Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in many food commodities and known to cause reproductive disorders. Several studies have shown that ZEN is hematotoxic and hepatotoxic and causes several alterations of immunological parameters. In the present study, we aimed to evaluate the protective effect of Crocin (CRO), a natural carotenoid, against ZEN-induced toxicity in both renal and hepatic tissues of Balb/c mice. We demonstrated that ZEN (40 mg/kg body weight (b.w.)) induced oxidative stress in both kidney and liver as monitored by measuring the malondialdehyde (MDA) level, the protein carbonyl generation, the catalase and superoxide dismutase activity, and the expression of the heat shock proteins (Hsp70). However, combined treatment of ZEN with different doses of CRO (50, 100, and 250 mg/kg b.w.) significantly reduced ZEN-induced alterations in all tested oxidative stress markers. It could be concluded that CRO was effective in the protection against ZEN-induced toxicity in the liver and kidney of Balb/c mice.

Zearalenone (ZEN) is a mycotoxin from Fusarium species commonly found in food commodities and is known to cause reproductive disorders. Several in vivo studies have shown that ZEN is haematotoxic and hepatotoxic and causes several alterations of immunological parameters. Meantime, the available information on the cardiotoxic effects of ZEN is very much limited. In the present study, we investigated the toxic effects of ZEN in heart tissues of Balb/c mice. We demonstrated that ZEN (40 mg kg(-1) body weight (b.w.)) increased creatine phosphokinase, lactate dehydrogenase, aspartate transaminase, alanine transaminase, total cholesterol and triglyceride levels and induced oxidative stress as monitored by measuring the malondialdehyde level, the generation of protein carbonyls, the catalase and superoxide dismutase activity and the expression of the heat shock proteins (Hsp 70). We also demonstrated that acute administration of ZEN triggers apoptosis in cardiac tissue. Furthermore, we aimed to evaluate the safety and efficacy of crocin (CRO), a natural carotenoid, to prevent ZEN-induced cardiotoxicity in mice. In fact, combined treatment of ZEN with different doses of CRO (50, 100, and 250 mg kg(-1) b.w.) showed a significant reduction of ZEN-induced toxicity for all tested markers in a dose-dependent manner. It could be concluded that CRO was effective in the protection against ZEN-induced toxicity in cardiac tissue.

Crocin and Quercetin protect HCT116 and HEK293 cells from Zearalenone-induced apoptosis by reducing endoplasmic reticulum stress.

Patulin (PAT) is a mycotoxin produced by several species of the genera ofPenicillium, Aspergillus, and Byssochlamys principally by Penicillium expansum. This mycotoxin is suspected to affect several organs including kidney and liver. However, its toxic effect on heart remains unknown. The present study investigated for the first time the cardiotoxic effect of PAT in mice. We demonstrated that PAT increased creatinin phosphokinase (CPK) level, induced lipoperoxydation and protein oxidation, and triggered the antioxidant enzymes such as superoxide dismutase and catalase activities. We also demonstrated that acute administration of PAT triggers apoptosis via P53 overexpression and caspase 3 activation. We further investigated the antioxidant efficiency of crocin (CRO), a carotenoid pigment, against PAT-induced cardiotoxicity. We found that pretreatment with CRO prevents cardiac impairment by reducing CPK levels, restoring the redox statute and suppressing apoptosis. Collectively, our data provide new preventive effect of CRO toward PAT-induced cardiotoxicity in mice.

Objective To investigate the protective effect of crocin on oxidative stress cell model of PC12 cell and the effect of crocin on PI3K/Akt signal pathway, as well as further explore the mechanism of protective effect on model cells. Methods Cells were divided into control group, model group, crocin group and VE group. The cell survival rate was detected by MTT method, and the expression of mRNA and protein of PI3K/Akt were detected by RT-PCR and Western blot. Results With the crocin concentration in 0.625 μM and 5 μM, the cell survival rate increased in a dose-dependent manner. The average optical density rate of PI3K and Akt mRNA were 0.435±0.044 and 0.375±0.034, and the PI3K and Akt protein were 0.378±0.038 and 0.386±0.043 of crocin group.Compared with the model group, the expression levels of PI3K/Akt increased in crocin group(P<0.05). Conclusion These results indicate that the antioxidant and antiapoptosis effects of crocin are induced via increasing expression of PI3K and pAkt. Key words: Crocin; Oxidative stress; Apoptosis; PI3K/AKT

The present study involves evaluation of antioxidant potential of Crocus sativus and its main constituents, safranal (SFN) and crocin (CRO), in bronchial epithelial cells, followed antiinflammatory potential of the active constituent safranal, in a murine model of asthma. To investigate the antioxidizing potential of Crocus sativus and its main constituents in bronchial epithelial cells, the stress was induced in these cells by a combination of different cytokines that resulted in an increase in nitric oxide production (NO), induced nitric oxide synthase (iNOS) levels, peroxynitrite ion generation, and cytochrome c release. Treatment with saffron and its constituents safranal and crocin resulted in a decrease of NO, iNOS levels, peroxynitrite ion generation, and prevented cytochrome c release. However, safranal significantly reduced oxidative stress in bronchial epithelial cells via iNOS reduction besides preventing apoptosis in these cells. In the murine model of asthma study, antiinflammatory role of safranal was characterized by increased airway hyper-responsiveness, airway cellular infiltration, and epithelial cell injury. Safranal pretreatment to these allergically inflamed mice lead to a significant decrease in airway hyper-responsiveness and airway cellular infiltration to the lungs. It also reduced iNOS production, bronchial epithelial cell apoptosis, and Th2 type cytokine production in the lungs.

Safranal, a Crocus sativus L constituent suppresses the growth of K-562 cells of chronic myelogenous leukemia. In silico and in vitro study

Crocus sativus L. extracts (saffron) are rich in carotenoids. Preclinical studies have shown that dietary intake of carotenoids has antitumor effects suggesting their potential preventive and/or therapeutic roles. We have recently reported that saffron (SE) and crocin (CR) exhibit anticancer activity by promoting cell cycle arrest in prostate cancer (PCa) cells. It has also been demonstrated that crocetin esters are produced after SE gastrointestinal digestion by CR hydrolysis. The aim of the present report was to investigate if SE, crocetin (CCT), and CR affected in vivo tumor growth of two aggressive PCa cell lines (PC3 and 22rv1) which were xenografted in male nude mice treated by oral gavage with SE, CR, and CCT. We demonstrated that the antitumor effects of CCT were higher when compared to CR and SE and treatments reverted the epithelial-mesenchymal transdifferentiation (EMT) as attested by the significant reduction of N-cadherin and beta-catenin expression and the increased expression of E-cadherin. Additionally, SE, CR, and CCT inhibited PCa cell invasion and migration through the downmodulation of metalloproteinase and urokinase expression/activity suggesting that these agents may affect metastatic processes. Our findings suggest that CR and CCT may be dietary phytochemicals with potential antitumor effects in biologically aggressive PCa cells.

Background: The aim of this study was to investigate the protective effects of crocin against tissue damages and leukocyte infiltration induced by renal ischemia/reperfusion. Methods: In this experimental study, 35 male Wister rats weighing 250-300 g were randomly divided into 5 groups; 1. Operated controls, but no vessel occlusion was considered and passed during equivalent reperfusion period for Sham group. The second group received solvent of crocin and went through surgery, bilateral renal ischemia for 30 min and 24-h reperfusion period (I/R). The rest (group 3-5) underwent Ischemia/reperfusion and received a dose of crocin with 100, 200 or 400 mg/kg,ip respectively. At the end of reperfusion period, the right kidney tissue were collected and after staining with haematoxylin-eosin, subjected to microscopic study. Results: Following ischemia/reperfusion, the Bowman's space size, cell necrosis in the tubules of the cortex and outer medulla, vascular congestion and tubular casts in the outer and inner medulla increased. However, RBCs number in glomerular capillaries decreased and all these injuries were improved considerably via taking crocin using every three doses. In addition, ischemia/reperfusion leads to partially severe infiltration of lymphocytes into interstitial space (P<0.001), and crocin could decrease their infiltration significantly. Conclusion: Intraperitoneal administration of Crocin has protective effects against 30 min ischemia and 24-h reperfusion induced tissue damages as well as leukocyte infiltration in kidneys.

The phenolic content and antioxidant activities of extracts from Quercus durifolia, Quercus resinosa, Quercus eduardii and Quercus sideroxyla leaves were studied. Extracts were obtained by successive extraction with aqueous acetone (70%) followed by methanol (50%). Antioxidant activities were determined using a single electron transfer (SET) and hydrogen atom transfer (HAT) based assays. For SET-based the assays performed were, 2,2-diphenyl-1-picryl hydrazine (DPPH) inhibition expressed as IC50, redox potential, as mV, inhibition of the low density lipoprotein (LDL) oxidation, as % of inhibition. For HAT-based assay, crocin bleaching technique was applied; a kinetics approach was used for the evaluation of total antioxidant capacity and the results presented in terms of equivalence by weight of a reference antioxidant. Acetone extracts (PFQA) from leaves of all Quercus species showed the best antioxidant capacity. Q. resinosa (PFQr) exhibited the best antioxidant capacity among the Quercus species analyzed either in acetone or methanol extracts (PFQM). Distinctive results are: DPPH IC50, PFQrA = 78.3 µg/g, PFQrM = 250.7 µg/g; In Redox potential, PFQrA = 147.0 mV, PFQrM = 201.6 mV; In LDL oxidation inhibition: 98.2% and in inhibition of crocin bleaching, PFQrA = 1.08 g, PFQrM = 0.98 g. In conclusion, Quercusleaves might be used as potential source of polyphenolic antioxidants. Key words: Quercus sp., SET-based assay, HAT-based assay, phenolic compounds, antioxidant activity.

Resveratrol (RES) and crocin (CRO) have shown some of the most promising anti-neurodegenerative activities. This study for the first time showed a synergistic effect between RES and CRO on SHSY-5Y neuroblastoma cells against oxidative damage (a model for Alzheimer's disease). After overnight growth of human SHSY-5Y cells, either RES, or CRO or their combination was added to the cell culture medium at increasing concentrations to assess toxicity. Cells were incubated for further 24 hours, and cell viability was measured by a colorimetric assay. To perform oxidative stress, 6-hydroxydopamine (6-OHDA, 30µM) or hydrogen peroxide (H2O2, 50µM) was added to the overnight-grown cell cultures after the addition of the protective compounds RES, or CRO or their combination. The cells were incubated for further 24 hours and cell viability was measured as above. Each experimental point was assessed in quadruplicate and each experiment was repeated at least twice. RES and CRO at concentrations that were not toxic to SHSY-5Y cells (RES; 0.5, 1 and 2µM, and CRO; 10, 20 and 30µM) displayed significant neuroprotection against 6-OHDA and H2O2. A positive synergistic effect between resveratrol (1µM) and crocin (10µM) against oxidative stress was observed that was more pronounced against 6-OHDA, in which the protection was near 100%. Experimental data on the synergistic effect and safety will be presented.

Objective To observe the effect of Crocin on structure and the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in rat retina after injury by ischemia-reperfusion.Methods A total of 80 Sprague-Dawley male rats at the age of 8-10 weeks were divided into control group,model group,low-dose Crocin group and high-dose Crocin group,with 20 rats in each group.The rats of control group were not treated.The rats in model,low-dose Crocin and high-dose Crocin group were induced with normal saline by anterior chamber perfusion creating a retinal ischemia-reperfusion (RIR) model.The rats of the low-dose Crocin and high-dose Crocin group received intraperitoneal injection with different doses of Crocin solution (5 mg/kg,or 50 mg/kg) 30 minutes prior to ischemic injury and one time per day after successful RIR.Optical microscopy was used to observe the retinal structure.Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression of TNF-α and IL-1β 6,12,24 and 48 hours after RIR.Results The retinal structure of control group was normal.Pathological changes were found in the RIR model and low-dose Crocin group,such as retinal edema,disorganized structure and loosely packed cells.The degree of pathological changes in low dose Crocin group was less than the RIR model group.The retinal structure of high-dose Crocin group was similar to the control group.The expression of TNF-α was the highest at 24 hours after modeling,while the expression of IL-1β was the highest at 12 and 48 hours after RIR modeling.Six,12,24 and 48 hours after RIR modeling,compared with the control group,the TNF-α expression of model (t=5.42,7.94,9.32,9.18; P＜0.05),low-dose Crocin (t=3.94,4.12,4.98,3.84; P＜0.05) and high-dose Crocin group (t=2.13,2.34,2.96,2.78;P＞0.05) were increased.Compared with the RIR model group,the TNF-α expression of low-dose Crocin (t=3.95,4.56,4.01,5.12) and high-dose Crocin group (t =5.23,7.65,7.74,7.63) was decreased.Compared with the control group,the IL-1β expression of model (t=7.23,7.87,7.15,15.60),low-dose Crocin (t 5.65,5.10,5.54,6.87;P＜0.05) and high-dose Crocin group (t=4.38,5.21,4.56,4.75) was increased (P＜0.05).Compared with the model group,the IL-1β expression of low-dose Crocin group was decreased significantly 48 hours after RIR modeling (t=7.56,P＜0.05) ; but it decreased significantly at each time point in high-dose Crocin group (t=6.94,5.36,6.05,10.50; P＜0.05).Conclusion Crocin can improve the retinal pathologic changes,while down regulating TNF-α and IL-1β expression in RIR rats. Key words: Reperfusion injury/drug therapy; Colchicum/drug effects; Tumor necrosis factor alpha; Interleukin-1beta; Animal experimentation

Recently, attention has been paid to the identification of natural antioxidants from the petals of Crocus S. flowers that are normally considered waste[1]. The antioxidant activities are mainly attributed to carotenoid compounds, like crocin. Scientific evidences demonstrate that this kind of compounds are among the most important natural plant sources of antioxidant activity in the human diet, protecting the body against damages caused by reactive oxygen species (ROS). This antioxidant was extracted using methanol and ethanol. In this experiment, it was used a new hydrogel consisting of three polymers - Polyvinyl pyrrolidone (PVP), Agar and Polyethylene glycol (PEG) – and Crocin, mixed, reticulated and then sterilized by gamma irradiation at 25 kGy. For the in vitro experimental protocol, it was used a primary culture of fibroblasts taken from the subcutaneous tissue of a newborn mice, seeding the cells on a little square (1cm2 area) of both kinds of hydrogel. As the ISO protocol prescribes, the experiments were repeated 3 times for each kind of hydrogel, stopping the culture at the 3rd, 7th and 14th day after the seeding. For all steps, 3 Petri dishes were used as controls without biomaterials. At fixed deadline, all Petri dishes were stained, using the Wright method for cell counting and morphological evaluations. The microscopic analysis revealed the complete biocompatibility of the hydrogel. The Petri dishes with this kind of new hydrogel has an overproduction of collagen from the fibroblasts. The presence of this natural important compound in saffron petals, now offers new possibilities for the best use of the hole flower. This study is still in progress.

Crocin is a chemical extraction from saffron and it is the most important kind of pigment of saffron.Recently,the research on cervical cancer,bladder cancer,leukemia,tongue squamous cell carcinoma,colon cancer,pancreatic cancer shows that crocin has a role in inducing apoptosis of the tumor cells by activating caspase-3,regulating the ratio of bcl-2/bax and repressing the expression of survivin.Crocin has a strong inhibitory effect on tumor proliferation by inducing cell cycle to arrest through down-regulating the expression of cyclin D1 and up-regulating the expression of p27kip1.Crocin can also exert antitumor effects by liposome encapsulation to enhance cytotoxic effect.Crocin is a kind of effective chemical for tumor treatment and prevention with no significant toxico-and by-effect in vivo,and is likely to be used as an anticancer drug clinically in the future. Key words: Neoplasms; Cell cycle; Apoptosis

C0135 In vitro and in silico study of the effect of crocin and safranal on human plasma coagulation

Synergistic cytoprotective effect of Crocin (CRN) and Resveratrol (RSV) on Primary Human Retinal Pigmentepithelium (RPE) cells

A simple, feasible and effective method of ultra performance liquid chromatography (UPLC) coupled with photo diode array (PDA) were established for fingerprint analysis and simultaneous quantification of three major classes of constitutions including iridoid glycosides, crocins and organic acids of fructus Gardeniae. Extraction method was optimized as 75% methanol ultrasonic extraction for 30 min. Acetonitrile-water (containing 0.2% formic acid) gradient elution on Waters Acquity BEH C18 column (50 × 2.1 mm, 1.7 μm) was used to obtain good chromatographic resolution. 24 characteristic peaks were comprised in the fingerprint common pattern. Among them, seven marked components,geniposide, shanzhiside methyl ester, geinpin, geniposidic acid, crocin I, crocin II, and chlorogenic acid, were quantified. Similarity evaluation, principal component analysis andhierachical cluster analysis were applied to demonstrate the distinction. It was concluded that chemical components of Gardenia jasminoides Eills and Gardenia jasminoides var. radicans Makino from different origins were similar. Other than determination of the contentof geniposide and crocin I, comparison of fingerprint atlas could be considered as a suitable quality control method for fructus Gardeniae. Key words: Fructus Gardeniae, chromatographic fingerprint, multi-component quantification, principal component analysis, hierachical cluster analysis.

The reputation of growing saffron is about 2500 years. Apparently saffron originally comes from Greece and Mediterranean region. There are some researchers who believe that origin of this plant comes from Iran. Iran is considered to be one of the greatest producers of saffron and nearly 90% of production of saffron is produced in Iran. There are varieties of chemical components present in the stigma of the saffron plant. These chemical components include carbohydrates, minerals, mucilage, vitamins such as riboflavin and thiamine, color pigments such as crocin, anthocyanin, carotene, lycopene, Zeaxanthin and aromatic terpenic essence called “safranal” and flavoring substances such as picrocrocin. Crocin (C44 H64 O24) is the most influential chemical in the coloring of saffron. Other than crocin, saffron is also made up of free aglycone crocin and a small number of anthocyanin pigments. The oil soluble color pigments include lycopene, alpha carotene, beta carotene and Zeaxanthin. Today, based on growing and effective applications of saffron in medical fields and in alternative medicine, it has attracted the attention of many researchers. Saffron may substitute chemical medicines. Some medical properties of saffron are as follows: helps digestion, strengthens the stomach and is anti-tympanites, activates the sexual desire, is analgesic, especially for colicky pains gingivitis, fights tumors and collection of free radicals (thus reacting against cancerous cells), is euphoriant and alleviates neuralgia, is a tranquilizer, cures insomnia, strengthens memory power, improve concentration, reacts against spasm, fights depression, the Alzheimer’s and Parkinson’s diseases, controls blood pressure disorders, lowers high cholesterol levels, cures iron deficiency (anemia) in girls, reduces chances of such heart diseases as arteriosclerosis, and helps improve heart conditions (due to the presence of thiamin, riboflavin and mineral components), cures respiratory disorders such as asthma, cough, influenza and cold, helps blood circulation in the retina, cures macula lutea and retinopathy ischemic caused by old age. Cures rheumatism and bruises when used externally, cures amebic dysentery, measles, and inflammation of the liver, splenomegaly and urogenital infections. The application of saffron in cancer-treatment experiments performed on laboratory animals has proved successful. Key words: Saffron, crocin, picrocrocin, carotene, iycopene, antitympanit, analgesic, fights tumor, antidepression.