Effects of limonin on oxidative stress and early apoptosis in oocytes during in vitro maturation.

To investigate the effects of limonin (Lim) on oxidative stress and early apoptosis in bovine oocytes during in vitro maturation (IVM), different concentrations of Lim (0, 10, 20, 50 µmol/L) were added to bovine IVM medium. Oocyte maturation rates and development 24 h after in vitro fertilization (IVF) were examined to determine the optimal Lim concentration. The optimal Lim concentration was added to the IVM medium, and 0 µmol/L Lim was used as the control. Immunofluorescence staining was used to detect the abnormal rate of spindle assembly, reactive oxygen species (ROS), glutathione (GSH), mitochondrial membrane potential (MMP) levels, mitochondrial distribution, and the fluorescence intensity of cathepsin B (CB)-active LC3 protein. RT‒qPCR was used to detect the mRNA expression levels of antioxidant-, apoptosis- and autophagy-related genes in oocytes. The total number of blastocysts and the proportion of apoptotic cells among blastocysts were detected. The results showed that the PBI ejection rate, cleavage rate and blastocyst rate of bovine oocytes in the 20 µmol/L Lim group were significantly higher than those in the control group (P < 0.05). Compared with those in the control group, ROS levels, abnormal mitochondrial distribution, the proportion of abnormal spindle assembly, CB activity and LC3 protein fluorescence intensity of oocytes in the 20 µmol/L Lim group were significantly decreased (P < 0.05), and GSH and MMP levels were significantly increased (P < 0.05). The expression of antioxidant genes (Prdx3, Prdx6, Sirt1) and antiapoptotic genes (Bcl-xl, Survivin) were significantly upregulated (P < 0.05), and the expression levels of proapoptotic genes (Caspase-4, BAX) and autophagy-related genes (LC3) were significantly downregulated (P < 0.05). The total number of cells among in vitro fertilized embryos was significantly increased (P < 0.05), and the apoptosis rate of blastocysts was significantly decreased (P < 0.05). Here, we show that Lim exerts positive effects on bovine oocyte IVM by regulating REDOX homeostasis, reducing spindle damage and enhancing mitochondrial function during IVM, thereby inhibiting oocyte apoptosis and autophagy.

Exploration of limonoids for their broad spectrum antiviral potential via DFT, molecular docking and molecular dynamics simulation approach.

Limonoids serve as vital secondary metabolites. Citrus limonoids show a wide range of pharmacological potential. As a result of which limonoids from citrus are of considerable research interest. Identification of new therapeutic molecules from natural origins has been widely adopted as a successful strategy in drug discovery. This work mainly focused on the high-throughput computational exploration of the antiviral potential of three vital limonoids, i.e. Obacunone, Limonin and Nomilin against spike proteins of SARS CoV-2 (PDB:6LZG), Zika virus NS3 helicase (PDB:5JMT), Serotype 2 RNA dependent RNA polymerase of dengue virus (PDB:5K5M). Herein we report the molecular docking, MD simulation studies of nine docked complexes, and density functional theory (DFT) of selected limonoids. The results of this study indicated that all three limonoids have good molecular features but out of these three obacunone exerted satisfactory results for DFT, docking and MD simulation study.

Nomilin and its analogue obacunone alleviate NASH and hepatic fibrosis in mice via enhancing antioxidant and anti-inflammation capacity.

Nonalcoholic steatohepatitis (NASH) and hepatic fibrosis are leading causes of cirrhosis with rising morbidity and mortality worldwide. Currently, there is no appropriate treatment for NASH and hepatic fibrosis. Many studies have shown that oxidative stress is a main factor inducing NASH. Nomilin (NML) and obacunone (OBA) are limonoid compounds naturally occurring in citrus fruits with various biological properties. However, whether OBA and NML have beneficial effects on NASH remains unclear. Here, we demonstrated that OBA and NML inhibited hepatic tissue necrosis, inflammatory infiltration and liver fibrosis progression in methionine and choline-deficient (MCD) diet, carbon tetrachloride (CCl4 )-treated and bile duct ligation (BDL) NASH and hepatic fibrosis mouse models. Mechanistic studies showed that NML and OBA enhanced anti-oxidative effects, including reduction of malondialdehyde (MDA) level, increase of catalase (CAT) activity and the gene expression of glutathione S-transferases (GSTs) and Nrf2-keap1 signaling. Additional, NML and OBA inhibited the expression of inflammatory gene interleukin 6 (Il-6), and regulated the bile acid metabolism genes Cyp3a11, Cyp7a1, multidrug resistance-associated protein 3 (Mrp3). Overall, these findings indicate that NML and OBA may alleviate NASH and liver fibrosis in mice via enhancing antioxidant and anti-inflammation capacity. Our study proposed that NML and OBA may be potential strategies for NASH treatment.

Complex scaffold remodeling in plant triterpene biosynthesis.

Triterpenes with complex scaffold modifications are widespread in the plant kingdom. Limonoids are an exemplary family that are responsible for the bitter taste in citrus (e.g., limonin) and the active constituents of neem oil, a widely used bioinsecticide (e.g., azadirachtin). Despite the commercial value of limonoids, a complete biosynthetic route has not been described. We report the discovery of 22 enzymes, including a pair of neofunctionalized sterol isomerases, that catalyze 12 distinct reactions in the total biosynthesis of kihadalactone A and azadirone, products that bear the signature limonoid furan. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations.

Limonin stabilises sirtuin 6 (SIRT6) by activating ubiquitin specific peptidase 10 (USP10) in cardiac hypertrophy.

BACKGROUND AND PURPOSE: Limonin, a naturally occurring tetracyclic triterpenoid, has extensive pharmacological effects. Its role in cardiac hypertrophy remains to be elucidated. We investigated its effects on cardiac hypertrophy along with the potential mechanisms involved. EXPERIMENTAL APPROACH: The effects of limonin on cardiac hypertrophy in C57/BL6 mice caused by aortic banding, plus neonatal rat cardiac myocytes (NRCMs) stimulated with phenylephrine to induce cardiomyocyte hypertrophy in vitro were investigated. KEY RESULTS: Limonin markedly improved the cardiac function and heart weight in aortic banded mice. Limonin-treated mice and NRCMs also produced fewer cardiac hypertrophy markers than those treated with the vehicle in the hypertrophic groups. Sustained aortic banding- or phenylephrine-stimulation impaired cardiac sirtuin 6 (SIRT6) protein levels, which were partially reversed by limonin associated with enhanced activity of PPARα. Sirt6 siRNA inhibited the anti-hypertrophic effects of limonin in vitro. Interestingly, limonin did not influence Sirt6 mRNA levels, but regulated ubiquitin levels. Thus, the protein biosynthesis inhibitor, cycloheximide and proteasome inhibitor, MG-132, were used to determine SIRT6 protein expression levels. Under phenylephrine stimulation, limonin increased SIRT6 protein levels in the presence of cycloheximide, but it did not influence SIRT6 expression in the presence of MG-132, suggesting that limonin promotes SIRT6 levels by inhibiting its ubiquitination degradation. Furthermore, limonin inhibited the degradation of SIRT6 by activating ubiquitin-specific peptidase 10 (USP10), while Usp10 siRNA prevented the beneficial effects of limonin. CONCLUSION AND IMPLICATIONS: Limonin mediates the ubiquitination and degradation of SIRT6 by activating USP10, providing an attractive therapeutic target for cardiac hypertrophy.

Co-treatment of Nimbolide augmented the anti-arthritic effects of methotrexate while protecting against organ toxicities.

Prolonged exposure to the pharmacological doses of disease-modifying anti-rheumatic drugs (DMARDs) often results in major organ toxicities resulting in poor patient compliance. Methotrexate (MTX) is one of the commonly prescribed DMARDs for the treatment of arthritis, which results in vital organ dysfunction. To retain the anti-arthritic activity of MTX with the reduction in toxicities, combination therapies are warranted. Nimbolide (NMB) is a potent anticancer, anti-inflammatory and anti-fibrotic agent whose potential has been demonstrated in various pre-clinical models. Monoarthritis was developed with Complete Freund's Adjuvant in the knees of Wistar rats and treatment was given with either NMB (3 mg/kg/day) or MTX (2 mg/kg/week) alone or combination therapy (NMB + MTX). The anti-arthritic effects were evaluated by arthritic scoring, radiological imaging, synovial tissue proteins analysis, and histopathological staining. While hepato-renal toxicity was assessed in serum by evaluating the kidney and liver functional parameters, in tissues by oxidative-nitrosative stress markers, and pro-inflammatory cytokines levels. Histopathological analysis was performed to study the extent of tissue damage. Molecular studies like immunoblotting and immunohistochemistry were performed to understand the effect of combination therapy. We thereby report that monotherapy with either NMB or MTX exhibited significant anti-arthritic effects, while combination therapy resulted in augmented anti-arthritic effects with significant reduction in hepato-renal toxicity produced by MTX probably through anti-inflammatory and anti-oxidant effects. Therefore, our proposed combination of NMB and MTX may serve as a potential strategy for the effective management of arthritis.

Nimbolide-encapsulated PLGA nanoparticles induces Mesenchymal-to-Epithelial Transition by dual inhibition of AKT and mTOR in pancreatic cancer stem cells.

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis and remains highly aggressive despite current advancements in therapies. Chemoresistance and high metastatic nature of PDAC is attributed to a small subset of stem-like cells within the tumor known as Cancer Stem Cells (CSCs). Here, we developed a strategy for targeting pancreatic CSCs through forceful induction of mesenchymal-to-epithelial transition driven by encapsulating a phytochemical Nimbolide in nanoparticles. Binding of Nimbolide with the key regulator proteins of CSCs were studied through molecular docking and molecular dynamic simulation studies, which revealed that it binds to AKT and mTOR with high affinity. Further, in vitro studies revealed that Nim NPs are capable of inducing forceful mesenchymal-to-epithelial transition of pancreatospheres that leads to loss of multidrug resistance and self-renewal properties of pancreatospheres. Our study gives a proof of concept that encapsulation of Nim in PLGA nanoparticles increases its therapeutic effect on pancreatospheres. Further, binding of Nim to AKT and mTOR negatively regulates their activity that ultimately leads to mesenchymal-to-epithelial transition of pancreatic CSCs.

ERK activation via A1542/3 limonoids attenuates erythroleukemia through transcriptional stimulation of cholesterol biosynthesis genes.

BACKGROUND: Cholesterol plays vital roles in human physiology; abnormal levels have deleterious pathological consequences. In cancer, elevated or reduced expression of cholesterol biosynthesis is associated with good or poor prognosis, but the underlying mechanisms are largely unknown. The limonoid compounds A1542 and A1543 stimulate ERK/MAPK by direct binding, leading to leukemic cell death and suppression of leukemia in mouse models. In this study, we investigated the downstream consequences of these ERK/MAPK agonists in leukemic cells. METHODS: We employed RNAseq analysis combined with Q-RT-PCR, western blot and bioinformatics to identify and confirm genes whose expression was altered by A1542 and A1543 in leukemic cells. ShRNA lentiviruses were used to silence gene expression. Cell culture and an animal model (BALB/c) of erythroleukemia induced by Friend virus were utilized to validate effects of cholesterol on leukemia progression. RESULTS: RNAseq analysis of A1542-treated cells revealed the induction of all 18 genes implicated in cholesterol biosynthesis. Expression of these cholesterol genes was blocked by cedrelone, an ERK inhibitor. The cholesterol inhibitor lovastatin diminished ERK/MAPK activation by A1542, thereby reducing leukemic cell death induced by this ERK1/2 agonist. Growth inhibition by cholesterol was observed both at the intracellular level, and when orally administrated into a leukemic mouse model. Both HDL and LDL also suppressed leukemogenesis, implicating these lipids as important prognostic markers for leukemia progression. Mechanistically, knockdown experiments revealed that the activation of SREBP1/2 by A1542-A1543 was responsible for induction of only a sub-set of cholesterol biosynthesis genes. Induction of other regulatory factors by A1542-A1543 including EGR1, AP1 (FOS + JUN) LDLR, IER2 and others may cooperate with SREBP1/2 to induce cholesterol genes. Indeed, pharmacological inhibition of AP1 significantly inhibited cholesterol gene expression induced by A1542. In addition to leukemia, high expression of cholesterol biosynthesis genes was found to correlate with better prognosis in renal cancer. CONCLUSIONS: This study demonstrates that ERK1/2 agonists suppress leukemia and possibly other types of cancer through transcriptional stimulation of cholesterol biosynthesis genes.

CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism.

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.

Microscale Thermophoresis (MST) to Detect the Interaction Between Purified Protein and Small Molecule.

Microscale thermophoresis (MST) is a biophysical assay to quantify the interaction between molecules, such as proteins and small molecules. In recent years, the MST assay has been used to detect protein-protein and protein-drug interactions. The assay detects the interaction between molecules by quantifying the thermophoretic movement of fluorescent molecules in response to a temperature gradient. In practice, the fluorescent molecule is mixed with different concentrations of the nonfluorescent ligand, and the mixture of molecules in solution is loaded to capillaries. A temperature gradient is applied to samples in the capillaries, and the movement of the fluorescent molecule in the temperature gradient is detected and recorded. The effect of different concentrations of the nonfluorescent ligand on the movement of the fluorescent molecule is quantified to test for the interaction between molecules. If the fluorescent molecule interacts with the ligand, the molecular properties of the molecules, such as charge, size, and hydration shell, will influence the molecular motility. MST has the advantages of being quantitative and robust. In this chapter, we will use Endosidin2 and its target protein Arabidopsis thaliana EXO70A1 (AtEXO70A1), as an example to show the procedure of using MST to test the interaction between a GFP-tagged protein and a small molecule.

Comprehensive Comparison on the Chemical Profile of Guang Chen Pi at Different Ripeness Stages Using Untargeted and Pseudotargeted Metabolomics.

The peel of Citrus reticulata 'Chachi' (GCP), which is highly valued in China for its health-promoting effects, is usually collected at different development stages to be processed into various functional foods. In the present work, a rapid method based on ultra-high-performance liquid chromatography Q Exactive Orbitrap mass spectrometry-incorporated untargeted and pseudotargeted metabolomics analysis was developed to investigate the chemical variations in GCP at different ripeness stages. Samples that originated from an individual tree were collected at immature, near mature, and mature stages. A total of 112 compounds were identified or tentatively identified, and flavonoids malonyl glycosides and polymethoxyfolavones glycosides were reported for the first time. Untargeted metabolomics analysis indicated the distinct chemical profiles and significant changes during ripeness stages. Then, a validated pseudotargeted metabolomics method based on parallel reaction monitoring was further applied with a wide coverage of targeted compounds. The GCP samples were found differing in the content variations of flavonoid aglycones, flavonoid O-/C-glycosides, polymethoxyfolavones, limonoids, alkaloids, and phenolic acid, which are important for phenotypic variations at different development stages. The present study is expected to provide new insight on comprehensive utilization of citrus peels at different ripeness stages.

Functional characterization and reclassification of an enzyme previously proposed to be a limonoid UDP-glucosyltransferase.

BACKGROUND: A major problem in the orange industry is 'delayed' bitterness, which is caused by limonin, a bitter compound developing from its non-bitter precursor limonoate A-ring lactone (LARL) during and after extraction of orange juice. The glucosidation of LARL by limonoid UDP-glucosyltransferase (LGT) to form non-bitter glycosyl-limonin during orange maturation has been demonstrated as a natural way to debitter by preventing the formation of limonin. RESULT: Here, the debittering potential of heterogeneously expressed glucosyltransferase, maltose-binding protein (MBP) fused to cuGT from Citrus unishiu Marc (MBP-cuGT), which was previously regarded as LGT, was evaluated. A liquid chromatography - mass spectrometry (LC-MS) method was established to determine the concentration of limonin and its derivatives. The protocols to obtain its potential substrates, LARL and limonoate (limonin with both A and D ring open), were also developed. Surprisingly, MBP-cuGT did not exhibit any detectable effect on limonin degradation when Navel orange juice was used as the substrate; MBP-cuGT was unable to biotransform either LARL or limonoate as purified substrates. However, it was found that MBP-cuGT displayed a broad activity spectrum towards flavonoids, confirming that the enzyme produced was active under the conditions evaluated in vitro. CONCLUSION: Our results based on LC-MS demonstrated that cuGT functionality was incorrectly identified. Its active substrates, including various flavonoids but not limonoids, highlight the need for further efforts to identify the enzyme responsible for LGT activity to develop biotechnology-based approaches for producing orange juice from varietals that traditionally have a delayed bitterness. © 2020 Society of Chemical Industry.

Melanogenesis-inhibitory activities of limonoids and tricyclic diterpenoids from Azadirachta indica.

The chemical constituents of the roots and bark of Azadirachta indica were investigated, leading to the isolation of six tricyclic diterpenoids and four limonoids including a new compound, azadirachtin J (4). The structures were elucidated on the basis of NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. Furthermore, melanogenesis-inhibitory activities of the isolated compounds were evaluated. As a result, compounds 1-3 and 10 exhibited superior inhibitory activities against melanogenesis with no, or almost no, toxicity to the cells (86.5-105.1% cell viability). Western blot analysis showed that compounds 1 and 3 exhibited melanogenesis inhibitory activities in α-MSH-stimulated B16 melanoma cells due to, at least in part, inhibition of the expression of MITF, followed by a decrease in the expression of tyrosinase, TRP-1, and TRP-2. Compounds 1 and 3 exhibited tyrosinase inhibitory activities (IC50 values of 44.86 µM and 69.85 µM respectively). Docking results confirm that the active inhibitors strongly interact with tyrosinase residues.

The gastrointestinal fate of limonin and its effect on gut microbiota in mice.

The gut microbiota plays a critical role in human health. Diets could modulate the gut microbiota, which in turn may contribute to altered health outcomes by way of changing the relative risk of chronic diseases. Limonin, widely found in citrus fruits, has been reported to possess multiple beneficial health effects. However, the gastrointestinal fate of limonin and its effect on gut microbiota remain unknown. Herein, mice were fed a diet containing 0.05% limonin (w/w) for 9 weeks. Liquid chromatography-mass spectrum analysis showed that limonin was concentrated along the gastrointestinal tract and reached 523.14 nmol g-1 in the colon lumen. Compared to control mice, colonic microbiota richness was significantly increased by limonin. Gut microbiota community was also clearly distinct from the control group as shown by Principle Coordinate Analysis. Additionally, the relative abundance of 22 genera (relative abundance >0.1%) was altered significantly. Among these, generally regarded probiotics (Lactobacillus and Bifidobacterium) were reduced, which was not due to direct inhibitory effect of limonin. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, amino acid metabolism, lipid, metabolism and immune system function were predicted to be upregulated, and immune system disease and infectious disease markers were predicted to be suppressed dramatically by limonin based on gut microbiota composition. Within the infectious disease category, bacterial toxin and Staphylococcus aureus infection markers were suppressed significantly with limonin treatment. Collectively, our study provides the first line of evidence that oral intake of limonin could shift gut microbiota composition and its functions, which warrants further investigation to determine its implication in human health.

Effect of Post-Harvest LED and UV Light Irradiation on the Accumulation of Flavonoids and Limonoids in the Segments of Newhall Navel Oranges (Citrus sinensis Osbeck).

To investigate the effect of post-harvest light irradiation on the accumulation of flavonoids and limonoids, harvested Newhall navel oranges were continuously exposed to light-emitting diode (LED) and ultraviolet (UV) light irradiation for 6 days, and the composition and content of flavonoids and limonoids in the segments were determined using UPLC-qTOF-MS at 0, 6, and 15 days after harvest. In total, six polymethoxylated flavonoids (PMFs), five flavone-O/C-glycosides, seven flavanone-O-glycosides, and three limonoids were identified in the segments. The accumulation of these components was altered by light irradiation. Red and blue light resulted in higher levels of PMFs during exposure periods. The accumulation of PMFs was also significantly induced after white light, UVB and UVC irradiation were removed. Red and UVC irradiation induced the accumulation of flavone and flavanone glycosides throughout the entire experimental period. Single light induced limonoid accumulation during exposure periods, but limonoid levels decreased significantly when irradiation was removed. Principal component analysis showed a clear correlation between PMFs and white light, between flavonoid glycosides and red light and UVC, and between limonoids and UVC. These results suggest that the accumulation of flavonoids and limonoids in citrus is regulated by light irradiation. White light, red light and UVC irradiation might be a good potential method for improving the nutrition and flavor quality of post-harvest citrus.

Endosidin2-14 Targets the Exocyst Complex in Plants and Fungal Pathogens to Inhibit Exocytosis.

The evolutionarily conserved octameric exocyst complex tethers secretory vesicles to the site of membrane fusion during exocytosis. The plant exocyst complex functions in cell wall biosynthesis, polarized growth, stress responses, and hormone signaling. In fungal pathogens, the exocyst complex is required for growth, development, and pathogenesis. Endosidin2 (ES2) is known to inhibit exocytosis in plant and mammalian cells by targeting the EXO70 subunit of the exocyst complex. Here we show that an analog of ES2, ES2-14, targets plant and two fungal EXO70s. A lower dosage of ES2-14 than of ES2 is required to inhibit plant growth, plant exocytic trafficking, and fungal growth. ES2-14 treatments inhibit appressorium formation and reduce lesion sizes caused by Magnaporthe oryzae Inhibition of EXO70 by ES2-14 in Botrytis cinerea also reduces its virulence in Arabidopsis (Arabidopsis thaliana). Interestingly, ES2-14 did not affect EXO70 localization or transferrin recycling in mammalian cells. Overall, our results indicate that a minor change in ES2 affects its specificity in targeting EXO70s in different organisms and they demonstrate the potential of using ES2-14 to study the mechanisms of plant and fungal exocytosis and the roles of exocytosis in fungus-plant interactions.

Developmental Stage- and Genotype-Dependent Regulation of Specialized Metabolite Accumulation in Fruit Tissues of Different Citrus Varieties.

Flavor traits in citrus are the result of a blend of low molecular weight metabolites including sugars, acids, flavonoids and limonoids, these latter being mainly responsible for the characteristic bitter flavor in citrus. In this work, the genotype- and developmental stage-dependent accumulation of flavonoids and limonoids is addressed. To fulfill this goal, three models for citrus bitterness: bitter Duncan grapefruit, bittersweet Thomson orange and sweet Wase mandarin were selected from a total of eight different varieties. Compounds were annotated from LC/ESI-QqTOF-MS non-targeted metabolite profiles from albedo and pulp tissues. Results indicated that the specific blend of compounds providing the characteristic flavor trait is genotype-specific and hence under genetic control, but it is also regulated at the developmental level. Metabolite profiles in albedo mirrored those found in pulp, the edible part of the fruit, despite differences in the concentration and accumulation/depletion rates being found. This is particularly relevant for polymethoxylated flavones and glycosylated limonoids that showed a clear partitioning towards albedo and pulp tissues, respectively. Fruit ripening was characterized by a reduction in flavonoids and the accumulation of limonoid glycosides. However, bitter grapefruit showed higher levels of limonin A-ring lactone and naringin in contrast to sweeter orange and mandarin. Data indicated that the accumulation profile was compound class-specific and conserved among the studied varieties despite differing in the respective accumulation and/or depletion rate, leading to different specialized metabolite concentration at the full ripe stage, consistent with the flavor trait output.

Biotransformation of (+)-isofraxinellone by Aspergillus niger and insect antifeedant activity.

The biotransformation of (+)-isofraxinellone (1) by Aspergillus niger was investigated. Compound 1 was transformed to only one new compound 2. The structure of 2 was identified as (-)-(4S)-4-hydroxyisofraxinellone which was regio- and stereo-selective hydroxylated at the C-4 position by IR, EI-MS 1D and 2D NMR. Absolute configuration of hydroxyl group at the C-4 position was detected by modified Mosher's method. Antifeedant activity of compounds 1 and 2 against larvae of Spodoptera litura was assayed. These compounds showed potent antifeedant activity and ED50 (50% of effective dose) values were 3.91 and 4.43 µg/cm2, respectively.

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.

Biotransformation and Metabolic Profile of Limonin in Rat Liver Microsomes, Bile, and Urine by High-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry.

Limonin is a triterpenoid in citrus seeds, which has significant biological activities. However, the metabolic profile of limonin has not been fully understood. To expound its metabolism in vivo and in vitro, the metabolites of limonin was studied by rat liver microsomes, urine, and bile. High-performance liquid chromatography/quadrupole time-of-flight mass spectrometry was used for identification. Among the metabolites, the structures of M1 and M3 were confirmed by chemical synthesis and nuclear magnetic resonance spectra analysis. Our results indicated that reduction and hydrolysis were the two major pathways during limonin metabolism in vivo and in vitro. The results from this work are valuable and important for understanding the metabolic process of limonin.