In vitro Metabolism of Grandisin, a Lignan with Anti-chagasic Activity

• Abbreviations
• Materials and Methods
• Supporting information
• Acknowledgements
• References

Abstract

Tetrahydrofuran lignans represent a well-known group of phenolic compounds capable of acting as antiparasitic agents. In the search for new medicines for the treatment of Chagas disease, one promising compound is grandisin which has shown significant activity on trypomastigote forms of Trypanosoma cruzi. In this work, the in vitro metabolism of grandisin was studied in the pig cecum model and by biomimetic phase I reactions, aiming at an ensuing a preclinical pharmacokinetic investigation. Although grandisin exhibited no metabolization by the pig microbiota, one putative metabolite was formed in a biomimetic model using Jacobsen catalyst. The putative metabolite was tested against T. cruzi revealing loss of activity in comparison to grandisin.

Abbreviations

CYP450: cytochrome P450

Mn(salen): Jacobsen catalyst

There are few studies on intestinal and phase I metabolites of natural products [1], [2], [3]. During the last years the “pig cecum model” was developed and further improved, showing good results for the metabolization of phenolic compounds [4], [5], [6], [7], [8], [9], [10]. For phase I metabolism, several in vitro biomimetic models were developed, while Mn(salen) appeared as an alternative to produce similar metabolites of CYP450 enzymes [11], [12] yielding a putative metabolite in a good scale [13], [14]. Previous investigations of the trypanocidal activity of tetrahydrofuran lignans showed several significant results [15], [16], [17] which were already registered as a patent [18]. Chagas disease affects more than 10 million people in the world and is concentrated in Latin America where it has an impact on the medical health system [19]. In this context, the aim of this work was to investigate the metabolism of grandisin in the pig cecum model by applying biomimetic reactions in order to improve information on its preclinical pharmacokinetic and also on the biological activity of the putative metabolite.

Under the chosen experimental conditions, no metabolization of grandisin was observed in the pig cecum model. Even though there are some studies showing fungal grandisin metabolism [20] and metabolism of lignans from flax by intestinal bacteria [21], the yield was very low and apparently the bacteria from the pig intestinal tract were not able to metabolize this lignan. The high recovery value observed in addition to the control metabolization using quercetin eliminates any possibility of technical problems. This result indicates high intestinal stability of grandisin, which is one prerequisite for good absorption in an oral administration.

Recently, the biotransformation of grandisin by rat liver microsomes showed a dehydro metabolite [22] suggesting that a possible oxidized metabolite may be formed by liver phase I reactions. To confirm this proposition, we applied a biomimetic oxidation of grandisin catalyzed by Mn(salen). The results demonstrate the presence of two products, a minor signal resulting from the oxidative cleavage of grandisin, as previously observed for caterpillarsʼ metabolism [23]. A second product was isolated by TLC and characterized as dehydro-grandisin: 3,4-dimethyl-2,5-bis(3,4,5-trimethoxyphenyl)-2,3-dihydrofuran (2) ([Fig. 1]).

The ESI-HRMS spectrum showed the [M + H]⁺ signal at m/z 431.2064, confirming the molecular formula as the same as observed for the product obtained after metabolization by liver microsomes [22]. The putative metabolite was submitted to ¹H NMR analysis, and the data was compared to grandisin. Grandisin has trans,trans,trans relative stereochemistry, so that the oxybenzylic and methynic protons are equivalent. In contrast, the putative metabolite lost this symmetry and appeared as two sets of signals with similar chemical shifts at δ _H 6.67 and δ _H 6.58 ppm for the aromatic protons H-2/H-6 and H-2′/H-6′, respectively. The same effect was observed for the –OCH₃ singlets confirming the symmetry loss and that no reaction occurred in any aromatic ring. The major difference was observed for the positions H-7/H-7′ and H-8/H-8′. The integration of the well-defined signals at δ _H 4.80 (H-7′, d, J = 8.6 Hz) and at δ _H 2.91 (H-8′, m) allowed to determine the occurrence of one proton per peak and also a deshielding effect in relation to the signals from grandisin [23]. The same deshielded effect was observed for the methyl protons at C-9 (1.84 d, J = 1.4 Hz). Finally, to confirm the stereochemistry of dehydro-grandisin, the CD spectra were recorded for the metabolite and grandisin. Both spectra showed two similar bands, one centered at about 208 (positive) and other at 243 nm (negative) for grandisin and the metabolite, proving that they have the same configuration (R,R) [24].

The final step was to define if the putative metabolite still had biological activity as observed with grandisin against the trypomastigote form of T. cruzi [2]. After isolation, dehydro-grandisin was evaluated against this parasite. The absence of activity indicates that the opposite spacial geometry of rings and methyl groups is important in the grandisin molecule to show an effect against this parasite.

Materials and Methods

Piper solmsianum C. DC. was collected in Ubatuba (São Paulo, Brazil) and identified by Dr. Elci Franklin Guimarães (Jardim Botânico do Rio de Janeiro, Brazil) where a voucher specimen (329676) is deposited. This work was carried out with the CGEN license number 005/2009. Dried and pulverized leaves were extracted as previously described, and (−)grandisin (98 %, determined by GC-MS) was obtained [25].

(−)Grandisin was assayed in the pig cecum model and the Mn(salen) oxidation procedure as reported before. The putative metabolite obtained using Mn(salen) was assayed against T. cruzi in a specific and sensitive test [26]. For a description of these methods, see Supporting Information.

3,4R-Dimethyl-2,5R-bis(3,4,5-trimethoxyphenyl)-2,3-dihydrofuran (2): white powder;[α]_D ²⁷ = − 69.1 (0.09 g/100 mL MeCN); UV (CH₃CN) λ _max 239, 270 nm; CD (CH₃CN) θ 207 + 4.8, θ 217 + 5.2, θ 241–4.4, θ 296 − 1.1, θ 318 − 1.2 mdeg (4.4 10⁻⁴ mol/L); ¹H NMR (CDCl₃, 300 MHz) δ _H 6.67 (s, H-2,6), 6.58 (s, H-2′,6′), 4.80 (d, J = 8.6, H-7′), 3.82 (s, OCH₃ at 4), 3.81(s, OCH₃ at 3,5), 3.80 (s, OCH₃ at 4′,3′,5′), 2,91 (m, H-8′), 1.84 (d, J = 1.4, H-9), 1.03 (d, J = 6.5, H-9′); ESI MS (pos. ion mode) m/z 431.2062 [M + H]⁺ (calcd. for C₂₄H₃₀O₇ 431.2064); EI MS m/z (% rel int): 430 ([M]⁺, 45), 348 (21), 347 (100), 247 (21), 235 (17), 219 (16), 204 (19), 195 (61).

Supporting information

The detailed information about grandisin assays, data for the pig cecum model, and dehydro-grandisin characterization spectra are available as Supporting Information.

Acknowledgements

The authors gratefully acknowledge FINEP, FAPESP, CAPES, and CNPq for financial support.

Conflict of Interest

Each author of this article has read this manuscript. We declare that there is no conflict of interest.

• References

• 1 Gertsch J, Tobler RT, Brun R, Sticher O, Heilmann J. Antifungal, antiprotozoal, cytotoxic and piscicidal properties of justicidin B and a new arylnaphthalide lignan from Phyllanthus piscatorum . Planta Med 2003; 69: 420-424
  Thieme ConnectPubMedSearch in Google Scholar
• 2 Emmanuel FM, Gentile D, Clive M, Eian DM. A comparative in vitro kinetic study of [14C]-eugenol and [14C]-methyleugenol activation and detoxification in human, mouse, and rat liver and lung fractions. Xenobiotica 2012; 42: 429-441
  CrossrefPubMedSearch in Google Scholar
• 3 Murray T, Kang J, Astheimer L, Price WE. Tissue distribution of lignans in rats in response to diet, dose-response, and competition with isoflavones. J Agric Food Chem 2007; 55: 4907-4912
  CrossrefPubMedSearch in Google Scholar
• 4 Engemann A, Hübner F, Rzeppa S, Humpf HU. Intestinal metabolism of two A-type procyanidins using the pig cecum model: Detailed structure elucidation of unknown catabolites with Fourier transform mass spectrometry (FTMS). J Agric Food Chem 2010; 60: 749-757
  PubMedSearch in Google Scholar
• 5 Hein EM, Rose K, vanʼt Slot G, Friedrich AW, Humpf H-U. Deconjugation and degradation of flavonol glycosides by pig cecal microbiota characterized by fluorescence in situ hybridization (FISH). J Agric Food Chem 2008; 56: 2281-2290
  CrossrefPubMedSearch in Google Scholar
• 6 Keppler K, Hein EM, Humpf HU. Metabolism of quercetin and rutin by the pig caecal microflora prepared by freeze preservation. Mol Nutr Food Res 2006; 50: 686-695
  CrossrefPubMedSearch in Google Scholar
• 7 Keppler K, Humpf HU. Metabolism of anthocyanins and their phenolic degradation products by the intestinal microflora. Bioorg Med Chem 2005; 13: 5195-5205
  CrossrefPubMedSearch in Google Scholar
• 8 Seefelder W. Fumonisine und deren Reaktionsprodukte in Lebensmitteln: Vorkommen, Bedeutung, biologische Aktivität und Metabolismus [dissertation]. Würzburg: Bayerische Julius-Maximilians-Universität; 2002
  PubMedSearch in Google Scholar
• 9 vanʼt Slot G, Humpf HU. Degradation of catechin, epigallocatechin-3-gallate (EGCG) and related compounds by the intestinal microbiota in the pig cecum-model. J Agric Food Chem 2009; 57: 8041-8048
  CrossrefPubMedSearch in Google Scholar
• 10 vanʼt Slot G, Mattern W, Rzeppa S, Grewe D, Humpf HU. Complex flavonoids in coca: synthesis and degradation by intestinal microbiota. J Agric Food Chem 2010; 58: 8879-8886
  CrossrefPubMedSearch in Google Scholar
• 11 Mac Leod TCO, Faria AL, Barros VP, Queiroz MEC, Assis MD. Primidone oxidation catalyzed by metalloporphyrins and Jacobsen catalyst. J Mol Catal A Chem 2008; 296: 54-60
  CrossrefPubMedSearch in Google Scholar
• 12 Mansuy D. Brief historical overview and recent progress on cytochromes P450: adaptation of aerobic organisms to their chemical environment and new mechanisms of prodrug bioactivation. Ann Pharm Fr 2011; 69: 62-69
  CrossrefPubMedSearch in Google Scholar
• 13 Niehues M, Barros VP, Emery FS, Dias-Baruffi M, Assis MD, Lopes NP. Biomimetic in vitro oxidation of lapachol: A model to predict and analyse the in vivo phase I metabolism of bioactive compounds. Eur J Med Chem 2012; 54: 804-812
  CrossrefPubMedSearch in Google Scholar
• 14 Pigatto MC, de Lima MDA, Galdino SL, Pitta ID, Vessecchi R, Assis MD, dos Santos JS, Costa TD, Lopes NP. Metabolism evaluation of the anticancer candidate AC04 by biomimetic oxidative model and rat liver microsomes. Eur J Med Chem 2011; 46: 4245-4251
  CrossrefPubMedSearch in Google Scholar
• 15 Lopes NP, Blumenthal EED, Cavalheiro AJ, Kato MJ, Yoshida M. Lignans, gamma-lactones and propiophenones of Virola surinamensis . Phytochemistry 1996; 43: 1089-1092
  CrossrefPubMedSearch in Google Scholar
• 16 Lopes NP, Chicaro P, Kato MJ, Albuquerque S, Yoshida M. Flavonoids and lignans from Virola surinamensis twigs and their in vitro activity against Trypanosoma cruzi . Planta Med 1998; 64: 667-669
  Thieme ConnectPubMedSearch in Google Scholar
• 17 Schmidt TJ, Khalid SA, Romanha AJ, Alves TMA, Biavatti MW, Brun R, Da Costa FB, de Castro SL, Ferreira VF, de Lacerda MVG, Lago JHG, Leon LL, Lopes NP, das Neves Amorim RC, Niehues M, Ogungbe IV, Pohlit AM, Scotti MT, Setzer WN, de NC Soeiro M, Steindel M, Tempone AG. The potential of secondary metabolites from plants as drugs/leads against protozoan neglected diseases – part II. Curr Med Chem 2012; 19: 2176-2228
  PubMedSearch in Google Scholar
• 18 Fundação de Amparo à Pesquisa do Estado de São Paulo. Acef SA, Silva MLA, Silva R, Rodrigues V, Pereira-Júnior OS, Silva-Filho AA, Donate PM, Albuquerque S, Bastos JK. Processo de obtenção de derivados sintéticos e semi-sintéticos de lignanas, suas atividades antiparasitárias e respectivas formulações farmacêuticas, englobando o método terapêutico utilizando tais lignanas no tratamento de parasitoses. BR Patent PI0503951-7; 2005.
  PubMed
• 19 WHO, Media Center. Chagas disease (American trypanosomiasis). Fact sheet N°340. Available at http://www.who.int/mediacentre/factsheets/fs340/en/index.html Accessed June 10, 2012
  PubMed
• 20 Verza M, Arakawa NS, Lopes NP, Kato MJ, Pupo MT, Said S, Carvalho I. Biotransformation of a tetrahydrofuran lignan by the endophytic fungus Phomopsis sp . J Braz Chem Soc 2009; 20: 195-200
  CrossrefPubMedSearch in Google Scholar
• 21 Côrtes C, Gagnon N, Benchaar C, da Silva D, Santos GTD, Petit HV. In vitro metabolism of flax lignans by ruminal and faecal microbiota of dairy cows. J Appl Microbiol 2008; 105: 1585-1594
  CrossrefPubMedSearch in Google Scholar
• 22 Messiano GB, Santos RAS, Ferreira LS, Simões RA, Kato MJ, Lopes NP, Pupo MT, Oliveira ARM. In vitro metabolism study of the bioactive lignan (−)-grandisin. Planta Med 2012; 78: 1139
  PubMedSearch in Google Scholar
• 23 Ramos CS, Vanin SA, Kato MJ. Metabolism of (−)-grandisin from Piper solmsianum in Coleoptera and Lepidoptera species. Phytochemistry 2008; 69: 2157-2161
  CrossrefPubMedSearch in Google Scholar
• 24 Saad JM, Soepadamo E, Fang XP, Mclaughlin JL, Fanwick PE. (−)-Grandisin from Cryptocarya crassinervia . J Nat Prod 1991; 54: 1681-1683
  CrossrefPubMedSearch in Google Scholar
• 25 Holloway D, Scheinmann F. Extractives from Litsea species. II. Two lignans from Litsea grandis and L. gracilipes . Phytochemistry 1974; 13: 1233-1236
  CrossrefPubMedSearch in Google Scholar
• 26 Martins RCC, Latorre LR, Sartorelli P, Kato MJ. Phenylpropanoids and tetrahydrofuran lignans from Piper solmsianum . Phytochemistry 2000; 55: 843-846
  CrossrefPubMedSearch in Google Scholar

Correspondence

• References

• 1 Gertsch J, Tobler RT, Brun R, Sticher O, Heilmann J. Antifungal, antiprotozoal, cytotoxic and piscicidal properties of justicidin B and a new arylnaphthalide lignan from Phyllanthus piscatorum . Planta Med 2003; 69: 420-424
  Thieme ConnectPubMedSearch in Google Scholar
• 2 Emmanuel FM, Gentile D, Clive M, Eian DM. A comparative in vitro kinetic study of [14C]-eugenol and [14C]-methyleugenol activation and detoxification in human, mouse, and rat liver and lung fractions. Xenobiotica 2012; 42: 429-441
  CrossrefPubMedSearch in Google Scholar
• 3 Murray T, Kang J, Astheimer L, Price WE. Tissue distribution of lignans in rats in response to diet, dose-response, and competition with isoflavones. J Agric Food Chem 2007; 55: 4907-4912
  CrossrefPubMedSearch in Google Scholar
• 4 Engemann A, Hübner F, Rzeppa S, Humpf HU. Intestinal metabolism of two A-type procyanidins using the pig cecum model: Detailed structure elucidation of unknown catabolites with Fourier transform mass spectrometry (FTMS). J Agric Food Chem 2010; 60: 749-757
  PubMedSearch in Google Scholar
• 5 Hein EM, Rose K, vanʼt Slot G, Friedrich AW, Humpf H-U. Deconjugation and degradation of flavonol glycosides by pig cecal microbiota characterized by fluorescence in situ hybridization (FISH). J Agric Food Chem 2008; 56: 2281-2290
  CrossrefPubMedSearch in Google Scholar
• 6 Keppler K, Hein EM, Humpf HU. Metabolism of quercetin and rutin by the pig caecal microflora prepared by freeze preservation. Mol Nutr Food Res 2006; 50: 686-695
  CrossrefPubMedSearch in Google Scholar
• 7 Keppler K, Humpf HU. Metabolism of anthocyanins and their phenolic degradation products by the intestinal microflora. Bioorg Med Chem 2005; 13: 5195-5205
  CrossrefPubMedSearch in Google Scholar
• 8 Seefelder W. Fumonisine und deren Reaktionsprodukte in Lebensmitteln: Vorkommen, Bedeutung, biologische Aktivität und Metabolismus [dissertation]. Würzburg: Bayerische Julius-Maximilians-Universität; 2002
  PubMedSearch in Google Scholar
• 9 vanʼt Slot G, Humpf HU. Degradation of catechin, epigallocatechin-3-gallate (EGCG) and related compounds by the intestinal microbiota in the pig cecum-model. J Agric Food Chem 2009; 57: 8041-8048
  CrossrefPubMedSearch in Google Scholar
• 10 vanʼt Slot G, Mattern W, Rzeppa S, Grewe D, Humpf HU. Complex flavonoids in coca: synthesis and degradation by intestinal microbiota. J Agric Food Chem 2010; 58: 8879-8886
  CrossrefPubMedSearch in Google Scholar
• 11 Mac Leod TCO, Faria AL, Barros VP, Queiroz MEC, Assis MD. Primidone oxidation catalyzed by metalloporphyrins and Jacobsen catalyst. J Mol Catal A Chem 2008; 296: 54-60
  CrossrefPubMedSearch in Google Scholar
• 12 Mansuy D. Brief historical overview and recent progress on cytochromes P450: adaptation of aerobic organisms to their chemical environment and new mechanisms of prodrug bioactivation. Ann Pharm Fr 2011; 69: 62-69
  CrossrefPubMedSearch in Google Scholar
• 13 Niehues M, Barros VP, Emery FS, Dias-Baruffi M, Assis MD, Lopes NP. Biomimetic in vitro oxidation of lapachol: A model to predict and analyse the in vivo phase I metabolism of bioactive compounds. Eur J Med Chem 2012; 54: 804-812
  CrossrefPubMedSearch in Google Scholar
• 14 Pigatto MC, de Lima MDA, Galdino SL, Pitta ID, Vessecchi R, Assis MD, dos Santos JS, Costa TD, Lopes NP. Metabolism evaluation of the anticancer candidate AC04 by biomimetic oxidative model and rat liver microsomes. Eur J Med Chem 2011; 46: 4245-4251
  CrossrefPubMedSearch in Google Scholar
• 15 Lopes NP, Blumenthal EED, Cavalheiro AJ, Kato MJ, Yoshida M. Lignans, gamma-lactones and propiophenones of Virola surinamensis . Phytochemistry 1996; 43: 1089-1092
  CrossrefPubMedSearch in Google Scholar
• 16 Lopes NP, Chicaro P, Kato MJ, Albuquerque S, Yoshida M. Flavonoids and lignans from Virola surinamensis twigs and their in vitro activity against Trypanosoma cruzi . Planta Med 1998; 64: 667-669
  Thieme ConnectPubMedSearch in Google Scholar
• 17 Schmidt TJ, Khalid SA, Romanha AJ, Alves TMA, Biavatti MW, Brun R, Da Costa FB, de Castro SL, Ferreira VF, de Lacerda MVG, Lago JHG, Leon LL, Lopes NP, das Neves Amorim RC, Niehues M, Ogungbe IV, Pohlit AM, Scotti MT, Setzer WN, de NC Soeiro M, Steindel M, Tempone AG. The potential of secondary metabolites from plants as drugs/leads against protozoan neglected diseases – part II. Curr Med Chem 2012; 19: 2176-2228
  PubMedSearch in Google Scholar
• 18 Fundação de Amparo à Pesquisa do Estado de São Paulo. Acef SA, Silva MLA, Silva R, Rodrigues V, Pereira-Júnior OS, Silva-Filho AA, Donate PM, Albuquerque S, Bastos JK. Processo de obtenção de derivados sintéticos e semi-sintéticos de lignanas, suas atividades antiparasitárias e respectivas formulações farmacêuticas, englobando o método terapêutico utilizando tais lignanas no tratamento de parasitoses. BR Patent PI0503951-7; 2005.
  PubMed
• 19 WHO, Media Center. Chagas disease (American trypanosomiasis). Fact sheet N°340. Available at http://www.who.int/mediacentre/factsheets/fs340/en/index.html Accessed June 10, 2012
  PubMed
• 20 Verza M, Arakawa NS, Lopes NP, Kato MJ, Pupo MT, Said S, Carvalho I. Biotransformation of a tetrahydrofuran lignan by the endophytic fungus Phomopsis sp . J Braz Chem Soc 2009; 20: 195-200
  CrossrefPubMedSearch in Google Scholar
• 21 Côrtes C, Gagnon N, Benchaar C, da Silva D, Santos GTD, Petit HV. In vitro metabolism of flax lignans by ruminal and faecal microbiota of dairy cows. J Appl Microbiol 2008; 105: 1585-1594
  CrossrefPubMedSearch in Google Scholar
• 22 Messiano GB, Santos RAS, Ferreira LS, Simões RA, Kato MJ, Lopes NP, Pupo MT, Oliveira ARM. In vitro metabolism study of the bioactive lignan (−)-grandisin. Planta Med 2012; 78: 1139
  PubMedSearch in Google Scholar
• 23 Ramos CS, Vanin SA, Kato MJ. Metabolism of (−)-grandisin from Piper solmsianum in Coleoptera and Lepidoptera species. Phytochemistry 2008; 69: 2157-2161
  CrossrefPubMedSearch in Google Scholar
• 24 Saad JM, Soepadamo E, Fang XP, Mclaughlin JL, Fanwick PE. (−)-Grandisin from Cryptocarya crassinervia . J Nat Prod 1991; 54: 1681-1683
  CrossrefPubMedSearch in Google Scholar
• 25 Holloway D, Scheinmann F. Extractives from Litsea species. II. Two lignans from Litsea grandis and L. gracilipes . Phytochemistry 1974; 13: 1233-1236
  CrossrefPubMedSearch in Google Scholar
• 26 Martins RCC, Latorre LR, Sartorelli P, Kato MJ. Phenylpropanoids and tetrahydrofuran lignans from Piper solmsianum . Phytochemistry 2000; 55: 843-846
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material