Evaluating the Efficacy of Effect of Eucalyptus camaldulensis Leaf Extracts Grown in Sandy Habitats on Histological and Gene Expression Changes in the Liver of Mice Infected with Plasmodium chabaudi

Plants function as living chemical factories, producing a diverse variety of secondary metabolites (SMs) that serve as essential components of many commercial pharmaceutical drugs (Hassan, 2012; Kandpal et al., 2023). Environmental conditions significantly affect the biosynthesis and variations of secondary metabolites in plants (Verma and Shukla, 2015). Different geographical and ecological harvest sites result in different chemical compositions of the same plants (Camara et al., 2021).
       
An important approach to fight against parasites is to promote the exploration of new antimalarial compounds from different origins, especially from traditional medicinal plants. The focus on research related to medicinal plants is increasing worldwide (Taek et al., 2018; Dakhil et al., 2021; Habibi et al., 2022; Sharma et al., 2021).
       
Medicinal plants have been used since ancient times to treat malaria and provide a good source for new antimalarial drugs (Dakhil et al., 2021; Al-Jawdah et al., 2022). According to the World Health Organization, malaria is still a serious disease with high morbidity and mortality, affecting 249 million people (World Health Organization, 2022).
       
According to a recent study by Anigboro et al., (2020), Eucalyptus camaldulensis can protect Plasmodium berghei-infected mice from malaria-induced liver and kidney function abnormalities. Furthermore, in mice infected with P. Chabaudi, our team documented the immunomodulatory and antioxidant properties of E. camaldulensis (Aljawdah et al., 2022).
       
Changes in chemical composition can affect the quality and bioactivity of plants (Tian et al., 2016). Several studies have investigated this topic or have recorded changes in the activity and composition of the same species in different regions. This study aims to fully clarify the differences in the chemical composition of E. camaldulensis collected in two distinct habitats (mud and sand) and to evaluate the antiparasitic activity of P. chabaudi.

Extraction of E. camaldulensis (ECE)
 
The leaves of the plant were collected from sandy habitats in Al-Qassim, Saudi Arabia on 6/15/2022. The plant was identified by a herbalist at King Saud University. Before turning into powder, the leaves were dried in the open air. Then, 70% methanol was used to extract the constituent parts of the leaves (Lubbad et al., 2015). For further studies, the ECE of sand samples (ECES) was diluted with distilled water.
 
Chemical analysis of plant extract (ECES)
 
In continuation of our previous studies (Dkhil et al., 2023 and Aljawdah et al., 2022) on the chemical analysis of plant extract (ECE), we used an optical spectrometer with infrared spectroscopy of transformation of Fourier (FT-IR) Nicolet 6700 to analyze the extract of plants that inhabit sand and mud habitats. In addition, antioxidant activity and phenolic and flavonoid compounds were evaluated. In this study, we used gas chromatography mass spectrometry to analyze the ECE of sand habitats (ECES) to verify the most significant differences between the chemical compounds in the E. camaldulensis extract. in different habitats (muddy and sandy) and evaluation of its antimalarial activity In the laboratories of the College of Science, King Saud University, during the period from 09/04/2023 to 01/01/2024.
 
Analysis for ECES use gas chromatography-mass spectrometry (GC-MS)
 
Kanthal et al., (2014) developed the protocol for the phytochemical analysis of ECES. The analysis was conducted using a GC-MS Triple Quad 7000D system from Agilent Technologies, USA, for gas chromatography coupled with mass spectrometry (GC-MS).
 
Infection and animals
 
Female C57BL/6 mice, aged 9–11 weeks, were provided with normal feed and water during the breeding period. The Research Ethics Committee for the Care of Laboratory Animals of King Saud University gave approval for the procedures (approval number: KSU-Se-21-77).
 
Mice infection and treatment
 
Introduction of P. chabaudi parasites into experimental mice was performed as reported in a previous study (Maksoud et al., 2019). The injection dose (106 parasitized erythrocytes) was determined using a Neubauer chamber and P. chabaudi parasitized erythrocytes were administered intraperitoneally to mice (Wunderlich et al., 1982). Blood smears were made from the mouse tails using Giemsa staining to assess the parasitemia that P. chabaudi induced.
       
Based on our recently published study (Aljawdah et  al., 2022), we determined the suitable dose and divided the mice into the following groups.
       
Group 1 represents the non-infected control group without treatment; Group 2 is the uninfected treated group that received 100 mg/kg ECES; Group 3 consists of the infected group that received no treatment; Group 4 includes the infected treated group that received 100 mg/kg ECES; and group 5 includes the infected group treated with chloroquine. Over the course of four days, Group 5 received 10 mg/kg of chloroquine phosphate (CQ) (Sigma-Aldrich, St. Louis, USA) (Abay et al., 2015).
 
Sample collection
 
Mice were sacrificed on day 7 postinfection and the livers were removed and minced. Some sections were stored at “80^oC for RNA extraction for subsequent gene expression studies and other sections were stored in neutral buffered formalin for histological examination.
 
Histopathology study
 
Liver samples were fixed in 10% neutral formalin and embedded in paraffin. Samples were cut into 5 μm thick sections and stained with hematoxylin and eosin (Drury and Wallington, 1980). Liver histology was performed according to the guidelines of Ishak et al., (1995). In summary, the quantitative measurements were based on the evaluation of liver sections at different magnifications. The quantity measure assigns scores from 1-3, 4-8, 9-12 and 13-18 for minimal, mild, moderate and severe liver damage, respectively, according to the severity of the injuries.
 
Gene expression
 
TRIzol (QIAGEN, Hilden, Germany) was used to extract hepatic RNA for quantitative real-time polymerase chain reaction (RT-qPCR). DNase (Applied Biosystems, Darmstadt, Germany) prepared the RNA and a reverse transcription kit (QIAGEN, Hilden, Germany) converted the samples to cDNA. The ABI Prism 7500HT Sequence Detection System (Applied Biosystems, Darmstadt, Germany) and the SYBR Green PCR Master Mix (QIAGEN, Hilden, Germany) were used for the PCR analysis. The levels of interleukin-IL-1β, IL-6 and IFN-γ were measured using primers from Macrogen Inc. (Seoul, South Korea). We followed the guidelines set out by Dkhil et al., (2019) for doing PCR. We calculated the fold change in mRNA expression using the 2-DDCT technique (Livak and Schmittgen, 2001).
 
Statistical analysis
 
Groups were statistically compared using SPSS version 17.0 and Duncan’s test. Significance was assessed using univariate analysis of variance. Standard deviations and means are presented for all values. All p values   were two-sided and statistical analyses were considered significant when p<0.05.

Phytochemical analysis performed by GC-mass revealed that ECES consisted of 33 phytochemicals (Table 1), each characterized by different peak areas and retention times. The compounds benzene, 1,2,4,5-tetramethyl-,hexadecanoic acid, ethyl ester (palmitic acid), methyl stearate, hexadecanoic acid and 2-hydroxy-1-(hydroxymethyl) ethyl ester are shown in Fig (1) with pointed peaks.




       
The results of the study showed tissue changes in the liver of mice. Significant liver damage characterized by the presence of inflammatory cells. Mice infected with P. chabaudi showed changes, while healthy controls and ECES-administered groups maintained normal liver architecture. Hepatic injury was attenuated and liver score decreased in mice treated with ECES and CQ, which correlated with reduced infiltration of inflammatory cells (Fig 2). The Ishak score revealed that the liver activity index of the infected group varied between 13 and 15. The administration of ECES to mice resulted in a reduction of the liver index to 5-8, while the chloroquine treatment gave a score of 6-8 (Fig 3).

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The levels of pro-inflammatory cytokines IL-1β, IL-6 and IFN-γ increased significantly (p<0.05) after P. chabaudi infection, suggesting the inflammation of the hepatic tissue (Fig 4). The preventive effect of ECES and CQ on the inflammatory events associated with the development of malaria is proven by the significant reduction of this inflammatory response after treatment with ECES or CQ.

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E. camaldulensis contains phenolic compounds and flavonoids, which are bioactive. These compounds may contribute to the antimalarial effect and the reduction of parasitemia that occurs during the treatment of infected animals. Anigboro et al., (2020) recently reported that the aqueous extract of E. camaldulensis leaves may be effective against P. berghei significantly reduces the metabolic defects caused by malaria in the liver and kidney, as well as serum electrolytes, thanks to the active phytochemical components in the extract.
       
GC mass analysis revealed that ECES contains several active compounds, including hexadecenoic acid and its methyl ester (palmitic acid), known for their antiseptic and anti-inflammatory intestinal properties (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002). Additionally, methyl stearate is employed in the management of neurological and cardiac disorders (Chen et al., 2020). Aissaoui et al., (2019) reported the antibacterial, antifungal, antioxidant and anticancer properties of 2,4-di-tert-butylphenol. Abbaszadeh et al., (2014) reported the potential activity of eugenol as antifungal, antibacterial, antiviral and antiparasitic. Phthalic acid-cyclobutyl nonyl ester (Kok et al., 2008), methyl tetradecanoate (Mallu et al., 2019), epizonarene (Mohammed et al., 2020), benzenamine, N,N,3-trimethyl- (Yang et al., 2021) and piperidine (Guo et al., 2022) have been documented for their therapeutic activity as antiviral and anti-cancer agents.
       
A limited number of studies have documented changes in species composition and/or activity in different geographic regions. Few studies have examined the antimalarial properties of natural resources in the region, although Saudi Arabia has many different plants and people have long used indigenous ethnic medicinal plants (Mothana et al., 2014; Al-Asmari et al., 2017; Aati et al., 2019). This study explores the antimalarial efficacy of ECE in two geographically distinct environments: sandy and muddy environments.
       
The difference in chemical constituents, their concentration and antimalarial efficacy between the ECEM and ECES samples can be attributed to the differences in the collection sites. ECEM derived from well-watered soil, while ECES derived from sand, which is less watered and therefore less favorable for optimal plant growth (Traore et al., 2013).
      
P. chabaudi infection induces an inflammatory response in the liver of mice. This response represents changes in the hepatic architecture resulting from the parasites consuming hemoglobin, which then cause the release of heme. This process leads to oxidative damage and histological changes in the liver (Kumar and Bandyopadhyay, 2005). The histological and biochemical results are consistent with recent studies (Lubbad et al., 2015; Dkhil et al., 2019) that examined the hepatoprotective effects of plant extracts related to P. chabaudi infection. The investigation showed that the ECES treatment improved the health of the host mice. Previous studies on other medicinal plants have shown similar results (Dkhil et al., 2021). The results show that the hepatic inflammation caused by P. chabaudi is associated with an inflammatory response in the liver. The results show that ECES effectively eradicates Plasmodium parasites in mouse models and has anti-inflammatory properties that maintain liver function.
       
The improvement in the structure of liver tissue indicates the potential protective effects of E. camaldulensis after malaria-induced infection, as shown by our results. In addition, E. camaldulensis reduced liver inflammation and restored the balance between antioxidants and antioxidants. The results show that E. Camaldulensis can act as a hepatoprotective agent in the context of malaria. Further study is needed to elucidate the mechanisms underlying the response liver of E. camaldulensis and P. chabaudi.
       
P. chabaudi infection can result in significant changes in the inflammatory cytokines IFN-, IL-1β and IL-6. There are early and powerful effector mechanisms mediated by cytokines that clear or eliminate parasite-infected cells. These include acquired and innate immune responses (Gowda and Wu, 2018). Furthermore, increased levels of IL-6 and IFN-γ are associated with hyperparasitemia (Angulo and Fresno, 2002). Hepatocytes, activated leukocytes and inflamed macrophages produce more cytokines and proinflammatory enzymes.in case of inflammation, which causes the initiation of the innate immune response (Wunderlich et al., 2014). IL-1 and IL-6 are proinflammatory cytokines that play a crucial role in inflammation and immunity, with elevated levels observed during Plasmodium infection (Dkhil et al., 2021). By decreasing cytokine production and altering oxidative changes in the liver, ECES treatment significantly slowed down the onset of inflammatory responses after P. chabaudi infection. Initially, we attributed the reduced inflammatory responses of ECES to the lower number of parasitized erythrocytes. Many studies support our findings that medicinal plants can reduce parasitemia and protect the liver from inflammation (Wunderlich et al., 2014; Dkhil et al., 2019; Dkhil et al., 2021).

The present study was supported by Researchers Supporting Project number (RSP2024R3), King Saud University, Riyadh, Saudi Arabia
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.