Anti-inflammatory and Quinone Reductase-Inducing Compounds from Beilschmiedia mannii

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Electronic circular dichroism calculation
• Quinone reductase induction activity
• Nuclear factor-kappa B inhibitory activity
• References

Abstract

Previous studies on the therapeutic potential of plant species found in the diet of chimpanzees living in Taï National Park have shown that they could be potential candidates for the search of new molecules useful for humans. Based on the screening of some of these plants, the fruits of Beilschmiedia mannii, whose dichloromethane extract showed cancer chemopreventive properties, were selected. Bioactivity-guided fractionation of the extract resulted in the isolation and identification of two γ-pyrones, including desmethoxydihydromethysticin (1), found in a natural source for the first time, and a new congener, beilschmiediapyrone (2), as well as five known alkamides (3 – 7). Their structures were established by using nuclear magnetic resonance spectroscopy and mass spectrometry methods. The isolated compounds were evaluated for their cancer chemopreventive potential by using quinone reductase induction and nuclear factor-kappa B inhibition tests in Hepa 1c1c7 and HEK-293/NF-κB-Luc cells, respectively. Among them, compounds 1 and 2 were the most active. The concentrations to double the quinone reductase activity were 7.5 µM for compound 1 and 6.1 µM for compound 2. Compounds 1 and 2 inhibited nuclear factor-kappa B with IC₅₀ values of 2.1 and 3.4 µM, respectively. These results are promising with regard to cancer chemoprevention, especially because this plant is also used for cooking by the local population around the Taï forest.

Introduction

Over the years, the burden of cancer has shifted to low and intermediate income countries, and approximately 70% of deaths from cancer occur in those countries [1]. According to the WHO, 600 000 new cases of cancer and 500 000 related deaths are declared each year in Africa [2]. In Côte dʼIvoire alone, the WHO estimated that there are between 15 000 and 20 000 new cases of cancer per year, with 80% of these cases at advanced stages necessitating invasive and costly treatments, such as surgery and chemotherapy, which often end up with a high mortality rate.

Following the therapeutic failures observed in many cases due to a high systemic toxicity and drug resistance, new strategies including chemoprevention [3] are being developed to overcome cancer by using natural or synthetic substances [4]. Plants are an inexhaustible source of natural substances with a wide variety of biological and pharmacological activities.

Previous studies on the therapeutic potential of some plant species found in the diet of chimpanzees (Pan troglodytes verus) in Taï National Park have shown that they possess strong antioxidant [5] and antimicrobial [6] properties and could be potential candidates for the search of new molecules useful for humans. Based on the screening of 18 of these plants, Beilschmiedia mannii (Meisn.) Robyns & Wilczek was selected for its cancer chemopreventive activities. This plant belongs to the Lauraceae family. It is used therapeutically in Côte dʼIvoire against lung affections in humans [7], and its seeds are used for cooking [8]. This study aimed at identifying cancer chemopreventive compounds from the dichloromethane extract of the fruits of B. mannii by using QR induction and NF-κB inhibition tests. Inflammation is an important factor for carcinogenesis, and NF-κB has been shown to be activated during the stages of promotion and progression. More than 20% of all reported cases of cancer are due to the activation of this protein [9]. On the other hand, QR, a phase II enzyme, can offer protection against toxic and reactive chemical species in the carcinogenesis process [10].

Results and Discussion

Bioactivity-guided fractionation of the dichloromethane extract of B. mannii fruits was performed based on QR induction and NF-κB inhibition activities (Table 1S, Supporting Information). This led to the isolation of seven compounds, two 5,6-dihydro-2H-pyran-2-ones (1 and 2) and five alkamides (3 – 7) ([Fig. 1]).

The UV spectrum of compound 1 showed two maxima of absorption at 231 and 285 nm, suggesting the presence of conjugated double bonds in the molecule [11]. HRESIMS in the positive ionization mode showed the presence of a molecular ion at m/z 247.0974 [M + H]⁺, corresponding to the molecular formula C₁₄H₁₅O₄. The ¹H and edited HSQC spectra showed the presence of four methylenes (including one methylenedioxy at δ _H 5.88, δ _C 101.9) and six methines (one oxygenated, two olefinic, and three aromatic). The HMBC experiment displayed the presence of four additional quaternary carbons, including two oxygenated aromatic carbons at δ _C 148.0 (C-11) and 145.7 (C-12), one aromatic carbon at δ _C 134.8 (C-9), and one ester carbon at δ _C 166.8 (C-2). The presence of a substituted 1,3-benzodioxole was identified by the aromatic protons at δ _H 6.73 (d, J = 1.7 Hz, H-10), 6.72 (d, J = 8.0 Hz, H-13), and 6.67 (dd, J = 8.0, 1.7 Hz, H-14) and the HMBC correlations of the methylenedioxy H-15 with the oxygenated aromatic carbons C-11 and C-12. The 5,6-dihydro-pyran-2-one moiety was established by the COSY correlations from the oxymethine H-6 (δ _H 4.42, ddt, J = 12.0, 8.4, 4.4 Hz) to the methylene H-5 (δ _H 2.44 and 2.35), from H-5 to the olefin H-4 (δ _H 7.02, ddd, J = 9.8, 5.7, 2.6 Hz), and from H-4 to H-3 (δ _H 5.97, ddd, J = 9.8, 2.6, 1.1 Hz). The HMBC correlations from H-3 and H-4 to the ester carbon C-2 confirmed the presence of this group. The HMBC correlations from the methylene H-8 (δ _H 2.76 and 2.67) to C-6, C-9, C-10, and C-14 and from the methylene H-7 (δ _H 2.02 and 1.92) to C-5, C-6 and C-9 allowed to link the 1,3-benzodioxole to the 5,6-dihydro-pyran-2-one by an ethyl chain. The structure of compound 1 was determined to be close to that of dihydromethysticin, a compound isolated from kava (Piper methysticum G. Forst.) [12], except for the lack of the methoxy group. The absolute configuration at C-6 was established by comparison of the experimental and calculated ECD spectra. The calculated ECD spectrum for the 6S-stereoisomer showed a good fit with the experimental data ([Fig. 2 A]). Therefore, compound 1 was defined as (6S)-desmethoxydihydromethysticin and the NMR data are summarized in [Table 1]. The semisynthetic preparation of this compound was mentioned in a patent [13].

The HRESIMS of compound 2 in the positive ionization mode showed the presence of a molecular ion at m/z 273.1119 [M + H]⁺, corresponding to the molecular formula C₁₆H₁₇O₄. The UV and NMR data ([Table 1]) were very similar to those of compound 1 except for the presence of two additional olefinic protons at δ _H 5.71 (dtt, J = 15.0, 6.7, 1.3 Hz, H-9) and 5.54 (dtt, J = 15.0, 7.0, 1.4, H-8), and the downfield shift of one of the methylene groups to δ _H 3.27 (d, J = 6.7 Hz, H-10). The HMBC correlations from H-9 to C-7 (δ _C, 38.7), C-8 (δ _C, 126.3), C-10 (δ _C, 39.6), and C-11 (δ _C, 135.5) and from H-8 to C-6 (δ _C,79.4), C-7, C-9 (δ _C, 134.9), and C-10 as well as the COSY correlations from H-6 (δ _H 4.50, dtd, J = 11.4, 6.2, 4.2 Hz) to H-7 (δ _H 2.49 and 2.43), from H-7 to H-8, from H-8 to H-9, and from H-9 to H-10 confirmed that the chain linking the 1,3-benzodioxole to the 5,6-dihydro-pyran-2-one was a butyl with an unsaturation in 2 instead of an ethyl in 1 ([Fig. 1]). The absolute configuration at C-6 was established by comparison of the experimental and calculated ECD spectra. The calculated ECD spectrum for the 6S-stereoisomer showed a good fit with the experimental data ([Fig. 2 B]). Thus, the structure of compound 2 was determined as (S)-6-(4-(benzo[d][1, 3]dioxol-5-yl)but-2-en-1-yl)-5,6-dihydro-2H-pyran-2-one and named beilschmiediapyrone.

In addition, five known compounds, trans-fagaramide (3) [11], [14], pipercallosidine (4) [15], piperine (5) [16], pellitorine (6) [17], and N-isobutyl-2E-decenamide (7) [18] were also identified based on comparison with spectroscopic data from the literature ([Fig. 1]).

The cancer chemopreventive activity of the dichloromethane extract, the fractions, and the seven compounds were evaluated. The extract induced QR activity more than fourfold, and inhibited 86.7% of NF-κB activity at 20 µg/mL (Table 1S, Supporting Information). Five compounds showed activity on both QR induction and NF-κB inhibition ([Table 2] and Figs. 1S and 2S, Supporting Information). Compounds 1 and 2 were the most potent with activity in the low µM range. The activity of desmethoxydihydromethysticin (1) towards NF-κB is consistent with previous reports revealing the NF-κB inhibitory properties of methysticin and dihydromethysticin isolated from kava (P. methysticum water extract), a traditional beverage from the South-Pacific islands with cancer chemopreventive activity [19]. Among the three previously mentioned compounds (methysticin, dihydromethysticin, and 1), methysticin was the most potent on NF-κB (IC₅₀ = 0.7 µM), highlighting the role of the C7 – C8 unsaturation for the activity. Interestingly, however, desmethoxydihydromethysticin (1) was over 30-fold more active than its methoxylated analogue dihydromethysticin (IC₅₀ = 73 µM [19]), suggesting a negative effect of the methoxy moiety on the activity. The similar activity between compounds 1 and 2 suggested that the length of the linker between the aromatic ring and the lactone did not play an important role in the NF-κB inhibitory activity. Overall, the higher potency of compounds 1 and 2, as well as the lack of activity of compounds 3 and 5, suggest a positive impact of the lactone on the activity.

This is the first time that pipercallosidine (4) and N-isobutyl-2E-decenamide (7) are shown to induce QR and inhibit NF-κB. Li et al. [20] reported that pipercallosidine (4) inhibited the growth of cancer cell lines such as WI38, VA13, and HepG2 with IC₅₀ values between 14.5 and 31.6 µM. In addition, consistent with the present results, the anti-inflammatory effect of pellitorine (6) was already described in the same concentration range [21]. Surprisingly, the results obtained with piperine (5) were different from what other authors have previously described. Pradeep and Kuttan [22] showed that piperine inhibited NF-κB and cytokine expression of some proinflammatory genes in melanoma B16F-10 cells at a concentration of 8.8 µM. This difference can be explained by the difference in the cells used. Similarly, the incubation time was relatively long (48 h) in the previous study, compared to 5 h in the current study.

In several Beilschmiedia species, compounds active at various stages of the carcinogenesis process have already been identified [23], [24]. For example, ferruginic acids B, C, and J isolated from Beilschmiedia ferruginea showed high binding affinity for two antiapoptotic proteins, namely, Mcl-1 and Bcl-xL, with K_i values between 6 and 20 µM [23]. Moreover, endiandric acid analogues and lignans isolated from Beilschmiedia tsangii showed anti-inflammatory activity measured through inducible nitric oxide synthase inhibition [24]. These results show that the genus Beilschmiedia is promising for the search of cancer chemopreventive compounds. Therefore, results obtained in this study could be the base for further investigations, especially because B. mannii is also used for cooking by the population around the Taï forest.

Materials and Methods

General experimental procedures

Optical rotation was measured on a Jasco P-1030 polarimeter (EtOH, c in g/100 mL). HPLC analysis was performed on a Hewlett Packard Series 1100 apparatus coupled with a diode array UV detector (190 – 500 nm). Extract fractionation was done using MPLC (Büchi, C series) and compound isolation was performed on a SpotPrep HPLC (Armen Instrument). UV spectra were recorded on a Perkin-Elmer Lambda-25 spectrophotometer. MS data were measured on LCT Premier micromass time-of-flight mass spectrometer. NMR spectroscopic data were recorded on a 500 MHz Varian INOVA NMR spectrometer. Chemical shifts are reported in parts per million (δ) using the residual CD₃OD signal (δ _H 3.31; δ _C 49.0) as internal standards for ¹H and ¹³C NMR, respectively, and coupling constants (J) are reported in Hz.

Plant material

The fruits of B. mannii were collected in December 2014 in Taï National Park, South-Western region of Côte dʼIvoire, and identified by Mr. Henri Téré at the botanic laboratory of the CSRS. The name of the plant species was confirmed by the Centre National de Floristique of Abidjan. A voucher specimen (N°1614) was deposited in the herbarium of the CSRS.

Extraction and isolation

The dichloromethane extract of the fruits of B. mannii was selected for further investigation. The whole fruits were washed after collection, lyophilized, and powdered. Six hundred grams were macerated in 6 L of dichloromethane under mechanical stirring (2 × 24 h) at room temperature and then filtered. The solution was evaporated at 40 °C and dried under a stream of nitrogen, yielding 22 g of crude extract. Ten grams of crude extract were fractionated into 53 fractions (F1 – F53) by MPLC, using a Scorpio silica column (460 × 49 mm i. d., 15 – 20 µm particle size) and a mobile phase made of hexane/EtOAc (15% EtOAc from 0 to 30 min; 15 – 70% EtOAc from 30 min to 5 h and 70 – 100% EtOAc from 5 h to 5 h 30 min) at a flow rate of 40 mL/min. Fractions were dissolved in HPLC grade methanol at 4 mg/mL and analyzed by HPLC using a C18-Xbridge (250 × 4.6 mm i. d., 5 µm) column with MeOH/H₂O (40% MeOH from 0 to 5 min; 40 – 100% MeOH from 5 to 25 min; 100% MeOH from 25 to 30 min and 100 – 40% MeOH from 30 to 35 min) at a flow rate of 1 mL/min. Fractions with a similar profile were gathered to obtain 20 fractions (FF1 – FF20). Based on the amount, the biological activity (Table 1S, Supporting Information), and the chemical profile, fractions FF9 (114.02 mg), FF10 (103.97 mg), FF14 (143.49 mg), and FF16 (116.02 mg) were selected for further fractionation using a C18 Xbridge column (250 × 10 mm i. d., 5 µm) with MeOH/H₂O at a flow rate of 4.7 mL/min. The separation conditions of the fractions where 35% MeOH from 0 to 6 min, 35 – 100% MeOH from 6 to 21 min, 100% MeOH from 21 to 26 min, 100 – 35% MeOH from 26 to 31 min for FF9; 40% MeOH from 0 to 6 min, 40 – 100% MeOH from 6 to 21 min, 100% MeOH from 21 to 26 min, 100 – 40% MeOH from 26 to 31 min for FF10 and FF14; and 65% MeOH from 0 to 6 min, 65 – 100% MeOH from 6 to 21 min, 100 – 65% MeOH from 21 to 26 min for FF16. Fraction FF9 provided compounds 2 (14 mg), 6 (5.2 mg), and 7 (9 mg), fraction FF10 afforded compound 1 (11.5 mg), fraction FF14 gave compounds 3 (15.9 mg) and 4 (3.2 mg), and fraction FF16 provided compound 5 (8.1 mg).

Electronic circular dichroism calculation

Conformational analysis of compounds 1 and 2 were performed with MacroModel 9.1 software (Schrödinger, LLC). The conformers within a 3 kcal/mol energy window from global minima were selected for geometrical optimization and ECD calculation using DFT/CAM-B3LYP/6-31G(d,p) and TD-DFT/CAM-B3LYP/6-31G(d,p) in MeOH as a solvent, respectively. All calculations were carried out using the Gaussian 09 package [25]. The reconstructed spectra were calculated using SpecDis V1.61 software [26]. The 2D and 3D structures of the compounds were drawn using ChemOffice Professional 17 package (CambridgeSoft, PerkinElmer).

Desmethoxydihydromethysticin (1): yellowish oily appearance; UV (MeOH) λ _max nm (log ε) 231 (3.77), 285 (3.53);[α]_D ²⁰ + 8.3 (c 0.1, EtOH); ¹H and ¹³C NMR data, see [Table 1]; HRESIMS m/z 247.0974 [M + H]⁺ (calcd. for C₁₄H₁₅O₄, 247.0970).

Beilschmiediapyrone (2): yellowish oil, UV (MeOH) λ _max nm (log ε) 231 (3.81), 285 (3.58); [α]_D ²⁰ + 59.5 (c 0.1, EtOH); ¹H and ¹³C NMR data, see [Table 1]; HRESIMS m/z 273.1119 [M + H]⁺ (calcd. for C₁₆H₁₇O₄, 273.1127).

Quinone reductase induction activity

The potential of the samples to induce QR was evaluated by the method used by Cuendet et al. [4], with small modifications. Hepa 1c1c7 cells (ATCC) were maintained in α-MEM culture medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin (Life Technologies) (100 U/mL), and streptomycin (Life Technologies) (100 µg/mL) at 37 °C under a CO₂ atmosphere (5%) to 80% confluence. One hundred µL of cell suspension were seeded in 96-well plates at a concentration of 2 × 10⁴ cells/mL and subsequently incubated for 24 h. Cells were then treated with the samples at various concentrations (dissolved in DMSO, 0.05% final concentration) and incubated for an additional 48 h. After incubation, cells were treated with 21.5 µM of Hoechst 3342 (Sigma-Aldrich) for 20 min at 37 °C and rinsed with PBS. Cell viability was evaluated by counting the stained nuclei using the fluorescent microscope of the Cytation 3 multimode plate reader (Biotek). Cells were then lysed by adding 50 µL of lysis buffer (40 mM Tricine-KOH, 2 mM Na₂EDTA-KOH, 0.1 mM PMSF, 20 mg/ml polidocanol in water at pH 7.4) and incubating for 10 min at 37 °C under stirring at 450 rpm. QR activity was then determined by measuring the NADPH dependent by menadiol mediated reduction of MTT (Sigma-Aldrich) in blue formazan. After 5 min incubation at room temperature, with stirring at 300 rpm, the absorbance was measured at 595 nm with the Cytation 3 multimode plate reader. The CD was calculated using GraphPad Prism 6. For each compound, the experiments were performed three times in triplicate and 4′-bromoflavone (Sigma-Aldrich) was used as a positive control.

Nuclear factor-kappa B inhibitory activity

The potential of samples to inhibit NF-κB was measured using genetically modified human kidney cells (HEK-293/NF-κB-Luc). The experiments were conducted according to the protocol described by Tran et al. [27], with slight modifications. Cells were cultured at 37 °C in a CO₂ atmosphere (5%) in Dulbeccoʼs modified glucose-enriched Eagleʼs culture medium (Invitrogen) supplemented with hygromycin B (Sigma-Aldrich) at 100 µg/mL, penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% deactivated FBS (Life Technologies). One day before the test, cells were pretreated for 1 h with a solution of 2.5 µM CMFDA (Invitrogen) (FBS free medium). One hundred µL of the cell suspension were seeded at a final concentration of 10 × 10⁴ cells/mL in a 96-well plate and then incubated overnight at 37 °C in 5% CO₂.

Cells were then treated with the samples (dissolved in DMSO, 0.05% final concentration) and stimulated by TNF-α (20 ng/mL; Biovision) for 5 h. After incubation, cells were lysed at room temperature for 10 min with lysis buffer (Reporter Lysis Buffer 1X; Promega). The fluorescence of the Cell Tracker Green CMFDA (cell viability) and the luminescence of luciferase were read with a Cytation 3 multimode plate reader. IC₅₀ values were determined using GraphPad Prism 6 software. The experiments were performed three times in duplicate. Parthenolide (Sigma-Aldrich) was used as a positive control.

Conflict of Interest

The authors declare no conflicts of interest.

Acknowledgements

The authors thank the Programme dʼAppui Stratégique à la Recherche Scientifique (PASRES) for the financial support for field research and the CSRS for providing various research and infrastructural facilities. We thank Mr. Henri Téré for botanical assistance. We are also grateful to the Swiss Confederation for a training fellowship in Geneva to ARCA, to Drs. Nicolas Martin and Sylvian Cretton for their technical assistance, and to Jasmina Saric for revising our manuscript. This article was prepared with the support of the DELTAS Africa Initiative [Afrique One-ASPIRE/DEL-15-008]. Afrique One-ASPIRE is funded by a consortium of donors including the African Academy of Sciences (AAS), the Alliance for Accelerating Excellence in Science in Africa (AESA), the New Partnership for Africaʼs Development Planning and Coordinating (NEPAD) Agency, the Wellcome Trust [107753/A/15/Z] and the UK government.

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Correspondence

• References

• 1 WHO. Cancer. Technical tips online. Available at: http://www.who.int/news-room/fact-sheets/detail/cancer Accessed October 17, 2018
  PubMed
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  CrossrefPubMedSearch in Google Scholar
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