Crocetin protects against mitochondrial damage induced by UV-A irradiation in corneal epithelial cell line HCE-T cells.

The corneal epithelium is located at the outermost layer of the ocular surface and continuously exposed to environmental factors, such as ultraviolet (UV) radiation from sunlight. UV irradiation causes excessive production of reactive oxygen species (ROS) in cells, which results in oxidative damage to membrane-bound organelles such as mitochondria, eventually leading to cell death. Crocetin, a natural carotenoid found in plants, has various biological properties including antioxidant activity. In this study, we investigated the effects of crocetin on UV-A-induced cell injury in the corneal epithelium. Using an in vitro system with the human corneal epithelial cell-transformed (HCE-T) cell line, pretreatment with 10 µM crocetin suppressed the reduction of cell viability induced by UV-A exposure. Crocetin ameliorated the decrease in oxygen consumption rates and the mitochondrial fragmentation that occurred following UV-A irradiation. Crocetin inhibited both ROS production and the activation of the apoptosis pathway; it also preserved the defects of epithelial cell polarity and barrier function in UV-A-irradiated HCE-T cells. The reduction in apical Mucin-16 expression was partially recovered in the presence of crocetin. Taking these findings together, we conclude that crocetin has a protective effect against UV-A irradiation-induced mitochondrial injury in corneal epithelial cells.

Effects of paprika carotenoid supplementation on bone turnover in postmenopausal women: a randomized, double-blind, placebo-controlled, parallel-group comparison study.

BACKGROUND: Paprika (Capsicum annuum L.) is a good source of carotenoids, including capsanthin, ß-carotene, ß-cryptoxanthin, and zeaxanthin. Several epidemiological studies have shown a beneficial association of intake of these carotenoids or their blood concentration with bone mineral density (BMD) and fracture risk. However, little information is available regarding the effect of intake of these carotenoids on bone metabolism in postmenopausal women. OBJECTIVE: The objective of the present study was to investigate the effects of paprika carotenoid extract (PCE) on bone turnover in healthy, postmenopausal women. DESIGN: We conducted a randomized, double-blind, placebo-controlled, parallel-group comparison study. One hundred participants were randomly assigned to PCE or placebo groups. Each group was given a 20 mg PCE (equivalent to 1.4 mg of carotenoids) a day or a placebo for 24 weeks. We measured bone resorption markers (tartrate-resistant acid phosphatase 5b [TRACP-5b] and serum type I collagen cross-linked N-telopeptide [sNTX]) at 12 and 24 weeks and bone formation markers (bone alkaline phosphatase and osteocalcin) at 24 weeks. RESULTS: The percentage decrease of TRACP-5b at 24 weeks was significantly higher for PCE than the placebo. There were no significant differences in sNTX or bone formation markers, although PCE decreased each marker compared with the placebo. CONCLUSION: Our findings suggest that PCE supplementation suppresses bone resorption and contributes to maintaining bone quality in postmenopausal women.

Effect of crocetin on quality of sleep: A randomized, double-blind, placebo-controlled, crossover study.

OBJECTIVES: The aim of the present study was to investigate the effect of crocetin on sleep architecture and subjective sleep parameters in healthy adult participants with mild sleep complaints. DESIGN: A randomized, double-blind, placebo-controlled, crossover study with two intervention periods of 14 days each, separated by a 14-day wash-out period. INTERVENTIONS: Thirty participants were randomly assigned to one of two sequence groups. Each group was given crocetin at 7.5 mg/day, or placebo. We measured objective sleep parameters using single-channel electroencephalography and assessed subjective sleep parameters using the Oguri-Shirakawa-Azumi Sleep Inventory, Middle-age and Aged version (OSA-MA). MAIN OUTCOME MEASURES: Differences between crocetin and placebo in an objective sleep parameter (delta power), and OSA-MA scores. RESULTS: Delta power was significantly increased with crocetin compared with placebo. There were no significant differences in the other sleep parameters, including sleep latency, sleep efficiency, total sleep time, and wake after sleep onset. Subjective scores for sleepiness on rising and feeling refreshed were significantly improved with crocetin compared with placebo. CONCLUSIONS: The findings of the present study suggest that crocetin supplementation contributes to sleep maintenance, leading to improved subjective sleep quality.

Safety Evaluation and Plasma Carotenoid Accumulation in Healthy Adult Subjects after 12 Weeks of Paprika Oleoresin Supplementation.

Paprika oleoresin is obtained by solvent extraction from Capsicum annuum L. fruits and contains multiple carotenoids, such as capsanthin, ß-carotene, zeaxanthin, and ß-cryptoxanthin, which are considered protective against various diseases. Herein, we investigated the effect of paprika oleoresin supplementation on plasma carotenoid accumulation and evaluated the safety of the oleoresin. We used a double-blinded, placebo-controlled comparative clinical study design and tested the effects of varying doses in healthy adult subjects. In total, 33 subjects were randomly divided into three groups to take capsules containing 0, 20, or 100 mg of paprika oleoresin daily for 12 consecutive weeks. Plasma carotenoid concentrations were measured at 0, 4, 8, and 12 weeks, and the safety of paprika oleoresin capsules was investigated using analyses of blood biochemistry, hematology, and urine contents. In these experiments, ß-cryptoxanthin and zeaxanthin dose-dependently accumulated in plasma within the dose range of the study over 12 consecutive weeks of paprika oleoresin supplementation. Moreover, ß-cryptoxanthin accumulated to higher levels than the other paprika oleoresin carotenoids. In contrast, capsanthin was not detected in plasma before or during the 12-week treatment period. Finally, no adverse events were associated with intake of paprika oleoresin (20 and 100 mg/day) in safety evaluations. Paprika oleoresin is a suitable source of carotenoids, especially ß-cryptoxanthin.

Crocetin protects ultraviolet A-induced oxidative stress and cell death in skin in vitro and in vivo.

Crocetin, the aglycone of crocin, is a carotenoid found in fruits of gardenia (Gardeina jasminoides Ellis) and saffron (Crocus sativus L.). We investigated the protective effects of crocetin against ultraviolet-A (UV-A)-induced skin damage and explored the underlying mechanism. Human skin-derived fibroblasts cells (NB1-RGB) were damaged by exposure to UV-A irradiation (10J/cm(2)). Crocetin protected these cells against cell death and reduced the production of reactive oxygen species induced by UV-A irradiation. Crocetin treatment also suppressed induction of caspase-3 activation by UV-A irradiation. The effects of crocetin against oxidative stress were also examined by imaging of Keap1-dependent oxidative stress detector (OKD) mice. UV-A irradiation upregulated oxidative stress in the OKD mice skin, while crocetin administration (100mg/kg, p.o.) ameliorated this oxidative stress. Crocetin administration also decreased lipid peroxidation in the skin. These findings suggest that crocetin its observed protective effects against UV-A induced skin damage by reducing reactive oxygen species production and cell apoptosis.

Crocetin, a carotenoid derivative, inhibits retinal ischemic damage in mice.

Crocetin, an aglycone of crocin, is found both in the saffron crocus (Crocus starus L.) and in gardenia fruit (Gardenia jasminoides Ellis). We evaluated the protective effects of crocetin against the retinal ischemia induced by 5h unilateral ligation of both the pterygopalatine artery (PPA) and the external carotid artery (ECA) in anesthetized mice. The effects of crocetin (20mg/kg, p.o.) on ischemia/reperfusion-induced retinal damage were examined by histological, electrophysiological, and anti-apoptotic analyses. Data for anti-apoptotic analysis was obtained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Using immunohistochemistry and immunoblotting, the protective mechanism mediating the effects of crocetin was evaluated by examining crocetin's effects on the expression of 8-hydroxy-2-deoxyguanosine (8-OHdG; used as a marker of oxidative stress) and on phosphorylations of mitogen-activated protein kinases [MAPK; viz. extracellular signal-regulated protein kinases (ERK), c-Jun N-terminal kinases (JNK) and p38], and the redox-sensitive transcription factors nuclear factor-kappa B (NF-κB) and c-Jun. The histological analysis revealed that ischemia/reperfusion (I/R) decreased the cell number in the ganglion cell layer (GCL) and the thickness of inner nuclear layer (INL), and that crocetin inhibited GCL and INL. ERG measurements revealed that crocetin prevented the I/R-induced reductions in a- and b-wave amplitudes seen at 5 days after I/R. In addition, crocetin decreased the numbers of TUNEL-positive cells and 8-OHdG-positive cells, and the phosphorylation levels of p38, JNK, NF-κB, and c-Jun present in the retina after I/R. These findings indicate that crocetin prevented ischemia-induced retinal damage through its inhibition of oxidative stress.

Oral administration of crocetin prevents inner retinal damage induced by N-methyl-D-aspartate in mice.

Crocetin, an aglycone of crocin, is found in stigmas of the saffron crocus (Crocus starus L.) and has been used in traditional medicine. We investigated the effects of oral administration of crocetin on damage induced by N-methyl-D-aspartate (NMDA) in the murine retina. Crocetin was orally administered before and after intravitreal injection of NMDA. A histological analysis was conducted by counting the cell number of ganglion cell layer (GCL). Cell apoptosis was assessed by counting cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Retinal functions were measured in terms of a- and b-wave amplitudes using an electroretinogram (ERG). Activation of caspase-3/7 and cleaved caspase-3 expression were assayed. Calpain activity was evaluated by immunoblotting assays for proteolysis of α-spectrin. NMDA injection decreased the cell number in the GCL, and crocetin at a dose of 100 mg/kg inhibited this reduction. TUNEL-positive cells were observed in both GCL and inner nuclear layer (INL) after NMDA injection, and crocetin inhibited the increase in number of TUNEL-positive cells. ERG analysis showed that both a- and b-wave amplitudes were decreased by NMDA injection. Crocetin inhibited the reduction in the b-wave amplitude, but not in the a-wave. NMDA injection activated caspase-3/7 and increased expression of cleaved caspsase-3 in the GCL and INL, but both of these processes were inhibited by crocetin. NMDA injection also induced cleavage of α-spectrin, but crocetin did not affect this process. These findings indicate that oral administration of crocetin prevented NMDA-induced retinal damage via inhibition of the caspase pathway.

Crocetin, a carotenoid derivative, inhibits VEGF-induced angiogenesis via suppression of p38 phosphorylation.

We evaluated the protective effects of crocetin against angiogenesis induced by vascular endothelial growth factor (VEGF). Crocetin, the aglycone of crocin carotenoids, is found in saffron crocus (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). The effects of crocetin on VEGF-induced angiogenesis were examined by in vitro tube formation assays and following 14-day co-culture of human umbilical vein endothelial cells (HUVECs) and fibroblasts. The anti-angiogenic mechanism of crocetin was evaluated by examining its effects on VEGF-induced proliferation and migration of human retinal microvascular endothelial cells (HRMECs) and phosphorylation of p38. Vascular endothelial (VE)-cadherin, zonula occludens (ZO-1) and occludin, which are adherens and tight junction proteins, respectively, play a major role in the control of vascular permeability. Therefore, we tested effects of crocetin on adhesion molecule dissociation induced by VEGF. Crocetin significantly suppressed VEGF-induced tube formation by HUVECs and migration of HRMECs. It also significantly inhibited phosphorylation of p38 and protected VE-cadherin expression. These findings indicate that crocetin suppresses the VEGF-induced angiogenesis by inhibiting migration and that the inhibition of phosphorylated-p38 and protection of VE-cadherin expression may be involved in its underlying mechanism of action.

Crocetin reduces the oxidative stress induced reactive oxygen species in the stroke-prone spontaneously hypertensive rats (SHRSPs) brain.

Crocetin is a natural carotenoid compound of gardenia fruits and saffron, which has various effects in biological systems. In this study, we investigated the antioxidant effects of crocetin on reactive oxygen species such as hydroxyl radical using in vitro X-band electron spin resonance and spin trapping. Crocetin significantly inhibited hydroxyl radical generation compared with the control. Moreover, we performed electron spin resonance computed tomography ex vivo with the L-band electron spin resonance imaging system and determined the electron spin resonance signal decay rate in the isolated brain of stroke-prone spontaneously hypertensive rats, a high-oxidative stress model. Crocetin significantly reduced oxidative stress in the isolated brain by acting as a scavenger of reactive oxygen species, especially hydroxyl radical, as demonstrated by in vitro and ex vivo electron spin resonance analysis. The distribution of crocetin was also determined in the plasma and the brain of stroke-prone spontaneously hypertensive rats using high-performance liquid chromatography. After oral administration, crocetin was detected at high levels in the plasma and the brain. Our results suggest that crocetin may participate in the prevention of reactive oxygen species-induced disease due to a reduction of oxidative stress induced by reactive oxygen species in the brain.

Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity.

Crocetin is a carotenoid that is the aglicone of crocin, which are found in saffron stigmas (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). In this study, we investigated the effects of crocetin on retinal damage. To examine whether crocetin affects stress pathways, we investigated intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, disruption of the mitochondrial membrane potential (ΔΨ(m)), and caspases activation. In vitro, we employed cultured retinal ganglion cells (RGC-5, a mouse ganglion cell-line transformed using E1A virus). Cell damage was induced by tunicamycin or hydrogen peroxide (H(2)O(2)) exposure. Crocetin at a concentration of 3µM showed the inhibitory effect of 50-60% against tunicamycin- and H(2)O(2)-induced cell death and inhibited increase in caspase-3 and -9 activity. Moreover, crocetin inhibited the enzymatic activity of caspase-9 in a cell-free system. In vivo, retinal damage in mice was induced by exposure to white light at 8000lx for 3h after dark adaptation. Photoreceptor damage was evaluated by measuring the outer nuclear layer thickness at 5days after light exposure and recording the electroretinogram (ERG). Retinal cell damage was also detected with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at 48h after light exposure. Crocetin at 100mg/kg, p.o. significantly inhibited photoreceptor degeneration and retinal dysfunction and halved the expression of TUNEL-positive cells. These results indicate that crocetin has protective effects against retinal damage in vitro and in vivo, suggesting that the mechanism may inhibit increase in caspase-3 and -9 activities after retinal damage.

Topogenesis of two transmembrane type K+ channels, Kir 2.1 and KcsA.

Potassium channels, which control the passage of K+ across cell membranes, have two transmembrane segments, M1 and M2, separated by a hydrophobic P region containing a highly conserved signature sequence. Here we analyzed the membrane topogenesis characteristics of the M1, M2, and P regions in two animal and bacterial two-transmembrane segment-type K+ channels, Kir 2.1 and KcsA, using an in vitro translation and translocation system. In contrast to the equivalent transmembrane segment, S5, in the voltage-dependent K+ channel, KAT1, the M1 segment in KcsA, was found to have a strong type II signal-anchor function, which favors the Ncyt/Cexo topology. The N-terminal cytoplasmic region was required for efficient, correctly orientated integration of M1 in Kir 2.1. Analysis of N-terminal modification by in vitro metabolic labeling showed that the N terminus in Kir 2.1 was acetylated. The hydrophobic P region showed no topogenic function, allowing it to form a loop, but not a transmembrane structure in the membrane; this region was transiently exposed in the endoplasmic reticulum lumen during the membrane integration process. M2 was found to possess a stop-transfer function and a type I signal-anchor function, enabling it to span the membrane. The C-terminal cytoplasmic region in KcsA was found to affect the efficiency with which the M2 achieved their final structure. Comparative topogenesis studies of Kir 2.1 and KcsA allowed quantification of the relative contributions of each segment and the cytoplasmic regions to the membrane topology of these two proteins. The membrane topogenesis of the pore-forming structure is discussed using results for Kir 2.1, KcsA, and KAT1.