Preparation method and application of genistein

Abstract

The application discloses a preparation method for separating and extracting genistein from carob pods, and specifically comprises the following steps: rough powder of carob pods is extracted with 80-95% and 60-80% ethanol successively to obtain an extract, and the extract is adsorbed by macroporous adsorption resin D101 to obtain non-sugar small molecules; the non-sugar small molecules are successively extracted by petroleum ether and ethyl acetate, and are separated and purified by silica gel column, ODS, Sephadex LH-20 column chromatography and semi-preparative liquid phase to obtain high-purity genistein; and the high-purity genistein has the effect of regulating estrogen and improving menopausal syndrome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extract preparation, in particular to a method for extracting genistein from seedless fruit pods of Caesalpinia spinosa Kuntze and application of the extracted genistein in female menopausal syndrome. BACKGROUND

[0002] Genistein is the main effective unit of soybean isoflavones. At present, the research on genistein mainly focuses on the role of isoflavones as plant hormones. Soybean isoflavones can not only replace estrogen and bind to receptors to exert estrogen-like effects, but also interfere with estrogen and receptor binding to exhibit anti-estrogen-like effects. For people with low estrogen levels, soybean isoflavones can prevent and treat menopausal syndrome, osteoporosis, elevated blood lipids and the like. For people with high estrogen levels, soybean isoflavones can prevent and treat breast cancer and endometritis, and has a two-way regulation and balance function. The component with estrogen-like effects in soybean isoflavones is mainly genistein. Genistein is mainly used in the form of soybean isoflavones in the market, but the effect is not obvious or ineffective when used. Investigation of literatures shows that only high-content soybean isoflavones with genistein are obtained in the prior art, and genistein is also reported to be purified from other plants, but the yield is not high. Therefore, there is great research potential for producing high-purity and high-content genistein.

[0003] Female menopausal syndrome is mainly caused by ovarian function decline, and further caused by estrogen deficiency in women. The main treatment method in clinical practice is estrogen replacement therapy, which has certain effects but has great side effects and cannot be used for a long time. Genistein in the present application is obtained by separation and purification from natural plant Caesalpinia spinosa Kuntze, has small side effects, raw materials are easy to obtain, and the extraction process is simple. SUMMARY

[0004] The present application first separates and extracts genistein from Caesalpinia spinosa Kuntze, and the method has the characteristics of easy-to-obtain raw materials, low price, simple and feasible process, low energy consumption, high purity of obtained genistein, and easy scale-up production and promotion.

[0005] The present application provides a preparation method and application of genistein, which comprises the following steps:

[0006] providing crushed seedless fruit pods of Caesalpinia spinosa Kuntze;

[0007] firstly extracting with 80-95% ethanol, and then extracting with 60-80% ethanol, and then concentrating the combined extract into extractum;

[0008] diluting and dispersing the obtained extractum with water, adsorbing through macroporous adsorption resin D101, firstly washing and eluting with distilled water to remove sugar impurities, and then washing and eluting with 95% ethanol, recovering the solvent of the obtained eluate, dispersing with methanol, and sequentially extracting with petroleum ether and ethyl acetate;

[0009] The ethyl acetate extraction part is recovered of solvent, and then separated and purified by silica gel column, ODS column (C18 column), Sephadex LH-20 column chromatography and semi-preparative liquid phase to obtain genistein. According to the embodiment of the present application, the carob tree is Ceratonia siliqua Linn. of the legume genus carob tree plant.

[0010] According to the embodiment of the present application, the seedless fruit pods of carob tree are crushed to 40-80 mesh, for example 60 mesh. It is found that the fruit pod cell structure can be fully broken within this particle size, which is beneficial to the full dissolution of genistein.

[0011] According to the embodiment of the present application, 80-95% ethanol is used for extraction first, and then 60-80% ethanol is used for extraction. It is found that this can increase the extraction rate of genistein.

[0012] According to the embodiment of the present application, when 80-95% ethanol is used for extraction for the first time, the weight ratio of 80-95% ethanol to seedless fruit pods of carob tree is 5-10:1, for example 5:1, 7:1, 8:1, 10:1. It is found that this is beneficial to the removal of part of impurities by precipitation.

[0013] According to the embodiment of the present application, 80-95% ethanol is used for extraction for 2-3 times for the first time, each time for 1-3 hours. According to the embodiment of the present application, when 60-80% ethanol is used for extraction for the second time, the weight ratio of 60-80% ethanol to seedless fruit pods of carob tree is 5-10:1. It is found that this is beneficial to the increase of solubility of genistein and the improvement of extraction rate.

[0014] According to the embodiment of the present application, 60-80% ethanol is used for extraction for 2-3 times for the second time, each time for 1-3 hours. It is found that macroporous adsorption resin D101 adsorption can separate ionizable substances, sugar substances and macromolecular substances.

[0015] It is found that the use of petroleum ether and ethyl acetate extraction can separate substances with different distribution coefficients.

[0016] According to the embodiment of the present application, when the ethyl acetate part is separated by silica gel column chromatography, petroleum ether-ethyl acetate (1:1-0:1, volume ratio) or dichloromethane-methanol (8:1-1:1, volume ratio) can be used for elution.

[0017] It is found that the use of silica gel column, ODS, Sephadex LH-20 column chromatography and semi-preparative liquid phase for separation and purification can obtain high-purity and high-yield genistein.

[0018] According to the embodiment of the present application, the semi-preparative liquid phase is Column I, methanol: purified water = 50:50 (volume ratio), absorption wavelength: 230 nm; Column II, methanol: purified water = 32:68 (volume ratio), absorption wavelength: 254 nm.

[0019] According to the embodiment of the present application, the method for extracting genistein from the legume of Caesalpinia seyal, comprising:

[0020] The legume of Caesalpinia seyal is taken out of seeds, crushed and passed through a 40-80 mesh sieve to obtain a coarse powder. Then 7 times the weight of 95% ethanol is added to the coarse powder to reflux extract 3 times (1.5 hours each time), and 75% ethanol is added to reflux extract 1 time (1.5 hours). The extract is concentrated to obtain an extract. The extract is dispersed in 5 times the weight of water, and adsorbed by a macroporous adsorption resin D101. The sugar and other impurities are removed by eluting 3 column volumes of distilled water, and then eluting 3 column volumes of 95% ethanol. After recovering the solvent, a non-sugar small molecule fraction is obtained. The non-sugar small molecule fraction is dispersed in 4 times the weight of 50% methanol, and then extracted with petroleum ether and ethyl acetate 5 times respectively. After recovering the solvent, a petroleum ether fraction, an ethyl acetate fraction and a water fraction are obtained. The ethyl acetate fraction is separated and purified by silica gel column chromatography, ODS column chromatography, Sephadex LH-20 column chromatography and semi-preparative liquid phase to obtain genistein.

[0021] After obtaining the ethyl acetate fraction, preferably, silica gel column chromatography is used for separation, a petroleum ether-ethyl acetate and / or dichloromethane-methanol elution system is used for gradient elution, TLC thin layer detection is used, and similar fractions are combined. The fraction is subjected to ODS column chromatography, methanol-water (15:85→100:0) elution, Sephadex LH-20, dichloromethane-methanol (1:1) elution, and then semi-preparative liquid phase (Column I, methanol: purified water = 50:50, absorption wavelength: 230 nm, Column II, methanol: purified water = 32:68, absorption wavelength: 254 nm) separation. The above operation is repeated 1-4 times to obtain genistein with a purity of 99.7%. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Effect of genistein in Caesalpinia seyal extract on estrogen.

[0023] Figure 2 Effect of genistein in Caesalpinia seyal extract on follicle stimulating hormone.

[0024] Figure 3 Effect of genistein in Caesalpinia seyal extract on bone density.

[0025] Figure 4 Chemical structural formula of genistein in Caesalpinia seyal extract. DETAILED DESCRIPTION

[0026] The following examples are intended to illustrate the present application but not to limit the scope of the present application.

[0027] The following examples are described by semi-preparative liquid chromatography, Column I is YMC-Pack ODS-A column with 20 mm i.d., Column II is YMC-Pack ODS-A column with 10 mm i.d.

[0028] Example 1

[0029] (1) Dry Alnus nepalensis fruit without seed, and pulverize to obtain 60 mesh coarse powder. Add 7 times the weight of 90% ethanol to the coarse powder, and reflux extract 3 times (1.5 hours each time). Then add 8 times the weight of 75% ethanol to the coarse powder, and reflux extract 1 time (1.5 hours). Combine the extract solutions, and concentrate to obtain an extract.

[0030] (2) Disperse the extract with 5 times the weight of water, and adsorb on macroporous adsorption resin D101. Elute with 3 column volumes of distilled water to remove sugars and other impurities, and then elute with 3 column volumes of 95% ethanol. Recover the solvent to obtain a non-sugar small molecule fraction. Disperse the obtained non-sugar small molecule fraction with 4 times the weight of 50% methanol, and sequentially extract with petroleum ether and ethyl acetate 5 times each. Recover the solvents to obtain a petroleum ether fraction, an ethyl acetate fraction, and a water fraction, respectively.

[0031] (3) Silica gel column chromatography gradient elution: recover the solvent of the ethyl acetate fraction, and separate by silica gel column chromatography with 200 mesh silica gel. Elute with petroleum ether-ethyl acetate (volume ratio 1:1, 1:3, 0:1) and dichloromethane-methanol (volume ratio 8:1, 7:1, 5:2, 1:1). TLC thin layer detection, and combine similar fractions to obtain 10 fractions (labeled A-J according to polarity from small to large).

[0032] (4) ODS column chromatography of fraction I, gradient elution with methanol-water (volume ratio 15:85→100:0). TLC thin layer detection of the obtained fractions, and combine similar fractions to obtain 5 fractions, labeled I1-I5 according to polarity from small to large.

[0033] (5) Sephadex LH-20 of fraction I3, elution with dichloromethane-methanol (1:1), then separation by semi-preparative liquid chromatography (Column I, methanol: purified water = 50:50, absorption wavelength: 230 nm), and purification by semi-preparative liquid chromatography (Column II, methanol: purified water = 32:68, absorption wavelength: 254 nm) (t R = 75.35 min) to obtain genistein with a purity of 99.7%.

[0034] Example 2

[0035] (1) Take dry Caesalpinia seeds to remove the seeds, and then crush to obtain 80 mesh coarse powder. Add 10 times the weight of 80% ethanol to the coarse powder, and reflux extract for 2 times, each for 1.5 hours. Then add 5 times the weight of 60% ethanol to the coarse powder, and reflux extract for 1 time, each for 1.5 hours. Combine the extract and concentrate to obtain the extract.

[0036] (2) Disperse the extract with 6 times the weight of water, and then adsorb it on a macroporous adsorption resin D101. Elute with distilled water for 3 column volumes to remove sugar and other impurities. Then elute with 95% ethanol for 3 column volumes. Recover the solvent to obtain the non-sugar small molecule fraction. Disperse the obtained non-sugar small molecule fraction with 5 times the weight of 50% methanol, and then sequentially extract with petroleum ether and ethyl acetate for 5 times, respectively. Recover the solvent to obtain the petroleum ether fraction, the ethyl acetate fraction, and the water fraction, respectively.

[0037] (3) Recover the solvent of the ethyl acetate fraction, and then separate it by using a 300 mesh silica gel column chromatography. Use petroleum ether-ethyl acetate (volume ratio 1:1, 1:2, 0:1) and dichloromethane-methanol (volume ratio 8:1, 7:1, 3:2, 1:1) as the elution system gradient elution. TLC thin layer detection, and then combine the similar fractions to obtain 10 fractions (labeled as A-J according to the polarity from small to large).

[0038] (4) Separate fraction I by ODS column chromatography, and gradient elute with methanol-water (volume ratio 15:85→100:0). TLC thin layer detection of the obtained fractions, and then combine the similar fractions to obtain 5 fractions, which are labeled as I1-I5 according to the polarity from small to large.

[0039] (5) Separate the obtained fraction by Sephadex LH-20, dichloromethane-methanol (1:1) elution, and then purify by semi-preparative liquid phase (column I, methanol: purified water = 50:50, absorption wavelength: 230 nm), and then purify by semi-preparative liquid phase (column II, methanol: purified water = 32:68, absorption wavelength: 254 nm) to obtain genistein with a purity of 98.5%.

[0040] Experimental Example 3

[0041] (1) Take Caesalpinia seed pods, remove the seeds, and then crush to obtain Caesalpinia seed pod coarse powder. Then add 8 times the weight of 85% ethanol to the coarse powder, and reflux extract for 4 times (each for 1.5 hours). Then add 10 times the weight of 75% ethanol to the coarse powder, and reflux extract for 2 times (1.5 hours). Combine the extract and concentrate to obtain the extract.

[0042] (2) The extract was dispersed with 5 parts by weight of water, adsorbed by macroporous adsorption resin D101, eluted with distilled water for 3 column volumes to remove sugar and other impurities, and then eluted with 95% ethanol for 3 column volumes. After recovering the solvent, a non-sugar small molecule fraction was obtained. The obtained non-sugar small molecule fraction was dispersed with 2 times by weight of 50% methanol, and then extracted with petroleum ether and ethyl acetate for 5 times, respectively. After recovering the solvent, a petroleum ether fraction, an ethyl acetate fraction, and a water fraction were obtained, respectively.

[0043] (3) The ethyl acetate fraction was recovered from the solvent, and then separated by silica gel column chromatography with a mesh of 300. Petroleum ether-ethyl acetate (volume ratio of 1:1, 1:2, 0:1) and dichloromethane-methanol (volume ratio of 8:1, 7:1, 3:2, 1:1) were used as elution systems for gradient elution. TLC thin layer detection was performed, and similar fractions were combined to obtain 10 fractions (labeled as A-J according to polarity from small to large).

[0044] (4) Fraction I was subjected to ODS column chromatography with gradient elution of methanol-water (volume ratio of 15:85→100:0). TLC thin layer detection was performed on the obtained fractions, and similar fractions were combined to obtain 5 fractions, which were labeled as I1-I5 according to polarity from small to large.

[0045] (5) The obtained fraction was subjected to Sephadex LH-20 with dichloromethane-methanol (1:1) elution, and then separated by semi-preparative liquid phase (column I, methanol: purified water = 50:50, absorption wavelength: 230 nm), and purified by semi-preparative liquid phase (column II, methanol: purified water = 32:68, absorption wavelength: 254 nm) to obtain genistein with a purity of 97.5%.

[0046] Comparative Example 1

[0047] Compared with Example 1, the only difference is that the concentration of ethanol in the first ethanol reflux extraction is 70%.

[0048] Genistein was obtained with a purity of 70.5%.

[0049] Comparative Example 2

[0050] Compared with Example 1, the only difference is that the concentration of ethanol in the second ethanol reflux extraction is 50%.

[0051] Genistein was obtained with a purity of 60.5%.

[0052] Comparative Example 3

[0053] Compared with Example 1, the only difference is that the obtained powder is 50 mesh.

[0054] Genistein was obtained with a purity of 50.5%.

[0055] Comparative Example 4

[0056] The difference from Example 1 is that petroleum ether-ethyl acetate (1:3), dichloromethane-methanol (5:2) is used for elution.

[0057] Genistein is obtained, and the purity reaches 60.5%.

[0058] Comparative Example 5

[0059] The difference from Example 1 is that only semi-preparative liquid chromatography (column I, methanol: purified water = 50:50, absorption wavelength: 230 nm) is used for separation, and genistein is obtained, and the purity reaches 69.5%.

[0060] Comparative Example 6

[0061] The difference from Example 1 is that only semi-preparative liquid chromatography (column II, methanol: purified water = 32:68, absorption wavelength: 254 nm) is used for separation, and genistein is obtained, and the purity reaches 79.5%.

[0062] Experimental Example Albizia Lebbeck Fruit Pod Extract Genistein Improving Female Menopausal Syndrome

[0063] (1) Effect of Genistein in Albizia Fruit Pod Extract on Estrogen and Follicle Stimulating HormoneSeventy female SD rats were selected and randomly divided into a sham operation group, a model group, a positive group (estradiol valerate), and Example 1-3 groups, and an Albizia extract group (prepared according to the procedure of Example 1, Step 4, fraction I4), 10 rats in each group. The body weight of the rats was 250-300 g. The rats were bred in an environment with a relative humidity of 50%-60% and a temperature of 18-22°C, with 12 h of day-night alternation, and free access to food and water. After one week of adaptive feeding, the modeling operation was performed. The sham operation group was subjected to removal of a small piece of fat, and the other groups were subjected to ovariectomy. The specific modeling method is described in reference to the Technical Specifications for Health Food Inspection and Evaluation (2003 edition).

[0064] The sham operation group and the model group were intragastrically administered with normal saline, the positive group was intragastrically administered with normal saline containing 1 mg / kg of body weight of estradiol valerate, the Example 1-3 groups were intragastrically administered with normal saline containing 1 mg / kg of body weight of the extracts of Example 1-3, and the Albizia extract group was intragastrically administered with normal saline containing 60 mg / kg of body weight of the extract I3 prepared according to the procedure of Example 1, Step 4 (the content of genistein was 0.35%), once a day, for 3 months of continuous administration. The water and food were not limited.

[0065] Twenty-four hours after the last administration, the rats in each group were sacrificed and blood was collected. The content of estradiol (E2) and follicle stimulating hormone (FSH) was detected by radioimmunoassay, and the results are shown in Table 1. Figure 1 、 Figure 2

[0066] The results show that the model group has a significant difference compared with the sham operation group, which indicates that the modeling is established. The example group can significantly reduce the content of estradiol (E2) in serum and can significantly increase the content of follicle stimulating hormone (FSH). The carob extract can also improve the content of E2 and FSH. It is shown that the genistein provided by the application can regulate estrogen.

[0067] (2) Effect of carob pod extract genistein on bone problems

[0068] The left femur of each group of rats in the above experiment (1) is taken out and placed under a dual-energy X-ray bone density measuring instrument to measure the bone density. The test results are shown in Table 2. Figure 3

[0069] The results show that the model group has a significant difference compared with the sham operation group, which indicates that the modeling is established. The example group can significantly reduce the content of estradiol (E2) in serum and can significantly increase the content of follicle stimulating hormone (FSH). The carob extract can also improve the content of E2 and FSH. It is shown that the genistein provided by the application can regulate estrogen.

[0070] In summary, the genistein provided by the application can regulate estrogen metabolism, and then improve female menopausal syndrome, including bone problems and the like.

[0071] Although the application has been described in detail in the foregoing with general description and specific embodiments, some modifications or improvements can be made on the basis of the application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the application, all belong to the scope of protection required by the application.​

Claims

1. A method for preparing genistein by isolating and extracting it from carob pods, characterized in that... Includes the following steps: (1) Extract the coarse powder of carob pods pulverized to 40-80 mesh using ethanol to obtain an extract; (2) The extract was adsorbed with macroporous adsorption resin D101 and eluted with distilled water and 95% ethanol for 3 column volumes to obtain non-sugar small molecules. (3) The non-carbohydrate small molecules are extracted sequentially with petroleum ether and ethyl acetate, and the ethyl acetate layer is recovered; (4) The ethyl acetate layer silica gel, ODS column chromatography, Sephadex LH-20 column chromatography and semi-preparative liquid chromatography are used to separate and purify the genistein.

2. The preparation method according to claim 1, characterized in that... Step (1) First, extract with 80-95% ethanol 2-3 times, 1-3 hours each time, with the weight ratio of 80-95% ethanol to the seedless carob pods being 5-10:1; then extract with 60-80% ethanol 2-3 times, 1-3 hours each time, with the weight ratio of 60-80% ethanol to the seedless carob pods being 5-10:

1.

3. The preparation method according to claim 1, characterized in that... In step (4), silica gel column chromatography was performed by gradient elution with two elution systems: petroleum ether-ethyl acetate and dichloromethane-methanol. The volume ratio of petroleum ether-ethyl acetate in the elution system was 1:1 → 0:1, and the volume ratio of dichloromethane-methanol in the elution system was 8:1 → 1:

1. After TLC thin-layer analysis, similar fractions were combined and numbered from smallest to largest polarity.

4. The preparation method according to claim 1, characterized in that... In step (4), during ODS column chromatography separation, the eluent is methanol-water with a volume ratio of 15:85→100:0; during Sephadex LH-20 column chromatography separation, the eluent is dichloromethane-methanol with a volume ratio of 1:

1.

5. The preparation method according to claim 1, characterized in that... In step (4), the semi-preparative liquid chromatography column I has a methanol:purified water volume ratio of 50:50 and an absorption wavelength of 230 nm, while column II has a methanol:purified water volume ratio of 32:68 and an absorption wavelength of 254 nm.

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