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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.
Table 1. The Collected Plant Parts, Collection Sites, Ethnobotanical Indications, and Percentage Yields of Methanolic Extracts
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] 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.
Table 2. Phenolic Compounds Identified in Methanolic Extracts by HPLC
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 -
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).
Table 3. Antibacterial Activity of Methanolic Extracts and Antibiotics Against four Bacterial Strains
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.
doi: 10.3967/bes2019.003
Antioxidant Potential of Four Species of Natural Product and Therapeutic Strategies for Cancer through Suppression of Viability in the Human Multiple Myeloma Cell Line U266
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Abstract:
Objective This research aimed to evaluate the protective effects of bioactive compounds such as phenolic acids, flavonoids, and tannins present in four species extracted with methanol. Methods The total phenolic content of the methanolic extracts was measured spectrophotometrically. The effect of the extracts on cell viability in U266 cells was measured. The effects of extracts on free radical scavenging were assessed by the DPPH test and FRAP assay. Antibacterial effects of the natural products in this report were investigated by using the disc diffusion method. Results Our Results clearly demonstrated that the methanolic extracts were characterized by a high amount of phenolic compounds. It has been speculated that ME-TA and ME-TAl exhibit a significant (P < 0.05) and dose-dependent antiradical potential. The exposure of cells to high doses of extracts almost completely suppressed cell growth in vitro. ME-TA and ME-TAl showed significant cytotoxic effects at a concentration of 100 μg/mL in the U266 cell line. ME-TAl and ME-CF inhibited the growth of B. subtilis and S. aureus, respectively, to the same extent as 10 μg/μL of chloramphenicol at a concentration of 1 mg/mL. Conclusion Overall, these Results suggest that plants used in traditional medicine have a novel application as free radical scavengers, bacterial inhibitors and tumor suppressors. -
Key words:
- Methanolic fractions /
- Phenolic content /
- DPPH /
- FRAP /
- U266 /
- Antibacterial effect
注释:1) DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST: -
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.
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.
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).
Table 1. The Collected Plant Parts, Collection Sites, Ethnobotanical Indications, and Percentage Yields of Methanolic Extracts
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 2. Phenolic Compounds Identified in Methanolic Extracts by HPLC
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 3. Antibacterial Activity of Methanolic Extracts and Antibiotics Against four Bacterial Strains
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. -
[1] Meher PK, Mishra KP. Radiation oxidative stress in cancer induction and prevention. J Radiat Cancer Res, 2017; 8, 44-52. doi: 10.4103/jrcr.jrcr_10_17 [2] Tariq A, Mussarat S, Adna M. Review on ethnomedicinal, phytochemical and pharmacological evidence of Himalayan anticancer plants. J Ethnopharmacol, 2015; 164, 96-119. doi: 10.1016/j.jep.2015.02.003 [3] Uysal S, Zengin G, Locatelli M, et al. Cytotoxic and Enzyme Inhibitory Potential of Two Potentilla species (P. speciosa L. and P. reptans Willd.) and Their Chemical Composition. Front Pharmacol, 2017; 8, 290. doi: 10.3389/fphar.2017.00290 [4] Bharti AC, Aggarwal BB. Nuclear factor-kappa B and cancer:its role in prevention and therapy. Biochem pharmacol, 2002; 64, 883-8. doi: 10.1016/S0006-2952(02)01154-1 [5] Millimouno FM, Dong J, Yang L, et al. Targeting Apoptosis Pathways in Cancer and Perspectives with Natural Compounds from Mother Nature. Cancer Prevent Res, 2014; 1081-7. [6] Clifford MN. Chlorogenic acids and other cinnamates. Nature, occurence, dietary burden, absorption and metabolism. J Sci Food Agric, 2000; 80, 103343. [7] Aggarwal BB, Prasad S, Sung B, et al. Prevention and Treatment of Colorectal Cancer by Natural Agents from Mother Nature. Curr Colorectal Cancer Rep, 2013; 9, 37-56. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=10.1080/019262301753386022 [8] Hecht F, Pessoa CF, Gentile LB, et al. The role of oxidative stress on breast cancer development and therapy. Tumour Biol, 2016; 37, 4281-91. doi: 10.1007/s13277-016-4873-9 [9] Abdel-Aziz MS, Shaheen MS, El-Nekeety AA, et al. Antioxidant and antibacterial activity of silver nanoparticles biosynthesized using Chenopodium murale leaf extract. J Saudi Chem Soc, 2014; 18, 356-63. doi: 10.1016/j.jscs.2013.09.011 [10] Tutin, TG, Wood D. Teucrium. In Flora Europaea, 1st ed.; Tutin TG, Heywood VH, Burges NA, Moore D, Valentine D, Walters S, Eds.; Cambridge University Press: Cambridge, UK, 1972; 3, 129-35. [11] Kästner A. Übersicht zur systematischen gliederung der gattung Teucrium L. Biocosme Mésogéen, 1989; 6, 63-78. [12] Guesmi F, Prasad S, Tyagi AK, et al. Antinflammatory and anticancer effects of terpenes from oily fractions of Teucruim alopecurus, blocker of IκBα kinase, through downregulation of NFκB activation, potentiation of apoptosis and suppression of NF-κB-regulated gene expression. Biomed. Pharmacother, 2017a; 95, 1876-85. doi: 10.1016/j.biopha.2017.09.115 [13] Guesmi F, Ben Hadj Ahmed S, Landoulsi A. Investigation of Extracts from Tunisian Ethnomedicinal Plants as Antioxidants, Cytotoxins, and Antimicrobials. Biomed Environ Sci, 2017; 30, 811-24. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=bes201711004 [14] Perry LM. Medicinal Plants of East and Southeast Asia:Attributed Properties and Uses, The MIT Press, Cambridge, Massachusetts and London, 1980; 334-60. [15] Gruenwald J, Brendler T, Jaenicke C. PDR for Herbal Medicine. Medical Economics Company, Montvale, N. J., USA, 2nd ed., 2000; 128, 194-5. [16] Nabavi SM, Marchesec A, Izadi M, et al. Plants belonging to the genus Thymus as antibacterial agents:From farm to pharmacy. Food Chem, 2015; 173, 339-47. doi: 10.1016/j.foodchem.2014.10.042 [17] Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am J Enol Vitic, 1965; 16, 144-58. [18] Dewanto V, Wu X, Adom KK, et al. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem, 2002; 50, 3010-4. doi: 10.1021/jf0115589 [19] Makkar HP, Becker K. Vanillin-HCl method for condensed tannins:effect of organic solvents used for extraction of tannins. J Chem Ecol, 1993; 19, 613-21. doi: 10.1007/BF00984996 [20] Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Food Sci Technol, 1995; 28, 25-30. doi: 10.1016-S0023-6438(95)80008-5/ [21] Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Japan J Nutrit, 1986; 44, 307. doi: 10.5264/eiyogakuzashi.44.307 [22] Takada Y, Aggarwal BB. Betulinic acid suppresses carcinogen-induced NF-kappa B activation through inhibition of I kappa B alpha kinase and p65 phosphorylation:abrogation of cyclooxygenase-2 and matrix metalloprotease-9. J Immunol, 2003; 171, 3278-86. doi: 10.4049/jimmunol.171.6.3278 [23] Pereira OR, Cardoso SM. Overview on Mentha and Thymus Polyphenols. Curr Anal Chem, 2012; 9, 1-15. doi: 10.2174/1573411011309010001 [24] Rana S, Rawat K, Mahendru M, et al. Screening of bioconstituents and in vitro cytotoxicity of Clematis gouriana leaves. Nat Prod Res, 2015; 29, 2242-6. doi: 10.1080/14786419.2014.1000891 [25] Atmani D, Chaher N, Berboucha M, et al. Antioxidant capacity and phenol content of selected Algerian medicinal plants. Food Chem, 2009; 112, 303-9. doi: 10.1016/j.foodchem.2008.05.077 [26] Adnan J, Al-Rehailya, Tawfeq A. Alkaloids from Haplophyllum tuberculatum. Phytochem, 2001; 57, 597-602. doi: 10.1016/S0031-9422(01)00041-3 [27] Guo X, Zhu K, Zhang H, et al. Anti-tumor activity of a novel protein obtained from Tartary Buckwheat. Int J Mol Sci, 2010; 11, 5201-11. doi: 10.3390/ijms11125201 [28] Athmouni K, Belghith T, El Fek A, et al. Phytochemical composition and antioxidant activity of extracts of some medicinal plants in Tunisia. Int J Pharmacol Toxicol, 2016; 4, 159-68. doi: 10.14419/ijpt.v4i2 [29] Petti S, Scully C. Polyphenols, oral health and disease:A review. J dent, 2009; 37, 413-23. doi: 10.1016/j.jdent.2009.02.003 [30] Guesmi F, Ben Farhat M, Mejri M, et al. In vitro assessment of antioxidant and antimicrobial activities of methanol extracts and essential oil of Thymus hirtus sp. algeriensis. Lipids Health Dis, 2014; 13, 114. doi: 10.1186/1476-511X-13-114 [31] Miri A, Moncef-Esfahani RH, Amini M, et al. Comparative Chemical Composition and Antioxidant Properties of the Essential Oils and Aromatic Water from Teucrium persicum Boiss. Iran J Pharm Res, 2012; 11, 573-81. http://d.old.wanfangdata.com.cn/OAPaper/oai_pubmedcentral.nih.gov_3832155 [32] Taghouti M, Martins-Gomes CA, Schäfer J, et al. Thymus pulegioides L. as a rich source of antioxidant, anti-proliferative and neuroprotective phenolic compounds. Food Funct, 2018; 9, 3617-29. doi: 10.1039/C8FO00456K [33] Rajabalian S. Methanolic extract of Teucrium polium L. potentiates the cytotoxic and apoptotic effects of anticancer drugs of vincristine, vinblastine and doxorubicin against a panel of cancerous cell lines. Exp Oncol, 2008; 30, 133-8. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=Open J-Gate000001495398 [34] Stankovic MS, Curcic MG, Zizic JB, et al. Teucrium Plant Species as Natural Sources of Novel Anticancer Compounds:Antiproliferative, Proapoptotic and Antioxidant Properties. Inter J Mol Sci, 2011; 12, 4190-205. doi: 10.3390/ijms12074190 [35] Gautam N, Mantha AK, Mittal S. Essential Oils and Their Constituents as Anticancer Agents:A Mechanistic View. BioMed Res Int, 2014; 2014, 23. http://d.old.wanfangdata.com.cn/NSTLQK/NSTL_QKJJ0233894421/ [36] Kamaraj S, Vinodhkumar R, Anandakumar P, et al. The effects of quercetin on antioxidant status and tumor markers in the lung and serum of mice treated with benzo(a)pyrene. Biol Pharm Bull, 2007; 30, 2268-73. doi: 10.1248/bpb.30.2268 [37] Al-Burtamani SK, Fatope MO, Marwah RG, et al. Chemical composition, antibacterial and antifungal activities of the essential oil of Haplophyllum tuberculatum from Oman. J Ethnopharmacol, 2005; 96, 107-12. doi: 10.1016/j.jep.2004.08.039 [38] Sharifi-Rad J, Sureda A, Tenore GC, et al. Biological Activities of Essential Oils:From Plant Chemoecology to Traditional Healing Systems. Molecules, 2017; 22, 70. doi: 10.3390/molecules22010070 [39] Ilić BS, Kocić BD, Ćirić VM, et al. An in vitro synergistic Interaction of combinations of Thymus glabrescens essential oil and its main constituents with chloramphenicol. Sci World J, 2014; 826219. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=Doaj000003718251 [40] Dob T, Dahmane D, Benabdelkader T, et al. Studies on the essential oil composition and antimicrobial activity of Thymus algeriensis Boiss. Et Reut. Int J Aromather, 2006; 16, 95-100. doi: 10.1016/j.ijat.2006.04.003 [41] Sfeir J, Lefrançois C, Baudoux D, et al. In vitro antibacterial activity of essential oils against Streptococcus pyogenes. Evid Based Complementary Altern Med, 2013; 269161. [42] Ait-Ouazzou A, Lorán S, Bakkali M, et al. Chemical composition and antimicrobial activity of essential oils of Thymus algeriensis, Eucalyptus globulus and Rosmarinus officinalis from Morocco. J Sci Food Agric, 2011; 91, 2643-51. doi: 10.1002/jsfa.v91.14