Sociedade Brasileira de Espectrometria de Massas – BrMASS
NAT- produtos Naturais e Sinteticos
Extration, purification and isolation of epiisopiloturine alkaloid
from leaves of jaborandi (Pilocarpus microphyllus)
Leiz Maria Costa Veras Miura1, Maria Adelaide Guimarães1*, Yuri Dias
Macedo Campelo1, Marianne Morais Vieira1, David Fernandes Lima2, Paulo
Robério Pinho2, Luciene Costa Vasconcelos2, José Roberto de Souza Almeida
Leite1
1Núcleo de Pesquisa em Biodiversidade e Biotecnologia, Universidade
Federal do Piauí, UFPI, Parnaíba, PI, Brasil;
2VEGEFLORA Extrações do Nordeste LTDA, Parnaíba, PI, Brasil.
*[email protected]
In last fifty years thousands of alkaloids were isolated, and actually, because from
advances of evaluation techniques to pharmacologic and toxicologic activities, new
applications to centenarians alkaloids were discovered, as like pilocarpine, it was
isolated in 1875 and used to glaucoma treatment, actually, it can be to xerostomy too.
Many alkaloids were isolated from Pilocarpus genus, but major of them are in assays
phase or they have unknown pharmacologic activity [1]. From of leaves of jaborandi
(Pilocarpus microphyllus) was found a imidazolic alkaloid called epiisopiloturine, this
alkaloid has schistosomal and leishmanicid activities [2]. The leaves of jaborandi were
alkalized with potassium carbonate at 5%, they extracted with toluene and methylene
chlorite, after one of part was filtrated and solid residue went to purification through
HPLC and another part was nitrated and submitted to HPLC. The way nitration that has
done
50 mg of epiisopiloturine base (EB) was dissolved under agitation with
10 ml of absolute ethanol P.A. and then added nitric acid P.A. until pH = 4. The
mixture (EB + ethanol + nitric acid) was evaporated until 2 mL and cooling between 0
– 5 ºC. The mixture was centrifuged and the epiisopiloturine nitrate was precipitated in
the tube. In high performance liquid chromatography were use (Shimadzu Prominence)
reverse phase column (LiChrospher 60, 5 µm i.d. – Merck), sodium phosphate at 5% in
isocratic gradient, 216 nm at detector and oven at 50ºC. The confirmation has done
through external standards. Mass spectrometry (Amazon SL, Bruker) was used to
confirm the weight molecular. Mass spectra were acquired in a mass range of m/z 220
to 400 Da. MS2 was carried out in manual mode with fragmentation of the precursor
ion by CID (collision-induced dissociation) using He (helium) as the collision gas.
Precursor ions were selected within an isolation width of 2 u and scans were
accumulated with variable RF signal amplitudes. The sample was diluted using
Water/ACN/Ac. Formic (8:200:2 µL). The chromatogram showed that epiisopiloturine
nitrate was isolated and purified with success. The mass spectrum showed that
epiisopiloturine nitrate was pure, there is not another substance and the nitration was
confirmed 347 Da. The process presented has success to extraction, isolation and
purification to epiisopiloturine alkaloid.
[1]Santos, A. P.; Moreno, P. R. H. Revista Brasileira de Ciências Farmacêuticas Brazilian Journal
of Pharmaceutical Sciences. 2004, 40, 2.
[2] Leite, J.R.S.A.; Miura, L.M.C.V.; Lima, D.F.; Carneiro, S.M.P.; Carvalho, F.A.A.; Moraes, J.;
Batista, M.C.S. Patente PI 0904110-9 A2 Revista de Propriedade Industrial, Instituto Nacional
de Prodiedade Industrial. 2011, 2108, 132.
4º Congresso BrMass – 10 a 13 de Dezembro de 2011