Browsing by Subject "Mycobacterium smegmatis"

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  • Kuosmanen, Soile (Helsingfors universitet, 2013)
    The lower respiratory infection tuberculosis (TB) has been the leading cause of death for centuries causing millions of deaths worldwide. The development of antibiotic therapy has reduced the morbidity and mortality during the 20th century, at least in the developed countries. However, tuberculosis is still the world's second leading cause of death from infectious diseases. Although TB can be treated and even cured with drug therapy, the treatment is extremely long and requires 6-9 months constant drug therapy. This prolonged treatment causes poor patient compliance, which is usually the reason for the selection of drug resistant and often multidrug (MDR-TB) or even extensively drug-resistant (XDR-TB) TB bacteria. Limitations of available therapies and the emergence of drug-resistant strains have intensified the search for new drugs from natural sources. Marine micro- and macro-organisms have proven to be an excellent source of structurally unique biologically active natural products. EU FP7 -funded MAREX project, launched in 2010, aims at identifying new biologically active compounds from marine sources. This Master's thesis was carried out as a part of the MAREX project. The aim of this study was to optimize and validate a reproducible method to determine antimicrobial activity of natural products against Mycobacterium smegmatis, which is a widely used non-pathogenic surrogate model for TB. In the present study, spectrophotometric microplate assay was optimized and validated using existing antibacterial agents ciprofloxacin and rifampicin as reference compounds. The assay was performed on 96-well plate by using two detection techniques, absorbance measurement and a colorimetric indicator, for the antibacterial MIC end-point determination. The results obtained by the described methods were compared with each other in order to achieve the most optimal assay conditions. The quality control parameters S/B, S/N and Z' factor were used in order to determine the optimal experimental conditions for the assay. Obtaining reliable results with the turbidimetric method required incubation for two days in the case of ciprofloxacin, and for five days with rifampicin. Colorimetric measurement led to similar results as the turbidimetric measurement for both of the reference compounds. The method was further used for the screening of a group of marine extracts. None of the 21 samples tested showed significant activity against M. smegmatis.
  • Salih, Enass Y. A.; Julkunen-Tiitto, Riitta; Luukkanen, Olavi; Sipi, Marketta; Fahmi, Mustafa K. M.; Fyhrquist, Pia Johanna (2020)
    In Sudanese traditional medicine, decoctions of the stem bark ofAnogeissus leiocarpaare used for the treatment of tuberculosis (TB). However, this plant has not been investigated before for its antimycobacterial effects. Our screening results show, for the first time, that many extracts of various parts ofA. leiocarpaexhibit growth inhibitory activity againstMycobacterium smegmatis. Minimum inhibitory concentration (MIC) values ranged between 625 and 5000 mu g/mL, with an ethyl acetate extract of the root showing the lowest MIC value. The good antimycobacterial effects of the root part could be due to its high concentration of ellagic acid derivatives, ellagitannins, and flavonoids. Thin layer chromatography (TLC) fractionation resulted in some fractions with better activity than the starting point crude methanol extract (MIC 2500 mu g/mL). Those fractions with the lowest MIC values contained a high number of antioxidant compounds. Fractions3and4(MIC 1500 and 1000 mu g/mL, respectively) contained high concentrations of di-methyl ellagic acid ([M-H](-)329.0318). Fraction6(MIC 2000 mu g/mL) contained a lower concentration of di-methyl ellagic acid and was not as growth inhibitory as fractions3and4. Moreover, in fraction3, an acetylated ellagic acid derivative ([M-H](-)343.0477) and di-methyl-ellagic acid xyloside ([M-H](-)461.0739) were tentatively characterized. Di-methyl ellagic acid xyloside was also present in fraction4and could strongly contribute to the antimycobacterial effect of this fraction. Additionally, protocatechuic acid ([M-H](-)at m/z 153.0196) was present in fraction4.Our antimycobacterial results obtained from this research justify the use ofA. leiocarpain Sudanese folk medicine against cough related to TB. Roots, stem bark, and leaves ofA. leiocarpaare sources for new potent anti-TB drug lead compounds.
  • Salih, Enass Y.A.; Julkunen-Tiitto, Riitta; Lampi, Anna-Maija; Kanninen, Markku; Luukkanen, Olavi; Sipi, Marketta; Lehtonen, Mari; Vuorela, Heikki; Fyhrquist, Pia (2018)
    AbstractEthnopharmacological relevance Terminalia laxiflora Engl. & Diels, (Sudanese Arabic name: Darout الدروت) and Terminalia brownii Fresen (Sudanese Arabic name: Alshaf ألشاف) (Combretaceae) are used in Sudanese traditional folk medicine and in other African countries for treatment of infectious diseases, TB and its symptoms, such as cough, bronchitis and chest pain. Aim of study Because of the frequent use of T. laxiflora and T. brownii in African traditional medicine and due to the absence of studies regarding their antimycobacterial potential there was a need to screen extracts of T. laxiflora and T. brownii for their growth inhibitory potential and to study the chemical composition and compounds in growth inhibitory extracts. Materials and methods The plant species were collected in Sudan (Blue Nile Forest, Ed Damazin Forestry areas) and selected according to their uses in traditional medicine for the treatment of bacterial infections, including TB. Eighty extracts and fractions of the stem bark, stem wood, roots, leaves and fruits of T. laxiflora and T. brownii and nine pure compounds present in the active extracts were screened against Mycobacterium smegmatis ATCC 14468 using agar diffusion and microplate dilution methods. Inhibition zones and MIC values were estimated and compared to rifampicin. HPLC-UV/DAD, GC/MS and UHPLC/Q-TOF MS were employed to identify the compounds in the growth inhibitory extracts. Results The roots of T. laxiflora and T. brownii gave the best antimycobacterial effects (IZ 22–27 mm) against Mycobacterium smegmatis. The lowest MIC of 625 µg/ml was observed for an acetone extract of the root of T. laxiflora followed by methanol and ethyl acetate extracts, both giving MIC values of 1250 µg/ml. Sephadex LH-20 column chromatography purification of T. brownii roots resulted in low MIC values of 62.5 µg/ml and 125 µg/ml for acetone and ethanol fractions, respectively, compared to 5000 µg/ml for the crude methanol extract. Methyl (S)-flavogallonate is suggested to be the main active compound in the Sephadex LH- 20 acetone fraction, while ellagic acid xyloside and methyl ellagic acid xyloside are suggested to give good antimycobacterial activity in the Sephadex LH-20 ethanol fraction. RP-18 TLC purifications of an ethyl acetate extract of T. laxiflora roots resulted in the enrichment of punicalagin in one of the fractions (Fr5). This fraction gave a five times smaller MIC (500 µg/ml) than the crude ethyl acetate extract (2500 µg/ml) and this improved activity is suggested to be mostly due to punicalagin. 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone and β-sitosterol were found in antimycobacterial hexane extracts of the stem bark of both studied species. Of these compounds, 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone have not been previously identified in T. brownii and T. laxiflora. Moreover, both plant species contained friedelin, betulinic acid, β-amyrine and two unknown oleanane-type triterpenoids. Of the listed compounds, friedelin, triacontanol and sitostenone gave a MIC of 250 µg/ml against M. smegmatis, whereas stigmasterol and β-sitosterol gave MIC values of 500 µg/ml. Conclusions Our results show that T. laxiflora and T. brownii contain antimycobacterial compounds of diverse polarities and support the traditional uses of various parts of T. laxiflora and T.brownii as decoctions for treatment of tuberculosis. Further investigations are warranted to explore additional (new) antimycobacterial compounds in the active extracts of T. laxiflora and T. brownii.