¹ Centre de Biotechnologie de Sfax (Universite de Sfax). Route de Sidi Mansour Km 6, BP 1177, 3018 Sfax, Tunisia;

² Chimie ParisTech (Ecole Nationale Superieure de Chimie de Paris), Laboratoire Charles Friedel, UMR CNRS 7223, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France

Corresponding Author: Mehdi El Arbi, Ph.D., Centre de Biotechnologie de Sfax (Universite de Sfax). Route de Sidi Mansour Km 6, BP 1177, 3018 Sfax, Tunisia; Chimie ParisTech (Ecole Nationale Superieure de Chimie de Paris), Laboratoire Charles Friedel, UMR CNRS 7223, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France. Tel: +216-74-871816; Fax: +216-74-875818; E-mail: mehdi_arbi@yahoo.fr.

Note: Both authors contributed equally to the completion of this work.

	ABSTRACT
	INTRODUCTION
	MATERIAL AND METHODS
	RESULTS
	DISCUSSION
	ACKNOWLEDGMENT
	CONFLICT OF INTEREST
	REFERENCES

	ABSTRACT

The crown gall induced in potato discs by Agrobacterium tumefaciens is becoming largely utilised in screening anti-tumor agents. The present work is showing that beet discs are more adequate for the anti-tumor screening test. In fact, maximal tumor induction was observed on beet discs (87.5%), followed by carrot discs (75%) and potato discs (68.5%). Beet discs present the most sensibility to crown gall disease with a fast expression of symptoms and more visible galls without any staining need. The beet discs bioassay was carried out by using some synthesized organometallics known for their antitumor activity in mammalian cells. We found significant crown gall inhibition (20.7% to 40.55%) of the tested compounds. Overall results supported that beet bioassay might be a potential prescreen system of anti-tumor molecules in mammalian cells.

	INTRODUCTION

   Bioassay methods offer special advantages in establishing the biological purposes (antitumor, antibacterial, antioxidant and phytotoxic properties. etc.). Bioassay is the preliminary step in drug discovery which allow the screening of biological and synthetic bioactive compounds (1). Potato disc assay was shown to be useful for checking known and novel antitumor molecules’ properties. This bioassay is based on Agrobacterium tumefaciens infection on potato disc (2).The validity for the use of such assay is that the tumorogenic mechanism initiated in plant tissues by A. tumefaciens is in many ways similar to that of animals (3). In fact, Kempf, et al., (4) showed that Bartonella henselae, a bacterium causing tumor in human shares a similar pathogenicity strategy, with plant pathogens A. tumefaciens. Several studies are finding numerous and significant areas of similarity among the mechanisms used by bacterial pathogens of plants and humans. These similarities include the use of common toxins, secretion system, adhesion mechanism, invasion and regulation (5).

This method is used during the last 15 years and it appears to be adaptable to the purpose of standardization or quality control of bioactive compounds (6). It has been shown that the inhibition ability of crown gall formation by A. tumefaciens on potato discs and subsequent growth was in good correlation with compounds and extracts, which are statistically much more predictive of in vivo and in vitro anti-leukemic activity (1, 7-9). Several studies have demonstrated that Podophyllin, Taxol, Camptothecin, Vincristine and Vinblastine have shown significant tumor inhibition in the 3PS leukemic mouse assay. These compounds have already been tested for their inhibitory action on tumor formation by potato disc tumor assay (7, 10-13).

A. tumefaciens, is a Gram-negative soil borne bacterium, rod–shaped and virulent that is the causative agent of Crown Gall Disease. Crown Gall is a neoplastic disease in which a mass of tissue bulging from stems and roots of woody and herbaceous plants is produced. The tumor masses could be spongy or hard, with or without a deleterious effect on the plant. The produced tumor is histologically similar to animal or human ones. The process of tumor induction by Ti-plasmid is the result of cell proliferation and blocking of apoptosis like in animal or human cancer cells (14). As a consequence, it was proposed to adopt the crown gall tumor (potato disc) assay as a prescreen for antitumor activity (14-15). Although aseptic technique is required, the methodology is simple and can be performed with minimal technical training. The authors have since used this assay to detect and isolate novel antitumor compounds.

The antitumor potato disc assay was shown to be sensitive for variable chemicals that interfere with cell cycle and have different modes of action (9, 16). This simple test that needs aseptic conditions has allowed the detection and isolation of many anti tumor compounds from plant microbial or biomolecules that were confirmed by in vivo animal tumor inhibition (6).

Considering the continuous demand for antitumor drugs in the area of tumor/cancer inhibition assay, the present study was undertaken to improve the screening system based on potato by the use of other vegetables such as beet, radish and carrot disc bioassay and to evaluate the activity of known antitumor organometallic molecules in the more appropriate vegetable disc system.

	MATERIAL AND METHODS

   Phytopathogenicity test

Phytopathogenicity tests were done using beet, radish, carrot (1, 17-18) and potato (19) disc bioassays. The strain C58 of A. tumefaciens was used for the tumor induction.

Potato, carrot, radish and beet disc bioassay

A. tumefaciens strains were cultured on Luria Bertani (LB) agar medium. A single colony was transferred into LB broth medium and incubated at 30°C for 24 h. Beets (Beta vulgaris L.), carrots (Daucas carota L.), potatoes (Solanum tuberosum L.) and radishes (Raphanus sativus L.) were disinfested by scrubbing under running water with a brush, then immersed in 2% Clorox for 5 min. Potato, carrot, radish and beet discs (5 mm x 8 mm) were made with cork borer and immersed in 2% Clorox for 30 min. Each disc was rinsed thrice in autoclaved distilled water for 15 min. After rinsing, the discs were removed from the distilled water, blotted on sterile paper towels. Sixteen discs were placed on Petri plates containing autoclaved agar medium (2%). Suspensions of A. tumefaciens on LB broth medium were standardized to 10⁷ CFU/ml as determined by an absorbance value of 0.96 ± 0.02 at 600 nm. Each disc was overlaid with 50 μL of bacterial suspension. Petri plates were sealed by parafilm and incubated at room temperature (25-30°C). Ten replications were used and experiment was repeated at least twice.

Carrot discs were checked after 10 days for young galls developing from meristematic tissue around vascular system. Beet discs were checked also after 10 days of tumors development on the entire disc surface. After 21 days, potato discs were stained with Lugol’s solution (10% KI + 5% I₂) and tumors were counted under dissecting microscope (19). Lugol’s reagent stains the starch in the potato tissue a dark blue to dark brown colour, but the tumors produced by A. tumefaciens will not take up the stain, and appear creamy to orange (6).

Chemical compounds

The compounds 1,1-Bis[4-(2-dimethylaminoethoxy)phenyl]-2-phenyl-but-1-ene [1] (20), 1,1-Bis[4-(3-dimethylaminopropoxy)phenyl]-2-phenyl-but-1-ene [2] (21), 1,1-Bis[4-(3-dimethylaminopropoxy)phenyl]-2-ferrocenyl-but-1-ene [4] (22), 1-bis[4-(3-dimethylamoniumpropoxy)phenyl]-2-ferrocenyl-but-1-ene citrate [5] (22), were synthesized as described in the above references and have already been reported for their antiproliferative activity on breast cancer cells. 1, 1-Bis [4-(4-dimethylaminobutoxy) phenyl]-2-phenyl-but-1-ene [3] is a newly synthesized compound (Figure 1).

Chemical experimental Section

THF and diethyl ether were obtained by distillation from sodium/benzophenone. Thin layer chromatography was performed on silica gel 60GF254. Column Chromatography purifications were performed by using silica gel with appropriate eluent. ¹H and ¹³C NMR spectra were recorded on a 300 MHz Bruker spectrometer. Mass spectrometry was performed with a Nermag R 10–10C spectrometer.

1,1-Bis[4-(4-bromobutoxy)phenyl]-2-phenyl-but-1-ene 7

Compound 6 (2.531 g, 8 mmoles), cesium carbonate (5.213 g, 16 mmoles), and potassium carbonate (11.057 g, 80 mmoles) where stirred in 100 ml of acetone, then 1,4-dibromobutane (8.637 g, 4.78 ml, 40 mmoles) was added. The mixture was refluxed overnight, cooled and evaporated. The residue was dissolved in a mixture of dichloromethane and water and decanted. After drying on magnesium sulfate, the solution was evaporated and the residue was chromatographed on silica gel column with a cyclohexane/ethyl acetate 1/1 solution as the eluent to yield product 7 in 86% yield. The product was utilized in the next step without further purification. ¹H NMR (CDCl₃) : δ 0.94 (t, J=7.5 Hz, 3 H, CH₃), 1.79-2.17 (m, 8 H, CH₂-CH₂), 2.50 (q, J=7.5 Hz, 2 H, CH₂), 3.45 (t, J=6.5 Hz, 2 H, CH₂Br), 3.52 (t, J=6.5 Hz, 2 H, CH₂Br), 3.86 (t, J=6.0 Hz, 2 H, CH₂O), 4.02 (t, J=6.0 Hz, 2 H, CH₂O), 6.54 (d, J=8.8 Hz, 2 H, C₆H₄), 6.78 (d, J=8.8 Hz, 2 H, C₆H₄), 6.88 (d, J=8.8 Hz, 2 H, C₆H₄), 7.07-7.22 (m, 7 H, C₆H₅+C₆H₄). ¹³C NMR (CDCl₃) : δ 13.7 (CH₃), 27.9 (CH₂), 28.0 (CH₂), 29.0 (CH₂), 29.5 (CH₂), 29.6 (CH₂), 33.5 (2 CH₂), 66.5 (CH₂), 66.7 (CH₂), 113.2 (2 CH C₆H₄), 113.9 (2 CH C₆H₄), 125.9 (CH C₆H₅), 127.9 (2 CH_arom), 129.7 (2 CH_arom), 130.6 (2 CH_arom), 132.0 (2 CH_arom), 135.9 (C), 136.4 (C), 137.8 (C), 141.0 (C), 142.6 (C), 156.7 (C), 157.6 (C). IR (KBr, ν cm^-1): 3033, 2959, 2928, 2870 (CH, CH₂, CH₃). MS (EI, 70 eV) m/z: 584 [M]^+., 504 [M-HBr]^+..

1, 1-Bis [4-(4-dimethylaminobutoxy) phenyl]-2-phenyl-but-1-ene 3

In a pressure tube, compound 7 was dissolved in 2.0 M dimethylamine solution in methanol. The reaction mixture was heated to 60°C under stirring for 1 day. The mixture was concentrated under reduced pressure. Dichloromethane was added and the solution was washed with a 1 M sodium hydroxide solution, then with water. After decantation, the organic layer was dried on magnesium sulfate and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography (Acetone/Et3N: 10/1) to give pure compound 3 in a 69% yield. ¹H NMR (CDCl₃) : δ 0.92 (t, J=7.4 Hz, 3 H, CH₃), 1.50-1.88 (m, 8 H, 2 CH₂-CH₂), 2.20 (s, 6 H, NMe₂), 2.24 (s, 6 H, NMe₂), 2.27 (t, J=7.4 Hz, 2 H, CH₂N), 2.33 (t, J=7.4 Hz, 2 H, CH₂N), 2.48 (q, J=7.4 Hz, 2 H, CH₂), 3.83 (t, J=6.2 Hz, 2 H, CH₂O), 3.99 (t, J=6.2 Hz, 2 H, CH₂O), 6.52 (d, J=8.8 Hz, 2 H, C₆H₄), 6.75 (d, J=8.8 Hz, 2 H, C₆H₄), 6.86 (d, J=8.8 Hz, 2 H, C₆H₄), 7.05-7.20 (m, 7 H, C₆H₅+C₆H₄). ¹³C NMR (CDCl₃) : δ 14.0 (CH₃), 24.6 (CH₂), 24.7 (CH₂), 27.5 (CH₂), 27.6 (CH₂), 29.4 (CH₂), 45.8 (NMe₂), 45.9 (NMe₂), 59.8 (2 CH₂), 67.7 (CH₂), 68.0 (CH₂), 113.5 (2 CH C₆H₄), 114.3 (2 CH C₆H₄), 126.2 (CH C₆H₅), 128.1 (2 CH_arom), 130.0 (2 CH_arom), 130.9 (2 CH_arom), 132.2 (2 CH_arom), 136.0 (C), 136.5 (C), 138.2 (C), 141.1 (C), 143.0 (C), 157.3 (C), 158.1 (C). IR (KBr, ν cm^-1): 3035, 2945, 2869, 2813, 2762 (CH, CH₂, CH₃).

Minimal inhibitory concentration

All synthesized compounds were tested against preliminary A. tumefaciens using microplate dilution method. Minimal inhibitory concentrations (MICs) of organic or organometallic molecules were determined according the National Committee for Clinical Laboratory Standard (NCCLS, 2002). The test was performed in sterile 96-well microplates. The inhibitory activity of synthesized compounds was properly prepared and transferred to each microplate well in order to obtain a twofold serial dilution of the original sample. The compounds were dissolved in dimethylsulfoxide (DMSO) or distilled water depending on their solubility. Serial twofold dilutions of each sample to be evaluated were made to yield volumes of 100 µl/well with final concentrations ranging from 2.54 × 10^-4 M to 1.98 × 10^-6 M. 100 µL of bacteria suspension with a concentration of 107 CFU/ml were added to each well. Negative control wells contained bacteria only in LB broth medium. After incubation at 30°C for 16 h, the minimal inhibitory concentrations (MICs) were recorded as the lowest concentration of compound in the medium that showed no microbial growth by determining of the optical density (OD) at 620 nm. The OD was measured with a Biohrom microplate reader. Solvent, media and positive growth controls were also run simultaneously. The inhibitory activity of the tested compounds was calculated according to the following formula:

IA (%) = 100 - 100 (OD 620 (x) / OD 620 (i))

where (x) is the microbial culture containing the inhibitor and (i) is the microbial culture without inhibitor (23).

Viability test of A. tumefaciens strains

Successful crown gall formation on a potato, radish, carrot and beet disc surface during an antitumor study is dependent on viable A. tumefaciens. It became necessary to avoid the lethal concentrations against A. tumefaciens (2, 19). Bacterial viability was determined by incubating each drug with 107 CFU/ml of bacterial suspension. At 30 and 60 min of inoculation, 10 μl of each solution was removed and plated on LB plates, and incubated for 24 h. Bacterial growth was evidenced by growth across the plates.

Antitumor Activity

For antitumor activity, the crown gall tumor inhibition assay (beet disc assay) was performed for compounds 2, 4 and 5 using A. tumefaciens (C58). Numbers of tumors per disc were counted and the percentage of tumor inhibition was calculated using the following formula (15).

Percentage inhibition = 100 - Number of tumor with sample / Number of tumor with control × 100.

More than 20% tumor inhibition was considered as significant (8). Ten replications were used for each treatment and experiment was repeated twice.

View larger version : [in a new window] Figure 1. Chemical structure of the tested compounds.

	RESULTS

   Chemical experimental Section

1, 1-Bis[4-(4-dimethylaminobutoxy)phenyl]-2-phenyl-but-1-ene [3] was synthesized starting from biphenol 6 (23). First, compound 6 was converted into the dibrominated compound 7 by action of 1,4-dibromobutane with a cesium carbonate/potassium carbonate mixture as the bases in refluxing dry acetone in an 86% yield. This latter compound was then converted into the diamine compound 3 by action of dimethylamine in methanol at 60°C in a 69% yield (Figure 2).

Phytopathogenicity test

The inoculation test on beet discs contained in Petri dishes gives the highest infection level and the fastest (8 days) followed by carrot, potato and radish discs.

The percentages of tumor induction were 87.5%, 75% and 68.5% for beet, carrot and potato discs, respectively (Figure 3). These percentages make them good candidates for determining the antitumor potentials of screened compounds. In the other hand the weak percentages of tumor induction of radish discs is not in favour of such application.

Moreover, the crown gall formation was observed after 8 days for both beet and carrot discs without any staining need. Meanwhile, the potato discs displayed the galls in 21 days after lugol staining. As a consequence the potato discs have a high risk of contamination and cost more for bioassay tests. The same conclusion was observed for radish discs with a higher risk of contamination which oblige the repetition of the test many times to get results (Figure 4).

These results allow selecting beet discs as the most sensitive to crown gall disease with a fast expression of symptoms, more visible galls without the need of any staining and homogeneous distributed galls on the entire surface of the disc. In fact, enumeration on carrot discs seems to be more difficult since the development of galls is limited in size and in space (around the vascular system) and originated from meristematic tissue taking its colour.

Antibacterial Assay

The tested compounds used in this study are organic or organometallic with different length of the amine chain. Stock solutions of the tested products were prepared in DMSO or water, depending on their solubility. We tested all synthesized compounds against A. tumefaciens to estimate the effect of the amine chain length and the conversion into salts of citric acid on the antimicrobial activities in order to select the more appropriate products for the antitumor activity test.

To evaluate the impact of the amine chain length on the antibacterial activity, organic compounds 1, 2 and 3, having an amino chain of 2, 3 and 4 carbons, respectively, were selected for the test with A. tumefaciens. At a concentration of 254 × 10-6 M A. tumefaciens was sensitive to the three compounds. Within these compounds, the compound with 3 carbons is more potent since the highest inhibitory activity was observed with compound 2 which inhibited bacterial growth at all concentrations (Figure 5).

As shown in Figure 6, all compounds are active against A. tumefaciens. Therefore, the organometallic compounds are more potent than their organic homologues. The highest inhibitory activity was observed with organometallic compound 4 and 5 at concentrations 15.9 × 10-6 M and 7.94 × 10-6 M. Moreover, the transformation to salts of citric acid preserves the same activity of our compounds with a slight increase (Figure 6).

Tumor activity on Beet-Disc Assay

Different levels of crown gall tumor inhibition (20.7% to 40.55%) by the organic or organometallic compounds were observed on the beet discs, depending on the synthesized molecules against A. tumefaciens strain. Doxycycline (positive control) showed 100% tumor inhibition (Figure 7).

Organometallic compounds were able to produce significant tumor inhibition 35.48 % to 40.55 %, while the organic compound produced 20.7 %. The highest tumor inhibition was observed with compound 5 (40.55 %) followed by compound 4 (35.48%). It has been shown that ferrocenyl compounds are more active than their organic analogues against cancer cells (21)  and our results confirmed these results.

Antiproliferative tests on hormone-independent breast cancer cells MDA-MB-231

Antitumor activity was evaluated using the antiproliferative assay as described by Mehdi et al. (22). The compound 4 shows a strong antiproliferative effect on MDA-MB-231 with an IC50 of 0.45 µM. After 6 days of incubation, compound 4 showed 75% inhibition on the cell at low concentration 1 × 10^-6. We have previously noted this effect on Beet-Disc Assay. We found significant crown gall inhibition 35.48% with this compound (considering more than 20% tumor inhibition is significant). As a consequence, the beet disc bioassay could be used effectively for screening different bioactive compounds in terms of antitumor activity (Figure 8).

View larger version : [in a new window] Figure 2. Chemical synthesis of compound 3.

 

View larger version : [in a new window] Figure 3. Percentage of tumor induction in Beet, Radish, Potato and Carrot discs.

 

View larger version : [in a new window] Figure 4. Phytopathogenicity test on different discs: (a) carrot (b) radish (c) beet (d) potato.

 

View larger version : [in a new window] Figure 5. Antibacterial activity of compounds 1, 2 and 3 against A.tumefaciens.

 

View larger version : [in a new window] Figure 6. Antibacterial activity of compounds 2, 4 and 5 against A.tumefaciens.

 

View larger version : [in a new window] Figure 7. Percentage of tumor inhibition by compound 2, 4 and 5 on beet disc.

 

View larger version : [in a new window] Figure 8. Effect of the compound 4 on the growth of hormone-independent breast cancer cells MDA-MB-231.

	DISCUSSION

   Bioassay tests have been playing an important role in the exploration of several clinically useful anti-cancer agents. Potato disc bioassay was based on A. tumefaciens infection on potato disc; it becomes useful for checking antitumor properties of biological and synthetic bioactive compounds. Coker et al. (9) examined several compounds known for their antitumor activity to be tested with the potato disc bioassay. This study was showing that camptothecin, palitaxel, podophyllin, vinblastine and vincristine have a significant inhibitory effect on the crown-gall tumor. As a consequence, the potato disc bioassay could be used effectively for screening different bioactive compounds in terms of antitumor activity. Nowadays several groups used potato disc bioassay for testing the antitumor properties of biological bioactive compounds (1-2, 16, 25-32). This assay has advantages of being rapid, inexpensive, simple and reliable pre-screen for antitumor activity. Meanwhile, potato disc method needs a lugol staining to display the crown gall formation. Many authors adopt other vegetables to test the antitumor activity such as carrot disc bioassay without any staining need (33). It became a necessity to compare several vegetables for selecting the best discs bioassay. In the present study, we found that the beet bioassay seems to be more adequate for the anti-tumor test screening. In fact, beet disc bioassay is more sensitive to crown gall disease with a fast expression of symptoms and more visible galls without any staining need.

Our selected beet disc bioassay allowed a significant crown gall tumor inhibition showing that the organic and organometallic compounds are a potential source of antitumor properties. This result is confirmed by testing compound 4 on MDA-MB-231 breast cancer cell line. Our results with the other compounds are in good agreement with previous findings dealing with significant antitumor activity of organic and organometallic compounds on hormone-dependent and hormone-independent breast cancer cells (21, 22).

	ACKNOWLEDGMENT

   This work was supported by the Ministry of Higher Education, Scientific Research and Technology of Tunisia and the Agence Nationale de la Recherche (Fance).

	CONFLICT OF INTEREST

   The authors declare no conflict of interest.

	REFERENCES

1. Islam MS, Akhtar MM, Parvez MS, Alam MJ, et al. Antitumor and antibacterial activity of a crude methanol leaf extract of Vitex negundo L. Archives of Biological Sciences. 2013; 65: 229-238.
2. Islam MS, Akhtar M, Rahman MM, Rahman MA, et al. Antitumor and phytotoxic activities of leaf methanol extract of Oldenlandia diffusa willd roxb. Global Journal of Pharmacology. 2009; 3: 99-106.
3. Srirama R, Ramesha G, Ravikanth RUS, Ganeshaiah KN. Are plants with anti-cancer activity resistant to crown gall? A test of hypothesis. Current Science.2007; 95, 10- 25.
4. Kempf VAJ, Hitziger N, Riess T, Autenrieth IB. Do plant and human pathogens have a common pathogenicity strategy? Trends in Microbiology. 2002; 10: 269-275.
5. David SG. Plants as models for the study of human pathogenesis. Biotechnology Advances. 2004; 22: 363-382.
6. Jerry LM, Lingling LR. The use of biological assays to evaluate botanicals. Drug Information Journal. 1998; 32: 513-524.
7. Galsky AG, Wilsey JP, Powell RG. Crown gall tumor disc bioassay: a possible aid in the detection of compounds with antitumor activity. Plant Physiology. 1980; 65: 184-185.
8. Ferigni NR, Putnam JE, Anderson B, et al. Modification and evaluation of the potato disc assay and antitumor screening of Euphorbiacae seeds. Journal of Natural Products. 1982; 45: 679-686.
9. Coker PS, Radecke J, Guy C, Camper ND. Potato disc tumor induction assay: a multiple mode of drug action assay. Phytomedicine. 2003; 10: 133-138.
10. Gordaliza M, Castro MA, Garcia-Gravalos MD, Ruiz P,Miguel del Corral JM, et al. Antineoplastic and antiviral activities of podophyllotoxin related lignans. Archiv der Pharmazie. 1994; 327: 175-179.
11. Kahl G. Molecular biology of wound healing: the conditioning phenomenon in Kahl G., Schell JS., ed. Molecular biology of plant tumors. New York Academic Press. 1982; p211-267.
12. Lewis WH, Elvin-Lewis MPF. Medical Botany: Plants Affecting Man's Health in John Wiley & Sons., ed. New York. 1977; p341.
13. Riley MR. Limitations of a random screen: Search for new anticancer drugs in higher plants inSt. Louis, MO., ed. Facts and Comparisons. Economic Botany. 1999; p266.
14. Agrios GN. Plant disease caused by prokaryotes: bacteria and mollicutes. San Diego: Plant Pathology Academic Press.1997; p407-470.
15. McLaughlin JL. Crown gall tumors on potato discs and bine shrimp lethality: Two single bioassays for plant screening and fractionation in: Hostettmann K, ed. Methods in plant Biochemistry. London Academic Press.1991;p1-31.
16. Islam MS, Rahman MM, Rahman MA, Qayum MA, et al. In vitro evaluation of Croton bonplandianum Baill.as potential antitumor properties using Agrobacterium tumefaciens. Journal of Agricultural Technology. 2010; 6: 79-86.
17. Chen FC, Hseu SH, Hung ST, Chen MC, et al. Leaf, stem and crown galls on perennial asters caused by Agrobacterium tumefaciens in Taiwan. Botanical Bulletin of Academia Sinica.1999; 40: 237-242.
18. Aysan Y, Sahin F, Mirik M, Donmez MF, et al. First report of crown gall of apricot (Prunus armeniaca) caused by Agrobacterium tumefaciens in Turkey. Plant Pathology. 2003; 52: 793-793.
19. Hussain A, Zia M, Mirza B. Cytotoxic and antitumor potential of Fagonia cretica L. Turkish Journal of Biology. 2007; 31: 19-24.
20. Isamu S, Yoshiyuki S, Kenya N, Takaaki K, et al. Synthesis and pharmacological evaluation of the novel pseudo-symmetrical tamoxifen derivatives as anti-tumor agents. Biochemical Pharmacology. 2008; 75: 1014-1026.
21. Pascal P, Siden T, Anne V, Michel H, et al. A new series of ferrocifene derivatives, bearing two aminoalkyl chains, with strong antiproliferative effects on breast cancer cells. New Journal of Chemistry. 2011; 35: 2212-2218.
22. Mehdi E, Pascal P, Siden T, Ali R, et al. Evaluation of bactericidal and fungicidal activity of ferrocenyl or phenyl derivatives in the diphenyl butene series. Journal of Organometallic Chemistry. 2011;696: 1038-1048.
23. Hammami R, Zouhir A, Hamida JB, Neffati M, et al. Antimicrobial properties of aqueous extracts from three medicinal plants growing wild in arid regions of Tunisia. Pharmaceutical Biology. 2009; 47: 452-457.
24. Yu DD, Forman BM. Simple and Efficient Production of (Z)-4-Hydroxytamoxifen, a Potent Estrogen Receptor Modulator. Journal of Organometallic Chemistry. 2003, 68: 9489-9491.
25. Das B, Kundu J, Bachar SC, Uddin MA, et al. Antitumor and antibacterial activity of ethylacetate extract of Ludwigia hyssopifolia linn and its active principle piperine. Pakistan Journal of Pharmaceutical Sciences. 2007; 20: 128-131.
26. Noudeh GD, Sharififar F, Noodeh, AD, Hassan M, et al. Antitumor and antibacterial activity of four fractions from Heracleum persicum Desf. and Cinnamomum zeylanicum Blume. Journal of Medicinal Plants Research. 2010; 4: 2176-2180.
27. Bibi G, Haq I, Ullah N, Mannan A, et al. Antitumor, cytotoxic and antioxidant potential of Asterthomsonii extracts. African Journal of Pharmacy and Pharmacology. 2011;5: 252-258.
28. Hosseinzadeh H, Behravan J, Ramezani M, Sarafraz S, et al. Study of antitumor effect of methanolicad aqueous extracts of Allium sativum L. (garlic) cloves using potato disc bioassay. Pharmacologyonline. 2011; 1: 881-888.
29. Mohammad MA, Lutfun N, Datta BK, Bashar KSA, et al. Potential antitumor activity of two Polygonum species. Archives of Biological Sciences. 2011; 63: 465-68.
30. Sarker MAQ, Mondol PC, Alam MJ, Parvez MS, et al. Comparative study on antitumor activity of three pteridophytes ethanol extracts. Journal of Agricultural Technology. 2011; 7: 1661-1671.
31. Jasmina C, Adisa P, Milka M, KasimB. Antioxidative and antitumor properties of in vitro-cultivated broccoli (Brassica oleracea var. italica). Pharmaceutical Biology. 2012; 50: 175-181.
32. Turker AU, KoyluogluH. Biological activities of some endemic plants in Turkey. Romanian Biotechnological Letters. 2012; 17: 6949-6961.
33. Mon M, Maw S, Oo ZK. Screening of Antioxidant, Anti-tumor and Antimicrobial Herbal Drugs/Diets from Some Myanmar Traditional Herbs. International Journal of Bioscience, Biochemistry and Bioinformatics. 2011; 1: 142-147.

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