阅读说明：本技术 桂花提取物在制备卵巢保养中药产品中的用途 (Application of sweet osmanthus extract in preparation of ovarian maintenance traditional Chinese medicine product ) 是由 陈心煜 宋萍萍 钱怡云 吕晔 韦敏 颜露 于 2021-10-30 设计创作，主要内容包括：本发明公开了桂花提取物在制备提高卵巢颗粒细胞活性的中药产品中的新用途。本发明根据卵巢功能失调和卵巢颗粒细胞生长不足的发病机制,充分利用中药资源,在制备桂花提取物的基础上,通过药理实验,开发出桂花提取物提高卵巢颗粒细胞活性的新用途,该提取物可以添加至不同剂型的产品中去,通过促进卵巢颗粒细胞增殖和提高卵巢颗粒细胞的雌孕激素分泌水平进而达到保养卵巢的活性,临床上用于治疗卵巢功能失调,具有疗效好、有效成分明确、副作用小、使用方便等优点。(The invention discloses a new application of an osmanthus fragrans extract in preparing a traditional Chinese medicine product for improving the activity of ovarian granulosa cells. According to the invention, according to the pathogenesis of ovarian dysfunction and insufficient growth of ovarian granulosa cells, traditional Chinese medicine resources are fully utilized, on the basis of preparing the osmanthus fragrans extract, a new application of the osmanthus fragrans extract in improving the activity of the ovarian granulosa cells is developed through pharmacological experiments, the extract can be added into products of different formulations, the activity of maintaining the ovaries is achieved by promoting the proliferation of the ovarian granulosa cells and improving the secretion level of estrogen and progestogen of the ovarian granulosa cells, and the osmanthus fragrans extract is clinically used for treating ovarian dysfunction and has the advantages of good curative effect, clear effective components, small side effect, convenience in use and the like.)

1. Application of osmanthus fragrans extract in preparing traditional Chinese medicine products for improving ovarian granulosa cell activity.

2. The use of claim 1, wherein the osmanthus fragrans extract is used for preparing a traditional Chinese medicine product for promoting the ovarian granulosa cell proliferation activity.

3. The use according to claim 1, characterized in that the osmanthus fragrans extract is used for preparing a traditional Chinese medicine product for improving the estrogen-progestogen secretion level of ovarian granulosa cells.

4. The use according to claim 1, characterized in that the osmanthus fragrans extract is used as an active ingredient and is prepared into any one dosage form of a traditional Chinese medicine product with common auxiliary materials; or can be used as an active ingredient, other traditional Chinese medicine extracts for regulating the ovarian function and common auxiliary materials to prepare any one of the allowable dosage forms of the traditional Chinese medicine preparation products.

5. The use of claim 4, wherein the dosage form is selected from the group consisting of tablets, gels, creams, pills, liquids, granules, soft capsules, drop pills, ointments, and the like.

6. The use according to claim 4, wherein the adjuvants can be common adjuvants for the preparation, and other Chinese medicinal extracts for combination are Chinese medicinal extracts (alcohol extract or water extract) for regulating ovarian function, such as radix Angelicae sinensis extract, fructus Ligustri Lucidi extract, radix Codonopsis extract, rehmanniae radix extract, semen Cuscutae extract, etc.

7. The osmanthus extract according to claim 1, wherein: the preparation method comprises extracting flos Osmanthi Fragrantis with water to obtain dried solid powder of water extract, wherein the extraction method can be decocting method, reflux extraction method, etc.

Technical Field

The invention belongs to the field of traditional Chinese medicine treatment, and particularly relates to a new application of an osmanthus fragrans extract in preparation of a traditional Chinese medicine product for improving ovarian granulosa cell activity.

Background

The physiological functions of the ovary comprise ovulation and endocrine functions, and have important significance for female reproductive function, physical and mental health and life quality. Ovarian dysfunction can cause ovulation dysfunction, corpus luteum dysfunction, endocrine disturbance and the like, so that various gynecological diseases such as uterine hypoplasia, infertility, premature ovarian failure, perimenopausal syndrome and the like can be caused, and physiological functions of other organs of the whole body can be influenced.

The ovary is an important reproductive organ of a female animal reproductive system and has two important roles, namely, the generation of mature ovum and the secretion of steroid hormone. Granulosa cells are the main functional cells of the ovary and are also important synthesis sites for estrogen and progestogen secreted by the ovary. The proliferation and differentiation of granulosa cells can be used as markers for follicular development. Granulosa cells are the largest cell population in mature follicles, the only cells expressed in females, and the metabolism of oocytes needs to be met by granulosa cells. Small molecule metabolites such as energy substrates, nucleotides, and amino acids are taken up from the surrounding granular cell media through gap junctions.

Premature ovarian failure means to some extent that atretic follicles increase and growing follicles decrease. And the ovarian Granulosa Cell (GC) is an important somatic cell in the follicle and is an important mark for the development of the follicle, and the apoptosis of the ovarian granulosa cell plays an important role in regulating the growth of the follicle, the maturation of the oocyte and the secretion of estrogen.

At present, western medicine mainly relies on means such as hormone replacement, ovulation promotion treatment and the like in promoting the proliferation of ovarian granulosa cells and regulating the ovarian function, achieves a certain curative effect, but has a plurality of side effects, and traditional Chinese medicine has the overall regulation effect of multiple systems, multiple links and multiple targets, has few side effects and has certain advantages in regulating the ovarian function. Therefore, it is necessary to develop a product for treating ovarian dysfunction and promoting the activity of ovarian granulosa cells to further maintain the ovaries, which has definite effective substances, definite curative effect, small side effect and convenient administration, under the guidance of the traditional Chinese medicine theory.

The flos Osmanthi Fragrantis is Olea officinalis of OleaceaeOsmanthus fragrans(Thunb.) dried flowers of lour. Collected when 9-10 months flowers bloom, and dried in the shade. Produced all over the country. Research reports that the sweet osmanthus has the effects of soothing liver, regulating qi, eliminating phlegm, relieving cough, smoothing lung and stimulating appetite. Literature research shows that the osmanthus fragrans contains phenols, iridoids, triterpenes, lignans, phenylpropanoids, phenethyl alcohol, alkaloids and other chemical components, so that the osmanthus fragrans has good medicinal value. Modern pharmacological research shows that the pharmacological activity of the osmanthus fragrans mainly comprises the following components: antioxidant, antibacterial, antiinflammatory, melanin generation inhibiting, antiaging, antitumor and neuroprotective effects. At present, the application of the osmanthus fragrans extract in preparing a traditional Chinese medicine product for improving the activity of ovarian granulosa cells is not reported.

Disclosure of Invention

The invention aims to explore the application of the osmanthus fragrans extract in preparing a traditional Chinese medicine product for improving the activity of ovarian granulosa cells.

In order to achieve the purpose, the invention adopts the technical scheme that: the preparation method of the osmanthus fragrans water extract comprises the following steps: pulverizing flos Osmanthi Fragrantis, sieving, adding appropriate amount of water, soaking, extracting under reflux for 2-4 hr, concentrating the extractive solution under reduced pressure to obtain solid powder, and drying to obtain flos Osmanthi Fragrantis water extract.

The osmanthus fragrans extract can be obtained by the preparation method, and can also be purchased commercially to obtain the osmanthus fragrans water extract.

The invention relates to an osmanthus fragrans extract, which mainly achieves the purpose of nourishing ovaries by improving the activity of ovarian granulosa cells and further regulating and controlling ovarian dysfunction by promoting the proliferation of the ovarian granulosa cells and improving the estrogen-progestogen secretion level of the ovarian granulosa cells.

The sweet osmanthus extract can be prepared into a product with any pharmaceutically allowed auxiliary material or pharmaceutical excipient, and the preparation can be any pharmaceutically allowed dosage form, including but not limited to tablets, gels, creams, pills, liquid preparations, granules, medicinal granules, soft capsules, dripping pills or ointments; or can be used as an active ingredient, other traditional Chinese medicine extracts for regulating the ovarian function and common auxiliary materials to prepare any allowable preparation formulation on the ovarian nourishing preparation. The adjuvants can be common adjuvants of the above dosage forms, and can be compounded with other Chinese medicinal extracts (such as ethanol extract or water extract), such as radix Angelicae sinensis extract, fructus Ligustri Lucidi extract, radix Codonopsis extract, rehmanniae radix extract, semen Cuscutae extract, etc.

Compared with the prior art, the invention has the beneficial effects that: the osmanthus fragrans extract has a remarkable proliferation effect on ovarian granulosa cells, and can remarkably improve the estrogen-progestogen secretion level of the ovarian granulosa cells so as to achieve the effect of nourishing ovaries. The sweet osmanthus extract can be used for preparing a traditional Chinese medicine product for improving the activity of ovarian granulosa cells and regulating the ovarian function. The invention provides a new application of the osmanthus extract and enlarges the application range of the osmanthus extract.

Detailed Description

The effects of the present invention are further illustrated by the following specific embodiments:

example 1: preparation of Osmanthus Water extract

Taking dry sweet osmanthus medicinal materials, crushing and sieving, adding 10 times of water for soaking, carrying out hot reflux extraction for 2-4 hours, and carrying out reduced pressure concentration on an extracting solution to obtain solid dry powder, namely the sweet osmanthus water extract, wherein the extraction rate is 10.07%.

Example 2: activity study of sweet osmanthus extract on ovarian granulosa cells

1 Material

1.1 Experimental materials

22-27 days old female rats and a laboratory animal feeding room have the advantages of room temperature (22 +/-2) DEG C, 55-65% of humidity, alternate illumination day and night and good ventilation. CO 2₂An incubator, a 0.22 mu m microporous filter membrane, a cell culture plate, a purification workbench, an enzyme linked immunosorbent assay detector, an electronic balance, an inverted biological microscope, an electric heating constant temperature water tank and a full-automatic centrifuge.

Pregnant Mare Serum Gonadotropin (PMSG), DMEM is a product of American GIBCO company, and calf serum (DBCS) is a product of JRH biosciences company; dimethylsulfoxide, Nanjing chemical reagents Ltd; MTT is prepared by subpackaging thiazole blue (MTT) by Ultrapuregrade and Amresco, preparing into 50mg/LMTT solution by Phosphate Buffer Solution (PBS) before use, and storing in a refrigerator at 4 ℃; penicillin; streptomycin; DMEM-F12 medium.

1.2 Experimental samples

Sample set 1 is example 1; sample group 2 is osmanthus fragrans water extract 10: 1 (extraction method is hot water extraction, spray drying the obtained extract dry powder, yield is 10%), purchased from Sikanbaichuan Biotech Co., Ltd.

2 Experimental methods and procedures

2.1 isolation and Primary culture of ovarian granulosa cells

Female rats of 22-27 days old (PMSG injected subcutaneously 48h in advance, 66U/rat) were taken, sacrificed by cervical dislocation after 48h of conventional feeding, bilateral ovaries of the rats were aseptically harvested, the ovaries were disrupted to release granulocytes, diluted with 2mL of DMEM-F12 medium containing penicillin and streptomycin, and gently blown to disperse into single cells. The granular cells were collected by centrifugal filtration.

2.2 ovarian granulosa cell proliferation function assay

The cell suspension with a certain concentration is inoculated in a 24-well plate, each well is 80 mul, each well is added with 120 mul of culture solution to adjust the cell density to 200 mul/well, and each group is provided with 6 parallel wells. It was placed in 5% CO₂Culturing in an incubator for 24 h. Taking CO₂The granules cultured in the incubator for 24 hours are fineAdding molding concentration cadmium chloride and sample (2 mg. mL) into each well of the cell suspension successively^-1) In parallel, a normal control group (physiological saline) and a model group (cadmium chloride and physiological saline) were set. After 20 h of conventional culture, MTT solution (5 mg. multidot.mL) was added to each well^-1) And continuously culturing for 4h by 20 muL, adding 80 muL of triple liquid into each hole, culturing overnight, selecting a wavelength of 570 nm, measuring the absorbance of each hole on an enzyme-linked immunosorbent assay, and expressing the experimental result in x +/-s. Data were processed using SPSS 16.0 statistical software and the comparisons between groups were performed using the t-test. P<0.05 is statistically significant.

2.3 analysis of ovarian granulosa cell Egestagen secretion Capacity

Samples and controls were treated as described in method 2.2, and after overnight incubation, radioimmunoassays were used (to determine the levels of estradiol (E2) and progesterone (P) in the cell suspension supernatants).

3 results and analysis of the experiments

3.1 proliferation of isolated ovarian granulosa cells by samples

For ovarian granulosa cells, 2 samples had proliferative effects, with very significant differences for the model group (p < 0.01). The water extract of the osmanthus has the activity of promoting the proliferation of ovarian granulosa cells.

TABLE 1 proliferation of isolated ovarian granulosa cells by extracts (n = 6)

*P<0.05，**P<0.01 vsAnd (4) model groups.

3.2 analysis of ovarian granulosa cell Egestagen secretion Capacity

The samples were treated in the same manner as the control group and after overnight incubation, radioimmunoassays were used (the contents of estradiol (E2) and progesterone (P) in the cell suspension supernatants were determined and the results are shown in Table 2.

TABLE 2 oestrogen secretion by extracts on isolated ovarian granulosa cells (n = 3)

*P<0.05，**P<0.01 vsAnd (4) model groups.

And (4) conclusion: for ovarian granulosa cells, 2 samples had the ability to increase estrogen secretion from ovarian granulosa cells, with very significant differences or significant differences for the model group (p <0.01 or p < 0.05). Therefore, the osmanthus fragrans extract can obviously improve the secretion capacity of the in vitro ovarian granular cell estrogen.

Example 3: osmanthus fragrans flower extract emulsifiable paste

1g of sweet osmanthus extract, 100g of stearic acid, 20g of cetyl alcohol, 10g of glycerin monostearate, 10g of liquid paraffin, 0.8g of methylparaben, 0.2g of butylparaben, 140g of glycerol, 5g of potassium hydroxide, 10g of ethanol and 1000g of distilled water.

Example 4: osmanthus fragrans extract gel

1g of sweet osmanthus extract, PEG 400450 g, 80g of hexanediol, 8020g of Tween, 30g of carbomer, 6ml of ethanolamine, 10g of benzyl alcohol and distilled water added to 1000 g.

Example 5: osmanthus fragrans extract capsule

100g of sweet osmanthus extract, 80g of starch, 24g of sodium carboxymethyl starch, 17g of dextrin and a proper amount of 70% ethanol, and 1000 granules (each granule contains 100mg of sweet osmanthus extract).

Example 6: osmanthus fragrans extract tablet

1000g of sweet osmanthus extract, 55g of lactose, 15g of compressible starch, 10g of hydroxypropyl cellulose, 5g of magnesium stearate and a proper amount of 70% ethanol, and 1000 tablets (each tablet contains 1000mg of sweet osmanthus extract) are prepared.

Example 7: osmanthus fragrans extract granule

10g of sweet osmanthus extract, 800g of lactose powder, 194g of magnesium stearate and a proper amount of 70% ethanol, and the sweet osmanthus extract is prepared into 1000g of granules (each g of granules contains 10mg of sweet osmanthus extract).

Example 8: osmanthus fragrans extract soft capsule

50g of sweet osmanthus extract, 10g of soybean oil, 5g of gelatin, 5g of glycerol and 7g of distilled water, and 1000 granules (each granule contains 50mg of sweet osmanthus extract) are prepared.

Example 9: sweet osmanthus extract and Chinese angelica extract composition cream

1g of sweet osmanthus extract, 1g of angelica extract, 100g of stearic acid, 20g of cetyl alcohol, 10g of glyceryl monostearate, 10g of liquid paraffin, 0.8g of methylparaben, 0.2g of butylparaben, 140g of glycerol, 5g of potassium hydroxide, 10g of ethanol and the total amount of distilled water is 1000 g.

Example 10: osmanthus fragrans flower extract and codonopsis pilosula extract composition gel

1g of sweet osmanthus extract, 1g of medlar water extract, PEG 400450 g, 80g of hexanediol, 8020g of Tween, 30g of carbomer, 6ml of ethanolamine, 10g of benzyl alcohol and 1000g of distilled water.

Example 11: osmanthus fragrans flower extract and privet extract composition gel

1g of sweet osmanthus extract, 1g of privet water extract, PEG 400450 g, 80g of hexanediol, 8020g of Tween, 30g of carbomer, 6ml of ethanolamine, 10g of benzyl alcohol and distilled water added to 1000 g.

Example 12: osmanthus fragrans flower extract and semen cuscutae extract composition gel

1g of sweet osmanthus extract, 1g of semen cuscutae extract, PEG 400450 g, 80g of hexanediol, 8020g of Tween, 30g of carbomer, 6ml of ethanolamine, 10g of benzyl alcohol and distilled water which are added to 1000 g.

Example 13: sweet osmanthus extract and rehmannia root extract composition gel

1g of sweet osmanthus extract, 1g of rehmannia root extract, PEG 400450 g, 80g of hexanediol, 8020g of Tween, 30g of carbomer, 6ml of ethanolamine, 10g of benzyl alcohol and distilled water which are added to 1000 g.