Acorus calamus stem-derived fungus and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a sea calamus stem-derived fungus and application thereof. According to the invention, a novel fungus strain Aspergillus fumigiffinis SYSU-6786 is obtained by separating the stem part of Semen Acori graminei, and the fermentation metabolite of the fungus strain is purified, so that the active super-sulfur compound is obtained. Research shows that the super-sulfur compound can remarkably promote proliferation and migration of human skin keratinocytes, shows a good skin wound repairing effect and has the potential of being developed into a skin repairing medicine. The fungus strain can be cultured on a large scale through a modern fermentation technology, raw material supply is stable, the ecological environment is not damaged, and industrial production is easy. As a sustainable development marine microorganism resource, the fungus strain provides an important raw material basis for research and development of marine microorganism drugs and functional products, and has a wide application prospect in the field of biological medicines.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a fungus derived from the stem of *Acorus calamus* and its applications. Background Technology

[0002] Sulfur is an essential element for the human body, playing a crucial role in maintaining healthy hair, nails, and skin. In recent years, a class of "supersulfides," composed of multiple sulfur atoms connected to carbon and other elements at both ends, has become a research hotspot due to their unique biological activities. Recent studies have found that these compounds have antioxidant capabilities far exceeding those of vitamin E, and also exhibit significant anti-inflammatory, antiviral, and other diverse physiological functions (Tsutsuki H, Zhang T, Akaike T, Sawa T. Regulation of innate immune and inflammatory responses by supersulfides. Int Immunol. 2024; 36(4):143-154). More notably, supersulfides also show outstanding effects in maintaining joint homeostasis and promoting bone regeneration (Maemura M, Morita M, Ogata S, et al. Supersulfides contribute to joint homeostasis and bone regeneration. Redox Biol. 2025; 81:103545.), which opens up new prospects for their application in the field of tissue repair.

[0003] However, the current acquisition of supersulfur compounds mainly relies on two routes: natural extraction and total chemical synthesis. The former is limited by the scarcity of raw materials, resulting in insufficient yield, while the latter suffers from technical bottlenecks such as cumbersome synthesis steps and low yields. This insufficient supply severely restricts the in-depth development of related research and the promotion of practical applications. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the limited sources of existing supersulfur compounds and to provide a fungus derived from the stem of the sea calamus.

[0005] The purpose of this invention is to provide the application of fungi derived from the stems of *Acorus calamus* in the preparation of supersulfur compounds.

[0006] Another object of the present invention is to provide the application of the fungus derived from the stem of the calamus in the preparation of a skin damage repair drug.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention protects a fungus derived from the stem of *Aspergillus fumigatiaffinis*, named *Aspergillus fumigatiaffinis* SYSU-6786, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 18, 2025, with accession number GDMCC No: 66541.

[0009] Furthermore, the fungus derived from the stem of *Acorus calamus* was isolated from the stem of *Acorus calamus* growing in seagrass beds of mangroves in the South China Sea, obtained by sampling during a scientific expedition to the South China Sea by Sun Yat-sen University.

[0010] Preferably, the culture medium for the fungi derived from the stem of the sea calamus is one or more of PDB medium, PDA medium, and rice medium.

[0011] Preferably, the culture conditions for the fungi derived from the stem of the sea calamus are cultured at 25-30°C for 3-5 days.

[0012] This invention protects the use of fungi derived from the stem of *Acorus calamus* in the preparation of supersulfur compounds, which have skin damage repair effects.

[0013] Preferably, the supersulfur compound comprises one or more having the structures shown in formula (I) and formula (II):

[0014]

[0015] This invention protects the use of fungi derived from the stem of *Acorus calamus* in the preparation of drugs for repairing skin damage.

[0016] Furthermore, the skin injury includes physical injury and / or chemical injury.

[0017] Furthermore, the physical injuries include mechanical injuries (such as abrasions, cuts, contusions, fractures, lacerations, punctures, and gunshot wounds), temperature-related injuries (burns, frostbite), and electrical injuries.

[0018] Furthermore, the chemical damage includes corrosive damage.

[0019] Furthermore, the repair is to promote the repair and / or regeneration of skin wounds or scars.

[0020] Specifically, the repair is to promote the migration and / or proliferation of HaCaT cells in the keratinocytes of the skin.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention isolated a novel fungal strain, *Aspergillus fumigatiaffinis* SYSU-6786, from the stem of *Acorus gramineus*. The fermentation metabolites of this strain were purified to obtain an active supersulfur compound. Studies have shown that the supersulfur compound significantly promotes the proliferation and migration of human skin keratinocytes, exhibiting excellent skin wound repair efficacy and possessing the potential to be developed into a skin repair drug. This fungal strain can be cultivated on a large scale using modern fermentation technology, ensuring a stable supply of raw materials without disrupting the ecological environment, and is easy to industrialize. As a sustainably developed marine microbial resource, this fungal strain provides an important raw material basis for the research and development of marine microbial drugs and functional products, and has broad application prospects in the biomedical field. Attached Figure Description

[0023] Figure 1 This is a plate colony diagram of fungi derived from the stem of *Acorus calamus* in Example 1 of the present invention.

[0024] Figure 2 This is a microscopic morphological image of Aspergillus fumigatiaffinis SYSU-6786, a fungus derived from the stem of Acorus calamus in Example 1 of this invention.

[0025] Figure 3 This is the carbon NMR spectrum of the supersulfur compound (I) obtained in Example 2 of the present invention.

[0026] Figure 4 This is the 1H NMR spectrum of the supersulfur compound (I) obtained in Example 2 of the present invention.

[0027] Figure 5 This is the carbon NMR spectrum of the supersulfur compound (II) obtained in Example 2 of the present invention.

[0028] Figure 6 This is the 1H NMR spectrum of the supersulfur compound (II) obtained in Example 2 of the present invention.

[0029] Figure 7 This is a statistical diagram (LSD, Mean±SD) of the cell scratch area after the supersulfur compound (I) obtained in Example 3 of the present invention was applied to human skin keratinocytes HaCaT cells.

[0030] Figure 8 This is a statistical diagram (LSD, Mean±SD) of the cell scratch area after the supersulfur compound (II) obtained in Example 3 of the present invention was applied to human skin keratinocytes HaCaT cells.

[0031] Figure 9 This is a diagram showing the changes in cell scratches after the supersulfur compound (I) obtained in Example 3 of this invention was applied to human skin keratinocytes (HaCaT cells).

[0032] Figure 10 This is a diagram showing the changes in cell scratches after the supersulfur compound (II) obtained in Example 3 of this invention was applied to human skin keratinocytes (HaCaT cells).

[0033] Figure 11 The cell activity statistics (K test, Mean±SD) of human skin keratinocytes after the supersulfur compounds (I) and (II) obtained in Example 3 of this invention were applied to human skin keratinocytes. Compared with the cn group, * indicates significant upregulation and # indicates significant downregulation (p<0.05). Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] PDB medium (short for Potato Dextrose Broth), commercially available.

[0037] PDA medium (short for Potato Dextrose Agar (Medium)) was purchased commercially and prepared according to the instructions. It was then sterilized in an autoclave at 121°C (0.1 MPa) for 25 minutes. Before it solidified, the plates were poured out and cooled to room temperature to obtain PDA culture plates for later use.

[0038] Seed culture medium: Prepared by adding 30g of sea salt and 24g of PDB culture medium powder per liter of water, sterilized in an autoclave at 121℃ (0.1MPa) for 25 minutes, and cooled to room temperature for later use.

[0039] The fermentation medium is rice culture medium, with the following composition: 50g rice, 50mL 3wt% sea salt water, mixed in a culture bottle, sealed, sterilized at 121℃ (0.1MPa) for 25min in a high-temperature sterilizer, and cooled to room temperature for later use.

[0040] Example 1: Obtaining Aspergillus fumigatiaffinis SYSU-6786, a fungus derived from the stem of Acorus calamus.

[0041] The method for obtaining the fungus Aspergillus fumigatiaffinis SYSU-6786 from the stem of the sea calamus is as follows:

[0042] 1. The fungus originating from the stem of *Acorus calamus* is an isolation of *Aspergillus fumigatiaffinis* SYSU-6786.

[0043] Wash the surface of the calamus stem with sterile water, then chop the stem and inoculate it into a PDA culture plate. Incubate at 25°C for 3-5 days. Pick fungal colonies with different morphologies and transfer them to a new PDA culture plate. Repeat the previous operation multiple times until a colony with a single morphology is obtained. Send the culture medium with a single colony to Beijing Qingke Biotechnology Co., Ltd. for identification and ITS sequencing.

[0044] 2. Identification of Aspergillus fumigatiaffinis SYSU-6786

[0045] (1) Culture characteristics, microscopic examination and morphological features

[0046] This strain is best cultured at 25–30°C with a humidity of around 80%. The colonies are fluffy or somewhat fibrous, darkening in color upon maturity and producing spores. The colonies turn bluish-gray, with conidia having columnar heads and flask-like apical structures (e.g., ...). Figures 1-2 (As shown).

[0047] (2) ITS gene sequence identification

[0048] The ITS-rRNA gene fragment sequence of this strain was obtained by sequencing, as shown in SEQ ID NO: 1, and is as follows:

[0049] .

[0050] After molecular biological identification, it was classified as Aspergillus fumigatiaffinis SYSU-6786 and deposited on June 18, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 66541.

[0051] Example 2 Extraction and characterization of supersulfur compounds (I) and (II)

[0052] This invention utilizes the fermentation broth of the fungus strain Aspergillus fumigatiaffinis SYSU-6786, derived from the stem of Acorus calamus, to isolate and purify supersulfur compounds (I) and (II). The specific acquisition process is as follows:

[0053] S1. Inoculate the fungus Aspergillus fumigatiaffinis SYSU-6786 into the seed culture medium and culture it on a shaker (25℃, 150-180rpm) to obtain the seed culture solution;

[0054] S2. Inoculate the seed culture medium obtained in step S1 into the fermentation medium and culture it at room temperature for 5 weeks (i.e. 35 days) to obtain the fermentation product.

[0055] S3. Soak the fermentation product obtained in step S2 in methanol for 3 days, filter, take the filtrate and concentrate under reduced pressure and extract with ethyl acetate, take the upper organic layer and concentrate under reduced pressure to obtain crude extract.

[0056] S4. The crude extract obtained in step S3 was adsorbed onto a silica gel column (specifically a Purospher STAR RP-18 reverse silica gel column, 10×30cm) and sequentially eluted with methanol-water solutions of volume ratios of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0. The fraction with a methanol-water volume ratio of 30:70 was collected.

[0057] S5. The methanol-water fraction obtained in step S3 with a volume ratio of 30:70 is separated and purified by high-performance liquid chromatography (HPLC). The HPLC conditions are as follows: mobile phase: acetonitrile-water solution containing 0.1 vol% acetic acid (the volume ratio of acetonitrile to the water solution containing 0.1 vol% acetic acid is (15-45):(85-55), and the acetonitrile content increases uniformly within 0-40 min); column: semi-preparative column Ultimate XB-C18, 10×250 mm, 5 μm; instrument: Essentia LC-16; flow rate: 3 mL / min; detection wavelength: 254 nm; components with retention times of 0-40 min are collected. The first peak appearing within the provided retention time (0-40 min) is supersulfur compound (I), and the third peak is supersulfur compound (II). That is, this invention distinguishes the supersulfur compounds by the elution order of the chromatographic peaks, wherein supersulfur compound (I) has a shorter retention time and elutes before supersulfur compound (II). Within a defined retention time range, the components corresponding to each chromatographic peak can be collected separately, and the structures of the obtained components can then be confirmed by nuclear magnetic resonance (NMR) technology.

[0058] The obtained supersulfur compound (I) was a yellow solid. Nuclear magnetic resonance (NMR) analysis of it yielded the following results: Figures 3-4 As shown, the physicochemical properties of this compound, as determined by analysis and testing, are as follows:

[0059] NMR data: 13C NMR(150MHz,DMSO-d6)δ:35.7,55.7,59.5,60.3,61.3,64.9,71.8,73.6,76.4 ,86.5,103.0,116.5,125.6,128.5,129.8,135.7,147.7,153.1,162.4,163.4.

[0060] 1 H NMR (600MHz, DMSO-d6) δ: 9.46 (s, 1H), 9.28 (d, J = 3.2Hz, 1H), 7.62 (d, J = 9.0Hz, 1H), 6.52 (d ,J=9.0Hz,1H),5.50(dt,J=2.5,10.3Hz,1H),5.43(d,J=10.3Hz,1H),5.29(d,J=5.2Hz,1H) ,5.28(s,1H),5.08(d,J=6.6Hz,1H),4.69(d,J=3.2Hz,1H),4.33(m,1H),4.20(m,1H),4.01 (d,J=7.1Hz,1H),3.78(s,3H),3.67(s,3H),2.43(d,J=15.2Hz,1H),1.96(d,J=15.2Hz,1H).

[0061] Analysis of the nuclear magnetic resonance (NMR) results confirmed the molecular formula of the obtained compound to be: C 20 H 22 N2O9S3 has the structure of formula (I):

[0062]

[0063] The obtained supersulfur compound (II) was a light yellow solid. Nuclear magnetic resonance (NMR) analysis of it yielded the following spectrum: Figures 5-6 As shown, the physicochemical properties of this compound, as determined by analysis and testing, are as follows:

[0064] NMR data: 13 C NMR(150MHz,DMSO-d6)δ:35.8,56.2,60.3,64.8,69.4,71.6,73.4,76.5,86.9 ,97.7,105.8,117.7,118.2,128.6,129.6,132.7,150.1,153.9,161.3,163.2.

[0065] 1H NMR(600MHz,DMSO-d6)δ:10.35(s,1H),6.73(d,J=8.2Hz,1H),6.59(d,J=8.2Hz ),5.50(s,1H),5.49(dt,J=10.4,2.5Hz,1H),5.43(d,J=10.4Hz,1H),5.33(d,J =5.4Hz,1H),5.29(d,J=6.6Hz,1H),4.29(m,1H),4.19(m,1H),4.03(d,J=7.1Hz ,1H),3.77(s,3H),3.73(s,3H),2.44(d,J=15.2Hz,1H),2.04(d,J=15.2Hz,1H).

[0066] Analysis of the nuclear magnetic resonance (NMR) results confirmed the molecular formula of the obtained compound to be: C 20 H 20 N2O9S3 has the structure of formula (II):

[0067]

[0068] Example 3: Activity Test of Supersulfur Compounds

[0069] 1. Experimental materials:

[0070] 12-well plates for counting, trypsin, double antibiotics (streptomycin / penicillin), fetal bovine serum, incubator, PBS buffer, cell counting chamber.

[0071] 2. Experimental methods:

[0072] (1) Cell culture: HaCaT cells were cultured in DEME medium containing double antibiotics (streptomycin / penicillin, 1%) and fetal bovine serum (10%). The culture conditions were 37°C and 5% CO2 constant temperature incubator.

[0073] (2) Cell passage: When the cell density reaches 80% to 100%, remove the culture medium, wash gently with preheated PBS, add 2 mL of 0.05% trypsin to digest for 2 to 5 minutes until the cell edges become rounded or partially and completely detach from the culture dish, add 5 mL of fresh complete culture medium to stop digestion, centrifuge at 1000 rpm for 2 minutes, discard the supernatant, add fresh complete culture medium to resuspend and passage culture at an appropriate ratio.

[0074] (3) Scratch test

[0075] DMSO was used as a solvent to prepare 10 mM mother liquors of supersulfur compound (I) and supersulfur compound (II).

[0076] Cells were seeded at a density of 15 mg / well in 12-well plates and incubated overnight in a 5% CO2 incubator at 37°C. The next day, cells were streaked, and each well was gently washed with 500 μL of pre-warmed PBS, followed by photographic recording. Using DMEM medium, 10 mM stock solutions of supersulfur compound (I) and supersulfur compound (II) were diluted to final concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM, and added to the 12-well plates, with photographic recording. After 48 h of incubation, the plates were photographed again.

[0077] (4) MTT cell viability / toxicity assay

[0078] Cells were seeded at a density of 5000 cells / well in 96-well plates (margin wells were filled with sterile PBS) and incubated overnight in a 5% CO2, 37°C incubator. The next day, the culture medium was discarded, and the cells were carefully rinsed 1-2 times with PBS. 100 μL of a compound diluted with DMEM medium was added to each experimental well (the compound was diluted with DMSO at a stock concentration of 10 mM, ensuring a final DMSO concentration <1%). Only 100 μL of DMEM medium was added to each negative control well (this was the untreated group, or CN group). Cells were incubated at this temperature for 48 hours, then culture medium containing 1% MTT (5 mg / mL) was added. After another 4 hours of incubation in the dark, the culture medium was carefully aspirated from each well, and 150 μL of DMSO was added to each well. The plates were shaken at 300 rpm for 10 minutes, and the absorbance was measured at 570 nm. Cell viability was calculated as [(experimental wells - blank wells) / (negative control wells - blank wells)] × 100%. The wells include negative control wells (containing cell-rich culture medium and MTT solution) and blank wells (containing no cells or test material, only culture medium, MTT solution, and DMSO).

[0079] 3. Experimental Results:

[0080] Cell scratch assay results are as follows Figures 7-10 As shown, two days after administration, the final concentrations of supersulfur compound (I) at 6.25 μM and 12.5 μM promoted the proliferation and migration of human keratinocytes (HaCaT cells), and the final concentrations of supersulfur compound (II) at 6.25 μM, 12.5 μM and 25 μM also promoted the proliferation and migration of human keratinocytes (HaCaT cells).

[0081] Depend on Figure 11 It is evident that supersulfur compound (I) at a final concentration ≤12.5 μM and supersulfur compound (II) at a final concentration ≤50 μM did not exhibit cytotoxicity against human keratinocyte HaCaT cells. Furthermore, supersulfur compound (II) significantly improved the survival rate of HaCaT cells at concentrations of 3.125 μM, 6.25 μM, and 12.5 μM.

[0082] In summary, this invention isolated a novel fungal strain, *Aspergillus fumigatiaffinis* SYSU-6786, from the stem of *Acorus gramineus*. The fermentation metabolites of this strain, after purification, yielded an active supersulfur compound. The above studies demonstrate that the supersulfur compound significantly promotes the proliferation and migration of human skin keratinocytes, possessing the potential to be developed into a skin repair drug. This fungal strain can be cultivated on a large scale using modern fermentation technology, ensuring a stable supply of raw materials without disrupting the ecological environment, and is easily suitable for industrial production. As a sustainably developed marine microbial resource, this fungal strain provides an important raw material basis for the research and development of marine microbial drugs and functional products, and has broad application prospects in the biomedical field.

[0083] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fungus derived from the stem of *Acorus calamus*, characterized in that, The fungus derived from the stem of the sea calamus is named Aspergillus fumigatiaffinis SYSU-6786, and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 18, 2025, with accession number GDMCC No: 66541.

2. The fungus derived from the stem of *Acorus calamus* according to claim 1, characterized in that, The *Acorus calamus* stem-derived fungus was isolated from the stem of *Acorus calamus* growing in seagrass beds of mangroves in the South China Sea.

3. The fungus derived from the stem of *Acorus calamus* according to claim 1, characterized in that, The culture medium for fungi derived from the stem of the calamus is one or more of PDB medium, PDA medium, and rice medium.

4. The fungus derived from the stem of *Acorus calamus* according to claim 1, characterized in that, The culture conditions for the fungi derived from the stem of the calamus were as follows: cultured at 25–30°C for 3–5 days.

5. The use of the fungus derived from the stem of *Acorus calamus* according to any one of claims 1 to 4 in the preparation of supersulfur compounds, characterized in that... The supersulfur compound has skin damage repair properties.

6. The application according to claim 5, characterized in that, The supersulfur compound includes one or more having the structures shown in formula (I) and formula (II):

7. The use of the fungus derived from the stem of *Acorus calamus* as described in any one of claims 1 to 4 in the preparation of a skin damage repair drug.

8. The application according to claim 5 or 7, characterized in that, The skin damage includes physical damage and / or chemical damage.

9. The application according to claim 5 or 7, characterized in that, The repair is to promote the repair and / or regeneration of skin wounds or scars.

10. The application according to claim 5 or 7, characterized in that, The repair is to promote the migration and / or proliferation of HaCaT cells in the keratinocyte epithelium of the skin.

Images