Composite of Polylactic Acid/Chitosan/Ag- Hydroxyapatite Synthesized Using Turmeric Leaves Extract-Mediated Silver Nanoparticle and Snail Shell as Antibacterial Material

The development of an antibacterial composite of polylactic acid/chitosan/silver nanoparticledoped hydroxyapatite has been synthesized. The composite was prepared using the silver nanoparticles (AgNPs) green synthesized by using turmeric (Curcuma longa Linn) leaves extract-mediated AgNPs and snail shell as biogenic calcium for hydroxyapatite synthesis. The precipitation method of hydroxyapatite by the doping of AgNPs was the first step, followed by composting with polylactic acid and chitosan as the polymer binder. Physicochemical characterization of the material was studied by using XRD, SEM, and FTIR analyses, and the antibacterial catalytic performance was examined against Escherichia coli (E. coli). The results showed that the synthesized AgNPs are within the <100 nm range in size and not significantly influence the crystallinity of the Ag/HAp. The composite materials maintained the antibacterial activity against E. coli.


Introduction
The development of biomaterials for medical applications highlighted the use of hydroxyapatite (HAp) in abundant demand [1]. Hydroxyapatite (HAp) is a compound formed from the elements calcium and phosphorus with the chemical formula Ca10(PO4)6(OH)2 or Ca5(PO4)3.OH. The use of hydroxyapatite in the biomedical field is very wide, including as a material for the repair or replacement of bone and tooth tissue [2]. HAp can be synthesized using biogenic calcium, namely calcium oxide derived from the calcination of animal materials by reaction with ammonium phosphate or phosphoric acid to produce HAp. Snail shell waste (Achatina fulica) has the potential to be developed as a source of Calcium Oxide (CaO). Waste snail shell has a mineral content of calcium carbonate (CaCO3) is high. CaCO3 can be decomposed into CaO on heating to high temperatures [3]. In addition, related to the antibacterial activity of biomaterial, some dopants to the HAp structure were utilized, and silver nanoparticles are the most popular dopant due to their high activity and biocompatibility. The development of Ag-doped HAP (Ag-HAp) by applying green chemistry methods such as the plant extract-mediated bioreduction was much selected [4].
Silver nanoparticles (AgNPs) have been shown to be most effective due to their good antimicrobial properties against bacteria, viruses, and other eukaryotic microorganisms. Silver nanoparticles are more effective because of their high surface area to volume ratio so that most of the silver nanoparticles are in direct contact with their environment [5]. In the ionic form, silver is a strong antibacterial agent and is toxic to cells. Several studies used a chemical reduction method with the silver metal precursor used is AgNO3 to produce AgNPs because it is relatively simple, easy, and effective. In the reduction process using the method, it is a green synthesis which is safe and not dangerous, so that it is environmentally friendly. The principle is to utilize the content of plant secondary metabolites as reducing agents. Bioreductors can be obtained from natural ingredients containing terpenoids, flavonoids, and tannins which have antioxidant activity that can reduce silver [6]. Several studies have investigated the physiochemical characteristics of turmeric leaf extract (Curcuma longa Linn) to show its functional effects such as antioxidant activity, these effects are mainly derived from curcumin, total phenolic compounds, and flavonoids in turmeric leaves [7]. Using this reason, turmeric leaves can be used as a bioreductant agent in metal nanoparticles and metal oxides.
The combination of silver-doped HAp with biocompatible polymers gives possibility to be more adaptive in the tissue engineering. Polylactic acid and chitosan were reported as potential polymers for several developments of biomaterials [8]. Such printed scaffold biomaterials were successfully produced by the combination of both polymers with better mechanical properties and stability [9] [10].
Based on these backgrounds, in this study, the synthesis of polylactic acid/chitosan/Ag-HAp Synthesized Using Turmeric Leaves Extract-Mediated Silver Nanoparticle and Snail Shell as Antibacterial Material was conducted. The physicochemical properties of the prepared composites were characterized by UV-Vis spectroscopy, Fourier transforms infrared spectroscopy (FTIR), particle size analyzer (PSA), and antibacterial activity was evaluated.

Materials
fresh Curcuma longa Linn leaves and Achatina fulica shell were collected from the traditional market in Sleman District, Yogyakarta Province, Indonesia. The aqueous leaf extract (henceforth called as CLE) was prepared by grinding 40 g of the fresh leaves followed by maceration in 100 mL of water for an hour, followed by filtration. The biogenic CaO from snail shells was obtained by calcining the crushed snail at 1000 •C for 4 h. Chemicals used in this research consist of pro analyst-grade of silver nitrate (Merck), ammonium diphosphate (Sigma-Aldrich), acetic acid, chitosan (Sigma-Aldrich), and polylactic acid (Sigma-Aldrich).

Synthesis of AgNPs
The AgNPs were synthesized using CLE as a bioreductor, using the method reported in previous work [4]. About 9 mL of AgNO3 10 -3 M with 1 mL of CLE, followed by microwave heating for 15 min. The reduction mechanism was monitored by UV-Visible spectrophotometry and particle size distribution identification.

Synthesis of Ag-HAp
The silver-doped hydroxyapatites were synthesized from CaO derived from snail shells and Ca(NO3)⋅2H2O by setting the atomic ratio of Ag/[Ag +Ca] at 0.2 and [Ca+Ag]/P at 1.67. The AgNPs and calcium source were dissolved in deionized water to obtain 250 mL [Ca + Ag]-containing solution and stirred at room temperature. Into the stirred solution, the (NH4)2HPO4 solution was added slowly until the [Ca + Ag]/P atomic ratio of 1.67 was obtained. The mixture was kept in an autoclave at 110 • C overnight. The resulting slurry was then dried in an electric oven at 80 • C before sintering at 900 • C for 1 h. The silver-doped hydroxyapatites were encoded as Ag/HA for the composite obtained from snail shells and Ca(NO3)⋅2H2O, respectively. As for comparisons, HA without silver doping was also synthesized.

Synthesis of PLA/Chitosan/Ag-HAp
PLA/Chitosan/Ag-HAp was synthesized by mixing PLA, chitosan gel, and Ag-HAP in the weight ratio of 1: 1: 1. The chitosan gel was prepared by diluted chitosan flakes in 2% of acetic acid.

Antibacterial Activity Test
Antibacterial activity of Ag/HAp was tested for Escherichia coli (ATCC 11303). A nutrient medium was prepared by suspending nutrient agar in distilled water and autoclaving before use. The tested bacterial was cultivated in a nutrient broth medium by incubation at 37 °C for 24 h. The bacterial culture was evenly spread throughout a Petri plate, and a 6-sterile filter disc was loaded with 0.02 g of Ag/HAp powder followed by incubation.   [6]. In addition, the synthesized AgNPs depict a maximum wavelength at 302 nm indicates the surface plasmon resonance of the metal nanoparticles [11]- [14].

Figure 2. FTIR spectra of AgNPs
The FTIR spectrum presented in Fig. 2. shows of AgNPs derived from CLE and AgNO3 showed that the absorbance peaks appeared at 1211, 1188, 1156, and 1100 cm -1 indicating the presence of C=O and CN groups. The active compounds of flavonols were also identified at the peaks of 1026 and 972 cm -1 [8]. The presence of an amine group in extract Curcuma longa Linn indicates that it is [7] involved in the reduction of silver ions to AgNPs because this plant acts as a bioreductor agent and it became the capping agent of the nanoparticles. The particle size distribution presented in Fig. 3. shows an uneven distribution because the peak increase and peak decrease are not the same. The highest peak is seen at 153 nm with a peak range of 50-1000 nm, indicating that the AgNPs obtained are still nanoparticles because they are still found in the <100 nm range. The XRD patterns of the samples presented in Fig. 4 show peaks characteristic of HAp at 2θ values of about 31.7o and 32.8o representing the reflection planes (211) and (301) respectively; these are consistent with JCPDS card No. 09-432 [9]. And the characteristics of the Hap-Ag peaks are consistent with JCPDS file no. 01-087-0717 indicate the presence of additional Ag which represents the reflection plane (210), (112), and (111) respectively [10] Further, the SEM profile confirms the morphology of HAp, and Ag/HAp has an open porous structure [ Fig. 5]. In Hap-Ag have higher porosity. The results of the EDX analysis showed the presence of Ag at 6.7% which indicated the success of Ag-HAp formation. In addition, there are peaks for the calcium, phosphorus, and oxygen (in hydroxyapatite crystals) atom [11]  The physical appearance of the prepared PLA/chitosan/Ag-HAP composite can be seen in Figure 6. From the images, it is seen a change in the morphology and color of the material, and a compact mixture of the component was identified.  The antibacterial activity test was carried out by measuring the inhibition zone in the antibacterial activity test of Curcuma longa Linn extract, HAp, Ag-HAp, and PLA/Chitosan/Ag-HAp at the incubation time of 24 using gram-negative bacteria (E. coli) are 12, 9, 11, and 9.5 mm, respectively. The inhibition zone, as presented in Figure 8, suggests that the Ag-HAp and the PLA/Chitosan/Ag-HAp are considered good enough for further antibacterial applications.

Conclusion
The composite of PLA/Chitosan/Ag-HAp was successfully synthesized using Curcuma longa Linn leaves extract for the green synthesis of AgNPs and snail shell as CaO source for HAp. The composite showed the nanoparticles dispersed in the composite giving influence for the homogeneous performance as well as the antibacterial activity. The nanocomposites exhibit remarkable antibacterial activity against E. coli provide a reference for designing and developing novel antibacterial materials for various applications, such as wound dressings, antibacterial surfaces, and biofilms.