international journal of pharma and bio sciences issn 0975 … · inhibition of group a...

Int J Pharm Bio Sci 2015 April; 6(2: (P) 85 - 98

This article can be downloaded from www.ijpbs.net

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Research Article Biotechnology

International Journal of Pharma and Bio Sciences ISSN

0975-6299

INHIBITION OF GROUP A STREPTOCOCCUS BY GREEN SYNTHESIZED ZINC

OXIDE NANOPARTICLES

AKASH S, SHANTHA KUMAR SS AND DHAMODHAR P*

Department of Biotechnology, M.S. Ramaiah Institute of Technology, Bangalore, Karnataka, India

ABSTRACT

Group A Streptococcus (GAS), an upper respiratory organism is becoming resistant to antibiotics and nanotechnology has shown tremendous applications as antibacterial agents.In the presentstudy, ZnO nanoparticles were synthesized using Punica granatam epicarp by pyrolysis method. ZnO nanoparticles were characterized by PXRD and were found to have hexagonal phase, wurtzite structure,the crystalline size of the nanoparticle was found to be ~60 nm. The UV-vis spectrum for ZnO nanoparticle showed a strong absorbance at 375 nm corresponding to the band gap energy of 3.25 eV. The FTIR spectrum showed a peak at 499 cm-1, which indicates Zn-O stretch bond. The SEM analysis of Zinc oxide nanoparticles showed the average size of 60 nm and EDS revealed the elemental composition of ZnO nanoparticles ZnO nanoparticles synthesized were tested for their antibacterial activity on GAS and were found to be susceptible. KEYWORDS: Group A Streptococcus, Nanotechnology, Zinc oxide, Punica granatam, pyrolysis

*Corresponding author

DHAMODHAR P

Department of Biotechnology, M.S. Ramaiah Institute of Technology,

Bangalore,Karnataka, India



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INTRODUCTION Streptococcus pyogenes is the organism belonging to Group A Streptococcus causing diverse range of diseases from mild infections like Pharyngotonsillitis and Impetigo to serious infections such as necrotizing fasciitis and Streptococcal toxic shock syndrome, if left untreated it may trigger autoimmune disease like acute post streptococcal glomerulonephritis, acute rheumatic fever , rheumatic heart disease and these diseases account for over half a million deaths per year globally1. Growing resistance to antibiotics makes the organism more vulnerable to survive and cause diseases2-3. Although penicillin remains the drug of choice4, but the patients who are sensitive to penicillin opt for macrolides. Current reports suggest that there is an increasing resistance to macrolides globally5-8, so it becomes necessary to find an alternative therapy to evade the infections caused by GAS. One such approach is by the application of nanotechnology, especially by the use of nanoparticle as antibacterial agents9. Nanoparticles exhibit unique mode of action in controlling the growth of bacteria, prevailing as an alternative therapy against drug resistant microorganisms10. Nanoparticles such as metal oxide exhibit selective toxicity to bacteria and minimal effects on human cells11.However, their effects on human pathogens such as GAS have not been addressed. ZnO nanoparticles are usually synthesized by thermal decomposition, thermolysis, chemical vapor deposition, sol–gel, spray pyrolysis, precipitationvapor phase oxidation, thermal vapor transport, condensation and hydrothermal12. These approaches use physical and chemical methods involving complex procedures, sophisticated equipment and rigorous experimental conditions, this pays a way to find an economically viable synthesis techniques. Green synthesis of nanoparticles provides advancement over other methods as itis simple, cost-effective, and relatively reproducible and often results in more stable materials13.The Plant parts such as leaf, root, latex, seed, stem and fruits are being used for synthesis of metal oxide nanoparticle. The compounds

namely polyphenols present in plant parts serve as reducing and capping agents14 can be considered as an attractive alternative for the synthesis of nanoparticles. In the present study we report the preparation of nanocrystalline ZnO nanoparticles by pyrolysis method using Punica granatum epicarp. The synthesized nano powders are characterized for their structure using Powder X-ray diffraction (PXRD). The morphological studies were carried out using Scanning Electron Microscopy (SEM). Fourier Transform Infrared spectroscopy (FTIR), UV–vis and Energy Dispersive X-ray Spectroscopy (EDS) were employed to investigate.Zinc oxide nanoparticles were further tested for their antibacterial activity on standards (MTCC 442 and ATCC 19615) and clinical isolates (six isolates) of Group A Streptococcus, upon varying the parameters like concentrations of Zinc oxide and dilutions of extract with water. The zone of Inhibition obtained were analyzed using two way ANOVA and was found to be statistically significant (p=0.007).From the present study we infer that Zinc oxide nanoparticles synthesized by pyrolysis using Punica granatum epicarp offers a valuable contribution in the area of nanotechnology without involving different chemical steps and the green synthesized Zinc oxide nanoparticles were found to be susceptible to Group A Streptococcus.

MATERIALS AND METHODS

(i) Preparation of the Plant extract Fresh native varieties of Punica granatum was purchased from safal market, Bangalore, India. The epicarp of Punica granatum was separated from mesocarp, endocarp and seeds. The Punica granatum epicarp was made into a fine paste by using sterile distilled water. 5 g of the extract was diluted in 5ml and 10 ml of deionized water, further 5ml and 10 ml of deionized water was diluted in 45 ml and 40 ml of sterile distilled water respectively, making the volumes to 50 ml and was taken for the synthesis of Zinc oxide nanoparticles.'



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(ii) Synthesis 1M Zinc nitrate (Zn (NO3)26H2O) was taken in a glass beaker and dilutions of dry extract of Punica granatum epicarp were added to the glass beaker. Continuous mixing of materials was done to ensure homogeneity of the solutions. Now the beaker was kept on water bath for 80 0C (pre-pyrolysis) for 1 hour. After pre-pyrolysis the extract was filtered and taken in a petri dish. The petri dishes containing the homogeneous mixture of Zinc nitrate and Punica granatum epicarp solutions were placed in a pre-heated muffle furnace which is maintained at 570 ± 10 0C for 2 hours. Initially the solution boils and undergoes dehydration, which was followed by decomposition accompanied by evolution of large amounts of gases (oxides of carbon and nitrogen) 15. (iii) Characterization of green synthesized

nanoparticles The phase purity and crystallinity of green synthesized Zinc oxide samples were examined by Powder X-ray diffractometer (Bruker-D2-Phaser) using Cu Ka (1.541 Å) radiation with a nickel filter. The surface morphology of the ZnO nanoparticles was examined using Scanning Electron Microscopy along with Energy Dispersive x-ray Spectroscopy (EDS) Zeiss (Gemini-Ultra 55). The SEM micrograph was recorded after vacuum dissection of ZnO nanoparticles and coating the samples with gold. The FTIR studies were performed on a Bruker-Alpha FT-IR Spectrometer with KBr pellets. The UV–Vis absorption of the samples was recorded on SL 159 ELICO UV–Vis Spectrophotometer. (iv) Isolation and Identification 20 pediatric cases of the age group 5 to 15 years suspected for pharyngotonsillitis infection and receiving no antibiotics in antecedent for 7 days visiting M S Ramaiah hospital were subjected for repeat throat samplings. All the isolates were taken for their identification study using routine lab diagnostic tools such as β – hemolysis on blood agar plates, gramstaining,

catalase test, bacitracin susceptibility and PYR test to prove the possibility as GAS. A standard Streptococcus pyogenes strain MTCC 442 received from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India and ATCC 19615 received from M S Ramaiah metro Politian hospital were also included as control16.

(v) Antibacterial Susceptibility All the isolates identified as GAS were tested for their sensitivity pattern with the green synthesized ZnO nanoparticles by agar well diffusion method on Mueller-Hinton agar (MHA) as recommended by the Clinical and Laboratory Standards Institute, Wayne, USA17.The anti-GAS activity at various concentrations (10, 20, 30 and 40mg/ml) and dilutions of synthesized ZnO nanoparticles was screened by well diffusion technique on sterile Muller Hinton Agar plates. Antibacterial activity in terms of zone of inhibition (mm) was recorded after 24 hours of incubation. Two factorial ANOVA was applied by taking GAS isolates, concentrations and dilutions of ZnO nanoparticles as categorical factors and zone of inhibition as the dependent variable and was analyzed (P < 0.05) using Statistica software (version 7.0; StatSoft, Inc.,USA).

RESULTS AND DISCUSSION

Among the 20 suspected cases with pharyngotonsillitis, only 6 children were confirmed to be harboring GAS by routine lab diagnostic tools. All the 6 GAS isolates were identified as Gram positive cocci in chains exhibiting β-hemolytic activity on blood agar plates and sensitive to Bacitracin (0.04U). Apart from that, the GAS isolates were catalase negative and showed positivity to PYR test. The Zinc oxide synthesized by Punica granatum epicarp of dilutions 40 ml of water + 10 ml of extract was further sent for characterization since both the methods showed equal zone of inhibition.



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1. Powder X-ray diffraction (PXRD)

Figure 1 PXRD pattern for ZnO synthesized using Punica granatum epicarp

The PXRD pattern for Zinc Oxide powder is shown in the Fig.1, studied at diffraction angles from 200-800 of 2θ. The peaks obtained confirm that the ZnO is of hexagonal phase having a wurtzite structure belonging to JCPDS(Joint Committee on Powder Diffraction Standards) no. 36-145118. The peaks also confirm that the synthesized ZnO nanoparticle doesn’t contain much of characteristic impurity.The green synthesized ZnO nanoparticle diameter was calculated using Debye-Scherrer formula shown in equation.1

(1)

Where, D is the average crystalline size, λ is the X-ray wavelength of 1.54 Ǻ, θ is the Bragg diffraction angle and βis the FWHM (0.2281) (Full Width Half Maximum). The ZnO nanoparticles synthezied by pyrolysis method of 40 ml dilution + 10 ml with extract showed the average crystilline size to be 63.87 nm corresponding to the plane 101 located at 35.860.Units Morphology Index (MI) was developed from FWHM (Full Width at Half Maximum) of XRD data. MI is obtained from Equation 2. The MI range of ZnO nanoparticle ranges from 0.5 to 0.6359 shown in table.1.19

(2)

Where, M.I is morphology index, FWHMh is highest FWHM value obtained from peaks and FWHMp is value of particulars peak’s FWHM for which M.I is to be calculated.

(3)



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(4)

Controlling X-ray intensities of diffraction angles needs to be monitored, so Lorentz-polarization factor was introduced. In the intensity calculations Lorentz factor is combined with the polarization factor and further the variation of the Lorentz’s factor with the Bragg angle (θ) is shown 20-21. The overall effect of Lorentz factor is to decrease the intensity of the reflections at intermediate angles compared to those in the forward or backward directions. Lorentz factor and Lorentz Polarization factor are calculated from Equations 3 and 4 and tabulated in table.1.

Table 1 Representing 2θ values, Miller indices, MI, LF, and LPF.

2θ h k l Morphology index Lorentz factor Lorentz polarization factor

31.09831 100 0.5802 3.6112 25.0359 33.76881 002 0.5817 3.0971 20.9494 35.60468 101 0.5809 2.8091 18.6641 46.94618 102 0.5652 1.7179 10.074 56.03922 110 0.5406 1.2832 6.7347 62.33633 103 0.5179 1.0908 5.3036 67.44761 112 0.5 0.9752 4.4744

2. UV- Visible spectroscopy (UV-vis spec) The green synthesized ZnO nanoparticles were characterized using UV-Visible spectroscopy, which showed a typical absorption at 375 nm shown in Fig.2.The spectrum also indicates that the sample produced in this experiment has a very high level of purity. The presence of any contaminants would have acted as dopants in the semiconductor material, causing lower energy (higher wavelength) transitions, which would appear as small peaks or drops above

the original drop of the band gap. The spectrum showed a smooth line, beyond the original drop. Band gap is another important factor that was measured, when photons of higher energy are larger than band gap of the semiconductor, an electron is transferred from the valence band to the conduction band where there occurs an abrupt increase in the absorbency of the material to the wavelength corresponding to the band gap energy 19.

Figure 2 UV- vis absorbance spectrum for green synthesized ZnO nanoparticle



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Typically, the absorbance measurements can be applied to obtain effective mass model to determine the particle size of the nanoparticles as opposed to concentrations22. The average particle size in a nanocolloid can be calculated from the absorption onset from UV-vis absorption spectra by using effective mass model shown below as equation, where the band gap E∗can be approximated by

(5)

Where, Eg* is the band gap energy of the nanoparticle, which will be determined from the UV-Visible absorbance spectrum, band gap energy of the bulk ZnO at room temperature, which has the value of 3.5eV, h is Planck’s constant, r is ZnOparticle radius (m), me= mass of a free electron(9.11*10-31)kg, is effective mass of a conduction band electron in ZnO (0.26), is effective mass of a valence band hole in ZnO (0.59), e is elementary charge(1.602*10-19),

0ε permittivity of free space (8.854*10-12C2N-1m-2) and ε is relative permittivity of ZnO (8.633).

Figure 3 Band gap energy for ZnO nanoparticles

The optical energy band gap Eg of samples was estimated using the Tauc relation by a software PARAV-V2

(6)

Where, hυ is the photon energy and α is the optical absorption coefficient near the fundamental absorption edge. The absorption coefficients were calculated from the optical absorption spectra. The optical band gap of ZnO is obtained by plotting 2 versus (equation 6) in the high-absorption range followed by extrapolating the linear region of the plots to 2 = 0 (Fig. 3). From the figure it is seen that the ZnO sample has an optical energy band gap of 3.25 eV that is quite nearer to the standard band gap energy of ZnO (3.3 eV). The following equation was derived from the effective mass model given above with small mathematical simplification which is used to find the size of the particle from the absorbance spectra as shown in the equation 723.



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(7)

Where, λp is peak absorbance wavelength in nm and r is the radius of the particle. From effective mass model, the diameter of the particle size was calculated and it was found to have an average size of 50 nm, which is similar to the size found by SEM.

3. Fourier Transform Infrared Spectroscopy (FTIR)

The Fourier transform infrared spectra of ZnO nanoparticle synthesized by Pyrolysis method is shown in the fig.4, the bands are acquired in the range of 400-4000 cm-1. A weak band at 3500 cm-1 attributes to the characteristic stretching mode of H2O

24. Small peaks at around 2300 cm-1 showed the presence of –CN nitrites25. The sharp peaks near 1600cm-1

showed trace amount of nitrates present26. This bands confirms the presence of precursor impurities in the sample, which is due to incomplete convertion of the constituents of Punica granatum epicarp. Bands ranging from 400 to 600 cm originate from metal to oxygen (M–O) groups. The band at 499 cm-1 corresponds to stretching frequency of Zn-O hexagonal phase27.

Figure 4

FTIR spectrum for ZnO nanoparticles synthesized by pyrolysis method 4. Scanning Electron Microscopy (SEM) The SEM morphology of the synthesized ZnO particles by pyrolysis methods showed the formation of agglomerated ZnO nanoparticles shown in fig.5.



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\

Figure 5 SEM images of green synthesized ZnO nanoparticles

ZnO synthesized showed the average particle size of 50 nm. These pictures substantiate the approximated shape of the nanoparticles and most of the particles exhibited some faceting. Characteristic impurities of Punica granatum epicarp was also found. It was also seen that the size of the nanoparticles were less than 100nm, which is in good agreement with the literature27.

5. Energy Dispersive x-ray Spectroscopy(EDS) The EDS of the ZnO sample carried out along with SEM revealed that the required composition ZnO were present. Elemental Composition of Zinc was found to be 51.78% and Oxygen was 48.22% (fig.6). This is in agreement with the ZnO nanoparticle which is in 1:1 ratio. The slight variation in the composition is due to the loss of x-rays being detected28.

Figure 6 EDS pattern for ZnO nanoparticles synthesized by pyrolysis method



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6. Univariate Tests of Significance for Zone of Inhibition (mm)

The zone of inhibition obtained for all the isolates with different concentrations and dilutions were subjected for Two – Way ANOVA. The ‘F’ distribution values obtained for the antibiotics and the isolates were compared with the theoretical F-value at F 7, 53 and F 2,53, calculated from the F distribution table. The F values for the antibiotics and the isolates were

F7, 53= 285.1516>2.188 and F2, 53= 5.3 >3.1716 respectively at α =0.05 level of significance (Table 2). Hence the null hypothesis was rejected and it was concluded that the effects of GAS isolates and concentrations of ZnO on zone of Inhibition were statistically significant and there was no statistical difference between the dilutions of Punica granatum epicarp extract.

Table 2

Two way Anova for zone of inhibition obtained for all the isolates with different concentrations and dilutions

Sigma-restricted parameterization Effective hypothesis decomposition

SS

Degrees of freedom

MS F p

Intercept 0

Isolate(no) 306.0000 7 43.71429 285.1516 0.000000

Concentrations (mg/ml) 1.6250 2 0.81250 5.3000 0.007974

Dilutions 0

Error 8.1250 53 0.15330 SS – Sum of squares; MS – Mean Square; DF – Degree of freedom;F– F-distribution Value at corresponding degree of freedom; P– pdistributionvalue at 0.05 level of significance

7. Effective hypothesis of Isolates The fig.7 shows a line plot of Isolate (no) vs. zone of Inhibition (mm) of Group A Streptococcus. The Standard isolate s1 and s2 are having a Zone size of 18mm and 19.5 mm, while the clinical isolates s3, s4, s5, s6, s7 and s8 are having the zone size of 18, 22.5, 22.8, 19.9, 17.1 and 15.8 mm respectively. The presence of inhibition zone clearly indicates that the mechanism of the biocidal action of ZnO nanoparticles, which involves disruption of the membrane with high rate of generation of surface Oxygen species and finally lead to the death of bacteria. Interestingly, the size of the inhibition zone was different according to the type of isolates, where a similar work has been reported29. ZnO powder has been used for a long time as an active ingredient for

dermatological applications in creams, lotions and ointments on account of its antibacterial properties30. However, nanoparticles of ZnO are much more effective agents in controlling the growth of various microorganisms. The studies suggested that synthesized ZnO nanoparticles are able to slow down the bacterial growth as a result of disorganization of the S.pyogenes membranes, which increases the membrane permeability leading to the accumulation of nanoparticles in the bacterial membrane and cytoplasm regions of the cells31.The present research reveals that the Isolate s4 is having the maximum zone size and Isolate s8 having the least. Overall results tell us that ZnO nanoparticles were effective in preventing the growth of Group A Streptococcus.



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Figure 7 Isolate (no) vs. Zone of Inhibition (mm) of Group A Streptococcus

8. Effective hypothesis of concentrations The fig.8 is a plot of concentrations of ZnO nanoparticles (mg/ml) vs. zone of Inhibition (mm) of GAS. The concentrations of 10, 20, 30, 40 mg/ml showed a zone size of 18.5, 18.8, 19.0 and 19.2 respectively. From the present study we found that by increasing the

concentration of ZnO nanoparticles, the growth of inhibition has also been increased consistently this was in agreement with the previous studies32-33. From the current findings it was concluded from the results that 40 mg/ml is showing large zone size when compared to other concentrations.

Figure 9 Concentrations of ZnO nanoparticles (mg/ml) vs. Zone of Inhibition (mm) of GAS.

9. Effective hypothesis of dilutions The fig.9 shows a plot of dilutions of Punica granatum epicarp extract with water and Zone of Inhibition of Group A Streptococcus. Punica granatum epicarp extract is primarily composed of alkaloids and polyphenols. The active

constituent that appears to be responsible for its multiple health benefits is ellagic acid. Pomegranate extract has demonstrated a variety of beneficial functions including antioxidant and anti-viral activity. Antioxidant action of phenolic compounds is due to their



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high tendency to chelate metals. Phenolics possess hydroxyl and carboxyl groups, able to bind to heavy metals. They may inactivate metal ions by chelating. When metal salts come in contact with the ellagic acid present in the peel extract the ester oxygen atom and the ortho-phenolic hydroxyl of the ellagic acid form p track conjugation effect. The esterification of the carboxyl and hydroxyl groups of ellagic acid make the ortho-phenolic hydroxyl loose the hydrogen much easily, forming a steadier semi-

quinone structure. Thus, ellagic acid has an easy electron loosing capacity which results in the formation of H+ radical, which reduces the size of ZnO particles to nanosize34. From the present work we can conclude that the dilutions containing 40 ml water + 10ml extract and 45 ml water + 5 ml extract showed a zone size at around 18.85 mm. Varying the dilutions did not make much difference towards synthesizing ZnO nanoparticles.

Figure 10 Dilutions of Punica granatumepicarp extract with water and

Zone of Inhibition of Group A Streptococcus

CONCLUSION Zinc oxide powder was successfully synthesized by the pyrolysis method using extracts of Punica granatum epicarp. Powder X-ray diffractionpatterns confirms the hexagonal phase and the average crystallite size calculated by the Debye−Scherer formula and were found to be in the range~60 nm which is in good agreement with SEM results. The transition of ZnO nanoparticles appeared at 375 nm and electronic band gap of ZnO formed is estimated to be 3.25 eV from UV−vis

spectroscopy. SEM analysis show the ZnO nanoparticles are in agglomerated form and characteristic impurities of Punica granatum epicarp were also observed. The Zinc oxide nanoparticles were tested for their antibacterial activity on Group A Streptococcus upon varying the parameters like concentrations of Zinc oxide and dilutions of extract with water while preparation. Among the clinical isolates, Isolate s4 showed maximum inhibition, 40mg/ml showed highest inhibition size and no difference was seen upon varying the dilutions in synthesizing ZnO nanoparticles.



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