Green synthesis of platinum nanoparticles using Saudi’s Dates extract and their usage on the cancer cell treatment

2.1 Materials

Dried hydrated hexachloroplantic acid (H₂PtCl₆·6(H₂O) was used as a chemical in the experiment. The chemical is derived from Sigma-Aldrich after drying it overnight at 90 °C. Further, a stock solution by dissolving 0.040981 g/100 ml ionized water in H₂PtCl₆ (0.1 × 10⁻³ M) was prepared.

Deionized water acquired from Sigma-Aldrich was used in the reaction, and the apparatus such as glassware was washed with distilled water and dilute HNO₃. After obtaining Ajwa and Barni from a local farm, impurities were removed by washing them with distilled water. The Dates were dried afterward in a hot air oven at 60 °C.

The stock solution of Ajwa and Barni was prepared from 50.60 g of Ajwa and 10.82 g of Barni after boiling them separately for 15 min until reduced to 100 ml. The obtained solution was then filtered and used as a reducing agent after keeping it in the dark at 100 °C. However, the filtrate was to be used within one day.

Further, for synthesizing the platinum nanoparticles, the stock solution prepared from Ajwa and Barni was mixed with 1–5 ml of H₂PtCl₆ solution. Finally, de-ionized water was mixed to make the solution 10 ml in volume. Depending on the parameters such as time, concentration, Ph, and temperature, when the solution changed its color to yellow or yellowish-brown, the reduction of Pt⁶⁺ to Pt⁰ nanoparticles was confirmed. HCl (0.1 M) or 0.1 M NaOH solution was used to adjust the pH of the solution as the nanoparticles were prepared and obtained using different pH values. To get solid PtNPs, the mixture of Dates namely Ajwa and Barni extract along with hexachloroplantic acid was boiled for 10 h until a black precipitn was obtained and the mixture was reduced to a quarter of its original volume. The mixture is then filtered by the process of centrifugation and washed with de-ionized water several times and then with ethyl alcohol.

2.2 Structural measurements

The structural characteristics of the investigated and platinum Nanoparticles powder have been examined by:

The platinum nanoparticles were characterized and investigated with methods like UV–Vis Absorption Spectrometry, X-ray Diffraction technique (XRD), EDX, Transmission Electron Microscope (TEM) technique, Thermal Gravimetric Analyzer (TGA), Fourier Transform Infrared Spectroscopy (FT-IR), High-Performance Liquid Chromatography (HPLC) which are discussed thoroughly in the section.

3.1 Analytical study of Dates extract

HPLC analysis of all the Dates from Al-Medina region was conducted to discover the amount of antioxidant. The antioxidants such as security acids, vitamins, phenols, and flavonoids were reportedly present in the Dates. Since the antioxidants in Ajwa and Barni are high, nanoparticles of these Dates were prepared. Out of 23, 19 varieties of amino acid, present in Dates, were studied. The amino acids that were studied are Serine, Glutamic acid, Asparagine, Isoleucine, Glutamine, Ornithine, Threonine, Alanine, Valine, Glycine, Leucine, Tyrosine Arginine, Cysteine, Lysine, Histidine, Methionine, Proline, and Phenylalanine.

The fruits of four cultivars, in the study, were a rich source of amino acids and each cultivar had marked the difference in the amino acid content. In the study, it was observed that the Dates Anbara and Ajwah had the highest amount of the major amino acid namely Proline (89.145 and 86.315 mg/100 g, respectively) (as depicted in Table 1 below).

The aqueous phase antioxidants in the study were Ascorbic acid (ASC) and Glutathione (GSH) (Table 2). The GHS value of Ajwa was 0.24 μmol·g⁻¹ FW, while that of Barni was 0.16 μmol·g⁻¹ FW. The ASC value of Ajwa was 0.47 μmol·g⁻¹ FW and that of Barni was 0.41 μmol·g⁻¹ FW. On the other hand, the lowest vale of GSH and ASC was recorded for Anbara (0.13 μmol·g⁻¹ FW) and Safawi (0.29 μmol·g⁻¹ FW) respectively.

The sugar content of all types of sugar (monosaccharide and disaccharide), namely, glucose, fructose, and sucrose were measured for the Dates of Al-Madinah region (shown in Table 3). It is noticed that the Dates are a rich source of sugar as a significant amount of sugar is present in the Dates. The highest content of glucose was present in Anbara and Barni (319.908 and 57.326 mg/100 g, respectively). Fructose was highest in Safawi and Ajwa (117.610 and 91.143 mg/100 g, respectively) while sucrose sugar was highest in Anbara and Safawi (95.157 and 42.294 mg/100 g, respectively) (see Table 4).

The organic acids present in the Dates were also studied. The concentration of Malic acid was the highest in the Dates (between 12 and 14 mg/100 g for all the varieties of Dates). Whereas, the other acids like succinic, oxalic, and formic acid were either present in a little amount or were present in traces.

The amount of total phenolic content such as Gallic, Resorcinol, Protocatechuic, Chlorogenic, Caffeic, Catechin, and Ferulic was between 30.553 and 41 mg/100 g FW. From the Table 5, it is evident that most dominant phenolic compounds were gallic, syringic, and coumaric respectively. The phenolic content of different Dates in decreasing order can be obtained from the table, which is Barni > Anbara > Ajwa (41.009 > 36.35 > 30.553 mg/100 g DW respectively).

Considering the content of flavonoids such as quercetin, luteolin, apigenin, isoquercitrin, and rutin, it was observed that the total flavonoid content was between 1.99 and 2.58 mg/100 g DW. The highest content of flavonoids was found in Safawi, which was 2.58 mg/100 g DW. Barni had the second highest content of flavonoids, and the lowest content was recorded in Anbara (see Table 6).

The mineral content of Barni was considerably high. Barni has enough amount of phosphorus, potassium, sodium, and metals like copper, manganese, zinc, and cadmium. All the Dates have a different amount of minerals. For instance, potassium content for all the cultivars was between 66.9 and 349.8 mg/100 g DW. Similarly, all the minerals were present in a different amount in the cultivars under study (see Tables 7 and 8).

Vitamins are essential for our body as they are required for digestion, immunity, and metabolism. Out of 13 vitamins, only A, B, C, D, E, and K are most important ones. Vitamin E (Tocopherol) and B3 (Niacin) was present in highest concentration in the Dates. The next abundant vitamin after E and B₃ was vitamin B₆ which was highest in Anbara (0.029 mg/100 g DW) and lowest in Safawi (0.018 mg/100 g DW). The amount of Riboflavin was highest in Barni (0.029 mg/100 g DW) and lowest in Safawi (0.018 mg/100 g DW). Furthermore, the concentration of other vitamins such as A, D, and K varied in every cultivar under consideration.

Variations in the values of proximate composition and energetic value were observed in the fruits from different cultivars as shown in Table 9. It was detected that Ajwa had the highest amount of protein, that was 4.27 g/100 g DW, while the lowest was detected in Safawi, which was 1.78 g/100 g DW. In addition to protein, the components like Ash, carbohydrates, and lipid were highest in Barni Date, which was 3.92, 58.8, and 0.53 g/100 g DW. Safawi had the highest moisture content which was equal to 21.2 g/100 g DW.

3.2 Synthesis of Pt NPs using dates water extract and its characterization under different conditions

PtNPs are useful for applications related to sensors, for example, the PtNPs can be used directly through SPR. PtNPs has indirect application also and can be used to enhance the sensitivity of molecular sensors based on fluorescence variation Platinum (0). The qualitative specific range of absorption peak is between (∼300–340 nm) (González et al., 2015; Nadaroglu et al., 2017; Riddin et al., 2010).

Surprising UV–Vis spectra was observed during the green synthesis of nanoparticles of Pt (0) under different conditions. The ranges of Ajwa and Barni’s SPR peak is from 321 to 329 nm. The absorbance of SPR peak increases with increase in the concentration of H₂PtCl₆ solution at constant pH and room temperature.

Under optimum condition, the difference in the absorbance peak for PtNPs was confirmed by TEM images. The absorbance spectrum of nanoparticles of platinum stretches across the visible, ultraviolet region (Dobrucka, 2016; Ioana-raluca et al., 2013; Pansare et al., 2016; Riddin et al., 2010; Thirumurugan et al., 2016). However, specific wavelength to determine the concentration of the Dates was present in Barni and absent in Ajwa.

3.3 UV–visible and TEM of Pt NPs formed at room temperature

UV–Vis spectrometer was used after the reduction process of Pt⁴⁺ to Pt⁰ nanoparticles. The change in color was monitored, and it was observed that the solution changed color from yellow to yellowish brown, which showed that platinum nanoparticles were formed due to the reduction of H₂PtCl₆. This change in the color of the solution indicates the formation of platinum nanoparticles (Karthik et al., 2016a, 2016b; Prabhu and Gajendran, 2017; Soundarrajan et al., 2012). Ajwa extract (in a different volume, between 1 and 5 ml) was mixed with 5 ml of H2PtCl6 (0.1 × 10⁻³ M) stock solution for synthesizing platinum nanoparticles. The volume of the solution was further made 10 ml by adding de-ionized water. However, the extract of Barni was added in a different amount (between 1 and 9 ml) in 1 ml of stock solution.

In the platinum particles, there was a record excitation of surface plasmon vibration. The value of Plasmon resonance (SPR) for platinum nanoparticles in Ajwa and Barni Dates (at ∼321, 329 nm respectively) depended on the shape and size of PtNPs (González et al., 2015; Huang et al., 2004; Nadaroglu et al., 2017).

The value of the absorbance peak at 321 and 329 nm for Ajwa and Barni respectively, can be used for Pt⁰ UV–Vis spectrum. Depending on the morphology and by annealing Pt thin films with different initial mass-thicknesses, UV–Vis spectrum, with localized-surface-plasmon (LSP) energy were prepared (Saafi et al., 2011; You et al., 2009).

For Ajwa extract (shown in Fig. 2(A)), the value of UV–Vis spectra was observed after mixing it in varying quantity between 1 and 5 ml with 5 ml of the platinum ion. The quantity of the solution was further made 10 ml by mixing de-ionized water. The solution was then kept for 10 h at room temperature. The sharpest peak for UV–Vis spectrum (at λ_max = 321 nm, SPR Pt (0)) was obtained while using 5 ml of the aqueous solution. At different concentrations of Barni extract (1–9 ml) and 1 ml of platinum ion stock solution, the UV–Vis spectra for PtNPs was measured, which is shown in Fig. 2(B). The solution was adjusted to 10 ml by mixing de-ionized water and kept for 10 h at room temperature before measurement. The shapest UV–Vis spectrum was obtained by mixing 9 ml of Barni extract at λ_max = 329 nm (SPR of Pt(0)).

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Fig. 2

(A) UV–visible spectra at different concentration of Ajwa extract (1–5 ml) and 5 ml of H₂PtCl₆ stock solutions after 10 h at the ordinary temperature. (B) UV–visible spectra at different concentration of Barni extract (1–9 ml) and 1 ml of H₂PtCl₆ stock solutions after 10 h at the ordinary temperature.

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When the concentration of Ajwa extract solution increases from 1 to 5 ml, the absorption spectra at λ_max = 321 increases gradually (shown in Fig. 2(A)). Fig. 2(A) also shows the shift in the wavelength towards red color, with an increase in the concentration of extract of Dates and smaller Pt (0) particles with sharper peak was obtained at 321 nm. Further, when 5 ml extract was added, more Pt (0) were formed, which was depicted by the shift in the wavelength (red shift).

The extract of Ajwa and Barni was added in different quantities in platinum ion solution. Smallest PtNPs of uniform spherical shape were formed (shown in Fig. 3(d)). Keeping the other condition standardized, 5 ml of Ajwa extract was added (see Fig. 3).

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Fig. 3

TEM micrograph of Pt NPs at different concentration of Ajwa extract (a) 2 ml (b) 3 ml (c) 4 ml (d) 5 ml and 5 ml of H₂PtCl₆ stock solutions after 10 h at the ordinary temperature.

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The UV–Vis absorbance of platinum ions (specific peak for Pt (IV)) is lowest at 261 nm and highest (specific peak for Pt(0)) at 321 nm (González et al., 2015; Riddin et al., 2010). The optimum volume of 5 ml for platinum ion solution was further confirmed by TEM image captured for different concentration.

3.4 The effect of contact time at room temperature

In the case, when the absorbance wavelength crosses the value of λ_max = 329 nm peak, it shows a non-quantitative indicator to demonstrate that formation of pt(0). There is an inverse correlation between mixing time and absorption time. With an increase in mixing time, the time of absorption either slightly decreases or remain stable. The time for attaining highest absorbance peak (at λ_max = 329 nm) was analyzed with this method. It was observed that within 10 h of preparation, and within homogenous shape and size, the highest concentration of PtNPs was achieved. The interpretation is shown in Fig. 4 with TEM.

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Fig. 4

TEM micrograph of Pt NPs at different mixing time (a) 7 h (b) 10 h (c) 13 h at the ordinary temperature.

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A solution of different pH (1.5–8.5) was used to analyze their effect on the reaction rate and the formation of PtNPs. It was observed that the rate of synthesis of PtNPs was faster in the basic medium than in acidic medium (Nadaroglu et al., 2017; Thesis et al., 2012).

The effect of the pH on the size and quantity of PtNPs is shown with the help of TEM image (in Fig. 5). The pH of the medium is affected by the particles in the medium. The formation of PtNPs increases with increase in alkalinity and inside the acidic medium, bundles of PtNPs are formed with particles of heterogeneous shape and size. However, in the normal pH (5.5), homogenous, spherical and small size particles are formed. For the formation of smaller nanoparticles, a higher value of pH is required as it can efficiently cap the nanoparticles.

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Fig. 5

TEM micrograph of Pt NPs at different pH (a) pH = 1.5 (b) pH = 3.5 (c) pH = 5.5 and (d) pH = 7.5.

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3.5 The temperature effect on Pt NPs synthesis

With an increase in temperature over 30 °C, the peak broadness of the spectra increases. This increase in peak broadness indicates that, with temperature, oxidation of phenolic compound in the extract of date occurs. If the oxidation is increased, the antioxidants in the Dates are decreased, which further decreases the synthesis of PtNPs. Moreover, if the temperature reaches below 30 °C, the peak of absorbance reduces which in turn lowers the process of PtNP synthesis. SPR value of 321 nm and 329 nm was observed in Ajwa and Barni Dates which showed the decrease in the PtNP synthesis. At different temperatures, the TEM images of PtNPs were captured, which are shown in Fig. 6 (Siddiqi and Husen, 2016).

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Fig. 6

TEM micrograph of Pt NPs at different temperature (a) 20 °C, (b) 30 °C, (c) 40 °C, (d) 50 °C (5 ml of H₂PtCl₆ stock solution and 5 ml Ajwa extract after 10 h).

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3.6 Thermal gravimetric analysis

Ajwa extract (5 ml) was used to plot thermal gravimetric analysis of the capped PtNPs (see Fig. 7). When the temperature is between 50 and 600 °C, a constant weight loss of nanopowder is observed. The thermal desorption in the bioorganic compounds of PtNPs reduced the weight of the Nano powder by 48%. In the remaining 52%, there is no bioorganic layer around the Pt (0), and hence the particles are in their pure form.

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Fig. 7

TGA of capped Pt NPs prepared using Ajwa extract.

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The 1:1 ratio of capped Nano Pt and pure Nano Pt is considered perfect for preparing platinum nanoparticle with the aforementioned conditions. The result with this condition shows the stability of PtNPs (Khalil et al., 2014).

3.7 Fourier transform infrared spectroscopy (FTIR)

Different types of polyphenols were present in the biosynthesized nanoparticles as revealed by FTIR spectra of Ajwa (dried) and PtNPs (capped) (see Fig. 8). When the dried Dates of Ajwa and Barni was put under FTIR, the characteristics of the bands observed at 1644 cm⁻¹, 1760, and 3360 were that of C—N(amide), C⚌O, and O—H respectively. The amino acids show the possible interaction of PTNPs and stretching modes for C⚌O and OH groups. The bio-reduction and capping of PtNPs through water-soluble polyphenols can be indicted with the help of disappearance of the strong band at 1760 cm⁻¹ in PtNPs. Moreover, the shift in the vibration to 1120 cm⁻¹ can be assigned to PtNPs, and the presence of a strong band at 1200 cm⁻¹ is due to C—O.

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Fig. 8

(A) FTIR spectra of the dried ajwa and capped PtNPs using the Ajwa extract. (B) FTIR spectra of the dried barni and capped PtNPs using the Barni extract.

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The bounding of biomolecules with Platinum nanoparticles through the group of amino acids can be represented by the shift from 2900 cm⁻¹ to 2790 cm⁻¹ in NH frequency.

The contribution of Protein amide in the bounding of platinum nanoparticles (PtNPs) can be illustrated with the help of disappeared C—N (amide) vibrations in the band observed at 1644 cm⁻¹ in the dried Ajwa and Barni. The coating of platinum nanoparticles, in the date extract, with antioxidant molecule C⚌O, C—N amid, and OH groups can be displayed with the help of appearance of IR band (C⚌O, amide NH) and shift of stretching vibrations of PtNPs from Ajwa and Barni (Dobrucka, 2016; Ioana-raluca et al., 2013; Siddiqi and Husen, 2016; Soundarrajan et al., 2012; Thirumurugan et al., 2016).

3.8 X-ray diffraction (XRD) of PtNPs

The X-ray diffraction patterns of platinum nanoparticles (dried) synthesized using Ajwa, and Barni Dates extract has been shown (Fig. 9). The XRD peak at 2θ showed different index plane for different values of 2 theta. For instance, at 39.38 the correspond index plane is 1 1 1, for 45.88 the corresponding index plane is 2 0 0, and for 67.33° the index value is 2 2 0 (Benaissi et al., 2010; Khalil et al., 2014; Pansare et al., 2016). The values were consistent with the face-centered cubic structure.

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Fig. 9

(A) X-ray diffraction pattern of PtNPs prepared with aqueous Ajwa extract. (B) X-ray diffraction pattern of PtNPs prepared with aqueous Barni extract.

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The crystalline structure of PtNPs formed using Ajwa was confirmed with the obtained XRD pattern. Debye-Scherrer formula, D = k ƛ /β cos θ (1) can be used to get an estimate of average particle sizes of PtNPs.

The relationship between the average particle size and broadening of the peak in XRD is provided by the equation given by Scherrer (1).

Where;

• ƛ is the wavelength of X-ray source (1.5406 nm),

• D is the diameter of the particle size,

• k = which is equal to 0.9, is a constant,

• β =Δ(2θ) is the full width at 1/2 maxima (FWHM),

3.9 EDX analysis

The purity of the platinum was confirmed by using X-ray (EDX) Spectrometers. The spectra illustrates a strong Pt signal (Fig. 10) (Nellore et al., 2013).

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Fig. 10

(A) EDX spectrum of PtNPs with Ajwa extract. (B) EDX spectrum of PtNPs with Barni extract.

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3.10 Biological activity

The in vitro antimicrobial activity has been scrutinized for the synthesized PtNPs. Diffusion Agar technique has been used for the investigation of these activities. Gram-negative Escherichia coli RCMB 010,052 and gram-positive Bacillus subtilis RCMB 010,067 has been analyzed using Gentamicin 5 mg/ml and ampicillin 5 mg/ml as a reference compound respectively. Table 10 shows the inhibition activities of PtNPs against these organisms.

Minimum inhibitory concentration values (MIC) were used to analyze the antibacterial activity of PtNPs. The results projected that PtNPs are potent antibacterial agents. Activities against Bacillis subtilis RCMB 010,067 are shown in Table 9 with the help of Fig. 11(A).

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Fig. 11

Inhibition zone of the synthesized PtNPs on (A) Bacillissubtilis RCMB 010067 (B) Escherichia coli RCMB 010052.

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The Fig. 11(B) shows that the growth of Gram negative bacteria Escherichia coli RCMB 010052 can be inhibited effectively by PtNPs (González et al., 2015).

3.11 Anticancer activity

The development of anti-microbial and anticancer therapeutic agents is the chief goal of medicinal chemistry. The platinum nanoparticles were evaluated for their anticancer activities against cancer cells in human especially, hepatocellular carcinoma (HePG-2), breast cells (MCF-7), and colon carcinoma cells (HCT-116) with the help of MTT method (Senthilraja and Kathiresan, 2015; Thesis et al., 2012). The Table 11 reveals that PtNPs are effective against the cancer cell line.

The values of half maximal inhibitory concentration IC₅₀ can be used to determine the behavior under these experimental conditions. The viability of the standard chemotherapy doses of doxorubicin HCl is assumed to be 0% (Table 10 and Fig. 12). The extract of Ajwa and Barni was tested for inhibitory activities against the cancerous cell MCF-7 (breast carcinoma cells). Sample concentration of 400 µg/ml of the platinum nanoparticle is taken keeping the value of IC₅ between 289 and 189 µg/ml. The tests showed that 61.53% and 67.15% of the breast carcinoma cells MCF-7 were obstructed from Ajwa and Barni extract respectively. The viability was this reduced to 38.47% and 32.85% making the extract an eco-friendly alternative for treating Cancer (Fig. 12).

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Fig. 12

(A) The photo image of breastcarcinoma cells (MCF-7) treatment by PtNPs of Ajwa (a) MCF control (b) 50ug of PtNPs (b) 100ug of PtNPs (c) 400ug of PtNPs (e) Evaluation of cytotoxicity against (MCF-7) cell line. (B) The photo image of breast carcinoma cells (MCF-7) treatment by PtNPs of Barni (a) MCF control (b) 50ug of PtNPs (b) 100ug of PtNPs (c) 400ug of PtNPs (e) Evaluation of cytotoxicity against (MCF-7) cell line.

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The dates of Ajwa and Barni were examined to analyze their inhibitory activities against colon carcinoma cells (HCT-116). During the experiment, 400 µg/ml platinum nanoparticle was taken as a sample and the value of IC₅₀ was kept between 94.2 and 175 µg/ml. The results showed that 76.82%, and 71.21% of the colon carcinoma cells (HCT-116) were inhibited as the viability for Ajwa and Barni became 23.18% and 28.79% respectively. The results reflect that plants extract of Ajwa and Barni are effective against the cancer cells (Fig. 13).

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Fig. 13

(A) The photo image of coloncarcinoma cells (HCT-116) treatment by PtNPs of Ajwa (a) HCT control (b) 50ug of PtNPs (c) 100ug of PtNPs (d) 400ug of PtNPs (e) Evaluation of cytotoxicity against (HCT-116) cell line. (B) The photo image of coloncarcinoma cells (HCT-116) treatment by PtNPs of Barni (a) HCT control (b) 50ug of PtNPs (c) 100ug of PtNPs (d) 400ug of PtNPs (e) Evaluation of cytotoxicity against (HCT-116) cell line.

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Additionally, the platinum nanoparticles of the Dates under consideration were examined for their effect against Hepatocellular carcinoma cells (HepG-2). Sample concentration of 400 µg/ml platinum nanoparticles was used in the experiment keeping IC₅₀ between 118 and 91 µg/ml. The results showed that 73.24% and 78.44% of the Hepatocellular carcinoma cells (HepG-2) were inhibited by Ajwa and Barni respectively, as the viability of the cells, after the experiment, was reduced to 26.76% and 21.56%. The outcomes also showed that use of Ajwa and Barni is an eco-friendly approach that can be adopted for treating Hepatocellular carcinoma cells (HepG-2) (shown in Fig. 14 Ajwa (A), Barni (B)).

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Fig. 14

(A) The photo image of Hepatocellular carcinoma cells (HepG-2) treatment by PtNPs of Ajwa (a) HepG control (b) 50ug of PtNPs (b) 100ug of PtNPs (c) 400ug of PtNPs (e) Evaluation of cytotoxicity against (HepG-2) cell line. (B) The photo image of Hepatocellular carcinoma cells (HepG-2) treatment by PtNPs of Barni (a) HepG control (b) 50ug of PtNPs (b) 100ug of PtNPs (c) 400ug of PtNPs (e) Evaluation of cytotoxicity against (HepG-2) cell line.

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The comparison of inhibition activities of Barni and Ajwa using their extract is shown in Fig. 15. Colon carcinoma cells were inhibited up to 76.82% by the extract of Ajwa, which is very satisfactory. Additionally, cells of HepG-2 were inhibited up to 73.24%, and the lowest inhibition of 61.53% was observed for MCF-7. The dates of Barni also showed promising results in inhibiting the cancer cells and inhibited about 75.44% of HepG-2, 71.21% HCT-116, and 67.15% of MCF-7 cells.

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Fig. 15

Inhibitory activity of synthesized PtNPs of Ajwa and PtNPs of Barni against human (HCT-116) cell line, (HepG-2) cell line and (MCF-7) cell line.

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