CN112876537A - Method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longilineans - Google Patents
Method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longilineans Download PDFInfo
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Abstract
The invention discloses a method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans, which is characterized by comprising the following steps: preparing a longitudinal stripe xylaria crassipes loading solution; pretreating D101 resin, filling the pretreated D101 resin into a column, and adding sample loading liquid for hydrodynamic adsorption; kinetic desorption of cyclopeptide Xylastranamide A from Xylastramide A in Xylastraria longissima by resin. By adopting the method, the component purity of the cyclopeptide Xylastramide A purified by the D101 resin is greatly improved, the enrichment and purification effects are obvious, the operation is simple and convenient, the separation steps are few, the organic solvent consumption is small, the time is short, and the enriched and purified cyclopeptide Xylastramide A has obvious antitumor and nematicidal activities and is suitable for related medicaments.
Description
Technical Field
The invention belongs to separation and purification of organic compounds, and relates to a method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans.
Background
Xylaria longissima (Xylaria striata Pat.1887) is a fungus of the phylum Ascomycota (Ascomycota) Xylariaceae (Xylariaceae) Xylaria (Xylaria Hill ex Schrank) and grows mainly on rotten bark and live roots. The xylaria fungi has great research, medicinal and edible values, for example, xylaria nigripes (commonly called Wulingshen) has the effects of dehumidifying, tranquilizing and allaying excitement, generating blood, improving the immune function of the organism and the like, and the health-care effect is excellent. The xylaria longipes as a fungus discovered by the applicant has been researched on the aspects of safety and sleep improvement of the xylaria longipes, the culture condition of the fruiting body of the xylaria longipes is optimized, the biological activity of the xylaria longipes is detected, and the xylaria longipes has good anti-tumor activity. In the prior art, rawland 2017 discloses a research on chemical components and biological activity of a xylaria longipes fruiting body, and the rawland discloses a novel cyclopeptide Xylastrimide A shown as a formula (I) separated from xylaria longipes, wherein the cyclopeptide A is used as a novel cyclopeptide substance and is found to have remarkable anti-tumor activity, certain anti-insect activity on caenorhabditis elegans and the like.
In the prior art, the separation method of cyclic peptide compounds generally adopts a traditional separation method, namely a series of chromatographic techniques are used for purification, so that monomer substances are obtained. In the prior art, high performance liquid chromatography (HPLC for short) is generally adopted to simply judge the purity of a compound, and after the sample liquid of xylaria longilineans obtained by the existing extraction is repeatedly detected by HPLC, the peak area ratio of xylastrinamide a is found to be 4.13% on average, namely the purity of xylastrinamide a is 4.13% on average.
The cyclopeptide Xylastrimide A is white needle-like fine crystal (the compound can be crystallized in a methanol solution), and the molecular formula is C45H64N6O9The chemical structural formula is shown as the following formula (I):
at present, no literature report of an adsorption-desorption method of cyclopeptide Xylastrimide A on macroporous adsorption resin in Xylastrium longilineatum is found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes. The invention obtains a mature process step and process parameters by analyzing and researching the adsorption kinetics of the D101 macroporous resin, thereby providing a method for enriching and purifying the cyclopeptide Xylastramide A from the xylaria longipes with less separation steps, less organic solvent consumption, short time and higher yield.
The content of the invention is as follows: a method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans is characterized by comprising the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the longitudinal stripe xylaria sporocarp with 20mL of ethanol water solution with the volume percentage concentration of 60-95% according to the proportion of adding the longitudinal stripe xylaria sporocarp into the 20mL of ethanol water solution with the volume percentage concentration of 60-95%, soaking for 2 hours at the temperature of 60-85 ℃, filtering, treating the solid for 1-4 times through the soaking process, and combining the filtrate; concentrating the filtrate by rotary evaporation to obtain an extract, and dissolving the extract in distilled water to obtain a solution with the concentration of 0.0010-0.0070 g/mL, namely the prepared xylaria longilineans sample solution for later use;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 80-95 vol% ethanol water solution for 15-30 h to fully swell the resin, filling the resin into a column by a wet method, washing the resin with ethanol until the effluent is not turbid, and washing the resin with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage can be 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent sodium hydroxide aqueous solution (the dosage can be 2BV) for 4 hours, and then washing the resin to be neutral by distilled water to obtain pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 50-300 mL of sample loading solution with the concentration of 0.0010-0.0070 g/mL, and dynamically adsorbing at the volume flow of 1-5 BV/h;
the BV, called volume, represents the resin bed volume, and for resins, BV is usually taken as a unit, so that the data can be changed with different resin bed volumes; 1BV means 1 time of the resin bed volume; the volume of the solution flowing per hour is 1-5 times of the volume of the resin bed by 1-5 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin:
and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step (b) reaches a leakage point), stopping adding the sample loading liquid, eluting for 1-5 BV by using an ethanol water solution with the volume percentage concentration of 10-50%, eluting for 1-5 BV by using an ethanol water solution with the volume percentage concentration of 60-100% (or an ethanol water solution with the volume percentage concentration of 60-95%), eluting at the rate of 1-5 BV/h, collecting the eluent, adding an ethanol water solution with the same volume percentage concentration, adjusting the volume of the eluent to be 150mL, and thus obtaining the desorption liquid after enrichment of the purified cyclopeptide Xylastrimide A.
The definition of the leak point is: the leak point is reached when the concentration of target in the effluent (in the context of the present invention the target is the cyclopeptide xylastramide a) reaches 10% of the target concentration in the loading solution.
After the longitudinal striated xylaria sporocarp is treated by the method for enriching and purifying the cyclopeptide Xylastrimide A from the longitudinal striated xylaria, a large amount of impurities can be removed, target components are reserved, and the purity of the target components (namely the cyclopeptide Xylastrimide A) is effectively improved; the pure cyclopeptide Xylastramide A can be obtained by further separation operation, and the separation step can be effectively simplified by adopting the macroporous resin purification method.
The content determination method of the cyclopeptide Xylastrimide A has the following set of chromatographic conditions: AgiLent ZORBAX EcLipse XDB-C18 (high performance liquid chromatography column), 4.6mm × 250mm, 5 um; mobile phase: the mobile phase A is acetonitrile, the mobile phase B is ultrapure water, and isocratic elution is carried out according to the procedures of: 0-20 min, 65% of A and 35% of B; detection wavelength: 216 nm; the column temperature is 35 ℃; flow rate: 1.0 mL/min; sample introduction amount: 15 uL; the peak-off time was 14.915 min.
In the context of the present invention, 1BV is 30 mL.
In the content of the invention, the production and provision enterprises of the D101 resin (or D101 macroporous resin) comprise: west reagent, Anhui Saxing resin science and technology, Inc., Tianjin Kai resin science and technology, Inc., and the like.
The invention comprises the following steps: the step a of preparing the xylaria longilineans sample loading liquid preferably comprises the following steps: taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the xylaria striata sporocarp with 20mL of 75-95 vol% ethanol water solution according to the proportion of 1g of xylaria striata sporocarp, soaking the xylaria striata sporocarp and the 75-95 vol% ethanol water solution for 2 hours at the temperature of 70-80 ℃, filtering, treating the solid for 1-3 times through the soaking process, and combining the filtrates; and (3) concentrating the filtrate by rotary evaporation to obtain an extract, and dissolving the extract in distilled water to prepare a solution with the concentration of 0.0010-0.0070 g/mL, namely the prepared xylaria striata sample solution for later use.
The invention comprises the following steps: the step a of preparing the xylaria longilineans sample loading liquid preferably comprises the following steps: taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the longitudinal stripe xylaria sporocarp with 90% ethanol water solution with volume percentage concentration of 20mL according to the proportion of adding the longitudinal stripe xylaria sporocarp into the 90% ethanol water solution with volume percentage concentration of 1g, soaking for 2h at the temperature of 80 ℃, filtering, treating the solid for 1-2 times through the soaking process, and combining the filtrates; and (3) concentrating the filtrate by rotary evaporation to obtain an extract, and dissolving the extract in distilled water to prepare a solution with the concentration of 0.0010-0.0070 g/mL, namely the prepared xylaria striata sample solution for later use.
The invention comprises the following steps: the resin in the step b has better dynamic adsorption to cyclopeptide Xylastrimide A in the xylaria longilineans:
1) pretreatment of D101 resin: soaking the D101 resin in 95% ethanol water solution for 24 hr to swell fully, loading into column by wet method, washing with ethanol until the effluent is not turbid, and washing with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage can be 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent sodium hydroxide aqueous solution (the dosage can be 2BV) for 4 hours, and then washing the resin to be neutral by distilled water to obtain pretreated D101 resin for later use;
2) dynamic adsorption: and (3) loading 18g of the pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 210mL of sample loading solution with the concentration of 0.0030g/mL, and dynamically adsorbing at the volume flow of 2 BV/h.
The invention comprises the following steps: the dynamic desorption of the cyclic peptide Xylastramide A in the xylaria longilineans by the resin in the step c is better as follows: and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 3BV by using an ethanol water solution with the volume percentage concentration of 40%, eluting for 3BV by using absolute ethyl alcohol, eluting at the rate of 2BV/h, collecting the part of eluent, adding the absolute ethyl alcohol, and adjusting the volume of the eluent to be 150mL to obtain the desorption solution after enriching and purifying the cyclopeptide Xylastramide A.
Compared with the prior art, the invention has the following characteristics and beneficial effects:
(1) according to the invention, a D101 resin (or called D101 macroporous resin) adsorption and purification technology is adopted, dynamic adsorption and desorption purification process parameters of the cyclopeptide Xylastramide A are optimized, HPLC (high performance liquid chromatography) is used as a detection means, the influence of the D101 resin on the purification effect of the cyclopeptide Xylastramide A is researched, and a practical technical scheme is provided for the development and utilization of Xylastramide A;
(2) the method takes the adsorption rate and the resolution rate of cyclopeptide Xylastramide A in the xylaria longilineans as investigation indexes, adopts a single-factor test combined with a D101 macroporous resin technology to analyze the xylaria longilineans medicinal material, and screens out the optimal enrichment and purification process conditions;
(3) according to the invention, by comparing the HPLC (high performance liquid chromatography) spectrum of the cyclopeptide Xylastramide A, the purity of the cyclopeptide Xylastramide A (generally, the purity of the compound is simply judged by adopting the high performance liquid chromatography, and the purity of the cyclopeptide Xylastramide A can be found according to the peak area proportion of the cyclopeptide Xylastramide A in the high performance liquid chromatography) is averagely improved to 39.63% from 4.13% in the prior art, so that the purity of the cyclopeptide Xylastramide A component purified by the D101 macroporous resin is greatly improved, and the enrichment and purification effects are obvious;
(4) the method is stable and feasible, simple and convenient to operate, less in separation steps, less in organic solvent consumption, short in time, higher in yield, suitable for enriching and purifying the cyclopeptide Xylastrimide A in the xylaria striata and high in practicability.
Drawings
FIG. 1 is an HPLC chart before purification of the cyclopeptide Xylastrimide A;
FIG. 2 is an HPLC chart of the purified cyclic peptide Xylastramide A obtained in example 3; the figure shows the ratio of xylastatriamide A after purification. From comparison of HPLC charts before and after purification, it can be seen that the ratio of the peak area of Xylastrimide A after purification is increased.
Detailed Description
The following examples are intended to further illustrate the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims appended hereto.
Example 1:
a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes comprises the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking 100g of Xylaria longipes sporocarp (from the microbiological laboratory of southwest science and technology university, the same way later), crushing, and sieving with a 40-mesh sieve; mixing the longitudinal stripe xylaria sporophore and 20mL of 90% ethanol water solution with volume percentage concentration according to the proportion that 1g of the longitudinal stripe xylaria sporophore is added with 20mL of 90% ethanol water solution with volume percentage concentration, soaking for 2 hours at the temperature of 80 ℃, filtering, treating the solid for 2 times through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0030g/mL, i.e. the obtained Xylaria longipes sample solution;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 95% ethanol water solution for 24 hr to swell fully, loading into column by wet method, washing with ethanol until the effluent is not turbid, and washing with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage is 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent by mass of sodium hydroxide aqueous solution (the dosage is 2BV) for 4 hours, and then washing the resin by distilled water to be neutral to prepare pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 210mL of sample loading solution with the concentration of 0.0030g/mL, and dynamically adsorbing at the volume flow of 2 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin: and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 3BV by using an ethanol water solution with the volume percentage concentration of 40%, eluting for 3BV by using absolute ethyl alcohol, eluting at the rate of 2BV/h, collecting the part of eluent, and adding the absolute ethyl alcohol to adjust the volume of the eluent to be 150mL, thus obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A. The Xylastramide A was 40.61% pure as determined by HPLC.
After the D101 resin is enriched and purified, a large amount of impurities can be obviously removed, target components are reserved, and the purity of the target components is effectively improved; then further separation operation is carried out, so that pure Xylastrimide A can be obtained, and the D101 resin purification method can effectively simplify the separation step.
Example 2:
a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes comprises the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking 100g of longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the Xylaria longissima sporophore with 80 vol% ethanol water solution at a ratio of 1g Xylaria longissima sporophore and 20mL 80 vol% ethanol water solution, soaking at 85 deg.C for 2h, filtering, treating the solid for 2 times, and mixing filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0050g/mL, and collecting the sample solution of Xylaria longissima;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 80 vol% ethanol water solution for 20h to fully swell the resin, packing the resin into a column by a wet method, washing the column with ethanol until the effluent is not turbid, and washing the column with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage is 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent by mass of sodium hydroxide aqueous solution (the dosage is 2BV) for 4 hours, and then washing the resin by distilled water to be neutral to prepare pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of a resin bed column is 30mL, adding 100mL of sample loading solution with the concentration of 0.0050g/mL, and dynamically adsorbing at the volume flow of 2 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin:
and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 5BV by using an ethanol water solution with the volume percentage concentration of 30%, eluting for 4BV by using an ethanol water solution with the volume percentage concentration of 90%, eluting at the rate of 2BV/h, collecting the part of eluent, adding an ethanol water solution with the same volume percentage concentration, adjusting the volume of the eluent to be 150mL, and thus obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A. The Xylastramide A was 36.58% pure as determined by HPLC.
Example 3:
a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes comprises the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking 100g of longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the longitudinal stripe xylaria sporophore and 20mL of 85% ethanol water solution with volume percentage concentration according to the proportion that 1g of the longitudinal stripe xylaria sporophore is added with 20mL of 85% ethanol water solution with volume percentage concentration, soaking for 2 hours at the temperature of 70 ℃, filtering, treating the solid for 3 times through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0010g/mL, and collecting the sample solution of Xylaria longissima;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 90% ethanol water solution for 18h to fully swell the resin, filling the resin into a column by a wet method, washing the resin with ethanol until the effluent is not turbid, and washing the resin with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage is 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent by mass of sodium hydroxide aqueous solution (the dosage is 2BV) for 4 hours, and then washing the resin by distilled water to be neutral to prepare pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 300mL of sample loading solution with the concentration of 0.0010g/mL, and dynamically adsorbing at the volume flow of 2 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin:
and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 4BV by using an ethanol water solution with the volume percentage concentration of 50%, eluting for 5BV by using an ethanol water solution with the volume percentage concentration of 90%, eluting at the rate of 2BV/h, collecting the part of eluent, adding an ethanol water solution with the same volume percentage concentration, adjusting the volume of the eluent to be 150mL, and thus obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A. The Xylastramide A was 38.45% pure by HPLC.
Example 4:
a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes comprises the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking 100g of longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the longitudinal stripe xylaria sporophore and 20mL of 90% ethanol water solution with volume percentage concentration according to the proportion that 1g of the longitudinal stripe xylaria sporophore is added with 20mL of 90% ethanol water solution with volume percentage concentration, soaking for 2 hours at the temperature of 80 ℃, filtering, treating the solid for 3 times through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0070g/mL, and collecting the sample solution of Xylaria longilineata;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 90% ethanol water solution for 20h to fully swell the resin, filling the resin into a column by a wet method, washing the resin with ethanol until the effluent is not turbid, and washing the resin with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage is 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent by mass of sodium hydroxide aqueous solution (the dosage is 2BV) for 4 hours, and then washing the resin by distilled water to be neutral to prepare pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 80mL of sample loading solution with the concentration of 0.0070g/mL, and dynamically adsorbing at the volume flow of 2 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin: and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 5BV by using ethanol water solution with the volume percentage concentration of 40%, eluting for 5BV by using ethanol water solution with the volume percentage concentration of 85%, eluting at the rate of 2BV/h, collecting the part of eluent, adding ethanol water solution with the same volume percentage concentration, and adjusting the volume of the eluent to be 150mL to obtain the desorption solution enriched with the purified cyclopeptide Xylastramide A.
The purity of xylastrtriamide a was 35.81% as determined by HPLC.
Example 5:
a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes comprises the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking 100g of longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the Xylaria longissima sporophore with 80 vol% ethanol water solution at a ratio of 1g Xylaria longissima sporophore and 20mL 80 vol% ethanol water solution, soaking at 80 deg.C for 2h, filtering, treating the solid for 4 times, and mixing filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0030g/mL, i.e. the obtained Xylaria longipes sample solution;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 90% ethanol water solution for 15h to fully swell the resin, filling the resin into a column by a wet method, washing the resin with ethanol until the effluent is not turbid, and washing the resin with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage is 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent by mass of sodium hydroxide aqueous solution (the dosage is 2BV) for 4 hours, and then washing the resin by distilled water to be neutral to prepare pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 210mL of sample loading solution with the concentration of 0.0030g/mL, and dynamically adsorbing at the volume flow of 2 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin:
and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 4BV by using an ethanol water solution with the volume percentage concentration of 45%, eluting for 4BV by using an ethanol water solution with the volume percentage concentration of 90%, eluting at the rate of 2BV/h, collecting the part of eluent, adding an ethanol water solution with the same volume percentage concentration, adjusting the volume of the eluent to be 150mL, and thus obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A.
The Xylastramide A was 38.27% pure as determined by HPLC.
Example 6:
a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes comprises the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the Xylaria longissima sporophore and 60% ethanol water solution with volume percentage concentration of 20mL according to the proportion of adding the Xylaria longissima sporophore of 1g into the 60% ethanol water solution with volume percentage concentration, soaking for 2h at the temperature of 80 ℃, filtering, treating the solid for 4 times through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0010g/mL, and collecting the sample solution of Xylaria longissima;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 80 vol% ethanol water solution for 30h to swell fully, packing into column by wet method, washing with ethanol until the effluent is not turbid, and washing with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage is 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent by mass of sodium hydroxide aqueous solution (the dosage is 2BV) for 4 hours, and then washing the resin by distilled water to be neutral to prepare pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 300mL of sample solution with the concentration of 0.0010g/mL, and dynamically adsorbing at the volume flow of 1 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin:
and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 1BV by using an ethanol water solution with the volume percentage concentration of 10%, eluting for 1BV by using an ethanol water solution with the volume percentage concentration of 60%, eluting at the rate of 1BV/h, collecting the part of eluent, adding an ethanol water solution with the same volume percentage concentration, adjusting the volume of the eluent to be 150mL, and thus obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A.
Example 7:
a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes comprises the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the Xylaria longissima sporophore and ethanol aqueous solution with the volume percentage concentration of 95% according to the proportion that the Xylaria longissima sporophore is added into 20mL of ethanol aqueous solution with the volume percentage concentration of 95%, soaking for 2h at the temperature of 60 ℃, filtering, treating the solid for 1 time through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0070g/mL, and collecting the sample solution of Xylaria longilineata;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 95% ethanol water solution for 15h to fully swell the resin, filling the resin into a column by a wet method, washing the resin with ethanol until the effluent is not turbid, and washing the resin with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage is 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent by mass of sodium hydroxide aqueous solution (the dosage is 2BV) for 4 hours, and then washing the resin by distilled water to be neutral to prepare pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 50mL of sample loading solution with the concentration of 0.0070g/mL, and dynamically adsorbing at the volume flow of 5 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin:
and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 5BV by using an ethanol water solution with the volume percentage concentration of 50%, eluting for 5BV by using an ethanol water solution with the volume percentage concentration of 95%, eluting at the rate of 5BV/h, collecting the part of eluent, adding an ethanol water solution with the same volume percentage concentration, adjusting the volume of the eluent to be 150mL, and thus obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A.
Example 8:
a method for enriching and purifying cyclopeptide Xylastrimide A from xylaria longipes comprises the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the Xylaria longissima sporophore and ethanol aqueous solution with the volume percentage concentration of 78% according to the proportion that 20mL of the ethanol aqueous solution with the volume percentage concentration of 78% is added into 1g of the Xylaria longissima sporophore, soaking for 2 hours at the temperature of 70 ℃, filtering, treating the solid for 3 times through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0050g/mL, and collecting the sample solution of Xylaria longissima;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 88% ethanol water solution for 22h to fully swell, packing into a column by a wet method, washing with ethanol until the effluent is not turbid, and washing with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in a hydrochloric acid aqueous solution with the mass percentage concentration of 5 percent for 4 hours and then washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent sodium hydroxide water solution for 4 hours, and then washing the resin to be neutral by distilled water to obtain pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 190mL of sample loading solution with the concentration of 0.0050g/mL, and dynamically adsorbing at the volume flow of 3 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin:
and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 3BV by using an ethanol water solution with the volume percentage concentration of 30%, eluting for 3BV by using an ethanol water solution with the volume percentage concentration of 80%, eluting at the rate of 3BV/h, collecting the part of eluent, adding an ethanol water solution with the same volume percentage concentration, adjusting the volume of the eluent to be 150mL, and thus obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A.
Example 9:
a method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans is disclosed, wherein the step a of preparing xylaria longilineans sample loading liquid is as follows: taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the Xylaria longissima sporophore and 75% ethanol water solution with volume percentage concentration of 20mL according to the proportion of adding the Xylaria longissima sporophore of 1g into 75% ethanol water solution with volume percentage concentration of 75%, soaking for 2h at the temperature of 80 ℃, filtering, treating the solid for 3 times through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0070g/mL, and collecting the sample solution of Xylaria longilineata; the rest of the procedures are the same as those in examples 6 to 8, and are omitted.
Example 10:
a method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans is disclosed, wherein the step a of preparing xylaria longilineans sample loading liquid is as follows: taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the Xylaria longissima sporocarp with a 95% ethanol aqueous solution with the volume percentage concentration of 20mL according to the proportion of adding the Xylaria longissima sporocarp into the 95% ethanol aqueous solution with the volume percentage concentration of 1g, soaking for 2 hours at the temperature of 75 ℃, filtering, treating the solid for 1 time through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0010g/mL, and collecting the sample solution of Xylaria longissima; the rest of the procedures are the same as those in examples 6 to 8, and are omitted.
Example 11:
a method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans is disclosed, wherein the step a of preparing xylaria longilineans sample loading liquid is as follows: taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the longitudinal stripe xylaria sporophore and 20mL of 90% ethanol water solution with volume percentage concentration according to the proportion that 1g of the longitudinal stripe xylaria sporophore is added with 20mL of 90% ethanol water solution with volume percentage concentration, soaking for 2 hours at the temperature of 80 ℃, filtering, treating the solid for 1 time through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0020g/mL, namely the prepared Xylaria longissima sample solution for later use; the rest of the procedures are the same as those in examples 6 to 8, and are omitted.
Example 12:
a method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans is disclosed, wherein the step a of preparing xylaria longilineans sample loading liquid is as follows: taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the longitudinal stripe xylaria sporophore and 20mL of 90% ethanol water solution with volume percentage concentration according to the proportion that 1g of the longitudinal stripe xylaria sporophore is added with 20mL of 90% ethanol water solution with volume percentage concentration, soaking for 2 hours at the temperature of 80 ℃, filtering, treating the solid for 2 times through the soaking process, and combining the filtrates; concentrating the filtrate by rotary evaporation to obtain extract, dissolving the extract in distilled water to obtain solution with concentration of 0.0060g/mL, and collecting the sample solution of Xylaria longissima; the rest of the procedures are the same as those in examples 6 to 8, and are omitted.
Example 13:
a method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans, wherein the dynamic adsorption of the cyclopeptide Xylastramide A in the xylaria longilineans by the resin in the step b is as follows:
1) pretreatment of D101 resin: soaking the D101 resin in 95% ethanol water solution for 24 hr to swell fully, loading into column by wet method, washing with ethanol until the effluent is not turbid, and washing with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in 5 percent hydrochloric acid aqueous solution (the dosage can be 2BV) for 4 hours, and then is washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent sodium hydroxide aqueous solution (the dosage can be 2BV) for 4 hours, and then washing the resin to be neutral by distilled water to obtain pretreated D101 resin for later use;
2) dynamic adsorption: and (3) loading 18g of the pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 210mL of sample loading solution with the concentration of 0.0030g/mL, and dynamically adsorbing at the volume flow of 2 BV/h.
The same as in any of examples 6 to 12, except that the above-mentioned process was omitted.
Example 14:
a method for enriching and purifying the cyclopeptide Xylastramide A from the xylaria longilineans, wherein the kinetic desorption of the cyclopeptide Xylastramide A from the xylaria longilineans by the resin in the step c is as follows: and (c) after dynamic adsorption balance (namely when the dynamic adsorption in the step b reaches a leakage point), stopping adding the sample solution, eluting for 3BV by using an ethanol water solution with the volume percentage concentration of 40%, eluting for 3BV by using absolute ethyl alcohol, eluting at the rate of 2BV/h, collecting the part of eluent, adding the absolute ethyl alcohol, and adjusting the volume of the eluent to be 150mL to obtain the desorption solution after enriching and purifying the cyclopeptide Xylastramide A.
Otherwise, the same as in any of examples 6 to 13 was omitted.
In the above embodiment: the BV, called volume, represents the resin bed volume, and for resins, BV is usually taken as a unit, so that the data can be changed with different resin bed volumes; 1BV means 1 time of the resin bed volume; the volume of the solution flowing per hour is 1-5 times of the volume of the resin bed by 1-5 BV/h;
in the above embodiment: the definition of the leak point is: the leak point is reached when the concentration of target in the effluent (in the context of the present invention the target is the cyclopeptide xylastramide a) reaches 10% of the target concentration in the loading solution.
In the above embodiment: the content determination method of the cyclopeptide Xylastrimide A has the following set of chromatographic conditions: AgiLent ZORBAX EcLipse XDB-C18 (high performance liquid chromatography column), 4.6mm × 250mm, 5 um; mobile phase: the mobile phase A is acetonitrile, the mobile phase B is ultrapure water, and isocratic elution is carried out according to the procedures of: 0-20 min, 65% of A and 35% of B; detection wavelength: 216 nm; the column temperature is 35 ℃; flow rate: 1.0 mL/min; sample introduction amount: 15 uL; the peak-off time was 14.915 min.
In the above embodiment: the 1BV is 30 mL.
In the above embodiment: the production and provision enterprises of the D101 resin (or D101 macroporous resin) are as follows: west reagent, Anhui Saxing resin science and technology, Inc., Tianjin Kai resin science and technology, Inc., and the like.
In the above embodiment: all the raw materials are commercially available products.
In the above embodiment: the percentages used, not specifically indicated, are percentages by weight or known to those skilled in the art; the parts by mass (by weight) may all be grams or kilograms.
In the above embodiment: the process parameters (temperature, time, concentration, etc.) and the amounts of the components in each step are within the range, and any point can be applicable.
The present invention and the technical contents not specifically described in the above embodiments are the same as the prior art.
After the longitudinal striated xylaria sporocarp is treated by the method for enriching and purifying the cyclopeptide Xylastrimide A from the longitudinal striated xylaria, a large amount of impurities can be removed, target components are reserved, and the purity of the target components (namely the cyclopeptide Xylastrimide A) is effectively improved; and further performing separation operation to obtain pure cyclopeptide Xylastramide A, wherein the macroporous resin purification method can effectively simplify the separation steps.
The present invention is not limited to the above-described embodiments, and the present invention can be implemented with the above-described advantageous effects.
Claims (6)
1. A method for enriching and purifying cyclopeptide Xylastramide A from xylaria longilineans is characterized by comprising the following steps:
a. preparing a longitudinal stripe xylaria sample loading solution:
taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the longitudinal stripe xylaria sporocarp with 20mL of ethanol water solution with the volume percentage concentration of 60-95% according to the proportion of adding the longitudinal stripe xylaria sporocarp into the 20mL of ethanol water solution with the volume percentage concentration of 60-95%, soaking for 2 hours at the temperature of 60-85 ℃, filtering, treating the solid for 1-4 times through the soaking process, and combining the filtrate; concentrating the filtrate by rotary evaporation to obtain an extract, and dissolving the extract in distilled water to obtain a solution with the concentration of 0.0010-0.0070 g/mL, namely the prepared xylaria longilineans sample solution for later use;
b. kinetic adsorption of cyclic peptide Xylastramide A in Xylastramide A by resin:
1) pretreatment of D101 resin: soaking the D101 resin in 80-95 vol% ethanol water for 15-30 h, packing the resin into a column by a wet method, washing the column with ethanol until the effluent liquid is not turbid, and washing the column with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in a hydrochloric acid aqueous solution with the mass percentage concentration of 5 percent for 4 hours and then washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent sodium hydroxide water solution for 4 hours, and then washing the resin to be neutral by distilled water to obtain pretreated D101 resin for later use;
2) dynamic adsorption: loading 18g of pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 50-300 mL of sample loading solution with the concentration of 0.0010-0.0070 g/mL, and dynamically adsorbing at the volume flow of 1-5 BV/h;
c. kinetic desorption of cyclic peptide xyloastramide a from xylaria longilineans by resin:
and after dynamic adsorption balance, stopping adding the sample solution, eluting for 1-5 BV by using an ethanol aqueous solution with the volume percentage concentration of 10-50%, eluting for 1-5 BV by using an ethanol aqueous solution with the volume percentage concentration of 60-100%, eluting at the rate of 1-5 BV/h, collecting the part of eluent, adding an ethanol aqueous solution with the same volume percentage concentration, adjusting the volume of the eluent to be 150mL, and thus obtaining the desorption solution enriched with the purified cyclopeptide Xylastrimide A.
2. The method for enriching and purifying the cyclopeptide Xylastrimide A from Xylastramide A as claimed in claim 1, wherein: the method for preparing the xylaria longilineans sample loading liquid in the step a comprises the following steps: taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the xylaria striata sporocarp with 20mL of 75-95 vol% ethanol water solution according to the proportion of 1g of xylaria striata sporocarp, soaking the xylaria striata sporocarp and the 75-95 vol% ethanol water solution for 2 hours at the temperature of 70-80 ℃, filtering, treating the solid for 1-3 times through the soaking process, and combining the filtrates; and (3) concentrating the filtrate by rotary evaporation to obtain an extract, and dissolving the extract in distilled water to prepare a solution with the concentration of 0.0010-0.0070 g/mL, namely the prepared xylaria striata sample solution for later use.
3. The method for enriching and purifying the cyclopeptide Xylastrimide A from Xylastramide A as claimed in claim 2, wherein: the method for preparing the xylaria longilineans sample loading liquid in the step a comprises the following steps: taking the longitudinal stripe xylaria sporocarp, crushing and sieving by a 40-mesh sieve; mixing the longitudinal stripe xylaria sporocarp with 90% ethanol water solution with volume percentage concentration of 20mL according to the proportion of adding the longitudinal stripe xylaria sporocarp into the 90% ethanol water solution with volume percentage concentration of 1g, soaking for 2h at the temperature of 80 ℃, filtering, treating the solid for 1-2 times through the soaking process, and combining the filtrates; and (3) concentrating the filtrate by rotary evaporation to obtain an extract, and dissolving the extract in distilled water to prepare a solution with the concentration of 0.0010-0.0070 g/mL, namely the prepared xylaria striata sample solution for later use.
4. The method for enriching and purifying the cyclopeptide Xylastrimide A from Xylastramide A as claimed in claim 1, 2 or 3, wherein: the dynamic adsorption of the resin on the cyclopeptide Xylastrimide A in the xylaria gracillima in the step b is as follows:
1) pretreatment of D101 resin: soaking the D101 resin in 95% ethanol water solution for 24h, packing the resin into a column by a wet method, washing the resin with ethanol until the effluent liquid is not turbid, and washing the resin with distilled water until no alcohol smell exists; then acid washing: namely, the mixture is soaked in a hydrochloric acid aqueous solution with the mass percentage concentration of 5 percent for 4 hours and then washed to be neutral by distilled water; then alkali washing: namely, soaking the resin in 2 percent sodium hydroxide water solution for 4 hours, and then washing the resin to be neutral by distilled water to obtain pretreated D101 resin for later use;
2) dynamic adsorption: and (3) loading 18g of the pretreated D101 resin into a column, wherein the volume of the resin bed column is 30mL, adding 210mL of sample loading solution with the concentration of 0.0030g/mL, and dynamically adsorbing at the volume flow of 2 BV/h.
5. The method for enriching and purifying the cyclopeptide Xylastrimide A from Xylastramide A as claimed in claim 1, 2 or 3, wherein: the dynamic desorption of the cyclic peptide Xylastramide A in the xylaria longilineans by the resin in the step c is as follows: and after dynamic adsorption balance, stopping adding the sample solution, eluting for 3BV by using an ethanol water solution with the volume percentage concentration of 40%, eluting for 3BV by using absolute ethanol, eluting at the rate of 2BV/h, collecting the part of eluent, adding the absolute ethanol, adjusting the volume of the eluent to be 150mL, and obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A.
6. The method for enriching and purifying the cyclopeptide Xylastrimide A from Xylastramide A as claimed in claim 4, wherein: the dynamic desorption of the cyclic peptide Xylastramide A in the xylaria longilineans by the resin in the step c is as follows: and after dynamic adsorption balance, stopping adding the sample solution, eluting for 3BV by using an ethanol water solution with the volume percentage concentration of 40%, eluting for 3BV by using absolute ethanol, eluting at the rate of 2BV/h, collecting the part of eluent, adding the absolute ethanol, adjusting the volume of the eluent to be 150mL, and obtaining the desorption solution after enriching and purifying the cyclopeptide Xylastramide A.
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