Paracoccus sp. strain isolated from sludge was identified and evaluated for catalytic activity in the degradation of penicillin G.The 1H NMR, and 13C NMR. Enzymes involved in the PAA pathway were proposed from a genomic analysis of KDSPL-02.High degradation efficiency and synergistic catalytic effects of the whole cell and visible light without additional catalysts were observed. The key factors influencing the degradation and kinetics of penicillin G were investigated. The results showed the phenylacetic acid, which was produced during penicillin G biodegradation, exhibited stronger inhibiting effects on KDSPL-02. However, this effect was reduced by visible light irradiation without any additional photocatalyst; furthermore, the rate of penicillin G biodegradation was accelerated, reaching a 100% rate in 12 h at a penicillin G concentration of 1.2 g/L. Four key intermediates produced during penicillin G degradation were isolated and identified by LC–MS, These results provide a new method for bio-degrading of penicillin or other antibiotic pollutants using photoaccelerating biocatalysts with greater efficiency and more environmentally friendly conditions.The online version contains supplementary material available at 10.1186/s13036-021-00275-4. In recent years, the irrational use of antibiotics has led to worldwide environmental pollution attributed to clinical prescriptions, animal husbandry abuse and fermentation fungi residue –3. Among1H NMR and 13C NMR spectroscopy, to confirm the structure of degradation products in the biodegradation of antibiotics.With respect to biodegradation mechanisms, most β-lactam antibiotic degradation studies have focused on the separation and characterization of key intermediate metabolites and not the whole pathway, including the mineralization products –24. HPLCRegarding the technology for β-lactam antibiotic degradation, various methods such as nanofiltration , ozonatiThe combination of biodegradation and photocatalysis is a promising technology that can achieve the complete mineralization of contaminants –37. The Paracoccus sp. strain cells isolated from sludge contaminated by antibiotics were utilized as biocatalysts; this strain has been shown to solely use penicillin G as an energy and carbon source. The effect of heterogeneous photocatalysis on the improvement in biodegradation efficiency of penicillin G was evaluated. Penicillin G degradation products were assessed and identified using HPLC, LC–MS, 1H NMR, and 13C NMR. This study describes an efficient photo-assisted biodegradation system that can be used for the complete mineralization and degradation of penicillin in the environment.In this work, we used penicillin G as a model contaminant for the biocatalytic mineralized degradation of penicillin G under visible light irradiation without any photocatalyst. Whole Sludge samples were collected from the district of Shijiazhuang, China. Penicillin G was provided by North China Pharmaceutical Group Corporation, China. All other chemicals used in this study were analytical grade and commercially available without further purification unless otherwise noted. Wastewater contained penicillin G was derived from North China Pharmaceutical Group Corporation.2HPO4, 0.40 g of KH2PO4, 0.20 g of MgSO4·7H2O, 0.03 g of CaCl2·2H2O, 0.02 g of FeCl3·6H2O, 0.50 g of NH4NO3, and 0.50 g of yeast extract per litre of water.The base mineral media (BMM) consisted of 1.60 g of KThe growth medium (GM) consisted of 2.40 g of yeast extract per litre of water in the base of BMM medium. The final pH was adjusted to 7.0.2HPO4, 0.40 g of KH2PO4, 0.20 g of MgSO4•7H2O, 0.03 g of CaCl2•2H2O, 0.02 g of FeCl3•6H2O, 0.50 g of NH4NO3 per litre of water; and 45 mg L− 1 penicillin G as the inducer.The fermentation medium (FM) was composed of the following: 10 mL of industrial liquid sugar; 2.40 g of yeast extract, 1.60 g of K4NO3 (1 g/L), NaCl (0.5 g/L), K2HPO4 (0.5 g/L), MgSO4 (0.5 g/L), and FeSO4 (0.01 g/L).To test whether strain KDSPL-02 can using penicillin as the sole carbon and energy source, varies carbon sources such as glucose (1.0 g/L), sucrose (1.0 g/L), maltose (1.0 g/L) and starch (1.0 g/L), were tested in addition to penicillin (0.1 g/L) in BMM. As a control, the base mineral media contained no carbon sources and comprised NHTo accumulate high titres for the biodegradation of penicillin G. the fermentation of strain KDSPL-02 was optimized. Inoculation medium was prepared with 0.01 mg of nickel chloride hexahydrate, 0.02 mg of sodium molybdate dihydrate, 0.01 mg of cobalt chloride, 0.1 mg of zinc sulphate heptahydrate, 0.005 mg of riboflavin, and 0.01 mg of pyridoxal hydrochloride per litre of water. The pH before autoclaving was within 6.8–7.0 (adjusted with 12% ammonia solution). Flasks were inoculated with the bacteria and placed on an orbital shaker. The agitation velocity was 200 rpm, and the volume of the flask was 250 mL. The activity (U) of penicillin G degraded by whole KDSPL-02 cells was defined as g/g·h, i.e., the wet cell of one gram degrading penicillin (g) over 1 hr. All experiments were conducted in triplicate biodegradation reactors and triplicate no-cell control reactors in dark to prevent photodegradation.− 1 penicillin G. The treatment was carried out at 120 rpm at 32 °C under visible light irradiation. Initially, persistent organic compounds were converted into more easily photodegradable compounds, thereby promoting photodegradation. The photocatalytic degradation of penicillin G was carried out in an annular reactor under visible light or LED irradiation with a medium mercury lamp (TQ 718 Z1 700 W) purchased from Heraeus at the reactor centre. Irradiation below 290 nm was filtered by a Duran cooling tube surrounding the lamp. The reaction was monitored via HPLC at 1 h intervals. Degradation was completed in approximately 12–14 h. Degradation intermediates were separated by HPLC.A suspension solution (0.2 mL) of whole KDSPL-02 cells with a degradation activity of 2.0–7.0 U was added to a 250-mL conical flask containing a 100-mL solution of 0.8–1.6 g LTo verify whether phenylacetic acid has an inhibitory effect on KSDPL-02, we conducted a phenylacetic acid inhibition test. Various concentrations of phenylacetic acid  were added to culture medium, and changes in OD600 were used to characterize the inhibition of phenylacetic acid.Furthermore, the degradation of phenylacetic acid by KDSPL-02 under light and non-light conditions was investigated. Culture medium was modified with 0.1 g/L of phenylacetic acid to investigate the clearance rate of phenylacetic acid at different times  under light and non-light conditions.1HNMR and 13CNMR were recorded on a Bruker Avance II 500 spectrometer in CDCl3 unless stated otherwise, using tetramethylsilane as an internal reference, operated at 500.13 MHz for 1H, and 125 MHz for 13C. J values are given in Hz. Penicillin and intermediate concentrations were measured via HPLC (Dionex and Agilent) with an UltimateR XB C18 column  at a flow rate of 1.0 mL min-1, column temperature of 30 °C, mobile phase of methanol and phosphate  at a ratio of 50:50, injection volume of 20 μL, and detection wavelength of 225 nm.The light absorption properties were measured using UV–vis diffuse reflectance spectrophotometer  with a wavelength range of 200–900 nm. A 300 W xenon lamp  was used as light source, and the average light intensity was 78.5mw/cm − 2 (UV-A radiation meter). LC-MS data were measured with Thermo Scientific ISQ QD. 600 after 6 h of growth reached 1.2, 0.9 and 0.9, respectively. When penicillin was used as the sole carbon and energy source, the growth of KDSPL-02 was relatively poor with the OD600 reaching 0.4 after 6 h of growth. Although the growth status of KDSPL-02 was poor when using penicillin, this showed that KDSPL-02 could still survive.First, the growth of KDSPL-02 when using penicillin as the sole carbon and energy source was investigated. As shown in Fig. Most microorganisms are not known to use antibiotics as sole carbon and energy sources; co-metabolism is often required to maintain growth . In our Because penicillin hydrolyses in water, penicillin biotransformation in solution cannot be directly measured. Hence, all experiments included control treatment reactors that contained no cells. Only hydrolysis occurred in the control reactors, whereas both hydrolysis and biotransformation occurred in the bacterial treatment reactors. Observed differences in penicillin concentrations with the control reactors were then attributed to biotransformation.Paracoccus sp. KDSPL-02 cell dry weight (5 g/L- 20 g/L). The research result showed that the Paracoccus sp. KDSPL-02 cell dry weight was related to the degradation efficiency. The degradation rate increased steadily with increasing cell dry weight. When the cell activity was above 10 g/L, the degradation rate reached to 100% within 12 h , the degradation ability of the strain lost gradually  was 353.2, and no fragment signal peaks showed a difference in LC–MS methyl)-5,5-dimethylthiazolidine-4-carboxylate (penicilloic acid)(2) and potassium 2--2-(2-phenylacetamido) acetate (potassium penicilloic acid)(3)  was 137.3   and potassium 2-amino-2- acetate (6).All intermediates attributed to peaks in HPLC were isolated and collected to yield purified samples. The structures of the compounds were determined via 5 and compound 6. Phenylacetic acid was oxidized by oxidases in the cell through oxophenylacetic acid 7 which coincide with the peak 150.1 in the MS of penicilloic acid degradation mixture to form inorganic mineralizers. Compound 5, which coincide with the peak 271.1 in the MS of its degradation mixture, was initially converted to the corresponding α-keto acid 8, which then underwent further decarboxylation, oxidation, and hydrolysis to yield small inorganic compounds.According to the HPLC, MS and HMR analysis of the intermediates, the possible pathway of complete degradation process catalyzed by KDSPL-02 whole cells was proposed, as shown in Fig. − 1 penicillin G was almost degraded completely by whole-cell catalysis at 30 °C. However, in the degradation of penicillin G, in solution or wastewater, the overall reaction rate was subject to secondary reactions. To improve the biodegradation efficiency, visible light irradiation was used to enhance the biocatalytic degradation.KDSPL-02 was a more effective catalyst of penicillin G degradation under the optimized conditions. Using the same methods for the treatment of penicillin G in solution, wastewater contained penicillin G was treated. The results showed that 0.8–1.6 g L− 1 are shown by HPLC data in Fig. − 1. However, the conversion rate of penicilloic acid intermediate was slow and required 24 h. Phenylacetic acid and compound 5 intermediates degraded more rapidly than penicilloic acid within 18 h. In the degradation process, the rate-limiting reaction was the conversion of penicilloic acid into phenylacetic acid and compound 5. To improve the degradation kinetics, we tested auxiliary strategies. The results indicated that visible light markedly accelerated the rate-limiting reaction. Based on HPLC data methyl)-5,5-dimethylthiazolidine-4-carboxylic acid, respectively. The high degradation performance could be attributed to the enhanced oxidation of phenylacetic acid by photocatalysis to reduce biodegradation inhibition. The novel heterogeneous photocatalysis of whole cell KDSPL-02 could provide a new method for the treatment of residual antibiotics and provide important experimental data on penicillin behaviour in nature.We identified and enriched the bacterial strain Additional file 1: Table S1. Degradation capability of Penicillin G by different strain species as reported from previous literatures and this study. Fig. S1a-c: Structure of the identified metabolites of peak Nos. 2 (a), 3 (a), 4 (b), and 5 (c) by LC–MS analysis. Peak Nos. 2 and 3 in HPLC: Potassium 2-(carboxy(2-phenylacetamido)methyl)-5,5-dimethylthiazolidine-4-carboxylate and potassium 2--2-(2-phenylacetamido) acetate. Peak No. 4 in HPLC: Phenylacetic acid. Peak No. 5 in HPLC: Mixture of two isomers, 2-(amino(carboxyl)methyl)-5,5-dimethylthiazolidine-4-carboxylic acid. Fig. S2a-b: 1H NMR (a) and 13C NMR (b) spectra of potassium 2-(carboxy(2-phenylacetamido)methyl)-5,5-dimethylthiazolidine-4-carboxylate and potassium 2--2-(2-phenylacetamido) acetate. 1H NMR  δ:7.34–7.45 ; 5.07–5.06 ; 5.05–5.06 ; 4.79–4.78 , 4.25–4.24 ; 3.81–3.80 ; 3.72–3.70 ; 3.43–3.42 ; 1.57 ; 1.51 ; 1.23 ; 1.05 .13C NMR  δ:176.17, 175.58, 175.24, 174.93, 174.86, 174.30, 135.01, 134.50, 129.65(2C), 129.49(2C), 129.27(2C), 129.04(2C), 127.61, 127.42, 75.84, 75.28, 67.00, 66.02, 60.01, 58.61, 58.45, 55.24, 42.51, 42.46, 27.98, 27.75, 26.75, 26.33. LC–MS (m/z): 391.3[M + 1]+ , 353.2[M` + 1]+ . Fig. S3a-b: 1H NMR (a) and 13C NMR (b) spectra of phenylacetic acid. 1H NMR  δppm: 7.42–7.32 ; 3.73 .13CNMR  δppm: 177.01, 134.17, 129.38(2C), 128.79(2C), 127.27, 40.54. LC–MS (m/z): 137.3[M + 1]+ . Fig. S4a-b: 1H NMR (a) and 13C NMR (b) spectra of 5,5-dimethylthiazolidine. 1H NMR  δ: 5.06–5.05 ; 4.85–4.83 ; 3.97–3.96 ; 3.64–3.62 ; 3.56 ; 3.47 ; 3.19 ; 1.45 ; 1.44 ; 1.19 ; 1.13. 13C NMR  δ: 176.34, 174.38, 171.85, 171.20, 74.47, 74.00, 63.59, 63.29, 59.31, 58.68, 56.80, 56.39, 29.29, 26.06, 26.02, 25.94. LC–MS (m/z): 235.1 [M + 1]+ .


The self-healing behavior of two supramolecular polymers based on π–π-interactions featuring different polymer backbones is presented. For this purpose, these polymers were synthesized utilizing a polycondensation of a perylene tetracarboxylic dianhydride with polyether-based diamines and the resulting materials were investigated using various analytical techniques. Thus, the molecular structure of the polymers could be correlated with the ability for self-healing. Moreover, the mechanical behavior was studied using rheology. The activation of the supramolecular interactions results in a breaking of these noncovalent bonds, which was investigated using IR spectroscopy, leading to a sufficient increase in mobility and, finally, a healing of the mechanical damage. This scratch-healing behavior was also quantified in detail using an indenter. Damage inflicted on different materials is omnipresent. Consequently, nature established a mechanism dealing with this problem . The regBesides the examples of self-healing/regeneration that exist in Nature, the general concept could also be transferred to different synthetic materials. Hereby, two concepts can be distinguished. In extrinsic self-healing materials, a material flow is achieved by the encapsulation of microcapsules  or microHowever, little is known about the exact healing mechanism on the molecular scale and the correlation to the macroscopic properties of such polymers. For this purpose, the current study will focus on the design of polymers containing π–π interactions and the quantification of the healing behavior as well as the in-depth characterization of the molecular behavior and the macroscopic properties, which reveals new insights into the self-healing materials based on π–π interactions.P1. In order to study the influence of the polymer backbone on the material’s properties, the diamine containing polymer was exchanged to a triblock copolymer of poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) (PPG3-PEG39-PPG3) featuring also two amine groups as end groups. The molar mass of this reactant was 1900 g/mol. The conversion with perylene-3,4,9,10-tetracarboxylic dianhydride resulted in polymer P2. In both synthesis protocols imidazole was applied as a catalyst in order to obtain higher molar masses.For the synthesis of supramolecular polymers based on π-π interactions a literature reported procedure was utilized see , which dMn = 11,400 g/mol for P1 and Mn = 17,400 g/mol for P2 with respect to a PEG-standard. The SEC traces of both polymers are depicted in Subsequently, both polymers were characterized regarding their structure. For this purpose, size exclusion chromatography (SEC) was performed revealing a molar mass of Tg) at −58 °C (P1) and −51 °C (P2), respectively. Furthermore, the polymers have an endothermic transition at 129 °C (P1) and 126 °C (P2). This transition is associated with the activation of the perylene domains, which was also reported in literature  with 1 mL/ min at 40 °C, poly(ethylene glycol) (standard).−1 (first and second heating cycle) and 10 K min−1 (third heating cycle). In general, the first cycle is used as annealing step, which deletes the thermal history of the sample, and thus is neglected.Differential scanning calorimetry was measured on a Netzsch DSC 204 F1 Phoenix instrument  under a nitrogen atmosphere with a heating rate of 20 K min−1 from 25 to 600 °C. The thermo gravimetric analysis revealed degradation temperatures above 370 °C for all synthesized polymers.Thermo gravimetric analysis was carried under nitrogen atmosphere using a Netzsch TG 209 F1 Iris  with a heating rate of 10 K minAll dynamic mechanical analysis (DMA) experiments were performed on a MCR 301 rheometer (SN80386674) from Anton Paar  using a convection temperature device CTD 450 which covers a broad temperature range of −100 to 450 °C. For measurements and the export of data the Rheocompas- software was used.P2) or 135 (P1) and 140 °C). Afterwards, the frequency was decreased in a logarithmic profile from 100 up to 0.01 Hz at a strain of 1%.The temperature sweeps (DMTA) and frequency sweeps (FS) were measured with a plate-plate setup (D-PP15-SN0). The sample was heated to 150 °C and the sample gap was set to 1 mm. For the DMTA, the samples were cooled to 25 °C and heated up to 200 °C with a heating rate of 2 °C/min under a frequency (f) of 1 Hz with 1% shear strain (γ). For the frequency sweeps, the sample was firstly annealed at the desired temperature  spectra were recorded on a Nicolet iS5 FTIR spectrometer , equipped with potassium bromide windows and beam splitter. The sample temperature was controlled with a temperature cell and temperature controller combination , which provides a heated sample chamber suitable for 12 mm windows.−1 spectral resolution was recorded. 1 mg of the respective sample was dissolved in 100 µL spectroscopic grade CHCl3  and 20 µL drop casted onto the window. To record the IR spectra of the samples, the temperature cell was heated to the respective temperature (30–150 °C in 20 °C steps) and left to equilibrize for 2 minutes. Afterwards, a sample spectrum with 32 scans was recorded.To collect IR spectra of the samples, first KBr windows were prepared directly before the measurement by pressing 200 mg dry spectroscopic grade KBr  into a ∅ 12 mm-form under vacuum and 7 MPa pressure. The window was subsequently transferred into the temperature cell and secured with a lockring and teflon seals. A background spectrum with 32 scans and 4 cmAll graphics were generated with GNU R (version 4.0.2)  without The preparation of the samples and the self-healing experiments  were performed according to literature . In the P1) or 600 µm (P2).The self-healing scratch tests were performed on an Anton Paar Micro scratch tester MST3 on a STeP 4 platform. The instrument was equipped with 10 µm and 50 µm Rockwell C indenters and the optical images were taken with the lenses MPlan N 5×/0.10/FN22. The scratches were performed with a 50 µm Rockwell indenter, 1500 mN normal force and 15 passages on a length of 2000 µm and a scratch speed of 30,000 µm/min. Subsequently, the scratch was imaged by the microscope in panorama mode. The sample was turned 90° in order to measure the profile of the scratch. Therefore, 150 scratches along the scratch (every 20 µm) were performed with a 10 µm Rockwell indenter with the following parameters: 3 mN normal force, 200 µm/min scratch speed, length 1600 µm , poly(propylene glycol) bis(2-aminopropyl ether)  and imidazole  were mixed in a round bottom flask under nitrogen atmosphere. Afterwards, the reaction mixture was heated to 150 °C for 17 h. After cooling to room temperature, water and chloroform were added to the mixture. The organic phase was washed two times with water, dried over sodium sulfate and concentrated. The residual was dissolved in tetrahydrofuran and dialyzed for three days with solvent exchange two times per day (MWCO: 3500 g/mol). After the solvent evaporation, a dark violet polymer could be obtained. 1H NMR  δ 8.74–8.45 , 4.27–3.12 , 1.46–0.81  ppm; SEC (PEG standard): Mn = 11,400 g/mol, Mw = 27,600 g/mol, Ð = 2.43 (RI detector), Mn = 11,800 g/mol, Mw = 25,000 g/mol, Ð = 2.12 (UV detector); elemental analysis: found: C: 63.81, H: 9.06, N: 1.48; expected: C: 64.04, H: 9.04, N: 1.18.P2: Perylene-3,4,9,10-tetracarboxylic dianhydride , poly(propylene glycol)-block-poly(propylene glycol)-block-poly(propylene glycol) bis(2-aminopropyl ether)  and imidazole  were mixed in a round bottom flask under nitrogen atmosphere. Afterwards, the reaction mixture was heated to 150 °C for 17 h. After cooling to room temperature, water and chloroform were added to the mixture. The organic phase was washed two times with water and one time with brine, dried over sodium sulfate and concentrated. The residual was dissolved in a 1:1 mixture of chloroform and tetrahydrofuran and dialyzed for two days with solvent exchange two times per day (MWCO: 3500 g/mol). After the solvent evaporation, a dark violet polymer could be obtained. 1H NMR  δ 8.86–8.31 , 4.29–3.15  PEG-H (OCH2)), 1.46–0.91  ppm; SEC (PEG standard): Mn = 17,400 g/mol, Mw = 34,100 g/mol, Ð = 1.95 (RI detector), Mn = 18,200 g/mol, Mw = 35,100 g/mol, Ð = 1.92 (UV detector); Elemental analysis: found: C: 57.94, H: 7.97, N: 1.50; expected: C: 58.71, H: 8.21, N: 1.14.File 1Additional data.


Aedes albopictus. Among the EOs, larvicidal activity against Ae. albopictus was strongest in those derived from massoia (Massoia aromatica) and nutmeg (Myristica fragrans). Larvicidal activities of massoia and nutmeg EOs against Ae. albopictus were 95.0% and 85.0% at 50 μg/mL, respectively. A total of 4 and 14 compounds were identified from massoia and nutmeg, respectively, and two massoia lactones, C10 and C12, were isolated from massoia EO. Among the identified compounds, benzyl salicylate, terpinolene, C12 massoia lactone, sabinene, benzyl benzoate, methyl eugenol, and C10 massoia lactone exhibited the strong larvicidal activity. Cellulose nanocrystal (CNC)-stabilized Pickering emulsions of massoia and nutmeg EOs were developed to overcome the insolubility of EOs in water. CNC/massoia and CNC/nutmeg emulsions were stable for at least 10 days, and larvicidal activities of CNC/massoia PE and CNC/nutmeg were higher than those of crude massoia and nutmeg EOs. This study presents a CNC-stabilized PE, a suitable formulation for EOs, as a potential larvicide against Ae. albopictus.We investigated the larvicidal potential of 10 plant essential oils (EOs) against the Asian tiger mosquito  Aedes albopictus transmits the dengue virus and chikungunya virus, both of which cause human diseases1. Aedes albopictus is also an insect vector of the Zika virus, which causes microcephaly and other congenital abnormalities in developing fetuses and newborns2. Although Ae. albopictus is native to East Asia, it has spread to Europe, North and South America, the Middle East, and Africa1. Insect growth regulators  and temephos are widely used to control Ae. albopictus larvae, but selection for resistance to some of current larvicides has been documented around the world6, creating a need for new control agents.The Asian tiger mosquito 9. Besides medicinal uses, many researchers have become interested in the insecticidal and repellent activities of EOs13. Larvicidal activity against mosquitoes such as Ae. albopictus and Ae. aegypti has been documented in many EOs, including Citrus and Cryptomeria japonica EOs18. However, many studies have not figured out the active components of EOs which have shown good activities against the mosquitos, despite the importance of the analysis. As to the physicochemical properties of EOs, high hydrophobicity makes it difficult to develop EOs that can serve as larvicides against mosquitoes. To overcome the insolubility of EOs in water, proper formulation is essential19. Emulsions are commonly used in the pesticide industry to incorporate hydrophobic materials into aqueous products20. However, synthetic and organic phase emulsions are associated with multiple undesirable side effects20. Recently, Pickering emulsions (PEs) stabilized by solid particles, and cellulose nanocrystals (CNCs) in particular, are being considered because of their low environmentally impact, bio-degradability, and low production cost21.Essential oils (EOs) have attracted attention for their potential medicinal applicationsAe. albopictus to identify new and effective larvicides and analyzed the chemical composition of the two most promising ones . Furthermore, the toxicity of the individual components of massoia (Massoia aromatica) and nutmeg (Myristica fragrans) EOs is described. Finally, formulations based on CNC-stabilized PEs were developed, and their larvicidal activities were compared with those of crude EOs.Here, we describe the larvicidal potential of 10 EOs against the third instar stage of Ae. albopictus are shown in Table Ae. albopictus at 50 μg/mL.The larvicidal activities of 10 EOs against α-pinene (12.21%), myristicin (7.38%), limonene (6.29%), and terpinen-4-ol (4.08%). All other constituents accounted for less than 4%. Four compounds, including C10 massoia lactone (79.97%), C12 massoia lactone (10.75%), benzyl benzoate (6.55%), and benzyl salicylate (1.13%) were identified in massoia EO.The chemical compositions of massoia and nutmeg EOs are supplied in Table 1H, 13C nuclear magnetic resonance (NMR) and mass spectrometer data. The purities of isolated C10 massoia lactone and C12 massoia lactone were 95% and 95%, respectively.C10 and C12 massoia lactones were identified and obtained using bioassay-guided isolation because two compounds were commercially unavailable. The chemical structures of the isolated compounds were confirmed by 10H16O2, electron ionization mass spectrometry (EI-MS) m/z: 168, 139, 122, 108, 97, 81, 68, 55, 41. 1H NMR : δ 0.91 , 1.28–1.35 , 1.39–1.42 , 1.50–1.53 , 1.61–1.67 , 1.77–1.88 , 2.23–2.34 , 4.40–4.44 , 6.02 , 6.86–6.98 . 13C NMR : δ 13.98 (C-10), 22.50 (C-9), 24.61 (C-7), 29.32 (C-4), 31.64 (C-8), 34.83 (C-6), 78.04 (C-5), 121.44 (C-2), 145.06 (C-3), 164.64 (C-1).C10 massoia lactone C12H20O2, electron ionization mass spectrometry (EI-MS) m/z: 196, 178, 136, 111, 97, 81, 68, 55, 41. 1H NMR : δ 0.91 , 1.27–1.35 , 1.39–1.42 , 1.50–1.53 , 1.61–1.67 , 1.77–1.83 , 2.31–2.36 , 4.40–4.44 , 6.02 , 6.86–6.89 . 13C NMR : δ 13.98 (C-12), 22.62 (C-11), 24.81 (C-7), 29.12 (C-8), 29.32 (C-9), 29.47 (C-4), 31.87 (C-10), 34.84 (C-6), 78.34 (C-5), 121.45 (C-2), 145.04 (C-3), 164.63 (C-1).C12 massoia lactone CAe. albopictus of the constituents derived from massoia and nutmeg EOs are shown in Table The larvicidal activities against massoia PEs. It resulted from insufficient CNC content for emulsifying the oils. In contrast, 270 mg of CNC/mLmassoia or more formed a stable emulsion state without phase separation for 10 days  followed by C12 massoia lactone (17.4%), benzyl benzoate (13.4%), β-bisabolene (1.4%), linalool (0.9%), and borneol (0.7%). Our chemical analysis of massoia EO was similar to that of a previous study33. Composition rates of C10 and C12 massoia lactones, benzyl benzoate, and benzyl salicylate were 79.97%, 10.75%, 6.55%, and 1.13%, respectively, but linalool and borneol were not detected in this study. Twenty-five compounds were identified as constituents of nutmeg EO in a previous study34, with the most abundant compound α-pinene (22.0%) followed by β-pinene (21.5%), sabinene (15.4%), myristicin (9.4%), terpinen-4-ol (5.7%), limonene (3.9%), myrcene (1.9%), γ-terpinene (1.8%), and α-thujene (1.2%). The contributions of other constituents were less than 1%. Fourteen compounds were found in nutmeg EO, with sabinene (50.44%) the most abundant. Detectable levels of α-thujene, camphene, β-pinene, δ-3-carene, p-cymene, linalool, borneol, α-terpineol, geraniol, bornyl acetate, eugenol, α-terpinyl acetate, neryl acetate, and methyl myristate were reported in a previous study34 but not in this study. Isoeugenol and elemicin were identified in this study but not in a previous study34. Harvest date, cultivation area, storage period, climate, and extraction method affected EO’s constituents and their ratios36. A chiral column could identify the enantiomers of two compounds of nutmeg EO, α-pinene and limonene, in this study. The composition ratio of ( +)-limonene was higher than that of (-)-limonene, but vice versa in α-pinene.Rali et al.Ae. albopictus have been documented in previous studies42. Lee et al.40 reported that the LC50 values of ( +)-α-pinene, (-)-α-pinene, myrcene, and ( +)-terpinen-4-ol against Ae. albopictus were 55.65, 28.61, and 35.98 mg/L, respectively. However, the values for ( +)-α-pinene, (-)-α-pinene, myrcene, and ( +)-terpinen-4-ol were > 100 μg/mL in this study. The larvicidal activities of ( +)-limonene, α-terpinene, α-phellendrene, and γ-terpinene reported in previous study40 were slightly stronger than those in this study, but the larvicidal activity of terpinolene was slightly weaker than that of this study. bin Jantan et al.37 reported that the LC50 values of benzayl salicylate, benzyl benzoate, and safrole were 5.5, 6.5, and 28.0 μg/mL, respectively. Larvicidal activity of benzyl salicylate was weaker than that of this study, but larvicidal activities of benzyl benzoate and safrole were slightly stronger. A previous research effort37 and this study discovered different larvicidal activities associated with eugenol and methyleugenol. Larvicidal activity of eugenol was stronger than that of methyleugenol in a previous study37 but the relationship was reversed in this study. In a report by Seo et al.41 the insecticidal activities of myristicin and elemicin were similar to those of this study. The differences in larvicidal activities of EOs constituents against Ae. albopictus may be attributable to the methodological differences, as bin Janten et al.37 used fourth instar larvae for larvicidal tests and ethanol as a solvent for EOs, unlike the method of this study.Larvicidal effect of EO constituents against 33, and they possess an α-β-unsaturated δ-lactone moiety. Massoia lactone groups have been reported to show biological activities, including antimicrobial, cytotoxic, anti-inflammatory and phytotoxic activities44. However, larvicidal activity against Ae. albopictus has not been reported for C10 and C12 massoia lactones.C10 and C12 massoia lactones are reportedly the main constituents of massoia EOAe. albopictus. Previous studies indicated that the chain length of compounds with similar chemical structures can play an important role in insecticidal and nematicidal activity46. Hammond and Kubo45 evaluated the larvicidal activity of alkanols with C1-C20 chain lengths against the mosquito Culiseta incidens. They found that larvicidal activities of dodecanol, tridecanol, and undecanol, with chain lengths of 12, 13, and 11, respectively, were stronger than those of alkanols with other chain lengths. Seo et al.46 also reported that an optimal chain length of aliphatic compounds was necessary for nematicidal activity against the pine wood nematode Bursaphelenchus xylophilu. Nematicidal activities of aliphatic compounds with a C9-C11 chain length were stronger than those of other aliphatic compounds with other chain lengths. Larvicidal activities of eugenol and its derivatives, such as methyleugenol, isoeugenol, and methylisoeugenol, showed that the methoxy group and a double-bond position affected activity. Larvicidal activities of eugenol and methyl eugenol with an allyl group were stronger than those of isoeugenol and methylisoeugenol with a propenyl group, and larvicidal activities of methyl eugenol and methylisoeugenol with two methoxy groups at a benzene ring were stronger than those of eugenol and isoeugenol with one methoxy group. Bhardwaj et al.47 reported that larvicidal activity of methyl eugenol was stronger than that of eugenol against the tobacco armyworm, Spodopter litura. Methyleugenol exhibited strong contact toxicity against the cigarette beetle, L. serricorne compared with methylisoeugenol32.Differences in the chemical structure of C10 and C12 massoia lactones include the aliphatic chain length at the C6 position, and this may be responsible for the difference in larvicidal activity against 48, and are used widely in the pesticide industry20. However, conventional emulsions made with synthetic emulsifiers such as alkylphenol ethoxylates and organic-phase emulsifiers such as toluene and xylene have many drawbacks20, requiring the development of environmentally friendly emulsions, and PEs stabilized by solid particles are considered alternatives for conventional emulsions52. CNCs are appropriate solid particles for manufacturing PEs due to their high aspect ratio of crystalline fibrils and amphiphilicity53. In this study, CNC-stabilized PEs of massoia and nutmeg EOs were made and their physiological properties and larvicidal activity were investigated. The PEs exhibited visual differences depending on CNC content after storage for 24 h  for EOs varied according to the EOs. Particle-size study of CNC-based PEs have also produced valuable information to determine the appropriate concentration of EOs. The mean diameter of droplets and distribution of the sizes of emulsions at different contents of CNCs indicated that 270 mg of CNC/mL of EO was the most effective concentration for massoia EO. In the case of nutmeg EO, 180 mg and 225 mg of CNC/mL of EO had a smaller emulsion size and narrower distribution than the other PEs. Based on this result, 270 mg and 180 mg of CNC/mL for massoia and nutmeg EOs were finally chosen to evaluate the larvicidal activity, respectively.The insolubility of plant EOs in water requires emulsion-based formula for the practical use of plant EOs as larvicides against mosquitoes. Emulsions generally comprise small spherical droplets of two liquids stabilized with surfactants or surface-active polymers h Figs. A, 3A. Th41. However, further studies about proper dilution of the formulation, safety to non-target organisms, radiation and temperature effect after application and other factors such as costs are required for practical application of CNC-based PEs of massoia and nutmeg EOs.The results of this study demonstrate that CNC-based PEs of massoia and nutmeg EOs as promising larvicides which can be applied in field, since the larvicidal activies of CNC/massoia and CNC/nutmeg PEs were stronger than those of crude massoia and nutmeg EOs. Also, CNC-based PEs have showed stability for at least 10 days after the production, with improved solubility of massoia and nutmeg EOs in water. Another advantage of CNC-based PEs may be controlled release of EOs, as EOs and their constituents easily evaporate when treated in waterThe information about EOs used in this experiment are shown in Table Ae. aegypti54. Eicosane (99% purity), heptadecane (99%), isoeugenol (98%), ( +)-limonene (97%), methyl eugenol (99%), myrcene (95%), myristicin (99%), nonadecane (99%), nonane (99%), octadecane (99%), octane (98%), S-( −)-α-pinene (99%), safrole (97%), temephos (95.6%), and tridecane (99%) were purchased from Sigma-Aldrich , and eugenol (99%), α-terpinene (85%), γ-terpinene (97%), and terpinen-4-ol (97%) from Fluka . Benzyl benzoate (99%), benzyl salicylate (95%), docosane (99%), heneicosane (99.5%), heptacosane (97%), hexacosane (99%), ( −)-limonene (95%), methyl isoeugenol (98%), nonacosane (98%), octacosane (98%), pentacosane (99%), α-phellandrene (65%), R-( +)-α-Pinene (> 95%), tetracosane (99%), and tricosane (95%) were obtained from Tokyo Chemical Industry . Decane (99.5%), dodecane (99%), hexadecane (97%), pentadecane (97%), tetradecane (99%), and undecane (99%) were purchased from Wako . Elemicin (99%) was supplied by Santa Cruz Biotechnology . C10 and C12 massoia lactones were isolated from massoia EO. Silica gel was purchased from Merck .Chemicals used in chemical analysis of the EOs and the components of EOs used in larvicidal activity test are listed below. Eugenol and methyl eugenol were used in larvicidal activity test based on the structure–activity relationship of eugenol derivatives against Ae. albopictus were supplied from Korean Disease Control and Prevention Agency . They were reared in an insectary at 26 ± 1 °C and a relative humidity of 60 ± 5% under a 16:8 h light/dark cycle. We supplied the larva with a sterilized diet composed of 40-mesh chick chow powder and yeast (4:1). All procedures involving the use of animals were performed in compliance with the ARRIVE guidelines for animal studies. A live mouse was provided as a blood-meal source, using a method approved by the Institutional Animal Care and Use Committee .Cultures of Ae. albopictus were used for each treatment55. 1 mL of acetone and CNC-stabilized PE without EOs was used for negative control, and temephos was used as a positive control. Treated and control larvae were kept at the same condition for maintenance. Larval mortality was recorded 48 h post-treatment.Each EO or compound (50 mg) was dissolved in 5 mL of acetone  to make a stock solution. For each EO or compound solution, up to 6 more concentrations  were prepared by serial dilution. Likewise, 0.5 g of CNC-stabilized PEs of massoia and nutmeg EOs (100 mg/g) were dissolved in 5 mL of double distilled water  to make a stock solution. For each CNC-stabilized PEs solution, up to 3 more concentrations  were prepared by serial dilution. Each test solution (1 mL) was suspended in 99 mL of water in 6.5-oz paper cups. 10 early-third-instar larvae of 8-C29; DB-5MS, HP-INNOWax) under the same GC operating conditions56. The oven was programmed to be isothermal at 40 °C for 1 min then, heat to 250 °C at the rate of 6 °C/min and hold at this temperature for 4 min. Nitrogen was used as the carrier gas at a rate of 1 mL/min. To determine the configurations of limonene and α-pinene, a Beta DEX 120 chiral column   was used. The oven was programmed to be isothermal at 40 °C for 5 min then, heat to 230 °C at 6 °C/ min and hold at this temperature for 10 min. Nitrogen flowing at 1 mL/min was used as a carrier gas.An Agilent 7890 N  gas chromatograph (GC) with a flame ionization detector was used to analyze the chemical composition of massoia and nutmeg EOs. We used DB-5MS columns  and HP-INNOWAX columns   to measure the retention time and retention index (RI) of EO components. The RI of each peak was determined by calculating the relationship to a homologous series of n-alkanes   was used, and the flow rate of the carrier gas (helium) was 1.0 mL/min. The oven was programmed to be isothermal at 40 °C for 5 min then, heat to 250 °C at the rate of 6 °C/min and hold at this temperature for 5 min. Ionization was obtained by electron impact , and the scan range was 41 − 400 amu. Most EO components were identified by comparing the mass spectra of each peak with those of standard compounds obtained from the National Institute of Standards and Technology mass spectrometry library.2 gel using column chromatography with hexane/diethyl ether solution (100:0 → 0:100) as the mobile phase. The fractions were combined and the solvent was removed using rotary a evaporator at 35 ℃. The concentrates were then used for bioassays and further purification of the active compounds. Pure C10 massoia lactone and C12 massoia lactone were isolated from hexane/diethyl ether fractions of 70:30 and 93:7, respectively. 1H and 13C NMR spectra were obtained on a Bruker Avance 600 NMR spectrometer  using CDCl3 as the solvent at the National Instrumentation Center for Environmental Management, College of Agricultural and Life Science, Seoul National University .Commercially unavailable C10 and C12 massoia lactones were obtained from massoia EO via a bioassay-guided isolation process. C10 massoia lactone (1.8 g) and C12 massoia lactone (87 mg) were isolated from 9.9 g of massoia EO on SiO57. Finely cut filter paper was mixed with a sulfuric acid solution and stirred at 45 °C for 60 min. The hydrolyzed CNC solution was centrifuged with de-ionized water at 6,000 rpm for 15 min. The well-dispersed CNC solution was dialyzed for 7 days using a cellulose dialysis membrane .Sulfated CNCs were prepared from 10 g of cotton pulp  and 100 mL of 60% (w/w) sulfuric acid solution 58. Size distribution of the PE droplets was characterized using ImageJ. The CNCs were stained with Calcofluor white dye  while massoia and nutmeg EOs were stained with Nile red  for confocal laser scanning microscopy .Massoia and nutmeg EO PEs were mixed with the CNC solution at a 10% ratio and tip-sonicated at a 60% amplitude for 1 min using an ultrasonic processor . The CNC contents were fixed at 135, 180, 270, 360 and 450 mg per 1 mL of massoia EO and 45, 90, 135, 180 and 225 mg per 1 mL of nutmeg EO. Each PE solution was stored for 10 days at room temperature to allow for observation of phase separation of the solution, and the height of phase separation was measured using ImageJ software . The fraction of each emulsion was calculated by dividing the height of the creaming layer by the height of the total solution. The droplet shape and the size of the PEs were observed by a polarized light microscope  in dark-field mode. The first droplet size was measured after diluting 20 times, and the second droplet size was measured after diluting the solution diluted 20 times again 2 times (40 times)Mortality data were transformed to arcsine square root values for one-way analysis of variance (ANOVA). Treatment means were compared and separated by Scheffe’s test. All statistical analysis was carried out in IBM SPSS Statistics 26.0 (2019).Our results showed that CNC-stabilized PEs of massoia and nutmeg EOs were stable and dispersed easily in water. In addition, larvicidal activities of the CNC-stabilized PEs of massoia and nutmeg EOs were stronger than those of crude EOs. This indicates that a CNC-stabilized PE is a suitable formulation for EOs as a larvicide against mosquitoes in field applications.


A new series of non-aqueous phthalocyanines having 3,4,5-trimethoxy phenyl group at peripheral positions in which the central cavity possessing Cu(II), Zn(II), and without metals has been synthesized, and its absorption, fluorescence (steady-state and excited state lifetimes), electrochemical, and third-order nonlinear optical (NLO) properties were evaluated. Absorption studies data suggest that all three phthalocyanines obey Beer–Lambert’s law, and the redox properties indicate that both oxidation and reduction reactions are macrocyclic centered. The singlet quantum yields were measured in different solvents and were found to be in the range of 0.2–0.5 in the case of free-base, whereas it was in the range of 0.1–0.5 in zinc derivative, while the time-resolved fluorescence data revealed lifetimes of typically a few ns. The third-order NLO properties were investigated using the Z-scan technique with kilohertz (for retrieving true electronic nonlinearities) and megahertz repetition rate femtosecond pulses at 800 nm. Intensity-dependent Z-scan studies revealed robust NLO coefficients for solutions and thin films  of these molecules suggesting a strong potential for optical switching, imaging, and optical limiting applications. Furthermore, the phthalocyanine macrocycle has rich electrochemistry, and it involves both oxidation and reduction reactions. The incorporation of alkyl or alkoxy groups as well as metals in its central cavity not only affects its optical but also its redox reactions.In the current era of optoelectronics, nonlinear optics (NLO) and related technologies play a significant role in its evolution . To revi1 to T1 states) phthalocyanines exhibit strong RSA when excited with long (nanosecond) laser pulses. In this study, we designed, synthesized, and characterized a series of phthalocyanines possessing the 3,4,5-trimethoxy phenol group at their peripheral positions and Cu(II) or Zn(II) metals at their central cavities (see 2), nonlinear absorption/two-photon absorption coefficients (β), third-order nonlinear optical susceptibility [χ(3)], and two-photon absorption cross-sections [σ2 or σTPA] were extracted from the Z-scan data.Recent research trends have undeniably demonstrated the need for the development of optical limiters and nonlinear materials for the protection of sensitive optical devices and components from laser-induced damage . The stuMaterials: 4-Nitrophthalonitrile, 3,4,5-trimethoxyphenol, 1-pentanol, and 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU) were procured from Sigma Aldrich and used without any further purification. Copper acetate, zinc acetate, and potassium carbonate were purchased from Finar Ltd. and used as such. All the solvents, dichloromethane (DCM), tetrahydrofuran (THF), dimethylformamide (DMF), toluene, n-hexane, acetonitrile, and methanol, were purchased from SD Fine Chemicals Ltd. and were dried before use.3,4,5-Trimethoxyphenoxy phthalonitrile (1): 4-Nitrophthalonitrile  and 3,4,5-trimethoxyphenol  were taken in a clean dry round bottom flask. To this, potassium carbonate  and dimethylformamide (25 ml) were added and stirred for 24 h at 60°C under N2 atmosphere. The reaction mixture was poured onto ice and the obtained product was filtered off. The obtained solid was further purified by silica gel column chromatography by eluting with hexane-ethyl acetate (85:15 v/v) to get the desired compound. Anal. calcd for C17H14N2O4%(310.10): C, 65.80; H, 4.55; N, 9.03. Found C, 65.84; H, 4.52; N, 9.01. 1H NMR : 7.75 , 8.20–8.40 , 3.90 , 3.85 .Synthesis of TmPc: 3,4,5-Trimethoxyphenoxy phthalonitrile  was taken in a clean dry 25 ml round bottom flask in which 1-pentanol was added and stirred at 200°C. After 15 min, DBU was added in catalytic amount and continued to reflux for 8 h under nitrogen atmosphere. The reaction was cooled down to room temperature and precipitated with methanol, and washed with hexane. It was further purified with silica gel column chromatography using dichloromethane. The obtained product was dried and the yield was 40%. Anal. calcd for C72H62N4O16%: C, 69.78; H, 5.04; N, 4.52. Found C, 69.80; H, 5.02; N, 5.51. MALDI-TOF MS calcd m/z (C72H62N4O16) 1,239.30, found 1,241.41.Synthesis of ZnTmPc: 3,4,5-Trimethoxyphenoxy phthalonitrile  and zinc acetate  were taken in a clean dry 25 ml round bottom flask in which 1-pentanol was added and stirred at 200°C. After 15 min, DBU was added in catalytic amount and continued to reflux for 6 h under nitrogen atmosphere. The reaction was cooled down to room temperature and precipitated with methanol, and washed with hexane. It was further purified with silica gel column chromatography using dichloromethane. The obtained product was dried and the yield was 45%. Anal. calcd for C72H60N4O16Zn %: C, 66.39; H, 4.64; N, 4.30. Found C, 66.40; H, 4.02; N, 4.31. MALDI-TOF MS calcd m/z (C72H60N4O16Zn) 1,302.66, found 1,304.49.Synthesis of CuTmPc: Similar procedure was followed as of Zn-TmPc; copper acetate was used instead of zinc acetate, and the yield was about 35%. Anal. calcd for C72H60N4O16Cu %: C, 66.48; H, 4.65; N, 4.31. Found C, 66.46; H, 4.62; N, 4.31. MALDI-TOF MS calcd m/z (C72H60N4O16Cu) 1,300.83, found 1,304.49.Thin films: The thin films were prepared by coating 2 mM concentration solution of Pc dissolved in DCM on a glass plate. Glass plates of size 2.5 × 2.5 mm each were cut and washed with water and acetone under sonication and dried at 100°C. A dried glass plate was inserted on the base of the spin coater and 0.1 ml of solution was added using a micropipette onto the glass plate to obtain the thin film at a speed of 2000 rpm. Later, the glass plate was dried in a hot furnace at 60°C to obtain the thin films. The thickness was monitored using a profilometer, and the obtained values were in the range of 47–56 µm. See (3)] in an ultrafast excitation regime. Now, the input excitation beam being a Gaussian profile, the normalized transmittance can be mathematically followed up properly by bringing in the multi-photon absorption theory. In an open and closed aperture configuration, the normalized transmittance turns out to be attending the mathematical expressions as per the following equations. The generic multi-photon absorption-influenced transmittance nature . Matrix-assisted laser desorption ionization time-of-flight (MALDI–TOF) mass spectrometry measurements were performed on Shimadzu Biotech AximaPerformance 2.9.3.20110624: Mode was Reflectron-Hi Res, Power: 85 using TMS as standard. FT-IR spectra (with KBr pellets) were recorded on a Bruker spectrometer.All the 2 values and the residual distribution.A UV-Visible-NIR spectrophotometer (Shimadzu UV-3600) was used to record the absorption spectra of the title compounds. A spectrofluorometer  was utilized to obtain the steady-state fluorescence spectra of solutions. The optical density at the excitation wavelength (λex) was ∼0.06. Zinc tert-butyl phthalocyanine (=0.37 in benzene) was used as a reference to calculate the fluorescence quantum yields (ϕ) by integrating their fluorescence bands . PicosecElectrochemical measurements were performed on a potentiostat that was computer controlled . The experiments were performed in solutions of respective phthalocyanines in DCM (using 1 mM concentration) and at a scan rate of 100 mV/s using 0.1 M tetrabutyl ammonium perchlorate as the supporting electrolyte. The working electrode was glassy carbon, and a standard calomel electrode was the reference electrode while a platinum wire was used as an auxiliary electrode. After cyclic voltammograms (CVs) have been recorded, ferrocene was added, and a second voltammogram was measured.By using the Gaussian 09 package, loaded on a personal computer, all the theoretical analyses were performed (following 1) was synthesized by the condensation of 3,4,5-trimethoxyphenol with 4-hydroxy phthalonitrile using K2CO3 in DMF. The cyclo tetramerization of 1 was achieved using DMF and pentanol method to get the free-base phthalocyanine (TmPc) and the corresponding metallo derivatives were achieved by using DBU, pentanol with respective metal acetates. All three phthalocyanines are characterized by various spectroscopic and electrochemical methods. The results obtained from the elemental analysis of all three compounds were found to be satisfactory. The presence of molecular ion peak in MALDI-Ms spectra of each compound confirmed the respective phthalocyanine  and logarithmic value of the molar extinction coefficients (log ε) are presented in CuTmPc and ZnTmPc that lead to further minimization of aggregation  from the Gaussian 09 package. The optimized structures of all three phthalocyanines obtained are displayed in TMPc, Cu-TMPc, Zn-TMPc) were synthesized in the form of macromolecules utilizing the matrix-assisted laser desorption ionization (MALDI) technique and extensive nonlinear optical measurements were carried out using the same.Promising novel organic molecules, possessing significant nonlinear optical (NLO) characteristics, are in high demand for various device applications in the fields of photonics, biomedical imaging, optical communications, etc., . All the−1), 2) two-photon absorption cross section σTPA (GM), 3) nonlinear refractive index n2 (cm2.W−1), and 4) nonlinear optical susceptibility index [χ(3)] by fitting the experimental data to mathematical/theoretical expressions. All the phthalocyanine molecules have exhibited purely two-photon absorbed reverse saturation absorption (RSA) nature in open aperture configuration while interacting with 80 MHz, 150 fs pulses at 800 nm. Intensity-dependent study has been carried out for all the samples, which clearly revealed an increase in two-photon absorption coefficient (β) with an increase in intensity order gradually. The RSA nature of the open aperture curves signifies the clear downfall in output transmittance, after the sample, with fine increase in intensity while approaching the focal point, clarifying the occurrence of nonlinear absorption. Whereas, in closed aperture mode, prominent peak-valley nature has been observed for all three phthalocyanines, which stands for a defocusing Kerr lens effect taking place due to high intensity dependence.To begin with, solution forms of these novel phthalocyanines were probed with MHz femtosecond pulses (at 800 nm) and the light-matter nonlinear interaction  were stuTMPc, Cu-TMPc, Zn-TMPc. The peak intensity has been varied from 8 MW/cm2 to 16 MW/cm2 and finally up to 24 MW/cm2, and we measured the corresponding normalized transmittance with the scanning position on and around the Rayleigh range. The estimated two–photon absorption coefficient (β) values for TMPc have been found increasing from (3.8–42) × 10−6 cm.W−1. Similarly, the β values corresponding to Cu-TMPc molecules have been found escalating from (4-11) × 10−6 cm.W−1, and the same for Zn-TMPc phthalocyanine has been observed to be in the range of (4.2–140) × 10−6 cm.W−1. Zn-TMPc has clearly shown significantly superior nonlinearity, making it a potential candidate for photonic applications. The two-photon absorption cross-sections (σ2) have also been calculated to be found in the order of ∼108 GM. From 2) values as of 3.6 × 10−11, 3.5 × 10−11, 5.7 × 10−11 cm2/W for TMPc, Cu-TMPc, Zn-TMPc according to the order. Overall, the nonlinear optical susceptibility [χ(3)] has also been determined to be in the order of ∼10−10e.s.u. All the detailed estimations of the NLO parameters have been tabulated in TMPc and Zn-TMPc, the study has revealed an exotic “M-shaped” open aperture Z-scan data, displaying combined nature of reverse saturation absorption (RSA) and saturation absorption (SA). For initial intensities, the samples depicted SA and for higher peak intensities it switched to RSA. Occurrence of SA shifting/switching to RSA phenomenon has been noticed in several works recently (s) where we observed this “M-shaped” behavior. In the case of Cu-TMPc, pure RSA nature has been observed, with decrease in transmission with increasing peak intensities. This kind of behavior was not observed with MHz fs pulses at 800 nm and this could possibly be explained based on the spectral bandwidth of both the pulses used. In the kHz case, the pulses were broad enough with a typical FWHM of >25 nm and an overall spectral width of >50 nm whereas in the case of MHz pulses, they had a FWHM of 8-10 nm with an overall spectral width of 25 nm. These molecules depicted small absorption close to 800 nm and the kHz pulses were able to access this small absorption and this resulted in saturation at initial (low) peak intensities. For higher peak intensities, the SA was overcome by the RSA as we have observed. These promising properties make these compounds potential candidates for optical switching applications. In the case of closed aperture Z-scan data, all three phthalocyanines have illustrated a focusing Kerr lens effect, revealing a downfall in the transmittance followed by a rise in it.High repetition rate fs, MHz pulses generally have contribution from thermal nonlinearity, leading to higher NLO coefficients of the system extracted from Z-scan data. To access the actual/electronic nonlinearity, we have performed the same measurements with the already mentioned solutions utilizing 50 fs, 1 kHz pulses of 800 nm single beam. For, recently . With deTMPc, Cu-TMPc, Zn-TMPc, respectively, with a gradual variation in peak power/intensity. The peak intensities where the measurement has been performed were within the range of 400–600 GW/cm2. In the case of kHz pulses, peak intensities turned out to be much higher compared to MHz pulses but we were sure of probing only the electronic nonlinearity because of the repetition rate. At the lowest peak intensity, two-photon absorption coefficients (β) value for TMPc, Cu-TMPc, Zn-TMPc revealed themselves to be 2.3 × 10−10 cm.W−1, 14 × 10−10 cm.W−1, and 1.1 × 10−10 cm.W−1 and the corresponding saturation intensity (Is) turned out to be 6 × 109 W/cm2, 1 × 109 W/cm2 for sample without central element (TMPc) and “Zn” central element incorporated phthalocyanine, respectively. In the case of pure RSA nature for Cu-TMPc molecules, the β values have been found to be in the range of (1-5)×10−10 cm.W−1. In this study also, Zn-TMPc has demonstrated superior (nonlinear) two-photon absorption compared to the other two molecules. Evidently, TmPc and Zn-TmPc data conspicuously unveiled a decreasing nature in their TPA coefficient with increasing input power of the laser excitation, we believe that the reason behind this is the inherent occurrence of stronger bleaching with growing laser excitation intensity, leading to prominence in enhanced transparency, i.e., saturation absorption (SA). Whereas, in the case of Cu-TmPc, the reason behind upsurge in the TPA coefficient with intensity can possibly be attributed to the near resonant excitation circumstances (800 nm laser excitation and two-photon states above 400 nm being available as evident from the absorption spectra). It is pertinent to note that in the case of TmPc and Zn-TMPc, an additional parameter of Is was used in the fittings along with β.2) has been extracted and found to be ∼103 GM. 2 values to be 4.9 × 10−16, 7.1 × 10−16, 4.7 × 10−16 cm2/W for without any component, and Cu, Zn incorporated central cavity phthalocyanines, respectively. The total χ(3) has been calculated of the order of 10−14 e.s.u., Zn-TMPc having the highest value of 20.9 × 10−14e.s.u. The NLO absorption cross sections  have been determined as 0.9 × 107 GM, 0.7 × 107 GM, and 1.5 × 107 GM. In 2 from the theoretical fits to experimental data turned out to be 1.85 × 10−11 cm2.W−1, 1.1 × 10−11 cm2.W−1, and 2.6 × 10−11 cm2.W−1 according to the same order of molecules. Finally, the order of magnitude of NLO susceptibilities [χ(3)] have been found to be ∼10−10e.s.u. The nonlinear absorption coefficients in thin films were an order of magnitude lower than in solution, while the n2 values were found to be similar in magnitude. This could possibly be attributed to the change in absorption spectra in thin films. In 2, due to high repetition rate inducing thermal nonlinearities, whereas the kHz studies depicted valley-peak structure or positive n2 in all the molecules . The reason behind the ascending order of magnitude of n2 for Cu-TMPc, TMPc, and Zn-TMPc could arise from the respective electronic distribution among the excited states or their orientation when the intense light pulse is incident. The absorption cross sections, imaginary as well as the real parts of the NLO susceptibility, are the fundamental parameters for any novel third-order NLO material developed, and these have given a promising clarity to reach out for a decisive conclusion that Zn-TMPc has turned out to be a potential compound by exhibiting significantly superior optical nonlinearity in comparison with Cu-TMPc and TMPc. Another crucial nature has got revealed corresponding to the Zn-TMPc and TMPc molecule, from their fine exotic “M curve nature”, as it stands for a tunable/shifting NLO absorption behavior depending on variable peak intensities. In regard of an unwavering structural-nonlinear property observation, usually revealed out from the complete study, “Zn” incorporation in the central cavity of tri-methoxy-phenoxy-phthalocyanine, has seemed to open up a possibility of much superior optical nonlinearity.To completely understand and confirm the origin of these fine optical nonlinearities, in each case, the NLO contribution from the corresponding solvents and/or base material (bare glass slide in thin films case) have been verified sincerely by recording their Z-scan data and they have been found to possess negligible magnitude  was prepared and the NLO properties were investigated using ∼280 fs pulses 2NO2, H2TPP(TPA)2CHO, ZnTPP(TPA)2NO2, and ZnTPP(TPA)2CHO]. The obtained β values were ∼10−8 cm/W, σTPA values of ∼105 GM, n2 values of ∼10−13 cm2/W, and χ(3) values of ∼10−11e.s.u. for those molecules. The coefficients retrieved in our present case . Another work reported 3] and phosphorus [P-(OH)2(C6F5)3] corroles. These data clearly indicate that the present molecules have strong NLO coefficients and potential for photonic applications .Our group has recently reported the NLO properties of two unsymmetrically substituted novel porphycenes, i.e., 2,7,12,13-tetraethyl-9,10,19,20-tetramethyl(OAPo-T) and 2,3,16,17-tetraethyl-9,10,19,20-tetramethyl (OAPo-C) porphycenes with TPA cross-sections of 159 and 145 GM, respectively, obtained with exact experimental conditions  used in the present study. Similarly, one of our earlier works  reportedcase see  are at lcase see  of ImCuPreported  the fs, me group  again re• Strong TPA coefficients and cross-sections . These coefficients were found to be on par or superior to some of the recently reported other phthalocyanine moieties.• A complicated nonlinear absorption behavior observed with kHz pulse excitation. This has been explained using the absorption profile and spectral bandwidths of the pulses used.• Thin film NLO coefficients were also evaluated because for practical applications, these are imperative.As concluding remarks, we state that we have synthesized a new series of non-aqueous phthalocyanines possessing the 3,4,5-trimethoxyphenyl group at peripheral positions. A thorough NLO investigation with femtosecond MHz and kHz pulses has been carried out, successfully probing into the molecules’ NLO coefficients to figure out their ultrafast interaction response nature. Significant achievements have been obtained in the NLO domain with both MHz and kHz pulse excitations. Some of the highlights are as follows:After extensive analysis, promising revelations have been clarified about optical nonlinearity being in deterministic relation molecular structural as well as central cavity components of the corresponding phthalocyanine molecules. This may definitely be an advantage in synthesizing novel phthalocyanine molecules according to desirable and tunable optical nonlinearity.


Eruca sativa Thell. In the present study, we explored the anti-cancer effect of 4-MTBITC against 7,12-dimethylbenz [a] anthracene (DMBA) induced breast cancer. Hypoxic conditions were developed using a single dose of 60 mg/kg DMBA. Hepatic and renal parameters were increased along with antioxidants in cancer-bearing rats which were lowered with the treatment of 4-MTBITC. Further, it inhibited the up-regulation of glycolytic enzymes caused by DMBA. The hypoxia pathway was evaluated using RT-PCR and it was found that the 40 mg/kg doses of 4-MTBITC statistically lowered the expression of HIF-1α. Akt/mTOR signaling pathway was one of the major pathways involved in 4-MTBITC-induced cell growth arrest by western blotting. Amino acid profiling serum-free plasma revealed the downregulation of specific amino acids required for vital components of fast-growing cancer cells. 4-MTBITC reduced the levels of serine, arginine, alanine, asparagines, and glutamic acid. Histological examination also showed neoplastic growth following DMBA doses. 4-MTBITC treated rats showed less infiltration and normal physiology. Our findings for the first time demonstrated the potential therapeutic significance of 4-MTBITC on modulation of glycolytic enzymes and hypoxia pathway in female rats.4-(methylthio)butyl isothiocyanate (4-MTBITC) is a hydrolytic product from the plant  Cancer cells exhibit high aerobic glycolytic rates and produce high levels of lactate and pyruvate (Warburg effect). Lactate and pyruvate regulate hypoxia-inducible gene expression by stimulating the accumulation of HIF-1α (Many factors such as glycolytic enzymes and hypoxia-inducible factor-1α (HIF-1α) are known to be responsible for causing cancer. Increased glycolysis has been known as an indicator of cancer . Cancer f HIF-1α . HIF-1α f HIF-1α . HIF-1α f HIF-1α . Clinicaf HIF-1α . HIF-1α f HIF-1α .The functional gain of oncogenes and loss of function of tumor suppressor genes are the main characteristic of cancer cells . This ca7,12-dimethylbenz [a]anthracene (DMBA) is commonly employed as a model of polycyclic aromatic hydrocarbons (PAHs)–induced breast carcinogenesis due to its powerful carcinogenic and immunosuppressive effects . It causUnderstanding therapy resistance and successful treatment require identifying oncogenes and their related potential pathways. Through receptor tyrosine kinases, activated PI3K phosphorylates PIP2 to PIP3 . PhosphoEruca sativa Thell. also known as rocket salad contains a high amount of 4-(methylthio)butyl isothiocyanate (4-MTBITC)  . Chemopr-MTBITC) . Followi studies . HoweverIn the present study, we sought to demonstrate if the 4-MTBITC could act as a novel anti-tumor agent by altering the glycolytic pathway and hypoxia-regulating genes in breast cancer-bearing rats.Eruca sativa using the method described by 4-MTBITC was isolated from the seeds of plant The rats were housed in the Central Animal Facility under standard animal husbandry conditions. The experimental protocol was approved by the Animal Ethical Committee of Guru Nanak Dev University (GNDU), Amritsar. The rules and regulations of control and supervision of experimental animals (CPCSEA) were followed according to the Ministry of Environment and Forests (File No. 226/CPCSEA/2014/17), Government of India. All experiments were performed following ethical standards.3 the animals were divided in experimental groups as follows: group A , group B (breast cancer-induced rats), group C , group D . 4-MTBITC was gavaged for total 15 days in 1 month. 40 mg/kg dose was selected from acute and subchronic toxicity studies and duration of repeated dose was selected from pharmacokinetic studies. Rats were palpated every day for mammary tumors. The quantitative measurements were done using caliper scale and equation: π/6(a)2*(b). Throughout the treatment, animals were observed for morbidity or mortality. The body weight of the animals was recorded before the start of the experiment, weekly during the experiment, and at the end of the experiment.Twenty-four female Sprague-Dawley rats were obtained from the National Institute of Pharmaceutical Education and Research, Mohali, Punjab. After 2 weeks of acclimatization, rats were divided randomly into four groups (n = 6). Initially twelve rats were administered with a single 60 mg/kg dose of DMBA, a dose sufficient to make 100% tumor incidence in the control group . After 9Blood samples were collected on the last day of the studies from the retro-orbital plexus and centrifuged at 3,000 rpm for 15 min. The serum was stored at -80°C for further analysis. Serum aliquots were used to estimate the different biochemical parameters like serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), direct bilirubin, total bilirubin, creatinine, triglycerides, and cholesterol using readymade ERBA kits.The weighed samples of liver were homogenized in ice-cold Tris-HCl buffer (pH 8.0) for 5 min and centrifuged at 8,000 rpm for 15 min at 4°C. The supernatant was stored at -80°C for enzyme analysis. Superoxide dismutase (SOD) was measured according to the method of Total RNA was isolated using TRIzol (Thermo Fisher). Briefly, 100 mg frozen tissue was weighed and minced in pestle mortar using liquid nitrogen. Instantly, TRIzol was added and centrifuged. Chloroform was used to induce phase separation. Isopropanol was used to precipitate the RNA. Isolated RNA was washed thrice with 70% ethanol. DNA was removed using DNase treatment. The extracted RNA was quantified and qualified using a NanoDrop spectrophotometer. Finally, total RNA was normalized to avoid false changes in gene expression. Using Luna One-Step RT-qPCR (New England BioLabs), the gene expression of hexokinase, phosphoglucose isomerase, aldolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate isomerase, phosphoglycerate mutase, enolase, pyruvate kinase, HIF-1α, HIF-1β, prolyl hydroxylase (PHD), mTOR, phosphatase and tensin homolog (PTEN), Heat shock protein-90α (HSP-90α), and succinate dehydrogenase was recorded. Primer sequences are enlisted in + according to the protocol of King . Approximately 40 μg protein was loaded on each well of polyacrylamide gel and separated by SDS-PAGE. The resolved proteins were transferred to the polyvinylidene difluoride membrane (0.2 μm) by electrophoresis. Then the membranes were blocked by 5% milk powder in 50 mM Tris-HCL, 150 mM NaCl, and 0.1% Tween 20 at room temperature for 1 h. The membrane was then immunoblotted for overnight at 4°C with either a rabbit monoclonal anti-HIF-1α , a rabbit polyclonal anti-Akt , a rabbit polyclonal anti-mTOR , a rabbit polyclonal anti-TNF-α (Tumor necrosis factor-α) , p21 , a rabbit monoclonal anti-PHD-2/Egln1 , a rabbit monoclonal anti-p53 , a rabbit monoclonal anti-NF-kB-p65 (Nuclear factor-kB)  or a rabbit polyclonal anti-β-Actin  diluted at 1/1,000–1/10,000 in TBS tween 0.1%. Subsequently, membranes were washed thrice with 50 mM phosphate-buffered saline and 0.1% tween-20. Following incubation with horseradish peroxidase-conjugated anti-rat secondary antibody diluted at 1/1,000 for 1 h at room temperature. The blots were developed using ECL chemilluminicance substrate solution. Autoradiographic signals were captured on a Genegenious imaging system using the Genesnap software.For the estimation of adiponectin, leptin, insulin, and IL6, fresh plasma was used. Respective rat ELISA kits  were used according to the manufacturer’s instructions. 96 well Elisa plate was read on a multimode microplate reader .Samples (liver) were prepared by digesting tissue homogenates in 6 N HCl along with 0.1% phenol in tightly closed 25 ml reagent bottles at 110°C for 24 h. Further, the samples were dried using a vacuum evaporator (Buchi) and reconstituted in 0.1 mol/L HCl solution for analysis. A total of 19 amino acid standards were analyzed including aspartic acid, glutamic acid, asparagines, serine, histidine, glutamine, glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, and lysine and their linearity of response across six-point serial dilution was plotted.The HPLC-based amino acid analyzer consisted of Nexera X2, Shimadzu coupled with Shimadzu RF-20A prominence fluorescence detector was used. The HPLC separation of derivatized amino acids required the two mobile phases. Mobile phase A consists of 20 mmol/L potassium phosphate buffer (pH 6.5) and mobile phase B consists of 45:40:15 acetonitrile:methanol: water. All buffers were filtered through 0.2 µm filtered and degassed by sonication. The derivatized amino acids were separated on a Phenomenex Luna C18 column . The chromatographic separation was performed at 35°C. Fluorometric measurement was conducted with an excitement wavelength of 350 nm and an emission wavelength of 450 nm and a gain at 100. The flow rate of the mobile phase was 0.95 ml/min throughout the analysis and sample injection volume for derivatization was 1 µL. The gradient conditions were as follows: initial conditions are 2% mobile phase A; from time 0.01–2 min the gradient changes to 90% solvent A and 10% solvent B; from 2 to 33.40 min the gradient changes to 43% solvent A and 51% solvent B; from 33.40 to 33.50 min the gradient changes to 100% solvent A and 0% solvent B; from 33.50 to 39.30 min the gradient remains same as 100% solvent A and 0% solvent B; from 39.30 to 39.40 min the gradient changes to 90% solvent A and 10% solvent B; from 39.4 to 45 min the gradient was maintained to 90% solvent A and 10% solvent B.O-phthalaldehyde (OPA) was prepared by dissolving 10 mg of OPA in 0.3 ml methanol and mixed completely by sonication. Then 0.7 ml borate buffer and 4 ml HPLC grade water were added to make a working solution. Mercapto propionic acid (MPA) was prepared by adding 10 µL in 10 ml of 0.1 mol/L borate buffer. 9-fluorenylmethyl chloroformate (FMOC) was prepared by adding 4 mg into 20 ml of acetonitrile. The derivatization of amino acids in the samples and standards was performed in an automated fashion using a Nexera SIL-30AC autosampler kept at 15°C. The needle of the autosampler was rinsed with 50% methanol/water between injections. During chromatography of the sample, the derivatization of the next sample is started so that there is no delay in the injection of the next sample.The HPLC run time for the separation of the derivatized amino acids in the sample or standard is 50 min. The amino acid derivatization reagents were prepared freshly each day. At the time of scheduled euthanasia, all animals underwent a full gross necropsy examination. Mammary tissue and tumor masses were preserved in 10% neutral buffered formalin (NBF) for each rat. All collected tissues were examined for histopathological changes with hematoxylin and eosin (H&E) staining after full fixation in 10% NBF. The tissues were trimmed, embedded in paraffin, sectioned, mounted on microscope slides, and stained with hematoxylin and eosin. The paraffin-embedded blocks were sectioned 3 μm thin and stained with H&E.p < 0.05 level.Data were analyzed statistically using a one-way analysis of variance (ANOVA) in GraphPad Prism 8. All results are presented as mean ± SEM from at least three independent experiments. Dunnett’s multiple comparison test was used to compare multiple groups. For amino acid analysis ANOVA with sidak multiple comparison test was used. A difference was considered significant at the p < 0.01), SGPT (87% at p < 0.01), direct bilirubin (133% at p < 0.01), total bilirubin (58% at p < 0.01)), renal (creatinine (71% at p < 0.01)), and lipid profile (triglyceride (63% at p < 0.01), cholesterol (115% at p < 0.01)) were significantly enhanced in rats treated with DMBA (group B) compared to control group. In group D the levels of SGOT were brought down by 59% (p < 0.01), SGPT by 30% (p < 0.01), direct bilirubin by 29% (p < 0.01), total bilirubin by 26% (p < 0.01), creatinine by 27% (p < 0.01), triglyceride by 36% (p < 0.01) and cholesterol by 30% (p < 0.01) compared to group B  and levels of reduced glutathione by 17% (p < 0.01) compared to normal rats. On the other hand, in group D the activity of SOD was increased by 112% (p < 0.01) and the levels of glutathione were increased by 112% (p < 0.01) compared to group B. DMBA treatment markedly increased the levels of MDA (122% at p < 0.01) and hydroxyl levels (73% at p < 0.01) in group B compared to group A. Oral doses of 4-MTBITC reduced the levels of MDA by 31% (p < 0.01) and hydroxyl radicals by 30% (p < 0.01) in group D compared to group A.p < 0.001), phosphoglucose isomerase (380% at p < 0.001), phosphofructokinase (76% at p < 0.01), aldolase (143% at p < 0.001), triosephosphate isomerase (119% at p < 0.05), GAPDH (219% at p < 0.001), phosphoglycerate kinase (57% at p < 0.02), phosphoglycerate mutase (102% at p < 0.01), enolase (60% at p < 0.01), and pyruvate kinase (897% at p < 0.001) was significantly increased after the treatment of DMBA as compared to control. The counter-treatment of 4-MTBITC significantly decreased the gene expression of hexokinase (59% at p < 0.01), phosphoglucose isomerase (66% at p < 0.01), aldolase (54% at p < 0.001), GAPDH (45% at p < 0.01), phosphoglycerate mutase (79% at p < 0.001), enolase (45% at p < 0.01) and pyruvate kinase (64% at p < 0.001) as compared to group B. 4-MTBITC treatment in group D non-significantly decreased the percent change in gene expression of phosphofructokinase (26%), triosephosphate isomerase (29%) and phosphoglycerate kinase (11%) as compared to group B.p < 0.001) and mTOR  and downregulation in PHD (79% at p < 0.01) was observed after the treatment of DMBA in group B as compared to group A while an insignificant increase in HIF-1β (60%) and HSP-90α (39%) and decrease in PTEN (44%) was observed. Following the treatment of 4-MTBITC in group D the upregulated expression of HIF-1α (86% at p < 0.001), and mTOR (5% at p < 0.001) was lowered while PHD (443% at p < 0.05) was upregulated  and 6-PGD (138% at p < 0.001) was increased in DMBA treated rats as compared to group A. Following the treatment of 4-MTBITC in group D, the activity of lactate dehydrogenase was decreased by 59% at p < 0.001 and 6-PGD by 48% at p < 0.001 as compared to group B (p < 0.05) in group B as compared to group A  under hypoxic conditions of group B compared to group A (p < 0.05) was observed in group D as compared to group B following the treatment of 4-MTBITC. Furthermore, the lactate content was quantified in all the experimental animals and it was found that the DMBA increased the lactate levels (214% at p < 0.001) which were then significantly reduced (27% at p < 0.001) by 4-MTBITC in group D as compared to group B (The activity of lactate dehydrogenase (145% at  group B . Succina group A . 4-MTBIT group A . A signi group B .p < 0.01) in serum leptin and a decrease in adiponectin levels (44% at p < 0.01) were observed in group B compared to vehicle control rats (p < 0.05) and adiponectin (52% at p < 0.01) levels compared to group B. It was observed that the levels of insulin were significantly increased (94% at p < 0.01) in cancer-bearing rats as compared to control (p < 0.05) the insulin significantly from group B. IL-6 a pleiotropic cytokine related to inflammation was seen significantly enhanced (241% at p < 0.01) in group B as compared to the control group (p < 0.01) the IL-6 levels significantly as compared to group B.Adiponectin is known for its overall energy homeostasis and metabolism. Significant elevation (200% at rol rats . 4-MTBIT control . Furtherol group . 4-MTBITp < 0.001), arginine (37% at p < 0.05), alanine (81% at p < 0.001), asparagine (43% at p < 0.01) and glutamic acid (145% at p < 0.001) were increased in animals bearing cancer (group B). Treatment of 4-MTBITC in group D decreased the levels of serine by 31% (p < 0.05), arginine by 54% (p < 0.001), alanine by 43% (p < 0.001), asparagines by 26% (p < 0.05) and glutamic acid by 24% (p < 0.05) as compared to group B (p < 0.01) were decreased in response to a carcinogen (group B) and increased by 4-MTBITC (53% at p < 0.05).The mean normalized amino acid concentrations of all the groups were investigated . The ave group B . On the The protein expression of Akt, mTOR, TNF-α, p21, PHD, p53, and NF-kB was evaluated using western blot analysis with normalization against Actin . DMBA inMammary tissue was sliced and washed with ice-cold PBS. Examination of group A showed dense collagenous tissue, blood vessels, and islands of glandular tissue surrounded by dense fibrous and adipose tissue . Adminis−1) from blood plasma and hence in the current study, the frequency of dose was lowered to reduce such side effects (Unpublished data).Chemoprevention is the use of plant, synthetic, or biological chemicals to prevent, kill, or reverse the progression of premalignant cells from their initial stages to invasive disease. Mammary tumor induction by DMBA is one of the most popular methods for researching different aspects of breast cancer among various animal models . The preTumor formation, development, angiogenesis, and metastasis are also thought to be influenced by inflammation. The main molecular players in the inflammation-to-cancer axis are proinflammatory mediators such as TNF-α and IL-6. TNF-α, an inflammatory cytokine that is strongly expressed in breast carcinomas, was reduced by 4-MTBITC. The inducible transcription factor Akt, mTOR, p21, and p53 regulates the expression of several genes involved in inflammation, proliferation, and apoptosis. 4-MTBITC inhibited the expression of genes NF-kB, and IL-6, which control the immune response to infection. It also inhibited the expression of cell proliferative markers such as Akt, mTOR, p21, and p53.A high level of plasma leptin and a low level of adiponectin was observed following the DMBA administration. Adiponectin represses proliferation in breast cancer cells through inactivation of p44/42 MAPK protein 1 and 3 expressions, a stimulation of AMPK activity by phosphorylation at Thr172, and a decrease in Akt phosphorylation (Thr308) associated with an increased expression of LKB1 leading to a reduction of mTOR activity as evidenced by reduced phosphorylation of S6K . Leptin There are multiple mechanisms by which HIF-1 mediates adaptive metabolic responses to hypoxia, such as increased glycolytic flux and decreased TCA flux, to minimize mitochondrial ROS production. HIF-1α a prime regulator of cell signaling in the absence of oxygen was upregulated in cancer-bearing rats. PHD an important gene that regulated the expression of genes involved in the hypoxia pathway was seen downregulated in cancer-bearing rats. In normal oxygen levels, glucose converts to pyruvate and enters the TCA cycle for the synthesis of ATP. As the oxygen level lowers the glucose converted to pyruvate will not enter the TCA cycle moreover lactate will be produced to lower the free radical formation from the electron transport chain. This lactate will serve as an instant energy source for neighboring cells. Lactic acid produced from lactate helps cancer cells to dissolve the extracellular matrix and finally metastasize to other organs. HIF-1α represses succinate dehydrogenase an important TCA enzyme that can limit the content of mitochondrial oxaloacetate required for aspartate biogenesis .Gluconeogenesis is the reverse of glycolysis through which glucose can be generated from a non-carbohydrate source. FBPase is a rate-limiting key enzyme that converts fructose-1,6-bisphosphate to fructose-6-phosphate. Loss of FBPase contributes to the development, promotion, and progression of multiple cancers, including basal-like breast cancer, whereas FBPase functions as a tumor suppressor by increasing oxidative phosphorylation and ROS levels, which are harmful to cancer cells . The actPPP pathway is critical for oxidative stress and the synthesis of nucleotides in the metabolism of breast cancer cells. 6PGD is a central enzyme in PPP’s oxidative branch that is active in nucleotide biosynthesis and redox status maintenance. DMBA induced the overactivation of this enzyme to withstand the increased requirement of nucleotides for proliferative cells which then reduced following the 4-MTBITC.Amino acids are readily measured in blood, saliva, and urine, they may be useful biomarkers for diagnosis and screening during treatment. Serine is synthesized from glycolysis intermediate 3-phosphoglycerate through several enzymatic reactions. Serine is also involved in the production of antioxidants, which help cancer cells survive in low-oxygen conditions. The removal of serine causes breast cancer cells to proliferate less, an effect that can be entirely reversed by reintroducing it. Out of other amino acids, glutamate is the one that is consumed by cancer cells at the highest rate. Glutamic acid is the precursor of glutamine converted by glutaminase synthetase.l-glutamine, a derivative of l-glutamic acid. Glutamine participates in the PPP pathway to withstand the high requirement of nucleotides by synthesizing purine and pyrimidines. It also transports to the mitochondrial matrix where glutaminase converts it into glutamate. This glutamate participates in the TCA cycle through conversion to α-ketoglutarate. Under hypoxic conditions, α-ketoglutarate can undergo reductive carboxylation to generate isocitrate which converts to citrate (Blockage of arginine uptake results in apoptotic cell death in cancer cells . Nitric  citrate . Citrate citrate . In the Based on these, we speculated that the 4-MTBITC modulate DMBA induced glycolytic pathway and hypoxia pathway by downregulating the expression of HIF-1α and mTOR along with some amino acids that are needed for cancer growth.in-vitro assay needs to be done. In-silico computational modeling may also help for further investigations.In conclusion, our study indicates for the first time that 4-MTBITC displays modulatory effect on DMBA induced key hypoxia-inducible pathway and metabolic pathway including glycolysis. It also modulates the expression of a central link of the proliferative pathway known as mTOR and Akt followed by modulation of p21, lactate production as well as amino acid metabolism involved in cancer cell proliferation. However, the direct interaction of 4-MTBITC with HIF-1α or glycolytic enzymes that may produce pharmacological response through


Rhodotorulamucilaginosa (Rho) can secrete large amounts of extracellular polymeric substances (EPS) to resist lead (Pb) toxicity. Phosphate is an effective material for the remediation of Pb. This study explored the Pb remediation by the combination of Rho and different types of phosphate in water. To do so, four phosphates, namely, ferric phosphate , aluminum phosphate , calcium phosphate , and phosphogypsum (PG) were employed along with Rho. Compared with Rho application, the addition of phosphate significantly promoted the secretion of EPS by Rho (21–25 vs 16 mg). The formed EPS-Pb contributes to the Pb immobilization in the combination of Rho and phosphate. After 6 days of incubation, Rho + phosphate treatments immobilized over 98% of Pb cations, which is significantly higher than Rho treatment (94%). Of all Rho + phosphate treatments, Ca-P and PG-amended Rho had higher secretion of EPS, resulting in higher Pb removal. Nevertheless, PG had the highest efficiency for Pb removal compared with other phosphates, which reached 99.9% after 6 days of incubation. Likewise, new Pb minerals, such as pyromorphite and lead sulfate, only appeared in Rho + PG treatment. Altogether, this study concludes on the combined application of Rho and phosphate as an efficient approach to promote Pb remediation, particularly using PG waste. Worldwide, lead (Pb) contamination has been recognized as a growing concern, especially in water bodies . The con2+) to form the highly insoluble pyromorphite (Pyro) [Pb5(PO4)3X] , which has a very low solubility (10(PO4)62], calcium phosphate 2), aluminum phosphate , and ferric phosphate  are the most common phosphate species in nature (via the secretion of secondary metabolites such as low-molecular weight organic acid (LMWOA) and extracellular polymeric substances (EPS)  .Aspergillus niger) can secrete large amounts of oxalic acid to dissolve apatite and promote the transfer of Pb2+ to pyro and lead oxalate  can resist high Pb levels  and adsorb Pb cations to form EPS-Pb . The composition of precipitates was analyzed by X-ray diffraction (XRD). The morphology of Rho and its mineral composition were observed by using a scanning electron microscope–energy dispersive spectrometer (SEM-EDS).Rhodotorula mucilaginosa (Rho) was isolated from orchard rhizosphere soil  (Red yeast versity) . Before 4, Fe-P), aluminum phosphate , calcium phosphate , and phosphogypsum (PG), were used for the remediation of Pb by Rho. Fe-P, Al-P, and Ca-P were supplied by Shanghai Macklin Biochemical Co. Ltd. PG was collected from a phosphate fertilizer production plant in Anhui Province, China. Before the experiment, all phosphate species were dried at 65°C for 24 h.Four phosphates, such as ferric phosphate (FePO3)2 powder (Xilong Scientific Ltd.). The initial Pb concentration in the medium was adjusted to 1,000 mg/L. Five treatments were performed in this experiment, that is, Rho, Rho + Fe-P, Rho + Al-P, Rho + Ca-P, and Rho + PG. Before the incubation, the 0.08 g Pb(NO3)2 powder and 0.5 g phosphate were added to 150-ml Erlenmeyer flasks with 50 ml PDB medium (sterilized at 121°C for 20 min) in a sterile environment. Then, 0.5 ml Rho suspension was added to each treatment. After sealing with parafilm , the flasks were incubated at 180 rpm, 28°C in a sterile condition. After incubating for 2, 4, and 6 days, the PDB medium was collected into 50-ml centrifugal tubes and centrifuged for 6 min at 5,000 rpm. Then, the supernatant liquid was filtered through a 0.45 μm polyethersulfone (PES) membrane. The filtrates were tested for pH value, P, and Pb content. The centrifugal precipitates were collected and dried at 55°C for 24 h to determine the biomass, XRD, and SEM characterization.The Pb contamination in water was prepared by Pb membrane and prepared for the test of P and Pb content. All treatments were performed with three replicates.After incubation, the supernatant was collected and centrifuged twice at 12,000 rpm at 4°C for 20 min. Then, a 3-fold volume of anhydrous ethanol was mixed with the supernatant to rest for 48 h at 4°C to collect extracellular secretions. Last, the collected crude extracts were transferred into 3,500 molecular dialysis bags and dipped in pure water for 72 h. The pure water was changed twice every 24 h. The last extracts were transferred into a 2-ml centrifuge tube and freeze-dried .via ICP-OES (PerkinElmer Avio 200). A calibration curve of P and Pb  was performed by the phosphorus and lead standard , respectively. The R square value of the external standard curves was 0.999.The pH value in each treatment was determined by a pH meter . The soluble P and Pb concentrations were analyzed −1). The XRD patterns were analyzed by MDI Jade 6.5 software for phase identification.The mineralogical characterization of the precipitates was examined using a Rigaku D/Max-2500 X-ray diffractometer , with an acceleration voltage of 3 kV. To enhance image quality, the samples were coated with a layer of gold for 1 min in a Hitachi E-1010 Sputter.The initial pH value in the medium was 5.7. After 2 days of incubation, the pH values of Rho and Rho + Fe-P treatments decreased to 4.8 and 5.4, respectively . In Rho The dry biomass in Rho treatment was 0.3, 0.3, and 0.6 g on days 2, 4, and 6 . In Rho The P contents in Rho and Rho + Fe-P treatments were lower, ranging from 0.4 to 0.5 mg/L and 0.5–3.5 mg/L, respectively, during the entire incubation time . In Rho The initial Pb concentration in the PDB medium was 1,000 mg/L. The Pb contents in Rho treatment were 53.5, 52.4, and 53.3 mg/L after 2, 4, and 6 days of incubation, respectively . In Rho Results of our parallel experiment indicated that the P content in Pb, Fe-P + Pb, Al-P + Pb, Ca-P + Pb, and PG + Pb treatments reached 0.02, 3.01, 93.6, 62.2, and 13.7 mg/L after 6 days of incubation . In Pb tThe Pb removal ratios in Rho and Pb treatment were 94.7 and 8.2% after 6 days of incubation, respectively . Compareo) can be observed in each treatment (o) was also clearly observed in the Rho treatment (o) appeared in Rho + Fe-P and Rho + Al-P treatments, respectively  and Ca-P (32.2o) were observed in Rho + PG treatment (o) were also identified in Rho + PG treatment (o) and lead sulfate (24.7o) were detected in this treatment , the Pb remediation by P. oxalicum was significantly limited by the high Pb toxicity , the trivalent cations  are more competitive for heavy metal cations in binding with EPS  (EPS has strong adsorption and binding capacity for Pb cations due to the complex molecular composition . The hig charges . Althoug charges . Comparewith EPS . In addind Cu2+) . TherefoIn this study, PG had the highest efficacy for Pb remediation by Rho. Except for EPS-Pb, the formed pyro only occurred in Rho + PG treatment . Since Pvia the formation of EPS-Pb, particularly in combination with Ca-P and PG. In addition, PG revealed the highest removal rate of Pb through the formation of pyro and lead sulfate. Altogether, our findings suggest that the combined application of PG and Rho is an economical and efficient approach for Pb remediation.This study concludes on the co-application of Rho and phosphate to significantly promote the Pb remediation in water. The secretion of EPS by Rho dominated the Pb removal