Acute and Subchronic Toxicity Assessment of Conventional Soxhlet Cymbopogon citratus Leaves Extracts in Sprague–Dawley Rats

Background In Ghana, Cymbopogon citratus leaves together with guava, pawpaw, and lime are processed into a decoction to treat fever. To encourage its usage, preclinical validation of the safety profile of the plant is required. The acute and subchronic toxicities of the conventional Soxhlet ethanolic Cymbopogon citratus leaves extract in Sprague–Dawley rats were investigated. Methods Pulverized Cymbopogon citratus leaves were extracted with 98% ethanol using the conventional Soxhlet extraction (CSE) method and dried. In the acute toxicity study, a single dose of 5000 mg/kg body weight was administered to six female Sprague–Dawley rats and 1 ml/100 g body weight normal saline to control (6) once, and signs of toxicity were observed every hour for the first 12 hr, 24 hr, and 48 hr through to 14 days. In the subchronic study, the treatment groups were administered 200 mg/kg, 600 mg/kg, and 1200 mg/kg, respectively, of the CSE C. citratus leaves extract for six weeks. Analyses were conducted on the blood, urine, and serum samples of the rats. Histopathological examination of the liver, heart, kidney, spleen, and lungs was carried out at termination. Analysis of variance (ANOVA) was performed to determine statistically significant differences between the test and control rats at P  <  0.05. Results The results revealed that there were no statistically significant differences (p  >  0.05) in the urinalysis and haematological analysis between control and test rats over the treatment period. Similarly, CSE C. citratus leaves extract did not induce any significant biochemical changes in the treatment group; however, there was a weight loss effect on the treated rats. There were no noticeable morphological changes in the heart, liver, spleen, lung, and kidney of the test rats compared to the control. Conclusion CSE ethanolic C. citratus leaves extract has a weight loss effect, and long-term administration of the extract may not cause any organ-specific toxicity to the consumers.


Introduction
Nowadays, pharmaceutical companies are extensively involved in the use of medicinal plants as antiparasitic, insecticidal, cosmetic, bactericidal, and fungicidal agents due to the nontoxic nature of secondary metabolites from such plants [1,2].Herbal medicines are alternatives to orthodox drugs due to their perceived low toxic efect on biological systems, low cost, and availability.It is believed that even if the expected therapeutic efcacy is not realized, their consumption is not dangerous.However, safety issues of herbal formulations have been raised by many due to poor scientifc validation of the safety profle of these herbal medicines and reports of illness and death from their consumption [3][4][5].Hepatotoxicity and nephrotoxicity are commonly reported toxic efects of herbal medicines due to the involvement of the liver and kidney in the metabolism and excretion of drugs [5,6].Tus, a comprehensive scientifc study of the toxicity of these herbal products will provide the needed scientifc evidence to support the argument that consumption of herbal concoctions is safe in humans.Tese studies are important for public health reasons to protect the user population from possible adverse side efects.Tere is a wide biodiversity of plant species with medicinal properties including Cymbopogon citratus.
Cymbopogon citratus with the common name lemongrass is a tufted, aromatic perennial grass with numerous stif stems arising from a common rhizome.It is a tropical plant cultivated in South Asia, South and Central America, and Africa [7,8] and used as an infusion or decoction in traditional medicine [8,9].C. citratus contains essential oils rich in ketones, alcohols, esters, terpenes, and aldehydes which vary according to the geographical origin of the plant [10].It is reported to have phenolic and favonoid, myrcene, and citral compounds.Tese compounds, depending on the concentration or dosage used, could be toxic to the user.For example, a study by Nogueira et al. [11] revealed that citral at 60 mg/ kg bw.t induced maternal and embryofoeto-toxicity in rats.Also, citral induced cytotoxicity and genotoxicity in NCTC 929 mouse fbroblast cell lines [12,13].Te LD 50 of lemongrass essential oils against Stitophilus granaries is 6.92 μg/insect [14].Plants naturally produce metabolites as defensive agents against predators in their ecosystem which have the potential to harm rats, humans, and the environment at large [15].
In Ghana, C. citratus leaves are used in a decoction along with guava, pawpaw, and lime leaves to treat fever and are generally regarded as safe.To encourage its continuous usage, however, preclinical validation of the plant dose is necessary because the lethal dose (LD 50 ) of the plant is unknown.Additionally, where the plant grows and how it is cultivated, harvested, and processed may change the chemical composition of the plant.Terefore, the purpose of this investigation was to ascertain the safety profle of C. citratus conventional Soxhlet ethanolic extracts when administered to Sprague-Dawley rats on an acute and subchronic basis.

Study Setting.
Te extraction was carried out at the Department of Science Laboratory Technology of Accra Technical University, Ghana and the animal experimentation study was conducted at the Centre for Plant Medicine Research, Akwapim Mampong, Ghana, between August, 2022 and February, 2023.

Plant Material.
Fresh leaves of matured C. citratus were harvested at Taifa Burkina, in the Ga East Municipal Assembly in the Greater Accra Region of Ghana in July 2022 and authenticated by a Botanist at the Plant Development Department at the Centre for Plant Medicine Research (CPMR).A voucher specimen of CPMR5167 was kept at the herbarium.Te C. citratus leaves were cut into pieces, washed with distilled water, allowed to air dry for three weeks at room temperature, and pulverized into powder.Te powder obtained was bagged into plastic zip lock bags and stored at room temperature until extraction.Te extraction of C. citratus crude extracts was carried out by the conventional Soxhlet extraction (CSE) method as described as follows.

Conventional Soxhlet Extraction (CSE) of C. citratus
Ethanolic Extract.Te CSE technique was carried out according to the method described in [20,21] with slight modifcations.Te extraction thimble was flled with a 15 g quantity of pulverized C. citratus powder, and the flled thimble was placed within the Soxhlet apparatus.A 250 ml of 98% ethanol was measured into a round bottom fask and connected to the extractor.Each cycle of extraction was performed for 3 hours.Te temperature of the extraction corresponded to the boiling point of ethanol.After the CSE was completed, a rotary evaporator (RE-52A, E. Track Scientifc Instruments, England) was used to concentrate the crude extract.Te concentrate was transferred into a stainless plate and dried in a water bath at 70 °C.

2
Journal of Toxicology

Sample Size Calculation.
Te sample size for the study was calculated using the resource equation approach and analysis of variance (ANOVA) as the expected statistical analysis [23,24].
where DF � degrees of freedom (range between 10 and 20), k � number of groups, and n � number of rats per group.For the four groups used for this study, a sample size of range between 4 and 6 rats per group was calculated.Tus, six rats per group were used for the study.[29].Te protocols used in [27,30] were used with slight modifcations.Sixty-day-old female (nulliparous and nonpregnant) Sprague-Dawley rats were randomly divided into four groups (n � 6) and allowed to acclimatize for one week.Tey were fed with rat chow and sterilized distilled water ad libitum.Te rats were grouped into the following treatment groups, respectively: Group I: negative control (sterilized distilled water) Group II: low dose (200 mg/kg bw.t) Group III: medium dose (600 mg/kg bw.t) Group IV: high dose (1200 mg/kg bw.t) Te extracts were orally administered to the rats daily for six weeks (42 days).Te rats were monitored closely daily for any signs of toxicity.Appearance and behavioural changes were also assessed, and any abnormalities in water and food intake were recorded according to the primary observation procedure of the Irwin test [28].Te rats were weighed at baseline, 2 nd , 3 rd , 4 th , 5 th , and 6 th weeks.Blood and urine samples were collected for analysis at baseline, 3 rd , and 6 th weeks.Te liver, kidney, heart, spleen, and lungs were excised at termination for histopathological examination.
2.10.Urinalysis.Fresh, clean urine samples for the analysis were collected by holding each rat over a tabletop and manually expressing its bladder by application of gentle transabdominal pressure and urine delivered onto the tabletop [31].A urine dipstick was immersed into the urine, and the strips were bloated with soft tissue to prevent crosscontamination.Te colour changes on the strips were compared with the urine colour chart.Te urinalysis was done at baseline, 3 rd , and 6 th weeks for glucose, proteins, ketones, leucocytes, nitrite, bilirubin, specifc gravity, pH, urobilinogen, and blood using urine biochemical strips (Urit Medical Electronics Co. Ltd., China).

Blood Sampling and Isolation of
Organs.Blood samples of the rats in each treatment group were obtained by tail strains (at baseline, 3 rd , and 6 th weeks) into EDTA anticoagulated tubes for haematological analysis and into serum gel separator tubes (SSTs) for serum biochemical analysis.Blood in the EDTA tubes was gently swirled and inverted about 5 to 10 times to ensure EDTA was well mixed with the blood to prevent clotting.Blood in the SST was allowed to clot for 20 minutes and centrifuged at 5000 g for 5 min.Te serum obtained was stored at −20 °C until analysis.
At termination, the rats in both the treatment and control groups were sacrifced by cervical dislocation and dissected.Te heart, liver, kidney, spleen, and lungs were excised, freed of fat and connective tissues, bloated on clean tissue paper, and weighed on an electronic balance.Te tissues were fxed in 10% neutral bufered formalin (pH � 7.2) for histopathological examination [32].

Efect of CSE C. citratus Ethanolic Extract on Body and
Organ Weights of Rats.Te body weights of the rats were recorded at baseline, 3 rd , and 6 th weeks.Te percentage change in body weight of the rats was calculated as follows: Percentage body weight gain � Weekly weight (g) − Baseline weight(g) Baseline Body Weight (g) x 100%.
Te relative organ weight (ROW) of each organ was calculated as follows: Relative Organ Weight � Absolute Organ Weight (g) Rat Body Weight (g) x 100%. (3)

Histopathological Examination.
Portions of the organs were excised and processed into parafn blocks in labeled tissue processing cassettes.Each was passed through ascending grades of alcohol (70%, 80%, 90%, and absolute).Tey were further dehydrated with two changes of absolute alcohol cleared in three changes of xylene and ultimately infltrated and embedded in parafn wax.Sections of 4 µm thickness were cut from each block, mounted on a microscope slide, and stained with haematoxylin and eosin stain [32].Te stained tissues were observed with an Olympus microscope for morphological changes and photographed.

Statistical Analysis. Te data were entered into
Microsoft Excel 365 for cleaning, analysis, and plotting of graphs and tables.Te results obtained were reported as mean ± standard error of the mean (SEM).Te data were further exported into GraphPad Prism version 8.4.2 (GraphPad Software, San Diego, CA, USA) for one-way analysis of variance (ANOVA) analysis.Te ANOVA was used to determine whether there was a statistically signifcant diference between the control and experimental groups followed by Tukey's multiple comparisons test.P < 0.05 was considered statistically signifcant.

Acute Toxicity Study.
All the rats were observed up to 12, 24, and 48 hr, 7 days, and 14 days after the treatment.No mortality was recorded (Table 1).No physical evidence of toxicity such as piloerection, diarrhoea, increased urination, salivation, lachrymatory, locomotory defects, difculty in breathing, or asthenia was recorded (Table 1).Tis, therefore, suggests that the oral median lethal dose (LD 50 ) of CSE C. citratus ethanolic extract is greater than 5000 mg/kg.

Percentage Body Weight Gain. Te efect of CSE
C. citratus ethanolic extract on the weekly percentage body weight gain of the rats is shown as follows (Figure 1).Both the control and the test rats gained weight steadily over the study period.However, the control rats gained more weight than the test rats, which was not statistically signifcant, p > 0.05.Te rats in the 600 mg/kg bw.t and 1200 mg/kg bw.t treatment groups gained comparatively the same amount of weight, which was higher than the weight gained by the 200 mg/kg, but this was not statistically signifcant, p > 0.05.Te area under the curve (AUC) analysis confrms this trend of growth of the rats (Figure 2).2).

Efect of CSE C. citratus Ethanolic Extract on Lipid
Profle.Table 2 shows the efect of the CSE ethanolic C. citratus extract on the lipid profle of the control and test rats at the termination of the experiment.Te total cholesterol and HDL-c were higher in the control than in the test rats, and for the treatment groups, the total cholesterol was higher in all test rats than HDL-c.However, triglycerides and LDL-c were higher in the test rats than in the control rats.Tere was no statistically signifcant diference between the control and test rats' total protein, albumin, and bilirubin concentrations.Te total protein was comparatively the same among the test groups and controls although concentration was decreased in this order: control >200 mg/kg > 1200 mg/kg > 200 mg/kg which was not statistically signifcant.For albumin, the concentrations were almost the same between control and test rats (Table 4).Total bilirubin was higher followed by indirect bilirubin and direct bilirubin was the least.Te direct bilirubin was comparable between control and test rats.However, total bilirubin concentration increased as the dosage of treatment increased (200 mg/kg < 600 mg/kg < 1200 mg/kg) in the treatment groups.Total bilirubin in the control rats was higher than that in the 200 mg/kg treatment group but less than in the 600 mg/kg and 1200 mg/kg treatment group.Te same trend was observed for indirect bilirubin.Tere was, however, no statistically signifcant diference (p > 0.05) (Table 4).
(2) Renal Function Test.Te serum concentrations of urea, creatinine, and electrolytes were measured after six weeks of oral administration of CSE extracts to control and test rats.Te serum urea concentration was comparatively the same among the control and test rats (Table 5).Te creatinine concentration of the 600 mg/kg bw.t treatment was the highest, followed by 1200 mg/kg bw.t, and then the control rats with the 200 mg/kg bw.t treatment were the least which was not statistically signifcant.Te serum electrolyte concentrations were relatively the same between the control and test rats, respectively.Serum sodium concentration was higher followed by chloride concentration and potassium concentration was the least between the control and test rats (Table 5).

Urinalysis.
Dipstick urinalysis data at the termination of the experiment following six weeks of oral administration of CSE C. citratus ethanolic extract is shown in Table 6.Te results indicate that there were no signifcant diferences in the levels of urine parameters between the control and test rats.

Histopathological Examination.
Te histopathological examination of the organs isolated from the rats after six weeks of subchronic toxicity studies revealed that there was no abnormal histologic fnding in sections of the liver, kidney, lung, heart, and spleen among the control and treatment groups (Figures 4-8).Te cellular integrity and topologies were intact when comparing all tissues in the treatment groups to their respective controls.

Discussion
Te increasing patronage of herbal preparations exposes consumers to toxicity due to poor scientifc validation of the efcacy and toxicity of these herbal preparations, poor monitoring and regulation by regulatory authorities, and misidentifcation of the right species of plant for the treatment of a specifc disease.C. citratus decoction is used in Ghana for the treatment of fever but the safety profle of the plant is not known.Tis study aimed to assess the safety profle of C. citratus extract by the Conventional Soxhlet ethanolic extraction method in Sprague-Dawley rats by acute and subchronic oral administration.
Te acute toxicity study of the extract showed that a single dose oral administration of CSE ethanolic C. citratus extract to the rats did not produce any adverse efects (i.e., asthenia, defaecation, salivation, abnormal respiration, Te clinical relevance of acute toxicity studies is limited since bioaccumulation of toxic substances can cause severe adverse efects even at very low doses.Tus, multiple-dose administration is critical in ascertaining the true safety profle of drugs of interest [33].Subchronic oral administration of the extracts was thus performed at dosages of 200 mg/kg bw.t, 600 mg/kg bw.t, and 1200 mg/kg bw.t, respectively.Treatment with CSE C. citratus ethanolic extract      resulted in decreased weight gain in the test rats compared to the control (Figure 1).Te weight loss efect of CSE C. citratus ethanolic extract could be due to appetite inhibiting or lipid-lowering efect.Te fndings reported here agree with [19].Te extract was shown to have cholesterol and HDL-c lowering efect (Table 2), but the hypothesis of appetite inhibiting efect requires validation.
Hypercholesterolaemia, obesity, and diabetes are closely linked to stroke and hypertension.Since the extract lowered the cholesterol levels in the test rats more than in the control, it lowered the cardiovascular risk of the test rats more than the control except for the 1200 mg/kg bw.t treatment group although LDL-c was higher in the test rats than in the control (Table 2).Tus, consumption of C. citratus extract has a low coronary risk.However, the borderline high triglyceride in the test rats than in the control suggests that continuous consumption of this extract could lead to pancreatitis and fatty liver disease due to triglyceride infltration of these organs.
Relative organ-to-body weight ratio is a more sensitive marker of specifc organ toxicity than absolute body weight, since the deleterious efect of a drug can be identifed when a sensitive organ is afected [27,32].Te results in this study revealed no signifcant change when the weight of the excised liver, heart, spleen, lungs, and kidney was expressed as a percentage relative to the mean body weight of the test rats.Te lack of signifcant diferences in the percentage relative organ weight to body weight afrms the safety profle of C. citratus CSE extracts (Figure 3).Te main transport system of drugs and xenobiotic products is the blood, and the cells of the blood interact with these substances during their stay and transport in the blood, making it one of the most vulnerable systems to pathological substances [34].White blood cells are the frst line of defense against infection, infammation, or cellular injury [34].Studies have reported that an increase in the level of WBC and its indices in test rats during the herbal extract subchronic administration results from the extract stimulating an immune response or inducing an infammatory condition in the treated rats.However, a signifcant decrease in WBC count will signify a decrease in the production of leukocytes termed leukopenia, which refects the reduced ability of the body to fght infection [35].As recorded in Table 3, since the WBC and its indices of the test rats were normal and lower than that of the control following subchronic oral administration of C. citratus extract suggests that the long-term administration of CSE ethanolic C. citratus extract protected the test rats against infections and did not elicit an infammatory response in the test rats.Tis fnding explains why C. citratus decoction is used as a fever tea in Ghana.Tis study contradicts a study by Sadi and Imam [9], who reported that the WBC and its indices of test rats treated with aqueous and ethanolic C. citratus extracts were comparatively the same as the control.Tis study however agrees with Nosiri et al. [36], who recorded a decrease in WBC and its index in test rats compared to the control.A similar observation was made by Ekpenyong et al. [10], in healthy volunteers who after 30 days of receiving oral infusion of C. citratus infusion had signifcantly low WBC diferential count.Te RBC, Hb, HCT, MCV, MCH, and MCHC count in both control and test rats were comparatively the same.It implies that CSE C. citratus ethanolic extract has no harmful efect on bone marrow function.Te normal count of the RBC indices (MCV, MCH, and MCHC) indicates that the RBCs were of normal morphology, and the extracts may be rich in vitamin B, folate, and Fe because a defciency in these minerals would have manifested in structural abnormalities of the RBCs [8].Tis study agrees with a study by Ayenew  ALT and AST are intrahepatic enzymes that escape out of the hepatocytes to the blood following altered permeability of the hepatocellular membrane.Tey are transaminases involved in amino acid and carbohydrate metabolism.
Elevation of the activities of these soluble cytosolic enzymes is a predictive marker of hepatocellular injury.Te results from this study revealed that there were no signifcant diferences between the activities of ALT and AST of the control and test rats (Table 4).Tis suggests that CSE ethanolic C. citratus extracts do not contain compounds that may have a membrane-damaging efect on the hepatocytes and thus adenosine triphosphate (ATP) synthesis, amino acids, and carbohydrate metabolism was not impaired [37,38] in the test rats.
ALP and GGT are cholestatic-inducing enzymes of hepatobiliary origin with normal activity in normal hepatic function [39,40].Tey are employed to diagnose biliary fow obstruction or cholestasis.Of course, there are four isoforms of ALP with diferent tissues of origin (i.e., bone, placenta, intestinal, and liver).An increase in the activity of these isoforms could cause an increase in the activity of ALP.Moreover, for GGT, aside from cholestatic induction of increased GGT activity, drugs such as warfarin, alcohol, phenytoin, phenobarbital, and other anticonvulsant drugs induce the synthesis and increased activity of GGT [41,42].Te absence of a nonsignifcant increase in GGT activity suggests that CSE ethanolic extracts of C. citratus have no secondary metabolite with GGT induction activity.Since GGT activity was normal, the increase in the activity of ALP in the test rats to the control is not of liver origin.Tere were no statistically signifcant diferences between the activities of ALP and GGT in the control and test rats (Table 4).Tis study agrees with the study [9] which also reported that C. citratus extract did not induce elevation of hepatic enzymes in albino rats.Indeed, reports in [43,44] indicate that C. Citratus has a hepatoprotective efect by signifcantly decreasing the activity of hepatic enzymes compared to controls.
Te concentrations of total protein and albumin were the same in both control and test rats as recorded in Table 4. Te liver synthesizes all kinds of plasma proteins such as acute phase proteins involved in the eliciting of an infammatory response, clotting factors, and intrinsic and extrinsic pathway factors [45].A measurable decrease in total protein and albumin concentration refects either impaired hepatocellular production, increased renal loss, or increased catabolism, which may occur in various pathological conditions [45].Te normal concentrations of total protein and albumin afrm the safety profle of C. citratus extract.Te Bilirubin is a product of the catalytic degradation of haemoglobin in red blood cells by haem oxygenase and biliverdin reductase [46].Increased bilirubin synthesis occurs in haemolytic conditions like glucose 6-phosphate dehydrogenase defciency (G6PD), sickle cell disease, ABO, and rhesus incompatibility, etc., resulting in anaemia [43,44].In this study, the total bilirubin for the control was higher than that for the 200 mg/kg bw.t treatment group, however, the total bilirubin for the 600 mg/kg bw.t and 1200 mg/kg bw.t treatment groups was higher than that for the control and 200 mg/kg bw.t treatment group.A similar trend was observed for indirect bilirubin (Table 4).Tis is suggestive that the increase in total and indirect bilirubin for 600 mg/kg and 1200 mg/kg bw.t is due to preanalytical errors but not pathological reasons because of the efect of the extract treated.Due to the tail strains method used, the RBCs could be easily haemolyzed, if the haemolysis was due to the extract, the control group should not have higher total and indirect bilirubin than the 200 mg/kg bw.t treatment.Te result shows that CSE ethanolic extract of C. citratus did not induce haemolysis and for that matter anaemia in the test rats.Te normal Hb levels measured in control and test rats in this study buttressed this point.Contrary to our report, Eraj et al. [43] reported a decreased level of bilirubin in test rats compared to controls when treated with C. citratus extract.
Serum urea, creatinine, and electrolytes are markers routinely measured to assess renal integrity.However, creatinine is a waste product of muscle metabolism and urea is a waste product of protein metabolism.Te accumulation of these metabolites in the serum refects renal impairment.Te result from this study shows that urea concentrations were almost the same in the control and test rats (Table 5).Tis is in contrast with a study by Tarkang et al. [47], who reported elevation of serum urea following 28 days of administration of C. citratus ethanolic and aqueous extracts to Swiss albino rats.Creatinine concentrations were nonsignifcantly higher in the 600 mg/kg and 1200 mg/kg treatment groups than in the control.Tese results suggest that C. citratus extract is not nephrotoxic.However, Ekpenyong et al. [8] reported that following 30 days of oral administration of C. citratus infusion, creatinine clearance (CCr), and estimated glomerulus fltration rate (eGFR) were decreased in healthy volunteers.Tey also observed a timeand dose-dependent decrease in CCr and eGFR, suggesting that prolonged and high-dose consumption of C. citratus extracts will be nephrotoxic to the consumer.
Te measured electrolytes, sodium, chloride, and potassium in this study did not vary between control and test rats (Table 5).Tis suggests that C. citratus did not cause electrolyte derangement in the test rats.Tese fndings agree with Christopher [48], who reported a nonelevation of serum electrolyte concentrations following 30 days of oral infusion of aqueous and ethanolic C. citratus extracts in healthy volunteers.Tey, however, reported a decrease in urinary and plasma pH at the end of the treatment compared to baseline and an increase in urinary excretion of electrolytes (i.e., Na + and K + ) suggesting that chronic consumption of C. citratus infusion could cause C. citratus associated acid-base derangement and electrolytes wastage.Tis report is contrary to our fndings in this study, the urine pH of control and test rats were averagely the same for both control and test rats (Table 6), and this explains why animal study results cannot be directly extrapolated to humans.
Te histological examination of the sections of the organs revealed that there were no abnormal morphological changes observed in the liver, kidney, heart, lung, and spleen tissues of the treatment groups compared to the control after six weeks of treatment with CSE C. citratus ethanolic extract (Figures 4-8).Tis confrms the results of the biochemical assays which are relatively normal comparing the treatment groups to the control.Saenthaweesuk et al. [44] reported that CSE extracts have a hepatoprotective efect against paracetamol-induced hepatotoxicity in rats.Pretreatment with CSE extracts showed less degree of cellular damage and healing of the hepatocytes following hepatoxicity induction with paracetamol.

Limitations of the Study.
Te resource equation method used for the sample size calculation is not robust and does not consider the factor of efect size.Te current study did not perform a biomarker assessment of cardiotoxicity, lung function, and clotting profle defciency efect of C. citratus CSE ethanolic extract.Moreover, the study did not assess serum pH and bicarbonate to see if there was any acid-base derangement following CSE ethanolic C. citratus oral administration.Besides that, urine electrolytes were not measured to assess electrolyte wastage.Te study, however, has unraveled the safety profle of C. citratus cultivated in Ghana.

. Conclusion
Te fndings from this study revealed the relative safety of C. citratus CSE extracts in Sprague-Dawley rats.However, these fndings cannot be extrapolated directly to humans, and people are therefore cautioned to use C. citratus infusion or decoction in moderation.

Figure 1 :Figure 2 :
Figure 1: Efect of oral administration of CSE C. citratus ethanolic extract on the percentage body weight of the rats over a six-week treatment period.Each data point represents mean ± SEM (n � 6).

Figure 3 :
Figure 3: Efect of oral administration of CSE C. citratus ethanolic extract on percentage organ/weight at the termination of the experiment.Each bar represents mean ± SEM (n � 6), (p > 0.05).

Figure 5 :
Figure 5: Representative micrographs of the lung tissue in Sprague-Dawley rats after six weeks of CSE C. citratus ethanolic extract administration (x100).Te micrographs show normal lung architecture with alveolar spaces (A), bronchiole (BC), and blood vessels (BV) with no infammation, fbrosis, or neoplastic change in all treatment groups.(a) Lung tissue of control rats treated with sterilized distilled, (b) lung tissue of rats treated with 200 mg/kg bw.t CSE C. citratus ethanolic extract, (c) lung tissue of rats treated with 600 mg/kg bw.t CSE C. citratus ethanolic extract, and (d) lung tissue of rats treated with 1200 mg/kg bw.t CSE C. citratus ethanolic extract.

Figure 6 :
Figure 6: Representative micrographs of the kidney tissue in Sprague-Dawley rats after six weeks of CSE C. citratus ethanolic extract administration (x100).Te micrographs show mainly glomeruli (G) and renal tubules (T) lined by a single layer of epithelial cells with regular nuclei.No tubular atrophy, interstitial fbrosis, or infammatory infltrates are noted.(a) Kidney tissue of control rats treated with sterilized distilled water.(b) Kidney tissue of rats treated with 200 mg/kg bw.t CSE C. citratus ethanolic extract.(c) Kidney tissue of rats treated with 600 mg/kg bw.t CSE C. citratus ethanolic extract.(d) Kidney tissue of rats treated with 1200 mg/kg bw.t CSE C. citratus ethanolic extract.

Figure 7 :
Figure 7: Representative micrographs of the liver tissue in Sprague-Dawley rats after six weeks of CSE C. citratus ethanolic extract administration (x100).Te micrographs show central veins (CV) surrounded by radiating plates of hepatocytes.Also shows blood vessels, including portal triads (PT) consisting of a branch of the hepatic artery, portal vein, and bile duct.No infammatory cells present and necrosis in all treatment groups.(a) Liver tissue of control rats treated with sterilized distilled water.(b) Liver tissue of rats treated with 200 mg/kg bw.t CSE C. citratus ethanolic extract.(c) Liver tissue of rats treated with 600 mg/kg bw.t CSE C. citratus ethanolic extract.(d) Liver tissue of rats treated with 1200 mg/kg bw.t CSE C. citratus ethanolic extract.

FFigure 8 :
Figure 8: Representative micrographs of the heart tissue in Sprague-Dawley rats after six weeks of CSE C. citratus ethanolic extract administration (x100).Micrographs showing the characteristic branching arrangement of myocardial fbres (F) with centrally placed nuclei.No infammatory cells are present and no myocardial infarcts.(a) Heart tissue of control rats treated with sterilized distilled water.(b) Heart tissue of rats treated with 200 mg/kg bw.t CSE C. citratus ethanolic extract.(c) Heart tissue of rats treated with 600 mg/kg bw.t CSE C. citratus ethanolic extract.(d) Heart tissue of rats treated with 1200 mg/kg bw.t CSE C. citratus ethanolic extract.

Table 2 :
Efect of six weeks of oral administration of CSE C. citratus ethanolic extract on lipid profle at termination of the experiment.
Values are mean ± SEM of n � 6, p > 0.05, compared to control.

Table 1 :
Acute toxicity study of the efect of conventional Soxhlet C. citratus leaves extract in Sprague-Dawley rats over 14 days.ethanolicconventionalSoxhletC.Citratus leaves extract was orally administered to six rats at a single dose of 5000 mg/kg bw.t.Observation for signs of toxicity was recorded after(12, 24, and 48)hr and(3, 4, 5, 6, 7, 8, 9,  10, 11, 12, 13, and 14)days after the treatment.Te control group was administered with 1 m/100 g bw.t of normal saline.0/6:nodeathsrecorded.changes(p>0.05) in the organ weights expressed as a percentage of body weight between the control and test rats for each treatment group.Te relative organ/body weight ratio (%) of the lungs decreased at 1200 mg/kg bw.t compared with the other treatment groups and control rats in the CSE treatment group.However, that of the heart increased in the 1200 mg/kg treatment compared to the control and other treatment groups in the CSE treatment group.Tese diferences were however not statistically signifcant (p > 0.05).3.2.4.Efect of CSE C. citratus Ethanolic Extract onHaematological Parameters.Table3shows the efect of CSE ethanolic C. citratus extracts on haematological indices of Sprague-Dawley rats at the termination of treatment.Te results show that there were no statistically signifcant differences in all parameters measured between the control and test rats (p > 0.05).Te WBC (18.6 × 10 9 )/L, Lymph# (11.98 × 10 9 )/L, Mid# (1.50 × 10 9 )/L, and Lymph% (68.05%) of the control group were higher than WBC (10.15-13.18) 10 9 /L, Lymph# (6.58-6.85)10 9, Mid# (0.88-1.13)10 9 /L, and Lymph% (58.73-64.68%) of the test rats.Gran% (23.58%) of the control rats was lower than the test rats' Gran %(26.55-32.48).However, for AST, its activity was relatively the same among the control and 600 mg/kg bw.t treatment groups.Moreover, the 200 mg/kg w.t and 1200 mg/kg bw.t was almost the same, which was lower than the control and 600 mg/kg bw.t.In the case of ALP, the activity of the enzyme was higher in the treatment group than the control with 200 mg/kg bw.t having the highest activity following 120 mg/kg bw.t and 600 mg/kg bw.t being the least. Fr GGT, on the other hand, the activity of the enzymes was almost the same for the control and test groups.Tere was no statistically signifcant diference (p > 0.05).
Te None: no signs of toxicity were observed over the study period.Latency: time to death (hours) after the treatment.-:latentperiod.signifcant3.2.5.Efect of CSE C. citratus Ethanolic Extract on Serum Biochemical Parameters(1) Liver Function Test.Te efect of subchronic oral administration of CSE extracts on rats' serum liver function test is shown in Table4.Te activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and c-glutamyl transferase (GGT) were determined.Te serum ALT activity was relatively the same for the control, 600 mg/kg bw.t, and 1200 mg/kg bw.t treatment groups.However, there was an increase in its activity in the 200 mg/kg bw.t treatment group than the control, 600 mg/kg bw.t, and 1200 mg/kg bw.t.

Table 3 :
Efect of six weeks of oral administration of CSE C. citratus ethanolic extract on haematological parameters at termination.

Table 5 :
Efect of six weeks of oral administration of CSE C. citratus ethanolic extract on renal function test at termination of the experiment.

Table 4 :
Efect of six weeks of oral administration of CSE C. citratus ethanolic extract on liver function test at termination of the experiment.SEM of n � 6, p > 0.05, compared to control.ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase.