Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

Crocetin inhibits choroidal neovascularization in both in vitro and in vivo models.

Choroidal neovascularization (CNV) is the primary pathogenic process underlying wet age-related macular degeneration, leading to severe vision loss. Despite current anti-vascular endothelial growth factor (VEGF) therapies, several limitations persist. Crocetin, a major bioactive constituent of saffron, exhibits multiple pharmacological activities, yet its role and mechanism in CNV remain unclear. Here, we investigated the potential effects of crocetin on CNV using in vitro and in vivo models. In human umbilical vein endothelial cells, crocetin demonstrated inhibition of VEGF-induced cell proliferation, migration, and tube formation in vitro, as assessed by CCK-8 and EdU assays, transwell and scratch assays, and tube formation analysis. Additionally, crocetin suppressed choroidal sprouting in ex vivo experiments. In the human retinal pigment epithelium (RPE) cell line ARPE-19, crocetin attenuated cobalt chloride-induced hypoxic cell injury, as evidenced by CCK-8 assay. As evaluated by quantitative PCR and Western blot assay, it also reduced hypoxia-induced expression of VEGF and hypoxia-inducible factor 1α (HIF-1α), while enhancing zonula occludens-1 expression. In a laser-induced CNV mouse model, intravitreal administration of crocetin significantly reduced CNV size and suppressed elevated expressions of VEGF, HIF-1α, TNFα, IL-1ß, and IL-6. Moreover, crocetin treatment attenuated the elevation of phospho-S6 in laser-induced CNV and hypoxia-induced RPE cells, suggesting its potential anti-angiogenic effects through antagonizing the mechanistic target of rapamycin complex 1 (mTORC1) signaling. Our findings indicate that crocetin may hold promise as an effective drug for the prevention and treatment of CNV.

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.

Complete microbial synthesis of crocetin and crocins from glycerol in Escherichia coli.

BACKGROUND: Crocin, a glycosylated apocarotenoid pigment predominantly found in saffron, has garnered significant interest in the field of biotechnology for its bioactive properties. Traditional production of crocins and their aglycone, crocetin, typically involves extraction from crocin-producing plants. This study aimed to develop an alternative biosynthetic method for these compounds by engineering the metabolic pathways of zeaxanthin, crocetin, and crocin in Escherichia coli strains. RESULTS: Employing a series of genetic modifications and the strategic overexpression of key enzymes, we successfully established a complete microbial pathway for synthesizing crocetin and four glycosylated derivatives of crocetin, utilizing glycerol as the primary carbon source. The overexpression of zeaxanthin cleavage dioxygenase and a novel variant of crocetin dialdehyde dehydrogenase resulted in a notable yield of crocetin (34.77 ± 1.03 mg/L). Further optimization involved the overexpression of new types of crocetin and crocin-2 glycosyltransferases, facilitating the production of crocin-1 (6.29 ± 0.19 mg/L), crocin-2 (5.29 ± 0.24 mg/L), crocin-3 (1.48 ± 0.10 mg/L), and crocin-4 (2.72 ± 0.13 mg/L). CONCLUSIONS: This investigation introduces a pioneering and integrated microbial synthesis method for generating crocin and its derivatives, employing glycerol as a sustainable carbon feedstock. The substantial yields achieved highlight the commercial potential of microbial-derived crocins as an eco-friendly alternative to plant extraction methods. The development of these microbial processes not only broadens the scope for crocin production but also suggests significant implications for the exploitation of bioengineered compounds in pharmaceutical and food industries.

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.

Therapeutic effects of saffron (Crocus sativus L) on female reproductive system disorders: A systematic review.

The effect of Crocus sativus on several disorders has been discussed or even confirmed, but the efficacy of this herb on the female reproductive system has not been well presented. In this regard, this systematic review comprehensively discussed the efficacy of C. sativus and its main phytochemical compounds on the female reproductive system and its disorders for the first time. In this systematic review, scientific databases, including PubMed, Web of Sciences, Google Scholar, Scopus, and Scientific Information Database, were explored profoundly. In vivo, in vitro, and human studies published until the end of July 2023, which had investigated the pharmacological properties of C. sativus, crocin, crocetin, safranal, or picrocrocin on the female reproductive system, were selected. A total of 50 studies conducted on the effect of C. sativus on the female reproductive system were acquired. These studies confirmed the efficacy of C. sativus or its main phytochemical ingredients in several aspects of the female reproductive system, including regulation of sex hormones, folliculogenesis, ovulation, and protection of the ovary and uterus against several oxidative stress. Several retrieved studies indicated that this herb also can alleviate the symptoms of patients suffering from dysmenorrhea, premenstrual syndrome, menopause, polycystic ovary disease (PCOD), and sexual dysfunction. Furthermore, it is a promising candidate for future studies or even trials regarding ovarian and cervical cancers. This review concluded that C. sativus can improve the symptoms of several female reproductive system disorders, which is particularly due to the presence of phytochemical ingredients, such as crocin, crocetin, and safranal.

Therapeutic potential of saffron in brain disorders: From bench to bedside.

Saffron is a spice derived from the flower of Crocus sativus L., which has been used for centuries as a coloring and flavoring agent, as well as a source of medicinal compounds. Saffron contains various bioactive constituents, such as crocin, crocetin, safranal, picrocrocin, and kaempferol, that have shown potential benefits for human health. Among them, crocin is the most abundant and characteristic constituent of saffron, responsible for its bright red color and antioxidant properties. One of the most promising applications of saffron and its constituents is in the prevention and treatment of neurological disorders, such as depression, anxiety, Alzheimer's disease, Parkinson's disease, and other brain disorders. Saffron and its constituents have been reported to exert neuroprotective effects through various mechanisms, such as modulating neurotransmitters, enhancing neurogenesis, reducing neuroinflammation, regulating oxidative stress, activating the Nrf2 signaling pathway, and modulating epigenetic factors. Several clinical and preclinical studies have demonstrated the efficacy and safety of saffron and its constituents in improving cognitive function, mood, and other neurological outcomes. In this review, we summarize the current evidence on the therapeutic potential of saffron and its constituents in neurological disorders, from bench to bedside. We also discuss the challenges and future directions for the development of saffron-based therapies for brain health.

Research progress on the pharmacological activity, biosynthetic pathways, and biosynthesis of crocins.

Crocins are water-soluble apocarotenoids isolated from the flowers of crocus and gardenia. They exhibit various pharmacological effects, including neuroprotection, anti-inflammatory properties, hepatorenal protection, and anticancer activity. They are often used as coloring and seasoning agents. Due to the limited content of crocins in plants and the high cost of chemical synthesis, the supply of crocins is insufficient to meet current demand. The biosynthetic pathways for crocins have been elucidated to date, which allows the heterologous production of these valuable compounds in microorganisms by fermentation. This review article provides a comprehensive overview of the chemistry, pharmacological activity, biosynthetic pathways, and heterologous production of crocins, aiming to lay the foundation for the large-scale production of these valuable natural products by using engineered microbial cell factories.

The effects of crocetin on oxidative stress induced ARPE-19 cells by H2O2.

The anti-apoptotic and antioxidant effects of crocetin was aimed to investigate on the oxidative damage model of ARPE-19 cells. The oxidative damage in ARPE cells was developed by H2O2 treatment at 800 µM. Different doses of crocetin (1-80 µM) were applied for 24 h, and the effects on viability were evaluated to find out the optimum drug dose. At first, three effective doses of crocetin (10, 20, 40 µM) on cell viability were selected for further analyses. The levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) were determined, and the expression of pro-apoptotic Bax gene and anti-apoptotic Bcl-2 gene were evaluated. The most effective crocetin dose on cell viability was found to be 10 µM. After the H2O2 treatment, SOD and GSH were decreased and MDA were increased significantly (p = 0.011, 0.037, 0.018, respectively). Following the crocetin treatment at 10 µM, SOD and GSH activities were improved compared to the no drug group; and MDA level was declined remarkably (p = 0.022, 0.019, 0.029, respectively). The Bcl-2 level was significantly decreased (p < 0.01), while the Bax1 and Nrf2 expression and ROS level was increased significantly in the damage model group (p < 0.01). After the drug treatment, the Bax1 and Nrf2 expression level were decreased in all groups (p < 0.01). The increase in Bcl-2 expression was significant in crocetin 40 µM (p < 0.05) and the decrease in ROS level were significant in 20 µM and 40 µM doses of crocetin (p < 0.05). It has been shown that crocetin might be used as an antioxidant and anti-apoptotic agent on the hindering the effect of the oxidative damage. Following the development of the oxidative stress in the cells, crocetin reversed the damage signals. By the in vitro tests, it was shown that crocetin might be considered as an effective molecule to be used in the AMD treatment.

Crocetin confers neuroprotection and is anti-inflammatory in rats with induced glaucoma.

BACKGROUND: Crocetin is a bioactive ingredient in saffron, derived from the Crocus sativus stigmas of the Iridaceae family. As a chemically carotenoid derivative, crocetin exhibites effects like anti-inflammatory, antioxidant, neuroprotective, etc. However, the protective effect of crocetin on glaucoma and its mechanism remains unclear. The current study assesed the neuroprotective and anti-inflammatory effects of crocetin on retinal neurons in glaucoma rats which were induced by 0.3% carbomer injection into the anterior chamber. METHODS AND RESULTS: The pathological structures on the retina and optic nerve were observed and examined by H&E staining and transmission electron microscopy. Immunohistochemical staining was used to detect the expression of TNF-α, IL-1ß, and IL-6 of the retina and the expression of a brain-derived neurotrophic factor (BDNF) in the primary visual cortex (PVC). Western blot was carried out to detect the expression of PI3K, Akt, and NF-κB in the retina. It was found that crocetin ameliorated the pathological changes of the retina and ON and reduced the number of apoptotic retinal ganglion cells. Immunohistochemical staining showed that crocetin could decrease the contents of TNF-α, IL-1ß, and IL-6 and increase the contents of BDNF. Western blot showed that crocetin was found to suppress the expression of PI3K, Akt, and NF-κB. CONCLUSION: The results obtained in this study have indicated that crocetin showes neuroprotective effects on retinal ganglion cells in glaucoma rats and inhibits retinal dysfunction. Meanwhile, crocetin exerted an anti-inflammatory effect to protect the retina by inhibiting the expression of the PI3K/Akt/NF-κB signaling pathway. This work provides substantial evidence that crocetin may be a potential drug for the treatment of glaucoma.

A countercurrent chromatography solvent system based on deep eutectic solvents for separation of cis- and trans-crocetin from Gardenia jasminoides Ellis.

Due to the structural similarity and large difference in concentration, the separation of trans- and cis-crocetin has been challenging, and the cis-crocetin is usually neglected. In this work, a countercurrent chromatography method was developed for the quick separation of trans-crocetin and cis-crocetin from the hydrolytic extract of Gardenia jasminoides Ellis. High purity of trans-crocetin (>95%) and cis-crocetin (>91%) were prepared simultaneously for the first time through a novel biphasic system based on deep eutectic solvents, n-heptane/n-butyl alcohol/13 mmol/L Na2 CO3 in water/acetamide-benzyltrimethylammonium chloride (4:1, mol/mol) (4:7:9:1, v/v). The addition of deep eutectic solvent significantly improved the separation efficiency. The two targets can be easily recovered from the separation system through simple acidification and precipitation. It has potential for preparative separations on a large scale.

Protective effect of saffron carotenoids against amyloid beta-induced neurotoxicity in differentiated PC12 cells via the unfolded protein response and autophagy.

The preventive effect of saffron against Alzheimer's disease (AD) has been reported. Herein, we studied the effect of Cro and Crt, saffron carotenoids, on the cellular model of AD. The MTT assay, flow cytometry, and elevated p-JNK, p-Bcl-2, and c-PARP indicated the AßOs-induced apoptosis in differentiated PC12 cells. Then, the protective effects of Cro/Crt on dPC12 cells against AßOs were investigated in preventive and therapeutic modalities. Starvation was used as a positive control. RT-PCR and Western blot results revealed the reduced eIF2α phosphorylation and increased spliced-XBP1, Beclin1, LC3II, and p62, which indicate the AßOs-induced autophagic flux defect, autophagosome accumulation, and apoptosis. Cro and Crt inhibited the JNK-Bcl-2-Beclin1 pathway. They altered Beclin1 and LC3II and decreased p62 expressions, leading cells to survival. Cro and Crt altered the autophagic flux by different mechanisms. So, Cro increased the rate of autophagosome degradation more than Crt, while Crt increased the rate of autophagosome formation more than Cro. The application of 4µ8C and chloroquine as the inhibitors of XBP1 and autophagy, respectively, confirmed these results. So, augmentation of the survival branches of UPR and autophagy is involved and may serve as an effective strategy to prevent the progression of AßOs toxicity.

Gardenia jasminoides Extract, with a Melatonin-like Activity, Protects against Digital Stress and Reverses Signs of Aging.

Digital stress is a newly identified cosmetic stress that is mainly characterized by blue light exposure. The effects of this stress have become increasingly important with the emergence of personal digital devices, and its deleterious effects on the body are now well-known. Blue light has been observed to cause perturbation of the natural melatonin cycle and skin damage similar to that from UVA exposure, thus leading to premature aging. "A melatonin-like ingredient" was discovered in the extract of Gardenia jasminoides, which acts as a filter against blue light and as a melatonin-like ingredient to prevent and stop premature aging. The extract showed significant protective effects on the mitochondrial network of primary fibroblasts, a significant decrease of -86% in oxidized proteins on skin explants, and preservation of the natural melatonin cycle in the co-cultures of sensory neurons and keratinocytes. Upon analysis using in silico methods, only the crocetin form, released through skin microbiota activation, was found to act as a melatonin-like molecule by interacting with the MT1-receptor, thus confirming its melatonin-like properties. Finally, clinical studies revealed a significant decrease in wrinkle number of -21% in comparison to the placebo. The extract showed strong protection against blue light damage and the prevention of premature aging through its melatonin-like properties.

Development and Evaluation of Crocetin-Functionalized Pegylated Magnetite Nanoparticles for Hepatocellular Carcinoma.

Liver cancer remains among the leading causes of cancer-related deaths worldwide. This is due to many reasons, including limitations of available drugs, late diagnosis due to the overlapping symptoms with many other liver diseases, and lack of effective screening modalities. Compared to conventional chemotherapy, targeted drug delivery systems are advantageous in many ways, as they minimize drug resistance and improve therapeutic value for cancer patients. Nanomaterials, in general, and nanoparticles, in particular, possess nm size, which provides a high surface area for a great extent of functionalization to be used for the targeted delivery of cancer drugs. Amongst the different formulations of nanoparticles, magnetic nanoparticles (MNPs) have unique chemical and physical characteristics and magnetic behavior, making them preferable candidates as a core for drug delivery systems. To maintain the nanosized structure of MNPs, a polymeric coating is usually applied to maintain the nanoparticles dispersed in the solution. Moreover, the polymeric coating provides a plate form for carrying drug molecules on its surface. In the present study, poly(ethylene glycol) (PEG)-coated MNPs were successfully synthesized, where the optimum concentration of PEG on the surface of the MNPs was investigated. The PEG-coated MNPs were further coated with crocetin at different concentrations. The crocetin-coated pegylated MNPs were evaluated in vitro using a hepatic cell line (HepG2) for up to 72 h. Results showed good release kinetics under acidic and neutral conditions. The optimally prepared drug delivery system showed a high potential for reducing the HepG2 cell proliferation in vitro using an MTT assay. The calculated IC50 for Cro-PEG-MNPs were 0.1019, 0.0903, and 0.0462 mg/mL of 5×, 10× and 20×, respectively.

Comparison of the pharmacokinetics of Crocin-I in normoxic and hypoxic rats.

An ultra-performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) method for the simultaneous determination of Crocin-I and its primary metabolites Crocetin was established, and a comparison of metabolic characteristics in vivo is made to Crocin-I and Crocetin in normoxic and hypoxic rats after intragastric administration. The acute hypoxic rat model was established by simulating high altitude environment in a hypobaric hypoxia animal experimental chamber. After intragastric administration of 400 mg•kg-1 Crocin-I. UPLC-Q-TOF-MS method was used to detect the plasma concentrations of Crocin-I and Crocetin in plasma at different times. Compared with normoxic rats, the area under the curve (AUC), mean residence time (MRT), time to peak (Tmax), half-life (T1/2) and plasma concentration (Cmax) of plasma Crocin-I in hypoxic rats were significantly increased (P < 0.01). The apparent distribution (Vz/F) and clearance (CLz/F) of plasma Crocin-I in hypoxic rats were significantly decreased (P < 0.01). Under hypoxic conditions, the pharmacokinetic parameters of Crocin-I and its metabolite Crocetin change significantly. The results provide a theoretical basis for the feasibility of Crocin-I for anti-hypoxia treatment in terms of pharmacokinetics and provide an essential experimental basis for optimizing the drug dosing regimen.

Progress in the treatment of drug-induced liver injury with natural products.

There are numerous prescription drugs and non-prescription drugs that cause drug-induced liver injury (DILI), which is the main cause of liver disease in humans around the globe. Its mechanism becomes clearer as the disease is studied further. For an instance, when acetaminophen (APAP) is taken in excess, it produces N-acetyl-p-benzoquinone imine (NAPQI) that binds to biomacromolecules in the liver causing liver injury. Treatment of DILI with traditional Chinese medicine (TCM) has shown to be effective. For example, activation of the Nrf2 signaling pathway as well as regulation of glutathione (GSH) synthesis, coupling, and excretion are the mechanisms by which ginsenoside Rg1 (Rg1) treats APAP-induced acute liver injury. Nevertheless, reducing the toxicity of TCM in treating DILI is still a problem to be overcome at present and in the future. Accumulated evidences show that hydrogel-based nanocomposite may be an excellent carrier for TCM. Therefore, we reviewed TCM with potential anti-DILI, focusing on the signaling pathway of these drugs' anti-DILI effect, as well as the possibility and prospect of treating DILI by TCM based on hydrogel materials in the future. In conclusion, this review provides new insights to further explore TCM in the treatment of DILI.

Saffron anti-metastatic properties, ancient spice novel application.

Crocus sativus L. (saffron), was applied as a spice, food colorant and medicine since four millennia ago and has been used as a remedy for various maladies. In the last three decades, the anti-primary tumor properties of saffron and its main carotenoids, crocin and crocetin, have been well explored. Despite the fact that metastasis is the leading cause of death in cancer patients, the anti-metastatic potential of saffron and its carotenoids has been surveyed only this decade. This review aims to provide an unprecedented overview of the anti-metastatic effects of saffron, crocin and crocetin, and the mechanisms underlying these effects. Investigations on various cancers demonstrated the anti-migratory, anti-invasion, anti-angiogenic potentials of saffron and its carotenoids, as well as their effects suppressing cell-ECM adhesion and enhancing cell-cell attachment. Saffron and its carotenoids exert their impact through different mechanisms such as reduction of CD34 and suppression of Wnt/ß-catenin, Ras/ERK, P38, DCLK1, EMT, matrix metalloproteinases and urokinases. Crocin displayed more effective anti-metastatic potency, in comparison with saffron extract and crocetin. The bioaccessibility/bioavailability, nontoxicity on normal cells, confirmed anti-tumor efficiency and the recent evidence on the anti-metastatic potential of saffron and its carotenoids, recommends them as a propitious multipotent dietary agent and herbal medicine.

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.

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.

The Mechanism of Crocetin Targeting Cardiovascular Disease Based on Network Pharmacology Constrained by Spectral Experiments.

The network pharmacology under conditions is a recent development trend. We use network pharmacology methods to analyze the mechanism of crocetin (CRO) that regulates cardiovascular diseases. In this work, the spectral experimental data of CRO-Protein interaction is first time combined with constraint conditions to solve the problems of targeting redundancy and lack of verification. CRO targets and cardiovascular disease targets were obtained by the target database. The STRING platform was used for PPI analysis. The GO and KEGG pathways of the target were analyzed using the Metascape platform; The core functional targets of CRO were screened by molecular docking techniques and the spectra of CRO and human serum albumin (HSA). Under the collaborative constraint conditions, the core targets of CRO that regulate cardiovascular diseases are ADRA1A, ADRA1B, CHRM1, CHRM2, GABRA1, and PTGS2; This study incorporates spectroscopy and molecular docking as constraints into the network pharmacological analysis, which significantly improves the credibility of network pharmacological analysis compared with unconstrained conditions. This method provides theoretical references for the in-depth study of the mechanism between active substances and protein targets for other medicines in network pharmacology.