Antibiofilm activity of carotenoid crocetin against Staphylococcal strains.

Staphylococcus aureus and Staphylococcus epidermidis stand as notorious threats to human beings owing to the myriad of infections they cause. The bacteria readily form biofilms that help in withstanding the effects of antibiotics and the immune system. Intending to combat the biofilm formation and reduce the virulence of the pathogens, we investigated the effects of carotenoids, crocetin, and crocin, on four Staphylococcal strains. Crocetin was found to be the most effective as it diminished the biofilm formation of S. aureus ATCC 6538 significantly at 50 µg/mL without exhibiting bactericidal effect (MIC >800 µg/mL) and also inhibited the formation of biofilm by MSSA 25923 and S. epidermidis at a concentration as low as 2 µg/mL, and that by methicillin-resistant S. aureus MW2 at 100 µg/mL. It displayed minimal to no antibiofilm efficacy on the Gram-negative strains Escherichia coli O157:H7 and Pseudomonas aeruginosa as well as a fungal strain of Candida albicans. It could also curb the formation of fibrils, which partly contributes to the biofilm formation in S. epidermidis. Additionally, the ADME analysis of crocetin proclaims how relatively non-toxic the chemical is. Also, crocetin displayed synergistic antibiofilm characteristics in combination with tobramycin. The presence of a polyene chain with carboxylic acid groups at its ends is hypothesized to contribute to the strong antibiofilm characteristics of crocetin. These findings suggest that using apocarotenoids, particularly crocetin might help curb the biofilm formation by S. aureus and S. epidermidis.

Spectral and mass characterization of kinetic conversion from retinoids to retinoic acid in an in vitro 3-D human skin equivalent model.

To investigate the effect of retinoids, such as retinol (ROL), retinal (RAL), and retinyl palmitate (RP), on epidermal integrity, skin deposition, and bioconversion to retinoic acid (RA). 3-D human skin equivalent model (EpiDermFT™) was used. Epidermal cellular integrity measured by TEER values was significantly higher for a topical treatment of ROL and RAL than RP (p < 0.05). The skin deposition (µM) of ROL and RAL was approximately 269.54 ± 73.94 and 211.35 ± 20.96, respectively, greater than that of RP (63.70 ± 37.97) over 2 h incubation. Spectral changes were revealed that the CO maximum absorbance occurred between 1600∼1800 cm-1 and was greater from ROL than that from RAL and RP, indicating conjugation of R-OH to R-CHO or R-COOH could strongly occur after ROL treatment. Subsequently, a metabolite from the bioconversion of ROL and RAL was identified as RA, which has a product ion of m/z 283.06, by using liquid a chromatography-mass spectrometry (LC-MS) - total ion chromatogram (TIC). The amount of bioconversion from ROL and RAL to RA in artificial skin was 0.68 ± 0.13 and 0.70 ± 0.10 µM at 2 h and 0.60 ± 0.04 and 0.57 ± 0.06 µM at 24 h, respectively. RA was not detected in the skin and the receiver compartment after RP treatment. ROL could be a useful dermatological ingredient to maintain epidermal integrity more effectively, more stably deposit on the skin, and more steadily metabolize to RA than other retinoids such as RAL and RP.

pH-Responsive Calcium Ions and Crocetin Releasing Hydrogel for Accelerating Skin Wound Healing.

The ideal and highly anticipated dressing for skin wounds should provide a moist environment, possess antibacterial properties, and ensure sustained drug release. In the present work, a hyaluronic acid-based hydrogel was formed by cross-linking crocetin and CaCO3@polyelectrolyte materials (CaCO3@PEM) microspheres with HA hydrogels via hydrogen bond and amido bonding (CaCO3@PEM@Cro@HA hydrogel, CPC@HA hydrogel). Moreover, the CPC@HA hydrogel had the capability of sustained, controlled release of calcium ions and crocetin via pH-sensitive and accelerated skin wound healing. The experiment results showed that the CPC@HA hydrogel exhibited porous network structures, stable physical properties, and had antibacterial properties and biocompatibility in vitro. In addition, the CPC@HA hydrogel covering on the skin wound could reduce inflammation and promote wound healing. The high expression of angiogenic cytokines (CD31) and epidermal terminal differentiation markers (Loricrin) of wound healing tissue suggested the CPC@HA hydrogel also had the function of promoting the remodeling of regenerated skin. Overall, CPC@HA hydrogel has promising potential for clinical applications in accelerating skin wound repair.

Biogenic crocetin-crosslinked chitosan nanoparticles with high stability and drug loading for efficient radioprotection.

The risk of radiation exposure increases with the development of nuclear energy and technology, and radiation protection receives more and more attention from public health and safety. However, the numerous adverse effects and low drug utilization limit the practical applications of radioprotective agents. In this study, we developed a biogenic crocetin-crosslinked chitosan nanoparticle with high stability and drug loading for efficient radioprotection. In detail, the nanoparticles were prepared using the natural antioxidant crocetin as a cross-linking reagent in amidation reactions of chitosan and mPEG-COOH. The nanoparticles exhibit a quick scavenging ability for common reactive oxygen species and reactive nitrogen in vitro. Meanwhile, cellular experiments demonstrate the good biocompatibility of the nanoparticles and the alleviation of radiation damage by scavenging reactive oxygen species, reducing apoptosis, and inhibiting DNA damage, etc. Importantly, the nanoparticles are effective in mitigating oxidative damage in major organs and maintaining peripheral blood cell content. In addition, they perform better radioprotective properties than free drug due to the significant extension of the blood half-life of crocetin in vivo from 10 min to 5 h. This work proposes a drug-crosslinking strategy for the design of a highly efficient radioprotective agent, which exhibits a promising prospect in the fields of nuclear emergency and public health.

Crocetin protects mouse brain from apoptosis in traumatic brain injury model through activation of autophagy.

BACKGROUND: Autophagy is recognized as a promising therapeutic target for traumatic brain injury (TBI). Crocetin is an aglycone of crocin naturally occurring in saffron and has been found to alleviate brain injury diseases. However, whether crocetin affects autophagy after TBI remains unknown. Therefore, we explore crocetin roles in autophagy after TBI. METHODS: We used a weight-dropped model to induce TBI in C57BL/6J mice. Neurological severity scoring (NSS) and grip tests were used to evaluate the neurological level of injury. Brain edema, neuronal apoptosis, neuroinflammation and autophagy were detected by measurements of brain water content, TUNEL staining, ELISA kits and western blotting. RESULTS: Crocetin ameliorated neurological dysfunctions and brain edema after TBI. Crocetin reduced neuronal apoptosis and neuroinflammation and enhanced autophagy after TBI. CONCLUSION: Crocetin alleviates TBI by inhibiting neuronal apoptosis and neuroinflammation and activating autophagy.

Autophagy regulation and redox perturbation by transcrocetin suppress the growth of endometriosis.

Until non-hormonal therapeutic targets for endometriosis are suggested, we focused on mitochondrial function and autophagy regulation in the disease. Transcrocetin is a carotenoid and retinoic acid with high antioxidant potency and antiproliferative effects in several diseases. In this study, we demonstrated the therapeutic mechanisms of transcrocetin in endometriosis using the End1/E6E7 and VK2/E6E7 cell lines. Transcrocetin suppressed the viability and proliferation of these cell lines and did not affect the proliferation of normal uterine stromal cells. p21 Waf1/Cip1 as a cell cycle regulator and target of p53, were increased by transcrocetin and caused the G1 arrest via inhibition of cyclin-dependent kinase activity, which might further cause cell death. Furthermore, we confirmed endoplasmic reticulum stress and calcium ion dysregulation in the cytosol and mitochondrial matrix, disrupting the mitochondrial membrane potential. Mitochondrial bioenergetics were suppressed by transcrocetin, and oxidative phosphorylation-related gene expression was downregulated. Moreover, the proliferation of End1/E6E7 and VK2/E6E7 cells was regulated by transcrocetin-induced oxidative stress. Finally, we verified the impairment of autophagic flux following pre-treatment with chloroquine. Therefore, transcrocetin may be a potent therapeutic alternative for endometriosis.

Crocetin inhibits mast cell-dependent immediate-type allergic reactions through Ca2+/PLC/IP3 and TNF pathway.

Crocetin is a kind of glycocone naturally occurring in Crocus sativus L.. It is an active metabolite produced by biohydrolysis of Crocus sativus L.. Crocetin has anti-cardiovascular diseases and antioxidant effects, but its anti-allergic effect has not been reported. In this study, the inhibitory effect of crocetin on immunoglobulin E (IgE) - mediated allergic reaction and the mechanism of action were investigated. The passive cutaneous anaphylaxis (PCA) was used to elucidate the anti-allergic effects of crocetin in vivo. Degranulation assay, calcium imaging, and cytokine release assay were to evaluate the anti-allergic effect of crocetin in vitro. We found that crocetin IgE-mediated RBL-2H3 cell degranulation and allergy both in vitro and in vivo. The TNF pathway was inhibited by crocetin in our RNA-seq sequences, Furthermore, crocetin inhibits IgE-mediated calcium influx, and PLC / IP3 phosphorylation in RBL-2H3 cells. Our findings suggested that crocetin revealed prominent anti-allergy activity through TNF and Ca2+/PLC/IP3 pathway.

Exploring the therapeutic efficacy of crocetin in oncology: an evidence-based review.

With cancer being a leading cause of death globally, there is an urgent need to improve therapeutic strategies and identify effective chemotherapeutics. This study aims to highlight the potential of crocetin, a natural product derived from certain plants, as an anticancer agent. It was  conducted an extensive review of the existing literature to gather and analyze the most recent data on the chemical properties of crocetin and its observed effects in various in vitro and in vivo studies. The study  particularly focused on studies that examined crocetin's impact on cell cycle dynamics, apoptosis, caspases and antioxidant enzyme levels, tumor angiogenesis, inflammation, and overall tumor growth. Crocetin exhibited diverse anti-tumorigenic activities including inhibition of tumor cell proliferation, apoptosis induction, angiogenesis suppression, and potentiation of chemotherapy. Multiple cellular and molecular pathways such as the PI3K/Akt, MAPK and NF-κB were modulated by it. Crocetin demonstrates promising anti-cancer properties and offers potential as an adjunctive or alternative therapy in oncology. More large-scale, rigorously designed clinical trials are needed to establish therapeutic protocols and ascertain the comprehensive benefits and safety profile of crocetin in diverse cancer types.

Involvement of PI3K/AKT Pathway in the Rapid Antidepressant Effects of Crocetin in Mice with Depression-Like Phenotypes.

The current first-line antidepressants have the drawback of slow onset, which greatly affects the treatment of depression. Crocetin, one of the main active ingredients in saffron (Crocus sativus L.), has been demonstrated to have antidepressant activities, but whether it has a rapid antidepressant effect remains unclear. This study aimed to investigate the onset, duration, and mechanisms of the rapid antidepressant activity of crocetin (20, 40 and 80 mg/kg, intraperitoneal injection) in male mice subjected to chronic restraint stress (CRS). The results of behavioral tests showed that crocetin exerted rapid antidepressant-like effect in mice with depression-like phenotypes, including rapid normalization of depressive-like behaviors within 3 h, and the effects could be maintained for 2 days. Hematoxylin-eosin (HE) and Nissl staining showed that crocetin ameliorated hippocampal neuroinflammation and nerve injuries in mice with depression-like phenotypes. The levels of inflammatory factors, corticosterone and pro brain-derived neurotrophic factor in crocetin-administrated mice serum were significantly reduced compared with those in the CRS group, as well as the levels of inflammatory factors in hippocampus. What's more, Western blot analyses showed that, compared to CRS-induced mice, the relative levels of mitogen-activated kinase phosphatase 1 and toll-like receptor 4 were significantly reduced after the administration of crocetin, and the relative expressions of extracellular signal-regulated kinase 1/2 (ERK1/2), cAMP-response element binding protein, phosphorylated phosphoinositide 3 kinase (p-PI3K)/PI3K, phosphorylated protein kinase B (p-AKT)/AKT, phosphorylated glycogen synthase kinase 3ß (p-GSK3ß)/GSK3ß, phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR were markedly upregulated. In conclusion, crocetin exerted rapid antidepressant effects via suppressing the expression of inflammatory cytokines and the apoptosis of neuronal cells through PI3K/AKT signaling pathways. The rapid antidepressant effect of crocetin (40 mg/kg) could be maintained for at least 2 days after single treatment.

Enhancement of dissolution and oral bioavailability by adjusting microenvironment pH in crocetin ternary solid dispersions: Optimization, characterization, in vitro evaluation, and pharmacokinetics.

The most promising active ingredient of Crocus sativus L., crocetin (CCT), has been demonstrated to possess many biological activities. However, only a few studies have been conducted on CCT formulation, especially in oral formulation, mainly due to its insolubility in water, which limits its application for oral administration. This article reports an equilibrium saturation solubility and single-pass intestinal perfusion studies conducted to classify the biopharmaceutics classification system (BCS) of CCT. To enhance in vitro dissolution and in vivo oral bioavailability, ternary solid dispersions of CCT (CCT-SDs) with soluplus (SOL) as hydrophilic carrier and meglumine (MEG) as alkalizer were optimized using response surface methodology (RSM) with central composite design (CCD) experiments. Four different preparation methods were evaluated using the optimal formulation, including solvent evaporation, ball milling, spray drying, and freeze-drying. Prepared formulations were characterized by TG-DSC, FTIR, X-RPD, and SEM; the pharmacokinetic studies were performed in rats after oral administration. The cumulative dissolution rate of CCT-SDs containing SOL and MEG prepared by the ball milling method was 97.1% at 15 min and remained at 95.6% at 480 min, which was significantly higher than that of untreated CCT. The lower crystallinity, smaller particle size, and higher microenvironment pH (pHM) were observed in CCT-SDs prepared by the ball milling method. In vivo absorption of CCT-SDs (Cmax = 52.789 ± 12.441 µg/mL and AUC0-12 = 191.748 ± 35.043 µg/mL·h) was greater than untreated CCT (Cmax = 5.918 ± 1.388 µg/mL and AUC0-12 = 44.309 ± 7.264 µg/mL·h). In conclusion, the current study provides ternary solid dispersion formulation of CCT to increase the in vitro dissolution and in vivo bioavailability, which will benefit the commercial production and future clinical applications of CCT.

[Rats' liver morphological features under conditions of different supply with vitamins and minerals].

The current practice of novel food safety assessment in the Russian Federation involves toxicological studies on the alimentary model of adaptation potential reduction of laboratory animals. Since vitamin and mineral deficiency can affect the size of structural elements of tissues, an objective estimation of the results obtained using this model is possible when determining the range of fluctuations of the studied morphometric parameters under conditions of different essential substances' supply, as well as under conditions of simulated toxic effects on the background of the corresponding supply. The purpose of the research was to investigate the morphological and morphometric features of the liver under the influence of reduced intake of vitamins and mineral elements in the combination with toxic effects of various nature, during growth and puberty of male Wistar rats. Material and methods. The article analyzed data of 4 model experiments on 140 animals that received semi-synthetic casein diet with different supply of vitamins B1, B2, B3, B6 and mineral elements Fe3+ and Mg2+, as well as data of 2 experiments on 180 animals with simulated toxic load of cadmium (Cd2+) salts and carbon tetrachloride. The animals were ~95 days old at the time of sampling, the duration of the experiments was ~65 days. For the analysis we used data on rats' body weight on the day of material sampling, absolute and relative liver weight, hepatocyte diameter, nucleus diameter and hepatocyte cytoplasm size in the central and peripheral zones of hepatic lobules. A total of 200 cells were analyzed in each group of animals. In accordance with the study design, all quantitative traits of the groups that received diets with an essential nutrient supply ranging from 75 to 2% were compared with the group that received a complete diet (100%). Results. Morphometric examination of hepatocytes revealed a linear decrease in the size of cell structural elements in the series of reducing the content of essential micronutrients in the diet. Under the conditions of 2-4% vitamin and mineral supply, cell and nucleus diameters as well as cytoplasm size were by ~16.8, 12.6 and 21.1% (p<0.05) lower respectively than in rats with optimal supply of these substances; under the conditions of 9-19% supply were by ~9.2, 9.7 and 8.7% lower (p<0.05); higher levels of supply caused reduction of hepatocyte, nucleus and cytoplasm sizes in a range not exceeding 5% (p>0.05). When comparing the size of hepatocytes of rats subjected to toxic load with the hepatocytes of rats referred to the reference standard, an increase in the size of hepatocytes under the action of carbon tetrachloride by 17.4% (p<0.05) on average and under the action of cadmium salts by 4.6% (p<0.05) was noted. Conclusion. Based on the analysis of liver morphological and morphometric studies' data, there were established sizes of hepatocytes structural elements in the rats kept on diets with decreasing supply with B group vitamins, iron and magnesium salts; the linear decrease in the sizes of structural elements of hepatocytes in the series of reduction of B group vitamins, iron and magnesium intake was revealed. Toxic exposures to carbon tetrachloride and cadmium salts against the background of a 19-30-75% supply with essential substances led to an increase in the hepatocytes size, the correlation between the degree of toxicant exposure and the supply level is not significant.

Crocetin Protected Human Hepatocyte LO2 Cell From TGF-ß-Induced Oxygen Stress and Apoptosis but Promoted Proliferation and Autophagy via AMPK/m-TOR Pathway.

Objective: To investigate the protective effects of crocetin against transforming growth factor-ß (TGF-ß)-induced injury in LO2 cells. Methods: Human hepatocyte LO2 cells were pre-treated with crocetin (10 µM) for 6, 12, and 24 h, and then induced by TGF-ß. Proliferation, oxidative stress, apoptosis, autophagy, and related proteins were assessed. Results: Crocetin pre-treating promoted proliferation but suppressed apoptosis in TGF-ß-induced LO2 cells. Crocetin protected LO2 cells from TGF-ß-induced inflammation and oxygen stress by reducing reactive oxygen species (ROS) and malondialdehyde (MDA) but enhancing superoxide dismutase (SOD) and glutathione (GSH). Autophagy was suppressed in TGF-ß but crocetin promoted autophagy in LO2 cells by mediating Adenosine 5'-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (m-TOR) signaling pathway via upregulating p-AMPK and p-Beclin-1 but downregulating p-mTOR. Conclusions: Crocetin protected LO2 cells from TGF-ß-induced damage by promoting proliferation and autophagy, and suppressing apoptosis and anti-inflammation via regulation of AMPK/m-TOR signaling pathway.

The mechanism by which crocetin regulates the lncRNA NEAT1/miR-125b-5p/SOX7 molecular axis to inhibit high glucose-induced diabetic retinopathy.

Diabetic retinopathy (DR) is a high-incidence microvascular complication with retinal neovascularization that generates irreversible visual impairment. However, the mechanism of DR is unclear and needs to be further explored. To explore the the effects of crocetin on expression of NEAT1 and miR-125b-5p and the proliferation activity, migration ability, and angiogenesis ability of human retinal microvascular endothelial cells (hRMECs), RT-qPCR, CCK-8, Transwell, and tube formation assays were performed. Additionally, Western blot was used to detect the expression of SOX7, VEGFA and CD31. Furthermore, a dual-luciferase reporter gene was used to verify the targeting connection. The DR mouse model was constructed by STZ. The effect of crocetin on DR angiogenesis was detected by hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), retinal digest preparations and Western blot. The results showed that crocetin inhibited the high-glucose (Hg)-induced upregulation of NEAT1 and SOX7 and the downregulation of miR-125b-5p. Crocetin inhibited Hg-induced proliferation, migration and angiogenesis by upregulating the targeted inhibition of SOX7 by miR-125b-5p through the inhibition of NEAT1. To summarize, our study revealed that crocetin has a protective effect on Hg-induced DR by regulating the lncRNA NEAT1/miR-125b-5p/SOX7 molecular axis.

The ameliorating effects of crocetin on frozen-thawed quality, and fertility via attenuating oxidative status of bubaline spermatozoa.

Current study was conducted to appraise the cryoprotective influence of crocetin on quality, oxidative status, and fertility potential of bubaline spermatozoa. Collected semen from four bulls was diluted in five aliquots with (10 µM, 5 µM, 2 µM, 1 µM, and control [0 µM] supplementation of crocetin). After gentle dilution (37 °C), cooling (4 °C, in 2 h), equilibration (4 °C, for 4 h) and packaging of samples was done in straws (polyvinyl French, 0.5 ml), and then frozen (programmable cell freezer). This study established that crocetin supplementation significantly (p < 0.05) improves CASA (Computer Assisted Sperm motion Analyzer) total motility (%), rapid velocity (%), average-path, and curved-line velocities (µm/sec, 10 µM vs. control), and progressive motility (%), straight-line velocity (µm/sec), total antioxidant capacity (TAC, µMol/l), ATP concentrations (nmol/million), and fertility potential (%) (10 µM vs. control, and 1 µM), and mitochondrial potential (%) of buffalo spermatozoa (5, and 10 µM vs. control). Crocetin supplementation significantly (p < 0.05) alleviates DNA fragmentation, seminal plasma ROS (104 RLU/20/25 million, RLU = Relative light unit) levels, and lipid peroxidation (LPO, µMol/ml) in buffalo spermatozoa (10 µM vs. control). In a nutshell, crocetin supplementation improves post-thaw quality by means of motility parameters, motion kinematics, TAC, and ATP concentrations, and fertility potential, and abolished DNA fragmentation parameters, seminal plasma ROS, and LPO concentrations of buffalo spermatozoa. The exact mechanism by which crocetin acts are not fully elucidated; however, it is probable to speculate that the reduction in ROS, and LPO recorded in this study may be related to scavenging ability of this antioxidant during cryopreservation.

Inhibitory effect of saffron, crocin, crocetin, and safranal against adipocyte differentiation in human adipose-derived stem cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron (Crocus sativus L.) has been introduced as a potential promising natural antioxidant with anti-obesity properties. In Persian Medicine, saffron has been used to control appetite and obesity. AIM OF THE STUDY: The present study aims to investigate the effect of saffron and its bioactive compounds on adipocyte differentiation in human adipose-derived stem cells (ADSCs). MATERIALS AND METHODS: Flow-Cytometric analysis was performed to quantify the cell surface markers. The extracts cytotoxicity on hASCs was measured using alamarBlue® assay whereas their activities against adipocyte differentiation were studied using Oil Red O staining. The level of Peroxisome proliferator-activated receptor-γ (PPARγ), Fatty Acid Synthetase (FAS), and Glyceraldehyde-3-phosphate dehydrogenase (GAPHD) which are key proteins in cell differentiation was investigated by western blot analysis. RESULTS: Flow-cytometry revealed the mesenchymal stem cells markers, CD44 and CD90, on ADSCs surface. The saffron, crocin, and crocetin significantly inhibited adipocyte differentiation while saffron up to 20 µg/mL and crocin, crocetin and safranal up to 20 µM did not exhibit cytotoxicity. The western blotting analysis revealed a remarkable reduction in the level of PPARγ, GAPDH, and FAS proteins by 10 and 20 µM of crocin and 2.5 and 5 µM of crocetin. CONCLUSION: It seems that saffron, crocin, and crocetin could efficiently inhibit the differentiation of hASCs with benefits for the treatment and prevention of obesity.

A strategy for attenuation of acute radiation-induced lung injury using crocetin from gardenia fruit.

PURPOSE: Radiation-induced lung injury limits the implementation of radiotherapy plans and severely impairs the quality of life. Crocetin has the capability to protect against radiation. This study is aimed at estimate the preventive effect and mechanism of crocetin on acute radiation induced lung injury. METHODS AND MATERIALS: In this study, we offer a strategy for radiation-induced lung injury by using crocetin, an extract of gardenia fruit. Histopathology, transcriptomics, flow cytometry, and other methods have served to examine the effect and mechanism of crocetin on acute radiation-induced lung injury. RESULTS: Crocetin effectively alleviates radiation-induced alveolar wall thickening and alveolar destruction. The number of normal alveoli and lung structure of mice is well protected by the prevention of crocetin. It is found that crocetin inhibits necroptosis to achieve effective radioprotection by down regulating the Tnfrsf10b gene in vitro. CONCLUSION: Crocetin inhibits necroptosis through transcriptional regulation of the Tnfrsf10b gene, thereby preventing radiation-induced lung injury. This work may provide a new strategy for the prevention of lung radiation injury by the extract from Chinese herbal medicine.

Crocetin Prolongs Recovery Period of DSS-Induced Colitis via Altering Intestinal Microbiome and Increasing Intestinal Permeability.

Crocetin is one of the major active constituents of saffron (Crocus sativus L.) which has a reputation for facilitating blood circulation and dispersing blood stasis in traditional Chinese medicine. However, there is little evidence showing the relationship between crocetin intake and the risk of gastrointestinal diseases such as colitis. In order to investigate the effect of crocetin on the regulation of intestinal barrier function and intestinal microbiota composition, mice were treated with crocetin after 3% dextran sulfate sodium (DSS) administration for one week. We found that crocetin intake at 10 mg/kg aggravated colitis in mice, showing increased weight loss and more serious histological abnormalities compared with the DSS group. The 16s rDNA sequencing analysis of the feces samples showed that mice treated with 10 mg/kg crocetin had lower species diversity and richness than those treated with DSS. At the genus level, a higher abundance of Akkermansia and Mediterraneibacter, and a lower abundance of Muribaculaceae, Dubosiella, Paramuribaculum, Parasutterella, Allobaculum, Duncaniella, Candidatus Stoquefichus, and Coriobacteriaceae UCG-002 were observed in the crocetin group. Untargeted metabolomic analyses revealed that crocetin reduced the levels of primary and secondary bile acids such as 12-ketodeoxycholic acid, 7-ketodeoxycholic acid, 3-sulfodeoxycholic acid, 6-ethylchenodeoxycholic acid, chenodeoxycholate, glycochenodeoxycholate-7-sulfate, glycocholate, and sulfolithocholic acid in the colon. In conclusion, crocetin intake disturbed intestinal homeostasis and prolonged recovery of colitis by promoting inflammation and altering gut microbiota composition and its metabolic products in mice. Our findings suggest that patients with gastrointestinal diseases such as inflammatory bowel disease should use crocetin with caution.

Crocetin alleviates the caerulein-induced apoptosis and inflammation in AR42J cells by activating SIRT1 via NF-κB.

The anti-inflammatory and anti-apoptotic properties of crocetin have been widely demonstrated in numerous diseases. However, the exact role and mechanism of crocetin in acute pancreatitis have not been elucidated. Thus, this paper aims at exploring whether crocetin could be used to alleviate acute pancreatitis and further demonstrating the underlying mechanisms. Cell viability of caerulein-induced pancreatic exocrine cell line AR42J treated with crocetin was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). Apoptosis and inflammation of these treated cells were detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), western blot and enzyme linked immunosorbent assay (ELISA). The expression of sirtuin-1 (SIRT1) was quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. After knockdown of SIRT1, cell viability, apoptosis and inflammation were measured again by corresponding kits. Finally, the NF-κB nuclear translocation and proteins in the NF-κB signaling were examined. Crocetin remarkably suppressed the apoptosis and inflammation of caerulein-induced AR42J cells. The decreased expression of SIRT1 was increased in caerulein-induced AR42J cells after exposure to crocetin. After knockdown of SIRT1, the alleviative effects of crocetin were found to be canceled in these cells. Furthermore, SIRT1 knockdown promoted the NF-κB signal transduction. On the whole, we presented the first evidence for the importance of SIRT1-NF-κB axis in acute pancreatitis and proposed that crocetin alleviates the caerulein-induced apoptosis and inflammation in AR42J cells by activating SIRT1 via NF-κB.

An easy way for the hydrolysis, pre-concentration, and chemical stabilization of crocetin from saffron powder.

To date there are no methods in the literature leading to crocetin selective concentration from saffron powder aqueous solutions. To this aim, we decided to test the performance of its heterogeneous extraction by means of a panel of 21 synthetic clays, 4 of which demonstrated to selectively retain crocetin in the solid phase after hydrolysis of its digentiobyosil ester (crocin) (and its isomers) and to its chemical stabilization (e.g., oxidation) over time. The best adsorption yield was obtained with zinc hydroxy chloride (66.18 ± 0.06 µg/g dry powder). This phenomenon was assessed by HPLC-DAD analyses after desorption of crocetin from the respective support and assessing its degradation along a period of 30 days. The method we established could represent a good mean to provide pure crocetin from saffron powder, preserving in the meantime its chemical properties for a concrete future exploitation for food pharmaceutical, and cosmetic purposes.