Soybean compound fermentation composition for treating climacteric syndrome as well as preparation method and application of soybean compound fermentation composition

Abstract

The invention discloses a soybean compound fermentation composition for treating climacteric syndrome as well as a preparation method and application thereof, relates to the technical field of special medical foods, and aims to solve the problem that a composition for treating climacteric syndrome, which is high in bioavailability and remarkable in effect, is lacked in the prior art. The preparation method is technically characterized by comprising the following steps: 1, respectively preparing a ginseng liquid medicine, a rehmannia liquid medicine, a rhizoma polygonati liquid medicine and a radix puerariae liquid medicine; 2, respectively adding soybeans into each liquid medicine, soaking until the liquid medicine is completely absorbed, sterilizing, respectively inoculating fermentation strains for fermentation, drying and crushing to obtain a ginseng and fermented soybean compound fermented product, a rehmannia and fermented soybean compound fermented product, a sealwort and fermented soybean compound fermented product and a kudzu vine root and fermented soybean compound fermented product; 3, soaking rhizoma polygonati and goat milk for multiple times, steaming, fermenting and drying to prepare a milk rhizoma polygonati fermented product; and 4, crushing and mixing the compound fermented product, the milk sealwort fermented product and the cucumber seeds to obtain the soybean compound fermented composition for treating the climacteric syndrome.

Description

Technical Field

[0001] This invention relates to the field of special medical food technology, and more specifically, to a soybean compound fermentation composition for treating menopausal syndrome, its preparation method, and its uses. Background Technology

[0002] Menopause is a crucial period in a woman's life, marking the end of her reproductive years, and is defined as the cessation of menstruation for 12 months. The average age of menopause is 51, with a normal range of 46 to 54. Menopause is accompanied by a series of estrogen changes. Starting in the early menopausal transition period, menstrual cycles become irregular and frequent. The ovaries' feedback to pituitary gonadotropins (GnRH), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) weakens, leading to a shortened follicular phase, reduced ovulation, decreased progesterone production, and a fluctuating, gradually declining estrogen levels. After menopause, estrogen levels tend to stabilize at a low level.

[0003] Menopausal syndrome, also known as perimenopausal syndrome, is a series of physical and psychological symptoms caused by fluctuations or decreases in estrogen levels. Approximately 70%-80% of women experience vasomotor symptoms (night sweats and hot flashes), as well as other symptoms such as palpitations, fatigue, autonomic nervous system dysfunction, dizziness, bladder irritation, and vaginal dryness. Studies show that during menopause, ovarian function declines, weakening the inhibitory negative feedback effect on the hypothalamus and pituitary gland, leading to abnormalities in the hypothalamic-pituitary-adrenal axis and a disordered neuroendocrine system. Decreased levels of neurotransmitters such as serotonin (5-HNT) and dopamine (DA) in the brain can easily cause menopausal anxiety, depression, impaired attention and memory, and insomnia. During the menopausal transition, hormonal imbalances and metabolic changes can lead to increased visceral fat deposition, dyslipidemia, increased insulin resistance, and postmenopausal osteoporosis and osteoarthritis. Estrogen therapy for menopause can alleviate some symptoms, but it also brings related side effects, leading to a general decline in its clinical use. Incorporating food-grade medicinal products into the daily diet can effectively avoid the side effects of hormone therapy while reducing the financial burden on patients.

[0004] Fermented soybeans are rich in important phytoestrogens—soy isoflavones, whose chemical structure is similar to that of the human estrogen 17-β-estradiol. Studies have reported that soy isoflavones not only exert estrogen-like effects but also have a bidirectional regulatory effect: when estrogen is insufficient, they exhibit estrogen-related functions; when estrogen is excessive, they can act as estrogen antagonists.

[0005] In recent years, there has been considerable research on the treatment of menopausal syndrome. For example, the existing technology with document number CN1961744A discloses a health food product using traditional Chinese medicine as raw materials, including 15-30 parts soybean meal powder, 15-30 parts pumpkin powder, 0.5-1.5 parts soy isoflavones, 2-5 parts soy saponins, 10-20 parts astragalus, 10-15 parts wolfberry, 10-15 parts kudzu root, 10-15 parts yam, 6-10 parts lotus seeds, 6-10 parts polygonatum, 6-10 parts cassia seeds, 5-8 parts lily bulb, 5-8 parts mulberry leaves, and 3-5 parts ginseng. It is said to lower lipids, lower blood pressure, have anticoagulant and antithrombotic effects, prevent osteoporosis, and prevent menopausal syndrome in women. However, the formulas disclosed in the above patents involve a wide variety of drugs, and the bioavailability of each raw material is low, making it difficult to achieve an effective treatment for menopausal syndrome. Furthermore, direct use of soy isoflavones may lead to related side effects or increase the burden on the liver. Summary of the Invention

[0006] The technical problem to be solved by this invention is:

[0007] There is a lack of bioavailable compositions for treating menopausal syndrome that have high bioavailability and significant efficacy.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0009] This invention provides a method for preparing a soybean compound fermentation composition for treating menopausal syndrome, comprising the following steps:

[0010] 1. Prepare ginseng extract, rehmannia extract, polygonatum extract, and kudzu root extract separately;

[0011] 2. Soybeans are added to ginseng medicinal liquid, rehmannia medicinal liquid, polygonatum medicinal liquid and kudzu root medicinal liquid respectively, soaked until the medicinal liquid is completely absorbed, sterilized, and inoculated with fermentation bacteria for fermentation. After fermentation, they are dried and crushed to obtain ginseng soybean compound fermented product, rehmannia soybean compound fermented product, polygonatum soybean compound fermented product and kudzu root soybean compound fermented product.

[0012] 3. Soak Polygonatum in fresh sheep milk for 5-7 hours to absorb the sheep milk, steam for 6-8 hours, inoculate with lactic acid bacteria for 24 hours, and dry; add fresh sheep milk again to soak, and repeat the above operation at least nine times to prepare fermented Polygonatum sibiricum.

[0013] 4. Mix the ginseng fermented soybean compound fermentation product, the rehmannia fermented soybean compound fermentation product, the polygonatum fermented soybean compound fermentation product, the kudzu root fermented soybean compound fermentation product, and the galangal fermentation product to obtain a mixture. Then, crush cucumber seeds are added to the mixture and mixed to obtain the soybean compound fermentation composition for treating menopausal syndrome.

[0014] In this invention, the soybean is the dried, mature seed of soybean (Glycine max (L.) Merr.), a legume.

[0015] Rehmannia glutinosa is the fresh or dried tuberous root of the plant Rehmannia glutinosa Libosch., belonging to the Scrophulariaceae family. It is harvested in autumn, with the rhizome, fibrous roots, and soil removed before use; alternatively, it can be slowly baked until about 80% dry. The former is commonly known as "fresh Rehmannia," and the latter as "raw Rehmannia." Fresh Rehmannia is sweet, bitter, and cold in nature; it enters the heart, liver, and kidney meridians; it has the effects of clearing heat and promoting body fluids, cooling the blood, and stopping bleeding; it is mainly used to treat yin deficiency due to febrile diseases, red tongue with thirst, febrile rashes, hematemesis, epistaxis, and sore throat. Raw Rehmannia is sweet and cold in nature, entering the heart, liver, and kidney meridians; it has the effects of clearing heat and cooling the blood, nourishing yin and promoting body fluids; it is mainly used to treat heat entering the blood level, febrile rashes, hematemesis, epistaxis, yin deficiency due to febrile diseases, red tongue with thirst, constipation due to fluid depletion, yin deficiency fever, bone steaming fever, and internal heat with thirst.

[0016] Polygonatum is the dried rhizome of *Polygonatum kingianum* Coll. et Hemsl., *Polygonatum sbiiricum* Red., or *Polygonatum cyrtonema* Hua, all belonging to the Liliaceae family. It is harvested in spring and autumn, the fibrous roots are removed, it is washed, briefly blanched in boiling water or steamed until thoroughly cooked, and then dried. Polygonatum is sweet and neutral in nature; it enters the spleen, lung, and kidney meridians; it has the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys; it is mainly used to treat spleen and stomach qi deficiency, fatigue, stomach yin deficiency, dry mouth and poor appetite, lung deficiency and dry cough, consumptive cough with hemoptysis, deficiency of essence and blood, soreness and weakness of the waist and knees, premature graying of hair, and internal heat and thirst. Milk Polygonatum is sweet, astringent, and warm. It warms the stomach, stimulates appetite, dries pus, strengthens the body, and promotes body fluid production; it is used to treat weakness, cold stomach, lower back and leg pain, indigestion, spermatorrhea, and impotence.

[0017] Ginseng is the dried root and rhizome of Panax ginseng CAMey, a plant belonging to the Araliaceae family. It is mostly harvested in autumn, washed, and then sun-dried or oven-dried. Ginseng is sweet, slightly bitter, and slightly warm in nature; it enters the spleen, lung, heart, and kidney meridians; it has the effects of greatly replenishing vital energy, restoring pulse and consolidating the body, tonifying the spleen and lungs, generating fluids and nourishing blood, calming the mind and improving intelligence; it is used for physical weakness and impending collapse, cold limbs and weak pulse, spleen deficiency and poor appetite, lung deficiency and cough, thirst due to fluid depletion, internal heat and thirst, deficiency of qi and blood, chronic illness and emaciation, palpitations and insomnia, impotence and cold uterus.

[0018] Kudzu root is the dried root of *Pueraria lobata* (Willd.) ohwi, a plant in the legume family. It is harvested in autumn and winter. While fresh, it is cut into thick slices or small pieces and then dried. Kudzu root is sweet, pungent, and cool in nature; it enters the spleen, stomach, and lung meridians. It has the effects of relieving muscle tension and reducing fever, promoting body fluid production and quenching thirst, promoting rash eruption, raising yang and stopping diarrhea, unblocking meridians and activating collaterals, and detoxifying alcohol. It is used for exogenous fever and headache, stiff neck and back pain, thirst, diabetes, incomplete measles eruption, dysentery, diarrhea, dizziness and headache, hemiplegia due to stroke, chest pain, and alcohol poisoning.

[0019] Cucumber seeds, also known as Halisu, are the seeds of the cucumber (Cucumis sativus L.), a plant in the Cucurbitaceae family. They are believed to have medicinal properties, including promoting bone healing, dispelling wind, and resolving phlegm. They are primarily used to treat fractures, muscle injuries, rheumatic pain, and phlegm-related asthma in the elderly.

[0020] Further, the preparation method of the ginseng extract, rehmannia extract, polygonatum extract and kudzu root extract mentioned in step one is as follows: according to the mass-volume ratio of ginseng, rehmannia, polygonatum or kudzu root and water of 1:(10-15), decoct and extract twice, each time for 1-2 hours, combine the filtrates after each decoction, and concentrate to (0.8-1.2) g / 12mL to obtain the extract.

[0021] Furthermore, in step two, the mass-to-volume ratio of soybeans to ginseng extract, rehmannia extract, polygonatum extract, and kudzu root extract is 100g:120mL.

[0022] Furthermore, the fermentation strain in step two is Bacillus subtilis, which was isolated from fermented soybean slices by the research team of this invention. It was deposited at the China Industrial Microbial Culture Collection Center on January 2, 2019, with the accession number CICC No. 24713. It has a high fibrinolytic enzyme production capacity. The ginseng fermented soybean compound fermented product, rehmannia fermented soybean compound fermented product, polygonatum fermented soybean compound fermented product, and kudzu root fermented soybean compound fermented product obtained by fermentation have high fibrinolytic enzyme activity.

[0023] Furthermore, in step four, the mass ratio of ginseng fermented soybean compound fermented product, rehmannia fermented soybean compound fermented product, polygonatum fermented soybean compound fermented product, kudzu root fermented soybean compound fermented product, cucumber seed powder and galangal fermented product is (10-11):(10-11):(10-11):(10-11):(10-11):1.

[0024] The present invention also provides a soybean compound fermentation composition for treating menopausal syndrome, wherein the composition is prepared by any of the above-described methods for preparing the soybean compound fermentation composition for treating menopausal syndrome.

[0025] The use of the composition in the preparation of medicines or foods for treating menopausal syndrome is also within the scope of protection of this invention.

[0026] The present invention also provides a pharmaceutical composition for treating menopausal syndrome, comprising the composition described in the above-described technical solution.

[0027] The pharmaceutical composition for treating menopausal syndrome provided by this invention is based on traditional Chinese medicine theory combined with modern research. The *Suwen* (Plain Questions) states: "When a person grows old and has no children, is it because their physical strength is exhausted, or is it simply the natural order of things? Qi Bo said: 'When a girl is seven years old, her kidney qi is abundant, her teeth change, and her hair grows longer;…at forty-nine, her Ren meridian is deficient, her Chong meridian is weak, her Tian Gui (menstrual blood) is exhausted, and her menstrual flow is blocked, therefore her form deteriorates and she has no children.'" *Fu Qingzhu's Gynecology* states: "When a couple reaches forty-nine, their Tian Gui is exhausted, and if they do not take medicines to nourish Yin and Yang, how can they regain their full essence and menstruation, like a young woman?" In the composition of this invention, Rehmannia glutinosa enters the kidney meridian and has the effect of nourishing yin and promoting body fluid production; Polygonatum sibiricum enters the spleen, lung, and kidney meridians and has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys; after Polygonatum sibiricum is added to sheep milk for fermentation, it has the effects of strengthening the body, relieving joint pain, and promoting body fluid production; Ginseng enters the spleen, lung, heart, and kidney meridians and has the effects of greatly replenishing vital energy, strengthening the spleen and benefiting the lungs, promoting body fluid production and nourishing blood, calming the mind and improving intelligence; Cucumber seeds have the effects of clearing the meridians, clearing heat and guiding downwards. The multiple drugs in the composition work together to achieve the effect of nourishing yin and replenishing yang.

[0028] The root cause of menopausal syndrome lies in kidney essence deficiency and yin-yang imbalance. The pharmaceutical composition of this invention uses ginseng and fermented soybean compound fermented product and rehmannia and fermented soybean compound fermented product as the principal ingredients. The ginseng and fermented soybean compound fermented product, in addition to its tonifying properties, also has a clearing effect, tonifying without causing stagnation. It is particularly effective for qi deficiency symptoms such as fatigue, weakness, shortness of breath, palpitations, and poor appetite due to spleen deficiency during menopause. The rehmannia and fermented soybean compound fermented product reduces the cloying nature of rehmannia, enhances its yin-tonifying effect, and is more easily absorbed, effectively improving symptoms of yin deficiency and internal heat such as hot flashes, night sweats, five-center heat (palms, soles, and chest), dizziness, and tinnitus. One tonifies qi, and the other nourishes yin; together, they tonify both qi and yin, serving as the principal ingredients to fundamentally regulate the physiological basis of menopausal syndrome. The fermented products of Polygonatum odoratum and fermented soybean, and Pueraria lobata and fermented soybean, are used as assistant herbs. The Polygonatum odoratum and fermented soybean compound fermented product can assist the ginseng and fermented soybean compound fermented product in replenishing qi, and also assist the Rehmannia glutinosa and fermented soybean compound fermented product in nourishing yin. At the same time, it strengthens the spleen and kidneys, and protects the acquired foundation to nourish the innate foundation. The Pueraria lobata and fermented soybean compound fermented product clears heat and generates fluids. Its ascending and dispersing properties are combined with the astringent properties of fermentation, making its effects more moderate. It assists the Rehmannia glutinosa and fermented soybean compound fermented product in clearing away deficiency heat. Its "raising yang" property can allow the tonifying effects of ginseng and fermented soybean and Rehmannia glutinosa and fermented soybean to reach the head and face, improving symptoms such as dizziness and headache. The fermented product of Calomel is used as an adjuvant herb. It strengthens the spleen and stomach, and prevents the principal and assistant herbs from being too rich and cloying, which would hinder the stomach and affect absorption. At the same time, its yin-nourishing and dryness-moistening properties further enhance the overall effect of replenishing water and controlling fire in the formula, improving concurrent symptoms such as dry skin and constipation. Cucumber seeds are used as an adjuvant in traditional Chinese medicine to promote blood circulation, clear heat and promote bowel movements, and synergistically improve symptoms of yin deficiency and dryness, as well as weak muscles and bones.

[0029] The present invention also provides a health food for treating menopausal syndrome, comprising the composition described in the above technical solution.

[0030] The present invention also provides a dietary supplement for treating menopausal syndrome, comprising the composition described in the above technical solution.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] In this invention, ginseng, rehmannia, polygonatum, and kudzu root are fermented with soybeans to prepare a compound fermented product, which is rich in the plant hormone soy isoflavones, exerting estrogen-like effects. Soy isoflavone glycosides decompose into aglycones, which further decompose into equol, not only improving bioavailability but also enhancing estrogen-like effects, thereby alleviating some symptoms caused by the decline in estrogen levels during menopause, while avoiding the side effects of direct estrogen supplementation. The composition of this invention produces high fibrinolytic activity through fermentation, which can lower blood lipids, reduce fat deposition, lower the risk of cardiovascular disease, and mitigate the problems of increased apolipoprotein secretion and elevated liver lipase activity caused by hormonal changes during the menopausal transition. Mongolian medicine cuscuta, after being soaked in milk, steamed thoroughly, and dried, has therapeutic effects on lower back and leg pain. This invention prepares cuscuta by repeatedly steaming and fermenting polygonatum with fresh goat milk, which not only enhances its tonic effects and reduces the irritation of polygonatum but also relieves joint pain. Simultaneously, the addition of cucumber seeds promotes bone formation, increases bone density, and improves postmenopausal osteoporosis and osteoarthritis.

[0033] Experiments have shown that the composition of the present invention can protect the morphological structure of the hippocampus, increase neurotransmitter secretion, and improve menopausal memory impairment, anxiety, and depression symptoms. The composition of the present invention can also promote osteoblast activity, increase bone density, and alleviate postmenopausal osteoporosis and osteoarthritis.

[0034] The pharmaceutical composition of the present invention can improve symptoms of anxiety, depression and memory impairment caused by decreased estrogen after menopause by increasing the secretion of neurotransmitters such as 5-HNT and DA in serum and brain tissue, and by protecting the hippocampus.

[0035] The medicinal and edible herbs in this invention work together to nourish Yin and replenish Yang, and are rich in various phytoestrogens, which can exert estrogen-like effects in the body; improve symptoms such as anxiety and depression caused by decreased estrogen after menopause; improve cardiovascular health and alleviate postmenopausal cardiovascular diseases by lowering blood lipids and resisting atherosclerosis; and promote bone formation and increase bone density to improve postmenopausal osteoporosis and arthritis, thus treating menopausal syndrome from multiple perspectives and with synergistic effects. Attached Figure Description

[0036] Figure 1 The HPLC chromatograms of daidzein (C), daidzein (A), genistein (D), and genistein (B) in the embodiments of the present invention are shown.

[0037] Figure 2 This is the HPLC chromatogram of equadol in an embodiment of the present invention;

[0038] Figure 3 The changes in soybean isoflavone content before and after fermentation in the compositions of this invention are shown in the embodiments of the present invention.

[0039] Figure 4 This is a diagram showing the effect of different concentrations of the drug on the morphology of the rat femur in an embodiment of the present invention;

[0040] Figure 5 This is a graph showing the effects of different drugs on serum BGP and ALP in osteoporosis model rats in this invention embodiment;

[0041] Figure 6 This is a diagram showing the effect of different concentrations of the drug on the morphology of rat hippocampal tissue in an embodiment of the present invention.

[0042] Figure 7 This is a diagram showing the effects of different drugs on 5-HNT and DA in the serum and brain tissue of a depressed rat model, as described in the embodiments of the present invention. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, exemplary embodiments or examples of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments or examples are merely some, not all, of the embodiments or examples of the present invention. All other embodiments or examples obtained by those skilled in the art based on the embodiments or examples of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0044] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0045] Example 1

[0046] A method for preparing a traditional Chinese medicine soybean compound fermentation composition for treating menopausal syndrome includes the following steps:

[0047] I. Weigh the raw materials according to the mass proportions and prepare the medicinal solution: Weigh 400g of soybean, 10g of ginseng, 20g of polygonatum, 10g of rehmannia, 10g of kudzu root and 100g of cucumber seed respectively; take 10g of ginseng, rehmannia, kudzu root and polygonatum into powder respectively, add 15 times the mass of distilled water to decoct and extract twice, each time for 1.5h, combine the filtrates after single decoction, concentrate to 1g / 12mL to obtain ginseng medicinal solution, rehmannia medicinal solution, polygonatum medicinal solution and kudzu root medicinal solution;

[0048] 2. Soak 100g of soybeans in the ginseng, rehmannia, polygonatum, and kudzu root extracts prepared in step 1 for 14 hours. After the homogenate is completely absorbed, sterilize at 121℃ to obtain ginseng fermentation substrate, rehmannia fermentation substrate, polygonatum fermentation substrate, and kudzu root fermentation substrate. Inoculate each fermentation substrate with Bacillus subtilis CICC24713, shake well, ferment at 35℃ for 48 hours, dry at 40℃, and pulverize to obtain ginseng-soybean compound fermented product, rehmannia-soybean compound fermented product, polygonatum-soybean compound fermented product, and kudzu root-soybean compound fermented product.

[0049] 4. Take 10g of Polygonatum sibiricum, cut it into pieces, soak it in fresh sheep milk for 6 hours, steam it for 7 hours, inoculate it with lactic acid bacteria for 24 hours, and dry it. Then soak it in fresh sheep milk again, and perform the above steaming, fermentation and drying operations. Continue to soak it in fresh sheep milk, and repeat this process nine times. Crush it to obtain fermented Polygonatum sibiricum.

[0050] 5. Crush 100g of cucumber seeds and mix them evenly with the ginseng fermented soybean compound fermented product, rehmannia fermented soybean compound fermented product, polygonatum fermented soybean compound fermented product, kudzu root fermented soybean compound fermented product obtained in step 3 and miltiorrhiza fermented product obtained in step 4 to obtain the soybean compound fermented composition for treating menopausal syndrome.

[0051] In step one of this embodiment, 50g of fresh Rehmannia glutinosa (equivalent to about 10g of dried Rehmannia glutinosa) can be juiced or mixed with a small amount of distilled water to prepare Rehmannia glutinosa liquid.

[0052] Prepare a medicinal solution by squeezing 80g of fresh Polygonatum (equivalent to about 20g of dried Polygonatum) or by adding a small amount of distilled water to homogenize it.

[0053] Prepare ginseng liquid by squeezing 30g of fresh ginseng (equivalent to about 10g of dried ginseng) or by adding a small amount of distilled water to homogenize it.

[0054] Experimental Example 2: Determination of Changes in the Content of Soy Isoflavones in Traditional Chinese Medicine Compositions

[0055] 1. Determination of the contents of daidzein, daidzein, genistein, genistein and equol in the composition.

[0056] 1.1 Preparation method of the test solution:

[0057] The preparation methods for the test solutions of daidzein, daidzein, genistein, and genistein are in accordance with the preparation methods for the test samples under the section on fermented soybean and soybean yellow volume in the Pharmacopoeia of the People's Republic of China (2020 edition).

[0058] Preparation of the test solution of the composition in Example 1: Accurately weigh about 2g of the powder of the composition in Example 1 (passed through a No. 2 sieve), place it in a stoppered conical flask, and accurately add 50mL of methanol solution (daidzein and aglycone), or 50mL of 70% methanol (daidzein and genistein), or 50mL of 80% methanol (estradiol). Weigh and record the weight, heat under reflux for 1 hour, and then cool. Make up the lost weight with methanol solution (daidzein and aglycone), or 70% methanol solution (daidzein and genistein), or 80% methanol solution (estradiol). Shake well, filter, and take the filtrate. Filter through a 0.45μm filter membrane to obtain the test solution.

[0059] Preparation of the test solution of the unfermented composition: Accurately weigh 40g of soybean, 1g of ginseng, 2g of polygonatum, 1g of rehmannia, 1g of kudzu root, and 10g of cucumber seed. After pulverizing, pass the drug powder through a No. 2 sieve and place it in a stoppered conical flask. Accurately add 50mL of methanol solution (daidzein and aglycone), or 50mL of 70% methanol (daidzein and genistein), or 50mL of 80% methanol (estradiol). Weigh and record the weight. Heat under reflux for 1 hour and then cool. Make up the lost weight with methanol solution (daidzein and aglycone), or 70% methanol solution (daidzein and genistein), or 80% methanol solution (estradiol). Shake well, filter, and take the filtrate. Filter through a 0.45μm filter membrane to obtain the final product.

[0060] 1.2 Preparation method of standard solution:

[0061] The preparation methods for the standard solutions of daidzein, daidzein, genistein, and genistein are in accordance with the preparation methods of the test samples under the section on fermented soybean and soybean yellow volume in the Pharmacopoeia of the People's Republic of China (2020 edition).

[0062] Equadol standard solution: Accurately weigh 1 mg of equadol standard, place it in a 1 mL volumetric flask, dissolve it in methanol, and dilute to the mark to obtain the standard stock solution. Store at 4℃. Accurately pipette 1 mL of the standard stock solution into a 10 mL volumetric flask and dilute to the mark with methanol to obtain a mass concentration of 100 μg / mL.

[0063] 1.3 Chromatographic conditions

[0064] Column: Symmetry C18 (5μm, 4.6×250mm).

[0065] Daidzin and genistein: Mobile phase: methanol solution (A) - 1% glacial acetic acid solution (B); Gradient elution: 0-25 min, 28% A; 25-33 min, 28-45% A; Flow rate: 1 mL / min; Detection wavelength: 260 nm; Injection volume: 10 μL.

[0066] Daidzein and genistein: Mobile phase: acetonitrile-1% glacial acetic acid (25:75), isocratic elution; Flow rate: 1 mL / min; Detection wavelength: 260 nm; Injection volume: 10 μL; Chromatographic peaks as shown Figure 1 As shown.

[0067] Estrol: Mobile phase: water-methanol-acetonitrile (5:3:2), isocratic elution; Flow rate: 0.8 mL / min; Detection wavelength: 205 nm; Injection volume: 10 μL; Chromatographic peaks as shown Figure 2 As shown.

[0068] 2 Experimental Results

[0069] like Figure 3 As shown, compared with the unfermented traditional Chinese medicine composition, the experimental group of traditional Chinese medicine compositions showed decreased contents of daidzein and genistein, and increased contents of daidzein, genistein, and equol. This indicates that, under the action of microorganisms, glycosides are decomposed into aglycones during the composition preparation process, and daidzein is further decomposed into equol. This suggests that the experimental group of drugs undergoes biotransformation through fermentation, thus better exerting estrogen-like effects.

[0070] Experimental Example 3: Determination of Anti-Osteoporosis Effect in Traditional Chinese Medicine Compositions

[0071] 1. Experimental materials:

[0072] Animals: Healthy female SD rats, clean grade, weighing 200±20g.

[0073] Drugs: The subjects were randomly divided into 3 experimental groups using a random number table method.

[0074] 2 methods

[0075] 2.1 Animal grouping and administration

[0076] Fifty rats were randomly divided into three groups: high-dose, medium-dose, and low-dose experimental groups, a model group, and a blank control group, for a total of five groups, with ten rats in each group, and were housed in separate cages.

[0077] 2.2 Dosage Design

[0078] The experimental group was given the clinically equivalent dosage of the Chinese herbal compound, namely, high, medium and low doses of 0.72, 0.36 and 0.18 g / kg, respectively, and the corresponding drug solution was prepared by gavage at a rate of 1 mL / 100 g. The blank control group was given the same dose of physiological saline.

[0079] 2.3 Test Methods:

[0080] 2.3.1 Methods for establishing animal models of osteoporosis

[0081] Female rats were castrated to simulate postmenopausal osteoporosis. Except for the control group, all other groups were castrated and fed for 8 weeks, followed by continuous drug administration for 12 weeks.

[0082] 2.3.2 HE staining method for observing pathological changes in bone tissue

[0083] After the rat model was established and the drugs were administered, the rats were euthanized, and the bilateral femurs of the rats were quickly removed, the surrounding muscle tissue was removed, and the femurs were fixed in 4% formaldehyde solution for 2-3 weeks. The femurs were then dehydrated, embedded in paraffin, and stained with hematoxylin and eosin (HE).

[0084] 2.3.3 Determination of osteocalcin and alkaline phosphatase activity in rat serum

[0085] According to the ELISA kit requirements, the activities of osteocalcin and ALP in serum were measured using an enzyme-linked immunosorbent assay (ELISA) reader. One-way ANOVA was performed between each experimental group and the model group.

[0086] 3 Experimental Results

[0087] 3.1 Effects of the composition on bone morphology in osteoporosis model rats

[0088] Results of HE staining of rat femur as follows Figure 4 As shown, after administering different doses of the composition of Example 1 to osteoporosis model rats for 12 weeks, the number of trabeculae increased and the connections became tighter and the gaps between trabeculae decreased with increasing dose. This indicates that the composition of the present invention has an ameliorative effect on postmenopausal osteoporosis.

[0089] 3.2 Effects of the experimental group drugs on the activity of osteocalcin and ALP in rat serum

[0090] like Figure 5 As shown, after administering different concentrations of the composition of Example 1 to castrated osteoporosis model rats for 12 weeks, the increase in serum BGP caused by bone resorption and osteolysis was reversed, while serum ALP activity was increased. This indicates that the drug of the present invention can promote bone formation and improve postmenopausal osteoporosis.

[0091] Example 4: Determination of the effect of traditional Chinese medicine composition on a rat model of depression

[0092] 1. Experimental materials:

[0093] Animals: Healthy female SD rats, clean grade, weighing 200±20g.

[0094] Drugs: The subjects were randomly divided into 3 experimental groups using a random number table method.

[0095] 2 methods

[0096] 2.1 Animal grouping and administration

[0097] Fifty rats were randomly divided into three experimental groups (high, medium, and low dose), a model group, and a blank control group, for a total of five groups, with ten rats in each group, and were housed in separate cages.

[0098] 2.2 Dosage Design

[0099] The experimental group was given the clinically equivalent dosage of the Chinese herbal compound, namely, high, medium and low doses of 0.72, 0.36 and 0.18 g / kg, respectively, and the corresponding drug solution was prepared by gavage at a rate of 1 mL / 100 g. The blank control group was given the same dose of physiological saline.

[0100] 2.3 Test Methods:

[0101] 2.3.1 Methods for establishing a rat model of depression

[0102] A rat depression model was established in female rats using a combination of CUMS (Cumulative, Microbial, and Orphanage) and isolation. Except for the control group, all other groups received various chronic stress stimuli over 6 weeks, including 10 minutes of cold water swimming, 24 hours of fasting, 24 hours of water deprivation, 24 hours of continuous lighting, 2 minutes of tail clamping, 24 hours of wet bedding, and 24 hours of tilted cages (7 stimuli in total). One stimulus was administered daily, with the same stimulus not appearing consecutively within 3 days. Two weeks after modeling, drug administration was administered concurrently for 4 weeks.

[0103] 2.3.2 Analysis of histopathological changes in rat hippocampus tissue using HE staining method

[0104] After the rat model was established and the drugs were administered, the rats were euthanized, and the hippocampus of the rat brain tissue was quickly removed, fixed in 4% formaldehyde solution for 1 week, dehydrated, embedded in paraffin, and stained with hematoxylin and eosin (HE).

[0105] 2.3.3 Determination of 5-hydroxytryptamine and dopamine levels in rat serum and hippocampus

[0106] The activities of 5-HT and DA in rat serum and hippocampus were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the requirements of the kit. One-way ANOVA was performed between the experimental group and the model group.

[0107] 3. Experimental Results

[0108] 3.1 Effects of the composition on hippocampal morphology in a depressed rat model

[0109] HE staining results of rat hippocampal tissue are as follows Figure 6 As shown, after 4 weeks of administration of different doses of the drug of this invention to a rat model of depression, the neurons showed varying degrees of recovery, characterized by an increase in the number of neurons, tighter arrangement, and basically normal morphology. This indicates that the composition of this invention has a repairing effect on damaged hippocampal neurons and can improve symptoms related to menopausal amnesia, insomnia, and memory impairment.

[0110] 3.2 Effects of the composition on the levels of 5-hydroxytryptamine and dopamine in rat serum and hippocampus

[0111] In rats with a depression model, after administration of different doses of the drug of this invention for 4 weeks, the levels of 5-HNT and DA in serum and brain tissue increased, as shown in the following results. Figure 7 As shown, the composition of the present invention can improve anxiety and depression symptoms caused by the reduction of neurotransmitters during menopause.

[0112] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A method for preparing a soybean compound fermentation composition for treating menopausal syndrome, characterized in that: Includes the following steps:

1. Prepare ginseng extract, rehmannia extract, polygonatum extract, and kudzu root extract separately; 2. Soybeans are added to ginseng medicinal liquid, rehmannia medicinal liquid, polygonatum medicinal liquid and kudzu root medicinal liquid respectively, soaked until the medicinal liquid is completely absorbed, sterilized, and inoculated with fermentation bacteria for fermentation. After fermentation, they are dried and crushed to obtain ginseng soybean compound fermented product, rehmannia soybean compound fermented product, polygonatum soybean compound fermented product and kudzu root soybean compound fermented product.

3. Soak the Polygonatum in fresh sheep milk to absorb the milk, then steam it, inoculate it with lactic acid bacteria for fermentation, and dry it; add fresh sheep milk again to soak, and repeat the above operation multiple times to prepare the fermented Polygonatum sibiricum product.

4. Mix the ginseng fermented soybean compound fermentation product, the rehmannia fermented soybean compound fermentation product, the polygonatum fermented soybean compound fermentation product, the kudzu root fermented soybean compound fermentation product, and the galangal fermentation product to obtain a mixture. Then, crush cucumber seeds are added to the mixture and mixed to obtain the soybean compound fermentation composition for treating menopausal syndrome.

2. The method for preparing the soybean compound fermentation composition for treating menopausal syndrome according to claim 1, characterized in that: The preparation methods of the ginseng extract, rehmannia extract, polygonatum extract and kudzu root extract mentioned in step one are as follows: the mass-volume ratio of ginseng, rehmannia, polygonatum or kudzu root to water is 1:(10-15), and the decoction is carried out twice, each time for 1-2 hours. The filtrates after each decoction are combined and concentrated to (0.8-1.2) g / 12 mL to obtain the extract.

3. The method for preparing the soybean compound fermentation composition for treating menopausal syndrome according to claim 2, characterized in that: In step two, the mass-to-volume ratio of soybeans to ginseng liquid, rehmannia liquid, polygonatum liquid, and kudzu root liquid is 100g:120mL.

4. The method for preparing the soybean compound fermentation composition for treating menopausal syndrome according to claim 3, characterized in that: The fermentation strain mentioned in step two is Bacillus subtilis, with accession number CICC No. 24713.

5. The method for preparing the soybean compound fermentation composition for treating menopausal syndrome according to claim 4, characterized in that: In step four, the mass ratio of ginseng fermented soybean compound fermented product, rehmannia fermented soybean compound fermented product, polygonatum fermented soybean compound fermented product, kudzu root fermented soybean compound fermented product, cucumber seed powder and galangal fermented product is (10-11):(10-11):(10-11):(10-11):(10-11):

1.

6. A soybean compound fermentation composition for treating menopausal syndrome, characterized in that: The composition is prepared by the method for preparing the soybean compound fermentation composition for treating menopausal syndrome according to any one of claims 1-5.

7. The use of the composition of claim 6 in the preparation of a medicine or food for treating menopausal syndrome.

8. A pharmaceutical composition for treating menopausal syndrome, characterized in that: Includes the composition of claim 6.

9. A health food product for treating menopausal syndrome, characterized in that: Includes the composition of claim 6.

10. A dietary supplement for treating menopausal syndrome, characterized in that: Includes the composition of claim 6.

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