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In this report, methanol was selected as the solvent to extract the bioactive compounds of four medicinal plants (Figure 1, Table 1). The mass yields of methanol extracts of the aerial parts of these species were found to be from 7.3% to 37% (w/w), respectively, with ME-CF being predominant (Figure 2A); the lowest yield was detected in ME-TAl.
Figure 1. Chemical structures of catechin (A), epicatechin (B), cinnamic acid (C), rutin (D), gallic acid (E), caffeic acid (F), vanillic acid (G), coumarin (H), coumaric acid (I), tyrosine (J), vanillin (K), flavone (L), catechin hydrate (M), hydroxyphenylacetic acid (N), ferulic acid (O), phenolic content present in collected plants with cytotoxic and antibacterial effects.
N° Code Scientific Name Family Voucher Specimen Local Name Localisation Part Used; Extraction and Solvent Used Traditional Therapeutic Indications References 1 ME-TAI Teucruim olopecurus Lablaceae n.1122 H'chichit Ben Salem Jbel Orbata-Gafsa-Tunisia Upper parts; Methanol Anti inflammatory drug (Guesmi et al., 2017)[12] 2 ME-TA Thymus hirtus sp.olgeriensis Lamiaceae n.1188 Moujecha Jbel Orbata-Gafsa-Tunisia Upper parts: Methanol Ulcers and testis toxicity (Guesmi etal., 2014; Guesmi et al., 2016)[13, 30] 3 ME-CF Clematis flommula Ranuncuiaceae Nar Berda Jbel Orbata-Gafsa-Tunisia Leaves; Methanol Rheumatism and anti inflammation (Guesmi et al., 2018; Reivera et al., 2008)[25] 4 ME-HT Hydrophyllum tuberculatum Rutaceae Mnitna ZannouchGafsa- Tunisia Upper parts; Methanol Anticancer, antioxydant, antibacterial, anti-HIV, uterus-relaxing activity (Raissi 2016; Adnan ct al., 2001)[37] Table 1. The Collected Plant Parts, Collection Sites, Ethnobotanical Indications, and Percentage Yields of Methanolic Extracts
Figure 2. (A) Yield of the methanolic extracts of species used in this study. TAME = Thymus algeriensis methanolic extracts, CFME = Clematis flammula methanolic extracts, TAlEM = Teucrium alopecurus methanolic extracts, HTEM = Hydrophyllum tuberculatum methanolic extracts. (B, C) HPLC chromatogram of phenolic compounds detected at 280 nm (Ⅰ) and flavonoids (Ⅱ), of methanolic extracts. (D) antiradical potential of methanolic extracts using DPPH (Ⅰ) and FRAP (Ⅱ) assay; (E) Cytotoxicity of different extracts obtained from TAEM, TAlEM, CFEM, and HTEM toward U266 cancer cell lines. Tumor cells were pre-treated with various concentrations of extracts and incubated for 72 h. Cell viability was analyzed with the MTT assay.
As shown in Figure 2B, the total phenolic contents of different methanolic extracts were determined from a gallic acid standard curve and expressed as μg GAE/mg DW. In this report, the phenolic content ranged from 250 ± 18 μg GAE/mg DW (C. flammula) to 500 ± 11 μg GAE/mg DW (T. algeriensis) for phenolic acids, 124 ± 4 to 180 ± 12 μg QE/mg DW for flavonoids, and 40 ± 7 to 94 ± 8 μg CE/mg DW for tannin content; the lowest mean values were obtained for ME-HT. In contrast, ME-TAl showed the highest total phenolic content. In turn, the latter showed a lower content of flavonoids and tannins in comparison with other plants. The obtained values of the flavonoid compounds in leaf extracts were found to range between 5.09 and 19.81 mg QE/g extract. Clematis flammula leaves revealed higher levels of tannin content (4.94-7280.52 mg tannic acid equivalents/g extract).
Based on the above results, the methanolic extracts of the four plants constitute a promising natural product, being particularly rich in phenolic acid derivatives, as well as rutin (Figure 2C). As shown in Table 2, we detected the presence of a high quantity of phenolic acids in ME-TAl and in ME-CF, including vanillic acids, coumarin, and epicatechin.
Compounds Methanolic Extracts ME-TA ME-TAI ME-CF Approximate RT (min) Content (μg/g dw) Area (counts) Width 1/2 (s) Approximate RT (min) Content (μg/g dw) Area (counts) Width 1/2 (s) Approximate RT (min) Content (Mg/g dw) Area (counts) Width 1/2 (s) Gallic acid 7.23 745 112 339229 11.7 6.00 266 ±23 289764 12.54 7.23 311±5 636005 10.9 Catechin 14.98 16±5 147892 22.7 15.08 18±3 159830 13.06 14.98 19 ±5 55709 12.8 Caffeic acid 16.64 26± 14 78109 9.0 - - - - 16.64 89 ±26 540775 30.9 Ferulic acid 17.83 42 ±6 115097 10.9 18.37 25 ±3 610987 19.45 - - - - Epicatechin 18.55 136 ±11 59027 11.2 18.50 162 ±47 524696 11.50 18.55 125 ±32 1479481 14.9 Rutin 20.01 89 ±3 256110 12.5 - - - - 20.01 45 ±15 89619 12.5 Vanillic acid 20.57 615±41 131110 11.4 15.96 792 ± 2 36126 14.90 20, 57 7182 ± 68 419659 13.0 Coumarin 27.70 234 ± 17 677045 12.2 - - - - 27.70 38 ±7 2211453 13.3 Coumaric acid 30.00 124 ±11 628065 11.8 30.00 22 ±15 233541 11.04 - - - - Flavone - - - - 9.16 66 ±10 679210 10.60 - - - - Quercetin 17.56 126 ±16 628017 11.3 - - - - - - - Cinnamic add 29.78 - 203634 11.5 - - — - 29.78 348 ± 56 500006 11.5 Table 2. Phenolic Compounds Identified in Methanolic Extracts by HPLC
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In the DPPH assay, the MeOH extract of Thymus algeriensis exhibited the highest reducing activity followed by ME-CF, ascorbic acid, ME-TAl, and ME-HT fractions at 100 and 200 μg/mL (Figure 2DⅠ). In the FRAP assay, ME-TA and ME-TAl displayed more potent radical scavenging activity than ME-CF and ME-HT (Figure 2DⅡ). BHT exhibited the highest radical scavenging activity among all samples.
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Our findings suggested that methanolic extracts of Thymus and Teucrium might act as chemopreventive agents with antioxidant properties, offering effective protection against in vitro U266 proliferation in a dose-dependent manner. Of the four methanolic extracts, two showed the potential to inhibit U266 cell viability (Figure 2E). Induction of apoptosis was increased significantly in tumor cells. Our results clearly demonstrated that the ME-TAl and ME-TA were the most active, whereas the ME-CF and ME-HT were the least active. These findings suggest that treatment with methanolic extracts rich in phenolic acids may provide an enhanced therapeutic response in human multiple myeloma cells. However, 50 μg/mL appeared to have no significant effect on cell proliferation in tumor cells. Moreover, the induction of apoptosis in tumor cells using the MTT assay was apparently associated with the antitumor activity of the respective natural preventive agents. Similar results were observed by several workers in different tumor cells exposed to natural products. Moreover, our results clearly show that Hydrophyllum tuberculatum extract significantly induced U266 cytotoxicity in a dose-dependent manner.
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Our findings, validated through bacterial inhibition, showed that methanolic extracts have potential novel applications as nutraceuticals and microbial inhibitors. Taken together, our results suggest that methanolic fractions (1 mg/mL) acted as bacterial inhibitors and suppressed the growth of bacterial strains when compared with antibiotics used in this report (Table 3), with a large inhibition zone detected in B. subtilis (24 mm), to the same extent as 0.10 μg of chloramphenicol, when bacteria were treated with ME-TA and in S. aueus (22 mm) after treatment with ME-TAl (Figure 3A). Furthermore, our data revealed that ME-HT showed a moderate effect on B. subtilis, S. aureus, and K. pneumoniae (Figure 3).
Figure 3. Antibacterial activity of the hydrophobic fraction of Teucrium alopecurus. Plant essential oils (1 μL) were applied to a filter disc (Whatman No 5 mm) onto the seeded top layer of the agar plates containing tested bacteria (E. coli, S. aureus, B. subtilis, and K. pneumoniae). Essential oil was tested with chloramphenicol, and ertapenem discs as positive controls. DMSO discs were used as negative controls (A). The plates were incubated at 37 ℃ for 24 h, and the zone of inhibition diameter was determined (B).
Methanolic Extracts and Antibiotics Inhibition Zones (mma) of Bacterial Strains in Presence of Me-OH Extracts and Antibiotics Gram + Gram - Plant name région S. aureus B. subtilis E. coli K. pneumoniae K. oxycota Control: DMSO * * * * - ME-CF Jbel Orbata-Gafsa 22 16 16 12 - ME-TAl Jbel Orbata-Gafsa 15 10 10 14 - ME-TA Jbel Orbata-Gafsa 10 24 7 10 - ME-HT Zannouch-Gafsa 7 8 14 11 - Antibiotics (μg/μL) ETP (10 μg/μL) * * * 32 24 CFM (10 μg/μL) 26 23 30 - * ATP10K (10 μg/μL) * * 18 * 21 CTX (30 μg/μL) * * 35 * 35 CIP (5 μg/μL) * * 37 * 40 CT (10 μg/μL) * * 24 * 19 CFM (5 μg/μL) * * 30 * 33 IPM (10 μg/μL) * * 33 * 30 CAZ (10 μg/μL) * * * * 24 TIC (75 μg/μL) * * * * * FOX (30 μg/μL) * * 25 * 37 CN (10 μg/μL) * * 19 * 22 TOB (10 μg/μL) * * 18 * 22 FF (200 μg/μL) * * 21 * 18 UA (30 μg/μL) * * 29 * 24 AMC (30 μg/μL) * * 21 * 25 AX (10 μg/μL) * * * * * Note. Me-OH: Methanol; S. aureus: Staphylococcus.auerus; E.coli: Eshershia.coli; K.pneumonie: Klebsiellla pneumoniae; B.subtilis: Bacillus subtilus; a: inhibition zone diameters; *: no activity detected; (-): not tested; AMC: Amoxicilline-acid clavulanic, PRL: Piperacillin; TIC: Ticarcillin; CFM: Cefixim (cystites); FOX: Cefoxitin; CTX: Cefotaxim, CAZ: Ceftazidim; ATM: Aztreonam; ETP: Ertapenem; IPM: Imipenem, NA: Nalidixic acid; NOR: Norfloxacin; CIP: Ciprofloxacin; AK: Amikacin; CN: Gentamicin; TOB: Tobramycin; TGC: Tigecyclin (E.coli); FF: Fosfomycin; CL: Cefalexin; TZP: Piperacillin-Tazobactam 1/2. Table 3. Antibacterial Activity of Methanolic Extracts and Antibiotics Against four Bacterial Strains