CN1305869C - Tech. for preparing isoflavone of red clover from red clover - Google Patents

Abstract

The present invention relates to a technology for preparing the isoflavone of red clover from red clover, particularly to a method for preparing the isoflavone of red clover from red clover. The method has the following concrete technical process: firstly, red clover alcohol liquid extract is prepared by the hot reflux extraction of low-carbon alcohol; then, hydrolytic treatment is carried out by mineral acid; the pH value of filtering liquid with the hydrolytic treatment is regulated to be neutral; the neutral filtering liquid is depressurized and condensed; high-temperature activated silica gel powder or high-temperature activated active aluminum oxide is added for absorption; after the washing and the impurity removal of absorbed substances, the absorbed substances are eluted; eluent is filtered; solvent is recovered, and after condensation and drying, total isoflavone preparation objects of the red clover are obtained. After the present invention is adopted, onospin and sissotorin which have very high content in the red clover are previously hydrolyzed and conversed into formoononetin and Abiochanin A; four kinds of effective component isoflavone, such as the formoononetin, the Abiochanin A, genistein and daidzein can be easily and effectively prepared from the red clover; the total isoflavone content of preparation products surpasses 60%, and accordingly, the isoflavone of red clover from red clover has the favorable value of medicinal foods or functional foods.

Description

A preparation process for preparing red clover isoflavones from red clover

Technical field The present invention relates to a method for preparing red clover isoflavones from red clover for clinical medicine or functional food.

Background technology Modern medical research has proved that estrogen metabolic disorders are closely related to menopausal syndrome, osteoporosis, cardiovascular disease, senile dementia, and estrogen-dependent tumors, such as uterine cancer, breast cancer, prostate cancer, and colon cancer. Estrogen replacement therapy is beneficial to the prevention and treatment of menopausal syndrome, osteoporosis, cardiovascular disease, senile dementia, prostate cancer, and colon cancer, but it may increase the incidence of uterine cancer and breast cancer. A large number of studies have proved that the supplementation of phytoestrogens is helpful to the prevention and treatment of the above diseases, but it does not have the disadvantages of human estrogen replacement therapy. Therefore, supplementary therapy of phytoestrogens has attracted more and more attention.

Red clover is a widely planted grass, also known as red clover and red clover. Isoflavones (Isoflavones) are a class of active substances that contain a structure similar to human estrogen, a non-steroidal structure, and have biphasic regulatory effects on the metabolism and expression of human estrogen and gonadotropins. It mainly includes "genistein", "daidzein", "chickpein A", "formononetin" and their glycosides, often called "phytoestrogens" (Phytoestrogens). Red clover is a rare plant containing four effective isoflavones. In recent years, advanced western countries have recommended the total isoflavones of red clover as a food supplement for menopausal and postmenopausal women and osteoporosis patients.

The preparation method of red clover isoflavones from red clover at present has the following several kinds:

1. Solvent method Thermal extraction with polar solvents such as water, methanol, and hydrous ethanol. Water and medium- and low-concentration ethanol are suitable for the extraction of flavonoid glycosides; methanol and high-concentration ethanol are used for the extraction of aglycones. The extract obtained by the solvent method has complex components, and it is often necessary to extract and separate the liquid extract of water or alcohol with different polar solvents, and the content of isoflavones in the extract is low, and the solvent consumption is large.

2. Precipitation method Extraction with water or alcohol, the extract is obtained by precipitation with lead acetate or polyvinylpyrrolidone to obtain flavonoid glycosides and flavonoids with o-diphenolic hydroxyl or carboxyl structures. Although this method is simple, the extraction of isoflavones is incomplete, and the content of isoflavones in the preparation is low.

3. Hydrolysis The crude isoflavone glycosides obtained by the solvent method or the precipitation method are hydrolyzed by heating with hydrochloric acid solution; or the polar solvent heat extraction solution is directly added with acid or enzyme hydrolyzed, and the aglycone is separated out by cooling. Although this method is simple, the impurity content of isoflavones in the preparation is very high.

4. Column chromatography Commonly used are polyamide, silica gel, alumina, dextran gel, and macroporous resin column chromatography. The product obtained by solvent method or precipitation method is adsorbed by the above-mentioned filler, and then eluted with multiple groups of multi-solvent systems with different polarity gradients, concentrated and dried to obtain various isoflavone glycosides and aglycones. This method can obtain isoflavone products with a relatively high content, but the operation of separating different isoflavone glucosides and aglycones is more complicated, the production cycle is long, the cost is high, and it is difficult to produce on a large scale.

5. Preparative high-performance liquid chromatography is to prepare a high-purity monomer of a certain isoflavone component by passing the crude isoflavone products obtained by column chromatography through preparative high-performance liquid chromatography, and it is not suitable for total isoflavone products preparation.

The isoflavone components in plants are very complex, and the physical and chemical properties of various isoflavone components are very different, so it is difficult to adopt a general method. At present, the production process of red clover isoflavone preparations generally adopts the solvent method to produce a crude product with relatively low isoflavone content——red clover extract; Higher products. In addition, isoflavone components with estrogen-like effects in plants generally include two major categories: isoflavone aglycone (element) and isoflavone glycoside. It is generally believed that the active part is aglycone, and its glycosides are not only low in activity but also difficult to absorb. The content of isoflavone glycosides in the plant is quite high, especially in the leaves and flowers. The content of isoflavone glycosides in red clover is more than 1/3 of its total isoflavones, and the target of total isoflavone products in red clover is high content of isoflavone aglycone.

"The method for preparing red clover extract" (patent application No. 03118845), the method comprises the following steps: extracting the whole herb of red clover with methanol or ethanol; filtering the extract and concentrating; centrifuging the concentrate to take slag; The residue is degreased and dried with petroleum ether or n-hexane; after extraction and concentration with aliphatic esters, crystallization is obtained to obtain extract a; the residue after extraction is extracted with 50-90% alcohol, and the alcohol is recovered to obtain a concentrated solution; After crystallization, the mother liquor and the concentrated liquor are adjusted to a pH value of 8-10, separated by polystyrene skeleton macroporous adsorption resin to obtain extract b, combining extracts a and b, and refining with alcohol to obtain a high-content extract. The application provides a combination of epimedium, vitamin B↓[1], B↓[6] and vitamins combined with red clover extract, which is used to nourish the kidney and supplement phytoestrogens, so as to better Prevention of breast cancer, prostate cancer, colon cancer and prevention of osteoporosis, menopausal syndrome.

SUMMARY OF THE INVENTION The purpose of the present invention is to address the above-mentioned status quo, and to provide a simple and efficient method for preparing four active ingredients isoflavones from red clover: formononetin, biochanin A, dye Lignin, daidzein, the total isoflavone content of the preparation exceeds 60%, and a preparation process for preparing red clover isoflavones from red clover with good medicinal or functional food value.

The realization mode of the object of the present invention is, a kind of preparation technology of red clover isoflavones is prepared from red clover grass, and its specific technological process is:

1) Chop the dried and stored medicinal materials into fine sections,

2) Preparation of red clover alcohol liquid extract: red clover thin section is extracted with 6-12 times the amount of low-carbon alcohol under heat reflux, and the solvent is recovered under reduced pressure and concentrated to obtain red clover containing 1 g ± 0.2 g of crude drug per ml liquid extract,

3) Hydrolysis treatment of clover clover fluid extract: fully stir and redissolve the fluid extract with 2 to 5 times the amount of 95% ethanol first, filter to remove precipitation, add mineral acid to the filtrate to a final concentration of 2 to 5%, heat Reflux hydrolysis treatment for 1.5 to 3 hours, the hydrolyzate was filtered to remove slag,

4) adding saturated sodium hydroxide or potassium hydroxide solution to the hydrolyzed filtrate to adjust the pH to neutral, filtering to remove slag,

5) After the neutral filtrate is concentrated under reduced pressure to 1/2 times the original volume, or add high-temperature activated silica gel powder to fully grind and absorb, the amount of silica gel is 1/2-1 times the solution volume, dry, and then perform subsequent washing and washing. De-treatment, or add high-temperature activated activated alumina particles and stir for 1/2 to 1 hour, the amount of alumina is 1/2 to 2 times the weight of the solution, and then filter to remove the filtrate.

6) Washing and removing impurities of the adsorbate: first use 2 to 5 times the amount of cold petroleum ether at a temperature lower than 15°C to stir and wash, then filter to remove the ether, and then use 5 to 20 times the amount of cold water at a temperature lower than 15°C Stirring, washing and suction filtration to remove water, remove impurities in silica gel or activated alumina adsorbate,

7) Elution of silica gel or activated alumina adsorbate: after washing, the silica gel adsorbate is fully eluted with water-saturated ethyl acetate at 50-60°C with hot stirring, and the amount of water-saturated ethyl acetate is 2-4% of the weight of the adsorbate. times, alumina adsorption, and the complex is eluted with 1000:1 water-saturated ethyl acetate-glacial acetic acid solution under hot reflux, the amount of water-saturated ethyl acetate is 2 to 4 times the weight of the adsorbate, and the eluent is filtered and recovered The preparation of total isoflavones of red clover is obtained by solvent, concentration and drying.

By adopting the present invention, the pre-hydrolyzation of Ononin and Sissoirin, which are quite high in red clover, into formononetin and chickpein A can be done more easily and efficiently. Four active ingredients isoflavones—Formonontin, Biochanin A, Genistein, and Daidzein—were prepared from red clover. The total isoflavone content exceeds 50% and has good medicinal or functional food value.

Description of drawings

Figure 1 HPLC analysis spectrum of red clover alcohol liquid extract before hydrolysis

Figure 2 HPLC analysis spectrum of red clover alcohol liquid extract after hydrolysis

Figure 3 High performance liquid chromatography of adsorbate cold petroleum ether washing solution

Figure 4 High performance liquid chromatogram of adsorbate cold water washing solution

Fig. 5 liquid chromatogram of red clover alcohol extract-hydrolysis-silica gel adsorption-ethyl acetate elution product

Fig. 6 liquid chromatogram of red clover alcohol extract-hydrolysis-alumina adsorption-acidified ethyl acetate elution product

The specific embodiment The specific technical process of the present invention is:

1) Chop the dried and stored medicinal materials into fine sections,

2) Preparation of red clover alcohol fluid extract: red clover thin section is extracted with 6-12 times the amount of low-carbon alcohol for 3 hours, 2-4 times, and low-carbon alcohol such as 40-95% ethanol, methanol, Water saturated n-butanol. The solvent is recovered under reduced pressure and concentrated to obtain red clover alcohol liquid extract containing 1 g ± 0.2 g of crude drug per milliliter, which is rich in formononetin, biochanin A, and awn Pine flower glycosides, Indian Dalbergia glycosides, and contain a small amount of soy isoflavones and genistein. Its HPLC analysis spectrum is shown in Figure 1

3) Hydrolysis treatment of red clover alcohol liquid extract: the red clover alcohol liquid extract is first fully stirred and redissolved with 2 to 5 times the amount of 95% ethanol, filtered to remove precipitation, and the filtrate is rich in formononetin, Biochanin A, formononetin, indosiderin, and contain a small amount of soy isoflavones and genistein, its HPLC analysis spectrum is shown in Figure 1. Add mineral acid to the filtrate to a final concentration of 2 to 5%, heat reflux hydrolysis for 1.5 to 3 hours, filter the hydrolyzate to remove slag, add 3 to 5 times The amount of the original solvent was stirred and diluted, filtered as above, and the precipitate was removed. . Add mineral acid to the filtrate, such as adding concentrated hydrochloric acid for hydrolysis. Filter to remove slag, the dried residue of the filtrate sample is dissolved in methanol and detected by high-performance liquid chromatography, and it is confirmed that the peaks of formononetin and indica glucoside have basically disappeared, and they are respectively converted into formonetin and biochanin A, which can be seen from Figure 2.

4) Add saturated sodium hydroxide or potassium hydroxide solution to the hydrolyzed filtrate to adjust the pH to neutral, filter to remove the slag, if direct concentration and adsorption without adding alkali to adjust the pH to neutral will lead to carbonization failure of some active ingredients,

5) Adsorption treatment of silica gel powder or activated alumina particles: After the neutral filtrate is concentrated under reduced pressure to about 1/2 of the original volume, or add high-temperature activated 100-200 mesh silica gel powder to fully grind and adsorb the silica gel. The dosage of silica gel is 1/2 of the solution volume 2-1, dry, then perform subsequent washing and elution treatment, or add activated alumina particles activated at high temperature to stir for 1/2 to 1 hour, and let stand for more than 2 hours, the amount of alumina is 1/2 to the weight of the solution 2 times, after full adsorption and complexation, the filtrate is removed by suction filtration. When using alumina particles for adsorption, some isoflavone components will form complexes with alumina.

6) Wash and remove impurities from silica gel or activated alumina adsorbate: First, stir and wash 2 times with 2 to 5 times the amount of cold petroleum ether at a temperature lower than 15°C, each time for 1/2 to 1 hour, and then filter to remove ether. Finally, use 5 to 20 times the amount of cold water at a temperature lower than 15°C to stir and wash 2 to 5 times, each time for 1/2 to 1 hour, filter off the water, and stir and wash with cold petroleum ether to remove non-polarity and low polarity. It can be confirmed from Figure 3 that it basically does not contain the required 6 kinds of isoflavone components. If it is stirred and washed with hot petroleum ether, part of the effective part of the isoflavones will be lost; a large amount of highly polar impurities can be removed by cold water stirring and washing. The high performance liquid chromatography of the water washing solution in Fig. 4 confirms that it does not substantially contain the required 6 isoflavone components; if it is not stirred and washed with cold water, the preparation is a high-gloss mud paste, which has strong moisture absorption and is difficult to pulverize and obviously reduces the total amount of the product. Isoflavone content.

7) Elution of silica gel or activated alumina adsorbate: after washing, the silica gel adsorbate is fully eluted with water-saturated ethyl acetate at 50-60°C with hot stirring, and the elution time of each stirring is not less than 1/2 hour. The number of elutions is 4-5 times, the amount of water-saturated ethyl acetate is 2-4 times the weight of the adsorbate, the alumina is adsorbed, and the complex is eluted with water-saturated ethyl acetate-glacial acetic acid solution (1000:1) under hot reflux , the amount of water-saturated ethyl acetate is 2 to 4 times the weight of the adsorbate, the elution time of each reflux is not less than 2 hours, the eluent is filtered, and the elution times are 2 to 4 times, then the solvent is recovered, concentrated, and dried. The preparation of total isoflavones of red clover was obtained. A certain amount of acetic acid contained in the eluent is helpful to decomplexation, which can obviously improve the yield.

Ultraviolet spectrophotometry detects that the total isoflavone content of red clover prepared by the method of the present invention will exceed 60% in terms of formononetin, and the high performance liquid chromatography detection of the preparation mainly contains formononetin and olecranon Bean sprouts A, and contains a small amount of soybean isoflavones and genistein. Among them, the finished product yield of silica gel adsorption ethyl acetate elution method is higher than that of activated alumina method; while the relative content of biochanin A in the finished product of activated alumina adsorption, complexation and acidified ethyl acetate elution method is higher. The total isoflavone content of the products obtained by the two methods is similar, which can be seen from Figures 5 and 6.

The 14-month-old climacteric female Wistar rats of the red clover total isoflavone products prepared by the method of the present invention were randomly divided into a negative control group, a diethylstilbestrol group, and three doses of large, medium, and small red clover ovaries one week after surgical removal of both ovaries. Trifolium total isoflavones group, 10 rats in each group, and 10 rats in the sham operation group. Orally administered once a day, solvent, diethylstilbestrol and different doses of total isoflavones of red clover were administered respectively for 90 consecutive days. The results of animal experiments are shown in Table 1:

Table 1 Effect of total isoflavones of red clover on estradiol, follicle-generating hormone and uterine index in castrated menopausal rats group dose Body weight (g) before and after the experiment Uterine index (%) Serum Estradiol (pg/L) Serum follicle generating hormone (pg/L) mock surgical group 268±11288±25 0.19±0.04 45.46±9.10 3.97±0.95 negative control group 272±16283±22 0.10±0.04 23.16±4.16 4.07±1.53 diethylstilbestrol group 20μg/kg 276±9276±17 0.14±0.03 31.45±8.50 5.66±1.37 Group 1 of this preparation 8mg/kg 263±14314±21 0.10±0.05 28.67±8.45 5.66±1.19 2 groups of this preparation 15mg/kg 265±15314±14 0.11±0.02 30.71±5.33 5.17±1.48 3 groups of this preparation 30mg/kg 270±18316±23 0.13±0.03 31.54±8.24 4.79±1.35

It can be seen from the table that the preparation has good phytoestrogens effect.

Claims (1)

1, a kind ofly prepare the preparation technology of red clover isoflavones, it is characterized in that its concrete technical process is from red clover:

1) will dry the storage medicinal material and cut into thin segment,

2) preparation red clover alcohol fluid extract: the red clover thin segment extracts with the thermal backflow of 6～12 times of amount low-carbon alcohol, and decompression and solvent recovery also concentrates and makes every milliliter of red clover fluid extract that contains crude drug 1 gram ± 0.2 gram,

3) hydrolysis treatment of pure red clover fluid extract: fluid extract is fully stirred redissolution with 2～5 times of amount 95% ethanol earlier, cross the elimination precipitation, it is 2～5% that filtrate adds ore deposit acid to final concentration, thermal backflow hydrolysis treatment 1.5～3 hours, and the hydrolyzed solution filter and remove residue,

4) filtrate of hydrolysis treatment is added saturated sodium hydroxide or potassium hydroxide solution and transfer pH to neutral, filter and remove residue,

5) after the neutral filtrate decompression is concentrated into 1/2 times of original volume, or increase temperature the activatory silica-gel powder and fully grind absorption, the silica gel consumption is 1/2-1 a times of solution amount, dry, carry out subsequent wash and wash-out again and handle, or increase temperature activatory activated alumina particle stirring 1/2～1 hour, the aluminum oxide consumption is 1/2～2 times of solution weight, suction filtration goes filtrate again

6) adsorptive washing impurity elimination: earlier with 2～5 times of amounts, temperature is lower than 15 ℃ cold petroleum ether and stirring washing, suction filtration removes ether, and the back is with 5～20 times of amounts, and temperature is lower than 15 ℃ cold water agitator treating suction filtration and anhydrates, slough the impurity in silica gel or the activated alumina adsorptive

7) silica gel or activated alumina adsorptive wash-out: the silica gel adsorption thing after the carrying out washing treatment with the water saturation vinyl acetic monomer at 50～60 ℃ of abundant wash-outs of heat agitated, water saturation vinyl acetic monomer consumption is 2～4 times of adsorptive weight, alumina adsorption, complex compound were with water saturation vinyl acetic monomer-Glacial acetic acid liquid thermal backflow wash-out of 1000: 1, water saturation vinyl acetic monomer consumption is 2～4 times of adsorptive weight, and elutriant filters, reclaims solvent, concentrates, is drying to obtain the red clover total isoflavones prepared product.

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