Journal of Materials Science and Engineering B 2 (12) (2012) 611-617

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Activity Test of Nanogold for Reduction of Free Radicals, a Pre-Assessment Utilization Nanogold in Pharmaceutical as Medicines and Cosmetics
Titik Taufikurohmah1, I Gusti Made Sanjaya1, Afaf Baktir2 and Achmad Syahrani3
1. Chemistry Department, the State University of Surabaya, Surabaya 60231, East Java, Indonesia 2. Chemistry Department, Airlangga University, Surabaya East Java, Indonesia 3. Pharmacy Department, Airlangga University, Surabaya East Java, Indonesia Received: September 14, 2012 / Accepted: September 29, 2012 / Published: December 25, 2012. Abstract: Gold has been used long times ago as a medicine and cosmetics. The development of materials has entered the nanomaterial era no exception of gold material that known as nanogold. The use of nanogold also entered the world of pharmacy as drugs or cosmetics. Nanogold has been used as a cure for cancer as well as antiaging material in cosmetics. This study aimed to test anticancer and antiaging nanogold activity. One of anticancer activity test is reduction of free radical activity. Antiaging activity assays can also use the free radical reduction activity test. In the reduction of free radical activity test using artificial free radical diphinylphikrylhidracyl (DPPH). DPPH compound has maximum absorption at UV-Vissible areas that will experience a decrease absorbance event damping. Nanogold concentrations used to reduce DPPH 6% (w / v) are 5, 10, 15, 20, 25, 30, 35 and 40 ppm. Percent reduction DPPH are 47.66%, 52.02%, 56.14%, 56.85%, 66.27%, 52.34%, 47.11% and 35,15%. It can be concluded thatif the concentration of nanogold is higher, the percent reduction of free radicals by nanogold is increase. But after the concentration 25 ppm, percent reduction of free radicals is decrease, because the colloidal nanogold system was damaged by the formation of sediment that has a particle size exceeds of colloidal particles range 1-100 nm. It also shows that the activity of nanogold particles greater than bulk gold activity. Keywords: Nanogold, anti aging, anti-cancer, free radicals, colloidal.

1. Introduction
Gold is classified as a Group IB metal in the periodic table. The most commonly recognized oxidation states are I, II, III, and V, although metal-metal bonds do exist in complexes for which it is difficult to assign a formal oxidation state to the gold atom. True salts of Au(I) such as the halides are unstable in the presence of water and disproportionate to Au(I) (metallic gold) and Au(III). However, Au(I) can be stabilized by the formation of complexes with soft ligands [1, 2], such as the thiolates and phosphines. All currently used anti-arthritic gold complexes exist as Au(I)-thiol or -phosphine compounds [2, 3]. The high toxicity of the
Corresponding author: Titik Taufikurohmah, research field: material. E-mail: ttaufikurohmah@yahoo.com.

Au(III) complexes such as chloroauric acid (HAuCl4), makes them unsuitable for human use. Elder and colleagues have shown that if gold sodium thiomalate is administered to laboratory animals, the gold recovered from the tissue and urine exists in the Au(I) oxidation state (i.e., the same oxidation state as the gold in gold sodium thiomalate and not the Au(III) oxidation state [2, 4] If AuCI3, is administered to laboratory animals, the gold state recovered in tissues and urine exists only as the Au(I) oxidation state. It is thus considered that Au(I) is the stable primary oxidation state in a biological milieu [2]. Metallic gold is one that has a unique metal that is not easily oxidized, even more likely to be reduced, this can be explained by thereduction potentialof

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Activity Test of Nanogold for Reduction of Free Radicals, a Pre-assessment Utilization Nanogoldin Pharmaceutical as Medicines and Cosmetics

thegoldpriceis positive. These unique characteristics of the under lying metal that is gold does not harm the body as platinum metal [5]. In the 20th century following the observations of Jacques Forestier, inject able gold compounds were successfully used for the treatment of rheumatoid arthritis. Of the many compounds developed, gold sodium thiomalate has been the most extensively studied by basic scientists and by clinicians. In the1980s, the oral gold compound auranofin showed promise as a therapeutic contender to injectable gold, but the clinical side effect profile and fear of long term effects of immune suppression by auran of in, resulted in gold sodium thiomalate continuing as the preferred gold compound for rheumatoid treatment. However, the increased use and demonstration of effectiveness of low dose Methotrexatein rheumatoid treatment over the last 20 years has resulted in a significant decline in the use of injectable gold sodium thiomalate, this despite the claims and evidence that it remains a useful agent in the management of rheumatoid arthritis [2]. The observations give the protein test results that the solution gives a negative result means a dilute solution of albumin did not experience denaturation with test solution include a solution of Au , Na solution and a solution of Au (nanogold). Albumin solution that remains transparent do not experience denaturation. This indicates that the presence of metal is safe in cosmetics because it will not harm the skin protein which is collagen [5]. Some research results related to the use of metallic gold has been patented, such as a dye that is safe for modern cosmetics. Patents related to the utilization of nanogold as dyes of various types of cosmetics that is U.S. patent number U.S. 0022765 in claims by Chung et al published 22 January 2009. Thus, it is precisely when the gold metal is used as a dye in modern cosmetics [6]. The size of nanogold cluster of synthesis is in the range 80-200 nm, where the material that was agreed as nano material has a size of 10-100 nm. Thus, the results
3+ +

of synthesis are partly already in the range of nanoscale materials and some larger than nanoscale materials. For use in cosmetics sizes 80-200 nm are still acceptable, because the pores of the skin has a much larger size of the micro or mesoscale. Particle size for cosmetics is in the range 10-800 nm, so that the result of the synthesis is still in accordance with the particle range of cosmetics. Nevertheless have to look for other matrices in the synthesis in order to obtain the results of synthesis in the nano size, so the catalytic effect is given to a maximum. Glyceryl monostearate has a small molecular size and shape of molecules that are less filled the space where only one fatty acid bound in glycerol. The shape and size of the matrix will determine the size of the resulting gold clusters [5]. It is time the modern cosmetic use of safe materials and provides great benefits to the compounds of the essential results of research development. Development of synthesis of nanogold becomes very important to be immediately applied in cosmetic products as an anti-aging material. Besides safe for health, nanogold also has activity as a cofactor of enzymes that help the formation of skin collagen and collagen in other tissues. Regeneration of collagen is an important process for maintaining youthful skin. Damage to collagen that occurs naturally, can be inhibited by the continuous regeneration of collagen. This is the basic use of nanogold as an important material in modern anti-aging cosmetics. Nanoscale forms also provide a catalytic effect that increased 20-200 times compared to the size of the micro-meter. Therefore, the development of the synthesis of nanoscale materials in the form of nanogold is becoming very important [7]. Nanogold also has a free radical reduction activity is very important as antiaging material in cosmetics. This study used an artificial (DPPH) free and radical nanogold diphinylpyikrylhidracyl

concentration of 20 ppm. Decrease inabsorbanceat a wavelength of 519.5 nm explain the magnitude of free

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radical reduction activity conducted by nanogold. Nanogold at a concentration of 20 ppm can be lowered absorbance from 0.631 to 0.412. Decrease of absorbance is explain that nanogold can reduce free radicals that can cause damage in various parts of the cell and cause various diseases such as tumors, cancer, arteriosclerosis, cataracts, wrinkles, aging and other [8]. Nanomaterial synthesis is generally divided into two kinds of processes, namely the top down and the bottom up. Top down process is the process of making nanomaterials from big initial material into small-sized nanomaterials. While the bottom up process is the process of making nanomaterial from small initial material, i.e., ionic or atomic-sized materials into big size nanomaterials. Nanomaterial synthesis in this research is to use the bottom up by changing the raw materials HAuCl4 into nanogold, which are bigger in size. Reducing reagent is sodium citrate with a concentration of 1% which is made by dissolving 1 g of sodium citrate in 100 ml of a quads. Gold solution (HAuCl4) with a concentrationof 1,000 ppm is made by dissolving 1 g of gold with 8 ml of strong acid (aquaregia) and diluted to be 1,000 ml of volume. Basically, the solution of HAuCl4 produces chloroauric ion or AuCl4 in solution which reduces to Au in the synthesis with the presence of sodium citrate as a reducing agent. Gold atoms are joined to each other and grow uncontrollably, so when it has achieved a big cluster and then falls as precipitation. When this occurs, the size of the nanomaterial is exceeded and is not produced the desired nano-scale materials. To solve this problem, use a matrix that would block the uncontrolled growth of these clusters [5]. Nanogold bound in this material will be used in modern material of anti-aging cosmetic along with other cosmetic. Nanogold serves as a catalyst the formation of collagen, helping enzymes that already exist in the system, metabolism of collagen formation by skin cells. Mechanism of the formation of collagen synthesis is based on the ability of gold or nanogold in
o

attractive electron-electron as the beginning of the chemical reaction. One species electron with another species in a system coupled to perform the reaction, henceforth when a bond has formed the gold or nanogold out of the reaction and will perform the same task for the next spec-spec. So forth so that all species in the reacting system becomes more complex material [5]. Modern cosmetic uses of gold as an anti-aging material mainly because gold becomes a catalyst in the formation of collagen. Collagen is one of the major protein constituent of skin tissue. Regeneration of collagen regularly makes collagen tissue of the skin to become solid so that skin becomes firmer and look younger. Conversely, if growth is not balanced with the damage of collagen, the skin looks wrinkled and old impressed. The catalyst used in the formation of collagen in the previous cosmetic is vitamin C, which has a weakness stinging on the skin and the activity easily decreased. The gold materials can overcome these weaknesses. Recent developments of anti-aging cosmetic by using gold nanoparticles need to be further developed to obtain optimum catalytic effect [5]. Previous studies using glyceryl monostearate matrix that has low solubility in water, so there are clumps separated from the matrix generated nanogold clusters. In this study the use of glycerin as a matrix in order to have a more stable colloid because glycerin is soluble in water. Thus expected to produce a more homogeneous nanogold cluster size in diameter. The problem in this research are: (1) How characterization nanogold is synthesized using glycerol as a matrix?. (2) Is nanogold synthesized using glycerol as a matrix able to reduce free radicals DPPH?. (3) What percentage of DPPH reduction by nanogold at different concentrations?

2. Experiments 2.1 Nanogold Synthesis

Starting materials in this research is a solution of HAuCl4 1,000 ppm are colored yellow, with reducing agent is sodium citrate. A total of (1,000-x) ml of

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Activity Test of Nanogold for Reduction of Free Radicals, a Pre-assessment Utilization Nanogoldin Pharmaceutical as Medicines and Cosmetics

distilled water in the 1000 ml beaker glass is heated to boiling. Sodium citrate 2 g added and stirred until homogeneous.HAuCl4 solutionx mlis added(x = 5, 10, 15, 20, 25, 30, 35 and 40). Stirring continued and at the solution became colorless, glycerin matrix incorporated and continue stirring until the color changes from colorless to dark blue, red and burgundy. Stirring and heating was stopped. Thus generated nanogold 5, 10, 15, 20, 25, 30, 35 and 40 ppm. Some nanogold colloids are then used in the reduction of free radical activity assay. Some were evaporated to dryness in a petri dish is then performed using SEM analysis instrument.

2.2 Nanogold Activity test to Reducing Free Radicals DPPH Beginsa 4%DPPH solutionwith dissolve 4 g DPPHto dissolve as much as 100 ml volume of the alcohol. Wanted maximum wavelength ofDPPH at UV-Vis spectrophotometer. An additional 2 ml of 4% DPPH solution was mixed with 2 ml of 5 ppm colloidal nanogold until homogeneous. Measured at a wavelength of maximum absorption of DPPH. Uptake measurements performed for colloidal nanogold at other concentration that is 10, 15, 20, 25, 30, 35 and 40 ppm. Calculated% DPPH reduction by nanogold at each concentration is given by:

(A DPPH - (A [DPPH + Nanogold]-A Nanogold) 100% , A= Absorbance (1) A DPPH

3. Results and Discussion
3.1 Nanogold Synthesis Nanogold synthesis results in a matrix or surfactant glycerin has a characteristic purple color and intensity of purple color increased at higher concentrations. Synthesis performed in the concentration range of 5-40 ppm, i.e., 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm, 30 ppm, 35 ppm and 40 ppm. At concentrations above 25 ppm, color intensity began drop to back because a precipitate is formed. Matrix glycerin only able to stabilize nanogold colloidal at limit concentration to 25 ppm. But glycerin matrix provides uniform pore or homogeneous spaces so that nanogold clusters formed also uniform diameter size. It is evident from the results of SEM analysis at various concentrations nanogold has an average cluster diameter of 20-21nm. Basically clusters grow and expand in accordance with the availability of empty spaces in the system. At concentrations greater nanogold the blank spaces provided more fully charged so that the intensity of the color is getting stronger. At low concentrations some of the unallocated space nanogold clusters so that the intensity of the color is also getting weak [9]. Characterization of the result synthesis with SEM analysis can be argued that the matrix of glycerin is very suitable for use in cosmetics because nanogold clusters have an average diameter of 20-21 nm that is expected to enter the pores of the skin and doing activity. Glycerin molecule is not large and wide thus less cluster so tightly spaced and not close the possibility of inter-cluster aggregation at concentrations above 25 ppm, making it less effective as a matrix in the synthesis of nanogold with concentrations above 25 ppm. From the data with a magnification 60,000 SEM image shows that the diameter of the cluster. Nanogold synthesized with a concentration of 5 ppm Au (in HAuCl4) produces clusters with a size of 15 nm, 17 nm

Fig. 1

Colloidalnanogold concentration of 5-40 ppm images from left to right.

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diameter of 27 nm, 13 nm and 25 nm. Variations in the size of clusters did not differ in the concentration range of 5-20 ppm, it is supported by physical observations. The resulting color is similar nanogold is purple with different concentrations where to 5 ppm brighter and rising at 10 ppm, 15 ppm and 20 ppm. Increasing the intensity of the color associated with the distance between the cluster where the density of the more dense concentration, the more narrow the distance between the cluster and the color intensity is getting stronger. The size of the clusters did not differ in concentrations of 5, 10, 15 and 20 ppm. Average cluster diameter is 21nm at 5 ppm, 10 ppm is 20nm, 15ppm is 20.75 nm and 20 ppm was 21.67 nm. The synthesis dasil 25-40 ppm color intensity decreased as beginning to look a colloidal precipitate indicating unstable again. DPPH solution of 6% (w/v) gave maximum absorption at a wavelength of 519.00 nm with the absorbance 1.263. Artificial Free radicals DPPH are
Fig. 2 SEM image of nanogold 20 ppm and 5 ppm magnification 60,000 .

suppressed by nanogold at various concentrations. After reduction process the absorbance price at a wavelength of 519.00 nm is expected to fall to or less than 1.263. Nanogold has a UV-Vis absorption region 500-600 nm is very close to the wavelength of maximum DPPH (519.00 nm). This needs to be taken into account in calculating the damping DPPH percent. Really need to reduce each sample uptake rates (DPPH + nanogold) to determine the uptake of nanogold DPPH absorbance decrease at having curbs by nanogold. Nanogold at various concentrations have a maximum wavelength and absorption vary prices. However, absorbances at various concentrations of

Fig. 3 UV-Viss absorbance of DPPH 6% (w/v).

nanogold considered is absorbance at a wavelength of 519.00 nm. It is intended for the calculation of DPPH absorbance decreased at maximum absorption 519.00 nm. Date of follows. nanogold absorbance at various concentrations at a wavelength of 519.00 nm are as

and 31 nm. While the concentration of 10 ppm produced clusters with size 21 nm, 13 nm and 23 nm. The concentration of 15 ppm produced clusters with a size of 25 ppm, 29 ppm, 19 ppm and 10 ppm. Recently at a concentration of 20 ppm clusters produced has a

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Activity Test of Nanogold for Reduction of Free Radicals, a Pre-assessment Utilization Nanogoldin Pharmaceutical as Medicines and Cosmetics

sediments with large size. The bigger the cluster nanogold more narrow so that the touch surface activity by DPPH reduction nanogold smaller anyway. Very consistent. with the the oy that ktivitas nanomaterial higher activity than solid material [10].

Fig. 4 UV-Vis absorbance of nanogold at various concentration. Table 1 UV-Vis Absorbance of nanogold at 519.00 nm. No. 1 2 3 4 5 6 7 8 Concentration of nanogold (pm) 5 10 15 20 25 30 35 40 Absorbance 0.133 0.210 0.349 0.380 0.630 0.301 0.301 0.128

Fig. 5 Graph of UV-Vis nanogold.

Further Table 1. Created plot a graph with x axis is nanogold concentration and y axis was UV-Vis absorption nanogold obtained Fig. 5. Maximum absorption at 25 ppm of concentration. Highest absorbance at a concentration of 25 ppm nanogold. Physically, the highest color intensity at 25 ppm. Concentrations above 25 ppm occurs aggregation between clusters nanogold or colloidal system damage. Damage colloidal system has an impact on the loss of colloidal color canthat be observed directly or with the aid of instruments. Further Table 2. Created plot a graph with x axis is nanogold concentration and y axis was UV-Vis absorption DPPH + nanogold obtained Fig. 6. Maximum absorption at 25ppm of concentration. Percent reduction in the highest DPPH nanogold concentration of 25 ppm. This is in accordance with the physical conditions of the synthesis results nanogold. At concentrations above 25 ppm occurs between clusters nanogold aggregation cause stimbut

Fig. 6 Uv-Viss absorbance of DPPH 6% (w/v) + nanogold at various concentration. Table 2 UV-Vis Absorbance of DPPH + Nanogold at 519.00 nm. No. 1 2 3 4 5 6 7 8 Concentration of nanogold (ppm) 5 10 15 20 25 30 35 40 Absorbance 0.794 0.816 0.903 0.925 1.056 0.903 0.969 0.947

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4. Conclusions
Nanogold were synthesized using glycerol matrix has a smooth and homogeneous surface with cluster size in the range of 10-25 nm. Nanogold able to reduce free radicals DPPH. Percent free radicals DPPH reduction by nanogold concentration 5, 10, 15, 20, 25, 30, 35 and 40 ppm, respectively 47.66%, 52.02%, 56.14%, 56.85%, 66.27%, 52.34%, 47.11% and 35.15 %.

References
Fig. 7 Graph of Absorbance DPPH + Nanogold. [1] A.S.K. Hashimi, R. Salath, T.M. Frost, L. Schwarz, J.H. Choi, Homogeneous catalysis by gold : The current status of C, H activation, J. Am. Chem. Soc. Appl. Catal. A 291 (1-2) (2005) 238-246. [2] W.F. Kean,L.I.R. Kean,Review clinical pharmacology of gold,UK Inflammopharmacology 16 (2008) 112-125. [3] P.J. Sadler, The comparative evaluation of the physical and chemical properties of gold compounds, J. Rheumato l9 (1982) 71-78. [4] R.C. Elder, M.K. Eidsness, M.J. Heeg, Gold-based antiarthritic drugs and metabolities, A.C.S. Symp. Ser. 209 (1983) 285-290. [5] T. Taufikurohmah, I.G.M. Sanjaya, A. Syahrani, Nanogold synthesis using matrix mono glyceryl stearate as antiaging compounds in modern cosmetics, Journal of Materials Science and Engineering A1 (2011) 857-864. [6] B.H. Chung, Y.T. Lim, J.K. Kim, J.Y. Jeong, T.H. Ha, Cosmetic pigment composition contaning gold or silver nano particles, United States, Patent, US20090022765A1, 2009. [7] A. Wolf, Y. Pouny, I. Marton,O. Dgany,A. Altman,O. Shoseyov,SP1 polypeptides, modified SP1 polypeptides and uses thereof,United States, Patent, US 2009/0062197, 2009. [8] Rhesma, T. Taufikurohmah, Synthesis and characterization of nanogold at various concentration HAuCl4 solution as antiaging material in cosmetic,in: National Conference of Chemistry, Sekarsari, 2011, pp. 55-64. [9] H. James, Nanogold synthesis in wool fibres: novel colourants, Gold Bull Springerlink.com (2011) 85-89. [10] J.K. Kenneth, M.R. Ryan, Nanoscale Materials in Chemistry, Hoboken, New Jersey, John Wiley & Sons, Inc, 2009.

Table 3 Uv-Viss absorbance of test results in reducing DPPH activity by nanogold. Absorbance Cons. of nanogold+DPPH nanogold at 519.00 (nm) (ppm) (A) 0 5 10 15 20 25 30 35 40 ppm 1.263 0.794 0.816 0.903 0.925 1.056 0.903 0.969 0.947 Absorbance nanogold at A-B 519.00 (nm) (B) % reduction of free radical DPPH (%) 47.66 52.02 56.14 56.85 66.27 52.34 47.11 35.15

0.133 0.210 0.349 0.380 0.630 0.301 0.301 0.128

0.661 0.606 0.554 0.545 0.426 0.602 0.668 0.819

Fig. 8 Graph Persentage of Reduction DPPH.