Application of ginseng neutral non-amyloid polysaccharide in treatment of premature ovarian failure

Abstract

The invention discloses application of ginseng neutral non-amyloid polysaccharide in treatment of premature ovarian failure, relates to the field of biological activity of traditional Chinese medicine plant polysaccharide, and aims to provide ginseng neutral non-amyloid polysaccharide with an effect of improving premature ovarian failure as well as a preparation method and application of the ginseng neutral non-amyloid polysaccharide. The ginseng neutral non-amyloid polysaccharide is applied to preparation of the medicine for treating premature ovarian failure. The invention provides an application of ginseng neutral non-amyloid polysaccharide in treatment of D-galactose induced premature ovarian failure, at present, no research on the improvement effect of the ginseng neutral non-amyloid polysaccharide on the premature ovarian failure exists, and a support is provided for revealing the improvement effect of ginseng polysaccharide on POF. The ginseng Zhongxing non-amyloid polysaccharide disclosed by the invention can regulate the hormone level of a D-galactose induced POF mouse in vivo. The invention is applied to the field of premature ovarian failure treatment.

Description

Technical Field

[0001] This invention belongs to the field of bioactivity of polysaccharides from traditional Chinese medicine plants, specifically relating to the application of ginseng neutral non-starch-like polysaccharide in the treatment of premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) refers to pathological amenorrhea before the age of 40, accompanied by clinical manifestations such as a rapid decrease in follicles, elevated gonadotropins, and genital atrophy. Oxidative stress is involved in the pathogenesis of POF, and its induced granulosa cell apoptosis is closely related to the occurrence of POF. When oxidative stress occurs, the body's ability to scavenge free radicals is insufficient, and excessive free radicals can cause granulosa cell apoptosis, thereby accelerating oocyte atresia and damaging ovarian structure and function.

[0003] Ginseng is a plant used both as food and medicine, with a long history and various biological activities. In traditional Chinese medicine, ginseng is used to "tonify qi and nourish blood" and can be used to treat infertility. Polysaccharides are the main active components of ginseng, with their biological activities primarily focused on immunomodulation, antioxidation, anti-aging, and anti-inflammation. They can also exert their biological activity by regulating the gut microbiota. However, few studies have focused on the role of ginseng polysaccharides in protecting female reproductive health. According to traditional Chinese medicine theory, the active components of natural drugs may play a role in regulating POF (Potentially Ovarian Failure). Currently, there are no reported studies on the regulatory effect of ginseng polysaccharides on POF, but it has been found that ginseng polysaccharides can induce the expression of estrogen receptor (ER) and can protect ovarian germ cell function by promoting progesterone secretion from granulosa cells and regulating the cAMP signaling system. Therefore, based on the antioxidant activity of ginseng polysaccharides, this study speculates that they may be used as a new drug for treating POF, providing a new strategy for the prevention and treatment of POF. Summary of the Invention

[0004] The purpose of this invention is to provide a ginseng neutral non-amyloid polysaccharide that can improve premature ovarian failure, its preparation method, and its application.

[0005] The present invention relates to the application of ginseng neutral non-starch polysaccharide in the preparation of a drug for treating premature ovarian failure; the ginseng neutral non-starch polysaccharide is composed of 94.43%-96.75% glucose, 2.23%-4.01% galactose and 1.02%-1.56% arabinose by molar percentage.

[0006] Furthermore, the application of the aforementioned ginseng neutral non-starch-like polysaccharide in the preparation of a drug for D-galactose-induced treatment of premature ovarian failure.

[0007] Furthermore, the ginseng neutral non-starch-like polysaccharide, by molar percentage, consists of 96.75% glucose, 2.23% galactose, and 1.02% arabinose.

[0008] Furthermore, the glucose is (1→)-Glc p (1→4)-Glc p and (1→4,6)-Glc p The galactose mentioned is (1→3,4)-Gal p The arabinose mentioned is (1→)-Ara f .

[0009] Furthermore, the (1→)-Ara f (1→)-Glc p (1→4)-Glc p (1→3,4)-Gal p and (1→4,6)-Glc p The molar ratio is 0.15:4.02:24.54:1:3.85.

[0010] Furthermore, the molecular weight of the ginseng neutral non-amyloid polysaccharide is 4.72 × 10⁻⁶. 4 Da.

[0011] Furthermore, the method for preparing the ginseng neutral non-amyloid polysaccharide is as follows: Step 1: Crush the dried ginseng into powder. Step 2: Extract the ginseng powder with water, then filter it to obtain the filtrate; Step 3: Centrifuge the filtrate, collect the supernatant and concentrate it to obtain a concentrated solution; Step 4: Add 95% ethanol to the concentrate, then centrifuge to obtain the residue; Step 5: Dissolve the residue in water and obtain the ginseng neutral non-starch polysaccharide by anion exchange chromatography, α-amylase enzymatic hydrolysis, and gel chromatography.

[0012] Further, in step two, the ginseng powder is subjected to water extraction. The specific operation is as follows: First, the ginseng powder is added to water with a weight of 20 times that of the ginseng powder and soaked for 1-2 hours. Then, it is extracted at 90°C for 180 minutes and repeated 3 times.

[0013] Further, in step five, the residue is dissolved in water, and the ginseng neutral non-starch-like polysaccharide is obtained by anion exchange chromatography, α-amylase enzymatic hydrolysis, and gel chromatography, respectively; specifically as follows: After dissolving the residue in water, the neutral polysaccharides were separated by DEAE-cellulose anion exchange chromatography with distilled water as the mobile phase. α-amylase was added to hydrolyze and remove starch. The non-starch-like polysaccharides were then purified by Sepharose CL-6B gel chromatography to obtain the ginseng neutral non-starch-like polysaccharides.

[0014] The present invention has the following beneficial effects: This invention proposes the application of ginseng neutral non-starch polysaccharide in the treatment of D-galactose-induced premature ovarian failure (POF). Currently, there is no research on the ameliorative effect of ginseng neutral non-starch polysaccharide on POF. This invention provides technical support for revealing the ameliorative effect of ginseng polysaccharide on POF. The ginseng neutral non-starch polysaccharide of this invention can regulate hormone levels in D-galactose-induced POF mice in vivo. Attached Figure Description

[0015] Figure 1 High-performance gel permeation chromatography and high-performance liquid chromatography (HPLC) chromatograms of ginseng neutral non-amyloid polysaccharides; Figure 2 A diagram showing the monosaccharide composition analysis of ginseng neutral non-amyloid polysaccharides; Figure 3 Fourier transform infrared (FT-IR) spectrum of ginseng neutral non-amyloid polysaccharide; Figure 4 Figure showing the effect of ginseng neutral non-amyloid polysaccharide on serum hormone levels in POF mice; Figure 5 Figure showing the effect of ginseng neutral non-amyloid polysaccharide on serum antioxidant enzymes and MDA levels in POF mice; Figure 6 HE staining (top image) and bar chart (bottom image) showing the effect of ginseng neutral non-amyloid polysaccharide on ovarian follicles in POF mice. Figure 7 Fluorescence micrograph (top) and bar chart (bottom) showing the effect of ginseng neutral non-amyloid polysaccharide on apoptosis of ovarian granulosa cells in POF mice. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0017] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0018] Example 1: The ginseng neutral non-amyloid polysaccharide in this example was prepared by the following specific method: 1. Extraction of crude ginseng polysaccharides: After washing and drying ginseng, pulverize it and weigh 1500 g. Add 30 L (solid-to-liquid ratio 1:20, w / v) of distilled water and boil at 90℃ for 3 h. After boiling, separate the extract from the residue. Repeat the boiling process with the residue three times. Collect the extracts from the three boilings, concentrate them to an appropriate volume, centrifuge at 4000 r / min for 10 min, remove the ginseng residue, and slowly add about 4 times the volume of anhydrous ethanol to the supernatant on a magnetic stirrer until the final ethanol concentration is 80%. Let it stand overnight at 4℃ for alcohol precipitation. Centrifuge this solution at 4000 r / min for 10 min to obtain crude polysaccharide precipitate. Wash it successively with 95% ethanol and anhydrous ethanol, and then vacuum dry to obtain crude ginseng polysaccharides.

[0019] 2. Weigh the crude polysaccharide sample and dissolve it in distilled water. Slowly add the solution dropwise to a pre-equilibrated DEAE-cellulose chromatography column and elute with distilled water at a flow rate of 10 mL / min. Use the phenol-sulfuric acid method for follow-up determination, measuring the OD value at λ490 nm every 20 mL. Plot the elution curve, collect the eluted neutral sugars, freeze-dry them, and weigh them to obtain ginseng neutral polysaccharide.

[0020] 3. A 5% polysaccharide solution of ginseng neutral polysaccharide was prepared, hydrolyzed with α-amylase, and stirred thoroughly in a 37°C water bath. The reaction solution was tested with KI-I reagent every 20 min until no color change was observed. The digest was then treated with boiling water for 10 min, cooled, and centrifuged at 3500 r / min for 15 min to remove α-amylase. The supernatant was freeze-dried and weighed to obtain ginseng neutral non-starch-like crude polysaccharide.

[0021] 4. Weigh the ginseng neutral non-starch-like crude polysaccharide and prepare a sample solution using distilled water. Centrifuge at 4000 r / min for 10 min. Gently add the supernatant to a Sepharose CL-6B gel column. After the sample has completely penetrated the gel, continue adding distilled water to the end of the column. Tighten the cap and elute with 0.15 mol / L NaCl solution as the mobile phase at a flow rate of 0.5 mL / min. Perform follow-up analysis using the phenol-sulfuric acid method, measuring the OD value at λ490 nm every 10 mL. Plot the elution curve, collect the main eluted components, and place them in a 3500 kDa dialysis bag. Dialyze with running water and distilled water for 24 h to remove salts. After freeze-drying, weigh to obtain the ginseng neutral non-starch-like polysaccharide.

[0022] The ginseng neutral non-starch-like polysaccharide obtained in this embodiment was analyzed using HPGPC under the following determination conditions, and the results are as follows: Figure 1 As shown, from Figure 1A single symmetrical peak was observed, with an elution time of 21.76 minutes. This indicates that the ginseng neutral non-amyloid polysaccharide has a uniform molecular weight. Based on the known molecular weight of dextran, the molecular weight of the ginseng neutral non-amyloid polysaccharide was calculated to be 4.72 × 10⁻⁶. 4 Da.

[0023] The HPGPC measurement conditions are as follows: Chromatographic column: TSK-G3000 PWXL (7.8 mm ID × 30.0 cm L) Detector: RID-10A Mobile phase: deionized water Flow rate: 0.6 mL / min Injection volume: 10 μL The ginseng neutral non-starch-like polysaccharide obtained in this embodiment was completely acid-hydrolyzed and PMP-derived, and analyzed by HPLC under the following determination conditions. The results are as follows: Figure 2 As shown, based on the relative retention time of the standard monosaccharides, the monosaccharides contained in this ginseng neutral non-starch polysaccharide are composed of glucose, galactose and arabinose, with molar percentage contents of 96.75%, 2.23% and 1.02%, respectively.

[0024] The HPLC determination conditions are as follows: Column: DIKMA Inertsil ODS-3 (4.6 mm × 150 mm) Detector: UV-VIS DAD Detection wavelength: 245 nm Mobile phase: PBS (0.1 M, pH 7.0):acetonitrile = 82:18 (v / v), pH 7.0 Flow rate: 1.0 mL / min Injection volume: 10 μL The ginseng neutral non-amyloid polysaccharide obtained in this embodiment was dried, mixed with KBr (1:100), and compressed into tablets at 400-4000 cm⁻¹. -1 Infrared spectral scanning was performed within the range (e.g.) Figure 3 As shown), the recorded infrared spectral data is as follows: 3410 cm⁻¹ -1 The strong absorption peak nearby represents the OH vibration peak. 2927 cm⁻¹ -1 and 1643 cm -1 The nearby absorption peaks are caused by the CH bending vibration peak and the OH stretching vibration peak, respectively. (1000–1200 cm⁻¹) -1 Absorption peaks within this range indicate the presence of a pyranose ring. (930 cm⁻¹) -1The characteristic absorption peak at 850 cm⁻¹ belongs to the β-glycosidic bond. Furthermore, the characteristic absorption peak at 850 cm⁻¹... -1 The characteristic absorption peak at this location belongs to α-glycosidic bonds. The results indicate that neutral non-amyloid polysaccharides exist simultaneously via both α-glycosidic and β-glycosidic bonds.

[0025] I. Determination of Glucuronic Acid The uronic acid content in the ginseng neutral non-starch polysaccharide obtained in this example was determined using the m-hydroxybiphenyl method. ① Preparation of 0.5% NaOH solution: Weigh 0.1 g NaOH and add distilled water to a final volume of 20 mL. Preparation of m-hydroxybiphenyl solution: Weigh 30 mg m-hydroxybiphenyl and add 0.5% NaOH solution to a final volume of 10 mL. Store at 4℃ protected from light. ② Preparation of saturated potassium hydroxide (KOH) solution: Weigh 5 g KOH and add distilled water to a final volume of 2 mL. Stir thoroughly and then remove the supernatant. Preparation of sulfamic acid solution: Weigh 3.9 g sulfamic acid and add 5 mL distilled water. Adjust the pH to 2.5 by adding saturated KOH solution dropwise, then add distilled water to a final volume of 10 mL.

[0026] Standard curve preparation: Weigh 10 mg of D-GalA and dilute to 100 mL with distilled water to prepare a 0.1 g / L D-GalA standard solution. Then prepare standard solutions with concentrations of 0.1, 0.075, 0.05, 0.025, 0.0125, and 0 mg / mL. Take 400 μL of each diluted standard solution, add 40 μL of sulfamic acid solution, mix well, add 2.5 mL of concentrated sulfuric acid, shake to mix, boil in a water bath for 20 min, cool to room temperature, add 40 μL of m-hydroxybiphenyl solution, react at room temperature for 15 min, and measure the absorbance at λ525 nm. Plot a standard curve with absorbance on the ordinate and standard solution concentration on the abscissa.

[0027] Determination of uronic acid content in samples: Acidic polysaccharides were prepared into a 1 mg / mL polysaccharide solution. The above procedure was repeated, and 400 µL of the solution was added to sulfamic acid solution, concentrated sulfuric acid, and m-hydroxybiphenyl solution, followed by incubation. The absorbance was measured and substituted into the standard curve (standard curve: y = 2.3703x + 0.0684(R)). 2 The content of uronic acid in the acidic polysaccharide was calculated using the formula (=0.9986). The results showed that all the detected values ​​for this sample were negative, and no uronic acid was detected.

[0028] II. Efficacy Test In vivo experiments were conducted using the ginseng neutral non-amyloid polysaccharide prepared in Example 1 to improve premature ovarian failure. 1. Model Establishment D-galactose (D-gal) has been widely used to construct mouse POF models. Its toxicity weakens the biological activity of follicle-stimulating hormone (FSH) and inhibits the production of estradiol (E2) by granulosa cells. The resulting oxidative stress reduces the number of follicles and oocytes and promotes granulosa cell apoptosis. When 10% of granulosa cells undergo apoptosis, the follicles will close.

[0029] Female C57BL / 6J mice (15±2g, 6 weeks old) were used in an environment with free access to food and water at 21±1℃ and 40-70% relative humidity, with a 12-hour light / dark cycle. The selected animals were randomly divided into 6 groups: control group, model group (D-gal, 1.35 g / kg), positive control group (Vc), and treatment groups: low-dose GPN-I group (GPN-I, 50 mg / kg), medium-dose GPN-I group (GPN-I, 100 mg / kg), and high-dose GPN-I group (GPN-I, 200 mg / kg). The model group received 0.2 mL of D-gal intraperitoneally daily, while the treatment groups received 0.2 mL of ginseng neutral non-starch-like polysaccharide prepared in Example 1 at the corresponding concentration daily by gavage, in addition to the treatment given to the model group. The control group received the same dose of physiological saline daily by gavage and intraperitoneal injection, for 6 consecutive weeks.

[0030] 1.1 Detection of serum LH, FSH, and E2 hormone levels in mice Twenty-four hours after the last administration, blood was collected from the eyeballs of all mice and centrifuged (4000 r / min, 10 min) to obtain serum. Serum levels of LH, FSH, and E2 were measured according to the kit instructions. Results showed abnormal hormone expression in POF mice. Compared with the control group, the D-gal group mice had elevated serum FSH levels and significantly decreased E2 and LH levels. P <0.001), compared with the D-gal group, GPN-I improved serum hormone disorders in POF mice, dose-dependently reducing FSH levels and increasing E2 and LH levels ( P <0.001, P <0.01 indicates that GPN-I has the effect of improving the disorder of serum hormone levels in mice. Figure 4 ).

[0031] 1.2 Detection of serum SOD, GSH-Px, CAT and MDA levels in mice Twenty-four hours after the last administration, blood was collected from the eyeballs of all mice and centrifuged (4000 r / min, 10 min) to obtain serum. The activities of SOD (#A001-3-2), GSH-Px (#A005-1-2), CAT (#A007-1-1), and the levels of MDA (#A003-4-1) in the serum were measured according to the kit instructions. The results showed that D-gal induced oxidative damage in mice, thereby affecting the expression of antioxidant enzymes. Compared with the control group, the D-gal group showed increased serum MDA levels and significantly decreased levels of SOD, GSH-Px, and CAT. P <0.001), compared with the D-gal group, GPN-I dose-dependently reduced serum MDA levels and increased SOD, GSH-Px, and CAT activities ( P <0.001, P <0.01), in the GPN-I 200 mg / kg dose group, the activities of SOD, GSH-Px and CAT were 16.36±1.18 U / mL, 347.83±18.14 µmol / L and 36.12±2.92 U / mL, respectively, and the MDA content was 3.63±1.67 nmol / mL. These results indicate that GPN-I can improve D-gal-induced oxidative damage in mice, increase antioxidant enzyme levels, improve lipid peroxidation, and reduce MDA expression (…). Figure 5 ).

[0032] 1.3 HE staining of mouse ovaries Twenty-four hours after the last drug administration, blood was collected from the eyeballs of all mice, and ovarian tissue was collected. After washing with PBS, the tissue was fixed with 4% paraformaldehyde. The fixed ovarian tissue was then dehydrated by immersion in ethanol of different concentrations, and finally immersed in xylene. After embedding in paraffin, the tissue was sectioned and stained according to the HE staining kit instructions. First, hematoxylin was stained for 30 min, followed by washing with distilled water to remove excess stain. Differentiation solution was added for 60 s, followed by immersion in distilled water, and then eosin staining for 2 min. After washing away the staining solution, the tissue was dehydrated and mounted. The staining results were observed under a microscope and photographed. In the HE staining results of the ovaries of mice in each group, brown arrows represent primordial follicles, black arrows represent primary follicles, red arrows represent secondary follicles, and blue arrows represent atretic follicles. Compared with the control group, the D-gal group of mice had a relatively reduced number of primordial follicles, primary follicles, secondary follicles, and corpora lutea. P <0.01), but the number of atretic follicles increased significantly ( P <0.01), at the same magnification, the follicle volume was significantly smaller, while the number of ovarian follicles and corpora lutea in the GPN-I group mice was significantly increased, and the number of atretic follicles was decreased. P <0.05, P <0.01)( Figure 6 ).

[0033] 1.4 TUNEL staining of mouse ovaries Twenty-four hours after the last drug administration, blood was collected from the eyeballs of all mice, and ovarian tissue was collected. After washing with PBS, the tissue was fixed with 4% paraformaldehyde. The fixed ovarian tissue was then dehydrated by immersion in ethanol of different concentrations, and finally immersed in xylene. After embedding in paraffin, the tissue was sectioned. After sectioning, the tissue was dewaxed with xylene and anhydrous ethanol, and 20 μg / mL of DNase-free proteinase K was added. The tissue was incubated at 37°C for 20 min, washed three times with PBS, and 50 μL of TUNEL detection solution was added to the sample. The tissue was incubated at 37°C in the dark for 60 min, washed three times with PBS, and then the cell nuclei were counterstained with DAPI for 10 min. After mounting with anti-fluorescence quenching mounting solution, positive apoptotic cells were observed and photographed under a fluorescence microscope. The results showed that, compared with the control group, D-gal induction increased the number of TUNEL-positive cells. GPN-I significantly reduced apoptosis of ovarian granulosa cells, indicating that GPN-I has a protective effect against D-gal-induced ovarian cell apoptosis. Figure 7 ).

Claims

1. The use of neutral non-soluble polysaccharides in ginseng for preparing a medicine for treating premature ovarian failure, characterized in that, The neutral non-amylodextrin polysaccharide in the ginseng consists of 94.43-96.75% glucose, 2.23-4.01% galactose and 1.02-1.56% arabinose in terms of molar percentage.

2. The use of the neutral non-amyloid polysaccharide in ginseng according to claim 1 in the preparation of a medicament for treating premature ovarian failure, characterized in that, The neutral non-amylodextrin polysaccharide in the ginseng is used for preparing a medicine for treating D-galactose-induced premature ovarian failure.

3. The use of the neutral non-amyloid polysaccharide in ginseng according to claim 1 in the preparation of a medicament for treating premature ovarian failure, characterized in that, The neutral non-amylodextrin polysaccharide in the ginseng consists of 96.75% glucose, 2.23% galactose and 1.02% arabinose in terms of molar percentage.

4. The use of the neutral non-amyloid polysaccharide in ginseng according to claim 1 or 3 in the preparation of a medicament for treating premature ovarian failure, characterized in that, The glucose is (1→)-Glcp, (1→4)-Glcp and (1→4,6)-Glcp; the galactose is (1→3,4)-Galp; and the arabinose is (1→)-Araf.

5. The use of the neutral non-amyloid polysaccharide in ginseng according to claim 4 for the preparation of a medicament for the treatment of premature ovarian failure, characterized in that, The molar ratio of (1→)-Araf, (1→)-Glcp, (1→4)-Glcp, (1→3,4)-Galp and (1→4,6)-Glcp is 0.15:4.02:24.54:1:3.

85.

6. The use of the ginseng neutral non-senile polysaccharide according to claim 1 or 2 in the preparation of a drug for treating premature ovarian failure, characterized in that, The molecular weight of the neutral non-amyloid polysaccharide in the ginseng is 4.72 x 10 4 Da.

7. The use of the ginseng neutral non-senile polysaccharide according to claim 1 or 2 in the preparation of a drug for treating premature ovarian failure, characterized in that, The preparation method of the neutral non-amylodextrin polysaccharide in the ginseng is as follows: Step one, grinding dried ginseng to obtain ginseng powder; Step two, water extraction of the ginseng powder, and then filtering to obtain a filtrate; Step three, centrifuging the filtrate, and taking supernatant to concentrate to obtain a concentrated solution; Step four, adding 95% ethanol to the concentrated solution, and then centrifuging to obtain a residue; Step five, dissolving the residue in water, and then respectively performing anion exchange chromatography, α-amylase enzymolysis and gel chromatography to obtain the neutral non-amylodextrin polysaccharide in the ginseng.

8. The use of the neutral non-amyloid polysaccharide in ginseng according to claim 7 for the preparation of a medicament for the treatment of premature ovarian failure, characterized in that, In step two, the ginseng powder is water extracted, and the specific operation is as follows: first, the ginseng powder is added into water with a weight of 20 times of the ginseng powder to soak for 1-2 h, and then extracted at 90 ℃ for 180 min, repeated for 3 times.

9. The use of the neutral non-amyloid polysaccharide in ginseng according to claim 7 for the preparation of a medicament for the treatment of premature ovarian failure, characterized in that, In step five, the residue is dissolved in water, and then respectively subjected to anion exchange chromatography, α-amylase enzymolysis and gel chromatography to obtain the neutral non-amylodextrin polysaccharide in the ginseng; the specific method is as follows: After the residue is dissolved in water, DEAE-cellulose anion exchange chromatography is used to separate the neutral polysaccharide with distilled water as the mobile phase, α-amylase is added to hydrolyze and remove starch, and then Sepharose CL-6B gel chromatography is used to purify the non-amylodextrin polysaccharide to obtain the neutral non-amylodextrin polysaccharide in the ginseng.

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