Cytotoxic, acute oral toxicity, genotoxic and mutagenic assessment of the essential oil from fresh leaves of Croton argyrophyllus (Kunth.).

ETHNOPHARMACOLOGICAL RELEVANCE: Croton argyrophyllus Kunth., commonly known as "marmeleiro" or "cassetinga," is widely distributed in the Brazilian Northeast region. Its leaves and flowers are used in traditional medicine as tranquilizers to treat flu and headaches. AIM OF THE STUDY: This study was conducted to determine the chemical composition and toxicological safety of essential oil from C. argyrophyllus leaves using in vitro and in vivo models. MATERIALS AND METHODS: The chemical composition of the essential oil was determined using a gas chromatograph coupled to a mass spectrometer. Cytotoxicity was tested in the HeLa, HT-29, and MCF-7 cell lines derived from human cells (Homo sapiens) and Vero cell lines derived from monkeys (Cercopithecus aethiops) using the MTT method. Acute toxicity, genotoxicity. Mutagenicity tests were performed in Swiss mice (Mus musculus), which were administered essential oil orally in a single dose of 2000 mg/kg by gavage. RESULTS: The main components of the essential oil were p-mentha-2-en-1-ol, α-terpineol, ß-caryophyllene, and ß-elemene. The essential oil exhibited more than 90% cytotoxicity in all cell lines tested. No deaths or behavioral, hematological, or biochemical changes were observed in mice, revealing no acute toxicity. In genotoxic and mutagenic analyses, there was no increase in micronuclei in polychromatic erythrocytes or in the damage and index in the comet assay. CONCLUSIONS: The essential oil was cytotoxic towards the tested cell lines but did not exert toxic effects or promote DNA damage when administered orally at a single dose of 2000 mg/kg in mice.

In vitro and in vivo genotoxicity evaluation of ALP1018, a nanomineral food supplement.

The use of nano-based dietary supplements is increasing around the world, as nanotechnology can help enhance nutrient bioavailability. ALP1018 is a newly developed iron-zinc complex supplement designed as a nanoformulation to improve the efficacy of iron and zinc supplementation. However, safety concerns have been raised, as there is no clear evaluation of ALP1018 toxicity. The goal of this study was to determine the potential mutagenicity and genotoxicity of ALP1018 through three standard screenings: the Ames test, which evaluates bacterial reverse mutations; the in vitro test of chromosomal aberration in Chinese hamster lung cells; and the in vivo micronucleus assay using ICR mice. ALP1018 showed no mutagenic effect, as no increase was observed in the presence or absence of metabolic activation (S9 mix) in revertant colonies on all the bacterial strains used in the Ames test. No structural chromosomal abnormalities were observed in the presence or absence of the S9 mix in mammalian cells used in the chromosomal aberration assay. In the micronucleus test, the frequency of micronucleated polychromatic erythrocytes was not significantly increased in mouse bone marrow cells. Based on these findings, we can conclude that ALP1018 is safe to use and has no mutagenic or genotoxic potential.

Tellimagrandin-I and camptothin a exhibit chemopreventive effects in Salmonella enterica serovar Typhimurium strains and human lymphocytes.

Tellimagrandin-I (TL) and camptothin A (CA) are ellagitannins widely found in diverse plant species. Numerous studies demonstrated their significant biological activities, which include antitumor, antioxidant, and hepatoprotective properties. Despite this protective profile, the effects of TL and CA on DNA have not been comprehensively investigated. Thus, the aim of this study was to determine the mutagenic and antimutagenic effects attributed to TL and CA exposure on Salmonella enterica serovar Typhimurium strains using the Ames test. In addition, the cytotoxic and genotoxic effects were examined on human lymphocytes, employing both trypan blue exclusion and CometChip assay. The antigenotoxic effect was determined following TL and CA exposure in the presence of co-treatment with doxorubicin (DXR). Our results from the Ames test indicated that TL or CA did not display marked mutagenic activity. However, TL or CA demonstrated an ability to protect DNA against the damaging effects of the mutagens 4-nitroquinoline-1-oxide and sodium azide, thereby exhibiting antimutagenic properties. In relation to human lymphocytes, TL or CA did not induce significant cytotoxic or genotoxic actions on these cells. Further, these ellagitannins exhibited an ability to protect DNA from damage induced by DOX during co-treatment, indicating their potential beneficial usefulness as antigenotoxic agents. In conclusion, the protective effects of TL or CA against mutagens, coupled with their absence of genotoxic and cytotoxic effects on human lymphocytes, emphasize their potential therapeutic value in chemopreventive strategies.

Cellular toxicity and DNA damage induced by Newbouldia laevis used for male infertility treatment in prokaryotic and eukaryotic models.

Leaves of Newbouldia laevis have been extensively used in solving problems associated with infertility and childbirth in many African countries. Yet, information is very limited on the DNA damaging potential of this plant. This study evaluated the cytogenotoxic effect of the aqueous extract of N. laevis leaf using prokaryotic models (Ames Salmonella fluctuation test using TA100 and TA98 strains of Salmonella typhimurium and SOS Chromotest with Escherichia coli PQ37) and eukaryotic model (Allium cepa root cells). Identification of the volatile organic compounds (VOCs) and phytochemical screening of the plant extract were also performed. Onion bulbs were grown on each concentration (1 to 50%; v/v, extract/tap water) of the extract for chromosomal aberrations and root growth analyses. Results of the Ames test indicated that the extract is mutagenic while the SOS Chromotest results showed good complementation to the Ames test results, although the E. coli PQ37 system showed slightly higher sensitivity in the detection of mutagenicity and genotoxicity of the extract. The plant extract was cytotoxic when compared to the control, inducing a significant (p < 0.05) concentration-dependent inhibition of root growth from 5 to 50% concentrations. At 50% concentration, the extract completely inhibited cell division in the A. cepa. Also, chromosomal aberration increased significantly (p < 0.05) in exposed onions from 5 to 20% concentrations. The mutagenicity and cytogenotoxicity recorded in this report were believed to be caused by the presence of VOCs such as 1,2,3-benzene-triol, 1,2-benzenediol, and 5-hydroxymethylfurfural, and alkaloids in the extract an indication of the cytogenotoxicity of the aqueous extract of N. laevis leaf even at low concentration.

Acute, subchronic toxicity and genotoxicity studies of JointAlive, a traditional Chinese medicine formulation for knee osteoarthritis.

AIM: In vivo and in vitro toxicity tests of JointAlive® were studied in animal models to support the safe use of JointAlive® as a drug for knee osteoarthritis treatment. METHODS: The acute toxicity study in Sprague Dawley (SD) rats was conducted at a 20 g/kg bw/day dose of JointAlive®. For 13-week subchronic toxicity tests, SD rats were orally dosed daily with 0.5, 1.5 and 5 g/kg bw/day of JointAlive®. To assess the potential genotoxicity, Ames test, cellular chromosome aberration and mouse micronucleus test in vivo were carried out. RESULTS: Based on a lack of notable findings other than histopathology finding of co-incidental prostate inflammation at the high dose, the "No Observed Adverse Effect Level (NOAEL)" of JointAlive® was concluded as 5 g/kg bw/day in males and females. Results also indicated that JointAlive® has no risk of genotoxicity. CONCLUSIONS: General toxicity and genotoxicity studies empirically demonstrated that JointAlive® poses a low risk of potential health risks, providing safety supports for the application of JointAlive® as a potential drug candidate to treat knee osteoarthritis.

Genotoxicity by rapeseed methyl ester and hydrogenated vegetable oil combustion exhaust products in lung epithelial (A549) cells.

Biofuel is an attractive substitute for petrodiesel because of its lower environmental footprint. For instance, the polycyclic aromatic hydrocarbons (PAH) emission per fuel energy content is lower for rapeseed methyl ester (RME) than for petrodiesel. This study assesses genotoxicity by extractable organic matter (EOM) of exhaust particles from the combustion of petrodiesel, RME, and hydrogenated vegetable oil (HVO) in lung epithelial (A549) cells. Genotoxicity was assessed as DNA strand breaks by the alkaline comet assay. EOM from the combustion of petrodiesel and RME generated the same level of DNA strand breaks based on the equal concentration of total PAH (i.e. net increases of 0.13 [95% confidence interval (CI): 0.002, 0.25, and 0.12 [95% CI: 0.01, 0.24] lesions per million base pairs, respectively). In comparison, the positive control (etoposide) generated a much higher level of DNA strand breaks (i.e. 0.84, 95% CI: 0.72, 0.97) lesions per million base pairs. Relatively low concentrations of EOM from RME and HVO combustion particles (<116 ng/ml total PAH) did not cause DNA strand breaks in A549 cells, whereas benzo[a]pyrene and PAH-rich EOM from petrodiesel combusted using low oxygen inlet concentration were genotoxic. The genotoxicity was attributed to high molecular weight PAH isomers with 5-6 rings. In summary, the results show that EOM from the combustion of petrodiesel and RME generate the same level of DNA strand breaks on an equal total PAH basis. However, the genotoxic hazard of engine exhaust from on-road vehicles is lower for RME than petrodiesel because of lower PAH emission per fuel energy content.

Reconstructed human intestinal comet assay, a possible alternative in vitro model for genotoxicity assessment.

The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet assay is a common follow-up genotoxicity test to confirm or supplement other genotoxicity data. Technically, it can be performed utilizing a range of in vitro and in vivo assay systems. Here, we have developed a new reconstructed human intestinal comet (RICom) assay protocol for the assessment of orally ingested materials. The human intestine is a major site of food digestion and adsorption, first-pass metabolism as well as an early site of toxicant first contact and thus is a key site for evaluation. Reconstructed intestinal tissues were dosed with eight test chemicals: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), phenformin hydrochloride (Phen HCl), benzo[a]pyrene (BaP), 1,2-dimethylhydrazine hydrochloride (DMH), potassium bromate (KBr), glycidamide (GA), and etoposide (Etop) over a span of 48 h. The RICom assay correctly identified the genotoxicity of EMS, ENU, KBr, and GA. Phen HCl, a known non-genotoxin, did not induce DNA damage in the 3D reconstructed intestinal tissues whilst showing high cytotoxicity as assessed by the assay. The 3D reconstructed intestinal tissues possess sufficient metabolic competency for the successful detection of genotoxicity elicited by BaP, without the use of an exogenous metabolic system. In contrast, DMH, a chemical that requires liver metabolism to exert genotoxicity, did not induce detectable DNA damage in the 3D reconstructed intestinal tissue system. The genotoxicity of Etop, which is dependent on cellular proliferation, was also undetectable. These results suggest the RICom assay protocol is a promising tool for further investigation and safety assessment of novel ingested materials. We recommend that further work will broaden the scope of the 3D reconstructed intestinal tissue comet assay and facilitate broader analyses of genotoxic compounds having more varied modes of actions.

Safety evaluation of kaempferol glycosides-rich standardized roasted goji berry leaf extract.

Goji berry leaf (GL) has been used for medicinal foods for its pharmacological effects, including anti-oxidative and anti-obesity activities. Nevertheless, toxicological information on GL is limited for developing health functional ingredient. The aim of the research was to evaluate the single dose acute, 14-day repeated oral toxicity, and genotoxicity of standardized roasted GL extract (rGL) rich in kaempferol-3-O-sophoroside-7-O-glucoside. Tested rGL was found to be stable as kaempferol-3-O-sophoroside-7-O-glucoside, showing 0.7-2.1% of analytical standard variance. According to the single dose toxicity for 14 days, the lethal dose of rGL was determined to be ≥ 2000 mg/kg. Repeated doses of 0-1000 mg/kg of rGL per day for 14 days did not show any toxicity signs or gross pathological abnormalities. No genotoxic signs for the rGL treatment appeared via bacterial reverse mutation up to 5000 µg/plate. There was no significant increase in chromosomal aberration of rGL irrespective of metabolic activation by using CHO-K1 cells (p > 0.05). Regarding carcinogenic toxicity, chromosomal aberrations were not induced at 2000 mg of rGL/kg by using the in vivo bone marrow micronucleus test (p > 0.05). Results from the current study suggest that rGL could be used as a functional ingredient to provide various effects with safety assurance.

Cytotoxicity, oral toxicity, genotoxicity, and mutagenicity evaluation of essential oil from Psidium glaziovianum Kiaersk leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Members of the Psidium genus have been suggested in ethnobotanical research for the treatment of various human diseases, and some studies have already proven their popular uses through research, such as Psidium glaziovianum, which is found in Brazil's northeast and southeast regions and has antinociceptive and anti-inflammatory properties; however, the safety of use has not yet been evaluated. AIM OF THE STUDY: This study investigated the safety of using essential oil obtained from P. glaziovianum leaves (PgEO) in vitro and in vivo models. MATERIALS AND METHODS: Cytotoxicity was evaluated in murine erythrocytes, while acute toxicity, genotoxicity (comet assay) and mutagenicity (micronucleus test) studies were performed using Swiss albino mice. RESULTS: In the cytotoxicity assay, the hemolysis rate indicated a low capacity of PgEO to cause cell lysis (0.33-1.78%). In the acute oral toxicity study, animals treated with up to up to 5000 mg/kg body weight did not observe mortality or physiological changes. Neither dosage caused behavioral problems or death in mice over 14 days. The control and 2,000 mg/kg groups had higher feed intake and body weight than the 5,000 mg/kg PgEO group. Erythrocyte count, hemoglobin level, mean corpuscular volume, and MCV decreased, but serum alanine and aspartate aminotransferases increased. In the genotoxic evaluation, 5000 mg/kg PgEO enhanced nucleated blood cell DI and DF. CONCLUSIONS: The present study describes that PgEO can be considered well tolerated in acute exposure at doses up to 2000 mg/kg, however the dose of 5000 mg/kg of PgEO should be used with caution.

Evaluation of genotoxicity and 13-week subchronic toxicity of root of Asarum heterotropoides var. seoulense (Nakai) Kitag.

ETHNOPHARMACOLOGICAL RELEVANCE: Asarum heterotropoides var. seoulense (Nakai) Kitag is a traditional herbal medicine used in Korea and China. It is effective in aphthous stomatitis, local anesthesia, headache, toothache, gingivitis, and inflammatory diseases. However, information on the toxicity of the root of Asarum heterotropoides var. seoulense (Nakai) Kitag (AR) is limited. Therefore, preclinical toxicity studies on AR are needed to reduce the risk of excessive intake. AIM OF THE STUDY: We aimed to evaluate genotoxicity and the potential toxicity due to repeated administration of AR powder. MATERIALS AND METHODS: In vitro bacterial reverse mutation assay (Ames), in vitro chromosomal aberration assay (CA), and in vivo micronucleus (MN) assay in ICR mice were conducted. As positive results were obtained in Ames and CA assays, alkaline comet assay and pig-a gene mutation test were conducted for confirmation. For evaluating the general toxicity of AR powder, a 13-week subchronic toxicity test was conducted, after determining the dose by performing a single and a 4-week dose range finding (DRF) test. A total of 152 Sprague-Dawley (SD) rats were orally administered AR powder at doses of 0, 150, 350, 500, 1000, and 2000 mg/kg/day in the 13-week subchronic toxicity test. Hematology, clinical chemistry, urinalysis, organ weight, macro-, and microscopic examination were conducted after rat necropsy. RESULTS: AR powder induced genotoxicity evidenced in the Ames test at 187.5, 750, 375, and 1500 µg/plate of TA100, TA98, TA1537, and E. coli WP2uvrA in the presence and absence of S9, respectively; CA test at 790 µg/mL for 6 h in the presence of S-9; 75 µg/mL for 6 h in the absence of S-9, and 70 µg/mL for 22 h in the absence of S-9 in the stomach in the comet assay but not in MN and pig-a assays. In the 13-week subchronic toxicity study, clinical signs including irregular respiration, noisy respiration, salivation, and decreased body weight or food consumption were observed in males and females in the 2000 mg/kg/day group. In hematology tests, clinical chemistry, urinalysis, organ weight, and macroscopic examination, changes were observed in the dose groups of 500 mg/kg/day and above. Microscopic examination revealed hyperplasia of the stomach as a test-related change. Hepatocellular adenoma and changes in liver-related clinical chemistry parameters were observed. The rat No Observed Adverse Effect Level (NOAEL) was 150 mg/kg/day in males and <150 mg/kg/day in females. CONCLUSIONS: AR powder is potentially toxic to the liver and stomach and should be used with caution in humans. A long-term study on carcinogenicity is necessitated because DNA damage or changes in tissue lesions were observed in SD rats.

Designed genotoxicity profiling detects genotoxic compounds in staple food such as healthy oils.

Current techniques used in food analysis overlook genotoxic compounds. This urgently calls for a paradigm shift in analytics towards non-target planar genotoxicity profiling that can detect genotoxins. Up to eight different genotoxins (i.e., genotoxic compound zones) have been detected in 33 oils used for healthy diets. A comparison of fresh oils with oils stored open and closed for one month identified genotoxic degradation products. Characterization of genotoxic zones via high-resolution mass spectrometry revealed oxidized linolenic acid as a source of genotoxicity in all samples. Detoxification via on-surface S9 liver metabolization was investigated, which showed a reduction in most, but not all, genotoxins. Food, feed, dietary supplements, and cosmetics as sources of genotoxicity can now be identified by combining separation, effect detection and optionally simulated metabolization on the same surface. The application of the planar genotoxicity profiling will improve the understanding on food and its impact as well as risk assessment and derived recommendations.

Comparative chemical analysis, mutagenicity, and genotoxicity of Petroleum refinery wastewater and its contaminated river using prokaryotic and eukaryotic assays.

Concern on the toxicity of final wastewater generated by the petroleum refining industry has increased in recent years due to the potential health threats associated with their release into the waterways. This study determined the mutagenic and genotoxic potential of petroleum refinery wastewater and a receiving river using the Ames fluctuation test on Salmonella typhimurium strains TA100 and TA98, SOS chromotest on Escherichia coli PQ37, and piscine peripheral micronucleus (MN) assay. Analyses of the physicochemical parameters, heavy metal, and organic contents of the samples were also performed. Ames test result showed that the two tested samples were mutagenic with TA100 strain as the more responsive strain for both the refinery wastewater and the river sample in terms of the calculated mutagenic index. A similar result was obtained in the SOS chromotest; however, the E. coli PQ37 system recorded a slightly higher sensitivity for detecting genotoxins than the Salmonella assay in the two samples. MN data showed induction of a concentration-dependent significant (p < 0.05) increase in the frequency of MN by both samples when compared with the negative control. Generally, the refinery wastewater induced the highest mutagenicity and genotoxicity compared to the river sample in the three assays used. Haemoglobin, platelets, red blood cells, mean corpuscular volume, total white blood cells, heterophils, haematocrit, and eosinophils reduced significantly with increased lymphocytes, basophils, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration in fishes exposed to both samples. Total petroleum hydrocarbon, benzene, toluene, phenol index, polycyclic aromatic hydrocarbons, cadmium, mercury, nickel, lead, and vanadium contents analysed in the samples were believed to be responsible for the observed genotoxicity and mutagenicity. The findings of this study revealed that petroleum refinery wastewater is a potential mutagenic and genotoxic risk to the environment.

Non-clinical safety evaluation of salvianolic acid A: acute, 4-week intravenous toxicities and genotoxicity evaluations.

BACKGROUND: Toxicological problem associated with herbal medicine is a significant public health problem. Hence, it is necessary to elaborate on the safety of herbal medicine. Salvianolic acid A (SAA) is a major active compound isolated from Danshen, a popular herbal drug and medicinal food plant in China. The aim of the present study was to explore the toxicological profile of SAA. METHODS: The acute toxicity studies were performed in mice and Beagle dogs with single administration with SAA. A 4-week subchronic toxicity was test in dogs. SAA was intravenously administered at doses of 20, 80 and 300 mg/kg. Clinical observation, laboratory testing and necropsy and histopathological examination were performed. The genotoxic potential of SAA was evaluated by 2 types of genotoxicity tests: a reverse mutation test in bacteria and bone marrow micronucleus test in mice. RESULTS: In acute toxicities, the LD50 of SAA is 1161.2 mg/kg in mice. The minimum lethal dose (MLD) and maximal non-lethal dose (MNLD) of SAA were 682 mg/kg and 455 mg/kg in dogs, respectively. The approximate lethal dose range was 455-682 mg/kg. In the study of 4-week repeated-dose toxicity in dogs, focal necrosis in liver and renal tubular epithelial cell, the decrease in relative thymus weight, as well as abnormal changes in biochemical parameters, were observed in SAA 80 or 300 mg/kg group. The no observed adverse effect level (NOAEL) of SAA was 20 mg/kg. Thymus, liver and kidneys were the toxic targets. These toxic effects were transient and reversible. These results indicated that it should note examination of liver and kidney function during the administration of SAA in clinic. Furthermore, SAA had no mutagenic effect at any tested doses. CONCLUSION: These results provide new toxicological information of SAA for its clinical application and functional food consumption.

Toxicological assessment of enzyme-treated Zizania latifolia extract: Oral toxicology and genotoxicity in rats.

Zizania latifolia Turcz. has long been used as a food source in Southeast Asia. The grains, stems, and leaves of Z. latifolia and its major component, tricin, have also been studied to determine their biological activities. Previously, we hydrolyzed the aerial part of Z. latifolia using an enzyme mixture to maximize the tricin content of the Z. latifolia extract. However, the safety of enzyme-treated Z. latifolia extract (ETZL; DermaNiA™) has not yet been determined. In this study, we performed an in vivo 90-day repeated-dose evaluation and genotoxicity study to assess the toxicological potential of ETZL. EZTL did not exhibit genotoxicity in the bacterial reverse mutation test, in vitro chromosomal aberration assay, or in vivo micronucleus test. Moreover, no changes in body weight or hematological and serum biological parameters were observed in male or female rats under high-dose EZTL treatment (5000 mg/kg body weight (bw)/day) for 90 days with a 4-week recovery period. Significant changes were noted in the forestomach, kidneys, and adrenal glands in the test groups, but these changes, or tendency for recovery, were not observed in the recovery group. Based on these data, the no adverse effect level was determined to be 1250 mg/kg bw/day in rats.

Safety assessment of marigold flavonoids from marigold inflorescence residue.

ETHNOPHARMACOLOGICAL RELEVANCE: Marigold flavonoids, extracted from marigold (Tagetes erecta L.) inflorescence residues, have attracted significant attention with respect to antioxidant, anti-inflammatory and chelating properties. However, the toxicity of marigold flavonoids have not yet been fully investigated. AIM OF THE STUDY: The main purpose of this study was to assess the safety of marigold flavonoids extracted from Marigold (Tagetes erecta L.) in order to provide information on its nonclinical safety. Thus, the acute oral toxicity, in vitro Ames test, sperm aberration study, bone marrow micronucleus test, subchronic oral toxicity test, and teratogenic potential were carried out in rats or mice. MATERIALS AND METHODS: For an acute oral toxicity test, SD rats and ICR mice (male and female, n = 5) orally received a single dose of 5000 mg/kg marigold flavonoids. Evaluation of marigold flavonoids genotoxic potential with a battery of tests, including an in vitro bacterial reverse mutation test using four mutant strains of Salmonella typhimurium (TA97、TA98、TA100、TA102), an sperm aberration test and an in vivo micronucleus test using bone marrow cells ICR mice that were orally administered marigold flavonoids, an subchronic oral toxicity study and teratogenic test employing male and female SD rats that were orally administered marigold flavonoids. All animals tests were completed in accordance with GB 15193 for toxicity tests. RESULTS: In the acute oral toxicity test, marigold flavonoids given at the dose of 5000 mg/kg body weight for 14 days didn't produce any abnormal clinical symptoms or mortality in SD rats and ICR mice (both sex, n = 5). There was no evidence of genotoxicity of marigold flavonoids based on the results of the in vitro bacterial reverse mutation test (up to 1250 µg/plate), the sperm aberration test (up to 5000 mg/kg body weight), the in vivo micronucleus test (up to 5000 mg/kg body weight), the subchronic oral toxicity study (up to 10 g/kg feed dose) and the teratogenic test (up to 1250 mg/kg body weight). CONCLUSIONS: We found that marigold flavonoids are safe with regard to acute toxicity in rats or mice as well as genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions. These results might support the safety of marigold flavonoids as a potential therapeutic material for the traditional use of herbal medicines and for the further development of novel antioxidant.

In Vitro and In Vivo Genotoxicity Assessments and Phytochemical Analysis of the Traditional Herbal Prescription Siryung-Tang.

Siryung-tang (SRT) is a traditional herbal prescription containing Oryeong-san and Soshiho-tang that is used to treat digestive system diseases. We performed safety evaluations of SRT based on genotoxicity and developed an assay for quality control using high-performance liquid chromatography with a photodiode array detector. Genotoxicity was evaluated based on bacterial reverse mutation (Salmonella typhimurium TA1535, TA98, TA100, and TA1537, and Escherichia coli WP2 uvrA), chromosomal aberration (Chinese hamster lung cells), and micronucleus (mouse) tests. Quality control analysis was conducted using a SunFire C18 column and gradient elution with a distilled water-acetonitrile mobile phase system containing 0.1% (v/v) formic acid for 12 markers (5-(hydroxy-methyl)furfural, 3,4-dihydroxybenzaldehyde, liquiritin apioside, liquiritin, coumarin, baicalin, wogonoside, cinnamaldehyde, baicalein, glycyrrhizin, wogonin, and atractylenolide III). SRT showed no genotoxicity in three tests. Ames tests showed that SRT at 313-5000 µg/plate did not significantly increase the number of revertant colonies with or without metabolic activation among five bacterial strains. Moreover, in vivo micronucleus testing showed that SRT did not increase the frequency of bone marrow micronuclei. The number of chromosomal aberrations associated with SRT was similar to that observed in the negative controls. The 12 markers were detected at 0.04-16.86 mg/g in a freeze-dried SRT sample and completely eluted within 45 min. The extraction recovery was 95.39-104.319% and the relative standard deviation value of the precision was ≤2.09%. Our study will be used as basic data for the safety and standardization of SRT.

Hepatotoxicity and Mutagenicity assessment during chronic in vivo exposure to aqueous extracts from Peperomia pellucida.

Studies on herbal medicine have exposed some toxic effects on humans. Peperomia pellucida (L.) HBK (P. pellucida) is one of the herbal medicines recommended as an alternative to synthetic medicine for diseases. Studies exist on the pharmacological activities of P. pellucida extracts, but studies on the potential hepatotoxic and mutagenic effects of subchronic administration of P. pellucida aqueous extracts, which is very important knowledge when we venture into alternative medicine, are lacking. In this study, two concentrations (60 mg/kg and 30 mg/kg) of P. pellucida aqueous extracts - decoction and freeze-dried extracts -were administered in vivo to BALB/c mice for nine (9) weeks. Significant differences were observed between the 60 mg/kg freeze-dried extract and the control in terms of mice weight and micronucleus frequency at 7-8 weeks of treatment. Also, no significant differences were found between groups in serum transaminases levels. Generally, there is no sufficient evidence to show that subchronic exposure to P. pellucida aqueous extracts is hepatotoxic though 60 mg/kg concentration may be mutagenic. This study suggests that although the herbal medicine is safe for prolonged consumption, users are advised to take precautions and moderations of its use due to the possibility of potential mutagenic effects.

Genotoxicity, acute and subchronic toxicity evaluation of fermented Morinda officinalis.

Morinda officinalis has diverse pharmacological effects and has the potential to be used as functional food and medicine. Fermentation is traditionally used to process Morinda officinalis. However, the toxicological profile of fermented Morinda officinalis (FMO) is not reported. In the present study, the toxicological characteristics of FMO were assessed for the first time. FMO did not show any genotoxicity based on the Ames test, mammalian erythrocyte micronucleus test, and mouse primary spermatocyte chromosome aberration test. FMO administered by gavage in mice and rats at a dose of 20 g/kg BW did not induce death or toxicity based on acute study, indicating that FMO could be regarded as non-toxic at the tested dose. In the 90-day subchronic toxicity study, rats fed with FMO at the maximum dose of 8 g/kg BW did not affect mortalities, BW, food consumption, organ weights, hematology, serum biochemistry, or urinalysis. The no observed adverse effect level of FMO in both sexes was not less than 8 g/kg BW/day based on subchronic toxicity. The obtained results support the safe use of FMO as functional food and medicine.

Safety Evaluation of Oleoresin-Based Turmeric Formulation: Assessment of Genotoxicity and Acute and Subchronic Oral Toxicity.

Turmeric rhizome (Curcuma longa L.) has been used without concern for safety as a culinary spice and traditional medicine under the ancient Ayurvedic medicinal system of India dating back nearly 4000 years. This preclinical safety evaluation was done to determine the safety of an oleoresin-based turmeric extract (CURCUGEN®). Guidelines from the Organization for Economic Co-operation and Development (OECD) directed the assessment of safety for the in vitro and in vivo application of CURCUGEN®. Safety of the herbal medicine was evaluated through the toxicological assessment of acute, oral, and 90-day repeated dosing, genotoxicity, and mutagenicity study. Genotoxicity tests included the in vitro bacterial reverse mutation test, chromosomal aberration test, and in vivo micronucleus test. The single dose of CURCUGEN® administered orally (gavage) to Sprague-Dawley (SD) rats resulted in a LD50 of >5000 mg/kg body weight. The subchronic assessment of CURCUGEN®, as administered to SD rats over 90 days resulted in a no observed adverse effect level (NOAEL) of 2000 mg/kg body weight/day. CURCUGEN® did not elicit any genotoxic or clastogenic effect in genotoxicity tests. The battery of safety studies carried out demonstrated that CURCUGEN® showed no evidence of general toxicity or genotoxicity.

The effect of alkyl substitution on the oxidative metabolism and mutagenicity of phenanthrene.

Alkyl-substituted PAHs may be present in certain petroleum-derived products and in the environment and may eventually end up in consumer products, such as foodstuffs, cosmetics and pharmaceuticals. Safety concerns over possible exposure to alkylated PAHs have emerged. Bioactivation is a prerequisite for the mutagenicity and carcinogenicity of PAHs and has been extensively studied for non-substituted PAHs, while data on the bioactivation of alkyl-substituted PAHs are scarce. The present study investigated the effect of alkyl substitution on the CYP 450-mediated metabolism of phenanthrene and eight of its alkylated congeners by quantifying metabolite formation in rat and human liver microsomal incubations. Furthermore, the mutagenicity of four selected methylated phenanthrenes was compared to that of phenanthrene using the Ames test. The obtained results support the hypothesis that alkyl substitution shifts the oxidative metabolism from the aromatic ring to the alkyl side chain. Increasing the length of the alkyl chain reduced overall metabolism with metabolic conversion for 1-n-dodecyl-phenanthrene (C12) being negligible. 1- and 9-methyl-phenanthrene, in which the methyl group generates an additional bay region-like structural motif, showed mutagenicity toward Salmonella typhimurium TA98 and TA 100, whereas phenanthrene and also 2- and 3-methyl-phenanthrene, without such an additional bay region-like structural motif, tested negative. It is concluded that the position of the alkylation affects the metabolism and resulting mutagenicity of phenanthrene with the mutagenicity increasing in cases where the alkyl substituent creates an additional bay region-like structural motif, in spite of the extra possibilities for side chain oxidation.