Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus.
ABSTRACT: Vitex agnus-castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus-castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus-castus. With the assistance of matrix-assisted laser desorption ionisation-mass spectrometry imaging (MALDI-MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome-specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus-castus. The identified CYP, VacCYP76BK1, was found to catalyse 16-hydroxylation of the diol-diterpene, peregrinol, to labd-13Z-ene-9,15,16-triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan- and lactone-containing diterpenoids that are present in this species.
Project description:A new labdane-diterpene, viteagnusin I (1), together with 23 known phytoconstituents were isolated from the fruits of Vitex agnus-castus L, and their structures characterized by spectroscopic methods (NMR and MS). The known compounds include ten flavonoids, five terpenoids, three neolignans, and four phenolic compounds, as well as one glyceride. Biological evaluation identified apigenin, 3-methylkaempferol, luteolin, and casticin as weak ligands of delta and mu opioid receptors, exhibiting dose-dependent receptor binding.
Project description:BNO 1095, a standardized dry extract from the fruits of <i>Vitex agnus-castus</i>, represents an approved herbal medicinal product for the treatment of premenstrual syndrome. Angiogenesis, the formation of new blood vessels from pre-existing capillaries, plays a major role in physiological situations, such as wound healing or tissue growth in female reproductive organs, but it is also of great importance in pathophysiological conditions such as chronic inflammatory diseases or cancer. Angiogenesis is a highly regulated multi-step process consisting of distinct key events that can be influenced pharmacologically. Few studies suggested anti-angiogenic actions of <i>V. agnus-castus</i> fruit extracts in <i>in vivo</i> and <i>ex vivo</i> models. Here, we provide for the first time profound <i>in vitro</i> data on BNO 1095-derived anti-angiogenic effects focusing on distinct angiogenesis-related endothelial cell functions that are inevitable for the process of new blood vessel formation. We found that <i>V. agnus-castus</i> extract significantly attenuated undirected and chemotactic migration of primary human endothelial cells. Moreover, the extract efficiently inhibited endothelial cell proliferation and reduced the formation of tube-like structures on Matrigel. Of note, the treatment of endothelial cell spheroids almost blocked endothelial sprouting in a 3D collagen gel. Our data present new and detailed insights into the anti-angiogenic actions of BNO 1095 and, therefore, suggest a novel scope of potential therapeutic applications of the extract for which these anti-angiogenic properties are required.
Project description:As part of our continuing efforts in the search for potential biologically active compounds from medicinal plants, we have isolated 18 compounds including two novel nitrogen containing diterpenes from extracts of the fruits of Vitex agnus-castus. These isolates, along with our previously obtained novel compound vitexlactam A (1), were evaluated for potential biological effects, including cancer chemoprevention. Chemically, the nitrogenous isolates were found to be two labdane diterpene alkaloids, each containing an ? , ? -unsaturated ? -lactam moiety. Structurally, they were elucidated to be 9 ? -hydroxy-13(14)-labden-16,15-amide (2) and 6 ? -acetoxy-9 ? -hydroxy-13(14)-labden-15,16-amide (3), which were named vitexlactams B and C, respectively. The 15 known isolates were identified as vitexilactone (4), rotundifuran (5), 8-epi-manoyl oxide (6), vitetrifolin D (7), spathulenol (8), cis-dihydro-dehydro-diconiferylalcohol-9-O- ? -D-glucoside (9), luteolin-7-O-glucoside (10), 5-hydroxy-3,6,7,4'-tetramethoxyflavone (11), casticin (12), artemetin (13), aucubin (14), agnuside (15), ? -sitosterol (16), p-hydroxybenzoic acid (17), and p-hydroxybenzoic acid glucose ester (18). All compound structures were determined/identified on the basis of 1D and/or 2D NMR and mass spectrometry techniques. Compounds 6, 8, 9, and 18 were reported from a Vitex spieces for the first time. The cancer chemopreventive potentials of these isolates were evaluated for NADP(H):quinone oxidoreductase type 1 (QR1) induction activity. Compound 7 demonstrated promising QR1 induction effect, while the new compound vitexlactam (3) was only slightly active.
Project description:<h4>Background</h4>Candida infections are becoming more drug resistant; it is necessary to search for alternative medications to treat them. Therefore, the present study estimates the anticandidal activity of <i>Vitex agnus-castus</i> (VA-C) leaf extracts.<h4>Methods</h4>We used the agar well diffusion method to assess the anticandidal activity of three different VA-C leaf extracts (ethanol, methanol, and water) against three <i>Candida</i> species (<i>Candida tropicalis</i>, <i>Candida albicans</i>, and <i>Candida ciferrii</i>). The minimum inhibitory concentration (MIC) was estimated using the two-fold dilution method and the minimum fungicidal concentration (MFC) was determined using the classic pour plate technique. The MFC/MIC ratio was calculated to estimate the microbicidal or microbiostatic activity. A gas chromatography mass spectrometer was used to screen the phytochemicals of the VA-C leaf extracts (ethanol, methanol, and water).<h4>Results</h4>All VA-C extracts ethanol, methanol, and water were significantly inhibited the growth of the test <i>Candida</i> species and the inhibition activity depended on the solvent used and the <i>Candida</i> species. The results showed that <i>C. tropicalis</i> was the most highly inhibited by all extracts followed by <i>C. albicans</i> and <i>C. ciferrii</i>. The MIC values were 12.5-25 µg/ml, and MFC values were 25-100 µg/ml. The ratios of MFC/MIC were two-fold to four-fold which was considered candidacidal activity. Ninety-five phytochemical compounds were identified by the GC-MS assay for the VA-C leaf extracts. The total number of compounds per extract differed. Methanol had 43 compounds, ethanol had 47 compounds, and water had 52 compounds. The highest compound concentrations were: 4,5-Dichloro-1,3-dioxolan-2-one in ethanol and methanol, 1H-Indene, 2,3-dihydro-1,1,2,3,3-pentamethyl in ethanol, Isobutyl 4-hydroxybenzoate in methanol, and Benzoic acid and 4-hydroxy- in water. These phytochemical compounds belong to different bioactive chemical group such as polyphenols, fatty acids, terpenes, terpenoids, steroids, aldehydes, alcohols, and esters, and most of which have anticandidal activity.<h4>Conclusions</h4>VA-C leaf extracts may be useful alternatives to anticandidal drugs, based on their effectiveness against all test <i>Candida</i> species at low concentrations. However, appropriate toxicology screening should be conducted before use.
Project description:BACKGROUND:Horehound (Marrubium vulgare) is a medicinal plant whose signature bioactive compounds, marrubiin and related furanoid diterpenoid lactones, have potential applications for the treatment of cardiovascular diseases and type II diabetes. Lack of scalable plant cultivation and the complex metabolite profile of M. vulgare limit access to marrubiin via extraction from plant biomass. Knowledge of the marrubiin-biosynthetic enzymes can enable the development of metabolic engineering platforms for marrubiin production. We previously identified two diterpene synthases, MvCPS1 and MvELS, that act sequentially to form 9,13-epoxy-labd-14-ene. Conversion of 9,13-epoxy-labd-14-ene by cytochrome P450 monooxygenase (P450) enzymes can be hypothesized to facilitate key functional modification reactions in the formation of marrubiin and related compounds. RESULTS:Mining a M. vulgare leaf transcriptome database identified 95 full-length P450 candidates. Cloning and functional analysis of select P450 candidates showing high transcript abundance revealed a member of the CYP71 family, CYP71AU87, that catalyzed the hydroxylation of 9,13-epoxy-labd-14-ene to yield two isomeric products, 9,13-epoxy labd-14-ene-18-ol and 9,13-epoxy labd-14-ene-19-ol, as verified by GC-MS and NMR analysis. Additional transient Nicotiana benthamiana co-expression assays of CYP71AU87 with different diterpene synthase pairs suggested that CYP71AU87 is specific to the sequential MvCPS1 and MvELS product 9,13-epoxy-labd-14-ene. Although the P450 products were not detectable in planta, high levels of CYP71AU87 gene expression in marrubiin-accumulating tissues supported a role in the formation of marrubiin and related diterpenoids in M. vulgare. CONCLUSIONS:In a sequential reaction with the diterpene synthase pair MvCPS1 and MvELS, CYP71AU87 forms the isomeric products 9,13-epoxy labd-14-ene-18/19-ol as probable intermediates in marrubiin biosynthesis. Although its metabolic relevance in planta will necessitate further genetic studies, identification of the CYP71AU87 catalytic activity expands our knowledge of the functional landscape of plant P450 enzymes involved in specialized diterpenoid metabolism and can provide a resource for the formulation of marrubiin and related bioactive natural products.
Project description:As a major staple food, maize (<i>Zea mays</i>) is critical to food security. Shifting environmental pressures increasingly hamper crop defense capacities, causing expanded harvest loss. Specialized labdane-type diterpenoids are key components of maize chemical defense and ecological adaptation. Labdane diterpenoid biosynthesis most commonly requires the pairwise activity of class II and class I diterpene synthases (diTPSs) that convert the central precursor geranylgeranyl diphosphate into distinct diterpenoid scaffolds. Two maize class II diTPSs, ANTHER EAR 1 and 2 (ZmAN1/2), have been previously identified as catalytically redundant <i>ent</i>-copalyl diphosphate (CPP) synthases. ZmAN1 is essential for gibberellin phytohormone biosynthesis, whereas ZmAN2 is stress-inducible and governs the formation of defensive kauralexin and dolabralexin diterpenoids. Here, we report the biochemical characterization of the two remaining class II diTPSs present in the maize genome, COPALYL DIPHOSPHATE SYNTHASE 3 (ZmCPS3) and COPALYL DIPHOSPHATE SYNTHASE 4 (ZmCPS4). Functional analysis via microbial co-expression assays identified ZmCPS3 as a (+)-CPP synthase, with functionally conserved orthologs occurring in wheat (<i>Triticum aestivum</i>) and numerous dicot species. ZmCPS4 formed the unusual prenyl diphosphate, 8,13-CPP (labda-8,13-dien-15-yl diphosphate), as verified by mass spectrometry and nuclear magnetic resonance. As a minor product, ZmCPS4 also produced labda-13-en-8-ol diphosphate (LPP). Root gene expression profiles did not indicate an inducible role of <i>ZmCPS3</i> in maize stress responses. By contrast, <i>ZmCPS4</i> showed a pattern of inducible gene expression in roots exposed to oxidative stress, supporting a possible role in abiotic stress responses. Identification of the catalytic activities of ZmCPS3 and ZmCPS4 clarifies the first committed reactions controlling the diversity of defensive diterpenoids in maize, and suggests the existence of additional yet undiscovered diterpenoid pathways.
Project description:Members of the mint family (Lamiaceae) accumulate a wide variety of industrially and medicinally relevant diterpenes. We recently sequenced leaf transcriptomes from 48 phylogenetically diverse Lamiaceae species. Here, we summarize the available chemotaxonomic and enzyme activity data for diterpene synthases (diTPSs) in the Lamiaceae and leverage the new transcriptomes to explore the diTPS sequence and functional space. Candidate genes were selected with an intent to evenly sample the sequence homology space and to focus on species in which diTPS transcripts were found, yet from which no diterpene structures have been previously reported. We functionally characterized nine class II diTPSs and 10 class I diTPSs from 11 distinct plant species and found five class II activities, including two novel activities, as well as a spectrum of class I activities. Among the class II diTPSs, we identified a neo-cleroda-4(18),13E-dienyl diphosphate synthase from Ajuga reptans, catalyzing the likely first step in the biosynthesis of a variety of insect-antifeedant compounds. Among the class I diTPSs was a palustradiene synthase from Origanum majorana, leading to the discovery of specialized diterpenes in that species. Our results provide insights into the diversification of diterpene biosynthesis in the mint family and establish a comprehensive foundation for continued investigation of diterpene biosynthesis in the Lamiaceae.
Project description:Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis.
Project description:While terpenoid production is generally associated with plants, a variety of fungi contain operons predicted to lead to such biosynthesis. Notably, fungi contain a number of cyclases characteristic of labdane-related diterpenoid metabolism, which have not been much explored. These also are often found near cytochrome P450 (CYP) mono-oxygenases that presumably further decorate the ensuing diterpene, suggesting that these fungi might produce more elaborate diterpenoids. To probe the functional diversity of such biosynthetic capacity, an investigation of the phylogenetically diverse cyclases and associated CYPs from the fungal genus Aspergillus was undertaken, revealing their ability to produce isopimaradiene-derived diterpenoids. Intriguingly, labdane-related diterpenoid biosynthetic genes are largely found in plant-associated fungi, hinting that these natural products may play a role in such interactions. Accordingly, it is hypothesized here that isopimarane production may assist the plant-saprophytic lifestyle of Aspergillus fungi.
Project description:The stolbur phytoplasma vector Hyalesthes obsoletus is generally considered as a polyphagous species associated with numerous wild and cultivated plants. However, recent research in southeastern Europe, the distribution centre of H. obsoletus and the area of most stolbur-inflicted crop diseases, points toward specific host-plant associations of the vector, indicating specific vector-based transmission routes. Here, we study the specificity of populations associated with four host-plants using mitochondrial and nuclear genetic markers, and we evaluate the evolution of host-shifts in H. obsoletus. Host-plant use was confirmed for Convolvulus arvensis, Urtica dioica, Vitex agnus-castus and Crepis foetida. Mitochondrial genetic analysis showed sympatric occurrence of three phylogenetic lineages that were ecologically delineated by host-plant preference, but were morphologically inseparable. Nuclear data supported the existence of three genetic groups (Evanno's ?K(3) = 803.72) with average genetic membership probabilities > 90%. While populations associated with C. arvensis and U. dioica form a homogenous group, populations affiliated with V. agnus-castus and C. foetida constitute two independent plant-associated lineages. The geographical signal permeating the surveyed populations indicated complex diversification processes associated with host-plant selection and likely derived from post-glacial refugia in the eastern Mediterranean. This study provides evidence for cryptic species diversification within H. obsoletus sensu lato: i) consistent mitochondrial differentiation (1.1-1.5%) among host-associated populations in syntopy and in geographically distant areas, ii) nuclear genetic variance supporting mitochondrial data, and iii) average mitochondrial genetic distances among host-associated meta-populations are comparable to the most closely related, morphologically distinguishable species, i.e., Hyalesthes thracicus (2.1-3.3%).