Population Pharmacokinetic of the Diterpenes ent-Polyalthic Acid and Dihydro-ent-Agathic Acid from Copaifera Duckei Oil Resin in Rats.

Copaifera duckei oleoresin is a plant product extensively used by the Brazilian population for multiple purposes, such as medicinal and cosmetic. Despite its ethnopharmacological relevance, there is no pharmacokinetic data on this important medicinal plant. Due to this, we determined the pharmacokinetic profile of the major nonvolatile compounds of C. duckei oleoresin. The diterpenes ent-polyalthic acid and dihydro-ent-agathic acid correspond to approximately 40% of the total oleoresin. Quantification was performed using LC-MS/MS, and the validated analytical method showed to be precise, accurate, robust, reliable, and linear between 0.57 and 114.74 µg/mL plasma and 0.09 to 18.85 µg/mL plasma, respectively, for ent-polyalthic acid and dihydro-ent-agathic acid, making it suitable for application in preclinical pharmacokinetic studies. Wistar rats received a single 200 mg/kg oral dose (gavage) of C. duckei oleoresin, and blood was collected from their caudal vein through 48 h. Population pharmacokinetics analysis of ent-polyalthic and dihydro-ent-agathic acids in rats was evaluated using nonlinear mixed-effects modeling conducted in NONMEN software. The pharmacokinetic parameters of ent-polyalthic acid were absorption constant rate = 0.47 h-1, central and peripheral apparent volume of distribution = 0.04 L and 2.48 L, respectively, apparent clearance = 0.15 L/h, and elimination half-life = 11.60 h. For dihydro-ent-agathic acid, absorption constant rate = 0.28 h-1, central and peripheral apparent volume of distribution = 0.01 L and 0.18 L, respectively, apparent clearance = 0.04 L/h, and elimination half-life = 3.49 h. The apparent clearance, central apparent volume of distribution, and peripheral apparent volume of distribution of ent-polyalthic acid were approximately 3.75, 4.00-, and 13.78-folds higher than those of dihydro-ent-agathic.

Ent-kaurenoic acid-enriched Mikania glomerata leaves-complexed ß-cyclodextrin: Pharmaceutical development and in vivo antitumor activity in a sarcoma 180 mouse model.

The extract obtained from Mikania glomerata leaves rich in ent-kaurenoic acid (ERKA) shows cytotoxic activity in vitro, but its hydrophobic nature and thermosensitivity are issues to be solved prior to in vivo antitumor studies. The purpose of this study was to investigate the antitumor activity of inclusion complexes formed between ERKA and ß-cyclodextrin (ERKA:ß-CD) in rodents. ERKA:ß-CD complexes obtained by malaxation (MX) and co-evaporation (CE) methods were firstly characterized regarding their physical properties, encapsulation efficiency, and cytotoxicity againts L929 cells. The antitumor activity study was then performed in mice with sarcoma 180 treated with saline, 5-fluouracil (5FU) and ERKA:ß-CD at 30, 100 and 300 µg/kg. The weight, volume, percentage of inhibition growth, gross and pathological features and positivity for TUNEL, ki67, NFκB and NRF2 in the tumors were assessed. Serum lactate-dehydrogenase activity (LDH), white blood cells count (WBC) and both gross and pathological features of the liver, kidneys and spleen were also evaluated. The formation of the inclusion complexes was confirmed by thermal analysis and FTIR, and they were non-toxic for L929 cells. The MX provided a better complexation efficiency. ERKA:ß-CD300 promoted significant tumor growth inhibition, and attenuated the tumor mitotic activity and necrosis content, comparable to 5-fluorouracil. ERKA:ß-CD300 also increased TUNEL-detected cell death, reduced Ki67 and NF-kB immunoexpression, and partially inhibited the serum LDH activity. No side effect was observed in ERKA:ß-CD300-treated animals. The ERKA:ß-CD inclusion complexes at 300 µg/kg displays antitumour activity in mice with low systemic toxicity, likely due to inhibition on the NF-kB signaling pathway and LDH activity.

A reliable validated high-performance liquid chromatography-photodiode array detection method for quantification of terpenes in Copaifera pubiflora, Copaifera trapezifolia, and Copaifera langsdorffii oleoresins.

The Copaifera oleoresins are widely used in folk medicine to treat various diseases. The goal of this study was to develop a validated reverse-phase high-performance liquid chromatography method with photodiode array detection (RP-HPLC-PDA) to quantify eight terpenes: ent-hardwickiic acid, ent-copalic acid, ent-7α-acetoxy hardwickiic acid, ent-16-hydroxy-3,13-clerodadiene-15,18-dioic acid, ent-5,13-labdadiene-15-oic acid, junenol, ent-kaurenoic acid, and 13E-ent-labda-7,13-dien-15-oic acid in the oleoresins of Copaifera pubiflora L. (OCP), Copaifera trapezifolia L. (OCT) and Copaifera langsdorffii L. (OCL). The linearity of the method was confirmed in the range of 20.00-500 µg.mL-1 (r2 > 0.999). The limit of quantification was between 1,05 and 16.89 µg.mL-1. Precision and accuracy ranges were found to be %RSD <0.2 and 96% to 110%, respectively. Based on the obtained results, the developed analytical method is rapid, precise, accurate, and sensitive for quantifying these terpenes in Copaifera's oleoresins.

Brazilian green propolis reduces worm burden and hepatic granuloma formation in a Schistosoma mansoni experimental murine model.

Characterized as an acute and chronic parasitic disease, schistosomiasis mansoni has as its central pathology the formation of hepatic granulomas in response to the parasite's eggs trapped in the host's liver. In recent years, research on propolis has grown; however, there is little anthelmintic work on this bee product. In the propolis scenario, Brazilian ones receive attention, with green and red propolis standing out. This study aims to evaluate in vivo the standardized extract of Brazilian green propolis (Pex) against Schistosoma mansoni. The in vivo antiparasitic activity of Pex was conducted in female BALB/c mice infected with S. mansoni and of the three groups treated with Pex (300 mg/kg); G2 (35th to 42nd dpi) reduced the total worm burden by 55.32%, followed by G3 (42nd to 49th dpi) and G4 (49th to 56th dpi), with about 46%. Furthermore, G2 significantly reduced the total egg load in the ileum (59.33%) and showed an increase in the dead eggs. Similarly, histological analysis of the livers showed a significant reduction in the number and diameter of the granulomas. Based on these results, there is an interesting schistosomicidal activity of Pex and its potential against the formation of hepatic granulomas, paving the way for more detailed studies of propolis in the animal model of schistosomiasis mansoni.

In vitro cytotoxicity and structure-activity relationship approaches of ent-kaurenoic acid derivatives against human breast carcinoma cell line.

In this study, ent-kaurenoic acid derivatives were obtained by microbial transformation methodologies and tested against breast cancer cell lines (MCF-7). A multivariate quantitative-structure activity relationship (QSAR) analysis was performed taking into account both microbial transformation derivatives and other analogues previously reported in literature to give some insight into the main features behind the cytotoxic activity displayed by kaurane-type diterpenes against MCF-7 cells. The partial least square regression (PLS) method was employed in the training set and the best PLS model was built with a factor describing 69.92% of variance and three descriptors (logP, εHOMO and εHOMO-1) selected by the Ordered Predictors Selection (OPS) algorithm. The QSAR model provided reasonable regression (Q2 = 0.64, R2 = 0.72, SEC = 0.29 and SEV = 0.33). The model was validated by leave-N-out cross-validation, y-randomization and external validation (R2pred = 0.89 and SEP = 0.27). The selected descriptors indicated that the activity was mainly related to electronic parameters (HOMO and HOMO-1 molecular orbital energies), as well as to logP. These findings suggest that higher activity values are directly related with both higher logP and frontier orbital energy values. The positive relationship between these orbitals and the activity suggests that the ent-kaurenoic acid analogues interaction with the target involves charge displacement, which is entirely consistent with the literature. Based on these findings, three compounds were proposed and one of them was synthesized and tested. The experimental result confirmed the activity predicted by the model.

Antibacterial Effect of Copaifera duckei Dwyer Oleoresin and Its Main Diterpenes against Oral Pathogens and Their Cytotoxic Effect.

This study evaluates the antibacterial activity of the Copaifera duckei Dwyer oleoresin and two isolated compounds [eperu-8(20)-15,18-dioic acid and polyalthic acid] against bacteria involved in primary endodontic infections and dental caries and assesses the cytotoxic effect of these substances against a normal cell line. MIC and MBC assays pointed out the most promising metabolites for further studies on bactericidal kinetics, antibiofilm activity, and synergistic antibacterial action. The oleoresin and polyalthic acid but not eperu-8(20)-15,18-dioic provided encouraging MIC and MBC results at concentrations lower than 100 µg mL-1. The oleoresin and polyalthic acid activities depended on the evaluated strain. A bactericidal effect on Lactobacillus casei (ATCC 11578 and clinical isolate) emerged before 8 h of incubation. For all the tested bacteria, the oleoresin and polyalthic acid inhibited biofilm formation by at least 50%. The oleoresin and polyalthic acid gave the best activity against Actinomyces naeslundii (ATCC 19039) and L. casei (ATCC 11578), respectively. The synergistic assays combining the oleoresin or polyalthic acid with chlorhexidine did not afford interesting results. We examined the cytotoxicity of C. duckei oleoresin, eperu-8(20)-15,18-dioic acid, and polyalthic acid against Chinese hamster lung fibroblasts. The oleoresin and polyalthic acid were cytotoxic at concentrations above 78.1 µg mL-1, whereas eperu-8(20)-15,18-dioic displayed cytotoxicity at concentrations above 312.5 µg mL-1. In conclusion, the oleoresin and polyalthic acid are potential sources of antibacterial agents against bacteria involved in primary endodontic infections and dental caries in both the sessile and the planktonic modes at concentrations that do not cause cytotoxicity.

Copaifera duckei Oleoresin and Its Main Nonvolatile Terpenes: In Vitro Schistosomicidal Properties.

In this article, the in vitro schistosomicidal effects of three Brazilian Copaifera oleoresins (C. duckei, C. langsdorffii, and C. reticulata) are reported. From these botanical sources, the oleoresin of C. duckei (OCd) demonstrated to be the most promising, displaying LC50 values of 75.8, 50.6, and 47.2 µg/ml at 24, 48, and 72 h of incubation, respectively, against adult worms of Schistosoma mansoni, with a selectivity index of 10.26. Therefore, the major compounds from OCd were isolated, and the diterpene, (-)-polyalthic acid (PA), showed to be active (LC50 values of 41.7, 36.2, and 33.4 µg/ml, respectively, at 24, 48, and 72 h of incubation). Moreover, OCd and PA affected the production and development of eggs, and OCd modified the functionality of the tegument of S. mansoni. Possible synergistic and/or additive effects of this balsam were also verified when a mixture of the two of its main compounds (PA and ent-labd-8(17)-en-15,18-dioic acid) in the specific proportion of 3:1 (w/w) was tested. The obtained results indicate that PA should be considered for further investigations against S. mansoni, such as, synergistic (combination with praziquantel (PZQ)) and in vivo studies. It also shows that diterpenes are an important class of natural compounds for the investigation of agents capable of fighting the parasite responsible for human schistosomiasis.

In vitro Evaluation of Copaifera oblongifolia Oleoresin Against Bacteria Causing Oral Infections and Assessment of Its Cytotoxic Potential.

The oral cavity, which harbors more than 750 bacterial species, is one of the most diverse sites of the human body. Some of these bacteria have been associated with oral diseases, such as dental caries and endodontic infections. We report on the antimicrobial and cytotoxic activities of Copaifera oblongifolia oleoresin against bacteria that cause caries and endodontic infections. The aim of this study is to determine the minimum (MIC) and the bactericidal (MBC) inhibitory concentrations as well as the biofilm inhibition ability (through determination of MBIC50) of the C. oblongifolia oleoresin. This study also investigated the bactericidal kinetics (time-kill curves) and the synergistic effect of the C. oblongifolia oleoresin. Additionally, this study evaluated the cytotoxic activity of the oleoresin toward V79 cells by means of the colony-forming assay. The C. oblongifolia oleoresin gave promising MIC and MBC values, which ranged from 25 to 200 µg/mL. Analysis of the MBIC50values of the oleoresin revealed it displayed biofilm inhibitory activity against all the assayed bacteria. Analysis of the bactericidal kinetics showed different behaviors of the oleoresin against the tested bacteria at the different time intervals and concentrations assayed in this study. An additive effect of the oleoresin with chlorhexidine dihydrochloride occurred only for S. mitis and A. actinomycetemcomitans. The C. oblongifolia oleoresin showed cytotoxic activity at concentrations ≥ 625 µg/mL.

Pimaradienoic Acid Inhibits Carrageenan-Induced Inflammatory Leukocyte Recruitment and Edema in Mice: Inhibition of Oxidative Stress, Nitric Oxide and Cytokine Production.

Pimaradienoic acid (PA; ent-pimara-8(14),15-dien-19-oic acid) is a pimarane diterpene found in plants such as Vigueira arenaria Baker (Asteraceae) in the Brazilian savannas. Although there is evidence on the analgesic and in vitro inhibition of inflammatory signaling pathways, and paw edema by PA, its anti-inflammatory effect deserves further investigation. Thus, the objective of present study was to investigate the anti-inflammatory effect of PA in carrageenan-induced peritoneal and paw inflammation in mice. Firstly, we assessed the effect of PA in carrageenan-induced leukocyte recruitment in the peritoneal cavity and paw edema and myeloperoxidase activity. Next, we investigated the mechanisms involved in the anti-inflammatory effect of PA. The effect of PA on carrageenan-induced oxidative stress in the paw skin and peritoneal cavity was assessed. We also tested the effect of PA on nitric oxide, superoxide anion, and inflammatory cytokine production in the peritoneal cavity. PA inhibited carrageenan-induced recruitment of total leukocytes and neutrophils to the peritoneal cavity in a dose-dependent manner. PA also inhibited carrageenan-induced paw edema and myeloperoxidase activity in the paw skin. The anti-inflammatory mechanism of PA depended on maintaining paw skin antioxidant activity as observed by the levels of reduced glutathione, ability to scavenge the ABTS cation and reduce iron as well as by the inhibition of superoxide anion and nitric oxide production in the peritoneal cavity. Furthermore, PA inhibited carrageenan-induced peritoneal production of inflammatory cytokines TNF-α and IL-1ß. PA presents prominent anti-inflammatory effect in carrageenan-induced inflammation by reducing oxidative stress, nitric oxide, and cytokine production. Therefore, it seems to be a promising anti-inflammatory molecule that merits further investigation.

RP-HPLC analysis of manool-rich Salvia officinalis extract and its antimicrobial activity against bacteria associated with dental caries

In this paper we screened the dichloromethane extract from the aerial parts of Salvia officinalis L., Lamiaceae, against a representative panel of microorganisms that cause caries, conducted a bioassay-guided fractionation to establish themselves the most active metabolite (manool) and determined the Salvia officinalis fraction with the manool highest concentration to be used to activate an ingredient in oral care products such as toothpastes and mouthwashes. Both manool and S. officinalis extract showed very promising minimal inhibitory concentration values (between 6.24 and 31.36 µg.ml-1) and time kill curves against the primary causative agents of dental caries (Streptococcus mutans) revealed that, at twice its minimal bactericidal concentration (12.48 µg.ml-1), manool required 6 h to completely kill the bacteria. Salvia officinalis extract at twice its minimal bactericidal concentration (31.36 µg.ml-1 ) needed 12 h. The results achieved with Salvia officinalis extract motivated us to develop and validate an analytical RP-HPLC method to detect and determine manool in this extract. The validation parameters were satisfactorily met and evaluated allows us to consider the developed method suitable for use in different labs. In conclusion, our results evidenced that the manool-rich S. officinalis extract can be considered an analytically validated alternative to develop novel and effective antimicrobial agents against the main bacteria responsible for dental caries.

Evaluation of antimicrobial activity of extracts of Tibouchina candolleana (melastomataceae), isolated compounds and semi-synthetic derivatives against endodontic bacteria

This work describes the phytochemical study of the extracts from aerial parts of Tibouchina candolleana as well as the evaluation of the antimicrobial activity of extracts, isolated compounds, and semi-synthetic derivatives of ursolic acid against endodontic bacteria. HRGC analysis of the n-hexane extract of T. candolleana allowed identification of b-amyrin, a-amyrin, and b-sitosterol as major constituents. The triterpenes ursolic acid and oleanolic acid were isolated from the methylene chloride extract and identified. In addition, the flavonoids luteolin and genistein were isolated from the ethanol extract and identified. The antimicrobial activity was investigated via determination of the minimum inhibitory concentration (MIC) using the broth microdilution method. Amongst the isolated compounds, ursolic acid was the most effective against the selected endodontic bacteria. As for the semi-synthetic ursolic acid derivatives, only the methyl ester derivative potentiated the activity against Bacteroides fragilis.

In vitro schistosomicidal effects of the essential oil of Tagetes erecta

The in vitro schistosomicidal effects of the essential oil obtained from Tagetes erecta L. Asteraceae, leaves (TE-EO) collected in Brazil against Schistosoma mansoni worms are reported in this paper. The oil caused a significant decrease in the motor activity at 50 µg/mL as minimal concentration after 24 h. This oil also caused death of all the parasites and the separation of coupled pairs into individual male and female at 100 µg/mL after 24 h. The viability of adult worm groups treated with the TE-EO at 100 µg/mL was similar to that of groups treated with praziquantel (positive control). In addition, the oil promoted the inhibition of eggs development at all the tested concentrations. These data indicate that the TE-EO could be considered as a promising source for the development of new schistosomicidal agents.

Antimicrobial evaluation of diterpenes from Copaifera langsdorffii oleoresin against periodontal anaerobic bacteria.

The antimicrobial activity of four labdane-type diterpenes isolated from the oleoresin of Copaifera langsdorffii as well as of two commercially available diterpenes (sclareol and manool) was investigated against a representative panel of microorganisms responsible for periodontitis. Among all the evaluated compounds, (-)-copalic acid (CA) was the most active, displaying a very promising MIC value (3.1 µg mL-1; 10.2 µM) against the key pathogen (Porphyromonas gingivalis) involved in this infectious disease. Moreover, CA did not exhibit cytotoxicity when tested in human fibroblasts. Time-kill curve assays performed with CA against P. gingivalis revealed that this compound only inhibited the growth of the inoculums in the first 12 h (bacteriostatic effect). However, its bactericidal effect was clearly noted thereafter (between 12 and 24 h). It was also possible to verify an additive effect when CA and chlorhexidine dihydrochloride (CHD, positive control) were associated at their MBC values. The time curve profile resulting from this combination showed that this association needed only six hours for the bactericidal effect to be noted. In summary, CA has shown to be an important metabolite for the control of periodontal diseases. Moreover, the use of standardized extracts based on copaiba oleoresin with high CA contents can be an important strategy in the development of novel oral care products.

Chemical composition and in vitro schistosomicidal activity of the essential oil of Plectranthus neochilus grown in Southeast Brazil.

The chemical composition and the in vitro schistosomicidal effects of the essential oil of Plectranthus neochilus (PN-EO) grown in Southeast Brazil was studied. ß-Caryophyllene (1; 28.23%), α-thujene (2; 12.22%), α-pinene (3; 12.63%), ß-pinene (4; 6.19%), germacrene D (5; 5.36%), and caryophyllene oxide (6; 5.37%) were the major essential oil constituents. This chemical composition differed from that previously reported for specimens harvested in Africa. Concerning the in vitro schistosomicidal activity against adult Schistosoma mansoni worms, PN-EO was considered to be active, but less effective than the positive control praziquantel (PZQ) in terms of separation of coupled pairs, mortality, decrease in the motor activity, and tegumental alterations. However, PN-EO caused an interesting dose-dependent reduction in the number and the percentage of developed S. mansoni eggs. These results suggest that PN-EO might be very promising for the development of new schistosomicidal agents.

Schistosomicidal activity of the essential oil of Ageratum conyzoides L. (Asteraceae) against adult Schistosoma mansoni worms.

The in vitro schistosomicidal effects of the essential oil of Ageratum conyzoides L. (Ac-EO) against adult worms of Schistosoma mansoni is reported in this paper. Concerning this activity, Ac-EO was considered to be active, but less effective than the positive control (praziquantel, PZQ) in terms of separation of coupled pairs, mortality, decrease in motor activity, and tegumental alterations. However, Ac-EO caused an interesting dose-dependent reduction in the number of eggs of S. mansoni. Precocene I (74.30%) and (E)-caryophyllene (14.23%) were identified as the two major constituents of Ac-EO. These compounds were tested individually and were found to be much less effective than Ac-EO and PZQ. A mixture of the two major compounds in a ratio similar to that found in the Ac-EO was also less effective than Ac-EO, thus revealing that there are no synergistic effects between these components. These results suggest that the essential oil of A. conyzoides is very promising for the development of new schistosomicidal agents.

Differences in secondary metabolites from leaf extracts of Mikania glomerata Sprengel obtained by micropropagation and cuttings / Diferenças nos metabólitos secundários acumulados nos extratos das folhas de Mikania glomerata Sprengel obtidos por micropropagação e estaquia

GC analysis of the dichloromethane extracts obtained from cultivated specimens of Mikania glomerata Sprengel possibilited to verify that cuttings technique led to production of kaurenoic acid and coumarin while the same results have not been observed by propagation process using in vitro techniques.

A análise realizada por CG dos extratos diclorometânicos das folhas de Mikania glomerata Sprengel mostrou que a planta propagada por estaca produziu cumarina e ácido caurenóico, enquanto que o material micropropagado acumulou apenas cumarina.

Variabilidade sazonal do teor de saponinas de Baccharis trimera (Less. ) DC (Carqueja) e isolamento de flavona / Seasonal variability in the content of saponins from Baccharis trimera (Less. ) DC (Carqueja) and isolation of flavone

O objetivo deste trabalho foi avaliar a sazonalidade sobre o teor de saponinas e o isolamento e elucidação estrutural de constituinte químico (flavona) de Baccharis trimera (Less.) DC (Carqueja). O cultivo de indivíduos estaminados de B. trimera foi realizado no Campo Experimental da Universidade de Ribeirão Preto - Unaerp, em Ribeirão Preto, no período de janeiro a dezembro de 1998 e foi conduzido em três blocos, sendo que cada um deles era constituído de 48 mudas. As amostras foram colhidas após seis meses do plantio (durante o inverno), três meses após a primeira colheita (durante a primavera) e três meses após a segunda colheita (durante o verão). Após processo de secagem e moagem, as drogas foram submetidas ao ensaio para obtenção do índice de espuma. Os resultados mostraram que não há variabilidade neste índice em função da época de colheita. Parte da droga colhida no verão sofreu análise química para isolamento e elucidação estrutural de componente químico. O isolamento a partir do extrato metanólico de partes aéreas de B. trimera foi realizado através de métodos cromatográficos (CC e CLAE) e a identificação estrutural da substância isolada, através de métodos espectrométricos (UV, RMN ¹H e 13C). Foi identificada a flavona denominada 5,6-diidroxi-7,3,4-trimetoxiflavona.

The aim of this study was to evaluate the seasonal effect on the foam index and the isolation and structural identification of a chemical constituent (flavone) of Baccharis trimera. The culture staminate samples of B. trimera were realized in the experimental cultivation area of the Universidade de Ribeirão Preto - Unaerp, in Ribeirão Preto, state of São Paulo, Brazil from January to December/1998 and were conduced in three blocks, which received 48 seedlings. The samples were harvested six months after installation (during the winter), three months after the first harvest (during the spring) and three months after the second harvest (during the summer). After the drying process and comminution, an assay to obtain the foam index was performed with the plant material. The results didn't show any alteration, independently of harvest times. Part of the dried plant that had been harvested during the summer was extracted to isolate and identify structurally the chemical compound. The isolation from the methanol extracts of the aerial parts of B. trimera has been performed through chromatographic methods (CC and HPLC) and the identification through spectrometric methods (UV, ¹H and 13C NMR). A flavone was identified as 5,6-dihidroxy-7,3,4-trimetoxiflavone.

Identification of biologically active triterpenes and sterols present in hexane extracts from Miconia species using high-resolution gas chromatography.

In this work we describe the identification of the biologically active triterpenes and sterols present in the hexane extracts of six species of Miconia using gas chromatography. The main compounds present in these extracts are beta-amyrin, alpha-amyrin, and beta-sitosterol. The technique employed herein is shown to be a valuable and rapid tool for determining biologically active triterpenes and sterols present in non-polar extracts.