0.35 g of dipotassium hydrogen phosphate, and 1 L of sterilized water at pH 7.0. The culture was shaken at 28°C (180 rpm). After 48 hours of culture in the seed medium, the culture was scaled up to a 30 L fermentation volume per 1 L of seed medium in H9 medium containing 20 g of soluble starch, 10 g of cottonseed meal, 5 g of yeast extract powder, 20 g of maltodextrin, 5 g of malt extract powder, 2 g of anhydrous magnesium sulfate, 2 g of sodium chloride, 2 g of calcium carbonate, and 1 L of sterilized water at pH 7.0-7.2. The culture conditions were 28°C with shaking (180 rpm) for 120 hours to obtain a fermentation broth, which was extracted with ethyl acetate and concentrated to obtain an extract. 60 L of the fermentation broth yielded 24.78 g of the extract.

[0048] The extract was subjected to ODS reverse-phase column chromatography (methanol / water, v / v, gradient elution: 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 9:1, 10:0) to sequentially yield eight fractions, SP1-SP8. Each crude fraction was dissolved in methanol to prepare a 100 mg / mL solution, which was then analyzed by HPLC (0-50 min, 5% acetonitrile; 50.1-55 min, 100% acetonitrile, 3 mL / min, C18 column, 6 μL). The HPLC analysis revealed that pieridinic acid was primarily concentrated in fractions SP5-7. The extract obtained by combining these three fractions was the active pieridinic acid glycoside fraction, yielding 2.82 g (60 L of fermentation broth), representing 11.4% of the extract weight.

[0049] Example 2 LCMS analysis of the active fraction and structural identification of pieridium glycosides

[0050] The effective fraction of pieridinium was dissolved in methanol to prepare a 100 mg / mL solution, and LCMS analysis was performed (chromatographic column brand: Phenomenex; model: Luna 5μm C18(2)100A, 250*4.6mm; 0-50min, 5% acetonitrile; 50.1-55min, 100% acetonitrile, 1mL / min, 10μL, detection wavelength 210, 260, 280nm, Bruker, Billerica, MA, USA). Based on the analysis results, 10 main pieridinium compounds were obtained.

[0051] Molecular ion peak of compound 1 m / z 756.3774 [M+H] + , the molecular formula is derived as C 37 H57 NO 15 , suggesting that it is a hydroxylated pieridin disaccharide; the molecular ion peak of compound 2 is m / z 902.4342[M+H] + , the molecular formula is derived as C 43 H 67 NO 19 , suggesting that it is pieridium trisaccharide; the molecular ion peak of compound 4 is m / z 740.3884[M+H] + , the molecular formula is derived as C 37 H 57 NO 14 , suggesting that it is pieridium disaccharide; the molecular ion peak of compound 5 is m / z 594.3264[M+H] + , the molecular formula is derived as C 31 H 47 NO 10 , suggesting that it is a hydroxylated monosaccharide of pieridin; the molecular ion peak of compound 6 is m / z 740.3852[M+H] + , the molecular formula is derived as C 37 H 57 NO 14 , suggesting that it is pieridialmycin disaccharide; the molecular ion peak of compound 7 is m / z564.3183[M+H] + , the molecular formula is derived as C 30 H 45 NO9, suggesting demethylated pieridin monosaccharide; molecular ion peak of compound 8 m / z 578.3369 [M+H] + , the molecular formula is derived as C 31 H 47 NO9, suggesting that it is a monosaccharide of pieridialmycin; the molecular ion peak of compound 9 is m / z 402.2631 [M+H] + , the molecule is deduced to be C 24 H 36 NO4, suggesting demethylated pieridin aglycone; molecular ion peak of compound 10 m / z 416.2795 [M+H] + , the molecular formula is derived as C 25 H 37 NO4 indicates that it is a pierithromycin aglycone. Compounds 1-8 are pierithromycin glycoside compounds.

[0052] The active fraction was further separated and purified by semi-preparative HPLC (Infinity 1260 liquid chromatograph, YMC-pack ODS-A column, 10 mm × 250 mm, 5 μm, mobile phase v / v, acetonitrile: water = 60:40, flow rate 3 mL / min) to obtain pure compound 1 (t R =22min),2(tR =23min),4(t R =26.4min),5(t R =27.5min),6(t R =28.9min),7(t R =32.5min),8(t R =34.8min),9(t R =41.0min) and 10(t R =43.9min).

[0053] Compounds 9 and 10 are pieridin aglycones. The remaining pieridin glycosides were characterized by H NMR and C NMR ( Figure 2-Figure 15 ) analysis, which was consistent with the piericidin glycosides previously discovered by the inventors. Compound 1 was identified as 13-hydroxypiericidinA10-O-α-D-glucose(1→6)-β-D-glucoside(6”Glc-13OH-GPA); Compound 2 was identified as 4'-O-β-D-glucose piericidin A 10-O-α-D-glucose(1→6)-β-D-glucoside(6”Gal-4'Glc GPA); Compound 4 was identified as piericidin A 10-O-β-D-glucoside(1→6)-α-D-galactose(6”Gal-GPA); Compound 5 was identified as 13-hydroxyglucopiericidinA(13OH-GPA); Compound 6 was identified as 4'-O-β-glucoseglucopiericidinA(4'Glc GPA); Compound 7 was identified as 7-demethyl-13-hydroxyglucopiericidinA (7DeMe-GPA); Compound 8 was identified as glucopiericidinA (GPA), and its specific structure is shown in Figure 1 shown.

[0054] The present invention carries out 60L fermentation under the above fermentation conditions, and obtains GPA, 13OH-GPA, 4'Glc-GPA, and 6"Gal-GPA yields of 355 mg, 162 mg, 212 mg, and 330 mg, respectively, all of which are greater than 150 mg / 60L. The effective fraction contains more than 8 types of pieridinic acid glycosides, and the contents of the four types of pieridinic acid glycosides, namely compound 4, compound 5, compound 6, and compound 8, are all greater than 5%.

[0055] Comparative Example 1

[0056] Reference is made to the fermentation method described in (Journal of Medicinal Chemistry, 2019, 62, 7058), wherein the seed culture medium is formulated as follows: 1 g mannitol, 0.5 g soy peptone, 0.125 g soybean oil, 0.02 g K₂HPO₃, pH 7.0, and 50 mL distilled water; the fermentation medium is formulated as follows: 2.5 g cottonseed meal, 1 g soluble starch, 1 g glucose, 0.3 g yeast extract, 0.5 g CaCO₃, 0.2 g sea salt, and 100 mL distilled water, pH 7.2. A 60 L fermentation was performed under the fermentation conditions of Example 1.

[0057] Experimental results demonstrate that the content of pieridin glycosides obtained from a 60-L fermentation under the aforementioned conditions is significantly higher than the yield from a 60-L fermentation previously reported by the applicant (Table 1). Under this fermentation method, the strain is the most abundant wild strain (not genetically engineered) producing pieridin glycosides reported both domestically and internationally.

[0058] Table 1. Yields of various pieritin glycosides in the present invention and original yields reported previously

[0059] code name Compound Original output The output of the present invention 1 6”Glc-13OH-GPA 4.1mg 25mg 2 6' Gal-4' GlcGPA 1.5mg 19mg 4 6”Gal-GPA 6.5mg 330mg 5 13OH-GPA 28mg 162mg 6 4'Glc-GPA 2.5mg 212mg 7 7DeMe-GPA 2.9mg 21mg 8 GPA 328mg 355mg

[0060] Example 3 Anti-acute kidney injury activity of compounds 1, 4, 5, 6, and 8

[0061] According to the literature (Du Yiting et al. Chinese Journal of Critical Care Medicine, 2005, 17: 619-622.), a HK-2 cell hypoxia / reoxygenation injury model was established. HK-2 cells were divided into hypoxia groups for 4, 12 and 24 hours and hypoxia for 24 hours followed by reoxygenation for 4, 12 and 24 hours. A blank control group was set up in each experimental group. The hypoxic environment was created by covering with liquid paraffin sterilized at high temperature; the trypan blue uptake method was used to count cells and detect cell viability, and the lactate dehydrogenase (LDH) content in the culture medium was detected by biochemical method. The increase in LDH content indicates that ischemia-reperfusion injury has led to the destruction of cell membrane integrity and even irreversible damage to the cells. The experimental results showed that compounds 1, 4, 5, 6, 8 at a concentration of 5 μM (non-toxic concentration) can significantly restore the cell viability of HK-2 cells after hypoxia ( Figure 16 ), showing activity against acute kidney injury at the cellular level.

Claims

1. A fermentation method for producing pieridium glycoside compounds, characterized in that: The method comprises inoculating Streptomyces psammoticus SCSIO NS126 into an H9 culture medium for fermentation and culture. The deposit number of Streptomyces psammoticus SCSIO NS126 is GDMCC No: 64524. The H9 culture medium comprises the following ingredients: 20 g / L soluble starch, 10 g / L cottonseed powder, 5 g / L yeast extract powder, 20 g / L maltodextrin, 5 g / L malt extract powder, 2 g / L anhydrous magnesium sulfate, 2 g / L sodium chloride, 2 g / L calcium carbonate, and water as a solvent.

2. The fermentation method according to claim 1, characterized in that The pieridin glycoside compounds include: 13-hydroxypiericidin A 10-O-α-D-glucose(1→6)-β-D-glucoside、4'-O-β-D-glucose piericid in A 10-O-α-D-glucose(1→6)-β-D-glucoside、piericidin A 10-O-β-D-glucoside(1→6)-α-D-gala ctose, 13-hydroxyglucopiericidin A, 4'-O-β-glucoseglucopiericidin A, 7-demethyl-13-hydroxyglucopiericidin A and glucopiericidin A.

3. The fermentation method according to claim 1, characterized in that The fermentation culture is cultured under the following conditions: shaking on a shaker at 28° C. and 180 rpm.

4. The fermentation method according to claim 1, characterized in that The method comprises the following steps: culturing Streptomyces psammoticus SCSIO NS126 in a seed liquid culture medium for 48 hours at a temperature of 28° C. and 180 rpm on a shaking table; and then carrying out a scaled-up fermentation in an H9 culture medium at a temperature of 28° C. and 180 rpm on a shaking table for 7 days. The seed liquid culture medium comprises 20 g / L of D-mannitol, 10 g / L of soy peptone, 2.5 g / L of soybean oil, and 0.35 g / L of dipotassium hydrogen phosphate, and the solvent is water.

5. The fermentation method according to claim 4, characterized in that The following steps are involved: (1) Streptomyces psammoticus SCSIO NS126 was cultured in a seed liquid medium for 48 hours at 28°C with shaking at 180 rpm; then, the fermentation was scaled up in H9 medium, with each liter of seed liquid scaled up to a fermentation volume of 30 liters at 28°C with shaking at 180 rpm; after 7 days of culture, the fermentation liquid was extracted with ethyl acetate, and the extract was concentrated to obtain an extract; (2) The extract obtained in step (1) was subjected to ODS reverse phase column chromatography to obtain 8 fractions SP1-SP8, under the following conditions: methanol / water, with a volume ratio of 1:9, 1:4, 3:7, 2:3, 1:1, 3:2, 9:1, 10:0 gradient elution, and SP1-8 fractions were obtained in sequence; the three fractions SP5-7 were combined and concentrated to obtain the effective part; (3) The active fraction was separated and purified by semi-preparative HPLC with a mobile phase of acetonitrile / water at a volume ratio of 60:40 and a flow rate of 3 mL / min. The compound 13-hydroxypiericidin A 10-O-α-D-glucose(1→6)-β-D-glucoside was obtained with a retention time of 22 min and a molecular formula of C 37 H 57 NO 15 The compound 4'-O-β-D-glucosepiericidinA 10-O-α-D-glucose(1→6)-β-D-glucoside with a retention time of 23 min has a molecular formula of C 43 H 67 NO 19 The compound piericidinA 10-O-β-D-glucoside(1→6)-α-D-galactose with a retention time of 26.4 min has a molecular formula of C 37 H 57 NO 14 The compound 13-hydroxyglucopiericidinA with a retention time of 27.5 min and a molecular formula of C 31 H 47 NO 10 The compound 4'-O-β-glucose glucopiericidinA with a retention time of 28.9 min and a molecular formula of C 37 H 57 NO 14 The compound 7-demethyl-13-hydroxyglucopiericidinA with a retention time of 32.5 min and a molecular formula of C 30 H 45 NO9; The compound glucopiericidinA with a retention time of 34.8 min and a molecular formula of C 31 H 47 NO9.

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