Tetrandrabroside, a New Cerebroside and Other Chemical Constituents with Antibacterial Activities from the Leaves of Strombosiopsis tetrandra Engl.

Introduction

The increased resistance of microorganisms like bacteria to drug resistant continuously lowers the efficiency of antibiotic therapy worldwide.¹ Bacterial strains that develop resistance include species of Staphylococcus, Escherichia, Klebsiella and Streptococcus. In most of developing countries, diseases caused by these resistant bacteria are primary problem in health. Despite the progress made in microbiological research to prevent or control infectious diseases, recurrent epidemics associated with drug resistant microorganisms still appear. The research of natural compounds that can effectively be used as therapeutic agents against drug resistant bacteria has seen renewed interest and has been subject of several studies.^2,3 Traditional African medicine, with its richness and diversity of the surrounding flora, offered a multitude of plants for health problems in general and infectious diseases in particular. Scientists believe in their potential as a major source for drug development. Strombosiopsis tetrandra is a tree of the dense and wet forest, living from Nigeria to Angola. This plant is commonly known as "edipmbazoa" in southern region of Cameroon and it is one such plant, commonly used to treat wounds. Bark decoction is used for scabies, dysentery and kidney ache.⁴ It is drunk or applied as a lotion to induce childbirth or treat menstrual problems as an enema, to treat elephantiasis and orally for bronchitis. It is also used in the management of diabetes and hypertension.⁵ Previous pharmacological studies carried out on plants of the family Olacaceae have evidenced anti-gonococci,⁶ anti-inflammatory⁷ and antimicrobial activities.⁸ S. tetrandra showed very good activity against methicillin-resistant strains, including Strombosiopsis aureus and Salmonella.⁴ Previous chemical works on this family found that it contains flavonoids,⁹ triterpenoids,⁹ isoprenoids⁶ and alkaloids.¹⁰ As part of interest in the isolation and characterization of new phytochemicals as well as new bioactive compounds of the Olacaceae family, the MeOH extract of the leaves of S. tetrandra was chromatographed to isolate fifteen compounds including a new cerebroside (1) while semi-synthetic approach yields new dibenzyled and tribenzyled flavonoid glycosides. The paper describes the isolation and structural characterization of isolated compounds; the antimicrobial activity of the extract, partitioned fractions, and that of the new cerebroside derivative.

Experimental

General experimental procedures ‒ IR spectrum was obtained on a PerkinElmer Spectrum Version 104.00. 1D NMR spectra were recorded at 400 or 600 MHz for ¹H and at 100 or 150 MHz for ¹³C while the DEPT 135 (100 or 150 MHz) and 2D data (COSY ¹H-¹H, HSQC, HMBC, and NOESY) were also recorded. The analysis at 400 MHz was done on a Jeol ECZ 400 NMR spectrometer and processed using Delta software version 5.3.1 (JEOL USA, Peabody, MA) while at 600 MHz, a Bruker Advance III 600 equipped with Xwin-NMR software version 2.1 (Bruker, Ettlingen, Germany) was used. CD₃OD, CDCl₃ and DMSO-d₆ were used as solubilizing solvents. Chemical shifts in ppm (parts per million) were in reference to tetramethylsilane (δ = 0, internal standard as internal reference, and coupling constants (J values) are expressed in Hertz (Hz). Merk silica gel 60 (70-230 mesh) was used for column chromatography, Sephadex LH-20 for size exclusion chromatography, while TLC was carried out on pre-coated silica gel GF₂₅₄ plates. After elution, TLC plates were sprayed with a solution of 30% H₂SO₄, heat (100^oC), and visualized at 254 and 365 nm under UV lamp. Melting points are taken on a Stuart SMP20 digital melting point apparatus. High resolution mass spectra were obtained with QTOF (Quadrupole Time of Flight) (Compact Spectrometer Agilent Trusted Answers, CANADA) equipped with a HR-ESI source. The spectrometer was operated in positive mode (mass range: 100–1000). Low resolution mass spectra were obtained with Spectrometer ADVION-CMS-ESI source quadrupole simple analyzer.

Plant materials ‒ The mature leaves of S. tetrandra Engl. were collected in the city of Dschang (West Province, Cameroon) in September 2019. They were authenticated by Victor Nana, a botanist at the National Herbarium, (Yaoundé, Cameroun). The voucher specimen 66369/NHC was deposited at the herbarium.

Extraction and isolation ‒ The mature leaves (4 Kg) were air-dried and powdered, followed by maceration, at normal pressure and room temperature with methanol (3 × 20 L, 72 h). The methanolic extracts were combined and dried with a Buchi rotatory evaporator under reduced pressure. In total 260 g of MeOH extract was obtained of which 250 g were dissolved in to distilled water (500 mL) and sequentially partitioned with EtOAc (2 × 600 mL) and n-BuOH (2 × 500 mL). The solvent removal yielded 73 g of the EtOAc fraction and 103.7 g of the n-BuOH fraction.

Compound 15 precipitated in the ethyl acetate fraction after the liquid-liquid extraction. After filtration, a mass of 100.1 mg was obtained. The dried filtrate (73 g) was fractionated on a silica gel column using increasing concentrations of EtOAc in n-hexane and then an increased concentration of MeOH in EtOAc. Fractions of 0.5 L were collected, concentrated and pooled into five sub-fractions (A to E) according to their TLC profiles. Further silica gel column separation of the sub-fraction A (10.5 g) provided A1 to A5 fractions. From A2 (1.78 g), compounds 8 (78.2 mg) and 12 (48.7 mg) were obtained after additional silica gel column purification using n-hexane-EtOAc (100:00 → 90:10, v/v). Compound 10 (18.2 mg) was filtered and recrystallized (using the mixture n-hexane-EtOAc (85:15)) from sub-fraction A5 (453.9 mg) and fraction B (31.3 g), respectively. The filtrate of fraction B (30.3 g) was loaded into a Sephadex LH-20 column with methylene chloride-methanol (1:1) to obtain sub-fractions B1 to B4. Compound 9 (12.8 mg) was isolated from B2 (1.1 g) in a silica gel column eluted with n-hexane-EtOAc (95:5 → 85:15). Sub-fraction D (4.1 g) yielded compounds 5 (19.8 mg), 6 (44.5 mg), 7 (22.6 mg), and 11 (7.1 mg) from an EtOAc-MeOH (70:30 → 20:80, v/v) eluted silica gel column while compound 13 (5.2 mg) was obtained from E (21.3 g) after elution with n-hexane-EtOAc (20:80).

The n-BuOH partitioned fraction (77 g) was chromatographed with gradients of n-hexane-EtOAc (20:80 → 0:100 v/v) and then EtOAc-MeOH (95:5 → 70:30, v/v). Fractions (500 mL each) were pooled into five sub-fractions (F – J) according to their TLC profiles. Gel permeation of F (13.3 g) on Sephadex LH-20 with methanol followed by silica gel column separation with EtOAc-MeOH (95:5 v/v) yielded compounds 1 (53.3 mg), 3 (37.7 mg), and 4 (6.4 mg). Compound 2 (247.3 mg) was obtained by permeation of G (31.3 g) on sephadex LH-20 and subsequent silica gel column purification with EtOAc-MeOH (95:5 → 80:20, v/v). Sub-fraction J (31.3 g) was treated on a Sephadex gel column chromatography with methanol as the eluent. Four sub-fractions were obtained and indexed from J1 to J4. Repeated silica gel column chromatography on sub-fraction J3 (2.51 g) with EtOAc-MeOH (90:10 → 85:25, v/v) as eluent led to compound 14 (21.0 mg).

1-O-(β-D-glucopyranosyl-(2S,3S,4E,8E)-2-{[(2R)-2-hydroxyheptadecanoyl]amino}heptadeca-4,8-diene-1,3-diol (tetrandrabroside) (1) ‒ White amorphous powder; Molecular formula: C₄₀H₇₅NO₉; HR-ESI-MS m/z 736.5331 [M+Na]⁺ (cald for C₄₀H₇₅NO₉Na⁺, 736.5334), m/z 758.5148 [M+2Na-H]⁺; LR-ESI-MS m/z 696.5 [M-H₂O+H]⁺; m/z 323.2 [M-C₁₇H₃₃O₂-C₁₀H₁₉+H₂O]⁺; m/z 288.2 [M-C₁₆H₃₃O-C₁₂H₂₃-H₂O]⁺; m/z 282.2 [M-C₁₇H₃₃O₂-C₆H₁₁O₅+H]⁺; m/z 115.1 [M-C₃₂H₅₈NO₉+H]⁺; IR (KBr): νmax 3361 (OH), 1643 (C=O), 1536 (C=C), 1467 and 1072 (C-O) cm^-1; ¹H-NMR (600 MHz, CD₃OD): 5.75 (1H, m, H-5), 5.49 (1H, m, H-4), 5.45 (1H, m, H-8), 5.44 (1H, m, H-9), 4.16 (1H, m, H-3), 4.12 (1H, m, H-1a), 4.01 (2H, m, H-2 and H-2'), 3.74 (1H, m, H-1b), 2.00 (2H, m, H-6), 2.10 (2H, m, H-7 and H-10), 1.72 (1H, m, H-3'a), 1.57 (1H, m, H-3'b), 1.43 (2H, m, H-4'), 1.40-1.23 (32H, brs, H-11 to H-15 and H-5' to H-15'), 0.92 (6H, t, J = 6.9 Hz, H-17 and H-17') for aglycone; 4.28 (1H, d, J = 7.8 Hz, H-1''), 3.88 (1H, m, H-6a''), 3.68 (1H, m, H-6b''), 3.37 (1H, m, H-3''), 3.30 (1H, m, H-5''), 3.29 (1H, m, H-4''), 3.21 (1H, m, H-2'') for sugar moieties; ¹³C-NMR (150 MHz, CD₃OD): 175.8 (C-1'), 133.0 (C-5), 130.6 (C-8), 129.8 (C-4), 129.3 (C-9), 71.5 (C-2'), 71.4 (C-3), 68.3 (C-1), 53.1 (C-2), 34.4 (C-3'), 32.3 (C-7), 32.2 (C-10), 31.6 (C-6), 29.6–28.8 (C-11 to C-15, and C-5' to C-15'), 24.8 (C-4'), 22.3 (C-16 and C-16'), 13.1 (C-17 and C-17') for aglycone; 103.3 (C-1''), 76.6 (C-5''), 76.5 (C-3''), 73.6 (C-2''), 70.2 (C-4''), 61.2 (C-6'') for sugar moieties. (Fig. 1)

Fig. 1.

Chemical structures of the isolated compounds.

Benzylation of compound 2 ‒ To 100 mg (0.136 mmol) of compound 2, 2 mL of DMSO, 56 mg of potassium carbonate and 5.18 µL of benzyl chloride were added. The obtained mixture was magnetically stirred at room temperature. The progress of the reaction was monitored by analytical TLC until the disappearance of the substrate after 6 h. After this, the reaction mixture was suspended in distilled water (10 mL) and extracted with n-BuOH (50 mL). The organic phase was evaporated to dryness to give a yellow residue which was purified on silica gel column chromatography with EtOAc-MeOH (80:20) to afford compounds 2a (71.8 mg) and 2b (9.8 mg). See Fig. 2.

Fig. 2.

Chemical structures of the semisynthetic compounds (2a and 2b).

Methanolysis of compound 1 ‒ Compound 1 (4.0 mg) was refluxed with 1.5 mL of 0.9 N HCl in 82% aq. methanol for 16 hours at 67^oC. The reaction mixture was left to cool, and dried under a vacuum, the residue was dissolved in distilled water (5 mL) and then fractionated with 10 mL n-hexane three times. The n-hexane layer concentrated afforded fatty acid unit, which was identified via ESI-MS analysis. (Fig. 3)

Fig. 3.

Methanolysis of compound 1 in 82% aq. methanol.

Microorganisms ‒ The studied microorganisms were two Gram-positive bacteria (Strombosiopsis aureus ATCC 25923 and Streptococcus pneumoniae ATCC 49619) and two Gram-negative bacteria (Escherichia coli ATCC 8739 and Klebsiella pneumoniae 109) taken on the basis of their relevance as human pathogens. Bacteria were stored at +4^oC and activated for 18 h on BBL® nutrient agar (NA, Conda, Madrid, Spain) before any antibacterial testing.

Preparation of bacterial inocula ‒ For the preparation of the bacterial inoculum, two bacterial colonies from the 18 h old culture were collected and diluted in sterile physiological solution (NaCl 0.9% w/v) to obtain a turbidity of 0.5 McFarland standards, approximately equal to 1.5 × 10⁸ CFU/mL. Dilutions were then carried out to obtain an absorbance of 0.100 at 600 nm, corresponding to a final concentration of 1.5 × 10⁶ CFU/mL.

Determination of minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) ‒ The Minimal inhibitory concentrations (MICs) were determined by the broth microdilution method.¹¹ 185 µL of Mueller Hinton broth (MHB) and 5 µL of inoculum were introduced into each microtiter plate (96 microwells). The MHB and bacterial suspension were used to perform the positive control, while the negative control was performed with the MHB and an aqueous solution of DMSO or 10% Tween 20. 10 µL of sample stock solution was then added to the first well to obtain a final volume of 200 µL and serial dilutions of 2 were performed. The plates were incubated under shaking at 35^oC for 24 h. Bacterial growth was detected by adding 5 µL of a 0.2 mg/mL para-iodonitrotetrazolium solution. Bacterial growth was demonstrated by the color change from yellow to purple. The MIC was defined as the lowest concentration of the substance that prevented this color change. The minimum bactericidal concentrations (MBCs) were defined as the lowest concentrations resulting in a negative subculture or a single colony on Mueller-Hinton agar medium. Three replicates were performed for each sample. Ciprofloxacin served as a reference antibiotic.

Results and Discussion

Chromatographic separations of the MeOH extract of S. tetrandra Engl. led to the isolation of tetrandrabroside (1) along with fourteen previously reported compounds (1–15) (Fig. 1). Compounds already available in the literature were identified as kaempferol 3-O-(2′′,6′′-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (2),¹² kaempferol 3-O-neohesperidoside (3),¹³ kaempferol 3-O-β-D-glucopyranoside (4),¹² kaempferol (5),¹⁴ quercetin (6),¹⁵ catechin (7),¹⁶ lupeol (8),¹⁷ lupenon (9),¹⁸ glochidon (10),¹⁸ glochidiol (11),¹⁹ oleanolic acid (12),²⁰ 2-ethylhexyl isonicotinate (13),²¹ phosphocholine (14)²² and β-sitosterol-3-O-β-D-glucopyranoside (15).²³

The isolated compound 1 had a white amorphous morphology and was soluble in methanol. Its positive-ion mode HR-ESI-MS displayed a molecular adduct [M+Na]⁺ at m/z 736.5331 corresponding to the molecular formula C₄₀H₇₅NO₉Na (calcd. 736.5334 for C₄₀H₇₅NO₉Na⁺). Its IR spectrum (Fig. S4) showed absorption bands at 3361 cm^-1 (br, OH and NH), 2954-2849 cm^-1 (aliphatic chain), 1643 cm^-1 (amide group CO), 1536 cm^-1 (carbon-carbon double bond), and 1072 cm^-1 (C-O-C). The analysis of one-dimensional (¹H and ¹³C) NMR data (Table S1), and two-dimensional spectra (¹H-¹H COSY, HSQC, and HMBC) (Fig. S7-S9) showed characteristic signals of sugar moieties, an amide linkage and aliphatic chains which suggested a glycosphingolipid skeleton. Its ¹H-NMR spectrum (Fig. S5) indicated resonance of terminal methyl (-CH₃) groups at δ_H 0.92 (t, J = 6.9 Hz, Me-17/Me-17') followed by a set of signals ranging from δ_H 1.40 to 1.23 ppm (brs, H-11 to H-16/H-4' to H-16') attributed to the methylenes of the two long chains. Allylic protons were observed at δ_H 2.00 (m, H-6), δ_H 2.10 (m, H-10/H-7). This spectrum exhibited also seven oxymethines and two oxymethylenes protons including those of the sugar unit at δ_H 4.28 (d, J = 7.8 Hz, H-1''), 4.16 (m, H-3), 4.12 (m, H-1a), 4.01 (m, H-2'), 3.88 (m, H-6''a), 3.74 (m, H-1b), 3.68 (m, H-6''b), 3.37 (m, H-3''), 3.30 (m, H-5''), δ_H 3.29 (m, H-4'') and 3.21 (m, H-2''). Signal at δ_H 4.01 (m, H-2) was attributed to the sphingosine aminomethine group.²⁴ The ¹H-NMR spectrum of 1 also exhibited signals of four olefinic protons at δ_H 5.75 (m, H-5), δ_H 5.49 (m, H-4), δ_H 5.45 (m, H-8) and 5.44 (m, H-9). All the above information revealed that 1 had a sphingosine type structure.²⁵

The analysis of ¹³C NMR and HSQC spectra of 1 concurs the structure type suggested by ¹H data. ¹³C NMR signal at δ_C 13.1 ppm confirmed the two terminal methyl groups (Me-17/Me-17') and the aliphatic methylenic carbons between δ_C 29.6-28.8 ppm. The chemical shifts of allylic carbons (C-6/C-7/C-10) at 31.6, 32.3 and 32.2 ppm agreed with (E)- or trans-configurations.^26–29 The conformation of aliphatic alkene is often identified by its coupling constant, 16 Hz (trans) and 8 Hz (cis) however, when olefinic protons are multiplets as in the case of compound 1, the determination is done using the ¹³C-NMR data which typically displayed δ_C more than 30 for (E) or trans-configuration, and δ_C at 26 for (Z)-or cis-configuration.^28–31 The signals observed at δ_C 53.1, 68.3 and 61.2 were assigned to C-2 of an aminomethine, C-1 and C-6'' of two oxymethylene respectively. The six sp³ oxymethines appeared at δ_C 76.6 (C-5''), 76.5 (C-3''), 73.6 (C-2''), 71.5 (C-2'), 71.4 (C-3), 70.2 (C-4''), and the four sp² methine corresponding to two carbon-carbon double bonds at δ_C 133.0 (C-5), 130.6 (C-8), 129.8 (C-4) and 129.3 (C-9). The anomeric carbon and the amide carbonyl carbon were detected at δ_C 103.3 (C-1'') and 175.8 (C-1') respectively. The ¹H-¹H correlation spectrum (COSY) of 1 showed linked of H-1a/H-1b with H-2, H-2 with H-3, H-3 with H-4, and H-4 with H-5 which allowed us to determine the location of one hydroxyl group and one double bond. This COSY ¹H-¹H spectrum showed also correlations of protons H-4 with H-5, H-5 with H-6, H-6 with H-7, H-7 with H-8, H-8 with H-9, and H-9 with H-10 which allowed the identification of the second double bond position. Further confirmation of the double bond position was obtained in the HMBC spectrum on which correlations between the proton at δ_H 2.00 (m, H-6) and the carbons at δ_C 133.0 (C-5), 129.8 (C-4), 130.6 (C-8), 129.3 (C-9) were observed. Other important HMBC correlations between the protons at δ_H 4.12 (H-1a)/3.74 (H-1b) and carbons at δ_C 71.4 (C-3), 53.1 (C-2); between the proton at δ_H 4.16 (H-3) and carbons at δ_C 133.0 (C-5), 129.8 (C-4), 68.3 (C-1), 53.1 (C-2); between the aminomethine proton at δ_H 4.01 (H-2) and carbons at δ_C 71.4 (C-3), 68.3 (C-1) and 175.8 (C-1') led to the conclusion that the basic and acidic chains were linked together. The position of sugar unit was determined through the correlations from the anomeric proton at δ_H 4.28 (H-1'') to the carbon at δ_C 68.3 (C-1), and from the protons at δ_H 4.12 (H-1a)/3.74 (H-1b) to the anomeric carbons at δ_C 103.3 (C-1''). The absolute configuration of the stereo centers (C-2, C-3, and C-2') determined to be 2S, 3S, 2'R was based on their carbon chemical shifts which are very close to those of triumfettoside Ic,³² and psychotramide.³³

The identity of the sugar moiety was confirmed after analysis of 2D NMR data including spin-spin coupling constants and chemical shifts, all which agreed with the literature data for a glucopyranosyl residue.³⁴ The identity of the sugar was further confirmed upon acid hydrolysis of compound 1 and TLC analysis of the digest and authentic glucose standard. The β configuration of the sugar was deduced from the coupling constant (J = 7.8 Hz) between H₁''-H₂'' while its D-configuration is due to it being the most prevalent in the plant kingdom.³⁵ The fragment ions at m/z: 115.1 [C₈H₁₈+H]⁺ in the LR-ESI-MS mass spectrum were used to confirm the location of the second bond. A methanolysis reaction followed by LC-MS analysis showed that compound (1) was converted to methyl (2R)-2-hydroxyheptadecanoate (1a) characterized by ion peaks observed at m/z 323.2 ([M+Na]⁺) and m/z 282.2 ([M−H₂O]⁺); and to (2S,3R,4E,8E)-2-aminoheptadeca-4,8-diene-1,3-diol (1b) ([M]⁺ at m/z 283.2). Accordingly, the new molecule (1) structure was ascertained as 1-O-(β-D-glucopyranosyl-(2S,3R,4E,8E)-2-{[(2R)-2-hydroxyheptadecanoyl]amino}heptadeca-4,8-diene-1,3-diol, trivially named tetrandrabroside.

Structural modifications often lead to derivatives with biological activities greater than those of substrate. In fact, Shamsudin et al 2022 have shown that the effect of different groups on biological activity depends on the flavonoid subclass and position of substituent.³⁶ To our knowledge, no results on the benzylation reaction of flavonols have been reported. To investigate the effect of benzyl groups, this reaction, performed on kaempferol-3-O-(2′′,6′′-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (2) afforded two new semisynthetic derivatives (2a and 2b) (Fig. 2).

The molecular formula C₅₄H₅₈O₁₉ of 2a was determined using HR-TOF-SIMS spectrum data which contained a [M+H]⁺ ion peak at m/z 1011.3659 (calcd. 1011.3645 for C₅₄H₅₉O₁₉⁺) which has 26 degrees of unsaturation. Compound 2b formula C₄₇H₅₂O₁₉ was determined based on its TOF-SIMS data on which the [M+H]⁺ ion peak was found at m/z 921.3194 (calcd. 921.3176 for C₄₇H₅₃O₁₉⁺, and corresponded to 22 degrees of unsaturation. A complementary analysis carried out on their NMR spectra (Fig. S15-S19, S21-S24) allowed to attribute to them the structures of kaempferol-4',5,7-tribenzyl-3-O-(2'',6''-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (2a) and kaempferol-4',7-dibenzyl-3-O-(2'',6''-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (2b). Indeed, ¹H-NMR spectra of 2a and 2b are almost superimposable on that of compound 2 except for the additional aromatic and aliphatic proton signals between δ_H 7.43–7.25 ppm and δ_H 5.17–5.08 ppm belonging to additional benzyl group. Almost identical information was observed on their ¹³C-NMR spectra. The carbon-proton correlation spectrum (HMBC) confirmed the presence of benzylic group through the links between benzylic protons and aromatic carbons of aglycone moieties.

The crude methanol extract, ethyl acetate and n-butanol fractions, some isolated compounds, and semisynthetic derivatives (2a, 2b) were assayed in vitro for their ability to inhibit the growth of four common pathogenic bacteria namely Streptococcus pneumoniae ATCC 49619 and Strombosiopsis tetrandra ATCC 25923 which are both Gram positive, the Gram negative Klebsiella pneumoniae 109 and Escherichia coli ATCC 8739. The experimentation was based on the broth microdilution procedure with ciprofloxacin as reference antibiotic. The results obtained were recorded in Table 1.

Table 1.

Antibacterial activities of extract, fractions, isolated and semisynthetic compounds and reference drug

The rational cutoff points for classification of antibacterial agents from natural sources against bacteria were established by Kuete, 2023.³⁷ According to this scale, the MeOH extract showed very good activity (MIC = 128 µg/mL) against E. coli, while the EtOAc fraction showed excellent activity (MIC = 32 µg/mL) against the same strain. This suggested that liquid-liquid extraction of the MeOH extract using EtOAc solvent increased activity. Similarly, the activity was maintained for the n-BuOH fraction with an MIC of 128 µg/mL.

Compound 1 showed average activity and was bacteriostatic with MIC equal to 64 µg/mL against E. coli and had weak activity against other species. This result was in agreement with those in literature, which indicated that cerebrosides have antibacterial and antimicrobial properties, respectively. Compounds 2 and 3 showed moderate activity (MIC = 64 µg/mL) against E. coli and S. aureus.^38,39 This is line with literature data as previous work on flavonoids have showed that glycosylation enhanced their water solubility and stability,³⁶ which may lead to improvement biological activities which can include better bioavailability but also a decrease acute toxicity or harmful effects.³⁶ In the case of microbes, glycosylation may enhance the interaction of flavonoids with membranes. The results obtained for flavonoids 5, 6 and 7, indicated that, quercetin (6) showed the best E. coli inhibition (MIC = 64 µg/mL), while kaempferol (5) and catechin (7) showed moderate activities with MIC up than 256 µg/mL. These data are in line with previous works on the antimicrobial activities of other flavonoids and their glycosides.² Secondary metabolites like flavonoids can exert their antibacterial activities through mechanisms such the suppression of the synthesis of nucleic acid, the disruption of the function of cytoplasmic membrane, and the disruption of energy metabolism.^40,41 Other mechanisms of flavonoids are linked to reducing adhesion and biofilm formation, and increasing membrane permeability, which are disadvantageous for bacterial to growth.⁴¹

Triterpenoids 8, 9, 10 and 12 inhibited the growth of the four tested bacteria. Indeed compounds 9 and 10 showed average activity (MIC = 64 µg/mL) against E. coli and weak activity (MIC = 128 µg/mL) against K. pneumoniae, E. coli, S. aureus and S. pneumoniae. This in vitro study suggested that the presence of ketone and functional groups at C-3 position of triterpenes increased their antibacterial activities. As for steroids, no activity was observed with MIC above 256 µg/mL.

The two hemisynthetic derivatives 2a and 2b as well as compound 2 showed average activities against E. coli (MIC = 64 µg/mL) and weak activities against S. aureus and K. pneumoniae (MIC = 128 µg/mL). Compounds 2 and 2a showed average activity (MIC = 64 µg/mL) against S. aureus while compound 2b showed weak activity (MIC = 128 µg/mL) against the same strain. These results corroborated a previous study that found that benzylation of flavone at positions C-5, C-7 and C-4' has no effect on their antibacterial activity.⁴²

The minimum bactericidal concentration (MBC) values of the MeOH extract, its partitioned fractions (EtOAc and n-BuOH) as well as compounds 1, 2, 2a, 2b, 6, 8, 9 and 10 were either equivalent or up to four times greater than their MICs values when considering the most sensitive bacterium (E. coli). A lower MBC (≤ 4) indicated the best antimicrobial activity with a minimum amount of an extract, fraction or pure molecule.⁴³ Decoctions of the S. tetrandra Engl. plant is used in traditional or alternative medicine to the treat of bacterial related diseases such as dysentery.⁴ Overall, data from this work partly support the use of S. tetrandra Engl. in alternative medicine.

In conclusion, Phytochemical investigations on the EtOAc and n-BuOH fractions of S. tetrandra Engl. leaves led to the isolation and characterization of fifteen secondary metabolites belonging to several classes, including one new cerebroside (tetrandrabroside), six flavonoids and their glycosides, five triterpenoids, one steroid glucoside, one phtatalate and one quaternary ammonium salt. Benzylation reaction, carried out on kaempferol 3-O-(2′′,6′′-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside, led to two new semisynthetic derivatives. The different fractions, some isolated compounds and the semisynthetic compounds were subjected to antibacterial evaluation against two Gram-positive and two Gram-negative. EtOAc fraction showed good activity against E. coli ATCC 8739 with a MIC and MBC equal to 32 µg/mL. The isolated compounds also showed moderate activity. The reaction had a positive impact as it improved the activity of the substrate. This phytochemical and biological investigation is the first of its kind on this plant and in the genus.

Acknowledgments

The authors are grateful to the University of Dschang for providing us with some consumables used in this work, to the “Institut de Chimie Moléculaire de Reims”, “Carleton University of Canada” and “University of Rennes” for the spectroscopic analysis on the NMR equipment of the PlAnet Platform and spectrometric analysis. We sincerely thank CRMPO (Centre de Mesures de Physique de l’Ouest de Rennes) for high resolution mass spectrometry data and ISCR (Institut des Sciences Chimiques de Rennes) for technical assistance for IR data.

References

• Falagas, M. E.; Bliziotis, I. A. Int. J. Antimicrob. Agents 2007, 29, 630‒636. [https://doi.org/10.1016/j.ijantimicag.2006.12.012]
• Jouogo, N. D. C.; Eckhardt, P.; Tamokou, J.-D.-D.; Matsuete Takongmo, G.; Voutquenne-Nazabadioko, L.; Opatz, T., Tapondjou, L. A.; Ngnokam, D.; Teponno, R. B. Nat. Prod. Res. 2024, 38, 2737‒2747. [https://doi.org/10.1080/14786419.2023.2232078]
• Mabou, F. D.; Tamokou, J.-D.-D.; Ngnokam, D.; Voutquenne-Nazabadioko, L.; Kuiate, J. R.; Bag, P. K. Drug Discov. Ther. 2016, 10, 141‒149. [https://doi.org/10.5582/ddt.2016.01040]
• Obiang-Obounou, B. W.; Kang, O.-H.; Choi, J.-G.; Keum, J.-H.; Kim, S.-B.; Kim, Y.-S.; Mun, S.-H.; Choi, M.-S.; Maroufath, L.; Kwon, D.-Y. Nat. Prod. Sci. 2011, 17, 33‒37.
• Tsabang, N.; Tsambang, W. L.; Tsambang, S. C.; Agbor, A. G. Endocrinol. Diabetes Metab. Case Rep. 2016, 1, 1‒9.
• Nair, J. J.; Mulaudzi, R. B.; Chukwujekwu, J. C.; Van Heerden, F. R.; Van Staden, J. S. Afr. J. Bot. 2013, 86, 111‒115. [https://doi.org/10.1016/j.sajb.2013.02.170]
• Soro, T. Y.; Néné-bi, A. S.; Zahoui, O. S.; Yapi, A.; Traoré, F. J. Anim. Plant Sci. 2015, 24, 3802‒3813.
• Ekalu, A.; Ayo, R. G.-O.; Habila, J. D.; Hamisu, I. Bull. Natl. Res. Cent. 2019, 43, 1‒7. [https://doi.org/10.1186/s42269-019-0159-x]
• Tsakem, B.; Tchuenguem, R. T.; Siwe-Noundou, X.; Kemvoufo, B. P.; Dzoyem, J. P.; Teponno, R. B.; Krause, R. W. M.; Tapondjou, A. L. Nat. Prod. Res. 2023, 37, 1641‒1650. [https://doi.org/10.1080/14786419.2022.2106566]
• Bitchi, M. B.; Magid, A. A.; Kabran, F. A.; Yao-Kouassi, P. A.; Harakat, D.; Morjani, H.; Tonzibo, F. Z.; Voutquenne-Nazabadioko, L. Phytochemistry 2019, 167, 112081. [https://doi.org/10.1016/j.phytochem.2019.112081]
• Nyaa, L. B. T.; Tapondjou, L. A.; Barboni, L.; Tamokou, J. D. D.; Kuiaté, J. R.; Tane, P.; Park, H.-J. Nat. Prod. Sci. 2009, 15, 76‒82.
• Chludil, H. D.; Corbino, G. B.; Leicach, S. R. J. Agric. Food Chem. 2008, 56, 5050‒5056. [https://doi.org/10.1021/jf800421j]
• Iwashina, T.; Yamaguchi, M.-A.; Nakayama, M.; Onozaki, T.; Yoshida, H.; Kawanobu, S.; Onoe, H.; Okamura, M. Nat. Prod. Commun. 2010, 5, 1903‒1906. [https://doi.org/10.1177/1934578X1000501213]
• Singh, R.; Singh, B.; Singh, S.; Kumar, N.; Kumar, S.; Arora, S. Toxicol. In Vitro 2008, 22, 1965‒1970. [https://doi.org/10.1016/j.tiv.2008.08.007]
• Dongmo, A. J. N.; Ekom, S. E.; Tamokou, J.-D.-D.; Tagousop, C. N.; Harakat, D.; Voutquenne-Nazabadioko, L.; Ngnokam, D. Nat. Prod. Sci. 2020, 26, 317‒325.
• Ismail, A.; Radwan, M. M.; Hassan, H. M.; Moawad, A. S.; Hetta, M. H. J. Appl. Pharm Sci. 2021, 11, 158–162.
• Wang, C.-M.; Chen, H.-T.; Wu, Z.-Y.; Jhan, Y.-L.; Shyu C.-L.; Chou, C.-H. Molecules 2016, 21, 139. [https://doi.org/10.3390/molecules21020139]
• Culhaoğlu, B.; Hatipoğlu, S. D.; Dönmez, A.; Topçu, G. Med. Chem. Res. 2015, 24, 3831‒3837. [https://doi.org/10.1007/s00044-015-1424-7]
• Srivastava, R.; Kulshreshtha, D. K. Phytochemistry 1988, 27, 3575‒3578. [https://doi.org/10.1016/0031-9422(88)80771-4]
• Cai, L.; Wu, C. D. J. Nat. Prod. 1996, 59, 987‒990. [https://doi.org/10.1021/np960451q]
• Lim, Q. Y. Faculty of Applied Sciences, Bachelor of Science (Honours) in Analytical Chemistry, Tunku Abdul Rahman University College, 2018.
• Sarkar, R.; Comment, A.; Vasos, P. R.; Jannin, S.; Gruetter, R. J. Am. Chem. Soc. 2009, 16014‒16015. [https://doi.org/10.1021/ja9021304]
• Molokoane, T. L.; Kemboi, D.; Siwe-Noundou, X.; Famuyide, I. M.; McGaw, L. J.; Tembu, V. J. Plants 2023, 12, 1‒15. [https://doi.org/10.3390/plants12193369]
• Garg, H. S.; Agrawal, S. J. Nat. Prod. 1995, 58, 442‒445. [https://doi.org/10.1021/np50117a016]
• Bankeu, K. J. J.; Mustafa, A. A. S.; Gojayev, S. A.; Lenta, D. B.; Noungoué, T. D.; Ngouela, S. A.; Asaad, K.; Choudhary, I. M.; Prigge, S.; Guliyev, A. A.; Nkengfack, E. A.; Tsamo, E.; Ali, S. M. Chem. Pharm. Bull. 2010, 58, 1661‒1665. [https://doi.org/10.1248/cpb.58.1661]
• Stothers, J. B. Appl. Spectrosc. 1972, 26, 1‒16. [https://doi.org/10.1366/000370272774352632]
• Jung, J. H.; Lee, C.-O.; Kim, Y. C.; Kang, S. S. J. Nat. Prod. 1996, 59, 319‒322. [https://doi.org/10.1021/np960201+]
• Kang, S. S.; Kim, J. S.; Xu, Y. N.; Kim, Y. H. J. Nat. Prod. 1999, 62, 1059‒1060. [https://doi.org/10.1021/np990018r]
• Chen, D.-Z.; Xiong, H.-B.; Tian, K.; Guo, J.-M.; Huang, X.-Z.; Jiang, Z.-Y. Molecules 2013, 18, 11241‒11249. [https://doi.org/10.3390/molecules180911241]
• Ishii, T.; Okino, T.; Mino, Y. J. Nat. Prod. 2006, 69, 1080‒1082. [https://doi.org/10.1021/np050530e]
• Tchoukoua, A.; Sandjo, L. P.; Keumedjio, F.; Ngadjui, B. T.; Kirsch, G. Chem. Nat. Compd. 2013, 49, 811‒814. [https://doi.org/10.1007/s10600-013-0753-3]
• Sandjo, L. P. Université de Yaoundé I, Université Paul-Verlaine de Metz, 2009.
• Wang, Y.-M.; Wu, A.-Z.; Zhong, Y.; Peng, G.-T.; Fan, Q.; Zhu, C.-C.; Zhang, C.-X. Nat. Prod. Res. 2019, 34, 2095‒2100. [https://doi.org/10.1080/14786419.2019.1574789]
• Agrawal, P. K. Phytochemistry 1992, 31, 3307‒3330. [https://doi.org/10.1016/0031-9422(92)83678-R]
• Nzowa, L. K.; Barboni, L.; Teponno, R. B.; Ricciutelli, M.; Lupidi, G.; Quassinti, L.; Bramucci, M.; Tapondjou, L. A. Phytochemistry 2010, 71, 254‒261. [https://doi.org/10.1016/j.phytochem.2009.10.004]
• Shamsudin, N. F.; Ahmed, Q. U.; Mahmood, S.; Shah, S. A. A.; Khatib, A.; Mukhtar, S.; Alsharif, M. A.; Parveen, H.; Zakaria, Z. A. Molecules 2022, 27, 1149. [https://doi.org/10.3390/molecules27041149]
• Kuete, V. In Potential of African medicinal plants against Enterobacteria: Classification of plants antibacterial agents; Academic Press Ed; Adv. Bot. Res. Publishing; Netherlands, 2023, pp 152‒328. [https://doi.org/10.1016/bs.abr.2022.08.006]
• Chen, J. H.; Cui, G. Y.; Liu, J. Y.; Tan, R. X. Phytochemistry 2003, 64, 903‒906. [https://doi.org/10.1016/S0031-9422(03)00421-7]
• Cateni, F.; Zilic, J.; Falsone, G.; Scialino, G.; Banfi, E. Bioorg. Med. Chem. Lett. 2003, 13, 4345‒4350. [https://doi.org/10.1016/j.bmcl.2003.09.044]
• Biharee, A.; Sharma, A.; Kumar, A.; Jaitak, V. Fitoterapia 2020, 146, 104720. [https://doi.org/10.1016/j.fitote.2020.104720]
• Xie, Y.; Yang, W.; Tang, F.; Chen, X.; Ren, L. Curr. Med. Chem. 2015, 22, 132‒149. [https://doi.org/10.2174/0929867321666140916113443]
• Chedjou, N. I.; Tchuenguem, T. R.; Tchegnitegni, T. B.; Ngouafong, T. F.; Dzoyem, P. J.; Ponou, K. B.; Teponno, B. R.; Barboni, L.; Tapondjou, A. L. Cameroon J. Exp. Biol. 2023, 17, 10‒15. [https://doi.org/10.4314/cajeb.v17i1.3]
• Djouossi, M. G.; Tamokou, J.-D.-D.; Ngnokam, D.; Kuiate, J. R.; Tapondjou, L. A.; Harakat, D.; Voutquenne-Nazabadioko, L. BMC Complement. Altern. Med. 2015, 15, 134. [https://doi.org/10.1186/s12906-015-0660-1]