A Comprehensive Review on Preclinical Safety and Toxicity of Medicinal Plants

Background:Globally,80%people use plant-derived products for treating or preventing diseases.One prevalent perception about medicinal plants is that they are safe and devoid of adverse effects.However,approximately 1,50,000 plants contain toxic substances.Objective:The present review focuses on medicinal plant extracts/fractions toxicity assessments made in preclin-ical models by oral route.Methods:Detail studies were searched from databases including PubMed and Google Scholar.A manual reference screening of the selected studies was done to identify relevant articles,with no language restriction being imposed at the time of searching.Results:The studies included were performed in rodents,and the test substances were administered orally.Our search revealed 33 widely used plants or products with significant toxicity,and phytochemicals from these plants have been summarized.Through a systematic review,we identified a plethora of medicinal plant extracts reporting safety and toxicity concerns.Conclusion:In the future,preclinical toxicokinetic studies of herbs and the determination of their no-observed-adverse-effect levels are required for a complete safety assessment.Finally,the interaction of herbs with com-monly used/over-the-counter drugs in terms of the latter’s metabolic profile should be undertaken.

Multi-omics:Powerful accelerator for uncovering plant specialized metabolic pathways:The case of leonurine

As sessile organisms,plants employ a unique adaptive strategy to survive harsh terrestrial environments and defend against or interact with coevolving animals and microorganisms.This strategy involves expanding their metabolic systems,resulting in the production of plant specialized metabolites(PSMs)or natural products.Chemically,PSMs constitute a highly diverse group of compounds based on their common core structure,comprising terpenoids,phenylpropanoids,benzenoids,alkaloids and nitrogen-containing compounds,glucosinolates,indoles and sulfur-containing indole compounds,phenolics,and fatty acid derivatives(Garagounis et al.,2021).

Identification of EST–SSRs and molecular diversity analysis in Mentha piperita

EST sequences of Mentha piperita available in the public domain(NCBI) were exploited to develop SSR markers. A total of 1316 ESTs were assembled into 155 contigs and 653 singletons and of these, 110 sequences were found to contain 130 SSRs, with a frequency of1 SSR/3.4 kb. Dinucleotide repeat SSRs were most frequent(72.3%) with the AG/CT(43.8%)repeat motif followed by AT/AT(16.2%). Primers were successfully designed for 68SSR-containing sequences(62.0%). The 68 primers amplified 13 accessions of M. piperita and 54 produced clear amplicons of the expected size. Of these 54, 33(61%) were found to be polymorphic among M. piperita accessions, showing from 2 to 4 alleles with an average of2.33 alleles/SSR, and the polymorphic information content(PIC) value varied between 0.13 and 0.51(average 0.25). All the amplified SSRs showed transferability among four different species of Mentha, with a highest in Mentha arvensis(87.0%) and minimum in Mentha citrata(37.0%). The newly developed SSRs markers were found to be useful for diversity analysis, as they successfully differentiated among species and accessions of Mentha.

A high methionine,low folate and vitamin B_6/B_(12) containing diet can be associated with memory loss by epigenetic silencing of netrin-1

Memory-epigenetics which is the loss of memory due to epigenetic modifications can be due to the silencing of genes involved in cognitive functions and this is the basis of the current study.We hypothesize that a diet containing high methionine and low vitamins can lead to memory impairment by increasing global DNA methylation and therefore,silencing the netrin-1 gene,which encodes the glycoprotein involved in neurogenesis,axonal guidance and maintenance of the synaptic plasticity.Wild type(C57 BL/6 J) mice were fed with a diet containing excess methionine(1.2%),low-folate(0.08 mg/kg),vitamin B_6(0.01 mg/kg),and B_(12)(10.4 mg/kg) for 6 weeks.Mice were examined weekly for the long-term memory function,using a passive avoidance test,which determined loss of fear-motivated long-term memory starting from the fourth week of diet.Similarly,an increase in brain %5-methyl cytosine was observed starting from the 4 th week of diet in mice.Mice fed with a high methionine,low folate and vitamins containing diet showed a decrease in netrin-1 protein expression and an increase in netrin-1 gene promotor methylation,as determined by methylation-sensitive restriction enzyme-polymerase chain reaction analysis.The increase in methylation of netrin-1 gene was validated by high-resolution melting and sequencing analysis.Furthermore,the association of netrin-1 with memory was established by administering netrin that considerably restored long-term fear motivated memory.Taken together,these results suggest that a diet rich in methionine and lacking in folate and vitamin B_6/B_(12) can induce defects in learning and memory.Furthermore,the data indicates that decrease in netrin-1 expression due to hyper-methylation of its gene can be associated with memory loss.The animal procedures were approved by the Institutional Animal Care and Use Committee,University of Louisville,USA(No.A3586-01) on February 2,2018.

Functional Role of miRNAs: Key Players in Soybean Improvement

Soybean(Glycine max(L.)Merr)is an agro-economic crop growing across the world to cater nutrition for both human and animal feed due to the high oil and protein content in its edible seeds.The genes and QTLs associated with important agronomic traits in this crop have already been identified and validated for soybean cyst nematode(SCN),Phytophthora root and stem rot,Pythium root rot and aphid resistance,seed quality,nutrient values,and also employed for genetic improvement in soybean.In the last decade,micro RNAs(miRNAs)have been considered the effector molecules,so the detection and characterization of novel miRNAs in soybean have been taken up by several workers.The advancement in the strategy of sequencing and tools of bioinformatics during last decade has contributed to the discovery of many soybean miRNAs,thus miRNA can be used as a tool in molecular breeding studies,and this has opened new vistas for miRNA mediated genetic improvement of soybean to augment crop productivity as well as nutritional quality.This review addresses the current state of understanding of miRNAmediated stress responses,nutrient acquisition,plant development and crop production processes in soybean.

Autochthonous halotolerant plant growth-promoting rhizobacteria promote bacoside A yield of Bacopa monnieri(L.)Nash and phytoextraction of salt-affected soil

Phytoremediation is a promising approach for reclamation of salt-affected soil.Phytoextraction is the most commonly used process,which exploits plants to absorb,immobilize,and accumulate salt in their shoots.In this study,halotolerant plant growth-promoting rhizobacteria(PGPR)were isolated from the rhizosphere of wild grasses growing naturally in salt-affected areas of Lucknow,Uttar Pradesh(India)and were tested for their efficacies of salt-tolerance and plant growth-promoting(PGP)abilities.Based on 16S rRNA sequences,the most efficient halotolerant isolates possessing PGP traits were identified as Pseudomonas plecoglossicida(KM 233646),Acinetobacter calcoaceticus(KM 233647),Bacillus flexus(KM 233648),and Bacillus safensis(KM 233652).Application of these isolates as bio-inoculants significantly(P<0.05)increased the growth and bacoside A yield of a medicinal plant,Bacopa monnieri(L.)Nash,grown on natural salt-affected soil.The phytoremediation of salt-affected soil was evident by the substantial increase in shoot Na^+:K^+ratio of bio-inoculant-treated plants.When compared to un-inoculated control plants,the soil physico-chemical properties of bio-inoculant-treated plants were improved.The shoot and root biomass(fresh and dry weights),soil enzymes,and soil nutrient parameters showed significant positive correlations with the shoot Na+:K+ratio.Consequently,the halotolerant PGPR screened in this study could be useful for the reclamation of saline soils concomitant with improved plant growth and bacoside A yield.

Heteromeric and Homomeric Geranyl Diphosphate Synthases from Catharanthus roseus and Their Role in Monoterpene Indole Alkaloid Biosynthesis

Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate （GPP）, the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate （IPP） and dimethylallyl diphosphate （DMAPP） by GPP synthase （GPPS）. Here, we report three genes encoding proteins with sequence similarity to large subunit （CrGPPS.LSU） and small subunit （CrGPPS.SSU） of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate （GGPP）, CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coil resulted in heteromeric enzyme with enhanced activity producing only GPR While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate （MelA） treatment of leaves sig- nificantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.

Identification, occurrence, and validation of DRE and ABRE Cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana

Plants posses a complex co-regulatory network which helps them to elicit a response under diverse adverse conditions. We used an in silico approach to identify the genes with both DRE and ABRE motifs in their promoter regions in Arabidopsis thaliana. Our results showed that Arabidopsis contains a set of 2,052 genes with ABRE and DRE motifs in their promoter regions. Approximately 72~o or more of the total predicted 2,052 genes had a gap distance of less than 40o bp between DRE and ABRE motifs. For positional orientation of the DRE and ABRE motifs, we found that the DR form （one in direct and the other one in reverse orientation） was more prevalent than other forms. These predicted 2,o52 genes include 155 transcription factors. Using microarray data from The Arabidopsis Information Resource （TAIR） database, we present 44 transcription factors out of 155 which are upregulated by more than twofold in response to osmotic stress and ABA treatment. Fifty-one transcripts from the one predicted above were validated using semiquantitative expression analysis to support the microarray data in TAIR. Taken together, we report a set of genes containing both DRE and ABRE motifs in their promoter regions in A. thaliana, which can be useful to understand the role of ABA under osmotic stress condition.

Exploring the benefits of biochar over other organic amendments for reducing of metal toxicity in Withania somnifera

Biochar is considered as a good metal sequester and ameliorates the metal toxicity and uptake in plants.However,its effec-tiveness over other organic amendments is not well discussed.The aim of this study is to compare the biochar amendments alleviating the levels of Pb and Cd in Withania somnifera L.Dunal with other organic manure.Farmyard manure,vermicom-post,and biochar were applied in the field spiked with highly available Pb and Cd.Metal bioavailability and accumulation,plant growth and plant physiology,antioxidant enzymes and metabolite content of W.somnifera along with soil properties were evaluated in all treatments.Study indicates that the application of organic manures significantly alleviated the metal levels(33-72%)in the plant in comparison with control.The herbage yield was significantly higher(33-69%)under different organic manures as compared to the metal treatments.Secondary metabolite contents and antioxidant enzymes were higher in metal treatments in comparison with control.Organic manures were not only able to restrain the Pb and Cd in soil but also enhanced the soil microbial activities.Results indicate that biochar amendments were more promising than farmyard manure and vermicompost due to the presence of more stable carbon in biochar and more alleviation in metal uptake.In addition,net profit in the cultivation of W.somnifera was higher for biochar amendments(50%higher)compared to control.The study recommended that biochar could be a better option for commercial and safer production of W.somnifera.

Comparative assessment for removal of anionic dye from water by different waste-derived biochar vis a vis reusability of generated sludge

In this study,four biochars prepared from different crop residue waste i.e.sugarcane bagasse(SBB),coconut shell(CNB),paddy straw(PDB),and distilled waste of lemongrass(LGB)were evaluated for removal of Remazol Brilliant Blue R from the aqueous system.The RBBR adsorption capacities of biochar were 97-79%for SBB,99.9-99.47%for CNB,66.1-48%for PDB,and 78-68%for LGB,dominantly controlled by their aromaticity and mineral content.The Langmuir and Freundlich isotherms and pseudo-second-order kinetic models have described the chemisorption of RBBR on biochar surfaces.The thermodynamic data suggested that adsorption was spontaneous and endothermic.These biochars demonstrated excellent reusability(till four cycles with 50-61%regeneration).The purified water and biochar dye sludge demonstrated no phytotoxicity.The findings obtained in this study may provide supports for the potential of biochars for anionic dye removal from water and utilization of generated sludge for zero waste-producing technologies in the future.