A sesquiterpene compound with anti-epileptic activity and a preparation method and use thereof

Abstract

The application discloses a sesquiterpene compound with anti-epilepsy activity, and a preparation method and application thereof, and has the characteristics that the structural formula of the compound is shown as I, and the preparation method comprises the following steps: firstly, obtaining a fermentation product of a marine fungus through fermentation culture; then, soaking and extracting the fermentation product with ethyl acetate to obtain a crude extract; and then, separating and purifying the crude extract through normal-phase silica gel column chromatography, reverse medium-pressure column chromatography and reverse-phase semi-preparative high performance liquid chromatography, so as to finally obtain the target sesquiterpene compound, and the advantages are that the sesquiterpene compound not only has a significant anti-epilepsy effect, but also shows a good development prospect, and can be used for drug research and development for preventing and treating epilepsy.

Description

TECHNICAL FIELD

[0001] The present application relates to a sesquiterpenoid compound, in particular to a compound with anti-epileptic activity extracted from the secondary metabolites of marine fungi and its preparation method and use. BACKGROUND

[0002] Epilepsy is a serious chronic neurological disease with the characteristics of spontaneity, uncontrollability and repeated seizures. This disease not only significantly increases the risk of various physiological complications in patients, but also greatly increases the probability of sudden death. The pathological mechanism of epilepsy is complex and diverse, usually involving abnormal discharge of neurons, pathological synchronization of brain electrical activity, and is closely related to ion channel dysfunction, neurotransmitter imbalance, abnormal neural network and genetic factors. Current anti-epileptic drugs (abbreviated as AEDs), such as phenytoin sodium, carbamazepine and valproate, mainly control seizures by regulating the above pathological mechanisms. However, the efficacy of these drugs is limited in some patient groups, and is often accompanied by serious adverse reactions such as hepatotoxicity, anemia, endocrine disorders and cognitive impairment. In addition, about 30% of epilepsy patients show resistance to existing drugs, leading to the occurrence of refractory epilepsy. An important mechanism of drug-resistant epilepsy is the overexpression of multidrug resistance (MDR) proteins in the blood-brain barrier, especially the overexpression of P-glycoprotein, which limits the penetration of anti-epileptic drugs into the central nervous system, reduces the bioavailability of the drugs, and thus weakens the control effect on seizures.

[0003] In view of the limitations of current anti-epileptic drugs in the treatment of drug-resistant epilepsy, there is an urgent need to develop new, more effective and less side-effect treatment strategies. In recent years, seaweed and its symbiotic fungi have become a hot research topic due to their complex and precise symbiotic relationship. Through chemical signal transmission, nutrient exchange and synergistic defense mechanisms, this symbiotic system can produce a variety of secondary metabolites with complex structures and diverse functions. These metabolites not only have unique structural diversity, but also exhibit a wide range of biological activities, including antibacterial, anticancer, antiviral and neuroregulatory functions, especially showing great application potential in the development of anti-epileptic drugs. Currently, there is no related research report on extracting compounds with anti-epileptic activity from the secondary metabolites of seaweed and its symbiotic fungi. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a sesquiterpenoid compound with anti-epileptic activity and its preparation method and use.

[0005] The technical scheme adopted by the present application to solve the above technical problem is:

[0006] 1. A sesquiterpenoid compound with anti-epileptic activity, the structure of the sesquiterpenoid compound is shown as formula (I):

[0007]

[0008] 2. The method for preparing the above-mentioned sesquiterpenoid compounds with antiepileptic activity includes the following steps:

[0009] (1) Fermentation production

[0010] Aspergillus pyrolyticus with accession number CCTCC NO: M2014086 was inoculated onto a plate containing potato dextrose agar solid medium (PDA solid medium). After activation by inverting the plate for 3 days at 28°C, the activated single colony was transferred to potato dextrose liquid medium (PDB liquid medium) that had been autoclaved and cooled. The plate was then cultured with shaking at 28°C and 150 rpm for 4 days to obtain the seed culture. 5–7 mL of the seed culture was inoculated into rice culture medium and incubated statically at 28°C for 30 days to obtain the fermentation product.

[0011] (2) Extraction of extract

[0012] Ethyl acetate was added to the fermentation product obtained in step (1), and the extraction was repeated 3 to 4 times. The ethyl acetate extract was then evaporated by rotary evaporation to obtain a crude extract.

[0013] (3) Isolation, purification and preparation of compounds

[0014] First, the crude extract obtained in step (2) was fully dissolved in a 1:1 mixture of dichloromethane and methanol. Then, 200-300 mesh silica gel was added and the mixture was stirred. Normal-phase medium-pressure column chromatography was performed, using a mixture of petroleum ether and ethyl acetate in a 2:3 volume ratio as the eluent. The eluent was collected and mixed with 100-200 mesh reversed-phase silica gel. Then, reversed-phase medium-pressure column chromatography was performed, using a methanol-water eluent with a methanol volume percentage of 20-100% for linear gradient elution over 120 minutes. The eluent fractions were collected and arranged in descending order of polarity, resulting in seven fractions. The seventh fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography using a 50:50 volume ratio mixture of acetonitrile and water as the mobile phase to obtain compound I, whose structure is shown in (I).

[0015]

[0016] Further, the method for preparing the PDA solid culture medium in step (1) is as follows: Boil 200g of peeled and diced potatoes for 30 minutes, filter, retain the filtrate, add 20g of glucose and 20g of agar to the filtrate, heat until completely dissolved, cool to below 50°C, adjust the volume to 1000mL with distilled water, autoclave at 121°C for 20 minutes, cool to 45-50°C, pour into sterile petri dishes, and wait for it to solidify before use.

[0017] Furthermore, the preparation method of the PDB liquid culture medium in step (1) is as follows: Take 200g of peeled and diced potatoes, add 1000mL of distilled water and boil for about 30 minutes, filter and retain the filtrate, add 20g of glucose to the filtrate, stir until completely dissolved, adjust the volume to 1000mL with distilled water, autoclave at 121℃ for 15-20mL and then cool, and it can be used for culture.

[0018] Furthermore, the rice culture medium prepared in step (1) is prepared as follows: Take 100g of rice, add 110mL of distilled water and 3g of sea salt, put the mixture into a culture bottle, and then sterilize it by high pressure steam at 121℃ for 20min and then cool it for inoculation and fermentation.

[0019] Furthermore, the flow rate of the mobile phase in the semi-preparative reversed-phase high-performance liquid chromatography described in step (3) is 2 mL / min.

[0020] The present invention also provides the use of the above-mentioned sesquiterpenoid compounds in the preparation of antiepileptic drugs.

[0021] Compared with the prior art, the advantages of the present invention are as follows: The present invention discloses a sesquiterpene compound with antiepileptic activity, its preparation method and uses. The fermentation product is obtained by microbial fermentation culture, and then the fermentation product is extracted by soaking in ethyl acetate to obtain a crude extract. The crude extract is then purified by medium-pressure normal-phase column chromatography, medium-pressure reverse-phase column chromatography and semi-preparative high-performance liquid chromatography. The compound has significant antiepileptic activity and can be used in the development of drugs to inhibit epilepsy-related diseases.

[0022] The aforementioned Aspergillus ustus strain, DJ003, is classified as Aspergillus ustus, with accession number CCTCCNO: M2014086. It was deposited at the China Center for Type Culture Collection on March 14, 2014, at Wuhan University, Wuhan, China. Attached Figure Description

[0023] Figure 1 High-resolution mass spectrometry (HR-ESI-MS) of compound I of the present invention;

[0024] Figure 2 The ultraviolet (UV) spectrum of compound I of the present invention is shown below.

[0025] Figure 3 The infrared (IR) spectrum of compound I of this invention is shown below.

[0026] Figure 4 The proton nuclear magnetic resonance spectrum of compound I of this invention ( 1 HNMR);

[0027] Figure 5 The carbon NMR spectrum of compound I of this invention ( 13 CNMR);

[0028] Figure 6 This is the DEPT-135 nuclear magnetic resonance spectrum of compound I of the present invention;

[0029] Figure 7 The COSY NMR spectrum of compound I of this invention;

[0030] Figure 8 The HSQC nuclear magnetic resonance spectrum of compound I of this invention;

[0031] Figure 9 The nuclear magnetic resonance HMBC spectrum of compound I of this invention;

[0032] Figure 10 The NMR NOESY spectrum of compound I of this invention;

[0033] Figure 11 The key to compound I of this invention 1 H- 1 Correlation between H COSY (thick blue line) and HMBC (red arrow);

[0034] Figure 12 Key NOE correlation of compound I in this invention;

[0035] Figure 13 Experimental and calculated values ​​of the ECD spectrum of compound I of the present invention;

[0036] Figure 14 Results of the antiepileptic activity of compound I of the present invention in a zebrafish model, wherein (A) typical movement trajectory of zebrafish at 6 dpf. (BE) total distance, average speed, high movement frequency and curvature. (F) AChE activity. (GI) level of epilepsy-related mRNA, compared with the control group, p # <0.05 (significant), compared with the model group, p * <0.05 (significant). Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:

[0038] Example 1: A sesquiterpene compound derived from marine fungi with antiepileptic activity, the structural formula of which is shown in (I):

[0039]

[0040] Example 2, the preparation method of the sesquiterpenoid compounds shown in Example 1, the specific steps are as follows:

[0041] Step 1: Fermentation Production

[0042] Aspergillus pyrolyticus with accession number CCTCC NO: M2014086 was streaked onto PDA solid medium (formula: 200g peeled and diced potato, 20g glucose, 15-20g agar, 1000mL distilled water). After activation at 28℃ for 3 days, the mycelium was cut into small pieces using a sterile inoculation loop. The mycelium was then transferred to 10 1L round-bottom conical flasks sterilized at 121℃, each containing 300mL of PDB liquid medium (formula: 200g peeled and diced potato, 20g glucose, 1000mL distilled water). After inoculation, the culture flasks were placed on a rotating shaker at 28℃ and 150rpm for 4 days to obtain seed culture. Then, 400 1L round-bottom conical flasks were prepared, each equipped with rice culture medium (formula: 100g rice, 110mL distilled water, 3.0g sea salt). After autoclaving at 121℃ and cooling, 5-7mL of seed culture was inoculated into each rice culture medium. The inoculated culture flasks were incubated at 28℃ under static conditions for 30 days to obtain fermentation products. The entire inoculation and fermentation process was carried out in a sterile environment to ensure no contamination.

[0043] Step 2: Extraction of the extract

[0044] Add sufficient ethyl acetate to the fermentation product obtained in step 1, and extract repeatedly 3-4 times. Then, use a rotary evaporator to vacuum evaporate the ethyl acetate extract to obtain a crude extract paste.

[0045] Step 3: Isolation and preparation of compounds

[0046] The crude extract obtained in step 2 was first thoroughly dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography (using a Silica Flash Column). 330g), was eluted using a mixture of petroleum ether and ethyl acetate in a volume ratio of 2:3. The eluent was collected and mixed with 100-200 mesh reversed-phase silica gel. Then, reversed-phase medium-pressure column chromatography was performed, using a methanol-water mixture with a methanol volume percentage of 20-100% as the eluent for linear gradient elution for 120 min. The eluted fractions were collected, arranged in descending order of polarity, and combined to obtain 7 components. The 7th component was purified by semi-preparative reversed-phase high-performance liquid chromatography (HPLC) using a mixture of acetonitrile and water in a volume ratio of 1:1 as the mobile phase at a flow rate of 2 mL / min to obtain compound I, whose structural formula is shown in Example 1 (I).

[0047] Structural identification of sesquiterpenoid compounds prepared by the methods in Example 3 and Example 2.

[0048] The sesquiterpene compound I prepared in Example 2 above was a white powder. Figure 1 The image shows the HR-ESI-MS spectrum of the compound of this invention, with the quasi-molecular ion peak at m / z 275.2376 [M+H]. + (The calculated molecular weight is 275.275.2369, C) 19 H 31 O), its molecular formula is determined to be C 19 H 30 O, with an unsaturation degree of 5. For example... Figure 4 As shown, compound I 1 The 1H NMR spectral data (Table 1) show the presence of three distinct olefin protons, located at δ1000 ppm and δ1000 ppm respectively. H 5.07 (1H, br s, H-4), δ H 7.15 (1H, d, J = 16.6 Hz, H⁻¹¹), and δ H 5.97 (1H, d, J = 16.6 Hz, H-12); In addition, doublet signals of three groups of methyl groups were observed, located at δ H 0.94(3H,d,J=7.0Hz,H-2'), δ H 0.80 (3H, d, J = 6.8 Hz, H-7') and δ H 0.85 (3H, d, J = 6.5 Hz, H-9'); and singlets for the three methyl groups, located at δ H 1.01(3H,s,H-1'), δ H1.61(3H,s,H-3') and δ H 2.21 (3H, s, H-14). Furthermore, a comprehensive analysis of the one-dimensional carbon spectrum of this compound was conducted. 13 C NMR (such as Figure 5 ), DEPT (e.g.) Figure 6 ) and HSQC spectrum (e.g. Figure 8 Compound I showed 19 carbon signals, including two methylene carbons located at δ. C 46.9 (C-8) and δ C 42.1(C-10); Five methyl carbon signals, located at δ C 49.2(C-2), δ C 39.6 (C-5), δ C 47.3 (C-6), δ C 34.8 (C-7) and δ C 32.5(C-9); a saturated quaternary carbon signal is located at δ C 41.3(C-1); An unsaturated quaternary carbon signal is located at δ C 135.3 (C-3); and a carbonyl carbon signal in a low-field region, located at δ C 198.0 (C-13). These data indicate that compound I contains two conjugated C=C double bonds and a carbonyl group, which contribute three degrees of unsaturation, while the other two degrees of unsaturation are attributed to the two fully saturated rings. Figure 2 As shown, the UV spectrum exhibits maximum absorption at 211 nm, indicating the presence of a conjugated π system in compound I. Figure 3 As shown, in the infrared spectrum at 1700 cm⁻¹ -1 Up to 1725cm -1 The characteristic absorption bands between them further confirmed the presence of the carbonyl group.

[0049] Table 1. Compound I 1 H and 13 C NMR data (600MHz, 150MHz, DMSO-d6)

[0050]

[0051] Note 1: s—single peak, d—double peak, q—quartet, m—multiple peak, overlapped—overlapped, br s—broad single peak.

[0052] Note 2: 1 H was obtained by 600MHz NMR; 13 C was obtained by 150MHz NMR.

[0053] Detailed analysis of the correlation between one-dimensional and two-dimensional NMR spectra further confirmed the planar structure of compound I. For example... Figure 7 As shown, 1 H- 1 In the H COSY spectrum, δ H 0.76 and δ H The cross signal between 1 and 80 indicates that the compound possesses a saturated closed ring structure CH(5)-CH(6)-CH(7)-CH2(8)-CH(9)-CH2(10)-CH(5), confirming the connection relationships between H-7 and H-7', and H-9 and H-9'. Figure 9 As shown in the HMBC spectrum, the correlations between H-7' and C-6 / C-7 / C-8, H-9' and C-8 / C-9 / C-10, and H-4 and C-5 / C-6 / C-10 support the attribution of two methyl groups (C-7' and C-9') and one olefin carbon (C-4). H-10a (δ... H 0.76, overlapped) and H-10b (δ H The correlation between 1.72,m) and C-6 / C-8, H-2 (δ) H The correlation between H-14 (1.45, q, J = 7.2, 6.6 Hz) and C-1 / C-2' / C-3' / C-3 / C-4 / C-6 further confirmed the presence of two six-membered rings (ring A and ring B) in the compound and clarified the positions of the two methyl groups (C-2' and C-3'). Combined with the analysis of unsaturation, this confirmed that compound I contains a two-ring structure. Furthermore, the HMBC correlation between H-14 and C-12 / C-13, the correlation between H-11 and C-1 / C-1' / C-6 / C-13, and the correlation between H-11 (δ... H 7.15, d, J = 16.6 Hz) and H-12 (δ H 5.97, d, J = 16.6 Hz) These two hydrogen atoms are in 1 H- 1 The correlation in the H COSY spectrum further confirmed a methyl (C-1') and a methylpropenone structural unit, which was attributed to the unit attached to C-1 (see schematic diagram of COSY and HMBC signals for compound I). Figure 11 (As shown).

[0054] The relative configuration of compound I was derived through analysis of coupling constants and NOESY spectra. The coupling constants of H-11 (J = 16.6 Hz) and H-12 (J = 16.6 Hz) indicate that the double bond is in trans configuration, with the two hydrogen atoms located on opposite sides of the double bond, corresponding to the E configuration. Figure 10As shown, the cross peaks between H-11 and H-6 / H-7' / H-9', H-12 and H-1' / H-2 / H-5 / H-7 in the NOESY spectrum, and the coupling constant (J = 7.00 Hz) between H-2 and H-2', indicate that there is an inverse fusion between ring A and ring B, establishing the β orientation of Me-2', Me-7', Me-9', and H-6, and the α orientation of Me-1', H-2, H-5, H-7, and H-9 (e.g., ...). Figure 12 (As shown).

[0055] The electronic excited states of compound I were predicted using a quantum chemical method based on TD-DFT, and its rotational intensity and transition energy were calculated. Two possible absolute configurations were proposed: (1S, 2R, 5S, 6R, 7S, 9R) and its enantiomers (1R, 2S, 5R, 6S, 7R, 9S). Quantum chemical TD-DFT calculations were then performed on these two structures to predict their ECD spectra. Figure 13 As shown, the calculated ECD curves for the (1R,2S,5R,6S,7R,9S) configuration agree well with the experimental results. Therefore, the structure of compound I was constructed.

[0056] Example 4: Antiepileptic activity of sesquiterpene compound I prepared by the method of Example 2.

[0057] 1. Preparation of experimental sample solutions: Pentyltetrazine (PTZ) was used as an epilepsy inducer to establish a zebrafish inflammation model. The test sample was the purified compound I isolated in Example 2 above. An appropriate amount of sample was accurately weighed and prepared into a solution of the required concentration (5 mg / L) with DMSO for testing antiepileptic activity. The zebrafish used in this experiment were housed in a flow-through tank, with the water temperature maintained at 28 ± 0.5℃ and a photoperiod of 14 hours light: 10 hours darkness. The zebrafish were fed newly hatched brine shrimp.

[0058] 2. Experimental Methods: Zebrafish larvae, 6 days post-fertilization, were randomly assigned to experimental groups, with 100 larvae in each group. The experimental groups included a control group, a PTZ treatment group, a compound I treatment group (5 mg / L), and a carbamazepine (CBZ) positive control group (50 μM CBZ). Except for the control group, all experimental groups were pre-treated with 10 mM PTZ for 30 minutes, and then the corresponding compound I and positive control CBZ were added to the compound treatment group and the positive control CBZ treatment group. The zebrafish larvae were placed in a dark environment for 10 minutes to acclimatize, followed by 15 minutes of behavioral recording. Behavioral data were analyzed using EthoVision XT 10.0 software, with the main parameters of interest being walking distance and epileptic-like behavior.

[0059] In gene expression analysis, 30 zebrafish larvae were randomly collected from each group and homogenized. Total RNA was extracted using an animal total RNA purification kit based on a centrifuge column. Subsequently, 1.0 μg of RNA was reverse transcribed into cDNA using cDNA synthesis premix. Quantitative real-time PCR (qPCR) was performed on a qTOWER 3G instrument using rapid SYBR mix to measure relative mRNA levels, with elongation factor 1α (ef1α) as an internal control gene. The qPCR cycling conditions were: initial denaturation at 94 °C for 180 seconds, followed by 42 cycles, each consisting of 5 seconds of denaturation at 94 °C and 30 seconds of annealing at 60 °C. Melting curve analysis was performed in increments of 1 °C from 60 °C to 95 °C. The primer sequences for the target genes are shown in Table 1.

[0060] Table 1. Primer sequences of epilepsy-related target genes used in the study

[0061]

[0062] Acetylcholinesterase (AChE) activity was detected according to the kit instructions. Total protein was extracted from 60 zebrafish embryos in each group, and protein quantification was performed using Coomassie Brilliant Blue G-250. Absorbance was measured on a PerkinElmer multi-plate reader, with each sample measured in triplicate. All data are expressed as mean ± standard error (SEM), and statistical analysis was performed using GraphPad Prism 8.0 software. Statistical significance was assessed by unpaired t-tests, with a significance level set at p < 0.05.

[0063] 3. Experimental Results: The antiepileptic activity of compound I in the zebrafish model is as follows: Figure 14 As shown in the figure, the horizontal axis 1 represents the compound.

[0064] (1) Behavioral improvement: such as Figure 14 As shown in A, B, C, D, and E, compound I effectively alleviated PTZ-induced hyperactivity in zebrafish, including reducing parameters such as total distance traveled, average speed, frequency of high-altitude movement, and tortuosity. The motor performance was almost restored to a level comparable to or even better than that of the CBZ-positive control group, indicating a significant reduction in seizure-related hyperactivity.

[0065] (2) Increase acetylcholinesterase (AChE) activity: such as Figure 14 As shown in Figure F, compared with the blank control group, compound I significantly increased AChE activity in zebrafish, suggesting that it may exert its anti-epileptic effect by regulating the cholinergic pathway. The increase in AChE activity was significantly different from that in the positive control group (p<0.05). Compared with the control group, p # <0.05 (significant), compared with the model group, p* <0.05 (significant).

[0066] (3) Regulating the expression of related genes: such as Figure 14 As shown in Figures G, H, and I, in the PTZ-induced epilepsy model, compound I effectively reversed the upregulation of c-fos and gria1b genes and the downregulation of gat1 gene, restoring the expression levels of these genes to near the control group. This indicates that compound I helps to rebalance excitatory glutamate and inhibitory GABAergic neurotransmission, maintain normal neuronal excitability, and prevent epileptic seizures. Compared with the control group, p # <0.05 (significant), compared with the model group, p * <0.05 (significant).

[0067] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A sesquiterpene compound with antiepileptic activity, characterized in that... This compound is a sesquiterpene compound derived from marine fungi, and its structural formula is shown in (I). （I）。 2. The method for preparing a sesquiterpene compound with antiepileptic activity as described in claim 1, characterized in that... Includes the following steps: (1) Fermentation production Aspergillus pyrolyticus with accession number CCTCC NO: M2014086 was inoculated onto a plate containing PDA solid medium and activated by inverted culture for 3 days at 28°C. The activated single colony was then transferred to PDB liquid medium that had been autoclaved and cooled, and cultured with shaking at 28°C and 150 rpm for 4 days to obtain seed culture. 5-7 mL of seed culture was inoculated into rice medium and incubated at 28°C for 30 days to obtain the fermentation product. (2) Extraction of extract Ethyl acetate was added to the fermentation product obtained in step (1), and the extraction was repeated 3 to 4 times. The ethyl acetate extract was then evaporated by rotary evaporation to obtain a crude extract. (3) Isolation, purification and preparation of compounds First, the crude extract obtained in step (2) was fully dissolved in a mixture of dichloromethane and methanol in a volume ratio of 1:

1. Then, 200-300 mesh silica gel was added and the mixture was stirred. Normal-phase medium-pressure column chromatography was performed, using a mixture of petroleum ether and ethyl acetate in a volume ratio of 2:3 as the eluent. The eluent was collected and mixed with 100-200 mesh reversed-phase silica gel. Then, reversed-phase medium-pressure column chromatography was performed, using a methanol-water eluent with a methanol volume percentage of 20-100% for linear gradient elution for 120 min. The eluted fractions were collected and arranged in descending order of polarity, and combined to obtain 7 components. The 7th component was purified by semi-preparative reversed-phase high-performance liquid chromatography using a mixture of acetonitrile and water in a volume ratio of 50:50 as the mobile phase to obtain compound I, the structure of which is shown in (I). （I）。 3. The method for preparing a sesquiterpene compound with antiepileptic activity according to claim 2, characterized in that... The method for preparing PDA solid culture medium in step (1) is as follows: Boil 200g of peeled and diced potatoes for 30 minutes, filter, retain the filtrate, add 20g of glucose and 20g of agar to the filtrate, heat until completely dissolved, cool to below 50℃, adjust the volume to 1000mL with distilled water, autoclave at 121℃ for 20 minutes, cool to 45-50℃, pour into sterile petri dishes, and wait for it to solidify before use.

4. The method for preparing a sesquiterpene compound with antiepileptic activity according to claim 2, characterized in that... The preparation method of PDB liquid culture medium in step (1) is as follows: Take 200g of peeled and diced potatoes, add 1000mL of distilled water and boil for 30min, filter and retain the filtrate, add 20g of glucose to the filtrate, stir until completely dissolved, adjust the volume to 1000mL with distilled water, autoclave at 121℃ for 15-20mL and then cool.

5. The method for preparing a sesquiterpene compound with antiepileptic activity according to claim 2, characterized in that... The method for preparing the rice culture medium in step (1) is as follows: Take 100g of rice, add 110mL of distilled water and 3g of sea salt, put the mixture into a culture bottle, and then sterilize it by high pressure steam at 121℃ for 20min and then cool it.

6. The method for preparing a sesquiterpene compound with antiepileptic activity according to claim 2, characterized in that: The flow rate for the semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) preparation of compounds in step (3) is 2.0 mL / min.

7. Use of the sesquiterpene compound of claim 1 in the preparation of an antiepileptic drug.

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