bioprospec*onforthesustainableuseofwetlands: acase!study ... presentation...
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Bioprospec*on 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
Interna@onal Wetlands Conference June 3-‐8, 2012 Orlando, Fl -‐ USA
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! The largest tropical wetland;
! Rich biological and cultural diversity; 865 birds species; 263 fish species;
1860 angiosperms species; etc…
Cultural contribu@on from Amerindian,
African and European popula@on
! THE PANTANAL
! HOWEVER: Economic development in highlands – significant impacts; Urgent ac@on to mi@gate.
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! PANTANAL
A rich tradi@on in the use of Medicinal Plants; e.g. Echinodorus macrophylus
Its leaves are used as Infusion for trea@ng:
Skin and venereal diseases, arthri@s, rheuma@sm, as a diure@c,
blood cleanser, an@-‐inflammatory, an@hypertensive, liver disease. BRANDÃO et al., 2009
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! Folk use:
Diure@c, an@-‐inflammatory, an@-‐rheuma@c, skin care, among
others;
! Chemistry: flavonoids, acids and terpenoids. GARCIA, 2010; SCHNITZLER et al., 2007; SHIGEMORI et al., 2002; COSTA, 2006; BEVILAQUA, 2001
www.discoverlife.org
! Echinodorus Genus ! Widespread in the Americas;
! Largest Alismataceae genus;
! 45 species; ! 17 in Brazil.
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! Echinodorus macrophyllus
! Difficult iden@fica@on;
! 70 cm long peciole ;
! Aqua@c plant; ! Grows in riverbanks and marshy
lowlands.
MATIAS, 2010; LORENZI & MATOS, 2002; Leite, 2007
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! Distributed in wetlands:
Caa@nga
Cerrado
Mata Atlân@ca
! Regions: North: RR
Northest: PI, BA
Midwest : MT, MS
Southeast: RJ, SP, MG
South: PR
MATIAS, 2010; LORENZI & MATOS, 2002
! Echinodorus macrophyllus ! Present in South America, mainly
in Brazil.
! Known as: Chapéu de couro, chá mineiro, erva de pântano and erva de brejo .
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1950 1960 1970 1980 1990 2000 2010 2011
Year
Publ
icat
ions
Key word: Echinodorus
Main constituents: Terpens (diterpenes) and flavonoids
Chemical-pharmacological studies: E. grandiflorus e macrophyllus;
43
11
! Echinodorus-‐ Publica@ons
SciFinder
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E. Grandiflorus -‐ Literature background
5 Flavones
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Echinodorus macrophyllus -‐ Literature Background
TANUS-RANGEL et al., 2010; COSENZA et al., 2010; Prabhakar et al., 1981 9
Flavones
Fenolic acid
4
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Echinodorus macrophyllus -‐ Literature Backgroud
KOBAYAHSI et al, 2000 10
6 Nitrogen-‐containing Clerodane Diterpenoids
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Echinodorus macrophyllus -‐ Literature Background
SHIGEMORI . et al., 2010; KOBAYASHI. et al, 2000 11
3 Labdane-‐like diterpenoids
2 Cembrane-‐like diterpenoids
Δ
Chapecoderins
Echinolides
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Echinodorus macrophyllus -‐ Literature Background
UFMT
2010 ! EtOH-H2O (7:3) extract caused
inhibition in rat paw edema (anti-
inflammatory activity);
O
OH
OOH
HO CH2OH
OHOH
HO
H
O
OH
OOHOH
HOH2C
HOHO
HO
H
TANUS-RANGEL et al., 2010 12
Isovitexin
Vitexin
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Objec*ves
General
• To develop an HPLC-‐based method for quality control of possible
an@-‐inflammatory phytomedicines produced from E. macrophyllus
leaves;
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Objec*ves
Specific
• To find suitable poten@al chemical markers from the EtOH-‐H2O (7:3)
extracts of E. macrophyllus leaves;
• To carry out quan@ta@ve HPLC-‐DAAD analyses of the poten@al chemical markers in order to study their spa@al varia@on;
• To verify a possible correla@on between the poten@al chemical
markers concentra@on and the an@-‐inflammatory ac@vity.
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Choice of Chemical Markers
Flavonoids: • Commercially available; • Chemically stable; • Present in rela@vely high concentra@on in the plant; • Easily detectable by ultraviolet detectors; • Described in the literature as an@-‐inflammatory;
Flavonoids x Diterpenoids
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! Experimental: Collec*on sites
Code Locality Coordenates Voucher 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 -‐-‐ -‐-‐
L7 Campo Grande -‐MS 20°30'25,79"S; 54°34'24,80”W 33665
L8 Campo Grande -‐MS 20°30'25,79"S; 54°34'24,87”W 33665
L9 Campo Grande -‐MS 20°28'41,28"S; 54°34'00,06“W 33665
L10 Campo Grande -‐MS 20°29'38,84"S; 54°35’02,52”W 33665
L11 Campo Grande -‐MS 20°30’04,02"S; 54°36’05,52”W 33665
Table 1 – Sites of collec*on of E. macrophyllus
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Equipment Varian Pro Star 5.5
Pump quaternary model 240
injector automa@c 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)
Stabiliza@on @me 2 min
Mobile phase flow 1 µL min-‐1
Injec@on volume 4 μL
Wavelengh 270 nm
Internal standard Catechin
Table 2 – Experimental HPLC-‐DAAD condi*ons
! Experimental: HPLC Method Development
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18
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
Table 3: Elu@on gradient
! Experimental: Method op*miza*on
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! Results and discussion Method op@miza@on
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.
Resolu@on:
Vit/Vitexin-‐2-‐O-‐rhamnoside: 0.62
Vitexin/Isovitexin: 1.16
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! Results and discussion Iden@fica@on 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)
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! Results and discussion Iden@fica@on 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).
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! Results and discussion Iden@fica@on 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].
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Ra@o
of the
st. areas
Concentra@on
Figure 8: Vitexin-‐2-‐O-‐rhaminoside calibra@on curve
y = 0.1025x + 0.0456 R2= 0.99934 0.5-‐8.0 µG.mL-‐1
y = 0.877x + 0.1651 R2 = 0.9999 8.0-‐20.0 µG.mL-‐1
! Results and discussion: Calibra@on curves
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0,0
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of the
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Concentra@on
Figure 9: Vitexin calibra@on curve
! Results and discussion: Calibra@on curves
y = 0.2242x + 0.0136 R² = 0.9993 0.5-8.0 µG.mL-1
y = 0.1562x + 0.574 R² = 0.9999
8.0-20.0 µG.mL-1
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0,0
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of the
st. areas
Concentra@on
Figure 10: Isovitexin calibra@on curve
! Results and discussion: Calibra@on curves
y = 0.2512x + 0.0072 R² = 0.9995
0.5-8.0 µG.mL-1
y = 0.2073x + 0.3473
R² = 0.9996 8.0-20.0 µG.mL-1
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! Results and discussion Quan@fica@on of vitexin, vitexin-‐2-‐O-‐rhamnoside e isovitexin.
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
Table 5:
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Conclusions • The chromatographic profile of samples collected at 11 different
sites showed large differences in the concentra@ons of the chemical markers, except for vitexin, which was detected in only 1 site;
• Isovitexin was the only flavonoid found in all sites, presen@ng higher
concentra@ons in sites 8-‐10 (Pantanal from MS);
• Vitexin-‐2-‐O-‐rhaminoside at higher concentra@ons 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 concentra@on x ac@vity correla@on; therefore, the flavonoids seem not to be have been a good choice as chemical markers.
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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. Tradi@onal uses of American plant species from the 1st
edi@on 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 characteriza@on of five Brazilian species of Echinodorus (Alismatales) with
chromosomal banding and 45S rDNA FISH. Plant Systema*cs and Evolu*on, 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.
An@edematogenic ac@vity and phytochemical composi@on of prepara@ons 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:
h�p://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 Inves@ga@ons on vitexin. Planta Med. 1981, 43, 396-‐403.
SCHNITZLER, M.; PETEREIT, F.; NAHRSTEDT, A. Trans-‐Aconi@c 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,
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