Antimicrobial Properties of Compounds Isolated from Syzygium malaccense (L.) Merr. and L.M. Perry and Medicinal Plants Used in French Polynesia

A preliminary ethnopharmacological survey, achieved in French Polynesia, led to the collection of the most cited plants among 63 species used to treat “infectious” diseases, with a description of their medicinal uses. Bibliographical investigations and antimicrobial screening permitted the selection of the botanical species Syzygium malaccense (Myrtaceae) for phytochemical analysis. Leaves of Syzygium malaccense were usually used in mixture with rhizomes of Curcuma longa to treat infectious diseases such as cystitis. The methanolic plant extracts were tested in vitro with an agar microdilution method on 33 bacteria strains and 1 yeast to obtain their Minimal Inhibitory Concentration (MIC), and cytotoxicity against HepG2 cells were evaluated. Antimicrobial synergistic effects of methanolic plant extracts from leaves of Syzygium malaccense and rhizomes from Curcuma longa were also evaluated. The bio-guided isolation of leaf extract from Syzygium malaccense led to the identification of seven alkyl-salicylic acids (anacardic acids or ginkgolic acids C15:0, C15:1, C17:0, C17:1, C17:2, C17:3 and C19:1) described for the first time in this species. All compounds were tested against Staphylococcus aureus (18.75 < MIC < 75.0 µg/mL), Streptococcus pyogenes (2.34 < MIC < 18.75 µg/mL) and Pseudomonas aeruginosa (MIC = 150 µg/mL), and their structure–activity relationships were discussed. The methanolic extract and salicylic derivatives from S. malaccense showed an interesting antimicrobial activity against Gram+ bacteria, without toxicity on hepG2 cells at 400 μg/mL. Moreover, these antibacterial compounds have already been studied for their anti-inflammatory activity, which supports the therapeutic interest of S. malaccense against infectious diseases.


Introduction
In 2011, the WHO (World Health Organization) asked for an increased search for new drugs as antibiotic resistance has increased dramatically, but only a few new molecules are in development. Plants can be a source of new antimicrobial drugs, hopefully safe both for human use and the environment [1]. An ethnopharmacological study performed in the Raiatea and Tahaa islands led to the collection of 30 of the most common plants and lichens among 63 species used to treat infectious diseases. In vitro assays revealed the antimicrobial activities of 13 plants and one lichen species with MIC ≤ 0.6 mg/mL for one or several of the 34 micro-organisms. Cytotoxic assays performed on HepG2 cells did not reveal growth inhibitions at MIC ≤ 0.4 mg/mL except for Curcuma longa (rhizome), Calophyllum inophyllum (bark and leaves) and Hibiscus tiliaceus (bark), which were known to have specific toxicity against hepatoma cell lines (HepG2) without any effects on healthy human cells [2,3].
Then, authors focussed on Syzygium malaccense, which was shown to be one of the most active of the tested plants and therefore deserves deeper investigation as it was little previously reported in literature.

Introduction
In 2011, the WHO (World Health Organization) asked for an new drugs as antibiotic resistance has increased dramatically, b molecules are in development. Plants can be a source of new a hopefully safe both for human use and the environment [1]. An et study performed in the Raiatea and Tahaa islands led to the collecti common plants and lichens among 63 species used to treat infectio assays revealed the antimicrobial activities of 13 plants and one lich ≤ 0.6 mg/mL for one or several of the 34 micro-organisms. Cytotoxic a HepG2 cells did not reveal growth inhibitions at MIC ≤ 0.4 mg/mL longa (rhizome), Calophyllum inophyllum (bark and leaves) and Hib which were known to have specific toxicity against hepatoma cell lin any effects on healthy human cells [2,3].
Then, authors focussed on Syzygium malaccense, which was sho most active of the tested plants and therefore deserves deeper investi previously reported in literature.
S. malaccense (Myrtaceae) is a cultivated fruit tree from French P of which are used in local medicine to treat infectious diseases like furunculosis, oral thrush, and vaginal mycosis. Scientific refere traditional use against a wide variety of inflammatory conditions in W Nonetheless, S. malaccense is still poorly studied regarding the bioacti leaves [6,7] and its antimicrobial activity [8].
This study reports the antimicrobial activity of methanolic extrac of 29 other species used against infectious diseases. It also revealed of the S. malaccense leaf extract with Curcuma longa rhizome extract.
Bio-guided isolation and identification of compounds were pe chromatographic methods and by spectroscopic analysis (UHPLC-UV NMR). Seven antibacterial compounds (6-alkenyl or 6-alkyl salicylic from S. malaccense methanolic leaf extract and were identified for species (Figure 1). The antimicrobial activity of some of these com been briefly mentioned in other studies against Gram-positive bact aureus [9], Bacillus subtilis, Brevibacterium ammoniagenes, Streptococcus m acnes [10] and Porphyromonas gingivalis [11] -and a Gram-negative ba pylori, [12], but not against most of the microorganisms cited in th allowed a discussion on the structure-antibacterial activity relationsh

Ethnopharmacological Survey
The survey was focused on plants and remedies used against i the Raiatea and Tahaa islands during 2017-2018. This prelimin identification of 63 species belonging to 36 botanical families with th most cited species by 18 healers (collected species with their tradition in Table 1 and other plant species were added in Supplementary dat

Ethnopharmacological Survey
The survey was focused on plants and remedies used against infectious diseases in the Raiatea and Tahaa islands during 2017-2018. This preliminary step led to the identification of 63 species belonging to 36 botanical families with the collection of the 30 most cited species by 18 healers (collected species with their traditional use are presented in Table 1 and other plant species were added in Supplementary data).  Thespesia populnea (L.) Sol. ex Corrêa "miro" (Malvaceae) (9) Pneumo. S. malaccense leaves were usually used to treat "cystitis" (83.3% of uses) in association with C. longa rhizome, suggesting synergistic effects of plant extracts.

Antimicrobial Activity and Cytotoxicity of Plant Methanolic Extracts
The antimicrobial activity of 33 extracts was evaluated against a panel of 34 pathogenic and multi-resistant microbes ( Table 2). As the authors are aware of the difficulties in ascribing any specific identified bacterial species to a so-called "traditional use", they decided to test plant extracts on a large panel of Gram-positive, Gram-negative bacteria and one yeast. According to previous publications concerning the anti-infective potential of natural products, plant extracts were considered to be interesting antimicrobial agents at MIC ≤ 0.30 mg/mL, as was observed eight 8 plant species: (Calophyllum inophyllum, Cordia subcordata, Curcuma longa, Psidium guajava, Syzygium cumini, Syzygium malaccense, Thespesia populnea, Torenia crustacea).      MIC ≤ 0.3 mg/mL; (-) no MIC or FIC determined at 1.2 mg/mL; (n = 3; with standard deviation for value: 1.2 ± 0.4 mg/mL; 0.6 ± 0.2 mg/mL; 0.3 ± 0.1 mg/mL; 0.15 ± 0.05 mg/mL; 0.07 ± 0.03 mg/mL).
The screening allowed the identification of 15 plant extracts, with antibacterial activities characterised by MIC ≤ 0.6 mg/mL (Tables 2 and 3). The higher antifungal activities against the yeast Candida albicans were observed for the bark extract of Syzygium cumini and the aerial part extract of Torenia crustacea (MIC = 0.15 mg/mL).

Antimicrobial Tests of Methanolic Extract from Syzygium malaccense and Curcuma longa
In vitro antimicrobial activities were characterized by the Minimal Inhibitory Concentration (MIC) and by the Fractional Inhibitory Concentration index (FIC index, Table 3).  Cytotoxic assays performed on HepG2 cells did not reveal growth inhibitions at MIC ≤ 0.4 mg/mL except for C. longa (rhizome, IC50 = 33 ± 9 μg/mL), C. inophyllum (bark and leaves; IC50 =59 ± 4 and 76 ± 5 μg/mL) and H. tiliaceus (bark, IC50 = 83 ± 10 μg/mL). While this assay was intended as a preliminary screening of nonspecific cell toxicity, its results should not discourage the use of these three plants, as their toxicity seems to be specific against tumoral cells like hepatoma cell lines (HepG2) without having any effects on healthy human cells [2,3].

Antimicrobial Tests of Methanolic Extract from Syzygium malaccense and Curcuma longa
In vitro antimicrobial activities were characterized by the Minimal Inhibitory Concentration (MIC) and by the Fractional Inhibitory Concentration index (FIC index, Table 3).  Cytotoxic assays performed on HepG2 cells did not reveal growth inhibitions at MIC ≤ 0.4 mg/mL except for C. longa (rhizome, IC50 = 33 ± 9 μg/mL), C. inophyllum (bark and leaves; IC50 =59 ± 4 and 76 ± 5 μg/mL) and H. tiliaceus (bark, IC50 = 83 ± 10 μg/mL). While this assay was intended as a preliminary screening of nonspecific cell toxicity, its results should not discourage the use of these three plants, as their toxicity seems to be specific against tumoral cells like hepatoma cell lines (HepG2) without having any effects on healthy human cells [2,3].

Antimicrobial Tests of Methanolic Extract from Syzygium malaccense and Curcuma longa
In vitro antimicrobial activities were characterized by the Minimal Inhibitory Concentration (MIC) and by the Fractional Inhibitory Concentration index (FIC index, Table 3). Regarding antibacterial effects, the best activities were observed for Calophyllum inophyllum bark extract, mainly against Gram-positive bacteria belonging to the genus Staphylococcus (0.07 < MIC < 0.30 mg/mL), Streptococcus (MIC = 0.15 mg/mL) and the species Corynebacterium striatum (MIC = 0.15 mg/mL). Moderate antimicrobial activities against Gram-positive bacteria (0.15 ≤ MIC ≤ 0.30 mg/mL) were also observed for Thespesia populnea bark extract (against Staphylococcus sp and Streptococcus agalactiae), Cordia subcordata and Syzygium cumini bark extracts (against Staphylococcus sp.), and with Psidium guajava leaf extract (against Staphylococcus epidermidis).
An antibiotic control using standard compounds had been previously performed and were available for each strain (Table S2 of Supplementary data), but it is important to remember that antibiotics are pure active compounds that do not have a vegetal origin; therefore, it cannot be expected that plant extracts could have the same efficiency as antibiotics [13]. Nevertheless, those tests were performed to obtain useful information for the choice of plant remedies against infectious diseases and to extend the scope of therapeutic applications.
While this assay was intended as a preliminary screening of nonspecific cell toxicity, its results should not discourage the use of these three plants, as their toxicity seems to be specific against tumoral cells like hepatoma cell lines (HepG2) without having any effects on healthy human cells [2,3].

Antimicrobial Tests of Methanolic Extract from Syzygium malaccense and Curcuma longa
In vitro antimicrobial activities were characterized by the Minimal Inhibitory Concentration (MIC) and by the Fractional Inhibitory Concentration index (FIC index, Table 3).
These results suggested the presence of antimicrobial compounds in S. malaccense leaf methanolic extracts, which could have synergic effects with compounds from the rhizome of Curcuma longa such as curcumin, and reinforce the therapeutic choice of this plant mixture in traditional medicine.

Structural Identification
Activity-guided isolation of S. malaccense leaf extract led to the identification of seven 6-alkenyl or 6-alkyl-salicylic acids (anacardic and ginkgolic acids) that were described for the first time in this species (Figure 1) The NMR spectra of these salicylic acid derivatives provided a common pattern of peaks characteristic of their aromatic structure (Table 4). In fact, the chemical shifts recorded for compound 1, at δ H : 6.89 ppm (d, J = 8. Regarding the olefinic chain unsaturations of all isolated compounds, the determination of the cis configuration was afforded by 13 C-NMR spectra that exhibited specific chemical shifts at 25.5 ≤ δC ≤ 27.3 ppm for the allylic methylene carbons of a cis geometry, whereas a trans stereochemistry would be indicated by a downfield shift of approximately 10 ppm [14,15]. Moreover, IR spectra of an unsaturated chain of phenolic acids showed an important absorbance at 695-699 cm −1 characterizing the cis configuration, whereas no trans configuration-specific absorption was observed around 960-980 cm −1 [16]. Localisation of the double bound was supported by NMR data (2D-HMBC correlations, COSY or selective TOCSY), and by mass spectrometry analysis of the thiomethylated derivatives (from compounds: 2, 4, 6 and 7). The position of the double bonds in the alkyl side chains was afforded by the EI-MS fragments of the α,β-bis-(methylthio)-derivatives produced by the reaction of the compounds with dimethyl disulfide [17]. The EI-MS spectra of the thiomethylated derivatives showed intense fragment ions [C n H 2n+1 S] + corresponding to the cleavage of the terminal aliphatic chain residues at the original site of the unsaturation. For example, the presence of a fragment ion at m/z: 117 corresponding to the cleavage of [C 6 H 13 S] + clearly revealed that the double bond on the alkyl side chain was originally located between the fifth and sixth carbons from the terminal methyl group, as observed for compounds 2, 4 and 7. (Figure 1). HNMR spectra revealed a 6-alkyl-salicylic acid moiety as previously described. The bidimensional HSQC and HMBC spectra combined with the molecular formula also demonstrated the presence of a linear saturated carbon chain composed of 15 carbons linked by the C-6 of the aromatic ring, corresponding to the 6-pentadecyl salicylic acid. The comparison of spectroscopic data (UHPLC-MS and NMR) with the standard of the ginkgolic acid C15:0 indicated a total similarity of the compounds structure. This anacardic acid (15:0) was originally identified in Amphipterygium adstringens (Schltdl.) Schiede ex Standl. (Anacardiaceae) stem bark [11] and NMR chemical shift were also consistent with previous spectroscopic data provided by the G. biloba leaves phytochemical analysis [18]. (C15:1, ω5) The molecular formula C 22  characterizing a [C4H9S] + moiety and corresponding to an unsaturation localized at ω3. Moreover, the selective TOCSY experiment performed on the signal at δH = 0.99 ppm exhibited correlations with the protons at δH = 2.09 and 2.82 ppm from the methylenes of the allylic groups, affording the position of double bonds in ω3 and ω6. The ω9 unsaturation was deduced from the chemical shift of the two methylenic protons at δH = 2.82 (carried by the allylic methylene carbon at δC = 25.5 ppm), which is specific of a methylene group wedged between two double bonds (ω6 and ω9), as it was also measured for the ω3-ω6 unsaturations [14]. Bibliographical data exhibited a similar compound previously isolated from the species Spondias mombin within double bonds located at ω3, ω6 and ω9 [22]. 2.4.6. Compound 6: Ginkgolic Acid (C17:2; ω6, ω9)

Compound 2: Ginkgolic Acid
The spectral NMR and the molecular formula C24H36O3 deduced from the HR El-MS spectra data highlighted the presence of a 6-alkenylsalicylic acid with a C17 diunsaturated side chain. The aromatic ring and carbonyl group accounted for five of the seven degrees of unsaturation from the molecular formula and the remaining two were attributed to the two olefinic double bonds with their methine protons (δ: 5.38 ppm, 4H).
Similar chemical shifts and correlations to those observed for compound 5 were observed, except for the aliphatic chain ending that did not exhibit any double bond in ω3. This compound was already identified in the aerial parts of Spondias mombin and their spectrometric data were corroborated [22].

Antimicrobical Activity of Compounds 1 to 7
MIC of isolated compounds and standard were determined on three strains: S. aureus 8241, S. pyogenes 19138 and P. aeruginosa 8129 (Table 5). Table 5. MIC (µg/mL) of compounds isolated from S. malaccense and standard molecules (n = 3); (NA: Not Active). The COSY and HMBC spectra showed correlations of protons from the methyl terminal of the aliphatic chain (δH = 0.99 ppm) with the protons δ H = 2.0 ppm (COSY) and with δ C = 20.6 ppm (alpha unsaturated carbon, HMBC) corresponding to the double bound in ω3. This localization of the unsaturation was afforded by GC-EI-MS analysis of the thiomethylated derivative, which exhibited the presence of a fragment ion at m/z: 89.2, characterizing a [C 4 H 9 S] + moiety and corresponding to an unsaturation localized at ω3. Moreover, the selective TOCSY experiment performed on the signal at δ H = 0.99 ppm exhibited correlations with the protons at δ H = 2.09 and 2.82 ppm from the methylenes of the allylic groups, affording the position of double bonds in ω3 and ω6. The ω9 unsaturation was deduced from the chemical shift of the two methylenic protons at δ H = 2.82 (carried by the allylic methylene carbon at δ C = 25.5 ppm), which is specific of a methylene group wedged between two double bonds (ω6 and ω9), as it was also measured for the ω3-ω6 unsaturations [14].
Bibliographical data exhibited a similar compound previously isolated from the species Spondias mombin within double bonds located at ω3, ω6 and ω9 [22].
2.4.6. Compound 6: Ginkgolic Acid (C17:2; ω6, ω9) The spectral NMR and the molecular formula C 24 H 36 O 3 deduced from the HR El-MS spectra data highlighted the presence of a 6-alkenylsalicylic acid with a C17 di-unsaturated side chain. The aromatic ring and carbonyl group accounted for five of the seven degrees of unsaturation from the molecular formula and the remaining two were attributed to the two olefinic double bonds with their methine protons (δ: 5.38 ppm, 4H).
Similar chemical shifts and correlations to those observed for compound 5 were observed, except for the aliphatic chain ending that did not exhibit any double bond in ω3. This compound was already identified in the aerial parts of Spondias mombin and their spectrometric data were corroborated [22].  (Table 5).

Synergistic Effects of Plant Extracts
The evaluation of the antibacterial synergy of curcumin (major compound from C. longa rhizome) with isolated compounds from S. malaccense were performed by a checkerboard method, but no particular effect was observed (in contrast with the synergy observed for the crude plant extracts in mixture), suggesting the presence of other bioactive compounds.

Structure-Activity Relationships
The best activities were observed against S. pyogenes. Bacteriological tests showed a decrease in the activity between C15:0 and C17:0 compounds. However, an improvement in the activity was observed between C15:0 and C15:1, and between C17:0, C17:1, C17:2 and C17:3, meaning an enhancement of antibacterial activity correlated with the number of unsaturations (the most unsaturated compound (5) was the most active against Grampositive bacteria). Moreover, the activity of compound C17:1 was comparable to those observed for C19:1 (CMI = 18.75 µg/mL). Therefore, experimental data underlined evidence of an optimal chain length and number of unsaturation, promoting the antibacterial activity against S. pyogenes. Similar observations were made for other Gram-positive bacteria (S. aureus). Those results were in accordance with previous data regarding the importance of the number of unsaturation even if optimal activity were also previously recorded against Gram-positive or -negative bacteria for saturated alkyl chains ranged between 6 and 13 carbons [10].
Nonetheless, no important variation in the activity against P. aeruginosa was observed for the different compounds, suggesting another pharmacological mechanisms against this Gram-negative bacterium (ginkgolic acids could restrict P. aeruginosa virulence by abolishing pyocyanin pigment production) [23].
Those results attested the importance of the aliphatic chain length and its unsaturation number or location for an optimal antimicrobial activity of these salicylic derivatives against Gram-positive bacteria and permitted us to explain the antimicrobial plant extract activity.

Conclusions
This preliminary ethnopharmacological survey was the first performed in the Leeward Islands of Society archipelago (most of our previous works were focussed on the Windward Islands archipelago) and led to the selection of antimicrobial plant extracts [24][25][26].
S. malaccense leaf methanolic extract was studied for its antimicrobial activities against Gram-positive bacteria (MIC < 0.6 mg/mL) with additive or synergistic effects with C. longa rhizome methanolic extract. However, no antibacterial synergy was observed between ginkgolic acids and curcumin, which are, respectively, major active compounds from these two plant extracts, suggesting the presence of other bioactive compounds. The 6-alkenyl or alkyl-salicylic acids (anacardic acids or ginkgolic acids C15:0, C15:1, C17:0, C17:1, C17:2, C17:3 and C19:1) were identified as the main antibacterial compounds from the methanolic extract of S. malaccense. The presence of these compounds can justify the medicinal uses of the plant species against infectious diseases and creates interesting anti-inflammatory and complementary effects to those observed for similar salicylic derivatives [27] and to those described in other traditional uses and experimental data [4].
Antimicrobial activity of the seven isolated compounds from S. malaccense was determined against three strains. The present study showed a good antimicrobial activity of all compounds on S. pyogenes, followed by S. aureus, and low activity against P. aeruginosa. The structure-activity relationship analyses concluded to the importance of the length of the side chain of molecules as well as the number of their unsaturations.
The methanolic extract from S. malaccense appeared to have interesting antimicrobial activity against Gram-positive bacteria and did not show toxicity on hepG2 cells at 400 µg/mL. Nonetheless, it is important to point out that the potential toxicity of these compounds may induce dermatitis and hepatic or renal disorders [28,29].

Ethnopharmacological Survey
The Tahaa and Raiatea islands are located in the Society archipelago (16 • 44 00 S; 151 • 27 00 W and 16 • 36 58 S; 151 • 30 00 W). The ethnopharmacological survey was carried out among the 18 traditional healers specialized in the treatment of "ma'i mau" or "real diseases" using semi-structured interviews. The study was focused on infectious diseases and was conducted with a rigorous ethical approach. The legal authorities of Polynesia were contacted and informed of the survey. The project was declared to the environment department of Polynesia (DIREN), with declaration to the Minister of Health and Social Services, and all informant consents were collected.

Plant Material and Preparation of Extract
The parts of the plants traditionally used were collected and dried in a dark room at 30 • C, then ground and macerated in methanol during 12 h (1 g/10 mL). Extracts were filtered, and the solvent was removed by evaporation under reduced pressure.

MIC and FIC Index Measures
The antimicrobial test of methanolic extract was carried out on Petri dishes containing Mueller-Hinton Agar (MHA) mixed with plant extract to obtain final concentrations ranged from 1.25 to 0.07 mg/mL. According to previous publications about the anti-infective potential of natural products, plant extracts were considered as active at MIC ≤ 0.30 mg/mL [33,34].
Extracts from S. malaccense and C. longa were tested against 34 clinical microbial strains. A multi-headed inoculator seeded the bacterial strains on prepared Petri dishes at 10 5 CFU/mL (Colony Forming Unit/mL) in Cysteine Ringer (CR) solution (Merck, Darmstadt, Germany). Minimal Inhibitory Concentrations (MIC) were visually determined after 24 h of incubation at 37 • C. MIC values of extracts and controls were recorded as the lowest concentrations of extracts, showing no growth of colonies. The results were obtained from the average of triplicate measurements.
Synergy was evaluated by a checkerboard method and determination of the FIC index (fractional inhibitory concentration). The final extract concentrations were ranged from 1.25 to 0.07 mg/mL both for vegetal extract or compounds (A and B). FIC index was calculated with FIC = (MIC of A in combination/MIC of A alone) + (MIC of B in combination / MIC of B alone). Results were interpreted as "synergistic activity" (FIC ≤ 0.5), "additional activity" (0.5 < FIC < 1), "indifferent activity" (1 ≤ FIC ≤ 2) and "antagonistic activity" (2 < FIC) [35].

MIC Determination of Isolated Compounds and Standard Molecules Using a Broth Microdilution Method
A serial dilution technique using 96-well microtiter plates was used to determine the MIC of the pure compounds against sensitive bacteria: Streptococcus pyogenes 19138, Staphylococcus aureus 8241 and Pseudomonas aeruginosa 8129. Nine concentrations of each compound, from 300 to 1.7 µg/mL, were used. They were serially twofold diluted with RC in nine wells. Two wells were represented as bacteria culture control (positive control) and medium sterility control (negative control). Then the wells were loaded with MH liquid medium and bacterial suspension (10 4 bacteria/mL), giving a final volume of 200 µL. The plates were incubated overnight at 37 • C. Bacterial growth was indicated visually and then by direct spray of 0.2 mg/mL INT to each well with incubation at 37 • C for 30 min. Bacterial growth was indicated by a reddish-pink colour. MIC values were determined as the lowest concentrations of compounds showing clear wells.

Cytotoxic Activity of Crude Plant Extracts
Hepatocellular carcinoma cell line (HepG2) and an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method were used to quantify cell viability. Cells were seeded into a 96-well microplate in a Gibco Dulbecco's Modified Eagles Medium (DMEM), with 10% FBS (Foetal Bovine Serum, ThermoFisher Scientific, Merelbeke, Belgium) and antibiotics (mixture penicillin/streptomycin 100 UI/mL, Sigma Aldrich, Machelen, Belgium). After two days at 37 • C (5% of CO 2 ), wells were emptied by suction and the cells were treated with plant extracts diluted in the culture medium (with 0.2% DMSO, at concentrations from 400 µg/mL to 25 µg/mL), to obtain a final volume of 100 µL. After two days, the medium was discarded and replaced with DMEM containing 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After 1 h 30 min of incubation at 37 • C and 5% CO 2 , the water-insoluble formazan was dissolved in 100 µL DMSO and the absorbance was measured at 550 nm using a UV-spectrometer (Tecan, Spark 10M). The cytotoxicity of the crude extracts and standard (camptothecin: IC 50 : 0.9 ± 0.1 µg/mL) was determined by comparing the absorbance of treated cells with the absorbance of control cells cultured in 0.2% DMSO. Data were expressed as a percentage of inhibition calculated according to the formula (% Cell viability = Abs treated cells × 100/Abs control cells) and were analysed by linear regression using GraphPad Prism (5.01).

4.4.
Bioautography and Isolation of Aliphatic-Salicylic Acids from Syzygium malaccense 4.4.1. Bioautography Bioautography of Syzygium malaccense leaf methanolic extract against Streptococcus pyogenes, Streptococcus pyogenes and Staphylococcus aureus enabled the activity-guided isolation of antibacterial compounds. TLC was carried out on silica gel 60 F254 aluminium plates (Merck) submitted to Tol/AcOEt/MeOH (7:2:1; v/v) eluent with UV detection 254 and 366 nm. The plates were covered by MHA containing the bacterial strain suspension in Petri dishes. After incubation (24 h at 37 • C), growth was revealed by iodonitrotetrazolium chloride (INT, 2 mg/mL) and inhibition zones were visually localized on the TLC.

Purification of Salicylic Acids Derivatives
The methanolic dried extract (50 g), obtained from 600 g of powdered leaf of S. malaccense, was subjected to column chromatography on silica gel (MN Kieselgel 60, 0.063-0.2 mm) and successively eluted with n-Hex, Tol, AcOEt and MeOH to afford 46 fractions. Fractions F26 to F43 were subjected to various column chromatography on silica gel (MN Kieselgel 60, 0.063-0.2 mm) and successively eluted with Tol, AcOEt and MeOH for the purification of active compounds monitored by UHPLC-UV-MS.

Spectrometric Analysis
Compounds isolated from S. malaccense were analysed by High Resolution Mass Spectrometry (HR-MS) using a Thermo Fisher Scientific Orbitrap Mass Spectrometer with an electrospray ion source (negative mode) and Xcalibur software (Arlington, VA, USA).
El-MS spectra of thiomethylated derivatives were realised on a GC-MS Trace DSQ (ThermoFisher in CHCl 3 , 70 eV, positive mode).

Thiomethylation
To a solution of alkenyl-salicylic acid (1 eq., 0.01 mmol) in diethyl ether (2 mL) was added iodine (9.5 eq., 24 mg, 0.095 mmol) and dimethyl disulfide (450 eq., 0.4 mL, 4.5 mmol) in a sealed tube. The reaction mixture was stirred at 50 • C for 5 h, then cooled to room temperature, poured into 5% aqueous sodium thiosulfate solution (5 mL) and extracted with diethyl ether (3 mL) three times. The combined organic layer was concentrated in vacuo to give a crude dimethyldisulfide (DMDS) adduct, which was addressed to EI MS analysis without further purification.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life12050733/s1, Table S1: Medicinal plants cited in the survey but not collected, with their medicinal uses; Table S2