FARMACIA, 2019, Vol. 67, 6 https://doi.org/10.31925/farmacia.2019.6.22 ORIGINAL ARTICLE CHROMATOGRAPHIC ANALYSIS AND ANTIBACTERIAL POTENTIAL OF EXTRACTS OF GNETUM AFRICANUM CRISTIAN SEBASTIAN VLAD 1, LAVINIA VLAIA 2, VICENŢIU VLAIA 2, VICTOR DUMITRAŞCU 1, MARIOARA NICOLETA FILIMON 3*, ROXANA POPESCU 4, ADINELA CIMPORESCU 5, CRISTINA DEHELEAN 2, CHINYERE EZINNE ONWUBIKO 6, CRISTINA VLAD DALIBORCA 1 “Victor Babeş” University of Medicine and Pharmacy, Faculty of Medicine, Pharmacology Department, 2 Eftimie Murgu Square, 300041, Timisoara, Romania 2 “Victor Babeş” University of Medicine and Pharmacy, Faculty of Pharmacy, 2 Eftimie Murgu Square, 300041, Timişoara, Romania 3 West University of Timişoara, Faculty of Chemistry, Biology, Geography, Biology-Chemistry Department, 16 Pestalozzi, 300115, Timişoara, Romania 4 “Victor Babeş” University of Medicine and Pharmacy, Faculty of Medicine, Cell and Molecular Biology Department, 14 Tudor Vladimirescu, 300173, Timişoara, Romania 5 Emergency Clinical Hospital “Pius Brinzeu”, Toxicology Department, 156 Liviu Rebreanu, 300723, Timişoara, Romania 6 Cardiotim Hospital, 6 Franyó Zoltán, 300014, Timişoara, Romania 1 *corresponding author: marioara.filimon@e-uvt.ro Manuscript received: June 2019 Abstract Traditionally, Gnetum africanum is being used widely for its nutritional value. In the present study, extracts of these plants were evaluated for their antimicrobial activity against some human pathogenic bacteria viz. S. aureus, B. cereus, E. coli, Klebsiella spp. and Enterobacter aerogenes. Gas-chromatographic results evidenced the presence of various bio-active compounds and the extracts applied on human pathogenic bacteria presented antibacterial effect, depending on the type of solvent used for the extraction and also the bacterial strain of concern, exception was made by the chloroformic extract which did not exhibit any antibacterial effect on the bacterial strains selected in the study. Rezumat În mod tradițional, Gnetum africanum este utilizat pe scară largă pentru valoarea sa nutritivă. În studiul de față, extracte din aceasta planta au fost evaluate pentru activitatea lor antimicrobiană împotriva unor bacterii patogene umane, si anume, S. aureus, B. cereus, E. coli, Klebsiella spp. și Enterobacter aerogene. Rezultatele cromatografice au evidențiat prezența diferiților compuși bioactivi, iar extractele aplicate pe bacteriile patogene umane au prezentat efect antibacterian, în funcție de tipul de solvent utilizat pentru extracție și, de asemenea, de tulpina luata in studiu, excepția a fost făcută de extractul cloroformic care nu a prezentat niciun efect antibacterian asupra tulpinilor selectate în studiu. Keywords: collagen, minocycline, spongious delivery systems, freeze-drying Introduction antibiotics as well as the resistance gained by many microbial strains. Many plant species (spontaneous, cultured, medicinal) showed pharmaceutical and antimicrobial properties [3-12] therefore testing the possible antimicrobial effects of different parts of plant extracts is recommended. Gnetum africanum is a wide use plant due to its nutritional value, high contents of proteins and minerals, thus it is used in food, animal feed and medical purposes for treating certain diseases. The antibacterial and antifungal properties of G. africanum extracts are based on the identified phytochemicals including tannins, flavonoids, terpenoids, alkaloids, saponins, and phenols [13]. The objectives of this study were to evaluate the phytochemical composition of Gnetum africanum Phytotherapy has become a resource in medicine, for its prevention purpose, and also for its use in the treatment of different affections. The use of plants and herbs for the purpose of cure has become attractive all over the world in the last decades [1]. Medicinal plants, and also spontaneous or crop plants are used as empirical therapy. Some active biological compounds from plants present antimicrobial effects; their action mechanisms sometimes assure a pathway to treat some infections determined by the antibioticresistant microorganisms [2]. An increased attention was focused on finding new “natural” sources due to the side effects caused by the administration of 1083 FARMACIA, 2019, Vol. 67, 6 hours in dark, at room temperature, and then the samples were submitted to GC-MS analysis. Gas-chromatography mass spectrometry (GC-MS) analysis GC-MS analysis of Gnetum africanum extract was achieved using a 450 GC – 240 MS (Varian, California USA). Separation of compounds was performed using a capillary column (VF-1ms), with 30 m long, an inner diameter of 0.25 μm and a film thickness of 0.25 μm. 1 μL of each extract was injected into the GC-MS using a micro syringe. Injection port temperature was set at 300ºC. Column temperature was set as follows: initial temperature was set at 60ºC maintained for 3 min, followed by 10ºC/min increase till 290ºC, with a stationary time of 6 min. Total run time was 30 min. Helium was used as carrier gas at a constant flow rate of 1.2 mL/min. Scanning was performed under 70 eV current emission, and fragments were monitored through 50 to 450 m/z. The ionized compounds were identified by comparing their spectra to those of the Wiley, PMW and NIST mass spectral libraries. For effective comparison of the obtained chromatographic data, and in order to eliminate factors that can mask the chemical fingerprint of bio-active compounds, a scaling step was performed on each chromatographic intensity across all extracts. Based on chemical information regarding the identification of bio-active compounds and also based on the abundance of chromatographic peaks corresponding to the compounds in the extracts, a stock solution corresponding to each extract was prepared and it was successively diluted to obtain 3 concentration levels for each extract. Antimicrobial susceptibility tests Microorganisms testing The antimicrobial activity was studied using Grampositive bacterial strains (Staphylococcus aureus, Bacillus cereus), as well as Gram-negative bacterial strains (Escherichia coli, Klebsiella spp., Enterobacter aerogenes). The antibiotic-resistant microorganisms used in the present study were isolated in the Microbiology Laboratory of Emergency County Hospital “Pius Brinzeu” Timişoara, Romania, from patients. Determination of Minimum Inhibitory Concentration (MIC) – Disc-diffusion method Antimicrobial tests for the selected microorganisms were carried out using a Kirby-Bauer disc-diffusion susceptibility test [15-16]. A small amount of each microbial culture was diluted in sterile 0.9% sodium chloride solution until the turbidity was equivalent to McFarland standard no. 0.5. The suspensions were further diluted 1:10 in medium CHROM agar (Oxoid) and then spread on sterile Petri plates. Blank sterile antimicrobial susceptibility discs were applied on the agar surface in Petri plates. Afterwards, 10 µL of each sample was added on leaves extracts in methanol, chloroform and n-hexane, and to highlight their possible antimicrobial effects on different antibiotic-resistant microorganisms. Materials and Methods Plant collection and identification G. africanum plants have been obtained from the market Eke-Awka (Anambra State, Southeastern Nigeria). G. africanum was brought from the origin country in the form of plant material. The leaves were removed from their stems and air dried under laboratory conditions, protected from light, for 14 days. After drying step, the plant material was milled into a fine powder. The obtained homogeneous mixture was further used for the extraction step. Extraction procedure In order to extract the desired chemical components from the plant material for further separation, the extraction of hydrophilic compounds uses polar solvents such as methanol (100 mL) and for the extraction of lipophilic compounds, solvents such as chloroform and hexane (100 mL) were used. The reaction was performed under magnetic stirring at room temperature for a period of 24 hours. The obtained extract was transferred to a clean tube and submitted to evaporation under nitrogen stream. The residue was filtrated using nylon membrane filter and reconstituted in 1 mL with the corresponding organic solvent for GC-MS analysis. The extraction efficiency has been calculated based on the dried weight. Efficiency (%) = (m1/m2) x 100, where m1 represents the weight of extract obtained after vaporization of solvent, and m2 represents the initial weight of fresh powder. Tested concentrations Methanol, chloroform and hexane extracts of G. africanum were divided in 3 experimental groups, each of them in 3 different concentrations of total phenol content determined according to Popescu R. et al. [14]: methanol extract of G. africanum (Group A) in concentrations of: 265.50 mg/mL (c1), 132.75 mg/mL (c2) and 66.37 mg/mL (c3); hexane extract of G. africanum (Group B) in concentrations of: 31.85 mg/mL (c1), 15.92 mg/mL (c2) and 7.96 mg/mL (c3); chloroform extract of G. africanum (Group C) in concentrations of: 44.25 mg/mL (c1), 22.10 mg/mL (c2) and 11.05 mg/mL (c3). Derivatization procedure Derivatization procedure was performed in order to increase the volatility and the thermal stability of analytes for chromatographic separation. Sylilation was the reaction of choice in this purpose. Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was applied to every extract. The reaction was allowed to perform 7 1084 FARMACIA, 2019, Vol. 67, 6 where Ac represents the absorbance of the control solution, and Ap, the absorbance of the sample. All experiments were performed in triplicate, the presented values are expressed as average and standard errors. One‑way ANOVA followed by Bonferroni's post‑ tests were used to determine the statistical difference between the effects of extracts vs. gentamicin (Gn) and sulfamethoxazole -trimethoprim (SXT) used as control: Gn for S. aureus and B. cereus and SXT for E. coli, Klebsiella spp., and Enterobacter aerogenes. *p < 0.05, **p < 0.01 and ***p < 0.001 disc surfaces. Commercially available antimicrobial susceptibility test discs were used as positive controls for antibiograms. The plates were incubated at 37°C for 24 h. After incubation, inhibitory areas around the discs were measured. Cellular viability test 100 μL of culture in Mueller Hinton broth with a turbidity equivalent to McFarland standard no. 0.5 was transferred to a 96-well plate. Following that, 50 μL of the extract was added. Samples were incubated at 37°C for 6 hours. 10 μL of 0.5% triphenyltrazolium chloride (TTC) 2,3,5-adduct were added and the samples were incubated at 37°C for another 2 hours. The samples analysis was performed at 460 nm with the TecamSunrise spectrophotometer. The rate of inhibition was determined using the following formula: Rate of inhibition: (%) = [(Ac - Ap)/(Ac)] x 100, Results and Discussion Chromatographic analysis Tables I, II and III present the volatile compounds obtained by extraction of Gnetum africanum with different solvents based on gas chromatography-mass spectrometry (GC-MS), using a 450-GC coupled with 240-ion trap MS. Table I Identified compounds in the derivatized chloroformic extract of Gnetum africanum Retention time [min] 4.822 5.107 7.128 7.637 8.663 9.530 9.821 10.477 13.998 14.450 15.499 16.007 16.092 17.057 17.983 18.628 18.866 19.205 19.494 19.736 20.060 20.427 Compound trifluoromethyl-bis-(trimethylsilyl)-methyl ketone Pentalin laevulic acid trimethylsilyl ether glucose 5-trimethylsilyl gluconic acid ɣ-lactone, 5methoxymine, tri(trimethylsilyl) glycerol, tris(trimethylsilyl ether) succinic acid trimethylsilyl ester nonanoic acid trimethylsilyl ester lauric acid trimethylsilyl ether suberic acid trimethylsilyl ester azelaic acid, bis-trimethylsilyl ester hexahydrofarnesyl acetone myristic acid trimethylsilyl ester n-pentadecanoic acid trimethylsilyl ester palmitic acid trimethylsilyl ester cis-10-heptadecanoic acid trimethylsilyl ester heptadecanoic acid trimethylsilyl ester phytol, trimethylsilyl ether trimethylsilyl 9E-9-octadecanat stearic acid trimethylsilyl ester cis-11-eicosenoic acid trimethylsilyl ester cis-10-nonadecenoic acid trimethylsilyl ester Area [%] 3.39 1.99 1.95 0.97 2.11 4.61 1.33 1.97 1.55 1.76 4.66 3.40 3.08 1.97 16.04 1.78 7.69 2.30 11.52 19.11 1.86 4.95 Table II Identified compounds in the derivatized hexane extract of Gnetum africanum Retention time [min] 4.828 7.126 8.661 8.732 9.184 9.812 10.474 11.712 13.997 14.441 15.059 Compound trifluoromethyl-bis-(trimethylsilyl)-methyl ketone laevuric acid trimethylsilyl ester gluconic acid, ɣ-lactone-5-methoximine, tri(trimethylsilyl) 2-phenylindolizine octanoic acid trimethylsilyl ester succinic acid trhymethylsilyl ester nonanoicaicdtrimethylsilyl ester decanoic acid trimethylsilyl ester dodecanoic acid trimethylsilyl ester suberic acid trimethylsilyl ester tridecanoic acid trimethylsilyl ester 1085 Area [%] 14.70 1.04 2.19 11.74 0.52 0.65 1.77 0.45 1.48 0.95 0.73 FARMACIA, 2019, Vol. 67, 6 Retention time [min] 15.273 15.412 16.489 17.049 17.405 17.655 19.501 20.058 20.427 21.350 22.575 22.898 24.428 Compound tetradecyltrimethylsilyl ether n-pentanoic acid trimethylsilyl ester palmitic acid trimethylsilyl ester n-pentadecanoic acid trimethylsilyl ester heptadecanoic acid trimethylsilyl ester 5-chloro-6-nitrocholestane-3-one cis-11-eicosenoic acid Trimethylsilyl ester (E)-3,7,11,15-tetramethylhexadec-2-enoic acid trimethylsilyl ester cis-10-nonadecenoic acid trimethylsilyl ester arachidic acid trimethylsilyl ester 1-monopalmitin trimethylsilyl ether docosanoic acid trimethylsilyl ester lanost-8-3,7-dione Area [%] 0.97 1.02 2.90 5.35 5.08 0.84 3.31 6.55 17.00 6.39 4.00 4.06 6.33 Table III Identified compounds in the derivatized methanolic extract of Gnetum africanum Retention time [min] 4.732 5.191 6.413 6.567 6.774 8.650 10.170 11.865 12.814 Compound 1,2-dimethylpirolydine 3-HO-5-N-pyrolydinomethyl-isoxazole piperidine, 1-(2-methylpropenyl) 2,5-bis-(1,1,3,3-tetramethylbutyl) thiophene piperidine, 3-dimethylamino-1-methyl 2,5-dimethyl-4-benzyl-pyridine 9,9-dimethyl-3,7-diazobicyclo[3.3.1]nanone 5,7-dimethyl-1,3-diazaadamantan-6-one hydrazone 1,8-dimethyl-3,6-diazahomoadamantan-9-spiro-2’-oxirane Microbiological analyses The bacterial strains used in the present study showed resistance to several antibiotics; the results of antibiograms are presented in Table IV. Table IV Sensibility and resistance to antibiotics of tested bacterial strains Bacteria S. aureus B. cereus E. coli Klebsiella spp. Enterobacter aerogenes Fox, E, Da, Gn, Cip, Sxt, Tec, Lzd Cro, Sam, Caz, Tob, Fep, Imi, Ak, Gn, Cip, Lev Imi, Ak, Net Sxt, Cs Tzp, Caz, Sam, Sxt, Mem, Imi, Ak, Lev Sensibility Sensitive Intermediary Resistant Area [%] 7.35 4.61 4.92 13.09 5.51 15.17 7.56 20.84 20.95 Sxt P Tzp, Sam, Gn, Sxt Cxm, Fep, Cro, Caz, Cip, Lev, Ak Mem, Imi, Gn, Cip, Cxm, Fep, Tzp, Cz, Caz, Sam Cxm, Cz, Pip, Cip, Gn Fox - Cefoxitin, E - Erythromycin, Da - Clindamycin, Gn - Gentamicin, Cip - Ciprofloxacin, Sxt – Trimethoprim-Sulfamethoxazole, Tec - Teicoplanin, Lzd - Linezolid, Cro - Ceftriaxone, Sam - Ampicillin + Sulbactam, Caz - Ceftazidime, Tob - Tobramycin, Fep - Cefepime, Imi - Imipenem, Ak - Amikacin, Lev - Levofloxacin, P - Penicillin, Net - Netilmicin, Tzp - Piperacillin + Tazobactam, Cxm - Cefuroxime, Cs - Colistin, Mem - Meropenem, Cz - Cefazolin, Pip - Piperacillin In the case of the G. africanum methanolic extract the inhibition rates ranged between 14.5 mm and 7 mm. In bacterial strains S. aureus and E. aerogenes at the concentration of c1 in the methanolic extract, inhibition values of 14.2 and 14.5 mm were recorded, indicating an intermediate susceptibility to the action of the extract according to Intorasoot A. et al. [17]. At the c2 concentration of the methanolic extract on S. aureus, B. cereus, E. coli, Klebsiella spp and E. aerogenes strains, the inhibition rates also indicated an intermediate sensitivity (compared to the values recorded for Gn and Sxt) (Figure 1a). Hexane extract The inhibition rate values determined for each bacterial strain and for each type of extract at each concentration were centralized and plotted. The values of the inhibition zones were obtained by comparatively testing of the extracts vs. gentamicin (Gn) and, respectively sulfamethoxazole-trimethoprim (SXT) which were used as references. The diameters of the inhibition zones differ depending on the species: for Gentamicin - S. aureus 15 mm and B. cereus 15 mm, respectively for the Trimethoprim/Sulfamethoxazole E. coli, Klebsiella spp and Enterobacter aerogenes 17 mm. 1086 FARMACIA, 2019, Vol. 67, 6 of bacterial strains hasn't been notified, the values of the inhibition ranges being of 7 mm or absent. The chloroform extract of G. africanum at the tested concentrations, didn't manifest any antibacterial effect, the values of inhibition ranges being absent or maximum at 10.8 mm (Figure 1c). of G. africanum presented an intermediate antibacterial action on the B. cereus strain at all 3 concentrations tested, with inhibition rates ranging from 14.5 to 11.2 mm. On the S. aureus strain, the hexane extract showed intermediate antibacterial action only at using the c1 concentration (Figure 1b). The antibacterial effect of hexane extract of G. africanum on the other types a b c Figure 1. Comparative inhibition of G. africanum extracts vs. Gn and SXT on the bacterial strains (a - methanolic extract; b - n-hexane extract; c - chloroformic extract) (*p < 0.05, **p < 0.01 and ***p < 0.001) G. africanum doesn't present an antibacterial effect on B. cereus, the inhibition values being below 50%. It was observed that E. coli exhibits an intermediary sensibility in the case of c1 and c2 concentrations of methanolic extract, respectively in the case of concentration c1 of the extract in hexane. The chloroformic extract of G. africanum, at the tested concentrations, doesn't present any antibacterial effect (Figure 2c). The inhibition values are below 50%, with the exception of the methanolic extract at c1 concentration for Klebsiella spp. Consequently, an intermediary sensibility is manifested by the Klebsiella spp. strain only in case of the c1 methanolic extract (Figure 2d). The values of the determined inhibition ranges at the application of the 3 types of extracts are under the level of 50%, in order to be considered as manifesting an antibacterial effect, excepting the methanolic extract of c1, where the bacterial strain E. aerogenes manifests an intermediary sensibility (Figure 2e). The use of the cellular viability test to the isolated bacterial strains, under the action of the G. africanum extracts, allowed the determination of the inhibition range and implicitly of the presence/absence of the antibacterial effects of these extracts. Against S. aureus, the extract of G. africanum in hexane, based on the value of the inhibition range, exhibits an intermediary antibacterial effect, at the use of concentrations c1 and c2. Intermediary sensibility presents also S. aureus at the application of the extract of G. africanum in methanol only at c1. The extract of G. africanum in chloroform doesn't exhibit an antibacterial effect on the S. aureus strain, the values of the inhibition range being under 50% (Figure 2a). The inhibition values of the hexane-based extract are between 50 - 80%; the concentration c1 can be considered as sensitive, while c2 and c3 exhibit an intermediary sensibility. The concentrations c1 and c2 of the chloroformic extractpresent intermediary sensibility (Figure 2b). The methanolic extract of 1087 FARMACIA, 2019, Vol. 67, 6 a b c d e Figure 2. Inhibition range (%) of the bacterial strains at the action of G. Africanum extracts (a- S. aureus, b- B. cereus, cE. coli, d- Klebsiella spp., e- E. aerogenes) The observed antimicrobial activity can represent a consequence of the rich phytochemistry of leaves, due to the fact that the preliminary phytochemical screening indicated the fact that leaves contain alkaloid, tannin, saponin, sterol, flavonoid, terpenoid, glycoside cyanogen and antraquinone [7]. The antibacterial and antifungal effects of flavonoids and tannins [18], terpenoids (acting on the integrity of cellular membranes and manifesting an inhibitor action on microorganisms) [19], saponins (a special category of glycosides used due to the large range of pharmacology and medicinal features) [20], and of sterols and phenols (which induce a mutagenicity at the level of the cellular DNA) are well known. Applying the cell viability test to the bacterial strains isolated under the action of G. africanum extracts allowed the determination of inhibition rate, and, implicitly, the presence or absence of the antibacterial effect on the tested extracts. On S. aureus bacterial strain, the hexane extract of G. africanum based on Previous studies have mentioned the antibacterial effect of aqueous and methanol extracts from Gnetum africanum leaves on standard microbial strains (Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923) and Candida albicans (ATCC 10231). The results have indicated the fact that both extracts had inhibitor effect dependent on the dose on the increase of S. aureus, with maximum inhibition areas of 13.30 and 13.10 mm at 200 mg/mL for the aqueous respectively ethanolic extracts. Although, the extracts didn't present antibacterial activity on the E. coli strain which might suggest the inefficiency against Gram-bacteria [7]. Comparing the results obtained by us with the ones presented in the previous study [7], we ascertain the fact that the determined values at the use of our extracts (methanolic, n-hexane and chloroform) are much lower, the possible explanations being the following: less concentrated extracts, the antibiotic-resistant microorganisms, the type of solvent used for extraction. 1088 FARMACIA, 2019, Vol. 67, 6 3. the inhibitory rate value, showed an intermediate antibacterial effect at the application of c1 and c2 concentrations. An intermediate sensitivity on S. aureus was shown only at c1 concentration of methanolic extract. During the mass analysis, there were identified saturated and unsaturated fatty acids, adamantan derivatives like 1,8-dimethyl-3,6-diazahomoadamantan-9-spiro2’-oxirane and 5,7-dimethyl-1,3-diazaadamantan-6one hydrazine, piperidine derivatives like 1-(2methylpropenyl) piperidine and, 3-dimethylamino1-methyl piperidine, steroids like 3,5-stigmastadien7-one, all of them presenting important biological activity as mentioned above. 4. 5. 6. Conclusions The results showed that different compounds were identified by applying different polarity solvents during the extraction step. The presence of various bio-active compounds was proven by GC-MS analysis. Based on our results, it could be concluded that G. africanum contains various bio-active compounds and it is recommended as a plant with a phytopharmaceutical importance. The extracts of Gnetum africanum showed different antibacterial effects, depending on the type of solvent and the tested concentration. Based on the inhibition values, the methanolic extract of G. africanum determined a decrease of sensibility as follows: Staphylococcus aureus > Enterobacter aerogenes > Bacillus cereus = Klebsiella spp. > Escherichia coli. Hexane-based extract of G. africanum at the tested concentrations, determined a decrease of sensibility of the tested strains, as follows: Bacillus cereus > Staphylococcus aureus > Enterobacter aerogenes = Escherichia coli > Klebsiella spp. The chloroformic extract of G. africanum at the tested concentrations didn't exhibit any antibacterial effect on the strains selected in the study. 7. 8. 9. 10. 11. 12. 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