Two New Flavonoids from the Leaves of Baccharis oblongifolia (Ruiz and Pav.) Pers. (Asteraceae).

In this work, two new flavonoids, oblongifolioside A (1) and oblongifolioside B (2), along with eight known compounds (3-10), are isolated from the leaves of Baccharis oblongifolia (Asteraceae). The new structures are established through spectroscopic data and the known compounds are identified by comparison with data reported in the literature. The compounds (1-10) are evaluated in relation to their antiradical properties. Compounds 1 and 2 are found to exhibit high antiradical activity compared to their respective non-acylated flavonoids.

Non-Clinical Studies for Evaluation of 8-C-Rhamnosyl Apigenin Purified from Peperomia obtusifolia against Acute Edema.

Compound 8-C-rhamnosyl apigenin (8CR) induced a moderate reduction in the enzymatic activity of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus and cytosolic phospholipase A2 (cPLA2), but the compound also significantly inhibited the enzymatic activity of the enzyme cyclooxygenase. In vitro assays showed that the compound induced a slight change in the secondary structure of sPLA2 from Crotalus durissus terrificus snake venom. In vivo assays were divided into two steps. In the first step, the 8CR compound was administered by intraperitoneal injections 30 min prior to administration of sPLA2. In this condition, 8CR inhibited edema and myonecrosis induced by the sPLA2 activity of Crotalus durissus terrificus in a dose-dependent manner by decreasing interleukin-1ß (IL-1ß), tumor necrosis factor α (TNF-α), prostaglandin E2 (PGE2), and lipid peroxidation. This has been demonstrated by monitoring the levels of malondialdehyde (MDA) in rat paws after the course of edema induced by sPLA2. These results, for the first time, show that sPLA2 of Crotalus durissus terrificus venom induces massive muscle damage, as well as significant edema by mobilization of cyclooxygenase enzymes. Additionally, its pharmacological activity involves increased lipid peroxidation as well as TNF-α and IL-1ß production. Previous administration by the peritoneal route has shown that dose-dependent 8CR significantly decreases the enzymatic activity of cyclooxygenase enzymes. This resulted in a decrease of the amount of bioactive lipids involved in inflammation; it also promoted a significant cellular protection against lipid peroxidation. In vivo experiments performed with 8CR at a concentration adjusted to 200 µg (8 mg/kg) of intraperitoneal injection 15 min after sPLA2 injection significantly reduced sPLA2 edema and the myotoxic effect induced by sPLA2 through the decrease in the enzymatic activity of cPLA2, cyclooxygenase, and a massive reduction of lipid peroxidation. These results clearly show that 8CR is a potent anti-inflammatory that inhibits cyclooxygenase-2 (COX-2), and it may modulate the enzymatic activity of sPLA2 and cPLA2. In addition, it was shown that Crotalus durissus terrificus sPLA2 increases cell oxidative stress during edema and myonecrosis, and the antioxidant properties of the polyphenolic compound may be significant in mitigating the pharmacological effect induced by sPLA2 and other snake venom toxins.

A trinuclear oxo-chromium(III) complex containing the natural flavonoid primuletin: synthesis, characterization, and antiradical properties.

A new trinuclear oxo-centered chromium(III) complex with formula [Cr3O(CH3CO2)6(L)(H2O)2] (L = 5-hydroxyflavone, known as primuletin) was synthetized and characterized by ESI mass spectrometry, thermogravimetry, and 1H-NMR, UV-Vis, and FTIR spectroscopies. In agreement with the experimental results, DFT calculations indicated that the flavonoid ligand is coordinated to one of the three Cr(III) centers in an O,O-bidentate mode through the 5-hydroxy/4-keto groups. In a comparative study involving the uncoordinated primuletin and its corresponding complex, systematic reactions with the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) showed that antiradical activity increases upon complexation.

Structural crystalline characterization of sakuranetin--an antimicrobial flavanone from twigs of Baccharis retusa (Asteraceae).

Bioactivity-guided fractionation of an antimicrobial active extract from twigs of Baccharis retusa C. DC. (Asteraceae) yielded the flavanone 5,4'-dihydroxy-7-methoxy-flavanone (sakuranetin) as responsible for the detected activity. The structure of the bioactive compound was established on the basis of spectroscopic data analysis, including NMR and MS. Additionally, the structure of a new crystal form of sakuranetin was confirmed by X-ray diffratometry. The minimum inhibitory concentrations (MIC) of isolated compound were determined against pathogenic yeast belonging to the genus Candida (six species), Cryptococcus (two species/four serotypes) and S. cerevisiae BY 4742 (S288c background) and ranged from 0.32 to 0.63 µg/µL. Our results showed that sakuranetin, which structure was fully characterized, could be used as a tool for the design of novel and more efficacious antifungal agents.

Chemical Constituents from Leaves of Baccharis sphenophylla (Asteraceae) and Their Antioxidant Effects.

Baccharis is one of the largest genera of Asteraceae and its species are used in folk medicine for several medicinal purposes due to the presence of bioactive compounds. We investigated the phytochemical composition of polar extracts of B. sphenophylla. Using chromatographic procedures, diterpenoids (ent-kaurenoic acid), flavonoids (hispidulin, eupafolin, isoquercitrin, quercitrin, biorobin, rutin, and vicenin-2), caffeic acid, and chlorogenic acid derivatives (5-O-caffeoylquinic acid and its methyl ester, 3,4-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and 3,5-di-O-caffeoylquinic acid and its methyl ester) were isolated from polar fractions and are described. The extract, polar fractions, and fifteen isolated compounds were evaluated in relation to radical scavenging activity using two assays. Chlorogenic acid derivatives and flavonols exhibited higher antioxidant effects, confirming that B. sphenophylla is an important source of phenolic compounds with antiradical properties.

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae).

Leishmaniasis and Chagas' are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 µg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 µg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4'-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4' associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas' disease.

Anti-malarial, anti-trypanosomal, and anti-leishmanial activities of jacaranone isolated from Pentacalia desiderabilis (Vell.) Cuatrec. (Asteraceae).

Leishmaniasis, Chagas disease, and malaria affect the poorest population around the world, with an elevated mortality and morbidity. In addition, the therapeutic alternatives are usually toxic or ineffective drugs especially those against the trypanosomatids. In the course of selection of new anti-protozoal compounds from Brazilian flora, the CH(2)C(l2) phase from MeOH extract obtained from the leaves of Pentacalia desiderabilis (Vell.) Cuatrec. (Asteraceae) showed in vitro anti-leishmanial, anti-malarial, and anti-trypanosomal activities. The chromatographic fractionation of the CH(2)Cl(2) phase led to the isolation of the bioactive compound, which was characterized as jacaranone [methyl (1-hydroxy-4-oxo-2,5-cyclohexandienyl)acetate], by spectroscopic methods. This compound showed activity against promastigotes of Leishmania (L.) chagasi, Leishmania (V.) braziliensis, and Leishmania (L.). amazonensis showing an IC(50) of 17.22, 12.93, and 11.86 µg/mL, respectively. Jacaranone was also tested in vitro against the Trypanosoma cruzi trypomastigotes and Plasmodium falciparum chloroquine-resistant parasites (K1 strain) showing an IC(50) of 13 and 7.82 µg/mL, respectively, and was 3.5-fold more effective than benznidazole in anti-Trypanosoma cruzi assay. However, despite of the potential against promatigotes forms, this compound was not effective against amastigotes of L. (L.) chagasi and T. cruzi. The cytotoxicity study using Kidney Rhesus monkey cells, demonstrated that jacaranone showed selectivity against P. falciparum (21.75 µg/mL) and a selectivity index of 3. The obtained results suggested that jacaranone, as other similar secondary metabolites or synthetic analogs, might be useful tolls for drug design for in vivo studies against protozoan diseases.

Anti-leishmanial and anti-trypanosomal potential of polygodial isolated from stem barks of Drimys brasiliensis Miers (Winteraceae).

Parasitic protozoan diseases affect the poorest population in developing countries. Leishmaniasis and Chagas disease have been included among the most important threats for public health in Central and South American continent, with few therapeutic alternatives and highly toxic drugs. In the course of selection of novel drug candidates, we studied the anti-protozoal potential of Drimys brasiliensis. Thus, the crude hexane extract from stem bark as well as its main derivative, the sesquiterpene polygodial, were tested using in vitro assays. The crude hexane extract and polygodial showed activity against Leishmania spp. in the range between 22 and 62 µg/mL, but polygodial demonstrated high parasite selectivity towards Trypanosoma cruzi trypomastigotes (2 µg/mL), with a selectivity index of 19. Finally, polygodial showed a leishmanicidal effect, inducing intense ultrastructural damages in Leishmania in short-time incubation. The obtained results suggested that polygodial could be used as a tool for drug design studies against protozoan diseases and as a candidate molecule for further in vivo studies against T. cruzi.

Anti-leishmanial effects of purified compounds from aerial parts of Baccharis uncinella C. DC. (Asteraceae).

Species of Baccharis exhibit antibiotic, antiseptic, wound-healing, and anti-protozoal properties, and have been used in the traditional medicine of South America for the treatment of several diseases. In the present work, the fractionation of EtOH extract from aerial parts of Baccharis uncinella indicated that the isolated compounds caffeic acid and pectolinaringenin showed inhibitory activity against Leishmania (L.) amazonensis and Leishmania (V.) braziliensis promastigotes, respectively. Moreover, amastigote forms of both species were highly sensible to the fraction composed by oleanolic + ursolic acids and pectolinaringenin. Caffeic acid also inhibited amastigote forms of L. (L.) amazonensis, but this effect was weak in L. (V.) braziliensis amastigotes. The treatment of infected macrophages with these compounds did not alter the levels of nitrates, indicating a direct effect of the compounds on amastigote stages. The results presented herein suggest that the active components from B. uncinella can be important to the design of new drugs against American tegumentar leishmaniases.

Evaluation of anti-inflammatory activity of derivatives from aerial parts of Baccharis uncinella.

CONTEXT: Species of Baccharis exhibit antibiotic, antiseptic, and wound-healing properties, and have been used in the traditional medicine of South America for the treatment of inflammation, headaches, diabetes, and hepatobiliary disorders. OBJECTIVE: To investigate the anti-inflammatory activity of organic phases from EtOH extract of the aerial parts of Baccharis uncinella DC (Asteraceae). MATERIALS AND METHODS: The crude EtOH extract from the aerial parts of B. uncinella was subjected to partition procedures and the corresponding CH(2)Cl(2) and EtOAc phases were subjected to several chromatographic separation procedures. Thus, these phases and their purified compounds were assayed for evaluation of anti-inflammatory activity. RESULTS: The CH(2)Cl(2) phase from EtOH extract from B. uncinella contained two triterpenoids (oleanolic and ursolic acids) and one flavonoid (pectolinaringenin), whereas the respective EtOAc phase showed to be composed mainly by two phenylpropanoid derivatives (caffeic and ferulic acids). The CH(2)Cl(2) and EtOAc phases as well as their isolated compounds exhibited anti-inflammatory effects against inflammatory reactions induced by phospholipase A2 (from Crotalus durissus terrificus venom) and by carrageenan. DISCUSSION AND CONCLUSION: The results suggested that the components obtained from partition phases of EtOH extract of B. uncinella could represent lead molecules for the development of anti-inflammatory agents. Additionally, the results confirmed the use of Baccharis genus in the traditional medicine of South America for the treatment of inflammation and other heath disorders. To date, the present work describes for the first time the anti-inflammatory effects of compounds isolated from B. uncinella.

Isolation of an antileishmanial and antitrypanosomal flavanone from the leaves of Baccharis retusa DC. (Asteraceae).

In the course of selection of new bioactive compounds from Brazilian flora, the crude MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) showed potential against Leishmania sp. and Trypanosoma cruzi. Chromatographic fractionation of the dichloromethane phase from MeOH extract yielded great amounts of the bioactive derivative, which was characterized as 5,6,7-trihydroxy-4'-methoxyflavanone. The structure of this compound was established on the basis of spectroscopic data analysis, mainly nuclear magnetic resonance and mass spectrometry.

Chemical composition, seasonal variation, and biosynthetic considerations of essential oils from Baccharis microdonta and B. elaeagnoides (Asteraceae).

The chemical composition and seasonal variation throughout one year of the essential oils from leaves of Baccharis microdonta and B. elaeagnoides, collected in Campos do Jordão, SP, were investigated. The composition of the latter species has been described for the first time. By GC (RI) and GC/MS analysis, 43 compounds were identified, and a predominance of oxygenated sesquiterpene derivatives was found in both species. The main components of the B. microdonta oils were elemol (31; 11.7-30.6%), spathulenol (34; 4.7-9.1%), ß-caryophyllene (19; 3.7-6.2%), and germacrene D (24; 2.9-12.2%), and those of the B. elaeagnoides oils were 34 (10.1-21.5%), viridiflorol (35; 3.6-18.4%), 24 (0.9-13.8%), and 19 (3.5-9.4%). The identified compounds were grouped according to their respective C-skeletons, and the percentages of occurrence of the C-skeletons in the essential oils of leaves collected in the four seasons allowed identifying the preferential accumulation of different types of C-skeletons as well as the seasonal variation of the biosynthetic routes over the studied period.

Antileishmanial activity and ultrastructural changes of related tetrahydrofuran dineolignans isolated from Saururus cernuus L. (Saururaceae).

OBJECTIVE: This work describes the isolation of anti-Leishmania amazonensis metabolites from Saururus cernuus (Saururaceae). Additionally, ultrastructural changes in promastigotes were evidenced by electron microscopy. METHODS: The MeOH extract from the leaves of S. cernuus was subjected to bioactivity-guided fractionation. Anti-L. amazonensis activity of purified compounds was performed in vitro against promastigote and amastigote forms. KEY FINDINGS: Bioactivity-guided fractionation of the MeOH extract from the leaves of S. cernuus afforded two related tetrahydrofuran dineolignans: threo,threo-manassantin A (1) and threo,erythro-manassantin A (2). Compounds 1 and 2 displayed activity against promastigotes (EC50 of 35.4 ± 7.7 and 17.6 ± 4.2 µm, respectively) and amastigotes (EC50 of 20.4 ± 1.9 and 16.0 ± 1.1 µm, respectively), superior to that determined for the positive control miltefosine (EC50 of 28.7 ± 3.5 µm). Reduced cytotoxicity for host cells was observed for both compounds. Additionally, ultrastructural changes in promastigotes leading to an alteration of structural morphology were observed, as evidenced by electron microscopy. Furthermore, these compounds altered the morphology and physiology of the plasmatic membrane of L. amazonensis. CONCLUSIONS: The obtained results indicated that dineolignans 1 and 2 could be considered as a scaffold for the design of novel and selective drug candidates for the treatment of leishmaniasis.

Antitrypanosomal activity and evaluation of the mechanism of action of diterpenes from aerial parts of Baccharis retusa (Asteraceae).

Baccharis retusa, a medicinal Brazilian plant from Asteraceae, has been used in Brazilian folk medicine to treatment of several illnesses, including parasitic diseases. Bioactivity-guided fractionation of the n-hexane extract from the aerial parts of B. retusa resulted in the isolation and characterization of three active related diterpenes: ent-15ß-senecioyl-oxy-kaur-16-en-19-oic acid (1), ent-kaur-16-en-19-oic (2) and ent-16-oxo-17-nor-kauran-19-oic (3) acids. The structures of isolated compounds were defined by spectroscopic analysis, including NMR and HRESIMS. Antitrypanosomal activity of 1-3 was performed against cell-derived trypomastigotes using the colorimetric resazurin assay. The obtained results demonstrated that isolated compounds displayed a reduced toxicity against NCTC cells and were effective against the trypomastigote forms of T. cruzi with IC50 values of 3.8µM (1), 75.3µM (2) and 44.2µM (3). Additionally, compound 3 displayed activity against amastigote forms of T. cruzi with IC50 of 83.2µM. Compound 1 displayed the highest selectivity index (SI) when considered the trypomastigote forms, and its effect in the plasma membrane of parasite was evaluated using the fluorescent probe SYTOX Green. A considerable permeabilization (57%) in the membrane of the parasite was observed when compared to the untreated trypomastigotes. These data demonstrate, for the first time, the antitrypanosomal activity and mechanism of action of 1 and related compounds 2 and 3, obtained from aerial parts of B. retusa.

Chromenes from Peperomia serpens (Sw.) Loudon (Piperaceae).

Chromatographic separation of the CH2Cl2 extract from leaves of Peperomia serpens yielded two chromenes [5-hydroxy-8-(3',7'-dimethylocta-2',6'-dienyl)-2,2,7-trimethyl-2H-1-chromene (1) and 5-hydroxy-8-(3'-methyl-2'-butenyl)-2,2,7-trimethyl-2H-1-chromene-6-carboxylic acid (2)], besides the known chromene [methyl 5-hydroxy-2,2,7-trimethyl-2H-1-chromene-6-carboxylate (3)] and the flavonoid, dihydrooroxylin (4). Their structural elucidation were achieved by spectroscopic analyses. The antifungal activities of the CH2Cl2 extract and the isolated chromenes were measured bioautographically against Cladosporium cladosporioides and C. sphaerospermum, when it was found that the crude extract showed higher activity as compared to the pure compounds.

Evaluation of antiradical capacity by H2O2-hemin-induced luminol chemiluminescence.

This paper describes a screening method for antioxidant potential determination based on luminol/hemin/hydrogen peroxide chemiluminescence. The emission depletion, caused by an antiradical compound added during the chemiluminescence decay, is proportional to the number of reactive species trapped. Therefore, the difference between the areas of the emission decay curves, obtained in the absence and in the presence of the potential antioxidant, is a measure for the antiradical capacity of the sample. The technique has been applied to measure the antiradical capacity of pure compounds and complex mixtures from natural origin, providing reliable results that indicate the method's feasibility.

Effect of chlorogenic acid (5-Caffeoylquinic Acid) isolated from Baccharis oxyodonta on the structure and pharmacological activities of secretory phospholipase A2 from Crotalus durissus terrificus.

The aim of this paper was to investigate the effect of chlorogenic acid (5-caffeoylquinic acid, 5CQA), isolated from Baccharis oxyodonta, on the structure and pharmacological effect of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus. All in vitro and in vivo experiments were conducted using a purified sPLA2 compared under the same experimental conditions with sPLA2 : 5CQA. 5CQA induced several discrete modifications in the secondary structure and the hydrophobic characteristics of native sPLA2 that induced slight changes in the α-helical content, increase in the random coil structure, and decrease of fluorescence of native sPLA2. Moreover, 5CQA significantly decreased the enzymatic activity and the oedema and myonecrosis induced by native sPLA2. As the catalytic activity of sPLA2 plays an important role in several of its biological and pharmacological properties, antibacterial activity was used to confirm the decrease in its enzymatic activity by 5CQA, which induced massive bacterial cell destruction. We found that 5CQA specifically abolished the enzymatic activity of sPLA2 and induced discrete protein unfolding that mainly involved the pharmacological site of sPLA2. These results showed the potential application of 5CQA in the snake poisoning treatment and modulation of the pathological effect of inflammation induced by secretory PLA2.

An evaluation of 3-rhamnosylquercetin, a glycosylated form of quercetin, against the myotoxic and edematogenic effects of sPLA 2 from Crotalus durissus terrificus.

This paper shows the results of quercitrin effects on the structure and biological activity of secretory phospholipase (sPLA2) from Crotalus durissus terrificus, which is the main toxin involved in the pharmacological effects of this snake venom. According to our mass spectrometry and circular dichroism results, quercetin was able to promote a chemical modification of some amino acid residues and modify the secondary structure of C. d. terrificus sPLA2. Moreover, molecular docking studies showed that quercitrin can establish chemical interactions with some of the crucial amino acid residues involved in the enzymatic activity of the sPLA2, indicating that this flavonoid could also physically impair substrate molecule access to the catalytic site of the toxin. Additionally, in vitro and in vivo assays showed that the quercitrin strongly diminished the catalytic activity of the protein, altered its Vmax and Km values, and presented a more potent inhibition of essential pharmacological activities in the C. d. terrificus sPLA2, such as its myotoxicity and edematogenic effect, in comparison to quercetin. Thus, we concluded that the rhamnose group found in quercitrin is most likely essential to the antivenom activities of this flavonoid against C. d. terrificus sPLA2.

Anti-trypanosomal phenolic derivatives from Baccharis uncinella.

Bioassay-guided fractionation of the EtOH extract of the aerial parts of Baccharis uncinella C. DC. (Asteraceae) led to identification of two cinnamic acid derivatives (caffeic and ferulic acids), two flavones (hispidulin and pectolinaringenin) and a mixture of three chlorogenic acids (3,4-, 3,5- and 4,5-O-dicaffeoylquinic acids), which displayed in vitro anti-trypanosomal activity. Pectolinaringenin, hispidulin and caffeic acid showed activity against trypomastigotes of Trypanosoma cruzi, exhibiting 50% inhibitory concentration (IC50) values of 52, 81 and 56 microg/mL, respectively, while the chlorogenic acid mixture showed an IC50 value of 61 microg/mL. The flavonoids and cinnamic acid derivatives were evaluated for cytotoxicity against NCTC cells resulting in a 50% cytotoxic concentration (CC50) ranging from 33.82 to 129.1 microg/mL while the chlorogenic acids did not display cytotoxicity (CC50 >150 microg/mL). This is the first report of anti-trypanosomal activity of compounds from B. uncinella.

Jacaranone induces apoptosis in melanoma cells via ROS-mediated downregulation of Akt and p38 MAPK activation and displays antitumor activity in vivo.

BACKGROUND: Malignant melanoma is a deadly type of metastatic skin cancer with increased incidence over the past 30 years. Despite the advanced knowledge on the biology, immunobiology and molecular genetics of melanoma, the alternatives of treatment are limited with poor prognosis. On clinical trials, natural products and among them redox-active quinones have been tested in the attempt to control the growth of cancer cells. Recently, we isolated jacaranone from Pentacalia desiderabilis, a benzoquinone derivative that showed a broad antitumor activity and protective anti-melanoma effect in a syngeneic model. The purified substance is active at micromolar concentrations, is not hemolytic, and is not toxic in naïve mice. METHODOLOGY/PRINCIPAL FINDINGS: The jacaranone antitumor activity was shown against several human cancer cell lines in vitro. Moreover, the induction of apoptosis in murine melanoma cells and jacaranone antitumor activity in vivo, in a melanoma experimental model, were also shown. Jacaranone renders antiproliferative and proapoptotic responses in tumor cells, by acting on Akt and p38 MAPK signaling pathways through generation of reactive oxygen species (ROS). The free radical scavenger N-acetyl-cysteine (NAC) was able to completely suppress cell death induced by jacaranone as it blocked Akt downregulation, p38 MAPK activation as well as upregulation of proapoptotic Bax. Notably, treatment of melanoma growing subcutaneously in mice with jacaranone significantly extended the mean survival times in a dose-dependent manner. CONCLUSIONS/SIGNIFICANCE: The results provide evidence for the mechanisms of action of jacaranone and emphasize the potential use of this quinone for the treatment of melanoma.