Clerodane-type Diterpenes Research Articles

Stereochemical assignment of clerodane-type diterpenes from the fruits of Casearia grewiifolia and their ability to inhibit PCSK9 expression

More than 20 natural products have been reported to modulate PCSK9-mediated cholesterol regulation, and small-molecule-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors continue to be developed and identified. Here, twelve undescribed clerodane-type diterpenes (1–9 and 12–14) and two known compounds were isolated from the chloroform-soluble extract of the dried fruits of Casearia grewiifolia Vent. using a PCSK9 mRNA expression monitoring assay. Among the undescribed compounds, the stereochemistry of two diastereomeric grewiifolins A and B (1 and 2) were extensively elucidated using 2D Nuclear Overhauser Effect Spectroscopy (NOESY) experiments, excitation-sculptured indirect detection experiments (EXSIDE), interproton distance analyses, and computational calculations that included quantum chemical shift calculations combined with DP4+ analysis. All isolates were assessed for their inhibitory activity against PCSK9 and IDOL mRNA expression. Among the compounds tested, compound 3 inhibited PCSK9 and IDOL mRNA expression.

Schistosomicidal activity of kaurane, labdane and clerodane-type diterpenes obtained by fungal transformation

Schistosomiasis continues to be a huge challenge for researchers, pharmaceutical companies, and governments in developing countries. Diterpene compounds are good source for the development of novel potential leading compounds to treat schistosomiasis. We are reporting herein the schistosomicidal activity of ent-kaurenoic acid, ent-copalic acid, ent-hardwickiic acid, isolated from oleoresins of Copaifera spp, and of their derivatives obtained by fungal transformation with strains of Aspergillus fumigatus, A. terreus, A. phoenicis and A. ochraceus, and of Cunninghamella echinulata e C. elegans. The in vitro antiparasitical assays were performed using adult worm pairs of Schistosoma mansoni for the evaluation of the worm pairing, egg production, and eggs development. Ten kaurane, labdane and clerodane-type diterpenes were obtained by fungal transformation and 7α-acetoxy-ent-kaur-16-en-19-oic acid and ent-hardwickiic acid were the most active ones by causing mortality of 100 % of the parasites within 24 h (concentrations of 100.0 and 200.0 μM) and displaying respective IC50 values for 24, 48 and 72 h of 56.7, 37.6 and 29.2 μM and 29.6, 30.8 and 25.7 μM. Additionally, 7α-acetoxy-ent-kaur-16-en-19-oic acid and ent-hardwickiic acid highly reduced the number of laid eggs at 6.25 and 12.5 μM, respectively. These diterpenes should be further investigated as potential candidates for antiparasitic drug discovery.

Fragmentation study of clerodane diterpenes from Casearia species by tandem mass spectrometry (quadrupole time-of-flight and ion trap).

Clerodane-type diterpenes from Casearia species show important pharmacological activites such as antitumor, antimicrobial and anti-inflamatory. There are several mass spectrometry (MS)-based methods for identification of diterpenes; however, there is still a lack of MS procedures capable of providing characteristic fragmentation pathways for a rapid and unambiguous elucidation of casearin-like compounds. Casearin-like compounds were investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The fragmentation studies were carried out by tandem mass spectrometry in space (quadrupole time-of-flight (QTOF)) using different collision energies and also by tandem mass spectrometry in time (QIT) by selective isolation of product ions. Casearin-like compounds presented a predominance of sodium- and potassium-cationized precursor ions. Both QIT and QTOF techniques provided sequential neutral losses of esters related to the R1 to R5 substituents linked to the nucleus of the clerodane diterpenes. The fragmentation pathway is initiated with a cleavage of the ester moieties R2 followed by the elimination of the ester groups R3 , both losing neutral carboxylic acids. Using QIT, it was also possible to observe the cleavage of the ester groups R1 or R5 by MS4 experiments. Through a rational analysis of the fragmentation mechanisms of Casearia diterpenes it was possible to suggest an annotation strategy based on the sequential cleavages of the ester groups related to the R2 , R3 and R5 substituents. These results will assist studies of the dereplication and metabolomics involving casearin-like compounds present in complex extracts of Casearia species.

Antimicrobial and antibiofilm activities of Casearia sylvestris extracts from distinct Brazilian biomes against Streptococcus mutans and Candida albicans

BackgroundDental caries is a biofilm-diet-dependent worldwide public health problem, and approaches against microorganisms in cariogenic biofilms are necessary.MethodsThe antimicrobial and antibiofilm activities of 12 Casearia sylvestris extracts (0.50 mg/mL) from different Brazilian biomes (Atlantic Forest, Cerrado, Caatinga, Pampa, and Pantanal) and varieties (sylvestris, lingua, and intermediate) were tested against two species found in cariogenic biofilms (Streptococcus mutans and Candida albicans). The extracts effective against S. mutans were used to evaluate the "adhesion strength" of this bacterium to the salivary pellicle and initial glucan matrix and the S. mutans-GtfB activity. Also, the antimicrobial activity against S. mutans of three fractions (methanol, ethyl acetate, and hexane; 0.25 mg/mL) from the extracts was evaluated.ResultsThree extracts from the Atlantic Forest variety sylvestris (FLO/SC, GUA/CE, PRE/SP) reduced ≥50% (> 3 logs) S. mutans viable population (p < 0.0001 vs. vehicle), while two extracts from the same biome and variety (PAC/CE, PRE/SP) decreased ≥50% of the viable counts of C. albicans (p < 0.0001 vs. vehicle). For S. mutans biofilms, three extracts (GUA/CE, PAC/CE, PRE/SP) reduced the biomass by ≥91% (p > 0.0001 vs. vehicle) and 100% of the microbial population (p < 0.0001 vs. vehicle). However, for the fungal biofilm, two extracts (PAC/CE, PRE/SP) reduced the viable counts by ≥52% (p < 0.0001 vs. vehicle), but none reduced biomass. The extracts with higher antimicrobial and antibiofilm activities presented higher content of clerodane-type diterpenes and lower content of glycosylated flavonoids than the less active extracts. The extracts had no effect on the removal of cells adhered to the pellicle (p > 0.05 vs. vehicle) while promoted the detachment of a larger number of S. mutans cells from GtfB-glucan matrix (p < 0.0031 vs. vehicle), and FLO/SC, GUA/CE and PRE/SP reduced the quantity of glucans (p < 0.0136 vs. vehicle). Only the ethyl acetate fractions reduced the microbial population of S. mutans (p < 0.0001 vs. vehicle), except for one (PAC/CE). Among the ethyl acetate fractions, three from var. lingua (two from Cerrado, and one from Cerrado/Caatinga) reduced ≥83% of the microbial population.ConclusionsC. sylvestris extracts from Atlantic Forest var. sylvestris and ethyl acetate fractions from Cerrado and Cerrado/Caatinga var. lingua may be used as a strategy against cariogenic microorganisms.

Non-polar and polar chemical profiling of six Casearia species (Salicaceae)

Six species of Casearia (C. decandra, C. grandiflora, C. javitensis, C. arborea, C. lasiophylla and C. ulmifolia) were chemically investigated in their non-polar and polar constituents. To this purpose, leaf extracts were analyzed by Gas- (GC) and Liquid-Chromatography (LC) coupled with Mass Spectrometry (MS). Twenty compounds, mainly identified as terpenes, fatty acids and hydrocarbons, and differentially expressed in the six species, were detected in the non-polar extracts. Fourteen compounds, among which glycosylated flavonoids and clerodane-type diterpenes, were tentatively identified by LC-MS/MS analysis in the leaf ethanol extracts.

English

The Casearia species (Salicaceae) occur in the tropics and subtropics and their extracts are rich in clerodane-type diterpenes, known as casearins. According to the literature, extracts from Casearia sylvestris exhibit cytotoxic and genotoxic effects in different tumor cell lines, possibly related to the casearins. On the other hand, there are few studies related to the DNA damages of the essential oils from this species. This study is aimed at evaluating DNA damages promoted by the essential oil from leaves of C. sylvestris collected in Rio de Janeiro. The essential oil was obtained from fresh leaves (1.5 kg) by hydrodistillation for 2 h in a Clevenger-type apparatus, and analyzed both by gas chromatography coupled to a mass spectrometer (GC-MS) and gas chromatography coupled to a flame ionization detector (GC-FID). These analyses revealed a very diversified (n = 21 compounds) volatile fraction composed mainly of non-oxygenated sesquiterpenes (72.1%), and the major component was identified as &alpha;-humulene (17.8%). Genotoxicity was evaluated by the comet assay, showing DNA damages, mainly of classes 3 and 4 at 4.0 &micro;g/ml (p &lt; 0.05) according to the damage index (DI). This is the first demonstration of DNA damages in response to the essential oil of C. sylvestris. Key words: A549, &alpha;-humulene, comet assay, sesquiterpenes.

Two New Clerodane Diterpenes from Tinospora sagittata.

Two new clerodane-type diterpenes, tinosporins C (1) and tinosporins D (2) were isolated from the stems of Tinospora sagittata (Oliv.), together with three known ones, columbin (3), tinophylloloside (4), and tinospinoside D (5). The structures of these compounds were determined on the basis of spectroscopic data interpretation, with that of the absolute configuration of compound 1 was assigned by experimental and calculated ECD spectra. The cytotoxicity and α-glucosidase inhibitory activities of isolated compounds were evaluated in vitro.

Antiamoebic and Antigiardial Activity of Clerodane Diterpenes from Mexican Salvia Species Used for the Treatment of Diarrhea.

Terpenoids from Salvia species have been identified to possess biological properties as antiprotozoal agents. Here, we evaluated the antiamoebic and antigiardial activities of 14 known clerodane and modified clerodane-type diterpenes isolated from five Mexican Salvia species against Entamoeba histolytica and Giardia lamblia, and analyzed the effects of the functionalities in decalin ring or in the whole clerodane framework to visualize the structural requirements necessary to produce an antiprotozoal activity. Among these, linearolactone was the most active clerodane diterpene against both protozoa with IC50 values of 22.9 μM for E. histolytica and of 28.2 μM in the case of G. lamblia. In this context it may be a lead compound for the development of novel therapeutic agent for the treatment of diarrhea and dysentery. The remaining diterpenes assayed showed moderate to weak activity against both protozoa. These findings give support to the use of Salvia species in the traditional medicine from México for the treatment of diarrhea.

Development of a comprehensive method for analyzing clerodane-type diterpenes and phenolic compounds from Casearia sylvestris Swartz (Salicaceae) based on ultra high performance liquid chromatography combined with chemometric tools.

This work describes the development and optimization of an analytical method utilizing liquid chromatography and chemometrics to evaluate and differentiate two varieties of Casearia sylvestris Swartz (Salicaceae) from São Paulo State (Brazil) based on their secondary metabolite profiles. Previously, analytical studies only concerned the analysis of clerodane-type diterpenes. Therefore, considering the importance of including phenolic compounds in such analysis, we used design of experiments to simultaneously extract and detect the largest number of compounds from both chemical classes. This new strategy allowed a comprehensive chromatographic analysis of C. sylvestris, and the results for the two varieties exhibited an interesting distribution according to their original ecosystems, suggesting a strong correlation to the main metabolites found in each species group. Besides their inherent morphological differences, C. sylvestris variety lingua, mainly found in Cerrado areas, predominantly contains phenolic compounds, while C. sylvestris variety sylvestris, mainly found in Atlantic Forest areas, contains mostly clerodane-type diterpenes. Finally, it was also possible to observe differences in the secondary metabolite composition within each group depending on the place where samples were collected.

Differential proteomics of two varieties of Casearia sylvestris Sw. (Salicaceae)

The Brazilian native plant species Casearia sylvestris SW. (Salicaceae) is an important representative of the Casearia Jacq. genus due to its antiulcerogenic, anti-inflammatory and cytotoxic activities. This species is divided into two varieties according to its morphology. Interestingly, C. sylvestris variety Sylvestris can be characterized by the production of clerodane-type diterpenes (casearins), while C. sylvestris variety Lingua produces mainly phenolic compounds [1]. Despite the existence of data concerning its pharmacological properties, secondary metabolite chemistry and morphological features, so far nothing is known about its expressed proteins. Thus, the aim of this work was to access the protein profile of C. sylvestris by selecting individuals that show differentiated production of diterpenes and phenolic compounds. To achieve this goal, five trees of each variety were sampled to compose a representative sampling and their metabolic profiles were analysed by ultra-performance liquid chromatography coupled to diode-array-detection (UPLC-DAD). Main peaks identification was achieved by liquid chromatography coupled to mass spectrometry (LC-MS) or by comparison using standard compounds. Protein extracts from both varieties were fractionated using gel-free digestion and fractionation (FASP) [2] followed by liquid chromatography coupled to tandem mass spectrometry analyses (LC-MS/MS). Protein identification and comparison between the two varieties were made in the peptide level using the platform Proteomatic® [3], running OMSSA and X! Tandem against the Populus trichocarpa protein data bank, and PEAKS for de novo amino acid sequencing from MS/MS data. Using this approach, it was possible to identify 820 peptide sequences and to annotate 547 proteins with high accuracy. In conclusion, a combination of metabolic profiling with large scale proteomic study of C. sylvestris allowed the detection of considerable differences between the two varieties.

ChemInform Abstract: Two New Diterpenes from Solidago canadensis

Abstractisolation and structure elucidation of solidagocanins A (Ia) and B (Ib)

Two New Diterpenes from Solidago canadensis

AbstractTwo new clerodane‐type diterpenes, solidagocanins A and B (1 and 2, resp.), as well as two known compounds, mellein (3) and physcion (4), were isolated from the aerial parts of Solidago canadensis. The structures of the new compounds were elucidated by spectroscopic analyses, including 2D‐NMR techniques.

Constituents of Polyalthia longifolia var. pendula

Three new clerodane-type diterpenes, 6alpha,16-dihydroxycleroda-3,13-dien-15-oic acid (1), 6alpha,16-dihydroxycleroda-4(18),13-dien-15-oic acid (2), and 4alpha,18beta-epoxy-16-hydroxyclerod-13-en-15-oic acid (3), and four new protoberberine alkaloids, (-)-8-oxo-10-hydroxy-2,3,9-trimethoxyberberine (4), (-)-8-oxo-2,11-dihydroxy-3,10-dimethoxyberberine (5), (-)-8-oxo-11-hydroxy-2,3,9,10-tetramethoxyberberine (6), and (-)-8-oxo-2,10-dihydroxy-3,9,11-trimethoxyberberine (7), together with 11 known substances, were isolated from a methanol extract of the stems of Polyalthia longifolia var. pendula. The structures of 1-7 were elucidated on the basis of spectroscopic data analysis. Compounds were evaluated for their antiproliferative activities against A549 and MCF-7 cancer cells, and among the substances tested, only 16-oxo-cleroda-3,13-dien-15-oic acid (8) exhibited cytotoxicity.

Inhibition of Leukotriene Biosynthesis by Secondary Plant Metabolites

Leukotrienes (LTs) are a group of biologically highly potent mediators of inflammation, synthesised in the first metabolic step by 5-lipoxygenase from 20 carbon fatty acids, predominantly arachidonic acid. They include the cysteinyl leukotrienes LTC4, LTD4, LTE4, which cause bronchoconstriction, chemotaxis and enhanced blood vessel-permeability, and therefore play a major role in asthma, and the dihydroxyeicosatetraenoate LTB4, an important mediator for acute and chronic inflammatory ailments. Inhibition of leukotriene biosynthesis therefore is a promising target for combating a number of diseases. Plant secondary metabolites offer a structurally very diverse potential source of new therapeutic leads. The aim of this review is to update the current knowledge of plant compounds with inhibitory effects on leukotriene metabolism, by presenting results published in the last seven years. Substances are presented with their IC50 values together with the activities of the positive controls to enable better comparability. Inhibitory effects of extracts are not discussed. Keywords: Kniphofia foliosa, Clerodane Type Diterpenes, FLAP inhibitors, Atractylodes lancea, Polyprenylated Hydroquinones, Maesanin

Parvitexins A–E, Clerodane-Type Diterpenes Isolated from thein Vitro-Cultured Liverwort,Scapania parvitexta

New clerodane-type diterpenes, designated as parvitexins A (1)-E (5), were isolated from the in vitro-cultured liverwort, Scapania parvitexta. These compounds were determined to be monoacetylated clerodane-type diterpenes based on spectroscopic evidence.

Anti-tumour clerodane-type diterpenes from Mitrephora thorelii

Bioassay-guided fractionation of the crude extract of Mitrephora thorelii (Annonaceae) led to the isolation of two clerodane-type diterpenes. Their structures were characterised on the basis of spectroscopic methods as 6α,16,18-trihydroxycleroda-3(4),13(14)-dien-15,16-olide (1) and 16-hydroxycleroda-3(4),13(14)-dien-15,16-olide (2). Compound 1 is a new compound. Compounds 1 and 2 exhibited inhibitory activity against the proliferation of human hepatoma BEL-7402 cells in vitro. Compound 2 also showed an in vivo anti-tumour effect against the growth of hepatoma H22 in mice.

Antifungal clerodane diterpenes from Macaranga monandra (L) Muell. et Arg. (Euphorbiaceae).

Hexane and ethyl acetate phases of the methanol extract of Macaranga monandra showed fungal growth inhibition of Colletotrichum acutatum, C. fragariae and C. gloeosporioides, Fusarium oxysporum, Botrytis cinerea, Phomopsis obscurans, and P. viticola. Bioassay-guided fractionation led to the isolation of two active clerodane-type diterpenes that were elucidated by spectroscopic methods (1D-, 2D-NMR, and MS) as kolavenic acid and 2-oxo-kolavenic acid. A 96-well microbioassay revealed that kolavenic acid and 2-oxo-kolavenic acid produced moderate growth inhibition in Phomopsis viticola and Botrytis cinerea.

Dytesinins A and B, New Clerodane-type Diterpenes with a Cyclopropane Ring from the Tunicate Cystodytes sp.

Two new clerodane-type diterpenes, dytesinins A ( 1) and B ( 2), with a cyclopropane ring have been isolated from the Okinawan marine tunicate Cystodytes sp., and the structures were elucidated on the basis of spectroscopic data including newly developed 2D NMR experiments such as CH-selected editing HSQC-NOESY and J-resolved HMBC.

Ethnopharmacology, phytochemistry and pharmacology: a successful combination in the study of Croton cajucara

Phytochemical and pharmacological studies of Croton cajucara were oriented by traditional medicine. The stem bark of the mature plant is a rich source of clerodane-type diterpenes: trans-dehydrocrotonin (DCTN), trans-crotonin (CTN), cis-cajucarin B, cajucarin A, cajucarinolide and two novel clerodanes, trans-cajucarin B and sacacarin. In young (18-month-old) plants, the triterpene acetyl aleuritolic acid (AAA) was the major stem bark component and in these the diterpene DCTN was not present. The highest concentration of DCTN (1.4% of dry bark) was detected in 4–6 year-old plants, while 3-year-old plants contained only 0.26% of this diterpene. Three steroids (β-sitosterol, stigmasterol and sitosterol-3- O-β-glucoside), two flavonoids (kaempferol 3,4′,7-trimethyl ether and 3,7-dimethyl ether) and one diterpene (cajucarinolide) were isolated from the leaves of this Croton. The main pharmacological activity was correlated with DCTN. This clerodane produced anti-inflammatory and antinociceptive effects and a significant hypoglycemia in alloxan-induced diabetic rats. The compound also reduced the index of gastric lesions induced by restraint-in-cold. Dose-related DCTN and CTN inhibited in vivo the basal acid secretion in pylorus-ligature rats and oxyntic glands isolated from rabbit gastric mucosa, DCTN, CTN or AAA decreased in vitro uptake basal acid secretion induced by histamine and measured with the 14C-aminopyrine uptake method. Uniquely DCTN inhibited 14C-AP uptake induced by bethanechol. The terpenoids, DCTN and AAA, and the chloroform extract of 6-month-old plants reduced gastrointestinal transit in mice. The effects of DCTN and CTN on the survival of mice bearing Sarcoma 180 and Ehrlich carcinoma ascitic tumors, on the proliferation of cultured cells and TNFα were determined. DCTN was also evaluated for a possible antioestrogenic activity using the immature rat as a model system for bioassay of oestrogen and for an anti-implantation effect in regularly cycling rats. The biological experiments, using the plant extracts and the terpenoids DCTN, CTN and AAA, are herein discussed.

Spiro-γ-lactone diterpenes from in vitro cultures of the liverwort, Heteroscyphus planus

Two new C-9-spiro-γ-lactones of clerodane-type diterpenes, named heteroscypholide A and B, and one new cleroda-15-oic acid methyl ester were isolated from the cells of suspension culture of the liverwort. Heteroscyphus planus. The structures of heteroscypholide A and B and the cleroda-15-oic acid methyl ester were established as 3,4-epoxy-6,13-diacetoxy-5,10- trans-cleroda-14-en-20,12-olide, 3,4-epoxy-6-acetoxy-13-hydroxy-5,10- trans-cleroda-14-en-20,12- olide and 18-hydroxy-5,10- trans-cleroda-3 E-dien-15-oic acid methyl ester, respectively. Their relative stereochemistry was determined on the basis of spectral analyses including X-ray crystallography and chemical correlation.