A method for continuous preparation and chromatography purification of small molecule active substances of Porites gracilis, and its products and applications

Abstract

The present invention discloses a method for continuous preparation chromatography to purify a small molecule active substance of Porosia leucophylla, and its products and applications, and relates to the field of food industry. The method comprises the following steps: (1) extracting the fruiting body of Porosia leucophylla to obtain a crude small molecule extract of Porosia leucophylla; (2) purifying and refining the crude small molecule extract of Porosia leucophylla by using a continuous preparation chromatography device to obtain a finished product of the small molecule active substance of Porosia leucophylla; the continuous preparation chromatography device comprises a purification chromatography column and a refining chromatography column; in step (2), the purification is performed by using the purification chromatography column, and the refining is performed by using the refining chromatography column. The method of the present invention can obtain a small molecule active substance of Porosia leucophylla with high purity and activity, and the method of the present invention also has the advantages of cost saving, simple process steps and continuous production.

Description

Technical Field

[0001] The invention relates to a method for continuously preparing chromatographically purified micromolecular active substances of Porites gracilis, and a product and application thereof. Background Art

[0002] Inonotus cuticularis, also known as rare needle-hole fungus and thin-skinned hair-back fungus, has the effects of soothing qi and benefiting the mind, removing evil wind, curing body odor, stopping bleeding, curing stomach diseases, curing leprosy, etc. The inhibition rate of sarcoma 180 and Ehrlich carcinoma in mice is 90% and 100%. Through experimental research, the small molecule compound of Inonotus cuticularis has a significant hypoglycemic effect, but there are few studies and applications on it at present, and there are few reports.

[0003] Chinese Patent 201910446467.1 discloses a new strain of Inonotus cuticularis and its artificial cultivation method and use. The patent protects a type of Inonotus cuticularis HMGIM-Z110188. Cultivation experiments show that it can be successfully cultivated artificially with a large yield. At the same time, the in vitro inhibition of α-glucosidase activity of its fermentation broth reached 94.7%, which is of great significance for the treatment of hyperglycemia-related diseases. This patent is only for the preservation of strains and preliminary activity research, and no purification research on the active substances of Inonotus cuticularis has been carried out. In order to further study and develop Inonotus cuticularis resources, efficient purification technology of small molecule compounds of Inonotus cuticularis is urgently needed.

[0004] Continuous preparative chromatography purification technology is an efficient purification method that can achieve continuous production and has good separation effect. At present, there is no research report on the purification technology of small molecule active substances from Porites gracilis. Summary of the invention

[0005] The purpose of the present invention is to provide a method for continuously preparing chromatographically purified microporous microorganism small molecule active substances and its products and applications, so as to solve the problems existing in the above-mentioned prior art. The method of the present invention can obtain microporous microorganism small molecule active substances with high purity and activity, and the method of the present invention also has the advantages of cost saving, simple process steps and continuous production.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for continuously preparing chromatographically purifying a small molecule active substance of Porites gracilis, comprising the following steps:

[0008] (1) extracting the fruiting bodies of Porifera gracilis to obtain a crude extract of Porifera gracilis small molecules;

[0009] (2) Purifying and refining the crude extract of the microporus microcarpa using a continuous preparative chromatography device to obtain a microporus microcarpa microcarpa small molecule active substance finished product;

[0010] The continuous preparative chromatography equipment comprises a purification chromatography column and a refinement chromatography column;

[0011] In step (2), the purification is performed using the purification chromatography column, and the refinement is performed using the refinement chromatography column.

[0012] Furthermore, the macroporous adsorption resin used in the purification chromatographic column is D101, AB-8, X-5 or HPD400.

[0013] Furthermore, the macroporous adsorption resin used in the refining chromatographic column is ADS-4, ADS-5 or ADS-8.

[0014] Furthermore, the continuous preparative chromatography equipment also includes a PLC control system.

[0015] Furthermore, in step (1), the solvent used for the extraction is ethanol solution.

[0016] Furthermore, in step (2), the feed flow rate of the purification is 1-5 mL / min, and the sample volume is 1-3 BV; the deionized water flow rate is 3-10 mL / min, and the elution volume is 2-5 BV; the eluent is an ethanol aqueous solution with a volume fraction of 60-80%, the flow rate is 2-8 mL / min, and the elution volume is 2-5 BV; the regenerated deionized water flow rate is 3-10 mL / min, and the elution volume is 2-5 BV.

[0017] Furthermore, in step (2), the feed concentration of the crude extract of the micromolecule of Porites gracilis in the purification chromatography column is 5-25 wt %.

[0018] The present invention also provides a micromolecular active substance of Porites thunbergii prepared according to the above method.

[0019] The present invention also provides the use of the above-mentioned Porites gracilis small molecule active substance in the preparation of antioxidant products.

[0020] The invention also provides an antioxidant product, comprising the above-mentioned Porites gracilis small molecule active substance.

[0021] The present invention discloses the following technical effects:

[0022] The present invention develops a technology for continuous preparation and chromatography purification of small molecule active substances of Poroides gracilis, and establishes a method for efficiently purifying small molecule active substances of Poroides gracilis. The method not only ensures the high purity and high activity of small molecule active substances of Poroides gracilis, but also saves the cost of the entire process, has simple process steps, and can be continuously produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a process flow chart for the continuous preparation and chromatography purification of small molecule active substances from Porites gracilis. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] like Figure 1 As shown, the continuous preparation chromatography equipment used in the present invention is composed of two chromatographic columns (inner diameter 35 mm, length 500 mm), and the feed, deionized water and eluent are controlled by a four-way injection valve.

[0031] The preparation method of the crude extract of the micromolecule of Porites gracilis used in the following examples is as follows:

[0032] The raw material of Microporus leucophyllus was low-temperature dried at 40°C for 12 hours, crushed and passed through an 80-mesh sieve, extracted with an ultrasonic-assisted ethanol solution (the volume fraction of 60-80% can achieve the same effect), centrifuged and filtered after extraction, and vacuum concentrated to obtain a Microporus leucophyllus small molecule crude extract, which was then freeze-dried to obtain a Microporus leucophyllus small molecule crude extract.

[0033] In the following examples, the purity of small molecule active substances was calculated according to the flavonoid detection method, and the antioxidant activity was calculated according to the DPPH clearance rate.

[0034] Flavonoids detection method:

[0035] (1) Drawing of standard curve

[0036] Weigh 1.0 mg of rutin standard dried to constant weight, dissolve it in 80% ethanol, transfer it to a 50 mL volumetric flask and make up to volume. The concentration of this standard solution is 0.2 mg / mL. Measure 0, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the standard solution in a 10 mL stoppered graduated test tube, add 80% (volume fraction) ethanol to 5 mL, add 0.3 mL of 5 wt% sodium nitrite solution, shake well and let stand for 6 min; add 0.3 mL of 10 wt% aluminum nitrate solution, shake well and let stand for 6 min; add 4 mL of 4 wt% sodium hydroxide solution, add 80% (volume fraction) ethanol to the scale, shake well and let stand for 10 min, measure the absorbance A at a wavelength of 510 nm, use rutin concentration as the horizontal axis and absorbance value as the vertical axis, and draw a standard curve according to the measured values.

[0037] (2) Flavonoids determination

[0038] Take 1mL of the test solution in a 10mL stoppered test tube, measure the absorbance value at a wavelength of 510nm according to the determination method and steps of the standard curve, substitute it into the standard curve equation to obtain the rutin content, and then substitute the rutin content into the yield calculation formula:

[0039]

[0040] Where C is the concentration (g mL -1 ); V is volume (mL); D is dilution factor; m is raw material mass (g); H is sample moisture content (%).

[0041] The yield was calculated, and then the total flavonoid content was calculated by m (mass of the sample) × ω (yield).

[0042] DPPH scavenging rate detection method: DPPH absorbs strongly at 517nm, and its ethanol solution is dark purple. When a free radical scavenger is present, its absorption gradually disappears due to pairing with its single electron. The degree of fading is stoichiometrically related to the number of paired electrons. Therefore, it can be quantitatively analyzed by spectrometry. The antioxidant capacity is expressed by the inhibition rate. The greater the inhibition rate, the stronger the antioxidant property. The specific operation is: take 0.2, 0.4, 0.6, 0.8, and 1.0mL of each test solution in a stoppered test tube, add 2mL of DPPH solution, and then use anhydrous ethanol to make up the system to 4mL, shake well, and after 30 minutes, use anhydrous ethanol as a reference to determine its absorbance A i ; At the same time, the absorbance A of 2 mL DPPH· solution mixed with 2 mL anhydrous ethanol was measured c ; Same method as A i After adding different volumes of the test solution, add anhydrous ethanol to 4 mL directly, mix and measure the absorbance of the system A j , the experiment used rutin as the control.

[0043] Inhibition rate = [1-(A i -A j ) / A c ]×100%

[0044] Among them: A i - absorbance of sample DPPH solution; A j - absorbance of the sample solution at the measurement wavelength; A c -Absorbance of DPPH· solution without adding antioxidant.

[0045] Example 1

[0046] (1) Resin loading: Prepare D101 macroporous adsorption resin, soak it in a 95% ethanol aqueous solution for 24 h, then rinse it with deionized water and wet-load it into a purification chromatographic column (3.5 cm × 50 cm). Treat ADS-4 macroporous adsorption resin in the same way and wet-load it into a refining chromatographic column (3.5 cm × 50 cm). Rinse the chromatographic columns with deionized water until the effluent has no alcohol smell.

[0047] (2) Purification of small molecules of Porospora serrata: The aqueous solution of crude extract of small molecules of Porospora serrata with a concentration of 5wt% was automatically pumped into the chromatographic column through the PLC control system. The raw material liquid was pumped into the purification chromatographic column by a constant flow pump, with a feed flow rate of 1mL / min and a sample volume of 3BV; then impurities such as proteins, polysaccharides, and ions were removed by deionized water, with an inlet flow rate of 3mL / min and an elution volume of 5BV; the small molecule active substances of Porospora serrata in the chromatographic column were eluted with an ethanol aqueous solution with a volume fraction of 60%, with a flow rate of 2mL / min and an elution volume of 5BV; finally, deionized water was used for regeneration, with a regeneration deionized water flow rate of 3mL / min and an elution volume of 5BV.

[0048] (3) Refining of small molecules of Porites thunbergii: When the purification preparation chromatographic column begins to elute, the solenoid valve is opened through the self-designed PLC control system, and the eluent automatically flows into the purification preparation chromatographic column for adsorption, decolorization and impurity removal. After 15 consecutive adsorption injection cycles, the purification preparation chromatographic column is regenerated during the regeneration-adsorption process of the purification preparation chromatographic column. 1wt% NaOH solution, 1wt% HCl solution and deionized water are pumped in turn for regeneration. The flow rate and elution volume are consistent with those in the regeneration, adsorption and elution areas.

[0049] (4) The purified microporus leucophylla small molecule active substance is vacuum concentrated at a concentration temperature of 45° C. and then freeze-dried to obtain the refined microporus leucophylla small molecule active substance.

[0050] The purified micromolecular active substance of Poroides gracilis prepared in this example has a purity increased by 38.5% and an antioxidant activity increased by 35.7% compared with the micromolecular crude extract of Poroides gracilis.

[0051] Example 2

[0052] (1) Resin loading: Prepare AB-8 macroporous adsorption resin, soak it in a 95% ethanol aqueous solution for 24 h, then rinse it with deionized water and wet-load it into a purification chromatographic column (3.5 cm × 50 cm). Treat ADS-5 macroporous adsorption resin in the same way and wet-load it into a refining chromatographic column (3.5 cm × 50 cm). Rinse the chromatographic columns with deionized water until the effluent has no alcohol smell.

[0053] (2) Purification of small molecules of Porospora leucoderma: A 20wt% aqueous solution of crude extract of small molecules of Porospora leucoderma is automatically pumped into the chromatographic column through a self-designed PLC control system. A constant flow pump is used to pump the raw liquid into the purification chromatographic column, with a feed flow rate of 5mL / min and a sample volume of 1BV; then deionized water is used to remove impurities such as proteins, polysaccharides, and ions, with an inflow rate of 10mL / min and an elution volume of 2BV; a 70% volume fraction ethanol aqueous solution is used to elute the small molecule active substances of Porospora leucoderma in the chromatographic column, with a flow rate of 8mL / min and an elution volume of 2BV; finally, deionized water is used for regeneration, with a regeneration deionized water flow rate of 10mL / min and an elution volume of 2BV;

[0054] (3) Refining of small molecules of Porites thunbergii: When the purification preparation chromatographic column begins to elute, the solenoid valve is opened through the self-designed PLC control system, and the eluent automatically flows into the purification preparation chromatographic column for adsorption, decolorization and impurity removal. After 20 consecutive adsorption injection cycles, the purification preparation chromatographic column is regenerated during the regeneration-adsorption process of the purification preparation chromatographic column. 1wt% NaOH solution, 1wt% HCl solution and deionized water are pumped in turn for regeneration. The flow rate and elution volume are consistent with those in the regeneration, adsorption and water washing areas.

[0055] (4) The purified microporus leucophylla small molecule active substance is vacuum concentrated at a concentration temperature of 50° C. and then freeze-dried to obtain the refined microporus leucophylla small molecule active substance.

[0056] The purified micromolecular active substance of Porifera gracilis prepared in this example has a purity increased by 32.2% and an antioxidant activity increased by 28.4% compared with the micromolecular crude extract of Porifera gracilis.

[0057] Example 3

[0058] (1) Resin loading: Prepare X-5 macroporous adsorption resin, soak it in a 95% by volume ethanol aqueous solution for 24 h, then rinse it with deionized water and wet-load it into a purification chromatographic column (3.5 cm × 50 cm). Treat ADS-8 macroporous adsorption resin in the same way and wet-load it into a refining chromatographic column (3.5 cm × 50 cm). Rinse the chromatographic columns with deionized water until the effluent has no alcohol taste.

[0059] (2) Purification of small molecules of Porospora leucophylla: The aqueous solution of crude extract of small molecules of Porospora leucophylla with a concentration of 25wt% is automatically pumped into the chromatographic column through the independently designed PLC control system. The raw material liquid is pumped into the purification chromatographic column by a constant flow pump, the feed flow rate is 2mL / min, and the sample volume is 2BV; then deionized water is used to remove impurities such as proteins, polysaccharides, and ions, the deionized water flow rate is 4mL / min, and the elution volume is 3BV; the small molecule active substances of Porospora leucophylla in the chromatographic column are eluted with an ethanol aqueous solution with a volume fraction of 80%, the flow rate is 2mL / min, and the elution volume is 4BV; finally, deionized water is used for regeneration, the regeneration deionized water flow rate is 6mL / min, and the elution volume is 4BV;

[0060] (3) Refining of small molecules of Porites thunbergii: When the purification preparation chromatographic column begins to elute, the solenoid valve is opened through the self-designed PLC control system, and the eluent automatically flows into the purification preparation chromatographic column for adsorption, decolorization and impurity removal. After 10 consecutive adsorption injection cycles, the purification preparation chromatographic column is regenerated during the regeneration-adsorption process of the purification preparation chromatographic column. 1wt% NaOH solution, 1wt% HCl solution and deionized water are pumped in turn for regeneration. The flow rate and elution volume are consistent with those in the regeneration, adsorption and water washing areas.

[0061] (4) The purified microporus leucophylla small molecule active substance is vacuum concentrated at a concentration temperature of 45° C. and then freeze-dried to obtain the refined microporus leucophylla small molecule active substance.

[0062] The purified micromolecular active substance of Porifera gracilis prepared in this example has a purity increased by 42.1% and an antioxidant activity increased by 40.6% compared with the micromolecular crude extract of Porifera gracilis.

[0063] Example 4

[0064] (1) Resin loading: Prepare HPD400 macroporous adsorption resin, soak it in a 95% ethanol aqueous solution for 24 h, then rinse it with deionized water and wet-load it into a purification chromatographic column (3.5 cm × 50 cm). Treat ADS-4 macroporous adsorption resin in the same way and wet-load it into a refining chromatographic column (3.5 cm × 50 cm). Rinse the chromatographic columns with deionized water until the effluent has no alcohol smell.

[0065] (2) Purification of small molecules of Porospora leucoderma: The aqueous solution of crude extract of small molecules of Porospora leucoderma with a concentration of 20wt% is automatically pumped into the chromatographic column through the independently designed PLC control system. The raw material liquid is pumped into the purification chromatographic column by a constant flow pump, the feed flow rate is 3mL / min, and the sample volume is 2BV; then deionized water is used to remove impurities such as proteins, polysaccharides, and ions, the deionized water flow rate is 5mL / min, and the elution volume is 4BV; the small molecule active substances of Porospora leucoderma in the chromatographic column are eluted with an ethanol aqueous solution with a volume fraction of 80%, the flow rate is 5mL / min, and the elution volume is 4BV; finally, deionized water is used for regeneration, the regeneration deionized water flow rate is 7mL / min, and the elution volume is 5BV;

[0066] (3) Refining of small molecules of Porites thunbergii: When the purification preparation chromatographic column begins to elute, the solenoid valve is opened through the self-designed PLC control system, and the eluent automatically flows into the purification preparation chromatographic column for adsorption, decolorization and impurity removal. After 20 consecutive adsorption injection cycles, the purification preparation chromatographic column is regenerated during the regeneration-adsorption process of the purification preparation chromatographic column. 1wt% NaOH solution, 1wt% HCl solution and deionized water are pumped in turn for regeneration. The flow rate and elution volume are consistent with those in the regeneration, adsorption and water washing areas.

[0067] (4) The purified microporus leucophylla small molecule active substance is vacuum concentrated at a concentration temperature of 50° C. and then freeze-dried to obtain the refined microporus leucophylla small molecule active substance.

[0068] The purified micromolecular active substance of Porifera gracilis prepared in this example has a purity increased by 25.1% and an antioxidant activity increased by 32.3% compared with the micromolecular crude extract of Porifera gracilis.

[0069] The purity and antioxidant activity test results of the refined Porites gracilis small molecule active substances prepared in Examples 1-4 are shown in Table 1.

[0070] Table 1

[0071]

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An application of a small molecule active substance of Porites thunbergii in the preparation of an antioxidant product, characterized in that: The method for preparing the micromolecular active substance of Porites thunbergii comprises the following steps: (1) extracting the fruiting bodies of Porifera gracilis to obtain a crude extract of Porifera gracilis small molecules; (2) Purifying and refining the crude extract of the microporus microcarpa using a continuous preparative chromatography device to obtain a microporus microcarpa microcarpa small molecule active substance finished product; The purification is carried out using a purification chromatographic column, and the refinement is carried out using a refinement chromatographic column; The macroporous adsorption resin used in the purification chromatographic column is D101, AB-8, X-5 or HPD400; The macroporous adsorption resin used in the refined chromatographic column is ADS-4, ADS-5 or ADS-8; In step (1), the solvent used for the leaching is an ethanol solution; In step (2), the feed flow rate of the purification is 1-5 mL / min, and the sample load is 1-3 BV; deionized water is used for impurity removal, and the flow rate is 3-10 mL / min, and the elution volume is 2-5 BV; 60-80% ethanol aqueous solution is used for elution, and the flow rate is 2-8 mL / min, and the elution volume is 2-5 BV; deionized water is used for regeneration, and the flow rate is 3-10 mL / min, and the elution volume is 2-5 BV.

2. The use according to claim 1, characterized in that: The continuous preparation chromatography equipment also includes a PLC control system.

3. The use according to claim 1, characterized in that: In step (2), the feed concentration of the crude extract of Porites gracilis small molecules in the purification chromatography column is 5-25 wt %.

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