A study on the stabilty of different frusemide liquid dosage formulas: Oral solution, syrup, Elixir, Suspension and emulsion Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity 63 Cytotoxic Assay of Nigella sativa Leaf Callus Extract (Thymol) on Hep-2 Cell Line Using ELISA Assay Zaynab S. Abdel Gany*,1 and Mayasaa F. Mahdi* * Iraqi Center for Cancer and Medical Genetics Researches, Al Mustansiriya University Baghdad , Iraq Abstract Extract from cell culture of medicinal plant like Nigella sativa have been assessed for its cytotoxic properties. Thymol is likely responsible for the theraputic effects of Nigella sativa leaf callus extract. In this short study the inhibitory effect of Nigella sativa leaf callus extract (Thymol) has been studied on Human Lorgnx Epidrmoid Carcinoma (Hep-2) cell line during different exposure period of time (24, 48 and 72 hrs.) using different concentration of the extract (1000, 500, 400, 300, 200 and 100 µg/ml). The optical density of the Hep-2 cells has been readed on 492 nm wave length. Thymol – induced cytotoxicity was (500 µg/ml) which inhibit cell growing compared to the control and this ratio increased at the 48 hrs of exopsure and stopped at 72 hrs. Key wards: Nigella sativa, callus extract, cell line, ELISA assay. ةصالخلا وه ربتعي لومياثلا .اياتءصلتهخ عماتهصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا لخسفت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ل ءيت مللمةت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا اليت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا المت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعت صممجمتننم نمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خصسمت مع سم . وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا نمسصمختسم لمت مت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا صخا ختءصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ؤسسم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاةت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا م وسمت للمةت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا المت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعتهصتهخ عماتا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا سم ا . وه ربتعي لومياثلا .اياتجيت ل ت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا رنسمتت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعمت وه ربتعي لومياثلا .ايؤسسمت تت 48 ,24) صصتا ممةت وه ربتعي لومياثلا .ايممستتهع منمت) Hep-2 وه ربتعي لومياثلا .ايعماتا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا تحم ات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا المت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعت) وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا نمسصمخ( ءماتصصمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ع سمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خمأمصسمتسم ت مع سمتءماتO.D( . وه ربتعي لومياثلا .اياتمسمست وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاخµg/ml 100ت 200 ,300 ,400 ,500 ,1000تعمءم( ) صلختسملمع عا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلامت وه ربتعي لومياثلا .ايم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاحسلتهع منمتهصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا صخ عمات)72 تتت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا لاتسليتصصمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ع سمت) صلختسملم صرم صمتµg/ml 500 . وات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا نمسصمختسسماتعمهمت مع سمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خمأمصسمتءلات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا محسلتتnm 492 وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا امخت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا صمويت تعمءمتهصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ممستت وه ربتعي لومياثلا .ايمملتءلات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا سممت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا نم تتهصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ممست.48هةتهممهممت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خسامتت ه وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ةت ل ت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا لخلمتجيت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا صاتت Introduction The main inspiration of black seed comes from the famous saying (Hadith) of our Prophet Mohammed; (God peace be upon him),تthatت“HabbatتAl-soda is remedy for all diseaseتexceptتdeath.”(1)ت.It is an annual herbaceous plant believed to be endogenous to the Mediteranean region but has been cultivated in other parts of the world including India and Pakistan (2). Black seed oil contains about 0.5-1.5 % volatile oil including nigellone and thymoquinone used as anti-histanimic, antioxidant, antiinfective and bronchodilating effects(3).Thymol, is one of the active compounds in N. sativa extract, plays important role in the inhibition of cancer cells, and can attach with the mutagenic substance, because thymol is one of the antioxidant phenolic compounds (4).Plant tissue culture techniques inters in several applications like plant micropropagation, genetic study, plant improvement, study of plant cell physiology, the production of secondary metabolites in addition to production of viruse free plant diseases (bacterial and fungal) (5). So, to increase the production of this compound (thymol) all the year round without depending on the mother plant, plant tissue culture techniques formed callus and then increased the production of thymol. The anticancer activity of N. sativa was first revealed by (6) who observed enhancement of natural killer (NK) cell activity ranging from 200-300% in advanced cancer patients receiving multimodality immunotherapy programme in which N. sativa was one of these components. Thymoquinone and dithymoquinone, active principles of N. sativa, had cytotoxic effect against parental and multi-drug resistant human tumour cell lines which were over 10- fold more resistant to doxorubicin and etoposide (7) . Radiation protection activity of N. sativa in mice against induction of chromosomal aberrations by gamma ray was also reported(8) 1Corresponding author : E-mail : zaynabsaad@yahoo.com Received : 14/6/2008 Accepted : 1/12/2008 Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity 64 The using of plant tissue culture techniques make the easy of pharmaceutical compounds production instead of depending on the mother plant and become possible to produce these compounds at high amount and at high rate of pure may be over than these isolated from the complete mother plant, and its production may be quickly and independent on the season, also limit the surface area that is used in the medicinal plant culturing (9).The objective of the present study was to assess the cytotoxic properties of this extract from cell culure of N. sativa leaf callus using against Hep-2 cell line using ELISA (enzyme linked immunosorbant assay) assay. Material and Methods: Collection of plant material: Seeds of Nigella sativa gotten from Dr. Aws Al-Ani (Directorate of Agriculture Research and Food Technology/ Ministry of Science and Technology/ Baghdad/ Iraq) to be used. Callus culture condition: The establishment and maintenance of callus were carried out using the procedures described before(10). Extraction prepration: For preliminary screening, the seeds were cultured and callus induced and material from callus culture were lyophilzed and extracted by a method described elsewhere (11). In short, one g of callus was mixed with 30 ml of NaOH solution 5% and then diethyl ether was then added in a ratio of (2:1) (v /v) and mixed well as described elsewhere (12). The extract was then filtered and concentrated in vacum at 45º C and then kept in the dark at 4º C until tested. Cytotoxicity test using ELISA assay: For this test, the extract were weighed (0.05 g) and dissolved in phosphate buffer saline (PBS) and dimethylsulphoxide (DMSO) to prepare extract solution at 1000 µg/ml. The following dilutions of extract were then prepared: 500, 400, 300, 200 and 100 µg/ml.Hep-2 cell line, obtained from Iraqi Center For Cancer and Medical Genetics Researches at the passage level 326 were used in this study. The origin and description of this cell line was mentioned by (13). It was a human laryngeal carcinoma excised from 57 years old man, then transplanted in immune suppressed rat by cortisone. After growth of the tumour in the rat, it was then excised and transplanted as an in vitro tissue culture. It was kept at -169̊Cت (in liquid nitrogen). In preparation to any in vitro assay, the frozen cell line was withdrawn and maintained in RPMI-1640 containing 10% bovine calf serum. When the in vitro cells culture forms a monolayer. These cells were treated with trypsin/ versine mixture in order to pursue subculture process.The percentage of inhibition was calculated according to the following equation: (14) Where : OC: optical density of control wells OT : optical density of test wells From the above calculation, a graph was plotted for the precentage of growth inhibition against each extract concentration. Activity against Hep-2 cell line was determined by the inhibition assay using an ELISA assay. In short, cell cultures in the microtitration plate were exposed to a range of plant extract concentrations during the log phase of growth and the effect determined after recovery time. The following protocol as descriped in (15) was performed the extracts of Nigella sativa leaf callus : a) After trypsinization, cell suspension seed in a micro titration plates at 50000 cells/ml RPMI-1640 growth medium with serum 5% was used for seeding. b) Plates then incubated for 24 hours. c) By using maintenance medium, two-fold serial dilution were prepared starting from .μg/mlت100تwithتendingتμg/mlت1000 d) After incubation for 24 hrs, cells exposed to different extract dilutions. Only 200 μlتofتeachتconcentrationتaddedتforتeachت well (6-replicates for each tested concentration).ت200تμlتofتmaintenanceت medium added to each well of control group.The times of exposure were (24, 48 and 72 hrs). The plates sealed with self adhesive film then returned to the incubator at 37º C.ْ. e) After the end of the exposure period, the medium and the cells decanted off and replaced byت200تμlتofت%0.01تcrystalتvioletت dye. After 20 min. the stain was washed gently with tap water for three times. The plate was left until become dry. The optical density of each well was read by using a micro-ELISA reader at 492 nm transmitting wave length (15 , 16). Statisitical analyses: A one-way analysis of variance was performed to test whether group variance was sifgnificant or not, the comparison between groups were used analyses of variance Least Significant Differences Test (L.S.D.)(16). Inhibition % = [(OC – OT) /OC] x 100 Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity 65 Results Cytotoxic effect of Nigella sativa leaf callus extract (thymol) on Hep-2 cell line: The result of the cytotoxic effect of the extrcat readed using ELISA micro reader with wave length 492 nm indicate the presence of a relation ship between color density of the stain and the number of the viable cells. The result showed presence of significant at level (p≤0.05)ت differencesت betweenت theت concentrations comparied with control started with the concentration (1000, 500, 400, 300, تvalueتinhibitionتwithت(μg/mlت100تandت200 ranged (67.2%, 75.4%, 74%, 75%, 72.2% and 74%) respectively at 48 hr of exposure periods, while there is no significant differences at level (p≤0.05)تbetweenتtheتconcentrationتitselfتasت shown in figure (1). 0 0.05 0.1 0.15 0.2 0.25 0.3 24 hr 48 hr 72 hr control 1000 500 400 300 200 100 Figure (1): Effect of extract concentrations on the growth of Hep-2 cell line during different exposure preiod. The results also showed the best exposure period was 48 hr than the other periods (24 and 72 hr), the inhbition to cell line begins at 48 hr, there is no significant differences at level (p≥0.05)تinتinhibitionتwhenتcomparedتwithتtheت period 72 hr. This means exposure the extract to cell line at 48 hr with lowest concentration showed significant differences. After that result we choose the inhibitory concentration which inhibit the growth of Hep-2 cell line depending on the changes that appears on the optical density and the changes in the color that appears on the plate itself from the stain reaction with the cells(17). So, the concentration 500 µg/ml has inhibitory effect compared to other concentration that also showed a minimum inhibition on the growth of Hep-2 cell line at 48 hr exposure period as shown in table (1), and the inhibition ratio was shown in table (2). Table 1: A comparison optical density of growth inhibition of Hep-2 cell line, by using different concentration of callus extracts of Nigella sativa during three periods of exposure. Type of extract Conc. (µg/ml) Optical density (OD) 24hrs 48hrs 72hrs Callus extract (thymol) 1000 0.071 0.092* 0.05 500 0.060 0.069* 0.05 400 0.063 0.073* 0.05 300 0.068 0.070* 0.05 200 0.067 0.078* 0.05 100 0.064 0.073* 0.05 control 0.115 0.281 0.16 )*):means the presence of siginificant differences atتlevelت(p≤0.05)تbetweenتtheتconcentrations. Table 2: A comparison of growth inhibition percentage of Hep-2 cell line, by using different concentration of callus extracts of Nigella sativa during three periods of exposure. Type of extract Conc. (µg/ml) Inhibition% 24hrs 48hrs 72hrs Callus extract (thymol) 1000 38.2 67.2 68.7 500 47.8 75.4 68.7 400 45.2 74.0 68.7 300 40.8 75.0 68.7 200 41.7 72.2 68.7 100 44.3 74.0 68.7 Discussion Although the quinone thymol has demonstrated significant in vitro and in vivo antineoplastic activities against different tumor cell lines(7,18). In this study , thymol demonstrated different cytotoxicity in vitro toward Hep-2 cell line according to its concentration. This study appeared that the concentration 500 µg/ml affact on the inhibition ratio when compared with the lowest concentrations which show a minimum inhibition compared with the control. This inhibition increased when reacrhed 48 hr of op ti ca l d en si ty extract concentrations µg/ml Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity 66 exopsure and stopped at the 72 hr of exposure. The using of these concentrations (1000, 500, 400, 300, 200 and 100 µg/ml) in this study affect on the growth of Hep-2 cell line with slight differences between them , the OD of Hep-2 cell line ratio was the lowest on the concentration 500 µg/ml compared with other concentrations. So, the extract make an inhibition on the growth of Hep-2 cell line compared the control depending on the concentration that is used and the length of incubation period. The result of this study suggest that thymol inhibited proliferation of tumor cell line by a mechanism that involves cytotoxicity, in fact, it is known that thymoquinone (a quinone from Nigella sativa) inhibited the proliferation of COS 31 (canine osteosarcoma) at concentration 100µM by inducing apoptosis and cell cycle arrest at G1. Non-cancerous cells are relatively resistance to thymoquinone (19) . Nigella sativa and other plants were tested on human hepatoma Hep G2 cell line, the effect were determined on 24 hr of incubation. The greatest inhibitory effects were observed on Nigella sativa plant extract even at low concentration (20) . Refrences 1. Abdullaev, I.F. Cancer chemoprevention and tumoricidal properties of Saffron (Crocus sativus L.). Experimental Biology and Medicine (2002). 227: 20-25. 2. Chakraverty, H.L. Plant Wealth of Iraqi Dictionary of Economic Plants, (1976). vol I. Baghdad p. 387-588. 3. Randhawa, M.A. and Al-Ghamdi, M.S. 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