Pyrrolizidine Alkaloids from Heliotropium indicum

Um novo alcalóide denominado Helindicina (1), e o conhecido licopsamina (2) foram isolados das raízes de Heliotropium indicum (Boraginaceae). As estruturas foram estabelecidas tendo como base um amplo estudo de 1D e 2D RMN (COSY, HMBC, HMQC, NOESY) e HREIMS. Este representa o primeiro relato de um alcalóide contendo um anel lacônico no gênero Heliotropium. Estes alcalóides mostraram moderada atividade antioxidante.


Introduction
5][6] The genus Heliotropium, a well-known source of such alkaloids [7][8][9] and other minor compounds, such as flavonoids and geranyl aromatic derivatives, is constituted of about 250 species represented by herbs and shrubs, distributed throughout the terrestrial globe. 10n the course of a search for novel and biologically active compounds from plants the EtOH extract from the roots of Heliotropium indicum L., popularly known as fedegoso, found abundantly in the region of northeast Brazil, was investigated.This species is widely used in folk medicine in the treatment of skin disease, and as a powerful expectorant. 11Based on a literature survey, the investigated species has been the subject of several previous studies, leading to the isolation of pyrrolizidine alkaloids. 3,12This paper describes the isolation and structure elucidation of the new pyrrolizidine alkaloid 1, and the known alkaloid lycopsamine (2).
It is worth noting that helindicine ( 1) is an unusual 9-membered lactone pyrrolizidine alkaloid, which is formed by an ether linkage between C-3' of the viridiflorate ester side chain and the C-7 of the retronecine moiety.Moreover, this is the first example  of a lactone pyrrolizidine alkaloid isolated from the genus Heliotropium.
The more polar isolated alkaloid was identified as lycopsamine (2), by comparison of its physical and spectral properties with literature data. 7,15he results of an extensive application of 1D ( 1 H NMR, proton broad-band decoupled and DEPT- 13 C NMR) and 2D ( 1 H-1 H-COSY, 1 H-1 H-NOESY, HMQC and HMBC) spectral techniques were also used to establish the complete 1 H and 13 C resonance assignments of these two pyrrolizidine alkaloids (1 and 2).
Structural examination of the these compounds in view of using biosynthetic arguments and application of a biosynthetic retroanalysis led us to suggest biogenetic route and, consequently, the alkaloid lycopsamine (2) may be postulated as precursor of helindicine (1) through a dehydration reaction.The antioxidant activities of 1 and 2, together with standard radical scavenging trolox and BHT were tested and compared (Table 2).As can be seen, 1 and 2 showed moderate free radical scavenging ability.The higher level of lycopsamine (2) as compared to helindicine (1) is probably due to the free hydroxyl group.
Alkaloids 1 and 2 were also tested for binding to estrogen receptor α and β, and alkaline phosphatase induction in Ishikawa cells.Neither alkaloid showed activity (>50%) in these assays.

General
The optical rotations were measured on a Perkin-Elmer 341 digital polarimeter.IR spectra were recorded on a Perkin-Elmer 1000 FT-IR spectrometer.Mass spectra data (EI-MS and HR-EIMS) were acquired on a Shimadzu QP5050A and a JEOL CGMate II instrument, through direct probe and operating at 70 eV.NMR

Plant material
The roots of H. indicum were harvested from Pentecoste County, State of Ceará -Brazil, in January 2003.The plant material was authenticated by Professor Edson P. Nunes, and a voucher specimen (# 31.610) has been deposited at the Herbário Prisco Bezerra (EAC) of the Departamento de Biologia, Universidade Federal do Ceará.

DPPH assay
The antioxidant activities of compounds 1 and 2 were assessed on the basis of the radical scavenging effect of the stable DPPH free radical.One milliliter of a 60 μmol L -1 DPPH ethanol solution was added to sample solutions at three different concentrations and allowed to react at room temperature.After 30 min, the absorbance values were measured at 520 nm using a spectrophotometer and converted into the percentage antioxidant activity using a known procedure. 17,18Blank experiment were also carried out to determine the absorbance of DPPH before interacting with the compounds.Trolox and BHT were used as positive control compounds.

Figure 1 .
Figure 1.Fragments proposed to justify peaks observed in the mass spectrum of 1.

Table 1 .
13 (400 MHz) and13C (100 MHz) NMR spectral data for helindicine(1)and lycopsamine (2) in CD 3 OD as solvent All assignments were based on DEPT, COSY, HMQC, HMBC and NOESY experiments.Coupling constants (J) in Hz for hydrogen atoms were obtained of the 1D 1 H NMR spectra.Superimposed 1 H signals are described without multiplicity and chemical shifts deduced by HMQC, HMBC and 1 H-1 H-COSY spectra. a