Ariaya Hymete

In vitro Evaluation of Antileishmanial Activity and Toxicity of Essential Oils of Artemisia absinthium and Echinops kebericho

Potential toxicity, costs, and drug‐resistant pathogens necessitate the development of new antileishmanial agents. Medicinal and aromatic plants constitute a major source of natural organic compounds. In this study, essential oils of Artemisia absinthium L. and Echinops kebericho Mesfin were investigated by GC and GC/MS analyses. Isolated oils were screened for antileishmanial activity against two Leishmania strains (L. aethiopica and L. donovani), and toxicity on the human monocytic leukemia (THP‐1) cell line and red blood cells in vitro. GC/MS Analysis revealed 65 compounds (93.74%) for Artemisia absinthium and 43 compounds (92.85%) for Echinops kebericho oil. The oils contained the oxygenated monoterpene camphor (27.40%) and the sesquiterpene lactone dehydrocostus lactone (41.83%) as major constituents, respectively. Both oils showed activity against promastigote (MIC 0.0097–0.1565 μl/ml) and axenic amastigote forms (EC50 0.24–42.00 nl/ml) of both leishmania species. Weak hemolytic effect was observed for both oils, showing a slightly decreased selectivity index (SI 0.8–19.2) against the THP‐1 cell line. Among the two oils tested, E. kebericho exerted strong antileishmanial activity that was even higher than that of amphotericin B with significant cytotoxicity. This study, therefore, demonstrated the potential use of both oils as source of novel agents for the treatment of leishmaniasis.

Essential-oil composition, antileishmanial, and toxicity study of Artemisia abyssinica and Satureja punctata ssp. punctata from Ethiopia.

Essential oils of Artemisia abyssinica and Satureja punctata ssp. punctata from Ethiopia were analyzed by GC and GC/MS, and screened for leishmanicidal activity against promastigote and axenic amastigotes of Leishmania donovani and L. aethiopica, including toxicity studies on human monocytic leukemia cells (THP‐1) and erythrocytes in vitro. GC/MS of A. abyssinica oil revealed 67 compounds (99.94%) with the major constituents yomogi alcohol (38.47%), artemisyl acetate (24.88%), and artemisia alcohol (6.70%), and oxygenated monoterpenes (84.00%) as the dominant group. The oil of S. punctata contained 67 compounds (99.49%) with the main constituents geranial (27.62%), neral (21.72%), α‐bisabolol (13.62%), and (E)‐nerolidol (4.82%), of which oxygenated mono‐ and sesquiterpenes (58.39 and 26.91%, resp.) showed highest abundance. Both oils showed effect on promastigotes (MIC 76.5 to 312.5 nl/ml) and amastigotes (EC50 4.06 to 131.00 nl/ml) of L. donovani and L. aethiopica, and varying toxicities on THP‐1 cells (CC50 0.013 to 350 nl/ml with selectivity index between 0.001 and 28) and erythrocytes (with LC50 0.35 to 1.52 μl/ml). S. punctata oil exerted highest activity against both Leishmania sp. and toxicity. The revealed antileishmanial activities support further isolation and investigation of oil constituents for in vitro/in vivo evaluation.

Synthesis and biological evaluation of some pyrazole derivatives as anti-malarial agents.

Novel series of pyrazole derivatives were synthesized and tested for their in vivo anti‐malarial activity using mice infected with chloroquine sensitive P. berghei at a dose level of 50 µmol/kg. The most active compounds were further tested in vitro against chloroquine resistant (RKL9) strain of P. falciparum. The in vivo anti‐malarial activity study indicated that compounds 2a, 2b, 8a and 8b had mean percent suppression of 85%, 83%, 95% and 97%, respectively at equimolar dose level of the standard drug chloroquine diphosphate. Moreover, compounds 2a, 2b, 8a and 8b showed in vitro IC50 values lower (p < 0.05) than that of the standard drug chloroquine phosphate (0.188 ± 0.003  µM) using the RKL9 strain. Compound 8b was the most active with IC50 of 0.033 ± 0.014  µM. Generally, among the tested compounds, those containing a free carboxylic acid functional group on the pyrazole ring were the most active and this finding was supported by the docking results performed for the active compounds. The acute toxicity studies of the active compounds revealed that they have a good safety profile.

Simultaneous Separation and Determination of Lamivudine and Zidovudine in Pharmaceutical Formulations Using the HPTLC Method

Abstract A high-performance thin-layer chromatographic method (HPTLC) for the simultaneous determination of lamivudine and zidovudine in a binary mixture has been developed. The method developed was based on HPTLC separation of the two drugs followed by densitometric measurements of spots at 276 and 271 nm for lamivudine and zidovudine, respectively. Separation was carried out on Merck HPTLC silica-gel 60 F254 plates, using toluene/chloroform/methanol (1:6:3 v:v) as the mobile phase. Validation of the method was performed based on The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Second-order polynomial equations were obtained for the regression line in the ranges of 250–1400 and 250–1700 ng/spot for lamivudine and zidovudine respectively. Correlation coefficient (r) values were 0.9998 for both analytes. The method provides sufficient accuracy as indicated by recovery percentages given for lamivudine and zidovudine. For system precision study, the low coefficient of variation values (<2%) for both lamivudine and zidovudine ensured reproducible performance of the instrument. In the method precision study, coefficients of variation <2% were obtained, which showed that the proposed method provides acceptable intraday and interday variation. The detection and quantification limits and were 3.06 and 9.28 ng/spot for lamivudine and 3.34 and 10.13 ng/spot for zidovudine, respectively. Parameters such as mobile-phase composition, volume of mobile phase, time from spotting to development, and time from development to scanning were employed while testing for robustness of the method, and the standard deviation of peak areas was calculated for each parameter. The low coefficient of variation values indicated the robustness of the method. Statistical manipulation did not show any significant effect of one parameter over the others on the robustness of the method.

DEVELOPMENT AND VALIDATION OF HPTLC ASSAY METHOD FOR SIMULTANEOUS QUANTIFICATION OF HYDROCORTISONE AND CLOTRIMAZOLE IN CREAM AND APPLYING FOR STABILITY INDICATING TEST

A simple, specific, precise and accurate simultaneous high-performance thin-layer chromatographic method of analysis for hydrocortisone and clotrimazole was developed and validated. The method employed HPTLC glass plates (20x10) mm precoated with silica gel 60 F 254 as the stationary phase. The solvent system consisted of Toluene: Propanol: Ammonia (13:3:0.1v/v) that gave compact spots with R f value of 0.27 ± 0.01 and 0.58 ± 0.02 for hydrocortisone and clotrimazole, respectively. Densitometric analysis of hydrocortisone and clotrimazole was carried out in the absorbance/reflectance mode at 226nm. The calibration curves showed good linear relationship with R2 = 0.996 + 0.003 and 0.996 + 0.002 in the concentration range of 200-1200 and 200-1000 ng/µl for hydrocortisone and clotrimazole, respectively. The method was validated for precision, recovery and robustness. The LOD & LOQ were found to be 35.31, 107.01 and 34.93, 105.87 ng/µl for hydrocortisone and clotrimazole respectively. Statistical analysis proved that the method is repeatable, specific and accurate for the estimation of the studied drugs. Resolution of hydrocortisone, clotrimazole and their degradation products formed under different stress conditions (acid-base hydrolysis, oxidation and thermal degradation) was successfully achieved with the developed method indicating that it can be employed as a stability indicating method.

Synthesis and biological screening of some pyridine derivatives as anti-malarial agents.

Two series of pyridine derivatives were synthesised and evaluated for their in vivo anti-malarial activity against Plasmodium berghei. The anti-malarial activity was determined in vivo by applying 4-day standard suppressive test using chloroquine (CQ)-sensitive P. berghei ANKA strain–infected mice. Compounds 2a, 2g and 2h showed inhibition of the parasite multiplication by 90, 91 and 80%, respectively, at a dose level of 50 µmol/kg. Moreover, The most active compounds (2a, 2g and 2h) were tested in vitro against CQ-resistant Plasmodium falciparum RKL9 strains where compound 2g showed promising activity with IC50 = 0.0402 µM. The compounds were non-toxic at 300 and 100 mg/kg through the oral and parenteral routes, respectively. The docking pose of the most active compounds (2a, 2g and 2h) in the active site of dihydrofolate reductase enzyme revealed several hydrogen and hydrophobic interactions that contribute to the observed anti-malarial activities.

Synthesis and antileishmanial evaluation of some 2,3-disubstituted-4(3H)-quinazolinone derivatives

Background Leishmaniasis is a neglected tropical parasitic diseases affecting millions of people around the globe. Quinazolines are a group of compounds with diverse pharmacological activities. Owing to their promising antileishmanial activities, some 3-aryl-2-(substitutedstyryl)-4(3H)-quinazolinones were synthesized in good yields (65.2% to 86.4%). Results The target compounds were synthesized by using cyclization, condensation, and hydrolysis reactions. The structures of the synthesized compounds were determined using elemental microanalysis, infrared (IR), and proton nuclear magnetic resonance (1H NMR). The in vitro antileishmanial activities of the synthesized compounds were evaluated using Leishmania donovani strain. All the synthesized compounds displayed appreciable antileishmanial activities (IC50 values, 0.0128 to 3.1085 μg/ml) as compared to the standard drug miltefosine (IC50 = 3.1911 μg/ml). (E)-2-(4-chlorostyryl)-3-p-tolyl-4(3H)-quinazolinone (7) is the compound with the most promising antileishmanial activities (IC50 = 0.0128 μg/ml) which is approximately 4 and 250 times more active than the standard drugs amphotericin B deoxycholate (IC50 = 0.0460 μg/ml) and miltefosine (IC50 = 3.1911 μg/ml), respectively. Conclusions The results obtained from this investigation indicate that the synthesized and biologically evaluated quinazoline compounds showed promising antileishmanial activities and are good scaffolds for the synthesis of different antileishmanial agents. Electronic supplementary material The online version of this article (doi:10.1186/s13588-014-0010-1) contains supplementary material, which is available to authorized users.

Quantification of total polyphenols, catechin, caffeine, L-theanine, determination of antioxidant activity and effect on antileishmanial drugs of ethiopian tea leaves extracts

Objective: In this study four tea samples Gumero black, Wushwush black and Wushwush green from Agri- Ceft Plc and East Africa black tea leaves from East African Agribusiness Plc were investigated for total polyphenols, caffeine, catechin and L-theanine content. Materials and Methods: The aqueous extracts were investigated for their antioxidant and antileishmanial property and effect on amphotericin B, miltefocine and sodium stibogluconate, the commonly used antileishmanial drugs. Antileishmanial studies were conducted on L. aethiopica. Results: Wushwush green tea had the highest content of polyphenol (19.98 ± 1.15 mg gallic acid equivalent /100 g dry leaf weight), catechin (37.06 mg/g) and L-theanine (48.54 mg/g but the lowest caffeine content). It exhibited the highest antioxidant activity. The highest antioxidant effect of Wushwush green tea may be attributed to the highest polyphenol content. East African black tea had the lowest L-theanine (20.72 mg/g) and antioxidant activity but the highest caffeine (16.60 mg/g) content. Conclusion: Wushwush green tea showed slight inhibitory effect on L. aethiopica while the lack tea extracts (Gumero, East Africa and Wushwush) exhibited no antileishmanial activity. Wushwush green tea did not show any synergistic or antagonistic effect on the antileishmanial drugs used in this study while Gumero, East Africa and Wushwush black tea extracts exhibited dose dependant inhibitory activity to the commonly used antileishmanial drugs included in this study.

Volatile constituents and biological activities of Pycnostachys abyssinica and Pycnostachys eminii extracts

Context: Pycnostachys abyssinica Fresen and Pycnostachys eminii Gürke (Lamiaceae) are used in traditional Ethiopian medicine against eye and skin infections, “Mitch disease”, and dysentery. Objective: Our study was aimed at characterizing essential oil (EO), phytochemical groups, and antimicrobial and anthelmintic activity of extracts to underscore the species’ indigenous medicinal use. Materials and methods: Plant organs of Pycnostachys species were subjected to hydrodistillation, and essential oils (EO) analyzed by GC-MS. Phytochemical compounds, antimicrobial (diffusion assay) and anthelmintic activity (bioassay) of gradient solvent extracts of different polarity were studied. Results: In the stem and root EO of P. abyssinica, 25 (99%) and 30 (99.79%) compounds were detected respectively, with estragole (70.4%) (stem) and exo-fenchyl acetate (30.6%) (root) as the most abundant compounds. In leaf, stem and root EO of P. eminii, 30 (90.66%), 27 (90.59%) and 27 (99.96%) compounds were detected, respectively, with high levels of β-caryophyllene (from 18.08% to 28.85%) and germacrene D (from 15.1% to 22.06%). Alkaloids, saponins, phytosterols, flavonoids, polyphenols, diterpenoids and carotenoids were detected in Pycnostachys. Petroleum ether, chloroform and methanol extracts showed distinct antimicrobial effects with generally higher potential activity of lipophilic and semi-lipophilic fractions. Leaf and root methanol extracts of both species showed lethal activity against earthworms. Discussion: Identified EO constituents and phytochemical groups underscore the observed antifungal, antibacterial and anthelmintic activity of Pycnostachys gradient solvent extracts. Conclusion: EO analysis, phytochemical screening, and antimicrobial and anthelmintic assays indicate the biological potential of Pycnostachys species from Ethiopia, and emphasize their pharmacological and indigenous applications.

Chapter 11 - Plant-Derived Natural Products for the Treatment of Leishmaniasis

Leishmaniasis is a vector-borne parasitic disease caused by protozoan parasites belonging to the family Trypanosomatidae and genus Leishmania. The disease prevails in 88 subtropical and tropical countries in five continents where about 350 million people live. Approximately two million incidences of new cases are recorded every year, causing high morbidity and mortality with a wide spectrum of clinical manifestations in humans. Treatment for leishmaniasis depends on pentavalent antimonials developed 50 years ago as first-line drugs, whereas a limited range of other drugs such as paromomycin, miltefosine, and amphotericin B exist to supplement them. However, potential toxicity, costs, and emergence of drug-resistant pathogens are the most serious obstacles for successful treatment of the disease in most endemic areas. This demands the development of new antileishmanial agents. In this regard, the search for new drugs from various synthetic products continues, and involves also compounds isolated from natural sources and drugs used for the treatment of other ailments (cancer, viral infections, TB, immunosuppression, etc.) in order to discover compounds with unknown chemical structures and with potential novel modes of action. Medicinal and aromatic plants are a major source of natural organic compounds which are widely used as medicine. The extensive ethnomedicinal knowledge, diversity of plant species, and the disease burden worldwide necessitates the status of natural products in treatments of leishmaniasis to be assessed. This chapter will review plant crude extracts and fractions/active principles obtained from medicinal plants which are used in or have potential for the treatment of leishmaniasis. Plant species are systematically presented by family, bioactive phytochemicals in various classes, and results obtained on specific organisms tested. Recent empirical and rationale approaches for antileishmanial drug targeting and development of novel drugs derived from natural products will be discussed.