Isoquinolines from the Roots of Thalictrum flavum L. and Their Evaluation as Antiparasitic Compounds

Alkaloids from Thalictrum flavum L. (Ranuculaceae) growing in the Loire valley (France) were isolated and evaluated for their antiplasmodial and leishmanicidal activities. Berberine was identified as a major component but its analogue, pseudoberberine, was isolated for the first time from this plant. As far as bisbenzylisoquinolines are concerned, thalfoetidine was also isolated and, besides, its nor- derivative, northalfoetidine, was identified as a new compound. Previously isolated alkaloids from Thalictrum species such as northalidasine, northalrugosidine, thaligosidine, thalicberine, thaliglucinone, preocoteine, O-methylcassythine and armepavine were newly described in the roots of T. flavum. Tertiary isoquinolines, and particularly bisbenzylisoquinolines, were found to be leishmanicidal against L. major. Thalfoetidine appeared as the most potent but its new nor- derivative northalfoetidine, as well as northalidasine, were of particular interest due to the fact that their potential leishmanicidal activity was not associated to a strong cytotoxicity.


Introduction
Widely distributed all over the world, Thalictrum species (Ranunculaceae) mainly grow in the temperate and cold zones of both hemispheres. Among secondary metabolites of these spp., isoquinoline alkaloids were the more investigated [1]. A variety of bisbenzylisoquinolines were isolated from roots and aerial parts of diverse Thalictrum [2]. These natural products are structurally and pharmacologically interesting since they exhibit specific antiparasitic properties [3], such as antimalarial [4,5] or antileishmanial activities [6][7][8]. Thalictrum flavum L., commonly known as "meadow-rue", is a 0.5 to 1.5 m height herb growing in tallgrass meadows, ditches, marshes and other habitats at the water's edge [9]. In spite of the fact that the alkaloid contents of T. flavum L. originating from Balkan countries [10][11][12] or Russia [13,14] were already investigated, no pharmacological study has been reported so far.
Tropical parasitic diseases affect hundreds of millions of people, mostly in the Third World. As resistance to conventional treatments emerges, new drugs are still necessary in the treatment of protozoan parasitic diseases, such as malaria or leishmaniasis [15]. Natural products tend inherently to interact with living organisms and so are still a good source for compound leads [16]. In the present study, we describe the alkaloids of T. flavum L., together with their antiparasitic potential.

Results and Discussion
While complex and pharmacologically active dimeric alkaloids such as bisbenzylisoquinolines were already isolated from T. flavum L., structurally simpler isoquinolines were also extracted from this species [17]. The dimeric bisbenzylisoquinolines thalidasine, hernandesine and thalfoetidine were previously extracted from the roots as well from the aerial parts of this plant, whereas thalicarpine was only found in the roots and O-methylthalicberine only in the aerial parts. Glaucine and thalicsimidine (aporphines), thalicsine and thaliglucine (phenanthrenes), cryptopine (protopine) and thalflavine (isoquinolone) were also purified from the roots of T. flavum [11,13,14]. The oxoaporphines glaucine and corunnine were finally identified in the aerial parts of the plant [10,12]. Isoquinolines are structurally and pharmacologically interesting, since they exhibit specific antiparasitic properties [3] such as antimalarial [4,5] or antileishmanial activities [6][7][8]. However, no antiparasitic study for alkaloids from T. flavum L. was previously reported.
In this work, as expected from a literature survey, all secondary metabolites isolated from T. flavum L. roots belong to the same isoquinoline group ( Figure 1). As already described elsewhere, berberine (11) was identified as a major component [11,13]. However its analogue, pseudoberberine (12), was isolated for the first time from this plant. As far as bisbenzylisoquinolines are concerned, thalfoetidine (4) was also isolated in large amount from the roots [11] and, besides, its nor-derivative, northalfoetidine (5), was identified as a new compound. Previously isolated alkaloids from other Thalictrum species such as northalidasine (2), northalrugosidine (3), thaligosidine (6) and thalicberine (8) were characterized in the roots of T. flavum L. for the first time. It may be noticed that thalidasine was already isolated from underground parts [11] whereas O-methylthalicberine was described in aerial parts [10]. When phenanthrene derivatives were previously identified in the plant [11,13], thaliglucinone (10) was here identified for the first time. Finally, the aporphines preocoteine (1) and Omethylcassythine (7), as well as the benzylisoquinoline armepavine (8), were newly described in this Thalictrum species.
Alkaloids isolated from T. flavum L. (Figure 1) were evaluated in assays evaluating their antiparasitic potential as well as their cytotoxicity. As depicted in Table 1, tertiary isoquinolines from T. flavum L. roots, and particularly bisbenzylisoquinolines, were found to be leishmanicidal against L. major. Thalfoetidine (4) appeared as the most potent but its new nor-derivative northalfoetidine (5), as well as northalidasine (2), were of particular interest due to the fact that their potential leishmanicidal activity was not associated to a strong cytotoxicity. As far as antiplasmodial activity was concerned, the aporphine preocoteine (1) and the bisbenzylisoquinoline thaligosidine (6) were more active but ten times less potent than the reference chloroquine. As expected, quaternary protoberberines 11 and 12 appeared as antiparasitic but cytotoxic [18].

Plant material
The roots of Thalictrum flavum L. were collected in May 2005 during flowering stage along the Loire river, near Angers (France). A voucher specimen was deposited in the Musée botanique d'Angers.

Extraction and isolation
Alkaloids of the air-dried and powdered roots of Thalictrum flavum L. were extracted following a classical protocol. Briefly, the plant material (300 g) was successively extracted with CH 2 Cl 2 /MeOH (1:1, 3 L), MeOH (3 L) and water (3 L). Organic layers were combined and evaporated under reduced pressure to yield 23.7 g of crude extract. This extract was solubilised in an acidic aqueous phase (HCl 0.1 N) then successively washed with cyclohexane and CH 2 Cl 2 . The aqueous phase was subsequently alkalinised using NH 4 OH, then extracted with CHCl 3 to give 1.63 g of tertiary alkaloids.

Evaluations of cytotoxicity towards MCR-5 and KB cells
Cytotoxicities were evaluated at the ICSN-CNRS (Gif sur Yvette, France) on MRC5 and KB cells, in DMSO, at 10 and 1 µg/mL according to the procedure described by Moret et al. [33].

In vitro P. falciparum culture and activity assays
Antimalarial activity was evaluated on FcB1 Colombia strain of Plasmodium falciparum according to a previously described procedure [34].

Conclusion
In summary, twelve isoquinolines were isolated from the roots of Thalictrum flavum L. As expected, berberine 11 was identified as the major component but its analogue, pseudoberberine 12, was isolated for the first time from this plant. Six bisbenzylisoquinolines (2-6 and 8) were identified: As previously described, thalfoetidine 4 was isolated in large amount but, more interesting, its nor-derivative, northalfoetidine 5, was identified as a new compound. Moreover, bisbenzylisoquinoline, aporphine, phenanthrene and benzylisoquinoline alkaloids, previously isolated from Thalictrum species, were newly described in the roots of T. flavum. This chemical composition is slightly different from those previously described. This could be due to either seasonal variations or a difference in the soil type. Indeed, previous studies were on T. flavum from Balkan countries or Russia, while we worked on a sample originating from the Loire valley. As far as antiparasitic activity was concerned, tertiary isoquinolines, especially bisbenzylisoquinolines, were found to be leishmanicidal against L. major. Thalfoetidine 4 appeared as the most potent but its nor-derivative northalfoetidine 5, as well as northalidasine 2, were of particular interest as their potential leishmanicidal activity was not associated to a strong cytotoxicity.