Use of in vitro assays to assess the potential antigenotoxic and cytotoxic effects of saffron (Crocus sativus L.).

Saffron is harvested from the dried, dark red stigmas of Crocus sativus L. flowers. It is used as a spice for flavoring and coloring food and as a perfume. It is often used for treating several diseases. We assessed the antimutagenic, comutagenic and cytotoxic effects of saffron and its main ingredients using the Ames/Salmonella test system, two well known mutagens (BP, 2AA), the in vitro colony formation assay and four different cultured human normal (CCD-18Lu) and malignant (HeLa, A-204 and HepG2) cells. When only using the TA98 strain in the Ames/Salmonella test system, saffron showed non-mutagenic, as well as non-antimutagenic activity against BP-induced mutagenicity, and demonstrated a dose-dependent co-mutagenic effect on 2-AA-induced mutagenicity. The saffron component responsible for this unusual comutagenic effect was safranal. In the in vitro colony formation test system, saffron displayed a dose-dependent inhibitory effect only against human malignant cells. All isolated carotenoid ingredients of saffron demonstrated cytotoxic activity against in vitro tumor cells. Saffron crocin derivatives possessed a stronger inhibitory effect on tumor cell colony formation. Overall, these results suggest that saffron itself, as well as its carotenoid components might be used as potential cancer chemopreventive agents.     

藏红花药理作用研究进展

藏红花是我国一种应用广泛的名贵中药与民族药,具有很好的活血化瘀、散郁开结作用。藏红花中主要含有苦藏花素、藏红花素等成分,藏红花及其提取物主要用于防治心血管疾病以及与衰老相关的疾病,并且有多种保健品和中成药在国内市场上销售。主要综述近年来有关藏红花及其有效成分防治动脉粥样硬化、保肝护胆、干预肿瘤、调节免疫功能、保护肾脏、防治骨质疏松等方面药理作用的研究进展,为更好地利用该药用植物资源、开发民族药提供一定的帮助。     

基于解吸电喷雾质谱成像技术分析番红花球茎中代谢物的组织分布

目的 探究不同产地番红花球茎中内源性代谢物的分布特征。方法 利用解吸电喷雾（DESI）质谱成像技术,通过优化样品前处理,建立了一种对番红花球茎内源性代谢物可视化分析的方法。结果 实现了黄酮、有机酸、氨基酸、类胡萝卜素和环烯醚萜苷等代谢物的原位表征;L-瓜氨酸、苯乙酰甘氨酸、紫苜蓿酚和栀子苷等特异性分布在球茎中;芹菜素7-（6''-O-乙酰基）-葡萄糖苷、异鼠李素-3-O-葡萄糖苷、蜀黍氰苷6''-葡萄糖苷、芹菜素7-O-二葡糖醛酸主要分布在顶芽中;对于在球茎部位分布的化合物,产于上海的番红花球茎丰度最高,浙江次之,安徽最低。结论 番红花球茎在不同产地及同一产地不同部位的代谢物分布存在显著差异,黄酮和黄酮衍生物如异鼠李素-3-O-葡萄糖苷、芹菜素衍生物主要分布于顶芽中,此外,天然植物保护剂蜀黍氰苷6''-葡萄糖苷也主要在顶芽分布,而作为能量和物质供应的氨基酸则主要积累在球茎。     

回归正交设计优化西红花苷的提取工艺

目的 研究从西红花中提取西红花苷的最佳工艺条件。 方法 以西红花苷的提取率为指标,在使用单因素试验方法考察乙醇浓度、提取时间、提取温度对西红花中西红花苷提取率的基础上,应用回归正交方法建立3元2次方程优化西红花苷的提取工艺。 结果 最佳的提取工艺是：乙醇浓度52%,提取时间30 min,提取温度30 ℃。此条件下,提取率为0.643 5 mg·g^-1。 结论 在优化的提取工艺下,西红花中西红花苷提取率较高。     

Use of in vitro assays to assess the potential cytotoxic,genotoxic and antigenotoxic effects of vanillic and cinnamic acid

Vanillic acid (VA) found in vanilla and cinnamic acid (CA) the precursor of flavonoids and found in cinnamon oil, are natural plant phenolic acids which are secondary aromatic plant products suggested to possess many physiological and pharmacological functions. In vitro and in vivo experiments have shown that phenolic acids exhibit powerful effects on biological responses by scavenging free radicals and eliciting antioxidant capacity. In the present study, we investigated the antioxidant capacity of VA and CA by the trolox equivalent antioxidant capacity (TEAC) assay, cytotoxicity by neutral red uptake (NRU) assay in Chinese Hamster Ovary (CHO) cells and also the genotoxic and antigenotoxic effects of these phenolic acids using the cytokinesis-blocked micronucleus (CBMN) and the alkaline comet assays in human peripheral blood lymphocytes. At all tested concentrations, VA (0.17–67.2?μg/ml) showed antioxidant activity but CA (0.15–59.2?μg/ml) did not show antioxidant activity against 2,2-azino-bis (3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS). VA (0.84, 4.2, 8.4, 16.8, 84 and 168?μg/ml) and CA (0.74, 3.7, 7.4, 14.8, 74, 148?μg/ml) did not have cytotoxic and genotoxic effects alone at the studied concentrations as compared with the controls. Both VA and CA seem to decrease DNA damage induced by H₂O₂ in human lymphocytes.     

Chemical composition and cytotoxic, mutagenic and genotoxic activities of the essential oil from Piper gaudichaudianum Kunth leaves

We have investigated the chemical composition of Piper gaudichaudianum essential oil, as well as its cytotoxic, mutagenic and genotoxic effects in V79 cells. The chemical analyses showed that the major compounds are (E)-nerolidol (22.4%), α-humulene (16.5%), (E)-caryophyllene (8.9%) and bicyclogermacrene (7.4%). Dose-dependent cytotoxic effects were observed in V79 cells treated with essential oil by using clonal survival, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide reduction assay (MTT) and trypan blue exclusion assay (TB), and a significant decrease in survival was observed at concentrations of 0.5 μg/mL and higher. The P. gaudichaudianum essential oil treatment caused DNA strand breaks in V79 cells at concentrations up to 2 μg/mL, as detected by the alkaline comet assay, but did not induce double-strand breaks, as verified by neutral comet assay. It induced a significant increase in the frequency of micronucleated cells at 4, 6 and 10 μg/mL. Moreover, P. gaudichaudianum essential oil significantly increased lipid peroxidation at doses of 0.5 μg/mL and higher, suggesting that the observed oxidant potential can be responsible, at least in part, for its cytotoxic and genotoxic effects.     

A new validated SPE-HPLC method for monitoring crocetin in human plasma--application after saffron tea consumption

Saffron (stigmas of Crocus sativus L.) is a well-known spice with many attributed therapeutic uses throughout centuries. Although studies have demonstrated that crocetin and crocins from saffron have various biological functions, issues concerning the route and way of saffron administration, the absorption and metabolism of saffron carotenoids in humans have not been answered yet. In the present study, an isocratic reversed-phase liquid chromatographic method was developed and validated for the determination of crocetin in plasma. Samples were pre-treated by solid phase extraction (recoveries >72%) and were chromatographed on a Luna C-18 column (4.6mm×250mm, 5μm) with a mobile phase consisting of methanol-water-trifluoroacetic acid (75.0:24.5:0.5, v/v/v) at a flow rate of 1.0mLmin(-1). The HPLC method developed resulted in sharp peaks at 10.7 (trans-crocetin) and 18.6min (cis-crocetin), whereas the calibration curve of total crocetin in plasma displayed a good linearity for concentrations of 0.020-20μM (R(2)=0.999). Specificity, precision, accuracy and stability were studied with spiked plasma samples and were acceptable. The developed method was applied to the determination of crocetin levels in plasma of four healthy human volunteers before and after consumption of one cup of saffron tea (200mg of saffron in 80°C water for 5min). Results showed that the concentration of crocetin was high after 2h (1.24-3.67μM) and still determined after 24h (0.10-0.24). Interestingly, the percentage of the cis-isomer ranges from 25 to 50%, suggesting in vivo isomerization.     

Sub-acute effects of diazinon on biochemical indices and specific biomarkers in rats: Protective effects of crocin and safranal

In this study, the effects of crocin and safranal were studied against sub-acute toxicity of diazinon (DZN) on specific biomarkers, biochemical indices and enzymes levels in rats. Vitamin E (200 IU/kg), safranal at doses 0.025, 0.05 and 0.1 ml/kg and crocin at doses 50, 100 and 200 mg/kg were injected intraperitoneally three times per week alone or with DZN (20 mg/kg/day, orally) for 4 weeks. The parameters were evaluated at the end of 4 weeks. Diazinon did not change serum urea, creatinine, cholesterol, triglyceride, total and direct bilirubin levels. Total protein and albumin concentrations were decreased by diazinon. Crocin, safranal and vitamin E prevented the effect of diazinon on some biochemical indices and enzymes levels. The levels of serum TNF-α, direct 8-iso-prostaglandin F_2α and soluble protein-100 β (S100β) were increased significantly by diazinon. The augmentation of direct 8-iso-prostaglandin F_2α and S100β levels by diazinon was significantly decreased by crocin, safranal and vitamin E. TNF-α level was significantly decreased in diazinon plus crocin 50 and 100 mg/kg treated groups compared to the diazinon group. This study showed that vitamin E, safranal and crocin could prevent diazinon induced enzymes elevation and augmentation of some specific biomarkers.     

Pharmacological activities of crocin in saffron

The effect of crocin on improving ethanol-induced impairment of learning behaviors of mice in passive avoidance tasks is reported. Based on these results, it became evident that crocin prevents the inhibitory effect of ethanol on long-term potentiation (LTP) in the dentate gyrus in vivo. We confirmed that crocin inhibits tumor necrosis factor (TNF)-α-induced apoptosis of PC-12 cells. PC-12 cells showed a rapid increase in cellular ceramide levels, followed by an increase in the phosphorylation of c-Jun kinase (JNK), leading to apoptosis by serum/glucose deprivation in the medium. The production of ceramide was dependent on the activation of magnesium-dependent neutral sphingomyelinase (N-SMase), but not on de novo synthesis. The oxidative stress also decreased the cellular levels of glutathione (GSH), which is the potent inhibitor of N-SMase. Crocin treatment resulted in the prevention of N-SMase activation, ceramide production and JNK phosphorylation. Exploration of the crocin’s preventive mechanism in oxidative stress-induced cell death revealed that the activities of GSH reductase and γ-glutamylcysteinyl synthase (γ-GCS) in the γ-glutamyl cycle affected the stable GSH supply that blocks the activation of N-SMase. These results strongly support the importance of the proposed GSH-dependent inhibitory mechanism in oxidative stress-mediated cell death.     

Evaluation of cytotoxic effects of fipronil on ovaries of semi-engorged Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) tick female

The ovary of the Rhipicephalus sanguineus dog tick female is structurally formed by oocytes in five different stages of development (from I to V) and attached to the gonad by the pedicel. The present study evaluated possible toxic effects of the acaricide fipronil (Frontline) on ovaries of semi-engorged ticks. Sixty partially fed females of R. sanguineus tick were distributed into four groups of 15 specimens each: I--non-treated; II, III and IV--treated with 1, 5 and 10 ppm of fipronil, respectively. The acaricide induced structural changes in the oocytes of individuals of the different treated groups ranging from the presence of a few small vacuoles to cellular death. In conclusion, germinative cells of semi-engorged R. sanguineus tick female are affected by different concentrations of fipronil leading to the reduction of tick fertility.     

Mutagenicity of Mouriri pusa Gardner and Mouriri elliptica Martius

Mouriri pusa Gardner and Mouriri elliptica Martius are fruit-bearing plants of the Melastomataceae family, popularly known in Brazil as puçá-preto or jaboticaba-do-cerrado, and they are used in folk medicine for the treatment of gastric ulcers. In this study, we employ the Ames test to assess the mutagenicity of compounds obtained from the leaves of these species. The methanol extract of the M. pusa was mutagenic to the Salmonella typhimurium strains TA98, TA97a and TA100, with or without metabolic activation. The methanol extract of M. elliptica induced mutagenic activity in TA98 when metabolized with S9 fraction and TA97a with and without S9, but with lower mutagenicity index (MI) and potencies values than those for M. pusa. Enriched fractions of flavonoids and tannins of M. pusa were also evaluated and they demonstrated positive mutagenicity. The highest values of MI and potency were obtained with the flavonoid fraction, which contains large amounts of quercetin, quercetin glycosides and myricetin. These compounds are probably related to the mutagenicity observed in the Ames test. The dichloromethane extract was not mutagenic in any of the test conditions employed.     

Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells

In the present study the chemopreventive effects of water soluble AquaROX^® 15 and oil soluble VivOX^® 40 rosemary extracts against 4-nitroquinoline-N-oxide (NQNO) and 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline (IQ) induced mutagenicity in the reverse mutation assays with Salmonella typhimurium TA98 and against t-butyl hydroperoxide (t-BOOH), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced DNA damage in HepG2 cells were studied, applying the comet assay. The results showed comparable protective effect of AquaROX and VivOX against oxidative DNA damage, whereas protection against indirect active genotoxic carcinogens was more efficient by VivOX.     

Black bean (Phaseolus vulgaris L.) as a protective agent against DNA damage in mice

This study was designed to evaluate the toxicogenetic or protective effect of cooked and dehydrated black beans (Phaseolus vulgaris L.) in bone marrow and peripheral blood cells of exposed mice. The frequency of micronuclei detected using the bone marrow erythrocyte micronucleus test and level of DNA lesions detected by the comet assay were chosen as end-points reflecting mutagenic and genotoxic damage, respectively. Initially, Swiss male mice were fed with a 20% black bean diet in order to detect mutagenic and genotoxic activity. However, no increase in the frequency of bone marrow micronucleated polychromatic erythrocytes (MN PCEs) or DNA lesion in leukocytes was observed. In contrast, received diets containing 1, 10 or 20% of black beans, a clear, but not dose-dependent reduction in the frequency of MN PCEs were observed in animals simultaneously treated with cyclophosphamide, an indirect acting mutagen. Similar results were observed in leukocytes by the comet assay. Commercial anthocyanin was also tested in an attempt to identify the bean components responsible for this protective effect. However, instead of being protective, the flavonoid, at the highest dose administered (50 mg/kg bw), induced primary DNA lesion, as detected by the comet assay. These data indicate the importance of food components in preventing genetic damage induced by chemical mutagens, and also reinforce the role of toxicogenetic techniques in protecting human health.     

In vitro antimicrobial activity of a chitooligosaccharide mixture against Actinobacillus actinomycetemcomitans and Streptococcus mutans

The purpose of this study was to evaluate the in vitro antibacterial activity of a chitooligosaccharide mixture (MW 2000–30 000 Da) with a deacetylation degree of 91.5% against two representative oral pathogens, Actinobacillus actinomycetemcomitans and Streptococcus mutans. A 0.1% concentration of the chitooligosaccharides (derived from the exoskeletons of marine crustaceans) was used to estimate antibacterial activity. Approximately 2 log colony forming units (CFU)/ml of A. actinomycetemcomitans were inactivated by 0.1% chitosan after 30 min, while 120 min exposure inactivated about 4.5 log CFU/ml of this organism. In contrast, the level of inactivation against S. mutans was less than 0.5 log CFU/ml after an exposure of up to 120 min. Electron microscopy showed that the exposure of A. actinomycetemcomitans to the chitooligosaccharides resulted in the disruption of cell membranes and that it could be considered for the treatment of periodontal diseases associated with A. actinomycetemcomitans.     

Transcript profiling and DNA damage in the European eel (Anguilla anguilla L.) exposed to 7,12-dimethylbenz[a]anthracene

The molecular responses induced during and after an acute exposure to 7,12-dimethylbenz[a]anthracene (DMBA) were analysed in liver, gill and blood cells of juvenile Anguilla anguilla with the aim of developing molecular biomarkers of environmental PAH pollution. Changes in the mRNA expression levels of the cell cycle checkpoint-related rad1 gene and the mRNAs of differentially expressed genes by suppression subtractive hybridization (SSH) were analysed in the liver, and related to well-established biomarkers: cyp1A1 mRNA expression and assessment of the DNA integrity using the comet assay and flow cytometry. DMBA exposure resulted in increased cyp1A1 mRNA levels, suggesting that cyp1A1 might be involved in the metabolism of DMBA. Global DNA damage, detected by the comet assay, was observed in the three tissues analysed but only blood cells showed chromosomal lesions as analysed by flow cytometry. Although DNA damage was found in the liver, no induction in rad1 gene was observed in this organ. The global SSH approach revealed that mRNAs of genes related to xenobiotic metabolism, immune processes and cytoskeleton dynamics were differentially expressed in DMBA-exposed eel livers, highlighting the complexity in the response observed in fish exposed to a genotoxic agent and providing directions for new biomarker development.     

知母盐炙前后体外降血糖作用的研究

目的利用Caco-2细胞模型比较知母盐炙前后的降血糖作用,并考察其作用机制,为盐知母炮制原理的阐明提供依据。方法建立Caco-2单层细胞模型,采用CCK-8细胞活力试剂盒检测5 000、2 500、500、250、50、25、5、2.5、0.5、0.25μg/m L生、盐知母水煎液细胞毒性,来确定药物最大毒性剂量。以蔗糖酶和麦芽糖酶底物,葡萄糖氧化酶法测定葡萄糖质量浓度,计算抑制率,比较其对Caco-2单层细胞上蔗糖酶和麦芽糖酶活力的影响。葡萄糖吸收实验测定剩余葡萄糖的质量浓度,比较生、盐知母对Caco-2细胞吸收和转运葡萄糖的影响。结果当生、盐知母质量浓度≤250μg/m L时,Caco-2细胞可进行细胞实验。实验选取了50、250μg/m L两个质量浓度。生、盐知母水煎液50、250μg/m L对蔗糖酶、麦芽糖酶活性均有抑制作用,且盐知母水煎液对蔗糖酶、麦芽糖酶的抑制作用均优于生知母。生、盐知母水煎液均有降低细胞对葡萄糖吸收的趋势,且呈现剂量相关性,但效果不显著。盐知母水煎液对葡萄糖吸收的抑制作用略好于生知母(P0.05)。结论盐知母对Caco-2细胞的降糖作用优于生知母,其降糖机制可能主要体现在α-葡萄糖苷酶上,而对葡萄糖吸收作用影响较小。     

Screening the toxic potential of Cylindrospermopsis raciborskii strains isolated from Lake Balaton, Hungary

Cylindrospermopsis raciborskii is becoming a major concern among cyanobacteria, due to its potential ability to produce toxic metabolites. We assessed the cytotoxic potential of four C. raciborskii strains (ACT 9502, ACT 9503, ACT 9504 and ACT 9505) isolated from Lake Balaton (Hungary), by lactate dehydrogenase (LDH) leakage measurements and by detecting morphological alterations in CHO-K1 (Chinese Hamster Ovary) cells. The Australian AQS (cylindrospermopsin producer) strain of C. raciborskii and purified cylindrospermopsin (CYN) were used as positive references in both the biochemical and morphological studies. Chemical analysis for known cyanotoxins was performed on aqueous extracts of ACT and AQS strains by the HPLC-MS technique.Comparing threshold values of LDH leakage data, different toxic potentials of cyanobacterial extracts are suggested in short term (3 h) and long (24 h) exposure regimes. In the acute (3 h) experiments the aqueous extract of the ACT 9505 strain proved to be most toxic (EC₅₀ = 7.4 mg mL⁻¹), while after 24 h the ACT 9504 extract was the most effective (EC₅₀ = 0.65 mg mL⁻¹). The extract of the AQS strain and the purified CYN exerted most of their toxic effects after 3 h exposure (EC₅₀ = 0.74 mg mL⁻¹, and 0.9 μg mL⁻¹ respectively).The morphological changes of CHO-K1 cells induced by the crude extracts of the ACT strains included fragmentation of the actin filaments then relocation of the depolymerized actin to the perinuclear region, resulting cell rounding and loss of adhesion. Exposure of CHO-K1 cells to the crude extract of the AQS strain, moreover, resulted cell shrinking and formation of filopodia, i.e. distinctly different cytological alterations from that induced by the ACT extracts and the purified CYN.Chemical analysis of the cyanobacterial crude extracts confirmed the presence of cylindrospermopsin in the extract of the AQS strain (8.5 mg CYN g⁻¹ dry weight), and none of the presently known cyanotoxins have been analytically confirmed in the extracts of the ACT strains isolated from the Lake Balaton.Although a significant toxicity of all four ACT C. raciborskii strains is confirmed by both biochemical and morphological studies, our results also pointed out the necessity of further studies to identify the toxic, but still unknown metabolic components produced by these cyanobacterial members of the phytoplankton communities.     

Genotyping and in vitro antifungal susceptibility of Neoscytalidium dimidiatum isolates from different origins

This study evaluated the genetic variability and in vitro susceptibility patterns of isolates of Neoscytalidium dimidiatum and Scytalidium hyalinum from different geographical origins. Partial sequences of four loci (the ITS region and D1/D2 domains of the 28S rRNA gene and the tubulin and chitin synthase genes) were analysed. Among a total of 1970 bp sequenced in 24 isolates, 7 polymorphic positions (0.36%) were detected, representing five different sequence types (ST1–ST5), from which two (ST2 and ST3) were detected exclusively in isolates from plants, two (ST1 and ST5) were found only in clinical isolates and one (ST4) was observed in isolates from humans and from a mango tree. We propose subordinating S. hyalinum as a variety of N. dimidiatum. Amphotericin B was the most active drug, but low minimum inhibitory concentrations were also detected for voriconazole, terbinafine and anidulafungin.     

Prenylated flavonoids and related compounds of the Indonesian Artocarpus (Moraceae)

Several species of the genus Artocarpus (Moraceae) have been investigated in our laboratories during the last decade. Over 60 phenolic constituents have been discovered and characterized, including 27 new compounds from 13 Indonesian taxa of Artocarpus, namely A. champeden, A. lanceifolius, A. teysmanii, A. scortechinii, A. rotunda, A. maingayi, A. kemando, A. bracteata, A. altilis, A. fretessi, A. gomezianus, A. reticulatus and A. glaucus. The principal and the most pronounced features of these phenolic constituents are the assembly of an isoprenyl substituent at C-3 of a flavone skeleton by closure of an ether bridge or a carbon–carbon linkage with the B ring of the skeleton, which may further rearrange into xanthone to produce various classes of natural products. The structures of the new and unusual natural products are presented. Many of the metabolites also exhibit cytotoxic effect against murine leukemia P388 cells.