Advances in sample preparation methods for pesticide residue analysis in medicinal plants: A focus on Nepal.

Medicinal plant safety is a rising challenge worldwide due to the continued overuse of pesticides to their maximum residue limits. Due to the high demand for medicinal plants, their production is being increased and sometimes protected by pesticide use. The analysis of these residues requires robust analytical methods to ensure the safety and quality of medicinal plants. Developing effective sample preparation for detecting pesticides is challenging, due to their diverse natures, classes, and physico-chemical characteristics. Hence, existing techniques and strategies are needed to improve the reliability of the results. The review discusses the current state of sample preparation techniques, analytical methods, and instrumental technologies employed in pesticide residue analysis in medicinal plants. It highlights the challenges, limitations, and advancements in the field, providing insights into the analytical strategies used to detect and quantify pesticide residues. Reliable, accessible, affordable, and high-resolution analytical procedures are essential to ensure that pesticide levels in medicinal plants are effectively regulated. By understanding the complexities of pesticide residue analysis in medicinal plants, this review article aims to support the conservation of medicinal plant resources, promote public health, and contribute to the development of sustainable strategies for ensuring the safety and quality of medicinal plants in Nepal. The findings of this review will benefit researchers, policymakers, and stakeholders involved in the conservation of medicinal plant resources and the promotion of public health.

Effect of exercise and pharmacotherapy on non-alcoholic fatty liver disease.

Objectives: Nonalcoholic fatty liver disease is one of the emerging liver diseases affecting 20%-30% of the population creating a burden on public health worldwide and has been associated with the causation of multiple diseases. Besides exercise, several drugs are being used in patients based on clinical evidence especially vitamin E, a potent antioxidant to reduce the oxidative stress responsible for the development and progression of nonalcoholic fatty liver disease. This study aims at evaluating the effect of exercise and pharmacotherapy on nonalcoholic fatty liver disease. Design: A prospective follow-up study with purposive sampling was done at a liver clinic for 3 months. Baseline characteristics such as anthropometric measurements and biochemical parameters were recorded and compared after 3 months to determine the effect of therapy. Descriptive analysis using a parametric test was used to assess the change in biochemical parameters and a non-parametric test was applied to find out the association between non-pharmacological and pharmacological approaches. Results: Out of 177 patients, 67.2 % were male and 32.8% were female with the mean age ±SD of 46.8 ± 12.06 years. Mean ± SD weight and body mass index of the patients were changed from 74.88 ± 11.61 kg to 72.37 ± 11.61 kg and from 28.41 ± 4.02 kg/m2 to 27.31 ± 4.58 kg/m2 respectively which was found to be statistically significant. There was a significant change in all the biochemical parameters with the p-value < 0.05 through both non-pharmacological and pharmacological approaches. Conclusion: Nonalcoholic fatty liver disease management through exercise and pharmacotherapy shows significant improvement in biochemical parameters indicating that alone or in combination with both approaches play an effective role in treating nonalcoholic fatty liver disease.

Self-medication practice in Kathmandu Metropolitan City: A cross-sectional study.

Objectives: Self-medication is increasingly popular among people in developing nations like Nepal where the expense of healthcare is relatively high and over-the-counter) medications are widely and easily accessible. Although this method has certain advantages, it is also clear that it has some drawbacks, including the potential for adverse drug reactions, drug resistance, medication interactions, and increased morbidity and mortality. This study aimed to evaluate the use of self-medication in nine wards of Kathmandu Metropolitan City (ward no. 4, 5, 13, 15, 16, 17, 23, 31, and 32). Methods: This cross-sectional descriptive survey was conducted in the chosen wards of Kathmandu Metropolitan city for 3 months from August to October 2021. A semi-structured questionnaire was used to collect data from 372 patients who were seeking self-medication. The participants were chosen randomly. Results: Self-medication was common among people (78%). The most frequent ailments for which participants self-medicated were the common cold (17.1%), headache (14.7%), fever (14.5%), and cough (7.6%). Anticold medications (18.2%), non-steroidal anti-inflammatory drugs (17.4%), antipyretics (15.2%), and analgesics (9.1%) were the most popular drug classes used in self-medication. The two most frequent justifications for self-medication were the absence of any major ailment (35%) and self-experience (22.7%). When symptoms started, the majority of patients started taking medication for themselves, and 47.7% of them got their prescriptions directly from the pharmacist by explaining their symptoms. When the symptoms were not relieved by the medication used for self-medication, it was found that the majority of participants (79.7%) stopped using the medication and visit the doctor. Conclusion: The prevalence of self-medication in Kathmandu city was ascertained by assessing the practice among residents of the Kathmandu Metropolitan city. The study showed self-medication is common among people and therefore, proper education about drug use and self-medication is needed.

The Influence of Environmental Conditions on Secondary Metabolites in Medicinal Plants: A Literature Review.

Medicinal plants, a source of different phytochemical compounds, are now subjected to a variety of environmental stresses during their growth and development. Different ecologically limiting factors including temperature, carbon dioxide, lighting, ozone, soil water, soil salinity and soil fertility has significant impact on medicinal plants' physiological and biochemical responses, as well as the secondary metabolic process. Secondary metabolites (SMs) are useful for assessing the quality of therapeutic ingredients and nowadays, these are used as important natural derived drugs such as immune suppressant, antibiotics, anti-diabetic, and anti-cancer. Plants have the ability to synthesize a variety of secondary metabolites to cope with the negative effects of stress. Here, we focus on how individual environmental variables influence the accumulation of plant secondary metabolites. A total of 48 articles were found to be relevant to the review topic during our systematic review. The review showed the influence of different environmental variables on SMs production and accumulation is complex suggesting the relationship are not only species-specific but also related to increases and decline in SMs by up to 50 %. Therefore, this review improves our understanding of plant SMs ability to adapt to key environmental factors. This can aid in the efficient and long-term optimization of cultivation techniques under ambient environmental conditions in order to maximize the quality and quantity of SMs in plants.

Sesquiterpene rich essential oil from Nepalese Bael tree (Aegle marmelos (L.) Correa) as potential antiproliferative agent.

Aegle marmelos (L.) Corr. (Rutaceae), also known as Bael tree, is an herbal traditional remedy in the South East Asia. In the present work, the leaf essential oil distilled from a population collected in Nepal was analyzed for the chemical composition by GC-MS showing different phytochemical constituents compared with literature data. The obtained oil was rich in sesquiterpenes, mainly ß-Caryophyllene (26%), whereas monoterpenes, known in literature as the major components, were present in little amounts. Due to richness in sesquiterpenes which are promising as anticancer drugs, the oil was tested against several human tumor cell lines namely pancreatic (PSN-1), colon (LoVo), lung (H157) and ovarian (2008) cells showing IC50 of 5.6 µg/mL, 6.5 µg/mL, 6.7 µg/mL and 2.3 µg/mL, respectively. In vivo distribution of oil was studied with a dose of 41.5 mg/kg in mice allowing the quantification of ß-Caryophyllene, α-Humulene, γ-Muurulene and ar-Curcumene at 30 and 60 min after oral administration. Sesquiterpene were found in higer amount in, liver, kidney and heart whereas lung and blood contained lower levels. The tissue distribution study demonstrated that active sesquiterpenoids of the oil can efficiently reach different organs.

Role of GhHDA5 in H3K9 deacetylation and fiber initiation in Gossypium hirsutum.

Cotton fibers are single-celled trichomes that initiate from the epidermal cells of the ovules at or before anthesis. Here, we identified that the histone deacetylase (HDAC) activity is essential for proper cotton fiber initiation. We further identified 15 HDACs homoeologs in each of the A- and D-subgenomes of Gossypium hirsutum. Few of these HDAC homoeologs expressed preferentially during the early stages of fiber development [-1, 0 and 6 days post-anthesis (DPA)]. Among them, GhHDA5 expressed significantly at the time of fiber initiation (-1 and 0 DPA). The in vitro assay for HDAC activity indicated that GhHDA5 primarily deacetylates H3K9 acetylation marks. Moreover, the reduced expression of GhHDA5 also suppresses fiber initiation and lint yield in the RNA interference (RNAi) lines. The 0 DPA ovules of GhHDA5RNAi lines also showed alterations in reactive oxygen species homeostasis and elevated autophagic cell death in the developing fibers. The differentially expressed genes (DEGs) identified through RNA-seq of RNAi line (DEP12) and their pathway analysis showed that GhHDA5 modulates expression of many stress and development-related genes involved in fiber development. The reduced expression of GhHDA5 in the RNAi lines also resulted in H3K9 hyper-acetylation on the promoter region of few DEGs assessed by chromatin immunoprecipitation assay. The positively co-expressed genes with GhHDA5 showed cumulative higher expression during fiber initiation, and gene ontology annotation suggests their involvement in fiber development. Furthermore, the predicted protein interaction network in the positively co-expressed genes indicates HDA5 modulates fiber initiation-specific gene expression through a complex involving reported repressors.

Genome-wide comparative and evolutionary analysis of Calmodulin-binding Transcription Activator (CAMTA) family in Gossypium species.

The CAMTA gene family is crucial in managing both biotic and abiotic stresses in plants. Our comprehensive analysis of this gene family in cotton resulted in the identification of 6, 7 and 9 CAMTAs in three sequenced cotton species, i.e., Gossypium arboreum, Gossypium raimondii, and Gossypium hirsutum, respectively. All cotton CAMTAs were localized in the nucleus and possessed calmodulin-binding domain (CaMBD) as identified computationally. Phylogenetically four significant groups of cotton CAMTAs were identified out of which, Group II CAMTAs experienced higher evolutionary pressure, leading to a faster evolution in diploid cotton. The expansion of cotton CAMTAs in the genome was mainly due to segmental duplication. Purifying selection played a significant role in the evolution of cotton CAMTAs. Expression profiles of GhCAMTAs revealed that GhCAMTA2A.2 and GhCAMTA7A express profoundly in different stages of cotton fiber development. Positive correlation between expression of these two CAMTAs and fiber strength confirmed their functional relevance in fiber development. The promoter region of co-expressing genes network of GhCAMTA2A.2 and GhCAMTA7A showed a higher frequency of occurrence of CAMTA binding motifs. Our present study thus contributes to broad probing into the structure and probable function of CAMTA genes in Gossypium species.

GhMYB1 regulates SCW stage-specific expression of the GhGDSL promoter in the fibres of Gossypium hirsutum L.

Secondary cell wall (SCW) biosynthesis is an important stage of the cotton fibre development, and its transcriptional regulation is poorly understood. We selected the Gossypium hirsutum GDSL (GhGDSL) lipase/hydrolase gene (CotAD_74480), which is expressed during SCW biosynthesis (19 through to 25 days postanthesis; DPA), for study. T1 -transgenic cotton lines expressing the ß-glucuronidase (gus) reporter under the control of a 1026-bp promoter fragment of GhGDSL (PGhGDSL ) showed 19 DPA stage-specific increase in GUS expression. 5' deletion indicated that the 194-bp fragment between -788 and -594 relative to the transcription start site was essential for this stage-specific expression. Site-directed mutagenesis of eight transcription factor binding sites within PGhGDSL demonstrated that the MYB1AT motif (AAACCA) at -603/-598 was critical for the 19 DPA-specific reporter gene expressions. Yeast one-hybrid (Y1H) analysis identified nine proteins, including GhMYB1 (CotAD_64719) that bound to the PGhGDSL promoter. Further, Y1H experiments using the 5' promoter deletions and individually mutated promoter motifs indicated that GhMYB1 interacted with PGhGDSL at MYB1AT sequence. GhMYB1 was expressed specifically in fibre from 19 DPA, overlapping with the sharp rise in GhGDSL expression, indicating that it could regulate GhGDSL during fibre development. Analysis of genes co-expressed with GhMYB1 showed that it potentially regulates a number of other 19-25 DPA-specific genes in networks including those functioning in the cell wall and precursor synthesis, but not the major polysaccharide and protein components of the fibre SCW. GhGDSL and its promoter are therefore potential tools for the improvement of cotton fibre quality traits.

Genome Sequence of Microbulbifer mangrovi DD-13T Reveals Its Versatility to Degrade Multiple Polysaccharides.

Microbulbifer mangrovi strain DD-13T is a novel-type species isolated from the mangroves of Goa, India. The draft genome sequence of strain DD-13 comprised 4,528,106 bp with G+C content of 57.15%. Out of 3479 open reading frames, functions for 3488 protein coding sequences were predicted on the basis of similarity with the cluster of orthologous groups. In addition to protein coding sequences, 34 tRNA genes and 3 rRNA genes were detected. Analysis of nucleotide sequence of predicted gene using a Carbohydrate-Active Enzymes (CAZymes) Analysis Toolkit indicates that strain DD-13 encodes a large set of CAZymes including 255 glycoside hydrolases, 76 carbohydrate esterases, 17 polysaccharide lyases, and 113 carbohydrate-binding modules (CBMs). Many genes from strain DD-13 were annotated as carbohydrases specific for degradation of agar, alginate, carrageenan, chitin, xylan, pullulan, cellulose, starch, ß-glucan, pectin, etc. Some of polysaccharide-degrading genes were highly modular and were appended at least with one CBM indicating the versatility of strain DD-13 to degrade complex polysaccharides. The cell growth of strain DD-13 was validated using pure polysaccharides such as agarose or alginate as carbon source as well as by using red and brown seaweed powder as substrate. The homologous carbohydrase produced by strain DD-13 during growth degraded the polysaccharide, ensuring the production of metabolizable reducing sugars. Additionally, several other polysaccharides such as carrageenan, xylan, pullulan, pectin, starch, and carboxymethyl cellulose were also corroborated as growth substrate for strain DD-13 and were associated with concomitant production of homologous carbohydrase.

Transcriptome sequencing of a thalloid bryophyte; Dumortiera hirsuta (Sw) Nees: assembly, annotation, and marker discovery.

Bryophytes are the first land plants but are scarcely studied at the molecular level. Here, we report transcriptome sequencing and functional annotation of Dumortiera hirsuta, as a representative bryophyte. Approximately 0.5 million reads with ~195 Mb data were generated by sequencing of mRNA using 454 pyrosequencer. De novo assembly of reads yielded 85,240 unigenes (12,439 contigs and 72,801 singletons). BlastX search at NCBI-NR database showed similarity of 33,662 unigenes with 10-(10) e-value. A total of 23,685 unigenes were annotated at TAIR10 protein database. The annotated unigenes were further classified using the Gene Ontology. Analysis at Kyoto Encyclopedia of Genes and Genomes pathway database identified 95 pathways with significant scores, among which metabolic and biosynthesis of secondary metabolite were the major ones. Phenylpropanoid pathway was elucidated and selected genes were characterized by real time qPCR. We identified 447 transcription factors belonging to 41 families and 1594 eSSRs in 1479 unigenes. D. hirsuta unigenes showed homology across the taxa from algae to angiosperm indicating their role as the connecting link between aquatic and terrestrial plants. This could be a valuable genomic resource for molecular and evolutionary studies. Further, it sheds light for the isolation and characterization of new genes with unique functions.

CAMTA 1 regulates drought responses in Arabidopsis thaliana.

BACKGROUND: Transcription factors (TF) play a crucial role in regulating gene expression and are fit to regulate diverse cellular processes by interacting with other proteins. A TF named calmodulin binding transcription activator (CAMTA) was identified in Arabidopsis thaliana (AtCAMTA1-6). To explore the role of CAMTA1 in drought response, the phenotypic differences and gene expression was studied between camta1 and Col-0 under drought condition. RESULTS: In camta1, root development was abolished showing high-susceptibility to induced osmotic stress resulting in small wrinkled rosette leaves and stunted primary root. In camta1 under drought condition, we identified growth retardation, poor WUE, low photosystem II efficiency, decline in RWC and higher sensitivity to drought with reduced survivability. The microarray analysis of drought treated camta1 revealed that CAMTA1 regulates "drought recovery" as most indicative pathway along with other stress response, osmotic balance, apoptosis, DNA methylation and photosynthesis. Interestingly, majority of positively regulated genes were related to plasma membrane and chloroplast. Further, our analysis indicates that CAMTA1 regulates several stress responsive genes including RD26, ERD7, RAB18, LTPs, COR78, CBF1, HSPs etc. and promoter of these genes were enriched with CAMTA recognition cis-element. CAMTA1 probably regulate drought recovery by regulating expression of AP2-EREBP transcription factors and Abscisic acid response. CONCLUSION: CAMTA1 rapidly changes broad spectrum of responsive genes of membrane integrity and photosynthetic machinery by generating ABA response for challenging drought stress. Our results demonstrate the important role of CAMTA1 in regulating drought response in Arabidopsis, thus could be genetically engineered for improving drought tolerance in crop.