J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
© Universiti Sultan Zainal Abidin ISSN 1985-5133 (Press)
ISSN 2180-1983 (Online)
Razali et al.
Assessment of Biosynthesis of Silver Nanoparticles
from Punica granum (Peels and Arils)
Assessment of Biosynthesis of Silver Nanoparticles from Punica granum (Peels and Arils)
Razifah Mohd Razali, Siti Aliyah Mohd Ismail and Fatimah Hashim School of Fundamental Science,
Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia
Corresponding author: Razifah Mohd Razali School of Fundamental Science,
Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia Corresponding author: Email: razifah@umt.edu.my
Keywords:
Antioxidant Antiamoebic Pomegranate Silver nanoparticles Cytotoxicity effect
2/ J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
ABSTRACT
In this study, a simple and eco-friendly biosynthesis of silver nanoparticles from peels and arils extract of Punica granatum (pomegranate) has been designed, which act as a reducing agent in AgNO3 solution. The formation of silver nanoparticles was spectroscopically characterized by UV-Vis spectroscopy analysis and results exhibited an absorbance spectrum peak between the range of 282-432 nm within 24 hours, when centred on the surface of silver nanoparticles. Further investigation on antioxidant activity via DPPH assay showed higher scavenging effect at 84.06% in AgNPs synthesized from peels extract compared to 80.16% for AgNPs synthesized from arils.
Both AgNPs also exhibited antibacterial activity against two species of Gram positive bacteria (Bacillus subtilis, Staphylococcus aureus) and two species of Gram negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa). These AgNPs were also assayed for their antiamoebic properties towards Acanthamoeba sp. in order to determine their cytotoxicity effect and the IC50 values obtained were 33.08% v/v and 2.33% v/v for peels and arils extracts respectively. These results indicate that both arils and peels of P. granatum have great potential for the synthesis of AgNPs that have antioxidant, antibacterial and antiamoebic activities.
Keywords: Antioxidant, antiamoebic, pomegranate, silver nanoparticles, Cytotoxicity effect
ABSTRAK
Di dalam kajian ini, satu biosintesis ringkas dan mesra alam melalui zarah perak berskala nano daripada ekstrak kulit dan biji buah delima (Punica granatum) telah direka, yang mana bertindak sebagai agen pengurangan di dalam larutan AgNO3. Pembentukan zarah perak berskala nano di kategorikan oleh analisis spektroskopi UV-Vis dan keputusan yang dipamerkan merupakan penyerapan puncak spektrum di antara 282-432 nm selama 24 jam apabila zarah perak berskala nano di ketengahkan. Siasatan lanjut ke atas aktiviti anti-oksida melalui DPPH esei menunjukkan kesan peningkatan yang lebih tinggi pada 84.06% dalam sintesis AgNPs daripada ekstrak kulit buah delima berbanding 80.16% dalam ekstrak biji buah delima. Kedua-dua sintesis AgNPs juga menunjukkan tindakan aktiviti anti bakteria dalam dua spesies bakteria bergram positif (Bacillus subtilis, Staphylococcus aureus) dan dua spesies bakteria bergram negatif (Klebsiella pneumoniae, Pseudomonas aeruginosa). Sintesis AgNPs ini juga menunjukkan keupayaan anti- ameobik terhadap Acanthamoeba sp. di dalam menentukan kesan sitotoksisiti dan nilai IC50 di perolehi daripada 33.08% v/v melalui ekstrak kulit dan 2.33% v/v melalui ekstrak biji. Kesemua keputusan yang diperolehi mempamerkan indikasi yang ke dua-dua kulit dan biji buah delima (P. granatum) mempunyai potensi yang besar ke atas aktiviti anti-oksida, anti-bakteria dan anti-amoebik melalui sintesis AgNPs.
Kata Kunci: Antioksidan, antiamoebic, delima, nanopartikel perak, kesan sitotoksik
INTRODUCTION
The field of nanotechnology is one of the areas of research that is being actively researched globally. One aspect of nanotechnology is the production of nanoparticles such as silver nanoparticles (AgNPs), which is typically conducted using a variety of chemical and physical methods. Residues that were produced after the synthesizing process are technically very harmful and energy consuming, thus, would lead to devastating effect to our environment. Due to these problems, researchers are investigating new biological approaches for the synthesis of metal nanoparticles as a reducing agent and a stabilizer in silver nitrate solution.
Silver nanoparticles (AgNPs) have high potential for the treatment of many parasites and diseases due to their antibacterial, antiplasmodial and antifungal activity and also for drug delivery. The biological synthesis of silver nanoparticles using natural plant extracts has gained attention due to it being more eco-friendly, low cost, and generate no hazardous waste. Different parts of plants such as stem, leaf, fruit, bark and seed extracts have been successfully utilized to synthesize AgNPs (Yugal et al., 2017). Biological synthesis of AgNPs require less energy, no toxic chemicals, lower temperatures and pressure (Fakhra and Shilpa, 2014). Lately, several researchers had tried to use microorganisms to produce nanoparticles but the rate of reaction for the solution to
3/ Razali et al.
form nanoparticles was much slower than using plants as the mediated source or reducing agent in silver nitrate solution (Ahmed, 2016). Nevertheless, there is a lack of research on using peels and arils of Punica granatum (pomegranate) fruit as a source to synthesize AgNPs and to characterize its antimicrobial activities.
Pomegranate (P. granatum) is a native plant from the Mediterranean region and is widely used for medicinal purposes in Asia and other countries around the world. It belongs to family of Punicacea and is the most significant horticultural member of this family (Solgi and Taghizadeh, 2012). The fruit has been used to treat stomach ache, fever, diarrhoea, inflammation, bronchitis and malaria. Furthermore, the fruit contains rich secondary metabolites such as flavonoids, anthocyanidins, crude fiber, pectin, estrogenic compound, sugars, polyphenols and several tannins (Akbarpour et al., 2009; Gil et al., 2000; Mori-Okamoto et al., 2004 as cited in Solgi and Taghizadeh, 2012). P. granatum was also reported as a nutritional, medicinal fruit due to its antibacterial, antihepatotoxic, antioxidant, antilipoperoxidative and antitumor properties (Bopitiya and Madhujith, 2012).
In this study, a biological synthesis route to produce silver nanoparticles (AgNPs) at room temperature by using P. granatum arils and peels extracts as a reducing agent in silver nitrate solution (AgNO3) was developed.
Furthermore, the antioxidant and the antibacterial activities of the AgNPs against four bacterial pathogens were evaluated. Lastly, inhibition of amoeba population using AgNPs was also accessed.
MATERIALS AND METHODS
Arils and peel water extraction of Punica granatum
Extraction of P. granatum was carried out using the method of AlSalhi et al. (2016). Pomegranate fruits were obtained from the local market and washed 10 times with distilled water to remove any dirt and contamination on the surface. Then, the peels and arils of the fruit were removed and were spread evenly on aluminium foil with the peels being cut into small portions to increase its surface area and to dry faster. Both peels and arils were oven-dried for two days at 31°C. After the peels were completely dried, they were ground to a powdered form using liquid nitrogen then quickly weighed because they became sticky even after drying. Next, the peels powder was filtered to separate the large clumps in order to obtain a smooth powder. Then, 10 g of each powder was precisely weighed and soaked in 250 mL of water for 24 hours. The soak water was then filtered using Whatman No.1 filter paper to produce a stock solution and stored at 4oC until further analysis.
Synthesis of AgNPs
A volume of 2.5 mL of each arils and peels stock solution were mixed with 250 mL of prepared AgNO3 (3 mM) solution in different conical flasks for the synthesis of AgNPs. Both solutions were then left at room temperature for 24 hours until there was a change of color from colorless to brown, which indicates the reduction of AgNO3.
In order to remove the excess silver ions, the solutions were then centrifuged at 4,000 rpm for 25 minutes. This centrifugation step was repeated five times in order to fully remove the excess silver colloids from the base of the centrifuge tube. Finally, the clear silver nanoparticles solutions were transferred into another tube in order to separate it from the silver residues.
Ultraviolet-visible (UV-VIS) spectroscopy
The reduction of Ag+ ions was monitored using a UV visible spectrophotometer (UV 1800, Shimadzu, Japan) with a scanning range of 200-800 nm. UV spectra for both arils and peels AgNPs solutions were recorded with periodic sampling, from the initial colourless color until the final brown color for 24 hours.
Determination of antioxidant activity DPPH assay
The DPPH assay was carried out based on the method described by Subbaiya et al. (2014) with modifications.
Exactly 50 µL of original arils and peels samples (without AgNO3) and AgNPs arils and peels samples (with AgNO3) were placed in test tubes in 3 replicates. Then, 6 mL of methanol were added to each tube and then incubated in a water bath for 10 minutes at 31oC. Subsequently, 1 mL of the each mixture was placed in 3 mL of
4/ J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
prepared DPPH solution and incubated in a room temperature for 30 minutes. Finally, the absorbance of each solution was monitored using UV visible spectroscopy at 517 nm wavelength. The radical scavenging activity was calculated using the following formula:
DPPH radical scavenging activity (%) = x100
) absorbance (Control
)) absorbance (Sample
) absorbance
((Control −
Antibacterial effect
The antibacterial assay was carried out on two species of Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and two species of Gram-negative bacteria (Klebsiella pneumoniae and Pseudomonas aeruginosa) by using disc diffusion method. Briefly, each bacterium was grown in nutrient broth and incubated in a shaker for 24 hours at 150 rpm at 37°C. The cultures were then centrifuged the bacteria were re-suspended in 5 mL of sterile distilled water. The suspension was adjusted to equalize the tube to 0.5 McFarland turbidity standard which equals to 1.5
× 108 colony forming units (CFU) per mL. Then, the bacterial cultures were inoculated in a lawn on prepared nutrient agar plates by using sterile cotton swabs. Sterile discs (5 mm in diameter, Whatman No. 1) which were impregnated with 10 µL of synthesized AgNPs from pomegranate peels and arils (with AgNO3) as well as peels and arils original extract (without AgNO3) were transferred onto each plate by using sterilized forceps. The antibiotic streptomycin was used as a positive control (10 µg/disc) and silver nitrate (AgNO3) as a negative control. The prepared plates of each bacterium with 3 replicates were incubated at 37oC for 24 hours. The diameter of the zone of inhibition of each disc on each plate was measured using a ruler.
Antiamoebic Effect
Determination of IC50 value of synthesized AgNPs from Punica granatum on Acanthamoeba sp. by using MTT assay
The MTT assay was performed according to Mossman (1983). 96-well plates were used to seed 40 µL of Acanthamoeba sp. suspension cell from Setiu wetland isolate (5cells/mL) which contained 80 µL of peptone- yeast-glucose (PYG) media. After 24 hours of incubation, the Acanthamoeba culture was drawn out from each well followed by seeding of 120 µL of different concentrations (3.125-100% v/v) of two dilutions factor peels and arils AgNPs (with AgNO3)and original extract (without AgNO3) in the 96-well with three replicates of each extraction. The treatment was then incubated for 24 hours. MTT powder was dissolved in PBS at 5 mg/mL for preparation of MTT stock solution. The next day, each well was emptied and 30 µL of MTT stock solution were seeded following incubation for 4 hours. Then, 100 µL of DMSO was added into the wells which contained MTT solutions and resuspended to ensure that all of the formazon blue crystals were dissolved. The mixtures were then incubated for another 30 minutes. To determine IC50 value, the 96-well plate was measured using a Dynatech MR580 MicroElisa reader at 570 nm.
The %absorbance was calculated using the following formula:
100 ) x
absorbance (Control
)) absorbance (Sample
) absorbance
((Control −
Statistical analysis
The data were analyzed using two tailed t-test to compare the activities between two solutions of original peels and arils extract of P. granatumas control with arils and peels synthesized AgNPs. Values were considered significantly different if the probability (p) is less than 0.05.
5/ Razali et al.
RESULTS AND DISCUSSION
Synthesis of silver nanoparticles from Punica granatum
The present study reports the biosynthesis of silver nanoparticles by using natural resources from the fruit of P.
granatum which acts as a reducing agent in silver nitrate solution (AgNO3). There are many chemical and physical methods of synthesizing nanoparticles, which utilise not only hazardous and toxic chemicals but are also very costly (Ali et al., 2016). Residues that were produced after the synthesizing process are very harmful thus, would lead to a devastating effect to our environment. Recently, the usage of plants in synthesizing AgNPs is becoming popular due to it being a safer and simpler approach.
The results revealed that the method used in this study allowed for cheap and reliable formation of AgNPs within 24 hours with no energy inputs or highly toxic chemicals were required. AgNPs were successfully synthesized from mixing aqueous silver nitrate with a small volume of peels and arils water extractions from pomegranate. This was proven by changes in colour of the silver nitrate solution, from colourless to a dark brownish suspension (Fig. 1A) after several hours of incubation with the peels extracts. Similar colour changes of AgNO3 solution were seen with arils extraction colour as shown in Figure 2(A). In the meantime, the control, original brown extract of peels and light pink extract of arils from P. granatum demonstrated no change in colour after 24 hours (Fig. 1B & Fig. 2B). This may be due to the higher amount of total phenolic and flavonoid compounds in the pomegranate peels than in arils, which lead to the excitation of surface plasmon vibrations in silver nanoparticles during the reaction period (Veerasamy et al., 2011 as cited in Ahmed et al., 2015). These particular changes of colour indicate that the formation of silver nanoparticles has occurred (Singh et al., 2010).
Figure 1 Biosynthesis of silver nanoparticles using the aqueous extract of P. granatum peels: (A) Peels extract post reaction with AgNO3; (B) P. granatum peels extract alone.
Figure 2 Biosynthesis of silver nanoparticles using the aqueous extract of P. granatum arils: (A) Arils extract postreaction with AgNO3 ; (B) P. granatum arils extract alone.
6/ J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
UV-Vis spectroscopy
UV-vis spectroscopy has been widely used in many studies to distinguish and detect the presence of AgNPs during biosynthesis. In particular, absorbance between the ranges of 420 nm - 450 nm has been used as an indicator to verify the reduction of Ag+ ion into metallic Ag. In this study, UV-Vis spectroscopy had been used to monitor the formation of AgNPs synthesized from peels and arils of P. granatum within 24 hours at room temperature (Fig. 3). The intensity of absorbance increases over time which indicates an escalating formation of AgNPs produced from both mixtures. In the peels mixture of AgNO3, there was a strong absorbance (Fig. 3; (I) A = 3.93) which peaked at 424 nm whereas in the arils mixture, the highest absorbance observed (Figure 3 (J) A
= 0.11) was at 420 nm in the spectra recorded within a time interval of 24 hours. Both peaks correspond to the surface plasmon resonance of AgNPs. The observations reveal that the increased of absorbance in those wavelengths was due to the formation of colloidal AgNPs (Ahmad et al., 2002).
The spectra reading revealed that the concentration of relative intensity ratios for peels AgNPs and arils AgNPswere increased from 0.92 to 3.93 and from 0.02 to 0.11 respectively. This clearly shows that P. granatum peels and arils extracts in the reaction medium have successfully interacted with the silver nitrate particles.
Mansouri and Ghauder, (2009) as cited in AlSalhi et al. (2016) had reported that absorption bands around 420 nm - 430 nm indicates to the spherical shape of AgNPs. Ultimately, the intensity increased and the peak became sharper due to the increase in nanoparticles produced in proportion to the reduction of Ag+ ions in the solution.
A1
Spectrum -
2.0 0.2 - 3.5
- 0.9
- 1.8 1.2
- 1.8 1.9
-
1.7 2.0 0.6 1.3 1.2 1.3 0.2 0 0 Nm 255 255 265 270 271 276 280 281 286 291 348 404 419 440 677 800 1000 A1: AgNPs (Peels) 1 hour (426 nm = 0.92)
-4 -3 -2 -1 0 1 2 3
0 200 400 600 800 1000 1200
Spectrum
nm
7/ Razali et al.
A2
Spectrum -2.9 -0.1 -0.5 0 -2.3 -0.4 -2.5 0.1 -2.1 0 -1.4 0.6 -0.4 0.1 0 0 0 nm 214 249 249 260 260 272 272 283 283 295 306 312 312 318 500 800 900 A2: AgNPs (Arils) 1 hours (404 nm = 0.02)
B1
Spectrum -1.7 0.6 -1.8 1.6 0.4 1.6 0.7 2.7 1.9 1.5 0.6 1.5 1.4 1.6 0.1 0 nm 262 273 275 293 298 314 319 334 336 349 365 427 447 468 685 800 B1: AgNPs (Peels) 2 hours (426 nm=1.45)
-3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1
0 100 200 300 400 500 600 700 800 900 1000
Spectrum
nm
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3
0 100 200 300 400 500 600 700 800 900
Spectrum
nm
8/ J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
B2
Spectrum -2.9 0 0.5 0.1 -2.5 -0.4 -2.7 0.2 -2 0 -1.1 0.5 -0.2 0.1 0 0 0 0 nm 214 226 237 249 249 260 260 273 273 284 284 290 295 295 366 400 800 900 B2: AgNPs (Arils) 2 hours (422 nm=0.03)
C1
Spectrum -2.2 -0.6 -2.5 0.6 -2.8 0 -1.4 -0.4 -2.4 1.4 -2.7 1.7 -1.4 3.4 0.8 2.0 1 0 nm 226 232 234 237 243 249 266 267 271 275 277 283 283 294 339 429 554 800
C1: AgNPs (Peels) 3 hours (426 nm=1.99) -3.5
-3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1
0 100 200 300 400 500 600 700 800 900 1000
Spectrum
nm
-4 -3 -2 -1 0 1 2 3 4
0 100 200 300 400 500 600 700 800 900 1000
Spectrum
nm
9/ Razali et al.
C2
Spec-
trum -1.2 -2.9 -1.1 -3.3 0.8 -1.8 -0.4 -1.8 1.2 -2.6 1.7 -1.3 0.6 0.1 0 0.1 0.05 0 0.05 0
nm 230 232 237 241 247 252 263 263 274 279 290 300 311 322 338 434 541 600 755 800
C2: AgNPs (Arils) 3 hours (428 nm=0.06)
D1
Spectrum -0.6 -3.4 -0.4 -3.4 -2.4 -3.4 -1.6 -2.8 0.6 -3.2 0 -1.8 3 0.8 2 2.4 0.4 0.4 nm 216 222 232 234 238 245 246 247 257 269 270 287 293 346 398 458 663 894 D1: AgNPs (Peels) 4 hours (428 nm=2.24)
-4 -3 -2 -1 0 1 2
0 200 400 600 800 1000 1200
Spectrum
nm
-4 -3 -2 -1 0 1 2 3 4
0 100 200 300 400 500 600 700 800 900 1000
Spectrum
nm
10/ J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
D2
Spectrum -3.8 -0.4 -3.4 -2 -2.5 -0.2 1.4 -1.6 -0.1 -1.6 0.4 0.1 0.1 0.1 0 0 0 nm 237 257 247 257 268 283 294 299 304 309 320 332 365 575 600 800 900 D2: AgNPs (Arils) 4 hours (432 nm=0.09)
E1
Spec- trum
-2.6 -0.6 -2.8 0.6 -1.2 0.8 -1.8 1.2 1 1.4 -0.7 2.3 -0.6 3.5 1 2.2 4.2 1.8 1 1
nm 224 229 235 235 237 245 255 256 261 266 271 276 287 297 360 381 465 611 769 863
E1: AgNPs (Peels) 24 hours (424 nm=3.93) -5
-4 -3 -2 -1 0 1 2
0 200 400 600 800 1000
Spectrum
nm
-4 -3 -2 -1 0 1 2 3 4 5
0 200 400 600 800 1000
Spectrum
nm
11/ Razali et al.
E2
Spec-
trum -0.65 -2 1.5 -1.3 -0.3 0.3 -0.4 0.5 -2.1 1 -1.5 1 0.3 -1.9 0.1 0.05 0.05 0.1 0.1 0 0 0 0
nm 226 236 247 247 257 262 268 278 288 294 299 304 309 320 330 352 393 424 466 497 521 600 800
E2: AgNPs (Arils) 24 hours(420 nm=0.11)
Figure 3 UV-Vis spectroscopy for monitoring the formation of AgNPs synthesized from peels and arils of P.
granatum within 24 hours at room temperature. A1), B1), C1), D1) and E1): UV-Vis absorbance of AgNPs (Peels) at 1,2,3,4 and 24 hours, respectively. A2), B2), C2), D2) and E2: is UV-Vis absorbance of AgNPs (Arils) at 1,2,3,4 and 24 hours respectively.
Antioxidant activities
The free radical scavenging effect antioxidant activity of AgNPs was investigated via its ability to bleach the stable DPPH radical. The DPPH scavenging activity is widely used and provides a simple assay to measure the ability of the compounds in extracts to act as radical scavengers or hydrogen donors in DPPH solution. In the DPPH assay, when the odd electron of extracts becomes paired in the presence of free radical scavengers, the deep violet DPPH solution will be decolorized to light yellow. The results of the present study are reported in Figure 4, where the control peels extract exhibited the highest radical scavenging effect (98.57%), followed by the peels AgNPs, arils extractand lastly, arils AgNPswith values of 84.06%, 82.18%, and 80.16% respectively. Other study by Murakami et al. (2015) indicated that quercetin effect based on radical scavenging effect is 2.2% at similar concentration of compounds used in this study. Which prove the efficiency of the all extracts from the P.
granatum in antioxidants activities. The higher color change observed in peels extracts may be due to higher amounts of volume and concentrations of secondary metabolites such as gallic acid, ellagic acid, flavonols, flavones, flavanones, anthocyanidins and ellagitannins (Jinnawat et al., 2012) compared to AgNPs peels extract that act as coating agent in silver nitrate. This is in agreement with previous research that also found peels extract possessed higher phenolics and flavanoids to facilitate better antioxidant activity than arils extract (Jinnawat et al., 2012). The 2-tailed t-test analyses of the present study showed that peels extract and AgNPs peels have statistically significant differences (p<0.05). However, for arils extract and AgNPs synthesized by arils, there were no significant differences. Supposedly, there should be a significance difference between these last two compounds but errors made during the experiment could have influenced the results.
-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2
0 200 400 600 800 1000 1200
Spectrum
nm
12/ J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
Figure 4 Peels and arils extracts of P. granatum (control) and synthesized AgNPs shows high scavenging effects on DPPH radicals. Error bar represent standard error.
Antibacterial Activity
Biosynthesized AgNPs were analyzed for antibacterial activity by using disc diffusion method. Table 1 shows that the widest zone of inhibition of peels AgNPs was observed against the Gram- negative bacterium, P.
aeruginosa while the smallest zone of inhibition was observed against B. subtilis. As for arils AgNPs, the widest zone of inhibition was observed against S. aureus and P. aeruginosa with the same diameter of inhibition (Fig. 5).
The special property of nanoparticles that imparts antibacterial activity is the small particles with large surface to volume ratios (Fakhra et al., 2014) which provides better contact with the cell wall of microorganisms (Haytham, 2015 as cited in Shakeel et al., 2016).
The results were compared with streptomycin and AgNO3 as a positive control and peels and arils plant extracts as the control. The results show that peels extract (control) of pomegranate had a large inhibition zone towards K. pneumoniae. Other than that, the control plant extract peels and arils showed no inhibition against other tested bacteria. AgNO3 (3 mM) exhibited considerable antibacterial activity. Peels of pomegranate are rich in tannins, phenols and flavonoids that might be responsible for the inhibitory action against bacteria (Suresh et al., 2011). Flavanoids was stated to inhibit spore germination from plant pathogen (Harborne and Williams, 2000 as cited in Bharani and Namasivayam, 2016). Likewise, flavonoids are formed by plants in retort to microbial infectivity. Phenolic compounds that have a C3 side chain at a lower level of oxidation which also contained no oxygen, also often exhibit antimicrobial activity (Berkada, 1978 as cited in Bharani and Namasivayam, 2016). The specific mechanism behind the antibacterial activity of nanoparticles remain unclear but it might be hypothesized that AgNPs interfere with the cell membrane of bacterial cells and also would combine with mesosome cell organelles in this manner, reducing mesosomal function. However, Nascimento et al., 2000 and Dahham et al., 2010 reported that both peels and arils have antibacterial activity towards all four bacteria. This may due to the low concentration used in the present study, which was at 40 mg/mL with a small volume. The extract might have diffused into the disc completely before the reaction with bacteria occurs.
As for AgNPs, the larger surface area and ultrafine size in particular, helps to terminate the cellular enzymes, while the positively charged Ag+ ions rupture the membrane of bacterial cells (Al Salhi et al., 2016). In addition, the presences of Ag+ ions are capable of denaturing proteins and eventually cause cell fatality (Ali et al., 2016). The antibacterial property of AgNPs might be boosted in the presence of secondary metabolites such as flavonoid, phenolics and tannins.
98.57
82.18 84.06 80.16
0 10 20 30 40 50 60 70 80 90 100
Peels Arils Peels Arils
Scavenging effect %
P. granatum extract
AgNPs synthesized from P.
granatum
13/ Razali et al.
Table 1 Average of inhibition zones of synthesized P. granatum peels and arils nanoparticles.
Inhibition zone diameter (mm) Positive Punica granatum control
Bacteria Peels
extract
Arils extract
AgNPs Peels
AgNPs Arils
Ag(NO3) (3 mM)
Streptomycin (10 g/disc)
S. aureus - - 10.0 11.0 12.0 19.3
B. subtilis - - 9.0 9.2 8.5 19.7
K. pneumoniae 15.0 - 10.0 10.0 10.0 23.0
P. aeruginosa - - 11.5 11.0 10.5 18.7
Figure 5 Anti-bacterial activity of AgNPs synthesized from P. granatum 1) Staphylococcus aureus 2) Bacillus subtilis 3) Klebsiella pneumoniae 4) Pseudomonas aeruginosa a) Peels extract (control) b) Arils extract (control) c) AgNO3 (positive control) d) AgNPs from arils e) AgNPs frompeels f) Streptomycin (positive control).
Antiamoebic activity
Inhibition of 50% of Acanthamoeba sp. population by P. granatum extracts as control and synthesized AgNPs were observed between a concentration ranges of 3.125-100% (v/v). Through the absorbance monitored, it was found that the percentage of amoeba population inhibition increased for all compounds as the concentrations of the compounds increased. Figurd 6 shows the IC50 values of synthesized AgNPs peels and aril from P. granatum and P. granatum extract as control against Acanthamoeba sp. (SW isolate). Differences in IC50 values between control and AgNPs may be due to the P. granatum fruit containing antibacterial properties of secondary metabolites. This means that peels and arils extract have higher antiamoebic activity against Acanthamoeba sp. The results also showed a higher toxicity level of arils synthesized AgNPs towards Acanthamoeba compared to peels synthesized AgNPs. Thus, higher concentrations of P. granatum peels are needed to synthesize AgNPs in order to inhibit the growth of Acanthamoeba sp. (SW isolate) as there will be more particles of secondary metabolites that can coat around the Ag+ ions. Thus, this will increase the efficiency of AgNPs antiamoebic properties.
14/ J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
0 5 0 1 0 0
0 5 0 1 0 0 1 5 0
C o n c e n tr a tio n s o f A r ils e x tr a c t (% v /v )
Percentage viability of cells (%)
a )
0 2 0 4 0 6 0 8 0 1 0 0
0 5 0 1 0 0 1 5 0
C o n c e n tr a tio n s o f P e e ls e x t r a c t (% v /v )
Numbers of viability cells (%)
b )
0 2 0 4 0 6 0 8 0 1 0 0
0 5 0 1 0 0 1 5 0
C o n c e n tra tio n s o f A rils A g N P s (% v /v )
Percentage viability of cells (%)
c )
0 2 0 4 0 6 0 8 0 1 0 0
0 5 0 1 0 0 1 5 0
C o n c e n tr a tio n s o f P e e ls A g N P s (% v /v )
Percentage viability of cells (%)
d )
Figure 6 The IC50 values of (a) arils extract as control (b) peels extract as control (c) synthesized AgNPs from arils (d) synthesized AgNPs from peels against Acanthamoeba sp. after 24 hours treatment.
Table 2 The IC50 values of each compounds obtained from MTT assay
Compounds IC50 value (% v/v)
P.granatum peels extract 2.72
P.granatum arils extract 24.76
AgNPs synthesized from peels extract 33.08
AgNPs synthesized from arils extract 2.33
All of the extracts and synthesized AgNPs used in this study have high anti-amoebic activity against Acanthamoeba with IC50 values for control peels and arils extracts at 2.72 % (v/v) and 24.76 % (v/v) respectively.
For synthesized peels and arils AgNPs, the IC50 values were 33.08 % (v/v) and 2.33 % (v/v) respectively (Table 2). The interactions and mechanisms between AgNPs and amoeba cells are still poorly understood due to lack of research. However, AgNPs are proven to have potential antimicrobial properties against amoeba.
CONCLUSIONS
In this study, pomegranate peels and arils extract play an important role in the stabilization and reduction of silver to silver nanoparticles (AgNPs). The synthesis of AgNPs using plant resources such as Punica granatum, has a high potential as an alternative to conventional methods since biosynthesis provides cheap, eco-friendly, and economic viability to fabricate nanoparticles. UV-Visible spectroscopy was the analytical technique used to detect
15/ Razali et al.
the presence of nanoparticles. The results revealed the presence of silver nanoparticles (AgNPs) in plants with AgNO3 mixture. In addition, the present study found that AgNPs biosynthesized from P. granatum exhibited antioxidant and also antibacterial activity towards Gram-positive and Gram-negative bacteria and this can be further explored for antimicrobial applications. The biosynthesized AgNPs also demonstrate cytotoxicity on Acanthamoeba sp. Most importantly, the biosynthesis reaction was simple to handle. Pomegranate is a safe source for AgNPs synthesis compared to chemical or physical methods and invites further investigation to confirm it.
ACKNOWLEDGEMENTS
The authors wish to express their gratitude to laboratory staff at School of Fundamental Science, Universiti Malaysia Terengganu for their invaluable assistance and hospitability throughout the study.
REFERENCES
Ahmed, S., Ahmad, M., Swami, B. L. & Ikram, S. (2015). Plants Extract Mediated Synthesis of Silver Nanoparticles for Antimicrobial Applications: A Green Expertise. Journal of Advanced Research 7(1): 17-28.
Ahmed, S., Ahmad, M., Swami, B. L. & Ikram, S. (2016). Green synthesis of silver nanoparticles using Azadirachta indica aqueous leaf extract. Journal of Radiation Research and Applied Sciences 9(1): 1-7.
Akbarpour, V., Hemmati, K. & Sharifani, M. (2009). Physical and Chemical Properties of Pomegranate (Punica granatum L.) Fruit in Maturation Stage. American- Eurasian Journal of Agricultural & Environmental Sciences 6: 411-416.
Ali, Z. A., Yahya, R., Sekaran, S. D. & Puteh, R. (2016). Green Synthesis of Silver Nanoparticles Using Apple Extract and Its Antibacterial Properties. Advances in Materials Science and Engineering, 6. article ID 4102196 AlSalhi, M. S., Devanesa, S., Alfuraydi, A. A., Vishnubalaji, R., Munusamy, M. A., Murugan, K., Nicoletti, M. &
Benelli, G. (2016). Green synthesis of silver nanoparticles using Pimpinella anisum seeds:
Antimicrobial Activity and Cytotoxicity on Human Neonatal Skin Stromal Cells and Colon Cancer Cells.
International Journal of Nanomedicine 11: 4439-4449.
Bharani, R. A., & Namasivayam, S. K. R. (2016). Pomegranate (Punica granatum L.) Peel Extract-A Study On Potential Source Of Pharmacological Activities. International Journal of Pharma and Bio Sciences 7(4): 282-290.
Bopitiya, D. & Madhujith, T. (2012). Antioxidant Potential of Pomegranate (Punica Granatum L.) Cultivars Grown in Sri Lanka. Tropical Agricultural Research 24(1): 71-81.
Fakhra, S. & Shilpa, S. (2014). Synthesis of Silver Nanoparticles using Costusspeciosus and study of its anti- microbial properties against urinary tract pathogens. International Journal of Current Microbial and Applied Science 3(1): 248-252.,
Gil, M. I., Tomás, B. F. A., Hess, P. B., Holcroft, D. M. & Kader, A. A. (2000). An Antioxidant Activity of Pomegranate Juice and Its Relationship with Phenolic Composition and Processing. Journal of Agricultural and For Chemistry 48: 4581-4589.
Harborne, J. B. & Williams, C. A. (2000). Advances in flavonoid research since 1992. Phytochemistry 55(6): 481-504.
Jinnawat, M., Korakod, I. & Kanok- Ornintarapichet. (2012). Antioxidant activity and Bioefficiency of Pomegranate peel and seed extract. Global Journal of Pharmacology 6: 131-141.
Mansouri, S. S. & Ghader, S. (2009). Experimental study on effect of different parameters on size and shape of triangular silver nanoparticles prepared by a simple and rapid method in aqueous solution. Arabian Journal of Chemistry 2(1): 47-53.
16/ J. Agrobiotech. Vol. 9 (1), 2018, p. 1–16.
Mittal, A. K., Chisti, Y. & Banerjee, U. C. (2013). Synthesis of metallic nanoparticles using plant extracts.
Biotechnology Advance 31: 346-356.
Mori, O. J., Otawara, H. Y. H. & Hiroyuki Y. Y. (2004). Pomegranate Extract Improves A Depressive State and Bone Properties in Menopausal Syndrome Model Ovariectomized Mice. Journal of Ethnopharmacology 92:
93-101.
Mossman, T. (1983). Rapid Colometric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assay. Journal of Immunological Methods 65(1-2): 701-710.
Mukarami, Y., Kawata, A., Ito, S., Katayama, T. & Fujisawa, S. (2015) Radical-scavenging and anti-inflammatory activity of quercetin and related compounds and their combinations against RAW264.7 cells stimulated with Porphyromonas gingivalis Fimbriae. Relationships between anti-inflammatory activity and quantum chemical parameters. In vivo 29: 55-63.
Nascimento, G. G., Locatelli, J., Freitas, P. C. & Silva, G. L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian Journal of Microbiology, 31(4), 247-256.Dahham, S.
S., Ali, M. N., Tabassum, H., & Khan, M. (2010). Studies on Antibacterial and Antifungal Activity of Pomegranate (Punica granatum L.). American-Eurasian Journal of Agricultural & Environmental Sciences 9(3):
273-281.
Singh, A., Jain, D., Upadhyay, M. K., Khandelwal, N. & Verma, H. N. (2010). Green synthesis of silver nanoparticles using Argemone mexicana leaf extracts and evaluation of their antimicrobial activities.
Digest Journal of Nanomaterials and Biostructure 5(2): 483-489.
Solgi, M. & Taghizadeh, M. (2012). Silver nanoparticles ecofriendly synthesis by two medicinal plants. International Journal of Nanomaterials and Biostructures 2(4): 60-64.
Subbaiya, R., Lavanya, R. S., Selvapriya, K. & Selvam, M. M. (2014). Green Synthesis of Silver Nanoparticles from Phyllanthus amarus and their Antibacterial and Antioxidant Properties. International Journal of Current Microbial and Applied Sciences 3(1): 600-606.
Suresh, K, D., Parnita, J., Shyam, N. J., Rishi, B. & Viswas, K. N. (2011). Antibacterial activity of aqueous extract of pomegranate peel against Pseudomonas stutzeri isolated from poultry meat. Journal of Food Science and Technology 50(3): 555-560.
Veerasamy, R., Xin, T. Z., Gunasagaran, S., Xiang, T. F. W., Yang, E. F. C., Jeyakumar, N. & Dhanaraj, S. A.
(2011). Biosynthesis of silver nanoparticles using mangosteen leaf extract and evaluation of their antimicrobial activities. Journal of Saudi Chemical Society 15(2): 113-120.
Yugal, K. M., Sujogya, K. P., Akshaya, K., Bastial & Tapan K. M. (2017). Biosynthesis of Silver Nanoparticles from Protium serratum and Investigation on their Potential Impacts on Food Safety and Control. Frontier in Microbiology. 8(626): 1-10.
