Abstract Oxidative stress (OS), resulting from an imbalance between reactive oxygen species (ROS) and antioxidant defences, contributes to numerous chronic and neurodegenerative diseases. Natural antioxidants, particularly from edible mushrooms, offer promising therapeutic potential. This study evaluated the antioxidant activity of Calocybe indica crude extract (CIC) and its submicron emulsion (CIC-SE) using in vitro biochemical assays and an in vivo Artemia salina model. FTIR and GC-MS analyses revealed the presences of polysaccharides, phenolics, flavonoid glycosides, fatty acids and sterols. The CIC-SE exhibited a hydrodynamic diameter of 431.8 nm and a zeta potential of -6.65 mV, indicating moderate stability. In vitro assays showed potent radical-scavenging activity: DPPH IC₅₀ of 30.12 ± 1.86 µg/mL and ABTS IC₅₀ of 22.19 ± 1.42 µg/mL, comparable to ascorbic acid (DPPH IC₅₀ = 21.35 ± 2.0 µg/mL; ABTS IC₅₀ = 18.31 ± 1.75 µg/mL). Acute toxicity experiments yielded a 24-h LC₅₀ of 63.82 µg/mL in A. salina, providing a preliminary assessment of toxicity under the tested conditions. The results demonstrate that CIC-SE exhibits promising antioxidant activity in the DPPH and ABTS radical-scavenging assays. Overall, the findings support further investigation of CIC-SE as a potential antioxidant formulation for nutraceutical applications.

Keywords: Calocybe indica crude extract (CIC), Submicron emulsion (CIC-SE), GC-MS pathway enrichment, FTIR spectroscopy, Free radical scavenging activity,  Artemia salina lethality assay

Citation:  Upadhyay, R. and Sevanan, M. 2026. Development and characterisation of a submicron emulsion of Calocybe indica extract: Physicochemical characterisation, antioxidant activity, and preliminary toxicity assessment. Natural and Life Sciences Communications. 26(1): e2027008.

Graphical Abstract:

INTRODUCTION

When there is an imbalance in the creation of ROS (reactive oxygen species) and antioxidant defence mechanisms in the body, it leads to oxidative stress. One of the conditions in the development and pathogenesis of many chronic diseases, such as cardiovascular conditions, neurodegenerative diseases, cancer, and diabetes, is imbalance (Jomova et al., 2023). Excessive ROS may destroy crucial biomolecules, such as lipids, proteins, and nucleic acids, which may interfere with the work of the cells and cause apoptosis or necrosis of the cell (Pizzino et al., 2017). In this regard, identification of natural antioxidants that could win the battle against oxidative stress has become a desirable approach in the field of pharmacology and nutraceutical formulation. For instance, an in vivo system, which has been widely used to evaluate the impact of oxidative stress and antioxidant capacity, and is relatively economical, extremely fecund and easy to maintain, is Artemia salina. Artemia has become an essential tool of ecotoxicology, drug screening, and biomedical research (Salay et al., 2024). The model was initially applied to general toxicity testing, yet it was also transformed to study oxidative stress and the protective qualities of bioactive compounds, in particular in the early stages of development (Turcov et al., 2023). In this model, the ROS inducer commonly used to simulate oxidative stress is hydrogen peroxide (H₂O₂). When exposed to sub-lethal levels of H₂O₂, it may cause higher mortality, delayed cyst hatching, abnormal morphological development, and impaired locomotor activity that can all be used as quantifiable endpoints for examining oxidative damage (Hernández-Calderón et al., 2025). A. salina nauplii’s transparency further facilitates direct visualisation of physiological and developmental anomalies, making it a robust model for antioxidant screening. Mushrooms have been of scientific interest in terms of nutritional and medicinal importance, especially with the high concentration of bioactive compounds. Okeke et al. (2025) suggested that advances in phytochemical profiling approaches, such as GC-MS, have enabled the identification of bioactive-rich extracts from natural sources. Consequently, a study by Narkpiban et al. (2026) demonstrated that the ethanolic extract of Nymphaea displayed significant biological activities, including antioxidant and antimicrobial properties, which are due to its rich phytochemical composition. It has been indicated in numerous studies that edible and medicinal mushrooms possess considerable antioxidant activity, which is explained by the presence of various phenolics, flavonoids, polysaccharides, sterols, carotenoids, vitamins, and trace elements in them (Chun et al., 2021; Al Qutaibi and Kagne, 2024).

Zahmoul et al. (2024) stated that Ganoderma adspersum crude extract indicated strong DPPH as well as ABTS radical scavenging activity, having IC₅₀ values of 49.42 and 9.75mg/mL, respectively, and was more effective than vitamin C. Similarly, Adamska and Felisiak (2025) ascribed the strong antioxidant activity of Laetiporus sulphureus to the existence of carotenoids, polyphenols, peptides, and polysaccharides, especially in its fresh fruiting bodies. Calocybe indica is an edible mushroom native to India and widely used for both culinary and medicinal purposes (Shashikant et al., 2022). Recent findings suggest that C. indica is rich in bioactive compounds, including flavonoids, phenolic acids, fatty acids, and polysaccharides, contributing to its reported anti-inflammatory and immunomodulatory properties (Shashikant et al., 2022). Despite increasing reports on the antioxidant properties of edible mushrooms, studies integrating phytochemical profiling, formulation strategies and pharmacokinetic prediction for Calocybe indica remain limited. Additionally, the extraction technique plays a crucial role in determining the yield and efficacy of the bioactive compound, with modern techniques such as ultrasonic and microwave-assisted extraction significantly enhancing phenolic recovery and antioxidant potential compared to conventional methods (Yulianti et al., 2026). Similarly, Auranwiwat et al. (2025) reported a comparative study on Garcinia mangostana that demonstrated that extraction techniques such as stirring and ultrasonic-assisted extraction significantly influence α-mangostin yield and antioxidant activity. Furthermore, Wongtrakul et al. (2026) suggested that recent advancements in formulation have shown emulsion-based delivery systems can improve the stability, bioavailability, and functional performance of plant-derived bioactive compounds in therapeutic applications. Therefore, this study aimed to develop a submicron emulsion formulation of C. indica crude extract and evaluate its physicochemical characteristics, antioxidant activity, toxicity profile, and predicted pharmacokinetic behaviour using an integrated experimental and computational approach.

MATERIALS AND METHODS

Calocybe indica crude extraction

Fresh, mature fruiting bodies of Calocybe indica were purchased from a local market in Coimbatore, Tamil Nadu, India. Approximately 2 kg of fresh mushrooms were collected for the extraction procedure. The mushrooms were visually inspected, and the pileus was separated from the stipe. Only the fresh pileus portion, including the attached gill region, was used for extraction, whereas the stipe was discarded. The pileus portions were thoroughly washed with distilled water to remove adhering soil and other surface impurities and were gently patted dry using clean tissue paper. 100 g of the fresh pileus material was then chopped into small pieces and immersed in 400 mL of distilled water. The mixture was heated at 50°C for 4 h. After extraction, the mixture was stored overnight at 4°C and subsequently processed after 24 h. The mushroom material was used in its fresh state and was not subjected to oven-drying or other drying treatment. Therefore, the extraction mass included the naturally occurring moisture present in the fresh pileus.

Fourier transform infrared (FTIR)

The functional groups in CIC, GEO and CIC-SE were analysed using FTIR spectroscopy (Thermo Scientific, Nicolet iS5 equipped with iD5 ATR accessory). Spectra were recorded in ATR mode using a diamond crystal. Samples were directly placed on the ATR crystal; moreover, spectra were acquired in the 4,000-400 cm⁻¹ range, with 4 cm⁻¹ resolution, averaging 16 scans. The characteristic absorption bands were analysed to identify functional groups in the extract by comparing with standard reference spectra.

Gas chromatography-mass spectrometry (GC-MS)

CIC was employed using a GC-MS system (Agilent 6890N GC coupled with a 5973 MSD). Compound separation was conducted over an HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness) using helium (at a flow rate of 1.0 mL/min) as carrier gas. Injection volume was 1µL in split mode (10:1), at 250°C injector temperature and 280°C detector temperature. Oven temperature was programmed from (2 min hold) 50 to 280°C (at 10°C/ min), further maintained for 10 min at 280°C. Mass spectra were acquired at 70 eV in EI (electron ionisation) mode, having a scan range of 50-550 m/z. The total run time was 22 min. Compounds were identified by comparing the obtained mass spectra with the NIST 2017 mass spectral library database.

Oil-binding properties

CIC oil-binding efficacy was assessed using the methodology established by Chawla et al. (2019). To evaluate this, pre-weighed falcon tubes (15 mL) were utilised; a 1 g sample was dispersed in castor oil, further sample was allowed to remain undisturbed for 30 min. Centrifugation (for 5 min) was performed at 5,000 rpm at 27°C (room temperature), then the oil supernatant was discarded. The tube weight containing pellets was further quantified, and the oil absorption capacity was measured as g of oil reserved/g of powdered sample.

Submicron emulsion synthesis

The submicron emulsion was prepared using a high-energy emulsification method. The oil phase containing the extract was mixed with the surfactant under continuous stirring. The mixture of garlic oil with Tween 80 and CIC (2:2:1) was subjected to high-speed homogenisation at 800 rpm for 15 minutes, followed by probe sonication for 10 minutes to obtain the CIC-SE formulation for subsequent physicochemical and stability characterisation.  The 2:2:1 ratio of garlic oil, Tween 80, and CIC was selected based on preliminary formulation trials, in which this composition provided satisfactory physical homogeneity and dispersion compared with the other compositions evaluated. The selected formulation was subsequently subjected to high-speed homogenisation and probe sonication for further characterisation.

Dynamic light scattering (DLS)

Hydrodynamic diameter as well as polydispersity index (PDI) of the CIC-SE were determined using a Malvern Zetasizer (Model: MAL1037088, Version 6.32, Malvern Instruments Ltd.). The sample had been dispersed in deionised water and analysed employing a disposable sizing cuvette. Experiments were performed at 25°C, utilising a dispersant refractive index=1.330; viscosity= 0.8872 cP. Samples were diluted 1:20 with deionised water for measurement to avoid multiple scattering effects.

Zeta potential

Zetasizer (Malvern Instruments Ltd) was used to measure the surface charge of CIC-SE. Dilution in deionised water was done, and the sample was then put in a clear disposable zeta cell to be analysed. Measurements were done at 25°C, and the viscosity= 0.8872 cP, dispersant refractive index= 1.330, dielectric constant= 78.5.

DPPH assay

It was utilised in establishing the free radical scavenging activity of the compound. Dissolution of 21 mg DPPH in 245 mL ethanol was used to prepare the DPPH free radical solution. The naturalisation colourimetric change was evaluated with the Tris-HCl buffer at pH of 7.4. A comparative analysis was done using extracted samples at different concentrations (1-5 mg/mL) in comparison with ascorbic acid. At the end of the 30 min incubation period, absorbance was recorded at 517 nm (Baliyan et al., 2022). The antioxidant activity % was determined according to Eq. (1).

Antioxidant Activity % = (A₀ -A/A₀) X 100                                                 (1)

A₀ = Optical density of the control without antioxidant

A = optical density of the extract diluted with DPPH/ ABTS/ H₂O₂

ABTS assay

To prepare solution A, 100 mL of acetic acid buffer (pH 4.5) was prepared, and 10 mg of potassium persulfate was dissolved to obtain a solution. To obtain solution B, 10 mg ABTS was dissolved in 10 mL of acetic acid buffer to obtain a solution at a 1:1 ABTS solution incubation was done in darkness at 4°C for 12-16 h. Samples at different concentrations (1-5 mg/mL) were extracted and compared to ascorbic acid (Lee et al., 2016). ABTS scavenging percentage analysis was employed as per the equation given in Eq. (1).

Brine shrimp culture and maintenance

Artemia salina cysts had been acquired at a local market in Coimbatore, Tamil Nadu, and kept at 4°C. Cysts had been hydrated in seawater with adequate aeration for 24 hours to hatch. A dark and light cycle of 8-16 hrs, respectively, was kept throughout this time. Hatched nauplii were gathered 24 hrs later and employed for conducting toxicity tests.

Acute toxicity assessment

Nauplii of the A. salina species were used in acute toxicity studies. Ranging concentrations of CIC and CIC-SE in seawater were added in 10 mL to 10 nauplii. Petri plates had been incubated for 24 hrs at room temperature, and the live and dead nauplii were counted. The experiment was repeated three times. Control was maintained to ensure that mortality observed after the treatment was not due to food restriction. The lethal concentration 50% (LC₅₀) value after 24 hours of interaction was determined by probit analysis (de Barros et al., 2021).

Statistical analysis

All experiments were performed in triplicate, and data are presented as mean ± standard deviation (SD). Statistical differences among experimental groups were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison post-hoc test. A p-value < 0.05 was considered statistically significant. IC₅₀ values were calculated for each replicate using piecewise cubic Hermite interpolation (PCHIP), and the resulting values are reported as mean ± SD. For the A. salina lethality assay, LC₅₀ values were estimated using probit regression. Linear regression was used to calculate R² values where applicable.

RESULTS

Functional group characterisation by FTIR

Fourier-transform infrared (FTIR) spectroscopy was employed to identify the principal functional groups present in CIC, GEO, and CIC-SE. The FTIR spectra revealed several prominent absorption bands corresponding to characteristic chemical moieties (Figure 1). The unencapsulated CIC extract exhibited a broad absorption band centred at 3,349.872 cm⁻¹ (3,200-3,400 cm⁻¹ region), corresponding to hydroxyl (O-H) and amine (N-H) stretching vibrations associated with hydroxyl- and nitrogen-containing constituents. Peaks observed at 2,975.473 and 2,896.970 cm⁻¹ were attributed to aliphatic C-H stretching vibrations. A signal at 1,646.816 cm⁻¹ was associated with C=O and/or C=C stretching vibrations. In addition, absorption bands at 1,085.921 and 1,043.805 cm⁻¹ were assigned to C-O and C-O-C stretching vibrations, consistent with carbohydrate and glycosidic functionalities. GEO exhibited characteristic bands at 3,356.857 cm⁻¹, 2,923.606 cm⁻¹, and 1,639.826 cm⁻¹, corresponding to O-H stretching, aliphatic C-H stretching, and C=O/C=C stretching vibrations, respectively. Additional bands at 1,085.029 and 1,045.019 cm⁻¹ were associated with C-O-containing functionalities, while low-frequency signals in the 406.048-518.274 cm⁻¹ region were observed in the GEO spectrum. The CIC-SE spectrum retained characteristic absorption features attributable to both CIC and the formulation components. The broad hydroxyl band shifted from 3,349.872 cm⁻¹ in CIC to 3,342.577 cm⁻¹ in CIC-SE, which may indicate changes in the hydrogen-bonding environment following formulation. The aliphatic C-H stretching band appeared at 2,926.389 cm⁻¹, while additional bands were observed at 1,459.322 and 1,349.815 cm⁻¹. The C-O/C-O-C absorption band was retained at 1,087.651 cm⁻¹ in CIC-SE. The overall retention of the characteristic CIC-associated bands after formulation indicates that the major functional-group features of the extract were preserved during the emulsification process.

Figure 1. Fourier-transform infrared (FTIR) spectra of Calocybe indica crude extract and emulsion formulations. CIC (a), GEO (b), and CIC-SE (c), showing the characteristic infrared absorption bands of the analysed samples.

GC-MS metabolite profiling

GC-MS analysis of CIC revealed a chemically diverse profile comprising predominantly oxygenated compounds, aromatic and phenolic derivatives, carbohydrate-related constituents, fatty acids and their esters, and a fungal sterol. Glycerin was the predominant detected compound, accounting for 41.86% of the reported chromatographic area, followed by benzeneacetaldehyde (2.18%), 2-methoxy-4-vinylphenol (1.27%), and phenol, 2-methoxy-3-(2-propenyl)- (1.05%). The detection of oxygenated heterocyclic compounds, including 4H-pyran-4-one derivatives and hydroxymethyl furanone, indicated the presence of chemically diverse low-molecular-weight metabolites. In addition, several lipid-associated constituents, including dodecanoic acid, tetradecanoic acid, n-hexadecanoic acid, and fatty acid esters, were tentatively identified. Importantly, ergosterol was detected at a retention time of 20.6626 min, supporting the fungal origin of the CIC sample. The presence of phenolic and oxygenated constituents may contribute to the antioxidant properties observed experimentally in the CIC sample (as shown in Table 1). 

Table 1. Major identified chemical constituents of Calocybe indica (CIC) by GC-MS. Major chemical constituents tentatively identified in the CIC sample by gas chromatography-mass spectrometry (GC-MS). Compound identification was based on comparison of mass spectra with the reference spectral library.

Development and characterisation of CIC submicron emulsion

Preliminary physicochemical analysis of the CIC extract demonstrated an oil-binding capacity of approximately 2 g/g, indicating a strong interaction between extract components and lipid phases. This property facilitated the formation of a water-in-oil (W/O) submicron emulsion. The emulsion was prepared using garlic essential oil as the continuous phase and Tween 80 as a stabilising surfactant. The formulation process yielded a visually homogeneous and stable dispersion without immediate phase separation for 15 days, as shown in Figure 2.

Figure 2. Physical appearance and storage stability of CIC-SE. Day 0 (a), Day 7 (b), Day 15 (c).

Dynamic light scattering (DLS) analysis (Figure 3) revealed a mean droplet size of 431.8 nm, with a relatively narrow size distribution, indicating uniform droplet formation. Although the droplet size exceeded the conventional nanoemulsion threshold (<200 nm), it remained well within the submicron range. Zeta potential analysis (Figure 4) showed a surface charge of -6.65 mV. This relatively low absolute value indicates weak electrostatic repulsion between droplets. However, no rapid aggregation or visible instability was observed immediately after formulation, suggesting the presence of stabilising mechanisms beyond electrostatic interactions. The formulation remained physically stable over the initial observation period, maintaining consistent turbidity and dispersion characteristics without creaming or coalescence.

Figure 3. Particle size distribution and zeta potential of CIC-SE determined by dynamic light scattering (DLS). Intensity-weighted particle size distribution (a) and zeta-potential distribution of the prepared submicron emulsion (b).

Figure 4. Antioxidant activity of Calocybe indica crude extract (CIC) and its submicron emulsion (CIC-SE) compared with garlic essential oil (GEO) and ascorbic acid. DPPH free radical scavenging activity (a) and ABTS radical scavenging activity at different concentrations (b). Data are presented as mean ± standard deviation (SD).

Antioxidant activity of CIC-SE

The antioxidant potential of the CIC-SE was quantitatively evaluated using DPPH and ABTS radical scavenging assays. In the DPPH assay, CIC-SE exhibited a clear concentration-dependent increase in radical scavenging activity (Figure 5). At lower concentrations, a gradual increase in inhibition was observed, followed by a steeper response at higher concentrations, indicating effective radical neutralisation kinetics. The IC₅₀ value was calculated as 30.12 ± 1.86 µg/mL.

The concentration-response curve demonstrated a consistent sigmoidal pattern, reflecting progressive saturation of radical scavenging sites. The relatively low standard deviation indicates good reproducibility of the assay. In the ABTS assay (Figure 6), CIC-SE showed even stronger radical scavenging efficiency, with an IC₅₀ value of 22.19 ± 1.42 µg/mL. The difference in IC₅₀ values between DPPH and ABTS assays suggests variability in interaction with different radical systems. The ABTS assay showed higher sensitivity, likely due to its compatibility with both hydrophilic and lipophilic antioxidant components present in the formulation. Overall, both assays consistently demonstrated strong antioxidant activity, with reproducible and concentration-dependent responses across replicates.

Figure 5. Probit dose–response analysis of CIC and CIC-SE. Dose–response relationships plotted as probit-transformed response against the logarithm of dose (µg/mL), illustrating the concentration-dependent response of the tested samples.

Figure 6. Antioxidant activity via ABTS radical scavenging activity at different concentrations of Calocybe indica crude extract (CIC) and its submicron emulsion (CIC-SE) compared with garlic essential oil (GEO) and ascorbic acid

Toxicity assessment using the brine shrimp model

The toxicity profile of CIC-SE was evaluated using the Artemia salina brine shrimp lethality assay. Exposure of nauplii to increasing concentrations of the formulation resulted in a clear concentration-dependent increase in mortality. At lower concentrations, minimal lethality was observed, while intermediate concentrations showed a gradual increase in mortality rates. At higher concentrations, a pronounced toxic effect was evident, with a substantial proportion of nauplii affected. Acute toxicity assessment of CIC-SE using A. salina nauplii showed a 24-h LC₅₀ of 63.82 µg/mL based on the observed mortality response (Figure 7). The reproducibility of the assay was confirmed by consistent mortality patterns across replicates. No abnormal behavioural responses, apart from lethality, were prominently noted during the observation period.

Figure 7. Probit dose-response analysis of CIC and CIC-SE. Dose-response relationships plotted as probit-transformed response against the logarithm of dose (µg/mL), illustrating the concentration-dependent response of the tested samples.

DISCUSSION

The FTIR analysis of CIC, GEO, and CIC-SE revealed characteristic hydroxyl, aliphatic C-H, carbonyl/C=C, and C-O/C-O-C functional groups. In CIC, the broad absorption band at 3,349.872 cm⁻¹ was associated with O-H and N-H stretching vibrations, while the bands at 2,975.473 and 2,896.970 cm⁻¹ corresponded to aliphatic C-H stretching. The signal at 1,646.816 cm⁻¹ was associated with C=O and/or C=C stretching, whereas the bands at 1,085.921 and 1,043.805 cm⁻¹ were consistent with C-O and C-O-C functionalities commonly found in carbohydrate-associated structures. These spectral features are compatible with the presence of hydroxyl-, nitrogen-, carbonyl-, and carbohydrate-containing constituents in mushroom extracts. Such functional groups may contribute to the chemical diversity of CIC, although FTIR alone cannot establish the identity or concentration of individual phenolics, polysaccharides, or other metabolites. Phenolic hydroxyl groups may participate in radical scavenging through hydrogen donation, while mushroom polysaccharides have been associated with antioxidant and immunomodulatory activities (Hassanpour and Doroudi, 2023; Singh et al., 2025). The observed spectral profile is also broadly consistent with functional-group patterns reported for bioactive edible mushrooms, including Ganoderma and Pleurotus species (Zahmoul et al., 2024). Similarly, the CIC-SE spectrum retained several characteristic absorption features of the extract and formulation components. The hydroxyl band shifted from 3,349.872 cm⁻¹ in CIC to 3,342.577 cm⁻¹ in CIC-SE, suggesting a change in the hydrogen-bonding environment following formulation. The aliphatic C-H band appeared at 2,926.389 cm⁻¹, while additional bands were observed at 1,459.322 and 1,349.815 cm⁻¹. The C-O/C-O-C band was retained at 1,087.651 cm⁻¹. These changes may reflect interactions between CIC constituents and the oil-surfactant-aqueous formulation environment. The retention of characteristic functional-group features indicates that the major chemical functionalities of the extract were preserved during emulsification (Liu et al., 2024).

The GC-MS profile of CIC demonstrated a chemically diverse composition consisting of oxygenated compounds, aromatic and phenolic derivatives, carbohydrate-related constituents, fatty acids and their esters, and a fungal sterol. Glycerin was the predominant detected compound, accounting for 41.86% of the reported chromatographic area, followed by benzeneacetaldehyde, 2-methoxy-4-vinylphenol, and phenol, 2-methoxy-3-(2-propenyl) (Tan et al., 2024; Xie et al., 2025). The detection of oxygenated heterocyclic compounds, including 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- and (S)-5-hydroxymethyl-2[5H]-furanone, suggests the presence of low-molecular-weight oxygen-containing metabolites that may be associated with carbohydrate transformation or other biochemical processes (Sakamoto et al., 2022; Żurawska et al., 2024). The detection of ethyl α-D-glucopyranoside further supports the presence of carbohydrate-related constituents. The phenolic and aromatic derivatives identified in CIC, including 2-methoxy-4-vinylphenol and phenol, 2-methoxy-3-(2-propenyl)-, may provide a plausible chemical basis for the antioxidant activity observed in the DPPH and ABTS assays (Rubab et al., 2020; Candido Júnior et al., 2022). Several lipid-associated compounds were also tentatively identified, including dodecanoic acid, tetradecanoic acid, hexadecanoic acid, hexadecanoic acid ethyl ester, and derivatives of hexadecanoic and octadecanoic acids. Fatty acids and their esters are important structural and metabolic components of fungal systems and may contribute to the physicochemical characteristics of the extract (Khoo et al., 2019; Ochoa-Ocampo et al., 2025). Overall, the GC-MS findings indicate that CIC contains a heterogeneous mixture of carbohydrate-related metabolites, oxygenated heterocyclic compounds, phenolic derivatives, fatty acids, lipid esters, and fungal sterol-associated constituents. These constituents may collectively contribute to the functional properties of CIC, particularly its antioxidant potential.

The development of CIC-SE provides a formulation approach for dispersing complex mushroom-derived constituents within an oil-surfactant system. The oil-binding capacity of approximately 2 g/g suggested an interaction between extract components and the lipid phase, supporting the selection of the formulation composition. The selected CIC-SE exhibited a mean hydrodynamic diameter of 431.8 nm. The zeta potential of -6.65 mV indicates limited electrostatic repulsion between droplets. The use of Tween 80 may provide additional steric stabilisation through the formation of a surfactant layer around the dispersed phase. Therefore, the observed formulation behaviour may involve both steric and electrostatic contributions (Mohammadi Taghiabadi and Saeedi Dehaghani, 2025). The absence of visible phase separation, creaming, or coalescence during the 15-day observation period indicates preliminary physical stability under the tested conditions. However, longer-term storage studies and additional stability parameters would be required to establish comprehensive formulation stability (Liu et al., 2022; Preeti et al., 2023; Giuliano et al., 2025).

CIC-SE exhibited concentration-dependent radical-scavenging activity in both DPPH and ABTS assays. The DPPH IC₅₀ was 30.12 ± 1.86 µg/mL, while the ABTS IC₅₀ was 22.19 ± 1.42 µg/mL. The lower IC₅₀ observed in the ABTS assay may reflect differences in the chemical characteristics of the two radical systems and their interactions with hydrophilic and lipophilic constituents. Similar antioxidant effects have been reported for extracts from other mushroom species, including Agaricus bisporus, Lentinus squarrosulus, and Heimioporus species (Batubara et al., 2025; Latif et al., 2025; Majumder et al., 2025). Previous studies have also highlighted the contribution of phenolic and flavonoid-associated constituents to antioxidant responses in natural products (Nguyen et al., 2021; Chaudhary et al., 2023; Al Qutaibi and Kagne, 2024). However, because the present Results do not include a direct comparison between unformulated CIC and CIC-SE under identical assay conditions, the data demonstrate antioxidant activity of CIC-SE but do not independently prove that emulsification enhanced the intrinsic antioxidant activity of CIC.

The Artemia salina lethality assay provided a preliminary assessment of the biological response to CIC-SE. Increasing concentrations produced a concentration-dependent increase in mortality, and the calculated 24-h LC₅₀ was 63.82 µg/mL. The brine shrimp assay is useful as an initial screening model for biological and cytotoxic effects, but it cannot replace mammalian toxicity studies or detailed mechanistic safety evaluation. The observed response may reflect the combined effects of multiple constituents present in CIC-SE. Consequently, further studies using mammalian cell models and appropriate in vivo toxicity protocols are required to clarify the safety profile and biological relevance of the formulation (Islam et al., 2017; Salay et al., 2024; Hasan et al., 2025).

CONCLUSION

The present study developed and characterised a submicron emulsion of Calocybe indica crude extract, designated CIC-SE. FTIR analysis demonstrated the retention of major functional-group features after formulation, while GC-MS profiling revealed a diverse mixture of oxygenated compounds, phenolic derivatives, carbohydrate-related constituents, fatty acids, lipid esters, and a fungal sterol. CIC-SE exhibited a mean droplet size of 431.8 nm, a zeta potential of -6.65 mV, preliminary physical stability during the 15-day observation period, and concentration-dependent antioxidant activity in the DPPH and ABTS assays. The formulation showed a 24-h A. salina LC₅₀ of 63.82 µg/mL under the tested conditions. These findings support CIC-SE as a preliminary antioxidant formulation candidate; however, further comparative, stability, toxicological, and in vivo studies are required before its nutraceutical or therapeutic potential can be established.

ABBREVIATIONS

ROS: Reactive Oxygen Species; CIC: Calocybe indica Crude Extract; CIC-SE: Calocybe indica Crude Extract Submicron emulsion; FTIR: Fourier Transform Infrared Spectroscopy; GC-MS: Gas Chromatography-Mass Spectrometry; DLS: Dynamic Light Scattering; PdI: Polydispersity Index; BBB: Blood-Brain Barrier; DPPH: 2,2-Diphenyl-1-picrylhydrazyl Radical Assay; ABTS: 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Assay; BSLT: Brine Shrimp Lethality Test; GI: Gastrointestinal.

ACKNOWLEDGEMENTS

The authors sincerely acknowledge the Division of Biotechnology, Karunya Institute of Technology and Sciences, for providing the necessary facilities and support that enabled the successful completion of this research.

AUTHOR CONTRIBUTIONS

Riddhi Upadhyay: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Lead), Validation (Lead), Resource (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Lead), Investigation (Equal); Murugan Sevanan: Conceptualization (Equal), Methodology (Lead), Formal Analysis (Equal), Validation (Lead), Resource (Equal), Writing – Original Draft (Equal), Writing – Review & Editing (Equal), Investigation (Equal), Supervision (Lead), Project administration (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

DATA AVAILABILITY

All the data have been included in the manuscript.

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