Evaluation of Bioactive Compounds, Antioxidant Activity, and Anticancer Potential of Wild Ganoderma lucidum Extracts from High-Altitude Regions of Nepal

1. Introduction

Ganoderma lucidum, commonly known as Lingzhi in China, Reishi in Japan, and Dadu chyau in Nepal, is a polypore mushroom with a long and storied history of use in East Asian medicine for promoting health and longevity [1]. It is often referred to as the “King of Herbs” or the “Mushroom of Immortality.” Its medicinal use dates back over 2,000 years, with its effects documented in ancient scripts like the Shen Nong Ben Cao Jing from China’s Eastern Han dynasty (25-220 AD) [2]. Traditionally, it has been used to treat a variety of ailments and is believed to enhance stamina, increase brain power, improve circulation, and strengthen the immune system [3].

Modern research has begun to validate these traditional claims, attributing the mushroom’s medicinal properties to its rich and varied chemical composition. Bioactive compounds such as polysaccharides, triterpenes, adenosine, organic germanium, phenolic compounds, flavonoids, and ergosterol contribute to its therapeutic effects, which include antioxidant, anti-cancer, anti-inflammatory, and antimicrobial activities [4,5,6,7,8]. For instance, triterpenic acids have demonstrated significant anti-cancer effects, while polysaccharides have shown anti-diabetic and antibiotic properties [9,10,11,12]. The antioxidant properties of G. lucidum are particularly noteworthy, as they neutralize reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are implicated in the development of chronic diseases such as cancer, aging, diabetes, cardiovascular disorders, and neurodegeneration [4,13]. Studies have shown that polysaccharides in G. lucidum exhibit potent ROS-scavenging activity and can inhibit tumor growth through immunomodulation and the induction of apoptosis [6,9,10]. Furthermore, its anti-inflammatory effects have been observed in animal models, suggesting significant therapeutic potential [14].

Nepal, with its diverse geography ranging from the Terai plains to the high Himalayas, provides a unique environment for a wide variety of mushrooms [15,16,17,18,19]. While approximately 1,300 mushroom species have been reported in Nepal, with 73 identified as having medicinal value, the therapeutic potential of most, including native G. lucidum, remains largely unexplored [20,21]. Research on Nepalese mushrooms, initiated by the Nepal Agricultural Research Council (NARC) in 1974, has historically focused more on cultivation and taxonomy rather than on the detailed analysis of their bioactive properties.

The unique environmental conditions of Nepal’s high-altitude regions—characterized by lower oxygen levels, intense UV radiation, and distinct soil compositions—may lead to the production of G. lucidum strains with enhanced or novel bioactive profiles [22,23]. Despite Nepal’s rich biodiversity, research on high-altitude G. lucidum is scarce, leaving a gap in our understanding of its full potential. This study, therefore, aims to investigate the bioactive constituents, antioxidant activities, and cytotoxic effects of wild G. lucidum collected from the high altitudes of Nepal, utilizing various solvent extracts to comprehensively characterize its mycochemical composition and therapeutic promise.

Figure 1. Overview of the study workflow, from G. lucidum collection and solvent extraction to bioactive compound screening, biological activity assays, and GC-MS profiling.

Figure 1. Overview of the study workflow, from G. lucidum collection and solvent extraction to bioactive compound screening, biological activity assays, and GC-MS profiling.

2. Materials and Methods

2.1. Collection and Identification

Fresh fruiting bodies of wild G. lucidum were collected from a dead trunk of Quercus lanata on Chandragiri Hill, Nepal (elevation 7482 ft; latitude 27.67402; longitude 85.19874) (Figure. 2). The specimens were identified based on detailed macro- and micro-morphological characteristics (Table 1).

Figure 2. Map of Nepal in a global context (A), G. lucidum collection site (B) and its fruiting body growing on Quercus lanata (C).

Figure 2. Map of Nepal in a global context (A), G. lucidum collection site (B) and its fruiting body growing on Quercus lanata (C).

2.2. Sample Preparation and Extraction

The samples were cleaned and oven-dried gradually from 45 °C to 60 °C for 3 days until a constant weight was attained. The dried mushroom was milled into a fine powder and stored in airtight containers for future use. 10 gm of G. lucidum were subjected for extraction. Solvent extraction was performed for 10 hrs in a Soxhlet apparatus with 250 mL each of water (GWE, 100 °C), 70% ethanol (GEE, 60 °C), 80% methanol (GME, 70 °C), and 50% acetone (GAE, 50 °C). Extracts were concentrated under vacuum in a rotary evaporator (50 °C) and stored in dark vials at 4 °C. The calculation of the % yield was done for each solvent using the formula,

$$\mathrm{\%}\mathrm{Y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}={\displaystyle \frac{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{G}.\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{u}\mathrm{m}\mathrm{p}\mathrm{o}\mathrm{w}\mathrm{d}\mathrm{e}\mathrm{r}\mathrm{b}\mathrm{e}\mathrm{f}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\mathrm{g}\mathrm{m}\right)}{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{o}\mathrm{b}\mathrm{t}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{d}\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\left(\mathrm{g}\mathrm{m}\right)}}\mathrm{X}100$$

2.3. Estimation of Total Phenolic, Flavonoid, β-carotene and Lycopene

2.3.1. Total Phenolic Content

Total phenolic content was determined using the Folin-Ciocalteu method with pyrogallol as the standard [24]. A calibration curve was established by measuring the absorbance of pyrogallol standards (10-100 µg/mL) at 760 nm. Sample solutions (1 mL) were reacted with 5 mL of 10% Folin-Ciocalteu reagent for 5 minutes at room temperature. Subsequently, 4 mL of Na₂CO₃ solution was added, and samples were vigorously mixed and incubated in the dark for 2 hours at room temperature. Absorbance was read at 760 nm, and results were expressed as pyrogallol equivalents (mg/g dry extract).

2.3.2. Total Flavonoid Content

Total flavonoid content was determined using the aluminum chloride colorimetric method, adapted from Shraim et al., 2021[25]. Quercetin standard curve was prepared by diluting a 10 mg quercetin stock in 50% methanol to concentrations ranging from 10-100 µg/mL, with absorbance measured at 415 nm. Sample aliquots (1.0 mL) were mixed with 0.5 mL of 1.2% aluminum chloride, 0.5 mL of 120 mM potassium acetate, and 1 mL of distilled water. After 30 minutes of incubation at room temperature, absorbance was read at 415 nm. Results were expressed as mg quercetin equivalents per gram of dry extract (mg QE/g dry weight).

2.3.3. Estimation of β-Carotene and Lycopene Content

β-carotene and lycopene were determined as per Prakash et al., 2016 [26]. Dried extracts (100 mg) were extracted with 10 mL of acetone-hexane (4:6) for 1 min, then filtered. Filtrate absorbance was measured at 453, 505, 645, and 663 nm. Carotenoid concentrations (mg/100 mL) were calculated using the following equations:

Lycopene = (−0.0458 × A₆₆₃) + (0.372 × A₅₀₅) + (0.0806 × A₄₅₃)

β-carotene = (0.216 × A₆₆₃) − (0.304 × A₅₀₅) + (0.452 × A₄₅₃)

Results are presented as mg carotenoid per gram of dry extract (mg carotenoid/g dry extract)

2.4. Determination of In Vitro Antioxidant Activities

2.4.1. DPPH (2, 2-diphenyl-1-picryl-hydrazyl) Assay

Different concentrations of extracts (20-100 µg/mL) were prepared. To 1 mL of each extract concentration, 2 mL of ice-cold 0.1 mM DPPH solution was added. The mixtures were incubated in the dark at room temperature for 30 min. Absorbance was then measured at 517 nm against a methanol blank [27]. A 3 mL DPPH solution was used as the control. Percentage inhibition was calculated using the formula:

% inhibition = [(A₀-A₁)/(A₀)] x 100

where A₀ is the absorbance of the control and A₁ is the absorbance of the sample.

2.4.2. Nitric Oxide Radical Scavenging Assay

Nitric oxide radical scavenging activity was determined with slight modification from Alam et al., 2013 [28]. Samples or ascorbic acid (20-100 µg/mL, 1 mL) were mixed with 2 mL of 10 mM sodium nitroprusside in phosphate buffer and incubated at 25°C for 2.5 hours. To 3 mL of the incubated solution, 3 mL of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride in 2% H3PO3) was added. Absorbance of the pink color was measured at 540 nm against a blank. Ascorbic acid served as a positive control. Percentage inhibition was calculated using:

% inhibition = [(A₀-A₁)/(A₀)] x 100

where A₀ is the absorbance of the control and A₁ is the absorbance of the sample.

2.4.3. Hydroxyl Radical Scavenging Assay

Hydroxyl radical scavenging activity was assessed by measuring the inhibition of salicylic acid hydroxylation [29]. The 7 mL reaction mixture contained 1 mL of sample/standard (100-500 µg/mL), 2 mL of 6 mM FeSO₄, 2 mL of 6 mM H₂O₂, and 2 mL of 6 mM salicylic acid. After incubation at 37 °C for 1 hour, absorbance was measured at 510 nm due to the color change of salicylic acid. Scavenging activity was calculated as follows:

% inhibition = [(A₀-A₁)/(A₀)] x 100

Where A₀ is the absorbance of the control and A₁ is the absorbance of the sample.

2.4.4. Superoxide Radical Scavenging Assay

Superoxide radical scavenging activity was assessed by a modified pyrogallol auto-oxidation method [29]. The reaction mixture contained 4.5 mL of 50 mM Tris-HCl buffer (pH 8.2), 0.4 mL of 25 mM pyrogallol, and 1 mL of sample (0.1-0.5 mg/mL). After 5 min incubation at 25°C, the reaction was terminated by adding 1 mL of 8 mM HCl. Absorbance was measured at 420 nm. Ascorbic acid served as the positive control. Superoxide radical scavenging activity was calculated using the formula:

% inhibition = [(A₀-A₁)/(A₀)] x 100

Where A₀ is the absorbance of the control and A₁ is the absorbance of the sample.

2.4.5. Reducing Power Assay

The reducing power of samples was assessed via the ferric reducing antioxidant power (FRAP) assay [30]. One mL of sample or standard (20-100 µg/mL) was combined with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% (w/v) K₃Fe(CN)₆. After 20 min incubation at 50 °C in a water bath, 2.5 mL of 10% (w/v) trichloroacetic acid was added to terminate the reaction. The mixture was centrifuged at 3000 rpm for 10 min. A 2.5 mL aliquot of the supernatant was then mixed with 2.5 mL distilled water and 0.5 mL of 0.1% (w/v) FeCl₃. Absorbance was measured at 700 nm against a blank.

2.5. Cytotoxicity Assay

HeLa cells were seeded in a 96-well plate. After 24 hours, cells were treated with dimethylsulfoxide or various extract concentrations for 48 hrs. Post-treatment, media were removed and replaced with 100 µL fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (final concentration 0.4 mg/mL). Plates were incubated at 37 °C for 3 hrs, during which intracellular purple formazan was observed. Next, 100 µL of solubilization solution (4 mM HCl, 0.1% NP40 in isopropanol) was added, and plates were kept in the dark for 15 minutes at room temperature. Absorbance was measured at 570 nm using a microplate reader [31].

2.6. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis

GC-MS analysis of G. lucidum extracts was conducted using a GCMS-QP2010 Ultra (Shimadzu, Kyoto, Japan) equipped with an Rtx-5 M5 capillary column (30 m × 0.02 mm i.d., 0.25 µm film thickness; Restek, Bellefonte, PA, USA). The operating conditions, including solvent cut-off, temperature program, and MS scan parameters, were identical to those described by Tiwari et al. 2023 [32]. Compounds were identified using NIST libraries (NIST 14, Gaithersburg, MD, USA).

2.7. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). Experiments were conducted in triplicate, expressed as mean ± SD. One-way ANOVA with Tukey’s post-hoc test compared extracts (p < 0.05).

3. Results

3.1. Extraction Yield

Extraction efficiency is affected by the chemical nature of bioactive compounds, the extraction method used, sample particle size, the solvent used, as well as the presence of interfering substances [33,34]. The yield of extraction depends on the solvent with varying polarity, temperature, pH, extraction time, and composition of the sample [33,34]. Extraction efficiency (% yield) varied significantly (p < 0.05) among solvents, with acetone yielding the highest crude extract (GAE; 5.01%), followed by ethanol (GEE; 3.43%), methanol (GME; 2.98%), and water (GWE; 2.29%) (Table 2).

3.1. Estimation of Total Phenolic and Flavonoid Content

Phenolic compounds are recognized as potent chain-breaking antioxidants due to the radical-scavenging capabilities of their hydroxyl groups [35]. The total phenolic content (TPC) exhibited significant variation among the tested solvents (Figure 3A). Ethanol extract (GEE) demonstrated the highest TPC (376.5 ± 9.3 mg PGE/g), which was significantly greater (p < 0.05) than that of methanol extract (GME; 97.3 ± 2.8 mg PGE/g), water extract (GWE; 96.6 ± 2.6 mg PGE/g), and acetone extract (GAE; 60.5 ± 7.4 mg PGE/g). Notably, the TPC of GEE exceeded values previously reported for 62 wild mushrooms from Nepal, including other Ganoderma species, and 29 other diverse mushroom species [16,17,36].

Flavonoids were quantified using the aluminum chloride method. GEE and GWE exhibited significantly higher TFCs, with values of 30.33 ± 0.5 mg QEs/g extract and 26.73 ± 0.6 mg QEs/g extract, respectively (Figure 3B). The low flavonoid content was observed in GME; 6.34 ± 0.4 mg QEs/g dry extract and GAE; 7.95 ± 0.23 mg QEs/g extract. Notably, GEE contained approximately 4.7-fold higher total flavonoids compared to GME.

3.2. Estimation of β-Carotene and Lycopene

The concentrations of lycopene and β-carotene in G. lucidum extracts were estimated spectrophotometrically. Carotenoid analysis demonstrated limited solvent efficacy. β-carotene content was highest in GME (0.4536 ± 0.000 mg/g), followed by GEE (0.1982 ± 0.006 mg/g), GWE (0.1595 ± 0.001 mg/g), and GAE (0.0944 ± 0.001 mg/g) (Figure 4A).

Similarly, lycopene content varied considerably among the extracts, ranging from 0.0163 ± 0.000 to 0.0670 ± 0.001 mg/g of dry extract (Figure 4B). The highest lycopene content was found in the methanolic extract (GME; 0.0670 ± 0.001 mg/g), followed by the ethanolic extract (GEE; 0.0308 ± 0.004 mg/g), water extract (GWE; 0.0175 ± 0.002 mg/g), and acetone extract (GAE; 0.0163 ± 0.000 mg/g). However, overall β-carotene and lycopene contents were comparatively low when contrasted with the phenolic and flavonoid contents of the same extracts.

3.3. Comparative In-Vitro Antioxidant Activities

Antioxidant activity cannot be definitively concluded from a single assay due to the diverse mechanisms involved and variations between in vitro test models. These diverse mechanims include free radical scavenging, metal ion chelation, reducing power, single electron transfer, and others [37,38]. Therefore, this study employed multiple in vitro antioxidant assays (DPPH radical scavenging, superoxide radical scavenging, hydroxyl radical scavenging, nitric oxide radical scavenging, and reducing power) to comprehensively evaluate and compare the antioxidant potential of G. lucidum solvent extracts.

3.3.1. DPPH Radical Scavenging Activity

The DPPH radical scavenging activity of the extracts (at 100 µg/mL) ranged from 86.58 ± 4.65% to 95.59 ± 0.17% (Figure 5). Methanolic extract (GME) exhibited the highest activity (95.59 ± 0.17%), followed by acetone extract (GAE; 92.13 ± 0.34%), ethanolic extract (GEE; 90.91 ± 0.06%), and water extract (GWE; 86.58 ± 4.65%). Ascorbic acid, as a standard, showed 97.26 ± 0.69% inhibition. All extracts demonstrated high antioxidant activity, scavenging over 80% of the DPPH radical even at 80 µg/mL. These findings indicate better scavenging activity compared to some previously reported wild mushrooms from Nepal, including G. lucidum [16,17,36].

3.3.2. Superoxide Radical Scavenging Activity

Superoxide radical scavenging activity was positively correlated with increasing extract concentrations (p < 0.05) (Figure 6). At 500 µg/mL, GEE exhibited the highest scavenging activity (72.24 ± 0.61%), followed by GME (54.84 ± 1.72%), GAE (39.37 ± 1.70%), and GWE (38.70 ± 0.65%). Ascorbic acid showed 98.80 ± 0.15% scavenging. The observed order of activity was: GEE > GME > GAE > GWE. These results suggest the extracts’ ability to scavenge superoxide anion radicals, potentially preventing oxidative damage, likely attributed to the electron-donating capacity of their phenolic hydroxyl groups [39].

3.3.3. Hydroxyl Radical Scavenging Activity

All samples exhibited significant dose-dependent hydroxyl radical scavenging activity. At tested concentrations, GME showed the highest activity (77.88 ± 0.15%), followed by GEE (76.62 ± 0.28%), and GWE (65.72 ± 0.13%), with GAE showing comparatively lower activity (42.12 ± 1.59%) (Figure 7). Ascorbic acid (47.73 ± 0.13%) served as a standard. Notably, the IC₅₀ values for GWE, GEE, and GME were lower than that of ascorbic acid, indicating their superior hydroxyl radical scavenging potential. The potent hydroxyl radical scavenging capacity of G. lucidum extracts suggests a preventive role against lipid peroxidation initiation and protection against DNA damage, mutagenesis, and cytotoxicity [40,41,42,43].

3.3.4. Nitric Oxide Radical Scavenging Activity

The extracts demonstrated good inhibition of nitric oxide radicals. At 100 µg/mL, GEE exhibited the highest scavenging potential (81.51%), followed by GME (63.81%), GAE (41.88%), and GWE (41.62%) (Figire 8). Ascorbic acid showed 97.02% inhibition. The order of activity was GEE > GME > GAE ≈ GWE. The ability of the extracts to scavenge nitric oxide suggests a potential to prevent peroxynitrite formation and a protective role against nitrosamine-mediated carcinogenesis in the digestive tract [44].

3.3.5. Reducing Power Assay

The reducing power assay (electron-donating capacity) further confirmed ethanol’s dominance (0.353 ± 0.003 at 100 µg/mL), aligning with radical scavenging trends (Figure 9). The reducing power of the extracts at 100 µg/mL followed the order: GEE (0.353 ± 0.002) > GME (0.176 ± 0.002) > GWE (0.164 ± 0.005) > GAE (0.158 ± 0.001). Standard ascorbic acid showed a reducing power of 0.493 ± 0.001 at the same concentration. The reducing powers of the ethanolic extracts were notably higher than those reported for other G. lucidum, Boletus edulis, and Pleurotus ostreatus [45,46,47,48]. This reducing capacity is likely due to their hydrogen-donating ability of the compounds present in the extracts, which can halt peroxide formation and terminate radical chain reactions [49,50].

3.4. MTT-Based Cytotoxicity Assay in HeLa Cells

Following the characterization of bioactive compounds and antioxidant activities, the cytotoxic potential of G. lucidum extracts was evaluated against human cervical cancer (HeLa) cells via MTT assay. Extracts were tested at concentrations of 100, 500, and 1000 µg/mL, and results are presented in Figure 10. All extracts exhibited a dose-dependent inhibition of HeLa cell viability. At the highest concentration tested (1000 µg/mL), GEE and GWE extracts demonstrated significantly higher (p < 0.05) cytotoxicity, suppressing cell proliferation by >65%. Specifically, GEE achieved 82.53 ± 1.46% inhibition and GWE 67.28 ± 1.39% inhibition. In comparison, GME and GAE showed more moderate inhibition at 1000 µg/mL, with 51.87 ± 2.1% and 34.78 ± 4.69 % inhibition, respectively.

3.5. IC₅₀ Comparison of Extraction Solvents for Antioxidant and Cytotoxicity Activities

The extraction efficiency and bioactive potential of G. lucidum were solvent-dependent, with GEE having the highest total phenolic (376.50 ± 9.32 PGE/g) and flavonoid (30.33 ± 0.50 QE/g) content, while GME showed higher carotenoids (lycopene: 0.067 ± 0.001 mg/g; β-carotene: 0.454 ± 0.000 mg/g) (Figure 11). Similarly, antioxidant assays revealed solvent-specific efficacy: GWE, GEE, GME, GAE exhibited an exceptional DPPH radical scavenging comparable to ascorbic acid, IC₅₀: 5.82 µg/mL), whereas GEE dominated in superoxide (IC₅₀: 328.95 µg/mL), nitric oxide (IC₅₀: 57.67 µg/mL), and reducing power (IC₅₀: 78.04 µg/mL) assays (Figure 11). Hydroxyl radical inhibition was strongest in GWE (IC₅₀: 237.89 µg/mL), closely followed by GEE (274.34 µg/mL). Cytotoxicity via MTT assay demonstrated dose-dependent inhibition, with GEE (IC₅₀: 520.19 µg/mL) and GWE (702.41 µg/mL) exhibiting moderate activity (Figure 11). Collectively, ethanol was found to be superior in extracting phenolic and flavonoid-rich fractions with high antioxidant capacity, despite GWE’s exceptional DPPH activity, likely due to ethanol extracting polar bioactive compounds with higher antioxidant capacity [51,52,53]. The correlation between ethanol’s high phenolic/flavonoid content and multi-target bioactivity positions it as the optimal solvent for extracting compounds with therapeutic potential against oxidative stress and cancer.

3.6. Solvent-Dependent Variation in Bioactive Compounds via GC-MS Profiling

GC-MS analysis of G. lucidum extracts showed solvent-dependent profiles of bioactive compounds, including sterols, triterpenoids, terpenoids, and polyphenols (Table 3). Fatty acids dominated ethanol (53.18%) and methanol (48.57%) extracts, with 9,12-octadecadienoic acid (linoleic derivative: 20.60% in ethanol, 14.05% in methanol) and pentadecanoic acid (14.52% in ethanol) as major constituents. Acetone exhibited the lowest fatty acid content (5.64%) but uniquely contained ergosterol (Vitamin D2 precursor) and retinoic acid. Polar protic solvents (ethanol, methanol) efficiently extracted free fatty acids and esters, including (E)-9-octadecenoic acid ethyl ester (oleic derivative: 3.86% in ethanol), while acetone’s mid-polarity favored sterols (7,22-ergostadienone, 9(11)-dehydroergosteryl benzoate) and triterpenoids (ergosta-4,6,8(14),22-tetraen-3-one). Similarly, Hinokione, an abietene diterpene, was detected across solvent extracts (0.9% in GAE, 2.9% in GEE, and 5.5% in GME) (Table 3).

Pharmacologically significant compounds included ferruginol (exclusive to ethanol), nerolidol acetate (methanol-specific), geranylgeraniol (anti-inflammatory terpenoid), and Hinokione (anti-inflammatory and anticancer) (Table 3). Ethanol and methanol extracts had the highest polyphenol, diterpenoid, and fatty acid content, whereas acetone had higher sterols and triterpenoids, demonstrating solvent polarity as a critical determinant of bioactive compound selectivity. These findings show G. lucidum’s diverse phytochemical composition and the impact of solvent choice in optimizing targeted metabolite extraction.

5. Conclusions

Our study aimed to investigate the therapeutic potential of G. lucidum from Nepal’s high-altitude regions, and our findings strongly confirms it as a key source of bioactive compounds. Through this work, we have shown that the choice of extraction solvent is critical, significantly impacting not only the yield but also the specific bioactive compounds obtained, and consequently, their biological activities. While acetone yielded the most crude extract, ethanol and methanol extract showed higher phenolic and flavonoid content, correlating with high antioxidant activity across a spectrum of in vitro assays. The ethanol and water extracts also demonstrated a powerful ability to inhibit HeLa cell growth. GC-MS analysis identified diverse array of beneficial compounds, including fatty acids, sterols like ergosterol, and various terpenoids (diterpenoids, triterpenoids). The specific distribution of these compounds varied depending on the extraction solvents, and they collectively contribute to the observed health benefits. Future research should focus on optimizing extraction methods and characterizing these individual compounds to maximize specific bioactivities, which will be critical for bridging the gap between traditional use and modern applications.