A new megastigmane,known porphyrinic and galloylated bioactive derivatives from the leaves of Gymnosporia senegalensis

Abstract

Investigation of the non-polar (rich in chlorophyll) and polar fractions of the ethyl acetate extract of Gymnosporia senegalensis (Lam.) Loes. leaves led to the isolation of nine compounds, including one new megastigmane derivative, (3R*,5S*,6R*,7E,9ξ)-7-megastigmene-3,6,9-triol-3-O-β-D-(6′-O-galloyl)glucopyranoside (1), four known galloylated derivatives (2, 3, 8, 9), three known chlorophyll derivatives (4–6), and one known fatty acid (7). The structures of the isolates were determined by means of spectroscopic and spectrometric data, as well as by comparison with literature values. The extracts and isolated compounds were tested for their antibacterial, antifungal, and antioxidant activities. The isolates showed weak to high antimicrobial activities with phaeophytin A (4) being the most active one whereas phaeophorbide-a (5) and Chlorine e₆ trimethyl ester (6) exhibited the highest antioxidant activities.

Introduction

The continuous dissemination of multi-drug-resistant (MDR) strains drastically reduces the efficiency of antibiotics and consequently increases the frequency of therapeutic failure. ¹ In many regions affected by these MDR strains, local and indigenous plants are often the only available means of treating such infections. Herbal antioxidant compounds play a defensive role by preventing the generation of free radicals. This action is therefore extremely beneficial to relieve infectious diseases generating free radicals as well as diseases caused by oxidative stress such as cardiovascular diseases, diabetes, inflammation, degenerative diseases, cancer, anemia, and ischemia.^2,3

Medicinal plants typically contain mixtures of different chemical compounds that may act individually, additively, or in synergy to improve health. It has been suggested that these compounds may have been synthesized by the plant as part of its defense system. Plants that survive under harsh and variable environmental conditions are expected to accumulate a wide range of secondary metabolites. Gymnosporia senegalensis (Lam.) Loes. (Celastraceae) is one of such a plant which is traditionally used as astringent, to treat toothaches, stomachaches, typhoid, malaria, and inflammatory disorders. It is widely distributed in semi-arid regions of Africa.^4,5 G. senegalensis (Lam.) Loes. is also called Maytenus senegalensis (Lam.) Exell. ⁶ Previous biological studies reported that extracts from various parts (stem, leaves, bark, and root) of G. senegalensis exhibited anti-inflammatory,^5,7,8 antimicrobial,^5,8 antiplasmodial, ⁹ antioxidant, ⁸ and antiproliferative ⁸ activities. In addition, compounds of different classes, including alkaloids, terpenoids, sugars, lignans, and flavonoids, have been reported from M. senegalensis.^5,10 Tatsimo et al. ¹¹ recently reported the isolation of glycerol, cyanogenic, and phenolic derivatives from leaves of G. senegalensis. In our effort to investigate the potential of G. senegalensis as source of lead compounds, we fractionated the non-polar (rich in chlorophyll) and polar fractions of ethyl acetate leaf extract. We report herein the isolation, antimicrobial, and antioxidant activities of one new megastigmane derivative (1) and eight known compounds (2–9).

Results and discussion

Chemical analysis

Phytochemical investigation of chlorophyll-rich and polar fractions from ethyl acetate extract of G. senegalensis leaves led to the isolation of nine compounds including one new megastigmane (1) and eight known secondary metabolites. The known constituents include 1-O-β-D-(6′-O-galloyl)-glucopyranosyl-3-methoxy-5-hydroxybenzene (2), ¹² 2,6-di-O-galloyl-β-D-glucose (3), ¹³ phaeophytin A (4), ¹⁴ phaeophorbide-a (5), ¹⁵ Chlorine e₆ trimethyl ester (6), ¹⁶ (4Z,7Z,10Z)-octadecatrienoic acid (7), ¹⁷ procyanidin B5 3,3′-di-O-gallate (8) ¹⁸ and procyanidin B2 3,3′-di-O-gallate (9) ¹⁸ (Figure 1).

Figure 1.

Structure of isolated compounds (1–9).

Compound 1, [α]_D ²³ −15.2 (c 0.50, MeOH), was obtained as a yellowish oil. The molecular formula, C₂₆H₃₈O₁₂, was established by HR-ESI-MS (-) (541.2300 [M-H]⁻, calcd for C₂₆H₃₇O₁₂ 541.2285) and HR-ESI-MS (+) m/z: 565.2274 [M+Na]⁺, calcd for C₂₆H₃₈O₁₂Na 565.2261. The IR spectrum displayed absorption bands characteristic of hydroxyl group (3372 cm⁻¹), carbonyl group (1623 cm⁻¹), and aromatic ring (1453, 1370 cm⁻¹), and the UV spectrum showed absorptions at λ_max 216 and 276 nm. The ¹³C NMR spectrum in association with DEPT-135 displayed 26 carbons, including 7 quaternary carbons, 12 methines, 3 methylenes, and 4 methyls. Analyses of 1D and 2D NMR (400 MHz, CD₃OD) spectra as outlined indicated that compound 1 is a galloylated megastigmane O-β-D-glucopyranoside. The ¹H NMR spectrum showed a singlet of two aromatic protons at δ_H 7.07 ppm, which in HMBC spectrum correlate with carbons at δ_c 167.0, 145.3, 138.5, 120.2, and 108.8, characteristic of a galloyl moiety.^19,20 The ¹H and ¹³C NMR spectra showed signals at δ_H 4.48 [1H, dd, J = 11.8, 7.1 Hz, (H_6′a); δ_C 63.6 (C_6′)], 4.43 [1H, dd, J = 11.8, 2.7 Hz, (H_6′b); δ_C 63.6 (C_6′)], 4.31 [1H, d, J = 7.8 Hz (C_1′); δ_C 101.7 (C_1′)], 3.55 [1H, m, (H_5′); δ_C 73.9 (C_5′)], 3.33 [2H, m, (H_3′ and H_4′); δ_C 76.8 (C_3′), 71.0 (C_4′)] and 3.15 [1H, m, (H_2′); δ_C 73.8 (H_2′)] indicative of a β-glucose moiety.^19,20 The nature of sugar was confirmed on NOESY spectrum where specific spatial correlations were observed between the anomeric proton δ_H 4.31 (H_1′) and protons at δ_H 3.55 (H_5′) and 3.33 (H_3′), between the proton at δ_H 3.33 (H_4′) and proton at δ_H 3.15 (H_2′) (Figure 2). Although the HMBC correlation of methylene group at C_6′ to carbonyl carbon at C_7′′ was missing in Figure S6, the glucose was suggested to be substituted at position 6′ due to the downfield shift of the methylene group at δ_H 4.48 (dd), 4.43 (dd), and δ_C 63.6.^19,20 Besides, the ¹H and ¹³C NMR data obtained with DMSO-d₆ (Figures S8 and S9) also confirmed the presence of 6′-O-galloyl-β-glucopyranosyl moiety in compound 1 by comparison of data with those of 2-butanone 4-glucopyranoside 6′-O-gallate ²¹ and (2R)-methyl [(6′-O-galloyl)-β-D-glucopyranosyloxy]phenylacetate. ¹¹ In addition, on the ¹H NMR spectrum (400 MHz, CD₃OD), signals of two doublet [δ_H 0.64 (J = 6.4 Hz; δ_C 15.0); δ_H 1.22 (J = 6.4 Hz; δ_C 22.8)], two singlet [δ_H 0.83 (δ_C 23.7 and 24.5)] methyls, and one pair of trans olefinic protons [δ_H 5.45 (d, J = 16.0 Hz; δ_C 132.6); δ_H 5.64 (dd, J = 16.0, 5.9 Hz; δ_C 134.0)] were observed. These data were characteristic of a megastigmane-type skeleton by comparison with literature values. ²² Further analyses of HMBC spectrum showed long-range correlations between the anomeric proton at δ_H 4.31 (H_1′) and the carbon at δ_C 74.8 ppm (C₃) (Figures S6 and 2). Thus, the megastigmane skeleton and galloyl group are linked to the glucose at position 1′ and 6′, respectively. This structure was also corroborated by MS/MS analysis which showed a pseudomolecular ion peak at m/z 565 [M+Na]⁺, and its product ion peaks at m/z 413 [M–C₇H₅O₄+Na+H]⁺ and m/z 355 [M–C₁₃H₂₃O₂+Na+H]⁺ (galloyl + glucose) (Figures 3 and S10c). Further analyses of NOESY spectrum indicated spatial correlations between the anomeric proton at δ_H 4.31 (H_1′) and proton at δ_H 3.73 (H₃); between the proton at δ_H 3.73 (H₃) and protons at δ_H 0.83 (H₁₂), 1.44 (H_2a) and 1.66 (H₅) which in turn correlated with the olefinic proton at δ_H 5.45 (H₇) (Figures 2 and S7). These spatial correlations indicated an α-orientation for H_1′, H₃, H_2a, H₅, H₇, and H₁₂, suggesting that the stereocenters have 3R*, 5S*, 6R* configurations. Therefore, compound 1 was elucidated to be (3R*,5S*,6R*,7E,9ξ)-7-megastigmene-3,6,9-triol-3-O-β-D-(6′-O-galloyl)glucopyranoside, as shown in Figure 1.

Figure 2.

Connectivities and correlations deduced from 2D spectra.

Figure 3.

MS/MS fragmentation pattern of compound 1 (m/z 565.23).

Antimicrobial and antioxidant activities

The extracts from G. senegalensis and their isolated compounds 1–9 were evaluated for their antibacterial, antifungal, and antioxidant activities (Table S1, Figures S36 and S37). The MeOH, EtOAc, and n-BuOH extracts, as well as compounds 2–6 and 8–9, were active against all the tested bacterial and fungal species (Table S1). The antimicrobial activity of plant extract was considered to be good if its minimum inhibitory concentration (MIC) value was less than 100.0 μg mL⁻¹, moderate if MIC was from 100.0 to 500.0 μg mL⁻¹, and poor over 500.0 μg mL⁻¹. ²³ Hence, the EtOAc extract exhibited good activity against Staphylococcus aureus and Pseudomonas aeruginosa, and moderate activity against the other microorganisms whereas the MeOH and n-BuOH extracts displayed moderate activities against all the tested fungal and bacterial strains. The n-hexane extract demonstrated poor activity. These findings suggest that the crude methanol extract contains several antibacterial and antifungal principles with different polarities.

Based on the antimicrobial cut-off points of pure compounds previously defined, ²⁴ compounds 2, 4, 5, 6, 8, and 9 can be considered moderately active against all the tested microorganisms whereas other compounds were in general, weakly active. The antimicrobial activities of compounds 4 and 8 against Escherichia coli and Shigella flexneri were equal to those of ciprofloxacin used as reference drug, highlighting their good antibacterial properties. Although polyphenolic compounds and chlorophyll derivatives have been reported to possess interesting activity against a wide range of microorganisms,^25,26 no study has been reported on the activity of compounds 1–3 and 5–9 against these types of pathogenic strains. The results of the minimum microbicidal concentration (MMC) values indicate that most of them are not more than fourfold their corresponding MICs. This proves that the killing effects of many tested samples could be expected on most of the sensitive microorganisms. ²⁶

The results of the radical-scavenging activity (Figure S36) demonstrated that compound 3 (EC₅₀ = 14.25 ± 1.15 µg mL⁻¹) had the highest EC₅₀ (i.e. the lowest activity) whereas compounds 5 (EC₅₀ = 7.58 ± 0.43 µg mL⁻¹) and 6 (EC₅₀ = 7.99 ± 0.11 µg mL⁻¹) had the lowest EC₅₀ values (i.e. the highest activity). Among the extracts, n-BuOH extract (EC₅₀ = 26.44 ± 0.56 µg mL⁻¹) displayed the highest activity followed in decreasing order by EtOAc extract (EC₅₀ = 37.03 ± 0.87 µg mL⁻¹), MeOH extract (EC₅₀ = 64.76 ± 0.74 µg mL⁻¹), and hexane extract (EC₅₀ = 78.11 ± 1.19 µg mL⁻¹). The DPPH free radical–scavenging activities of compounds 5 and 6 were lower than that of the standard antioxidant vitamin C. No antioxidant activity was observed with compound 7. The investigated samples also displayed concentration-dependent ferric iron reducing power (Figure S37). Indeed, for all the tested extracts/compounds, the ferric ion reducing activities increase as the concentrations increase. The EtOAc and n-BuOH extracts from G. senegalensis showed the largest reductive abilities when compared with the MeOH extract, indicating that the fractionation enhanced the antioxidant activity of these extracts and diluted that of hexane extract. Interestingly, compound 3 showed the lowest ferric iron reducing power whereas compounds 5 and 6 exhibited the highest ferric iron reducing activity at the different tested concentrations. These results corroborate the DPPH assay, where compounds 5 and 6 exhibited the best radical-scavenging activities. However, the antioxidant power of compounds 5 and 6 is lower than that of butylated hydroxytoluene (BHT) used as standard antioxidant. To the best of our knowledge, this is the first report on the antioxidant activities of compounds 3, 5, and 6. No antioxidant activity was observed with compound 7. Compounds 1, 2, 4, 8, and 9 obtained in small quantities were not tested. The antioxidant activity of compound 3 corroborated those of galloylated derivatives. ²⁷

Experimental

General experimental procedures

A JEOL ECX400 (¹H, 400 MH_Z; ¹³C, 100 MH_Z) Delta spectrometer (JEOL, Tokyo, Japan) with TMS as an internal standard was used to measure NMR spectra. HR-MS and LC-MS spectra were recorded on a hybrid ion trap time-of-flight (IT-TOF) mass spectrometer (Shimadzu, Kyoto, Japan). A JASCO P2100 digital polarimeter (JASCO International Co. Ltd., Tokyo, Japan) and a JASCO FT/IR-460 Plus spectrophotometer (JASCO International Co. Ltd.) were used to measure optical rotations and IR spectra, respectively. The stationary phases for column chromatography (CC) were either Sephadex LH-20 (GE) or silica gel (Wakogel® C-200 or 400HG, Wako Pure Chemical Industries, Ltd., Osaka, Japan), while Biotage® SNAP Ultra 10 or 100 g (25 µm) Cartridge pack (silica gel packed column) were used for medium pressure liquid chromatography (MPLC) on a Biotage Isolera™ One apparatus (Biotage, Uppsala, Sweden). TLCs were carried out on precoated glass TLC silica gel 60F₂₅₄ or RP-18 F₂₅₄s plates (0.25 mm thickness, Merck, Darmstadt, Germany). Precoated glass PLC silica gel 60F₂₅₄ plates (0.5 mm thickness) were used for Preparative TLCs. MPLC was performed using the following solvent systems (detection at 210 and 254 nm) as described by Tatsimo et al., 2019; Y₁ (flow rate: 12 mL min⁻¹; volume: 8 mL): water (A)-methanol (B):30% B for 8:45 min, 30% B to 100% for 23:45 min, and 100% B for 25 min; Y₂ (flow rate: 12 mL min⁻¹; volume of each fraction collected: 15 mL); H₂O (A)-MeOH (B): 40% B for 7:00 min, 40% B to 100% for 10:30 min, and 100% B for 10 min.

Sample collection

Leaves of G. senegalensis were collected in November 2015 at Mindif, Far North, Cameroon, and identified by Mr Tapsou, a botanist at IRAD (Institut de Recherche Agricole et de Développement) of Maroua, Cameroon. A voucher number No 22314/SRCam was obtained at the National Herbarium, Yaoundé, Cameroon.

Extraction and isolation of constituents

The extraction and fractionation were carried out as previously described. ¹¹ Previously unstudied chlorophyll fractions, A (476.2 mg) and B (277.8 mg), and polar subfractions G [G₅ (200.9 mg) and G₆ (211.5 mg)] of ethyl acetate extract were investigated. Fraction A was subjected to SiO₂ gel CC eluted with hexane-acetone gradient to give 150 subfractions of 12 mL each. Subfraction A121-150 (21.2 mg) was purified on reverse phase MPLC using system Y₁ described above to give 4 (9.2 mg). Fraction B was subjected to reverse phase MPLC using system Y₂ described above. Subfraction B15-16 (35.0 mg) was loaded on SiO₂ gel CC eluted with hexane-acetone gradient [95:5 (200 mL), 92.5:7.5 (1000 mL), 90:10 (300 mL), 80:20 (100 mL), and 50:50 (200 mL)] to give compound 7 (8.4 mg). Subfractions B17-18 (55.5 mg) and B19-28 (75.0 mg) followed the same process but fractions obtained at hexane-acetone (92.5:7.5) were purified on preparative-TLC eluted with hexane-acetone 30% to yield compounds 5 (21.6 mg) and 6 (13.6 mg). G₅ (200.9 mg) was loaded on Sephadex column eluted with a step gradient of H₂O-MeOH(v/v): 1:1 (100 mL), 3:7 (100 mL), 1:9 (100 mL), and 0:1 (500 mL); and subfractions were purified by normal or reverse phase TLC eluted with CHCl₃-MeOH-H₂O (70-30-5) and H₂O-MeOH (1:1) to give 1 (4.9 mg) and 8 (9.8 mg). G₆ (211.5 mg) underwent the same process as G5 to yield 3 (13.5 mg) and 9 (8.1 mg).

(3R*,5S*,6R*,7E,9ξ)-7-megastigmene-3,6,9-triol-3-O-β-D-(6′-O-galloyl) glucopyranoside (1): yellowish oil; [α]_D ²³ −15.2 (c 0.50, MeOH). UV(PDA): λ_max 216, 276 nm. IR (KBr)ν_max: 3372, 2922, 2363, 1623, 1453, 1370, 1235, 1075 cm⁻¹. ¹H NMR (400 MHz, CD₃OD): δ 7.07 (2H, s, H_2′′ and H_6′′), 5.64 (1H, dd, J = 16.0, 5.9 Hz, H₈), 5.45 (1H, d, J = 16.0 Hz, H₇), 4.48 (1H, dd, J = 11.8, 7.1 Hz; H_6′a), 4.43 (1H, dd, J = 11.8, 2.7 Hz, H_6′b), 4.31 (1H, d, J = 7.8 Hz, H_1′), 4.26 (1H, m, H₉), 3.73 (1H, m, H₃), 3.55 (1H, m, H_5′), 3.33 (2H, m, H_3′ and H_4′), 3.15 (1H, t, J = 8.2 Hz, H_2′), 1.66 (1H, m, H₅), 1.64 (1H, m, H_4b), 1.62 (1H, m, H_2b), 1.44 (1H, m, H_2a), 1.38 (1H, m, H_4a), 1.22 (3H, d, J = 6.4 Hz, H₁₀), 0.83 (6H, s, H₁₁ and H₁₂), and 0.64 (3H, d, J = 6.4 Hz, H₁₃). ¹H NMR (400 MHz, DMSO-d₆): δ 6.92 (2H, s, H_2′′ and H_6′′), 5.55 (1H, dd, J = 15.6, 5.7 Hz, H₈), 5.30 (1H, d, J = 15.6 Hz, H₇), 4.33 (1H, dd, J = 11.6, 1.8 Hz; H_6′a), 4.22 (1H, dd, J = 11.6, 6.4 Hz, H_6′b), 4.09 (1H, m, H₉), 3.67 (1H, m, H₃), 1.06 (3H, d, J = 6.5 Hz, H₁₀), 0.77 (3H, s, H₁₁), 0.73 (3H, s, H₁₂), and 0.54 (3H, d, J = 6.6 Hz, H₁₃). ¹³C NMR (100 MHz, CD₃OD): δ 167.0 (C_7′′), 145.3 (C_{3′′/5′′}), 138.5 (C_4′′), 134.0 (C₈), 132.6 (C₇), 120.2 (C_1′′), 108.8 (C_{2′′/6′′}), 101.7 (C_1′), 76.9 (C₆), 76.8 (C_3′), 74.8 (C₃), 73.9 (C_5′), 73.8 (C_2′), 71.0 (C_4′), 68.0 (C₉), 63.6 (C_6′), 41.3 (C₂), 39.0 (C₁), 36.9 (C₄), 34.0 (C₅), 24.5 (C₁₁), 23.7 (C₁₂), 22.8 (C₁₀), and 15.0 (C₁₃). ¹³C NMR (100 MHz, DMSO-d₆): δ 166.3 (C_7′′), 146.1 (C_{3′′/5′′}), 138.9 (C_4′′), 135.2 (C₈), 131.8 (C₇), 119.9 (C_1′′), 109.1 (C_{2′′/6′′}), 77.2 (C₆), 76.3 (C_3′), 74.6 (C₃), 74.1 (C_5′), 74.0 (C_2′), 70.8 (C_4′), 67.1 (C₉), 64.3 (C_6′), 41.9 (C₂), 37.9 (C₄), 34.1 (C₅), 25.9 (C₁₁), 25.0 (C₁₂), 24.7 (C₁₀), and 16.5 (C₁₃). HR-ESI-MS (-) m/z: 541.2300 [M-H]⁻, calcd for C₂₆H₃₇O₁₂ 541.2285. HR-ESI-MS (+) m/z: 565.2274 [M+Na]⁺, calcd for C₂₆H₃₈O₁₂Na 565.2261. HR-ESI-MS/MS (+) from precursor m/z: 565 [M+Na]⁺, product ions m/z 413.2159 [M–C₇H₅O₄+Na+H]⁺, calcd for C₁₉H₃₄NaO₈ 413.2151, and m/z 355.0626 [M–C₁₃H₂₃O₂+Na+H]⁺, calcd for C₁₃H₁₆NaO₁₀ 355.0641.

Antimicrobial evaluation

The antimicrobial activity was performed against one Gram-positive (Staphylococcus aureus ATCC25923) and three Gram-negative (Pseudomonas aeruginosa ATCC27853, Escherichia coli S2(1), Shigella flexneri SDINT) bacteria, and three yeast strains of Candida albicans ATCC10231, C. tropicalis PK233, and Cryptococcus neoformans H99. These microorganisms were taken from our local stocks. The broth microdilution method was used to determine the MICs and MMC as previously described. ²⁸ Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) and nystatin (Merck, Darmstadt, Germany) were used as positive controls for bacteria and yeasts, respectively.

Antioxidant assay

Ferric reducing antioxidant power (FRAP) assay

The FRAP was performed by the Fe³⁺–Fe²⁺ transformation in the presence of extracts and compounds as previously described. ²⁹ BHT was used as a standard antioxidant.

2,2'-diphenyl-1-picrylhydrazyl (DPPH) free radical–scavenging assay

The free radical–scavenging activity of extracts and isolates was carried out as previously described. ³⁰ Vitamin C was used as a positive control.

Statistical analysis

The experimental results were analyzed by one-way analysis of variance followed by Waller-Duncan post hoc test. The results were expressed as the mean of three replicates ± standard deviation (SD). Differences between means were considered significant at p-value <0.05. The mean comparisons were performed using the Statistical Package for Social Sciences (SPSS, version 12.0) software.

Conclusion

The chemical investigation of ethyl acetate extract of G. senegalensis leaves led to the isolation of nine compounds, including one new megastigmane derivative. The isolates showed weak to high antimicrobial activities, with phaeophytin A (4) being the most active one whereas compounds 5 and 6 exhibited the highest antioxidant activities. These constituents may further serve as important taxonomic markers for the genus and may also be contributing to the efficiency of this plant extract in traditional medicine.

Supplemental Material

sj-doc-1-chl-10.1177_17475198211072498 – Supplemental material for A new megastigmane, known porphyrinic and galloylated bioactive derivatives from the leaves of Gymnosporia senegalensis

Supplemental material, sj-doc-1-chl-10.1177_17475198211072498 for A new megastigmane, known porphyrinic and galloylated bioactive derivatives from the leaves of Gymnosporia senegalensis by Simplice Jöel Tatsimo Ndendoung, Jean-De-Dieu Tamokou, Kazufumi Toume, Léopold Havyarimana, Steve Endeguele Ekom and Katsuko Komatsu in Journal of Chemical Research