Open Access Short Communication

Evaluation of Four Assays to Determine Cytotoxicity of Selected Crude Medicinal Plant Extracts In vitro

Werner Cordier, Vanessa Steenkamp

Journal of Pharmaceutical Research International, Page 16-21
DOI: 10.9734/BJPR/2015/16906

Aims: Cytotoxic assessment of plant extracts is crucial during the pre-clinical screening of medicinal preparations selected for further development. The aim of this study was to compare four assays used to test the cytotoxicity of eight African plant extracts, as well as to determine whether the antioxidant properties of the extracts potentially diminished the reliability of the resazurin conversion assay.
Methodology: HepG2 cells were exposed to hot water or methanol extracts of Acokanthera oppositifolia, Boophane disticha, Solanum aculeastrum and Tabernaemontana elegans for 72 h. Cell viability was determined using neutral red uptake, sulphorhodamine B staining, MTT and resazurin conversion assays. Phytochemical interference in the resazurin conversion assay was assessed in a cell-free environment and antioxidant activity of the crude extracts was determined using the Trolox Equivalence Antioxidant Capacity assay.
Results: Compared to the other three assays, the resazurin conversion assay failed to detect cytotoxicity, even at the highest concentration tested. The sulphorhodamine B staining assay showed the highest reproducibility, and compared well to the neutral red uptake and MTT conversion assays. Although extracts possessed moderate antioxidant activity, this did not contribute to the spontaneous conversion of resazurin in the cell-free environment, inferring that cellular conversion of resazurin was up-regulated by the crude extracts, leading to the false negative result of cytotoxicity.
Conclusion: Due to the potential interference between samples and substrates used in cytotoxicity assays, assessment of the assay’s suitability should always be conducted. It is possible that the crude extracts increased resazurin-specific enzyme activity through up-regulation, and as such led to higher conversion. Due to the interference, the resazurin conversion assay should not be used when assessing cytotoxicity of plant extracts. It is recommended that in vitro toxicological evaluation be performed using multiple cytotoxicity assay, preferably those based on different mechanistic principles to ensure higher accuracy.

Open Access Original Research Article

Synergistic Antibacterial Activity of Pleurotus Species (Mushroom) and Psychotria microphylla (Herb) against Some Clinical Isolates

O. E. Udu-ibiam, O. Ogbu, U. A. Ibiam, A. U. Nnachi

Journal of Pharmaceutical Research International, Page 1-8
DOI: 10.9734/BJPR/2015/17603

Aims: This study investigated the synergistic antimicrobial activity of the extracts of Pleurotus species and Psychotria microphylla against five clinical bacterial isolates: Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhi, Klebsiella pneumoniae and Staphylococcus aureus from Federal Teaching Hospital, Abakaliki (FETHA), Ebonyi State, Nigeria.
Study Design: This was an experimental study.
Place and Duration of Study: This study was carried out in the Department of Applied Microbiology Laboratory, Ebonyi State University, Abakaliki, Nigeria between October, 2014 and December, 2014.
Methodology: Antimicrobial components from the mushroom and herb were extracted with water and ethanol. In this, the antimicrobial activities were examined by agar well diffusion method adapted from Kirby Bauer disk diffusion technique against confirmed isolates of Escherichia coli, Pseudomonas aeruginosa, Salmonella Typhi, Klebsiella pneumoniae and Staphylococcus aureus. Mixtures of the different extracts (mushroom and herb) at varying ratios (of 1:1, 1:2, 2:1) were also used to determine their synergistic inhibitory effects at 37ºC and 24 hours incubation. Inhibition zone diameters were measured and recorded.
Results: The result revealed antibacterial activity of the extracts although at varying degrees. All the isolates (except Klebsiella pneumoniae) were sensitive to Psychotria microphylla. The highest sensitivity (7.0 mm) with ethanol extract of the herb was observed against Pseudomonas aeruginosa, closely followed by Salmonella Typhi (5.0 mm) and then Escherichia coli and Staphylococcus aureus (4.0 mm each). Although the herb showed a wider range of antimicrobial activity against the tested isolates than the mushroom, Klebsiella pneumoniae was sensitive (6.0 mm). The herb-mushroom extract combination produced a better antimicrobial activity against the isolates with a very high sensitivity of 16.0 mm recorded against S. aureus as compared to the highest sensitivity (10.00 mm) produced by Ampiclox against S. Typhi. The activity of Pleurotus sp was independent of the extract (p-value=0.189), meaning that the difference in zones of inhibition in relation to extract types was not significant (p>0.05). P. microphylla activities was dependent on the type of extract (p-value=0.031). ); that is, the difference in zones of inhibition produced by P. microphylla in relation to extract types was significant (p<0.05). The most efficient ratio of synergy was the 1:2 ratios which involved one part of Pleurotus species and two parts of P. microphylla.
Conclusion: The synergistic effect produced by Psychotria microphylla and Pleurotus species holds a good promise in the treatment of infections caused by tested bacteria and should be harnessed and patented.

Open Access Original Research Article

Phytochemistry, Phenolic Contents and Free Radical Scavenging of Bauhinia thoningii kurtz and Ricinus communis L Extracts as Possible Contribution to Medicinal Effects

Ojezele Matthew Obaineh, Abatan Matthew Oluwole, Onifade Abdulfatah Adekunle

Journal of Pharmaceutical Research International, Page 9-15
DOI: 10.9734/BJPR/2015/13476

Aims: Current research places premium on isolation bioactive constituents and antioxidants activities of plants. However, the chemical constituents and antioxidant potential of many local medicinal plants are still unknown. This study sought to assay the phytochemistry and in vitro antioxidant potential of Bauhinia thoningii (leaf) and Ricinus communis (root) as a likely justification of scientifically proved and folklore uses.
Study Design: Air-dried parts of the plants were extracted in solvents and evaporated in-vacuo. The extracts were analysed for secondary metabolites.
Place and Duration of Study: Sample: Department of Veterinary Physiology, Biochemistry and Pharmacology, University of Ibadan, Ibadan, Nigeria (FHI 108864). The confirmatory identification of the plants was done at Forestry Research Institute of Nigeria (FRIN) with voucher deposited, between 2012 and 2013.
Methodology: Standard procedures were used to carry out phytochemical analyses of aqueous and methanol extracts of the plants. In vitro antioxidant effects of the plants were tested by ferrous reducing antioxidant property (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability. The total phenol contents in the extracts were determined by the modified Folin-Ciocalteu method.
Results: Anthraquinone, phenols and cardenolide were detected in all the plants. The FRAP values of Ricinus communis and Bauhinia thoningii were 915.3 µmol/g and 620.4 µmol/g while their DPPH scavenging ability was 93.6% and 71.4%. R. communis which showed higher phenolic, 90.33 mg/g, content as against 62.14 mg/g for B. thoningii.
Conclusion: Results from this study showed a direct relationship between the total phenol constituents and antioxidant activities of the plants. The high phenolic content and the observed radical scavenging activities could be some of the factors responsible for medicinal activities. This may also be clue to the likely mechanism of action of the plants in managing the disease conditions.

Open Access Original Research Article

Assessment of Mutagenic Effects of Sulfadoxine-Pyrimethamine (SP) on Animal Model

N. J. Chukwurah, O. O. Aina, O. A. Otubanjo

Journal of Pharmaceutical Research International, Page 22-33
DOI: 10.9734/BJPR/2015/18164

Introduction: The use of medicines in pregnancy has always been a source of concern because of adverse effects on the foetus.
Aim: The study therefore investigated the mutagenic potential of sulfadoxine-pyrimethamine (SP) in Swiss albino mice.
Study Design: Comparative study.
Place and duration of the study: The study was carried out at Nigerian Institute of Medical Research, Yaba within 4weeks.
Methodology: Four groups of pregnant female laboratory bred virgin mice were used. Group 1 received the normal human therapeutic dose (HTD) of SP. Group 2 received 1½ X HTD group while group 3 received 2X HTD. Group 4 received only water and served as the control. Assessment of sperm head abnormality was conducted using four groups of laboratory bred male Swiss albino mice. Group 1 received the HTD; group 2, 1½ while group 3 received 2 X HTD. The fourth group received only water and served as the control.
Results: The result showed no abnormality when the fetuses were examined and there was no significant association between the test mice and the control mice, P>0.05. Analysis of the sperm head abnormality showed no significant association between the test and control male mice.
Conclusion: Inference can be made from this study that that SP may not be adjudged to induce sperm head abnormalities and may not be mutagenic. It can therefore be concluded that SP is safe for use in pregnancy after quickening.

Open Access Original Research Article

Design, Synthesis, Characterization and Cancer Cell Growth-Inhibitory Properties of Novel Derivatives of 2-(4-Fluoro-phenyl)-5-(5-Aryl Substituted-1, 3, 4-Oxadiazol-2-yl) Pyridine

Adimule Vinayak, Medapa Sudha, Adarsha Haramballi Jagadeesha, Kumar Sanjeev Lalita

Journal of Pharmaceutical Research International, Page 34-43
DOI: 10.9734/BJPR/2015/15486

Aims: A series of novel 2-(4-Fluoro-phenyl)-5-(5-aryl substituted-1, 3, 4-oxadiazol-2-yl) pyridine derivatives 7a-7e were synthesized and evaluated for their cancer cell growth inhibitory properties.
Study Design: Designed and synthesized the novel derivatives of pyridine containing 1, 3, 4-oxadiazole ring based on the initial SAR studies of pyridine and their diversified biological properties.
Place and Duration of the Study: Synthesis, purification was performed at centre for scientific research and advanced learning, Mount Carmel College, Autonomous, Bangalore, India (between June 2013 to March 2014), characterization using 1H-NMR and 13C NMR was done at Indian Institute of Science (IISc), Bangalore, India. Cytotoxic evaluation was performed at Genelon Institute of Life Sciences, Yalahanka, Bangalore, India.
Methodology: A mixture of 6-Bromo-nicotinic acid ethyl ester with 4-Fluoro-phenyl boronic acid in ethyl alcohol afforded the 6-(4-Fluoro-phenyl)-nicotinic acid ethyl ester (4). Subsequent reaction of (4) with hydrazine hydrate in ethanol afforded 6-(4-Fluoro-phenyl)-nicotinic acid hydrazide. Reaction with compound (5) and various aldehydes furnished the novel Schiff base derivatives (6a-6e). Schiff base derivatives were cyclized using Chloramine-T under microwave irradiation and obtained novel derivatives 7a-7e. All the reagents, chemicals and solvents were purchased from S-d fine and spectro chem Ltd. Bengaluru. India. 1H-NMR and 13CNMR were recorded by Brucker 400 MHz spectrophotometer. Melting points were determined using Buchi melting point 545. Mass spectra were recorded by Agilent 1200 series. TLC was done on F254 grade silica 60 from Merck. IR spectra were recorded by using FTIR (1800S) series. Microwave used was whirlpool semi-automated specially designed. The human cancer cell lines were purchased from NCCS (National Centre for Cell Science), Pune, India.
Results: Compounds 7b and 7d showed better activity than 5-Fluorouracil against MCF7 cell line. Title compounds 7a-7e is prepared by the oxidative cyclization reaction of the corresponding Schiff base compounds 6a-6e using chloramine-T as promoter. Cytotoxicity of the 1, 3, 4-oxadiazole compounds were obtained by screening the compounds against human cancer cell lines using MTT assay. Among the various derivatives (7a-7e), compounds bearing 5-bromo-2-fluoro-phenyl ring and 3-methyl thiophene ring demonstrated potent antiproliferative activity. In this research work compounds 7b and 7d showed good cytotoxicity against MCF7 cell line.
Conclusion: Novel derivatives of 1, 3, 4-oxadiazole compounds namely, 5-[5-(5-Bromo-2-fluoro phenyl)-[1, 3, 4] oxadiazol-2-yl]-2-(4-fluoro-phenyl)-pyridine (7b) and 2-(4-Fluoro-phenyl)-5-[5-(3-methyl-thiophen-2-yl)-[1, 3, 4] oxadiazol-2-yl]-pyridine (7d) showed better anticancer properties against MCF7 cell line with IC50 of 6.9 µM and 3.8 µM respectively. Cytotoxicity of the compounds (7c), (7d) against Caco-2 cell line with IC50 of 23.6 µM and 56.5 µM respectively. Rest all the compounds showed more resistance against all the cell lines.

Open Access Original Research Article

In vitro Doses and Incubations Dependent Thrombolytic Potential Study of Edible Mushrooms Pleurotus ostreatus, Ganoderma lucidum and Lentinula edodes Available in Bangladesh

Maafi Rizwana Islam, M. Moyen Uddin Pk

Journal of Pharmaceutical Research International, Page 44-51
DOI: 10.9734/10.9734/BJPR/2015/18227

Aims: The study was carried out to investigate the thrombolytic potential of methanolic extracts of edible mushrooms.
Study Design: Experimental study.
Place and Duration of Study: Department of Biochemistry, Primeasia University, August 2014 to December 2014
Methodology: An in vitro thrombolytic model was used to check the thrombolytic effect of Pleurotus ostreatus, Ganoderma lucidum and Lentinula edodes along with Streptokinase as a positive control and water as a negative control. In this study two concentrations of the extracts i.e. 400µg/ml and 800 µg/ml were tested at three time intervals, 24 hrs, 48 hrs and 72 hrs duration of incubation at 37ºC for observing maximum clot lysis.
Results: At 400 µg/ml dose, Pleurotus ostreatus showed highest clot lysis activity in 72 hrs of incubation, 18.62% while Ganoderma lucidum, and Lentinula edodes showed 17.01% and 9.02% in the same incubation. Similarly, at 800 µg/ml dose, the thrombolytic activities were 63.35%, 34.16%, and 29.00% respectively for Pleurotus ostreatus, Ganoderma lucidum, and Lentinula edodes. Our study clearly demonstrated that Pleurotus ostreatus exhibits maximum 63.37% clot lysis at 800 µg/ml concentration in 72 hrs of incubation with reference to Streptokinase 86.43%. The thrombolytic nature of the mushroom extracts was found to be significant (P<0.0001), when compared with the negative control (water) at different doses. The relative thrombolytic activity of Pleurotus ostreatus, Ganoderma lucidum and Lentinula edodes was found to significant at different doses (P<0.0001) and at various incubation periods (P<0.0001).
Conclusion: The result of our findings indicated that percentage of clot lysis is directly proportional to the concentration and incubation time of the extracts.

Open Access Original Research Article

Antioxidant Activity, Total Phenolic and Flavonoid Contents of Essential Oils of Three Cyperus Species (Cyperaceae)

Oladipupo A. Lawal, Anthony B. Ojekale, Olugbenga S. Oladimeji, Titilola S. Osinaike, Adetayo A. Sanni, Mthokozisi B. C. Simelane, Rebamang A. Mosa, R. Opoku

Journal of Pharmaceutical Research International, Page 52-62
DOI: 10.9734/BJPR/2015/18631

Aims: To determine the in vitro antioxidant activity, total phenolic and flavonoid contents of essential oils from the rhizomes of Cyperus distans, Cyperus papyrus and Cyperus rotundus collected from different locations.
Study Design: The study was design to investigate the total phenolic and flavonoid contents and antioxidant properties of essential oils from the rhizomes of Cyperus distans, Cyperus papyrus and Cyperus rotundus in order to assess their medicinal values.
Place and Duration of Study: Fresh plant materials of Cyperus distans, Cyperus papyrus and Cyperus rotundus were randomly collected at the flowering stage between October, 2006 and September, 2007 from different locations in KwaZulu-Natal Province, South Africa.
Methodology: The antioxidant potential of the oils of C. distans, C. papyrus and C. rotundus was evaluated by 1,1-diphenylpicryl-hydrazyl (DPPH), hydroxyl radical, nitric oxide, metal chelating and reducing potential methods. The total phenolic and flavonoid contents of the oils were determined by spectrophotometer using Gallic acid and Quercetin equivalents, respectively.
Results: The essential oils of C. distans, C. papyrus and C. rotundus showed no metal chelating activity and poor aptitude to scavenge 1,1-diphenyl-2-picrylhydrazyl radical with inhibitory concentration (IC50) >173 µg/mL, when compared to standard antioxidants; butyl hydroxyl anisole (BHA), butyl hydroxyl toluene (BHT), ascorbic acid (Vitamin C) and α-tocopherol with IC50 ≤10.70 µg/ mL. But, displayed significant antioxidant effects, when tested with other methodologies. The total phenolic content and the concentrations of flavonoid in the oils varied from (0.032 to 0.085) mg (Gallic acid)/100 µL and (0.027 to 0.072) mg (Quercetin)/ 100 µL equivalents, respectively.
Conclusion: The observed difference in antioxidant activities of the essential oils of C. distans, C. papyrus and C. rotundus may be due to climatic, seasonal and geographic conditions, harvest period, distillation technique, plant maturity and the presence of some major components or synergy between compounds present in the essential oils.