Acute, subacute oral toxicity and Ames test of Py-mulin: an antibacterial drug candidate

Background Py-mulin is a new pleuromutilin derivative with potent antibacterial activities in vitro and in vivo, suggesting this compound may lead to a promising antibacterial drug after further development. The present study is aimed to evaluate the acute and subacute oral toxicity, and the genotoxicity with the standard Ames test according to standard protocols. Methods Acute oral toxicity of Py-mulin was determined using Kunming mice. The 28-day repeated dose oral toxicity study in SD rats was performed according to OECD guideline No. 407. The bacterial reverse mutation (Ames test) was carried out using four Salmonella typhimurium (S. typhimurium) strains TA97, TA98, TA100 and TA1535 with and without S9 metabolic activation. Results The LD50 values in acute oral toxicity were 2973 mg/kg (female mice) and 3891 mg/kg (male mice) calculated by the Bliss method. In subacute toxicity study, 50 mg/kg Py-mulin did not induce any abnormality in body weight, food consumption, clinical sign, hematology, clinical chemistry, organ weight, and histopathology in all of the treatment groups. However, high doses of Py-mulin (100 and 300 mg/kg) displayed slightly hepatotoxicity to female rats. Furthermore, Py-mulin did not significantly increase the number of revertant colonies of four standard S. typhimurium strains with the doses of 0.16–1000 μg/plate in the Ames study. Conclusions Based on our findings, our study provides some information for the safety profile of Py-mulin.

Molecular Docking Senyawa Ekstrak Etanol Daun Kenikir (Cosmos caudatus) Sebagai Inhibitor Il-6 Dalam Respon Inflamasi

Inflamasi merupakan reaksi alami tubuh untuk melindungi diri dari patogen menyebab penyakit. Interleukin-6 (IL-6) berperan sebagai sitokin pro-inflamasi dan antiinflamasi yang dikeluarkan oleh sel T dan makrofag untuk merangsang respon imun saat terjadi infeksi. Pada penelitian ini, dilakukan studi in silico ekstrak etanol daun kenikir (Cosmos caudatus) sebagai anti-inflamasi yang didasarkan pada afinitas terhadap Interleukin-6 (IL-6) dengan menggunakan metode molecular docking secara in silico. Penelitian ini meliputi simulasi penambatan molekuler kandungan senyawa ekstrak etanol daun kenikir terhadap reseptor IL-6 (PDB ID : 1ALU) dan prediksi profil farmakokinetik dan toksisitas menggunakan server Pre-ADMET. Hasil penelitian menunjukkan bahwa diperoleh empat senyawa potensial, yaitu C1 (Kaempferol), C2 (1-Caffeoylquinic acid, C3 (Procyanidin B1), dan C5 (Quercetin-3-O-pentoside).Keempat senyawa tersebut memiliki nilai ?G dan Ki yang lebih baik dibandingkan dengan ligan alami. Nilai ?G; Ki senyawa uji tersebut, yaitu berturut-turut (-5,29 Kkal/mol; 133,31 uM), (-6,02 Kkal/mol; 38,44 uM), (-5,38 Kkal/mol; 114,24 uM), dan (-5,46 Kkal/mol; 99,50 uM). Hasil PreADMET keempat senyawa tersebut memiliki daya adsorpsi yang baik sehingga dapat dipertimbangkan dalam pembuatan sediaan rute oral. Hasil ames test menunjukkan bahwa C1 dan C2 bersifat mutagenik, sedangkan C3 dan C4 non mutagenik.

Evaluation of the genotoxic and mutagenic potentials of a living nosode compounded with Influenza A virus

Background: The influenza virus has been responsible for contagious respiratory diseases with high mortality rates [1]. Some drugs have been used to treat human influenza. However, these drugs cause many common side effects and induce the appearance of resistant viral strains [2]. The impact caused by the influenza virus has motivated the development of new approaches for the prevention and control of influenza [3]. Therefore, a new homeopathic medicine was developed using, as a starting point, the infectious influenza virus [4]. This belongs to a group called living nosodes [5]. However, its mutagenic and genotoxic potentials, especially when used in low dilutions, has not yet been evaluated and it is important because this biotherapic is prepared from living microorganisms. Different methods can be used to detect mutagenic and genotoxicic effects. Aims: This study aims to evaluate the genotoxic and mutagenic potentials of influenza A living nosode at different homeopathic potencies. Methodology: 1 ml of purified viral suspension was diluted in 9 ml of sterile distilled water. This sample was submitted to 100 mechanical succussions (approximately 3 Hz), using Autic® Brazilian machine, originating the first dilution, named decimal (1x). 1 ml of this solution was diluted in 9 ml of solvent and was submitted to 100 sucussions, generating biotherapic 2x. This procedure was successively repeated, according to Brazilian Homeopathic Pharmacopoeia, to obtain the biotherapic 30x [6]. By the same technique, water vehicle was prepared until 30x potency to be used as control. All samples were prepared in sterile and under aseptic conditions, using laminar flow cabinet, class II, and were stored in the refrigerator (8ºC). The samples 1x, 6x, 12x, 18x, 24x and 30x and water 30x (vehicle control) were analysed by: the Inductest, which assesses the ability of physical or chemical agents to promote lysogenic induction as a reflection of damage in DNA molecules in lysogenic bacteria, and the Ames test, which uses indicator strains of Salmonella typhimurium, sensitive to substances that can induce different types of mutation. Results: The Inductest showed no decrease in the survival fraction of the bacteria used, and no increase in the formation of lysogenic induction, in any tested potency. The same profile was obtained after the Ames test, with similar results to negative control. Conclusion: We can conclude that this living nosode compounded with Influenza A virus is not able to induce DNA damage in prokaryotic cells. This result permits us to conclude that patients who use this medicine have no side effects related to mutagenesis and genotoxicity.

Evaluation of the genotoxic and mutagenic potentials of phytotherapic and homeopathic solutions of Euphorbia tirucalli Lineu (Aveloz)

Introduction: Euphorbia tirucalli Lineu, commonly known as Aveloz, is a very common plant found in tropical regions [1]. The ingestion or contact with its latex causes symptoms such as vomiting and diarrhea, pallor, skin irritation, hepatotoxicity as well as carcinogenesis [2]. Moreover, the Aveloz latex is also responsible for a few important activities against some infectious and neoplastic diseases. Aveloz latex phytochemical composition may vary according to seasonal aspects and geographic location [3], and it is used either orally or topically in traditional medicine. Popularly known as an antitumoral agent (breast, prostate, lung, kidney), it is used not only in Brazil, but in several other countries. According to the literature, the latex could have a dual behaviour, activating or inhibiting tumoral events [3-6]. However, there are few reports discussing these mechanisms. Besides, the mutagenic and genotoxic potentials of phytochemical and homeopathic Aveloz have not yet been described. Several experimental methods have been used to evaluate the mutagenic and genotoxic effects, such as Inductest, the Ames test and the chromotest. Objective: This study aims to evaluate the genotoxic and mutagenic potentials of Aveloz latex and Aveloz phytotherapic and homeopathic solutions. Methodology: In this study, Aveloz 5 and 30cH are prepared according to Brazilian Homeopathic Pharmacopoeia [7], from Aveloz latex collected in the Center for Natural Products Research (NPPN) at the Universidade Federal do Rio de Janeiro [8]. The Aveloz phytochemical solution was prepared following the doses used in folk medicine: 2 drops diluted in 250ml of water and 2 drops diluted in 25 ml of water. All test solutions were submitted to the following methodologies: (a) Inductest: assesses the ability of physical or chemical agents to promote lysogenic induction as a response to DNA damage in lysogenic bacteria; (b) The Ames test: uses indicator strains of Salmonella typhimurium, which are sensitive to substances that can induce different types of mutation; (c) Chromotest: evaluates the genotoxicity of chemicals through the induction of the bacterial SOS system. Results: In the Inductest there was no decrease in bacterial survival fraction and no increase in lysogenic cycle. As measured by The Ames test and the chromotest no mutagenic or genotoxic potentials were detected. Discussion: The homeopathic and the phytochemical Aveloz solutions were unable to produce lysogenic induction or mutagenesis in bacterial cells and they were also unable to produce genotoxic effects, as measured by chromotest. Conclusion: Our results showed that no mutagenic or genotoxic damages were induced by all Aveloz preparations studied, thus we are led to believe that patients using Aveloz as a complementary therapy present no side effects in relation to mutagenesis and genotoxicity.

Evaluation of the genotoxic and mutagenic potentials of homeopathic Candida albicans solutions

Background: Candida spp is naturally found in humans’ flora of skin, gastrointestinal and genitourinary tracts and, in general, up to 75% of the population does not have any symptom [1]. However, oral candidiasis is very common among HIV patients and patients undergoing chemotherapy. The treatment of oral candidiasis is necessary once the disease causes discomfort and dysphagia, resulting in poor nutrition, slow recovery, and prolonged hospital stay [2,3]. Preliminary results obtained by our group with a new biotherapic prepared from Candida albicans (Candida 30x) showed a great potential to reduce the candida yeast adhesion rate when the epithelial cells were pre-treated. This study is currently being developed with the evaluation of mutagenic and genotoxic potentials of several homeopathic solutions. Aims: The goal of this study was to assess the genotoxic and mutagenic potentials of different homeopathic potencies of C. albicans. Methodology: One part of C. albicans yeast obtained from Brazilian patient’s blood [4] was diluted in 9 parts of sterile water. This sample was submitted to 100 mechanical succussions (approximately 3 Hz), using Autic® Brazilian machine, originating the first dilution (1x). Then, 1 ml of this solution was diluted in 9 ml of solvent, submitted to 100 succussions, obtaining 2x potency. This procedure was successively repeated to obtain 30x potency, according to Brazilian Homeopathic Pharmacopoeia [5]. By the same technique, water vehicle was prepared until 30x to be used as control. All samples were prepared in sterile and aseptic conditions, using laminar flow cabinet, class II and were stored in the refrigerator (8ºC). The samples 1x, 6x, 12x, 18x, 24x and 30x of C. albicans and water 30x (vehicle control) were analysed by: the Inductest, which assesses the ability of physical or chemical agents to promote lysogenic induction as a reflection of damage in DNA molecules in lysogenic bacteria, and the Ames test, which uses indicator strains of Salmonella typhimurium, sensitive to substances that can induce different types of mutation. Results: In the Inductest no decrease in survival fraction of bacteria and no increase in the formation of lysogenic induction were detected independently of the homeopathic potency employed. The same profile was obtained after the Ames test, with similar results to negative control. Conclusion: Afterwards, we can conclude that these samples are not able to induce DNA damage in the cells tested. So, the use of this medicine does not present any side effects related to mutagenesis and genotoxicity.

Assessment of genotoxicity of Ssanghwa-tang, an herbal formula, by using bacterial reverse mutation, chromosome aberration, and in vivo micronucleus tests

Objectives: Ssanghwa-tang (SHT) is a traditional herbal formula comprising nine medicinal herbs, and it is used for reducing fatigue in Korea. SHT exerts various effects such as anti-inflammatory, antioxidant, and anti-aging activities, and protection against acute hepatotoxicity. However, the genotoxicity of SHT has not yet been established.Methods: Ten components were identified in SHT water extract by using high-performance liquid chromatography analysis. We assessed the genotoxicity of SHT by using bacterial reverse mutation (Ames test), chromosome aberration, and in vivo micronucleus tests.Results: The contents of paeoniflorin, glycyrrhizin, and liquiritin apioside in SHT were 15.57, 6.94, and 3.48 mg/g extract, respectively. SHT did not increase the revertant colonies of Salmonella typhimurium and Escherichia coli strains in the presence or absence of metabolic activity. Although SHT did not induce structurally abnormal chromosomes in Chinese hamster lung (CHL) cells in the presence of metabolic activity, the number of structurally aberrated chromosomes increased dose-dependently in the absence of metabolic activity. In the in vivo micronucleus test, SHT did not affect the formation of micronuclei compared with the vehicle control.Conclusions: Genotoxicity of SHT was not observed in the Ames test and in vivo micronucleus test. However, based on the results of chromosome aberration test, it can be presumed that SHT has the potential to induce genotoxicity because it induced structurally abnormal chromosomes in the absence of metabolic activity.

Evaluation of mutagenicity, genotoxicity and chronic toxicity of antiviral drug imidazolyl ethanamide pentandioic acid in in vitro and in vivo test systems

Introduction. The antiviral properties of imidazolyl ethanamide pentandioic acid (IPA), the active compound of the drug product, has been proven in various experimental models. However, the literature data on the toxicological properties of IPA are limited.Purpose. To evaluate mutagenic and genotoxic properties in in vitro and in vivo models, as well as to study the toxicity of IPA following chronic oral administration to rats and dogs.Materials and methods. Mutagenic and genotoxic properties of IPA were assessed using the Ames test, the test of chromosomal aberrations in human lymphocytes, and the micronucleus test in rats. The chronic toxicity of IPA was studied in Sprague Dawley rats and beagle dogs of both sexes, to which IPA was administered orally at doses of 30-300 mg/kg/day for 26 and 39 weeks, respectively.Results and discussion. In the Ames test, the addition of IPA up to the maximum dose (5000 mcg/plate) did not result in the increase in the number of revertant colonies. At a concentration of up to 5000 mcg/ml, IPA did not cause chromosomal aberrations in human leukocytes. At doses doses ≤ 2000 mg/kg, IPA did not increase the amount of micronuclei in the bone marrow of rats. In chronic experiments, animals tolerated the administration of IPA well: the dose without an observed effect (NOEL) for rats and dogs was 300 mg/kg/day.Conclusion. IPA did not show mutagenic and genotoxic properties in standard in vitro and in vivo tests. With chronic oral administration to rats and dogs, NOEL IPA equal to 300 mg/kg/day provided a systemic exposure that was 8-10 and 41-65 times higher than that in humans, respectively. The results obtained allow us to consider the safety profile of the prolonged use in humans as favorable.

In Vitro Genotoxicity Assessment from the Glycyrrhiza New Variety Extract

The various species that comprise the genus Glycyrrhiza (Licorice) have long been used as oriental herbal medicines in Asian countries. Wongam (WG), which is a new variety of Glycyrrhiza, was developed in Korea to overcome the limitations of low productivity, environmental restrictions, and an insufficient presence of glycyrrhizic acid and liquiritigenin. In this study, we evaluated WG extract’s genotoxicity through an in vitro bacterial reverse mutation (AMES) test, an in vitro chromosome aberration test, and an in vivo mouse bone marrow micronucleus test. In the AMES test, WG extract at concentrations of up to 5000 µg/plate showed no genotoxicity regardless of S9 mix. No chromosome aberrations appeared after 6 h in 1400 µg/mL WG extract regardless of S9 mix or in 1100 µg/mL WG extract after 24 h without S9 mix. Nor was there a significant increase in the number of micronucleated polychromatic erythrocytes to total erythrocytes up to 5000 mg/kg/day for 2 days detected in the micronucleus test. These results confirm that WG extract is safe for use as an herbal medicine, as it precipitates no detectable genotoxic effects.

Teaching during the COVID-19 Pandemic: Sharing Results and Data Obtained from the Ames Test

We present a resource for instructors that contains results and data sets from the Ames test. Our aim is to share the results we have collected in previous semesters with other instructors, so they will be able to “conduct” the Ames test without the need to set foot in a laboratory classroom.