Divergence of Secondary Metabolism in Cell Suspension Cultures and Differentiated Plants of Piper cernuum and P . crassinervium

Os principais metabólitos secundários das folhas de Piper cernuum são derivados de ácidos cinâmico e di-hidrocinâmicos, além da lignana cubebina. No caso de P. crassinervium, há um predomínio de flavonóides, ácidos benzóicos e hidroquinonas preniladas. As culturas celulares de P. cernuum produziram as feniletilaminas dopamina e tiramina enquanto que, no caso de P. crassinervium, foram isoladas quatro alcamidas, incluindo as novas 2,3,4-trimetoxi-N-metilaristolactama e 3-hidroxi-2-metoxi-N-metil-aristolactama.


Introduction
The Piper species are mostly tropical plants of worldwide occurrence and are represented by ca.700 species. 1 Their phytochemical investigation have led to the isolation of a number of physiologically active compounds viz.][4][5][6][7][8][9][10][11][12] Piper cernuum Vell., an imposing, very large-leave shrub up to 6 m in height, is widely spread in Amazon, South-eastern and North-eastern regions of Brazil. 13Piper crassinervium is a shrub frequently found in Atlantic Forest (Brazil), besides Peru, Colombia and Equador. 13Although there is no report for medicinal use of P. cernuum and P. crassinervium their phytochemical investigation were undertaken as part of our study of Piperaceae species looking at the search for bioactive compounds, chemosystematic study and also the study of regulatory process of the secondary metabolism that takes place during the differentiation process.
The regeneration or micropropagation of Piper species have been found in literature only for few species such as P. longum, [14][15][16] , P. colubrinum, 17 and P. nigrum. 18evertheless, the establishment of cell suspension cultures and investigation of associated secondary metabolism in Piperaceae species have so far remained untouched.Previous analysis of leaves of P. crassinervium have showed the occurrence of antifungal flavanones, benzoic acids and prenylated hydroquinones. 10,19The only studies carried out on P. cernuum were addressed to investigate the composition of its essential oil 20,21 and its antimicrobial activity. 22Herein we wish to describe the occurrence of four cinnamic acid derivatives (1-4) together the lignan cubebin (5) in the leaves of P. cernuum.Additionally, its callus and suspension cultures were showed to contain phenylethylamines tyramine (6) and dopamine (7) as major compounds.In case of P. crassinervium the major compounds were identified as the aristolactams (8-11), including the new natural product 2,3,4-trimethoxy-N-methyl-aristolactam (10) and 3-hydroxy-3-methoxy-Nmethyl-aristolactam (11).
HPLC analysis and 1 H NMR of extracts from callus and cell suspension cultures (cells and media) of P. cernuun showed the presence of two major compounds that were isolated and identified as tyramine (6) 25 and dopamine (7). 3 Their content as dansyl derivatives was determined during the cell growth and increased along the development (Figure 4).Dopamine has been previously identified in Piper amalago 3 and also in suspension cultures of Mucuna pruriens. 26The analysis of the extracts by HPLC indicated that no other alkaloids was produced either in cell, excreted into the liquid media or in the differentiated tissues of the P. cernuum plants.Both phenylethylamines have been frequently found in plants as conjugates such as N-trans (-coumaroyl)-tyramine in P. sanctum, 27 as N-feruroyltyramines in P. argyrophylum, 28 and as N-benzoyltyramine in Aniba riparia. 25Tyramine amide is wound inducible phytoalexin on tobacco (Nicotiana tabacum) 29 and is directly involved in the suberization process of wounded potato (Solanum tuberosum). 30Together dopamine are important intermediates in the biosynthesis of benzylisoquinoline alkaloids. 31he extract from cell suspensions of Piper crassinervium was analyzed by 1 H NMR whose signals showed the presence of several methoxyl and aromatic hydrogens.The extract was fractionated by several chromatographic steps yielding four compounds (8-11).Compounds 8 and 9 were identified as goniopedaline C and piperolactam C, which were previously isolated from Goniothalamus sesquipedalis 32 and P. argyrophylum, P. boehimerifolium, P. longum, P. wightii besides P. acutisleginum, 33 respectively.
Compound 10 was showed to be has similar spectrometric data as compound 9, but was recognized as its N-methyl derivative since the 1 H and 13 C NMR data showed a singlet at δ 3.48 and 26.4,respectively.The molecular ion in the mass spectrum at m/z 323 Da was compatible with the molecular formula C 19 H 17 NO 4 .
All 13 C NMR data could be assigned based on the similarities with 9, thus 10 could be determined as 2,3,4trimethoxy-N-methyl-aristolactam.
Compound 11 showed a molecular ion at 279 Da.Its 1 H NMR showed a N-methyl signal at 3.49 and a methoxyl group at 4.13 and could be determined as N-methyl-3-methyl-cepharanone B by comparison of spectrometric data 31 and it has not been previously described elsewhere.The determination of two major aristolactams in the suspension cells during the growth curve showed that 9 is converted to its N-methyl derivative 10 (Figure 5).
Such type of alkaloids groups are relatively restrict in plant kingdom and has been found in Aristolochiaceae, Piperaceae, Annonaceae, Menispermaceae and Monimiaceae species. 34he production of phenylethylamines or aristolactams in cell suspension cultures of P. cernuum and P. crassinervium, respectively, is a metabolic drift that requires the predominance of tyrosine decarboxylase (TYDC) over    phenylalanine ammonia lyase (PAL).This branch point in the differentiated tissue is followed by additional steps of general phenylpropanoid pathway including the oxidation and methylations steps and/or hydrogenation yielding the cinnamic and dihydrocinnamic acid derivatives in P. cernuum.Since only one lignan (cubebin) was isolated, further reductions of cinnamoyl-SCoA esters to the cinnamyl alcohols followed by oxidative coupling to yield lignans are not fully expressed such as in other Piper species. 3Such divergence was also observed in suspension cells of P. crassinervium, but in this case, phenylethylamines are supposedly consumed in the formation of aristolactams 8-11.Apparently, the Mannich condensation is the limiting step in P. cernuum and no aristolactams could be detected in its suspension cells.The modulation of alkaloid metabolism in plant suspension culture of alkaloid-free species have been previously 35

Instrumental and chromatographic materials
Silica gel (Merck 230-400 mesh) and Sephadex LH-20 (Pharmacia) were used for column chromatographic separation.Silica gel PF254 (Merck) was used to TLC preparative purification.The NMR spectra (300 and 200 MHz) and 13 C NMR (75 and 50 MHz) were recorded on Bruker DRX-300 using CDCl 3 as solvents or otherwise stated and TMS as internal standard.IR spectra were obtained on a FT-IT 510 Nicolet spectrometer.LREIMS were measured at 70 eV on a HP 5990/5988A spectrometer.IR: KBr; EIMS 70 eV.HPLC analysis was performed on a HP-1050 using C-18 Econosil, (Alltech) (5 μm, 250 X 4.6 mm column with UV detection at 254 nm.
Isolations of secondary compounds from cells of P. cernuum 30.0 g of fresh cells were extracted with MeOH yielding 170 mg of crude extract.The extract was suspended in H 2 O and extracted with n-BuOH.The n-BuOH fraction (82.3 mg) was dissolved in MeOH and filtered over silanized silica gel to retain the apolar compounds and the yielding fraction (46 mg) was subjected to prepared TLC with CHCl 3 -MeOH (7:3) yielding 6 (23 mg) and 7 (15 mg).

Quantification of tyramine and dopamine
2.0 g of fresh cells were extracted and derivatized to dansyl derivatives according to procedure described 37 but with dihydrochloride putrescine as internal standard.The dansyl derivatives were determined by HPLC as described. 38Standard curves of tyramine and dopamine hydrochloride (Sigma) were prepared similarly.
Cell suspensions of 15-20 days of age were used for determination of growth curves.In 50 mL flasks contained the medium prepared for the cell suspensions, were inoculated 5 g of fresh weight of P. crassinervium cells.This procedure was performed in triplicate.The cells were separated from cell suspensions by vacuum filtration and had their fresh weight determined.Then cells were kept at 50 °C, until constant weight.

Isolation of aristolactams from cells of P. crassinervium
Cells (100 g of fresh cell) obtained of cell suspensions by vacuum filtration, were extracted with MeOH under sonication bath (15 min).After elimination of solvent the residue was submitted to EtOAc partition, and concentrated under vacuum to yield the cell extract (40 mg).The extract was purified by preparative TLC (hexane: EtOAc 7:3) to yield four fractions.Fraction 2 was submitted to preparative TLC (CHCl 3 :MeOH 9:1), then by HPLC using a RP-18 column eluted with a gradient MeOH:H 2 O, yielding the aristolactams 8 (1.0 mg) and 11 (1.1 mg).Fractions 3 and 4 consisted of the pure aristolactams 9 (4.1 mg) and 10 (5.3 mg).

Figure 4 .
Figure 4. Changes in the content of tyramine and dopamine (determined as dansyl derivatives) during the growth cycle of P. cernuum cell suspension.

Figure 5 .
Figure 5. Changes in the content of aristolactams 9 and 10 during the growth of cell suspension of P. crassinervium.