scholarly journals ANTIBACTERIAL ACTIVITY OF GOLDEN BERRY (PHYSALIS PERUVIANA) EXTRACT AGAINST ESCHERICHIA COLISPP. ISOLATES FROM MEATS IN ECUADOR

2020 ◽  
pp. 115-118
Author(s):  
FAVIAN BAYAS-MOREJON ◽  
ANGELICA TIGRE-LEON ◽  
MARCELO TAPIA-VERDEZOTO ◽  
FABIAN FLORES-RIBADENEIRA
Keyword(s):  
Public Health ◽  
Antibacterial Activity ◽  
Food Safety ◽  
Ethyl Alcohol ◽  
Pathogenic Bacteria ◽  
Antibacterial Effect ◽  
E Coli ◽  
Physalis Peruviana ◽  
Berry Extracts ◽  
Global Threat

Objective: The resistance of pathogenic bacteria to multiple antimicrobials has become a real global threat in terms of food safety and public health. To Study of antibacterial activity of golden berry extracts against E. coli isolated from meats in Ecuador. Methods: Two solvents (water and ethyl alcohol) were used as well as three parts of the Goldenberry (mature berries, semi-mature berries and leaves) for its transformation into extracts. The extracts obtained were used in tests of anti-E. coli activity, contrasting with Ampicillin and gentamicin by means of the disk difusión method. Results: Extracts in ethyl alcohol from semi-ripe berries and leaves were more effective at inhibiting the development of E. coli, with A2B1 and A2B3 treatments being those with the best antibacterial effect with 35.3% and 50% respectively. These results were close to those obtained with the antibiotics under study. Conclusion: The use of uvilla extracts could be a viable natural alternative to act against pathogens.

scholarly journals Uji Efek Antibakteri Jamur Endofit Akar Bakau Rhizophora stylosa Terhadap Bakteri Staphylococcus aureus dan Escherichia coli

2014 ◽  
Vol 2 (1) ◽  
Author(s):  
Michele Sumampouw
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Endophytic Fungi ◽  
Pathogenic Bacteria ◽  
Antibacterial Effect ◽  
Inhibition Zone ◽  
Potato Dextrose Agar ◽  
Rhizophora Stylosa ◽  
E Coli

Abstract: The aim of this study is to investigate the antibacterial effect of endophytic fungi originated from the root of mangrove Rhizophora stylosa growing on Mangrove Plantation Area around DAS Simpang 5 Jl. Piere Tendean Manado. The method of this research was using combination of PDA and NA media. Two species of endophytic fungi were extracted from the root of R. stylosa, black mycelium fungi as isolate A and brown mycelium fungi as isolate B. The activities of both isolates were tested against pathogenic bacteria Staphylococcus aureus and Escherichia coli. The result showed the same inhibition zone of isolate A and B to S. aureus, while, isolate A showed bigger the inhibition zone in comparison to isolate B against E. coli. As a conclusion, the endophytic fungi taken from the root of R. stylosa have antibacterial activity toward S. aureus and E. coli. Keywords: endophytic fungi, Rhizophora stylosa, antibacterial, Staphylococcus aureus, Escherichia coli.   Abstrak: Penelitian ini bertujuan untuk melihat adanya efek antibakteri dari jamur endofit yang diambil dari akar bakau Rhizophora stylosa yang ditanam di sekitar Perairan Daerah Aliran Sungai Simpang Lima Jl. Piere Tendean Manado. Metode penelitian dilakukan dengan menggunakan kombinasi media Potato Dextrose Agar (PDA) dan Nutrient Agar (NA). Dari akar bakau R. stylosa diperoleh dua  jenis jamur endofit yaitu isolat A dengan karateristik miselium jamur berwarna hitam dan isolat B yang dengan karakteristik miselium berwarna coklat. Kedua isolat ini selanjutnya diujikan aktivitasnya terhadap bakteri Staphylococcus aureus dan Escherichia coli. Hasil penelitian menunjukkan isolat A dan B memberikan  zona hambat yang sama terhadap bakteri Staphylococcus aureus dan pada bakteri Escherichia coli isolat A menunjukkan diameter zona hambat yang lebih besar dibandingkan isolat B. Kesimpulan, Jamur endofit yang diisolasi dari akar bakau R. Stylosa memiliki efek antibakteri terhadap pertumbuhan bakteri S. aureus dan E. coli. Kata kunci: jamur endofit, Rhizophopra stylosa, antibakteri, Staphylococcus aureus, Escherichia coli.

scholarly journals UJI EFEK ANTIBAKTERI JAMUR ENDOFIT PADA TUMBUHAN SEREH (Cymbopogon citratus) PADA BAKTERI UJI Staphylococcus aureus DAN Escherichia coli

PHARMACON ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 705
Author(s):  
Fathia Falugah ◽  
Jimmy Posangi ◽  
Paulina V. Y. Yamlean
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Endophytic Fungi ◽  
Pathogenic Bacteria ◽  
Antibacterial Effect ◽  
Cymbopogon Citratus ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Escherichia Coli Bacteria

ABSTRACT Endophytic fungi are fungi that live in plant tissues and do not harm these plants. Endophytic fungi can produce compounds that have the potential as antibacterial. This study aimed to examine the antibacterial effects of endophytic fungi isolated from the lemongrass (Cymbopogon citratus) against Staphylococcus aureus and Escherichia coli bacteria. Lemongrass is used to inhibit or kill pathogenic bacteria because it contains essential oils that function as an antifungal and antibacterial against several pathogenic bacteria such as Staphylococcus aureus and Escherichia coli. The laboratory experiment method tested the antibacterial activity of endophytic fungi isolates obtained from lemongrass (Cymbopogon citratus) against Staphylococcus aureus and Escherichia coli using hole / well method. From the results of the study, four endophytic fungi were isolated from the roots and leaves of lemongrass (Cymbopogon citratus). Antibacterial results showed that endophytic fungi extract isolated from lemongrass plants was more effective in inhibiting Staphylococcus aureus compared to Escherichia coli bacteria. In Staphylococcus aureus bacteria the average inhibitory value is 5-9 mm while in Escherichia coli bacteria the average inhibitory value is only 3 mm. Endophytic fungi from lemongrass roots have a better antibacterial effect compared to endophytic fungi from lemongrass leaves. Conclusion, endophytic fungi isolated from the roots and leaves of lemongrass (Cymbopogon citratus) have an antibacterial effect on the growth of S. aureus bacteria and are not effective against E. coli bacteria. This endophytic fungus isolate is more active against Gram positive (+) bacteria and less active against Gram negative (-) bacteria. Keywords: Lemongrass plants, Endophytic Fungi, Antibacterial Activity, Staphylococcus aureus,  Escherichia coli. ABSTRAK Jamur endofit ialah jamur yang hidup di dalam jaringan tumbuhan dan tidak membahayakan tumbuhan tersebut. Jamur endofit dapat menghasilkan senyawa yang berpotensi sebagai antibakteri. Penelitian ini bertujuan untuk menguji efek antibakteri jamur endofit yang diisolasi dari akar,batang dan daun tumbuhan sereh (Cymbopogon citratus)  terhadap bakteri Staphylococcus aureus dan Escherichia coli. Sereh digunakan untuk menghambat atau membunuh bakteri patogen karena mengandung minyak atsiri yang berfungsi sebagai antijamur dan antibakteri terhadap beberapa bakteri patogen seperti Staphylococcus aureus dan  Escherichia coli.  Metode penelitian secara eksperimen laboratorium menguji aktivitas antibakteri dari isolat jamur endofit  yang diperoleh dari tumbuhan sereh (Cymbopogon citratus) pada bakteri Staphylococcus Aureus dan Escherichia Coli menggunakan metode lubang/sumuran. Dari hasil penelitan diperoleh empat jenis jamur endofit yang diisolasi dari akar dan daun  tumbuhan sereh (Cymbopogon citratus).  Hasil penelitian antibakteri menunjukkan bahwa ekstrak jamur endofit yang diisolasi dari tumbuhan sereh lebih efektif menghambat  bakteri Staphylococcus aureus dibandingkan dengan bakteri Escherichia coli. Pada bakteri  Staphylococcus aureus nilai rata-rata daya hambat yaitu 5-9 mm sedangkan pada bakteri Escherichia coli nilai rata-rata daya hambat yaitu hanya 3 mm. Jamur endofit dari akar sereh memiliki efek antibakteri yang lebih baik dibandingkan dengan jamur endofit dari daun sereh. Kesimpulan, Jamur endofit yang diisolasi dari akar  dan daun sereh (Cymbopogon citratus) memiliki efek antibakteri terhadap pertumbuhan bakteri S. aureus dan tidak efektiv terhadap bakteri E. coli. Isolat jamur endofit ini lebih aktif pada bakteri Gram positif (+) dan kurang  aktif pada bakteri negatif (-). Kata Kunci : Tumbuhan Sereh, Jamur Endofit, Aktivitas Antibakteri, Staphylococcus aureus, Escherichia coli

Photocatalytically Enhanced Inactivation of Internalized Pathogenic Bacteria in Fresh Produce using Ultraviolet Irradiation with Nano-titanium Dioxide

2020 ◽  
Author(s):  
Seungjun Lee ◽  
Chulkyoon Mok ◽  
Jiyoung Lee
Keyword(s):  
Public Health ◽  
Titanium Dioxide ◽  
Food Safety ◽  
Uv Irradiation ◽  
Foodborne Pathogens ◽  
Pathogenic Bacteria ◽  
Safety Issue ◽  
Fresh Produce ◽  
Future Research ◽  
E Coli

Once pathogens are internalized in fresh produce, they pose a challenging food safety issue since they are not effectively inactivated by conventional rinsing or sanitization. To protect food safety and public health, the objectives were to examine internalized levels of foodborne pathogens in different types of fresh produce and to investigate the effectiveness of photocatalytically enhanced inactivation of internalized pathogens in fresh produce using UV irradiation with titanium dioxide (TiO 2 ). For this, green fluorescent protein-labeled S. Typhimurium and E. coli O157:H7 were inoculated on the leaf surface of four types of fresh produce (~10 8 CFU (colony-forming unit)/leaf) and varying concentrations of TiO 2 suspension (0.50, 0.75, 1.00, 1.25, and 1.50 µg/ml)) were applied to the surface of contaminated leaves. Depending on the nature of each vegetable, the internalized bacterial level differed (log 2 – 5 CFU/g of leaf). When UV irradiation (6,000 J/m 2 ) was applied, the internalized S. Typhimurium and E. coli levels were reduced by 0.8 – 2.4 log CFU/leaf and was with TiO 2 , the reduction was 1.1 – 3.7 log CFU/leaf. The inactivation efficiency increased as the TiO 2 concentration (up to 1.50 μg/leaf). These results indicate that the TiO 2 application enhanced the photocatalytic inactivation of internalized foodborne pathogens. The application of TiO2 would be most practical before UV irradiation and before distributing the produce. This study established a platform for future research on the inactivation of various internalized pathogens for protecting public health and scaling up fresh produce treatments by the food industry.

Bioenhancing Effect of Cow Urine Distillate and Pepper Extract on Antibacterial Activity of Azadirachta Indica Leaves

1970 ◽  
Vol 9 (2) ◽  
pp. 3212-3214
Author(s):  
Pramod Dhakal ◽  
Ankit a Achary ◽  
Vedamurthy Joshi
Keyword(s):  
Antibacterial Activity ◽  
Azadirachta Indica ◽  
Pathogenic Bacteria ◽  
Ethanol Extract ◽  
Watery Diarrhea ◽  
Diffusion Method ◽  
E Coli ◽  
Drug Molecules ◽  
Cow Urine

Bioenhancers are drug facilitator which do not show the typical drug activity but in combination to enhance the activity of other molecule in several way including increase the bioavailability of drug across the membrane, potentiating the drug molecules by conformational interaction, acting as receptor for drug molecules and making target cell more receptive to drugs and promote and increase the bioactivity or bioavailability or the uptake of drugs in combination therapy. The objective of the present study was to evaluate the antibacterial and activity of combination in Azadirachta indica extract with cow urine distillate and pepper extract against common pathogenic bacteria, a causative agent of watery diarrhea. It has been found that Indian indigenous cow urine and its distillate also possess bioenhancing ability. Bioenhancing role of cow urine distillate (CUD) and pepper extract was investigated on antibacterial activity of ethanol extract of Azadirachta indica. Antibacterial activity of ethanol extract neem alone and in combination with CUD and pepper extract were determined the ATCC strains against Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and E-coli by cup plate diffusion method. Ethanol extract of neem has showed more effect on P. aeruginosa, E-coli than S. aureus and K. pneumonia with combination of CUD and pepper extract. CUD and pepper did not show any inhibition of test bacteria in low concentration. The antibacterial effect of combination of extract and CUD was higher than the inhibition caused by extract alone and is suggestive of the bioenhancing role of cow urine distillate and pepper. Moreover, inhibition of test bacteria was observed with less concentration of extract on combining with CUD

scholarly journals Alkaloid-Rich Crude Extracts, Fractions and Piperamide Alkaloids of Piper guineense Possess Promising Antibacterial Effects

Antibiotics ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 98 ◽  
Author(s):  
Eunice Mgbeahuruike ◽  
Pia Fyhrquist ◽  
Heikki Vuorela ◽  
Riitta Julkunen-Tiitto ◽  
Yvonne Holm
Keyword(s):  
Antibacterial Activity ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Hot Water ◽  
Bacterial Strains ◽  
Inhibitory Effects ◽  
Piper Guineense ◽  
E Coli ◽  
Growth Inhibitory ◽  
Derivatives Of

Piper guineense is a food and medicinal plant commonly used to treat infectious diseases in West-African traditional medicine. In a bid to identify new antibacterial compounds due to bacterial resistance to antibiotics, twelve extracts of P. guineense fruits and leaves, obtained by sequential extraction, as well as the piperine and piperlongumine commercial compounds were evaluated for antibacterial activity against human pathogenic bacteria. HPLC-DAD and UHPLC/Q-TOF MS analysis were conducted to characterize and identify the compounds present in the extracts with promising antibacterial activity. The extracts, with the exception of the hot water decoctions and macerations, contained piperamide alkaloids as their main constituents. Piperine, dihydropiperine, piperylin, dihydropiperylin or piperlonguminine, dihydropiperlonguminine, wisanine, dihydrowisanine and derivatives of piperine and piperidine were identified in a hexane extract of the leaf. In addition, some new piperamide alkaloids were identified, such as a piperine and a piperidine alkaloid derivative and two unknown piperamide alkaloids. To the best of our knowledge, there are no piperamides reported in the literature with similar UVλ absorption maxima and masses. A piperamide alkaloid-rich hexane leaf extract recorded the lowest MIC of 19 µg/mL against Sarcina sp. and gave promising growth inhibitory effects against S. aureus and E. aerogenes as well, inhibiting the growth of both bacteria with a MIC of 78 µg/mL. Moreover, this is the first report of the antibacterial activity of P. guineense extracts against Sarcina sp. and E. aerogenes. Marked growth inhibition was also obtained for chloroform extracts of the leaves and fruits against P. aeruginosa with a MIC value of 78 µg/mL. Piperine and piperlongumine were active against E. aerogenes, S. aureus, E. coli, S. enterica, P. mirabilis and B. cereus with MIC values ranging from 39–1250 µg/mL. Notably, the water extracts, which were almost devoid of piperamide alkaloids, were not active against the bacterial strains. Our results demonstrate that P. guineense contains antibacterial alkaloids that could be relevant for the discovery of new natural antibiotics.

Reducing Pathogens by Using Zinc Oxide Nanoparticles and Acetic Acid in Sheep Meat

2014 ◽  
Vol 77 (9) ◽  
pp. 1599-1604 ◽  
Author(s):  
MAHBOUBEH MIRHOSSEINI ◽  
VAHID ARJMAND
Keyword(s):  
Zinc Oxide ◽  
Antibacterial Activity ◽  
Acetic Acid ◽  
Zno Nanoparticles ◽  
Pathogenic Bacteria ◽  
Inhibitory Effect ◽  
Initial Growth ◽  
Practical Applications ◽  
E Coli ◽  
Sheep Meat

Practical applications of different concentrations (0, 1, 2, 4, 6, and 8 mM) of zinc oxide (ZnO) suspensions containing 1% acetic acid were investigated against the pathogenic bacteria Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Bacillus cereus. ZnO suspensions (0, 1, 3, 6, and 8 mM) containing acetic acid had a significant inhibitory effect on the growth of L. monocytogenes, E. coli, and S. aureus during 12 h of incubation, and the 8 mM suspensions of ZnO were the most effective against all the strains. These data suggested that the antibacterial activity of ZnO was concentration dependent. Thus, 6 and 8 mM ZnO were selected for further studies in meat. ZnO nanoparticles reduced initial growth of all inoculated strains in meat. To our knowledge, this is the first report describing the antibacterial activity of ZnO nanoparticles in meat and indicates the potential of these nanoparticles as an antibacterial agent in the food industry.

Phytochemical Screening, Antibacterial and Synergistic activity of Dittrichia Viscosa Extracts against Multi-Resistant Pathogenic Bacteria

2019 ◽  
Vol 2 (1) ◽  
pp. 01-06
Author(s):  
Rhazi Fouzia
Keyword(s):  
Antibacterial Activity ◽  
Pathogenic Bacteria ◽  
Antibacterial Effect ◽  
Synergistic Activity ◽  
Phytochemical Screening ◽  
Bacterial Strains ◽  
Dittrichia Viscosa ◽  
Study Results ◽  
Ethanolic Extracts

Study contextual: Faced with the global problem of antimicrobial resistance, and the use of traditional medicine for the research of antibacterial biomolecules. Aim: our work focused on the valorization of a medicinal plant Dittrichia viscosa which has many therapeutic and culinary virtues worldwide. Methods: To do this, a phytochemical screening of the leafy stems of the plant is carried out according to a set of physicochemical reactions, as well as an in vitro evaluation of the antibacterial activity, of the aqueous and ethanolic extracts against multi-resistant bacterial strains, by microdilution technique on microplates. An evaluation of the synergistic interaction between extracts and weakened antibiotics against pathogenic bacteria was also highlighted in this study. Results: The tests revealed the richness of Dittrichia viscosa species by tannins, flavonoids, saponosides, sterols and triterpenes. As for the antibacterial effect, the MICs range from 0.858±0.29 to 66.66 ± 0.00 mg / ml and the MBCs from 4.300 ± 1.01 to 11.610 ± 2.31 mg / ml is an interesting antibacterial activity. Regarding the combination of extracts with antibiotics tested, it revealed a synergistic action inducing an amplification of the antibacterial power of Penicillin, Imipenem and Erythromycin with a rate that reaches 471%. Conclusion: The results of this study show that the aqueous and ethanolic extracts of Dittrichia viscosa have interesting and promising antibacterial activity in the pharmaceutical, food and cosmetic industries.

Inhibition of Food-Related Pathogenic Bacteria by god-Transformed Penicillium nalgiovense Strains

1999 ◽  
Vol 62 (8) ◽  
pp. 940-943 ◽  
Author(s):  
ROLF GEISEN
Keyword(s):  
Antibacterial Activity ◽  
Inhibitory Activity ◽  
Type Strain ◽  
Salmonella Enteritidis ◽  
Pathogenic Bacteria ◽  
Wild Type Strain ◽  
Antibacterial Effect ◽  
Wild Type ◽  
Penicillium Nalgiovense ◽  
And Staphylococcus Aureus

Two strains of Penicillium nalgiovense, which carried the god gene of Aspergillus niger and had increased glucose oxidase (GOD) activity compared with the wild-type strain, were tested for their ability to suppress the growth of certain food-related pathogenic bacteria. In contrast to the wild type, which showed no antibacterial effect when grown in mixed culture with different bacteria, the two tranformed strains were highly antagonistic. The strain that expressed higher amounts of GOD in general had higher inhibitory activity. Both strains showed antibacterial activity against Listeria monocytogenes, Salmonella Enteritidis, and Staphylococcus aureus. The inhibitory activity was dependent on the glucose concentration in the medium. S. aureus was completely inhibited at 1% glucose in the presence of the higher GOD-producing transformant. In contrast, if arabinose was used as a carbon source, no inhibition occurred. If catalase was added to the medium, the inhibitory activity of the transformants was completely inactivated, indicating that the hydrogen peroxide produced was responsible for the antibacterial activity of the transformants.

Rapid and Sensitive Detection of Escherichia coli O157:H7 in Milk and Ground Beef Using Magnetic Bead–Based Immunoassay Coupled with Tyramide Signal Amplification

2014 ◽  
Vol 77 (1) ◽  
pp. 100-105 ◽  
Author(s):  
MUHSIN AYDIN ◽  
GENE P. D. HERZIG ◽  
KWANG CHEOL JEONG ◽  
SAMANTHA DUNIGAN ◽  
PARTH SHAH ◽  
...  
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Food Safety ◽  
Foodborne Pathogens ◽  
Signal Amplification ◽  
Magnetic Bead ◽  
Escherichia Coli O157 ◽  
Tyramide Signal Amplification ◽  
E Coli ◽  
Enrichment Step

Escherichia coli O157:H7 is a major foodborne pathogen that has posed serious problems for food safety and public health. Recent outbreaks and recalls associated with various foods contaminated by E. coli O157:H7 clearly indicate its deleterious effect on food safety. A rapid and sensitive detection assay is needed for this harmful organism to prevent foodborne illnesses and control outbreaks in a timely manner. We developed a magnetic bead–based immunoassay for detection of E. coli O157:H7 (the most well-known Shiga toxigenic E. coli strain) with a 96-well microplate as an assay platform. Immunomagnetic separation (IMS) and tyramide signal amplification were coupled to the assay to increase its sensitivity and specificity. This immunoassay was able to detect E. coli O157:H7 in pure culture with a detection limit of 50 CFU/ml in less than 3 h without an enrichment step. The detection limit was decreased 10-fold to 5 CFU/ml with addition of a 3-h enrichment step. When this assay was tested with other nontarget foodborne pathogens and common enteric bacteria, no cross-reactivity was found. When tested with artificially contaminated ground beef and milk samples, the assay sensitivity decreased two- to fivefold, with detection limits of 250 and 100 CFU/ml, respectively, probably because of the food matrix effect. The assay results also were compared with those of a sandwich-type enzyme-linked immunosorbent assay (ELISA) and an ELISA coupled with IMS; the developed assay was 25 times and 4 times more sensitive than the standard ELISA and the IMS-ELISA, respectively. Tyramide signal amplification combined with IMS can improve sensitivity and specificity for detection of E. coli O157:H7. The developed assay could be easily adapted for other foodborne pathogens and will contribute to improved food safety and public health.

Synthesis Antimicrobial and Anticancer Activity of 1-[(arylalkylidene)amino]-3-(4H-1,2,4-triazol-4-yl)thiourea

2014 ◽  
Vol 1 (1) ◽  
pp. 5
Author(s):  
Umasankar Kulandaivelu ◽  
Bhawatha Chawada ◽  
Shireesha Boyapati ◽  
Alavala Rajasekhar Reddy
Keyword(s):  
Antibacterial Activity ◽  
Anticancer Activity ◽  
Cell Lines ◽  
Cancer Cell ◽  
Pathogenic Bacteria ◽  
Inhibitory Concentration ◽  
E Coli ◽  
Derivatives Of ◽  
Ht 29 ◽  
Better Than

Arylalkylidene derivatives of aminotriazoles (3a-3j) were synthesized and tested for their antimicrobial and anticancer activity. Four non-pathogenic bacteria [E. coli (NCIM 2068), K. pneumoniae (NCIM 2957), S. aureus (NCIM 2079), B. subtilis (NCIM 2921)] two fungi [C. albicans, A. niger] and two cancer cell lines [HBL-100 and HT-29] were employed in the study. All the compounds were found to have better antibacterial activity against B. subtilis than Ciprofloxacin (standard) and compound 3i was equivalent to Ciprofloxacin in inhibiting S. aureas. Similarly all the compounds inhibited the growth of A. niger better than Fluconazole and compound 3c was equivalent to Fluconazole (standard) in inhibiting C. albicans. In case of anticancer activity none of the molecule exhibited activity better than the standard used (Methotrexate), though they have inhibitory concentration at submicromolar level.

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