Modulatory Efficiency of Echinacea purpurea Extract on Hyperthyroidism Modeled Rats

Background and objective: Thyroid hormones are crucial for growth, metabolism, thermoregulation, and development. Hyperthyroidism is a thyroid disorder characterized by


INTRODUCTION
Thyroid hormones are crucial for growth, metabolism, thermoregulation, and development (Lazar, 1993;Mo Kim et al.,2012). Thyroid and pituitary gland tumors, however, cause thyroid cells to generate extra hormones under several pathological situations, such as Graves' disease, which leads to a hyperthyroid state (Gulcelik et al., 2006;Kim et al., 2012). Changes in these hormone levels cause a variety of health issues in addition to changed basal metabolic rate, more specifically, hyperthyroidism (Kim et al., 2012). Background and objective:Thyroid hormones are crucial for growth, metabolism, thermoregulation, and development. Hyperthyroidism is a thyroid disorder characterized by over secretion of thyroid hormones that lead to raise in the metabolic rate and oxygen utilization in several tissues. Echinacea purpurea (EchEE) is a significant medicinal plant with several pharmacological benefits. Aim of the work: This study was conducted to evaluate the modulatory efficiency of Echinacea purpurea ethanolic extract on hyperthyroidism-modeled rats. Materials and methods: Both normal and hyperthyroidism animals were classified into four groups (10 rats each) as follows: 1) normal animals without any treatments and served as normal control; 2) normal animals orally administrated with EchEE (465 mg/kg/day) dissolved in water for 30 days; 3) untreated hyperthyroidism-modeled animals; 4) hyperthyroidism-modeled animals treated with EchEE for a similar period. Results: Treatment of the hyperthyroidism-modeled animals with EchEE, resulted in a significant reduction in the serum level of total T3, total T4, free T3 and free T4 hormones coupled with a remarkable elevation in TSH level. EchEE succeeded also in ameliorating the histological and immunohistochemical picture of the thyroid gland. Also, it improved liver and kidney functions and decreased blood sugar levels. Conclusions: This study concluded that EchEE efficiently improved the pathophysiological architecture of the hyperthyroid gland, this was achieved from the marked decline in its hormone with a notable raise in TSH. This effect could be mechanized through the antioxidant characteristics of EchEE constituents. This extract could be a promising medical agent for human health; additional research is needed.
The endocrine condition known as hyperthyroidism is defined by increased thyroid hormone (T3 and T4) release, which causes a rise in basal metabolic rate and oxygen consumption in many tissues (Mannaa et al., 2021).
The hypermetabolic state resulting from hyperthyroidism is associated with various degrees of oxidative stress in the organism. Oxidative damage to lipids, proteins, and DNA, indicative of oxidative stress, was found in hyperthyroid rats (Duntas, 2002;Venditti and Meo, 2006).
In the pharmaceutical business, medicinal plants are known to offer a variety of biological and pharmacological properties that are extremely valuable for the discovery and development of medications with notable efficacy and tolerable side effects. Echinacea purpurea, a member of the Asteraceae family, is a significant medicinal plant with several pharmacological benefits (Lee et al., 2009;Khalaf et al., 2019).The active components of Echinacea purpurea include carbohydrates, glycosides, alkaloids, alkyl amides, and polyacetylene (Lalone et al., 2007;EL-Sherbiny et al.,2021), sitosterol, cynarine, caffeic acid, chlorogenic acid, echinacoside, and cichoric acid. They are all potent antioxidants that effectively scavenge free radicals and reduce the production of nitric oxide free radicals (Zhao et al., 2010;Motamedi et al., 2018). The choice of anti-hyperthyroid drugs is not based on their relative treatment efficacy, but instead on consideration of side effects and risk of profound clinical consequences, in particular teratogenicity and serious liver injury. Therefore, the main goal of the present study was to explore the possible modulatory potential of Echinacea purpurea extract on hyperthyroidmodeled adult rats.

MATERIALS AND METHODS Plant and Ethanolic Extraction:
Moench (Echinacea purpurea L; family: Asteraceae) flowers were obtained from a local supplier in Cairo, Egypt; The plant was identified and was found carrying taxonomy serial number 3728. The dry flowers were grinded and soaked in ethanol (70%) at ratio of (1:5 w/v) for seven days. Then, the mixture was filtered using Whatman 1 filter paper. The solvent was evaporated using a rotatory evaporator, while the moisture residuals were removed through lyophilization process with freeze drier (Todd et al., 2015). Dry Echinacea purpurea ethanolic extract (EchEE) was calculated for its yield (g extract /100g crude powdered flowers), however, the obtained dry EchEE was stored at 4 -20 °C until in vitro assessments and further use. EchEE Radical Scavenging Activity Using DPPH Assay: Radical scavenging activity (RSA) of EchEE was estimated as previously described (Nogala-Kalucka et al., 2005). Certain amount of EchEE was dissolved in methanol to obtain a concentration of 200 ppm. Then a 200µL of this solution was completed to 4 ml methanol, then 1 ml DPPH methanolic solution (6.09 x 10 -5 mol/L) was added; 10 min later, the absorbance of this mixture was recorded at 516 nm against the absorbance of the blank (1 ml of DPPH solution plus 4 ml methanol) was measured. Both test and blank samples were measured in triplicates. RSA was calculated according to the equation: (RSA%= ).

EchEE Total Phenolics Content:
Total phenolic content of EchEE was evaluated according to Jayaprakasha et al. (2003). In brief, 5 mg of the extract was dissolved in a 10 ml mixture of acetone and water (6:4 v/v). Samples (0.2 ml) were mixed with 1.0 ml of 10-fold diluted Folin-Ciocalteu reagent and 0.8 ml of sodium carbonate solution (7.5%). After 30 minutes at room temperature, the absorbance was measured at 765 nm using V-530 UV/visible spectrophotometer. Estimation of phenolic compounds as catechin equivalents was carried out using standard curve of catechin.

EchEE Reducing Power:
Ferric-reducing power of EchEE was determined according to the method described by Sethiya et al. (2014). The reducing power of the extract was calculated as equivalent to ascorbic acid from the standard curve of ascorbic acid.

Experimental Design:
Adult male albino rats (180-250g) were obtained from the animal colony, National Research Centre, and placed in suitable polyethylene plastic cages with free access to food and water one week before the experiment began. All animals received human care in compliance with the standard institutional criteria for the care and use of experimental animals according to standard guidelines of the ethical committee of National Research Centre. Hyperthyroidism rats were modeled as described by Carageorgiou et al. (2007) by daily subcutaneous injection of L-thyroxine (Eltroxin TM 100mg, GlaxoSmithKline Co, Bomerange Building, Cairo, Egypt, dissolved in saline) at dose of 250 microg/kg/day for 14 consecutive days. After induction of hyperthyroidism, both normal and modeled animals were classified into the four groups (10 rats each) as follows: I) normal animals without any treatments and acting as normal control; 2) normal animals orally administrated with EchEE (465 mg/kg/day) dissolved in water (Mao et al., 2021) for 30 days; 3) untreated hyperthyroidism modeled animals; 4) hyperthyroidism modeled animals treated with EchEE for a similar period. Blood and Tissue Sampling: All animals were weighed at the end of the experiment, then fasted overnight, and then blood samples were collected from the retroorbital venous plexus following inhalation of diethyl ether anesthesia, and divided into 2 portions per each sample: heparinized portions for the determination of hemoglobin, and the other for clear serum which was separated, divided into aliquots, and stored at -80ºC until biochemical determinations.
After blood collection, each animal was sacrificed by sudden decapitation, then a part of the liver was dissected out each animal, washed with saline, dried on filter paper, and homogenized in 50 mmol/L phosphate buffer (ice-cold) solution (pH 7.4) to give 10% w/v homogenate. The homogenate was coolly centrifuged at 3000 rpm, and the clear supernatant was separated, divided into aliquots, and stored at -80 ºC for further determination of the oxidative stress markers. Immediately, the thyroid gland was dissected out and kept in buffered formalin-saline solution (10%) for the histopathological and immunohistochemical examination which was carried out by cutting 5µm thick paraffin sections and then stain with hematoxylin and eosin (Drury and Wallington, 1980) and examined with a light microscope.
Serum liver and kidney functions were determined spectrophotometrically to evaluate the safety of EchEE. The concentrations of creatinine and serum urea were assessed using reagent kits obtained from Biodiagnostic Co. (Egypt). The activity of the serum enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) were measure using reagent kits purchased from Human Gesell Schaft fur Biochemical und Diagnostica mbH, Germany.
Hepatic glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities, as well as the levels of glutathione (GSH) and nitric oxide (NO) were determined using reagent kits obtained from Biodiagnostic Co., Egypt. Whereas liver lipid peroxidation level was estimated chemically according to the method described by Ruiz-Larrea et al. (1994) on the base of malondialdehyde (MDA) reaction with thiobarbituric acid (TBA) which forms a pink complex that can be measured photometrically.

Histopathological Examination:
General histological examination: The procedure for histological preparations is as described by Bancroft and Stevens (2013). Thyroid tissues were thinly sliced to a thickness of 3-4 mm, then fixed in 10% neutral buffered formalin (10% NBF), dehydrated in ethanol at varying percentages, cleaned in xylene, and finally embedded in paraffin. To analyse overall tissue structure, the paraffin blocks were cut into sections using a microtome at a thickness of (4-6 m). Using a Leica microscope, H&Estained slices were examined. (CH9435 Hee56rbrugg) (Leica Microsystems, Switzerland).

Immunohistochemistry:
Immunohistochemistry Staining Protocol was carried out on paraffin sections and mounted on positively charged slides by using avidin-biotin-peroxidase complex (ABC) method (Hsu et al., 1981). Anti-Rabbit Calcitonin Receptor Antibody (ThermoFisher Scientific, Cat# PA1-84457, Polyclonal, Dil.: 1:1000) was used in this IHC examination. Sections from each group were incubated with the previously mentioned antibodies, then the reagents required for the ABC method (Vectastain ABC-HRP kit, Vector laboratories) were added. Marker expression was labeled with peroxidase and colored with diaminobenzidine (DAB, produced by Sigma) to detect antigen-antibody complex. Negative controls were included using nonimmune serum in place of the primary or secondary antibodies. IHC stained sections were examined using a Leica microscope (CH9435 Hee56rbrugg) (Leica Microsystems, Switzerland) in Faculty of Veterinary Medicine, Cairo University.

Statistical Analysis:
According to Steel and Torrie (1980), using a statistical analysis system (SAS) program software, the collected data were subjected to an ANOVA followed by Duncan multiple post hoc tests at the level of p≤0.05. Copyright (c) 1998 by SAS Institute Inc., Cary, NC, USA.
RESULTS Results of in vitro assessments (yields, total phenolics content, radical scavenging activity and reducing power) of the EchEE are illustrated in Figures (1&2). It can be clearly noticed that EchEE possesses considerable amounts of total phenolic compounds and higher antioxidant capacity achieved from the great ability to scavenge 77.61±2.55 % of DPPH • radical that confirms the resultant reducing power which reflected a concentration-dependent relation.
Ingestion of healthy rats with EchEE didn't affect the percent of body weight gain (BWG), while hyperthyroidism-modeled animals recorded a significant decrease in BWG when both groups were compared with the control group. Favorably, treatment of hyper-thyroidal animals with EchEE significantly restored BWG close to that of healthy animals (Fig. 3).  The current study indicated that administration of EchEE to healthy animals didn't affect the hormonal profile of thyroid gland reflecting its safe effect on thyroid gland of healthy animals at the used level, while the serum level of total T3, total T4, free T3 and free T4 levels hormones was markedly increased matched with noticeable decrease in TSH level of hyperthyroidism modeled animals. Interestingly, administration of hyperthyroidism-modeled animals with EchEE, resulted in a significant reduction in the serum total T3, total T4, free T3 and free T4 levels coupled with a remarkable elevation in TSH level (Table 1).   Data are presented as mean ± standard error for 10 rats. ( A ) is statistically significant from control group while ( B ) is statistically significant from hyperthyroid group at p≤0.05, EchEE (Echinacea purpurea ethanolic extract)., Hyper (hyperthyroidism model), Significant increases in serum TNFα, IL-4, and IL-13 coupled with marked drop in IL-10 levels were detected regarding hyperthyroidism model.
Favorably, hyperthyroid animals that received EchEE revealed downregulation in TNFα, IL-4 and IL-13, associated with clear upregulation of the anti-inflammatory IL-10 close to the controls; (Fig. 5).

Fig. 5.
Effect of Echinacea purpurea ethanolic extract (EchEE) on serum cytokine levels of hyperthyroidism modeled and treated rats. Data are presented as mean ± standard error for 10 rats. ( A ) is statistically significant from the control group while ( B ) is statistically significant from hyperthyroid group at p≤0.05, EchEE (Echinacea purpurea ethanolic extract)., Hyper (hyperthyroidism model).

Histopathological Examination:
Histological examination of thyroid sections Figure (6) from control rats detected the basic histological structure of thyroid follicles with their lining of simple cuboidal follicular epithelial cells which are characterized by eosinophilic cytoplasm as well as hyperchromatic spherical nuclei. Inside lumen of these follicles, a colloid is observed as consistent with pink color. In between thyroid follicles, polyhedral parafollicular cells in addition to intralobular fibrous connective tissue were noticed (A). Thyroid gland sections of Echinacea purpurea ethanolic extract group emphasized the same structure of thyroid follicles in negative control group involving; follicular epithelial cells, colloid, parafollicular cells as well as intra lobular fibrous connective tissue (B). Thyroid gland sections of positive hyperthyroidism group underscored a variety of deteriorating changes along thyroid gland (C): Thyroid follicles presented with hyperplasia, flattening of epithelial follicular cells, detachment of basement membrane, epithelial desquamation, as well as scalloped colloids with a prominent expanding amount. Also, an obvious increase in parafollicular cells numbers, congested blood vessels, severe amount of fibrous connective tissue and leucocytic infiltration was detected (D). thyroid gland section of treated group revealed noticeable development in the thyroid gland structure. Most thyroid follicles existed with normal assembly of their follicular cells lining and slightly scalloped colloid.
Diminished intralobular fibrous connective tissue along with congested blood vessels were also observed (E).

Immunohistochemical Findings:
Immunohistochemical examination demonstrated the reactivity of Calcitonin antibodies in thyroid gland tissue sections including researched groups (Figure 7). Thyroid gland section of the negative control group presented positive brown expression in a few amounts to Calcitonin antibody along parafollicular cells (A). Thyroid gland section of Echinacea purpurea extract group exhibited slight expression of parafollicular cells to Calcitonin antibody like negative control group (B). Thyroid gland section of hyperthyroidism group underscored intense expression of Calcitonin antibodies along parafollicular cells as well as follicular ones (C). Thyroid gland section of treated group denoted moderate reactivity with brown positive expression to Calcitonin Antibody (D) .   Fig. 6. Photomicrographs displayed the histopathological differences in thyroid gland tissue sections among analyzed groups as follows: (A) Thyroid gland sections of negative control group presented the basic histological structure of thyroid follicles with their lining of simple cuboidal follicular epithelial cells was characterized by eosinophilic cytoplasm as well as hyperchromatic spherical nuclei (thick arrow). Inside lumen of these follicles, colloid is observed as consistent with pink color (wave arrow). In between thyroid follicles, polyhedral parafollicular cells (thin arrow) in addition to intra-lobular fibrous connective tissue (star) were noticed. (B) Thyroid gland sections of EchEE group emphasized the same structure of thyroid follicles in negative control group involving; follicular epithelial cells (thick arrow), colloid (wave arrow), parafollicular cells (thin arrow) as well as intra lobular fibrous connective tissue (star). (C & D) Thyroid gland sections of Positive Hyperthyroidism Group underscored a variety of deteriorating changes along thyroid gland. (C): Thyroid follicles presented with hyperplasia (thick arrow), flattening of epithelial follicular cells, detachment of basement membrane (thin arrow), epithelial desquamation (circle), as well as scalloped colloid (wave arrow) with a prominent expanding amount (curved arrow). Also, an obvious increase in parafollicular cells numbers (star), congested blood vessels (cube), severe amount of fibrous connective tissue and leucocytic infiltration (arrowhead) were detected. (E) Thyroid gland section of treated Group revealed noticeable development in the thyroid gland structure. Most thyroid follicles existed with normal assembly of their follicular cells lining (circle) and slightly scalloped colloid (wave arrow). Diminished intralobular fibrous connective tissue (cube) along with congested blood vessels (thin arrow) were also observed. (Hematoxylin & Eosin Stain, Magnification Power= x400 & Scale Bar= 50μm).

DISCUSSION
The hypermetabolic state resulting from hyperthyroidism is associated with various degrees of oxidative stress in the organism. Oxidative damage to lipids, proteins, and DNA, indicative of oxidative stress, was found in hyperthyroid rats (Duntas, 2002;Venditti and Meo, 2006). As the choice of antihyperthyroid drugs is not based on their relative treatment efficacy, but instead on consideration of side effects and risk of profound clinical consequences, in particular teratogenicity and serious injuries, therefore, the objective of the present study was to explore the possible modulatory potential of Echinacea purpurea extract, enhanced with its safe effect, on hyperthyroid modeled adult rats. The current study revealed that modeled hyperthyroidism was associated with a remarkable increase in serum total T3, FT3, total T4, and FT3 levels together with a notable drop in serum TSH level; In addition, hyperthyroidism animals were asserted also by a decreased body weight gain. These results are consistent with many previous reports (Ourique et al., 2013;Asker et al., 2015;and Mannaa et al., 2021).
The thyroid hormones significantly improved after EchEEtherapy in the hyperthyroidismmodeled rats, demonstrating the antihyperthyroidism potential of EchEE by balancing the levels of T3 and T4 and preventing the resulting loss in body weight.
Significant increases in serum TNFα, IL-4, and IL-13 coupled with significant decreases in IL-10 levels were detected regarding hyperthyroidism model, this result goes in line with that of Isaacs (1995) and Mannaa et al. (2021). Favorably, hyperthyroid animals that received EchEE revealed downregulation in TNFα, IL-4 and IL-13, associated with clear upregulation of the antiinflammatory IL-10 close to the controls; this result agonists the finding of Kishazi et al. (2018). It was reported that Echinacea purpurea extract is a promising immunomodulatory agent with a potent therapeutic value in stimulating the immune response (El-Sherniny et al., 2021). Cytokines are glycosylated proteins that are involved in cell-cell communications and are often categorized as pro-inflammatory and anti-inflammatory. The antiinflammatory IL-10 is well recognized as an important negative regulator of pro-inflammatory gene expression in mononuclear phagocytes; also, IL-10 is an immunosuppressive glycoprotein. It had been clear that IL-10 primarily acts on activated macrophages to suppress the secretion of IL-1, IL-12, TNF-α, and reactive oxygen radicals (Isaacs 1995).
Moreover, the hyperthyroidism model used in this study displayed a clear decrease in hepatic values of the antioxidative agents along with a marked elevation in the hepatic oxidative voltage These deteriorations were improved by posttherapeutic doses of EchEE; this effect is evidenced by the marked elevation in the antioxidant markers (GSH, GPx, and SOD) coupled with significant reduction in the oxidative products (MDA and NO).
However, in the current study, the urea and creatinine levels unchanged in the hyperthyroid rat model. These findings are consistent with earlier studies that demonstrated that administering L-thyroxin did not affect serum urea and creatinine levels (Mannaa et al., 2021).
Our findings regarding hyperthyroidism resulted in an increase in serum ASAT and ALAT activity, along with a significant decrease in Hb and FBS values. Previous studies have shown that hyperthyroidism causes the liver to undergo apoptosis, which leads to hepatic dysfunction (Huang and Liaw, 1995;Giris et al., 2010), and is associated with an increase in liver enzymes like ALAT, ASAT, and alkaline phosphatase in serum (Huang and Liaw, 1995;Mannaa et al.,2021). Messarah et al. (2011) found that rats given L-thyroxine sodium salt (0.0012%) in drinking water had significantly lower levels of red blood cells, hemoglobin, and hematocrit.
The values of Hb and FBS considerably increased after EchEE treatment for the hyperthyroidism group. EchEE therapy improved liver enzymes and reduced oxidative stress, consequently, there is decreased leakage of hepatic ASAT and ALAT leakage into the bloodstream as EchEE contains antioxidant components like phenolic compounds that can suppress lipid peroxidation, stabilize cell membranes, and prevent the oxidation of membrane lipids, and restore the cellular integrity consequently.
By studying the thyroid gland tissue histopathologically, our biochemical findings were supported. The thyroid follicles displayed hyperplasia, flattening of the epithelial follicular cells, detachment of the basement membrane, desquamation of the epithelium, and scalloped colloid with a clearly increasing quantity. Additionally, there is a clear rise in the number of parafollicular cells, clogged blood arteries, a significant amount of fibrous connective tissue, and leucocytic infiltration. However, animals treated with EchEE resulted in an improvement in thyroid gland tissue as compared with the group of hyperthyroidisms, as EchEE indicated improvement in pathological changes. Previous research reported that vacuolated colloid was present inside the thyroid follicles of hyperthyroid rats. There was also an increase in interstitial tissue as well as the proliferation of bands of fibrous tissue and several layers of follicular cells. Reactive oxygen species, which are physiologically necessary for the synthesis of thyroid hormone and are produced in moderate amounts by thyroid epithelial cells in basal conditions, are continuously detoxified either during the hormone-synthesis process or by endogenous antioxidant systems, making them non-toxic. (Mogulkoc et al., 2005;Mannaa et al.,2021). But when compared to the hyperthyroidism group, animals given EchEE showed improvement in thyroid gland tissue because EchEE showed improvement in the degenerative alterations. This improvement most likely resulted from reducing oxidative stress and improving thyroid hormones (Mannaa et al., 2021). The thyroid gland tissue was found to be reactive to the Calcitonin Antibody by immunohistochemical analysis. The hyperthyroidism group's thyroid gland portion highlighted strong expression of calcitonin antibody along parafollicular cells. With brown positive expression to calcitonin antibody, the thyroid gland region of the EchEE Group indicated moderate reactivity.

CONCLUSIONS
The outcomes of the present study showed that EchEE therapy efficiently regulated hyperthyroid hormones, improved liver and kidney functions and restored blood sugar and hemoglobin levels. The high concentration of antioxidant components in this extract may be responsible for its positive effects in treating hyperthyroidism and its accompanying problems. To support the significance of EchEE in treating human hyperthyroidism, more research is needed.