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
! 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.
! 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
! 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.
! 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
! 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;
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11
! Echinodorus-‐ Publica@ons
SciFinder
E. Grandiflorus -‐ Literature background
5 Flavones
Echinodorus macrophyllus -‐ Literature Background
TANUS-RANGEL et al., 2010; COSENZA et al., 2010; Prabhakar et al., 1981 9
Flavones
Fenolic acid
4
Echinodorus macrophyllus -‐ Literature Backgroud
KOBAYAHSI et al, 2000 10
6 Nitrogen-‐containing Clerodane Diterpenoids
Echinodorus macrophyllus -‐ Literature Background
SHIGEMORI . et al., 2010; KOBAYASHI. et al, 2000 11
3 Labdane-‐like diterpenoids
2 Cembrane-‐like diterpenoids
Δ
Chapecoderins
Echinolides
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
Objec*ves
General
• To develop an HPLC-‐based method for quality control of possible
an@-‐inflammatory phytomedicines produced from E. macrophyllus
leaves;
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.
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
! 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
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
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
! 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
! 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)
! 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).
! 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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0,00 5,00 10,00 15,00 20,00 25,00
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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of the
st. areas
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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Ra@o
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
! 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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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
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