UTILIZATION OF ETHYL ACETATE FRACTION KETAPANG LEAF (Terminalia catappa) AS BIOREDUCTOR IN SYNTHESIS GOLD NANOPARTICLES AND ANALYSIS ANTIBACTERIAL PROPERTIES
UTILIZATION OF ETHYL ACETATE FRACTION KETAPANG LEAF (Terminalia catappa) AS BIOREDUCTOR IN SYNTHESIS GOLD NANOPARTICLES AND ANALYSIS ANTIBACTERIAL PROPERTIES 1 Sarwina Hafid , Muhammad Zakir, Seniwati Dali Chemistry Departement, Faculty of Mathematics and Natural Sciences, Hasanuddin University, Makassar
90245 Abstrak. Nanopartikel emas disintesis dengan metode bioreduksi menggunakan fraksi etil asetat daun ketapang (Terminalia catappa). Nanopartikel yang terbentuk dikarakterisasi menggunakan spektrofotometer UV-Vis, Fourier Transform Infra Red (FTIR) dan X-Ray Diffraction (XRD). Pada analisis spektrofotometer UV-Vis nanopartikel emas dengan konsentrasi larutan prekursor 0,5 mM dan 1 mM masing-masing memiliki serapan antara 533,5-540 nm dan 539-587 nm. Analisis gugus fungsi dalam sintesis nanopartikel emas dianalisis menggunakan Fourier Transform Infra Red (FTIR). Sistem kristal dari nanopartikel emas adalah kubik dengan ukuran nanopartikel 17,13 nm. Pengujian aktivitas antibakteri dilakukan menggunakan beberapa bakteri uji yaitu Staphylococcus aureus , Escherichia coli, dan Bacillus subtilis. Nanopartikel emas tidak dapat menghambat aktivitas bakteri uji Staphylococcus aureus, Escherichia coli, dan Bacillus subtilis sehingga perbedaan konsentrasi nanopartikel emas tidak mempengaruhi daya hambat bakteri.
Kata kunci: antibakteri, biosintesis, karakterisasi n anopartikel, nanopartikel emas, Terminalia catappa .
Abstract. Gold nanoparticles was synthesized by bioreduction method using ethyl acetate fraction of ketapang leaf (Terminalia catappa). Nanoparticles formed were characterized using spectrophotometer UV-Vis, Fourier Transform Infra Red (FTIR) and X-Ray Diffraction (XRD). In the analysis of spectrophotometer UV-Vis gold nanoparticles with a precursor solution concentration of 0.5 mM and 1 mM each has an absorption between 533.5-540 nm and 539-587 nm.
Analysis of functional groups of gold nanoparticles was analyzed by Fourier Transform Infra Red (FTIR). Crystal system of gold nanoparticles is cubic with nanoparticles size of 17.13 nm. The evaluation of test antibacterial activity was performed using several bacteria test such as Staphylococcus aureus, Escherichia coli , and Bacillus subtilis. Gold nanoparticles can not inhibit the activity of a test bacterium Staphylococcus aureus, Escherichia coli, and Bacillus subtilis so that differences in the concentration of gold nanoparticles did not affect the inhibition of bacteria.
Keyword: antibacterial, biosynthesis, characterization of nanoparticles, gold nanoparticles, Terminalia catappa .
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INTRODUCTION
Nanotechnology is a branch of science that is growing very rapidly and is an interesting technological innovations in the field of production, size and shape. Nanoparticles are part of nanotechnology which is very popular and increasingly rapid development since the beginning of 2000. This is due to the benefits and applications of nanoparticles are very broad for human life, among others in the field of environmental, biomedical, healthcare, and industrial (Tsuzuki, 2009).
Some research techniques are available for the synthesis of metal nanoparticles, both methods of chemical and physical methods. However, the synthesis method involving a variety of chemicals can cause the presence of a toxic chemical species which can give side effects on biological applications (Mittal et al., 2014). Because of this, various methods have been developed so called experts emerging nanotechnology based green plants as bioreduktor for the synthesis of gold nanoparticles (Singh et al., 2012). Ketapang is a medicinal plant that grows in Indonesia and has been traditionally used to treat cardiovascular disease, skin, liver, respiratory, stomach, gonorrhea and insomnia (Pauly, 2001). Ketapang known to contain medicinal compounds such as flavonoids (Lin et al., 2000), triterpenoids (Gao et al., 2004), tannins (Ankamwar 2010), alkaloids (Mandasari, 2006), steroids (Babayi et al., 2004) and fatty acids (Jaziroh, 2008). Extracts are used in the synthesis of nanoparticles is ethyl acetate fraction of leaf ketapang. Acetate ethyl is a good solvent used for extraction because it can be easily evaporated, not hygroscopic and has a low toxicity (Ward and Sulistyani, 2012). Ethyl acetate is a solvent that may be interesting compounds alkaloids, flavonoids, tannins and phenols in leaves ketapang (Packirisamy and Krishnamorthi, 2014).
Gold nanoparticles can be applied in the development of antibacterial strategies. This is because the gold nanoparticles are non-toxic and flexibility in surface modification (Cui et al., 2012). Nanoparticles of gold is a metal that is unique among other metals because it is resistant to oxidation and corrosion. Gold tends to be reduced so that in the long term, gold is embedded in the body does not have an adverse effect (Fatimah and Hidajati, 2012) because of the size of the gold nanoparticles are very small be compared with the body's cells, the nanoparticles can exit and enter easily into the cells without interfere with the workings of the cell (Abdullah, 2010).
Based on the description above, conducted research on the synthesis of gold nanoparticles by using ethyl acetate fraction compound leaves of Ketapang
(Terminalia catappa) as bioreduktor and
analysis of antibacterial properties of gold nanoparticles were tested against
Staphylococcus aureus , Escherichia coli
and Bacillus subtilis. The third of these bacteria are toxic harmful bacteria and resistant to antibiotics, so it is necessary to control the lives of the bacteria.
MATERIALS and METHODS Materials Research
Materials used gold, ketapang leaf (Terminalia catappa), HCl pa, HNO
3 pa,
distilled water, akuabides, NaCl 0.9%, poly acrylic acid (PAA), methanol pa, ethyl acetate pa, Bovine Serum Albumin (BSA), Nutrient Agar (NA), Medium Muller Hinton Agar (MHA), klorampenikol, cultures of bacteria
Staphylococcus aureus , cultured
The synthesis of gold nanoparticles is done by adding a fraction of ethyl acetate ketapang leaves into the solution HAuCl
Staphylococcus aureus, Escherichia coli
Testing inhibition of gold nanoparticles on the growth of
Testing the Antibacterial Activity
Paper discs soaked in colloidal gold nanoparticles using a magnetic stirrer, stirring for 5 minutes and then allowed to stand for 1 day. After that, the disc Paper dried in an oven at a temperature of 70 °C for 5 minutes.
Coatings Gold Nanoparticles on Paper Disc Paper disc sterilized in the oven.
(0.5 and 1 mM) and added PAA 1%. Characterization of the mixed solution in the form of color, pH and UV-Vis spectra. Once characterized, the mixed solution is dried with a spray dryer to obtain a powder of gold nanoparticles. powder gold nanoparticles were characterized by FTIR and XRD.
4
Synthesis of Gold Nanoparticles With Ethyl Acetate Fraction Ketapnag Leaf
bacterium Bacillus subtilis, bacterial culture of Escherichia coli, ethanol 95%, paper disc, paper pH universal, aluminum foil, and Whatman filter paper 42.
Extract thick ketapang leaf in the partition with ethyl acetate (3 x 100 mL). Furthermore, separated between the organic phase and the water phase. The organic phase was concentrated using a rotary evaporator.
The Solvent Partition from Extract Ketapang Leaf
Furthermore, sonicated for 1 hour. After the suspension was filtered, and then the methanol evaporated from the filtrate by using a rotary evaporator to obtain a thick extract ketapang leaf (Terminalia catappa ).
Plants used in this study is Ketapang (Terminalia catappa). Part of the plant used in this study are the leaves in fresh condition. The leaves were dried at room temperature. Once dried then crushed and sieved. Furthermore weighed. Ketapang leaf dry powder weighed 50 g, was added a mixture of 50 mL MeOH and 50 mL akuabides.
PROCEDURE Preparation Extract Ketapang Leaf
IRPrestige-21 Shimadzu, incubator, autoclave, tweezers, magnetic stirrer, funnel , vacuum filters, rotary evaporator Heidolph Hei-Vap Value, spray dryer Armfield, a petri dish (diameter 8.5 cm), 30 mL vial bottle, desiccator, rod stirrer, spatula, spray bottle and scissors.
The tools used are glass tools commonly used in the laboratory, oven SPNI 505 FD, analytical balance, spectrophotometer UV-Vis 2600 Shimadzu, XRD 700 Shimadzu, FTIR
Research Tools
and Bacillus subtilis performed with agar diffusion method using a paper disc. MHA sterile medium cooled to a temperature of 40-45 °C. Then aseptically poured into the petri dish and put the bacterial suspension test. After the four paper disc placed aseptically using sterile tweezers on the surface of the medium. Subsequently incubated for 24 hours and 48 hours at 37 ° C and then observed and measured its resistance zone with a sliding crossbar.
RESULTS AND DISCUSSION Characterization Color and pH
Characterization color of the solution was conducted to determine the formation of gold nanoparticles
Figure 1. Changes the color in of gold nanoparticles from the first minute until 1 day
Burgundy color that forms indicate the occurrence of reduction of Au
3+
into Au . The color formation caused by the excitation of surface plasmon nanoparticles. The changes that occur during the synthesis of the color indicates the growth of clusters generated even greater, at which time the gold atom has not interact with each other (colorless solution). In a certain amount of gold clusters provide a purple color is followed into the red, while the larger clusters and upon entering the nano-size, gold became red wine. Au atoms will interact with each other and produce metal bonding cluster in very large quantities.
Characterization of the pH measured for increasing time. PH value measurement is done to observe any possible changes in pH during the reaction process. PH measurement starts for a precursor solution, stabilizers, and bioreduktor with each pH value is 2, 4 and 5. The pH value of the gold nanoparticles is pH 1, even after the seventh day the pH is not changed. This shows the increase in acidity of the gold nanoparticles is characterized by a decrease in pH value.
Characterization Using UV-Vis Spectrophotometer The Formation of Gold Nanoparticles
Based on the UV-Vis spectra (Table 1) wavelength spectrum measurement results, obtained pattern and the wavelength of maximum absorbance which became the basis of the formation of gold nanoparticles. Gold nanoparticles has a wavelength of 500- 600 nm. The process of formation of gold nanoparticles can be seen from the shift of the maximum wavelength is 313.00 nm solution HAuCl
4 be 540.00 nm and 587.00 nm as shown in Table 1.
Table 1. Results of UV-Vis spectrum analyzer Samples Wavelength (nm) Absorbance
HAuCl
Gold nanoparticles 1 mM 313,00 540,00 587,00
2,412 1,635 0,862
4 Gold nanoparticles 0,5 mM
Based on the results of the infrared spectrum (FTIR), obtained by
, 1037 cm
) has been reduced into a wave number 1035 cm-1. In addition, there is also a shift in the wave number 3448 cm- 1 to 3439 cm-1 due to the binding of the hydroxyl group with gold nanoparticles.
. Resonance may be due to binding of gold nanoparticles on the surface. Shifting the CO group (1037 cm
shifted into 1716 cm
. Carbonyl group in 1707 cm
and 1035 cm
, 3439 cm
which is a characterization of the CO group. Figure 3 (b) is an FTIR spectrum of ethyl acetate fraction ketapang reducing gold leaf at wave number 1716 cm
arises because of the free OH in phenol, besides there are local wave number 1037 cm
are characteristic of the carbonyl group of carboxylic acids and phenols. vast stretches in the wave number 3448 cm
. At wave number 1707 cm
and 3448 cm
Figure 3 (a) is an FTIR spectrum of ethyl acetate fraction ketapang leaf (Terminalia catappa) with the addition of poly acrylic acid (PAA) shows a prominent absorption band at 1707 cm
Figure 3. FTIR spectra of (a) Fraction ethyl acetate ketapang leaves + PAA, (b) gold nanoparticles.
- 1
- 1
- 1
Phytochemical test on leaves ketapang showed phenolic compounds are compounds that dominant. Phenolic compounds indicated are tannins. Chen et al., (2000) reported that ketapang leaf contains 21% of tannin.
4
FTIR measurements conducted to identify biomolecules that may play a role in the ethyl acetate fraction of leaf ketapang to reduce ion AuCl
Characterization of Gold Nanoparticles with FTIR
In this study poly acrylic acid is used as a stabilizer. PAA can be used as a stabilizing agent it can be seen from PAA structure that has a -OH group. O atoms in the PAA likes to stick to the gold atom as O atom has a lone pair, while the acrylate groups that have a long chain would be ligands away from the surface of the gold and gold shield so as not to form aggregates.
nanoparticles (a) AuNp 0.5 mM (b) AuNp 1 mM.
(a) (b) Figure 2. Spectrum stability of gold
The stability of the colloidal solution of nanoparticles can be seen from the changes in absorption peak. Gold nanoparticles have a tendency to form larger clusters. Therefore, it takes a stabilizer to prevent aggregation process.
The Stability of Gold Nanoparticles
- 1
- 1
- 1
- 1
- 1
- 1
- 1
- 1
- 1
- into Au .
OH OH HO
HO O OH O OH HO O HO O O OH O OH OH O OH HO O O O HO O O O O OH
OH HO O - H HO O OH O O
OH OH
O TaninHO
HO
OHOH
HO HO O O OH OH HO O O HO O OH2+ O
OH OH
OH- Au
HO O O O O HO O O O OH O OH HO 3+ O HO O O OH O Au O OH O Au HO
HO OH
OH
HO HO O OH O HO O OHHO O
O OH OH O HO OOH OH O O O O O H 2+
HO
- HO
Au O O O
O O
- Au- Au + Au + H
O OH O Au
HO O HO O O OH HO
Au
2
2 Au gold nanoparticles
Figure 4. Estimated reaction mechanism of synthesis of gold nanoparticles using
ethyl acetate fraction ketapang leaf (Terminalia catappa) In addition to the involvement of hydrolyzed to form the core of Au as in bioactive compounds from ethyl acetate the following scheme. fraction ketapang leaf (Terminalia
Au-Au Nucleud Au AuNp Coloid
catappa ), the possibility of
(4) disproportionation reaction to produce Colloid formation associated with gold nanoparticles. Disproportionation the emergence of a core in saturated reaction is a redox reaction in which an conditions. Once it is formed Au oxidant and reduktornya the same nanoparticles would grow into colloids substance (Shriver and Atkins, 1999). (Zakir, 2005). Thus, some of the substances that undergo oxidation and partly reduced.
Characterization Gold Nanoparticles 3+ 2+
by XRD
Au + e- Au
2+ 2+ 3+
Powders from synthesis results
- Au + Au Au
- 2+ further characterization using XRD.
Au + Au Au + Au (3) Analysis using XDR conducted to
A process that may occur in the determine the shape and size of the formation of gold nanoparticles is the crystals of gold nanoparticles. Gold formation of polymer Au then nanoparticles diffraction pattern shown in
Figure 5.
4 BSA (-) - - - - - -
E. Coli B. subtilis.
11 10 27,3 28,3 24,7 24,9
3 Kloramfenikol (+)
1 mM)
3+
2 AuNp (Au
0,5 mM)
3+
1 AuNp (Au
48 hours
24 hours
48 hours
24 hours
48 hours
24 hours
No AuNp Average - average Barriers Diameter (mm) S. aureus
The peaks of the diffraction pattern of the gold nanoparticles is clearly shown in
Table 2. The average diameter obstacle gold nanoparticles against some bacteria test for 1 x 24 hours and 2 x 24 hours.
the concentration of gold nanoparticles, has no inhibitory zone of incubation time 24 to 48 hours. This shows that the gold nanoparticles are not resistant to test bacteria Staphylococcus aureus , Escherichia coli and Bacillus subtilis.
Escherichia coli and Bacillus subtilis to
Based on data from Table 2, the test bacteria Staphylococcus aureus,
Testing inhibition of gold nanoparticles on the growth of bacteria is done by using the agar diffusion method using MHA media as a growth medium.
Escherichia coli and Bacillus subtilis.
This study uses a bacterial test consisting of Staphylococcus aureus,
Testing the Antibacterial Activity
Lembang (2013) managed to synthesize gold nanoparticles of decoction of the leaves extract ketapang with the size of 18.91 nm. It showed that gold nanoparticles were synthesized using a fraction of ethyl acetate resulted in a smaller particle size than the extract of decoction of the ketapang leaves.
2θ FWHM 0.97 38.1750 with a value that can be used as a reference for the calculation of the size of the gold nanoparticles. Based on the XRD results obtained gold nanoparticle size is 17.13 nm.
2θ produce several peaks of gold nanoparticles. But which has a high intensity is at an angle
nanoparticles At Angle
Figure 5. XRD patterns of gold
2θ value is 38.1750, 34.2975 and 44.3150 respectively with FWHM value of 0.97, 0.745 and 1.11. In addition, Miller index of each peak is (111), (200), (202) and (311). At the orientation (202) and (311) peak looks weak and is much stronger on the orientation (111) and (200). This suggests that the gold nanocrystal dominant on the orientation (111) and (200).
A B
Figure 6. Photo inhibitory zone diameter gold nanoparticles against some bacteria test for 1 x 24 hours (A) and 2 x 24 hours (B).
Label : 1. gold nanoparticles 0,5 mM
3. BSA (-) 2. gold nanoparticles 1 mM
4. Kloramfenikol (+)
2. Ankamwar, B., 2010, Biosynthesis
CONCLUSION
Based on research that has been of Gold Nanoparticles (Green- Gold) done, it can be concluded that: Using Leaf Extract of Terminalia
1. The gold nanoparticles can be Catappa , E. J. Chem., 7(4): 1334- synthesized by the method of 1339. reduction using ethyl acetate fraction
3. Babayi, H., Kolo, I., Okogun, J.I., bioreduktor ketapang leaf Ijah, and U.J.J., 2004, The (Terminalia catappa) with a size of antimicrobial Activities of gold nanoparticles is 17.13 nm. Methanolic Extract of Eucalyptus
2. The gold nanoparticles can not camaldulensis and Terminalia inhibit the activity of the bacteria catappa Againt some Pathogenic
Staphylococcus aureus , Escherichia Microorganisms, An Int. J. Niger. coli, and Bacillus subtilis. Soc. Exp. Bio ., 16(2): 106-111.
3. The difference in the concentration
4. Chen, P.S., Li, J.H., Liu, T.Y., and of gold nanoparticles did not affect Lin, T.C., 2000, Cancer Letters., the activity of the bacteria 152(2): 115-122.
Staphylococcus aureus, Escherichia
5. Cui, Y., Zhao, Y., Tian, Y., Zhang,
coli, and Bacillus subtilis. W., Lü, X., and Jiang, X., 2012, The
Molecular Mechanism of Action of
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