In vitro toxicity of latex, its terpenoidal fractions and isolated phorbol esters from Euphorbia umbellata (Pax) Bruyns on monocytic and melanoma cells.

In Brazil, latex from Euphorbia umbellata (African milk tree) has been increasingly used in folk medicine to treat several types of cancer, including melanoma. The effect of lyophilized latex (LL), its hydroethanolic extract (E80), triterpene (F-TRI)- and diterpene (F-DIT)-enriched fractions, along with six isolated phorbol esters from LL and phorbol 12-myristate 13-acetate (PMA) on J774A.1, THP-1, SK-MEL-28, and B16-F10 cell line viability were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The compounds were identified by 2D-NMR and HRESIMS. The effect of the LL, extract and fractions on cell viability was also assessed through a resazurin reduction assay. At 100 µg/ml, LL, and its fractions moderately inhibited J774A.1 (37.5-59.5%) and THP-1 (12.6-43.6%) metabolism. LL (IC50 70 µg/ml) and F-TRI (IC50 68 µg/ml) were barely more effective against B16-F10 cells, and only F-TRI exerted an inhibitory effect on SK-MEL-28 cells (IC50 66-75 µg/ml). The samples did not effectively inhibit THP-1 growth (IC50 69-87 µg/ml, assessed by MTT). B16-F10 was susceptible to PMA (IC50 53 µM) and two 12-phenylacetate esters (IC50 56-60 µM), while SK-MEL-28 growth was inhibited (IC50 58 µM) by one of these kinds of esters with an additional 4ß-deoxy structure. Synagrantol A (IC50 39 µM) was as effective as PMA (IC50 47 µM) in inhibiting J774A.1 growth in a dose-dependent manner. Furthermore, an in silico study with target receptors indicated a high interaction of the compounds with the PKC proteins. These results provide useful knowledge on the effect of tigliane-type diterpenes on tumor cell from the perspective of medicinal chemistry.

Phorbol Esters from the Latex of Euphorbia umbellata: Bioguided Isolation of Highly Potent HIV-1 Latency Interrupters in Virus Reservoir Cells.

Three known compounds, 20-deoxyphorbol-5ß-hydroxy-12-tiglate-13-isobutyrate (1), 20-deoxyphorbol-5ß-hydroxy-12-tiglate-13-phenylacetate (2), and 4-deoxy-4ß-phorbol-12-tiglate-13-phenylacetate (3), were reisolated from the latex of Euphorbia umbellata through a bioguided fractionation process to target HIV-1 latency reactivation. The in vitro bioassay using infected T-cell lymphoblasts (J-Lat 10.6), complemented with surface CD4 receptor downregulation assessment, led to isolation of the compounds as a highly active ternary mixture. Effective purification of the individual compounds was achieved by first subjecting a phorbol-enriched fraction (previously prepared from crude latex) to MPLC, followed by semipreparative HPLC and characterization by 1D and 2D NMR spectroscopy and (+)-HRESIMS. Compared with a positive control, the isolated compounds were effective in reactivating 68-75% of the virus latency in the range of 9.7-0.097 µM for compound 1, 8.85-0.088 µM for compound 2, and 9.1-0.091 µM for compound 3, with the latter maintaining steady effectiveness down to a 10-5 dilution. Accordingly, compound 3 may serve as a promising lead compound for the development of anti-HIV drugs based on latency reactivation therapy.

Production of antiretroviral drugs in middle- and low-income countries.

This review outlines the main issues concerning the production of antiretroviral (ARV) drugs in middle- and low-income countries and the relevant political, legal and technical requirements for supporting such production. The requirements for efficient local production, including the manufacture of generic and branded products and public demand, have been considered from economic, market and socio-political perspectives. A steady and consistent government policy is crucial to success. Additional crucial factors in establishing local production are adequate infrastructure, qualified human resources in technical and managerial areas, and production-distribution logistics systems. The creation or strengthening of a national drug regulatory agency is a basic requirement. Production of ARVs relies on the structure of the international market for active pharmaceutical ingredients (APIs), which are highly monopolized for inclusion in branded or patented drugs, or are concentrated in a few Asian generic companies. Countries seeking to begin local production must develop strategies to overcome the various barriers. For instance, sub-Saharan African countries may benefit from developing multilateral health agreements with neighbouring countries. Such agreements are recommended and should be complemented by technology transfers, especially for the manufacture of APIs. Achieving a production level that is sustainable in the long term is crucial to maintaining patients' access to ARVs.

Efficiency and selectivity of triterpene acid extraction from decoctions and tinctures prepared from apple peels.

BACKGROUND: This study assessed the extraction efficiency of ursolic (UA) and oleanolic acids (OA), as well as the total phenols in aqueous and hydroethanolic extracts of dry apple peels at room temperature. MATERIALS AND METHODS: AFTER RUNNING PRELIMINARY ASSAYS ON DECOCTIONS AND TINCTURES (ETHANOL: water 7:3 v/v), the extracts from dried apple (cv. Fuji) peels were obtained by static maceration over varied intervals (2 to 180 days). The UA and OA content in the extracts was quantified by High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD) with a reversed phase column and isocratic elution (CH3CN/H2O/H3PO4) against calibration curves (R(2) > 0.9995). The total phenol content in the extracts was evaluated spectrophotometrically at 760 nm using the Folin-Ciocalteau method referencing gallic acid. RESULTS: UA and OA in the hydroethanolic extracts ranged from 3.63-6.12 mg/g and 2.12-3.30 mg/g, corresponding to 1.72-3.07 and 1.00-1.66 mg/g in the raw material, respectively. Higher values of triterpene acid content corresponded to maceration periods of 10 or 30 days. The residual phenol and polyphenol content ranged from 6.97 to 11.6 mg/g. The UA and OA yields, as well as the total phenol content, versus the maceration time were plotted in Control Charts within confidence intervals (95%) and were unaffected during the assayed period. CONCLUSION: Apple peel tinctures from 10% solids obtained at room temperature exhibited the highest content of triterpene acids when employing a maceration period of 10 to 30 days. Extracts prepared using this procedure contained an average of 7.33 mg/g of total triterpene acids and 10.6 mg/g phenolic compounds. These results establish supporting data for apple peel tinctures and their derived phytopharmaceuticals that are standardized on the ursolic-oleanolic acid content.

Anti-inflammatory effects of methyl ursolate obtained from a chemically derived crude extract of apple peels: potential use in rheumatoid arthritis.

Ursolic acid (UA), a pentacyclic triterpene acid found in apple peels (Malus domestica, Borkh, Rosaceae), has a large spectrum of pharmacological effects. However, the vegetal matrix usually produces highly viscous and poorly soluble extracts that hamper the isolation of this compound. To overcome this problem, the crude EtOH-AcOEt extract of commercial apple peels was exhaustively treated with diazomethane, after which methyl ursolate (MU) was purified by column chromatography and characterized spectrometrically. The anti-inflammatory effects of UA and MU (50 mg/kg) were analyzed by zymosan-induced paw edema, pleurisy and in an experimental arthritis model. After 4 h of treatment with UA and MU, paw edema was reduced by 46 and 44 %, respectively. Both UA and MU inhibited protein extravasation into the thoracic cavity; tibio-femoral edema by 40 and 48 %, respectively; and leukocyte influx into the synovial cavity after 6 h by 52 and 73 %, respectively. Additionally, both UA and MU decreased the levels of mediators related to synovial inflammation, such as KC/CXCL-1 levels by 95 and 90 %, TNF-α levels by 76 and 71 %, and IL-1ß levels by 57 and 53 %, respectively. Both the compounds were equally effective when assayed in different inflammatory models, including experimental arthritis. Hence, MU may be considered to be a useful anti-inflammatory derivative to overcome the inherent poor solubility of UA for formulating pharmaceutical products.

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 ºC) in a reaction medium containing 0.5% v/v limonene and 10 gµL of stationary phase cells (dry weight). The best results, corresponding to 564 mgµL of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 ºC for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mgµL, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.

Trans- ß -Caryophyllene: An Effective Antileishmanial Compound Found in Commercial Copaiba Oil (Copaifera spp.).

This study investigated the leishmanicidal activity against Leishmania amazonensis of four commercial oils from Copaifera spp. named as C1, C2, C3, and C4, the sesquiterpene and diterpene pools obtained from distilling C4, and isolated ß -caryophyllene (CAR). Copaiba oils chemical compositions were analyzed by gas chromatography and correlated with biological activities. Diterpenes-rich oils C2 and C3 showed antipromastigote activity. Sesquiterpenes-rich C1 and C4, and isolated CAR presented a dose-dependent activity against intracellular amastigotes, with IC50s of 2.9 µ g/mL, 2.3 µ g/mL, and 1.3 µ g/mL (6.4 µ M), respectively. Based on the highest antiamastigote activity and the low toxicity to the host cells, C4 was steamdistillated to separate pools of sesquiterpenes and diterpenes. Both pools were less active against L. amazonensis and more toxic for the macrophages than the whole C4 oil. The leishmanicidal activity of C3 and C4 oils, as well as C4 fractions and CAR, appears to be independent of nitric oxide production by macrophages. This study pointed out ß -caryophyllene as an effective antileishmanial compound and also to its role as potential chemical marker in copaiba oils or fractions derived thereof, aiming further development of this rainforest raw material for leishmaniasis therapy.

Anti-inflammatory activity of essential oils from Syzygium cumini and Psidium guajava.

CONTEXT: Despite the many biological activities reported for essential oils, their anti-inflammatory ability is relatively underexplored considering the wide variation in plant sources and in their volatile composition. Oils from Syzygium cumini Skells (SC) and Psidium guajava L. (PG) (Myrtaceae) have been described as having diverse pharmacological activities. OBJECTIVE: The current study seeks to evaluate the anti-inflammatory activity of the essential oils from the leaves of SC and PG, as well as some of their terpene-enriched fractions (+V = more volatile and -V = less volatile) obtained by vacuum distillation. Both the pharmacological responses and chemical compositions were correlated. MATERIALS AND METHODS: The relative contents of the oils and their fractions were evaluated by gas chromatography. Individual constituents in the oils were characterized by gas chromatography coupled to mass spectrometry. Anti-inflammatory activity was accessed in the lipopolysaccharide-induced pleurisy model, by measuring the inhibition of total leukocyte, neutrophil and eosinophil migration in the mice pleural lavage, after oil treatment with the oils at 100 mg/kg. RESULTS: Eosinophil migration was inhibited by SC (67%), SC (+V) (63%), PG (76%), PG (+V) (67%) and PG (-V) (74%). This efficacy was correlated with the presence of ß-pinene and ß-caryophyllene in the oils, a result that was reinforced by evaluating both these pure components (38 and 50% inhibition, respectively). Synergistic effects associated with the presence of α-pinene were speculated. DISCUSSION AND CONCLUSION: Essential oils from SC and PG may be useful to treat inflammatory diseases by mechanisms that include the inhibition of eosinophil migration.

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica.

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 °C) in a reaction medium containing 0.5% v/v limonene and 10 g/L of stationary phase cells (dry weight). The best results, corresponding to 564 mg/L of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 °C for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mg/L, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.

Flavonoids bearing an O-arabinofuranosyl-(1-->3)-rhamnoside moiety from Cladocolea micrantha: inhibitory effect on human melanoma cells.

Eleven known triterpenes (alpha-amyrin, beta-amyrin, lupeol, and their respective acetates, 3-O-acetyl derivatives of betulinic, oleanolic, and ursolic acids, cycloartenol, and tirucall-7,24-dienol), two new flavonols presenting an uncommon interglycosidic O-(1-->3) linkage (kaempferol 3-O-alpha-L-arabinofuranosyl(1-->3)-alpha-L-rhamnoside and quercetin 3-O-alpha-L-arabinofuranosyl-(1-->3)-alpha-L-rhamnoside), beta-sitosterol, stigmasterol, quercetin, and gallic acid were isolated from the Amazonian medicinal mistletoe, Cladocolea micrantha Kuijt (Loranthaceae). Their structures were established by spectral methods and eventual chromatographic comparisons. The quercetin derivative was not cytotoxic to MV3 human melanoma cells, but was able, when administered at 1 microg/mL, to promote a twofold inhibition of the migration of the cells through the transwell system when compared with paclitaxel at 5 microM.

Immunomodulating and antiviral activities of Uncaria tomentosa on human monocytes infected with Dengue Virus-2.

Uncaria tomentosa (Willd.) DC., a large woody vine native to the Amazon and Central American rainforests has been used medicinally by indigenous peoples since ancient times and has scientifically proven immunomodulating, anti-inflammatory, cytotoxic and antioxidant activities. Several inflammatory mediators that are implicated in vascular permeability and shock are produced after Dengue Virus (DENV) infection by monocytes, the primary targets for virus replication. Here we assessed the immunoregulatory and antiviral activities from U. tomentosa-derived samples, which were tested in an in vitro DENV infection model. DENV-2 infected human monocytes were incubated with U. tomentosa hydro-alcoholic extract or either its pentacyclic oxindole alkaloid-enriched or non-alkaloid fractions. The antiviral activity was determined by viral antigen (DENV-Ag) detection in monocytes by flow cytometry. Our results demonstrated an in vitro inhibitory activity by both extract and alkaloidal fraction, reducing DENV-Ag+ cell rates in treated monocytes. A multiple microbead immunoassay was applied for cytokine determination (TNF-alpha, IFN-alpha, IL-6 and IL-10) in infected monocyte culture supernatants. The alkaloidal fraction induced a strong immunomodulation: TNF-alpha and IFN-alpha levels were significantly decreased and there was a tendency towards IL-10 modulation. We conclude that the alkaloidal fraction was the most effective in reducing monocyte infection rates and cytokine levels. The antiviral and immunomodulating in vitro effects from U. tomentosa pentacyclic oxindole alkaloids displayed novel properties regarding therapeutic procedures in Dengue Fever and might be further investigated as a promising candidate for clinical application.

Desenvolvimento e aplicação de metodologia por cromatografia em camada delgada para determinação do perfil de alcalóides oxindólicos pentacíclicos nas espécies sul-americanas do gênero Uncaria / Development and application of a thin layer chromatographic method for the determination of the pentacyclic oxindole alkaloid profile in South-American species of the genus Uncaria

O gênero Uncaria (Rubiaceae) é representado na América do Sul e Central por duas espécies: U. tomentosa (Willd.) DC. e U. guianensis (Aubl.) Gmel., conhecidas popularmente como unha-de-gato. Ambas são trepadeiras perenes, sendo empregadas na prevenção e cura de várias doenças. Nessas plantas são encontrados alcalóides oxindólicos e indólicos, triterpenos glicosilados, taninos e flavonóides. Seis alcalóides oxindólicos pentacíclicos, considerados seus marcadores: especiofilina, mitrafilina, uncarina F, isomitrafilina, pteropodina e isopteropodina, são usados na padronização do material vegetal e fitoterápicos derivados. O presente trabalho descreve o desenvolvimento de metodologia analítica qualitativa utilizando cromatografia em camada delgada (CCD) para determinação do perfil dos seis alcalóides oxindólicos pentacíclicos marcadores das espécies. O desenvolvimento do método incluiu a comparação entre o uso do extrato metanólico bruto, e de frações enriquecidas obtidas por partição ácido-base clássica ou pelo uso de resina básica Poliamida 6. Utilizou-se gel de sílica como fase estacionária, e variaram-se alguns parâmetros como: eluentes, concentração da amostra, espaço de eluição e tipos de reveladores. O método desenvolvido em CCD mostrou-se confiável, reprodutível e seletivo para os alcalóides alvos, sendo aplicado na análise de amostras de folhas e caule das duas espécies e também de fitoterápicos comerciais à base de U. tomentosa.

The species Uncaria tomentosa (Willd.) DC. and U. guianensis Gmel. (Rubiaceae), known as cat's claw, are large woody vines occurring in the Amazon rain forest and other tropical areas of South and Central America. It has been used medicinally by indigenous peoples for at least 2,000 years for several diseases. Tetra- and pentacyclic oxindole alkaloids, triterpenoid glycosides, sterols and flavonoids are found in these plants. Among these metabolites, six pentacyclic oxindole alkaloids, speciophylline, mitraphylline, pteropodine, uncarine F, isopteropodine and isomitraphylline, are considered to be the biochemical markers and are used to standardize commercial herbal medicines. The present study describes the development of an analytical methodology to determine the profile of these alkaloid markers through thin layer chromatography (TLC). This development has also included a comparison among the use of the crude methanol extract and fractions obtained through the classical acid-base partition or by using the basic resin Polyamide 6. Silica gel was used as stationary phase with the variation of some parameters such as solvent systems, sample concentration, distance of development and detection method. The TLC method developed was shown to be reliable, reproducible and selective for the target alkaloids. It has been applied to the analysis of leaves and stems from both species as well as phytopharmaceutical derivatives based on U. tomentosa.

5-Methoxypropacin, a novel coumarinolignoid from Protium unifoliolatum.

The novel coumarinolignoid 5-methoxypropacin was isolated from the ethereal extract of the wood of the Amazonian species Protium unifoliolatum. Its structure was elucidated by spectroscopic methods as IR, 1D and 2D (1)H, (13)C-NMR and mass spectrometry. The regioisomer and the position of coumarin and arylpropanoid coupling were determined by long-distance C-H correlation (HMBC) and NOE experiments. Scopoletin and beta-sitosterol were also isolated and identified by the same physical methods.

Linalool from Lippia alba: study of the reproducibility of the essential oil profile and the enantiomeric purity.

A new chemotype of the aromatic Verbenaceae species Lippia alba Mill. N. E. Br. from southeastern Brazil has recently been shown to have a high content of linalool in the leaf essential oil. Vegetative propagation of this chemotype was conducted at six different locations in Brazil, and the variation of the content and the optical purity of linalool in the oils were verified. Yields (0.6-0.9%, hydrodistillation), chemical composition, linalool content, and optical purity of the oils from all the plants were compared, using GC-FID, GC-MS, chiral chromatography, and retention index calculation. No plant exceeded the matrix in linalool content (46.5 to 90.7%), and the chemical profile of the oils was the same for all the samples. Purification of linalool to a content close to 100% was effected by vacuum distillation of the crude oil. Chiral analysis showed exclusively the presence of S-linalool in all the crude oils and in the distilled samples.