Gold Nanoparticles from Ostruthin: Green Synthesis, a Stable Mechanism, Antitumor, and Antibacterial Activity

The Ostruthin was extracted and identified its structure from rhizomes of Luvunga scandens located at Ta Cu Mountain, Binh Thuan province, Vietnam for the first time. Gold nanoparticles (AuNPs) were synthesized by the green method. The AuNPs acted as antitumor against breast cancer cell line (MCF-7), human liver cancer cell line (HepG2), and Non-Small Cell Lung (NCI-H460). They showed the potential antitumor activity against MCF-7 with the IC50 value of 65.47±3.09 μM. The antitumor activity of the AuNPs was also compared with the extracted constituents from the root of Luvunga Scandens in a previous article. The AuNPs were exposed to high antitumor activity against MCF-7 and Hep G2, human cancer cell lines. The AuNPs have also been tested the antibacterial activity and shown the moderate antibacterial activity on both Salmonella enterica and Bacillus subtilis at a concentration of 0.25 mM.


Introduction
The metallic nanoparticle has been found in the medical section due to multiple advantages: the usage of nanoparticles as drug delivery systems to potential the therapeutic action of a phytochemical agent, myriad applications in cancer therapy [1,2]. The AuNPs are precious, inert, and not oxidized easily being exposed to oxygen or high acidity environments [3]. There are many applications in the biomedical field as antimicrobial activities, stabilizing with proteins, and peptides [4]. The synthesis of AuNPs can perform on using green route methods according to their cytotoxicity and electrochemical activity [5][6][7][8], inflammation [9], and antimicrobial activities [10]. Many articles have been reported to synthesize gold and silver nano from extracted plants and gold and silver ions and apply in medical chemistry [11][12][13]. Many chemical methods conducted on the synthesis involving reduction of gold cations (Au 3+ ) to Au with chemical reducing agents or other organic compounds. These methods were either expensive or environmentally hazardous, but they were easier to control the shape and size by varying synthesis conditions, stability, and catalyst applications [14,15]. Recent, several studies have shown approaching green chemistry, which was a type of reducing agent from plant extracts [16,17]. The mechanism of the biochemistry method showed that Au 3+ ions (oxidizing agent) reacted with biological molecules from plant extracts, which provided proteins, amino acids, enzymes, polysaccharide, alkaloid, tannin, phenol, saponin, terpenoids, and vitamins. These molecules added with both electrostatic stability and reducing agents at the biochemistry synthesis [18,19]. Besides, the properties of AuNPs were determined according to some parameters like the forming time of the AuNPs, temperature, pH, the concentration of gold ions, [20,21] nature and morphology of Au structures [22]. The Than Xa Huong plant was named scientific Luvunga scandens (Roxb.) Buch. Ham and the traditional medicinal plants have been conducted in the treatments of cirrhosis, rheumatism, scorpion bite, hepatitis, sinusitis, pain in bone joints, and muscles.

Extraction Ostruthin from rhizomes of Luvunga scandens
The powdered rhizomes of L. scandens (5.0 kg) were extracted three times with 30 L of 96% ethanol at room temperature. The residue was filtered, and the solvent was evaporated to obtain the crude extract (350 g). The crude extract was dissolved in water and successively partitioned into nhexane (120 g, 2.4%), chloroform (80 g, 1.6%), ethyl acetate (30 g, 0.6%) and the aqueous portion (110 g). The n-hexane extract (120 g) was fractionated on a silica gel column with n-hexane: ethyl acetate (v: v = 50:1, 20:1, 10:1, 5:1 and 1:1) by increase the amount to 100% of ethyl acetate to give six fractions which were labeled F.1 to F.6. The fraction (F.2) collected the crude solid, which was washed with n-hexane: acetone (v: v= 1:1) to obtain the light-yellow crude solid after filtration. The 31.25g of crude solid recrystallized by adding 40 mL of dichloromethane and 40 mL of n-hexane sequence and collected 27.34 g (0.55%) of the impure solid after completion filtration. The 27.34 g of impure solid was continued to separate by normal phase chromatography column with a dynamic phase, which was n-hexane: ethyl acetate (v/v = 5:1). The pure solid was obtained 18.59 g (0.38%) of pure Ostruthin.

The green synthesis of the AuNPs
The 6.70 mM of the pure Ostruthin was prepared by adding 0.1 g of the Ostruthin to 50 mL of 95% ethanol. The 5 mL of 6.70 mM Ostruthin was added to 50 mL of 1 mM Gold (III) chloride trihydrate solution. The temperature of Gold (III) chloride trihydrate solution in Ostruthin was increased from 30 to 70 o C with multiple of 10 o C for every temperature reading. The changes in the colour of the solution observed at (5,10,20,30, and 60 min) of every reaction time.

Antimicrobial activity
The antibacterial activity was assessed based on the previous articles without significant changes [27,28]. The AuNPs were tested for antimicrobial activity by disk-diffusion assay against Salmonella enterica, Bacillus substilis, Escherichia coli, and Bacillus cereus bacterium. The 300 µL of test organisms was added in 10 mL of the nutrient broth solution, which obtained 1x 10 6 CFU mL -1 of each organism. They were grown in an incubator for 24 h at 37°C. Muller-Hinton agar (MHA) was prepared media using bacteria. The wells of 5 mm diameter were made on MHA gel disks. Each strain was swabbed uniformly into the individual plate using a sterile cotton swab. The AuNPs were prepared in concentrations of 0.125, 0.25, 0.5, and 1 mM and Gentamicin at a concentration of 1 mg. L -1 ) for testing antimicrobial activity. Using a sterile micropipette 30 μL of the sample of the AuNPs or standard drug poured onto each of a well. After incubation at 35 o C for 24 h, the different levels of a zone of inhibition were measured using the meter scale.

Antitumor activity of AuNPs
The antitumor of AuNPs was screened based on Sulforhodamine B colorimetric assay (SRB) [29]. The sulforhodamine B (SRB) assay is conducted for cell density determination, based on the measurement of cellular protein content. The method has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. The method not only allows many samples to be tested within a few days but also requires only simple equipment and inexpensive reagents. The SRB assay is, therefore, an efficient and highly cost-effective method for screening. The assay was performed as previously described with some modifications. Cells, seeded at a density of 10,000 cells/well (MCF-7, Hep G2) or 7,500 cells/well (NCI-H460) in 96-well plates were cultured for 24 h before being incubated with AuNPs at different concentrations for 48 h. Treated cells were fixed with cold 50 % (w/v) trichloroacetic acid (Merck) solution for 1-3 h, washed and stained with 0.2 % (w/v) SRB (Sigma) for 20 min. After five times washed with 1 % acetic acid (Merck), the protein-bound dye was solubilized in 10 mM Tris base solution (Promega). Optical density values were determined with a 96-well microtiter plate reader (Synergy HT, Biotek Instruments) at the wavelengths of 492 nm and 620 nm. The percentage of growth inhibition (Inh %) was calculated according to the formula: Inh % = (1-[ODt/ODc]) x 100 %, [30][31][32], in which ODt and ODc were the optical density value of the test sample and the control sample, respectively. Camptothecin was used as a positive control. The antitumor activity in vitro of AuNPs was performed at differential concentrations against breast cancer cell line (MCF-7), NCI H460 (Non-Small Cell Lung), and Hep G2 (human liver cancer cell line) by RSB assay. Figure S1 showed the FT-IR spectra displayed the infrared absorption peaks at 3156 cm -1 (OH), 2968 cm -1 , 2917 cm -1 (C-H), 1684 cm -1 , 1605 cm -1 (C=O), 1570 cm -1 , 1445 cm -1 and 1390 cm -1 (C=C), 1271 cm -1 and 1237 cm -1 (C-O).

1 H-NMR spectrum
As indicated in Figure S2-4, the 1 H-NMR spectrum appeared 13 resonance signals which are related to 22 protons. One resonance signal of the singlet hydroxyl group appeared at 8.16 ppm (1H, brs, 7-OH) and there were eight resonance signals of geranyl group including 2 protons of 2 alkene groups at 5.34 (t, J =7. 3

The HMBC, HSQC and HR-MS spectra
As shown in Figure S9 Table S1 provided the Ostruthin in Figure 1.

The characterization of AuNPs 3.2.1. Ultraviolet-visible (UV-vis) spectroscopy for determination of formation of AuNPs
The UV-vis spectrum determined the formation and size of gold nanoparticles in aqueous solution. The UV-vis spectrum of the AuNPs indicated the Ostruthin solution before reacting with gold salts did  Figure 2b with a yellow line in graph and appeared one peak of 535 nm in the wavelength range of 300 to 800 nm in Figure 2b, black line. The maximum absorption of the solution was at 548 nm being demonstrated colloidal dispersion system of the AuNPs formed. The synthesized AuNPs made a surface Plasmon Resonance (SPR) which caused the color change and also appeared a maximum absorption peak in the visible region. The absorption peak that caused the plasmon resonance peak of AuNPs depended on the shape, size, crystallinity, and nature of the nanoparticles. As presented in Figure 2b, the highest absorbance at a wavelength of 548 nm and the intensity of the absorption band increased with an increasing time period from 15 to 40 min and displayed without significant difference of absorption band from above 40 to 60 min. In the first step, the effect of reaction times was determined and they fully completed during a reaction time of 30 min.

Effects of temperatures on the biosynthesis of the AuNPs
The color of the AuNPs changed when temperatures change between 30 and 70 o C in Figure 3. They were initiated with the light color, aggregation, and sedimentation at 70 o C. The spectrum of them shifted absorbance peak as the temperature change. The intensity of the absorption band increased with increasing temperature from 30 to 50 o C being made increasing in the reaction rate of the conversion of the Au 3+ ion to Au atom due to the decreasing active energy reaction. The intensity of the absorption band decreased on which the temperature of reaction increased from about 60 to 70 o C. When the temperature installed higher than 50 o C, the Brown motion was stronger, which made random motions of the AuNPs to increase the probability of collisions between colloidal particles and possibly more aggregation and sedimentation. When the temperature increased, the widths of diffuse layers also extended, and the Zeta potential increased to the range of -30 mV to +30 mV leading to the sedimentation of the AuNPs.

Figue 4a. The effects of times on forming the AuNPs
The UV-vis spectrum showed that Osthuthin solution before adding HAuCl4 did not appear peak and after adding HAuCl4 solution and exposing to the sunlight appeared the maximum absorption, λmax in the wavelength range of 500-550 nm. The absorption rate (Amax) at the λmax in the range of 500 to 550 nm increased rapidly up to 30 min and from above 30 to 70 min of reaction time, as shown in Figure 4a, the color of the AuNPs has not changed too much, bold purple with the constant absorption wavelength. The synthesized AuNPs nanoparticles created a surface plasmon resonance (SPR), which made the effect on the color change of the AuNPs, the darker the color, the more forming AuNPs, smaller the particle size, as shown in Figure 4b. As shown in Figure 5, the UV-vis spectrum of AuNPs indicated the absorption rates at concentrations of 0.5, 1, 2, and 3 mM. The maximum UV absorption performed at 1mM, 548 nm, exposure time under the sunlight of 40 min. The optimum UV-vis spectrum created the optimal AuNPs for 30 min, pH 4, and a concentration of the AuNPs of 1mM, which was dark purple. The FT-IR spectrum of the AuNPs as shown the infrared absorption peaks in Figure S18. The presences of characteristic functional groups which formed in reduction between Ostruthin and Au (III) were peaks at 3420 cm -1 (OH of alcohol, phenol or OH of water), 1651 cm -1 (C=O, lactone), and 741 cm -1 (aromatic bearing substitution at C-7, C-6). The absence of absorption peaks at 1589 cm -1 , 1445 cm -1 , and 1390 cm -1 (C=C) cm -1 in Ostruthin proved to transform functional groups of two alkene groups to functional groups of a tertiary alcohol in water media.

The XRD of the AuNPs
The XRD of the AuNPs: The extracted Ostruthin solid carried out in the green redox reaction for the transform of Au 3+ ion to Au atoms which were verified by Bragg peaks at 38.  Figure 6. They were indexed as face-centered cubic (FCC) structures of gold. The X-ray diffraction (XRD) results clearly indicated the AuNPs formed by the reduction of Au 3+ ions to Au ions by reduction Ostruthin compound [33].

The Zeta potential and stable dispersion system of AuNPs
In Figure 7, the AuNPs were synthesized and kept in 14 days. The Zeta potential value was measured +30.3 mV at that time, which identified out of range of [-30 mV, + 30 mV] to prove the stable thermodynamic of the dispersion system of the AuNPs. The AuNPs conducted high stability because they were protected by compound (2) as shown in (Figure 9) after storage 14 days as a surfactant agent, which occupied in a colloidal system.

Proposed mechanics for forming and the stable thermodynamic of AuNPs
As shown in Figure 6-10 and Figure S.18, the AuNPs formed in hydrophobic colloidal system and occupied the organic molecules, which transformed during the reaction between the HAuCl4 and Ostruthin in Figure 9, and made the role of a surfactant agent (wetting agent), which protected a colloidal system of AuNPs. The IR spectrum as shown in Figure S18, the two alkene groups in the structure of Ostruthin were absent so the Ostruthin molecules reacted with HAuCl4 and formed two tertiary alcohol groups (structure (2) in Figure 9). The structure (2) connected to an aliphatic hydrocarbon chain of eight carbons (as a hydrophobic group) and a coumarin skeleton (as a hydrophilic group), which was the role of the surface-active molecules. They formed the colloidal micelles that covered the AuNPs outside. This also proved in the TEM image of the AuNPs as shown in Figure 8a-b. The proposed mechanics of stable colloid of the AuNPs was illustrated clearly in Figure 10, which led to an increase in the electrostatic repulsions between the AuNPs and made high stability of nanoparticles of the AuNPs.

Figure. 12
The antitumor activity of the AuNPs against three human cancer cell lines

Transmission electron microscopy (TEM)
The photo of the TEM image as shown in Figure 8a-b, the physical characteristics of AuNPs have been identified as the spherical, triangular, hexagonal distribution and sizes from 10-20 nm. The AuNPs have been covered by molecules (6-(3,7-dihydroxy-3,7-dimethyloctyl)-7-hydroxy -2Hchromen-2-one), molecules (2) as a surfactant agent which yielded during a reduction reaction. This proved new results in AuNPs about synthesizing of green gold nano via using a reducing agent that previous research articles were only used plant extracts, which included many constituents (crude extracts).

Energy dispersive X-ray (EDX) spectrum
EDX spectrum, as shown in Figure 11 and S19, indicated the colloidal dispersion system of the AuNPs was 100% of gold atoms. Figure 11. The EDX spectrum of the AuNPs

Antitumor and antibacterial activity of the AuNPs 3.3.1. Antitumor activity
The AuNPs were conducted again for three human cancer cell lines MCF-7, NCI H460, and Hep G2. The results presented in Figure 12-13 and Table S2. The AuNPs indicated the highest antitumor activity with the IC50 value of 65.47±3.09 µM against MCF-7, moderate activity with NCI H460, and Hep G2 at different concentrations. The antitumor activity of the AuNPs was also compared with it of the extracted constituents from the Root of Luvunga Scandens in a previous article [23]. The AuNPs were exposed to higher activity than compounds, which extracted from Root of Luvunga Scandens against MCF-7 and Hep G2. The IC50 values of Camptothecin against MCF-7, NCI H460, and Hep G2 was reported 1.81±0.09, 9.17±0.41, and 7.43±0.55 µM, respectively. The AuNPs can be well delivered, but the HAuCl4 contained the chlorine ions. They have affected cells and made dead of cancer cell lines.  Table 1, the AuNPs exposed the moderate inhibition against Salmonella enterica and Bacillus substilis with the inhibition zones of 10 mm, and 10 mm, respectively at a concentration of 0.25 mM. The data, compared to the inhibition zones of standard drug, Gentamicin were measured 16 and 18 mm, respectively. The AuNPs had the less or no activity against Escherichia Coli and Bacillus cereus bacteria in Figure 14-15 and Table 1 at all of the concentrations.

Conclusions
The AuNPs were synthesized by biochemistry approaching via green reduce reaction between HAuCl4 and Ostruthin, which was extracted, isolated, and resoled structure. The AuNPs were characterized by UV-vis, FT-IR, TEM, EDX, Zeta potential, performed the antitumor activity against three human cancer cell lines, and tested antibacterial activity. The AuNPs exposed the potential antitumor activity against MCF-7 with the IC50 value of 65.47±3.09 µM. They also showed higher antitumor activity than that of the compounds extracted from the root of Luvunga Scandens. They indicated potential activity against Salmonella enterica and Bacillus substilis at a concentration of 0.25 mM. In addition, the SEM image exhibited AuNPs were covered by organic molecules as the surfactant molecules being formed in the reaction between HAuCl4 and Ostruthin. Those molecules (2) made the AuNPs more stable because the Zeta potential value was conducted +30,3 mV after storage in 14 days. Their size and shape were 10-20 nm and face-centered-cubic, spherical, triangular, and hexagonal distribution. The best conditions for synthesized the AuNPs carried out at the concentration of 1mM, pH 4, at 50 o C, and for 30 min. The EDX spectrum proved 100 % of Gold (III) chloride trihydrate reducing to Au atoms by Ostruthin was the benefit approach to comply with a reduced method, being used the extract to plants forming nanoscale.