Rodrigo Rodrigues de Oliveira

Gradient Elution for Triterpene Separation from Cecropia lyratiloba Miquel by HSCCC

Abstract The triterpenoid pool from the dichloromethane fraction obtained from the methanolic extract of roots of Cecropia lyratiloba Miquel was submitted to CCC using a gradient elution consisting of Hex/EtOAc/MeOH/H2O—1/2/X/1 (X=0.5 (A); 0.75 (B); 1.0 (C); 1.5 (D); 2.0 (E)) in five steps. The lower aqueous phase was used as mobile phase, 2 mL/min at 850 rpm. This procedure led to the isolation of tormentic acid and a mixture of tormentic and euscaphic acids. In order to improve the triterpene separation the fractions Fr 31–49 were submitted to a new CCC run using a fine adjustment of the methanol concentration in the gradient elution system. This separation procedure led to the isolation of euscaphic acid, 3‐acetyl tormentic acid and a mixture of tormentic and isoarjunolic acids.

Activity of Cecropia lyratiloba extract on contractility of cardiac and smooth muscles in Wistar rats.

1 Brazilian forests show high diversity of medicinal plants and several are used in folk medicine for the treatment of hypertension and asthma. The aim of the present study was to investigate the effects of a methanol extract (ME) of Cecropia lyratiloba and its flavonoid fraction (FF) on the contractility of cardiac, vascular and tracheal smooth muscles. 2 Twitches of rat papillary muscles were obtained with electrical stimulation and were recorded before and after exposure to increasing concentrations of ME and FF. 3 Cardiac depression was induced by FF. At 500 mg/mL FF, the amplitude of twitches was reduced to 56.7 5.1% of control values (P < 0.05). 4 The contractile response to a single concentration of adrenaline (10 mmol/L) was measured before and after exposure to ME and FF in rat aorta rings with intact endothelium. Both ME and FF inhibited adrenaline‐induced contractions of the aorta in a concentration‐dependent manner. Adrenaline‐induced contrations were reduced to 46.4 9.9 and 34.2 6.9% (P < 0.05) of control in the presence of 500 mg/mL ME and FF, respectively. 5 The flavonoids isolated from FF, namely isoorientin and a mixture of orientin and isovitexin, were also tested in the aorta. These flavonoid do not seem to be responsible for the vasorelaxant effects of ME and FF. 6 No changes were observed in acetylcholine‐precontracted trachea when exposed to ME or FF. 7 Endothelium‐dependent vasodilation induced by FF is likely to be mediated by the release of nitric oxide because vascular relaxation was abolished in the presence of Nw‐nitro‐l‐arginine methyl ester, an inhibitor of nitric oxide synthase. 8 In conclusion, vascular relaxation induced by ME and FF could explain the traditional use of the extract of C. lyratiloba for treatment of arterial hypertension.

3β-acetyl tormentic acid induces apoptosis of resistant leukemia cells independently of P-gp/ABCB1 activity or expression

SummaryChronic Myeloid Leukemia (CML) is a potentially fatal stem-cell cancer. P-glycoprotein (P-gp/ABCB1) activity has been described as a relevant factor in the chemotherapeutic failure and correlated to a poor prognosis in these malignancies. In the present study, we investigated the mechanism of the antineoplastic activity of 3β-acetyl tormentic acid (3ATA), a triterpene isolated from C. lyratiloba, on Lucena-1, an MDR leukemia cell line, that overexpressed P-gp/ABCB1. Results showing that this triterpene induced DNA-fragmentation, activation of caspase-3 and cytochrome c release indicated that its activity is mediated by the activation of the intrinsic pathway of apoptosis. Interestingly, this triterpene did not interfere with P-gp/ABCB1 expression or activity, indicating that induction of death is not mediated by any effect on this protein. Moreover, the results show that none of the others triterpenes from C. lyratiloba were able to modulate the activity of P-gp/ABCB1. Together these results suggest 3ATA and the other triterpenes as a promising material for the development of anti-neoplastic drugs for leukemia and other tumors independent of P-gp/ABCB1 activity or expression.

3β-Acetyl tormentic acid reverts MRP1/ABCC1 mediated cancer resistance through modulation of intracellular levels of GSH and inhibition of GST activity

ABC transporter overexpression is an important mechanism of multidrug resistance (MDR) and one of the main obstacles to successful cancer treatment. As these proteins actively remove chemotherapeutics from the tumor cells, the pharmacological inhibition of their activity is a possible strategy to revert drug resistance. Moreover, the ability of MDR inhibitors to sensitize resistant cells to conventional drugs is important for their clinical use. Evidence has shown that the multidrug resistance protein 1 (MRP1/ABCC1) is a negative prognostic marker in patients with lung, gastric, or breast cancers or neuroblastoma. Previous data have shown that 3β-acetyl tormentic acid (3ATA) inhibits the transport activity of the protein MRP1/ABCC1. In this study, we evaluated the ability of 3ATA to sensitize an MDR cell line (GLC4/ADR), which overexpresses MRP1, and investigated the anti-MRP1 mechanisms activated by 3ATA. The results showed that 3ATA is able to reverse the resistance of the MDR cell line to doxorubicin and vincristine, two drugs that are commonly used in cancer chemotherapy. Regarding the sensitizing mechanism induced by 3ATA, this work shows that the triterpene does not modulate the expression of MRP1/ABCC1 but is able to reduce total intracellular glutathione (GSH) levels and decrease the activity of glutathione-s-transferase (GST), the enzyme responsible for the glutathione conjugation of xenobiotics. Together, these results show that 3ATA sensitizes the MDR cell line overexpressing MRP1/ABCC1 to antineoplastic drugs and that this effect is mediated by the modulation of intracellular levels of GSH and GST activity.

Effects of 3β-Acethyl Tormentic Acid (3ATA) on ABCC Proteins Activity

Multidrug resistance (MDR) is considered the main cause of cancer chemotherapy failure and patient relapse. The active drug efflux mediated by transporter proteins of the ABC (ATP-binding cassette) family is the most investigated mechanism leading to MDR. With the aim of inhibiting this transport and circumventing MDR, a great amount of work has been dedicated to identifying pharmacological inhibitors of specific ABC transporters. We recently showed that 3β-acetyl tormentic acid (3ATA) had no effect on P-gp/ABCB1 activity. Herein, we show that 3ATA strongly inhibited the activity of MRP1/ABCC1. In the B16/F10 and Ma104 cell lines, this effect was either 20X higher or similar to that observed with MK571, respectively. Nevertheless, the low inhibitory effect of 3ATA on A549, a cell line that expresses MRP1-5, suggests that it may not inhibit other MRPs. The use of cells transfected with ABCC2, ABCC3 or ABCC4 showed that 3ATA was also able to modulate these transporters, though with an inhibition ratio lower than that observed for MRP1/ABCC1. These data point to 3ATA as a new ABCC inhibitor and call attention to its potential use as a tool to investigate the function of MRP/ABCC proteins or as a co-adjuvant in the treatment of MDR tumors.

Application of preparative high-speed counter-current chromatography for the separation of two alkaloids from the roots of Tabernaemontana catharinensis (Apocynaceae).

The methanolic extract of Tabernaemontana catharinensis (Apocynaceae) roots, which contains alkaloids with several biological activities, was separated on a preparative scale using high-speed counter-current chromatography. The optimum solvent system was found to be a mixture of CHCl3-MeOH-H2O [5:10:6 (v/v/v)] and led to a successful separation of two monoterpenic indole alkaloids, voachalotine (1) and 12-methoxy-Nb-methylvoachalotine (2) in approximately 4.0 hours. The alkaloids were all isolated at purities over 95%, and their structures were established on the basis of spectroscopic methods, including 1D and 2D NMR and EI/MS.

A new alkaloid and flavonoids isolated from Solanum cernuum leaves by high-performance countercurrent chromatography

Abstract Solanum cernuum is a medicinal plant widely distributed in south-east regions of Brazil and popularly used to treat several disorders. Despite their utilisation as a medicine, few studies on its chemical composition are reported. An efficient separation method for the ethyl acetate fraction was developed by high-performance countercurrent chromatography using n-butanol/chloroform/methanol/water (3:7:3:4, v/v/v/v) as the solvent system, affording five compounds (1–5) in one-step separation. A new cyclic guanidine alkaloid named cernidine (5) was obtained, besides four glycosylated flavonoids: afzelin (1), astragalin (2), kaempferol 3-O-α[apiofuranosyl-(1 → 2)]-α-rhamnopyranoside (3) and kaempferol 3-O-α[apiofuranosyl-(1→2)]-β-galactopyranoside (4). A further purification step afforded a mixture of trans- and cis-tiliroside (6–7). Countercurrent chromatography proved itself as a powerful tool for the isolation of similar compounds since compounds 1–2 and 3–4 possess little structural differences.

One-step separation of terpenoids from leaves extracts of Solanum cernuum by high-performance countercurrent chromatography

ABSTRACT Solanum cernuum is a native shrub from Brazil that has been used as medicine since the nineteenth century. Although it has been utilized for a long time, there are few studies regarding its chemical composition and it motivated us to investigate this species. The dried leaves of S. cernuum were extracted with methanol and the obtained extract was further partitioned with different organic solvents. As part of our investigation, the n-hexane fraction was submitted to CCC with a nonaqueous solvent system composed of hexane, acetonitrile, and ethyl acetate (5:5:1, v/v/v). Three terpenoids were obtained by one-step separation and identified as 24-oxo-31-norcycloartanone (22.8 mg), lupeol (9.4 mg), and cycloeucalenone (13.4 mg). Complementarily, the solvents system used on the separation have had their compositions determined by gas chromotography–flame ionization detector (GC-FID). GRAPHICAL ABSTRACT

LIGNANS AND FLAVONOIDS ISOLATED FROM CUSCUTA RACEMOSA MART. & HUMB (CONVOLVULACEAE) BY DROPLET COUNTER-CURRENT CHROMATOGRAPHY

Lignans and flavonoids were isolated from Cuscuta racemosa Mart. & Humb (Convolvulaceae) by Droplet Counter-Current Chromatography (DCCC). The solvent system was composed of hexane/ethyl acetate/methanol/water (1:2:1:1, v/v). In this experiment it was possible to isolate six substances: 9α-hydroxysesamin, 9β-hydroxysesamin, kaempherol, acuminatolide, quercetin, and pinoresinol. The chemical structures of the six substances were elucidated by NMR and GC-MS.

Essential Oil Composition of Brazilian Pourouma Species

Abstract The chemical composition of essential oils isolated by hydrodistillation from ripe fruits and leaves of Pourouma cecropiifolia Martius, Pourouma mollis Trécul and Pourouma velutina Martius ex Miguel was determined by GC and GC/MS. From ripe fruits of P. cecropiifolici Martius, 24 compounds were identified, accounting for 73.8% of the oil. Oxygenated monoterpenes were found to be an important group of constituents with linalool (3.1%) predominating. A new monoterpene, 2-methoxγ-1,7,7-trimethylbicyclo[2.2.1]hept-2-ene (2-methoxybornylene), was tentatively identified by mass spectrometry. In addition, 29, 37 and 26 constituents were identified in the leaf oils of P. cecropiifolia, P. mollis and P. velutina, respectively. Aliphatic alcohols, aldehydes and esters (C3-C9) were quantitatively the most significant components. (Z)-3-Hexenol was clearly the dominant product occurring in contents of 26.7%, 22.3% and 3.2%, respectively. The major sesquiterpene hydrocarbons in the analyzed oils were β-caryophyllene (9.1%) and (E,E)-α-farnesene (4.0%) from P. cecropiifolia oil; (E,E)-farnesene (1.7%) from P. mollis oil; and (1.5%) from P. velutina oil. Linalool (26.1%) was the major compound identified in the volatile fraction of P. mollis. It was also identified in the oils of P. cecropiifolia (1.2%) and P. velutina (1.7%). Phytol was the main constituent of the leaf oil of P. velutina. Methyl salicylate was also identified in the leaf oils of P. cecropiifolia (1.6%), P. mollis (1.4%) and P. velutina (2.8%).