Conseguences of soil crude oil pollution on some wood properties of olive trees Biology | 10 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 Study the Effect of Alcoholic Extract of Nigella sativa Seeds on Trichomomas vaginalis In Vitro Maroof Sabti Juma Al-Ammash Dept. of Pathological Analysis/College of Applied Science/University of Samarraa Received in :25/April /2017 , Accepted in : 15 /October/ 2017 Abstract The present study included experimental effect of Metronidazole drug and Alcoholic extract of Nigella sativa seeds on T. vaginalis that cultivated on i T i Results showed that the numbers of parasite began increasing during a period after 24-48 hrs then began decreasing after 72-96 hrs, so that 72 hrs from growth considered logarithmic phase of T. vaginalis growth. Present results showed poisonous effect of N. sativa alcoholic extract that was prepared in laboratory and imported at concentrations (450, 550, 650 and 750) mg/ml on T. vaginalis by observing gradual decrease of trophozoite numbers with concentrate increase of extracts during growth periods (24, 48, 72 and 96) hrs. Different concentrations of Metronidazole were used as chemical therapy and control model, the results of the drug effects were followed-up daily, where current results showed (after 24 hours of concentration added) a high reduction in number and activity of the parasites at all concentrations of the drug were , p ci lly 150 200 μg/ l, th parasites disappeared completely after 96 hours, also the inhibitory concentration50 (IC50) of the parasit w 100 μg/ l ( ft r 48 h r incubation). The inhibitory effect of N. sativa alcoholic extract on the number and activity of T. vaginalis parasite was noted that the concentrations (450 and 550 mg/ml) were approximated in their effect on parasite growth, while the concentrations 650 and 750 mg/ml were the best in their inhibitory activity for parasite growth after 96 hours of addition. The concentration that causes 50% of death inculture (IC50) was 550 mg/ml (after 72 hours of concentration addition). Key words: Trichomonas vaginalis, Nigella sativa, Treatment, In vitro. Biology | 11 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 Introduction Trichomonas vaginalis is a cosmopolitan, it is a parasite that is found in the reproductive tract of both men and women. It lives in the vagina and urethra of women, prostate gland and seminal vesicles of men, its transmission was done through sexual intercourse mainly [1]. Trichomoniasis has a worldwide importance especially in recent years [2], The infection causes a range of symptoms and it also has long-term effects, especially in women and it is associated with a variety of serious complications including preterm labour, low birth weight of newborns, cervical cancer and implicated in amplifying in human immunodeficiency virus (HIV) transmission [3,4,5]. Parent et al. [6] referred to T. vaginalis causes the infection of 250 million new cases annually throughout the world. Al-Juwary [7] indicated to the biological and poisonous effect of the aqueous extract of Nerium oleander leaves and Melia azedarach fruits on T. vaginalis growing on i T i , There extracts caused gradual inhibition of trichomonads number with the increase of concentration during different growth periods (24, 48 and 72) hours. AL-mbashaa [8] used crude aqueous extracts (hot and cold) to each Seidlitzia resmarinus and Ziziphus spina- christi plants and she indicated to the effect of both plants extracts on T. vaginalis growing on i TYM medium and CPLM medium and gave 100% inhibition at concentration of 100 mg/ ml within 24 hours. The aim of the present study was to focus on the in vitro anti parasitic activity of N. sativa on the T. vaginalis growth. Materials and Methods Twenty five samples were collected during august and September /2016 from women those underwent to the Primary Health Care Centers, the samples were collected by speculum and cotton swab by Gynecologist and then added to each sample 2ml of normal saline and were quickly transported to laboratory to make direct smear, the parasite has been diagnosed by its jerky movement flagella motile and undulating membrane among epithelial cells [9]. The stains that mentioned below were used in microscopical examination for diagnosis. Buffer solution and normal saline It was prepared according to WHO [10]. Lugol’s iodine stain It was prepared according to WHO [9]. Eosin stain It was prepared according to Smyth and Barret [11]. Method of staining of T. vaginalis by Giemsa stain T. vaginalis smears were stained according to Manson–Bahar and Bell [12]. Preparation of alcoholic extracts of N. sativa seeds It was prepared according to Harborne [13]. Methode of culturing and detection of T. vaginalis in vitro The samples were c lti t y i T i 14]. The number of parasites count The number of generations and generation time was counted by the following two equations [15]: Include: n= Number of generation N= Number of cells at time (t) ) 0.301 No) Log N Log ( 2 Log No) Log N Log ( ( n Biology | 12 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 No= Number of start cells 2.02×10 5 cell/ml. n t g  Include: g= Generation time (hours) t= Incubation period n= Number of generation. Evaluation of efficacy of Metronidazole and N. sativa extracts on cultured T. vaginalis trophozoites. Metronidazole (MTZ) was produced by Sanofi-aventis company from Egypt under licence of Sanofi-aventis/France (50, 100, 150 and 200) μg/ml and N. sativa extract (450, 550, 650 and 750) mg/ml were used. Percentage of growth and inhibition were detected and inhibitory concentration50 (IC50) detected also through logarithmic phase according to the following two equations [7]: (Percentage of growth= 100 - Percentage of inhibition) Statistical analysis St ti tic l pr gr ( i it ) w f r lyz t y i g c ʼ ltipl r g test, analysis performed probable values less than 0.05 which were considered statistically significant [16]. Results and Discussion Chemical detection of some active chemical groups in alcoholic extract of N. sativa seeds Present results showed alcoholic extract of N. sativa contain some active compounds (Table 1), include flavonoids, glycosides, phenols and resins. These results agreed with the results of Chakravarty [17], about N. sativa contents. Also they agreed with results of Al-Ani [18], about N. sativa alcoholic extract contain on flavonoids, glycosides and resins, and not agreed about alkaloids and tannins. Determination of logaritmic phase of T. vaginalis growth T. vaginalis numbers were counted by using counting slide of blood cells (hemocytometer) after every 24 hours of growth for four days, thus the log. phase was identified of the parasite developing in Diamond's TYM medium (Figure 1). T. vaginalis numbers began multiplying and increasing during the period 24-48 hours of growth and then it began decreasing during the period 72-96 hours, so that the period 72 hours was considerd the log. phase of the parasite growth. The table (2) revealed the generations number for T. vaginalis in a log. phase after 48 hours pass was 48.28  5.93, also it revealed the generation time after 48 hours was 0.99  0.14. Mean of parasite numbers in treated group - Mean of parasite numbers in control group (Percent inhibition=-------------------------------------------------------------------------------------------------------×100) of growth % Mean of parasite numbers in control group Biology | 13 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 This result is an approach to report by Belding [19], where he referred to the log. phase of T. vaginalis limited between 24-96 hours of growth in vitro, the present results agreed with results of Castro-Garza et al. [20], who confirmed that the log. phase of T. vaginalis developing up to 72 hours, either Kostara et al. [21] pointed out that the log. phase does not exceed 144 hours, and the period determination of the log. phase depends on several factors, including growth medium acidic, primary number of cultivate and temperature. Effect of Metronidazole drug and N. sativa alcoholic extract (praperation in lab. and import) on T. vaginalis In vitro Different concentrations were used of the drug as chemical therapy and control model, the results of the drug effect on the parasite the follow-up daily, where present results were shown (after 24 hours of concentration added) a high reduction in number and activity of the p r it t ll c c tr ti f th r g , p ci lly 150 200 μg/ l, th p r it disappeared completely after 96 hours (table 3), also the inhibitory concentration50 (IC50) of th p r it w 100 μg/ l ( ft r 48 h r f c c tr ti ) Table (3) shows the effect of alcohol extract on the number and activity of T. vaginalis parasite. It is noted that the concentrations 450 and 550 mg/ml were approach in their effect on parasite growth, while the concentrations 650 and 750 mg/ml were the best in their inhibitory activity for parasite growth after 96 hours of addition. The concentration that causes the death of 50% of T. vaginalis parasites (IC50) was 550 mg/ml (after 72 hours of concentration addition). Table (3) revealed significant statistical differences between some groups treated on the one hand and between them and the control group on the other hand, especially after 72 hours of added concentrations under study. Metronidazole belongs to the 5-nitroimidazoles group and it is considered first choice for trichomoniasis treatment, this drug is administered orally or intravenously. A bioavailability of 93-100% because metronidazole does not bind to serum proteins and enter the cell and its organelles via facilated diffusion [22,23], then the drug was activated by the reduction of hydrogenosomal ferredoxin via reduction of the 5-nitro group [24,25]. The efficiency of this drug is high, with a cure rate of 85-95% of treated patients and re-infections can be avoided through simultaneous treatment of sexual partners [26]. Chemotherapy commonly using tr i z l c il i ff ct , ch r ct riz y th y’ f c ch i against toxic substances, like nausea, vomiting, diarrhoea, dizziness and headache. More serious side effects, like anorexia, hypersensitivity, leukopenia, palpitation, confusion, encephalopathy, and peripheral neuropathy are clinically rarely observed [27]. The present r lt ʼt agree with the results that reported by Ahmed [28], where he indicated to reduction a ratio 100% of T. vaginalis In vitro (after 48 hours of MTZ concentrations added) t c c tr ti f 50 100 μg/ l A th pr t r lt t gr with th r lt f Sulyman [29], where he stated the percentage of inhibition of MTZ was 100% for some concentrations (1.25%, 2.5%, 0.5% and 10%) used after 48 hours of added and the percentage of inhibition was 100%. Al-Banea [30] confirmed on importance of the N. sativa in the treatment of parasitic infections. It was observed the decrease of Entamoeba histolytica parasites number with mice feces after (8, 6 and 5 days) from the administration of the mice with the oil extract by concentrations (0.04, 0.05 and 0.06) ml respectively, Al-Khuzaay [31] pointed to the importance of the alcoholic extract of the N. sativa plant in inhibiting of protoscolices growth of Echinoccocus granulosus in vitro. He used concentrations (5%, 10% and 15%). The last concentration (15%) was the best in inhibiting, where it causes a viability low of protoscolices to 15% after 48 hours of concentration Biology | 14 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 addition. The effect was attributed to the plant extracts containment on the active compounds (alkaloids, flavonoids and tannins). The alkaloids, flavonoids and tannins interfere in the chain of proteins necessary metabolites for the survival of the parasite, or it may have the ability to break down the cell wall, stimulating proteins and fats and thus causing kill of the parasite [30]. Conclusions 1-Alcohol extract of Nigella sativa seeds which has therapeutic efficiency was close to the efficiency of Metronidazole. 2-The plant extract under study took a period more than Metronidazole in inhibiting the growth of T. vaginalis trophozoites In vitro. 3-Alcohol extract of N. sativa seeds can be used as a natural alternative instead of drugs that used in treatment of T. vaginalis, as N. sativa seeds are available on the market and easy to obtain. References 1-Roberts, L.S. and Janovy, J.R. (2009). Schmidt and Roberts Foundation of Parasitology. 8 th ed. McGraw-Hill Companies, Inc., New York, 701pp. 2-Lazenby, G.; Soper, D. and Nolte, F. (2013). Correlation of Leukorrhea and Trichomonas vaginalis Infection. J. Clin. Microbiol., 51(7): 2323-2327. 3-Kissinger, P.; Amedee, A.; Clark, R.A.; Dumestre, J.; Theall, K.P.; Myers, L.; Hagensee, M.E.; Farley, T.A. and Martin, D.H. (2009). Trichomonas vaginalis treatment reduces vaginal HIV-1 shedding. Sex Transm. Dis. 36(1): 11-16. 4-Hillier, S.L. (2013). Prevalent, treatable and significant: barriers to the control of Trichomonas vaginalis in women. Sex Transm. Infect. 89(6):415pp. 5-Kissinger, P. and Adamski, A. (2013). Trichomoniasis and HIV interactions: A review. Sex Transm Infect. 89(6):426-433. 6-Parent, K.N.; Takagi, Y.; Cardone, G.; Olson, N.H.; Ericsson, M.; Yang, M.; Lee, Y.; Asara, J.M.; Fichorova, R.N.; Baker, T.S. and Nibert, M.L. (2013). Structure of a Protozoan Virus from the Human Genitourinary Parasite Trichomonas vaginalis. mBio. 4(2): e00056-13. 7-Al-Juwary, R. S. A. (2006). The Aqueous Extracts Effect of Nerium oleander and Melia azedarach on Growth and Metabolism of Trichomonas vaginalis In vitro. M.Sc. thesis, Sci. Coll. Univ. Mosul. 111pp. 8-AL-mbashaa, N.A.H. (2014). Isolation and Cultivation of Trichomonas vaginalis and study the effect of Seidlizia rosmarinus and Ziziphus spina-christi extracts on the parasite and some physiological parameters of infection women in Samara city. M.Sc. thesis, Coll. Edu. For women Univ. of Tikrit. 130pp. 9-WHO. (1991). Basic laboratory methods in medical parasitology. World Health Organ, Geneva. 122pp. 10-WHO. (2003). Manual of basic techniques for a health laboratory. 2 nd ed. World Health Organ. Geneva. 384pp. 11-Smyth, J.D. and Barrett, N.J. (1980). Procedure for testing the viability of human hydatid cysts following surgical removal, especially after chemotherapy. Trans. Roy. Soci. Trop. Med. Hyg., 74(5): 649-652. 12-Manson-B h r, P B ll, R (1987) “ ' Tr pic l i ” 9 th ed. Baillier Tindall. London. 1557pp. 13-Harborne, J.B. (1984). Phytochemical methods: A guide to modern techniques of plant analysis. 2 nd edn. Chapman and Hall Ltd., London. 288pp. https://www.ncbi.nlm.nih.gov/pubmed/?term=Dumestre%20J%5BAuthor%5D&cauthor=true&cauthor_uid=19008776 https://www.ncbi.nlm.nih.gov/pubmed/?term=Theall%20KP%5BAuthor%5D&cauthor=true&cauthor_uid=19008776 https://www.ncbi.nlm.nih.gov/pubmed/?term=Myers%20L%5BAuthor%5D&cauthor=true&cauthor_uid=19008776 https://www.ncbi.nlm.nih.gov/pubmed/?term=Hagensee%20ME%5BAuthor%5D&cauthor=true&cauthor_uid=19008776 https://www.ncbi.nlm.nih.gov/pubmed/?term=Hagensee%20ME%5BAuthor%5D&cauthor=true&cauthor_uid=19008776 https://www.ncbi.nlm.nih.gov/pubmed/?term=Farley%20TA%5BAuthor%5D&cauthor=true&cauthor_uid=19008776 https://www.ncbi.nlm.nih.gov/pubmed/?term=Martin%20DH%5BAuthor%5D&cauthor=true&cauthor_uid=19008776 Biology | 15 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 14-Diamond, L.S. (1957). The establishment of various trichomonads of animals in axenic cultures. J. Parasitol. 43: 488-490. 15-Benjamin, C.L. and German, G.R. (1993). Students study guide microbiology. Concepts and Application. McGraw-Hill. London: 254-248. 16-Elsahookie, M. and Waheeb, K.M. (1990). Applications in design of experiments. Dar- Alhikma for printing. Univ. Mosul. 488pp. 17-Chakravarty, H.L. (1976). Plant wealth of Iraq. A dictionary of economic plants. Ministry of Agriculture and Agrarian reform. (1): 81-82 18-AL-Ani, A.H.J. (1998). A study on the chemical components of local black seed Nigella sativa L. and antimicrobial effect of its extracts on some Microorganisms. M.Sc. thesis, Sci. Coll. Univ. AL-Mustansiriya. 82pp. 19-Belding, D.L. (1965). Textbook of parasitology. 3 rd ed. Appleton - century- croft, New York. 1374pp. 20-Castro-Garza, J.; Anaya-Velazquez, F.; Said-Fernandez, S. and Gonzalez-Garza, M.T. (1996). Comparable growth of a Trichomonas vaginalis strain in PEHPS and TYI-S-33 media. J. Arch. Med. Res., 27(4): 567-570. 21-Kostara, I.; Carageorgiou, H.; Varonos, D. and Tzannetis, S. (1998). Growth and survival of Trichomonas vaginalis. J. Med. Microbiol., 47(6): 555-560. 22-Houghton, G.W.; Smith, J.; Thorne, P.S. and Templeton, R. (1979). The pharmacokinetics of oral and intravenous metronidazole in man. J. Antimicrob. Chemother., 5(5): 621-623. 23-Harris, J.C., Cottrell, S.L., Plunmer S. and Lloyd, D. (2001). Antimicrobial properties of Allium sativum (garlic). Appl. Microbiol. Biotechnol., 57: 282-86. 24-Lindmark, D.G. (1980). Energy metabolism of the anaerobic protozoon Giardia lamblia. Mol. Biochem. Parasitol., 1(1): 1-12. 25-Muller, M. (1986). Reductive activation of nitroimidazoles in anaerobic microorganisms. Biochem. Pharmacol., 35(1), 37-41. 26-Lossick, J.G. (1990). In Trichomonads Parasitic in Humans. Honiberg, B.M., Ed.; Springer-Verlag: New York: 324-341. 27-Lossick, J.G. (1990). Treatment of sexually transmitted vaginosis/vaginitis. Rev. Infect. Dis., 12(Suppl 6), S665-S681. 28-Ahmed, S.A. (2010). In vitro Effects of Aqueous Extracts of Garlic (Allium sativum) and Onion (Allium cepa) on Trichomonas vaginalis. Parasitol. Unit. J. 3(1&2): 45-54. 29-Sulyman, M.A. (2008). Epidemiological study on Trichomonas vaginalis & some associated bacteria that causing sexual transmitted diseases & Effect of some herbal extraction on the parasite in vitro in Kirkuk city. M.Sc. thesis .Coll. Sci. Univ. Tikrit. 143pp. 30-Al-Banea, S.Z.M. (2006). Effect of Nigella sativa oil on pathogenicity of Entamoeba histolytica. M.Sc. thesis .Coll. Sci. Univ. AL-Mustansiriya. 84pp. 31-AL-Khuzaay, J.H.R. (2005). Evaluation of the effectiveness of some plant extracts, drugs and direct electric current on the viability of the protoscolecies of Echinococcus granulosus in vitro and in vivo in white mice. M.Sc. thesis .Coll. Edu. Univ. AL-Qadisiya. 100pp. Biology | 16 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 Table (1):Chemical determinat of some active compounds in alcoholic extract of N. sativa seeds. N Type of test Alcoholic extract 1 Alkaloids -- 2 Flavonoids + 3 Glycosides + 4 Phenols + 5 Resins + 6 Tannins -- Table (2): Mean of parasites number (×10 5 ), Mean of generations number and Mean of generation time  SD. of control group. The period (hour) Number of parasites ×10 5 /ml Mean  SD Number of generations Mean  SD Generation time Mean  SD 24 4.04  0.96 27.14  6.46 0.92  0.23 48 7.19  0.88 48.28  5.93 0.99  0.14 72 5.55  1.26 37.23  8.42 1.88  0.37 96 4.23  0.73 28.41  4.90 3.46  0.74 Biology | 17 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 Table (3): Mean  SD and percentage of growth inhibition of T. vaginalis per culture after exposure to various concentration of MTZ and alcoholic N. sativa extract in comparison to control. Incubation Period (hour) Dosage Treated (μg/ml) 24 48 72 96 Mean  SD Growth (%) Inhibition (%) Mean  SD Growth (%) Inhibition (%) Mean  SD Growth (%) Inhibition (%) Mean  SD Growth (%) Inhibition (%) Control 4.04 0.96 ± E 100 ----- 7.19 0.88 ± A 100 ----- 5.54 1.25 ± A 100 ----- 4.23 0.73 ± A 100 ----- The MTZ (µg/ml) 55 4.12 0.58 ± E 98.02 1.98 2.76 0.66 ± GH 38.39 61.61 1.06 0.51 ± DE 19.14 80.86 0.00 0.00 ± C 0.00 100 100 5.19 1.08 ± CD 71.54 28.46 3.17 0.35 ± EFGH 44.09 55.91 0.70 0.15 ± EF 12.64 87.36 0.00 0.00 ± C 0.00 100 150 4.37 1.15 ± DE 91.83 8.16 2.48 0.65 ± H 34.50 65.50 0.14 0.04 ± G 2.53 97.47 0.00 0.00 ± C 0.00 100 200 3.83 0.80 ± E 94.81 5.19 0.53 0.18 ± I 7.38 92.62 0.00 0.00 ± G 0.00 100 0.00 0.00 ± C 0.00 100 Alcoholic Extract of N. sativa seeds prepared in lab. (mg/ml) 455 6.23 0.32 ± AB 45.79 54.20 5.03 0.47 ± BC 69.96 30.04 3.56 0.27 ± B 64.26 35.74 1.93 0.28 ± B 45.63 54.37 555 5.57 0.89 ± BC 62.12 37.87 4.30 0.44 ± CD 59.81 40.19 2.84 0.36 ± C 51.27 48.73 1.13 0.32 ± C 26.72 73.28 650 5.11 0.72 ± C 73.51 26.48 3.30 0.62 ± EFG 45.90 54.10 1.45 0.35 ± D 26.18 73.82 0.00 0.00 ± C 0.00 100 750 4.45 0.62 ± DE 89.85 10.14 3.06 0.38 ± FG 42.56 57.44 0.31 0.15 ± FG 5.60 94.40 0.00 0.00 ± C 0.00 100 Import alcoholic extract of N. sativa (mg/ml) 455 7.13 0.90 ± A 23.52 76.48 5.57 1.06 ± B 77.47 22.53 3.74 0.95 ± B 67.51 32.49 1.76 0.33 ± B 41.61 58.39 555 6.97 1.33 ± A 27.48 72.52 5.12 1.24 ± B 71.22 28.78 3.11 0.56 ± C 56.14 43.86 1.04 0.27 ± C 24.59 75.41 650 6.25 1.24 ± AB 45.30 54.70 3.89 1.25 ± DE 54.11 45.89 1.35 0.33 ± D 24.37 75.63 0.00 0.00 ± C 0.00 100 750 5.15 1.31 ± CD 72.53 27.47 2.52 0.61 ± H 35.15 64.95 0.18 0.08 ± FG 3.25 96.75 0.00 0.00 ± C 0.00 100 Similar letters indicate to non-significant differences (P > 0.05) among groups (vertical compare). Different letters indicate to significant differences (P ≤ 0.05) among groups (vertical compare). Biology | 18 https://doi.org/10.30526/30.3.1596 7102( عام 3( العدد ) 30مجلة إبن الهيثم للعلوم الصرفة و التطبيقية المجلد ) Ibn Al-Haitham J. for Pure & Appl. Sci. Vol.03 (3) 2017 Figure (1): Normal curve of the growth T. vaginalis trophozoites in Diamondʼs TYM medium.