Hydroazulene type sesquiterpene compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of chemistry, and particularly relates to a hydrodroazulene type sesquiterpene compound as well as a preparation method and application thereof. The invention relates to a preparation method of a hydroazulene type sesquiterpene compound, which is characterized in that the hydroazulene type sesquiterpene compound is a colorless oily substance, the molecular formula of the hydroazulene type sesquiterpene compound is C15H20O4, the hydroazulene type sesquiterpene compound is named as yunnadol A, the English name of the hydroazulene type sesquiterpene compound is ((3R, 4S, 6aR, 7R, 9aS)-7-hydroxy-6a-methyl-4-(prop-1-en-2-yl) octahydro-1H-3, 9a-methanocyclopenta [c] oxocine-1, 10-dione), and the hydroazulene type sesquiterpene compound has the following structure: # imgabs0 #. The hydroazulene type sesquiterpene is a natural organic compound, has high neuroanalgesic activity, and is used as an active component to be prepared into an anti-migraine medicine or a medicine composition together with a pharmaceutically acceptable carrier or an auxiliary material, and the anti-migraine medicine or the medicine composition is prepared from the hydroazulene type sesquiterpene and the pharmaceutically acceptable carrier or the auxiliary material.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant chemistry, and in particular relates to a hydroazulene-type sesquiterpene compound and a preparation method and application thereof. Background Art

[0002] Migraine is a common, recurrent primary headache disorder characterized by severe, throbbing headaches on one or both sides, often accompanied by nausea, vomiting, and hypersensitivity to sound and light stimulation. High-frequency attacks and chronic migraines seriously affect patients' quality of life. Currently, the pathogenesis of migraine is not fully understood, and there are many limitations in its treatment. For example, triptans, a drug used to treat migraine attacks, have drawbacks that cannot be ignored: less than 50% of patients have a good effect; the 24-hour recurrence rate is as high as 40%; and due to serotonin (5-HT) 1B The vasoconstriction caused by receptor stimulation makes it contraindicated for patients with cardiovascular diseases. The main preventive drugs for chronic migraine include beta-blockers, calcium ion antagonists, anti-epileptic drugs, and antidepressants. These drugs were not originally designed to target the pathogenic mechanism of migraine itself, and their adverse reactions are significant, and their therapeutic effects are limited, so they are often not widely accepted by patients. The incidence of migraine varies greatly in different countries, regions, and different epidemiological studies, ranging from approximately 2.6% to 21.7%. Among them, women aged 25 to 55 are the most susceptible group to migraine (Yeh WZ, Blizzard L, Taylor BV. What is the actual prevalence of migraine?. Brain and Behavior, 2018, 8(6):e00950). Data from the Global Burden of Disease show that migraine ranks third among the causes of disabling diseases in people aged 15 to 49 years (Deuschl G, Beghi E, Fazekas F, et al. The burden of neurological diseases in Europe: an analysis for the Global Burden of Disease Study 2017. Lancet Public Health, 2020, 5(10): e551-e567).

[0003] Migraine medication is divided into acute treatment and preventive treatment. Non-specific medications for acute treatment include nonsteroidal anti-inflammatory drugs, acetaminophen, and caffeine-containing compound preparations. NSAIDs are the most widely used medications for acute migraine treatment, but they may cause adverse reactions such as gastrointestinal problems. Specific medications for acute treatment include triptans and ergotamines. When using triptans, attention should be paid to their contraindications. Patients with coronary heart disease, ischemic stroke, ischemic peripheral vascular disease, and severe liver damage should not use them. Ergotamines are currently less used due to their cardiac, hepato-renal toxicity (Dong Xuejia, Jiang Mingfang. New Progress in Migraine Pharmacological Treatment. Chinese Journal of Neurology, 2020, 53(5):385-386). Preventive treatment of migraine can reduce the frequency, duration and severity of migraine attacks and improve the quality of life of patients. Traditional commonly used drugs include calcium channel antagonists, antiepileptic drugs, beta-blockers, calcium channel modulators and antidepressants (Neurologists Branch of the Chinese Medical Doctor Association, Headache and Sensory Disorders Professional Committee of the Chinese Association of Research Hospitals. Guidelines for the Diagnosis and Treatment of Migraine in China (2022 Edition). Chinese Journal of Pain Medicine, 2022, 28(12):881-898). These are all non-specific drugs. Long-term use may cause central or peripheral adverse reactions. For example, long-term use of flunarizine may cause sedation and weight gain, and long-term, high-dose use may cause extrapyramidal reactions. Therefore, there is an urgent need to develop safe, effective and long-term migraine treatment drugs to improve the quality of life of patients. Summary of the Invention

[0004] The first purpose of the present invention is to provide a hydroazulene-type sesquiterpene compound; the second purpose is to provide a method for preparing the hydroazulene-type sesquiterpene compound; and the third purpose is to provide an application of the hydroazulene-type sesquiterpene compound.

[0005] The first object of the present invention is achieved in this way. The hydroazulene type sesquiterpene compound is a colorless oil with the molecular formula: 15 H 20 O4, named yunnadolin A, English name: ((3 R ,4 S , 6a R ,7 R ,9a S )-7-hydroxy-6a-methyl-4-(prop-1-en-2-yl)octahydro-1 H -3,9a-methano cyclopenta[ c]oxocine-1,10-dione), having the following structure: .

[0006] The second object of the present invention is achieved by using the root of Aristolochiaceae Aristolochiae genus as raw material, and preparing the product through pretreatment, extract extraction, organic solvent extraction, silica gel column chromatography and purification and separation steps, specifically comprising the following steps: A. Pretreatment: The root of Yunnan Acanthopanax yunnanensis was dried and crushed through a 10-mesh sieve to obtain material a; B. Extraction: Add 5 to 8 times the mass of material a to material a with a 95% ethanol aqueous solution by volume for extraction. The extraction time is 1 to 2 hours and the number of extractions is 1 to 3. Combine the extracts to obtain extract b. C. Organic solvent extraction: extract b was suspended in water, extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and concentrated under reduced pressure to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water extract respectively; D. Silica gel column chromatography: 1) The ethyl acetate fraction was gradient eluted with a dichloromethane-methanol solution in a volume ratio of 1:0 to 0:1. After TLC analysis, the same fractions were combined to obtain five components, Fr.A to Fr.E: Fr.A (1:0, v / v), Fr.B (50:1, v / v), Fr.C (20:1, v / v), Fr.D (10:1, v / v), and Fr.E (0:1, v / v). 2) Fr. A (fractions obtained by eluting with a 1:0 dichloromethane-methanol solution) was gradient eluted with petroleum ether-ethyl acetate solutions in volume ratios of 50:1, 25:1, and 0:1. After TLC analysis, the same fractions were combined to obtain three fractions: Fr. A1 (50:1, v / v), Fr. A2 (25:1, v / v), and Fr. A3 (0:1, v / v). 3) Fr.A2 (i.e., the eluate fraction obtained by eluting with a 25:1 petroleum ether-ethyl acetate solution) was gradient eluted with a petroleum ether-ethyl acetate solution in a volume ratio of 80:1 to 0:1. After TLC analysis, the same fractions were combined to obtain five fractions, Fr.A2a to Fr.A2e: Fr.A2a (80:1, v / v), Fr.A2b (50:1, v / v), Fr.A2c (15:1, v / v), Fr.A2d (5:1, v / v), and Fr.A2e (0:1, v / v). E. Purification and separation: The Fr.A2e portion (i.e., the eluate fraction obtained by eluting with a 0:1 petroleum ether-ethyl acetate solution) was purified and separated by semi-preparative HPLC to obtain yunnadolin A, i.e., the target compound, a hydroazulene-type sesquiterpene compound.

[0007] The third object of the present invention is achieved by using the hydroazulene-type sesquiterpene compound in the preparation of a drug for treating migraine.

[0008] Yunnan Acanthopanax Isotrema yunnanensis (Franch.) belongs to the genus Aristolochiaceae, and is endemic to southwest my country. Its dried rhizome is a Chinese herbal medicine called Xiaonanmuxiang. Xiaonanmuxiang is also a traditional ethnic minority medicinal material with the effect of relieving pain, treating stomachache, abdominal pain, bruises, fractures, and rheumatoid arthritis. According to historical records: "Yunnan Aristolochia, pungent in taste and warm in nature, is mainly used to treat gastritis, abdominal cold pain, biliary colic, indigestion, gastrointestinal disharmony, abdominal pain and diarrhea, rheumatic bone pain, bruises, and malaria." The chemical components of Yunnan Aristolochia mainly include various types of sesquiterpenoid compounds, such as humulene, 12-membered macrocyclic, isocaryophyllene, germarane, and hydroazulene (Cheng ZB, Shao WW, Liu YN, et al. Extracellular signal-regulated kinases (ERK) inhibitors from Aristolochia yunnanensis . J. Nat. Prod. 2013,76(4), 664-671; Chen SR, Zhang WP, BaoJ.M., et al. Aristoyunnolin H attenuates extracellular matrix secretion incardiac fibroblasts by inhibiting calcium influx. Arch. Pharmacal Res. 2017,40(1),122-130; Lou LL, Li W., Zhou BH, et al. (+)-Isobicyclogermacrenaland spathulenol from Aristolochia yunnanensis alleviate cardiac fibrosis by inhibiting transforming growth factor β / small mother against decapentaplegicsignaling pathway. Phytother. Res. 2019,33(1),214-223; Du Z., Wen FN, ZhangJ.P., et al. Two rare hydroazulene-type sesquiterpenes from the roots of Aristolochia yunnanensis . Zeitschrift für Naturforschung B. 2014, 69(6), 742-746). Pharmacological studies have shown that the plant has cytotoxic and anti-cardiac fibrosis activity. Although Yunnan Aristolochia has analgesic effects and is widely used among the people, there are no reports on the analgesic activity of its monomeric components, and its mechanism of action is still unclear. The present invention conducts in-depth research on compounds with anti-migraine effects in Aristolochia plants, in order to discover natural products with anti-migraine activity and provide a basis for screening highly effective and low-toxic anti-migraine drugs.

[0009] The present invention discovered that a solvent (e.g., ethanol solution) extract of the Aristolochia yunnanensis plant (Aristolochia yunnanensis) exhibits significant analgesic activity. The chemical composition of the extract, guided by biological activity testing, was studied, resulting in the identification of a hydroazulene-type sesquiterpenoid compound, which exhibits a potent inhibitory effect on writhing in mice. Further experiments revealed that the hydroazulene-type sesquiterpenoid compound exhibited significant anti-migraine activity in a nitroglycerin-induced migraine mouse experiment. The hydroazulene-type sesquiterpenoid compound is also used in the preparation of an anti-migraine drug or pharmaceutical composition for the treatment of acute or preventive migraines.

[0010] The present invention uses the acetic acid writhing method of mice, which is currently recognized both domestically and internationally, to test the analgesic activity of the hydroazulene-type sesquiterpenoid compound of the present invention, and calculates the median effective dose (ED50) of the hydroazulene-type sesquiterpenoid compound for inhibiting the writhing reaction in mice. The calculation results show that the median effective dose (ED50) of yunnadolin A for inhibiting the writhing reaction in mice is 1. 50 The above results show that yunnadolin A has a good analgesic effect, and its analgesic effect is higher than that of the positive control aspirin (ED 50 is 4.38 times that of 79.61 mg / kg).

[0011] Furthermore, the present invention uses the nitroglycerin-induced migraine mouse model, which is currently recognized both domestically and internationally, to test the anti-migraine activity of the hydroazulene-type sesquiterpenoid compound of the present invention. The results show that the hydroazulene-type sesquiterpenoid compound can significantly reduce the number of times mice scratch their heads, reduce the level of nitric oxide (NO) in serum, downregulate the level of calcitonin gene-related peptide (CGRP) in mouse plasma, and increase the levels of 5-hydroxytryptamine (5-HT) and β Endorphins ( β -EP) content, indicating that the hydroazulene-type sesquiterpenoid compound has good anti-migraine activity.

[0012] The beneficial effects of the present invention are: 1) The hydroazulene-type sesquiterpenoid compound of the present invention can significantly reduce the number of head scratching in mice in the nitroglycerin-induced migraine experiment, reduce the NO content in serum, down-regulate the CGRP content in mouse plasma, and increase the 5-HT and β -EP content suggests that yunnadolin A has good anti-migraine activity and can be used as an anti-migraine active ingredient or lead compound with good application prospects.

[0013] 2) The Aristolochia plants that can be used in the preparation method of the hydroazulene-type sesquiterpene compound of the present invention are abundant in resources and have a simple source of raw materials. Moreover, the hydroazulene-type sesquiterpene compound of the present invention is easily obtained from Aristolochia plants, especially Aristolochia yunnanensis.

[0014] 3) The preparation method of the hydroazulene-type sesquiterpene compound of the present invention can adopt a conventional column chromatography preparation method. The compound preparation operation process is simple, the obtained compound has high purity, and subsequent industrial production is easy to achieve. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a flow chart for the activity tracking and separation of the hydroazulene-type sesquiterpenoid compounds of the present invention; Figure 2 is the structural formula of the hydroazulene-type sesquiterpene compound of the present invention; Figure 3 This is the high-resolution mass spectrum (HRESI-MS) of the hydroazulene-type sesquiterpenoid compound prepared in Example 3 of the present invention; Figure 4 This is the H NMR spectrum of the hydroazulene-type sesquiterpene compound prepared in Example 3 of the present invention ( 1H NMR); Figure 5 This is the C NMR spectrum of the hydroazulene sesquiterpene compound prepared in Example 3 of the present invention ( 13 C NMR) and DEPT spectra; Figure 6 This is the COSY correlation spectrum of the hydroazulene-type sesquiterpene compound prepared in Example 3 of the present invention; Figure 7 This is the HSQC correlation spectrum of the hydroazulene-type sesquiterpene compound prepared in Example 3 of the present invention; Figure 8 This is the HMBC correlation spectrum of the hydroazulene-type sesquiterpene compound prepared in Example 3 of the present invention; Figure 9 This is the NOESY correlation spectrum of the hydroazulene-type sesquiterpene compound prepared in Example 3 of the present invention; Figure 10 Comparison of the simulated CD spectrum and the measured spectrum of the hydroazulene-type sesquiterpene compound prepared in Example 3 of the present invention; Figure 11 This is a graph showing the inhibitory effect of the hydroazulene-type sesquiterpenoid compound prepared in Example 3 of the present invention on the writhing reaction of mice; the figure shows the mean and standard deviation of 8 replicate samples;

[0016] Figure 12 The hydroazulene sesquiterpenoid compound prepared in Example 3 of the present invention has an effect on the levels of NO in serum and 5-HT, CGRP and β -EP content; the figure shows the mean and standard deviation of 8 repeated samples. DETAILED DESCRIPTION

[0017] The present invention is further described below with reference to the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0018] The hydroazulene sesquiterpene compound of the present invention is a colorless oil with the molecular formula: C 15 H 20 O4, named yunnadolin A, English name: ((3 R ,4 S ,6a R ,7 R ,9a S)-7-hydroxy-6a- methyl-4-(prop-1-en-2-yl)octahydro-1 H -3,9a-methanocyclopenta[ c ]oxocine-1,10-dione), having the following structure: .

[0019] The preparation method of the hydroazulene-type sesquiterpene compound of the present invention uses the root of Aristolochiaceae (Aristolochiaceae) as raw material, and prepares the compound through pretreatment, extract extraction, organic solvent extraction, silica gel column chromatography, and purification and separation steps, specifically comprising the following steps: A. Pretreatment: The root of Yunnan Acanthopanax yunnanensis was dried and crushed through a 10-mesh sieve to obtain material a; B. Extraction: Add 5 to 8 times the mass of material a to material a with a 95% ethanol aqueous solution by volume for extraction. The extraction time is 1 to 2 hours and the number of extractions is 1 to 3. Combine the extracts to obtain extract b. C. Organic solvent extraction: extract b was suspended in water, extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and concentrated under reduced pressure to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water extract respectively; D. Silica gel column chromatography: 1) The ethyl acetate fraction was gradient eluted with a dichloromethane-methanol solution in a volume ratio of 1:0 to 0:1. After TLC analysis, the same fractions were combined to obtain five components, Fr.A to Fr.E: Fr.A (1:0, v / v), Fr.B (50:1, v / v), Fr.C (20:1, v / v), Fr.D (10:1, v / v), and Fr.E (0:1, v / v). 2) Fr. A (i.e., the eluate fraction obtained by eluting with a 1:0 dichloromethane-methanol solution) was gradient eluted with petroleum ether-ethyl acetate solutions in volume ratios of 50:1, 25:1, and 0:1. After TLC analysis, the same fractions were combined to obtain three fractions: Fr. A1 to Fr. A3; Fr. A1 (50:1, v / v), Fr. A2 (25:1, v / v), and Fr. A3 (0:1, v / v). 3) Fr.A2 (i.e., the eluate fraction obtained by eluting with a 25:1 petroleum ether-ethyl acetate solution) was gradient eluted with a petroleum ether-ethyl acetate solution in a volume ratio of 80:1 to 0:1. After TLC analysis, the same fractions were combined to obtain five fractions, Fr.A2a to Fr.A2e: Fr.A2a (80:1, v / v), Fr.A2b (50:1, v / v), Fr.A2c (15:1, v / v), Fr.A2d (5:1, v / v), and Fr.A2e (0:1, v / v). E. Purification and separation: The Fr.A2e portion (i.e., the eluate fraction obtained by eluting with a 0:1 petroleum ether-ethyl acetate solution) was purified and separated by semi-preparative HPLC to obtain yunnadolin A, i.e., the target compound, a hydroazulene-type sesquiterpene compound.

[0020] D. The volume ratios of the dichloromethane-methanol solution in step 1) are 1:0, 50:1, 20:1, 10:1 and 0:1.

[0021] D. The volume ratios of the petroleum ether-ethyl acetate solution in step 3) are 80:1, 50:1, 15:1, 5:1 and 0:1.

[0022] The semi-preparative HPLC purification and separation in step E was performed using a 21.2 mm × 25 cm, 5 μ m's C 18 The chromatographic column used a methanol-water solution with a volume ratio of (45-49): (55-51) as the mobile phase at a flow rate of 3 mL / min. The UV detector was used at a detection wavelength of 210 nm, and the chromatographic peak was collected for 20.6 min.

[0023] The application of the present invention is the application of the hydroazulene type sesquiterpene compound in the preparation of a medicine for treating migraine.

[0024] The present invention will be further described below with specific embodiments: Example 1

[0025] Inhibitory activity of Yunnan Aristolochia chinensis extract on acetic acid writhing response in mice Source of material: The plant of the genus Aristolochia was collected in Kunming, Yunnan, and identified by Professor Yang Qingsong of the School of Ethnic Medicine of Yunnan University for Nationalities. Isotrema yunnanensis (Franch.) XX Zhu, S. Liao, and J.S. Ma. The specimens are deposited in the Herbarium of the College of Ethnic Medicine, Yunnan Nationalities University.

[0026] Preparation of Yunnan Aristolochia strychnifolia extract: The dried roots of Yunnan Aristolochia strychnifolia are crushed to obtain Yunnan Aristolochia strychnifolia root fragments; the Yunnan Aristolochia strychnifolia fragments are then extracted three times with 95% by volume ethanol for 2 hours each time to obtain an extract; the Yunnan Aristolochia strychnifolia extracts are filtered and concentrated under reduced pressure using a rotary evaporator to obtain an extract for later use.

[0027] The experimental model of acetic acid writhing inhibition in mice was used (see scientific literature: Hayashi G., Takemori AE The type of analgesic-receptor interaction involved in certain analgesic assays. Eur. J. Pharmacol The ability of a Yunnan Amur Caulis extract to inhibit acetic acid writhing in mice was tested. Forty healthy female mice weighing 18-22 g were randomly divided into five groups, each consisting of eight mice. The groups included a negative control (0.9% sodium chloride solution), a positive control (aspirin, 0.1 g / kg), and high-, medium-, and low-dose Yunnan Amur Caulis groups (2 g / kg, 1 g / kg, and 0.5 g / kg). Thirty minutes after drug administration, each mouse received an injection of 0.2 mL of 0.6% acetic acid solution. The number of writhing episodes within 15 minutes after acetic acid injection was observed and recorded. The number of writhing episodes among the test groups was compared, and the inhibitory rate of the drug on writhing reactions was calculated.

[0028] Inhibition rate = (average number of writhings in the control group - average number of writhings in the drug-treated group) / average number of writhings in the control group × 100%).

[0029] The results showed that under the above concentrations, the extract of Aristolochia yunnanensis had the ability to significantly inhibit the acetic acid writhing reaction of mice. The results are shown in Table 1.

[0030] Example 2

[0031] Further experiments with Aristolochia yunnanensis A 95% ethanol extract of Caulis Aristolochiae Yunnanensis was suspended in water and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol, followed by concentration under reduced pressure. This yielded four extracts: a petroleum ether extract, an ethyl acetate extract, an n-butanol extract, and an aqueous extract. Example 1 was repeated. The analgesic effects of the petroleum ether extract, the ethyl acetate extract, the n-butanol extract, and the aqueous extract were tested using an acetic acid writhing reaction inhibition test in mice. The results demonstrated that the ethyl acetate extract and the aqueous extract significantly inhibited the acetic acid writhing reaction in mice. The results are shown in Table 1.

[0032] Example 3

[0033] Isolation and identification of analgesic compounds from the roots of Aristolochia yunnanensis (1) Dry 7 kg of Yunnan Acanthus root and crush it into particles with a size of 0.1 cm to obtain Yunnan Acanthus powder. Extract the Yunnan Acanthus powder with 40 kg of 95% ethanol at room temperature for 3 times, each time for 2 hours. Combine the ethanol extracts and set aside. (2) Filter the ethanol extract obtained in step (1) through 80-120 μm filter paper and concentrate under reduced pressure using a rotary evaporator at 50°C until the specific gravity reaches 1.2, obtaining 440 g of extract for later use; (3) 440 g of the extract from (2) was suspended in 1000 mL of water and extracted three times with 1000 mL of petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate fraction (60 g) was separated by silica gel column chromatography and eluted with dichloromethane-methanol (volume ratio 1:0 to 0:1) to obtain five components (Fr. A to Fr. E). Fr. A (7.5 g) was subjected to silica gel column chromatography and eluted with a gradient of petroleum ether-ethyl acetate with a volume ratio of 50:1, 25:1, and 0:1 to obtain three components, Fr. A1 to Fr. A3, with Fr. A1 being 2.1 g, Fr. A2 being 3.6 g, and Fr. A3 being 1.6 g. Fr. A2 was subjected to silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate (volume ratios of 80:1, 50:1, 15:1, 5:1, and 0:1) to yield five components (Fr. A2a-Fr. A2e). Fr. A2e (1.2 g) was subjected to semi-preparative HPLC using a mobile phase of methanol-water (volume ratio of 47:53) at a flow rate of 3 mL / min to yield yunnadolin A (108.6 mg). The isolation and identification process for the analgesic active ingredients in the root of Aristolochia yunnanensis is described in [ 1 ]. Figure 1 .

[0034] Example 4

[0035] Structural identification of the hydroazulene-type sesquiterpenoid compound obtained in Example 3 The hydroazulene type sesquiterpene compound is a white crystal (the solvent is chloroform), and the high-resolution mass spectrum shows that its molecular ion peak is m / z 263.1290 [MH] - (calculated value 263.1289), indicating that the molecular formula of the compound is C 15 H 20 O4, its unsaturation is calculated to be 6. 1 The signal of a terminal double bond was read in the H-NMR spectrum [ δ H 4.93 (2H,H-12)], 3 methine proton signals [ δ H 4.70 (1H, s, H-6), 3.83 (1H, dd, J = 11.0, 7.2Hz, H-1), 2.37 (1H, dd, J = 11.5, 4.7 Hz, H-7)], four methylene signals [( δ H 2.13 / 1.78 (2H, H-2), 2.07 / 1.91 (2H, H-3), 1.94 / 1.23 (2H, H-9), and 1.78 / 1.73 (2H, H-8)] and the singlet proton signals of two methyl groups [ δ H 1.83 (3H, s, H-13) and 1.08 (3H, s, H-14)]. In the total carbon spectrum and DEPT spectrum, it was found that yunnadolin A contained 15 carbon signals, including 2 methyl δ C :22.0,15.1;5 methylene groups δ C :113.4, 36.9, 30.3, 21.6, 21.5; 3 methines δ C : 85.4, 79.6, 49.9; 5 quarterly carbon δ C : 208.8, 174.8, 144.2, 59.3, 50.6; From this, we can first infer that yunnadolin A is a compound with a hydroazulene-type sesquiterpenoid skeleton, which contains a carbonyl group, a double bond and a lactone ring, and there are 2 degrees of unsaturation left. The chemical shifts of the remaining carbon signals are mostly between 20-90 ppm, and the remaining fragments can be inferred from the ring formation. From the HMBC spectrum, we can see that H-1 and C-9, C-10, C-14; H-2 and C-3, C-4, C-6, C-9, C-10; H-3 and C-1, C-4, C-5; H-9 and C-2, C-7, C-14, C-4; H-8 and C-1, C-6, C-10, these five groups of signals have long-range correlations, and it is inferred that the compound is likely to be a combination of a five-membered ring and a seven-membered ring. The hydroazulene-type sesquiterpenoid compound and the known compound versicolactone C (Zhang J., He L. Determination of the structures of versicolactone B and versicolactone C in the root of Aristolochia versicolar SM Hwang. Acta Pharm. Sin. 1986, 21(04), 273-278) NMR data were very similar, except that the chemical shift of the 5-carbon was very different. The carbon of the hydroazulene type sesquiterpenoid compound is a quaternary carbon ( δ C 208.8), while the carbon of silver bag lactone propionate is a methine carbon ( δ C 80.1), the chemical shifts of the 6-, 7-, and 8-carbon positions of the hydroazulene sesquiterpenoid compound are all shifted 2 to 4 ppm downfield, indicating that the linking group on the 5-carbon position is a carbonyl group. Through ECD simulation calculations, it was found that the CD spectrum of the hydroazulene sesquiterpenoid compound is similar to that of (3 R ,4 S ,6a R ,7 R ,9a S )-1 is similar to the simulated spectrum ( Figure 10 Finally, the hydroazulene-type sesquiterpenoid compound was identified as yunnadolin A ((3 R ,4 S ,6a R ,7 R ,9a S )-7-hydroxy-6a-methyl-4-(prop-1-en-2-yl)octahydro-1 H -3,9a- methanocyclopenta[ c ]oxocine-1,10-dione).

[0036] Physicochemical data of hydroazulene-type sesquiterpenoid compounds: Compound yunnadolin A is a colorless oil, HR-ESI-MS m / z : 263.1290 [MH] – , molecular formula C 15 H 20 O4. 1 H NMR (400 MHz, CDCl3) δ H : 4.93(2H, m, H-12), 4.70 (1H, s, H-6), 3.83 (1H, dd, J = 11.0, 7.2 Hz, H-1), 2.37(1H, dd, J= 11.5, 4.7 Hz, H-7), 2.13 (1H, m, H-2a), 2.07 (1H, m, H-3a), 1.94(1H, m, H-9a), 1.91 (1H, m, H-3b), 1.83 (3H, s, H-13), 1.78 (2H, m, H-2b / 8a),1.73 (1H, m, H-8b), 1.23 (1H, m, H-9b), 1.08 (3H, s, H-14); 13 C NMR (100 MHz,CDCl3) δ C : 208.8 (C-5), 174.8 (C-15), 144.2 (C-11), 113.4 (C-12), 85.4 (C-6), 79.6 (C-1), 59.3 (C-4), 50.6 (C-10), 49.9 (C-7), 36.9 (C-9), 30.3 (C-2), 22.0(C-13), 21.6 (C-8), 21.5 (C-3), 15.1 (C-14). Example 5

[0037] Detection of analgesic activity of the hydroazulene-type sesquiterpenoid compound The experimental model of acetic acid writhing inhibition in mice was used (see scientific literature: Hayashi G., Takemori AE The type of analgesic-receptor interaction involved in certain analgesic assays. Eur. J. Pharmacol The ability of yunnadolin A to inhibit acetic acid writhing in mice was tested. Healthy female Kunming mice weighing 18-22 g were randomly divided into groups of 8 each. The groups included a negative control (0.9% sodium chloride solution), a positive control (aspirin at 100 mg / kg), and sample groups at doses of 40 mg / kg, 20 mg / kg, 10 mg / kg, 5 mg / kg, and 2.5 mg / kg, respectively. Thirty minutes after administration, each mouse received an injection of 0.2 mL of 0.6% acetic acid solution. The number of writhing episodes within 15 minutes of acetic acid injection was observed and recorded. The number of writhing episodes among the test groups was compared, and the inhibitory rate of the drug on the writhing reaction was calculated.

[0038] Inhibition rate = (average number of writhings in the control group - average number of writhings in the drug-treated group) / average number of writhings in the control group × 100%).

[0039] Calculate the median effective dose (ED) 50 The results of the determination of the median effective dose of aspirin for inhibiting the acetic acid writhing reaction in mice were 79.61±3.74 mg / kg, and the median effective dose (ED50) of yunnadolin A for inhibiting the acetic acid writhing reaction in mice was 79.61±3.74 mg / kg. 50 ) was 18.19 ± 3.22 mg / kg, indicating that the compound of the present invention has the ability to significantly inhibit the acetic acid writhing reaction in mice.

[0040] Example 6

[0041] Anti-migraine activity detection of the hydroazulene-type sesquiterpenoid compound The anti-migraine activity of hydroazulene-type sesquiterpenoids was tested using a nitroglycerin-induced migraine mouse model (see the scientific literature: Chen Y., Cheng Q., Zeng S., et al. Potential analgesic effect of Foshousan oil-loaded chitosan-alginate nanoparticles on the treatment of migraine. Front Pharmacol. 2023, 14, 1190-920). Male Kunming mice weighing 18-22 g were randomly divided into groups of eight. The groups were: blank control (distilled water), negative control (distilled water), sample group (yunnadolin A), and positive control group (ibuprofen tablets at a dose of 80 mg / kg). Treatment was continued for 7 consecutive days. Thirty minutes after the last dose, nitroglycerin (10 mg / kg) was injected subcutaneously into the right shoulder of each group, except for the blank group, which received normal saline. 30 minutes after the injection of nitroglycerin, the number of times the mice scratched their heads in 6 time periods after modeling was observed and recorded. 4 hours after the subcutaneous injection of nitroglycerin, the mice were anesthetized and blood was collected from the orbital vein. The blood was stored at room temperature and centrifuged at 4°C for 15 minutes (3500 r / min). The supernatant was collected for further testing. The content of nitric oxide (NO) in the serum of mice was detected by colorimetry, and the content of 5-hydroxytryptamine (5-HT), calcitonin gene-related peptide (CGRP) and β-catenin in the plasma of mice was detected by enzyme-linked immunosorbent assay (ELISA). β -Endorphins ( β -EP) content. Measurement data are expressed as mean ± standard deviation ( ±S) and statistical processing was performed using Prism 6 statistical software. The experimental results are shown in Table 2 and Figure 12 The experimental results showed that hydroazulene-type sesquiterpenoid compounds can significantly reduce the number of times mice scratch their heads, reduce the content of NO in serum, downregulate the content of CGRP in mouse plasma, and increase the levels of 5-HT and β -EP content, suggesting that hydroazulene-type sesquiterpenoid compounds have good anti-migraine activity and can be used as anti-migraine ingredients or lead compounds.

[0042] Note: n=8, ### Compared with the blank control group, P <0.001, ## Compared with the blank control group, P <0.01, # Compared with the blank control group, P <0.05, *** Compared with the negative control group, P <0.001, ** Compared with the negative control group, P <0.01, * Compared with the negative control group, P <0.05.

[0043] Example 7

[0044] Yunnadolin A prepared according to the method described in Example 3 was added with common excipients for tablets and prepared into tablets according to conventional preparation technology.

[0045] Example 8

[0046] Yunnadolin A prepared according to the method described in Example 3 was added with common excipients for injection and prepared into an injection according to conventional preparation technology.

[0047] Example 9

[0048] Yunnadolin A prepared according to the method described in Example 3 was added with common excipients for capsules and prepared into capsules according to conventional preparation technology.

[0049] Example 10

[0050] The yunnadolin A prepared according to the method described in Example 3 was added with common excipients for cataplasms and prepared into cataplasms according to conventional preparation technology.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A hydroazulene-type sesquiterpene compound, characterized in that: The hydroazulene type sesquiterpenoid compound is a colorless oil with the molecular formula: C 15 H 20 O4, named yunnadolin A, English name: ((3 R ,4 S ,6a R ,7 R ,9a S )-7-hydroxy-6a-methyl-4-(prop-1-en-2-yl)octahydro-1 H -3,9a-methanocyclopenta[ c ]oxocine-1,10-dione), having the following structure: 。 2. A method for preparing the hydroazulene-type sesquiterpene compound according to claim 1, characterized in that: The method is prepared from the root of Aristolochiaceae, a plant of the genus Aristolochiaceae, as a raw material, through pretreatment, extract extraction, organic solvent extraction, silica gel column chromatography and purification and separation steps, specifically comprising the following steps: A. Pretreatment: The root of Yunnan Acanthopanax yunnanensis was dried and crushed through a 10-mesh sieve to obtain material a; B. Extraction: Add 5 to 8 times the mass of material a to material a with a 95% ethanol aqueous solution by volume for extraction. The extraction time is 1 to 2 hours and the number of extractions is 1 to 3. Combine the extracts to obtain extract b. C. Organic solvent extraction: extract b was suspended in water, extracted with petroleum ether, ethyl acetate and n-butanol in sequence, and concentrated under reduced pressure to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water extract respectively; D. Silica gel column chromatography: 1) The ethyl acetate fraction was gradient eluted with a dichloromethane-methanol solution in a volume ratio of 1:0 to 0:

1. After TLC analysis, the same fractions were combined to obtain five components, Fr.A to Fr.E: Fr.A (1:0, v / v), Fr.B (50:1, v / v), Fr.C (20:1, v / v), Fr.D (10:1, v / v), and Fr.E (0:1, v / v). 2) Fr. A (i.e., the eluate fraction obtained by eluting with a 1:0 dichloromethane-methanol solution) was gradient eluted with petroleum ether-ethyl acetate solutions in volume ratios of 50:1, 25:1, and 0:

1. After TLC analysis, the same fractions were combined to obtain three fractions: Fr. A1 (50:1, v / v), Fr. A2 (25:1, v / v), and Fr. A3 (0:1, v / v). 3) Fr.A2 (i.e., the eluate fraction obtained by eluting with a 25:1 petroleum ether-ethyl acetate solution) was gradient eluted with a petroleum ether-ethyl acetate solution in a volume ratio of 80:1 to 0:

1. After TLC analysis, the same fractions were combined to obtain five fractions, Fr.A2a to Fr.A2e: Fr.A2a (80:1, v / v), Fr.A2b (50:1, v / v), Fr.A2c (15:1, v / v), Fr.A2d (5:1, v / v), and Fr.A2e (0:1, v / v). E. Purification and separation: The Fr.A2e portion (i.e., the eluate fraction obtained by eluting with a 0:1 petroleum ether-ethyl acetate solution) was purified and separated by semi-preparative HPLC to obtain yunnadolin A, i.e., the target compound, a hydroazulene-type sesquiterpene compound.

3. The preparation method according to claim 2, characterized in that D. The volume ratios of the dichloromethane-methanol solution in step 1) are 1:0, 50:1, 20:1, 10:1 and 0:

1.

4. The preparation method according to claim 2, characterized in that D. The volume ratios of the petroleum ether-ethyl acetate solution in step 3) are 80:1, 50:1, 15:1, 5:1 and 0:

1.

5. The preparation method according to claim 2, characterized in that The semi-preparative HPLC purification and separation in step E was performed using a 21.2 mm × 25 cm, 5 μ m's C 18 The chromatographic column was used with a methanol-water solution in a volume ratio of (45-49): (55-51) as the mobile phase at a flow rate of 3 mL / min. The detection wavelength of the UV detector was 210 nm, and the chromatographic peak at 20.6 min was collected.

6. Use of the hydroazulene sesquiterpene compound according to claim 1, characterized in that: The invention relates to an application of the hydroazulene type sesquiterpene compound in the preparation of a medicine for treating migraine.

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