Introduction

In the concept of nutrition, the connection between diet and health is a major area of research; however, enhancing immunity is a major interest in the human diet. Indeed, an array of plants and their components hold immunomodulatory properties¹. Plants containing functional ingredients can be utilized as prophylaxis for preventing infections².

An emerging epidemic of unexplainable pneumonia was recorded³, named COVID-19 by World Health Organization. Unfortunately, specific antiviral drugs currently have not been available for treatment⁴, and the vaccines are not accurate 100% besides it is expensive for many countries⁵. To overcome these obstacles, the authors at Shanghai Changhai Hospital announced that it should speed up research performance on traditional Chinese medicine (TCM) which is used as an alternative remedy⁶. Immunocompromised patients are at high risk for COVID-19 infection which underlies the use of the immunomodulatory plant for prophylaxis and prevention of the disease⁷. Recently, they found that TCM has antivirus, anti-inflammation, through balancing the immune system regulation^8,9.

Achillea fragrantissima (Forssk.) Sch. Bip.  (A. fragrantissima) is related to the Asteraceae family in Arabic called ‘’Qaysoom’’. Traditionally, it is used as a medical plant in Arabian countries for the treatment of dysfunction of the liver and kidneys, gastrointestinal tract, as well as wound healing due to its antiseptic properties¹⁰. Achillea fragrantissima is used in the treatment of common health problems such as respiratory disease, eye infections, smallpox, fever, diabetes, dysmenorrhea, headache, or fatigue¹¹. Achillea fragrantissima oil used in many countries as a stomachic, diuretic, anthelmintic, and antispasmodic¹². It has antimicrobial, anti-inflammatory, anticancer, antifungal, and antibacterial effects. Nevertheless, till now no clinical uses for A. fragrantissima are described¹³.

Endoxan (cyclophosphamide) belongs to a group of medicines called antineoplastic or cytotoxic medicines used routinely for chemotherapy and in hematopoietic cell transplantation usually exerts side effects¹⁴, especially immunosuppressive impacts¹⁵, and oxidative stress¹⁶. It causes a sudden and adverse change of proinflammatory and anti-inflammatory cytokines (Th1/Th2) balance¹⁷, and immunoglobulins¹⁸.

On the other hand, Echinaid (Echinacea purpurea) supplement boosts the immune system. Echinaid has been used to prevent and treat upper respiratory infections¹⁹. Recently, many researchers settled that Echinacea purpurea could modulate immune functions in both animals and humans, because of its glycoproteins, alkyl amides, and polysaccharides compounds which have antioxidative, anti-inflammatory, and immunomodulatory effects²⁰. 

Anyway, most of the results published with A. fragrantissima extract have been conducted in vitro, little information exists in vivo and with oral administration. According to our knowledge, no study recorded the immune responses of the experimental animal that received A. fragrantissima taking into its consideration a comparison with immunosuppressive and immune-stimulating standards. Therefore, in the present study, it is wise to take into consideration, the detection of bioactive phytochemical constituents and antioxidant activities of A. fragrantissima extract. Furthermore, the immune modulation of A. fragrantissima extract will standardize with a positive immune stimulant (Echinaid) and negative immune suppressive (Endoxan). In this context, the current work is concerned with serum immunoglobulins: total immunoglobulins (Igs), immunoglobulins M (IgM), and immunoglobulins G (IgG) as well as cytokines (TNF-α, IL4, and IL10) changes with oral administration of A. fragrantissima extract. Histopathological investigation of the spleen, as one of the main organs involved in immune response, will be performed.

Materials and Methods

Ethical Standard

Qassim University Committee, Kingdom of Saudi Arabia, approved the current work as planned by the International Animal Ethics Committee under the number “cavm-2018-1-14-S-3478”.

Preparation of Achillea fragrantissima extract for oral administration

The Aerial parts of A. fragrantissima with a voucher specimen (AF-2008-51) were dried for 10 days at room temperature. The dried plant was ground into powder, then extracted with 80% ethanol (Arkan, Germany): and 20% distilled water using the Soxhlet apparatus (Shanghai Heqi Glassware Co., Ltd. China) for 18 hours²¹. The ethanol extract was evaporated and concentrated in a rotary evaporator (KNF Technology Shanghai Co., Ltd. China). The crude extract was diluted with distilled water to a concentration of 100 mg/ml.

Preparation of sheep erythrocytes (SRBCs)

Erythrocytes were extracted from healthy sheep blood as previously collected in sterile Alsevar’s solution (20.0 gm D-glucose, 8.0 gm sodium chloride, 4.2 gm sodium citrate, and distilled water up to 1000.0 ml.). The mixture was centrifuged 3 times at low speed for isolation of erythrocytes²². The obtained cells were adapted to be in a concentration equivalent to 100 µl phosphate buffer saline (PPS) pH 7.2 containing 1×10⁵/mm3 cells. Cells were adjusted by using a hemocytometer.

Animals

Adult healthy Wistar rats about 6 weeks of age weighed (170–200 g) were get from the laboratory center of the University of King Saud, Riyadh, KSA. Rats were put in a fitted rearing room, College of Agriculture and Veterinary Medicine, Qassim University, KSA. Animal acclimatization was performed by keeping rats in suitable cages at stable room temperature (22±2 ᵒC) under a light/dark cycle photoperiod 12 hrs. with free available fresh water and a commercial diet purchased from General Company of Feed Mills. The rat’s diet was formulated to be furnished with the nutrients recommended by National Research Council (NRC)²³. We did our best to minimize animal’s suffering and reduce pain. After one week of acclimatization, the experiment was started.

Experimental design and sampling 

Fifty animals were assigned into five groups ten per each as follows; Group (1): untreated control group. Rats in all other groups (2-5) were injected intraperitoneal (i/p) with Endoxan containing 200 mg cyclophosphamide (Baxter Oncology GmbH- kantstrassa 2- D-33790 Halle, Germany) as a standard immuno-suppressive, in a dose of 90 mg/kg for three successive days¹⁸. Group (2): Endoxan only. Group (3): Endoxan plus Echinaid (ESI srl Via delle industrie 1 Albissola Marina (SV) ITALY esi.it) containing Echinacea purpurea dry powder 375 mg/capsule as a standard immuno-stimulant, in a dose of 300 mg/kg/day orally²⁴. Groups (4 and 5): Endoxan and administrated with 300 and 500 mg/kg body weight per day A. fragrantissima extract orally suspended in saline respectively. A. fragrantissima extract doses were chosen to be within the safe and common range without any toxicity^21,25. On day 17 of the experiment rats in all groups (1-5) were challenged with the first dose of SRBCs (0.5 ml SRBCs suspensions containing 1×10⁸/mm³ cells i/p) followed by a second booster dose after 2 weeks i.e., at day 31 of the experiment (Table 1).

Table 1: Grouping and designing of the current experiment.

The experiment was conducted for 38 days. Rats were anesthetized with ether to collect blood samples pre-and post- the second dose of SRBC challenge at one-week intervals in all experimental groups, i.e., on days 24 and 38 of an experiment to follow up antibodies’ titers. Whilst the blood collected post-second dose of SRBC challenge was used to determine the biochemical analysis suggested in the present study. The spleen was gently removed from 3 animals in each group, for the determination of splenic non-viable cell numbers in percentage and counting the score of microscopic immune reaction of the spleen as well as for histopathological examination as mentioned below.

Measurements 

Gas chromatography-mass spectral analysis (GC/MS)

The aerial parts of A. fragrantissima were subjected to extraction using the same steps previously mentioned in preparation of A. fragrantissima extract for oral administration but using methanol 99.9% instead of 80% ethanol. The methanolic extract of the plant was analyzed qualitative and quantitative separately to identify the bioactive constituents of A. fragrantissima²⁶. The process was done using Agilent Gas Chromatography (Model 6890N coupled to 5973 Mass Selective Detector (MSD), (USA). The relative percentage amount of each component was calculated by comparing its average peak area to the total areas using software adopted to handle mass spectra and chromatograms (Turbo Mass Version 5.2).

Antioxidant activities of Achillea fragrantissima

The plant was extracted with 50% aqueous ethanol by stirring for 3 minutes at 25,000 rpm using a homogenizer (IKA, Germany). Samples were then centrifuged at 3500 rpm for 10 min to get the supernatants which were used for antioxidant analyses²⁷. Antioxidant activities of plant extract were detected for total phenolic content (TPC), 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), and 2, 2′-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS). The total phenolic content was estimated by the Folin-Ciocalteu method using gallic acid as the standard²⁸. The 2, 2-Diphenyl-1-picrylhydrazyl was determined using a modified method of Brand-Williams to determine antioxidant activity²⁹. The 2, 2′-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid was estimated using a modified method of Re to determine antioxidant activity using Trolox as the standard³⁰.

Table 2: Gas chromatography-mass spectral (GC/MS) components of Achillea fragrantissima.

Table 3: Antioxidant activity of the Achillea fragrantissima.

Total Phenolic Content (TPC) expressed as mg gallic acid equivalents (GAE) per 100 g, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) express percentage inhibition of the DPPH radical, and 2, 2′-Azino- Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) express mg Trolox equivalents. Mean± Standard error (SE).

Serum Antioxidant Activities and Lipid Peroxidation Biomarker

The antioxidant activities of serum were assayed by detecting the total antioxidant capacity (TAC), superoxide dismutase enzyme (SOD), and catalase enzyme (CAT) contents. Meanwhile, the lipid peroxidation biomarker is represented by malondialdehyde (MDA). The TAC, SOD, CAT, and MDA were assessed using Biodiagnostic kits (Cairo, Egypt) Cat. No. (TA 26 14, SD 26 22, CA 26 18, and MD 26 30 respectively). The absorbencies of TAC, SOD, CAT, and MDA were spectrophotometrically measured at 340 nm, 560 nm, 520 nm, and 534 nm respectively.

Serum total immunoglobulins (Igs), immunoglobulins M (IgM), and immunoglobulins G (IgG) titers (log2)

Detecting the antibodies against SRBC was performed using the ELISA method to measure the humoral immune response. ELISA was used to measure total Igs, IgM, and IgG anti-SRBC membrane antibody titers in sera³¹. Hemoglobin-free SRBC membranes were prepared, and the protein content of the solution was measured using SPECTRUM kits Cat. No. 08-700-131. ELISA plates were coated with prepared SRBC membrane³². Each serum sample was then added and incubated at 4°C for 12 hs. The use of mercaptoethanol-resistant IgG and sensitive IgM was followed as previously described³³.  

Cytokines assay (TNF-α, IL-4, and IL-10)

The pro-inflammatory cytokine determination, represented by TNF–α was performed using ELISA kits (Assaypro, 30 Triad South Drive, St Charles MO 63305, USA). The anti-inflammatory cytokines including Interleukin-4 (IL4) and Interleukin-10 (IL10) were assayed by ELI-SA kit (Cusabio Biotech Co., Ltd. Lot: 004152652, Wuhan, China). The manufacturer’s instructions were followed, and the color change was measured spectrophotometrically at a wavelength of 450 nm.

Spleen non-cellular viability %

Spleen specimens collected at the end of the experiment were divided into two parts. The first part was exposed to erythrocytes lysis with warm isotonic ammonium chloride lysing solution to use for determination of non-viable cell number % using trypan blue dye exclusion method³⁴. Following the next equation:

Histopathology of the spleen

The second part of spleen specimens was preserved using a 10% neutral buffered formalin aqueous solution and processed using routine paraffin wax, then stained with H & E (Hematoxylin, and Eosin)³⁵ for histological observations. The score of the microscopic immune reaction of the spleen which is expressed in (– and +) marks has been counted.

Statistical analysis

Values of data were illustrated as means ± standard errors. A simple one-way analysis of variances (ANOVA) test was used for each measured parameter. Post hoc analysis using the Mann-Whitney test was performed to compare the control with the Endoxan group and between the Endoxan and other experimental groups with P<0.05 reflecting a statistical difference.

Results 

Chromatographic analysis (GC–MS) of Achillea fragrantissima

The major phytochemical constituents of A. fragrantissima extract were determined by GC–MS found in (Table 2). Camphor, 1,8-Cineole, Artemisia ketone, Thujone, and Cyclohexene, 3-(1,5-dimethyl-4-hexenyl)-6-methylene were the main bioactive constituents of A. fragrantissima.

Antioxidant activities of Achillea fragrantissima 

The antioxidant activities of A. fragrantissima were 1446.59±40.51, 77.58±7.10, and 67.57±4.17 for TPC, DPPH, and ABTS respectively (Table 3).

Serum antioxidants enzyme activities and malondialdehyde

Rats treated with Endoxan without plant extract and challenged with SRBC (group 2) exert a significant (P<0.05) reduction in the TAC, SOD, and CAT comparable to the control group. On the contrary, Echinaid which was supplemented as an immune stimulant standard recorded significant (P<0.05) alleviation of the adverse effect of Endoxan. Echinaid and A. fragrantissima extracts in both doses (300 and 500) mg/kg showed significant (P<0.05) elevation in SOD. A high dose of A. fragrantissima extract (500 mg/kg) ameliorated TAC significantly (P<0.05) in comparison to the Endoxan group, where it showed non-significant improvement in CAT enzyme (Table 4). Nevertheless, a low dose of A. fragrantissima (300 mg/kg) extract supplementation success to increase CAT significantly (P<0.05) whereas, failed to improve TAC significantly.

Serum MDA, which is the final product of lipid peroxidation exerted significant (P<0.05) elevation in group 2, whereas exhibited significant (P<0.05) improvement by Echinaid and 500 mg/kg A. fragrantissima extract supplementations. Serum TAC and MDA did not affect by a low dose of A. fragrantissima ((300 mg/kg).

Table 4: Effect of Achillea fragrantissima extract on serum antioxidants enzyme activities and malondialdehyde.

Means having mark * in the same column have significant values at (P<0.05) compared to the control SRBC group. Means having letter ^a in the same column has significant values at (P<0.05) compared to the Endoxan group challenged with SRBC. Total antioxidant capacity (TAC); Superoxide dismutase enzyme (SOD); Catalase enzyme (CAT); Malondialdehyde (MDA). mean ± Standard error (SE)

Serum total immunoglobulins (Igs), immunoglobulins M (IgM), and immunoglobulins G (IgG) titers titer 

There was a drop in all antibodies titer measured which were significant in Igs and IgM pre- 2ry dose of SRBC challenge (at day 24) and in Igs post- 2ry dose of SRBC challenge (at day 38) for the group treated with Endoxan without plant extract comparable to SRBC control. Echinaid and A. fragrantissima succeeded in rising IgM (on day 24) of the experiment and Igs and IgG (on day 38) of the experiment significantly (P<0.05) (Table 5). Total Igs significantly (P<0.05) increased because of pre- 2ry dose of SRBC challenge (day 24) in groups received Echinaid, and 500 mg/kg A. fragrantissim although this increase was non-significant at a low dose (300 mg/kg). IgG and IgM did not exert any significant increase for pre and post 2ry doses of SRBC challenge for all experimental treatments.

Serum cytokines (TNF-α, IL-4, and IL-10)

There was a significant elevation in TNF-α level at (P<0.01) in the group treated with Endoxan without plant extract comparable to SRBC control. However, TNF-α levels showed a non-significant reduction with Echinaid and A. fragrantissima at two doses (300 and 500mg/kg in comparison to group (2) which received Endoxan only. The level of cytokine IL-4 was significantly (P<0.01) declined in animals receiving Endoxan compared to the control SRBC challenged group. On the other hand, the level of IL-4 was significant (P<0.05) elevated in rats offered to Echinaid and 300 mg/kg A. fragrantissima compared to those subjected to Endoxan and challenged with SRBC group. In addition, IL-10 level showed a significant decline (P<0.01) for rats treated with Endoxan and challenged with SRBC comparable with the control SRBC challenged group (P<0.05). However, the level of IL-10 exerted significant elevation for rats treated with A. fragrantissima at two doses (300 and 500) mg/kg (Table 6 & Fig. 1).

Table 5: Effect of Achillea fragrantissima extract on total anti-SRBC antibodies (Igs), IgM, and IgG titers (log2) 

Means having mark * in the same column have significant values at (P<0.05) compared to the control SRBC group. Means having letter ^a in the same column have significant values at (P<0.05) compared to the Endoxan group challenged with SRBC. Total immunoglobulins (Igs), immunoglobulins M (IgM), immunoglobulins G (IgG). mean ± Standard error (SE)

Table 6: Effect of Achillea fragrantissima extract on serum TNF-a, IL-4, and IL-10 cytokines

Means having mark * in the same column have significant values at (P<0.05) compared to the control SRBC group. Means having letter ^a in the same column has significant values at (P<0.05) compared to the Endoxan group challenged with SRBC. Tumor necrosis factor-a (TNF-a), Interlukine-4 (IL-4), and Interlukine-10 (IL-10). mean ± Standard error (SE).

Figure 1: Combo clustered column line combination chart of TNF-a, IL-4, and IL-10. Click here to view Figure

Spleen non-cellular viability% 

Spleen cellular non-viability % values achieved a significant (P≤0.05) increase in group (2) offered Endoxan without plant extract comparable to SRBC control. Whereas there was a significant (P≤0.05) decrease in non-cellular viability % in the rats who received either Echinaid or 500 mg/kg A. fragrantissima extract, they recorded values near to that found in the SRBC control group (7.38±1.12 and 8.12±0.94, vs 8.25±1.25) respectively (Table 7). Nevertheless, A. fragrantissima at a low dose (300 mg/kg) showed a non-significant reduction in this parameter.

Scores of the microscopic immune reaction of the spleen

Regarding the scores of the encountered microscopic immune reaction of the spleen (Table 7), the obtained results signified those rats in the group treated with Endoxan and challenged by SRBC showed lymphoid cell depletion and white pulp atrophy (score of immunosuppression -). Echinaid and A. fragrantissima at both doses exerted improvement in the scores of the microscopic immune reaction of the spleen in various degrees. The best improvement recorded in the group received 500 mg/kg A. fragrantissima extract which showed prominent lymphoid cell hyperplasia and white pulp hypertrophy (the highest score of immunostimulation ++++) followed by those who received Echinaid that showed moderate lymphoid cell hyperplasia and white pulp hypertrophy (moderate degree of immunostimulation +++) and finally a low dose of A. fragrantissima (300 mg/kg) extract which showed mild lymphoid cell hyperplasia and white pulp hypertrophy (mild degree of immunostimulation ++).

Table 7: Effect of Achillea fragrantissima extract on splenic non-cellular viability % and microscopic immune reaction score.

Means having mark * in the same column have significant values at (P<0.05) compared to the control SRBC group. Means having letter ^a in the same column have significant values at (P<0.05) compared to the Endoxan group challenged with SRBC. mean ± Standard error (SE). Normal spleen (+), Lymphoid cell depletion (-) Mild lymphoid cell hyperplasia (++), Moderate lymphoid cell hyperplasia (+++), Prominent lymphoid cell hyperplasia (++++).

Figure 2: The Control group revealed mild immunostimulant criteria of the spleen (H&E 40X). MZ: marginal zone; MS: marginal sinus region; G: germinal center; RP: red pulp; WP: white pulp; A: splenic artery. Click here to view Figure

Figure 3: Endoxan group showed the severest immunosuppressive reaction of the spleen. (H&E 40X). MZ: marginal zone; MS: marginal sinus region; G: germinal center; RP: red pulp; WP: white pulp; A: splenic artery Click here to view Figure

Figure 4: Echinaid group showed enhancement in the immunostimulant criteria of the spleen, (H&E 40X). MZ: marginal zone; MS: marginal sinus region; G: germinal center; RP: red pulp; WP: white pulp; A: splenic artery. Click here to view Figure

Figure 5: Achillea fragrantissima (300 mg/kg) group showed improvement in the immunostimulant criteria of the spleen (H&E 40X). MZ: marginal zone; G: germinal center; RP: red pulp; WP: white pulp; A: splenic artery. Click here to view Figure

Figure 6: Achillea fragrantissima (500 mg/kg) group showed more improvement in the immunostimulant criteria of the spleen. (H&E 40X). MZ: marginal zone; G: germinal center; RP: red pulp; WP: white pulp; A: splenic artery. Click here to view Figure

 
Histopathological observation 

The spleen specimens in the control group showed a mild immunostimulant characterized by slight enlargement of the white pulp (WP) and normal red pulp (RP). Endoxan group had an immunosuppressive effect which manifested by severe lymphoid cell necrosis and depletion giving the splenic parenchyma the classic moth-eaten appearance characteristic of immunosuppression besides decreased size and density of WP. Echinaid group showed enhancement of the immunostimulant criteria of the spleen. There were hyperplasia and hypertrophy of lymphoid cells and WP with high lymphocytes respectively. The size and density of G have increased. Administration of A. fragrantissima showed improvement of the immunostimulant criteria of the spleen which was clearer with the high dose represented by lymphoid cell hyperplasia and hypertrophy of the WP with high lymphocytes. The hyperplastic lymphoid cells invaded the red pulp zones. The best degree of immunostimulant among all the tested rats was shown in a high dose of A. fragrantissima which encountered in both the size and density of the follicular germinal center has increased. the hyperplastic lymphoid cells invaded the RP zones (Fig. 2-6).

Discussion

Most of the results previously published with A. fragrantissima extract have been conducted in vitro, little information exists in vivo and with oral administration. The current work was designed to evaluate the immune response of A. fragrantissima extract in vivo using the SRBC challenge. Highlighting the immunoglobulins and cytokines in comparison with Endoxan and Echinaid as standard immuno-suppressive and immuno-stimulative respectively.

The main bioactive constituents of A. fragrantissima obtained in the present study were corresponding with many types of research with some differences in percentage and content. Such variation may be due to some factors, for example, the geographical area that is affected by climate³⁶. The major bioactive constituent of A. fragrantissima obtained in the present study was camphor as previously recorded³⁷ The recorded antioxidant results of A. fragrantissima confirmed that it has antioxidant properties³⁸. The phenolic compounds have a higher activity of antioxidants and confirmed that A. fragrantissima has a radical scavenging activity³⁹. In any case, the finding indicated that A. fragrantissima has a high total phenolic content corresponding to fruits rich in ascorbic acid and other antioxidants⁴⁰.

Endoxan administration had a harmful effect on serum TAC, SOD, and CAT. Moreover, it elevated serum MDA significantly, which is the final product of lipid peroxidation, due to the formation of reactive oxygen species (ROS)⁴¹. The oxidative stress was dramatically increased in mice when received cyclophosphamide⁴². The oxidative stress could be due to metabolic conversion of Endoxan to several types of toxic metabolites, such as acrolein which reacts with cellular nucleophiles, leading to induce the oxidative stress consequence, inhibition of antioxidant enzymes⁴³. On the other side, Echinaid extract, and A. fragrantissima supplementations significantly alleviated the adverse effect of Endoxan in various manners. They achieved a significant increase in serum SOD, whereas A. fragrantissima recorded an improvement in TAC, CAT, and MDA with the dose depending. Administration of ethanol extract of A. fragrantissima caused a significant decrease in MDA²¹. Echinacea purpurea supplementation regulates the activities of MDA, SOD, and CAT levels²⁰. A. fragrantissima has biologically active constituents which fight the oxidative stress caused by scavenging of (ROS) and blocking of H2O2-induced mitogen-activated protein kinase (MAPK) pathway⁴⁴. The SOD considers the first-line defense system against reactive oxygen species. It catalyzes to break the two molecules of superoxide into hydrogen peroxide and molecular oxygen so that superoxide becomes less harmful. The CAT enzyme catalyzes the degradation of the hydrogen peroxide to water and oxygen so that completing the detoxification process is initiated by SOD⁴⁵. The antioxidants of the Echinaid and A. fragrantissima seem to protect the white blood cells responsible for immunity (neutrophils and lymphocytes) from oxidative stress, preventing their apoptosis⁴⁶. The essential oil of A. fragrantissima can be used safely as an antioxidant³⁸.

Endoxan administration caused a drop in all antibodies titer measured which were significant for Igs and IgM peri the second dose of SRBC challenge and in Igs and IgG post the second booster dose of SRBC challenge by one week. The obtained finding might be attributed to cyclophosphamide damaging the DNA of the immune cells, interfering with the proliferation and differentiation of B cells, and subsequence the humoral immune depression occurred¹⁸. Administration of Echinaid and, A. fragrantissima succeeded in rising Igs and IgM titer for the first dose of SRBC and Igs and IgG for the second dose of SRBC significantly. The present results agree with Mathivanan and Kalaiarasi⁴⁷ who concluded that medicinal plants increased antibody titer against SRBC more than virginiamycin. Unfortunately, little research has studied the relationship between A. fragrantissima and humoral immunity, whereas many papers studied the effect of the genus Achillea which is all from the same family, Asteraceae. Increasing IgG and IgM titer is an indication for enhancement of B lymphocyte production that is involved in the antibody synthesis consequently, improving humoral immune response to SRBCs⁴⁸. A. millefolium L. achieved high IgG and IgM titers against SRBC⁴⁹. In Contrast, inhibition of antibody production was recorded with A. talagonica administration⁵⁰. The conflict in the mentioned results is required further studies to solve that dilemma and connect between plant phytochemical constituents and humoral immunity.

Cytokines consider an inflammation regulator and a cornerstone in the pathophysiology of disease inside the human body. One of the immune response measures was depending on the balance between Th1 and Th2²⁵. Th1 pro-inflammatory cytokines are represented in the current study by TNF-α. Th2 anti-inflammatory cytokines which represented herein by IL4, and IL10. The present results revealed that rats treated with Endoxan and challenged with SRBC showed disturbances in the inflammatory cytokines indicating a low humoral immune response. These disturbances were reported previously in some studies¹⁵. Echinaid and A. fragrantissima extract mitigated the inflammatory cytokines. There was a reduction in TNF-α, a significant elevation in IL-4, and an increase in IL-10 in the rats who offer Echinaid. A low dose of A. fragrantissima (300 mg/kg) showed a decrease in TNF-α and significant elevation in IL-4 and IL-10. Likewise, a high dose of A. fragrantissima (500 mg/kg) caused a modulation in cytokines which was significant in IL-10. The cytokine IL-10 is a key anti-inflammatory regulator ensuring the protection of a host from exaggerated responses to SRBS⁵¹. The findings are in accordance with many studies that deal with other Achillea species⁵². Few studies were recorded on A. fragrantissima, which could cause a significant reduction in proinflammatory cytokines, suggesting a possible cytoprotective effect against cytokines disturbances²⁵. Treatment with ethanol and ethyl acetate extracts of A. fragrantissima restores the levels of the inflammatory cytokine in the serum to a normal state due to its anti-inflammatory activity²¹. Mostly A. fragrantissima extract could inhibit lipopolysaccharide (LPS)-induced expression of TNF-α and downregulated ROS production that leading to its anti-inflammatory effect⁴⁴. In the same context, A. fragrantissima extract might be to inhibit Nuclear Factor Kappa B (NFkB) activation subsequence produces pro-inflammation of protein kinase C or p38 MAPK⁵³.

The spleen is an essential organ in mediating the immune status of the body through its filtering of blood-borne pathogens and antigens. The increase in spleen cellular non-viability % caused by Endoxan without plant administration agrees with many types of research^15,17. Endoxan always has immune-suppressive, oxidative stress effects, and considers a life risk agent¹⁶. The significant reduction in spleen non-cellular viability % recorded with Echinaid and 500 mg/kg A. fragrantissima indicates that Echinaid and A. fragrantissima at a high dose success to modulate the immunosuppressive caused with Endoxan. This finding coincided with a recent study, which concluded that A. fragrantissima extract has an immunostimulant effect concerning humoral and cell-mediated immunity⁵⁴. Echinaid maybe deals with macrophages and T cells by stimulating phagocytosis, and lymphocytic activity to activate the immune response and protect the organism from infection^20,55.

Histopathological observation of the spleen confirmed the results obtained in an immune response manner. The immune response based upon the score of the microscopic immune reaction of the spleen signified that those rats in the group treated with Endoxan and challenged by SRBC showed lymphoid cell depletion and white pulp atrophy indicating an immunosuppression effect. This finding may be due to Endoxan causing damage in the spleen and thymus tissues, the main organs in the immune stimulation⁵⁶. Whereas Echinaid and 500 mg/kg A. fragrantissima, exerted prominent lymphoid cell hyperplasia and white pulp hypertrophy ranging from moderate (immunostimulation +++) to high (immunostimulation ++++) scores of the microscopic immune reaction respectively which indicates that A. fragrantissima could restore immune response as previously observed⁵⁴.

The beneficial effect of A. fragrantissima on immune responses recorded in the current study may be attributed to its antioxidant properties and phytochemical constituents (Camphor, 1,8-Cineole, Artemisia ketone, Thujone, and Cyclohexene, 3-(1,5-dimethyl-4-hexenyl)-6-methylene) detected in the present study.

Conclusion

Finally, the results of the current study support the hypothesis that it is possible for individuals who suffer from immunosuppressive, administration of A. fragrantissima may be useful as an alternative remedy for prophylaxis for some fatal diseases such as COVID-19 before infection that needs further research to detect the exact dose in humans. Inconclusively the current study highlights that a high dose of A. fragrantissima extract is preferred over a low dose to restore and modulate the immune response in vivo.   

 Acknowledement

Authors would like to thank the Reviewers for taking the time and effort necessary to review the manuscript. We sincerely appreciate all valuable comments and suggestions, which helped us to improve the quality of the manuscript.

Conflicts of interests 

The authors declare no conflict of interest in conducting this study.

Funding Sources

The authors received no financial support for the research, authorship, and publication of this article.

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