Evaluation of the Acute Toxicity of the Ethanolic Extract of the Rhizome of Zingiber officinale Roscoe in Rats

. Evaluation of the Acute Toxicity of the Ethanolic Extract of the Rhizome of Zingiber officinale Roscoe in Rats. Pharmacogn J. 2024;16(2): 323-331

macerated for 7 days in a hermetically sealed amber bottle, and the mixture was shaken daily for 7 days.At the end of this period, it was filtered and concentrated in a rotary evaporator, then taken to an oven at a temperature of 40°C to dry and obtain the dry extract. 18

Experimental animals and housing
Both female and male Holtzman albino rats (250 -300 g), aged 16 to 20 weeks, were used in this study.Rats were maintained in individual cages and were exposed to a 12-hour light/12-hour dark cycle, with a temperature of 22 ± 3 °C.They were provided with a standard laboratory diet and water administered ad libitum.The rats underwent an acclimatization period for 2 weeks before carrying out the study. 19

Acute toxicity test
The test was carried out according to the method described in OECD regulation No. 423.Experimental groups were formed, each with 10 rats (5 males and 5 females).The Control Group was administered 2 mL of physiological saline solution (SSF 0.9%).Problem Group I was administered the ethanolic extract of Zingiber officinale Roscoe rhizomes in a single dose of 300 mg/Kg/day.Problem Group II was administered the ethanolic extract of Zingiber officinale Roscoe rhizomes in a single dose of 2000 mg/Kg/day.The body weight of the rats was measured three times, once before administering the extract and on days 7 and 14 of observation. 19e animal study protocol was approved by the Ethics Committee of the Faculty of Medicine the Universidad Nacional de Trujillo (Approval Certificate No.: 372 -2022/ UNT-FM-C.E.).

Blood biochemistry evaluation
On day fourteen, after the treatments were administered, blood was obtained from the specimens through cardiac puncture.Plasma was separated by centrifugation at 2000 rpm for 15 minutes in a refrigerated centrifuge (4°C) and stored at −80°C.The biochemical parameters that were measured are urea, creatinine, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), cholesterol, and triglycerides. 19

Histopathological analysis
The animals were euthanized using sodium pentobarbital 60 mg/kg intraperitoneally.The brain, heart, lung, kidney, liver, stomach, uterus, ovary, prostate, and testicles were extracted, preserved in 10% formalin, and then taken to the corresponding histological study. 19

Statistical analysis
Statistical analysis was performed using the R-4.1.1 program (free software) for Windows®, and the data were subjected to analysis of variance (ANOVA) followed by posthoc (Tukey's test).The values were considered statistically significant when p < 0.05.

Changes in body weight
The determination of the body weight of the rats was carried out after the administration of the Zingiber officinale extract, as well as on days 7 and 14, observing variation in the weights on day 14 post-treatment, although this variation is not statistically significant.

Biochemical parameters
In Figure 2, a significant difference is observed in the AST values between the Control, Zingiber officinale 300, and Zingiber officinale 2000 groups in both males and females, with the AST concentration increasing as the dose of Zingiber officinale extract increases (p<0.05).In the ALT and ALP values, only the Zingiber officinale 2000 group is statistically different, with the Control group and Zingiber officinale 300 being statistically similar to each other (p<0.05).Cholesterol, triglycerides, urea, and creatinine were measured, observing significant differences in all these parameters in the Zingiber officinale 2000 group (p<0.05) in both males and females (Figure 3).

Histopathological analysis
The liver tissue of the group that received the Z. officinale extract shows mild lymphocytic portal inflammation, hepatocytes with condensed nuclei, and the presence of eosinophilia, as well as moderate hepatic steatosis; this effect is observed at the dose of 2000 mg/kg, in comparison to the control group.The kidneys at the same dose show a slight infiltrate with the presence of lymphocytes and some polymorphonuclear cells around renal tubules and glomeruli in the region of the renal cortex, as well as in the brain tissue in which a slight increase in the number of astrocytes with reaction mild focal glial is observed (Figure 4).The lung tissue does not show histological alterations in bronchi or bronchioles in both males and females.The fundic gastric mucosa shows normal glandular cells: mucinous, parietal, and main cells distributed normally in its three regions, with a slight inflammatory infiltrate between them made up of some lymphocytes and polymorphonuclear cells at the dose of 2000 mg/kg, as well as in the cardiac tissue, which shows in   both males and females some polymorphonuclear cells between the cardiomyocytes between the cardiac fibers.All these effects are dosedependent (Figure 5).
In male rats, prostate glands vary slightly in shape and size, are observed in moderate quantity, and are covered by simple pseudostratified epithelium without atypia.They have oval nuclei, separated by a moderate amount of stroma, which does not show alterations at the dose of 300 mg/day/kg.At the dose of 2000 mg/kg, the prostate shows mild inflammation containing some polymorphonuclear cells and lymphocytes in its lumen.The testes in the experimental groups do not show damage, with the presence of testicular parenchyma with seminiferous tubules of normal conformation, containing spermatogenic cells in different phases of differentiation, the presence of Sertoli cells, Spermatogonia, secondary spermatocytes, spermatids and mature spermatozoa in the tubular lumen, the stroma shows a moderate amount of Leydig cells, with preserved vascularization and no inflammatory infiltrate or neoplastic changes are observed.While in female rats at doses of 300 mg/kg and 2000 mg/kg, the uterine wall is observed with a normal conformation of its three layers: endometrium, myometrium, and perimetrium, the smooth muscle fibers of the myometrium have a normal arrangement, tapered shapes with oval nuclei without atypia, surrounded by a stroma with adequate vascularization.The ovaries at both doses show ovarian follicles in different phases of differentiation, with primary and secondary follicles and corpora lutea with normal characteristics (Figure 6).

DISCUSSION
The rhizome of Zingiber officinale is very useful in the culinary field; however, the doses usually used are low compared to therapeutic uses, which is why a toxicity study is of great importance despite being a highly consumed product over time.The experimental animals' weights are observed to 14 days of treatment.Although a slight increase in this is observed at 7 and 14 days of treatment, it is not statistically significant.If the treatment had been for a longer time, higher variation could have been observed and determined if the consumption of the ethanolic extract of Zingiber officinale produces changes in the weight of the experimental animals, as mentioned by Attari E. et al (2016), a study in which Zingiber officinale was administered to obese women for 12 weeks and a decrease in weight was observed (Figure 1). 20e measurement of the liver markers AST, ALT, and ALP was carried out.2] The analysis of liver function is important for evaluating the toxicity of plant extracts. 23In both the female and male groups, a statistically significant elevation of the three parameters is observed in the group that received the Zingiber officinale 2000 extract.This may be due to the high concentration of the extract (Figure 2). It is shown that urea and creatinine levels increase in the Zingiber officinale 2000 group (Figure 3) compared to the control group (p<0.05).These parameters are of greater importance to be measured since they are considered sensitive biomarkers of kidney damage. 21Plasma urea generally increases in kidney diseases, and its clearance decreases when the kidney fails, while creatinine is produced endogenously, released into body fluids at a constant rate, and its plasma concentration is maintained by glomerular filtration.Serum urea values of normal adult rats are measured between 15 and 45 mg/dl, and creatinine in adult rats is approximately 0.2 to 0.8 mg/dl 26 , so in this study, the mean values of urea would be within the normal range, but creatinine levels would show a slight significant increase.
Histological changes in the liver show a dose-dependent toxic effect, with the presence of mild liver damage in the liver parenchyma and mild lymphocytic portal inflammation, showing mild steatosis at the dose of 2000 mg/kg (Figure 4).This is related to the parameters of AST, ALT, and ALP that were also observed to increase (Figure 2).A previous study has shown that Z. officinale extract at doses of 300 and 600 mg/kg prevents liver injury in a model of liver fibrosis induced by carbon tetrachloride in male Wistar rats, observing a decrease in inflammation, modulating the TGF-β1/Smad3 and NF-κB/IκB signaling pathways. 27Furthermore, the Z. officinale extract has higher antioxidant capacity thanks to the bioactive compounds that this plant has 28 , so the damage observed in this study at the dose of 2000 mg/kg may be caused by a prooxidant effect of the antioxidants that depend on the dose of these. 29e renal tissue shows a slight inflammatory infiltrate with the presence of lymphocytes and some polymorphonuclear cells around renal tubules and glomeruli at the dose of 2000 mg/kg, compared to the control group, which shows renal parenchyma without histological alterations (Figure 4).This effect is related to the levels of urea and creatinine that were also observed to be increased in the group that received doses of 2000 mg/kg (Figure 3).Studies show nephroprotective effects in rats treated with Z. officinale extract with reversal of histomorphological changes in kidney tissues in animals exposed to fluoride. 30The nephroprotective and antioxidant potential of ginger against kidney lesions in osteoporotic rats was also confirmed.Analysis by HPLC revealed that Z. officinale contained appreciable amounts of phenols and flavonoids and exhibited high purifying activity 31 .Regarding high doses of Z. officinale extract (2000 mg/Kg), it has been shown that these large amounts of antioxidant substances can cause damage due to antioxidative stress. 29e brain tissue of the experimental groups shows normal histological characteristics, with a slight increase in the number of astrocytes and glial response to the dose of 2000 mg/kg, while the rest of the brain tissue shows conservation of its normal architectural conformation.The Zingiberaceae family has several bioactive compounds with significant systemic activities in the brain; these compounds can affect the expression of neurotrophic agents. 32Z. officinale extract protects lipid peroxidation in brain homogenate; this protection mechanism is correlated with the property of this extract to eliminate free radicals. 33ikewise, Z. officinale was effective in ameliorating and protecting against the neurotoxic effects of reserpine in the hippocampus. 34One of the bioactive compounds is 6-shogaol, which showed significant neuroprotective effects in an in vivo model of transient global ischemia through inhibition of microglia by repressing the production of prostaglandin E2, interleukin-1β, and tumor necrosis factor-α, and by downregulating cyclooxygenase-2, p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB) expression. 35 the other hand, in brain samples with doses of 2000 mg/Kg, the excess of bioactive antioxidant compounds may be the cause of the glial reaction and inflammation.Reductive stress (oxidative stress) is harmful to the structure and neuronal function, which can affect neurogenesis through protein misfolding and endoplasmic reticulum dysfunction, promoting protein aggregation and proteotoxicity. 36The use of excessive antioxidants causes reductive stress, which disturbs the functionality of the blood-brain barrier and angiogenic properties and has adverse implications for the regulation of the brain's homeostatic environment.Thus, irresponsible treatment with antioxidants tends to mitigate reactive oxygen species (ROS)-induced stimulation of normal cellular mechanisms, including cell proliferation, membrane permeability and transport, and mitochondrial function. 37 the histological analysis of lung tissue, no histopathological changes were observed in the structures of bronchi and bronchioles (Figure 5).No changes were observed in the gastric and cardiac tissues at the dose of 300 mg/Kg, likely due to the antioxidant properties of Z. officinale, which prevent oxidative stress and inflammation.This antioxidant capacity is attributed to compounds present in this root, such as 10-gingerol and 6-shogaol. 38However, at the dose of 2000 mg/ Kg (Figure 5), a slight inflammatory process is evident in both gastric and cardiac tissues.This effect is most likely caused by an excess of the same substances that, at a lower concentration (300 mg/Kg), provide the antioxidant characteristic of Z. officinale.Some food components, such as polyphenols, flavonoids, and carotenoids, among other compounds, have cytoprotective and antioxidant activity. 39However, these can also exhibit prooxidant activities when consumed in high doses or when metal ions are present.1] This prooxidant activity of the substances contained in the extract of Z. officinale at high doses (2000 mg/Kg) could be involved in the inflammation observed in gastric and cardiac tissues.Thus, high levels of flavonoids can cause cytotoxicity, DNA damage, apoptosis, and the presence of ROS due to autooxidation. 42Likewise, phenolic antioxidants show prooxidant activities in the presence of transition metal ions such as iron and copper at high concentrations, forming chelators and reducing antioxidant capacity. 43e Z. Officinale extract does not generate any change in the testicular parenchyma, which is composed of normally conformed seminiferous tubules.These tubules contain spermatogenic cells in different phases of differentiation, with the presence of Sertoli cells, spermatogonia, secondary spermatocytes, spermatids, and mature spermatozoa in the tubular lumen.The testicle shows its thin albugineal layer with normal characteristics.No inflammatory infiltrate or neoplastic changes were observed.In contrast, the prostate shows acute inflammation and suppurative cryptitis, with some prostate glands containing polymorphonuclear cells and lymphocytes in their lumen, but only at the dose of 2000 mg/kg of the Z. Officinale extract.This is in comparison to the control group, which shows a simple pseudostratified epithelium without atypia, with oval nuclei, separated by a moderate amount of stroma (Figure 6).
In female rats, the ovary displays ovarian follicles in different phases of differentiation, including primary and secondary follicles, as well as corpora lutea with normal characteristics.The endometrium and endometrial glands exhibit a ciliated pseudostratified epithelial lining without atypia, along with smooth muscle fibers.The myometrium has a normal arrangement, with tapered shapes and oval nuclei without atypia, surrounded by a stroma with adequate vascularization.It is only at the dose of 2000 mg/kg that the endometrial stroma shows a slight inflammatory infiltrate (Figure 6).

CONCLUSIONS
The acute toxicity evaluation of the ethanolic extract of the rhizome of Zingiber officinale Roscoe in rats revealed histopathological changes, mainly at the dose of 2000 mg/kg.The liver showed mild lymphocytic portal inflammation, condensed hepatocyte nuclei, eosinophilia, and moderate hepatic steatosis.The kidneys exhibited a mild infiltration of lymphocytes and some polymorphonuclear cells around the renal tubules and glomeruli at the same dose.The brain showed a slight increase in the number of astrocytes with focal glial reaction, and the stomach and heart also showed mild inflammatory processes.Biochemical parameters such as cholesterol, triglycerides, AST, ALT, ALP, urea, and creatinine in rat serum showed statistically significant differences between the dose of 2000 mg/Kg/day and the control group.

Figure 1 :
Figure 1: Body weight of rat in the treated and control groups during the 14-day acute toxicity assessment

Figure 2 :Figure 3 :
Figure 2: Biochemical parameters (AST, ALT and ALP) of rat serum after acute oral administration of extracts of Zingiber officinale Roscoe.#: A statistically significant difference was observed with Control female group; *: A statistically significant difference was observed with Control male group, according to the ANOVA statistical analysis (p<0.05) and Tukey's post hoc test

Figure 6 :
Figure 6: Photomicrographs of histological sections from prostate, testicle, uterus, and ovary of Holtzman rats treated with aqueous extract of officinale Roscoe