Bisphenol A (BPA) is a synthetic organic compound that acts as a monomer for the production of polycarbonate plastic and is widely used in food and beverage packaging, medical devices, thermal paper and dental materials and can contaminate food, beverages, air and soil. It accumulates in many human tissues and organs, so it is harmful to human health due to its hormone-like properties and also binds to estrogen receptors, which affects body weight and is a cause of tumor formation and also affects metabolism and cancer and may impair male reproductive function [1]. 

Nanotechnology is the science that is concerned with studying molecules within a scale of 1-100 nanometers, as there is a difference in the properties of the material in its nano state compared to its natural, non-nano state [2]. Changes occur in the properties of materials in their nano state, i.e., with dimensions less than 100 nanometers than they are in their natural state, i.e., higher than 100 nanometers in terms of their physical and chemical properties, as the change of surface mechanics and the anisotropy of the nanoparticles and other anomalies occurs in the nanoparticles. The basic units of nanotechnology are prepared by physical methods, which depend on top-down technology. As for chemical and biological methods, their approach is technical from bottom-up in preparation [3]. 

Foeniculum vulgare Mill Known as fennel, it is a member of the Apiaceae family. This plant is native to the Mediterranean regions and is widely cultivated all over the world [4]. This plant has many different applications in many areas of the food and pharmaceutical industries and can be used as a substance that increases the flavor of food due to its sweet smell and taste and in the manufacture of perfumes, soaps, cosmetics and spices. Many commercial pharmaceuticals are expressed based on the compounds of fennel essential oil [5] It was found that the essential oil of fennel is rich in trans-anethole, estragol, fenugreek and limonene [6].       

Among the medicinal plants, the astragalus plant is among the top 50 essential herbs used in the field of medicine, as it has many pharmaceutical properties. It is an antioxidant, anti-inflammatory and an immune booster, all of which help improve the general health of humans and help prevent diseases and sometimes help in disease treatment [7]. It also has an anti-aging property that prevents or slows down the aging process in cells and organs [8]. It can be used as a drug to protect neurons and prevent inflammation and thus, has the potential to prevent chronic neurodegenerative diseases such as Alzheimer's disease [9]. 

Probiotics are defined as substances secreted by bacteria that encourage the multiplication of other beneficial bacteria in the body, which are often known as the natural flora, or they are types of beneficial bacteria that are cultivated in the laboratory and preserved in a live form in special containers amounting to millions or billions and sometimes to billions of Live bacterial cells in a single dose and they do the opposite of what antibiotics do to kill bacteria and they were discovered in 1953 and proven effective in treating gastrointestinal diseases in particular. Also, probiotics help to absorb food well, prevent inflammatory bowel diseases, lactose intolerance or lactose sensitivity, especially in infants and prevent irritable bowel disease and colon cancer, reduce gas in the stomach and intestines, treat constipation and colic and treat nutritional deficiencies. It helps to produce several vitamins in the digestive system and reduce the infection of fungi in the vagina in women, especially from Candida albicans, which causes many problems, especially for pregnant women. As well as when eaten in certain numbers, it has health benefits on humans [10]. 

This study aimed to find out the effectiveness of laboratory-synthesized zinc nanoparticles, medicinal herbs and bio-enhancer in reducing the effects of bisphenol in the liver and the body of the organism and its effect on the level of liver enzymes and total protein.

Laboratory animals were purchased from animal house - College of Veterinary Medicine - Tikrit University and used in this study 50 male laboratory white rats of age 8 weeks and weight (200-220 g) and they were divided into ten groups and placed in separate cages that were examined The male rats were infected with the new bisphenol, then they were dosed with zinc nanoparticles solution, herbal extract and bio-enhancer. Water and food were also given until they start feeding on the previous ration and conduct the required analyzes. 

Male Rats were Divided into 10 Groups, which are as Follows :

• The First Group (M1): It is the healthy control group, which included 5 healthy rats and they were fed a normal diet and normal water 

• The Second Group (M2): It is the group infected with the newly developed bisphenol and it was fed the normal diet and normal water 

• The Third Group (M3): The group infected with the new bisphenol A, which was treated with zinc nanoparticles (ZnNPs) at a concentration of 30% 

• The Fourth Group (M4): The group infected with the new bisphenol, which was treated with fennel extract 

• The Fifth Group (M5): The group infected with the new bisphenol, which was treated with astragalus extract 

• The Sixth Group (M6): The group infected with the new bisphenol, which was treated with the use of the probiotic 

• Group VII (M7): It is the group infected with the new bisphenol A, which was treated with ZnNPs at a concentration of 30% + fennel extract 1 g / 10 ml distilled water

• The Eighth Group (M8): The group infected with the new bisphenol A, which was treated with ZnNPs at a concentration of 30% + Astragalus extract 2 g / 10 ml of distilled water

• The Ninth Group (M9): The group infected with the new bisphenol A, which was treated with the probiotic 2 ml + fennel extract 1 g/10 ml distilled water 

• The Tenth Group (M10): The group infected with the new bisphenol, which was treated with the probiotic 2 ml + Astragalus extract 2 g/10 ml distilled water 

Carry out Measurements and Checks

Determination of the activity of aspartate aminotransferase in serum 

Aspartate Amino Transferase (AST) 

The enzymatic method was used to estimate the activity of the AST enzyme in the blood serum. The method included the use of a kit supplied by the British company Randox. The method is based on the ability of the enzyme to act on the base substance (aspartic acid and alpha-ketoglutaric acid) and convert the amino-aspartic acid to alpha-ketone acid. (Pyruvic), which can be converted into a reddish brown derivative compound by adding the reagent 2,4-Di-Nitrophenyl hydrazine.

The absorbance is read using a spectrophotometer at a wavelength of 505 nm. As in the following equation:

Alanine Amino Transferase (ALT)

The enzymatic method was used as the analysis kit (Kit) supplied by the British company Randox was used and the ALT enzyme works on the base material (alanine and alpha-ketoglutaric acid) by converting the amino acid alanine to pyruvic acid directly, which easily turns into a compound derived from hydrazine with a reddish-brown color Reddish Brown by adding the reagent 2,4 Di-Nitrophenyl hydrazine and read the absorbance on a spectrophotometer at a wavelength of 505 nm depending on as in the following equation: 

Determination of basal phosphatase activity in serum Alkaline Phosphatas (ALP) 

The activity of the enzyme Alkaline phosphatase (ALP) was estimated using a standard kit supplied by ROCHE (Germany) and the results were read using the Swiss-origin Reflotron device according to the instructions of the processing companies.

Blood Biochemical Assay

Determination of Total Protein in Serum: The Biuret method was used to estimate the total protein in the blood serum I used the ready-made kit from the Spanish company SPINREACT, The samples were read with a spectrophotometer at a wavelength of 540 nm, The following equation is applied in measuring the amount of total protein. 

Serum Albumin Determination

The bromocresol green method was used; For the determination of albumin in the blood serum, using a ready-made kit, The models were read by a spectrophotometer at a wavelength of 630 nm, The following equation is applied to measure the amount of albumin. 

Determination of Globulin in Serum

The immunoglobulin concentration was calculated by the difference between total protein and albumin as stated in. Globulin concentration = total protein concentration - albumin concentration (g/dL)

Statistical Analysis 

The statistical program sciences social package Statistical (SPSS) version 16 was used in order to analyze the primary data of the results of the current study, as the analysis of variance (ANOVA) test was used and the level of significant differences between the rates of the criteria included in the current study was extracted using the least significant difference at the statistical level. (p<0.05, p<0.01). 

Effect of Oral Administration of Znnps Complex, Herbal, Probiotic to Rats after 28 Days on the Activity of Liver Enzymes

The liver is one of the organs of the body responsible for the production of many enzymes that have an important role in catalyzing and regulating many chemical reactions, including the enzymes Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Alkaline Phosphatase (ALP), which were estimated in groups of animals given concentrations of Zinc nanoparticles, herbs and probiotic. The results in Table 1 showed that the AST enzyme showed a significant increase (p<0.05) in the M2 treatment group) compared to the healthy control group M1, reaching (77, 58) (IU/ L) respectively, while it was noticed from the results of the table that there were significant decrease (p˂0.05) in the values of the coefficients M3, M4, M5, M6, M7, M8, M9, M10 as their values were 70, 44.3, 58, 55, 73.3, 76.6, 71.3, 54.3 (IU/L) respectively compared to the treatment groupM2, while the enzyme recorded the lowest level in the treatment M4 in contrast to the treatment M2 which was the highest in the enzyme level. 

The results showed that the ALT enzyme showed a significant increase in the value of its concentration, as the rate of increase in the treatment group M2 was 36.3 (IU/L) compared with the healthy control group M1, which amounted to 19.3 (IU/L). From observing the results of the transactions, it was found that there was a significant decrease in the values of all transactions M3, M4, M5, M6, M7, M9, M10, as the rate of decline reached (26.6, 27, 20.9, 31, 31, 19, 24.3 (IU/L). respectively compared with the treatment group M2, while it was found that treatment M8 whose value was 42.6 (IU/L), which represented the oral administration of ZnNPs at 30% and astragalus extract, was the highest and significantly superior to the treatment group M2. 

It was also shown from the results in the table that there was a significant increase in the ALP enzyme values for the treatment group M2 compared to the healthy control group M1, which amounted to 121.3, 71.6 (IU/L), respectively and it appeared from the results of the table that there was a significant decrease in the values of the enzyme In groups M3, M4, M5, M6, M7, M8, M9 and M10, the rate of decrease in them was 50.6, 66, 74.6, 85.6, 79, 72, 52.3, 106 (IU/L) respectively while It was found that the highest concentration was in the share of treatment M2, which represented oral administration of bisphenol A and treatment M3, which included oral administration of ZnNPs at 30%, gave the lowest concentration compared to treatments, the results agreed with what was found by Naeem et al. [11]. These results agreed with the results obtained by Nizar et al. who confirmed that the use of aqueous extract of fennel led to a decrease in the level of liver enzymes. The effect of the extract may be attributed to an increase in metabolic activity and an increase in glucose metabolism. These results also agreed with what was found by Fei Yana et al. [12] who confirmed that oral administration of Astragalus led to a decrease in the level of liver enzymes, whose levels were high as a result of calcium tetrachloride CC14 poisoning. 

Table 1: Effect of Oral Administration of Znnps Complex, Herbs and Probiotic on Liver Enzyme Values of Rats after 28 Days

Table 2: Effect of Oral Administration of Znnps Complex, Herbs and Probiotic on Protein Values of Laboratory Rats after 28 Days

The different letters in the same column indicate the presence of significant differences between the treatments at the level of significance (p˂0.05).

M1: group of healthy control animals, M2: group of animals treated with BPA, M3: group of animals treated with ZnNPs, M4: group of animals treated with fennel extract, M5: group of animals treated with astragalus extract, M6: group of animals treated with probiotic, M7: group of animals treated ZnNPs + fennel extract, M8: group of animals treated with ZnNPs + asparagus extract, M9: group of animals treated with probiotic + fennel extract, M10: group of animals treated with probiotic + asparagus extract. 

Effect of Oral Administration of Znnps Complex, Herbs, As a Probiotic on Protein Parameters in Rats after 28 Days

Table 2 shows the effect of oral administration of ZnNPs, herbs and the probiotic separately or together on the value of protein parameters in the serum of rats treated for 28 days. The results showed that the total protein concentration showed a significant decrease in the treatment group M2 compared to the healthy control treatment M1, as its values reached 5.76, 9.36 g/dl, respectively. While there was a significant increase at (p˂0.05) up to their normal rates in the group of coefficients M4, M3, M5, M6, M7, M8, M9 and M10, which amounted to 778, 7.28, 8.09, 7.61, 6.63, 8.13, 8.2, 8.7 g/dL, respectively, compared to treatment M2, which recorded a higher decrease in total protein concentration and that the highest value appeared in the healthy control treatment M1. 

The results in the table also indicated that the albumin concentration showed a significant decrease in the treatment group M2 compared to the healthy control treatment M1, which amounted to 4.56, 7.33 g/dL, respectively, while a significant increase appeared at (p˂0.05). In the treatments M4, M3, M5, M6, M7, M8, M9 and M10 compared to the control treatment M2, which amounted to 5.36, 6.36, 6.36, 4.7, 5.33, 6, 5.4, 7.4 g/dL, respectively. M10 as the highest content of the two albums and in contrast, M2 recorded the lowest value of it. As for the values of globulin protein, the control treatment M2 showed a significant decrease at (p˂0.05) compared to the healthy control treatment M1, which amounted to 1.2, 2.02 g/dL, respectively, while a significant increase appeared at (p˂0.05). In treatments M3, M5, M6, M7, M8, M9 and M10, they reached (2.42, 1.73, 2.91, 1.3, 2.13, 2.8, 1.6) g/dL, respectively, compared to the control treatment M2, which recorded the lowest level. 

The results agreed with what was found by Shen et al. [13], who confirmed that the use of astragalus extract had a significant role in influencing the level of total protein and albumin to their normal levels. These results are also in agreement with what was found by Assia et al. [14], who confirmed that the use of the probiotic had a significant role in the significant increase in the level of total protein and albumin due to the increase in globulin, which was recently found to be associated with the development of diabetes mellitus as it has been suggested as biomarkers for the development of fibrosis. Hepatotoxicity in patients with nonalcoholic fatty liver disease. These results are in agreement with what was mentioned by Khalaf N et al. [15], who indicated that oral administration of zinc nanoparticles to male albino rats led to globulin and albumin levels reaching their normal levels.

Blood proteins are one of the most important components of blood serum that have important functions in the body. They are the building unit of many organs, hormones and enzymes. It is also important in enhancing the immunity and health of the body and the total blood serum protein test can be used to know the amount of albumin and globulin proteins. and antibodies from immunoglobulins. 

The different letters in the same column indicate the presence of significant differences between the treatments at the level of significance (p˂0.05).

M1: group of healthy control animals, M2: group of animals treated with BPA, M3: group of animals treated with ZnNPs, M4: group of animals treated with fennel extract, M5: group of animals treated with astragalus extract, M6: group of animals treated with probiotic, M7: group of animals treated ZnNPs + fennel extract, M8: group of animals treated with ZnNPs + asparagus extract, M9: group of animals treated with probiotic + fennel extract, M10: group of animals treated with probiotic + asparagus extract. 

1. Cimmino, Ilaria et al. “Potential mechanisms of bisphenol A (BPA) contributing to human disease.” International Journal of Molecular Sciences, vol. 21, 2020, pp. 5761, https://doi.org/10.3390/ijms21165761.

2. Abbas, A.H. “Biosynthesis of titanium dioxide nanoparticles by fungi isolated from soil.” Journal of the College of Basic Education, vol. 23, no. 98, 2017, pp. 205–220.

3. Ahmed, B.N. Chemical and biological evaluation of some synthetic nanoparticles and nisin loaded on gelatin for use as a film in the preservation of soft cheese. PhD thesis, Tikrit University, College of Agriculture, 2020.

4. Bahmani, K. et al. “Agro-morphological and phytochemical diversity of various Iranian fennel landraces.” Industrial Crops and Products, vol. 77, 2015, pp. 282–294.

5. Napoli, E.M. et al. “Screening the essential oil composition of wild Sicilian fennel.” Biochemical Systematics and Ecology, vol. 38, no. 2, 2010, pp. 213–223.

6. Ahmed, A.F. et al. “Comparative analysis of antioxidant activities of essential oils and extracts of fennel (Foeniculum vulgare Mill.) seeds from Egypt and China.” Food Science and Human Wellness, vol. 8, no. 1, 2019, pp. 67–73.

7. Li, W. et al. “Anti-tumor potential of Astragalus polysaccharides on breast cancer cell line mediated by macrophage activation.” Materials Science and Engineering C, vol. 98, 2019, pp. 685–695, https://doi.org/10.1016/j.msec.2019.01.025.

8. Liu, P. et al. “Anti-aging implications of Astragalus membranaceus (Huangqi): A well-known Chinese tonic.” Aging and Disease, vol. 8, no. 6, 2017, pp. 868–886, https://doi.org/10.3892/etm.2018.6501.

9. Singh, A.K. et al. “Medicinal plants for curing Alzheimer’s disease.” International Journal of Pharmaceutical and Biological Archive, vol. 1, no. 2, 2010, pp. 108–114.

10. Ranadheera, R.D.C.S. et al. “Importance of food in probiotic efficacy.” Food Research International, vol. 43, 2010, pp. 1–7.

11. Rabeh, M.N. and A.O. Aboraya. “Hepatoprotective effect of dill (Anethum graveolens L.) And fennel (Foeniculum vulgare) oil on hepatotoxic rats.” Pakistan Journal of Nutrition, vol. 13, no. 6, 2014, pp. 303–309.

12. Yan, F. et al. “Synergistic hepatoprotective effect of Schisandrae lignans with Astragalus polysaccharides on chronic liver injury in rats.” Phytomedicine, vol. 16, 2009, pp. 805–813.

13. Shen, P. et al. “Effect of Astragali and Angelica particles on proteinuria in Chinese patients with primary glomerulonephritis.” Journal of Traditional Chinese Medicine, vol. 36, no. 3, June 2016, pp. 299–306.

14. Bouaziz, Assia et al. “Study of probiotic effects of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus plantarum 299v strains on biochemical and morphometric parameters of rabbits after obesity induction.” Biology, vol. 10, 2021, pp. 131, https://doi.org/10.3390/biology10020131.

15. Ahmed, K.N. et al. “Estimation of Ag, Zn nanoparticles and nisin effects on biological and immunological parameters in male rats.” Journal of Critical Reviews, vol. 7, no. 4, 2020, pp. 1–10.