Comparison of the cytotoxic effects of Juniperus sabina and Zataria multiflora extracts with Taxus baccata extract and Cisplatin on normal and cancer cell lines
M Shokrzadeh1, M Azadbakht2, N Ahangar1, H Naderi1, SS Saeedi Saravi1
1 Department of Toxicology-Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Mazandaran Pharmaceutical Sciences Research Center, Iran
2 Department of Pharmacognosy, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Mazandaran Pharmaceutical Sciences Research Center, Iran
|Date of Submission||15-Jan-2010|
|Date of Acceptance||19-Jan-2010|
|Date of Web Publication||30-Apr-2010|
18th Km of Khazarabad Road, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari
Source of Support: Grant from the research council of Mazandaran University of Medical Sciences, Sari, Iran, Conflict of Interest: None
| Abstract|| |
Isolation and identification of some potent anti-tumor compounds from medicinal plants has motivated researchers to screen different parts of plant species for the determination of anti-tumor effects. In this study, cytotoxic effects and IC 50 of specific concentrations of hydro-alcoholic extracts of fruits of Juniperus sabina and leaves of Zataria multiflora were compared with hydro-alcoholic extract of bark of Taxus baccata and Cisplatin, well-known anticancer compounds, on normal (CHO and rat fibroblast) and cancer (HepG2 and SKOV3) cell lines. The hydro-alcoholic extracts of the plants were prepared by percolation. The cytotoxic effects and IC 50 of the extracts on the cell lines were studied followed by colonogenic assay after 72 h incubation. The results showed that the extract of Juniperus sabina possesses lower IC 50 in comparison with Zataria multiflora extract on all 4 normal and cancer cell lines (P < 0.05); but, IC 50 of the Juniperus sabina extract was significantly higher than the Taxus baccata extract and Cisplatin on all 4 normal and cancer cell lines (P < 0.05). As a result, it is concluded that the extract of J. sabina has almost similar cytotoxicity with the extract of Taxus baccata on cancer cells.
Keywords: Cell line, colonogenic assay, cytotoxicity, Juniperus sabina, Zataria multiflora
|How to cite this article:|
Shokrzadeh M, Azadbakht M, Ahangar N, Naderi H, Saeedi Saravi S S. Comparison of the cytotoxic effects of Juniperus sabina and Zataria multiflora extracts with Taxus baccata extract and Cisplatin on normal and cancer cell lines. Phcog Mag 2010;6:102-5
|How to cite this URL:|
Shokrzadeh M, Azadbakht M, Ahangar N, Naderi H, Saeedi Saravi S S. Comparison of the cytotoxic effects of Juniperus sabina and Zataria multiflora extracts with Taxus baccata extract and Cisplatin on normal and cancer cell lines. Phcog Mag [serial online] 2010 [cited 2021 May 6];6:102-5. Available from: http://www.phcog.com/text.asp?2010/6/22/102/62894
| Introduction|| |
Isolation and identification of some potent anti-tumor compounds, such as colchicine, Vinca alkaloids, and also docetaxol and paclitaxel (taxol), as one of the most consumed natural anticancer compounds have encouraged scientists to study the toxico-pharmacological effects of different parts of plant species against cancer cell lines. ,,,,,,,
It has been previously reported that all parts of the yew plant contain poisonous taxine alkaloids. Taxines save their toxic effects during the year,  with its maximal concentration during winter.  However, previous investigations revealed that different parts of some species of Iranian Juniperus sabina possess cytotoxic effects on some human cancer cell lines.  The potent compound of the species of this plant is podophyllotoxin, but active ingredients of other species are lignan, silicicolin called desoxypodophyllotoxin. Further investigations on the leaves of several genera of Juniperus sabina (Taxus, P., Libocedrus, Podocarpus, Chamacyparis, and Callitris) showed the presence of cytotoxic compounds or tumor necrotizing substances. , Taxol is an intense anti-tumor compound extracted from Taxus species. However, difficulty of obtaining this compound from yew trees has limited its clinical use. 
Zataria multiflora Boiss belongs to the family Laminaceae that geographically grows in Iran, Pakistan and Afqanistan. , This plant (vernacular name of Avishan Shirazi, in Iran) has been traditionally used as an antiseptic, anesthetic and anti-spasmodic drug.  Also, this plant is extensively used as a flavor ingredient in Iranian food. The main constituents of its essential oil are phenolic compounds such as carvacrol and thymol.  In a study, Basti, Misaghi and Khaschabi reported that essential oil of Z. multiflora showed inhibitory effects on Salmonella More Details typhimurium and Staphylococcus aureus in brain heart infusion (BHI) broth medium. 
Cisplatin (cis-dichlorodiammineplatinum-II) gained a widespread use against various malignant tumors in experimental animals , and various human malignancies.  Most of the biological effects of Cisplatin have been well documented , with numerous reports indicating that the cellular DNA could be the primary target in exposure to Cisplatin. , However, the therapeutic efficacy of Cisplatin is limited due to cellular drug resistance  and its side effects, such as delayed nausea, vomiting and nephrotoxicity.  Also, an increased risk of development of secondary malignancies in animals/patients treated with Cisplatin has also been reported. , To develop therapeutic effects and diminish the side effects, new analogs of Cisplatin are produced  and combination therapy of Cisplatin and its analogs has been tried. 
In this investigation, cytotoxic effects and IC 50 of specific concentrations of hydro-alcoholic extracts of fruits of Juniperus sabina and leaves of Zataria multiflora were compared with hydro-alcoholic extract of bark of Taxus baccata and Cisplatin, as well as known herbal and chemical anticancer compounds on normal (CHO and rat fibroblast) and cancer (HepG2 and SKOV3) cell lines.
| Materials and Methods|| |
Fruits of J. Sabina and bark of T. baccata were collected from the northern regions of the Iran (Galugah and Neka in Mazandaran province) in September 2007. Also, leaves of Z. multiflora were collected from downtown of Shiraz (Fars province). The plant specimen was identified by the Department of Pharmacognosy, Sari faculty of Pharmacy, and stored at -20°C.
Extraction and isolation
A measured quantity of 50 g of dried and powdered parts of each plant was chopped and soaked in 75 ml of ethanol (80% v/v) for 24 h and then percolated (5 h, 30 drops/min).  The extracts were separately concentrated by rotary evaporator, dried in oven at 45° C and dissolved in 500 ml of filtered and sterilized water (using 0.22 μm microbiological filters) containing 0.1% ethanol. The specific concentrations of the hydro-alcoholic extracts (5, 25, 50, 100 and 150 μg/ml) were prepared using phosphate buffer (pH=7.4).
CHO (Normal human ovarian cells), normal rat fibroblast, HepG2 (Human hepatocarcinoma) and SKOV3 (Human ovary carcinoma) cell lines were purchased from Pasture Institute (Tehran, Iran).
The completed media were sterilized by 0.22 μ microbiological filters and kept at 4°C before use.
In Colonogenic assay, 50 μl of DMEM/F12 including 500-700 cells were added to 3 wells of 6well/plates for each concentration of the extracts and Cisplatin. Then, they were incubated for 48 h. After incubation, the cell lines were exposed to 50 μl of 0 (phosphate buffer), 5, 25, 50, 100, 150 μg/ml of hydro-alcoholic extracts of Juniperus Sabina , Zataria multiflora and Taxus baccata0, and 50 μl of 0, 2.5, 5, 10, 25 μg/ml of Cisplatin for 2 h; and then washed using sterile normal saline 0.09%.
Then, 4 ml of fresh culture media was added to the wells, and incubated for seven days. After this period, the contents of wells were excluded; the cells were fixed by formalin 9%, and dyed by trypan blue 4% (w/v) for 20 min. Then, trypan blue was excluded, and the six well/plates were washed using sterile normal saline 0.09%. At the end, the dyed colonies were counted by light microscope.
Prism ver.3 Software was used to perform statistical analysis. One-way ANOVA method followed by Tukey test was used to determine the differences among the groups (p0 <0.05).
| Results|| |
The results showed that the extract of Juniperus sabina possesses lower IC 50 in comparison with the Zataria multiflora extract on all 4 normal and cancer cell lines (P<0.05). But, IC 50 of the Juniperus sabina extract was significantly higher than the extract of Taxus baccata and Cisplatin on all 4 normal and cancer cell lines (P<0.05) [Table 1]. The lower IC 50 represent the higher potency of a compound to inhibit the growth of cells and cause toxicity and death of cells.
Comparison of the evaluated IC 50 of the Juniperus Sabina and Zataria multiflora extracts with the Taxus baccata extract and Cisplatin on normal and cancer cell lines are showed at [Figure 1]. However, the lowest and highest IC 50 was related to cisplatin and hydro-alcoholic extract of Zataria multiflora in all cell lines. IC 50 of the compounds on the 4 cell lines increased according to the rank order of cells Cisplatin < Taxus baccata < Juniperus sabina < Zataria multiflora.
On the other hand, the highest and lowest cytotoxicity of Cisplatin was related to HepG2 (IC 50 = 0.87±0.07 μg/ml) and CHO cell lines (IC 50 = 5.5±0.21 μg/ml).
The IC 50 of cisplatin on the 4 cell lines increased according to the rank order of cells CHO > Fibroblast > SKOV3 > HepG2.
On the other hand, the IC 50 of hydro-alcoholic extract of Taxus baccata on the 4 cell lines increased according to the rank order of cells CHO > Fibroblast > HepG2 > SKOV3.
Also, the IC 50 of hydro-alcoholic extract of Juniperus sabina on the 4 cell lines decreased according to the rank order of cells CHO < Fibroblast < HepG2 < SKOV3.
The IC 50 of hydro-alcoholic extract of Zataria multiflora on the 4 cell lines decreased according to the rank order of cells Fibroblast < CHO < HepG2 < SKOV3.
| Discussion|| |
According to the results, IC 50 of the Juniperus sabina and Zataria multiflora extracts, and Cisplatin, as drug control positive compound and Taxus baccata extract, as plant control positive on normal cell lines were higher than that on cancer cell lines. This difference can be resulted from dysfunction of cellular organisms following cancer incidence which cause higher rate of proliferation and increased cellular intake. Also, defensive disorders and effusion insufficiency to escape toxic substances from cells can lead to lower necessity to amounts of cytotoxic compounds to inhibit the growth of cancer cells in comparison with normal cells. ,
Determination of viability percent of the cells treated with the extract of Juniperus sabina and its insignificant differences with IC 50 of the extract of Taxus baccata, as a common natural anticancer product, allows us to conclude that extracts of different parts of Juniperus sabina are good candidates for further studies of activity-monitored fractionation to identify their active components.
| Conclusion|| |
In this study, we have determined the cytotoxic activity of hydroalcoholic extracts of Juniperus Sabina and Zataria multiflora on cancer cell lines and compared their IC 50 with the hydroalcoholic extract of Taxus baccata and Cisplatin. We have observed that Juniperus sabina has insignificant differences with IC 50 of the extract of Taxus baccata on HepG2 and SKOV3 cancer cells. This result presented another novel approach for the treatment ofsome cancers.
| Acknowledgments|| |
This study was supported by a grant from the research council of Mazandaran University of Medical Sciences, Sari, Iran.
| References|| |
|1.||Huang CH, Kingston DG, Magri NF, Samaranayake G, Boettner FE. New taxanes from Taxus brevifolia. J Nat Prod 1986;49:665-9. [PUBMED] |
|2.||Van Uden W, Homan B, Woerdenbag HJ, Pras N, Malingre TM, Wichers HJ, et al. Isolation, purification, and cytotoxicity of 5-methoxypodophyllotoxin: A lignan from a root culture of Linum flavum. J Nat Prod 1992;55:102-10. |
|3.||Prasain JK, Stefanowicz P, Kiyota T, Habeichi F, Konishi Y. Taxines from the needles of Taxus wallichiana. Phytochem 2001;58:1167-70. |
|4.||Jafarian-Dehkordi A, Emami SA, Saeidi M, Sadeghi H. Cytotoxicologic studies of the extracts of iranian Juniperus sabina and Platycladus orientalis on cancer cells. J Res Med Sci 2004;5:205-9. |
|5.||Saeedi Saravi SS, Shokrzadeh M. The study of hepatic and renal disorders in mice which were administered ethyl acetate extract of plant Sambucus ebulus Intraperituenally (IP) and effect of vitamins C and E on prevention of its disorders. Toxicol Lett 2008;180S:S57-8. |
|6.||Shokrzadeh M, Saeedi Saravi SS, Mirzayi M. Cytotoxic effects of ethyl acetate extract of sambucus ebulus compared with etoposide on normal and cancer cell lines. Phcog Mag 2009a;5:316-9. |
|7.||Saeedi Saravi SS, Shokrzadeh M. Histopathological and biochemical disorders following administration of Sambucus ebulus extract on mice and rats and preventive effects of vitamins C and E on renal and hepatic disorders. Phcog Mag 2009b;5:131-5. |
|8.||Shokrzadeh M, Azadbakht M, Ahangar N, Naderi H, Saeedi Saravi SS. Cytotoxic effects of hydroalcoholic extracts of Cucurbita pepo and Solanum nigrum compared with hydroalcoholic extract of Taxus baccata and cisplatin on normal and cancer cell lines. Planta Med 2009;75:1077. |
|9.||Alden CL, Fosnaugh CJ, Smith JB, Mohan R. Japanese yew poisoning of large domestic animals in the Midwest. J Am Vet Med Assoc 1977;170:314-6. [PUBMED] |
|10.||Watt JM, Breye-Brandwijk MG. The medicinal and poisonous plants of Southern and Eastern Africa. Edinburgh: Livingstone; 1962. p. 1019-22. |
|11.||Jafarian A, Emami SA, Saeidi M, Sadeghi H. Cytotoxic effects of the extract of Iranian Taxus baccata and Cupresus horizentalic on cancer cells. Iranian J Pharmaceut Res 2003;2:107-10. |
|12.||Bonfill M, Expσsito O, Onrubia M, Janι A, Cusidσ RM, Palazσn J. Effect of external factors on the production of taxol and other taxanes in cell cultures of Taxus baccata. J Biotechnol 2007;131:S45. |
|13.||Ali MS, Saleem M, Ali Z, Ahmad VU. Chemistry of Zataria multiflora (Lamiaceae). Phytochem 2000;55:933-6. |
|14.||Hosseinzadeh H, Ramezani M, Salmani G. Antinociceptive, anti-inflammatory and acute toxicity effects of Zataria multiflora Boiss. extracts in mice and rats. J Ethnopharmacol 2000;73:379-85. [PUBMED] [FULLTEXT] |
|15.||Shaffiee A, Javidnia K. Composition of essential oil of Zataria multiflora. Planta Med 1997;63:371-2. |
|16.||Misaghi A, Basti AA. Effects of Zataria multiflora Boiss. essential oil and nisin on Bacillus cereus ATCC 11778. Food Control 2007;18:1043-9. |
|17.||Cummings BS, Schnellmann GR. Cisplatin-induced renal cell apoptosis: Caspase 3-Dependent and-independent pathways. J Pharmacol Exp Ther 2002;302:8-17. |
|18.||Prasad SB, Giri A. Antitumor effect of cisplatin against murine ascites Dalton's lymphoma. Indian J Exp Biol 1994;32:57-62. |
|19.||Fokkema E, Groen HJ, Helder MN, de Vries EG, Meijer C. JM216-, JM118-, and cisplatin-induced cytotoxicity in relation to platinum-DNA adduct formation, glutathione levels and p53 status in human tumour cell line with different sensitivities to cisplatin. Biochem Pharmacol 2002;63:1989-96. [PUBMED] [FULLTEXT] |
|20.||Rosenberg B. Fundamental studies with cisplatin. Cancer 1985;55:2303-16. [PUBMED] |
|21.||Pinto AL, Lippard SJ. Binding of the antitumor drug cis-diamminedichloroplatinum (II) (cisplatin) to DNA. Biochim Biophys Acta 1985;780:167-80. [PUBMED] |
|22.||Blasiak J, Gloc E, Wozniak K, Mlynarski W, Stolarska M, Skorski T, et al. Genotoxicity of idarubicin and its modulation by vitamins C and E and amifostine. Chemico Biol Interact 2002;140:1-18. |
|23.||Zamble BD, Lippard SJ. Cisplatin and DNA repair in cancer chemotherapy. Trends Biochem Sci 1995;20:435-9. |
|24.||Timmer-Bosscha H, Mulder NH, de Vries EG. Modulation of cis-diamminedichloroplatinum (II) resistance: A re-view. Br J Cancer 1992;66:227-38. [PUBMED] [FULLTEXT] |
|25.||Krakoff H. Nephrotoxicity of cis-dichlorodiamminep-latinum. Cancer Treat Reprod 1979;63:1523-5. |
|26.||Kempf SR, Ivankovic S. Carcinogenic effect of cisplatin (cis-diamminedichloroplatinum-II, CDDP) in BD IX rats. J Cancer Res Clin Oncol 1986;111:133-6. [PUBMED] |
|27.||Greene MH. Is cisplatin a human carcinogen. J Nat Cancer Inst 1992;84:306-12. [PUBMED] [FULLTEXT] |
|28.||Christian M. The current status of new platinum analogs. Semin Oncol 1992;19:720-33. |
|29.||Treskes M, Van der Vijgh WJF. WR2721 as a modulator of cisplatin-and chemopreventive agents: A molecular approach. Cancer Chemother Pharmacol 1993;33:93-106. |
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