Chemical Proﬁle and In Vitro Evaluation of the Antibacterial Activity of Dioscorea communis Berry Juice

: Within the large family of Dioscoreaceae, Dioscorea communis (L.) Caddick & Wilkin (syn. Tamus communis L.) is considered among the four most widespread representatives in Europe, and it is commonly known under the name black bryony or bryonia. To date, reports have revealed several chemical components from the leaves and tubers of this plant. Nevertheless, an extensive phytochemical investigation has not been performed on its berry juice. In the present study, metabolite proﬁling procedures, using LC-MS, GC-MS, and NMR approaches, were applied to investigate the chemical proﬁle of the D. communis berries. This work reveals the presence of several metabolites belonging to different phytochemical groups, such as fatty acid esters, alkylamides, phenolic derivatives, and organic acids, with lactic acid being predominant. In parallel, based on orally transmitted traditional uses, the initial extract and selected fractions were tested in vitro for their antibacterial effects and exhibited good activity against two bacterial strains related to skin infections: methicillin-resistant Staphylococcus aureus and Cutibacterium acnes. The MIC and MBC values of the extract were determined at 1.56% w / v against both bacteria. The results of this study provide important information on the chemical characterization of the D. communis berry juice, unveiling the presence of 71 metabolites, which might contribute to and further explain its speciﬁc antibacterial activity and its occasional toxicity.


Introduction
The genus Dioscorea L. is the largest representative of the Dioscoreaceae family, consisting of ≤600 species [1]. The name of the genus, as well as the whole family, was given by the French botanist Charles Plumier in honor of the famous Greek physician Pedanios Dioscorides [2]. Dioscorea species are mainly distributed across wet and periodically dry tropical regions, whereas some of them are extended from temperate to alpine climates [3]. In European countries, the Dioscoreaceae family is represented only by four species: Dioscorea balcanica Košanin and the formerly known Borderea pyrenaica Miégeville
Moreover, two equal amounts (4.0 g) of the lyophilized berry juice (A1 and A2) were subjected to liquid-liquid extraction to extract the non-polar constituents in two different ways. A1 was dissolved in H 2 O (10 mL), and the aqueous layer was extracted with diethyl ether (Et 2 O; 10 mL × 3; A1A) and then with CH 2 Cl 2 (10 mL × 3; A1B). A2 was first subjected to acid hydrolysis by boiling under reflux with 10% w/v hydrochloric acid (37% w/w) for 120 min, and then it was extracted as described above with Et 2 O (A2A) and CH 2 Cl 2 (A2B). The organic layers of all extractions were concentrated to dryness, and afterward, they were analyzed by GC-MS and 1 H-NMR. The 1 H-NMR analyses revealed the presence of compound 1 in all obtained extracts, while compound 2 was found only in A2A and A2B. In addition, 80.0 mg of the lyophilized berry juice (A3) was extracted with n-butanol, and the organic phase, after evaporation, was analyzed by LC-MS. The flow chart of the isolation procedures is shown in Figure S40 [23].

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
Fractions A1A, A1B, A2A, and A2B, as well as the less polar AE, AG, and AI, were subjected to GC-MS analyses using a Hewlett-Packard 7820A-5977B MSD system (Agilent Technologies, Santa Clara, CA, USA) operating in EI mode (70 eV), equipped with an HP-5MS-fused silica capillary column (30 m × 0.25 mm; film thickness: 0.25 µm) and a split-splitless injector. The temperature program was, from 60 • C at the time of injection, raised to 300 • C at a rate of 3 • C/min and subsequently held at 300 • C for 10 min. Helium was used as a carrier gas at a flow rate of 2.0 mL/min. The injected volume of the samples was 2 µL [24].
The retention indices for all compounds were determined according to the Van der Dool approach [25], with reference to a homologous series of n-alkanes from C 9 to C 25 . The identification of the chemical components was based on a comparison of both relative retention times and mass spectra with those reported by Adams [26] and the NIST/NBS and Wiley libraries. The component relative percentages were calculated based on the GC peak areas without using correction factors [24].

Nuclear Magnetic Resonance Spectroscopy (NMR) Spectroscopy
During the whole analysis course, all extracts and obtained subfractions were continuously monitored and traced down using an NMR metabolomic strategy, which permitted detailed characterization thereof. Furthermore, the NMR spectra of compounds 1-5 were measured ( Figures S19-S33), as well as of the fractions with low complexity (AE, AG, and AI).

Identification of Cutibacterium acnes Strain ATCC 6919 by 16S rRNA Gene Sequencing
Genomic DNA extraction from Pure BHI broth and Anaerobe CDC Blood agar cultures of Cutibacterium acnes strain ATCC 6919 was performed using an ExtractMe Genomic DNA Kit (Blirt, Gdánsk, Poland). Universal primers 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) [28] and 1492R (5 -GGTTACCTTGTTACGACTT-3 ) [29] (Eurofins Genomics, Germany) were used to amplify the 16S rRNA gene by PCR. The reaction mixture contained the following: 1 U FastGene Taq DNA Polymerase (NIPPON Genetics, Tokyo, Japan), 1 × PCR buffer A,  25 pmol of each primer, 1 mM dNTPs, a 3µL DNA template, and deionized sterile water at a final volume of 50 µL. The thermal cycler Primus 25 (PEQLAB Biotechnologie, Erlangen, Germany) was used in the following PCR conditions: initialization at 95 • C for 3 min, followed by 35 cycles of denaturation at 95 • C for 30 s, annealing at 50 • C for 30 s, and elongation at 72 • C for 2 min. A final elongation step at 72 • C for 5 min was added.
Amplicons of C. acnes were purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, Düren, Germany) and then directly sequenced via the Sanger dideoxy termination method by Cemia (Larissa, Greece). Chromas (Version 2.6.6 Software, Technelysium Pty Ltd., South Brisbane, Australia, www.technelysium.com.au, accessed on 20 October 2021) was used to check the quality of the obtained sequencing results. The sequences were assembled into a single sequence via MEGA X (Version 10.1.6 Software) [30] and Gene Runner (Version 6.5 Software, Inc., Hudson, NY, USA, www.generunner.net accessed on 20 October 2021) and subjected to a BlastN (Megablast) (Bethesda, MD, USA, https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 20 October 2021 ) search in the 16S rRNA Database-GENEBANK to identify the sequences with the highest similarity.

Bacterial Strains and Growth Conditions
The antibacterial activity of D. communis berry juice was determined against MRSA strain 1552 and Cutibacterium acnes strain ATCC 6919. MRSA strain 1552 was isolated from the clinical samples, and the identification and characterization were conducted by standard laboratory methods (kindly provided by Prof. Spyros Pournaras, School of Medicine, NKUA). MRSA was routinely grown in Müller-Hinton broth (Lab M, Bury, UK) or Müller-Hinton agar (Lab M, Bury, UK) at 37 • C aerobically and C. acnes in Brain Heart Infusion (BHI) broth (Condalab S.A., Spain) or BHI agar (Condalab S.A., Spain) at 37 • C anaerobically.

Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) of the berry juice and AC-AG fractions were determined in sterile 96-well polystyrene microtiter plates (Kisker Biotech GmbH & Co. KG, Steinfurt, Germany) using a spectrophotometric bioassay as previously described [31], with some modifications. Briefly, 0.25 g of berry juice was suspended in sterile ddH 2 O (2-mL final volume) for 1 h at room temperature with occasional vortexing and then centrifuged at 10,000× g for 7 min. The aqueous phase was filtered through a 0.22-µm syringe filter and used for serial dilutions (in Müller-Hinton and BHI broth for MRSA and C. acnes, respectively), corresponding from 25 to 0.39% w/v. The weighed part of the AC-AG fractions was suspended in sterile ddH 2 O containing 1.5% DMSO (2.5-mL final volume) and then centrifuged at 5000× g for 3 min. The aqueous phase was filtered through a 0.22-µm syringe filter and used for serial dilutions as described above. Overnight bacterial cultures of MRSA (grown in Müller-Hinton) were adjusted to a 0.5 McFarland turbidity standard (~1.5 × 10 8 CFU/mL). For 3 days, the old bacterial cultures of C. acnes (grown in BHI broth) were adjusted to a 0.5 McFarland turbidity standard (~1.5 × 10 8 CFU/mL). A 10-µL broth, containing approximately 5 × 10 4 CFUs, was added to 190 µL of the tested twofold sample dilutions.
The positive control wells, containing broth, were inoculated with MRSA or C. acnes to test the growth of the pathogen. The negative control wells contained dilutions of berry juice or fractions in Müller-Hinton or BHI broth without bacteria. The Müller-Hinton or BHI broth control wells without bacteria were used to test for any possible contamination.
The optical density (OD) was determined at 600 nm using an EL × 808 absorbance microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) just prior to incubation (t = 0) and 24 h after incubation (t = 24 h) at 37 • C aerobically for MRSA (t = 0) and 5 days after incubation (t = 5 d) at 37 • C for C. acnes under anaerobic conditions. The OD for each negative control replicate well (containing sample) at t = 24 or t = 5 d for MRSA and C. acnes was subtracted from the OD of the same replicate test well with bacteria at t = 24 or t = 5 d for MRSA and C. acnes, respectively. The growth inhibition at each sample dilution was determined using the formula Sci 2022, 4, 21 6 of 16 % inhibition = [1 − (OD test well − OD of corresponding negative control well)] × 100. The MIC was determined as the lowest sample concentration which resulted in 100% growth inhibition. The MIC values of the berry juice and AC-AG fractions were expressed as w/v and mg/mL, respectively.

Determination of Minimum Bactericidal Concentration (MBC)
The MBC was determined by transferring a small quantity of the sample contained in each replicate well of the microtiter plates to Müller-Hinton agar plates for MRSA and BHI agar for C. acnes by using a microplate replicator (Boekel Scientific, Feasterville-Trevose, PA, USA). The plates were incubated for 24 h aerobically for MRSA and 5 days anaerobically for C. acnes at 37 • C. The MBC was determined as the lowest concentration of the initial extract and fractions at which no grown colonies were observed [32].

Results and Discussion
Previous studies on the title plant showed that root tubers have been the most intensively investigated plant part, with triterpenoids, sterols, and saponins as well as phenanthrenes and furanocoumarins being reported [11,12,33,34]. Similarly, the aerial parts (leaves and shoots) have resulted in a different yield of phenolic derivatives and flavonoids, saponins, sterols, triterpenoids, carotenoids, tocopherols, fatty acids, and organic acids [11,15,35,36]. Nevertheless, to the best of our knowledge, extracts from the berry juice led to the identification of sterols and flavonoids in the Oand C-glucoside forms, though no extensive phytochemical investigation has been performed [16,37]. In southern Central Greece, D. communis berry juice has been reported (by oral sources) as a traditional remedy for skin-related ailments, as well as for oral infections. Taking into consideration the above, our study was oriented to the investigation of the berry juice, targeting its phytochemical content and its potential biological effects on bacteria related to skin infections.
The phytochemical analysis of D. communis berry juice was processed by different chromatographic techniques. Through GC-MS and LC-MS/MS chromatographies aided by NMR spectroscopy, a great number of compounds belonging to different phytochemical groups was identified. The results are presented in Tables 1-8.        Components are listed in all tables according to their elution from an HP-5MS column. KI = Kováts indices calculated against C 9 -C 25 n-alkanes on the HP-5MS column; AI = arithmetic indices; and tr = traces.

Chemical Composition by GC-MS Analyses in Various Fractions of Dioscorea communis Berry Juice
At first, the chemical composition of the berry juice was achieved by two approaches. Extracts with different polarities were obtained through liquid-liquid extraction (see Section 2.3 and Figures S1-S18). The untreated (A1A and A1B) and after acid hydrolysis non-polar extracts (A2A and A2B) were submitted to GC-MS analyses, revealing the presence of several fatty acid esters (Tables 1-4). It is noteworthy that both qualitative and quantitative differences were observed after acid hydrolysis, which could be attributed to the hydrolysis of fatty acid esters, triglycerides, or phospholipids [38]. The chemical fingerprints of the untreated Et 2 O (A1A) and CH 2 Cl 2 (A1B) extracts were quite similar, with ethyl linoleate (45.2% and 46.7%, respectively) and ethyl linolenate (36.9% and 38.1%, respectively) being their main metabolites. After acid hydrolysis, the obtained Et 2 O extract (A2A) was characterized by the presence of methyl esters of palmitic (53.3%), linoleic (20.3%), and linolenic (14.8%) acid, while the CH 2 Cl 2 extract (A2B) was abundant in methyl palmitate (33.2%), ethyl linoleate (28.1%), and ethyl linolenate (24.2%). It was noticed that the ethyl esters of linoleic and linolenic acid were present in all extracts, while methyl linolenate was absent in the untreated Et 2 O extract (A1A). Phthalates, such as dibutyl phthalate (Table 1, compound 1), are used as plasticizer solvents. Thus far, they have been previously described from the genus Dioscorea and the family Dioscoreaceae, as well as from other natural sources [39]. However, their presence as natural products is controversial, as they could be either stored from the environment or co-extracted using solvents during the handling of the plant material [40].
For a more detailed analysis, part of the lyophilized berry juice was subjected to RP 18 -MPLC, and the yielded fractions were screened by 1 H-NMR. Based on the obtained spectra, three fractions (AI, AE, and AG) were selected and further analyzed by GC-MS (Tables 5-7). Briefly, the fractions AI, AE, and AG were mixtures of fatty acid esters, ketones, aldehydes, alcohols, and hydrocarbons. In detail, 23 compounds were detected in fraction AI, with 2-octadecanone (34.0%) and 2E-nonadecene (9.4%) being the main constituents.
In fraction AE, 10 compounds were detected, and once more the main ingredient proved to be 2-octadecanone (49.4%), while methyl stearate was also abundant (20.8%). Fraction AG featured 19 compounds, and again the predominant compound was 2-octadecanone (59.7%), followed by methyl stearate (11.5%) and an unknown compound (11.6%), with m/z = 313.3. The available GC-MS libraries do not include data regarding Nand Pcontaining compounds. However, the odd m/z values suggested the presence of such compounds, which was further supported by the LC-MS analysis.

LC-MS/MS Analysis of n-Butanol Extract of Dioscorea communis Berry Juice
The n-butanol extract (A3) of the berry juice obtained after liquid-liquid extraction (Section 2.3) was submitted to LC-MS/MS analysis. The putative identification of these compounds is summarized in Table 8, where the compounds are listed according to their retention times in the total ion chromatogram (TIC) (Figures S34 and S37). Its 1 H-NMR spectrum was also measured ( Figure S33). Based on these results, the main constituent of A3 was lactic acid. Moreover, more than 45 compounds were tentatively identified by LC-MS analysis, including amino acids, organic acids, sugars, fatty acid derivatives, Ncontaining derivatives, flavonoids, phenolic acids, and other phenolic derivatives. The In agreement with the GC-MS analyses, both Et 2 O extracts (A1A and A2A) were mainly characterized by the presence of ethyl esters. In addition, the Et 2 O extract after acid hydrolysis (A2A) revealed the presence of methyl esters, while the CH 2 Cl 2 extract (A2B) consisted of methyl and ethyl esters. It is worth mentioning that the CH 2 Cl 2 extract after acid hydrolysis (A2B) was remarkably different, since the fatty acid esters and unsaturated derivatives were minor metabolites. The main compounds of this extract were lactic (1) and levulinic acids (2) not detected through the GC-MS analyses. Concerning the polar n-butanol extract (A3), the main signals of the 1 H-NMR spectrum were assigned to lactic acid ( Figure S33).

LC-MS/MS Analysis of n-Butanol Extract of Dioscorea communis Berry Juice
The n-butanol extract (A3) of the berry juice obtained after liquid-liquid extraction (Section 2.3) was submitted to LC-MS/MS analysis. The putative identification of these compounds is summarized in Table 8, where the compounds are listed according to their retention times in the total ion chromatogram (TIC) (Figures S34 and S37). Its 1 H-NMR spectrum was also measured ( Figure S33). Based on these results, the main constituent of A3 was lactic acid. Moreover, more than 45 compounds were tentatively identified by LC-MS analysis, including amino acids, organic acids, sugars, fatty acid derivatives, N-containing derivatives, flavonoids, phenolic acids, and other phenolic derivatives. The molecular formulas were established based on high-precision quasi-molecular ions such as [ (Table 8). The chemical evaluation was in agreement with previous reports on the genus Dioscorea and the Dioscoreaceae family.
It is noteworthy that the accumulation of fatty acid derivatives and phytosterols is essential during fruit development and ripening. For example, stearic acid, although more abundant in animals, can also be found in vegetable fat. Linolenic acid is mostly found in seeds and berries, while ethyl palmitate is among the most common saturated fatty acid esters in plants [41]. Other commonly detected metabolites in plant extracts, like phytosterols (β-sitosterol) and triterpenes (amyrin), also have physiological roles in plants.
For example, phytosterols are naturally present in plant cell membranes, and triterpenes are associated with plant defense [42]. Furthermore, N-alkylamides are essential for plant immunity, usually being produced as a response to abiotic (non-pathogen-induced) and biotic (pathogen-induced) stress. Such compounds act as a chemical defense against phytopathogens and herbivorous predators. Many pathways lead to the expression of defense-related genes, including the production of anti-microbial secondary metabolites like alkylamides [43]. The monitoring of amides in LC-MS was found to be more effectively performed in a positive mode where the carboxamide group is protonated. However, both positive and negative ionization modes were used in the current study, as the negative mode was reported to be more sensitive in the analysis of phenolics and other compounds [44].

Antibacterial Activity of Dioscorea communis Berry Juice and Selected Fractions
To the best of our knowledge, D. communis berry juice has been assessed regarding its antibacterial activity for the first time. Thus far, previous studies on D. pentaphylla and D. bulbifera extracts and fractions from different plant parts revealed their antibacterial activity [21,50,51].
In the present study, D. communis berry juice exhibited bactericidal activity against MRSA and C. acnes. Its MIC and MBC values were determined to be 1.56% w/v against both bacteria. These results indicate that berry juice might be considered a novel source of antibacterial substances against these two bacteria, which are often implicated in dermatological infections and acne. Moreover, the fraction AD exhibited bacteriostatic activity against C. acnes, with an MIC at 6.6 mg/mL. Based on our chemical analyses, the effect could be attributed to 2-octadecanone (compound 3), methyl stearate, and tris-(2,4-ditert-butylphenyl)phosphate (compound 4), which were identified in fraction AD (Figures S12-S18 and Table S1).

Conclusions
In this study, GC-MS analysis offered influence measurements on the non-and lesspolar components with a key role in the characterization of 22 fatty acid derivatives. On the other hand, LC-MS analysis comprises a wide variety of compounds predominant as primary or secondary metabolites, such as amino acids (1), organic acids (3), lipids (14), terpenes-sterols (6), sugars, and phenolics (8), and NMR offers the structure elucidation of 5 individual components, as well as the metabolite fingerprinting. These methods were equally adapted in order to provide both an inclusive impression and complete analysis of the critical components existing in the plant material. The antibacterial activity of D. communis berry juice against pathogens often implicated in dermatological infections has been reported herein for the first time. MRSA and C. acnes were used, showing MIC and MBC values at 1.56% w/v against both bacteria, which warrants further investigation as this may lead to medical applications. It is notable that these bacteria are resistant to several antibiotics, and treatments that target multiple pathological processes of skin abnormalities are accompanied by side effects [52,53]. Therefore, alternative therapies are urgently needed. Nevertheless, future studies for the evaluation of the acute and sub-acute toxicity effects of the berry juice extract should be conducted.  Table S1: Chemical composition of fraction AD B . Table S2