Ziziphora clinopodioides Lam leaf aqueous extract mediated novel green synthesis of iron nanoparticles and its anti-hemolytic anemia potential: A chemobiological study

2.1 Preparation and extraction of Z. Clinopodioides

Five hundred grams of the powdered Z. clinopodioides leaf was soaked in 5 L of distilled water and swirled regularly for 24 hrs. The extract was decanted, filtered and concentrated under reduced pressure using a rotary evaporator at 40 °C to obtain a semi-solid residue.

2.2 Preparation and synthesis of FeNPs

The green synthesis of FeNPs was carried out according to the previous study with some modification (Wei et al., 2016). A 40 ml of aqueous extract solution (100 µg/mL) was added to 10 ml of FeCl₃·6H₂O in the concentration of 0.05 M (deionized water was used for the all steps of this section). The mixture was refluxed for 1 h at 50 °C. The color-changing from yellow to black indicated the formation of iron nanoparticles. The precipitate was triplet washed with water and centrifuged at 10000 rpm for 15 min subsequently. The obtained black powder was kept in a vial for the chemical characterization and evaluation of its biological activity.

2.3 Analysis of antioxidant properties of FeNPs by DPPH

Free radicals are unstable atoms that have one or more unpaired electrons. These active species are very harmful due to their high reactivity. They are most often formed when oxygen molecules in the body split into separate unstable atoms. This process can turn into a chain reaction. Free radicals excessive production in the body causes cell damage and oxidative stress. Genetics and the environment affect the extent of free radical damage in individuals. These active molecules are produced as part of the body's natural biological processes. One of the most important free radicals is DPPH. DPPH is widely used to study the antioxidant activities of natural compounds and nanoparticles (Hosseinimehr et al., 2011).

In this method, the antioxidant activity of nanoparticles is measured for DPPH radical scavenging. The basis of the action is the reduction of the alcoholic solution of DPPH in the presence of hydrogen-giving antioxidants, especially phenolic compounds. To achieve the IC50 of the samples, 11 different concentrations of nanoparticles were prepared and the percentage of inhibitory versus concentration was used to plot. In practice, 300 μl of 1 M DPPH was combined with 100 μl of diluted sample to a final volume of 2 ml using methanol. After half an hour in the dark, the absorbance was read at 517 nm and the inhibitory percentage was obtained using the following formula: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{A}.}x100$$

In this formula, “Control A” shows the negative control of light absorption that lacks nanoparticles, and “Sample A” expresses the amount of light absorption of different concentrations of nanoparticles (Hosseinimehr et al., 2011).

2.4 Analysis of cell toxicity of FeNPs

Investigation of cell proliferation and survival is one of the most important and basic techniques in cell laboratories. This study requires accurate quantification of the number of living cells in the cell culture medium. Therefore, cell survival calculation methods are necessary to optimize cell culture conditions, evaluate cell growth factors, detect antibiotics and anticancer drugs, evaluate the toxic effects of environmental pollutants, and study apoptosis. Many methods can be used for such purposes, but indirect methods using fluorescent or dye (chromogenic) markers provide very fast large-scale methods. Among these methods, measurement of cell survival by MTT method is the most widely used method. This method is a colorimetric method to study cell proliferation and survival, introduced in 1983 by Mossman. The method basis is based on mitochondrial activity. Mitochondrial activity in living cells is stable and therefore a raise or reduce in the living cells number is linearly related to mitochondrial activity (Arulmozhi et al., 2013). Mitochondrial dehydrogenases in living cells break the tetrazolium ring and yield NADPH and NADH, leading to forming a purple insoluble deposit called formazan. This precipitate can be dissolved by dimethyl sulfoxide or isopropanol. Dye formation is used as a marker of living cells. The color intensity produced is measured at a 540 to 630 nm wavelength and is directly proportional to the living cells number. High safety and providing a colorimetric and non-radioactive system are important advantages of this method. This kit is very easy to use, has high sensitivity and accuracy and can detect less than 950 cells. On the other hand, it has high efficiency for measuring cell proliferation, survival and mortality, and its implementation method does not require time-consuming washing steps and transfer from one plate to another. Examples studied in this method are adhesive or suspended cells and proliferating or non-proliferating cells (Arulmozhi et al., 2013).

In this research, we used the following cell line to evaluating cytotoxicity effects of FeCl₃, Z. clinopodioides, and FeNPs using an MTT method.

Because nanoparticles is not soluble directly in 1640-RPMI medium and also the solvent of dimethyl sulfoxide nanoparticles (DMSO) itself has cytotoxic effects, to eliminate the effect of this substance on treated cells (Normal cell line (HUVEC)), its amount in the final solution is considered less than 1%. Dimethyl sulfoxide is not toxic to concentrations less than 1% and the concentration of this solvent is important in this regard. For this purpose, 1000 µg of nanoparticle was dissolved in 100 μl of dimethyl sulfoxide solvent after weighing. Then 1 ml of culture medium was added for better dissolution and finally the volume of solution was increased to 24 ml using culture medium: Then, successive dilutions of this stock were used in the proportions of 1–1000 μg/ml. Eleven concentrations were used for the cell lines (Arulmozhi et al., 2013).

In this study, 100 µl of culture medium containing 10⁴ cells per plate 96 were placed. After 24 h, incubation of concentrations of 1–1000 μg/ml of nanoparticles was added to the cells, and incubated for 24, 48, and 72 h, respectively. After these times, 20 μl of MTT plate with a concentration of 5 mg/ml was added to each cell and incubated in the dark for another 4 h. After some time, the MTT medium was carefully removed, and 200 µl of acidified isopropanol were added to each plate to remove the purple formanes. After 15 min of incubation at room temperature, the light absorption of each well was read using an ELISA at 570 nm against a reference wavelength of 690 nm. The findings were reported as cell survival and IC50 (concentration that inhibits cell growth up to 50%) based on the concentration curve (μg/ml) (Arulmozhi et al., 2013).

It should be noted that the effect of each concentration of the nanoparticles on cell lines was investigated in five independent experiments. According to the values of light absorption obtained by the ELISA reader, the percentage of growth inhibition related to each concentration was calculated using the following formula: $$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{A}.}{\mathit{ControlA}.}\times 100$$

Finally, linear regression was done to gain IC50, which indicates the nanoparticles concentration, which causes 50% cancer cell growth inhibition. Using the curve, the line equation for cancer cells was obtained, respectively, then by replacing 50% inhibition in the equation, the IC50 value for cancer cells was obtained (Arulmozhi et al., 2013).

2.5 In vivo design

In this study, fifty male, twelve-week-old mice weighing 39–40 g were obtained. At the beginning of the study, hemolytic anemia induction was done with three doses of 20 mg/kg body weight intravenous injection of phenylhydrazine in 40 animals on days 1, 3, and 5. One group was considered as a healthy control and injected with normal saline. On day 5, the 40 phenylhydrazine-treated mice were randomly divided into four subgroups, including untreated receiving distilled water, and three groups receiving 2 mg/kg body weight intravenous injection of FeCl₃, Z. clinopodioides, and FeNPs. During this period, the healthy control group received normal saline. The mice underwent injection treatment for 15 days. The mice were weighed nine times during the study. At the end of day 16 of treatment after anesthetizing the animals, the blood samples were drawn immediately from the animals’ hearts. Also, the mice livers and spleens were harvested to analyze the biochemical parameters (Zangeneh et al., 2019).

Immunological (Pro-inflammatory (IL1, IL6, IL12, IL18, IFNY, and TNFα) and anti-inflammatory (IL4, IL5, IL10, IL13, and IFNα) cytokines) and biochemical (ALP, AST, ALT, GGT, cholesterol, LDL, HDL, triglyceride, total protein, albumin, total and conjugated bilirubin, urea, creatinine, ferrous, ferritin, and erythropoietin) parameters concentration were measured in the serum by ELISA procedure and determined using kits (Ziest Chem Diagnostics) according to the manufacturers’ instructions.

The capacity of antioxidant enzymes in serum, spleens, and livers of each group was evaluated by determining the activity of SOD, CAT, GR, and GPx by ELISA procedure and determined using kits (Ziest Chem Diagnostics) according to the manufacturers’ instructions.

The number of total WBC, lymphocyte, monocyte, neutrophil, eosinophil, basophil, platelet, RBC, and nRBC, and the level of HB, PCV, MCV, MCH, and MCHC were analyzed in the plasma by automatic hematology analyzer (Sysmex XS 800i).

All hematological, immunological, and biochemical analyses were done in duplicate (Zangeneh et al., 2019).

2.6 Statistical analysis

SPSS statistical software version 22 was used for data analysis and the findings were determined as the mean standard deviation of 5 replications. Data were analyzed using one-way analysis of variance and Duncan post hoc test and the significance level in the test was considered 0.01.

3.1 Characterization of FeNPs

3.1.1

3.1.1 UV–Visible spectroscopy analysis

The UV–Vis. spectrum of the biosynthetic nanoparticles of iron is presented in Fig. 1. The surface plasmon resonance (SPR) of FeNPs was completed using UV–Vis. spectroscopy. The peaks at 221 and 298 nm approved the creation of FeNPs. In differentiation with former reports, the presence of the mentioned band proves the creation of iron nanoparticles (Harshiny et al., 2015; Katata-Seru et al., 2018).

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Fig. 1

UV–Vis. The spectrum of biosynthesized FeNPs.

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3.1.2

3.1.2 FT-IR analysis

The FT-IR spectra of the green synthetic of FeNPs and Z. clinopodioides are shown in Fig. 2. For the FT-IR analysis of metallic nanoparticles, the peaks between 400 and 700 cm⁻¹ usually M—O vibration. The FeNPs formation was confirmed by the the peaks at 457, 524, and 638 cm⁻¹ that is in relation with vibration of Fe—O. However, it is obvious that many organic molecules of the extract strongly protected the surface of iron nanoparticles in within the synthesis step.The similarity has been observed for the FT-IR spectrums of the Z. clinopodioides extract and FeNPs, that could be proved the biosynthetic nanoparticles of Iron . Existences of different functional groups in Z. clinopodioides extract reveal different-IR bands. Such as the peaks in 3371 and 2939 cm⁻¹ are contributed to O—H and C—H stretching; the bands from 1421 to 1749 cm⁻¹ belong to C⚌C and C⚌O, and the peaks at 1261 and 1081 cm⁻¹ could be considered for —C—O and —C—O—C bonds. These peaks confirmed the presence of various organic in the plant extract which have been reported previously (Oganesvan et al., 1991; Tian et al., 2010). According to a previous study, the hydroxyl and carbonyl group of the organic compounds in the herbal extracts linked to the metallic nanoparticles (Ghidan et al., 2016).

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Fig. 2

FT-IR spectra of biosynthesized FeNPs and aqueous extract of Z. clinopodioides leaf.

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3.1.3

3.1.3 XRD analysis

The XRD diffraction patterns of FeNPs evaluated its crystallinity. The pattern of diffractogram is showed in Fig. 3. The creation of iron nanoparticles were approved to this result. Despite the small size of FeNPs, the pattern of XRD indicated well crystallizing. The achieved data were compared with the usual database ICDD PDF card no. 96-900-5813. The peaks at positions of 35.50, 37.27, 53.95, 62.28, and 75.36 are indexed to (3 1 1), (2 2 2), (4 2 2), (4 0 4), and (5 3 3) planes which are in good agreement with those of magnetite (Fe₃O₄). These peaks are also reported previously (Patra and Baek, 2017; Kuang et al., 2013; Yew et al., 2016). The average crystal size of FeNPs was calculated using the Scherrer equation. The FeNPs had an average crystal size of 29.91 nm. In our literature review, A few studies have reported various crystalline size for the green-synthesized FeNPs by plants extracts. For example, 14 nm has been reported for the estimated crystallite size of the biosynthesized magnetite iron oxide for Trigonella foenum-graecum (Radini et al., 2018); 48.91 nm for Brassica rapa (Patra and Baek, 2017); 2.3 nm for Platanus orientalis (Nurbas et al., 2017); and 68.17 nm for Cynometra ramiflora fruit extract (Bishnoi et al., 2018).

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Fig. 3

XRD pattern of FeNPs.

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3.1.4

3.1.4 FE-SEM analysis

FE-SEM imaging investigated the morphology of surface and size of FeNPs. Fig. 4 (a-d) presents the FE-SEM of the green synthetic of iron nanoparticles. According to the result the FeNPs are in spherical morphology. The displaying of the images of synthesis of FeNPs with spherical morphology have been reported previously (Devatha et al., 2016). The images show a tendency for accumulation of the green-synthetic FeNPs and changeable size, which was reported for the other green synthetic metallic nanoparticles (Harshiny et al., 2015; Ghidan et al., 2016; Rao and Pennathur, 2017; Baghayeri et al., 2018; Seydi et al., 2019). As shown in Fig. 5d and according to the findings, the range of 13.27–43.10 nm was indicated for the diameter size of FeNPs. A 30.04 nm can be considered for the FeNPs of this study. In the literature, different sizes have been reported for the green synthesized FeNPs. The size of the iron nanoparticles was from 7.69 to 110 nm (Yew et al., 2016; Gautam et al., 2018; Zhang et al., 2017; Devatha et al., 2018; Wu et al., 2015; Huang et al., 2014).

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Fig. 4

FE-SEM image of FeNPs.

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Fig. 5

EDS analysis of FeNPs.

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3.2 Antioxidant and cytotoxicity properties of FeNPs

In this study, we assessed the antioxidant properties of Z. clinopodioides leaf aqueous extract green-synthesized FeNPs by using the DPPH test as a common free radical. Oxidation is the electrons transfer from an atom and is the aerobic life and metabolism part of living organisms. Oxygen is the receptor for electrons in the electron transport system, which yields energy from ATP (Adenosine triphosphate) in the body. Under certain conditions, oxygen may become a single electron and release free radicals. When oxygen becomes a single electron, it is called reactive oxygen species (ROS). Oxidative loss to proteins, DNA, and other macromolecules is one of the internal causes of degenerative diseases such as aging, cardiovascular disease, cancer, immune system deficiency, cataracts, and abnormal brain function. Single oxygen, high-energy, mutagenic oxygen, can be produced by lipid peroxidation by the transmission of energy from light or the respiratory tract of neutrophils (Zangeneh et al., 2019201820192019). Some free radicals have positive roles such as regulating cell growth, phagocytosis, energy production, intracellular signals, or the important biological compounds synthesis. Antioxidants produced in the body fight free radicals with two systems: enzymatic defense and non-enzymatic defense. Superoxide dismutase, catalase, and glutathione peroxidase metabolize lipid peroxide, hydrogen peroxide, and superoxide and prevent the production of toxic hydroxyl radicals (Zangeneh et al., 20192019202120202019201920192020). In non-enzymatic defense, there are two classes of fat-soluble antioxidants (such as carotenoids and vitamin E) and water-soluble (glutathione and vitamin C) that trap free radicals. These two systems help neutralize oxidants. However, oxidants can escape from antioxidants and damage tissues. In this case, the activated antioxidant repair system (which is the enzymes lipase, protease, transferase and DNA repair enzymes), counteract the oxidant effects. However, due to deficiencies in the production of antioxidants in the body or due to physiopathological factors and situations (such as smoking, air pollution, UV radiation, diets containing high unsaturated fatty acids, inflammation, ischemia, bleeding, etc) that ROS are yielded in large quantities at the wrong place and time, oral antioxidants are needed to counteract the oxidative damage cumulative effects (Zangeneh et al., 2019201820192019; Zangeneh et al., 20192019202120202019201920192020; Zangeneh et al., 2019).

In the present survey, the best result for the antioxidant activity was observed at dose 1000 µg/mL. The IC50 of BHT, Z. clinopodioides, and FeNPs were 394, 309, and 203 µg/mL, respectively (Fig. 6). About the antioxidant properties of BHT, in the previous study has been indicated that the inhibition percentage of BHT against DPPH is about 404 µg/mL (Zangeneh et al., 2019). Also, in the other studies presented that the inhibition percentage of BHT against DPPH was about 300–500 µg/mL (Zangeneh et al., 20192019202120202019201920192020).

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Fig. 6

Antioxidant potential of FeCl₃, Z. clinopodioides, FeNPs, and BHT. BHT: Butylated hydroxyl toluene.

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In the current study, the cytotoxicity properties of FeCl₃, Z. clinopodioides, and FeNPs were measured against HUVEC cells (Fig. 7). HUVECs are the human normal cells that are separated from umbilical vein endothelial cells. These cells are used for investigating the cytotoxicity effects of metallic nanoparticles such as iron nanoparticles (Zangeneh et al., 20192019202120202019201920192020).

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Fig. 7

Percent viability measured on HUVEC after treatment with present FeCl₃, Z. clinopodioides, and FeNPs.

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The absorbance rate was determined at 570 nm, which indicated extraordinary viability on normal cell line (HUVEC) even up to 1000 μg/mL for Z. clinopodioides and FeNPs. The IC50 of FeCl₃ was 859 μg/mL. The absence of any significant toxicity of Z. clinopodioides and FeNPs has numerous safe applications in pharmaceutical domains. In the previous study has been indicated when FeCl₃ is combined with plant extracts, its cytotoxicity capacity significantly reduces against HUVECs (Zangeneh et al., 2019).

3.3 Anti-hemolytic anemia potential of FeNPs

Many factors such as industrial chemicals, some commercial pesticides or improper use of some drugs or some natural factors such as animal toxins or some parasites lead to red blood cell lysis and hemolytic anemia. One of the most obvious side effects of this type of anemia is an increase in the amount of tissue iron due to the lysis of red blood cells and a decrease in oxygen (hypoxia) caused by anemia (Zangeneh et al., 2019; Shukla et al., 2012). The amount of intracellular iron is precisely regulated and it is believed that increasing the amount of this substance outside the cell causes oxidative stress conditions and damage to the fat of cell membranes and organs (Mahdavi et al., 2019; Belyaev and Demeubaeva, 1999; Shukla et al., 2012). It can also be stated that about 20 million people live at altitudes of more than 3,000 m above sea level and under hypoxic conditions. Recently, the population of people working in the highlands has also increased. In the last decade, a number of studies on growth, development and production have been performed under hypoxic conditions (Mahdavi et al., 2019; Belyaev and Demeubaeva, 1999; Zangeneh et al., 2019; Shukla et al., 2012).

Hydrazines are colorless liquid and oxidant compounds with hemolytic properties that cause toxicity in various tissues and at different levels - which are widely used in industry, industrial pesticides, pesticides, explosives, chemical, pharmaceutical, agricultural and laboratory industries (Mahdavi et al., 2019; Belyaev and Demeubaeva, 1999). These compounds are recognized by the World Health Organization as carcinogenic pollutants that can cause many public health problems and cause irreversible cellular damage (Belyaev and Demeubaeva, 1999; Shukla et al., 2012). Phenylhydrazine was first developed from hydrazine by Fischer Emil in 1875. Phenylhydrazine is used to prepare indoles, which is an intermediate substance for the production of various dyes and drugs. Phenylhydrazine is also used to produce phenylhydrazones from natural compounds that are used to distinguish simple sugars from each other (Mahdavi et al., 2019; Belyaev and Demeubaeva, 1999; Zangeneh et al., 2019; Shukla et al., 2012). According to studies, phenylhydrazine exerts its effects through direct damage to the erythrocyte membrane. Phenylhydrazine, which is also used clinically in the treatment of polycythemia due to its hemolytic effects, it causes a wide range of side effects such as hemolytic anemia, hypoxia, vascular disorders, liver, kidney, respiratory, central and autoimmune nervous system, malignancies, genetic abnormalities and inflammatory reactions (Shukla et al., 2012). Phenylhydrazine auto-oxidation results in the production of reactive oxygen species and phenylhydrazine-derived radicals such as phenylhydrazyl radicals. In addition to reactive oxygen species, phenylhydrazine metabolites can also react with cell membranes and cause red blood cell destruction and hemolytic anemia due to lipid peroxidation and protein oxidation (Mahdavi et al., 2019; Shukla et al., 2012). On the other hand, increased hematopoietic activity and iron absorption following severe phenylhydrazine-induced severe hemolytic anemia increase iron storage in tissues, that high levels of tissue iron also increase the production of reactive oxygen species and cause several pathophysiological changes (Mahdavi et al., 2019; Belyaev and Demeubaeva, 1999; Zangeneh et al., 2019; Shukla et al., 2012). In this regard, several experimental studies have confirmed the role of hemolytic anemia and phenylhydrazine-induced vascular disorders in the development of systemic inflammatory reactions and histopathological changes in various organs such as the heart (Shukla et al., 2012).

In our experiment, the number of total WBC, lymphocyte, monocyte, neutrophil, eosinophil, basophil, RBC, platelet counts and the other RBC parameters including Hb, PCV, MCV, MCH, and MCHC decreased significantly (p ≤ 0.01) and the number of nRBC counts raised significantly (p ≤ 0.01) in the phenylhydrazine-treated group (Fig. 8,9). Above results show that phenylhydrazine successfully has induced hemolytic anemia in mice. Phenylthydrazine is known to induce hemolytic anemia mixed from insufficiency of many organs such as heart, testis, spleen, stomach, kidney, and liver (Shukla et al., 2012). According to our study, in similar studies indicated that phenylhydrazine with decreasing the number of RBC to 4 × 10¹²/L induced severe hemolytic anemia. In the previous studies, the levels of Hb, PCV, MCV, MCH, MCHC reduced to 8 g/dL, 29%, 49fL, 18 pg, and 20 g/dL, respectively. About the number of total WBC and platelet, these parameters also reduced in the phenylhydrazine-treated group (Zangeneh et al., 2019; Zangeneh et al., 2017). Z. clinopodioides and FeNPs improved significantly (p ≤ 0.01) the hematological parameters, so that they could significantly (p ≤ 0.01) enhance the number of total WBC, lymphocyte, monocyte, neutrophil, eosinophil, basophil, RBC, platelet counts and the other RBC parameters including Hb, PCV, MCV, MCH, and MCHC and reduce the number of nRBC in comparison to untreated group. There isn’t any significant difference (p ≤ 0.01) between Z. clinopodioides, FeNPs, and control groups in the number of nRBC. In the previous studies, the hematoprotective and anti-hemolytic anemia potentials of aqueous extract of medicinal plants have been related to their antioxidant compounds. (Zangeneh et al., 2019; Zangeneh et al., 2017) Due to Z. clinopodioides is rich of the antioxidant compounds including cineol, sis-isopulegone, thymol, terpenoides, α and β pinen, flavonoids, piperitenone, and pulegone, so its hematoprotective and anti-hemolytic anemia potentials are normal [Behravan et al., 2007; Salehi et al., 2005; Mahdavi et al., 2019; Belyaev and Demeubaeva, 1999; Zangeneh et al., 2019, Zangeneh et al., 2017; Saravanan and Ponmurugan, 2013; Hoseinpouran et al., 2015; Ogunlade et al., 2012]. Fig. 9

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Fig. 8

The number of platelet, RBC, nRBC, and WBC in the experimental groups.

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Fig. 9

The levels of RBC parameters in tested groups.

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About immunosuppressive property of phenylhydrazine, the anti-inflammatory cytokines including IL4, IL5, IL10, IL13, and IFNα reduced significantly (p ≤ 0.01) and the pro-inflammatory cytokines including IL1, IL6, IL12, IL18, IFNY, and TNFα increased significantly (p ≤ 0.01) in the phenylhydrazine-treated group (Fig. 10). Agreement with our study, in a similar study has been indicated that phenylhydrazine as inflammation inducing agent, increases the concentration of pro-inflammatory cytokines and decreases the concentration of anti-inflammatory cytokines in serum (Zangeneh et al., 2017). Both groups of Z. clinopodioides leaf aqueous extract and FeNPs significantly (p ≤ 0.01) ameliorated the levels of anti-inflammatory and pro-inflammatory cytokines near to normal. The best results have been observed in FeNPs-treated group. No significant difference (p ≤ 0.01) was seen between FeNPs and control groups in the concentrations of IL6, IL13, IL18, and IFNα. In this study, FeNPs were introduced as immunoprotective nanoparticles.

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Fig. 10

The levels of immunological parameters in tested groups.

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In the current research, the levels of serum, liver, and spleen SOD, CAT, and GPx increased and GR reduced significantly (p ≤ 0.01) at Z. clinopodioides leaf aqueous extract and FeNPs-treated mice in comparison to the untreated group. No significant differences (p ≤ 0.01) were found between Z. clinopodioides, FeNPs and control groups in the concentration of serum GR and liver GPx (Fig. 11). In the study of Saravanan and Ponmurugan (Saravanan and Ponmurugan, 2013), it revealed the strong antioxidant effect of medicinal plants with ameliorating the concentrations of SOD, CAT, and GPx (Saravanan and Ponmurugan, 2013).

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Fig. 11

The levels of antioxidant enzymes of serum (A), liver (B), and spleen (C) in tested groups.

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In our study, phenylhydrazine-induced hemolytic anemia significantly (p ≤ 0.01) decreased the concentrations of HDL, total protein, and albumin and raised ALP, AST, ALT, GGT, cholesterol, LDL, triglyceride, total and conjugated bilirubin, urea, creatinine, ferrous, ferritin, and erythropoietin in serum. Z. clinopodioides leaf aqueous extract and FeNPs significantly (p ≤ 0.01) ameliorated the above parameters. There were no significant changes (p ≤ 0.01) between Z. clinopodioides, FeNPs, and control groups in conjugated bilirubin, triglyceride, and GGT levels. The best results in improving the biochemical parameters were seen in FeNPs-treated group (Fig. 12, Fig. 13.). In this regards, in a study of Dkhil et al. (2015) has been indicated that metallic nanoparticles such as iron nanoparticles have an excellent protective role against free radicals, so that they could regulate the concentrations of biochemical and immunological parameters of serum near to normal (Dkhi et al., 2015).

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Fig. 12

The levels of hepatic biochemical parameters in tested groups.

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Fig. 13

The levels of Fe, ferritin, erythropoietin, urea, and creatinine in tested groups.

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In Figs. 14 and 15, phenylhydrazine increased significantly (p ≤ 0.01) the weights and volumes of spleen and liver and reduced the weight of the body. Treatment with Z. clinopodioides leaf aqueous extract and FeNPs reduced significantly (p ≤ 0.01) the above parameters in liver and spleen and increased significantly (p ≤ 0.01) the weight of the body. The best results were seen in FeNPs-treated group.

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Fig. 14

The weight of the body in tested groups.

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Fig. 15

The weights and volumes of liver and spleen in tested groups.

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