Dioscorea alata l. oxylipins with aromatase inhibitory activity, application and analysis and identification method thereof

Abstract

This invention discloses a yam oxylipoic acid compound with aromatase inhibitory activity, its application, and analytical identification methods, belonging to the field of medicinal chemistry. The yam oxylipoic acid compound is (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid, a novel compound. This compound can significantly inhibit aromatase activity and possesses the ability to inhibit excessive estrogen production, thus showing promising application prospects and value in diseases related to estrogen imbalance regulation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a yam oxygen lipid with aromatase inhibitory activity, application thereof and an analysis and identification method. BACKGROUND

[0002] Aromatase (CYP19A1) is an important member of the cytochrome P450 enzyme superfamily, which catalyzes the aromatization of androgens (such as testosterone, androstenedione) to estrogens (such as estradiol, estrone), and this conversion process is a key rate-limiting step in the biosynthetic pathway of endogenous estrogens. The enzyme is widely distributed in human ovaries, placenta, adipose tissue, skin, bone and brain, etc. and fine-tunes the endocrine homeostasis by maintaining the dynamic balance between androgens and estrogens.

[0003] The physiological synthesis of estrogen plays an indispensable role in the development of the female reproductive system, the regulation of the menstrual cycle, the maintenance of bone health and the protection of neural function. However, the excessive production of local or systemic estrogen caused by abnormal up-regulation of aromatase expression or activity is closely related to the pathogenesis of various hormone-dependent diseases. In particular, in postmenopausal women, the increased aromatase activity in peripheral tissues (such as adipose tissue and skin) promotes the conversion of androgens to estrogens, and this continuous stimulation of estrogen is considered to be an important driving factor for the occurrence and development of estrogen receptor-positive breast cancer. In addition, abnormal aromatase activity is also involved in the pathological process of endometrial hyperplasia, polycystic ovary syndrome, endometriosis and male prostate hyperplasia, etc.

[0004] Therefore, targeting the inhibition of aromatase activity has become one of the core strategies for the treatment of hormone-dependent breast cancer and the chemical prevention of high-risk populations. The third-generation aromatase inhibitors such as letrozole, anastrozole and exemestane, which are widely used in clinical practice, have shown significant efficacy in endocrine therapy for breast cancer. However, there are still certain limitations in long-term use, including increased risk of osteoporosis caused by decreased bone density, joint pain, abnormal blood lipid metabolism and increased risk of cardiovascular events. At the same time, in the field of health management and aesthetic medicine, there is an increasing demand for regulating local estrogen levels to maintain skin health and improve hormone-related skin problems. However, the high selectivity and potential systemic effects of existing synthetic drugs make it difficult to directly apply them to such non-indicated scenarios.

[0005] Based on the above background, exploring new aromatase inhibitors with novel structures and unique mechanisms from natural product resources is expected to provide safer auxiliary or alternative solutions for the treatment of hormone-dependent diseases such as breast cancer, and opens up a new research direction for the development of health products targeting hormone-related skin problems and endocrine regulation, which has important clinical translation value and broad application prospects. Summary of the Invention

[0006] This invention provides a novel compound with aromatase inhibitory activity, belonging to the oxolipases of yam. The compound is (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid, and its structural formula is as follows: .

[0007] This invention provides the use of the above-mentioned compounds in the preparation of drugs with aromatase inhibitory activity.

[0008] This invention provides the use of the above-mentioned compound in the preparation of a drug that inhibits excessive estrogen production.

[0009] This invention provides the use of the above-mentioned compound in the preparation of a medicament for treating estrogen-dependent breast cancer.

[0010] The present invention provides an estrogen-dependent breast cancer drug, the drug containing the above-mentioned compound having aromatase inhibitory activity.

[0011] This invention provides a method for preparing the above-mentioned compound, the steps of which are as follows: Yam powder was extracted three times with 70% ethanol aqueous solution. The extracts were combined and then concentrated under reduced pressure to remove organic solvents, yielding yam extract. The yam extract was subjected to normal-phase silica gel column chromatography, eluting with a chloroform / methanol gradient from a volume ratio of 100:0 to 60:40. The fraction eluted at a chloroform / methanol volume ratio of 80:20 was collected. Then, medium-pressure column chromatography was performed, eluting with a methanol / water gradient from a volume ratio of 35:65 to 95:5. The fraction eluted at a methanol / water volume ratio of 95:5 was collected. The fraction was purified by high-performance liquid chromatography (HPLC) under the following conditions: 45% acetonitrile, flow rate 5 mL / min. The fraction with a retention time of 67 min was collected to obtain the compound.

[0012] The present invention provides a method for analyzing and identifying the above-mentioned compounds in yam, wherein the method includes at least one of thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance.

[0013] The above thin-layer chromatography identification method has the following steps: Take 10 g of yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, soak at room temperature for 30 min, filter and concentrate the extract under reduced pressure, and make up to 10 mL with anhydrous ethanol to prepare the test solution; prepare a 1 mg / mL reference solution of the compound with anhydrous ethanol; take 3 µL of the test solution and the reference solution respectively, and spot them on the same silica gel G thin layer plate, using a chloroform-methanol-water mixture of 30:3:1 as the developing solvent, develop, remove, air dry, heat on a 105℃ hot plate for 1 h, spray with 10% sulfuric acid ethanol solution, and bake at 200℃ for 1 min until the color is clear; if the test sample chromatogram shows a spot of the same color at the corresponding position as the reference chromatogram, the yam contains the compound.

[0014] The above high-performance liquid chromatography (HPLC) analysis method comprises the following steps: Take 10 g of yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. After extraction, filter and concentrate under reduced pressure, dissolve in chromatographic grade methanol and make up to 10 mL. Filter through a 0.45 µm filter membrane to prepare the test solution. Prepare a 1 mg / mL reference solution of the compound with anhydrous ethanol. Use an Agilent Zorbax SB-C18 column at 30 ℃, with 0.1% formic acid-acetonitrile as the mobile phase, and elute isocratically at the following conditions: 0–30 min, 40% acetonitrile, flow rate 1 mL / min; detection wavelength 210 nm, injection volume 10 μL. If the chromatogram of the test sample shows a peak with the same retention time as the compound, the yam is identified as containing the compound.

[0015] The above-mentioned liquid chromatography-mass spectrometry (LC-MS) analysis and identification method comprises the following steps: Take 10 g of yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Filter and concentrate the extract under reduced pressure, dissolve in chromatographic grade methanol and bring the volume to 25 mL. Filter through a 0.22 µm filter membrane to prepare the test solution. Prepare a 1 mg / mL reference solution of the compound using anhydrous ethanol. Use a Hypersil GOLD column at 35℃, a flow rate of 0.3 mL / min, and an injection volume of 5 µL. The mobile phase consists of 0.1% acetic acid solution and methanol, with a gradient elution program of: 0–2 min, 10% methanol; 2–15 min, 10%–40% methanol; 15–30 min, 40%–75% methanol. A triple quadrupole mass spectrometer equipped with an electrospray ionization source is used in positive ion mode under the following conditions: spray voltage, 3500 V (ESI+); sheath gas pressure set to 40 units (positive ion mode); auxiliary gas pressure set to 10 units; vaporization chamber temperature set to 330 °C. °C (positive ion mode); if the ESI-MS m / z 381.26 [M+Na] appears at approximately 17.64 min in the LC-MS ion chromatogram of the test sample... + The presence of a characteristic quasi-molecular ion peak indicates the presence of this compound in yam.

[0016] The above-mentioned nuclear magnetic resonance (NMR) analysis identification method has the following steps: Take 10 g of yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Concentrate the extract under reduced pressure, and dissolve 30 mg of the extract in deuterated methanol to obtain the test solution. Perform 1H NMR spectroscopy. If the following characteristic signals appear simultaneously in the 1H NMR spectrum of the test sample: aromatic region (δ¹²) H Multiple cleavage olefin proton signals appear in the region of 5.5-6.0, and the oxygen-chain proton region (δ) H If two sets of multiple splitting characteristic signals appear (4.06 (q) and 3.56 (m)), the presence of this compound in yam can be identified.

[0017] This invention provides the application of the above-mentioned analytical identification method in the quality control of yam; the quality of yam refers to (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid in yam.

[0018] The beneficial effects of this invention are as follows: This invention extracts a novel compound, (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid, from yam. This compound can significantly inhibit the activity of aromatase and has the ability to inhibit excessive estrogen production, thus showing good application prospects and value in diseases related to estrogen imbalance regulation. Attached Figure Description

[0019] Figure 1This is the 1H NMR spectrum of compound A.

[0020] Figure 2 This is the carbon NMR spectrum of compound A.

[0021] Figure 3 Nuclear magnetic resonance of compound A 1 H- 1 H COSY diagram.

[0022] Figure 4 This is the HSQC NMR spectrum of compound A.

[0023] Figure 5 The image shows the nuclear magnetic resonance (HMBC) spectrum of compound A.

[0024] Figure 6 Comparison of thin-layer chromatograms from different developing systems.

[0025] Figure 7 Comparison of thin-layer chromatograms of representative yam varieties.

[0026] Figure 8 High-performance liquid chromatography (HPLC) identification of compound A in Chinese yam extract.

[0027] Figure 9 Characteristic NMR signal analysis of compound A in Chinese yam extract identifies the compound. Detailed Implementation

[0028] The materials used in this invention are as follows: The aromatase (CYP19A) activity assay kit was purchased from BD Biosciences, Inc. (USA); dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, Inc. (USA).

[0029] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0030] I. Isolation and Identification of Compound A This invention provides a novel compound, named (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid, which is obtained by the following method: 51 kg of dried yam powder (Huai yam) was extracted three times with an ethanol-water solution (70%). The extracts were combined and then concentrated under reduced pressure to remove the organic solvent, yielding the yam extract. The yam extract was subjected to normal-phase silica gel column chromatography, eluted with a chloroform / methanol gradient from 100:0 to 60:40 (v / v), and the fraction Fr5 (24.05 g) eluted at a chloroform / methanol volume ratio of 80:20 was collected. Fraction Fr5 was then subjected to ODS-A medium-pressure column chromatography, eluted with a methanol / water gradient from 35:65 to 95:5 (v / v), and the fraction eluted at a methanol / water volume ratio of 95:5 was collected. The fraction was purified by high-performance liquid chromatography (YMC-Pack ODS-A, 250 × 20.0 mm, S-5 μm; 45% acetonitrile, flow rate 5 mL / min, retention time 67 min) to obtain compound A (15.1 mg).

[0031] Structural identification Compound A is a pale yellow oil, ESI-MS m / z 357.20 [MH] - 381.26 [M+Na] + It is speculated that the molecular formula of compound A is C. 20 H 38 O5.

[0032] One-dimensional nuclear magnetic resonance hydrogen and carbon spectra, such as Figure 1 and Figure 2 As shown, its nuclear magnetic resonance data are as follows: 1 H NMR (CD3OD, 500 MHz) δ: 5.67 (1H, dd, J = 15.6, 6.4 Hz, H-7), 5.58(1H, ddd, J = 15.6, 7.7, 0.9 Hz, H-8), 4.06 (1H, q, J = 6.4 Hz, H-6), 3.60 (2H,m, H-9, H-19a), 3.56 (1H, m, H-10), 3.38 (1H, dq, J = 9.6, 7.0 Hz, H-19b), 2.26(2H, t, J = 7.4 Hz, H-2), 1.60 (2H, m, H-3), 1.55 (1H, m, H-5a), 1.50 (3H, m,H-5b, H-11a, H-12), 1.37 (1H, m, H-11b), 1.34 (10H, m, H-4, H-13, H-14, H-15,H-16, H-17), 1.17 (3H, t,J = 7.0 Hz, H-20), 0.91 (3H, t, J = 6.9 Hz, H-18).

[0033] 13 C NMR (CD3OD, 125 MHz) δ: 178.3 (C-1), 139.2 (C-7), 128.7 (C-8), 84.9 (C-9), 74.6 (C-10), 73.2 (C-6), 65.0 (C-19), 38.4 (C-5), 35.4 (C-2),33.8 (C-11), 33.0 (C-16), 30.7 (C-13), 30.4 (C-14), 30.3 (C-4, C-15), 26.8(C-12), 26.3 (C-3), 23.7 (C-17), 15.6 (C-20), 14.4 (C-18).

[0034] Nuclear magnetic resonance 1 H- 1 The two-dimensional correlation spectra of H COSY, HSQC, and HMBC are respectively as follows: Figures 3-5 As shown.

[0035] Based on the above spectral data, the structure of compound A can be identified as (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid. This compound is a new compound, and its chemical structure is as follows: .

[0036] II. Aromatase Inhibition Activity Test 1. In vitro activity test The inhibition rate (IC50) of the test compounds against aromatase was determined using BD Gentest's commercial CYP19 / MFC high-throughput screening kit. 50 The determination process is as follows: Add 100 μL of sample solution containing serially diluted analyte compounds (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78 μM) and NADPH-cofactor mixture to a 96-well plate and incubate at 37°C for 10 min. Then, initiate the reaction by adding 100 μL of enzyme-substrate mixture (recombinant human aromatase; 50 μM MFC; 20 mM phosphate buffer, pH 7.4) and incubate at 37°C for 30 min. Terminate all reactions by adding 0.1 M Tris base dissolved in acetonitrile. Measure fluorescence at 409 nm excitation and 530 nm emission wavelengths. Calculate the percentage inhibition of each inhibitor concentration relative to the well without inhibitor. Obtain the IC50 for each compound using SPSS software. 50 value.

[0037] The test results are shown in Table 1: Table 1. Half-maximal inhibitory concentrations of drugs

[0038] Finally, the inhibitory activity (IC50) of compound (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid against aromatase was determined. 50 The concentration was 14.7 µM, and the positive control aminoglutethimide showed inhibitory activity (IC50) against aromatase. 50 The concentration was 0.74 µM. As a prodrug compound, compound A already exhibits excellent inhibitory effects on aromatase, and through pharmacological design, it is expected to become an aromatase inhibitor with significant medical value.

[0039] 2. Cell viability test MCF-7 BUS cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. During the experiment, cells in logarithmic growth phase were seeded at a density of 6000 cells / well in 96-well plates and cultured for 48 hours to allow adhesion. Subsequently, the medium was replaced with phenol red-free DMEM (containing 10% dextran-coated activated charcoal-treated fetal bovine serum, 1% sodium pyruvate, and 1% non-essential amino acids), and different treatments were added according to the experimental groups: the control group received no drugs; the testosterone group received 10 μmol / L testosterone; the drug treatment group received 10 μmol / L testosterone and different concentrations of the test compound; and the positive control group received testosterone and a known aromatase inhibitor. After culturing cells for another 5 days, cell viability was assessed using the MTT assay: 100 μL of MTT solution (1 mg / mL) was added to each well, and the cells were incubated at 37°C for 4 hours. After discarding the supernatant, 150 μL of dimethyl sulfoxide was added to dissolve the formazan crystals, and the absorbance was measured at 550 nm. Cell viability was expressed as a percentage of the absorbance of the treatment group to that of the control group.

[0040] The test results are shown in Table 2: Table 2. Cell inhibition rate of drugs at various concentrations

[0041] It is evident that compound (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid possesses excellent aromatase inhibitory activity and exhibits superior aromatase inhibitory effects in estrogen-dependent human breast cancer cells, demonstrating remarkable application prospects and value.

[0042] III. Analytical and Identification Methods for Compound A Given that compound A has good aromatase inhibitory activity and has excellent application prospects and value in estrogen-dependent human breast cancer cell therapy, this invention further provides and optimizes analytical identification methods for compound A, including thin-layer chromatography identification method, high-performance liquid chromatography analysis method, liquid chromatography-mass spectrometry analysis identification method, and nuclear magnetic resonance analysis identification method.

[0043] The accumulation of secondary metabolites in medicinal plants exhibits significant chemical diversity, with their synthesis and expression strictly regulated by both genetic factors (variety) and environmental factors (origin ecology). Different yam varieties show differences in secondary metabolic pathways, and ecological factors such as soil and climate directly influence the expression of relevant functional genes, leading to significant intraspecific variations in the types and contents of active ingredients. Therefore, isolation results based on a single-source sample can only represent the chemical characteristics of that specific sample under specific conditions and cannot be directly inferred as common components of all yams. Given the significant chemical variations in active ingredients among different yam varieties and origins, establishing an analytical identification method for compound A is particularly necessary. This method can not only provide a scientific basis for tracing the origin and identifying varieties of yams, but also effectively control the batch-to-batch consistency of raw materials and products, ensuring the reproducibility of subsequent pharmacological studies and the quality stability of end products.

[0044] 1. Thin-layer chromatography identification method Example 1 Take 10 g of Chinese yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Filter and concentrate the extract under reduced pressure, then dilute to 10 mL with anhydrous ethanol to prepare the test solution. Prepare a 1 mg / mL reference solution of compound A with anhydrous ethanol. According to thin-layer chromatography, apply 3 µL of the test solution and 3 µL of the reference solution separately to the same silica gel G thin-layer plate. Develop using chloroform-methanol-water (30:3:1) as the developing solvent. Remove the plate, air dry, heat at 105℃ for 1 hour, spray with 10% sulfuric acid ethanol solution, and bake at 200℃ for 1 minute until the color is clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0045] Example 2 Take 10 g of Chinese yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Filter the extract and concentrate under reduced pressure, then dilute to 10 mL with anhydrous ethanol to prepare the test solution. Prepare a 1 mg / mL reference solution of compound A with anhydrous ethanol. According to thin-layer chromatography, apply 3 µL of the test solution and 3 µL of the reference solution separately to the same silica gel G thin-layer plate. Develop using chloroform-methanol-water (40:3:1) as the developing solvent. Remove the plate, air dry, heat at 105℃ for 1 hour, spray with 10% sulfuric acid ethanol solution, and bake at 200℃ for 1 minute until the color is clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0046] Example 3 Take 10 g of Chinese yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Filter and concentrate the extract under reduced pressure, then dilute to 10 mL with anhydrous ethanol to prepare the test solution. Prepare a 1 mg / mL reference solution of compound A with anhydrous ethanol. According to thin-layer chromatography, apply 3 µL of the test solution and 3 µL of the reference solution separately to the same silica gel G thin-layer plate. Develop with petroleum ether:acetone (7:3) as the developing solvent. Remove the plate, air dry, heat at 105℃ for 1 hour, spray with 10% sulfuric acid ethanol solution, and bake at 200℃ for 1 minute until the color is clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0047] Analysis results as follows Figure 6 As shown: A comprehensive comparison of the development effects of three thin-layer chromatography (TLC) solvent systems on the extract of Chinese yam and compound A revealed that the petroleum ether-acetone system was unsatisfactory. Under these chromatographic conditions, the spots of the target compound exhibited severe tailing and significant edge diffusion, resulting in blurred outlines after development. This indicates that this solvent system is unsuitable for the analysis and identification of compound A. After optimization, chloroform-methanol-water was selected as the development system, significantly improving the chromatographic behavior of the compound. The spots showed good symmetry and sharp edges. Among these, the chloroform-methanol-water (30:3:1) system demonstrated the best separation effect with a suitable development position.

[0048] Example 4 Take 10 g of powder from different varieties of yam (iron stick yam, Huai yam, and glutinous rice yam), add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Filter the extract and concentrate under reduced pressure, then dilute to 10 mL with anhydrous ethanol to prepare the test solution. Prepare a 1 mg / mL reference solution of compound A with anhydrous ethanol. According to thin-layer chromatography, apply 3 µL of the test solution and 3 µL of the reference solution separately to the same silica gel G thin-layer plate. Develop using chloroform-methanol-water (30:3:1) as the developing solvent. Remove the plate, air dry, heat at 105℃ for 1 hour, spray with 10% sulfuric acid ethanol solution, and bake at 200℃ for 1 minute until the color is clear. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0049] Analysis results as follows Figure 7 As shown: Among the three representative yam varieties, only iron yam and Huai yam contain compound A. Based on the area and color development of the spots in thin-layer chromatography, it can be determined that the content of compound A in Huai yam is significantly higher than that in iron yam.

[0050] The thin-layer chromatography identification method established in this invention can achieve rapid analysis and identification of compound A in extracts of different varieties of yam, thus providing a rapid and efficient technical means for the analysis of the nutritional and functional quality of yam with compound A as the characteristic component.

[0051] 2. High Performance Liquid Chromatography (HPLC) Analysis Method Take 10 g of Chinese yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Filter and concentrate the extract under reduced pressure, dissolve in chromatographic grade methanol and bring the volume to 10 mL. Filter through a 0.45 µm filter membrane to prepare the test solution. Prepare a 1 mg / mL reference solution of compound A with anhydrous ethanol. Use an Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5.0 μm), column temperature 30 ℃, with 0.1% formic acid water (A)-acetonitrile (B) as the mobile phase, isocratic elution (0–30 min, 40% B), flow rate 1 mL / min, detection wavelength 210 nm, and injection volume 10 μL. The test sample chromatogram should show a peak with the same retention time as the peak of compound A.

[0052] Analysis results as follows Figure 8 As shown: Under high-performance liquid chromatography (HPLC) conditions, the elution time of standard compound A is 9.5 min. If the extract of the test sample shows a chromatographic peak near this retention time, it can be determined that it contains compound A.

[0053] 3. Liquid chromatography-mass spectrometry (LC-MS) analysis and identification method Take 10 g of Chinese yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Filter and concentrate the extract under reduced pressure, dissolve in chromatographic grade methanol and bring the volume to 25 mL. Filter through a 0.22 µm filter membrane to prepare the test solution. Compound A is prepared as a 1 mg / mL reference solution using anhydrous ethanol. A Hypersil GOLD column (100 × 2.1 mm, 1.9 μm) was used. The column temperature was 35℃; the flow rate was 0.3 mL / min; and the injection volume was 5 µL. The mobile phase consisted of (A) 0.1% acetic acid solution and (B) methanol, with a gradient elution program of: 0–2 min, 10% B; 2–15 min, 10%–40% B; 15–30 min, 40%–75% B. The triple quadrupole mass spectrometer (TSQ Endura, Thermo Fisher Scientific, Inc.) is equipped with an electrospray ionization (ESI) source and operates in positive ion mode under the following optimal conditions: spray voltage, 3500V (ESI+); sheath gas pressure set to 40 units (positive ion mode); auxiliary gas pressure set to 10 units; vaporization chamber temperature set to 330 °C (positive ion mode).

[0054] The analysis results are shown below: The ESI-MS m / z reading of the test sample should appear at approximately 17.64 minutes after liquid chromatography-mass spectrometry (LC-MS) analysis, showing 381.26 [M+Na] m / z. + Characteristic quasi-molecular ion peak.

[0055] 4. Nuclear magnetic resonance analysis identification method Take 10 g of Chinese yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, soak at room temperature for 30 min, concentrate the extract under reduced pressure, take 30 mg and dissolve in deuterated methanol as the test solution, and perform nuclear magnetic resonance 1H and 1C spectroscopy. 1 H NMR 500 MHz, 13 C NMR 125 MHz). For example Figure 9 As shown, the following characteristic signals should appear simultaneously in the proton NMR spectrum of the test sample: aromatic region (δ¹²). H Multiple cleavage olefin proton signals appear in the region of 5.5-6.0, and the oxygen-chain proton region (δ) H Two sets of multiple splitting characteristic signals appeared at 4.06 (q) and 3.56 (m).

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A compound having aromatase inhibitory activity, characterized in that, The compound is (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid, and its structural formula is as follows: 。 2. The use of the compound of claim 1 in the preparation of a medicament having aromatase inhibitory activity.

3. The use of the compound of claim 1 in the preparation of a medicament for inhibiting excessive estrogen production.

4. The use of the compound of claim 1 in the preparation of a medicament for treating estrogen-dependent breast cancer.

5. An estrogen-dependent breast cancer drug, characterized in that, The drug contains the compound with aromatase inhibitory activity as described in claim 1.

6. The method for preparing the compound according to claim 1, characterized in that, The steps are as follows: Yam powder was extracted three times with 70% ethanol aqueous solution. The extracts were combined and then concentrated under reduced pressure to remove organic solvents, yielding yam extract. The yam extract was subjected to normal-phase silica gel column chromatography, eluting with a chloroform / methanol gradient from a volume ratio of 100:0 to 60:

40. The fraction eluted at a chloroform / methanol volume ratio of 80:20 was collected. Then, medium-pressure column chromatography was performed, eluting with a methanol / water gradient from a volume ratio of 35:65 to 95:

5. The fraction eluted at a methanol / water volume ratio of 95:5 was collected. The fraction was purified by high-performance liquid chromatography (HPLC) under the following conditions: 45% acetonitrile, flow rate 5 mL / min. The fraction with a retention time of 67 min was collected to obtain the compound.

7. The method for analyzing and identifying the compound of claim 1 in yam, characterized in that, The analytical identification method includes at least one of thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance.

8. The analytical identification method according to claim 7, characterized in that: The thin-layer chromatography identification method comprises the following steps: Take 10 g of yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, soak at room temperature for 30 min, filter and concentrate the extract under reduced pressure, and make up to 10 mL with anhydrous ethanol to prepare the test solution; prepare a 1 mg / mL reference solution of the compound with anhydrous ethanol; take 3 µL of the test solution and the reference solution respectively, and spot them on the same silica gel G thin layer plate, using a chloroform-methanol-water mixture of 30:3:1 as the developing solvent, develop, remove, air dry, heat on a 105℃ hot plate for 1 h, spray with 10% sulfuric acid ethanol solution, and bake at 200℃ for 1 min until the color is clear; if the test sample chromatogram shows a spot of the same color at the corresponding position as the reference chromatogram, then the yam contains the compound; The high-performance liquid chromatography (HPLC) analysis method comprises the following steps: Take 10 g of yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. After extraction, filter and concentrate under reduced pressure, dissolve in chromatographic grade methanol and make up to 10 mL. Filter through a 0.45 µm filter membrane to prepare the test solution. Prepare a 1 mg / mL reference solution of the compound with anhydrous ethanol. Use an Agilent Zorbax SB-C18 column at 30 ℃, with 0.1% formic acid-acetonitrile as the mobile phase, and elute isocratically at the following conditions: 0–30 min, 40% acetonitrile, flow rate 1 mL / min; detection wavelength 210 nm, injection volume 10 μL. If the chromatogram of the test sample shows a peak with the same retention time as the compound, the yam is identified as containing the compound. The liquid chromatography-mass spectrometry (LC-MS) analysis and identification method comprises the following steps: Take 10 g of yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Filter and concentrate the extract under reduced pressure, dissolve in chromatographic grade methanol and bring the volume to 25 mL. Filter through a 0.22 µm filter membrane to prepare the test solution. Prepare a 1 mg / mL reference solution of the compound using anhydrous ethanol. Use a Hypersil GOLD column at 35℃, a flow rate of 0.3 mL / min, and an injection volume of 5 µL. The mobile phase consists of 0.1% acetic acid solution and methanol, with a gradient elution program of: 0–2 min, 10% methanol; 2–15 min, 10%–40% methanol; 15–30 min, 40%–75% methanol. If the ESI-MS m / z 381.26 [M+Na] appears at approximately 17.64 min in the LC-MS ion chromatogram of the test sample... + The presence of a characteristic quasi-molecular ion peak indicates the presence of this compound in yam. The nuclear magnetic resonance analysis identification method comprises the following steps: Take 10 g of yam powder, add 50 mL of 95% ethanol aqueous solution for extraction, and extract at room temperature for 30 min. Concentrate the extract under reduced pressure, take 30 mg of the extract, dissolve it in deuterated methanol, and use it as the test solution for nuclear magnetic resonance (NMR) 1H NMR spectroscopy. If the following characteristic signals appear simultaneously in the NMR 1H NMR spectrum of the test sample: Aromatic zone (δ H Multiple cleavage olefin proton signals appear in the region of 5.5-6.0, and the oxygen-chain carbon proton region (δ) H If two sets of multiple splitting characteristic signals appear (4.06, 3.56), the presence of this compound in yam can be identified.

9. The application of the analytical identification method described in claim 7 in the quality control of yam.

10. The application according to claim 9, characterized in that, The quality of yam refers to (Z)-9-ethoxy-6,10-dihydroxyoctadec-7-enoic acid in yam.

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