Fed-batch cultivation of Euglena gracilis for the high-yield production and GPC-assisted molecular weight determination of paramylon.

Paramylon, a starch-like carbohydrate accumulated in Euglena gracilis cells, could be a potential source as a raw material for chemical products; its high-yield production would thus be highly desired. Although the molecular weight and polymerization degree of paramylon are important properties for its use as a raw material for chemical products, the available information about paramylon molecular weight remains insufficient. Therefore, in this study, we investigated a high-density E. gracilis culture approach and how culture conditions affect paramylon molecular weight. The nitrogen source, cultivation temperature, and nutrient feeding were optimized for maximum biomass and paramylon productivity. The maximum dry cell weight and paramylon content yields reached 108.9 g/L and 87.2%, respectively. Paramylon molecular weight was in the range of 220 000-320 000 Da. Our gel permeation chromatography analysis showed that the cells with a higher paramylon content tended to contain paramylon of a higher molecular weight.

Characterization of chloroplastic thioredoxin dependent glutathione peroxidase like protein in Euglena gracilis: biochemical and functional perspectives.

Euglena gracilis, a fascinating organism in the scientific realm, exhibits characteristics of both animals and plants. It maintains redox homeostasis through a variety of enzymatic and nonenzymatic antioxidant molecules. In contrast to mammals, Euglena possesses non-selenocysteine glutathione peroxidase homologues that regulate its intracellular pools of reactive oxygen species. In the present study, a full-length cDNA of chloroplastic EgGPXL-1 was isolated and subjected to biochemical and functional characterization. Recombinant EgGPXL-1 scavenged H2O2 and t-BOOH utilizing thioredoxin as an electron donor rather than glutathione. Despite its monomeric nature, EgGPXL-1 exhibits allosteric behavior with H2O2 as the electron acceptor and follows typical Michaelis-Menten kinetics with t-BOOH. Suppression of EgGPXL-1 gene expression under normal and high-light conditions did not induce critical situations in E. gracilis, suggesting the involvement of compensatory mechanisms in restoring normal conditions.

Development of a new sterilization method for microalgae media using calcium hypochlorite as the sterilant.

Calcium hypochlorite (Ca(ClO)2), which can be stably stored in powder form for a long period, is widely used as a disinfectant in various fields. A new sterilization process was developed in the present study, where a microalgal medium was sterilized using 0.02% Ca(ClO)2, followed by complete neutralization of the Ca(ClO)2 within 8 h through catalytic reaction of an MnCl2-Na2EDTA complex and a synergistic effect of glucose. When comparing the growth of Chlorella vulgaris in the autoclaved medium, a 2.65 times greater maximum cell growth was observed in cells grown in the medium prepared by treatment of Ca(ClO)2. This result indicates that denaturation of the medium by heat can hinder the growth of some microorganisms. In the case of cultivation of Euglena gracilis, successful culture growth was achieved without growth inhibition or contamination on a medium prepared in the same manner.

18S rDNA sequence-structure phylogeny of the Euglenophyceae (Euglenozoa, Euglenida).

The phylogeny of Euglenophyceae (Euglenozoa, Euglenida) has been discussed for decades with new genera being described in the last few years. In this study, we reconstruct a phylogeny using 18S rDNA sequence and structural data simultaneously. Using homology modeling, individual secondary structures were predicted. Sequence-structure data are encoded and automatically aligned. Here, we present a sequence-structure neighbor-joining tree of more than 300 taxa classified as Euglenophyceae. Profile neighbor-joining was used to resolve the basal branching pattern. Neighbor-joining, maximum parsimony, and maximum likelihood analyses were performed using sequence-structure information for manually chosen subsets. All analyses supported the monophyly of Eutreptiella, Discoplastis, Lepocinclis, Strombomonas, Cryptoglena, Monomorphina, Euglenaria, and Colacium. Well-supported topologies were generally consistent with previous studies using a combined dataset of genetic markers. Our study supports the simultaneous use of sequence and structural data to reconstruct more accurate and robust trees. The average bootstrap value is significantly higher than the average bootstrap value obtained from sequence-only analyses, which is promising for resolving relationships between more closely related taxa.

Gravity sedimentation of eukaryotic algae Euglena gracilis accelerated by ethanol cultivation.

Euglena gracilis (E. gracilis) is a unicellular microalga with various applications in medicine, agriculture, aquaculture, health supplement, and jet fuel production. Euglena possibly solves population growth and exhaustion of fossil resources. Efficient cell harvesting is needed for the industry, and the gravity sedimentation method is low cost and does not require any equipment, although it has low efficiency. This study showed that the gravity sedimentation of E. gracilis cells is improved by cultivation in the presence of ethanol (EtOH). The gravity sedimentation of E. gracilis cells cultivated under 0.5% or 1.0% EtOH conditions was faster than that cultivated without EtOH. The mean calculated cell diameter was also found to be largest in cells cultivated under 0.5% or 1.0% EtOH conditions compared to that in cells cultivated without EtOH. Intracellular paramylon content, cell shapes, and motility differed between cells cultivated under 0.5% or 1.0% EtOH conditions and in the absence of EtOH. The results suggest that E. gracilis cultivation with EtOH leads to increased cell productivity, paramylon production, and efficient cell harvesting. KEY POINTS: • Euglena gracilis is an edible microalga producing value-added metabolites. • Ethanol addition upregulates E. gracilis growth and paramylon accumulation. • Gravity sedimentation is accelerated by ethanol-grown E. gracilis cells.

Chemical and genetic carotenoid deficiency delays growth in dark-grown Euglena gracilis.

Light-independent functions of carotenoids in photosynthetic organisms are poorly understood. Here, we investigated the growth properties of microalga, Euglena gracilis, under altered light and temperature using norflurazon-treated carotenoid-deficient cells and genetically modified strains, including nonphotosynthetic SM-ZK and colorless cl4. Norflurazon treatment decreased carotenoid and chlorophyll contents, causing cell bleaching. SM-ZK strain had lower carotenoid content than wild-type (WT) strain, and it was below the detectable level in the cl4 strain. Norflurazon treatment decreased phytoene synthase EgCrtB levels, although EgcrtB was transcriptionally induced. Carotenoid deficiency in norflurazon-treated cells and the cl4 strain caused similar extents of delayed growth under light and dark conditions at 25 °C, indicating that carotenoids promote growth in darkness. Both WT and SM-ZK strains exhibited similar growth rates. Dark conditions at 20 °C enhanced the growth delay of norflurazon-treated cells and the cl4 strain. These results indicate that carotenoids impart environmental stress tolerance to E. gracilis in light-dependent and light-independent manners.

Optimization of Heterotrophic Culture Conditions for the Microalgae Euglena gracilis to Produce Proteins.

Euglena gracilis is one of the few permitted edible microalgae. Considering consumer acceptance, E. gracilis grown heterotrophically with yellow appearances have wider food industrial applications such as producing meat analogs than green cells. However, there is much room to improve the protein content of heterotrophic culture cells. In this study, the effects of nitrogen sources, temperature, initial pH, and C/N ratios on the protein production of E. gracilis were evaluated under heterotrophic cultivation. These results indicated that ammonium sulfate was the optimal nitrogen source for protein production. The protein content of E. gracilis cultured by ammonium sulfate increased by 113% and 44.7% compared with that cultured by yeast extract and monosodium glutamate, respectively. The manipulation of the low C/N ratio further improved E. gracilis protein content to 66.10% (w/w), which was 1.6-fold of that in the C/N = 25 group. Additionally, amino acid analysis revealed that the nitrogen-to-protein conversion factor (NTP) could be affected by nitrogen sources. A superior essential amino acid index (EAAI) of 1.62 and a balanced amino acid profile further confirmed the high nutritional value of E. gracilis protein fed by ammonium sulfate. This study highlighted the vast potency of heterotrophic cultured E. gracilis as an alternative dietary protein source.

Advanced Microscopy Techniques for Molecular Biophysics.

Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids).

Microbe Profile: Euglena gracilis: photogenic, flexible and hardy.

Euglena gracilis is a unicellular photosynthetic eukaryotic flagellate of the Discoba supergroup, which also encompasses Kinetoplastida and Diplonema. Plastids have green algal origin and are secondarily acquired. The nuclear genome is extremely large and many genes suggest multiple endosymbiotic/gene transfer events, i.e. derivation from prokaryotes of various lineages. E. gracilis is remarkably robust and can proliferate in environments contaminated with heavy metals and acids. Extraordinary metabolic plasticity and a mixotrophic lifestyle confers an ability to thrive in a broad range of environments, as well as facilitating production of many novel metabolites, making Euglena of considerable biotechnological importance.

Discrimination of Euglena gracilis strains Z and bacillaris by MALDI-TOF MS.

AIMS: Euglena gracilis is used as model organism for various microbiological, molecular biological and biotechnological studies. Its most studied wild-type strains are Z and bacillaris, but their discrimination by standard molecular methods is difficult. Therefore, we decided to test the suitability of MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) for identification of E. gracilis and for discrimination of these two strains possessing functional chloroplasts. MALDI-TOF MS profiling was also tested for two white (non-photosynthetic) stable E. gracilis mutant strains Wgm ZOflL and W10 BSmL. METHODS AND RESULTS: We have successfully obtained main spectrum profiles (MSPs) of E. gracilis strains Z, SAG 1224-5/25 and bacillaris, SAG 1224-5/15 using protein extraction procedure. Subsequent MALDI-TOF MS profiling of a number of tested samples and the comparison of the obtained protein profiles with our in-house database including MSPs of both strains have revealed that these two strains can be easily distinguished by MALDI-TOF MS based on score values over two in most cases. This method has also confirmed the ancestry of white mutant strains Wgm ZOflL and W10 BSmL, originally derived from strains Z and bacillaris, respectively. CONCLUSIONS: MALDI-TOF MS is suitable, accurate and rapid method for discrimination of E. gracilis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results can have broad practical implications for laboratories cultivating various strains of euglenids, and they can be applied for their discrimination by MALDI-TOF MS.

Dectin-1 Reactivity to Paramylon Derived from Euglena gracilis EOD-1.

Euglena gracilis is a microalga that has recently attracted attention because of its bioactivities. Paramylon (PM), a major ß-1,3-glucan, constitutes 70-80% of the cells of the E. gracilis EOD-1 strain. Dectin-1 is a pattern recognition receptor that recognizes ß-glucan. However, it is unclear whether PM binds to dectin-1. In this study, we investigated the reactivity of EOD1PM with dectin-1 by analyzing the binding of soluble murine and human dectin-1-Fc fusion protein (m dectin-1 Fc, h dectin-1 Fc) to EOD1PM using flow cytometry and enzyme-linked immunosorbent assay (ELISA). m Dectin-1 Fc bound to EOD1PM particles when m dectin-1-Fc is added. Furthermore, the binding specificity was examined in a competitive reaction following addition of a soluble antigen. It was found that the binding of m dectin-1-Fc to EOD1PM was not inhibited by the addition of dextran or ovalbumin but by the addition of solubilized EOD1PM or Candida cell wall- solubilized ß-glucan. In addition, the h dectin-1-Fc fusion protein was found to specifically bind to EOD1PM. These results suggest that dectin-1 recognizes and binds to the ß-glucan structure of EOD1PM. Dectin-1 is expressed in leukocytes as a ß-glucan receptor and is involved in the expression of various biological activities; therefore, the dectin-1 pathway may be involved in the biological activity of EOD1PM.

Carotenoids Biosynthesis, Accumulation, and Applications of a Model Microalga Euglenagracilis.

The carotenoids, including lycopene, lutein, astaxanthin, and zeaxanthin belong to the isoprenoids, whose basic structure is made up of eight isoprene units, resulting in a C40 backbone, though some of them are only trace components in Euglena. They are essential to all photosynthetic organisms due to their superior photoprotective and antioxidant properties. Their dietary functions decrease the risk of breast, cervical, vaginal, and colorectal cancers and cardiovascular and eye diseases. Antioxidant functions of carotenoids are based on mechanisms such as quenching free radicals, mitigating damage from reactive oxidant species, and hindering lipid peroxidation. With the development of carotenoid studies, their distribution, functions, and composition have been identified in microalgae and higher plants. Although bleached or achlorophyllous mutants of Euglena were among the earliest carotenoid-related microalgae under investigation, current knowledge on the composition and biosynthesis of these compounds in Euglena is still elusive. This review aims to overview what is known about carotenoid metabolism in Euglena, focusing on the carotenoid distribution and structure, biosynthesis pathway, and accumulation in Euglena strains and mutants under environmental stresses and different culture conditions. Moreover, we also summarize the potential applications in therapy preventing carcinogenesis, cosmetic industries, food industries, and animal feed.

Mixotrophic Cultivation Optimization of Microalga Euglena pisciformis AEW501 for Paramylon Production.

Euglena, a flagellated unicellular protist, has recently received widespread attention for various high-value metabolites, especially paramylon, which was only found in Euglenophyta. The limited species and low biomass of Euglena has impeded paramylon exploitation and utilization. This study established an optimal cultivation method of Euglena pisciformis AEW501 for paramylon production under mixotrophic cultivation. The results showed that the optimum mixotrophic conditions were 20 °C, pH 7.0, and 63 µmol photons m-2∙s-1, and the concentrations of sodium acetate and diammonium hydrogen phosphate were 0.98 g L-1 and 0.79 g L-1, respectively. The maximal biomass and paramylon content were 0.72 g L-1 and 71.39% of dry weight. The algal powder contained more than 16 amino acids, 6 vitamins, and 10 unsaturated fatty acids under the optimal cultivation. E. pisciformis paramylon was pure ß-1,3-glucan-type polysaccharide (the purity was up to 99.13 ± 0.61%) composed of linear glucose chains linked together by ß-1,3-glycosidic bonds. These findings present a valuable basis for the industrial exploitation of paramylon with E. pisciformis AEW501.

Effect of supplementation with algae ß-glucans on performance, health, and blood metabolites of Holstein dairy calves.

Studies have shown that ß-glucans extracted from the cell wall of cereals, algae, and yeasts have been associated with improved immune function. However, it is unknown whether algae ß-glucan supplementation affects the performance, blood metabolites, or cell counts of immune cells in dairy calves. The objective of this randomized clinical trial was to evaluate whether supplementation of ß-glucans to milk replacer in dairy calves fed 6 L/d improved growth performance and fecal status and altered the blood metabolite profile. In this trial, we enrolled Holstein calves (n = 34) at birth (body weight 36.38 ± 1.33 kg; mean ± standard deviation) to receive, from 1 d of age, either 2 g/d algae ß-glucans mixed into 6 L/d of milk replacer (22.4% crude protein and 16.2% fat) or an unsupplemented milk replacer (control). The calves were blocked in pairs according to birth weight, sex, and date of birth (up to 5 d difference). Calves were housed individually, and calf starter (24.7% crude protein and 13.9% neutral detergent fiber) was offered ad libitum based on orts of the previous day until 56 d of age (end of the trial). Body weight was measured weekly, and health checks and daily fecal consistency were evaluated daily in every calf by the same observer. Calves with 2 consecutive days of loose feces that sifted through bedding were considered diarrhea positive. We used a linear mixed effects model to evaluate the effects of ß-glucan supplementation fed during the preweaning period on performance (average daily gain), final weight, feed efficiency (FE), white blood cell count, and selected blood metabolites, repeated by time. A generalized linear mixed effects model was also run to evaluate the likelihood of a diarrhea bout in the first 28 d of life, controlling for the calf as the subject with a logistic distribution. We included age, serum total protein at 48 h, and birth weight as covariates. At 56 d, ß-glucan-supplemented calves weighed more than control calves (56.3 vs. 51.5 kg). Treatment had no effect on total starter intake, but there was a treatment by age interaction for FE, with greater FE for ß-glucan-supplemented calves in wk 3 and 5 of age. There was only a tendency for average daily gain to be greater in supplemented calves than in control calves for the duration of the study. Furthermore, control calves had 14.66 [95% confidence interval (95% CI): 9.87-21.77] times greater odds of having a diarrheal bout than ß-glucan-supplemented calves. Control calves had 12.70 (95% CI: 8.82-18.28) times greater odds of having an additional day with an abnormal fecal score compared with ß-glucan-supplemented calves, suggesting that supplementation ameliorated diarrhea severity. We found no association of treatment with concentrations of serum total protein, albumin, creatinine, or glucose during the preweaning period. Our findings suggest that dietary supplementation of 2 g/d of algae ß-glucans to milk replacer improved fecal status and may affect growth, as evidenced by a higher weaning weight, compared with control calves. Future studies should explore the effect of algae ß-glucans on lower-gut physiology and digestibility in dairy calves.

Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

Paramylon and Other Bioactive Molecules in Micro and Macroalgae.

Many algae synthesize compounds that have exceptional properties of nutraceutical, pharmacological, and biomedical interest. Pigments, fatty acids, phenols, and polysaccharides are among the main compounds investigated so far. Polysaccharides are the most exploited compounds, widely used in pharmaceutical, food, and chemical industries, which are at present entering into more advanced applications by gaining importance, from a therapeutic point of view, as antioxidant, antimicrobial, antitumor, and immunomodulatory agents. Establishing algae as an alternative supplement would complement the sustainable and environmental requirements in the framework of human health and well-being. This review focuses on the proprieties and uses of the main micro- and macroalgae metabolites, describing their potential for application in the different industrial sectors, from food/feed to chemical and pharmacological. Further, current technologies involved in bioactive molecule extraction strategies are documented.

Molecular Cross-Talk between Gravity- and Light-Sensing Mechanisms in Euglena gracilis.

Euglena gracilis is a photosynthetic flagellate. To acquire a suitable position in its surrounding aquatic environment, it exploits light and gravity primarily as environmental cues. Several physiological studies have indicated a fine-tuned relationship between gravity sensing (gravitaxis) and light sensing in E. gracilis. However, the underlying molecular mechanism is largely unknown. The photoreceptor photoactivated adenylyl cyclase (PAC) has been studied for over a decade. Nevertheless, no direct/indirect interaction partner (upstream/downstream) has been reported for PAC. It has been shown that a specific protein, kinase A (PKA), showed to be involved in phototaxis and gravitaxis. The current study reports the localization of the specific PKA and its relationship with PAC.

Heterotrophic Cultivation of Euglena gracilis in Stirred Tank Bioreactor: A Promising Bioprocess for Sustainable Paramylon Production.

Paramylon is a valuable intracellular product of the microalgae Euglena gracilis, and it can accumulate in Euglena cells according to the cultivation conditions. For the sustainable production of paramylon and appropriate cell growth, different bioreactor processes and industrial byproducts can be considered as substrates. In this study, a complex medium with corn steep solid (CSS) was used, and various bioreactor processes (batch, fed batch, semicontinuous and continuous) were performed in order to maximize paramylon production in the microalgae Euglena gracilis. Compared to the batch, fed batch and repeated batch bioprocesses, during the continuous bioprocess in a stirred tank bioreactor (STR) with a complex medium containing 20 g/L of glucose and 25 g/L of CSS, E. gracilis accumulated a competitive paramylon content (67.0%), and the highest paramylon productivity of 0.189 g/Lh was observed. This demonstrated that the application of a continuous bioprocess, with corn steep solid as an industrial byproduct, can be a successful strategy for efficient and economical paramylon production.

Metabolomics revealed the photosynthetic performance and metabolomic characteristics of Euglena gracilis under autotrophic and mixotrophic conditions.

Photosynthetic and metabolomic performance of Euglena gracilis was examined and compared under autotrophic and mixotrophic conditions. Autotrophic protozoa (AP) obtained greater biomass (about 33% higher) than the mixotrophic protozoa (MP) after 12 days of growth. AP maintained steady photosynthesis, while MP showed a remarkable decrease in photosynthetic efficiency and dropped to an extremely low level at day 12. In MP, low light absorption and photosynthetic electron transport efficiency, and high energy dissipation were reflected by the chlorophyll (chl a) fluorescence (OJIP) of the protozoa. The values of ΨO, ΦEo, and ETO/RC of MP decreased to extremely low levels, to 1/15, 1/46, and 1/9 those of AP, respectively, while DIO/RC increased to approximately 16 times that of AP. A total of 137 metabolites were showed significant differences between AP and MP. AP accumulated more monosaccharide, lipids, and alkaloids, while MP produced more amino acids, peptides, and long-chain fatty acids including poly-unsaturated fatty acids. The top nine most important enriched pathways obtained from KEGG mapping were related to ABC transporters, biosynthesis of amino acids, purine metabolism, and carbohydrate metabolism. There were significant differences between AP and MP in photosynthetic activity, metabolites, and metabolic pathways. This work presented useful information for the production of high value bioproducts in E. gracilis cultured under different nutritional conditions.

Protective Effect of Methanolic Extract of Euglena tuba Against Dalton Lymphoma Induced Oxidative Stress in BALB/c Mice.

The identification and pharmacological validation of plant-based lead compounds for the cure of different diseases including cancer have always been globally strived. In addition to possessing numerous medicinal properties, many of the phytochemicals display antioxidant potential activities. Reactive oxygen species (ROS) causeoxidative stress leading to several severe diseases such as cancer. The antioxidants are substances that fight against ROS to protect the cells from their damaging effects. In the present study, the effects of methanol extract of Euglena tuba(ETME) have been evaluated for its antioxidant and antitumor potential against Dalton's lymphoma (DL) introduced in BALB/cmice. After 24 h of intraperitoneal inoculation of DL cells in mice, ETME (300 mg kg-1 body weight) was administered intraperitoneally upto18 alternative days. On the 18th day, the mice were sacrificed; the blood and tissues (liver and brain) were collected to determine the tumor growth parameters including morphological, behavioural, haematological profile, and antioxidant indices. The results indicated that ETME exhibited significant antioxidative and antitumor properties when compared with the data from DL bearing mice. The results from the present study indicated that ETME contained remarkable antitumor efficacy, which was mediated through amelioration of oxidative stress. The data suggested that ETME could be used as a potential natural anticancer agent.