Preclinical safety assessment of the crude extract from Sida rhombifolia L. aerial parts in experimental models of acute and repeated-dose 28 days toxicity in rats.

Sida rhombifolia (Malvaceae) is popularly used as a treatment for several pathological conditions; however, there is a lack of studies that identify its compounds and that evaluate comprehensively the safety of its consumption. Therefore, the aim of this study was to determinate the phytochemical constitution of the crude extract of Sida rhombifolia (CESR), and its safety in models of acute and repeated doses (28 days) toxicity. The tested dose for the model of acute toxicity was 2000 mg/kg doses for the repeated dose model were 150, 300 e 600 mg/kg. Hematological, biochemical, histopathological and oxidative markers were investigated. HPLC-DAD-MS analysis evidenced the presence of caffeic acid, coumarin, and rutin. In the acute toxicity model the only altered parameters were tissue ROS, and AST and BUN in serum. As for the repeated dose experiment both hematological and biochemical markers remained within the values of reference for the species. Obtained results demonstrate that the CESR did not present significant toxic effects when administrated orally to male and female rats in acute and repeated doses.

Caffeic acid phenethyl ester, a coffee polyphenol, inhibits DNA methylation in vitro and in vivo.

DNA methylation represents an important epigenetic regulation of the genome. Earlier studies have suggested that dietary phenolic compounds including those contained in coffee, tea and soy products may modulate the level of DNA methylation. In this study, we first characterize the effect of caffeic acid phenethyl ester (CAPE) and other dietary phenolic compounds on DNA methylation in vitro. The IC50 values of CAPE, daidzein, isorhamnetin and genistein are 7.6, 6.9, 6.2, and 4.3 µM, respectively, in an in-vitro enzymatic assay system. Computational analysis indicates that CAPE, daidzein, isorhamnetin and genistein can bind inside the DNA substrate-binding site in human DNMT1 with a favorable binding energy. In an animal study, we find that maternal CAPE treatment shifts the coat color distribution of the 21-day-old Avy/a offspring towards the yellow phenotype, indicating that CAPE inhibits the methylation of the agouti gene promoter sequence in vivo. The results from this study may shed light on the potential epigenetic effect in the offspring resulting from maternal intake of certain coffee phenolics during pregnancy.

Cells Deficient in the Fanconi Anemia Protein FANCD2 are Hypersensitive to the Cytotoxicity and DNA Damage Induced by Coffee and Caffeic Acid.

Epidemiological studies have found a positive association between coffee consumption and a lower risk of cardiovascular disorders, some cancers, diabetes, Parkinson and Alzheimer disease. Coffee consumption, however, has also been linked to an increased risk of developing some types of cancer, including bladder cancer in adults and leukemia in children of mothers who drink coffee during pregnancy. Since cancer is driven by the accumulation of DNA alterations, the ability of the coffee constituent caffeic acid to induce DNA damage in cells may play a role in the carcinogenic potential of this beverage. This carcinogenic potential may be exacerbated in cells with DNA repair defects. People with the genetic disease Fanconi Anemia have DNA repair deficiencies and are predisposed to several cancers, particularly acute myeloid leukemia. Defects in the DNA repair protein Fanconi Anemia D2 (FANCD2) also play an important role in the development of a variety of cancers (e.g., bladder cancer) in people without this genetic disease. This communication shows that cells deficient in FANCD2 are hypersensitive to the cytotoxicity (clonogenic assay) and DNA damage (γ-H2AX and 53BP1 focus assay) induced by caffeic acid and by a commercial lyophilized coffee extract. These data suggest that people with Fanconi Anemia, or healthy people who develop sporadic mutations in FANCD2, may be hypersensitive to the carcinogenic activity of coffee.

In vitro and in vivo toxicological evaluations of methyl ferulate, methyl p-coumarate, and pulegone 1,2-epoxide.

CONTEXT: Toxicological screening of natural compounds for medicinal purposes. OBJECTIVES: The objective of this study is to evaluate the toxicity of methyl ferulate (MF), methyl p-coumarate (MpC), and pulegone 1,2-epoxide (PE) with in vitro and in vivo assays. MATERIALS AND METHODS: The in vitro toxicity of MF, MpC, and PE was assessed at a concentration of 10 mg/ml with the Ames assay using two strains of Salmonella typhimurium TA98 and TA100. Human red blood cells (RBC) were used to determine the hemolytic activity of these compounds. The cytotoxicity of above compounds was determined with brine shrimp lethality bioassay (BSLB) at the concentrations of 0.1-20 mg/ml. While dermal and ocular irritation studies were conducted on healthy rabbits (n = 8) for 96 and 12 h post-topical application of test compounds, respectively. RESULTS: PE produced 6-8% hemolysis of RBCs at all the tested concentrations while MF and MpC produced 10-5% hemolysis up to 20 mg/ml, and 50-85% hemolysis at concentrations of 40 and 80 mg/ml, respectively. The Ames assay indicated that MF, MpC, and PE were non-mutagenic as the test values were not significantly higher as compared with background values of the assay. BSLB suggested the lethal concentration (LC50) values of MF, MpC, and PE as 4.38, 6.74, and 25.91 mg/ml, respectively. In vivo ocular and dermal irritation scores of MF, MpC, and PE were comparable with ethanol (control) in rabbits indicating the non-irritant nature of these natural compounds. CONCLUSION: The present studies suggest that these compounds are non-toxic/non-irritant and might be used for medicinal purposes.

Methylation and its role in the disposition of tanshinol, a cardiovascular carboxylic catechol from Salvia miltiorrhiza roots (Danshen).

AIM: Tanshinol is an important catechol in the antianginal herb Salvia miltiorrhiza roots (Danshen). This study aimed to characterize tanshinol methylation. METHODS: Metabolites of tanshinol were analyzed by liquid chromatography/mass spectrometry. Metabolism was assessed in vitro with rat and human enzymes. The major metabolites were synthesized for studying their interactions with drug metabolizing enzymes and transporters and their vasodilatory properties. Dose-related tanshinol methylation and its influences on tanshinol pharmacokinetics were also studied in rats. RESULTS: Methylation, preferentially in the 3-hydroxyl group, was the major metabolic pathway of tanshinol. In rats, tanshinol also underwent considerable 3-O-sulfation, which appeared to be poor in human liver. These metabolites were mainly eliminated via renal excretion, which involved tubular secretion mainly by organic anion transporter (OAT) 1. The methylated metabolites had no vasodilatory activity. Entacapone-impaired methylation did not considerably increase systemic exposure to tanshinol in rats. The saturation of tanshinol methylation in rat liver could be predicted from the Michaelis constant of tanshinol for catechol-O-methyltransferase (COMT). Tanshinol had low affinity for human COMT and OATs; its methylated metabolites also had low affinity for the transporters. Tanshinol and its major human metabolite (3-O-methyltanshinol) exhibited negligible inhibitory activities against human cytochrome P450 enzymes, organic anion transporting polypeptides 1B1/1B3, multidrug resistance protein 1, multidrug resistance-associated protein 2, and breast cancer resistance protein. CONCLUSION: Tanshinol is mainly metabolized via methylation. Tanshinol and its major human metabolite have low potential for pharmacokinetic interactions with synthetic antianginal agents. This study will help define the risk of hyperhomocysteinemia related to tanshinol methylation.

[Optimization of processing technology for xanthii fructus by UPLC fingerprint technique and contents of toxicity ingredient].

The experiment's aim was to optimize the processing technology of Xanthii Fructus which through comparing the difference of UPLC fingerprint and contents of toxicity ingredient in water extract of 16 batches of processed sample. The determination condition of UPLC chromatographic and contents of toxicity ingredient were as follows. UPLC chromatographic: ACQUITY BEH C18 column (2.1 mm x 100 mm, 1.7 microm) eluted with the mobile phases of acetonitrile and 0.1% phosphoric acidwater in gradient mode, the flow rate was 0.25 mL x min(-1) and the detection wavelength was set at 327 nm. Contents of toxicity ingredient: Agilent TC-C18 column (4.6 mm x 250 mm, 5 microm), mobile phase was methanol-0.01 mol x L(-1) sodium dihydrogen phosphate (35: 65), flow rate was 1.0 mL x min(-1), and detection wavelength was 203 nm. The chromatographic fingerprints 16 batches of samples were analyzed in using the similarity evaluation system of chromatographic, fingerprint of traditional Chinese medicine, SPSS16.0 and SIMCA13.0 software, respectively. The similarity degrees of the 16 batches samples were more than 0.97, all the samples were classified into four categories, and the PCA showed that the peak area of chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeic acid were significantly effect index in fingerprint of processed Xanthii Fructus sample. The outcome of determination showed that the toxicity ingredient contents of all samples reduced significantly after processing. This method can be used in optimizing the processing technology of Xanthii Fructus.

Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NF-κB and MAPK activation in macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis is a bee product with numerous biological and pharmacological properties, such as immunomodulatory and anti-inflammatory activities. It has been used in folk medicine as a healthy drink and in food to improve health and prevent inflammatory diseases. However, little is known about its mechanism of action. Thus, the goal of this study was to verify the antioxidant activity and to explore the anti-inflammatory properties of propolis by addressing its intracellular mechanism of action. Caffeic acid was investigated as a possible compound responsible for propolis action. MATERIALS AND METHODS: The antioxidant properties of propolis and caffeic acid were evaluated by using the 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging method. To analyze the anti-inflammatory activity, Raw 264.7 macrophages were treated with different concentrations of propolis or caffeic acid, and nitric oxide (NO) production, a strong pro-inflammatory mediator, was evaluated by the Griess reaction. The concentrations of propolis and caffeic acid that inhibited NO production were evaluated on intracellular signaling pathways triggered during inflammation, namely p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK1/2), the transcription nuclear factor (NF)-κB and extracellular signal-regulated kinase (ERK1/2), through Western blot using specific antibodies. A possible effect of propolis on the cytotoxicity of hepatocytes was also evaluated, since this product can be used in human diets. RESULTS: Caffeic acid showed a higher antioxidant activity than propolis extract. Propolis and caffeic acid inhibited NO production in macrophages, at concentrations without cytotoxicity. Furthermore, both propolis and caffeic acid suppressed LPS-induced signaling pathways, namely p38 MAPK, JNK1/2 and NF-κB. ERK1/2 was not affected by propolis extract and caffeic acid. In addition, propolis and caffeic acid did not induce hepatotoxicity at concentrations with strong anti-inflammatory potential. CONCLUSIONS: Propolis exerted an antioxidant and anti-inflammatory action and caffeic acid may be involved in its inhibitory effects on NO production and intracellular signaling cascades, suggesting its use as a natural source of safe anti-inflammatory drugs.

Administration of caffeic acid worsened bone mechanical properties in female rats.

Natural phenolic acids, commonly present in plants that are normally consumed in the diet, have been reported to exert antiresorptive and/or bone formation increasing activity. The aim of the present study was to investigate the effects of ferulic, caffeic, P-coumaric, and chlorogenic acids on the skeletal system of normal, mature female rats. The phenolic acids (10 mg/kg p. o. daily for 4 weeks) were administered to 3-month-old female Wistar Cmd:(WI)WU rats. Bone mass, mineral and calcium content, macrometric and histomorphometric parameters, and mechanical properties were examined. Phenolic acids had differential effects on the rat skeletal system. Although none of them affected bone macrometric parameters, mass and mineralization, all of them increased the width of femoral trabeculae. Administration of caffeic acid worsened bone mechanical properties (decreasing ultimate load sustained by the femur in three-point bending test). In conclusion, high intake of caffeic acid may unfavorably affect the skeletal system.

Mechanism of toxicity of esters of caffeic and dihydrocaffeic acids.

Ten esters each of caffeic acid and dihydrocaffeic acid have recently been synthesized. Cytotoxicity evaluations of these esters versus L1210 leukemia and MCF-7 breast cancer cells in culture have led to the delineation of substantially different QSAR for each series. The L1210 QSAR for dihydrocaffeic acid esters resembles the QSAR obtained for simple phenols and estrogenic phenols. However, the QSAR pertaining to the caffeic acid esters differs considerably from its sister QSAR. This difference may be attributed to the presence of the olefinic linkage in the side chain. The octyl ester of caffeic acid is nearly ten times as toxic to the leukemia cells than the widely studied phenethyl ester, CAPE.

Can the concept of hormesis Be generalized to carcinogenesis?

The concept of hormesis (i.e., low-dose stimulation/high-dose inhibition) has been shown to be widely generalizable with respect to chemical class, animal model, gender, and biological end point. The public health implication of this lack of linearity in the low-dose area of the dose-response curve raises the question of whether low doses of carcinogens will reduce cancer risk. Articles relating to the process of carcinogenesis (i.e., initiation, promotion, tumor development, and progression) were obtained from a recently developed chemical hormesis database and evaluated for their evidence of hormesis. Numerous examples in well-designed studies indicate that U- or J-shaped dose-response relationships exist with respect to various biomarkers of carcinogenesis in different animal models of both sexes. Examples of such J-shaped dose-response relationships in each stage of the process of carcinogenesis were selected for detailed toxicological examination. These results have important implications for both the hazard assessment of carcinogens and cancer risk assessment procedures.

Apoptosis mediates the selective toxicity of caffeic acid phenethyl ester (CAPE) toward oncogene-transformed rat embryo fibroblast cells.

The active component of the folk medicine propolis, caffeic acid phenethyl ester (CAPE), displays selective toxicity toward cloned rat embryo fibroblast (CREF) cells transformed by a spectrum of diverse acting oncogenes. Identification of the mode of action of CAPE should provide useful information for possible applications of this compound for cancer therapy. The present study uses a series of oncogene transformed, oncogene-reverted and CAPE-resistant oncogene transformed CREF cells to investigate the mechanism underlying the increased sensitivity of transformed cells to CAPE. A direct relationship exists between the cytotoxic effects of CAPE and the induction of DNA fragmentation and apoptosis. DNA degradation into nucleosomal fragments and apoptotic shifts in DNA cell cycle profiles occur in CAPE-treated CREF cells transformed by wild-type 5 adenovirus (Ad5), a mutant Ad5 (H5hr1), the wild-type Ad5 E1A transforming gene, v-src, Ha-ras and the human papilloma virus type 18 transforming genes (HPV-18). In contrast, untransformed CREF cells, human fibroblast expression library-induced morphological revertants of Ad5- and v-src-transformed CREF cells, and Krev-1 expressing revertant Ha-ras-transformed CREF cells are resistant to CAPE-induced toxicity and apoptosis. Similarly, mutant Ad5-transformed CREF cells selected by step-wise growth in increasing concentrations of CAPE are resistant to growth inhibition and apoptosis induced by CAPE. These findings indicate that expression of the transformed phenotype by rodent cells evokes sensitivity to CAPE induced toxicity through apoptosis. The acquisition of CAPE sensitivity in rodent cells is independent of the mode of action of the oncogenic agent. CAPE may prove useful as an antiproliferative agent in cancer cells transformed by mechanistically diverse acting oncogenes.

Modifying effects of components of plant essence on the induction of sister-chromatid exchanges in cultured Chinese hamster ovary cells.

The influence of 21 kinds of components of plant essence on sister-chromatid exchanges (SCEs) induced by mitomycin C was investigated in cultured Chinese hamster CHO K-1 cells. Posttreatment with scopoletin, jasmone, caffeic acid and ferulic acid significantly increased the frequency of SCEs. Further investigation of the SCE-enhancing effect of analogues of caffeic acid and ferulic acid revealed that an alpha,beta-unsaturated carbonyl group may be necessary for SCE-enhancing effects. The influence of caffeic acid and ferulic acid on X-ray- or UV-induced SCEs was also studied. The frequencies of SCEs induced by UV were increased by treatment with these compounds. This increasing effect was observed in the S phase of the cell cycle. On the contrary, X-ray-induced SCEs were reduced by the treatment with these compounds. The decreasing effect was observed in the G1 phase but not in the S or G2 phase. To explain these contradictory results, we assumed that caffeic acid and ferulic acid may modify the repair of DNA strand breaks.

Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis.

The honeybee hive product, propolis, is a folk medicine employed for treating various ailments. Many important pharmaceutical properties have been ascribed to propolis, including anti-inflammatory, antiviral, immunostimulatory and carcinostatic activities. Propolis extracts have provided an active component identified as caffeic acid phenethyl ester (CAPE), which was readily prepared in one step. Differential cytotoxicity has been observed in normal rat/human versus transformed rat/human melanoma and breast carcinoma cell lines in the presence of CAPE.