Synthesis and biological activities of new azacrown ether Schiff bases ...

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Muhammad Ashrama, Ahmed Maslatb* and Shehadeh Mizyedc. aDepartment of .... and different hosts in solution has already been reported (Ashram 2002).
Toxicological & Environmental Chemistry Vol. 91, No. 6, August 2009, 1095–1104

Synthesis and biological activities of new azacrown ether Schiff bases and spectrophotometric studies of their complexation with [60]fullerene Muhammad Ashrama, Ahmed Maslatb* and Shehadeh Mizyedc a Department of Chemistry, Mutah University, Al-Karak, Jordan; bDepartment of Biological Sciences, Yarmouk University, Irbid, Jordan; cDepartment of Chemistry, Yarmouk University, Irbid, Jordan

(Received 29 October 2007; final version received 21 May 2008) A series of novel azacrown ether Schiff bases 1–3 have been synthesized in good yield and in a simple way. Their host–guest interaction with [60]fullerene has been studied in toluene by absorption spectroscopic method. All the complexes are found to be stable with 1:1 stoichiometry. Because of their potential applications in industry, agriculture and medicine, they were investigated for their mutagenic and antimutagenic activities using the spot test and the plate incorporation assay of Ames. Compounds 1, 2 and 3 were found to be nonmutagenic in the Ames test using strains TA 1535, TA100 and TA97a of Salmonella typhimurium. However, using strain TA102 revealed that, although both compounds 1 and 2 were nonmutagenic, compound 2 gave a positive response indicating that it acts as an oxidative mutagen. The structure-activity relationship may throw some light on the biological activity of such series of compounds. Keywords: azacrown, ether Schiff bases; mutation

Introduction Since the discovery and establishment of large scale synthesis of [60]- and [70]fullerenes, a lot of work has been done on these two novel new generation of carbon clusters. Most of the work is directed toward their chemical transformations and isolation of C60 and C70 from fullerite selectively. One of these methods is related to their selective complexation with a wide variety of macrocycle molecules. For example, it has been shown that selective extraction of C60 into a water layer was achieved by using -cyclodextrin (Andersson et al. 1992) and water-soluble calix[8]arene (Williams and Verhoeven 1992), while p-tertbutylcalix[n]arenas (Ikeda et al. 1998) (n ¼ 5, 6, 8), p-tert-butylhomoxacalix[3]arenes (Ikeda et al. 2000) and cyclotriveratrylenecan (Steed et al. 1994) encapsulate C60 preferentially to C70 in solution. However, p-halohomoxacalix[3] arenes (Komatsu 2003) and (thia)calix[4]arene–prophyrin conjucates (Dudicˇ et al. 2004) were found to include C70 over C60 in solution. It has also been shown that dibenzo-24-crown-8 forms an inclusion complex with C60 in preference (Saha et al. 2003) to C70, while azacrown ether (Datta et al. 2004) form a complex with C70 in preference to C60. In the present study, we synthesized three novel azacrown ether Schiff bases 1–3 and investigated their [60]fullerene binding behavior in order to evaluate their potential as

*Corresponding author. Email: [email protected] ISSN 0277–2248 print/ISSN 1029–0486 online ß 2009 Taylor & Francis DOI: 10.1080/02772240802541361 http://www.informaworld.com

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suitable new supramolecular hosts for fullerenes The compounds were further analyzed for some biological activities, namely mutagenicity and antimutagenicity.

Results and discussion It has been shown that macrocyles of large ring size, deep cavity and presence of multiple – vander Waals interaction between the electron-rich aromatic ring(s) and the electronpoor fullerenes are of interest in host–guest chemistry (Shinkai and Ikeda 1999). Therefore, in order to evaluate these factors, several variables were considered in our synthesis such as: (1) introducing electron-withdrawing substituents (nitro group) into the benzene rings as shown for crown ether 2; (2) introducing an additional – interaction and deep cavity, which are represented by the naphthalene units; (3) introducing electronrich hetero-atoms (nitrogen atoms) at the periphery of the macrocycles. For ligand 2, additional benefits can be obtained from the presence of the nitro group as a substituent on the benzene rings. Reduction of the nitro group would provide a potential attachment site for a chromogenic group (Takagi, Nakamura, and Ueno 1977) or modification for coupling with a monoclonal antibody (Moi et al. 1987). As shown in Scheme 1, preparation of macrocycles 1–3 was achieved in two simple steps. Alkylation of salicylaldehyde, 5-nitrosalicylaldehyde or 3-hydroxy-2-naphthaldehyde with an excess of 1,2-dibromoethane (10 equiv.) in the presence of two equivalents of anhydrous K2CO3 in refluxing anhydrous CH3CN afforded, after column chromatographic purification products 4–6 as yellow solid, 66, 40 and 78% yield, respectively. Condensation of aldehydes 4–6 with ethylenediamine in the presence of anhydrous K2CO3 and anhydrous CH3CN at reflux temperature for 24 h afforded Schiff bases 1–3 in 70, 72 and 81% yield, respectively. Figure 1 shows the absorption spectra of the titration of C60 and 3 in toluene; the intensity of the shoulder at 300–340 nm region is increasing with the increasing concentration of 3. Similar spectral changes were observed in the titration experiments with 1 and 2. The increase in the observed absorbance is associated with the formation of donor–acceptor molecular complex between compounds 1–3 and C60 in toluene solution. The stoichiometry of the complex in the concentration range was determined to be 1:1 using the Jobs plot. It is noteworthy that a 1:1 stoichiometry of the complexes between C60 and different hosts in solution has already been reported (Ashram 2002). Formation constants of the resulting 1:1 donor–acceptor complexes were determined by measuring the absorbance at 330–340 nm for a series of solutions with varying concentrations of 1–3 and constant C60 concentration. The following modified form of the

O R1

H

+ H2NCH2CH2NH2

O

R2

K2CO3, CH3CN reflux

R1 N

O

O

R2

N H

Br 4 R1 = R2 = H 5 R1 = NO2, R2 = H 6 R1+R2 = CH=CH-CH=CH

R1

N

R2

N H

1 R1 = R2 = H 2 R1 = NO2, R2 = H 3 R1+R2 = CH=CH-CH=CH

Scheme 1. The synthetic route for the preparation of ligands 1–3.

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Benesi–Hildebrand equation was used to calculate the formation constants (Mizyed et al. 2001): ½C60 o =A ¼ 1="½3K

þ 1=",

ð1Þ

where [C60]o is the initial concentration of C60, K is the formation constant, A is the absorbance change due to Schiff base [3] addition, and " is the change in molar absorbtivity. According to Equation (1), a plot of [C60]o/A against 1/[3] will result in a straight line, shown in Figure 2, from the slope intercept, from which the K and " values can be computed. Also, Figure 2 further supports the 1:1 stoichiometric ratio. All the formation constants evaluated for compounds 1–3 with C60 complexes are listed in Table 1. As expected, the results in Table 1 show that the stability constant of the complex of compound 2 is smaller than that of compound 1, which means that adding the nitro

1.2 1.0

Abs.

0.8 0.6 0.4 0.2 0.0 300

400 l (nm)

Figure 1. (Color online). Absorption spectra of C60 (1.00e4 M) after the addition of 3 in toluene at 25 C.

0.002

[C60]/ΔA

0.0015 0.001 0.0005 0 0.0E+00

2.0E+03

4.0E+03

6.0E+03

8.0E+03

1/[3]

Figure 2. Benesi–Hildebrand plot of data for the C60–3 system in toluene at 25 C.

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M. Ashram et al. Table 1. The formation constant values for C60 complexes with 1–3 in toluene at 25 C. Schiff base 1 2 3

K (M1) 646.6  6.2 114.0  2.5 35.0  2.0

groups to the aromatic rings (compound 2) decreases the stability of the complex. This is due to the electronic effect of the nitro-groups as an electron with drawing groups, which will decrease the electron density on the cavity. On the other hand, adding extra aromatic rings does not enhance the stability of the complex. This could be due to steric effect, which rules out the effect of naphthyl groups as an electron-rich extra binding site. A similar result was seen with some corannulene complexes with C60 (Mizyed et al. 2001). It was suggested that the naphthyl groups experience an ‘‘edge-to-face’’ interaction with C60, in contrast to the more extensive ‘‘face-to-face’’ – stacking interactions possible with the smaller phenyl groups.

Biological activity of the synthesized compounds The three derivatives in this study have an interesting structure which allowed us to investigate some of their biological effects.

Mutagenic activity The spot test In the spot test, most compounds can be applied directly on the agar surface. As the test compound diffuses out from the central spot, a range of concentrations are tested simultaneously (Maron and Ames 1983). This test is a qualitative one used to determine the dose range of toxicity and/or mutagenicity that may be exhibited by the tested compounds. Tables 2 and 3 summarize the results of this test for compounds 1–3; þ ¼ 20–100; þþ ¼ 100–200; þþþ ¼ 200–500; þþþþ ¼ 4500 and  ¼ 520 revertants plate1. The initial assessment of the genotoxic potential of 1–3 in Ames Salmonella spot test revealed that in the absence and presence of metabolic activation, although compounds 1 and 3 did not show any mutagenic activity in strain TA102, compound 2 induced 57, 73 and 97 hisþ revertants in such a strain at concentrations 10, 20 and 40 mg/plate, respectively. This result may indicate that compound 2 has at least a weak oxidative mutagenic activity. Also, it means that S9mix did not lead to any increase in the mutagenic activity of that compound. However, all tested compounds did not induce more than 20 hisþ revertants in the other strains, namely, TA1535, TA 100 and TA 97a. It should be clarified that a typical positive mutagenic activity of a chemical compound in the spot test is indicated by clustering of hisþ revertants with or without the presence of an inhibition zone. Such a result could not be observed for the three compounds, suggesting that they are most probably nonmutagenic. The use of S9mix did not lead to conversion of the tested compounds into mutagenic ones. It is worth mentioning that the presence of 20-–00 revertant colonies over the control values in the spot test is of no value concerning any decision about the mutagenicity of

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Toxicological & Environmental Chemistry Table 2. Activity of compounds 1 and 2 in the Ames Salmonella spot test. TA1535

TA97a

TA102

TA100

Concentration 1 2 1 2 1 2 1 2 (mg plate1) S9/þS9 S9/þS9 S9/þS9 S9/þS9 S9/þS9 S9/þS9 S9/þS9 S9/þS9 5 10 20 40 SA NPD NQNO

/ / / / þþ þ N.T. N.T.

/ / / / þþ þ N.T. N.T.

/ / / / N.T. þþ þ N.T.

/ / / / N.T. þþ þ N.T.

/ / / / N.T. N.T. þþþ þ

/ /þ /þ /þ N.T. N.T. þþþ þ

/ / / / þþþ þ N.T. N.T.

/ / / / þþþ þ N.T. N.T.

Note: N.T.: Not tested, since they are not diagnostic mutagens for the specified strains.

Table 3. Activity of compound 3 in the Ames Salmonella spot test. TA1535 Concentration (mg plate1) 5 10 20 40 SA NPD NQNO

TA97a

TA102

TA100

S9

þS9

S9

þS9

S9

þS9

S9

þS9

    þþ þ N.T. N.T.

    þþ þ N.T. N.T.

    N.T. þþ þ N.T.

    N.T. þþ þ N.T.

    N.T. N.T. þþþ þ

    N.T. N.T. þþþ þ

    þþþ þ N.T. N.T.

    þþþ þ N.T. N.T.

Note: N.T.: Not tested, since they are not diagnostic mutagens for the specified strains.

a chemical compound. It should be clear that the sensitivity of the spot test is less than the standard plate incorporation assay. Accordingly, we decided to further analyze the ability of the three compounds to induce revertants in the four Salmonella strains using the standard plate incorporation and the pre-incubation assays. The standard plate incorporation assay The mutagenicity assays were carried out in the presence and absence of S9 mixture. Positive result are indicated by more than a two-fold increase in the number of revertants over the number of spontaneous revertants (Maron and Ames 1983; Ono, Norimatsu, and Yoshimura 1994). The three derivatives showed no mutagenic activity in the three Salmonella strains TA1535, TA 100 and TA 97a without metabolic activation. Bacterial background lawn examination revealed no toxicity of the three compounds in the tested dose range. No dose-response increase could be detected. The response was negative, therefore, the compounds were retested using the pre-incubation assay procedure with S9mix. The use of the S9mix did not convert the tested compounds into mutagenic ones. The number of revertants did not reach double the number of spontaneous revertants using

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solvent control; they were considered to be nonmutagenic in the three previously mentioned Salmonella strains and so they can be classified as causing neither base-pair substitutions in TA1535 and TA100, nor frameshift mutations in TA97a. However, results detected with strain TA102 indicated that, while compounds 1 and 3 were inactive as oxidative mutagens, compound 2 showed a typical dose-response curve for a mutagenic compound. This result is presented in Figure 3. The peak of revertant colonies was obtained at a concentration of 165 mg plate1. The structure-activity relationship may throw some light on the obtained results.

Antimutagenicity assay The highest used concentration 660 mg plate1 for the compounds 1–3 did not inhibit the growth of bacterial cells. The results are presented in Table 4. In general, the effects of 1–3 on the standard mutagens (SA (sodium azide), NQNO (nitroquinoline-N-oxide) and H2O2) were similar. The three compounds showed no mutagenic repression activity against hydrogen peroxide using TA102, NQNO using TA97a, and SA using TA100. This implies

Number of His + revertants/plate

800 700 600 500 400 300 200 100 0 0

50

100 150 200 250 300 350 400 450 500 550 600 650 700 Chemical conc. (μg plate−1)

Figure 3. Dose-response curve for compound 2 using TA102 strain.

Table 4. Antimutagenicity test for compounds 1–3 on bacterial strains TA102, TA100 and TA97a. TA102 Strain Compound 1 2 3 DMSO SA NQNO NPD

TA100

TA97a

þS9

S9

þS9

S9

þS9

S9

– –

– –

– –

– –

– –

– –

120 N.T. – N.T.

105 N.T. – N.T.

22 – N.T. N.T.

35 – N.T. N.T.

20 N.T. N.T. –

23 N.T. N.T. –

Note: N.T.: Not tested, since they are not diagnostic mutagens for the specified strains.

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that they could not act as desmutagens. Accordingly, we can say that the tested compounds were unable to: (1) inhibit the metabolic activation of H2O2, nitroo-phenylenediamine (NPD), or SA in vitro, (2) deactivate the above mutagens themselves, or (3) act on DNA repair systems.

Experimental General: NMR spectra were recorded on a Bruker instrument at 200 MHz for 1H NMR and 50.33 MHz for 13C NMR. Chemical shifts are reported relative to TMS as an internal standard. Mass spectra were determined by VG7070E spectrometer with uncertainty of 1. Chromatographic separations were performed on silica gel columns (60–120 mesh, CDH). Unless otherwise noted, all reactions were carried out under dry nitrogen. o-(2-bromoethoxy)benzaldehyde (4), prepared according to literature conditions (Ashram 2002).

Synthesis of 5-nitro-o-(2-bromoethoxy)benzaldehyde (5) and 3-(2-bromoethoxy)2-naphthaldehyde (6) They were prepared according to general literature conditions (Ikeda et al. 1998) as 4 to give, after column chromatographic purification using ethyl acetate: hexane (1:4); the following: 5-Nitro-o-(2-bromoethoxy)benzaldehyde (5). Pale yellow solid in 40% yield, m.p. 75–76 C, H NMR H ¼ 3.80 (t, J ¼ 6 Hz, 2H), 4.59 (t, J ¼ 6 Hz, 2H), 7.11 (d, t, J ¼ 6 Hz, 1H), 8.46 (d, J ¼ 7 Hz, 1H), 8.75 (s, 1H), 10.52 (s, 1H); 13C NMR C ¼ 28.0, 69.1, 112.4, 124.5, 130.1, 164.0, 187.4, m/z ¼ 275.4 (Mþ, Br81), 273.4 (Mþ, Br79). 1

3-(2-Bromoethoxy)-2-naphthaldehyde (6). Yellow solid in 78% yield, m.p. 81–82 C, H NMR H ¼ 3.73 (t, J ¼ 6 Hz, 2H), 4.45 (t, J ¼ 6 Hz, 2H), 7.11 (s, 1H), 7.37 (t, J ¼ 7 Hz, 1H), 7.52 (t, J ¼ 6 Hz, 1H), 7.69 (d, J ¼ 9 Hz, 1H), 7.85 (d, J ¼ 9 Hz, 1H), 8.34 (s, 1H); 13 C NMR C ¼ 28.9, 68.1, 107.4, 125.1, 125.6, 126.7, 128.1, 129.4, 130.0, 130.7,137.3, 156.1, 190.0, m/z ¼ 280.4 (Mþ, Br81), 278.4(Mþ, Br79). 1

General procedure for synthesis of azacrown ether Schiff bases 1–3 In a 250 mL three-necked flask equipped with a magnetic stirrer bar and a reflux condenser and a gas line to maintain a nitrogen atmosphere, anhydrous K2CO3 (0.11 g, 3.6 mmol) was suspended in anhydrous CH3CN (100 mL). To this well-stirred solution at reflux temperature was added, simultaneously, drop wise over a period of 12 h, a solution of aldehyde (1.8 mmol) in dry CH3CN (50 mL) and a solution of ethylenediamine (0.11 g, 1.8 mmol) in dry CH3CN (50 mL). The reaction mixture was further refluxed with stirring overnight. The reaction mixture was filtered and the solvent was evaporated. The residue was washed with ethyl acetate to give pure products of the following: Azacrown ether Schiff base (1). Pale yellow solid (0.25 g, 70%), m.p. 78.5–80 C, 1H NMR H ¼ 2.36 (s, 2H), 2.63 (br, 2H), 2.99 (br, 2H), 3.28 (br, 8H), 3.89 (br, 2H), 4.32 (br, 2H), 4.51 (s, 2H), 6.98–7.22 (m, 6H), 7.51 (d, J ¼ 5 Hz, 2H); 13C NMR C ¼ 44.1, 56.0, 56.1, 72.1, 78.7, 121.0, 123.9, 126.0, 128.8, 133.7, 158.3, m/z (M  2) ¼ 189.

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Azacrown ether Schiff base (2). Pale red solid (0.3 g, 72%), m.p. 138–140 C, 1H NMR H ¼ 2.38 (br, 2H), 2.64–2.75 (m, 2H), 2.95–3.08 (m, 2H), 3.20–3.38 (m, 8H), 3.90–4.02 (m, 2H), 4.40–4.50 (m, 2H), 4.60 (s, 2H), 7.10 (d, J ¼ 8 Hz, 2H), 8.08 (dd, J ¼ 2 Hz, 8 Hz, 2H), 8.62 (d, J ¼ 2 Hz, 2H); 13C NMR C ¼ 44.6, 55.1, 56.5, 72.9, 122.0, 123.4, 124.7, 136.0, 143.9, 164.0, m/z (M  2) ¼ 234. Azacrown ether Schiff base (3). Pale yellow solid (0.35 g, 81%), m.p. 71–72 C, 1H NMR H ¼ 2.43 (br, 2H), 2.64–2.69 (m, 2H), 2.96–3.04 (m, 2H), 3.20–3.37 (m, 8H), 3.84–3.92 (m, 2H), 4.39–4.44 (m, 2H), 4.64 (s, 2H), 7.34–7.41 (m, 4H), 7.43 (s, 2H), 7.70 (d, J ¼ 9 Hz, 2H), 7.80 (d, J ¼ 9 Hz, 2H), 8.02 (s, 2H); 13C NMR C ¼ 44.3, 56.0, 56.5, 73.1, 78.9, 117.5, 124.9, 125.6, 126.3, 126.8, 128.1, 130.5, 133.8, 134.5, 156.7, m/z (M2) ¼ 239.5.

Complexation measurements C60 (99.5%) was purchased from Sigma-Aldrich. All UV–Vis spectra were recorded at 25 C on a Unicam UV2 UV–Vis spectrophotometer. The solvent was toluene (HPLC grade, Scharlau, assay 99.6%) purchased from Charlie and used without any further purifications. To obtain the association constant K corresponding to complex formation, changes in the absorbance A as a function of the concentration of Schiff bases were determined. An increment of Schiff base solutions were added to the C60 solution, then the absorbance was recorded after each addition. The same C60 solution is used as a working solution and as a solvent to rule out the dilution effect. To calculate K values, the slopes and the intercepts obtained from linear regression analyses of the Benesi–Hildebrand plots were used. The uncertainties in K values were calculated from the uncertainties in the slopes and intercepts of these plots. We found that the formation constants listed in Table 1 are low in magnitude compared to usual host–guest complexes of [60]fullerene. This may be due to the use of toluene as a solvent which also complexes with [60]fullerene and thus solubilizes it. So, there is competition between the salvation and the complexation of the synthesized hosts with [60]fullerene (Bhattacharya, Banerjee, and Mukherjee 2003).

Biological studies Chemicals The test compounds were synthesized and characterized as previously described under the experimental part. For the preparation of bacterial growth media, the Oxoid nutrient broth no. 2 (Oxoid, Basingstoke, UK), the Difco Bacto nutrient broth (Difco, Detroit, MI, USA), and agar powder (Gainland Chemical Company, Deeside, UK) were used. Dextrose anhydrous extrapure (Fine-Chem Ltd. Biosar, India), and -naphthoflavone were purchased from Aldrich Chemical Company (Milweukee, WI, USA). NADP and glucose-6-phosphate were purchased from Boehringer Mannheim, GmbH, (Mannheim, Germany). Phenobarbital was purchased from BDH Chemicals Ltd. (Poole, England). NPD, D (þ) Biotin and dimethyl sulfoxide (DMSO) were obtained from Janssen Chimica, (Beerse, Belgium). Hydrogen peroxide (H2O2) was purchased from CBH Lab Chemicals (Nottingham, UK); ampicillin from NenTech Ltd. (UK); and L-histidine, tetracycline and SA were from Sigma Chemical Company (St. Louis, MO, USA). All other chemicals were of analytical grade.

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Bacterial strains The Salmonella typhimurium strains TA97a, TA100, TA102 and TA1535 used in this study were kindly supplied by Prof. B.N. Ames (University of California, Department of Biochemistry). The bacterial strains were maintained on master plates as described by Maron and Ames (1983). The Salmonella mutagenicity assay The spot test (with and without S9mix), and the standard plate incorporation assay (without S9mix) were carried out as described by Maron and Ames (1983). The rat-liver S9 fraction was prepared as described by Ono, Norimatsu and Yoshimura (1994). Young male Sprague–Dawley rats, weighing approximately 200 g were supplied by Yarmouk University, Animal House Unit (Jordan), and were used after induction with phenobarbital and -naphthoflavone. The S9mix (50 mL) contained 5 mL of induced rat liver S9. This corresponds to a protein concentration of approximately 40 mg mL1 (Maron and Ames 1983). For the spot test, four concentrations of the tested chemicals were used, while for the standard plate incorporation assay, at least 10 concentrations were used. All concentrations were spaced at about one-third intervals. The highest concentration was restricted by the solubility of the tested nucleoside derivatives in DMSO. Triplicate plates were made for each dose, and each experiment was repeated independently at least twice on separate days. The antimutagenicity assay The antimutagenic activity of 1–3 was analyzed using a modified pre-incubation assay (Gichner et al. 1994). The principle of the test was to incubate the tested compound with the authentic or diagnostic mutagen for 30 min at 37 C in a shaker incubator at 150 rpm. Then the bacterial strain was added and incubated for a further 30 min. For each test chemical, six doses starting from 660 mg plate1 were tested, separated by about one-third intervals or a multiple of that. In the present investigation, NaN3: 4 mg plate1, NQNO: 8 mg plate1 and H2O2: 20 mL plate1 were used with strains TA100, TA97a and TA102, respectively. Triplicate plates were poured for each dose, and each experiment was repeated at least twice on separate days. In this test, three types of control were made (negative, positive and chemical). The negative control contained the buffer with the bacterial suspension only. The positive control contained the buffer, the bacterial suspension, and the mutagen. The chemical control contained the buffer, the bacterial suspension, and the tested chemical at its highest nontoxic concentration.

Acknowledgments This work is financially supported by Yarmouk University (Project No. 24/2004) and Mu’tah University. The financial support is greatly acknowledged.

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