Incorporation of Biotransformed Silver Nanoparticles in ... - Springer Link

ISSN 1560-0904, Polymer Science, Series B, 2016, Vol. 58, No. 1, pp. 61–72. © Pleiades Publishing, Ltd., 2016.

COMPOSITES

Incorporation of Biotransformed Silver Nanoparticles in Plant Polysaccarides Resin and Their Effect on Sustained Drug Release1 V. Subhaa, Preethi Ramadossb, and S. Renganathana,* a

Centre for Biotechnology, Alagappa College of Technology, Anna University, Chennai-600025, India b Crystal Growth Centre, Anna University, Chennai-600025, India *e-mail: [email protected] Received April 26, 2015; Revised Manuscript Received September 24, 2015

Abstract—The intension of current study was to determine antibacterial and drug releasing capacity of green synthesized silver nanoparticles (AgNps) with Moringa oleifera resin in the presence of Montelukast sodium and Ibuprofen. This plant gum is economic, easily available, biodegradable, safe and potential tablet binder. There was no significant study reported on the incorporation of green synthesized silver nanoparticle with plant resin in drug release. The aqueous extract of Clerodendron phlomoides was used for the bioreduction of silver nanoparticles as well as a capping agent. This green synthesized AgNps was observed in UV at 489 nm due to the SPR (Surface Plasmon Effect) effect, and the presence of protein and polyol compounds was identified by FTIR. The crystalline structure of AgNps was analyzed by XRD, elemental silver composition was measured by EDAX, morphological structure and size was revealed by SEM and TEM analysis. The antibacterial effect of green synthesized AgNps was analyzed by zone of inhibition method. Silver nanoparticles incorporated in M. oleifera plant resin and its functional groups and thermal degradation properties were characterized by FTIR and TGA, respectively. The drug release properties of the AgNps incorporated with plant resin were evaluated for the sustained release and compared with raw plant gum without AgNps consistency. DOI: 10.1134/S1560090416010073

Metal nanoparticles are very small in size (around 10 nm), but can carry high dosage of therapeutic agents (target molecules) [12]. Drugs used in the targeted-drug delivery method are more effective and convenient to overcome patient compliance, increase the half-life of bioactive molecule and decrease healthcare expenses [13]. Till date, silver could be implemented for inhibition of microbe colonization on the surgical instruments and silicone rubber gaskets [14]. The antimicrobial activity of colloidal silver nanoparticles depend upon the dimensions of the silver nanoparticles that small particle size has increased antibacterial activity [15]. The anti-bacterial properties of green synthesized silver nanoparticles could be studied by directly exposing bacterial species to colloidal silver nanoparticles [16]. Clear mechanism behind the antibacterial activity properties of AgNPs is unknown, but one of the mechanisms to kill the bacteria is attained due to the electrostatic attraction between negatively charged bacterial cell wall to the positively charged silver nanoparticles [17, 18]. There are many articles which have reported that the nano silver particle can interact with various macromolecules [19] such as metabolic enzymes and DNA by electron-release mechanism for antibacterial activity [20]. It is believed that AgNPs

INTRODUCTION Although a lot of drug delivery and targeting methods are available in medical field for the sustained release of bioactive molecules, plant gum with silver nanoparticles are entirely new for drug delivery field. Nanotechnology is the under-standing and control of matter at dimensions of roughly 1 to 100 nm [1]. In recent days, 95% of drugs have low pharmacokinetics as well as biopharmaceutical properties [2]. Nanomedicine helps to overcome poor pharmacokinetics, bio distribution and high dosage toxicity of therapeutic drugs, non-specific site targeting, improves the efficacy of therapeutic agents by increasing solubility of hydrophobic compounds, maintain sustained availability of bioactive therapeutic molecules and safety features of the new drugs [3–6]. Drug delivery is a process, in which bioactive medicine can be delivered to the target site at specific rate with safe and effective delivery system [7]. Nanomedicines are especially designed for targeting tumor and inflammatory cells when compared to normal cells. Delivery system advances the stability of the therapeutic molecules such as small peptides, proteins, oligonucleotides and hydrophobic molecules [8–11]. 1The article is published in the original.

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inhibits the DNA multiplication capacity, enhances loss in cellular protein action and denaturation of enzyme of the bacteria and also binding of silver nanoparticles with some functional groups of enzyme [20]. Reports have shown the deposition of AgNPs on the cell wall of certain microbes, especially sulphide group of the cystine amino acids, which plays a vital role in bacterial metabolism, and thereby inhibiting the growth of bacterial pathogens. However AgNPs generate reactive oxygen species that are also responsible for the bactericidal activity of the same. Polymers were used as solid, liquid or semisolid in pharmaceutical encapsulating material and formulation in ancient days [21]. Plant excipients are excellent natural polymers for the drug formulation when compared to synthetic polymers. Plant resins are biodegradable, economic, easily available, non-toxic, have ability to react with chemicals and biocompatible. Plant polymers have been used in the pharmaceutical field as buccal film, microsphere, film coating purposes, eyedrops, suspensions, colloids and viscous formulations and tablet controlled systems [22, 23]. Natural gums made up of polysaccharides, resins and tannins that were responsible for the drug-retardant-release property. Burst release of drug creates several side effects, that include neuro, musculo toxicity, neutronpenia, and therefore sustained (controlled release) release of drug can overcome these major problems. Vegetable gum is used as potential hydrophilic polymer in pharmaceutical preparation as a drug binder and releasing agent. Gum from Moringa oleifera (M. oleifera) tree has been reported to make gel for topical application [24]. This is naturally and easily available gum that is used as drug release retardant agent in tablet fabrication because it is sparingly soluble in water but it gets imbibe once it contacts with water. It also contains polyuronide that in turn consists of arabinose, galactose and glucoronic acid in the proportion of 10 : 7 : 2 with traces of rhamnose [25]. Synthesis of metallicnano silver from various routes with various sizes, morphologies and their antibacterial properties has been already reported [26–30]. Many plants were used for the synthesis of silver nanoparticles through green synthesis methodology. Limited research work has been established with Clerodendron phlomoides as a plant for the synthesis of silver nanoparticles. Dhanabal et al. reported that the ethanol extracts of Clerodendron phlomoides plant contains alkaloids, phytosterols, glycosides, saponins, phenolic compounds, proteins and flavonoids and their proteins amino acids patterns are similar to that of fenugreek [31]. In literature, a lot of plant gums were used for the drug delivery study, but not done with the combination of both the above mentioned green synthesized silver nanoparticles and M. oleifera gum. Montelukast sodium is used for the treatment of asthma and allergy. Ibuprofen is

the common medicine used for the pain relief. So both medicines were used for our drug release study. In the present investigation, bioreduction of Ag+ species by Clerodendron phlomoides was established for the first time. For the first time the analysis of drug release capacity of silver nanoparticles incorporated in the gum along with Montelukast sodium and Ibuprofen was done separately. Characterization of synthesized AgNPs was incorporated in the present investigation such as UV–Vis spectrometry (UV), Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) with EDAX, Transmission electron microscopy (TEM), zetapotential analysis and particle size analyzer. Antibacterial activity study was carried out for the synthesized nanoparticles. Plant gum materials thermostability was characterized using Thermo gravimetric analysis (TGA). Silver nanoparticle incorporated with plant gum material was studied for drug releasing profile of each drug separately. EXPERIMENTAL Materials Chemicals used in the present investigation such as Silver Nitrate (AgNO3), Muller Hinton agar, Ibuprofen and montelukast sodium were purchased from Himedia Laboratories, Mumbai, Ltd., and used without further modification. Clerodendron phlomoides plant and resin of Moringa oleifera were collected from the Mannargudi, Tamilnadu, India. Methods Moringa oleifera gum isolation. This plant gum was gathered from injured site of M. oleifera trees. The collected plant gum was dried and ground into powder. Then, 10 gm of powdered gum was taken along with 250 mL of distilled water in a beaker and it was stirred for 10 h at room temperature. Supernatant of gum was collected by centrifugation method. The remaining residue was washed thrice with water and added to the supernatant. This mixture was stirred with acetone, twice the volume of gum. The settled materials were collected, autoclaved and dried at 60°C and were used further for protocols and analysis. Preparation of plant extract. Finely ground Clerodendron phlomoides dried plant of 5 g was soaked in 100 mL of water for 3 h and then boiled for 20 min. Then the boiled mixture was brought to room temperature which was then filtered with Whatman no: 1 filter paper and the resultant filtrate were stored at 4°C for further use. Green synthesis of silver nanoparticles. 1 mM of silver nitrate was dissolved in 100 mL of double distilled water and 10 mL of aqueous plant extract was mixed with silver nitrate solution under continuous stirring at 70 rpm with 50°C with neutral pH. Synthesized silver

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INCORPORATION OF BIOTRANSFORMED SILVER NANOPARTICLES

nanoparticles were separated by centrifugation at 12000 rpm. Isolated silver nanoparticles were freezedried by lyophilizer and the dried silver nanoparticles were used for further characterization. Characterization Methods UV–Vis spectroscopy is a basic analysis method for Surface Plasmon resonance (SPR) effect nanoparticles. (Perkin-Elmer Lambda 2 Spectrophotometer Bremen, Germany in the range of 300–700 nm). The reactive groups present in the plant extract were responsible for the bioreduction of AgNO3 into silver nanoparticles and for the stabilization of synthesized silver nanoparticles. The reactive groups present in the plant extracts were analyzed by Fourier transform infrared spectroscopy (FTIR) (Perkin-Elmer, Shelton, Connecticut). The structural morphological, size and elemental composition of the nanoparticles could be characterized by Scanning Electron Microscope (SEM) with EDAX, (Hitachi S-4500 SEM machine) and Transmission Electron Microscope (TEM) (Hitachi H-7650 BIOTEM 120 Kv Tungsten filament instrument). Crystalline nature of the silver nanoparticles was analyzed by XRD analyzer (INEL X-ray diffractometer). Zetapotential of silver nanoparticles was analyzed by Zeta Plus Analyzer (Brookhaven, Instrument Corp., Holtsville, New York). From this analysis, surface charge of silver nanoparticles could be analyzed. According to electrophoretic mobility (EPM), the Zetapotential of the particles were determined. Nanoparticles possess either charge that pulls the oppositely charged ions towards itself. These nanoparticles along with double layer of ions are moved though the solution and an electric potential is created on the edge of the double ion layer which is called Zetapotential. When an electric field was applied, the EPM effect of the particles in water was analyzed by Phase Analysis Light Scattering method (PALS). Particle size analyzer (Malvar 200 particle analyzer) was used to determine the monodispersity of the nanoparticles. Antibacterial Effect of Silver Nanoparticles Muller Hinton medium was used for culturing bacteria. Media of 14 g with 2 g of agar in 500 mL of water was sterilized and poured in a Petri plate that was allowed to solidify for few minutes in microbe free environment. 50 μL of bacterial species was applied on the solidified media using spread plate method by the L-rod technique. Then the antibacterial reference Gentamicin disc was placed on the center of the medium along with 25, 50, 75, and 100 μL of colloidal silver nanoparticles with water as negative control. These petriplates were covered by paraffin wax film and kept in incubator at 37°C for 24 h. POLYMER SCIENCE, SERIES B

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Incorporation of Silver Nanoparticle in M.oleifera Resin 5 g of plant gum was taken in a beaker and 5 mL of silver nanoparticles were added drop by drop into it under stirring condition. The resultant mixture was dried at room temperature and the dried samples were further characterized using FTIR to find functional groups and TGA to study the thermal behavior of both the pure gum and gum with AgNPs. Loading of Drugs in Gum Along with Silver Nanoparticles 100 mg of either gum was soaked with 10 mg of montelukast sodium and Ibuprofen separately for 10 h. Then the gums were isolated from soaking and remaining solutions were taken for UV-spectroscopy to determine the drug absorption concentration in each gums. TGA (Thermogravimetric Analysis) Thermogravimetric analysis is done to study the thermal behavior of both the plant gum and gum along with silver nanoparticles. Thermal properties were measured in a TG/DTA instrument (TGA-50, Shimadzu) from 30 to 900°C with 20 grad/min under nitrogen atmosphere. In vitro Drug Release In vitro drug release capacity of raw M. oleifera gum and silver nanoparticles incorporated gum were evaluated for different time from 1 to 72 h on phosphate buffered saline (PBS) at pH 7.4. 100 mg of gums were placed in a dialysis bag and suspended in 20 mL of PBS at 37°C under slow magnetic agitation at 100 rpm. The dialysis bag is made up of cellulose membrane (mw cut off 12.400) and pre-treated with PBS solution over night with mild agitation. After the pre-treatment, the dialysis bag with 5 mL capacity was used for the experiment. 20 mL of phosphate buffered saline was used as dissolution medium used for drug release analysis. 4mL of medium (PBS) was withdrawn periodically and replaced by equal volume of fresh medium. The amount of montelukast sodium and ibuprofen released was measured by UV- spectrophotometer. All these studies were repeated triplicate and the releasing ability of M. oleifera plant resin was analyzed in the same way. The cumulative drug release (CR) can be calculated by CR% = (Mt/Mi)/100,

(1)

where Mi is the initial concentration of drug and Mt is the amount of drug released from the silver nanoparticles incorporated with plant gum at time t respectively.

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FTIR Analysis

Absorbance 4

3

489 nm

2

1 400

500

600

700 800 Wavelength, nm

Fig. 1. UV–Vis spectrum of bio reduced silver nanoparticles by Clerodendron phlomoides.

RESULTS AND DISCUSSION UV-Spectrophotoscopy In this analysis, the light absorbed and scattered by the sample in the range of 200 to 800 nm was considered. It was observed that, when 10 mL of plant extract was mixed with 1 mM of AgNO3 solution, the colorless solution changes to brown color within 3 min after the addition. The plant extract contains the flavonoids such as pectolinaringenin, clerodendrin, scutellarein might reduce the AgNO3 into silver nanoparticles. Optical properties and refractive index of plasmonic silver nanoparticles which are sensitive to size, shape, concentration and agglomeration was identified using UV-spectrophotometer. UV-spectrum otherwise known as surface Plasmon resonance absorption band shows different values for individual nanoparticle and agglomerated nanoparticles. If nanoparticles agglomerate, the band will be shifted to red (longer wavelength) when compared to Surface Plasmon absorption band (SPR) of individual nanoparticles [32]. From the obtained results (Fig. 1), it was found the confirmation of silver nanoparticles at 489 nm. The same trend of result was previously reported by Sivakumar et al. [33] for the synthesis of silver nanoparticles using Peltophorum pterocarphum plant in the 390 to 490 nm range. After the addition of plant extract, the colorless silver nitrate solution was change into brown color due to the bioreduction of silver nanoparticles, these silver nanoparticles possess free electrons which gives the SPR band that collective vibration of free electrons in the silver nanoparticles. The synthesized nanoparticles absorb the SPR exactly at 489 nm and the ranges started from 360–550 nm. The wavelength of SPR peaks is inversely proportional to the particle size [34].

Figure 2 shows the FTIR spectrum of both plant extract and silver nanoparticles respectively. FTIR plays a vital role in comprehending the functional groups that were responsible for the biotransformation of AgNO3 into Ag(0) species. FTIR report acts upon to spot out the possible macromolecules accountable for capping and stabilizing the silver nanoparticles. Figure 2 shows the FTIR peaks in the range, such as 3922, 3479, 2097 and 1639 cm–1. The peaks at 3479 and 1639 cm–1 represents the presence of N–H stretching and bending vibrations via amines from the proteins group of the plant extracts. Thirunavoukkarasu et al. reported the same trend of the result for the nanoparticles synthesized from Desmodiumgangetic [35]. Plant extracts that shows characteristic peak at 1639 cm–1 are found to be responsible for NH2 groups in aminoacid. Jagtap and Bapatalready reported the similar type of result for the synthesized nanoparticles from Artocarpus heterophyllus that shows the peak at 1632–1638 cm–1 [36]. The FTIR spectrum at 3922 cm–1 shows the presence of O–H stretching of hydroxyl groups. When compared to FTIR spectrum for plant extract, spectrum for silver nanoparticles were found to be shifted towards higher ranges. Theses O–H groups may come from the polyphenol compounds of the plant which could be used for the reduction of the metal silver ions. This type of shift leads to binding of silver ion with free –NH groups in plant extract interacted with silver nanoparticles for stabilization [37]. The Prominent shift appeared on the FTIR spectrum are presented as 1639–1642 cm–1 (protein groups), 2097–2103 and 3479–3516 cm–1 (amide I group). The peak at 2071 cm–1 shows the representation of C–H stretching in aldehyde group. FTIR Analysis of Raw Gum with Silver Nanoparticles Scheme 1 shows the schematic representation of possibility carbohydrates polymers in M. oleifera gum which contains arabinose, galactose, glucuronic acid with small amount of mannose, xylose and rhamnose. Scheme 2 shows the chemical structure of the (a) Ibuprofen and (b) Montelukast sodium and the FTIR spectrum of raw M. oleifera resin and blend of green synthesized silver nanoparticles were observed and are shown in Fig. 3 (curves 1, 2). Peaks were observed at 530, 1020, 1219, 1369, 1601, 1739, 2361, and 3353 cm–1 for raw M. oleifera resin and observed at 527, 1018, 1213, 1367, 1736, 2359, 2970, and 3262 cm–1 for blend of green synthesized silver nanoparticles, respectively. The peak at 1601 cm–1 could be due to the O–H bending of water from the combined vibration stretches of free inter and intra molecular hydroxyl groups in the gross link of carbohydrates of the plant gum [38]. The broad peak recorded at 3353–3262 cm–1 could be a stretching band of O–H. The analogous type of results


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T, % 100 2097

80

2103

3922

1642

1 2

3904

1639

60

40 3516

20 3479

0

1000

4000 3000 Wavenumber, cm−1

2000

Fig. 2. (1) FTIR vibration of plant extract and (2) green synthesized silver nanoparticles.

tose, and leucoanthocyanin [40, 41]. M. oleifera gum which comes under glucuronoarabino-galactan type, when gets hydrolyzed by autoclave, will give polyhydroxylated reducing aldose sugars like arabinose, galactose, glucoronic acid and trace amount of rhamnose. The vibrations at 530–527 represented the Ag(0) metal stretching in the plant resin. When compared to the band vibrations of raw plant resin, silver nanoparticles incorporated with plant resins were slightly shifted. Similar sort of result was previously reported for the AgNps synthesized from M. oleifera gum [42].

was previously reported by Martins Emeje et al., for the natural plant gum from Cissusrefescence [39]. The band at 2967 cm–1 represented the presence of symmetry and asymmetry stretching vibration of CH3 (methylene) group. The sharp peak found at 1739 cm–1 could be carbonyl stretching of aldehyde, ketone and carboxylic groups present in the polysaccharides. The vibration at 2932 cm–1 was due to the presence of carbonyl group in the aldehyde and the presence of bands at 1020–1018 cm–1 was due to the C–N stretching. This gum exudates contain D-glucuronic acid, L-rhamnose, D-mannose, D-xylose, L-arabinose, D-galac-

H OH

O H OH

H

H

COOH

CH2OH O

O

O

OH

1

H

H H OH Arabinose

O

4

O

OH H

H (n)

OH Glucoronic acid

OH Galactose Scheme 1.

(a)

(b)

CH3 OH

CH3

Cl

O

H3C

N HO H3C H3 C

Scheme 2. POLYMER SCIENCE, SERIES B

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T, %

4

965 677 760 836

1068 862

1070

1420

1602

3759

2363

2931

2361

1232 1420

3867

1708 2954

527

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3436

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2359

1018 1213 1367

2970

1736

3262

1 530

1020 1219 1369

1000

1601

2361

1739

3353

2967

2000

3000

4000 Wavenumber, cm−1

Fig. 3. FTIR absorption of Moringa oleifera gum and along with silver nanoparticles incorporated gum and AgNps blended gum with ibuprofen and montelukast sodium. (1) Moringa oleifera gum; (2) silver nanoparticles blended Moringa oleifera gum; (3) MOgum+AgNps+Ibuprofen; (4) MOgum+AgNps+Montelukast sodium.

(a)

(b) Ag

1000 800 600

Ag

400 O

200 5 μm

0

2

4

6

8

10 V, keV

Fig. 4. (a) SEM analysis of green synthesized nanosilver. (b). Elemental composition of silver nanoparticles.

Ibuprofen coated silver nanoparticles blended with M. Oleifera gum observed FTIR spectrum at 1070, 1232, 1420, 1708, 2361, 2954, 3448, 3867 cm–1 and these above band shown the presence of Ibuprofen in gum but it does not alter the functional groups of the gum (Fig. 3 (curve 3)). The Ibuprofen FTIR band possesses an intense defined band at 1708 cm–1 attributed to the stretching of carboxyl acid group (COOH) C=O group of the standard ibuprofen [43]. Other smaller peaks are in the region 1232–1070 cm–1 contributes from the aromatic benzene ring (Fig. 4a) [44]. The silver nanoparticles coated with Montelukast sodium blended with M. oleifera gum absorbed

the FTIR bands at 677, 760, 836, 862, 965, 1068, 1420, 1608, 2363, 2931, 3436, 3759 cm–1 shown in Fig. 3 (curve 4). The stretches were at 677, 760, 836 cm–1 shown the aromatic C–H vibration of the montelukast sodium compounds [45]. The band at 2363, 2931 cm–1 denotes the methyl groups in the drug and the band at 1608 cm–1 represents the carbonyl group of the montelukast sodium (Fig. 3 (curve 2)). SEM with EDX Analysis The SEM images shown in Fig. 4a represent the structure and morphology of the green synthesized sil-


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Intensity (311) 60

40 (220)

(100) (111)

20

0 10

20

30

40

50

60

70 2θ

Fig. 5. X-Ray diffraction analysis for silver nanoparticles.

ver nanoparticles. From the SEM result, the spherical and ovoid structure of nano silver was confirmed. This similar agreement was previously reported by Sivakumar et al. [33] for the nanoparticles synthesized from Peltophorum pterocarphum plant. From the EDAX results, the elemental compositions of green synthesized nano silver were observed. In this present investigation, silver nanoparticles synthesized yield was found to be 28.7%. When compared to Ag and O, the presence of other elements such as C and N were found in negligible amount, so it was not taken into account. The composition of C and N present in EDAX were found to be too less when compared Ag and O. So the composition of C and N could not be shown in the EDAX result. Figure 4a shows the SEM image of silver nanoparticles and Figure 4b shows the EDAX of silver nanoparticles. Table 1 shows the elemental composition of silver nanoparticles. XRD Analysis The crystalline nature of the silver nanoparticles was confirmed by XRD analysis. The 2θ values of the freeze-dried AgNPs were found to be 27.9°, 32.8°, 38.14° and 46.2°, which corresponds to (220), (311), (111) and (100) miller indices plane of pure silver. JCPDS file no. 01-1167 matched with these 2θ values of XRD. Figure 5 shows the XRD value of silver nanoparticle. The crystalline size of AgNPs was deterTable 1. Quantitative analysis for silver nanoparticles Element O Ag Total

Net counts

Weight, %

Atom, %

4462

29.06

73.42

29051

70.94 100.00

26.58 100.00


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mined as 2 nm using XRD analysis. Less intensity peaks were observed in the area other than AgNPs, this may be obtained due to the presence of organic macromolecules in the plant Clerodendron phlomoides. This type of XRD results were previously reported by [37, 46] where it was shown the similar type of XRD pattern for green synthesized silver nanoparticles with Face-centered cubic structure. The 2θ value observed at 13 represents the organic macromolecule present on the silver nanoparticles [47]. From the XRD value, it could be confirmed that the silver nanoparticles were bioreduced by Clerodendron phlomoides plant extract effectively in crystalline nature. The nano crystalline size of the silver particles was determined by Scherrer’s formulae [48] D = kλ/βcosθ, where D is the particle diameter, k is constant equal to 1, λ is wavelength of X-ray light (0.1541 nm) and beta is (FWHM) full width half maximum of high intensity peak respectively. Theta is diffraction angle equal to lattice plane (hkl) value (111). From the Scherrer’s equation, the average crystal size was found to be 4 nm. TEM Analysis The TEM image shows a perfect morphology and distribution of AgNPs size. Figure 6a shows the TEM image of silver nanoparticles. The spherical shaped Ag(0) NPs were observed from TEM image. Figure 6 shows the small and monodispersed nano sized spherical shaped silver nanoparticles. The particle size was obtained as 14.1 ± 51 nm. A variation in the AgNPs size depends upon the particle aggregation during the sample preparation [49]. Figures 6c, 6d denote the TEM images of Ibuprofen and montelukast sodium coated silver nanoparticles respectively. The TEM images imply that the drugs that are coating AgNps do not change the size and morphology of the nanoparticles much when compared to as-prepared silver

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Total counts 500000

(b)

300000 100000 −100 100 nm

(а)

100 nm

(c)

0 100 200 Apparent zeta potential, mV

100 nm

(d)

Fig. 6. (a) TEM image of the green synthesized nanosilver. (b) Zeta potential values of silver nanoparticles. (c) TEM image of Ibuprofen coated AgNps, (d) TEM image of Montelukast sodium coated AgNps.

nanoparticles. The size of Ibuprofen and montelukast sodium coated AgNps were in the ranges of 50 ± 60 nm and the 75 ± 90 nm respectively. Zetapotential Analysis Figure 6b shows zeta potential for biologically reduced AgNPs. Zeta potential is an important instrument to analyze the stability and state of silver nanoparticle surface. Nanoparticles with zeta potential values greater than +25 mV or less than –25 mV typically have high degrees of stability. The zetapotential value of the nano particle highly depends upon the pH and electrolyte concentration [50]. In our study, AgNPs have shown the negative zeta potential value. The zeta potential value was –6.02 mV. From the analysis, it was observed that the green synthesized nanoparticles were found to be stable at –6.02 mV. Antibacterial Mechanism of Silver Nanoparticles Figure 7 shows the antibacterial effect of silver nanoparticles against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli. Antimicrobial properties of the nanoparticles can be achieved by the following methods such as Diffusion (Kirby-Bauer and Stokes), dilution (Minimal Inhibitory Concentration), diffusion and dilution (E-test). According to CLSI (Clinical and Laboratory Standards Institute) standard recommendation, the Kirby-Bauer and Stokes methods are employed to show the antibacte-

rial properties of bioactive molecules [51, 52]. Silver has a very good antibacterial property and silver formulated compounds are highly toxic for microorganisms at lower concentration. The bactericidal susceptibility of AgNPs is due to the plasmolysis of bacterial cell or separation of cell organelles from the cell wall [53], There are many possible mechanisms that were available for the bactericidal action of colloidal nanosilver such as (1) disturbance in the cell wall synthesis, (2) interaction in the protein synthesis, (3) intrusion with nucleic acid synthesis and (4) disturbance in the metabolic pathway [53]. Table 2 shows the zone of inhibition against the bacterial species with various dilution concentrations such as 25, 50, 75, and 100 μL. Gentamicin was used as positive control which was noted in Fig. 4. Water control did not give any zone of inhibition. Maximum zone of inhibition was found to be in gram positive Staphylococcus aureus species at minimum doses of 75 μL of AgNPs. The bactericidal effect of silver nanoparticles was dose-dependent and was more obvious against gram positive bacteria. According to the results, bio reduced silver nanoparticles have been found to have great antibacterial activity on both gram positive and gram negative bacteria. Thermal Behavior of Plant Gum Figure 8 has shown thermal degradation properties of raw M. oleifera gum and raw gum with silver nanoparticles. Thermal degradation properties of M. oleifera gum were analyzed by thermo gravimetric


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(а)

(b)

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(c)

Fig. 7. Bactericidal effect of silver nanoparticles by disc diffusion method for (a) Staphylococcus aureus, (b) Klebsiellapneumoniae, (c) Escherichia coli and Gentamicin used as reference drug and water used as a negative control.

Loss of mass, % 100

80

60 2

40 1 20 0

200

400

600

800 Temp., °C

Fig. 8. TGA results for the (1) Moringa oleifera resin and (2) resin with silver nanoparticles.

analysis in which 2.679 mg samples were taken in an alumina crucible under nitrogen atmosphere. Samples were analyzed at a heating rate of 20 grad/min. The result shows that 10% mass loss was observed around 100°C (Fig. 8a). This mass loss is due to the removal of moisture content at initial decomposition temperature. Polysaccharides decay consists of 4 phases which depends on the decay pattern of polysaccharide. As a result of Dehydration, depolymerisation of gum took place. CO, CO2 and H2O present in the gum were evaporated due to the breakage of C–O and C–C bonds present in the ring. Aromatic and graphic car-

bon structure was formed as a result of C–O and C–C bonds breakage [54]. The AgNPs impregnated with gum sample had good thermal stability when compared with raw gum due to the synergistic effect of silver nanoparticles. Due to the presence of phosphorous in the raw gum (4.34%) w/w, thermal stability of the raw gum along with nanoparticles was found to increase further [55–57]. Totally 76.7 and 78.8% mass loss was observed in raw gum and Ag nanoparticles impregnated with plant gum in the temperature range from 30 to 900°C respectively. For raw gum and gum with AgNPs, mass loss was observed at 59.1 and 48.9%

Table 2. Antibacterial analysis of silver nanoparticles against few pathogenic bacteria Zone of inhibition for biosynthesized AgNp from Clerodendron Phlomoides against bacterial species, mm. Name of the bacteria’s Staphylococcus aureus Klebsiella pneumoniae Escherichia coli

Water control

Gentamicin

3 3 3

27 ± 2 27 ± 3 30 ± 1


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22 ± 2 21 ± 1 21 ± 1

24 ± 1 21 ± 1 22 ± 1

24 ± 2 20 ± 2 23 ± 1

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60 40 20 0

15

100

30

45

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1 2

80 60 40 20 0

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80 Time, h

Fig. 9. Drug releasing profile of silver nanoparticles blended Moringa oleifera resin. (a) Drug releasing profiles of (1) Moringa resin with montelukast sodium, (2) silver nanoparticles blended Moringa resin with and montelukast sodium drug; (b) Ibuprofen with (1) raw Moringa resin, (2) silver nanoparticles blended Moringa resin with Ibuprofen drug.

respectively in the range from 100 to 400°C due to the decomposition of carbohydrates. 10.2% of difference in mass loss was recorded in gum when compared to raw gum. This means that silver nanoparticles impregnated gum has good thermal stability (Fig. 8b). In vitro Drug Release 100 mg of raw gum and raw gum with silver nanoparticles were soaked in 10 mg of Montelukast sodium and 10 mg of Ibuprofen drugs separately along with 4 mL of methanol for 10 h to determine the drug loading capacity of the raw gum. After 10 h, the experimental solutions were taken for UV–Vis spectrometry analysis. The same procedure was followed for the raw gum with silver nanoparticles. From the UV analysis result, it was observed that 65.7% of montelukast sodium was found to be absorbed in raw gum and 80% of the same drug was found to be absorbed in raw gum with silver nanoparticles. 60% of Ibuprofen was found to be absorbed in raw gum and 70% was found to be absorbed in raw gum with silver nanoparticles. In vitro drug release capacity of raw gum and raw gum with AgNps are shown in Figs. 9a, 9b. There are two mechanisms behind the drug/bioactive materials release from the films such as usual diffusion through the polymeric network pores because of the density gradient and release by the rupture of the films. From Figs. 9a, 9b, the release of montelukast sodium (Fig. 9a, curves 1, 2) and ibuprofen from both gums

was indicated with various time intervals. It revealed the release of Montelukast sodium and ibuprofen from the both gums. In the case of raw gum, 100% of montelukast sodium was observed to be released within 24 h and for AgNPs incorporated with raw gum, 70% of the same drug was observed within 72 h. Raw gum initially showed fast release due to few factors, they are (1) diffusion of free drugs on the raw gum, (2) charges of the drug and polymer or presence of salts in PBS and (3) pH 7.4 may reduce the charge state of the raw gum and drugs and makes the fast release possible [58]. Ibuprofen in raw gum was found to be observed as 96% of release at 18 h duration and 80% of the same drug release was observed for the silver nanoparticles along with raw gum within 72 h (Fig. 9b, curves 1, 2). It is observed that the AgNPs incorporated gum shows retardant release than raw gum. Silver nanoparticles are zerovalent, hence the drugs molecules could adsorb on the surface of the silver nanoparticles and there were no strong interaction between the nanoparticles and drugs and the gum slowly release the drugs to the target site. AgNps have the ability to penetrate the blood small capillaries and can be excreted by liver and kidneys because of this properties, possibility of delivering of nanomedicine to the target tissue. AgNPs do not show considerable cytotoxicity so we can use it in effective drug delivery [59]. Silver nanoparticles may carry the drugs to the liver and there it can accumulate and deliver the bioactive molecules to the liver cells by cellular engulf mechanism. Silver nanoparti-


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cles of the small size of 50 nm can more easily penetrate the cell than nanoparticles of the other size. In this study, gum was used as the carrier for the drug release. The nanoparticles were used to analyze the delay and sustained release of the drug. CONCLUSIONS In the present investigation, Silver nanoparticles (AgNPs) were green synthesized using novel medicinal plant Clerodendron Phlomoides and analyzed using UV spectroscopy, FTIR, SEM, TEM and XRD. The AgNPs was found to be 51 nm in size with spherical shape and cubical structure. The antibacterial study was established for the green synthesized AgNPs using various bacterial species such as Staphylococcus aureus, Klebsiella pneumoniae and E. coli. Synthesized AgNPs were incorporated in M. oleifera gum for drug release study. Thermal stability was found to be more in AgNPs incorporated with raw gum when compared to raw M. oleifera gum. Drug delivery study was conducted for the drugs montelukast sodium and Ibuprofen using the raw gum and raw gum incorporated with AgNPs. The absorption capacity and drug releasing capacity of montelukast sodium using both raw gum and raw gum with AgNPs were found to be more when compared with Ibuprofen. The absorption capacity and sustained drug releasing capacity of both drugs using raw gum with AgNPs was found to be more when compared with raw gum. With this present investigation, medicinal plant was used for the synthesis of AgNPs and plant gum M. oleifera was used along with green synthesized silver nanoparticles for sustained drug releasing applications. ACKNOWLEDGMENTS We would like to express our sincere thanks for ACRF (Anna Centenary Research Fellowship), Anna University Chennai-600 025. REFERENCES 1. The National Nanotechnology Initiative: Research and Development Leading to a Revolution in Technology and Industry (National Science and Technology Council Committee on Technology, Washington (DC), Office of Science and Technology Policy, 2005). 2. D. J. Brayden, Drug. Discovery Today 8, 976 (2003). 3. J. L. Au, S. H. Jang, J. Zheng, C. T. Chen, S. Song, L. Hu, and M. G. Wientjes, J. Controlled Release 74, 31 (2001). 4. G. J. Fetterly and R. M. Straubinger, AAPS PharmSci 5 (4), E32 (2003). 5. D. Hoarau, P. Delmas, S. David, E. Roux, and J. C. Leroux, Pharm. Res. 21, 1789 (2004). 6. S. M. Moghimi and J. Szebeni, Prog. Lipid Res. 42, 478 (2003). 7. R. Langer, Science 249, 1527 (1990). POLYMER SCIENCE, SERIES B

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