The signal intensity of MG showed good linearity over the concentration range of 10–1000 μg·L−1. Measurement of practical water samples fortified with MG at 0.01, 0.1 and 1.0 mg·L−1 gave a good validation of the established calibration curve. The average recoveries and relative standard deviation (RSD) of malachite green in lake water and Carassius carassius fish farm effluent water were 115% (6.64% RSD), 85.4% (9.17% RSD) and 96.0% (7.44% RSD), respectively. Overall, the established EESI-MS/MS method has been demonstrated suitable for sensitive and rapid (<2 min per sample) quantitative detection of malachite green in various aqueous media, indicating its potential for online real-time monitoring of real life samples.Exposure to malachite green (MG) may pose great health risks to humans; thus, it is of prime importance to develop fast and robust methods to quantitatively screen the presence of malachite green in water. Herein the application of extractive electrospray ionization mass spectrometry (EESI-MS) has been extended to the trace detection of MG within lake water and aquiculture water, due to the intensive use of MG as a biocide in fisheries. This method has the advantage of obviating offline liquid-liquid extraction or tedious matrix separation prior to the measurement of malachite green in native aqueous medium. The experimental results indicate that the extrapolated detection limit for MG was ~3.8 μg·L In the EU, the use of MG for food fish was banned in 2000 [−1 [Malachite green (MG) is a cationic triarylmethane dye that is commonly used as a biocide in aquaculture worldwide. It provides efficient defense against fungal attacks, protozoan infections and other diseases in aquatic organisms, e.g., caused by helminths . Besides in 2000 . Inspect in 2000 , posing n) [−1 after the complicated pre-concentration processes , which took more than 2.5 min per sample [−1 [Over the years, an increasing number of analytical techniques such as spectrophotometry ,7,8, hign) ,13,14 ann)  have been) . The limr sample . Temperample [−1 . A long Liquid samples can be directly analyzed by extractive electrospray ionization mass spectrometry (EESI-MS) without sample pretreatment ,18,19,20Carassius carassius (pH 6.0), respectively.Malachite green was purchased from Tianjin Fuchen Chemical Reagent Factory. Methanol (HPLC grade) was provided by ROE Company . Ultrapure water (resistivity 18.2 MΩ·cm) was supplied by a Barnstead Nanopure ultrapure water purification system . Environmental water samples were obtained from a man-made lake (pH 5.5) and aquariums for feeding m/z. The ESI voltage was set at 3.5 kV; the temperature of the ion-transport capillary was 400 °C; the injection rates of ESI solvent and sample solution was set at 3 μL·min−1 and 5 μL·min−1, respectively; high purity nitrogen gas (purity ≥ 99.999%) from a gas cylinder are used for nebulizing the ESI solvent  and sample solution ; and the pressure was 1.4 MPa. The EESI assembly was mounted on a 3-D adjustable stage  equipped with a home-made EESI source ,22,23. Tshown in . The dism/z 329 was selected as the parent ion, and the isolation width and activation time were set at 1.5 Da and 30 ms, respectively. CID was set with 30% collision energy, and other parameters were automatically optimized by LTQ-MS system. All the mass spectra were recorded with an average duration time of 0.2 min, followed by background subtraction.The full scan mass spectra were recorded using Xcalibur software of the LTQ-MS instrument. In collision induced dissociation (CID) experiments, the ion at −1) was prepared by dissolving 1 g of malachite green in the 1 L volumetric flask of ultrapure water, and stored in dark. Standard working solutions  were prepared by serially diluting the malachite green stock solution with lake water. Stock solution of malachite green (1 g·L−1 MG. In the MS2 spectrum of the MG cation (m/z 329), major fragments at m/z 313, 285, 251, 237, 208 were recorded upon collision activation at the energy of 30%  pressure, were systematically optimized.In order to achieve the best extraction and ionization efficiency of malachite green within aqueous samples using our EESI source, several experimental parameters, including ESI voltage, sample injection rate, ion-transport capillary temperature and sheath gas  and relative standard deviation (RSD) as error bars were 0.50 , 2.4 , 3.9 , 12 , 23 , 1.9 × 102  and 1.1 × 103 , respectively, for lake water samples. As shown in 2 = 0.998 at 95% confidence limits was obtained for MG in the range of 0.01–1.0 mg·L−1. Since the points correspond to the concentrations of 5 and 10 mg·L−1 beyond the linear range, the two points have been excluded in the fitting process. The LOD for the lake water sample was extrapolated as 3.8 μg·L−1 (S/N = 3), while the value for the spiked ultrapure water batch was estimated to be ~0.5 μg·L−1 (S/N = 3) (data not shown). While MG can be determined in water samples by LC-vis/FLD and LC-MS/MS with lower LODs of 50 ng·L−1 and 40 ng·L−1, respectively [Under the optimized experimental parameters, lake water samples spiked with 0.001, 0.01, 0.1, 0.5, 1, 5 and 10 mg·LCarassius carassius, were quantitatively analyzed using the developed method. Although the background in the mass spectra of the lake water is neater than that of the fish water due to the more complex inclusions of the latter, MG was not detected in any of these water samples, indicating that the levels of MG in these water samples were below the detection limit. In order to calculate the recovery, spiked samples were prepared at the MG concentrations of 0.1 mg·L−1, 0.01 mg·L−1 and 1.0 mg·L−1. Due to the matrix effect and system error, the recovery rates deviated 100% and were close to 100%, which shows the selectivity and roughness of our method. In addition, due to the same reason mentioned above, the recovery rate of the fish water deviated 100% further. These results are listed in Two different water samples, lake and aquarium water used to feed Low sample consumption is of great value for the analysis of real-life samples that are difficult to obtain. In this study, the minimum volume of the sampled solution was below 1 mL. The measurement time in a typical experiment was less than 2 min. Due to the minimal requirements for the sample’s pretreatment, the measurement time has been greatly reduced in comparison with LC-MS. This shows that the EESI-MS/MS method has several advantages such as high accuracy, high sensitivity and low sample consumption for rapidly quantifying trace analytes in a complex matrix.In this work, a novel method based on EESI-MS/MS has been developed and applied to detect the trace levels of malachite green in different types of aquiculture waters without sample pre-treatment. The analysis does not require tedious sample preparation and can be accomplished within less than 2 min per sample. The method can potentially be applied for the detection of MG and its metabolites from a variety of complex matrices  with high throughput.


Cytochalasin B is a potentially novel microfilament-directed chemotherapeutic agent that prevents actin polymerization, thereby inhibiting cytokinesis. Although cytochalasin B has been extensively studied in vitro, only limited data are available to assess its in vivo potential. Cytochalasin B was administered to Balb/c mice challenged i.d. with M109 murine lung carcinoma to determine whether the agent could affect an established i.d. tumor when the compound is administered s.c. in the region of the i.d. tumor, but not in direct contact with it. Cytochalasin B was also administered either i.p. or s.c. at a distant site or i.v. to determine whether it could affect the long-term development of an established i.d. tumor. Cytochalasin B was then liposome encapsulated to determine whether the maximum tolerated dose (MTD) of the compound could be increased, while reducing immunosuppression that we have previously characterized. Liposomal cytochalasin B was also administered to mice challenged i.d. with M109 lung carcinoma to assess its chemotherapeutic efficacy. The results can be summarized as follows: 1) cytochalasin B substantially delayed the growth of i.d. M109 tumor nodules, inhibited metastatic progression in surrounding tissues, and produced long-term cures in treated mice; 2) liposomal cytochalasin B increased the i.p. MTD by more than 3-fold, produced a different distribution in tissue concentrations, and displayed antitumor effects against M109 lung carcinoma similar to non-encapsulated cytochalasin B. These data show that cytochalasin B exploits unique chemotherapeutic mechanisms and is an effective antineoplastic agent in vivo in pre-clinical models, either in bolus form or after liposome encapsulation. Cytochalasins are mycogenic toxins known to disrupt the formation of actin polymers, thereby preventing the formation of functional microfilaments. These congeners are characterized by a highly substituted perhydro-isoindolone structure that is typically attached to a macrocyclic ring . More th0 resting state until sufficient actin levels have been gathered for successful cytokinesis, malignant cells typically continue to progress through the cell cycle, resulting in the formation of enlarged, multinucleated cells. The multinucleation phenomenon is almost exclusively observed in neoplastic cells, potentially representing a neoplastic-targeting feature of cytochalasin B, as well as other congeners  × 100. The percentage of specific Cr release represents values obtained with immune effectors minus values obtained from control lymphocytes cultured for 4 days in the absence of P815 antigen. Control lymphocytes were prepared from each of the groups treated at various cytochalasin B or liposomal cytochalasin B concentrations, or with the vehicle.To determine whether liposomal cytochalasin B had less of an immunosuppressive effect than its natural counterpart, dose–response and time-course study of the influence that i.p. bolus injection had on spleen response to an allogeneic sensitizer and target tumor cell in vitro were performed as previously described . Splenict-test.Survival analysis used the Cox-Mantel test as detailed by Lee . Effects4 cells/cm2 grew as attached cells and reached confluency at 2 × 105 cells/cm2 after 5 days. The attached cells were compared to the in vivo passaged cells for tumorigenicity defined as fraction of animals developing tumor and time to appearance of palpable tumor nodules, growth rate, invasion, and formation of spontaneous metastases. Growth curves in vivo for i.d. tumors produced by challenge with 2 × 105 M109c cells were fully superimposable for 19 days with growth curves obtained in mice challenged with 4 × 105 M109c trypan blue negative in vivo passaged M109 cells. After 21 days, spontaneous lung metastases had a mean value of 80.3 nodules per mouse (SEM = 19.7) for tumors produced by the attached cells, compared to a mean of 86.6 nodules per mouse (SEM = 13.6) for tumors produced from in vivo passaged cells. Neither of the tumor sources produced significant numbers of metastases to the liver, kidney, spleen, or pancreas. Tumors grew and invaded in all animals challenged i.d. with either tumor source.M109c cells seeded at 4 × 10U-test. The mean number of metastases to the lungs at the respective terminal stages was not significantly affected.Cytochalasin B administered s.c. as a single bolus dose at 100 mg/kg Fig. , or threP < 0.001 between subject effects; p, 0.01 time vs. group interaction by repeated measures analysis of variance) (data not shown). Spontaneous lung metastases determined on day 17 showed a mean number of lung nodules in controls of 23.5 nodules per mouse (SEM = 9.7) compared to 8.3 nodules per mouse (SEM = 4.1) in the treated group (n = 10). This inhibition of spontaneous lung metastases formation was significant at the P < 0.05 level.Treatment of established i.d. tumor nodules of M109c on days 6, 8, and 10 after tumor implantation with bolus cytochalasin B under conditions identical to those used in Fig. Animals challenged with M109c cells i.d. (day 0) were treated daily with i.v. cytochalasin B solution in ethanol-saline at 5 mg/kg/day for 4 or 5 successive days beginning 1 day after tumor implantation. Figure P < 0.05). When all 10 treated animals are used in the determination of “terminal” lung metastases, including the 4 long-term tumor-free survivors, the mean becomes 13.6 nodules per mouse (SEM = 4.3) an 82 % reduction compared to the vehicle controls.As detailed in Fig. p < 0.07 and p < 0.09 respectively). Combined i.p. and s.c. treated groups compared to controls to day 24 was statistically significant (between subject effects p < 0.05).Mice were challenged with M109ni cells i.d., and then stratified into two treatment and one control groups, 10 animals per group, according to tumor size on day 3. Treatments were given on days 4, 7, 10, and 13; 50 mg/kg/day i.p., or 100 mg/kg/day s.c., with cytochalasin B in carboxymethylcellulose-tween suspension as detailed in the materials and methods section. The i.d. tumor challenge was in the lower left anterior quadrant over the peritoneal cavity. Cytochalasin B treated i.p. or s.c. was monitored to day 35. Figure As previously shown  50 mg/kgOne hundred eighty milligram/kilogram liposomal cytochalasin B s.c. had considerable in vivo anticancer activity against M109 lung carcinoma. There was a 66.7 % survival rate at least up to day 62 for mice challenged with two i.d. tumor nodules in the cohort treated with liposomal cytochalasin B on day 8 Fig. . This isThe novel and pleotypic effects of cytochalasins on cells and tissues in vitro have prompted us to predict that these agents will exhibit measurable antitumor activity in vivo by mechanisms which do not depend extensively on gross toxicity . Further, or systemically i.p., s.c., or i.v. is active against established tumor nodules, whereas it is inactive when administered i.p. against early i.p. challenges with ascites cancers [Cytochalasin B administered peritumorally in the same compartment , locally cancers . This suIn our previous study , we obseThe s.c. peritumoral activity of cytochalasin B against s.c. nodules of M109 lung carcinoma has now been extended to established i.d. nodules of M109 tumor where the agent is administered s.c. peritumorally, and to models where direct contact between s.c. cytochalasin B and the i.d. tumor nodule does not occur Fig. . CytochaSystemic antitumor activity elicited by cytochalasin B against nascent M109 i.d. tumor nodules is confirmed in Fig. Although the anticancer activity of cytochalasin B is substantial, it may be beneficial to encapsulate the agent in liposomes before it is administered in vivo. A three-fold increase in the i.p. MTD compared with non-encapsulated cytochalasin B was achieved by the liposomal derivative, with less immunosuppression being observed at this elevated dose Fig. . In addiThis study demonstrates that cytochalasin B produces both localized, separate compartment, and systemic antitumor activity against established tumor nodules. The activity is evidenced by significant alterations to tumor growth rate, invasion, metastases, host survival time, and in some cases, complete tumor regression. Direct gross toxicity is unlikely to be responsible for the effects observed, although toxic effects on sensitive clonogenic subpopulations of tumor cells are not excluded by these data. The localized and systemic perturbations of solid tumors, and the long-delayed influence observed with i.v. drug treatment suggest that direct toxicity is not the sole, or even the most important mechanism by which cytochalasin B exerts in vivo activity. Therefore, it appears plausible that the effects of cytochalasin B in vivo depend on features of tumor tissue physiology, as well as the host response to solid tumors. In addition, liposomal cytochalasin B appears to be an attractive formulation of the agent, exhibiting decreased host toxicity and increased efficacy with favorable effects on host immune response. The liposomal formulation of cytochalasin B and of other cytochalasin congeners may increase the opportunity to develop microfilament-directed agents that are of value in the clinical management of neoplasms.


Morinda lucida was tested on pulse beetle Callosobruchus maculatus, which is a pest that causes serious damage to several pulses. The insecticidal activity was compared with two pesticides, Phostoxin and Primo-ban-20. 120 mixed sex adult C. maculatus were introduced, along with 30 g of cowpeas. Four concentrations  of the M. lucida essential oil, Phostoxin, and Primo-ban-20 were tested. Essential oil chemical composition was analyzed by GC-MS. M. lucida essential oil showed a high toxicological effect, producing 100% mortality after 72 hours at a dose of 0.20 μg/mL. M. lucida essential oil had a potent insecticidal activity (LC90 = 0.629 μg/mL) compared to both pesticides, Phostoxin (LC90 = 0.652 μg/mL) and Primo-ban-20 (LC90 = 0.726 μg/mL), at 24 h. The main compounds of the essential oil were the oxygenated monoterpenoids, 1,8-cineole (43.4%), and α-terpinyl acetate (14.5%), and the monoterpene hydrocarbons, mostly sabinene (8.2%) and β-pinene (4.0%). Results clearly indicate that M. lucida essential oil can be used as an effective alternative for pulse beetle C. maculatus control, and it could be tested against other pulse beetles affecting Asia and Africa and throughout the world, thereby reducing use of synthetic pesticides.Insecticidal activity of essential oil extracted from  Callosobruchus maculatus is an economically important pest of several pulses, including leguminous grains such as lentils, cowpeas, green gram, chickpea, black gram, soybean, and haricot beans [ C. maculatus larvae bore into pulse grain or into stored crop, making it unfit for human consumption and unviable for replanting [ot beans , 2. Thesplanting –5.Control of insect pestsrelies heavily on the use of synthetic insecticides such as methyl bromide or phosphine. However, their intensive use has led to the development of genetic resistance by insect species, pest resurgence, residual toxicity, environmental hazards, and serious problems arising from factors such as direct toxicity to predators, pollinators, fish, and man . As suchBecause of their high volatility, plant extracts or plant compounds and the use of natural compounds with insecticidal activity provide a potential biodegradable alternative to synthetic pesticides –12. The  Morinda lucida (Benth), a member of Rubiaceaefamily, is a medicinal plant of about 15 m tall with a dense crown of slender crooked branches approximately 20–30 cm in diameter. M. lucida commonly known by the Yoruba in southwestern part of Nigeria as “Oruwo” is widely distributed in West Africa and is used in African folk medicine to treat several diseases [Several botanical families for pest control are described, such as Meliaceae, Rutaceae, Asteraceae, Labiatae, and Malvaceae. Rubiaceae is a family of 630 genera and about 13,000 species . Morindadiseases . The leadiseases , 23. M. citrifolia, showed larvicidal activity against three species of mosquito vectors [ M. lucida suggested the need to search for new properties of this plant; therefore the present study was conducted to determine the insecticidal activity of the essential oil of M. lucida against C. maculatus by dose response bioassays and to characterize its chemical composition for consideration as an alternative insecticide for pulse beetle.In a previous study, another species of the same genus, vectors  and the  M. lucida were collected from Apomu , which is located in Osun State, Nigeria, and identified at the Herbarium of the Botany Department, University of Lagos, Nigeria, where voucher specimen number LUH 2637 was kept. The samples were air-dried, pulverized, and stored in polythene bag to reduce evaporation of the volatile oil.The aerial parts of theTo extract the essential oil, 500 g of pulverized sample was placed in a 5 L flask and distilled water was added to cover sample. Essential oil was obtained by hydrodistillation using glass modified Clevenger-type apparatus; the process ran for 4 h at normal atmospheric pressure and at 96-97°C inside the extractor. After the essential oil isolation, residual water was removed by filtration with anhydrous sodium sulphate, and then the essential oil was stored in an amber vial at 4°C for future analysis.μm film thickness), working with the following temperature program: 60°C for 10 min, rising at 5°C/min to 220°C. The injector and detector temperatures were maintained at 250°C; carrier gas nitrogen (2 mL/min); detector dual, FID; split ratio 1 : 30. The volume injected was 0.5 μL. The relative proportions of the oil constituents were percentages obtained by FID peak-area normalization without the use of response factor.GC analysis was performed with a HP-5890 Series II instrument equipped with a HP-Wax and HP-5 capillary columns (both 30 m × 0.25 mm ID × 0.25 μm film thickness) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions were injector and transfer line temperature 220°C and 240°C, respectively; oven temperature was programmed from 60 to 240°C at 3°C/min. Carrier gas was helium at 1.0 mL/min flow rate. Injection volume was 0.2 μL (10% hexane solution) using a 1 : 30 split ratio. The mass spectrometer was operated at 70-eV ionization voltage. The acquisition mass range was 30 to 300 m/z at 1.0 scan/s.GC-MS analysis was performed with a Varian CP-3800 gas chromatograph equipped with a HP-5 capillary column  comparison of mass spectra with the Wiley library spectra 275 L (Rev.C.00.00) electronic database; (2) injection of authentic compounds from Sigma-Aldrich with a 95% minimum purity under the same analytical conditions; and (3) comparison of the retention index (RI) obtained by GC-MS with the RI theoretical in similar phases. The molecular weights of all the identified substances were confirmed by GC-MS, using MeOH as CI ionizing gas.Callosobruchus maculatus were reared on cowpea seeds (Vigna unguiculata) under controlled temperature and humidity at 28 ± 1°C, 58 ± 5% RH (12 h light cycle) and 25.5 ± 1°C, 45 ± 5% RH (12 h dark cycle). Adult insects of 8 days old were used for insecticidal test.All experiments were carried out under the same environmental conditions as described above. C. maculatus (8 days old) were put into a 500 mL glass bottle with 30 g of cowpea seeds and kept in the laboratory at 28 ± 1°C, 58 ± 5% RH (12 h light cycle) and 25.5 ± 1°C, 45 ± 5% RH (12 h dark cycle) for 72 h. Concentrations of the M. lucida essential oil diluted in acetone were tested on C. maculatus . The appropriate concentrations were applied to filter paper  and immediately introduced into a glass bottle that was then hermetically sealed. For the control group, the insects were placed in the glass bottles under the same conditions but without the essential oil. Each concentration and control was replicated three times. Insect mortality was determined by observing the recovery of immobilized insects in 12 h intervals up to 72 h. When no antennal or leg movements were observed, insects were considered dead.The insecticidal activity was evaluated as described by Ilboudo et al. . One hunμg/mL. Both pesticides were evaluated at the same concentrations as the essential oil in triplicate.Two synthetic pesticides were purchased from Chemical and Allied company, Lagos, Nigeria. Phostoxin (55% Aluminium phosphide) and Primo-ban-20 . The first is converted to phosphine gas, which is taken by the insect through its respiratory system and the second is able to penetrate the insect cuticle. Pellets of Phostoxin were divided into small pieces and weighed to achieve the appropriate concentrations and were applied according to manufacturer's directions . Primo-b C. maculatus were released each 12 h after exposure of essential oil up to 72 h. After a 12 h exposure period the containers were opened and the dead beetles were removed and recorded. Knocked-down adults were regarded as alive if they showed continued movement of their appendages by the touch of a fine brush. After observation, the containers were tightly closed. After every 12 h, new batches of twenty insects were released and the numbers of dead insects were recorded and the percentage of mortality was calculated and corrected using Abbott's formula [Ten pairs of adult formula .50  and LC90  values at 24 and 48 h with Probit analysis [Average percentage insect mortality was calculated from three replicates. Dosage-mortality responses were used to estimate LCanalysis  using St M. lucida essential oil, containing mainly 1,8-cineole (43.4%) followed by α-terpinyl acetate (14.5%), sabinene (8.2%), and β-pinene  essential oil. The insecticidal bioassay of the essential oil of M. lucida against C. maculatus  at 24 h, while Phostoxin had a 46.7% and 86.7% mortality after 24 h and 48 h at 0.20 μg/mL, respectively  similar to Phostoxin (LC90 = 0.652 μg/mL) and Primo-ban-20 (LC90 = 0.726 μg/mL). At 24 h essential oil of M. lucida (LC50 = 0.298 μg/mL) and Phostoxin (LC50 = 0.295 μg/mL) were more efficient than Primo-ban-20 (LC50 = 0.393 μg/mL). At 48 h after exposure, there was no significant difference in LC50 between M. lucida essential oil compared to Primo-ban-20 and Phostoxin -myroxide, was shown to be toxic to both adults and eggs of C. maculatus. Likewise, Cinnamomum aromaticum bark oil, rich in cis-cinnamaldehyde, was insecticidal with an LC50 of 27.6 μg/cm2 after 24 h [ Cymbopogon giganteus, rich in limonene and p-mentha-1(7),8-dien-2-ol, demonstrated insecticidal activity against C. maculatus and C. subinnotatus [ M. lucida essential oil are consistent with use of this plant as an insecticide and insect repellent.Several essential oils components have shown insecticidal activity against6.7 μL/L . Plectrater 24 h . The essnnotatus . The con M. lucida essential oil could be attributed to those known major components of oxygenated monoterpenes: 1,8-cineole (43.4%), α-terpinyl acetate (14.5%), 4-terpinen-4-ol (3.4%), α-terpinol (3.4%), and monoterpene hydrocarbons: sabinene (8.2%) and β-pinene (4.0%). These are compounds whose insecticide and repellent activities have been reported previously [α-phellandrene, and Camphene.The insecticidal activity of theeviously , 43, 44.eviously  such as  M. lucida represents a botanical insecticide source, since it showed a potent insecticidal activity comparable to both pesticides, Phostoxin, and Primo-ban-20 ( M. lucida essential oil and two commercial pesticides is a positive result and demonstrates their equivalence, though we cannot conclude that M. lucida is more effective.The intensive use of synthetic insecticides not only produces genetic resistance by insect species but also causes serious problems of toxicity to humans and other animal species , 7. Ther M. lucida essential oil was biocidal even at lower concentration and may, therefore, be considered to be a useful alternative to synthetic insecticides.The results obtained indicate that M. lucida was toxic to pulse beetle C. maculatus. Therefore, it could be used as an alternative strategy for C. maculatus control and as a substitute for synthetic pesticides. However, further studies are necessary to elucidate the mode of action and their environmental impact and develop formulations to improve the insecticidal efficacy. This would benefit agricultural sectors of developing countries as these essential oils are readily available and biodegradable. The problem of volatility can be resolved through a controlled release formulation of their active chemical compositions.This study demonstrated that essential oil extracted from


Aspergillus species. In mycelial growth assay, nanocomposite was more potent, as fungicidal effect of 1.04/0.6 μg mL−1 was obtained in all but one of the isolates (A. niger), while proportional concentration of pure biocide (0.6 μg mL−1) completely inhibited mycelial growth of only three Aspergillus species. However, conidia appear to be less susceptible to nanocomposite treatment, as lower fungistatic (MIC) and fungicidal (MFC) concentrations were obtained with biocide alone (MIC in range from 0.03 to 0.15 μg mL−1 and MFC from 0.075 to 0.45 μg mL−1). To a different degree, both substances stimulated aflatoxin B1 production and inhibited ochratoxin A synthesis. Very low mycelium biomass yield, in range from 1.0 to 3.0 mg dry weight, was documented in both biocide and nanocomposite enriched medium.A comprehensive comparative analysis of antifungal potential of benzalkonium chloride and newly synthesized fullerenol/benzalkonium chloride nanocomposite was conducted to assess the possible impact of carbon-based nanocarrier on antimicrobial properties of the commonly used biocide. Physical characterization of synthesized nanocomposite showed zeta potential of +37.4 mV and inhomogeneous particles size distribution, with nanocomposite particles' dimensions within 30–143 nm and maximum number of particles at 44 nm. The effect of pure and fullerenol nanocarrier-bound biocide was evaluated in eight  Aspergillus are one of the primary contaminants of indoor air and surfaces worldwide, as well as causative agents of a wide assortment of diseases collectively dubbed aspergilloses [The health implications associated with indoor surface fungal proliferation have always been an important issue. However, only recently has the topic of the environmental fungal contamination and consequential health risk gained increased public concern. Generally, though not a norm, fungi are of interest only in water damaged buildings with excess moisture, possibly a result of leaks in the envelope, plumbing failures, or condensation. Under such conditions, exposure to indoor fungal growth can cause or exacerbate a variety of health problems, from minor allergic reactions and respiratory obstructions to severe systemic mycoses and mycotoxicoses. With more than 180 currently recognized species, fungi of the genusSince first introduced as antimicrobial agents by Domagk over seventy years ago, to date, many alterations to basic quaternary ammonium compounds (QACs) formulas have been made hoping to augment their already potent antimicrobial activity . As a re60(OH)n), polyhydroxyl water-soluble fullerenes, in form of polyanionic nanoparticles, have been examined in numerous in vitro and in vivo model systems [n > 16), since they are nontoxic for the most of examined biological model systems, possess significant antioxidative properties [Fullerenols : Aspergillus flavipes (Bainier & R. Sartory) Thom & Church, Aspergillus flavus Link, Aspergillus fumigatus Fresen., Aspergillus nidulans (Eidam) G. Winter, Aspergillus niger Tiegh., Aspergillus ochraceus Wilh., Aspergillus parasiticus Speare, and Aspergillus terreus Thom. Isolated fungi were deposited to the Mycotheca of the Department for Algology, Mycology and Lichenology, Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade. Selected isolates were stored in cryovials with 1.5 mL of 30% glycerol at −75°C.Morphophysiological changes, induced by BAC, FNP, and fullerenol/benzalkonium chloride nanocomposite (FNP/BAC), were investigated on eight2 surface and the concentrations of conidia in the suspensions were calculated per formula: 5. The inocula were stored at −20°C and prior to experiments, dilutions of the inocula were cultured on solid MEA to verify the absence of contamination and check the validity of the inocula.Conidial suspensions of selected fungi were prepared by washing conidia from the surface of the 7-day-old Malt Extract Agar (MEA) slants with sterile saline  containing 0.1% Tween 20 (Sinex Laboratory). Using a hemocytometer  conidia were counted on a 1 mmFor mycotoxicological analysis, conidial suspensions were prepared per given procedure, with the exception of using sterile distilled water, instead of saline solution, in the preparation process.An aqueous solution of the quaternary ammonium compound, benzalkonium chloride (50 v/v), was obtained from a team of expert chemists and restorers of the Institute for Protection of Cultural Monuments in Serbia. Prior to experiments, biocide was diluted in sterile deionized water to make a stock solution of final concentration 3 v/v %.60(OH)24 was obtained by complete substitution of bromine atoms of polybromine derivative C60Br24 with hydroxyl groups [−1, which was sonicated 15 min prior to further FNP/BAC nanocomposite preparation. To synthesize FNP/BAC nanocomposite, 6.93 mL of 5 v/v % BAC was mixed with 4.95 mL of FNP solution. Final concentrations of components in nanocomposite were as follows: 3 v/v % for BAC and 0.052 mg mL−1 for FNP. Nanocomposite mixture was stored in dark and was characterized as stable for more than several weeks. Size distribution of FNP and nanocomposite particles was measured after 48 h by Dynamic Light Scattering (DLS) on Zetasizer Nano ZS .Fullerenol Cl groups , 18. ThuFor scanning electron microscopy, stable nanocomposite in water was evaporated under reduced pressure on Al substrate and was steamed with gold. Electron images were obtained using JEOL JSM 6460 LV microscope. Morphology and structure of FNP/BAC nanocomposite in solution were evaluated via atomic force microscopy (AFM). Surface topography and phase images were simultaneously acquired by standard AFM tapping mode using a commercial SNC (Solid Nitride Cone) AFM probe (NanoScience-Team Nanotec GmbH), with the tip radius lower than 10 nm. Highly orientated pyrolytic graphite (HOPG) was used as a surface. Multimode quadrex SPM with a Nanoscope IIIa controller , operated under ambient conditions, was used.Ø 50 mm) to form final concentrations in range from 0.03 to 0.6 μg mL−1, 0.05 to 1.04 μg mL−1, and 0.05/0.03 to 1.04/0.6 μg mL−1 for BAC, FNP, and FNP/BAC, respectively. The fungi were inoculated at the center of the MEA, via sterile cork borer. The inoculated plates were incubated in a thermostat at 25 ± 2°C for 21 days, during which the growth of fungal colonies was observed daily. In addition, on weekly basis, mycelial growth dynamics were monitored by means of colony diameter measurement. Mycelial growth inhibition (%) was calculated per formula of Pandey et al. [To elucidate the susceptibility of the somatic phase (mycelium) of tested isolates to BAC, FNP, and FNP/BAC nanocomposites, the modified macrodilution method, utilizing MEA nutrient media, was used . The stoy et al. : (2)MyceμL of conidial suspension, to make the final concentrations in range from 20.1 to 0.03 μg mL−1, 34.84 to 0.05 μg mL−1, and 34.84/20.1 to 0.05/0.03 μg mL−1 for BAC, FNP, and FNP/BAC, respectively. The microplates were incubated for 72 h at 25 ± 2°C. After incubation period, the lowest concentrations without visible growth, under a binocular microscope , were defined as the minimum inhibitory concentrations (MICs), concentrations that completely inhibited fungal growth. The minimum fungicidal concentrations (MFCs) were determined by serial subcultivation of 2 μL into microtiter plates containing 100 μL of MEB. The lowest concentrations with no visible growth were defined as the MFCs, indicating 99.5% killing of the original inoculums.Sensitivity of the asexual reproductive stage of the fungal life cycle (conidia), to the BAC, FNP, and FNP/BAC treatment, was evaluated via modified microdilution technique , 22. In  Aspergillus flavus, A. ochraceus, and A. parasiticus, were chosen to study the effects of BAC, FNP, and FNP/BAC treatment on aflatoxin B1 (AFB1) and ochratoxin A (OTA) production. Selected isolates were cultivated in 250 mL of semisynthetic glucose medium (GPK) for 5 days at room temperature (22 ± 2°C) on a rotary shaker  set to 200 rpm. Prior to incubation, inoculated media were enriched with different volumes of tested substances to make final concentrations of 0.003 μg mL−1 for BAC, 0.0052 μg mL−1 for FNP, and 0.0052/0.003 μg mL−1 for FNP/BAC. After cultivation period, mycelial biomasses were filtered through Whatman No. 4 filter paper, dried for 24 h at 70°C, and measured. Filtrates (100 mL) were dissolved in 150 mL of methanol , concentrated under reduced pressure in a rotary evaporator  at 90°C to dryness, measured, and redissolved in fivefold greater mass of 70% methanol. Quantitative (ELISA) analyses of AFB1 and OTA in samples were performed per protocol of enzyme-linked immunosorbent assay kits Celer AFLA B1 (code MA220/MA221) and I'screen OCHRA (code OR360/OR361) (Tecna s.r.l.). The detection limit for both methods is 1 μg kg−1 of given mycotoxins.Among tested micromycetes, three well-known mycotoxin producers,For microscopic observations of induced morphophysiological changes in tested microfungi, samples of mycelium and/or conidiogenous apparatus were stained with few drops of Crystal Violet or Lactophenol Cotton Blue, put on slides, and analyzed with optical microscope Zeiss AxioImager M1, using software AxioVision Release 4.6. Results were documented with original micrographs.p values less than 0.05 were considered statistically significant.The results were expressed as the mean ± standard error of data obtained from three measurements. To test any significant differences, one-way analysis of variance (ANOVA) was performed using Microsoft Excel 2010 . Size distributions by number and zeta potentials of FNP and FNP/BAC nanocomposite are presented in  Aspergillus niger, with inhibition of mycelium growth observed in range from 4.08 ± 2.97% to 16.33 ± 2.97% in BAC treatment and between 3.06 ± 2.22% and 14.29 ± 1.86% in FNP/BAC treatment. Compared to the pure biocide, fullerenol bound form was more potent, as fungicidal effect of 1.04/0.6 μg mL−1 was obtained in all of the tested isolates, with the exception of A. niger. On the other hand, proportional concentration of pure biocide completely inhibited mycelial growth in only three fungal species . Furthermore, A. flavipes was also the most sensitive isolate, with mycelium growth completely suppressed at 0.26/0.15 μg mL−1 of FNP/BAC.Different susceptibility levels of tested isolates to biocide BAC and FNP/BAC nanocomposite treatments were documented in mycelial growth assay . In both A. flavus  and A. parasiticus (2.04 ± 1.43% at 0.03 μg mL−1 of BAC). On the other hand, no inhibition of mycelium growth and/or morphophysiological changes were observed in any of the FNP treated cultures.With increase in both BAC and FNP/BAC concentrations, a higher percentage of mycelial growth inhibition was achieved . In most A. ochraceus colonies, grown in the presence of 0.15 μg mL−1 of benzalkonium chloride, were consistently characterized by apparent morphological variations, compared to control  and MFC (in range from 0.075 to 0.45 μg mL−1 for BAC and 0.13/0.075 to 0.52/0.3 μg mL−1 for FNP/BAC) values (Aspergillus flavipes), whereas MFC values, although generally lower for BAC, were the same or higher as in FNP/BAC treatment for three of the eight tested microfungi . Isolates A. nidulans, A. ochraceus, and A. terreus were the most susceptible to BAC treatment, with fungicidal effect achieved at 0.075 μg mL−1, while the most resistant isolate was A. parasiticus (MFC 0.45 μg mL−1). In regard to FNP/BAC, A. flavipes was the most sensitive isolate and growth was completely suppressed with 0.13/0.075 μg mL−1. On the other hand, five tested microfungi displayed the same, highest level of resistance to FNP/BAC treatment (MFC 0.52/0.3 μg mL−1) . Reinoculation of albino colonies, via single spore transfer, onto the sterile MEA resulted in formation of colonies with typical species morphology, suggesting reversibility of induced morphophysiological changes. No statistically significant inhibition of conidial germination (p < 0.05) was observed for any of the isolates cultured on MEB enriched with pure FNP nanoparticles; hence, no data regarding FNP are presented in High fungistatic and fungicidal activities were obtained in conidia germination assay for both BAC and FNP/BAC nanocomposite, as demonstrated by very low MIC (in range from 0.03 to 0.15 ) values . Lower Mμg mL−1) . Distinc Aspergillus flavus and A. ochraceus control samples were characterized by the lack of and/or immeasurable quantities (<1 μg kg−1) of AFB1, while in A. parasiticus detectable mycotoxin level was present (1.201 ± 0.6). In general, investigated substances, to a varying degree, stimulated AFB1 synthesis in all but one case. Apparently, FNP has no effect on aflatoxin B1 production in A. ochraceus, conclusion further supported by approximately the same concentrations of AFB1 detected in both BAC (1.422 ± 0.1) and FNP/BAC (1.518 ± 0.01) treated A. ochraceus cultures. On the other hand, in A. parasiticus, FNP and FNP/BAC induced nearly twofold higher AFB1 production  compared to the control and roughly 30% higher than pure biocide. However, in A. flavus BAC stimulated AFB1 production the most (2.095 ± 0.68), whereas the concentrations in FNP (1.562 ± 0.29) and FNP/BAC (1.505 ± 0.15) were smaller and practically indistinguishable.The results of biomass production and quantitative AFB1 and OTA analyses are presented in  A. parasiticus and A. ochraceus cultures resulted in considerable reduction of OTA concentrations. In A. ochraceus, FNP reduced OTA concentration slightly (30.51 ± 2.9), while BAC and FNP/BAC treatments caused significant inhibition of ochratoxin A synthesis . Similar results, with FNP being the weakest (30.15 ± 1.95) and BAC (8.01 ± 1.01) and FNP/BAC (13.48 ± 2.73) the strongest OTA inhibitors, were documented in A. parasiticus. On the other hand, in BAC and FNP/BAC treated A. flavus cultures, OTA concentrations were in the level of the control (12.08 ± 0.58). Interestingly, however, FNP treatment did not inhibit but stimulated OTA production twofold (24.95 ± 0.05), a trait, based on the results, more closely associated with AFB1 than OTA metabolism.Unlike AFB1, ochratoxin A, in range from 12.08 ± 0.58 to 45.85 ± 2.35, was detected in all control samples. Likewise, opposite to the aflatoxin B1, used treatments inMycelium biomass yield ranged from 0.851 ± 0.06 to 1.291 ± 0.04 g dry weight in the controls and 0.011 ± 0.01 to 0.294 ± 0.03 g in the FNP treatment, while very low production, in range from 0.001 ± 0.0 to 0.003 ± 0.0 g dry weight, was documented in both BAC and FNP/BAC enriched medium .μg mL−1) and FNP/BAC (0.6 μg mL−1 of BAC bound to 1.04 μg mL−1 of FNP) had different levels of impact, since nanocomposite induced fungicidal effect in all but one of the tested isolates (Aspergillus niger), while BAC merely inhibited growth of the majority of the isolates. The mechanisms thought to be responsible for nanocomposite's increased toxicity against fungi may be linked with C60(OH)x-triggered process of programmed cell death, caspase-dependent apoptosis, characterized by chromatin condensation, activation of caspases, and fragmentation of DNA without membrane breakdown, inter alia [1O2 and O2• [1O2 can start a chain reaction in which a more dangerous reactive oxygen species is produced, resulting in cell damage, disruption in the DNA chains, and cell death [ Aspergillus section Flavi (A. flavus and A. parasiticus), while in other tested isolates, fungal growth evidently continued midst the incubation period, albeit at a much slower rate. Fungi are apparently able to cope with BAC and FNP/BAC's very low dose induced toxicity and gradually, over time, entirely suppress biocides inhibitory effect on mycelium growth. In both treatments, the most resistant isolate was A. niger, on whom fungicidal effect was not achieved at all. However, this is not unexpected as fungal melanins, abundant in cell walls of this fungus, have a protective role and are probably responsible for the higher resistance documented. As for A. ochraceus, complete mycelium growth inhibition (100%) was not accomplished even with the highest concentration of BAC used in the experiment (0.6 μg mL−1), although observed morphophysiological variations indicate interference of biocide with fungal cellular metabolism. Aberrant changes in somatic  and reproductive structures morphology  were most likely a result of partial dissociation of cellular membrane phospholipid bilayers, caused by the biocides disruption of intermolecular interactions or denaturation/inactivation of structural proteins and enzymes essential for the developmental processes [Strong antifungal activity of biocide BAC and FNP/BAC nanocomposite, estimated via microdilution and macrodilution assays, suggests pronounced capability of tested agents to interfere with various stages of the fungal life cycle. Obtained results demonstrate that even low concentrations of pure and nanocarrier-bound benzalkonium chloride can hinder conidia germination and achieve a high percentage of mycelium growth inhibition. In mycelial growth assay, proportional concentrations of BAC  or higher (A. flavipes and A. parasiticus), compared to pure biocide, emphasizing the already well-known significant role of individual fungal physiology in species-specific susceptibility to biocide treatment. Inhibition of melanin synthesis and subsequent formation of A. niger and A. flavipes albino phenotype in cultures treated with subinhibitory FNP/BAC concentrations are a nanocomposite property most likely associated with the nanocarrier, rather than biocide, given the fact that fullerenol nanoparticles were already known to induce depigmentation of the melanized conidiogenous apparatus in A. niger [As demonstrated by the very low MIC and MFC values, both BAC and FNP/BAC possess strong potential to interfere with the first step in the fungal asexual life cycle, that is, conidia germination. However, higher fungistatic and fungicidal activities, for example, up to tenfold forA. niger . MelaninA. niger  and are,A. niger . Albeit A. niger , substan Aspergillus species in glucose-rich liquid medium was severely inhibited by suboptimal inhibitory biocide and nanocomposite concentrations, yet only AFB1 concentrations were notably increased. Apparently, used BAC and FNP/BAC concentrations, although much lower  compared to documented MIC values, nonetheless, created stress conditions responsible for the increased AFB1 production, probably a form of a defense mechanism. Furthermore, such results are fully consistent with earlier findings that low fungicide doses stimulate mycotoxin deoxynivalenol  production in Fusarium species [Up to date, numerous studies have demonstrated the role of various stress factors in mycotoxin production of many filamentous fungi . In thi species . On the  species . At any  species . It was  species .Aspergillus properties of nitrogen-based quaternary ammonium compound, that is, benzalkonium chloride 24, in form of polyanionic nanoparticles, to synthesize stable nanocomposite and evaluate its antifungal potential on selected Aspergillus species. Our research confirms well-established antimicrobial activity of BAC but also emphasizes greater potency of FNP/BAC nanocomposite in suppression of fungal somatic growth. Moreover, even though nanocomposite treatment had a weaker effect on conidia germination, reduction of fungal pathogenicity, via inhibition of melanin synthesis, is a feature that should not be overlooked. We also point out the need for careful evaluation of BAC and FNP/BAC doses, before application, as treatments with subinhibitory concentrations of both substances stimulate aflatoxin B1 production. In conclusion, the preliminary results of our study suggest that novel-synthesized nanocomposite might be used as an effective antifungal alternative to benzalkonium chloride in treating various fungal contaminated surfaces. Further in-depth investigations on common contaminants from other fungal genera, as well as prospective evaluation of safety, will be necessary prior to in situ application.Several studies to date have been engaged in research on anti-e e.g., , 39). Ho. HoAsper


Trametes species represent promising degraders due to a well-developed ligninolytic enzyme system. Although numerous studies have confirmed that low molecular weight compounds can induce the production and activity of ligninolytic enzymes it is not clear how this reflects on the extent of delignification. The aim of the study was to assess the capacity of p-anisidine and veratryl alcohol to induce the production and activity of Mn-oxidizing peroxidases and laccases, and wheat straw delignification by six Trametes species. Significant inter- and intraspecific variations in activity and features of these enzymes were found, as well as differences in the potential of lignocellulose degradation in the presence or absence of inducers. Differences in the catalytic properties of synthesized enzyme isoforms strongly affected lignin degradation. Apart from enhanced lignin degradation, the addition of p-anisidine could significantly improve the selectivity of wheat straw ligninolysis, which was especially evident for T. hirsuta strains.Wheat straw is the major crop residue in European countries which makes it the most promising material for bioconversion into biofuels. However, cellulose and hemicellulose are protected with lignin, so delignification is an inevitable phase in lignocellulose processing. The organisms predominantly responsible for its degradation are white-rot fungi and among them  Additiot effect . On the EOFB 321 , the strT. multicolor HAI 540 (6160.9 ± 70.7 U L−1)  . p-anisi HAI 540 .T. multicolor HAI 540 it was the highest, as was the case with peroxidases   .32,132 = 38.45, P < 0.001; for MnIP was F32,132 = 38.45, P < 0.001 and for laccase was F32,132 = 38.45, P < 0.001.Two-way analysis of variance of MnP, MnIP and laccase activities showed that strain, inducer type, as well as their interaction were significant factors which affected the activities of these enzymes. The F statistic for MnP was FT. pubescens BEOFB 330 and T. sualveolens HAI 300 (12.0 ± 1.6 mg mL−1) which had a 6-fold higher concentration than under control conditions (2.0 ± 0.1 mg mL−1). Under control conditions, the highest specific MnP activity was present in T. gibbosa BEOFB 311 (656.6 ± 5.6 U mg−1), slightly lower in T. sualveolens HAI 300 (432.2 ± 63.5 U mg−1), while in all other strains MnP activities were below 100.0 U mg−1. The two inducers had no significant effect, except in the case of p-anisidine which showed a positive effect in T. gibbosa BEOFB 311 (431.9 ± 30.4 U mg−1) and T. multicolor HAI 540 and HAI 426 .The two inducers significantly affected total protein production which was reflected in specific activities of the enzymes studied . Total pT. multicolor HAI 540, where the maximum activity was found (95.6 ± 3.9 U mg−1), T. versicolor BEOFB 322 (41.7 ± 7.5 U mg−1) and T. suaveolens HAI 300 (19.4 ± 4.2 U mg−1). p-anisidine stimulated specific MnIP activity only in T. multicolor HAI 540 (135.9 ± 21.8 U mg−1) which was about 30% higher than in the control, while in all other strains its effect was either absent or inhibitory, as in the cases of T. versicolor BEOFB 322 and T. suaveolens HAI 300. Veratryl alcohol showed similar effects, and expressed the enzyme only in the case of T. hirsuta BEOFB 301, where the specific activity was 60% higher than in the control (54.9 ± 10.3 U mg−1) , T. hirsuta BEOFB 30 and T. versicolor BEOFB 320 (about 90 U mg−1)  . In the  U mg−1) .Trametes in the presence and the absence of inducers. Significant interspecific differences were primarily present in MnP production. Namely, contrary to T. multicolor where this enzyme was the most active, the activity obtained in fermentation of wheat straw/(NH4)2SO4/wheat bran medium with T. hirsuta was about 30-fold lower, which is in accordance with results reported by Erden et al.T. versicolor and T. hirsuta.Significant inter- and intraspecific variations were found in the activities of Mn-oxidizing peroxidases and laccase within the genus et al.p-anisidine on laccase activity in T. pubescens and veratryl alcohol in T. versicolor. However, these findings were not in accordance with results of Galhaup et al.et al.et al.et al.Phanerochaete chrysosporium and an asparagine-succinate medium with Physisporinus rivulosus regulating the transcription of MnP genes.The effect of inducers on the activities of ligninolytic enzymes has been the subject of numerous studies910111516819202223244)2SO4/wheat bran medium, strains of Trametes species synthesized one MnP isoform of pI around 4.1, except T. suaveolens HAI 300 where the pI was 4.4 , in T. pubescens BEOFB 330 veratryl alcohol stimulated the production of an additional isoform (pI 3.8) and completely inhibited the enzyme in T. hirsuta BEOFB 301. The induced isoform of pI 4.6 in T. hirsuta BEOFB 30 and T. suaveolens HAI 300 showed increased levels of enzyme activity, while at the same time less of it was produced , caused the production of an isoform with the highest pI in T. suaveolens HAI 300 (pI 4.8), and an isoform with a lower pI in T. hirsuta BEOFB 301 (pI around 3.8)  . The incund 3.8) b and 2.T. hirsuta BEOFB 30, which produced an isoform with a pI of 4.4, and in T. versicolor BEOFB 322 which synthesized three additional isoforms . p-anisidine completely inhibited synthesis of the enzyme in T. hirsuta BEOFB 301 and T. versicolor BEOFB 322, while in T. suaveolens HAI 300 production of an additional isoform (pI 4.4) was stimulated  in the presence of the inducer. The appearance of wide bands is likely to be the result of point changes in amino acid sequence or some post-translational modifications of laccase molecules. This effect is expected because laccase synthesis is controlled by families of closely-located genes originating from gene duplication2728T. multicolor and T. versicolor BEOFB 321. As there were no functional changes, a point mutation could have been the cause of small differences in pI values of laccases encoded by gene families, and this would be reflected through the densely-packed protein bands on the gel . Only T. versicolor BEOFB 322 showed a significant difference, which was the same with all other strains (P < 0.01).The number of laccase isoforms was best-fitted with two factors in the GLM: treatment and strain . Tukey’s multiple post hoc tests showed a significant difference between veratryl alcohol treatment and the control (P < 0.001), and no difference between −10).The level of enzymatic activity was not significantly correlated with all isoforms. The isoform present in the control group (base expression), pI 4.0–4.3, was the one with the weakest effect on the laccase activity detected . On the other hand, isoforms synthesized in the treatment with veratryl alcohol (pI 4.3–4.5 and pI 6.7) strongly and positively affected the enzyme activity  (p-anisidine increased the rate of lignin degradation (from 3.3% to 16.4%) in T. gibbosa BEOFB 310, T. hirsuta BEOFB 30 and BEOFB 301, T. multicolor HAI 540 and T. versicolor BEOFB 322, decreased it in T. gibbosa BEOFB 311, T. pubescens BEOFB 330 and T. suaveolens HAI 300, while there was no effect of this inducer on T. multicolor HAI 426 and T. versicolor BEOFB 321 , or decreased in other strains (ranging from 4.2% to 15.9%), having no effect on T. multicolor HAI 426 and T. versicolor BEOFB 320 (ctively) . The addEOFB 321 . In the EOFB 320 .T. gibbosa BEOFB 310 had the highest hemicellulose degradation (54.9%) and T. suaveolens HAI 300 the lowest (38.3%) (p-anisidine increased hemicellulose degradation only in T. versicolor BEOFB 321 (7.4%), T. hirsuta BEOFB 301 (5.9%), with no significant effect in T. gibbosa BEOFB 311 (0.6%), while all other strains were inhibited. Veratryl alcohol increased the amount of degraded hemicellulose from 1.8% to 4.9% in T. hirsuta BEOFB 301, T. versicolor BEOFB 321 and BEOFB 322 and T. suaveolens HAI 300, while in other strains degradation of this polymer was suppressed  . Compareppressed .T. multicolor HAI 540 (40.2%) and lowest with T. pubescens BEOFB 330 (17.2%)  to 18.9% (T. pubescens BEOFB 330)  and T. hirsuta BEOFB 301 the weakest one .Capacities of all the species and strains to degrade cellulose were significantly lower in comparison with their capacities for hemicellulose and lignin depolymerisation. Under control conditions, the highest extent of cellulose degradation was noted in substrate fermentation with  (17.2%) . p-anisiOFB 330) . T. mult32,132 = 105.3, P < 0.001, for hemicellulose F32,132 = 118.9, P < 0.001, and for cellulose F32,132 = 329.7, P < 0.001. Tukey’s post-hoc test revealed significant differences (P < 0.05) between strains in all treatments for each polymer degradation, except for the pair veratryl alcohol – p-anisidine in hemicellulose analysis.Two-way analysis of variance of the lignin, hemicellulose and cellulose degradations demonstrated that the strain, inducer and their interaction were statistically significant factors which affected depolymerisation. The overall F statistic for lignin degradation was FT. versicolor varied between 12.0% after 16 d of cultivation and 42.0% after 35 d323+, while laccase has limited diffusion into the non-modified plant cell wall due to its molecule size. However, in numerous studies of Trametes spp. strains, the extent of delignification was not correlated with the level of enzyme activity. Thus, a high capacity of lignin degradation in the presence of p-anisidine characterised T. hirsuta BEOFB 30, where a low MnP activity and the absence of MnIP and laccase were noted, while T. multicolor BEOFB 540 was the weakest lignin degrader both under control conditions and in the presence of inducers, though activities of the enzymes were high. The significant degradation of hemicellulose (32.0–54.9%) and cellulose (14.7–48.7%) is an expected result of substrate fermentation with these white-rot fungal species and strains as they simultaneously degrade lignin and the other two polymersPrevious studies showed that the amount of degraded lignin during wheat straw fermentation with Trametes species identified five main factors  and two significant two-way interactions among them . However, in the presence of p-anisidine, both T. hirsuta strains were the most selective in the sense that cellulose degradation was lower than 20% .Looking overall at the efficiency of ligninolysis, Trametes in the presence or absence of inducers. p-anizidine and veratryl alcohol can be considered as efficient inducers of these ligninolytic enzymes in Trametes species. Differences in the catalytic properties of enzyme isoforms is a factor which strongly affects lignin degradation and this study indicated the most active isoforms. Apart from enhanced lignin degradation, the addition of p-anisidine can significantly improve the selectivity of wheat straw ligninolysis which was especially demonstrated for T. hirsuta strains. This study demonstrates not only a method to optimize the system for the degradation of wheat straw but also clarifies the role of individual enzymes in lignin degradation by different Trametes species and strains.Our results clearly showed significant inter- and intraspecific variations in the activities and characteristics of Mn-oxidizing peroxidases and laccases, as well as the potential of lignocellulose degradation within the genus Trametes species with 11 strains were isolated from fruiting bodies collected from Serbia and Russia or obtained from the culture collection of the Institute of Evolution, University of Haifa, Israel (HAI), and maintained on malt agar medium in the culture collection of the Institute of Botany, Faculty of Biology, University of Belgrade (BEOFB) (Cultures of six  (BEOFB) .−1; NH4NO3, 2.0 g L−1; K2HPO4, 1.0 g L−1; NaH2PO4 × H2O, 0.4 g L−1; MgSO4 × 7H2O, 0.5 g L−1; yeast extract, 2.0 g L−1; pH 6.5) with 25 mycelial discs , incubation at room temperature (22 ± 2 °C) on a rotary shaker (100 rpm) for 7 d, washing of the obtained biomass with sterile distilled water (dH2O), and its homogenization with 100.0 mL of dH2O in a laboratory blender.The inoculum was prepared by inoculating 100.0 mL of synthetic medium 2SO4 at a previously-defined optimum nitrogen concentration of 10.0 mM). The medium with dH2O instead of the modified synthetic medium was used as the control. The obtained inoculum (3.0 mL per flask) was used for inoculation. p-anisidine or veratryl alcohol were added to the medium 72 h after inoculation to final concentrations of 1.0 mM and 0.5%, respectively, to test their potentials to induce ligninolytic enzyme activity and enhance delignification of wheat straw.Solid-state cultivation was carried out at 25 °C in 100-mL flasks containing 1.8 g of wheat straw as the carbon source, 0.2 g of wheat bran as an organic nitrogen source and 10.0 mL of the modified synthetic medium  and the supernatants obtained were used for determining Mn-oxidizing peroxidases and laccases activities spectrophotometrically (CECIL CE2501 (BioQuest)), as well as total protein content.Ligninolytic enzymes synthesised during incubation were extracted after solid-state fermentation of wheat straw/wheat bran medium by stirring samples with 50.0 mL of dH610 = 22,000 M−1 cm−1) as a substrate, in a succinate buffer pH 4.5  and DL-lactic acid sodium salt). The reaction mixture (Vtot = 1.0 mL) contained buffer, sample, 2.0 mM H2O2 and phenol red, with or without 2.0 mM MnSO4 . The reaction was stopped by adding 2.0 M NaOH. The reaction mixture using 2.0 M NaOH added before phenol red was used as the blank.Activities of Mn-oxidizing peroxidases  were determined with 3.0 mM phenol red  (ε436 = 29300 M−1 cm−1) in 0.1 M phosphate buffer (pH 6.0) at 35 °C. The reaction mixture (Vtot = 1.0 mL) contained buffer, ABTS and sample. The mixture without sample was used as the blank.Activity of laccase (EC 1.10.3.2) was determined by monitoring the AEnzymatic activity is expressed in U and 1 U is defined as the amount of enzyme that transforms 1.0 μmol of substrate per min.et al.−1, was used for determining specific enzymatic activity (U mg−1).The amount of total proteins was determined according to the method of Bradford Trametes species and strains were determined by isoelectric focusing (IEF) using the Mini IEF Cell-Model 111 (BIO-RAD). Focusing was carried out in 7.5% polyacrylamide gel with 5.0% ampholyte on a pH gradient from 3.0 to 10.0, in three phases: (i) at 100 V, for 15 min; (ii) at 200 V, for 15 min; (iii) at 450 V, for 60 min. An IEF marker for the pI range from 3.6 to 9.3 (Sigma-Aldrich) was used. Mn-oxidizing peroxidase bands were located by incubating the gel in a solution of 4-Cl-1-naphthol (0.1 mg mL−1), 0.05 mM H2O2 and 0.1 M sodium acetate buffer (pH 4.5), with or without MnSO4 , at room temperature till the appearance of dark-brown bands. Protein bands corresponding to laccases were located by gel incubation in visualization solution (10.0 mM ABTS and 200.0 mM phosphate buffer pH 5.0) at room temperature. After focusing, the gel was fixed in 12.0% trichloroacetic acid (TCA) and stained with a solution of 0.1% Coomassie brilliant blue R-250 in fixative .The profiles of Mn-oxidizing peroxidases and laccases of the six −1; SDS, 30.0 g L−1; 2-ethoxyethanol, 10.0 mL; NaH2PO4 × H2O, 4.56 g L−1; Na2B4O7 × 10H2O, 6.81 g L−1; pH 6.9–7.1), 0.5 g Na2SO3 and a few drops of 1-octanol was heated to boiling and then refluxed for an hour to remove soluble sugars, proteins, pectin, lipids and vitamins from the sample. The sample was filtered, washed three times with boiled water and twice with cold acetone, dried at 105 °C for 8 h and weighed as neutral detergent fibres (NDF). The samples were further treated with acidic detergent solution (ADS) (CTAB (20.0 g) dissolved in 1000.0 mL of 0.5 M H2SO4; pH 6.9–7.1), heated to boiling, refluxed for an hour, filtered and washed with boiled water and a few times with acetone. Thereafter samples were dried at 105 °C overnight and weighed. Weight of acidic detergent fibres (ADF) was determined gravimetrically as the residue remaining after extraction. The hemicellulose content was expressed as ADF-NDF.Content of hemicellulose was determined by a modified Van Soest method362SO4 method2SO4 was added for each 100.0 mg of sample. The mixture was incubated at 30 ± 0.5 °C in a water bath for an hour, stirred frequently and after that diluted using 28.0 mL of water for each 1.0 mL of acid. Secondary hydrolysis was performed in an autoclave at 120 °C for 1 h. The hot solution was filtered through a tared Gooch crucible and the Klason lignin residues were washed with hot water. The sample was dried at 105 °C till constant weight and lignin content (LC) is expressed as a percentage of that present in the original sample. Cellulose content is presented as ADF-LC.Acidic detergent fibres were used for determining cellulose and lignin contents. Lignin content was defined by the Klason or 72% HThe assays were carried out in five replicates for each measurement occasion and results correspond to the mean ± standard error. Two-way analysis of variance (ANOVA) and Tukey’s HSD post-hoc test were performed to test the significance of differences among mean values of enzyme activities and polymer degradation rates.Numerical variation in the isoenzyme profiles, as well as in the efficiency of lignin degradation were analysed by generalized linear models (GLM). The difference between percentage of degraded lignin and hydrolyzed cellulose was taken as a measure of the efficiency of ligninolysis for all strains. The full GLM model for isoenzyme profile contained strain, treatment and their interaction as exploratory variables, while full GLM model for efficiency of ligninolysis contained strain, treatment, and activity of ligninolytic enzymes, as well as interactions among these factors. The minimal best fitting model was determined by backward selection, as the model with the lowest Akaike information criterion (AIC). All statistical analyses were performed in R software, ver. 3.2.1.How to cite this article: Knežević, A. et al. Induction of wheat straw delignification by Trametes species. Sci. Rep.6, 26529; doi: 10.1038/srep26529 (2016).


Background and Aims: Phosphate (Pi) is one of the most limiting nutrients for agricultural production in Brazilian soils due to low soil Pi concentrations and rapid fixation of fertilizer Pi by adsorption to oxidic minerals and/or precipitation by iron and aluminum ions. The objectives of this study were to quantify phosphorus (P) uptake and use efficiency in cultivars of the species Coffea arabica L. and Coffea canephora L., and group them in terms of efficiency and response to Pi availability.Methods: Plants of 21 cultivars of C. arabica and four cultivars of C. canephora were grown under contrasting soil Pi availabilities. Biomass accumulation, tissue P concentration and accumulation and efficiency indices for P use were measured.Key Results: Coffee plant growth was significantly reduced under low Pi availability, and P concentration was higher in cultivars of C. canephora. The young leaves accumulated more P than any other tissue. The cultivars of C. canephora had a higher root/shoot ratio and were significantly more efficient in P uptake, while the cultivars of C. arabica were more efficient in P utilization. Agronomic P use efficiency varied among coffee cultivars and E16 Shoa, E22 Sidamo, Iêmen and Acaiá cultivars were classified as the most efficient and responsive to Pi supply. A positive correlation between P uptake efficiency and root to shoot ratio was observed across all cultivars at low Pi supply. These data identify Coffea genotypes better adapted to low soil Pi availabilities, and the traits that contribute to improved P uptake and use efficiency. These data could be used to select current genotypes with improved P uptake or utilization efficiencies for use on soils with low Pi availability and also provide potential breeding material and targets for breeding new cultivars better adapted to the low Pi status of Brazilian soils. This could ultimately reduce the use of Pi fertilizers in tropical soils, and contribute to more sustainable coffee production. Thus, Pi fertilizers are necessary to maintain crop production. Fertilizer Pi can be adsorbed by clay minerals or precipitated by iron  related traits in plants  before the emergence of cotyledons. Pots were filled with soil, classified as Oxisol with medium texture, collected in Piracicaba, São Paulo State, at 20 cm below the surface to eliminate the effect of previous fertilizations (Supplementary Table −3 - resin extraction). The resin Pi extraction procedure was based upon ion exchange using an ion exchange resin to measure plant available Pi  and 1.56 g monobasic potassium phosphate (KH2PO4) per pot, to give a soil resin extractable Pi concentration of 120 mg Pi dm−3, considered high for coffee production . The limestone and fertilizers were mixed into the soil for each pot and incubated for 20 days, with moisture maintained at 60% of water retention capacity. Limestone was added to correct soil acidity. The remaining nutrients were provided in the following concentrations (mg dm−3): N (−3): N-50, K-50, and S-20. Pots were irrigated daily by means drip irrigation and the volume of water was calculated so that there was no runoff of water.All seeds were germinated in washed sand and irrigated with deionized water. Seedlings were transplanted into 9 dmThe experimental design consisted of randomized blocks, in a factorial design: 25 (cultivars) × 2 (Pi concentrations: low and high Pi), with five repetitions. Glasshouse average temperature was 23 ± 3.35°C (mean ± SD) and relative humidity 70% ± 12.04 (mean ± SD).Plant height, stem diameter, and number of branches were measured every month. The height was obtained from the base to the apex of the orthotropic branch, and the stem diameter at 2 cm from the stem base. The orthotropic branch is the branch that grows vertically and supports the side branches. At harvest, 9 months after germination, the number of young and mature leaves, leaf area, and the dry mass of young leaves, mature leaves, branches, stem and roots were determined. Mature leaves were considered those fully expanded, with intense green color, and young leaves were considered as still expanding, with a pale green color. Leaf area was determined with a LiCor 3100 leaf area meter .Plant material was rinsed in deionized water. Plagiotropic branches stem, and roots were dried in an oven with air circulation at 65°C for 72 h. Plagiotropic branches are the reproductive side branches where the beans are produced. After drying, dry mass (DM) was obtained for each tissue, and the materials were finely milled in a Wiley mill.The P concentration in roots, stem, branches, mature, and young leaves were determined by X-ray fluorescence spectroscopy of dispersive energy (EDXRF). Briefly, 1 g of dried plant material was packed into a polyethylene cup of 20 mm internal diameter and covered with 6-μm-thick polypropylene film (Mylar®). The samples were irradiated in triplicate for 300 s under vacuum using an energy dispersive X-ray fluorescence spectrometer . The intensity of element Kα counts per second (cps/μA) was obtained from the sample X-ray spectrum deconvolution, and P concentration calculated, according methodology proposed by Tezotto et al. . The P cThe relative efficiency of phosphorus use  of cultivars was calculated as the ratio between the plant DM under low Pi and DM under high Pi, as described by Ozturk et al. :−1 Pi) was obtained by expression adapted from Oliveira et al.  eff−1 Pi) and P utilization efficiency . The PUpE was obtained by the ratio of P uptake in plant and the amount of Pi applied and PUtP represents the production of plant biomass per unit P accumulated in the plant:P use efficiency was separated into P uptake efficiency ) corresponds to DM produced for a given plant P concentration . Significant correlations were identified at a significance level of P < 0.05.Data were analyzed by analysis of variance using SAS  and means were compared by Tukey test at C. canephora produced more DM in the roots, regardless of the Pi treatment, compared to cultivars of C. arabica. Consequently, cultivars of C. canephora had higher root/shoot ratios  to 0.90 g kg−1 (Apoatã) in low P, and from 1.03 g kg−1 (E22 Sídamo) to 1.96 g kg−1 (Guarini) at high Pi. In turn, P content ranged from 14.8 g plant−1 (Catuaí Amarelo) to 25.7 g plant−1 (Geisha) in low Pi, and from 49.2 g plant−1 (Obatã) to 92.3 g plant−−1 (Guarini) in high P, with an increase of between 160 and 459% with Pi supply (Table There was significant variation in plant P concentration and P content among the cultivars Table . The P c−1 E2 Sídamo −1 (Catuaí Amarelo) to 0.60 g kg−1 (Acaiá), stem+branches from 0.43 g kg−1 (Geisha) to 0.89 g kg−1 (Apoatã); in mature leaves from 0.62 g kg−1 (Acaiá) to 0.99 g kg−1 (Guarini) and in young leaves from 0.91 g kg−1  to 1.85 g kg−1 (Guarini). At high Pi, P concentration in roots ranged from 0.73 g kg−1  to 1.00 g kg−1 (Bukobensis), stem+branches from 0.73 g kg−1 (E22 Sídamo) to 2.28 g kg−1 (Guarini), in mature leaves from 1.17 g kg−1 (E22 Sídamo) to 2.14 g kg−1 (Guarini) and in young leaves from 1.68 g kg−1 (Geisha) to 2.96 g kg−1  in biomass accumulation, obtained by the ratio between DM at low Pi and DM at high Pi, was 61% on average for cultivars of ra Table . The var−1 P, while the average for non-responsive cultivars was 15.99 g DM g−1 P. Of the 25 lines studied, only four C. arabica lines were classified as being efficient and responsive  and agronomic P use efficiency (APE) index (axis y) of each cultivar Gerloff, . The aveC. canephora showed higher PUpE compared to C. arabica in both Pi treatments (Table −1 Pi (cv. Catuaí Yellow ) to 319 mg P g−1 Pi (Apoatã) at low Pi and between 46 mg P g−1 Pi (Obatã) and 85 mg P g−1 Pi (Guarini) at high Pi (Table C. arabica in both Pi treatments. At low Pi PUtE ranged from 1.12 g DM mg−1 P (Apoatã) to 1.66 g DM mg−1 P (E22 Sidamo), and at high Pi, from 0.52 g DM mg−1 P (Guarini) to 0.97 g DM mg−1 P  to 64.9 g2 DM mg−1 P (Geisha) at low Pi, and from 24.4 (Guarini) to 51.6 g2 DM mg−1 P (E22 Sidamo) under high Pi  positively correlated with PUpE, but significantly (P = < 0.001) negatively correlated with PUtE. Root to shoot ratio was also significantly positively correlated with aboveground tissue P concentrations under both low and high Pi supplies. Root to shoot ratio was significantly (P = < 0.001) negatively correlated with both PUtE and PPUE under high Pi supply  Resource for Project Execution Grant number 2010/11744-2 and Scholarship Grant number 2010/11745-9.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BD and handling Editor RB declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Moringa oleifera (MO) leaf extract as a dietary supplement on the growth performance and antioxidant parameters was evaluated on broiler meat and the compounds responsible for the corresponding antioxidant activity were identified. 0.5%, 1.0%, and 1.5% w/v of MO leaf aqueous extracts (MOLE) were prepared, and nutritional feed supplemented with 0%, 0.5%, 1.0%, and 1.5% w/w of MO leaf meal (MOLM) extracts were also prepared and analysed for their in vitro antioxidant potential. Furthermore, the treated broiler groups (control (T1) and treatment ) were evaluated for performance, meat quality, and antioxidant status. The results of this study revealed that, among the broilers fed MOLM, the broilers fed 0.5% w/w MOLM (T2) exhibited enhanced meat quality and antioxidant status (P < 0.05). However, the antioxidant activity of the MOLE is greater than that of the MOLM. The LC-MS/MS analysis of MOLM showed high expression of isoflavones and fatty acids from soy and corn source, which antagonistically inhibit the expression of the flavonoids/phenols in the MO leaves thereby masking its antioxidant effects. Thus, altering the soy and corn gradients in conventional nutrition feed with 0.5% w/w MO leaves supplement would provide an efficient and cost-effective feed supplement.The influence of  Poultry farming has grown since 2000, and the demand for broiler chicken meat has increased recently. From 2000 to 2010, both Africa and Asia have recorded increases of broiler meat production approximately 4.5 per cent a year, whereas growth in Europe has averaged 3.9 per cent, 3.7 per cent in Oceania and 3.5 per cent in the Americas [The composition of the nutritional feed plays a major role in poultry meat production, which in general includes soy and corn as sources of energy and protein. In addition, the formulation of nutritional feed includes essential dietary supplements, which are important for improving the health and performance of the birds and also for enhancing the meat quality of human consumption . In term Miracle Tree or Moringa oleifera Lam. (MO) is postulated to have the highest antioxidant content in food and also has a remarkable range of medicinal uses and high nutritional value [A major natural source of antioxidants is plants. Theal value –14. The al value , 16. MO al value . Kakengial value  reportedal value . Althougal value –22, the  M. oleifera were harvested locally from garden-2 of Universiti Putra Malaysia and were first air-dried for 12 h at room temperature (24°C) and then oven-dried for two consecutive days, ground, and stored in air tight plastic bags until further processing. Initially, three various aqueous concentrations of MO leaf extracts (MOLE) were prepared ; 1 g : 1 L (1.0% w/v); and 1.5% g : 1 L (1.5 w/v)) followed by maceration of the leaf powder in 3 different bottles for two days at room temperature. The corresponding residues were further condensed with a rotary evaporator at 40°C, freeze-dried, and stored at −80°C individually until further use. Subordinately, four concentrations of MO leaf meal (MOLM) extracts were prepared ; 0.5 g : 99.5 g (0.5% w/w); 1.0 g : 99 g (1.0% w/w); and 1.5 g : 98.5 g (1.5% w/w)). The MOLM feed formulations with concentrations are listed in Fresh and mature leaves ofL: 0.5 m, W: 0.9 m, and H: 0.6 m/compartment) in a trial room with controlled climate and a light regimen of 16 h light : 8 h dark. The feed and water were provided ad libitum. The chickens were randomly divided into the following four groups: three treatment groups (T2 (0.5% w/w), T3 (1.0% w/w), and T4 (1.5% w/w)) and one control group ). Each group had five replicates of 8 broilers per group. The trial lasted for 42 days, and, for the first 21 days, the birds were fed a commercial starter ration, followed by the experimental diet until day 42. The experimental diet contains 20 mg crude protein (CP) and 3050 kcal metabolisable energy (ME)/kg supplemented with 0%, 0.5%, 1.0%, and 1.5% MOLM, and the chickens were fed as shown in A feeding trial using Cobb 500 breed broiler chickens (23 weeks) was carried out on 160 birds retained in 6-tier battery cages (W1). The samples were then dried carefully with absorbent paper and weighed again (W2) to calculate the drip loss using the formula ( = W1 − W2/W1∗100). Two samples from each replicate of the meat samples were preweighed (w1) and then cooked in a water bath at 80°C for 20 min and cooled for 30 min to obtain the weight of the cooked sample (w2). The cooking loss was calculated with the formula ( = w1 − w2/w1∗100). The meat color and tenderness were measured on three samples from each replicate based on the L (lightness), a (redness), and b (yellowness) using the HunterLab system . For the measurement of meat tenderness, three cores were obtained from each sample and sheared at two locations using a texture analyser  with a Warner-Bratzler blade. The shear force values were then reported.On the 42nd day, the experimental birds were weighed and sacrificed using the halal procedure by severing the jugular veins. The liver, kidney, breast, and thigh muscles were carefully excised, packed in polyethylene bags, flash-frozen in liquid nitrogen, and stored at −80°C until use. The carcasses were dressed by removing the head, feet, skin, and viscera, and the dressing percentage was calculated using the formula ( = carcass weight  (CW)/live weight  (LW)∗100). The bones, muscles (meat), and fat were carefully trimmed from the carcass and weighed accordingly to calculate the meat : bone : fat ratio for each dietary treatment. Two samples from each replicate of the meat samples stored at −80°C were removed and weighed immediately  and MOLM  using DPPH radical scavenging and phosphomolybdenum total antioxidant capacity assays as described by Karthivashan et al. [A comparative analysis was performed for then et al. . The livμm) Phenomenex column  on an Agilent 1100 series HPLC system  equipped with a binary pump, diode array detector (DAD) (200 to 600 nm), and autosampler. The sample injection volume was 20 μL with a mobile phase flow rate of 1.0 mL/min monitored at a wavelength of 254 nm. The mobile phase consisted of solvent A, distilled water, and solvent B, methanol : distilled water 70 : 30 (v/v). The gradient program profile was a combination of solvents A and B as follows: 0 to 10 min, 30% solvent B; 10 to 20 min, 40% solvent B; 20 to 35 min, 50% solvent B; 35 to 40 min, 60% solvent B; 40 to 45 min, 70% solvent B; and 45 to 50 min, 0% solvent B.Chromatographic separation of the compounds in the 0.5% w/v, 1.0% w/v, and 1.5% w/v aqueous MO leaf extracts (MOLE) was performed with a LUNA C18  at a scan rate of 0.5 Hz. The system was supported with mass spectrometry software and a spectral library provided by ACD labs . Analyte separation was performed on a C18 column  with a gradient mobile phase consisting of water (solvent A) and methanol with 1% acetonitrile (solvent B), each containing 0.1% formic acid and 5 mM ammonium format. The gradient program was 40% solvent B to 50% solvent B over 11.00 min at a flow rate of 1.0 mL/min. The sample injection volume was 20 μL. All chromatographic procedures were performed at ambient temperature, and the corresponding peaks from the QTrap LCMS/MS analysis of both the 0.5 w/v MOLE and 0.5 w/w MOLM were identified by comparison with the literature/ACD labs mass spectral library.The compounds in the 0.5 w/v aqueous MO leaf extracts (MOLE) and 0.5 w/w MO leaf meal (MOLM) extracts were identified with accurate mass detection using an AB Sciex 3200 QTrap LCMS/MS with a Perkin Elmer FX 15 UHPLC system . The negative ion mass spectra were obtained with a LC QTrap MS/MS detector in full ion scan mode  followed by Tukey's test using the IBM-SPSS Statistics software, version 20 . The significance level was set at P < 0.05.All data from the in vitro antioxidant activity of the MO leaf extracts (MOLE) and MO leaf meal (MOLM) extracts were comparatively evaluated with DPPH, nitric oxide (NO) radical scavenging assay, and phosphomolybdenum (PMO) total antioxidant capacity assays. For the DPPH and NO assays, the changes in the ability of the MO leaf extracts (MOLE) and MO leaf meal (MOLM) extracts to scavenge free DPPH and NO radicals were calculated as the percentage inhibition and are shown in Figures P < 0.05) free radical scavenging activity, with the lowest IC50 values of 154.5 and 56.4 for DPPH and NO, respectively, compared to the other extracts. For the MOLM extracts, 0.5% w/w MOLM has the highest scavenging activity with IC50 values of 229.4 and 134.5 for DPPH and NO, respectively, but these values are not as low as the 0.5% w/v MOLE. The nutritional feed alone (0% w/w MOLM) shows the least scavenging activity with IC50 values of 378 and 298 for DPPH and NO, respectively, although the IC50 values for 1.5% w/w MOLM are higher than 0 w/w MOLM (control) for NO radical scavenging activity. For the PMO total antioxidant capacity assay, we determined whether the MO leaf extract (MOLE) and MO leaf meal (MOLM) extracts reduce phosphomolybdic acid to phosphomolybdic blue (Mo+6 → Mo+5), and this was expressed quantitatively in terms of ascorbic acid equivalent μg/g of extract in P < 0.05) of all concentrations, with 36.26 ascorbic acid equivalent μg/g of extract. However, for the other MOLM concentration, 0.5% w/w MOLM has the highest antioxidant capacity (P < 0.05), with 34.66 ascorbic acid equivalent μg/g of extract, and the control (0% w/w MOLM) has the lowest activity, with 22.63 ascorbic acid equivalent μg/g of extract.TheP < 0.05) weight gain compared to broilers fed nutritional feed only (T1), although there were no significant differences in weight gain for the dietary treatments.The average weight gain, feed intake (FI), feed conversion ratio (FCR), and mortality for the treatment period of 22–42 days are reported in P < 0.05) highest FCR value of 2.67 ± 0.052 compared to all the dietary treatment groups . Throughout the experimental period, approximately 2% mortality was observed for all groups, and there were no significant differences, except for group T2 with 8% mortality, which caused the anomaly.The feed conversion ratio (FCR) data from P < 0.05) higher dressing percentages than the broilers fed nutritional feed alone (T1: control). Of the MOLM concentrations , the dressing percentages were significantly (P < 0.05) different, ranging from 67 to 70%, and T3 (1.0% w/w MOLM) showed the highest dressing percentage of 70%. The meat : bone and meat : fat ratios were significantly (P < 0.05) different among the treated birds, with values ranging 3.459–3.814 and 6.31–8.43, respectively.P < 0.05) for cooking loss and drip loss. The treatment groups  exhibited significantly higher drip loss values compared to the control group (T1), which had the lowest drip loss value. By contrast, the control group (T1), T3, and T4 exhibited the highest cooking loss without any significant difference among the groups, compared to T2 with the lowest cooking loss value of 16.62%. The percentage of cooking loss ranged from 16.62 to 21.99%. *), redness (a*), and yellowness (b*) of broiler chickens fed MOLM supplements. As expected, the broiler chicken breast meat exhibited greater yellowness (b*) than redness (a*). For lightness, there was no significant difference between T2 and T4. However, T3 showed a significantly (P < 0.05) higher lightness value of 51.70. For redness, there was no significant difference between T1 and T4; however, T2 is significantly different (P < 0.05) from T3 and possesses the highest value of 7.93. Although there are slight differences among all the treatments, T4 exhibited the highest yellowness value of 14.24. P < 0.05) lower than the control group (T1). The results were also significantly different among all the treatment groups (P < 0.05) except for kidney. Group T3 was not significantly different from the control group. However, T2 (0.5% w/w MOLM) showed the lowest MDA formation in both the muscles and organs, indicating suppressed lipid peroxidation activity. Changes in the activities of the antioxidant enzymes SOD, CAT, and GPx in the muscles (breast and thigh) and organs (liver and kidney) of broilers fed MOLM feed supplement are shown in P < 0.05) and control, no significant differences were observed in the breast and liver. Overall, T2 (0.5% w/w MOLM) showed significantly high levels of catalase in both the muscles and organs compared to the control group (T1), except the kidney of T3, possibly because of an anomaly. P < 0.05) increase in the level of GPx activity in both the kidney and the liver of T2 (0.5% w/w MOLM) and T3 (1.0% MOLM) and a nonsignificant decrease in the GPx level in T4 (1.5% w/w MOLM) compared to the control group (T1). However, T2 (0.5% w/w MOLM) showed the highest GPx levels in both the muscles and the organs compared to the control.The impact of MOLM feed supplements on the lipid peroxidation (MDA) levels in the liver, kidney, breast, and thigh muscles of treated broilers is shown in The HPLC fingerprints of 0.5% w/v, 1.0% w/v, and 1.5% w/v MOLE showed the same profile of peaks Figures . A few p M. oleifera leaf meal added to broiler diets significantly increased the average daily weight gain of the broilers. The feed conversion ratio is used to determine the performance of the animals and is obtained by dividing the feed intake by the weight gained. A low FCR is a good indication of high quality feed [Growth performance is a primary factor for determining the productivity of broiler chickens. The results indicate that MOLM significantly improves the growth performance of broilers, which is consistent with Ayssiwede et al.  who repoity feed . In accoity feed . Approxiity feed . Thus, tThe dressing percentage is a trait of economic importance, and the higher the dressing percentage the better the economic returns . HoweverMeat tenderness is a function of collagen content, heat stability, and myofibrillar structure of the muscle. Meat tenderness significantly improves with muscle aging because of the breakdown of myofibrillar proteins. Tenderness is an important attribute that consumers consider when purchasing chicken meat . The sheP < 0.05) improvement in health status, performance, and meat quality compared to the control group fed only conventional feed. However, consistently varying results  were obtained for the MOLM treated groups . This makes it difficult to select the optimal concentration of MOLM based on the growth performance, carcass, and meat quality. However, 0.5% w/w MOLM fed group retained the meat tenderness, which is an important factor for commercialization. Furthermore, the efficiency of the MO supplement was evaluated.Cumulatively, the results of the growth performance, carcass, and meat quality assays indicate that broilers fed MOLM feed supplement show significant . These results support a previous report indicating aqueous MOLE exhibit high antioxidant activity that may be attributed to phytoconstituents, such as polyphenols, tannins, anthocyanin, glycosides, and thiocarbamates that scavenge free radicals, activate antioxidant enzymes, and inhibit oxidases [The results of theoxidases . In the oxidases  and Elofoxidases . In addioxidases . However in vitro results, 0.5 w/w MOLM exhibited lowest MDA level and highest SOD, CAT, and GPx activity in both liver and tissue. This is due to the MOLM supplement, enriched with phenolics, flavonoids, and vitamin C, inhibits the formation of free radicals, and contributes to the retention of muscles and organs and lipid stabilization. This result is consistent with the data for meat quality improvement, in which the T2 group (0.5% w/w) has a significantly higher meat tenderness value compared to the other groups. Therefore, the addition of 0.5% w/w MO with conventional feed (MOLM) retains meat quality by reducing the activity of lipid peroxidation.In accordance with the in vitro and in vivo antioxidant activity, which is consistent with increased meat quality.Overall, the results indicated that broilers fed the MOLM supplement have significantly better performance and antioxidant status compared to the control fed only nutritional feed. For the percentage of MOLM added to the conventional meal, although the results of the growth performance and carcass and meat quality show some inconsistencies, the T2 (0.5% w/w MOLM) group showed significantly higher in vitro results specified that generally MOLE exhibit higher antioxidant activity than MOLM, and this drives us to analyze the role of constituents in the activity. In accordance, firstly the comparative analysis of the 0.5% w/v, 1.0% w/v, and 1.5% w/v MOLE using HPLC revealed that 0.5% w/v exhibit few unique peaks further confirmed as flavonoids using LC-MS/MS. In the previous study, the presence of apigenin, quercetin, and kaempferol has been reported in M. oleifera leaves, which are responsible for its antioxidant activity [Though antioxidant assay results convincingly revealed 0.5% w/w MOLM as the optimal gradient for feed supplement, theactivity . This waactivity . Thus, M M. oleifera leaf extract and identified their antidiabetic property as restoring the antioxidant status in streptozotocin-induced diabetic rats. Qwele [ M. oleifera leaves and categorized them as antioxidant compounds because they enhance endogenous antioxidant enzyme levels. Coppin [ M. oleifera leaves, which may prevent hepatic lipid peroxidation by scavenging free radicals. Together, these results indicate that 0.5% w/v MOLE are enriched with antioxidant compounds, such as flavonoids, vitamins, carotenoids, and a few organic acids, which may be responsible for its enhanced antioxidant activity.Secondly, LC-MS/MS was further used to compare and identify compounds in the 0.5% w/v MOLE and 0.5% w/w MOLM (T2) and their role in meat antioxidant status. As anticipated, 0.5% w/v MOLE exhibited high expression of flavonoids. Previously Gupta et al.  isolateds. Qwele  also rep. Coppin  reported. Coppin , reporte M. oleifera leaves, soy, and corn and are consistent with a previous report [On contrast, 0.5% w/w MOLM possesses a high amount of isoflavones, phospholipids, and fatty acids, possibly from the soy and corn ingredients of feed. In a previous study, He and Chen  reporteds report . The LC Conclusively, during MOLM preparation, the compounds in the conventional feed, such as the isoflavones, phospholipids, and fatty acids, antagonistically inhibit the expression/activity of the flavonoids and phenols in the MO leaves. Therefore, 0.5% w/w MOLM showed relatively low antioxidant activity compared to the 0.5% w/v MOLE. However, isoflavones, phospholipids, and fatty acids also possess antioxidant effects, relatively less than flavonoids. Consequently, the chickens fed 0.5% w/w MOLM feed supplement showed the highest antioxidant activity compared to the other treatment and control groups. Thus, optimizing the gradient of soy and corn in the conventional nutrition feed with 0.5% w/w of MOLE might help the researchers/nutritional feed corps/farmers to provide an efficient and cost-effective feed for broilers.