Potentilla fruticosa extract for preventing and treating ovarian function decline as well as preparation method and application of Potentilla fruticosa extract

Abstract

The invention provides a potentilla fruticosa extract for preventing and treating ovarian function decline as well as a preparation method and application of the potentilla fruticosa extract, and belongs to the field of traditional Chinese medicines. The preparation method comprises the following steps: crushing the potentilla fruticosa, adding an ethanol water solution, extracting, refluxing, filtering, and removing ethanol and water from filtrate to obtain a potentilla fruticosa crude extract; extracting the crude extract with petroleum ether, and removing the petroleum ether to obtain a refined product of the potentilla fruticosa; and carrying out spray drying on the refined product to obtain the potentilla fruticosa extract. The extract prepared by the method is stable in physical property, easy to store and transport, simple in preparation process and convenient to operate; meanwhile, in the production process, no 1-3 types of organic solvents are used, so that the method is green, environment-friendly and good in safety. The potentilla fruticosa extract has a good effect of preventing and treating ovarian function decline.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and particularly relates to a Potentilla fruticosa extract for preventing and treating ovarian dysfunction, a preparation method thereof and an application thereof. Background Art

[0002] Ovarian failure refers to the persistent amenorrhea caused by ovarian atrophy in women, accompanied by the regression of secondary sexual characteristics and the appearance of menopausal symptoms. Common specific diseases include diminished ovarian reserve (DOR), premature ovarian insufficiency (POI), ovarian failure (POF), menopausal syndrome and other diseases. Ovarian failure is characterized by very low levels of estrogen produced by the ovaries. At present, there is still a lack of ideal treatment methods. Western medicine treatment mainly supplements estrogen, using estrogen and progesterone. Although it can improve symptoms, it is not ideal for the recovery of ovarian function. After stopping the drug, the symptoms reappear. Hormone therapy can only improve menopausal symptoms, but it is not effective in restoring fertility and improving sexual function. In addition, long-term and large-scale supplementation of exogenous estrogen can easily increase the incidence of endometrial cancer.

[0003] Potentilla fruticosa L. is a plant of the genus Potentilla in the family Rosaceae, mainly distributed in the Qinghai-Tibet Plateau. In the Qinghai-Tibet Plateau of my country, Potentilla fruticosa is called "Ge Sang Mei Duo" by the Tibetan people. It is a kind of ethnic medicine commonly used in Tibetan medicine. According to the Tibetan medicine classic "Jingzhu Bencao", it has the effects of strengthening the spleen, relieving heat and regulating menstruation. Potentilla fruticosa was also developed by the Siberian Branch of the Russian Academy of Sciences into black tea and green tea representing "Siberian tea". Modern research has determined its components and found that Potentilla fruticosa contains dozens of compounds such as flavonoids, tannins, terpenes, steroids, coumarins, tannins and lignans. It is recorded in the classics that Potentilla fruticosa extract has the effect of regulating menstruation, but there is no literature report on the prevention and treatment of ovarian dysfunction. Therefore, it is of great practical significance to separate and extract the active ingredients in Potentilla fruticosa and apply them to the prevention and treatment of ovarian dysfunction.

[0004] The present invention provides a preparation method and application of a Herba Citri Reticulatae Extract for preventing and treating ovarian dysfunction. The Herba Citri Reticulatae Extract has obvious activity and excellent effect, and can effectively slow down the estrogen (E 2 ) and gonadotropin (GnRH) are attenuated, follicle-stimulating hormone (FSH) is increased, dependence on exogenous estrogen is reduced, and the incidence of endometrial cancer is reduced. The extract prepared by this method has stable physical and chemical properties, is easy to store and transport, has a simple preparation process, and is easy to operate; at the same time, no 1-3 type organic solvents are used in the production process, which is green, environmentally friendly, and has good safety. At the same time, the present invention provides methods for preparing different dosage forms of the extract.

[0005] Regarding the extract and application of Herba Citri Reticulatae, the Chinese patent application with authorization announcement number CN103961427B provides a scheme for extracting an extract for treating type 2 diabetes from Herba Citri Reticulatae branches and leaves, using Herba Citri Reticulatae branches and leaves as raw materials, crushing and then passing through a 20-mesh sieve, reflux extraction with 5 times the volume of 95% ethanol for 3 times, each time for 1 hour, suction filtration, combining the 3 filtrates, vacuum concentrating the filtrate and dispersing it in water, extracting and stratifying with 5 times the volume of petroleum ether, extracting and stratifying the obtained water layer with 5 times the volume of ethyl acetate, obtaining an ethyl acetate extract, and vacuum drying to obtain a Herba Citri Reticulatae extract for treating type 2 diabetes. And the extract is used to treat type 2 diabetes, and good technical effects have been achieved. The Chinese patent application with authorization announcement number CN107029028B provides a Herba Citri Reticulatae extract and a preparation method thereof, which is to cut the Herba Citri Reticulatae into pieces to obtain the Herba Citri Reticulatae powder, and then extract, filter, and dry to obtain the extract. The extract is used to inhibit human neuroblastoma cells, and good technical effects have been achieved. The Chinese patent application with application publication number CN112999273A obtained a water extract of Herba Citri Reticulatae Fruticosae, and the extract was used to prepare a drug for preventing, treating or inhibiting apoptosis of eye cells. In the prior art, Herba Citri Reticulatae Fruticosa ... Summary of the invention

[0006] The technical problem to be solved by the present invention is to invent a Herba Citri Reticulatae Extract for preventing and treating ovarian dysfunction. Studies have shown that the extract has a significant effect on preventing and treating ovarian dysfunction and has no side effects.

[0007] The second object of the present invention is to invent a method for preparing the above-mentioned Herba Citri Reticulatae Extract. Preferably, the method comprises the following steps: taking Herba Citri Reticulatae Dry Materials, crushing them with a grinder, and sieving them; adding ethanol aqueous solution, and decocting them in a decoction machine; solid-liquid separation, removing ethanol and water to obtain a crude extract; diluting the obtained crude extract with water, extracting it with petroleum ether, removing the petroleum ether part, and spray drying to obtain the extract powder.

[0008] Preferably, the extraction solvent concentration of the method is 48-52 v / v%, and the mass volume ratio (g:mL) of Potentilla fruticosa to ethanol aqueous solution is 1:6 to 1:10.

[0009] Preferably, the extraction temperature of the method is 40-80°C and the extraction time is 4-8h.

[0010] The third object of the present invention is to invent the use of the above-mentioned Herba Citri Reticulatae Extract in preventing and treating ovarian dysfunction. Preferably, the ovarian dysfunction is a disease such as diminished ovarian reserve (DOR), premature ovarian failure (POF), and menopausal syndrome.

[0011] The beneficial effects of using the extract of Fructus Aurantii in improving ovarian dysfunction are as follows: the classic cyclophosphamide-induced mouse ovarian dysfunction model was used to observe the improvement effect of different doses of Fructus Aurantii Extract on mouse uterine and ovarian dysfunction. The study showed that: (1) Fructus Aurantii Extract increased the ovarian and uterine indexes of mice in the cyclophosphamide-induced ovarian dysfunction model. (2) Fructus Aurantii Extract significantly increased the levels of estradiol and gonadotropin, decreased follicle-stimulating hormone, thickened endometrium, and increased body weight compared with the model group, and slightly decreased compared with the blank group.

[0012] The fourth object of the present invention is to invent the health care products and pharmaceutical preparations of the above-mentioned Potentilla fruticosa extract, and the specific dosage forms are granules, tablets, capsules, pills, oral liquids, etc.

[0013] Preferably, the granules are prepared by mixing the Herba Citri Reticulatae Extract and other active ingredients, fillers, and binders to form granules, wherein the Herba Citri Reticulatae Extract is 4-8 parts, the other active ingredients are 1-2 parts, the fillers are 2-4 parts, and the binder is appropriate.

[0014] Preferably, the tablet is prepared by adding a lubricant to the granules and pressing the granules into tablets, wherein the amount of the lubricant added is 0.3-0.6%.

[0015] Preferably, the capsule is prepared by: encapsulating the granules into capsules.

[0016] Preferably, the pills are prepared by mixing the Herba Citri Reticulatae Extract and other active ingredients, fillers, and adhesives to prepare a soft material, and then extruding and spheronizing the mixture, wherein the Herba Citri Reticulatae Extract is 4-8 parts, other active ingredients are 1-2 parts, fillers are 2-4 parts, and adhesives are appropriate.

[0017] Preferably, the oral liquid is prepared by dissolving the Herba Citris Fruticosae Extract and other active ingredients in a solvent, with or without preservatives, wherein the Herba Citris Fruticosae Extract comprises 4-8 parts and the other active ingredients comprise 1-2 parts.

[0018] Preferably, the fillers for granules, tablets, capsules and pills are sucrose, dextrin, cyclodextrin, microcrystalline cellulose and the like.

[0019] Preferably, the binder for granules, tablets, capsules and pills is starch slurry, PVP aqueous solution, alcohol solution and the like.

[0020] Preferably, the lubricant for the tablet is silicon dioxide, micro powder silica gel, etc.

[0021] Preferably, the solvent of the oral solution is water, alcohol solution or the like.

[0022] In the present invention, the amount of binder in the granules and pills is appropriate, which means that the amount used should meet the amount used in the preparation of the granules and pills, and the present invention does not limit the specific amount.

[0023] Preferably, in the use of the Herba Citri Reticulatae Extract of the present invention in preventing and treating ovarian dysfunction, the dosage of the Herba Citri Reticulatae Extract is 450-600 mg / kg.

[0024] The beneficial effects of the present invention are as follows: (1) a Herba Citri Reticulatae Extract for preventing and treating ovarian dysfunction is invented, the Herba Citri Reticulatae Extract has obvious activity and excellent effect; (2) a method for preparing the extract is invented, the preparation process is simple and the operation is convenient; at the same time, no 1-3 organic solvents are used in the production process, which is green, environmentally friendly and safe. The extract prepared by the method has stable physical and chemical properties and is easy to store and transport; (3) the application of the extract is invented, the extract can effectively slow down the estrogen (E 2 ) and gonadotropin (GnRH) are attenuated, follicle-stimulating hormone (FSH) is increased, dependence on exogenous estrogen is reduced, and the incidence of endometrial cancer is reduced. It is suitable for diseases caused by ovarian dysfunction such as diminished ovarian reserve (DOR), premature ovarian failure (POF), and menopausal syndrome. (4) Provide methods for preparing different dosage forms of the extract. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] Example 1 Extraction and quality evaluation of Potentilla fruticosa

[0027] (1) Extract preparation

[0028] Take the dried medicinal material of Herba Citri Reticulatae and grind it, and pass it through a No. 10 sieve. Add ethanol aqueous solution (ethanol: water = 1:1) according to Table 1, decoct in a Chinese medicine decoction machine, the extraction temperature and extraction time are shown in Table 1, filter, take the filtrate, remove ethanol and water, and obtain a crude extract; dilute the crude extract with water, separate it with petroleum ether, and then spray dry it to obtain Herba Citri Reticulatae extract powder.

[0029] Table 1 Preparation parameters of Potentilla fruticosa extract

[0030]

[0031] (2) Quality evaluation

[0032] The quality of the above-mentioned Potentilla fruticosa extract was evaluated, and the specific results are shown in Table 2:

[0033] Table 2 Quality indexes of extracts from Potentilla fruticosa

[0034] Serial number ① ② ③ Extraction rate, % 8.5 8.6 8.3 Polysaccharide content, % 10.0 10.2 10.1 Polyphenol content, % 15.0 15.2 14.9 Flavonoids and flavonoid glycosides content 13.2 13.3 13.1

[0035] It can be seen from the above table that the extracts prepared under various parameters have stable physical and chemical properties and uniform quality.

[0036] Example 2 Evaluation of the Effect of Potentilla Fruticosa Extract

[0037] Normal female Kunming mice were divided into five groups, namely, a young control group, an aging model group, a low-dose group, a high-dose group and a positive drug group, with 10 mice in each group. Except for the control group, which was injected with normal saline every day, the other experimental groups were intraperitoneally injected with cyclophosphamide (60 mg / kg) every 2 days within 10 days to prepare an ovarian dysfunction model. The golden plum extract in Example 1 was taken into the low-dose group, the high-dose group and the positive drug group, and 450 mg / kg and 600 mg / kg of golden plum extract and 0.085 mg / kg of estradiol were administered by gavage for 8 consecutive weeks. Blood was collected from the mouse eyeballs in the fourth and eighth weeks respectively for the detection of estradiol (E 2 ), gonadotropin (GnRH) and follicle stimulating hormone (FSH) levels. After the eighth week of drug administration, the rats were killed by cervical dislocation, and the ovaries and uterus were removed and weighed quickly to calculate the organ indexes. The left uterus and ovaries were fixed with 10% formalin, paraffin sliced, and HE stained. The average thickness of the endometrium was measured with a micrometer under a microscope. The number of total ovarian follicles was counted, and the morphology and number of layers of granulosa cells were observed, as well as the presence of fibrosis and necrosis in the tissue.

[0038] The effects of the extract of the present invention on the uterine and ovarian indexes of female mice, the serum (E 2 ), the levels of gonadotropin (GnRH) and follicle stimulating hormone (FSH), and the average thickness of the endometrium in female mice are shown in Tables 3 to 5.

[0039] As shown in Table 3, compared with the blank group, the uterus and ovary indexes of the model group mice were significantly reduced. Compared with the model group, the uterus and ovary indexes of the Herba Fruticosae Extract Group and the estradiol Group of the present invention were significantly increased. Compared with the model group, the body weight was increased, and compared with the blank group, it was slightly decreased.

[0040] Table 3 Effect of the extract of Potentilla fruticosa on body weight, ovary and uterine index of mice

[0041] Group Mouse weight gain or loss / g Ovarian index / % Uterine index / % blank +3.52g±0.40 35.12±3.05％ 145.00±1.02％ Model -3.52g±0.80 18.75±2.04％ 70.10±2.35％ High dose group +1.14±0.99 34.82±1.11％ 144.95±1.33％ Low dose group +1.45±0.45 26.18±2.00％ 108.98±1.91％ Estradiol +1.53±0.98 32.10±1.15％ 140.10±1.71％

[0042] From the above data, it can be seen that the estradiol hormone in the mouse ovarian index and uterine index in the high-dose group is stronger than that in the positive control group, which is beneficial to the repair of ovarian function decline.

[0043] As shown in Table 4, compared with the blank group, the E 2 , GnRH and FSH levels were significantly decreased, and the E 2 and GnRH levels were significantly reduced. Compared with the model group, the FSH level of the mice in the model group was significantly increased. The present invention shows that the Herba Citri Reticulatae Extract Group and the Estradiol Group significantly increase the levels of estradiol and GnRH in the serum of mice and significantly reduce the FSH level.

[0044] Table 4 E in the serum of mice in each drug administration group 2 , GnRH and FSH content determination results

[0045]

[0046] Compared with the blank group, in the high-dose group, the estrogen level of mice increased with the length of time of drug feeding, while gonadotropin and follicle-stimulating hormone remained basically unchanged, and the results were better than the positive control.

[0047] As shown in Table 5, the average endometrial thickness of the group containing the extract of the present invention is significantly higher than that of the model group, and the difference is significant.

[0048] Table 5 Effect of the extract of the present invention on the average thickness of the mouse endometrium

[0049] Group Endometrial thickness (um) blank 399.93±13.34 Model 236.84±17.35 Extract high dose 434.84±6.91 Extract low dose 355.31±16.63 Estradiol 344.43±10.45

[0050] The high-dose group of Potentilla fruticosa extract is beneficial to the repair of endometrial thickness in mice.

[0051] Example 3

[0052] Different from Example 1, in the preparation method of the Herba Citri Reticulatae Extract of this embodiment, the Herba Citri Reticulatae Extract and the ethanol aqueous solution can be arbitrarily selected in the range of 1g:6-10mL by mass volume ratio, wherein the concentration of the ethanol aqueous solution can be arbitrarily selected in the range of 48-52v / v%. The extraction temperature is 40-80°C, the extraction time is 4-8h, and the rest of the method is the same, and the obtained Herba Citri Reticulatae Extract has the same effect as that of Example 1.

[0053] Example 4 Preparation of Different Formulations of Potentilla Fruticosa Extract

[0054] (1) Granules

[0055] According to the following proportions: 6 parts of Herba Citri Reticulatae Extract, 1 part of Vitamin B1 and 3 parts of cyclodextrin are mixed and then an appropriate amount of starch slurry is added to prepare a soft material, and the soft material is granulated to obtain Herba Citri Reticulatae Granules.

[0056] (2) Tablets

[0057] Take the granules described in step (1), add 0.5% silicon dioxide, and compress them into tablets to prepare Herba Citri Reticulatae Fruticosae tablets.

[0058] (3) Capsules

[0059] The granules described in step (1) are taken and filled into capsules to prepare Herba Citri Reticulatae Fructus Capsules.

[0060] (4) Pills

[0061] The following proportions are used: 6 parts of Herba Citri Reticulatae Extract, 1 part of Vitamin B1, and 3 parts of cyclodextrin are mixed, and then an appropriate amount of starch slurry is added to prepare a soft material, and the soft material is extruded and rolled into a round shape to obtain Herba Citri Reticulatae Pills.

[0062] (5) Oral solution

[0063] Add purified water in the following proportions: 6 parts of Potentilla fruticosa extract and 11 parts of vitamin B1, and sterilize at 121°C for 15 minutes.

[0064] Example 5 Preparation of different dosage forms of Herba Citri Reticulatae Extract

[0065] (1) Granules

[0066] According to the following proportions: 4 parts of Herba Citri Reticulatae Extract, 1 part of Vitamin B1 and 2 parts of sucrose are mixed and then an appropriate amount of PVP aqueous solution is added to prepare a soft material, and the soft material is granulated to obtain Herba Citri Reticulatae Granules.

[0067] (2) Tablets

[0068] Take the particles described in step (1), add 0.3% of micro-powdered silica gel, and compress them into tablets to prepare Herba Citri Reticulatae Fruticosae tablets.

[0069] (3) Capsules

[0070] The granules described in step (1) are taken and filled into capsules to prepare Herba Citri Reticulatae Fructus Capsules.

[0071] (4) Pills

[0072] The following proportions are used: 8 parts of Herba Citri Reticulatae Extract, 2 parts of Vitamin B12, and 4 parts of sucrose are mixed, and then an appropriate amount of PVP aqueous solution is added to prepare a soft material, and the soft material is extruded and rolled into a round shape to obtain Herba Citri Reticulatae Pills.

[0073] (5) Oral solution

[0074] Add purified water in the following proportions: 4 parts of Potentilla fruticosa extract and 1 part of vitamin B1, and sterilize at 121°C for 15 minutes.

[0075] Example 6 Preparation of different dosage forms of Potentilla fruticosa extract

[0076] (1) Granules

[0077] According to the following proportions: 8 parts of Herba Citri Reticulatae Extract, 2 parts of Vitamin B1, and 4 parts of cyclodextrin are mixed, and then an appropriate amount of PVP aqueous solution is added to prepare a soft material, and the soft material is granulated to obtain Herba Citri Reticulatae Granules.

[0078] (2) Tablets

[0079] Take the particles described in step (1), add 0.6% silicon dioxide, and compress them into tablets to prepare Herba Citri Reticulatae Fruticosae tablets.

[0080] (3) Capsules

[0081] The granules described in step (1) are taken and filled into capsules to prepare Herba Citri Reticulatae Fructus Capsules.

[0082] (4) Pills

[0083] The following proportions are used: 4 parts of Herba Citri Reticulatae Extract, 2 parts of Vitamin B12, and 2 parts of Microcrystalline Cellulose are mixed and then an appropriate amount of starch slurry is added to prepare a soft material, and the soft material is extruded and rolled into a round shape to obtain Herba Citri Reticulatae Pills.

[0084] (5) Oral solution

[0085] Add purified water in the following proportions: 8 parts of Potentilla fruticosa extract and 2 parts of vitamin B1, and sterilize at 121°C for 15 minutes.

[0086] Based on the above embodiments, the present invention discloses a method for preparing a Herba Citri Reticulatae Extract, verifies the components of the Herba Citri Reticulatae Extract obtained, and evaluates the effects of the extract on the uterus and ovaries. The Herba Citri Reticulatae Extract obtained by the method of the present invention has stable physical and chemical properties and obvious activity, and can effectively slow down the estrogen (E 2 ) and gonadotropin (GnRH) are attenuated, follicle-stimulating hormone (FSH) is increased, dependence on exogenous estrogen is reduced, and the incidence of endometrial cancer is reduced. The present invention further uses the obtained golden plum extract to prepare health products and pharmaceutical preparations for preventing and treating ovarian dysfunction. After reasonable combination verification, granules, tablets, capsules, pills or oral liquids can be prepared for preventing and treating ovarian dysfunction, with outstanding effects and safety.

[0087] It should be further explained that the above implementation modes are only used for understanding the technical solutions of the present invention, and are not used to limit the protection scope of the present invention. Any obvious adjustments and modifications made to the technical solutions within the technical concept of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for preparing a Herba Citri Reticulatae Extract for preventing and treating ovarian dysfunction, characterized in that: The method comprises the following steps: (1) crushing Potentilla fruticosa at high speed and collecting the crushed powder; (2) taking the powder of step (1), adding 48-52 v / v% ethanol aqueous solution at a mass volume ratio of 1 g: 6-10 mL, extracting and refluxing at a temperature of 40-80° C. for 4-8 h to obtain an extract; (3) taking the extract from step (2), filtering, and removing ethanol and water from the filtrate to obtain a crude extract; (4) taking the crude extract of step (3), adding petroleum ether for extraction, removing the petroleum ether, and the lower layer is the refined product; (5) taking the refined product of step (4), spray drying it to obtain a Potentilla fruticosa extract.

2. A Herba Citri Reticulatae Extract for preventing and treating ovarian dysfunction, characterized in that: It is prepared by the method according to claim 1.

3. Use of the Potentilla fruticosa extract for preventing and treating ovarian dysfunction as claimed in claim 2 in the preparation of health products and pharmaceutical preparations for preventing and treating ovarian dysfunction.

4. The use according to claim 3, characterized in that It includes health products and pharmaceutical preparations containing single or compound extracts of Potentilla fruticosa. The specific dosage form is but not limited to granules, tablets, capsules, pills or oral liquids.

5. The use according to claim 4, characterized in that The granules are obtained by mixing and granulating the Herba Citri Reticulatae Extract and other active ingredients, fillers and adhesives, wherein the Herba Citri Reticulatae Extract is 4-8 parts, the other active ingredients are 1-2 parts, the fillers are 2-4 parts and the adhesive is in an appropriate amount; the other active ingredients are vitamin B1; the fillers are sucrose, dextrin, cyclodextrin or microcrystalline cellulose; and the adhesive is starch slurry, PVP aqueous solution or alcohol solution.

6. The use according to claim 4, characterized in that The tablet is prepared by adding 0.3-0.6% of lubricant to the granules according to claim 5 and pressing the tablet, wherein the lubricant is silicon dioxide or micro-powder silica gel.

7. The use according to claim 4, characterized in that The capsule is prepared by encapsulating the granule according to claim 5.

8. The use according to claim 4, characterized in that The pills are prepared by mixing the Herba Citri Reticulatae Extract and other active ingredients, a filler and an adhesive to prepare a soft material, and then extruding and spheronizing the mixture, wherein the Herba Citri Reticulatae Extract comprises 4-8 parts, other active ingredients comprise 1-2 parts, a filler comprises 2-4 parts, and an appropriate amount of an adhesive; the other active ingredient is vitamin B1; the filler is sucrose, dextrin, cyclodextrin or microcrystalline cellulose; and the adhesive is starch slurry, a PVP aqueous solution or an alcohol solution.

9. The use according to claim 4, characterized in that The oral liquid is prepared by dissolving the Herba Citri Reticulatae Extract and other active ingredients in a solvent, with or without adding preservatives, wherein the Herba Citri Reticulatae Extract is 4-8 parts and the other active ingredients are 1-2 parts; the other active ingredient is vitamin B1.

10. The use according to any one of claims 3 to 9, characterized in that: The dosage of the extract of Herba Citri Reticulatae is 450-600 mg / kg.

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