Subchronic toxicity evaluation of ethanol extract of Cassia tora L. seeds in rats.

Cassia tora Linn. is an annual or perennial plant of the Fabaceae/Leguminosae family. It is used in traditional medicine for various biological activities including anti-constipation, anti-inflammatory, visual acuity, and hepato-protective activities. The present study was carried out to investigate the potential toxicity of C. tora L. seed ethanol extract (CTSEE) following a 13-week repeated oral administration to Sprague-Dawley rats. CTSEE was administered orally to male and female rats for 13 weeks at 0 (control), 500, 1000, and 2000 mg/kg/day (n = 10, for male and female rats for each dose). Additional recovery groups from the control group and high dose group were observed for a 4-week recovery period. At the end of the treatment and recovery periods, animals were sacrificed, and their organs were weighed and blood samples collected. There were no treatment-related adverse effects in clinical signs, body weight, food consumption, estrous cycle, sperm parameters, urinalysis, hematology, serum biochemistry, necropsy findings, organ weight, and histopathology at any doses tested. Under the present experimental conditions, the no-observed-adverse-effect level of the CTSEE was >2000 mg/kg/day in both genders, and no target organs were identified.

Effect of Saururus chinensis leaves extract on type II collagen-induced arthritis mouse model.

BACKGROUND: Saururus chinensis leaves have been used as traditional medicine in Korea for pain, intoxication, edema, and furuncle. According to previous reports, these leaves exert renoprotective, neuroprotective, and antioxidant effects by attenuating inflammatory responses. However, the beneficial effect of Saururus chinensis leaves on arthritis has not been elucidated. Thus, we evaluated the water extract of Saururus chinensis leaves (SHW) using type II collagen-induced arthritis (CIA) mice models. METHODS: Quantitative analysis of major components from SHW was performed by HPLC. Arthritis was induced by injection of type II collagen. Each group was orally administered SHW (100 mg/kg and 500 mg/kg). Methotrexate (MTX) was used as a positive control. Serum levels of interleukin-6, TNF-alpha, and type II collagen IgG in the animal models were measured using ELISA. Histological features were observed by H&E staining. RESULTS: Quantitative analysis of SHW showed the contents as 56.4 ± 0.52 mg/g of miquelianin, 7.75 ± 0.08 mg/g of quercetin 3-O-(2"-O-ß -glucopyranosyl)-α-rhamnopyranoside, and 3.17 ± 0.02 mg/g of quercitrin. Treatment with 500 mg/kg SHW decreased the serum level of Interleukin-6 (IL-6), TNF-alpha, and collagen IgG in the CIA model. Moreover, SHW treatment diminished the swelling of hind limbs and monocyte infiltration in blood vessels in CIA animal models. The results indicate that SHW could decrease CIA-induced arthritis in vivo. CONCLUSIONS: The results indicate that SHW could be used to improving arthritis by reducing inflammatory factors (IL-6 and TNF-alpha). However, further experiments are required to determine how SHW influences signal transduction in animal models.

Antioxidant and Antimelanogenic Activities of Compounds Isolated from the Aerial Parts of Achillea alpina L.

Achillea alpina is widely distributed in Korea and is often used as a folk medicine for stomach disorders. Although a previous study isolated antioxidant compounds (flavonoid O-glucoside, sesquiterpene) from this plant, no systematic study of its chemical constituents had been reported. The present study aimed to identify the phytochemicals present in a methanol extract of A. alpina, assess their potential antioxidant activities in vitro, and determine their effects on melanogenesis in B16F10 melanoma cells. Column chromatographic separation of aqueous fractions of A. alpina led to the isolation of 17 compounds. The chemical structures of these compounds were determined using spectroscopic data from electrospray ionization-mass spectrometry and nuclear magnetic resonance. To the best of our knowledge, the present study is the first to identify compounds 2-10 and 12-17 in A. alpina. Furthermore, compound 6 possessed powerful antioxidant activity, while compound 15 suppressed intracellular tyrosinase activity and thus reduced melanogenesis in B16F10 cells. Therefore, our research suggested that these naturally occurring compounds have the potential to reduce oxidative stress and promote skin whitening. Further investigations will be required to elucidate the mechanisms underlying the antioxidant and antityrosinase activities of these compounds.

The mixture of different parts of Nelumbo nucifera and two bioactive components inhibited tyrosinase activity and melanogenesis.

Melanin is the pigment responsible for the color of the eyes, hair, and skin in humans. Tyrosinase is well known to be the key enzyme in melanin biosynthesis. JKTM-12 is composed of the flowers, roots, seeds, and receptacles of Nelumbo nucifera (lotus). In this study, JKTM-12 was investigated for its inhibitory effects on tyrosinase activity and melanin biosynthesis in B16F10 melanoma cells. Moreover, two main bioactive compounds (hyperoside and astragalin) were found from the receptacles of N. nucifera, which are used as the main material of JKTM-12. JKTM-12 was shown to inhibit tyrosinase activity and melanin biosynthesis in alpha-melanocyte-stimulating hormone-stimulated B16F10 melanoma cells. Hyperoside and astragalin, which are the main bioactive compounds of JKTM-12, not only inhibited tyrosinase activity and melanogenesis but also tyrosinase-related protein 1 and tyrosinase-related protein 2 mRNA expression without cytotoxicity at various experiment doses (0.1, 1, and 10 µg/ml). These results suggest that JKTM-12 has the potential for skin whitening with hyperoside and astragalin as the main bioactive compounds.

The complete chloroplast genome of Bupleurum euphorbioides, a traditional medicinal plant.

Bupleurum euphorbioides is a rare native plant attributed with analgesic, gallbladder-supportive, and other functions in China and the Republic of Korea. However, the complete chloroplast genome sequence of the native plant B. euphorbioides has not been determined. In this study, we sequenced the complete chloroplast genome sequence, and examined the molecular phylogeny and genetic information of B. euphorbioides. The total chloroplast genome of B. euphorbioides was 154,871 bp in length with a large single-copy region (85,089 bp), small single-copy region (17,714 bp), and pair of inverted repeats regions (26,034 bp). The chloroplast genome encoded a total of 176 genes, including 131 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic tree indicated that B. euphorbioides was the most closely related to B. latissimum.

The complete chloroplast genome of Bupleurum euphorbioides, a traditional medicinal plant.

Bupleurum euphorbioides is a rare native plant attributed with analgesic, gallbladder-supportive, and other functions in China and the Republic of Korea. However, the complete chloroplast genome sequence of the native plant B. euphorbioides has not been determined. In this study, we sequenced the complete chloroplast genome sequence, and examined the molecular phylogeny and genetic information of B. euphorbioides. The total chloroplast genome of B. euphorbioides was 154,871 bp in length with a large single-copy region (85,089 bp), small single-copy region (17,714 bp), and pair of inverted repeats regions (26,034 bp). The chloroplast genome encoded a total of 176 genes, including 131 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The phylogenetic tree indicated that B. euphorbioides was most closely related to B. latissimum.

Characteristics and phylogenetic analysis of the complete chloroplast genome of Paeonia japonica (Paeoniaceae).

Paeonia japonica, distributed throughout Asia, is a traditional medicinal herb in Korea, with many potential beneficial effects including pain-relieving, anti-inflammatory, and anti-cancer activities. Despite its high pharmacological value, the genetic information on Paeonia japonica remains limited. In this study, the chloroplast genome of P. japonica was sequenced using next-generation sequencing (NGS) technology and genome and phylogeny were analyzed using multiple tools. The chloroplast genome of P. japonica was 152,731 bp in length with an inverted repeat region of 26,656 bp, including a large single-copy region of 84,389 bp and a small single copy region of 17,030 bp. The P. japonica chloroplast genome included 113 genes comprising 80 protein-coding genes, 27 tRNA, and 5 rRNA genes. Phylogenetic analysis indicated that P. japonica and P. obovata share a close evolutionary relationship.

Characterization of the complete chloroplast genome of Liriope platyphylla (Asparagaceae: Nolinoideae) isolated in Korea.

Liriope platyphylla is used as an important medicinal plant for fatigue, cough, and inflammation in South Korea. Here, we report the complete chloroplast genome of L. platyphylla. The total genome size of the chloroplast is 157,076 bp with a large single-copy region (LSC: 85,374 bp), a small single-copy region (SSC: 18,748 bp), and inverted repeat regions (IRa and IRb: 26,477 bp). The GC content of the L. platyphylla chloroplast was 37.6%. The cp genome encoded a set of 129 genes, including 83 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The phylogenetic tree analysis indicated that L. platyphylla is closely related to L. spicata.

A 13-week repeated oral dose toxicity evaluation and a 4-week recovery evaluation of the Sam So Eum (SSE) in male and female rats.

ETHNOPHAMACOLOGICAL RELEVANCE: Sam So Eum (SSE), used in traditional Korean medicine, has been prescribed for the treatment of various ailments including emesis, and fever for centuries. SSE is known by several different names (Shen Su Yin in traditional Chinese medicine; Jin So In traditional Japanese Kampo medicine). It is a mixture of medicinal plants including Panax ginseng C. A. Mey., Perilla frutescens (L.) Britton, and Peucedanum praeruptorum Dunn. Studies have revealed that SSE has many pharmacological effects including anti-inflammatory, anti-cancer, and anti-allergic properties, but its toxic effects have not been evaluated in vivo. Recently, the use of traditional medicinal herbs to treat various diseases has increased, owing to increased number of studies supporting their efficacy. However, safety evaluations for toxicity and other adverse effects have not been extensive. It is commonly considered that natural products extracted from traditional medicinal herbs are safer than synthetic drugs, but this lacks a scientific basis. Thus, in this study, we evaluated the toxicity of SSE in male and female rats. AIM OF THE STUDY: To evaluated the safety of SSE in male and female rats. MATERIALS AND METHODS: SSE was administered orally for 13 weeks at 1000, 2000, and 4000 mg kg-1·day-1, and then the rats were maintained for 4 weeks without SSE administration (recovery evaluation). RESULTS: We observed the animals for changes in clinical signs, including hematological parameters, and food consumption; serum chemistry profiling and urinalysis were also carried out. Creatinine levels in the serum were significantly increased following oral administration of SSE at 2000 and 4000 mg kg-1·day-1 in male and female rats, but returned to the normal levels during the recovery period. In addition, SSE administration does not cause kidney and liver toxicity. Thus, we determined that the no-observed-adverse-effect level of SSE is 4000 mg kg-1·day-1. The no-observed-effect level of SSE was determined to be 1000 mg kg-1·day-1, because serum creatinine was increased by oral administration of SSE at 2000 and 4000 mg kg-1·day-1 in male and female rats. CONCLUSIONS: SSE administration does not cause toxicity at 4000 mg kg-1·day-1 in male and female rats.

6'-O-acetyl mangiferin from Iris rossii Baker inhibits lipid accumulation partly via AMPK activation in adipogenesis.

Effective control of white adipose tissue accumulation would provide a therapeutic strategy for obesity, which poses a growing global problem. The plant chemical mangiferin stimulates adenosine monophosphate-activated protein kinase (AMPK), which inhibits adipogenesis and has therefore been considered a therapeutic target for obesity and related diseases. We previously reported the anti-inflammatory properties of 6'-O-acetyl mangiferin (OAM). In this study, we evaluated the potential of OAM as an AMPK activator in vitro in 3T3-L1 preadipocytes. OAM inhibited adipogenesis as indicated by lower intracellular lipid and triglyceride accumulation as well as reduced adipogenic gene and protein expression upon treatment. OAM-treated 3T3-L1 cells excreted more glycerol, indicating increased lipolysis, which was supported by increased expression of lipolysis-related genes, including adipose triglyceride lipase and hormone-sensitive lipase. We determined that OAM upregulates lipolysis via phosphorylation-dependent activation of AMPK. Further, OAM upregulated the ß-oxidation pathway as indicated by enhanced expression of phosphorylated acetyl-CoA-carboxylase and long-chain acyl-CoA synthetase 1. In conclusion, OAM markedly decreased intracellular lipid accumulation by enhancing lipolysis via AMPK activation and by upregulating ß-oxidation. Thus, OAM has potential as a drug for the prevention and/or improvement of obesity and related diseases and deserves further study.

Water Extract of Acori Graminei Rhizoma Attenuates Features of Rheumatoid Arthritis in DBA/1 Mice.

The dry rhizome of Acorus gramineus Solander, known as Acori Graminei Rhizoma, is used to treat dementia, stroke, eczema, and indigestion in traditional Chinese medicine, traditional Korean medicine, and traditional Japanese Kampo medicine. Previous studies have reported that Acori Graminei Rhizoma extract ameliorated cognitive impairment in Aß1-42 injected mice. However, the effect of Acori Graminei Rhizoma on type II collagen induced arthritis (CIA) has not been elucidated. Thus, we evaluated the water extract of Acori Graminei Rhizoma (WAG) in CIA mice models. Male DBA/1 mice were separated into five groups (NOR; n=10, CON; n=10, CIA + methotrexate (MTX); n=10, CIA + 100 mg/kg WAG; n=10, CIA + 500 mg/kg WAG; n=10). CIA was induced by injecting the mice with bovine type II collagen, after which the mice were treated with WAG and/or MTX. Hematological parameters and liver and kidney serum toxicity markers were analyzed. Further, serum levels of interleukin (IL)-6, TNF-α, and type II collagen IgG were analyzed via enzyme-linked immunosorbent assay (ELISA). Treatment with 500 mg/kg WAG decreased serum levels of IL-6, TNF-α, and collagen IgG in a CIA model. Moreover, WAG treatment decreased CIA-induced swelling of mouse hind legs, infiltration of inflammatory cells into the synovial membrane, and blood neutrophil levels. WAG administration did not influence hematological parameters or kidneys and liver toxicity markers. WAG may be used to treat arthritis by reducing the inflammation indicators. However, further experiments are required to determine how WAG affects inflammation mechanisms in vitro and in vivo.

A 90-Day Repeated Oral Dose Toxicity Study of Alismatis Rhizoma Aqueous Extract in Rats.

Alismatis rhizoma (AR), the dried rhizome of Alisma orientale (Sam.) Juzep, is a well-known, traditional medicine that is used for the various biological activities including as a diuretic, to lower cholesterol and as an anti-inflammatory agent. The present study was carried out to investigate the potential toxicity of the Alismatis rhizoma aqueous extract (ARAE) following 90-day repeated oral administration to Sprague-Dawley rats. ARAE was administered orally to male and female rats for 90 days at 0 (control), 500, 1,000 and 2,000 mg/kg/day (n = 10 for male and female rats for each dose). Additional recovery groups from the control group and high dose group were observed for a 28-day recovery period. Chromatograms of ARAE detected main compounds with four peaks. Treatment-related effects including an increase in the red blood cells, hemoglobin, hematocrit, albumin, total protein, and urine volume were observed in males of the 2,000 mg/kg/day group (p < 0.05). However, the diuretic effect of ARAE was considered, a major cause of hematological and serum biochemical changes. The oral no-observed-adverse-effect level (NOAEL) of the ARAE was > 2,000 mg/kg/day in both genders, and no target organs were identified.

Beneficial Effects of Cynaroside on Cisplatin-Induced Kidney Injury In Vitro and In Vivo.

Anti-cancer drugs such as cisplatin and doxorubicin are effectively used more than radiotherapy. Cisplatin is a chemotherapeutic drug, used for treatment of various forms of cancer. However, it has side effects such as ototoxicity and nephrotoxicity. Cisplatin-induced nephrotoxicity increases tubular damage and renal dysfunction. Consequently, we investigated the beneficial effect of cynaroside on cisplatin-induced kidney injury using HK-2 cell (human proximal tubule cell line) and an animal model. Results indicated that 10 µM cynaroside diminished cisplatin-induced apoptosis, mitochondrial dysfunction and caspase-3 activation, cisplatin-induced upregulation of caspase-3/MST-1 pathway decreased by treatment of cynaroside in HK-2 cells. To confirm the effect of cynaroside on cisplatin-induced kidney injury in vivo, we used cisplatin exposure animal model (20 mg/kg, balb/c mice, i.p., once a day for 3 days). Renal dysfunction, tubular damage and neutrophilia induced by cisplatin injection were decreased by cynaroside (10 mg/kg, i.p., once a day for 3 days). Results indicated that cynaroside decreased cisplatin-induced kidney injury in vitro and in vivo, and it could be used for improving cisplatin-induced side effects. However, further experiments are required regarding toxicity by high dose cynaroside and caspase-3/MST-1-linked signal transduction in the animal model.

Antioxidant effects of Geranium nepalense ethanol extract on H2O2-induced cytotoxicity in H9c2, SH-SY5Y, BEAS-2B, and HEK293.

Oxidative damage leads to many diseases. In this study, we evaluated the antioxidant effects of 70% ethanol extract of Geranium nepalense (GNE) on hydrogen peroxide-induced cytotoxicity in cell lines: H9c2, SH-SY5Y, HEK293, and BEAS-2B. We determined the free radical scavenging activity of GNE using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and superoxide dismutase-like activities, as well as the polyphenol and flavonoid contents of GNE. The results showed that GNE scavenged DPPH and ABTS radicals in a dose-dependent manner. In addition, it contained abundant contents of total polyphenol and flavonoid contents and strongly suppressed cellular reactive oxygen species, thereby protecting H2O2-induced cytotoxicity in H9c2, SH-SY5Y, HEK293, and BEAS-2B cell lines. The powerful antioxidant activity exhibited by GNE, both in vitro and in cell systems, was attributed to its free radical scavenging activity. Therefore, GNE may be useful in preventing oxidative stress-induced diseases including Alzheimer's disease, respiratory inflammatory disease, and chronic kidney diseases.

Anti-inflammatory effect of tricin isolated from Alopecurus aequalis Sobol. on the LPS-induced inflammatory response in RAW 264.7 cells.

The aim of this study was to identify major anti-inflammatory compounds in Alopecurus aequalis Sobol. (A. aequalis). The ethanol extract and the hexane-, dichloromethane-, ethyl acetate- and n-butanol-soluble fractions derived from A. aequalis were evaluated in order to determine their inhibitory effects on nitric oxide (NO) production in RAW 264.7 cells stimulated with lipopolysaccharide (LPS). The ethanol extract decreased NO production in a dose-dependent manner without any evidence of cytotoxicity at a concentration range of 0-200 µg/ml. The ethyl acetate soluble fraction was the most potent among the four soluble fractions. A compound was isolated by reversed-phase high-performance liquid chromatography from the ethyl acetate soluble fraction and this was identified to be tricin. Tricin inhibited the LPS-induced NO production in a dose-dependent manner without any evidence of cytotoxity at a concentration range of 1-100 µg/ml. Tricin also inhibited the LPS-induced production of prostaglandin E2. Western blot analysis indicated that tricin decreased the LPS-induced increase in the protein levels of inducible NO synthase and cyclooxygenase. In addition, tricin suppressed the production of intracellular reactive oxygen species in the LPS-stimulated RAW 264.7 cells, as measured by flow cytometry. Taken together, our results clearly indicate that tricin is a major functional anti-inflammatory compound which can be isolated from A. aequalis extracts.

Antioxidant effects of Cirsium setidens extract on oxidative stress in human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs) may be used in cell-based therapy to promote neovascularization for the treatment of ischemic diseases. However, high levels of reactive oxygen species (ROS) derived from the pathophysiological ischemic environment induce senescence and apoptosis of MSCs, resulting in reduced functionality and defective neovascularization. Therefore, the present study aimed to determine the protective effects of Cirsium setidens, a natural product, on oxidative stress­induced apoptosis in MSCs. The present study investigated for the change of ROS levels in MSCs using ROS assays. In addition, cell viability determined by MTT and TUNEL assays. Western blot analysis was performed to investigate the change of apoptosis­associated proteins in MSCs. Treatment of MSCs with hydrogen peroxide (H2O2; 200 µM) significantly increased intracellular ROS levels and cell death; however, pretreatment with C. setidens (100 µg/ml) suppressed H2O2­induced ROS generation and increased the survival of MSCs. H2O2­induced ROS production increased the levels of phosphorylated­p38 mitogen activated protein kinase, c­Jun N­terminal kinase, ataxia telangiectasia mutated and p53; these increases were inhibited by pretreatment with C. setidens. In addition, C. setidens inhibited ROS­induced apoptosis of MSCs by increasing the expression levels of the anti­apoptotic protein B­cell lymphoma 2 (BCL­2), and decreasing the expression levels of the proapoptotic protein BCL­2­associated X protein. These findings indicated that pretreatment of MSCs with C. setidens may prevent ROS­induced oxidative injury by regulating the oxidative stress­associated signaling pathway, and suppressing the apoptosis­associated signal pathway. Therefore, C. setidens may be developed as a beneficial broad­spectrum agent for enhancing the effectiveness of MSC transplantation in the treatment of ischemic diseases.

Anti-inflammatory effects of 6'-O-acetyl mangiferin from Iris rossii Baker via NF-κb signal blocking in lipopolysaccharide-stimulated RAW 264.7 cells.

A series of acylated xanthone C-glucosides were identified from the methanolic extract of whole Iris rossii Baker. The major constituent was characterized as 6'-O-acetyl mangiferin (OAM), and complete structure elucidation was carried out using 2D NMR (COSY, HSQC, and HMBC) and LC-IT-TOF-MS analyses. The present study is the first to report the anti-inflammatory effects of 6'-O-acetyl mangiferin from Iris rossii Baker on the lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 macrophage cells. OAM strongly suppressed protein expression of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2), thereby inhibiting the production of nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). Furthermore, OAM inhibited the LPS-induced phosphorylation of ERK, JNK, and p38, which led to the blockade of nuclear factor-kappa B (NF-κB) and inhibitor kappa B (IκB)-α activation. These results suggest that the anti-inflammatory effects of OAM may be attributed to the downregulation of COX-2 and iNOS via the suppression of NF-κB and the MAPK signaling pathway in RAW264.7 macrophages.

Acute toxicity and cytotoxicity evaluation of Dendrobium moniliforme aqueous extract in vivo and in vitro.

Dendrobium moniliforme (L.) Sw., an herb of the Orchidaceae family, has long been used in traditional medicine to strengthen bones, nourish the stomach, and promote the production of bodily fluid. Recently, polysaccharides isolated from Dendrobium have been used in functional foods and nutraceutical products. A traditional method to process Dendrobium is to soak fresh stems in an ethanol solution, which is the most important factor to ensure high yields of aqueous-extractable polysaccharides. The present study was carried out to investigate the potential acute toxicity of D. moniliforme aqueous extract (DMAE), by a single oral dose in Sprague-Dawley rats. The test article was orally administered once by gavage to male and female rats at doses of 0, 2,500, and 5,000 mg/kg body weight (n=5 male and female rats for each dose). Throughout the study period, no treatment-related deaths were observed and no adverse effects were noted in clinical signs, body weight, food consumption, serum biochemistry, organ weight, or gross findings at any dose tested. The results show that a single oral administration of DMAE did not induce any toxic effects at a dose below 5,000 mg/kg in rats, and the minimal lethal dose was considered to be over 5,000 mg/kg body weight for both sexes. With respect to cytotoxicity, the cell viability of human embryonic kidney (HEK293) cells was less than 50% when the cells were treated with 10 mg/mL aqueous extract for 24 h.