Evaluating the toxicity of the roots of Asarum heterotropoides var. mandshuricum extracted using the decoction method: Genotoxicity, single-dose toxicity, and 13-week repeated-dose toxicity studies.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Asarum heterotropoides F. Maekawa var. mandshuricum F. Maekawa (AR) is a traditional herbal medicine used across Asia, including Korea, China, and Japan. AR exhibits a range of biological activities, such as anti-inflammatory, anti-cancer, cold treatment, and anti-nociceptive effects. Various extraction methods, including decoction, which utilizes traditional knowledge and techniques. The AR decoction extract expected to contain fewer toxicants and have reduced toxicity due to the use of hot water in the extraction process. However, scientific evidence on the toxicity of AR decoction extracts is lacking, necessitating further studies for safe usage. AIM OF THE STUDY: This study aimed to evaluate the genotoxicity and toxicity of single and repeated administration of AR decoction extracts. MATERIALS AND METHODS: The genotoxicity was assessed using a bacterial reverse mutation (Ames test), an in vitro mammalian chromosome aberration test (CA test), and an in vivo micronucleus test (MN test) in Sprague-Dawley (SD) rats. The general toxicity was evaluated through single-dose and 13-week repeated-dose toxicity studies. In the single-dose toxicity study, 40 SD rats were orally administered AR decoction extract at doses of 1000, 2000, and 5000 mg/kg. In the 13-week repeated-dose toxicity study, 140 SD rats received daily oral doses of 0, 250, 500, 1000, 2000, and 5000 mg/kg of AR decoction extract. RESULTS: The genotoxicity tests revealed that AR decoction extract was not genotoxic. The single-dose toxicity study showed no changes in body weight, clinical pathology, or macroscopic findings, with the approximate lethal dose (ALD) exceeding 5000 mg/kg. The 13-week repeated-dose toxicity study demonstrated no treatment-related changes in body weight, general symptoms, hematology, clinical chemistry, or urinalysis. Histopathological findings revealed hyperplasia of squamous cells in the forestomach after AR decoction extract administration, a treatment-related effect that resolved during the recovery period. The no observed adverse effect level (NOAEL) for both male and female rats was estimated to be 2000 mg/kg. CONCLUSIONS: This study establishes the non-toxic dose of AR decoction extract, providing a foundation for further non-clinical and clinical evaluations AR safety.

Genotoxicity assessment of root extracts of Paeonia lactiflora Pall.

The roots of Paeonia lactiflora Pall., (Paeoniae Radix, PL) are a well-known herbal remedy used to treat fever, rheumatoid arthritis, systemic lupus erythematosus, hepatitis, and gynecological disorders in East Asia. Here we evaluated the genetic toxicity of PL extracts (as a powder [PL-P] and hot-water extract [PL-W]) in accordance with the Organization for Economic Co-operation and Development guidelines. The Ames test revealed that PL-W was not toxic to S. typhimurium strains and E. coli in absence and presence of the S9 metabolic activation system at concentrations up to 5000 µg/plate, but PL-P produced a mutagenic response to TA100 in the absence of S9 mix. PL-P was cytotoxic in in vitro chromosomal aberrations (more than a 50 % decrease in cell population doubling time), and it increased the frequency of structural and numerical aberrations in absence and presence of S9 mix in a concentration-dependent manner. PL-W was cytotoxic in the in vitro chromosomal aberration tests (more than a 50 % decrease in cell population doubling time) only in the absence of S9 mix, and it induced structural aberrations only in the presence of S9 mix. PL-P and PL-W did not produce toxic response during the in vivo micronucleus test after oral administration to ICR mice and did not induce positive results in the in vivo Pig-a gene mutation and comet assays after oral administration to SD rats. Although PL-P showed genotoxic in two in vitro tests, the results from physiologically relevant in vivo Pig-a gene mutation and comet assays illustrated that PL-P and PL-W does not cause genotoxic effects in rodents.

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.

An International Collaborative Animal Study of the Carcinogenicity of Mobile Phone Radiofrequency Radiation: Considerations for Preparation of a Global Project.

Radiofrequency radiation (RFR) was classified as a "possible" human carcinogen in 2011, which caused great public concern. A carcinogenicity study by the National Toxicology Program (NTP) found Code Division Multiple Access-and Global System for Mobile Communications-modulated mobile phone RFR to be carcinogenic to the brain and heart of male rats. As part of an investigation of mobile phone carcinogenesis, and to verify the NTP study results, a 5-year collaborative animal project was started in Korea and Japan in 2019. An international animal study of this type has two prerequisites: use of the same study protocol and the same RF-exposure system. This article discusses our experience in the design of this global study on radiofrequency electromagnetic fields (RF-EMFs).© 2022 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.

The increased amount of coffee consumption lowers the incidence of fatty liver disease in Korean men.

BACKGROUND AND AIMS: Coffee is known to have a beneficial effect on various liver diseases. The aim of this retrospective longitudinal study was to investigate an association between the amount of coffee consumption and the incidence of fatty liver disease in Korean adults. METHODS AND RESULTS: Data from a total of 91,436 male and female subjects with the mean follow-up period of 2.8 years were analyzed. The incidence of fatty liver was not associated with the amount of coffee consumption at baseline, but it was associated with the change in the amount of coffee consumption at the follow-up period. Multiple linear regression analyses showed that hazard ratios for incidence of fatty liver disease were significantly low in "increase" group comparing with "no change" group in fully adjusted model. When a subgroup analysis by gender was conducted, similar significant results were observed in male subjects, but not in females. CONCLUSIONS: The increment in the amount of coffee consumption is associated with the lower incidence of fatty liver in Korean men and suggests that increasing the coffee consumption may have a protective effect on fatty liver.

Safety assessment of Oryeong-san, a traditional herbal formula: Study of subacute toxicity and influence of cytochrome P450s and UDP-glucuronosyltransferases.

Oryeong-san is a traditional herbal formula that is used for the treatment of common genitourinary diseases in Korea and other Asian countries. However, little is known about its safety and influence on drug metabolism. In the present study, we investigated the subacute toxicity of an Oryeong-san water extract (OSWE) in rats and its effects on activities of drug-metabolizing enzymes. Subacute toxicity was modeled in animals exposed to treatment with the extract at multiple doses. Rats were given OSWE by oral gavage at 0, 1000, 2000 and 5000 mg/kg/day for 4 weeks. We checked general observations and investigated any changes of body/organ weight, food consumption, hematology, serum biochemistry, and urinalysis in vivo; and the activities of human microsomal cytochrome P450s (CYP450s) and UDP-glucuronosyltransferase (UGT) isozymes in vitro. We found that OSWE caused no significant toxicological changes at the doses tested. Therefore, the no observed adverse effect level of OSWE was more than 5000 mg/kg/day for male and female rats. OSWE inhibited the activities of CYP2C19 (IC50: 737.69 µg/mL) and CYP2E1 (IC50: 177.77 µg/mL). These results indicate that OSWE may be safe with no drug-related toxicity for up to 4 weeks and provide useful information concerning its potential to interact with conventional drugs or other herbal medicines.

Continuous cropping of endangered therapeutic plants via electron beam soil-treatment and neutron tomography.

Various medicinal plants are threatened with extinction owing to their over-exploitation and the prevalence of soil borne pathogens. In this study, soils infected with root-rot pathogens, which prevent continuous-cropping, were treated with an electron beam. The level of soil-borne fungus was reduced to ≤0.01% by soil electron beam treatment without appreciable effects on the levels of antagonistic microorganism or on the physicochemical properties of the soil. The survival rate of 4-year-old plant was higher in electron beam-treated soil (81.0%) than in fumigated (62.5%), virgin (78%), or untreated-replanting soil (0%). Additionally, under various soils conditions, neutron tomography permitted the monitoring of plant health and the detection of root pathological changes over a period of 4-6 years by quantitatively measuring root water content in situ. These methods allow continual cropping on the same soil without pesticide treatment. This is a major step toward the environmentally friendly production of endangered therapeutic herbs.

A subchronic toxicity study of Radix Dipsaci water extract by oral administration in F344 rats.

Radix Dipsaci, the dried root of Dipsacus asperoides C.Y. Cheng & T.M.Ai, has therapeutic effects on various disorders, and in particular, bone and joint disease. Despite such ethnomedicinal benefits, there is very little information regarding its in vivo toxicity or adverse effects. This study was conducted to evaluate the potential toxicity of the Radix Dipsaci water Extract (RD-wE) by using F344 rats. The RD-wE was administered orally to rats at doses of 0, 125, 250, 500, 1000, and 2000 mg/kg body weight (bw)/day for 13 weeks. During the treatment period there were no mortalities attributed to RD-wE. Moreover, no toxic effects were observed with regard to body weight, clinical pathology (hematology, clinical biochemistry, and urinalysis), and anatomic pathology (gross findings, organ weight, and microscopic examination). The changes related to the treatment were excessive salivation at the mouth and soft feces, observed in male and female rats at 1000 or 2000 mg/kg bw/day, but these were not accompanied by any microscopic correlate or other pathophysiological changes. Based on these results, the oral no-observed-adverse-effect level of the RD-wE was considered to be 2000 mg/kg bw/day in both genders, although the target organs were not determined under the current experimental conditions.

Safety assessment of Guibi-tang: Subchronic toxicity study in Crl:CD SD rats.

Guibi-tang (Kihi-To in Japanese and Qui-Pi-Tang in Chinese) is a multiherbal traditional Korean medicinal formula used for treatment of amnesia, fatigue, poor memory or forgetfulness, anemia, insomnia, and necrosis. The aim of the present study was to investigate potential safety, if any, of subchronic administration of Guibi-tang aqueous extract (GBT) in laboratory animals. For this study, 0, 1000, 2000, and 5000 mg/kg/day of GBT was administered to Crl:CD Sprague Dawley rats (10/sex/group) for 13 weeks via oral gavage. Administration of the GBT did not result in any mortality, body weight, food consumption, hematology, serum biochemistry, clinical or ophthalmological signs or changes in urinalysis, gross findings, or organ weight. However, histopathology revealed forestomach hyperplasia and duodenum mucosal hyperplasia in rats of both sexes at the highest dose of GBT, 5000 mg/kg/day. Therefore, the no-observed-adverse-effect level in rats was established for GBT at 2000 mg/kg/day under the conditions of this study.

Serum Vitamin D Status as a Predictor of Prognosis in Patients with Acute Ischemic Stroke.

BACKGROUND: Low 25-hydroxyvitamin D (25(OH)D) concentrations have been shown to predict risk of cardiovascular disease and all-cause mortality. Although the prevalence of 25(OH)D deficiency is high in patients with acute stroke, the prognostic value of 25(OH)D in stroke has not been clearly established. The purpose of this study was to determine whether the baseline serum 25(OH)D level was associated with the functional outcome in patients with acute ischemic stroke. METHODS: From June 2011 to January 2014, consecutive patients with acute ischemic stroke within 7 days of symptom onset were enrolled in this study from a prospectively maintained stroke registry. Serum 25(OH)D level was measured at admission. Clinical and laboratory data including stroke severity using the National Institute of Health Stroke Scale (NIHSS) score were collected during admission, and the functional outcome at 3 months was assessed by modified Rankin scale (mRS). The association between the baseline 25(OH)D level and a good functional outcome (mRS 0-2) at 3 months was analyzed by multiple logistic regression models. RESULTS: A total of 818 patients were enrolled in this study. Mean age was 66.2 (±12.9) years, and 40.5% were female. The mean 25(OH)D level was 47.2 ± 31.7 nmol/l, and the majority of patients met vitamin D deficient status (<50 nmol/l; 68.8%), while an optimal vitamin D level (≥75 nmol/l) was present in only 13.6% of the patients, and 436 (53.3%) patients showed good functional outcome at 3 months. Serum 25(OH)D levels in patients with good outcomes were significantly higher than those with poor outcome (50.2 ± 32.7 vs. 43.9 ± 30.0 nmol/l, p = 0.007). The 3-month functional outcome was significantly associated with month-specific 25(OH)D quartiles in multivariable logistic regression analysis. After adjustment for age and sex, the highest 25(OH)D quartile group had higher tendency for good functional outcome at 3 months (odds ratio (OR) = 1.68, 95% confidence interval (CI) = 1.13-2.51). After fully adjusting for other potential confounders, such as stroke severity and vascular risk factors, the association was further strengthened with an OR (95% CI) of 1.90 (1.14-3.16). Other factors associated with good functional outcome in multivariable analysis were younger age, lower initial NIHSS score and absence of diabetes. CONCLUSIONS: This study suggests that serum 25(OH)D level is an independent predictor of functional outcome in patients with acute ischemic stroke. Further studies are required to determine whether vitamin D supplementation could improve functional outcome in patients with ischemic stroke.

25-hydroxyvitamin D status is associated with chronic cerebral small vessel disease.

BACKGROUND AND PURPOSE: The aim of this study was to determine the association between 25-hydroxyvitamin D (25(OH)D) and neuroimaging correlates of cerebral small vessel disease. METHODS: We identified 759 consecutive patients with acute ischemic stroke or transient ischemic attack. Lacunes, white matter hyperintensity, and cerebral microbleed (CMB) were assessed using MR images. Deep CMB was defined as the presence of CMB in basal ganglia, thalamus, or brain stem. The association between 25(OH)D and small vessel disease was tested using linear and logistic regression analyses. RESULTS: Mean age was 68 (±13) years. Mean level of 25(OH)D was 34.1±17.8 nmol/L. On bivariate analysis, a 25-nmol/L decrease in 25(OH)D was associated with lacunes (regression coefficient, 0.23; 95% confidence interval [CI], 0.02-0.45), severe white matter hyperintensity (odds ratio, 2.05; 95% CI, 1.41-3.08), and deep CMB (odds ratio, 1.28; 95% CI, 1.01-1.63). Also, 25(OH)D deficiency (≤25 nmol/L) was associated with lacunes (regression coefficient, 0.5; 95% CI, 0.04-0.95), severe white matter hyperintensity (odds ratio, 2.74; 95% CI, 1.31-6.45), and deep CMB (odds ratio, 1.68; 95% CI, 1.03-2.78). The association remained significant even after multivariable adjustment and in the subgroup of previously healthy patients. CONCLUSIONS: 25(OH)D is inversely associated with lacunes, white matter hyperintensity, and deep CMB. Our findings suggest that 25(OH)D is linked to small vessel disease, and in future trials it should be tested whether 25(OH)D supplementation can prevent small vessel disease.

Comparison of the toxicity of aluminum oxide nanorods with different aspect ratio.

Aluminum oxide nanoparticles are listed among 14 high-priority nanomaterials published by the Organization for Economic Co-operation and Development, but limited information is available on their potential hazards. In this study, we compared the toxicity of two different aluminum oxide nanorods (AlNRs) commercially available in vivo and in vitro. Considering aspect ratio, one was 6.2 ± 0.6 (long-AlNRs) and the other was 2.1 ± 0.4 (short-AlNRs). In mice, long-AlNRs induced longer and stronger inflammatory responses than short-AlNRs, and the degree reached the maximum on day 7 for both types and decreased with time. In addition, in vitro tests were performed on six cell lines derived from potential target organs for AlNPs, HEK-293 (kidney), HACAT (skin), Chang (liver), BEAS-2B (lung), T98G (brain), and H9C2 (heart), using MTT assay, ATP assay, LDH release, and xCELLigence system. Long-AlNRs generally produced stronger toxicity than short-AlNRs, and HEK-293 cells were the most sensitive for both AlNRs, followed by BEAS-2B cells, although results from 4 kinds of toxicity tests conflicted among the cell lines. Based on these results, we suggest that toxicity of AlNRs may be related to aspect ratio (and resultant surface area). Furthermore, novel in vitro toxicity testing methods are needed to resolve questionable results caused by the unique properties of nanoparticles.

Procyanidins from the stem wood of Machilus japonica and their inhibitory effect on LDL oxidation.

The stem wood of Machilus japonica Siebold & Zucc were extracted with 80 % aqueous MeOH, and the concentrated extract was successively partitioned with ethyl acetate (EtOAc), normal butanol, and water. From the EtOAc fraction, five procyanidins, procyanidin A1 (1), procyanidin A2 (2), procyanidin B7 (3), cinnamtannin B1 (4), and aesculitannin B (5), were isolated. Their chemical structures were identified through spectroscopic data analyses including NMR, MS, and IR. This is the first time any of these compounds have been isolated from this plant. The compounds were evaluated for inhibition activity on LDL oxidation. All of these compounds and the positive control, BHT, showed a very high inhibition effect with IC50 values of 0.94, 2.1, 1.8, 1.1, 1.0, and 1.9 µM, respectively.

Quality evaluation of Panax ginseng roots using a rapid resolution LC-QTOF/MS-based metabolomics approach.

Korean ginseng (Panax ginseng C.A. Meyer) contains several types of ginsenosides, which are considered the major active medicinal components of ginseng. The types and quantities of ginsenosides found in ginseng may differ, depending on the location of cultivation, making it necessary to establish a reliable method for distinguishing cultivation locations of ginseng roots. P. ginseng roots produced in different regions of Korea, China, and Japan have been unintentionally confused in herbal markets owing to their complicated plant sources. PCA and PLS-DA using RRLC-QTOF/MS data was able to differentiate between ginsengs cultivated in Korea, China, and Japan. The chemical markers accountable for such variations were identified through a PCA loadings plot, tentatively identified by RRLC-QTOF/MS and partially verified by available reference standards. The classification result can be used to identify P. ginseng origin.

Production and characterization of methyl ester sophorolipids with 22-carbon-fatty acids.

In this study, various fatty acids of 18 and 22 carbons were used as substrates to produce sophorolipids in Candida bombicola ATCC 22214. Methyl esterification of erucic acid and C22-enriched rapeseed oil generated mainly acidic sophorolipids with different degrees of saturation in the fatty acid chain, which was demonstrated by structural analysis using HPLC, LC-MS and NMR spectroscopy. Regardless of methyl esterification, the rapeseed oil served as the best substrate for high production of sophorolipids among the tested. Methyl esterification also had no noticeable effect on the interfacial properties of sophorolipids. However, 22 carbons introduced in the fatty acid chain increased the hydrophobicity of sophorolipids, and therefore improved surface-active properties and biodegradability.

AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus.

Obesity is an epidemic in Western society, and causes rapidly accelerating rates of type 2 diabetes and cardiovascular disease. The evolutionarily conserved serine/threonine kinase, AMP-activated protein kinase (AMPK), functions as a 'fuel gauge' to monitor cellular energy status. We investigated the potential role of AMPK in the hypothalamus in the regulation of food intake. Here we report that AMPK activity is inhibited in arcuate and paraventricular hypothalamus (PVH) by the anorexigenic hormone leptin, and in multiple hypothalamic regions by insulin, high glucose and refeeding. A melanocortin receptor agonist, a potent anorexigen, decreases AMPK activity in PVH, whereas agouti-related protein, an orexigen, increases AMPK activity. Melanocortin receptor signalling is required for leptin and refeeding effects on AMPK in PVH. Dominant negative AMPK expression in the hypothalamus is sufficient to reduce food intake and body weight, whereas constitutively active AMPK increases both. Alterations of hypothalamic AMPK activity augment changes in arcuate neuropeptide expression induced by fasting and feeding. Furthermore, inhibition of hypothalamic AMPK is necessary for leptin's effects on food intake and body weight, as constitutively active AMPK blocks these effects. Thus, hypothalamic AMPK plays a critical role in hormonal and nutrient-derived anorexigenic and orexigenic signals and in energy balance.

Thioredoxin h overexpressed in barley seeds enhances selenite resistance and uptake during germination and early seedling development.

The uptake, distribution and metabolism of selenite were examined in germinating homozygous barley ( Hordeum vulgare L.) grain with thioredoxin h overexpressed in starchy endosperm. Results were related to the null segregant in which the transgene had segregated out during crossing. Compared with the null segregant, the homozygote showed enhanced germination and root and shoot growth in the presence of 1 and 2 mM sodium selenite. The rate of incorporation of selenite by the homozygote was approximately twice that of the null segregant. Based on X-ray absorption spectroscopy, the major products in both cases were selenomethionine-like species and the red, monoclinic form of elemental selenium, a derivative not previously reported in green plants. Selenite and selenate made up the balance. The distribution of the products formed differed as to the tissue - root, shoot, aleurone, endosperm - but the ratios were similar in the homozygote and null segregant. The results provide evidence that, in addition to the accelerated germination observed previously in water, barley grain overexpressing thioredoxin h are resistant to the inhibitory effects of selenite. These properties raise the possibility that plants overexpressing thioredoxin h could find application in the remediation of polluted environments.