Search

A known kaurenoic acid (1), together with a new kaurane derivative named 3α-isobutyryloxykaurenoic acid (2), was isolated from the roots of Lasianthaea aurea, a plant endemic to Mexico. The compounds were characterized using their physical and spectroscopic data. In order to verify the structure and absolute configuration of 2, the derived methyl ester 3, obtained after diazomethane treatment, was subjected to single-crystal X-ray diffraction analysis, whose Flack and Hooft parameter calculations evidenced that the new natural product is an ent-kaurane derivative.

The forest products industry has a very important role in the development toward a sustainable, biobased society. However, many fungi that deteriorate the main structural components of wood, such as cellulose, hemicellulose and lignin, are able to cause decay in standing trees, leading to significant losses in forest output due to impacts on biomass production. Trametes versicolor (L.: Fr) Pilát is a fungus able to attack the trees by saprotrophic and parasitic life strategies, causing white-rot of wood species, a serious problem for the industry that is focused on wood and its uses. The objectives of the work were the synthesis of organotin(IV) carboxylate of a natural product (ent-kaurenoic acid), characterization of the structures and anti-fungal evaluation against T. versicolor, as a contribution of new alternatives for the wood preservation. The synthesis of organotin(IV) complexes derived from the diterpene tetracyclic ent-kaur-16-en-19-oic acid (ent-kaurenoic acid, KA) gave three new complexes potentially active against T. versicolor. The complexes were characterized by FTIR and NMR spectroscopy. The biological activity was evaluated by a gel dilution method with superficialplate inoculation, using 60 and 120 μg/mL concentration, for the KA and the three organotin(IV) complexes. A considerably higher bioactivity was observed to the 120 μg/mL concentration, and for the complex with greater molecular weight.

Made available in DSpace on 2019-10-06T15:33:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-05-10 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Ethnopharmacological relevance: Copaiba oleoresin has been used in folk medicine in the treatment of bronchitis, syphilis, skin diseases and ulcers due to its anti-inflammatory and antiseptic activities, but there is no information about major compounds oral absorption to support the traditional use. Aim of study: Considering the potential of copalic (CA) and kaurenoic acid (KA) – major biological activity (in vitro) diterpenes found in the oleoresin, this study aimed to evaluate the intestinal permeability of CA and KA using Caco-2 cells model as predictive test for oral drug absorption. Materials and methods: Chemical stability at pH 1.2 and 7.4 and plasma stability were evaluated to mimic physiological conditions of the gastrointestinal tract. The intestinal permeability of CA and KA was evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. Results: CA and KA were rapidly degraded at pH 1.2 (0.2 M Clark-Lubs buffer). At pH 7.4 (0.1 M phosphate buffer), CA was stable for up to 24 h and KA for up to 6 h. In human plasma, CA and KA can be considered stable for 24 h and 12 h at 37 °C, respectively. Caco-2 cells were considered viable when incubated with CA or KA in the range of 3.9–250 μM for 24 h. CA and KA exhibited moderate apparent permeability (P app ) of 4.67 (±0.08) × 10 −6 cm/s and 4.66 (±0.04) × 10 −6 cm/s, respectively. Simultaneous incubation with verapamil showed that P-glycoprotein does not play a relevant role on CA and KA oral absorption, with P app of 4.48 (±0.26) × 10 −6 cm/s and 5.37 (±0.72) × 10 −6 cm/s observed for CA and KA, respectively. Conclusion: The oral absorption of both CA and KA is driven by mainly passive permeability, is not limited by p-glycoprotein, but enteric-coated dosage forms should be used to avoid chemical instability in the gastric pH. São Paulo State University (UNESP) School of Pharmaceutical Sciences Federal University of Mato Grosso University of Sao Paulo (USP) NPPNS School of Pharmaceutical Sciences of Ribeirao Preto São Paulo State University (UNESP) School of Pharmaceutical Sciences FAPESP: 14/50265-3

Ferreira NH, Ribeiro AB, Morais MD, Peixoto AM, Bernardino MA, Moreira MR, Soares ACF, Heleno VCG, Veneziani RCS, Tavares DC. 2018. Cytotoxic and genotoxic effects of the ent-kaurenoic acid and ent-kaurenoic acid-enriched Mikania glomerata extract in V79. Biofarmasi J Nat Prod Biochem 17: xxxx. The ent-kaurenoic acid-rich extract from Mikania glomerata Sprengel was effective to inhibit the formation of Streptococcus mutans biofilm. In view of the biological potential of this extract and its major component, the present study was carried out to evaluate the safety of the ent-kaurenoic acid-rich extract from Mikania glomerata Sprengel and ent-kaurenoic acid alone in an in vitro test system. The results showed that the ent-kaurenoic acid-rich extract from Mikania glomerata Sprengel was cytotoxic at concentrations up to 40.0 μg/mL. Genotoxic effects were observed in cell cultures treated with the highest concentrations tested of ent-kaurenoic acid-rich extract from Mikania glomerata Sprengel (10.0 and 15.0 µg/mL) and ent-kaurenoic acid alone (2.5, 5.0 and 7.5 µg/mL) when compared to the control group. Therefore, the ent-kaurenoic acid-rich extract from Mikania glomerata Sprengel demonstrated cytotoxicity and genotoxicity effects at the highest concentrations tested, while ent-kaurenoic acid showed to be genotoxic at the same concentrations present in ent-kaurenoic acid-rich extract from Mikania glomerata Sprengel in V79 cells. These results demonstrate that the ent-kaurenoic acid should be responsible, at least in part, of the genotoxicity of ent-kaurenoic acid-rich extract from Mikania glomerata Sprengel.

BACKGROUND Parasitic weeds are widespread and they cause significant losses in important crops. Their germination requires the detection of crop-derived molecules such as strigolactones. Strigolactone mimics are germination-inducing molecules with potential to apply the suicidal germination strategy for seed bank control of parasitic weeds. RESULTS Attachment of the D-ring, which is instrumental in the germination process of seeds of parasitic weeds, has been carried out to gibberellin (GA3) and kaurenoic as the scaffold. It was shown that indeed strigolactone mimics prepared from GA3 and kaurenoic acid are active as stimulant when a D-ring is present, some of the mimics are as active as GR24. CONCLUSIONS The starting molecules were plant hormones that had previous growth regulating activity in other organisms and the products showed enhanced activity towards parasitic weeds. The information generated may contribute to a better understanding of the germination biochemistry of the weed species used. Further research is required in this area but it is clear that the results are promising.

Background Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry. Recently, efforts were made to produce these natural sweeteners in microorganisms using metabolic engineering. Nonetheless, the steviol titer is relatively low in metabolically engineered microorganisms, and therefore a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The purpose of this study was to redesign and reconstruct a steviol-biosynthetic pathway via synthetic-biology approaches in order to overproduce steviol in Escherichia coli. Results A genome-engineered E. coli strain, which coexpressed 5′ untranslated region (UTR)-engineered geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, and kaurene synthase, produced 623.6 ± 3.0 mg/L ent-kaurene in batch fermentation. Overexpression of 5′-UTR–engineered, N-terminally modified kaurene oxidase of Arabidopsis thaliana yielded 41.4 ± 5 mg/L ent-kaurenoic acid. Enhanced ent-kaurenoic acid production (50.7 ± 9.8 mg/L) was achieved by increasing the cellular NADPH/NADP+ ratio. The expression of a fusion protein, UtrCYP714A2-AtCPR2 derived from A. thaliana, where trCYP714A2 was 5′-UTR–engineered and N-terminally modified, gave 38.4 ± 1.7 mg/L steviol in batch fermentation. Conclusions 5′-UTR engineering, the fusion protein approach, and redox balancing improved the steviol titer in flask fermentation and bioreactor fermentation. The expression engineering of steviol-biosynthetic enzymes and the genome engineering described here can serve as the basis for producing terpenoids—including steviol glycosides and carotenoids—in microorganisms.

The aerial parts of Sphagneticola trilobata (L.) Pruski (Asteraceae) are popularly used to treat topical inflammation, but have not been fully investigated.To identify polar compounds in S. trilobata extracts and develop a new topical phytomedicine based on the kaurenoic acid (KA) content while monitoring and demonstrating its topical anti-inflammatory activity.Ethanol spray-dried extract of S. trilobata was analysed by LC-MS while the KA content from semisolid was analysed by LC-UV. The extent of ear edema induced by applying 20 μL of croton oil (2.5%), arachidonic acid (AA; 2 mg/ear) and decanoylphorbol-13-acetate (TPA; 2.5 mg/ear) in mice was used to evaluate the biological activity of the semisolids, which were applied 30 min before the phlogistic agents.Eight phenylpropanoids and four oleanane-type triterpenoid saponins were identified, majority of them reported for the first time in this species, in addition to KA. The semisolid containing 1.0% of dried extract reduced the ear edema induced by croton oil [77.2 ± 4.5%; ID50 = 0.49 (0.28-0.87%)], TPA (81.5 ± 2.4%) and AA (39.1 ± 6.9%), with decreasing effect at higher KA concentrations. This was accompanied by neutrophil migration inhibition as investigated by biochemical and histological assays.The anti-inflammatory effects were (at least in part) due to the interference in protein kinase C (PKC) activation, AA-cascade products and neutrophil migration inhibition, demonstrating the efficacy of the folk topical usage of this plant. The results support the development of a novel topical anti-inflammatory phytomedicine properly standardized to treat inflammatory dermatological diseases.

Ethnopharmacological relevance The oleoresin of Brazilian Copaifera reticulata is a traditional remedy used for the treatment of skin and urinary tract infections, respiratory diseases, rheumatism, ulcer and tumours; thus, playing an important role in the primary health care of the indigenous population. Aim As most previous pharmacological tests used the crude oleoresin and only a few studies so far dealt with enriched fractions or pure chemically defined compounds, the aim of this study was to systematically evaluate the antimicrobial and cytotoxic properties of the Copaifera reticulata oleoresin and to assign traditional uses to specific secondary metabolites. Materials and methods The oleoresin, as well as its neutral and acidic fractions were tested for their activity against six cancer cell lines, two clinically relevant bacterial strains, and two dermatophytes. Both fractions were analysed by GC-MS and UHPLC-ELSD, respectively. The antibacterial acidic phase was further fractionated by preparative chromatography to purify and characterize the compounds responsible for the observed pharmacological effect. Results Whereas no cytotoxic activity was detected, the crude oleoresin and its acidic fraction showed antibacterial activity against gram-positive bacteria Enterococcus faecium (IC50 values 4.2 and 4.8 µg/mL, respectively) and methicillin-resistant Staphylococcus aureus (MRSA, IC50 values 5.3 and 7.2 µg/mL, respectively). Purification of the acidic fraction of the C. reticulata oleoresin yielded two dicarboxylic diterpene acids and the four main diterpene acids, comprising three different diterpene scaffolds. Interestingly, the activity was not restricted to a particular diterpene-type but rather depended on the compounds’ lipophilicity, with the most active constituent showing IC50 values of 1.6 (E. faecium) and 2.5 µg/mL (MRSA), respectively. Furthermore, ent-polyalthic acid, the major diterpenoid, was significantly active against dermatophytes with IC50 values of 6.8 µg/mL (Trichophyton rubrum) and 4.3 µg/mL against (T. mentagrophytes). Conclusion The present study proved the antimicrobial effects of the C. reticulata oleoresin and its diterpenoid constituents, confirming its wide use in folk medicine for the treatment of skin and urinary tract infections. The inhibitory activity of copaiba diterpenoids against dermatophytic fungi as well as the gram-positive bacteria E. faecium and MRSA is being reported for the first time, providing potential lead structures for the treatment of these clinically relevant bacterial strains.

Trees of the Copaifera genus are native to the tropical regions of Latin America and Western Africa. Copaifera sp is widely used as a popular medicine and it has various ethnopharmacological indications, including gonorrhea, bronchitis, asthma, skin ulcers, ulcers, sore throat, uterine infections, general inflammations, cancer, and leishmanioses. Kaurenoic acid is a naturally occurring diterpene found in Copaifera and has been used as an anti-inflammatory, treatment of ulcer, leishmaniasis, and cancer. Bearing in mind the fact that the Ames test is an excellent tool to assess the safety of extracts, oils, and phytochemicals isolated from medicinal plants, from it, we evaluate the mutagenic potential of four species, between oleoresins (C. oblongifolia; C. langsdorffii) and leaves extracts (C. lucens; C. multijuga), of the Copaifera genus and also of kaurenoic acid, which is one of its major compounds. The results showed that the Copaifera spp. and kaurenoic acid did not induce an increase in the number of revertant colonies, without mutagenic effect in experiments, in the all concentrations evaluated by Ames test. The results obtained in our study support the safe use of the Copaifera genus medicinal plants selected and of kaurenoic acid.

Cantinoa althaeifolia, C. heterodon, C. mutabilis, C. stricta and C. sylvularum were investigated for the essential oil and exudate composition. The essential oils of these species, previously placed in the genus Hyptis, demonstrated the presence of at least 46 compounds, being β-pinene, δ-3-carene, β-caryophyllene, bicyclogermacrene, caryophyllene oxide and globulol the compounds present in higher amounts. Besides monoterpenes and sesquiterpenes, three species presented diterpenes. Kaurenoic acid was isolated from C. heterodon exudate and detected by gas chromatography in C. stricta and C. mutabilis. Although kaurane diterpenes are widely found in nature, their occurrence in Lamiaceae seems to be restricted to few genera.

In this study, we present the quantitation of eight diterpene acids in the oleoresin of Copaifera reticulata Ducke by UHPLC-ELSD and quantitative HSQC (heteronuclear single quantum correlation spectroscopy). UHPLC was performed using reversed phase material and external calibration and showed RSD values of ≤ 3% (repeatability) and ≤ 4% (precision), and mean recovery rates of 91.2–104.8%. LOQs were determined with 10 and 20 μg/mL, and LODs with 4 and 8 μg/mL, respectively. For the qHSQC method, calibration curves of eight different NMR cross-peaks (furylic, endo- and exocyclic methine signals, exocyclic methylene and methyl signals) were established and normalized with dimethyl terephthalate, which served as internal standard. This approach allowed the direct quantification of four major and one minor diterpene, whereas simple calculation procedures led to the contents of the remaining minor compounds. Comparison with the results of the UHPLC assay showed good agreement for seven of the eight diterpene acids. In terms of precision, the qHSQC method was advantageous for the quantification of the three main compounds, whereas UHPLC-ELSD was superior in the determination of the minor components. In contrast to previous reports, kolavenic acid was identified as a major diterpene acid in the oleoresin of Copaifera reticulata, with amounts of 4.0 ± 0.3%.

Ursolic acid with large amount (0.67% of dried plant weight) along with 7 compounds, namely as spatozoate (1), kaurenoic acid (2), ursonic acid (3), 3-hydroxy-11-ursen-28,13-olide (4), ursolic acid (5), vindoline (6) and mixture of β-sitosterol and stigmasterol were isolated from dichloromethane and ethyl acetate extracts which have shown anti-glucosidase activity of the whole plant of C.roseus. Some isolated compounds and their derivatives were also tested for anti-glucosidase and cytotoxicity.Ursolic acid was examined for hypoglycemic activity in alloxan-induced diabetic mice with dose of 200 and 300 mg/kg/day, respectively. The results have shown that the blood glucose level were reduced by 45.75% and 51.31% to compare with the control group. This study has confirmed that the main component of Vietnamese C. roseus has had significant anti-hyperglycemia activity.

The cytotoxic activities of the diterpene kaurenoic acid ( 1 ) and its 15 semi-synthesis derivatives were assessed on human cell cultures. The human tumor cells used comprised colon (SW620 and SW480), pancreatic (PANC-1 and BxPC-3), stomach (SGC-7901), esophageal (Eca-109), and leukemia (K562 and HL-60). Kaurenoic acid was inactive against the tumor cell lines; however, its derivatives which contain α,β -unsaturated ketone rendered compounds with cytotoxic activity. Compounds 5 – 14, 17 – 19 , and 24 with a substitution at the C-4 position showed significant inhibitory activity against the tested cell lines, while compound 3 , without a substitution at the C-4 position, was slightly less active in these cell lines. The SW620 colon cancer cell was highly susceptible to all of the tested derivatives.

Kaurenoic acid (KA) is a kaurane diterpene found in several medicinal plants that displays biological activities, such as anti-inflammatory, smooth muscle relaxant and hypotensive response. However, there are no pharmacokinetic data available about this molecule. The purpose of the study was to determine the pharmacokinetic profile and the oral bioavailability of KA in rats. Wistar rats submitted to jugular vein cannulation received 50 mg/kg of KA by intravenous or oral route. The implanted cannula allowed intravenous administration and serial blood collection along 10 h. Analytical quantification was performed by reversed phase HPLC-UV and mobile phase composed by acetonitrile:acidified water (70:30 v/v). The validated analytical method showed precision, accuracy, robustness, reliability and linearity between 0.75 and 100 μg/mL. KA administered intravenously showed a linear and two-compartment kinetic behavior at the tested dose. The following pharmacokinetic parameters were determined: Cmax = 22.17 ± 1.65 mg/L; V = 14.53 ± 1.47 L/kg; CL = 17.67 ± 1.50 mL/min/kg; AUC0-∞ = 2859.65 ± 278.42 mg·min/L, K = 0.073 ± 0.005 h-1 and t1/2β = 9.52 ± 0.61 h. Oral treatment did not provide detectable plasma levels of KA, avoiding the determination of its bioavailability.

Kaurenoic acid has been displaying anti-inflammatory effect described in different models. However, the per se immunomodulatory effects of kaurenoic acid remain to be investigated. Thus, the immunomodulatory and antioxidant effects of kaurenoic acid were investigated in vitro on peritoneal macrophages from BALB/c mice. Kaurenoic acid induced per se the production of pro-inflammatory cytokines such as TNFI±, IL-1I² and IFN-I³ while also increased the levels of IL-10. There was also reduction of NO production and induction of anti-oxidant profile. Therefore, in addition to inhibiting inflammation, kaurenoic acid presents immunomodulatory effects per se.

Aims: Cancer is one of the leading causes of death worldwide with an estimated 6.7 million deaths and 24.6 million people living with cancer in 2002. Presently, there is a global increase in prevalence, mortality and health burden of various malignancies. World Health Organization (WHO) report projected that cancer prevalence rates could further increase by 50% to 15 million new cases in the year 2020. The bioactivity guided isolation of the bioactive constituent and its characterization, responsible for the anticonvulsant effects of the root bark extract of

Coumarin, o-coumaric, and kaurenoic acid are bioactive compounds usually found in the leaves of Mikania laevigata. Genetic and environmental variations in the secondary metabolites of plants may have implications for their biological effects. Three different accessions of M. laevigata cultivated in four sites between the Equator and the Tropic of Capricorn in Brazil were evaluated aiming to present potential raw materials and discuss relationships among these three bioactive compounds. The results revealed effects of plant accessions and environmental factors and suggested two contrasting chemical phenotypes of M. laevigata. The first phenotype presented the highest levels of kaurenoic acid (2283 ± 316 mg/100 g) besides lower levels of coumarin (716 ± 61 mg/100 g), which was also stimulated by the environment and mild climate at the site nearest to the Tropic of Capricorn. The other phenotype presented the lowest levels of kaurenoic acid (137 ± 17 mg/100 g) besides higher levels of coumarin (1362 ± 108 mg/100 g), which was also stimulated by the environment and tropical climate at the site nearest to the Equatorial beach.

Guadeloupean Parkinsonism has been linked epidemiologically to the consumption of Annonaceae fruits. These were proposed to be etiological agents for sporadic atypical Parkinsonism worldwide, because of their content of neurotoxins such as isoquinolinic alkaloids and Annonaceous acetogenins. The pulp of Annona cherimolia Mill. from Spain was screened for these toxic molecules using Matrix-Assisted Laser Desorption Ionisation - Time of Flight mass spectrometry (MALDI-TOF MS) and it was found not to be a source of exposure. However, kaurenoic acid, a diterpene considered to be cytotoxic, was detected in high amounts (66 mg/fresh fruit). Treatment of rat embryonic striatal primary cultures, up to a high concentration (50 µM), did not cause neuronal death nor astrogliosis, suggesting that this molecule is not at risk of implication in human neurodegenerative diseases.

A series of new thiosemicarbazones derived from natural diterpene kaurenoic acid were synthesized and tested against the epimastigote forms of Trypanosoma cruzi to evaluate their antitrypanosomal potential. Seven of the synthesized thiosemicarbazones were more active than kaurenoic acid with IC50 values between 2-24.0 mM. The o-nitro-benzaldehyde-thiosemicarbazone derivative was the most active compound with IC50 of 2.0 mM. The results show that the structural modifications accomplished enhanced the antitrypanosomal activity of these compounds. Besides, the thiocyanate, thiosemicarbazide and the p- methyl, p-methoxy, p-dimethylamine, m-nitro and o-chlorobenzaldehyde-thiosemicarbazone derivatives displayed lower toxicity for LLMCK2 cells than kaurenoic acid, exhibing an IC50 of 59.5 mM.

Hypoglycaemic activity was observed in normoglycaemic mice orally administered with the aqueous Smallanthus sonchifolius leaf tea extract, alloxan-induced diabetic mice orally administered with ent-kaurenoic acid (1), and normoglycaemic mice intraperitoneally administered with 1 from S. sonchifolius leaves. A single dose administration of 50 mg kg(-1) BW yacon leaf tea extract demonstrated immediate but relatively short hypoglycaemic activity, with significant effects observed during 1-2 h. Similarly, administration with 100 mg kg(-1) BW yacon leaf tea extract obtained by heavy stirring in hot water demonstrated a more potent activity compared to the positive control at 1.5-2.0 h. Oral administration of 1 did not affect the blood glucose level of the alloxan-induced diabetic mice, but a single intraperitonial injection of 10 mg kg(-1) BW in normoglycaemic mice had consistent percent blood glucose reduction persisting from 1 to 2 h observation periods.

Annona glabra Linneau, Annonaceae, é uma árvore de pequeno porte encontrada em todo território brasileiro, principalmente nas áreas costeiras e conhecida popularmente como araticum-do-brejo e araticum-bravo. Este trabalho teve como objetivos investigar os efeitos do extrato de A. glabra e do ácido caurenóico dele purificado sobre a migração de granulócitos humanos e seu potencial imunomodulatório. Os resultados demonstraram que o extrato de A. glabra inibe a migração natural de granulócitos, de acordo com a dose, sugerindo potencial antiinflamatório, enquanto o ácido caurenóico demonstrou estimulá-la de forma significativa. Em contraste, nenhum efeito foi observado com relação a imunomodulação. Os efeitos apresentados ainda não foram descritos e, dessa forma, contribuem para ampliar a lista de atividades biológicas descritas não só do extrato de A. glabra, como também para o ácido caurenóico.

Wedelia paludosa D.C. (Asteraceae) is an ornamental species occurring in many regions of Brazil. Aiming to find new cytotoxic compounds, the hydromethanol extract of W. paludosa (HME), as well as the dichloromethane (DF) and water (WF) fractions resulting from its partition, were submitted to the brine shrimp lethality bioassay (BSLB) in order to evaluate their cytotoxicity. Dichloromethane fraction (DF) was shown to be the most cytotoxic fraction (LC50 = 140.6 μg/mL), and its analysis by reversed phase high performance liquid chromatography (RP-HPLC) revealed ent-kaurenoic (1, 6.22 ± 0.23%) and grandiflorenic (2, 3.22 ± 0.31%) acids as important constituents. HME (LC50 = 980 μg/mL), DF (LC50 = 140.6 μg/mL), 1 (LC50 = 15.9 μg/mL) and 2 (LC50 = 29.8 μg/mL) were found to be cytotoxic, while the water fraction (WF, LC50 >> 1000 μg/mL) was inactive. As conclusion, the cytotoxicity observed for HME and DF is mainly due to the presence of 1 and 2 in their constitution.

The lipophilic and phenolic extractives of the ripe mesocarp of four cherimoya cultivars ('Perry Vidal', 'Mateus I', 'Mateus III' and 'Funchal') from Madeira Island, were studied for the first time. The predominant lipophilic compounds are kaurene diterpenes (42.2-59.6%), fatty acids (18.0-35.6%) and sterols (9.6-23.7%). Kaur-16-en-19-oic acid is the major lipophilic component of all cultivars accounting between 554 and 1350 mg kg(-1) of dry material. The studied fruits also contain a high variety of flavan-3-ols, including galloylated and non-galloylated compounds. Five phenolic compounds were identified for the first time: catechin, (epi) catechin-(epi) gal locatechin, (epi) gallocatechin, (epi) afzelechin-(epi) catechin and procyanidin tetramer. 'Mateus I' and 'Mateus III' cultivars present the highest content of phenolic compounds (6299 and 9603 mg kg(-1) of dry weight, respectively). These results support the use of this fruit as a rich source of health-promoting components, with the capacity to prevent or delay the progress of oxidative-stress related disorders. (C) 2016 Elsevier Ltd. All rights reserved.

Copaifera langsdorffii is a native Brazilian plant containing important amounts of polyphenols and diterpenes with biological activity. To obtain extracts enriched in these two types of compounds, supercritical fluid extraction (SFE) conditions have been optimized by using a response surface methodology (RSM) and considering as factors: extraction pressure and temperature and percentage of co-solvent (ethanol). The response variables selected were the concentration of the two most representative compounds in C. langsdorffii, kaurenoic acid (diterpenic acid) and quercitrin (glycosylated flavonol), total flavonoids content, and antioxidant activity (measured by DPPH and TEAC assays). According to the statistical analysis of the experimental design, extraction temperature and ethanol percentage were the main factors influencing the selectivity towards the extraction of the target compounds. Two optimized extracts were obtained: (1) containing high amount of total flavonoids, high concentration of quercitrin and low EC50 for the DPPH assay, named HQE - High Quercitrin Extract (100 bar, 70 °C and 50% of ethanol, in the region of gas-expanded liquids, GXLs) and (2) containing high value of kaurenoic acid extract, named HKAE-High Kaurenoic Acid Extract, (390 bar, 70 °C and pure CO2). Chemical characterization by LC/MS of HQE showed the presence of galloylquinic acids and flavonoids while, on the other hand, HKAE presented only nonpolar compounds such as kaurenoic acid. Moreover, a solid-liquid extraction (SLE) with methanol was carried out as a benchmark protocol for comparative purposes. SFE presented approximately 10-fold higher content of total flavonoids than the extracts obtained by SLE., The authors are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) grant number 2013/16268-2 for the financial support.

We herein report the synthesis of six diterpene derivatives, three of which are 23 new, generated through known organic chemistry reactions that allowed structural 24 modification of the existing natural products kaurenoic acid (1) and copalic acid (2). The 25 new compounds were fully characterized using high resolution mass spectrometry, infra-26 red spectroscopy, 1H and 13C NMR experiments. We also report the evaluation of the anti-27 tuberculosis potential for all compounds, which showed some promising results for 28 Micobacterium tuberculosis inhibition. Moreover, the toxicity for each of the most active 29 compounds was also assessed.

Kaurenoic acid, a kauranic diterpenoid, presents in vitro activity against trypomastigote forms of Trypanosoma cruzi, showing, however, lytic activity on blood erythrocytes, as a side effect. The syntheses of twelve new derivatives of kaurenoic acid, four amides, four amines (and three hydroclorides) and four oximes, was carried out aiming at the improvement of the therapeutic activity and without the side effect. Among the derivatives prepared, one compound showed enhanced trypanocidal activity in vitro towards Trypanosoma cruzi trypomastigote erythrocytic forms, when compared to kaurenoic acid, but continued to show discrete lytic activity on erythrocytes; another compound showed a level of activity similar to that of kaurenoic acid, but without lysis. O ácido caurenóico, um diterpeno caurânico, mostrou-se ativo in vitro contra formas tripomastigotas do Trypanosoma cruzi. Uma ação lítica sobre os eritrócitos foi uma das limitações encontradas para esta atividade. A síntese de doze derivados deste ácido: quatro amidas, quatro aminas (e três cloridratos) e quatro oximas foi realizada, com o objetivo de se tentar diminuir ou eliminar esse efeito secundário e, se possível, aumentar a atividade em relação ao material de partida. Dentre esses compostos, um mostrou-se mais ativo que o ácido caurenóico, mas também apresentou lise discreta de eritrócitos; outro não apresentou este efeito, mas a atividade não foi aumentada em relação àquela apresentada pelo ácido caurenóico.

Wedelia paludosa is frequently employed in popular medicine to treat several diseases. This work describes a comparative study of different parts (stems, leaves, flowers and roots) of this plant. The results showed that kaurenoic acid, the main active compound present in this plant, is located in all parts, but it is more concentrated in roots and stems. Such results could be useful in the development of a suitable method of standardization of the extracts of this plant and their phytopreparations.

We propose that Arabidopsis has a vernalization (low temperature) dependent and a vernalization-independent induction pathway for the initiation of inflorescence development in the shoot apex. Our data suggest that the vernalization-dependent pathway is normally blocked by methylation of the promoter of a gene critical for floral induction and that the low temperature treatment activates the pathway by reducing the methylation status of the gene(s). Kaurenoic acid hydroxylase (KAH), a gene early in the GA biosynthesis pathway is suggested as the methylation controlled gene. Vernalization results in expression of KAH in the shoot apex and the synthesis of the bioactive GAs which promote flowering. We propose that the vernalization independent pathway involves the supply and transport of carbohydrate to the shoot apex.

We isolated a single chemical compound fromA. continentalisand identified it to be kaurenoic acid (KA) and investigated the influence of anticariogenic properties. Inhibitory effects of KA on cariogenic properties such as growth, acid production, biofilm formation, and the adherence ofS. mutanswere evaluated. Furthermore, real-time PCR analysis was performed to evaluate the influence of KA on the genetic expression of virulence factors. KA significantly inhibited the growth and acid production ofS. mutansat 2–4 μg/mL and 4 μg/mL of KA, respectively. Furthermore, the adherence onto S-HAs was inhibited at 3-4 μg/mL of KA and biofilm formation was significantly inhibited when treated with 3 μg/mL KA and completely inhibited at 4 μg/mL. Also, the inhibitory effect of KA on biofilm formation was confirmed by SEM. In confocal laser scanning microscopy, bacterial viability gradually decreased by KA in a dose dependent manner. Real-time PCR analysis showed that the expressions ofgtfB, gtfC, gbpB, spaP, brpA, relA, andvicRwere significantly decreased inS. mutanswhen it was treated with KA. These results suggest that KA fromA. continentalismay be a useful agent for inhibiting the cariogenic properties ofS. mutans.

Lipophilic extracts from the roots of 16 species of Alepidea were studied by GC-MS for the presence of kaurenoic acids, dehydrokaurenoic acids, kaurenic lactones, hydroxykaurenoic acids and other kaurene derivatives. Various mixtures of these compounds are present in yields of up to 11.8% dry wt. in the rhizomes and roots. Two different isomers of kaurenoic acid occur in Alepidea , the one as a major compound in all the species, the other as a major compound only in some of them. The first isomer is clearly a useful chemical marker for the genus Alepidea , but the apparent chemical dichotomy is not obviously correlated with morphological patterns. Alepidea amatymbica , however, is part of a morphologically isolated group within the genus and also differs from other species in its chemical complexity and in the unique presence of kaurenoic lactones.

Coumarin (1) and kaurane-type diterpenes are considered the bioactive constituents of Mikania glomerata and M. laevigata, used in Brazil to treat respiratory affective disorders. The seasonal variation of 1, ortho-coumaric acid (2), benzoylgrandifloric acid (3), cinnamoylgrandifloric acid (4), and kaurenoic acid (5) in leaves of both species, cultivated in full sunlight and under shade levels of 40 and 80%, was quantified by HPLC. Compound 2 was detected solely in M. laevigata in concentrations below the limit of quantification. Coumarin was not found in M. glomerata, whereas its concentration reached 0.94±0.24% (w/w) in M. laevigata farmed in summer under 80% shading. Both Mikania species produced higher amounts of kaurane diterpenes when cultivated in plenty of sunlight. Hence, maximum contents of 1 are reached in M. laevigata cultivated under high shading, but with reduced concentrations of 3-5. Conversely, M. glomerata should be cultivated under full sunlight and harvested in winter for highest concentrations of kaurane-type diterpenes.

Mikania laevigata and M. glomerata, commonly known as guaco, are important medicinal plant species used in South America for the treatment of respiratory diseases. In folk medicine, their leaves have ample use due to their balsamic, antiophidic, appetite stimulant, antispasmodic, expectorant, and antimalarial properties, among others (Coimbra, 1942; Lucas, 1942; Neves & Sa, 1991; Alice et al., 1995; Gasparetto et al., 2010; Napimoga & Yatsuda, 2010). There is also pre-clinical evidence of the anti-inflammatory, anti-allergy, and bronchodilation activities of these species (Fierro et al., 1999; Moura et al., 2002; Suyenaga et al., 2002; Graca et al., 2007a). Due to their important effects, pharmaceutical preparations, including syrup and oral solutions, are freely distributed through various government phytotherapy programs and, thus, are widely used by the population (Gasparetto et al., 2010). The pharmacological effects of guaco are attributed mainly to the presence of coumarin (1,2benzopyrone); however, other metabolites have been shown to produce significant pharmacological effects. Studies that have evaluated isolated markers in the mouse model of allergic pneumonitis have demonstrated that coumarin and o-coumaric acid are part of the phytocomplex that is responsible for the therapeutic activities of guaco species (Santos et al., 2006). In addition, dihydrocoumarin and syringaldehyde have antioxidant, immunologic and anti-inflammatory properties (Farah & Samuelsson, 1992; Hoult & Paya, 1996; Bortolomeazzi et al., 2007; Stanikunaite et al., 2009; Gu & Xue, 2010). Finally, kaurenoic acid, isolated in high quantities from both species (Fierro et al., 1999; Veneziani et al., 1999; Yatsuda et al., 2005), has been shown to contribute to the effects of guaco through its antimicrobial, antinociceptive, anti-inflammatory and smooth muscle relaxant activities (Block et al., 1998; Costa-Lotufo et al., 2002; Wilkens et al., 2002; Cunha et al., 2003; Cotoras et al., 2004; Tirapelli et al., 2004; Cavalcanti et al., 2006). The presence of these metabolites is directly related to the benefits of guaco, but studies have shown them to be toxic. Dihydrocoumarin administered to groups of rodents led to carcinogenic activity, ulcers, forestomach inflammation, parathyroid gland hyperplasia and increased nephropathy (National Toxicology Program, 1993a). Kaurenoic acid has been shown to kill sea urchin embryos and to cause hemolysis in mouse and human erythrocytes

A recent reinvestigation of aerial parts of Wedelia paludosa D.C. is described and reports, for the first time, the isolation of iso-kaurenoic acid from this species.Uma recente reinvestigação das partes aéreas de Wedelia paludosa D.C. é descrita e relata, pela primeira vez, o isolamento do ácido iso-caurenóico desta espécie.

The Mikania genus is widely known as "guaco" and is used to treat fever, rheumatism, flu and respiratory diseases. Our previous work evidenced the synergism among M. laevigata extract components to produce desirable effects, and included the coumarin precursor, o-coumaric acid as marker. Many Mikania species are producers of ent-kaurene diterpenes which presented antiespasmodic and relaxant activities on smooth muscle. Seeking to standardize the guaco extract, which is registered in the Brazilian Pharmacopoea, this paper deals with the determination of kaurenoic acid through LC-PDA and the isolation through LC of syringaldehyde. Kaurenoic acid was not found in the extract, and syringaldehyde is one of the major compounds of pharmacopoeal extract, together with coumarin and o-coumaric acid. Samples from the lung and liver of Balb-C isogenic allergic pneumonitis bearing mice, treated with the same extract, were analyzed through GC-FID, and the fatty acid content was determined and analyzed. The results obtained by measuring the arachidonic acid (ARA) and docosahexaenoic acid (DHA) in the liver and lung of treated animals demonstrated that the fatty acid composition is distinct in both tissues, and that in the liver, only the DHA was altered as a result of the treatments. DHA is absent in the lung and in both organs, no significant difference in ARA production was observed. The aqueous extract, coumarin and o-coumaric acid stimulated DHA synthesis in the liver (p < 0.05). O gênero Mikania é popularmente conhecido como "guaco" e é utilizado para tratar febre, reumatismo, resfriados e afecções respiratórias. Em trabalho prévio demonstramos sinergismo entre os componentes do extrato de M. laevigata para produzir os efeitos farmacológicos esperados, incluindo a cumarina e seu precursor ácido o-cumárico como marcadores. Muitas espécies de Mikania são produtoras de diterpenos ent-caurenos que apresentam atividade antispasmódica e relaxante da musculatura lisa. Buscando a padronização do extrato medicinal de guaco (preparado segundo a farmacopéia brasileira 1ª edição), este trabalho visou determinar a presença de ácido caurenóico através de CLAE-DAD e isolou siringaldeído através de CLAE semipreparativa, sendo que o primeiro não foi encontrado no extrato e o siringaldeído é um dos seus componentes majoritários. Camundongos isogênicos Balb-C portadores de pneumonite alérgica foram tratados com este extrato, e amostras de pulmão e fígado foram analisadas por CG-DIC quanto ao seu conteúdo de ácidos graxos. A quantidade de ácido araquidônico (ARA) e de ácido docosahexaenóico (DHA) encontrada demonstrou que a composição é distinta em ambos tecidos, e apenas a concentração de DHA hepático foi alterado em função do tratamento, o qual não foi encontrado no pulmão. Não foi detectada diferença significativa na produção de ARA. Tanto o extrato aquoso, quanto a cumarina e o ácido o-cumárico, estimularam a síntese de DHA no fígado (p < 0.05).

Novel ent-kaurane glucosides were synthezised by a Koenigs–Knorr reaction between C17 and C19 alcohols derived from kaurenoic acid and 2,3,4,6-tetra-O-acetyl-glucopyranosyl bromide, followed by the hydrolysis of the acetates. Main products were assayed in vitro and in vivo against blood trypomastigote forms of Trypanosoma cruzi, the aetiological agent of Chagas’ disease (American trypanosomiasis). The results allowed to establish structure–activity relationships among these derivatives, as well as pointed out the C19-methylester-C17-O-glucoside as a potential trypanocidal agent, whose trypanocidal profile was shown to be comparable to those of gentian violet and benznidazole.

We evaluated the variation of the concentration of kaurenoic acid (1), which is a bioactive diterpene, in leaves, flowers, stems and roots from Wedelia paludosa (Acmela brasiliensis) for different seasons using the HRGC/FID method. The results indicated that the concentration of 1 is higher in the roots and stems during the autumn. The pharmacological results suggested that kaurenoic acid is responsible, at least in part, for the hypoglycemic potential detected in this plant.

Sitosterol and six diterpenes were isolated from green fruit, stem bark and leaves of Xylopia frutescens and characterized as ent-15α-acetoxy-kaur-16-en-19-oic acid (xylopic acid), ent-kaur-16-en-19-oic acid (kaurenoic acid), ent-16β-hydroxy-kaurane, ent-kaur-16-en-19-ol, ent-16β-hydroxy-kauran-19-oic acid and ent-16β,17-dihydroxy-kauran-19-oic acid. Acutifloric acid, a diterpenic dimer, and its 7β-hydroxy-derivative, a novel compound named frutoic acid, were also isolated.

The isolation of ent-kaur-16-en-19-oic acid (1) from the leaves and bark of Annona glabra and its determination by reversed-phase HPLC with a photodiode array detector is described. The results indicate that 1 accumulates in the bark of the plant with only very low concentrations in the leaves. Levels of 1 varied throughout the year being highest for bark and leaves in the autumn (5.29 +/- 0.23 and 0.27 +/- 0.01 mg/g dry weight, respectively) and lowest in the spring for bark and in the winter for leaves (4.00 +/- 0.25 and 0.11 +/- 0.01 mg/g dry weight, respectively).

Ent-kaur-16(17)-en-19-oic acid (kaurenoic acid, KA) is a tetracyclic diterpene prototype for natural anticaries agents. Six KA derivatives were prepared and their antimicrobial activity against the main microorganisms involved in the caries process evaluated. The sodium salt of KA (KA-Na) was the most active, displaying very promising MIC values for most pathogens. Time-kill assays against the primary causative agent of caries ( Streptococcus mutans) indicated that KA and KA-Na only inhibited growth in the first 12 h, suggesting a bacteriostatic effect. After this period (12-24 h), their bactericidal effect was clearly noted. KA and KA-Na showed no synergy when combined with the gold standard anticariogenic (chlorhexidine dihydrochloride, CHD) in the checkerboard assays against S. mutans.

The title compound, C26H42O9·1.5H2O, was extracted from the leaves and flowers of Ageratina vacciniaefolia, a Colombian native plant, which is called `chilca' by locals. The mol­ecule consists a kauranol group connected to a glucopyranosyl group by an O-atom bridge adjacent to a β-type anomeric center. In the crystal structure, inter­molecular O—H⋯O hydrogen bonds link mol­ecules to form a three-dimensional network. One solvent water mol­ecule lies on a crystallographic twofold axis, while another is disordered over three sites with equal occupancies.

Zoapatle aqueous crude extract (ZACE) from Montanoa tomentosa has been used as an oral contraceptive in traditional Mexican medicine for centuries. Kaurenoic acid, as isolated from Montanoa frutescens, and the methyl esters of 15-hydroxy-dihydro-kaurenoic acid and 15-keto-dihydro-kaurenoic acid were tested in vitro on human sperm motility and viability. The estimated ED50 concentrations immobilization were 374, 126 and 58 micrograms/ml, respectively using 15 X 10(6) sperms/500 microliter. Kaurenoic acid and both of its derivatives displayed only weak to negligible capacity for killing human sperms.The effects on human sperm motility and viability of 3 compounds isolated from an aqueous extract of Montanoa frutescens Cerv (Compositae, Heliantheae) were tested. The plant is related to the zoapatle, traditionally used as a contraceptive, and known to have uterotonic and luteolytic activity. Kaurenoic acid, its 15 hydroxy- and its methyl ester were prepared from the polar fraction, and dissolved in 95% ethanol at a concentration of 10 mg/ml. Human sperm, suspended in Bigger's Whitten and Wittingham medium at a final concentration of 15 million sperm per 500 mcl were incubated at 37 degrees C. for 60 seconds. Judging sperm motility under phase contrast microscopy, kaurenoic acid was 0.89 times less effective, and the hydroxy kaurenoic acid 2.6 times more effective than verapamil, while the keto derivative was 5.7 times more potent than verapamil at the same concentration. Sperm viability assessed by vital staining was hampered only about 10% by the keto-kaurenoic acid, 21% by kaurenoic acid, and 33% by hydroxy-kaurenoic acid. Doses required for immobilization of 50% of human sperms ranged from 58 to 374 mcg/ml. This is 225 times lower than that reported for gossypol in vitro, and much lower than the millimolar range needed to inactivate sperm with nonoxynol. Thus these drugs may prompt a search for analogs useful in vaginal contraceptives.

The incorporation of ent -kaurenoic acid into the derived hydroxy acid and dihydroxy acid by Beyeria calycina has been studied. The biosynthesis of the hydroxy acid involves a hydrogen 1,2-shift from the C-17 position of ent -kaurenoic acid.

A cell-free system from immature pea seeds converts 14 C-labelled ent -kaurene to ent -kaurenol, ent -kaurenal, ent -kaurenoic acid, ent -7α-hydroxykaurenoic acid, and gibberellin A 12 -aldehyde. The latter becomes converted further to 13-hydroxygibberellin A 12 , gibberellin A44, gibberellin A 12 -alcohol, and several unidentified products. Thus the biosynthesis of gibberellins via ent -kaurene is now established for a member of the Leguminosae . It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A 12 -alcohol has been obtained in any biological system.

A cell-free system capable of gibberellin (GA) biosynthesis has been prepared from immature seed of Phaseolus coccineus . This system converted [ 14 C]mevalonic acid (MVA) to ent -kaurene, ent -kaurenoic acid, ent -Kauradienoic acid, ent -7α-hydroxykaurenoic acid, ent -6α,7α-dihydroxykaurenoic acid, GA 12 -aldehyde and GA 12 ;. ent -[ 14 C]Kaurene was converted to ent -kaurenol, ent -kaurenal, ent -kaurenoic acid and ent -7α-hydroxykaurenoic acid. All identifications were by GC/MS. The pathway from MVA to GA 12 -aldehyde in this species thus appears to be the same as that found in cell-free preparations derived from immature seed of other species.

Three new heliangolides (niveusin-A, -B and -C) and three known diterpene carboxylic acids were isolated and characterized from a CHCl 3 , extract of Helianthus niveus . The structures of the three new lactones were deduced by 1 H and 13 C NMR as well as extensive decoupling experiments and derivatizations.

Subcellular fractions from germinated barley embryos, chloroplast preparations and whole germinating barley grains are able to carry out the conversions ent -kaurenol → ent -kaurenal → ent -kaurenoic acid → ent -hydroxykaurenoic acid, the initial steps of the biosynthetic pathway to gibberellins. Whole grains, and chloroplasts to a slight extent, incorporate radioactivity from ent -kaurenol-[17- 14 C] and ent -kaurenoic acid-[17- 14 C] into materials with similar but distinct properties from the gibberellins GA 1 , GA 3 , GA 4 and GA 7 .