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Stability and Enzyme Inhibition Activities of Au Nanoparticles using an Aqueous Extract of Clove as a Reducing and Stabilizing Agent.

Byline: Abdul Hameed, Ibrahim Khan, Andaleeb Azam, Syeda Sohaila Naz, Ajmal Khan, Muhammad Saleem, Mohammad Raza Shah and Nazar Ul Islam

Summary: Gold nanoparticles (AuNPs) were synthesized in one pot using aqueous extract of clove buds (CB) to reduce HAuCl4 and stabilize gold in its atomic form at room temperature. To determine the potential of gold nanoparticles with clove buds (AuCB) for in vivo applications, the stability of the nanoparticles was explored as a function of temperature, pH and salt concentration. The suspensions were found to be stable for salt concentrations up to 1 mol/L, temperatures of up to 1000C and a pH range of 2-13. Our results indicate that CB exhibited comparable activities to standardsof urease and carbonic anhydrase, but its conjugation to Au knocks out the enzyme inhibition activity by about two times. In case of xanthine oxidase activity, CB and its gold Au bio-conjugates (AuCB) are found to be absolutely inactive.

Keywords: Clove buds extract, gold nanoparticles, stability and enzyme inhibition activities, comparison.


Now a day's nanotechnology, nanostructure and nanoparticles are most commonly used words in scientific literature. Nano size materials are very attractive since they can move inside the body to repair damaged tissues [1] and super computer which are small enough to be kept in a pocket. Besides these, there are various applications of nanomaterials such as biological detection, optical filters, controlled drug delivery, sensors, therapeutic and medicinal science [2]. Nanotechnology finds useful application in the field of chemistry, biotechnology, medicine, and electronics. Biological systems such as plant, algae and fungi are also involved in the synthesis of nanoparticles in an easy and safe way. There are certain facts which highlights that plant materials have a strong affinity to reduce the noble metals (gold and silver) from their higher oxidation to zerooxidation state and leads to the formation ofnanoparticles [3, 4]. This reducing property of theclove is being used as analgesic and antiseptic in dentistry Fig. 1. It is also used as a home recipe for dental pain (toothache). The oils obtained from clove are used for many purposes i.e. from flavoring medicine to the remedies of bronchitis, common cold, sore throat, fever and anesthetic agent in high concentration. Clove oil is also an important ingredient in weed killer and herbicides.A large number of metal containing proteins, such as hemocyanin (a Cu metal containingprotein present in some invertebrate animals which helps them in the transport of O2;like hemoglobin in humans) retain metal ions in their active sites so a great interest is developed in such metal containing enzymes which are ureases, laccase, tyrosinase and ascorbate oxidase [5-9] containing enzyme urease (urea amidohydrolase EC 3.5.15) act as a catalyst to hydrolyse urea to ammonia and carbon dioxide. It enables an organism to use urea as a nitrogen source [10]. Urease is the enzyme produced by Helicobacter pylori, which allows them to survive at a low pH of the stomach, resulting in peptic ulcers and, peptic cancer. Urease is responsible for the formation of stones and pathogenesis of uroliothiasis, pyelonephritis and hepatic encephalopathy [11, 12]. The different functional groups present in this enzyme and their interaction with potent specific compounds will make a valuable count to the treatment of infections.

Carbonic anhydrase (EC CA-II) catalyzes the interconversion of carbon dioxide and bicarbonate ion. Certain physiological processes involed in respiration and transport of CO2/bicarbonate in between metabolizing tissues and lungs are controlled by it. Its key role is also played in gluconeogenesis, lipogensis and ureogensis, bone resporbtion, calcification, tumorigenicity, in addition to many other physiological or pathological processesThe majority of carbonic anhydrase inhibitors eitherbelong to sulfonamide or sulphamate class of organic compounds. In the prevention and treatment of disorders e.g. glaucoma, pH imbalance disequilibria, epilepsy, anticancer, and other minor neuromuscular disorders these inhibitors are clinically used.Xanthine oxidase contains molybdenum metal which is responsible for the production of uric acid from purines, hypoxanthine and xanthine. This is also responsible for the formation of reactive oxygen species. Excessive accumulation of uric acid in joints causes gout (also known as podagra) whereas free radicals are responsible for the pathological processes of inflammation, atherosclerosis, cancer and mutagenesis [14, 15]. One of the approaches to treat gout is to inhibit XO which catalyze the formation of uric acid. The standard inhibitor of xanthine oxidase is allopurinol and is clinically used for three decades.[16]. Severe side effects e.g. 6- mercaptopurine toxicity, nephropathy, hepatitis and allergic reactions has been observed by this drug.[17,18, 19]. Thus, there is a need for new drugs for the inhibition of xanthine oxidase.

Results and Discussion

It is observed that the absorption peak occurs at 530 nm in the visible part of the spectrum as shown in Fig. 2. It may be due to the excitation of surface plasmon vibrations of gold nanoparticles in the aqueous solution. In the clove buds, hydroquinones having redox properties can play a vital role in metal reduction. Thus, hydroquinones or other reducing agents present in clove buds are responsible for the reduction of Au3+ ions to AuWhile such reduction of Au3+ ions does not occur in the absence of the extract of clove buds. Evidence suggests that the reducing agents present in the clove buds solution are involved in the reduction of gold ion to its metallic state forming AuNPs [20].

The gold nanoparticles formed were confirmed initially by UV-Vis spectroscopy at a wavelength of 530 nm which is an absorption band for gold nanoparticles. The results are depicted in Fig. 2. (UV-Vis spectra) and Fig. 3. (Color of solutions).

Stability of AuCB was checked against pHand was found to be stable at all pH values (i.e. 2-13). The results are shown in Fig. 4 and Fig. 5.The pH of gold nanoparticles solution was adjusted in the range of 2-14 and its effect was studied with the help of UV-Vis spectra. It was found that the gold nanoparticles of clove were quite stable at all pH values. The pH of optimized gold nanoparticles was found to be 2.9, which may be due to the release of proton from eugenol's by oxidation. Moreover the sizes of gold nanoparticles were not affected by changes in pH.

The effect of NaCl solution was also studied. No effect was noticed by increasing the concentration of NaCl from 0.1 M to 1 M NaCl solution for couple of week as shown in Fig. 6 and Fig. 7.The stability was also checked by boiling the nanoparticles solution for 30 min and it was found that some changes were occurred in the absorption band, thus suggesting variations in the shapes of nanoparticles as shown in Fig. 8.IR spectra suggested that the peak intensities decreased in the spectrum of AuCB which is a clue that some specific groups are involved in the reduction and stabilization of nanoparticles. Most probably the ketonic and carbonyl groups of esters are responsible for such stabilization which is present in the clove buds Fig. 9.The sizes and shapes were studied using atomic force microscope (AFM) which indicated that the particles were in the range of 22-25 nm and of almost spherical shapes (Fig. 10.)Comparative Enzyme Inhibition Assays of CB, AuCBand Au

The gold nanoparticles (AuCB) were screened for enzymatic activities against Urease, Xanthine oxidase, and Carbonic anhydrase-II. The results were compared with pure CB, pure Au and standard drugs. CB was found active against urease and carbonic anhydrase-II enzymes having IC50 values of 41.920.94 and 89.502.34 M respectively (Fig. 11. and Fig. 12.). But conjugation of CB to Au knocks out the enzyme inhibition activity by approximately two times. This decrease in enzyme inhibition activities indicates that the active ingredients in CB are used for the stabilization of gold, which may be the possible reason for the reduction in the activity of AuCB. In case of xanthine oxidase inhibition both CB and AuCB were inactive (Fig. 13.).Experimental

Materials and Method

Syzgium aromaticum (clove) flowering buds were obtained from the local market of district Peshawar, Khyber Pukhtunkhawa, Pakistan, in the month of February 2012. Tetrachloroaurate trihydrate (HAuCl4.3H2O) was obtained from Merck. Doubly distilled water was used throughout the experiments.


Flowering buds of clove (5 g) were taken and soaked in 100 mL of distilled water. The mixture was boiled for 30 min. Filtered to remove any suspended particles and diluted to a final volume of100 mL. This was used as a source extract for the synthesis of Au nanoparticles.

Green Synthesis of Gold Nanoparticles

Nanoparticles stabilized with clove which is already reported [20] were synthesized with slight modifications. For synthesis of Au nanoparticle 60mL of 1 mM gold solution (HAuCl4) was taken in a titration flask and kept on stirring, than 1mL of plant extract (CB) was added drop wise. After 20 min the solution started changing its color from yellowish to light red and finally a dark red color was obtained. The resulting mixture was stirred for 4 h at room temperature.

Enzyme Inhibition Assays

The nanoparticles stabilized with clove buds were used for various activities. The nanoparticles along with an extract of clove bud were screened for the 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 [18]. The determination of xanthine oxidase inhibitory activity of test samples was measured by the rate of hydroxylation of the substrate (xanthine) and subsequent formation of uric acid, which is a colorless product absorbing at 295 nm [21]. 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, purchased from Sigma Aldrich, was used as a standard. Analysis was carried out in triplicate [22].


Gold nanoparticles stabilized with aqueous extracts of clove buds were synthesized. CB extract were found active in inhibiting Urease and Carbonic anhydrase II enzymes while AuCB was inactive. Both CB and AuCB were inactive in inhibiting xanthine oxidase enzyme.

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Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Jun 30, 2014
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