Oxidation Products from the Neolignan Licarin A by Biomimetic Reactions and Assessment of in vivo Acute Toxicity

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Chemicals
• Characterization of licarin A
• Biomimetic oxidation with [Fe(TPP)Cl] and Mn(Salen)
• Identification of products
• In vitro metabolism by rat liver microsomes (RLM) and human liver microsomes (HLM)
• Gas chromatography–mass spectrometry analysis (GC-MS)
• Ultra-performance liquid chromatography–tandem mass spectrometry analysis (UPLC-MS/MS)
• Sequential mass spectrometry analysis (LC-MSn)
• High-resolution mass spectrometry analysis
• In vivo oral acute toxicity
• Contributorsʼ Statement
• References

Abstract

Licarin A, a dihydrobenzofuranic neolignan presents in several medicinal plants and seeds of nutmeg, exhibits strong activity against protozoans responsible for Chagas disease and leishmaniasis. From biomimetic reactions by metalloporphyrin and Jacobsen catalysts, seven products were determined: four isomeric products yielded by epoxidation from licarin A, besides a new product yielded by a vicinal diol, a benzylic aldehyde, and an unsaturated aldehyde in the structure of the licarin A. The incubation with rat and human liver microsomes partially reproduced the biomimetic reactions by the production of the same epoxidized product of m/z 343 [M + H]⁺. In vivo acute toxicity assays of licarin A suggested liver toxicity based on biomarker enzymatic changes. However, microscopic analysis of tissues sections did not show any tissue damage as indicative of toxicity after 14 days of exposure. New metabolic pathways of the licarin A were identified after in vitro biomimetic oxidation reaction and in vitro metabolism by rat or human liver microsomes.

Introduction

Licarin A, a dihydrobenzofuran neolignan, is reported from several medicinal plants and the seeds of nutmeg (Myristica fragrans Houtt, Myristicaceae) and used as a flavoring agent in cuisine. This neolignan has been semi-synthetically produced from isoeugenol [1] and several biological properties have been described from it, such as being neuroprotective, anti-inflammatory, cytotoxic against cancer cells, and antimicrobial [1], [2], [3], [4], [5]. In addition, potent activities of licarin A against protozoans responsible for Chagas disease and leishmaniases were also reported [6], [7].

The development of a new drug requires preclinical evaluations regarding its metabolism and toxicologic evaluations for a better understanding of its action mechanisms in humans. The biotransformation of xenobiotics is a process of elimination mediated by cytochrome P450 (CYP) and other xenobiotic metabolizing phase I or phase II enzymes, which are responsible for metabolism in living organisms. The phase I metabolism consists in hydrolysis, oxidation, or reduction reactions that increase the polarity of drugs to promote their excretion, mainly by the kidney, through urine [8]. These reactions can be reproduced in vitro by biomimetic chemical models based on synthetic metalloporphyrins and metal–salen complexes, such as the Jacobsen catalyst [9].

The metalloporphyrins ([Fe(TPP)Cl]) exhibit a unit of iron (III) porphyrins; they are able to mimic the CYP enzymes and have shown similar products compared to in vivo experiments. Biomimetic oxidation systems reveal advantages in relation to other in vitro models because they minimize the use of animals for metabolism experiments, and high amounts of the oxidized products can be obtained, which improve its isolation at acceptable costs and can assist to understand the products yielded in vivo [9], [10]. Moreover, this alternative in vitro metabolism model allows comparison and identification of products generated in vivo in preclinical or clinical phases of drug development [11], [12]. The chemical structure determination of products can be assessed by the application of the mass spectrometry technique for systematic investigation based on gas-phase fragmentation reactions, such as the electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled to liquid chromatography, which is an useful tool to analyze in vitro metabolism reactions [9].

The aim of the present study was to investigate different metabolic pathways for licarin A by the biomimetic oxidative model, in comparison with the rat and human liver microsomes models, and to apply ESI-MS/MS fragmentation studies and NMR techniques to determine the oxidative products. In addition, in vivo acute toxicity was evaluated, as well as the biochemical and histological parameters.

Results and Discussion

Licarin A showed 98% chromatographic purity (Fig. 1S, Supplementary Material), its specific optical rotation revealed a value of − 5.5 (c 0.79, MeOH). According to Lee et al. [13], (+) – and (−) – licarin A presented [α]_D ²⁵ of + 52 (c 0.79) and − 52 (c 0.79 MeOH), respectively, suggesting that licarin A synthesized is a non-racemic mixture. The chemical shifts of ¹H and ¹³C NMR were compatible with data reported for licarin A [14] (Table 1S, Fig. 2S–6S, Supporting Information). The MS/MS of licarin A, obtained by high-resolution ESI-MS, revealed the fragment ions of m/z 297, 267, 203, 188, 171, 163, 151, and 137, which were yielded from its protonated ion m/z 327 [M+H]⁺ (Fig. 7S).

In order to propose the fragmentation pathways of the licarin A, computational calculations of the probable protonation site were performed based on the gas-phase basicity (GB) and proton affinity (PA) studies (Fig. 8S). GB and PA are suitable descriptors for the protonation site, since it combines the energy variations between the neutral molecule and its protonated forms and can describe the protonation in electrospray ionization mass spectrometry [15], [16]. Calculations have shown that the oxygen atom of the furan ring of licarin A is the most probable site of protonation (Fig. 9S), with the higher GB and PA values. Thereby, the fragmentation profile of protonated licarin A was proposed as represented in [Fig. 1]. To produce products from licarin A, different catalysis conditions were evaluated applying four solvents (MeOH, DCM, ACN, and EtOAc) and two oxygen donors (PhIO and mCPBA) and under the action of two catalysts ([Fe(TTP)Cl] or Mn(Salen)) in all possible combinations. The Jacobsen catalyst Mn(Salen) has an electronic structure and catalytic activities similar to metalloporphyrins, generally catalyzing epoxidation reactions [17]. The variation in oxidant concentration and reaction time was first evaluated, and the most suitable molar ratio of reagents was 1 : 30 : 30 (catalyst : substrate : oxidant), with the reactions kept under stirring for 24 h.

Initially, the reactions were monitored by GC-MS and showed higher efficiency to oxidate licarin A with Jacobsenʼs catalyst on reaction solvents of lower polarity, such as DCM and EtOAc ([Fig. 2]). The reaction employing Jacobsenʼs catalyst and PhIO in DCM showed better results for the production of products, as well as a greater number of them. These products comprised the additions of 16 and 34 u in licarin A, which are relative to the addition of one oxygen atom and two hydroxyl groups, respectively, besides the elimination of 2 u from licarin A that was also observed by the formation of a double bond (Fig. 11S, Table 2S).

The reaction promoted with Jacobsenʼs catalyst, mCPBA, and EtOAc (solvent) was also analyzed by UPLC-MS/MS and revealed the production of seven oxidation products from licarin A ([Fig. 3], [Tables 1] and 3S). Due to a greater diversity of products detected in this reaction, it was applied to determine their chemical structures. The spectral data of these products are summarized in [Table 1] and the mass spectra (MS and MS/MS) are illustrated in the Supporting Information (Fig. 12S-26S).

The protonation sites were obtained for oxidation products by the same computational methodology employed for licarin A studies. Gas-phase basicities were calculated and can be assessed in Supporting Information. Product 1 showed intense ions at m/z 361 [M+H]⁺ and 343 [M+H – H₂O]⁺ (Fig. 12S). The fragmentation of the ion m/z 361 yielded the product ions at m/z 343 and 325 ions from losses two successive water molecules ([Table 1], Fig. 10S). For product 1, the protonation takes place at hydroxyl ([Fig. 4]), which can contribute to the interpretation of the fragmentation spectra by H₂O elimination from the protonated molecule. In addition, the fragment ions m/z 219, 163, and 137 were also observed. The ion m/z 137 was also observed from licarin A ([Fig. 1]), which indicates no change in the furan ring. After the first loss of H₂O, the fragmentation of the m/z 343 ion resulted in the m/z 219 ion by the elimination of the benzene ring, corroborating with the addition of 16 u compared to the m/z 203 ion fragment of licarin A ([Fig. 1]). Therefore, data obtained for product 1 demonstrated oxidation of the double bond in the licarin A chain with the formation of a vicinal diol ([Fig. 4]).

Products 2 – 3 and 6 – 7 were yielded from the epoxidation of the double bond between the C-7′ and C-8′ positions of the licarin A, yielding diastereomers since the synthetized licarin A is an enantiomeric mixture ([Fig. 4]). The mass spectra of these products (Fig. 13S-14S, 17S-18S) exhibited ions with the addition of 16 u (m/z 343 [M+H]⁺ and 341 [M–H]⁻), suggesting the addition of one oxygen atom in licarin A. For product 2, the protonation occurs at the oxygen atom of epoxide ([Fig. 4]), and it led to the ring opening and subsequent H₂O molecule elimination. The MS/MS of these products showed similarities in their fragmentation profiles ([Table 1], Fig. 20S-21S and 25S-26S). Product 3 was isolated by HPLC and analyzed by ESI-MS/MS and NMR. The MS/MS from m/z 343 [M+H]⁺ of product 3 produced the m/z 325, 219, 163, and 137 fragments ions. As shown in its proposed fragmentation ([Fig. 5]), the m/z 325 fragment ion originated from the loss of 18 u (H₂O), while the product ion m/z 219 was yielded from the elimination of phenol. The ion m/z 137 is similar to the fragmentation of licarin A ([Fig. 1]). The high-resolution mass spectrum of product 3 exhibited an intense peak of m/z 343.1522 [M + H]⁺ in positive ionization mode, which is compatible with C₂₀H₂₃O₅ ⁺ (error of 5.2 ppm).

It is important to note that the epoxidation in the carbon-carbon double bond present in the side chain of the licarin A leads to the formation of a three-membered ring with high tension and consequently high instability [18], [19]. The peaks collected by HPLC on semipreparative scale were monitored by MS, which was possibly isolated and confirmed in products 3 and 4. However, due to the time required for sample drying and NMR analysis, it was only possible to detect signals of the degradation product from 4. For products 4 and 5, the protonation sites, determined by computational method, exhibited the carbonyl as the most basic site ([Fig. 4]).

The ¹H NMR analysis from compound 3 confirmed the absence of the carbon-carbon double bond signals between the C-7′ and C-8′ from licarin A at δ 6.38 (d) and 6.13 (dq) (Fig. 27S-34S). In addition, the correlations observed in the COSY, HMQC, and HMBC spectra, the signals of the furan ring, were determined (Fig. 27S) in its degradation product, confirming the absence of change in this part of the structure of licarin A.

According to the biomimetic reaction analysis by UPLC-MS/MS ([Fig. 3]), product 4 ([Fig. 4]) revealed the ions at m/z 315 [M+H]⁺ and 313 [M–H]⁻ ([Table 1]), and the fragment ions m/z 191, 163, and 137 (Fig. 15S, 23S). The formation of a benzoic aldehyde was confirmed by NMR, which showed signals very similar to those observed for licarin A, mainly the signals of the aromatic ring containing the hydroxyl group (Table 4S, Fig. 35S-39S). The hydrogens observed in δ 5.22 (d, J = 9.3 Hz), 3.53 (m), and 1.42 (d, J = 6.8 Hz) are attributed to hydrogen H-7, H-8, and H-9 of the furan ring, suggesting no change in this part of the licarin A chemical structure. The chemical shifts of ¹H and ¹³C of the benzofuran aromatic ring were changed (Fig. 35S), as well as the C-7′ and C-8′ double bond of licarin A, since these signals were no longer observed, along with H-9′. Taken together with the chemical shift of the carbonyl group, we can suggest a conjugated aldehyde, which is bound directly to the benzofuran aromatic ring. This proposal was confirmed by the correlations observed in the HMBC spectrum (Fig. 38S-39S). All the chemical shifts and correlations from product 4 are listed in Table 4S. In addition, the high resolution MS analyses (Fig. 40S-42S) exhibited a more intense peak of m/z 315.1232 [M+H]⁺ in positive ionization mode, which is compatible with the molecular formula C₁₈H₁₉O₅ ⁺(error of 1.6 ppm). Therefore, it was possible to confirm other products, which have not been described in the literature, yielded by oxidation and formation of a benzoic aldehyde ([Fig. 4]).

Product 5 presented the ions m/z 341 [M+H]⁺ and m/z 339 [M–H]⁻, which suggested the formation of an aldehyde in C-9′ olefinic methyl. Li and Yang [20] reported this product as an aldehyde yielded by the oxidation of a terminal hydroxyl, generated from the rat liver microsome model. From the ion m/z 341 [M+H]⁺, it was observed the product ions at m/z 323 and 308 were yielded by losses of a water molecule (18 u) and CH₃ ̇ (15 u), corroborating the data described by Li and Yang [20] (Fig. 24S). In addition, losses of subsequent methyl radicals (CH₃ ̇, 15 u) were observed in negative ion mode. These deletions occurred probably because of the radical loss of the methyl groups from the methoxyl substituents to carbons 3 and 3′, resulting from the weakening of the O-C bound after protonation, as discussed by Cardozo et al. [21]. After several in vitro metabolism assays under different conditions, the reaction conditions were defined as follows: substrate concentration and incubation time of 77 µM and 1 h, respectively, for RLM and 306 µM and 2 h, respectively, for HLM. The technique of sample preparation was also previously evaluated, and liquid-liquid extraction with ethyl acetate was the most adequate methodology.

The samples obtained by metabolism with RLM were analyzed by UPLC-DAD-MS/MS, and the chromatogram showed a product with similar retention time and spectral data observed for product 2 from biomimetic reactions (Fig. 43S). The MS showed the ions m/z 343 [M+H]⁺, 365 [M+Na]⁺, 381 [M+K]⁺, and 325 [M+H – H₂O]⁺ in positive ionization mode (Fig. 44S-45S) and the ion m/z 341 [M–H]⁻ in the negative ion mode (Fig. 46S). The metabolism of licarin A has been investigated by in vitro assays employing rat or human liver microsomes and 13 products were identified, but only oxidation product 5 reported in our study was similar with those described by Lv et al. [22]. Differences in the number of products have been related to the application of different in vitro metabolism protocols, especially with regard to RLM protein concentration, which was 16.6-fold higher in the study performed by Lv and collaborators [22].

The samples obtained after metabolism with HLM were analyzed by LC-ESI-IT-MS/MS. Similarly to that observed from oxidation of licarin A by RLM, the in vitro metabolization by HLM also indicated that product 2 (m/z 343 [M+H]⁺) yielded by epoxidation of licarin A ([Fig. 6 a – b]), by which was observed the ions m/z 365 [M+Na]⁺, 343 [M+H]⁺, and 325 [M+H – H₂O]⁺ in the MS ([Fig. 6 b]). The fragmentation of the m/z 343 ion produced the m/z 325 ion (Fig. 45S). The products found in the HLM and RLM reactions of licarin A were also analyzed by high-resolution MS and confirmed the epoxidized product of licarin A from both reactions (Table 5S).

Among the in vitro methods employed in preclinical studies, microsomes are the most often used for metabolism of drug molecules by CYP enzymes. However, this model comprises a complex biological matrix, including coenzymes that must be regenerated, and is more suitable for quantitative analysis, such as the determination of intrinsic clearance [23]. On the other hand, biomimetic models are simple, with fewer interferences in the reaction medium, allowing the detection of minority products. This model has been applied to understand the phase I metabolism of several natural products, such as lapachol [12], piplartine [24], [25], grandisin [26], [27], [28], monensin [29], and kaurenoic acid [9], [30], in which some products were also observed using microsomal models or in vivo models. In this work, it was demonstrated that the biomimetic oxidation of licarin A by Jacobsenʼs catalyst was able to reproduce the oxidation system with RLM and HLM. As expected, other biomimetic oxidation products unknown to the natural system were also observed [31], which opens the possibility of investigating the therapeutic or toxicological actions of these compounds.

From an in vivo acute toxicity assay, the serum biochemical parameters related to lipid, renal, and hepatic profiles were evaluated 24 h after the treatment with licarin A at 300 mg/kg. Regarding the lipid profile, the total cholesterol dosage did not show variations between treated and untreated animals, but a reduction in LDL (low-density lipoprotein) cholesterol was observed in the treated group (Fig. 48S). The HDL (high-density lipoprotein) cholesterol remained unchanged, but the levels of triglycerides and VLDL (very-low-density lipoprotein) cholesterol had an increase. Excess blood glucose could accelerate the production of triglycerides, but this was not the case, since the animals treated with licarin A presented a decrease in the glycemia, compared to the untreated and control groups.

In addition, there was no variation in relation to urea, but the treated animals exhibited a decrease in blood creatinine levels and a small increase in uric acid levels (Fig. 48S). In relation to the hepatic profile, increases in serum lactate dehydrogenase (LDH), ALT (alanine amino transferase), and AST (aspartate amino transferase) levels were observed. Transaminases (AST and ALT) are enzymes especially indicative of cell death [32], [33]. These preliminary results suggest that licarin A could induce liver changes. However, histological analysis showed that the tissue organization of the kidney, heart, and liver was preserved, and no tissue lesions were observed, such as necrosis, leukocyte infiltrate, and fibrosis, after 14 days of exposure ([Fig. 7]). Since it is important to consider the time and frequency of exposure to the compound, chronic toxicity studies are required to assess the long-term effects of licarin A, especially in relation to the liver function markers. In addition, (+)-licarin A has demonstrated in pharmacokinetic studies that it is adequately absorbed and distributed in the body [20]. In conclusion, this work demonstrated the ability of the biomimetic oxidation model to mimic the CYP metabolism of licarin A with the formation of seven products such as the vicinal diol and epoxide formed at C-7′/C-8′ and a benzoic aldehyde formed on the aromatic ring benzofuran. The epoxidated product was also produced by in vitro metabolism by rat or human liver microsomes, which demonstrates the usefulness of biomimetic reactions as a CYP enzyme chemical model for preclinical studies of bioactive molecules. Finally, this work proved the importance of the in vivo toxicity studies in which 300 mg/kg licarin A showed signs of acute toxicity in the liver biochemical parameters, although without any type of tissue lesion in the histopathological analyses after 14 days of exposure. These findings should be taken into consideration for further chronic toxicity investigations in order to advance the preclinical evaluations of licarin A.

Materials and Methods

Chemicals

Jacobsenʼs catalyst (S,S-(+)-N,N′-Bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexane diaminomanganese(III) chloride) was purchased from Sigma-Aldrich, as well as the first generation metalloporphyrin [Fe(TPP)Cl] and the oxidizing meta-chloroperbenzoic acid (mCPBA). The oxidizing iodosylbenzene (PhIO) was synthesized [34] from idosylbenzene diacetate and shows a purity of 90 – 95% as determined by iodometric titration. Solvents used for metabolism assay and isolation of products were HPLC-grade. The licarin A used for experiments was synthesized according to the methodology described by Leopold [35].

Characterization of licarin A

The synthesized licarin A was analyzed by ¹H NMR (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) (Bruker). A digital polarimeter (Jasco DIP-370) was used for the determination of specific optical rotation of the licarin A, with measurements performed in methanol at 25 °C.

The fragmentation studies were performed by direct infusion in a high-resolution mass spectrometer with electrospray ionization source and analyzers quadrupole and time-of-flight (MicrOTOF, Bruker Daltonics) and also another mass spectrometer with an ion trap analyzer (Amazon IES-SL – Bruker Daltonics). The B3LYP/6 – 31+G(d,p) computational model [36], [37] was used to obtain the protonation sites of licarin A from the gas-phase basicity (GB) calculations using the Gaussian 03 software [38].

Biomimetic oxidation with [Fe(TPP)Cl] and Mn(Salen)

The reactions of homogeneous catalysis were carried out in vials containing screw caps and protection from the light. Licarin A (30 µmol) was weighed and reserved. The catalysis reactions were assayed in different conditions, using the catalyst iron (III) tetraphenylporphyrin chloride ([Fe(TPP)Cl]) or Jacobsenʼs catalyst (Mn(Salen)) (1 µmol) dissolved in 1 mL of the following solvents: methanol (MeOH), dichloromethane (DCM), acetonitrile (ACN), and ethyl acetate (EtOAc). Finally, the oxidizing agent iodosybenzene (PhIO) or m-chloroperbenzoic acid (mCPBA) (30 µmol) was added and the final volume was adjusted to 4 mL. Reaction mixtures were stirred in constant temperature over a period of 24 h. The samples were analyzed by gas chromatography–mass spectrometry (GC-MS). The reaction with the highest amount of products formed was selected and analyzed by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS).

Identification of products

The isolation of products was achieved by semipreparative HPLC-DAD analysis, applying a C₁₈ chromatography column (Shim-pack Prep-ODS, 20 mm × 25 cm × 5 µm, Shimadzu), and using the flow rate of 9 mL/min and a mobile phase composed by ultrapure water (A) and ACN (B). The elution gradient profile was as follows: 0 – 20 min of 20% B, 20 – 35 min of 50% B, 50 min of 80% B, 57 – 66 min of 100% B, 67 – 70 min of 20% B. The isolated products were analyzed by NMR (500 MHz, Bruker DRX500, CDCl₃, δ 3.31) and high-resolution ESI-MS (MicroOTOF, Bruker Daltonics).

In vitro metabolism by rat liver microsomes (RLM) and human liver microsomes (HLM)

Rat liver microsomes (RLM) were obtained “in house”. Male Wistar rats weighing 180 – 120 g, acquired from the bioterium of Faculdade de Ciências Farmacêuticas de Ribeirão Preto – Universidade de São Paulo (FCFRP-USP), were maintained in a controlled room with a 12 h light/dark cycle at a temperature of 23 ± 2 °C, with food and tap water provided ad libitum. Animals were sacrificed by decapitation, and the livers were removed and processed according to a protocol for the isolation of microsomes by differential centrifugation and stored at − 80 °C until use [25]. Human liver microsomes (HLMs), a pool of 150 donors, were acquired from Corning Life Sciences (Phoenix, AZ, USA) and stored at − 80 °C.

For the evaluation of in vitro metabolization of licarin A by microsomal models, the incubation mixtures were prepared with 77 µM licarin A with 0.3 mg/mL microsomal proteins of rat liver microsomes (RLM) or 306 µM licarin A with 1 mg/mL microsomal proteins of human liver microsomes (HLM) in 100 mM phosphate buffer at pH 7.4. After preincubation for 5 minutes with shaking at 37 °C, the reactions were initiated by the addition of NADPH-generating system (0.25 mM NADP⁺, 5 mM glucose-6-phosphate, 0.5 units glucose-6-phosphate dehydrogenase) prepared in Tris-HCl buffer (0.05 M Tris-HCl/0.15 M KCl, pH 7.4) corresponding to 25% of the reaction medium. The in vitro metabolization assay with RLM was performed with a reaction volume of 1 mL. After 2 h, the reactions were quenched by the addition of ethyl acetate (EtOAc, 4 mL). The samples were mixed for 15 minutes and centrifuged at 2000 × g for 7 min at 4 °C. Subsequently, 3 mL of the organic phase were collected, dried, and resuspended in 100 µL ACN and H₂O (8 : 2, v/v) and analyzed by UPLC-MS/MS as described subsequently here.

The in vitro metabolism assay with HLM was performed with 10 samples in a reaction volume of 200 µL. The same procedures for sample preparation described above were adopted, except for the volume of EtOAc that was 1 mL, and the evaporation of the organic phase, which was unified in a conical bottom tube and evaporated under a stream of compressed air. The pooled sample was analyzed by sequential mass spectrometry (LC-MSⁿ) and high-resolution mass spectrometry (LC-ESI-Q-TOF). Control samples, containing only licarin A or only RLM or HLM in phosphate buffer were assayed in the same conditions and analyzed concomitantly to guarantee the selectivity of the analysis.

Gas chromatography–mass spectrometry analysis (GC-MS)

Biomimetic reactions were analyzed by gas chromatography (GC-MS-QP-2010, Shimadzu) coupled to a quadrupole mass spectrometer with electron impact (EI) ionization at 70 eV. The gas chromatography was equipped with an auto-sampler AOC-20i. Separation was carried out on a 5% phenyl methyl siloxane (DB-5MS) chromatographic column (30 m × 0.25 mm × 0.25 µm). The oven temperature gradient used was 120 °C for 4 min, followed by temperature increases to 300 °C at 8 °C/min, where it was kept constant for 12 min. The injection (split mode) and oven temperature were 250 and 120 °C, respectively, with gas (helium) pressure of 80.6 kPa and flow of 1 mL/min.

Ultra-performance liquid chromatography–tandem mass spectrometry analysis (UPLC-MS/MS)

Biomimetic reactions, at conditions with highest yields of metabolization products, were also analyzed by UPLC-MS/MS by an Acquity UPLC (Waters) coupled to an Acquity TQD triple quadrupole analyzer equipped with an electrospray ionization (ESI) interface. The column used was ACQUITY UPLC BEH C₁₈ (50 mm × 2.1 mm × 1.7 microns, Waters). The mobile phase was composed of ultrapure water (A) and ACN (B) with the following gradient elution profile: 0 min (20% B), 5 min (40% B), 11 min (80% B), 11.5 – 13.5 min (100% B), and 14 – 15 min (20% B), applying a flow rate of 0.3 mL/min. The electrospray ionization source was operated in both positive and negative mode. Nitrogen (N₂) was used as nebulization gas at the drying temperature of 350 °C and argon was used as collision gas, with a flow rate of 0.15 mL/min and cone voltage of 25 eV. Data acquisition and analysis were performed using MassLynx V4.1 software (Waters).

Sequential mass spectrometry analysis (LC-MSⁿ)

Chromatographic analysis was performed using an HPLC system (Shimadzu) comprised of a solvent pump unit LC-20AD, degasser DGU-20A, auto-injector SIL-20AHT, diode array detector SPD-M20A, column oven CTO-20A, and communication module CBM-20A coupled to ion trap mass spectrometer (AmaZon SL, Bruker Daltonics). An Ascentis Express Fused Core C18 (100 × 4.6 mm, 2.7 µm) chromatographic column (Supelco) was used and maintained at 40 °C during the analyses. The mobile phase was composed of ultrapure water (A) and MeOH (B); both added 0.1% formic acid, and the linear gradient profile was 10 to 90% of B in 60 min, subsequently 10% of B in 60 – 61 min, and 10% of B in 61 – 66 min. The flow rate was 1 mL/min, and 8 µL of sample was injected. The spectra were acquired in positive mode employing electrospray ionization (ESI). N₂ was used as nebulization gas at the drying temperature of 300 °C, with a flow rate of 9 L/min, and a pressure of 40 psi. The fragmentation amplitude was 0.6 V.

High-resolution mass spectrometry analysis

For determination of the exact mass of the licarin A products, the samples were analyzed by the same chromatographic conditions described above using the equipment MicrOTOF (ESI-Q-TOF, Bruker Daltonics). The voltage of the capillary was maintained at 3500 mV, in positive ionization mode, with a drying gas (N₂) temperature of 220 °C, a flow rate of 9 mL/min, and a pressure of 6 bar. For internal calibration, a solution of sodium trifluoroacetic acid at a concentration of 10 mg/mL was used.

In vivo oral acute toxicity

For acute toxicity experiments, male mice of Balb/c were used, weighing between 28 – 32 g. These animals were acquired from the bioterium of Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (FCFRP-USP) and kept under controlled temperature conditions (22 °C ± 3), relative humidity (50 – 60%), light/dark cycle, and access to food and water ad libitum. This study was approved by the Ethical Committee for the animal experiments (CEUA) at number 09.1.112.53.4 (May 31, 2011).

The treatments with licarin A were performed at doses of 5, 50, 300, and 2000 mg/kg, as preconized by OECD 423, 2002 [39], for the acute toxicity assay. The biochemical parameters in the serum of mice treated with licarin A by gavage were evaluated from 18 animals [three groups (n = 6): control (vehicle administration), untreated (no administration) and treated (300 mg/kg licarin A in PEG)], since the treatment with this dose presented toxicity events. This dosage was previously determined by a toxicity assay performed according to OECD 423, 2002 [39]. After 24 h, the blood was collected from the retro-orbital plexus. Then the animals were anesthetized (500 µL ketamine, 125 µL xylazine to 2 mL of water) and euthanized. The blood serum obtained was separated by centrifugation (21 382 xg – relative centrifugal force for 20 min) and sent to the clinical analyses service of the university FCFRP-USP for the dosage of the biochemical parameters. To another group of six mice (and one vehicle control), 300 mg/kg licarin A was administered and, after 14 days, the animals were euthanized for the histopathological analysis of the liver, kidney, and heart.

Contributorsʼ Statement

Design and conception of the study: J. N. P. Souza, N. P. Lopes, D. B. Silva. Writing the manuscript: J. N. P. Souza, RM Silva, S. S. Fortes, A. R. M. Oliveira, L. S. Ferreira, R. Vessecchi, NP Lopes, DB Silva. Data collection: J. N. P. Souza, RM Silva, S. S. Fortes. Analysis and interpretation of data: N. P. Souza, RM Silva, S. S. Fortes, A. R. M. Oliveira, L. S. Ferreira, R. Vessecchi, NP Lopes, DB Silva. All the authors revised the manuscript and approved.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process number 313047/2020 – 0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

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• 2 Ma CJ, Sung SH, Kim YC. Neuroprotective Lignans from the bark of the Machilus thunbergii . Planta Med 2004; 70: 78-80
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• 3 Paiva MRB, Vasconcelos-Santos DV, Coelho MM, Machado RR, Lopes NP, Silva-Cunha A, Fialho SL. Licarin A as a Novel Drug for Inflammatory Eye Diseases. J Ocul Pharmacol Ther 2021; 37: 290-300
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• 4 Murakami Y, Shoji M, Hirata A, Tanaka S, Yokoe I, Fujisawa S. Dehydrodiisoeugenol, an isoeugenol dimer, inhibits lipopolysaccharide-stimulated nuclear factor kappa B activation and cyclooxygenase-2 expression in macrophages. Arch Biochem Biophys 2005; 434: 326-332
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• 6 Néris PLN, Caldas JPA, Rogrigues YKS, Amorim FM, Patrícia LN, Leite JA, Mascarenhas SR, Barbosa-Filho JM, Rodrigues LC, Oliveira MR. Neolignan Licarin A presents effect against Leishmania (Leishmania) major associated with immunomodulation in vitro . Exp Parasitol 2013; 35: 307-313
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• 7 Pereira AC, Magalhães LG, Gonçalves UO, Luz PP, Moraes ACG, Rodrigues V, Guedes PMM, Silva Filho AA, Cunha WR, Bastos JK, Nanayakkara NPD, Silva MLA. Schistosomicidal and trypanocidal structure–activity relationships for (±)-licarin A and its (−)- and (+)-enantiomers. Phytochemistry 2011; 72: 1424-1430
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• 8 Yadav J, Hassani ME, Sodhi J, Lauschke VM, Hartman JM, Russell LE. Recent developments in vitro and in vivo models for improved translation of preclinical pharmacokinetics and pharmacodynamics data. Drug Metab Rev 2021; 53: 207-233
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• 9 Fernandes EFA, Oliveira ARM, Barros VP, Guaratini T, Lopes NP. Biomimetic metabolism of kaurenoic acid validated by microsomal reactions. Rev Bras Farmacog 2020; 30: 551-558
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• 10 Bernadou J, Meunier B. Biomimetic chemical catalysts in the oxidative activation of drugs. Adv Synth Catal 2004; 346: 171-184
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• 11 Yan X, Lu N, Gu Y, Li C, Zhang T, Liu H, Zhang Z, Zhai S. Catalytic activity of biomimetic model of cytochrome P450 in oxidation of dopamine. Talanta 2018; 179: 401-408
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• 12 Nieheus M, Barros VP, Emery SF, Dias-Baruffi M, Assis DM, Lopes PN. 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
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• 13 Lee SU, Shim KS, Ryu SY, Min YK, Kim SH. Machilin A isolated from Myristica fragrans stimulates osteoblast differentiation. Planta Med 2009; 75: 152-157
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• 14 Barbosa-Filho MJ, Da-Cunha LVE, Silva SM. Complete assignment of the ¹H and ¹³C spectra of some lignoids from Lauraceae. Magn Reson Chem 1998; 36: 929-935
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• 15 Moser A, Range K, York DM. Accurate proton affinity and gas-phase basicity values for molecules important in biocatalysis. J Phys Chem B 2010; 114: 13911-13921
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• 16 Amad MH, Cech NB, Jackson GS, Enke CG. Importance of gas-phase proton affinities in determining the electrospray ionization response for analytes and solvents. J Mass Spectrom 2000; 35: 784-789
  CrossrefPubMedSearch in Google Scholar
• 17 Bolzon LB, Bindeiro AKS, Souza LMO, Zanatta LD, Paula R, Cerqueira BC, Santos JS. rhodamine B oxidation promoted by P450- bioinspired Jacobsen catalysts/cellulose systems. RSC Adv 2021; 11: 33823-33834
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• 18 Han HJ, Hong JS, Lee YE, Lee HJ, Kim JH, Kwak H. Conversion of epoxides into trans-diols or trans-diol mono-ethers by iron (III) porphyrin complex. Bull Korean Chem Soc 2005; 26: 1434-1436
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• 19 Yoo KS, Han HJ, Lee JS, Ryu YJ, Kim WS, Jin WS, Kim Y, Nam W. Conversion of olefins into trans-diols or trans-diol mono-ethers by using iron porphyrin(III) complex and H₂O₂ . Inorg Chem Commun 2003; 6: 1148-1151
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• 20 Li F, Yang WX. Simultaneous determination of diastereomers (+)-licarin A and isolicarin A from Myristica fragrans in rat plasma by HPLC and its application to their pharmacokinetics. Planta Med 2008; 74: 880-884
  Thieme ConnectPubMedSearch in Google Scholar
• 21 Cardozo KHM, Vessecchi R, Carvalho VM, Pinto E, Gates PJ, Colepicolo P, Galembeck SE, Lopes NP. A theorical and mass spetrometry sutdy of the fragmentation of mycosporine – like amino acids. Int J Mass Spectrom 2008; 273: 11-19
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• 22 Lv QQ, Yang XN, Yan DM, Liang WQ, Liu HN, Yang XW, Li F. Metabolomic profiling of dehydrodiisoeugenol using xenobiotic metabolomics. J Pharmaceut Biomed 2017; 145: 725-733
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• 23 Fasinu P, Bouic PJ, Rosenkranz B. Liver-based in vitro technologies for drug biotranformation studies – a review. Curr Drug Metab 2012; 13: 215-224
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• 24 Shaab EH, Crotti AEM, Iamamoto Y, Kato MJ, Lotufo LVC, Lopes NP. Biomimetic oxidation of piperine and piplartine catalyzed by iron (III) and manganese (III) porphyrins. Biol Pharm Bull 2010; 5: 912-916
  CrossrefPubMedSearch in Google Scholar
• 25 Marques LMM, Silva-Junior EA, Gouvea DR, Vessecchi R, Pupo MT, Lopes NP, Kato MJ, Oliveira ARM. In vitro metabolism of the alkaloid piplartine by rat liver microsomes. J Pharm Biomed Anal 2014; 95: 113-120
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• 26 Ferreira LS, Callejon DR, Engemann A, Cramber B, Humpf HU, Barros VP, Assis MD, Silva DB, Albuquerque S, Okano LT, Kato MJ, Lopes NP. In vitro metabolismo of grandisin, a lignan with anti-chagasic activity. Planta Med 2012; 78: 1939-1941
  Thieme ConnectPubMedSearch in Google Scholar
• 27 Messiano GB, Santos RAS, Ferreira LDS, Simões RA, Jabor VAP, Kato MJ, Lopes NP, Pupo MT, Oliveira ARM. In vitro metabolism study of the promissing anticâncer agent the lignan (−)-grandisin. J Pharm Biomed Anal 2013; 72: 240-244
  CrossrefPubMedSearch in Google Scholar
• 28 Barth T, Habenschus MD, Moreira FL, Ferreira LDS, Lopes NP, Oliveira ARM. In vitro metabolismo of the lignan (−)-grandisin, an anticancer drug candidate, by human liver microsomes. Drug Test Anal 2015; 7: 780-786
  CrossrefPubMedSearch in Google Scholar
• 29 Rocha BA, Oliveira ARM, Pazin M, Dorta DJ, Rodrigues APN, Berretta AA, Peti APF, Moraes LAB, Lopes NP, Pospisil S, Gates PJ, Assis MD. Jacobsen catalyst as a cytochrome P450 biomimetic model for the metabolismo of monensin A. Biomed Res Int 2014; 2014: 152102
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• 30 Mauro M, Silva RM, Campos ML, Bauermeister A, Lopes NP, Moraes NV. In vitro metabolism of copalic and kaurenoic acid in rat and human liver microsomes. Quim Nova 2021; 6: 700-708
  PubMedSearch in Google Scholar
• 31 Simões MMQ, Paula RD, Neves MGPMS, Cavaleiro JAS. Metalloporphyrins in the biomimetic oxidative valorization of natural and other organic substrates. J Porphyr Phthalocyanines 2009; 13: 589-596
  CrossrefPubMedSearch in Google Scholar
• 32 Lehmann-Werman R, Magenheim J, Moss J, Neiman D, Abraham O, Piyanzin S, Zemmour H, Fox I, Dor T, Grompe M, Landesberg G, Loza BL, Shaked A, Olthoff K, Glaser B, Shemer R, Dor Y. Monitoring liver damage using hepatocyte-specific methylation markers in cell-free circulating DNA. JCI Insight 2018; 3: e120687
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• 33 Ozardalı I, Bitiren M, Karakılcık AZ, Zerin M, Aksoy N, Musa D. Effects of selenium on histopathological and enzymatic changes in experimental liver injury of rats. Exp Toxicol Pathol 2004; 56: 59-64
  CrossrefPubMedSearch in Google Scholar
• 34 Lucas HJ, Kennedy ER, Formo MW, Baungratem HE. Organic Synthesis Collective. New York: John Willey & Sons; 1955; 3: 483
  PubMedSearch in Google Scholar
• 35 Leopold B. Aromatic keto and hydroxyl-polyethers as lignin models III. Acta Chem Scand 1950; 4: 1523-1537
  CrossrefPubMedSearch in Google Scholar
• 36 Becke AD. A new mixing of hartree-fock and local density-functional theories. J Chem Phys 1993; 98: 1372
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• 37 Lee CT, Yang W, Parr RG. Development of the colle-salvetti correlation-energy formula into a functional of the electron-density. Phys Rev B Condens Matter 1988; 37: 785-789
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• 38 Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Montgomery jr. JA, Vreven T, Kudin KN, Burant JC, Millam JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi M, Scalmani G, Rega N, Petersson GA, Nakatsuji H, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Klene M, Li X, Knox JE, Hratchian HP, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Ayala PY, Morokuma K, Voth GA, Salvador P, Dannenberg JJ, Zakrzewski VG, Dapprich S, Daniels AD, Strain MC, Farkas O, Malick DK, Rabuck AD, Raghavachari K, Foresman JB, Ortiz JV, Cui Q, Baboul AG, Clifford S, Cioslowski J, Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Martin RL, Fox DJ, Keith T, Al-Laham MA, Peng CY, Nanayakkara A, Challacombe M, Gill PMW, Johnson B, Chen W, Wong MW, Gonzalez C, Pople JA. Gaussian 03, Revision D. 01. Wallingford CT: Gaussian Inc.; 2004
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• 39 Organisation for Economic Co-operation and Development (OECD). Test N°423 – Acute Oral Toxicity – Acute Toxic Class Method, OECD Guidelines for the Testing of Chemicals, Section 4. Paris: OECD Publishing; 2002
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Correspondence

Publication History

Received: 27 July 2022

Accepted: 18 December 2022

Article published online:
08 March 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Alvarenga DJ, Matias LMF, Oliveira LM, Leão LPMO, Hawkes JA, Raimundo BVB, Castro LFD, Campos MMA, Siqueira FS, Santos T, Carvalho DT. Exploring how structural changes to new licarin A derivatives effects their bioactive properties against rapid growing mycobacteria and biofilm formation. Microb Pathog 2020; 144: 104203
  CrossrefPubMedSearch in Google Scholar
• 2 Ma CJ, Sung SH, Kim YC. Neuroprotective Lignans from the bark of the Machilus thunbergii . Planta Med 2004; 70: 78-80
  PubMedSearch in Google Scholar
• 3 Paiva MRB, Vasconcelos-Santos DV, Coelho MM, Machado RR, Lopes NP, Silva-Cunha A, Fialho SL. Licarin A as a Novel Drug for Inflammatory Eye Diseases. J Ocul Pharmacol Ther 2021; 37: 290-300
  CrossrefPubMedSearch in Google Scholar
• 4 Murakami Y, Shoji M, Hirata A, Tanaka S, Yokoe I, Fujisawa S. Dehydrodiisoeugenol, an isoeugenol dimer, inhibits lipopolysaccharide-stimulated nuclear factor kappa B activation and cyclooxygenase-2 expression in macrophages. Arch Biochem Biophys 2005; 434: 326-332
  CrossrefPubMedSearch in Google Scholar
• 5 Maheswari U, Ghosh K, Sadras SR. Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells. Apoptosis 2018; 23: 210-225
  CrossrefPubMedSearch in Google Scholar
• 6 Néris PLN, Caldas JPA, Rogrigues YKS, Amorim FM, Patrícia LN, Leite JA, Mascarenhas SR, Barbosa-Filho JM, Rodrigues LC, Oliveira MR. Neolignan Licarin A presents effect against Leishmania (Leishmania) major associated with immunomodulation in vitro . Exp Parasitol 2013; 35: 307-313
  CrossrefPubMedSearch in Google Scholar
• 7 Pereira AC, Magalhães LG, Gonçalves UO, Luz PP, Moraes ACG, Rodrigues V, Guedes PMM, Silva Filho AA, Cunha WR, Bastos JK, Nanayakkara NPD, Silva MLA. Schistosomicidal and trypanocidal structure–activity relationships for (±)-licarin A and its (−)- and (+)-enantiomers. Phytochemistry 2011; 72: 1424-1430
  CrossrefPubMedSearch in Google Scholar
• 8 Yadav J, Hassani ME, Sodhi J, Lauschke VM, Hartman JM, Russell LE. Recent developments in vitro and in vivo models for improved translation of preclinical pharmacokinetics and pharmacodynamics data. Drug Metab Rev 2021; 53: 207-233
  CrossrefPubMedSearch in Google Scholar
• 9 Fernandes EFA, Oliveira ARM, Barros VP, Guaratini T, Lopes NP. Biomimetic metabolism of kaurenoic acid validated by microsomal reactions. Rev Bras Farmacog 2020; 30: 551-558
  CrossrefPubMedSearch in Google Scholar
• 10 Bernadou J, Meunier B. Biomimetic chemical catalysts in the oxidative activation of drugs. Adv Synth Catal 2004; 346: 171-184
  CrossrefPubMedSearch in Google Scholar
• 11 Yan X, Lu N, Gu Y, Li C, Zhang T, Liu H, Zhang Z, Zhai S. Catalytic activity of biomimetic model of cytochrome P450 in oxidation of dopamine. Talanta 2018; 179: 401-408
  CrossrefPubMedSearch in Google Scholar
• 12 Nieheus M, Barros VP, Emery SF, Dias-Baruffi M, Assis DM, Lopes PN. 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
• 13 Lee SU, Shim KS, Ryu SY, Min YK, Kim SH. Machilin A isolated from Myristica fragrans stimulates osteoblast differentiation. Planta Med 2009; 75: 152-157
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Barbosa-Filho MJ, Da-Cunha LVE, Silva SM. Complete assignment of the ¹H and ¹³C spectra of some lignoids from Lauraceae. Magn Reson Chem 1998; 36: 929-935
  CrossrefPubMedSearch in Google Scholar
• 15 Moser A, Range K, York DM. Accurate proton affinity and gas-phase basicity values for molecules important in biocatalysis. J Phys Chem B 2010; 114: 13911-13921
  CrossrefPubMedSearch in Google Scholar
• 16 Amad MH, Cech NB, Jackson GS, Enke CG. Importance of gas-phase proton affinities in determining the electrospray ionization response for analytes and solvents. J Mass Spectrom 2000; 35: 784-789
  CrossrefPubMedSearch in Google Scholar
• 17 Bolzon LB, Bindeiro AKS, Souza LMO, Zanatta LD, Paula R, Cerqueira BC, Santos JS. rhodamine B oxidation promoted by P450- bioinspired Jacobsen catalysts/cellulose systems. RSC Adv 2021; 11: 33823-33834
  CrossrefPubMedSearch in Google Scholar
• 18 Han HJ, Hong JS, Lee YE, Lee HJ, Kim JH, Kwak H. Conversion of epoxides into trans-diols or trans-diol mono-ethers by iron (III) porphyrin complex. Bull Korean Chem Soc 2005; 26: 1434-1436
  CrossrefPubMedSearch in Google Scholar
• 19 Yoo KS, Han HJ, Lee JS, Ryu YJ, Kim WS, Jin WS, Kim Y, Nam W. Conversion of olefins into trans-diols or trans-diol mono-ethers by using iron porphyrin(III) complex and H₂O₂ . Inorg Chem Commun 2003; 6: 1148-1151
  CrossrefPubMedSearch in Google Scholar
• 20 Li F, Yang WX. Simultaneous determination of diastereomers (+)-licarin A and isolicarin A from Myristica fragrans in rat plasma by HPLC and its application to their pharmacokinetics. Planta Med 2008; 74: 880-884
  Thieme ConnectPubMedSearch in Google Scholar
• 21 Cardozo KHM, Vessecchi R, Carvalho VM, Pinto E, Gates PJ, Colepicolo P, Galembeck SE, Lopes NP. A theorical and mass spetrometry sutdy of the fragmentation of mycosporine – like amino acids. Int J Mass Spectrom 2008; 273: 11-19
  CrossrefPubMedSearch in Google Scholar
• 22 Lv QQ, Yang XN, Yan DM, Liang WQ, Liu HN, Yang XW, Li F. Metabolomic profiling of dehydrodiisoeugenol using xenobiotic metabolomics. J Pharmaceut Biomed 2017; 145: 725-733
  CrossrefPubMedSearch in Google Scholar
• 23 Fasinu P, Bouic PJ, Rosenkranz B. Liver-based in vitro technologies for drug biotranformation studies – a review. Curr Drug Metab 2012; 13: 215-224
  CrossrefPubMedSearch in Google Scholar
• 24 Shaab EH, Crotti AEM, Iamamoto Y, Kato MJ, Lotufo LVC, Lopes NP. Biomimetic oxidation of piperine and piplartine catalyzed by iron (III) and manganese (III) porphyrins. Biol Pharm Bull 2010; 5: 912-916
  CrossrefPubMedSearch in Google Scholar
• 25 Marques LMM, Silva-Junior EA, Gouvea DR, Vessecchi R, Pupo MT, Lopes NP, Kato MJ, Oliveira ARM. In vitro metabolism of the alkaloid piplartine by rat liver microsomes. J Pharm Biomed Anal 2014; 95: 113-120
  CrossrefPubMedSearch in Google Scholar
• 26 Ferreira LS, Callejon DR, Engemann A, Cramber B, Humpf HU, Barros VP, Assis MD, Silva DB, Albuquerque S, Okano LT, Kato MJ, Lopes NP. In vitro metabolismo of grandisin, a lignan with anti-chagasic activity. Planta Med 2012; 78: 1939-1941
  Thieme ConnectPubMedSearch in Google Scholar
• 27 Messiano GB, Santos RAS, Ferreira LDS, Simões RA, Jabor VAP, Kato MJ, Lopes NP, Pupo MT, Oliveira ARM. In vitro metabolism study of the promissing anticâncer agent the lignan (−)-grandisin. J Pharm Biomed Anal 2013; 72: 240-244
  CrossrefPubMedSearch in Google Scholar
• 28 Barth T, Habenschus MD, Moreira FL, Ferreira LDS, Lopes NP, Oliveira ARM. In vitro metabolismo of the lignan (−)-grandisin, an anticancer drug candidate, by human liver microsomes. Drug Test Anal 2015; 7: 780-786
  CrossrefPubMedSearch in Google Scholar
• 29 Rocha BA, Oliveira ARM, Pazin M, Dorta DJ, Rodrigues APN, Berretta AA, Peti APF, Moraes LAB, Lopes NP, Pospisil S, Gates PJ, Assis MD. Jacobsen catalyst as a cytochrome P450 biomimetic model for the metabolismo of monensin A. Biomed Res Int 2014; 2014: 152102
  CrossrefPubMedSearch in Google Scholar
• 30 Mauro M, Silva RM, Campos ML, Bauermeister A, Lopes NP, Moraes NV. In vitro metabolism of copalic and kaurenoic acid in rat and human liver microsomes. Quim Nova 2021; 6: 700-708
  PubMedSearch in Google Scholar
• 31 Simões MMQ, Paula RD, Neves MGPMS, Cavaleiro JAS. Metalloporphyrins in the biomimetic oxidative valorization of natural and other organic substrates. J Porphyr Phthalocyanines 2009; 13: 589-596
  CrossrefPubMedSearch in Google Scholar
• 32 Lehmann-Werman R, Magenheim J, Moss J, Neiman D, Abraham O, Piyanzin S, Zemmour H, Fox I, Dor T, Grompe M, Landesberg G, Loza BL, Shaked A, Olthoff K, Glaser B, Shemer R, Dor Y. Monitoring liver damage using hepatocyte-specific methylation markers in cell-free circulating DNA. JCI Insight 2018; 3: e120687
  CrossrefPubMedSearch in Google Scholar
• 33 Ozardalı I, Bitiren M, Karakılcık AZ, Zerin M, Aksoy N, Musa D. Effects of selenium on histopathological and enzymatic changes in experimental liver injury of rats. Exp Toxicol Pathol 2004; 56: 59-64
  CrossrefPubMedSearch in Google Scholar
• 34 Lucas HJ, Kennedy ER, Formo MW, Baungratem HE. Organic Synthesis Collective. New York: John Willey & Sons; 1955; 3: 483
  PubMedSearch in Google Scholar
• 35 Leopold B. Aromatic keto and hydroxyl-polyethers as lignin models III. Acta Chem Scand 1950; 4: 1523-1537
  CrossrefPubMedSearch in Google Scholar
• 36 Becke AD. A new mixing of hartree-fock and local density-functional theories. J Chem Phys 1993; 98: 1372
  CrossrefPubMedSearch in Google Scholar
• 37 Lee CT, Yang W, Parr RG. Development of the colle-salvetti correlation-energy formula into a functional of the electron-density. Phys Rev B Condens Matter 1988; 37: 785-789
  CrossrefPubMedSearch in Google Scholar
• 38 Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Montgomery jr. JA, Vreven T, Kudin KN, Burant JC, Millam JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi M, Scalmani G, Rega N, Petersson GA, Nakatsuji H, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Klene M, Li X, Knox JE, Hratchian HP, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Ayala PY, Morokuma K, Voth GA, Salvador P, Dannenberg JJ, Zakrzewski VG, Dapprich S, Daniels AD, Strain MC, Farkas O, Malick DK, Rabuck AD, Raghavachari K, Foresman JB, Ortiz JV, Cui Q, Baboul AG, Clifford S, Cioslowski J, Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Martin RL, Fox DJ, Keith T, Al-Laham MA, Peng CY, Nanayakkara A, Challacombe M, Gill PMW, Johnson B, Chen W, Wong MW, Gonzalez C, Pople JA. Gaussian 03, Revision D. 01. Wallingford CT: Gaussian Inc.; 2004
  Search in Google Scholar
• 39 Organisation for Economic Co-operation and Development (OECD). Test N°423 – Acute Oral Toxicity – Acute Toxic Class Method, OECD Guidelines for the Testing of Chemicals, Section 4. Paris: OECD Publishing; 2002
  Search in Google Scholar

• Supplementary Material