Traditional Chinese medicine composition for premature ovarian failure as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition for premature ovarian failure as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10-40 parts of prepared rehmannia root, 2-8 parts of leech, 2-8 parts of rheum officinale, 8-20 parts of radix paeoniae alba, 10-30 parts of angelica sinensis and 8-16 parts of ligusticum wallichii. The preparation method comprises the following steps: mixing prepared rehmannia root, leech, rheum officinale, radix paeoniae alba, angelica sinensis and ligusticum wallichii according to the formula ratio, adding water, decocting, combining decoction, and concentrating to obtain clear paste. According to the traditional Chinese medicine composition, six traditional Chinese medicine raw materials including prepared rehmannia root, leech, rheum officinale, radix paeoniae alba, angelica sinensis and ligusticum wallichii are compounded, and the effect of relieving chemotherapy-damaged ovary can be achieved by promoting SIRT1 protein expression, inhibiting ovarian tissue apoptosis, promoting primary follicle development, increasing the content of estradiol in serum and reducing the levels of follicle-stimulating hormone and luteinizing hormone in the serum.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a traditional Chinese medicine composition for premature ovarian failure, its preparation method and application. Background Art

[0002] Premature ovarian failure (POF) refers to the premature failure of ovarian function in women with established regular menstruation due to exhaustion of ovarian follicles or iatrogenic injury. The main clinical manifestations are secondary amenorrhea before the age of 40, decreased estrogen and increased gonadotropin, often accompanied by perimenopausal symptoms.

[0003] Although traditional Chinese medicine has no record of premature ovarian failure, its pathogenesis is similar to "early cessation of menstruation" and "premature closure of monthly menstruation" in traditional Chinese medicine, and it should belong to the categories of "amenorrhea", "blood depletion", "blood separation", "infertility", "syndrome of pre- and post-menopause" in traditional Chinese medicine. Traditional Chinese medicine generally believes that kidney deficiency is the main cause of this disease, and the main treatment principle is to tonify the kidney.

[0004] CN106075361A discloses a traditional Chinese medicine composition, which is made from the following raw materials in the following mass ratio: Morinda officinalis 1 - 20 parts, stir-fried Atractylodes macrocephala 1 - 20 parts, Ligusticum chuanxiong 1 - 20 parts, stir-fried Angelica sinensis 1 - 30 parts, bran-fried Paeonia lactiflora 1 - 50 parts, Phellodendron amurense 1 - 20 parts, Rehmannia glutinosa 1 - 30 parts, Glossy privet fruit 1 - 30 parts, Epimedium brevicornu 1 - 50 parts, stir-fried Eucommia ulmoides 1 - 30 parts, roasted Plastrum Testudinis 1 - 30 parts, Photinia serratifolia 1 - 50 parts, Polygonatum odoratum 1 - 20 parts, Astragalus membranaceus 1 - 50 parts, stir-fried Dipsacus asperoides 1 - 30 parts, stir-fried Citrus reticulata Blanco 1 - 20 parts, Radix Aconiti Lateralis Preparata 1 - 20 parts and dried ginger 1 - 20 parts. This invention can promote the development of ovarian follicles in female rats and maintain the normal physiological function of the ovaries.

[0005] CN107753707A discloses a traditional Chinese medicine composition for preventing premature ovarian failure, which is composed of the following raw materials in parts by weight: Angelica sinensis 4 - 15 parts, Paeonia lactiflora 8 - 20 parts, Rehmannia glutinosa 20 - 30 parts, Dioscorea opposita 20 - 30 parts, Cornus officinalis 10 - 20 parts, Glossy privet fruit 10 - 20 parts, Bupleurum chinense 4 - 15 parts, Gentiana scabra 8 - 20 parts, Phellodendron amurense 7 - 17 parts, Cuscuta chinensis 8 - 20 parts, Poria cocos 5 - 13 parts, Alisma orientale 10 - 25 parts, Carthamus tinctorius 4 - 15 parts, Ligusticum chuanxiong 4 - 15 parts. The traditional Chinese medicine provided by this invention can nourish the ovaries, balance estrogen and progesterone, regulate reproductive endocrinology, reduce immune damage and improve the endocrine function of the ovaries, and has no obvious toxic and side effects, is safe and effective, and can prevent premature ovarian failure.

[0006] CN110742952A discloses a traditional Chinese medicine prescription for treating amenorrhea. The composition and weight ratio of the traditional Chinese medicine are as follows: 30 grams of Codonopsis pilosula, 10 grams of stir-fried Atractylodes macrocephala, 10 grams of Poria cocos, 30 grams of Glycyrrhiza uralensis, 30 grams of Angelica sinensis, 30 grams of Paeonia lactiflora, 6 grams of Ligusticum wallichii, 30 grams of Rehmannia glutinosa, 10 grams of Euonymus alatus, 10 grams of Clerodendrum trichotomum, 6 grams of Squama Manitis, 2 grams of Scorpio, 1 gram of Scolopendra subspinipes, 6 grams of Hirudo, 10 grams of Leonurus japonicus, 10 grams of Cyperus rotundus and 15 grams of Rubia cordifolia. The prescription of the invention has no side effects and can improve amenorrhea caused by deficiency of both qi and blood through treatment.

[0007] The existing traditional Chinese medicine formula has overly complex components, unclear action mechanisms, and does not disclose the impact on premature ovarian failure caused by iatrogenic injury. Therefore, there is an urgent need to develop a traditional Chinese medicine composition for premature ovarian failure caused by iatrogenic injury. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a traditional Chinese medicine composition for premature ovarian failure, its preparation method and application. The traditional Chinese medicine composition obtained by the optimized compounding of raw materials in the present invention has simple components and has a good therapeutic effect on premature ovarian failure caused by iatrogenic injury, especially chemotherapy-induced premature ovarian failure.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] In the first aspect, the present invention provides a traditional Chinese medicine composition for premature ovarian failure, which is composed of the following raw materials: Rehmannia glutinosa, Hirudo, Rheum palmatum, Paeonia lactiflora, Angelica sinensis and Ligusticum wallichii.

[0011] In some embodiments, the weight ratio of Rehmannia glutinosa, Hirudo and Rheum palmatum is 5-20:1-4:1-4; preferably 10:3:3.

[0012] In some embodiments, the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 10-40 parts of Rehmannia glutinosa, 2-8 parts of Hirudo, 2-8 parts of Rheum palmatum, 8-20 parts of Paeonia lactiflora, 10-30 parts of Angelica sinensis and 8-16 parts of Ligusticum wallichii.

[0013] Preferably, the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 10-20 parts of Rehmannia glutinosa, 3-6 parts of Hirudo, 3-6 parts of Rheum palmatum, 8-12 parts of Paeonia lactiflora, 15-30 parts of Angelica sinensis and 10-16 parts of Ligusticum wallichii.

[0014] Further preferably, the traditional Chinese medicine composition is composed of the following raw materials in parts by weight: 20 parts of Rehmannia glutinosa, 6 parts of Hirudo, 6 parts of Rheum palmatum, 12 parts of Paeonia lactiflora, 15 parts of Angelica sinensis and 10 parts of Ligusticum wallichii.

[0015] In the second aspect, the present invention provides a preparation method of the above traditional Chinese medicine composition, including the following steps:

[0016] Add the formula amounts of Rehmannia glutinosa, Hirudo, Rheum palmatum, Paeonia lactiflora, Angelica sinensis, and Ligusticum chuanxiong, mix them, add water, and decoct. Combine the decoction liquids and concentrate to a clear extract to obtain the Chinese medicine composition.

[0017] In some embodiments, the amount of water added is 4 - 10 times the total weight of Rehmannia glutinosa, Hirudo, Rheum palmatum, Paeonia lactiflora, Angelica sinensis, and Ligusticum chuanxiong, preferably 6 - 8 times.

[0018] In some embodiments, the number of decoction times is 1 - 5 times, and the decoction time each time is 1 - 3 h, preferably 2 - 3 times, 1 - 2 h each time.

[0019] In some embodiments, the concentration is by reduced pressure concentration to a relative density of 1.18 - 1.22 (60 °C).

[0020] In the third aspect, the present invention provides the application of the above Chinese medicine composition in the preparation of a drug for treating premature ovarian failure.

[0021] In some embodiments, the premature ovarian failure is iatrogenic premature ovarian failure, including premature ovarian failure caused by drug treatments such as chemotherapeutic drugs, radiotherapy, immunosuppressants, GnRH analogs, PARP inhibitors, aromatase inhibitors, etc.

[0022] In the fourth aspect, the present invention provides the application of the above Chinese medicine composition in the preparation of a SIRT1 activator.

[0023] In the fifth aspect, the present invention provides a pharmaceutical preparation made from the above Chinese medicine composition and pharmaceutically acceptable carrier excipients.

[0024] In some embodiments, the pharmaceutical preparation is a granule, powder, oral liquid, tablet, capsule, or ointment.

[0025] In some embodiments, the pharmaceutical preparation contains the Chinese medicine composition and at least one excipient, and the excipient is selected from at least one of solvents, colorants, lubricants, diluents, and disintegrants.

[0026] Preferably, the solvent is selected from at least one of water, ethanol, and isopropanol; the colorant is selected from at least one of iron oxide yellow NF, brilliant blue Supra, FD and C green 3, and sunset yellow; the lubricant is selected from at least one of magnesium stearate, talc, silicon dioxide, and stearic acid; the disintegrant is selected from at least one of carboxymethyl cellulose and hydroxypropyl methylcellulose; the diluent is selected from at least one of polyethylene glycol, dimethyl sulfoxide, ethyl lactate, and a mixture of D - mannitol - xylitol - microcrystalline cellulose - crosslinked polyvinylpyrrolidone - anhydrous calcium hydrogen phosphate.

[0027] The beneficial effects of the present invention are:

[0028] The present invention utilizes the compounding of six traditional Chinese medicine raw materials, namely Rehmannia glutinosa, Hirudo, Rheum palmatum, Paeonia lactiflora, Angelica sinensis, and Ligusticum chuanxiong, which can promote the expression of SIRT1 (silent information regulator 1) protein, inhibit ovarian tissue apoptosis, promote the development of primary follicles, increase the content of serum estradiol, and reduce the levels of serum follicle-stimulating hormone and luteinizing hormone, so as to achieve the effect of alleviating chemotherapy-induced ovarian damage.

[0029] Meanwhile, SIRT1 can regulate the downstream regulator FOXO3 through the P13K / AKT pathway to exert an inhibitory effect on the adverse effects of oxidative stress on oocytes, thereby playing a protective role against premature ovarian failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the HE result of ovarian tissue, and the scale of the figures is the same among the same rows.

[0031] Figure 2 It is the screening result of Tμnel.

[0032] Figure 3 It is the qPCR result of rat ovarian tissue. Among them, ** indicates P < 0.01 compared with the model group, and N / A indicates P > 0.05 compared with the model group.

[0033] Figure 4 It is the WB figure of rat ovary.

[0034] Figure 5 It is the result of the apoptosis experiment.

[0035] Figure 6 It is the qPCR result of cells. Among them, *** indicates P < 0.001 compared with the model group.

[0036] Figure 7 It is the WB figure of COV434 cells. DETAILED DESCRIPTION OF THE INVENTION

[0037] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific implementation manners and are not intended to limit the protection scope of the present invention.

[0039] When embodiments provide a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0040] The present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercially available products in this technical field.

[0041] As used herein, the "pharmaceutical preparation" and "SIRT1 activator" of the present invention include traditional Chinese medicine compositions and pharmaceutically acceptable excipients. In specific embodiments, the traditional Chinese medicine compositions described in the present invention are provided in the "drug" and "SIRT1 activator" in an effective amount (e.g., a therapeutically effective amount).

[0042] As used herein, the "pharmaceutically acceptable excipients" include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients (such as cocoa butter and suppository wax), colorants, coating agents, sweeteners, flavoring agents and fragrances may also be present in the pharmaceutical compositions.

[0043] As used herein, the "pharmaceutical preparation" and "SIRT1 activator" of the present invention can be prepared by any method known in pharmacy. Generally, these preparation methods include associating the traditional Chinese medicine composition (i.e., the first active ingredient) with a carrier or excipient and / or one or more other auxiliary components, and then, if necessary and / or desired, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0044] The "drugs" and "SIRT1 activators" of the present invention can be prepared according to known methods, such as the methods described in the general rules of preparation in the 2020 Edition of the Chinese Pharmacopoeia, the 16th Edition of the Japanese Pharmacopoeia, the United States Pharmacopoeia, and the 9th Edition of the European Pharmacopoeia. The preparation method depends on the dosage form.

[0045] As used herein, the first active ingredient, pharmaceutically acceptable excipients in the "pharmaceutical preparation" and "SIRT1 activator" will vary according to the identity, body size and / or condition of the subject being treated and further according to the route of administration of the composition. The "pharmaceutical preparation" and "SIRT1 activator" may contain from 0.1% to 100% (w / w) of the first active ingredient.

[0046] As used herein, the term "treatment", unless otherwise indicated, means reversing, alleviating a disorder or condition to which the term applies or one or more symptoms of such a disorder or condition, inhibiting the progression of the disorder or condition or one or more symptoms thereof, or preventing the disorder or condition or one or more symptoms thereof. The term "treatment" as used in the invention refers to a treatment act as "treatment" is defined just above.

[0047] As used herein, the term "effective amount" means an amount sufficient to elicit a desired biological response. The effective amount of the active ingredient of the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disorder being treated, the mode of administration, and the age and health status of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. The effective amount is the amount of the first active ingredient described in the present invention in a single dose. In certain embodiments, the effective amount is the combined amount of the active ingredients described in the present invention in multiple doses.

[0048] As used herein, the term "therapeutically effective amount" means an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or sufficient to delay or minimize one or more symptoms associated with the disorder. The therapeutically effective amount of the traditional Chinese medicine composition means the amount of the therapeutic agent alone or in combination with other therapies that provides a therapeutic benefit in the treatment of a disorder. The term "therapeutically effective amount" may encompass an amount that improves overall therapy, reduces or avoids the symptoms, signs or causes of a disorder, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, the therapeutically effective amount is an amount sufficient to treat any disease or disorder described.

[0049] Example 1 Traditional Chinese Medicine Composition

[0050] The formula of the traditional Chinese medicine composition is as follows: 10 g of Rehmannia glutinosa, 3 g of Hirudo, 3 g of Rheum palmatum, 8 g of Paeonia lactiflora, 30 g of Angelica sinensis, and 16 g of Ligusticum chuanxiong.

[0051] The preparation method is as follows: Mix Rehmannia glutinosa, Hirudo, Rheum palmatum, Paeonia lactiflora, Angelica sinensis, and Ligusticum chuanxiong according to the formula amount, decoct twice with water. For the first time, use 8 times the amount of water (the total weight of Rehmannia glutinosa, Hirudo, Rheum palmatum, Paeonia lactiflora, Angelica sinensis, and Ligusticum chuanxiong) and decoct for 2 hours. For the second time, use 6 times the amount of water (the total weight of Rehmannia glutinosa, Hirudo, Rheum palmatum, Paeonia lactiflora, Angelica sinensis, and Ligusticum chuanxiong) and decoct for 1 hour. Combine the decoction liquids, filter, and concentrate under reduced pressure to obtain a clear paste with a relative density of 1.18 - 1.22 (60 °C), thus obtaining the traditional Chinese medicine composition.

[0052] Example 2 Traditional Chinese Medicine Composition

[0053] The formula of the traditional Chinese medicine composition is as follows: 20 g of Rehmannia glutinosa, 6 g of Hirudo, 6 g of Rheum palmatum, 12 g of Paeonia lactiflora, 15 g of Angelica sinensis, and 10 g of Ligusticum chuanxiong.

[0054] The preparation method is the same as that of Example 1.

[0055] Chinese medicine composition of comparative example

[0056] The difference between this comparative example and Example 2 is that the mass ratios of Rehmannia glutinosa, Hirudo, and Rheum palmatum are different.

[0057] Specifically: the formula of the Chinese medicine composition is: 17 g of Rehmannia glutinosa, 3 g of Hirudo, 12 g of Rheum palmatum, 12 g of Paeonia lactiflora, 15 g of Angelica sinensis, and 10 g of Ligusticum chuanxiong.

[0058] The preparation method is the same as that of Example 1.

[0059] I. Protective effect of Chinese medicine composition on premature ovarian failure

[0060] 1. Experimental animals

[0061] SPF-grade SD rats, 10 weeks old, female.

[0062] 2. Experimental samples

[0063] Chinese medicine compositions prepared in Examples 1-2 and the comparative example.

[0064] 3. Modeling and administration

[0065] The rats were randomly divided into 5 groups: normal control group (CON), model group (MOD), Example 1 group (ZY1), Example 2 group (ZY2), and comparative example group (DB). Except for the normal control group, the other 4 groups of rats were established with premature ovarian failure animal models. On the 14th and 21st days, cisplatin (4 mg / kg, 20 mg dissolved in 20 ml of 0.9% sodium chloride injection, injected after giving the Chinese medicine composition) was intraperitoneally injected. The rats in the Example 1 group, Example 2 group, and control group were intragastrically administered 6.21 g of crude drug / kg / d (the intragastric administration volume was 18 mL / kg / d), and the normal control group and the model group were intragastrically administered an equal volume of normal saline. Gastric gavage was performed on the first day of the experiment (day 0), once a day, until the end of the experiment on the 28th day. After fasting for 12 hours without water, on the 29th day, all rats were weighed and anesthetized, and blood was taken from the abdominal aorta and sacrificed. Plasma was used for the evaluation of ovarian reserve function, and then the ipsilateral ovary was taken for observing ovarian tissue morphology and follicle counting, and the other ovary was taken for the determination of SIRTsmRNA and related apoptotic proteins.

[0066] 4. Detection indexes and results

[0067] 4.1 Observation of ovarian tissue morphology by HE staining

[0068] After each tissue was trimmed, dewaxed with xylene, dehydrated with gradient alcohol, and embedded in paraffin, sections were made, stained with HE, and read under a LEIKA400B upright fluorescence microscope. The results are as Figure 1 shown.

[0069] Results showed that the ovarian tissue morphology of rats in the normal control group was normal. Follicles and corpus luteum tissues at various stages could be seen under the microscope. There were many primordial follicle cells. Most of the growing follicle cells had larger cell bodies, fine granular nuclear chromatin, and reticular nucleoli could be seen. The ovarian tissue of rats in the model group decreased, and obvious pathological changes were observed. Under the microscope, the ovarian cortex became thinner, the primordial cells increased, and typical growing follicles were rare; the atretic cells increased significantly, and a large number of interstitial glands could be seen. The pathological changes of the ovaries of rats in the control group were not obvious compared with those in the model group. The number of growing follicle cells in the rats of the Example 2 group increased and the number of atretic cells decreased compared with those in the model group.

[0070] 4.2 Detection of blood FSH, E2, and LH levels using ELISA kits

[0071] According to the instructions of the ELISA kits for E2, LH, and FSH, the OD values of the absorption of E2, LH, and FSH in the sera of rats in each group were detected at a wavelength of 450 nm. The Logistic standard curve was drawn using the ELISAcalc program and the regression equation y = (A - D) / [1 + (x / C)^B] + D was fitted. The concentrations of E2, LH, and FSH in each serum sample were calculated according to the regression equation.

[0072] The results are shown in Table 1.

[0073] Table 1 Serum E2, LH, and FSH levels of rats in each group

[0074] Group E2 (pg / mL) LH (ng / mL) FSH (ng / mL) Normal control group 569.7±15.73** 40.41±0.5713** 44.92±1.049** Model group 377.3±8.385## 60.66±1.069## 53.31±1.238## Comparative example group 427.5±9.934*# 51.72±0.8124**# 51.19±1.341## Example 2 group 565±13.45** 41.07±0.9264** 45.00±1.497**

[0075] Note: * Compared with the model group, p < 0.05; ** compared with the model group, p < 0.01, # compared with the normal control group, p < 0.05; ## compared with the model group, p < 0.01.

[0076] Results showed that the serum E2 level of rats in the model group was significantly lower than that in the normal control group, and the LH and FSH levels were significantly higher than those in the normal control group. The serum E2 levels of rats in the comparative example group and the Example 2 group were significantly higher than those in the model group, and the improvement effect of the Example 1 group was better. There was no statistical significance in the serum E2 level of its rats compared with the normal control group.

[0077] Both the comparative example group and the Example 2 group could reduce the serum LH level of rats in the model group, and the effect of the Example 2 group was better. There was no statistical significance in the serum LH level of its rats compared with the normal control group.

[0078] Compared with the model group, the serum FSH level of rats in the Example 2 group decreased significantly, while there was no statistical significance in the serum FSH level of rats in the comparative example group.

[0079] 4.3 Detection of apoptosis in ovarian tissue by TμNEL staining

[0080] The ovarian tissue sections were dewaxed with xylene, dehydrated with gradient ethanol, rinsed with distilled water, incubated with protease K solution at room temperature, subjected to the TμNEL reaction, rinsed with PBS, observed under a microscope, and finally stained with DAB to observe the apoptosis distribution.

[0081] Refer to Jiangsu Kaygee Biotechnology: One-step TμNEL In Situ Apoptosis Detection Kit, upgraded version (green FITC-labeled fluorescence detection method, universal type).

[0082] The results were as Figure 2 shown. The results showed that compared with the model group, the apoptosis of ovarian tissue in the Example 2 group could be significantly improved, and the effect was significantly better than that of the comparative example group.

[0083] 4.4 Determination of the expression of Sirt1 mRNA in ovarian tissue by qPCR

[0084] Add 1 mL of Trizol reagent to the rat ovarian tissue, break it with a homogenizer, let it stand on ice for 5 min to lyse the cells, add 200 μL of chloroform to the lysed sample, shake it for 15 s to mix well, let it stand at room temperature for 10 min, centrifuge at 4°C and 12,000 g for 15 min, take 400 μL of the supernatant into a new 1.5 mL enzyme-free tube, add 400 μL of pre-cooled isopropanol, gently mix well, let it stand at room temperature for 5 min, centrifuge at 4°C and 12,000 g for 10 min, discard the supernatant, add 1 mL of pre-cooled 80% ethanol, gently mix well, centrifuge at 4°C and 8,000 g for 5 min, discard the supernatant, air-dry the precipitate, add 30 μL of enzyme-free water to the tissue sample and 15 μL of enzyme-free water to the cell sample, mix well, dissolve it at 4°C for 1 h, quantify with Nanodorp2000, and record the results.

[0085] Operate according to the reverse transcription kit and qPCR kit instructions. The qPCR results of rat ovarian tissue were as Figure 3 shown.

[0086] The results showed that the expression level of Sirt1 mRNA in the ovarian tissue of the model group rats was significantly lower than that of the normal control group (P < 0.01). Both the Example 1 group and the Example 2 group could significantly increase the expression level of Sirt1 mRNA in the ovarian tissue of the model group rats (P < 0.01), while the comparative example group could not increase the expression level of Sirt1 mRNA in the ovarian tissue of the model group rats (P > 0.05).

[0087] 4.5 Determination of the protein contents of SIRT1 and β-actin in ovarian tissue by Western blot

[0088] Add 400 μL of RIPA working solution (prepared with 10X RIPA, added with 1% of three protease inhibitors, stored at 2 - 8°C) to the rat ovarian tissue. After disrupting with a homogenizer, let it stand on ice for 5 min to lyse the cells. Centrifuge at 4°C, 8000 g for 5 min, and transfer the supernatant to a new 1.5 mL enzyme-free tube for use on ice.

[0089] Use Western Blot technology to detect the protein expression levels of SIRT1 and β-actin in the ovarian tissue.

[0090] The results are as Figure 4 shown. The results show that compared with the model group, the protein expression of SIRT1 in the ovarian tissue of the Example 1 group, Example 2 group, and control group was significantly increased. Among them, the effects of the Example 1 group and Example 2 group were better than those of the control group.

[0091] II. Effects of the traditional Chinese medicine composition on the expression of PI3K pathway-related factors in ovarian granulosa cells after cisplatin administration

[0092] After cisplatin damaged the ovarian granulosa cells, observe the expression of PI3K / AKT pathway-related factors in the ovarian granulosa cells after chemotherapy with the drug-containing serum.

[0093] 1. Preparation of drug-containing serum

[0094] Randomly divide 10-week-old female SD rats into 5 groups: blank control group (CON), model group (MOD), control group (DB), Example 1 group (ZY1), and Example 2 group (ZY2). The administration groups were given the drug at 10 times the adult dose (1 g of crude drug per ml, 2 ml of the traditional Chinese medicine composition per 100 g body weight of the rat). Administer by gavage once every 12 h for 3 consecutive days. The rats in the blank control group and the model group were gavaged with 2 ml / 100 g of pure water. On the 4th day, collect blood from the abdominal aorta. Let the blood of each group stand at 4°C for 4 h, centrifuge at low temperature, take the supernatant, filter and sterilize it with a 0.22 μm filter, inactivate it in a 56°C water bath for 30 min, then aliquot and store it in a -20°C refrigerator.

[0095] 2. Culture and grouping of ovarian granulosa cells

[0096] 2.1 Cell culture

[0097] (1) Resuscitation: Take out the cryopreserved cells from liquid nitrogen and continuously shake them in a 37°C water bath to promote melting. Transfer them to a 15 mL centrifuge tube, add 5 mL of pre-warmed complete medium (DMEM / H + 10% FBS + 1% double antibody 100X), gently blow and mix evenly, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add 10 mL of complete medium, gently pipette, and inoculate into a 10 cm dish, and culture in a 5% CO2, 37°C incubator.

[0098] (2) Passage: When the cell density reaches 80%-90%, remove the culture medium and wash twice with 10 mL of PBS. Add 1 mL of trypsin containing 0.25% EDTA, place it in a 37°C incubator for 2 min (observe under an inverted microscope until the cells become round and detached), add 3 mL of complete medium to terminate the trypsin digestion, gently pipette until all the cells fall off, and transfer them to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min, discard the supernatant. Add 2 mL of complete medium, resuspend, and take 10 μL for counting. Inoculate at 1×10 6 / dish and continue culturing in a cell incubator containing 5% CO 2 .

[0099] (3) Cryopreservation: When the cell density reaches 80%-90%, remove the culture medium and wash twice with 10 mL of PBS. Add 1 mL of trypsin containing 0.25% EDTA, place it in a cell incubator for 1-3 min (observe under an inverted microscope until the cells become round and detached). Add 3 mL of DMEM complete medium to terminate the trypsin digestion, gently pipette until the cells fall off, and transfer them to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min at RT, discard the supernatant. Add 1 mL of cryopreservation solution, resuspend, place it in a gradient freezing box, immediately put it into a -80°C refrigerator overnight, and transfer it to liquid nitrogen for storage the next day.

[0100] 2.2 Cell Seeding

[0101] MTT: Seed 5000 cells / well in a 96-well plate and incubate overnight; Flow cytometry apoptosis / WB / PCR: Seed 2*10^5 cells / well in a 6-well plate and incubate overnight.

[0102] 2.3 MTT Assay Method

[0103] Remove the culture supernatant from the cells that have adhered overnight, add the drug at the corresponding concentration and continue culturing for 24 h, 48 h, and 72 h, and detect by the MTT method. Take out the incubated plate, add 20 μL of MTT solution (5 mg / mL, prepared with PBS) to each well, incubate at 37°C for 4 h, pat dry the supernatant, quickly add 150 μL of DMSO to lyse the cells, shake at 100 rpm for 5 min to mix evenly, and detect the OD value at 490 nm with an ELISA reader.

[0104] The results of the survival rate of COV434 cells at different times in the MTT assay are shown in Table 2.

[0105] Table 2 Survival rate of COV434 cells at different times in the MTT assay (%)

[0106] Time h Blank control group % Model group % Comparative example group % Example 1 group % Example 2 group % 24 100.00 81.8* <![CDATA[83.17 NA > <![CDATA[81.76 NA > 86.94NA 48 100.00 56.27*** <![CDATA[60.24 NA > 73.26*** 75.45*** 72 100.00 33.44*** 43.07** 54.61*** 57.46***

[0107] Note: "NA" indicates P>0.05, "*" indicates P≤0.05, "**" indicates P≤0.01, "***" indicates P≤0.001; the comparison object of the model group is the blank control group, and the comparison object of each drug administration group is the model group.

[0108] The results showed that the survival rates at 48 h and 72 h in the Example 1 group and the Example 2 group were significantly increased; the survival rate at 72 h was only significantly increased in the Comparative Example group.

[0109] 2.4 Detection of granulosa cell apoptosis by flow cytometry

[0110] After collecting cells with a cell scraper, wash once with PBS and count 10^5 cells / tube. Resuspend with 100 μL Binding Buffer, make a negative control tube, stain with Annexin V-FITC and PI, incubate in the dark at room temperature for 15 min, resuspend with 400 μL PBS, and detect on the machine.

[0111] The results are as Figure 5 shown. The results showed that the number of apoptotic cells in the model group was significantly increased compared with the blank control group. Compared with the model group, the Example 1 group could significantly reduce the number of apoptotic cells; while there was no significant difference in the change of the number of apoptotic cells in the Comparative Example group.

[0112] 2.5 Detection of Foxo3a mRNA expression by qPCR

[0113] For COV434 cells: After scraping the cells, centrifuge at 400 g for 5 min, collect the precipitate, completely lyse it with 1 mL of Trizol reagent, transfer it to a 1.5 mL enzyme-free tube, and lyse at 4 °C for 5 min for standby.

[0114] The results are as Figure 6 shown. The results showed that the Foxo3a mRNA expression in the cells of the model group was significantly decreased compared with the blank control group. Compared with the model group, the Foxo3a mRNA expression in the cells of each drug administration group could be significantly increased; at the same time, the effects of the Example 1 group and the Example 2 group were significantly better than those of the Comparative Example group.

[0115] 2.6 Detection of the protein expressions of Foxo3a, AKT, p-AKT, and β-actin by Western Blot

[0116] After collecting COV434 cells with a cell scraper, wash once with PBS, add 100 μL of RIPA working solution (prepared with 10X RIPA, added with 1% protease inhibitor, stored at 2-8 °C) to the precipitate, blow and mix well, and let it stand on ice for 10 min to lyse the cells. Adjust the 4 °C centrifuge to 12000 xg, centrifuge the lysate for 10 min, transfer the supernatant to a new 1.5 mL enzyme-free tube, and keep it on ice for standby.

[0117] The results are as Figure 7 shown.

[0118] The above is a further description of the present invention in combination with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

Claims

1. A Chinese medicine composition for premature ovarian failure, characterized in that: The invention is composed of the following raw materials in parts by weight: 10-40 parts of Radix Rehmanniae Preparata, 2-8 parts of Hirudo, 2-8 parts of Radix Rhizoma Rhei, 8-20 parts of Radix Paeoniae Alba, 10-30 parts of Radix Angelicae Sinensis and 8-16 parts of Rhizoma Chuanxiong.

2. The Chinese medicine composition according to claim 1, characterized in that: The weight ratio of the Radix Rehmanniae Preparata, the leech and the rhubarb is 10:3:

3.

3. The Chinese medicine composition according to claim 2, characterized in that: The invention is composed of the following raw materials in parts by weight: 10-20 parts of Radix Rehmanniae Preparata, 3-6 parts of Hirudo, 3-6 parts of Radix Rhizoma Rhei, 8-12 parts of Radix Paeoniae Alba, 15-30 parts of Radix Angelicae Sinensis and 10-16 parts of Rhizoma Chuanxiong.

4. The Chinese medicine composition according to claim 1, characterized in that: The invention is composed of the following raw materials in parts by weight: 20 parts of Radix Rehmanniae Preparata, 6 parts of Hirudo, 6 parts of Rhubarb, 12 parts of White Peony Root, 15 parts of Angelica Sinensis and 10 parts of Ligusticum chuanxiong.

5. The method for preparing the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The steps include: The Chinese medicine composition is prepared by mixing the prepared rehmannia root, leech, rhubarb, white peony root, angelica root and chuanxiong in the prescribed amount, boiling with water, combining the boiling liquids and concentrating them into a clear paste.

6. The preparation method according to claim 5, characterized in that: The amount of water added is 4-10 times the total weight of Rehmannia root, Hirudo, Rhubarb, White Peony Root, Angelica Sinensis and Ligusticum chuanxiong; And / or the concentration is concentrated under reduced pressure to a relative density of 1.18-1.

22.

7. Use of the Chinese medicine composition according to any one of claims 1 to 4 or the Chinese medicine composition prepared by the preparation method according to any one of claims 5 to 6 in preparing a drug for treating premature ovarian failure, wherein the premature ovarian failure is iatrogenic premature ovarian failure.

8. Use of the traditional Chinese medicine composition according to any one of claims 1 to 4 or the traditional Chinese medicine composition prepared by the preparation method according to any one of claims 5 to 6 in the preparation of a SIRT1 activator.

9. A pharmaceutical preparation, characterized in that The invention comprises the Chinese medicine composition according to any one of claims 1 to 4 and at least one excipient, wherein the excipient is selected from at least one of a solvent, a colorant, a lubricant, a diluent and a disintegrant.

10. The pharmaceutical preparation according to claim 9, characterized in that The pharmaceutical preparation is granules, powders, oral liquids, tablets, capsules or pastes.

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