Study of routine metabolism and acute toxicity of mycogenic silver nanoparticles on Palaemon pandaliformis (shrimp)

Palaemon pandaliformis is a shrimp species considered as an important indicator of environmental conditions. In this study, the toxicological effects of mycogenic silver nanoparticles obtained using the fungus Aspergillus tubingensis (AgNP-AT) were evaluated on P. pandaliformis. The AgNP-AT were spherical and homogeneous in size. Compared with the untreated group, at 10 μM AgNP-AT, there was a reduction of 60 and 87% for oxygen consumption and ammonia excretion, respectively. In addition to the untreated group, silver nitrate (AgNO3) was also used as a control, where concentrations at least 10 times lower than those of the AgNPs also reduced the oxygen consumption by 54% and increased the ammonia excretion by 33%, demonstrating its high toxicity. At 24, 48, 72 and 96 h, the AgNP-AT showed lethal concentrations (LC50) of 17.4, 14.4, 12.6 and 0.5 μM, while AgNO3 showed LC50 values of 2.5, 1.0, 0.02 and 0.01 μM, respectively. For all groups exposed to AgNPs, histological analysis showed that, in relation to the untreated group, there was a significant increase in the mean area of the shrimps' gills, indicating hypertrophy. All the changes observed in the hepatopancreatic and gill tissue, mainly for AgNPs at the highest concentration of 10 μM, are described as reversible. The results indicated that the AgNP-AT toxicity to the shrimps increased according to the time of exposure. However, it is important to highlight that the AgNP-AT were less toxic than AgNO3 to the shrimps, probably due to the presence of a protein coat in these AgNPs, which is able to control the release of Ag+ ions, avoiding high toxicity to the shrimps.

Janibacter sp. isolated from deschampsia antarctica rhizosphere as a potential source of antimicrobial compounds

In this study, 106 bacterial strains were isolated from rhizosphere of Deschampsia antarctica and used for the screening of potential antimicrobial compounds. Among all the bacterial strains, 61 (57.55%) were classified as Gram-positive, 34 (32.08%) as Gram-negative, and 11 (10.37%) did not grown under the laboratory culture conditions and were not classified. Organic crude extracts were analysed for potential antimicrobial activity and the B-22-EA ethyl acetate extract was the most effective inhibiting 84% of Escherichia coli growth, and Staphylococcus aureus, Pseudomonas aeruginosa and Micrococcus luteus by 31, 17 and 5%, respectively. The B-22-EA extract showed a MIC of 187 µg/mL on Cryptococcus neoformans, and of 438 µg/mL on Trichophyton rubrum. The strain, coded as B22, was classified as Gram-positive and the taxonomic analysis indicated that this strain belongs to the genera Janibacter, with 98.66% of similarity with Janibacter hoylei PVAS-1 and Janibacter limosus DSM 11140; 98.51% with Janibacter cremeus HR08-44, and 98.36% with Janibacter indicus 0704P10-1, Janibacter anophelis H2.16B and Janibacter terrae CS12. The cellular fatty acids (CFA) composition for B22 (Janibacter sp.) was determined by fatty acid methyl esters (FAME) using gas chromatography and compared with those species for which the B22 strain has a high level of similarity. The comparison among the different species from the genus Janibacter shows that there is a difference in their CFA composition and in their optimum temperature for growth, showing the high biodiversity, even for the same species. The data from this study are important and B-22 (Janibacter sp.) is an interesting source of antimicrobial compounds.

Effect of monocerin, a fungal secondary metabolite, on endothelial cells

This study reports the isolation and identification of the endophytic fungus Exserohilum rostratum through molecular and morphological analysis using optical and transmission electron microscopy (TEM), as well as the procurement of its secondary metabolite monocerin, an isocoumarin derivative. Considering the previously observed biological activities of monocerin, this study was performed on human umbilical vein endothelial cells (HUVECs) that are widely used as an in vitro model for several different purposes. Important parameters, such as cell viability, senescence-associated β-galactosidase, cellular proliferation by using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), apoptosis analysis with annexin, cellular morphology through scanning electron microscopy (SEM), and laser confocal analysis were evaluated after exposing the cells to monocerin. After 24 h of exposure to monocerin at 1.25 mM, there was more than 80% of cell viability and a low percentage of cells in the early and late apoptosis and necrosis. Monocerin increased cell proliferation and did not induce cell senescence. Morphological analysis showed cellular integrity. The study demonstrates aspects of the mechanism of action of monocerin on endothelial cell proliferation, suggesting the possibility of its pharmaceutical application, such as in regenerative medicine.

Antifungal activity of mycogenic silver nanoparticles on clinical yeasts and phytopathogens

In this study, seven different silver nanoparticles (AgNPs) were obtained using the fungi species from the phylum Ascomycota, Aspergillus tubingensis, Aspergillus spp., Cladosporium pini-ponderosae, Fusarium proliferatum, Epicoccum nigrum, Exserohilum rostratum, and Bionectria ochroleuca, isolated from the Brazilian biodiversity, particularly from the mangrove and Caatinga biomes. The nanoparticles were coded as AgNP-AT, AgNP-Asp, AgNP-CPP, AgNP-FP, AgNP-EN, AgNP-ER, and AgNP-BO and characterized using spectrophotometry (UV-Vis), dynamic light scattering (DLS), zeta potential, transmission electron microcopy (TEM), and Fourier-transform infrared (FTIR) spectroscopy. All the AgNPs presented homogeneous size in the range from 43.4 to 120.6 nm (DLS) and from 21.8 to 35.8 nm (TEM), pH from 4.5 to 7.5, negative charge, and presence of protein coating on their surface. The antifungal activity of the AgNPs was evaluated on clinical strains of Candida albicans, and on the non-albicans species, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Candida guilliermondii, common in hospital infections, and against the phytopathogens Fusarium oxysporum, Fusarium phaseoli, Fusarium sacchari, Fusarium subglutinans, Fusarium verticillioides, and Curvularia lunata, which are species responsible for serious damage to agriculture production. The AgNPs were effective against the yeasts with MICs ranging from 1.25 to 40 µM and on the phytopathogens with MICs from 4 to 250 µM, indicating the promising possibility of application of these AgNPs as antifungal agents. The results indicated that the physicochemical parameters of the AgNPs, including the functional groups present on their surface, interfered with their antifungal activity. Overall, the results indicate that there is no specificity of the AgNPs for the yeasts or for the phytopathogens, which can be an advantage, increasing the possibility of application in different areas.

Overview of nanomaterials and cellular interactions

In the last three decades, there has been wide progress in nanomaterials development, and several studies are being performed to show its biological effects and cellular interaction for biomedical applications. Due to the exponential increase in nanomaterial diversity, production, and possibilities of applications in different areas, there is an important concern about its toxicity for humans, animals, and ecosystems. There is a great effort to minimize experimental assays in animals, and this is a commendable initiative. Several alternatives in vitro assays are available; however, several new protocols have been introduced to elucidate the mechanisms of cell-nanomaterial interaction. Wide and fast progress in nanotechnology has been observed. Nonetheless, the nanomaterial interaction with cells or biological systems is still not totally described. In this aspect, this paper is a brief overview of nanomaterials and cellular interactions (nano-bio interaction).

Overview of bioactive fungal secondary metabolites: cytotoxic and antimicrobial compounds

Microorganisms are known as important sources of natural compounds that have been studied and applied for different purposes in distinct areas. Specifically, in the pharmaceutical area, fungi have been explored mainly as sources of antibiotics, antiviral, anti-inflammatory, enzyme inhibitors, hypercholesteremic, antineoplastic/antitumor, immunomodulators, and immunosuppressants agents. However, historically, the high demand for new antimicrobial and antitumor agents has not been sufficiently attended by the drug discovery process, highlighting the relevance of intensifying studies to reach sustainable employment of the huge world biodiversity, including the microorganisms. Therefore, this review describes the main approaches and tools applied in the search for bioactive secondary metabolites, as well as presents several examples of compounds produced by different fungi species with proven pharmacological effects and additional examples of fungal cytotoxic and antimicrobial molecules. The review does not cover all fungal secondary metabolites already described; however, it presents some reports that can be useful at any phase of the drug discovery process, mainly for pharmaceutical applications.

Biosynthesis of silver nanoparticles using actinomycetes, phytotoxicity on rice seeds, and potential application in the biocontrol of phytopathogens

To find effective silver nanoparticles (AgNPs) for control of phytopathogens, inthis study, two strains of actinomycetes isolated from the soil of the Brazilianbiome Caatinga (Caat5–35) and from mangrove sediment (Canv1–58) wereutilized. The strains were identified by using the 16S rRNA gene sequencing asStreptomycessp., related toStreptomyces mimosusspecies. The obtained AgNPswere coded as AgNPs35and AgNPs58and characterized by size andmorphology using dynamic light scattering, zeta potential, transmissionelectron microscopy, and Fourier transformed infrared (FTIR). The antifungalactivity of the AgNPs35and AgNPs58was evaluatedin vitroby the minimalinhibitory concentration (MIC) assay on the phytopathogens,Alternariasolani,Alternaria alternata, andColletotrichum gloeosporioides. The phytotoxiceffect was evaluated by the germination rate and seedling growth of rice(Oryza sativa). AgNPs35and AgNPs58showed surface plasmon resonance andaverage sizes of 30 and 60 nm, respectively. Both AgNPs presented sphericalshape and the FTIR analysis confirmed the presence of functional groups suchas free amines and hydroxyls of biomolecules bounded to the external layer ofthe nanoparticles. Both AgNPs inhibited the growth of the three phytopatho-gens tested, andA. alternatewas the most sensible (MIC≤4 μM). Moreover,the AgNPs35and AgNPs58did not induce phytotoxic effects on thegermination and development of rice seedlings. In conclusion, these AgNPsare promising candidates to biocontrol of these phytopathogens withoutendangering rice plants.

The bioflavonoids rutin and rutin succinate neutralize the toxins of B. jararaca venom and inhibit its lethality

The venom of the Brazilian pit viper Bothrops jararaca (BjV) is a complex mixture of molecules, and snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) are the most abundant protein families found therein. Toxins present in BjV trigger most of the deleterious disturbances in hemostasis observed in snakebites, i.e., thrombocytopenia, hypofibrinogenemia and bleedings. The treatment of patients bitten by snakes still poses challenges and the bioflavonoid rutin has already been shown to improve hemostasis in an experimental model of snakebite envenomation. However, rutin is poorly soluble in water; in this study, it was succinylated to generate its water-soluble form, rutin succinate (RS), which was analyzed comparatively regarding the chemical structure and characteristic features of rutin. Biological activities of rutin and RS were compared on hemostatic parameters, and against toxic activities of crude BjV in vitro. In vivo, C57BL/6 mice were injected i.p. with either BjV alone or pre-incubated with rutin, RS or 1,10-phenanthroline (o-phe, an SVMP inhibitor), and the survival rates and hemostatic parameters were analyzed 48 h after envenomation. RS showed the characteristic activities described for rutin – i.e., antioxidant and inhibitor of protein disulfide isomerase – but also prolonged the clotting time of fibrinogen and plasma in vitro. Differently from rutin, RS inhibited typical proteolytic activities of SVMP, as well as the coagulant activity of BjV. Importantly, both rutin and RS completely abrogated the lethal activity of BjV, in the same degree as o-phe. BjV induced hemorrhages, falls in RBC counts, thrombocytopenia and hypofibrinogenemia in mice. Rutin and RS also improved the recovery of platelet counts and fibrinogen levels, and the development of hemorrhages was totally blocked in mice injected with BjV incubated with RS. In conclusion, RS has anticoagulant properties and is a novel SVMP inhibitor. Rutin and RS showed different mechanisms of action on hemostasis. Only RS inhibited directly BjV biological activities, even though both flavonoids neutralized B. jararaca toxicity in vivo. Our results showed clearly that rutin and RS show a great potential to be used as therapeutic compounds for snakebite envenomation.

Antimicrobial activity of snake β-defensins and derived peptides

β-defensins are antimicrobial peptides presenting in vertebrate animals. They participate in innate immunity, but little is known about them in reptiles, including snakes. Although several β-defensin genes were described in Brazilian snakes, their function is still unknown. The peptide sequence from these genes was deduced, and synthetic peptides (with approximately 40 amino acids and derived peptides) were tested against pathogenic bacteria and fungi using microbroth dilution assays. The linear peptides, derived from β-defensins, were designed applying the bioisosterism strategy. The linear β-defensins were more active against Escherichia coli, Micrococcus luteus, Citrobacter freundii, and Staphylococcus aureus. The derived peptides (7–14 mer) showed antibacterial activity against those bacteria and on Klebsiella pneumoniae. Nonetheless, they did not present activity against Candida albicans, Cryptococcus neoformans, Trychophyton rubrum, and Aspergillus fumigatus showing that the cysteine substitution to serine is deleterious to antifungal properties. Tryptophan residue showed to be necessary to improve antibacterial activity. Even though the studied snake β-defensins do not have high antimicrobial activity, they proved to be attractive as template molecules for the development of antibiotics.

Wide visible-range activatable fluorescence ZnSe:Eu3+/Mn2+@ZnS quantum dots: local atomic structure order and application as a nanoprobe for bioimaging

The development of QDs-based fluorescent bionanoprobe for cellular imaging fundamentally relies upon the precise knowledge of particle–cell interaction, optical properties of QDs inside and outside of the cell, movement of a particle in and out of the cell, and the fate of particle. We reported engineering and physicochemical characterization of water-dispersible Eu3+/Mn2+ co-doped ZnSe@ZnS core/shell QDs and studied their potential as a bionanoprobe for biomedical applications, evaluating their biocompatibility, fluorescence behaviour by CytoViva dual mode fluorescence imaging, time-dependent uptake, endocytosis and exocytosis in RAW 264.7 macrophages. The oxidation state and local atomic structure of the Eu dopant studied by X-ray absorption fine structure (XAFS) analysis manifested that the Eu3+ ions occupied sites in both ZnSe and ZnS lattices for the core/shell QDs. A novel approach was developed to relieve the excitation constraint of wide bandgap ZnSe by co-incorporation of Eu3+/Mn2+ codopants, enabling the QDs to be excited at a wide UV-visible range. The QDs displayed tunable emission colors by a gradual increase in Eu3+ concentration at a fixed amount of Mn2+, systematically enhancing the Mn2+ emission intensity via energy transfer from the Eu3+ to Mn2+ ion. The ZnSe:Eu3+/Mn2+@ZnS QDs presented high cell viability above 85% and induced no cell activation. The detailed analyses of QDs-treated cells by dual mode fluorescence CytoViva microscopy confirmed the systematic color-tunable fluorescence and its intensity enhances as a function of incubation time. The QDs were internalized by the cells predominantly via macropinocytosis and other lipid raft-mediated endocytic pathways, retaining an efficient amount for 24 h. The unique color tunability and consistent high intensity emission make these QDs useful for developing a multiplex fluorescent bionanoprobe, activatable in wide-visible region.

Anti-Biofilm action of biological silver nanoparticles produced by aspergillus tubingensis and antimicrobial activity of fabrics carrying it

Biological silver nanoparticles (AgNPs) were synthesized using the marine endophytic fungus Aspergillus tubingensis and inhibited Bacillus subtilis biofilm formation at low concentrations. Cotton and polyester fabrics impregnated with AgNPs were analyzed by transmission electron microscopy (TEM), and the concentration of AgNPs in both fabrics was determined using inductivelycoupled plasma atomic emission spectrometry (ICP-AES). The fabrics carrying the AgNPs inhibited the Staphylococcus aureus and Escherichia coli growth by 100%. Both fabrics impregnated one time with AgNPs inhibited yeasts' clinical species' growth, Candida albicans, Candida glabrata, and Candida parapsilosis, from 80.1% to approximately 98.0%. Besides the anti-biofilm effect, the AgNPs impregnation process on cotton and polyester fabrics was highly efficient, and both fabrics presented antimicrobial effects against clinically relevant bacteria and yeast species. The results evidence that functionalized textiles containing these biological AgNPs can play an essential role in combating pathogenic microorganisms. Thereby offering an alternative to design effective solutions, mainly for hospital garments and biomedical devices, to avoid microorganisms transmissions and hospitalacquired nosocomial infections.

Differential physiological responses of a biogenic silver nanoparticle and its production matrix silver nitrate in Sorghum bicolor

Silver nanoparticles (AgNP) have been extensively applied in different industrial areas, mainly due to their antibiotic properties. One of the environmental concerns with AgNP is its incorrect disposal, which might lead to severe environmental pollution. The interplay between AgNP and plants is receiving increasing attention. However, little is known regarding the phytotoxic effects of biogenic AgNP on terrestrial plants. This study aimed to compare the effects of a biogenic AgNP and AgNO3 in Sorghum bicolor seedlings. Seeds were germinated in increasing concentrations of a biogenic AgNP and AgNO3 (0, 10, 100, 500, and 1000 μM) in a growth chamber with controlled conditions. The establishment and development of the seedlings were evaluated for 15 days. Physiological and morpho-anatomical indicators of stress, enzymatic, and non-enzymatic antioxidants and photosynthetic yields were assessed. The results showed that both AgNP and AgNO3 disturbed germination and the establishment of sorghum seedlings. AgNO3 released more free Ag+ spontaneously compared to AgNP, promoting increased Ag+ toxicity. Furthermore, plants exposed to AgNP triggered more efficient protective mechanisms compared with plants exposed to AgNO3. Also, the topology and connectivity of the correlation-based networks were more impacted by the exposure of AgNO3 than AgNP. In conclusion, it is plausible to say that the biogenic AgNP is less toxic to sorghum than its matrix AgNO3.

Orange-Emitting ZnSe: Mn2+ quantum dots as nanoprobes for macrophages

The biocompatibility, bionanointeraction, uptake efficiency, and entry pathway of luminescent nanomaterials are the key factors to understand development of an efficient bionanoprobe. The foremost objective of this work is to explore the potential of 3-mercaptopropionic acid (3-MPA) capped ZnSe:xMn2+ (x = 5, 10, and 15 mol %) quantum dots (QDs) for the development of bionanoprobe used in future biological and clinical applications. For this purpose, highly intense orange-emitting activator Mn2+ ion doped ZnSe QDs were synthesized via a high-temperature organometallic method and rendered water-soluble by a ligand exchange approach. The morphological and physicochemical characterizations displayed the ultrasmall zinc-blend cubic crystal structure of QDs with an elliptical shape nanocrystals and average diameter of 4 nm. The luminescent nanomaterials exhibited orange emission centered at 584 nm under excitation at 385 nm. The biocompatibility, time-dependent cellular uptake, and the uptake mechanism of QDs were studied in RAW 264.7 macrophages, accomplished by various cytotoxicity assays, CytoViva hyperspectral enhanced dark-field and dual-mode fluorescence (DMF) microscopy, and transmission electron microscopy (TEM) images. The cytotoxicity study did not confirm any noticeable deleterious effect of QDs within incubation for 6 h. The fluorescence images of cells incubated with QDs showed efficient emission, which is a manifestation that QDs are photochemically stable in the intracellular environment. The cellular uptake findings demonstrated that the QDs were predominantly internalized via clathrin- and caveolae-mediated pathways. After the uptake, QDs aggregates appeared inside the vesicles in the cytoplasm, and their number and size gradually increased as a function of time. Nevertheless, the fluorescent QDs presented remarkable colloidal stability in various media, biocompatibility within the designated time, efficient time-dependent uptake, and distinct entry pathway in RAW macrophages, suggesting promising candidates to explore for the development of future bionanoprobes.

Environmental impact of biogenic silver nanoparticles in soil and aquatic organisms

Synthetic silver nanoparticles (AgNPs) are being extensively used in our daily lives; however, they may also pose a risk to public health and environment. Nowadays, biological AgNPs are considered an excellent alternative, since their synthesis occurs by a green technology of low cost and easy scaling. However, studies with these biological nanomaterials (NM) are still limited. Thus, a more careful assessment of their industrial application, economic feasibility and ecotoxicological impacts is crucial. The aim of this study was to investigate the effects of different concentrations of mangrove fungus Aspergillus tubingensis AgNPs on the aerobic heterotrophs soil microorganisms, rice seeds (Oryza sativa) and zebrafish (Danio rerio). Biogenic AgNPs were less harmful for soil microbiota compared to AgNO3. On rice seeds, the AgNPs displayed a dose-dependent inhibitory effect on germination and their subsequent growth and development. The percentage of inhibition of rice seed germination was 30, 69 and 80% for 0.01, 0.1 and 0.5 mM AgNPs, respectively. After 24h of AgNPs exposition at a limit concentration of 0.2 mM, it did not induce mortality of the zebrafish D. rerio. Overall, A. tubingensis AgNPs can be considered as a suitable alternative to synthetic nanoparticles.

Biogenic Aspergillus tubingensis silver nanoparticles’ in vitro effects on human umbilical vein endothelial cells, normal human fibroblasts, HEPG2, and Galleria mellonella

Silver nanoparticles (AgNPs) are widely incorporated into different hygiene, personal care, and healthcare products. However, few studies have been undertaken to determine the effects of biogenic AgNPs on human health. The effect of biosynthesized AgNPs using the fungus Aspergillus tubingensis culture was evaluated on human umbilical vein endothelial cells (HUVECs), normal human fibroblasts (FN1), human hepatoma cells (HEPG2) and a Galleria mellonella model. HUVECs were more susceptible to biogenic AgNPs than normal fibroblasts FN1 and intense cytotoxicity was observed only for very high concentrations at and above 2.5 µM for both cells. Normal human fibroblasts FN1 exposed to AgNPs for 24 h showed viability of 98.83 ± 8.40% and 94.86 ± 5.50% for 1.25 and 2.5 µM, respectively. At 5 and 10 µM, related to the control, an increase in cell viability was observed being 112.66 ± 9.94% and 117.86 ± 8.86%, respectively. Similar results were obtained for treatment for 48 and 72 h. At 1.25, 2.5, 5 and 10 µM of AgNPs, at 24 h, HUVECs showed 51.34 ± 7.47%, 27.01 ± 5.77%, 26.00 ± 3.03% and 27.64 ± 5.85% of viability, respectively. No alteration in cell distribution among different cycle phases was observed after HUVEC and normal fibroblast FN1 exposure to AgNPs from 0.01 to 1 µM for 24, 48 and 72 h. Based on the clonogenic assay, nanoparticles successfully inhibited HEPG2 cell proliferation when exposed to concentrations up to 1 µM. In addition to that, AgNPs did not induce senescence and no morphological alteration was observed by scanning electron microscopy on the endothelial cells. In the larvae of the wax moth, Galleria mellonella, a model for toxicity, AgNPs showed no significant effects, which corroborates to the safety of their use in mammalian cells. These results demonstrate that the use of A. tubingensis AgNPs is a promising biotechnological approach and these AgNPs can be applied in several biomedical situations.

Functional textiles impregnated with biogenic silver nanoparticles from Bionectria ochroleuca and its antimicrobial activity

Biogenic silver nanoparticles (AgNPs) were obtained throughout the fungal biosynthesis using extracellular filtrate of the epiphytic fungus B. ochroleuca and were incorporated in cotton and polyester fabrics by common impregnation procedure that was repeated once, twice or four times. Both fabrics were analyzed by scanning electron microscopy (SEM), and the effectiveness of impregnation was determined using inductively coupled plasma optical emission spectrometry (ICP OES). The AgNPs loaded fabrics showed potent antimicrobial activity on Staphylococcus aureus and Escherichia coli as well as on clinically relevant Candida albicans, Candida glabrata, and Candida parapsilosis, indicating that the AgNPs impregnation of cotton and polyester fabrics was efficient. AgNPs effectively inhibited the biofilm formation by Pseudomonas aeruginosa and was not toxic to Galleria mellonella larvae indicating a promising probability of biotechnological application.

Chemical composition and histochemistry of Sphagneticola trilobata essential oil

Anatomical and histochemical investigations of Sphagneticola trilobata (L.) Pruski, Asteraceae, secretory structures in leaves and stems and the seasonal variation of essential oils were carried out. Histochemical techniques enabled the specific location of the essential oil accumulation in the internal (canals) and external structures (trichomes). Histochemical analysis showed that the secretory trichomes produced steroids. The highest yield was obtained from plants collected in winter, when it was registered low temperature and precipitation. The essential oil was characterized by high percentage of hydrocarbon sesquiterpenes, hydrocarbon monoterpenes and low levels of oxygenated sesquiterpenes. The major components were germacrene D (11.9-35.8%), α-phellandrene (1.4-28.5%), α-pinene (7.3-23.8%), E-caryophyllene (4.6-19.0%), bicyclogermacrene (6.0-17.0%), limonene (1.8-15.1%) and α-humulene (4.0-11.6%). The percentage of most of the individual constituents present in S. trilobata essential oil changed significantly during the months.