A new phenanthrene derivative from Entada abyssinica with antimicrobial and antioxidant properties

2.1 Phytochemical analysis

The methanol crude extract of E. abyssinica root bark were subjected repeatedly to column chromatography over silica gel to afford eight compounds (1–8, Figure 1). The known compounds (2–8) were identified by comparison of their spectroscopic data with those reported in literature. However, the structure elucidation of the new compound (1) is reported below.

Figure 1:

Structures of compounds 1–8 isolated from E. abyssinica.

Compound (1) was obtained as a white amorphous powder and was positive to the ferric chloride test indicating its phenolic nature. Its molecular formula C₁₇H₁₆O₅ was determined through the use of HR-ESI-MS which showed the protonated molecular ion pic at m/z = 301.1047 ([M+H]⁺, C₁₇H₁₇O₅ ⁺, calcd. m/z = 301.1076) and elementary analysis indicate 10° of unsaturation. The IR spectrum of (1) showed strong absorption bands associated with a hydroxyl (3382 cm⁻¹), together with the one of C–O (1023 cm⁻¹), while its UV spectrum showed maxima absorption bands at λ _max 224 and 280 nm characteristic of phenanthrene derivative [11].

Moreover, the ¹H NMR spectrum (Table 1) of (1) exhibited the signals of six aromatic protons among which the signal of one pair of ortho-coupled aromatic protons δ _H = 8.91 ppm (1H, d, J = 9.1 Hz, H-9) and δ _H = 8.30 ppm (1H, d, J = 9.1 Hz, H-10) [12] and four singlets at δ _H = 8.32 ppm (1H, s, H-4), 7.89 ppm (1H, s, H-5), 8.38 ppm (1H, s, H-8) and 7.76 ppm (1H, s, H-1) characteristics of a phenanthrene monosubstituted skeleton [13, 14]. It also showed a singlet of two protons at δ _H = 6.36 ppm (2H, s) suggesting a methylenedioxy moiety [15] and two singlets of three protons at δ _H = 4.05 ppm (3H, s, 6-OMe) and 4.24 ppm (3H, s, 7-OMe) indicating the presence of two methoxy groups. The decoupled ¹³C NMR spectrum of (1) exhibited 17 carbon signals (Table 1) including those of two methoxy groups at δ _C = 56.8 ppm (6-OMe) and 57.8 ppm (7-OMe), one of a methylenedioxy unit at δ _C = 103.2 ppm (2-O-CH₂-O) [15, 16] and of 14 tertiary and quaternary aromatic carbons signals at δ _C = 148.9 ppm (C-2), 149.4 ppm (C-3), 105.1 ppm (C-4), 132.6 ppm (C4a), 132.9 ppm (C-4b), 109.2 ppm (C-5), 152.0 ppm (C-6), 158.7 ppm (C-7), 103.8 ppm (C-8), 124.6 ppm (C-8a), 119.8 ppm (C-9), 130.5 ppm (C-10), 120.4 ppm (C-10a) and 106.2 ppm (C-1) confirming the phenanthrene skeleton [13]. Moreover, extensive analysis of ¹H–¹H COSY, HSQC and HMBC spectra (Figure 2) led to the assignment of ¹H and ¹³C resonances. The location of the methylenedioxy group was confirmed by the HMBC correlations observed between its protons at δ _H = 6.36 ppm and the O-bearing aromatic carbon at C-2 (δ _C = 148.9 ppm), this carbon is also having correlations with protons at δ _H = 7.79 ppm (H-1) and 8.32 ppm (H-4). The cross peak correlation observed between the methoxy protons at δ _H = 4.05 ppm (6-OMe) and 4.24 ppm (7-OMe) with carbons at δ _C = 152.0 ppm (C-6) and 158.7 ppm (C-7) respectively show that they are linked at C-6 and C-7. Consequently, the structure of (1) was elucidated as a new phenanthrene derivative named 2-(hydroxymethoxy)-6,7-dimethoxyphenanthren-3-ol to which we gave the trivial name phenentada.

Figure 2:

¹H-¹H COSY and HMBC correlations observed in compound 1.

The structures of known compounds were identified by comparison of their spectroscopic data with those reported in the literature as (8 S, 13E)-kolavic acid 15-methyl ester (2) and 8 S-kolavic acid 15-methyl ester (3) [6]; 8 S-kolavic acid 15-methyl ester (3) [7]; 8 S-kolavic acid 18-methyl ester (4) [6, 7]; 13,14,15,16-tetranorclerod-3-ene-12,18-dioic acid (5) [10]; 1′,26′-bis-[(S)-2,3-dihydroxypropyl] hexacosanedioate (6) [7]; campesterol (7) [17] and 3-O-β -d-glucopyranosylstigmasterol (8) [18] (Figure 1). Kolavic acid and derivates exhibit anti-inflammatory properties through various mechanisms contributing the anti-inflammatory properties of E. abyssinica [9, 19] and also inhibit Trypanosoma brucei [5], howewer, campesterol and derivates possess various pharmacological properties including cholesterol lowering effects, anti-inflammatory, antibacterial and antifungal activities and also have chemopreventive effects against cancer [18]. The identification of these compounds contributes to the verification of some therapeutic virtues of this species.

2.2 Antimicrobial activity

E. abyssinica is traditionally used in Cameroon to treat coughs, rheumatism, skin infection, diarrhoea and fever [5, 6]. The results of in vitro antimicrobial activities of the MeOH extract of root bark as well as compounds 1, 3–6 and the mixture of 2/3 against pathogenic bacteria and yeast are presented in Table 2. This table present the inhibition parameters (MIC, MBC and MBC/MIC ratio) of the crude extract and tested compounds from E. abyssinica. The crude extract and isolates showed varying levels of antimicrobial properties against yeast and bacteria strains tested. Indeed, the antimicrobial activity of plant extracts can be classified as significant (MIC < 100 μg mL⁻¹), moderate (100 < MIC ≤ 625 μg mL⁻¹) and weak (MIC > 625 μg mL⁻¹) [20]. According to this classification, the inhibition potential of the methanol crude extract of E. abyssinica could be considered as significant against yeasts (MIC ranging from 15.6–31.3 μg mL⁻¹) and bacteria (MIC ranging from 7.81–31.3 μg mL⁻¹) except on Salmonella enterica strain. Furthermore, the gram (+) S. epidermis was the most sensitive with a MIC value of 7.81 μg mL⁻¹ followed by Candida albicans (MIC = 15.6 μg mL⁻¹) and the Candida parapsilosis (MIC = 32.3 μg mL⁻¹). Additionally, the results obtained from the crude extract in this study were in line with those obtained by others authors in the literature. In fact, Harun et al. [21] demonstrated that the methanol extract of Eleocharis spiralis stem bark exhibited promising antifungal activity against three dermatophytes strains. While, Baidoo et al. and Dzoyem et al. [22, 23] showed antibacterial potential of methanol extract of Epimyrma africana root bark and E. abyssinica leaves respectively. According to the criteria used by Gatsing and Adoga [24], the antibacterial substance is considered as bactericidal, when the ratio MBC/MIC ≤ 4 and bacteriostatic when MBC/MIC > 4 [24]. Base on those criteria, the crude extract acted as good bactericide and fungicide against these pathogens suggesting that they could be potent candidates for the treatment of skin and other infectious diseases. Regarding pure compounds, antimicrobial cut-off points have been defined in the literature to enable the understanding of their effectiveness as follows: highly active (MIC < 1 μg mL⁻¹), significantly active (1 = MIC ≤ 10 μg mL⁻¹), moderately active (10 < MIC ≤ 100 mL⁻¹) and weakly active (100 < MIC ≤ 1000 μg mL⁻¹) [25]. Based on this cut-off, compounds 1, 4 and the mixture of 2/3 showed significant activity against C. albicans with MIC ranging from 3.12–6.25 μg mL⁻¹ while, compounds 4, 5, 6 and the mixture of 2/3 inhibited significantly Candida parasilosis with MIC values of 3.12 μg mL⁻¹ each. The values of MIC of all the tested compounds in comparison to those of the crude extract against yeasts could justify his significant activity and suggesting that many of them should proceed by synergism to enable the higher activity of the crude extract. Regarding the antibacterial test, all the screened compounds showed high to significant activity (MIC values between 0.78 and 1.56 μg mL⁻¹) against the gram (+) bacteria S. epidermis with the best activity observed to the mixture 2/3 (MIC = 0.78 μg mL⁻¹) compare to ciprofloxacin used as reference drug. Furthermore, compound 5 showed the best activity against the gram (−) bacteria Escherichia coli with MIC = 1.56 μg mL⁻¹ while compound 1 showed a significant activity against all the selected gram (−) bacteria with MIC values ranging from 3.12–6.25 μg mL⁻¹ and then confirm the antimicrobial potential of phenanthrene derivatives against a wide range of human pathogens [13]. All these compounds acted as bactericidal (MBC/MIC ≤ 4). Moreover, these results are in accordance with those obtained by Tchinda et al. [6] and Dzoyem et al. [6, 23] which have evaluated the antimicrobial activities of compounds 2, 3 and 4, and which also assumed that the presence and position of ester and carboxylic groups in isolated diterpenoids played an important role in their antimicrobial activity.

2.3 Antioxidant activity

The in vitro antioxidant properties of crude extract and some of the isolated compounds along with standard drug (Gallic acid), using DPPH assays were reported in Table 3. Crude extract as well as compounds 1, 3–6 and the mixture of 2/3 exerted good scavenging capacities toward DPPH radical but not really significant with comparison to gallic acid. The scavenging capacity of each compound was similar except those of the compound 1 which had a SC₅₀ of 4.72 ± 0.28 μg mL⁻¹ which is similar to the gallic acid with a SC₅₀ value of 2.86 ± 0.53 μg mL⁻¹. This compound belongs to the phenolic group which is well known for their antioxidant capacities due to the formation of stable radical when they give hydrogen to the radical DPPH [21, 26]. The scavenging capacity of the other compounds may be attributed to the presence of carboxyl group that classifies them as organic acids which can share their hydrogen with the radical DPPH. Relation between phenolic and organic acids with antioxidant capacities has been reported earlier [13, 21, 22, 27].

In conclusion, the phytochemical investigation of the MeOH extract from the root bark of E. abyssinica led to the isolation and characterization of eight compounds including one new phenanthrene derivative trivially name phenentada. It appears that the methanol extract, phenentada (1) and the mixture of 2/3 possess the most antifungal and antibacterial properties. Regarding the antioxidant activity, compound 1 exhibited an activity similar to that of gallic acid. In order to verify the synergetic and/or antagonism effect of different constituents in the mixture 2/3 which were more active than ciprofloxacin used as reference drug, we will focused our future investigation on isolation of compound 2 followed by evaluation of the antibacterial activity on the same strain. Additionally, these results highlight the traditional use of E. abyssinica in the treatment of infectious diseases, especially those caused by the tested microorganisms. Therefore, further studies should be conducted such as the evaluation of the toxicity of the extracts and the isolated compounds with the elucidation of their mechanism of action in order to propose a plant-based preparation for phytomedicine.

3.1 General experimental procedures

Gravitational column chromatography (CC) was performed using silica gel (Macherey-Nagel) (Si gel) 60 (70–230 mesh), 60 (240–400 mesh) while precoated Si gel 60 F254 (Merck) aluminum plates were used for thin layer chromatography (TLC). Si gel CC was eluted with n-hexane, to which EtOAc and MeOH were gradually added to increase the polarity while TLC plate was eluted with mixtures of solvents such as. TLC plates were sprayed with 50% diluted sulfuric acid and heated at about 96 °C or visualized under UV lamp at 254 and 365 nm. All the reagents were of analytical grade. The HR-ESI-MS spectra were performed with a spectrometer (QTOF Bruker, Germany) equipped with a HR-ESI source. The spectrometer operates in positive ion mode (mass range: 100–1500, with a scan rate of 1.00) with automatic gain control to provide high-accuracy mass measurements within 0.40 ppm deviation using Na formate as calibrant. The following parameters were used for experiments: spray voltage of 4.5 kV, capillary temperature of 200 °C. Nitrogen was used as sheath gas (10 L/min). The spectrometer was attached to an Ultimate 3000 (Thermo Fisher, Germany) UHPLC system consisting of LC pump, Diode Array Detector (DAD) (λ = 190–600 nm), auto-sampler (injection volume 10 μL) and column oven (40 °C). The separations were performed using a Synergi MAX-RP 100 A (50 × 2 mm, 2.5 μm particle size) with a H₂O (+0.1% HCOOH) (A)/acetonitrile (+0.1% HCOOH) (B) gradient (flow rate 500 μL/min, injection volume 5 μL). Samples were analyzed using a gradient program as follows: 95% A isocratic for 1.5 min, linear gradient to 100% B over 6 min, after 100% B isocratic for 2 min, the system returned to its initial condition (90% A) within 1 min and was equilibrated for 1 min. Infra-Red (IR) spectroscopy was performed on an Alpha spectrometer (Bruker, MA, USA). The spectra were recorded from 4000 to 600 cm⁻¹ in attenuated total reflectance (ATR) mode on a diamond crystal. The NMR experiments were performed on Bruker Avance 600 and Bruker Avance 500 spectrometers (Bruker, Belguim) in CD₃OD and in CDCl₃, DMSO-d ₆, and Pyridine-d ₅ respectively containing tetramethylsilane (TMS) as the internal standard. The resulting solution will be filtered and then concentrated by the rotary evaporator (Rotavapor (BUCHI) R II) to give the crude extract.

3.2 Collection and identification

The roots bark of E. abyssinica Steud. ex A. Rich were collected separately in October 2019 from Babadjou (Mbouda), in the Western Region of Cameroon. Identification was made by Mr. Victor Nana, a botanist from the National Herbarium of Cameroon in Yaoundé by comparison to the existing voucher specimen HNC 10672/SFR/CAM.

3.3 Extraction and isolation

The roots bark of E. abyssinica was chopped, air-dried and ground. The resulting powder (2 kg) was extracted by maceration using MeOH (100%) for 72 h and then filtered. The filtrate

The 40 g of the crude extract was assessed on silica gel column chromatography method (70–230 μm (Merck)) eluted with a gradient mixture of n-hexane-EtOAc (from 100/0 v/v to 0/100 v/v) followed by a gradient mixture of EtOAc/MeOH (from 100/0 v/v to 0/100 v/v). Collected fractions were pooled out according to their TLC profiles: F1 (14.7 g), F2 (2.4 g), F3 (10.6 g) and F4 (5.3 g). Fraction F1 (14.7 g) was subjected to column chromatography and eluted with hexane/EtOAc (9/1–7/3, v/v) to yield three subfractions (F1A – F1C). F1A was assessed on silica gel column chromatography eluted with hexane/EtOAc (7/3, v/v) to afford compound 1 (15 mg) and compound 2/3 (10 mg). From sub-fractions F1B and F1C, compound 3 (20 mg) precipitated as white powder and purified. Based on their TLC profiles, fractions F2 and F3 were combined and subjected to silica gel CC using a gradient elution of hexane/EtOAc (100:0–90:10, v/v) to afford four subfractions (F2,3A–F2,3D). Compound 4 (8 mg) was isolated from subfraction F2,3D after repeated silica gel and Sephadex column chromatography and compounds 5 (18 mg), 6 (12 mg), 7 (10 mg) and 8 (5 mg) were isolated from subfraction F2,3A. The chemical structure was elucidated by spectrometric method with comparison with literature data.

3.4 Antimicrobial activities assessment