A traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness and a preparation method thereof

Abstract

The application provides a preparation method of a traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type. The application is based on Linggui Zhugan Decoction, and through long-term screening of effective substances and active ingredients, each medicinal material is extracted, separated and purified in a modern scientific way, and the extract is screened in combination with pharmacology and efficacy to form a new prescription, so that a new prescription with clear material basis and more accurate and significant curative effect is obtained. Through pharmacodynamic comparative study, it is determined that the effect of the traditional Chinese medicine composition provided by the application is obviously better than that of the control group. The traditional Chinese medicine composition can achieve the purposes of tonifying the kidney and invigorating the spleen, and resolving phlegm and dampness, can obviously regulate the endocrine and metabolic levels of PCOS patients of spleen deficiency and phlegm dampness type, and significantly improve the clinical symptoms. Further, the traditional Chinese medicine composition degrades fat through multiple pathways and multiple targets, promotes the recovery of ovarian function, promotes the growth and development of follicles, induces ovulation and the like, and has a significant effect in treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine composition technology, and in particular to a traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type and its preparation method. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women of reproductive age, characterized by persistent anovulation, hyperandrogenemia, and insulin resistance. Clinically, it often manifests as obesity, acne, oligomenorrhea, or even amenorrhea. In severe cases, it can lead to infertility. While occasional ovulation may result in pregnancy, the miscarriage rate is high, and there is a risk of developing secondary diseases such as diabetes, cardiovascular disease, endometrial cancer, and breast cancer. Without timely and effective treatment, it can seriously impact a woman's health and quality of life.

[0003] Due to environmental pollution, unhealthy diets, and lifestyles, the incidence of polycystic ovary syndrome (PCOS) has been on the rise in recent years. Statistics show that the prevalence of this disease among women of reproductive age is 5%–10%. This disease is currently a hot topic in both Western and traditional Chinese medicine gynecology research. Western medicine mainly uses hormones and other drugs to regulate the menstrual cycle and induce ovulation. While Western medicine treatment for PCOS has some efficacy, it has many side effects. Hormonal drugs can increase weight, and rebound phenomena are common after intermittent use. Missed or delayed doses can cause vaginal bleeding, and in rare cases, pigmentation. Many patients find this difficult to accept and therefore seek treatment from traditional Chinese medicine (TCM). TCM treats the disease by identifying the "syndrome" rather than just the "symptom," using a holistic approach that focuses on regulating the balance of Yin and Yang, examining the symptoms to determine the underlying cause, and achieving a comprehensive cure. The basic pathogenesis of PCOS is spleen deficiency and phlegm-dampness. Fu Qingzhu's *Gynecology* believes that female infertility is closely related to spleen deficiency and phlegm-dampness.

[0004] Linggui Zhugan Decoction is a classic formula for treating spleen deficiency with phlegm-dampness, demonstrating significant clinical efficacy. Currently, Linggui Zhugan Decoction is primarily available as a decoction, although some are prepared as granules or pills. However, the processing methods are relatively outdated, relying on traditional Chinese medicine extraction methods, resulting in low levels of effective components, unclear drug activity, uncertain efficacy, slow onset of action, and large dosages. Absorption is relatively poor, and the slow effect makes it unsuitable for acute conditions, leading to poor patient compliance and numerous inconveniences. Therefore, developing new Chinese medicines for treating spleen deficiency with phlegm-dampness type polycystic ovary syndrome is a goal pursued by many medical professionals. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type and its preparation method, which has significant effects in treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0006] This invention provides a method for preparing a traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm-dampness type, comprising the following steps:

[0007] S1) Preparation of Chinese herbal extract A: Mix 1-3 parts by weight of Atractylodes macrocephala and 2-5 parts by weight of Glycyrrhiza uralensis, pulverize and steam distill to obtain volatile oil, distillate and residue;

[0008] The drug residue was mixed with the compound biological enzyme in an aqueous solution and enzymatically hydrolyzed under pH 3–5.5 conditions. After neutralization of the enzymatic hydrolysate, water extraction was performed. The extract was concentrated and combined with the distillate. After concentration, a clear extract was obtained, which was freeze-dried and pulverized to obtain drug powder Aa.

[0009] The volatile oil was dissolved in ethanol solvent, then mixed with hydroxypropyl-β-cyclodextrin solution, and a solid was precipitated. The solid was dried and pulverized to obtain drug powder Ab.

[0010] Mix the medicinal powder Aa and medicinal powder Ab to obtain the traditional Chinese medicine extract A;

[0011] S2) Preparation of Traditional Chinese Medicine Extract B:

[0012] 2-8 parts by weight of licorice were extracted with water. The extract was filtered, concentrated, and then purified by AB-8 macroporous resin and D201 ion exchange resin in sequence. The eluent was concentrated to obtain a thick paste, which was freeze-dried and pulverized to obtain the medicinal powder Ba.

[0013] 1-7 parts by weight of Poria cocos were pulverized, extracted with alcohol, and the extract and residue were obtained. The extract was concentrated to obtain a clear paste, which was freeze-dried and pulverized to obtain medicinal powder Bb. The residue was boiled with water, filtered to obtain a filtrate, which was concentrated to obtain a clear paste, freeze-dried and pulverized to obtain medicinal powder Bc.

[0014] Mixing medicinal powders Ba, Bb, and Bc yields traditional Chinese medicine extract B.

[0015] S3) Preparation of Traditional Chinese Medicine Extract C:

[0016] 3-10 parts by weight of Atractylodes macrocephala were extracted with alcohol to obtain an extract and residue. The extract was concentrated to obtain a clear paste. The clear paste was precipitated with distilled water, and then the pH value was adjusted to 1.0-2.0. After standing, centrifugation was performed, and the pH value was adjusted to 8 with sodium hydroxide. The mixture was filtered, and sulfuric acid was added to the filtrate to adjust the pH value to 1-3. Sodium carbonate was added, the mixture was left to stand, filtered, freeze-dried, and pulverized to obtain powder Ca.

[0017] The dregs were boiled in water, filtered to obtain filtrate, and the filtrate was concentrated to obtain clear extract. Ethanol was added and the pH of the system was adjusted to 5.0. After standing, the extract was concentrated under reduced pressure and the concentrate was freeze-dried to obtain powder Cb.

[0018] Mixing the powdered herbs Ca and Cb yields the herbal extract C.

[0019] S4) Preparation of Traditional Chinese Medicine Extract D:

[0020] 1-9 parts by weight of cinnamon twigs were pulverized and extracted with alcohol to obtain an extract and volatile oil. The extract was concentrated and purified with polyamide resin, eluted with water, the eluent was discarded, and then eluted with ethanol. The ethanol eluent was collected, concentrated under reduced pressure to obtain a thick paste, freeze-dried, and pulverized to obtain the medicinal powder Da.

[0021] The volatile oil was dissolved in an ethanol solution, then mixed with a hydroxypropyl-β-cyclodextrin solution to precipitate a solid. The solid was dried and pulverized to obtain the drug powder Db.

[0022] Mixing the powdered herbs Da and Db yields the herbal extract D.

[0023] Chinese herbal extracts A, B, C, and D were mixed to obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0024] The traditional Chinese medicine composition provided by this invention is based on the classic formula Linggui Zhugan Decoction. Through the enrichment, separation, and purification of the effective components of each medicinal material, and after extensive component screening research and experiments, a new prescription was finally obtained by combining effective components extracted by different methods. This led to the development of a traditional Chinese medicine with a clear material basis, a clear mechanism of action, and more accurate clinical indications. Pharmacodynamic experiments have confirmed that the traditional Chinese medicine composition prepared by this invention has significant efficacy, high bioavailability, stable quality, reliability, and effectiveness. This traditional Chinese medicine composition can be prepared in different dosage forms to meet patient needs, is convenient to carry, easy to take, and suitable for a wide range of people. The advantages of the composition obtained by this invention are: it uses extracts as medicine, the active parts are relatively complete, the effective components are clearly defined, the proportion of effective components is scientifically sound, and the material basis is clear.

[0025] This invention prepares a traditional Chinese medicine composition using Poria cocos, Atractylodes macrocephala, Cinnamomum cassia, and Glycyrrhiza uralensis.

[0026] Poria cocos: It has the effects of promoting diuresis and eliminating dampness, strengthening the spleen and calming the mind. It is used for edema with scanty urine, phlegm retention with dizziness and palpitations, spleen deficiency with poor appetite, loose stools and diarrhea, restlessness, palpitations and insomnia.

[0027] Atractylodes macrocephala: It has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. It is used for symptoms such as spleen deficiency with poor appetite, abdominal distension and diarrhea, phlegm retention, dizziness and palpitations, edema, spontaneous sweating, and threatened abortion.

[0028] Cinnamon twig: It has the effects of inducing sweating and relieving muscle tension, warming and unblocking the meridians, and assisting yang and transforming qi. It is used for wind-cold exterior syndrome, pain syndromes caused by cold stagnation and blood stasis, phlegm retention, water retention, etc.

[0029] Licorice has the effects of tonifying the spleen and replenishing qi, clearing heat and detoxifying, eliminating phlegm and relieving cough, relieving spasms and pain, and harmonizing various medicines. It is used for spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough with phlegm, abdominal and limb spasms and pain, carbuncles and boils, and to relieve the toxicity and harshness of drugs.

[0030] This invention first prepares a traditional Chinese medicine extract A by mixing 1-3 parts by weight of Atractylodes macrocephala and 2-5 parts by weight of Glycyrrhiza uralensis.

[0031] Preferably, herbal extract A is prepared by mixing 2 parts by weight of Atractylodes macrocephala and 3 parts by weight of Glycyrrhiza uralensis.

[0032] In step S1), before steam distillation, 2 parts by weight of Atractylodes macrocephala and 3 parts by weight of Glycyrrhiza uralensis are mixed, pulverized, and then soaked in 5 to 8 times the amount of water for 0.5 to 2 hours.

[0033] The preferred steam distillation time is 4 to 10 hours.

[0034] The residue is then mixed with the compound bio-enzyme in an aqueous solution. The water is preferably 4 to 9 times the weight of the residue, more preferably 7 times. The mixing temperature is preferably 39 to 50°C, more preferably 46°C.

[0035] The composite bioenzyme is preferably a mixture of cellulase, xylanase, and pectinase.

[0036] The preferred mass ratio of cellulase, xylanase, and pectinase is 3:4:(1-9), and more preferably 3:4:1.

[0037] The preferred dosage of the compound bio-enzyme is 1 wt% to 3 wt% of the medicinal residue.

[0038] The enzymatic hydrolysis time is preferably 1 to 5 hours, more preferably 2 hours.

[0039] The preferred temperature for enzymatic hydrolysis is 39–50°C, more preferably 46°C.

[0040] The pH value for the enzymatic hydrolysis is preferably 3 to 5.5, more preferably 4.5.

[0041] The preferred temperature for water extraction is 65–93°C, more preferably 80°C.

[0042] The water extraction time is preferably 1 to 3.5 hours, more preferably 1.5 hours.

[0043] Preferably, the extract after water extraction is concentrated to a density of 1.10-1.16 (60℃) and combined with the distillate.

[0044] Preferably, the combined system is concentrated under reduced pressure to a clear paste with a relative density of 1.16-1.24 (60°C).

[0045] Preferably, in the hydroxypropyl-β-cyclodextrin solution, the ratio of water volume (ml) to hydroxypropyl-β-cyclodextrin weight (g) is 7:1 to 18:1.

[0046] Preferably, the volume ratio (ml) of the volatile oil to the weight ratio (g) of the hydroxypropyl-β-cyclodextrin is 1:5 to 1:9.

[0047] Preferably, the mixing temperature with the hydroxypropyl-β-cyclodextrin solution is 35–60°C, and the mixing time is 3–5 hours.

[0048] The preferred specific step S1) is as follows:

[0049] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder. Soak in 5-8 times the amount of water for 0.5-2 hours, then steam distill for 4-10 hours. Collect the volatile oil and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 1%-3% of compound biological enzyme, adjust the pH to 4.5 with acetic acid, stir evenly, and maintain the temperature at 46℃ for 2 hours of enzymatic hydrolysis. After neutralization, raise the temperature to 80℃ and extract for 1.5 hours. Combine the extracts and concentrate to a density of 1.10-1.16 (60℃). Combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.16-1.24 (60℃), freeze-dry, and pulverize to obtain herbal powder Aa.

[0050] Step A-2: Take the volatile oil from Step A-1, dissolve it in 10-20 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) in the hydroxypropyl-β-cyclodextrin solution is 7:1-18:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:5-1:9. Stir at 35-60℃ for 3-5 hours, refrigerate at 4℃ for 24 hours, filter, vacuum dry the precipitate at 40℃ for 4-6 hours, pulverize, and obtain the drug powder Ab.

[0051] Step A-3: Mix the medicinal powder Aa and medicinal powder Ab to obtain the traditional Chinese medicine extract A.

[0052] Then, herbal extract B was prepared using licorice and poria.

[0053] Two to eight parts by weight of licorice are extracted with water. More preferably, four parts by weight of licorice are extracted.

[0054] The preferred amount of water used for water extraction is 6 to 10 times the weight of the licorice.

[0055] The preferred water extraction time is 1 to 3 hours.

[0056] The water extraction is preferably performed 2 to 4 times.

[0057] After water extraction, the extract is preferably filtered and concentrated, preferably to a solid-liquid ratio of 1:(1 to 1.9).

[0058] Then it was purified sequentially by AB-8 macroporous resin and D201 ion exchange resin.

[0059] Preferably, the concentration is 0.8–1.2 BV·h. -1 The solution was passed through AB-8 macroporous resin at a certain speed and eluted sequentially with 10%–20% ethanol aqueous solution and 40%–60% ethanol aqueous solution.

[0060] The eluent obtained from the above elution was purified using a D201 ion exchange resin. Preferably, the collected eluent had a pH of 3.4–4.1 and a loading concentration of 25–30 mg / mL. -1 The flow rate is 2.6–3.1 BV·h. -1 .

[0061] Preferably, the solution is eluted with a 25%–35% aqueous ethanol solution containing 1.5%–2.5% sodium hydroxide and a 45%–65% aqueous ethanol solution containing 2.0%–3.0% sodium hydroxide, and the eluent is collected.

[0062] Preferably, the eluent is concentrated to a thick paste with a relative density of 1.18-1.24 (60°C), freeze-dried, and pulverized to obtain the pharmaceutical powder Ba.

[0063] Then take 1 to 7 parts by weight of Poria cocos, preferably 3 parts by weight, pulverize it, and preferably extract it by reflux extraction with 5 to 9 times the amount of 65% to 85% ethanol 1 to 3 times, with the extraction time preferably 1 to 3 hours.

[0064] Preferably, after filtering the extract, the filtrates are combined, and the filtrates are concentrated to a clear extract with a relative density of 1.14-1.22 (60℃), freeze-dried, and pulverized to obtain drug powder Bb.

[0065] Then, the dregs are boiled with water. The amount of water is preferably 5 to 9 times the weight of the dregs, the number of times of boiling is preferably 1 to 3, and the boiling time is preferably 0.5 to 1.5 hours.

[0066] The filtrate was then filtered, the filtrates were combined, and concentrated to a clear extract with a relative density of 1.18-1.24 (60℃). The extract was then freeze-dried, pulverized, and the resulting powder was Bc.

[0067] Preferably, step S2) specifically comprises:

[0068] Step B-1: Take 4 parts of licorice and reflux extract 2-4 times, each time using 6-10 times its weight of water for 1-3 hours. Combine the extracts, filter, and concentrate under reduced pressure until the solid-liquid ratio is 1:1.4, at a rate of 0.8-1.2 BV·h. -1 The sample was passed through AB-8 macroporous resin and eluted sequentially with 10%–20% ethanol aqueous solution and 40%–60% ethanol aqueous solution. Each eluted fraction was then passed through D201 ion exchange resin at pH 3.4–4.1 at a loading concentration of 25–30 mg / mL. -1 Flow velocity 2.6–3.1 BV·h -1 The sample was eluted with a 25%–35% aqueous ethanol solution containing 1.5%–2.5% sodium hydroxide and a 45%–65% aqueous ethanol solution containing 2.0%–3.0% sodium hydroxide, respectively. The eluent was collected, concentrated to a thick paste with a relative density of 1.18–1.24 (60°C), freeze-dried, and pulverized to obtain the drug powder Ba.

[0069] Step B-2: Take 3 portions of Poria cocos, crush them into coarse powder, and extract them three times by reflux with 5 to 9 times the amount of 65% to 85% ethanol, each time for 1 to 3 hours. Filter the mixture, combine the filtrates, and keep the residue for later use. Concentrate the filtrate to a clear extract with a relative density of 1.14 to 1.22 (60℃), freeze-dry it, crush it, and obtain medicinal powder Bb.

[0070] Step B-3: Take the dregs from step B-2, add 5 to 9 times the amount of water and decoct 1 to 3 times, each time for 0.5 to 1.5 hours. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.18-1.24 (60℃), freeze-dry, pulverize, and obtain medicinal powder Bc.

[0071] Mixing medicinal powders Ba, Bb, and Bc yields traditional Chinese medicine extract B.

[0072] Then, the traditional Chinese medicine extract C was prepared.

[0073] Extract 3-10 parts by weight of Atractylodes macrocephala with alcohol, preferably 6 parts by weight, to obtain the extract and residue.

[0074] The ethanol used for the alcohol extraction is preferably ethanol with a concentration of 60% to 85%.

[0075] The alcohol extraction is preferably performed 1 to 3 times, and the extraction time is preferably 1 to 2 hours.

[0076] Preferably, the extract is condensed to a clear paste with a relative density of 1.16-1.24 (60°C).

[0077] The ointment was precipitated by adding distilled water, and the preferred solid-liquid ratio of the system after adding water was 1:7 to 1:20.

[0078] Then adjust the pH value to 1.0-2.0 with 2 mol / L hydrochloric acid solution, let it stand, centrifuge, add 1%-2% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 1-3, add 4%-7% sodium carbonate of the total drug solution, let it stand, filter, freeze dry, pulverize, and obtain drug powder Ca.

[0079] The dregs of the medicine are boiled in water, the amount of water used for boiling is preferably 5 to 9 times that of the dregs of the medicine, the number of times the medicine is boiled is preferably 1 to 3 times, and the boiling time is preferably 0.5 to 1.5 hours.

[0080] Preferably, the filtrate is concentrated to a clear extract with a relative density of 1.18-1.24 (60°C), ethanol is added, preferably to a concentration of 65%, the pH of the solution is adjusted to 5.0, and then allowed to stand.

[0081] The preferred settling time is 17 to 30 hours.

[0082] Preferably, step S3) specifically comprises:

[0083] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice, each time for 1-2 hours. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.16-1.24 (60℃), add distilled water for precipitation, with a solid-liquid ratio of 1:15. Adjust the pH to 1.0-2.0 with 2mol / L hydrochloric acid solution, let stand, centrifuge, add 1%-2% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 1-3, add 4%-7% sodium carbonate of the total liquid volume, let stand, filter, freeze dry, and pulverize to obtain the medicinal powder Ca.

[0084] Step C-2: Take the dregs from Step C-1, add 5 to 9 times the amount of water and decoct 1 to 3 times, each time for 0.5 to 1.5 hours. Filter, combine the filtrates, and concentrate to a clear extract with a relative density of 1.18-1.24 (60℃). Add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, and freeze-dry the concentrate to obtain powder Cb.

[0085] Mix the medicinal powders Ca and Cb to obtain the traditional Chinese medicine extract C.

[0086] Then, the herbal extract D was prepared.

[0087] 1 to 9 parts by weight of cinnamon twigs, preferably 4 parts by weight, are pulverized and extracted with alcohol to obtain an extract and volatile oil.

[0088] The ethanol used for the alcohol extraction is preferably 20% to 25% ethanol. The number of alcohol extractions is preferably 1 to 4 times. Preferably, the alcohol extraction time is 1 to 3 hours, and more preferably, the alcohol extraction is performed 3 times, with the first extraction lasting 3 hours, the second lasting 2 hours, and the third lasting 1 hour.

[0089] Preferably, the extract is concentrated to a material-to-liquid ratio of 1:3 and then purified with polyamide resin.

[0090] Preferably, the polyamide resin is a pretreated polyamide resin.

[0091] The preferred pretreatment method is to elute with 2-4 volumes of 90%-95% ethanol after column packing, followed by 1-3 volumes of 5% NaOH aqueous solution, 1-3 volumes of distilled water, and 2-3 volumes of 5%-10% acetic acid aqueous solution, and finally elute with distilled water to pH 6-6.5 for later use.

[0092] Then, preferably, elute with 3 to 5 column volumes of water, discard the eluent, and then elute with 6 to 8 column volumes of 40% to 60% ethanol. Collect the ethanol eluent, concentrate under reduced pressure, and concentrate to a thick paste with a relative density of 1.18 to 1.25 (60°C).

[0093] The volatile oil was dissolved in an ethanol solution and then mixed with a hydroxypropyl-β-cyclodextrin solution.

[0094] Preferably, in the hydroxypropyl-β-cyclodextrin solution, the ratio of water volume (ml) to hydroxypropyl-β-cyclodextrin weight (g) is 7:1 to 18:1.

[0095] Preferably, the volume ratio (ml) of the volatile oil to the weight ratio (g) of the hydroxypropyl-β-cyclodextrin is 1:5 to 1:9.

[0096] The mixing temperature is preferably 35–60°C, and the mixing time is preferably 3–5 hours.

[0097] Preferably, step S4) specifically comprises:

[0098] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 20%-25% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 3-5 column volumes of water. Discard the eluent, and then elute with 6-8 column volumes of 40%-60% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.18-1.25 (60℃). Dry the paste under cold pressure, crush it, and obtain the medicinal powder Da.

[0099] Step D-2: Take the volatile oil from step D-1, dissolve it in 10-20 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) in the hydroxypropyl-β-cyclodextrin solution is 7:1 to 18:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:5 to 1:9. Stir at 35-60℃ for 3-5 hours, refrigerate at 4℃ for 24 hours, filter, vacuum dry the precipitate at 40℃ for 4-6 hours, pulverize, and obtain drug powder Db.

[0100] Mixing the powdered medicines Da and Db yields the traditional Chinese medicine extract D.

[0101] The above-mentioned Chinese herbal extracts A, B, C, and D are mixed to obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0102] The traditional Chinese medicine extracts A, B, C, and D prepared according to the preparation method described in this invention were tested for their main active ingredients, volatile components, flavonoids, triterpenoid saponins, organic acids, and other effective substances. The above-mentioned traditional Chinese medicine extracts contain the following active ingredients: In traditional Chinese medicine extract A: component I is atractylone, and component II is glycyrrhizic acid; In traditional Chinese medicine extract B: component III is glycyrrhizin, component IV is pamoic acid, and component V is dehydrotomolic acid; In traditional Chinese medicine extract C: component VI is atractylenolide I, component VII is atractylenolide II, and component VIII is atractylenolide III; In traditional Chinese medicine extract D: component IX is cinnamaldehyde, and component X is cinnamic acid.

[0103] In herbal extract A, the content of atractylone is ≥0.8mg / g and the content of glycyrrhizic acid is ≥6.8mg / g;

[0104] In herbal extract B, the content of glycyrrhizin is ≥3.5mg / g, the content of pamoic acid is ≥2.3mg / g, and the content of dehydrotemoic acid is ≥0.7mg / g.

[0105] In herbal extract C, the total lactone content of Atractylodes macrocephala is ≥3.1 mg / g;

[0106] In the herbal extract D, the content of cinnamaldehyde is ≥1.6mg / g and the content of cinnamic acid is ≥0.8mg / g.

[0107] Preferably, the amounts of the above-mentioned Chinese herbal extracts A, B, C, and D are 1-16 parts by weight, 2-24 parts by weight, 2-15 parts by weight, and 1-20 parts by weight, respectively.

[0108] More preferably, the amount of the herbal extract A is 2 to 14 parts by weight, and even more preferably 2 parts by weight, 4 parts by weight, 6 parts by weight, 8 parts by weight, 10 parts by weight, 12 parts by weight or 14 parts by weight, or any of the above values ​​as the upper or lower limit.

[0109] More preferably, the amount of the herbal extract B is 2 to 12 parts by weight, more preferably 2 parts by weight, 4 parts by weight, 6 parts by weight, 8 parts by weight, 10 parts by weight or 12 parts by weight, or any of the above values ​​as the upper or lower limit.

[0110] More preferably, the amount of the herbal extract C is 2 to 8 parts by weight, more preferably 2 parts by weight, 4 parts by weight, 6 parts by weight or 8 parts by weight, or any of the above values ​​as the upper or lower limit.

[0111] More preferably, the amount of the herbal extract D is 1 to 6 parts by weight, more preferably 1 part by weight, 2 parts by weight, 3 parts by weight, 4 parts by weight, 5 parts by weight or 6 parts by weight, or any of the above values ​​as the upper or lower limit.

[0112] More preferably, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 2-14 parts by weight, 2-12 parts by weight, 2-8 parts by weight, and 1-6 parts by weight, respectively.

[0113] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 2 parts by weight, 6 parts by weight, 2 parts by weight, and 1 part by weight, respectively.

[0114] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 2 parts by weight, 2 parts by weight, 8 parts by weight, and 3 parts by weight, respectively.

[0115] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 2 parts by weight, 12 parts by weight, 4 parts by weight, and 6 parts by weight, respectively.

[0116] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 2 parts by weight, 2 parts by weight, 4 parts by weight, and 1 part by weight, respectively.

[0117] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 8 parts by weight, 2 parts by weight, 2 parts by weight, and 3 parts by weight, respectively.

[0118] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 14 parts by weight, 6 parts by weight, 2 parts by weight, and 1 part by weight, respectively.

[0119] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 2 parts by weight, 12 parts by weight, 2 parts by weight, and 1 part by weight, respectively.

[0120] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 2 parts by weight, 6 parts by weight, 8 parts by weight, and 3 parts by weight, respectively.

[0121] In some specific embodiments of the present invention, the amounts of the above-mentioned Chinese herbal extract A, Chinese herbal extract B, Chinese herbal extract C, and Chinese herbal extract D are 2 parts by weight, 2 parts by weight, 4 parts by weight, and 6 parts by weight, respectively.

[0122] The most preferred dosages of the above-mentioned Chinese herbal extracts A, B, C, and D are 2 parts by weight, 12 parts by weight, 4 parts by weight, and 6 parts by weight, respectively.

[0123] This invention provides a traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type, which is prepared by the above preparation method.

[0124] Compared with existing technologies, this invention provides a method for preparing a traditional Chinese medicine composition for treating polycystic ovary syndrome (PCOS) of the spleen deficiency and phlegm-dampness type. Based on Linggui Zhugan Decoction, this invention involves long-term screening of effective substances and active ingredients, extraction, separation, and purification of various medicinal materials using modern scientific methods, and component screening of the extracts combined with pharmacological efficacy to form a new prescription. This new formula has a clear material basis and more definite and significant therapeutic effects. Pharmacodynamic comparative studies have determined that the traditional Chinese medicine composition provided by this invention has significantly better effects than the control group. This composition can achieve the purpose of tonifying the kidney and spleen, resolving phlegm and eliminating dampness, and can significantly regulate the endocrine and metabolic levels of PCOS patients with spleen deficiency and phlegm-dampness type, significantly improving their clinical symptoms. Furthermore, through multi-pathway and multi-target fat degradation, it promotes ovarian function recovery, promotes follicle growth and development, and induces ovulation, demonstrating significant effects in treating PCOS of the spleen deficiency and phlegm-dampness type. Attached Figure Description

[0125] Figure 1 These are the Western blot results of p38 MAPK / NF-κB pathway proteins in rat ovarian tissue from each group. Detailed Implementation

[0126] To further illustrate the present invention, the following describes in detail, with reference to embodiments, the traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type and its preparation method.

[0127] Example 1

[0128] 1. Preparation of herbal extract A:

[0129] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 4 times the amount of water for 1 hour, steam distill for 5 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 2.5wt% (by weight of the residue) of a compound biological enzyme (cellulase:xylanase:pectinase in a mass ratio of 3:4:1), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.12 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.17 (60℃), freeze dry, and pulverize to obtain powder a.

[0130] Step A-2: Take the volatile oil from Step A-1, dissolve it in 12-20 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) in the hydroxypropyl-β-cyclodextrin solution is 7.2:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6. Stir at 38°C for 3.5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 4.5 hours, pulverize, and obtain drug powder b.

[0131] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0132] Tests showed that the main component I, atractylone, in herbal extract A was 0.82 mg / g, and the main component II, glycyrrhizic acid, was 6.41 mg / g.

[0133] 2. Preparation of herbal extract B:

[0134] Step B-1: Take 4 parts of licorice, reflux extract 3 times, each time using 7 times its weight of water for 1.5 hours, combine the extracts, filter, concentrate under reduced pressure until the solid-liquid ratio is 1:1.4, and concentrate at 0.9 BV·h -1 The sample was passed through AB-8 macroporous resin and eluted sequentially with 12% ethanol aqueous solution and 42% ethanol aqueous solution. The eluents were then passed through D201 ion exchange resin at pH 3.5 at a loading concentration of 26 mg / mL. -1 Flow velocity 2.7 BV·h -1The sample was eluted with a 26% ethanol aqueous solution containing 1.6% sodium hydroxide and a 46% ethanol aqueous solution containing 2.2% sodium hydroxide, respectively. The eluent was collected, concentrated to a thick paste with a relative density of 1.19, freeze-dried, and pulverized to obtain drug powder a.

[0135] Step B-2: Take 3 portions of Poria cocos, grind them into coarse powder, and extract them three times with 6 times the amount of 68% ethanol, each time for 1.5 hours. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.15 (60℃), freeze-dry it, and grind it to obtain powder b.

[0136] Step B-3: Take the dregs from Step B-2, add 6 times the amount of water and decoct twice, 1 hour each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.19 (60℃), freeze-dry, pulverize, and obtain powder c.

[0137] Step B-4: Mix powder a with powder b and powder c to obtain traditional Chinese medicine extract B.

[0138] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.41 mg / g, the main component IV pamoic acid was 2.28 mg / g, and the main component V dehydrotomolic acid was 0.69 mg / g.

[0139] 3. Preparation of herbal extract C:

[0140] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice, 1.5 hours each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.17 (60℃), add distilled water to precipitate, with a solid-liquid ratio of 1:15. Adjust the pH to 1.2 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 1.2% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 1.2, add 5% sodium carbonate of the total liquid volume, let stand, filter, freeze dry, and pulverize to obtain powder a.

[0141] Step C-2: Take the dregs from step C-1, add 6 times the amount of water and decoct twice, 1 hour each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.19 (60℃), add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, freeze dry the concentrate to obtain powder b.

[0142] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0143] The test results showed that the main component VI, atractylodes lactone I, in the herbal extract C was 0.89 mg / g, the main component VII, atractylodes lactone II was 0.58 mg / g, and the main component VIII, atractylodes lactone III was 1.35 mg / g.

[0144] 4. Preparation of herbal extract D:

[0145] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 21% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 3.5 column volumes of water. Discard the eluent and elute with 7 column volumes of 42% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.19 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0146] Step D-2: Take the volatile oil from step C-1, dissolve it in 12 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) in the hydroxypropyl-β-cyclodextrin solution is 7.5:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6. Stir at 37°C for 3.5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 4.5 hours, pulverize, and obtain powder b.

[0147] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0148] Tests showed that the main component IX cinnamaldehyde in the herbal extract D was 1.51 mg / g, and the main component X cinnamic acid was 0.79 mg / g.

[0149] 5. Preparation of Traditional Chinese Medicine Compositions:

[0150] Take 2 parts of the above-mentioned Chinese herbal extract A, 6 parts of Chinese herbal extract B, 2 parts of Chinese herbal extract C, and 1 part of Chinese herbal extract D, mix them evenly, and obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0151] Example 2

[0152] 1. Preparation of herbal extract A:

[0153] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 5 times the amount of water for 1 hour, steam distill for 6 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 2% of a compound biological enzyme (cellulase: xylanase: pectinase in a ratio of 3:4:3), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.13 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.19 (60℃), freeze dry, and pulverize to obtain medicinal powder a.

[0154] Step A-2: Take the volatile oil from Step A-1, dissolve it in 13 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 8:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:7. Stir at 38°C for 4 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5 hours, pulverize, and obtain powder b.

[0155] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0156] Tests showed that the main component I, atractylone, in herbal extract A was 0.78 mg / g, and the main component II, glycyrrhizic acid, was 6.17 mg / g.

[0157] 2. Preparation of herbal extract B:

[0158] Step B-1: Take 4 portions of licorice and reflux extract 4 times, each time using 8 times its weight of water for 2.5 hours. Combine the extracts, filter, concentrate under reduced pressure until the material-to-liquid ratio is 1:1.4, and pass through AB-8 macroporous resin at a rate of 1.1 BV·h-1. Elute sequentially with 13% ethanol aqueous solution and 44% ethanol aqueous solution. Each eluent is then passed through D201 ion exchange resin at pH 3.6, with a loading concentration of 27 mg·mL-1 and a flow rate of 2.8 BV·h-1, and eluted with 28% ethanol aqueous solution containing 1.7% sodium hydroxide and 49% ethanol aqueous solution containing 2.3% sodium hydroxide, respectively. Collect the eluent, concentrate to a thick paste with a relative density of 1.18 (60℃), freeze-dry, and pulverize to obtain powder a.

[0159] Step B-2: Take 3 portions of Poria cocos, grind them into coarse powder, and extract them three times with 8 times the amount of 69% ethanol for 2 hours each time. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.17 (60℃), freeze-dry it, and grind it to obtain powder b.

[0160] Step B-3: Take the dregs from Step B-2, add 7 times the amount of water and decoct 3 times, 1 hour each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.20 (60℃), freeze dry, pulverize, and obtain powder C.

[0161] Step B-4: Mix powder a with powder b and powder C to obtain traditional Chinese medicine extract B.

[0162] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.49 mg / g, the main component IV pamoic acid was 2.17 mg / g, and the main component V dehydrotomolic acid was 0.71 mg / g.

[0163] 3. Preparation of herbal extract C:

[0164] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice, 1.5 hours each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.19 (60℃), add distilled water to settle, with a solid-liquid ratio of 1:13, then adjust the pH to 1.4 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 1.4% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 1.3-3, add sodium carbonate (4.6% of the total liquid volume), let stand, filter, freeze dry, and pulverize to obtain powder a.

[0165] Step C-2: Take the dregs from Step C-1, add 7 times the amount of water and decoct twice, 1 hour each time. Filter, combine the filtrates, and concentrate to a clear extract with a relative density of 1.21 (60℃). Add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, and freeze-dry the concentrate to obtain powder b.

[0166] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0167] The test results showed that the main component VI atractylodes lactone I in the herbal extract C was 0.91 mg / g, the main component VII atractylodes lactone II was 0.62 mg / g, and the main component VIII atractylodes lactone III was 1.41 mg / g.

[0168] 4. Preparation of herbal extract D:

[0169] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 23% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 4.5 column volumes of water. Discard the eluent and elute with 6.8 column volumes of 46% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.20 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0170] Step D-2: Take the volatile oil from step C-1, dissolve it in 10 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 8:5, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6.5. Stir at 39°C for 3.5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5.5 hours, pulverize, and obtain powder b.

[0171] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0172] Tests showed that the main component IX cinnamaldehyde in the herbal extract D was 1.53 mg / g, and the main component X cinnamic acid was 0.81 mg / g.

[0173] 5. Preparation of Traditional Chinese Medicine Compositions:

[0174] Take 2 parts of Chinese herbal extract A, 2 parts of Chinese herbal extract B, 8 parts of Chinese herbal extract C, and 3 parts of Chinese herbal extract D, mix them evenly, and obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0175] Example 3

[0176] 1. Preparation of herbal extract A:

[0177] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 8 times the amount of water for 2 hours, steam distill for 10 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 3% of a compound biological enzyme (cellulase: xylanase: pectinase in a ratio of 3:4:5), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.16 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.24 (60℃), freeze dry, and pulverize to obtain medicinal powder a.

[0178] Step A-2: Take the volatile oil from Step A-1, dissolve it in 20 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 18:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:9. Stir at 60°C for 5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 6 hours, and pulverize to obtain drug powder b.

[0179] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0180] Tests showed that the main component I, atractylone, in herbal extract A was 0.86 mg / g, and the main component II, glycyrrhizic acid, was 6.83 mg / g.

[0181] 2. Preparation of herbal extract B:

[0182] Step B-1: Take 4 portions of licorice and reflux extract 4 times, each time using 10 times its weight of water for 3 hours. Combine the extracts, filter, concentrate under reduced pressure until the material-to-liquid ratio is 1:1.4, and pass through AB-8 macroporous resin at a rate of 1.2 BV·h-1. Elute sequentially with 20% ethanol aqueous solution and 60% ethanol aqueous solution. Each eluent is then passed through D201 ion exchange resin at pH 4.1, with a loading concentration of 30 mg·mL-1 and a flow rate of 3.1 BV·h-1, and eluted with 35% ethanol aqueous solution containing 2.5% sodium hydroxide and 65% ethanol aqueous solution containing 3.0% sodium hydroxide, respectively. Collect the eluent, concentrate to a thick paste with a relative density of 1.24 (60℃), freeze-dry, and pulverize to obtain powder a.

[0183] Step B-2: Take 3 portions of Poria cocos, grind them into coarse powder, and extract them three times with 9 times the amount of 85% ethanol for 3 hours each time. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.22 (60℃), freeze-dry it, and grind it to obtain powder b.

[0184] Step B-3: Take the dregs from step B-2, add 9 times the amount of water and decoct 3 times, 1.5 hours each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.24 (60℃), freeze dry, pulverize, and obtain powder c.

[0185] Step B-4: Mix powder a with powder b and powder c to obtain traditional Chinese medicine extract B.

[0186] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.54 mg / g, the main component IV poria cocos acid was 2.31 mg / g, and the main component V dehydrotomoglycic acid was 0.72 mg / g.

[0187] 3. Preparation of herbal extract C:

[0188] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice for 2 hours each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.24 (60℃), add distilled water to settle, with a solid-liquid ratio of 1:15. Adjust the pH to 2.0 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 2% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 3, add sodium carbonate (7% of the total liquid volume), let stand, filter, freeze dry, and pulverize to obtain powder a.

[0189] Step C-2: Take the dregs from step C-1, add 9 times the amount of water and decoct 3 times, 1.5 hours each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.24, add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, freeze dry the concentrate to obtain powder b.

[0190] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0191] The test results showed that the main component VI, atractylodes lactone I, in the herbal extract C was 0.93 mg / g, the main component VII, atractylodes lactone II was 0.75 mg / g, and the main component VIII, atractylodes lactone III was 1.45 mg / g.

[0192] 4. Preparation of herbal extract D:

[0193] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 25% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 5 column volumes of water. Discard the eluent and elute with 8 column volumes of 60% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.25 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0194] Step D-2: Take the volatile oil from step C-1, dissolve it in 20 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 18:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:9. Stir at 60°C for 5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 6 hours, and pulverize to obtain powder b.

[0195] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0196] Tests showed that the main component IX cinnamaldehyde in the herbal extract D was 1.61 mg / g, and the main component X cinnamic acid was 0.82 mg / g.

[0197] 5. Preparation of Traditional Chinese Medicine Compositions:

[0198] Take 2 parts of the above-mentioned Chinese herbal extract A, 12 parts of Chinese herbal extract B, 4 parts of Chinese herbal extract C, and 6 parts of Chinese herbal extract D, mix them evenly, and obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0199] Example 4

[0200] 1. Preparation of herbal extract A:

[0201] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 8 times the amount of water for 2 hours, steam distill for 10 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 2.5% of a compound biological enzyme (cellulase: xylanase: pectinase in a ratio of 3:4:2), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.14 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.18 (60℃), freeze dry, and pulverize to obtain medicinal powder a.

[0202] Step A-2: Take the volatile oil from Step A-1, dissolve it in 20 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 18:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:7.5. Stir at 55°C for 5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5.5 hours, pulverize, and obtain powder b.

[0203] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0204] Tests showed that the main component I, atractylone, in herbal extract A was 0.76 mg / g, and the main component II, glycyrrhizic acid, was 6.28 mg / g.

[0205] 2. Preparation of herbal extract B:

[0206] Step B-1: Take 4 portions of licorice and reflux extract 3 times, each time using 8 times its weight of water for 2 hours. Combine the extracts, filter, concentrate under reduced pressure until the material-to-liquid ratio is 1:1.4, and pass through AB-8 macroporous resin at a rate of 1.1 BV·h-1. Elute sequentially with 17% ethanol aqueous solution and 60% ethanol aqueous solution. Each eluent is then passed through D201 ion exchange resin at pH 4.0, with a loading concentration of 30 mg·mL-1 and a flow rate of 3.1 BV·h-1, and eluted with 31% ethanol aqueous solution containing 2.1% sodium hydroxide and 62% ethanol aqueous solution containing 3.0% sodium hydroxide, respectively. Collect the eluent, concentrate to a thick paste with a relative density of 1.24 (60℃), freeze-dry, and pulverize to obtain powder a.

[0207] Step B-2: Take 3 portions of Poria cocos, crush them into coarse powder, and extract them three times with 9 times the amount of 80% ethanol for 3 hours each time. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.18 (60℃), freeze-dry it, crush it, and obtain powder b.

[0208] Step B-3: Take the dregs from Step B-2, add 7 times the amount of water and decoct 3 times, 1 hour each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.21 (60℃), freeze-dry, pulverize, and obtain powder c.

[0209] Step B-4: Mix powder a with powder b and powder c to obtain traditional Chinese medicine extract B.

[0210] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.51 mg / g, the main component IV poria cocos acid was 2.19 mg / g, and the main component V dehydrotomoglycic acid was 0.64 mg / g.

[0211] 3. Preparation of herbal extract C:

[0212] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice for 2 hours each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.24 (60℃), add distilled water to settle, with a solid-liquid ratio of 1:15, then adjust the pH to 1.7 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 1.4% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 2, add sodium carbonate (5.5% of the total liquid volume), let stand, filter, freeze dry, and pulverize to obtain powder a.

[0213] Step C-2: Take the dregs from step C-1, add 6 times the amount of water and decoct twice, 1 hour each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.19 (60℃), add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, freeze dry the concentrate to obtain powder b.

[0214] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0215] The test results showed that the main component VI atractylodes lactone I in the herbal extract C was 0.84 mg / g, the main component VII atractylodes lactone II was 0.63 mg / g, and the main component VIII atractylodes lactone III was 1.47 mg / g.

[0216] 4. Preparation of herbal extract D:

[0217] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 24% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 4.5 column volumes of water. Discard the eluent and elute with 7.2 column volumes of 48% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.19 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0218] Step D-2: Take the volatile oil from step C-1, dissolve it in 14 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 10:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6. Stir at 55°C for 3.5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5 hours, and pulverize to obtain powder b.

[0219] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0220] Tests showed that the main component IX cinnamaldehyde in the herbal extract D was 1.59 mg / g, and the main component X cinnamic acid was 0.75 mg / g.

[0221] 5. Traditional Chinese medicine composition:

[0222] Take 2 parts of the above-mentioned Chinese herbal extract A, 2 parts of Chinese herbal extract B, 4 parts of Chinese herbal extract C, and 1 part of Chinese herbal extract D, mix them evenly, and obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0223] Example 5

[0224] 1. Preparation of herbal extract A:

[0225] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 6 times the amount of water for 1 hour, steam distill for 7.5 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 1.9% of a compound biological enzyme (cellulase: xylanase: pectinase in a ratio of 3:4:7), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.12 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.19 (60℃), freeze dry, and pulverize to obtain medicinal powder a.

[0226] Step A-2: Take the volatile oil from Step A-1, dissolve it in 16 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 11:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:7. Stir at 39°C for 4 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5 hours, and pulverize to obtain powder b.

[0227] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0228] Tests showed that the main component I, atractylone, in herbal extract A was 0.81 mg / g, and the main component II, glycyrrhizic acid, was 6.05 mg / g.

[0229] 2. Preparation of herbal extract B:

[0230] Step B-1: Take 4 portions of licorice and reflux extract 3 times, each time using 7 times its weight of water for 1 hour. Combine the extracts, filter, concentrate under reduced pressure until the material-to-liquid ratio is 1:1.4, and pass through AB-8 macroporous resin at a rate of 1.0 BV·h-1. Elute sequentially with 17% ethanol aqueous solution and 52% ethanol aqueous solution. Each eluent is then passed through D201 ion exchange resin at pH 3.9, a loading concentration of 27 mg·mL-1, and a flow rate of 2.8 BV·h-1, and eluted with 28% ethanol aqueous solution containing 1.8% sodium hydroxide and 52% ethanol aqueous solution containing 2.1% sodium hydroxide, respectively. Collect the eluent, concentrate it to a thick paste with a relative density of 1.20 (60℃), freeze-dry, and pulverize to obtain powder a.

[0231] Step B-2: Take 3 portions of Poria cocos, grind them into coarse powder, and extract them three times with 8 times the amount of 69% ethanol for 2 hours each time. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.19 (60℃), freeze-dry it, and grind it to obtain powder b.

[0232] Step B-3: Take the dregs from Step B-2, add 5 times the amount of water and decoct 3 times, 1 hour each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.22 (60℃), freeze dry, pulverize, and obtain powder c.

[0233] Step B-4: Mix powder a with powder b and powder c to obtain traditional Chinese medicine extract B.

[0234] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.61 mg / g, the main component IV poria cocos acid was 2.30 mg / g, and the main component V dehydrotomoglycic acid was 0.69 mg / g.

[0235] 3. Preparation method of traditional Chinese medicine extract C:

[0236] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice, 1 hour each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.19 (60℃), add distilled water to precipitate, with a solid-liquid ratio of 1:11. Adjust the pH to 1.7 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 1.8% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 1.9, add sodium carbonate (4.7% of the total liquid volume), let stand, filter, freeze dry, and pulverize to obtain powder a.

[0237] Step C-2: Take the dregs from step C-1, add 8 times the amount of water and decoct twice, 1 hour each time. Filter, combine the filtrates, and concentrate to a clear extract with a relative density of 1.21 (60℃). Add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, freeze-dry the concentrate to obtain powder b.

[0238] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0239] The test results showed that the main component VI, atractylodes lactone I, in the herbal extract C was 0.84 mg / g, the main component VII, atractylodes lactone II was 0.70 mg / g, and the main component VIII, atractylodes lactone III was 1.32 mg / g.

[0240] 4. Preparation method of traditional Chinese medicine extract D:

[0241] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 23% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 4.3 column volumes of water. Discard the eluent and elute with 6.7 column volumes of 49% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.19 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0242] Step D-2: Take the volatile oil from step C-1, dissolve it in 16 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 9:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:5. Stir at 42°C for 3 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5.5 hours, pulverize, and obtain powder b.

[0243] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0244] Tests showed that the main component IX cinnamaldehyde in the herbal extract D was 1.52 mg / g, and the main component X cinnamic acid was 0.70 mg / g.

[0245] 5. Traditional Chinese medicine composition:

[0246] Take 8 parts of the above-mentioned Chinese herbal extract A, 2 parts of Chinese herbal extract B, 2 parts of Chinese herbal extract C, and 3 parts of Chinese herbal extract D, mix them evenly, and obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0247] Example 6

[0248] 1. Preparation of herbal extract A:

[0249] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 6 times the amount of water for 0.5 hours, steam distill for 7 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 2% of a compound biological enzyme (cellulase: xylanase: pectinase in a ratio of 3:4:3), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.13 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.18 (60℃), freeze dry, and pulverize to obtain medicinal powder a.

[0250] Step A-2: Take the volatile oil from Step A-1, dissolve it in 16 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 12:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6. Stir at 52°C for 3.5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 4.5 hours, pulverize, and obtain powder b.

[0251] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0252] Tests showed that the main component I, atractylone, in herbal extract A was 0.78 mg / g, and the main component II, glycyrrhizic acid, was 6.72 mg / g.

[0253] 2. Preparation of herbal extract B:

[0254] Step B-1: Take 4 portions of licorice, reflux extract twice, each time using 7 times its weight of water for 1 hour, combine the extracts, filter, concentrate under reduced pressure until the material-to-liquid ratio is 1:2.5, pass through AB-8 macroporous resin at a rate of 0.9 BV·h-1, and elute sequentially with 13% ethanol aqueous solution and 55% ethanol aqueous solution. Each eluent fraction is then passed through D201 ion exchange resin at pH 3.6, loading concentration of 27 mg·mL-1, flow rate of 2.7 BV·h-1, and eluted with 30% ethanol aqueous solution containing 2.1% sodium hydroxide and 58% ethanol aqueous solution containing 2.4% sodium hydroxide, respectively. Collect the eluent, concentrate to a thick paste with a relative density of 1.21 (60℃), freeze-dry, and pulverize to obtain powder a.

[0255] Step B-2: Take 3 portions of Poria cocos, grind them into coarse powder, and extract them three times with 6 times the amount of 70% ethanol, each time for 1 hour. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.15 (60℃), freeze-dry it, and grind it to obtain powder b.

[0256] Step B-3: Take the dregs from step B-2, add 5.5 times the amount of water and decoct twice, 1 hour each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.21 (60℃), freeze-dry, pulverize, and obtain powder c.

[0257] Step B-4: Mix powder a with powder b and powder c to obtain traditional Chinese medicine extract B.

[0258] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.59 mg / g, the main component IV poria cocos acid was 2.09 mg / g, and the main component V dehydrotomolic acid was 0.65 mg / g.

[0259] 3. Preparation of herbal extract C:

[0260] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice, 1.5 hours each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.19, add distilled water to settle, with a solid-liquid ratio of 1:13. Adjust the pH to 1.6 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 1.4% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 2.2, add sodium carbonate (6.1% of the total liquid volume), let stand, filter, freeze dry, and pulverize to obtain powder a.

[0261] Step C-2: Take the dregs from Step C-1, add 5 times the amount of water and decoct once, 0.5 hours each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.18 (60℃), add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, freeze dry the concentrate to obtain powder b.

[0262] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0263] The test results showed that the main component VI, atractylodes lactone I, in the herbal extract C was 0.84 mg / g, the main component VII, atractylodes lactone II was 0.69 mg / g, and the main component VIII, atractylodes lactone III was 1.45 mg / g.

[0264] 4. Preparation of herbal extract D:

[0265] Step D-1: Take 4 portions of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 22% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:2.5. Pass the filtrate through a treated polyamide resin and elute with 3.5 column volumes of water. Discard the eluent and elute with 6.5 column volumes of 51% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.20 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0266] Step D-2: Take the volatile oil from step C-1, dissolve it in 13 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 13:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6. Stir at 42°C for 3 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5 hours, and pulverize to obtain drug powder b.

[0267] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0268] Tests showed that the main component IX cinnamaldehyde in the herbal extract D was 1.54 mg / g, and the main component X cinnamic acid was 0.80 mg / g.

[0269] 5. Traditional Chinese medicine composition:

[0270] Take 14 parts of the above-mentioned Chinese herbal extract A, 6 parts of Chinese herbal extract B, 2 parts of Chinese herbal extract C, and 1 part of Chinese herbal extract D, mix them evenly, and obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0271] Example 7

[0272] 1. Preparation of herbal extract A:

[0273] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 7 times the amount of water for 1.5 hours, steam distill for 8 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 2.5% of a compound biological enzyme (cellulase: xylanase: pectinase in a ratio of 3:4:4), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.11 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.17 (60℃), freeze dry, and pulverize to obtain medicinal powder a.

[0274] Step A-2: Take the volatile oil from Step A-1, dissolve it in 14 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 13:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6. Stir at 48°C for 5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5 hours, and pulverize to obtain drug powder b.

[0275] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0276] Tests showed that the main component I, atractylone, in herbal extract A was 0.82 mg / g, and the main component II, glycyrrhizic acid, was 6.28 mg / g.

[0277] 2. Preparation of herbal extract B:

[0278] Step B-1: Take 4 portions of licorice, reflux extract twice, each time using 6 times its weight of water for 2 hours, combine the extracts, filter, concentrate under reduced pressure until the material-to-liquid ratio is 1:1.4, pass through AB-8 macroporous resin at a rate of 1.1 BV·h-1, and elute sequentially with 17% ethanol aqueous solution and 54% ethanol aqueous solution. Each eluent fraction is then passed through D201 ion exchange resin at pH 3.9, loading concentration of 28 mg·mL-1, flow rate of 2.9 BV·h-1, and eluted with 30% ethanol aqueous solution containing 2.2% sodium hydroxide and 58% ethanol aqueous solution containing 2.5% sodium hydroxide, respectively. Collect the eluent, concentrate to a thick paste with a relative density of 1.19 (60℃), freeze-dry, and pulverize to obtain powder a.

[0279] Step B-2: Take 3 portions of Poria cocos, grind them into coarse powder, and extract them three times with 6 times the amount of 83% ethanol, each time for 1 hour. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.18 (60℃), freeze-dry it, and grind it to obtain powder b.

[0280] Step B-3: Take the dregs from Step B-2, add 6 times the amount of water and decoct once, 1 hour each time, filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.21 (60℃), freeze dry, pulverize, and obtain powder c.

[0281] Step B-4: Mix powder a with powder b and powder c to obtain traditional Chinese medicine extract B.

[0282] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.47 mg / g, the main component IV pamoic acid was 2.14 mg / g, and the main component V dehydrotomolic acid was 0.57 mg / g.

[0283] 3. Preparation of herbal extract C:

[0284] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice, 2 hours each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.20 (60℃), add distilled water for precipitation, with a solid-liquid ratio of 1:15. Adjust the pH to 1.0-2.0 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 1.8% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 2.4, add sodium carbonate (4.3% of the total liquid volume), let stand, filter, freeze dry, and pulverize to obtain powder a.

[0285] Step C-2: Take the dregs from step C-1, add 8 times the amount of water and decoct twice, 0.5 hours each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.19 (60℃), add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, freeze dry the concentrate to obtain powder b.

[0286] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0287] The test results showed that the main component VI, atractylodes lactone I, in the herbal extract C was 0.86 mg / g, the main component VII, atractylodes lactone II was 0.69 mg / g, and the main component VIII, atractylodes lactone III was 1.49 mg / g.

[0288] 4. Preparation method of traditional Chinese medicine extract D:

[0289] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 24% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 3.5 column volumes of water. Discard the eluent and elute with 7.5 column volumes of 47% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.22 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0290] Step D-2: Take the volatile oil from step C-1, dissolve it in 13 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 16:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6. Stir at 54°C for 4 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 4.5 hours, pulverize, and obtain powder b.

[0291] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0292] Tests showed that the main component IX cinnamaldehyde in the herbal extract D was 1.55 mg / g, and the main component X cinnamic acid was 0.72 mg / g.

[0293] 5. Traditional Chinese medicine composition:

[0294] Take 2 parts of the above-mentioned Chinese herbal extract A, 12 parts of Chinese herbal extract B, 2 parts of Chinese herbal extract C, and 1 part of Chinese herbal extract D, mix them evenly, and obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0295] Example 8

[0296] 1. Preparation of herbal extract A:

[0297] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 6 times the amount of water for 1.5 hours, steam distill for 7 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 1.5% of a compound biological enzyme (cellulase: xylanase: pectinase in a ratio of 3:4:8), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.16 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.24 (60℃), freeze dry, and pulverize to obtain medicinal powder a.

[0298] Step A-2: Take the volatile oil from Step A-1, dissolve it in 14 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 14:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6.5. Stir at 35°C for 3 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5.5 hours, pulverize, and obtain powder b.

[0299] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0300] Tests showed that the main component I, atractylone, in herbal extract A was 0.79 mg / g, and the main component II, glycyrrhizic acid, was 6.35 mg / g.

[0301] 2. Preparation of herbal extract B:

[0302] Step B-1: Take 4 portions of licorice and reflux extract 3 times, each time using 7 times its weight of water for 2.5 hours. Combine the extracts, filter, concentrate under reduced pressure until the material-to-liquid ratio is 1:1.2, and pass through AB-8 macroporous resin at a rate of 1.1 BV·h-1. Elute sequentially with 14% ethanol aqueous solution and 50% ethanol aqueous solution. Each eluent is then passed through D201 ion exchange resin at pH 3.8, a loading concentration of 29 mg·mL-1, and a flow rate of 3.0 BV·h-1, and eluted with 29% ethanol aqueous solution containing 2.1% sodium hydroxide and 46% ethanol aqueous solution containing 2.4% sodium hydroxide, respectively. Collect the eluent, concentrate it to a thick paste with a relative density of 1.19 (60℃), freeze-dry, and pulverize to obtain powder a.

[0303] Step B-2: Take 3 portions of Poria cocos, grind them into coarse powder, and extract them three times with 5 times the amount of 68% ethanol for 3 hours each time. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.21 (60℃), freeze-dry it, and grind it to obtain powder b.

[0304] Step B-3: Take the dregs from step B-2, add 5 times the amount of water and decoct once, 1.5 hours each time, filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.24 (60℃), freeze dry, pulverize, and obtain powder c.

[0305] Step B-4: Mix powder a with powder b and powder c to obtain traditional Chinese medicine extract B.

[0306] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.42 mg / g, the main component IV poria cocos acid was 2.26 mg / g, and the main component V dehydrotomoglycic acid was 0.63 mg / g.

[0307] 3. Preparation of herbal extract C:

[0308] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice, 1 hour each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.22 (60℃), add distilled water to settle, with a solid-liquid ratio of 1:15, then adjust the pH to 1.5 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 1.5% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 1.8, add sodium carbonate (4.7% of the total liquid volume), let stand, filter, freeze dry, and pulverize to obtain powder a.

[0309] Step C-2: Take the dregs from Step C-1, add 7 times the amount of water and decoct twice, 1.0 hour each time. Filter, combine the filtrates, and concentrate to a clear extract with a relative density of 1.23 (60℃). Add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, and freeze-dry the concentrate to obtain powder b.

[0310] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0311] The test results showed that the main component VI, atractylodes lactone I, in the herbal extract C was 0.87 mg / g, the main component VII, atractylodes lactone II was 0.67 mg / g, and the main component VIII, atractylodes lactone III was 1.41 mg / g.

[0312] 4. Preparation of herbal extract D:

[0313] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 22% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 5 column volumes of water. Discard the eluent and elute with 6.5 column volumes of 58% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.19 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0314] Step D-2: Take the volatile oil from step C-1, dissolve it in 16 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 13:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:6. Stir at 42°C for 3-5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 6 hours, and pulverize to obtain powder b.

[0315] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0316] Tests showed that the main component IX cinnamaldehyde in the herbal extract D was 1.51 mg / g, and the main component X cinnamic acid was 0.74 mg / g.

[0317] 5. Traditional Chinese medicine composition:

[0318] Take 2 parts of the above-mentioned Chinese herbal extract A, 6 parts of Chinese herbal extract B, 8 parts of Chinese herbal extract C, and 3 parts of Chinese herbal extract D, mix them evenly, and obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type.

[0319] Example 9

[0320] 1. Preparation of herbal extract A:

[0321] Step A-1: ​​Crush 2 parts of Atractylodes macrocephala and 3 parts of Glycyrrhiza uralensis into coarse powder, soak in 5 times the amount of water for 1.5 hours, steam distill for 7 hours, collect the volatile oil, and reserve the distillate. Add 7 times the amount of water to the residue, raise the temperature to 46℃, add 1.3% of a compound biological enzyme (cellulase: xylanase: pectinase in a ratio of 3:4:3), adjust the pH to 4.5 with acetic acid, stir evenly, maintain 46℃, enzymatically hydrolyze for 2 hours, neutralize, raise the temperature to 80℃, extract for 1.5 hours, combine the extracts, concentrate to a density of 1.14 (60℃), combine with the distillate, concentrate under reduced pressure to a clear extract with a relative density of 1.20 (60℃), freeze dry, and pulverize to obtain medicinal powder a.

[0322] Step A-2: Take the volatile oil from Step A-1, dissolve it in 15 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 15:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:8. Stir at 56°C for 5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 5 hours, and pulverize to obtain drug powder b.

[0323] Step A-3: Mix powder a and powder b thoroughly to obtain herbal extract A;

[0324] Tests showed that the main component I, atractylone, in herbal extract A was 0.77 mg / g, and the main component II, glycyrrhizic acid, was 6.31 mg / g.

[0325] 2. Preparation of herbal extract B:

[0326] Step B-1: Take 4 portions of licorice and reflux extract 4 times, each time using 10 times its weight of water for 3 hours. Combine the extracts, filter, concentrate under reduced pressure until the material-to-liquid ratio is 1:1.4, and pass through AB-8 macroporous resin at a rate of 1.0 BV·h-1. Elute sequentially with 19% ethanol aqueous solution and 52% ethanol aqueous solution. Each eluent is then passed through D201 ion exchange resin at pH 3.9, with a loading concentration of 28 mg·mL-1 and a flow rate of 3.0 BV·h-1, and eluted with 30% ethanol aqueous solution containing 1.9% sodium hydroxide and 51% ethanol aqueous solution containing 2.7% sodium hydroxide, respectively. Collect the eluent, concentrate to a thick paste with a relative density of 1.23 (60℃), freeze-dry, and pulverize to obtain powder a.

[0327] Step B-2: Take 3 portions of Poria cocos, grind them into coarse powder, and extract them three times with 7 times the amount of 69% ethanol, each time for 2 hours. Filter the mixture, combine the filtrates, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.17 (60℃), freeze-dry it, and grind it to obtain powder b.

[0328] Step B-3: Take the dregs from step B-2, add 7 times the amount of water and decoct 3 times, 1.5 hours each time. Filter, combine the filtrates, concentrate to a clear extract with a relative density of 1.18 (60℃), freeze dry, pulverize, and obtain powder c.

[0329] Step B-4: Mix powder a with powder b and powder c to obtain traditional Chinese medicine extract B.

[0330] The test results showed that the main component III glycyrrhizin in the herbal extract B was 3.46 mg / g, the main component IV pamoic acid was 2.07 mg / g, and the main component V dehydrotomolic acid was 0.68 mg / g.

[0331] 3. Preparation of herbal extract C:

[0332] Step C-1: Take 6 portions of Atractylodes macrocephala, add 75% ethanol and reflux twice, 1 hour each time. Combine the extracts, filter, and set aside the residue. Concentrate the filtrate to a clear extract with a relative density of 1.21 (60℃), add distilled water to settle, with a solid-liquid ratio of 1:15. Adjust the pH to 1.4 with 2 mol / L hydrochloric acid solution, let stand, centrifuge, add 1.7% sodium hydroxide to adjust the pH to 8, filter, add sulfuric acid to the filtrate to adjust the pH to 2.5, add sodium carbonate (6.2% of the total liquid volume), let stand, filter, freeze dry, and pulverize to obtain powder a.

[0333] Step C-2: Take the dregs from step C-1, add 8 times the amount of water and decoct twice, 1 hour each time. Filter, combine the filtrates, and concentrate to a clear extract with a relative density of 1.21 (60℃). Add ethanol to make the ethanol concentration 65%, adjust the pH of the decoction to 5.0, let stand for 24 hours, recover the ethanol, concentrate under reduced pressure, freeze-dry the concentrate to obtain powder b.

[0334] Step C-3: Mix powder a and powder b thoroughly to obtain traditional Chinese medicine extract C;

[0335] The test results showed that the main component VI, atractylodes lactone I, in the herbal extract C was 0.83 mg / g, the main component VII, atractylodes lactone II was 0.61 mg / g, and the main component VIII, atractylodes lactone III was 1.48 mg / g.

[0336] 4. Preparation of herbal extract D:

[0337] Step D-1: Take 4 parts of cinnamon twigs, crush them into coarse powder, and extract them 3 times with 24% ethanol (collecting volatile oil at the same time). The first extraction takes 3 hours, the second takes 2 hours, and the third takes 1 hour. Combine the extracts, filter them, and concentrate the filtrate to a material-to-liquid ratio of 1:3. Pass the filtrate through a treated polyamide resin and elute with 3 column volumes of water. Discard the eluent and elute with 6 column volumes of 60% ethanol. Collect the ethanol eluent, concentrate it under reduced pressure, and concentrate it to a thick paste with a relative density of 1.24 (60℃). Dry the paste under cold pressure, crush it, and obtain powder a.

[0338] Step D-2: Take the volatile oil from step C-1, dissolve it in 11 ml of anhydrous ethanol, and add it dropwise to the hydroxypropyl-β-cyclodextrin solution. The volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 9.5:1, and the volume ratio of volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:9. Stir at 54°C for 3.5 hours, refrigerate at 4°C for 24 hours, filter, vacuum dry the precipitate at 40°C for 4.5 hours, pulverize, and obtain powder b.

[0339] Step D-3: Mix powder a and powder b to obtain traditional Chinese medicine extract D.

[0340] After detection, the main component IX, cinnamaldehyde, in the traditional Chinese medicine extract D was 1.62 mg / g, and the main component X, cinnamic acid, was 0.78 mg / g.

[0341] 5. Traditional Chinese medicine composition:

[0342] Take 2 parts of the above-mentioned traditional Chinese medicine extract A, 2 parts of traditional Chinese medicine extract B, 4 parts of traditional Chinese medicine extract C, and 6 parts of traditional Chinese medicine extract D, mix them evenly to obtain a traditional Chinese medicine composition for treating polycystic ovary syndrome with spleen deficiency and phlegm dampness.

[0343] Pharmacodynamic comparison experimental study of nine groups of extracts

[0344] 1.1 Test substances

[0345] The traditional Chinese medicine composition was prepared from the above-mentioned examples.

[0346] 1.2 Animals

[0347] 80 Wistar rats, half male and half female, were of SPF grade. The animal production license number was SCXK(Ji)-2020-0002. The SPF rat maintenance feed was provided by Changchun Yisi Experimental Animal Technology Co., Ltd.

[0348] 1.3 Reagents

[0349] Follicle-stimulating hormone (FSH) kit, luteinizing hormone (LH) kit, testosterone (T) kit, tumor necrosis factor α (TNF-α) kit, interleukin 6 (IL-6) kit, purchased from Nanjing Jiancheng Bioengineering Co., Ltd.

[0350] 2 Experimental methods

[0351] 2.1 Construction and grouping of polycystic ovary syndrome (PCOS) model

[0352] Establishment of PCOS model: Rats were subcutaneously injected with dehydroepiandrosterone at 60 mg / kg (diluted with 0.2 mL injection oil), once a day. After 28 days of injection, the vaginal smear of rats was observed continuously for 4 days (one estrous cycle). If the vaginal epithelium of rats showed continuous keratinization and lost the estrous cycle, it indicated that the model was successfully established. The 66 successfully modeled rats were randomly divided into a model group, a CN112294869A preparation group (15.0 g crude drug / kg), and traditional Chinese medicine composition groups I - IX (15.0 g crude drug / kg), with 6 rats in each group. Another 6 rats were subcutaneously injected with 60 mg / kg normal saline as the blank group. On the first day after successful modeling, the rats in each administration group were given intragastric administration, and the intragastric volume was 10 mL / kg; the rats in the blank group and the model group were given intragastric administration of an equal volume of normal saline; each group was intervened once a day for 28 consecutive days.

[0353] 2.2 Detection of serum markers in rats

[0354] After administration, the patient was kept on a fasting diet but allowed free access to water for 24 hours. The next day, the patient was anesthetized with sodium pentobarbital and blood was drawn from the abdominal aorta. After centrifugation at 4000 r / min for 15 min, the supernatant was collected. The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured according to the ELISA kit instructions.

[0355] 3 Experimental Results

[0356] 3.1 Comparison of serum levels of FSH, LH, T, TNF-α and IL-6 in each group

[0357] Compared with the control group, the serum levels of LH, T, TNF-α, and IL-6 in the model group were significantly increased, while the FSH level was significantly decreased (P<0.001). Compared with the model group, the serum levels of LH, T, TNF-α, and IL-6 in each treatment group were decreased to varying degrees, while the FSH level was increased to varying degrees (P<0.01, P<0.001). Compared with the CN112294869A preparation group, the serum levels of LH and TNF-α in the traditional Chinese medicine composition II group were decreased to varying degrees (P<0.01), while the serum levels of LH, T, TNF-α, and IL-6 in the traditional Chinese medicine composition III group were decreased to varying degrees, while the FSH level was significantly increased (P<0.05). (P<0.01, P<0.001) Serum levels of LH, T, TNF-α, and IL-6 in group IV of traditional Chinese medicine composition decreased to varying degrees (P<0.05, P<0.01); serum levels of LH and T in group V of traditional Chinese medicine composition decreased to varying degrees (P<0.05, P<0.01); serum levels of LH, T, and TNF-α in group VI of traditional Chinese medicine composition decreased significantly (P<0.05); serum levels of TNF-α in group VII of traditional Chinese medicine composition decreased significantly (P<0.01); serum levels of LH in group VIII of traditional Chinese medicine composition decreased extremely significantly (P<0.001); and serum levels of TNF-α in group IX of traditional Chinese medicine composition decreased significantly (P<0.01). See Table 1 for details.

[0358] Table 1 Comparison of serum levels of FSH, LH, T, TNF-α, and IL-6 in each group

[0359]

[0360]

[0361] Note: The formulation group in CN112294869A is prepared by the method disclosed in Example 2 of CN112294869A;

[0362] Compared with the blank group, ***P < 0.001; compared with the model group, ## P < 0.01, ### P < 0.001; compared with the CN112294869 preparation group, ▲ P < 0.05, ▲▲ P < 0.01, ▲▲▲ P < 0.001.

[0363] 3.2 Comparison of the expression of p38 MAPK / NF-κB pathway proteins in ovarian tissues of each group

[0364] Compared with the blank group, the expression of p38 MAPK and NF-κB p65 proteins in the ovarian tissues of the model group increased; compared with the model group, the expression of p38 MAPK and NF-κB p65 proteins in the ovarian tissues of each drug administration group decreased significantly; compared with the CN112294869A preparation group, the expression of p38 MAPK and NF-κB p65 proteins in the traditional Chinese medicine composition III group decreased.

[0365] The results are shown in Figure 1 . Figure 1 It is the Western blot results of the p38 MAPK / NF-κB pathway proteins in the ovarian tissues of rats in each group. Among them, CK: blank group; M: model group; PC: CN112294869A preparation group; I-IX: traditional Chinese medicine composition I-IX groups.

[0366] Conclusion:

[0367] Traditional Chinese medicine composition III promotes the recovery of sex hormone levels and improves ovarian function in PCOS rats by inhibiting the p38 MAPK / NF-κB inflammatory pathway and reducing the expression of inflammatory factors, and its effect is better than that of CN112294869A preparation and other traditional Chinese medicine compositions.

[0368] According to the results of the pharmacodynamic comparison experiment research, traditional Chinese medicine composition III was screened out to carry out the overall pharmacodynamic research.

[0369] Overall pharmacodynamic research

[0370] 1.1 Test substances

[0371] The traditional Chinese medicine composition was prepared by the above Example 3.

[0372] 1.2 Animals

[0373] 42 Wistar rats, half male and half female, were of SPF grade. The animal production license number was SCXK(Ji)-2020-0002. The SPF rat maintenance feed was provided by Changchun Yisi Experimental Animal Technology Co., Ltd.

[0374] 1.3 Reagents

[0375] Follicle-stimulating hormone (FSH) kit, luteinizing hormone (LH) kit, testosterone (T) kit, tumor necrosis factor α (TNF-α) kit, and interleukin-6 (IL-6) kit were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0376] 2 Experimental Methods

[0377] 2.1 Effects on hormones and inflammatory factors in a rat model of polycystic ovary syndrome

[0378] 2.1.1 Construction and grouping of polycystic ovary syndrome (PCOS) model

[0379] PCOS model establishment: Rats were subcutaneously injected with dehydroepiandrosterone (DHEA) 60 mg / kg (diluted with 0.2 mL injection oil) once daily for 28 days. Vaginal smears were observed for 4 consecutive days (one estrous cycle) after 28 days of injection. Successful model establishment was indicated by persistent keratinization of the vaginal epithelium and loss of estrous cycles. Thirty-six successfully modeled rats were randomly divided into three groups: the model group, the Dingkun Pill group (2.2 g / kg), the CN112294869A preparation group (15.0 g crude drug / kg), and high, medium, and low dose groups of the traditional Chinese medicine composition (30.0, 15.0, and 7.5 g crude drug / kg, respectively), with six rats in each group. Six rats were subcutaneously injected with 60 mg / kg physiological saline as a control group. On the first day after successful modeling, rats in the Dingkun Pill group, CN112294869A preparation group, and high, medium, and low dose groups of traditional Chinese medicine composition were administered the drugs by gavage, with a gavage volume of 10 mL / kg. Rats in the blank group and model group were administered an equal volume of physiological saline by gavage. All groups were treated once a day for 28 consecutive days.

[0380] 2.1.2 Detection of serum markers in rats

[0381] After administration, the patient was kept on a fasting diet but allowed free access to water for 24 hours. The next day, the patient was anesthetized with sodium pentobarbital and blood was drawn from the abdominal aorta. After centrifugation at 4000 r / min for 15 min, the supernatant was collected. The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured according to the ELISA kit instructions.

[0382] 2.2 Effects on T lymphocyte proliferation

[0383] Rat spleens were harvested, ground on sterile petri dishes, and passed through a 100-mesh sieve to obtain single spleen cells. These cells were then added to a lymphocyte separation medium with a specific gravity of 1.08 and centrifuged using a density gradient at 2000 rpm for 20 minutes. The intermediate cloud-like cell layer was collected to obtain mononuclear cells. The cells were washed twice with RPMI-1640 incomplete nutrient solution, centrifuged at 1000 rpm for 10 minutes, and then prepared with complete nutrient solution to a concentration of 5 × 10⁶ cells / mL. 6Cells / ml. 1 ml of the solution was added to each well of a 24-well cell culture plate (5 × 10⁶ cells / ml). ConA was added to a final concentration of 5 μg / ml. The plate was incubated at 37°C with 5% CO₂ for 48 hours. Then, 100 μl of MTT solution (0.5 mg / ml) was added to each well, and the plate was incubated for another 12 hours. The plate was removed, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. 1 ml of DMSO was added to each well, and the plate was shaken thoroughly for 30 s. After standing for 20 min, the cells were analyzed at 560 nm. The stimulation index was calculated using a formula to determine lymphocyte proliferation.

[0384]

[0385] 3 Experimental Results

[0386] 3.1 Comparison of serum levels of FSH, LH, T, TNF-α and IL-6 in each group

[0387] Compared with the control group, the serum levels of LH, T, TNF-α, and IL-6 in the model group were significantly increased, while the FSH level was significantly decreased (P<0.001). Compared with the model group, the serum levels of LH, T, TNF-α, and IL-6 in each treatment group were decreased to varying degrees, while the FSH level was increased to varying degrees (P<0.01, P<0.001). Compared with the CN112294869A preparation group, the serum levels of LH, T, TNF-α, and IL-6 in the high-dose group of the traditional Chinese medicine composition were decreased to varying degrees (P<0.05, P<0.01), while the FSH level was significantly increased (P<0.001). The serum levels of TNF-α and IL-6 in the medium-dose group of the traditional Chinese medicine composition were significantly decreased (P<0.05), while the FSH level was significantly increased (P<0.05). See Table 2 for details.

[0388] Table 2 Comparison of serum levels of FSH, LH, T, TNF-α, and IL-6 in each group

[0389]

[0390] Note: Compared with the blank group, *** P < 0.001; compared with the model group, ## P<0.01, ### P<0.001; compared with the Dingkun Pill group,

[0391] P<0.01; compared with the CN112294869A formulation group, ▲ P<0.05, ▲▲ P<0.01, ▲▲▲ P<0.001.

[0392] 3.2 Effects on splenic lymphocyte proliferation

[0393] Compared with the blank control group, the stimulation index of the model control group was significantly reduced (P<0.001), indicating that ConA-induced proliferation of mouse lymphocytes was significantly inhibited. Compared with the model group, the stimulation index of each treatment group increased to varying degrees (P<0.05, P<0.01, P<0.001). Compared with the CN112294869A preparation group, the stimulation index of the high-dose group of the traditional Chinese medicine composition was significantly increased (P<0.05). See Table 3 for details.

[0394] Table 3 Effects on lymphocyte proliferation

[0395]

[0396] Note: Compared with the blank group, *** P < 0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001; compared with the CN112294869A formulation group, ▲ P<0.05.

[0397] in conclusion

[0398] The above experiments show that the traditional Chinese medicine composition provided by the present invention can effectively improve the sex hormone levels and inflammatory factor expression in polycystic ovary syndrome model rats, enhance the proliferation capacity of spleen cells under ConA stimulation, and thus regulate immunity.

[0399] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm-dampness type, comprising the following steps: S1) Preparation of Chinese herbal extract A: Mix 2 parts by weight of Atractylodes macrocephala and 3 parts by weight of Glycyrrhiza uralensis, pulverize and steam distill to obtain volatile oil, distillate and residue; The drug residue was mixed with the compound biological enzyme in an aqueous solution and enzymatically hydrolyzed under pH 3-5.5 conditions. After neutralization of the enzymatic hydrolysate, water extraction was performed. The extract was concentrated and combined with the distillate. After concentration, a clear extract was obtained, which was freeze-dried and pulverized to obtain drug powder Aa. The volatile oil was dissolved in ethanol solvent, then mixed with hydroxypropyl-β-cyclodextrin solution, and a solid was precipitated. The solid was dried and pulverized to obtain drug powder Ab. Mix the medicinal powder Aa and medicinal powder Ab to obtain the traditional Chinese medicine extract A; S2) Preparation of Traditional Chinese Medicine Extract B: Four parts by weight of licorice were extracted with water. The extract was filtered, concentrated, and then purified sequentially through AB-8 macroporous resin and D201 ion exchange resin. The eluent was concentrated to obtain a thick paste, which was freeze-dried and pulverized to obtain the medicinal powder Ba. The purification process using AB-8 macroporous resin and D201 ion exchange resin specifically involved using 0.8–1.2 BV•h. -1 The sample was passed through AB-8 macroporous resin and eluted sequentially with 10%–20% ethanol aqueous solution and 40%–60% ethanol aqueous solution. The eluent was purified by D201 ion exchange resin. The collected eluent had a pH of 3.4–4.1, and the loading concentration was 25–30 mg / mL. -1 The flow rate is 2.6–3.1 BV•h. -1 Elute with 25%–35% aqueous ethanol solution containing 1.5%–2.5% sodium hydroxide and 45%–65% aqueous ethanol solution containing 2.0%–3.0% sodium hydroxide, respectively, and collect the eluent. Three parts by weight of Poria cocos were pulverized and extracted with 65% to 85% ethanol to obtain an extract and residue. The extract was concentrated to obtain a clear paste, which was freeze-dried and pulverized to obtain powder Bb. The residue was boiled with water, filtered to obtain a filtrate, which was concentrated to obtain a clear paste, freeze-dried and pulverized to obtain powder Bc. Mixing medicinal powders Ba, Bb, and Bc yields traditional Chinese medicine extract B. S3) Preparation of Traditional Chinese Medicine Extract C: Six parts by weight of Atractylodes macrocephala were extracted with ethanol to obtain an extract and residue; the ethanol used for the extraction of Atractylodes macrocephala was 60%~85% ethanol; the extract was concentrated to obtain a clear extract, the clear extract was precipitated with distilled water, then the pH value was adjusted to 1.0~2.0, allowed to stand, centrifuged, and then the pH value was adjusted to 8 with sodium hydroxide, filtered, sulfuric acid was added to the filtrate to adjust the pH value to 1~3, sodium carbonate was added, allowed to stand, filtered, freeze-dried, and pulverized to obtain medicinal powder Ca; The dregs were boiled in water, filtered to obtain filtrate, and the filtrate was concentrated to obtain clear extract. Ethanol was added and the pH of the system was adjusted to 5.

0. After standing, the extract was concentrated under reduced pressure and the concentrate was freeze-dried to obtain powder Cb. Mixing the powdered herbs Ca and Cb yields the herbal extract C. S4) Preparation of Traditional Chinese Medicine Extract D: Four parts by weight of cinnamon twigs were pulverized and extracted with alcohol to obtain an extract and volatile oil. The alcohol used for the extraction was 20% to 25% ethanol. The extract was concentrated and purified with polyamide resin, eluted with water, the eluent was discarded, and then eluted with ethanol. The ethanol eluent was collected, concentrated under reduced pressure to obtain a thick paste, freeze-dried, and pulverized to obtain the medicinal powder Da. The volatile oil was dissolved in an ethanol solution, then mixed with a hydroxypropyl-β-cyclodextrin solution to precipitate a solid. The solid was dried and pulverized to obtain the drug powder Db. Mixing the powdered herbs Da and Db yields the herbal extract D. Chinese herbal extracts A, B, C, and D are mixed to obtain a Chinese herbal composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type. The dosages of the above-mentioned Chinese herbal extracts A, B, C, and D are 2 parts by weight, 2 parts by weight, 8 parts by weight, 3 parts by weight, or 2 parts by weight, 12 parts by weight, 4 parts by weight, 6 parts by weight, or 2 parts by weight, 2 parts by weight, 4 parts by weight, 1 part by weight, or 8 parts by weight, 2 parts by weight, 2 parts by weight, 3 parts by weight, or 14 parts by weight, 6 parts by weight, 2 parts by weight, 1 part by weight, or 2 parts by weight, 12 parts by weight, 2 parts by weight, 1 part by weight, or 2 parts by weight, 6 parts by weight, 8 parts by weight, 3 parts by weight, or 2 parts by weight, 2 parts by weight, 4 parts by weight, 6 parts by weight. The composite bioenzyme is a mixture of cellulase, xylanase, and pectinase; The mass ratio of cellulase, xylanase and pectinase is 3:4:(1~9); The enzymatic hydrolysis time is 1-5 hours; The enzymatic hydrolysis temperature is 39~50℃; When the medicinal residue and the compound bio-enzyme are mixed in an aqueous solution, the weight of the water is 4 to 9 times that of the medicinal residue; the mixing temperature is 39 to 50°C; and the amount of the compound bio-enzyme is 1 wt% to 3 wt% of the medicinal residue.

2. The preparation method according to claim 1, characterized in that, In step S1), before steam distillation, 2 parts by weight of Atractylodes macrocephala and 3 parts by weight of Glycyrrhiza uralensis are mixed, pulverized, and then soaked in 5 to 8 times the amount of water for 0.5 to 2 hours; the steam distillation time is 4 to 10 hours. The enzymatic hydrolysis time is 1-5 hours; the enzymatic hydrolysis temperature is 39-50℃; The water extraction temperature is 65~93℃; the water extraction time is 1~3.5 hours; After water extraction, the extract is concentrated to a density of 1.10-1.16 and combined with the distillate; the combined system is then concentrated under reduced pressure to a clear extract with a relative density of 1.16-1.24; the density is the density at 60°C. In the hydroxypropyl-β-cyclodextrin solution, the volume ratio of water (ml) to hydroxypropyl-β-cyclodextrin (g) is 7:1 to 18:1; the volume ratio of the volatile oil (ml) to hydroxypropyl-β-cyclodextrin (g) is 1:5 to 1:

9. The temperature for mixing with the hydroxypropyl-β-cyclodextrin solution is 35–60°C, and the mixing time is 3–5 hours.

3. The preparation method according to claim 1, characterized in that, In step S2), the amount of water used for water extraction is 6 to 10 times the weight of the licorice. The water extraction time is 1-3 hours; The water extraction is performed 2 to 4 times; After water extraction, the extract is filtered and concentrated until the solid-liquid ratio is 1:(1~1.9). The eluent was concentrated to a thick paste with a relative density of 1.18-1.24, freeze-dried, and pulverized to obtain pharmaceutical powder Ba; the density is the density at 60°C. The ethanol extraction of the pulverized Poria cocos is specifically carried out by reflux extraction with 5 to 9 times the amount of 65% to 85% ethanol; the number of extractions is 1 to 3; and the extraction time is 1 to 3 hours. The extract obtained from the above alcohol extraction was filtered, and the filtrates were combined. The filtrates were concentrated to a clear extract with a relative density of 1.14-1.22, freeze-dried, and pulverized to obtain pharmaceutical powder Bb; the density is the density at 60°C. In the process of decocting the medicinal residue with water, the amount of water used is 5 to 9 times the weight of the medicinal residue; the number of times of decoction is 1 to 3, and the decoction time is 0.5 to 1.5 hours. After decoction, the filtrate is filtered, the filtrates are combined, and concentrated to a clear extract with a relative density of 1.18-1.

24. The extract is then freeze-dried, pulverized, and the resulting powder is Bc; the density is the density at 60°C.

4. The preparation method according to claim 1, characterized in that, In step S3), the alcohol extraction is performed 1 to 3 times, and the extraction time is 1 to 2 hours. The extract obtained from alcohol extraction is concentrated to a clear paste with a relative density of 1.16-1.24; the density is the density at 60°C. The ointment was added to distilled water and settled, and the solid-liquid ratio of the system after adding water was 1:(7~20). The amount of sodium carbonate added is 4% to 7% of the total drug solution volume; In the process of decocting the dregs with water, the amount of water used for decocting is 5 to 9 times that of the dregs, the number of times of decocting is 1 to 3, and the decocting time is 0.5 to 1.5 hours. The filtrate, after concentration, yields a clear extract with a density of 1.18-1.24; this density is the density at 60°C.

5. The preparation method according to claim 1, characterized in that, In step S4), the alcohol extraction is performed 1 to 4 times; the alcohol extraction time is 1 to 3 hours. The extract is concentrated to a material-to-liquid ratio of 1:3; The polyamide resin is a pretreated polyamide resin; In the water elution process, the amount of water used is 3 to 5 times the column volume; In the elution with ethanol, the amount of ethanol used is 6 to 8 times the column volume; the ethanol is 40% to 60% ethanol. The density of the paste is 1.18-1.25, and the density is the density at 60°C. In the hydroxypropyl-β-cyclodextrin solution, the ratio of water volume (ml) to hydroxypropyl-β-cyclodextrin weight (g) is 7:1 to 18:

1. The volume ratio (ml) of the volatile oil to the weight ratio (g) of the hydroxypropyl-β-cyclodextrin is 1:5 to 1:

9. The mixing temperature with the hydroxypropyl-β-cyclodextrin solution is 35–60°C, and the mixing time is 3–5 hours.

6. A traditional Chinese medicine composition for treating polycystic ovary syndrome of spleen deficiency and phlegm dampness type, prepared by the preparation method according to any one of claims 1 to 5.

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