A Perspective on Crocus sativus L. (Saffron) Constituent Crocin: A

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Perspective

A perspective on Crocus sativus L (Saffron). Constituent Corcin: a potent water soluble antioxidant and potential therapy for Alzheimer’s Disease John W. Finley, and Song Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04398 • Publication Date (Web): 18 Jan 2017 Downloaded from http://pubs.acs.org on January 20, 2017

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Journal of Agricultural and Food Chemistry

A Perspective on Crocus sativus L. (Saffron) Constituent Crocin: a Potent Water Soluble Antioxidant and Potential Therapy for Alzheimer’s Disease John W Finley 1* and Song Gao2 1

Adjunct Professor Louisiana State University Department of Nutrition and Food Science, 14719 Secret Harbor Pl. Lakewood Ranch, FL. 34202, Phone: 225 571 2711, Email: [email protected] 2

Quality Phytochemicals LLC, 13 Dexter Road, East Brunswick, NJ 08816

*

Corresponding Author

Running title: Crocin for Alzheimer’s Prevention and Treatment

ACS Paragon Plus Environment


ABSTRACT Alzheimer’s Disease (AD) is the most common form of dementia in which the death of brain cells causes memory loss and cognitive decline. Several factors are thought to play roles in the development and course of AD. Existing medical therapies only modestly alleviate and delay cognitive symptoms. Current research has been focused on developing antibodies to remove the aggregates of amyloid-β (Aβ) and tau protein. This approach has achieved removal of Aβ; however, no cognitive improvement to AD patients has been reported. The biological properties of saffron, the dry stigma of the plant Crocus sativus L., and particularly its main constituent crocin, have been studied extensively for many conditions including dementia and traumatic brain injury. Crocin is a unique antioxidant because it is a water soluble carotenoid. Crocin has shown potential to improve learning and memory as well as protection of brain cells. A search of the studies on saffron and crocin which has been published in recent years for its impact on AD as well as crocin’s effects on Aβ and tau protein has been conducted. This review demonstrates that crocin exhibits multifunctional protective activities in the brain and could be a promising agent applied in supplement or drug for prevention or treatment of AD. Key Words: Alzheimer’s disease, crocin, crocetin, saffron, Crocus sativus


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INTRODUCTION

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Alzheimer's disease (AD) is the most common form of dementia among people over the age of 65 years old and accounts for 60 to 80 percent of dementia cases. The global prevalence of dementia is estimated to be 24 million, and is predicted to double every 20 years through to 20401. More than 5 million Americans may have various stages of AD, according to Alzheimer’s Disease Fact Sheet published by National Institute on Aging. This irreversible and progressive brain disorder destroys memory and thinking skills of its patients and has a devastating impact on both patients and their families. Although the greatest known risk factor is increasing age, less than 5 percent of people with the disease have early onset in age 30 to 60 with increasing numbers appearing in the early 60s. . Damage to the brain likely starts years before memory and other cognitive symptoms appear. Damage to the brain is a gradual progression leading to memory and other cognitive symptoms. Previously, the most common belief of early symptom pattern is a gradual increase in difficulty remembering new information, followed by gradual progression to losses in cognitive functions including thinking, remembering, and reasoning and changes in behaviour. Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog), Clinical Dementia Rating Scale Sums of Boxes (CDR-SOB) and Mini–Mental State Examination (MMSE) are the most frequently used tests to detect memory and cognitive impairments and AD. Scores of these tests help assess severity and progression of AD. ADAS-Cog and CDR-SOB are more thorough and accurate than MMSE to estimate the severity and progression of cognitive impairment and to follow the course of cognitive changes in an individual over time. Therefore, ADAS-Cog and CDR-SOB are believed to be the better choices to document an individual's response to treatment. Recently, researchers at the Alzheimer's Association International Conference 2016 (AAIC 2016) in Toronto introduced a patient status, known as Mild Behavioural Impairment (MBI), and believed MBI may be a forerunner of the neurodegeneration and progression to AD or dementia. A MBI check list was proposed to evaluate five categories of behavioural symptoms of a patient and the evaluation may help capture changes signalling the beginnings of neurodegeneration. AD is the sixth leading cause of death in the United States. Patients with AD live an average of eight years after their symptoms become obvious to others. However, survival may range from four to 20 years, depending on age, care and other health conditions of individual patients.

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Currently, there is no cure for AD. Five drugs have been developed and several more are being tested to alleviate cognitive symptoms. Leading drugs are the four acetylcholinesterase inhibitors (AChEIs), donepezil, rivastigmine, galantamine, tacrine and one N-methyl-D-aspartate (NMDA) glutamate receptor blocker, memantine2-5. Tacrine, the first drug approved for AD treatment, has been largely abandoned due to its poor efficacy and adverse side effects. Recent new drugs approved by FDA for AD are still combinations of donepezil and memantine in either regular or slow-releasing forms. These drugs attempt to prolong cognitive function through restoration of neurotransmission, increasing synaptic activity, but they do not provide neuroprotection. AChEI works to prevent the breakdown of acetylcholine, a brain chemical believed to be important for memory and thinking. However, as AD progresses, the brain



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produces less and less acetylcholine and AChEI gradually losses its effectiveness. The NMDA receptor antagonist, memantine, is believed to work by regulating the activity of glutamate, an important neurotransmitter in the brain functions involved in learning and memory. In AD patients, excessive glutamate is released which leads to cellular damage and cell death. Memantine helps control the level of glutamate by partially blocking the NMDA receptors. Prolonged treatments with donepezil, rivastigmine, galantamine, memantine, or combination of donepezil and memantine are currently favoured therapies for AD treatment. These drugs have modest efficacy but can induce severe side effects. AD is pathologically characterized by formation and deposition of extracellular amyloid-β (Aβ) peptide as plaque and intracellular aggregation of microtubule associated tau protein as neurofibrillary tangles as well as loss of neurons and synapses 6, 7. In recent years, tremendous efforts have been focused on Aβ and tau protein to develop immunization therapies. However, the immunization therapies have failed to clearly improve cognition in AD patients in clinical studies.

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Although mechanisms leading to the development of AD are not clearly defined, risk factors are believed to include genetic predisposition, oxidative stress, inflammation, environmental stress, history of traumatic head injuries, depression or hypertension. Clearly, there is a need to develop practical and safe means or medications to delay or prevent the cognitive, behavioural and psychological symptoms of dementia associated with AD and in the meantime preserve or protect neurons during aging. Two of the approved drugs, galantamine and rivastigmine, are derived from natural compounds. Many studies with herbal plants or natural ingredients have also been carried out in recent years in searching alternatives for AD prevention and treatment8. Among these herbal plants including Ginkgo biloba, Huperzia Serrata, Salvia officinalis, Melissa officinalis and Crocus sativus (Saffron) that are traditionally used for memory and brain health, saffron and its main constituent crocin have been extensively studied. Crocin is believed primarily responsible for saffron’s various beneficial effects. Preliminary evidence from a few clinical studies indicate that crocin rich saffron provides cognition improving effects to patients with mild to moderate AD and moderate to severe AD comparable to donepezil and memantine, respectively. In vitro and animal studies indicate crocin could provide effects of neuroprotection and memory and cognition improvement. This review presents studies of Crocus sativus L. (Saffron), its main constituent crocin, in the past 20-25 years and support of crocin in preventive and therapeutic use for AD.

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SAFFRON CHARACTERISTICS

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Saffron is the dried stigma of flowers of Crocus sativus L. according to the 29th Joint FAO/WHO Expert Committee on Food Additives (JECFA). For over 3,000 years, saffron is considered the most expensive spice and is widely used in Persian, Indian, European, Arab, and Turkish cuisines as a food colorant as well as in traditional medicine for the treatment of some 90 illnesses9-11. Saffron contains more than 150 volatile and aroma-yielding compounds. Safranal is the most abundant component of the saffron volatile compounds. Safranal is the constituent primarily


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responsible for the aroma of saffron. Safranal is thought to be a degradation product of the carotenoid zeaxanthin in a pathway where picrocrocin is an intermediate. Non-volatile constituents of saffron include crocins, crocetin, picrocrocin, flavonoids (quercetin and kaempferol), isophorones and low levels of other carotenoids, including zeaxanthin, lycopene, and various α- and β-carotenes 11-15. The color of saffron is primarily associated with the crocins and crocetin. Traditionally, saffron has been used to provide benefits in multiple therapeutic areas16. More recently, saffron extracts and particularly crocin have been shown to potentially provide significant antitumor, antidepressant, neuroprotective, memory and cognition improvement, and a few other effects 12, 13, 17-24.

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Crocin is the unique water soluble carotenoid found in saffron and the primary component contributing to the bright red color of saffron. Most of carotenoids in human diet including αCarotene, β-carotene, β-cryptoxanthin, lutein, lycopene, zeaxanthin, and astaxanthin, come from fruits, vegetables and sea foods. These intense chromophoric molecules exhibit yellow, orange and red colors in many plants and shellfishes25, but they are not water soluble. Carotenoids including α-Carotene, β-carotene and β-cryptoxanthin exhibit provitamin A activity because they are converted to the active form retinol in the body. Lutein, lycopene, astaxanthin, and zeaxanthin cannot be converted to retinol and do not exhibit vitamin A activity. All carotenoids can provide antioxidant protection in the body. Chemically the common carotenoids can be broadly classified into two classes, carotenes (α-carotene, β-carotene, and lycopene) and xanthophylls (β-cryptoxanthin, lutein, astaxanthin and zeaxanthin). Crocin is unique in comparison to most other carotenoids because of its water solubility which can be attributed to sugar moieties bound to the carboxylic acid groups appended on crocetin. The structure of crocin is shown in Figure 1.

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Crocin is a collective term of a series of hydrophilic carotenoids that are either monoglycosyl or diglycosyl polyene esters of crocetin11. Crocetin is a conjugated polyene dicarboxylic acid that is hydrophobic. When crocetin is esterified with water-soluble gentiobiose(s) or other sugar units, the resulting adducts are water-soluble. Crocetin is also the metabolite or product of crocin hydrolysis, either through acid or enzymatic hydrolysis. Sugars bound to the two acidic groups of the aglycone crocetin are provided in Table 126.

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Contents of crocin in saffron extracts are influenced by the geographical location where the Crocus sativus L. is grown and processing methods utilized to produce the extracts. Crocin contents of saffron have been reported ranging from less than 1% to over 30%27-30. Two studies reported that crocin content in Spanish saffron ranges between (0.01–9.44%) and (0.46– 12.12%) respectively 31, 32. Crocin and crocetin mainly exist in trans-form; however cis-crocetin and its glycosides have been reported as minor constituents in saffron33. One study reported that all crocin derivatives occur as pairs of cis–trans isomers except crocin-134. Speranza et al. found that trans-crocin undergo photoisomerization resulting in conversion to cis-crocin. This isomerization varies depending on the agricultural and environmental conditions in the area of the plants origin35. In saffron, trans-crocetin di-β-D-gentiobiosyl ester which exerts the highest



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coloring capacity in the extracts has high water solubility36. The water insoluble carotenoids make comparatively minor contributions to the red color of the extracts. Crocus sativus L is grown in many different regions and a wide range of altitudes. The altitude of the growth region has a positive correlation with crocin content 30. Variations in the crocin content of saffron has also attributed to differing drying processes and storage conditions29. Several authors have reported that trans-crocetin di-β-D-gentiobiosyl ester, which is also described as crocin or α-crocin or crocin I, represents about 10% of saffron's dry mass14. The JECFA document describes that saffron normally contains between 4-6% of crocin and crocetin 37.

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In the past 20-25 years, extensive studies on saffron extracts and crocin have evaluated the potent antioxidant and bioactivities of this unique water soluble carotenoid. It appears crocin may be a multi-functional therapeutic agent, including neuroprotective functions that may have preventive and therapeutic values for AD38.

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CROCIN BIOAVAILABILITY

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Many of saffron’s biological activities have been attributed to the presence of crocin39. Orally administered crocin in animals has been shown to provide protective and immunostimulatory activities in animals against chemical induced cancer, oxidative damage/inflammation, and organ toxicity in various animal models39-43. To this point, systematic studies of crocin bioavailability and metabolism in humans are very limited. Several animal studies have shown that orally administered crocin is not absorbed at any significant level from the gastrointestinal tract. Crocin has been administered in both single dose and repeated doses. In either case, crocin was not detected in animal blood44-46. The aglycone crocetin was detected at low level in plasma. Xi et al 46 suggested that intestine is the primary site for crocin hydrolysis. In a rat study, Zhang et al confirmed that crocin was hydrolysed in the intestine and absorbed as crocetin47. As a result of these studies it was postulated that the in vivo pharmacological activities of crocin may be primarily caused by crocetin 48. Asai et al compared the absorption of orally administered crocetin and crocin in mice49. They demonstrated that both orally administered crocin and crocetin were absorbed as free crocetin into blood plasma where they were found as the glucuronide conjugates (crocetin -monoglucuronide and crocetin diglucuronide). Crocetin, which was rapidly absorbed, and its glucuronide conjugates were found in mouse plasma after crocin administration. Similar to previous studies, no intact crocins were detected44-47. These results supported the hypothesis that orally administered crocin was hydrolysed to crocetin before or during intestinal absorption and the absorbed crocetin was partially metabolized to mono- and di-glucuronide conjugates. In 2011, Chryssanthi et al developed a validated SPE-HPLC method to monitor crocetin in human plasma after volunteers consumed saffron tea50. Four healthy volunteers (25-35 years old, 3 female and 1 male, all with normal BMI) participated in the study. Blood samples were drawn at 0(hr), 2hr and 24hr after the subjects consumed saffron tea (200mg of saffron in 80 ◦C 150 ml water within 5 min). High

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concentrations of crocetin (1.24-3.67 µM) were detected in blood plasma 2 hr after the subjects consumed the saffron tea. Crocetin (0.10-0.24 µM) was also detected after 24 hr. Umigai et al studied crocetin pharmacokinetics in healthy adult human volunteers (5 men and 5 women)51. They found that crocetin was absorbed more rapidly than other carotenoids, such as βcarotene, lutein and lycopene after single dose oral administration. Crocetin concentrations in plasma were proportional to doses administered in the study. Crocetin was absorbed and detected within one hour after oral administration, the mean time to reach maximum concentration (Tmax) for crocetin ranged from 4.0 to 4.8 hr. The mean elimination half-life for crocetin from human plasma (T1/2) was 6.1 to 7.5 hr. Lautenschläger et al used in vitro Caco-2 monolayer cell culture as a model to evaluate the intestinal permeation of crocin and crocetin52. Crocin at concentrations up to 1000 μM did not permeate through Caco-2 monolayer in relevant amounts. Conversely crocetin permeated the Caco-2 monolayer in a concentration-independent manner (10-114μM) with about 32% of the crocetin being transported across the monolayer within 2h. Through incubation of crocin enriched saffron extract with freshly prepared tissue homogenate from purged small mouse intestine, they found that generation of crocetin from deglycosidation of crocin was mediated by enzymatic activity of either esterases or β-glycosidases in the intestinal cells. They further demonstrated crocetin was able to penetrate the blood brain barrier. Two in vitro blood brain barrier models; 1) porcine brain capillary endothelial cells (BCEC) and 2) blood cerebrospinal fluid barrier (BCSFB) were applied in their tests to evaluate crocetin capable of reaching the central nervous system. These studies indicate orally administered crocin is hydrolysed to crocetin in the intestine, crocetin is absorbed into the blood stream, and then crocetin passes through blood brain barrier. In an in vivo study53, Yoshino et al demonstrated that in a short time after oral administration, crocetin was detected at high levels in the rat plasma and the brain. They observed that crocetin exhibited antioxidant properties in the stroke prone spontaneously hypertensive rats. However, Ochiai et al demonstrated that intravenous injected crocin passed through the blood-brain barrier in mice and prevented neuron death caused by ischemic stress54. Based on these results, there is a clear need for more animal and human studies to assess the bioavailability, pharmacokinetics, and effects of crocin that is administered orally, intravenously or by other means.

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CROCIN SAFETY Saffron has been put into the list of substance as Generally Recognized As Safe (GRAS) [21CFR182.10] by FDA since 2012. Several studies have reported that saffron and crocin have an attractive safety profile with few side effects noted55-57. Saffron which is rich in crocin has been consumed for thousands of years as spice and food colorant, and for its medicinal qualities58. According to the WHO monograph in 2007, saffron daily doses of up to 1.5 g are considered to be safe. When saffron doses of 5.0 g or more a day are consumed, serious



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adverse reactions occur and overdoses (12.0-20.0 g/day) may be fatal59. The adverse effects may include vomiting, uterine bleeding, bloody diarrhoea, haematuria, bleeding from the nose, lips and eyelids, vertigo, numbness and yellowing of the skin and mucous membranes. Safety studies reported that daily doses of 200 and 400 mg of saffron to healthy volunteers for one week had no significantly adverse effects60, 61. Crocin has no Vitamin A activity; however, it provides antioxidant protection and is not toxic at low doses of less than 5.0 g/day. Pharmacological levels of crocin do not cause damage to any major organ in experimental animals62. The safety of crocin was evaluated in a randomized, double-blind, placebo-controlled study with healthy volunteers for one month63. 44 volunteers were randomly distributed into 2 groups of 22 (males and females). Each in one group was given 20 mg crocin tablets a day and the other group was given a placebo. Clinical examination and laboratory tests (hematology, blood biochemistry and hormone and urine analysis) were conducted before and after the treatment period. Volunteers in both groups were monitored for adverse drug reactions throughout the trial. No major adverse effects were reported by any of the volunteers. The consumption of crocin (20 mg/day, one month) resulted in a decrease the level of serum amylase, partial reduction in thromboplastin time and lower levels of basophils, eosinophils and monocytes. There were no effects on total white blood cells count, neutrophils or lymphocytes in these healthy adult volunteers. It was concluded that crocin presented a relatively safe and normal profile through the clinical evaluation. Two papers reported results of a 12-week study of 66 schizophrenia patients evaluating the effects of consumption of 30 mg crocin per day64, 65. Patients diagnosed with schizophrenia who were receiving olanzapine treatment (5-20 mg daily) were randomly divided into three 22patient groups. Group 1 received capsules of saffron aqueous extract (SAE) (15 mg twice daily), group 2 received crocin (15 mg twice daily) and group 3 were given a placebo. Sixty-one subjects completed the trial and no serious side effects were observed. White blood cell counts, other hematologic components, markers of thyroid, liver and kidney or inflammation markers were all normal. Both SAE and crocin prevented increase of fasting blood sugar which was observed in the placebo group. Both SAE and crocin were tolerated during the 12 weeks. These study results further confirmed crocin is relatively safe. Additionally, after a careful review under the new dietary ingredient guideline, FDA issued no objection to a high purity crocin ingredient for supplement application in October 2015.

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CROCIN EFFECTS ON CHOLINERGIC METABOLISM INHIBITOR, NMDA RECEPTOR, LEARNING AND MEMORY Inhibition of acetylcholine breakdown is one of the primary therapeutic approaches for AD treatment4, 66, 67. Abnormalities of cholinergic metabolism in AD patients are not considered to be the primary the cause of the disorder; however, cholinergic changes are significant because


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they are closely correlated with cognitive defects. Treatment with AChE inhibitor drugs, including donepezil, rivastigmine, and galantamine, have been shown clinically to exert beneficial effects on cognition among AD patients, however the longer term benefits have been limited68-71. Limited studies have been conducted to assess the activities of saffron and its components on AChE activity. Geromichalos et al applied an AChE inhibition assay to demonstrate that an aqueous methanolic saffron extract exhibited moderate inhibition of acetylcholine esterase68. Crocetin, dimethylcrocetin and safranal all exhibited dose-dependent AChE inhibition with IC50 values of 96.33, 107.1, and 21.09 μM, respectively. The interactions of crocin or crocetin with AChE protein were evaluated in an attempt to identify potential mechanisms of action. Crocetin was found to bind simultaneously to catalytic and peripheral anionic sites of AChE presenting a mixed-type of inhibition in kinetic analysis. The results indicated potential beneficial effects of crocin and crocetin probably through action of crocetin on cholinergic activity. The inhibitory effects of crocin and saffron extract were also demonstrated in a study that used a Drosophila model of Parksonism72. In this study, Rotenone was applied to flies to induced oxidative stress, neurotoxicity, locomotor phenotype dysfunction, and mortality responses. The activity levels of AChE were significantly elevated in the head (40%) and body (37%) regions of the Rotenone treated flies, while in the co-exposure groups the activity levels of AChE were significantly inhibited and restored to normalcy by saffron methanolic extract (SME) (35-31%) and crocin (19-21%) in a concentration-dependent manner. The results suggested that crocin may be an effective strategy in mitigating cholinergic dysfunction. Similarly significant indication to benefit Parkinson’s disease, the diet with crocin enrichment improved locomotor ability of flies by up to 25%. SME and crocin were also found to lower oxidative stress, raised antioxidant enzyme activities and significantly improved survival rate in the Rotenone challenged flies. The median life span of the flies under their regular conditions is 80 days and maximum life span is 94 days. Feeding with a diet supplemented with SEM or crocin extended the maximum life span to the flies by 10-20%.

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Glutamate is considered to be the most important excitatory neurotransmitter involved in learning and memory within the central nervous system (CNS)73. NMDA receptors play important roles in regulating the levels of glutamate in brain. Chronic activation of NMDA receptors causes excessive glutamate production which leads to neuronal damage and brain cell death. Conversely, complete NMDA receptor blockage also impairs neuronal plasticity. Therefore, both hypo- and hyperactivity of the glutamatergic system lead to dysfunction. Memantine is a moderate affinity, uncompetitive NMDA receptor antagonist with strong voltage-dependency and fast kinetics. In studies of saffron and its constituents, Lechtenberg et al demonstrated that saffron extracts and crocetin exhibited a clear binding capacity at the phencyclidine binding site of the NMDA receptor and at the σ1 receptor. The results also demonstrated that crocins and picrocrocin were not effective agonists74. In vivo and in vitro studies by Abe et al demonstrated that crocin alone did not affect either NMDA receptormediated synaptic potential or non-NMDA receptor-mediated synaptic potential75. Although not effective in binding NMDA receptor, crocin has been found to specifically antagonize the



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inhibitory effect of ethanol on NMDA receptor and induction of long-term potentiation, an activity involved in process of learning and memory, in the rat hippocampus75, 76. Oral preadministration of crocin (50 to 200 mg/kg) improved the impairment of memory acquisition in ethanol-treated mice in a dose-dependent manner. Crocin (50 to 200 mg/kg) also provided beneficial effects on ethanol-induced memory retrieval deficit77. Sub-chronic treatment with the non-competitive NMDA receptor antagonist ketamine induced cognitive deficits and significantly reduced the time spent in social interaction and motor activity levels in rats. A single injection of crocins (15-30mg/kg, i.p.) reversed recognition memory deficits produced by ketamine in rats78. The effects of saffron extract and crocin on learning, memory improvement and long term potentiation (LTP), which is a form of activity-dependent synaptic plasticity that may underlay learning and memory, have been extensively studied21, 79, 80. Table 2 listed more studies of crocin/crocetin effects on learning and memory.

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The studies in Table 2 demonstrated that saffron and crocin ameliorated impairments of learning and memory in rats and mice in a wide range of tasks and conditions. One exception described in the study by Linardaki et al97 where saffron extract was co-administered with Al during the last 6 days of a 5 week treatment period. Saffron extract was not effective in reversing aluminium-induced cognitive impairment. This was likely due to administration of saffron extract only at the latest stage of the impairment. However, saffron extract exhibited neuroprotective potential by significantly reducing oxidative stress in mouse brain and liver and significantly lowering AChE activity. The consensus of all the study results listed in Table 2 supported the therapeutic potential of crocin in aging and age-related neurodegenerative disorders where cognitive impairment is involved21, 38, 79, 80, 86-88.

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EFFECTS OF CROCIN ON OXIDATIVE STRESS Oxidative stress has been implicated in the progression of a number of neurodegenerative diseases, including AD. A correlation was observed between cognitive impairment and plasma antioxidant capacity (AOC) of patients. The degree of oxidative stress is directly related to the advance of cognitive impairment and AD100, 101. Crocin was found as a potent neuronal antioxidant102, 103 and was believed responsible for much of the antioxidant activity of saffron extract104, 105. Ochiai et al106 demonstrated that crocin acted as a unique and potent antioxidant in neurons. Crocin protected neuronally differentiated pheochromocytoma (PC-12) cells from serum/glucose derivation caused peroxidation of their cell membrane lipids and decreased intercellular superoxide dismutase (SOD) activity. The antioxidant effects of crocin were more effective than α-tocopherol at the same concentration. More studies, both in vitro and in vivo, listed in Table 3, confirmed that crocin suppressed oxidative stress and exhibited neuroprotective activities.

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CROCIN EFFECTS ON ΑMYLOID PEPTIDE AND TAU PROTEIN


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Amyloid-β (Aβ) peptide, Tau proteins and their associated signalling pathways are potentially important therapeutic targets for AD intervention. Aβ is the primary constituent of the amyloid plaque found in the brains of patients with AD. The increased levels of extracellular Aβ and the aggregation of Aβ result in synaptic loss and cell apoptosis. There are two common isoforms of Aβ: the more common form, Aβ40, and the less common but more amyloidogenic form, Aβ42. The effects of saffron extract and crocin on aggregation were investigated in vitro utilizing thioflavine T-based fluorescence assay and the DNA binding shift assay. Papandreou et al evaluated the inhibitory effects of saffron extract and crocin on fibrillogenesis of Aβ40 in vitro102. Saffron extract and crocin both exhibited inhibitory effects on the fibrillogenesis of Aβ40 in a concentration and time-dependent manner. The interaction between crocin and Aβ40 which results in the inhibition of Aβ aggregation and fibril formation have also been observed in vitro by other investigators107. Ghahghaei et al further investigated the effects of crocin on fibrillogenesis of Aβ42 by thioflavine T-based fluorescence assay, DNA binding shift assay, CD spectroscope and Transmission electron microscopy. They found that crocin prevented Aβ42mediated amyloid fibril formation in vitro, probably through the stabilization of the helical structure, and resulted in the dissolution of previously formed aggregates108. Furthermore, Ahn et al109 reported that crocetin, was capable of passing through blood brain barrier and impacting the Aβ aggregation. Their results demonstrated that crocetin inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils. Crocetin also caused stabilization of Aβ oligomers and prevented their conversion into Aβ fibrils.

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A major factor in the progression of AD is the accumulation of Aβ peptide which induces neuronal loss and cognitive impairments. Yoshino et al. investigated the effect of crocetin on death induced by Aβ42 in mouse hippocampal HT22 cells110. Treatment of mouse hippocampal HT22 cells with Aβ42 at 0.2 to 20 μM induced cell death in a concentration-dependent manner. Crocetin at low concentrations (1 - 10 µM) protected HT22 cells against Aβ42-induced neuronal cell death as well as H2O2 (200 μM) induced cell death. Based on previous studies51, 53 it is conceivable that the 1 to 10 μM concentrations are attainable in brain through oral administration. Morelli et al 111 evaluated crocin’s neuroprotective activity against Aβ induced toxicity in a neuronal membrane bioreactor. The bioreactor reconstructed neuronal network with synaptic formation in vitro. Administration of the Aβ produced a dramatic decrease in cell viability, induced the reactive oxidative species generation and apoptosis. When Aβ was administered together with crocin, a significant dose-dependent inhibition of Aβ-induced apoptosis and ROS production was observed. This supports the hypothesis that crocin has potential to prevent the aggregation of Aβ peptide and the subsequent neurotoxicity. The results are consistent with the previously reported work by Asadi et al, who reported that crocin is effective in improving rat memory impairment as a result of Aβ injection38. Their proteomic studies also demonstrated crocin administration significantly restored apoptotic biomarkers caused by Aβ.



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Another marker of AD is the abnormal accumulation of hyperphosphorylated tau protein and intracellular aggregation of the microtubules associated tau protein. Orderly assembled microtubules and stabilization of the microtubule network are believed to serve an important role in signal transmission. In normal adult human brain, tau is phosphorylated at 2–3 moles phosphate per mole of tau protein. In AD brain tau is about three to four-fold more hyperphosphorylated than the normal adult brain tau112. Hyperphosphorylation of tau proteins causes the formation of neurofibrillary tangles through polymerization into paired helical filaments admixed with straight filaments which leads to disruption in neuronal cells. Inhibition of abnormal hyperphosphorylation of tau may provide a promising therapeutic target for AD and related tauopathologies112. Karakani et al investigated the inhibitory effects of crocin on aggregation of recombinant human tau protein 1N/4R isoform using both biochemical methods and cell culture113. Under conditions that would normally lead to fibrillation with tau protein, addition of crocin stabilizes the system by preventing fibrillation and lowering the tendency for aggregation. The suppression of forming tau protein filaments was confirmed by transmission electron microscopy images. In an in vitro study, crocin increased the orderly microtubule assembly in a dose dependent manner 114. Fluorescence spectroscopic data also pointed to significant conformational changes of tubulin as a consequence of crocin interaction. It was also observed that when crocin was present no aggregation of tau protein occurred at any concentrations studied. If this were to occur in neuronal cells, this effect would be beneficial. In an animal model study, Rashedinia et al115 investigated the effect of oral administration of crocin against acrolein induced tau-phosphorylation in the rat cerebral cortex. Acrolein, a byproduct of lipid peroxidation and toxic chemical, has been implicated in brain aging and in the pathogenesis of oxidative stress mediated neurodegenerative disorders such as AD. In AD brain tissue, acrolein, increased levels of Aβ, phosphorylated-tau, malondialdehyde (MDA) and decreased concentration of glutathione (GSH). They found co-administration of crocin significantly attenuated MDA, Aβ and phosphorylated-tau levels. The results demonstrated that crocin provided neuroprotection by reducing acrolein-induced Aβ, tau phosphorylation and oxidative stress.

375

CROCIN PROTECTIVE EFFECTS ON BRAIN CELLS/TISSUES

376 377 378 379 380 381 382 383 384 385 386

AD is characterized by the death of brain cells and loss of synaptic activity. Current thinking is that multiple risk factors beyond genetics play roles in the development of AD. Oxidative stress, infection, inflammation, environmental stress, brain injury, sleep disorder, hypertension, etc. are all considered risk factors for AD. Compelling evidence showed the impact of oxidative processes in AD pathogenesis100. The degree of oxidative stress was directly related to the advance of cognitive impairment and AD101, 116. Endoplasmic reticulum (ER) stress is a homeostatic mechanism, which is used by cells to adapt to intercellular and intracellular changes. ER stress is closely linked to neuroinflammation and contributes to the development of neurological diseases117. Pro-inflammatory and neurotoxic factors aiding neuronal damage and apoptosis are also produced by chronic microglial activation or brain injuries. Therefore, if crocin acts to reduce oxidative stress, ER stress and inflammation and provide neuroprotective


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387 388 389 390 391

activity, it could be beneficial for AD. In the past 20-25 years, saffron extract, crocin and crocetin have been evaluated and published by many researchers for the antioxidative, antiinflammatory and neuroprotective activities. Several studies have shown that crocin exhibited preventive effects on neuron/brain against oxidative stress, cell death/damage induced by internal and external apoptotic factors. Summaries of these studies are included in Table 3.

392

PROTECTIVE EFFECTS OF CROCIN IN THE HIPPOCAMPUS

393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415

In the early stages of AD the initial damage in a patient’s brain appears to take place in the hippocampus which is the part of the brain essential in learning and forming memories. As the disease progresses more neurons die, additional parts of the brain are impacted, and the brain begins to shrink. By the final stages of Alzheimer’s, damage is widespread, and brain tissue shows significant shrinkage. In an animal model utilizing streptozotocin-induced diabetic rats, Tamaddonfard et al94 demonstrated that crocin prevented the loss in numbers of hippocampal neurons in the diabetic rats. Learning and memory impairments and changes of blood total antioxidant capacity (TAC), MDA, glucose, and insulin levels induced by streptozotocin were improved in the rats with long-term intraperitoneal injections of crocin at doses of 15 and 30 mg/kg bw94. Crocin was also found effective in improving the impairment of learning and memory and preventing oxidative stress damage to the hippocampus region of the brain which was induced by chronic stress90. Aging is associated with diminished blood flow, this pathology is known as cerebral hypoperfusion and it is also a major cause of hippocampal damage, cognitive deficits and memory impairment. In a study with rat model for chronic cerebral hypoperfusion, the effects of crocetin on spatial learning and memory function were tested using Morris water maze, after permanent occlusion of common carotids. Histopathological changes in cerebral cortex and hippocampus were examined. Results demonstrated that neuronal damage in regions of hippocampus was widespread in the control ischemic group and treatment of crocetin (8 mg/kg) effectively protected cerebrocortical and hippocampus neurons against ischemia. Crocetin improved spatial learning memory in rats after chronic cerebral hypoperfusion95. It may be concluded that the antioxidant activities of crocin and crocetin could be significant factors responsible for their neuroprotective and learning and memory improving effects.

416

CROCIN EFFECTS ON BRAIN INJURY

417 418 419 420 421 422 423 424 425

Traumatic brain injury (TBI) is thought to lead to AD and similar conditions. Athletes, such as boxers and football players, are prone to repetitive TBI which results in greater incidences of neurodegenerative diseases including AD and Parkinson disease (PD). There is growing evidence that increased levels of Aβ and tau protein accumulate in human brains after traumatic brain injury 125-126. However, thus far there are no published studies demonstrating impact of crocin for inhibitory effects on brain injury induced Aβ42 or tau protein in humans. TBI leads to a complex neurodegenerative process, involving many cellular and molecular events, including neuronal inflammation and apoptosis. Wang et al studied the neuroprotective effects of crocin against traumatic brain injury in mice127. Mice were subjected to controlled cortical impact to



426 427 428 429 430 431 432 433 434 435 436 437

induce TBI. Pretreatment with crocin (20 mg/kg) which was administered 30min before TBI demonstrated that crocin was effective in reduction of TBI induced damage. The protective effects were demonstrated by improved neurological severity score (NSS) and brain edema, decreased microglial activation, release of several pro-inflammatory cytokines, and cell apoptosis. In another study, the protective role of crocetin following cerebral trauma and its effects on the enhancement of angiogenesis in rats were investigated128. The results indicated that neurological function of crocetin treated group as measured by modified Neurological Severity Scores showed significant recovery at seven days and fifteen days after the trauma. Crocetin inhibition of neuronal apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and electron microscopy as well as analysis of B cell lymphoma/leukemia-2 (Bcl-2) protein expression. These observations showed that crocetin has neuroprotective effects against brain injury.

438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465

Cerebral ischemia and TBI lead to brain damage by similar mechanisms including among others excitotoxicity, over production of free radicals, inflammation and apoptosis. It has been speculated that, neuroprotective compounds found to be active against one of these conditions may have protective effects in the other conditions129. As listed in Table 3, Zhang et al103 studied the protective effects of crocin with a mouse model for transient global ischemia. A pre-treatment with orally administered crocin markedly inhibited oxidizing reactions and to a large extent helped maintain the ultrastructure of cortical microvascular endothelial cells in mice after a 20 min of bilateral common carotid artery occlusion followed by 24 h of reperfusion. Crocin reversed the induced increase in MDA levels and the decreased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities. These results confirm the protective role of crocin in brain ischemia/reperfusion (I/R). Similarly, Oruc et al investigated the antioxidant, anti-apoptotic and protective effects of crocin on the global cerebral I/R injury induced by four-vessel occlusion in rats130. This technique consists of a two stage surgery within a 24-h interval that provides a more consistent reduction in cerebral blood flow and production of preconditioning effects. Subsequent to pre-treatment with crocin (40 mg/kg/day orally for 10 days) followed by I/R procedure, oxidative stress parameters and apoptotic parameters were measured. Results demonstrated that pre-treatment with crocin reduced the oxidative and apoptotic parameter levels back to the baseline values. Immunohistochemical staining of hippocampal CA1 regions of brain cornu ammon confirmed that crocin has neuroprotective and anti-apoptotic abilities. In another study, the protective effects of crocin in a rat I/R model was investigated with a single dose at different time of ischemia131. Transient focal cerebral ischemia was induced to rats by 60-minute middle cerebral artery occlusion (MCAO), followed by 23-hour reperfusion. Instead of a period of pre-treatment, crocin was only injected at the start of ischemia. Crocin at doses of 15, 30, 60, and 120 mg/kg were intraperitoneally injected at the start of ischemia. A single dose of crocin (60mg/kg) was also given 1 hour before, at the start or 1, 3, and 6 hours after ischemia to evaluate the therapeutic time window. Infarct volume and neurologic outcome were evaluated 24 hours after MCAO. Treatment with crocin at doses of 30, 60, and 120 mg/kg was found to significantly decrease infarct volume by 64%,


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74%, and 73%, respectively. Administration of crocin (60 mg/kg) 1 hour before, at the start, or 1 hour after ischemia reduced brain edema by 48%, 52%, and 51%, respectively. Results further revealed crocin (60 mg/kg) significantly reduced MDA levels and increased activity of SOD and GPx in the ischemic cortex. Similar studies and results of crocin protective effects were also reported by Sarshoori et al132. These studies showed that crocin could effectively reduce TBI and ischemia-induced damage and improve neurological outcome. Crocin, as a powerful antioxidant and neuroprotective factor, could be a potential agent to lower risks of developing neurodegenerative disorders induced by TBI or ischemic stroke.

474 475

CLINICAL STUDIES OF SAFFRON AND CROCIN

476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504

Saffron as the major source of crocin was reported in a few clinical studies and found comparable to AChEI drug donepezil and NMDA receptor blocker drug memantine in treating patients with AD or mild cognitive impairment. In a 22-week, multicenter, randomized, doubleblind controlled clinical study133, saffron at 30 mg/day (15 mg twice daily) or donepezil (10mg/day) was administered to 54 patients of age 55 years and older with mild-to-moderate AD. Patients were assayed by Alzheimer’s Disease Assessment Scale-cognitive subscale (ADAScog) and Clinical Dementia Rating Scale-Sums of Boxes scores. Saffron was found as effective as donepezil in the treatment of patients with mild-to-moderate AD. Frequency of saffron extract side effects was similar to those of donepezil; however patients in saffron extract group experienced less frequent vomiting than those in donepezil group. The efficacy and safety of saffron were also compared with memantine in a randomized double-blind parallel-group study134. In this trial, 68 patients with moderate to severe AD received memantine (20 mg/day) or saffron extract (30mg/day) capsules for 12 months. Participants were evaluated every month by Severe Cognitive Impairment Rating Scale (SCIRS) and Functional Assessment Staging (FAST), along with recording of probable adverse events. Results revealed that saffron treatment (30mg/day) was as effective as memantine in prevention of cognitive decline in patients with moderate to severe AD. There were no significant differences in adverse effects between the two treatments. In another 16 weeks study135, 46 patients with mild-to-moderate AD were randomly assigned to receive saffron 30 mg/day (15 mg/capsule, twice per day) or placebo (two capsules per day). Global cognitive and clinical profiles of the patients were evaluated by psychometric measures, including ADAS-cog, and clinical dementia rating scale-sums of boxes. The results showed that the cognitive functions in saffron-treated group were significantly improved compared to the placebo and there was no significant difference in adverse effects between the two groups. Another one-year efficacy and safety study of saffron in patients with mild cognitive impairment was recently reported136. In this one-year study, 17 of 35 patients were assigned to saffron group and 18 were in a parallel control group. Participants were examined with a short neuropsychological battery, 3Tesla Magnetic Imaging (MRI 3T), while some patients were examined via 256-channel electroencephalogram (HD-EEG) at baseline and after 12 months. Results showed that patients on saffron improved cognitive impairment



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505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524

measured by Mini-Mental State Examination scores (p = 0.015), while the control group continued deterioration. Additionally, improvement in specific domains was also observed by MRI, EEG and event-related potentials (ERP) in the saffron group. These findings demonstrated that saffron could be effective in improvement of mild cognitive impairment and AD. In traditional medicine, combinations of a few herbal ingredients are frequently used. One standardized herbal formulation Sailuotong (SLT) containing saffron based crocin, ginseng and ginkgo was evaluated in a one-week study for its efficacy of improving memory in healthy adults137. In this one week randomised, double blind, placebo controlled crossover pilot trial, 16 participants were treated with a daily dose of 10.92 mg of crocins from saffron, 54.54 mg of ginsenosides from Panax ginseng, and 27.27 mg total ginkgo flavone-glycosides from Ginkgo biloba. Neurocognitive and cardiovascular function were measured before and after each of the interventions, using a Computerised Mental Performance Assessment System (Compass) neuropsychological test battery which measured a range of cognitive abilities including attention, episodic memory, and working memory as well as cardiovascular measures. In comparison to placebo, SLT resulted in a trend towards improvements of alphabetic working memory and visual working memory in the one week intervention, while no significant changes of the cardiovascular parameters were detected in these healthy adults. Thus, crocin or saffron could be potentially applied alone or in combination with other herbal ingredients or drugs for improvement of memory and cognitive deficits or AD. Clinical studies of saffron or crocin on improvement of memory or cognition were summarized and listed in Table 4.

525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544

In our search, although pure crocin has not been found directly evaluated in clinical studies to treat AD, three clinical studies reported the effects of crocin on prooxidant/antioxidant balance (PAB) in patients with metabolic syndrome, on prevention of metabolic syndrome in patients with Schizophrenia on Olanzapine treatment, and on anti-depression in patients with major depressive disorders65, 138, 139. In the randomized, placebo-controlled clinical trial to evaluate the influence of crocin on PAB in patients with metabolic syndrome, 60 participants were randomly assigned to two groups of 30 subjects as the intervention group (15mg crocin twice a day) or control group (given placebo) for 8 weeks. Blood samples were taken before and after the intervention period and assayed for PAB. Results showed that mean serum PAB fell by 11.7% in the intervention group and no significant change of serum PAB in the control group. This study demonstrated crocin ingestion at a dose of 30 mg/d significantly reduced oxidative stress in individuals with metabolic syndrome138. In the randomized triple blind placebo controlled study to evaluate effects of saffron extract (SEA) and crocin on prevention of antipsychotics drug olanzapine induced metabolic syndrome65, SEA (30mg/day) and crocin (30mg/day) mitigated olanzapine induced metabolic syndrome compared to placebo and prevented an increase in FBS during the 12 weeks of treatment. In the randomized, double-blind, placebocontrolled, pilot clinical trial using crocin as an adjunctive treatment for major depressive disorders139, 40 major depressive disorder patients between 24 and 50 years old were divided into two groups which were treated for 4 weeks. Crocin group (n= 20) received one selective serotonin reuptake inhibitor (SSRI) drug (fluoxetine 20mg/day or sertraline 50mg/day or


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545 546 547 548 549 550 551 552 553 554 555 556 557 558

citalopram 20mg/day) plus crocin tablets (15mg twice a day); the placebo group (n= 20) was administered one SSRI (fluoxetine 20mg/day or sertraline 50mg/day or citalopram 20 mg/day) plus placebo (two placebo tablets per day). Both groups were evaluated by Beck depression inventory (BDI), Beck anxiety inventory (BAI), general health questionnaire (GHQ), the mood disorder questionnaire (MDQ), side effect evaluation questionnaire, and demographic questionnaire before and after the one month intervention. Results showed crocin group presented significantly improved scores on BDI, BAI and GHQ compared to placebo group. No serious side effects were reported with crocin consumption. The averages of decrease in BDI, BAI and GHQ scores in placebo group were 6.15, 2.6 and 10.3 respectively, whereas the values in crocin group were 17.6, 12.7 and 17.2. The results suggested crocin could be effective as adjunctive treatment for major depressive disorder139. As it is known that oxidative damage progressively increases with age and a high ratio of AD patients suffers from significant depression, the multi-functions of crocin could be particularly effective in preventing and treating brain aging and AD.

559

DISCUSSION

560 561 562 563 564 565 566 567

The progression of AD involves many factors including cholinesterase activity and Aβ and tau proteins. Current therapies with AChE inhibitors and NMDA glutamate receptor blocker only provide modest benefits on delaying or preventing symptoms of AD from progressing for a limited time frame. Recent research targeting Aβ and tau protein with immunotherapies for treatment of AD is promising. However, the efficacy on improving cognition so far has not been validated in human studies. In pursuing treatment and prevention of AD, new strategy should be developed to address the complicate factors that influence the degeneration of neurons and the progression of AD.

568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583

Natural alternatives which exhibit beneficial effects to multiple targets and pathways could be valuable options and applied in conjunction with drug therapies for AD prevention and management140. Studies presented in this review provided evidence that saffron and particularly crocin could exert positive effects in delaying progression of the disease 80,140. More importantly, crocin appears to be multifunctional in protecting brain cells, modulating aggregation of Aβ and tau protein, and attenuating cognitive and memory impairments. As a water soluble carotenoid with relatively safe profile, crocin could be particularly beneficial as it also exhibited supportive effects in improving depression, anxiety and oxidative stress, which are symptoms frequently associated with AD patients. So far, the number of saffron clinical studies in the treatment of AD is limited and the patient number involved in these studies is also relative small. But findings from animal studies of crocin as well as the clinical studies of saffron support further studies of crocin as a promising natural alternative in prevention and treatment of AD. Clinical studies of crocin at different doses by itself or in combination with other AD beneficial natural ingredients or chemical drugs, including donepezil and memantine, should be conducted. Results from these crocin studies will potentially benefit the battle against AD through either crocin supplement or drug development.



REFERENCES 1. Reitz, C.; Brayne, C.; Mayeux, R. Epidemiology of Alzheimer disease. Nat Rev Neurol 2011, 7: 137-152. 2. Hsiung, G. Y. R.; Feldman, H. H. Pharmacological treatment in moderate-to-severe Alzheimer’s disease. Expert Opin Pharmaco 2008, 9: 2575-2582. 3. Rodda, J.; Walker, Z. Ten years of cholinesterase inhibitors. Int. J. Geriatr Psych. 2009, 24: 437-442. 4. Nygaard, H. B. Current and Emerging Therapies for Alzheimer’s Disease. Clin Ther 2013, 35: 1480-1489. 5. Forchetti, C. M. Treating patients with moderate to severe Alzheimer’s Disease: implications of recent pharmacologic studies. Primary Care Comp. J. Clin Psych 2005 7(4): 155161. 6. Nisbet, R. M.; Polanco, J. C.; Ittner, L. M.; Götz, J. Tau aggregation and its interplay with amyloid-β. Acta Neuropathol 2015, 129: 207-220. 7. Mondragón-Rodríguez, S.; Perry, G.; Zhu, X.; Boehm, J. Amyloid Beta and Tau Proteins as Therapeutic Targets for Alzheimer’s Disease Treatment: Rethinking the Current Strategy. Intl J Alzheimer's Dis 2012, Article ID 630182, 7 pages ttp://dx.doi.org/10.1155/2012/630182. 8. Howes, M. J.; Perry, E. The role of phytochemicals in the treatment and prevention of dementia. Drugs Aging 2011, 28: 439-468. 9. Mousavi, S. Z.; Bathaie, S. Z. Historical uses of saffron: Identifying potential new avenues for modern research. Avicenna J Phytomed 2011, 1: 27-66. 10.

Basker, D.; Negbi, M. Uses of saffron. J Econ Bot 1983, 37 (2): 228-236.

11. Abdullaev, F. I. Cancer Chemopreventive and Tumoricidal Properties of Saffron (Crocus sativus L. Exp Biol Med (Maywood) 2002, 227: 20-5. 12. Bathaie, S. Z.; Mousavi, S. Z. New applications and mechanisms of action of saffron and its important ingredients. Crit Rev Food Sci Nutr. 2010, 50: 761-86. 13. Hosseinzadeh, H.; Nassiri-Asl M. Avicenna’s (Ibn Sina) the Canon of Medicine and Saffron (Crocus sativus): A Review. Phytother Res 2013, 27: 475-483. 14. Willard, P. Secrets of Saffron: The Vagabond Life of the World's Most Seductive Spice, Beacon Press (published 11 April 2002), ISBN 978-0-8070-5009-5 15. Melnyk, J. P.; Wang, S.; Marcone, M. F. Chemical and biological properties of the world's most expensive spice: Saffron. Food Res Int 2010, 43: 1981-1989.


Page 18 of 41

Page 19 of 41


16. Nemati, H.; Boskabady, M. H.; Ahmadzadef Vostakolaei, H. Stimulatory effect of crocus sativus (saffron) on beta2-adrenoceptors of guinea pig tracheal chains. Phytomed 2008, 15: 1038-1045. 17.

Abdullaev, F. I. (1993) Biological effects of Safron. BioFactors 1993, 4: 83-86.

18. Asdaq, S. M. B.; Inamdar, M. N. (2010). Potential of crocus sativus (saffron) and its constituent, crocin, as hypolipidemic and antioxidant in rats. Appl Bioch Biotech 2010 162: 358372. 19. Nam, K. N.; Park, Y. M.; Jung, H. J.; Lee, J. Y.; Min, B. D.; Park, S. U.; Jung, W. S.; Cho, K. H.; Park, J. H.; Kang, I.; Hong, J.W.; Lee, E. H. Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells. Eur J Pharmacol. 2010, 648: 110-116. 20. Rios, J. L.; Recio, M. C.; Giner, R. M.; Manez, S.(1996) An update review of saffron and its active constituents. Phytother Res 1996, 10:189-193. 21. Abe, K.; Saito, H. Effects of saffron extract and its constituent crocin on learning behaviour and long-term potentiation. Phytother Res 2000, 14:149-152. 22. Soeda, S.; Ochiai, T.; Paopong, L.; Tanaka, H.; Shoyama, Y.; Shimeno, H. Crocin suppresses tumor necrosis factor-alpha-induced cell death of neuronally differentiated PC-12 cells. Life Sci 2001 69:2887-2898. 23. Bhargava, V. K. Medicinal uses and pharmacological properties of crocus sativus Linn. (saffron). Int. J Pharm Pharm Sci 2011, 3: 22-26. 24. Hosseinzadeh, H.; Sadeghnia, H. R.; Ghaeni, F. A.; Motamedshariaty, V. S.; Mohajeri, S. A. Effects of Saffron (Crocus sativus L.) and its Active Constituent, Crocin, on Recognition and Spatial Memory after Chronic Cerebral Hypoperfusion in Rats. Phytother Res 2012, 26: 381– 386. 25. Johnson, I. T. IARC handbooks of cancer prevention volume 2: carotenoids and volume 3: vitamin A. Eur J Clin Nutr. 1999, 53: 830-4. 26. Liakopoulou-Kyriakides, M.; Kyriakidis, D. Crocus sativus-biological active constituents. Stud. Nat Prod Chem 2002, 26: 293-312. 27. Alonso, G. L.; Salinas, M. R.; Garijo, J.; Sánchez-Fernández, M. A. Composition of crocins and picrocrocin from Spanish saffron (Crocus sativus L.). J Food Qual 2001, 24: 219-233. 28. Caballero-Ortega, H.; Salinas, M. R.; Garijo, J.; Sanchez-Fernandez, M. A. Composition of Crocins and Picocrocin from Spanish Saffron (Crocus sativus L.). J. Food Qual 2004, 24:219-233. 29. Caballero-Ortega, H.; Pereda-Miranda, R.; Abdullaev, F. I. HPLC quantification of major active components from 11 different saffron (Crocus sativus L.) sources. Food Chem 2007, 100: 1126-1131.



30. Lage, M.; Cantrell, C. L. Quantification of saffron (Crocus sativus L.) metabolites crocins, picrocrocin and safranal for quality determination of the spice grown under different environmental Moroccan conditions. Scientia Hortic-Amsterdam 2009, 121: 366-373. 31. Alonso, G. L.; Carmona, M.; Zalacain, A.; Gonzalez, L. V.; Gonzalez, M. L.; Sarasa-Delgado, F. (1999). Study of saffron adulteration by increasing its colouring strength. In: Proceedings of 1st International Congress PFT Pigments in Food Technology, Sevilla, Spain 1999, 24-26, pp. 341-346. 32. Pfander, H.; Wittwer, F. Carotenoid composition in safran. Helv. Chim Acta 1975, 58: 2233-2236. 33. Li, N.; Lin, G.; Kwan, Y. W.; Min, Z. D. Simultaneous quantification of five major biologically active ingredients of saffron by high-performance liquid chromatography. J Chromatogr A 1999, 849: 349-355. 34. Dhingra, V.; Seshadri, T.; Mukerjee, S. Minor carotenoid glycosides from saffron (Crocus sativus). Ind J Chem 1975, 13: 339-341. 35. Speranza, G.; Dada, G.; Manitto, P.; Monti, D.; Gramatica, P. 13-cis-crocin: a new crocinoid of saffron. Gazz. Chim Ital 1984, 114: 189-192. 36. Tarantilis, P. A; Tsoupras, G.; Polissiou, M. Determination of saffron (Crocus sativus L.) components in crude plant extract using high-performance liquid chromatography-UV-visible photodiode-array detection-mass spectrometry. J Chromatogr A. 1995, 699: 107-118. 37.

fao.org/ag/agn/jecfa-additives/specs/Monograph1/Additive-381.pdf

38. Asadi, F.; Abdollahi, M.; Ghahremani, M. H.; Jamshidi, A. H.; Khodagholi, F.; Yans, A.; Azimi, L.; Faizi, M.; Vali, L.; Sharifzadeh, M. Reversal effects of crocin on amyloid β-induced memory deficit: Modification of autophagy or apoptosis markers. Pharmacol Biochem Behavior 2015 139(Pt A): 47-58. 39. Khajuria, D. K.; Asad, M.; Asdaq, S. M. B.; Kumar, P. The Potency of Crocus sativus (Saffron) and its Constituent Crocin as an Immunomodulator in Animals. Lat Am J Pharm 2010, 29: 713-8. 40. Konoshima, T.; Takasaki, M.; Tokuda, H.; Morimoto, S.; Tanaka, H.; Kawata, E.; Xuan, L. J.; Saito, H.; Sugiura, M.; Molnar, J.; Shoyama, Y. Crocin and Crocetin Derivatives Inhibit Skin Tumour Promotion in Mice. Phytother Res 1998, 12: 400–404. 41. Kawabata, K.; Tung, N. H.; Shoyama, Y.; Sugie, S.; Mori, T.; Tanaka, T. Dietary Crocin Inhibits Colitis and Colitis-Associated Colorectal Carcinogenesis in Male ICR Mice. EvidenceBased Complement Alter Med 2012, Article ID 820415, doi:10.1155/2012/820415.


Page 20 of 41

Page 21 of 41


42. Jnaneshwari, S.; Hemshekhar, M.; Santhosh, M. S.; Sunitha, K.; Thushara, R.; Thirunavukkarasu, C.; Kemparaju, K.; Girish, K. S. Crocin, a dietary colorant mitigates cyclophosphamide-induced organ toxicity by modulating antioxidant status and inflammatory cytokines. J. Pharm. Pharmacol. 2013, 65: 604-614. 43. Tanaka, T.; Mori, T.; Yuan, C. S.; Soed, S.; Tung, N. H.; Shoyama, Y. The Function of Saffron and its Constituent in Gastroenterological Tissues. Global J Gastroenterol Hepatol 2015, 3: 31-38. 44. Jin, Y. Y.; Zhang, J. S.; Zhang, Y.; Zhang, Y. H. Studies on the intestinal absorption of crocin in rats and determination of the partition coefficient. J China Pharmac Univ. 2004, 35:283-284. 45. Hirota, S.; Takahama, U. Starch can inhibit nitrite dependent oxidation of crocin in gastric lumen increasing bioavailability of carotenoids. Food Sci Technol Res 2013, 19:11211126. 46. Xi, L.; Qian, Z.; Du, P.; Fu, J. Pharmacokinetic properties of crocin (crocetin digentiobiose ester) following oral administration in rats. Phytomed 2007, 14: 633-636. 47. Zhang, Y.; Liu, J. X.; Lin, L.; Li, L. Q. Pharmacokinetics of crocin-1 after oral administration in rats. Chin Pharm J 2012, 47: 136-140. 48. Li, X. Y.; Feng, W. L.; Zhu, J. B.; Nima, C. R.; Ge, R. L. Effect of Tibetan medicine Zuotai on in vivo pharmacokinetics of crocin-1 in rats. Zhong Cao Yao 2009, 40: 1425-1428. 49. Asai, A.; Nakano, T.; Takahashi, M.; Nagao A. Orally administered crocetin and crocins are absorbed into blood plasma as crocetin and its glucuronide conjugates in mice. J Agr Food Chem 2005, 53: 7302-7306. 50. Chryssanthi, D. G.; Lamari, F. N.; Georgakopoulos, C. D.; Cordopatis, P. A new validated SPE-HPLC method for monitoring crocetin in human plasma-application after saffron tea consumption. J Pharm Biomed Anal. 2011, 55: 563-568. 51. Umigai, N.; Murakami, K.; Ulit, M. V.; Antonio, L. S.; Shirotori, M.; Morikawa, H.; Nakano, T. The pharmacokinetic profile of crocetin in healthy adult human volunteers after a single oral administration. Phytomed 2011, 18: 575-578. 52. Lautenschläger, M.; Sendker, J.; Hüwel, S.; Galla, H. J.; Brandt, S.; Düfer, M.; Riehemann, K.; Hensel, A. Intestinal formation of trans-crocetin from saffron extract (CrocussativusL.) and in vitro permeation through intestinal and blood brain barrier. Phytomed 2015, 22: 36–44. 53. Yoshino, F.; Yoshida, A.; Umigai, N.; Kubo, K.; Lee, M. C. Crocetin reduces the oxidative stress induced reactive oxygen species in the stroke-prone spontaneously hypertensive rats (SHRSPs) brain. J Clin Biochem Nutr. 2011, 49: 182–187.



54. Ochiai, T.; Shimeno, H.; Mishima, K.; Iwasaki, K.; Fujiwara, M.; Tanaka, H.; Shoyama, Y.; Toda, A.; Eyanagi, R.; Soeda, S. Protective effects of carotenoids from saffron on neuronal injury in vitro and in vivo. Biochim Biophys Acta 2007, 1770: 578-584. Moshiri, M.; Vahabzadeh, M.; Hosseinzadeh, H. Clinical Applications of Saffron (Crocus 55. sativus) and its Constituents: A Review. Drug Res (Stuttg), 2015, 65: 287-295. 56. Alavizadeh, S. H.; Hosseinzadeh, H. Bioactivity assessment and toxicity of crocin: A comprehensive review. Food Chem. Tox. 2014, 64: 65-80. 57. Milajerdi, A.; Djafarian, K.; Hosseini, B. The toxicity of saffron (Crocus sativus L.) and its constitutes against normal and cancer cells. J Nutr Interm Metab 2016, 3:23-32. 58. Bolhassani, A.; Khavari, A.; Bathaie, S. Z. Saffron and natural carotenoids: Biochemical activities and anti-tumor effects. Biochim Biophys Acta 2014, 1845:1:20-30. 59. WHO Monographs on Selected Medicinal Plants, World Health Organization, 2007, 3: 126–135. 60. Modaghegh, M. H.; Shahabian, M.; Esmaelli, H. A.; Rajbai, O.; Hosseinzadeh, H. Safety evaluation of saffron (Crocus sativus) tablets in healthy volunteers. Phytomed 2008, 15: 10321037. 61. Ayatollahi, H.; Javan, A. O.; Khajedaluee, M.; Shahroodian, M.; Hosseinzadeh, H. Effect of Crocus sativus L.(Saffron) on Coagulation and Anticoagulation Systems in Healthy Volunteers. Phytother Res 2014, 28: 539-43. 62. Hosseinzadeh, H.; Jahanian, Z. Effect of Crocus sativus L. (saffron) stigma and its constituents, crocin and safranal, on morphine withdrawal syndrome in mice. Phytother Res 2010, 24: 726-30. 63. Mohamadpour, A. H.; Ayati, Z.; Parizadeh, M. R.; Rajbai, O.; Hosseinzadeh, H. Safety Evaluation of Crocin (a constituent of saffron) Tablets in Healthy Volunteers. Iran J Basic Med Sci. 2013, 16: 39-46. 64. Mousavi, B.; Bathaie, S. Z.; Fadai, F.; Ashtari, Z.; Alibeigi, N.; Farhang, S.; Hashempour, S.; Shahhamzei, N.; Heidarzadeh, H. Safety evaluation of saffron stigma (Crocus sativus L.) aqueous extract and crocin in patients with schizophrenia. Avicenna J Phytomed. 2015, 5: 413-419. 65. Fadai, F.; Mousavi, B.; Ashtari, Z.; Ali beige, N.; Farhang, S.; Hashempour, S.; Shahhamzei, N.; Bathaie, S. Z. Saffron Aqueous Extract Prevents Metabolic Syndrome in Patients with Schizophrenia on Olanzapine Treatment: A Randomized Triple Blind Placebo Controlled Study. Pharmacopsychiatry 2014, 47: 156-161.


Page 22 of 41

Page 23 of 41


66. Khazdair, M. R.; Boskabady, M. H.; Hosseini, M.; Rezaee, R. M.; Tsatsakis, A. The effects of Crocus sativus (saffron) and its constituents on nervous system: A review. Avicenna J Phytomed 2015, 5: 376-391. 67. Terry, A. V. Jr; Buccafusco, J. J. The cholinergic hypothesis of age and Alzheimer's disease-related cognitive deficits: recent challenges and their implications for novel drug development. J Pharmacol Exp Ther. 2003, 306: 821-7. Epub 2003 Jun 12. Review. 68. Geromichalos, G. D.; Lamari, F. N.; Papandreou, M. A.; Trafalis, D. T.; Margarity, M.; Papageorgiou, A.; Sinakos, Z. Saffron as a source of novel acetylcholinesterase inhibitors: molecular docking and in vitro enzymatic studies. J. Agr Food Chem. 2012, 60: 6131-6138. 69. Massoud, F.; Leger, G. C. Pharmacological treatment of Alzheimer disease. Can. J. Psychiatry 2011, 56: 579-588. 70. Wallin, A. K.; Wattmo, C.; Minthon, L. Galanthamine treatment in Alzheimer’s disease: response and long-term outcome in a routine clinical setting. Neuropsychiatr Dis Treat 2011, 7: 565-576. 71. Lockhart, I. A.; Mitchell, S. A.; Kelly, S. Safety and tolerability of donepezil, rivastigmine and galantamine for patients with Alzheimer’s disease: systematic review of the “real world” evidence. Dement Geriatr Cogn Disord 2009, 28: 389-403. Rao, S. V.; Muralidhara; Yenisetti, S. C.; Rajini, P. S. Evidence of neuroprotective effects 72. of saffron and crocin in a Drosophila model of parkinsonism. NeuroToxicology 2016, 52: 230242. 73. Parsons, C. G.; Stöffler, A.; Danysz, W. Memantine: a NMDA receptor antagonist that improves memory by restoration of homeostasis in the glutamatergic system - too little activation is bad, too much is even worse. Neuropharmacol 2007, 53: 699-723. 74. Lechtenberg, M.; Schepmann, D.; Niehues, M.; Hellen- brand, N.; Wunsch, B.;Hensel, A. Quality and Function- ality of Saffron: Quality Control, Species Assortment and Affinity of Extract and Isolated Saffron Compounds to NMDA and U1 (Sigma-1) Receptors. Planta Medica 2008, 74: 764-772. 75. Abe, K.; Sugiura, M.; Shoyama, Y.; Saito, H. Crocin antagonizes ethanol inhibition of NMDA receptor-mediated responses in rat hippocampal neurons. Brain Res. 1998, 787: 132138. 76. Sugiura, M.; Shoyama, Y.; Saito, H.; Abe, K. Crocin (Crocetin Di-Gentiobiose Ester) Prevents the Inhibitory Effect of Ethanol on Long-Term Potentiation in the Dentate Gyrus in Vivo J Pharm Exp Ther 1994, 271: 703-707.



77. Sugiura, M.; Shoyama, Y.; Saito, H.; Nishiyama, N. Crocin improves the ethanol-induced impairment of learning behaviors of mice in passive avoidance tasks. Proc Jpn. Acad. 1995, 71: 319-24. 78. Georgiadou, G.; Grivas, V.; Tarantilis, P.; Pitsikas, N. Crocins, the active constituents of Crocus Sativus L., counteracted ketamine-induced behavioural deficits in rats. Psychopharmacol 2014, 231: 717. 79. Soeda, S.; Ochiai, T.; Shimeno, H.; Saito, H.; Abe, K.; Tanaka, H.; Shoyama, Y. Pharmacological activities of crocin in saffron. J Nat Med 2007, 61: 102-111. Pitsikas, N. The Effect of Crocus sativus L. and Its Constituents on Memory: Basic Studies 80. and Clinical Applications. Evidence-Based Complementary and Alternative Medicine 2015, 2015, Article ID 926284, 7 pages. 81. Zhang, Y.; Shoyama, Y.; Sugiura, M.; Saito, H. Effects of Crocus sativus L. on the ethanolinduced impairment of passive avoidance performances in mice. Biol Pharm Bull. 1994, 17: 217221. 82. Sugiura, M.; Shoyama, Y.; Saito, H.; Abe, K. The effects of ethanol and crocin on the induction of long-term potentiation in the CA1 region of rat hippocampal slices, Jpn J Pharmacol. 1995, 67: 395-397. 83. Sugiura, M.; Shoyama, Y.; Saito, H.; Abe, K. Ethanol extract of Crocus sativus L. antagonizes the inhibitory action of ethanol on hippocampal long-term potentiation in vivo, Phytother Res 1995, 9: 100–104. 84. Abe, K.; Sugiura, M.; Yamaguchi, S.; Shoyama, Y.; Saito, H. Saffron extract prevents acetaldehyde-induced inhibition of long-term potentiation in the rat dentate gyrus in vivo. Brain Res. 1999, 851: 287-9. 85. Hosseinzadeh, H.; Ziaei, T. Effects of Crocus sativus stigma extract and its constituents, crosin and safranal, on intact memory and scopolamine -indused learning deficits in rats performing the Morris water maze task. J Med Plants 2006, 5: 40-50. 86. Pitsikas, N.; Zisopoulou, S.; Tarantilis, P. A.; Kanakis, C. D.; Polissiou, M. G.; Sakellaridis, N. Effects of the active constituents of Crocus sativus L., crocins on recognition and spatial rats’ memory. Behavioural Brain Research 2007, 183: 141-146. 87. Ghadami, M. R.; Pourmotabbed, A. The effect of Crocin on scopolamine induced spatial learning and memory deficits in rats. Physiol Pharmacol 2009, 12: 287-295. 88. Khalili, M.; Hamzeh, F. Effects of active constituents of Crocus sativus L., crocin on streptozocin-induced model of sporadic Alzheimer's disease in male rats. Iran Biomed J. 2010, 14: 59-65.


Page 24 of 41

Page 25 of 41


89. Papandreou, M. A.; Tsachaki, M.; Efthimiopoulos, S.; Cordopatis, P.; Lamari, F.; Margarity, M. Memory enhancing effects of saffron in aged mice are correlated with antioxidant protection. Behavioural Brain Res 2011, 219: 197-204. 90. Ghadrdoost, B.; Vafaei, A. A.; Rashidy-Pour, A.; Hajisoltani, R.; Bandegi, A. R.; Motamedi, F.; Haghighi, S.; Sameni, H. R.; Pahlvan, S. Protective effects of saffron extract and its active constituent crocin against oxidative stress and spatial learning and memory deficits induced by chronic stress in rats. Eur J Pharmacol. 2011, 667: 222-9. 91. Khan, M. B.; Hoda, M. N.; Ishrat, T.; Ahmad, S.; Moshahid Khan, M.; Ahmad, A.; Yusuf, S.; Islam, F. Neuroprotective efficacy of Nardostachys jatamansi and crocetin in conjunction with selenium in cognitive impairment. Neurol Sci. 2012, 33: 1011-20. 92. Naghibi, S. M.; Hosseini, M.; Khani, F.; Rahimi, M.; Vafaee, F.; Rakhshandeh, H.; Aghaie, A. Effect of Aqueous Extract of Crocus sativus L. on Morphine-InducedMemory Impairment. Adv Pharmacol Sci 2012, Article ID 494367, 7 pages, doi:10.1155/2012/494367. 93. Dashti-r, M. H.; Zeinalib, F.; Anvaric, M.; Hosseinid, S. M. Saffron (Crocus sativus L.) Extract prevents and improves D-galactose and NaNO2 induced memory impairment in mice. EXCLI Journal 2012, 11: 328-337. 94. Tamaddonfard, E.; Farshid, A. A.; Asri-Rezaee, S.; Javadi, S.; Khosravi, V.; Rahman, B.; Mirfakhraee, Z. Crocin Improved Learning and Memory Impairments in Streptozotocin-Induced Diabetic Rats. Iran J Basic Med Sci. 2013, 16: 91-100. 95. Tashakori-Sabzevar, F.; Hosseinzadeh, H.; Motamedshariaty, V. S.; Movassaghi, A. R.; Mohajeri, S. A. Crocetin Attenuates Spatial Learning Dysfunction and Hippocampal Injury in a Model of Vascular Dementia. Curr Neurovasc Res 2013, 10(4): 325-334. 96. Naghizadeh, B.; Mansouri, M. T.; Ghorbanzadeh, B.; Farbood, Y.; Sarkaki, A. Protective effects of oral crocin against intracerebroventricular streptozotocin-induced spatial memory deficit and oxidative stress in rats. Phytomed 2013, 20: 537-542. 97. Linardaki, Z. I.; Orkoula, M. G.; Kokkosis, A. G.; Lamari, F. N.; Margarity, M. Investigation of the neuroprotective action of saffron (Crocus sativus L.) in aluminum-exposed adult mice through behavioral and neurobiochemical assessment. Food Chem Toxicol. 2013, 52: 163-70. 98. Sreenu, G.; Banala, R. R.; Reddy, K. P. Saffron Extract’s Protective Effects against Arsenic Induced Excitotoxicity and Learning Disabilities in Male Wistar Rats. Intl J Bioassay 2015, 4: 4223-4229. 99. Ghaffari, S.; Hatami, H.; Dehghan, G. Saffron ethanolic extract attenuates oxidative stress, spatial learning, and memory impairments induced by local injection of ethidium bromide. Res Pharm Sci. 2015, 10: 222-232.



100. Perry, G.; Cash, A. D.; Smith, M. A. Alzheimer Disease and Oxidative Stress. J. Biomed Biotech 2002, 2: 120-123. 101. Sekler, A.; Jiménez, J. M.; Rojo, L.; Pastene, E.; Fuentes, P.; Slachevsky, A.; Maccioni, R. B. Cognitive impairment and Alzheimer’s disease: Links with oxidative stress and cholesterol metabolism. Neuropsych Dis Treat 2008, 4:715-722. 102. Papandreou, M. A.; Kanakis, C. D.; Polissiou, M. G.; Efthimiopoulos, S.; Cordopatis, P.; Margarito, M.; Lamari, F. N. Inhibitory activity on amyloid-beta aggregation and antioxidant properties of Crocus sativus stigmas extract and its crocin constituents. J Agr Food Chem. 2006, 54 : 8762-8. 103. Zheng, Y. Q.; Liu, J. X.; Wang, J. N.; Xu, L. Effects of crocin on reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia. Brain Res 2007, 1138: 86-94. 104. Hosseinzadeh, H.; Shamsaie, F.; Mehri, S. Antioxidant activity of aqueous and ethanolic extracts of Crocus sativus L. stigma and its bioactive constituents, crocin and safranal. Phcog Mag. 2009, 5, Suppl S1:419-24. 105. Ordoudi, S. A.; Befani, C. D.; Nenadis, N.; Koliakos, G. G.; Tsimidou, M. Z. Further examination of antiradical properties of Crocus sativus stigmas extract rich in crocins. J. Agr Food Chem. 2009, 57, 3080−3086. 106. Ochiai, T.; Ohno, S.; Soeda, S.; Tanaka, H.; Shoyama, Y.; Shimeno, H. Crocin prevents the death of rat pheochromyctoma (PC-12) cells by its antioxidant effects stronger than those of atocopherol. Neurosci Let 2004, 362: 61-64. 107. Ghahghaei , A.; Bathaie, Z.; Bahraminejad, E. Mechanisms of the Effects of Crocin on Aggregation and Deposition of Ab1–40 Fibrils in Alzheimer’s Disease. Int. J Pept Res Ther. 2012 18: 347-351. 108. Ghahghaei, A.; Bathaie, S. Z.; Kheirkhah, H.; Bahraminejad, E. The protective effect of crocin on the amyloid fibril Formation of aβ42 peptide in vitro. Cell Molec Biol. 2013, 18: 328339. 109. Ahn, J. H.; Hu, Y.; Hernandez, M.; Kim, J. R. Crocetin inhibits beta-amyloid fibrillization and stabilizes beta-amyloid oligomers. Biochem Biophy Res Com. 2011, 414: 79-83. 110. Yoshino, Y.; Ishisaka, M.; Umigai, N.; Shimazawa, M.; Tsuruma, K.; Hara, H. Crocetin Prevents Amyloid β1-42-Induced Cell Death in Murine Hippocampal Cells. Pharmacology Pharm. 2014, 5: 37-42. 111. Morelli, S.; Salerno, S.; Piscioneri, A.; Tasselli, F.; Drioli, E.; De Bartolo, L. Neuronal membrane bioreactor as a tool for testing crocin neuroprotective effect in Alzheimer's disease.


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Chemical Engineering Journal 2016, Available online 22 January, http://dx.doi.org/10.1016/j.cej.2016.01.035 112. Iqbal, K.; Liu, F.; Gong, C. X.; Grundke-Iqbal, I. Tau in Alzheimer Disease and Related Tauopathies. Curr Alzheimer Res. 2010, 7: 656-664. 113. Karakani, A. M.; Riazi, G.; Ghaffari, S. M.; Ahmadian, S.; Mokhtari, F.; Firuzi, M. J.; Bathaie, S. Z. Inhibitory effect of corcin on aggregation of 1N/4R human tau protein in vitro. Iranian J Basic Med Sci. 2015, 18: 485-492. 114. Jaliani, H. Z.; Riazi, G. H.; Ghaffari, S. M.; Karima, O.; Rahmani, A. The Effect of the Crocus Sativus L. Carotenoid, Crocin, on the Polymerization of Microtubules, in Vitro. Iran J Basic Med Sci. 2013, 16: 101-107. 115. Rashedinia, M.; Lari, P.; Abnous, K.; Hosseinzadeh, H. Protective effect of crocin on acrolein-induced tau phosphorylation in the rat brain. Acta Neurobiol Exp. 2015, 75: 208-219. 116. Torres, L. L.; Quaglio, N. B.; de Souza, G. T.; Garcia, R. T.; Dati, L. M.; Moreira, W. L.; Loureiro, A. P.; de Souza-Talarico, J. N.; Smid, J.; Porto, C. S.; Bottino, C. M.; Nitrini, R.; Barros, S. B.; Camarini, R.; Marcourakis, T. Peripheral oxidative stress biomarkers in mild cognitive impairment and Alzheimer's disease. J Alzheimers Dis. 2011, 26: 59-68. 117. Deslauriers, A. M.; Afkhami-Goli, A.; Paul, A. M.; Bhat, R. K.; Acharjee, S.; Ellestad, K. K.; Noorbakhsh, F.; Michalak, M.; Power, C. Neuroinflammation and Endoplasmic Reticulum Stress Are Coregulated by Crocin To Prevent Demyelination and Neurodegeneration. Journal of Immunology 2011, 187: 4788-4799. 118. Ochiai, T.; Soeda, S.; Ohno, S.; Tanaka, H.; Shoyama, Y.; Shimeno, H. Crocin prevents the death of PC-12 cells through sphingomyelinase-ceramide signaling by increasing glutathione synthesis. Neurochemistry International 2004, 44: 321-330. 119. Mousavi, S. H.; Tayarani, N. Z.; Parsaee, H. Protective effect of saffron extract and crocin on reactive oxygen species-mediated high glucose-induced toxicity in pc12 cells. Cell. Mol. Neurobiol. 2010, 30: 185-191. 120. Mehri, S.; Abnous, K.; Mousavi, S. H.; Shariaty, V. M.; Hosseinzadeh, H. Neuroprotective Effect of Crocin on Acrylamide-induced Cytotoxicity in PC12 cells. Cell Mol Neurobiol. 2012, 32: 227-235. 121. El-Beshbishy, H. A.; Hassan, M. H.; Aly, H. A. A.; Doghish, A. S.; Alghaithy, A. A. A. Crocin ‘‘saffron’’ protects against beryllium chloride toxicity in rats through diminution of oxidative stress and enhancing gene expression of antioxidant enzymes. Ecotoxicol. Environ. Saf. 2012, 83: 47-54. 122. Naghizadeh, B.; Mansouri, M. T.; Ghorbanzadeh, B. Protective effects of crocin against streptozotocin-induced oxidative damage in rat striatum. Acta Med Iran 2014, 52: 101-5.



123. Bandegi, A. R.; Rashidy-Pour, A.; Vafaei, A. A.; Ghadrdoost, B. Protective Effects of Crocus Sativus L. Extract and Crocin against Chronic-Stress Induced Oxidative Damage of Brain, Liver and Kidneys in Rats. Adv Pharm Bull, 2014, 4(Suppl 2), doi: 10.5681/apb.2014.073. 124. Zhang, G. F.; Zhang, Y.; Zhao, G. Crocin protects PC12 cells against MPPþ-induced injury through inhibition of mitochondrial dysfunction and ER stress. Neurochem Internat. 2015, 89: 101-110. 125. McKee, A. C.; Cantu, R. C.; Nowinski, C. J.; Hedley-Whyte, E. T.; Gavett, B. E.; Budson, A. E.; Santini, V. E.; Lee, H. S.; Kubilus, C. A.; Stern, R. A. Chronic Traumatic Encephalopathy in Athletes: Progressive Tauopathy After Repetitive Head Injury. J Neuropath Experimental Neurol. 2009, 68: 709 -735. 126. Marklund, N.; Blennow, K. B.; Zetterberg, H., Ronne-Engström, E.; Enblad, P.; Hillered, L. Monitoring of brain interstitial total tau and beta amyloid proteins by microdialysis in patients with traumatic brain injury. J Neurosurgery 2009, 110: 1227-1237. 127. Wang, K.; Zhang, L.; Rao, W.; Su, N.; Hui, H.; Wang, L.; Peng, C.; Tu, Y.; Zhang, S.; Fei, Z. Neuroprotective effects of crocin against traumatic brain injury in mice: Involvement of notch signalling pathway. Neurosci Lett. 2015, 591: 53-8. 128. Bie, X.; Chen, Y.; Zheng, X.; Dai, H. The role of crocetin in protection following cerebral contusion and in the enhancement of angiogenesis in rats. Fitoterapia 2011, 82: 997-1002. 129. Leker, R. R.; Shohami, E. Cerebral ischemia and trauma-different etiologies yet similar mechanisms: neuroprotective opportunities. Brain Res Rev. 2002, 39: 55-73. 130. Oruc, S.; Gönül, Y.; Tunay, K.; Oruc, O. A.; Bozkurt, M. F.; Karavelioğlu, E.; Bağcıoğlu, E.; Coşkun, K. S.; Celik, S. The antioxidant and antiapoptotic effects of crocin pretreatment on global cerebral ischemia reperfusion injury induced by four vessels occlusion in rats. Life Sci. 2016, 154: 79-86. 131. Vakili, A.; Einali, M. R.; Bandegi, A. R. Protective Effect of Crocin against Cerebral Ischemia in a Dose-Dependent Manner in a Rat Model of Ischemic Stroke. J Stroke Cerebrovasc 2014, 23: 106-113. 132. Sarshoori, J. R.; Asadi, M. H.; Mohammadi, M. T. Neuroprotective effects of crocin on the histopathological alterations following brain ischemia-reperfusion injury in rat. Iran J Basic Med Sci. 2014, 17: 895-902. 133. Akhondzadeh, S.; Shafiee-Sabet, M.; Harirchian, M. H.; Togha, M.; Cheraghmakani, H.; Almardani, R.; Jamshidi, A.; Rezasadeh, S-A.; Yousefi, A.; Zare, F.; Moradi, A.; Vossoughi, A. A 22-week, multicentre randomized, double-blind controlled trial of Crocus sativus L., in the treatment of mild-to-moderate Alzheimer’s disease, Psychopharmacol. 2010, 207: 637-643.


Page 28 of 41

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134. Farokhnia, M.; Sabet, M. S.; Iranpour, N.; Gougol, A.; Yekehtaz, H.; Alimardani, R.; Farsad, F.; Kamalipour, M.; Akhondzadeh, S. Comparing the efficacy and safety of Crocus sativus L. with memantine in patients with moderate to severe Alzheimer's disease: a double-blind randomized clinical trial. Hum Psychopharm Clin. 2014, 29: 351-359. 135. Akhondzadeh, S.; Sabet, M. S.; Harirchian, M. H.; Togha, M.; Cheraghmakani, H.; Razeghi, S.; Hejazi, S. S.; Yousefi, M. H.; Alimardani, R.; Jamshidi, A.; Zare, F.; Moradi, A. Saffron in the treatment of patients with mild to moderate Alzheimer’s disease: a 16-week, randomized and placebo-controlled trial. J. Clin Pharm Ther 2010, 35: 581-588. 136. Tsolaki, M.; Karathanasi, E.; Lazarou, I.; Dovas, K.; Verykouki, E.; Karakostas, A.; Georgiadis, K.; Tsolaki, A.; Adam, K.; Kompatsiaris, I.; Sinakos, Z. Efficacy and Safety of Crocus sativus L. in Patients with Mild Cognitive Impairment: One Year Single-Blind Randomized, with Parallel Groups, Clinical Trial. J Alzheimer's Dis. 2016, 54: 129-133. 137. Steiner, G. Z.; Yeung, A.; Liu, J. X.; Camfield, D. A.; de Blasio, F. M.; Pipingas, A.; Scholey, A. B.; Stough, C.; Chang, D. H. The effect of Sailuotong (SLT) on neurocognitive and cardiovascular function in healthy adults: a randomised, double blind, placebo controlled crossover pilot trial. BMC Compl Alter Med 2016, 16:15, DOI: 10.1186/s12906-016-0989-0. 138. Nikbakht-Jama, I.; Khademib, M.; Nosratia, M.; Eslamic, S.; Foroutan-Tanhac, M.; Sahebkara, A.; Tavalaiea, S.; Ghayour-Mobarhana, M.; Fernse, G. A. A.; Hadizadehf, F.; Tabassig, S. A. S.; Mohajerig, S. A.; Emamiana, M. Effect of crocin extracted from saffron on pro-oxidant– anti-oxidant balance in subjects with metabolic syndrome: A randomized, placebo-controlled clinical trial. Eur J Integr Med. 2016, 8: 307-312. 139. Talaei, A.; Moghadam, M. H.; Tabassi, S. A. S.; Mohajeri, S. A. Crocin, the main active saffron constituent, as an adjunctive treatment in major depressive disorder: A randomized, double-blind, placebo-controlled, pilot clinical trial. J. Affective Disorders 2015, 174: 51-56. 140. Russo, P.; Frustaci, A.; Del Bufalo, A.; Fini, M.; Cesario, A. Multitarget Drugs of Plants Origin Acting on Alzheimer's Disease. Current Med Chem 2013, 20: 1686-1693.



Figure Legend Figure 1. Structure of carotenoids which are converted to Retinol with Vitamin A activity and carotenoids including Crocin which are antioxidants but not converted to Vitamin A.


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26

Table1. Crocetin and crocin derivatives of saffron . Compound

Sugar moieties

Chemical formula

Isomer occurrence in saffron

Crocetin

R1 = R2 = OH

C20H24O4

cis–trans

Crocin 1

R1 = b-D-glucosyl R2 = H

C26H34O9

Trans

Crocin 2

R1 = b-D-gentiobiosyl R2 = H

C32H44O14

cis–trans

Crocin 20

R1 = R2 = b-D-glucosyl

C32H44O14

cis–trans

Crocin 3

R1 = b-D-gentiobiosyl R2 = b-D-glucosyl

C38H54O19

cis–trans

Crocin 4

R1 = R2 = b-D-gentiobiosyl

C44H64O24

cis–trans

Crocin 5

RI = 3 b-D-glucosyl R2 = b-D-gentiobiosyl

C50H24O29

cis–trans




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Table 2. Crocin Effects on Learning and Memory Species impairment cause/agent Mouse Ethanol Scopolamine Saffron extract (SE)

Dose 30% ethanol (10 ml/kg) 10mg/kg; 150-500 mg/kg 30% ethanol(2ml), 10.2-51.2 nmol

Route Oral IP, Oral

Memory Task passive avoidance

IV ICV

LTP by 30 pulses at 60 Hz

LTP by 11-51 pulses at 100 Hz, 30 pulses at 60 Hz passive avoidance, step through and step down

Rat

Ethanol Crocin

Rat/hippocam pal slices.

Ethanol Crocin, Saffron extract

10-50, 50-75 μM 100mM, 125 & 250 mg/kg

in vitro, oral

Mouse

Ethanol Crocin

30% ethanol (10 ml/kg), crocin (50 to 200 mg/kg)

Oral, oral

Rat hippocampal

Ethanol Crocin

50, 100 mM 10 μM

in vitro

electricity current

Rat

Acetaldehyde Saffron extract

60mg/kg 62.5-250mg/kg

IV, oral

LTP (Tetanic stimulation 30 pulses at 60 Hz)


Effects SE revered ethanol and scopolamine caused impirment of cognition and memory. Pre-administred crocin prevent the LTP blocking effect of ethanol. SE and crocin revesed the inhibitory response of ethanol on hippocampal LTP. Crocin prevented EtOH induced learning and memory impairment in a dose-dependent manner. Crocin also improve memory retrieval deficit. Crocin selectively antagonized the inhibitory effect of ethanol on NMDA receptor-mediated responses in hippocampal neurons. Orally administered saffron extract antagonized the action of acetaldehyde on the central nervous system.

Reference Zhang 1994, (81)

Sugiura 1994, (76)

Sugiura 1995a,b, (82) (83) Sugiura 1995, (77)

Abe 1998, (75)

Abe 1999, (84)


Rat

Scopolamine Saffron aqueous extract, Crocin

1-500mg/kg 2.5-560mg/kg, 50-200mg/kg

IP IP, IP

Morris water maze

Rat

Scopolamine Crocin

0.2 mg/kg, 15 & 30 mg/kg

SC, IP

Object recognition, Radial water maze

Rat

Scopolamine Crocin

0.5 mg/kg, 1, 5 or 10 mg/kg

IP, IP

Morris water maze

Rat

Streptozocin (STZ) Crocin

3mg/kg, 15-30mg/kg

ICV, IP

Y-maze, passive avoidance

Mouse

Aged Saffron extract (in crocetin)

IP

passive avoidance

60 mg/kg


Page 34 of 41

Saffron aqueous extract and crocin inhibited the scopolamine impaired acquisition/performan ce activity. Scopolamine impaired learning and memory. Crocin benefited cognition of new objects and ameliorated the effects of scopolamine. scopolamine impaired learning and memory. Crocin in a dosedependent manner ameliorated effects of scopolamine. Crocin significantly improved learning, memory, and spatial cognitive deficitd resulted from STZ. SE facilitated learning in both adult and aged mice. SE and crocetin provided antioxidant protection and attenuated caspase-3 activity.

Hosseinzad eh 2006, (85)

Pitsikas 2007, (86)

Ghadami 2009, (87)

Khalili 2010, (88)

Papandreo u 2011, (89)

Page 35 of 41


Rat

Stress Saffron extract, crocin

30mg/kg; 15-30mg/kg

IP IP

Streptozotocin combination of Nardosatchys jatamansi extract (N), crocetin (C) and selenium(Se)

3 mg/kg in 5µl NCSe (N =200 mg/kg, C = 25 lg/kg, Na2SeO3 = 0.05 mg/kg)

ICV,

oral

passive avoidance, Morris water maze

Mouse

Morphine Saffron aqueous extract

5mg/kg; 50, 150 and 450 mg/kg

SC, IP

passive avoidance

Mouse

D-galactose+NaNO2 Saffron extract

120mg/kg+90mg/kg; 30mg/kg

IP IP

Active+passive avoidance

Rat

Surgical ischemia Saffron extract, Crocin

50-250mg/kg, 5-25mg/kg

IP IP

Rat

water maze

Morris water maze


Saffron extract and crocin prevented chronic-stress induced deficits in learning and memory. Crocetin combination prevented STZ induced loss of cognition & oxidative stress in aged rats. NCSe pretreatment reversed STZ infused impairments in learning and memory performance in old rats. Saffron aqueous extracts attenuated morphine-induced memory impairment. SE prevented and treated learning impairment and memory dificit induced by Dgalactose+NaNO2. Ischemia led to impairment in cognition and memory. Crocin and saffron extract reversed impairment.

Ghadrdoost 2011, (90)

Khan 2011, (91)

Naghibi 2012, (92)

Dashti-r 2012, (93)

Hosseinzad eh 2012, (24)


Rat

Streptozotocin Crocin

60 mg/kg, 7.5-30mg/kg

IP IP acute

elevated plus maze

Rat

Surgical hypoperfusion Crocetin

2, 4, & 8mg/kg

IP post 7 days

Rat

Streptozotocin Crocin

3 mg/kg 100 mg/kg

IP oral

Morris water maze

Mouse

Aluminum Saffron extract

50 mg AlCl3/kg/day 60mg/kg

passive avoidance

Rat

Ketamine Crocin

3-25mg/kg 15-50mg/kg

Oral (5 weeks) IP (last 6 days) IP IP

Rat

beta-Amyloid (1-42) Crocin

100 ng/μL 150, 300 and 600 nmol

IP & IH IP & IH

Morris water maze

Rat

Arsenic Saffron extract

100mg/kg 100mg/kg

Oral oral

maze

Morris water maze

novel object recognition


Page 36 of 41

Crocin reversed the impairment, glucose level and prevent loss of neuron in dose dependent manner. Crocetin effectively protected neurons against ischemia and improved spatial learning memory. STZ induced memory deficit and oxidative stress. Crocin improved cognition and antioxidant status. SE coadministration in the last 6 days had no effect on cognitive performance of mice. Crocin reversed ketamine induced memory deficit. Crocin inhibited death of brain cells and improved the memory impairment caused by Aβ. Intake of Arsenic (30d) led to severly deteration of memory and cognition. SE kept body, brain weights and learning behavior.

Tamaddonf ard 2013, (94)

TashakoriSabzevar 2013, (95)

Naghizadeh 2013, (96)

Linardaki 2013, (97)

Georgiadou 2014, (78) Asadi 2015, (38)

Sreenu 2015, (98)

Page 37 of 41


Rat

Ethidium bromide

3 μl of 0.01% EB

Saffron ethanolic extract

5-10 µg

intrahip pocamp al injectio n, IH

Morris water maze

SE ameliorated the impairment of learning and memory, and oxidative stress in brain.

LTP: Long term potentiation, a form of activity-dependent synaptic plasticity that may underlie learning and memory IP: intraperitoneal; IV: intravenous; ICV: intracerebroventricular; SC: subcutaneous; IH: intra-hippocampal.


Ghaffari 2015, (99)


Table 3. Studies of crocin protective effects against brain cells/tissue injury/death Cell/tissue/animal Type Original Toxicin/inducer Protectant & dose PC12 neuronally TNF-α (500U/ml), Crocin (1-10 differentiated daunorubicin (50 µM) mM) PC12

neuronally differentiated

Cerebral microvessels PC12, mouse

neuronally differentiated

Depriving of serum/glucose

Crocin (10 µM)

mouse

Global cerebral ischemia (20min)reperfusion (24 hr)

Crocin (20, 10 mg/kg, PO)

mouse

Serum-deprived and hypoxic, cerebral infarction

Crocin (10 µM), (10mg/kg, IV) Saffron extract (5, 25 µg/ml), crocin (10, 50µM) Crocin, crocetin

PC12

High glucose (4.5, 13.5 and 27 mg/ml)

Microglial cells, hippocampal slice cultures

BV2 organotypic

mouse rat

Lipopolysaccharide (LPS)

HFA, oligodendrocyte, mouse

primary organotypic EAE

human Rat mouse

Syncytin-1, sodium nitroprusside

Crocin


Page 38 of 41

Effects

Ref

Crocin inhibits neuronal cell death induced by both internal (TNF-α) and external (daunorubicin) apoptotic stimuli. Crocin inhibited lipid oxidation, partly restored antioxidative enzyme activities, and maintained the neuron’s morphology. Crocin protected brain cells from edema, damage and injuries, and inhibited oxidizing reactions.

Soeda, 2001 (22)

Crocin maintained neuronal cell normal morphology and prevented neuron death caused by ischemic stress. Saffron and crocin protected cells against high glucose induced toxicity and radical oxidative species (ROS).

Ochiai, 2007 (54)

Crocin and crocetin showed neuroprotective effects by blocking LPS induced hippocampal cell death through antioxidative and antiinflammatory effects. Crocin demonstrated neuroprotective effects and protected in HFAs and oligodendrocytes by suppressing ER stress and inflammatory gene expression. Crocin improved animal

Nam, 2010 (19)

Ochiai, 2004a,b (106), (118) Zhang, 2007 (103)

Mousavi, 2010 (119)

Deslauriers, 2011 (117)

Page 39 of 41


neurobehaviour.

PC12

Acrylamide (5 mM)

Crocin, 1050 µM

Brain

rat

BeCl2 (86 mg/kg, oral 5 days)

Crocin 200mg/kg IP, -7 day

Striatum

rat

Streptozotocin (3 mg/kg, ICV), 1st,3rd day

Crocin (100 mg/kg, PO), 21 days

Brain, liver, kidneys

rat

Chronic restraint stress (6 h/day), 121d

21 days, saffron ext (30mg/kg IP), Crocin (30mg/kg, IP) Crocin

PC12

1-Methyl-4phenylpyridinium (MPP+)

Crocin inhibited acrylamide induced cell death through reducing ACRincreased Bax/Bcl-2 ratio and oxidative stress. Crocin normalized oxidative status and antioxidant enzyme levels, and prevented BeCl2 induced brain and liver damages. Crocin significantly reduced oxidative stress and elevated antioxidant enzyme activities.

Mehri, 2012 (120)

El-Beshbishy, 2012 (121)

Naghizadeh, 2014 (122)

saffron and crocin prevented Bandegi, chronic stress-induced oxidative 2014 (123) stress damage of the brain, liver and kidneys.

Crocin significantly attenuated Zhang, 2015 + MPP -induced cell injury and death. (124) Crocin inhibited MPP+-induced mitochondrial dysfunction and ER stress . PC12: neuronally differentiated pheochromocytoma; BV2: BV2 mouse microglial cells; HFA: human fetal astrocytes; EAE: experimental autoimmune encephalomy PO: orally; IV: intravenous; IP: intraperitoneal; ICV: intracerebroventricular ER: endoplasmic reticulum; ACR: acrylamide.



Table 4. Summary of Clinical Studies of Saffron/Crocin on Memory/Cognition Number of Duration & Disease Treatment Subjects Type of study 46 16 weeks Mild-toSaffron extract double moderate AD blind

Page 40 of 41

Dose

Route

Effects

References

30mg/day

oral

Saffron significantly improved cognitive function in patients in comparison to placebo.

135

Saffron was as effective as donepezil and slightly less side effects in the treatment of patients. Saffron exhibited similar efficacy and safety profile to memantine in prevention of cognitive decline. Patients on saffron had improved cognitive impairment, while the control group presented continued deterioration.

133

Improved alphabetic working memory and visual working memory.

137

54

22 weeks double blind

Mild-tomoderate AD

Donepezil Saffron extract

10mg/day 30mg/day

oral

68

12 months double blind

Moderate-to severe AD

Memantine Saffron extract

20mg/day 30mg/day

oral

35

12 months single blind

Mild cognitive impairment

Saffron extract

125mg/da y

oral

16

1 week of 3 double blind crossover

Healthy adults

SLT formulation of crocins of saffron, Ginsenosides, & ginkgo flavoneglycosides

10.92mg, 54.54mg, 54.54mg

oral

SLT: Sailuotong, product name of the formulation


134

136

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TOC Graphic


A Perspective on Crocus sativus L. (Saffron) Constituent Crocin: A

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