Acute dysfunctional status of hepatorenal tissues of rats administered with leaf extracts ofOcimumgratissimum L. (Lamiaceae)

Use your smartphone to scan this QR code and download this article ABSTRACT Background: Given the vast medicinal properties of Ocimumgratissimum, the present study evaluated, in comparative terms, the acute dysfunctional status of hepatorenal tissues of Wistar rats administeredwith petroleumether (PE) and ethyl acetate (EA) leaf extracts ofO.gratissimum. Methods: Grouping of the experimental rats was assigned according to the treatments given, in which graded doses (200, 400, 600 and 800mg/kg body weight (b.w.)) of PE and EA fractions ofO. gratissimum leaf extractwere administered to the rats by oral gavage on a daily basis for a period of 21 days. Serum levels of hepatorenal tissues biomarkers weremeasured using standard spectrophotometric methods. The organ-to-body weight ratio of the rats was measured on the 21st day of the experiment. Results: Serum aspartate aminotransferase (AST) to alanine aminotransferase (ALT) ratio (i.e. AST/ALT) of the experimental rat groups was found to be within the range of 0.919 – 1.022 unit. The experimental rat groups administered with PE and EA fractions of O. gratissimum leaf extract showed dose-dependent increasing levels of serum alkaline phosphatase (ALP) activity. Likewise, rat groups administered with the herbal extracts exhibited increasing serum total bilirubin, urea and creatinine concentrations, in a dose-dependent manner. At the end of the 21-day treatment period, all the experimental rat groups showed increase in body weight, ranging from 0.79 – 1.98% increase. The liver weight and kidneyweight to bodyweight ratios werewithin the range of (0.0468 ± 0.02 – 0.0981± 0.04) unit and (0.00245± 0.002 – 0.01968± 0.007) unit, respectively. Conclusion: The results showed that doses of PE and EA fractions ofO. gratissimum leaf extract greater than 400 mg/kg b.w. induced dose-dependent hepatorenal toxicity, with the EA fraction provoking greater toxicity than the PE fraction ofO. gratissimum leaf extract.


INTRODUCTION
In general terms, metabolic events within the liver and kidney are essential to ensure constancy in the internal environment of vertebrates 1,2 . The control mechanisms of metabolic events in the hepatocytes are regulated at the molecular, organelle, cellular and organ levels 3,4 . Endogenous metabolic control mechanisms of hepatocytes involve the actions of regulatory enzymes, organelles responsible for protein and lipid biosynthesis, as well as interactions of the hepatocytes with sinusoidal and Kupffer cells. Meanwhile, exogenous control mechanisms are accomplished by biochemical interactions between the liver and the musculature, as well as interactions among the renal, enteric and endocrine systems 3 . The metabolic heterogeneity of hepatocytes in health and disease is summarized elsewhere [5][6][7] . Routine clinical evaluation of the functional status of hepatocytes, the socalled liver function test (LFT)/biliary integrity test (BIT), is established by evaluating activities of nonfunctional plasma enzyme indicators, namely aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and the inducible hepatocyte smooth endoplasmic reticulum (SER) specific enzyme, γ-glutamyl transferase (γ-GT). As well, albumin, total bilirubin and total protein concentrations in blood samples are examined [8][9][10][11][12] . The nephron is the functional unit of the kidneys. The renal tissues are primarily involved in the removal of low plasma threshold substances, such as urea, creatinine and uric acid. The renal tissues also regulate blood electrolyte concentrations and, by extension, osmolality, extracellular fluid volume and acidbase balance of the vascular system. Furthermore, the kidneys are sites for the biosynthesis of steroid and polypeptide hormones, such as 1, 25 dihydroxyvitamin D, erythropoietin, and renin 13,14 . Elevations of plasma low threshold substances in the blood are indicative of compromised renal function. The kidney function test (KFT) measures plasma creatinine and blood urea nitrogen (BUN) levels, among other blood indicators, such that their raised levels in the blood are diagnostic of presentation and progression of renal disease 15 . The renal/kidney function test indicators are applied in monitoring the efficacy of therapeutic intervention against compromised renal function 14 .
Ocimum gratissimum L. belongs to the family Lamiaceae 16 . The plant is a perennial herb widely distributed in warm and temperate regions of the world 17 . The phytochemical compositions of diethyl ether, ethyl acetate, ethanol and aqueous leaf extracts of O. gratissimum have been exhaustively reported elsewhere [18][19][20][21][22][23] , in which it was noted that O. gratissimum contained relatively high quantities of alkaloids, flavonoids, saponins, methyl cinnamate, camphor, thymol, eugenol, linalool, xanthones, citral, terpenes and lactones 17,18,[20][21][22][23][24] . Traditional medicine practitioners administer O. gratissimum extracts for the treatment and management of fever, rheumatism, paralysis, epilepsy, high fever, diarrhea, sunstroke, influenza, gonorrhea and mental illness 25,26 .The use of O. gratissimum extracts by folklore medicine practitioners for the treatment of microbial infections has been validated by empirical investigations 20,22,23 . There are empirical evidence that edible vegetables and medicinal plants contain deleterious phytochemicals which are usually eliminated by traditional and conventional processing methods prior to consumption of the plant materials 27,28 . Some of these edible and medicinal plants have been reported to provoke organ dysfunction and systemic toxicity, especially when ingested in large quantities and unprocessed forms [29][30][31] . However, the susceptibility of animal models to chemical-induced hepatic or systemic toxicity is regulated by genetic, environmental, dietary and pathophysiological factors 2 . The vast medicinal properties of O. gratissimum notwithstanding, the present study ascertained, in comparative terms, the acute dysfunctional status of hepatorenal tissues using blood levels of enzyme activities and metabolite profiles of hepatorenal origin, as well as organ/body weight indicator in Wistar rats administered with petroleum ether (PE) and ethyl acetate (EA) leaf extracts of O. gratissimum.

Collection and identification of leaf samples
Fresh and healthy leaves of O. gratissimum were collected between the period of August 9 th and September 2 nd , 2019-from a private botanical garden located within Imo State University, Owerri, Nigeria (Latitude 5 • 30.2237'N; Longitude 7 • 2.6277'E). The leaves were identified and authenticated by a botanist. A voucher number (IMSUH: 021) was assigned to the leaf samples and, thereafter, deposited in the herbarium for reference purposes.

Preparation of leaf samples
The collected leaves of O. gratissimum were washed using tap water and then transferred into an oven (WTC BINDER-7200 Oven, Tuttlingen, Germany). The leaves were dried to constant weight at 50 • C for 10-12 h 32 . The dried leaf samples were pulverized and subsequently stored for use as previously described 33 .

Extraction and fractionation of leaf extracts
A 500 g part of the pulverized leaf sample of O. gratissimum was subjected to repeated hydro-ethanol (ratio: 2:3 v/v) extraction for 24 h using Soxhlet apparatus. The hydro-ethanolic leaf extract was fractionated according the methods previously described 34

Animal handling and experimental design
The male Wistar rats, within the ages of 7 -9 weeks old and of average weight of 109.74 ± 2.81 g, were obtained from the Animal House of Imo State University, Owerri, Nigeria. Handling of the animals was performed according to the methods previously described 33 . The Ethical Committee on the use of animals for research, Department of Biochemistry, Imo State University, Owerri, Nigeria (Ethics Approval Number: ODVC/REN/1232/19) approved the present study. Handling of the rats was in accordance with the standard principles of laboratory animal care of the United States National Institutes of Health (NIH, 1978). A total of 54 rats were divided into 9 groups of 6 rats each. The rats were deprived of pelletized standard guinea feed (PSGF) (United Africa Company Nigeria Plc., Jos, Nigeria) and water 16 h prior to the commencement of treatment 35 . The grouping of the experimental rats was assigned according to the treatments given, in which the PE and EA fractions of O. gratissimum leaf extract were administered to the rats by oral gavage on a daily basis for a period of 21 days. All the experimental rat groups received water ad libitum.

Collection and preparation of blood, liver and kidneys
At the end of the experimental time of 21 days, the 12 h post-fasted rats were killed by cervical dislocation. Blood volumes of 0.5 mL were drawn from the orbital sinus of rats and allowed to clot. The serum was measured for hepatorenal tissues biomarkers. The collection and preparation of the liver and kidneys, in order to ascertain their respective weights, were done according to the methods previously described 33 .

Hepatorenal tissues biomarkers
Serum levels of hepatorenal tissues biomarkers were measured; serum AST and ALT activities were assessed according to the methods of Henry et al., 36 as described 37 ,and by serum ALP activity 38 ,serum total bilirubin concentration 39 , serum urea concentration 40 , and serum creatinine concentration 41 .

Body weight of rats
The body weight of the rats was measured using electronic weighing balance {Digital Precision Weighing Balance (JCS-QC03) -China}, on the 1 st and 21 st days of the experiment 33 . Thus:

Liver and kidney weights to body weight ratios
The liver and right and left kidneys weights were measured on day 21. The organ weight and body weight were reported in grams 33 .Thus:

Data and statistical analyses
The data collected were analyzed by the ANOVA procedure while treatment means were separated by the least significance difference (LSD) incorporated in the statistical analysis system package of Version 9.1 of 2006.

Yield of the fractionated leaf extract of O. gratissimum
The yields of PE and EA fractions of O. gratissimum leaf extract were 8.03 ± 0.07 g per 100 g and 7.56 ± 0.05 g per 100 g of dry leaf sample, respectively.   Specifically, serum ALT activity of the rat groups administered with herbal extract was such that Group 8 EA600 = 80.03 ± 3.38 U/L > Group 4 PE600 = 64.08 ± 2.32 U/L, and Group 9 EA800 = 87.6 ± 3.85 U/L > Group 5 PE800 = 76.41 ± 2.02 U/L; p < 0.05. Furthermore, Figure 2 showed dose-dependent increasing levels of serum ALT activities of the rat groups administered with herbal extract. Table 1 showed that the serum AST/ALT ratio of the experimental rat groups was within the range of 0.919 -1.022 unit. Furthermore, an overview of Table 1 showed that serum AST/ALT ratios of Group 1 CONT ROL , as well as Group 2 PE200 -Group 4 PE600 , were less than 1.0 unit, whereas those of Group 5 PE800 -Group 9 EA800 were greater than 1.0 unit. Serum ALP activity of Group 4 PE600 (153.19 ± 6.74 U/L) was not significantly different (p < 0.05) from that of Group 8 EA600 (167.48 ± 6.88 U/L). Likewise, serum ALP activity of Group 5 PE800 (158.98 ± 5.28 U/L) and Group 9 EA800 (171.68 ± 6.93 U/L) showed no significant difference (p > 0.05). Figure 3 showed dose-dependent increasing levels of serum ALP activities of experimental rat groups administered with PE and EA fractions of O. gratissimum leaf extract.

Body weights and organ-to-body weight ratios of rats administered with fractions of O. gratissimum leaf extract
At the end of the 21-day treatment period, all the experimental rat groups exhibited increase in body weight within the range of 109.11 ± 1.03 to 112.2.65 ± 1.01 g ( Table 2). Additionally, Table 2 showed that Group 2 PE200 (110.98 ± 1.08 g) exhibited comparatively the highest gain in body weight after treatment, whereas Group 8 EA600 (110.37 ± 1.04 g) gave the lowest gain in body weight. Overall, the gain in body weight of the experimental rat groups varied within the range of 0.79 -1.98%. Specifically, the cumulative gain in body weight of the herbal extract-treated rat groups was such that Group 2 PE200 -Group 5 PE800 (1.98 -1.03%) was greater than that of Group 6 EA200 -Group 9 EA800 (0.95 -0.79%). The relative gain in body weight of the Group 1 CONT ROL (112.65 ± 1.01 g) was greater than that of the herbal treated rat groups, except that of Group 2 PE200 (110.98 ± 1.08 g). Table 2 showed that the liver weight to body weight ratios of Group 2 PE200 (0.0542 ± 0.02) and Group 3 PE400 (0.0582 ± 0.02) were not significantly different (p > 0.05) from that of Group 1 CONT ROL (0.0468 ± 0.02). The cumulative liver weight to body weight ratio of Group 2 PE200 -Group 5 PE800 was comparatively greater than those of Group 6 EA200 -Group 9 EA800 . Similarly, there was no significant difference (p > 0.05) between the kidney weight to body weight ratio of Group 2 PE200 (0.00260 ± 0.002) and Group 3 PE400 (0.00379 ± 0.003) ( Table 2). However, further increase in the experimental dose of the herbal extract caused increased kidney weight to body weight ratio. The liver weight and kidney weight to body weight ratios of Group 2 PE200 were significantly different (p < 0.05) from the corresponding Group 2 EA200 .

DISCUSSION
The combinations of distinctive molecular species present in PE and EA fractions of O. gratissimum leaf extract obviously dictated the toxic outcomes in the experimental rats. Chemical-induced hepatorenal injuries and resultant dysfunction is often initiated by metabolic transformation of molecular species to reactive intermediate species, such as electrophiles, which alter the function and structure of cellular macromolecules 2,42 . Measurement and evaluation of blood indices are fundamental in establishing the pathological and physiological statuses relevant to the clinician, nutritionist and toxicologist 43 The liver is primarily rich in aminotransferases, namely AST and ALT, such that their presence in the blood system indicates hepatic necrosis as well as extrahepatic tissue damage or both 8,12,44,45 . The findings of the present study suggest that doses of PE (≥ 600 mg/kg b.w.) and EA (≥ 400 mg/kg b.w.) fractions of O. gratissimum leaf extract administered to the rats provoked hepatic tissue injuries by virtue of the reported serum AST activities of the experimental rat groups. Likewise, the pattern of serum ALT activities of the experimental rat groups administered with PE and EA fractions of O. gratissimum leaf extract exhibited a mutual relationship with serum AST activities in terms of the dose-depended elevation of serum AST activity. Extrahepatic tissues contain appreciable quantities of the aminotransferases, whereby their raised levels in serum are also diagnostic of extrahepatic tissues necrosis. However, the measure of elevated serum AST and ALT activities are non-specific confirmatory tests for hepatic functional status. Accordingly, for the purpose of differential diagnosis, evaluation of serum AST/ALT ratio is applied in order to ascertain the severity and pathologic status of the animal, as well as to identify and confirm the organ of pathologic interest 12,46 . For instance, serum AST/ALT ratio > 1 unit indicates advanced liver fibrosis and chronic hepatitis, whereas serum AST/ALT ratio of 0.9 is diagnostic of nonalcoholic steatohepatitis 12,46,47 . Based on serum aminotransferases indicators, the present study showed that administration of PE and EA fractions of O. gratissimum leaf extract did not substantially cause hepatic dysfunction at relatively low dose of less than 400 mg/kg b.w.; however, the rats exhibited acute hepatic dysfunction following the administration of relatively higher doses of PE and EA fractions of O. gratissimum leaf extract. Elevation of ALP in the blood is linked to pathology of the liver as well as the mucosal epithelia of small intestine, proximal convoluted tubule of kidney, bone, and placenta. Serum ALP activities of experimental rat groups administered with PE and EA fractions of O. gratissimum leaf extract at doses greater than 600 mg/kg b.w. were substantially higher than the control rat group, which further confirmed a compromised hepatobiliary function and was in agreement with previous reports 8, 10,12,43,45 . Additionally, previous studies had noted that mild elevation of ALP activity in the blood was indicative of cirrhosis, hepatitis, and congestive cardiac failure 48 . Serum total bilirubin concentrations of the experimental rat groups also confirmed that at relatively low dose, PE and EA fractions of O. gratissimum leaf extract did not provoke hepatic dysfunction. Hyperbilirubinemia, which is diagnostic of hepatic dysfunction and hemolytic disorders, is diagnostic when blood serum total bilirubin concentration is greater than 1.0 mg/dL 8,49 . Accordingly, serum total bilirubin concentrations of the experimental rat groups administered with 200 mg/dL PE and EA fractions of O. gratissimum leaf extract did not exhibit hepatic dysfunction and hemolytic disorders. Nevertheless, higher doses of PE and EA fractions of O. gratissimum leaf extract elicited hyperbilirubinemia, which was indicative of compromised hepatic dysfunction in the rats. On the contrary, aqueous leaf extract of O. gratissimum was reported to enhance hematological parameters following oral administration to experimental rats 24 . It therefore implies that the phy-tocomponents from aqueous leaf extract of O. gratissimum, as compared to PE and EA fractions of O. gratissimum, did not provoke hemolytic disorders and hepatic dysfunction; the blood bilirubin concentration was greater than the upper normal limit of the reference range of blood bilirubin concentration.
The findings of the present study showed that the pattern of renal tissue dysfunction appeared to correspond to that of hepatic tissues following the administration of PE and EA fractions of O. gratissimum leaf extract. Specifically, elevation of serum urea and creatinine concentrations of the experimental rat groups suggest that the severity of compromised renal function was dose-dependent on the administered PE and EA fractions of O. gratissimum leaf extract. In a related research finding, Goniothalamin (GTN), which is a phytocompound from several plants of the genus Goniothalamus, engendered dose-dependent renal dysfunction in Sprague-Dawley rats 45 . Contrary to the outcome of the present study, Ogundipe et al. 16 reported that aqueous leaf extract of O. gratissimum ameliorated gentamicin-induced renal tissues injury in rats. However, based on empirical evidence of low creatinine clearance after 28 days of treatment, they noted that the risk profile of renal dysfunction is not unlikely following the administration of aqueous leaf extract of O. gratissimum 16 .
Another report 50 showed that aqueous leaf extract of O. basilicum reversed δ -methrin-induced nephrotoxicity in albino rats. Accordingly, the present research findings appeared to suggest that the molecular species that provoked hepatorenal tissue dysfunction, for the most part, were not associated with the aqueous fraction of O. gratissimum leaf extract. Furthermore, the molecular species from O. gratissimum leaf extract that caused dose-dependent hepatorenal toxicity in the experimental rat groups were hydrophobic in character in view of the fact that they tended to associate with solvents of low polarity, namely, the PE and EA fractions of O. gratissimum leaf extract. Alteration in visceral organ weight, which precedes morphological changes, is a sensitive indicator of systemic toxicity 33,51 . The alteration of visceral organ weight, which is indicative of pathology or compensatory changes in response to stress to the organ, is often reported in relation to the body weight of the experimental animals in toxicological studies 43,[51][52][53] . Notable factors that influence adverse change in visceral organ weight are strain, age and sex of the animal, as well as environmental and experimental conditions 51,54 . The results of body weight indicator suggest that molecular species from the EA fraction of O. gratissimum leaf extract caused greater tendency to retard gain in body weight of rats than the corresponding PE fraction. Thus, the EA fraction of O. gratissimum leaf extract appeared to exhibit a greater toxicological score than PE fraction. Furthermore, the pattern of alteration in body weights paralleled the changes in organ-to-body weight ratio of the experimental rat groups. The increase in organ-to-body weight ratio is a reliable indicator of inflammatory response of hepatorenal tissues following the administration of PE and EA fractions of O. gratissimum leaf extract to the experimental rat groups, as previously described 33,51 . Hypertrophy of the hepatic tissues was evident in the experimental rat groups administered with comparatively high doses of PE and EA fractions of O. gratissimum leaf extract. Specifically, the increase in liverbody weight ratio of the experimental rat groups administered with PE and EA fractions of O. gratissimum leaf extract was an indication of hepatocellular hypertrophy, inflammation and fibrosis, with resultant hepatic dysfunction as previously described 15,55 . Empirical investigations showed that hepatic hypertrophy is primarily the manifestation of accumulation of lipids as well as other connecting pathologic factors 55 . The increase in kidney-to-body weight ratio of the experimental rat groups was a reflection of renal toxicity, tubular hypertrophy, and/or chronic progressive nephropathy as previously reported 33,56 .

CONCLUSION
For the most part, the administration of PE and EA fractions of O. gratissimum leaf extract at a dose less than 200 mg/kg b.w. did not cause hepatorenal toxicity in the experimental rats. On the contrary, doses of PE and EA fractions of O. gratissimum leaf extract greater than 400 mg/kg b.w. caused dose-dependent hepatorenal toxicity, with the EA fraction provoking greater toxicity than the PE fraction of O. gratissimum leaf extract. Further investigations are required in order to identify, quantify, and characterize the molecular species present in the PE and EA fractions of O. gratissimum leaf extract that elicited the toxic outcomes in the experimental rats.