Phytochemical Profile and Antioxidant Potential of Endophytic Fungi Isolated from the Stem Bark of Oroxylum indicum (L.) Kurz

INTRODUCTION

Recently, there has been a growing awareness of the importance of a healthy lifestyle, leading to an increase in the consumption of nutrient-dense, antioxidant-rich organic natural foods instead of synthetic and processed alternatives. These natural foods can help restore the body’s ability to combat various stresses, infections, and related health conditions. As a result, the identification and development of new antioxidants from nature could play a crucial role in mitigating the harmful effects of oxidative stress in different diseases. This approach allows us to explore both past and ongoing research aimed at identifying natural antioxidants and understanding their mechanisms, with the potential for future therapeutic advancements.^[1]

Natural antioxidant compounds play a crucial role in mitigating oxidative stress and enhancing immune function by neutralising free radicals.^[2] Synthetic antioxidants like butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary butyl hydroquinone (TBHQ) are commonly used as food preservatives to prevent oxidation.^[3] However, due to their potential toxicity and carcinogenic effects, the use of these synthetic antioxidants in food is not advisable.^[4] Therefore, antioxidants derived from natural sources hold promise for disease prevention and treatment, as they can protect against oxidative damage caused by reactive oxygen species (ROS). Increasing evidence indicates that ROS, including superoxide (O₂⁻) and hydroxyl radicals (OH⁻), along with free radical-induced reactions, can damage vital biomolecules such as lipids, proteins, and DNA. This oxidative damage is associated with a range of health conditions, including aging, cancer, atherosclerosis, coronary heart disease, diabetes, Alzheimer’s disease, and other neurodegenerative disorders.

Antioxidants are considered effective in counteracting ROS-induced tissue damage. Many antioxidant compounds also possess anti-inflammatory, anti-atherosclerotic, antitumour, antimutagenic, anticancer, antibacterial, and antiviral properties, to varying extents. Recently, there has been significant interest in naturally derived antioxidants.

Endophytes, which are fungi or bacteria that live within healthy plant tissues without causing visible infection, may represent a promising source of novel natural products for medicinal, agricultural, and industrial applications. Although relatively underexplored, considerable attention is now being focused on endophyte biodiversity, the chemical composition and bioactivity of their metabolites, and the interactions between these microorganisms and their host plants. Endophytes are known to produce a wide range of structurally diverse bioactive natural products, including alkaloids, benzopyranones, quinones, flavonoids, phenolic acids, steroids, terpenoids, xanthones, etc. These compounds have been reported to exhibit various activities, such as antibiotic, antiviral, anticancer, insecticidal, antidiabetic, immunosuppressive, and antioxidant effects. Medicinal plants, in particular, have been recognised as a valuable source of endophytes that yield novel metabolites with potential pharmaceutical applications.^[5]

Some findings claim that endophytic fungi stimulate the development of these bioactive secondary metabolites in medicinal plants, and that a close connection between these endophytic fungi and host plants will increase the production of these secondary metabolites.^[6,7] Fascinatingly, the isolation of these endophytic fungi linked to medicinal plants will be very advantageous since it will support the large-scale synthesis of bioactive secondary metabolites with therapeutic value.^[8] Oroxylum indicum (L.) Kurz is a member of the Bignoniaceae family. In ancient times, this plant was employed for its medicinal properties. This plant is also referred to as the trumpet tree or Shyonaka in Sanskrit. This plant exhibits anti-hyperglycaemic, cardioprotective, antibacterial, anti-inflammatory, anticancer, pro-neurogenesis, and anti-adipogenesis qualities.^[9] In light of this, we assessed the antioxidant capacity of fungal endophytes that were isolated from the stem bark of Oroxylum indicum.

MATERIAL AND METHODS

RESULTS

Morphological identification of the isolated endophytic fungal strain

Using a culture-dependent method, three fungal endophytes were extracted from the stem bark of O. indicum. The codes for them were Endophytic fungi-1 (EF-1), EF-2, and EF-3. The isolated strains were identified using a light microscope, based on colony colour and the morphological characteristics of hyphae, setae, conidia, and spores^[23-25] [Figure 1].

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Figure 1:

The endophytic fungal morphology (colony appearance, hypha, and conidia) of O. indicum L. Kurr. stem bark. Strains of fungi EF1 (a-front view, b- rare view, c-microscopic view), EF2 (a-front view, b-rare view, c-microscopic view), EF3 (a-front view, b-rare view, c-microscopic view). After seven days, the colonies on PDA revealed the generative hyphae, conidia, and both the top and reverse sides of each isolate.

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The EF-1 strain was found to be of the filamentous fungus Simplicillium species, which has morphological characteristics including being white and floss-shaped [Figure 1 EF1(a)] and having a citron-yellow reverse side 10 days after being cultured at 25°C [Figure 1 EF1(b)]. Dense colonies were found there. Optical microscopy revealed septate hyphae and conidia that were usually obclavate to ellipsoidal, dark taupe [Figure 1 EF1(c)].

EF-2 strain was determined to be a Neopestalotiopsis species with white, cottony, and flocculent characteristics. It had dense aerial mycelium on its surface, undulate edges, and superficially formed black conidiomata that were dispersed over the PDA ten days after being incubated at room temperature. They also contained slimy black conidial mass [Figure 1 EF2(c)]. Conidia were somewhat curved and ranged from fusoid to ellipsoidal.

The EF-3 strain exhibited the physical traits of the mature fruiting body, which featured a reniform to subcircular basidiocarp. It had a creamy top with no stipe, delicate, silky hairs, and radial furrows [Figure 1 EF3(a), (b)]. The curved, cylindrical, sausage-like basidiospores have smooth walls and a white spore print [Figure 1 EF3(c)]. These traits indicated that the EF3 isolate was a member of the Trametes genus.

DISCUSSION

Numerous antioxidants are recognised to offer defence against a variety of illnesses. Epidemiological research has shown that increased antioxidant consumption leads to decreased risk of numerous illnesses, including heart disease. This explains why natural antioxidants and their significance for human nutrition and health are of such great interest.^[27]

Research has explored various medicinal herbs, spices, fruits, vegetables, and fungi as potential natural sources of safe antioxidants. Many compounds, particularly polyphenols, have demonstrated strong antioxidant properties. Recently, studies have shown that different mushrooms, endophytes, and fungi also exhibit antioxidant activity. These organisms produce unique metabolites with antioxidant effects, making them as effective as synthetic antioxidants and other phytochemicals. Species such as Chaetomium sp., Cladosporium sp., Torula sp., Phoma sp., and Penicillium roqueforti generate secondary metabolites like phenolic acid derivatives, terpenoids, benzoic acid, and rutin, which not only possess antioxidant properties but also exhibit a range of biological activities, including antibacterial, antiviral, antimutagenic, and immunomodulatory effects.^[28,29]

The phytochemical components of the plants determine their therapeutic potency.^[30] Alkaloids, flavonoids, phenolics, tannins, saponins, steroids, glycosides, terpenes, and other significant phytochemicals are found in different parts of the plants.^[31] The metabolites synthesised by the plants as a defence system against biotic and abiotic stresses have the medicinal potential that humans can utilise to treat various ailments.^[32,33] The phytochemicals present in the isolated endophytic fungi associated with the O. indicum stem bark (Simplicillium, Neopestalotiopsis, and Trametes) have shown the presence of the phytoconstituents, namely saponins, alkaloids, flavonoids, and tannins, which are known to exhibit biological activities.

Kempegowda and Maddaya, in their preliminary qualitative phytochemical analysis and secondary phytochemical screening on the endophytic fungal extract using standard qualitative procedures, revealed the presence of phenols and flavonoids. Among these, the crude extract of N. clavispora showed maximum flavonoid and phenol content, which is similar to our study findings.^[34]

According to the study by Tang et al., it was observed that various solvents can recover different compounds with varying contents and components.^[35] Polar and medium polarity solvents were more effective in extracting low and high-molecular-weight polyphenols from the endophytic fungus fermentation broth compared to nonpolar solvents. The Neopestalotiopsis extract exhibited the highest antioxidant activity, which may be attributed to the concentration and composition of the chemical compounds identified in the endophytic fungal extract.

In the present study, Neopestalotiopsis extract exhibited strong antioxidant activity compared to Simplicillium and Trametes extracts. The percentage of DPPH radical scavenging potential of Neopestalotiopsis extract was found to be 31.77 ± 0.36 compared to Simplicillium (21.27 ± 0.55) and Trametes (19.02 ± 0.01) extracts [Figure 2a]. The suppression of chain initiation, binding of transition metal ion catalysts, peroxide breakdown, prevention of continuous hydrogen abstraction, and radical scavenging are some of the processes that have been linked to the antioxidant activity of putative antioxidants.^[36] The results of different assays conducted demonstrate the Neopestalotiopsis extract’s strong, broad-ranging antioxidant activity. The capacity of the endophytic fungi to donate hydrogen may be the reason for their antioxidant effectiveness against DPPH free radicals. The DPPH is an unstable free radical that transforms into a stable diamagnetic molecule by accepting an electron or hydrogen radical.^[37]

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Figure 2:

The free-radical scavenging activity of methanol endophytic fungal extract. (a) DPPH and (b) superoxide radicals. Black: AA - Ascorbic acid; Light Grey: S - Simplicillium extract and Grey: N -Neopestalotiopsis extract; White: T- Trametes extract. The data have been expressed as mean ± standard deviation. The concentration range was taken at (0-100 µg/mL). At 100 µg/mL, the DPPH and SOD radical scavenging potential of Neopestalotiopsis was found to be significantly different compared to Simplicillium and Trametes extracts. (**** - p < 0.0001, ** - p < 0.01, * - p < 0.05). DPPH: (2,2-diphenyl-1-picrylhydrazyl) assay, SOD: Superoxide dismutase.

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The percentage of SOD radical scavenging potential of Neopestalotiopsis extract was found to be 35.41 ± 0.41 compared to Simplicillium (29.40± 0.54) and Trametes (31.03 ± 0.11) extracts [Figure 2b]. Superoxide radicals must thus be neutralised or eliminated to shield cells from their damaging effects. The production of O₂•- was suppressed in a concentration-dependent manner by the endophytic fungal extracts. The examination of total phenolics and total flavonoid components confirms Neopestalotiopsis extract has a greater phenolic and flavonoid content, which may be the cause of its enhanced superoxide free radical scavenging ability.^[38]

The FRAP value of Neopestalotiopsis extract was found to be 0.15 ± 0.003 compared to Simplicillium (0.10 ± 0.002) and Trametes (0.09 ± 0.00) extracts [Figure 3]. A rapid and easy way to measure antioxidant activity is the FRAP assay.^[39,40] The FRAP value increased in a concentration-dependent manner in all three of the isolated endophytic fungal extracts. Numerous plant extracts have been shown to have significant FRAP values in vitro, indicating antioxidant activity. According to the FRAP assay, the isolated fungal endophytes have antioxidative properties because they have reducing capability. The TAC value of Neopestalotiopsis extract was found to be 30.03 ± 0.02, compared with Simplicillium (14.15± 0.11) and Trametes (12.28 ± 0.38) extracts [Figure 4].

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Figure 3:

Ferric Reducing Antioxidant Power Assay (FRAP). The data have been expressed as mean ± standard deviation. Black: AA - Ascorbic acid; Light Grey: S - Simplicillium extract and Grey: N -Neopestalotiopsis extract; White: T- Trametes extract. The data are expressed as mean ± standard deviation. The concentration range was taken at (0-100 µg/mL). At 100 µg/mL, the FRAP value of Neopestalotiopsis was found to be significantly different compared to Simplicillium and Trametes extracts. (**** - p < 0.0001).

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Figure 4:

Total antioxidant capacity (TAC) of methanol endophytic fungal extract. Light Grey: S - Simplicillium extract and Grey: N - Neopestalotiopsis extract; White: T - Trametes extract. The data have been expressed as mean ± standard deviation. The concentration range was taken at (0-100 µg/mL). At 100 µg/mL, the TAC value of Neopestalotiopsis was found to be significantly different compared to Simplicillium and Trametes extract. (**** - p < 0.0001).

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The total phenolic content of Neopestalotiopsis, Simplicillium, and Trametes extracts was found to be 37.89 ± 0.42, 35.12 ± 0.47, and 29.41± 0.22, respectively. The total flavonoid content of Neopestalotiopsis, Simplicillium, and Trametes extracts was found to be 31.24 ± 0.42, 22.39±0.49, and 20.41± 0.12, respectively [Figures 5a-b]. Plant phenols are significant substances with redox characteristics that contribute to antioxidant activity because their hydroxyl groups enable the scavenging of free radicals.^[41] Phenolic chemicals account for a significant amount of the antioxidant capacity found in a variety of medicinal plants.^[42] The TAC observed in the endophytic fungal extracts may be attributed to the presence of phytoconstituents.

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Figure 5:

The total phenol and flavonoid contents of methanol endophytic fungal extract. Light Grey: S - Simplicillium extract and Grey: N - Neopestalotiopsis extract; White: T - Trametes extract. (a) Total phenolic content (TPC assay) (b) Total flavonoid content (TFC assay). The data have been expressed as mean ± standard deviation. The concentration range was taken at (0-100 µg/mL). At 100 µg/mL, the TPC and TFC value of Neopestalotiopsis was found to be significantly different compared to Simplicillium and Trametes extract. (**** - p < 0.0001).

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Free radicals, reactive oxygen, and nitrogen species significantly influence cancer and other oxidative stress-mediated diseases. However, the phenolic and flavonoid chemicals scavenge the free radicals, preventing oxidative damage to the cells.^[43] The analysis of total phenolics and total flavonoid components confirms that the isolated endophytic fungi from O. indicum stem bark can scavenge free radicals, which may be due to their greater phenolic and flavonoid content. The presence of phytoconstituents with antioxidant activity could be useful in the prevention of diseases in which free radicals are involved. If the non-toxicity of the antioxidant compounds and the biological properties of the endophytic fungi are proven in vivo, these could be suggested as possible natural sources of antioxidants to prevent many free radical-mediated diseases.

CONCLUSION

The present study revealed that the endophytic fungi isolated from O. indicum stem bark can produce compounds having significant antioxidant efficacy. The synthesis of phenolic and flavonoid compounds by endophytic fungi will be helpful in the biotechnological mass production of safe alternative sources of antioxidants. Furthermore, to identify the active compounds, the crude extracts are being subjected to a purification process, which may provide a better source for developing new pharmacological agents. These active natural compounds have a potential pharmaceutical application as antioxidants and antimicrobial agents. But unless their mechanisms of action are elucidated through pharmacodynamic, biochemical, bioinformatics, and pre-clinical approaches, the biological and clinical applications of endophytic fungi will be limited.