Preparation method and application of malus hallasisiana endophytic fungal metabolites

Abstract

The application discloses a preparation method and application of a malus sieversii endophytic fungus metabolite, and comprises the following steps: (1) inoculating a strain of the malus sieversii endophytic fungus CL-4 into a fermentation medium, and obtaining a fermentation liquor after fermentation culture; (2) filtering and separating the fermentation liquor to obtain a bacterial liquid and wet bacterial bodies, and obtaining a paste after extraction and concentration; (3) separation and purification: the paste is separated by adopting a silica gel column chromatography, a mixed liquid of petroleum ether and ethyl acetate is used as an eluent, the volume ratio of the petroleum ether to the ethyl acetate is 10-5:1, two column volumes are eluted, elution is collected, then a gel chromatography column is used, anhydrous methanol is used as an eluent to elute, elution of 10-15 hours is collected, and finally, the malus sieversii endophytic fungus metabolite is obtained by using semi-preparative liquid chromatography separation and purification. The malus sieversii endophytic fungus metabolite with a novel structure is found, and has unique anti-inflammatory activity, thereby providing a new way for research and development of new drugs.

Description

Technical Field

[0001] This invention relates to the field of natural compound production technology, and in particular to a method for preparing and applying metabolites of endophytic fungi from prickly pear. Background Technology

[0002] Rosa rugosa is a perennial deciduous shrub belonging to the genus Rosa in the family Rosaceae, widely distributed in the Yunnan-Guizhou Plateau and western Sichuan in my country. It is renowned for its exceptionally high vitamin C content (earning it the title of "King of Vitamin C") and its rich content of flavonoids, polysaccharides, and superoxide dismutase. Endophytic fungi within Rosa rugosa are considered a potentially vast "new continent of microorganisms" and a "treasure trove of natural products." Living for a long time in the unique internal environment of plants, these endophytic fungi have evolved unique metabolic pathways to adapt to survival, interact with the host, or defend against other microorganisms. They are capable of producing novel and diverse secondary metabolites, making them an important source for new drug development.

[0003] The prickly pear boasts a rich diversity of endophytic fungi. 401 strains of endophytic fungi have been reported isolated from the roots, stems, leaves, flowers, fruits, and seeds of the prickly pear, with some strains exhibiting significant antibacterial activity and possessing high development and utilization value. Compared to directly extracting active ingredients from plants (which may be affected by resources, season, and environment), producing target products using microbial fermentation technology offers advantages such as shorter cycle time, lower cost, ease of industrial scale-up, and no limitations imposed by natural conditions, making it an effective approach to protecting wild prickly pear resources and achieving sustainable development. Current research on "prickly pear endophytic fungi" focuses primarily on the isolation of endophytic fungal resources, with limited research on their metabolites. One paper reports the discovery of an antibacterial compound from these fungi. The research scope for prickly pear endophytic fungal metabolites is vast, with enormous potential for developing lead compounds. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying a metabolite of endophytic fungi from prickly pear. A novel structure of the metabolite of endophytic fungi from prickly pear has been discovered, which has unique anti-inflammatory activity, providing a new approach for the development of new drugs.

[0005] The technical solution adopted by this invention to solve its technical problem is: A method for preparing metabolites of endophytic fungi from prickly pear includes the following steps: (1) The endophytic fungus CL-4 of prickly pear was inoculated into the fermentation medium and fermented to obtain the fermentation broth; (2) The fermentation broth was filtered and separated to obtain bacterial liquid and wet bacterial cells. The bacterial liquid was extracted with ethyl acetate to obtain bacterial liquid extract. The wet bacterial cells were soaked in anhydrous methanol, concentrated under reduced pressure to remove methanol, and then extracted with ethyl acetate to obtain the bacterial cell extract. The bacterial broth extract and the bacterial cell extract were combined and concentrated under reduced pressure to remove ethyl acetate, yielding a paste. (3) Separation and purification: The paste was separated by silica gel column chromatography, using a mixture of petroleum ether and ethyl acetate at a volume ratio of 10⁻⁵:1 as the eluent. Two column volumes of eluent were collected, and then the eluent was loaded onto a gel chromatography column (using Sephadex LH-20 as the filter medium) and eluted with anhydrous methanol (flow rate 0.5 mL / min). The eluent was collected for 10-15 hours, and finally purified by semi-preparative liquid chromatography (flow rate 3.0 mL / min, isocratic elution with 75% methanol, detection wavelength 210 nm) to obtain the metabolites of the prickly pear endophytic fungus. The obtained prickly pear endophytic fungal metabolites included two novel compounds, QCF-4 and QCF-5.

[0006] The endophytic fungus CL-4 of prickly pear, classified as Aspergillus japonicus, is deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 4, 2026, with accession number GDMCC No: 67878.

[0007] The metabolite of the endophytic fungus *Prickly pear* is QCF-4, and the chemical structural formula of QCF-4 is: .

[0008] The metabolite of the endophytic fungus *Prickly pear* is QCF-5, and the chemical structural formula of QCF-5 is: .

[0009] An anti-inflammatory drug comprising an effective dose of a metabolite of the endophytic fungus *Rhizopus pilosa*, and pharmaceutically acceptable excipients.

[0010] The metabolites of the endophytic fungi of prickly pear are QCF-4, QCF-5, or a combination of both.

[0011] The anti-inflammatory drug is prepared into an oral dosage form.

[0012] Preferably, the oral dosage form is a tablet, capsule, granule, powder, pill, oral liquid, or dry suspension.

[0013] The anti-inflammatory drug is prepared as an injectable dosage form.

[0014] Preferably, the dosage form for injection is an injection solution or a lyophilized powder for injection.

[0015] The beneficial effects of this invention are: this invention screened out new endophytic fungi of prickly pear, and through fermentation culture, discovered compounds with novel structures in their metabolites, which have unique anti-inflammatory activities, providing a new approach for the development of new drugs. Attached Figure Description

[0016] Figure 1 This is a diagram showing the results of CL-4 fungal PDA plate culture; Figure 2 This is a graph showing the effects of different concentrations of QCF-4 and QCF-5 on LPS-induced inflammatory responses. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0018] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0019] Example 1: Isolation of endophytic fungus CL-4 from prickly pear In July 2025, wild prickly pear samples were purchased from the Qixingguan District Farmers Market in Bijie City, Guizhou Province, as experimental materials.

[0020] Cell isolation: The prickly pear peel was disinfected and placed on a sterile fume hood tray. The peel was scraped off with a clean scraper, and the pulp was placed in a clean mortar and pestle. The pulp was ground, and the resulting liquid was inoculated onto PDA agar plates at dilutions of 1×, 10×, 100×, and 1000×. The plates were incubated statically at 30°C for 3 days. Single, scattered, uniformly colored cells were picked and inoculated a second time onto PDA plates. After 3 days of incubation, CL-4 fungus was obtained. Figure 1 Mycelia were extracted from PDA plates using toothpicks, and the cell walls were broken by liquid nitrogen grinding to extract the bacterial DNA. The DNA was identified by Qingke Biotechnology (Guangzhou, China) Co., Ltd. using 16S rDNA sequence analysis. The 16S rDNA sequence (SEQ ID No. 1) was compared with the GenBank database using the BLAST (nucleotide sequence comparison) method. The BLAST search results for CL-4 showed that the sequence of this strain had a 99.43% similarity to Aspergillus japonicus. CBS 114.51 (accession number 1448312), indicating that this strain is a new subspecies of Aspergillus japonicus. It was named CL-4, an endophytic fungus of prickly pear, and classified as Aspergillus japonicus. It is deposited at the Guangdong Provincial Microbial Culture Collection Center on March 4, 2026, with accession number GDMCC No: 67878.

[0021] SEQ ID No. 1: .

[0022] Example 2: Preparation of metabolites from endophytic fungi of Prickly Pear Take the CL-4 endophytic fungus strain of prickly pear from a -80℃ freezer, thaw it in an ice box, and after thawing, use a toothpick to pick the mycelium from the cryovial and inoculate it onto a PDA plate. Incubate at a constant temperature of 30℃ for 3 days. Pick the mycelium into PDB liquid medium and place it on a shaker at 30℃ and 280 rpm for 7 days. After 7 days, filter and separate the fermentation broth to obtain bacterial solution and wet mycelium. Extract the bacterial solution with ethyl acetate to obtain the bacterial solution extract. Soak the wet mycelium in anhydrous methanol, concentrate it under vacuum to remove methanol, and then extract it with ethyl acetate to obtain the mycelium extract. Combine the bacterial solution extract and the mycelium extract, concentrate them under vacuum to remove ethyl acetate, and obtain 9.7g of extract.

[0023] Separation and purification: The extract was separated by silica gel column chromatography (200-300 μm normal silica gel) using a petroleum ether:ethyl acetate mixture at a volume ratio of 5:1 as the eluent. The eluent was collected and then eluted onto a gel chromatography column (Sephadex LH-20 as the filter medium) using anhydrous methanol as the eluent (flow rate 0.5 mL / min). The eluent was collected for 10-15 hours and finally purified by semi-preparative liquid chromatography (column: YMC-Pack ODS-AQ, 10 mm × 250 mm, 5 μm; mobile phase: 75% methanol aqueous solution; flow rate 3.0 mL / min; detection wavelength 210 nm, LC-52, Saipu Ruisi (Beijing) Technology Co., Ltd.) to obtain the metabolite of the endophytic fungus *Rhizopus pilosa*: QCF-4 (19.8 mg, t R = 13.7 min), QCF-5 (21.4 mg, t R = 17.1 min).

[0024] Structural analysis: New compound QCF-4, C 23 H 34 O7, A pale yellow oily substance, 1 H NMR (400 MHz, CDCl3) δ H 5.79 (dt, J = 13.8, 6.2 Hz, 1H),5.37 (tdd, J = 23.6, 13.2, 5.6 Hz, 5H), 5.01 (q, J = 7.0 Hz, 1H), 3.81 (s, 3H), 3.64 (d, J = 17.2 Hz, 1H), 3.02 (d, J = 17.2 Hz, 1H), 2.88 (dd, J = 9.7, 5.8 Hz,1H), V), 2.34 (dt, J = 13.4, 6.7 Hz, 2H), 2.15 (dd, J = 10.9, 5.5 Hz, 2H), 2.11(d, J = 6.0 Hz, 1H), 2.04 (d, J = 7.0 Hz, 1H), 2.03 – 1.91 (m, 1H), 1.30 (ddt, J=14.8, 11.4, 7.7 Hz, 8H), 0.87 (m, 3H). 13 C NMR (150 MH Z , CDCl3) δ C : 175.11,175.11, 173.63, 135.18, 130.61, 129.14, 128.63, 128.00, 127.67, 79.90,75.80,53.69, 46.34, 40.62, 32.20, 31.64, 30.80, 29.43, 27.35, 26.57, 25.79, 22.63,14.20.

[0025] New compound QCF-5, C 23 H 36 O7, A pale yellow oily substance, 1 H NMR (400 MHz, CDCl3) δ H : 5.72 (m, 1H), 5.35 (dddd, J =38.8, 17.6, 13.4, 6.8 Hz, 3H), 4.99 (q, J = 7.0 Hz, 1H), 3.79 (s, 3H), 3.61 (d, J = 17.2 Hz, 1H), 3.13 (dq, J = 11.5, 6.6 Hz, 1H), 3.00 (d, J = 17.2 Hz, 1H), 2.87(dd, J = 9.7, 5.8 Hz, 1H), 2.33 (dt, J = 13.3, 6.7 Hz, 1H), 2.09 (t, J = 3.7 Hz,4H), 2.01–1.90 (m, 3H), 1.39 (t, J = 7.2 Hz, 2H), 1.29 (d, J = 11.9 Hz, 4H), 0.86(t, J = 6.6 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ C: 175.20, 174.62, 173.60, 135.33, 131.01, 128.27, 127.81, 79.97, 75.80, 53.59, 46.26, 40.57, 32.26, 31.97, 30.76, 29.73, 29.58, 29.39, 29.37, 27.36, 26.52, 22.74, 14.19.

[0026] 1 H and 13 The data corresponding to the C spectrum are detailed in the table below: .

[0027] QCF-4 Spectral Analysis: HSQC spectrum information confirms the presence of a long-chain structure containing 9 methylene groups and 6 olefinic carbons, confirming the correct conclusion from the C-ray spectral analysis. Analysis of the HMBC spectrum reveals a correlation between the carbonyl signal at C1 and C2, C3, and C4, with a relatively large chemical shift signal at these three carbon atoms, indicating a cyclic structure with C1. Combined with the mass spectrometry information showing an unsaturation degree of 7, the compound is presumed to form a five-membered lactone structure. The correlation between the methoxy group and C22 indicates that the methoxy group forms an ester group with the carbonyl carbon. No common correlation signals exist between C22, C1, and C20. However, similar signals exist between any two groups: H21 correlates with C20 and C22, and H2 further confirms a correlation only with C1 and C20, indicating that C2 is located at the end furthest from C22. C2 and C1 form a five-membered lactone ring structure, and only H2 correlates with C20, indicating the presence of a carbon atom between C2 and C20. Further investigation of the H2 signal reveals the presence of C19 between C2 and C20. C19 is a quaternary carbon, and H2 correlates with C1, C20, and C19. This confirms that C2 is adjacent to C19, and C19 is adjacent to C20 and C21. C22 forms a terminal ester group at one end and is adjacent to C20 at the other. The H4 signal correlates with C5 and C6, confirming the double bond is adjacent to the five-membered ring. The H3 signal only correlates with C5, confirming that C4 is adjacent to C5, and C5 forms a double bond with C6. The remaining signals showed no correlation with the carbonyl signal, and only the methylene hydrogen signal correlated with the alkenyl signal. This confirmed that C5-C18 is a long-chain structure. Starting from the signal correlation at the terminal H18 position, it was determined that C16 is not an alkenyl carbon. Double bonds were identified between C16 and C16 via H11 and H14, with positions at C12 and C13. A double bond was also identified between H11 and H8, with positions at C9 and C10. A signal correlated with C6 but not with C5, indicating the presence of C7 between C6 and C8. This was confirmed by the correlation with H7, showing that C7 and C8 are located between the two double bonds, and only these two carbons are present. By analyzing the NMR and mass spectrometry data, two hydroxyl groups were identified. Comparison of the HSQC of the quaternary carbon signal confirmed that one hydroxyl group is located at C20, forming a carboxyl group, and the other is located at the quaternary carbon C19 position. This confirmed the final structure.

[0028] Spectral Analysis of QCF-5: HSQC and HMBC spectra confirmed that the only differences between QCF-5 and QCF-4 are the positions and number of double bonds. Comparison of the HSQC and HMBC spectra of QCF-4 confirmed the presence of the five-membered lactone ring, with C5 connected to C4 and a double bond between C5 and C6. The right-hand structures of QCF-4 and QCF-5 are identical, with the only difference being the long chain structure after C6. Comparison of HMBC and HSQC spectra and proton NMR spectral differences revealed that QCF-5 lacks the double bonds at positions C12 and C13 found in compound QCF-4; otherwise, they are identical.

[0029] Example 3: Anti-inflammatory experiment RAW264.7 cells were seeded in 24-well plates, with 1 × 10⁶ cells per well. 5 Cells were incubated for 2 hours, followed by stimulation with LPS (lipopolysaccharide) (1 μg / ml) for 1 hour to establish an inflammation model simulating in vitro inflammatory responses. The experiment was divided into four groups: ① Control group: no LPS or drugs were added, representing the baseline state; ② LPS + blank control group: LPS was added to induce inflammation, but no drugs were added, representing the model group; ③ LPS + QCF-4 group, with three doses of QCF-4: 50 μM, 75 μM, and 100 μM; ④ LPS + QCF-5 group, with three doses of QCF-5: 50 μM, 75 μM, and 100 μM. QCF-4 and QCF-5 were incubated for 24 hours. Samples were then collected, proteins were extracted, and Western blotting was used to detect the protein expression of iNOS (inducible nitric oxide synthase), a key enzyme in the inflammatory response. β-tubulin was used as an internal control for standardization. Figure 2 The results showed that LPS stimulation significantly upregulated iNOS protein expression in RAW264.7 cells compared with the control group (p<0.001), indicating that the inflammation model was successfully established. Compared with the LPS-only treatment group, iNOS protein expression decreased to varying degrees after treatment with different concentrations of QCF-4 and QCF-5. Among them, QCF-4 inhibited iNOS expression in a concentration-dependent manner, with inhibition rates of 26.82%, 51.89%, and 62.30% at concentrations of 50, 75, and 100 μM, respectively. All concentration groups showed statistically significant differences compared with the LPS control group (50 μM: p<0.05; 75 μM and 100 μM: p<0.001). QCF-5 significantly inhibited iNOS expression at concentrations of 75 μM and 100 μM, with inhibition rates of 36.82% and 38.82%, respectively (75 μM: p<0.05; 100 μM: p<0.01). While the 50 μM QCF-5 treatment group showed a certain decreasing trend, the effect was not statistically significant (p>0.05). These results indicate that both QCF-4 and QCF-5 inhibit LPS-induced inflammatory responses, with QCF-4 exhibiting stronger and concentration-dependent anti-inflammatory activity.

[0030] Example 4: The difference between this embodiment and Embodiment 2 is that: Separation and purification: The extract was separated by silica gel column chromatography (200-300 micrometer normal silica gel) using a mixture of petroleum ether and ethyl acetate in a volume ratio of 10:1 as the eluent.

[0031] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for preparing metabolites of endophytic fungi from prickly pear, characterized in that, Includes the following steps: (1) The endophytic fungus CL-4 of prickly pear was inoculated into the fermentation medium and fermented to obtain the fermentation broth; (2) The fermentation broth was filtered and separated to obtain bacterial liquid and wet bacterial cells. The bacterial liquid was extracted with ethyl acetate to obtain bacterial liquid extract. The wet bacterial cells were soaked in anhydrous methanol, concentrated under reduced pressure to remove methanol, and then extracted with ethyl acetate to obtain the bacterial cell extract. The bacterial broth extract and the bacterial cell extract were combined and concentrated under reduced pressure to remove ethyl acetate, yielding a paste. (3) Separation and purification: The paste was separated by silica gel column chromatography, using a mixture of petroleum ether and ethyl acetate at a volume ratio of 10-5:1 as the eluent. Two column volumes were eluted, and the eluent was collected. Then, it was loaded onto a gel chromatography column and eluted with anhydrous methanol as the eluent. The eluent was collected for 10-15 hours, and finally, the metabolites of the endophytic fungus of Prickly pear were separated and purified by semi-preparative liquid chromatography.

2. The preparation method according to claim 1, characterized in that, The endophytic fungus CL-4 of prickly pear, classified as Aspergillus japonicus, is deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 4, 2026, with accession number GDMCC No: 67878.

3. The application of prickly pear endophytic fungal metabolites as raw materials for the preparation of anti-inflammatory drugs, characterized in that, The metabolite of the endophytic fungus *Prickly pear* is QCF-4, and the chemical structural formula of QCF-4 is: 。 4. The application of prickly pear endophytic fungal metabolites as raw materials for the preparation of anti-inflammatory drugs, characterized in that, The metabolite of the endophytic fungus *Prickly pear* is QCF-5, and the chemical structural formula of QCF-5 is: 。 5. An anti-inflammatory drug, characterized in that, It contains an effective dose of prickly pear endophytic fungal metabolites, as well as pharmaceutically acceptable excipients.

6. The anti-inflammatory drug according to claim 5, characterized in that, The metabolites of the endophytic fungi of prickly pear are QCF-4, QCF-5, or a combination of both.

7. The anti-inflammatory drug according to claim 5, characterized in that, The anti-inflammatory drug is prepared into an oral dosage form.

8. The anti-inflammatory drug according to claim 7, characterized in that, The oral dosage forms are tablets, capsules, granules, powders, pills, oral liquids, or dry suspensions.

9. The anti-inflammatory drug according to claim 5, characterized in that, The anti-inflammatory drug is prepared as an injectable dosage form.

10. The anti-inflammatory drug according to claim 9, characterized in that, The dosage form for injection is either an injection solution or a lyophilized powder for injection.

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