2011 - Cláudio Roberto Novello

Ph.D. Thesis

Author: Cláudio Roberto Novello

Postgraduate Program in Chemistry - State University of Maringá

Area of Knowledge: Phytochemistry

Date of Defense: December 19, 2011

Advisor: Prof. Dr.  Profa. Dra. Maria Helena Sarragiotto

Co-advisor: Prof. Dr. João Carlos Palazzo de Mello

Examination Board:  Profa. Dra. Maria Helena Sarragiotto

                            Profa. Dra. Débora Cristina Baldoqui

                            Profa. Dra. Andrea Diniz

                            Prof. Dr. Wagner Villegas

                            Prof. Dr. Fernando Batista da Costa

Title: Phytochemical study and evaluation of cytotoxic, antioxidant and adaptogenic activity of Croton echioides Baill. - Euphorbiaceae

Abstract: Croton echioides Baill. (Euphorbiaceae) is a medicinal plant found in semi-arid brazilian areas, known popularly as “velame”. In folk medicine it is renowned for its tonic and aphrodisiac properties. Its stem bark has been widely sold as an aphrodisiac and tonic, as a substitute for the roots of Ptychopetalum olacoides  Benth. (Olacaceae), the Amazon Marapuama. Plants of the Croton genus are rich in diterpenes, alkaloids and flavonoids, which are active principles associated with many biological activities such as antitumor and antioxidant. In this study stem bark of C. echioides was used to perform quality control physico-chemical tests and optimization of the solvent extractor. To choose the best solvent extractor dry residue content and antioxidant capacity of extracts obtained were determined with several solvents of different polarity. The mixture of ethanol/water at 70% was the most effective solvent in the parameters tested being used for the production of crude extract (EB) through Ultra-Turrax process. The crude extract was suspended in water and partitioned into five fractions with n-hexane, dichloromethane, ethyl acetate and n-butanol. The fractions obtained with n-hexane (FH) and ethyl acetate (FA) were subjected to successive chromatographic separation processes. Through spectroscopic and spectrometric analysis and comparison with literature data were isolated and identified, from the FH fraction, the diterpenes of the type clerodane 15,16-epoxy-3,13(16),14-neo-clerodatrien-17,18-methyl dicarboxylate (nasimalun B) (CEH-1), 15,16-epoxy-3,13(16),14-neo-clerodatrien-17-carboxy-18-methyl carboxylate (CEH-2), 3-oxo-15,16-epoxy-13(16),14-neo-clerodadien-17,18-methyl dicarboxylate (CEH-3), ptychonal hemiacetal (CEH-4a) and ptychonal (CEH-4b) (in mixture), and hardwickiic acid methyl ester (CEH-5); the sesquiterpene trans-1(10)-epoxy-4(15)-caryophyllene (CEH-6), the triterpene lupeol (CEH-7); the phytosteroids β-sitosterol (CEH-8) and stigmasterol (CEH-9) (in mixture) and β-sitosterol glycoside(CEH-10). From the FA fraction were isolated and identified the indole alkaloids N-trans-p-coumaroyl-tryptamine (CEA-1), N-trans-p-coumaroyl-5-hydroxytryptamine (CEA-2), N-trans-4-methoxy-cinnamoyl-5-hydroxytryptamine (CEA-3), N-trans-feruloyl-5-hydroxytryptamine (moschamine) (CEA-4) and N-trans-feruloyl-3,5-dihydroxyindolin-2-one (CEA-5); the flavonoids 3-O-methyl kaempferol (CEA-6), 3-O-methyl quercetin (CEA-7), 3,7-di-O-methyl quercetin (CEA-8) and 3,3′-di-O-methyl quercetin (CEA-9) and the benzoic acid derivatives 4-hydroxybenzoic acid (CEA-10), 4-hydroxy-3-methoxybenzoic acid (CEA-11) and 4-hydroxy-3,5-dimethoxybenzoic acid (CEA-12). The CEH-2, CEH-3 and CEA-5 are new compounds in the literature while CEA-3 was isolated from a natural product for the first time. Behavioral tests were performed with the crude extract in young mice: pharmacological screening, motor activity, hexabarbital-induced sleeping time, motor coordination and elevated plus maze. In pharmacological screening the extract showed stimulating effect at low doses by both intraperitoneal and oral administration and toxicity evident in larger intraperitoneal doses. The motor activity evaluation results have not confirmed the stimulating action found in the screening and indicated a depressing effect at a 50 mg/kg dose. Significant differences were not found in the evaluated parameters in the motor coordination and hexabarbital-induced sleeping time tests, but only signs of depressing effect at a 50 mg/kg dose in this last test. The elevated plus maze test showed contradictory results without the prevalence of depressing or stimulating effect. The antioxidant capacity of the crude extract was determined for the fractions obtained from the liquid/liquid partition and from the FA fraction isolated compounds. The IC50 value for EB was 167.6 mg/mL and 48.9 mg/mL for FA, the most active fraction. The most active compounds were CEA-2 (IC50 of 10.7±0.4 mmol/L), CEA-3 (IC50 of 17.5±0.4 mmol/L), CEA-4 (IC50 of 14.5±0.4 mmol/L), CEA-7 (IC50 of 9.2±0.3 mmol/L), CEA-8 (IC50 of 11.0±0.6 mmol/L) and CEA-12 (IC50 of 20.2±0.7 mmol/L). The indole alkaloids isolated from the FA fraction were tested against a strain of human colon tumor cells HCT-116 by sulforhodamine assay, highlighting the N-trans-4-methoxy-cinnamoyl-5-hydroxytryptamine with IC50 of 29.3 mg/mL and death of 90% of tumor cells at a concentration of 72 mg/mL.

Keywords: Croton echioides, marapuma, clerodane diterpenes, alkaloids, flavonoids.

Thesis PDF:  Claudio Roberto Novello

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