A pharmaceutical composition for treating dysfunctional uterine bleeding, its preparation method and application

Abstract

This invention relates to the field of pharmaceutical technology, specifically to a pharmaceutical composition for treating functional uterine bleeding, its preparation method, and its application. The pharmaceutical composition consists of *Rheum palmatum*, *Panax ginseng*, *Smilax china*, *Achyranthes bidentata*, and *Gentiana scabra*. The preparation method involves weighing the five herbs according to the prescribed amounts, extracting with ethanol, filtering, and recovering the ethanol. The residue after ethanol extraction is then soaked in water and with cellulase under specific conditions, boiled, and filtered to obtain an enzymatic extract. The ethanol extract and the enzymatic extract are combined and concentrated to a thick paste. Appropriate excipients are added to the thick paste to formulate the corresponding dosage form. This drug has the functions of promoting blood circulation, regulating qi, stopping bleeding, and regulating menstruation, and is used to treat functional uterine bleeding. Furthermore, the pharmaceutical composition is formulated into appropriate dosage forms using modern pharmaceutical preparation technology, making its clinical efficacy in treating functional uterine bleeding more significant and its onset of action faster.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pharmaceutical composition for treating functional uterine bleeding, its preparation method, and its application. Background Technology

[0002] Dysfunctional uterine bleeding (DUB), also known as dysfunctional uterine bleeding or dysfunctional uterine bleeding, is abnormal uterine bleeding caused by neuroendocrine dysfunction due to abnormalities in the hypothalamic-pituitary-ovarian axis, affecting the levels of estrogen and progesterone in the body. DUB is a common gynecological condition, accounting for 10% to 15% of cases, with 50% occurring during menopause, 30% during the reproductive years, and 20% during puberty.

[0003] In recent years, the number of patients with diabetic uterine bleeding (DUB) has been gradually increasing. This not only affects patients' lives and work, but in severe cases, excessive bleeding can lead to shock and endanger their lives, attracting widespread attention from society and clinicians. Furthermore, the etiology and pathogenesis of DUB are not yet fully understood. Modern medicine believes that DUB is mainly related to excessive mental stress, fatigue, malnutrition or metabolic disorders, changes in environment and climate, or endocrine disorders caused by factors such as the ovaries or corpus luteum, which in turn cause uterine bleeding symptoms.

[0004] Traditional Chinese medicine considers DUB to fall under the category of "metrorrhagia". The "Treatise on the Causes and Symptoms of Various Diseases" states that "when the Chong and Ren meridians are deficient, they cannot control the menstrual blood, so blood flows out at irregular times." Its main pathogenesis is that the Chong and Ren meridians are not stable and cannot control the menstrual blood, causing the uterus to have abnormal storage and discharge. Its characteristics are mostly a mixture of deficiency and excess, with blood heat, blood deficiency, and blood stasis coexisting. Therefore, the treatment should combine cooling blood, nourishing blood, and promoting blood circulation to stabilize the Chong and Ren meridians, replenish the blood sea, and achieve the goal of blood returning to its proper channels.

[0005] Currently, the main goal of clinical treatment for DUB is hemostasis. The common method is curettage combined with hormone therapy. Curettage is used to achieve rapid hemostasis, supplemented by endocrine regulation. However, the efficacy and recovery are not satisfactory. It can only temporarily suppress the development or worsening of clinical symptoms. Relapse often occurs due to incomplete prevention and treatment. Therefore, there is an urgent need for effective treatment drugs.

[0006] Rhubarb root (Rubus terrestris) refers to the fresh or dried roots and rhizomes of *Rumex nepalensis* Spreng., *Rumex dentatus* L., or *Rumex japonicus* Houtt., all belonging to the Polygonaceae family. It is harvested in spring and summer, washed, and used fresh or dried. It is bitter, slightly astringent, and cold in nature. It enters the heart, liver, and large intestine meridians. It has the effects of clearing heat and detoxifying, astringing blood and stopping bleeding. It is used for various inflammations, conjunctivitis, constipation, etc.

[0007] Blood ginseng is the dried root of *Indigofera stachyoides* Lindl., a plant in the legume family. It can be harvested year-round, cleaned of dirt and sand, and then sun-dried. It is sweet, slightly bitter, and warm in nature. It enters the liver, kidney, and large intestine meridians. It has the effects of nourishing both qi and blood, promoting qi circulation and blood flow, soothing the meridians and activating collaterals, and nourishing yin and tonifying the kidneys. It is used for deficiency of both qi and blood.

[0008] Ardisia punctata Lindl., a plant in the Myrsinaceae family, is the root or whole plant of this species. It can be harvested year-round, washed, and used fresh or dried. It is bitter, pungent, and warm in nature. It has the effects of promoting blood circulation and regulating menstruation, dispelling wind and dampness, and is used for amenorrhea, dysmenorrhea, and rheumatic pain.

[0009] Achyranthes aspera L., a plant in the Amaranthaceae family, is the dried root of the plant. It is harvested in winter after the stems and leaves have withered and turned yellow. The remaining stems and fibrous roots are removed, and the roots are washed and dried. It is bitter, sour, and slightly cold in nature. It enters the liver, lung, and bladder meridians. It has the effects of promoting blood circulation and removing blood stasis, promoting diuresis and eliminating dampness, and clearing heat and detoxifying. It is used for amenorrhea, urinary tract infections, and rheumatoid arthritis.

[0010] Corallodiscus cordatulus (Craib) burtt., a plant in the Gesneriaceae family, is a fresh or dried whole herb. It is harvested in autumn and winter, cleaned of dirt and sand, and used fresh or dried. It is bitter, pungent, and cold in nature. It enters the liver, heart, and lung meridians. It has the effects of clearing heat and detoxifying, removing dampness and relieving pain, promoting blood circulation and reducing swelling. It is used for irregular menstruation, leukorrhea, etc.

[0011] Therefore, this invention uses traditional Chinese medicine as the main ingredient. After long-term experimentation and repeated research, a pharmaceutical composition for treating functional uterine bleeding has been developed. This composition consists of rhubarb, ginseng, small umbellatus, achyranthes bidentata, and gentiana. Rhubarb clears heat and detoxifies, stops bleeding and astringes, and has a unique therapeutic effect on functional uterine bleeding; it is used in large quantities as the principal ingredient. Ginseng and small umbellatus nourish blood and promote blood circulation, remove blood stasis, regulate menstruation and relieve pain, enhancing the efficacy of the principal ingredient; they are assistant ingredients. Achyranthes bidentata promotes blood circulation and removes blood stasis, guiding blood downwards. Gentiana regulates qi and promotes blood circulation; they are adjuvant ingredients. The combined effects of these herbs promote blood circulation, regulate qi, stop bleeding and regulate menstruation. Furthermore, this pharmaceutical composition has been formulated into appropriate dosage forms using modern pharmaceutical technology, making the clinical treatment of functional uterine bleeding more effective, faster-acting, and more convenient. Summary of the Invention

[0012] The object of this invention is to provide a pharmaceutical composition for treating dysfunctional uterine bleeding;

[0013] Another object of the present invention is to provide a method for preparing a pharmaceutical composition for treating dysfunctional uterine bleeding;

[0014] Another object of the present invention is to provide the use of a pharmaceutical composition for treating dysfunctional uterine bleeding in the preparation of a pharmaceutical formulation for treating dysfunctional uterine bleeding;

[0015] The pharmaceutical composition for treating functional uterine bleeding described in this invention consists of 10-20 parts of Rheum palmatum, 6-14 parts of Panax ginseng, 6-14 parts of Angelica dahurica, 4-12 parts of Achyranthes bidentata, and 4-12 parts of Gentiana scabra.

[0016] Furthermore, the drug composition consists of 12-18 parts of Rheum palmatum, 8-12 parts of Panax ginseng, 8-12 parts of Angelica dahurica, 6-10 parts of Achyranthes bidentata, and 6-10 parts of Gentiana scabra.

[0017] Furthermore, the drug composition consists of 15 parts of Rheum palmatum, 10 parts of Panax ginseng, 10 parts of Angelica dahurica, 8 parts of Achyranthes bidentata, and 8 parts of Gentiana scabra.

[0018] The preparation method of the pharmaceutical composition for treating dysfunctional uterine bleeding according to the present invention is as follows:

[0019] (1) Ethanol extraction: Weigh out the five medicinal materials, namely Rheum palmatum, Panax ginseng, Smilax china, Achyranthes bidentata and Gentiana scabra, according to the formula amount. Add 6-10 times the total amount of medicinal materials in 60% ethanol. Heat at 70-80℃ for 1-3 times, each extraction for 0.5-2 hours. Filter and recover the ethanol to obtain the ethanol extract.

[0020] (2) Enzymatic extraction: Add 8-12 times the amount of water to the residue after ethanol extraction, add 1.0-3.0% cellulase, and extract at 50℃ and pH 5.0 for 1-2 hours. After boiling, filter to obtain the enzymatic extract.

[0021] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0022] (4) Prepare the corresponding dosage form: Add an appropriate amount of excipients to the thick paste to prepare the corresponding dosage form.

[0023] Furthermore, the preparation method of the pharmaceutical composition for treating dysfunctional uterine bleeding according to the present invention is as follows:

[0024] (1) Ethanol extraction: Weigh out five medicinal materials according to the formula: Rhubarb, Ginseng, Small Angelica, Achyranthes bidentata, and Gentiana scabra. Add 60% ethanol at 8 times the total amount of medicinal materials, heat at 75℃ once for 1 hour, filter, recover ethanol, and obtain ethanol extract.

[0025] (2) Enzymatic extraction: Add 10 times the amount of water to the residue after ethanol extraction, add 1.0% cellulase, and extract for 1.5 h at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0026] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0027] (4) Prepare the corresponding dosage form: Add an appropriate amount of excipients to the thick paste to prepare the corresponding dosage form.

[0028] The excipients described in this invention are pharmaceutically acceptable excipients.

[0029] The dosage forms described in this invention are: solid dosage forms and liquid dosage forms.

[0030] The solid dosage forms described in this invention are: granules, capsules, tablets, and pellets.

[0031] The liquid preparations described in this invention are: mixtures and syrups.

[0032] The use of the pharmaceutical composition described in this invention in the preparation of a drug for treating dysfunctional uterine bleeding.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. The product of this invention has significant therapeutic effects. Clinical efficacy trials of this invention show that when used in combination with Western medicine, the total effective rate reaches 98.0%, which is significantly different from the 89.0% total effective rate of the control group. This indicates that this invention can effectively improve patients' clinical symptoms and enhance their quality of life, demonstrating its unique advantages.

[0035] 2. The preparation process of this invention has high extraction efficiency and is reasonable and feasible. This invention investigates the preparation process, using the extraction rate of total flavonoids (quercetin, hydroxyemodin, cassia codone, emodin methyl ether, and rhein, all flavonoid components) in the prescription as the evaluation index. Through experiments comparing three different extraction processes—water extraction, alcohol extraction, and alcohol extraction with enzymatic hydrolysis—the results show that the combined alcohol extraction and enzymatic hydrolysis extraction process significantly improves the extraction efficiency of total flavonoids in the prescription, achieving an extraction rate of 67.12%, which is high, indicating that this process is reasonable and feasible.

[0036] 3. The preparation process of this invention can better enrich the effective components. This invention uses alcohol extraction and enzymatic hydrolysis to extract the effective components in the formula. This process has a high enrichment rate of effective components (total flavonoid extraction rate of 67.12%), which is significantly better than water extraction (total flavonoid extraction rate of 18.18%), alcohol extraction (total flavonoid extraction rate of 41.11%), and enzymatic hydrolysis (total flavonoid extraction rate of 23.34%).

[0037] 4. The products prepared by this invention are convenient to use, safe and effective, and have few adverse reactions. After the pharmaceutical compositions of this invention are formulated into preparations (granules, oral liquids, etc.), they are more convenient for patients to take, carry, and store compared to decoction.

[0038] 5. The product prepared by this invention has low cost, which can reduce the economic burden on patients and has broad application value. The pharmaceutical composition of this invention contains only 5 medicinal materials, which are inexpensive and readily available. The prescription quantity is small and the preparation process is simple, so the cost is very low, which can significantly reduce the economic burden on patients and has broad market application value. Detailed Implementation

[0039] Example 1

[0040] Formula: Rhubarb 15g, Ginseng 10g, Ulmus pumila 10g, Achyranthes bidentata 8g, Gentiana scabra 8g.

[0041] Example 2

[0042] Formula: Rhubarb 10g, Ginseng 6g, Ulmus pumila 6g, Achyranthes bidentata 4g, Gentiana scabra 4g.

[0043] Example 3

[0044] Formula: Rhubarb 20g, Ginseng 14g, Ulmus pumila 14g, Achyranthes bidentata 12g, Gentiana scabra 12g.

[0045] Example 4

[0046] Formula: Rhubarb 10g, Ginseng 10g, Ulmus pumila 8g, Achyranthes bidentata 8g, Gentiana scabra 10g.

[0047] Example 5

[0048] Formula: Rhubarb 15g, Ginseng 8g, Ulmus pumila 8g, Achyranthes bidentata 10g, Gentiana scabra 10g.

[0049] Example 6

[0050] Formula: Rhubarb 10g, Ginseng 14g, Ulmus pumila 14g, Achyranthes bidentata 4g, Gentiana scabra 4g.

[0051] Example 7

[0052] Formula: Rhubarb 10g, Ginseng 6g, Ulmus pumila 14g, Achyranthes bidentata 12g, Gentiana scabra 4g.

[0053] Example 8

[0054] Formula: Rhubarb 12g, Ginseng 8g, Ulmus pumila 8g, Achyranthes bidentata 6g, Gentiana scabra 6g.

[0055] Example 9

[0056] Formula: Rhubarb 18g, Ginseng 12g, Ulmus pumila 12g, Achyranthes bidentata 10g, Gentiana scabra 10g.

[0057] Example 10

[0058] Formula: Rhubarb 10g, Ginseng 10g, Ulmus pumila 10g, Achyranthes bidentata 12g, Gentiana scabra 12g.

[0059] Example 11

[0060] Formula: Rhubarb 20g, Ginseng 14g, Ulmus pumila 10g, Achyranthes bidentata 10g, Gentiana scabra 8g.

[0061] Example 12

[0062] Formula: Rhubarb 15g, Ginseng 10g, Ulmus pumila 8g, Achyranthes bidentata 10g, Gentiana scabra 10g.

[0063] Example 13

[0064] Formula: Rhubarb 15g, Ginseng 14g, Ulmus pumila 14g, Achyranthes bidentata 8g, Gentiana scabra 8g.

[0065] Example 14

[0066] Formula: Rhubarb 18g, Ginseng 8g, Ulmus pumila 8g, Achyranthes bidentata 10g, Gentiana scabra 6g.

[0067] The pharmaceutical compositions from Examples 1-14 were used in the preparation methods of the following examples.

[0068] Example 15

[0069] (1) Ethanol extraction: Weigh out five medicinal materials according to the formula: Rhubarb, Ginseng, Small Angelica, Achyranthes bidentata, and Gentiana scabra. Add 60% ethanol at 8 times the total amount of medicinal materials, heat at 75℃ once for 1 hour, filter, recover ethanol, and obtain ethanol extract.

[0070] (2) Enzymatic extraction: Add 10 times the amount of water to the residue after ethanol extraction, add 1.0% cellulase, and extract for 1.5 h at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0071] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0072] (4) Preparation of dosage form: Add an appropriate amount of soluble starch to the thick paste, granulate, dry, and make 30g granules to obtain granules.

[0073] Example 16

[0074] (1) Ethanol extraction: Weigh out the five medicinal materials, namely Rheum palmatum, Panax ginseng, Smilax china, Achyranthes bidentata and Gentiana scabra, according to the formula amount. Add 60% ethanol, which is 6 times the total amount of medicinal materials. Heat at 70℃ twice for 0.5h each time. Filter and recover the ethanol to obtain ethanol extract.

[0075] (2) Enzymatic extraction: Add 8 times the amount of water to the residue after ethanol extraction, add 1.0% cellulase, and extract for 1 hour at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0076] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0077] (4) Preparation of dosage form: Add an appropriate amount of soluble starch to the thick paste, granulate, dry, and make 30g granules to obtain granules.

[0078] Example 17

[0079] (1) Ethanol extraction: Weigh out five medicinal materials according to the formula: Rhubarb, Ginseng, Small Angelica, Achyranthes bidentata, and Gentiana scabra. Add 60% ethanol at 10 times the total amount of medicinal materials. Heat at 80℃ for 3 times, each extraction for 2 hours. Filter and recover the ethanol to obtain ethanol extract.

[0080] (2) Enzymatic extraction: Add 12 times the amount of water to the residue after ethanol extraction, add 3.0% cellulase, and extract for 2 hours at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0081] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0082] (4) Preparation of dosage form: Add an appropriate amount of soluble starch to the thick paste, granulate, dry, and make 30g granules to obtain granules.

[0083] Example 18

[0084] (1) Ethanol extraction: Weigh out five medicinal materials according to the formula: Rhubarb, Ginseng, Small Angelica, Achyranthes bidentata, and Gentiana scabra. Add 60% ethanol at 8 times the total amount of medicinal materials. Heat at 75℃ twice for 1 hour each time. Filter and recover the ethanol to obtain ethanol extract.

[0085] (2) Enzymatic extraction: Add 10 times the amount of water to the residue after ethanol extraction, add 2.0% cellulase, and extract for 1.5 hours at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0086] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0087] (4) Prepare the corresponding dosage form: Add an appropriate amount of refined honey to the thick paste, mix well, and make 30g of paste.

[0088] Example 19

[0089] (1) Ethanol extraction: Weigh out five medicinal materials according to the formula: Rhubarb, Ginseng, Small Angelica, Achyranthes bidentata, and Gentiana scabra. Add 60% ethanol at 8 times the total amount of medicinal materials, heat at 75℃ once for 1 hour, filter, recover ethanol, and obtain ethanol extract.

[0090] (2) Enzymatic extraction: Add 10 times the amount of water to the residue after ethanol extraction, add 1.0% cellulase, and extract for 1.5 h at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0091] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0092] (4) Prepare the corresponding dosage form: Add an appropriate amount of refined honey to the thick paste, mix well, and make 30g of paste.

[0093] Example 20

[0094] (1) Ethanol extraction: Weigh out five medicinal materials according to the formula: Rhubarb, Ginseng, Small Angelica, Achyranthes bidentata, and Gentiana scabra. Add 60% ethanol at 10 times the total amount of medicinal materials. Heat at 75℃ for 3 times, each extraction for 2 hours. Filter and recover the ethanol to obtain ethanol extract.

[0095] (2) Enzymatic extraction: Add 12 times the amount of water to the residue after ethanol extraction, add 3.0% cellulase, and extract for 2 hours at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0096] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0097] (4) Prepare the corresponding dosage form: Add an appropriate amount of refined honey to the thick paste, mix well, and make 30g of paste.

[0098] Example 21

[0099] (1) Ethanol extraction: Weigh out five medicinal materials according to the formula: Rhubarb, Ginseng, Small Angelica, Achyranthes bidentata, and Gentiana scabra. Add 60% ethanol at 8 times the total amount of medicinal materials, heat at 75℃ once for 1 hour, filter, recover ethanol, and obtain ethanol extract.

[0100] (2) Enzymatic extraction: Add 10 times the amount of water to the residue after ethanol extraction, add 1.0% cellulase, and extract for 1.5 h at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0101] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0102] (4) Prepare the corresponding dosage form: Add an appropriate amount of starch and dextrin to the thick paste to make 30g tablets.

[0103] Example 22

[0104] (1) Ethanol extraction: Weigh out the five medicinal materials, namely, rhubarb, ginseng, small umbrella, achyranthes, and gentian, according to the formula. Add 60% ethanol, which is 6 times the total amount of medicinal materials. Heat at 75°C for extraction twice, each time for 0.5 h. Filter and recover the ethanol to obtain the ethanol extract.

[0105] (2) Enzymatic extraction: Add 8 times the amount of water to the residue after ethanol extraction, add 1.0% cellulase, and extract for 1 hour at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0106] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0107] (4) Prepare the corresponding dosage form: Add an appropriate amount of polyethylene glycol 4000 to the thick paste and drop it into 30g pills.

[0108] Example 23

[0109] (1) Ethanol extraction: Weigh out five medicinal materials according to the formula: Rhubarb, Ginseng, Small Angelica, Achyranthes bidentata, and Gentiana scabra. Add 60% ethanol at 10 times the total amount of medicinal materials. Heat at 75℃ for 3 times, each extraction for 2 hours. Filter and recover the ethanol to obtain ethanol extract.

[0110] (2) Enzymatic extraction: Add 12 times the amount of water to the residue after ethanol extraction, add 3.0% cellulase, and extract for 2 hours at 50℃ and pH 5.0. After boiling, filter to obtain the enzymatic extract.

[0111] (3) Concentration: Combine the ethanol extract and the enzymatic extract and concentrate them into a thick paste;

[0112] (4) Prepare the corresponding dosage form: Add an appropriate amount of water to the thick paste to dilute it, add 1g of sodium benzoate, stir and mix thoroughly, and then adjust the volume to 50mL to prepare an oral liquid.

[0113] To further verify the effectiveness of the present invention, the inventors conducted a series of verification experiments, as follows:

[0114] I. Prescription Composition and Compatibility

[0115] The traditional Chinese medicine components selected for this invention are: Rhubarb, Ginseng, Ulmus pumila, Achyranthes bidentata, and Gentiana scabra. Rhubarb clears heat and detoxifies, stops bleeding and astringes, and has a unique therapeutic effect on functional uterine bleeding; it is used in large quantities as the principal herb. Ginseng and Ulmus pumila nourish blood and qi, invigorate blood circulation, remove blood stasis, regulate menstruation and relieve pain, enhancing the efficacy of the principal herb; Achyranthes bidentata invigorates blood circulation, removes blood stasis, and guides blood downwards; Gentiana scabra regulates qi to aid blood circulation; both are used as adjuvant herbs. The combined effects of these herbs are to invigorate blood circulation, regulate qi, stop bleeding, and regulate menstruation.

[0116] II. Optimization Experiment of Preparation Process

[0117] This invention's prescription consists of five herbs: Rheum palmatum, Panax ginseng, Angelica dahurica, Achyranthes bidentata, and Gentiana scabra. Rheum palmatum is the principal herb. Our team previously analyzed the active components, targets, and pathways of action of Rheum palmatum (for the prevention and treatment of dysfunctional uterine bleeding, DUB) using network pharmacology and molecular docking techniques. The study revealed nine core active components in Rheum palmatum that contribute to the prevention and treatment of DUB: quercetin, ferulic acid, myristic acid, hydroxyemodin, cassia codon, emodin methyl ether, potassium oleracea acid, rhein, and ethyl 3,4-dihydroxybenzoate. Literature review indicates that these nine components have poor water solubility but good alcohol solubility; therefore, ethanol extraction was prioritized in the extraction process design.

[0118] In order to find the best extraction method, the team of this invention used the extraction rate of total flavonoids (quercetin, hydroxyemodin, cassia codone, emodin methyl ether, and rhein are all flavonoid components) in the prescription as the evaluation index. Through experiments, they compared three different extraction processes, namely water extraction, alcohol extraction, and alcohol extraction enzymatic hydrolysis, and finally selected the best extraction process.

[0119] 3.1 Method for determining the total flavonoid extraction rate

[0120] 3.1.1 The determination was performed using ultraviolet spectrophotometry.

[0121] (1) Determination of detection wavelength

[0122] Accurately measure 2 ml of rutin reference standard and place it in a 25 ml volumetric flask. Add water to a final volume of 6.0 ml, add 1 ml of 5% sodium nitrite solution, mix well, and let stand for 6 minutes. Add 1 ml of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes. Add 10 ml of sodium hydroxide test solution, then add water to the mark, shake well, and let stand for 15 minutes. Using the corresponding reagent as a blank, perform a spectral scan at wavelengths of 400–800 nm. The results showed that the reference standard solution had a large absorption at 500 nm, therefore, the detection wavelength was determined to be 500 nm.

[0123] (2) Preparation of reference solution

[0124] Accurately weigh 50 mg of rutin reference standard and place it in a 25 ml volumetric flask. Add an appropriate amount of methanol and heat gently in a water bath to dissolve. Cool and add methanol to the mark, then shake well. Accurately measure 10 ml of the solution and place it in a 100 ml volumetric flask. Add water to the mark and shake well to obtain the solution (each 1 ml contains 0.2 mg of rutin).

[0125] (3) Preparation of standard curve

[0126] Accurately measure 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, and 6 ml of the reference solution and place them in separate 25 ml volumetric flasks. Add water to each flask to a final volume of 6.0 ml. Add 1 ml of 5% sodium nitrite solution, mix well, and let stand for 6 minutes. Add 1 ml of 10% aluminum nitrate solution, shake well, and let stand for 6 minutes. Add 10 ml of sodium hydroxide solution, then add water to the mark, shake well, and let stand for 15 minutes. Using the corresponding reagents as blanks, measure the absorbance at a wavelength of 500 nm using ultraviolet-visible spectrophotometry. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa.

[0127] (4) Determination method

[0128] A: Total flavonoid content before extraction

[0129] Accurately weigh approximately 2g of the crude powder according to the prescription ratio, place it in a Soxhlet extractor, add an appropriate amount of ether, heat under reflux until the extract is colorless, cool, and discard the ether solution. Add 90ml of methanol, heat under reflux until the extract is colorless, transfer to a 100ml volumetric flask, wash the container with a small amount of methanol, add the washings to the same volumetric flask, add methanol to the mark, and shake well. Accurately measure 10ml, place it in a 100ml volumetric flask, add water to the mark, and shake well. Accurately measure 3ml, place it in a 25ml volumetric flask, and following the method under the standard curve preparation section, starting from "add water to 6.0ml", determine the absorbance according to the method, read the weight (μg) of rutin in the test solution from the standard curve, and calculate to obtain the total flavonoid content before extraction.

[0130] B: Total flavonoid content after extraction

[0131] Accurately measure 5 ml of the extract from each extraction method and place it in a 25 ml volumetric flask. Following the method under the preparation of the standard curve, starting from "add 1 ml of 5% sodium nitrite solution", determine the absorbance according to the method. Read the weight (μg) of rutin in the test solution from the standard curve and calculate to obtain the total flavonoid content after extraction.

[0132] (5) Total flavonoid extraction rate

[0133] Total flavonoid extraction rate = (Total flavonoid content after extraction ÷ Total flavonoid content before extraction) × 100%

[0134] 3.2 Research on water extraction technology

[0135] 3.2.1 Orthogonal Experiment Optimization

[0136] The factors of water addition (factor A), extraction time (factor B), and number of extractions (factor C) were selected as the factors of investigation. The optimal water extraction process parameters were screened by orthogonal experiment. The factors and levels are shown in Table 1, the orthogonal design and experimental results are shown in Table 2, and the results of analysis of variance are shown in Table 3.

[0137] Table 1. Factors and Levels in the Orthogonal Experiment of Water Extraction Process

[0138]

[0139]

[0140] Table 2. Orthogonal experimental design and results of water extraction process

[0141] serial number Factor A Factor B Factor C Error D Total flavonoid extraction rate (%) 1 1 1 1 1 11.5 2 1 2 2 2 15.28 3 1 3 3 3 18.42 4 2 1 2 3 19.95 5 2 2 3 1 21.77 6 2 3 1 2 20.93 7 3 1 3 2 17.15 8 3 2 1 3 19.03 9 3 3 2 1 21.35 K1 45.20 48.60 51.46 54.62 K2 62.65 56.08 56.58 53.36 K3 57.53 60.70 57.34 57.40 R 17.45 12.10 5.88 4.04

[0142] Table 3. Results of ANOVA for water extraction process

[0143] Source of variance Sum of Squares of Deviations Degrees of freedom Mean Square F value Significance A 53.64 2 26.82 18.83 Not significant B 24.86 2 12.43 8.73 Not significant C 6.82 2 3.41 2.39 Not significant D (error) 2.85 2 1.42

[0144] Note: F 0.05 (2, 2) = 19

[0145] As shown in Table 2 of the orthogonal experiment results, the influence of each factor on the water extraction effect is A > B > C, and A2 > A3 > A1, B3 > B2 > B1, C3 > C2 > C1. The optimal water extraction process parameters are A2B3C3. According to the analysis of variance in Table 3, factors A, B, and C have no significant effect. However, although factor A has no significant difference, A2 is significantly higher than A1. Therefore, from the perspective of cost saving, the optimal water extraction process parameters can be adjusted to A2B1C1, that is, add 8 times the amount of water and decoct once for 0.5 hours.

[0146] 3.2.2 Verification of optimal parameters for water extraction process

[0147] Three portions of Rheum palmatum, Panax ginseng, Angelica dahurica, Achyranthes bidentata, and Gentiana scabra were taken according to the prescription ratio. Each portion was extracted by decoction using the optimal water extraction parameters selected above. The extracts were then diluted with water and brought to a final volume of 1000 ml. The total flavonoid extraction rate was determined, and the results are shown in Table 4.

[0148] Table 4. Validation Results of Optimal Water Extraction Process Parameters

[0149]

[0150]

[0151] The verification results showed that the optimal water extraction process parameters obtained through orthogonal experiments resulted in a total flavonoid extraction rate of 18.18% in the formulation.

[0152] 3.3 Study on alcohol extraction process

[0153] 3.3.1 Investigation of Ethanol Concentration

[0154] Four portions of Rheum palmatum, Panax ginseng, Angelica dahurica, Achyranthes bidentata, and Gentiana scabra were taken according to the prescription ratio. These were extracted with 10 times the amount of 40%, 60%, 80%, and 90% ethanol, respectively, at 75°C for 1 hour. The extracts were filtered and diluted with the corresponding concentrations of ethanol to a final volume. The total flavonoid extraction rate was determined, and the results are shown in Table 5.

[0155] Table 5 Results of Ethanol Concentration Investigation

[0156] Test No. Ethanol concentration (%) Alcohol addition (times) Extraction time (hours) Total flavonoid extraction rate (%) 1 40 10 1 27.58 2 60 10 1 39.20 3 80 10 1 41.48 4 90 10 1 43.22

[0157] The experimental results show that when the ethanol concentration is greater than 60%, the extraction rate of flavonoids in the prescription does not increase much. In order to save production costs and reduce the amount of ethanol used, 60% ethanol was selected as the extraction solvent.

[0158] 3.3.2 Orthogonal Experiment Optimization

[0159] The amount of alcohol added (factor A), extraction time (factor B), and number of extractions (factor C) were selected as the factors to be investigated. Orthogonal experiments were used to screen the optimal alcohol extraction process parameters. The factors and levels are shown in Table 6, the orthogonal design and experimental results are shown in Table 7, and the results of the analysis of variance are shown in Table 8.

[0160] Table 6. Factors and Levels in the Orthogonal Experiment for Alcohol Extraction Process

[0161] level Factor A (times) Factor B (hours) Factor C (times) 1 6 0.5 1 2 8 1 2 3 10 2 3

[0162] Table 7. Orthogonal experimental design and results for alcohol extraction process.

[0163]

[0164]

[0165] Table 8. Results of ANOVA for Alcohol Extraction Process

[0166] Source of variance Sum of Squares of Deviations Degrees of freedom Mean Square F value Significance A 124.89 2 62.45 27.44 Significant B 96.54 2 48.27 21.21 Significant C 18.33 2 9.16 4.03 Not significant D (error) 4.55 2 2.28

[0167] Note: F 0.05 (2, 2) = 19

[0168] As shown in Table 7 of the orthogonal experiment results, the influence of each factor on the alcohol extraction effect is A > B > C, and A2 > A3 > A1, B3 > B2 > B1, C3 > C2 > C1. The optimal alcohol extraction process parameters are A2B3C3. According to the analysis of variance in Table 8, A and B have significant differences, while C has no significant effect. Since B3 and B2 in factor B are close, from the perspective of cost saving, the optimal alcohol extraction process parameters can be adjusted to A2B2C1, that is, extract once with 8 times 60% ethanol for 1 hour.

[0169] 3.3.3 Validation of Optimal Parameters for Alcohol Extraction Process

[0170] Three portions of Rheum palmatum, Panax ginseng, Smilax china, Achyranthes bidentata, and Gentiana scabra were taken according to the prescription ratio. Each portion was extracted using the optimal alcohol extraction parameters selected above. The extracts were then diluted with 60% ethanol and brought to a final volume. The total flavonoid extraction rate was determined, and the results are shown in Table 9.

[0171] Table 9 Validation Results of Optimal Alcohol Extraction Process Parameters

[0172]

[0173] The verification results showed that the optimal alcohol extraction process parameters obtained through orthogonal experiments yielded a total flavonoid extraction rate of 41.11% for the lipid-lowering formula.

[0174] 3.4 Study on enzymatic extraction process

[0175] The results of water extraction and alcohol extraction studies show that the extraction rate of total flavonoids in the prescription using water extraction is only 18.18%, which is relatively low. Although the extraction rate can be increased to 38.56% using alcohol extraction, there is still considerable room for improvement. Other extraction methods should be considered to enhance the extraction efficiency of total flavonoids in the prescription. Literature review indicates that enzymatic extraction, as a novel extraction method for traditional Chinese medicine, involves enzymatically hydrolyzing the raw materials. Because cellulase disrupts the cell walls of the substances, releasing intracellular substances, the extraction efficiency of the target extract can be significantly increased. Therefore, our team conducted preliminary experiments on the extraction effect of total flavonoids in the prescription using enzymatic extraction.

[0176] Take the medicinal materials according to the prescription ratio, mix them well, add 8 times the amount of water, add 2.0% cellulase, and extract for 120 minutes at 50℃ and pH 5.0. After boiling, filter, concentrate and make up to a fixed volume, and then determine the total flavonoid extraction rate. The result shows that the total flavonoid extraction rate is 23.34%.

[0177] The experimental results show that the total flavonoid extraction rate in the prescription is not ideal when using only enzymatic extraction.

[0178] 3.5 Study on the combined extraction process of alcohol extraction and enzymatic hydrolysis

[0179] Among the three extraction processes mentioned above, the total flavonoid extraction rates, from highest to lowest, are ethanol extraction, enzymatic extraction, and water extraction. The total flavonoid extraction rates in single extraction method formulations do not exceed 40%. In order to increase the total flavonoid extraction rate, a combined extraction method can be considered, that is, combining the two extraction methods with higher extraction efficiency mentioned above: ethanol extraction and enzymatic extraction.

[0180] After extraction under the aforementioned optimal ethanol extraction conditions, the research team added 8 times the amount of water to the residue, along with 2.0% cellulase, and extracted for 120 minutes at 50°C and pH 5.0. The mixture was then boiled and filtered. The filtrate and the extract obtained from the ethanol extraction were combined, concentrated, and brought to a fixed volume before determining the total flavonoid extraction rate. The result showed a total flavonoid extraction rate of 73.82%. The experimental results indicate that the combined ethanol extraction and enzymatic hydrolysis extraction process significantly improved the extraction efficiency of total flavonoids in the formula, demonstrating the rationality and feasibility of this process. Further refinement of the enzymatic hydrolysis process parameters is possible in this direction.

[0181] 3.5.1 Orthogonal Experiment Optimization

[0182] The medicinal materials in the prescription were first extracted using the aforementioned optimal ethanol extraction process, and the residue was then subjected to a secondary extraction using enzymatic hydrolysis. Water addition (Factor A), cellulase dosage (Factor B), and hydrolysis time (Factor C) were selected as the factors to be investigated. An orthogonal experiment was used to screen the optimal enzymatic hydrolysis extraction conditions (50℃, pH 5.0). The factors and levels are shown in Table 10, the orthogonal design and experimental results are shown in Table 11, and the results of the analysis of variance are shown in Table 12.

[0183] Table 10. Factors and Levels in the Orthogonal Experiment for Alcohol Extraction and Enzymatic Hydrolysis Process

[0184] level Factor A (times) Factor B (%) Factor C (min) 1 8 1.0 60 2 10 2.0 90 3 12 3.0 120

[0185] Table 11 Orthogonal experimental design and results of alcohol extraction and enzymatic hydrolysis process

[0186]

[0187]

[0188] Table 12 Results of ANOVA for Alcohol Extraction and Enzymatic Hydrolysis Process

[0189] Source of variance Sum of Squares of Deviations Degrees of freedom Mean Square F value Significance A 933.88 2 466.94 64.06 Significant B 119.94 2 59.97 8.23 Not significant C 478.47 2 239.23 32.82 Significant D (error) 14.58 2 7.29

[0190] Note: F 0.05 (2, 2) = 19

[0191] As shown in Table 11 of the orthogonal experiment results, the influence of each factor on the extraction effect in the enzymatic extraction process is A > C > B, and A3 > A2 > A1, C3 > C2 > C1, B3 > B2 > B1. As shown in Table 12 of the variance analysis, factors A and C have a significant impact on the enzymatic extraction process, while factor B has no significant impact. Considering the production cycle and production cost, the optimal process parameters can be selected as A2B1C2, that is, adding 10 times the amount of water of the medicinal materials, adding 1.0% cellulase, and extracting for 90 minutes at 50℃ and pH 5.0.

[0192] 3.5.2 Validation of Optimal Parameters for Alcohol Extraction and Enzymatic Hydrolysis Combined Extraction Process

[0193] Three portions of Rheum palmatum, Panax ginseng, Smilax china, Achyranthes bidentata, and Gentiana scabra were taken according to the prescription ratio. Each portion was extracted using the optimal alcohol extraction parameters selected above (extracted with 8 times the volume of 60% ethanol for 1 hour). After filtration, the ethanol was recovered from the filtrate to obtain the ethanol extract, which was then set aside. The residue was added to 10 times the volume of water, along with 1.0% cellulase. The mixture was extracted at 50℃ and pH 5.0 for 90 minutes, boiled, and then filtered. The filtrate and ethanol extract were combined, concentrated, and brought to a final volume. The total flavonoid extraction rate was determined, and the results are shown in Table 13.

[0194] Table 13 Validation Results of Optimal Alcohol Extraction and Enzymatic Hydrolysis Combined Extraction Process Parameters

[0195]

[0196] The verification results show that the optimal alcohol extraction and enzymatic hydrolysis process selected by orthogonal experiment yielded a total flavonoid extraction rate of 67.12% in the prescription, which is highly efficient and reasonable. The process is feasible and can be used as the optimal extraction process for this prescription.

[0197] IV. Comparative Study of Pharmacological Effects of Formulations Prepared by Different Methods

[0198] To verify the efficacy of preparations obtained by different extraction processes, the team of this invention prepared granules by extracting the same proportion of the formula with water, ethanol, enzymatic hydrolysis, and a combination of ethanol and enzymatic hydrolysis. The pharmacodynamic comparative study was carried out under the condition that the raw drug content of the granules obtained by each process was comparable.

[0199] 4.1 Materials

[0200] 4.1.1 Drug testing

[0201] Water-extracted granules (self-made, each gram equivalent to 1.7g of raw medicinal material), alcohol-extracted granules (self-made, each gram equivalent to 1.7g of raw medicinal material), enzymatically hydrolyzed granules (self-made, each gram equivalent to 1.7g of raw medicinal material), alcohol-extracted enzymatically hydrolyzed granules (self-made, each gram equivalent to 1.7g of raw medicinal material), Gongxue Ning capsules (batch number: ZJC2204, Yunnan Baiyao Group Co., Ltd.), adrenaline (product number: L137185-1g, Aladdin Biochemical Technology Co., Ltd.), mifepristone tablets (specification: 25mg / tablet, batch number: C019210304, Shanghai Xinhua Lian Pharmaceutical Co., Ltd.), misoprostol tablets (specification: 0.2mg / tablet, batch number: 02201201, Wuhan Jiulong Renfu Pharmaceutical Co., Ltd.). The estradiol (E2) ELISA kit (catalog number: E-OSEL-R0001) and progesterone (P) ELISA kit (catalog number: E-EL-0154c) were used (Elabscience). All other reagents were domestically produced analytical grade reagents.

[0202] 4.1.2 Animals

[0203] SPF-grade female ICR mice, weighing 18–22 g; SPF-grade healthy female nonpregnant SD rats, weighing 180–220 g; and SPF-grade male SD rats, weighing 200–220 g. Animals were housed in an indoor temperature of 22–26°C with a relative humidity of 40%–70%, with free access to food and water. Experiments were conducted after one week of acclimatization.

[0204] 4.1.3 Instruments

[0205] 5810R benchtop high-speed refrigerated centrifuge (Eppendor, Germany); blood coagulation analyzer (STAGO, France, reagents were original imported); fully automated blood rheometer (SA-6000, Beijing Saike Xide Co., Ltd.); BSA124S electronic analytical balance (Sartorius Scientific Instruments Co., Ltd., Beijing); inverted biological microscope (Jiangnan Yongxin XD202); UV-Vis spectrophotometer (Beijing Purkinje Instruments Co., Ltd.); full-wavelength microplate reader (Thermo Scientific, USA).

[0206] 4.2 Statistical Methods

[0207] Statistical analysis was performed using Graphpad Prism 7.00 software. All data are expressed as mean ± standard deviation. The data indicate that they conform to a normal distribution and have homogeneous variances. One-way ANOVA was used, and Tukey's test was used for further pairwise comparisons. P < 0.05 was considered statistically significant.

[0208] 4.3 Methods and Results

[0209] 4.3.1 Effects of particles prepared by different processes on bleeding time and clotting time in normal mice

[0210] Sixty female ICR mice were randomly divided into six groups: a control group, a Gongxuening capsule group (0.13 g / kg), a water-extracted granule group (2.2 g / kg), an alcohol-extracted granule group (2.2 g / kg), an enzymatically hydrolyzed granule group (2.2 g / kg), and an alcohol-extracted enzymatically hydrolyzed granule group (2.2 g / kg). The dosage was calculated based on the clinical dosage, with a human-to-mouse dosage ratio of 0.11:1. The control group received physiological saline. The mice were administered the medication by gavage for 7 consecutive days. One hour after the last administration, the bleeding time was measured: the mouse tail was cut transversely 3 mm from the tip using dissecting scissors. The bleeding was timed after the blood spontaneously overflowed, and the blood was aspirated with filter paper every 30 seconds until the bleeding stopped naturally. Coagulation time determination: Blood was collected from the inner canthus of the eye using a glass capillary tube. After the blood collection was complete, the capillary tube was laid flat on a table. Every 30 seconds, both ends of the capillary tube were broken by 0.5 cm, and the tube was slowly pulled apart to the left and right. The presence of coagulation streaks was observed at the break points. The time elapsed from when the blood flowed into the capillary tube until the appearance of coagulation streaks was recorded as the coagulation time. The experimental results are shown in Table 14.

[0211] Table 14 Effects of particles prepared by different processes on bleeding time and clotting time in normal mice.

[0212] Group Dosage (g / kg) Bleeding time (min) Clotting time (min) Blank group 0 18.7±2.9 3.7±0.5 Gongxue Ning Capsule Group 0.13 13.2±2.8* 2.8±0.4* Water-extracted granules 2.2 14.8±2.6* 2.7±0.5* Alcohol Extract Granules 2.2 12.8±2.2* 2.4±0.6* Enzymatic hydrolysis of granules 2.2 12.9±2.5* 2.0±0.5** Alcohol-extracted enzymatic hydrolysis granules 2.2 9.2±1.7** 1.6±0.3**

[0213] Note: Compared with the control group, *P<0.05, **P<0.01.

[0214] Experimental results showed that, compared with the blank group, all particle groups prepared by different processes could significantly shorten the bleeding time and clotting time in mice. Among the particle groups prepared by different processes, the alcohol-extracted and enzymatically hydrolyzed particle group had the shortest bleeding time and clotting time in mice, suggesting that the alcohol-extracted and enzymatically hydrolyzed particle group had better hemostatic effect than other preparation processes.

[0215] 4.3.2 Effects of particles prepared by different processes on coagulation function in normal rats

[0216] Forty-eight female SD rats were randomly divided into six groups: a blank control group, a Gongxuening capsule group (0.091 g / kg), a water-extracted granule group (1.5 g / kg), an alcohol-extracted granule group (1.5 g / kg), an enzymatically hydrolyzed granule group (1.5 g / kg), and an alcohol-extracted enzymatically hydrolyzed granule group (1.5 g / kg). The dosage was calculated based on the clinical dosage, with a human-to-rat ratio of 1:7. The blank control group received normal saline via gavage for 7 consecutive days. One hour after the last administration, rats were anesthetized with 10% chloral hydrate (300 mg / kg intraperitoneally), and blood was collected using 3.2% sodium citrate at a 1:9 anticoagulation ratio. Plasma samples were used for parameter testing. The experimental results are shown in Table 15.

[0217] Table 15 Effects of particles prepared by different processes on coagulation function in normal rats

[0218] Group Dosage (g / kg) PT(s) APTT(s) FIB(g / L) Blank group 0 16.21±0.73 38.32±6.55 2.24±0.97 Gongxue Ning Capsule Group 0.091 14.81±0.68* 27.93±4.26* 3.67±1.12* Water-extracted granules 1.5 15.32±0.64 35.44±6.75 3.48±0.92* Alcohol Extract Granules 1.5 14.71±0.71* 29.30±5.58* 3.58±0.86* Enzymatic hydrolysis of granules 1.5 13.56±0.60** 30.39±5.56* 3.41±0.93* Alcohol-extracted enzymatic hydrolysis granules 1.5 12.88±0.57** 24.38±5.11** 3.73±1.08*

[0219] Note: Compared with the control group, *P<0.05, **P<0.01.

[0220] The experimental results showed that, compared with the control group, the prothrombin time (PT) and activated partial thromboplastin time (APTT) of the granules prepared by different processes were significantly shortened (P < 0.05, P < 0.01), while the fibrinogen (FIB) content was significantly increased (P < 0.05). This suggests that granules prepared by different processes all have a certain promoting effect on the coagulation system, and the alcohol-extracted and enzymatically hydrolyzed granule group showed the strongest promoting effect.

[0221] 4.3.3 Effects of particles prepared by different processes on bleeding volume in rats with functional uterine bleeding model

[0222] Female rats in estrus were housed together at a female-to-male ratio of 2:1 at 5 PM daily. Vaginal smears were examined the following morning, and the presence of sperm under a microscope was considered day 1 of pregnancy. Seventy successfully conceived female rats were selected. Ten rats were used as a blank control group, and the remaining 60 were randomly divided into six groups: a model control group, a Gongxue Ning capsule group (0.091 g / kg), a water-extracted granule group (1.5 g / kg), an alcohol-extracted granule group (1.5 g / kg), an enzymatically hydrolyzed granule group (1.5 g / kg), and an alcohol-extracted enzymatically hydrolyzed granule group (1.5 g / kg). Except for the blank control group, the other groups were administered mifepristone 8.8 mg / kg by gavage at 8 AM on day 7 of pregnancy and misoprostol 100 μg / kg by gavage at 6 PM to establish a uterine bleeding model of incomplete abortion in early pregnancy. A quantitative amount of sterile cotton balls was inserted into the rat's vagina; the cotton balls were removed the following day, and the presence of blood indicated successful modeling. On day 8 of pregnancy, all groups began gavage administration of the drug. The blank control group and the model control group were given physiological saline for 7 consecutive days. A fixed amount of sterile cotton balls were changed in the rat's vagina every morning and evening. The cotton balls were removed the following morning and evening and stored in sealed bags under cold storage. One hour after the last administration, the rats were anesthetized with 10% chloral hydrate, and blood was collected from the abdominal aorta. 0.02 mL of blood was added to 4 mL of 50 g / L NaOH solution and mixed well. The collected uterine bleeding cotton balls from each rat were placed in a beaker, and an appropriate amount of 50 g / L NaOH was added for extraction for 24 hours, depending on the amount of bleeding. The absorbance (A) of the abdominal aortic blood NaOH solution and the filtered extract was measured at 546 nm using a UV spectrophotometer, with 50 g / L NaOH as a blank control, to calculate the amount of uterine bleeding (mL). The results are shown in Table 16.

[0223] Table 16 Effects of Kuning Granules on Bleeding Volume in Rats with Functional Uterine Bleeding Model

[0224] Group Dosage (g / kg) Uterine bleeding volume (mL) Blank group, control group 0 0 Model control group 0 0.55±0.03 Gongxue Ning Capsule Group 0.091 0.35±0.02** Water-extracted granules 1.5 0.39±0.06* Alcohol Extract Granules 1.5 0.34±0.05** Enzymatic hydrolysis of granules 1.5 0.35±0.03** Alcohol-extracted enzymatic hydrolysis granules 1.5 0.30±0.04**

[0225] Note: Compared with the model control group, *P<0.05, **P<0.01

[0226] The experimental results showed that, compared with the model control group, the amount of uterine bleeding in the different preparation process particle groups was significantly reduced (P<0.05, P<0.01), among which the alcohol-extracted enzymatic hydrolysis particle group had the least amount of bleeding.

[0227] 4.3.4 Effects of particles prepared by different processes on serum E2 and P levels in rats with functional uterine bleeding model

[0228] The blood samples taken in the previous experiment were centrifuged at 3000 r / min for 15 min using a high-speed refrigerated centrifuge to separate the serum. The levels of E2 and P in the serum were measured using an ELISA kit. The results are shown in Table 17.

[0229] Table 17 Effects of particles prepared by different processes on bleeding time and clotting time in normal mice.

[0230]

[0231]

[0232] Note: Compared with the blank control group, *P<0.05; compared with the model control group, # P < 0.05.

[0233] The experimental results showed that, compared with the blank control group, the E2 and P contents in the model control group were significantly reduced (P < 0.05); compared with the model control group, the P contents in the particle groups prepared by different processes were significantly increased (P < 0.05). This suggests that the particle groups prepared by different processes all have a neuroendocrine-improving effect.

[0234] 4.3.5 Effects of particles prepared by different processes on the histopathology of uterine tissue in rats with functional uterine bleeding model

[0235] Rats were euthanized by cervical dislocation, and their uterine tissue was collected and fixed in 4% paraformaldehyde for 24 hours. The tissue was then cleared, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). In the normal control group, the uterine tissue structure was intact, the stroma was tightly packed, and blood vessels and glands were clearly visible. In the model control group, there were more residual villi and decidua, congested and dilated endothelium, and fewer blood vessels and glands. The groups with particles prepared using different processes showed a slight increase in the number of blood vessels and glands, a tighter arrangement of epithelial cells, and varying degrees of improvement in the degree of uterine damage.

[0236] 4.3.6 Conclusion

[0237] Based on the aforementioned pharmacodynamic studies, it can be concluded that among various preparation processes, the ethanol enzymatic hydrolysis combined extraction method yields superior overall pharmacodynamic results compared to other preparation methods.

[0238] V. Clinical efficacy verification

[0239] To verify the clinical efficacy of the preparations obtained by the ethanol enzymatic hydrolysis and compound extraction process, the invention team further conducted clinical efficacy verification experiments.

[0240] 1. General Clinical Data

[0241] Two hundred patients with dysfunctional uterine bleeding treated at the Department of Gynecology of XX Traditional Chinese Medicine Hospital from January 2021 to November 2023 were selected as the study subjects. The patients were aged 25 to 50 years, with a mean age of (36.52±1.65) years; the duration of the disease ranged from 2 to 12 months, with a mean duration of (6.53±1.52) months. Inclusion criteria: (1) meeting the diagnostic criteria of the "Clinical Diagnosis and Treatment Guidelines for Dysfunctional Uterine Bleeding"; (2) being in a bleeding state at the time of consultation; (3) the patient gave consent and signed an informed consent form. Exclusion criteria: (1) patients with serious diseases of organs such as heart, liver, spleen, and kidney; (2) history of drug allergy; (3) patients with bleeding caused by other reasons; (4) patients with hematological diseases; (5) patients with mental illness.

[0242] 2. Grouping and Treatment Methods

[0243] Patients were randomly divided into a control group (n=100) and a treatment group (n=100) using a random number method. The control group patients were aged 25–49 years, with a mean age of (36.13±1.43) years; their disease duration ranged from 2 to 10 months, with a mean duration of (6.11±1.63) months. The treatment group patients were aged 25–50 years, with a mean age of (38.50±1.54) years; their disease duration ranged from 4 to 12 months, with a mean duration of (8.43±1.62) months. There were no statistically significant differences in age, disease duration, or other general characteristics between the two groups, making them comparable.

[0244] The control group received oral drospirenone and ethinylestradiol tablets (manufactured by Bayer Weimar GmbH & Co. KG, 3mg / tablet, batch number 202011028), 1 tablet once daily. The treatment group, in addition to the treatment given to the control group, received oral granules of the drug combination of this invention prepared according to the formulation of Example 1 and the preparation method of Example 15, 1 sachet three times daily. Both groups received the medication for 7 weeks.

[0245] 3. Efficacy evaluation criteria

[0246] Significant effect: Symptoms such as bleeding, dizziness, fatigue, pale complexion, and menstrual irregularities basically disappear; Effective: Symptoms such as bleeding, dizziness, fatigue, pale complexion, and menstrual irregularities improve; Ineffective: The above symptoms remain unchanged, and in some cases, they worsen.

[0247] Overall effectiveness = (Number of cases with significant effects + Number of cases with positive results) / Total number of cases

[0248] 4. Observation Indicators

[0249] (1) Improvement of clinical symptoms

[0250] During the treatment with the two medications, the patient and their family recorded any bleeding, dizziness, weakness, paleness, menstrual irregularities, etc., and also recorded the time when the corresponding symptoms improved, and informed the same doctor for analysis.

[0251] (2) Sex hormone levels

[0252] All subjects had 3 mL of venous blood collected before and after treatment. The levels of pituitary follicle-stimulating hormone (FSH), estradiol (E2), luteinizing hormone (LH), and testosterone (T) were measured using a fully automated chemiluminescence immunoassay analyzer. All procedures were performed in accordance with the kit standards.

[0253] (3) Serum inflammatory factors

[0254] Five mL of venous blood was drawn from the upper limb in the morning on an empty stomach and placed in a sterile glass tube. The serum was centrifuged and stored at -50°C for later testing. The levels of interleukin-2 (IL-2), vascular endothelial growth factor A (VEGFA), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) were measured using enzyme-linked immunosorbent assay (ELISA) according to the kit instructions.

[0255] 5. Adverse drug reactions

[0256] During treatment, adverse reactions such as nausea, vomiting, loss of appetite, general weakness, and drug rash that occurred in patients while taking the medication were compared.

[0257] 6. Statistical Analysis

[0258] Data were processed using SPSS 19.0 software. Categorical data were analyzed using the χ² test and expressed as percentages; continuous data at the levels of IL-2, VEGFA, TNF-α, and CRP were analyzed using the t-test. express.

[0259] 7. Results

[0260] (1) Comparison of clinical efficacy between the two groups

[0261] After treatment, in the control group, 52 cases showed significant improvement, 37 cases showed improvement, and 11 cases showed no improvement; in the treatment group, 75 cases showed significant improvement, 23 cases showed improvement, and 2 cases showed no improvement; the effective rate of the treatment group was higher than that of the control group (P<0.05), as shown in Table 18.

[0262] Table 18 Clinical efficacy trial results

[0263]

[0264]

[0265] Note: Compared with the control group: *P<0.05

[0266] (2) Comparison of symptom improvement time between the two groups

[0267] After treatment, the symptoms such as bleeding, dizziness, fatigue, pale complexion, and menstrual irregularities in the treatment group improved significantly earlier than those in the control group (P<0.05), as shown in Table 19.

[0268] Table 19 Comparison of symptom improvement time between the two groups

[0269]

[0270] Note: Compared with the control group: *P<0.05

[0271] (3) Comparison of sex hormone levels between the two groups of patients

[0272] After treatment, the levels of hormone FSH in both groups increased significantly, while the levels of E2, LH and T decreased significantly (P<0.05); and the hormone FSH level in the treatment group was higher than that in the control group, while the levels of E2, LH and T were significantly lower than those in the control group (P<0.05), as shown in Table 20.

[0273] Table 20 Comparison of sex hormone levels between the two groups

[0274]

[0275] Note: *P<0.05 for comparisons before and after treatment within the same group; #P<0.05 for comparisons after treatment with the control group.

[0276] (4) Comparison of serological levels between the two groups

[0277] After treatment, the serum levels of IL-2, VEGFA, TNF-α, and CRP in both groups of patients decreased significantly (P<0.05), and the levels of IL-2, VEGFA, TNF-α, and CRP in the treatment group were significantly lower than those in the control group (P<0.05), as shown in Table 21.

[0278] Table 21 Comparison of serum factor levels between the two groups

[0279]

[0280] Note: *P<0.05 for comparisons before and after treatment within the same group; #P<0.05 for comparisons after treatment with the control group.

[0281] (5) Comparison of adverse reactions between the two groups

[0282] During the treatment period, 2 patients in the control group experienced nausea and vomiting, 4 experienced anorexia, 3 experienced general weakness, and 2 experienced drug rash; 1 patient in the treatment group experienced nausea and vomiting, 1 experienced anorexia, 0 experienced general weakness, and 0 experienced drug rash; the incidence of adverse events in the treatment group (2.00%) was significantly lower than that in the control group (11.00%, P<0.05), as shown in Table 22.

[0283] Table 22 Comparison of adverse reactions between the two groups

[0284]

[0285] Note: Compared with the control group: *P<0.05.

[0286] 8. Conclusion

[0287] In summary, the drug combination of the present invention, after being prepared into granules through alcohol extraction and enzymatic hydrolysis, combined with drospirenone and ethinylestradiol to treat functional uterine bleeding, can shorten the hemostasis time, significantly improve the patient's sex hormone levels, and reduce inflammatory factors. It is safe and effective, and can improve clinical efficacy.

[0288] VI. Collection of Typical Cases

[0289] 1. Ms. Que, 50 years old, XX Traditional Chinese Medicine Hospital

[0290] Admission details: The patient was admitted on August 30, 2023, due to "a pelvic mass discovered 2+ years ago and irregular vaginal bleeding for 20+ days". The patient presented with pale complexion, dizziness, fatigue, pale and dark tongue with a thin white coating, and a thready and hesitant pulse.

[0291] Intraoperative diagnoses: 1. Abnormal uterine bleeding; 2. Adenomyosis; 3. Pelvic mass: 1) Ovarian tumor; 2) Other; 4. Hydrosalpinx; 5. Grade 2 high-risk group of essential hypertension.

[0292] Treatment: In addition to the treatment for the control group, patients were given oral administration of the granules prepared according to the preparation method of Example 1 of this invention, one sachet per dose, three times a day. Treatment lasted for 7 weeks. A second consultation was subsequently conducted.

[0293] Second consultation results: After integrated traditional Chinese and Western medicine treatment, the patient's condition has significantly improved compared to before. The patient reported slight vaginal bleeding, with spotting visible during urination; the blood was dark red, without clots, and she reported no lower abdominal pain, no rectal pressure, no chills or fever, no nausea or vomiting, no dizziness or fatigue, no palpitations or chest tightness, and no urinary frequency, urgency, or pain. Her mental state, appetite, and sleep were good, and her bowel movements were normal. Her complexion was rosy. Physical examination: Vital signs were stable, heart and lung sounds were negative, and there was no tenderness, rebound tenderness, or muscle guarding in the lower abdomen.

[0294] 2. Jiang XX, female, 30 years old, XX Traditional Chinese Medicine Hospital

[0295] Admission details: The patient was admitted on March 15, 2023, due to "irregular vaginal bleeding for 15 days." The patient presented with dizziness and weakness, a dark red tongue with a thin white coating, and a wiry and hesitant pulse.

[0296] Intraoperative diagnosis: 1. Abnormal uterine bleeding; 2. Cervical polyps; 3. Pelvic inflammatory disease.

[0297] Treatment: In addition to the treatment for the control group, patients were given oral administration of the granules prepared according to the preparation method of Example 1 of this invention, one sachet per dose, three times a day. Treatment lasted for 7 weeks. A second consultation was subsequently conducted.

[0298] Second visit results: After integrated traditional Chinese and Western medicine treatment, the patient's condition has significantly improved compared to before. The patient reported no abdominal pain or lumbosacral soreness, no radiating pain, no pulling pain, no anal tenesmus, no abnormal vaginal bleeding, no fever, no sweating, no nausea, no vomiting, no dizziness, no weakness, no palpitations, no chest tightness, no diarrhea, no constipation, etc. Her mental state, appetite, and sleep were good, and bowel movements were normal. Physical examination: Vital signs were stable, heart and lungs were negative, abdomen was soft, without tenderness, rebound tenderness, or muscle guarding. Complexion was rosy. Physical examination: Vital signs were stable, heart and lungs were negative, no lower abdominal tenderness, no rebound tenderness, or muscle guarding.

[0299] 3. Fu X, female, 30 years old, XX Traditional Chinese Medicine Hospital

[0300] Admission status: The patient was admitted on September 3, 2023 at 12:36 PM due to "irregular vaginal bleeding and lower abdominal pain for 5 days on the 19th day of menstruation." The patient presented with amenorrhea, irregular vaginal bleeding (small amount, brownish in color, no blood clots, no fleshy or vesicular tissue discharge), lower abdominal distending pain (intermittent, worse with activity, relieved by rest), no lumbosacral pain, no radiating or radiating pain, no anal tenesmus, no nausea or vomiting, no palpitations or chest tightness, no urinary frequency, urgency, or dysuria, and good mental state, appetite, and sleep. Bowel movements were normal. The tongue was pale and dark with a thin white coating, and the pulse was wiry and hesitant.

[0301] Traditional Chinese Medicine diagnosis: Ectopic pregnancy (unruptured type) (fetal obstruction); Western medicine diagnosis: 1. Abnormal uterine bleeding; 2. Miscarriage; 3. Bacterial vaginosis.

[0302] Treatment: In addition to the treatment for the control group, patients were given oral administration of the granules prepared according to the preparation method of Example 1 of this invention, one sachet per dose, three times a day. Treatment lasted for 7 weeks. A second consultation was subsequently conducted.

[0303] Second visit results: After integrated traditional Chinese and Western medicine treatment, the patient's condition has significantly improved compared to before. The patient reported no significant lower abdominal pain, no anal tenesmus, no vaginal bleeding, no fleshy or vesicular tissue discharge, no chills or fever, no nausea or vomiting, no dizziness or fatigue, no palpitations or chest tightness, no diarrhea or constipation, no urinary frequency, urgency, or dysuria, and her mental state, appetite, and sleep were good, and bowel movements were normal. Physical examination: Vital signs were stable, heart and lungs were unremarkable, there was no significant lower abdominal tenderness, rebound tenderness, or muscle guarding.

[0304] 4. Lin X, female, 44 years old, XX Traditional Chinese Medicine Hospital

[0305] Admission status: Admitted on 2023-09-16 due to "discovery of cervical polyps 19 days ago".

[0306] Surgical Name: Hysteroscopic Removal of Cervical Polyps + Fractional Curettage. Surgery Date: 2023-09-18; Surgical Method: Hysteroscopy; Anesthesia: General Anesthesia; Intraoperative Diagnosis: 1. Cervical polyps; 2. Cervical human papillomavirus infection; 3. Pelvic inflammatory disease. Postoperative...

[0307] On the first day after surgery, the patient reported slight vaginal bleeding, which was light red in color. She also experienced occasional soreness and pain in the lower abdomen and lumbosacral region, which was mild. She had no chills, fever, nausea, vomiting, dizziness, fatigue, palpitations, chest tightness, or other discomfort. Her mental state and sleep were normal. Her tongue was dark with a thin white coating, and her pulse was thready. Physical examination revealed stable vital signs, negative heart and lung sounds, and mild tenderness in the lower abdomen.

[0308] Treatment: In addition to the treatment for the control group, patients were given oral administration of the granules prepared according to the preparation method of Example 1 of this invention, one sachet per dose, three times a day. Treatment lasted for 7 weeks. A second consultation was subsequently conducted.

[0309] Second visit results: After integrated traditional Chinese and Western medicine treatment, the patient's condition has significantly improved compared to before. The patient reported no significant abdominal pain, no dry mouth or bitter taste, no radiating or pulling pain, no vaginal bleeding, no rectal tenesmus, no chills or fever, no palpitations, no chest tightness, no dizziness or weakness, no nausea or vomiting, and good mental state and sleep. Her tongue was dark with a thin white coating, and her pulse was thready. Physical examination: Vital signs were stable, heart and lungs were unremarkable, the abdomen was soft with mild tenderness, and there was no rebound tenderness or muscle guarding.

[0310] 5. Chen X, female, 27 years old, XX Traditional Chinese Medicine Hospital

[0311] Admission details: The patient was admitted on June 25, 2023 at 16:42 due to "51 days of amenorrhea and vaginal bleeding for 2+ hours". The patient presented with amenorrhea, vaginal bleeding (small amount, coffee-colored discharge), no lower abdominal pain or rectal pressure, no chills, fever, diarrhea, constipation, or other discomfort. The patient's mental state, appetite, and sleep were normal, and bowel movements were regular.

[0312] The patient presented with the main symptom of "51 days of amenorrhea and vaginal bleeding for 2+ hours," falling under the category of "fetal restlessness" in Traditional Chinese Medicine (TCM). The patient is a woman of childbearing age; during the gestation period, Yin and Yang are in harmony, and the two sexes are in accumulating, leading to pregnancy. The patient is generally weak, with kidney deficiency damaging the Chong and Ren meridians and the uterus, resulting in an unstable fetus and fetal restlessness. Kidney deficiency and instability of the Chong and Ren meridians cause blood to not return to its proper channels, hence the vaginal bleeding. A pale red tongue with a thin white coating and a thready, slippery pulse are signs of pregnancy. Considering the tongue, pulse, and symptoms, this condition is a kidney-deficiency type of threatened abortion and fetal restlessness, with the disease location in the Chong and Ren meridians and the uterus; it is a deficiency syndrome with a short course.

[0313] Treatment: Due to the patient's abdominal pain, even accompanied by vaginal bleeding, and increased vaginal bleeding on the basis of threatened abortion and fetal restlessness, combined with the patient's tongue and pulse symptoms, the patient was given oral administration of the product granules prepared according to the preparation method of Example 1 of this invention, one sachet / time, three times / day, in addition to the treatment of the control group. The medication was taken for 7 weeks. A second visit was subsequently conducted.

[0314] Second visit results: After integrated traditional Chinese and Western medicine treatment, the patient's condition has significantly improved compared to before. The patient has no vaginal bleeding, no abdominal pain, no fever, chills, abdominal distension, diarrhea, rectal tenesmus, etc. Her mental state, appetite, and sleep are good, and her bowel movements are normal. Physical examination: Vital signs are stable, and the heart, lungs, and abdomen are negative.

[0315] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A pharmaceutical composition for treating dysfunctional uterine bleeding, characterized in that, The pharmaceutical composition is composed of 10-20 parts of Radix Rumicis, 6-14 parts of Radix Salviae Miltiorrhizae, 6-14 parts of Radix Astragali, 4-12 parts of Radix Astragali seu Hedysari, and 4-12 parts of Radix Rubi.

2. The pharmaceutical composition for treating functional uterine bleeding according to claim 1, wherein The pharmaceutical composition is composed of 12-18 parts of Radix Rumicis, 8-12 parts of Radix Salviae Miltiorrhizae, 8-12 parts of Radix Astragali, 6-10 parts of Radix Astragali seu Hedysari, and 6-10 parts of Radix Rubi.

3. The pharmaceutical composition for treating functional uterine bleeding according to claim 2, characterized by, The pharmaceutical composition is composed of 15 parts of Radix Rumicis, 10 parts of Radix Salviae Miltiorrhizae, 10 parts of Radix Astragali, 8 parts of Radix Astragali seu Hedysari, and 8 parts of Radix Rubi.

4. A process for the preparation of a pharmaceutical composition as claimed in any one of claims 1 to 3, characterized in that, The preparation method is as follows: (1) ethanol extraction: according to the prescription amount, the five medicinal materials of Radix Rumicis, Radix Salviae Miltiorrhizae, Radix Astragali, Radix Astragali seu Hedysari, and Radix Rubi are weighed, 60% ethanol is added at 6-10 times the total amount of medicinal materials, and heated at 70-80°C for 1-3 times, each time for 0.5-2h, filtered, and the ethanol is recovered to obtain the ethanol extract; (2) enzyme extraction: the medicinal residue after ethanol extraction is added with 8-12 times water, 1.0-3.0% cellulase is added, and the mixture is soaked at 50°C and PH 5.0 for 1-2h, then boiled and filtered to obtain the enzyme extract; (3) concentration: the ethanol extract and the enzyme extract are combined and concentrated to a thick paste; (4) preparation of corresponding dosage form: the thick paste is added with appropriate amount of excipients for compounding, and the corresponding dosage form is prepared to obtain the product.

5. The method of claim 4, wherein the pharmaceutical composition is prepared by, The preparation method is as follows: (1) ethanol extraction: according to the prescription amount, the five medicinal materials of Radix Rumicis, Radix Salviae Miltiorrhizae, Radix Astragali, Radix Astragali seu Hedysari, and Radix Rubi are weighed, 60% ethanol is added at 8 times the total amount of medicinal materials, and heated at 75°C for 1h, filtered, and the ethanol is recovered to obtain the ethanol extract; (2) enzyme extraction: the medicinal residue after ethanol extraction is added with 10 times water, 1.0% cellulase is added, and the mixture is soaked at 50°C and PH 5.0 for 1.5h, then boiled and filtered to obtain the enzyme extract; (3) concentration: the ethanol extract and the enzyme extract are combined and concentrated to a thick paste; (4) preparation of corresponding dosage form: the thick paste is added with appropriate amount of excipients for compounding, and the corresponding dosage form is prepared to obtain the product.

6. Process for the preparation of a pharmaceutical composition according to any of claims 4 or 5, characterized in that, The excipient is a pharmaceutically acceptable excipient.

7. A process for the preparation of a pharmaceutical composition according to any of claims 4 or 5, characterized in that, The dosage form is: solid preparation and liquid preparation.

8. The pharmaceutical composition of claim 7, wherein, The solid preparation is: granules, capsules, tablets, and dripping pills.

9. The pharmaceutical composition of claim 7, wherein, The liquid preparation is: mixture and syrup.

10. The use of the pharmaceutical composition of any one of claims 1-3 or the pharmaceutical composition prepared by the preparation method of any one of claims 4-9 in the preparation of a drug for treating functional uterine bleeding.

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