Evaluation of the possible Immunomodulatory and Neuroprotective effects of γ-irradiated basil (Ocimum basilicum) against arsenic toxicity in rats

In view of the widespread incidence of arsenic poisoning around the world, it was necessary to study this phenomenon and analyze it to nd out how to treat it through the application of alternative medicine, which has spread its use signicantly in the world using herbs and plants. Gamma irradiation as a phytosanitary treatment of food and herbal materials is increasingly recognized throughout the world by improving their hygienic quality. The aims of this study to evaluate the therapeutic effect of raw or irradiated basil on rats exposed to arsenic toxicity. Basil was irradiated by gamma rays at dose 10 KGy. Forty-eight adult Wistar albino rats were divided into six groups as follows group-1: Control group, group-2: received 400 mg/ kg of aqueous extract of basil, group-3: received 400 mg/ kg of aqueous extract of irradiated basil, group- 4: received 10 mg/kg of sodium arsenate solution, group- 5: received 10 mg/kg of sodium arsenate solution and 400 mg/ kg of aqueous extract of basil, and group- 6: received 10 mg/kg of sodium arsenate solution and 400 mg/ kg of aqueous extract of irradiated basil. At the end of the experiment (5 weeks), the rats were sacriced, blood and brain tissue samples were subjected to estimate of the following: CBC, inammatory markers (CRP, TNF-α, and IL6), immunoglobulin markers (IgA, IgG, IgM), and the levels of oxidative stress and antioxidant in brain tissue (MDA, CAT, SOD, and GSH). The results showed a rise in the antioxidant of basil after the irradiation process. The arsenic caused a signicant decreased in levels of HB, RBC, LYM, NEU and PLT, while the increased levels of WBC and reticulocyte count as compared to the control group. Also, the rats exposed to arsenic showed a signicant increase in inammatory markers, immunoglobulin markers in serum, and oxidative stress accompanied with signicant decreased in antioxidant in the brain. In contrast, the administration of basil extract along


Introduction
Arsenic is a toxic substance that occurs naturally and is found in water, rock soil and many foods (Duker et al., 2018). Arsenic toxicity affects millions of people in different parts of the world through the ingestion of arsenic-contaminated drinking water and food (Huq et al., 2006; Flanagan, Johnston and Zheng, 2012). In recent years, arsenic toxicity has created signi cant public concern (Duker et al., 2018). It has no taste or smell, which makes it particularly hazardous, so you can be exposed to it without knowing it (ATSDR, 2017). So, the increasing presence of an amount of arsenic in the environment present major risks, as exposure through inhalation, ingestion and dermal contact can cause various adverse effects on health (Vimercati et al., 2017). From these adverse health effects, the disorders of the nervous system (Mochizuki et al., 2019), immune system (Tutkun et al., 2019), digestive system (Chiocchetti, Vélez and Devesa, 2018), and respiratory system (Zhao et al., 2019). To treat these disorders has suggesting used complementary and alternative medicine (CAM) that may be as helpful.
The National Center for CAM (NCCAM) describes (complementary and alternative medicine) as a category of healthcare and medical systems (NCCIH, 2018). A systemic review conducted by Eardley et al. (2012) found that, for many reasons, people are using CAM, including its availability, perceived health, and disease prevention. As one of the main forms of CAM is herbal medicines, which uses parts of a plant or whole plants to avoid and cure diseases (Bent, 2008;Pan et al., 2014). The herbal medicinal products are characterized by the presence of complex chemical compounds responsible for the pharmacological activities that contribute to health bene ts (Bent, 2008). Basil plants considered from herbal medicine that has eminence value as a cure for various diseases (Patel et al., 2018).
Basil (Ocimum basilicum L.) is an herbaceous and aromatic plant worldwide cultivated which belongs to the Lamiaceae family (Jakovljević et al., 2016). It has several therapeutic properties including antioxidant, anti-aging, anticancer, antiviral, antimicrobial, anti-genotoxic, and anti-in ammatory ( (Singh, Krishan and Shri, 2018). Based on the pharmacological and therapeutic properties, basil has played an important role in both traditional pharmaceutical products and contemporary pharmacological and clinical science (Shirazi et al., 2014). But there may be concerns about the use of herbs, which is that herbs are susceptible often to contamination during processing or storage by micro-organisms and insect pests.
This leads to shortens their shelf life and triggering serious illness in some cases, particularly if the herbs contaminated with Salmonella and Staphylococcus aureus (Chatterjee et al., 2016). Thus, herbs should be subjected to sterilization or microbial treatment before use.
There are various techniques for the decontamination of medicinal plants, including irradiation (Garg and Gupta, 2016). This is a physical process that applies high-energy ionizing radiation to the plants in order to enhance their safety and shelf-life (Byun et al., 1999;SádECká, 2007;Alothman, Bhat and Karim, 2009). In particular, gamma irradiation seemed to be the best way to decontaminate herbs from microbes without triggering quality changes (Lee, Lee and Kim, 2005). As the absorbed energy of radiation can break the bonds of DNA molecules in microorganisms present in the product and inactivates certain enzymes, this greatly reduced its damaging impact on products. Not only microorganisms are destroyed, but even gametes, insects and parasites are prevented from reproducing, resulting in various preservative effects (Farkas, 2006). Moreover, irradiation serves as a safe technique in food processing supported by many internationally recognized organizations, where joint (Food and Agriculture Organization/ International Atomic Energy Agency/World Health Organization) Expert Committee on the Wholesomeness of Irradiation of Food has ruled that food subject to low irradiation dosage (up to 10 kGy) is safe and not need any testing of toxicology (Wen et al., 2006).
The present study was therefore designed to evaluate the possible immunomodulatory and antiin ammatory effects of raw and irradiated basil (Ocimum basilicum) against arsenic toxicity in rats.

Plant material
The basil (Ocimum basilicum) leaves were purchased from the local traditional market in Jeddah, Saudi Arabia.

Gamma Irradiation treatment
The samples of dry basil powder were irradiated with 10 KGy of gamma rays after the leaves were transferred into polyethylene bags, using a Cobalt-60 source at a dose rate of 4.75 KGy/h at the National Centre for Radiation Research and Technology (NCRRT), Nasr City, Cairo, Egypt.

Experimental animals
The study will be carried out using adult female Wistar rats weighing (150-200 g); they were obtained from faculty of pharmacy at King Abdulaziz University. The animals were housed in cages and received normal rat chow and tap water in a constant environment (room temperature 28 ± 2 °C, room humidity 60 ± 5%) in a 12 h light and 12 h dark cycle. Rats were kept under supervision for two weeks before the experiments started and during all stages of the whole experiment. Animal's procedures were performed in accordance with the Ethics Committee of the King Fahad Medical Research Center and in accordance with the recommendations for the proper care and use of laboratory animals.

Preparation of basil extracts
The water extracts of raw or irradiated dried basil leaves were prepared according to the method described by Ghazwani, Osman and Balamash (2020).
The total antioxidant capacity was measured in basil extracts using the Total Capacity Assay kit with CAT# STA-360.

Antioxidant in irradiated plant leaves
In the experiment, 48 rats were divided into six groups each of 8 rats as follows: Group 1: normal control rats were given only distilled water. All doses were given through an oral gastric tube daily for ve weeks.
At the end of the experiment, rats fasted overnight for scari cation. Blood samples were withdrawn by a heparinized capillary tube from the retro-orbital plexus of each rat under anesthesia with diethyl ether, it is put into two tubes, one is ethylenediamine tetra-acetic acid (EDTA) tube and the other is a serumseparating tube. The EDTA tube immediately turned to lab analysis while serum-separating tube centrifuged at 3000 rpm for 15 min to separate serum and then stored at -40° C until the biochemical analysis is done. Directly after preparing the blood sample, rats sacri ced and the brain was kept in ice for homogenate preparation.

Biochemical analysis
The biochemical analysis for complete blood count (CBC) were done in Kingdoms Labs. The blood serum IgG, IgM and IgA, TNFα, IL-6 and CRP were identi ed using the kits with CAT# ab189578, ab157735, ab157738, ab46070, ab119548, and ab108827, respectively. For tissue analysis, the brain was homogenized and estimated each of SOD, GSH, CAT and MDA according to the kits with CAT# ab65354, ab138881, ab83464, and ab118970, respectively, while the protein concentration estimated in the brain was used Kits with CAT# 23225.

Statistical analysis
The data of each group were analyzed using MegaStat 9.4 (add-in for Excel). The data were expressed as arithmetic mean and standard deviation of the mean (SD). Differences between groups were analyzed for parametric parameters using one-way variance analysis (ANOVA), the least signi cant difference equation (LSD). A P value below or equal to 0.05 was considered signi cant.
The effect of irradiation on antioxidants is showed in Fig. 1. Where leaves treated with gamma irradiation (10 kGy) showed a change, a 10.3% increase in the content of total antioxidants compared to raw leaves not treated with radiation.

Complete blood count (CBC)
The effects of aqueous extracts of raw basil or irradiated basil for 5 weeks on the levels of hemoglobin (HB), red blood cells (RBC), hematocrit (HCT), platelets (PLT), White blood cells (WBC), WBC differential and reticulocyte count in rats exposed to arsenic are present in Tables 1 and 2.
No effect of raw or irradiated basil administration in normal animals on all previous parameters was noted. Rats, which were exposed to arsenic showed a signi cant decrease in levels of HB, RBC, PLT, neutrophils (NEU), and lymphocytes (LYM) compared to the control group. While there was a marked increase in the levels of HCT, WBC, monocytes (MON) and reticulocyte count compared to the control group. Administration of raw and irradiated basil along with arsenic signi cantly reduced arsenic toxicity effect by the improvement levels of these parameters. Table 1 Effect of basil extracts on hematological parameters of rats exposed to arsenic

Antibodies
The extract of each raw and irradiated basil had distinct effects on antibodies in rats exposed to arsenic that are showed in Table 3.
The treatment with raw or irradiated basil to normal animals not producing an effect on levels of serum IgG, IgM and IgA. The arsenic group showed a signi cant decrease in IgG, IgM and IgA levels compared to the control group. The treatment with basil extract as raw or irradiated along with arsenic dose was showed a signi cant increase in those antibodies' levels, compared with the group of rats given only arsenic.

In ammatory markers
The level of markers of in ammation in rats exposed to arsenic which received raw or irradiated basil as a treatment showed in Table 4.
In normal animals, the administration of raw or irradiated basil has not had an effect on the levels of serum TNF, IL-6 and CPR. The rats exposed to arsenic showed a signi cant increase in levels of TNF, IL-6, and CPR as compared to the control group. While giving raw or irradiated basil extract was noted signi cantly reduced in these in ammatory markers induced by arsenic.

Antioxidants and oxidative damage
The results of determining the content of SOD, GSH, CAT and MDA in rats given arsenic along with raw basil or irradiated basil were shown in Table 5.
The normal rats treated with irradiated basil showed an increase in the level of SOD than rats treated with raw basil. The level of GSH was showed an increase in normal rats of both of raw basil group and irradiated basil group as compared to the control group. Arsenic-exposed rats showed a signi cant increase in MDA levels accompanied by a signi cant decrease in SOD, GSH and CAT compared to a control group. The management of raw or irradiated basil along with arsenic alleviated the effects of arsenic and resulted in a signi cantly decreased MDA with signi cantly increased SOD, GSH, CAT.  Basil contains phenolic compounds and avonoids (Bahcesular et al., 2020), that are considered as natural antioxidants. These biomolecules exhibit their activity through various mechanisms, including inhibiting enzymes that inducing free radical produce, increasing endogenous antioxidants, removing free radicals, and inducing the expression of the numerous genes responsible for enzyme synthesis that inhibit oxidative stress (Primiano, Sutter and Kensler, 1997). Ghazwani, Osman and Balamash (2020) reported that the Fourier-transform infrared (FTIR) analysis indicated to increase the content of phenolic acids and avonoids in basil leaves after treated with 10 kGy of gamma-ray. Moreover, Maraei, Khaled and Elsawy (2017) reported that the gamma irradiation-induced the biosynthesis of certain phenolic compounds. Also, it seems that gamma irradiation with 10 kGy might stimulate some chemical reactions in basil, which perhaps increase in phenolic content by the breakdown of covalence bonds among phenolic components and, free phenolic components with low molecular weight are increasing (Jamshidi, Barzegar and Sahari, 2014).

Complete blood count (CBC)
A complete blood count test is a blood test used to assess general health and detect a range of disorders in hematological parameters. A complete blood count test measures many blood components and features, including red blood cells that carry oxygen, white blood cells that ght infection, hemoglobin that oxygen-carrying protein in red blood cells, hematocrit that indicate to the ratio of red blood cells to the liquid or plasma component of the blood and platelets that help blood clot, and that any change whether an abnormal rise or decrease in the census, indicate the incidence of diseases or disorders requiring medical procedures (Clinic, 2018).
In our study, it has been observed that the levels of HB, RBC, PLT, NEU, and LYM are decreased signi cantly with a marked increase in the HCT, WBC, MON and reticulocyte count in rats exposed to arsenic compared to the control group. The results are consistent with some studies ( Moreover, arsenic exposure can also cause a range of changes, such as increasing ceramide formation, membrane disintegration, cytosolic calcium levels, besides decreasing in adenosine triphosphate (ATP) levels, cell membrane integrity affecting erythrocyte lifespan (Abdul et al., 2015).
Regarded the change in platelet count, this con rmed that arsenic inhibited platelet differentiation within the hematopoietic system of bone marrow, leading to reduced platelet production (Wu et al., 2014).
The white blood cell level was decreased in arsenic feed groups. It might be due to the impact of arsenic which induced apoptotic effect on plasma cells as noted by Rousselot et al. (2004). WBC may generally be divided into ve classes, based on their function, morphology and origin: LYM, MON and NEU (Villa et al., 2003). The changes in the LYM and NEU populations present in this study may be due to arsenic caused immune inhibition in rats (Taheri et al., 2016).
Our ndings demonstrated that extracts of basil caused improved the disorders that occur in CBC. This is in agreement with the results of previous researches (Ofem, Ani and Eno, 2012;Zangeneh et al., 2019). This effect may due to basil which contains a proportion of iron (Nworgu, Yekini and Oduola, 2013), that contributes to improving the level of HB in the blood and has the ability to stimulate production and increase of RBC to treat de ciency caused by arsenic. Furthermore, in normal, the lack of oxygen in the local tissue appears to lead to the production of glycoprotein known as erythropoietin, which induces increased erythrocyte output (Bowman and Rand, 1980). Basil leaves extract is very likely to contain erythropoietin-like agents that are responsible for increased erythrocyte production (Ofem, Ani and Eno, 2012). Saha et al. (2012) reported that secondary metabolites of basil, consisting of important elements include essential oil geraniol, a monoterpene and citral, play a role as the modulator in hematological abnormalities (Ofem, Ani and Eno, 2012). Moreover, in results about the increased lymphocyte count after basil administration, it has been reported that Ocimum basilicum modulates the cell-mediated as well as a humoral immune response that could be due to the presence of avonoids and terpenoids (Mediratta, Sharma and Singh, 2002).

Antibodies
Antibodies also are known as immunoglobulins, are substances made by the body's immune system in response to foreign substances. Antibodies bind to these foreign substances and they can be killed by the

In ammatory markers
In ammation is a biological response of the immune system which can be induced by damaged cells, toxic compounds or pathogens (Medzhitov, 2010). It is part of the body's defense mechanism. one of the major aims of in ammation is to bring immune cells to the area of concern as well as to inactivate or destroy any injurious stimuli and to also begin the repair (Ferrero-Miliani et al., 2007;Medzhitov, 2010).
The in ammation response is caused by speci c immune factors released from the damaged cells.
Where, the damaged cells release cytokines, including interleukins, such as IL-6, IL-8, and tumor necrosis factor-α, that are responsible for communication between white blood cells. Interleukins also stimulate the production and release of CRP from the liver; an important component of the innate immune system (Sinclair, Wang and Tetrick, 2012).
Usually, molecular and cellular activities and interactions e ciently alleviate inevitable infection or damage, during acute in ammatory responses. This effect helps restore homeostasis in the tissue and overcome the acute in ammation. Uncontrolled acute in ammation can become chronic, however, and can lead to a number of chronic in ammatory diseases (Zhou, Hong and Huang, 2016). The elevated levels of in ammatory markers are expected to be associated with toxic metals exposure.
Results of this study showed that serum IL-6, TNF-α and CRP level in rats exposed to arsenic was highly signi cantly elevated. Our ndings agree with the results of the previous study on the association between arsenic and ability to cause chronic in ammation by demonstrating the increased proin ammatory mediators like TNF-α, IL-6 and CRP in the arsenic exposed group in comparison to the control group (Prasad and Sinha, 2017). In ammation considered to be one of the main arsenic toxicity mechanisms that can be correlated with increasing cellular damages ,oxidative stress and lipid peroxidation (Bhadauria and Flora, 2007).
In this work also, it was demonstrated that oral treatment with basil extracts diminished in ammations in

Antioxidants and oxidative damage
Antioxidants are considered the enzymes of the body's protection, are able to stabilize free radicals before attacking components of the cell. They work to diminishing free radicals through reducing their energy or donate electrons to them, thus making it stable (Krishnamurthy and Wadhwani, 2012). While, "oxidative stress, de ned as a disturbance in the balance between the production of reactive oxygen species (free radicals) and antioxidant defenses" (Betteridge, 2000).
In the present study, observed a very highly signi cant decrease in GSH, SOD and CAT accompanied by increased MDA of the rat's brain in the arsenic group as compared to the normal control group. This result is similar to the study of Sun et al. (2018) that reported that arsenic caused signi cantly decreased GSH, SOD and CAT with increased MDA content in the brain tissues of chickens. This may be attributed to the toxic effect of arsenic that may induce oxidative stress by interacting with antioxidants, resulting in the accumulation of free radicals in cells (Bonetto, Villaamil Lepori and Puntarulo, 2014).
In contrast, basil giving a positive effect on the brain by improving the levels of antioxidant antioxidants, and fat oxidation (MDA). These results were in line with recent studies showed that the water extract of gamma-irradiated basil contributes to improving the oxidative stress induced by arsenic exposure in rats (Ghazwani, Osman and Balamash, 2020; Osman, Ghazwani and Balamash, 2020). Also agree with the results of Khodabakhshi et al. (2017), who proved that the increased level of MDA in the mice brain tissue following seizures was prevented by basil extract. Moreover, Khaki (2016) demonstrated that the basil extract protects brain cells from the harmful effects by regulating the antioxidant enzymes in the serum.
This saves the neurons from irreversible cell injury. The antioxidant effect is due primarily to phenolic elements, such as, phenolic acids and avonoids, which have redox properties and ability to neutralize free radicals (Shahidi, Janitha and Wanasundara, 1992).

Conclusion
In this study, we demonstrated that gamma radiations have the ability to increases antioxidants contents in basil leaves. Moreover, the water extracts obtained from Ocimum basilicum can be successful in diminished arsenic toxic effect through the improvement of the homeostasis of CBC and immunoglobins level, reduced in ammatory markers and, enhancement ability of antioxidants to be overcome oxidative stress in the brain.

Declarations
Ethical approval and consent to participate:  Total antioxidants in raw and irradiated basil

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Chapter4.pdf