Gold is a precious coinage metal usedas jewelryFrom ancient times[1
Gold is a precious coinage metal usedas jewelryFrom ancient times1,2.
1 Daniel, M. C., &Astruc, D. (2004). Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chemical reviews, 104(1), 293-346.
2 Eustis, S., & El-Sayed, M. A. (2006). Molecular mechanism of the photochemical generation of gold nanoparticles in ethylene glycol: Support for the disproportionation mechanism. The Journal of Physical Chemistry B, 110(29), 14014-14019.
it is a soft, dense and bright yellow metal. The gold nanoparticles are biocompatible with large surface area and high dispersion owingto their very small size. The synthesis, characterization and their use in biologyand medicine has produced significant results 3–5
3Husen, A., & Siddiqi, K. S. (2014). Carbon and fullerene nanomaterials in plant system. Journal of nanobiotechnology, 12(1), 16.
4 Husen, A., & Siddiqi, K. S. (2014). Phytosynthesis of nanoparticles: concept, controversy and application. Nanoscale research letters, 9(1), 229.
5 Siddiqi, K. S., &Husen, A. (2016). Fabrication of metal nanoparticles from fungi and metal salts: scope and application. Nanoscale research letters, 11(1), 98.
Gold nanoparticles (AuNPs) have certain characteristics and properties those can be used in biomedics, drug delivery agents, and sensors. AuNPs has several advantages compared other nanoparticles namely inert, resistance to oxidation by moistureand air and unaffected by mild acids,biologically nonhazardous and compatible 6.
6 Tomar, A., & Garg, G. (2013). Short review on application of gold nanoparticles. Global Journal of Pharmacology, 7(1), 34-38.
Conventionally, synthesis of AuNPs canbe performed by many physical, chemical and green synthesis methods 7. However, high cost andtoxic chemicals are major challenges for applying physical and chemical methods therefore biosynthesis got edge due to its simple, eco-friendly and low cost process 8. Phytochemicals present inbiomassare efficient to reduce chloroauric acid to form AuNPs. In fact, synthesis of nanomaterials.using biosynthesis approach has provided a common junction between nanotechnology and biotechnology which led to the fabrication of unique materials with tunable size and shapes.
7Ahmed, S., & Ikram, S. (2016). Biosynthesis of gold nanoparticles: a green approach. Journal of Photochemistry and Photobiology B: Biology, 161, 141-153.
8Umer, A., Naveed, S., Ramzan, N., & Rafique, M. S. (2012). Selection of a suitable method for the synthesis of copper nanoparticles. Nano, 7(05), 1230005.
Plant-mediatedsynthesis of metal nanoparticles has become Widely useddue to ease and availability of plant material which contain reducing compounds such as sugars,proteins, phenols, ketones, aldehydes, carboxylic acids etc. 9–11.
.9Husen, A., & Siddiqi, K. S. (2014). Plants and microbes assisted selenium nanoparticles: characterization and application. Journal of nanobiotechnology, 12(1), 28.
10Khan, M., Al-Marri, A. H., Khan, M., Shaik, M. R., Mohri, N., Adil, S. F., … & Tahir, M. N. (2015). Green approach for the effective reduction of graphene oxide using Salvadorapersica L. root (Miswak) extract. Nanoscale research letters, 10(1), 281.
.
11Wadhwani, S. A., Shedbalkar, U. U., Singh, R., &Chopade, B. A. (2016). Biogenic selenium nanoparticles: current status and future prospects. Applied microbiology and biotechnology, 100(6), 2555-2566.
it has been reported that GNPs functionalized with chitosan andliposomes are highly stable in gastric acid, and capable of fusingwith bacteria at physiological pH, making them suitable to treatgastric pathogens such as Helicobacter pylori infections12.
12Thamphiwatana, S., Gao, W., Pornpattananangkul, D., Zhang, Q., Fu, V., Li, J., … & Zhang, L. (2014). Phospholipase A2-responsive antibiotic delivery via nanoparticle-stabilized liposomes for the treatment of bacterial infection. Journal of Materials Chemistry B, 2(46), 8201-8207.
Previously few studies have been carried
out to explore the involvement of reactive oxygen species (ROS)mediated DNA damage by exposure to GNP in different cell types13
13 KhaingOo, M. K., Yang, Y., Hu, Y., Gomez, M., Du, H., & Wang, H. (2012). Gold nanoparticle-enhanced and size-dependent generation of reactive oxygen species from protoporphyrin IX. ACS nano, 6(3), 1939-1947
Several studies reported that GNPs cause cellulardamage to mammalian cells through unintended mechanismsincluding induction of necrosis and apoptosis 14,15
14Pan, Y., Neuss, S., Leifert, A., Fischler, M., Wen, F., Simon, U., … &Jahnen?Dechent, W. (2007). Size?dependent cytotoxicity of gold nanoparticles. Small, 3(11), 1941-1949.
15Leite, P. E. C., Pereira, M. R., do Nascimento Santos, C. A., Campos, A. P. C., Esteves, T. M., &Granjeiro, J. M. (2015). Gold nanoparticles do not induce myotube cytotoxicity but increase the susceptibility to cell death. Toxicology in Vitro, 29(5), 819-827.
, oxidative stress, inflammation, DNA damageand alterations in gene expression 16
16Li, J. J., Hartono, D., Ong, C. N., Bay, B. H., & Yung, L. Y. L. (2010). Autophagy and oxidative stress associated with gold nanoparticles. Biomaterials, 31(23), 5996-6003.
However, thereare some contradictory reports on non-toxicity of GNPs to variousmammalian cells under ambient conditions. Moreover, there is limitedreport on GNP toxicity on gastric cell lines, where H. pyloripredominately cause peptic ulcers in immune-compromised patients.
Presently, the major concern around the worldin medical science is that H. pylori has developed resistanceagainst the standard antibiotics being used in the clinical
practices 17.Therefore, there is a need for the developmentof novel antimicrobial agents possessing superior effectivenessagainst H. pylori with reduced toxicity for human cells.So far, metals including zinc 18, bismuth 19, and silver-
NPs 11
17Mégraud, F., Lehn, N., Lind, T., Bayerdörffer, E., O’Morain, C., Spiller, R., … ; Burman, C. F. (1999). Antimicrobial Susceptibility Testing ofHelicobacter pylori in a Large Multicenter Trial: the MACH 2 Study. Antimicrobial agents and chemotherapy, 43(11), 2747-2752.
18Amin, M., Iqbal, M. S., Hughes, R. W., Khan, S. A., Reynolds, P. A., Enne, V. I., … ; Mirza, A. S. (2010). Mechanochemical synthesis and in vitro anti-Helicobacter pylori and uresase inhibitory activities of novel zinc (II)–famotidine complex. Journal of Enzyme Inhibition and Medicinal Chemistry, 25(3), 383-390.
19 JGisbert, J. P., ;Calvet, X. (2011). non?bismuth quadruple (concomitant) therapy for eradication of Helicobater pylori. Alimentary pharmacology ; therapeutics, 34(6), 604-617.
uptake, membrane interaction and cellular toxicity 20
20Schaeublin, N. M., Braydich-Stolle, L. K., Maurer, E. I., Park, K., MacCuspie, R. I., Afrooz, A. N., … ; Hussain, S. M. (2012). Does shape matter? Bioeffects of gold nanomaterials in a human skin cell model. Langmuir, 28(6), 3248-3258.
many plants from Apiaceae family(Umbelliferae) have been used in traditional medicine to treat various diseases, including asthma, gastrointestinal disorders, intestinal parasites, and have been known to possess antifungal, anti-diabetic, anti inflammatory, antimutagenic and antiviral activities(21).
21 Iranshahy, M., ;Iranshahi, M. (2011). Traditional uses, phytochemistry and pharmacology of asafoetida (Ferulaassa-foetida oleo-gum-resin)—A review. Journal of ethnopharmacology, 134(1), 1-10.
Pituranthostortuosus(Desf.) (family Umbelliferae), smells very strong aromatic ordour in thefruiting stage..Pituranthostortuosus(Desf.) is known in arabic as Shabat El-Gabal and is usedin folk medicine as diuretic, diuretic analgesic, it is also used to relief stomach pain and against intestinal parasites(22). There is phytochemical study on this plant revealed the presence of flavonoidal glycosides, steroids and furanocoumarins which are known to exhibit valuable biological properties such as analgesic, anti-inflammatory, antibacterial, antiviral, antiproliferative,
22,23.
22 Abdel Ghani, A.???? ??? ?????, ; Hafez, S. S. (1995). Gc-ms analysis and antimicrobial activity of volatile oil of Pituranthostortuosus (desf.).
23 El-Mousallamy, A.M. D., 1992. J. Egypt. Soc. Toxicol.,
9: 81 .
Materials: HAuCl4.H2O 99.9% was purchased from Aldrich.Pituranthos tortuosus.arial parts ware was collected at full flowering stage from Ras-ELHekma, Mersa-Mattruh-North Westeron coast at April 2016, then the Pituranthos tortuosus aerial parts were air dried, then grounded to fine powder and kept to be used for different analysis.
2.1.Preparation of Pituranthos tortuosus extract:
The plant extract (2% w/v) was prepared by boiling 2.0g of dried,well grinded Pituranthos tortuosus arial parts for 20 min, filtrating, and completing to 100 ml deionized water.The extract was freshly prepared for each experiment.
2.2.Preparation of Essential Oil of Pituranthos tortuosus
prepared by desolving 0.5 ml in 100 ml ethanol .
2.3.. Phytochemical screening
Freshly collected plant Pituranthos tortuosus arial parts were dried and then coarsely powdered. One hundred gm of the coarse powder were extracted using deionized water till clearness. The extract was filtered and subjected to qualitative tests for the identification of various phytochemical constituents (Brinda et al., 1981; Anonymous, 1990 and Lala, 1993)24-26. Presence of Alkaloids were assessed using Dragendorff’s test 27(Adegoke et al., 2010), while carbohydrates and proteins using Molisch and Biuret tests, respectively 28(Boxiet al., 2010). In addition, cardiacglycosides were assessed using Concentrated H2SO4 test(Obianime and Uche, 2008), coumarinusing alcoholic sodium hydroxide, flavonoids by Pew’s tests(Peach et al., 1965)29, saponinusing Foam test (Adegoke et al., 2010)27, tannins by Ferric chloride test and terpenes using Salkowski’s test (Obianime and Uche, 2008)30
. Moreover, volatile oils were assessed using oil distillation method. All these procedures are underlined by Allen (1989)31.
24Brinda P., Sasikala P. and Purushothaman K.K. (1981):Pharmacognostic studies on Merugankizhangu. Bulletin in Medical Ethnobotanical arch, 3: 84 – 96.
25Anonymous. (1990): Phytochemical investigation of certain medicinal plants used in Ayurveda. Central Council for Research in Ayurveda and Siddha, New Delhi, India.
26Lala P.K.(1993): Lab manuals of Pharmacognosy. CSI Publishers and Distributers, Calcutta, 226.
27 Adegoke A.A., Iberi P.A., Akinpelu D.A., Aiyegoro O.A. andMboto C.I. (2010): studies on phytochemical screening and antimicrobial potentials of Phyllanthusamarus against multiple antibiotic resistant bacteria. International J. Appl. Res. Nat. Prod. 3(3):6-12.
28 Boxi M., Rajesh Y., Kumar V. Raja, Praveen B. andMangamma K. (2010): Extraction, phytochemical screening and in-vitro evaluation of anti-oxidant properties of Commicarpuschinesis (aqueous leaf extract). Int. J. Pharm. Biosci., 1: 537–547.
29PeachK.and Tracey M.V. (1956): Modern methods of plant analysis. Vol. 3, Springer Verlag, Berlin.
30ObianimeA.W.andUche F.I. (2008): The phytochemical screening and effects of methanolic extract of Phyllanthusamarus leaf on the biochemical parameters of male guinea pigs.Journal of Applied Sciences and Environmental Management 12 (4): 73-77.
31Allen S.E. (1989): Chemical Analysis of Ecological Materials. (Seconded.) Blackwell Scientific Publications, Oxford. 368pp.
Phytochemical compound Results
Alkaloids +
Carbohydrates +
Glycosides +
Flavonoids +
Tannins +
Cumarines +
Protein +
Saponins –
Terpenes +
Fatty acids +
Volatile oil +
2.4.Synthesis of AuNPs using Pituranthos tortuosus aqueous extract:
A 2.8×10-2 M stock solution of hydrogentetrachloroaurate (III) was prepared by dissolving 1.0 g of the hydrogen tetrachloroaurate (III) in 100 mL
deionized water. To synthesize nanoparticles using Pituranthos tortuosus aqueous, a certain volume of extract was added to 0.05ml of HAuCl4 at room temperature and the mixture was shacked well to forma colloidal solution then completed to 10 ml with deionized water. The final concentration of Au was 1.4
x 10-4 M. The reduction process of Au3+ to AuNPs was followed by the change in the color of the solutionfrom yellow to violet to dark pink and green depending on the extract concentration. The nanoparticles
prepared at different pH values, the pH of the solutions (1.4×10-4 M AuCl4- and2.6ml extract in 10 ml
flask) were adjusted using 0.1 N HCl or 0.1 N NaOH solutions.
2.5.Synthesis of AuNPs using Essential Oil of Pituranthos tortuosus
a certain volume of oil desolving in ethanol
was added to 0.05ml of HAuCl4 at room temperature and the mixture was shacked well to forma colloidal solution then completed to 10 ml with deionized water and adjust pH at 7
3Characterization of AuNPs
3.1.UV–visible spectral analysis: The bioreduction of AuCl4
– ions in solution was monitored by measuring the UV–vis spectra of the reacted mixtures at wavelengths between 300-800 nm on a ?-Helios SP Pye-Unicam UV-Vis spectrophotometer using 10 mm optical path length quartz cuvettes.
3.2.Transmission electron microscopy (TEM): The size and morphology of the nanoparticles were examined and the TEM images were obtained on a JEOL-1200JEM. Transmission electron microscopy samples of AuNPs were conducted by placing a drop of the bio-synthesized AuNPs suspension on to carbon coated copper grids and allowing the solvent to evaporate in air.
3.3.X-Ray Diffraction: XRD measurement of the AuNPs was done on a Shimadzu XRD-6000 diffractometer operating at a voltage of 40 KV and current of 20 mA with Cu K? radiation.(?= 1.54 Å).
3.4.Fourier transform infra-red (FTIR) spectroscopy:
Nicolet 6700 FTIR spectrometer was used to obtain FTIR spectra at room temperature. The bio-reduced chloroauric acid solution was centrifuged at 10,000 rpm for 15 min and the sample was dried and grinded with KBr in the form of round disk and it was analyzed to get FTIR of capped nanogold. FTIR of Pituranthos tortuosus was obtained by grinding dried leaves with KBr.
3.5.Thermogravimetric analysis: Thermal study was carried out to determine the amount of capping phytochemicals on the AuNPs. A Shimadzu DT-50 thermal analyzer was used for Thermogravimetric analyses with a heating rate of 10 C/min.
2-2 Anti-diabetic potential Evolution
Determination of ?-glucosidase inhibitory activity: The? -glucosidase inhibitory activity was measured according to the method described by 32. Briefly 0.1 mg of each sample or acarbose at different concentration (1000- 1.95 µg/ml) was incubated with 500 µl of 1.0 U/ml ?-glucosidase solution in100 mM phosphate buffer (100 mM, pH 6.8) at 37 0C for 15 minutes. Thereafter, 250 µl of p- nitrophenyl glucopyranoside (pNPG) (5 mM) in the same buffer was added and the reaction mixture was further incubated at 37 0C for 20 min. The absorbance of the released p-nitrophenol was measured at 405 nm using a microplate reader (BioTeK Instruments, Inc., Winooski, VT). The inhibition percentage was calculated using as follow:% Inhibition percentage= Abscontol- Abssample Abscontrol*100Where, abs control is the absorbance of the control reaction (containing all reagents except the test sample) and abs sample is the absorbance of the test sample. The IC50 value was defined as the concentration of -glucoside inhibitor to inhibit 50%of its activity under the assay condition.
32You, Q., Chen, F., Wang, X., Luo, P. G., & Jiang, Y. (2011). Inhibitory effects of muscadine anthocyanins on ?-glucosidase and pancreatic lipase activities. Journal of agricultural and food chemistry, 59(17), 9506-9511
2-3 Helicobacter pylori activity assay
Determination of the minimal inhibitory concentration (MIC)
Antibacterial activity of tested compound against helicobacter pylori was determined by a micro-well dilution method described by 33. The inoculum of helicobacter pylori was prepared and the suspension was adjusted to 106 CFU? ml. The compounds under investigation and standard drug (Clarithromycin) were prepared in dimethyl sulfoxide (DMSO) and subsequent twofold dilutions (1000-0.03 µg g) were prepared in a 96 – well plate. Each well of the microplate included 40 µl of the growth medium (Brain Heart (BHI) plus 10% fetal bovine serum (FBS), 10 µl of inoculum and 50 µl of the diluted compounds. The Clarithromycin and DMSO are used as positive controls, respectively. The plates were incubated at 37 0C for 3 days, in 5% O2,10%CO2, and 85% N2 atmosphere. After that ,40 µl of 3-(4,5-dimethyl-thiazol-2-yl) – 2,5- diphenyl- tetrazolium bromide(MTT) at a final concentration 0.5 mg/ml freshly prepared in water was added to each well and incubated for 30 min. The change to purple color indicated that the bacteria were biologically active. The inhibition percentage was calculated using the formula:
% % Inhibition percentage= Abscontol- Abs samplelAbscontrol*100The concentration of samples (Inhibitor) required for 90%of inhibition (MIC 90) was determined form corresponding – dose -response curves. The MIC was taken to the lowest concentration, where no change of color of MTT was determined using an automatic reader at 620 nm. TheMITT values were done in triplicate.
33 Bonacorsi, C., Raddi, M. S. G., Carlos, I. Z., Sannomiya, M., & Vilegas, W. (2009). Anti-Helicobacter pylori activity and immunostimulatory effect of extracts from Byrsonima crassa Nied.(Malpighiaceae). BMC complementary and alternative medicine, 9(1), 2.
2-4 The cytotoxicity assay
The cytotoxicity assay performed according to the micro culture MTT method with slight modifications 34. The cells were harvested (1.5 × 104 cells/well) and inoculated in 96-well microtiter plates. They werewashed with phosphate-buffered saline (PBS) and the cultured cells were then inoculated with and without the sample (1 mg/ml). After 72 h of incubation, the medium was aspirated. Ten microliters of MTT solution (5 mg/ml in PBS, pH 7.2) was added to each well and the plates were incubated for 4 h at 37 °C. After incubation, 100 ?l of dimethyl sulfoxide (DMSO) was added to the wells followed by gentle shaking to solubilize the formazan dye for 15 min. Absorbance was read at 540 nm and the surviving cell fraction was calculated. Suramin (100 ?M) was used as the reference standard for anticancer activity, and H2O2 (1 mM) was used as the cytotoxic agent against normal liver cell lines. The inhibition of cell viability and COX was calculated using the formula: % viability = /Ac x100Where At = Absorbance of the test sample and Ac = Absorbance of the control sample
34Kawase, M., Motohashi, N., Satoh, K., Sakagami, H., Nakashima, H., Tani, S., … &Molnár, J. (2003). Biological activity of persimmon (Diospyros kaki) peel extracts. Phytotherapy Research, 17(5), 495-500.
2-5 Synthesis of AuNPs Pituranthos tortuosus aqueous extract:
To synthesize nanoparticles using Pituranthos tortuosus acertain volume of Pituranthos tortuosus extract was added to 0.05ml of HAuCl4 at room temperature and the mixture was shacked well then completed to 10 ml with deionized water. The final concentration of Au was 1.4 x 10-4 M. The reduction process of Au3+ to AuNPs was followed by the change in the color of the solution andUV–visible spectroscopy. The nanoparticles prepared at different pH values, the pH of the solutions (1.4×10-4 M AuCl4-and 2.6ml extract in 10 ml flask) were adjusted using 0.1 N HCl or 0.1 N NaOH solutions.
3.1.UV–visible spectroscopy and TEM Studies
The formation and stability of gold nanoparticles was followed by Uv-visible spectrophotometry.
Figure 1 shows the Uv-visible spectra of gold nanoparticles formation using constant HAuCl4 concentration (1.4 x 10-4 M) with different concentrations of extract from 0.2 to 4 ml. The formation of AuNPs was confirmed by the visual color change from yellow into mauve, purple (or brilliant red color) and green according to size and shape of formed AuNPs .As the concentration of the extract increases, SPR band intensity increase with a blue shift in the band from 544 nm to 531 nm,this blue shift suggests that by increasing the extract concentration, the particle size decreases. 2.6 ml of extract give strong SPR band absorption peak centered at about ?max 531 nm this beak is considered the best peak.This sharp narrow shape SPR band indicating the formation of spherical and homogeneous distribution of gold nanoparticles was observed. Further addition of higher amounts of the extract , the ?max was shifted to longer wavelengths with a slight decrease in absorbance 35(Khalil M.M.H. et al., 2014). This is most likely due to changes in the dielectric properties of the layer immediately surrounding the gold nanoparticles (Mulvaney P, 1996)36 and to some small amount of particle aggregation.
35Mulvaney, P. (1996). Surface plasmon spectroscopy of nanosized metal particles. Langmuir, 12(3), 788-800.
36 Khalil, M. M., Ismail, E. H., El-Baghdady, K. Z., & Mohamed, D. (2014). Green synthesis of silver nanoparticles using olive leaf extract and its antibacterial activity. Arabian Journal of Chemistry, 7(6), 1131-1139.
Fig.1Uv-vis spectra of gold nanoparticles using constant HAuCl4concentration 1.4x 10-4M with different concentrations of extract from 0.2 to 4 ml
The nanoparticles products were further characterized using Transmission Electron Microscopy (TEM). The TEM images confirm the formation of the nanoparticles, the gold nanoparticles quasi spherical nanoparticles were formed with an average size of 20 -50 nm (Fig. 2a), At lower extract concentrations(0.4 ml extract), On the other hand, at higher extract concentrations (2.6ml), the majority of the Au nanoparticles were in the range of 5–20 nm (Fig. 2b).This indicates that low quantities of the extract can reduce gold
ions, but do not protect most of the quasi-spherical nanoparticles from aggregating because of the deficiency of biomolecules to act as protecting agents.
Fig. (2): Effect of the arial part extract quantity on the size and shape of the AuNPs. a) TEM image measured at 0.4 ml extract s; b) TEM image measure at 2.6 ml.
3.2. Effect of contact time
The kinetic of AuNPs formation was followed at different contact time using UV-VIS spectroscopy. It was noted that with increasing in contact time, the peak at 531 nm becomes sharper (Fig.3). Formation of AuNPs started within 2 min and increased up to 36 min but after that only slight variation could be observed. It was observed that the gold SPR band is centered at about 531 nm and the reduction of HAuCl4 reached saturation within 36 min of reaction.
Fig. (3): Effect of contact time on AuNPs formation using Pituranthos tortuosus arial part extract
4.3. Effect of pH on nanoparticles synthesis
The nanoparticles prepared at different pH values, the pH of the extract were adjusted using 0.1N HCl or 0.1N NaOH solutions.The color of gold nanoparticles solutions changed from pale red to pink to green.This change would be an indication of gold reduction by Pituranthos tortuosus arial part extract, increasing of extract pH enhanced proton removal from polyphenol which might facilitate capping and stabilizing and consequently the growth of nanoparticles.So that, the particles size at acidic pH was expected to be larger than the size at the basic pH Kuyucak and Volesky, 1989):37
37 Kuyucak, N. (1989). Accumulation of gold by algal biosorbent. Biorecovery, 1, 189-204.
By increasing the pH of the extract form pH (2 -7 )the SPR absorption band increases (Fig. 4a) accompanied by a blue shift in the SPR band from 550 nm to 531 nm indicating the formation of smaller size uniform nanoparticles38.
38 Husseiny, M. I., El-Aziz, M. A., Badr, Y., & Mahmoud, M. A. (2007). Biosynthesis of gold nanoparticles using Pseudomonas aeruginosa. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 67(3), 1003-1006
Increasing alkalinity of the surrounding media induced change in electron density on the surface so it was affected on surface plasmon band and band intensity decreased(Ma and Han, 2008)39.
39 Ma, Z., & Han, H. (2008). One-step synthesis of cystine-coated gold nanoparticles in aqueous solution. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 317(1-3), 229-233.
Extract pH7 is taken as the optimum pH where the smallest (5-15 nm)monodispersed AuNPs are obtained at this pH.
using acidic extract pH a broad SPR band centered at 550 nm is observed indicating the formation of large (35-50 nm) aggregated nanoparticles.This was confirmed by TEM images taken when extract of pH 2 in used for the synthesis of AuNPs as shown in (Fig. 5)
Fig. (4) Uv-vis spectra of gold nanoparticles formation at different extract pH
.
TEM images gave a clear view about pH effect (Fig. (4.10), Fig (4.11)). size of gold nanoparticles that formed through using acidic media (pH2) ranged from 20 to 50 nm whereas in basic media (pH7), gold nanoparticles size ranged from 5 to 15 nm were formed.
Figure (5):TEM images of (a)AuNPs at pH 2(b)Au NPs at pH 7
Effect of reaction temperature on AuNPs synthesis
Temperature is play one of the important physical parameter on the synthesis of god nanoparticles. Fig. 4showsthe effect of temperature in the nanoparticles synthesis. The synthesis of nanoparticles was increases while increasing the reaction temperature. Different absorbance spectra of GNPs were taken at different temperature. The peak sharpness increases with an increase in the reaction temperature). Most likely that occurs due to increase in reaction rate of the conversion of the metal ion to nanoparticles at higher temperature 40.
40 Huang, J., Lin, L., Li, Q., Sun, D., Wang, Y., Lu, Y., … & Wang, W. (2008). Continuous-flow biosynthesis of silver nanoparticles by lixivium of sundried Cinnamomum camphora leaf in tubular microreactors. Industrial & Engineering Chemistry Research, 47(16), 6081-6090.
The SPR bands of gold colloids synthesized at the highest temperature (90 °C) was comparatively smaller than those synthesized at lower temperatures. Broader SPR bands have been linked to increased particle sizes 41.
41 Aromal, S. A., Vidhu, V. K., & Philip, D. (2012). Green synthesis of well-dispersed gold nanoparticles using Macrotyloma uniflorum. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 85(1), 99-104.
The absorbanceband was broadened and positioned at 568nm at the temperature of 30 °C,
(Fig. 6). The higher rate of reduction was occurred at higher temperature due to the
consumption of gold ions in the formation of nuclei whereas the secondary reduction was stopped on thesurface performed nuclei1. The broadening peak was obtained at low temperature shows formation oflarge sized nanoparticles and the narrow peak was obtained at high temperature, which indicates theformed nanoparticles are small in size and the higher rate of reduction of gold ions was occurred in the90.
Fig. (6): UV–vis spectra of gold nanoparticles formation at different reaction temperature.
.5. The X-Ray Diffraction Study
XRD analysis of the nanoparticles, prepared using 1.4×10-4 M Au3+and 3ml extract at room temperature, prominent Bragg reflections obtained at 2?= 38.06 °(1 1 1), 44.35° (2 0 0), 64.65° (2 2 0), and 77.43° (3 11) are identical with those reported for the standard gold metal (Au0) (Joint Committee on Powder Diffraction Standards-JCPDS, USA). The data revealed crystalline nature of AuNPs.Thebroadening of Bragg’s peaks indicated the formation of nanoparticles. The mean size of gold nanoparticles was calculated using the Debye–Scherrer’s equation by determiningthe width of the (111) Bragg’s reflection (Borchert et al., 2005)42.
42 Borchert, H., Shevchenko, E. V., Robert, A., Mekis, I., Kornowski, A., Grübel, G., ; Weller, H. (2005). Determination of nanocrystal sizes: a comparison of TEM, SAXS, and XRD studies of highly monodisperse CoPt3 particles. Langmuir, 21(5), 1931-1936.
The size of the nanoparticles was thus determined to be about 12 nm for gold nanoparticles. This size was in good agreement with the particle size obtained from the TEM image at 2.6 ml of extract concentration and 1.4×10-4 M Au3+ at room temperature and pH of extract (7).TheXRD of gold nanoparticles showed strong signals for gold atoms along with weak signals. These weak signals could have been arisen from X-ray emission from macromolecules like proteins/enzymes bound to the nanoparticles or in the vicinity ofthe particles.
Fig.(7): XRD pattern of dried powder of gold nanoparticles synthesized using Pituranthos tortuosus arial part extract.
4.6. Fourier transform infra-red spectroscopy (FTIR)
As shown in Scheme 1, number of significant bands are observed in the region 500 – 4000 cm-1 centered at 3420.6, 2922.8, 1736.7, and 1639.5, which are attributed to hydroxyl, aliphatic –CH–, C=O stretch and C=O(amide ) groups, respectively. By comparing FTIR spectra of both dried Pituranthos tortuosus arial part extract and capped AuNPs, a shift in the hydroxyl peak from 3420.6 to 3395.5 cm-1 is observed suggesting that -OH of Pituranthos tortuosus polyphenols are involved in the reduction of Au+3. The peak due to carbonyl group became weaker in the FTIR spectrum of stabilized AuNPs, indicating the interaction of carbonyl groups with the AuNPs surface stabilizing it. The band observed due to the stretching of C=O of the amide groups of protein in plant at 1639.5 cm-1 was shifted to 1626.6 cm-1 revealing that proteins are involved in the capping and stabilization of AuNPs. carbonyl group was found at 1736.7 in extract shifted to 1717.5
This phytochemicals might be responsible for bioreduction of Au+3(Isorhamnetin,KaempferolandQuerectin)(Saleh et al.,1983)43.
Saleh, N. A., El-Negoumy, S. I., El-Hadidi, M. N., ; Hosni, H. A. (1983). Comparative study of the flavonoids of some local members of the Umbelliferae. Phytochemistry, 22(6), 1417-1420
Fig.(8):FTIR spectra of Pituranthostortuosusextractand AuNPs
.7. Thermal gravimetric analysis
The thermal stability of NPs was studied using TGA which Also, there is a steady weight loss until 500°C. The total weight loss up to 500°C for AuNPs (Fig. 10) is about 61 % for AuNPs. The observed behavior is most likely as a consequence of the surface desorption of bio-organic compounds present in nano particle powder. Thus, plant leaf extract-stabilized AuNPs are expected to be made up of molecules responsible for the reduction of metal ion and stabilizing the particles in the solution (J. Kasthuriet al., 2009)44
44 Kasthuri, J., Kathiravan, K., ; Rajendiran, N. (2009). Phyllanthin-assisted biosynthesis of silver and gold nanoparticles: a novel biological approach. Journal of Nanoparticle Research, 11(5), 1075-1085.
.
(4.18): TGA of the capped AuNPs prepared using Pituranthos tortuosus extract
Inhibition of AuNPs on ?-glucosidase
The carbohydrates digestive enzymes are hydrolyzed by intestinal ?-glucosidase liable for the breakdown of oligosaccharides and disaccharides into monosaccharides suitable for absorption( O?wieja, et al.,2012)45
45 O?wieja, M., Adamczyk, Z., ; Kubiak, K. (2012). Tuning properties of silver particle monolayers via controlled adsorption–desorption processes. Journal of colloid and interface science, 376(1), 1-11.
The inhibition of this digestive enzymes is specifically useful forthe treatment of non insulin diabetes because it will delaythe release of glucose in theblood(Podsedek et al.,2014) 46.
46 Podsedek, A., Majewska, I., Redzynia, M., Sosnowska, D., ;Kozio?kiewicz, M. (2014). In vitro inhibitory effect on digestive enzymes and antioxidant potential of commonly consumed fruits. Journal of agricultural and food chemistry, 62(20), 4610-4617
As shown in Fig the results indicated that ?-glucosidase was significantly inhibited in a concentration dependent manner following incubation with various concentrations of AuNPs. The increasing concentration of AuNPs level, the enzymaticactivity level was reduced remarkably Table .
.According to numerous in vivo studies, inhibition of ?-glucosidase isbelieved to be one of the most effective approaches for diabetes care 47,48(Balan et al.,2015; Balan et al.,2015).
47 Balan, K., Perumal, P., Sundarabaalaji, N., ;Palvannan, T. (2015). Synthesis, molecular modeling and biological evaluation of novel 2-allyl amino 4-methyl sulfanyl butyric acid as ?-amylase and ?-glucosidase inhibitor. Journal of Molecular Structure, 1081, 62-68.
48 Balan, K., Qing, W., Wang, Y., Liu, X., Palvannan, T., Wang, Y., … ; Zhang, Y. (2015). Antidiabetic activity of green synthesis silver nanoparticles using Lonicera japonica leaves extract.Arabian J chem, DOI: http://dx.doi.org/10.1016/j.arabjc.2014.07.002…
The results indicate that AuNPs exhibited well anti ?- glucosidase activity for all concentration variation.We observed. The inhibition activity of AuNPs is nearly the same commercial drug, Acarbose and much higher compared to using extract alone. The drug showed (IC50 = 30.57 ?g/ml), where extract exhibited ( IC50 = 813.5 ?g/ml) and biosynthesized AuNPs (IC50 = 77.41 ?g/ml) (Fig 4). Most interestingly. So that AuNPs can be easily reduced to control the blood sugar level.
Table2: Anti-diabetic activity (IC50) of Acarbose , extract and AuNPs
Sample code: IC50
Acarbose 30.57
extract 813.50
AuNPs 77.41
Figure11: Anti-diabetic activity ofAcarbose , Pituranthos tortuosus extract and AuNPs
In Vitro Anti-Helicobacter pylori Activity.
MIC of AuNPs synthesized using Pituranthos tortuosus for their in vitro anti-H. pylori activity revealed that inhibits the bacterial growth relatively at low concentration than extract at 15.63 and ;1000 for AuNPS and extract respectively for H. pylori . The AuNPs are effective againstH. pylori reflecting that AuNPs could be used as potent anti-H. pylori agents. current studies is correlated with earlier studies (Gopinath et al.,2016)49
Table 3: MIC of Clarithromycin ,extract andAuNPs
Sample code: MIC
Clarithromycin 1.95
Extract ;1000
AuNPs 15.63)
Figure12: Anti-Helicobacter pylori Activity of Clarithromycin, Pituranthos tortuosus extract and AuNPs
Cell Viability
Gold nanoparticles as novel agents for cancer therapy are gaining greater demand in medical
applications. At the same time, there are only limited studies on the cytotoxic activity of
biosynthesized gold nanoparticles against cancer cell lines. MTT assay was used to assess the effect ofgold nanoparticles on the proliferation of HepG2 andHCT-116.In the present study, we investigated that the(IC50) value of biosynthesized gold nanoparticles againstHCT-116 and HepG2, (IC50)= 23.60µg/ml and IC50 = 158.00µg/mlfor colon carcinoma(HCT-116), IC50 =6.27 µg/ml and IC50 =102.00µg/ml. for Hepatocellular carcinoma (HepG-2) respectively.
The experimental results clearly proved the excellent anticancer activityof gold nanoparticles against the colon carcinoma and Hepatocellular carcinomacell line
Table4. Cytotoxicity (IC50) of aqueous extract of Pituranthostortuosusand the nanoparticles
Sample code: IC50= µg/ml ((HCT-116) IC50 = µg/ml (HepG-2)
Extract 158.00 102.00
AuNPs 23.60 6.27
Figure14:Cell viability% of AuNPs prepared using Pituranthostortuosusextract for colon carcinoma (HCT-116)
Figure13:Cell viability% of AuNPs prepared using Pituranthostortuosusextract for Hepatocellular carcinoma (HepG-2)
