Bioprospecon for the Sustainable Use of Wetlands: A Case Study from the Pantanal

Claudia L. Strada, ELiana F.C. Dores, E.L. Dall’Oglio, V.C.Silva and Paulo Teixeira de Sousa Jr*

9th INTECOL Internaonal Wetlands Conference June 3-8, 2012 Orlando, Fl - USA

THE PANTANAL The largest tropical wetland; Rich biological and cultural diversity; 865 birds species; 263 fish species; 1860 angiosperms species; etc… Cultural contribuon from Amerindian, African and European populaon

HOWEVER: Economic development in highlands – significant impacts; Urgent acon to migate. PANTANAL

A rich tradion in the use of Medicinal ;

e.g. macrophylus

Its leaves are used as Infusion for treang:

Skin and venereal diseases, arthris, rheumasm, as a diurec, blood cleanser, an-inflammatory, anhypertensive, liver disease.

BRANDÃO et al., 2009 Echinodorus Genus

Widespread in the Americas; Largest genus; 45 species; 17 in Brazil.

www.discoverlife.org Folk use:

Diurec, an-inflammatory, an-rheumac, skin care, among others; Chemistry: flavonoids, acids and terpenoids.

GARCIA, 2010; SCHNITZLER et al., 2007; SHIGEMORI et al., 2002; COSTA, 2006; BEVILAQUA, 2001 Echinodorus macrophyllus

Aquac ; Grows in riverbanks and marshy lowlands.

Difficult idenficaon; 70 cm long peciole ;

MATIAS, 2010; LORENZI & MATOS, 2002; Leite, 2007 Echinodorus macrophyllus Present in South America, mainly Distributed in wetlands: in Brazil. Caanga

Known as: Chapéu de couro, Cerrado chá mineiro, erva de pântano and Mata Atlânca erva de brejo . Regions: North: RR Northest: PI, BA Midwest : MT, MS

MATIAS, 2010; LORENZI & MATOS, 2002 Southeast: RJ, SP, MG South: PR Echinodorus- Publicaons

Chemical-pharmacological studies: E. grandiflorus e macrophyllus;

Main constituents: Terpens (diterpenes) and flavonoids

Key word: Echinodorus 50 43 45 40 35 30 25 20

Publications Publications 11 15 10 5 0 1950 1960 1970 1980 1990 2000 2010 2011 Year

SciFinder E. Grandiflorus - Literature background

5 Flavones Echinodorus macrophyllus - Literature Background 4 Flavones

Fenolic acid

9 TANUS-RANGEL et al., 2010; COSENZA et al., 2010; Prabhakar et al., 1981 Echinodorus macrophyllus - Literature Backgroud

6 Nitrogen-containing Clerodane Diterpenoids

10 KOBAYAHSI et al, 2000 Echinodorus macrophyllus - Literature Background

2 Cembrane-like diterpenoids Echinolides

Δ 3 Labdane-like diterpenoids Chapecoderins

11 SHIGEMORI . et al., 2010; KOBAYASHI. et al, 2000 Echinodorus macrophyllus - Literature Background

UFMT EtOH-H2O (7:3) extract caused 2010 inhibition in rat paw edema (anti- inflammatory activity); H OH

HO O

HOH2C

HO HO OH OH O OHOH

H CH OH Isovitexin HO 2 OH

HO O

OH O Vitexin 12 TANUS-RANGEL et al., 2010 Objecves

General • To develop an HPLC-based method for quality control of possible an-inflammatory phytomedicines produced from E. macrophyllus leaves; Objecves

Specific

• To find suitable potenal chemical markers from the EtOH-H2O (7:3) extracts of E. macrophyllus leaves; • To carry out quantave HPLC-DAAD analyses of the potenal chemical markers in order to study their spaal variaon; • To verify a possible correlaon between the potenal chemical markers concentraon and the an-inflammatory acvity.

Choice of Chemical Markers Flavonoids x Diterpenoids Flavonoids:

• Commercially available; • Chemically stable; • Present in relavely high concentraon in the plant; • Easily detectable by ultraviolet detectors; • Described in the literature as an-inflammatory; Experimental: Collecon sites Table 1 – Sites of collecon of E. macrophyllus Voucher Code Locality Coordenates specimens L1 Juína-MT 15°56’02,37”S; 56°36’08,97”W 31645 L2 Poconé-MT 16º31'57,40”S; 56º43'53,00“W 33635 L3 Chapada dos Guimarães-MT 15°36'02,30“S; 56º03'44,00“W 33637 L4 Dom Aquino-MT 15°48'20,40“S; 54º55'00,20“W 33638 L5 Cuiabá-MT 15°42'03,80“S; 55º53'11,20”W 33639 L6 Market place-Chapada dos Guimarães -- -- 20°30'25,79"S; 54°34'24,80”W L7 Campo Grande -MS 33665

20°30'25,79"S; 54°34'24,87”W L8 Campo Grande -MS 33665

20°28'41,28"S; 54°34'00,06“W L9 Campo Grande -MS 33665

20°29'38,84"S; 54°35’02,52”W L10 Campo Grande -MS 33665

20°30’04,02"S; 54°36’05,52”W L11 Campo Grande -MS 33665

Experimental: HPLC Method Development Table 2 – Experimental HPLC-DAAD condions

Equipment Varian Pro Star 5.5 Pump quaternary model 240 injector automac model 410 detector absorbance UV-DAD model 330 e

Estacionary phase C18 (250 x 4.6 mm D.I., 5 μm; HiChrom)

Mobile phase CH3OH; 0.05% aq. TFA and CH3CN (gradient) Stabilizaon me 2 min

Mobile phase flow 1 µL min-1 Injecon volume 4 μL

Wavelengh 270 nm

Internal standard Catechin Experimental: Method opmizaon

Table 3: Eluon gradient

Time (min) %MeCN %Aq. TFA ( 0,05%) %MeOH 0 10 75 15 1 10 75 15 20 15 65 20 25 20 55 25 30 10 75 15

18 Results and discussion Method opmizaon Resoluon: Vit/Vitexin-2-O-rhamnoside: 0.62 Vitexin/Isovitexin: 1.16

Figure 1: Chromatogram – Standards Catechin (IS), Vitexin-2-O-rhamnoside, vitexin and isovitexin .

Figure 2: UV from IS (catechin). Figure 3: UV from Vitexin-2-O-rhamnoside, vitexin and isovitexin. Results and discussion Idenficaon of Vitexin in EmE

Figure 4: 5a) chromatogram of EmE (from site 1 – L1); 5b) chromatogram of EmE (L1) co-injected with vitexin [0.8 µg.mL-1) Results and discussion Idenficaon of Vitexin-2-O-rhamnoside

Figure 5: 6a) chromatogram of EmE (from site 6 - L6); 6b) chromatogram of EmE (L6) co-injected with vitexin-2-O-rhaminoside [0.8 µg.mL-1). Results and discussion Idenficaon of Isovitexin

Figure 6: 7a) chromatogram of EmE (from site 4- L4); 7b) chromatogram of EmE (L4) co-injected with isovitexin [0.8 µg.mL-1]. Results and discussion: Calibraon curves

Figure 8: Vitexin-2-O-rhaminoside calibraon curve

1 2,5

0,9 y = 0.1025x + 0.0456

0,8 2 2

areas R = 0.99934

0,7 -1 st. 0,6 1,5 0.5-8.0 µG.mL 0,5 the

0,4 1 of

0,3

0,2 0,5

0,1 Rao 0 0 0,00 5,00 10,00 15,00 20,00 25,00 Concentraon y = 0.877x + 0.1651 R2 = 0.9999 8.0-20.0 µG.mL-1 Results and discussion: Calibraon curves Figure 9: Vitexin calibraon curve

2,0 4,0

1,8 3,5 y = 0.2242x + 0.0136 1,6 3,0 R² = 0.9993 areas

1,4 2,5 -1

st. 1,2 0.5-8.0 µG.mL 1,0 2,0

the 0,8 1,5 of 0,6 1,0 y = 0.1562x + 0.574 0,4 0,5 Rao 0,2 R² = 0.9999

0,0 0,0 -1 0 5 10 15 20 25 8.0-20.0 µG.mL Concentraon Results and discussion: Calibraon curves

Figure 10: Isovitexin calibraon curve

2,5 5,0 y = 0.2512x + 0.0072 4,5

2 4,0 R² = 0.9995

areas 3,5 -1 0.5-8.0 µG.mL

st. 1,5 3,0

2,5 the 1 2,0 of

y = 0.2073x + 0.3473 1,5 0,5 1,0 R² = 0.9996 Rao 0,5 -1 0 0,0 8.0-20.0 µG.mL 0,0 5,0 10,0 15,0 20,0 25,0 Concentraon Results and discussion Quanficaon of vitexin, vitexin-2-O-rhamnoside e isovitexin.

Table 5: Site Vitexin-2-O-rhamnoside (µg/g) Vitexin (µg/g) Isovitexin (µG/G)

1 Not detected 0.004 0.024 2 13.460 Not detected 0.720 3 Not detected Not detected 1.060 4 0.128 Not detected 0.620 5 5.430 Not detected 0.470 6 33.130 Not detected 2.440 7 0.036 Not detected 6.750 8 0.089 Not detected 14.68 9 0.028 Not detected 9.220 10 0.220 Not detected 6.990 11 0.051 Not detected 5.710 Conclusions • The chromatographic profile of samples collected at 11 different sites showed large differences in the concentraons of the chemical markers, except for vitexin, which was detected in only 1 site;

• Isovitexin was the only flavonoid found in all sites, presenng higher concentraons in sites 8-10 (Pantanal from MS);

• Vitexin-2-O-rhaminoside at higher concentraons was detected in samples from sites 2, 5 and 6 (market place), collected in the Pantanal from MT;

• To the best of our knowledge, vitexin-2-O-rhaminoside was detected for the fist me in E. macrophylus;

• Preliminary pharmacological assays did not show a concentraon x acvity correlaon; therefore, the flavonoids seem not to be have been a good choice as chemical markers.

27 Thank you! Visit us [email protected]

28 References BEVILAQUA, G. A. P.; NEDEL, J. J.; ZUANAZZI, A. Z.; C. T. Distribuição geográfica e composição química de chapéu de couro (Echinodorus spp.) No rio grande do sul. Ciência Rural. v.31, n.2, 2001. BRANDÃO, M. G. L; COSENZA, G. P; GRAEL, C.F; NETTO JÚNIOR, N.L; MONTEMOR, R. Tradional uses of American plant species from the 1st edion of Brazilian Official Pharmacopoeia. Revista Brasileira de Farmacognosia. v 19, p. 478-4, 2009. COSTA, Y. J.; FORNI-MARTINS, E. R.; VANZELA, A. L. L. Karyotype characterizaon of five Brazilian species of Echinodorus () with chromosomal banding and 45S rDNA FISH. Plant Systemacs and Evoluon, v. 257, p.119-127, 2006. GARCIA, E. F.; ASSIRIA, O. M.A; GODIN, A. M.; FERREIRA, W. C.; BASTOS, L. F. S.; COELHO, M. M.; BRAGA, F. C. Anedematogenic acvity and phytochemical composion of preparaons from Echinodorus grandiflorus leaves. Phytomedicine. v. 18, n. 1, p. 80, 2010. LEITE, J. P. V.; PIMENTA, D. S.; GOMES, R. S. D.; DANTAS-BARROS, A. M..Contribuição ao estudo farmacobotânico da Echinodorus macrophyllus (Kunth) Micheli (chapéu-de-couro) – Alismataceae. Brazilian Journal of Pharmacognosy. v. 17, n. 2, p. 242, 2007. MATIAS, L.Q. 2010. Alismataceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. Disponível em: hp://floradobrasil.jbrj.gov.br/2010/FB004264. Acessado em: 18/10/ 2011. Prabahakar M.M.; Bano H.K.; Kumar, I. Shamsi, M.A., Khan, S.Y. Pharmacological Invesgaons on vitexin. Planta Med. 1981, 43, 396-403. SCHNITZLER, M.; PETEREIT, F.; NAHRSTEDT, A. Trans-Aconic acid, glucosylfl avones and hydroxycinnamoyltartaric acids from the leaves of Echinodorus grandiflorus ssp. aureus, a Brazilian medicinal plant. Brazilian Journal of Pharmacognosy. v.17, n. 2, p. 149, 2007. SHIGEMORI, H.; SHIMAMOTO, S.; SEKIGUCHI, M.; OHSAKI, A.; KOBAYASHI, J. Echinodolides A and B, New Cembrane Diterpenoids with an Eight- Membered Lactone Ring from the Leaves of Echinodorus macrophyllus. Jounal Nature Products, v. 65, p. 82, 2002. TANUS-RANGEL,E.; SANTOS, S. R.; LIMA, J. C. S.; LOPES, L.; NOLDIN, V.; MONACHE, F. D.; CECHINEL-FILHO, V.; MARTINS, D. T. O. Topical and Systemic An-Inflammatory Effects of Echinodorus macrophyllus (Kunth) Micheli (Alismataceae). Journal of medicinal food. v. 13, n. 5, p. 1161, 2010. 29