Constituents of the Stem Bark of Symphonia globulifera Linn. f. with Antileishmanial and Antibacterial Activities

The chemical investigation of the n-hexane fraction from the methanol extract of the stem bark of Symphonia globulifera Linn f., which displayed good in vitro activity against Leishmania donovani NR-48822 promastigotes (IC50 43.11 µg/mL), led to the isolation of three previously unreported polyprenylated benzophenones, guttiferone U (1), V (2)/W (3), and a new tocotrienol derivative named globuliferanol (4), along with 11 known compounds (5–15). Their structures were elucidated based on their NMR and MS data. Some isolated compounds were assessed for both their antileishmanial and cytotoxic activities against L. donovani and Vero cells, respectively. Guttiferone K (5) exhibited the best potency (IC50 3.30 μg/mL), but with low selectivity to Vero cells. The n-hexane fraction and some compounds were also assessed in vitro for their antibacterial activity against seven bacterial strains. All the samples exhibited moderate to potent antibacterial activity (MICs ≤ 15.6 µg/mL) against at least one of the tested strains.


Introduction
Leishmaniasis is a neglected infectious vector-borne disease caused by the protozoan parasite Leishmania, transmitted to humans and animals through the bite of infected female phlebotomine sandflies during their blood meals [1,2]. Visceral leishmaniasis remains the most lethal if left untreated, with the most severe and underreported form of the disease representing up to 95% of cases [3][4][5][6]. Based on the most recent available data on this disease, between 498,000 and 862,000 new cases of all forms of leishmaniasis occur annually, resulting in up to 18,700 deaths and approximately 1.6 million disability-adjusted life years lost [7]. Moreover, the number of imported infection cases in non-endemic areas is increasing [8][9][10]. Leishmaniasis is, however, largely ignored and faces problems of resistance of the parasite to the available therapeutic molecules. The goal of the WHO
Compounds 2 and 3 were obtained as an optically active mixture of a yellow amorphous solid with the same Rf on thin-layer chromatography (TLC) in different solvent systems. They were present as a 1:1 mixture based on their NMR peak intensities. The HR-ESIMS showed a sodium adduct peak at m/z 693.4141 [M + Na] + (calcd for C43H58O6Na + , 693.4126) corresponding to the molecular formula, C43H58O6, a mass which was 68 a.m.u higher than that of compound 1, suggesting the presence of an additional prenyl side chain (C5H9) when compared to 1. The IR spectrum exhibited strong absorption bands at 3300 (hydroxy groups), 1729, and 1669 (for non-conjugated and conjugated carbonyl groups), and 1699 cm −1 (C=C). The UV spectrum displayed characteristic absorptions at λmax 340 and 370 nm for the aromatic ring and conjugated carbonyl chromophores, respectively [38]. The analysis of the NMR spectra confirmed 2/3 to be polyprenylated benzophenone derivatives. The 1 H NMR spectrum (Table 1)  This information suggested that the relative stereochemistry of compound 1 could be identical to that of guttiferone A. This was further confirmed by the comparison of their optical rotation signs. Therefore, compound 1 was concluded to be a new polyprenylated acylphloroglucinol derivative named guttiferone U with the structure as shown.

Antileishmanial and Antibacterial Activities
Compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) were assessed in vitro for their antileishmanial activity against L. donovani NR-48822 promastigotes and for their cytotoxicity toward Raw 264.7 macrophage cells (Table 3). Guttiferone K (5) exhibited the best antileishmanial activity against the parasite with an IC 50 value of 3.30 ± 0.51 µg.mL −1 but with weak selectivity toward Raw 264.7 macrophage cells (SI = 1.57), while compounds 1-4, 6-8, and 10 showed moderate activity with IC 50 values ranging from 10.80 to 15.98 µg.mL −1 . The assessed compounds were more active than the n-hexane soluble fraction from which they were obtained. The inactivity of the MeOH crude extract and EtOAc fraction may be due to the antagonistic effect of its constituents. The majority of the active compounds were xanthones or benzophenones, which are known to possess antileishmanial activity. Table 3. Antileishmanial and cytotoxic activities of extract, fractions, and compounds from the stem bark of S. globulifera.

Extracts/ Compounds
Antileishmanial Activity IC 50  The methanol crude extract, the n-hexane, EtOAc, and n-butanol soluble fractions along with some the isolates were assessed for their antibacterial activity against seven bacterial strains: Salmonella typhi CPC, S. enterica NR13555, Staphylococcus aureus ATCC43300, S. aureus ATCC25923, Klebsiella pneumoniae clinical isolate, K. pneumoniae NR41388, and Pseudomonas aeruginosa HM801 (Table 4). The MeOH crude extract and the n-BuOH fraction exhibited moderate activity, while the n-hexane and EtOAc fractions exhibited good antibacterial activities on at least two strains with MIC values ranging from 15.7 to 31.2 µg.mL −1 , except on S. aureus ATCC43300, which was not susceptible to the EtOAc fraction. Compounds 1 and 2-5 displayed good to moderate activity against these strains, with MIC values ranging from 3.9 to 62.5 µg.mL −1 , except for S. aureus ATCC43300, which was not susceptible.
These results highlight the knowledge on the potential of guttiferone derivatives as potent antileishmanial and antibacterial agents [15,49], and thus justify the use of this plant in traditional medicine to treat skin and bacterial diseases [19,23].

General Experimental Procedures
Column chromatography (CC) separations were carried out with silica gel (230-400, 70-230 mesh Merck, Darmstadt, Germany) and Sephadex LH-20 (Sigma-Aldrich, Munich, Germany). Pre-coated aluminum-backed silica gel 60 F254 sheets were used for thin-layer chromatography. Spots were visualized under UV light (254 nm and 366 nm) or using a diluted solution of sulfuric acid followed by heating at about 80 • C. Optical rotation was determined by using a JASCO DIP-3600 polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded on a UV-3100 PC spectrophotometer. The 1 H and 13 C NMR spectra were recorded at 500 MHz or 600 MHz and 125 MHz or 150 MHz, respectively. The spectrometers were Bruker AM Avance DRX 500 (Rheinstetten, Germany; 1 H NMR, 500 MHz and 13 C NMR, 125 MHz) and Bruker Avance 600 ( 1 H NMR, 600 MHz and 13 C NMR, 150 MHz). Infrared (IR) spectra (KBr tablet or film) were recorded on a Bruker Tensor 27 FTIR-spectrometer equipped with a diamond ATR. High-resolution mass spectra were recorded on a Bruker QTOF compact spectrometer equipped with an ESI source.

Plant Material and Identification
The stem bark of S. globulifera was harvested in May 2016 in Bangangte (west region of Cameroon) and identified by Mr. Nana Victor, a retired botanist at the National Herbarium of Cameroon, where a voucher specimen (29529 SRFK) was already available.

Antileishmanial and Cytotoxicity Assays
The antileishmanial activity on cultured L. donovani 1S (MHOM/SD/62/1S) promastigotes was evaluated using the resazurin colorimetric method as described by Siqueira-Neto et al. (2010) [50] with little modifications. They were all assessed in triplicate at concentrations ranging from 100 to 0.16 µg/mL for the extract and 50-0.08 µg/mL for the compounds. Test plates were incubated for 28 h at 28 • C, followed by the addition of 1 mg/mL resazurin. The negative and positive controls were 0.1% DMSO and amphotericin B (10-0.016 µg/mL), respectively. The cytotoxicity profile of the crude extract and compounds was assessed using the Alamar blue assay (Mosman, 1983) against Raw 264.7 macrophage cells.

Antibacterial Bioassay
The minimum inhibitory concentration (MIC) of the samples was evaluated following the broth microdilution method as described by Eloff, with light modifications [51]. The extracts, compounds, and reference drug were dissolved in DMSO-MHB. The strain inocula were prepared and their turbidity was adjusted to 0.5 McFarland standard to give an approximate 1.5 × 10 8 CFU/mL. Gentamicin was used as the positive control. Briefly, one hundred microliters of Mueller Hinton Broth was added to all wells of the 96-well plate, and 100 µL of the compounds/extracts was introduced into the wells in the first row (A) and mixed thoroughly. The sample mixture (100 µL) was removed from the well from row A to perform a twofold serial dilution down the rows (B-H). The last 100 µL was discarded. Then, 100 µL of the inoculum was introduced into the corresponding wells. The final volume in each well was 200 µL. Each extract concentration was assayed in triplicate and each test was performed twice. After an incubation period of 18 h at 37 • C, 20 µL of Alamar Blue was added to each well. The plates were then re-incubated for 30 min at 37 • C. A blue color in the well was scored as "no bacterial growth", while a pink color was scored as a "growth occurrence". MIC values were read at those concentrations where a pronounced change in color formation was noticed (from blue to pink).

Conclusions
In addition to enriching the knowledge on the chemistry of S. globulifera, this work represents a significant chemophenetic contribution to this species. It has provided further information with regard to possible chemophenetic markers of S. globulifera and showed the presence of uncommon metabolites encountered in this species. Moreover, the results obtained for the biological evaluation of isolated compounds support the use of S. globulifera in folk medicine.