Leishmanicidal and antimalarial activities of crude extracts from aerial parts of Copaifera langsdorffii and isolation of secondary metabolites

João Paulo Barreto Sousa et al. / Journal of Pharmacy Research 2012,5(8),4103-4107

Research Article ISSN: 0974-6943

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Leishmanicidal and antimalarial activities of crude extracts from aerial parts of Copaifera langsdorffii and isolation of secondary metabolites a

João Paulo Barreto Sousa, bDhammika Nanayakkara, cAndresa Aparecida Berretta Silva, aJairo Kenupp Bastos* a Laboratório de Farmacognosia, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil, b National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, MS, USA, c Apis Flora Industrial e Comercial Co. Rua Triunfo, 945, 14020-670, Ribeirão Preto, SP, Brazil.

Received on:09-05-2012; Revised on: 14-06-2012; Accepted on:22-07-2012 ABSTRACT Copaifera langsdorffii Desf. is a medicinal plant found in all regions of Brazil. Copaiba oleoresin is widely trade around the world and well-known for its distinct biological properties. The present work reports the isolation of compounds from leaves, fruits and flowers of Copaifera langsdorffii, as well as in vitro leishmanicidal and antimalarial assays with the crude hydroalcoholic extracts from leaves, fruits and flowers. The hydroalcoholic crude extracts were chromatographed using silica gel by classic chromatography, and purified by preparative TLC. HPLC-DAD was used to purify the polar compounds. The thermally stable compounds were evaluated by GC/MS. NMR and mass spectrometry data were used to establish the chemical structures. Nine compounds were isolated and identified: 1: kaurenoic acid, 2: ent-labda-7,13-dien-15-oic acid, 3: (E)-2-ethoxyvinylphenol, 4: 2-hydroxy-ent-labda-7,13-dien-15-oic acid, 5: caryophyllene oxide, 6: kaurenol, 7: ethyl 4-hydroxylcinnamate (semisynthetic 4-methylated form of compound 7 was isolated), 8: quercetin-3-O-α-Lrhamnopyranoside (quercitrin), and 9: kaempferol-3-O-α-L-rhamnopyranoside (afzelin), and a new compound (3) from fruits. Compounds 4 and 7 were reported for the first time in the aerial parts of Copaifera genus. Kaurenoic acid and β-caryophyllene were major compounds in all samples studied. Flavonoids 8 and 9 were major phenolics in the leaves hydroalcoholic extract. The Fruits extract displayed antileishmanial activity against Leishmania donovani with IC50 of 40 µg/mL. Leaves and flowers extract displayed antimalarial activity with IC50 of 3.4 and 6.2 µg/mL, respectively for different clones of Plasmodium falciparum. In addition, none of the tested samples displayed cytotoxicity against mammalian Vero cells. Keywords: Copaifera langsdorffii, secondary metabolites, antiprotozoal assays.

INTRODUCTION Copaifera L. genus (Leguminosae), popularly known as “copaiba,” is a large tree that grows mainly in Northern Brazil.[1] Copaifera langsdorffii is a tree found in all regions of Brazil. It can reach 5 to 40 meters in height and live up to 400 years. The trunk is rough and dark-colored, and its diameter varies from 0.4 to 4 meters.[1] The leaves are alternate, petiolate and feathered. Each fruit contains one seed that is enclosed in a colorful aril.[2] The flowers are small, without petals, hermaphroditic and arranged in axillary panicles.[2] Copaiba oil has been used in folk medicine as anti-inflammatory[3], urinary antiseptic[4], and for skin diseases[5], gastroprotective[6], analgesic[3], wound healing[7], antinociceptive[8] and antimicrobial[9]. According to last edition of the Index Kewensis, there are 72 species belonging to genus Copaifera.[1] Only 17 species of Copaifera have been chemically studied and the analyses are restricted to the oil.[10] The volatile compounds of the oil were characterized by GC/MS showing the following sesquiterpenes: β-caryophyllene, caryophyllene oxide, α-copaene, α-humulene, γ-muurolene, and ß-bisabolol.[5] The main non-volatile components found in copaiba oil belong to diterpene class, including kaurenoic acid, kaurenol, copalic acid, agathic acid, and hardwiickic acid[2, 10]. Analyses of other plant parts allowed the isolation of coumarin from seeds and sesquiterpenes and amino acids from leaves.[11] Palmitic and oleic acids were quantified by GC/MS from seeds non-polar extract.[11] The occurrence of colleters on the adaxial face of the leaves of this

*Corresponding author. J. K. Bastos, Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café s/n, CEP 14040-903, Ribeirão Preto, SP, Brazil.

plant has been reported. [12] The effect of hydroalcoholic extract of C. langsdorffii leaves on the crystal deposition in experimental urolithiasis has been previously evaluated.[13] However, there are very few reports in the literature regarding the chemical composition of aerial parts of Copaifera. The analytical and chemistry standardization of species of Copaifera are essential to correlate chemistry composition with biological activities, to determine the extraction conditions and drying process, to develop an adequate formulation able to deliver the active compounds, to quantify the analytes during the production processes, to analyze the final product and determine its shelf life. Therefore, the present work reports compounds and antiprotozoal assays from aerial parts of Copaifera langsdorffii, because either copaiba oil or compounds from aerial parts has potential for the development of new medicines. MATERIAL AND METHODS General procedures The crude extracts were chromatographed on silica gel 60 (Merck, art. 7734) using open columns. The obtained fractions were analyzed by thin-layer chromatography (TLC) and some were purified by preparative TLC (Merck art. 9385; 1 mm thickness). High performance liquid chromatography (HPLC) was used to purify polar compounds. The analyses were performed using a Shimadzu liquid chromatography (Kyoto, Japan), equipped with an LC6AD solvent pump unit, an SCL-10A system controller and an SPD-M10A diode array detector Shimadzu, operating with Shimadzu Chromatopak CR6A integrator (Kyoto, Japan) and a reverse-phase Shim-Pack CLC-ODS (C18) column (Shimadzu, Tokyo, Japan; 250 × 20 mm i.d.). The thermally stable compounds detected in aerial parts of C. langsdorffii were evaluated

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João Paulo Barreto Sousa et al. / Journal of Pharmacy Research 2012,5(8),4103-4107 by GC/MS (Shimadzu – QP 2010 equipped with an automatic injector AOC – 20Si) and retention indices (RI relative to C9-C25 n-alkanes). High-resolution ESITOFMS data of main compounds were obtained on an Agilent 6210 mass spectrometer. NMR spectra were recorded on a Varian Mercury 400BB spectrometer (400 MHz for 1H and 100 MHz for 13C). Samples were dissolved in CDCl3 or DMSO-d depending on polarity of the isolated compounds. TMS was used as internal reference.

solvent with hexane-ether 95:5 % giving compound 7 (ethyl 4methoxycinnamate; 10 mg). Fraction VII (1 g) was purified by semipreparative HPLC. The mobile phase consisted of water (A) and methanol (B). The elution program was 30 % B, followed by a linear gradient to 100 % of B after 30 min, at a flow rate of 6.0 mL/min. Two compounds were isolated: 8 (quercetin-3-O-a-L-rhamnopyranoside (quercitrin); 100 mg) and 9 (kaempferol-3-O-a-L-rhamnopyranoside (afzelin); 50 mg).

Copaifera aerial parts samples and extracts preparation C. langsdorffii leaves were collected in November 2008 in the campus of Ribeirão Preto of the University of São Paulo (21º10’S, 47º50’W, altitude 748 m), Brazil. The flowers were collected between January and February 2009 and the fruits were collected between August and September 2009, from the same plants. Professor Milton Groppo Junior authenticated the plant material, and a voucher specimen (SPFR 10120) was deposited in the herbarium of the School of Sciences, Philosophy and Education of Ribeirão Preto of the University of São Paulo-USP, Ribeirão Preto, Brazil. Leaves, flowers and fruits were dried under air circulation (40°C), powdered (35 meshes), conditioned in hermetically sealed glass flasks and kept under refrigeration at 8°C until they were used.

Antiprotozoal screening assays The in vitro antimalarial assay procedure utilized was an adaptation of the parasite lactate dehydrogenase (pLDH) assay, according to literature[15], using a 96-well microplate assay protocol with two P. falciparum clones [Sierra Leone D6 (chloroquine-sensitive) and Indochina W2 (chloroquine-resistant)]. The primary screening involved determination of pLDH inhibition (percentage) of each sample tested at 15.9 µg/mL for extracts. The IC50 values were determined only for samples that inhibited parasite growth by >50% for one of the clones. IC50 values were calculated from dose-response inhibition graphs. The antimalarial agents chloroquine and artemisinin were used as positive controls, with DMSO as the negative (vehicle) control.

The aliquots of dried and powdered leaves (2.0 kg), flowers (0.5 kg), and fruits (0.3 kg) of C. langsdorffii were individually macerated for 72 h with 70 % aqueous ethanol at room temperature, followed by percolation. These procedures were repeated three times, and the combined hydroalcoholic extracts were filtered and concentrated under reduced pressure at 60 °C to give the crude extracts. The yielding of these extracts were 300 g (15 %) from leaves; 30 g (6 %) from flowers; and 21 g (7 %) from fruits. Isolation of compounds Crude hydroalcoholic extract from fruits (4 g) was submitted to classic chromatography on silica gel 60 using mixtures of hexane-ethyl acetate 98:2 % and increasing the polarity with ethyl acetate (EtOAc). Twenty fractions (I – XX) of 300 mL each were collected and monitored by TLC. The fraction IV (500 mg) was re-chromatographed on silica gel column chromatography by collecting fractions of 50 mL using isocratic solvent system with hexaneEtOAc 95:5 %. The fractions IV.3-IV.6 were combined to furnish compound 1 (kaurenoic acid; 40 mg). The fraction V (400 mg) was re-chromatographed as for fraction IV. Compound 2 (ent-labda-7,13-dien-15-oic acid; 7 mg) was obtained combining the V.9-V.15 fractions. Fractions V.19-V.30 were combined and submitted to preparative TLC using hexane-ether-CHCl3 ; 70:10:20% as eluent, furnishing compound 3 ((E)-2-ethoxyvinylphenol; 2 mg). Fractions XII, XIII and XIV were combined and re-chromatographed on classic silica gel column using mixtures of hexane-EtOAc in increasing polarities, starting with 80:20 % furnishing compound 4 (2-hydroxy-entlabda-7,13-dien-15-oic acid; 20 mg). Compounds 1 (20 mg), 2 (10 mg) and 4 (12 mg) were also isolated from the flowers crude hydroalcoholic extract (6 g) using the same chromatographic procedures. The crude hydroalcoholic extract from leaves (50 g) were dissolved in 1 L of 90 % aqueous methanol and sequentially partitioned with 400 mL of hexane, chloroform and ethyl acetate. The ethyl acetate crude fraction (10 g) was subjected to a classic chromatography column using mixtures of hexaneEtOAc in increasing polarities, stating with 90:10 %. The obtained fractions were combined into fractions I – X based on their similarities by thin layer chromatography. Both I (3 g) and III (1 g) fractions were re-chromatographed according to procedures carried out with fruit extract. The separation process involving the fraction I led to the isolation of three compounds: 1 (680 mg), 5 (caryophyllene oxide; 20 mg), and 6 (kaurenol; 15 mg). The purification of fraction III gave compound 4 (30 mg). Aiming to improve the isolation process, fraction V (0.3 g) was treated with ethereal diazomethane [14]. The methylated sample was submitted to classic column using isocratic system

A transgenic cell line of L. donovani promastigotes showing stable expression of luciferase was used as the test organism. Cells in 200 µL of growth medium (L-15 with 10% FCS) were plated at a density of 2 x 106 cells per mL in a clear 96-well microplate. Stock solutions of the standards and test compounds/extracts were prepared in DMSO. Culture medium without cells and the controls were incubated (at 26 °C for 72 h) simultaneously, in duplicate, at six concentrations of the test compounds. An aliquot of 50 µL was transferred from each well to a fresh opaque/black microplate, and 40 µL of Steadyglo reagent was added to each well. The plates were read immediately in a Polar Star galaxy microplate luminometer. IC50 and IC90 values were calculated from dose-response inhibition graphs. Pentamidine and amphotericin B were tested as standard antileishmanial agents.[15] The mammalian cells Vero (African green monkey kidney fibroblast) used in this study were obtained from ATCC (Manassas, VA). Cytotoxicity was determined by the neutral red method according to procedure previously described.[15] The IC50 value for value for each compound was computed from the growth inhibition curve. RESULTS AND DISCUSSION Chemical structures, MS data and molecular formula of the isolated compounds (Figure 1) and the spectroscopic data of the compounds from the aerial parts of C. langsdorffii are in agreement with those reported previously in the literature: 1: kaurenoic acid[16], 2: ent-labda-7,13-dien-15-oic acid[17], 3: (E)-2-ethoxyvinylphenol, 4: 2-hydroxy-ent-labda-7,13-dien-15oic acid [18], 5: caryophyllene oxide [14], 6: kaurenol [16], 7: ethyl 4methoxycinnamate[19], 8: quercitrin[20], and 9: afzelin[20]. We included 1H- and 13 C-NMR spectra of (E)-2-ethoxyvinylphenol (3) in the supplementary material, because compound 3 was not found in literature. The isolated compounds were evaluated by TLC using different solvent systems, as well as by HPLC-DAD and MS, and in addition with their 1H and 13C NMR data, the purities of such compounds were considered suitable to structural proposition. Analyses by GC/MS of the non-polar samples obtained from aerial parts of C. langdorffii allowed the identification of 12 sesquiterpenes, including α-copaene, α-bergamotene, β-caryophyllene, α-humulene, bicyclogermacrene, germacrene D, germacrene B, δ-cadinene and α- cadinol, as the major volatiles in these samples. Kaurenoic acid and β-caryophyllene are widely reported in the literature as major compounds in copaiba oil.[21] Kaurenoic acid was also the major diterpene isolated from aerial parts in our study. β-caryophyllene was a major compound in all non-polar fractions obtained from fruits, flowers and

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R= H: 2: ent-Labda-7,13-dien-15-oic acid.

R= OH:4:2-Hydroxy-ent-labda-7,13-dien-15-oic acid

Figure 1: Compounds isolated and identified from aerial parts of C. langsdorffii.

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João Paulo Barreto Sousa et al. / Journal of Pharmacy Research 2012,5(8),4103-4107 leaves. Moreover, caryophyllene oxide was purified during separation process of the leaves extract. These compounds could be used as chemical markers of copaiba extracts. The diterpenes 2 and 4 are structurally similar to ent-labd-8(17)-en-15-oic acid (eperuic acid), which was isolated for the first time by Djerassi and Marshall[22], and are known as eperuanes derivatives.[22] Compound 4: 2hydroxy-ent-labda-7,13-dien-15-oic acid was identified by mass spectrometry from Dodonaea spp. samples.[18] The 1H and 13C NMR of 2-hydroxyent-labda-7,13-dien-15-oic acid revealed the presence of 5 methyl groups (δ 16.1, 19.6, 22.5, 24.0, 34.1); 5 sp3 methylenes (δ 24.7, 25.8, 43.8, 44.7, 47.0); 1 hydroxylated sp3 methine (δ 68.4); 2 sp3 methines (δ 49.6 and 55.2); 2 sp2 methines (δ 115.6 and 123.3); 2 sp3 quaternary carbons (δ 32.6 and 36.6); 2 sp2 quaternary carbon (δ 134.6 and 163.2); and 1 carboxylic carbon (δ 171.7). The position of attachment of the hydroxyl group was confirmed by the correlation between H-2 (δ 4.2; sl) and C-2 (δ 68.4) observed in the HMQC, and the correlation between H-20 (δ 0.9; s) and C-2 was verified in the HMBC spectrum. The 13 C-NMR spectroscopic data of the compound 3: (E)-2ethoxyvinylphenol displayed 8 sp2 carbon identified from their chemical shifts: at δ 158.3 (C-2), 143.7 (C-7), 132.1(C-4), 128.1 (C-6), 124.6 (C-5), 119.0 (C-1), 117.1 (C-3), and 116.9 (C-8). The chemical shift at δ 158.3 was assigned to a hydroxylated carbon. Also, there are 2 sp3 carbons at δ 66.1 (C1’) and 15.5 (C-2’) enabling a chemistry structure with 10 carbon. In the 1HNMR spectra of this compound the observed signals in the aromatic region suggest two doublet groups: between δ 7.5 and 7.6 (2H, J = 8.0 Hz, H-3 and H-5), and between δ 7.3 and 7.4 (2H, J = 8.0 Hz, H-4 and H-6). The doublets at δ 7.7 and 6.4 (1H, J = 8.0 Hz, H-7 and H-8, respectively) are related to trans vinyl carbon bond. The double of doublet at δ 3.5 (2H, J = 4.0 and 8.0 Hz) and singlet at δ 1.26 (3H) are in agreement with H-1’ and H-2’, respectively. A comparison of the 1H- and 13C-NMR spectra of (E)-o-coumaric acid[23] with NMR data acquired for compound 3 demonstrated that the chemical shifts are similar taking into account both phenol and trans vinyl groups. This comparative study along with MS data, in addition with NMR analysis allowed to establish the chemical structure of compound 3 as (E)-2ethoxyvinylphenol, which have not been described in the literature. Therefore, it is a novel isolated compound from aerial parts of C. langsdorffii. The NMR data of compound 7 (ethyl 4-methoxycinnamate) have not been reported from Copaifera species. The 1H-NMR spectra of this compound presented two doublets, typical of the p-substituted aromatic ring at δ 7.5 (2H, J = 8.0 Hz, H-3 and H-5) and 6.9 (2H, J = 8.0 Hz, H-2 and H-6); olefinic protons at δ 6.3 (1H, d, J = 16 Hz, H-8) and δ 7.7 (1H, d, J = 16 Hz, H-7); double of doublet at δ 4.3 (2H, J = 4.0 and 8.0 Hz, H-1’); methyl at δ 1.3 (3H, t, J = 4.0 and 8.0 Hz, H-2’) and methoxyl group at δ 3.8 (3H, s, H-3’). In the 13C NMR spectra was observed 10 signals: at δ 114.8 and 130.2 assigned to C-2/C-6 and C-3/C-5, respectively, in addition to the signals at δ 127.7 (C-1); δ 161.9 (C-4); δ 144.8 (C-7); δ 116.2 (C-8); δ 167.8 (C-9); δ 60.7 (C-1’); δ 14.9 (C-2’) and δ 55.8 (C-3’). To get this component, the sample leaves extract was methylated by the treatment with ethereal diazomethane.[14] This derivative reaction priority attacks hydrogen acid in the chemical structure. Thus, the hydroxyl was methylated resulting in the methoxyl group. Therefore, the isolation of the ethyl 4-methoxycinnamate confirms the presence of the ethyl 4-hydroxylcinnamate in Copaifera for the first time. The flavonoid heterosides 8 and 9 are very common in plant kingdom, and phytochemical investigation of the leaves hydroalcoholic extract of the C. langsdorffii, combined with HPLC-DAD analyses, allowed the detection and quantitation of these two major flavonoids monthly for 14 months in plant leaves.[24]

Leishmaniasis is a disease regarded as a major public health problem that affects approximately 12 million people in 80 countries and causes morbidity and mortality mainly in Africa, Asia, and Latin America.[25] Malaria is another important tropical disease which has the potential to affect nearly 40% of the world’s population.[25] In the antileishmanial assay, the crude extract of fruits showed IC50 and IC90 values of 40 and 85 µg/mL, respectively (Table 1). With respect to antimalarial assay, the leaves extract displayed IC50 mean values of 3.4 µg/mL (D6 and W2 clones). An IC50 of 6.2 µg/ mL (D6 clone) and 5.0 µg/mL (W2 clone) were obtained from flowers extract (Table 1). The observed antiparasitical activities should be related to the presence of main compounds reported in this work. It should be pointed out that none of the tested samples displayed cytotoxicity against mammalian Vero cells, and the select indexes were greater than 7.7. Table 1: In vitro antileishmanial, antimalarial and cytotoxic activities of crude extracts from aerial parts of the C. langsdorffii Samples

Leaves extract Flowers extract Fruits extract Chloroquine Artemisinin Pentamidine Amphotericin B

L. donovani (µg/mL)

P. falciparum ( g/mL) D6 clonea) W2 cloneb)

µ

IC 50

IC 90

IC 50

SIc)

IC 50

SIc)

Cytotoxicity (Vero cells) IC50 (µg/mL)

NA NA 40 1.2 0.2

NA NA 85 6 0.4

3.4 6.2 NA 0.015 0.014 -

>14 >7.7 NA >556 >669 -

3.3 5.0 NA 0.160 0.007 -

>14 >9.5 NA >56 >1176 -

NC NC NC NC NC -

NA= Not active (concentration for pure compounds: P. falciparum 4.7 µg/mL; L. donovani 40 µg/mL). NC= not cytotoxic (up to the maximum dose tested). a Chloroquine-sensitive clone. b Chloroquine-resistant clone. c Selectivity index = IC50 Vero cells/IC50 P. falciparum.

CONCLUSION The reported work shows that aerial parts from C. langsdorffii have the potential for the development of new medicines. The phytochemical study using leaves, fruits and flowers extracts led to the isolation and identification of nine compounds including: sesquiterpene, diterpene and phenolics. The (E)-2-ethoxyvinylphenol is a novel compound from fruits extract. Compounds 2-hydroxy- ent-labda-7,13-dien-15-oic acid and ethyl 4methoxycinnamate are described for the first time. Kaurenoic acid and βcaryophyllene can be considered major compounds in the samples studied, along with flavonoid heterosides quercitrin and afzelin. Fruits extract displayed antileishmanial activity against Leishmania donovani with IC50 of 40 µg/mL. Leaves and flowers extract displayed antimalarial activity with IC50 of 3.4 and 6.2 µg/mL, respectively for different clones of Plasmodium falciparum. ACKNOWLEDGMENTS The authors are thankful to FAPESP for financial support (grant FAPESP no. 08/57775-6) and provided fellowships (FAPESP 04/13005-1; FAPESP 06/59893-0). We are also grateful to Dr. Milton Groppo, School of Sciences, Philosophy and Education-USP for plant identification. João Paulo B. Sousa is thankful to the Research team of the National Center for Natural Products Research, Ole Miss-USA, for the assistance in the Lab and good friendships. REFERENCES 1. Veiga Junior VF, Pinto AC. The Copaifera L. genus. Quim Nova 25, 2002, 273-86. 2. Braga WF, Patitucci ML, Antunes OAC, Veiga Junior VF, Bergter L, Garrido FMS, Pinto AC. Separation of acid diterpenes of Copaifera cearensis Huber ex Ducke by flash chromatography using potassium hydroxide impregnated silica gel. J. Braz. Chem. Soc. 11, 2000, 355-360.

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14. Souza AB, Martins CHG, Souza MGM, Furtado NAJC, Heleno VCG, Sousa JPB, Rocha EMP, Bastos JK, Cunha WR, Veneziani RCS, Ambrósio SR. Antimicrobial activity of terpenoids from Copaifera langsdorffii Desf. against cariogenic bacteria. Phytother. Res. 25, 2011, 215-220. 15. Parreira NA, Magalhães LG, Morais DR, Caixeta SC, Sousa JPB, Bastos JK, Cunha WR, Silva MLA, Nanayakkara NPD, Rodrigues V, Da Silva Filho AA. Antiprotozoal, schistosomicidal, and antimicrobial acitivities of the essential oil from the leaves of Baccharis dracunculifolia. Chem. Biodivers. 7, 2010, 993-1001. 16. Batista R, Humberto JL, Chiari E, Oliveira AB. Synthesis and trypanocidal activity of ent-kaurane glycosides. Bioorg. Med. Chem. 15, 2007, 381-391. 17. Xiang W, Li RT, Song QS, Na Z, Sun HD. ent-Clerodanoids from Isodon scoparius. Helvet. Chim. Acta 87, 2004, 2860-2865. 18. Jefferies PR, Knox JR, Scaf B. The chemistry of Dodonaea spp. VII* ent-Labdanes from D. microzyga (Sapindaceae). Aust. J. Chem. 27, 1974, 1097-1102. 19. Huang SH, Chen JR, Tsai FY. Palladium (II)/Cationic 2,2’-bipyridyl system as a highly efficient and reusable catalyst for the MizorokiHeck reaction in water. Molecules 15, 2010, 315-330. 20. Agrawal PK. Carbon-13 NMR of Flavonoids, 1 ed. The Netherlands, Elsevier 1989. 21. Sousa JPB, Brancalion APS, Souza AB, Turatti IC, Ambrósio SR, Furtado NAJ, Lopes NP, Bastos JK. Validation of a gas chromatographic method to quantify sesquiterpenes in copaíba oils. J. Pharm. Biomed. Anal. 54, 2011, 653-659. 22. Djerassi C, Marshall D. Optical rotator dispersion studies – XII: absolute configuration of eperuic and labdanolic acids. Tetrahedron 1, 1957, 238-240. 23. Canuto KM. Chemical Aspects of Interdisciplinary Studies (Chemistry-Pharmacology and Agronomy) of Amburana cearensis A.C. Smith. P.h.D. Thesis: Federal University of Ceará-CE, 2007, Brazil. 24. Sousa JPB, Brancalion APS, Groppo M, Bastos JK. A validated chromatographic method for the determination of flavonoids in Copaifera langsdorffii by HPLC. Nat. Prod. Commun. 7, 2012, 25-28. 25. Da Silva Filho AA, Resende DO, Fukui MJ, Santos FF, Pauletti PM, Cunha WR, Silva MLA, Gregório LE, Bastos JK, Nanayakkara NPD. In vitro antileishmanial, antiplasmodial and cytotoxic activities of phenolics and triterpenoids from Baccharis dracunculifolia D. C. (Asteraceae). Fitoterapia 80, 2009, 478-482.

Source of support: Nil, Conflict of interest: None Declared

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