Hepatotoxicity evaluation of an aqueous extract of Clinopodium vimineum (L.) Kuntze in female Wistar rats

Clinopodium vimineum has been used to relieve gastrointestinal symptoms due to its carminative and antispasmodic properties. Besides, it has been associated with antimicrobial, sedative, analgesic, anti-inflammatory, and healing effects. Nevertheless, its essential oil has hepatotoxic compounds such as pulegone, so the plant's possible hepatotoxic effect on aqueous extract was evaluated with a female Wistar rats model. An aqueous extract of the vegetal material was prepared


Introduction
Clinopodium vimineum (known in Costa Rica as menta de palo) [1] is an aromatic shrub native to Central America and the Caribbean.It is described as a plant species with woody stems of vertical growth and multiple branches.The height is calculated between 15 and 20 cm.Its leaves have oval and opposite shapes, with toothed margins, a lime green upper side, engraved veins, and covered by hairiness.Furthermore, its flowers are small, tubular, and with white or pink/purple tones [2,3].
It has been traditionally utilized to relieve gastrointestinal tract symptoms, as it has carminative and antispasmodic properties.Other described assets are a healing agent, antimicrobial, sedative, analgesic, and anti-inflammatory [2,3].
Given this situation, this work aimed to evaluate the possible hepatotoxic effect of an aqueous extract of C. vimineum in a female Wistar rats model.

Vegetal material
Flowers, buds, and stems of C. vimineum were collected at the School of Agriculture of the Humid Tropical Region (EARTH University) (10°13'09" N 83°35'33" W) located in Guácimo, Limón.The sample was identified and deposited in the Dr. Luis A. Fournier Origgi Herbarium of the School of Biology of the Universidad de Costa Rica (UCR, for its Spanish acronym).The material was dried at room temperature and pulverized until achieving an acceptable material for its preservation.

Preparation of the plant's aqueous extract
An infusion was produced with 45 g of the powdered plant material, adding boiling water.The mixture was left to rest for 30 minutes in a container with a lid.Later, it was diminished by reduced pressure at 50 °C, using a Büchi® R-134 rotary evaporator (Büchi® B-480 water bath and Büchi® B-721 vacuum controller).The extract was lyophilized for four days (LABCONCO® FreeZone 6) to facilitate its administration.The final product was stored at -80 °C.

Experimental animals
Adult Wistar female rats (HsdBrlHan: WIST) with 207.80 ± 3.33 g of body weight, between eight and nine weeks of age, from the Laboratory of Biological Assays (LEBi, for its Spanish acronym) of the UCR were employed.
Fifteen subjects were considered.They were kept in polycarbonate boxes in the laboratory for in vivo work of the Phytopharmacology and Pharmaceutical and Cosmetic Technology Laboratory (LAFITEC, for its Spanish acronym) of the Instituto de Investigaciones Farmacéuticas (INIFAR, for its Spanish acronym) under the following conditions: temperature: 23.2 °C; relative humidity: 73 %; and periods of light and darkness: 12 hours.Bottled drinking water (Members Selection®) and rodent food (Aguilar & Solís®) were provided ad libitum.They were at the laboratory for seven days without manipulation to allow their adaptation.

Experimental design
Random block modeling was established with a total of three groups of five experimental subjects each: group 1 (control, which was administered water by gastric cannulation instead of the extract), group 2, which received extract for four days, starting with a loading dose of 300 mg/kg/day on the first day and then continuing with a dose of 150 mg/kg/day for three more days by intragastric cannula; and group 3, whose subjects received a daily dose of 150 mg/kg/day for four days by the same route mentioned above.
The trial lasted five days, ensuring the animals had access to food and drinking water ad libitum.On the fifth day, each animal was euthanized using the decapitation technique to get whole blood, and a general necropsy was performed to collect liver tissue.Plasma samples were obtained by centrifugation at 3000 rpm for 10 minutes (HettichZentrigugen® Universal 320 R model) at LAFITEC of the Faculty of Pharmacy of the UCR.

Analysis of biochemical liver function parameters
The serum samples were sent to the Clinical Analysis Laboratory at the Faculty of Microbiology of the UCR, and the hepatic enzymes analysis was done (Roche Cobas® c111 equipment).The enzymes analyzed were alkaline phosphatase (AP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT).

Hepatic histopathological analysis
The collected organs were fixed with 10 % buffered formalin.Tissues were then dehydrated with increasing concentrations of ethanol, cleared with xylene, and embedded in paraffin.A rotary microtome (Leica® RM2245) was employed to make 4 to 5 μm thick sections stained with hematoxylin-eosin.Through a Zeiss® AxioLab A1 microscope, morphological and structural changes in hepatocytes were analyzed, along with the physical characterization of sinusoids and vascular structures of said organ (hepatic portal vein, renal artery, and bile ducts).

Statistical analysis
The results were expressed as the mean together with its relative standard deviation.Unifactorial statistical analysis (ANOVA) was contemplated to settle group differences.The comparison between the means of the groups was elaborated through Tukey's multiple comparison analysis with a significance level of 95 % (p < 0.05), utilizing the software GraphPadPrism® version 6.01 for Windows.

Analysis of biochemical liver function parameters
AST and ALT are the most sensitive cell necrosis or hepatocellular injury indicators [6].Damage to the hepatocyte membrane provokes an elevation in the transaminases serum levels above the typical values because of their escape into circulation [7].The quantification usually has high sensitivity but low specificity [8].ALT is a more specific indicator.Both are at high levels in the liver cytosol.However, AST is also found in the liver mitochondria and has activities in muscle, brain, pancreas, lungs, kidneys, erythrocytes, and leukocytes [9].
As a complement, FA is the best indicator parameter of biliary obstruction.Nonetheless, this enzyme does not differentiate between intrahepatic or extrahepatic cholestasis clinical pictures.Given this, the enzyme is not a conclusive parameter to determine the existence of liver damage [10].
For the in vivo model, the changes in the serum levels of AST, ALT, and FA were jointly considered biomarkers of cell damage in hepatocytes.Table 1 shows the results achieved for the serum parameters of liver function.For FA, there was a significant reduction in groups 2 and 3 compared to the control group (p < 0.05).
Table 1 Biochemical parameters of the liver function obtained at the end of the in vivo model with an aqueous extract of C. vimineum.Data were expressed as the mean along with their respective relative standard error.Concentrations were compared to the control, n = 5 animals per group, * p < 0.05.Regarding AST, a significant increase was appreciated in the group without a loading dose, compared to the control (p < 0.05), rising 1.4 times in five days.Though, this enzyme is not tissue-specific [9].

Experimental
Finally, there were no appreciable differences between the groups for ALT.Since these results were not conclusive, the hepatic histopathological analysis of each experimental group was made.

Hepatic histopathological analysis
For the histopathological analysis (Figure 1), there were no relevant structural or morphological alterations in the hepatic tissue when comparing the animals exposed to the extract.The main morphological alterations located were the isolated presence of infiltrate with macrophages (Kuffer cells) [11], a slight increase in the size of the bile ducts [12], the widening of the sinusoids oriented towards the hepatic portal vein [13], and the pyknotic nuclei presence with concentrated chromatin [14].These findings suggest some particular infectious processes.With the histopathological analysis of the liver tissue and the changes in the serum parameters of liver function (FA, AST, and ALT), it can be assured that after the administration of the aqueous plant extract, there was no liver toxicity induced by characteristic metabolites of the genus Clinopodium, such as the pulegone.No indications of cell damage in the liver sections or significant alteration in the transaminase values were found.

Conclusion
The evaluation of the serum to study hepatic function parameters showed an appreciable but non-specific increase in AST values.No alterations or considerable changes were found for ALT and FA, which did not suggest liver damage because of the aqueous plant extract.Moreover, there were no structural or morphological alterations in the histopathological analysis for the liver sections that would indicate toxicity in said organ due to the treatment administration.Therefore, the information acquired reveals that the aqueous extract of C. vimineum did not generate a hepatotoxic effect in female Wistar rats.

Figure 1
Figure 1 Sagittal sections of livers taken from female Wistar rats after the in vivo model.A and B. Control group.C and D. Group exposed to the extract without a loading dose.E and F. Group exposed to the extract with a loading dose.IM: infiltration of macrophages.BD: opening of bile ducts oriented towards the central hepatic vein.Arrow: leukocyte infiltration oriented towards the central hepatic vein.Asterisk: bile aggregation in bile ducts.Hematoxylin and eosin staining.Objectives: 10X and 40X.