Traditional Chinese medicine composition for treating dysfunctional uterine bleeding, pharmaceutical preparation and preparation method thereof

Abstract

The invention provides a traditional Chinese medicine composition for treating dysfunctional uterine bleeding, a pharmaceutical preparation and a preparation method thereof, and belongs to the technical field of medicines. The traditional Chinese medicine composition comprises the following components: sun-dried ginseng, radix rehmanniae, garden burnet, eclipta alba, madder, fried pollen typhae, cuttlebone, teasel root, dogwood and herba cepbalanoplosis segeti. The traditional Chinese medicine composition and the medicinal preparation provided by the invention have the effects of tonifying qi, controlling blood, cooling blood and reinforcing the kidney, and can be used for treating dysfunctional uterine bleeding symptoms such as liver-kidney yin deficiency, bleeding due to blood heat, preceded menorrhagia caused by blood heat, intermenstrual bleeding and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a traditional Chinese medicine composition, pharmaceutical preparation and preparation method for treating dysfunctional uterine bleeding. Background Technology

[0002] Menstrual irregularities are a common gynecological condition, characterized by abnormalities in the menstrual cycle (e.g., early menstruation, delayed menstruation, prolonged menstruation), amount of bleeding, color of blood and consistency of menstrual blood, or abdominal pain and systemic symptoms before or during menstruation.

[0003] Western medicine treatments for menstrual disorders often have significant side effects and high dependency rates. Traditional Chinese medicine (TCM) treatments for menstrual disorders, on the other hand, focus on regulating the body, replenishing qi and blood, and nourishing the liver and kidneys, making them gentler on the body. There are many existing TCM remedies for menstrual disorders, such as: Baogong Zhixue Granules, composed of oyster shell, white peony root, rehmannia root, arborvitae leaf, rosehip, and vinegar-processed bupleurum; this medicine's effects focus on nourishing yin and consolidating the Chong meridian, as well as astringing and stopping bleeding. Oyster shell and rosehip, while astringent and hemostatic, may affect the discharge of stagnant blood, while white peony root softens the liver but has weak qi-replenishing power; it is suitable for yin deficiency and blood heat type bleeding: heavy menstrual flow, prolonged menstrual period accompanied by five-center heat (heat in the palms, soles, and chest), but without obvious qi or kidney deficiency. Gongxue Ting Granules, composed of astragalus root, codonopsis root, motherwort, atractylodes rhizome, yam, and cimicifuga rhizome; this medicine's effects focus on tonifying the spleen and kidneys, resolving blood stasis and stopping bleeding, with tonifying as the main function and weaker hemostatic power; it is suitable for spleen and kidney deficiency type menorrhagia (pale menstrual blood, fatigue, lower abdominal cold pain).

[0004] Because the causes and symptoms of menstrual disorders vary, and the focus of each Chinese medicine treatment is different, there is still a need to develop targeted treatment drugs for dysfunctional uterine bleeding. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a traditional Chinese medicine composition, pharmaceutical preparation, and preparation method for treating dysfunctional uterine bleeding. This traditional Chinese medicine composition and pharmaceutical preparation have the effects of tonifying qi and stopping bleeding, cooling blood and strengthening the kidneys, combining tonification and purgation, and treating both the root cause and symptoms. It can be used to treat dysfunctional uterine bleeding symptoms such as early and heavy menstrual bleeding, and intermenstrual bleeding caused by liver and kidney yin deficiency and blood heat.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a traditional Chinese medicine composition for treating dysfunctional uterine bleeding, the traditional Chinese medicine composition comprising the following components:

[0008] Sun-dried ginseng, Rehmannia glutinosa, Sanguisorba officinalis, Eclipta prostrata, Rubia cordifolia, stir-fried Typha pollen, Cuttlebone, Dipsacus asper, Cornus officinalis, and Cirsium japonicum.

[0009] In some preferred embodiments of the present invention, the traditional Chinese medicine composition comprises the following components in parts by weight:

[0010] Sun-dried ginseng 13-16 parts, Rehmannia glutinosa 19-22 parts, Sanguisorba officinalis 19-22 parts, Eclipta prostrata 16-18 parts, Rubia cordifolia 16-19 parts, stir-fried Typha orientalis pollen 13-16 parts, Cuttlebone 19-22 parts, Dipsacus asper 13-16 parts, Cornus officinalis 19-22 parts, Cirsium japonicum 28-32 parts.

[0011] The weight of sun-dried ginseng can be any value within the range of 13-16 parts, such as 13 parts, 13.2 parts, 13.5 parts, 13.8 parts, 14 parts, 14.2 parts, 14.5 parts, 14.8 parts, 15 parts, 15.2 parts, 15.5 parts, 15.8 parts, or 16 parts, etc.

[0012] The weight of raw rehmannia root can be any value within the range of 19-22 parts, such as 19 parts, 19.2 parts, 19.5 parts, 19.8 parts, 20 parts, 20.2 parts, 20.5 parts, 20.8 parts, 21 parts, 21.2 parts, 21.5 parts, 21.8 parts, or 22 parts, etc.

[0013] The weight of Sanguisorba officinalis can be any value within the range of 19-22 parts, such as 19 parts, 19.2 parts, 19.5 parts, 19.8 parts, 20 parts, 20.2 parts, 20.5 parts, 20.8 parts, 21 parts, 21.2 parts, 21.5 parts, 21.8 parts, or 22 parts, etc.

[0014] The weight percentage of Eclipta prostrata can be any value within the range of 16-18 parts, such as 16 parts, 16.2 parts, 16.3 parts, 16.5 parts, 16.6 parts, 16.8 parts, 17 parts, 17.2 parts, 17.3 parts, 17.5 parts, 17.6 parts, 17.8 parts, or 18 parts, etc.

[0015] The weight of madder can be any value in the range of 16-19 parts, such as 16 parts, 16.2 parts, 16.5 parts, 16.8 parts, 17 parts, 17.2 parts, 17.5 parts, 17.8 parts, 18 parts, 18.2 parts, 18.5 parts, 18.8 parts, or 19 parts, etc.

[0016] The weight of stir-fried cattail pollen can be any value within the range of 13-16 parts, such as 13 parts, 13.2 parts, 13.5 parts, 13.8 parts, 14 parts, 14.2 parts, 14.5 parts, 14.8 parts, 15 parts, 15.2 parts, 15.5 parts, 15.8 parts, or 16 parts, etc.

[0017] The weight of cuttlebone can be any value within the range of 19-22 parts, such as 19 parts, 19.2 parts, 19.5 parts, 19.8 parts, 20 parts, 20.2 parts, 20.5 parts, 20.8 parts, 21 parts, 21.2 parts, 21.5 parts, 21.8 parts, or 22 parts, etc.

[0018] The weight of Dipsacus asperoides can be any value within the range of 13-16 parts, such as 13 parts, 13.2 parts, 13.5 parts, 13.8 parts, 14 parts, 14.2 parts, 14.5 parts, 14.8 parts, 15 parts, 15.2 parts, 15.5 parts, 15.8 parts, or 16 parts, etc.

[0019] The weight of Cornus officinalis can be any value within the range of 19-22 parts, such as 19 parts, 19.2 parts, 19.5 parts, 19.8 parts, 20 parts, 20.2 parts, 20.5 parts, 20.8 parts, 21 parts, 21.2 parts, 21.5 parts, 21.8 parts, or 22 parts, etc.

[0020] The weight of thistle can be any value within the range of 28-32 parts, such as 28 parts, 28.2 parts, 28.5 parts, 28.8 parts, 29 parts, 29.2 parts, 29.5 parts, 29.8 parts, 30 parts, 30.2 parts, 30.5 parts, 30.8 parts, 31 parts, 31.2 parts, 31.5 parts, 31.8 parts, or 32 parts, etc.

[0021] In some preferred embodiments of the present invention, the traditional Chinese medicine composition comprises the following components in parts by weight:

[0022] 15 parts of sun-dried ginseng, 20 parts of raw rehmannia root, 20 parts of burnet root, 18 parts of eclipta prostrata, 18 parts of madder root, 15 parts of stir-fried cattail pollen, 20 parts of cuttlebone, 15 parts of dipsacus root, 20 parts of cornus officinalis, and 30 parts of small thistle.

[0023] The efficacy of each component in the traditional Chinese medicine composition provided by this invention is as follows:

[0024] Sun-dried ginseng: greatly replenishes vital energy, restores pulse and prevents collapse;

[0025] Rehmannia glutinosa: Clears heat and cools the blood, nourishes yin and promotes the production of body fluids;

[0026] Sanguisorba officinalis: cools the blood and stops bleeding, detoxifies and astringes sores;

[0027] Eclipta prostrata: Nourishes the liver and kidneys, cools the blood and stops bleeding;

[0028] Madder root: Cools the blood, removes blood stasis, stops bleeding, and promotes menstruation;

[0029] Fried cattail pollen: stops bleeding, removes blood stasis, and promotes urination;

[0030] Cuttlebone: Astringent and hemostatic, astringent and stops leukorrhea;

[0031] Dipsacus: Nourishes the liver and kidneys, strengthens tendons and bones, and helps heal fractures;

[0032] Cornus officinalis: Tonifies the liver and kidneys, astringes and consolidates the body;

[0033] Small thistle: cools the blood and stops bleeding, disperses blood stasis and detoxifies.

[0034] The compatibility mechanism of each component is as follows:

[0035] The principal herbs are: sun-dried ginseng and rehmannia root. Sun-dried ginseng greatly replenishes vital energy, enabling qi to control blood; rehmannia root clears heat, cools blood, nourishes yin and blood. Together, they serve as the principal herbs, targeting the pathogenesis of uterine bleeding that may involve deficiency of both qi and yin, and reckless bleeding due to heat.

[0036] Assistant herbs: Sanguisorba officinalis, Eclipta prostrata, Rubia cordifolia, and stir-fried Typha pollen. Sanguisorba officinalis, Eclipta prostrata, Rubia cordifolia, and stir-fried Typha pollen all have the effects of cooling the blood, stopping bleeding, and removing blood stasis. They assist the principal herbs in strengthening the hemostatic and blood stasis-removing effects, and are therefore assistant herbs.

[0037] Adjuvant herbs: Cuttlebone and Cornus officinalis. Cuttlebone has astringent and hemostatic properties, while Cornus officinalis tonifies the liver and kidneys, astringes and consolidates the body, and can prevent excessive bleeding from depleting vital energy, thus serving as an adjuvant herb.

[0038] Guide herbs: Dipsacus asper and Cirsium japonicum. Dipsacus asper tonifies the liver and kidneys, strengthens tendons and bones, and guides the medicine into the liver and kidney meridians; Cirsium japonicum can both cool the blood and stop bleeding, and guide heat downwards, guiding the other medicines to exert their effects, thus serving as the guide herbs.

[0039] This formula combines three methods: cooling the blood and stopping bleeding (Rehmannia glutinosa, Sanguisorba officinalis, Cirsium japonicum), resolving blood stasis (Rubia cordifolia, Typha orientalis), and astringing (Cuttlebone). It stops bleeding while preventing blood stasis, making it suitable for complex bleeding syndromes involving both deficiency and excess. It simultaneously nourishes Qi, Yin, Liver, and Kidney; ginseng tonifies Qi and stops bleeding; Rehmannia glutinosa and Eclipta prostrata nourish Yin; Dipsacus asper and Cornus officinalis tonify the Kidney and strengthen the Chong and Ren meridians, forming a "tonifying Qi-nourishing Yin-strengthening Kidney" chain to regulate the Chong and Ren meridians from the source. The herbal composition provided by this invention works synergistically to tonify Qi, stop bleeding, cool the blood, and strengthen the Kidneys, addressing both the root cause and symptoms. It can be used to treat dysfunctional uterine bleeding symptoms such as early and heavy menstruation, and intermenstrual bleeding caused by Liver and Kidney Yin deficiency and blood heat.

[0040] In a second aspect, the present invention provides a pharmaceutical preparation for treating dysfunctional uterine bleeding, the pharmaceutical preparation comprising the effective components of the traditional Chinese medicine composition as described in the first aspect.

[0041] In some embodiments of the present invention, the pharmaceutical preparation further includes excipients.

[0042] In some embodiments of the present invention, the dosage form of the pharmaceutical preparation is granules, tablets, capsules, or decoction.

[0043] Thirdly, the present invention provides a method for preparing a pharmaceutical preparation as described in the second aspect, the method comprising the following steps:

[0044] (1) The traditional Chinese medicine composition described in the first aspect is subjected to water extraction to obtain an aqueous extract;

[0045] (2) The aqueous extract is prepared into the dosage form of the drug preparation.

[0046] In some embodiments of the present invention, the water extraction step includes:

[0047] The herbal composition is decocted with water at least twice (e.g., twice, three times, four times, or five times), each time for 1-4 hours (e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours). After each decoction, the mixture is filtered, and the filtrates are combined to obtain the aqueous extract.

[0048] In some embodiments of the present invention, in the water extraction step, the total mass of water is 18-35 times the mass of the traditional Chinese medicine composition; for example, it can be 18 times, 19 times, 20 times, 22 times, 23 times, 25 times, 26 times, 28 times, 30 times, 32 times, 33 times or 35 times, etc.

[0049] In some embodiments of the present invention, the water extraction step involves two decoctions. For the first decoction, the mass of water is 10-20 times the mass of the herbal composition (e.g., 10, 12, 13, 15, 16, 18, or 20 times, etc.), and the decoction time is 2-3 hours (e.g., 2 hours, 2.2 hours, 2.3 hours, 2.5 hours, 2.6 hours, 2.8 hours, or 3 hours, etc.). For the second decoction, the mass of water is 8-15 times the mass of the herbal composition (e.g., 8, 9, 10, 11, 12, 13, 14, or 15 times, etc.), and the decoction time is 1-2 hours (e.g., 1 hour, 1.2 hours, 1.3 hours, 1.5 hours, 1.6 hours, 1.8 hours, or 2 hours, etc.).

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] This invention utilizes a scientific combination of raw ginseng, raw rehmannia root, burnet root, eclipta prostrata, madder root, stir-fried cattail pollen, cuttlebone, dipsacus root, cornus officinalis, and small thistle to obtain a traditional Chinese medicine composition and pharmaceutical preparation with the effects of tonifying qi and blood, cooling blood and strengthening the kidneys. It combines tonification and purgation, treating both the root cause and the symptoms, and can be used to treat dysfunctional uterine bleeding symptoms such as early menstruation and heavy bleeding between menstruation caused by liver and kidney yin deficiency and blood heat. Attached Figure Description

[0052] Figure 1A This is a bar chart showing the amount of uterine bleeding in each group of rats in the embodiments of the present invention;

[0053] Figure 1B This is a bar chart showing the number of days of uterine bleeding in each group of rats in this embodiment of the invention;

[0054] Figure 2 These are photographs of the uterus of rats in each group in the embodiments of the present invention;

[0055] Figure 3A This is a bar chart showing the coefficients of uterine organs in each group of rats in the embodiments of the present invention;

[0056] Figure 3B This is a bar chart showing the coefficients of ovarian organs in each group of rats in the embodiments of the present invention;

[0057] Figure 4 HE staining images of the rat uterus in each group in the embodiments of the present invention. Detailed Implementation

[0058] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the specific embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0059] Example 1

[0060] This embodiment provides a traditional Chinese medicine composition for treating dysfunctional uterine bleeding, comprising the following components in parts by weight:

[0061] 13 parts of raw ginseng, 22 parts of raw rehmannia root, 19 parts of burnet root, 18 parts of eclipta prostrata, 16 parts of madder root, 16 parts of stir-fried cattail pollen, 19 parts of cuttlebone, 16 parts of dipsacus root, 19 parts of cornus officinalis, and 32 parts of small thistle.

[0062] This embodiment also provides a decoction for treating dysfunctional uterine bleeding, the preparation method of which is as follows:

[0063] Weigh the medicinal materials according to the proportions of the Chinese herbal composition, add water and decoct twice. For the first decoction, add 20 times the amount of water as the medicinal materials and decoct for 3 hours. For the second decoction, add 15 times the amount of water as the medicinal materials and decoct for 2 hours. After each decoction, filter, combine the filtrates, and concentrate under reduced pressure to obtain a decoction for treating dysfunctional uterine bleeding.

[0064] Example 2

[0065] This embodiment provides a traditional Chinese medicine composition for treating dysfunctional uterine bleeding, comprising the following components in parts by weight:

[0066] 16 parts of raw ginseng, 19 parts of raw rehmannia root, 22 parts of burnet root, 16 parts of eclipta prostrata, 19 parts of madder root, 13 parts of stir-fried cattail pollen, 22 parts of cuttlebone, 13 parts of dipsacus root, 22 parts of cornus officinalis, and 28 parts of small thistle.

[0067] This embodiment also provides a decoction for treating dysfunctional uterine bleeding, the preparation method of which is as follows:

[0068] Weigh the medicinal materials according to the ratio of the traditional Chinese medicine composition, decoct them with water twice. For the first decoction, add 10 times the mass of the medicinal materials in water and decoct for 2 hours; for the second decoction, add 8 times the mass of the medicinal materials in water and decoct for 1 hour. Filter after each decoction, combine the filtrates, and obtain the decoction for treating dysfunctional uterine bleeding after concentration under reduced pressure.

[0069] Example 3

[0070] This example provides a traditional Chinese medicine composition for treating dysfunctional uterine bleeding, including the following components in parts by weight:

[0071] 15 parts of dried ginseng, 20 parts of rehmannia root, 20 parts of sanguisorba root, 18 parts of eclipta prostrata, 18 parts of rubia cordifolia, 15 parts of stir-fried cattail pollen, 20 parts of cuttlefish bone, 15 parts of dipsacus asper, 20 parts of cornel fruit, and 30 parts of cephalanoplos segetum.

[0072] This example also provides a decoction for treating dysfunctional uterine bleeding, and its preparation method is as follows:

[0073] Weigh the medicinal materials according to the ratio of the traditional Chinese medicine composition, decoct them with water twice. For the first decoction, add 15 times the mass of the medicinal materials in water and decoct for 2.5 hours; for the second decoction, add 10 times the mass of the medicinal materials in water and decoct for 1.5 hours. Filter after each decoction, combine the filtrates, and concentrate under reduced pressure to 2.51 g of the traditional Chinese medicine composition / mL to obtain the decoction for treating dysfunctional uterine bleeding, and store it at 4°C for later use.

[0074] Therapeutic efficacy test

[0075] Use the decoction for treating dysfunctional uterine bleeding prepared in Example 3 for animal experiments to confirm its therapeutic effect on the rat uterine bleeding model.

[0076] 1. Test materials

[0077] 1.1. Test animals

[0078] 60 SPF-grade female SD rats, weighing 180 - 200 g, were purchased from Spf (Suzhou) Biotechnology Co., Ltd., with the production license number SCXK (Su) 2022 - 0006 and the certificate number A202411260182. Feeding environmental conditions: The feeding environmental conditions are standard, referring to the national standard GB14925 - 2010 of the People's Republic of China; the temperature is 20 - 26°C (daily temperature difference ≤ 4°C); the relative humidity is 40 - 70%; the pressure in the feeding room ≥ 10 Pa; 12 / 12 hour day / night light / dark cycle.

[0079] 1.2. Test reagents

[0080] The decoction for treating dysfunctional uterine bleeding prepared in Example 3 was diluted with sterile water for injection to 0.979 g / mL, 1.958 g / mL, and 3.916 g / mL, respectively. This concentration refers to the mass of the traditional Chinese medicine composition corresponding to the decoction contained in 1 mL of solution.

[0081] Gongxue Ning Capsules, specification 0.13g / capsule, batch number ZFC2401, should be stored in a tightly closed container. The manufacturer is Yunnan Baiyao Group Co., Ltd.; product approval number is Z20020087. Preparation method: Dilute the capsule contents with 0.5wt% sodium carboxymethyl cellulose to 8.12mg / mL.

[0082] Mifepristone tablets, 25mg / tablet, batch number 57240302, should be stored in a tightly closed container. The manufacturer is Wuhan Jiulong Renfu Pharmaceutical Co., Ltd.; product approval number is H20033551. Preparation method: Grind the tablets into powder and dilute with 0.5wt% sodium carboxymethyl cellulose to the required concentration.

[0083] Misoprostol tablets, 0.2 mg / tablet, batch number 66240103, should be stored in a tightly closed container. The manufacturer is Wuhan Jiulong Renfu Pharmaceutical Co., Ltd.; product approval number is H20073696. Preparation method: Grind the tablets into powder and dilute with 0.5 wt% sodium carboxymethyl cellulose to the required concentration.

[0084] Sodium carboxymethyl cellulose, 500g / bottle, batch number 20210402, store in a tightly closed container, manufactured by Sinopharm Chemical Reagent Co., Ltd.; product number 30036328. Preparation method: Dilute sodium carboxymethyl cellulose to 0.5wt% with sterile water for injection.

[0085] Sterile water for injection, 500ml / bottle, batch number 240621, to be stored in a sealed container, manufactured by Shaanxi Shengao Animal Pharmaceutical Co., Ltd.; product approval number is Veterinary Drug No. 270071791.

[0086] This is a general-purpose tissue fixative, available in 500ml vials, batch number GP24103081634. Store at room temperature. The manufacturer is Wuhan Saiweier Biotechnology Co., Ltd.

[0087] 1.3. Instruments

[0088] The pipette is manufactured by Eppendorf and has a model number of 200μL.

[0089] The electronic balance is manufactured by Shanghai Yaoxin Electronic Technology Co., Ltd., and its model number is LQ-C12001.

[0090] The benchtop high-speed refrigerated microcentrifuge is manufactured by Thermo and its model number is Sorvall Legand Micro 17R.

[0091] Mini mixing centrifuge, manufacturer: LABGIC, model: L-CM-MINI;

[0092] The microplate reader is manufactured by Thermo and is model VARIOSKAN LUX.

[0093] The dehydrator is manufactured by DIAPATH and its model is Donatello.

[0094] The embedding machine is manufactured by Wuhan Junjie Electronics Co., Ltd., and its model number is JB-P5.

[0095] The freezing station is manufactured by Wuhan Junjie Electronics Co., Ltd., and its model number is JB-L5.

[0096] The pathology slider is manufactured by Leica Instruments Shanghai Co., Ltd., and its model number is RM2016.

[0097] The sheet spreader is manufactured by Zhejiang Jinhua Kedi Instrument Equipment Co., Ltd., and its model is KD-P.

[0098] The oven is manufactured by Tianjin Laiborui Instrument Equipment Co., Ltd., and its model is GFL-230.

[0099] An upright optical microscope, manufactured by Nikon Japan, model Nikon Eclipse E100;

[0100] The imaging system is manufactured by Nikon of Japan, and its model is NIKON DS-U3.

[0101] An upright white light photographic microscope, manufactured by Nikon Japan, model Eclipse Ci-L.

[0102] 2. Test Methods

[0103] 2.1. Modeling, grouping, and drug administration

[0104] Female and male rats were acclimatized for 7 days under the same conditions, with free access to food and water, a room temperature of (25±1)℃, an air humidity of 55-65%, and a 12-hour light-dark cycle. They were mixed-sex at a female:male ratio of 2:1. The presence of vaginal plugs in female rats the following morning indicated day 1 of pregnancy. Successfully pregnant female rats were randomly divided into 6 groups: a control group, a model group, a low-dose prescription group, a medium-dose prescription group, a high-dose prescription group, and a uterine bleeding treatment group, with 8 rats in each group. Except for the control group, all other groups were administered mifepristone (8:00 am, 12.4 mg / kg) and misoprostol (6:00 pm, 0.1 mg / kg) by gavage, successfully establishing a uterine bleeding model of incomplete abortion in early pregnancy rats.

[0105] After successful modeling, each group of rats was administered the drug twice daily by gavage for 14 consecutive days. The types and dosages of drugs administered to each group of rats are as follows:

[0106] Blank group and model group: physiological saline, administration volume 100μL / 10g;

[0107] Low-dose prescription group: a diluted solution of the decoction prepared in Example 3 with a concentration of 0.979 g / mL, administered at a dose of 9.79 g / kg, with an administration volume of 100 μL / 10 g;

[0108] The dosage group in the prescription was a diluted solution of the decoction prepared in Example 3 with a concentration of 1.958 g / mL, with a dosage of 19.58 g / kg and a dosage volume of 100 μL / 10 g.

[0109] High-dose prescription group: a diluted solution of the decoction prepared in Example 3 with a concentration of 3.916 g / mL, administered at a dose of 39.16 g / kg, with an administration volume of 100 μL / 10 g;

[0110] The Gongxue Ning group: a diluted solution of Gongxue Ning capsules with a concentration of 8.12 mg / mL was administered at a dose of 0.0812 g / kg and a volume of 100 μL / 10 g.

[0111] 2.2. Detection Indicators and Methods

[0112] 2.2.1. Observation of uterine bleeding volume, number of days of uterine bleeding, and uterine morphology

[0113] On the 7th day after animal modeling, a measured amount of cotton ball (weighing 85-90 mg, with one side wrapped in plastic film to prevent blood leakage and urine reflux) was inserted into the vagina simultaneously with drug administration. The cotton ball was removed at 8:00 and 18:00 the following day, placed in a sealed plastic bag and refrigerated, and a new cotton ball was inserted into the vagina at the same time to observe vaginal bleeding. This was continued until the 14th day, at which time the animal was sacrificed, and the pathological morphological changes of the uterus were observed. The amount of bleeding was measured from the collected cotton balls.

[0114] On day 14, 20 μL of blood was collected from the tail vein of rats in each experimental group, and 4 mL of 5% NaOH solution was added. The mixture was mixed with a pipette, and the absorbance value A was measured at 546 nm using a UV spectrophotometer with 5% NaOH solution as the zero point.

[0115] Collect uterine bleeding cotton balls from each rat daily and place them in a beaker. Add 5% NaOH solution as needed based on the amount of bleeding. After 24 hours of extraction, pour the extracted solution into a new beaker for storage. Add an appropriate amount of 5% NaOH solution again to soak and rinse the blood-stained cotton balls. Combine the extracted solutions into a new beaker and record the volume of the extract. Generally, 1-2 extractions are performed; if there are many bleeding cotton balls, 3-4 extractions can be performed. The extraction standard is that the blood-stained cotton balls are washed until they retain their original color and no bloodstains remain. Filter the extract and take 4 mL, placing it in a cuvette. Measure the absorbance of the extract at a wavelength of 546 nm. Calculate the amount of uterine bleeding using the following formula.

[0116] Uterine bleeding volume (mL) = venous blood volume × (A2 × V2) / (A1 × V1);

[0117] Wherein, V1 is the volume of NaOH solution used to dilute venous blood (4 mL), V2 is the volume of NaOH solution used to extract uterine bleeding from cotton balls, A1 is the absorbance value of venous blood, and A2 is the absorbance value of the uterine bleeding cotton ball extract.

[0118] 2.2.2. Ovarian and left uterine organ coefficients

[0119] After measuring the rat's weight and euthanizing it by cervical dislocation, the ovary and left uterus were immediately removed. The adipose connective tissue surrounding these organs was removed, the blood on the surface of the organs was aspirated, and the rats were weighed using an electronic balance. The organ coefficient was then calculated (organ coefficient = organ weight / body weight × 100%).

[0120] 2.2.3. HE staining of the left uterus

[0121] (1) Paraffin slice preparation

[0122] 1) Tissue collection: Fresh left uterine tissue was fixed with fixative for at least 24 hours. The tissue was removed from the fixative and trimmed in a fume hood using a scalpel. The trimmed tissue and corresponding labels were then placed in a dehydration box.

[0123] 2) Dehydration and wax impregnation: The dehydration box is placed in a dehydrator for gradient alcohol dehydration. The steps are as follows: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzyl alcohol for 10 minutes, xylene I for 10 minutes, xylene II for 10 minutes, 65℃ melted paraffin I for 1 hour, 65℃ melted paraffin II for 1 hour, and 65℃ melted paraffin III for 1 hour.

[0124] 3) Embedding: The paraffin-impregnated tissue is embedded in an embedding machine. First, the molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box, placed into the embedding frame according to the embedding surface requirements, and labeled accordingly. The tissue is then cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed.

[0125] 4) Sectioning: Place the trimmed wax block on a -20℃ freezing stage to cool, then place the cooled wax block on a paraffin microtome to section to a thickness of 4μm. Float the sections on 40℃ warm water in a slide to flatten the tissue, then lift the tissue onto a glass slide and bake in a 60℃ oven. After the wax has melted in the water, remove the slide and store it at room temperature for later use.

[0126] (2) Staining

[0127] 1) Dewaxing paraffin sections to water: Place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% ethanol for 5 min, and then wash with tap water.

[0128] 2) Hematoxylin staining: After dewaxing, the sections were stained with hematoxylin solution for 5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue solution, and rinsed with running water.

[0129] 3) Eosin staining: After hematoxylin staining, the sections were dehydrated in 85% and 95% alcohol for 5 minutes each, and then stained in eosin staining solution for 5 minutes.

[0130] 4) Dehydration and mounting: After eosin staining, the sections were sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, xylene II for 5 min, and then mounted with clear neutral resin.

[0131] 5) Microscopic examination, image acquisition and analysis.

[0132] 2.3. Data Analysis and Processing

[0133] GraphPad 9.5.0 and SPSS 26 software were used for plotting and statistical analysis. Data are expressed as mean ± SEM. One-way ANOVA was used for statistical analysis to test for significant differences between the two groups. P < 0.05 was considered statistically significant.

[0134] 3. Test Results

[0135] 3.1. Effects of the prescription on the amount and duration of uterine bleeding in a rat model of uterine bleeding.

[0136] Figure 1A A bar chart showing the amount of uterine bleeding in each group of rats. Figure 1B A bar chart showing the number of days of uterine bleeding in each group of rats. Compared to the model group, * p < 0.05 ** p < 0.01; compared with the Gongxueling group, # p < 0.05.

[0137] from Figure 1A and Figure 1B It can be seen that the amount of uterine bleeding and the number of days of uterine bleeding in rats in each prescription dosage group were significantly reduced compared with those in the model group (P<0.05, P<0.01), while the amount of uterine bleeding in rats in the positive drug Gongxue Ning group was not significantly reduced compared with those in the model group (P>0.05). The hemostatic effect of the traditional Chinese medicine prescription of this invention is significantly better than that of Gongxue Ning (P<0.05).

[0138] 3.2. Effects of the prescription on uterine morphology in a rat model of uterine hemorrhage

[0139] Figure 2 The images show photographs of the uterus of rats in each group. A represents the control group, B the model group, C the low-dose prescription group, D the medium-dose prescription group, E the high-dose prescription group, and F the Gongxue Ning group.

[0140] Macroscopic observation of rat uterine morphology revealed that in the control group, the uterine tissue surface was smooth, light pink, and Y-shaped, with uniform thickness throughout the uterine segment, and no obvious nodules, thickening, enlargement, congestion, or edema. In the model group, nodules of varying sizes were visible on both sides of the uterus, with a dark red tissue color and severe tissue residue, exhibiting ecchymosis, irregular and asymmetrical nodular enlargement, and significant uterine congestion and edema. In the groups of rats treated with the herbal prescription of this invention, the uterus was rosy in color, with a significant reduction in nodular enlargement and no obvious tissue residue. In the positive control group treated with Gongxue Ning, the uterus was slightly thickened and edematous, with no obvious blood stasis residue.

[0141] 3.3. Effects of the prescription on organ coefficients in a rat model of uterine hemorrhage

[0142] Figure 3A Bar chart showing the coefficients of uterine organs in each group of rats. Figure 3BA bar chart showing the coefficients of ovarian organs in each group of rats. Compared to the control group, ** p < 0.01; compared with the model group, ## p < 0.01, ### p < 0.001.

[0143] from Figure 3A and Figure 3B It can be seen that, compared with the blank group, the uterine organ coefficient of rats in the model group was significantly increased (P<0.01); compared with the model group, the uterine organ coefficient of rats in each prescription dose group and the positive drug Gongxuening group was significantly decreased (P<0.01, P<0.001). There was no significant change in the ovarian organ coefficient of rats in each group (P>0.05).

[0144] 3.4. Effects of the prescription on uterine tissue in a rat model of uterine bleeding

[0145] Figure 4 HE staining images of the uterus of rats in each group. A represents the blank group, B the model group, C the low-dose prescription group, D the medium-dose prescription group, E the high-dose prescription group, and F the Gongxue Ning group.

[0146] from Figure 4 It can be seen that the epithelial cells of the uterine tissue in the blank group rats have a clear morphology and structure; the lamina propria contains abundant stromal cells and capillaries, and the epithelium invaginates into the lamina propria to form many tubular uterine glands, with no abnormalities observed in the glandular epithelial cells; the smooth muscle of the myometrium is neatly arranged. In the model group rats, the uterine tissue shows a moderate degree of absence of endometrial epithelial cells; the lamina propria contains abundant stromal cells and capillaries, and the epithelium invaginates into the lamina propria to form many tubular uterine glands, with many dilated uterine glands visible, eosinophilic flocculent material visible in the lumen, and edema of endometrial and glandular epithelial cells, cell swelling, and loose, pale cytoplasm. In the uterine tissue of the Gongxuening group rats, the epithelium invaginates into the lamina propria to form many tubular uterine glands, with a large number of uterine glands, and edema of endometrial and glandular epithelial cells, cell swelling, and loose, pale cytoplasm; the myometrium shows abundant angiogenesis and a small amount of vascular dilation. The morphology and structure of the uterine epithelial cells in the rat uterine tissue of each dosage group of the traditional Chinese medicine prescription of this invention are clear; the lamina propria contains abundant stromal cells and capillaries, and the epithelium invaginates into the lamina propria to form many tubular uterine glands. The number of uterine glands is large, cell edema is reduced, inflammatory cells are few, and a small number of neovascularizations are visible; the smooth muscle of the myometrium is arranged neatly.

[0147] In summary, low, medium, and high doses of the formulation of this invention can effectively reduce the amount and duration of uterine bleeding in rats with uterine bleeding models, alleviate nodular uterine distension, reduce uterine organ coefficient, reduce uterine tissue cell edema, and reduce inflammatory cells. Moreover, the efficacy is superior to that of the positive control drug, Gongxue Ning.

[0148] The above description is merely a specific embodiment of this disclosure, enabling those skilled in the art to understand or implement it. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this disclosure. Therefore, this disclosure is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A traditional Chinese medicine composition for treating dysfunctional uterine bleeding, characterized in that, The traditional Chinese medicine composition includes the following components: Sun-dried ginseng, Rehmannia glutinosa, Sanguisorba officinalis, Eclipta prostrata, Rubia cordifolia, stir-fried Typha pollen, Cuttlebone, Dipsacus asper, Cornus officinalis, and Cirsium japonicum.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following components in parts by weight: Sun-dried ginseng 13-16 parts, Rehmannia glutinosa 19-22 parts, Sanguisorba officinalis 19-22 parts, Eclipta prostrata 16-18 parts, Rubia cordifolia 16-19 parts, stir-fried Typha orientalis pollen 13-16 parts, Cuttlebone 19-22 parts, Dipsacus asper 13-16 parts, Cornus officinalis 19-22 parts, Cirsium japonicum 28-32 parts.

3. The traditional Chinese medicine composition according to claim 1 or 2, characterized in that, The traditional Chinese medicine composition comprises the following components in parts by weight: 15 parts of sun-dried ginseng, 20 parts of raw rehmannia root, 20 parts of burnet root, 18 parts of eclipta prostrata, 18 parts of madder root, 15 parts of stir-fried cattail pollen, 20 parts of cuttlebone, 15 parts of dipsacus root, 20 parts of cornus officinalis, and 30 parts of small thistle.

4. A pharmaceutical preparation for treating dysfunctional uterine bleeding, characterized in that, The pharmaceutical preparation contains the active ingredient of the traditional Chinese medicine composition as described in any one of claims 1-3.

5. The pharmaceutical preparation according to claim 4, characterized in that, The pharmaceutical preparation also includes excipients.

6. The pharmaceutical preparation according to any one of claims 4-5, characterized in that, The dosage form of the pharmaceutical preparation is granules, tablets, capsules, or decoction.

7. A method for preparing a pharmaceutical formulation as described in any one of claims 4-6, characterized in that, The preparation method includes the following steps: (1) The traditional Chinese medicine composition according to any one of claims 1-3 is subjected to water extraction to obtain an aqueous extract; (2) The aqueous extract is prepared into the dosage form of the drug preparation.

8. The preparation method according to claim 7, characterized in that, The water extraction step includes: The herbal composition is decocted with water at least twice, each time for 1-4 hours. After each decoction, the mixture is filtered, and the filtrates are combined to obtain the aqueous extract.

9. The preparation method according to claim 7 or 8, characterized in that, In the water extraction step, the total mass of water is 18-35 times the mass of the traditional Chinese medicine composition.

10. The preparation method according to claim 8, characterized in that, In the water extraction step, the decoction is performed twice. In the first decoction, the mass of water is 10-20 times the mass of the Chinese herbal composition, and the decoction time is 2-3 hours. In the second decoction, the mass of water is 8-15 times the mass of the Chinese herbal composition, and the decoction time is 1-2 hours.

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