Preparation method and application of a compound polysaccharide composition for delaying reproductive senescence

Abstract

The application provides a preparation method of a compound polysaccharide composition for delaying reproductive aging, and belongs to the technical field of traditional Chinese medicine extracts. The preparation method comprises the following steps: in step one, the raw materials are weighed and mixed, then soaked in an organic solvent to remove the fat-soluble part, and then filtered to obtain the residue and the filtrate; in step two, water is added to the residue of step one to perform reflux extraction, and the extraction liquid is combined; in step three, the extraction liquid of step two is concentrated, then ethanol is added to perform alcohol precipitation, and the precipitate is obtained by filtration; in step four, the precipitate obtained in step three is dissolved with water, then purified with ethanol to obtain a crude extract; and in step five, the protein in the crude extract is removed by the Sevag method, and the compound polysaccharide composition is obtained by freeze-drying. The compound polysaccharide composition obtained by the application mainly comprises mannose, glucose, galactose, arabinose, a small amount of rhamnose and galacturonic acid, and research shows that the compound polysaccharide composition can effectively delay reproductive aging.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extract technology, specifically relating to a method for preparing and applying a compound polysaccharide composition for delaying reproductive aging. Background Technology

[0002] Due to environmental and social factors, the incidence of premature decline in female reproductive function (early-onset ovarian insufficiency and premature menopause) has been on the rise in recent years. Female reproductive aging leads to a series of health problems. First, there is a decline in fertility and reproductive quality. Second, reproductive aging initiates the aging process in the female body. With the significant increase in human lifespan, women spend one-third to one-half of their lives after reproductive function declines. The cardiovascular, skeletal, cognitive, and metabolic disorders caused by reproductive aging seriously affect women's health and quality of life. Reproductive aging is a continuous and gradual process, related to genetic, socio-psychological, environmental, immune, and inflammatory factors. How to provide effective protection during reproductive aging to slow down its progression is an urgent need in today's society.

[0003] Current strategies for preventing and treating reproductive aging mainly include antioxidants, small molecule compounds or plant extracts that improve mitochondrial function, stem cell therapy, and hormone supplementation. Traditional Chinese medicine (TCM) has shown efficacy in anti-oxidation, anti-apoptosis, regulation of the female reproductive endocrine axis, and improving pregnancy rates in older women using assisted reproductive technologies; therefore, its role in delaying reproductive aging is receiving increasing attention.

[0004] Prior patent CN107213311A discloses a traditional Chinese medicine composition for treating premature ovarian insufficiency, comprising the following raw materials in parts by weight: Astragalus membranaceus 150 parts, Angelica sinensis 30-45 parts, Dioscorea opposita 30-45 parts, Rehmannia glutinosa 30-45 parts, Epimedium brevicornu 30-45 parts, Cuscuta chinensis 30-45 parts, and Adenophora stricta 30-45 parts. This invention's traditional Chinese medicine composition for treating premature ovarian insufficiency addresses both the congenital and acquired deficiencies, harmonizing Qi and blood, and achieving the effects of "strengthening the spleen and kidneys, replenishing Qi and nourishing blood," thus improving menstrual conditions and serum endocrine levels in patients with premature ovarian insufficiency. A systematic understanding of the active substances in traditional Chinese medicine (TCM) is key to elucidating the essence of its effects. Based on this, the inventors' team then conducted research on the active substance basis of TCM compound formulas. They discovered that TCM compound polysaccharide compositions obtained by changing the preparation method have better effects on promoting the recovery of residual follicles and improving declining ovarian function, and the effects are superior to those of TCM compositions disclosed in prior patents. Furthermore, no research has been found on the efficacy of TCM compound polysaccharides in protecting female fertility. Therefore, they proposed a preparation method and application of a compound polysaccharide composition for delaying reproductive aging. Summary of the Invention

[0005] Based on the above, the purpose of this invention is to provide a method for preparing and applying a compound polysaccharide composition for delaying reproductive aging.

[0006] On one hand, the present invention provides a method for preparing a compound polysaccharide composition for delaying reproductive aging, comprising the following steps:

[0007] Step 1: Weigh all the raw materials, mix them, soak them in an organic solvent to remove the fat-soluble parts, then filter, and discard the liquid and remove the residue;

[0008] Step 2: Add water to the residue from Step 1 and reflux for extraction. After boiling, continue extraction for 30-120 minutes. Repeat the extraction 2-5 times and combine the extracts.

[0009] Step 3: Concentrate the extract from Step 2 to a crude drug content of 3-5 g / mL, add ethanol for alcohol precipitation, and filter to obtain the precipitate;

[0010] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with ethanol to obtain the crude extract;

[0011] Step 5: Remove proteins from the crude extract using the Sevag method, and freeze-dry to obtain the compound polysaccharide composition.

[0012] Preferably, the raw materials mentioned in step one include, by weight, the following: 25-50 parts of Astragalus membranaceus, 5-20 parts of Angelica sinensis, 5-15 parts of Dioscorea opposita, 5-20 parts of Rehmannia glutinosa, 1-20 parts of Epimedium brevicornu, 5-25 parts of Cuscuta chinensis, and 5-25 parts of Adenophora stricta.

[0013] More preferably, the raw materials mentioned in step one include, by weight, 50 parts of Astragalus membranaceus, 10 parts of Angelica sinensis, 10 parts of Dioscorea opposita, 10 parts of Rehmannia glutinosa, 10 parts of Epimedium brevicornu, 10 parts of Cuscuta chinensis, and 10 parts of Adenophora stricta.

[0014] Preferably, the organic solvent in step one is 90-95% ethanol, the soaking time is 0.5-1 h, and the soaking temperature is 20-40℃.

[0015] Preferably, the process of removing residue and discarding liquid in step one involves collecting the residue and discarding the liquid; the residue is then dried at 40°C.

[0016] Preferably, the amount of water added in step two is 6-20 times the water volume, more preferably 12 times the water volume; the number of extractions is 2-5 times, and the extraction time is 60-120 minutes; even more preferably, the number of extractions is 3 times, and the extraction time is 90 minutes.

[0017] Preferably, the concentration in step three is achieved by rotary evaporation.

[0018] Preferably, the ethanol concentration in steps three and four is 75-85%; more preferably 80%.

[0019] Preferably, the mixture after the alcohol precipitation step in step three needs to be left to stand overnight at 4°C.

[0020] Preferably, the reagent used in the Sevag method in step five is a mixed solution of chloroform and n-butanol, with a volume ratio of 4:1; the volume ratio of the Sevag reagent to the crude extract solution is 1:4, and the process is repeated 3-6 times except for proteins.

[0021] Preferably, the Sevag method for removing proteins from the crude extract in step five is as follows: thoroughly mix using a vortex mixer, centrifuge at 3000 r / min for 15 minutes to remove the intermediate protein layer and the lower solvent layer.

[0022] On the other hand, the present invention also provides the application of the above-mentioned compound polysaccharide composition in the preparation of pharmaceutical formulations for delaying female reproductive aging.

[0023] In another aspect, the present invention provides a pharmaceutical preparation for delaying female reproductive aging, the pharmaceutical preparation comprising the above-mentioned compound polysaccharide composition and a pharmaceutically acceptable carrier.

[0024] Furthermore, the pharmaceutically acceptable carrier refers to conventional drug carriers in the pharmaceutical field, such as: diluents, excipients like water, fillers like starch and sucrose; humectants like glycerin; binders like cellulose derivatives, alginate, and gelatin; absorption enhancers like quaternary ammonium compounds; surfactants like hexadecyl alcohol; adsorbents like kaolin and soap clay; disintegrants like agar, calcium carbonate, and sodium bicarbonate; and lubricants like talc, calcium stearate, and polyethylene glycol. Sweeteners and flavorings may also be used.

[0025] Furthermore, the dosage forms of the pharmaceutical preparations include aqueous solutions, granules, powders, tablets, capsules, pastes, mixtures, or pills.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) The compound polysaccharide composition of the present invention is mainly composed of mannose, glucose, galactose, arabinose and a small amount of rhamnose and galacturonic acid. The compound polysaccharide composition obtained by using an extraction time of 90 min, an alcohol precipitation concentration of 80%, and an extraction water volume multiple of 12 times has a better effect on scavenging DPPH free radicals.

[0028] (2) The compound polysaccharide composition obtained in this invention can effectively improve the atrophy of reproductive organs in naturally aging rats; effectively improve the disordered estrous cycle in aging rats; effectively delay the decline of AMH level in aging rats and increase estrogen and testosterone levels; significantly increase the number of primitive, primary, dominant and total follicles in the ovaries of aging rats; significantly increase SOD and GSH-Px levels and decrease MDA level; and significantly reduce the expression levels of apoptosis-related proteins p53, BAX and BAK. Attached Figure Description

[0029] Figure 1 The freeze-dried powder of the traditional Chinese medicine polysaccharide composition of this invention;

[0030] Figure 2 Molecular weight range distribution diagram of the traditional Chinese medicine polysaccharide composition of this invention;

[0031] Figure 3 Chromatogram of the monosaccharide composition of the polysaccharide composition;

[0032] Wherein, A is the polysaccharide composition spectrum; B is the mixed standard spectrum; 1-PMP; 2-mannose; 3-ribose; 4-rhamnose; 5-glucuronic acid; 6-galacturonic acid; 7-glucose; 8-galactose; 9-arabinose;

[0033] Figure 4 Infrared scanning spectrum of the traditional Chinese medicine polysaccharide composition of this invention;

[0034] Figure 5 The effect of the traditional Chinese medicine polysaccharide composition of this invention on the morphology and size of the ovaries in naturally aging rats;

[0035] Figure 6 The effects of the herbal polysaccharide composition of this invention on changes in the estrous cycle of naturally aging rats;

[0036] Figure 7 Pathological section of rat ovary;

[0037] Figure 8 Expression of Bax, Bak, and p53 proteins in rat ovaries;

[0038] Figure 9 Expression levels of NQO-1 protein in rat ovaries (n=3). Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Basic Example: A method for preparing a compound polysaccharide composition for delaying reproductive aging

[0041] Includes the following steps:

[0042] Step 1: Weigh out 25-50 parts of Astragalus membranaceus, 5-20 parts of Angelica sinensis, 5-15 parts of Dioscorea opposita, 5-20 parts of Rehmannia glutinosa, 1-20 parts of Epimedium brevicornu, 5-25 parts of Cuscuta chinensis, and 5-25 parts of Adenophora stricta. Mix them together and soak them in 90-95% ethanol for 0.5-1 hour at a temperature of 20-40℃ to remove the fat-soluble parts. Then filter the mixture, remove the liquid and dry the residue at 40℃.

[0043] Step 2: Add 6-20 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction time is 60-120 minutes. After boiling, continue extraction for 30-120 minutes. Repeat the extraction 2-5 times and combine the extracts.

[0044] Step 3: Concentrate the extract from Step 2 by rotary evaporation until the crude drug content is 3-5 g / mL, add 75-85% ethanol for alcohol precipitation for 12-24 hours, overnight at 4°C, and filter to obtain the precipitate.

[0045] Step 4: Dissolve the precipitate obtained in Step 3 with water, and then purify it with 75-85% ethanol to obtain the crude extract;

[0046] Step 5: Remove proteins from the crude extract using the Sevag method, and freeze-dry to obtain the compound polysaccharide composition; the reagent used in the Sevag method is a mixed solution of chloroform and n-butanol, with a volume ratio of 4:1.

[0047] The Sevag method for removing proteins from crude extract involves: thoroughly mixing the extract using a vortex mixer, centrifuging at 3000 r / min for 15 minutes to remove the intermediate protein layer and the lower solvent layer.

[0048] Example 1: A method for preparing a compound polysaccharide composition for delaying reproductive aging

[0049] Includes the following steps:

[0050] Step 1: Weigh out 50 parts of Astragalus membranaceus, 10 parts of Angelica sinensis, 10 parts of Dioscorea opposita, 10 parts of Rehmannia glutinosa, 10 parts of Epimedium brevicornu, 10 parts of Cuscuta chinensis and 10 parts of Adenophora stricta, mix them together, soak them in 95% ethanol for 0.5 hours at 40°C to remove the fat-soluble parts, then filter, collect the residue and discard the liquid, and dry the residue at 40°C.

[0051] Step 2: Add 10 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction time is 90 minutes. After boiling, continue extraction for another 90 minutes. Repeat the extraction 3 times and combine the extracts.

[0052] Step 3: The extract from Step 2 was concentrated by rotary evaporation to a crude drug content of 3 g / mL. 80% ethanol was added for alcohol precipitation for 20 hours, overnight at 4°C, and the precipitate was obtained by filtration.

[0053] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 80% ethanol to obtain the crude extract;

[0054] Step 5: Add polysaccharide aqueous solution and Sevag reagent (chloroform: n-butanol = 4:1) at a volume ratio of 4:1, mix thoroughly with a vortex mixer, centrifuge at 3000 r / min for 15 minutes to remove the intermediate protein layer and the lower solvent layer, repeat the process several times to remove all protein, and freeze-dry to obtain the compound polysaccharide composition.

[0055] Example 2

[0056] The difference from Example 1 is as follows:

[0057] Step 2: Add 12 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction is performed 3 times for a total extraction time of 120 minutes. After boiling, continue extraction for another 90 minutes and combine the extracts.

[0058] Step 3: The extract from Step 2 was concentrated by rotary evaporation to a crude drug content of 3 g / mL. 75% ethanol was added for alcohol precipitation. The mixture was left to stand overnight at 4°C and then filtered to obtain the precipitate.

[0059] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 75% ethanol to obtain the crude extract;

[0060] The other steps are the same as in Example 1.

[0061] Example 3

[0062] The difference from Example 1 is as follows:

[0063] Step 2: Add 8 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction is performed 3 times for 90 minutes each time. After boiling, continue extraction for another 90 minutes and combine the extracts.

[0064] Step 3: The extract from Step 2 was concentrated by rotary evaporation to a crude drug content of 3 g / mL. 85% ethanol was added for alcohol precipitation. The mixture was left to stand overnight at 4°C and then filtered to obtain the precipitate.

[0065] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 85% ethanol to obtain the crude extract;

[0066] The other steps are the same as in Example 1.

[0067] Example 4

[0068] The difference from Example 1 is as follows:

[0069] Step 2: Add 12 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction is performed 3 times for 60 minutes each time. After boiling, continue extraction for 90 minutes and combine the extracts.

[0070] Step 3: The extract from Step 2 was concentrated by rotary evaporation to a crude drug content of 3 g / mL. 85% ethanol was added for alcohol precipitation. The mixture was left to stand overnight at 4°C and then filtered to obtain the precipitate.

[0071] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 85% ethanol to obtain the crude extract;

[0072] The other steps are the same as in Example 1.

[0073] Example 5

[0074] The difference from Example 1 is as follows:

[0075] Step 2: Add 8 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction is performed 3 times for 120 minutes. After boiling, continue extraction for 90 minutes and combine the extracts.

[0076] Step 3: The extract from Step 2 was concentrated by rotary evaporation to a crude drug content of 3 g / mL. 80% ethanol was added for alcohol precipitation. The mixture was left to stand overnight at 4°C and then filtered to obtain the precipitate.

[0077] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 80% ethanol to obtain the crude extract;

[0078] The other steps are the same as in Example 1.

[0079] Example 6

[0080] The difference from Example 1 is as follows:

[0081] Step 2: Add 10 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction is performed 3 times for a total extraction time of 120 minutes. After boiling, continue extraction for another 90 minutes and combine the extracts.

[0082] Step 3: The extract from Step 2 was concentrated by rotary evaporation to a crude drug content of 3 g / mL. 85% ethanol was added for alcohol precipitation. The mixture was left to stand overnight at 4°C and then filtered to obtain the precipitate.

[0083] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 85% ethanol to obtain the crude extract;

[0084] The other steps are the same as in Example 1.

[0085] Example 7

[0086] The difference from Example 1 is as follows:

[0087] Step 2: Add 10 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction is performed 3 times for 90 minutes each time. After boiling, continue extraction for another 90 minutes and combine the extracts.

[0088] Step 3: The extract from Step 2 was concentrated by rotary evaporation to a crude drug content of 3 g / mL. 75% ethanol was added for alcohol precipitation. The mixture was left to stand overnight at 4°C and then filtered to obtain the precipitate.

[0089] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 75% ethanol to obtain the crude extract;

[0090] The other steps are the same as in Example 1.

[0091] Example 8

[0092] The difference from Example 1 is as follows:

[0093] Step 2: Add 10 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction is performed 3 times for 60 minutes each time. After boiling, continue extraction for 90 minutes and combine the extracts.

[0094] Step 3: The extract from Step 2 is concentrated by rotary evaporation to a crude drug content of 3 g / mL. 80% ethanol is added for alcohol precipitation. The mixture is left overnight at 4°C and then filtered to obtain the precipitate.

[0095] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 80% ethanol to obtain the crude extract;

[0096] The other steps are the same as in Example 1.

[0097] Example 9

[0098] The difference from Example 1 is as follows:

[0099] Step 2: Add 8 times the volume of water to the residue from Step 1 and reflux for extraction. The extraction is performed 3 times for 60 minutes each time. After boiling, continue extraction for 90 minutes and combine the extracts.

[0100] Step 3: The extract from Step 2 was concentrated by rotary evaporation to a crude drug content of 3 g / mL. 75% ethanol was added for alcohol precipitation. The mixture was left to stand overnight at 4°C and then filtered to obtain the precipitate.

[0101] Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with 75% ethanol to obtain the crude extract;

[0102] The other steps are the same as in Example 1.

[0103] Effect Experiment

[0104] The yield and DPPH scavenging effect of the compound polysaccharide compositions prepared in Examples 1-9 are shown in Table 1 below.

[0105] DPPH scavenging rate: The antioxidant activity of the polysaccharides extracted in Examples 1-9 above was preliminarily determined using the 1,1-diphenyl-2-trinitrophenylhydrazine radical method (DPPH). The determination of DPPH radical scavenging rate is a widely used method for screening and evaluating the antioxidant capacity of antioxidants. The higher the DPPH scavenging rate, the stronger the antioxidant capacity. Vitamin C was used as a positive control, and the DPPH scavenging rate of 1 mg / mL Vitamin C was 78.3%.

[0106] Calculation formula: DPPH free radical scavenging rate (%) = (A0 - A sample) / A0 × 100. (A0 is the absorbance of DPPH at 517 nm without sample; A sample is the absorbance of DPPH at 517 nm with sample added).

[0107] Table 1

[0108] Example 1 40.3 54.6 Example 2 39.1 47.8 Example 3 42.1 52.4 Example 4 43.7 53.7 Example 5 34.6 39.4 Example 6 38.2 43.5 Example 7 37.7 44.8 Example 8 38.9 50.2 Example 9 35.2 43.9

[0109] As can be seen from the test data in Table 1 above, extraction time has the greatest impact on yield, followed by alcohol precipitation concentration and water volume ratio. The compound polysaccharide compositions prepared using the methods in Examples 1, 4, and 5 showed better DPPH scavenging effects. In Example 1, the compound polysaccharide composition obtained with an extraction water volume ratio of 12, an extraction time of 90 min, and an alcohol precipitation concentration of 80% exhibited both high yield and high DPPH scavenging effect.

[0110] Experimental Example 1: Detection of Total Sugar Content

[0111] Determination of polysaccharide content in the compound polysaccharide composition samples obtained in Example 1 using the phenol-sulfuric acid method: Using distilled water as a blank, accurately pipette 200 μL each of anhydrous glucose standard solution or polysaccharide solution with concentrations of 0, 20, 40, 60, 80, and 100 μg / mL into 2 mL stoppered centrifuge tubes. Add 200 μL of 5% phenol solution and 700 μL of concentrated sulfuric acid respectively, shake well, and let stand at room temperature for 15 minutes. Accurately pipette 200 μL of each reaction solution into a 96-well plate, with 5 replicates for each sample. Measure the OD at 490 nm using an ELISA reader. Plot a standard curve with glucose concentration on the x-axis and OD at 490 nm on the y-axis.

[0112] The average total sugar content of the three batches of compound polysaccharide composition samples was 366.56 mg / g, with an RSD of 10.20%.

[0113] Test Example 2: Detection of Average Acidic Sugar Content

[0114] Accurately weigh 1 mg of the compound polysaccharide composition sample obtained in Example 1, and dilute to 10 mL in a volumetric flask. Invert the flask several times to mix thoroughly, and dilute with distilled water to a concentration of 100 μg / mL. Accurately weigh 25.00 mg of D-galacturonic acid standard into a 25 mL volumetric flask, dilute to volume, and mix thoroughly to prepare a standard solution with a concentration of 1 mg / mL. Dilute with ultrapure water to prepare standard curve solutions with concentrations of 250, 125, 62.5, 31.25, 15.6, 7.8, and 3.9 μg / mL. Take 100 μL of the sample solution and 600 μL of sodium tetraborate sulfuric acid solution, vortex thoroughly, heat in a boiling water bath for 12 min, cool in an ice bath, add 10 μL of 1.5 mg / mL m-hydroxybiphenyl solution, vortex to mix thoroughly, and measure OD 532 nm using an ELISA reader. Plot a standard curve with uronic acid concentration on the x-axis and OD 532 nm on the y-axis.

[0115] The average acid sugar content of the three batches of compound polysaccharide composition samples was 170.58 mg / g, with an RSD of 15.58%.

[0116] Test Example 3: Detection of Average Protein Content

[0117] The protein content in the compound polysaccharide composition sample obtained in Example 1 was determined using a Thermo Fisher Scientific protein quantification kit. The concentrations of the standard series working solutions were 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 0 μg / mL, respectively. The compound polysaccharide composition was dissolved in ultrapure water to prepare a concentration of 250 μg / mL. The OD562nm was measured according to the instructions. A standard curve was plotted with the standard concentration on the x-axis and OD562nm on the y-axis.

[0118] The average protein content of the three batches of compound polysaccharide composition samples was 28.20 mg / g, with an RSD of 0.40%.

[0119] Experimental Example 4: Determination of Molecular Weight Distribution of Compound Polysaccharide Composition

[0120] Accurately weigh 5 mg of dextran standards of different molecular weights and the compound polysaccharide composition sample obtained in Example 1, and dilute to a final concentration of 1 mg / mL in a volumetric flask with ultrapure water. Filter the solution through a 0.22 μm aqueous filter membrane for later use. A PDA detector and a RID detector are connected in series.

[0121] Column: TSKgel G5000PWXL (7.8mm×300mm);

[0122] Mobile phase: ultrapure water;

[0123] Column temperature and differential refractive index detector temperature: 40℃;

[0124] Flow rate: 0.3 mL / min;

[0125] Injection volume: 10 μL.

[0126] A linear regression equation was calculated using the retention time (tR) of the standard chromatographic peak as the x-axis and the logarithm of the molecular weight (logMw) as the y-axis. The retention times of the chromatographic peaks were recorded and substituted into the regression equation to calculate the molecular weight distribution range of the polysaccharide.

[0127] Based on the molecular weight of polysaccharides and the chromatographic peaks ( Figure 1 The retention time can be used to determine () Figure 2 The molecular weight distribution of the polysaccharide samples can be divided into five main parts: <4 kDa; 4 kDa-9 kDa; 9 kDa-19 kDa; 19 kDa-47 kDa; and 47 kDa-806 kDa, with 47 kDa-806 kDa being the most predominant molecular weight distribution range. Example 5: Component Detection of the Compound Polysaccharide Composition

[0128] Accurately weigh 25 mg of the compound polysaccharide composition sample obtained in Example 1 into a 50 mL round-bottom flask, add 25 mL of 2 mol / L trifluoroacetic acid (TFA), hydrolyze at 110 °C for 6 h under nitrogen protection, concentrate under reduced pressure at 50 °C until dry, wash with 5 mL of methanol, repeat 5-7 times to remove residual trifluoroacetic acid, and prepare a 1.0 mg / mL sample aqueous solution for later use. Accurately weigh 5.00 mg of each monosaccharide standard: D-mannose (Man), D-ribose, D-anhydrous glucose (Glc), D-galactose (Gal), L-arabinose (Ara), L-rhamnose (Rha), D-galacturonic acid (Gala), and D-glucuronic acid (Glca), dissolve thoroughly in ultrapure water, and dilute to 5 mL in a volumetric flask. The mixed monosaccharide standard solution was prepared by mixing the various monosaccharide standard solutions and sequentially diluting them by half to prepare standard curve working solutions with concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8 μg / mL. Accurately transfer 200 μL each of the mixed reference solution, test solution, negative control solution, and blank control solution into separate EP tubes. Add 200 μL of 0.3 mol / L NaOH solution and 200 μL of 0.6 mol / L PMP solution sequentially, mix thoroughly, and then derivatize in a 70°C water bath for 100 min. After cooling to room temperature, neutralize the system with 200 μL of 0.3 mol / L HCl solution. Extract thoroughly with 0.6 mL of dichloromethane, centrifuge at 4000 rpm for 10 min, repeat three times, and filter the supernatant through a 0.22 μm aqueous filter membrane for HPLC analysis.

[0129] like Figure 3 As shown, the negative control did not interfere with the sample determination. In the HPLC chromatogram, excluding the PMP solvent peak, the standard mixture showed a total of 8 peaks with retention times of 18.606, 24.179, 26.138, 28.119, 32.510, 39.310, 44.744, and 49.298 min, corresponding to D-(+)-Man, L-(+)-Rha, D-(+)-Rib, D-(+)-GlcA, D-(+)-GalA, D-(+)-Glc, D-(+)-Gal, and L-(+)-Ara, respectively. It can be seen that the compound polysaccharide composition mainly consists of mannose, glucose, galactose, arabinose and a small amount of rhamnose and galacturonic acid, with a molar ratio of Man:Rha:GalA:Glu:Gal:Ara=27.72:1.57:1:70.25:13.61:6.91.

[0130] Infrared spectral detection of compound polysaccharide composition in Experiment Example 6

[0131] Take an appropriate amount of the compound polysaccharide composition sample obtained in Example 1, and use a Fourier transform infrared scanner to measure the infrared wavelength at 4000-500 cm⁻¹. -1 A wide range of infrared spectral scans were performed to record the infrared spectral images.

[0132] like Figure 4 As shown, intermolecular and intramolecular hydrogen bonds on the sugar bond cause the sugar hydroxyl group to be at 3271.98 cm⁻¹ -1 A broad peak appears at 2931.5 cm⁻¹, which is the stretching vibration of OH. -1 The area near the point is the stretching vibration of CH, a characteristic absorption peak of carbohydrates; at 1600.05 cm⁻¹. -1 The absorption peak at 1408.65 cm⁻¹ represents the asymmetric vibrational absorption peak of uronic acid COO-. -1 The nearby absorption peaks are absorption peaks of the CH angular vibration; 1100-1010 cm⁻¹ -1 The presence of two absorption peaks suggests the possible presence of furanyl glycosides.

[0133] Efficacy Study - Phenotypic Study of Reproductive Effects of Compound Polysaccharide Composition on Naturally Aged Female Rats

[0134] The animal experimental protocol underwent ethical review by the Animal Ethics Committee of the Guangdong Provincial Center for Experimental Animals of Traditional Chinese Medicine, with ethical approval number 2021096. SPF-grade female SD rats were used as experimental animals. The entire experiment was conducted at the Animal Experiment Center of the Guangdong Academy of Traditional Chinese Medicine. Nine-month-old rats were quarantined for one week and then acclimatized until they reached 14 months of age before the start of the experiment.

[0135] Rats were randomly divided into four stratified groups: a natural aging model group, a traditional Chinese medicine decoction group, a compound polysaccharide composition group prepared in Example 1, and a compound small molecule group, with eight rats in each group. Three-month-old young female SD rats were also purchased as a young control group (n=8). The compound decoction group, compound polysaccharide group, and compound small molecule group were given clinically equivalent doses, i.e., the corresponding drug at a crude drug concentration of 1.1 g / mL. The drug administration groups were administered 1 ml / 100 g / day by gavage for 90 consecutive days; the natural aging model group and the young group were administered 1 ml / 100 g / day of physiological saline by gavage.

[0136] Preparation of the decoction of traditional Chinese medicine compound: Astragalus membranaceus, Angelica sinensis, Dioscorea opposita, Rehmannia glutinosa (processed), Adenophora stricta, Cuscuta chinensis (processed with salt), and Epimedium brevicornu were placed in a decoction pot in a weight ratio of 5:1:1:1:1:1:1. Pure water was added at a ratio of 1:12 (g:ml) and the mixture was soaked for 30 minutes. After the water boiled, heating was continued for 90 minutes. The decoction was collected, and the process was repeated once. The two decoctions were collected and combined. The decoction was then concentrated to a final concentration of 1.1 g / mL using a rotary evaporator.

[0137] Preparation of compound small molecules: Add 95% ethanol to the HYF compound aqueous extract (final concentration 1.1 g / mL) to adjust the ethanol concentration to 80%, stir well, and let stand overnight. Separate the small molecules from the supernatant and freeze-dry. Weigh and dissolve in water to a final concentration of 1.1 g / mL of crude drug in the compound small molecule solution.

[0138] The estrous cycle of rats was determined based on the type and proportion of exfoliated vaginal cells. Vaginal cell smears were prepared daily from 09:00 to 10:00 AM during the experiment to observe the estrous cycle. Twenty-four hours after the last drug administration, the animals were anesthetized and euthanized, and blood, ovaries, uterus, and other organs were collected for relevant parameter testing.

[0139] 1. Effects of compound polysaccharide composition on reproductive-related indicators in naturally aging rats

[0140] 1.1 Effect on ovarian volume

[0141] The sampling revealed significant differences in ovarian size among the groups, such as... Figure 5 As shown in Table 2, ovarian volume was calculated using the formula: V = L * S² / 2 (ovarian volume V: unit mm³, ovarian long diameter L: unit mm, ovarian short diameter S: unit mm). Compared with the young control group, the ovarian volume of rats in all groups decreased, with the natural aging group and the compound small molecule group showing particularly significant decreases (P<0.001). After intervention with the compound decoction and compound polysaccharide composition, the ovarian volume of all groups increased compared with the natural aging group, but there was no statistically significant difference.

[0142] Table 2 Comparison of ovarian volume and length and short diameter of rats in each group.

[0143]

[0144] Note: n = 5-8, X ± S. Compared with the young control group, ***P < 0.001, **P < 0.01, *P < 0.05; there was no statistically significant difference compared with the naturally aging group.

[0145] 1.2 Effects on the estrous cycle of rats

[0146] Throughout the experiment, vaginal smears of rats were taken at fixed times daily. To clarify the changes in the estrous cycle of rats during the drug administration period, the estrous cycle was divided according to the grouping method described above. The estrous phase was taken as the starting point. If a rat was in the interestrous or postestrous phase on a given day, it was considered an estrous change and recorded as "1". A line graph of the estrous cycle of rats during the experiment was plotted based on the registration results.

[0147] like Figure 6As shown, the estrous cycle recordings of naturally aging rats exhibited a prolonged "plateau phase," indicating that aging rats lost the regularity of their estrous cycle. After drug intervention, the estrous cycles of rats in the compound decoction group and the compound polysaccharide combination group showed significant changes compared to the aging group, manifested in the increased frequency of interestrus and postestrus phases, restoring the rhythm of the estrous cycle. These results indicate that the traditional Chinese medicine compound and the compound polysaccharide combination group can effectively improve the disordered estrous cycle in aging rats.

[0148] 1.3 Effects on serum sex hormones

[0149] Serum AMH, testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured.

[0150] To clarify the changes in serum AMH levels in rats during the drug administration period, blood samples were collected and serum AMH levels were measured at three time points: before drug administration, day 60, and day 90. As shown in Table 3, the AMH levels in the naturally aging group rats continued to decrease over the 90 days of drug administration, indicating that rat follicles were continuously consumed and ovarian function was continuously weakened. After intervention with the compound decoction and the compound polysaccharide composition, AMH levels decreased in all groups during the first 60 days, with the decrease in the compound polysaccharide composition group being slower than that in the compound decoction group. From day 60 to day 90 of drug administration, AMH levels remained basically stable and no longer continued to decrease. This indicates that the traditional Chinese medicine compound and the compound polysaccharide composition have the effect of maintaining ovarian function and slowing down ovarian function decline, and the rate of AMH decrease in the compound polysaccharide composition group was slower than that in the decoction group.

[0151] The ovary is a crucial site for the production of androgens and estrogens, with androgens serving as precursors for estrogen synthesis. With aging, the number of follicles decreases, leading to a decline in the levels of androgens and estrogens produced. The experiment monitored changes in testosterone (T) at two time points: before drug administration and 90 days after administration. The results are shown in Table 4. In aging rats, testosterone levels continued to decline. Administration of the compound decoction and the compound polysaccharide composition both slowed the rate of decline, with the compound polysaccharide composition showing the most significant improvement.

[0152] E2 plays an important regulatory role in the growth, development, and differentiation of the reproductive system. The E2 content was measured 90 days after administration, and the results are shown in Table 4. Compared with naturally aging rats, the E2 content increased after administration of the traditional Chinese medicine compound and the compound polysaccharide combination. Compared with the compound decoction group, the E2 level in the compound polysaccharide combination group was significantly increased.

[0153] Pituitary gonadotropins FSH and LH are glycoproteins composed of two glycosylated, non-covalently linked polypeptide subunits. Their levels are regulated through the negative feedback of AMH and estradiol produced by follicles. During ovarian aging, AMH and E2 levels gradually decrease, while FSH and LH levels show an age-related increase. To clarify the changes in FSH and LH during drug administration, levels were measured at two time points: 90 days after administration. As shown in Table 4, compared with the naturally aging group, LH levels decreased in all drug-treated groups. Regarding FSH levels, compared with the naturally aging group, the compound decoction group and the compound polysaccharide composition group showed decreased levels.

[0154] Table 3 Comparison of AMH levels in rats at different administration times between groups

[0155]

[0156] Note: n = 5-8, DAY0 = before administration, DAY60, DAY90 = 60 and 90 days after administration.

[0157] Table 4. E2, FSH, T and LH levels in rats of each group

[0158]

[0159] Note: n = 5-8, VS natural aging group, *P < 0.05, VS decoction group, #P < 0.05.

[0160] 1.4 Effect on the number of follicles

[0161] The number of follicles is the most direct and classic indicator of ovarian reserve and function. Therefore, follicles at various stages were counted in rats, and the results are shown in Table 5.

[0162] Compared with the young control group, the number of follicles at all stages was significantly reduced in aging rats, specifically in the number of primordial follicles, primary follicles, small antral follicles, and total follicles (P < 0.001, P < 0.01, P < 0.001, P < 0.001), indicating a significant decrease in ovarian reserve in aging rats. This is corroborated by experimentally measured ovarian atrophy and pathological section results. After drug intervention, compared with the naturally aging group, the compound decoction group and the compound polysaccharide composition group showed significant reductions at different developmental stages. The number of follicles increased in all groups. Specifically, the number of primordial follicles, secondary follicles, and small antral follicles in the compound decoction group was significantly increased (P<0.01, P<0.05, P<0.05). Compared with the naturally aged group, the compound polysaccharide composition significantly increased the number of secondary follicles, small antral follicles, mature follicles, and total follicles (P<0.05, P<0.05, P<0.05, P<0.01, P<0.05). The total number of follicles in the compound polysaccharide composition group was slightly higher than that in the decoction group.

[0163] Furthermore, experiments revealed that the number of mature follicles in aging rats significantly increased after intervention with the compound polysaccharide composition. This suggests that the compound polysaccharide composition can protect ovarian function, delay its decline, and promote follicle development in aging ovaries.

[0164] Table 5 Follicle grading and counting

[0165]

[0166] Note: n=3, VS young control group, *P<0.05, **P<0.01, ***P<0.001; VS natural aging group, #P<0.05, ##P<0.01.

[0167] 1.5 Effects on ovarian histopathological changes

[0168] Depend on Figure 7 It is evident that, compared to the young control group, the ovaries in the naturally aging group began to atrophy, and the number of follicles at each stage was extremely low. After intervention with the compound decoction and the compound polysaccharide composition, a greater number of follicles at different developmental stages were visible under the microscope. Among them, the number of mature follicles was particularly significant in the compound polysaccharide composition group. The ovaries in the compound small molecule group were severely deformed, with almost no developing follicles. The experiment shows that ovarian reserve in aging rats is significantly reduced, and intervention with the compound polysaccharide composition can effectively slow down the loss of ovarian reserve in rats, as reflected in the appearance of a greater number of follicles at different developmental stages under the microscope.

[0169] 2. Effects of polysaccharides on ovarian apoptosis indices in naturally aging rats

[0170] p53 is a key regulator of cellular senescence; elevated p53 levels are a cause of cellular senescence. p53 expression is very low in normal cells without external stimulation. When cells are under stress, p53 levels increase dramatically, and when damage cannot be repaired, p53 induces apoptosis. Figure 8 It was found that, compared with the young control group, the expression of p53 in the ovaries of naturally aging rats was significantly increased. After drug intervention, the expression of p53 in the ovaries of all groups decreased, with the compound polysaccharide combination group showing the most significant effect. BAX and BAK are proteins that exert pro-apoptotic effects. BAX and BAK bind to the mitochondrial membrane to form the MOMP complex, which regulates mitochondrial apoptosis by altering mitochondrial membrane permeability and releasing cytochrome C. Figure 8 It can be seen that, compared with the young control group, the expression of pro-apoptotic proteins BAX and BAK in the ovaries of naturally aging rats was significantly increased. After drug intervention, the expression of BAX and BAK in the ovaries of all groups decreased, with the compound polysaccharide combination group showing the most significant effect.

[0171] 3. Effects of polysaccharides on oxidative stress indices in naturally aging rats

[0172] Superoxide dismutase (SOD) is one of the most important antioxidant enzymes in living organisms. It protects the integrity of cell structure by blocking the damage of superoxide anions to cells, and its activity level is an important indicator reflecting the body's antioxidant capacity. Metabolic acid (MDA) is an end product of lipid peroxidation, and its level is a typical indicator for assessing the degree of oxidative damage to cell membranes under oxidative stress, and is one of the most important biomarkers of aging. GSH-Px is an important selenium-containing antioxidant enzyme in the body. It protects the body from oxidative damage by reducing H2O2 to H2O and lipid hydrogen peroxide to the corresponding alcohols. Currently, the measurement of its activity is considered one of the important indicators for anti-aging or dementia drugs.

[0173] As shown in Table 6, compared with the young control group, the SOD activity and GSH-Px content in the serum of rats in the naturally aging group were significantly decreased (P < 0.001), while the MDA level was significantly increased (P < 0.001), indicating that aging leads to a significant decline in the body's antioxidant capacity. Compared with the naturally aging group, the SOD activity and GSH-Px content in the serum of rats in the compound decoction group and the compound polysaccharide composition group were both increased, while the MDA level was decreased. In terms of SOD activity, there was a significant difference between the compound polysaccharide composition group and the naturally aging group (P < 0.01); in terms of MDA level, there was a significant difference between the compound decoction group and the naturally aging group (P < 0.01), while the difference between the compound polysaccharide composition group and the naturally aging group was more significant (P < 0.001); in terms of GSH-Px content, there was a significant difference between the compound polysaccharide composition group and the naturally aging group (P < 0.05). The above results indicate that the compound decoction and its compound polysaccharide composition can increase the activity of SOD and GSH-Px antioxidant enzymes and reduce the level of lipid peroxidation end product MDA, thus exhibiting certain antioxidant effects. Among them, the compound polysaccharide composition has a more significant antioxidant effect.

[0174] Table 6 Comparison of serum SOD, MDA and GSH-Px activities in each group

[0175]

[0176]

[0177] Note: n = 5-8, X ± S, compared with the young control group, ***P < 0.001, *P < 0.05; compared with the naturally aging group, ###P < 0.001, ##P < 0.01.

[0178] NQO1 (NAD(P)H:quinone oxidoreductase 1) is an enzyme that plays a vital role in cells, specifically in protecting endogenous antioxidants. It protects cells from oxidative stress by maintaining the reduced forms of ubiquinone and α-tocopherol quinone. Figure 9It was found that, compared with the young control group, the expression of NQO-1 protein in the ovaries of naturally aging rats was significantly decreased (P < 0.05). Intervention with the compound polysaccharide composition significantly increased the expression of NQO-1 protein (P < 0.05), suggesting that the compound polysaccharide composition can directly or indirectly activate the NQO-1 signaling pathway in ovarian tissue under aging stress, thereby exerting an antioxidant effect.

[0179] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a compound polysaccharide composition for delaying reproductive aging, characterized in that: Includes the following steps: Step 1: Weigh all the raw materials, mix them, soak them in an organic solvent to remove the fat-soluble parts, then filter, and discard the liquid and remove the residue. Step 2: Add water to the residue from Step 1 and reflux for extraction. After boiling, continue extraction for 30-120 minutes. Repeat the extraction three times and combine the extracts. Step 3: Concentrate the extract from Step 2 to a crude drug content of 3-5 g / mL, add ethanol for alcohol precipitation, and filter to obtain the precipitate; Step 4: Dissolve the precipitate obtained in Step 3 with water and then purify it with ethanol to obtain the crude extract; Step 5: Remove proteins from the crude extract using the Sevag method, and freeze-dry to obtain the compound polysaccharide composition; The raw materials mentioned in step one, by weight, include: 25-50 parts of Astragalus membranaceus, 5-20 parts of Angelica sinensis, 5-15 parts of Dioscorea opposita, 5-20 parts of Rehmannia glutinosa, 1-20 parts of Epimedium brevicornu, 5-25 parts of Cuscuta chinensis, and 5-25 parts of Adenophora stricta.

2. The preparation method according to claim 1, characterized in that: The organic solvent mentioned in step one is 90-95% ethanol, the soaking time is 0.5-1h, and the soaking temperature is 20-40℃; the amount of water added in step two is 6-20 times the water volume; the number of extractions is 2-5 times, and the extraction time is 60-120min.

3. The preparation method according to claim 2, characterized in that: The amount of water added in step two is 12 times the water volume; the extraction is performed 3 times, and the extraction time is 90 minutes.

4. The preparation method according to claim 1, characterized in that: The concentration in step three is achieved by rotary evaporation; the ethanol concentration in steps three and four is 75-85%; and the alcohol precipitation time in step three is 12-24 hours.

5. The preparation method according to claim 4, characterized in that: The ethanol concentration mentioned in steps three and four is 80%.

6. The preparation method according to claim 1, characterized in that: In step five, the reagent used in the Sevag method is a mixed solution of chloroform and n-butanol in a volume ratio of 4:

1. The volume ratio of the Sevag reagent to the crude extract solution is 1:

4. This is repeated 3-6 times, except for proteins.

7. The preparation method according to claim 1, characterized in that: The Sevag method for removing proteins from the crude extract described in step five involves: thoroughly mixing the extract using a vortex mixer, centrifuging at 3000 r / min for 15 minutes to remove the intermediate protein layer and the lower solvent layer.

8. The use of the compound polysaccharide composition prepared by the preparation method according to any one of claims 1-7 in the preparation of a drug formulation for delaying female reproductive aging.

9. A pharmaceutical preparation for delaying female reproductive aging, characterized in that: The pharmaceutical preparations include the compound polysaccharide composition prepared by the preparation method according to any one of claims 1-7 and a pharmaceutically acceptable carrier.

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