Phytol from Scoparia dulcis prevents NF-κB-mediated inflammatory responses during macrophage polarization.

Macrophages are primary immune cells that mediate a wide range of inflammatory diseases through their polarization potential. In this study, phytol isolated from Scoparia dulcis has been explored against 7-ketocholesterol and bacterial lipopolysaccharide-induced macrophage polarization in IC-21 cells. Isolated phytol has been characterized using GC-MS, TLC, HPTLC, FTIR, 1H-NMR, and HPLC analyses. The immunomodulatory effects of viable concentrations of phytol were tested on oxidative stress, arginase activity, nuclear and mitochondrial membrane potentials in IC-21 cells in addition to the modulation of calcium and lipids. Further, gene and protein expression of atherogenic markers were studied. Results showed that the isolated phytol at a viable concentration of 400 µg/ml effectively reduced the production of nitric oxide, superoxide anion (ROS generation), calcium and lipid accumulation, stabilized nuclear and mitochondrial membranes, and increased arginase activity. The atherogenic markers including iNOS, COX-2, IL-6, IL-1ß, MMP-9, CD36, and NF-κB were significantly downregulated at the levels of gene and protein expression, while macrophage surface and nuclear receptor markers (CD206, CD163, and PPAR-γ) were significantly upregulated by phytol pre-treatment in macrophages. Therefore, the present pharmacognostic study supports the role of phytol isolated from Scoparia dulcis in preventing M2-M1 macrophage polarization under inflammatory conditions, making it a promising compound. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03924-9.

Potential of insect endogenous cellulases for lignocellulosic break down deciphered using molecular docking studies.

Insects possess cellulolytic system capable of producing variegate enzymes with multifarious specificities to break down complex lignocellulosic products. Astonishingly, endoglucanases, exoglucanases and ß-glycosidases act sequentially in a synergistic system to facilitate the breakdown of cellulose to utilisable energy source glucose. In silico docking studies of endo-ß-1,4-glucanase from 19 different insects belonging to six different orders identified that it possesses high affinity for all the six substrates, including CMC, cellulose, cellotriose, cellotetraose, cellopentose and cellohexaose. Additionally, ß-glucosidase from nearly all the reported insect sources also showed considerable affinity towards cellobiose. Van der Waals, conventional hydrogen bonds and carbon-hydrogen bonds stabilise the interaction between the enzyme and different substrates. Molecular dynamics simulations also held up the stability of various complexes. Efficient breakdown of lignocelluloses-based substrates becoming a major focus of industrial and academic communities worldwide, this study can perhaps complement the propensity of insect cellulases for prospected applications.

Detection and substrate portrayal on the serum phenoloxidase activity from the grub of rhinoceros beetle, Oryctes rhinoceros.

Phenoloxidase (PO) is a significant biomolecule involved in humoral defence mechanism of invertebrates. Spontaneous melanization of insect haemolymph is the major hinderance for studying PO activity, as haemolymph was collected devoid of phenylthiourea. In the study, no visible melanization was observed in crude serum from the grub of Oryctes rhinoceros up to 30 min of incubation amongst crude haemolymph, diluted haemolymph, crude serum and diluted serum that were subjected to visual observation for spontaneous melanization reaction. Accordingly, crude serum was taken for evaluating PO activity. At the same time, as PO substrates tend to auto-oxidize and provide false optical density value, tris-buffered saline devoid of any substrates were used as blank for PO assays. The ideal wavelength at which maximum PO activity occurred for each substrate, namely, tyrosine, tyramine, dopamine, L-dopa, DL-dopa, catechol, protocatechuic acid and pyrogallol was determined as 407, 410, 429, 465, 403, 466, 428 and 400 nm, respectively. Additionally, time course of oxidation for each phenolic substrate by the serum PO were examined and DL-dopa was identified as the specific substrate for serum PO in the grub of O. rhinoceros. Furthermore, maximum PO activity was observed at 5 min of incubation for 10 mM of DL-dopa that was considered as optimum concentration. The ideal pH and temperature for serum PO activity was observed as 7.5 and 20°C, respectively. These results suggested that standardizing a suitable substrate is an essential prerequisite to evaluate the real PO activity of serum which might significantly fluctuate in each insect model.

Anti-infective potential of plant-derived quorum sensing inhibitors against multi-drug resistant human and aquatic bacterial pathogens.

The present study intended to decipher the anti-infective potential of bioactive phytocompounds, such as rosmarinic acid, morin, naringin, chlorogenic acid, and mangiferin, against aquatic and human bacterial pathogens using Artemia spp. nauplii and Caenorhabditis elegans as animal models, respectively. Initially, the test compounds were screened against the QS traits in Vibrio spp., such as bioluminescence production and biofilm formation. The test compounds effectively inhibited the bioluminescence in V. harveyi. Further, the confocal laser scanning microscopic analysis revealed that these natural compounds could efficiently reduce the clumping morphology, a characteristic biofilm formation in Vibrio spp., without inhibiting bacterial growth. The results of in vivo analysis showed a significant increase in the survival of Artemia spp. nauplii infected with Vibrio spp. upon exposure to these compounds. Moreover, the compounds used in this study were already proven and reported for their quorum sensing inhibitory efficacy against Pseudomonas aeruginosa. Hence, the anti-infective efficacy of these compounds against P. aeruginosa (PAO1) and its clinical isolates (AS1 and AS2) was studied using C. elegans as a live animal model system. The results of time-killing assay deciphered that rosmarinic acid and naringin are being the most effective ones in rescuing the animals from P. aeruginosa infection followed by morin, mangiferin, and chlorogenic acid. Further, the toxicity results revealed that these compounds did not show any lethal effect on C. elegans and Artemia spp. nauplii at the tested concentrations. In conclusion, the phytochemicals used in this study were effective in controlling the QS-regulated virulence traits in Vibrio spp. and P. aeruginosa infections in Artemia spp. nauplii and C. elegans animal model systems, respectively.

An insight on 7- ketocholesterol mediated inflammation in atherosclerosis and potential therapeutics.

7-ketocholesterol, a toxic oxidative product of oxysterol is a causative agent of several diseases and disabilities concomitant to aging including cardiovascular diseases like atherosclerosis. Auto-oxidation of cholesterol esters present in low-density lipoprotein (LDL) deposits lead to the formation of oxidized LDL (Ox-LDL) along with its byproducts, namely 7KCh. It is predominantly found in atherosclerotic plaque and also found to be more atherogenic than cholesterol by being cytotoxic, interfering with cellular homeostasis. This makes it a serious threat by being the foremost cause of morbidity and mortality worldwide and is likely to become more serious during forth coming years. It involves in mediating inflammatory mechanisms characterized by the advancement of fibroatheroma plaques. The atherosclerotic lesion is composed of Ox-LDL along with fibrotic mass consisting of immune cells and molecules. Macrophages being the specialized phagocytic cells, contribute to removal of detrimental contents of the lesion along with accumulated lipids leading to alteration of its biology and functionality due to its plasticity. Here, we have explored the known as well as proposed mechanisms involved with 7KCh associated atherogenesis along with potential therapeutic strategies for targeting 7KCh as a diagnostic and target in medicine.

A serine protease-associated lectin in the cytolytic system of blowfly (Chrysomya megacephala) larvae: Evidence and characterization.

Cytolytic activity against invading microorganisms is one of the innate forms of immunity in invertebrates. A serine protease-associated sialic acid-specific cytolytic lectin was purified using glutaraldehyde-fixed ox erythrocytes from the larval extract of blowfly (Chrysomya megacephala). The purified lectin lysed vertebrate erythrocytes with effective haemolysis of ox red blood cells (RBCs) in an isotonic medium. The degree of haemolytic (HL) activity of the purified cytolytic lectin depended on its concentration, pH, temperature, and calcium ions. It was sensitive to ethylenediaminetetraacetic acid. The native molecular mass of the C-type lectin was 260 ± 26 kDa, comprising four different polypeptide subunits of 75 kDa (pI ~8), 69 kDa (pI ~7.0), 61 kDa (pI ~5.3), and 55 kDa (pI ~4.6). The association between the C-type lectin and serine protease was confirmed by MALDI-TOF-MS analysis that revealed its homology in the same spectral peak as well as the proteases and phenylmethylsulphonyl fluoride inhibition of HL activity. Haemolysis inhibition by N-acetylneuraminic acid and other sugars revealed the properties of the lectin. The purified lectin distorted the integrity of ox RBCs and Paenalcaligenes hermetiae. This in vitro study documents the presence of a cytolytic system in blowfly (C. megacephala) larvae for the clearance of invading microbial pathogens in their feeding niche.

Antiviral activity of seselin from Aegle marmelos against nuclear polyhedrosis virus infection in the larvae of silkworm, Bombyx mori.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Indian medicine has utilized Aeglemarmelos (L.) Corr. commonly called as bael in several indigenous systems against various diseases. Bioactive components isolated from various plant parts of A. marmelos were used in ethno-medicine. More precisely they are known for its antiviral property against various human and animal viruses. AIM OF THE STUDY: The study was conducted to investigate the antiviral activity of A.marmelos against Bombyx mori nucleopolyhedrovirus (BmNPV). MATERIALS AND METHODS: Among the various crude extracts tested, hexane extracts of leaves of A. marmelos with promising anti-BmNPV activity was subjected to bioactivity guided fractionation based on column chromatography. Out of 40 fractions obtained from the fractionation, fractions showing similar TLC profiles were pooled into 14 fractions. A fraction with potential activity was used to purify a molecule with anti-BmNPV activity. This molecule was characterized through structural and functional analyses. RESULTS: The functionally and structurally characterized molecule in the fraction with prospective anti-BmNPV activity revealed a single crystal compound 'seselin' (8, 8-dimethyl pyrido oxazine-2-one). CONCLUSION: It is therefore understood that this seselin compound could be used as a natural medicine for the management of NPV infection in the silkworm larvae under commercial conditions after suitable field evaluations.

ß-Galactoside binding lectin from caddisfly larvae, Stenopsyche kodaikanalensis with selective modes of antibacterial activity: Purification and characterization.

Insects sustain the invading bacterial pathogens by inducing the production of lectin which participates in surveillance of non-self molecules. The antibacterial property of lectin is an inevitable aspect of innate immune system especially for the insects feeding the detritus organic matter. ß-galactoside binding lectin possessing antibacterial property was detected and purified from the hemolymph of larvae of caddisfly, Stenopsyche kodaikanalensis using affinity chromatography. The purified lectin exhibited highest hemagglutination titer value against buffalo erythrocytes and has affinity to lactose and fetuin which contains ß-galactoside linkages. It was found to be calcium independent, EDTA insensitive and heat labile. These reveal the characteristics features of S-Lac lectin. The molecular weight of lectin was 360 kDa with five distinct subunits such as 95, 90, 66, 62 and 47 kDa. The sequences acquired through MALDI-TOF-MS analysis shared homologies to the putative conserved region of leguminous lectin. Antibacterial studies were carried out with native soil bacterial isolates. It revealed that the lectin possessed the specific modes of action against bacteria that it can agglutinate the Bacillus subtilis and lyse the Bacillus flexus.

Population genetic structure and molecular diversity of Leucinodes orbonalis based on mitochondrial COI gene sequences.

Leucinodes orbonalis is a destructive pest found throughout eggplant cultivating fields of Tamil Nadu, India. The genetic diversity and its population structure were investigated in this pest using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences from 20 populations of L. orbonalis collected from various agro-climatic conditions. The study indicated almost no genetic diversity among various populations. The COI nucleotide sequence based haplotype analysis also revealed no significant genetic variation among various populations. However, haplotype network analysis with three clades was nearly matching with the structure of phylogenetic analysis that showed geographical separations induced distribution of some genetic variation. The PCA and nMDS Shephard's plot analyses were also illustrated that the populations sampled were nearly matched with phylogenetic tree and haplotype network. This study on phylogeographical structure using the mitochondrial COI sequence diversity of L. orbonalis therefore suggested presence of few genetically distinct populations due to some specific habitat requirements.

Purification and functional characterization of lectin with phenoloxidase activity from the hemolymph of cockroach, Periplaneta americana.

Lectins also identified as hemagglutinins are multivalent proteins and on account of their fine sugar-binding specificity play an important role in immune system of invertebrates. The present study was carried out on the hemolymph lectin of cockroach, Periplaneta americana with appropriate screening and purification to understand its molecular as well as functional nature. The lectin from the hemolymph was purified using ion-exchange chromatography. The approximate molecular weight of purified lectin was 340 kDa as determined by FPLC analysis. Rabbit erythrocytes were highly agglutinated with purified lectin from the hemolymph of P. americana. The hemagglutination activity (HA) of lectin was specifically inhibited by fucose. Glycoproteins also inhibited the HA activity of lectin. The amino acid sequences of the purified lectin revealed homology with amino acid sequences of allergen proteins from P. americana. Purified lectin showed the highest phenoloxidase activity against dopamine. The activators such as exogenous proteases and LPS from Escherichia coli and Salmonella minnesota significantly enhanced the PO activity of the purified lectin. Besides, the presence of copper and hemocyanin conserved domain in the purified lectin provided a new facet that insects belonging to the ancient clade such as cockroaches retained some traces of evolutionary resemblance in possessing lectin of ancient origin.

Isolation and analysis of mannose/trehalose/maltose specific lectin from jack bean with antibruchid activity.

A lectin with insecticidal property against the stored product pest, Callosobruchus maculatus was successfully isolated from the seeds of Canavalia virosa using standard affinity chromatography. The isolated molecule typically behaved like a lectin in its characteristics. It agglutinated indicator red blood cells (RBC) in its native as well as enzyme treated conditions. The enzyme treated RBC types exhibited a very high hemagglutination (HA) titre values and this property of isolated molecule behaved like arcelin, the lectin-like molecules reported from several species of Phaseolus. As a characteristic feature of a lectin, the isolated molecule effectively inhibited the agglutination of indicator RBC types with simple and complex carbohydrates including glycoproteins. This nature of the isolated molecule also relate with characteristic feature of arcelin isoforms in inhibiting HA activity with complex glycoproteins as reported in many studies. Most interestingly, the present study disclosed trehalose as a potent inhibitor of C. virosa lectin. Therefore, feeding insect pests on the lectin like arcelin could serve as antibiosis factor/anti-insect activity. The molecular characteristics of this isolated molecule and its mass studies too revealed its homology with arcelin, arcelin-1, 2 and 6 isoforms of P. vulgaris and lectin from Canavalia cathartica, C. lineata and C. brasiliensis.

Detection of genetic variability in various isolates of cattle tick, Boophilus microplus from Tamil Nadu, India using PCR-RAPD analysis.

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.