Monomeric compound separated from alpine yarrow herb extract, preparation method of monomeric compound and application of monomeric compound in delaying ovarian senescence and improving fertility

Abstract

The invention discloses a monomeric compound separated from an alpine yarrow herb extract, a preparation method of the monomeric compound and an application of the monomeric compound in delaying ovarian aging and improving fertility. The monomeric compound is prepared from the following raw materials: acesulfamide E, acesulfamide F and acesulfamide A; the preparation method of the alpine yarrow herb extract comprises the following steps: taking the overground part of the alpine yarrow herb, carrying out reflux extraction for 2 hours by using 80% ethanol, repeating for three times, combining extracting solutions, filtering, concentrating the extracting solutions until no alcohol smell exists, and carrying out reduced pressure freeze drying to obtain the alpine yarrow herb extract. Research finds that the monomeric compound has relatively good protection activity on primary ovarian granular cells, so that the monomeric compound can be used for preparing medicines or / and health care products for delaying ovarian senescence, the fertility age is prolonged, and the problem of reduction of the fertility rate caused by postponing of the fertility age is solved.

Description

Technical Field

[0001] The present invention relates to the field of natural product extraction, to Achilleae Herba extract, and particularly to a monomeric compound isolated from Achilleae Herba extract, its preparation method, and its application in delaying ovarian senescence and improving fertility ability. Background Art

[0002] During the aging process of the human body, the reproductive system is the first to decline, and among the aging of the female reproductive system, the ovary is the first organ to age. The decline of its function will lead to the acceleration of the aging of other organs. Ovarian senescence not only affects fertility ability, but also causes a decrease in estrogen levels, which in turn triggers a series of systemic health problems such as cardiovascular diseases, osteoporosis, and psychological problems. Currently, the starting age of ovarian senescence is about 35 years old, and by the age of 50, drastic changes occur at multiple levels such as organs, cells, and molecules in the female aging process. With the influence of factors such as environment and genetics, the ovarian senescence process accelerates.

[0003] Currently, there is no clear drug for treating ovarian senescence. Clinically, hormone replacement therapy (HDR) is mostly used to relieve the related symptoms of low estrogen, but long-term use will increase the risk of cancer and other diseases. Traditional Chinese medicine has the advantages of multiple components, multiple targets, and small side effects. Achilleae Herba is a traditional Chinese medicine in China. It is the dried aerial part of the plant Achillea alpina L. of the Compositae family, mainly distributed in Northeast China, Inner Mongolia, Hebei, Shanxi, Ningxia, Gansu and other places. It has the effects of detoxifying dampness, promoting blood circulation and relieving pain, and is used to treat sore throat, diarrhea, dysentery, dampness and heat leukorrhea, snake and insect bites, etc. Modern medicine has less research on the chemical components and corresponding activities of Achilleae Herba, and the exploration of its biological activity potential is insufficient. There is no literature report on the delaying effect of Achilleae Herba on physiological ovarian senescence in rats. Summary of the Invention

[0004] The first object of the present invention is to provide a monomeric compound isolated from Achilleae Herba extract, and the monomeric compound is achilleamide E, achilleamide F, achillneoside A; wherein, the structural formulas of achilleamide E, achilleamide F, and achillneoside A are as follows:

[0005]

[0006] As a preference of the present invention, the preparation method of the Achilleae Herba extract is: take the aerial part of Achilleae Herba, reflux extract with 80% ethanol for 2 h, repeat three times, combine the extraction solutions, filter and concentrate the extraction solution until there is no alcohol smell, and obtain the Achilleae Herba extract by vacuum freeze-drying.

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

[0008] Step S1: Take the extract of Achillea millefolium and separate it by a macroporous resin column. The separation system is water - ethanol = 1:0 to 0:1, with gradient elution to obtain the 40% ethanol elution fraction. After concentration until the alcohol smell disappears and freeze - drying under reduced pressure, an extract of the 40% ethanol elution fraction of Achillea millefolium is obtained;

[0009] Step S2: Take the 40% ethanol elution fraction of Achillea millefolium and separate it by normal - phase silica gel column chromatography. The separation system is dichloromethane - methanol = 1:0 to 0:1, with gradient elution to obtain the dichloromethane - methanol 10:1 elution component. Concentrate it and freeze - dry under reduced pressure;

[0010] Step S3: Take the dichloromethane - methanol 10:1 elution component and prepare it by semi - preparative liquid phase. The mobile phase is methanol - water = 73:27 with isocratic elution, and the DAD detection wavelengths are 210 and 254 nm to obtain monomeric compounds achilleamide E, achilleamide F, and achillneoside A.

[0011] As a preference of the present invention, when the extract of Achillea millefolium is separated by a macroporous resin column in Step S1, an atmospheric - pressure glass chromatography column is used: with a height of 130 cm, an inner diameter of 15 cm, column temperature: room temperature, flow rate: 1.3 L / h, pressure: atmospheric pressure; the specific conditions are: water - ethanol = 100:0 5 BV, water - ethanol = 80:20 5 BV, water - ethanol = 60:40 5 BV, water - ethanol = 20:80 5 BV, water - ethanol = 0:100 5 BV.

[0012] As a preference of the present invention, when the 40% ethanol elution fraction of Achillea millefolium is separated by normal - phase silica gel column chromatography in Step S2, an atmospheric - pressure glass chromatography column is used: with a height of 60 cm, an inner diameter of 8 cm, column temperature: room temperature, flow rate: 5 mL / min, pressure: atmospheric pressure; the specific conditions are: dichloromethane - methanol = 10:1 5 BV, dichloromethane - methanol = 5:1 5 BV, dichloromethane - methanol = 2:1 5 BV, dichloromethane - methanol = 1:1 5 BV, dichloromethane - methanol = 0:1 5 BV.

[0013] As a preference of the present invention, for the chromatographic column of the semi - preparative liquid phase in Step S3: an Agilent XDB - C18 preparative chromatographic column with a column length of 250 mm, a diameter of 9.4 mm, and a packing of 5 μm, column temperature: room temperature, flow rate: 3 mL / min, pressure: 10 MPa.

[0014] The present invention first isolates new monomeric compounds achilleamide E, achilleamide F, and achillneoside A from Achillea alpina L., and first discloses that the monomeric compounds have good protective activity against primary ovarian granulosa cells. Therefore, the monomeric compounds achilleamide E, achilleamide F, and achillneoside A provided by the present invention can be used in the preparation of drugs and / or health products for treating or delaying ovarian aging and improving fertility.

[0015] The present invention also provides a drug for delaying ovarian aging and improving fertility. The active ingredient in the drug is any one or a mixture of any two or more of the above monomeric compounds achilleamide E, achilleamide F, and achillneoside A, or the above-mentioned Achillea alpina L. extract. The active ingredient is used to delay physiological ovarian aging and extend the female reproductive age.

[0016] Advantages and beneficial effects of the present invention:

[0017] (1) The present invention first isolates new monomeric compounds achilleamide E, achilleamide F, and achillneoside A from Achillea alpina L., and first discloses the NMR data of achilleamide E, achilleamide F, and achillneoside A.

[0018] (2) The present invention first discloses that the monomeric compounds achilleamide E, achilleamide F, and achillneoside A have good protective activity against primary ovarian granulosa cells, and can be used in the preparation of drugs and / or health products for delaying ovarian aging and improving fertility, providing a new means for delaying ovarian aging.

[0019] (3) The Achillea alpina L. extract containing the monomeric compounds achilleamide E, achilleamide F, and achillneoside A provided by the present invention can significantly improve the number, morphology, and peripheral granulosa cell count of ovarian follicles in rats with ovarian aging, and can play a role in improving ovarian aging by improving follicular development and granulosa cell status, thereby delaying the process of ovarian aging.

[0020] (4) The Achillea alpina L. extract containing the monomeric compounds achilleamide E, achilleamide F, and achillneoside A provided by the present invention can improve the ovarian hormone levels in aging rats, improve the oxidative stress of the ovaries in aging rats, and enhance the fertility of rats with physiological ovarian aging.

[0021] (5) Any one or a mixture of two or more of the monomer compounds of achilleamide E, achilleamide F, and achillneoside A provided by the present invention, or the achillea extract containing the monomer compounds of achilleamide E, achilleamide F, and achillneoside A can be used as the active ingredient of a drug for delaying ovarian aging and improving fertility. This active ingredient can delay physiological ovarian aging, thereby extending the female childbearing age and solving the problem of the decline in fertility rate caused by the postponement of the childbearing age. Description of the Drawings

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

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

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

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

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

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

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

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

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

[0031] Figure 10 It is of the compound achilleamide F1 H- 1 H COSY NMR spectrum;

[0032] Figure 11 is the HSQC NMR spectrum of compound achilleamide F;

[0033] Figure 12 is the HMBC NMR spectrum of compound achilleamide F

[0034] Figure 13 is the MS spectrum of compound achillneoside A;

[0035] Figure 14 is of compound achillneoside A 1 H-NMR spectrum;

[0036] Figure 15 is of compound achillneoside A 13 C-NMR spectrum;

[0037] Figure 16 is the HSQC NMR spectrum of compound achillneoside A;

[0038] Figure 17 is the HMBC NMR spectrum of compound achillneoside A;

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

[0040] Figure 19 is the HPLC result chart of the derivatization of reference substance L-arabinose;

[0041] Figure 20 is the HPLC result chart of the derivatization of reference substance D-glucose;

[0042] Figure 21 is the HE staining chart of the ovary; among them, A, E are the normal groups; B, F are the model groups; C, G are the low-dose groups; D, H are the high-dose groups; A, B, C, D are the 5X magnification of the ovary HE staining; E, F, G, H are the 10X magnification of the ovary HE staining;

[0043] Figure 22 Chart of the pregnancy rate and litter size of rats;

[0044] Figure 23 is the chart of the levels of serum anti-Müllerian hormone (AMH) and estradiol (E2);

[0045] Figure 24 Graph of serum reactive oxygen species (ROS), reduced glutathione (GSH), and malondialdehyde (MDA) levels. Detailed implementation

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

[0047] The present invention will be further described below in conjunction with embodiments:

[0048] Preparation of monomeric compounds achilleamide E, achilleamide F, and achillneoside A in Achillea extract and experiments on their protective effects on primary ovarian granulosa cells, as well as experiments on the protective effects of Achillea extract containing the above monomeric compounds on the ovaries of rats with physiological ovarian aging.

[0049] 1. Reagents and Instruments

[0050] 1.1 Reagents and Medicinal Materials

[0051] Silica gel GF254 thin layer plate (Qingdao Ocean Chemical Co., Ltd.); Macroporous adsorption resin D101 (Sinopharm Chemical Reagent Co., Ltd.); Normal phase silica gel packing (Qingdao Ocean Chemical Co., Ltd.); Sephadex LH-20 (GE Healthcare Bio-Science AB); Chromatographic methanol (Thermo Fisher, USA, chromatographic grade); Dichloromethane, methanol and other reagents are all of analytical grade (Tianjin Zhiyuan Chemical Reagent Co., Ltd.); 0.25% trypsin (containing EDTA) (Gibco, USA); DMEM high glucose culture medium (Gibco, USA); Fetal bovine serum (FBS) (Gibco, USA); Penicillin-streptomycin mixture (Jiangsu KeyGen Biotech Co., Ltd.), Phosphate buffered solution (PBS) (Hyclone, USA); Dimethyl sulfoxide (DMSO) (Sigma, USA); CCK8 cell proliferation detection kit (Lanbolide Biotech Co., Ltd.); Sodium carboxymethyl cellulose (CMC-Na, Merck, USA); Superoxide dismutase (SOD) test kit (Nanjing Jiancheng Bioengineering Institute); Malondialdehyde (MDA) assay kit (Nanjing Jiancheng Bioengineering Institute); Reduced glutathione (GSH) assay kit (Nanjing Jiancheng Bioengineering Institute); Rat anti-Müllerian hormone (AMH) enzyme-linked immunosorbent assay kit (Enzyme Immuno-Bio-Technology Co., Ltd.); Rat estradiol (E2) enzyme-linked immunosorbent assay kit (Enzyme Immuno-Bio-Technology Co., Ltd.); Yarrow was purchased from Anguo Chinese herbal medicine market in Hebei Province and identified by Associate Researcher ZAN Ke of the National Institutes for Food and Drug Control as Achillea alpina L. of the genus Achillea in the Compositae family.

[0052] 1.2 Instruments

[0053] NE-2001 Rotary Evaporator (Eyela Co., Ltd., Japan); GB11240-89 Electrothermal Constant Temperature Water Bath (Beijing Medical Equipment Factory), Vacuum Pump (Zhengzhou Great Wall Science & Industry and Trade Co., Ltd.); CAV412 One-in-ten-thousandth Electronic Analytical Balance (Shanghai Ohaus Instruments Co., Ltd.); ME235S One-in-hundred-thousandth Electronic Analytical Balance (Sartorius Scientific Instruments Co., Ltd., Germany); KQ5200DV CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); LC-20AD Semi-preparative High Performance Liquid Chromatograph (Shimadzu Technology Co., Ltd., Japan); Agilent XDB-C18 Preparation Chromatographic Column (9.4mm×250mm, 5μm) (Waters Corporation, USA); Av III 500MHz Nuclear Magnetic Resonance Spectrometer (Bruker Co., Ltd., Germany), Av III 300MHz Nuclear Magnetic Resonance Spectrometer (Bruker Co., Ltd., Germany), Agilent 8453 UV-Visible Spectrophotometer (Agilent Technologies, USA), ThermoFisher FT-IR iS7 Infrared Spectrometer (Thermo Fisher Scientific, USA), Bruker Fourier Cyclotron Resonance High Resolution Mass Spectrometer (Bruker Co., Ltd., Germany); ESCO AC2-4S1 Ultra-clean Biosafety Cabinet (Thermo Fisher Scientific, USA); 371 Carbon Dioxide Incubator (Thermo Fisher Scientific, USA); MSL-3020 Autoclave (Sanyo Co., Ltd., Japan).

[0054] 1.3 Animal grouping and administration

[0055] Normal female SD rats were used. Among them, the normal group (Control) was 3 months old, and 6 rats were raised conventionally; the rats with physiological ovarian failure were 10 months old, and 18 rats were randomly divided into a model group, a low-dose group and a high-dose group, with 6 rats in each group. The model group (Model) was given an equal volume of normal saline, and the ethanol extract of Achillea alpina was dissolved in 0.5% CMC-Na solution. The high-dose group (High) was administered 360 mg / kg, and the low-dose group (Low) was administered 120 mg / kg. The administration dose was calculated from the results of preliminary experiments and clinical dosages in the early stage of the experiment. Among them, the administration dose of the high-dose group of 360 mg / kg was equivalent to the maximum dosage of Achillea alpina of 45 g specified in the pharmacopoeia.

[0056] 2 Experimental methods

[0057] 2.1 Preparation of Achillea alpina extract

[0058] An Achillea alpina extract, and the preparation method of the Achillea alpina extract is as follows: Take the upper part of Achillea alpina, reflux extract with 10 times the amount of 80% ethanol for 2 h, repeat three times, combine the extractive solutions, filter and concentrate the extractive solution until there is no alcohol smell, and obtain the Achillea alpina extract by vacuum freeze-drying.

[0059] 2.2 Isolation of monomeric compounds from Achillea extract

[0060] In this example, new monomeric compounds are isolated from the above-mentioned Achillea extract, and the monomeric compounds are achilleamide E, achilleamide F, and achillneoside A.

[0061] Specifically, in this example, the method for isolating the monomeric compounds achilleamide E, achilleamide F, and achillneoside A from the Achillea extract is as follows:

[0062] Step S1: Take 964 g of Achillea extract, dissolve it in ethanol, mix it with 1 kg of D101 macroporous resin, and dry it in a fume hood. Take 5 kg of macroporous resin and pack it into a column by the wet method, tap the packing tightly and firmly, and then slowly add the dried and mixed macroporous resin into the column; the conditions for column chromatography analysis are that the eluent is water-ethanol; column separation system: atmospheric pressure glass chromatography column: 130 cm high, 15 cm inner diameter, column temperature: room temperature, flow rate: 1.3 L / h, pressure: atmospheric pressure, gradient elution with water-ethanol system (100:0 - 0:100), specific conditions: water-ethanol (100:0) 5 BV, water-ethanol (80:20) 5 BV, water-ethanol (60:40) 5 BV, water-ethanol (20:80) 5 BV, water-ethanol (0:100) 5 BV, and a total of 5 elution fractions are obtained.

[0063] Step S2: Mix 64 g of the water-ethanol (60:40) elution fraction with 80 g of normal-phase silica gel (80 - 100 mesh), take 2 kg of normal-phase silica gel (200 - 300 mesh) and pack it into a column by the wet method, tap the packing tightly and firmly, and then slowly add the dried and mixed normal-phase silica gel into the column. The eluent for column chromatography analysis is petroleum ether-ethyl acetate. Column separation system: atmospheric pressure glass chromatography column: 60 cm high, 8 cm inner diameter, column temperature: room temperature, flow rate: 5 mL / min, pressure: atmospheric pressure, gradient elution with dichloromethane-methanol system (100:0 - 0:100), specific conditions: dichloromethane-methanol (10:1) 5 BV, dichloromethane-methanol (4:1) 5 BV, dichloromethane-methanol (2:1) 5 BV, dichloromethane-methanol (1:1) 5 BV, dichloromethane-methanol (0:1) 5 BV, and a total of 5 elution fractions are obtained.

[0064] Step S3: The 6 g of dichloromethane-methanol (10:1) elution fraction was prepared using preparative liquid chromatography. The column analysis conditions were as follows: the mobile phase was methanol-water. Column separation system: Agilent XDB-C18 preparative chromatography column, column length 250 mm, diameter 9.4 mm, packing 5 μm, column temperature: room temperature, flow rate: 3 mL / min, pressure: 10 MPa, isocratic elution with methanol-water (73:27), DAD detection wavelengths: 210, 254 nm, to obtain monomeric 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 spectroscopy, infrared spectroscopy, nuclear magnetic resonance hydrogen spectroscopy, nuclear magnetic resonance carbon spectroscopy, mass spectrometry, etc. (see Figures 1 to 17 ), and the structural formulas were determined as follows:

[0066]

[0067] The results of the structural identification are as follows:

[0068] It was determined that achilleamide E is a pale yellow oily compound. (+)HR-ESI-MS gave the quasi-molecular ion peak [M+Na] + m / z 262.1388, and its molecular formula was speculated to be C 13 H 21 NO 3 , with an unsaturation degree of 4. The infrared spectrum showed absorption peaks at 1656 cm -1 and 1733 cm -1 , indicating the presence of two carbonyl groups. 13 In the C-NMR spectrum, the signals at δ C 167.6 (C-1) and 175.5 (C-7) indicated the presence of amide and ester groups, suggesting that the compound is an alkane amide compound. In 1 H-NMR and 1 H- 1 H-COSY spectra, the 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') belonged to the same spin-coupling system and were typical piperidine ring signals [1] . In addition, 13 the 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') in the C-NMR spectrum also confirmed the presence of the piperidine ring.1 The signals at δ in the 1H-NMR spectrum and 13 in the 13C-NMR spectrum 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 The signals at 146.2 (C-3) and 122.4 (C-2) indicate the presence of a trans double bond. In the HMBC spectrum, the correlation peaks between the signals of H-2, H-3 and C-1 indicate that the trans double bond is connected to C-1. In 1 The 1H-NMR spectrum gives 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), which prove the existence of three -CH 2 - groups, and 1 H- 1 The H-H COSY spectrum suggests that H-2 to H-6 are in the same coupling system, indicating that these three methylene groups are connected. According to the correlation peak between H-6 and C-7 in the HMBC spectrum, the carbonyl group is located at C-7, and the correlation peak from H-1" to C-7 indicates that the methoxy group is connected to C-1". Therefore, the structure of achilleamide E is determined to be methyl (E)-1-oxo-1-(piperidin-7-yl)hept-2-enoate.

[0069] Achilleamide F was determined to be a pale yellow oily compound. By (+)HR-ESI-MS determination, [M+Na] + m / z 262.1388, and its molecular formula was speculated to be C 13 H 21 NO 3 , and the degree of unsaturation is 4. 13 The 13C-NMR data show δ C 169.0 (C-1) and 174.7 (C-8), indicating the presence of amide and ester groups in achilleamide F. 1 The 1H-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') are typical signals of isobutylamine groups, and δ C 48.0 (C-2'), 29.7 (C-3') and 20.5 (C-4', 5') also prove this [2] . 1 H- 1Two spin systems were observed in the H-COSY spectrum, namely from H-2 to H-7 and from H-2' to H-5'. 1 The H-NMR spectrum gave δ H Four vinylic hydrogen signals at 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), suggesting the presence of a conjugated diene, and its configuration was determined to be trans for both with coupling constants of 15.0 and 14.9 Hz. The connection 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 methylene groups, and the CH 2 -CH 2 connected to C-5 was determined by the absorption between H-6 and C-5 in the HMBC spectrum. In addition, the connection of the carbonyl group at C-8 was confirmed by the correlation peak between H-7 and C-8 in the HMBC spectrum, and the connection of the methoxy group at C-8 was proven by the correlation peak between H-1” and C-8. Therefore, the structural formula of achilleamide F was determined to be methyl (2E,4E)-1-(isobutylamino)-1-oxoocta-2,4-dienoate.

[0070] The 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 CD 3 OD

[0072]

[0073] Achillneoside A was determined to be a yellow powder, and its molecular formula C + was determined to be C 21 H 28 O 10 with 8 degrees of unsaturation by (+)HR-ESI-MS measurement at [M+Na] max m / z 463.1600. Characteristic signals of typical polyynes were observed in the ultraviolet absorption spectrum at λ 13 232, 244, 270, 290, 309, 330 nm. C79.9 (C-4), 67.0 (C-5), 60.3 (C-6), 61.2 (C-7), 64.3 (C-8) and 76.1 (C-9) also suggest the presence of polyynes. [3] 。 1 In the 1H-NMR spectrum, the signals at δ H 3.65 (1H, m, H-1), 3.93 (1H, m, H-1), 1.85 (2H, m, H-2), 2.54 (2H, m, H-3) suggest the presence of three methylene groups and the methyl signal at 1.94 (3H, s, H-10). The HMBC spectrum shows the correlation between H-10 and C-9, indicating that the methyl is attached to the polyyne. Further comparison with the [1] 1H-NMR 1 and 13 13C-NMR spectral data in the literature reveals that the aglycone nucleus of achillneoside A is identical to that of kamiohyneoside A. 13 In the 13C-NMR spectrum, the signals at δ C 103.2 (C-1') and 106.1 (C-1”) suggest the presence of two anomeric carbon atoms. After acid hydrolysis, it is found that the linked sugars of compound 3 consist of L-arabinose and D-glucose. The HMBC spectrum shows the correlation peak between H-1' and C-1, indicating that L-arabinose is attached to C-1 of the aglycone, while the correlation peak between H-1” and C-2' indicates that D-glucose is attached to C-2' of L-arabinose. From the coupling constants of the anomeric protons ( 3 J H-1' / H-2 ' = 7.7 Hz, 3 J H-1″ / H-2″ = 6.6 Hz), it is proven that the glycosidic bond configurations of L-arabinose and D-glucose are α and β, respectively. Therefore, the structure of achillneoside A is determined to be deca-4,6,8-triyn-1-O-α-L-arabinopyranoside-(1→2)-β-D-glucopyranoside.

[0074] The spectral data are as follows:

[0075] Table 2 1 1H NMR (500 MHz) and 13 13C NMR (125 MHz) data for achillneoside A in CD 3 OD

[0076]

[0077]

[0078] 2.3 Protective Experiments of Monomeric Compounds Achilleamide E, Achilleamide F and Achillneoside A in Achillea Extract on Primary Ovarian Granulosa Cells

[0079] 2.3.1 Extraction and Culture of Primary Ovarian Granulosa Cells

[0080] Three-week-old female SD rats were sacrificed after being anesthetized with excessive CO 2 and then disinfected by soaking in 75% alcohol. In a laminar flow hood, the lower abdominal tissues were exposed through a V-shaped incision, and the Y-shaped uterus and ovaries were separated, washed and placed in pre-cooled PBS solution. Then the ovaries were transferred into DMEM medium containing 1% double antibiotics, the follicles were punctured, and the granulosa cells were obtained by filtration. The remaining tissues were digested with 0.25% trypsin, filtered and centrifuged again, and finally the cells were resuspended with cell culture medium. After counting, the cells were inoculated into T25 culture flasks at an appropriate concentration. 5 mL of cell culture medium was added, mixed well and placed in a cell culture incubator for culture (37 °C, 5% carbon dioxide CO 2 2). The culture medium was changed every two days, and the cell morphology was observed to keep the cells in the logarithmic growth phase for standby.

[0081] 2.3.2 Activity Screening

[0082] 10,000 primary ovarian granulosa cells were inoculated into each well of a 96-well plate and cultured for 24 h. When the cells grew to 70% confluence, VCD with a concentration of 0.5 mM was added to establish a model. Each group had 3 replicate wells. After 24 h, the culture medium was changed, and 20 μM of achilleamide E, achilleamide F and achillneoside A were added respectively and cultured for 24 h. Then the culture medium was changed again, and 10 μL / well of CCK-8 was added respectively and cultured for 1 h. The absorbance value at a wavelength of 450 nm was read by an enzyme-linked immunosorbent assay reader. 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 monomeric compounds in Achillea extract on primary ovarian granulosa cells are shown in Table 3. It can be seen from Table 3 that the cell survival rates of monomeric compounds achilleamide E, achilleamide F and achillneoside A in Achillea extract at 20 μM were 87.69 ± 5.93%, 92.40 ± 3.72% and 83.13 ± 5.29% respectively. It can be seen that monomeric compounds achilleamide E, achilleamide F and achillneoside A in Achillea extract have good protective activity on primary ovarian granulosa cells.

[0085] Table 3 Improvement of the damage of primary ovarian granulosa cells by monomeric compounds (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 state of the ovary to a certain extent. The monomeric compounds achilleamide E, achilleamide F and achillneoside A provided in this example have good protective activity against primary ovarian granulosa cells. Therefore, it can be proved that the above monomeric compounds can be used in the preparation of drugs and / or health products for delaying ovarian aging and improving fertility, providing a new means for delaying ovarian aging.

[0088] 2.4 Effects of the Achillea extract containing the above three monomeric compounds (the Achillea extract prepared in 2.1) on the ovaries of aging rats

[0089] 2.4.1 Sample collection and index determination

[0090] Normal female SD rats were used. The normal group (Control) was 3 months old and 6 rats were fed conventionally; the rats with physiological ovarian aging 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 intragastrically administered with the Achillea extract or an equal proportion of normal saline for 6 weeks, with a 1-day drug withdrawal every week. After the administration, they were fasted for 12 hours, and samples were collected the next day. Blood was collected from the abdominal aorta, serum was separated, and the levels of SOD, GSH, MDA, AMH and E2 in the serum were measured using a kit. Statistical analysis was performed using Graphpad Pism software. One-way ANOVA was used for comparison among multiple groups, and P < 0.05 was considered statistically significant. The ovaries were fixed and paraffin sections were prepared and stained with HE.

[0091] 2.4.2 HE staining of ovaries

[0092] The results are as Figure 21As shown, there are more normal mature follicles and fewer atretic follicles. The follicles are smaller and round in shape, and there are more granulosa cells in the periphery of the follicles. In the model group, there are almost no mature follicles, more atretic follicles, the follicles are vacuolated in shape, and there are fewer granulosa cells in the periphery. Compared with the model group, in the low-dose Achillea extract administration group of rats, fewer mature follicles can be seen in the ovaries, with a relatively round shape and more granulosa cells in the periphery; more atretic follicles can be seen, with an irregular shape and fewer granulosa cells in the periphery. In the high-dose Achillea extract administration group of rats, the number of mature follicles increases significantly and the shape is round, with many granulosa cells, while the atretic follicles are relatively few and begin to show an irregular shape and a larger volume. Compared with the model group, after administration of Achillea extract, from the perspective of follicle status, the process of ovarian senescence is significantly delayed. Compared with the model group, each administration group of Achillea extract can significantly improve the number, morphology and the number of granulosa cells in the periphery of follicles in ovariectomized rats, indicating that Achillea extract may play a role in improving ovarian senescence by improving follicular development and granulosa cell status.

[0093] 2.4.3 Fertility of Rats

[0094] After 6 weeks of administration, each group of rats was caged with age-appropriate male rats adapted to the environment (female to male ratio of 2:1), with a 1-day rest every 4 days, and then different male rats were replaced to cage with the female rats again. The vaginal plugs and vaginal smears were examined for sperm at 7 o'clock every morning. The day when the vaginal plug and sperm were found was taken as the 0th day of pregnancy. The pregnancy rate (%) and litter size (average number of offspring) on the 1st - 10th day after caging were calculated to evaluate their fertility. The average gestation period of pregnant rats is 20 days (19 - 22 days). The results are as Figure 22 shown. Compared with the rats in the model group, the fertility rate and litter size of the rats in the Achillea extract administration group were significantly improved, enhancing the fertility of physiologically ovariectomized rats.

[0095] 2.4.4 Serum Hormone Levels

[0096] The results are as Figure 23 shown. Compared with the normal group, AMH and E2 in the model group decreased significantly, indicating a decline in ovarian function in rats. Compared with the model group, AMH and E2 in the Achillea extract administration group increased significantly, with a statistically significant difference (P < 0.01), suggesting that Achillea extract can improve the ovarian hormone levels in senescent rats.

[0097] 2.4.5 Serum Oxidative Stress Indexes

[0098] The results are as Figure 24As shown, compared with the normal group, the levels of SOD and GSH in the model group decreased significantly, while the level of MDA increased significantly. Compared with the model group, the levels of SOD, GSH, and MDA in the Achillea extract administration group improved significantly, suggesting that the Achillea extract can improve the oxidative stress of the ovaries in aging rats, enhance the antioxidant capacity of the ovaries in aging rats, and thus 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 Achillea extract containing monomeric compounds achilleamide E, achilleamide F, and achillneoside A provided by the present invention can be used in the preparation of drugs and / or health products for delaying ovarian aging and improving fertility.

[0101] References:

[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 A and B,two new polyacetylene glycosides fromflowers of edible Chrysanthemum"Kamiohno"[J].J Nat.Med.2021,75,167-172.

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

Claims

1. A monomer compound isolated from an achillea extract, characterized in that: The monomer compounds are achilleamide E, achilleamide F, and achillneoside A; wherein the structural formulas of achilleamide E, achilleamide F, and achillneoside A are as follows:

2. A monomer compound isolated from an achillea extract according to claim 1, characterized in that: The preparation method of the Achillea extract is as follows: taking the aboveground part of Achillea, extracting with 80% ethanol under reflux for 2 hours, repeating the process three times, combining the extracts, filtering and concentrating the extracts until there is no alcohol taste, and freeze-drying under reduced pressure to obtain the Achillea extract.

3. A method for separating monomeric compounds achilleamide E, achilleamide F and achillneoside A from the yarrow extract of claim 2, characterized in that: The method comprises the following steps: Step S1, taking an achillea extract, separating it through a macroporous resin column, using a separation system of water-ethanol = 1:0 to 0:1, gradient elution, obtaining a 40% ethanol elution fraction, concentrating it until there is no alcohol taste, and freeze-drying it under reduced pressure to obtain an achillea extract of the 40% ethanol elution fraction; Step S2, taking the 40% ethanol elution fraction of Achillea millefolium, separating it by normal silica gel column chromatography, using a separation system of dichloromethane-methanol = 1:0 to 0:1, gradient elution, obtaining a dichloromethane-methanol 10:1 elution fraction, concentrating it, and freeze-drying it under reduced pressure; Step S3, taking the elution component of dichloromethane-methanol 10:1, and preparing it by semi-preparative liquid phase, using methanol-water = 73:27 as the mobile phase for isocratic elution, and detecting the components at wavelengths of 210 and 254 nm to obtain monomer compounds achilleamide E, achilleamide F, and achillneoside A.

4. The method according to claim 3, characterized in that Step S1: When the yarrow extract 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; specific conditions are: water-ethanol = 100:0 5BV, water-ethanol = 80:20 5BV, water-ethanol = 60:40 5BV, water-ethanol = 20:805BV, water-ethanol = 0:100 5BV.

5. The method according to claim 3, characterized in that: When the 40% ethanol elution portion of step S2 yarrow is separated by normal silica gel column chromatography, 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; specific conditions are: dichloromethane-methanol = 10:1 5BV, dichloromethane-methanol = 5:1 5BV, dichloromethane-methanol = 2:1 5BV, dichloromethane-methanol = 1:15BV, dichloromethane-methanol = 0:1 5BV.

6. The method according to claim 3, characterized in that The chromatographic column for semi-preparative liquid phase in step S3 was 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.

7. Use of the monomer compound achilleamide E according to claim 1 or 2 in the preparation of medicines and / or health products for delaying ovarian aging and improving fertility.

8. Use of the monomer compound achilleamide F according to claim 1 or 2 in the preparation of medicines and / or health products 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 medicines and / or health products for delaying ovarian aging and improving fertility.

10. A drug for delaying ovarian aging and improving fertility, characterized in that: The active ingredient in the drug is any one of the monomer compounds achilleamide E, achilleamide F, achillneoside A described in claim 1 or a mixture of any two or more thereof or the achillea extract described in claim 2, and the active ingredient is used to delay physiological ovarian aging and extend the reproductive age of females.

Images