Bioactivities of Rubidium Chloride-Poly (4-Vinylpyridine)

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Bioactivities of Rubidium Chloride-Poly (4-Vinylpyridine) Composite Fabricated Via Sol Gel Technique Rehana Rashid1, Sadullah Mir1, Majid Hussain1, Atiya Zahra1, Ghulam Murtaza2,*, Ehsan ul Haq4, Saqib Ali3 and Abida Kalsoom Khan1,* 1

Department of Chemistry, 2Department of Pharmacy COMSATS Institute of Information Technology, Abbottabad, Pakistan; 3Department of Chemistry, 4Department of Pharmacy Quaid-i-Azam University, Islamabad, Pakistan Abstract: Objective: The aim of this study was to fabricate the composite of poly(4-vinylpyridine) (P4VP) with rubidium metal salt to improve polymeric properties including thermal stability as well as the pathogen and enzyme inhibition activity. Methods: The composites were synthesized via sol gel technique. The resulting product was studied Ghulam Murtaza through various analytical techniques including fourier transform infrared spectroscopy, x-ray diffractometry (XRD), thermal gravimetric analysis (TGA), differential thermal analysis (DTA) and differential scanning calorimetry (DSC). Bioactivity of the rubidium chloride/polymer matrix composite (RbCl-PMC) was assessed by performing different bioassays like antioxidant activity (DPPH radical scavenging activity), anti-microbial activity (antifungal and antibacterial), enzyme inhibition activity against protein kinase and -amylase enzymes and cytotoxicity by Brine Shrimp method. Results: The XRD data depicts that the amorphous nature of polymer is reduced by incorporating rubidium chloride and the resulting composite is well crystalline in nature. Grain size, strain, dislocation line density, molecular packing in crystal lattice (face-centered cubic), unit cell structure, Bravia’s lattice parameters, and the volume of the unit cell were determined from XRD data, while, thermal degradation pattern illustrates that thermal stability of the synthesized composite enhanced as compared to P4VP. Moreover, P4VP-rubidium chloride composite exhibited good antioxidant, antibacterial, and cytotoxic activities. Conclusion: P4VP-rubidium chloride composite is found to be an active pharmaceutical.

Keywords: Poly(4-vinylpyridine) composite, rubidium chloride, FTIR, XRD, thermal studies, biocompatible behavior of RbCl-PMC. Received: December 24, 2015

Revised: March 14, 2016

INTRODUCTION Polymer metal composites (PMCs) are one of the most important, smart and light-weight materials among composite class of materials. The intermolecular linkages between components in composite materials or other complexes play an important role in controlling the physical properties of the system. Hybrid materials of inorganic polymers have various applications such as optical glasses, laser systems, fluorescent applications and photonic crystals [1]. Poly(4-vinylpyridine) (P4VP) is an interesting polymer having pyridine and vinyl groups. Pyridine is a strong ligand that is able to make coordination bonds due to its good electron donor capability. Polymers such as P4VP having the properties of sensitivity to temperature, pH and electrical *Address correspondence to this author at the Department of Chemistry, Department of Pharmacy COMSATS Institute of Information Technology, Abbottabad, Pakistan; E-mails: [email protected], Ghulam [email protected]

1573-4129/16 $58.00+.00

Accepted: April 4, 2016

fields have suggested their use in biotechnological and biomedical fields, e.g., in enzyme immobilization studies [2-5]. Composites of polymer and metal salts, in particular, a salt having polyvalent metal ion such as rubidium chloride, have properties similar to both the inorganic components of the metal and organic components of the polymer [6]. In polymer matrix composites, polymers have been used as matrix. Thin fibers of polymer or metal have been incorporated into the matrix. Organic-inorganic hybrid composites have interesting properties because they have characteristics of both organic polymer matrix as well as inorganic reinforcement. Different kinds of structures can be incorporated into these hybrid materials via physical mixing, blending or chemical linkage to get desirable properties of the product. Properties of the polymer matrix and reinforcement are abruptly changed by mixing both of them [7]. This work is a continuation of our previously conducted study [8], which involved the synthesis of composites of poly(4-vinylpyridine) through sol-gel method. Different rare © 2016 Bentham Science Publishers

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earth inorganic metal salts, including cerium chloride, lanthanum chloride, samarium chloride, and titanium chloride were incorporated into the polymer for enhancing thermal stability of the composites. Thermal degradation and the XRD data proved that thermal stability of the synthesized composites was better than that of pure P4VP. For the synthesis of composites, various physical, chemical, biological and hybrid methods have been reported. Solgel approach, a chemical method, is a liquid phase technique for thesynthesis of composites either using a chemical solution or colloidal particles [1]. The aim of this study was to fabricate the composite of poly(4-vinylpyridine) (P4VP) with rubidium salt, a model salt having polyvalent metal ion, to improve the polymeric properties including thermal stability as well as the pathogen and enzyme inhibition activity. EXPERIMENTAL Materials 4-Vinyl pyridine (4VP), rubidium chloride (RbCl), Benzophenone (C6H5)2CO, ammonium molybdate ((NH4)6Mo7O24.4H2O), sodium dihydrogen phosphate (NaH2PO4), sulphuric acid (H2SO4), sodium dihydrogen Phosphate (anhydrous) (NaH2PO4), trichloroacetic acid, sodium hydrogen phosphate dihydrate (Na2HPO4.2H2O), ferric chloride (FeCl3), potassium ferricyanide (K3Fe(CN)6), ascorbic acid (C6H8O6), HCl, methanol, Iodine (I2), Potassium Iodide (KI), and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Aldrich through commercial sources. -Amylase enzyme was purchased from Unichem Laboratories, India. Acarbose (Glucobay® 100) was obtained from Bayer, Pakistan. Preparation of Poly (4-Vinylpyridine) Composite of Rubidium Chloride Poly(4-vinyl pyridine) was synthesized through reflux of 4-vinyl pyridine monomer using methanol as solvent and benzoyl peroxide as an initiator. The resulting mixture was stirred at STP for 24 hours until thick jelly was obtained. The resulting thick jelly of P4VP polymer was washed with excess of the extra-dried methanol solvent to get rid of any impurities. Then, 2 g of P4VP was dissolved in about 80 ml of methanol, while, 0.7 g of RbCl (molecular weight = 120.92 g/mol) was dissolved in about 60 ml volume of methanol and stirred for 40 minutes to obtain a clear solution. The salt solution was added drop-wise into polymer solution with continuous gentle stirring for 12 hours. The resultant composite was rotary evaporated, dried, powdered, collected and stored in desiccator under inert atmosphere. FTIR, TGA and XRD Studies The composite of P4VP with rubidium was characterized by FTIR, TGA and XRD techniques. FTIR spectrum was recorded through Thermo Scientific Nicolet 6700 spectrometer in the region of 400-4000 cm-1 using KBr pellets. Thermogravimetric analysis (TGA) was done to know about the degradation behavior and thermal stability of the synthesized composite. Moreover, TGA was done using “Shimadzu DTG-60H Differential Thermal Gravimetric Analyzer in-

Rashid et al.

strument. Powder X-ray diffraction study was also accomplished for P4VP and its composite with rubidium salt using “PANalytical "X"pertPRO” instrument. Antioxidant Assays For the determination of antioxidant activity of different samples, there are various assay techniques. Out of which, total antioxidant capacity assay, reducing power assay, and DPPH antioxidant capacity assay were performed. Total antioxidant capacity assay was performed to determine total antioxidant (TAO) ability of the samples through phosphomolybdenum method [9]. Stock solution of ascorbic acid was prepared by dissolving 4 mg ascorbic acid in 1 ml of DMSO. 0.1 ml of test sample was mixed with 1 ml of reagent solution containing ammonium-molybdate and NaH2PO4. This mixture was incubated at 95ºC for 90 minutes. After cooling up to room temperature, absorbance was taken at 695 nm through microplate reader. Ascorbic acid was used as an equivalent to determine the antioxidant capacity of the samples. Reducing Power Assay Reducing power assay of metal composite was determined by using the previously reported procedure [9]. 4 mg/ml solution of ascorbic acid was used as a standard while DMSO was used as blank solution. Subsequently, 10 l of each test sample was taken in separate eppendorf tube. Then, 500 l of 1% potassium ferricyanide was added to each sample and these reaction mixtures were incubated at 50 ºC for about 20 min. After incubation, 500 l trichloroacetic acid was added to these reaction mixtures separately. Reaction mixtures were centrifuged at 3000 rpm for 10 min. After this, 100 l layer of each centrifuged reaction mixture was separated and poured in respective 96 well-plate. Then 0.1% of ferric chloride was added to each well. At last, 20 l of distilled water was added. Each sample was analyzed on microplate at 630 nm. DPPH Free Radical Scavenging Assay A free radical scavenging assay using 2,2-diphenyl-1picrylhydrazyl (DPPH) is the most adaptable method to determine the antioxidant activity of the samples [10]. Then, 20 l of each test sample was taken in 96 well microplate and added 180 l of DPPH reagent in each sample. These sample plates were incubated at 37°C for 1 hour and then incubation absorbance was measured at 517 nm through microplate reader. The experiment was accomplished three times on each sample and final percentage scavenging was calculated by using following formula: Inhibition (%) = (Ac-As/Ac) 100 Where, As = absorbance of the sample while Ac = the absorbance of the control [9]. Amylase Inhibition Assay This assay was performed to calculate antidiabetic activity of P4VP and its composite with rubidium chloride. For this study, 0.5 mg of -amylase (28 U/mg) was dissolved in 1 ml of phosphate buffer pH 6.8. This stock solution was further diluted to 0.12 U/ml. Stock solutions of samples were

Bioactivities of Rubidium Chloride-Poly (4-Vinylpyridine) Composite

prepared by dissolving 4 mg/ml, iodine reagent containing 5 mM iodine, starch 2 g/L, 5 mM KI, and Acarbose 8.64 mg/ml in DMSO which were used as solvent. Then, 15 μl of phosphate buffer pH 6.8 was mixed with 25 μl of -amylase enzyme, followed by the addition of 10 μl (100 μg/ml final concentration) and 40 μl starch. This reaction mixture was incubated for 30 min at 50 °C. After incubation, the reaction mixture was cooled down to room temperature and then 20 μl of 1 M HCl was added to stop further reaction. At last, 90 μl of iodine solution was added. Addition of iodine imparted colour change in the reaction mixture where starch was used due to inhibition of amylase enzyme. A blank experiment was performed to compare the results of the samples. In blank experiment, buffer solution, starch and DMSO were run as negative control, while for positive control, a solution containing only DMSO and acarbose was used. Results were calculated as percentage -amylase inhibition/mg. Blue colour of the reaction mixture indicated the activity. Maximum blue colour appeared in the blank experiment because it contained starch and enzyme only [11]. Antifungal Assay Disc diffusion method was used to determine the antifungal activity of samples [12]. Standard terbinafine solution was prepared by dissolving 4 mg terbinafine in 1 ml of DMSO and about 20 g/disc concentration of this solution was used. Sample solution was prepared by dissolving 20 mg of sample in 1 ml of DMSO. Sabouraud dextrose agar (SDA) was used to grow fungal cultures. To prepare fungal cultures, 62 g of SDA was dissolved in 1 L of distilled water. The pH of culture was maintained at 6.5 by using phosphate buffer. Then, 25 ml of the media was poured to each petri-plate which was autoclaved earlier. The mixture was then left to solidify for few minutes. After that, already prepared fungal solution of each fungus was moistened on the media surface of each plate. Then, 5 l of each sample solution was transferred to filter paper disc and then the disc was placed in the respective labeled petri-plates. These plates were incubated at 28°C for 24 hours. After incubation, the plates were cooled down to room temperature and inhibition zone was measured by Vernier caliper. Antibacterial Assay Disc diffusion method was used to determine the antibacterial activity of each sample [12]. About 4 mg of standard drug, cefotaxime, was dissolved in 1 mL of DMSO. It was used as positive control while DMSO was used as a negative control. Stock solution of each sample was prepared by dissolving 20 mg of each sample in 1 ml of DMSO. The nutrient agar media was transferred into already sterilized petriplates, and left for few minutes to solidify and then moistened it with an already prepared bacterium inoculum. Each plate was then properly labelled. About 2.5 l of each sample solution was poured on filter paper and placed in respective petri-plates. Each plate contained positive control and incubated them at 37°C for 24 hours. After incubation, the zone of inhibition was measured with vernier caliper. This experiment was also conducted in triplicate.

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Protein Kinase Inhibition Assay Stock solutions of samples were prepared by dissolving 20 mg sample in 1 ml DMSO. Surfactin stock solution was prepared at the concentration of 4 mg/ml DMSO. The final concentrations of samples and standard on the disc were 100 and 20 g/disc, respectively. Sterile loopful Streptomyces 85E strain spores were inoculated into previously sterile TSB and then incubated for 24 hours at 37oC. The protein kinase inhibition assay was done by detecting the formation of hyphae in purified isolates of Streptomyces 85E strain [13]. Bacterial colonies were permitted to grow by spreading mycelia fragments of Streptomyces on sterile agar plates containing mineral ISP4 medium. About 5 l of each sample having concentration of 20 mg/ml in DMSO was loaded on already sterilized 6 mm filter paper discs. The soaked filter paper discs with a final concentration of 100 g/disc were applied directly on the surface of the agar plates loaded with Streptomyces 85E. Then, 0.02% of tween 80 and DMSO infused discs were used as positive and negative control. Then the plates were incubated at 30°C for 72-96 hours (time required for hyphae formation in Streptomyces 85E) and the results were interpreted as bold zone of inhibition around samples and controls infused discs. RESULTS AND DISCUSSION FTIR Spectroscopy Fig. (1a) shows the FTIR spectrum of synthesized polymer P4VP. A characteristic absorption peak of pyridine moiety is shown at 1595 cm-1. This indicates that the pyridine ring is uncoordinated. The peaks at positions 717.8 cm-1 and 819.1cm-1 were observed due to “out of plane” bending of the aromatic ring. Peak at 992.6 cm-1 attributed to the ring breath. The peaks that appeared at 1170.2 cm-1 and 1217.9 cm-1 wererelated to sigma C-H vibration modes. Two peaks for C=N stretching appeared at 1414.2 cm-1 and 1555.5 cm1 . Peak at position 2929.2 cm-1 showed vibration of CH 2 group and another peak appeared at 3025.6 cm-1 showed aromatic and vinyl rocking vibration of C-H group present in the polymer chain. In rubidium chloride-P4VP composite, the peaks at less than 500 cm-1 were also observed which showed metal chloride bonding. Pyridine peak was observed at 1595.5 cm-1 and C=N stretching vibration peak was observed at 1414 cm-1 . The peaks were relatively weak when compared to the polymer (Fig. 1b) and Table 1. Thermogravimetric Analysis (TGA) Thermal behavior of Rubidium chloride-polymer composite was examined through TGA analysis, executed under inert atmosphere of nitrogen (25 ml/min) using heating rate of 10°C/min. Fig. (2a) illustrates that the degradation of polymer started at 350°C and at 400°C more than 90 percent of polymer was degraded. The remaining polymer was decomposed to almost zero mass at 500°C. The primary weight loss in the polymer and also in composite was because of the loss of moisture present in the sample.


Rashid et al.

a. FTIR spectra of P4VP

Fig. (2). TGA curve of P4VP (a) and its Rb-P4VP composite (b).

Degradation of metal composite took place in three steps as shown in Fig. (2a,b). In the first step, degradation occurred till 100°C. It was due to the loss of moisture and the bonded water. The second step started at about 300°C. It was due to the decomposition of polymer chains into its individual groups. Third step at about 450°C attributed the decomposition of metal chloride bonds and it went on till 550°C. Thermal behavior of rubidium composite was similar to the polymer (Fig. 2b and Table 2). The only difference observed was that the rubidium composite was not fully degraded, since the metal particles were still observed at 500 °C. Rubidium composite showed decline in the initial thermal behavior.

b. FTIR spectra of rubidium chloride- P4VP composite Fig. (1). FTIR spectra of P4VP and composite.

Table 1.

FTIR spectra of P4VP and composite Peak position

In the second step, a reduction in the composite’s degradation was observed at 300 °C. It may be attributed to the development of coordination between ring and metal center, leading to the change in the electronic density of the ring. It resulted in the weakening of the bonds in the polymer chain, which led to the lowered thermal stability. In the second step, degradation of metal chloride bonds was noted at or near 450°C.

Functional Group

Fig. 1 (a) P4VP 1595 cm-1 -1

Pyridine

717.8 cm and 819.1 cm 992.6cm

-1

Out of plane bending of aromatic ring

-1

Ring breath

1170.2 cm-1 and 1217.9 cm-1 -1

1414.2 cm and 1555.5 cm

Sigma C-H vibrations

-1

X-Ray Diffraction (XRD) Studies

C=N

2929.2 cm-1

CH2

3025.6 cm-1

C-H rocking of vinyl

Powder XRD spectrum of P4VP is shown in Fig. (3a). In this spectrum, a broad peak was obtained around the angle of 21°. This is the characteristic pattern of P4VP polymer which indicated that the polymer was in amorphous form.

Fig. 1 (b) Rubidium chloride polymer composite Peaks at less than 500 cm

Table 2.

-1

The composite of P4VP with rubidium chloride salt showed obvious changes in the XRD diffraction pattern. Different peaks were observed at 23.5°, 27°, 38.5°, 45.5°, 48°, 56°, 61° and 63°. This pattern was the same as mentioned in the literature [14,15]. The most intense peak was the one at 27° and the second intense peak was at 38.5°.

Rb-Cl

1595.5 cm-1

Pyridine

1414 cm-1

C=N

Thermal behavior of polymer and its composite

Polymer metal Composite

Degradation temp. initial (°C)

Degradation temp. final (°C)

Percent weight degraded

Weight remaining after (550°C)

P4VP

331

402

89.89

0.12 mg

Rb-P4VP

321

392

71

1.71 mg


Other peaks were short and less in intensity. This observation shows that crystalline nature of the composite was higher than its parent compounds. This can be explained by the fact that chains in polymer can move and fold to form crystallization regions. The inter- or intra-molecular interactions can cause the formation of regular crystallite structures. The XRD pattern of rubidium chloride-P4VP composite is shown above in Fig. (3b).


S = 0.9/4D [17] Where S= strain, = wavelength, D = Grain size D= 1/D2 [18] D = Dislocation line density The different material parameters are summarized in Table 3. Table 3.

Intensity (counts)

(a) X-ray Diffraction patterns of P4VP.

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XRD Parameters of Rubidium-P4VP

2º

Grain Size (nm)

Dislocation density (lines/cm2)

Strain (lines-2 cm-4)

28.92

23.3955

53.86

0.0033

0.0850

100

27.1014

48.21

0.0032

0.0324

73.04

38.6372

55.88

0.0020

0.0106

13.24

45.6512

28.6

0.0033

0.0173

20.1

47.7776

57.68

0.0016

0.0067

9.31

55.7069

47.7

0.0016

0.0069

4.9

61.2826

30.63

0.0023

0.0045

20.1

63.0181

33.8

0.0021

0.0047

A comparison between material parameters verses 2º can be represented as shown in Fig (4).

(b) XRD spectra of P4VPcomposite with rubidium chloride

(a) 2 verses intensity and grain size

Fig. (3). XRD pattern of P4VP and its metal composites.

Different material parameters like grain size, dislocation line density and strain were calculated using different relations as follows: Grain sizes of crystalline particles were determined using the following formula: D = 0.9/Cos [16] Where D= Grain size of particles = wavelength of Cu source 1.54 cm-1 = full wavelength at half maxima (FWHM) = angle of diffraction (Bragg’s angle) The strain and dislocation line density were calculated as follow.

(b) 2 verses dislocation density and strain Fig. (4). Graphical Representation of Materials Parameters verses 2.


Table 4.

Rashid et al.

Calculations of Lattice Parameters Sin2 1

Sin2 2

Sin2 3

Sin2min

Sin2min

Sin2min

0.041

1.000

2.000

27.1014

0.055

1.341

3

38.6372

0.109

4

45.6512

5

Peak No.

2º

Sin2

1

23.3955

2

h2 +k2+l2

hkl

a (A°)

3.000

3

111

6.57

2.683

4.024

4

200

6.57

2.658

5.317

7.976

8

220

6.58

0.150

3.658

7.317

10.976

11

311

6.58

47.7776

0.164

4.000

8.000

12.000

12

222

6.58

6

55.7069

0.218

5.317

10.634

15.951

16

400

6.59

7

61.2826

0.259

6.317

12.634

18.951

19

331

6.58

8

63.0181

0.273

6.658

13.317

19.975

20

420

6.58

Note: Lattice parameter = 6.578 A°, Bravais lattice is Face-Centered Cubic.

The parameter values show that the particle size of synthesized material was in the range of 33 nm to 57 nm, which lies in nano-size range. The variation of size was observed due to nucleation of particles as well as re-arrangement of grains during the material formation. The variation of diffraction intensity was also observed. It might be due to the crystal imperfections and texture effects. The values of strain and dislocation line density observed were very smaller as compared to the grain size and intensity due to polgonization at high temperature. Lattice parameter and cubic system was also calculated using appropriate relations as shown in Table 4 [19]. Lattice parameter “a” was calculated through following formula: a = /2 Sin h2 +k2+ l2 Volume of cell was calculated by using following relation: V = a3 V = (6.578) 3 V = 284.63 (A°)

g/ml as compared to rubidium composite with P4VP which showed 17% inhibition at 100 g/ml. In case of Staphylococcus aureus, P4VP was inactive while its composite with rubidium showed 7% inhibition at 100 g/ml. Overall results indicate that P4VP polymer had significant antibacterial activity against Pseudomonas aurignosa while its composite with rubidium had low antibacterial activity against both bacterial strains. Antifungal Activity Antifungal activity was accomplished on three fungal strains named aspergillus niger, Aspergillus flavus and Fusarium solani. In case of Aspergillus niger and Fusarium solani, both P4VP and its composite with rubidium was inactive and did not show any result. In case of Aspergillus flavus, data obtained for rubidium composite with P4VP indicated that it had some antifungal activity with 8 mm zone of inhibition at the sample of 100 g/disc while the polymer P4VP showed no activity. Enzymatic Activities

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Antioxidant Activity Antioxidant activity of P4VP and rubidium composite with P4VP was measured by using DPPH as a free radical. P4VP and its composite were treated and percent scavenging at 200 g/ml concentration was measured for each sample. P4VP showed some antioxidant activity at IC50 value of 17.04 ± 1.39 g/ml. Rubidium composite with P4VP also showed significant activity with an IC50 value of 19.16 ± 2.75 g/ml, which is non-significantly (P > 0.05) higher as compared to that of P4VP. Antibacterial Activity Antibacterial assay of P4VP and its composite was performed on two bacterial strains named Pseudomonas aurignosa and Staphylococcus aureus. The data obtained for Pseudomonas aurignosa indicated that P4VP showed significant antibacterial activity with 92% inhibition at 100

Alpha-Amylase Activity Alpha-amylase is a major enzyme found in the human pancreatic juice and saliva. This enzyme plays a vital role in the hydrolysis of polysaccharides and insoluble starch to form small absorbable molecules such as glucose and maltose. Diabetes mellitus is a metabolic disease characterized by hyperglycemia in which blood sugar level increases due to defects in functioning of pancreas that do not secrete enough insulin. Diabetes can be treated by retarding the absorption of glucose and alpha-amylase to hydrolyze the carbohydrates as amylase is a carbohydrate hydrolyzing enzyme [21]. Poly 4-vinyl pyridine and its composite with rubidium showed the remarkable values of alpha-amylase activity. Amylase was used as positive control. An IC50 value for P4VP was 168 g/ml while rubidium composite with P4VP showed the value of 153 g/ml. Percentage inhibition at 200 g/ml was 98 for P4VP while 81 for metal polymer com-


Table 5.


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Brine shrimp activity

Sample

Percent death

LD50

200 (g/ml)

100 (g/ml)

50 (g/ml)

25 (g/ml)

(g/ml)

Rb-P4VP

100

50

30

20

104

P4VP

30

30

30

10

>200

posite. Sample concentrations of 200.00 g/ml, 66.66 g/ml, 22.00 g/ml and 7.41 g/ml were used. This data shows that P4VP had better amylase activity than its metal polymer composite. Protein kinase Activity In protein kinase assay, a kinase enzyme imparts a phosphate group of ATP to various amino acids like threonine, tyrosine or serine. As a result of this reaction, a phosphorylated protein and ADP were generated. Protein kinase activity has the tendency to cure diseases like cancer and other viral infections. Classical method to determine the kinase activity is the quantification study of transfer of phosphate group to the protein and from the conversion of ADP from ATP [22] (Fig. 5). Results of kinase activity of samples show that P4VP had 7 zones of inhibition (mm) at 100 ug/disc while the rubidium composite with P4VP was active towards protein kinase assay. Sample concentration was 5 l (20 mg/ml) per disk while final concentration was 100 g/ml per disc. Although P4VP has not been an efficient protein kinase activity but as compared to polymer metal composite, it is somehow active towards protein kinase bioassay.

Fig. (5). Protein kinase activity.

P4VP-rubidium chloride composite has also been found to be an active pharmaceutical. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.

Brine Shrimp Activity

ACKNOWLEDGEMENTS

Brine shrimp lethality bioassay was used for the determination of the cytotoxicity of samples depending on the capability to kill the laboratory test compounds on a simple organism brine shrimp (Artemia salina) [23]. The brine shrimp activity data for P4VP and its composite with rubidium showed significant results. The LD50 (lethality death) values were measured at different concentrations and compared with one another. Results are shown below in Table 5.

The authors thank Professor Zareen Akhtar and Professor Saqib Ali, Department of Chemistry, Quaid-i-Azam University, Islamabad and Associate Professor, Uzma Yunus Department of Chemistry, Allama Iqbal Open University for providing research facilities.

These results showed that P4VP was less toxic as compared to its composite with rubidium This was due to the toxic nature of the metal. Addition of the metal to P4VP made the composite toxic.

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