Genome-wide identification and characterization of NBLRR genes in finger millet (Eleusine coracana L.) and their expression in response to Magnaporthe grisea infection.

BACKGROUND: The nucleotide binding site leucine rich repeat (NBLRR) genes significantly regulate defences against phytopathogens in plants. The genome-wide identification and analysis of NBLRR genes have been performed in several species. However, the detailed evolution, structure, expression of NBLRRs and functional response to Magnaporthe grisea are unknown in finger millet (Eleusine coracana (L.) Gaertn.). RESULTS: The genome-wide scanning of the finger millet genome resulted in 116 NBLRR (EcNBLRRs1-116) encompassing 64 CC-NB-LRR, 47 NB-LRR and 5 CCR-NB-LRR types. The evolutionary studies among the NBLRRs of five Gramineae species, viz., purple false brome (Brachypodium distachyon (L.) P.Beauv.), finger millet (E. coracana), rice (Oryza sativa L.), sorghum (Sorghum bicolor L. (Moench)) and foxtail millet (Setaria italica (L.) P.Beauv.) showed the evolution of NBLRRs in the ancestral lineage of the target species and subsequent divergence through gene-loss events. The purifying selection (Ka/Ks < 1) shaped the expansions of NBLRRs paralogs in finger millet and orthologs among the target Gramineae species. The promoter sequence analysis showed various stress- and phytohormone-responsive cis-acting elements besides growth and development, indicating their potential role in disease defence and regulatory mechanisms. The expression analysis of 22 EcNBLRRs in the genotypes showing contrasting responses to Magnaporthe grisea infection revealed four and five EcNBLRRs in early and late infection stages, respectively. The six of these nine candidate EcNBLRRs proteins, viz., EcNBLRR21, EcNBLRR26, EcNBLRR30, EcNBLRR45, EcNBLRR55 and EcNBLRR76 showed CC, NB and LRR domains, whereas the EcNBLRR23, EcNBLRR32 and EcNBLRR83 showed NB and LRR somains. CONCLUSION: The identification and expression analysis of EcNBLRRs showed the role of EcNBLRR genes in assigning blast resistance in finger millet. These results pave the foundation for in-depth and targeted functional analysis of EcNBLRRs through genome editing and transgenic approaches.

Bottle gourd IC-0262269, a super-susceptible genotype to tomato leaf curl Palampur virus.

While conducting field trial of 82 genotypes of bottle gourd at Delhi during 2020-2021, a particular genotype, IC-0262269 was found to be affected by chlorotic curly stunt disease (CCSD). The affected plants were severely stunted and bearing very small chlorotic and crinkle leaves. The disease incidence in the said genotype was as high as 80% among different replicated trial blocks. The application of PCR using a generic primers specific to begomoviruses, as well as species-specific PCR diagnostics to six tomato-infecting begomoviruses: tomato leaf curl New Delhi virus (ToLCNDV), tomato leaf curl Palampur virus (ToLCPalV), tomato leaf curl Joydebpur virus (ToLCJoV), tomato leaf curl Gujrat virus (ToLCGuV), tomato leaf curl Bangalore virus (ToLCBV), and chilli leaf curl virus (ChiLCV) showed that, only ToLCPalV could be detected in the genotype IC-0262269. Following, rolling circle amplification, cloning and sequencing of full-length DNA-A and DNA-B genome of an isolate BoG1-ND from the genotype IC-0262269 revealed association of ToLCPalV with the disease. The successful agro-infection of the cloned genome of BoG1-ND (DNA-A and DNA-B) in the plants of Nicotiana benthamiana and bottle gourd demonstrated that ToLCPalV is the causal begomovirus of CCSD. The study provides the first evidence of the natural occurrence of ToLCPalV in bottle gourd crop and also showed that the bottle gourd genotype IC-0262269 is super-susceptible to ToLCPalV. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03838-y.

Geographical distribution of tomato-infecting begomoviruses in major cucurbits in India: a diagnostic analysis using begomovirus species specific PCR.

Cucurbits are an essential summer-season vegetable crops, but they are highly vulnerable from a range of abiotic and biotic factors. One of the significant biotic factors posing a growing menace to the production of major cucurbits in India is the emergence of tomato-infecting begomoviruses. In this study, we utilized PCR-based species-specific primers, developed earlier in our laboratory for the detection of begomoviruses infecting tomato and chilli plants, to identify begomoviruses in cucurbits across various regions of India. Leaf samples from major cucurbits were collected from different regions of Haryana, Delhi, Uttar Pradesh, Chhattisgarh, Maharashtra, Telangana and Karnataka, during the year 2020-2021. Total nucleic acid (TNA) was extracted from the samples and subjected to PCR using a generic primer specific to begomoviruses. The samples that exhibited positive amplification were further tested using six different species-specific primers targeting specific begomovirus species, namely Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPalV), Tomato leaf curl Bangalore virus (ToLCBV), Tomato leaf curl Joydebpur virus (ToLCJoV), Tomato leaf curl Gujarat virus (ToLCGuV), and Chilli leaf curl virus (ChiLCV). The PCR analysis revealed that among the 551 plant samples tested, a total of 124 samples exhibited positive amplification using the universal begomovirus PCR. Specifically, 47 samples tested positive for ToLCNDV, 73 samples were positive for ToLCPalV and only one sample showed positive amplification for ChiLCV. However, none of the samples tested positive for ToLCJoV, ToLCGuV and ToLCBV. These findings from our study indicate the prevalence of ToLCNDV and ToLCPalV in major cucurbits across India. Furthermore, the study highlights the varied distribution of begomoviruses in major cucurbits between northern and southern regions of India.

Delivery of progeny virus from the infectious clone of cucumber green mottle mosaic virus and quantification of the viral load in different host plants.

Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is a widely occurring tobamovirus in cucurbits. The genome of CGMMV has been used previously for the expression of foreign genes in the plant. High throughput delivery and high viral titer are important requirements of foreign protein expression in plant through virus genome-based vector, in this study, Agrobacterium containing infectious construct of CGMMV was infiltrated through syringe, vacuum and high-speed spray to N. benthamiana, cucumber and bottle gourd leaves. The success rate of systemic infection of CGMMV agro-construct through all three methods was higher (80-100%) in N. benthamiana compared to the cucurbits (40-73.3%). To determine the high-throughput delivery of CGMMV in the plant system, four delivery methods viz. rubbing, syringe infiltration, vacuum infiltration and high-speed spray using the progeny virus derived through CGMMV agro-construct were compared in the three different plant species. Based on the rate of systemic infection and time required to perform delivery by different methods, vacuum infiltration was found most efficient for the high-throughput delivery of CGMMV. The quantification of CGMMV through qPCR revealed that CGMMV load varied considerably in leaf and fruit tissues depending with the time of infection. Immediately after expression of symptoms, a high load of CGMMV (~ 1 µg/100 mg of tissues) was noticed in young leaves of N. benthamiana and cucumber. In bottle gourd leaves, the CGMMV load was far low compared to N. benthamiana and cucumber plants. In the fruit tissues of cucumber and bottle gourd higher virus load was observed in mature fruit but not in immature fruit. The findings of the present study will serve as an important base line information to produce foreign protein through CGMMV genome-vector. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03630-y.

Exploring the diversity of virulence genes in the Magnaporthe population infecting millets and rice in India.

Blast pathogen, Magnaporthe spp., that infects ancient millet crops such pearl millet, finger millet, foxtail millet, barnyard millet, and rice was isolated from different locations of blast hotspots in India using single spore isolation technique and 136 pure isolates were established. Numerous growth characteristics were captured via morphogenesis analysis. Among the 10 investigated virulent genes, we could amplify MPS1 (TTK Protein Kinase) and Mlc (Myosin Regulatory Light Chain edc4) in majority of tested isolates, regardless of the crop and region where they were collected, indicating that these may be crucial for their virulence. Additionally, among the four avirulence (Avr) genes studied, Avr-Pizt had the highest frequency of occurrence, followed by Avr-Pia. It is noteworthy to mention that Avr-Pik was present in the least number of isolates (9) and was completely absent from the blast isolates from finger millet, foxtail millet, and barnyard millet. A comparison at the molecular level between virulent and avirulent isolates indicated observably large variation both across (44%) and within (56%) them. The 136 Magnaporthe spp isolates were divided into four groups using molecular markers. Regardless of their geographic distribution, host plants, or tissues affected, the data indicate that the prevalence of numerous pathotypes and virulence factors at the field level, which may lead to a high degree of pathogenic variation. This research could be used for the strategic deployment of resistant genes to develop blast disease-resistant cultivars in rice, pearl millet, finger millet, foxtail millet, and barnyard millet.

Composition and Codon Usage Pattern Results in Divergence of the Zinc Binuclear Cluster (Zn(II)2Cys6) Sequences among Ascomycetes Plant Pathogenic Fungi.

Zinc binuclear cluster proteins (ZBC; Zn(II)2Cys6) are unique to the fungi kingdom and associated with a series of functions, viz., the utilization of macromolecules, stress tolerance, and most importantly, host-pathogen interactions by imparting virulence to the pathogen. Codon usage bias (CUB) is the phenomenon of using synonymous codons in a non-uniform fashion during the translation event, which has arisen because of interactions among evolutionary forces. The Zn(II)2Cys6 coding sequences from nine Ascomycetes plant pathogenic species and model system yeast were analysed for compositional and codon usage bias patterns. The clustering analysis diverged the Ascomycetes fungi into two clusters. The nucleotide compositional and relative synonymous codon usage (RSCU) analysis indicated GC biasness toward Ascomycetes fungi compared with the model system S. cerevisiae, which tends to be AT-rich. Further, plant pathogenic Ascomycetes fungi belonging to cluster-2 showed a higher number of GC-rich high-frequency codons than cluster-1 and was exclusively AT-rich in S. cerevisiae. The current investigation also showed the mutual effect of the two evolutionary forces, viz. natural selection and compositional constraints, on the CUB of Zn(II)2Cys6 genes. The perseverance of GC-rich codons of Zn(II)2Cys6 in Ascomycetes could facilitate the invasion process. The findings of the current investigation show the role of CUB and nucleotide composition in the evolutionary divergence of Ascomycetes plant pathogens and paves the way to target specific codons and sequences to modulate host-pathogen interactions through genome editing and functional genomics tools.

Targeting STAT3 signaling pathway in cancer by agents derived from Mother Nature.

Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that reside in the cytoplasm of several types of cells. In canonical signaling, upon stimulation by cytokines and growth factors, STATs get activated and translocate into the nucleus to transcribe target genes. Among STATs, the STAT3 variant has been studied extensively and implicated in diverse human malignancies. Transcriptionally, STAT3 can upregulate the expression of genes associated with cell proliferation, antiapoptosis, prosurvival, angiogenesis, metastasis, and immune evasion. STAT3 can be constitutively activated in a broad range of human cancers including solid as well as hematological tumors and overexpression of STAT3 has been observed in a wide-range of patient-derived tumor tissue samples that may contribute to dismal prognosis. In contrast, blockade of STAT3 activation using inhibitors or knockdown systems can markedly suppress tumor progression, thus highlighting the significance of abrogating STAT3 signaling cascade in cancer therapy. In this review, we have provided a comprehensive overview of mechanisms of STAT3 signal transduction and its endogenous negative modulators, the role of STAT3 in oncogenesis, the interplay of miRNAs in STAT3 signaling, and mechanisms involved in persistent activation of STAT3. Furthermore, the review also provides a detailed overview of STAT3 signaling inhibition by selected natural compounds, which have displayed potent activity in various preclinical cancer model.

Fusarium oxysporum f. sp. lycopersici causal agent of vascular wilt disease of tomato: Biology to diversity- A review.

Tomato (Lycopersicon esculentum) is one of the widely grown vegetables worldwide. Fusarium oxysporum f. sp. lycopersici (FOL) is the significant contributory pathogen of tomato vascular wilt. The initial symptoms of the disease appear in the lower leaves gradually, trail by wilting of the plants. It has been reported that FOL penetrates the tomato plant, colonizing and leaving the vascular tissue dark brown, and this discoloration extends to the apex, leading to the plants wilting, collapsing and dying. Therefore, it has been widely accepted that wilting caused by this fungus is the result of a combination of various physiological activities, including the accumulation of fungal mycelia in and around xylem, mycotoxin production, inactivation of host defense, and the production of tyloses; however, wilting symptoms are variable. Therefore, the selection of molecular markers may be a more effective means of screening tomato races. Several studies on the detection of FOL have been carried out and have suggested the potency of the technique for diagnosing FOL. This review focuses on biology and variability of FOL, understanding and presenting a holistic picture of the vascular wilt disease of tomato in relation to disease model, biology, virulence. We conclude that genomic and proteomic approachesare greater tools for identification of informative candidates involved in pathogenicity, which can be considered as one of the approaches in managing the disease.

Specific PCR-based detection of Phomopsis vexans the cause of leaf blight and fruit rot pathogen of Solanum melongena L.

Leaf blight and fruit rot disease caused by Phomopsis vexans is a devastating disease of brinjal. The detection of P. vexans in plant parts and seeds of brinjal can be complicated, mainly when the inoculum is present at low levels and/or overgrown by fast-growing saprophytic fungi or other seed-borne fungi. A PCR-based diagnostic method was developed with specific primers designed based on sequence data of a region consisting of the 5·8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans. The efficiency and specificity of primer pairs PvexF/PvexR designed were established by PCR analysis of DNA from P. vexans strains isolated from India and fungal isolates of other genera. A single amplification product of 323-bp was detected from DNA of P. vexans isolates. No cross-reaction was observed with any of the other isolates tested. The specific primers designed and employed in PCR detected P. vexans up to 10 pg from DNA isolated from pure culture. This is the first report on the development of species-specific PCR assay for identification and detection of P. vexans. Thus, PCR-based assay developed is very specific, rapid, confirmatory and sensitive tool for the detection of pathogen P. vexans at early stages. SIGNIFICANCE AND IMPACT OF THE STUDY: Phomopsis vexans is an important seed-borne pathogenic fungus responsible for leaf blight and fruit rot in brinjal. Current detection methods, based on culture and morphological identification is time consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on sequence data of a region consisting of the 5·8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans.

Lipopolysaccharide-induced priming enhances NO-mediated activation of defense responses in pearl millet challenged with Sclerospora graminicola.

Lipopolysaccharide (LPS) elicitors isolated from Pseudomonas fluorescens UOM SAR 14 effectively induced systemic and durable resistance against pearl millet downy mildew disease caused by the oomycete Sclerospora graminicola. Rapid and increased callose deposition and H2O2 accumulation were evidenced in downy mildew susceptible seeds pre-treated with LPS (SLPS) in comparison with the control seedlings, which also correlated with expression of various other defense responses. Biochemical analysis of enzymes and quantitative real-time polymerase chain reaction data suggested that LPS protects pearl millet against downy mildew through the activation of plant defense mechanisms such as generation of nitric oxide (NO), increased expression, and activities of defense enzymes and proteins. Elevation of NO concentrations was shown to be essential for LPS-mediated defense manifestation in pearl millet and had an impact on the other downstream defense responses like enhanced activation of enzymes and pathogen-related (PR) proteins. Temporal expression analysis of defense enzymes and PR-proteins in SLPS seedlings challenged with the downy mildew pathogen revealed that the activity and expression of peroxidase, phenylalanine ammonia lyase, and the PR-proteins (PR-1 and PR-5) were significantly enhanced compared to untreated control. Higher gene expression and protein activities of hydroxyproline-rich glycoproteins (HRGPs) were observed in SLPS seedlings which were similar to that of the resistant check. Collectively, our results suggest that, in pearl millet-downy mildew interaction, LPS pre-treatment affects defense signaling through the central regulator NO which triggers the activities of PAL, POX, PR-1, PR-5, and HRGPs.

Draft genome sequence of Sclerospora graminicola, the pearl millet downy mildew pathogen.

Sclerospora graminicola pathogen is the most important biotic production constraints of pearl millet in India, Africa and other parts of the world. We report a de novo whole genome assembly and analysis of pathotype 1, one of the most virulent pathotypes of S. graminicola from India. The draft genome assembly contained 299,901,251 bp with 65,404 genes. This study may help understand the evolutionary pattern of pathogen and aid elucidation of effector evolution for devising effective durable resistance breeding strategies in pearl millet.

Molecular phylogeny, pathogenicity and toxigenicity of Fusarium oxysporum f. sp. lycopersici.

The present study aimed at the molecular characterization of pathogenic and non pathogenic F. oxysporum f. sp. lycopersici strains isolated from tomato. The causal agent isolated from symptomatic plants and soil samples was identified based on morphological and molecular analyses. Pathogenicity testing of 69 strains on five susceptible tomato varieties showed 45% of the strains were highly virulent and 30% were moderately virulent. Molecular analysis based on the fingerprints obtained through ISSR indicated the presence of wide genetic diversity among the strains. Phylogenetic analysis based on ITS sequences showed the presence of at least four evolutionary lineages of the pathogen. The clustering of F. oxysporum with non pathogenic isolates and with the members of other formae speciales indicated polyphyletic origin of F. oxysporum f. sp. lycopersici. Further analysis revealed intraspecies variability and nucleotide insertions or deletions in the ITS region among the strains in the study and the observed variations were found to be clade specific. The high genetic diversity in the pathogen population demands for development of effective resistance breeding programs in tomato. Among the pathogenic strains tested, toxigenic strains harbored the Fum1 gene clearly indicating that the strains infecting tomato crops have the potential to produce Fumonisin.

Evaluation of fatty acid profile and biodiesel properties of microalga Scenedesmus abundans under the influence of phosphorus, pH and light intensities.

The present study dealt with biomass, lipid concentration, fatty acid profile and biodiesel properties of microalga Scenedesmus abundans under different phosphate concentrations, pH and light intensities, one at a time. Among different phosphate concentrations, higher biomass (770.10±11.0mg/L) and lipid concentration (176.87±4.6mg/L) were at the concentration of 60mg/L. Light intensity at 6000lux yielded higher biomass and lipid concentration of 742.0±9.7 and 243.15±9.1mg/L, respectively. The biomass (769.0±12.3mg/L) and lipid (179.47±5.5mg/L) concentration were highest at pH 8 and pH 6, respectively. All the culture treatments showed marked effect on the fatty acid profile and biodiesel properties of the extracted oil. FAME derived biodiesel properties were compared with European biodiesel standards (EN 14214), Indian biodiesel standards (IS 15607) and American biodiesel standards (ASTM D 6751-08) to assess the suitability of algal oil as biodiesel feedstock.

Analysis of genetic and aflatoxin diversity among Aspergillus flavus isolates collected from sorghum seeds.

Thirty-four Aspergillus flavus isolates were recovered from sorghum seeds sampled across five states in India. Our study included (1) species confirmation through PCR assay, (2) quantification of total aflatoxin concentrations by the indirect competitive-ELISA (ic-ELISA) method, and (3) analysis of molecular diversity among the A. flavus isolates using ß-tubulin, ITS, and ISSR markers. Among the isolates studied, 28 were found to be positive for the production of aflatoxins. ITS and ß-tubulin phylogenetic analysis segregated the A. flavus sample population into two major groups or clades with little to no subdivision based on geography. In contrast, ISSR analysis also separated the A. flavus isolates into two main clusters, showing a distance of 0.0-0.5, with one cluster exhibiting a high level of diversity though no geographic or chemotype subdivision could be observed. The majority of sampled A. flavus isolates were highly toxigenic, and also highly diversified in terms of toxin-producing potential in-vitro. Genetic diversity among the sorghum isolates of A. flavus further warrants the development of appropriate farming management practices as well as improved aflatoxin detection measures in India.

Rosmarinic acid mediated neuroprotective effects against H2O2-induced neuronal cell damage in N2A cells.

AIMS: Oxidative stress plays a key role in several ailments including neurodegenerative conditions. The aim of the study was to demonstrate the effect of rosmarinic acid (RA) in preventing oxidative stress related death of neuronal cell lines. MAIN METHODS: In the present study, we demonstrated direct neuroprotective effect of RA using H2O2-induced oxidative challenge in N2A mouse neuroblastoma cells. The mechanism of neutralization of H2O2-induced toxicity by RA was evaluated using MTT, lactate dehydrogenase, mitochondrial membrane potential (MMP), intracellular ROS, and comet assays. Up-regulation of brain neuronal markers at molecular level was performed by RT-PCR. KEY FINDINGS: Results presented in the paper indicate that H2O2-induced cytotoxicity in N2A cells was suppressed by treatment with RA. Moreover, RA is very effective in attenuating the disruption of lactate dehydrogenase, mitochondrial membrane potential and intracellular ROS. Pretreatment with RA significantly prevents genotoxicity (3.7-fold, p<0.01) and promotes the up-regulation of tyrosine hydroxylase (TH) (4.5-fold, p<0.01), and brain-derived neurotrophic factor (BDNF) genes (5.4-fold, p<0.01) against H2O2-induced cytotoxicity in N2A cells. SIGNIFICANCE: Our results revealed that N2A cells are suitable cellular models to evaluate neuroprotective effects of RA, and suggest that RA may potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress.

Zearalenone induced toxicity in SHSY-5Y cells: The role of oxidative stress evidenced by N-acetyl cysteine.

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and are known to cause reproductive disorders, genotoxic and immunosuppressive effects. Although many studies have demonstrated the cytotoxic effects of ZEN, the mechanisms by which ZEN mediates its cytotoxic effects appear to differ according to cell type and route of exposure. Meantime, the available information on the neurotoxic effects of ZEN is very much limited. In the present study we evaluated the role of oxidative stress in ZEN mediated neurotoxicity in SH-SY5Y cells and investigated the possible underlying mechanism. ZEN induced ROS formation and elevated levels of MDA, loss of mitochondrial membrane potential (MMP) and increase in DNA damage in a dose dependent manner as assessed by COMET assay and agarose gel electrophoresis. However, there was no DNA damage by plasmid breakage assay at 6, 12 and 24h time points. DAPI staining showed apoptotic nuclei at 12 and 24h. Further, ZEN treated SH-SY5Y cells showed a marked suppressive effect on the neuronal gene expression. Use of an antioxidant N-acetylcysteine (NAC) reversed the toxin-induced generation of ROS and also attenuated loss of MMP. Collectively, these results suggest that ROS is the main upstream signal leading to increased ZEN mediated neurotoxicity in SH-SY5Y cells.

Antioxidant and neuroprotective activities of Hyptis suaveolens (L.) Poit. against oxidative stress-induced neurotoxicity.

The present study was carried out to investigate the antioxidant and neuroprotective effects of Hyptis suaveolens methanol extract (HSME) using various in vitro systems. The total phenol and flavonoids contents of the HSME were quantified by colorimetric methods. The HSME extract exhibited potent antioxidant activity as determined by 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 2,2-diphenyl-1-picrylhydrazyl, and ferric reducing antioxidant power assays. The neuroprotective activity of HSME was determined on mouse N2A neuroblastoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lactate dehydrogenase, intracellular ROS assays, and upregulation of brain neuronal markers at genetic level. The N2A cells were pretreated with different concentrations (0.5, 1, 1.5, and 2 mg/ml) of the extract and then exposed to H2O2 to induce oxidative stress and neurotoxicity. The survival of the cells treated with different concentrations of HSME and H2O2 increased as compared to cells exposed only to H2O2 (47.3 %) (p < 0.05). The HSME also dose-dependently reduced LDH leakage and intracellular ROS production (p < 0.05). Pretreatment with HSME promotes the upregulation of tyrosine hydroxylase (2.41-fold, p < 0.05), and brain-derived neurotrophic factor genes (2.15-fold, p < 0.05) against H2O2-induced cytotoxicity in N2A cells. Moreover, the HSME showed antioxidant activity and decreased neurotoxicity. These observations suggest that HSME have marked antioxidant and neuroprotective activities.

Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: an in vivo and in vitro evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Preparations of Orthosiphon diffusus (Benth.) have been used by folk medicinal practitioners in the Western Ghats of India for treating inflammation, hepatitis and jaundice for many years and their effectiveness is widely acclaimed among the tribal communities. AIM OF THE STUDY: To evaluate the mechanisms behind the antioxidant and hepatoprotective potential of Orthosiphon diffusus methanol active fraction (MAF) using in vivo (rat) and in vitro (cell culture) models. MATERIALS AND METHODS: Neutralization of CCl4-induced hepatotoxicity by MAF was evaluated in rats. Towards this, serum levels of hepatic injury markers (lactate dehydrogenase and alkaline phosphatase), antioxidant enzymes in the liver homogenates, and histological examination were performed. In in vitro studies, mechanisms of neutralization of H2O2-induced toxicity by MAF using MTT, Comet assay and up-regulation of antioxidant enzymes at genetic level (RT-PCR) was performed in HepG2 cells. RESULTS: Rats pre-treated with Orthosiphon diffusus MAF demonstrated significantly reduced levels of serum LDH (1.3-fold, p<0.05) and ALP (1.6-fold, p<0.05). Similarly, multiple dose MAF administration demonstrated significantly enhanced levels (p<0.05) of antioxidant enzymes in the liver homogenates. Histological analysis revealed complete neutralization of CCl4-induced liver injury by the extract. The in vitro studies demonstrated that, pre-treatment of MAF effectively prevented H2O2-induced oxidative stress, genotoxicity and significantly enhanced (~6-fold, p<0.01) expression of genes for antioxidant enzymes. CONCLUSIONS: Orthosiphon diffusus MAF demonstrated significant hepatoprotection against CCl4-induced hepatotoxicity by antioxidant mechanisms comparable to silymarin. H2O2-induced oxidative stress was completely neutralized by MAF through enhanced expression of genes for antioxidant enzymes. Therefore, this study validates the use of Orthosiphon diffusus by folk medicinal practitioners in India. Further, MAF of Orthosiphon diffusus can serve as a strong candidate for the development of herbal hepatoprotective agents.

Crocin, a dietary additive protects platelets from oxidative stress-induced apoptosis and inhibits platelet aggregation.

Platelets are the key players in the development of cardiovascular diseases as the microparticles generated by apoptotic platelets and platelet aggregation contribute actively towards the disease propagation. Thus, the aim of this study was to demonstrate the effect of a phytochemical which can prevent these two processes and thereby project it as a cardio-protective compound. Crocin, a natural carotenoid exhibits a wide spectrum of therapeutic potentials through its antioxidant property. The study demonstrated its effects on cytoplasmic apoptotic events of mitochondrial pathway in platelets. Collagen/calcium ionophore-A23187 stimulated platelets were treated with crocin and endogenous generation of reactive oxygen species (ROS) and hydrogen peroxide (H(2)O(2)) were measured. H(2)O(2)-induced changes in crocin-pretreated platelets such as intracellular calcium, mitochondrial membrane potential (ΔΨm), caspase activity, phosphatidylserine exposure and cytochrome c translocation were determined. Crocin dose-dependently ameliorated collagen- and A23187-induced endogenous generation of ROS and H(2)O(2). It also abolished the H(2)O(2)-induced events of intrinsic pathway of apoptosis. Further, it hindered collagen-induced platelet aggregation and adhesion. The current piece of work clearly suggests its anti-apoptotic effect as well as inhibitory effects on platelet aggregation. Thus, crocin can be deemed as a prospective candidate in the treatment regime of platelet-associated diseases.

Specific PCR-based detection of Alternaria helianthi: the cause of blight and leaf spot in sunflower.

Alternaria helianthi is an important seed-borne pathogenic fungus responsible for blight disease in sunflower. The current detection methods, which are based on culture and morphological identification, are time-consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on the sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers-ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of A. helianthi. The specificity of the primer pairs AhN1F and AhN1R designed was verified by PCR analysis of DNA from 18 Alternaria helianthi strains isolated from India, 14 non-target Alternaria spp. and 11 fungal isolates of other genera. A single amplification product of 357-bp was detected from DNA of A. helianthi isolates. No cross-reaction was observed with any of the other isolates tested. The detection limit of the PCR method was of 10 pg from template DNA. The primers could also detect the pathogen in infected sunflower seed. This species-specific PCR method provides a quick, simple, powerful and reliable alternative to conventional methods in the detection and identification of A. helianthi. This is the first report of an A. helianthi-specific primer set.