A heteroterpene compound from pythiopsora genus, a preparation method and application in anti-inflammation

Abstract

The application belongs to the technical field of pharmaceutical compounds, and particularly relates to a heteroterpene compound from a fungus of the genus Pythiopsis, a preparation method and application in anti-inflammation. Dothiorella A series of new heteroterpene compounds are extracted from fermentation broth of the fungus of the genus Pythiopsis (Pythiopsis sp.) QQYZ-1. In vitro cell experiments prove that the compounds have significant anti-inflammatory activity. The compounds are novel in structure, and the anti-inflammatory activity is reported for the first time. The compounds are potential anti-inflammatory drug lead compounds, and provide a new direction and selection for development of anti-inflammatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical compound technology, specifically relating to a heteroterpenoid compound derived from the genus *Cryptococcus*, its preparation method, and its application in anti-inflammatory effects. Background Technology

[0002] Inflammation is a biological response of the immune system. It is both a key barrier to maintaining homeostasis and a core driving factor for many chronic diseases. It can be triggered by various factors, including pathogens, damaged cells, and toxic compounds. These factors can induce acute or chronic inflammatory responses in the heart, pancreas, liver, kidneys, lungs, brain, intestines, and reproductive system, leading to tissue damage and even disease. Inflammation is typically characterized by redness, swelling, heat, and pain, and is mainly divided into two categories: acute inflammation and chronic inflammation. Acute inflammation-related conditions include acute pancreatitis, acute kidney injury, and acute asthmatic inflammation; chronic inflammation-related conditions include psoriasis, rheumatoid arthritis, and ankylosing spondylitis.

[0003] Since the discovery and application of the anti-inflammatory drug aspirin over a century ago, the range of anti-inflammatory drugs available in clinical practice has become increasingly diverse. For example, non-terpenoid anti-inflammatory drugs (such as etoricoxib) that can reduce gastrointestinal reactions and specifically act on cyclooxygenase-2 (COX-2), corticosteroids, statins, and some monoclonal antibody drugs have all been used to treat common inflammatory diseases and have effectively relieved patients' pain.

[0004] Inflammation is a dynamic physiological process in which pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and vascular endothelial growth factor (VEGF) play a central role. Currently, anti-cytokine therapy is a common treatment for autoimmune diseases; for example, natezumab, developed for psoriasis, exerts its anti-inflammatory effect by blocking inflammatory factors. Small molecule inhibitors targeting pathway signals can inhibit common inflammatory pathways such as oxidative stress, JAK / STAT, NF-κB, MAPK, and inflammasomes. However, currently used anti-inflammatory drugs generally have side effects or drug resistance issues, making the development of novel, safe, low-toxicity, or even non-toxic anti-inflammatory drugs an urgent priority.

[0005] Natural products are an important source for the development of drugs and drug lead compounds. Statistics show that from January 1981 to September 2019, 1394 small molecule drugs were approved for marketing by the U.S. Food and Drug Administration (FDA), of which 441 were derived from natural products and their derivatives, accounting for approximately 31.6% of the total. Microbial natural products, due to their unique advantages such as short culture cycles, high reproducibility, and abundant secondary metabolites, have become a hot area of ​​current natural product research. The chemical composition of microbial natural products encompasses terpenes, alkaloids, macrolides, polyketides, cyclic peptides, etc., and they possess a wide range of biological activities, including anti-inflammatory, antitumor, antiviral, antimalarial, and antibacterial activities. Therefore, discovering novel and highly active anti-inflammatory lead compounds from microbial natural products has significant research value and application prospects. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention first provides a heteroterpene compound derived from the genus *Cryptocoryne*, characterized in that the structural formula of the heteroterpene compound is shown in Formula A: A; In the formula, R1 is Cl, Br or H. When R1 is Cl, R2 is CH3. When R1 is Br, R2 is CH3 or H. When R1 is H, R2 is CH3.

[0007] The present invention further provides a method for preparing heteroterpenoid compounds derived from fungi of the genus *Cryptocoryne*, wherein the heteroterpenoid compounds are from the genus *Cryptocoryne* (…). Dothiorella (sp.) Secondary metabolites of fungus QQYZ-1, which was deposited on September 19, 2025 at Guangdong Microbial Culture Collection Center, located at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with accession number GDMCC No:67002.

[0008] Furthermore, the preparation method includes the following steps: S1. Fungal culture: After activating the target fungus, it was inoculated into PDB medium and cultured in a constant temperature shaker at 27℃ for 3-5 days to obtain seed liquid. The seed liquid was transferred to rice medium and cultured at room temperature for 28±2 days to obtain fermentation broth. S2. Crude extraction: After fermentation, methanol solution was added to the rice culture medium and soaked for 24 h. Then, the extraction was carried out by filtration three times. The filtrates were combined and concentrated to obtain crude extract. Then, it was extracted with ethyl acetate three times and concentrated under reduced pressure to obtain ethyl acetate extract. S3. Separation and purification: The extract was mixed with silica gel and separated using a normal-phase silica gel column with gradient elution using ethyl acetate-petroleum ether as the starting eluent to obtain fractions Fr.1-Fr.10, wherein... 1) Collect the eluted fraction Fr.2 and further separate it by silica gel column chromatography. The eluent is dichloromethane-methanol with a volume ratio of 200:1. After spotting, select the fraction that absorbs at a wavelength of 254 nm and turns gray after being developed with concentrated vanillin sulfuric acid. Combine and concentrate to obtain compound 1. 2) Collect the eluted fraction Fr.3 and further separate it using a silica gel column with dichloromethane-methanol at a volume ratio of 150:1. The purified product was further purified using a Sephadex LH-20 gel chromatography column with dichloromethane-methanol at a volume ratio of 1:1. The fraction that absorbs at a wavelength of 254 nm and turns gray after being developed with concentrated vanillin sulfate was selected, and the fractions were combined and concentrated to obtain compounds 2 and 3. 3) Collect the eluted fraction Fr.4 and further separate it using a silica gel column with dichloromethane-methanol at a volume ratio of 100:1. The purified product was further purified using a Sephadex LH-20 gel chromatography column with dichloromethane-methanol at a volume ratio of 1:1. The fraction that absorbs at a wavelength of 254 nm and turns gray after being developed with concentrated vanillin sulfate was selected, and the fractions were combined and concentrated to obtain compound 4. Compound 1 is a heteroterpene compound with R1 being Cl and R2 being CH3; compound 2 is a heteroterpene compound with R1 being Br and R2 being CH3; compound 3 is a heteroterpene compound with R1 being Br and R2 being H; and compound 4 is a heteroterpene compound with R1 being H and R2 being CH3.

[0009] Preferably, in step S1, the rice culture medium is prepared by mixing rice, NaCl, NaBr and water in a ratio of 100 g : 3 g : 3 g : 80 mL.

[0010] Preferably, in step S3, gradient elution is performed using a system of 0 / 1 ethyl acetate-petroleum ether: 1 / 0 ethyl acetate-petroleum ether. The second component Fr.2 is obtained by elution in a 2 / 8 ethyl acetate-petroleum ether system, the third component Fr.3 is obtained by elution in a 3 / 7 ethyl acetate-petroleum ether system, the fourth component Fr.4 is obtained by elution in a 4 / 6 ethyl acetate-petroleum ether system, and the fifth component Fr.5 is obtained by elution in a 1 / 1 ethyl acetate-petroleum ether system.

[0011] This invention further provides the application of heteroterpenoid compounds derived from the genus *Cryptocera* as described above in the preparation of anti-inflammatory drugs.

[0012] The present invention provides an anti-inflammatory drug comprising a pharmaceutically effective dose of a heteroterpenoid compound derived from a fungus of the genus *Cryptocoryne* as described in claim 1.

[0013] Preferably, the heteroterpenoids derived from the fungi of the genus *Cryptocoryne* account for 60-90% of the total mass or volume of the drug.

[0014] Preferably, the heteroterpenoids derived from the fungi of the genus *Cryptocoryne* account for 62%, 65%, 70%, 72%, 75%, 78%, 80%, 85%, and 88% of the total mass or volume of the drug.

[0015] Preferably, the anti-inflammatory drug further includes a pharmaceutically acceptable carrier.

[0016] Preferably, the pharmaceutically acceptable carrier includes one or more excipients with functions such as excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents; such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.

[0017] Preferably, the drug is any one of injection, tablet, granule, pill, capsule, suspension or emulsion.

[0018] The beneficial effects of this application are as follows: 1. This application is based on fungi of the genus *Sclerotium* (…). Dothiorella A series of novel heteroterpenoid compounds, Phomernoids, were successfully extracted, isolated, and identified from the fermentation broth of *Phomernoids* sp. In vitro cell experiments demonstrated that these compounds possess significant anti-inflammatory activity. These compounds exhibit novel structures, and their anti-inflammatory activity is reported for the first time. They represent promising lead compounds for anti-inflammatory drugs, providing new directions and options for the development of anti-inflammatory drugs.

[0019] 2. An LPS (lipopolysaccharide)-induced mouse macrophage RAW264.7 cell model showed that the heteroterpenoid compounds 1-4 provided in this application effectively inhibited NO release, with an IC50 value of [missing information]. 50 The values ​​ranged from 5.8 to 12.1 μM, with compound 2 having an IC50 value of [missing value]. 50 The concentrations were 5.8 μM, which was stronger than the positive control drug (L-NMMA: 17.75 μM). Furthermore, compound 2 significantly inhibited the expression of inflammatory cytokines TNF-α, IL-1β, and IL-6, indicating that the heteroterpenoid compounds provided in this application have the potential to be used as anti-inflammatory drugs.

[0020] 3. The heteroterpenoids provided in this application are secondary metabolites of fungi in the genus *Cryptocoryne*, which can be prepared by microbial fermentation. The preparation method is simple, has a short cycle, mild culture conditions, few byproducts, and low cost. It can be obtained by unlimited fermentation, is not limited by resource shortages, is green and environmentally friendly, and is easy to industrialize. It not only meets the needs of modern green, low-carbon and environmentally friendly economy, but also provides a new way for the discovery of potential anti-inflammatory drugs. Attached Figure Description

[0021] Figure 1 Compound 1 provided in this application 1 H NMR spectrum.

[0022] Figure 2 Compound 1 provided in this application 13 C NMR spectrum.

[0023] Figure 3 Compound 1 provided in this application 1 H- 1 H COSY spectrum.

[0024] Figure 4 The HSQC spectrum of compound 1 provided in this application.

[0025] Figure 5 The HMBC spectrum of compound 1 provided in this application.

[0026] Figure 6 The NOESY spectrum of compound 1 provided in this application.

[0027] Figure 7 The HR-ESI-MS spectrum of compound 1 provided in this application.

[0028] Figure 8 Compound 2 provided in this application 1 H NMR spectrum.

[0029] Figure 9 Compound 2 provided in this application 13 C NMR spectrum.

[0030] Figure 10 Compound 2 provided in this application 1 H- 1 H COSY spectrum.

[0031] Figure 11 The HSQC spectrum of compound 2 provided in this application.

[0032] Figure 12 The HMBC spectrum of compound 2 provided in this application.

[0033] Figure 13 The NOESY spectrum of compound 2 provided in this application.

[0034] Figure 14 The HR-ESI-MS spectrum of compound 2 provided in this application.

[0035] Figure 15 Compound 3 provided in this application1 H NMR spectrum.

[0036] Figure 16 Compound 3 provided in this application 13 C NMR spectrum.

[0037] Figure 17 Compound 3 provided in this application 1 H- 1 H COSY spectrum.

[0038] Figure 18 The HSQC spectrum of compound 3 provided in this application.

[0039] Figure 19 The HMBC spectrum of compound 3 provided in this application.

[0040] Figure 20 The NOESY spectrum of compound 3 provided in this application.

[0041] Figure 21 The HR-ESI-MS spectrum of compound 3 provided in this application.

[0042] Figure 22 Compound 4 provided in this application 1 H NMR spectrum.

[0043] Figure 23 Compound 4 provided in this application 13 C NMR spectrum.

[0044] Figure 24 Compound 4 provided in this application 1 H- 1 H COSY spectrum.

[0045] Figure 25 The HSQC spectrum of compound 4 provided in this application.

[0046] Figure 26 The HMBC spectrum of compound 4 provided in this application.

[0047] Figure 27 The NOESY spectrum of compound 4 provided in this application.

[0048] Figure 28 The HR-ESI-MS spectrum of compound 4 provided in this application.

[0049] Figure 29 This is a schematic diagram of the spatial structure of compounds 1-4 provided in this application.

[0050] Figure 30 ECD spectra of compounds 1-4 provided in this application.

[0051] Figure 31 The effects of compounds 1-4 provided in this application on the viability of RAW264.7 cells, where ns indicates non-toxicity. p <0.05, p < 0.01, p < 0.0001. Detailed Implementation

[0052] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art.

[0053] The technical solution of the present invention will be described in more detail below with reference to the embodiments.

[0054] Example 1

[0055] Extraction of heteroterpenoids

[0056] 1. Fungi of the genus *Cryptocoryne* ( Dothiorella Acquisition and fermentation culture of sp.

[0057] Roots of *Kandelia candel* were obtained from the Dongzhaigang Mangrove Nature Reserve in Haikou City. After rinsing with sterile water, the roots were surface-sterilized in 75% ethanol for 1 minute in a 200 mL beaker. The samples were then immersed in 5% sodium hypochlorite for 1 minute, followed by rinsing with sterile water. The roots were cut into 3 mm segments and inoculated onto potato dextrose agar (PDA) containing penicillin (100 IU / mL) and streptomycin (0.08 mg / mL). The culture dishes were incubated at 25 ± 1 °C. Ten endophytic fungi were isolated and identified through transfer purification and were stored in triplicate on PDA slant agar at 4 °C. The strain labeled QQYZ-1 showed vigorous growth on PDA plates, exhibiting a brown, velvety appearance and abundant secondary metabolites, and was selected as the target strain for further research. The target strain was identified using a molecular biology protocol involving DNA amplification and ITS region sequencing. BLAST search results showed that the sequence had the highest sequence similarity (100%) to the genus *Cryptocoryne* (compared to PQ559842.1), thus identifying it as belonging to the genus *Cryptocoryne*. Dothiorella The fungus (sp.) was named QQYZ-1, and its gene sequence information is shown in the sequence listing SEQ ID NO:1.

[0058] The QQYZ-1 strain was deposited on September 19, 2025, at the Guangdong Microbial Culture Collection Center, located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No:67002.

[0059] The selected QQYZ-1 strain was activated and subjected to primary culture.

[0060] The activation method is as follows: inoculate QQYZ-1 strain onto PDA medium and incubate at room temperature for 3 days; The primary culture is as follows: the activated strain is inoculated onto PDB medium and cultured in a shaker (160 r / min) at 27°C for 4-5 days to obtain fungal seed culture.

[0061] The PDA medium formula is: potato extract 300.0 g / L, glucose 20.0 g / L, agar 15.0 g / L, and chloramphenicol 0.1 g / L.

[0062] The PDB medium formula is: potato extract powder 300.0 g / L, glucose 20.0 g / L.

[0063] Then, fermentation culture was carried out: an appropriate amount of fungal seed liquid was transferred to rice culture medium (60×1000 mL conical flask, each flask containing 100 g of rice, 3 g of NaCl, 3 g of NaBr and 80 mL of water), and placed at room temperature for 25±1 days to obtain fermentation broth.

[0064] 2. Isolation and purification of compounds 1-4

[0065] After fermentation, methanol solution was added to the conical flask and soaked for 24 hours before extraction. The extraction was repeated three times. After filtration, the organic phases were combined and concentrated to obtain a crude extract, which is the secondary metabolite of the strain. Then, the extract was extracted three times with ethyl acetate and concentrated under reduced pressure to obtain the ethyl acetate fraction, which is the lipid-soluble part of the secondary metabolite.

[0066] The extract was mixed with silica gel and separated using a normal-phase silica gel column with gradient elution using ethyl acetate-petroleum ether as the starting eluent (petroleum ether / ethyl acetate: 10 / 0–0 / 10) to obtain fractions Fr.1–Fr.10. 1) The second fraction Fr.2 was obtained by elution in a 2 / 8 ethyl acetate-petroleum ether system. Fr.2 was further separated by silica gel column chromatography with dichloromethane-methanol in a volume ratio of 200:1. After spotting, the fraction that absorbed at a wavelength of 254 nm and turned gray after being developed with concentrated vanillin sulfuric acid was selected, and the fractions were combined and concentrated to obtain compound 1. 2) The fraction Fr.3 obtained by elution in a 3 / 7 ethyl acetate-petroleum ether system was further separated by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 150:1. The purified product was further purified by Sephadex LH-20 gel chromatography with dichloromethane-methanol at a volume ratio of 1:1. The fraction that absorbed at a wavelength of 254 nm and turned gray after being developed with concentrated vanillin sulfate was selected, and the fractions were combined and concentrated to obtain compounds 2 and 3. 3) The fraction Fr.4 obtained by elution in a 4 / 6 ethyl acetate-petroleum ether system was further separated by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 100:1. The purified product was further purified by Sephadex LH-20 gel chromatography with dichloromethane-methanol at a volume ratio of 1:1. The fraction that absorbed at a wavelength of 254 nm and turned gray after being developed with concentrated vanillin sulfate was selected, and the fractions were combined and concentrated to obtain compound 4. 3. Characterization of compounds 1-4 The instruments used for identification included a Bruker 500 MHz nuclear magnetic resonance spectrometer, a Thermo Scientific Q Exactive Orbitrap triple quadrupole high-resolution mass spectrometer, and CDCl3 as the deuterated reagent.

[0067] The structures of compounds 1-4 were determined by NMR and HR-ESI-MS spectra. Compound 1... 1 H NMR spectrum, 13 CNMR spectrum, 1 H- 1 For H COSY, HSQC, HMBC, NOESY, and HR-ESI-MS spectra, please refer to [reference needed]. Figures 1-7 Compounds 2-4 1 H NMR spectrum, 13 C NMR spectrum, 1 H- 1 For H COSY, HSQC, HMBC, NOESY, and HR-ESI-MS spectra, please refer to [reference needed]. Figures 8-28 Among them, using NMR ( 1 H, 13 C) The basic skeleton and molecular formula of the compound were determined by high-resolution mass spectrometry (HR-ESI-MS), the connection relationship between protons and carbons was confirmed by COSY and HMBC, the relative configuration was determined by NOESY spectrum, and the absolute configuration was determined by theoretical calculation (ECD, electronic circular dichroism).

[0068] The specific allocation (assignment) of all NMR experimental data (chemical shift, J value, etc.) is shown in Table 1.

[0069] Table 1. NMR data (CDCl3, TMS, ppm) of compounds 1-4

[0070]

[0071] See Figure 29 , Figure 30Finally, the structures of compounds 1-4 were confirmed as shown in Formula A, wherein compound 1 is a heteroterpene compound with R1 being Cl and R2 being CH3; compound 2 is a heteroterpene compound with R1 being Br and R2 being CH3; compound 3 is a heteroterpene compound with R1 being Br and R2 being H; and compound 4 is a heteroterpene compound with R1 being H and R2 being CH3.

[0072] A.

[0073] Example 2

[0074] Anti-inflammatory activity studies of compounds 1-4

[0075] Experimental materials: mouse macrophage cell line RAW 264.7 cells, fetal bovine serum (FBS), penicillin-streptomycin mixture, DMEM high glucose medium, carbon dioxide incubator, centrifuge, electrophoresis apparatus, real-time quantitative PCR instrument, inverted microscope, cell culture plates, lipopolysaccharide, PBS buffer, thiazolyl blue, cell lysis buffer, NO reagent kit, etc.

[0076] 1. Cell experiments

[0077] Cell culture: Mouse macrophage cell line RAW 264.7 was cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin, and incubated at 37 ℃ in a 5% CO2 incubator. When the cells reached 70%-80% confluence, they were passaged at a ratio of 1:3, and cells in the logarithmic growth phase were used for experiments.

[0078] 1) Cell viability assay: Macrophages in the logarithmic growth phase were digested, counted, and seeded into 96-well plates, 1×10⁶ cells per well. 4 Cells were cultured for 20 hours, and the original culture medium was aspirated. A blank control group and drug treatment groups were set up. The blank control group received normal complete culture medium, while the drug treatment groups received DMEM containing 50, 25, 12.5, 6.25, and 3.125 μM of drug, respectively. Each group had 6 replicates, and the experiment was repeated 3 times. After 48 hours of treatment, the supernatant was aspirated from each treatment group, and then 10 μL of basal culture medium containing MTT (0.5 µg / ml) was added. The cells were incubated at 37°C for 4 hours, and then the supernatant was carefully aspirated. Then, 10 μL of DMSO was added to each well, and the cells were shaken on a shaker for 10 min in the dark. The OD value at 490 nm was measured using a microplate reader. The effect of the drug on the viability of RAW 264.7 macrophages was investigated.

[0079] See results Figure 31Compared to the control group, compounds 2 and 3 showed no cytotoxicity at a concentration of 25 μM, compound 1 did not significantly reduce cell viability at 50 μM, and compound 4 was at a safe concentration of 12.5 μM. Compounds 1-3 showed no toxic effect on RAW264.7 macrophages at a concentration of 6.25 µM. Therefore, the maximum concentrations of compounds 1-4 were selected as 25, 25, 25, 50, 12.5, and 50 μM for subsequent experiments.

[0080] 2) Nitric oxide (NO) content determination: The nitric oxide content in cell supernatant was determined using the nitrate reduction method. Logarithmic growth phase mouse RAW 264.7 cells were seeded into 24-well plates and cultured for 24 h. Compounds containing different concentration gradients were added, and 1 h later, LPS was added to each well. The experiment was divided into three groups: a blank control group (fresh culture medium only), an LPS model group (fresh culture medium + LPS 1 μg / mL), and a control group (fresh culture medium + LPS 1 μg / mL). μ The test compound group (compound diluted in fresh medium + LPS 1 μg / mL) was stimulated with LPS for 24 h, and then 200 µL of medium containing different concentrations of the compound (3.125~50 µM) or L-NMMA (3.125~50 µM) / LPS (1 µg / mL) was added, and the mixture was incubated again for 24 h. After incubation, 50 µL of the supernatant from each well was transferred to another 96-well plate, and 50 µL of NO kit solution (Beyotime, Shanghai, China) I (Sulfanilamide) and N-(1-naphthyl)ethylenediamine dihydrochloride were added sequentially.

[0081] Collect and centrifuge the cell supernatant, and use a nitric oxide assay kit to detect and calculate the concentration of NO in the cell supernatant.

[0082] The detection steps were performed according to the reagent instructions: Griess Reagent I and II and cell supernatant were removed and allowed to return to room temperature. (1) Prepare a standard curve: Dilute the standard concentrations to 1, 5, 10, 20, 40, 60, and 100 μM with fresh culture medium. (2) Add the standard and cell supernatant to each well of a 96-well plate at a concentration of 50 μL. (3) After returning to room temperature, add 50 μL of Griess Reagent I and 50 μL of Griess Reagent II, respectively. (4) Measure the absorbance at 540 nm using a microplate reader. (5) Calculate the NO concentration in the cell supernatant based on the standard curve.

[0083] Referring to Table 2, at the experimental concentrations, compounds 1-4 and L-NMMA (positive control, 17.75 µM) showed significant inhibitory activity against LPS-induced NO release from RAW264.7 macrophages. Compounds 1-4 exhibited superior inhibitory activity compared to the positive control drug L-NMMA. Compound 2 showed the most significant inhibitory effect, with an IC50 concentration of [missing value]. 50 The values ​​are 5.8 μM.

[0084] Table 2. Inhibitory activity of compounds 1-4 against NO

[0085] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A heteroterpene compound derived from a fungus of the genus *Cryptocoryne*, characterized in that, The structural formula of the heteroterpenoid compound is shown in formula (A): （A） In the formula, R1 is Cl, Br, or H; when R1 is Cl, R2 is CH3; when R1 is Br, R2 is CH3 or H; when R1 is H, R2 is CH3.

2. The method for preparing heteroterpenoid compounds derived from *Cryptocoryne* fungi as described in claim 1, characterized in that, The heteroterpenoids are from the genus *Cryptococcus* ( Dothiorella (sp.) Secondary metabolites of fungus QQYZ-1, which was deposited at Guangdong Microbial Culture Collection Center on September 19, 2025, with accession number GDMCC No:67002.

3. The preparation method according to claim 2, characterized in that, The preparation method includes the following steps: S1. Fungal culture: After activating the QQYZ-1 strain, it was inoculated into PDB medium and cultured in a constant temperature shaker at 27℃ for 3-5 days to obtain seed liquid. The seed liquid was transferred to rice medium and cultured at room temperature for 28±2 days to obtain fermentation broth. S2. Crude extraction: After fermentation, methanol solution was added to the rice culture medium and soaked for 24 h. Then, the extraction was carried out by filtration three times. The filtrates were combined and concentrated to obtain crude extract. Then, it was extracted with ethyl acetate three times and concentrated under reduced pressure to obtain ethyl acetate extract. S3. Separation and purification: The extract was mixed with silica gel and separated using a normal-phase silica gel column with gradient elution using ethyl acetate-petroleum ether as the starting eluent to obtain fractions Fr.1-Fr.10, wherein... 1) Collect the eluted fraction Fr.2 and further separate it by silica gel column chromatography. The eluent is dichloromethane-methanol with a volume ratio of 200:

1. After spotting, select the fraction that absorbs at a wavelength of 254 nm and turns gray after being developed with concentrated vanillin sulfuric acid. Combine and concentrate to obtain compound 1. 2) Collect the eluted fraction Fr.3 and further separate it using a silica gel column with dichloromethane-methanol at a volume ratio of 150:

1. The purified product was further purified using a Sephadex LH-20 gel chromatography column with dichloromethane-methanol at a volume ratio of 1:

1. The fraction that absorbs at a wavelength of 254 nm and turns gray after being developed with concentrated vanillin sulfate was selected, and the fractions were combined and concentrated to obtain compounds 2 and 3. 3) Collect the eluted fraction Fr.4 and further separate it using a silica gel column with dichloromethane-methanol at a volume ratio of 100:

1. The purified product was further purified using a Sephadex LH-20 gel chromatography column with dichloromethane-methanol at a volume ratio of 1:

1. The fraction that absorbs at a wavelength of 254 nm and turns gray after being developed with concentrated vanillin sulfate was selected, and the fractions were combined and concentrated to obtain compound 4. Compound 1 is a heteroterpene compound with R1 being Cl and R2 being CH3; compound 2 is a heteroterpene compound with R1 being Br and R2 being CH3; compound 3 is a heteroterpene compound with R1 being Br and R2 being H; and compound 4 is a heteroterpene compound with R1 being H and R2 being CH3.

4. The method for preparing heteroterpenoid compounds derived from *Cryptocoryne* fungi as described in claim 3, characterized in that, In step S1, the rice culture medium is prepared by mixing rice, NaCl, NaBr and water in a ratio of 100 g : 3 g : 3 g : 80 mL.

5. The method for preparing heteroterpenoid compounds derived from *Cryptocoryne* fungi as described in claim 3, characterized in that, In step S3, a gradient elution is performed using a system of 0 / 1 ethyl acetate-petroleum ether: 1 / 0 ethyl acetate-petroleum ether. The second component Fr.2 is obtained by elution in a 2 / 8 ethyl acetate-petroleum ether system, the third component Fr.3 is obtained by elution in a 3 / 7 ethyl acetate-petroleum ether system, the fourth component Fr.4 is obtained by elution in a 4 / 6 ethyl acetate-petroleum ether system, and the fifth component Fr.5 is obtained by elution in a 1 / 1 ethyl acetate-petroleum ether system.

6. The use of a heteroterpene compound derived from a fungus of the genus *Cryptocoryne* as described in claim 1 in the preparation of an anti-inflammatory drug.

7. An anti-inflammatory drug, characterized in that, Contains a pharmaceutically effective dose of a heteroterpene compound derived from a fungus of the genus *Cryptocoryne* as described in claim 1.

8. The anti-inflammatory drug as described in claim 7, characterized in that, The heteroterpenoids derived from the fungi of the genus *Cryptocoryne* account for 60-90% of the total mass or volume of the drug.

9. The anti-inflammatory drug as described in claim 8, characterized in that, It also includes pharmaceutically acceptable carriers, which include carriers that function as one or more of the following: excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents.

10. The anti-inflammatory drug as described in claim 9, characterized in that, The drug is any one of the following: injection, tablet, granule, pill, capsule, suspension, or emulsion.

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