Composition and Evaluation of Acute, Subacute, and Developmental Toxicity of Essential oil and Developmental Toxicity of aqueous crude extracts of Thymus schimperi in Wistar Albino rats; In vivo and In-Silico Toxicity Studies.

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Addis Abeba University


ABSTRACT Background:In Ethiopian traditional medicine, the aerial part of Thymus schimperi is widely used to treat diseases such as gonorrhea, cough, liver disease, kidney disease, hypertension, stomach pain, and fungal skin infections. In addition, theyhave been used as vegetables to flavor a broad variety of food products. However, there is an insufficient investigation of the toxic effect of T. schimperi. The aim of this study was, therefore, to evaluate the acute, sub-acute, developmental, and in-silico toxicity of the essential oil,as well as the developmental toxicity of the aqueous crude extract of T. schimperi on the Wistar albino rats. Method: The aqueous extracts of T. schimperi leaves were prepared.Essential oil of the aerial part of T. schimperiwas extracted by hydrodistillation and was analyzed by GC-MS. The oil was subjected to toxicity studies. In the acute toxicity study, rats were randomly divided into seven groups (n=5). The control group received the vehicle (distilled water and 2% tween 80)whereas the experimental groups received single doses of 300, 600, 900, 1200, 1500, and 2 000 mg/kg of vehicle dissolved essential oil. In the sub-acute toxicity study, rats were randomly divided into four groups (n=10). The control group received the vehicle whereas the experimental groups received vehicle-dissolved doses of 65 mg/kg, 130 mg/kg, and 260 mg/kg of oil orally for 28 days. At the end of the experiment, blood samples were collected for hematology and clinical chemistry evaluation. Gross pathology and histopathology of the liver and the kidneys were also evaluated. For the in-silico toxicity study, PubChem CID numbers of GC-MS identified bioactive compounds in essential oils of T. schimperi have been obtained from PubChem. Chemdraw (8.0) was used to construct the two-dimensional structure of the compounds. The Swiss ADMET web tool was used to convert the two-dimensional structures into a simplified molecular-input line input system (SMILES). Furthermore, the toxicity parameters were predicted via Protox II, vNN, and ADMET servers. For aqueous and essential oil extracts developmental toxicity experiments, five groups of Wistar albino rats, each consisting of ten pregnant rats, were used as experimental animals. For the aqueous crude extract developmental toxicity study, the rats in groups III-V were given 500 mg/kg, 1000 mg/kg, and 2000 mg/kg extract of T.schimper, respectively. On the other hand, in the essential oil developmental toxicity study, the doses 65 mg/kg, 130mg/kg, and 260 mg/kg of the essential extract of T. schimperi were administered forIII-V groups, respectively. Group I and II were negative and ad libitum controls for both experiments. Similarly, Embryos and fetuses were revealed on days 12 and 20 of gestation, respectively. The embryos were examined for developmental delays or growth retardation. Gross external, skeletal, and visceral anomalies in the fetuses were examined. Histopathological examination was carried out on the placenta from both the treatment and control groups. Results: In this study, the LD of the essential oil of T. schimperi was found to be 1284.2 mg/kg. According to the World Health Organization, the oil is classified as moderately hazardous in its oral administration. In the subacute toxicity study, rats showed no significant changes in behavioral indices, gross pathology, body weight, biochemical, and in most hematological parameters. However, hematological profiles showed a significant decrement in 50WBC counts and a significant increment of MCV in high dose (260 mg/kg) groups as compared to the control group. Furthermore, no significant differences were observed between the control and essential oil-treated groups, observed in the gross and histopathology of the liver and the kidneys. In the in-silico toxicity study, all compounds derived from essential oil showed no cardiac toxicity (h-ERG Blocker), AMES (Ames Mutagenicity), and cytotoxicity via Pro Tox II, ADMET, and vNN-ADMET toxicity predictors. However, by using these servers, from the total 57 compounds, around 21% showed carcinogenicity, 8.8% showed hepatotoxicity, 3.5% caused drug-induced liver injury, 3.5% showed immunotoxicity, and only 1.75 % were potentially toxic to the mitochondrial membrane. In the aqueous crude extract developmental toxicity study, on embryo day 12, the number of somites and the morphological scores in the high-dose treatment group were significantly lower than the control groups. Similarly, the number of implantation sites, fetal weight, fetal resorption, CRL, and placental weightwere also significantly lower in the high dose (2000 mg/kg)treatment group. The mean numbers of implantation sites in the pair-fed control group and the high dose(2000 mg/kg)group were 11.1 ± 0.76 and 8.01 ± 0.45, respectively. Similarly, in the middle dose (1000 mg/kg)and high dose (2000 mg/kg)groups, the developments of the otic and olfactory systems were significantly delayed. Furthermore, in the high dose group (2000 mg/kg), the developmental score of optic system, the number of branchial bars, and the maxillary and mandibular processes were significantly lower than the control groups. Treatment with the aqueous extract of the T.schimpericaused no skeletal or soft tissue malformations. In an essential oil developmental toxicity study, the developmental socres of fetal resorptions, crown-rump length, the number of somites, and morphological scores were significantly lower in 12-day-old rat embryos treated with 260 mg/kg of the extract. There was also a significant delay in the developments of the otic system, olfactory system, and a reduction in the number of branchial bars in day-12 embryos treated with 260 mg/kg of the oil.However, external morphological examinations of rat fetuses revealed no detectable structural abnormalities. The fetal skull, vertebrae, hyoid, forelimb, and hindlimb ossification centers did not differ significantly across all groups. Moreover, treatment with the essential oil caused no skeletal or soft tissue malformations. Although the difference was not statistically significant, fetuses of high-dose treated rats had a reduced number of ossification centers in the caudal vertebrae and hind limp phalanges.There were no significant histopathological changes in placentas in either the crude aqueous extract or the essential oil experiments. Although the difference was not statistically significant, placentas from high-dose essential oil treatment rats had increased decidual cystic degeneration, thrombosis in the intervillous spaces, and decidual cellular apoptosis. Similarly, in the essential oil experiment, capillary dilation and terminal villi proliferation increased dosedependently. Conclusion: From this study, oral administration of the essential oil T. shimperi up to a dose of 130 mg/kg is not harmful. However, in the high-dose (260 mg/kg) group, the WBC count was significantly decreased and the MCV was significantly increased. In the in-silico toxicity study,most of the components of the oil were found to be nontoxic although a few of the compounds showed carcinogenicity, hepatotoxicity,immunotoxicity and mitochondrial membrane potential toxicity.The crude aqueous and essential oil extracts of T. shimperi at high doses have a detrimental effect on the development of rat embryos and fetuses. Its developmental toxicity is evidenced by significant delays in fetal and embryonic development, a decrease in the number of implantation sites, and an increase in fetal resorption. Furthermore, administration of the aqueous crude and essential oil extracts in higher doses resulted in a significant decrease in placenta weight, and litter weight. It is, therefore, essential to conducting chronic toxicity of the essential oil as well as its components which showed toxicity in the in- silico study before using preparations containing T. schimperi essential oil as drugs. In addition, the present study provided evidence that using the T.schimperi extracts in a high dose could affect the developing embryo and fetus. Thus, it is recommended to discourage the use of crude and essential oil extracts in high doses.



Thymus schimperi; Aqueous crude extract;Essential oil; Acute toxicity; Sub-acute toxicity;Developmental toxicity;Insilico toxicity; and Wistar albino rats.