Determination of Methylene Blue and Its Metabolite Residues in Aquatic Products by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

A sensitive and reliable method was developed to determine methylene blue (MB) and its metabolite residues, including azure A (AZA), azure B (AZB), and azure C (AZC) in aquatic products by HPLC–MS/MS. The samples were extracted by acetonitrile and cleaned up by alumina-neutral (ALN) cartridges. The analytes were separated on a Sunfire C18 column (150 mm × 2.1 mm, 5 µm). The method was validated according to the European criteria of Commission Decision 2002/657/CE. Good linearity between 1–500 µg/L was obtained with correlation coefficients (R²) greater than 0.99. The limit of quantification (LOQ) was 1.0 µg/kg. The average recoveries at three levels of each compound (1, 5, and 10 µg/kg) were demonstrated to be in the range of 71.8–97.5%, with relative standard deviations (RSDs) from 1.05% to 8.63%. This method was suitable for the detection of methylene blue and its metabolite residues in aquatic products.     

Which Extraction Solvents and Methods Are More Effective in Terms of Chemical Composition and Biological Activity of Alcea fasciculiflora from Turkey?

The bioactive content, antioxidant properties, and enzyme inhibition properties of extracts of Alcea fasciculiflora from Turkey prepared with different solvents (water, methanol, ethyl acetate) and extraction methods (maceration, soxhlet, homogenizer assisted extraction, and ultrasound assisted extraction) were examined in this study. UHPLC-HRMS analysis detected or annotated a total of 50 compounds in A. fasciculiflora extracts, including 18 hydroxybenzoic and hydroxycinnamic acids, 7 Hexaric acids, 7 Coumarins, 15 Flavonoids, and 3 hydroxycinnamic acid amides. The extracts had phenolic and flavonoid levels ranging from 14.25 to 24.87 mg GAE/g and 1.68 to 25.26 mg RE/g, respectively, in the analysis. Both DPPH and ABTS tests revealed radical scavenging capabilities (between 2.63 and 35.33 mg TE/g and between 13.46 and 76.27 mg TE/g, respectively). The extracts had reducing properties (CUPRAC: 40.38–78 TE/g and FRAP: 17.51–42.58 TE/g). The extracts showed metal chelating activity (18.28–46.71 mg EDTAE/g) as well as total antioxidant capacity (phosphomolybdenum test) (0.90–2.12 mmol TE/g). DPPH, ABTS, FRAP, and metal chelating tests indicated the water extracts to be the best antioxidants, while the ethyl acetate extracts had the highest overall antioxidant capacity regardless of the extraction technique. Furthermore, anti-acetylcholinesterase activity was identified in all extracts (0.17–2.80 mg GALAE/g). The water extracts and the ultrasound-assisted ethyl acetate extract were inert against butyrylcholinesterase, but the other extracts showed anti-butyrylcholinesterase activity (1.17–5.80 mg GALAE/g). Tyrosine inhibitory action was identified in all extracts (1.79–58.93 mg KAE/g), with the most effective methanolic extracts. Only the ethyl acetate and methanolic extracts produced by maceration and homogenizer aided extraction showed glucosidase inhibition (0.11–1.11 mmol ACAE/g). These findings showed the overall bioactivity of the different extracts of A. fasciculiflora and provided an overview of the combination of solvent type and extraction method that could yield bioactive profile and pharmacological properties of interest and hence, could be a useful reference for future studies on this species.     

Purification and Biochemical Characterization of Polyphenol Oxidases from Embryogenic and Nonembryogenic Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) Cells

Polyphenol oxidases (PPOs) were isolated from cell suspensions of two cultivars of cotton (Gossypium hirsutum L.), and their biochemical characteristics were studied. PPO from Coker 312, an embryogenic cultivar, showed a highest affinity to catechol 20 mM, and PPO from R405-2000, a nonembryogenic cultivar, showed a highest affinity to 4-methylcatechol 20 mM. The optimal pH for PPO activity was 7.0 and 6.0 for Coker 312 and R405-2000, respectively. The enzyme had an optimal temperature of 25 °C and was relatively stable at 20–30 °C. Reducing sodium metabisulfite, ascorbic acid, dithiothreitol, SnCl₂, and FeCl₃ markedly inhibited PPO activity, whereas its activity was highly enhanced by Mg²⁺, Ca²⁺, and Mn²⁺ and was moderately inhibited by Ba²⁺, Cu²⁺, and Zn²⁺. The analysis revealed a single band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis which corresponded to a molecular weight of 55 kDa for Coker 312 and 42 kDa for R405-2000.     

Purification and Characterization of Cell Suspensions Peroxidase from Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Two peroxidases, cPOD-I and rPOD-II, have been isolated and purified from cotton cell suspension and their biochemical characteristics studied. rPOD-II from R405-2000, a non-embryogenic cultivar, has higher activity than cPOD-I derived from Coker 312, which developed an embryogenic structure. The cPOD-I and rPOD-II had molecular mass of 39.1 and 64 kDa respectively, as determined by SDS-PAGE. Both enzymes showed high efficiency of interaction with the guaiacol at 25 mM. The optimal pH for cPOD-I and rPOD-II activity was 5.0 and 6.0, respectively. The enzyme had an optimum temperature of 25 °C and was relatively stable at 20–30 °C. The isoenzymes were highly inhibited by ascorbic acid, dithiothreitol, sodium metabisulfite, and β-mercaptoethanol. Their activities were highly enhanced by Al³⁺, Fe³⁺, Ca²⁺, and Ni²⁺, but they were moderately inhibited by Mn²⁺ and K⁺. The enzyme lost 50% to 62% of its activity in the presence of Zn²⁺ and Hg²⁺.     

Field development of oil palms (Elaeis guineensis jacq) originating from cryopreserved stabilized polyembryonic cultures

In this paper, the long term observation of plants originating from control and cryopreserved stabilized polyembryonic cultures (SPCs) of six elite oil palm clones was carried out. Survival of plantlets in the nursery was monitored, then a series of vegetative and floral characteristics of over 440 palms were studied for up to 12 years after field transfer in C?te D'Ivoire. The six clones tested showed an average recovery of 34% after freezing in liquid nitrogen. The average survival in the nursery of plantlets originating from pretreated and dehydrated and from cryopreserved SPCs was higher than that of control SPCs. Palm trees originating from control SPCs were found to flower earlier than those originating from pretreated and dehydrated and from cryopreserved SPCs. This delay in flowering disappeared progressively and all palms had flowered 3 years after planting regardless of the SPC treatment. Abnormal palms were observed in one clone only. With this clone, the percentage of abnormal palms originating from cryopreserved SPCs was significantly lower (5%) than that measured on palms originating from control SPCs (29%).     

Towards the Pharmacological Validation and Phytochemical Profiling of the Decoction and Maceration of Bruguiera gymnorhiza (L.) Lam.—A Traditionally Used Medicinal Halophyte

Decoctions (leaves and roots) of Bruguiera gymnorhiza (L.) Lam. are traditionally used against diabetes in many countries, including Mauritius. This study endeavoured to evaluate the inhibitory potential of leaves, roots, twigs and fruits extracts (decoction and maceration) of B. gymnorhiza against key enzymes relevant to diabetes. Considering complications related to diabetes, other clinical enzymes, namely, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, elastase and pancreatic lipase, were used. Identification of compounds was carried out using ultra-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Antioxidant capacities were assessed using DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum, metal chelating. The relationship between mode of extraction, plant parts and biological activities was determined using multivariate analysis. Macerated fruits, rich in phytochemicals (phenolic, flavanol, tannin, and triterpenoid), exhibited substantially high antioxidant capacities related to radical scavenging (DPPH: 547.75 ± 10.99 and ABTS: 439.59 ± 19.13 mg TE/g, respectively) and reducing potential (CUPRAC: 956.04 ± 11.90 and FRAP: 577.26 ± 4.55 mg TE/g, respectively). Additionally, the same extract significantly depressed AChE and BChE (3.75 ± 0.03 and 2.19 ± 0.13 mg GALAE/g, respectively), tyrosinase (147.01 ± 0.78 mg KAE/g), elastase (3.14 ± 0.08 mg OE/g) and amylase (1.22 ± 0.01 mmol ACAE/g) enzymatic activities. Phytochemical results confirmed the presence of 119 compounds in all maceration and 163 compounds in all decoction samples. The screening also revealed important compounds in the extracts, namely, quinic acid, brugierol, bruguierol A, epigallocatechin, chlorogenic acid, to name a few. Multivariate analysis reported that the plant parts of B. gymnorhiza greatly influenced the observed biological activities in contrast to the types of extraction methods employed. Docking calculations have supported the findings of the experimental part through the high binding affinity and strong interactions of some compounds against tyrosinase, AChE, BChE and elastase enzymes. The decocted root and leaf of B. gymnorhiza showed low to moderate antidiabetic activity, thereby partially supporting its traditional uses in the management of diabetes. However, the fruit, the most active organ, can be used as a diet supplement to reduce the risk of diabetes complications after evaluating its cytotoxic effects.     

Chromatographic Separation of Breynia retusa (Dennst.) Alston Bark,Fruit and Leaf Constituents from Bioactive Extracts

Breynia retusa (Dennst.) Alston (also known as Cup Saucer plant) is a food plant with wide applications in traditional medicine, particularly in Ayurveda. Extracts obtained with four solvents (dichloromethane, methanol, ethyl acetate and water), from three plant parts, (fruit, leaf and bark) were obtained. Extracts were tested for total phenolic, flavonoid content and antioxidant activities using a battery of assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), total antioxidant capacity (TAC) (phosphomolybdenum) and metal chelating. Enzyme inhibitory effects were investigated using acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase and α-glucosidase as target enzymes. Results showed that the methanolic bark extract exhibited significant radical scavenging activity (DPPH: 202.09 ± 0.15; ABTS: 490.12 ± 0.18 mg Trolox equivalent (TE)/g), reducing potential (FRAP: 325.86 ± 4.36: CUPRAC: 661.82 ± 0.40 mg TE/g) and possessed the highest TAC (3.33 ± 0.13 mmol TE/g). The methanolic extracts were subjected to LC-DAD-MSⁿ and NMR analysis. A two-column LC method was developed to separate constituents, allowing to identify and quantify forty-four and fifteen constituents in bark and fruits, respectively. Main compound in bark was epicatechin-3-O-sulphate and isolation of compound was performed to confirm its identity. Bark extract contained catechins, procyanidins, gallic acid derivatives and the sulfur containing spiroketal named breynins. Aerial parts mostly contained flavonoid glycosides. Considering the bioassays, the methanolic bark extract resulted a potent tyrosinase (152.79 ± 0.27 mg kojic acid equivalent/g), α-amylase (0.99 ± 0.01 mmol acarbose equivalent ACAE/g) and α-glucosidase (2.16 ± 0.01 mmol ACAE/g) inhibitor. In conclusion, methanol is able to extract the efficiently the phytoconstituents of B. retusa and the bark is the most valuable source of compounds.     

Evaluation of Antioxidant and Enzyme Inhibition Properties of Croton hirtus L’Hér. Extracts Obtained with Different Solvents

Croton hirtus L’Hér methanol extract was studied by NMR and two different LC-DAD-MSⁿ using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources to obtain a quali-quantitative fingerprint. Forty different phytochemicals were identified, and twenty of them were quantified, whereas the main constituents were dihydro α ionol-O-[arabinosil(1-6) glucoside] (133 mg/g), dihydro β ionol-O-[arabinosil(1-6) glucoside] (80 mg/g), β-sitosterol (49 mg/g), and isorhamnetin-3-O-rutinoside (26 mg/g). C. hirtus was extracted with different solvents—namely, water, methanol, dichloromethane, and ethyl acetate—and the extracts were assayed using different in vitro tests. The methanolic extracts presented the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) values. All the tested extracts exhibited inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with a higher activity observed for dichloromethane (AChE: 5.03 and BChE: 16.41 mgGALAE/g), while the methanolic extract showed highest impact against tyrosinase (49.83 mgKAE/g). Taken together, these findings suggest C. hirtus as a novel source of bioactive phytochemicals with potential for commercial development.     

Pyrethroid and Etofenprox Resistance in Anopheles gambiae and Anopheles coluzzii from Vegetable Farms in Yaoundé, Cameroon: Dynamics,Intensity and Molecular Basis

Previous studies have indicated widespread insecticide resistance in malaria vector populations from Cameroon. However, the intensity of this resistance and underlying mechanisms are poorly known. Therefore, we conducted three cross-sectional resistance surveys between April 2018 and October 2019, using the revised World Health Organization protocol, which includes resistance incidences and intensity assessments. Field-collected Anopheles gambiae s.l. populations from Nkolondom, Nkolbisson and Ekié vegetable farms in the city of Yaoundé were tested with deltamethrin, permethrin, alpha-cypermethrin and etofenprox, using 1× insecticide diagnostic concentrations for resistance incidence, then 5× and 10× concentrations for resistance intensity. Subsamples were analyzed for species identification and the detection of resistance-associated molecular markers using TaqMan® qPCR assays. In Nkolbisson, both An. coluzzii (96%) and An. gambiae s.s. (4%) were found together, whereas only An. gambiae s.s. was present in Nkolondom, and only An. coluzzii was present in Ekié. All three populations were resistant to the four insecticides (<75% mortality rates―MR1×), with intensity generally fluctuating over the time between mod-erate (<98%―MR5×; ≥98%―MR10×) and high (76–97%―MR10×). The kdr L995F, L995S, and N1570Y, and the Ace-1 G280S-resistant alleles were found in An. gambiae from Nkolondom, at 73%, 1%, 16% and 13% frequencies, respectively, whereas only the kdr L995F was found in An. gambiae s.s. from Nkolbisson at a 50% frequency. In An. coluzzii from Nkolbisson and Ekié, we detected only the kdr L995F allele at 65% and 60% frequencies, respectively. Furthermore, expression levels of Cyp6m2, Cyp9k1, and Gste2 metabolic genes were highly upregulated (over fivefold) in Nkolondom and Nkolbisson. Pyrethroid and etofenprox-based vector control interventions may be jeopardized in the prospected areas, due to high resistance intensity, with multiple mechanisms in An. gambiae s.s. and An. coluzzii.