Inhibition of desmoglein-1 by aspirin leads to synthetic lethality of keratinocytes in Shuanghuanglian-induced cutaneous eruption response.

Cutaneous eruptions caused by the combination of Chinese and Western medicine have attracted widespread attention; however, the underlying mechanism remains unclear. This study aimed to evaluate the potential mechanism of cutaneous eruptions in vivo and in vitro using the combination of Shuanghuanglian injection powder (SHL) and aspirin (ASA) as an example. ASA and SHL co-administration induced inflammatory responses in HaCat cells, as evidenced by marked increases in the expression of IL-4 and TNF-α, and the level of apoptosis. Additionally, histopathological investigation of mice skin tissues showed local inflammatory cell infiltration. Western boltting was used to detect the effects of ASA on desmoglein-1 (DSG1) expression; we found that DSG1 expression was down-regulated in vivo and in vitro. Finally, the key components of SHL were administered to HaCat cells with down-regulated DSG1; it was seen that neochlorogenic acid and rutin have a significant effect on HaCat cell apoptosis. These results demonstrate that DSG1 deficiency is a potential cause of cutaneous eruptions caused by the combination of SHL and ASA, and neochlorogenic acid and rutin are the main allergenic components. This study provides a new research strategy for the safety evaluation of integrated traditional Chinese and Western medicine.

In vivo and in vitro toxicological evaluations of aqueous extract from Cecropia pachystachya leaves.

CECROPIA PACHYSTACHYA: leaves are popularly used to treat asthma and diabetes. Despite the widespread consumption of this plant, there are few scientific studies regarding its toxicological potential. In order to conduct a thorough study concerning the potential adverse effects, the aim of this study was to assess acute and subacute toxicity tests of crude aqueous extract from C. pachystachya leaves (CAE-Cp) using in vivomodel, as well as in vitro cytotoxicity, genotoxicity and antioxidant activity. In addition, genotoxicity, and cytotoxicity of chlorogenic acid (CGA) and cytotoxicity of isoorientin (ISOO) were also evaluated. The antioxidant activity was verified by DPPH, cytotoxicity using sulforhodamine B (SRB) assay and genotoxicity by comet assay on V79 cells. The phytochemical analysis of CAE-Cp detected flavonoids and tannins, CGA and ISOO as the major compounds utilizing HPLC. The total flavonoid content (6.52 mg/g EQ) and antioxidant activity (EC50 = 62.15 µg/ml) of CAE-Cp were determined. In vitro evaluations with CAE-Cp showed genotoxic effects at 0.31 to 2.5 mg/ml and an expressive cytotoxicity on HT-29 (IC50 = 4.43 µg/ml) cells. CGA was genotoxic against V79 cells at 0.07 mg/ml and cytotoxic against to HT-29 (IC50 = 71.70 µg/ml), OVCAR-3 (IC50 = 80.07 µg/ml), MCF-7 (IC50 = 45.58 µg/ml) and, NCI-H460 (IC50 = 71.89 µg/ml) cancer cell lines. Wistar rats treated with a single dose (2,000 mg/kg) CAE-Cp decreased hemoglobin levels after 14 days, although no significant toxicity was observed in animals after 28 days. In view of the in vitro cytotoxicity and genotoxicity detected, further studies are necessary to establish the safe use of CAE-Cp.

Antibacterial activity of chlorogenic acid-loaded SiO2 nanoparticles caused by accumulation of reactive oxygen species.

Diseases caused by pathogenic bacilli pose an increasing threat to human health. A common feature of these bacteria is a complete cell wall; therefore, drugs that can penetrate this protective barrier could be used as a novel approach for treating these infections. Here we present a simple method for synthesizing a silica mesoporous material loaded with cadmium selenide (CdSe) and chlorogenic acid. Using UV-visible, fluorescence, and infrared imaging in combination with transmission electron microscopy, it was shown that CdSe and chlorogenic acid could be successfully embedded in the mesopores of silica nanoparticles (CSC NPs), and these NPs presented with a strong fluorescence, uniform size, and good dispersion. Additionally, the results of these analyses indicated that the fluorescence of the CSC NPs was localized within the cells of Escherichia coli and Bacillus subtilis, signifying that these NPs could breach the cell wall and enter the cells of these two bacilli. Additional assessments found that these CSC NPs inhibited the proliferation of the bacteria by disrupting the cell wall, and this was most likely due to the overproduction of reactive oxygen species induced by chlorogenic acid. Importantly, histopathology analysis indicated that the CSC NPs had limited side effects and high biocompatibility.

Chlorogenic Acid and Its Microbial Metabolites Exert Anti-Proliferative Effects, S-Phase Cell-Cycle Arrest and Apoptosis in Human Colon Cancer Caco-2 Cells.

Chlorogenic acid (CGA) decreases colon cancer-cell proliferation but the combined anti-cancer effects of CGA with its major colonic microbial metabolites, caffeic acid (CA), 3-phenylpropionic acid (3-PPA) and benzoic acid (BA), needs elucidation as they occur together in colonic digesta. Caco-2 cancer cells were treated for 24 h with the four compounds individually (50-1000 µM) and as an equimolar ratio (1:1:1:1; MIX). The effective concentration to decrease cell proliferation by 50% (EC50) was lower for MIX (431 ± 51.84 µM) and CA (460 ± 21.88) versus CGA (758 ± 19.09 µM). The EC50 for cytotoxicity measured by lactate dehydrogenase release in MIX (527 ± 75.34 µM) showed more potency than CA (740 ± 38.68 µM). Cell proliferation was decreased by 3-PPA and BA at 1000 µM with no cytotoxicity. Cell-cycle arrest was induced at the S-phase by CA (100 µM), MIX (100 µM), CGA (250 µM) and 3-PPA (500 µM) with activation of caspase-3 by CGA, CA, MIX (500 and 1000 µM). Mitochondrial DNA content was reduced by 3-PPA (1000 µM). The anti-cancer effects occurred at markedly lower concentrations of each compound within MIX than when provided singly, indicating that they function together to enhance anti-colon cancer activities.

Roasted and green coffee extracts show antioxidant and cytotoxic activity in myoblast and endothelial cell lines in a cell specific manner.

Coffee is one of the most highly consumed beverages with potential beneficial health implications, however its molecular mechanism of action has not been completely elucidated yet. To that cause, the polyphenolic composition of different coffee extracts (from Light, Medium and Dark roasts as well as green beans) was examined by UHPLC-HRMS analysis, indicating chlorogenic acids isomers as the main constituents. In the following step, the toxicity of the extracts was tested in myoblasts and endothelial cells and differential toxicity of green and roasted samples was displayed as the myoblasts were more sensitive to green coffee extracts, in contrast to the endothelial cells. Subsequently, biologically relevant, non-cytotoxic extract concentrations were administered to explore their potential effect on cell redox status using flow cytometry and spectrophotometric assays. The results indicated that all coffee extracts improved cell redox status, however differences were observed between the two different cell lines tested, implying that coffee compounds display cell- and tissue-specificity. Glutathione levels were increased in almost all cases up to 70%, while the roasting degree affected the free radical scavenging potential of the extracts and their ability to protect from macromolecular oxidation as exhibited by the differences in ROS, CARB and TBARS levels, especially in the myoblasts.

Graphene oxide as a nanocarrier for controlled release and targeted delivery of an anticancer active agent, chlorogenic acid.

We have synthesized graphene oxide using improved Hummer's method in order to explore the potential use of the resulting graphene oxide as a nanocarrier for an active anticancer agent, chlorogenic acid (CA). The synthesized graphene oxide and chlorogenic acid-graphene oxide nanocomposite (CAGO) were characterized using Fourier transform infrared (FTIR) spectroscopy, thermogravimetry and differential thermogravimetry analysis, Raman spectroscopy, powder X-ray diffraction (PXRD), UV-vis spectroscopy and high resolution transmission electron microscopy (HRTEM) techniques. The successful conjugation of chlorogenic acid onto graphene oxide through hydrogen bonding and π-π interaction was confirmed by Raman spectroscopy, FTIR analysis and X-ray diffraction patterns. The loading of CA in the nanohybrid was estimated to be around 13.1% by UV-vis spectroscopy. The release profiles showed favourable, sustained and pH-dependent release of CA from CAGO nanocomposite and conformed well to the pseudo-second order kinetic model. Furthermore, the designed anticancer nanohybrid was thermally more stable than its counterpart. The in vitro cytotoxicity results revealed insignificant toxicity effect towards normal cell line, with a viability of >80% even at higher concentration of 50µg/mL. Contrarily, CAGO nanocomposite revealed enhanced toxic effect towards evaluated cancer cell lines (HepG2 human liver hepatocellular carcinoma cell line, A549 human lung adenocarcinoma epithelial cell line, and HeLa human cervical cancer cell line) compared to its free form.

Comparative cytotoxicity of artemisinin and cisplatin and their interactions with chlorogenic acids in MCF7 breast cancer cells.

In parts of Africa and Asia, self-medication with a hot water infusion of Artemisia annua (Artemisia tea) is a common practice for a number of ailments including malaria and cancer. In our earlier work, such an extract showed better potency than artemisinin alone against both chloroquine-sensitive and -resistant parasites. In this study, in vitro tests of the infusion in MCF7 cells showed high IC50 values (>200 µM). The combination of artemisinin and 3-caffeoylquinic acid (3CA), two major components in the extract, was strongly antagonistic and gave a near total loss of cytotoxicity for artemisinin. We observed that the interaction of 3CAs with another cytotoxic compound, cisplatin, showed potentiation of activity by 2.5-fold. The chelation of cellular iron by 3CA is hypothesized as a possible explanation for the loss of artemisinin activity.

Café: uma bebida rica em substâncias com efeitos clínicos importantes, em especial a cafeína / Coffee: a beverage rich in substances with important clinical effects, especially caffeine

O café é uma das bebidas mais populares do mundo, chegando ao consumo aproximado de 6,7 milhões de toneladas por ano. Há certo tempo, alguns dos seus efeitos fisiológicos, relacionados a uma gama de substâncias encontradas na bebida, estão sendo amplamente estudados. Alguns estudos destacam a cafeína como uma substância fundamental para os efeitos estudados desta bebida. O trabalho objetivou discernir e ressaltar alguns efeitos clínicos relevantes da cafeína. Nesse sentido, foi realizada uma busca de trabalhos que valorizam as propriedades clínicas do café, que ressaltam algumas de suas substâncias, e estudos específicos sobre a cafeína, que a atribuem uma abordagem clínica. Foram definidos pelos autores alguns aspectos positivos e negativos dos efeitos clínicos provocados pela cafeína. Assim, reforça-se a discussão sob as perspectivas de uso da cafeína, seja na alimentação, como medicamento ou em estudos de parâmetros clínicos para diabetes tipo 2, arritmias, parada cardíaca, infarto agudo não fatal do miocárdio, Parkinson e Alzheimer. É preciso atribuir, nesse contexto, certa ponderação ao seu uso, relevando a vulnerabilidade do indivíduo e as manifestações clínicas atribuídas à cafeína...

Coffee is one of the most popular beverages in theworld, with an approximate consumption of 6.7 million tons per year. Some of the physiological effects of a variety of substances found in the beverage are being widely studied. Some research highlights caffeine as a substance crucial to coffee?s biological effects. The aim of this study was to discern and highlight some of the relevant clinical effects of caffeine. To this end, we made a search for studies related to the clinical properties of coffee, which highlighted some of its main substances, and studies specifically about caffeine, which followed a clinical approach. The authors defined some positive and negative features of the clinical effects provoked by caffeine. Thus, the prospects of using caffeine, in food, as a medicine or in clinical parameter studies of type 2 diabetes, arrhythmia, cardiac arrest, nonfatal acute myocardial infarction, Parkinson and Alzheimers disease, were well discussed. In this context, it is very important to give responsible consideration to theuse of caffeine, keeping in mind the vulnerability of the individual and the clinical manifestations of this substance...

Pharmacologic overview of systemic chlorogenic acid therapy on experimental wound healing.

Chlorogenic acid (CGA) is a well-known natural antioxidant in human diet. To understand the effects of CGA on wound healing by enhancing antioxidant defense in the body, the present study sought to investigate the potential role of systemic CGA therapy on wound healing and oxidative stress markers of the skin. We also aimed to understand whether chronic CGA treatment has side effects on pivotal organs or rat bone marrow during therapy. Full-thickness experimental wounds were created on the backs of rats. CGA (25, 50, 100, 200 mg/kg) or vehicle was administered intraperitoneally for 15 days. All rats were sacrificed on the 16th day. Biochemical, histopathological, and immunohistochemical examinations were performed. Possible side effects were also investigated. The results suggested that CGA accelerated wound healing in a dose-dependent manner. CGA enhanced hydroxyproline content, decreased malondialdehyde and nitric oxide levels. and elevated reduced glutathione, superoxide dismutase, and catalase levels in wound tissues. Epithelialization, angiogenesis, fibroblast proliferation, and collagen formation increased by CGA while polymorph nuclear leukocytes infiltration decreased. CGA modulated matrix metalloproteinase-9 and tissue inhibitor-2 expression in biopsies. Otherwise, high dose of CGA increased lipid peroxidation of liver and kidney without affecting the heart and muscle samples. Chronic CGA increased micronuclei formation and induced cytotoxicity in the bone marrow. In conclusion, systemic CGA has beneficial effects in improving wound repair. Antioxidant, free radical scavenger, angiogenesis, and anti-inflammatory effects of CGA may ameliorate wound healing. High dose of CGA may induce side effects. In light of these observations, CGA supplementation or dietary CGA may have benefit on wound healing.

Repeated-dose toxicological studies of Tithonia diversifolia (Hemsl.) A. gray and identification of the toxic compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Tithonia diversifolia (Hemsl.) A. Gray has been commonly used in folk medicine to treat abscesses, microbiological infections, snake bites, malaria and diabetes. Both anti-inflammatory and anti-malarial properties have been identified using appropriate assays, but the effective doses have demonstrated toxic effects for the experimental animals. Most of the pharmacological activities have been attributed to sesquiterpene lactones (STLs) and some chlorogenic acid derivatives (CAs) in the leaves of this species. This work aimed to evaluate the repeated-dose toxicity of an aqueous extract (AE) from Tithonia diversifolia leaves and to compare the results with an extract rich in STLs (LRE) and a polar extract (PE) without STLs but rich in CAs. The purpose of this work was to provide insights into the identity of the compounds responsible for the toxic effects of Tithonia diversifolia. MATERIALS AND METHODS: The major classes of compounds were confirmed in each extract by IR spectra and HPLC-UV-DAD profiling using previously isolated or standard compounds. The toxicity of each extract was evaluated in a repeated-dose toxicity study in Wistar rats for 90 days. RESULTS: The AE is composed of both STLs and CAs, the LRE is rich in STLs, and the PE is rich in CAs. The AE caused alterations in haematological parameters but few alterations in biochemical parameters and was relatively safe at doses lower than 100mg/kg. However, the PE and LRE demonstrated several adverse effects by damaging the liver and kidneys, respectively. CONCLUSION: STLs and CAs can be toxic in prolonged use at higher doses in extracts prepared from Tithonia diversifolia by affecting the kidneys and liver.

High-dose chlorogenic acid induces inflammation reactions and oxidative stress injury in rats without implication of mast cell degranulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chlorogenic acid (CA) exits widely in those Chinese herbal injections that have antibacterial and antiphlogistic effects and belongs to the ethnopharmacological family of medicines. Chinese herbal injections containing high levels of CA have been reported to increase the adverse drug reactions, but the mechanism for which is still unclear. In this study, we investigated the mechanism of the CA derived adverse drug reactions. AIM OF THE STUDY: The present study was to explore the potential role of CA in initiating inflammatory reaction and the underlying mechanism. MATERIALS AND METHODS: Male Wistar rats were treated with different dosages of CA for different time period. The variables examined included microcirculation by intravital microscopy, histology of ileum tissue, expression of adhesion molecules CD11b and CD18 on leukocytes by flow cytometry, myeloperoxidase activity and maleic dialdehyde content in ileum tissue by spectrophotometry, activity of superoxide dismutase and catalase in serum by ELISA, and expression of NADPH oxidase subunits by PCR and Western blot. RESULTS: High-dose CA increased the number of adherent leukocytes, generation of peroxides in the venular walls and induced albumin leakage from mesentery venules. High-dose CA induced changes also included an increase in maleic dialdehyde, myeloperoxidase, inflammatory cytokines and NADPH oxidase activities, and a decline in activity of superoxide dismutase and catalase. CONCLUSION: High-dose, but not Low-dose CA induced inflammation reaction, and in this process an imbalance between oxidant and antioxidant mechanism may be involved, providing more information for better understanding the rationale behind the adverse effects of CA.

Renal toxicity caused by oral use of medicinal plants: the yacon example.

AIM OF THE STUDY: Yacon [Smallanthus sonchifolius (Poepp. & Endl.) H. Robinson, Asteraceae] is an Andean species that has traditionally been used as an anti-diabetic herb in several countries around the world, including Brazil. Its hypoglycaemic action has recently been demonstrated in normal and diabetic rats. However, studies about the safety of prolonged oral consumption of yacon leaf extracts are lacking. Thus, this work was undertaken to evaluate the repeated-dose toxicity of three extracts from yacon leaves: the aqueous extract (AE) prepared as a tea infusion; the leaf-rinse extract (LRE), which is rich in sesquiterpene lactones (STLs); and a polar extract from leaves without trichomes, or polar extract (PE), which lacks STLs but is rich in chlorogenic acids (CGAs). MATERIALS AND METHODS: The major classes of the compounds were confirmed in each extract by IR spectra and HPLC-UV-DAD profiling as well as comparison to standard compounds. The toxicity of each extract was evaluated in a repeated-dose toxicity study in Wistar rats for 90 days. RESULTS: The PE was rich in CGAs, but we did not detect any STLs. The AE and LRE showed the presence of STLs. The polar extract caused alterations in some biochemical parameters, but the animals did not show signs of behavioural toxicity or serious lesions in organs. Alterations of specific biochemical parameters in the blood (creatinine 7.0 mg/dL, glucose 212.0 mg/dL, albumin 2.8 g/dL) of rats treated with AE (10, 50 and 100 mg/kg) and LRE (10 and 100 mg/kg) pointed to renal damage, which was confirmed by histological analysis of the kidneys. CONCLUSIONS: The renal damage was associated with increased blood glucose levels after prolonged oral administration of the AE. This observation suggested that the hypoglycaemic effect observed after treatment for 30 days in an earlier study is reversible and was likely the result of renal injury caused by the toxicity of yacon. Because STLs were detected in both AE and LRE, there is strong evidence that these terpenoids are the main toxic compounds in the leaves of the yacon. Based on our results, we do not recommend the oral use of yacon leaves to treat diabetes.

Mast cell degranulation induced by chlorogenic acid.

AIM: To investigate the mechanism of chlorogenic acid (CA)-induced anaphylactoid reactions. METHODS: Degranulation of peritoneal mast cells was assayed by using alcian blue staining in guinea pigs, and the degranulation index (DI) was calculated. CA-induced degranulation of RBL-2H3 cells was also observed and assayed using light microscopy, transmission electron microscopy, flow cytometry, and beta-hexosaminidase release. RESULTS: CA 0.2, 1.0, and 5.0 mmol/L was able to promote degranulation of peritoneal mast cells in guinea pigs in vitro, but it did not increase the degranulation of peritoneal mast cells in CA-sensitized guinea pigs compared with control (P>0.05). Treatment with CA 0.2, 1.0, and 5.0 mmol/L for 30, 60, and 120 min induced degranulation in RBL-2H3 cells in a dose- and time-dependent manner (P<0.01). Under transmission electron microscope typical characteristics of degranulation, including migration of granular vesicles toward the plasma membrane and integration combined with exocytosis, were observed, after CA or C48/80 treatment. Fluorescent microscopy and flow cytometric analysis showed that CA induced concentration-dependent translocation of phosphatidylserine in RBL-2H3 cells. beta-hexosaminidase release in RBL-2H3 cells was significantly increased after incubation with 1 mmol/L CA for 60 min and 5 mmol/L CA for 30 min (P<0.01). CONCLUSION: CA induces degranulation of peritoneal mast cells and RBL-2H3 cells in guinea pigs, which might be one of the mechanisms of the generation of anaphylactoid reactions induced by CA.

Role of chlorogenic acid in the toxicity induced by Chinese herbal injections.

Adverse reactions induced by Chinese herbal injections have been frequently reported. However, the precise causes of these adverse reactions are not yet fully understood. The aim of the present study was to determine the role of chlorogenic acid (a ubiquitous component of Chinese herbs) in the toxicity of Chinese herbal injections. Beagle dogs were given chlorogenic acid, Yuxingcao injection, or Qingkailing injection (the latter two both containing chlorogenic acid) by intravenous (i.v.) injection, once a day for 7 or 9 days. The systemic toxicity was evaluated. An additional ultrastructural observation on liver and kidney was performed. Anaphylactoid reactions were obvious in dogs treated with Yuxingcao injection. Varying degrees of ultrastructural changes in liver and kidney were observed in the treated dogs, especially in dogs treated with Chinese herbal injections. Our study has led to the view that chlorogenic acid is not an allergen when administrated by i.v. injection, but liver and kidney injury induced by Chinese herbal injections can be partly attributed to chlorogenic acid.

Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice.

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA-PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP-OVA or TNP-Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.

Inhibitory effect of 3-caffeoyl-4-dicaffeoylquinic acid from Salicornia herbacea against phorbol ester-induced cyclooxygenase-2 expression in macrophages.

Salicornia herbacea (S. herbacea), an annual herb that grows in the salt marshes of the Korean peninsula, has been used as a folk medicine to treat a variety of diseases such as constipation, obesity, diabetes, and cancer. However, the effect of S. herbacea on inflammation is unclear. In the present study, we investigated the effects of a novel chlorogenic acid, 3-caffeoyl-4-dicaffeoylquinic acid (CDCQ), isolated from S. herbacea, on cyclooxygenase-2 (COX-2) expression in murine macrophage RAW 264.7 cells. Phorbol 12-myristate 13-acetate (PMA) induces COX-2 expression and production of prostaglandin E(2) (PGE(2)). PMA-induced COX-2 protein, gene expression and PGE(2) production were significantly inhibited by CDCQ in a dose-dependent manner. Transfection of hCOX-2, as well as of deletion and mutation promoter constructs, revealed that the CCAAT/enhancer-binding protein (C/EBP) and activator protein-1 (AP-1) predominantly contributed to the effects of CDCQ. In addition, electrophoretic mobility shift assays and transfection results showed that CDCQ directly inhibited PMA-induced C/EBP and AP-1 transcription and binding activity. CDCQ also remarkably reduced PMA-induced C/EBPbeta and c-jun protein expression. Furthermore, CDCQ significantly inhibited PMA-induced activation of the mitogen-activated protein kinases (MAP kinases), JNK and p38. These findings demonstrate that CDCQ effectively attenuates COX-2 production, and enhance our understanding of the anti-inflammatory properties of CDCQ.

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.

Sequential fractionation with concurrent chemical and toxicological characterization of the combustion products of chlorogenic acid.

Chlorogenic acid is the most abundant polyphenol found in the tobacco plant. The biological effects of its combustion products remain largely unknown. In this study, chlorogenic acid was burned at 640 degrees C for 2 min and the particulate matter of the smoke was collected onto Cambridge filter pads followed by selective extraction in five different solvents. Various fractions of the chlorogenic acid combustion products were tested for induction of micronuclei in V79 Chinese hamster fibroblast cells. Over 40 compounds were identified in the dimethyl sulfoxide (DMSO) extract by high-performance liquid chromatography coupled to electrospray time-of-flight mass spectrometry (HPLC/TOF-MS). The DMSO extract was then fractionated into three major fractions by preparative LC. The fraction inducing the highest degree of toxicity was further separated into four sub-fractions. The sub-fraction responsible for the most toxic response was determined to contain catechol as its major component. The overall reproducibility of the combustion, the extraction procedure and the chemical characterization of the compounds responsible for the toxicity in the chlorogenic acid smoke were evaluated by LC/TOF-MS.

A flavanone and two phenolic acids from Chrysanthemum morifolium with phytotoxic and insect growth regulating activity.

Leaves of Chrysanthemum morifolium cv. Ramat were extracted sequentially with hexane, ethyl acetate, and methanol. The methanol fraction, when incorporated into artificial diet was found to reduce the growth of cabbage looper (Trichoplusia ni Hubner) larvae at concentrations between 500 and 5000 ppm of diet. Fractionation of the methanol extract on a Sephadex column yielded five fractions, three of which reduced the weight of larvae relative to the control. One fraction was analyzed using high performance liquid chromatography (HPLC) and found to contain three main constituents. These compounds were purified using a combination of gel permeation chromatography on Sephadex LH20 and HPLC, and analyzed by 1H and 13CNMR as well as undergoing chemical and physical analyses. The compounds were identified as: 1, chlorogenic acid (5-O-caffeoylquinic acid); 2, 3,5-O-dicaffeoylquinic acid; and 3, 3', 4',5-trihydroxyflavanone7-O-glucuronide (eriodictyol7-O-glucuronide). At concentrations between 100 to 1000 ppm these compounds reduced both growth and photosynthesis of Lemna gibba L. with the order of efficacy being: flavanone > chlorogenic acid > 3,5-O-dicaffeoylquinic acid. Furthermore, when incorporated separately into artificial diet these compounds, at 10 to 1000 ppm, enhanced or reduced growth of the cabbage looper (Trichoplusia ni) and gypsy moth (Lymantria dispar L.).

Dicaffeoyltartaric acid analogues inhibit human immunodeficiency virus type 1 (HIV-1) integrase and HIV-1 replication at nontoxic concentrations.

The human immunodeficiency virus type 1 (HIV-1) is a major health problem worldwide. In this study, 17 analogues of L-chicoric acid, a potent inhibitor of HIV integrase, were studied. Of these analogues, five submicromolar inhibitors of integrase were discovered and 13 compounds with activity against integrase at less than 10 microM were identified. Six demonstrated greater than 10-fold selectivity for HIV replication over cellular toxicity. Ten analogues inhibited HIV replication at nontoxic concentrations. Alteration of the linkages between the two bis-catechol rings, including the use of amides, mixed amide esters, cholate, and alkyl bridges, was explored. Amides were as active as esters but were more toxic in tissue culture. Alkyl and cholate bridges were significantly less potent against HIV-1 integrase in vitro and were inactive against HIV-1 replication. Two amino acid derivates and one digalloylderivative of L-chicoric acid (L-CA) showed improved selectivity over L-CA against integration in cell culture. These data suggest that in addition to the bis-catechols and free carboxylic acid groups reported previously, polar linkages are important constituents for optimal activity against HIV-1 integrase and that new derivatives can be developed with increased specificity for integration over HIV entry in vivo.