Fungus-source anti-enterovirus sesquiterpene compound as well as preparation and application thereof

Abstract

The invention belongs to the technical field of medical chemistry, and particularly relates to a fungal anti-enterovirus sesquiterpene compound as well as preparation and application thereof. A compound with anti-EV71 activity is screened from natural products, the sesquiterpenoids are extracted from a fermentation culture of plant endophytic fungi Paramrothecium sp. Q021, the sesquiterpenoids have remarkable anti-EV71 virus activity, the half effective concentration (EC50) of the sesquiterpenoids is remarkably lower than that of ribavirin, and the sesquiterpenoids have the advantages that the sesquiterpenoids have obvious anti-EV71 virus activity; meanwhile, the half cytotoxicity concentration (CC50) and the selection index (SI index) of the ribavirin are higher than those of the ribavirin. The results show that the sesquiterpenoids have remarkable anti-EV71 virus activity and excellent safety, a candidate compound with great potential is provided for developing a novel medicine for preventing and treating the infant hand-foot-and-mouth disease caused by the EV71 virus, and the sesquiterpenoids have a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to a fungal-derived anti-enterovirus sesquiterpene compound and its preparation and application. Background Technology

[0002] Enterovirus 71 (EV71) is one of the main pathogens of hand-foot-and-mouth disease (HFMD) in infants and young children. This virus is highly contagious and has a high pathogenicity, spreading rapidly through multiple routes including fecal-oral, respiratory, and contact routes, and easily causing neurological complications. Children of all ages are generally susceptible, with the highest incidence in children under 3 years old. Some infected children may develop severe illness, with clinical manifestations including aseptic meningitis, brainstem encephalitis, and neurogenic pulmonary edema; some cases are even life-threatening, and survivors may suffer long-term sequelae. Therefore, EV71 infection is considered a disease with a significant public health threat, often referred to as the "polio of the 21st century." Statistics show that HFMD caused by EV71 exhibits a periodic epidemic pattern every 2-3 years, making it a global public health concern.

[0003] EV71 belongs to the genus Enterovirus of the family Picornaviridae in the order Picornaviridae. Its genome is a single-stranded positive-sense RNA, approximately 7500 bases in length, containing only one open reading frame (ORF) encoding a polyprotein. This polyprotein, upon hydrolysis, yields four structural proteins (VP1-VP4) and seven non-structural proteins (2A-2C, 3A-3D), flanked by 5' and 3' untranslated regions, respectively. At the onset of viral infection, the structural proteins undergo conformational changes, facilitating viral binding to host cells. Subsequently, as the virus initiates replication, the structural proteins undergo further conformational changes, releasing the viral single-stranded RNA into the host cell. The viral RNA then replicates and translates to generate a polyprotein precursor, which is ultimately processed and cleaved by the virus's own functional proteases to form the mature EV71 viral protein, completing viral assembly and enabling further infection of other cells.

[0004] Currently, clinically available drugs for treating EV71 infection (such as ribavirin and acyclovir) have limited efficacy, and long-term use may cause adverse reactions such as liver damage. Therefore, developing highly specific, effective, and low-toxicity anti-EV71 drugs is of urgent practical significance for protecting infant and child health and promoting social development. Natural products, due to their diverse structures and broad biological activities, have always been an important resource for drug development. Discovering natural components with anti-EV71 activity from natural resources not only has significant scientific research value but also demonstrates broad development prospects. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a fungal sesquiterpene compound with significant anti-EV71 activity and higher safety, which can be used as a lead compound and drug candidate for anti-EV71, and contribute to the development of new safe anti-EV71 drugs.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a sesquiterpene compound, the structural formula of which is shown in Formula 1: .

[0007] The second aspect of the present invention also provides the use of the sesquiterpenoid compounds described in the first aspect in the preparation of medicaments against EV71 virus.

[0008] The sesquiterpenoid compounds provided by this invention, after 48 h of EV71 (MOI=0.1) infection, exhibited EC50... 50 The concentration was 3.817 μg / mL [the positive control drug ribavirin (Rb) was 45.31 μg / mL]; simultaneously, this sesquiterpene compound showed increased CC in RD cells. 50 The concentration was 263.1 μg / mL (compared to 225.8 μg / mL for the positive control drug Rb); additionally, the SI index of this sesquiterpene compound was 68.83 (compared to 4.98 for the positive control drug Rb). This indicates that under the same infection conditions, the EC50 of this sesquiterpene compound is significantly higher than that of the positive control drug Rb. 50 Biribavirin is small, and CC 50 Both its activity and SI were higher than those of ribavirin. This indicates that it has significant anti-EV71 virus activity and a safety profile superior to the positive control drug ribavirin. It can be used to prepare anti-EV71 lead compounds and antiviral drugs, providing candidate compounds for the research and development of safer new anti-EV71 drugs.

[0009] A third aspect of the present invention also provides an anti-EV71 virus drug, the drug comprising a sesquiterpene compound as the main active ingredient as described in the first aspect.

[0010] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0011] More preferably, the excipients include at least one of the following: solubilizer, emulsifier, colorant, binder, disintegrant, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, coating material, pH adjuster, absorbent, and antioxidant.

[0012] Preferably, the dosage form of the drug includes powder, tablet, capsule, granule, syrup, and oral liquid preparation.

[0013] Preferably, the sesquiterpenoid compound is isolated from the fermentation culture of the plant endophytic fungus Paramyrothecium sp. Q021, whose preservation number is GDMCC No: 67350.

[0014] More preferably, the method for separating the sesquiterpenoid compounds includes the following steps: S1. Activate the plant endophytic fungus Paramyrothecium sp. Q021; S2. Preparation of fermentation culture by fermentation: Activated Paramyrothecium sp. Q021 was inoculated into PDB medium and fermented by shaking to obtain a bacterial mixture; then the mixture was inoculated into rice medium and cultured in the dark to obtain the fermentation culture. S3. Extraction and extraction of fermentation culture to obtain enrichment: Add ethyl acetate to fermentation culture, and after soaking and ultrasonic extraction, combine the ethyl acetate extract and concentrate under reduced pressure for later use; collect the solid culture medium remaining after extraction, add ethyl acetate submerged in the culture medium and let stand for extraction, collect the extract and concentrate under reduced pressure; add ethyl acetate submerged in the culture medium again to the above solid culture medium, extract under ultrasonication, collect the extract and concentrate under reduced pressure, combine all concentrated extracts to obtain enrichment; S4. Systematic separation and purification of the enriched product yielded the sesquiterpenoid compounds: The aforementioned enriched product was separated by silica gel column chromatography, using a gradient elution with petroleum ether-ethyl acetate (v / v) and dichloromethane-methanol (v / v) at ratios of 80:1 to 0:1. Fractions with the same eluent were combined with the results of thin-layer chromatography, yielding a total of 11 fractions Fr.1-Fr.11. Fraction Fr.7 was further separated using a Sephadex HW-40 dextran gel column with pure methanol as the eluent, yielding 8 subfractions Fr.7.1-Fr.7.8. Subfraction Fr.7.5 was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) under the following conditions: Welch Materials, Inc. RP-C18 column (5 μm, 250 mm × 10 mm), mobile phase acetonitrile-water (v / v) of 10:90, flow rate of 1-3 mL / min, and collection retention time t. R The chromatographic peak at 24.7 min was concentrated under reduced pressure to obtain the compound shown in Formula 1.

[0015] More preferably, the activation is performed under constant temperature conditions of 28-30 °C for 3-7 days.

[0016] More preferably, the rice culture medium is cultured in the dark at a temperature of 20-40 ℃ for 30-40 days.

[0017] More preferably, the activation is performed on a PDA tablet.

[0018] More preferably, the plant endophytic fungus Paramyrothecium sp. Q021 is stored in a 20% glycerol aqueous solution at -80 °C before activation.

[0019] More preferably, the temperature for shaking fermentation of PDB medium is 28-30 ℃, and the time is 5-7 days.

[0020] Compared with the prior art, the beneficial effects of the present invention are: To screen compounds with anti-EV71 activity from natural products, this invention discloses a sesquiterpene compound that exhibits significant inhibitory activity against enterovirus 71. Analysis of its anti-EV71 activity, cytotoxicity, and selectivity index indicates that, under the same infection conditions, this sesquiterpene compound has a high half-maximal effective concentration (EC50). 50 Its cytotoxic concentration (CC) was significantly lower than that of ribavirin, while its half-maximal cytotoxic concentration (CC) was also significantly lower. 50 Both the antiviral activity and the selectivity index (SI index) were higher than those of ribavirin. These results indicate that this sesquiterpene compound possesses significant antiviral activity against EV71 virus and exhibits excellent safety, showing promising application prospects in the development of novel drugs for the prevention and treatment of hand-foot-mouth disease induced by EV71 virus in infants and young children. Furthermore, this invention first activates the plant endophytic fungus Paramyrothecium sp. Q021, then prepares a fermentation culture of this strain using a fermentation method, and finally extracts the sesquiterpene compound from it using separation and purification techniques. The preparation method described in this invention is simple and easy to implement, possessing good operational feasibility. Attached Figure Description

[0021] Figure 1 The proton NMR spectrum of compound 1 ( 1 (H NMR spectrum); Figure 2 The carbon NMR spectrum of compound 1 ( 13 (C NMR spectrum) Figure 3 The distortion-free polarization transfer technique (DEPT) spectrum of compound 1 is shown. Figure 4 The hydrogen-hydrogen chemical shift correlation spectrum of compound 1 ( 1 H– 1 H COSY spectrum); Figure 5 The heteronuclear single quantum correlation spectrum (HSQC spectrum) of compound 1. Figure 6 The heteronuclear multibond correlation spectrum (HMBC spectrum) of compound 1. Figure 7 This is the single-crystal structure analysis data for compound 1; Figure 8 Determination of EC of compound 1 by CCK8 method 50 Resulting image; Figure 9 EC50 of ribavirin as a positive control drug was determined using the CCK8 assay. 50 Resulting image; Figure 10 CCK8 method for determining the C2O2 of compound 1 50 Resulting image; Figure 11 CCK8 assay for the determination of the C10 concentration of ribavirin, a positive control drug 50 Result image. Detailed Implementation

[0022] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0024] Instructions regarding strains and culture media: (1) Isolation, purification, and identification of the fungus Paramyrothecium sp. Q021: This strain was isolated from fresh leaves of Rhododendron racemosum collected in Panzhou City, Guizhou Province in June 2023. ITS sequence analysis and BLAST alignment confirmed that the strain was a fungus belonging to the genus Paramyrothecium sp., with the accession number Q021. Its gene sequence is registered in GenBank under accession number OW984150.1. The ITS sequence of this strain is shown in SEQ ID NO: 1. The strain Paramyrothecium sp. Q021 was deposited on November 25, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67350.

[0025] Paramyrothecium sp.Q021 ITS (SEQ ID NO: 1): .

[0026] (2) Preparation method of rice culture medium: Weigh 300 g of rice, add 350 mL of double-distilled water, and sterilize at 121 °C for 30 min under natural pH conditions.

[0027] Example 1: Isolation and Identification of Sesquiterpenoids 1. Reagents and instruments used in the experiment (1) (HCB-1300V) Ultra-clean workbench; (Yamato SQ810C) Vertical autoclave; (ZQZY-CF8) Three-layer constant temperature shaking culture incubator; (GHP-9080) Water-jacketed incubator; (DW-25L262) Ultra-low temperature freezer; (2) Potato glucose agar (PDA) medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd.; Potato glucose water (PDB) medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd. (3) Ethanol, ethyl acetate, dichloromethane and methanol were purchased from Guangzhou Bell Biotechnology Co., Ltd.; chromatographic grade methanol was purchased from TEDIA Inc.; water for high performance liquid chromatography was Wahaha purified water (Hangzhou Wahaha Company); (4) Silica gel for column chromatography (80-120 mesh), thin layer chromatography silica gel plate (GF254) was purchased from Shanxi Nuotai Biotechnology Co., Ltd.; (5) Analytical column XB-C18 5 μM (250 mm × 4.6 mm), semi-preparative column XB-C18 5 μM (250 mm × 10 mm), Sephadex TM The LH-20 was purchased from Merck, a German company.

[0028] (6) Thin-layer chromatography (TLC) uses a 10% sulfuric acid-ethanol solution as the colorimetric reagent.

[0029] (7) Others: (WHF-203B) UV spectrophotometer; (EYEKA N-1300) rotary evaporator; (XD-5210A) rotary evaporator; Agilent 1120 high performance liquid chromatograph (HPLC); Waters e2695 (HPLC); Bruker AM-500 nuclear magnetic resonance spectrometer (Bruker, Karlsruhe, Germany).

[0030] 2. Isolation of sesquiterpenoids: (1) Take the strain Paramyrothecium sp. Q021 preserved at -80℃ with 20% glycerol aqueous solution, inoculate the strain onto PDA plate, and activate it at 30℃ for 5 days; (2) Cut the activated plates (90 mm id) covered with bacteria into uniform small pieces and inoculate them into PDB liquid medium, shaking gently to mix. Inoculate 3-4 pieces of bacterial plates into each bottle of PDB medium (250 mL), and ferment on a shaker for 7 days (28 ℃, 180 rpm) to obtain a bacterial mixture. Then, inoculate 15-20 mL of the bacterial mixture into each bottle of rice medium (500 mL), for a total of 50 bottles. Incubate at room temperature in the dark for 40 days to obtain the fermentation culture.

[0031] (3) Add 2 L of ethyl acetate to each bottle of fermentation culture, soak for 12 hours, and then extract by sonication for 30 min. After extraction, combine the ethyl acetate extracts and concentrate under reduced pressure for later use. At the same time, combine the remaining solid culture medium after extraction, add an appropriate amount of ethyl acetate (just enough to submerge), let stand for 24 hours, collect the extract and concentrate under reduced pressure; repeat this standing extraction operation twice. Finally, add an appropriate amount of ethyl acetate (just enough to submerge) to the extracted solid culture medium, extract by sonication for 30 min, collect the extract and concentrate under reduced pressure, combine all the above concentrated extracts to obtain the total extract. Add water to suspend the total extract at a material-to-liquid ratio of 1:12 (g / mL), add an equal volume of petroleum ether for extraction, and repeat the extraction 3 times; discard the petroleum ether layer, take the remaining aqueous layer, add an equal volume of ethyl acetate for extraction 3 more times, combine the ethyl acetate extracts, concentrate under reduced pressure to obtain the target enrichment.

[0032] (4) The above-mentioned enriched fractions were subjected to silica gel column chromatography for separation. Gradient elution was performed sequentially using petroleum ether-ethyl acetate (volume ratios: petroleum ether:ethyl acetate = 80:1, 40:1, 20:1, 10:1, 5:1, 4:1, 2:1, 1:1, 1:2, 1:5, 1:20, 0:1) and dichloromethane-methanol (volume ratios: dichloromethane:methanol = 80:1, 10:1, 5:1, 3:1, 1:1, 0:1). Thin-layer chromatography was used to detect and combine fractions with the same eluent, yielding a total of 11 fractions (Fr.1-Fr.11). Fraction Fr.7 was separated using a Sephadex HW-40 dextran gel column (pure methanol as eluent) to obtain 8 subfractions (Fr.7.1-Fr.7.8). Subfraction Fr.7.5 was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) under the following chromatographic conditions: Welch column. Materials, Inc. RP-C 18 Column (5 μm, 250 mm × 10 mm), mobile phase acetonitrile-water (v / v 10:90), flow rate 3 mL / min, collection retention time t R The chromatographic peak corresponding to 24.7 min was concentrated under reduced pressure to prepare compound 1.

[0033] Structural analysis was performed on compound 1, and the physicochemical properties obtained are as follows: Compound 1 is a colorless crystal; its 500 MHz concentration was determined using deuterated methanol (CD3OD) as solvent. 1 H NMR spectrum and 125MHz 13 C NMR spectra, spectral data can be found in [reference]. Figure 1-3 Simultaneously, single-crystal diffraction experiments of this compound were performed, and its... 1 H- 1 HCOSY, HSQC, and HMBC spectra; detailed spectral results can be found in [link to relevant data]. Figure 4-6The combined spectral data clearly identified the signals from hydrogen and carbon atoms, fully elucidating the interatomic connections and the positions of functional groups, thus confirming the planar structure of compound 1.

[0034] Single crystal data of compound 1 (Table 1, Figure 7 ) is: C 15 H 18 O4, M = 262.29, a = 17.8236(5) Å, b= 6.6556(2) Å, c = 18.2739(5) Å, α = 90°, β = 106.0170(10)°, γ = 90°, V =2083.62(10) Å 3 , T = 150.(2) K, space group P1211, Z = 6, μ(Cu Kα) = 0.742 mm -1 , 35287 reflections measured, 7565 independent reflections (R int = 0.0568).The final R1 values ​​were 0.0328 (I > 2σ(I)). The final wR(F 2 ) values ​​were0.0835 (I > 2σ(I)). The final R1 values ​​were 0.0345 (all data). The final wR(F 2 ) values ​​were 0.0844 (all data). The goodness of fit on F 2 was 1.067. Flackparameter = 0.03(6).

[0035] Table 1. Crystallographic data and structural refinement of compound 1 Continued from the previous table: Finally, the molecular structure of this sesquiterpene compound (compound 1) was determined to be as follows: .

[0036] Experimental Example 1: Anti-EV71 Activity Experiment of Sesquiterpene Compound 1 1. Experimental Materials (1) Main reagents: Ribavirin (Rb) was purchased from Selleck; DMEM (high glucose) medium was purchased from Sangon Biotech Co., Ltd.; penicillin and streptomycin were purchased from Nanjing Shenghang Biotechnology Co., Ltd.; fetal bovine serum (FBS) was purchased from ZETA Life; 0.25% trypsin (containing EDTA) and CCK-8 were purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.

[0037] (2) Test cells and virus strains: RD cells and EV71 strains were obtained from the Shanghai Institute of Major Infectious Diseases and Biosafety.

[0038] 2. Experimental Methods (1) In vitro anti-EV71 activity experiment: RD cells were seeded in 96-well cell culture plates overnight (DMEM medium containing 10% fetal bovine serum + 1% penicillin and streptomycin). When the cells reached approximately 90% confluence, a 1:1 volume ratio of a fractionally diluted test compound 1 (0.4 μg / mL-50 μg / mL) and EV71 virus dilution (MOI=0.1) was added to the cell wells and incubated at 37 ℃ in a 5% CO2 incubator. After 2 h, the cell supernatant was aspirated, the cells were washed with PBS, and the medium was replaced with fresh medium containing the appropriate concentration of compound 1. Cells were then incubated at 37 ℃ in a 5% CO2 incubator for another 48 h. Cell viability was then assessed using a CCK-8 assay: 10 μL of CCK-8 reagent was added to each well, and after incubation at 37 ℃ for 1.5 h, the OD value at 450 nm was measured using a microplate reader. Three replicates were set up for each sample, along with control wells (without the drug) and virus-infected control wells. Cell viability (%) in the drug-treated group = (OD value of experimental wells) / (OD value of experimental wells). 450nm -Virus control well OD 450nm / Cell control well OD 450nm -Virus control well OD 450nm () × 100%. Graph Pad Prism 9.5.1 software was used for plotting.

[0039] (2) In vitro toxicity test: RD cells were seeded in 96-well cell culture plates and incubated overnight. Once the cells reached approximately 90% confluence, compound 1 was diluted sequentially (3.125 μg / mL - 400 μg / mL) and added to the wells. Cells were then cultured for another 48 hours. Cell viability was then assessed using a CCK-8 assay: 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37 °C for 1.5 hours. The OD value at 450 nm was then measured using a microplate reader. Three replicates were performed for each sample, along with control wells containing no drug and pure culture medium. Cell viability was represented by cell survival rate. The cell viability rate (%) of the drug-treated group was calculated as follows: (OD value of experimental wells) / (OD value of experimental wells) 450nm -Culture control well OD 450nm / Cell control well OD 450nm -Culture control well OD 450nm () × 100%. Graph Pad Prism 9.5.1 software was used for plotting.

[0040] 3. Experimental Results Compound 1 and (Ribavirin, Rb) showed EC activity 48 h after EV71 (MOI=0.1) infection. 50 The concentrations were 3.817 μg / mL and 45.31 μg / mL, respectively. Figure 8 , 9 Compound 1 and the positive drug Rb on RD cells (CC) 50 The concentrations were 263.1 μg / mL and 225.8 μg / mL, respectively. Figure 10 , 11 ); SI index (CC) of compound 1 and positive drug Rb 50 / EC 50 The EC values ​​were 68.83 and 4.98, respectively. These results indicate that, under the same infection conditions, the EC values ​​of the sesquiterpene compound (compound 1) were... 50 Biribavirin is small, and CC 50 Both the SI (antiviral activity) and the activity level were higher than those of ribavirin. This indicates that the compound has significant antiviral activity against EV71 virus and a safety profile superior to the positive control drug ribavirin.

[0041] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A sesquiterpene compound, characterized in that, The structural formula of the sesquiterpene compound is shown in Formula 1: 。 2. The use of the sesquiterpene compound of claim 1 in the preparation of a drug for treating EV71 virus.

3. An anti-EV71 virus drug, characterized in that, The drug comprises a sesquiterpene compound as described in claim 1 as the main active ingredient.

4. The anti-EV71 virus drug according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The anti-EV71 virus drug according to claim 4, characterized in that, The excipients include at least one of the following: solubilizer, emulsifier, colorant, binder, disintegrant, lubricant, wetting agent, osmotic pressure regulator, stabilizer, flow aid, flavoring agent, preservative, coating material, pH adjuster, absorbent, and antioxidant.

6. The anti-EV71 virus drug according to claim 4, characterized in that, The dosage forms of the drug include powder, tablets, capsules, granules, syrups, and oral liquid preparations.

7. A sesquiterpene compound according to claim 1, characterized in that, The sesquiterpenoid compounds were isolated from the fermentation culture of the plant endophytic fungus Paramyrothecium sp. Q021, whose accession number is GDMCC No: 67350.

8. A sesquiterpene compound according to claim 7, characterized in that, The method for separating the sesquiterpenoids includes the following steps: S1. Activate the plant endophytic fungus Paramyrothecium sp. Q021; S2. Preparation of fermentation culture by fermentation: Activated Paramyrothecium sp. Q021 was inoculated into PDB medium and fermented by shaking to obtain a bacterial mixture; then the mixture was inoculated into rice medium and cultured in the dark to obtain the fermentation culture. S3. Extraction and extraction of fermentation culture to obtain enrichment: Add ethyl acetate to fermentation culture, and after soaking and ultrasonic extraction, combine the ethyl acetate extract and concentrate under reduced pressure for later use; collect the solid culture medium remaining after extraction, add ethyl acetate submerged in the culture medium and let stand for extraction, collect the extract and concentrate under reduced pressure; add ethyl acetate submerged in the culture medium again to the above solid culture medium, extract under ultrasonication, collect the extract and concentrate under reduced pressure, combine all concentrated extracts to obtain enrichment; S4. Systematic separation and purification of the enriched product yielded the sesquiterpenoid compounds: The aforementioned enriched product was separated by silica gel column chromatography, using a gradient elution with petroleum ether-ethyl acetate (v / v) and dichloromethane-methanol (v / v) at ratios of 80:1 to 0:

1. Fractions with the same eluent were combined with the results of thin-layer chromatography, yielding a total of 11 fractions Fr.1-Fr.

11. Fraction Fr.7 was further separated using a Sephadex HW-40 dextran gel column with pure methanol as the eluent, yielding 8 subfractions Fr.7.1-Fr.7.

8. Subfraction Fr.7.5 was purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) under the following conditions: Welch Materials, Inc. RP-C18 column (5 μm, 250 mm × 10 mm), mobile phase acetonitrile-water (v / v) of 10:90, flow rate of 1-3 mL / min, and collection retention time t. R The chromatographic peak at 24.7 min was concentrated under reduced pressure to obtain the compound shown in Formula 1.

9. A sesquiterpene compound according to claim 8, characterized in that, The activation is carried out under constant temperature conditions of 28-30 ℃ for 3-7 days.

10. A sesquiterpene compound according to claim 8, characterized in that, The rice culture medium should be cultured in the dark at a temperature of 20-40 ℃ for 30-40 days.

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