Manjari Medika Grape Seed Extract Protects Methotrexate-Induced Hepatic Inflammation: Involvement of NF-κB/NLRP3 and Nrf2/HO-1 Signaling System.

Objective: Grape Seed Extract is a natural source of various polyphenols, which have been shown to possess potent antioxidant and free radical-scavenging activities. The earlier studies have reported that grape seed extract exhibits broad-spectrum pharmacological activities. Therefore, studying the hepatoprotective effects and elucidation of mechanisms of action of the Indian Variety, Manjari Medika grape seed extract (GSE), may give an insight into therapeutic benefits. Methotrexate (MTX) is the first-line pharmacological therapy for different rheumatic diseases. The major adverse events such as hepatotoxicity are evident even in the low doses used for the treatment. The present study investigated the role of MTX on hepatic damage in murine liver and the plausible protective effects of the Indian grape variety, Manjari Medika grape seed extract, in ameliorating it. Methods and Results: To assess the hepatological modulation, mice were divided into eight groups to investigate the ameliorative potential of this GSE (75 and 125 mg/kg) and correlate the experimental findings. The active components of the extract were assessed through UPLC-(ESI)-QToF-MS analysis. On the other hand, various biochemical and immunological indices were carried out to correlate the experimental data. The result demonstrated that the prophylactic administration of GSE reduced MTX-induced hepatic toxicity indices, which subsequently restored the hepatic morphological architecture. Moreover, the application of GSE in a dual dosage (75 and 125 mg/kg) suppressed MTX-induced reactive oxygen species generation, followed by lipid peroxidation and cellular nitrite formation. MTX-induced inflammasome activation through the redox-assisted cascade of TLR4/NF-κB signaling was further reduced by applying the GSE. The results showed that the activation of cytoprotective transcription factor Nrf2 enhanced the level of endogenous antioxidants. Furthermore, through the regulation of TLR4/NF-κB and Nrf2/HO-1 axis, this extract could reduce the MTX-mediated hepatic damage. Conclusion: Our findings suggest that Manjari Medika seed extract could be used as a therapeutic agent to relieve the side effects of MTX and other hepatic disorders.

Elucidation of False Detection of Pesticides During Residue Analysis in Indian Tobacco by Multidimensional GC-MS.

BACKGROUND: False detection of pesticides in agricultural produce may raise serious questions regarding both consumer safety and trade. High levels of delta-hexachlorocyclohexane (δ-HCH; 11.7-22.3 mg/kg) were detected in some tobacco samples in a retention time-based GC analysis. Hence, the selection of an appropriate analytical method is an uncompromisable necessity. OBJECTIVES: This research work aimed to elucidate false detection of pesticides along with identification of coeluting tobacco matrix compounds to understand the dynamics of false detection with an increase in the number of analyzed pesticides and to screen suitable analytical methods. METHODS: Initially, retention time-based GC analysis was performed for monitoring of 10 pesticide residues in tobacco leaf matrix, followed by GC-MS/selected-ion monitoring (SIM) analysis. Then, the total number of pesticides to be analyzed was increased to 47, and residue analysis was performed by involving GC-MS/SIM and multidimensional (MD) GC-MS. RESULTS: A false-positive detection of δ-HCH due to a coeluting tobacco aroma compound, neophytadiene, during residue analysis of 10 pesticides in tobacco (Nicotiana tabacum L.) leaf was observed. This problem was resolved by employing the unique quantifier and qualifier ions in SIM mode. However, with 47 pesticides, neophytadiene completely masked the signal of δ-HCH, which resulted in an impure spectrum of δ-HCH (<30% similarity match) even after application of selective quantifier and qualifier ions. Finally, MDGC-MS analysis could resolve it by chromatographic separation of the said analyte from the coeluting matrix compound. CONCLUSIONS: The findings of this work offer the potential to minimize false reporting of target pesticides to comply with consumer safety and trade standards. HIGHLIGHTS: The study identifies various tobacco matrix compounds coeluting with pesticides during multiresidue analysis. Neophytadiene, a tobacco aroma compound, resulted in false-positive detection of δ-HCH. The MDGC-MS could be effectively used as a confirmatory analysis tool for reliable detection of pesticide residue in tobacco leaf matrix.

Rapid and sensitive multiresidue analysis of pesticides in tobacco using low pressure and traditional gas chromatography tandem mass spectrometry.

Public exposure to pesticide residues through the main/side-stream smoke of tobacco (Nicotiana tabacum L.) is an international concern. This article reports optimization and validation of large-scale multiresidue analysis methods involving low pressure and traditional gas chromatography tandem mass spectrometry in compliance with the guidance residue levels (GRLs) of Cooperative Centre for Scientific Research Relative to Tobacco (CORESTA). Analysis by low pressure GC-MS/MS offered three times rapid turn around time over the traditional GC-MS/MS with limits of quantifications (LOQs) less than 2µg/L for all the 259 test compounds and the recoveries in the range of 70-118% (±20%) at 10 and 20µg/kg levels of fortification.

Assessment of organochlorine pesticide residues in Indian flue-cured tobacco with gas chromatography-single quadrupole mass spectrometer.

Presence of pesticide residues in tobacco increases health risk of both active and passive smokers, apart from the imminent potential health problems associated with it. Thus, monitoring of pesticide residue is an important issue in terms of formulating stringent policies, enabling global trade and safeguarding the consumer's safety. In this study, a gas chromatography-single quadrupole mass spectrometry (GC-MS) method based upon quantifier-qualifier ions (m/z) ratio was employed for detecting and assessing ten organochlorine pesticide residues (α-HCH, ß-HCH, γ-HCH, δ-HCH, 2,4-DDT, 4,4-DDT, endrin, α-endosulfan, ß-endosulfan and endosulfan sulphate) in 152 flue-cured (FC) tobacco leave samples from two major tobacco growing states, Karnataka and Andhra Pradesh, of India. In the majority of samples, pesticide residue levels were below the limit of quantification (LOQ). In few samples, pesticide residues were detected and they found to comply with the guidance residue levels (GRL) specifications of the Cooperation Center for Scientific Research Relative to Tobacco (CORESTA). Detection of the phase out pesticides like DDT/HCH might be due to transfer of persistent residues from the environmental components to the plant. This is the first report on these ten organochlorine pesticide residues in Indian FC tobacco.

Optimization of a sample preparation method for multiresidue analysis of pesticides in tobacco by single and multi-dimensional gas chromatography-mass spectrometry.

A selective and sensitive multiresidue analysis method, comprising 4 7pesticides, was developed and validated in tobacco matrix. The optimized sample preparation procedure in combination with gas chromatography mass spectrometry in selected-ion-monitoring (GC-MS/SIM) mode offered limits of detection (LOD) and quantification (LOQ) in the range of 3-5 and 7.5-15ng/g, respectively, with recoveries between 70 and 119% at 50-100ng/g fortifications. In comparison to the modified QuEChERS (Quick-Easy-Cheap-Effective-Rugged-Safe method: 2g tobacco+10ml water+10ml acetonitrile, 30min vortexing, followed by dispersive solid phase extraction cleanup), the method performed better in minimizing matrix co-extractives e.g. nicotine and megastigmatrienone. Ambiguity in analysis due to co-elution of target analytes (e.g. transfluthrin-heptachlor) and with matrix co-extractives (e.g. δ-HCH-neophytadiene, 2,4-DDE-linolenic acid) could be resolved by selective multi-dimensional (MD)GC heart-cuts. The method holds promise in routine analysis owing to noticeable efficiency of 27 samples/person/day.

Ensuring selectivity and sensitivity by timed- and ultra-selective reaction monitoring during gas chromatography-tandem mass spectrometric determination of pesticides.

In multiresidue analysis, as the complexity of matrix increases, matrix co-extractives might co-elute and interfere with the detection of target analytes, and thereby result in false positives and erroneous quantifications. This paper aims to evaluate the combination of enhanced mass resolution and concurrent selectivity and sensitivity in analyzing a complex mixture of 341 pesticides in grape, orange, tomato, okra and spinach by gas chromatography triple quadrupole mass spectrometry (GC-MS/MS). Timed-selective reaction monitoring (t-SRM) was found advantageous over segment SRM in terms of ease of method optimization and sensitivity. The optimized t-SRM method was used to test the applicability of ultra-SRM (USRM) in filtering out co-eluting interfering matrix compounds. False detections of benfluralin, dimethoate, etc. could be avoided when mass resolution was increased from 0.7 to 0.4 and 0.2Da (full width at half maximum). Similar observations were noted for chlorobenzilate, spiromesifen, tebuconazole, etc., in grape; omethoate, bendiocarb, monocrotophos, etc. in orange; omethoate, dimethoate, ethoxyquin, atrazine, etc., in tomato; and dichlobenil, omethoate, propoxur, monocrotophos, etc., in okra. The analysis at higher mass resolution could significantly minimize matrix effects (ME), e.g., 34% at 0.7Da for spiroxamine in grape to 0.6% (0.4Da) and -44% (0.7Da) for dichlorvos in okra to 8.8% (0.1Da). A feature called "quantitation enhanced data-dependent (QED) scan feature" was found effective in targeted screening to confirm the detection of atrazine, azoxystrobin, λ-cyhalothrin, etc. at trace quantities in incurred samples and avoid false detection of chlorpyrifos.

A novel HLA (HLA-A*0201) transgenic rabbit model for preclinical evaluation of human CD8+ T cell epitope-based vaccines against ocular herpes.

We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8(+) T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8(+) T cell peptide epitopes from HSV-1 glycoprotein D (gD(53-61), gD(70-78), and gD(278-286)) were joined with a promiscuous human CD4(+) T cell peptide epitope (gD(49-82)) to construct three separate pairs of CD4-CD8 peptides. Each CD4-CD8 peptide pair was then covalently linked to an N(epsilon)-palmitoyl-lysine residue via a functional base lysine amino group to construct CD4-CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4-CD8 HSV-1 gD lipopeptides. The HSV-gD-specific T cell responses induced by the mixture of CD4-CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD(49-82)-specific CD4(+) T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD(53-61), gD(70-78), and gD(278-286)-specific CD8(+) T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8(+) T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8(+) T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4-CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed.

Nasolacrimal duct closure modulates ocular mucosal and systemic CD4(+) T-cell responses induced following topical ocular or intranasal immunization.

Both topical ocular and topical intranasal immunizations have been reported to stimulate the ocular mucosal immune system (OMIS) and the systemic immune system. Nasolacrimal ducts (NLDs) are the connecting bridges between the OMIS and nasal cavity-associated lymphoid tissue (NALT). These ducts drain topical ocularly administrated solutions into the inferior meatus of the nose to reach the NALT. Inversely, NLDs also drain intranasally administrated solutions to the mucosal surface of the eye and thus the OMIS. This unique anatomical connection between the OMIS and NALT systems provoked us to test whether the OMIS and NALT are immunologically interdependent. In this report, we show that both topical ocular administration and topical intranasal administration of a mixture of immunodominant CD4(+) T-cell epitope peptides from herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) emulsified with the CpG(2007) mucosal adjuvant are capable of inducing local (in conjunctiva) as well as systemic (in spleen) HSV-peptide-specific CD4(+) T-cell responses. Interestingly, surgical closure of NLDs did not significantly alter local ocular mucosal CD4(+) T-cell responses induced following topical ocular immunization but did significantly enhance systemic CD4(+) T-cell responses (as measured by both T-cell proliferation and gamma interferon (IFN-gamma) production; P < 0.005). In contrast, NLD closure significantly decreased ocular mucosal, but not systemic, CD4(+) T-cell responses following intranasal administration of the same vaccine solution (P < 0.001). The study suggests that NALT and the OMIS are immunologically interconnected.

Streptococcal antibodies among rural school children in Bangladesh.

The upper limit of normal values of group A streptococcal antibodies should be known for a population concerned because it is influenced by many local conditions. As yet the reference value of the these antibodies has not been determined by using a quantitative method among Bangladeshi children. We determined the reference value of anti-streptolysin O and anti-deoxyribonuclease B among 361 apparently healthy rural Bangladeshi primary school children (aged 5 to 14 years, mean 9.2 years). Anti-streptolysin O was measured by an auto-analyzer and antideoxyribonuclease B was measured by microtitre method. The geometric mean titres for the entire group was 241 IU/ml and 222 IU/ml for anti-streptolysin O and anti-deoxyribonuclease B respectively. The upper limit of normal values (80th percentile) was 390 and 340 for anti-streptolysin O and anti-deoxyribonuclease B, respectively. These limits should be of value to physicians, epidemiologists and clinical laboratory personnel as well.

New antioxidant and antimicrobial ellagic acid derivatives from Pteleopsis hylodendron.

Bioassay-guided isolation of two new compounds, 3,4-methylenedioxy-3'-O-methyl-4'-O-glucoside ellagic acid (1) and the pteleoellagic acid derivative (2), from the stem bark of Pteleopsis hylodendron is reported along with 3,4-methylenedioxy-3'-O-methyl ellagic acid (3), 3,3'-di-O-methyl ellagic acid (4) and 3,3',4'-tri-O-methyl ellagic acid (5), which were obtained for the first time from this plant. The structures of these compounds were elucidated with the help of spectroscopic studies. Compounds 1 and 4 were found to have significant antioxidant activity, while compounds 1-4 showed antibacterial activity against different pathogenic bacteria.

Reference value of immunoglobulins in healthy school children of Bangladesh.

The reference value of immunoglobulins (Igs) should be known for a population concerned because it is influenced by many clinical and local conditions. As yet the reference value of the Igs have not been determined in Bangladeshi children. This study determined the reference value of Igs in apparently healthy 261 rural Bangladeshi primary school children (aged 5 to 14 years, mean 9.3 years). IgG, IgM and IgA were determined by an auto-analyzer. The mean (standard deviation) value of IgG was 1728 (344) mg/dl. The corresponding values for IgM and IgA were 200 (88) and 163 (63) mg/dl, respectively. The 95% reference value calculation in all subjects showed that the range for IgG was 1103 to 2524, IgM was 92 to 390, and IgA was 72 to 325 mg/dl. These values could be used to evaluate Ig status in children with a variety of clinical conditions.

Erythrocyte sedimentation rate in healthy school children of Bangladesh.

The reference value of erythrocyte sedimentation rate (ESR) should be established for a population concerned because it is influenced by many local conditions. It has not yet been determined in healthy children of Bangladesh. This study was done to determine the reference value of ESR in Bangladeshi children. ESR was determined in a sample of 413 'healthy' primary school students (259 boys and 154 girls, mean age 8.7 years). Ninety-five percent of them had ESR < or = 30 mm (Westergren 1 h). Our findings suggest that ESR value > 30 mm (Westergren 1 h) should be considered high in Bangladeshi primary school children.