Selective transformations of friedelanes isolated from cork smoker wash solids.

Friedelin (1) and 3-acetoxyfriedel-3-en-2-one (4), commonly known as friedelane triterpenoids, have been isolated from cork smoker wash solids (also known as black wax) on a multi-gram scale. These compounds are valuable starting materials for the synthesis of new friedelane derivatives. Stereoselective reduction of friedelin by treatment with LiAlH4, sodium, or catalytic hydrogenation results in the formation of both isomers of friedelinol (5 and 7) in excellent yields. Similarly, the reduction of 3-acetoxyfriedel-3-en-2-one gave epi-cerin (14) and a series of isomeric 2,3-diols or α-hydroxyketones. These transformations provide the most straightforward and convenient methods for the synthesis of A-ring functionalised friedelane derivatives using easily accessible starting materials.

Friedelin: Structure, Biosynthesis, Extraction, and Its Potential Health Impact.

Pharmaceutical companies are investigating more source matrices for natural bioactive chemicals. Friedelin (friedelan-3-one) is a pentacyclic triterpene isolated from various plant species from different families as well as mosses and lichen. The fundamental compounds of these friedelane triterpenoids are abundantly found in cork tissues and leaf materials of diverse plant genera such as Celastraceae, Asteraceae, Fabaceae, and Myrtaceae. They possess many pharmacological effects, including anti-inflammatory, antioxidant, anticancer, and antimicrobial activities. Friedelin also has an anti-insect effect and the ability to alter the soil microbial ecology, making it vital to agriculture. Ultrasound, microwave, supercritical fluid, ionic liquid, and acid hydrolysis extract friedelin with reduced environmental impact. Recently, the high demand for friedelin has led to the development of CRISPR/Cas9 technology and gene overexpression plasmids to produce friedelin using genetically engineered yeast. Friedelin with low cytotoxicity to normal cells can be the best phytochemical for the drug of choice. The review summarizes the structural interpretation, biosynthesis, physicochemical properties, quantification, and various forms of pharmacological significance.

Friedelin and Glutinol induce neuroprotection against ethanol induced neurotoxicity in pup's brain through reduction of TNF-α, NF-kB, caspase-3 and PARP-1.

Ethanol administration triggers an inflammatory response that leads to a complex series of immune responses including the release of an excessive amount of inflammatory mediators particularly tumor necrosis factor (TNF-α) and nuclear factor-kB (NF-KB) which produce a large amount of reactive oxygen species. The inflammatory-induced cytotoxicity is increased when the PI3-kinase/Akt pathway is inhibited. Some studies have also shown that ethanol suppresses the PI3-kinase signaling pathway induced by receptor activation. Friedelin and Glutinol belong to pentacyclic triterpenoid class and are known for their anti-inflammatory and antioxidant properties. The present study was aimed to elucidate the effects of these phytoconstituents on one of the key ethanol-induced neuronal damage pathways. The pups having (5-7 g average body weight) were used and randomly divided into groups. The control and ethanol treated pups were administered 0.9% normal saline while treated pups received glutinol and friedelin (30 mg/kg subcutaneously) respectively. After four hours all the experimental animals were sacrificed and their brains were collected carefully for protein expression analysis of p-Akt, TNF-α, NF-KB, caspase-3 and PARP-1 employing immunoblotting technique. Hemolytic, DNA protection, chelating power and ß-carotene assays results revealed that freidelin and glutinol are safe for parenteral administration. Glutinol administration with ethanol significantly abridged the ethanol induced over expression of TNF-α, caspase-3 and PARP-1 in pup's brain. Similarly, freidelin attenuated the neurodegeneration by inhibiting the ethanol induced p-JNK and NF-kB expression in pups' brain. This protection may be attributed to the revival of p-Akt signaling for cell survival. It is concluded that the present study demonstrates the neuro-protective effects of friedelin and glutinol via modulating the capase-3 and PARP-1 expression and modulating the neuronal apoptotic pathways.

Friedelin, a novel inhibitor of CYP17A1 in prostate cancer from Cassia tora.

In prostate cancer (PC), drugs targeting CYP17A1 have shown great success in regulating PC progression. However, successful drug molecules show adverse side effects and therapeutic resistance in PC. Therefore, we proposed to discover the potent phytochemical-based inhibitor against CYP17A1 using virtual screening. In this study, a phytochemicals library of ∼13800 molecules was selected to screen the best possible inhibitors against CYP17A1. A molecular modelling approach investigated detailed intermolecular interactions, their structural stability, and binding affinity. Further, in vitro and in vivo studies were performed to confirm the anticancer activity of identified potential inhibitor against CYP17A1. Friedelin from Cassia tora (CT) is identified as the best possible inhibitor from the screened library. MD simulation study reveals stable binding of Friedelin to conserved binding pocket of CYP17A1 with higher binding affinity than studied control, that is, Orteronel. Friedelin was tested on hormone-sensitive (22Rv1) and insensitive (DU145) cell lines and the IC50 value was found to be 72.025 and 81.766 µg/ml, respectively. CT extract showed a 25.28% IC50 value against 22Rv1, ∼92.6% increase in late Apoptosis/Necrosis, and three folds decrease in early apoptosis in treated cells compared to untreated cells. Further, animal studies show a marked decrease in prostate weight by 39.6% and prostate index by 36.5%, along with a reduction in serum PSA level by 71.7% and testosterone level by 92.4% compared to the testosterone group, which was further validated with histopathological studies. Thus, we propose Friedelin and CT extract as potential leads, which could be taken further for drug development in PC.[Figure: see text]Communicated by Ramaswamy H. Sarma.

Effect of Holoptelea integrifolia (Roxb.) Planch. n-Hexane Extract and Its Bioactive Compounds on Wound Healing and Anti-Inflammatory Activity.

The stem bark of Holoptelea integrifolia (Roxb.) Planch. has been applied for the treatment of human cutaneous diseases as well as canine demodicosis in several countries. However, no detailed mechanistic studies have been reported to support their use. In this study, thin-layer chromatography and gas chromatography were used to screen phytochemicals from the fresh stem bark extract of H. integrifolia. We found the two major bioactive compounds, friedelin and lupeol, and their activity on wound healing was further investigated in keratinocytes. Both bioactive compounds significantly reduced wound area and increased keratinocyte migration by increasing matrix metalloproteinases-9 production. Subsequently, we found that the mRNA gene expressions of cadherin 1 and desmoglobin 1 significantly decreased, whereas the gene expression involved in keratinocyte proliferation and homeostasis (keratin-17) increased in compound-treated human immortalized keratinocytes cells. The expression of inflammatory genes (cyclooxygenase-2 and inducible nitric oxide synthase) and pro-inflammatory cytokine genes (tumor necrosis factor-alpha and interleukin-6) was reduced by treatment with n-hexane extract of H. integrifolia and its bioactive compounds. Our results revealed that H. integrifolia extract and its bioactive compounds, friedelin and lupeol, exhibit wound-healing activity with anti-inflammatory properties, mediated by regulating the gene expression involved in skin re-epithelialization.

Friedelin Attenuates Neuronal Dysfunction and Memory Impairment by Inhibition of the Activated JNK/NF-κB Signalling Pathway in Scopolamine-Induced Mice Model of Neurodegeneration.

Oxidative stress (OS) and c-Jun N-terminal kinase (JNK) are both key indicators implicated in neuro-inflammatory signalling pathways and their respective neurodegenerative diseases. Drugs targeting these factors can be considered as suitable candidates for treatment of neuronal dysfunction and memory impairment. The present study encompasses beneficial effects of a naturally occurring triterpenoid, friedelin, against scopolamine-induced oxidative stress and neurodegenerative pathologies in mice models. The treated animals were subjected to behavioural tests i.e., Y-maze and Morris water maze (MWM) for memory dysfunction. The underlying mechanism was determined via western blotting, antioxidant enzymes and lipid profile analyses. Molecular docking studies were carried out to predict the binding modes of friedelin in the binding pocket of p-JNK protein. The results reveal that scopolamine caused oxidative stress by (1) inhibiting catalase (CAT), peroxidase enzyme (POD), superoxide dismutase (SOD), and reduced glutathione enzyme (GSH); (2) the up-regulation of thiobarbituric acid reactive substances (TBARS) in mice brain; and (3) affecting the neuronal synapse (both pre- and post-synapse) followed by associated memory dysfunction. In contrast, friedelin administration not only abolished scopolamine-induced oxidative stress, glial cell activation, and neuro-inflammation but also inhibited p-JNK and NF-κB and their downstream signaling molecules. Moreover, friedelin administration improved neuronal synapse and reversed scopolamine-induced memory impairment accompanied by the inhibition of ß-secretase enzyme (BACE-1) to halt amyloidogenic pathways of amyloid-ß production. In summary, all of the results show that friedelin is a potent naturally isolated neuro-therapeutic agent to reverse scopolamine-induced neuropathology, which is characteristic of Alzheimer's disease.

Characterization of the Biological Activity of the Ethanolic Extract from the Roots of Cannabis sativa L. Grown in Aeroponics.

Cannabis sativa var. Kompolti, a variety routinely used for food production purposes, is characterized by a low concentration of psychoactive molecules, although containing many other biologically attractive metabolites in all parts of the plant, including the roots. In the present work, we evaluate the specific biological activities of the roots' extract from plants cultivated through aeroponics, an affordable and reliable method facilitating the isolation and processing of roots, with the advantage of being suitable for industrial scale-up. Furthermore, aeroponics results in an increased net accumulation of the most biologically attractive constituents (ß-sitosterol, friedelin and epi-friedelanol) found in the roots. The ethanolic extract of the aeroponic roots of C. sativa (APEX) and its separate components are studied to evaluate their anti-inflammatory (modulation of the expression level of specific markers upon LPS stimulation in U937 cells, such as IL-6, IL-8, TNF-α, IkB-α, iNOS, IRAK-1 and miR-146a) and antioxidant (in either acellular or cellular settings) activities. The APEX anti-inflammatory and antioxidant capacities are also functionally benchmarked using the wound-healing assay. On the whole, the data obtained show that APEX and its main components showed significant anti-inflammatory and antioxidant activities, which may render the exploitation of roots as a source of natural antioxidants and anti-inflammatory agents highly attractive, with the additional technical and economic advantages of aeroponics compared to soil cultivation.

Friedelin Alleviates the Pathogenesis of Collagenase-Induced Tendinopathy in Mice by Promoting the Selective Autophagic Degradation of p65.

With the development of an aging population, tendinopathy has become a common musculoskeletal disease in the elderly with a high recurrence rate and no curative treatment. The inflammation mediated by NF-κB signaling plays an important role in tendon senescence and degeneration. Friedelin (FR) is a triterpenoid derived from green plants, which has a variety of pharmacological functions, such as analgesia, anti-inflammation, antioxidation, and anti-tumor functions. However, the role and mechanism of FR in tendinopathy are unclear. Here, we found that FR improved the mechanical strength of the Achilles tendon, restored the orderly arrangement of collagen fibers, reduced inflammatory cell infiltration, and promoted tenogenesis, thereby blocking the progression of tendinopathy. Mechanistically, FR promoted the autophagic degradation of p65 by enhancing the interaction between p62 and p65 and effectively inhibited the activation of the NF-κB pathway, thus alleviating the inflammatory response of tenocytes. In addition, FR recruited E3 ubiquitin enzyme RNF182 to increase the K48-linked ubiquitination of p65 and promoted p62-mediated autophagic degradation. Furthermore, blocking ubiquitination reversed the degradation of p65 by FR. Therefore, these findings identify the new pharmacological mechanism of the anti-inflammatory effect of FR and provide a new candidate drug for the treatment of tendinopathy.

The influence of friedelin, resinone, tingenone and betulin of compounds on chondrogenic differentiation of porcine adipose-derived mesenchymal stem cells (pADMSCs).

The study investigated the influence of friedelin, resinone, tingenone and betulin plant-based secondary metabolite compounds on cellular proliferation, extracellular matrix (ECM) components synthesis, expression of chondrogenic markers and maturation of differentiated chondrocytes (cell proliferation and hypertrophy) in porcine adipose-derived mesenchymal stem cells (pADMSCs) undergoing chondrogenic differentiation. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Cyquant assays were used to determine cell proliferation, viability, and total cellular DNA, DMMB (Dimethyl methylene blue) was used for glycosaminoglycan (GAG) synthesis, RT-qPCR for gene expression and histology combined with immunohistochemistry for cartilage ECM proteoglycan deposition. The MTT results showed that friedelin at 37 µM, resinone at 36 µM and betulin at 18 µM with cell viability of above 100% compared to control. Tingenone at 37 µM showed cell viability of about 76%. These concentrations were considered the most effective with no toxicity effect on the cells and were further analysed with TGF-ß3 (10 ng/mL) as a positive control. The results showed a high synthesis of DNA with friedelin on day 14. There was up-regulation of SOX 9, Col II and Col X with friedelin and resinone at day 14 with the significance of p < 0.01. Pellet from friedelin, resinone and tingenone showed more staining of the matrix for Safranin-O and Toluidine blue at day 14. Immunohistostaining of collagen type X (COL-10) showed more stain intensity at friedelin and resinone on day 21. These results provided new knowledge on the potential use of natural isolated secondary metabolites compounds as inducers for chondrogenic and bone differentiation.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Friedelin via Genetic Manipulation.

Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant Tripterygium wilfordii, is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered Saccharomyces cerevisiae, strains capable of producing high-level friedelin have not been stably engineered. In this study, a combined strategy was employed with integration of endogenous pathway genes into the genome and knockout of inhibiting genes by CRISPR/Cas9 technology, which successfully engineered multiple strains. After introducing an efficient TwOSC1T502E, all strains with genetic integration (tHMG1, ERG1, ERG20, ERG9, POS5, or UPC2.1) showed a 3.0∼6.8-fold increase in friedelin production compared with strain BY4741. Through further double knockout of inhibiting genes, only strains GD1 and GD3 produced higher yields. Moreover, strains GQ1 and GQ3 with quadruple mutants (bts1; rox1; ypl062w; yjl064w) displayed similar increases. Finally, the dominant strain GQ1 with TwOSC1T502E was cultured in an optimized medium in shake flasks, and the final yield of friedelin reached 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain BY4741 and 229% higher than that in ordinary SD-His-Ura medium. It was the highest titer for friedelin production to date. Our work provides a good example for triterpenoid production in microbial cell factories and lays a solid foundation for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

The Methionine 549 and Leucine 552 Residues of Friedelin Synthase from Maytenus ilicifolia Are Important for Substrate Binding Specificity.

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and ß-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

Yield, Characterization, and Possible Exploitation of Cannabis Sativa L. Roots Grown under Aeroponics Cultivation.

Cannabis sativa L. has been used for a long time to obtain food, fiber, and as a medicinal and psychoactive plant. Today, the nutraceutical potential of C.sativa is being increasingly reappraised; however, C. sativa roots remain poorly studied, despite citations in the scientific literature. In this direction, we identified and quantified the presence of valuable bioactives (namely, ß-sitosterol, stigmasterol, campesterol, friedelin, and epi-friedelanol) in the root extracts of C. sativa, a finding which might pave the way to the exploitation of the therapeutic potential of all parts of the C. sativa plant. To facilitate root harvesting and processing, aeroponic (AP) and aeroponic-elicited cultures (AEP) were established and compared to soil-cultivated plants (SP). Interestingly, considerably increased plant growth-particularly of the roots-and a significant increase (up to 20-fold in the case of ß-sitosterol) in the total content of the aforementioned roots' bioactive molecules were observed in AP and AEP. In conclusion, aeroponics, an easy, standardized, contaminant-free cultivation technique, facilitates the harvesting/processing of roots along with a greater production of their secondary bioactive metabolites, which could be utilized in the formulation of health-promoting and health-care products.

Friedelin exhibits antidiabetic effect in diabetic rats via modulation of glucose metabolism in liver and muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: Demand for plant-based medications and therapeutics is increasing worldwide as of its potential effects and no toxic. Traditionally, so many medicinal plants are used to treat diabetes. Subsequently, investigation on medicinal plants was enduring to discover potential antidiabetic drugs. A. tetracantha is used traditionally to cure diabetes mellitus, cough, dropsy, chronic diarrhea, rheumatism, phthisis and smallpox. Scientifically, A. tetracantha has been reported as an antidiabetic agent. Friedelin, the isolated compound has been reported as hypolipidemic, antioxidant, scavenging of free radicals, antiulcer, anti-inflammatory, analgesic and antipyretic agent. AIM OF THE STUDY: To scrutinize the mechanism of antidiabetic activity of friedelin isolated from the leaves of A. tetracantha. MATERIALS AND METHODS: A. tetracantha leaves powder (5 kg) was soaked in hexane (15 L) to obtain hexane extract. Using column chromatography, the hexane extract was fractionated using a combination of solvents like hexane and ethyl acetate. 25 fractions were obtained and the fractions 13 and 14 yielded the compound, friedelin. Friedelin at the doses of 20 and 40 mg/kg was used to treated STZ -induced diabetic rats for 28 days. Later 28 days of treatment, the bodyweight changes, levels of blood glucose, insulin, SGOT, SGPT, SALP, liver glycogen and total protein were assessed. RESULTS: Friedelin significantly brought these altered levels to near normal. Moreover, friedelin also enhanced the translocation as well as activation of GLUT2 and GLUT4 through PI3K/p-Akt signaling cascade in skeletal muscles and liver on diabetic rats. CONCLUSION: This finding proved that friedelin has an anti-diabetic effect through insulin-dependent signaling cascade mechanism, thus it may lead to establishing a drug to treat type 2 diabetes mellitus.

Studies on chemical constituents of Chloranthus fortunei / 中草药

Objective: To study the antitumor constituents from Chloranthus fortunei. Methods: Various chromatographic techniques and spectroscopic methods were applied to investigate the chemical constituents from C. fortunei, and some of the compounds were screened for their antitumor activities by MTT method. Results: Sixteen compounds were obtained from the whole plants of C. fortunei and identified as rosmarinic acid (1), 2’-hydroxy-4,3’,4’,6’-tetramethoxychalcone (2), flavokawain A (3), cycloshizukaol A (4), atractylenolide III (5), 4β-hydroxy-8,12-epoxyeudesma-7,11-diene-1,6-dione (6), (8α)-6,8-dihydroxycadina-7 (11),10 (15)-dien-12-oic acid γ-lactone (7), curcolonol (8), 11-hydroxyldrim-8,12-en-14-oic acid (9), friedelin (10), isovanillic acid (11), 6β-hydroxystigmast-4-en-3-one (12), 3,4-dihydroxybenzoic acid (13), shikimic acid (14), scopolin (15) and N-acetyltyramine 1-O-β-D-glucoside (16). Compounds 4 and 5 showed weak cytotoxicity with IC50 ranged from 46 to 85 μmol/L. Conclusion: Compounds 2, 10, 11, and 13-15 are obtained from the genus Chloranthus for the first time and compounds 1-3 and 6-16 are isolated from C. fortunei for the first time. Some sesquiterpenoids from C. fortunei exhibited weak antitumor activities.

CYP712K4 Catalyzes the C-29 Oxidation of Friedelin in the Maytenus ilicifolia Quinone Methide Triterpenoid Biosynthesis Pathway.

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.

Functional characterization of an oxidosqualene cyclase (PdFRS) encoding a monofunctional friedelin synthase in Populus davidiana.

MAIN CONCLUSION: An oxidosqualene cyclase (PdFRS) from Populus davidiana was characterized as a monofunctional friedelin synthase by its heterologous expression in yeast and overexpression in plants. Triterpenes are one of the largest classes of plant chemical compounds composed of three terpene units, which form the basic skeleton of all sterols and saponins. Friedelin (friedelan-3-one), a pentacyclic triterpene, occurs in many plant families and is particularly present in rich amounts in cork tissues from trees. The biosynthesis of friedelin occurs through the oxidosqualene cyclase (OSC) enzyme that generates friedelin from 2,3-oxidosqualene after the maximum rearrangement of a triterpene skeleton. Populus davidiana is called Korean aspen and grows in northern East Asia. From 57,322 unique sequences generated from the P. davidiana transcriptome database, one complete coding sequence (PdFRS) was obtained from a contig, which showed 74% identity to Betula platyphylla ß-amyrin synthase and 73% identity with friedelin synthase from Maytenus ilicifolia. The open reading frame (ORF) region of the PdFRS sequence was 2280 bp long and composed a 759 amino acid protein with a predicted molecular mass of 87.81 kDa. qPCR analysis revealed that methyl jasmonate treatments strongly upregulated PdFRS gene expression and resulted in enhanced friedelin accumulation in leaves. Heterologous expression of the PdFRS gene in yeast resulted in the production of friedelin triterpene as a single product, which was confirmed by comparison with the mass fragmentation pattern from an authentic friedelin standard by GC/MS analysis. Transgenic P. davidiana overexpressing the PdFRS gene was constructed via Agrobacterium-mediated transformation. Overexpression of PdFRS in transgenic P. davidiana lines resulted in enhanced friedelin production.

Characterization of chemical compounds susceptible to be extracted from cork by the wine using GC-MS and 1H NMR metabolomic approaches.

This work presents a metabolomics study of cork by gas chromatography-mass spectrometry (GC-MS) and 1H nuclear magnetic resonance (NMR) spectroscopy to characterize compounds susceptible to be extracted from cork by the wine in an attempt to find a relationship between the content of these compounds and the geographical origin of cork. Cork from eleven geographical regions was studied, five from Portugal and six from Spain. Unsupervised pattern recognition techniques unveiled three main clusters of regions according to their chemical similarity but not related with geographical proximity. Nineteen compounds were found to be responsible for the clusters, including terpenes (trans-squalene, friedelin, camphene, trans-3-pinanone, 1-terpinen-4-ol, two sesquiterpenes), polyphenols (vescalagin, castalagin), among others (pyrogallol, glucosan, sitost-4-en-3-one, o-cymene, quinic acid, five unknowns). These preliminary results unveiled the potential for a more efficient selection of cork planks for stoppers production based on the compounds susceptible to be extracted from cork by the wine.

1H NMR-based metabolomics of antimalarial plant species traditionally used by Vha-Venda people in Limpopo Province, South Africa and isolation of antiplasmodial compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: The Vha-Venda people living in rural areas of Limpopo Province of South Africa regularly use traditional plant-based medicines to treat malaria. In our earlier publication, twenty indigenous plant species used to treat malaria or its symptoms by Vha-Venda people were evaluated for antiplasmodial activity. The main objective of the current study was to assess the robustness of NMR-based metabolomics in discriminating classes of secondary compounds that are responsible for the observed antimalarial activity and the isolation of antiplasmodial compounds. MATERIALS AND METHODS: Twenty dichloromethane extracts were reconstituted in CDCl3, subjected to 1H NMR-based metabolomic analysis on a Varian 600 MHz spectrometer and the acquired 1H NMR spectra were then evaluated collectively using multivariate data analysis (MDA). Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA) were used to 'globally' discern antiplasmodial profiles. A contribution plot was then generated from the OPLS-DA scoring plot in an attempt to determine the classes of compounds that are responsible for the observed grouping. Further phytochemical analyses were conducted on the lipophilic extracts of Tabernaemontana elegans and Vangueria infausta subsp. infausta. These best candidates were fractionated, purified and their isolated compounds identified based on conventional chromatographic and spectroscopic techniques. RESULTS: The PCA did not separate the acquired profiles according to the detected antiplasmodial bioactivity. Application of a supervised OPLS-DA on the 1H NMR profiles resulted in a discrimination pattern that could be correlated to the observed antimalarial bioactivity. A contribution plot generated from the OPLS-DA scoring plot illustrated the classes of compounds responsible for the observed grouping. Prominent peaks were observed in the aromatic, sugar-based/N-containing and aliphatic spectral regions of the contribution plot. Two known indole alkaloids were isolated from T. elegans, and identified as tabernaemontanine (IC50 = 12.0 ±â€¯0.8 µM) and dregamine (IC50 = 62.0 ±â€¯2.4 µM). Friedelin (IC50 = 7.20 ±â€¯0.5 µM) and morindolide (IC50 = 107.1 ±â€¯0.6 µM) were isolated from V. infausta subsp. infausta. This is the first report of the rare iridoid lactone, morindolide's antimalarial activity. While these two compounds have been previously identified, this is the first account of their occurrence in the genus Vangueria. CONCLUSION: The study illustrated the potential of NMR-based metabolomics in discriminating classes of compounds that may be attributed to antiplasmodial activity. Additionally, the study demonstrated the potential of discovering novel antiplasmodial scaffolds from medicinal plants and the rationale for the bioprospecting antimalarial plant species used by Vha-Venda people.