Comprehensive analysis of physiological and metabolomic responses to drought reveals specific modulation of acquired tolerance mechanisms in rice.

Mild stresses induce "acquired tolerance traits" (ATTs) that provide tolerance when stress becomes severe. Here, we identified the genetic variability in ATTs among a panel of rice germplasm accessions and demonstrated their relevance in protecting growth and productivity under water-limited conditions. Diverse approaches, including physiological screens, association mapping and metabolomics, were adopted and revealed 43 significant marker-trait associations. Nontargeted metabolomic profiling of contrasting genotypes revealed 26 "tolerance-related-induced" primary and secondary metabolites in the tolerant genotypes (AC-39000 and AC-39020) compared to the susceptible one (BPT-5204) under water-limited condition. Metabolites that help maintain cellular functions, especially Calvin cycle processes, significantly accumulated more in tolerant genotypes, which resulted in superior photosynthetic capacity and hence water use efficiency. Upregulation of the glutathione cycle intermediates explains the ROS homeostasis among the tolerant genotypes, maintaining spikelet fertility, and grain yield under stress. Bioinformatic dissection of a major effect quantitative trait locus on chromosome 8 revealed genes controlling metabolic pathways leading to the production of osmolites and antioxidants, such as GABA and raffinose. The study also led to the identification of specific trait donor genotypes that can be effectively used in translational crop improvement activities.

Ultra-high performance liquid chromatography Q-Orbitrap MS/MS-based profiling and quantification of limonoids in Meliaceae plants.

Meliaceae plants have been extensively used in agriculture, folklore, and traditional medicine. They are the major storehouses for structurally diverse limonoids (meliacins) possessing various bioactivities like antifeedant, insecticidal, antimicrobial, etc. However accurate detection of these tetranortriterpenes from the vast pool of metabolites in plant tissue extracts or biological sample is a crucial challenge. Though the mass spectrum (MS) provides the molecular mass and the corresponding elemental composition, it cannot be relied precisely. The exact identification of a specific metabolite demands the MS/MS spectrum containing the signature product ions. In the present study, we have developed the UHPLC Q-Orbitrap-based method for identification, quantification, and characterization of limonoids in different plant tissue extracts requiring minimum plant material. Using this method, we carried out the limonoid profiling in different tissue extracts of sixteen Meliaceae plants and the identification of limonoids was performed by comparing the retention time (RT), ESI-( +)-MS spectrum, and HCD-MS/MS of the purified fifteen limonoids used as reference standards. Our results revealed that early intermediates of the limonoid biosynthetic pathway such as azadiradione, epoxyazadiradione, and gedunin occurred more commonly in Meliaceae plants. The MS/MS spectrum library of the fifteen limonoids generated in this study can be utilized for identification of these limonoids in other plant tissue extracts, botanical fertilizers, agrochemical formulations, and bio pesticides.

Dynamic coupling analysis on plant sesquiterpene synthases provides leads for the identification of product specificity determinants.

Sesquiterpene synthases catalyse cyclisation of farnesyl pyrophosphate to produce diverse sesquiterpenes. Despite utilising the same substrate and exhibiting significant sequence and structural homology, these enzymes form different products. Previous efforts were based on identifying the effect of divergent residues present at the catalytic binding pocket on the product specificity of these enzymes. However, the rationales deduced for the product specificity from these studies were not generic enough to be applicable to other phylogenetically distant members of this family. To address this problem, we have developed a novel approach combining sequence, structural and dynamical information of plant sesquiterpene synthases (SSQs) to predict product modulating residues (PMRs). We tested this approach on the SSQs with known PMRs and also on sesquisabinene synthase 1 (SaSQS1), a SSQ from Indian sandalwood. Our results show that the dynamical sectors of SSQs obtained from molecular dynamics simulation and their hydrophobicity and vicinity indices together provide leads for the identification of PMRs. The efficacy of the technique was tested on SaSQS1 using mutagenesis. To the best of our knowledge, this is a first technique of this kind which provides cues on PMRs of SSQs, with divergent phylogenetic relationship.

Structural studies on 10-hydroxygeraniol dehydrogenase: A novel linear substrate-specific dehydrogenase from Catharanthus roseus.

Conversion of 10-hydroxygeraniol to 10-oxogeranial is a crucial step in iridoid biosynthesis. This reaction is catalyzed by a zinc-dependent alcohol dehydrogenase, 10-hydroxygeraniol dehydrogenase, belonging to the family of medium-chain dehydrogenase/reductase (MDR). Here, we report the crystal structures of a novel 10-hydroxygeraniol dehydrogenase from Catharanthus roseus in its apo and nicotinamide adenine dinucleotide phosphate (NADP+ ) bound forms. Structural analysis and docking studies reveal how subtle conformational differences of loops L1, L2, L3, and helix α9' at the orifice of the catalytic site confer differential activity of the enzyme toward various substrates, by modulating the binding pocket shape and volume. The present study, first of its kind, provides insights into the structural basis of substrate specificity of MDRs specific to linear substrates. Furthermore, comparison of apo and NADP+ bound structures suggests that the enzyme adopts open and closed states to facilitate cofactor binding.

Estrogen receptor activation in response to Azadirachtin A stimulates osteoblast differentiation and bone formation in mice.

The positive effects of the sex hormone in sustaining bone homeostasis are exercised by maintaining the equilibrium between cell activity and apoptosis. In this regard, the importance of estrogen receptors in maintaining the bone is that it is an attractive drug target, if devoid of known side effects. In this study, we show that a natural pure compound Azadirachtin A (Aza A) isolated from Azadirachta indica binds selectively to a site in the estrogen receptor, identifying itself to be a selective tissue modifier. Using computational and medicinal chemistry, we show that Aza A binds potentially and selectively to estrogen receptor-α (ERα) as compared with ERß. This preferential binding of Aza A to ERα with good pharmacokinetic distribution in the body forms metabolites, showing that it is well absorbed. In in vivo estrogen deficiency models for osteoporosis, Aza A at a much lower dose enhances new bone formation at both sites of the trabecular and cortical bone with increased bone strength and presents with no hyperplastic effect in the uterus.

Terpene profiling, transcriptome analysis and characterization of cis-ß-terpineol synthase from Ocimum.

Ocimum species produces a varied mix of different metabolites that imparts immense medicinal properties. To explore this chemo-diversity, we initially carried out metabolite profiling of different tissues of five Ocimum species and identified the major terpenes. This analysis broadly classified these five Ocimum species into two distinct chemotypes namely, phenylpropanoid-rich and terpene-rich. In particular, ß-caryophyllene, myrcene, limonene, camphor, borneol and selinene were major terpenes present in these Ocimum species. Subsequently, transcriptomic analysis of pooled RNA samples from different tissues of Ocimum gratissimum, O. tenuiflorum and O. kilimandscharicum identified 38 unique transcripts of terpene synthase (TPS) gene family. Full-length gene cloning, followed by sequencing and phylogenetic analysis of three TPS transcripts were carried out along with their expression in various tissues. Terpenoid metabolite and expression profiling of candidate TPS genes in various tissues of Ocimum species revealed spatial variances. Further, putative TPS contig 19414 (TPS1) was selected to corroborate its role in terpene biosynthesis. Agrobacterium-mediated transient over-expression assay of TPS1 in the leaves of O. kilimandscharicum and subsequent metabolic and gene expression analyses indicated it as a cis-ß-terpineol synthase. Overall, present study provided deeper understanding of terpene diversity in Ocimum species and might help in the enhancement of their terpene content through advanced biotechnological approaches.

Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension.

BACKGROUND: Neem tree serves as a cornucopia for triterpenoids called limonoids that are of profound interest to humans due to their diverse biological activities. However, the biosynthetic pathway that plant employs for the production of limonoids remains unexplored for this wonder tree. RESULTS: Herein, we report the tracing of limonoid biosynthetic pathway through feeding experiments using 13C isotopologues of glucose in neem cell suspension. Growth and development specific limonoid spectrum of neem seedling and time dependent limonoid biosynthetic characteristics of cell lines were established. Further to understand the role of mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways in limonoid biosynthesis, Ultra Performance Liquid Chromatography (UPLC)- tandem mass spectrometry based structure-fragment relationship developed for limonoids and their isotopologues have been utilized. Analyses of labeled limonoid extract lead to the identification of signature isoprenoid units involved in azadirachtin and other limonoid biosynthesis, which are found to be formed through mevalonate pathway. This was further confirmed by treatment of cell suspension with mevinolin, a specific inhibitor for MVA pathway, which resulted in drastic decrease in limonoid levels whereas their biosynthesis was unaffected with fosmidomycin mediated plastidial methylerythritol 4-phosphate (MEP) pathway inhibition. This was also conspicuous, as the expression level of genes encoding for the rate-limiting enzyme of MVA pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) was comparatively higher to that of deoxyxylulose-phosphate synthase (DXS) of MEP pathway in different tissues and also in the in vitro grown cells. Thus, this study will give a comprehensive understanding of limonoid biosynthetic pathway with differential contribution of MVA and MEP pathways. CONCLUSIONS: Limonoid biosynthesis of neem tree and cell lines have been unraveled through comparative quantification of limonoids with that of neem tree and through 13C limonoid isotopologues analysis. The undifferentiated cell lines of neem suspension produced a spectrum of C-seco limonoids, similar to parental tissue, kernel. Azadirachtin, a C-seco limonoid is produced in young tender leaves of plant whereas in the hard mature leaves of tree, ring intact limonoid nimocinol accumulates in high level. Furthermore, mevalonate pathway exclusively contributes for isoprene units of limonoids as evidenced through stable isotope labeling and no complementation of MEP pathway was observed with mevalonate pathway dysfunction, using chemical inhibitors.

MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato.

To combat pathogen infection, plants employ local defenses in infected sites and elicit systemic acquired resistance (SAR) in distant tissues. MicroRNAs have been shown to play a significant role in local defense, but their association with SAR is unknown. In addition, no such studies of the interaction between potato and Phytophthora infestans have been reported. We investigated the role of miR160 in local and SAR responses to P. infestans infection in potato. Expression analysis revealed induced levels of miR160 in both local and systemic leaves of infected wild-type plants. miR160 overexpression and knockdown plants exhibited increased susceptibility to infection, suggesting that miR160 levels equivalent to those of wild-type plants may be necessary for mounting local defense responses. Additionally, miR160 knockdown lines failed to elicit SAR, and grafting assays indicated that miR160 is required in both local and systemic leaves to trigger SAR. Consistently, SAR-associated signals and genes were dysregulated in miR160 knockdown lines. Furthermore, analysis of the expression of defense and auxin pathway genes and direct regulation of StGH3.6, a mediator of salicylic acid-auxin cross-talk, by the miR160 target StARF10 revealed the involvement of miR160 in antagonistic cross-talk between salicylic acid-mediated defense and auxin-mediated growth pathways. Overall, our study demonstrates that miR160 plays a crucial role in local defense and SAR responses during the interaction between potato and P. infestans.

Epoxyazadiradione suppresses breast tumor growth through mitochondrial depolarization and caspase-dependent apoptosis by targeting PI3K/Akt pathway.

BACKGROUND: Breast cancer is one of the most commonly diagnosed invasive cancers among women around the world. Among several subtypes, triple negative breast cancer (TNBC) is highly aggressive and chemoresistant. Treatment of TNBC patients has been challenging due to heterogeneity and devoid of well-defined molecular targets. Thus, identification of novel effective and selective agents against TNBC is essential. METHODS: We used epoxyazadiradione to assess the cell viability, mitochondrial potential, ROS level, cell migration, apoptosis and protein expression in cell culture models of TNBC MDA-MB-231 and ER+ MCF-7 breast cancer cells. The molecular mechanism was examined in two different type of breast cancer cells in response to epoxyazadiradione. We have also analyzed the effect of epoxyazadiradione on breast tumor growth using in vivo mice model. RESULTS: In this study, we for the first time investigated that out of 10 major limonoids isolated from Azadirachta indica, epoxyazadiradione exhibits most potent anti-cancer activity in both TNBC and ER+ breast cancer cells. Epoxyazadiradione induces apoptosis and inhibits PI3K/Akt-mediated mitochondrial potential, cell viability, migration and angiogenesis. It also inhibits the expression of pro-angiogenic and pro-metastatic genes such as Cox2, OPN, VEGF and MMP-9 in these cells. Furthermore, epoxyazadiradione attenuates PI3K/Akt-mediated AP-1 activation. Our in vivo data revealed that epoxyazadiradione suppresses breast tumor growth and angiogenesis in orthotopic NOD/SCID mice model. CONCLUSION: Our findings demonstrate that epoxyazadiradione inhibits PI3K/Akt-dependent mitochondrial depolarisation, induces apoptosis and attenuates cell migration, angiogenesis and breast tumor growth suggesting that this compound may act as a potent therapeutic agent for the management of breast cancer.

Regioselective and efficient enzymatic synthesis of antimicrobial andrographolide derivatives.

Labdane diterpene andrographolide (1) is a major constituent of Andrographis paniculata and known to exhibit wide spectrum of biological activities. In this study, regioselective monoesters of (1) have been synthesized by using Amano lipase AK (Pseudomonas fluorescens) as a biocatalyst. Amano lipase AK was able to execute highly efficient esterification of hydroxyl group attached to C-14 carbon of (1) in presence of acyl donors. Among the various synthesized derivatives including two novel compounds such as andrographolide-14-propionate (3) and andrographolide-14-caproate (5) displayed antimicrobial activity against Staphylococcus aureus with low minimal inhibitory concentration (MIC) 4 µg/mL and 16 µg/mL respectively. Furthermore, they have shown low hemolysis activity at their respective MIC and increase in the permeability of the bacterial cell membrane as delineated by FITC uptake and SEM imaging studies.

Ion pairing in 1-butyl-3-methylpyridinium halide ionic liquids studied using NMR and DFT calculations.

We present the 1H, 13C and 15N NMR chemical shifts of bulk ionic liquids based on 1-butyl-3-methylimidazolium (the cation also known as 1-butyl-3-picolinium) halides (Cl-, Br- and I-) and tribromide (Br3-) salts. A characterization in solution of the analogous ICl2- and I3- salts is also reported. A series of DFT calculations has been run to predict the features of the NMR spectra of the pure ILs based on a few selected supramolecular ionic aggregates. To test the effect of temperature, and vibrational and conformational motions, only for the chloride salt, we also run first-principles molecular dynamics simulations of the ion pair in the gas phase, using the ADMP scheme (Atom Centered Density Matrix Propagation molecular dynamics model). The aim of our investigation is to test whether a simple DFT based approach of ion-pairing in ionic liquids is capable of providing reliable results and under which conditions the protocol is robust. We obtained a very good agreement between the calculated and experimental spectra for the three halides, where the bulk structure of the ILs is dominated by H-bond interactions between the X- anion (X = Cl, Br and I) and the ortho protons of the pyridinium ring (a structural arrangement not too different from the solid-state structure of pyridinium halides). In contrast, when the H-bond is weak, as in the Br3- case, a number of supramolecular arrangements exist in solution and the simple DFT calculations of a few selected cases cannot exhaustively explore the complete energy landscape. Moreover, the dynamic effects due to thermal motion, evaluated by ADMP MD simulations of the chloride salt, appear to be not very significant.

Comparative functional characterization of eugenol synthase from four different Ocimum species: Implications on eugenol accumulation.

Isoprenoids and phenylpropanoids are the major secondary metabolite constituents in Ocimum genus. Though enzymes from phenylpropanoid pathway have been characterized from few plants, limited information exists on how they modulate levels of secondary metabolites. Here, we performed phenylpropanoid profiling in different tissues from five Ocimum species, which revealed significant variations in secondary metabolites including eugenol, eugenol methyl ether, estragole and methyl cinnamate levels. Expression analysis of eugenol synthase (EGS) gene showed higher transcript levels especially in young leaves and inflorescence; and were positively correlated with eugenol contents. Additionally, transcript levels of coniferyl alcohol acyl transferase, a key enzyme diverting pool of substrate to phenylpropanoids, were in accordance with their abundance in respective species. In particular, eugenol methyl transferase expression positively correlated with higher levels of eugenol methyl ether in Ocimum tenuiflorum. Further, EGSs were functionally characterized from four Ocimum species varying in their eugenol contents. Kinetic and expression analyses indicated, higher enzyme turnover and transcripts levels, in species accumulating more eugenol. Moreover, biochemical and bioinformatics studies demonstrated that coniferyl acetate was the preferred substrate over coumaryl acetate when used, individually or together, in the enzyme assay. Overall, this study revealed the preliminary evidence for varied accumulation of eugenol and its abundance over chavicol in these Ocimum species. Current findings could potentially provide novel insights for metabolic modulations in medicinal and aromatic plants.

Functional characterization and transient expression manipulation of a new sesquiterpene synthase involved in ß-caryophyllene accumulation in Ocimum.

The genus Ocimum has a unique blend of diverse secondary metabolites, with major proportion of terpenoids including mono- and sesquiterpenes. Although, ß-Caryophyllene, bicyclic sesquiterpene, is one of the major terpene found in Ocimum species and known to possess several biological activities, not much is known about its biosynthesis in Ocimum. Here, we describe isolation and characterization of ß-caryophyllene synthase gene from Ocimum kilimandscharicum Gürke (OkBCS- GenBank accession no. KP226502). The open reading frame of 1629 bp encoded a protein of 542 amino acids with molecular mass of 63.6 kDa and pI value of 5.66. The deduced amino acid sequence revealed 50-70% similarity with known sesquiterpene synthases from angiosperms. Recombinant OkBCS converted farnesyl diphosphate to ß-caryophyllene as a major product (94%) and 6% α-humulene. Expression variation of OkBCS well corroborated with ß-caryophyllene levels in different tissues from five Ocimum species. OkBCS transcript revealed higher expression in leaves and flowers. Further, agro-infiltration based transient expression manipulation with OkBCS over-expression and silencing confirmed its role in ß-caryophyllene biosynthesis. These findings may potentially be further utilized to improve plant defense against insect pests.

Azadirachta indica triterpenoids promote osteoblast differentiation and mineralization in vitro and in vivo.

Terpenoids were isolated using chromatographic purification through solvent purification technique and identified as Azadirone (1), Epoxyazadiradione (2) Azadiradione (3) Gedunin (4) Nimbin (5) Salannin (6) Azadirachtin A (7) and Azadirachtin B (8) from Azadirachta indica. Out of eight compounds, only three compounds had osteogenic activity and enhanced osteoblast proliferation, differentiation and mineralization in osteoblast cells. Active compounds stimulated osteogenic genes ALP, RunX-2 and OCN expressions in vitro, but Azadirachtin A had a maximum ability to stimulate osteoblast differentiation and mineralization compared to other two active compounds. For in vivo study, Azadirachtin A injected subcutaneously in pups, which enhanced osteogenic gene expressions and promoted bone formation rate significantly. Here, we conclude that active compounds of Azadirachta indica have osteogenic activity and Azadirachtin A has a beneficial effects on bone.

Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica).

BACKGROUND: Neem tree (Azadirachta indica) is one of the richest sources of skeletally diverse triterpenoids and they are well-known for their broad-spectrum pharmacological and insecticidal properties. However, the abundance of Neem triterpenoids varies among the tissues. Here, we delineate quantitative profiling of fifteen major triterpenoids across various tissues including developmental stages of kernel and pericarp, flower, leaf, stem and bark using UPLC-ESI(+)-HRMS based profiling. Transcriptome analysis was used to identify the initial genes involved in isoprenoid biosynthesis. Based on transcriptome analysis, two short-chain prenyltransferases and squalene synthase (AiSQS) were cloned and functionally characterized. RESULTS: Quantitative profiling revealed differential abundance of both total and individual triterpenoid content across various tissues. RNA from tissues with high triterpenoid content (fruit, flower and leaf) were pooled to generate 79.08 million paired-end reads using Illumina GA ΙΙ platform. 41,140 transcripts were generated by d e novo assembly. Transcriptome annotation led to the identification of the putative genes involved in isoprenoid biosynthesis. Two short-chain prenyltransferases, geranyl diphosphate synthase (AiGDS) and farnesyl diphosphate synthase (AiFDS) and squalene synthase (AiSQS) were cloned and functionally characterized using transcriptome data. RT-PCR studies indicated five-fold and ten-fold higher relative expression level of AiSQS in fruits as compared to leaves and flowers, respectively. CONCLUSIONS: Triterpenoid profiling indicated that there is tissue specific variation in their abundance. The mature seed kernel and initial stages of pericarp were found to contain the highest amount of limonoids. Furthermore, a wide diversity of triterpenoids, especially C-seco triterpenoids were observed in kernel as compared to the other tissues. Pericarp, flower and leaf contained mainly ring-intact triterpenoids. The initial genes such as AiGDS, AiFDS and AiSQS involved in the isoprenoids biosynthesis have been functionally characterized. The expression levels of AiFDS and AiSQS were found to be in correlation with the total triterpenoid content in individual tissues.

MicroRNA156: a potential graft-transmissible microRNA that modulates plant architecture and tuberization in Solanum tuberosum ssp. andigena.

MicroRNA156 (miR156) functions in maintaining the juvenile phase in plants. However, the mobility of this microRNA has not been demonstrated. So far, only three microRNAs, miR399, miR395, and miR172, have been shown to be mobile. We demonstrate here that miR156 is a potential graft-transmissible signal that affects plant architecture and tuberization in potato (Solanum tuberosum). Under tuber-noninductive (long-day) conditions, miR156 shows higher abundance in leaves and stems, whereas an increase in abundance of miR156 has been observed in stolons under tuber-inductive (short-day) conditions, indicative of a photoperiodic control. Detection of miR156 in phloem cells of wild-type plants and mobility assays in heterografts suggest that miR156 is a graft-transmissible signal. This movement was correlated with changes in leaf morphology and longer trichomes in leaves. Overexpression of miR156 in potato caused a drastic phenotype resulting in altered plant architecture and reduced tuber yield. miR156 overexpression plants also exhibited altered levels of cytokinin and strigolactone along with increased levels of LONELY GUY1 and StCyclin D3.1 transcripts as compared with wild-type plants. RNA ligase-mediated rapid amplification of complementary DNA ends analysis validated SQUAMOSA PROMOTER BINDING-LIKE3 (StSPL3), StSPL6, StSPL9, StSPL13, and StLIGULELESS1 as targets of miR156. Gel-shift assays indicate the regulation of miR172 by miR156 through StSPL9. miR156-resistant SPL9 overexpression lines exhibited increased miR172 levels under a short-day photoperiod, supporting miR172 regulation via the miR156-SPL9 module. Overall, our results strongly suggest that miR156 is a phloem-mobile signal regulating potato development.

Whole-Cell Mediated 11ß-Hydroxylation on the Basic Limonoid Skeleton by Cunninghamella echinulata.

Regio- and stereoselective 11ß-hydroxylation was achieved on the basic limonoid skeleton through microbial transformation. Whole cells of Cunninghamella echinulata efficiently converted basic limonoids such as epoxyazadiradione, azadiradione, and gedunin to their 11ß-hydroxy analogues as the sole metabolite. Fermentation conditions affecting the efficiency (96%) of biotransformation including substrate concentration, incubation period, pH, and temperature were optimized. The position and stereochemistry of hydroxyl functionality on the isolated metabolites were established through extensive spectroscopic and spectrometric studies (1D, 2D NMR, ESI-MS, and MS/MS).

Biocatalyst mediated regio- and stereo-selective hydroxylation and epoxidation of (Z)-α-santalol.

Biocatalyst mediated regio- and stereo-selective hydroxylation and epoxidation on (Z)-α-santalol were achieved for the first time, using a fungal strain Mucor piriformis. Four novel metabolites were characterized as 10,11-cis-ß-epoxy-α-santalol, 5α-hydroxy-(Z)-α-santalol, 10,11-dihydroxy-α-santalol and 5α-hydroxy-10,11-cis-ß-epoxy-α-santalol. Using Amano PS lipase from Burkholderia cepacia, α- and ß-isomers of 10,11-cis-epoxy-α-santalol were resolved efficiently.

Antimicrobial flavonoid: in silico targeting Escherichia coli DNA gyrase adeptly.

A phytochemical investigation of Leucas aspera involved the isolation of flavones and steroids, along with triterpenoids. The identified chemical constituents were characterized as baicalein, baicalein-7-O-ß-D-glucuronide, lup-20(29)-ene-3α,23-diol, ß-sitosterol, and stigmasterol. Notably, baicalein (4) and baicalein-7-O-ß-D-glucuronide (5) demonstrated significant antimicrobial activity at a low minimum inhibitory concentration against Staphylococcus aureus, Pseudomonas fluorescens, and Escherichia coli. Meanwhile, lup-20(29)-ene-3α,23-diol, ß-sitosterol, and stigmasterol displayed moderate antimicrobial activity against all the screened microorganisms. Further analysis showed that baicalein-7-O-ß-D-glucuronide had a strong docking score of -5.78 kcal/mol, indicating its robust interaction with E. coli DNA gyrase, which aligns with the in vitro findings. Additionally, baicalein-7-O-ß-D-glucuronide exhibited in silico ADME (absorption, distribution, metabolites, and excretion) properties similar to those of tetracycline.

Novel anti-inflammatory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (K(i), 2.11-5.23 µm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1ß, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.