Chemical and Bioactive Evaluation of Essential Oils from Edible and Aromatic Mediterranean Lamiaceae Plants.

The Lamiaceae family, which includes several well-known aromatic plants, is scientifically relevant due to its essential oils (EOs). In this work, four EOs from Mediterranean species, namely Origanum vulgare L., Rosmarinus officinalis L., Salvia officinalis L., and Thymus vulgaris L., were evaluated for their volatile profiles and the biological activity in vitro to assess their potential use in the food and cosmetic sector. GC/MS analysis revealed dominant compounds, such as carvacrol, thymol, and eucalyptol. Regarding biological action, the samples exhibited antioxidant, cytotoxic, anti-inflammatory, antimicrobial, and antifungal activities, with O. vulgare and T. officinalis standing out. T. vulgaris showed the lowest EC50 in the reducing power assay, and O. vulgare had the lowest EC50 in the DPPH assay. Most EOs also displayed excellent anti-inflammatory responses and antifungal properties, with O. vulgare and T. vulgaris also demonstrating antibacterial activity. All EOs from Mediterranean species showed cytotoxicity against tumoral cell lines. Overall, the selected EOs stood out for their interesting bioactivities, with the obtained results underscoring their potential as natural preservatives and bioactive agents in various industrial applications, including food, pharmaceuticals, and cosmetics.

Polyphenol Composition by HPLC-DAD-(ESI-)MS/MS and Bioactivities of Extracts from Grape Agri-Food Wastes.

BACKGROUND: Grape agri-food wastes, such as skin, seeds, and other discarded by-products, contain phytochemical compounds that offer potential health benefits. METHODS: This study aimed to investigate the polyphenol composition and bioactivities of different extracts obtained from grape marc and seeds, with the goal of exploring their potential for application as natural food additives. RESULTS: Regardless of the extraction method used (dynamic maceration, ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE)), all extracts exhibited relatively high concentrations of phenolic compounds. The chemical characterization of the extracts revealed the presence of specific compounds and chemical groups associated with each extraction methodology. Moreover, the extracts displayed satisfactory antioxidant activities, especially in inhibiting lipoperoxidation as assessed by the TBARS assay. Additionally, the extracts demonstrated effective inhibition against different strains of bacteria and fungi known as food contaminants. Taken together, these findings indicate that those extracts have the potential to be tested as natural antioxidants and preservatives with sustainable origins in food and beverage systems. Among the extraction methods evaluated, traditional maceration and UAE provided extracts with the highest antioxidant and antimicrobial activities. CONCLUSIONS: Our results suggest the opportunity to explore grape marc and seeds discarded by the winery industry in Portugal as natural sources of bioactive compounds, which could be employed as functional food ingredients or technological additives. The valorization of grape biowastes offers a promising strategy to reduce waste and harness their potential health benefits.

Assessment of the Use of Common Juniper (Juniperus communis L.) Foliage following the Cascade Principle.

Juniperus communis L. is a species commonly grown in regions of the Northern Hemisphere, and is a good candidate to be cultivated in marginal lands. Plants coming from a pruning performed in a natural population located in Spain were used to assess the yield and quality of different products obtained following the cascade principle. A total of 1050 kg of foliage biomass were crushed, steam-distilled, and separated into fractions to produce biochar and absorbents for the pet industry using pilot plants. The obtained products were analysed. The essential oil, with a yield of 0.45% dry basis and a qualitative chemical composition similar to that described for the berries in international standards or monographs, showed antioxidant activity with promising CAA results (inhibition of 89% of the cell's oxidation). However, regarding antibacterial and antifungal activities, it only inhibited the growth of microorganisms at the maximum concentration tested, 2.5%. Concerning the hydrolate, it did not show bioactivity. Regarding the biochar, whose yield was 28.79% dry basis, interesting results were obtained for its characterisation as a possible soil improver for agronomic purposes (PFC 3(A)). Finally, promising results were obtained regarding the use of common juniper as absorbent, taking into account the physical characterisation and odour control capacity.

Chemical and Bioactive Characterization of the Essential Oils Obtained from Three Mediterranean Plants.

Cupressus sempervirens L., Juniperus communis L. and Cistus ladanifer L. are Mediterranean arboreal and shrub species that possess essential oils (EO) in their leaves and branches. This study aimed at characterizing the EOs obtained by steam distillation from the three species collected in different locations from Spain (Almazán, Andévalo, Barriomartín, Cerezal, Ermitas and Huéscar). For this purpose, volatiles composition was determined by GC-MS, and different bioactivities were evaluated. The highest content in terpenes was observed in C. sempervirens (Huéscar origin) followed by J. communis (Almazán origin), corresponding to 92% and 91.9% of total compounds, respectively. With exception of C. ladanifer from Cerezal that presented viridiflorol as the most abundant compound, all the three species presented in common the α-pinene as the major compound. The EOs from C. ladanifer showed high antibacterial potential, presenting MIC values from 0.3 to 1.25 mg/mL. Concerning other bioactivities, C. ladanifer EO revealed an oxidation inhibition of 83%, while J. communis showed cytotoxicity in the MCF-7 cell line, and C. sempervirens and C. ladanifer EOs exhibited the highest potential on NCI-H460 cell lines. Nevertheless, some EOs revealed toxicity against non-tumoral cells but generally presented a GI50 value higher than that of the tumor cell lines.

Infusions of Herbal Blends as Promising Sources of Phenolic Compounds and Bioactive Properties.

Several plants have been used for medicinal applications and have been traditionally consumed as decoctions and infusions. Although some herbs are used alone as a beverage, they are often blended in mixtures to maximize their effects. Herein, the nutritional characterization of six infusions from herbal blends was evaluated using the official methods of analysis (AOAC international). A further characterization of the individual phenolic profile was also performed by HPLC-DAD/ESI-MSn, and finally bioactive potential was determined by evaluating the antioxidant, cytotoxic, anti-inflammatory, and antimicrobial activities of each blend. The wide variety of plants in each sample led to variability in the results for all analyzed parameters. However, blends containing 15% Laurus nobilis L. and 15% Juglan regia L. in their composition showed higher sugar content and energy contribution; higher concentration of phenolic compounds (phenolic acids and flavonoids); greater antioxidant, cytotoxic, and anti-inflammatory capacity; and also better antimicrobial effects against all the tested bacterial and fungal strains. Further studies will be necessary to evaluate the real synergistic effects that these two species show in the presence of other plants, and to evaluate their potential for application in various food, pharmaceutical, and nutraceutical products as infusion preparations.

Magnesium and manganese induced changes on chemical, nutritional, antioxidant and antimicrobial properties of the pansy and Viola edible flowers.

Pansy and viola edible flowers were grown hydroponically with different levels of Mg and Mn. The nutritional composition was determined using standard methods. Free sugars, fatty acids, organic acids, tocopherols, and phenolic compounds were analyzed using various HPLC and GC devises. The extract's antimicrobial, antioxidant, cytotoxicity, and anti-inflammatory activity were assessed. The results indicated that Mg enrichment negatively affected plant growth and mineral accumulation but improved photosynthetic performance. The edible flowers contained significant amounts of protein, low levels of fat, and varying sugar contents, such as glucose and fructose. Various fatty acids and phenolic compounds were identified, with different concentrations depending on the treatment. The flowers exhibited antioxidant potential, antimicrobial activity, cytotoxic effects, and anti-inflammatory properties. The correlations between the investigated parameters not only expand knowledge on Mg and Mn interaction but also catalyze significant advancements in sustainable agriculture and food health, fostering a healthier and more conscious future.

Cistus, Acacia, and Lemon verbena Valorization through Response Surface Methodology: Optimization Studies and Potential Application in the Pharmaceutical and Nutraceutical Industries.

Cistus ladanifer L., Acacia dealbata L., and Aloysia citrodora Paláu were subject to an optimization procedure for two extraction techniques (heat-assisted extraction (HAE) and ultrasound-assisted extraction (UAE)). The extracts were then analyzed by HPLC-DAD-ESI/MS for their phenolic profile (cistus-15 compounds, acacia-21 compounds, and lemon verbena-9 compounds). The response surface methodology was applied, considering four varying factors: ethanol percentage; extraction time; temperature/power; and S/L ratio, generating two responses (the major phenolic compound, or family of compounds, and the extraction yield). For cistus, both techniques optimized the extraction yield of punicalagins, with UAE proving to be the most efficient extraction method (3.22% ethanol, 22 min, 171 W, and 35 g/L). For acacia, HAE maximized the extraction of procyanidin (74% ethanol, 86 min, 24 °C, and 50 g/L), and UAE maximized the content of myricetin (65% ethanol, 8 min, 50 W, and 50 g/L). For lemon verbena, HAE favored the extraction of martynoside (13% ethanol, 96 min, 49 °C and 17 g/L) and forsythiaside UAE (94% ethanol, 25 min, 399 W, and 29 g/L). The optimal conditions for the extraction of compounds with high added value and potential for use in pharmaceuticals and nutraceuticals were defined.

Biological Activity of 6,7-Dehydroxyroyleanone and Derivatives Obtained from Plectranthus aliciae (Codd) A.J.Paton.

The Plectranthus genus (Lamiaceae) is known to be rich in abietane diterpenes. The bioactive 6,7-dehydroxyroyleanone (DHR, 1) was previously isolated from Plectranthus madagascariensis var. madagascariensis and var. aliciae. This study aimed to explore the occurrence of DHR, 1, in P. aliciae and the potential bioactivities of new semisynthetic derivatives from DHR, 1. Several extraction methods were evaluated, and the hydrodistillation, using a Clevenger apparatus, afforded the highest yield (77.8 mg/g of 1 in the essential oil). Three new acyl derivatives (2-4) were successfully prepared from 1 (yields of 86-95%). Compounds 1-4 showed antioxidant activity, antibacterial effects, potent cytotoxic activity against several cell lines, and enhanced anti-inflammatory activity that surpassed dexamethasone (positive control). These findings encourage further exploration of derivatives 2-4 for potential mechanisms of antitumoral, antioxidant, and anti-inflammatory capabilities, studying both safety and efficacy.

The generally recognized as safe (GRAS) microalgae Haematococcus pluvialis (wet) as a multifunctional additive for coloring and improving the organoleptic and functional properties of foods.

This work proposes the application of astaxanthin-rich H. pluvialis wet paste (HPW) as a partial substitute for wheat flour in the preparation of filloas, a dish that combines the basic ingredients of industrial bakery. The nutritional and color profile of HPW-enriched samples was evaluated by comparative analysis with a mixture of synthetic food dyes. The highest content of carotenoids (798 ± 12 µg g-1) and fatty acids (76 ± 2 mg g-1) was obtained for a filloa fortified with H. pluvialis in contrast to a non-significant dye response. Subsequently, the color stability of the fortified filloa was evaluated over time (3, 6 and 9 days), as well as its physicochemical properties and microbiological profile. As a result, HPW provided filloas with a longer shelf life, brightness (*L), and texture, in comparison with a mixture of synthetic dyes. In addition, an inhibitory effect of HPW towards mesophilic aerobic microorganisms in the food was obtained.

Valorization of Punica granatum L. Leaves Extracts as a Source of Bioactive Molecules.

Due to a lack of innovative valorization strategies, pomegranate processing generates a significant amount of residues with a negative environmental footprint. These by-products are a rich source of bioactive compounds with functional and medicinal benefits. This study reports the valorization of pomegranate leaves as a source of bioactive ingredients using maceration, ultrasound, and microwave-assisted extraction techniques. The phenolic composition of the leaf extracts was analyzed using an HPLC-DAD-ESI/MSn system. The extracts' antioxidant, antimicrobial, cytotoxic, anti-inflammatory, and skin-beneficial properties were determined using validated in vitro methodologies. The results showed that gallic acid, (-)-epicatechin, and granatin B were the most abundant compounds in the three hydroethanolic extracts (between 0.95 and 1.45, 0.7 and 2.4, and 0.133 and 3.0 mg/g, respectively). The leaf extracts revealed broad-spectrum antimicrobial effects against clinical and food pathogens. They also presented antioxidant potential and cytotoxic effects against all tested cancer cell lines. In addition, tyrosinase activity was also verified. The tested concentrations (50-400 µg/mL) ensured a cellular viability higher than 70% in both keratinocyte and fibroblast skin cell lines. The obtained results indicate that the pomegranate leaves could be used as a low-cost source of value-added functional ingredients for potential nutraceutical and cosmeceutical applications.

Chemical Characterization, Bioactivity and Toxicity of European Flora Plant Extracts in Search for Potential Natural Origin Preservatives.

Consumer demand for natural and healthier products has led to an increasing interest in the bioactive and therapeutic properties of plant extracts. In this study, we evaluated the phenolic compounds profile, bioactivities, and toxicities of plant extracts from eight European flora species, including Calendula officinalis L., Calluna vulgaris (L.) Hull, Hippophae rhamnoides L., Juglans regia L., Mentha cervina L., Rubus idaeus L., Sambucus nigra L., and Vitis vinifera L. The aim was to identify potential preservatives of natural origin. Phenolic compounds were identified by HPLC-DAD-ESI-MS. Caffeic acid derivatives, ellagitannins, flavonols, and flavones were the major phenolic compounds identified. The total phenolic content varied from 16.0 ± 0.2 (V. vinifera) to 123 ± 2 mg/g (H. rhamnoides) of dry extract. All extracts showed antioxidant potential and exhibited activity against some of the microorganisms tested. S. nigra showed the highest activity in the inhibition of oxidative hemolysis (OxHLIA) assay and H. rhamnoides, notably, had the lowest IC50 values in TBARS and DPPH assays, as well as the lowest minimum inhibitory concentration (MIC) values. Regarding in vitro cytotoxicity, in tumor and non-tumor cell lines, although some extracts revealed toxicity against normal cells, it was found that the samples C. vulgaris, V. vinifera and R. idaeus might be used against tumor cells since the active concentration is much lower than the one causing toxicity. In vivo acute toxicity tests using Artemia franciscana suggest low toxicity for most extracts, with LC50 > 400 mg/L. These results showed the potential of the studied extracts as natural preservatives, given their richness in compounds with bioactive properties, highlight their potential value to the production chain.

Brazilian berry waste as a source of bioactive compounds: grumixama (Eugenia brasiliensis Lam.) as a case study.

Grumixama, Eugenia brasiliensis Lam., is a Brazilian berry little explored commercially and scientifically. However, local small producers market this fruit in the form of frozen pulp, which generates bioresidues, composed of seeds and peels. With the view to propose strategies for valuing grumixama, this study aimed to determine the chemical composition and assess the bioactivities of the hydroethanolic extracts of the whole residue (GR), seed (GS) and peel (GP) fractions of E. brasiliensis. From the results, GP had the highest concentration of organic acids (oxalic, malic, ascorbic and citric acids), total tocopherols, condensed tannins, anthocyanins, and other flavonoids. On the other hand, GS showed the highest content of monounsaturated fatty acids and hydrolysable tannins, whereas GR displayed a mixture of compounds detected in each of its parts. Regarding the bioactivities, low extract concentrations were required in two in vitro antioxidant assays, namely TBARS (EC50 = 0.90-1.34 µg mL-1) and OxHLIA (IC50 = 21-65 µg mL-1). Furthermore, GP had the highest inhibition activity of cellular oxidation in the CAA assay (80 ± 0.6%), while GS showed the highest anti-inflammatory activity via nitric oxide production inhibition (EC50 = 98.0 ± 0.5 µg mL-1). All samples induced cell growth inhibition of the tested tumor cells (GI50 = 14.7-186 µg mL-1) besides antibacterial and antifungal effects at low concentrations, but all samples were harmful to normal cells at moderate concentrations (GI50 = 145-268 µg mL-1). Therefore, E. brasiliensis residue could be a good source of bioactive compounds to be used in several areas. However, additional studies are needed to confirm its safety as well as to unravel the mechanisms behind its biological activities.

Sequential steps of the incorporation of bioactive plant extracts from wild Italian Plantago coronopus L. and Cichorium intybus L. leaves in fresh egg pasta.

The application of bioactive extracts from Cichorium intybus L. and Plantago coronopus L. species were incorporated as a functional ingredient in fresh egg pasta (Fettuccine). In that sense, a pasta making procedure was accessed using different concentrations of the plant extracts (0.25-0.63 mg/g), drying times (20-420 min) and drying temperatures (40-90 °C; only for P. coronopus enriched pasta), to screen an optimal factor selection in the pasta making procedure and to enhance the bioactive properties of the final product. In the chemical characterisation of the plant extracts, twenty-five phenolic compounds were tentatively identified (twenty compounds belonging to phenolic acid and phenylpropanoid classes and five belonging to the flavonoid sub-class) and a strong synergy between the plant extract concentration and the drying time was showed. The analysed antioxidant properties were enhanced by the phenolic compounds of the extracts and a new functional food with higher bioactive quality was developed.

Neuroprotective Profile of Edible Flowers of Borage (Borago officinalis L.) in Two Different Models: Caenorhabditis elegans and Neuro-2a Cells.

The flowers of Borago officinalis L. (Boraginaceae), commonly known as borage, are widely used as a culinary ingredient. The aim of this study was to assess the potential benefits of fresh borage flower extract related to antioxidant, neuroprotective and anti-aging properties. The extract was obtained by Soxhlet extraction with ethanol as a solvent, and fatty acids were detected by GC-FID. The antioxidant activity was evaluated in vitro through the DPPH, FRAP and ORAC assays. Regarding the fatty acid (FA) composition, the extract showed high amounts of polyunsaturated FA. The Neuro-2a cell line was used to determine the cytoprotective capacity of the extract subjected to oxidative stress (H2O2). Moreover, the model organism Caenorhabditis elegans was used to assess antioxidant activity, delayed ageing as well as cytoprotection and reduced ß-amyloid toxicity. Cells treated with the extract and H2O2 showed a better response to oxidative stress than the control group, particularly in terms of mitochondrial activity (MTT assay), redox state (ROS formation) and the activity of antioxidant enzymes (catalase and superoxide dismutase). B. officinalis flower extract showed promising antioxidant activity in the selected models, without causing toxicity. Hence, the results obtained support the antioxidant properties of borage flowers in different bioassays using living organisms.

Pineapple by-products as a source of bioactive compounds with potential for industrial food application.

Pineapple is a tropical fruit consumed fresh or processed into various food products. However, the peel and crown of this fruit are not industrially exploited, thus generating tons of by-products that represent an economic and environmental concern. In order to promote the upcycling of these by-products, this work aimed to characterize the phenolic profile of its hydroethanolic extracts obtained from pineapple peel and crown leaves and to evaluate their in vitro bioactivity. The HPLC-DAD-ESI/MS analysis allowed the identification of 25 phenolic compounds, including phenolic acids and flavonoids. The antioxidant, cytotoxic, and antimicrobial activity assays highlighted the peel extract as the most promising and, therefore, it was incorporated into a traditional Portuguese pastry cake as a functional ingredient. The nutritional parameters of the developed food were not affected by the incorporation of the extract, but it promoted the antioxidant activity during its shelf-life. Overall, pineapple peel and crown appeared as promising by-products to be exploited by the food industry, which can be achieved through a circular economy approach.

Comparative study on the phenolic composition and in vitro bioactivity of medicinal and aromatic plants from the Lamiaceae family.

Medicinal and aromatic plants (MAP) have been described as a source of phenolic compounds with potential as antioxidant, antiproliferative and antimicrobial agents. MAP from the Lamiaceae family (Origanum vulgare L., Thymus vulgaris L., Ocimum basilicum L., Salvia officinalis L., Melissa officinalis L., and MatricariachamomillaL.) were selected to perform a phytochemical and biological screening for their further exploitation as natural bioactive ingredients. The total content of phenolic compounds varied from 184.02 mg/g extract in M. officinalis to 17.97 mg/g extract in M. chamomilla. Caffeic and rosmarinic acids were the main phenolic acids found in the respective hydroalcoholic extracts. The extracts showed a promising antioxidant activity in vitro, being related the phenolic compositions of the extracts, furthermore, all extracts being able to combat lipid peroxidation in TBARS assays with an IC50 under 26 µg/mL, moreover all the plant extract has prevented the oxidative haemolysis in OxHLIA assays at concentrations below 67 µg/mL in a Δt 60 min and under 118 µg/mL for a Δt 120 min. Regarding to the bactericidal and fungicidal action the plant extracts were able to inhibit growth against bacteria associated with food hazards, such as Salmonella typhimurium (MIC < 1) and Listeria monocytogenes (MIC < 1), regarding to fungicidal activity it can be highlighted the MIC values under to 0.25 for Aspergillus versicolor and Trichoderma viride. Overall, the selected Lamiaceae plants stood out as a source of active phytochemicals that can be used by different industries, such as food and cosmetics.

Bioactive properties of Sanguisorba minor L. cultivated in central Greece under different fertilization regimes.

In this study, the chemical characterization and bioactive properties of S. minor cultivated under different fertilization rates (control, half rate and full rate) were evaluated. Twenty-two phenolic compounds were identified, including five phenolic acids, seven flavonoids and ten tannins. Hydrolysable tannins were prevalent, namely Sanguiin H-10, especially in leaves without fertilization (control). Roots of full-rate fertilizer (660 Kg/ha) presented the highest flavonoid content, mainly catechin and its isomers, whereas half-rate fertilizer (330 Kg/ha), presented the highest content of total phenolic compounds, due to the higher amount of ellagitannins (lambertianin C: 84 ± 1 mg/g of dry extract). Antimicrobial activities were also promising, especially against Salmonella typhimurium (MBC = 0.44 mg/mL). Moreover, root samples revealed activity against all tested cell lines regardless of fertilization rate, whereas leaves were effective only against HeLa cell line. In conclusion, S. minor could be a source of natural bioactive compounds, while fertilization could increase phenolic compounds content.

Echinacea purpurea (L.) Moench: Chemical Characterization and Bioactivity of Its Extracts and Fractions.

Echinacea purpurea (L.) Moench is widely known for its medicinal properties, being one of the most used medicinal plants for its immunostimulant properties. Nevertheless, there is still scarce information on its cytotoxic activity. Thus, this study aims at evaluating the cytotoxicity and antimicrobial activity of several aqueous and organic extracts of the aerial parts of this plant and chemically characterizing the obtained extracts. The analysis was performed by HPLC-DAD-ESI/MS. Fifteen compounds were identified; of these, seven were phenolic acids and eight were flavonoids. Non-polar compounds were evaluated by GC/MS, with a total of sixty-four compounds identified, and the most abundant groups were the sterols, fatty acids and long-chain hydrocarbons. The highest antimicrobial activity was exhibited by the dichloromethane, ethyl acetate, and acetone extracts. Dichloromethane and n-hexane extracts showed the highest cytotoxic activity. Therefore, they were fractionated, and the obtained fractions were also assessed for their cytotoxicity. Notwithstanding, the cytotoxicity of the extracts was superior to that of the obtained fractions, evidencing a possible synergistic effect of different compounds in the whole extracts.

Challenges of traditional herbal teas: plant infusions and their mixtures with bioactive properties.

In order to characterize and study the bioactivities of individual plant species and to determine how these characteristics are modified when preparing blends, five different plant species were selected: Erica australis L., Genista tridentata L., Melissa officinalis L., Mentha spicata L., and Prunella vulgaris L. Infusions prepared from each plant species and from three selected mixtures were analyzed in terms of nutritional values, phenolic compositions, and bioactive properties (antioxidant, antimicrobial, cytotoxic, and anti-inflammatory activities). The major compound detected in M. officinalis, M. spicata, and P. vulgaris infusions was rosmarinic acid, whilst in E. australis and G. tridentata flavonoid derivatives such as quercetin and genistein were identified. P. vulgaris and M. officinalis presented the best results in TBARS and OxHLIA assays, respectively. M. spicata and all mixtures presented anti-inflammatory activity. M. spicata showed the best cytotoxic properties and antimicrobial activity, and none of the infusions showed hepatotoxicity for non-tumour cells.

Extrinsic and Intrinsic Apoptotic Responses Induced by Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Aqueous Extract against a Larynx Carcinoma Cell Line.

Cumulative evidence from research studies has shown that the shiitake culinary-medicinal mushroom, Lentinus edodes, is an excellent source of natural antitumor agents and is capable of inhibiting cancer cell growth. However, the cell signaling pathway that leads tumor cells to apoptosis is not well understood because many chemical compounds may be acting. This study investigated the chemopreventive effects of an L. edodes aqueous extract on human HEp-2 epithelial larynx carcinoma cells and normal human MRC-5 lung fibroblasts by identifying proliferative and apoptotic pathways. The chemical characterization of the dry powder was assessed by high-performance liquid chromatography. Antiproliferative and proapoptotic effects induced by the extract were evaluated by assessing proliferative markers, cell sorting through flow cytometry, and expression levels of apoptotic proteins with Western blotting. The results suggest that inhibition of cell proliferation was more prominent in HEp-2 than in MRC-5 cells. Cell death analysis showed the appearance of cell populations in the sub-G1 phase, with late apoptotic signal increased in a dose-dependent manner. In addition, the aqueous extract induced depolarization of mitochondria, activating the generation of intracellular reactive oxygen species in HEp-2 cells. These observations suggest that L. edodes extract may exert a chemopreventive effect, regulating mitotic induction of apoptogenic signals. These findings highlight the mushroom's pharmacological potential in cancer treatment.