Preventive effects of crocin, a natural compound from saffron, against nicotine­induced oxidative stress and neurobehavioral disturbances.

Nicotine is a psychostimulant that induces neurochemical and behavioral changes upon chronic administration, leading to neurodegenerative conditions associated with smoking. As of now, no preventive or therapeutic strategies are known to counteract nicotine­induced neurodegeneration. In this study, we explore the neuroprotective effects of crocin, a bioactive agent commonly found in saffron - a spice derived from the flower of Crocus sativus - using a rat model. The dose­dependent effects of crocin were evaluated in nicotine­induced neurodegeneration and compared with a control group. Neurobehavioral changes, assessed through the elevated plus maze, the open field test, the forced swim test, and the Morris water maze, as well as oxidative stress in the hippocampus, were evaluated. Interestingly, nicotine administration resulted in depression, anxiety, and abnormal motor and cognitive functions, while crocin treatment protected the rat brain from these abnormalities. The beneficial effects of crocin were associated with reduced oxidative stress biomarkers such as malondialdehyde, along with increases in superoxide dismutase, glutathione peroxidase, and glutathione reductase activities. These results demonstrate that crocin can mitigate nicotine­induced neurodegeneration by reducing oxidative stress, potentially offering a protective measure against neurodegenerative effects in smokers.

Transcriptional dynamics in source-sink tissues identifies molecular factors regulating the corm development process in saffron (Crocus sativus L.).

AIMS: Geophytic plants have evolved to develop underground storage organs (USO) in the active growing season to withstand harsh environments as well as to coordinate growth and reproduction when conditions are favourable. Saffron is an autumn flowering geophyte and an expensive spice crop restricted to certain geographical locations in the world. Saffron, being sterile, does not produce seeds and thus propagates only through corms, the quality of which determines its yield. Corm development in saffron is unexplored and the underlying molecular mechanism is still elusive. In this study, we performed an extensive characterisation of the transcriptional dynamics in the source (leaf) and sink (corm) tissues during corm development in saffron. KEY RESULTS: Via morphological and transcriptome studies, we identified molecular factors regulating corm development process in saffron, which defined corm development into three stages: the initiation stage demonstrates enhanced vegetative growth aboveground and swelling of shoot base belowground due to active cell division & carbohydrate storage; the bulking stage comprises of increased source and sink strength, active photosynthesis, circadian gating and starch accumulation; the maturation stage represents reduced source and sink strength, lowered photosynthesis, sugar transport, starch synthesis and cell cycle arrest. UTILITY: The global view of transcriptional changes in source and sink identifies similar and new molecular factors involved in the saffron corm development process compared to USO formation in other geophytes and provides a valuable resource for dissecting the molecular network underlying the corm development. We propose a hypothetical model based on data analysis, of how molecular factors via environmental cues can regulate the corm development process in saffron.

Hepatoprotective Effect of Medicine Food Homology Flower Saffron against CCl4-Induced Liver Fibrosis in Mice via the Akt/HIF-1α/VEGF Signaling Pathway.

Liver fibrosis refers to a complex inflammatory response caused by multiple factors, which is a known cause of liver cirrhosis and even liver cancer. As a valuable medicine food homology herb, saffron has been widely used in the world. Saffron is commonly used in liver-related diseases and has rich therapeutic and health benefits. The therapeutic effect is satisfactory, but its mechanism is still unclear. In order to clarify these problems, we planned to determine the pharmacological effects and mechanisms of saffron extract in preventing and treating liver fibrosis through network pharmacology analysis combined with in vivo validation experiments. Through UPLC-Q-Exactive-MS analysis, a total of fifty-six nutrients and active ingredients were identified, and nine of them were screened to predict their therapeutic targets for liver fibrosis. Then, network pharmacology analysis was applied to identify 321 targets for saffron extract to alleviate liver fibrosis. Functional and pathway enrichment analysis showed that the putative targets of saffron for the treatment of hepatic fibrosis are mainly involved in the calcium signaling pathway, the HIF-1 signaling pathway, endocrine resistance, the PI3K/Akt signaling pathway, lipid and atherosclerosis, and the cAMP signaling pathway. Based on the CCl4-induced liver fibrosis mice model, we experimentally confirmed that saffron extract can alleviate the severity and pathological changes during the progression of liver fibrosis. RT-PCR and Western blotting analysis confirmed that saffron treatment can prevent the CCl4-induced upregulation of HIF-1α, VEGFA, AKT, and PI3K, suggesting that saffron may regulate AKT/HIF-1α/VEGF and alleviate liver fibrosis.

The effects of Crocus sativus extract on inhaled paraquat-induced lung inflammation, oxidative stress, pathological changes and tracheal responsiveness in rats.

Paraquat is a green liquid toxin that is used in agriculture and can induce multi-organ including lung injury. Various pharmacological effects of Crocus sativus (C. sativus) were indicated in previous studies. In this research, the effects of C. sativus extract and pioglitazone on inhaled paraquat-induced lung inflammation, oxidative stress, pathological changes, and tracheal responsiveness were studied in rats. Eight groups of rats (n = 7 in each) including control (Ctrl), untreated paraquat aerosol exposed group (54 mg/m3, 8 times in alternate days), paraquat treated groups with dexamethasone (0.03 mg/kg/day, Dexa) as positive control, two doses of C. sativus extract (20 and 80 mg/kg/day, CS-20 and CS-80), pioglitazone (5 and 10 mg/kg/day, Pio-5 and Pio-10), and the combination of CS-20 + Pio-5 were studied. Total and differential WBC, levels of oxidant and antioxidant biomarkers in the BALF, lung tissue cytokine levels, tracheal responsiveness (TR), and pathological changes were measured. The levels of IFN-γ, IL-10, SOD, CAT, thiol, and EC50 were reduced, but MDA level, total and differential WBC count in the BALF and lung pathological changes were increased in the paraquat group (all, p < 0.001). The levels of IFN-γ, IL-10, SOD, CAT, thiol and EC50 were increased but BALF MDA level, lung pathological changes, total and differential WBC counts were reduced in all treated groups. The effects of C. sativus high dose and combination groups on measured parameters were equal or even higher than dexamethasone (p < 0.05 to p < 0.001). The effects of the combination of CS-20 + Pio-5 on most variables were significantly higher than CS-20 and Pio-5 alone (p < 0.05 to p < 0.001). C. sativus treatment improved inhaled paraquat-induced lung injury similar to dexamethasone and showed a synergistic effect with pioglitazone, suggesting possible PPAR-γ receptor-mediated effects of the plant.

CstPIF4 Integrates Temperature and Circadian Signals and Interacts with CstMYB16 to Repress Anthocyanins in Crocus.

Crocus sativus has emerged as an important crop because it is the only commercial source of saffron that contains unique apocarotenoids. Saffron is composed of dried stigmas of Crocus flower and constitutes the most priced spice of the world. Crocus floral organs are dominated by different classes of metabolites. While stigmas are characterized by the presence of apocarotenoids, tepals are rich in flavonoids and anthocyanins. Therefore, an intricate regulatory network might play a role in allowing different compounds to dominate in different organs. Work so far done on Crocus is focussed on apocarotenoid metabolism and its regulation. There are no reports describing the regulation of flavonoids and anthocyanins in Crocus tepals. In this context, we identified an R2R3 transcription factor, CstMYB16, which resembles subgroup 4 (SG4) repressors of Arabidopsis. CstMYB16 is nuclear localized and acts as a repressor. Overexpression of CstMYB16 in Crocus downregulated anthocyanin biosynthesis. The C2/EAR motif was responsible for the repressor activity of CstMYB16. CstMYB16 binds to the promoter of the anthocyanin biosynthetic pathway gene (LDOX) and reduces its expression. CstMYB16 also physically interacts with CstPIF4, which in turn is regulated by temperature and circadian clock. Thus, CstPIF4 integrates these signals and forms a repressor complex with CstMYB16, which is involved in the negative regulation of anthocyanin biosynthesis in Crocus. Independent of CstPIF4, CstMYB16 also represses CstPAP1 expression, which is a component of the MYB-bHLH-WD40 (MBW) complex and positively controls anthocyanin biosynthesis. This is the first report on identifying and describing regulators of anthocyanin biosynthesis in Crocus.

Network pharmacology-based strategic prediction and target identification of apocarotenoids and carotenoids from standardized Kashmir saffron (Crocus sativus L.) extract against polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a hormonal disorder that affects women of reproductive age, characterized by a range of symptoms, including irregular menstrual cycles, excess male hormones (androgens), metabolic abnormalities such as hyperinsulinemia, hyperlipidemia, and metabolic disturbances like glucose imbalance. Botanical supplements are perceived first and safe choice over available regimens to regulate PCOS. There are several reports available stating that apocarotenoids, carotenoids, and whole extracts of Crocus sativus were identified to have a potential role in the management of women health. This study aimed to propose a network pharmacology-based method to determine the potential therapeutic pathways of phytoconstituents (apocarotenoids and carotenoids) of UHPLC-PDA standardized stigma-based Crocus sativus extract (CSE) for the management of PCOS. Furthermore, to validate the potential targets and signaling pathways, these apocarotenoids, and carotenoids were screened for molecular docking and in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions. The information regarding PCOS-related genes was retrieved from the PCOS knowledge database (PCOSKB), resulting in an established network between putative targets of PCOS and Crocus sativus extract phytochemicals to prevail the mechanism of action. Based on the screening conditions, 4 prominent targets namely, serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription (STAT3), mitogen-activated protein kinase 3 (MAPK3), and mitogen-activated protein kinase 1 (MAPK1), were identified through network analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that MAP kinase and serine-threonine pathways were found prominent targets in PCOS. Further, a molecular docking study shows that crocetin, picrocrocin, and safranal had the best binding affinity for the identified targets. In silico ADMET results revealed that carotenoids and apocarotenoids were found to have the maximum bioavailability and were able to cross the blood-brain barrier without any toxic effects. The combined results revealed that the apocarotenoids and carotenoids of Crocus sativus extract could act on various targets to regulate multiple pathways related to PCOS.

Saffron and crocin ameliorate prenatal valproic acid-induced autistic-like behaviors and brain oxidative stress in the male offspring rats.

Autism is a neurobehavioral disease that induces cognitive and behavioral alterations, usually accompanied by oxidative stress in the brain. Crocus sativus (saffron) and its active ingredient, crocin, have potent antioxidative effects that may benefit autistic behaviors. This study aimed to determine the effects of saffron extract and crocin against brain oxidative stress and behavioral, motor, and cognitive deficits in an animal model of autism in male offspring rats. 14 female rats were randomly divided into the saline and valproic acid (VPA) groups. Then, they were placed with mature male rats to mate and produce offspring. VPA (500 mg/kg, i.p.) was injected on day 12.5 of pregnancy (gestational day, GD 12.5) to induce an experimental model of autism. 48 male pups were left undisturbed for 29 days. First-round behavioral tests (before treatments) were performed on 30-33 post-natal days (PND), followed by 28 days of treatment (PND 34-61) with saffron (30 mg/kg, IP), crocin (15 or 30 mg/kg, i.p.), or saline (2 ml/kg, i.p.). The second round of behavioral tests (after treatments) was performed on PND 62-65 to assess the effects of the treatments on behavioral and cognitive features. In the end, animals were sacrificed under deep anesthesia, and their brains were dissected to evaluate the brain oxidative stress parameters, including malondialdehyde (MDA), glutathione (GSH), and catalase (CAT). VPA injection into female rats increased anxiety-like behaviors, enhanced pain threshold, impaired motor functions, disturbed balance power, increased MDA, and decreased GSH and CAT in their male offspring. 28 days of treatment with saffron or crocin significantly ameliorated behavioral abnormalities, reduced MDA, and increased GSH and CAT levels. Brain oxidative stress has been implicated in the pathophysiology of autistic-like behaviors. Saffron and crocin ameliorate anxiety-like behaviors, pain responses, motor functions, and brain oxidative stress parameters in an experimental model of autism. Saffron and crocin may hold promise as herbal-based pharmacological treatments for individuals with autism. However, further histological evidence is needed to confirm their efficacy.

Prevention of neurotoxicity and cognitive impairment induced by zinc nanoparticles by oral administration of saffron extract.

The accumulation of relatively higher dose of zinc oxide nanoparticles in brain was reported to produce neurotoxicity. Indeed, nanoparticles have a high ability to penetrate biological membranes and be uptaken by cells, which may cause cell disorders and physiological dysfunctions. The aim of the current study was to evaluate, whether oral administration of saffron extract, in rats, can protect from neurotoxicity and behavioural disturbances induced by chronic administration of ZnO-NPs. Daily oral administration of ZnO-NPs was performed for 21 consecutive days to induce oxidative stress-like situation. Then after the saffron extract was concomitantly administrated in several rat groups to overcome the nanotoxicological effect induced by ZnO-NPs. In the frontal cortex, the hippocampus and the cerebellum, ZnO-NPs induced a H2 O2 -oxydative stress-like effect reflected in reduced enzymatic activities of catalase, superoxide dismutase and glutathione S-transferase, and decreased acetylcholinesterase activity. In addition, increased levels of proinflammatory interleukins IL-6 and IL-1-⍺ occurred in the hippocampus, reveal the existence of brain inflammation. The concomitant administration of saffron extract to animals exposed to ZnO-NPs prevented the enhanced anxiety-related to the behaviour in the elevated plus-maze test, the open field test and preserved spatial learning abilities in the Morris water maze. Moreover, animals exposed to ZnO-NPs and saffron showed abnormal activity of several antioxidant enzymes as well as acetylcholinesterase activity, an effect that may underly the preserved anxiety-like behaviour and spatial learning abilities observed in these animals. Saffron extract has a potential beneficial therapeutic effect: antioxidant, anti-inflammatory and neuroprotective agent.

Effect of saffron, black seed, and their main constituents on inflammatory cytokine response (mainly TNF-α) and oxidative stress status: an aspect on pharmacological insights.

Tumor necrosis factor-α (TNF-α), an inflammatory cytokine, is produced by monocytes and macrophages. It is known as a 'double-edged sword' because it is responsible for advantageous and disadvantageous events in the body system. The unfavorable incident includes inflammation, which induces some diseases such as rheumatoid arthritis, obesity, cancer, and diabetes. Many medicinal plants have been found to prevent inflammation, such as saffron (Crocus sativus L.) and black seed (Nigella sativa). Therefore, the purpose of this review was to assess the pharmacological effects of saffron and black seed on TNF-α and diseases related to its imbalance. Different databases without time limitations were investigated up to 2022, including PubMed, Scopus, Medline, and Web of Science. All the original articles (in vitro, in vivo, and clinical studies) were collected on the effects of black seed and saffron on TNF-α. Black seed and saffron have therapeutic effects against many disorders, such as hepatotoxicity, cancer, ischemia, and non-alcoholic fatty liver, by decreasing TNF-α levels based on their anti-inflammatory, anticancer, and antioxidant properties. Saffron and black seed can treat a variety of diseases by suppressing TNF-α and exhibiting a variety of activities such as neuroprotective, gastroprotective, immunomodulatory, antimicrobial, analgesic, antitussive, bronchodilator, antidiabetic activity, anticancer, and antioxidant effects. To uncover the beneficial underlying mechanisms of black seed and saffron, more clinical trials and phytochemical research are required. Also, these two plants affect other inflammatory cytokines, hormones, and enzymes, implying that they could be used to treat a variety of diseases.

SAFFRON-103: a phase 1b study of the safety and efficacy of sitravatinib combined with tislelizumab in patients with locally advanced or metastatic non-small cell lung cancer.

BACKGROUND: Some patients with locally advanced/metastatic non-small cell lung cancer (NSCLC) respond poorly to anti-programmed cell death protein 1 (PD-1)/anti-programmed death-ligand 1 (PD-L1) treatments. Combination with other agents may improve the outcomes. This open-label, multicenter, phase 1b trial investigated the combination of sitravatinib, a spectrum-selective tyrosine kinase inhibitor, plus anti-PD-1 antibody tislelizumab. METHODS: Patients with locally advanced/metastatic NSCLC were enrolled (Cohorts A, B, F, H, and I; N=22-24 per cohort). Cohorts A and F included patients previously treated with systemic therapy, with anti-PD-(L)1-resistant/refractory non-squamous (cohort A) or squamous (cohort F) disease. Cohort B included patients previously treated with systemic therapy, with anti-PD-(L)1-naïve non-squamous disease. Cohorts H and I included patients without prior systemic therapy for metastatic disease, no prior anti-PD-(L)1/immunotherapy, with PD-L1-positive non-squamous (cohort H) or squamous (cohort I) histology. Patients received sitravatinib 120 mg orally one time per day plus tislelizumab 200 mg intravenously every 3 weeks, until study withdrawal, disease progression, unacceptable toxicity, or death. The primary endpoint was safety/tolerability among all treated patients (N=122). Secondary endpoints included investigator-assessed tumor responses and progression-free survival (PFS). RESULTS: Median follow-up was 10.9 months (range: 0.4-30.6). Treatment-related adverse events (TRAEs) occurred in 98.4% of the patients, with ≥Grade 3 TRAEs in 51.6%. TRAEs led to discontinuation of either drug in 23.0% of the patients. Overall response rate was 8.7% (n/N: 2/23; 95% CI: 1.1% to 28.0%), 18.2% (4/22; 95% CI: 5.2% to 40.3%), 23.8% (5/21; 95% CI: 8.2% to 47.2%), 57.1% (12/21; 95% CI: 34.0% to 78.2%), and 30.4% (7/23; 95% CI: 13.2% to 52.9%) in cohorts A, F, B, H, and I, respectively. Median duration of response was not reached in cohort A and ranged from 6.9 to 17.9 months across other cohorts. Disease control was achieved in 78.3-90.9% of the patients. Median PFS ranged from 4.2 (cohort A) to 11.1 months (cohort H). CONCLUSIONS: In patients with locally advanced/metastatic NSCLC, sitravatinib plus tislelizumab was tolerable for most patients, with no new safety signals and overall safety profiles consistent with known profiles of these agents. Objective responses were observed in all cohorts, including in patients naïve to systemic and anti-PD-(L)1 treatments, or with anti-PD-(L)1 resistant/refractory disease. Results support further investigation in selected NSCLC populations. TRIAL REGISTRATION NUMBER: NCT03666143.

Efficacy of Hydroponically Cultivated Saffron in the Preservation of Retinal Pigment Epithelium.

Saffron treatment is a broad-spectrum therapy used for several retinal diseases, and its effectiveness depends on a particular molecular composition (REPRON® saffron). Its production requires specific crops and procedures that, together with low yields, make this spice expensive. To reduce costs, the use of hydroponic crops is gradually increasing. In this study, we tested the protective properties of a hydroponic saffron (sH) batch in models of retinal pigmented epithelium (RPE) degeneration. ARPE-19 cells were pretreated with 40 µg/mL saffron and exposed to different types of damage: excess light and retinol (LE + RET) or oxidative stress (H2O2). After analyzing the composition of all saffron types with spectroscopy, we performed cell viability and immunofluorescence analysis for both protocols. We compared the sH results with those of a validated batch of saffron REPRON® (sR) and those of a saffron non-REPRON® (sNR) batch. sH and sR, which we found had the same chemical composition, were more effective than sNR in increasing cell survival and attenuating the morphological changes related to apoptosis. In conclusion, hydroponic culturing is a suitable strategy to produce high-quality saffron to reduce costs and increase the accessibility of this promising treatment for retinal degeneration.

Saffron essential oil ameliorates CUMS-induced depression-like behavior in mice via the MAPK-CREB1-BDNF signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron, the dried stigma of Crocus sativus L., has a long history of use in the treatment of depression in traditional Chinese medicine and Islamic medicine. The unique aroma of saffron, primarily derived from its volatile oil, has been widely used by folk to mitigate anxiety and depression via sniffing because the aroma of saffron has a pleasant and invigorating effect. AIM OF THE STUDY: This study aimed to investigate the antidepressant effect and the underlying mechanism of saffron essential oil (SEO) in mice exposed to chronic unpredictable mild stress (CUMS). MATERIALS AND METHODS: In this study, compounds of SEO were identified using gas chromatography-mass spectrometry analysis, while network pharmacology was used to predict potential active compounds, antidepressant targets, and related signaling pathways of SEO. The CUMS depression model was further used to explore the therapeutic effect and possible mechanism of SEO. During the modeling period, mice were regularly administered fluoxetine (3.6 mg/kg, i.g.) or diluted SEO (2%, 4%, and 6% SEO, inhalation). The antidepressant and neuroprotective effects of SEO were evaluated by behavior tests (the open field test, the sucrose preference test, the tail suspension test, and the forced swimming test), hematoxylin-eosin staining, and Nissl staining. The enzyme-linked immunosorbent assay kits were used to measure dopamine (DA), 5-serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA) levels in serum. The relative abundance of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B in the hippocampus was determined using western blot (WB). RESULTS: According to the network pharmacology analysis, seven active SEO compounds mediated 113 targets related to depression treatment, most of which were enriched in the 5-HT synapse, calcium signaling pathway, and cAMP signaling pathway. In vivo experiments indicated that fluoxetine and SEO improved depression-like behaviors in depressed mice. The levels of 5-HT, DA, BDNF, and GABA in serum increased significantly. Histopathological examinations revealed that fluoxetine and SEO ameliorated neuronal damage in the hippocampus. WB analysis showed that the relative expressions of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B were significantly higher in the fluoxetine and SEO groups than in the CUMS group. CONCLUSION: Overall, these findings suggest that SEO significantly alleviates the depressive symptoms in CUMS exposed mice and partially restores hippocampal neuronal damage. Meanwhile, the best efficacy was observed in 4% SEO. Furthermore, the antidepressant mechanism of SEO is primarily dependent on the regulation of the MAPK-CREB1-BDNF signaling pathway.

Saffron extract attenuates Sofosbuvir-induced retinal neurodegeneration in albino rat.

Sofosbuvir is a novel drug candidate for the treatment of hepatitis C viral infection; however, vision loss is one of its growing adverse effects. Saffron is a natural biomolecule with a high antioxidant potential that has been efficiently used in some diseases caused by oxidative stress. This study evaluated Sofosbuvir's neurodegenerative effect on the retina of albino rat and examined the potential protective role of saffron aqueous extract. Twenty-one adult male albino rats were randomly divided into three groups: Control, Sofosbuvir-treated (41.1 mg/kg /day for 6 weeks), and Sofosbuvir + Saffron co-treated groups. Retinal specimens were biochemically analyzed for malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels. In addition, light and transmission electron microscopic examination, as well as immunohistochemical staining for Caspase-3, COX-2, and GFAP were performed. Sofosbuvir treatment caused a significant increase in retinal MDA, IL-6, and TNF-α levels coupling with a significant decrease in retinal total antioxidant capacity level. Histopathological findings revealed disturbed retinal architecture, detached pigment epithelium, vacuolated photoreceptors, in addition to a significant decrease in the thicknesses of both outer and inner nuclear layers, and the number of ganglionic cells. Ultrastructural examination revealed extensive degenerative changes in all retinal layers. Caspase-3, COX-2, and GFAP immunohistochemical expressions were significantly increased. Meanwhile, concomitant treatment with Saffron significantly improved retinal redox status, inflammation, histological, and ultrastructural parameters. Saffron may protect the retina from the hazardous effects of Sofosbuvir. Saffron could be used as an adjuvant therapy to protect patients receiving Sofosbuvir from retinal damage.

Saffron as a promising therapy for diabetes and Alzheimer's disease: mechanistic insights.

The prevalence of both Alzheimer's disease (AD) and diabetes mellitus is increasing with the societies' aging and has become an essential social concern worldwide. Accumulation of amyloid plaques and neurofibrillary tangles (NFTs) of tau proteins in the brain are hallmarks of AD. Diabetes is an underlying risk factor for AD. Insulin resistance has been proposed to be involved in amyloid-beta (Aß) aggregation in the brain. It seems that diabetic conditions can result in AD pathology by setting off a cascade of processes, including inflammation, mitochondrial dysfunction, and ROS and advanced glycation end products (AGEs) synthesis. Due to the several side effects of chemical drugs and their high cost, using herbal medicine has recently attracted attention for the treatment of diabetes and AD. Saffron and its active ingredients have been used for its anti-inflammatory, anti-oxidant, anti-diabetic, and anti-AD properties. Therefore, in the present review paper, we take account of the clinical, in vivo and in vitro evidence regarding the anti-diabetic and anti-AD effects of saffron and discuss the preventive or postponing properties of saffron or its components on AD development via its anti-diabetic effects.

Subfunctionalization of D27 Isomerase Genes in Saffron.

Chromoplasts and chloroplasts contain carotenoid pigments as all-trans- and cis-isomers, which function as accessory light-harvesting pigments, antioxidant and photoprotective agents, and precursors of signaling molecules and plant hormones. The carotenoid pathway involves the participation of different carotenoid isomerases. Among them, D27 is a ß-carotene isomerase showing high specificity for the C9-C10 double bond catalyzing the interconversion of all-trans- into 9-cis-ß-carotene, the precursor of strigolactones. We have identified one D27 (CsD27-1) and two D27-like (CsD27-2 and CsD27-3) genes in saffron, with CsD27-1 and CsD27-3, clearly differing in their expression patterns; specifically, CsD27-1 was mainly expressed in the undeveloped stigma and roots, where it is induced by Rhizobium colonization. On the contrary, CsD27-2 and CsD27-3 were mainly expressed in leaves, with a preferential expression of CsD27-3 in this tissue. In vivo assays show that CsD27-1 catalyzes the isomerization of all-trans- to 9-cis-ß-carotene, and could be involved in the isomerization of zeaxanthin, while CsD27-3 catalyzes the isomerization of all-trans- to cis-ζ-carotene and all-trans- to cis-neurosporene. Our data show that CsD27-1 and CsD27-3 enzymes are both involved in carotenoid isomerization, with CsD27-1 being specific to chromoplast/amyloplast-containing tissue, and CsD27-3 more specific to chloroplast-containing tissues. Additionally, we show that CsD27-1 is co-expressed with CCD7 and CCD8 mycorrhized roots, whereas CsD27-3 is expressed at higher levels than CRTISO and Z-ISO and showed circadian regulation in leaves. Overall, our data extend the knowledge about carotenoid isomerization and their implications in several physiological and ecological processes.

Isolation of high-quality RNA for high throughput applications from secondary metabolite-rich Crocus sativus L.

OBJECTIVE: Isolating high-quality RNA is a basic requirement while performing high throughput sequencing, microarray, and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides, and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction including CTAB, TriZol, and SDS-based methods, which invariably yield less and poor quality RNA and hence it necessitated the isolation of high-quality RNA suitable for high throughput applications. RESULTS: In the present study we made certain adjustments to the available protocols including modifications in the extraction buffer itself and the procedure employed. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) value greater than 7.5. The quality of the RNA was further assessed by qPCR-based amplification of mRNA and mature miRNAs such as Cs-MIR166c and Cs-MIR396a.

CstMYB14 links ROS signaling, apocarotenoid metabolism, and stress response in Crocus sativus L.

Reactive oxygen species (ROS) behave as signaling molecules and induce biosynthesis of many secondary metabolites, including apocarotenoids, which play critical roles in stress tolerance through radical scavenging. However, the mechanism that regulates ROS responsive apocarotenoid metabolism and subsequent stress response is unknown. In this study, an R2R3-MYB transcription factor (CstMYB14) was identified from Crocus sativus L., which acts as a regulator of apocarotenoid biosynthesis. CstMYB14 expression increases in response to H2 O2 in a concentration and time-dependent manner. CstMYB14 localizes to the nucleus and acts as a transcriptional activator. Over-expression of CstMYB14 in Crocus stigmas enhanced apocarotenoid biosynthesis. Yeast-one-hybrid demonstrated binding of CstMYB14 to promoters of two apocarotenoid pathway genes (phytoene synthase and carotenoid cleavage dioxygenase 2). Nicotiana benthamiana plants overexpressing CstMYB14 showed better growth and higher stress tolerance than wild type plants. Higher antioxidant activity in CstMYB14-Ox plants indicated that stress tolerance might be due to ROS scavenging. These results establish a molecular link between ROS signaling, apocarotenoid metabolism and stress tolerance. Further, CstMYB14 is shown to act as a key regulator which modulates ROS responsive biosynthesis of apocarotenoids which in turn impart stress tolerance through ROS scavenging.

Understanding saffron biology using omics- and bioinformatics tools: stepping towards a better Crocus phenome.

Saffron is a unique plant in many aspects, and its cellular processes are regulated at multiple levels. The genetic makeup in the form of eight chromosome triplets (2n = 3x = 24) with a haploid genetic content (genome size) of 3.45 Gbp is decoded into different types of RNA by transcription. The RNA then translates into peptides and functional proteins, sometimes involving post-translational modifications too. The interactions of the genome, transcriptome, proteome and other regulatory molecules ultimately result in the complex set of primary and secondary metabolites of saffron metabolome. These complex interactions manifest in the form of a set of traits 'phenome' peculiar to saffron. The phenome responds to the environmental changes occurring in and around saffron and modify its response in respect of growth, development, disease response, stigma quality, apocarotenoid biosynthesis, and other processes. Understanding these complex relations between different yet interconnected biological activities is quite challenging in saffron where classical genetics has a very limited role owing to its sterility, and the absence of a whole-genome sequence. Omics-based technologies are immensely helpful in overcoming these limitations and developing a better understanding of saffron biology. In addition to creating a comprehensive picture of the molecular mechanisms involved in apocarotenoid synthesis, stigma biogenesis, corm activity, and flower development, omics-technologies will ultimately lead to the engineering of saffron plants with improved phenome.

Comparative analysis of apocarotenoids and phenolic constituents of Crocus sativus stigmas from 11 countries: Ecological impact.

The chemical compositions of 15 saffron samples from 11 countries (Morocco, India, Italy, Spain, Germany, Switzerland, Iran, Lithuania, Ukraine, Australia, and Azerbaijan) were evaluated. The samples were analyzed regarding the impact of environmental factors on the composition of apocarotenoids and phenolic constituents. Quantification of saffron metabolites was carried out using high-performance liquid chromatography. It was found that the high content of chlorogenic acid (0.2 mg/g, Ukraine) and ferulic acid (0.28 mg/g, India) was controlled by the duration of solar radiation during plant development. The accumulation of caffeic acid (the higher content 4.88 mg/g, Ukraine) in stigmas depended on the average air temperature. In contrast, the total crocins content according to the correlation analysis depended on the duration of solar radiation, the solar UV index, and the soil type. Rutin was found in all samples (0.83-8.74 mg/g). The highest amount of crocins (average 382.45 mg/g) accumulated in saffron from Italy and Ukraine. Crocins, picrocrocin, safranal, and rutin can further serve as saffron quality markers. All validation parameters were satisfactory and high-performance liquid chromatography methods could be successfully applied for the composition assessment of saffron metabolites. Saffron extracts showed the highest antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli (MICs 62.5-125 µg/ml).