Antisalmonellal Activities of Extracts, Fractions, Compounds and Semi-synthetic Flavonoid Derivatives from Tristemma hirtum P. Beauv (Melastomataceae)

The development and spread of resistance to currently available antibiotics is a major drawback in the treatment of microbial infections. Salmonellosis for example remains among the most common cause of morbidity and mortality in developing countries. This study aimed to evaluate the antisalmonellal potential of extracts, fractions, isolated compounds and semi-synthetic flavonoids from Tristemma hirtum P. Beauv. Bioguided fractionation by column chromatography of the EtOAc and n-BuOH fractions led to the isolation of eleven compounds including two new esterified glucuronide flavonoids namely: luteolin-3′-O-β-D-glucuronopyranosylbutyl ester (1), a mixture of compound 1 and quercetin-3-O-β-D-glucuronopyranosylbutyl ester (2). Chemical transformation mainly based on the prenylation of 6-hydroxyapigenin-7-O-β-D-glucopyranoside (5) afforded four new semi-synthetic flavonoid derivatives namely: 6, 4'-O-diprenylapigenin-7-O-β-D-glucopyranoside (5a), 8-Cprenyl-6, 4'-O-diprenylapigenin-7-O-β-D-glucopyranoside (5b), 8-C-prenyl-4'-O-prenylapigenin-7-O-β-D-glucopyranoside (5c), 4'-O-prenylapigenin-7-O-β-D-glucopyranoside (5d). The chemical structures of these compounds were assigned using NMR techniques, mass spectrometry and by comparison of their data with reported ones. The antisalmonellal activity was assessed by determining the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) using serial microdilution methods. The results showed that the MeOH extract and EtOAc fraction were active against all the bacteria tested with MICs ranging from 24 to 1536 μg/mL. Seven isolated compounds and three semi-synthetic compounds tested showed MIC values ranging from 16 to 256 μg/mL. Compounds 1, 3, 5a, 5c and 11 displayed the most potent antisalmonellal properties but were generally less potent than those of reference drugs. The activity of extracts and isolated compounds could be used as the starting point for the development of alternative phytodrugs against salmonellosis.


Introduction
According to the World Health Organization, there is an estimated 22 million cases of typhoid fever causing 216 500 deaths each year in the world [1]. Despite the multiple use of antibiotics couple to the advanced research and development of new products, typhoid and paratyphoid remain important problems in developing countries, particularly in Asia and in sub-Saharan Africa [2][3][4]. Also, the non-respect of medical prescriptions and the practice of auto medication cause resistance to common antibiotics including third generation quinolones [5]. Salmonella or bacteria causing salmonellosis are intracellular Gram-negative pathogens. These genera include Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B and Salmonella typhimurium [6,7]. The global emergence of multi-drug resistant bacteria invokes a necessity to identify new antibacterial therapy. Thus, secondary metabolites from plant origin appear to be an alternative source of new efficient and active compounds.
The Melastomataceae family is the seventh largest flowering plants and is known as an important source of terpenoids, phenolic compounds, quinones, lignans, tannins as well as their glycoside derivatives [8,9] that may be responsible for their traditional medicinal applications [10]. Previous studies carried out on extracts of some plants belonging to Melastomataceae family as Dissotis perkinsiae, Dissotis thollonii and Tristemma mauritianum proved their anti-oxidant and antimicrobial activities [9,11,12 ]. As a rare species, Tristemma hirtum is a herb or small forest bush of 1.25 m high, which grows in marsh usually found in tropical Africa, especially in Ivory Coast, Equatorial Guinea and Cameroon [13]. It is widely found in the West Region of Cameroon where it is used in folk medicine, associated with other plants for the treatment of hemorrhoids, reproductive problems, skin diseases and typhoid fever [14,15]. Previously, researchers have reported antibacterial activities of Tristemma hirtum extracts [16]. As part of our ongoing phytochemical studies of medicinal plants growing in Cameroon, we have previously reported the isolation and characterization of ten compounds including flavonoids, terpenoids, glycerol derivatives and hydrolysable tannins from some fractions of the methanol extract from Tristemma hirtum [17]. In continuation, this work aimed to isolate compounds from T. hirtum capable to inhibit Salmonella bacteria supporting its traditional usage against typhoid fever. The chemical prenylation of 6-hydroxyapigenin-7-O-β-Dglucopyranoside afforded four new semi-synthetic derivatives that were also evaluated for their antisalmonellal activity.

General Experimental Procedures
Optical rotations were determined using a JASCO digital polarimeter (Model DIP-3600). 1 H NMR, 13 C NMR, COSY, HMQC and HMBC spectra were performed in deuterated solvents on a Bruker DRX 500 Spectrometer at 500 MHz and on a Bruker AVANCE III 600 spectrometer (Bruker, Germany) at 600 MHz. All chemical shifts (δ) are given in ppm with reference to tetramethylsilane (TMS) as internal standard or by using the remaining protonated solvent as an internal standard and the coupling constants (J) are in Hz. High resolution mass spectra were obtained with a QTOF Compact Spectrometer (Bruker, Germany) equipped with a HRESI source. The spectrometer was operated in positive and negative modes (mass range: 50-1500, with a scan rate of 1.00 Hz) with automatic gain control to provide high-accuracy mass measurements within 0.4 ppm deviation using Na Formate as calibrant. Column chromatography was performed using 70-230 mesh and 230-400 mesh silica gel 60 (Merck) and sephadex LH-20. TLC was carried out on precoated silica gel 60 F 254 (Merck) plates and spots were visualized by a UV lamp multiband UV-254/365 nm (Model UVGL-58 Upland CA 91786, USA) and by spraying with 50% H 2 SO 4 and heating for 10 min at 110˚C.

Plant Material
The aerial parts of Tristemma hirtum P. Beauv. were collected in Bangang, Bamboutos Division, Western Region of Cameroon in January 2015. The plant material was identified by Mr Flubert TADJOUTEU, botanist at the Cameroon National Herbarium, Yaoundé, where a voucher specimen was kept (reference number (33937/HNC)).

Extraction and Bioactivity-guided Fractionation of
Tristemma Hirtum Dried aerial parts (3 kg) were extracted with MeOH at room temperature for 3 days, and the extract was concentrated to dryness under reduced pressure to yield a dark crude extract (543.3 g). After a preliminary test that revealed the antisalmonellal activity of the methanol extract, 519 g were suspended in distilled water (500 mL) and successively extracted with EtOAc and n-BuOH. The organic phases were concentrated to dryness under reduced pressure, yielding 130 g and 136 g of EtOAc and n-BuOH extract, respectively as well as aqueous residual fraction. The assessment of the antisalmonellal activities of the fractions showed the EtOAc fraction to be more active than the n-BuOH, while no activity was observed for the aqueous residual fraction.

Preparation of Semi-synthetic Derivatives (5a-5d) from 6-Hydroxyapigenin-7-O-β-D -Glucopyranoside (5)
A portion 200 mg 6-hydroxyapigenin-7-O-β-Dglucopyranoside (5) (0.446 mol) was dissolved in 30 mL acetone and 64.8 mg K 2 CO 3 (0.469 mol) and 54.1 µL prenyl bromide (0.459 mol) were added successively to the resulting solution. The mixture was magnetically stirred at room temperature (25˚C) and monitored by TLC until complete disappearance of the starting material (12 hours). Afterwards, distilled water was added and the mixture was extracted with n-BuOH. The obtained organic phase was washed with water, dried over anhydrous Na 2 SO 4 and the solvent was evaporated under vacuum. The obtained mixture was chromatographed over silica gel using an isocratic solvent system of n-hexane-EtOAc (60:40) as the eluent and then purified over multiple step of sephadex LH-20 gel column chromatography with MeOH as the eluent to yield four compounds 54.1 mg of 5a (20.75%), 36.8 mg of 5b (12.64%), 44.5 mg of 5c (17.06%) and 25.7 mg of 5d (11.15%).

Bacteria Used and Culture Media
Microorganisms used for antimicrobial activities included four isolates namely Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B and Salmonella typhimurium. These isolates were obtained from the Medical Bacteriology Laboratory of the Centre Pasteur, Yaoundé, Cameroon. One strain of Salmonella typhi ATCC6539 obtained from American Type Culture Collection was used as reference strain. They were maintained in Mueller Hinton Broth (MHB)/Glycerol (1:1) at -20°C and subcultured on fresh appropriate agar plates 24 h prior to any antimicrobial test. Culture media used were Salmonella-Shigella Agar (SSA) for activation and maintenance of Salmonella strain/isolates and Mueller Hinton Broth (MHB) for sensibility test (MICs) and (MBCs).

In vitro Antisalmonellal Assay
The MICs of extracts, fractions and isolated compounds were determined using serial microdilution using rapid 2-(4iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) (Sigma Aldrich, St Quentin Fallavier, France) colorimetric assay. The samples were carried out in 96-micro well sterile plates as previously described [18]. For this, the test extracts/fractions were dissolved in dimethyl sulfoxide (DMSO)/Mueller Hinton Broth (MHB) (v/v, 5%). This solution was then serially introduced in the different wells of the microplate, which contains 100 µL/well of MHB. One hundred microliters of inoculum (1.5 × 10 6 CFU/mL) prepared in MHB were added to the respective wells. The plates were covered with a sterile plate sealer and incubated at 37°C for 18 h. Wells containing MHB, 100 µL of bacterial suspensions and DMSO at a final concentration of 2.5% served as a negative control. Ciprofloxacin was used as reference antibiotic. The MICs of samples were detected after 18 h of incubation at room temperature (25°C), following addition of 40 µL of 0.2 mg/mL INT and incubation at 37°C for 30 min [19]. Viable bacteria reduced the yellow color of INT into pink. The MIC was defined as the lowest sample concentration that prevented this colour change and exhibited inhibition of microbial growth. The MBCs were determined by adding 50 µL aliquots of the preparations (without INT), which did not show any visible colour change after incubation during MIC assays, into 150 µL of fresh Mueller Hinton broth. These preparations were further incubated at 37°C for 48 h and bacterial growth was revealed by the addition of INT as above. The lowest concentration at which no visible colour change was observed was considered as the MBC. These tests were performed in triplicates at three different occasions.

Chemical Analysis
The dry aerial parts of T. hirtum (3 kg) were extracted with methanol. Part of the crude extract (519 g) was suspended in water and successively extracted with EtOAc and n-BuOH to yield 130 g and 136 g of EtOAc and n-BuOH extracts, respectively. Parts of EtOAc and n-BuOH fractions were subjected to column chromatography over silica gel and sephadex LH-20 gel to yield eleven secondary metabolites Flavonoid Derivatives from Tristemma hirtum P. Beauv (Melastomataceae) ( Figure 1). Known compounds were identified as: mixture of arjunolic acid-28-β-D -glucopyranosyl ester (Arjunglucoside II) (8a) and asiatic acid-28-β-D -glucopyranosyl ester (Quadranoside IV) (8b) [20], β-sitosterol (6), oleanolic acid (7), ellagic acid (10) [21]. The 1 H NMR spectrum also exhibited an anomeric proton signal at δ H 5.27 (1H, d, J = 7.5 Hz, H-1′′) showing HSQC correlation with the corresponding anomeric carbon at δ C 100.8 (C-1′′). Complete assignment of the protons and carbons of the sugar unit was achieved by analysis of 1 H-1 H COSY, HSQC and HMBC spectra. The sugar was identified as a glucuronic acid and this was further confirmed by its quaternary carbonyl carbon (C-6′′) at δ C 169.2 [24], and the β configuration deduced from the coupling constant of 7.5 Hz [25]. The D -configuration of the glucuronyl unit was assumed according to the one most encountered in plant glycosides [26]. Moreover, two oxymethylene protons with acyl induced downfield shifts appearing at δ H 4.05 and 4.13 ppm showed correlations in the HSQC spectrum with the carbon at δ C 64.8 (C-1′′′).  [27,28]. The presence of this group was confirmed by four signals on the 13 C NMR (DMSO-d 6 ; 150 MHz) spectrum at δ C 64.8, 30.5, 18.9, 13.9 ppm corresponding to C-1′′′, C-2′′′, C-3′′′, and C-4′′′, respectively. The total assignment of protons and carbons of this spin system was achieved by 1 H-1 H COSY, HSQC and HMBC correlations. The connectivity of the flavonoid aglycone, glucuronic acid moiety and butyl ester group was determined by HMBC correlations between the anomeric proton at δ H 5.27 (H-1′′) and carbon C-3′ at δ C 145.5, and also between the oxymethylene protons of the butyl ester group (H-1′′′a/ Hb-1′′′b) at δ H 4.05/4.13 ppm and the glucuronic acid moiety carbonyl C-6′′ at δ C 169.2 ppm (Figure 2). The structure of 1 was thus concluded to be luteolin-3′-O-β-Dglucuronopyranosylbutyl ester. Compound 2 was obtained as a yellow amorphous powder. It appeared as a mixture of two related secondary metabolites despite repeated column chromatographic separation and the apparent homogeneity on TLC. The negative mode HRESI-MS showed two pseudomolecular ion peaks at m/z 517.   (Table 1) corresponding to C-3 of the flavonol skeleton [23]. The difference in the molecular weight of 1 and 2 was 16 a. m. u. which indicates the presence of an additional oxygen atom in compound 2 having a quercetin skeleton as the aglycone [29]. The 1 H NMR spectrum also showed the anomeric proton signal of an additional sugar unit at δ H 5.31 (1H, d, J = 7.8 Hz, H-1′′) which had HSQC correlation with the corresponding anomeric carbon at δ C 102.9 ppm (C-1′′). The complete assignment of the protons and carbons of the glucuronide butyl ester moiety with its carbonyl signal at δ C 168.7 (C-6′′) and its butyl signals at δ C 64.8 (C-1′′′), 30.1 (C-2′′′), 18.5 (C-3′′′), 12.5 (C-4′′′) was achieved as for compound 1 [27,28]. The connectivity of the aglycone, glucuronic acid and butyl ester group was also determined by the HMBC correlations between the anomeric proton H-1′′′ (δ H 5.31) and C-3 (δ C 133.9 ppm) of the aglycone, and between the oxymethylene protons of the butyl ester group (H-1′′′) at δ H 4.08 and a carbonyl carbon C-6′′ of glucuronic acid moiety at δ C 168.7 ( Figure 2). Consequently, the structure of compound 2 was found to be quercetin-3-O-β-D -glucuronopyranosylbutyl ester.
In order to verify if compounds 1 and 2 are natural products and not artifacts formed during extraction, comparative TLC was performed and revealed their presence Flavonoid Derivatives from Tristemma hirtum P. Beauv (Melastomataceae) in the MeOH extract. This is in agreement with other β-Dglucuronopyranoside 6′′-O-butyl ester derivatives previously reported from Araliaceae [30], Aquifoliaceae [31] and Cucurbitaceae [32] plant species as naturally occurring compounds.

Characterization of Semi-synthetic Derivatives from Compound 5
The O-alkylation using prenyl bromide afforded four semisynthetic flavanone derivatives ( Figure 3 Table 2 and Table 3.   Table 2 and Table 3.   Table 2 and Table 3.

Discussion
The antibacterial activity of a plant extract is considered to be highly active if the MIC < 100 µg/mL, significantly active when 100 ≤ MIC ≤ 512 µg/mL, moderately active when 512 < MIC ≤ 2048 µg/mL, weakly active if MIC > 2048 µg/mL and not active when MIC > 10,000 µg/mL [33]. The antibacterial activity of extracts and fractions showed MICs varying from 24 to 1536 µg/mL against five bacteria strains of the Salmonella genus ( Table 4). The MeOH extract of Tristemma hirtum was highly active (MIC < 100 µg/mL) against two bacterial species (Salmonella typhi, Salmonella paratyphi B) and significantly active (100 ≤ MIC ≤ 512 µg/mL) against Salmonella paratyphi A and Salmonella typhimurium. The EtOAc fraction also was highly active against Salmonella typhi ATCC6539 and Salmonella paratyphi A; significantly active against Salmonella typhi, Salmonella paratyphi B and Salmonella typhimurium. Despite the significant activity on just two bacterial strains (Salmonella paratyphi A, Salmonella typhimurium), the n-BuOH fraction was also purified according to its abundance of phenolic components which were shown to be active against many bacterial strains [12].
Antimicrobial cut-off points have been defined in the literature to enable the understanding of the effectiveness of pure compounds as follows: highly active when MIC < 1 µg/mL (or 2.5 µM), significantly active for 1 ≤ MIC ≤ 10 µg/mL (or 2.5 ≤ MIC < 25 µM), moderately active when 10 < MIC ≤ 100 µg/mL (or 25 < MIC ≤ 250 µM), weakly active for 100 < MIC ≤ 1000 µg/mL (or 250 < MIC ≤ 2500 µM) and not active when MIC > 1000 µg/mL (or > 2500 µM) [33]. Based on this cut-off, the antibacterial activities of all the tested isolated compounds could be considered as significant or moderate against the specific microorganisms. They showed MIC values from 16 to 256 µg/mL. Compounds 3 and 11 were moderately active (MIC =16 µg/mL) against Salmonella paratyphi A and Salmonella typhi ATCC6539, respectively. Compounds 1, 4, 5, 9 and 10 are also moderately active (MIC from 16 to 64 µg/mL) and then, could justify the activity shown by the original fraction, suggesting that many of them should proceed by synergism to enable the higher activity of the extract. This potentiating effect of the methanol extract from leaves of T. hirtum against Multi-Drugs Resistant Gram-Negative Bacteria have been reported when it was combined with other antibiotics [16]. Compound 5 as a substrate of prenylation reaction showed moderate activity against three bacteria strains tested while two of its semi-synthetic derivatives, 5a and 5c showed the same activity against four various bacterial strains. This observed activity may be due to the prenylated chain because it is reported that the addition of a prenyl groups to an aromatic secondary metabolite often results in a derivative with improved or modified pharmacological activity [34]. These ''hybrid'' natural products nowadays represent a new frontier for the development of novel drugs, and particularly as antibacterial agents [34].
According to the criteria used by Gatsing and Adoga, the antibacterial substance is considered as bactericidal, when MBC/MIC ≤ 4 and bacteriostatic, when MBC/MIC > 4 [35]. Based on those criteria, the large majority of these extracts and isolated compounds are bactericidal. Bactericidal activity of a Tristemma mauritianum extract was already presented by Ngoudjou [12]. In addition, many plant extracts and isolated compounds from the Melastomataceae family are known for their antibacterial activity [9,11]. The isolated compounds found to be active in the present study are members of the triterpenoid, flavonoid, and phenolic acid groups. Although triterpenoid, flavonoid, and phenolic acid compounds have been reported to possess antibacterial activity [36], no study has reported the activity of these compounds about pathogenic bacterial strains used in the present study.
The activity of flavonoids is probably due to their ability to form complexes with extracellular and soluble proteins and to form complexes with bacterial cell wall components as previously described by Cowan [37]. In addition, previous studies revealed that, lipophilic flavonoids may also upset microbial membranes, hence a possible mechanism by which a prenyl group may modify the biological activity of flavonoids is through enhancement of lipophilicity [38]. Furthermore, they notice that insertion of a prenyl chain into a molecule increases its lipophilicity which could modify its biological activity through improve approach, affinity and interaction with the lipophilic membrane [38]. However, further studies are needed to show the detailed mechanisms by which the prenyl group influences biological activity.

Conclusion
Eleven compounds were isolated from the aerial parts of Tristemma hirtum including two new esterified glucuronic flavonoids. Furthermore, four new semi synthetic derivatives Flavonoid Derivatives from Tristemma hirtum P. Beauv (Melastomataceae) were obtained from prenylation of 6-hydroxyapigenin-7-O-β-Dglucopyranoside. The structures of all the compounds were elucidated mainly by NMR and mass spectrometric data. It appears that the MeOH extract and the EtOAc fraction from Tristemma hirtum, as well as luteolin-3′-O-β-Dglucuronopyranosylbutyl ester (1), luteolin (3) and casuarinine (11) possess the most potent antibacterial property. These results highlight the traditional use of Tristemma hirtum in the treatment of infectious diseases, especially those caused by the tested microorganisms. Therefore, Tristemma hirtum may be a good candidate for the search of phytodrugs against salmonellosis.