Mustafa K. Mushatet^1*, Asaad Abbas khalaf¹, Doaa A. Hamad¹ and Thikra Abd Jary²

¹University of Kerbala, College of Nursing, Branch of basic sciences, Kerbala, Iraq,

²University of Al-Ameed, College of Medicine, Branch of Biochemistry, Kerbala, Iraq,

Corresponding Author E-mail: mustafa.k@uokerbala.edu.iq

Abstract

This research looked at how an aquatic Eucalyptus globulus extract (ECP) affected inflammation and oxidative stress in male rats that were given an experimental form of carrageenan (CRG). The study randomly assigned twenty-four male Wistar rats. In the study, 2 ml of physiological solution was administered to the control group, 0.6 g/kg of ECP BW was given to the ECP group orally, 100 μl of the CRG solution dissolved in 1 milliliter of distilled water was injected intraperitoneally to the CRG group for 30 days, and 0.6 g/kg of BW of ECP was given orally to the C&E group for 15 days after 15 days of 100 μl of the carrageenan solution dissolved in 1 milliliter of distilled water intraperitoneal injection. We sacrificed the rats after 30 days and analyzed the serum samples to measure white blood cell count (WBC), lymphocytes, monocytes, neutrophils, C-reactive protein (CRP), and tumor necrosis factor (TNF). The study found that the CRG and C&E groups had higher levels of WBC, lymphocytes, monocytes, neutrophils, CRP, and TNF than the ECP and control groups. The decrease in inflammation markers CRP and TNF in the serum of E&C group rats demonstrates that ECP's aqueous extract has anti-inflammatory properties. However, further clinical and histological research is necessary to validate its potential as a therapeutic agent.

Keywords

Carrageenan; C- Reactive Protein; Eucalyptus Globulus; Inflammatory; Lymphocytes; Monocytes; Necrosis Factor; Neutrophils and Tumor

Copy the following to cite this article: Mushatet M. K, Khalaf A. A, Hamad D. A, Jary T. A. The Effect of an Aquatic Extract of Eucalyptus globulus Leaves on Reducing the Inflammation Parameters Caused by Carrageenan in Male Wistar Rats. Biomed Pharmacol J 2024;17(3).

Copy the following to cite this URL: Mushatet M. K, Khalaf A. A, Hamad D. A, Jary T. A. The Effect of an Aquatic Extract of Eucalyptus globulus Leaves on Reducing the Inflammation Parameters Caused by Carrageenan in Male Wistar Rats. Biomed Pharmacol J 2024;17(3). Available from: https://bit.ly/3XzTtyU

Introduction

In this study, we investigated the anti-inflammatory properties of an aquatic extract of Eucalyptus globulus (ECP) by inducing inflammatory indices using carrageenan. Herbal products are essential sources of herbal extracts and other pharmacological agents. Since ancient times, people have used the leaves of various Eucalyptus species as traditional medicines to treat fever, colds, and other ailments¹.

ECP leaves are utilized locally to treat burns, dermatitis, scabies, tonsillitis, and dysentery; they are also taken orally to treat articular pain, tonsillitis, dysentery, influenza, and cystitis. Research has shown that using ECP leaves orally can reduce cold symptoms during flu-episodes^2,3. Eucalyptus globulus possesses remarkable pharmacological activities, including antioxidant and anti-inflammatory effects⁴. The naturally oils in Eucalyptus globulus include 1-eucalyptol, which has a variety of pharmacological actions, including insecticidal, gastrointestinal, dermatological, anti-inflammatory, analgesic, antidiabetic, antioxidant, anticancer, and antibacterial properties^5–7.

Concisely, Eucalyptus leaves have the ability to protect the liver from damage and reduce oxidative stress due to reactive oxygen species (ROS) produced during metabolism⁸, It has previously been shown to be a source of bioactive chemicals, namely phenolic compounds like phenolic acids, flavonoids, or hydrolyzable tannins. E. globulus leaves have long been used to treat respiratory issues^9,10.

Eucalyptus leaves have antioxidant properties, as evidenced by their ability to scavenge free radicals, activate antioxidant enzymes, and suppress inflammation by inhibiting lipoxygenase and lowering nitric oxide (NO) levels¹⁰. It was demonstrated that their ethanolic extracts reduced the levels of pro-inflammatory mediators and nitric oxide (NO)¹¹, and that they had the ability to scavenge free radicals¹².

A basic defense mechanism of the host in reaction to damaging stimuli such as, illness, and/or irritation is inflammation¹³. The symptoms of acute inflammation manifest suddenly and include edema, leukocyte emigration, enhanced vascular permeability, and vasodilation¹⁴. A series of cytokines governs the inflammatory process to a significant extent and is essential for cell-to-cell contacts and communications¹⁵.

Carrageenan (E407) is a polysaccharide extracted from red algae (Chondrus crispus) that induces inflammation induced inflammatory in the rat16,17, Acute inflammation leads to the development of biphasic inflammatory responses in the tissue of the paw18. The main objective of the current research is to ascertain the therapeutic properties of Eucalyptus leaves and their role in mitigating the inflammatory activity of carrageenan.

Research design and methods

The current study aims to identify the therapeutic qualities of eucalyptus leaves and how they contribute to reducing carrageenan’s inflammatory activity. We obtained 24 male Albino rats, aged 12 to 14 weeks, from the Animal House, College of Pharmacy / University of Karbala. The Institutional Animal Ethics Committee, University of Kerbala, Iraq, assisted in reviewing and approving all animal experimentation protocols, with the approval of experimental research. Animal maintenance therapies, as instructed by animal care experts, adhere to the International Animal Care and Use Guidelines¹⁹. We randomly divided these rats into four groups, each containing six rats, kept them in a separate cage, and gave them two weeks to acclimate to the water and food supplies, following the American Institute of Nutrition’s semi-purified diet (AIN).

Group 1: control, which receives an oral dose of 2 ml of a normal saline physiological solution for 30 days.

Group 2: is the positive control، receiving 0.6 g/kg of Eucalyptus globulus extract (ECP) orally at a dose of 2 ml for 30 days. The (LD₅₀) of eucalyptus oil is 2770-4500 mg/kg (rat)²⁰.

Group 3: For 30 days, the Carrageenan group will receive an intraperitoneal injection of 100 μl of the carrageenan solution (CRG) dissolved in 1 milliliter of distilled water.

Group 4: CRG and ECP Group (C&E): For fifteen days, this group receives an intraperitoneal injection of 100 μl of the carrageenan solution dissolved in 1 milliliter, followed by an oral dose of ECP after two hours, to follow up on the inflammation status in the C&E group, in which inflammation was decreasing compared to the CRG group, in which inflammation was ascending (inflammation is directly proportional to the time of carrageenan injection) in the same period, which 30 days. We assumed that a period of 15 days is sufficient to stimulate an inflammatory state using intraperitoneal carrageenan²¹, followed by an equal period of treatment using ECP to try to restore inflammatory factors to their normal levels as in the control group.

Following a 12-hour fast, we sacrificed the rats after 30 days and extracted seven ml of blood directly from the heart for a complete blood count (CBC). We recorded the results and separated the blood serum using an Inflammation Panel 1 Rat Kit to measure the percentage of oxidants, antioxidants, and inflammation. We assessed and contrasted the proportion of inflammation in the treated group with that of the control group.

Plant Materials Collection and Extraction Procedure

We collected the Eucalyptus globulus leaves from our garden in Karbala, Iraq, in February 2024. The National Herbarium. We cut and divided the plant leaves into small pieces, dried them in the air for a few days, and ground them to powder using a motorized grinder (beerfingo, SR01, Algeria). We used a weighing machine (Suyue, China) to powder the plant materials into 250 g, which we then submerged in 750 ml of distillation water in airtight containers for three days. We then filtered the powder through a fresh cotton bed and No. 2 filter paper, respectively. We stored the crude extracts at 25 °C in well-closed containers for future use.

Devices and Substances Used in the Study

To prepare Carrageenan, type IV (Sigma, USA), dissolve 0.01 g in 1 ml of distilled water. We measured the white blood cell (WBC) leucocyte, lymphocyte, monocyte, and neutrophil count by used BC-3000 hematology machines from Mindray (India). We used Rat CRP detection kits from Chondrex (USA) to measure the C-reactive protein (CRP) and a Rat TNF enzyme-linked immunosorbent assay kit (ELISA) for the TNF assay.

Data Analysis

We display each result as the mean and standard deviation. ANOVA analysis was utilized to determine the experimental data’s statistical significance, with a significance threshold of P <.05We used the Excel 2013 program as a numerical instrument to carry out this statistical study.

Results

Tables 1 and 2 displays the noteworthy variations in blood parameter values between groups. Carrageenan has been altering the levels of WBC, lymphocytes, monocytes, neutrophils, CRP, and TNF. The levels of monocytes, neutrophils, CRP, and TNF were all the same between the ECP and the control groups (P = 0.05), but there was a remarkable rise (P< 0.05) between the CRG and the C&E group in contrast to the group of control. Carrageenan has caused significant Inflammation in the CRG group. The table also clearly showed that Carrageenan impacted WBC, lymphocytes, monocytes, neutrophils, CRP, and TNF. The CRG group showed a remarkable rise in contrast to the control and ECP groups. This result aligns with numerous previous studies^22,23. This is due to the inflammation induced by carrageenan in the experiment animals^24,25, based on the findings of previous studies^22,23,25. Additionally, Table 1 demonstrated that there was a considerable rise in the ECP group’s WBC count as a result of ECP (P< 0.05) in contrast to the control group and a significant decrease (P< 0.05) compared to the CRG and C&E groups. In other words, the ECP reduced the count of WBC in the C&E group in contrast to the CRG group and caused a slight increase in the ECP group compared to the control group. The effect of ECP on lymphocyte count increased in the ECP group as shown in Table 1, with a significant increase (P< 0.05) in contrast to the control group and a significant decrease (P< 0.05) compared to the CRG and C&E groups. In addition, there are no significant differences. (P = 0.05) There is a significant difference (P = 0.05) in monocyte counts between the ECP and control groups. Table 2 showed a significant increase (P< 0.05) in the level of CRP in the serum of rats belonging to the CRG group compared to the all-experimental group. There are no significant differences (P = 0.05) between the ECP and control groups. These results are consistent with Study²⁶. Finally, Table 2 shows a descending order of TNF levels in rats’ serum among the experimental groups. The highest level (243.50 ± 18.59 Pg/mL) was recorded by Up, with highly significant differences (P< 0.05) compared to all other experimental groups, including the control group. It was followed by the treatment group that was given CRG for fifteen days, followed by the treatment period with ECP for fifteen days (186.00 ± 3.85 Pg/mL), then the ECP and the control groups (133.50 ± 3.51 Pg/mL) and (127.83 ± 8.33 Pg/mL), respectively, between which there were no notable variations (P = 0.05).

Discussion

The Effect of Carrageenan on the Blood Parameters Levels

According to experimental data in Tables 1 and 2, CRG changes the microbiota, stimulates the release of pro-inflammatory cytokines, activates innate immunity pathways, and initiates or intensifies the inflammatory response²⁷.

In general, WBC count increases in many inflammation conditions²⁸, such COVID-19²⁹, Parkinson’s disease³⁰, rheumatism, lymphoid inflammation³¹, liver diseases³², and  urinary tract inflammation³³, induced colitis³⁴. In this study, increases of peripheral Leucocytes, that suggest because of carrageenan²⁴, by stimulated macrophages and the release of IL-1β that have a major part in the inflammatory reaction³⁵. CRG can elicit an acute inflammatory response with polymorphonuclear neutrophils infiltration and exudate increasing in cells³⁶.

Carrageenan stimulated the production of cytokines/chemokines by neutrophils or macrophages³⁷, resulting tissue damage³⁸, and this leads to estimate the extent of damage caused by carrageenan³⁹. Increase level of C-reactive protein (CRP) in the CRG group⁴⁰.

Moreover, CRG reported to affect the oxidative burst activity in neutrophils. The oxidative burst is a crucial process by which neutrophils eliminate ingested pathogens by the formation of reactive oxygen species (ROS)⁴¹.

It seems that the mechanism responsible for this impact is the activation of certain signaling pathways linked to inflammation. CRG attaches to immune cells’ toll-like receptors (TLRs)⁴², setting off a series of events that lead to increased neutrophil synthesis and recruitment. This mechanism is necessary to create a successful immune response, but it can also result in excessive inflammation if it is not control appropriately⁴³. That demonstrate of induced inflammation⁴⁴, as the best-known inflammatory marker in an oncological situation, CRP, represents an inflammatory agent⁴⁵.

In addition to the WBC, lymphocyte, monocyte, neutrophils, and CRP, the significant increase in the levels of TNF as inflammatory mediator in the CRG group it is consider another evidence of inflammation in this group^34,46.  One of the most effective medications for the management of autoimmune and chronic inflammatory diseases is a biologic that neutralizes TNF, a key cytokine in inflammatory responses⁴⁷

Inflammatory reactions can be triggered directly through the production of inflammatory genes or indirectly through the induction of cell death⁴⁷.

Consequently, it shown that in a range of animal models of TNF-induced inflammatory diseases, genetically guided cell death may be able to reverse the inflammatory phenotype⁴⁸. Cells do not always react to TNF by dying. In order to shield the organism from possible harm, protective brakes, also known as cell death checkpoints, often actively inhibit TNF cytotoxicity, Although TNF can cause cell death, this reaction only happens when one of the checkpoints for cell death deactivated⁴⁹.

The effect of ECP on WBC count, Lymphocytes, Monocytes and Neutrophils

In this study we investigation to determine the ant-inflammatory of aqueous extract of Eucalyptus globulus (ECP) by the inflammatory indices. The data of this study in the Table 1, it is attributed to the presence of active substances in the ECP that have a protective role against inflammatory factors caused by CRG^50,51. Most cases of inflammation are accompanied by a significant increase in the of WBC count⁵², This is because these cells have primary functions in eliminating foreign bodies such as bacteria⁵³, cancer cells ⁵⁴, and cell debris⁵⁵. ECP has antibacterial⁵⁶, anti-inflammatory properties⁵⁷, therefore played a major role in reducing inflammation caused by the effect of CRG in the C&E group.

Lymphocytes and monocytes are types of WBC with Polymorphonuclear, Lymphocytes play a major role in correlating at a cellular level with the accumulation in lymphoid tissues⁵⁸. As an illustration in Table 1, the evidence for clonal expansion in innate lymphocytes—which has largely been observed in cytotoxic natural killer (NK) cells responding to CMV infection is gathered, in response to this viral infection, NK cells in particular go through clonal growth in a manner specific to each antigen on their route to developing memory characteristics⁵⁹.

Aside from the fact that macrophages are the most prevalent immune cell type in adipose tissue, other immune cells, such as B cells, are also present and play significant roles in regulating adipose tissue inflammation. These factors explain the immune system’s involvement in obesity-induced adipose tissue inflammation and the ensuing metabolic dysfunction⁶⁰.

Since it proven in this study that CRG caused inflammation in experimental animals in the CRG and C&E groups, which was evident through the increase in the inflammatory indicators mentioned above, including WBC, monocytes and neutrophils as clear in the data of the Table 1, ECP make to maintain the integrity of the cells exposed to inflammation and reduce the indicators of inflammation caused by CRG. This was evident in the decrease in the levels of these cells of all types in the C&E and ECP groups, while the rest of the levels of these cells were high in the CRG group. This confirms the presence of anti-inflammatory substances in the aqueous extract of Eucalyptus leaves^61,62.

CRG-induced inflammation allows for the evaluation of cell migration, the involvement of cytokines and chemical mediators, and protein leakage. While ECP did decrease the overall leukocyte count in the peripheral blood, it is worth noting that neutrophils comprised the largest proportion of leukocytes in the group treated with CRG. We can see that this ECP can lower the amounts of inflammatory substances made by immune cells because the ratio of neutrophils went down and the levels of monocytes, lymphocytes, CRP, and TNF went down in the blood of the test animals⁶³.

The effect of ECP on CRP

The liver produces CRP in response to inflammatory cytokines⁶⁴, CRP levels rise when the liver is exposed to oxidative stress⁶⁵, indicating systemic inflammation and linked to chronic diseases like cardiovascular disorders and autoimmune conditions⁶⁶. Since CRG enhances levels of inflammation that in turn increase CRP levels, which is what was observed in the CRG group, ECP reduces the level of inflammation that in turn decrease CRP levels (Table 2), which is what we observed in the C&E group⁶⁷. Eucalyptus globulus leaves’ essential oils, particularly eucalyptol, have anti-inflammatory properties, preventing pro-inflammatory cytokine synthesis and encouraging anti-inflammatory mediator release, potentially lowering CRP levels⁶⁸. Eucalyptus globulus extracts enhance antioxidant activity, reduce oxidative stress, lower inflammatory responses, and reduce CRP levels⁶⁹. Essential oils are known to possess significant antioxidant action, which is related to phenolic groups like thymol, carvacrol, and maybe 1,8 cineole⁷⁰.

The Effect of ECP on TNF

The cytokine TNF, which is generated by lymphocytes (T-cell)⁷¹, and monocytes (macrophages)⁷², plays a crucial role in immune cell control and systemic inflammation⁷³, triggering a range of immunological responses⁷⁴. Although cytokines are not the only routes or substances that cause inflammation⁷⁵, TNF plays a critical role as a mediator in the start and spread of inflammatory reactions⁷⁶. The CRG showed higher TNF levels due to abnormally increased lymphocytes and monocytes, which are key secretors of TNF. Conversely, a decrease these cells in C&E group resulted in a decrease in TNF levels in this group (Table 2). The antioxidant property of Eucalyptus globulus  leaves is due to the following monoterpenes⁷⁷: α pinene, β pinene, limonene, β myrcene, sabinene and terpinolene have antioxidant properties⁵⁶.

Table 1: Evaluation of Blood parameters in different experimental configurations

Groups		WBC (x103/μL)	Lymphocytes (x103/μL)	Monocytes (x103/μL)	Neutrophils (x103/μL)
Control	04.00 ± 0.00		07.08 ± 0.15	00.25 ± 0.03	01.55 ± 0.19
ECP	04.85 ± 0.31 a		07.36 ± 0.20 a	00.20 ± 0.02	01.63 ± 0.09
CRG	13.50 ± 0.98 ab		11.10 ± 0.13 ab	01.97 ± 0.08 ab	04.77 ± 0.12 ab
C&E	08.83 ± 0.75 ab		09.42 ± 0.09  ab	01.16 ± 0.08 ab	03.79 ± 0.09 ab

 

The values are mean ± SD value, n = six in each group, ^a show the difference in statistics. With a control group, ^b statistical disparity according to illness group, (P < 0.05). ECP: Group of aqueous extract of Eucalyptus globulus leaves, CRG: group of Carrageenan, C&E: group of carrageenan then aqueous extract of Eucalyptus globulus leaves.

Table 2: Evaluation of Serum parameters in different experimental configurations

Groups	CRP Mg/dl	TNF Pg/mL
Control	0.10 ± 0.10	127.83 ± 8.33
ECP	0.12 ± 0.10	133.50 ± 3.51
CRG	0.60 ± 0.33 ab	243.50 ± 18.59 ab
C&E	0.37 ± 0.21 ab	186.00 ± 3.85 ab

 

The values are mean ± SD value, n = six in each group, ^a show the difference in statistics. With a control group, ^b statistical disparity according to illness group, (P < 0.05). ECP: Group of aqueous extract of Eucalyptus globulus leaves, CRG: group of Carrageenan, C&E: group of carrageenan then aqueous extract of Eucalyptus globulus leaves.

Conclusion

A simple method similar to Iraqi folk medicine was used to get antioxidant and anti-inflammatory compounds from Eucalyptus globulus leaves. The study found that rats exposed to carrageenan for 30 and 15 days had much lower levels of inflammation than rats in the control group.

The study identifies parameters like WBC, lymphocytes, monocytes, neutrophils, CRP, and TNF to establish relationships between them. It recommends research on leaf extract’s effects on the respiratory system, bronchi, and bronchioles.

Acknowledgement

I would like to thank the Faculty of Nursing, Faculty of pharmacology / University of Kerbala, Iraq.

Funding Source

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

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required

Author’s contribution

Mustafa K. Mushatet: Conceptualization, Methodology, Writing – Original Draft.

Asaad Abbas khalaf: Analysis, Writing – Review and Editing.

Doaa A. Hamad: Visualization, Supervision, Project Administration.

Thikra Abd Jary: Data Collection, Funding Acquisition, Resources, Supervision

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