Acute and Subacute Toxicity Study of Essential Oil of Cymbopogon Martini in Mice

Background Local Ethiopians regularly use Cymbopogon martini for cosmetic purposes. The plant's safety, however, is not supported by any solid facts. This investigation aimed to evaluate the acute and subacute toxicities of C. martini essential oil in mice. Methods The essential oil was analyzed using GC-MS. The approach outlined by Chinedu et al., 2013 has been used to calculate the median lethal dose. According to organization for economic cooperation and development (OECD) 407 standard, a 28-day repeated dose oral toxicity study was carried out on female mice. Three groups of ten experimental mice each were distributed at random. Group I received the same saline volume and was considered the control. Groups II and III were treated with doses of C. martini of 500 mg/kg and 1000 mg/kg, respectively, of body weight. Hematological and biochemical markers were assessed. The liver and kidney were taken out after the sacrifice using sodium pentobarbital for pathological analysis. Results Geraniol (40.89%) was the predominant component in the essential oil composition of C. martini with cyclofenchene (13.91%), myrcene (9.34%), 2, 4, 6, octatriene, 2, 6, dimethyl (8.20%), and ocimene (5.93%) being present in small amounts. The LD50 of C. martini essential oil was discovered to be greater than 5000 mg/kg body weight. During a 4-week follow-up period, mice treated with C. martini, the essential oil, at doses of 500 mg/kg or 1000 mg/kg body weight showed no evidence of toxicity or mortality. Biochemical and hematological parameters were not significantly altered in mice treated with the essential oil of C. martini compared with the control group. Histopathological evaluation of the liver and kidney did not exhibit any adverse results. Conclusions The essential oil of C. martini from Ethiopia is considered relatively safe and nontoxic.


Introduction
Herbal remedies have been used extensively to cure illnesses and as a source of contemporary medications [1]. Around 80% of the world's population heavily relies on herbal medicine, and its use is expanding globally [2,3]. Due to the various limitations of contemporary medications, attention is being paid to herbal remedies.
About 70% of the active components used today or in clinical trials are derived from natural sources, making them a rich source of biodiversity for identifying pharmaceutical lead compounds and developing new drugs [4,5]. Not only do pharmacodynamic and pharmacokinetic parameters need to be known but also the drug's safety [6].
Te primary reasons for discontinuing medications from clinical studies are hepatotoxicity and nephrotoxicity [7,8]. Aminotransferases, alkaline phosphatase, sorbitol dehydrogenase, glutamate dehydrogenase, gamma-glutamyltransferase, total bilirubin, total bile acids, and 5′nucleotidase are some of the common biomarkers used to assess the safety of drugs for hepatotoxicity [9].
A signifcant volume of blood in circulation (approximately 25% of cardiac output) may be related to the kidneys' high susceptibility to active substances or their metabolites.
More signifcantly, the concentration of toxins in the blood in the renal tubular fuid is linked to the role of nephrons in the processes of urine generation [7]. Blood urea nitrogen and serum creatinine in experimental animals have been suggested as markers for nephrotoxicity assessment [10].
90% of the population in Ethiopia relies on traditional medicine [11]. Local Ethiopians utilize Cymbopogon martini for cosmetic purposes.
C. martini belongs to the family of Poaceae. It is commonly called palmarosa which has historically been used as a culinary ingredient and for fumigation. Essential oils from C. martini have been shown to have cytotoxic, antibacterial, and antioxidant efects [12].
Despite being widely regarded as safe medications with a high therapeutic index, medicinal plants naturally contain metabolites that have the potential to harm humans, animals, and the environment [13].
C. martini ethnopharmacology has been documented. However, studies on safety profles are lacking. Tis study aimed to examine the safety of C. martini essential oil in mice.

Plant Material Collection and Identifcation.
Fresh leaves of C. martini were collected from the botanical garden of the Debre Berhan University-Ankober Herbal Medicine Research Center at Chefa Kebele in the North Shewa zone of the Amhara region, Ethiopia. Te Botanist identifed and authenticated the leaves in the Botanical Garden of Debre Berhan University, Ethiopia. For future use, voucher specimen No. CM 2012 was deposited in the herbarium of Addis Ababa University, Addis Ababa, Ethiopia.

Method of Extraction.
Te leaves were steam distilled to produce C. martini essential oil. In order to ensure that the steam was delivered properly, the plant materials were placed into the still and stacked in layers. Tis approach was centered on putting the fragrant plant via steam. Te plant's volatile substances were collected by steam, which washed them away after passing through a serpentine that was chilled with cold water. A separator funnel was used to separate the distillate which contained a mixture of hydrosol vapor and essential oil. After drying over sodium sulfate, the essential oil was put into clean brown glass bottles to be kept until required [14].

Phytochemical
Analysis. GC-MS determined the phytochemical analysis of the essential oil composition of C. martini according to the method described previously [15,16].

Experimental Animals.
In this investigation, threemonth-old mice were employed. Tey were obtained from the pharmacology department of Addis Ababa University, Addis Ababa, Ethiopia. Mice ranged in weight from 25 to 35 grams. Tey were given regular pellets to eat, and unlimited tap water was available. Te procedure involved keeping mice in a sanitary cage at ambient temperature [17]. Te institutional review board of Debre Berhan University in Debre Berhan, Ethiopia, granted ethical approval for the use of experimental mice. Mice were treated and handled in compliance with ethical standards adopted globally.

Determination of the Median Lethal Dose (LD 50 ).
A limit dose of 5000 mg/kg body weight was used to determine the LD 50 of the essential oil of C. martini. Te LD 50 was calculated according to the new approach outlined by Chinedu et al., 2013 [18]. Four female mice were employed in the study's early phase. Tere were a total of four groups, each with one mouse. C. martini was administered in doses of 10 mg/kg and 100 mg/kg body weight (b.w) to Groups I and II, respectively. Groups III and IV were treated with 300 mg/ kg and 600 mg/kg b.w., respectively. Mice were observed for an hour after the administration, then for ten minutes every two hours for the next 24 hours. In the second step, three female mice were used. Tey were randomly allocated into three groups. Te doses given to Groups I and II were 1000 mg/kg and 1500 mg/kg b.w. Group III received treatment with 2000 mg/kg b.w. of C. martini essential oil. After treatment, animals were observed.
In the last round of LD 50 determination, three female mice were also used. Te placement of the animals into three groups of one was random. Te essential oil of C. martini was administered at doses of 3000 mg/kg, 4000 mg/kg, and 5000 mg/kg b.w. to Groups I, II, and III, respectively. Following administration, meticulous observation was carried out for one hour, then for ten minutes every two hours for the following twenty-four hours. Two mice were used in a confrmatory test at 5000 mg/kg b.w.

Sub Acute Toxicity Study.
According to the Organization for Economic Cooperation and Development's (OECD) 407 standard, the repeated dose 28-day oral toxicity study was carried out on female mice [19]. Since the responses are usually similar, the available literature suggests that any sex is used for subacute toxicity. When diferences do exist, however, female mice are generally more sensitive [20,21]. Tree groups of ten experimental mice were randomly assigned into three groups of 10 animals each. Te exact amount of saline was administered daily to Group I as the control group. Te doses of 500 mg/kg and 1000 mg/kg b.w. of C. martini essential oil were given once daily to Groups II and III, respectively. Tese doses were established based on the fndings of acute toxicity and the limit dose concept described by the European Chemical Agency in 2022 [22].
Te animals were observed daily for toxicity, death, abnormal behavior, and other signs throughout the trial.
Clinical observations were gathered and recorded prior to the test chemical's distribution and each day after. Te number of mice in each cage, their weights, and how much food they ate each week were recorded. On the 28 th day, the only thing mice were allowed to drink was water. Te next morning, mice were weighed, and blood was taken from a common carotid artery for hematological and biochemical examinations. Sodium pentobarbital was used to fnish the experiment by killing all mice from both experimental groups.
Blood was extracted into a precalibrated tube containing EDTA for hematological assays. Hematological parameters including mean cell hemoglobin (MCH), mean cell volume (MCV), mean cell hemoglobin concentration (MCHC), hemoglobin (HB), hematocrit (HCT), and platelet count (PC) were analyzed using a hematological analyzer. For biochemical examination, the serum was separated, and samples were centrifuged at 3000 rpm for 10 minutes. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphate (ALP), total cholesterol (TC), blood urea nitrogen (BUN), and creatinine (CREA) were measured using an automated chemical analyzer. All animals had their livers and kidneys removed for gross anatomical and histological examinations. Formalin bufered at 10% was used to preserve the organs. Hematoxylin and eosin were used to stain the tissues, which were subsequently sliced to a thickness of 5 μm, placed on glass slides, and examined under a light microscope. Photomicro-graphs were taken at 4 and 40 times [23].

Statistical Analysis.
Results are presented in terms of the mean and standard errors of the mean (SEM). A one-way analysis of variance (ANOVA) and the Bonferroni multiple comparison tests were used to assess the data. At P < 0.05, diferences were deemed statistically signifcant.

Results
Te mice used in the experiment were all healthy. In the acute toxicity investigation, 12 female mice in total were employed. In subacute toxicity investigations, 30 female mice were used, and every animal followed the prescribed treatment plan.

Efect on Acute Toxicity.
Mice receiving oral doses of the essential oils of C. martini up to 5000 mg/kg did not exhibit treatment-related mortality, and no symptoms of toxicity or morbidity were noticed. Te LD 50 of C. martini essential oils was found to be greater than 5000 mg/kg b.w.

Efect on Subacute
Toxicity. During a 4-week follow-up period, female mice treated with 500 mg/kg and 1000 mg/kg b.w. of the essential oils of C. martini showed no evidence of toxicity or death due to the treatment. As can be observed in Table 1, there were no appreciable variations in body weight between the mice treated with C. martini essential oils at doses of 500 mg/kg and 1000 mg/kg b.w. and the control group.
When compared to mice that received normal saline, mice were treated with essential oils of C. martini at doses of 500 mg/kg and 1000 mg/kg b.w. did not consume food in signifcantly diferent ways, as shown in Table 2.
No appreciable variations were seen between mice given C. martini essential oil and the control group's hematological parameters, which were all within the normal range ( Table 3).
As shown in Table 4, compared to the control group, subacute administration of C. martini essential oil in mice at doses of 500 mg/kg and 1000 mg/kg b.w. exhibited no signifcant changes in all biochemical markers assessed.

Efect on the Histology of the Liver and Kidney.
Te liver and kidneys of the control mice were healthy, as seen in Figure 1. Both the cellular topologies and the integrity of the cells were unafected when comparing the two tissues in the experimental groups to their respective controls. Figures 2  and 3 show that neither the kidneys nor the liver of the experimental animals showed any histological abnormalities.

Discussion
Cosmetics sectors all utilize essential oils often. Te toxicity of these products is becoming a topic of growing study [24]. Te primary reasons for discontinuing efective medications from clinical studies are hepatotoxicity and nephrotoxicity [7,8].
Up to a dose of 5000 mg/kg b.w., mice receiving oral administration of C. martini essential oils did not exhibit treatment-related mortality, and no symptoms of toxicity or morbidity were noticed. Te results of the investigation on acute toxicity revealed that the LD 50 of C. martini essential oil was more than 5000 mg/kg b.w. Based on this fnding, the general harmonized system (GHS) of the United Nations has classifed the essential oil of C. martini as belonging to the class of chemicals with a low acute oral toxicity, or the 5 th class of toxicity [26].
In line with the fndings of Andrade et al., subacute treatment of essential C. martini at 500 mg/kg and 1000 mg/ kg b.w had no discernible impact on the body weight and food intake of mice [25].       According to this study's evaluation, RBC, WBC, PC, HB, MCH, MCV, and MCHC did not substantially alter in mice following treatment with C. martini essential oil. Tis discovery has not yet been reported so far.
Te release of cytosolic enzymes into the circulation happens in conjunction with plasma membrane damage from hepatic cells under various pathological circumstances. After exposure to harmful agents, the aminotransferases ALT and AST are utilized to diagnose liver damage [27]. Following therapy with C. martini essential oil, these parameters in mice did not alter appreciably. In this instance, Andrade et al. reported a similar outcome [25].

Conclusions
Te essential oil of C. martini was shown to be neither acutely poisonous nor sub acutely harmful in mice. Using this oil for cosmetic reasons is safe, according to the information from the present study. Finally, it can be claimed that the essential oil of Ethiopian C. martini is nontoxic. Future research should be done to corroborate the current fndings, and studies on long-term toxicity should be utilized to determine how safe it is for humans.

ALT:
Alanine transaminase ALP: Alkaline phosphatase AST: Aspartate transaminase BUN: Blood urea nitrogen LD 50 : Median lethal dose MCH: Mean cell hemoglobin MCHC: Mean cell hemoglobin concentration MCV: Mean cell volume OECD: Organization for Economic Cooperation and Development PC: Platelet count RBC: Red blood cell count WBC: White blood cell count.

Data Availability
Te majority of the data are included in this manuscript. Further data can be found from the corresponding author based on reasonable request.

Ethical Approval
Te study was approved by the Institutional Review Board of Debre Berhan University, Ethiopia, before the commencement of the study. A letter of clearance was obtained. In addition, all possible steps were taken to avoid animal sufering during the experiment.

Conflicts of Interest
Te authors declare that they have no conficts of interest in this work.