Preparation method of flaxseed extract for bidirectional regulation of estrogen and its product

Abstract

The present invention relates to the field of plant extraction and fermentation, and in particular to a preparation method of a flaxseed extract capable of bidirectionally regulating estrogen and a product thereof. The present invention selects specific parts of flaxseed, and adopts microbial fermentation combined with a specific method for extraction to obtain a product rich in flax lignans, and the extraction purity and extraction rate are high. At the same time, the product has been tested on mice, and it is shown that the product has a significant bidirectional regulating effect on estrogen levels.

Description

Technical Field

[0001] The invention relates to the field of plant extraction and fermentation, and in particular to a preparation method of a flaxseed extract capable of bidirectionally regulating estrogen and a product thereof. Background Art

[0002] Flax is widely planted in temperate regions around the world, making the raw materials for lignan extraction sufficient. In my country alone, the annual planting area of ​​flax has reached 3-4 million mu, and 620,000 tons are exported annually. The planting and product processing industry of flax plays an important role in the development of the national economy. The development and utilization of lignans has been highly valued. Domestic and foreign studies have found that lignans, as natural plant extracts, are inactive substances. They become active substances only after they produce enterodiol and enterolactone under the action of mammalian intestinal microorganisms. The high content of lignans in flax and its estrogen effects, anti-cancer, anti-tumor and diabetes prevention effects have become one of the focuses of natural product research. The United States and Canada have developed rapidly in the development of lignan products, and have developed pure flaxseed shells containing lignans, lignan powder products, capsules, and flaxseed oil capsules containing lignans. Lignans are also added to cosmetics, and their antioxidant properties are used to prevent and treat eczema, acne, dry skin, etc. Flaxseed lignans are redeveloped and utilized based on the waste residues of extracting flax gum and squeezing flax oil.

[0003] Flaxseed mainly contains secoisolariciresinol (SECO) and wutairesinol (MAT). SECO is usually in the form of secoisolariciresinol diglucoside SDG (see Figure). Figure 1 ) exists in the form of SDG. SDG exists in two spatial isomers. Lignans are colorless crystals, soluble in water, methanol, aqueous ethanol and propanol, and are easily hydrolyzed by enzymes and acids.

[0004] Flax lignans are mainly secoisolariciresinoldiglucoside (SDG) and secoisolarciresinol (SECO). Flax lignans are mainly found in the secondary cell walls of flax seed exocarp cells, and only about 8% are found in endosperm cells.

[0005] Flax lignans, as a natural phytoestrogen, have multiple biological effects and significant efficacy in anti-cancer, anti-tumor, anti-cardiovascular disease, etc. They have important utilization value and broad market prospects.

[0006] SECO (or SDG) and MAT are converted into active animal lignans that can be absorbed and utilized by the human body by facultative aerobic microorganisms in the gastrointestinal tract of humans or animals: enterolactone and animal lignan enterodiol, such as Figure 2 shown.

[0007] Lignans can reduce the incidence of breast cancer in postmenopausal women.

[0008] Estrogen can alleviate the hormone metabolism of menopausal women. Hutchins et al. found that supplementing flax lignans in the food of menopausal women can reduce the content of estradiol and estrone sulfate, thereby increasing the concentration of prolactin in the blood. Abe et al. found that menopausal women can greatly improve osteoporosis, hyperlipidemia, hypertension, obesity and other symptoms caused by menopause by eating foods supplemented with flax lignans.

[0009] Therefore, it is necessary to develop related products containing high content of flax lignans. Summary of the invention

[0010] In view of the above problems, the present invention provides a method for preparing a flaxseed extract capable of bidirectionally regulating estrogen and a product thereof, and the present invention adopts the following technical solutions to achieve the above problems:

[0011] A method for preparing a flaxseed extract comprises the following steps:

[0012] Flax seeds are taken, aerobically fermented, the fermented liquid is extracted with an alkaline aqueous solution, then filtered, an acidic substance is added to adjust the pH to 6.5-7.5, and then dried to obtain a flax seed extract.

[0013] Preferably, the part of flax seed is flax seed husk and / or flax seed meal.

[0014] Preferably, the liquid aerobic fermentation bacteria are yeast and bacillus; the liquid aerobic fermentation conditions are: oxygenation, temperature of 25-37°C, time of 12-24h; the mass ratio of the yeast and bacillus is 1:1-3; the addition amount of the bacteria is 0.5-2wt% of the fermentation substrate mass.

[0015] Preferably, the acidic substance is at least one of the following substances:

[0016] Citric acid, acetic acid, vitamin C.

[0017] Preferably, the conditions for alkaline aqueous solution extraction are alkaline aqueous solution with a pH of 9-12 and a temperature of 50-70°C;

[0018] The alkaline aqueous solution is a solution formed by dissolving sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate in water.

[0019] Preferably, the filtration is firstly filtering through a fiber membrane and then ultrafiltration through an ultrafiltration membrane, and then concentrating after ultrafiltration.

[0020] Preferably, the drying is spray drying or freeze drying.

[0021] A product is prepared by the above-mentioned preparation method.

[0022] In the present invention:

[0023] Fermentation process: Using aerobic fermentation to increase the content of flax lignans: The content of flax lignans in flax seeds can be increased through microbial fermentation. The combined action of yeast and Bacillus may activate the enzyme system in flax seeds, promote the release and synthesis of flax lignans, and accelerate hydrolysis to release the flax lignans in flax seeds themselves.

[0024] Optimization of fermentation conditions: By controlling oxygenation, temperature and time as well as the ratio of yeast and Bacillus, the fermentation process can be optimized and the yield and purity of flax lignans can be increased.

[0025] The alkaline aqueous solution has high extraction efficiency: it can effectively dissolve the flax lignans, while the lower temperature is conducive to reducing the damage of heat-sensitive substances.

[0026] Double filtration process: first use fiber membrane filtration to remove large particle impurities, and then use ultrafiltration membrane for further purification, which can effectively separate the target components while retaining the activity of flax lignans.

[0027] Concentration process: The concentration step after ultrafiltration can increase the concentration of the extract and reduce the energy consumption in the subsequent drying process.

[0028] Diversity of drying methods: Spray drying or freeze drying can be used. The appropriate drying method can be selected according to the needs of the final product. Spray drying is suitable for large-scale production, while freeze drying can better maintain the activity and stability of flax lignans.

[0029] Extracting non-traditional parts of flaxseed: Flaxseed oil is usually widely extracted and utilized, but the present invention uses flaxseed husk and / or flaxseed meal, which may contain underutilized flax lignans. Through a specific extraction process, flaxseed resources can be more fully utilized.

[0030] The beneficial technical effects of the present invention are embodied in the following aspects:

[0031] The invention selects specific parts of flax seeds and uses microbial fermentation combined with a specific method to extract, thereby obtaining a product rich in flax lignans with high extraction purity and extraction rate. At the same time, the product has been shown to have significant bidirectional estrogen regulation effects through mouse experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the chemical structural formula of flax lignan (SDG).

[0033] Figure 2The present invention is the pathway of the metabolism of secoisobutyric acid ester (SECO) and wutai resin phenol (MAT) into enterolactone and animal lignan enterodiol in human body.

[0034] Figure 3 This is the HPLC spectrum of SDG standard - 2ul - 280nm.

[0035] Figure 4 This is the DAD spectrum of SDG standard. DETAILED DESCRIPTION

[0036] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples, wherein the terms used in the examples are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0037] The sources of some ingredients in the present invention are as follows

[0038] Candida albicans, number CICC1965, purchased from China Industrial Microbiological Culture Collection Center;

[0039] Bacillus subtilis, number CICC10732, purchased from China Industrial Microbiological Culture Collection Center;

[0040] Bacillus licheniformis, number CICC10084, was purchased from China Industrial Microbiological Culture Collection Center;

[0041] Lactobacillus plantarum: number CICC21790, purchased from China Industrial Microbiological Culture Collection Center;

[0042] Aspergillus oryzae, number CICC2001, was purchased from China Industrial Microbiological Culture Collection Center;

[0043] Flaxseed (whole), flaxseed husks, flaxseed meal: marketed.

[0044] A method for preparing a flaxseed extract comprises the following steps:

[0045] (1) taking flax seed raw material and crushing it to obtain raw material;

[0046] (2) adding bacteria and flaxseed raw material with water in an amount of 5 wt% by weight; stirring, keeping warm and providing oxygen for fermentation to obtain a fermentation liquid;

[0047] (3) extracting the fermented liquid by stirring with an alkaline aqueous solution in a water bath to obtain an extract, wherein the alkaline aqueous solution is a sodium hydroxide aqueous solution;

[0048] (4) filtering the extract through a fiber membrane, ultrafiltration through a 5000 Dalton molecular weight cutoff ultrafiltration membrane, and concentrating the ultrafiltration membrane permeate through a 200 Dalton nanofiltration membrane to obtain a concentrated solution;

[0049] (5) Add citric acid, acetic acid or vitamin C to the concentrate to adjust the pH to the range of 6.5-7.5.

[0050] (6) The concentrated solution is spray-dried or freeze-dried to obtain a hemp seed extract.

[0051] The specific parameters and composition ratios are shown in Table 1 below.

[0052] Table 1 Preparation parameters of flaxseed extract

[0053]

[0054]

[0055] Table 1 Preparation parameters of flaxseed extract

[0056]

[0057] In Table 1, “-” means no addition or no relevant steps; the fermentation temperature is the initial fermentation temperature, and the temperature of the entire fermentation process is controlled within ±1°C of the initial fermentation temperature.

[0058] The difference between Comparative Example 4 and Example 2 is that flaxseed (whole) is used as the fermentation raw material.

[0059] The difference between Comparative Example 5 and Example 2 is that a single yeast was used for fermentation.

[0060] The difference between Comparative Example 6 and Example 2 is that a single Bacillus sp. was used for fermentation.

[0061] The difference between Comparative Example 7 and Example 2 is that a single lactic acid bacteria was used for fermentation.

[0062] The difference between Comparative Example 8 and Example 2 is that a single Aspergillus niger fermentation was used.

[0063] The difference between Comparative Example 9 and Example 2 is that a lower fermentation temperature was used.

[0064] The difference between Comparative Example 10 and Example 2 is that the mass ratio of yeast to Bacillus is 2:1.

[0065] Content and extraction rate test:

[0066] The flax lignan content of each comparative example and example was measured by HPLC, and the yield was calculated based on the mass ratio of the raw material to the product.

[0067] (1) Chromatographic conditions

[0068] Waterse 2695 model HPLC, 2998 PDA detector, chromatographic column: Altalantis TMT3 (250 mm*4.6 mm, 5 um); column temperature 30°C; flow rate 1.0 ml / min; detection wavelength 254 nm, 327, 220, 280 nm; mobile phase gradient table is as follows:

[0069] Table 2 Mobile phase gradient

[0070] Time / min 0.1% Formic acid acetonitrile / % 0.1% Formic acid / % 0 20 80 10 35 65 12 20 80 15 20 80

[0071] (2) Solution preparation

[0072] Preparation of reference solution: Accurately weigh about 5 mg of SDG reference substance into a 10 mL volumetric flask, and add methanol to dissolve and dilute to the mark.

[0073] Preparation of sample solution: Accurately weigh about 10 mg of flax lignan sample into a 25 mL volumetric flask, and add methanol to dissolve and dilute to the mark.

[0074] (3) Sample measurement

[0075] Under the above chromatographic conditions, the sample was injected and measured, and the content of SDG in the sample was calculated by the external standard method.

[0076] (4) The results are as follows:

[0077] Example 1: yield 6.68%, purity 43.7%;

[0078] Example 2: yield 6.11%, purity 41.2%;

[0079] Example 3: yield 7.02%, purity 42.6%;

[0080] Comparative Example 4: yield 3.68%, purity 29.4%;

[0081] Comparative Example 5: yield 4.91%, purity 36.2%;

[0082] Comparative Example 6: yield 3.76%, purity 32.6%;

[0083] Comparative Example 7: yield 2.24%, purity 41.7%;

[0084] Comparative Example 8: yield 3.45%, purity 28.4%;

[0085] Comparative Example 9: yield 3.12%, purity 26.2%;

[0086] Comparative Example 10: yield 5.16%, purity 31.9%;

[0087] In Comparative Example 4, flaxseed (whole) was used as the fermentation raw material. Since the raw material contained a large amount of oil, the yield and purity of lipid-insoluble flax lignans were seriously affected.

[0088] Comparative Examples 5-8 used different strains for fermentation, and the results showed that the effects were not as good as the mixed fermentation of yeast and Bacillus.

[0089] Comparative Example 9 uses a relatively low temperature fermentation, and its strain is not active, and the yield cannot be improved; at the same time, it is also impossible to release flax lignans by fermentation to accelerate hydrolysis, resulting in lower purity.

[0090] Examples 1-3 and Comparative Example 10 use different ratios of yeast and Bacillus, and the effects are different, which proves that the yield can not be further improved by mixing yeast and Bacillus in any ratio.

[0091] Estrogen Regulation Tests:

[0092] 1. Reagents and medicines

[0093] Luteinizing hormone radioimmunoassay kit, interleukin-2 radioimmunoassay kit, estradiol radioimmunoassay kit, follicle-stimulating hormone radioimmunoassay kit, nialestradiol tablets (2 mg / tablet): commercially available.

[0094] Flaxseed extract: sample prepared in Example 2;

[0095] 2. Instruments

[0096] Model 1470 Wizard fully automatic gamma counter;

[0097] DK-type electric constant temperature water tank;

[0098] LDZ5-2 low speed automatic balancing centrifuge;

[0099] Leica TP1020 automatic water processor.

[0100] 3. Animals

[0101] 40 female SD rats, weighing 250-280 g, half of them were male and half were female.

[0102] 4. Experimental methods

[0103] 4.1. Reproduction of the menopausal syndrome model in ovariectomized rats

[0104] 40 female SD rats weighing 250-280g were selected. The experimental rats were anesthetized with ether, fixed on the rat fixing board in the ventral position, and a cotton ball soaked in ether was placed in the mouth and nose of the animal to maintain anesthesia. Then, under the last rib, at the intersection of the midaxillary line and about 2cm from the outside of the spine, the long hair was cut, and the skin and back muscle were cut about 1.5-2cm. A milky white fat mass was visible in the incision field, and the ovary was embedded in it. The fallopian tube under the ovary was ligated, and the bilateral ovaries were removed. After the operation, the uterine horns were returned to the abdominal cavity and sutured. Vaginal smears were made 5 days after the operation to observe the estrous cycle of the animals after modeling, once a day for 5 consecutive days, and rats with no obvious cyclical changes (i.e., the ovaries were completely removed) were selected as rats with successful modeling.

[0105] Evaluation metrics

[0106] 4.2.1. Vaginal smear cytological changes during the estrous cycle

[0107] (1) Diestrus: Mainly a large number of multinucleated white blood cells, and a small number of epithelial cells.

[0108] (2) Proestrus: There are a large number of epithelial cells with granular cytoplasm and a small number of keratinized (anucleated) epithelial cells, but no leukocytes.

[0109] (3) Estrus period: mainly a large number of keratinized epithelial cells, which are large and irregular in shape, with a small number of epithelial cells in between.

[0110] (4) Metestrus: There are a large number of white blood cells and fused keratinized epithelial cells.

[0111] 4.2.2. Reference results of successful modeling:

[0112] All experimental animals entered the estrus from the 5th day after the operation. The shedding cells decreased day by day. By the 9th day, only a small number of white blood cells were seen, and occasionally nucleated epithelial cells were seen. However, the animals were still lively and active, with smooth fur and normal muscle tension.

[0113] 4.3. Grouped Dosing

[0114] The rats with successful modeling were randomly divided into 4 groups: (1) sham operation group: distilled water 10 ml / kg; (2) model group: distilled water 10 ml / kg; (3) nialestradiol group: 0.42 mg / kg; (4) flaxseed extract group: 8.0 g (raw drug) / kg.

[0115] 4.4. Detection indicators

[0116] (1) Vaginal smear cytological observation: Vaginal smears were performed on the 10th day after oral administration to observe the estrous cycle of rats. A sterile cotton swab was inserted into the rat's vagina for about 1-2 cm, rotated along the vaginal wall once, and then evenly smeared on a slide. After slightly air-drying, it was fixed with 10% formaldehyde solution. The samples were routinely collected, dehydrated, paraffin-embedded, and sliced. After HE staining, light microscopy examination was performed once a day for 4 consecutive days.

[0117] (2) Determination of sex hormone levels: Blood was collected from the rats’ orbits 24 h after the last administration to detect serum gonadotropin and sex hormone levels: estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), interleukin-2 (IL-2) and other indicators were determined by radioimmunoassay.

[0118] Statistical methods

[0119] The quantitative response was analyzed by Student-t test using Excel software. The qualitative response was analyzed by χ test using SPSS software. 2 (Chi-square) test.

[0120] 5. Experimental results

[0121] 5.1 Effects on vaginal smear and estrous cycle results in rats

[0122] The results showed that the number of rats with a large number of keratinized epithelial cells in the model group was significantly lower than that in the sham operation group, and the difference was significant (p<0.01); the number of animals with a large number of keratinized epithelial cells in each drug-treated group was increased compared with the model group, among which the flaxseed extract group had a significant difference compared with the model group (p<0.05), but the extract I group had a very significant difference compared with the model group (p<0.01). This suggests that the drug can improve the changes in the estrous cycle of the menopausal rat model; as shown in Table 3.

[0123] 5.2. Effects on serum IL-2, LH, E2, and FSH levels in rats

[0124] The results showed that after modeling, the levels of interleukin-2 (IL-2) and estradiol (E2) in rats were lower than those in the sham operation group, while the levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were higher than those in the sham operation group.

[0125] Table 3 Effects of rat vaginal smear

[0126]

[0127] Table 4 Effects of serum hormone levels in rats

[0128]

[0129] Compared with the sham operation group in Tables 3 and 4, △△p<0.01; compared with the model group, **p<0.01, *p<0.05.

[0130] 6. Experimental conclusion

[0131] The above research results show that the extract obtained by the present invention has a certain effect on regulating estrogen and can alleviate the hormone metabolism of menopausal women after menopause.

[0132] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing flaxseed extract, characterized in that: The following steps are involved: Taking flax seeds, fermenting them aerobically in liquid form, extracting the fermented liquid with an alkaline aqueous solution, filtering it, adding an acidic substance to adjust the pH to 6.5-7.5, and drying it to obtain a flax seed extract; The part of flax seed is flax seed husk and / or flax seed meal; The bacterial species for the liquid aerobic fermentation are yeast and bacillus; The conditions of the liquid aerobic fermentation are: oxygenation, temperature of 25-37°C, and time of 12-24h; The mass ratio of the yeast to the bacillus is 1:1-3; The amount of the bacteria added is 0.5-2wt% of the fermentation substrate mass; The conditions for the alkaline aqueous solution extraction are an alkaline aqueous solution with a pH of 9-12 and a temperature of 50-70°C; The alkaline aqueous solution is a solution formed by dissolving sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate in water; The filtration is firstly filtering through a fiber membrane and then filtering through an ultrafiltration membrane, and then concentrating after ultrafiltration.

2. The method for preparing the flaxseed extract according to claim 1, characterized in that: The acidic substance is at least one of the following substances: Citric acid, acetic acid, vitamin C.

3. The method for preparing the flaxseed extract according to claim 1, characterized in that: The drying is spray drying or freeze drying.

4. A product, characterized in that The product is prepared by the preparation method of the flaxseed extract according to any one of claims 1 to 3.

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