Safety assessment and in vitro cytotoxicity profiling of fruiting bodies from Lentinula edodes (shiitake) strains in HeLa and MCF-7 cell lines

Abstract

Medicinal mushrooms offer diverse therapeutic benefits, but proper administration is crucial to avoid long-term health issues linked to fungal toxicity. This study evaluated the safety and in vitro cytotoxic potential of Lentinula edodes (shiitake) mushrooms for broader use in food and health supplements. Two strains, DMRO-34 and DMRO-356, were tested for cytotoxic and nutraceutical properties. In vitro analysis using MTT and LDH assays on human cervical (HeLa) and breast cancer (MCF-7) cell lines showed that DMRO-356 exhibited greater anti-proliferative effects compared to DMRO-34. β-glucan content was quantified in both strains using a Megazyme assay as part of the compositional profile. In vivo studies with a limit dose of 5000 mg/kg body weight assessed acute and sub-acute toxicity over 14 and 28 days. Both strains showed no observable adverse effects (NOAEL) in rats, confirming their safety at the tested dose. The results indicate that L. edodes may possess bioactive properties and are well-tolerated in animal models. While the in vitro cytotoxic effects are promising, the in vivo results presented here establish their safety profile; however, further in vivo efficacy investigations are required to validate their therapeutic potential. Overall, these findings suggest that DMRO-34 and DMRO-356 are safe for use and may act as natural therapeutic agents, with DMRO-356 showing greater efficacy. This study supports the safe incorporation of L. edodes mushrooms into dietary supplements and functional foods, and highlights their potential role in adjunct strategies for cancer prevention and immune support.

Introduction

Mushrooms have long played an essential role in global healthcare systems, being used across many civilizations and cultures, not only in human nutrition but also in medicine¹. Many mushroom species are non-toxic and have been consumed as natural foods since ancient times². In recent decades, mushrooms and their extracts have gained worldwide popularity as nutritious and flavorful food³.

Among them, Lentinula edodes (Berk.) Pegler, commonly known as shiitake, is one of the most extensively cultivated species, accounting for approximately 22% of global mushroom production—second only to Pleurotus spp.⁴. Widely consumed in East Asian countries, L. edodes is valued for its rich nutritional composition, including essential vitamins, minerals, antioxidants, unsaturated fatty acids, and high-quality proteins⁵. It also exhibits a wide range of medicinal properties, including antimicrobial, antiviral, antioxidant, cholesterol-lowering, anti-hypertensive, cardiovascular protective, hepatoprotective, anti-inflammatory, anti-fibrotic, anti-diabetic, and anti-cancer properties⁶. Classic works by Mori⁷ and Breene⁸ provided comprehensive accounts of its traditional uses and nutritional value. While many natural products are now widely used for health benefits, unregulated or excessive consumption without evidence-based dosing may pose safety risks. Therefore, it is increasingly important to evaluate both efficacy and toxicity through rigorous scientific investigation.

Several studies suggest that the health-promoting effects of L. edodes arise from synergistic actions among its diverse bioactive constituents⁹. Methanolic extracts are particularly suitable for experimental evaluation because they effectively solubilize a wide range of bioactive compounds, resembling the composition of fractions typically ingested through dietary intake. Thus, assessing the biological activity of the whole mushroom extract is considered more representative than analyzing isolated compounds alone¹⁰. Furthermore, although L. edodes is typically consumed cooked, raw extracts were used in this study to account for potential retention or loss of bioactivity during thermal processing^11,12.

This study investigates the safety and in vitro cytotoxic potential of two L. edodes strains, DMRO-34 and DMRO-356. Cytotoxicity was evaluated using human cancer cell models (HeLa and MCF-7), and safety was assessed via acute and sub-acute oral toxicity studies in rodents. By addressing both biological activity and toxicological safety, this research support the responsible use of L. edodes in the development of functional foods and nutraceuticals, bridging traditional applications with modern scientific validation.

Results

In vitro toxicity study of L. edodes mushroom

Analysis of cell viability via MTT assay

In the present study, cytotoxic activity were assessed using different concentrations (10 µg/mL to 600 µg/mL) of DMRO-34 and DMRO-356 strains of L. edodes against the human cervical cancer cell line (HeLa) and breast cancer cell line (MCF-7) via the MTT assay. The results revealed that the effects of DMRO-34 and DMRO-356 strains on HeLa and MCF-7 cell lines followed a dose-dependent pattern. Specifically, in HeLa cells, the DMRO-356 strain at 500 µg/mL exhibited a 7.17% higher cytotoxic effect than DMRO-34, while in MCF-7 cells, the DMRO-356 strain at 500 µg/mL showed an 8.28% greater cytotoxic effect compared to DMRO-34.

The study observed a decrease in cancer cell viability as the mushroom concentration increased from 10 µg/mL to 500 µg/mL, followed by a slight increase in viability at the highest tested concentration (600 µg/mL). The mushroom extracts exhibited low cytotoxicity on cells at lower doses (10 µg/mL), but this effect intensified at higher doses (500 µg/mL), resulting in a percentage cell viabilities of 18.22 ± 0.24% for DMRO-34 and 12.35 ± 0.61% for DMRO-356 on HeLa cell lines at a concentration of 500 µg/mL, respectively (as shown in Table 1). In MCF-7 cells, the percentage of cell viability was higher, approximately 28.69 ± 0.68% for DMRO-34 versus 22.78 ± 0.59% for DMRO-356 at a concentration of 500 µg/mL.

Table 1.

Cytotoxic effects of L. edodes strains DMRO-34 and DMRO-356 on HeLa and MCF-7 cell lines assessed using the MTT assay.

Concentration in µg/mL	HeLa cell line		MCF-7 cell line
	DMRO-34	DMRO-356	DMRO-34	DMRO-356
	% Cell viability	% Cell viability	% Cell viability	% Cell viability
10	95.67 ± 0.36	92.44 ± 0.85	96.67 ± 0.51	93.45 ± 0.34
25	90.24 ± 0.37	89.67 ± 0.49	91.78 ± 0.08	91.23 ± 0.34
50	84.66 ± 0.36	80.55 ± 0.37	86.45 ± 0.51	80.44 ± 0.60
100	64.22 ± 0.49	61.45 ± 0.61	82.98 ± 0.68	72.98 ± 0.77
200	48.79 ± 0.60	44.23 ± 0.36	67.34 ± 0.69	51.76 ± 0.76
300	39.80 ± 0.36	29.98 ± 0.12	49.21 ± 0.42	45.01 ± 0.85
400	25.66 ± 0.73	23.87 ± 0.85	44.22 ± 0.52	42.11 ± 0.34
500	18.22 ± 0.24	12.35 ± 0.61	28.69 ± 0.68	22.78 ± 0.59
600	28.78 ± 0.45	24.23 ± 0.25	40.99 ± 0.52	34.58 ± 0.22

Values are presented as mean ± standard deviation (SD) from triplicate samples.

The IC₅₀ value was determined to be 200 µg/mL for DMRO-34 and 150 µg/mL for DMRO-356 in HeLa cells, while in MCF-7 cells, the IC₅₀ value were found to be 300 µg/mL for DMRO-34 and 250 µg/mL for DMRO-356 are within the expected range for crude natural extracts^13,14.

Assessment of cytotoxicity using LDH leakage assay

The LDH leakage assay was conducted to assess the cytotoxic activity of different concentrations (10–600 µg/mL) of DMRO-34 and DMRO-356 strains of L. edodes on HeLa and MCF-7 cell lines. HeLa cells treated with concentrations of 500 µg/mL of both DMRO-34 and DMRO-356 strains for 48 h showed significantly higher LDH release values of 77.48 ± 0.49% and 84.22 ± 0.45%, respectively. The DMRO-356 strain demonstrated greater effectiveness in inducing cytotoxicity against HeLa cells compared to DMRO-34 (Table 2).

Table 2.

Assessment of cytotoxic effects of L. edodes strains DMRO-34 and DMRO-356 on HeLa and MCF-7 cell lines using the LDH leakage.

Concentration in µg/mL	HeLa cell line		MCF-7 cell line
	DMRO-34 (% LDH)	DMRO-356 (% LDH)	DMRO-34 (% LDH)	DMRO-356 (% LDH)
10	–	–	–	–
25	10.77 ± 0.34	6.34 ± 0.38	7.45 ± 0.36	14.33 ± 0.35
50	15.67 ± 0.21	16.88 ± 0.24	15.33 ± 0.76	26.98 ± 0.28
100	39.45 ± 0.67	18.97 ± 0.55	19.76 ± 0.45	29.34 ± 0.37
200	56.39 ± 0.55	34.56 ± 0.44	20.44 ± 0.39	36.45 ± 0.49
300	66.19 ± 0.42	63.28 ± 0.76	23.84 ± 0.36	42.33 ± 0.61
400	72.61 ± 0.39	75.44 ± 0.65	28.67 ± 0.44	45.63 ± 0.54
500	77.48 ± 0.49	84.22 ± 0.45	32.33 ± 0.21	52.25 ± 0.32
600	74.15 ± 0.34	80.33 ± 0.67	29.03 ± 0.23	48.04 ± 0.52

Results are presented as Mean ± SD values obtained from triplicate samples.

In MCF-7 cell lines, the DMRO-34 strain extract showed a significant increase in % LDH leakage, indicating cell membrane damage, with a value of 32.33 ± 0.21%. In comparison, DMRO-356 exhibited the highest % LDH leakage at 52.25 ± 0.32%, implying that the DMRO-356 sample had a more pronounced cytotoxic effect than DMRO-34.

Apoptotic effect of L. edodes mushroom on HeLa and MCF-7 cell lines

Cancer cell apoptosis was studied using nuclear morphology to assess cell viability, apoptosis, and necrosis. In this method, viable cells are characterized by an intact green nucleus, apoptotic cells exhibit a condensed green nucleus with a compact or scattered structure, and necrotic cells display a red nucleus. Based on MTT and LDH assay results, a concentration of 500 µg/mL was selected for further analysis due to its significant inhibitory effect on cancer cells. HeLa and MCF-7 cells treated with both extracts of DMRO-34 and DMRO-356 strains at this concentration were stained with acridine orange and ethidium bromide and visualized under a fluorescence microscope.

The red/orange fluorescence signal from EtBr—expected in apoptotic or necrotic cells—was not prominently captured in the presented images, likely due to suboptimal imaging conditions such as dye concentration, exposure time, camera sensitivity, or overlay settings. Nevertheless, morphological features consistent with apoptosis, including cell shrinkage, chromatin condensation, and apoptotic body formation, were clearly visible, particularly in DMRO-356-treated cells.

In HeLa cells, DMRO-356 treatment induced a higher apoptosis index (63%) compared to DMRO-34 (48%), suggesting about 31% greater apoptotic effect. Cells treated with DMRO-356 exhibited more pronounced apoptotic features, such as chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. The positive control group showed 61% apoptosis, while the normal cell group exhibited minimal apoptosis (3%) (Figs. 1, 3).

Fig. 1.

Apoptosis induction in HeLa cells was observed by fluorescence microscopy following treatment with methanolic extracts of L. edodes strains (500 µg/mL). (A) Normal (untreated) cells with minimal fluorescence. (B) Positive control: staurosporine-treated HeLa cells showing marked apoptotic nuclear changes and strong fluorescence. (C) HeLa cells treated with DMRO-34 extract exhibiting moderate nuclear condensation and apoptotic morphology. (D) HeLa cells treated with DMRO-356 extract exhibited pronounced nuclear changes and enhanced fluorescence indicative of apoptosis. Cells were stained with acridine orange (AO) and ethidium bromide (EtBr). The red/orange EtBr signal expected in apoptotic or necrotic cells was not prominently captured in the images—likely due to imaging limitations such as dye concentration or exposure settings—however, characteristic apoptotic morphology was evident, particularly in DMRO-356-treated cells. All images were captured at a 100 μm scale using identical fluorescence microscopy settings.

Fig. 3.

Comparison of apoptosis index in HeLa and MCF-7 cells following treatment with methanolic extracts of L. edodes strains (500 µg/mL). The bar graph shows the percentage of apoptotic cells in normal (untreated), positive control (staurosporine-treated), and extract-treated groups (DMRO-34 and DMRO-356). Both HeLa and MCF-7 cell lines exhibited increased apoptosis following treatment, with the DMRO-356 extract inducing the highest apoptosis index (63% in HeLa and 56% in MCF-7 cells). Data represent mean ± SD from three independent experiments (P < 0.05 compared to untreated control).

Similarly, MCF-7 cells treated with DMRO-356 displayed a significantly higher apoptosis index (56%) compared to those treated with DMRO-34 (43%), demonstrating approximately 30% greater induction of apoptosis. These results were consistent with the HeLa cell observations. The positive control for MCF-7 showed 59% apoptosis, whereas normal cells showed a baseline level of 2% (Figs. 2, 3).

Fig. 2.

Fluorescence microscopy analysis of apoptosis induction in MCF-7 cells treated with methanolic extracts of L. edodes strains (500 µg/mL). (A) Untreated control cells show minimal fluorescence. (B) Positive control (staurosporine-treated cells) exhibiting pronounced nuclear fluorescence. (C) MCF-7 cells treated with DMRO-34 extract showing moderate nuclear condensation and apoptotic morphology. (D) MCF-7 cells treated with the DMRO-356 extract displayed enhanced fluorescence and features consistent with apoptosis. Cells were stained with acridine orange (AO) and ethidium bromide (EtBr). The red/orange EtBr signal expected in apoptotic or necrotic cells was not prominently captured in the images—likely due to imaging limitations such as dye concentration, exposure time, or camera sensitivity—however, characteristic apoptotic morphology was evident, particularly in DMRO-356-treated cells. All images were captured using identical fluorescence microscopy settings at a 100 μm scale.

Quantitative apoptosis index data from replicate images confirmed the higher apoptotic activity of DMRO-356 in both cell lines, despite the imaging limitation, as illustrated in the bar graph (Fig. 3). Overall, these results demonstrate that DMRO-356 is more effective than DMRO-34 in promoting apoptosis in both HeLa and MCF-7 cancer cell lines.

Measurement of beta-glucan content in DMRO-34 and DMRO-356 strains of L. edodes

To further investigate the potential basis for the observed differences in cytotoxicity, β-glucan (lentinan) levels were quantified in both strains. The DMRO-356 strain exhibited a significantly higher β-glucan content (30.369 ± 0.009 %) compared to the DMRO-34 strain (15.423 ± 0.032 %) on a dry weight basis (P < 0.0001). This increased lentinan concentration may contribute to the enhanced cytotoxic effects observed in DMRO-356. The results are summarized in Table 3.

Table 3.

Determination of β-glucan levels in L. edodes strains DMRO-34 and DMRO-356.

Strains	DMRO-34	DMRO-356
Lentinan Content (%)	15.423 ± 0.032	30.369 ± 0.009***

Dunnett’s test was performed for statistical analysis, where *** indicates highly significant (P < 0.0001), **significant (P < 0.001), *less significant (P < 0.05), and ns indicates non-significant (P > 0.05).

In vivo toxicity study of L. edodes mushroom

Acute oral toxicity study of L. edodes mushroom

Body weight, feed, and water consumption The mean body weight of all rats steadily increased throughout the 14‑day study (Fig. 4). A one‑way analysis of variance (ANOVA) of final body weight on day 14 revealed no significant differences among the three groups (F = 0.27, P = 0.764), and post hoc Tukey’s tests confirmed no significant pairwise differences (P > 0.05).

Fig. 4.

Graph showing changes in body weight of rats treated with 5000 mg/kg of L.edodes mushroom sample (Groups II and III) over a 14 day period, compared with the control Group (Group I).

To further evaluate longitudinal changes, a mixed between–within subjects ANOVA was performed. This analysis revealed a significant main effect of time (P < 0.001), indicating progressive weight gain across all groups, but no significant main effect of group (P > 0.25) and no group × time interaction (P > 0.05). These findings indicate that L. edodes supplementation did not affect body weight or overall health during the study.

Error bars represent the mean ± standard deviation of five rats per group. The control group (Group I) received CMC, while Groups II and III were treated with DMRO-34 and DMRO-356 strains of L. edodes mushroom, respectively.

Following a single-dose treatment of 5000 mg/kg of both strains of L. edodes, feed consumption by male rats showed no substantial difference from the untreated (control) Group over the 14-day (Fig. 5). To capture the temporal pattern of consumption, a mixed between-within subjects ANOVA was conducted. This analysis revealed a significant main effect of time (P < 0.05), indicating natural fluctuations in feed intake over the study duration. However, no significant main effect of group or group × time interaction was observed (P > 0.05), suggesting that feed intake patterns were consistent across all treatment groups.

Fig. 5.

Graph showing feed intake in rats treated with 5000 mg/kg of L.edodes mushroom samples for 14 days (Groups II and III), compared with the control Group (Group I).

On day 14, mean feed consumption was 78.47 g in the control group, 89.63 g in the DMRO-34 group, and 91.56 g in the DMRO-356 group. These findings confirm that dietary behaviour was unaffected by mushroom sample administration, further supporting the absence of treatment-related toxicity or metabolic disruption.

All data represent the mean ± SD of five rats per group. The control group (Group I) received CMC, while Groups II and III were treated with DMRO-34 and DMRO-356 strains of L. edodes mushrooms, respectively.

At the dose level of 5000 mg/kg, the water intake of rats in both treatment groups gradually increased. However, this increase in water intake was within the normal range and did not reach statistical significance compared with the untreated group (Fig. 6).

Fig. 6.

Graph showing water intake of rats in Groups II and III treated with 5000 mg/kg of L.edodes mushroom samples for 14 days, compared with the control Group (Group I).

A mixed between-within subjects ANOVA showed a significant main effect of time (P < 0.05), indicating natural variation in water intake across the 14-day period. However, there was no significant main effect of treatment group (P > 0.05) or group × time interaction, suggesting that water consumption trends were similar across all groups regardless of treatment. These results confirm that administration of L. edodes samples did not lead to abnormal shifts in hydration patterns, supporting their non-toxicological profile under the tested conditions.

All data represent the mean ± SD of five rats per group. The control group (Group I) received CMC, while Groups II and III were treated with DMRO-34 and DMRO-356 strains of L. edodes mushrooms, respectively.

Mortality and Clinical signs of toxicity: The treated rats were monitored for clinical signs of intoxication or behavioral changes for the first four hours after dosing, then at one-hour intervals for the next 24 h, and subsequently twice a day for the next 14 days. At the dose level of 5000 mg/kg body weight, no abnormalities in mortality or clinical signs—such as changes in skin and fur, eyes, and mucous membranes, salivation, lacrimation, pale mucous membrane, diarrhoeal faeces, hunched posture, scratching, polyuria, hypoactivity, and other indicators — were observed in Groups II and III. All clinical signs were graded as normal.

Necropsy following treatment with L. edodes mushroom: At the dose level of 5000 mg/kg, necropsy revealed no gross pathological lesions in any examined organ—including the liver, kidney, heart, spleen, testis, lungs, and brain—of treated rats. All organs appeared normal in size, shape, and colour.

Sub-acute oral toxicity study of L. edodes mushroom

Effect of treatment on male and female body weight

Rats in Groups II and III, treated with the DMRO-34 and DMRO-356 strains of mushrooms at a dose levels of 5000 mg/kg, exhibited a higher mean male body weight than rats in Group I (control) from Day 0 to Day 28. In comparison with the control group I, the percent enhancement was 31.93 % in Group III and 23.40 % in Group II. Although body weight increased in the treated groups, the change was not statistically significant and remained within the normal range.

Throughout the observation period, male rats in both treatment groups consistently demonstrated differences in average body weight. Specifically, on Days 7, 14, 21, and 28 post-treatment, rats in Group III consistently displayed higher body weights compared with the control Group, while rats in Group II exhibited slightly lower body weights. The results are presented in Fig. 7.

Fig. 7.

Graph showing mean body weight changes in male and female rats treated with 5000 mg/kg of DMRO-34 and DMRO-356 strains of L. edodes mushroom.

A mixed between-within subjects ANOVA revealed a significant main effect of group (F(5, 162) = 5.979, P = 4.19 × 10⁻⁵, η² = 0.156), indicating overall differences in mean body weight across the six treatment groups. There was also a significant main effect of time (F(3, 486) = 112.34, P < 0.001, η² = 0.410), suggesting that body weight changed significantly over time in all groups. Furthermore, a significant group × time interaction was observed (F(15, 486) = 2.89, P = 0.001, η² = 0.082), indicating that the trajectory of weight change over time differed among groups.

Group I (control) received CMC, while Groups II and III were treated with DMRO-34 and DMRO-356, respectively. Similarly, Group IV (control) received CMC, while Groups V and VI were treated with DMRO-34 and DMRO-356 strains, respectively. GI, GII, and GIII represent male rats, while GIV, GV, and GVI represent female rats.

Female rats in Groups V and VI (5000 mg/kg) exhibited a higher mean body weight from Day 0 to Day 28 compared with Group IV (control). The percentage enhancement was 34.95 % in Group V and 30.30 % in Group VI compared control Group IV, as shown in Fig. 7. The increase in body weight for the treated groups was not significant and remained within the normal range.

Feed and water intake measurements

At the 5000 mg/kg treatment level, feed intake gradually increased in both male and female rats over the 28 days. Feed intake remained within the normal range in all treatment groups, with no substantial changes observed between treated and control groups. Although male rats in the control group experienced reduced food consumption during the middle of the study, this decrease recovered by the end of the observation period.

A mixed between-within subjects ANOVA revealed a statistically significant main effect of group (F(5, 162) = 9.536, P = 1.24 × 10⁻⁷, η² = 0.263), indicating overall differences in feed intake among the treatment groups. There was also a significant main effect of time (P < 0.001), suggesting that feed intake changed significantly over the 28-day period in all groups. Furthermore, a significant group × time interaction (P < 0.05) indicated that the pattern of change in feed intake across time points varied between groups (Fig. 8).

Fig. 8.

Graph showing feed intake in rats treated with 5000 mg/kg of L. edodes mushroom samples across all groups. Groups I and IV (control) received CMC, Groups II and V were treated with DMRO-34, and Groups III and VI were treated with DMRO-356. GI, GII, and GIII represent male rats, while GIV, GV, and GVI represent female rats.

Rats in Groups II, III, V, and VI of both sexes, were administered 5000 mg/kg of mushroom sample, and their water intake gradually increased over 28 days. All treated groups exhibited normal water intake compared with the control CMC-treated Groups I and IV.

A mixed between-within subjects ANOVA revealed a statistically significant main effect of group (F = 3.08, P = 0.0109, η² = 0.0183), suggesting that water intake differed between treatment groups. There was also a significant main effect of time (P < 0.05), indicating that water intake changed over the 28-day period in all groups. However, the group × time interaction was modest, suggesting relatively consistent patterns of change across groups (Fig. 9).

Fig. 9.

Graph showing water intake across all groups of rats treated with 5000 mg/kg of L. edodes mushroom samples. Groups I and IV (control) received CMC, while Groups II and V were treated with DMRO-34, and Groups III and VI were treated with DMRO-356. GI, GII, and GIII represent male rats, while GIV, GV, and GVI represent female rats.

Assessment of toxicity symptoms and mortality rates

No clinical symptoms of toxicity or mortality were observed in rats of both sexes treated with both strains of L. edodes at a dose levels of 5000 mg/kg body weight. Clinical observations were recorded for the first four hours post-dosing, then hourly for the next 24 h, and subsequently twice daily for 28 days. Parameters included skin and fur condition, mucosal integrity, salivation, lacrimation, posture, activity levels, and excretory behaviour (including diarrhoea or polyuria). Diarrhoea was identified by the presence of loose or watery faecal matter in the bedding.

Clinical signs were graded using a standardized scale: 0 = Normal, + = Mild, ++ = Moderate, +++ = High, ++++ = Severe. Based on these assessments, moderate diarrhoea (++D) was observed in one male rat from Group II on Day 4, and mild diarrhoea (+D) in one female rat from Group V on Day 9. All other animals appeared normal, and no mortality was recorded in any group (Table 4).

Table 4.

Assessment of toxicity-related clinical signs and mortality in male and female rats across groups treated with  5000 mg/kg of L. edodes mushroom samples.

Groups	Sex	No. of rats	% Mortality (28 days)	Clinical Sign
G I (Control)	M	5	Nil	NAD
G II (DMRO-34)	M	5	Nil	++ D
G III (DMRO-356)	M	5	Nil	NAD
G IV (Control)	F	5	Nil	NAD
G V (DMRO-34)	F	5	Nil	+D
G VI (DMRO-356)	F	5	Nil	NAD

Clinical signs were graded as 0 = Normal, + = Mild, ++ = Moderate, +++ = High, and ++++ = Severe. “Nil” indicates no observed mortality, “NAD” = no adverse effect detected, “+D” = mild diarrhoea, and “++D” = moderate diarrhoea. GI, GII, and GIII represent male rats; while GIV, GV, and GVI represent female rats.

Assessment of hematological parameters

Male and female rats in Groups II, III, V, and VI showed no significant changes in haemoglobin (Hb), red blood cell (RBC), white blood cell (WBC), mean corpuscular haemoglobin (MCH), platelet count (PLT), lymphocyte, percentage (Lym %), or other measured indices after receiving DMRO-34 and DMRO-356 strains of L. edodes mushroom powder (5000 mg/kg) for 28 days. None of the parameters differed significantly (P > 0.05) compared with their respective control groups (I and IV) (Table 5).

Table 5.

Haematological parameters in male and female rats after 28 days of treatment with 5000 mg/kg of L. edodes mushroom samples.

Sex	Male			Female
Parameters	G I (control)	G II (DMRO-34)	G III (DMRO-356)	G IV (control)	G V (DMRO-34)	G VI (DMRO-356)
Hb (g %)	11.78 ± 0.55	11.90 ± 0.12	11.98 ± 0.10	11.85 ± 0.35	11.70 ± 0.45	12.00 ± 0.48
WBC (x 103cmm)	5.83 ± 0.16	5.83 ± 0.09	5.70 ± 0.26	5.53 ± 0.32	5.62 ± 0.27	5.32 ± 0.13
RBC (x 106/cmm)	6.79 ± 0.14	6.84 ± 0.11	6.95 ± 0.37	6.56 ± 0.20	6.79 ± 0.22	6.90 ± 0.44
Lym (%)	63.00 ± 0.31	63.47 ± 0.35	63.20 ± 0.37	63.09 ± 0.20	63.44 ± 0.27	63.070 ± 1.32
MID (%)	7.74 ± 0.36	7.76 ± 0.34	7.48 ± 0.33	7.64 ± 0.31	7.89 ± 0.25	7.42 ± 0.37
Gran (%)	28.79 ± 0.74	29.11 ± 0.29	28.75 ± 0.28	28.42 ± 0.27	28.92 ± 0.16	28.42 ± 0.24
PLT	495.0 ± 10.0	463.7 ± 8.02	481.7 ± 29.8	495.0 ± 9.52	478.6 ± 38.28	495.3 ± 15.95
MCH (pg)	15.1 ± 0.55	15.16 ± 0.16	15.10 ± 0.18	15.19 ± 0.16	15.15 ± 0.16	15.27 ± 0.25
MCHC (%)	28.60 ± 0.3	28.30 ± 0.21	28.8 ± 0.29	28.63 ± 0.20	28.26 ± 0.25	28.46 ± 0.19
MCV (fL)	51.86 ± 1.82	51.02 ± 1.02	50.55 ± 0.60	51.86 ± 1.44	50.94 ± 0.83	50.75 ± 1.35
HCT (%)	35.76 ± 0.88	35.92 ± 0.46	36.03 ± 0.05	35.66 ± 0.76	36.10 ± 0.64	36.26 ± 0.64

Group I & VI—male & female control groups; Group II & V—male & female rats treated with the DMRO-34 strain of L. edodes; Group III & VI—male & female rats treated with the DMRO-356 strain of L. edodes. Abbreviations: Hb = hemoglobin; WBC = white blood cells; RBC = red blood cells; Lym% = lymphocyte percentage; MID% = percentage of lymphocytes, neutrophils, monocytes, eosinophils, and basophils; Gran% = granulocyte percentage; PLT = platelet count; MCH (pg) = mean corpuscular hemoglobin (picogram, 10^-12 g); MCHC (%) = mean corpuscular hemoglobin concentration (%); MCV (fL) = mean corpuscular volume (femtoliter, 10^− 15 L); HCT (%) = hematocrit; (cmm): cells per cubic millimeter.

Analysis of biochemical parameters

Comparison with control groups (Groups I and IV), male and female rats (Groups II, III, V, and VI) treated with DMRO-34 and DMRO-356 strains of L. edodes mushroom powder (5000 mg/kg body weight for 28 days) showed no signs of changes in biochemical parameters, including total protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine, bilirubin, tissue transglutaminase IgA (TGA), cholesterol, and blood urea nitrogen (BUN). Only cholesterol showed a slight increase (P < 0.05) in female rats treated with DMRO-34, but it remained within the normal range (47–88 mg/dL)¹⁵. No other parameters exhibited significant changes (P > 0.05) when compared with the control groups. The results are presented in Table 6.

Table 6.

Biochemical parameters in male and female rats after 28 days of treatment with 5000 mg/kg of L. edodes mushroom samples.

Sex	Male			Female
Parameters	G I (control)	G II (DMRO-34)	G III (DMRO-356)	G IV (control)	G V (DMRO-34)	G VI (DMRO-356)
AST (IU/L)	45.93 ± 3.66	46.52 ± 3.48	46.38 ± 2.18	46.85 ± 2.52	45.61 ± 1.90	47.48 ± 1.79
ALT (IU/L)	44.66 ± 1.13	43.32 ± 1.45	43.28 ± 1.68	44.75 ± 0.98	46.27 ± 2.00	45.86 ± 0.46
ALP (IU/L)	106.40 ± 6.94	105.40 ± 2.0	105.44 ± 1.04	110.90 ± 2.72	112.7 ± 2.02	109.24 ± 2.05
CRE (mg/dL)	0.372 ± 0.025	0.36 ± 0.041	0.32 ± 0.03	0.312 ± 0.025	0.28 ± 0.021	0.28 ± 0.024
TB (mg/dL)	0.24 ± 0.031	0.25 ± 0.040	0.21 ± 0.041	0.184 ± 0.011	0.178 ± 0.019	0.184 ± 0.011
TP (mg/dL)	4.72 ± 0.26	4.71 ± 0.24	4.99 ± 0.184	5.38 ± 0.32	5.21 ± 0.14	5.09 ± 0.098
CHO (mg/dL)	63.01 ± 0.79	63.99 ± 0.84	63.59 ± 1.64	65.13 ± 0.68	68.50 ± 0.87	66.10 ± 1.01
TGA (mg/dL)	95.78 ± 2.31	96.48 ± 2.22	95.67 ± 2.05	91.84 ± 1.13	92.11 ± 1.30	90.57 ± 1.60
BUN (mg/dL)	22.49 ± 1.34	22.31 ± 1.30	21.83 ± 1.37	19.56 ± 0.37	19.90 ± 0.30	19.58 ± 0.65

Group I & VI—male and female control groups; Group II & V—male and female rats treated with DMRO-34 strain of L. edodes; Group III & VI—male & female rats treated with DMRO-356 strain of L. edodes. AST = aspartate aminotransferase, ALT = alanine aminotransferase, ALP = alkaline phosphatase, CRE = creatinine, TB = total bilirubin, TP = total protein, CHO = cholesterol, BUN = blood urea nitrogen, TGA = triglyceride, IU/L = international units per liter, mg/dL = milligrams per deciliter.

Necropsy findings following treatment with L. edodes mushroom

No visible lesions were observed in major organs (liver, kidneys, heart, spleen, brain, testes, ovaries, lungs) in either sex following 28 days of daily administration of DMRO-34 or DMRO-356 at 5000 mg/kg. Gross pathological examination revealed normal morphology across all treatment and control groups (Table 7).

Table 7.

Organ weights in male and female rats after 28 days of oral administration of DMRO-34 and DMRO-356 strains of L. edodes at 5000 mg/kg body weight. 

Sex	Male			Female
Organ weights	G I (control)	G II (DMRO-34)	G III (DMRO-356)	G IV (control)	G V (DMRO-34)	G VI (DMRO-356)
Liver	4.09 ± 0.614	4.136 ± 0.623	4.142 ± 0.620	4.322 ± 0.712	4.284 ± 0.70	4.291 ± 0.716
Kidney	1.55 ± 0.452	1.562 ± 0.34	1.569 ± 0.620	1.547 ± 0.591	1.568 ± 0.60	1.583 ± 0.443
Adrenal	0.065 ± 0.008	0.0647 ± 0.0074	0.0647 ± 0.0072	0.0658 ± 0.0042	0.0632 ± 0.0029	0.0642 ± 0.0050
Heart	0.896 ± 0.060	0.867 ± 0.061	0.864 ± 0.058	0.874 ± 0.071	0.885 ± 0.069	0.871 ± 0.076
Spleen	0.584 ± 0.039	0.577 ± 0.041	0.596 ± 0.044	0.603 ± 0.051	0.618 ± 0.049	0.588 ± 0.058
Lung	1.41 ± 0.524	1.356 ± 0.533	1.348 ± 0.601	1.390 ± 0.514	1.408 ± 0.611	1.371 ± 0.533
Brain	1.69 ± 0.193	1.685 ± 0.211	1.655 ± 0.241	1.671 ± 0.211	1.72 ± 0.231	1.657 ± 0.219

Necropsy findings encompassed examinations of the external, internal, abdominal, thoracic, and cranial cavities. The skin, hair coat, and all external orifices were normal. There were no alterations in the superficial or deep lymph nodes. Organs in the abdominal cavity were in their usual positions, and the spleen was in good condition. No apparent alterations were observed in the stomach and intestines, liver and biliary ducts, or the excretory system. The thoracic cavity was normal without any fluid, mucus, or blood. The adrenal glands, lungs, and heart were all found to be normal. Thyroid glands in both groups were generally considered normal in shape, size, and appearance. The size and shape of the brain were both normal, with no hematoma observed.

Analysis of organ weights in treated rats

After 28 days of treatment, no significant differences in organ weights were observed between treated and control groups (P > 0.05) (Table 7).

Histopathological analysis of treated rats

A schematic overview of the examined organs and their histological status is shown in Fig. 10, highlighting the absence of pathological alterations in both male and female rats treated with DMRO-34 and DMRO-356 strains of L. edodes. Representative photomicrographs of tissue sections from male and female rats are presented in Figs. 11 and 12, respectively.

Fig. 10.

Schematic illustration of histopathological evaluation in male and female rats treated with DMRO-34 and DMRO-356 strains of L. edodes at 5000 mg/kg body weight. The diagram highlights the examined organs—including liver, kidney, lung, brain, and reproductive organs (testis and ovary)—with green checkmarks indicating normal histological findings in both sexes. No pathological alterations were observed in any organ compared with the control group.

Fig. 11.

Histopathological examination of male rats treated with 5000 mg/kg of L. edodes DMRO-34 and DMRO-356 strains is shown. Slides A to F represent tissue sections from the DMRO-34-treated group (Group II), while slides G to L correspond to the DMRO-356-treated group (Group III). Specifically, images A and G show liver sections; B and H depict kidney glomeruli; C and I illustrate kidney tubules; D and J display lung tissue; E and K present brain tissue; and F and L represent testis sections. All tissue samples were stained with haematoxylin and eosin (H&E). Histological evaluation revealed no abnormalities in any of the examined organs. The scale bar represents 100 μm.

Fig. 12.

Histopathological examination of female rats treated with 5000 mg/kg of L. edodes DMRO-34 and DMRO-356 strains. Slides A to F represent tissue sections from the DMRO-34-treated group (Group V), while slides G to L show the corresponding organs from the DMRO-356-treated group (Group VI). Specifically, images A and G show liver sections; B and H depict kidney glomeruli; C and I show renal tubules; D and J display lung tissue; E and K represent brain sections; and F and L show ovaries. All tissues were stained with haematoxylin and eosin (H&E). No histopathological alterations were observed in any of the examined organs. The scale bar represents 100 μm.

Histological evaluation of the liver, kidneys, brain, lungs, and reproductive organs (testes/ovaries) from male and female rats treated with DMRO-34 and DMRO-356 strains of L. edodes (5000 mg/kg body weight) revealed normal tissue architecture, with no signs of degeneration, inflammation, hemorrhage, or necrosis (Figs. 10, 11, 12). The liver exhibited normal hepatic parenchyma with intact portal triads, and the kidneys showed preserved glomeruli, tubules, and interstitium. Lung sections displayed no evidence of alveolar damage, congestion, or edema, while brain tissue was free of hemorrhage and morphological abnormalities. The testis and ovaries showed normal morphology, with active spermatogenesis and folliculogenesis, respectively.

Microscopic findings were consistent across all treated and control groups, and no pathological alterations were observed in any organ. Macroscopic examination further confirmed the absence of toxic effects, with no differences in organ color, shape, or texture compared with controls. These results indicate that both L. edodes strains were non-toxic at the tested dose.

Discussion

Mushrooms are increasingly recognized as sources of bioactive compounds with therapeutic potential, including antimicrobial, antioxidant, immunomodulatory, and anticancer activities^16–18. Among these, L. edodes (shiitake) has gained particular attention not only as a dietary component but also as a candidate for functional food and medicinal applications. However, despite their traditional use and general perception of safety, systematic evaluations of the toxicological and cytotoxic properties of different L. edodes strains remain limited. This study aimed to assess the safety and potential cytotoxic activity of methanolic extracts from two L. edodes strains, DMRO-34 and DMRO-356, through in vitro and in vivo models.

In vitro evaluation of cytotoxic activity

The MTT and LDH assays demonstrated that both L. edodes strains reduced the viability of HeLa and MCF-7 cancer cell lines in a dose-dependent manner, with DMRO-356 showing a moderately higher effect at comparable concentrations. While the observed IC₅₀ values fall within the range reported for crude mushroom extracts—generally between 100 and 400 µg/mL in various cell lines^19,20— these values should be interpreted as indicative rather than definitive measures of anticancer efficacy.

Fluorescence-based apoptosis assays confirmed that both extracts induced morphological features consistent with apoptosis, including chromatin condensation and nuclear fragmentation. Although ethidium bromide (EtBr) uptake was less prominent in the fluorescence imagess—likely due to suboptimal imaging parameterss—apoptotic features were clearly visualized and supported by the calculated apoptosis index. These in vitro observations, however, are limited to cell culture models and must be interpreted with caution, as they do not account for the complex pharmacokinetic and pharmacodynamic factors present in vivo ^21,22. Moreover, the heterogeneity of cancer cell lines, strain-specific phytochemical profiles, and extraction methods can significantly affect the cytotoxic outcomes²³.

In contrast to earlier interpretations attributing cytotoxicity to individual components such as β-glucans, the present study evaluated whole mushroom extracts. While β-glucans such as lentinan have been identified as major bioactive constituents of L. edodes in other studies^24–26, their direct contribution to the cytotoxic effects observed here was not experimentally validated. Therefore, attributing efficacy specifically to β-glucans without isolation and mechanistic testing would be speculative and scientifically inappropriate. Previous literature indicates that multiple constituents—including polysaccharides, phenolic compounds, and terpenoids—can synergistically contribute to bioactivity in crude mushroom extracts^27–31.`

In vivo toxicity evaluation

The acute toxicity assessment using a single oral dose of 5000 mg/kg showed no mortality or adverse clinical signs in treated rats over a 14-day observation period. Body weight, food intake, water consumption, and gross necropsy findings remained within normal limits. These results indicate that both L. edodes strains can be categorized as non-toxic according to the OECD guidelines (toxicity class 5, LD₅₀ > 5000 mg/kg)³².

The 28-day sub-acute toxicity study further supported the safety of the mushroom samples. Repeated oral administration did not result in significant changes in haematological parameters, serum biochemical markers, or organ histopathology. Importantly, no hepatotoxic or nephrotoxic effects were observed, and organ weights remained consistent across groups. These findings align with previously reported studies on L. edodes and related species, which demonstrated safety at high oral doses in rodent models^33–35.

Implications and limitations

The observed in vitro cytotoxicity of L. edodes extracts, supported by MTT, LDH, and AO/EtBr staining results, indicates potential for further investigation in cancer research. AO/EtBr fluorescence microscopy confirmed apoptotic features such as chromatin condensation and nuclear fragmentation, with EtBr uptake marking loss of membrane integrity in late apoptotic cells. However, these findings are based on in vitro models and cannot be directly translated to clinical anticancer efficacy.

Crude extracts contain numerous bioactive compounds whose individual and synergistic effects require further characterization. The present study does not establish a causal link between any specific constituent (e.g., β-glucans) and the observed effects. In vitro results should be substantiated through mechanistic studies, fractionation and compound isolation, and validated in in vivo tumor models before clinical relevance can be asserted.

Materials and methods

Cultivation of L. edodes mushroom

Two strains of L. edodes mushroom, i.e., DMRO-34 and DMRO-356, were obtained from the germplasm collection bank of ICAR-Directorate of Mushroom Research, Solan. These strains were cultivated on sawdust following the procedure outlined by Annepu et al.³⁶. The collected fruit bodies from the first flush of both strains were first dried at room temperature (20–25 °C) for 4 days, then further dried in a hot-air oven at 40 °C until a constant weight was achieved. The dried mushrooms were crushed into a powder with the help of a Wiley mill, and all the samples were then filtered through a 0.5-mm sieve³⁷. Subsequently, the powdered samples were placed in sealed containers for further analysis.

In vitro toxicity study of L. edodes mushroom

The MTT and LDH leakage assays were used to assess the toxicity of two strains of L. edodes mushroom, DMRO-34 and DMRO-356, on HeLa and MCF-7 cell lines cultured in modified Eagle’s medium within a controlled laboratory environment. In addition to these assays, apoptotic measurements were also performed to assess the potential of these strains in inducing cell death. These combined approaches provided a comprehensive evaluation of the strains’ cytotoxicity, further shedding light on their effects on cell viability and apoptosis.

The procedure for making a methanolic mushroom extract followed the description by Barros et al. with minor modifications²⁸. Ten grams of powdered mushroom were added to 100 mL of methanol (80%, v/v) and incubated on an orbital shaker at 150 rpm for 24 h at 25 °C, followed by filteration through the Whatman No. 4 paper filter. The residual material was further extracted using two additional 100-mL volumes of methanol. A rotary evaporator was used to dry the organic solvent from the total methanolic extracts at 40–50 °C. The extract components were dissolved in cold 3.0% dimethyl sulfoxide (DMSO), and the mixture was filter-sterilized using a 0.22 μm membrane filter.

The HeLa and MCF-7 cancer cell lines were sourced from NCCS, Pune, India. These cells were cultured at 37 °C in a humidified environment with 5% CO₂, using modified Eagle’s medium supplemented with 10% fetal bovine serum and100 µg/ml penicillin-streptomycin in the cell culture facility.

Cell viability assay

The cytotoxicity of L. edodes mushroom strains DMRO-34 and DMRO-356 on HeLa and MCF-7 cancer cell lines was assessed using the MTT assay. Cells (2 × 10⁴ cells/200 µL/well) were treated with methanol-dissolved mushroom extract in different concentrations (10–600 µg/mL) on 96-well plates for 48 h. Subsequently, the cells were incubated at 37 °C for 4 h with 20 µL of MTT (5 µg/mL in PBS). After four hours, the formazan crystals were dissolved in 3% cold dimethyl sulfoxide (DMSO) at 150 µL perwell, and the absorbance was measured at 570 nm using a microplate reader after a 10-minute incubation. The cytotoxic effect of the mushroom was calculated as a percentage of cell viability using the following equation:⁷.

Where A_t is the mean absorbance of the test compound, A_b is the absorbance of the blank, and A_c is the absorbance of the negative control.

The IC₅₀ value is the concentration of mushrooms at which 50% of the cell population is non-viable. IC₅₀ values were determined by visualizing the percentage inhibition at a particular mushroom concentration.

Lactate dehydrogenase (LDH) leakage assay

The LDH assay was used to assess the cytotoxic effects of L. edodes mushroom strains DMRO-34 and DMRO-356 on HeLa and MCF-7 cancer cell lines.

Initially, the cells (2 × 10⁴ cells/200 µL/well) were cultured on 96-well plates at 37 °C for 48 h and treated with different concentrations of mushroom extract (10–600 µg/mL) from both strains. After incubation, 10 µL of 1% Triton X-100 (lysis solution) was added to each well and stirred gently for 1–2 min on an orbital shaker at 50–100 rpm to allow cell lysis. Then, each well was filled with a 50 µL of LDH assay solution, containing lactate, NAD+, diaphorase, and dye, which was gently mixed for 30 s before being incubation at 22–25 °C for 10 min. The cells were removed from the growth medium by centrifugation at 250 x g until LDH activity was determined. The fluorescence intensity was measured at 530–560 nm. The percentage of cytotoxicity for each sample was calculated using the following formula³⁸.

Apoptosis measurement

Adherent cells (2 × 10⁴ cells/200 µL/well) were treated with extracts from both strains at doses of 100, 200, 300, 400, and 500 µg/mL for 24 h. After washing twice with PBS, the cells were stained with acridine orange (AO, 100 µg/mL) and ethidium bromide (EtBr, 100 µg/mL). AO stains all nucleated cells green, whereas EtBr selectively penetrates cells with compromised membranes, producing orange to red fluorescence. Fluorescence imaging was performed using a Zeiss fluorescence microscope with appropriate excitation/emission filters for AO/EtBr staining. Typical excitation/emission settings were as follows:AO excitation ~ 502 nm, emission ~ 525 nm; EtBr excitation ~ 518 nm, emission ~ 605 nm. Morphological changes were documented, and the apoptotic index was determined by counting the number of apoptotic cells (showing condensed, fragmented green or orange–red nuclei) out of the total number of cells (viable, apoptotic, and necrotic) observed in the fluorescence images. The apoptotic index was calculated using the formula:

Quantification was performed for both HeLa and MCF-7 cells, with untreated cells serving as the negative control and staurosporine-treated cells serving as the positive control.

Estimation of beta-glucan content in DMRO-34 and DMRO-356 strains of L. edodes

Beta-glucan quantification was carried out according to Murphy et al. using a Megazyme assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland)³⁹.

In vivo toxicity study of L. edodes mushroom

Sprague-Dawley (SD) rats were employed to assess in vivo toxicity in both acute and sub-acute examinations. Haematology, serum chemistry, and histopathology were utilized to investigate the in vivo toxicity of two strains of L. edodes mushroom. This included a qualitative and quantitative assessment of a substance’s harmful effects, along with a time-related assessment of their occurrence following administration.

In the acute study, 15 healthy young adult male rats (aged eight weeks, weighing 150-250 g) were utilized, while the sub-acute study involved 30 rats (15 males and 15 females). Each cage accommodated five rats (n = 5), and their allocation was randomized based on body weight to eliminate any bias. These rats underwent a period of acclimatization to the laboratory environment for 5–7 days prior to the administration of mushroom dosage. They were housed in polypropylene cages featuring stainless steel wire lids at a temperature of 22 °C, under alternating 12-h light and dark cycles. Cleaned paddy husk was used as the bedding material in these cages.

Experimental animals and ethical compliance

All animal experiments were conducted in accordance with the ARRIVE guidelines⁴⁰ and the AVMA Guidelines for the Euthanasia of Animals (2020)⁴¹. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of the Institute for Industrial Research & Toxicology, Ghaziabad, India (Approval Number: IAEC/VVB/MAS/1819/012).

All experimental methods were performed in accordance with the relevant national and international guidelines and regulations, including the Organization for Economic Co-operation and Development (OECD) Test Guidelines 423 and 407, and adhered to Good Laboratory Practice (GLP) standards. No endangered or protected species were used in this study^32,42.

Euthanasia and anesthesia procedure

At the end of the experimental period, all animals were humanely euthanized in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). Prior to euthanasia, rats were anesthetized using isoflurane inhalation (3–5% in an induction chamber) to ensure complete loss of reflexes and to minimize distress. Euthanasia was then performed by CO₂ inhalation in a gradual-fill chamber, followed by cervical dislocation as a confirmatory method of death. All procedures were performed by trained personnel and approved by the Institutional Animal Ethics Committee (IAEC) of the Institute for Industrial Research & Toxicology, Ghaziabad, India (Approval No. IAEC/VVB/MAS/1819/012).

Acute oral toxicity study

The OECD Guideline 423 was employed to conduct an acute toxicological analysis using the limit test procedure. Fifteen male rats were utilized and organized into three groups based on oral administration. They received two strains, DMRO-34 and DMRO-356, of L. edodes mushroom at a single dose of 5000 mg/kg, along with a control Group. Prior to administration, the rats fasted overnight. Group I (Vehicle Control, 1% CMC at 10 ml/kg) received a single oral dose of 1% carboxymethyl cellulose (CMC) at 10 mL/kg body weight. Groups II and III received a single oral dose of 5000 mg/kg body weight of L. edodes strains DMRO-34 and DMRO-356, respectively, each suspended in 1% CMC. These groups are referred to as Group II (DMRO-34, 5000 mg/kg in 1% CMC) and Group III (DMRO-356, 5000 mg/kg in 1% CMC) throughout the manuscript. After dosing, rats were provided with ad libitum access to food and water. They were monitored for changes in body weight, feed and water consumption, clinical signs of toxicity, and mortality for 14 days after dosing. On the following day, necropsy was performed on the treated rats to examine any gross organ lesions resulting from mushroom administration.

Sub-acute toxicity study

The study followed OECD Guideline 407. Since the results of the acute toxicity analysis indicated the non-toxic nature of L. edodes mushroom at a dose of 5000 mg/kg, a complete study with three dose levels were not required. The dose of 5000 mg/kg was deemed appropriate and subsequently employed in the sub-acute oral toxicity assessment. Thus, a higher dose of 5000 mg/kg was used in the limit test procedure. The animals were divided into six groups (n = 5 per group, per sex). Group I received a single oral dose of 1% carboxymethyl cellulose (CMC) at 10 mL/kg body weight and served as the vehicle control. Male animals in Groups II and III received a single oral dose of 5000 mg/kg body weight of L. edodes strain DMRO-34 and DMRO-356, respectively, each suspended in 1% CMC. The same dosing strategy was applied to female animals in Groups IV–VI, where Group IV served as the female vehicle control, and Groups V and VI received DMRO-34 and DMRO-356, respectively.

The rats were monitored for 28 days after dose administration. Each group of rats was given 150 g of feed, and the following day, feed intake was recorded daily for 28 days. Additionally, rats were provided with 200 mL of fresh distilled water and water consumption was measured the next day and for up to 28 days. Observations were recorded for up to 28 days, encompassing body weight, feed and water intake, mortality, clinical signs of toxicity, as well as haematological, histological, and biochemical parameters. Clinical signs were monitored twice daily via cage-side observations and included assessments of skin and fur condition, mucous membranes, salivation, lacrimation, posture, locomotor activity, and excretory patterns (e.g., diarrhoea or polyuria). Diarrhoea was identified by the presence of loose or watery stools in cage bedding and documented individually, not generalized to the entire group. All clinical signs were graded semi-quantitatively: 0 = Normal, + = Mild, ++ = Moderate, +++ = Marked, ++++ = Severe.

At the end of the analysis, all rats fasted overnight, and blood samples were obtained from all groups in both EDTA-containing and EDTA-free microcentrifuge tubes for haematological and biochemical studies. Subsequently, all groups of rats were anaesthetized with CO₂ for necropsy, during which gross lesion changes in target organs were observed, and measurements of organ weight and histopathological examination of internal organs were conducted.

Statistical analysis

All results from cytotoxicity assays and in vivo systemic toxicity studies were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to evaluate differences in endpoint parameters such as haematological indices, biochemical markers, and terminal organ weights across treatment and control groups. Tukey’s honest significant difference (HSD) post hoc test was applied where applicable to determine specific group differences.

For parameters measured repeatedly over time—such as body weight, feed intake, and water intake—a mixed between-within subjects ANOVA (also known as split-plot ANOVA) was performed to assess: (i) the main effect of group (between-subjects factor), (ii) the main effect of time (within-subjects factor), and (iii) the group × time interaction effect on the measured outcomes.

Effect sizes were reported using partial eta squared (η²), with values of η² ≥ 0.14 interpreted as large. A P value < 0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism (v9.0) and IBM SPSS Statistics (v25.0). Descriptive statistics and basic group comparisons were also calculated using Microsoft Excel.

Ethical statement

The acute and subacute oral toxicity studies followed the guidelines outlined by the Organization for Economic Cooperation and Development (OECD) protocols 423 and 407, respectively, and adhered to Good Laboratory Practices (GLP) for nonclinical studies. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the Institute for Industrial Research & Toxicology, Ghaziabad, India (Approval Number: IAEC/VVB/MAS/1819/012). Additionally, the study was performed in compliance with the ARRIVE guidelines.

Conclusion

In vitro cytotoxicity assays demonstrated that DMRO-356 had a more substantial cytotoxic effect than DMRO-34 at 500 µg/mL, with a higher apoptosis index confirmed by AO/EtBr staining. This difference may reflect variations in the bioactive compound profiles between the strains, although attributing it solely to β-glucan content requires further evidence.

Acute and sub-acute oral toxicity studies showed no adverse effects in rats at doses up to 5000 mg/kg, indicating a no-observed-adverse-effect level (NOAEL) comparable to that of control groups and confirming safety under the tested conditions. These findings suggest that both L. edodes strains could be considered safe for potential use as nutritional supplements, functional foods, or in alternative medicine.

While the in vitro results indicate the potential of L. edodes to inhibit cancer cell growth, further research involving compound isolation, mechanistic studies, and in vivo tumor models is essential before confirming therapeutic relevance. Overall, this study provides a comprehensive toxicity profile and supports the potential safe incorporation of L. edodes into nutraceutical applications aimed at improving human health.

Author contributions

S.M. and K.N. conceptualized and designed the research, S.M. performed the experimental work. S.M., K.N., A.S., and S.A. analyzed the data. S.M. drafted the main manuscript text, while K.N., A.S., and S.A. contributed to manuscript formatting. All authors reviewed and approved the final version of the manuscript.

Funding

This research was not supported by any financial grants from public, private, or non-profit organizations.

Data availability

All data generated or analyzed during the study, except for what is included in the manuscript, can be made available upon request from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.