Purification, molecular docking and in vivo analyses of novel angiotensin-converting enzyme inhibitory peptides from protein hydrolysate of moth bean (Vigna aconitifolia (Jacq.) Màrechal) seeds.

The moth bean is a high-protein food legume. Enzymatic hydrolysates of food proteins demostrate health benefits. Search for diet related food protein hydrolysates is therefore within the scope of functional foods. Present study asertains to produce, screen and identify natural ACE-I inhibitory peptides derived from moth bean seed protein hydrolysates. The extracted protein was hydrolysed using alcalase, chymotrypsin, flavourzyme, papain, pepsin and trypsin respectively. Alcalase achieved the greatest degree of hydrolysis and ACE inhibition. The highest ACE-I inhibitory activity was exhibited by the peptide with the lowest molecular weight i.e. <3 kDa (IC50 11.19 ± 0.15 µg/mL). This was further separated by FPLC, followed by mass spectrometry. Molecular docking analysis showed the peptides IAWDFR and ADLPGLK bind to active sites whereas DKPWWPK and AVIPNAPNLR to non-active sites of the ACE molecule. In vivo administration of MBP hydrolysate to dexamethasone-induced hypertensive rats reduced their systolic blood pressure (125 ± 0.76 mmHg) compared with positive control (155 ± 3.13 mmHg). Moth bean protein peptides exhibit functional nutraceutical properties with adequate antihypertensive activity.

Deltamethrin Contact Exposure Mediated Toxicity and Histopathological Aberrations in Tissue Systems of Public Health Importance Cockroach Species Periplaneta americana and Blattella germanica.

Cockroach species Periplaneta americana and Blattella germanica potentially survive in locations close to human activity. Besides spoiling food material, cockroaches also transfer pathogens of different diseases among human beings. Since the insecticides have been used extensively to control cockroaches, information on their insecticide susceptibility and toxicity at the cellular level may be crucial. In the study, deltamethrin toxicity as well as the deltamethrin-mediated cytomorphological changes in the brain, ovary and midgut of the two important cockroach species have been assessed. Different concentrations [0.00025% (0.0025 mg/ml), 0.0025% (0.025 mg/ml), 0.025% (0.25 mg/ml), 0.25% (2.5 mg/ml), 0.5% (5 mg/ml), 1% (10 mg/ml)] of deltamethrin in acetone were used to expose test species in WHO bottle assay. Knockdown was recorded after 5 min interval while delayed mortality was observed after 24 h. Brain, ovary and gut were dissected post 1 h exposure and 24 h holding (for 0.25, 0.5 and 1% concentration), and tissues were processed for microscopic analysis. Deltamethrin exposed cockroaches and dissected tissues were used to estimate deltamethrin using HPLC. At 0.00025% (lowest concentration), the percentage knock-down observed was 66.7% for P. americana and 80% B. germanica respectively (R 2 = 0.78; p = 0.0001) in 1 h. KDT50 value was found to be 8.7 min (95% CI: 7.3-10.2), while KDT99 was 20.7 min (95% CI: 16.0-35.7) in P. americana at 1% concentration. Whereas, the KDT50 and KDT99 values for B. germanica were 7.4 min (95% CI: 5.4-9.1) and 27.4 min (95% CI: 18.2-80.0) at a similar concentration. LD50 and LD95 values (for 60 min standard exposure) were 0.0006% (95% CI: 0.00-0.001) and 0.034% (95% CI: 0.013-0.49) respectively for P. americana, while these values were 0.0005 (95% CI: 0.00-0.001) and 0.04 (95% CI: 0.01-0.23) for B. germanica. Exposure to 1% deltamethrin induced a considerable toxic effect in the epithelial cells in the midgut. HPLC estimated 0.21 ± 0.05 mg (95% CI: 0.18-0.25; CoV 23.9%) deltamethrin in P. americana post 1% exposure. Even short term exposure to a low concentration of synthetic pyrethroid deltamethrin displayed immediate knockdown and delayed mortality in both the test species. Considerable histological damage was observed in both the insects at 1% exposure. In India, resistance to deltamethrin may have been reported among different insects due to its extensive use. However, the formulations such as insecticide paints, attractant baits etc. developed using deltamethrin as an active ingredient could be useful in cockroach control operations.

Silibinin ameliorates abrin induced hepatotoxicity by attenuating oxidative stress, inflammation and inhibiting Fas pathway.

Abrin is a toxin from the seeds of Abrus precatorius. Abrin is considerably more toxic than ricin and a potent bio-warfare agent. The mechanism of abrin induced hepatotoxicity remains unclear. Silibinin has antioxidant, anti-inflammatory and hepatoprotective activities. But, its therapeutic potential in abrin toxicity is unknown. In view of these facts, the purpose of this study was to delineate the mechanisms and ameliorative role of silibinin against abrin induced hepatotoxicity. Parameters related to liver functions, oxidative stress, inflammation, Fas pathway and histopathology were evaluated in the liver of BALB/c mice after abrin exposure. Abrin intoxication resulted in hepatotoxicity, oxidative stress, inflammation, altered histopathology and increased Fas pathway signaling. Silibinin improves survival of abrin-exposed mice by decreasing serum liver enzymes and reinstating the antioxidant capacity. Silibinin also inhibits abrin-induced inflammation and Fas pathway. Present study for the first time demonstrates the hepatoprotective potential of silibinin against abrin toxicity.

Emergence and expansion of highly infectious spike protein D614G mutant SARS-CoV-2 in central India.

COVID-19 has emerged as global pandemic with largest damage to the public health, economy and human psyche.The genome sequence data obtained during the ongoing pandemic are valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating SARS-CoV-2 strains in India will enable the scientific community to understand the emergence of new variants and their impact on human health. The first case of COVID-19 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of cases within next three months. More than 5000 COVID-19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh were analyzed during the nation -wide lockdown and unlock period. A total of 136 cases were found positive over a span of three months that included virus introduction to the region and its further spread. Whole genome sequences employing Oxford nanopore technology were generated for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh state of India. This period witnessed index cases with multiple travel histories responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were identified. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades including A2a, A4 and B. The cluster-wise segregation was observed, suggesting multiple introduction links and subsequent evolution of virus in the region. This is the first comprehensive whole genome sequence analysis from central India, which revealed the emergence and evolution of SARS-CoV-2 during thenation-wide lockdown and unlock.

Pulmonary protective efficacy of S-2[2-aminoethylamino] ethyl phenyl sulphide (DRDE-07) and its analogues against sulfur mustard induced toxicity in mice.

Our previous study showed that percutaneous sulfur mustard (SM) exposure induced pulmonary toxicity, which was attenuated by DRDE-07 (S-2[2-aminoethylamino] ethyl phenyl sulphide) pretreatment. The present study aimed to evaluate the protective efficacy of DRDE-07 and its analogues viz., DRDE-30 (S-2(2-aminoethyl amino)ethyl propyl sulphide) and DRDE-35 (S-2(2-aminoethyl amino)ethyl butyl sulphide) against SM. Thirty minutes before percutaneous SM (0.8 LD50) exposure, female Swiss mice were orally gavaged with DRDE-07 and its analogues(0.2 LD50). Animals were sacrificed on day 3 and 7, BAL fluid (BALF) and lung tissue were collected for biochemical, histopathological studies. As results, DRDE-07 and its analogues were beneficial in reducing the number of BALF inflammatory cells, protein level, lactate dehydrogenase (LDH) activity, myeloperoxidase (MPO) and ß-glucuronidase activity, while content of BALF and lung reduced glutathione level (GSH) were significantly protected. The pretreatment of DRDE-07 and its analogues inhibited the recruitment of inflammatory cells into the lung. The beneficial effects of DRDE-07 and its analogues were attributed to their antioxidant and anti-inflammatory activity. Among the analogues, DRDE-30 exhibited significant beneficial effects as compared to the other two compounds. These analogues may be considered as prototype candidate molecules as there is no effective antidote for SM toxicity.

Effect of fentanyl and its three novel analogues on biochemical, oxidative, histological, and neuroadaptive markers after sub-acute exposure in mice.

AIMS: Comparative sub-acute toxicity, including tolerance and dependence potential of fentanyl and its three novel and potent analogues was determined in mice. MAIN METHODS: Comparative sub-acute (21 d, intraperitoneal; 1/10 LD50) toxicity of fentanyl and its three novel analogues viz., N-(1-(2-phenoxyethyl)-4-piperidinyl) propionanilide (2), N-isopropyl-3-(4-(N-phenylpropionamido)piperidin-1-yl)propanamide (5), and N-t-butyl-3-(4-(N-phenylpropionamido)piperidin-1-yl)propanamide (6) was determined in mice. Animals were observed for additional seven days to assess the recovery. The brain, liver and kidney toxicity was assessed on the basis of various biochemical, oxidative, histological, and neuroadaptive markers. The expression levels of key neuronal markers associated with drug tolerance and dependence were investigated by western blot and immunohistochemistry. KEY FINDINGS: Fentanyl and its analogues caused abnormal levels of liver and kidney specific biomarkers in plasma. Brain malondialdehyde (MDA) levels were raised by all the treatments and kidney MDA level by analogue 6 (21 d). Reduced glutathione levels in brain, liver, and kidney were diminished by all the treatments (21 & 28 d) and a significant change in the levels of antioxidant enzymes was also produced mainly after 21 d. The deleterious effects of fentanyl and its analogues were further substantiated by corresponding histopathological changes in brain, liver and kidney (21 & 28 d). These compounds were also found to produce neuroadaptive changes as evidenced by the increased expression levels of c-Fos, glucocorticoid receptor, N-methyl-d-aspartate receptor1 and µ-opioid receptor (21 & 28 d). SIGNIFICANCE: Three novel analogues of fentanyl were envisaged to have alternative therapeutic potentials. However, their comparative sub-acute toxicity revealed undesirable side effects, thereby masking their therapeutic ability.

Development of a novel chimeric PA-LF antigen of Bacillus anthracis, its immunological characterization and evaluation as a future vaccine candidate in mouse model.

Tremendous efforts are being made to develop an anthrax vaccine with long term protection. The main component of traditional anthrax vaccine is protective antigen (PA) with the trace amount of other proteins and bacterial components. In this study, we developed a recombinant PA-LF chimera antigen of Bacillus anthracis by fusing the PA domain 2-4 with lethal factor (LF) domain 1 and evaluated its protective potential against B. anthracis in mouse model. The anti-PA-LF chimera serum reacted with both PA and LF antigen, individually. The chimera elicited a strong antibody titer in mice with predominance of IgG1 isotype followed by IgG2b, IgG2a and IgG3. Cytokines were assessed in splenocytes of immunized mice and a significant up-regulation in the expression of IL-4, IL-10, IFN-γ and TNF-α was observed. The PA-LF chimera immunized mice exhibited 80% survival after challenge with virulent spores of B. anthracis. Pathological studies showed normal architecture in vital organs (spleen, lung, liver and kidney) of recovered immunized mice on 20 DPI after spore challenge. These findings suggested that PA-LF chimera of B. anthracis elicited good humoral as well as cell mediated immune response in mice, and thus, can be a potent vaccine candidate against anthrax.

Neutrophil mediated inflammatory lung damage following single Sub lethal inhalation exposure to plant protein toxin abrin in mice.

Abrin, a highly toxic plant protein found in the seeds of Abrus precatorius plant. To date, there is no antidote against abrin intoxication. Abrin is toxic by all routes of exposure, but inhalation exposure is the most toxic of all routes. Present study was conducted to evaluate the acute inhalation toxicity of aerosolized abrin in BALB/c mice. Animals were exposed to 0.2 and 0.8LC50 doses of aerosolized abrin and evaluated at 1 and 3 day post toxin exposure. Bronchoalveolar fluid from lungs was used for evaluation of markers for lung injury. Abrin inhalation exposure caused rise in LDH activity, protein content, increase in ß-glucuronidase and myeloperoxidase activity. Increase in CRP activity, MMP-9 expression and recruitment of CD11b + inflammatory cells in lungs was also observed which was associated with severe inflammation and lung damage. Histopathological findings support the lung damage after abrin exposure. Our results indicate lung injury after single aerosol inhalation exposure, associated with excessive inflammation, oxidative stress, pulmonary edema followed by lung damage. These results could supplement treatment strategies and planning for therapeutic approaches against aerosolized abrin inhalation exposure.

Oxidative and histopathological alterations after sub-acute exposure of diisopropyl phosphorofluoridate in mice: Beneficial effect of N­acetylcysteine.

AIMS: Protective efficacy of N­acetylcysteine (NAC) was assessed against sub-acute diisopropyl phosphorofluoridate (DFP) poisoning in mice. MAIN METHODS: Mice were allocated into nine groups of six each: vehicle control; DFP (0.125 LD50 ≈ 0.483 mg/kg bwt, s.c.); DFP + Atropine (ATR, 10 mg/kg bwt, i.p., 0 min); DFP + Pralidoxime (2-PAM, 30 mg/kg bwt, i.m., 0 min); DFP + NAC (150 mg/kg bwt, i.p., -60 min); DFP + ATR + NAC; DFP + 2-PAM + NAC; DFP + ATR + 2-PAM; and DFP + ATR + 2-PAM + NAC. Animals received various treatments for 21 d daily. Plasma butyrylcholinesterase (BChE) was measured after 7, 14 and 21 d of exposure. Brain acetylcholinesterase (AChE) and reduced glutathione (GSH), malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD) were measured (brain, liver and kidney) after 21 d of exposure. Histopathology, immunohistochemistry, and Western blot for inducible nitric oxide synthase (iNOS) and c-fos were also performed. KEY FINDINGS: DFP significantly reduced BChE and AChE levels. Diminished GSH, CAT, SOD (brain and liver), GPx, GR, and elevated MDA (Brain) levels were also observed. DFP caused notable histopathology (brain, liver and kidney) and over expression of iNOS, and c-fos proteins (brain). NAC enhanced the protective efficacy of ATR and 2-PAM in most parameters, without any appreciable protection in iNOS and c-fos expression. SIGNIFICANCE: NAC as an adjunct with ATR and 2-PAM, exhibited marked beneficial effects against sub-acute DFP poisoning, indicating its possible implications in the management of OP poisoning.

Comparative evaluation of antidotal efficacy of 2-PAM and HNK-102 oximes during inhalation of sarin vapor in Swiss albino mice.

Efficacy of two oximes treatments evaluated during inhalation of sarin vapor (LCt50, 755.9 mg/min/m3) in simulated real scenario in vivo. Majority of mice either became moribund or died within 1-2 min during exposure to multifold-lethal concentrations of sarin vapor. Protection indices were determined by exposing to sarin vapor in two sessions, 1 min exposure followed by treatments with or without HNK-102 (56.56 mg/kg, im) or 2-PAM (30 mg/kg, im) and atropine (10 mg/kg, ip), and again exposed for remaining 14 min. Protection offered by HNK-102 was found to be four folds higher compared to 2-PAM in the same toxic environment. Secondly, sub-lethal concentration of sarin vapor (0.8 × LCt50 or 605 mg/min/m3), 24 h post investigations revealed that the oximes could not reactivate brain and serum acetylcholinesterase (AChE) activity. The treatments prevented increase in protein concentration (p < .05) and macrophages infiltration compared to sarin alone group in broncho-alveolar lavage fluid. Lung histopathology showed intense peribronchial infiltration and edema with desquamating epithelial lining and mild to moderate alveolar septal infiltration in sarin and atropine groups, respectively. Noticeable peeling-off observed in epithelial lining and sporadic mild infiltration of epithelial cells at bronchiolar region in 2-PAM and HNK-102 groups, respectively. The oximes failed to reactivate AChE activity; however, the mice survived up to 6.0 × LCt50, proved involvement of non-AChE targets in sarin toxicity. Atropine alone treatment was found to be either ineffective or increased the toxicity. HNK-102, exhibited better survivability with lung protection, can be considered as a better replacement for 2-PAM to treat sarin inhalation induced poisoning.

Biochemical and functional properties of a lectin purified from the seeds of Cicer arietinum L.

A 35 kDa rabbit erythrocyte agglutinating lectin from the seeds of Cicer arietinum was purified and designated as CAL. The lectin was inhibited by fetuin and N-acetyl-d-galactosamine at a concentration of 20 and 50 mM respectively, but not by simple mono or oligosaccharides. CAL is active between pH 5 and 10 presented thermo stability up to 50 °C and demonstrated DNA damage inhibition at 30 µg concentration. The lectin elicited maximum mitogenic activity towards mice splenocytes at 7.5 µg ml- 1. CAL exerted an inhibitory activity on HIV-1 reverse transcriptase with IC50 of 180 µM. CAL abilities in animal bioassay resulted decreased levels of total triglyceride and creatinine. In vitro and in vivo studies revealed that CAL may constitute an important role impending biomedical applications.

Comparative safety evaluation of riot control agents of synthetic and natural origin.

Riot control agents (RCA) are lachrymatory, irritating compounds which temporarily incapacitate the uncontainable crowd. Ortho-Chlorobenzylidene-malononitrile (CS), 2-chloroacetophenone (CN), dibenz[b,f]1:4-oxazepine (CR), and nonivamide (PAVA) are synthetic RCAs, while oleoresin extract of chili known as oleoresin capsicum (OC) a natural irritant has been in use by various law enforcement agencies. Though efficacy of these agents is beyond doubt, they suffer from certain drawbacks including toxicity, production cost, and ecological compatibility. Presently, we have evaluated the safety of CR, OC, and PAVA on inhalation variables along with oral lethality. Additionally, the liver function test (LFT) in serum and lungs function was evaluated in broncho-alveolar-lavage fluid (BALF), both collected on the 14th day after RCA exposure. Animals then sacrificed and histopathology of liver and lungs was carried out. Results showed OC and PAVA to be more toxic than CR with an oral LD50 of 150 and 200 mg/kg body weight, respectively, while CR was safe at >3 g/kg body weight. All three agents caused severe impairment of respiratory variables bringing down normal respiration by >80% with rise in sensory irritation. Recovery from the irritating effect of CR was more rapid than OC and PAVA. LFT and BALF variables were not significantly different from that of control. There were no remarkable histopathological changes in liver and lungs. Hence, as per results, CR is safest among all synthetic and natural origin RCAs and can be safely used for effective dispersion of disobedient mob.

Evaluation of abrin induced nephrotoxicity by using novel renal injury markers.

Abrin is a potent plant toxin analogous to ricin that is derived from the seeds of Abrus precatorius plant. It belongs to the family of type II ribosome-inactivating proteins and causes cell death by irreversibly inactivating ribosomes through site-specific depurination. In this study we examined the in vivo nephrotoxicity potential of abrin toxin in terms of oxidative stress, inflammation, histopathological changes and biomarkers of kidney injury. Animals were exposed to 0.5 and 1.0 LD50 dose of abrin by intraperitoneal route and observed for 1, 3, and 7 day post-toxin exposure. Depletion of reduced glutathione and increased lipid peroxidation levels were observed in abrin treated mice. In addition, abrin also induced inflammation in the kidneys as observed through expression of MMP-9 and MMP-9/NGAL complex in abrin treated groups by using zymography method. Nephrotoxicity was also evaluated by western blot analysis of kidney injury biomarkers including Clusterin, Cystatin C and NGAL, and their results indicate severity of kidney injury in abrin treated groups. Kidney histology confirmed inflammatory changes due to abrin. The data generated in the present study clearly prove the nephrotoxicity potential of abrin.

In vivo protection studies of bis-quaternary 2-(hydroxyimino)- N-(pyridin-3-yl) acetamide derivatives (HNK oximes) against tabun and soman poisoning in Swiss albino mice.

The study reports antidotal efficacy of three HNK [ bis quaternary 2-(hydroxyimino)-N-(pyridin-3yl) acetamide derivatives] and pralidoxime (2-PAM), against soman and tabun poisoning in Swiss albino mice. Protection index (PI) was determined (treatment doses: HNK oximes, ×0.20 of their median lethal dose (LD50) and 2-PAM, 30 mg/kg, intramuscularly (im)) together with atropine (10 mg/kg, intraperitoneally). Probit log doses with difference of 0.301 log of LD50 of the nerve agents administered and inhibition of acetylcholinesterase (AChE) activity by 50% (IC50) was calculated at optimized time in brain and serum. Using various doses of tabun and soman (subcutaneously (sc)), in multiples of their IC50, AChE reactivation ability of the oximes was studied. Besides, acute toxicity (0.8× LD50, im, 24 h postexposure) of HNK-102 and 2-PAM was also compared by determining biochemical, hematological variables and making histopathological observations. Protection offered by HNK-102 against tabun poisoning was found to be four times higher compared to 2-PAM. However, nearly equal protection was noted with all the four oximes against soman poisoning. HNK-102 reactivated brain AChE activity by 1.5 times more than 2-PAM at IC50 dose of soman and tabun. Acute toxicity studies of HNK-102 and 2-PAM showed sporadic changes in urea, uric acid, aspartate aminotransferase, and so on compared to control group, however, not supported by histopathological investigations. The present investigation showed superiority of newly synthesized HNK-102 oxime over standard 2-PAM, as a better antidote, against acute poisoning of tabun (4.00 times) and soman (1.04 times), in Swiss albino mice.

Prophylactic efficacy of S-2(2-aminoethylamino)ethyl phenyl sulfide (DRDE-07) against sulfur mustard induced lung toxicity in mice.

OBJECTIVE: The present study was planned to investigate the prophylactic efficacy of S-2(2-aminoethylamino)ethyl phenyl sulfide (DRDE-07), against topically applied SM induced pulmonary toxicity in mouse. MATERIALS AND METHODS: Animals were pretreated with S-2(2-aminoethylamino)ethyl phenyl sulfide (DRDE-07) (249.4 mg/kg by oral gavage) 30 minutes before SM exposure. The SM (6.48 mg/kg) was applied on hair clipped dorsocaudal region (percutaneous) of the animal. The animals were sacrificed on day 1, 3, 5 and 7. The biochemical changes those were observed in the bronchoalveolar lavage (BAL) fluid and lung tissue included protein, LDH, MPO, ß-glucuronidase, MMP-2, MMP-9, activated macrophages, reduced glutathione and lipid peroxidation level. RESULTS AND DISCUSSION: Pretreatment with DRDE-07 (0.2 LD50) attenuated SM-induced changes at all time point tested. BAL fluid biochemical endpoints indicated epithelial and endothelial cell damages as evidenced by increase in BAL protein, LDH level and increased number of activated macrophages. The increased myeloperoxidase activity and ß-glucuronidase level exhibited the degranulation of neutrophils due to SM toxicity in lung. The zymogrphy analysis of BAL fluid showed a significant increase in matrix metalloproteases (MMP) activity due to inflammatory cells accumulation. CONCLUSION: Thirty minutes pretreatment with DRDE-07 decreased vascular permeability reduced the inflammation and oxidative stress, hence may be recommended as a potential prophylactic agent for SM intoxication.