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Full Research Article

The Effect of Epigenetic Modifiers on the Production of Lipase-inhibiting Secondary Metabolites in Aspergillus fischeri VO1R, An Endophyte from Viola odorata

R
Ruziyeva Dilaram Mutalibovna1
G
Gulyamova Tashkhan Gafurovna1
Y
Yoldosheva Maftuna Mengbotayevna2,*
K
Kondrasheva Kseniya Valentinovna1
O
Odilova Xosiyat Abdusamat qizi1
A
Abdulmyanova Liliya Ilyasovna3
N
Nasmetova Saodat Mamajanovna3
T
Tangirov Roziboy Xudoyshukurovich4
1Institute of Microbiology, Academy of Sciences, Republic of Uzbekistan, 100128, Uzbekistan, Tashkent. Laboratory of Biochemistry and Biotechnology of Biologically Active Substances.
2University of Business and Science. Non-state Higher Education Institution Department of Natural Sciences Chilonzor dist., Yakkabog MFY Gavhar St., 1, Tashkent, Uzbekistan.
3Institute of Microbiology, Academy of Sciences, Republic of Uzbekistan, 100128, Uzbekistan, Tashkent.
4Termez University of Economics and Service, 190100, Republic of Uzbekistan, Termiz.

ABSTRACT

Background: This study investigated the impact of epigenetic modifiers (5-azacytidine, nicotinamide and quercetin) on the growth, secondary metabolite production and lipase-inhibitory activity of the endophytic fungus Aspergillus fischeri VO1R, isolated from the roots of Viola odorata. The study’s findings demonstrate that the addition of modifiers reduced overall biomass and metabolite production, while maintaining the initial, relatively high level of lipase-inhibitory activity. A pronounced effect on biomass production was observed with the use of 5-azacytidine. These results shed light on the potential of epigenetic regulators to activate silent genes and stimulate the synthesis of more effective inhibitors, thereby enhancing our understanding of natural enzyme inhibitors.

Methods: During the study the dishes were incubated for 7 days at 28°C. Cultivation was performed in liquid Czapek-Dox medium supplemented with 5-azacytidine, nicotinamide and quercetin at a final concentration of 100 µL on a shaker at 180 rpm and 28°C for seven days. The culture biomass was separated by centrifugation at 6,000 rpm, weighed and stored at -4°C until use.

Result: Thus, the specific activity of metabolites, or the proportion of inhibitory metabolites, increased in the presence of azacitidine, quercetin and nicotinamide at different times of cultivation. For example, with the addition of 5-azacytidine on the seventh day of cultivation, the inhibitory activity of the extract was 94.6% given that the amount of dry extract of metabolites was 14.9 mg. In comparison, in the control, the inhibitory activity was 91.5% and the amount of extract was almost three times greater (42 mg). Thus, a significant increase in the specific inhibitory activity of the compounds was produced in the presence of the epigenetic modifier.

 

INTRODUCTION

In recent years, the potential of endophytic fungi, residing in the tissues of medicinal plants, has been a subject of significant interest (Strobel, 2018; Aly et al., 2011; Tiwari and Bae, 2022; Slama et al., 2021; Burragoni and Jeon, 2021). These fungi have the remarkable ability to produce a diverse array of biologically active secondary metabolites with antibacterial, antifungal, antioxidant, cytotoxic and enzyme-inhibitory activity, including amylase, urease and lipase inhibitors (Meshram et al., 2018). This potential makes endophytic fungi a promising frontier for the development of new drugs to combat metabolic and infectious diseases. However, maintaining and enhancing productivity during cultivation under axenic conditions remains a key area of research (Brakhage and Schroeckh, 2011; Cichewicz, 2010; Henrikson et al., 2009; Islam et al., 2024).

For instance, the taxol-producing endophytic fungus Periconia sp. was isolated from the plant Torreya grandifolia. Transfer of the fungus to a semisynthetic medium resulted in a gradual decrease in taxol production until its level became minimal, although fungal growth was relatively unaffected. Taxol production was completely restored after the addition of a plant extract to the culture medium (Ben ami et al., 2010; Loura et al., 2023).

In addition to plant metabolites, the aim of inducing the biosynthesis of new or previously inactive secondary metabolites in endophytic fungi is to enhance the production of these compounds. In recent years, increasing attention has been paid to the use of epigenetic modifiers, such as histone deacetylase (HDAC) inhibitors, including 5-azacytidine and quercetin, as well as DNA methyltransferase inhibitors and nicotinamide, which serves as both a deacetylase inhibitor and a DNA methyltransferase (DNMT) inhibitor (Li et al., 1998). These compounds, added to the nutrient medium, alter the epigenetic status of the producer cells, which can lead to the activation of “silent” biosynthetic genes (Kim and Kaang, 2016).

Thus, epigenetic chromatin remodeling represents a promising strategy for expanding the metabolic potential of endophytes and producing new biologically active compounds (Xue et al., 2023; Verma et al., 2023).

In the course of screening endophytic fungi isolated from medicinal plants in Uzbekistan, several endophytes were identified that produced secondary metabolites inhibiting pancreatic lipase activity. The Aspergillus fischeri VO1R strain, isolated from Viola odorata and exhibiting lipase-inhibitory activity greater than 90%, was selected as the most active (Gulyamova et al., 2022). However, during submerged cultivation, a gradual decrease in secondary metabolite production was observed. In this regard, this study aimed to investigate the effect of plant extracts and epigenetic modifiers-5-azacytidine, nicotinamide and quercetin-on the production level of lipase-inhibitory metabolites by A.fischeri VO1R, as possible factors in increasing the metabolic potential of endophytes.

MATERIALS AND METHODS

Objects and subject of the study
 
The endophytic fungus Aspergillus fischeri VO1R, isolated from Viola odorata, was the subject of the study.
 
Isolation and cultivation of fungal endophytes
 
Individual endophyte colonies that grew after incubation were removed with a fine needle, transferred to agar tubes and incubated at 28°C for seven days. Endophytic fungi were isolated from Viola odorata roots, as described by Hazalin et al., (2009). The roots were surface disinfected using 70% ethanol, followed by a 1-3 minute ethanol treatment and washing with sterile water, the leaves were aseptically chopped into pieces no larger than 0.5 cm and placed in Petri dishes with Czapek-Dox agar containing chlortetracycline at a concentration of 50 mg/l and streptomycin sulfate at a concentration of 250 mg/l to suppress bacterial growth.

Czapek-Dox medium (g/l): NaNO3 - 2 g, MgSO4 - 0.5 g, KCL - 0.5 g, FeSO4 - 10 mg, KH2PO4 - 1 g, sucrose - 20 g, agar-agar - 20 g, distilled water. - up to 1000 ml, pH 6-6.5.

Extraction of metabolites from the biomass of the endophyte fungus A. fischeri VO1R was carried out according to the method of Hazalin et al., (2009).

Obtaining the plant extract
 
The V. rosea extract was obtained by maceration of 10 g of crushed (no more than 3 mm) crude biomass of fresh plants (stems, leaves) with 100 ml of 96% ethanol for 24 hours at room temperature on an orbital shaker at 180 rpm. After settling for 2 days at a temperature not exceeding 10°C, the extract was separated by filtration through filter paper (Whatman No. 1) to obtain a clear liquid. The extract was then evaporated on a rotary evaporator, diluted with sterile water to the required concentration and passed through a polytetrafluoroethylene syringe filter with a pore size of 0.45 μm. The resulting plant extract was added to the nutrient medium to a final volume ratio of 2.0% before inoculation (Qing et al., 2018).
 
The lipase-inhibitory activity of extracts
 
50 mg of lipase (Sigma, 100 U/ml) was suspended in 10 ml of Tris-HCl buffer containing 2.5 mM Tris and 2.5 mM NaCl, pH 7.4. The solution was vigorously shaken for 15 min, followed by centrifugation (4000 rpm for 10 min). The supernatant was collected and reused as the enzyme solution. Stock solutions of extracts and Xenical were prepared in DMSO at a concentration of 10 mg/mL. The final reaction mixture consisted of 875 μL buffer, 100 μL enzyme and 20 μL extract, which were pre-incubated for 5 min at 37°C (Shilpa and Shukla, 2025). Then, 10 μL of substrate (4-nitrophenyl palmitate, 10 mM in acetonitrile) was added. The optical density of the final mixture was measured using a SPECOL-1300 spectrophotometer at a wavelength of 405 nm after 5 minutes (Azmir et al., 2013). The assay was performed in triplicate and the percentage of inhibition was calculated using the formula: 
 
 
 
Where,
Ae (enzyme control) =  Optical density of the enzyme control (without inhibitor).
At (test sample) = Difference between the optical density of the test sample with and without the substrate.

RESULTS AND DISCUSSION

The effect of plant extracts on endophytes was observed by Zhao et al., (2013) when growing the endophytic fungus Colletotrichum gloeosporioides ES026, isolated from Huperzia serrata, which produces the acetylcholinesterase inhibitor hupercin A. It was found that at the seventh passage, growth and alkaloid content decreased. At the ninth passage, the mycelium turned from white to yellow, with mycelial yield and hupercin A content significantly lower than the initial values. To overcome this problem, cells were first grown in the host plant extract and then in basal or enriched media. This strategy helped maintain fungal viability and stable alkaloid production at the initial level for 3 years (Zhao et al., 2013). The addition of 2% host plant extract to the medium on the seventh day of A. fischeri cultivation resulted in noticeable morphological and cultural changes, including green pigmentation, hyphal thickening and compaction (Fig 1, 2).

Fig 1: Morfological structure of the endophytic fungus A. fischeri VO1R.



Fig 2: Morphological changes in the endophytic fungi A. fischeri VO1R (with addition of Viola odorata extract).



A slight increase in the mycelial mass of A.fischeri and the amount of extracted secondary metabolites increased twofold compared to the control. In contrast, the inhibitory activity of the extract remained at the initial level at 92% (Fig 3).

Fig 3: Effect of V. odorata plant extract (2%) on the inhibitory activity of A.fischeri VO1R.



Thus, in contrast to the data with Colletotrichum gloeosporioides ES026, under experimental conditions, host plant metabolites promote increased production of inhibitory metabolites by A. fischeri VO1R.

Epigenetic modifiers added to the endophyte culture medium help activate dormant or weakly active genes responsible for secondary metabolite production. These substances do not alter DNA but affect its activity by influencing chromatin structure, for example, by altering histone status or DNA methylation (Gupta et al., 2020; Verma et al., 2023).

5-azacytidine and quercetin were used as DNA methyltransferase inhibitors, while nicotinamide was used as a deacetylase inhibitor as epigenetic modifiers. The ability of 5-azacytidine to induce the expression of biosynthetic clusters in fungi, including the genera Aspergillus and some others, has been repeatedly noted in the literature (Ben Ami et al., 2010; Abdelhamid et al., 2025; Jia et al., 2024; Pissios, 2017). Nicotinamide acts as an inhibitor of NADz -dependent histone deacetylases, particularly enzymes of the SIRT (sirtuins) family, leading to histone hyperacetylation and transcriptional activation. Nicotinamide can also suppress the activity of DNA methyltransferases, making it a universal epigenetic modifier with a combined mechanism of action (Saldiyar et al., 2018). Quercetin is a natural flavonoid known as a selective inhibitor of DNMTs and histone deacetylases of classes I and II. Additionally, quercetin exhibits antioxidant and antimicrobial activity, which may contribute to further stimulation of stress-induced metabolism in endophytes (Zhu et al., 2025).

The effect of modifiers on growth and the formation of inhibitory metabolites was studied dynamically under submerged cultivation conditions of A.fischeri VO1R. As can be seen from the data presented below, the addition of epigenetic modifiers significantly reduced the biomass and secondary metabolite levels compared to the control throughout the entire cultivation period. At the same time, PL inhibitory activity remained at a level comparable to the control (Fig 4). Thus, the addition of epigenetic modifiers-nicotinamide (NAD), quercetin and 5-azacytidine-to the nutrient medium did not increase the amount of secondary metabolites produced by Aspergillus fischeri VO1R, but, on the contrary, significantly reduced their content. However, in the presence of 5-azacytidine, the inhibitory activity of the extracts in the dynamics on days 5-11 of cultivation is a significant value from 56.6 to 94.6% and secondary metabolites from 10.4 to 14.9 mg, in the presence of quercetin, the highest inhibitory activity is also observed on days 5-11 and is from 46 to 82% with a content of secondary metabolites from 7.5 to 14.8 mg, in the presence of nicotinamide, the highest inhibitory activity and metabolite content are observed on days 5-9. They are 76 to 84.9% and 12.7 to 14.3 mg, respectively.

Fig 4: Effect of epigenetic modifiers on growth, inhibitory activity and accumulation of secondary metabolites in A.fischeri VO1R during growth dynamics.


  
It should be noted that, unlike the control, the presence of modifiers did not show a direct dependence of inhibitory activity on the amount of secondary metabolites. Moreover, the inhibitory activity of the extracts was almost equal to that of the control extracts, but with a twofold lower amount of metabolites.

CONCLUSION

The obtained results indicate that the use of epigenetic modifiers (5-azacytidine, nicotinamide and quercetin) during the cultivation of the A. fischeri VO1R strain results in a decrease in total biomass and total yield of secondary metabolites compared to control conditions, while maintaining inhibitory activity at a level comparable to the control. A redistribution of the metabolic profile of the fungus toward an increase in the level of metabolites characterized by lipase-inhibitory activity could be proposed.

Among the epigenetic modifiers tested, treatment with 5-azacytidine resulted in the highest pancreatic lipase inhibitory activity, reaching 94.6%, compared with 91.5% in the control group. However, biomass production and, consequently, the yield of secondary metabolites were reduced relative to the control. Further studies of the composition and nature of the compounds produced are clearly needed to more thoroughly determine the effect of epigenetic modifiers on the metabolic spectrum.

ACKNOWLEDGEMENT

The authors express their gratitude to the leadership and scientific team of the Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan for collecting scientific sources for the article, processing them in-house and conducting scientific experiments.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
Our experiments did not involve animals and the experimental animal care committee approved the work and the animal care committee of the Technical University did not approve the work. Our article mainly focuses on plants.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

REFERENCES


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    Full Research Article

    The Effect of Epigenetic Modifiers on the Production of Lipase-inhibiting Secondary Metabolites in Aspergillus fischeri VO1R, An Endophyte from Viola odorata

    R
    Ruziyeva Dilaram Mutalibovna1
    G
    Gulyamova Tashkhan Gafurovna1
    Y
    Yoldosheva Maftuna Mengbotayevna2,*
    K
    Kondrasheva Kseniya Valentinovna1
    O
    Odilova Xosiyat Abdusamat qizi1
    A
    Abdulmyanova Liliya Ilyasovna3
    N
    Nasmetova Saodat Mamajanovna3
    T
    Tangirov Roziboy Xudoyshukurovich4
    1Institute of Microbiology, Academy of Sciences, Republic of Uzbekistan, 100128, Uzbekistan, Tashkent. Laboratory of Biochemistry and Biotechnology of Biologically Active Substances.
    2University of Business and Science. Non-state Higher Education Institution Department of Natural Sciences Chilonzor dist., Yakkabog MFY Gavhar St., 1, Tashkent, Uzbekistan.
    3Institute of Microbiology, Academy of Sciences, Republic of Uzbekistan, 100128, Uzbekistan, Tashkent.
    4Termez University of Economics and Service, 190100, Republic of Uzbekistan, Termiz.

    ABSTRACT

    Background: This study investigated the impact of epigenetic modifiers (5-azacytidine, nicotinamide and quercetin) on the growth, secondary metabolite production and lipase-inhibitory activity of the endophytic fungus Aspergillus fischeri VO1R, isolated from the roots of Viola odorata. The study’s findings demonstrate that the addition of modifiers reduced overall biomass and metabolite production, while maintaining the initial, relatively high level of lipase-inhibitory activity. A pronounced effect on biomass production was observed with the use of 5-azacytidine. These results shed light on the potential of epigenetic regulators to activate silent genes and stimulate the synthesis of more effective inhibitors, thereby enhancing our understanding of natural enzyme inhibitors.

    Methods: During the study the dishes were incubated for 7 days at 28°C. Cultivation was performed in liquid Czapek-Dox medium supplemented with 5-azacytidine, nicotinamide and quercetin at a final concentration of 100 µL on a shaker at 180 rpm and 28°C for seven days. The culture biomass was separated by centrifugation at 6,000 rpm, weighed and stored at -4°C until use.

    Result: Thus, the specific activity of metabolites, or the proportion of inhibitory metabolites, increased in the presence of azacitidine, quercetin and nicotinamide at different times of cultivation. For example, with the addition of 5-azacytidine on the seventh day of cultivation, the inhibitory activity of the extract was 94.6% given that the amount of dry extract of metabolites was 14.9 mg. In comparison, in the control, the inhibitory activity was 91.5% and the amount of extract was almost three times greater (42 mg). Thus, a significant increase in the specific inhibitory activity of the compounds was produced in the presence of the epigenetic modifier.

     

    INTRODUCTION

    In recent years, the potential of endophytic fungi, residing in the tissues of medicinal plants, has been a subject of significant interest (Strobel, 2018; Aly et al., 2011; Tiwari and Bae, 2022; Slama et al., 2021; Burragoni and Jeon, 2021). These fungi have the remarkable ability to produce a diverse array of biologically active secondary metabolites with antibacterial, antifungal, antioxidant, cytotoxic and enzyme-inhibitory activity, including amylase, urease and lipase inhibitors (Meshram et al., 2018). This potential makes endophytic fungi a promising frontier for the development of new drugs to combat metabolic and infectious diseases. However, maintaining and enhancing productivity during cultivation under axenic conditions remains a key area of research (Brakhage and Schroeckh, 2011; Cichewicz, 2010; Henrikson et al., 2009; Islam et al., 2024).

    For instance, the taxol-producing endophytic fungus Periconia sp. was isolated from the plant Torreya grandifolia. Transfer of the fungus to a semisynthetic medium resulted in a gradual decrease in taxol production until its level became minimal, although fungal growth was relatively unaffected. Taxol production was completely restored after the addition of a plant extract to the culture medium (Ben ami et al., 2010; Loura et al., 2023).

    In addition to plant metabolites, the aim of inducing the biosynthesis of new or previously inactive secondary metabolites in endophytic fungi is to enhance the production of these compounds. In recent years, increasing attention has been paid to the use of epigenetic modifiers, such as histone deacetylase (HDAC) inhibitors, including 5-azacytidine and quercetin, as well as DNA methyltransferase inhibitors and nicotinamide, which serves as both a deacetylase inhibitor and a DNA methyltransferase (DNMT) inhibitor (Li et al., 1998). These compounds, added to the nutrient medium, alter the epigenetic status of the producer cells, which can lead to the activation of “silent” biosynthetic genes (Kim and Kaang, 2016).

    Thus, epigenetic chromatin remodeling represents a promising strategy for expanding the metabolic potential of endophytes and producing new biologically active compounds (Xue et al., 2023; Verma et al., 2023).

    In the course of screening endophytic fungi isolated from medicinal plants in Uzbekistan, several endophytes were identified that produced secondary metabolites inhibiting pancreatic lipase activity. The Aspergillus fischeri VO1R strain, isolated from Viola odorata and exhibiting lipase-inhibitory activity greater than 90%, was selected as the most active (Gulyamova et al., 2022). However, during submerged cultivation, a gradual decrease in secondary metabolite production was observed. In this regard, this study aimed to investigate the effect of plant extracts and epigenetic modifiers-5-azacytidine, nicotinamide and quercetin-on the production level of lipase-inhibitory metabolites by A.fischeri VO1R, as possible factors in increasing the metabolic potential of endophytes.

    MATERIALS AND METHODS

    Objects and subject of the study
     
    The endophytic fungus Aspergillus fischeri VO1R, isolated from Viola odorata, was the subject of the study.
     
    Isolation and cultivation of fungal endophytes
     
    Individual endophyte colonies that grew after incubation were removed with a fine needle, transferred to agar tubes and incubated at 28°C for seven days. Endophytic fungi were isolated from Viola odorata roots, as described by Hazalin et al., (2009). The roots were surface disinfected using 70% ethanol, followed by a 1-3 minute ethanol treatment and washing with sterile water, the leaves were aseptically chopped into pieces no larger than 0.5 cm and placed in Petri dishes with Czapek-Dox agar containing chlortetracycline at a concentration of 50 mg/l and streptomycin sulfate at a concentration of 250 mg/l to suppress bacterial growth.

    Czapek-Dox medium (g/l): NaNO3 - 2 g, MgSO4 - 0.5 g, KCL - 0.5 g, FeSO4 - 10 mg, KH2PO4 - 1 g, sucrose - 20 g, agar-agar - 20 g, distilled water. - up to 1000 ml, pH 6-6.5.

    Extraction of metabolites from the biomass of the endophyte fungus A. fischeri VO1R was carried out according to the method of Hazalin et al., (2009).

    Obtaining the plant extract
     
    The V. rosea extract was obtained by maceration of 10 g of crushed (no more than 3 mm) crude biomass of fresh plants (stems, leaves) with 100 ml of 96% ethanol for 24 hours at room temperature on an orbital shaker at 180 rpm. After settling for 2 days at a temperature not exceeding 10°C, the extract was separated by filtration through filter paper (Whatman No. 1) to obtain a clear liquid. The extract was then evaporated on a rotary evaporator, diluted with sterile water to the required concentration and passed through a polytetrafluoroethylene syringe filter with a pore size of 0.45 μm. The resulting plant extract was added to the nutrient medium to a final volume ratio of 2.0% before inoculation (Qing et al., 2018).
     
    The lipase-inhibitory activity of extracts
     
    50 mg of lipase (Sigma, 100 U/ml) was suspended in 10 ml of Tris-HCl buffer containing 2.5 mM Tris and 2.5 mM NaCl, pH 7.4. The solution was vigorously shaken for 15 min, followed by centrifugation (4000 rpm for 10 min). The supernatant was collected and reused as the enzyme solution. Stock solutions of extracts and Xenical were prepared in DMSO at a concentration of 10 mg/mL. The final reaction mixture consisted of 875 μL buffer, 100 μL enzyme and 20 μL extract, which were pre-incubated for 5 min at 37°C (Shilpa and Shukla, 2025). Then, 10 μL of substrate (4-nitrophenyl palmitate, 10 mM in acetonitrile) was added. The optical density of the final mixture was measured using a SPECOL-1300 spectrophotometer at a wavelength of 405 nm after 5 minutes (Azmir et al., 2013). The assay was performed in triplicate and the percentage of inhibition was calculated using the formula: 
     
     
     
    Where,
    Ae (enzyme control) =  Optical density of the enzyme control (without inhibitor).
    At (test sample) = Difference between the optical density of the test sample with and without the substrate.

    RESULTS AND DISCUSSION

    The effect of plant extracts on endophytes was observed by Zhao et al., (2013) when growing the endophytic fungus Colletotrichum gloeosporioides ES026, isolated from Huperzia serrata, which produces the acetylcholinesterase inhibitor hupercin A. It was found that at the seventh passage, growth and alkaloid content decreased. At the ninth passage, the mycelium turned from white to yellow, with mycelial yield and hupercin A content significantly lower than the initial values. To overcome this problem, cells were first grown in the host plant extract and then in basal or enriched media. This strategy helped maintain fungal viability and stable alkaloid production at the initial level for 3 years (Zhao et al., 2013). The addition of 2% host plant extract to the medium on the seventh day of A. fischeri cultivation resulted in noticeable morphological and cultural changes, including green pigmentation, hyphal thickening and compaction (Fig 1, 2).

    Fig 1: Morfological structure of the endophytic fungus A. fischeri VO1R.



    Fig 2: Morphological changes in the endophytic fungi A. fischeri VO1R (with addition of Viola odorata extract).



    A slight increase in the mycelial mass of A.fischeri and the amount of extracted secondary metabolites increased twofold compared to the control. In contrast, the inhibitory activity of the extract remained at the initial level at 92% (Fig 3).

    Fig 3: Effect of V. odorata plant extract (2%) on the inhibitory activity of A.fischeri VO1R.



    Thus, in contrast to the data with Colletotrichum gloeosporioides ES026, under experimental conditions, host plant metabolites promote increased production of inhibitory metabolites by A. fischeri VO1R.

    Epigenetic modifiers added to the endophyte culture medium help activate dormant or weakly active genes responsible for secondary metabolite production. These substances do not alter DNA but affect its activity by influencing chromatin structure, for example, by altering histone status or DNA methylation (Gupta et al., 2020; Verma et al., 2023).

    5-azacytidine and quercetin were used as DNA methyltransferase inhibitors, while nicotinamide was used as a deacetylase inhibitor as epigenetic modifiers. The ability of 5-azacytidine to induce the expression of biosynthetic clusters in fungi, including the genera Aspergillus and some others, has been repeatedly noted in the literature (Ben Ami et al., 2010; Abdelhamid et al., 2025; Jia et al., 2024; Pissios, 2017). Nicotinamide acts as an inhibitor of NADz -dependent histone deacetylases, particularly enzymes of the SIRT (sirtuins) family, leading to histone hyperacetylation and transcriptional activation. Nicotinamide can also suppress the activity of DNA methyltransferases, making it a universal epigenetic modifier with a combined mechanism of action (Saldiyar et al., 2018). Quercetin is a natural flavonoid known as a selective inhibitor of DNMTs and histone deacetylases of classes I and II. Additionally, quercetin exhibits antioxidant and antimicrobial activity, which may contribute to further stimulation of stress-induced metabolism in endophytes (Zhu et al., 2025).

    The effect of modifiers on growth and the formation of inhibitory metabolites was studied dynamically under submerged cultivation conditions of A.fischeri VO1R. As can be seen from the data presented below, the addition of epigenetic modifiers significantly reduced the biomass and secondary metabolite levels compared to the control throughout the entire cultivation period. At the same time, PL inhibitory activity remained at a level comparable to the control (Fig 4). Thus, the addition of epigenetic modifiers-nicotinamide (NAD), quercetin and 5-azacytidine-to the nutrient medium did not increase the amount of secondary metabolites produced by Aspergillus fischeri VO1R, but, on the contrary, significantly reduced their content. However, in the presence of 5-azacytidine, the inhibitory activity of the extracts in the dynamics on days 5-11 of cultivation is a significant value from 56.6 to 94.6% and secondary metabolites from 10.4 to 14.9 mg, in the presence of quercetin, the highest inhibitory activity is also observed on days 5-11 and is from 46 to 82% with a content of secondary metabolites from 7.5 to 14.8 mg, in the presence of nicotinamide, the highest inhibitory activity and metabolite content are observed on days 5-9. They are 76 to 84.9% and 12.7 to 14.3 mg, respectively.

    Fig 4: Effect of epigenetic modifiers on growth, inhibitory activity and accumulation of secondary metabolites in A.fischeri VO1R during growth dynamics.


      
    It should be noted that, unlike the control, the presence of modifiers did not show a direct dependence of inhibitory activity on the amount of secondary metabolites. Moreover, the inhibitory activity of the extracts was almost equal to that of the control extracts, but with a twofold lower amount of metabolites.

    CONCLUSION

    The obtained results indicate that the use of epigenetic modifiers (5-azacytidine, nicotinamide and quercetin) during the cultivation of the A. fischeri VO1R strain results in a decrease in total biomass and total yield of secondary metabolites compared to control conditions, while maintaining inhibitory activity at a level comparable to the control. A redistribution of the metabolic profile of the fungus toward an increase in the level of metabolites characterized by lipase-inhibitory activity could be proposed.

    Among the epigenetic modifiers tested, treatment with 5-azacytidine resulted in the highest pancreatic lipase inhibitory activity, reaching 94.6%, compared with 91.5% in the control group. However, biomass production and, consequently, the yield of secondary metabolites were reduced relative to the control. Further studies of the composition and nature of the compounds produced are clearly needed to more thoroughly determine the effect of epigenetic modifiers on the metabolic spectrum.

    ACKNOWLEDGEMENT

    The authors express their gratitude to the leadership and scientific team of the Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan for collecting scientific sources for the article, processing them in-house and conducting scientific experiments.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    Our experiments did not involve animals and the experimental animal care committee approved the work and the animal care committee of the Technical University did not approve the work. Our article mainly focuses on plants.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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