A sesquiterpene lactone compound extracted and separated from wild horsetail, and a method and application thereof

Abstract

The present invention discloses a sesquiterpene lactone compound extracted and separated from wild horsetail, and a method and application thereof, wherein the structural formula thereof is shown in formula (I). The sesquiterpene lactone compound is extracted and separated from dried wild horsetail by ethanol reflux extraction, ethyl acetate extraction, silica gel column chromatography, C18 reverse phase chromatography separation and other steps, and is a new sesquiterpene lactone compound with a new structure and pharmacological activity, which can be used in the preparation of anti-lung cancer drugs, and provides a reliable basis for the preparation of new anti-cancer drugs and the medicinal research of sesquiterpene lactone substances. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the technical field of plant chemistry, and specifically relates to a sesquiterpene lactone compound with anti-cancer activity extracted and separated from wild horse mackerel, a preparation method thereof, and application in anti-cancer drugs. Background Art

[0002] Eupatorium verticillatum Eupatorium lindleyanum The whole plant of DC. Wild horsetail is bitter and neutral in nature. It enters the liver and spleen meridians. It has the effects of clearing away heat and detoxification, removing phlegm and relieving asthma, and lowering blood pressure. It is often used in the clinic to treat chronic tracheitis, bronchitis, and hypertension. The branches and leaves are used as medicine to relieve dampness and dispel dampness. It is used for cough caused by overwork, vomiting blood and coughing, as well as turbid leucorrhea, and nameless swelling and pain. In recent years, it has been found that wild horsetail also has anti-tumor effects.

[0003] The document "Research Progress on Chemical Components and Pharmacological Actions of Chinese Medicine Herba Eupatorii" (Lu Mengyuan; Chinese Ethnic Folk Medicine; Vol. 28, No. 9, May 2019) reviews the research results on the chemical components and pharmacological activities of Herba Eupatorii in recent years. So far, the chemical components of Herba Eupatorii have been studied relatively systematically at home and abroad, and it has been found that they are mainly sesquiterpenoid lactones, flavonoids, volatile oils, steroids, organic acids, etc. In addition, it also contains diterpenoids, triterpenoids, trace elements, amino acids and some other components. Sesquiterpenoid lactones Sesquiterpenoid lactones are the most important active ingredients in Herba Eupatorii, mainly including guaiacane type, germarane type, eucalyptane type, and cadinane type, among which germarane type and guaiacane type are the most common. In 1979, Ito K et al. first isolated four germarane-type compounds from Herba Eupatorii: Eupalinin A, Eupalinin B, Eupalinin C, and Eupalinin D. Subsequently, Huo J et al. isolated 13 guaiacane-type, 7 germarane-type and 1 eucalyptane-type sesquiterpene lactones from the whole herb of Y. Yang Nianyun et al. obtained 9 germazine-type sesquiterpene lactone compounds, including Eupalinolide A, Eupalinolide B, Eupalinolide C, Eupalinolide D, and Eupalinolide E, from the whole herb of wild horse chase; Ye G et al. isolated a cadinane-type sesquiterpene lactone, Eupalinilide M, from the 95% ethanol extract of the whole herb of wild horse chase in 2008; Yan G, Wu Shuangqing et al. isolated 7 germazine-type sesquiterpene lactones, including 3β-hydroxy-8β-[4′-hydroxytigloyloxy]-costunolide, Eupalinolide F, Eupalinolide G, Eupalinolide H, Eupalinolide I, Eupalinolide J, and Eupalinolide K, from the ethanol extract of wild horse chase in 2012; from 2016 to 2017, Zhong Huanhuan, Yang B et al. isolated 2 germazine-type sesquiterpene lactones, including Eupalinolide L, Eupalinolide M and a new germarane-type sesquiterpene lactone: Eupalinolide O.

[0004] Regarding anti-tumor pharmacological effects, the literature records that Eupalinolide J sesquiterpene lactone compound can inhibit the activity of triple-negative breast cancer cells. It is a typical natural MRA that has a significant inhibitory effect on STAT3 activity and has potential cytotoxic activity against triple-negative breast cancer (TNBC) cells.

[0005] It can be seen that there is still a lack of in-depth research on most sesquiterpene lactone compounds, and the pharmacological activity of each compound is not clear, especially the detailed components that really play a therapeutic role in anti-tumor activity are still unclear. Therefore, it is necessary to further explore and study its effective parts or effective ingredients, which has great practical value for improving drug efficacy and reducing side effects.

[0006] The present invention is based on the discovery and separation of a new medicinal compound obtained when studying the active ingredients of the dry aerial parts of the Asteraceae plant Eupatorium lindleyanum DC. The compound is a sesquiterpene lactone compound. Pharmacological activity studies have shown that the compound has certain anti-lung cancer activity, providing a scientific basis for the research and development of new anti-lung cancer drugs. Summary of the invention

[0007] One of the purposes of the present invention is to provide a sesquiterpene lactone compound extracted and separated from the wild horsetail. By extracting and separating from the wild horsetail medicinal material, a new compound with a novel structure and pharmacological activity is obtained, which provides a reference for the chemical composition and pharmacological effects of the wild horsetail.

[0008] The second object of the present invention is to provide a method for extracting and separating sesquiterpene lactone compounds from wild horsetail. It uses dried wild horsetail as a raw material, and obtains a new medicinal compound with pharmacological activity through ethanol reflux extraction, ethyl acetate extraction, silica gel column chromatography separation, C18 reverse phase chromatography filler high pressure preparative separation and other process steps, and obtains a sesquiterpene lactone compound with a new structure and pharmacological activity. The method has simple operating steps and is easy to control, which can ensure that the purity of the new compound reaches more than 99%, and the entire production process is short in time, which is suitable for industrial production.

[0009] The third object of the present invention is to provide an application of sesquiterpene lactone compounds extracted and separated from Rhizoma Cibotii in the preparation of anti-lung cancer drugs, so as to provide a reliable basis for the preparation of new anti-lung cancer drugs.

[0010] The object of the present invention is achieved by the following technical scheme: a sesquiterpene lactone compound extracted and separated from wild horsetail, the structural formula of which is shown in formula (I):

[0011]

[0012] The sesquiterpene lactone compound with the structural formula shown in formula (I) is obtained by extraction and separation from dried wild horsetail, and its Chinese name is: 3aR, 4R, 6Z, 9S, 10Z, 11aS)-6-(acetoxymethyl)-9-hydroxy-10-methyl-3-methylene-2-oxo-2,3,3a,4,5,8,9,11aS octahydrocyclodecan[b]furan-4-yl (E)-4-hydroxy-2-methylbut-2-enoate, which belongs to the sesquiterpene lactone compound and is named: wild horsetail lactone H2.

[0013] The wild horsetail is the dried aerial part of Eupatorium lindleyanumDC. of the Asteraceae family.

[0014] The molecular weight of the yamachu lactone H2 is 420, and the molecular formula is C 22 H 28 O8.

[0015] The structure of the compound has not been reported in the literature, and pharmacological experiments show that the compound has inhibitory activity on the proliferation of A1975 cells cultured in vitro and has certain anti-lung cancer activity. Therefore, the yemachus lactone H2 compound extracted and separated from yemachus provided by the present invention can be used in the preparation of anti-lung cancer drugs.

[0016] The present invention discloses a method for extracting and separating sesquiterpene lactone compounds from wild horsetail, comprising the following steps:

[0017] A. Ethanol Reflux Extraction

[0018] The dried wild horsetail was crushed into 60-80 meshes, and then extracted by heating and refluxing with 90wt% ethanol.

[0019] After concentrating under reduced pressure until there is no alcohol, a concentrated extract is obtained;

[0020] B. Ethyl acetate extraction

[0021] The concentrated extract obtained in step A is dispersed by adding water to obtain an aqueous dispersion, and the aqueous dispersion is extracted 3 to 5 times with an equal volume of ethyl acetate, and the solvent is recovered to obtain an ethyl acetate extract and an aqueous phase;

[0022] C. Silica gel column chromatography

[0023] a. The ethyl acetate extract obtained in step B was dissolved in methanol, filtered, and the filtrate was mixed with silica gel and dried to obtain a mixed sample;

[0024] b. The mixed sample was wet-mixed on a silica gel column for column chromatography and purification, and the fractions containing sesquiterpene lactone compounds were collected and concentrated to dryness under reduced pressure to obtain the target product;

[0025] D. Separation by C18 reverse phase chromatography column: The target product obtained in step C is dissolved in methanol, filtered, and the filtrate is separated by high pressure preparative separation using C18 reverse phase chromatography filler to obtain product collection liquid, and the collection liquid is concentrated to dryness to obtain

[0026] The white powder product is a sesquiterpene lactone compound with the structural formula shown in formula (I).

[0027] In step A, the amount of ethanol used is 8 to 10 times the weight of the crushed dry wild horsetail.

[0028] In step A, the reflux extraction is performed 3 to 5 times, each time for 2 hours.

[0029] In step B, the concentrated extract is dispersed by adding water at a volume ratio of 1:6 to 1:10.

[0030] In step C, the silica gel column chromatography separation uses petroleum ether:ethyl acetate = 10:1 V / V as the mobile phase.

[0031] In step D, the C18 reverse phase chromatography column is used for separation: A: acetonitrile B: water, A:B = 34:66 V / V is used as the mobile phase; the detection wavelength is 220 nm.

[0032] The white powder obtained by extraction and separation of the present invention turns red in a 10wt% sulfuric acid ethanol solution, which further proves that the white powder is a sesquiterpene lactone compound.

[0033] The beneficial effects of the present invention are:

[0034] 1. The new sesquiterpene lactone compound provided by the present invention is obtained by extraction and separation from dried Herba Eupatoriae. The compound has a structural formula shown in formula (I) and its structure is determined. At the same time, the anti-lung cancer activity experiment of the compound shows that it has inhibitory activity on the proliferation of A1975 cells cultured in vitro and has certain anti-lung cancer activity, which provides a scientific basis for the systematic study of the chemical components and pharmacological effects of Herba Eupatoriae.

[0035] 2. The present invention uses the dried aerial parts of Eupatorium lindleyanumDC. of the Asteraceae plant as raw materials, and obtains sesquiterpene lactone compounds through ethanol reflux extraction, silica gel column chromatography separation and purification, C18 reverse phase chromatography filler high pressure preparative separation and other process steps. The method has simple operation steps and is easy to control, which can ensure that the purity of yemazhui lactone H2 reaches more than 99%, and the entire production process is short in time, which is suitable for industrial production.

[0036] 3. The present invention reports the structure of yemachu lactone A2 for the first time. Its relative configuration is determined based on plant sources, nuclear magnetic resonance data and mass spectrometry data. It is a new type of sesquiterpene lactone compound. The anti-lung cancer activity experiment of the compound proves that yemachu lactone H2 has inhibitory activity on the proliferation of PC-9 cells cultured in vitro. The maximum inhibition rate within the research dose is 52.48%, and the IC50 is 82.676 (65.858-178.850) μmol / ml. Therefore, the compound can be developed and utilized as a latent structure, and provides a reliable basis for the research and development of new anticancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a chromatogram of the scutellaria lactone H2 in the scutellaria raw material of the present invention.

[0038] Figure 2 The figure is a chromatogram of yemachalactone H2 extracted and separated by the present invention.

[0039] Figure 3 The carbon nuclear magnetic resonance spectrum of the compound of the structure represented by formula (I) of the present invention.

[0040] Figure 4 The hydrogen nuclear magnetic resonance spectrum of the compound of the structure represented by formula (I) of the present invention.

[0041] Figure 5 This is the HSQC spectrum of the compound of the present invention having the structure represented by formula (I).

[0042] Figure 6 This is the HMBC spectrum of the compound of the structure represented by formula (I) of the present invention.

[0043] Figure 7 This is the NOESY spectrum of the compound represented by formula (I) of the present invention. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with the embodiments. However, it is necessary to point out that the following embodiments cannot be understood as limiting the scope of protection of the present invention, and if some non-essential improvements and adjustments made by technicians in this field according to the above content of the present invention still fall within the scope of protection of the present invention.

[0045] Embodiment 1:

[0046] A sesquiterpene lactone compound extracted and separated from wild horsetail, the compound is a white powder, 10wt% sulfuric acid ethanol solution is red, the molecular weight is 420, the molecular formula is C 22 H 28 O8, the structural formula is shown in formula (I):

[0047]

[0048] The specific steps of extraction and separation are as follows:

[0049] A. Ethanol Reflux Extraction

[0050] The dried wild horsetail was crushed into 60-80 meshes, and then extracted by heating and refluxing with 90wt% ethanol.

[0051] After concentrating under reduced pressure until there is no alcohol, a concentrated extract is obtained;

[0052] Since 90% ethanol has good solubility for sesquiterpene lactone compounds and a high extraction rate, reflux extraction can further shorten the extraction time and improve the extraction efficiency.

[0053] B. Ethyl acetate extraction

[0054] The concentrated extract obtained in step A is dispersed by adding water to obtain an aqueous dispersion, and the aqueous dispersion is extracted 3 to 5 times with an equal volume of ethyl acetate, and the solvent is recovered to obtain an ethyl acetate extract and an aqueous phase;

[0055] By adding water for dispersion treatment, the water-soluble components in the concentrated extract can be dissolved and the influence of the organic reagents in the concentrated extract during the extraction process can be reduced. Since hemiterpenoid lactone compounds are soluble in ethyl acetate, ethyl acetate can be used to extract and separate hemiterpenoid lactone compounds from the concentrated solution, and the water-soluble components are in the water phase, thereby achieving the effect of primary separation.

[0056] C. Silica gel column chromatography

[0057] a. The ethyl acetate extract obtained in step B was dissolved in methanol (methanol can dissolve the target compound), filtered, and the filtrate was mixed with silica gel and dried to obtain a mixed sample;

[0058] b. The mixed sample was wet-mixed on a silica gel column for column chromatography and purification, and the fractions containing sesquiterpene lactone compounds were collected and concentrated to dryness under reduced pressure to obtain the target product;

[0059] The extract is dissolved in methanol and filtered to remove the methanol insoluble matter. The large amount of methanol solvent contained in the filtrate is removed by drying to ensure the separation effect of silica gel column chromatography. Other impurities with large polarity differences in hemiterpene lactone compounds can be further separated by silica gel column chromatography, which is beneficial to the next step of preparation and separation.

[0060] D. Separation by C18 reverse phase chromatography column: The target product obtained in step C is dissolved in methanol, filtered, and the filtrate is separated by high pressure preparative separation using C18 reverse phase chromatography filler to obtain product collection liquid, and the collection liquid is concentrated to dryness to obtain

[0061] The white powder product is a sesquiterpene lactone compound with the structural formula shown in formula (I).

[0062] The target product (mixture of safflower lactone H2) after separation by silica gel column chromatography also contains a large number of compounds with similar structures and small polarity differences. By using a high-pressure preparative separation system with C18 filler and an acetonitrile-water mobile phase system, impurities can be effectively removed. By collecting the preparation liquid of safflower lactone H2, the target component of safflower lactone H2 with a purity of more than 99% can be separated.

[0063] Embodiment 2:

[0064] A. Take 10kg of dried wild horsetail, crush it into 60 mesh, and then use 8 times the weight of 90wt% ethanol to heat and reflux for extraction 3 times, 2 hours each time, and concentrate under reduced pressure until there is no alcohol, and obtain 5L of concentrated extract;

[0065] B. Add water to the concentrated extract obtained in step A at a volume ratio of 1:6 to obtain a water dispersion, extract the water dispersion with an equal volume of ethyl acetate for 5 times, recover the solvent, and obtain an ethyl acetate extract and 30L of water phase;

[0066] C. Dissolve the ethyl acetate extract obtained in step B with methanol, filter, mix the filtrate with silica gel, dry, and purify the obtained mixture by column chromatography on a silica gel column (petroleum ether: ethyl acetate = 10:1 V / V as the mobile phase), collect the fraction containing sesquiterpene lactone compounds, and concentrate under reduced pressure to dryness to obtain the target product;

[0067] D. Dissolve the target product obtained in step C in methanol, filter, and separate the filtrate using C18 reverse phase chromatography packing material under high pressure preparation (A: acetonitrile B: water, A:B = 34:66 V / V as mobile phase; detection wavelength 220 nm). Collect Figure 1 The corresponding chromatographic peak product is shown in the figure. The product liquid is collected until dry to obtain 5g of white powder product, which is a sesquiterpene lactone compound with the structure shown in formula (I), which is yamachu lactone H2. Figure 2 shown.

[0068] Embodiment 3:

[0069] A. Take 20kg of dried wild horsetail, crush it to 70 mesh, and then use 10 times the weight of 90wt% ethanol to heat and reflux for 5 times, 2 hours each time, and concentrate it under reduced pressure until there is no alcohol, and obtain 9L of concentrated extract;

[0070] B. Add water to the concentrated extract obtained in step A at a volume ratio of 1:10 to obtain a water dispersion, extract the water dispersion with an equal volume of ethyl acetate for 5 times, recover the solvent, and obtain an ethyl acetate extract and 90L of water phase;

[0071] C. Dissolve the ethyl acetate extract obtained in step B with methanol, filter, mix the filtrate with silica gel, dry, and purify the obtained mixture by column chromatography on a silica gel column (petroleum ether: ethyl acetate = 10:1 V / V as the mobile phase), collect the fraction containing sesquiterpene lactone compounds, and concentrate under reduced pressure to dryness to obtain the target product;

[0072] D. Dissolve the target product obtained in step C in methanol, filter, and separate the filtrate using C18 reverse phase chromatography packing material under high pressure preparation (A: acetonitrile B: water, A:B = 34:66 V / V as mobile phase; detection wavelength 220 nm). Collect Figure 1 The corresponding chromatographic peak product is shown in the figure. The product liquid is collected to dryness to obtain 11g of white powder product, which is a sesquiterpene lactone compound with the structure shown in formula (I), which is yamachu lactone H2. Figure 2 shown.

[0073] Embodiment 4:

[0074] A. Take 30kg of dried wild horsetail, crush it into 80 meshes, and then use 9 times the weight of 90wt% ethanol to heat and reflux and extract 4 times, 2 hours each time, and then concentrate it under reduced pressure until there is no alcohol, and obtain 11L of concentrated extract;

[0075] B. Add water to the concentrated extract obtained in step A at a volume ratio of 1:8 to obtain a water dispersion, extract the water dispersion with an equal volume of ethyl acetate for 4 times, recover the solvent, and obtain an ethyl acetate extract and 90L of water phase;

[0076] C. Dissolve the ethyl acetate extract obtained in step B with methanol, filter, mix the filtrate with silica gel, dry, and purify the obtained mixture by column chromatography on a silica gel column (petroleum ether: ethyl acetate = 10:1 V / V as the mobile phase), collect the fraction containing sesquiterpene lactone compounds, and concentrate under reduced pressure to dryness to obtain the target product;

[0077] D. Dissolve the target product obtained in step C in methanol, filter, and separate the filtrate using C18 reverse phase chromatography packing material under high pressure preparation (A: acetonitrile B: water, A:B = 34:66 V / V as mobile phase; detection wavelength 220 nm). Collect Figure 1 The corresponding chromatographic peak product is shown in the figure. The product liquid is collected to dryness to obtain 16g of white powder product, which is a sesquiterpene lactone compound with the structure shown in formula (I), which is yamachu lactone H2. Figure 2 shown.

[0078] Embodiment 5:

[0079] A. Take 50kg of dried wild horsetail, crush it to 60 mesh, and then use 8 times the weight of 90wt% ethanol to heat and reflux for 4 times, 2 hours each time, and concentrate it under reduced pressure until there is no alcohol, and obtain 20L of concentrated extract;

[0080] B. Add water to the concentrated extract obtained in step A at a volume ratio of 1:6 to obtain a water dispersion, extract the water dispersion with an equal volume of ethyl acetate for 4 times, recover the solvent, and obtain an ethyl acetate extract and 90L of water phase;

[0081] C. Dissolve the ethyl acetate extract obtained in step B with methanol, filter, mix the filtrate with silica gel, dry, and purify the obtained mixture by column chromatography on a silica gel column (petroleum ether: ethyl acetate = 10:1 V / V as the mobile phase), collect the fraction containing sesquiterpene lactone compounds, and concentrate under reduced pressure to dryness to obtain the target product;

[0082] D. Dissolve the target product obtained in step C in methanol, filter, and separate the filtrate using C18 reverse phase chromatography packing material under high pressure preparation (A: acetonitrile B: water, A:B = 34:66 V / V as mobile phase; detection wavelength 220 nm). Collect Figure 1 The corresponding chromatographic peak product is shown in the figure. The product liquid is collected to dryness to obtain 32g of white powder product, which is a sesquiterpene lactone compound with the structure shown in formula (I), namely, yamachu lactone H2. Figure 2 shown.

[0083] The entire production process takes about 7 days;

[0084] By replacing the mobile phase components, the product purity was rechecked using reverse phase analytical liquid chromatography (RP-HPLC), and the result was 99.21%.

[0085] Embodiment 6:

[0086] A. Take 50kg of dried wild horsetail, crush it to 70 mesh, and then use 10 times the weight of 90wt% ethanol to heat and reflux for 5 times, 2 hours each time, and then concentrate it under reduced pressure until there is no alcohol, and obtain 35L of concentrated extract;

[0087] B. Add water to the concentrated extract obtained in step A at a volume ratio of 1:8 to obtain a water dispersion, extract the water dispersion with an equal volume of ethyl acetate for 5 times, recover the solvent, and obtain an ethyl acetate extract and 120L of an aqueous phase;

[0088] C. Dissolve the ethyl acetate extract obtained in step B with methanol (methanol can dissolve the target compound), filter, mix the filtrate with silica gel, dry, and separate and purify the mixed sample by column chromatography on a silica gel column (petroleum ether: ethyl acetate = 10:1 V / V as the mobile phase), collect the fraction containing sesquiterpene lactone compounds, and concentrate under reduced pressure to dryness to obtain the target product;

[0089] D. Dissolve the target product obtained in step C in methanol, filter, and separate the filtrate using C18 reverse phase chromatography packing material under high pressure preparation (A: acetonitrile B: water, A:B = 34:66 V / V as mobile phase; detection wavelength 220 nm). Collect Figure 1 The corresponding chromatographic peak product is shown in the figure. The product liquid is collected to dryness to obtain 38g of white powder product, which is a sesquiterpene lactone compound with the structure shown in formula (I), namely, yamachu lactone H2. Figure 2 shown.

[0090] The entire production process takes about 9 days;

[0091] By replacing the mobile phase components, the product purity was rechecked using reverse phase analytical liquid chromatography (RP-HPLC), and the result was 99.34%.

[0092] Embodiment 7:

[0093] A. Take 50kg of dried wild horsetail, crush it to 80 mesh, and then use 9 times the weight of 90wt% ethanol to heat and reflux for extraction 3 times, 2 hours each time, and concentrate it under reduced pressure until there is no alcohol, and obtain 25L of concentrated extract;

[0094] B. Add water to the concentrated extract obtained in step A at a volume ratio of 1:10 to obtain a water dispersion, extract the water dispersion three times with an equal volume of ethyl acetate, recover the solvent, and obtain an ethyl acetate extract and 100 L of water phase;

[0095] C. Dissolve the ethyl acetate extract obtained in step B with methanol, filter, mix the filtrate with silica gel, dry, and purify the obtained mixture by column chromatography on a silica gel column (petroleum ether: ethyl acetate = 10:1 V / V as the mobile phase), collect the fraction containing sesquiterpene lactone compounds, and concentrate under reduced pressure to dryness to obtain the target product;

[0096] D. Dissolve the target product obtained in step C in methanol, filter, and separate the filtrate using C18 reverse phase chromatography packing material under high pressure preparation (A: acetonitrile B: water, A:B = 34:66 V / V as mobile phase; detection wavelength 220 nm). Collect Figure 1 The corresponding chromatographic peak product is shown in the figure. The product liquid is collected to dryness to obtain 35g of white powder product, which is a sesquiterpene lactone compound with the structure shown in formula (I), namely, yamachu lactone H2. Figure 2shown.

[0097] The entire production process takes about 8 days;

[0098] By replacing the mobile phase components, the product purity was rechecked using reverse phase analytical liquid chromatography (RP-HPLC), and the result was 99.19%.

[0099] Embodiment 8:

[0100] The structure of the sesquiterpene lactone compound of formula (I) obtained by extraction and separation of the present invention, namely, yemachu lactone H2, is identified as follows:

[0101] Electrospray ionization mass spectrometry (ESI-MS) showed: m / z 443.0 [M+Na] + ; 419.0 [MH] ─ , indicating that the molecular weight of the compound is 420 and the molecular formula is C 22 H 28 O8.

[0102] Combining the plant source, NMR data and mass spectrometry data, it can be preliminarily determined that the compound is a sesquiterpene lactone compound. Its molecular weight is the same as that of Eupalinolide H, indicating that it is its isomer. Comparison of carbon spectra ( Figure 3 As shown in Figure 2, some carbon chemical shifts are basically consistent, indicating that the structure is similar to Eupalinolide H. By HSQC ( Figure 5 As shown in the figure, the carbon and hydrogen are fully assigned by HMBC ( Figure 6 As shown) found δ H 5.23 (H-8) and δ C 166.1 (C-1″) correlation, while δ H 4.92, 4.64 (H-14) in addition to δ C 171.3 (C-1′) related, indicating that the acetyl group is attached to the 14-carbon; by NOESY ( Figure 7 As shown) found δ H 2.99 (H-7) and δ H4.64 (H-3) and 5.23 (H-8), indicating that the 3-hydroxyl group is in β configuration. Combined with the literature NMR data, the compound was determined to be (3aR, 4R, 6Z, 9S, 10Z, 11aS)-6-(acetoxymethyl)-9-hydroxy-10-methyl-3-methylene-2-oxo-2,3,3a,4,5,8,9,11a-octahydrocyclodeca[b]furan-4-yl(E)-4-hydroxy-2-methylbut-2-enoate. Chinese name: 3aR, 4R, 6Z, 9S, 10Z, 11aS)-6-(acetoxymethyl)-9-hydroxy-10-methyl-3-methylene-2-oxo-2,3,3a,4,5,8,9,11aS octahydrocyclodeca[b]furan-4-yl(E)-4-hydroxy-2-methylbut-2-enoate. No relevant reports on this compound were found through scifinder search. 1 H-NMR, 13 C-NMR, DEPT135°, NMR two-dimensional HSQC, HMBC, H-HCOSY, NOESY and other analytical techniques confirmed that the compound was a new sesquiterpene lactone structure with the chemical structure shown in formula (I).

[0103] 1 H-NMR and 13 C-NMR data are shown in Table 1 below.

[0104]

[0105] Embodiment 9:

[0106] Anti-lung cancer activity experiment of compounds

[0107] The white powder compound having the structure shown in formula (I) obtained by extraction and separation in Example 5 above (namely, yamachu lactone H2 in the following experiments) was randomly selected for the following experiment:

[0108] (1) Experimental materials and instruments

[0109] Cells: A1975 cells (Wuhan Pronocell Life Science Co., Ltd.)

[0110] Drugs and reagents: Mustangolide H2 (purity > 99.0%, homemade); dissolved in DMSO solution, the prepared stock solution concentration is 200 μmol / L, 1 μL is taken each time and diluted to 1 ml with serum-free culture medium, and diluted at a ratio of 1:4. RPMI-1640 medium (Gibco, USA, batch number 8120139), FBS (Procell, USA, batch number SA201126), PBS buffer powder (Wuxi Aorui Dongyuan Biotechnology Co., Ltd.), trypsin (biofrox, batch number: EZ6688C183), penicillin-streptomycin solution (biosharp, batch number: 1334GR005), MTT (biofrox, batch number: EZ6688D183), DMSO (Chengdu Cologne Chemical Co., Ltd., batch number: 67-68-5) Instruments: Thermo Varlosks microplate reader (Thermo Fisher Scientific, USA), Allegra X-30R centrifuge (Beckman Coulter, USA), clean bench (Changhong Meiling Co., Ltd.), inverted microscope (Zeiss, Germany), CO2 constant temperature incubator (Suzhou Jiemei Electronics Co., Ltd.).

[0111] (2) Experimental methods

[0112] The cells were cultured in 10% 1640 complete medium at 37°C and 5% CO2. The cells in the logarithmic growth phase were digested to prepare a cell suspension and then cultured at 1×10 4 100 μL / ml was inoculated into a 96-well plate and cultured in a CO2 constant temperature incubator for 24 hours. The culture medium was discarded, washed once with PBS, and then different concentrations of yamachai lactone H2 were added (5 replicates were set for each group), and 3 wells of blank culture medium were given as a normal control group; they were returned to the CO2 constant temperature incubator for 24 hours. Subsequently, 20 μL of MTT solution was added to each well, cultured in the culture medium for 4 hours, the culture medium was discarded, 150 μL / well of DMSO solution was added, and the plate was placed in an ELISA reader for 5 minutes of shaking, and the absorbance value (OD) was detected at a wavelength of 490 nm.

[0113] SPSS 26.0 statistical software was used to process the data, and the results were expressed as mean ± standard deviation (). One-way-ANOVA was used for comparison among multiple groups.

[0114] (3) Experimental results

[0115] As shown in Table 2 below, yamachus lactone H2 has inhibitory activity on the proliferation of A1975 cells cultured in vitro, with the maximum inhibition rate within the studied dose being 52.48% and the IC50 being 82.676 (65.858-178.850) μmol / ml.

[0116]

[0117] From the above anti-lung cancer activity experimental results, it can be known that: Yemazhui lactone H2 has inhibitory activity on the proliferation of A1975 cells cultured in vitro, the maximum inhibition rate within the research dose is 52.48%, and the IC50 is 82.676 (65.858-178.850) μmol / ml. Therefore, the sesquiterpene lactone compound of the structure shown in formula (I) obtained by extraction and separation of the present invention can be used in the preparation of anti-lung cancer drugs.

Claims

1. A sesquiterpene lactone compound extracted and separated from Psoralea corylifolia, characterized in that: The compound has the structural formula shown in formula (I): Its Chinese name is: (3aR, 4R, 6Z, 9S, 10Z, 11aS)-6-(acetoxymethyl)-9-hydroxy-10-methyl-3-methylene-2-oxo-2,3,3a,4,5,8,9,11aS octahydrocyclodecan[b]furan-4-yl (E)-4-hydroxy-2-methylbut-2-enoate, which belongs to the sesquiterpene lactone compound and is named: Yemazhui lactone H2.

2. The sesquiterpene lactone compound extracted and separated from the wild horsetail according to claim 1, characterized in that: The sesquiterpene lactone compound represented by formula (I) is a white powder, and a 10wt% sulfuric acid ethanol solution is red.

3. The sesquiterpene lactone compound extracted and separated from the wild horsetail according to claim 1, characterized in that: The electrospray ionization mass spectrum ESI-MS of the sesquiterpene lactone compound represented by the formula (I) shows: m / z 443.0 [M+Na] + ; 419.0 [MH] ─ The molecular weight of the compound is 420 and the molecular formula is C 22 H 28 O8.

4. The method for extracting and separating sesquiterpene lactone compounds from Rhizoma Cibotii according to claim 1, characterized in that: The following steps are involved: A. Ethanol Reflux Extraction The dried wild horsetail was crushed into 60-80 meshes, and then extracted by heating and refluxing with 90wt% ethanol. After concentrating under reduced pressure until there is no alcohol, a concentrated extract is obtained; B. Ethyl acetate extraction The concentrated extract obtained in step A is dispersed by adding water to obtain an aqueous dispersion, and the aqueous dispersion is extracted 3 to 5 times with an equal volume of ethyl acetate, and the solvent is recovered to obtain an ethyl acetate extract and an aqueous phase; C. Silica gel column chromatography a. The ethyl acetate extract obtained in step B was dissolved in methanol, filtered, the filtrate was mixed with silica gel, and dried to obtain a mixed sample; b. The mixed sample was wet-mixed on a silica gel column for column chromatography and purification, and the fractions containing sesquiterpene lactone compounds were collected and concentrated to dryness under reduced pressure to obtain the target product; The silica gel column chromatography separation was performed with petroleum ether:ethyl acetate = 10:1 V / V as the mobile phase; D. Separation by C18 reverse phase chromatography column: The target product obtained in step C is dissolved in methanol, filtered, and the filtrate is separated by high pressure preparative separation using C18 reverse phase chromatography filler to obtain product collection liquid, and the collection liquid is concentrated to dryness to obtain White powder product, sesquiterpene lactone compound of the structural formula shown in (Ⅰ); The C18 reverse phase chromatographic column was used for separation: A: acetonitrile B: water, A:B = 34:66 V / V as the mobile phase; the detection wavelength was 220 nm.

5. The method for extracting and separating sesquiterpene lactone compounds from Rhizoma Cibotii according to claim 4, characterized in that: In step A, the amount of ethanol used is 8 to 10 times the weight of the crushed dry wild horsetail; the reflux extraction is 3 to 5 times, each time for 2 hours.

6. The method for extracting and separating sesquiterpene lactone compounds from Rhizoma Cibotii according to claim 4, characterized in that: In step B, the concentrated extract is dispersed by adding water at a volume ratio of 1:6 to 1:

10.

7. Use of the sesquiterpene lactone compounds extracted and separated from Rhizoma Cibotii according to claim 1 in the preparation of anti-lung cancer drugs.

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