New ent-Kaurane and cleistanthane diterpenoids with potential cytotoxicity from Phyllanthus acidus (L.) Skeels.

Six diterpenoids including three ent-kauranes (1-2, 4) and three cleistanthanes (3, 5-6) were isolated from the roots and stems of Phyllanthus acidus (L.) Skeels. Of them, (16S)-ent-16,17,18-tri-hydroxy-19-nor-kaur-4-en-3-one (1), phyllanthone A (2), and 6-hydroxycleistanthol (3) are new compounds, while the ent-kaurane diterpenoids were reported from the titled plant for the first time. Their structures were elucidated on the basis of the extensive spectroscopic analyses. Compounds 2 and 4-6 displayed cytotoxic potential with IC50 values ranging from 1.96 to 29.15 µM. They also showed moderate anti-inflammatory activities (IC50 = 6.30-12.05 µM). Particularly, the new ent-kaurane 2 displayed cytotoxic potential against HL-60 (IC50 = 2.00 µM) and MCF-7 (IC50 = 3.55 µM) cells, and anti-inflammatory activity (IC50 = 6.47 µM).

Preliminary safety assessment of oridonin in zebrafish.

Context: Oridonin, isolated from the leaves of Isodon rubescens (Hemsl.) H.Hara (Lamiaceae), has good antitumor activity. However, its safety in vivo is still unclear. Objective: To investigate the preliminary safety of oridonin in zebrafish. Materials and methods: Embryo, larvae and adult zebrafish (n = 40) were used. Low, medium and high oridonin concentrations (100, 200 and 400 mg/L for embryo; 150, 300 and 600 mg/L for larvae; 200, 400 and 800 mg/L for adult zebrafish) and blank samples were administered. At specific stages of zebrafish development, spontaneous movement, heartbeat, hatching rate, etc., were recorded to assess the developmental effects of oridonin. VEGFA, VEGFR2 and VEGFR3 gene expression were also examined. Results: Low-dose oridonin increased spontaneous movement and hatching rate with median effective doses (ED50) of 115.17 mg/L at 24 h post-fertilization (hpf) and 188.59 mg/L at 54 hpf, but these values decreased at high doses with half maximal inhibitory concentrations (IC50) of 209.11 and 607.84 mg/L. Oridonin decreased heartbeat with IC50 of 285.76 mg/L at 48 hpf, and induced malformation at 120 hpf with half maximal effective concentration (EC50) of 411.94 mg/L. Oridonin also decreased body length with IC50 of 324.78 mg/L at 144 hpf, and increased swimming speed with ED50 of 190.98 mg/L at 120 hpf. The effects of oridonin on zebrafish embryo development may be attributed to the downregulation of VEGFR3 gene expression. Discussions and conclusions: Oridonin showed adverse effects at early stages of zebrafish development. We will perform additional studies on mechanism of oridonin based on VEGFR3.

Oridonin, A natural diterpenoid, protected NGF-differentiated PC12 cells against MPP+- and kainic acid-induced injury.

Oridonin (ORI) is a natural diterpenoid presented in some medicinal plants. The effects of pre-treatments from ORI against MPP+- or kainic acid (KA)-induced damage in nerve growth factor (NGF)-differentiated PC12 cells were investigated. Results showed that pre-treatments of ORI at 0.25-2 µM enhanced the viability and plasma membrane integrity of NGF-differentiated PC12 cells. MPP+ or KA exposure down-regulated Bcl-2 mRNA expression, up-regulated Bax mRNA expression, increased caspase-3 activity and decreased Na+-K+ ATPase activity. ORI pre-treatments at test concentrations reversed these changes. ORI pre-treatments decreased reactive oxygen species production, raised glutathione level, and increased glutathione peroxidase, glutathione reductase and catalase activities in MPP+ or KA treated cells. ORI pre-treatments lowered tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and prostaglandin E2 levels in MPP+ or KA treated cells. ORI also diminished MPP+ or KA induced increase in nuclear factor-κB binding activity. MPP+ exposure suppressed tyrosine hydroxylase (TH) mRNA expression and decreased dopamine content. KA exposure reduced glutamine synthetase (GS) mRNA expression, raised glutamate level and lowered glutamine level. ORI pre-treatments at 0.5-2 µM up-regulated mRNA expression of TH and GS, restored DA and glutamine content. These findings suggested that oridonin was a potent neuro-protective agent against Parkinson's disease and seizure.

A research on the genotoxicity of stevia in human lymphocytes.

Stevia extracts are obtained from Stevia rebaudiana commonly used as natural sweeteners. It is ∼250-300 times sweeter than sucrose. Common use of stevia prompted us to investigate its genotoxicity in human peripheral blood lymphocytes. Stevia (active ingredient steviol glycoside) was dissolved in pure water. Dose selection was done using ADI (acceptable daily intake) value. Negative control (pure water), 1, 2, 4, 8 and 16 µg/ml concentrations which were equivalent to ADI/4, ADI/2, ADI, ADI × 2 and ADI × 4 of Stevia were added to whole-blood culture. Two repetitive experiments were conducted. Our results showed that there was no significant difference in the induction of chromosomal aberrations and micronuclei between the groups treated with the concentrations of Stevia and the negative control at 24 and 48 h treatment periods. The data showed that stevia (active ingredient steviol glycosides) has no genotoxic activity in both test systems. Our results clearly supports previous findings.

Toxicological evaluation of ethanolic extract from Stevia rebaudiana Bertoni leaves: Genotoxicity and subchronic oral toxicity.

Stevia rebaudiana Bertoni leaves have a long history of use as an abundant source of sweetener. The aqueous extract of stevia leaves and the predominant constitutes steviol glycosides have been intensively investigated. However, rare studies provided toxicological evaluation of bioactive components in the polar extract regarding their safety on human health. This study aimed to evaluate the toxicity of ethanolic extract of Stevia rebaudiana Bertoni leaves through a battery of in vitro and in vivo tests. Negative results were unanimously obtained from bacterial reverse mutation assay, mouse bone marrow micronucleus assay and mouse sperm malformation assay. Oral administration at dietary levels of 1.04%, 2.08% and 3.12% for 90 days did not induce significant behavioral, hematological, clinical, or histopathological changes in rats. Significant reduction of cholesterol, total protein and albumin was observed in female animals only at high dose level. The results demonstrated that Stevia rebaudiana Bertoni leaves ethanolic extract, which is rich in isochlorogenic acids, does not possess adverse effects through oral administration in this study. Our data provided supportive evidence for the safety of Stevia rebaudiana Bertoni leaves that may potentially be used in functional foods as well as nutritional supplements beyond sweetner.

A Review on the Pharmacology and Toxicology of Steviol Glycosides Extracted from Stevia rebaudiana.

Stevia rebaudiana Bertoni is a sweet and nutrient-rich plant belonging to the Asteraceae family. Stevia leaves contain steviol glycosides including stevioside, rebaudioside (A to F), steviolbioside, and isosteviol, which are responsible for the plant's sweet taste, and have commercial value all over the world as a sugar substitute in foods, beverages and medicines. Among the various steviol glycosides, stevioside, rebaudioside A and rebaudioside C are the major metabolites and these compounds are on average 250-300 times sweeter than sucrose. Steviol is the final product of Stevia metabolism. The metabolized components essentially leave the body and there is no accumulation. Beyond their value as sweeteners, Stevia and its glycosdies possess therapeutic effects against several diseases such as cancer, diabetes mellitus, hypertension, inflammation, cystic fibrosis, obesity and tooth decay. Studies have shown that steviol glycosides found in Stevia are not teratogenic, mutagenic or carcinogenic and cause no acute and subacute toxicity. The present review provides a summary on the biological and pharmacological properties of steviol glycosides that might be relevant for the treatment of human diseases.

Cytotoxic ent-kaurane diterpenoids from Isodon nervosus.

Four new ent-kaurane diterpenoids, rabdonervosins G-J (1-4, resp.), were isolated from the leaves and stems of Isodon nervosus. Their structures were elucidated by extensive spectroscopic analyses, including 1D-, 2D-NMR and HR mass spectra. Compound 2 showed potent cytotoxicity against the HepG2 and PC-9/ZD cell lines with IC50 values of 2.36 and 6.07 µM, respectively, and compound 3 exhibited cytotoxicity against the HepG2 and CNE2 cell lines with IC50 values of 8.64 and 9.77 µM, respectively.

Glycosylation of ent-kaurene derivatives and an evaluation of their cytotoxic activities.

AIM: To discover more active and water-soluble derivatives of tetracyclic diterpenoids containing an exo-methylene cyclopentanone or an α-methylenelactone moiety. METHODS: All of the key intermediates were synthesized from stevioside, and the target compounds were obtained through glycosylation of the 4-carboxyl group. The cytotoxicity of the target compounds against six human cancer cell lines, HepG2, Bel-7402, A549, U251, MCF-7 and MDA-MB-231, were evaluated by the MTT assay. RESULTS: Compound 1b was more effective than the positive control adriamycin against the HepG2, Bel-7402, A549, MCF-7, and MDA-MB-231 cell lines with IC50 values of 0.12, 0.91, 0.35, 0.08, and 0.07 µmol·L(-1), respectively. Moreover, compound 3c exhibited the most potent and selective cytotoxic activity against the HepG2 cell line (IC50, 0.01 µmol·L(-1)). CONCLUSION: Compounds 1b and 3c could be considered as potential anticancer candidates for further study.

Effect of some ent-kaurenes on the viability of human peripheral blood mononuclear cells.

The possible cytotoxic activity of some ent-kaurenes on human mononuclear cells, obtained from peripheral blood, was studied having in mind future studies on their antitumor activity. The cells were obtained using the Ficoll-Hypaque method, adjusted to 2 x 10(6) cells/mL, and incubated with kaurenes for 48 hours at 3 x 10(-5), 30 x 10(-5), 300 x 10(-1) and 3000 x 10(-5) micromol/well. Ent-kaurenic acid showed no toxicity at all concentrations studied. The least toxic of all the kaurene derivatives studied was ent-15,16-epoxy-17-acetoxy-(-)-kauran-19-oic acid, with a cellular viability of 99% at 3 x l0(-5) micromol/well, and 94% at 30 x 10(-1) micromol/well. Another compound that showed low toxicity was the 2,3,4,6-tetra-acetyl-alpha-D-pyranosyl ester of ent-15-oxo-(-)-kaur-16-en-19-oic acid with 44% viability at 3000 x 10(-5) micromol/well. The most toxic compounds at all concentrations tested were ent-kaur-16-en-19-ol acetate and ent-16alpha-hydroxy-(-)-kauran-19-oic acid. On the other hand, ent-kaur-9(11)16-dien-19-oic acid, ent-kauran-19-oic acid, and ent-kaur-16-en-19-ol were toxic only at the highest concentration studied. According to these results, and considering the concentrations employed, ent-kaur-16-en-19-oic acid and ent-15,16-epoxy-17-acetoxy-(-)-kauran-19-oic acid could be used for in vivo experiments and possibly for therapeutic purposes on humans, without much risk.

Hepatotoxicity of kaurene glycosides from Xanthium strumarium L. fruits in mice.

The fruit of Xanthium strumarium L. (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. However, clinical utilization of Xanthium strumarium is relatively limited because of its toxicity. The present investigation was carried out to evaluate the toxic effects on acute liver injury in mice of the two kaurene glycosides (atractyloside and carbxyatractyloside), which are main toxic constituents isolated from Fructus Xanthii on acute liver injury in mice. Histopathological examinations revealed that there were not obviously visible injury in lungs, heart, spleen, and the central nervous system in the mice by intraperitoneal injection of atractyloside (ATR, at the doses 50,125 and 200 mg/kg) and carbxyatractyloside (CATR, at the doses 50,100 and 150 mg/kg) for 5 days. However, it revealed extensive liver injuries compared with the normal group. In the determination of enzyme levels in serum, intraperitoneal injection of ATR and CATR resulted in significantly elevated serum alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP) activities compared to controls. In the hepatic oxidative stress level, antioxidant-related enzyme activity assays showed that ATR and CATR administration significantly increased hepatic malondialdehyde (MDA) concentration, as well as decreased superoxide dismutase (SOD), catalase (CAT) activities and glutathione (GSH) concentration, and this was in good agreement with the results of serum aminotransferase activity and histopathological examinations. Taken together, our results demonstrate that kaurene glycosides induce hepatotoxicity in mice by way of its induction of oxidative stress as lipid peroxidation in liver, which merited further studies. Therefore, these toxic constituents explain, at least in part, the hepatotoxicity of X. strumarium L. in traditional medicine.

6,7-seco-ent-kaurane diterpenoids from Isodon sculponeatus with cytotoxic activity.

Six new 6,7-seco-ent-kaurane diterpenoids, sculponeatins N-S (1-6, resp.), together with eleven known analogues, 7-17, were isolated from the aerial parts of Isodon sculponeatus. The structures of compounds 1-6 were elucidated by spectroscopic methods including extensive 1D- and 2D-NMR experiments, as well as HR-ESI-MS analysis. All diterpenoids obtained were assayed for their cytotoxic activity against K562 and HepG2 human tumor cell lines. Among them, compound 1 showed the most significant cytotoxicity with the IC50 values of 0.21 and 0.29 µM, respectively. The structure-activity relationships are discussed.

Anxiolytic and sedative-hypnotic activities of polygalasaponins from Polygala tenuifolia in mice.

In the present study, the anxiolytic and sedative-hypnotic activities of polygalasaponins extracted from Polygala tenuifolia Willdenow (Polygalaceae) were determined in mice using hole-board, elevated plus maze, open field, and sodium pentobarbital-induced hypnosis tests. Moreover, the acute toxicity of polygalasaponins was also estimated in mice. Sixty minutes after p.o. administration of polygalasaponins (40, 80, 160 mg/kg) in mice, the central crossing counts and percentage of central/total ambulation significantly increased and the number of rearings and defecations was evidently inhibited in the open field test. Polygalasaponins also increased the head-dips of mice in the hole-board test and the time spent by mice in the open arms of the X-maze, prolonged sleep duration and shortened sleep latency in the test of synergetic effect on sodium pentobarbital (45 and 25 mg/kg, respectively). Acute toxic study showed the oral median lethal dose (LD(50)) of polygalasaponins was 3.95 g/kg and 0% lethal dose 2.6 g/kg. These results suggest that polygalasaponin possesses evident anxiolytic and sedative-hypnotic activities and has a relatively safe dose range, which supports the use of Polygala tenuifolia root as an anxiolytic and sedative-hypnotic drug in folk medicine.

Cytotoxic and anticarcinogenic activity of the ent-kaurene diterpenoid, melissoidesin, from Isodon wightii (Bentham) H. Hara.

Isodon wightii is a common plant found in the Western Ghats of South India. The ent-kaurene diterpenoid melissoidesin, isolated from the leaves of I. wightii, showed significant cytotoxicity against lung cancer and neuroblastoma cell lines with IC(50) values of 6 and 4.2 microg mL(-1), respectively. The prevention of deoxyribose degradation activity of melissoidesin was significant and the IC(50) value was calculated to be 163.1 microg mL(-1). The anticarcinogenic activity of melissoidesin by comet assay showed significant DNA damage protecting activity in a dose-dependent manner.

Stevioside and related compounds: therapeutic benefits beyond sweetness.

Stevioside, an abundant component of Stevia rebaudiana leaf, has become well-known for its intense sweetness (250-300 times sweeter than sucrose) and is used as a non-caloric sweetener in several countries. A number of studies have suggested that, beside sweetness, stevioside along with related compounds, which include rebaudioside A (second most abundant component of S. rebaudiana leaf), steviol and isosteviol (metabolic components of stevioside) may also offer therapeutic benefits, as they have anti-hyperglycemic, anti-hypertensive, anti-inflammatory, anti-tumor, anti-diarrheal, diuretic, and immunomodulatory actions. It is of interest to note that their effects on plasma glucose level and blood pressure are only observed when these parameters are higher than normal. As steviol can interact with drug transporters, its role as a drug modulator is proposed. This review summarizes the current knowledge of the pharmacological actions, therapeutic applications, pharmacokinetics and safety of stevioside and related compounds. Although much progress has been made concerning their biological and pharmacological effects, questions regarding chemical purity and safety remain unsolved. These issues are discussed to help guide future research directions.

Subchronic toxicity of rebaudioside A.

The safety of the stevia-derived sweetener, rebaudioside A (CAS No. 58543-16-1), was evaluated in two oral toxicity studies. In a 4-week study, Wistar rats were administered rebaudioside A at dietary concentrations of 0, 25,000, 50,000, 75,000 and 100,000ppm. The NOAEL, including an evaluation of testes histopathology, was determined to be 100,000 ppm. In the 13-week study, Wistar rats were administered rebaudioside A at dietary concentrations of 0, 12,500, 25,000 and 50,000ppm. Reductions in body weight gain attributable to initial taste aversion and lower caloric density of the diet were observed in high-dose male and females groups. Inconsistent reductions in serum bile acids and cholesterol were attributed to physiological changes in bile acid metabolism due to excretion of high levels of rebaudioside A via the liver. All other hepatic function test results and liver histopathology were within normal limits. Significant changes in other clinical pathology results, organ weights and functional observational battery test results were not observed. Macroscopic and microscopic examinations of all organs, including testes and kidneys, were unremarkable with respect to treatment-related findings. The NOAEL in the 13-week toxicity study was considered to be 50,000ppm or approximately 4161 and 4645mg/kg body weight/day in male and female rats, respectively.

Cytotoxic diterpenoids from Vietnamese medicinal plant Croton tonkinensis GAGNEP.

Six new ent-kaurane-type diterpenoids were isolated from the leaves of the endemic Vietnamese medicinal plant Croton tonkinensis GAGNEP. (Euphorbiaceae) together with three known ent-11alpha-acetoxy-7beta,14alpha-dihydroxykaur-16-en-15-one (1), ent-kaur-16-en-15-one 18-oic acid (5) and ent-18-hydroxykaur-16-ene (7). Their structures were determined by spectroscopic analyses to be ent-7beta-acetoxy-11alpha-hydroxykaur-16-en-15-one (2), ent-18-acetoxy-11alpha-hydroxykaur-16-en-15-one (3), ent-11alpha-acetoxykaur-16-en-18-oic acid (4), ent-15alpha,18-dihydroxykaur-16-ene (6), ent-11alpha,18-diacetoxy-7beta-hydroxykaur-16-en-15-one (8), and ent-(16S)-1alpha,14alpha-diacetoxy-7beta-hydroxy-17-methoxykauran-15-one (14). ent-Kaurane-type diterpenoids from Croton tonkinensis 2-4, 6, and 9-13, were tested for toxicity in the brine shrimp lethality assay. Compounds 9, 10, and 12 demonstrated significant activity, compounds 2, 3, 6, and 11 showed weak activity, and compounds 4 and 13 were inactive.

New ent-kaurene-type diterpenoids possessing cytotoxicity from the New Zealand liverwort Jungermannia species.

Two new ent-kaurene-type and a new rearranged ent-kaurene-type diterpenoids possessing cytotoxicity against a human leukemia cell line have been isolated from the New Zealand liverwort Jungermannia species, together with previously known ent-kaurene-type diterpenoids. Their structures were established based on extensive NMR techniques.