WO2023020501A1 - Method for extracting fr901464 from burkholderia fermentation broth - Google Patents

Abstract

Provided in the present invention is a method for extracting FR901464 from a fermentation broth. The fermentation broth is obtained by means of fermenting Burkholderia. A fermentation method comprises: adding a macroporous resin to the fermentation broth, and subjecting the mixture to adsorption and then filtration; soaking and separating the filtered macroporous resin with an organic solvent, and concentrating the separated organic solvent; dissolving the concentrated product with a mixed solvent of n-hexane and ethyl acetate, performing chromatographic separation by means of silica gel, and then performing elution with ethyl acetate to obtain an eluent; concentrating the eluent into a paste, and then formulating same into a column loading solution which is separated and purified by means of a high-pressure liquid chromatograph, wherein the filler is a C18 filler, and performing elution with a liquid-phase eluent to obtain an eluent; and extracting the eluent and then collecting an organic phase, and evaporating same until dry to form a solid powder.

Description

A method for extracting FR901464 from Burkholderia fermented liquid technical field

The invention relates to the technical field of fermentation broth purification, in particular to a method for extracting FR901464 from Burkholderia fermentation broth.

Background technique

FR901464 is a natural compound with high anti-tumor activity. It was originally isolated and purified from Pseudomonas sp. by fermentation in 1996 by Nakajima et al. (Nakajima, H.; Sato, B.; Fujita, T. ; Takase, S.; Terano, H.; Okuhara, M.Antitumor substances, FR901463, FR901464 and FR901465.I. taxonomy, fermentation, isolation, physico-chemical properties and biological activities[J].J.Antibiot.,1996, 49:1196-1203.), wherein, the Pseudomonas sp.No.2663 fermentation output described in the text is about 360mg/L. Its chemical structural formula is shown in Formula 1.

As a splicing inhibitor of precursor mRNA in eukaryotic cells, FR901464 has a strong inhibitory effect on the growth of tumor cells. Activity tests show that FR901464 has a very significant inhibitory effect on human solid tumor cells. The IC50 value of the experimental test is 0.3 -3.4nM. Because FR901464 exhibits extremely high anti-tumor activity and its unique physiological activity, it has attracted widespread attention from scientists as soon as it was discovered.

Although Nakajima H etc. disclosed the purification method in "New antitumor substances, FR901463, FR901464 and FR901465 I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities", this method needs to consume a large amount of ethyl acetate for extraction, and High purity FR901464 could not be obtained through the applicant's attempts.

Contents of the invention

In order to overcome the above problems, the present invention provides a method for extracting FR901464 from fermentation broth, characterized in that:

The fermented liquid is fermented by Burkholderia, and the extraction method comprises the following steps:

Step 1: Add the macroporous resin to the fermentation broth, mix and absorb and filter;

Step 2: Soak and separate the macroporous resin obtained by filtration with an organic solvent, and concentrate the separated organic solvent;

Step 3: dissolving the product concentrated in step 2 with a mixed solvent, separating and filtering in a silica gel column, and rinsing with eluent A to obtain an eluent;

Step 4: Concentrate the eluate to a creamy state, and then make it into a column liquid, and use eluent B to separate and purify by high-pressure liquid chromatography.

Preferably, step 4 is followed by step 5: the eluate in step 4 is extracted and the organic phase is collected and evaporated to dryness to form a solid powder.

Preferably, the packing of the high pressure liquid chromatograph is C18 packing.

Preferably, in step 1, the macroporous resin is added to the fermentation broth and then stirred.

Preferably, in step 1, the adsorption time is not less than 3h, or 4h.

Preferably, in step 2, the soaking time is not less than 1h, or 2h.

Preferably, in step 2, the macroporous resin is soaked with organic solvent twice or more.

Preferably, the mixed solvent is formed by mixing a polar solvent and a non-polar solvent.

Preferably, the polar solvent is selected from alcohols, acids, fats, nitriles, water, preferably isopropanol, n-butanol, tetrahydrofuran, chloroform, ethanol, ethyl acetate, methanol, acetone, acetonitrile, acetic acid , more preferably ethyl acetate; the non-polar solvent is selected from saturated hydrocarbons, benzene, preferably n-hexane.

Preferably, n-hexane and ethyl acetate are mixed to form the mixed solvent, wherein the volume fraction of ethyl acetate is not greater than 70%.

Preferably, in step 4, the upper column solution is prepared with 30% volume fraction of acetonitrile.

Preferably, the organic solvent in step 2 is selected from ethyl acetate, dichloromethane, and chloroform.

Preferably, the eluent A is ethyl acetate, and the eluent B is selected from acetonitrile or methanol in water.

Preferably, the eluent B is an acetonitrile solution with a volume concentration of 30%-45%, or 30%-40%.

Preferably, step 3.1 is included between step 3 and step 4: concentrate the eluate obtained in step 3 to a paste, and repeat step 3 to obtain the eluate.

Preferably, the specification of the silica gel filler in the silica gel column is 100-200 mesh, and the pore diameter is

Preferably, in step 4, the flow rate of the upper column liquid in the high pressure liquid chromatograph is 50-70ml/min, preferably 70ml/min.

Preferably, in step 5, the collected organic phase is dehydrated with anhydrous magnesium sulfate, filtered and then evaporated to dryness to form a solid powder.

The present invention is applicable to the fermentation broth obtained by fermentation of any Burkholderia sp. available on the market, preferably, the Burkholderia sp. HDCC00024, preserved In the General Microorganism Center (CGMCC) of the China Committee for the Collection of Microorganisms, the preservation number is CGMCC NO.22290, and the preservation date is May 08, 2021.

Beneficial effect:

1. By adopting the purification method of the present invention, the purity of FR901464 in the final product is high;

2. The operation steps of the purification method of the present invention are simple. Suitable for industrial production;

3. Using high-pressure preparative liquid chromatography and mobile phase with specific solvent and concentration can effectively remove FR901464 analogues with similar structures and difficult to separate;

4. Most of the more polar and pigment impurities can be removed through the silica gel filler.

Description of drawings

Fig. 1 is the liquid chromatogram of fermented liquid;

Fig. 2 is the liquid chromatogram of finished powder of FR901464 in embodiment 8.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Unless otherwise specified, the materials and reagents used in the following examples are commercially available and can be purchased in the market.

The present invention will be further described through examples below, and these descriptions are not intended to further limit the content of the present invention. Those skilled in the art should understand that equivalent replacements or corresponding improvements made to the contents of the present invention still fall within the protection scope of the present invention.

The purity of acetonitrile, ethyl acetate, dichloromethane, chloroform, n-hexane and methanol used in the purification is industrial grade.

The fermentation broth involved in the following examples was obtained according to the patent application with the application number CN202110681279.4, and the strain used was Burkholderia sp. HDCC00024 (CGMCC NO.22290). The specific fermentation method is:

(1) Preparation and cultivation of slant strains:

Slant medium formula (g/L): yeast extract powder 4.0g/L, malt extract 10.0g/L, glucose 4.0g/L, agar 20.0g/L, pH before elimination 7.2～7.4, test tube 30× 200mm, filling volume 15mL, sterilized at 121°C for 20 minutes, cooled to about 55-60°C and placed on a slope, after cooling and solidification, inoculated on the slope, cultivated at 28±1°C for 3 days, the strains matured.

(2) Preparation and cultivation of seed solution:

Seed medium formula (g/L): glucose 30g/L, sorbitol 5g/L, cottonseed cake powder 10g/L, yeast extract powder 20g/L, calcium chloride 10g/L, magnesium sulfate 10g/L, phosphoric acid Potassium dihydrogen 1g/L, pH 7.0 before disinfection; 250mL Erlenmeyer shaker flask, filled with 50mL, sterilized at 121°C for 20min. Inoculate 10 ⁷ ～10 ⁸ cfu/mL into the seed medium, culture at 28±1°C, shake at 250 rpm for 24 hours, at this time, the pH of the culture solution is 6.8-7.2, and the OD600 of the bacteria is 15-20.

(3) Preparation and cultivation of fermentation medium:

Corn starch 20g/L, glucose 30g/L, sorbitol 10g/L, mannitol 10g/L, yeast extract powder 6g/L, soybean cake powder 8g/L, cottonseed cake powder 7g/L, magnesium sulfate 3g/L , Potassium dihydrogen phosphate 6g/L, potassium chloride 3g/L, calcium chloride 3g/L. The pH before disinfection is 6.0. A 250mL Erlenmeyer shaker flask should contain 20mL and be sterilized at 121°C for 20min. The seed solution was added with an inoculation amount of 10% (volume ratio). Culture at 26±1° C. with shaking at 250 rpm for 96 hours.

Embodiment 1 macroporous resin adsorption

Take four groups of fermentation broths, add macroporous resins of different specifications according to the following table respectively, after stirring and absorbing, take the fermentation supernatant, and detect the titer of FR901464 in the supernatant by HPLC method.

Adsorption rate (%) = 100% - supernatant titer/fermentation pot titer * 100%

Embodiment 2 solvent extraction

Obtain the mixture of fermented liquid and macroporous resin LX-30 according to the adsorption method of the first group in the above-mentioned Example 1, and obtain the macroporous resin LX-30 through vibrating sieve separation, and add the extraction solvent to the macroporous resin according to the following table After stirring in LX-30 for 2h, separate and collect the extract. The potency of FR901464 in the extract was detected by HPLC.

Extraction yield = extraction solution titer * extraction solution volume / (resin weight * resin titer) * 100%

Embodiment 3 Silica gel separation

Referring to Example 2, in 1.27 kg of adsorbed macroporous resin LX-30, 2.5 L of ethyl acetate was added for extraction, after stirring for 2 hours, the extraction solution was separated, and the extraction solution was co-extracted twice to obtain 4.7 L of mixed extraction solution. , after standing still for liquid separation, 4.5 L of organic phase was obtained. At 40 °C, the organic phase was concentrated to a paste by a rotary evaporator.

Take the above-mentioned paste, dissolve and filter according to the mixed solvent of n-hexane and ethyl acetate in the following table, and prepare the paste with a mixed solvent to about 3g/L. The solution is filled with silica gel (100-200 mesh, pore size

) for chromatographic separation.

n-Hexane: ethyl acetate (v:v)	Adsorption rate(%)
30:70	98.9
40:60	100
50:50	100

The results showed that during the chromatography process, when the ratio of ethyl acetate in the upper column solution increased, the silica gel adsorption rate decreased. When the ratio was 30:70, FR901464 leaked out from the upper column filtration.

Through this step, the less polar impurities flow out with the liquid, and FR901464 and more polar substances will be adsorbed in the silica gel.

Embodiment 4 Silica gel separation

Referring to Example 3, the paste was dissolved in a mixed solvent of n-hexane:ethyl acetate (v:v) of 50:50, filtered and then chromatographically separated with a silica gel filler, and then the FR901464 adsorbed on the silica gel was separated with ethyl acetate Eluted off, while more polar impurities and pigments are adsorbed on silica gel. The purity of FR901464 in the eluent was measured by the peak area distribution of HPLC (ie the peak area of FR901464 accounted for the percentage of the total peak area, the purity in the following examples were all obtained by this method), and the result was 58.25%. The eluate was concentrated to a paste by a rotary evaporator at 40 °C. The above steps were repeated to obtain an eluate with a purity of 75.68%, which was concentrated to a paste and prepared as a column liquid with 30% volume fraction of acetonitrile.

Embodiment 5 High pressure liquid chromatography separation and purification

With the upper column liquid in embodiment 4, carry out high-pressure liquid chromatography separation and purification, filler is Huapu C18 filler, and liquid phase eluent uses acetonitrile and methanol respectively, and solvent volume concentration is 35% in the eluent, and flow velocity is 70ml/min, the column temperature is room temperature. The experimental data are shown in the table below, and the amount of eluent in the table is the multiple of the packing volume.

eluent	Qualified component purity (%)	Eluent volume (BV)
35% acetonitrile	95.68	1

35% methanol

86.30

3

Embodiment 6 High pressure liquid chromatography separation and purification

Carry out the separation and purification of high pressure liquid chromatography with the upper column liquid in embodiment 4, filler is Huapu C18 filler, use the acetonitrile of 4 kinds of different volume concentrations respectively as eluent, flow velocity is 70ml/min, and column temperature is room temperature . The results show that the concentration of acetonitrile is 30%-40%, and the effect is better. The experimental results are as follows:

Acetonitrile concentration (%)	Qualified component purity (%)	Yield (%)
30	97.08	74.34
35	95.91	69.74
40	95.44	62.73
45	92.01	40.36

Embodiment 7 high pressure liquid chromatograph separation and purification

Using the upper column liquid in Example 4, carry out separation and purification by high-pressure liquid chromatography, the eluent is 35% acetonitrile, and separate with 3 kinds of C18 fillers (Nanomicro C18 fillers, Huapu C18 fillers, Kromasil C18 fillers) respectively In the purification experiment, the flow rate was 70ml/min, and the column temperature was room temperature. The experimental results are as follows:

Packing type	Qualified component purity (%)	Yield (%)
Nano micro C18 filler	95.55	65.81
Huapu C18 filler	95.91	69.74
Kromasil C18 filler	96.03	63.18

Embodiment 8 High-pressure liquid chromatograph separation and purification

Using the upper column liquid in Example 4, carry out separation and purification by high-pressure liquid chromatography, the eluent is 35% acetonitrile, the filler is Huapu C18 filler, the flow rate is 70ml/min, and the column temperature is room temperature to obtain a purity of 96.14%. eluent. The eluent was extracted with ethyl acetate, and the organic phase was obtained by static separation. The organic phase was dehydrated with anhydrous magnesium sulfate and filtered. The dehydrated organic phase was concentrated to a solid powder with a rotary evaporator at 40° C., with a purity of 96.75% and a total yield of 26.6%.

Example 9

Step 1: Take 25L of FR901464 fermentation broth, add 2.5kg of macroporous resin HP20, stir for 12 hours, take a sample and detect that the content of FR901464 in the fermentation broth is 11.34ug/ml. Then separate fermentation broth and macroporous resin with vibrating sieve, obtain 2.3kg macroporous resin HP20.

Step 2: Add 4.2 L of chloroform to the separated 2.3 kg macroporous resin HP20 for leaching. The leaching time is 4 hours, and leaching is performed twice to obtain 8.2 L of mixed extracts.

Step 3: The mixed extracts were left to stand for liquid separation to obtain 8 L of organic phase. The organic phase was concentrated by a rotary evaporator at 40° C., and the mixed extract was concentrated to a paste.

Step 4: Dissolve the paste with a mixed solvent of n-hexane and ethyl acetate (volume ratio 50:50) and filter. The filtrate is chromatographically separated with a silica gel packing to obtain an eluent with a purity of 57.22%. The eluate was concentrated by a rotary evaporator at 40° C., and the eluate was concentrated to a paste.

Step 5: Step 4 was repeated to obtain an eluate with a purity of 68.68%. The eluate was concentrated by a rotary evaporator at 40° C., and the eluate was concentrated to a paste.

Step 6: The above paste was prepared as a column liquid with 30% volume concentration of acetonitrile. The upper column liquid is separated and purified by high-pressure liquid chromatography, and the filler is Kromasil C18 filler. Elution with 40% volume concentration of acetonitrile in water gave a purity of 95.68%.

Step 7: The eluate in step 6 was extracted with ethyl acetate, and left to separate liquids to obtain an organic phase. The organic phase was dehydrated with anhydrous magnesium sulfate and filtered. The dehydrated organic phase was concentrated to a solid powder with a rotary evaporator at 40° C., with a purity of 95.66% and a total yield of 25.1%.

Example 10:

Step 1: Take 25L of FR901464 fermentation broth, add 2kg of macroporous resin HZ818, stir for 8 hours, take a sample to detect that the content of FR901464 in the fermentation broth is 126ug/ml. Then, separate the fermented liquid and the macroporous resin with a vibrating sieve to obtain 1.8 kg of macroporous resin HZ818.

Step 2: Add 3.6 L of dichloromethane to 1.8 kg of macroporous resin HZ818 for leaching. The leaching time is 2 hours, and leaching is performed twice to obtain 7.1 L of mixed extracts.

Step 3: The mixed extracts were left to stand for liquid separation to obtain 7L of organic phase. The organic phase was concentrated by a rotary evaporator at 40° C., and the mixed extract was concentrated to a paste.

Step 4: Dissolve the paste with a mixed solvent of n-hexane and ethyl acetate (volume ratio 40:60) and filter. The filtrate was chromatographed with a silica gel packing to obtain an eluate with a purity of 52.23%. The eluate was concentrated by a rotary evaporator at 40° C., and the eluate was concentrated to a paste.

Step 5: Step 4 was repeated to obtain an eluate with a purity of 68.37%. The eluate was concentrated by a rotary evaporator at 40° C., and the eluate was concentrated to a paste.

Step 6: The above-mentioned paste is prepared into a column liquid with 30% volume fraction of acetonitrile. The upper column liquid is separated and purified by high-pressure liquid chromatography, and the filler is nano-micro C18 filler. Elution with 35% volume concentration of acetonitrile in water gave a purity of 96.30%.

Step 7: The eluate in step 6 was extracted with ethyl acetate, and left to separate liquids to obtain an organic phase. The organic phase was dehydrated with anhydrous magnesium sulfate and filtered. The dehydrated organic phase was concentrated to a solid powder with a rotary evaporator at 40° C., with a purity of 96.02% and a total yield of 27.2%.

Claims (10)

1. A method for extracting FR901464 from fermentation broth, characterized in that:

   The fermented liquid is fermented by Burkholderia, and the extraction method comprises the following steps:

   Step 1: Add the macroporous resin to the fermentation broth, mix and absorb and filter;

   Step 2: Soak and separate the macroporous resin obtained by filtration with an organic solvent, and concentrate the separated organic solvent;

   Step 3: dissolving the product concentrated in step 2 with a mixed solvent, separating and filtering in a silica gel column, and rinsing with eluent A to obtain an eluent;

   Step 4: Concentrate the eluate to a creamy state, and then make it into a column liquid, and use eluent B to separate and purify by high-pressure liquid chromatography.
2. The method according to claim 1, characterized in that:

   The mixed solvent is formed by mixing polar solvents and non-polar solvents.
3. The method according to claim 2, characterized in that:

   The polar solvent is selected from alcohol, acid, fat, nitrile, water, preferably selected from isopropanol, n-butanol, tetrahydrofuran, chloroform, ethanol, ethyl acetate, methanol, acetone, acetonitrile, acetic acid, more preferably ground is ethyl acetate;

   The non-polar solvent is selected from saturated hydrocarbons, benzene, preferably n-hexane.
4. The method according to claim 3, characterized in that:

   The mixed solvent is formed by mixing n-hexane and ethyl acetate, wherein the volume fraction of ethyl acetate is not more than 70%.
5. The method according to claim 4, characterized in that:

   In step 4, use 30% volume fraction of acetonitrile to prepare the upper column solution.
6. The method according to claim 5, characterized in that:

   Eluent A is ethyl acetate,

   Eluent B is selected from acetonitrile or methanol in water.
7. The method according to claim 6, characterized in that:

   The organic solvent in step 2 is selected from ethyl acetate, dichloromethane, chloroform.
8. The method according to claim 7, characterized in that:

   The eluent B is an acetonitrile solution with a volume concentration of 30%-45%, or 30%-40%.
9. The method according to claim 8, characterized in that:

   The specification of the silica gel packing in the silica gel column is 100-200 mesh, the pore diameter

   
10. The method according to claim 8, characterized in that:

    In step 4, the flow rate of the upper column liquid in the high pressure liquid chromatograph is 50-70ml/min, preferably 70ml/min.

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