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Pistacia integerrima gall extract mediated green synthesis of gold nanoparticles and their biological activities
⁎Corresponding author at: Department of Pharmacy, Sarhad University of Science and Information Technology, Peshawar 25000, Pakistan. Tel.: +92 919239305. islanaz@yahoo.com (Nazar Ul Islam)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
This paper reports a rapid, facile and one-pot synthesis of environmentally safe gold nanoparticles capped and stabilized with galls extract of Pistacia integerrima. The aqueous gold ions when exposed to P. integerrima galls extract were rapidly reduced as evident from abrupt color change to ruby red, suggesting the biosynthesis of gold nanoparticles (Au-NPs) which were further characterized by UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). Their stability was evaluated against varying pH and different volumes of sodium chloride (NaCl) as well as at a range of temperature (20–80 °C). Au-NPs were tested for enzyme inhibition, antibacterial, antifungal, antinociceptive, muscle relaxant and sedative activities. The UV–Vis spectra of the gold nanoparticles gave surface plasmon resonance at 540 nm while the SEM analysis revealed the particle size in the range of 20–200 nm. FTIR spectra confirmed the involvement of amines, amide groups and alcohols in capping and reduction of gold nanoparticles. Au-NPs showed remarkable stability in different NaCl and pH solutions as well as at elevated temperature. Au-NPs have good antifungal activity and possessed antinociceptive and muscle relaxant properties as observed from their zone of inhibition and significant attenuation of acetic acid induced writhing and reduction of time spent on the rota rod respectively. These results concluded that the gall extract of P. integerrima is a very good bioreductant for the synthesis of gold nanoparticles that have potential for various biomedical and pharmaceutical applications.
Keywords
Gold nanoparticles
Pistacia integerrima
Stability
Biological activities
1 Introduction
Nanotechnology is playing an increasingly important role for improving the design and performance of products in areas as diverse as electronics, biomedical devices, food and agriculture, cosmetics and energy (Jain et al., 2006; Wong and Karn, 2012). In recent years, synthesis of metallic nanoparticles has gained significant importance due to their catalytic, optical and electrical properties (Daniel and Astruc, 2004). Nanoparticles of gold are being used to increase electroluminescent and quantum efficiency in organic light emitting diodes (Park et al., 2004). Palladium and platinum nanoparticles are utilized as proficient catalyst (Wang et al., 2010) and glucose sensors (Zhong, 2009). Moreover, nanoparticles of iron oxide are used in magnetic resonance imaging for cancer diagnosing (Rivière et al., 2005). Nanoproducts have the ability to prevent harm from known pollutants and are incorporated into environmental technologies to remediate hazardous waste sites, clean up polluted streams and desalinate water (Bystrzejewska-Piotrowska et al., 2009).
Plant mediated biological synthesis of nanoparticles is gaining importance due to its simplicity and eco-friendliness. Plants have a strong ability to reduce heavy metals from their higher oxidation to zero oxidation state which may be attributed to the presence of different phytochemicals (Jha et al., 2009; Arya, 2010). Because of their strong antioxidant properties, these phytochemicals are considered as a reservoir of electrons. The electron rich phytochemicals may serve as building blocks for conversion of metallic precursors into their corresponding nanoparticles, thus replacing the use of more toxic external reducing agents in the overall nanoparticles production processes (Dauthal and Mukhopadhyay, 2013). Green nanotechnology therefore provides opportunities for designing of engineered nanomaterials in medicine, energy, electronics and communication sectors (Vaseashta, 2008). Application of nanotechnology to herbal drugs may lead to the development of nanoherbal products possessing high bioavailability and less toxicity, which consequently will open a new era of herbal drug discovery (Ansari and Farha Islam, 2012). Plants like Aloe vera, Brassica juncea, Emblica officinalis, Azadirachta indica, Cinnamommum camphora (Kumar and Yadav, 2009), Tanacetum vulgare, Hibiscus rosa sinensis (Philip, 2010), Chenopodium album (Dwivedi and Gopal, 2010), Memecylon edule (Elavazhagan and Arunachalam, 2011), Camellia sinensis (Vilchis-Nestor et al., 2008), Mentha piperita (MubarakAli et al., 2011) and Diopyros kaki (Song and Kim, 2008; Song et al., 2010) have been utilized for the synthesis of silver, gold and platinum nanoparticles. Due to the unique and tunable surface plasmon resonance, gold nanoparticles find wide applications in biomedical sciences including drug delivery, tissue/tumor imaging, photothermal therapy and immunochromatographic identification of pathogens in clinical specimens (Sahoo and Labhasetwar, 2003; Huang, 2006).
Pistacia integerrima which is commonly known as Kakarshringi (Shani) and belongs to family Anacardiacea, is a moderate size deciduous tree widely distributed at an altitude of 350–400 m in the sub-alpine regions of Himalaya ranging from Indus to Kumaun (Uddin et al., 2012). P. integerrima has many ethobotanical uses and is being used for the treatment of diarrhea, dysentery, fever, vomiting, skin diseases, respiratory ailments and psoriasis and marketed in a traditional dosage form ‘habb-e-suranjan’ by Hamdard Laboratories (WAQF) Pakistan (Ahmad et al., 2008). P. integerrima has been widely studied for its analgesic, anti-inflammatory (Ahmad et al., 2010a), anti-gout (Ahmad et al., 2008), anti-asthmatic (Shirole et al., 2014), antimicrobial (Shafiq et al., 2011), hepatoprotective (Khan et al., 2011) and antioxidant (Rauf et al., 2013) properties. The major phytochemical constituents isolated from P. integerrima are monoterpenes, triterpenoids, sterols, dihydromalvalic acid and flavonoids (Ahmad et al., 2010b). Although nanoparticles of other pistacia species have been reported (Molaie et al., 2012; El-Chaghaby and Ahmad, 2011; Sadeghi et al., 2015) however, synthesis of nanoparticles using P. integerrima is not yet revealed.
In the present study we report the synthesis of gold nanoparticles (Au-NPs) by reduction of gold ions using P. integerrima extract. The method is simple, rapid, inexpensive, eco-friendly and reproducible. Au-NPs were characterized with UV–visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). The effects of salt, pH and temperature on the stability of Au-NPs were studied. Moreover Au-NPs were screened for enzyme inhibition, antibacterial, antifungal, antinociceptive, muscle relaxant and sedative activities.
2 Materials and methods
2.1 Materials
All chemicals and reagents used in this study were of analytical grade. Hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O); obtained from Merck was used as a source of AuIII ions for the synthesis of gold nanoparticles. Double distilled water was used throughout the experiment. Galls of P. integerrima were collected from Toormang, Razagram area of district Dir, KPK, Pakistan. After identification by Barakat Ullah (taxonomist), a specimen was deposited with a voucher No. Bot.20037(PUP) in the herbarium of the Department of Botany, University of Peshawar. The shade dried and crushed galls of P. integerrima were repeatedly extracted with methanol for 7 days at room temperature. The methanol extract was then concentrated under reduced pressure at 50 °C to yield a dark brown residue.
2.2 Synthesis and characterization of gold nanoparticles
A source extract solution of 100 ml was prepared by mixing 2 g of plant extract with absolute ethanol. For the formation of Au-NPs the volume of the plant extract was kept constant while changing the volume of 1 mM gold solution. The bioreduction of the AuCl4− ions was monitored by UV–Vis spectra of the solution in 10 mm optical path length quartz cuvettes with a UV–Vis spectrophotometer (Hitachi U-3200, Japan) at a wavelength between 200 and 900 nm. The size of gold nanoparticles was characterized by SEM (JSM-5910-JEOL, Japan). For FTIR measurements, the bioreduced chloroauric solution was centrifuged at 10,000 rpm for 15 min to remove free proteins or other components present in the solution. The samples were vacuum dried and grinded with finally divided KBr in a mortar that resulted in the formation of KBr pellets which were placed in the sample holder of FTIR instrument (Prestige 21 Shimadzu) for measurement.
2.3 Stability of gold nanoparticles
The effect of sodium chloride (NaCl) on the stability of Au-NPs was determined by adding varying volume (50–200 μl) of 0.1 M NaCl to the biosynthesized gold nanoparticles (3 ml). In order to ascertain the pH effect on the stability of Au-NPs, the pH of the gold nanoparticles solutions was adjusted between 2 and 14 by drop wise addition of 1 M HCl or NaOH solution. The effect of temperature on the stability of Au-NPs was studied by heating the gold nanoparticles each time for 30 min at a temperature of 20, 40, 60 and 80 °C in a water bath.
2.4 Biological activities
2.4.1 Enzyme inhibition activities
Au-NPs along with an extract of P. integerrima were screened for enzyme inhibition activities against urease, xanthine oxidase and carbonic anhydrase-II enzymes. For urease inhibition assay, the sample solutions were incubated with urea. Indophenol method was used to measure ammonia production as urease activity. Thiourea was used as the standard inhibitor (Sumi, 1996). The xanthine oxidase inhibitory activity of test samples was determined by measuring the rate of hydroxylation of the substrate (xanthine) and the subsequent formation of uric acid, which is a colorless end product of the reaction and showed absorption at 295 nm (Lee et al., 1998). The inhibitory activities of the test samples were compared with allopurinol, used as standard. Carbonic anhydrase-II inhibitory activity of the test samples was investigated using 4-NPA (4-Nitrophenyl acetate), which is colorless. Upon hydrolysis it is converted to 4-nitrophenol and carbon dioxide. The formation of 4-nitrophenol, a yellow colored compound, was monitored during the experiment. The reaction was carried out at 25–28 °C. Acetazolamide was used as standard (Arslan, 2001). The experiments were performed in triplicate.
2.4.2 Antimicrobial activities
The antibacterial and antifungal activities were performed by using the well diffusion method (Boyanova et al., 2005; Uddin and Rauf, 2012). Mueller Hinton agar was used as medium. The cultures were taken in triplicates at an incubation temperature of 37 °C for 24–72 h. The broth culture (0.6 ml) of the test organism was placed in a sterile petri dish to which 20 ml of the sterile molten Mueller Hinton Broth (MHB) was added. Holes were bored into the medium using 0.2 ml of oil. Inoculation was done for 1 h to make possible the diffusion of the antimicrobial agent into the medium. After incubation for 24 h at 37 °C, the diameters of the zone of inhibition of microbial growth were measured in the plate in millimeter (mm). The activities were performed against ATCC bacterial strains of Klebsiella pneumonia, Bacillus subtillis and Staphylococcus aureus and fungal strains of Alternaria solani, Aspergillus niger and Aspergillus flavus. The bioassays were performed in triplicate.
2.4.3 Animals
BALB/c mice of either sex weighing 25–30 g were purchased from NIH Islamabad. The animals were maintained in a 12 h light/dark cycle at 22 ± 2 °C for one week prior to experimentation. Access to food and water was ad libitum. Experiments on animals were performed according to the NIH guidelines for the care and use of laboratory animals.
2.4.4 Antinociceptive activity
Animals were withdrawn from food 2 h before the start of experiment. The animals were divided into four groups. Group I was injected with normal saline (i.p) and served as control while group II was injected with standard diclofenac sodium (10 mg/kg, i.p). The remaining groups were injected (i.p) with 10 and 20 mg/kg of Au-NPs. After 30 min of treatment the animals were injected (i.p) with 1% acetic acid. Writhing was counted after 5 min of acetic acid injection and was continued for 10 min (Muhammad et al., 2012).
2.4.5 Muscle relaxant activity
The rota rod used in this test was a metallic rod (3 cm diameter) coated with rubber and connected to a motor. The rod was rotating at a constant velocity, i.e. 9 rpm and was about 60 cm above the table top in order to prevent the mice from jumping off the roller. Mice were exposed to the rota rod as a pre-test before the experiment and only those mice included in the study that remained on the rod for 5 min at a speed of 9 rpm. All the groups (n = 6) were treated (i.p.) with diazepam (1 mg/kg), saline (10 ml/kg) and Au-NPs (10 and 20 mg/kg) before 30, 60 and 90 min of exposure to rota rod. Each mouse was allowed for 5 min on the revolving rod and time spent on the rod was recorded (Muhammad et al., 2013).
2.4.6 Sedative activity
The apparatus used for this activity consists of an area of white wood (150 cm diameter) enclosed by stainless steel walls and divided into 19 squares by black lines. The open field was placed in light and sound attenuated room. Animals were acclimatized under red light (40 W red bulb) 1 h before the commencement of experiment. Animals were administered with saline (10 ml/kg), diazepam (0.5 mg/kg, i.p) and Au-NPs (10 and 20 mg/kg, i.p). After 30 min each animal was placed in the center of box and the numbers of lines crossed were counted for each mouse (Goyal et al., 2009).
3 Statistical analysis
Data were expressed as mean ± standard error of the mean (SEM). Significance of difference among mean values was evaluated by ANOVA followed by Dunnett’s post test using GraphPad Prism 5 (GraphPad Software Inc. San Diego CA, USA).
4 Results
4.1 Characterization of P. integerrima gold nanoparticles
It was observed that the color of Au-NPs changed as the volume of the gold solution varied; keeping the volume of P. integerrima extract constant. The change in color from purple blue to pink and then finally to ruby red indicated the formation of gold nanoparticles. As shown in Fig. 1, no peak at a wavelength of 540 nm appeared for the extract; however in comparison, the gold nanoparticles functionalized with P. integerrima extract gave uniform and sharp peaks with varying intensities. Gold-Pistacia solutions were used in a ratio of 4:1, 5:1, 8:1, 10:1 and 12:1. The 10 ml 1 mM gold solution and 1 ml plant extract (i.e. 10:1) gave a uniform and sharp peak at 540 nm. With this ratio a bulk solution was made for further studies.
The FTIR spectra of the crude extract as compared to that of Au-NPs are shown in Fig. 2. P. integerrima extract showed bands at 3275 cm−1 which is a characteristics region for hydroxyl functional group of alcohols, phenol or amine. Similarly a band at 1016 cm−1 corresponds to the C—N stretching vibration of aliphatic amines, alcohols or phenols, and a band at 1687 cm−1 indicated the presence of carbonyl functional group of an amide linkage of proteins.
As shown in Fig. 3, the SEM images revealed the size of Au-NPs in the range of 20–200 nm.
4.2 Stability of P. integerrima gold nanoparticles
Au-NPs were stable at an extreme of pH i.e. 4–5 and 10–11. However enhanced stability was observed at alkaline pH. Moderate stability of the gold nanoparticles was observed at other pH values i.e. 8–9, where the nanoparticles showed red shift and peak broadening. The UV–Vis spectra of Au-NPs at varying pH are shown in Fig. 4.
The effect of NaCl on the stability of Au-NPs was determined by using different volumes of NaCl. It was observed that by changing the volume of 0.1 M NaCl from 50 μl to 300 μl there was a very little effect on the gold nanoparticles as suggested by a slight change in the color from ruby red to dark brown. Fig. 5 shows that there is very slight aggregation of nanoparticles and is confirmed from a decrease in absorption of the UV–visible spectra. Moreover, a gradual change in color of Au-NPs is observed with an increase in the volume of 0.1 M NaCl from 50 μl to 300 μl.
The stability of Au-NPs was also checked by heating the nanoparticles solution for 30 min at a range of temperature i.e. 20–80 °C. As shown in Fig. 6, Au-NPs show remarkable thermal stability at elevated temperature. The slight change in the intensities of the UV–Vis spectral peaks was might be due to variations in the size of the biosynthesized nanoparticles with a change in temperature.
4.3 Enzyme inhibition activities
Tables 1–3 show the enzyme inhibition activities of Au-NPs. P. integerrima extract possessed excellent enzymes inhibition activity against urease, xanthine oxidase and carbonic anhydrase with an IC50 values of 96.3, 21.45 and 23.45 respectively. However Au-NPs were found inactive against these enzymes.
| Sample | Concentration (mg/ml) | Percent inhibition (%) | IC50 ± SEM (μg/ml) |
|---|---|---|---|
| Pistacia integerrima extract | 0.2 | 81.8 | 96.3 ± 1.41 |
| Au-NPs | 0.2 | 29.6 | – |
| Thiourea | 0.2 | 98.2 | 21 ± 0.11 |
Values are expressed as mean ± SEM of three different experiments. NA = Not active.
| Sample | Concentration (mM) | Percent inhibition | IC50 ± SEM (μg/ml) |
|---|---|---|---|
| Pistacia integerrima extract | 0.25 | 74.2 | 21.45 ± 0.26 |
| Au-NPs | 0.25 | NA | – |
| Allopurinol | 0.25 | 98.6 | 2.0 ± 0.01 |
Values are expressed as mean ± SEM of three different experiments. NA = Not active.
| Sample | Concentration (mM) | Percent inhibition | IC50 ± SEM (μg/ml) |
|---|---|---|---|
| Pistacia integerrima extract | 0.25 | 78.2 | 23.45 ± 0.41 |
| Au-NPs | 0.25 | NA | – |
| Acetazolamide | 0.25 | 89.0 | 0.12 ± 0.03 |
Values are expressed as mean ± SEM of three different experiments. NA = Not active.
4.4 Antimicrobial activities
Au-NPs were tested for antibacterial activity against K. pneumonia, B. subtillis and S. aureus as well as antifungal activity against A. solani, A. niger and A. flavus using the agar well-diffusion method. P. integerrima extract was inactive against B. subtillis and S. aureus except K. pneumonia. Similarly Au-NPs were inactive against the three studied bacterial strains (Table 4). As shown in Table 5, no antifungal activity is observed with P. integerrima extract, however Au-NPs possess moderate antifungal activity as compared to control.
| Sample | Klebsiella pneumonia | Bacillus subtillis | Staphylococcus aureus |
|---|---|---|---|
| Pistacia integerrima extract | 10 ± 0.76 | NA | NA |
| Au-NPs | NA | NA | NA |
| DMSO negative control | NA | NA | NA |
| Streptomycin | 28 ± 0.24 | 30 ± 0.19 | 28 ± 0.28 |
Values are expressed as mean ± SEM of three different experiments. NA = Not active (no zone of inhibition).
| Sample | Alternaria solani | Aspergillus niger | Aspergillus flavus |
|---|---|---|---|
| Pistacia integerrima extract | NA | NA | NA |
| Au-NPs | 20 ± 0.27 | 20 ± 0.29 | 10 ± 0.39 |
| Miconazole | 100 ± 0.22 | 100 ± 0.22 | 100 ± 0.23 |
Values are expressed as mean ± SEM of three different experiments. NA = Not active (no zone of inhibition).
4.5 Writhing test
As shown in Table 6 significant attenuation (p < 0.01) of acetic acid induced writing is demonstrated in a dose dependent manner by the test doses (10 and 20 mg/kg) of Au-NPs comparable to that of the standard diclofenac sodium. Maximum writhing inhibition was observed at a dose of 20 mg/kg (80.76%) as compared to diclofenac sodium (82.54%).
| Treatment | Dose | No. of writhing (10 min) |
|---|---|---|
| Saline | 10 ml/kg | – |
| Diclofenac sodium | 10 mg/kg | 82.54 ± 0.56⁎⁎ |
| Au-NPs | 10 mg/kg | 65.98 ± 2.34⁎⁎ |
| 20 mg/kg | 80.76 ± 2.67⁎⁎ |
Values are reported as mean ± SEM (n = 6). Data were analyzed by ANOVA followed by Dunnett’s test.
4.6 Muscle relaxant effect
The rota rod activity of the synthesized gold nanoparticles is shown in Fig. 7. The time spent on the rota rod was significantly reduced by Au-NPs at 10 mg/kg (p < 0.05) and 20 mg/kg (p < 0.01) compared to saline. The positive control, diazepam was found to be more significant (p < 0.001) than the test doses of Au-NPs.
4.7 Sedative effect
As shown in Table 7, the synthesized gold nanoparticles were inactive in producing a significant sedative effect in comparison to the standard, diazepam which significantly reduced (p < 0.001) the number of lines crossed in 10 min.
5 Discussion
Application of gold nanoparticles in electronics, cosmetics, coatings, packaging, catalysis, photonics, biomedicine and optics depends on the ability to synthesize particles having uniform chemical composition, shape, size and should be chemically stable without undergoing degradation (Daniel and Astruc, 2004). Nanoparticles are known to be synthesized by leaf, fruit and seed extracts of various plants (Kumar and Yadav, 2009). The present work proved that P. integerrima extract is able to reduce gold ions and produced gold nanoparticles. The reduction of gold ions to gold nanoparticles during exposure to P. integerrima extract could be detected by color change and may be easily followed by UV–Vis spectroscopy. UV–Vis spectroscopy is one of the important techniques to ascertain the formation and stability of metal nanoparticles in aqueous solution (Kasthuri et al., 2009). Based on the UV–Vis spectra it was observed that the sharpness of the absorption peak is dependent on the volume ratio of extract, thus being sharper with a higher ratio. It was noticed that the 10:1 ratio of gold solution and galls extract solution in the reaction mixture is effective for the generation of gold nanoparticles as the UV–Vis peak become sharper with SEM particle size ranging between 20 and 200 nm. Surface plasmon resonance band, intensity maximum and band width are influenced by the particle shape, dielectric constant of the medium and temperature (Link and El-Sayed, 1999). The color of gold nanoparticles stabilized with P. integerrima extract changes from purple blue to pink and then finally to ruby red thus indicating the formation of gold nanoparticles. It is well known that gold nanoparticles exhibit ruby red color, arising due to excitation of surface plasmon vibrations in the gold nanoparticles which give rise to surface plasmon resonance band centered at about 536 nm (Eustis and El-Sayed, 2006). The appearance of the ruddiness color and the maximum absorbance was indications of formation of P. integerrima mediated colloidal gold nanoparticles in the medium. Moreover, the UV–Vis spectra of Au-NPs at other ratios have broad peaks with low intensities which might be due to the formation of large anisotropic particles. The surface plasmon resonance absorbance is extremely sensitive to the nature, size and shapes of the particles formed and their inter-particle distances (Sun and Xia, 2002). Furthermore, the size and the concentration of gold nanoparticles can be determined directly from UV–Vis spectra (Haiss et al., 2007; Murphy et al., 2008). The rapid synthesis of gold nanoparticles at room temperature without utilizing any external reducing agent suggests that the P. integerrima extract contained certain compounds like flavonoids and phenolic constituents that might be responsible for the reduction of HAuCl4 complex as phytochemical analysis showed the presence of monoterpenes, triterpenoids and flavonoids in P. integerrima (Ahmad et al., 2010b).
FTIR measurements were carried out to identify the possible biomolecules in P. integerrima galls extract responsible for capping which leads to efficient stabilization of gold nanoparticles. The FTIR spectra of P. integerrima extract before and after bioreduction do not show any significant changes. The FTIR spectrum of P. integerrima alcoholic extract showed broad band at 3365, 3275 cm−1 which corresponds to hydroxyl functional group of alcohols, polyphenolic or amine. Similarly a band at 1016 cm−1 corresponds to the C—N stretching vibration of aliphatic amines, alcohols or phenols, and a band at 1687 cm−1 indicated the presence of chelated carbonyl functional group of carboxylic acid in pentacyclic triterpenes. The FTIR spectrum of AuNPs revealed a slight shift in the OH stretching band from 3365 to 3375, NH2 band from 3275 to 3263 cm−1 and 1687 to 1635 cm−1 (C⚌O). The band at 1338 and 1016 cm−1 for C—N stretching is very weak in gold nanoparticles. From these observations it is clear that polyphenols, hydroxy groups, chelated carboxylic acids and C—N groups biomolecules present in P. integerrima galls extract are responsible for reduction and stabilization of gold nanoparticles. Moreover, there is a slight change in band positions of various functional groups in the spectrum of gold nanoparticles synthesized and stabilized with P. integerrima extract which indicates the presence of some proteins and metabolites, such as terpenoids having functional groups of amines, alcohol, phenol, aldehyde, ketones and carboxylic acid. Hence, it may be inferred that these bio-molecules are responsible for capping and efficient stabilization of gold nanoparticles. Moreover, one or more of these proteins may be enzymes that reduced chloroaurate ions and capped the gold nanoparticles formed by the reduction process. The adsorption on the surface of metal nanoparticles is a characteristic of flavanones and terpenoids, which may be able possibly by interaction through carbonyl groups in the absence of other strong ligating agents in sufficient concentration. It is also possible that the terpenoids play a role in the reduction of metal ions by oxidation of aldehyde groups in the molecules to carboxylic acids (Iravani, 2011; Mittal et al., 2013).
The stability of nanoparticles under a wide range of environmental conditions, such as high salt concentration, extremes of pH, high temperature and buffer solutions, is important in determining their environmental implication and potential risk to human health (Levard et al., 2012). Nanoparticles stability can be monitored by UV–Vis spectroscopy because aggregation, precipitation and decomposition each lead to distinctive changes in the UV–Vis spectra. To study the effects of pH and ionic strength on nanoparticles stability, the gold nanoparticles were exposed to a range of pH from 2 to 12 and to different volumes of 0.1 M NaCl. It was observed that the nanoparticles were stable over a wide range of pH as there were less significant shifts in the UV–Vis absorption spectra indicating that the resulting nanoparticles are stable at these conditions. Furthermore by increasing the volume of 0.1 M NaCl from 50–200 μl, the size and the number of gold particles changed very slightly. This extremely high stability of the gold nanoparticles may be due to the coordination of sterically bulky organic molecules that act as protective shields on the nanoparticles surface. When exposed to various pH and salt concentrations, the nanoparticles respond with physicochemical changes to their material structure and surface characteristics and can be manifested as swelling, dissociating or surface charge switching, in a manner that favors drug release at the target site over surrounding tissues (Gao et al., 2010). Therefore, the surface composition of gold nanoparticles played an important role in the stability of nanoparticles. Temperature is one of the important environmental factors that influence the stability, activity and chemical characteristics of nanoparticles (Link and El-Sayed, 1999). The effect of temperature on the stability of gold nanoparticles was investigated by heating the nanoparticles solution for 30 min at 20–80 °C. A slight change in the absorbance was observed that may indicate temperature facilitated aggregation of nanoparticles. However, the particles still showed the typical absorption peak at 540 nm. Chemically stable metallic nanoparticles have no significant cellular toxicity whereas nanoparticles able to be reduced, oxidized or dissolved are cytotoxic and even genotoxic for cellular organisms (Auffan et al., 2009b). Colloidal stability is a function of many factors including the type of capping agent, surrounding environmental conditions like pH, ionic strength and the background electrolyte composition (Badawy et al., 2010). Unstable nanoparticles aggregates may significantly contribute to dissolution of ions from nanoparticles, which increase during particle storage. Such coexistence of the nanoparticles and its ionic forms may induce toxic pathway (Asharani et al., 2008; Auffan et al., 2009a). In the current study, we have demonstrated that the gold nanoparticles were found most stable in an acidic pH range of 4–5 and basic pH range of 10–11. However these were unstable at pH 8–9 whereas at other pH values these nanoparticles were moderately stable. The overall stability of nanoparticles is crucial, which determines their mobility, bioavailability and toxicity in any ecosystem (Fabrega et al., 2011). Hence it is essential to synthesize stable nanoparticles, which can minimize ion dissolution and retain their physicochemical properties. The enhanced stability toward varying pH, ionic strength and temperature will enable us to explore different formulations of P. integerrima gold nanoparticles for potentially effective and safe herbal therapy.
Literature review indicates that in most of the plant mediated synthesis of nanoparticles, research has stopped at the level of basic material characterization without exploring their biological applications. In this study the biosynthesized nanoparticles were studied for their enzyme inhibition, antimicrobial, antinociceptive, muscle relaxant and sedative activities. The gold nanoparticles were devoid of enzyme inhibition activities as compared to P. integerrima extract alone having an IC50 of 96.3 μg/ml (urease), 21.45 μg/ml (carbonic anhydrase) and 23.45 μg/ml (xanthine oxidase). Furthermore, the gold nanoparticles were found inactive against B. subtillis, S. aureus and K. pneumonia while P. integerrima extract has some antibacterial activity only against K. pneumonia. Moderate antifungal activity was observed with gold nanoparticles while P. integerrima extract was inactive against all the three studied fungal strains i.e. A. solani, A. niger and A. flavus. Gold nanoparticles alone do not have any microbial activity. The importance of using gold is that the large surface area allows it to carry a large number of drugs (De Jong and Borm, 2008). Gold nanoparticles possess well developed surface chemistry, chemical stability and a large surface to volume ratio due to which more number of drug molecules gets adsorbed on their surfaces via electrostatic attraction between the amino group of drugs and nanoparticles. The gold nanoparticles surrounded by a number of drug moieties now act as a single group against the microbial organisms thereby increasing the microbial activity (Burygin et al., 2009).
Acetic acid-induced writhing test is a well recommended protocol in evaluating medicinal agents for their antinociceptive property. The nociceptive response in the acetic acid induced abdominal constriction assay stems from the synthesis of prostaglandins via the action of the constitutive enzyme cyclooxygenase-1 (COX-1) and its isoform COX-2 which produce hyperalgesia in sensory nerve endings and pain. The acetic acid induced abdominal constriction assay is sensitive to analgesics and sensory afferents in the peritoneum carry α-1/2-adrenoceptors, β-adrenoceptors and opioid receptors on their terminals. When activated by appropriate agonists, these receptors depress the generation of pain impulses (Collier et al., 1968; Georgieva and Georgiev, 1999). In this study significant attenuation of acetic acid induced abdominal constriction was demonstrated at all the tested doses of gold nanoparticles as compared to diclofenac sodium. In the muscle coordination test, significant skeletal muscle relaxation was produced at 10 mg/kg (p < 0.05) and 20 mg/kg (p < 0.01) after 30, 60 and 90 min. Demonstration of marked muscle relaxant effect by the rota-rod study indicated that gold nanoparticles induced neurological deficit accompanied with taming or calming effect in mice, supporting its CNS depressant effect. However, no significant effect in the locomotor activity was observed with gold nanoparticles as compared to diazepam suggesting that the nanoparticles do not sufficiently depress the CNS. Nanotechnological strategies change a substance’s properties and behavior in a biological environment and can potentiate the actions of plant extracts, promote sustained release of active ingredients, reduce the required dose, decrease side effects and improve activity (Bonifácio and da Silva, 2014). Nanoscience and nanotechnology are emerging technologies that have great potential for delivering bioactive compounds to improve human health (Patil et al., 2009).
6 Conclusion
We have developed a green nanotechnological method to synthesize gold nanoparticles using the galls extract of P. integerrima that act both as reducing and stabilizing agent by avoiding the use of hazardous and toxic solvents. The nanoparticles were mostly in the size of 20–200 nm. FTIR analysis indicated the possible involvement of carbonyl and other groups in the reduction process and may be carboxylate ions act as shielding agent. Phytochemicals capping the nanoparticles makes them colloidally stable in different media such as electrolyte and in pH solutions as well as at extreme of temperature as revealed from UV–Vis spectra. The good antifungal activity along with significant attenuation of pain as well as muscle relaxant effect of the nanoparticles can potentially be applied in various products such as topical preparations. The process for the synthesis of nanoparticles in large scale using P. integerrima galls extract may have several advantages such as cost-effectiveness and compatibility for biomedical and pharmaceutical applications.
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