Introduction

Superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH) are reactive oxygen species that include very toxic components [1]. The presence of excessive reactive oxygen species (ROS) generated as byproducts of aerobic metabolism leads to mutations and cell death by damaging proteins, lipids, and DNA in the human body [2]. In biological systems, there are defense mechanisms called simply antioxidants to prevent the damages such as aging, cancer, cardiovascular diseases, and Alzheimer’s caused by ROS and free radicals. Although natural antioxidants are widely used to prevent these damages, their use there is inhibited by factors including low solubility, sensitivity to light, low levels of oxygen and pH, poor target specificity, poor bioavailability, and high side effects when used at high levels [3,4,5]. In the last decade, to overcome these limitations, new materials such as nanostructures and hybrid materials which were synthesized through new technology and methods started to be used instead of traditional antioxidants. Organic–inorganic hybrid nanoflowers exhibit improved bioactivity and antioxidant activity compared to their free form due to their ability to encapsulate high amounts of organic molecules in the structure.

Organic–inorganic hybrid nanoflowers as an important biomolecule immobilization method were first reported by Zare and co-workers [6]. The hybrid nanoflowers have gained tremendous attention from researchers [7, 8]. Until now, different kinds of hybrid nanoflowers have been synthesized for several applications such as biosensors [9], dye decolorization [10], catalysis [11], biological analysis [12], and pharmaceuticals [13]. In addition, various biomolecules such as protein [14], enzyme [15], DNA [16,17,18], antibody [19], plant extracts [20, 21], and amino acids [22] have been utilized as organic components for the synthesis of hybrid nanoflowers. When proteins or enzymes are used as the organic component, the hybrid nanoflowers show increased activities compared to free molecules [10]. The synthesis conditions, particularly the type and amount of both organic and inorganic components, affect the composition and morphology of the hybrid materials and ultimately the functions of hybrid materials [23].

As it is known, proteins are biopolymers of amino acids (Aa) [24,25,26]. Amino acids can be divided into three main groups: acidic, basic, and neutral [24]. Their characteristics vary according to their groups. Shi and co-workers reported that L-Glu and L-Asp exhibited an intrinsic peroxidase-like activity against the TMB substrate [24]. They used these amino acids to detect H2O2 and glucose and to measure the anti-oxidant behaviors. They particularly investigated the side-chain effect on the peroxidase-like activity and reported that when the side-chain had -COOH, peroxidase-like activity was seen [24], and if the side-chain was replaced with -H, -OH, -SH or CONH2, mimic activity could hardly be seen. Singh and co-workers developed asparagine functionalized magnetic nanoparticles to apply for Ni (II) adsorption [27]. Chandra and Singh reported a green approach to the biosynthesis of amino acid-functionalized silver nanoparticles using neem gum which can be promising for a natural mechanism of oxygen sensing [26]. In addition, Sun and co-workers reported that the C=O groups were the catalytically active sites, whereas the O=CO groups acted as substrate-binding sites, and COH groups could inhibit the peroxidase-like activity [28].

Amino acids are strongly bound to some transition metal ions (especially Cu2+) to generate complexes, and their types of side chains play an important role in the complex formation [25, 29,30,31]. Under certain conditions, the interaction between amino acids and metal ions allows the formation of hybrid structures with flower-like shapes. Amino acids incorporated hybrid nanoflowers were firstly reported by Wu and co-workers. Twenty standard amino acids as the organic component and Cu2+ ion as the inorganic component were used by the researchers for the synthesis of the hybrid nanoflowers [22]. They investigated the peroxidase-like activity of the synthesized hybrid structures against ABTS and Rhodamine B as substrates and suggested that peroxidase-like activity follows a Fenton-like reaction mechanism. Several related studies claiming that this mechanism is followed in organic–inorganic hybrid nanoflowers have also been reported [32,33,34]. In one study regarding the peroxidase activity of amino acids, it was shown that the type of side chains of amino acids directly affect the peroxidase-like activity of free amino acids [35], while in another study side chains in the hybrid structure caused a change in this type of activity as follows: positively charged R groups > nonpolar, aliphatic R groups > aromatic R groups > polar, uncharged R groups > negatively charged R groups [22].

As mentioned before, for the synthesis of organic–inorganic hybrid nanoflowers, various types of organic molecules were used as the organic component and some divalent metal ions (Ni2+, Ca2+, Mn2+, Co2+, and Zn2+, etc.) particularly Cu2+ ion as the inorganic component [36, 37, 39, 40].

To the best of our knowledge, no investigation was reported on the synthesis of amino acid incorporated hybrid nanoflowers using divalent metal ions (Mn2+, Ni2+, Co2+, and Zn2+) except Cu2+. In this study, we synthesized amino acid-metal phosphate hybrid nanoflowers (AaHNFs) using some metal ions (Cu2+, Mn2+, Ni2+, Co2+, and Zn2+) and selected amino acids (His, Cys, Asn, and Asp) which contain an imidazole ring, sulfhydryl, carboxamide and carboxylate groups. Unlike the previous study reported by Wu et al., we used four novel metal ions (Mn2+, Ni2+, Co2+, and Zn2+) besides the Cu2+ in our study to synthesize AaHNFs for the first time. Wu and co-workers reported in their study that the synthesized amino acid-Cu2+ HNFs showed enhanced peroxidase-like activity and these activities proceed through the Fenton mechanism [22]. Fenton reagents act as radical scavengers [38]. This study also aims to investigate the anti-oxidant capacity of AaHNFs. For this purpose, we applied different analytical measurement methods such as hydrogen peroxide scavenging activity assay of AaHNFs, and the assays of DPPH and ABTS radical scavenging activities for detecting the anti-oxidant capacity of AaHNFs.

Experimental

Materials

L-Cysteine hydrochloride monohydrate, ascorbic acid, copper sulfate pentahydrate (CuSO4·5H2O), cobalt (II) sulfate heptahydrate (CoSO4·7H2O), zinc acetate dihydrate (Zn(CH3COO)2·2H2O), sodium dihydrogen phosphate (NaH2PO4), calcium chloride dihydrate (CaCl2·2H2O), magnesium chloride (MgCl2), potassium chloride (KCl), sodium hydroxide (NaOH), 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate (K2S2O8), ethanol, hydrochloric acid (HCl) and guaiacol were purchased from Sigma-Aldrich (USA) and zinc acetate dihydrate (Zn(CH3COO)2·2H2O) from Carlo Erba. Nickel (II) sulfate hexahydrate (NiSO4·6H2O), manganese sulfate monohydrate (MnSO4·H2O), sodium chloride (NaCl), potassium phosphate dibasic (KH2PO4), disodium hydrogen phosphate (Na2HPO4), and hydrogen peroxide (H2O2) were obtained from Merck. All the reagents used for the experiments were of analytical grade. The substrate solutions used for each experiment were freshly prepared in sufficient amounts.

Preparation of AaHNFs

The preparation procedure of the AaHNFs was carried out as described previously [15, 16]. For Aa-Cu2+, Aa-Mn2+, and Aa-Ni2+ HNFs; a 120 mM metal ion solution was prepared. A certain amount of metal ion stock solution was mixed with the 10 mM PBS at different pH levels (pH 5–9) containing 0.02 mg/mL amino acids. Then, the mixtures were vortexed vigorously and incubated without disturbing at RT for 3 days. At the end of the incubation period, the precipitates were centrifuged at 5000 rpm for 10 min to remove unbounded molecules. Finally, the synthesized AaHNFs were washed with pure water at least 3 times and dried at 30 °C.

Aa-Co2+ and Aa-Zn2+ HNFs were synthesized following the previous works with slight modifications [4, 22, 34]. 1.6 mL of Cobalt (II) sulfate heptahydrate solution (0.05 g/mL) and zinc acetate (0.05 g/mL) were added separately into 20 mL of PBS (10 mM) at different pH levels containing 16 mg amino acid. Each mixture was stirred on a magnetic stirrer at RT for 3 h and 3 days, respectively. At the end of the incubation period, the solutions were centrifuged at 5000 rpm for 10 min to remove unbounded molecules. The synthesized AaHNFs were washed with pure water at least 3 times and dried at 30 °C.

Characterization of AaHNFs

Some devices and techniques were used to characterize the synthesized AaHNFs. The SEM images and EDX analyses of AaHNFs were obtained by using SEM (Leo 440 computer controlled digitally, Leica, Jena, Germany). FTIR analyses were conducted on a FTIR spectrometer (PerkinElmer 400, USA). Bruker X-ray diffraction spectroscopy (Bruker AXS D8 Advance Model, Germany) was used for the XRD analyses of AaHNFs.

Evaluation of peroxidase mimic activity of AaHNFs

The method is based on measuring by catalyzing the dehydrogenation of guaiacol as substrate [40]. In peroxidase mimic activity assays of AaHNFs, first, 1 mL of 45 mM guaiacol, 1 mL of 22.5 mM H2O2, and 1 mL of PBS buffer at different pH levels (pH 5–9) were mixed separately. Then, 3 mg of AaHNFs was added into the experiment tubes to start the reaction. Next, the reaction tubes were incubated for 25 min at RT. After that, at the end of the incubation period, the mixture was centrifuged at 5000 rpm for 5 min. Finally, the supernatants were separated and read at 470 nm. The experiment was repeated three times.

Hydrogen peroxide scavenging activity of AaHNFs

The hydrogen peroxide scavenging ability of the AaHNFs was studied with the method described by Ruch et al. [41]. In the first step, 43 mM H2O2 stock solution was prepared in 0.1 M of phosphate buffer (pH 7.4) which contained 0.2 M KH2PO4 and 0.1 M NaOH. After the AaHNFs were dispersed in a 3.4 mL of phosphate buffer solution, 0.6 mL of 43 mM H2O2 stock solution was also added. These mixtures were incubated for 10 min and then centrifuged at 5000 rpm for 3 min. The absorbance values of supernatants were recorded at 230 nm. Hydrogen peroxide scavenging activity was calculated by the equation of % H2O2 scavenging activity = {1-(Abs Sample/Abs Control} × 100, where Abs Control was the absorbance of reaction solution without sample and the blank solution was phosphate buffer. The experiment was repeated three times at each concentration.

DPPH radical scavenging activity of AaHNFs

DPPH free radical scavenging ability of the AaHNFs was determined using the method proposed by Blois [42] with some modifications. First, sample solutions at different concentrations (1000 ppm-50 ppm) were prepared by adding AaHNFs in 1.5 mL ethanol. Then, 0.5 mL of 0.1 mM DPPH (in ethanol) was added to the 1.5 mL sample solution. Next, the reaction solution was vortexed and incubated in a dark place for 30 min. After the incubation period, the reaction solution was centrifuged at 5000 rpm. After that, the absorbance of the supernatant was recorded spectrophotometrically at 517 nm. DPPH free radical scavenging ability was calculated by using the equation of % DPPH radical scavenging activity = {1-(Abs Sample/Abs Control)} × 100, where Abs Control was the absorbance of reaction solution without sample and the blank solution was ethanol. Finally, the experiment was repeated three times at each concentration.

ABTS radical scavenging activity of AaHNFs

ABTS free radical scavenging ability of the AaHNFs was carried out by the method proposed by Re et al. [43]. Firstly, ABTS radical was obtained by mixing 2 mM of ABTS (in ethanol) and 2.45 mM of K2S2O8 and this solution was incubated in a dark place for 4 h. Then, the ABTS radical solution was diluted to form a 0.75 Abs at 734 nm by using 0.1 M Na phosphate buffer (pH 7.4). Next, 1 mL of ABTS radical solution and 3 mL of sample solution (in H2O) were mixed and incubated for 30 min. After the incubation period, the solution was centrifuged at 5000 rpm. After that, the supernatant was measured spectrophotometrically at 734 nm. ABTS radical scavenging activity of AaNHFs was calculated using the formula of % ABTS+ radical scavenging activity = [1-(Abs Sample/Abs Control] × 100, where Abs Control was the absorbance of reaction solution without sample and the blank solution was H2O. Finally, the experiment was repeated three times at each concentration.

Results and discussion

Preparation and characterization of AaHNFs

It’s well known that certain biomolecules such as proteins and enzymes can easily coordinate and make complexes with some metal ions [44] because of their strong affinity thanks to the amide groups in their backbones. Amino acids can also make complexes with the metal ions and under certain conditions, the interaction between the amino acids and some metal ions can allow the formation of hybrid structures with flower-like shapes. The organic–inorganic HNFs are prepared simply by mixing organic and inorganic components. In the synthesis procedure, the types and concentrations of both organic and inorganic components affect the composition, morphology, and ultimately function of HNFs. Several comprehensive studies were performed to shed light on the formation mechanism of organic–inorganic HNFs [7, 13, 15, 45].

The formation mechanism of AaHNFs is shown in Fig. 1. Firstly, primary metal phosphate nanocrystals are formed in the nucleation step. Then, these primary metal phosphate nanocrystals interact simultaneously with the amino acids. These interactions between metal ions of the primary metal phosphate nanocrystals and N atoms of the amino acids form leaf-like amino acid metal phosphate nanoplates. Finally, the multi-layered flower-like structures are obtained as a result of the anisotropic growth of the nanoplates.

Fig. 1
figure 1

The formation mechanism of AaHNFs

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Until now, there is only one study about amino acid incorporated HNFs reported by Wu et al. They reported that the synthesized amino acid-Cu2+ HNFs showed enhanced peroxidase-like activity [22]. To the best of our knowledge, there is no information about the formation of amino acid-metal phosphate hybrid nanoflowers (AaHNFs) using different metal ions except copper. Therefore, in our study, we synthesized AaHNFs by using four more metal ions called Mn2+, Ni2+, Co2+, Zn2+ besides Cu2+ and selected amino acids (His, Cys, Asn, and Asp) which contain imidazole ring, sulfhydryl, carboxamide, and carboxylate groups. As mentioned before, types and concentrations of organic and also inorganic components affect the formation of flower-like morphology and function of HNFs. For this reason, AaHNFs were synthesized in the pH range from 5 to 9 by using some amino acids and metal ions as described in the experimental procedure section above. The product formation results of AaHNFs were presented in Table S1. As shown in Table S1, for all amino acids and metal ions used in the synthesis, no product was obtained at pH 5 because of the strong repulsion between amino acids and metal ions. SEM images of Aa-Cu2+ HNFs synthesized at pH 7 are given in Fig. 2.

Fig. 2
figure 2

SEM images of Aa-Cu2+ HNFs; a Asn-Cu2+, b Asp-Cu2+, c Cys-Cu2+, and d His-Cu2+ hybrid nanoflowers

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Asn-Cu2+, Asp-Cu2+, and His-Cu2+ HNFs had a ball-shaped and uniformly distributed morphology, while Cys-Cu2+ hybrid nanoflowers had a slightly different shape and morphology (Fig. 2). It is well known that the changes in surface morphology and area affect the biological activity of the hybrid material [23]. Wu and co-workers synthesized the amino acids incorporated HNFs at only one pH level (pH 7.4) [22]. Unlike their study, both the effect of pH on the morphologies of AaHNFs and the formation of Aa-Cu2+ HNFs at different pH values were evaluated in our study. SEM images of Aa-Cu2+ HNFs synthesized at different pH levels are given in Fig S1-S4.

SEM images of Aa-Co2+ HNFs synthesized under optimum conditions are given in Fig. 3.

Fig. 3
figure 3

SEM images of Aa-Co2+ HNFs; a Asn-Co2+, b Asp-Co2+, c Cys-Co2+, and d His-Co2+ HNFs

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As seen in Fig. 3, when we use Co2+ as the inorganic part, the hybrid morphology looked slightly different from the structure obtained with Cu2+. The optimum synthesis pH levels of Asn-Co2+, Asp-Co2+, Cys-Co2+, and His-Co2+ HNFs were determined to be pH 7, pH 8, pH 7, and pH 6, respectively (Fig. 2). While Asn-Co2+ and Asp-Co2+ HNFs were shaped rose flower-like, the plates of Asn-Co2+ HNFs were thinner than Asp-Co2+ HNFs. The morphology of His-Co2+ HNFs was similar to that of the Cu2+ synthesized one. Unlike these, the leaves of Cys-Co2+ HNFs were thicker than the leaves of the other three and anchored at the center of the nanoflower. There was no product obtained at pH 5 and pH 6 for Asp-Co2+ HNFs (Table S1). SEM images of Aa-Co2+ HNFs obtained at different pH levels are shown in Figures S5–S8.

When Mn2+ ion was used, no product formation was observed at pH 5 and pH 6 (Table S1). At higher pH values, although product formation was observed, no Aa-Mn2+ HNFs formation could be observed. SEM images of Aa-Mn2+ hybrid structures are shown in Figures S9–S12.

When the Zn2+ ion was used as the inorganic part, the obtained morphologies were generally similar to the rose shape. The optimum pH levels of Asn-Zn2+, Asp-Zn2+, Cys-Zn2+, and His-Zn2+ HNFs were determined to be pH 6, pH 8, pH 7, and pH 7, respectively (Fig. 4). Cys-Zn2+ HNFs had plates thicker than those in the other three nanostructures (Fig. 4c).

Fig. 4
figure 4

SEM images of Aa-Zn2+ HNFs; a Asn-Zn2+, b Asp-Zn2+, c Cys-Zn2+, and d His-Zn2+ HNFs

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When Ni2+ ion was used as the inorganic part, no product was observed at different pH levels (Table S1). After determining the optimum synthesis pHs of AaHNFs, EDX analysis was performed to confirm the presence of the respective metal ions, C, P, N, and O (Figure S17).

XRD analysis in Figures S18–S20 proved the crystal structures of the nanoflowers and metal-phosphates. All diffraction peaks of metal-phosphates matched well with those obtained from the JCPDS cards. The formed crystals of metal phosphate provided the major skeleton of the nanoflower.

The results of the FTIR analysis of AaHNFs and free amino acids are presented in Figures S21–S24. The characteristic absorbance bands of amino acids can be observed clearly in the nanoflowers.

Evaluation of peroxidase mimic activities of AaHNFs

The peroxidase mimic activities of AaHNFs were examined with the peroxidase substrate, Guaiacol, in the presence of H2O2. The AaHNFs were obtained in the optimum conditions. The AaHNFs can act as a Fenton-like reaction [9, 33]. The parameters at different pH values were scanned to determine the optimum peroxidase mimic activity of AaHNFs. The peroxidase mimic activity results of the AaHNFs obtained by using copper ion are given in Fig. 5. According to this result, His-Cu2+ hybrid nanoflowers showed the highest peroxidase activity value at pH 8. This value was followed by Asn-Cu2+ hybrid nanoflowers at pH 8 as 84.4%. The highest peroxidase activities for Cys-Cu2+ and Asp-Cu2+ hybrid nanoflowers were determined at pH 7. Asp-Cu2+ and Cys-Cu2+ hybrid nanoflowers exhibited 67.5% and 52.9% higher activity than phosphate crystals and other hybrid nanoflowers, respectively. Shi and co-workers found that free amino acids such as L-Glu and L-Asp displayed peroxidase mimic activity like HRP [24]. They used the peroxidase mimic properties of these amino acids in a colorimetric glucose test system to determine in the range of 0.1–10 µM. In the first work related to amino acid-incorporated nanoflowers, Wu and co-workers employed all amino acids with copper ions to prepare hybrid nanoflowers for peroxidase mimics [22]. Using ABTS as substrate, they found that peroxidase mimic activities of Asp nanoflower, Asparagine nanoflower, Cysteine nanoflower, and Histidine nanoflower were 0.00799, 0.00889, 0.00901, and 0.01347 µmol/mg/min, respectively. As we know, these amino acids have different side chains. Histidine has an imidazole ring, cysteine has a sulfhydryl group, asparagine has a carboxamide group, and finally, aspartate has an acidic side chain (CH2COOH). As shown in Fig. 5, when the peroxidase mimics of amino acid-metal phosphate hybrid nanoflowers are compared, it is seen that those activities were in a decreasing manner as follows: His > Asn > Asp > Cys. When they are evaluated according to the type of amino acid side groups, peroxidase mimic activities were in a manner changing from + charged to -charged as follows: positively charged R group > polar > negatively charged groups. We confirmed the similar results reported in the article by Wu and co-workers [22]. The positively charged R groups of amino acids created more hydroxyl radicals and increased their peroxidase mimic level. Additionally, we examined how the other AaHNFs influenced the peroxidase mimics on the same chart. We just separated the metal types for a clearer understanding of the data.

Fig. 5
figure 5

Enzymatic activity values of AaHNFs and Cu3(PO4)2 crystal in different pH conditions

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The highest peroxidase activities for Asn-Co2+, Asp-Co2+, Cys-Co2+, and His-Co2+ HNFs were determined to be at pH 6, pH 7, pH 8, and pH 5, respectively (Fig. 6). The peroxidase mimics of amino acid-Co2+ phosphate HNFs were determined to be 59.16%, 53.63%, 48.78%, and 49.82% for Asn-Co2+ HNFs, Asp-Co2+ HNFs, Cys-Co2+ HNFs, and His-Co2+ HNFs, respectively. The highest peroxidase activities for His-Zn2+, Asp-Zn2+, Cys-Zn2+, and Asn-Zn2+ HNFs were determined to be at pH 7, pH 7, pH 9, and pH 5, respectively (Fig. 7). For amino acid-Zn2+ phosphate HNFs, the values of peroxidase mimics were determined to be 35.29% for His-Zn2+ HNFs, 30% for Asp-Zn2+ HNFs, 29.75% for Cys-Zn2+ HNFs, and 27.33% for Asn-Zn2+ HNFs. Interestingly, the peroxidase mimic activity of His-Cu2+ and His-Zn2+ HNFs was higher within their group, but they showed the lowest activity among the structures obtained with Co2+ metal. To compare the activities of metal crystals with AaHNFs, the activities of metal phosphate crystals were very low.

Fig. 6
figure 6

Enzymatic activity values of AaHNFs and Co3(PO4)2 crystal in different pH conditions

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Fig. 7
figure 7

Enzymatic activity values of AaHNFs and Zn3(PO4)2 crystal at different pHs

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Hydrogen peroxide scavenging activity of AaHNFs

Although hydrogen peroxide is not a free radical, it is very important due to its oxidizing properties. H2O2 inactivates several cell enzymes by penetration to the biological membranes in the human body. Inactivated enzymes can bring on cell damage. Due to its oxidizing property, H2O2 plays a mediator role in the production of ROS species through Fenton (1) and Haber- Weiss (2) reactions [46]. Decomposition of H2O2 causes a decrease in the level of oxidative stress in vivo depending on the reduction in ROS level.

$${\text{Fe}}^{{{2} + }} + {\text{ H}}_{{2}} {\text{O}}_{{2}} - - - - - - - {\text{ Fe}}^{{{3} + }} + {\text{ OH}}^{ - } + {\text{ OH}}^{.}$$
(1)
$${\text{O}}_{{2}}^{. - } + {\text{ H}}_{{2}} {\text{O}}_{{2}} - - - - - - - - - - {\text{ O}}_{{2}} + {\text{ OH}}^{ - } + {\text{ OH}}$$
(2)

As shown in Table 1, we compared the Aa-metal phosphate hybrid nanoflower’s H2O2 scavenging activities of the metal phosphate crystals, Cys, Asp, Asn, His, and standard ascorbic acid at different concentrations (50–1000 ppm).

Table 1 H2O2 scavenging activity (%) of AaHNFs at different concentrations
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H2O2 scavenging activities of all samples showed an increase from 50 ppm concentration to 1000 ppm. At all concentrations, the Hydrogen peroxide scavenging activity of ascorbic acid which was used as the positive control was the highest (97.01%, at 1000 ppm) among all samples. According to Table 1, H2O2 scavenging activities of amino acid-metal phosphate HNFs are higher than those of Cys, Asp, Asn, His, and metal phosphate crystals. Chemical formations on surfaces and the large surface area of Aa-metal phosphate HNFs were attributed to the decrease in H2O2 scavenging activities. Their results at 1000 ppm were as follows: Asn-Cu2+ HNFs, (48.03%), Asp-Cu2+ HNFs (59.84%), Cys-Cu2+ HNFs (54.20%), His-Cu2+ HNFs (64.00%), Asn-Co2+ HNFs (57.22%), Asp-Co2+ HNFs (52.15%), Cys-Co2+ HNFs (50.88%); His-Co2+ HNFs (44.62%), Asn-Zn2+ HNFs (39.65%), Asp-Zn2+ HNFs (42.4%), Cys-Zn2+ HNFs (40.04%) and His-Zn2+ HNFs (45.76%). H2O2 scavenging activities of AaHNFs varied depending on the metal in the structure: Aa- Cu2+  > Aa- Co2+  > Aa- Zn2+. Hydrogen peroxide scavenging results of metal phosphate crystals at 1000 ppm were as follows: Cu3(PO4)2 crystal (24.55%), Co3(PO4)2 crystal (19.14%), and Zn3(PO4)2 crystal (15.83%). The interaction with amino acids could have increased the hydrogen peroxide scavenging activity of those metal ions [47]. Hydrogen peroxide scavenging activity of amino acids was lower. Their results at 1000 ppm were as follows: Asp (17.50%), Asn (15.60%), His (14.91%), and Cys (10.09%). Their side chain negatively affected their hydrogen peroxide scavenging activity. According to Kim et al.; the thiol group in the Cys may serve as a substrate for peroxidase in generating H2O2 [48]. H2O2 scavenging activity of cysteamine obtained by the degradation of cysteine was analyzed by Aruamo et al. They reported that the thiol group in structure negatively affected the H2O2 scavenging activity [49].

DPPH and ABTS radical scavenging activity of AaHNFs

Antioxidants play an important role against oxidative damage caused by free radicals and reactive oxygen species (ROS) which are thought to cause diseases. Various antioxidant defense mechanisms are available to counter the damage caused by ROS and free radicals. They reduce or eliminate the harmful effects of ROS or free radicals. Many methods are used to estimate antioxidant activities. DPPH and ABTS radical scavenging activities [2,2′-diphenylpicrylhydrazyl and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] are the most used ones among these methods due to their ease of measurement, short test time, and cheapness [50].

DPPH, firstly introduced by Blois in 1958 [42], and ABTS, developed by Re et al. [43], are methods in which color changes are used to determine free radical scavenging activity of Aa-metal phosphate HNFs. DPPH and ABTS scavenging activities of AaHNFs compared with the metal phosphate crystals, Cys, Asp, Asn, His, and standard ascorbic acid at different concentrations (25–500 ppm) are shown, respectively, in Tables 2, 3. Decreases in the absorption of DPPH and ABTS radicals were monitored in all samples. Their DPPH and ABTS radical scavenging activities increased as the concentrations increased.

Table 2 Effect of DPPH radical scavenging activity of AaHNFs at different concentrations
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Table 3 Effect of ABTS radical scavenging activity of AaHNFs at different concentrations
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According to our assays, ascorbic acid which was used as the positive control displayed the most powerful DPPH radical scavenging activity at all concentrations among all of the samples. Cys, Asp, His, and Asn showed the strongest activity after ascorbic acid. For example, Ascorbic acid, Cys, Asp, His, and Asn exhibited 96.44%, 92.35%, 84.70%, 72.53%, and 66.55% at the same concentration (500 ppm), respectively. DPPH absorption of Aa-metal phosphate hybrid nanoflowers was higher than metal crystals but lower than amino acids. While the maximum DPPH radical scavenging activities of AaHNFs were calculated as 56.66% at 500 ppm, the maximum activities of metal crystals were calculated as 12.43%.

In comparison with all Aa-metal phosphate hybrid nanoflowers, it was highlighted by Guide et al. that some of the amino acids containing heteroatoms in their side chains such as Cys and Asp showed strong DPPH radical scavenging activities [51]. DPPH radical scavenging activity of AaHNFs was lower than amino acids due to the interaction of active side groups in amino acids with metal phosphate crystals during the formation of hybrid nanoflowers. Cys-Zn2+ hybrid nanoflowers exhibited the most powerful DPPH radical scavenging activities with 56.66%. DPPH radical scavenging activities of AaHNFs changed depending on the metal and amino acid in the structure. We evaluated the AaHNFs among themselves at the same concentration (500 ppm): The DPPH radical scavenging activities of Cys-Cu2+ HNFs, Asn-Cu2+ HNFs, Asp-Cu2+ HNFs, and His-Cu2+ HNFs were 49.66%, 28.19%, 43.99%, and 36.26%, respectively; the activities of Cys-Co2+ HNFs, Asn-Co2+ HNFs, Asp-Co2+ HNFs, and His-Co2+ HNFs were calculated to be 46.57%, 26.15%, 38.03%, and 30.23%, respectively; and the activities of Cys-Zn2+ HNFs, Asn-Zn2+ HNFs, Asp-Zn2+ HNFs, and His-Zn2+ HNFs were 56.66%, 32.49%, 40.02%, and 36.02%, respectively. DPPH radical scavenging activities of Cys-metal phosphate HNFs in the different amino acid- same metal groups exhibited the highest one. Cys-metal phosphate HNFs ​​were followed by Asp-metal phosphate HNFs. These high activities depended on the structure of Cys and Asp. Some AaHNFs such as Asn-Zn2+ HNFs and Asn-Cu2+ HNFs showed no ABTS radical scavenging activity at some concentrations.

Cys and ascorbic acid inhibited ABTS free radicals at the rates of 96.20% and 90.05%, respectively, at 500 ppm (Table 3). Contrary to these results, inhibitions of ABTS free radicals by His, Asp, and Asn were very low being 13.60%, 4.12%, and 18.33% for each, respectively. DPPH radical scavenging activities of Asp, His, and Asn were higher than their ABTS radical scavenging activities. Generally, the ABTS radical scavenging activity of AaHNFs was more powerful than those of amino acids and metal crystals due to the interaction of active side groups in amino acids with metal phosphate crystals during the formation of hybrid nanoflowers. To compare the AaHNFs among themselves at 500 ppm; the activities of Cys-Cu2+ HNFs, Asn-Cu2+ HNFs, Asp-Cu2+ HNFs, and His-Cu2+ HNFs were 71.20%, 38.63%, 13.44% and 35.80%, respectively; the activities of Cys-Co2+ HNFs, Asn-Co2+ HNFs, Asp-Co2+ HNFs and His-Co2+ HNFs were 67.71%, 33.86%, 10.94% and 32.01%, respectively; and the activities of Cys-Zn2+ HNFs, Asn-Zn2+ HNFs, Asp-Zn2+ HNFs and His-Zn2+ HNFs were 61.69%, 30.17%, 9.64%, and 29.09%, respectively. Some Aa-metal phosphate HNFs such as Asp-Zn2+ HNFs and Asp-Cu2+ showed no ABTS radical scavenging activity at some concentrations. The phenolic group in His structure had limited ABTS radical scavenging activity because of the domination of hydrogen atom transfer.

Ascorbic acid is converted to ascorbate radical by giving an electron to lipid radical in order to finish the oxidative chain reaction. Ascorbate radicals react to form dehydroascorbate molecules that exhibit no antioxidant activity [52]. Due to the existence of thiol groups in its structure, a stronger antioxidant activity of Cys is observed. According to Guidea et al., amino acids like His or Asp which have amino [(–NH2) or (–NH–)] or carboxyl (–COOH) groups in their side chains display powerful antioxidant behavior toward radicals because of their H+ donating capability [51]. ABTS is based on electron transfer, while the DPPH method is based on H+ transfer.

In summary, we developed the synthesis of AaHNFs considering Cu2+, Mn2+, Ni2+, Co2+, and Zn2+ metal ions and some amino acids which consist of imidazole ring, sulfhydryl group, carboxamide group, and acidic side chain (CH2COOH). The characteristic properties of AaHNFs proved that amino acids are verified in the HNFs. AaHNFs exhibited a more effective peroxidase-like activity by using Fenton's principle than free amino acids. In addition, AaHNFs can be evaluated as an effective anti-oxidant scavenger material due to their superior properties. Antioxidants play an important role in inhibiting the oxidative damage caused by free radicals and reactive oxygen species (ROS), which are thought to cause diseases. Various antioxidant defense mechanisms are available to counter the damage caused by ROS and free radicals. They reduce or eliminate the harmful effects of ROS or free radicals. The application of natural antioxidants is conventionally limited due to susceptibility to light, oxygen, and pH, poor solubility in physiological fluid, low bioavailability, and improper delivery in undesired cellular compartments. To overcome traditional problems, it is important to develop materials that form strong complexes with organic molecules and to determine their antioxidant capacity. Thus, it has been of our interest, to make and evaluate the synthesis of amino acid-metal phosphate hybrid nanoflowers which could serve as a potential application for procedures such as biosensing, bioassay, biomedicine, pharmaceutics, and biocatalysis to be able to personalize the biomaterials for a specific therapeutic agent and use these novel materials as drug carriers in different devices.