A monomer compound isolated from yarrow extract and its preparation method and application in delaying ovarian aging and improving fertility

Abstract

The application discloses a monomer compound separated from achillea extract and a preparation method and application thereof in delaying ovary aging and improving fertility, wherein the monomer compound is achilleamide E, achilleamide F and achillneoside A; the structural formulae of achilleamide E, achilleamide F and achillneoside A are as follows: the preparation method of the achillea extract is as follows: taking the above-ground part of achillea, extracting the above-ground part by refluxing with 80% ethanol for 2 hours, repeating the extraction for three times, combining the extraction liquid, filtering and concentrating the extraction liquid until no alcohol smell is left, and freeze-drying the extraction liquid under reduced pressure to obtain the achillea extract. Researches show that the monomer compound has good protective activity on primary ovary granulosa cells, and therefore can be used for preparing a medicine for delaying ovary aging, thereby prolonging the fertility age and solving the problem of the decline of fertility rate caused by the delay of the fertility age.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of natural product extraction, and relates to Achillea extract, and in particular to a monomer compound isolated from Achillea extract and a preparation method thereof and application thereof in delaying ovary aging and improving fertility. BACKGROUND

[0002] In the aging process of the human body, the reproductive system is the first system to decline, and in the aging of the female reproductive system, the ovary is the first organ to age, and its functional decline will lead to accelerated aging of other organs. Ovarian aging not only affects fertility, but also leads to a decrease in estrogen levels, which in turn triggers a series of systemic health problems such as cardiovascular disease, osteoporosis, and psychological problems. At present, the average life expectancy of Chinese women in 2024 is 76.0 years, which has increased significantly compared to the past, but the age at which ovarian aging begins is still around 35 years old. By the time women reach 50 years old, the aging process at the organ, cell, and molecular levels has undergone dramatic changes, and with the influence of environmental and genetic factors, the ovarian aging process accelerates. With the aging of the population in China and the complete relaxation of the two-child or even three-child policy, delaying ovarian aging in women has become a major concern in terms of both women's health and fertility rates.

[0003] There is currently no clear drug for treating ovarian aging, and hormone replacement therapy (HDR) is commonly used in clinical practice to alleviate the symptoms of low estrogen, but long-term use can increase the risk of cancer. Traditional Chinese medicine has the advantages of multiple components and multiple targets, and small side effects, Achilleae Herba is a traditional Chinese medicinal material, which is the dried aboveground part of Achillea alpina L. of the Compositae family, mainly distributed in Northeast China, Inner Mongolia, Hebei, Shanxi, Ningxia, Gansu and other places, and has the effects of detoxifying and relieving dampness, promoting blood circulation and relieving pain, and is used to treat sore throat, diarrhea, dysentery, wet and hot lower body, snake and insect bites, etc. Modern medicine has less research on the chemical components and corresponding activities of Achillea, and the biological activity potential of Achillea has not been fully explored. There is no literature reporting the delaying effect of Achillea on physiological rat ovarian aging. SUMMARY

[0004] A first object of the present application is to provide a monomer compound isolated from Achillea extract, the monomer compound being achilleamide E, achilleamide F, and achillneoside A; wherein the structural formulae of achilleamide E, achilleamide F, and achillneoside A are as follows:

[0005]

[0006] As a preferred embodiment of the present application, the preparation method of the extract of Achillea yamaguhi is as follows: the aboveground parts of Achillea yamaguhi are extracted with 80% ethanol by reflux extraction for 2 hours, and the extraction is repeated three times; the extracts are combined, filtered, and concentrated to remove the alcohol taste; and the extract is freeze-dried under reduced pressure to obtain the extract of Achillea yamaguhi.

[0007] A second object of the present application is to provide a method for separating monomeric compounds achilleamide E, achilleamide F and achillneoside A from the extract of Achillea yamaguhi, which comprises the following steps:

[0008] Step S1: the extract of Achillea yamaguhi is separated by a macroporous resin column, the separation system is water-ethanol=1:0-0:1, gradient elution, and the 40% ethanol elution part is obtained; the extract is concentrated to remove the alcohol taste, and freeze-dried under reduced pressure to obtain the 40% ethanol elution part extract of Achillea yamaguhi;

[0009] Step S2: the 40% ethanol elution part of Achillea yamaguhi is separated by a normal silica gel column, the separation system is dichloromethane-methanol=1:0-0:1, gradient elution, and the dichloromethane-methanol 10:1 elution component is obtained; the extract is concentrated and freeze-dried under reduced pressure;

[0010] Step S3: the dichloromethane-methanol 10:1 elution component is prepared by semi-preparative liquid phase preparation, the mobile phase is methanol-water=73:27 isocratic elution, and DAD detection wavelength is 210, 254 nm; and the monomeric compounds achilleamide E, achilleamide F and achillneoside A are obtained.

[0011] As a preferred embodiment of the present application, when the extract of Achillea yamaguhi in step S1 is separated by a macroporous resin column, a normal pressure glass chromatography column is used: height 130 cm, inner diameter 15 cm, column temperature: room temperature, flow rate: 1.3 L / h, pressure: normal pressure; and the specific conditions are as follows: water-ethanol=100:05 BV, water-ethanol=80:20 5BV, water-ethanol=60:40 5BV, water-ethanol=20:80 5BV, and water-ethanol=0:100 5BV.

[0012] As a preferred embodiment of the present application, when the 40% ethanol elution part of Achillea yamaguhi in step S2 is separated by a normal silica gel column, a normal pressure glass chromatography column is used: height 60 cm, inner diameter 8 cm, column temperature: room temperature, flow rate: 5 mL / min, pressure: normal pressure; and the specific conditions are as follows: dichloromethane-methanol=10:1 5BV, dichloromethane-methanol=5:1 5BV, dichloromethane-methanol=2:1 5BV, dichloromethane-methanol=1:1 5BV, and dichloromethane-methanol=0:1 5BV.

[0013] As a preferred embodiment of the present application, the chromatographic column for semi-preparing the liquid phase in step S3 is an Agilent XDB-C18 preparative chromatographic column with a column length of 250 mm, a diameter of 9.4 mm, a filler of 5 μm, a column temperature of room temperature, a flow rate of 3 mL / min, and a pressure of 10 MPa.

[0014] The present application first separates new monomer compounds achilleamide E, achilleamide F and achillneoside A from achillea, and first discloses that the monomer compounds have good protective activity on primary ovarian granulosa cells. Therefore, the monomer compounds achilleamide E, achilleamide F and achillneoside A provided by the present application can be applied in the preparation of drugs for treating or delaying ovarian aging and improving fertility.

[0015] The present application further provides a drug for delaying ovarian aging and improving fertility, wherein the active ingredient in the drug is any one of the monomer compounds achilleamide E, achilleamide F and achillneoside A or a mixture of two or more thereof or the achillea extract described above, and the active ingredient is used for delaying physiological ovarian aging and prolonging the female fertile age.

[0016] The present application has the following advantages and beneficial effects:

[0017] (1) The present application first separates new monomer compounds achilleamide E, achilleamide F and achillneoside A from achillea, and first discloses the nuclear magnetic resonance data of achilleamide E, achilleamide F and achillneoside A.

[0018] (2) The present application first discloses that the monomer compounds achilleamide E, achilleamide F and achillneoside A have good protective activity on primary ovarian granulosa cells, can be applied in the preparation of drugs for delaying ovarian aging and improving fertility, and provides a new means for delaying ovarian aging.

[0019] (3) The achillea extract containing the monomer compounds achilleamide E, achilleamide F and achillneoside A provided by the present application can obviously improve the number, morphology of ovarian follicles and the number of peripheral granulosa cells in ovarian aging rats, can improve the follicle development and granulosa cell state, and play a role in improving ovarian aging, thereby delaying the progress of ovarian aging.

[0020] (4) The achillea extract containing achilleamide E, achilleamide F and achillneoside A monomer compounds provided by the application can improve the ovarian hormone level of aging rats, improve the oxidative stress of the ovaries of aging rats, and improve the fertility of physiologically aging rats.

[0021] (5) Any one of the achilleamide E, achilleamide F and achillneoside A monomer compounds or a mixture of two or more thereof or the achillea extract containing the achilleamide E, achilleamide F and achillneoside A monomer compounds provided by the application can be used as an active ingredient of a drug for delaying ovarian aging and improving fertility. The active ingredient can delay physiologically ovarian aging, thereby prolonging the female fertile age and solving the problem of the decline of fertility rate caused by the delay of the fertile age. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the MS spectrum of the compound achilleamide E;

[0023] Figure 2 is the MS spectrum of the compound achilleamide E 1 H-NMR nuclear magnetic resonance spectrum;

[0024] Figure 3 is the MS spectrum of the compound achilleamide E 13 C-NMR nuclear magnetic resonance spectrum;

[0025] Figure 4 is the MS spectrum of the compound achilleamide E 1 H- 1 H COSY nuclear magnetic resonance spectrum;

[0026] Figure 5 is the HSQC nuclear magnetic resonance spectrum of the compound achilleamide E;

[0027] Figure 6 is the HMBC nuclear magnetic resonance spectrum of the compound achilleamide E;

[0028] Figure 7 is the MS spectrum of the compound achilleamide F;

[0029] Figure 8 is the MS spectrum of the compound achilleamide F 1 H-NMR nuclear magnetic resonance spectrum;

[0030] Figure 9 is compound achilleamide F 13 C-NMR nuclear magnetic resonance spectrum;

[0031] Figure 10 is compound achilleamide F 1 H- 1 H COSY nuclear magnetic resonance spectrum;

[0032] Figure 11 HSQC nuclear magnetic resonance spectrum of compound achilleamide F

[0033] Figure 12 HMBC nuclear magnetic resonance spectrum of compound achilleamide F

[0034] Figure 13 MS spectrum of compound achillneoside A

[0035] Figure 14 is compound achillneoside A 1 H-NMR nuclear magnetic resonance spectrum;

[0036] Figure 15 is compound achillneoside A 13 C-NMR nuclear magnetic resonance spectrum;

[0037] Figure 16 HSQC nuclear magnetic resonance spectrum of compound achillneoside A

[0038] Figure 17 HMBC nuclear magnetic resonance spectrum of compound achillneoside A

[0039] Figure 18 acid hydrolysis HPLC result chart of compound achillneoside A

[0040] Figure 19 HPLC result chart of control L-arabinose derivatization

[0041] Figure 20 HPLC result chart of control D-glucose derivatization

[0042] Figure 21 ovarian HE staining chart; wherein, A, E normal group; B, F model group; C, G low-dose group; D, H high-dose group; A, B, C, D ovarian HE staining 5X magnification; E, F, G, H ovarian HE staining 10X magnification

[0043] Figure 22 Figure 2 shows the pregnancy rate and the number of offspring of rats;

[0044] Figure 23 Figure 4 shows the serum anti-mullerian hormone (AMH) and estradiol (E2) levels;

[0045] Figure 24 Figure 6 shows the serum reactive oxygen species (ROS), reduced glutathione (GSH) and malondialdehyde (MDA) levels. DETAILED DESCRIPTION

[0046] The present application is not limited by the following examples, and the specific embodiments can be determined according to the technical solutions of the present application and the actual situation. The various chemical reagents and chemical products mentioned in the present application are well-known and commonly used in the prior art unless otherwise specified; the percentages in the present application are mass percentages unless otherwise specified; the solutions in the present application are aqueous solutions with water as the solvent unless otherwise specified, for example, an ethanol solution is an aqueous ethanol solution; the normal temperature and room temperature in the present application generally refer to a temperature of 15-25℃, and are generally defined as 25℃.

[0047] The present application is further described below in conjunction with examples:

[0048] Preparation of monomer compounds achilleamide E, achilleamide F and achillneoside A in yarrow extract and experiment on the protective effect of the monomer compounds on primary ovarian granulosa cells, and experiment on the protective effect of yarrow extract containing the monomer compounds on the ovaries of physiologically aged rats.

[0049] 1. Reagents and instruments

[0050] 1.1 Reagents and medicinal materials

[0051] Silica gel GF254 thin layer plate (Qingdao Haigong Chemical Co., Ltd.); D101 macroporous adsorption resin (National Pharmaceutical Group Chemical Reagent Co., Ltd.); normal phase silica gel filler (Qingdao Haigong Chemical Co., Ltd.); sephadex LH-20 (GE Healthcare Bio-Science AB Co.); chromatographic methanol (Thermo Fisher Co., USA, chromatographically pure); dichloromethane, methanol, and other reagents were all analytical pure (Tianjin Zhiyuan Chemical Reagent Co., Ltd.); 0.25% trypsin (containing EDTA) (Gibco Co., USA); DMEM high-sugar culture medium (Gibco Co., USA); fetal bovine serum (FBS) (Gibco Co., USA); streptomycin mixture (Jiangsu Kaikai Biotechnology Co., Ltd.); phosphate buffered solution (PBS) (Hyclone Co., USA); dimethyl sulfoxide (DMSO) (Sigma Co., USA); CCK8 cell proliferation detection kit (Lambdabio Technology Co., Ltd.); sodium carboxymethylcellulose (CMC-Na, Merck Co., USA); superoxide dismutase (SOD) test kit (Nanjing Jiancheng Biological Engineering Institute); malondialdehyde (MDA) assay kit (Nanjing Jiancheng Biological Engineering Institute); reduced glutathione (GSH) assay kit (Nanjing Jiancheng Biological Engineering Institute); rat anti-mullerian hormone (AMH) enzyme-linked immunoassay kit (Enzyme Immune Biotechnology Co., Ltd.); rat estradiol (E2) enzyme-linked immunoassay kit (Enzyme Immune Biotechnology Co., Ltd.); yarrow was purchased from Hebei Anguo Medicinal Material Market, and was identified as Achillea alpina L. of the family Asteraceae by Associate Researcher Shan Mo of China Food and Drug Inspection Research Institute.

[0052] 1.2 Instruments

[0053] NE-2001 rotary evaporator (Japan Eyela Co., Ltd.); GB11240-89 type electric heating constant temperature water bath (Beijing Medical Equipment Factory), vacuum pump (Zhengzhou Great Wall Science and Technology Co., Ltd.); CAV412 type one ten-thousandth electronic balance (Shanghai Ohaus Instrument Co., Ltd.); ME235S ten-thousandth electronic analytical balance (Sartorius Scientific Instrument Co., Ltd. of Germany); KQ5200DV digital control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); LC-20AD semi-preparative high performance liquid chromatograph (Japan Shimadzu Technology Co., Ltd.); Agilent XDB-C18 preparative chromatographic column (9.4mm×250mm, 5μm) (Waters Corporation, USA); Av III 500MHz nuclear magnetic resonance spectrometer (Germany Bruker Company), Av III 300MHz nuclear magnetic resonance spectrometer (Germany Bruker Company), Agilent 8453 ultraviolet visible spectrophotometer (Agilent Technology Co., Ltd. of USA), ThermoFisher FT-IR iS7 infrared spectrometer (Thermo Fisher Corporation of USA), Bruker Fourier transform rotational high-resolution mass spectrometer (Germany Bruker Company); ESCO AC2-4S1 super-clean biological safety cabinet (Thermo Fisher Corporation of USA); 371 type carbon dioxide constant temperature incubator (Thermo Fisher Corporation of USA); MSL-3020 autoclave (Japan Sanyo Company).

[0054] 1.3 Animal grouping and administration

[0055] Normal female SD rats were used, wherein the normal group (Control) was 3 months old, and 6 were routinely fed; the physiological ovarian aging rats were 10 months old, and 18 were randomly divided into a model group, a low-dose group and a high-dose group, each group of 6, the model group (Model) was given an equal volume of normal saline, the yarrow ethanol extract was dissolved in a 0.5% CMC-Na solution, the high-dose group was administered (High) 360mg / kg, and the low-dose group was administered (Low) 120mg / kg. The administration dose was obtained by converting the pre-experimental results and the clinical usage before the experiment, wherein the administration dose of the high-dose group 360mg / kg was equivalent to the maximum usage of yarrow 45g specified in the pharmacopoeia.

[0056] 2 Experimental method

[0057] 2.1 Preparation of yarrow extract

[0058] A yarrow extract, the preparation method of the yarrow extract is: taking the aboveground part of yarrow, refluxing and extracting with 10 times the amount of 80% ethanol for 2h, repeating three times, combining the extract, filtering and concentrating the extract to no alcohol taste, and freeze-drying under reduced pressure to obtain the yarrow extract.

[0059] 2.2 Isolation of monomer compounds from the extract of achillea

[0060] This embodiment isolates new monomer compounds, achilleamide E, achilleamide F, achillneoside A, from the achillea extract as described above.

[0061] Specifically, in this embodiment, the method for isolating monomer compounds achilleamide E, achilleamide F, achillneoside A from the achillea extract is as follows:

[0062] Step S1, take 964g of achillea extract, dissolve in ethanol, mix with 1kg of D101 macroporous resin, and place in a fume hood to dry. Take 5kg of macroporous resin and wet pack the column, then slowly add the macroporous resin that has been dried and mixed to the column; the analysis conditions for passing through the column are water-ethanol as the developing agent; the column separation system: normal pressure glass chromatography column: height 130cm, inner diameter 15cm, column temperature: room temperature, flow rate: 1.3L / h, pressure: normal pressure, water-ethanol system gradient elution (100:0~0:100), specific conditions: water-ethanol (100:0) 5BV, water-ethanol (80:20) 5BV, water-ethanol (60:40) 5BV, water-ethanol (20:80) 5BV, water-ethanol (0:100) 5BV, a total of 5 elution components.

[0063] Step S2, mix 64g of water-ethanol (60:40) elution component with 80g (80-100 mesh) normal phase silica gel, take 2kg (200-300 mesh) normal phase silica gel and wet pack the column, then slowly add the normal phase silica gel that has been dried and mixed to the column. The developing agent for passing through the column is petroleum ether-ethyl acetate. The column separation system: normal pressure glass chromatography column: height 60cm, inner diameter 8cm, column temperature: room temperature, flow rate: 5mL / min, pressure: normal pressure, dichloromethane-methanol system gradient elution (100:0~0:100), specific conditions: dichloromethane-methanol (10:1) 5BV, dichloromethane-methanol (4:1) 5BV, dichloromethane-methanol (2:1) 5BV, dichloromethane-methanol (1:1) 5BV, dichloromethane-methanol (0:1) 5BV, a total of 5 elution components.

[0064] Step S3, the components eluted by 6 g dichloromethane-methanol (10:1) were prepared by preparative liquid chromatography, and the column analysis conditions were that the mobile phase was methanol-water. The column separation system: Agilent XDB-C18 preparative chromatographic column, column length 250 mm, diameter 9.4 mm, filler 5 μm, column temperature: room temperature, flow rate: 3 mL / min, pressure: 10 MPa, methanol-water isocratic elution (73:27), DAD detection wavelength: 210, 254 nm, to obtain monomer compounds achilleamide E, achilleamide F and achillneoside A.

[0065] The structures of the obtained compounds achilleamide E, achilleamide F and achillneoside A were identified by ultraviolet spectrum, infrared spectrum, nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, mass spectrum and the like (see Figures 1 to 17 ), and the structural formulas are as follows:

[0066]

[0067] The structure identification results are as follows:

[0068] It is determined that achilleamide E is a light yellow oily compound, and (+) HR-ESI-MS gives a quasi-molecular ion peak [M+Na] + m / z 262.1388, and the molecular formula is speculated to be C 13 H 21 NO3, and the unsaturation degree is 4. The infrared spectrum shows absorption peaks at 1656 cm -1 and 1733 cm -1 , indicating the presence of two carbonyl groups. 13 In the C-NMR spectrum, signals at δ C 167.6 (C-1) and 175.5 (C-7) indicate the presence of amide and ester groups, indicating that the compound is an alkanamide compound. In the 1 H-NMR and 1 H- 1 H-COSY spectrum, signals at δ H 3.58 (4H, m, H-2', 6'), 1.81 (2H, m, H-4') and 1.59 (4H, m, H-3', 5') belong to the same spin coupling system, which are typical piperidine ring signals [1] In addition, 13 In the C-NMR spectrum, signals at δ C 48.1 (C-6'), 44.4 (C-2'), 27.8 (C-5'), 26.8 (C-3') and 25.5 (C-4') also confirm the presence of a piperidine ring. 1 H-NMR spectrum and13 C-NMR spectrum δ H 6.74 (1H, dt, J = 15.1, 7.1 Hz, H-3), 6.44 (1H, d, J = 15.1 Hz, H-2) and δ C 146.2 (C-3), 122.4 (C-2) indicated the presence of a trans double bond. In the HMBC spectrum, the correlation between the signals of H-2, H-3 and C-1 indicated that the trans double bond was attached at C-1. In the 1 H-NMR spectrum gave δ H 2.29 (2H, dt, J = 7.4, 7.1 Hz, H-4), 1.69 (2H, tt, J = 7.4, 7.4 Hz, H-5) and 2.38 (2H, t, J = 7.4 Hz, H-6) signals proved the presence of three -CH2- groups, and 1 H- 1 H-COSY spectrum suggested that from H-2 to H-6 were in the same coupling system, indicating that the three methylene groups were connected. According to the correlation between H-6 and C-7 in the HMBC spectrum, it indicated that the carbonyl group was located at C-7, and the correlation from H-1" to C-7 indicated that the methoxy group was connected to C-1". Thus the structure of achilleamide E was determined as methyl (E)-1-oxo-1-(piperidin-7-yl)hept-2-enoate.

[0069] Achilleamide F was determined as a yellowish oil compound, and the (+) HR-ESI-MS determined [M+Na] + m / z 262.1388, the molecular formula was speculated as C 13 H 21 NO3, and the unsaturation was 4. 13 C-NMR data showed δ C 169.0 (C-1) and 174.7 (C-8) indicated the presence of amide and ester groups in achilleamide F. 1 H-NMR data δ H 3.11 (2H, d, J = 6.9 Hz, H-2'), 1.86 (1H, m, H-3') and 0.96 (6H, d, J = 6.7 Hz, H-4', 5') were the typical signals of isobutylamine group, while δ C 48.0 (C-2'), 29.7 (C-3') and 20.5 (C-4', 5') also proved this [2] . 1 H- 1 H-COSY spectrum observed two spin systems, from H-2 to H-7 and H-2' to H-5', respectively. 1H-NMR spectrum gave δ H 6.00 (1H, d, J = 15.0 Hz, H-2), 7.15 (1H, dd, J = 15.0, 10.8 Hz, H-3), 6.30 (1H, dd, J = 14.9, 11.1 Hz, H-4) and 6.13 (1H, m, H-5) four olefinic hydrogen signals, indicating the presence of a conjugated diene, the configuration of which was determined to be all trans by the coupling constants 15.0 and 14.9 Hz. The attachment of the diene to C-1 was confirmed by the correlation between H-2 and C-1 in the HMBC spectrum. δ H 2.50 (4H, m, H-6,7) indicated the presence of two methylenes, the attachment of CH2-CH2to C-5 was determined by the absorption between H-6 and C-5 in the HMBC spectrum. In addition, the attachment of the carbonyl group at C-8 was confirmed by the correlation peak between H-7 and C-8 in the HMBC spectrum, while the methoxy group was attached at C-8 according to the correlation peak between H-1" and C-8. Thus the structure of achilleamide F was determined to be methyl (2E,4E)-1-(isobutylamino)-1-oxoocta-2,4-dienoate.

[0070] Spectral data are as follows:

[0071] Table 1 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data for achilleamide E and F in CD3OD

[0072]

[0073] Achillneoside A was determined to be a yellow powder, its molecular formula was determined to be C + by (+) HR-ESI-MS [M + Na] 21 H 28 O 10 with 8 degrees of unsaturation. The UV absorption spectrum showed typical polyene characteristic signals at λ max 232, 244, 270, 290, 309, 330 nm. 13 The six quaternary carbon signals in the C-NMR spectrum appeared at δ C 79.9 (C-4), 67.0 (C-5), 60.3 (C-6), 61.2 (C-7), 64.3 (C-8) and 76.1 (C-9), again suggesting the presence of a polyene [3] . 1 The H-NMR spectrum gave δ H3.65 (1H, m, H-1), 3.93 (1H, m, H-1), 1.85 (2H, m, H-2), 2.54 (2H, m, H-3) signals indicated the presence of three methylene groups and a methyl signal at 1.94 (3H, s, H-10), HMBC spectrum gave the correlation of H-10 with C-9 indicated the methyl group attached to the polyacetylenic chain. Further comparison with the literature [1 ]in 1 H-NMR and 13 C-NMR spectral data, it was found that the aglycone of achillneoside A was identical to that of kamiohyneoside A. 13 C-NMR spectrum showed signals at δ C 103.2 (C-1') and 106.1 (C-1") indicated the presence of two sugar anomeric carbon atoms. After acid hydrolysis, it was found that the attached sugars of compound 3 consisted of L-arabinose and D-glucose. HMBC spectrum showed the correlation of H-1' with C-1 indicated the L-arabinose attached at C-1 position of the aglycone, while the correlation of H-1" with C-2' indicated the D-glucose attached through C-2' position of the L-arabinose. The coupling constants (J 3 J H-1' / H-2 = 7.7 Hz, 3 J H-1″ / H-2″ = 6.6 Hz) of the anomeric protons of L-arabinose and D-glucose proved the glycosidic linkage configurations to be α and β, respectively. Thus the structure of achillneoside A was determined as deca-4,6,8-triyn-1-O-α-L-arabinopyranoside-(1→2)-β-D-glucopyranoside.

[0074] Spectral data are as follows:

[0075] Table 2 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data for achillneoside A in CD3OD

[0076]

[0077]

[0078] 2.3 Protection of primary ovarian granulosa cells by monomeric compounds achilleamide E, achilleamide F and achillneoside A in Yarrowia extract

[0079] 2.3.1 Extraction and culture of primary ovarian granulosa cells

[0080] 3-week-old female SD rats were sacrificed after excessive CO2 anesthesia, 75% alcohol immersion disinfection; in the clean bench, the lower abdominal tissue was exposed by V-shaped incision, the Y-shaped uterus and ovary were separated, and after washing, they were placed in pre-cooled PBS solution; then the ovary was transferred into DMEM medium containing 1% double antibody, the follicle was punctured, and the granulosa cells were obtained by filtration; the remaining tissue was digested with 0.25% trypsin, then filtered and centrifuged again, and finally the cells were resuspended in cell culture medium, counted and inoculated into T25 culture bottles at an appropriate concentration. Add 5 mL of cell culture medium, mix well and put into the cell culture box for culture (37℃, 5% carbon dioxide CO2). Replace the culture medium every two days, observe the cell morphology, and maintain the cells in the logarithmic growth phase for standby.

[0081] 2.3.2 Activity screening

[0082] In 96-well plates, 10000 primary ovarian granulosa cells were inoculated in each well, cultured for 24h, and when the cells grew to 70% confluence, VCD modeling was added at a concentration of 0.5mM, 3 replicates were set in each group, 24h later, the medium was replaced, and achilleamide E, achilleamide F and achillneoside A 20μM were added respectively, and cultured for 24h, the medium was replaced, CCK-8 10μL / well was added, and cultured for 1h, then the absorbance value at 450nm wavelength was read by microplate reader, and the cell survival rate = (OD experimental group-OD blank group) / (OD control group-OD blank group).

[0083] 2.3.3 Results

[0084] The results of the protective activity screening of monomer compounds in Yarrowia extract on primary ovarian granulosa cells are shown in Table 3. As shown in Table 3, the cell survival rates of monomer compounds achilleamide E, achilleamide F and achillneoside A in Yarrowia extract at 20μM were 87.69±5.93%, 92.40±3.72% and 83.13±5.29% respectively, indicating that monomer compounds achilleamide E, achilleamide F and achillneoside A in Yarrowia extract had good protective activity on primary ovarian granulosa cells.

[0085] Table 3 Improvement of monomer compounds on primary ovarian granulosa cell damage (n=3, *** P<0.001 vs normal group; ## P<0.01 vs model group; ### P<0.001 vs model group)

[0086]

[0087] Primary ovarian granulosa cells play an important role in the process of ovarian aging, they not only support the growth and development of oocytes, but also reflect the functional status of the ovary to some extent. The monomer compounds achilleamide E, achilleamide F and achillneoside A provided in the embodiment have good protective activity on primary ovarian granulosa cells, and therefore it can be proved that the above monomer compounds can be applied in the preparation of drugs for delaying ovarian aging and improving fertility, providing a new means for delaying ovarian aging.

[0088] 2.4 Effect of the Yarrowia extract (Yarrowia extract prepared in 2.1) containing the above three monomer compounds on the ovaries of aging rats

[0089] 2.4.1 Sampling and index determination

[0090] Normal female SD rats were used, wherein the normal group (Control) was 3 months old, and 6 rats were routinely fed; the physiologically aging rats were 9 months old, and were randomly divided into a model group (Model), a low-dose group (Low) and a high-dose group (High). All rats were continuously administered with yarrowia extract or an equal proportion of normal saline for 6 weeks, with 1 day of drug withdrawal per week. At 12 hours after fasting after the end of administration, the rats were sampled the next day. Blood was taken from the abdominal aorta, and serum was separated. The levels of SOD, GSH, MDA, AMH and E2 in the serum were determined using a kit. Statistical analysis was performed using Graphpad Pism software, and single factor analysis of variance was used for comparison among multiple groups, with P<0.05 being statistically significant. The ovaries were fixed to prepare paraffin sections and were stained with HE.

[0091] 2.4.2 Ovary HE staining

[0092] The results are as follows Figure 21As shown, the normal mature follicle is more, the atretic follicle is less, the follicle is small and round in shape, and the peripheral granulosa cells are more; the model group has almost no mature follicle, more atretic follicles, and the follicle shape is empty, and the peripheral granulosa cells are less; compared with the model group, the low-dose acacia extract administration group has fewer mature follicles in the rat ovary, the shape is more round, and the number of peripheral granulosa cells is more; a large number of atretic follicles can be seen, and the shape is irregular, and the number of peripheral granulosa cells is less; the high-dose acacia extract administration group has a significant increase in the number of mature follicles in the rat ovary and the shape is round, and the peripheral granulosa cells are more, while the atretic follicles are relatively less, and begin to show irregular shape and larger volume. Compared with the model group, after administration of acacia extract, the progression of ovarian aging was significantly delayed from the follicle state. Compared with the model group, the acacia extract administration groups can significantly improve the number, shape and peripheral granulosa cell count of ovarian follicles in rats with ovarian aging, indicating that acacia extract may improve ovarian aging by improving follicular development and granulosa cell state.

[0093] 2.4.3 Fertility of rats

[0094] After 6 weeks of administration, each group of rats was caged with an adapted age-appropriate male rat (2:1 female to male), with a 1-day rest every 4 days, and then different male rats were caged with female rats again. The vaginal swabs and smears were checked every morning at 7 o'clock to find sperm, and the day when the vaginal swabs and sperm were found was taken as the 0th day of pregnancy. The success of pregnancy was determined by the delivery of female rats, and the pregnancy rate (%) and litter size (average number of offspring) were calculated 1-10 days after caging. The average gestation period of pregnant rats was 20 days (19-22 days). The results are shown in Figure 22 As shown, compared with the model group, the fertility rate and litter size of the acacia extract administration group were significantly improved, and the fertility of the physiologically aged ovarian rats was improved.

[0095] 2.4.4 Serum hormone levels

[0096] The results are shown in Figure 23 As shown, compared with the normal group, the AMH and E2 of the model group decreased significantly, indicating that the ovarian function of rats decreased; compared with the model group, the AMH and E2 of the acacia extract administration group were significantly improved, and the difference was statistically significant (P<0.01), indicating that acacia extract can improve the ovarian hormone level of aged rats.

[0097] 2.4.5 Serum oxidative stress indicators

[0098] The results are shown in Figure 24Compared with the normal group, the SOD and GSH levels of the model group were significantly decreased, and the MDA was significantly increased; compared with the model group, the SOD, GSH and MDA levels of the extract group were significantly improved, indicating that the extract can improve the oxidative stress of the ovary of the aging rat, and improve the antioxidant capacity of the ovary of the aging rat to resist aging.

[0099] Note: *** P < 0.001 vs normal group; ## P < 0.01 vs model group; ### P < 0.001 vs model group

[0100] In summary, the extract containing monomer compounds achilleamide E, achilleamide F and achillneoside A can be applied to the preparation of a drug for delaying ovarian aging and improving fertility.

[0101] Reference:

[0102] [1] Hofer O., Grege H., Robien W., 13C-NMR and 1H lanthanide induced shifts of naturally occurring alkamides with cyclic amide moieties-amides from Achillea falcata [J]. Tetrahedron, 1986, 42, 2707-2716.

[0103] [2] Li X.W., Yue H.C., Wu X., Guo Q., Tian J.Y., Liu Z.Y., Xiao M., Li X.X., Yu L., Li A., Ning Z.Q., Zan K., Chen X.Q., Neuroprotective Alkamides from the Aerial Parts of Achillea alpina L. [J]. Chem. Biodivers. 2022, 19, e202200218.

[0104] [3]Kurimoto S.I.,.Fujita H.,Kawaguchi S.,Sasaki Y.F.,Nakamura T.,Kubota T.,Kamiohnoyneosides Aand B,two new polyacetylene glycosides from flowers of edible Chrysanthemum"Kamiohno"[J].J Nat.Med.2021,75,167-172.

[0105] The above technical features constitute embodiments of the present application, which have strong adaptability and implementation effect. Non-essential technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A monomeric compound isolated from extract of yarrow, characterized in that, The monomer compound is achilleamide E, achilleamide F, achillneoside A; wherein, the structural formula of achilleamide E, achilleamide F, achillneoside A is as follows:

2. A monomeric compound isolated from the extract of the yarrow plant as claimed in claim 1, wherein, The preparation method of the extract of the achillea yamauhaw is as follows: the above-ground parts of achillea yamauhaw are taken, and 80% ethanol is used to reflux extract for 2 hours, which is repeated for three times; the extract solutions are combined, filtered, and concentrated to remove alcohol taste; and the extract solution is freeze-dried under reduced pressure to obtain the achillea yamauhaw extract.

3. A process for isolating the monomeric compounds achilleamide E, achilleamide F, achillneoside A from the extract of Yarrowia herba of claim 2, characterized by, The method comprises the following steps: Step S1, the extract of achillea yamauhaw is separated by a macroporous resin column, the separation system is water-ethanol=1:0-0:1, gradient elution is adopted, 40% ethanol elution part is obtained, and the extract is concentrated to remove alcohol taste, and freeze-dried under reduced pressure to obtain the 40% ethanol elution part extract of achillea yamauhaw; Step S2, the 40% ethanol elution part of achillea yamauhaw is separated by a normal silica gel column, the separation system is dichloromethane-methanol=1:0-0:1, gradient elution is adopted, dichloromethane-methanol 10:1 elution component is obtained, concentrated, and freeze-dried under reduced pressure; Step S3, the dichloromethane-methanol 10:1 elution component is prepared by semi-preparative liquid phase, the mobile phase is methanol-water=73:27 isocratic elution, and the detection wavelength is 210, 254 nm, monomer compounds achilleamide E, achilleamide F, achillneoside A are obtained.

4. The method of claim 3, wherein, When the extract of achillea yamauhaw in step S1 is separated by a macroporous resin column, a normal pressure glass chromatographic column is used: high 130 cm, inner diameter 15 cm, column temperature: room temperature, flow rate: 1.3 L / h, pressure: normal pressure; the specific conditions are as follows: water-ethanol=100:0 5BV, water-ethanol=80:20 5BV, water-ethanol=60:40 5BV, water-ethanol=20:80 5BV, water-ethanol=0:100 5BV.

5. The method of claim 3, wherein, When the 40% ethanol elution part of achillea yamauhaw in step S2 is separated by a normal silica gel column, a normal pressure glass chromatographic column is used: high 60 cm, inner diameter 8 cm, column temperature: room temperature, flow rate: 5 mL / min, pressure: normal pressure; the specific conditions are as follows: dichloromethane-methanol=10:1 5BV, dichloromethane-methanol=5:1 5BV, dichloromethane-methanol=2:1 5BV, dichloromethane-methanol=1:1 5BV, dichloromethane-methanol=0:1 5BV.

6. The method of claim 3, wherein, The chromatographic column of semi-preparative liquid phase in step S3 is Agilent XDB-C18 preparative chromatographic column, column length 250 mm, diameter 9.4 mm, filler 5 μm, column temperature: room temperature, flow rate: 3 mL / min, pressure: 10 MPa.

7. The monomer compound achilleamide E of claim 1 or 2 is used in the preparation of a medicine for delaying ovarian aging and improving fertility.

8. The monomer compound achilleamide F of claim 1 or 2 is used in the preparation of a medicine for delaying ovarian aging and improving fertility.

9. Use of the monomer compound achillneoside A according to claim 1 or 2 in the preparation of a medicament for delaying the physiological ovarian aging and improving the fertility.

10. A medicament for delaying ovarian aging and improving fertility, characterized by comprising the compound of claim 1. The active ingredient in the medicament is any one of the monomer compounds achilleamide E, achilleamide F, achillneoside A according to claim 1 or a mixture of two or more thereof or the extract of achillea according to claim 2, and the active ingredient is used for delaying the physiological ovarian aging and extending the female fertile age.

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