Method for extracting chrysin from fructus alpiniae oxyphyllae and application

Abstract

The invention discloses a method for extracting chrysin from fructus alpiniae oxyphyllae and application. Belongs to the technical field of plant active ingredient extraction. According to the extraction method disclosed by the invention, the fructus alpiniae oxyphyllae is uniformly stirred in the saline water and then is stir-fried with slow fire, so that the conversion of bioactive components in the fructus alpiniae oxyphyllae can be promoted in the processing process, and the content of chrysin is increased; and bromelain and papain are added for enzymolysis treatment, so that the extraction efficiency of the chrysin is improved, the extracted chrysin extract has better biological activity, such as anti-hypoxia capability, and the treatment effect is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant active ingredient extraction, and more particularly to a method for extracting chrysin from Alpinia oxyphylla seeds and application thereof. Background Art

[0002] Alpinia oxyphylla (Alpinia oxyphylla) is the dried, mature fruit of the plant Alpinia oxyphylla, which belongs to the genus Alpinia in the Zingiberaceae family. According to records, Alpinia oxyphylla has various benefits, including warming the kidneys and strengthening essence, stopping diarrhea, reducing urination, and warming the spleen. Since ancient times, it has been used in Traditional Chinese Medicine for symptoms such as kidney deficiency, spermatorrhea, diarrhea, and abdominal pain.

[0003] The chemical composition of Alpinia oxyphylla is complex and difficult to separate. There are limited reports on its chemical composition in domestic and foreign literature. Chrysin is a 5,7-dihydroxyflavone and is one of the effective active ingredients. According to research, it has multiple pharmacological activities such as anti-tumor, anti-anxiety, anti-inflammatory, antioxidant, and anti-hypoxia, and belongs to the flavonoids.

[0004] Existing methods for extracting chrysin often rely on solvent extraction, using common solvents such as ethanol and acetone for extraction and adsorption with macroporous resins. However, the chrysin produced by these methods has a low extraction yield, low purity, and high levels of impurities, resulting in a dark color and poor efficacy.

[0005] Therefore, how to provide a method for extracting chrysin with high yield, pure quality and excellent effect is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for extracting chrysin from Alpinia oxyphylla seeds and its application. Chrysin in the Alpinia oxyphylla seeds is extracted by the above method, the Alpinia oxyphylla seeds are moistened with salt water and then stir-fried over low heat to effectively transform the biologically active components in the Alpinia oxyphylla seeds, and the method is combined with the enzymatic hydrolysis of bromelain and papain, and finally the enzymatic hydrolyzate is subjected to ethanol reflux extraction and macroporous resin adsorption to prepare a chrysin extract, thereby promoting the dissolution of the effective components to the greatest extent, significantly improving the yield and purity of chrysin, and improving the therapeutic efficacy of chrysin.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for extracting chrysin from Alpinia oxyphylla seeds comprises the following steps:

[0009] (1) Add salt water to the Alpinia oxyphylla seeds, mix well, and stir-fry over low heat. After stir-frying, let it cool and set aside;

[0010] (2) Grinding the cooled Alpinia oxyphylla seeds, adding an acidic aqueous solution and mixing well, and then adding a composite enzyme consisting of bromelain and papain for enzymolysis to obtain an enzymatic solution;

[0011] (3) adding 10-15 times of ethanol to the enzymatic hydrolyzate obtained in step (2), adjusting the pH to 9-10, reflux extraction, combining the extracts, and distilling under reduced pressure to remove the ethanol for later use;

[0012] (4) The extract obtained in step (3) is adsorbed on a macroporous resin, washed with water to further remove impurities, eluted with ethanol, and distilled under reduced pressure to remove ethanol to obtain an eluate;

[0013] (5) The eluate is decompressed, concentrated, crystallized, and dried to extract chrysin.

[0014] Preferably, the addition ratio of the Alpinia oxyphylla kernel and the saline in step (1) is 500-1000 g / 200-400 ml; and the concentration of the saline is 0.5-2 g / ml.

[0015] Preferably, the frying time in step (1) is 30-60 minutes.

[0016] Preferably, the crushed particle size in step (2) is 20-60 mesh; an acidic aqueous solution is added to adjust the pH to 5-6; the added amount of the complex enzyme is 2-5% of the weight of the Alpinia oxyphylla kernel; and the added ratio of bromelain and papain is 2-3:2-4.

[0017] Preferably, the enzymatic hydrolysis temperature in step (2) is 50-60° C., and the enzymatic hydrolysis time is 2-6 h.

[0018] Preferably, the reflux extraction in step (3) is performed 2-3 times, each time for 2-3 hours.

[0019] Preferably, the model of the macroporous resin in step (4) is HPD-300, HPD-100 or D101; and 70-80% ethanol is used for elution.

[0020] Another object of the present invention is to provide application of the above method in extracting chrysin.

[0021] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0022] According to the method of the present invention, the Alpinia oxyphylla seeds are placed in salt water, mixed evenly, and then stir-fried over a low fire. This processing process can promote the conversion of bioactive components in the Alpinia oxyphylla seeds and increase the content of chrysin. Simultaneously, bromelain and papain are added for enzymatic hydrolysis, which not only improves the extraction efficiency of chrysin, but also makes the extracted chrysin extract have better biological activity, such as anti-hypoxia ability. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] Example 1

[0025] A method for extracting chrysin from Alpinia oxyphylla seeds comprises the following steps:

[0026] (1) Take 500g of Alpinia oxyphylla seeds, add 200ml of saline solution with a concentration of 2g / ml, mix well, stir-fry over low heat for 30min, and let cool for later use;

[0027] (2) Grind the cooled Alpinia oxyphylla seeds into a 20-mesh particle size, add an acidic aqueous solution to adjust the pH to 5, mix well, add 1 g of bromelain and 1 g of papain, and perform enzymatic hydrolysis at 50°C for 6 h to obtain an enzymatic hydrolyzate;

[0028] (3) Add 10 times the amount of ethanol to the enzymatic hydrolyzate obtained in step (2), adjust the pH to 9, perform reflux extraction twice, each time for 3 hours, combine the extracts, and distill under reduced pressure to remove the ethanol for later use;

[0029] (4) The extract obtained in step (3) was adsorbed on HPD-300 macroporous resin, washed with water to further remove impurities, eluted with 70% ethanol, and distilled under reduced pressure to remove ethanol to obtain an eluate;

[0030] (5) The eluate is decompressed, concentrated, crystallized, and dried to extract chrysin.

[0031] Example 2

[0032] A method for extracting chrysin from Alpinia oxyphylla seeds comprises the following steps:

[0033] (1) Take 500 g of Alpinia oxyphylla seeds, add 400 ml of saline solution with a concentration of 0.5 g / ml, mix well, stir-fry over low heat for 40 min, and let cool for later use;

[0034] (2) Grind the cooled Alpinia oxyphylla seeds to a particle size of 60 mesh, add an acidic aqueous solution to adjust the pH to 6, mix well, add 3 g of bromelain and 2 g of papain, and perform enzymolysis at 60°C for 2 h to obtain an enzymatic hydrolyzate;

[0035] (3) Add 15 times the amount of ethanol to the enzymatic hydrolyzate obtained in step (2), adjust the pH to 10, and reflux extract 3 times, each time for 2 hours. Combine the extracts, distill under reduced pressure, and remove the ethanol for later use;

[0036] (4) The extract obtained in step (3) was adsorbed on HPD-100 macroporous resin, washed with water to further remove impurities, eluted with 75% ethanol, and distilled under reduced pressure to remove ethanol to obtain an eluate;

[0037] (5) The eluate is decompressed, concentrated, crystallized, and dried to extract chrysin.

[0038] Example 3

[0039] A method for extracting chrysin from Alpinia oxyphylla seeds comprises the following steps:

[0040] (1) Take 500g of Alpinia oxyphylla seeds, add 300ml of saline solution with a concentration of 1g / ml, mix well, stir-fry over low heat for 60min, and let cool for later use;

[0041] (2) Grind the cooled Alpinia oxyphylla seeds to a particle size of 40 mesh, add an acidic aqueous solution to adjust the pH to 5.5, mix well, add 1 g of bromelain and 2 g of papain, and perform enzymatic hydrolysis at 55°C for 4 h to obtain an enzymatic hydrolyzate;

[0042] (3) Add 10 times the amount of ethanol to the enzymatic hydrolyzate obtained in step (2), adjust the pH to 9, perform reflux extraction twice, each time for 3 hours, combine the extracts, and distill under reduced pressure to remove the ethanol for later use;

[0043] (4) The extract obtained in step (3) was adsorbed on D101 macroporous resin, washed with water to further remove impurities, eluted with 75% ethanol, and distilled under reduced pressure to remove ethanol to obtain an eluate;

[0044] (5) The eluate is decompressed, concentrated, crystallized, and dried to extract chrysin.

[0045] Comparative Example 1

[0046] A method for extracting chrysin from Alpinia oxyphylla seeds comprises the following steps:

[0047] (1) 500 g of Alpinia oxyphylla seeds were crushed to a particle size of 40 mesh, an acidic aqueous solution was added to adjust the pH to 5.5, and after mixing well, 1 g of bromelain and 2 g of papain were added, and enzymatic hydrolysis was carried out at 55° C. for 4 h to obtain an enzymatic hydrolyzate.

[0048] (2) Add 10 times the amount of ethanol to the enzymatic hydrolyzate obtained in step (1), adjust the pH to 9, perform reflux extraction twice, each time for 3 hours, combine the extracts, and distill under reduced pressure to remove the ethanol for later use;

[0049] (3) The extract obtained in step (2) was adsorbed on D101 macroporous resin, washed with water to further remove impurities, eluted with 75% ethanol, and distilled under reduced pressure to remove ethanol to obtain an eluate;

[0050] (4) The eluate is decompressed, concentrated, crystallized, and dried to obtain chrysin.

[0051] Comparative Example 2

[0052] A method for extracting chrysin from Alpinia oxyphylla seeds comprises the following steps:

[0053] (1) Take 500g of Alpinia oxyphylla seeds, add 300ml of saline solution with a concentration of 1g / ml, mix well, stir-fry over low heat for 60min, and let cool for later use;

[0054] (2) Grind the cooled Alpinia oxyphylla seeds to a particle size of 40 mesh, add an acidic aqueous solution to adjust the pH to 5.5, mix well, add 3 g of cellulase, and perform enzymatic hydrolysis at 55°C for 4 h to obtain an enzymatic hydrolyzate;

[0055] (3) Add 10 times the amount of ethanol to the enzymatic hydrolyzate obtained in step (2), adjust the pH to 9, perform reflux extraction twice, each time for 3 hours, combine the extracts, and distill under reduced pressure to remove the ethanol for later use;

[0056] (4) The extract obtained in step (3) was adsorbed on D101 macroporous resin, washed with water to further remove impurities, eluted with 75% ethanol, and distilled under reduced pressure to remove ethanol to obtain an eluate;

[0057] (5) The eluate is decompressed, concentrated, crystallized, and dried to extract chrysin.

[0058] The extraction rates of chrysin extracted by different methods in Examples 1-3 and Comparative Examples 1-2 were statistically analyzed, and the purity of the chrysin extracts was tested by high performance liquid chromatography. The extraction rates were calculated using the following formula:

[0059] Chrysin extraction rate = Chrysin mass / Alpinia oxyphylla mass. The statistical results are shown in Table 1:

[0060] Table 1 Extraction rate and purity of chrysin extracts from different groups of methods

[0061]

[0062] Drug efficacy trials

[0063] Pharmacodynamic experiments were conducted on the extracts of Example 3 and Comparative Example 2 to investigate the effects of the chrysin extracts obtained from different groups on the hypoxia tolerance of mice.

[0064] Mouse confined hypoxia tolerance experiment

[0065] Experimental method: 70 adult healthy mice, half male and half female, 10 mice in each group, were taken. After adaptive feeding for 3 days, they were randomly divided into 7 groups. According to the treatment methods of different groups, they were orally gavaged for 5 days, with a dosage volume of 0.4mL / 20g. The hypoxia model group was given an equal volume of normal saline. 50 minutes after administration, the mice were placed in a 250mL wide-mouth bottle containing 5g of soda lime (only one mouse was placed in each bottle), the bottle mouth was smeared with vaseline, sealed to prevent air leakage, and timing was immediately started until breathing stopped. The survival time of mice in the closed wide-mouth bottle was used as an indicator to compare the hypoxia tolerance time of each drug group. The specific groups, dosage and hypoxia tolerance are shown in Table 2:

[0066] Table 2 Treatment methods and hypoxia tolerance of mice in different groups

[0067]

[0068]

[0069] Note: Commercially available chrysin: Purity greater than 95%, purchased from Shaanxi Ciyuan Biotechnology Co., Ltd.

[0070] Results analysis: As shown in Table 2, compared with the hypoxia model group, the low, medium, and high dose groups of the chrysin extract of Example 3 were able to prolong the survival time of mice in a closed hypoxic environment at normal pressure in a dose-dependent manner. The survival time extension rate of the high dose chrysin group reached 80.1%, which was significantly higher than that of the commercially available chrysin group and the acetazolamide group. A comparison of the high dose chrysin extract of Example 3 and the chrysin extract of Comparative Example 2 showed significant differences in the average extension time and extension rate. This indicates that the combination of bromelain and papain used in the enzymatic hydrolysis of Alpinia oxyphylla and extraction of chrysin in the present invention significantly improved the hypoxia tolerance of mice and enhanced the efficacy compared to cellulase.

[0071] Acute decompression and hypoxia tolerance experiment in mice

[0072] Experimental Methods: Fifty healthy adult mice, half male and half female, with 10 mice in each group, were randomly divided into five groups after three days of adaptive feeding. Oral gavage was administered for five days according to the treatment methods of the different groups, with a dosage volume of 0.4 mL / 20 g. The hypoxia model group was given an equal volume of normal saline. Twenty minutes after administration, the animals were placed in a hypobaric chamber with the door sealed. The animals were decompressed at a rate of 1000 m / min and ascended to an altitude of 5000 m and 8000 m, respectively, with a 5-minute dwell time. Finally, they ascended to an altitude of 10000 m. After maintaining this altitude for one hour, the inlet valve was adjusted and the animals were slowly descended to normal altitude. The door was opened and the animal mortality rate within one hour was observed and counted. The experimental results are shown in Table 3:

[0073] Table 3 Treatment methods and acute hypoxia tolerance of mice in different groups

[0074]

[0075] Note: Commercially available chrysin: Purity greater than 95%, purchased from Shaanxi Ciyuan Biotechnology Co., Ltd.

[0076] Analysis of results: As shown in Table 3, compared with the hypoxia model group, acetazolamide group and commercially available chrysin group, the chrysin extract of Example 3 can prolong the survival time of mice under reduced pressure and airtight hypoxia, with a mortality rate of 30% within 1 hour, indicating that the chrysin extract obtained by the method of the present invention can effectively reduce the mortality rate of mice under acute hypoxia conditions.

[0077] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0078] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting chrysin from Alpinia oxyphylla seeds, characterized in that: The steps include: (1) Add salt water to the Alpinia oxyphylla seeds, mix well, and stir-fry over low heat. After stir-frying, let it cool and set aside; (2) Grinding the cooled Alpinia oxyphylla seeds, adding an acidic aqueous solution and mixing well, and then adding a composite enzyme consisting of bromelain and papain for enzymolysis to obtain an enzymatic solution; (3) adding 10-15 times of ethanol to the enzymatic hydrolyzate obtained in step (2), adjusting the pH to 9-10, reflux extraction, combining the extracts, and distilling under reduced pressure to remove the ethanol for later use; (4) The extract obtained in step (3) is adsorbed on a macroporous resin, washed with water to further remove impurities, eluted with ethanol, and distilled under reduced pressure to remove ethanol to obtain an eluate; (5) The eluate is decompressed, concentrated, crystallized, and dried to extract chrysin.

2. The method for extracting chrysin from Alpinia oxyphylla seeds according to claim 1, characterized in that: In step (1), the addition ratio of the Alpinia oxyphylla kernel and the saline solution is 500-1000 g / 200-400 ml; and the concentration of the saline solution is 0.5-2 g / ml.

3. The method for extracting chrysin from Alpinia oxyphylla seeds according to claim 1, characterized in that: The frying time in step (1) is 30-60 minutes.

4. The method for extracting chrysin from Alpinia oxyphylla seeds according to claim 1, characterized in that: The crushed particles in step (2) are 20-60 meshes; an acidic aqueous solution is added to adjust the pH to 5-6; the amount of the complex enzyme added is 2-5% of the weight of the Alpinia oxyphylla kernel; and the addition ratio of bromelain and papain is 2-3:2-4.

5. The method for extracting chrysin from Alpinia oxyphylla seeds according to claim 1, characterized in that: The enzymolysis temperature in step (2) is 50-60° C., and the enzymolysis time is 2-6 h.

6. The method for extracting chrysin from Alpinia oxyphylla seeds according to claim 1, characterized in that: The reflux extraction in step (3) is performed 2-3 times, each time for 2-3 hours.

7. The method for extracting chrysin from Alpinia oxyphylla seeds according to claim 1, characterized in that: The model of the macroporous resin in step (4) is HPD-300, HPD-100 or D101; 70-80% ethanol is used for elution.

8. Use of the method according to any one of claims 1 to 7 in the extraction of chrysin.

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