Diabetes mellitus (DM) is a chronic metabolic disease characterized by persistent high blood glucose levels and accompanied by impaired metabolic pathways of carbohydrates, proteins, and lipids. Diabetes is essentially caused either by the loss of β-cells, or of their ability to produce insulin (type I). Type II diabetes refers to the inability of the organism to properly regulate and sense insulin, known as resistance to insulin. Hyperglycemia is the result of both types. Since insulin is the main transporter, responsible for removing glucose from the blood-stream, its deficiency can inescapably lead to elevated blood glucose levels. Persistent hyperglycemia can lead to severe micro and macrovascular complications causing long-term damage to nerves and blood vessels, affecting different body organs. In this regard, the management of blood glucose levels in diabetic patients is the main focus of the available antidiabetic treatment including either administration of insulin and/or synthetic drugs [1
Numerous medicines have been developed over the past years to alleviate symptoms, increase life expectancy, and maintain the progression of the disease in remission. Medical treatments through synthetic formulations can overcome risk factors. There are several cases where synthetic formulations lead to severe side effects that are not associated with the disease itself. In order to avoid treatment related side effects, scientists are driven towards the development of new therapeutic molecules, able to replace current therapeutic strategies.
Phytochemicals are emerging as powerful alternatives towards the fight against chronic diseases, including diabetes. These plant-derived molecules used as a mixture or as a single compound, contribute to the prevention and treatment of several chronic disorders such as cancer, neurodegenerative and metabolic diseases. It has been proposed that their efficacy is mainly due to their antioxidant activity [2
]. Treatment with natural products is yet preliminary and much information is still needed regarding their mechanism of action. These include pharmacological parameters such as pharmacokinetics and pharmacodynamics, drug tolerance, and possible side effects. Accumulating data of several in vitro, in vivo, and clinical trial studies provide evidence that these substances are of great therapeutic importance and may consist a new era of treatment against the above-mentioned long-term conditions [4
L. is a stemless, perennial plant belonging to the Iridaceae family and the genus Crocus. The plant is commonly known as saffron, a name which is referred to its dried red stigmas. Stigmas are considered the pharmacologically active part of the plant and their chemical composition has been widely studied [7
]. More than 150 volatile and non-volatile compounds have been identified. Crocins (CRCs) are the predominant constituents of the stigmas and give them their characteristic deep red color [10
]. On the contrary to other carotenoids, CRCs due to their glycosylated terminals, are water soluble molecules.
Zebrafish, Danio rerio
, a small tropical fish, has become a popular model for studying a wide range of human diseases. These include cancer [14
], cardiovascular [15
], neurodegenerative [16
] and metabolic diseases [17
]. This is due to the high genetic homology with humans and the similarities in organ physiology and metabolism, offering several unique advantages. Among those, most useful for this study are the external fertilization, development, and transparency of the embryos allowing non-invasive in vivo imaging, as well as the plethora of synchronized progeny. In vivo phenotypic screens using zebrafish embryos have been particularly valuable in identifying novel bioactive natural compounds or optimizing the activity of lead compounds [19
]. Chemical library screens using zebrafish embryos for the identification of melanogenesis inhibitors [20
] and or transgenic-based screens for angiogenesis [21
] or neoglucogenesis [18
] have been very productive.
In addition, zebrafish has become a popular experimental model regarding the study of metabolic diseases, including diabetes. Similarities regarding the exocrine and endocrine pancreas between mammals and zebrafish [22
], the conservation of key proteins that control glucose metabolism [18
], the capability of measuring glucose levels in larvae and adult zebrafish, as well as the unique capacity of this model to recover its β-cells [23
], make zebrafish a promising experimental model for testing compounds that alter glucose metabolism or regulate glucose levels [25
We aimed to investigate the effect of CRCs isolated from Crocus sativus L., on the control of glucose levels using zebrafish as an animal model. We also examined whether the addition of crocins would regulate the levels of phosphoenolpyruvate carboxykinase 1 (pck1), a key regulatory gene for the gluconeogenesis process that contributes to the maintenance of normoglycemia. Finally, we evaluated the effect of crocins on β-pancreatic cells.
L., is a plant with rich pharmacologic activities. It has been studied in the context of several diseases such as cancer, neurodegenerative disorders, inflammation, heart related, and metabolic disorders. These properties are mainly attributed to the stigmas of the plant and specifically to its main constituents, crocins (CRCs), picrocrocin, and safranal [31
However, growing, harvesting, storage, and environmental conditions, in addition to the extraction procedure, are important criteria to consider for the quality of the raw material. In herbal medicine, the quality of the material is directly linked to the efficacy of the natural product when tested in either in vivo or in vitro experiments and has a high impact on the study results. However, environmental conditions are rather uncontrolled and thus, a quality control test is always needed in order to guarantee the presence of the desired compounds. Furthermore, storage conditions including temperature, humidity, and light strongly affect the stability of the compounds presented. The above-mentioned quality criteria for the raw material used in this study are followed identically for each harvesting crop.
Saffron safety has been evaluated in clinical trials and in vivo studies [35
]. Generally, saffron extracts as well as its constituents are relatively safe. For example, the intraperitoneal administration of stigmas ethanolic extract in rats at a range of a concentration between 1–5 g/kg bw (body weight), caused lethal effects at the highest administered dose (100% mortality), whereas the lowest dose resulted in no death at the end of 48 h. Authors estimated the LD50 value at 3.5 g kg−1
]. Similarly, Hosseinzadeh et al., (2010) [36
] examined the safety of an extract of CRCs in mice and rats, after oral or intraperitoneal administration. No toxic effect was stated by the authors at mice treated with the extract since no mortality was observed at 2 h and after 48 h of treatment. The oral administrated dose was 3 g/kg, while the intraperitoneal injection was within the range of 0.5–3 g/kg. According to these data, stigmas of Crocus sativus
L., and its active constituent CRCs, show a safe profile, since, an LD50 value within the range of 1–5 g/kg is attributed to low-toxic chemicals according to the toxicity classification [38
In our experiments, the administration of CRCs is performed via immersion of the embryos in an aqueous solution of the tested compound. Even though the exact amount of crocin uptake is not known, this is a standard method for toxicity evaluation of compounds in zebrafish. Since, to our knowledge, this is the first time that a purified extract of crocins is tested in zebrafish, we investigated a range of concentrations up to 2 mg/mL in order to evaluate the toxicity. To investigate the effect of crocins on the control of glucose levels, we performed all the experiments at a concentration that corresponds to 1/3 of the LC50, and did not result in any phenotypic abnormalities. In this way, we are ensured that all measurements are not affected by any toxic response of the developing larvae.
The antidiabetic and hypoglycemic potential of Crocus sativus
L., is supported by many studies and saffron is nowadays considered as a promising candidate in the field of metabolic diseases. For example, Kianbakht and Hajiaghaee, 2011 [39
] discussed the anti-glycemic effect of crocins, safranal, and saffron extracts in diabetic rats. Their results indicated that these compounds managed to control glycemia without triggering any toxic effect in the liver or the kidney. Another study performed by Kang et al., 2012 [40
] demonstrated the in vitro capacity of saffron extract to activate glucose uptake and ameliorate insulin sensitivity in skeletal muscle cells. Furthermore, Dehghan et al., 2016 [41
] showed the effectiveness of saffron to improve diabetic biochemical markers, such as blood glucose and glycosylated hemoglobin, in in vivo and in vitro models. In addition, several clinical trials have confirmed the antidiabetic effect of saffron in type II diabetes patients. Milajerdi et al., 2018 [42
] performed a triple-blinded randomized clinical trial in type II diabetic patients and suggested that saffron extracts, administrated twice a day and used in combination with current antidiabetic therapy, lowered blood glucose levels. In addition, the administration of saffron in overweight/obese patients diminished both sugar levels and hemoglobin A1c [43
Streptozotocin-induced diabetic models have been used to evaluate the effect of CRCs on glucose levels and pancreatic function. Rajaei et al., 2013 [45
] evaluated serum glucose levels of hyperglycemic rats treated intraperitoneally with crocins. Lower glucose levels observed in the study with respect to the control group were attributed to higher insulin production or to the strong antioxidant activity of the extract used. The latter antidiabetic mechanism was further investigated by Yaribeygi et al., 2019 [46
], who measured SOD and catalase levels of pancreas in diabetic rats. In this study, they found that CRCs significantly increased both enzymes and thus, harmful effects caused by oxidative stress due to hyperglycemia were avoided.
In another study, the hypoglycemic and hypolipidemic effects of CRCs were evaluated in streptozotocin-induced type II diabetes rats, where advanced glycation products, glucose and HbA1c levels, and fasting insulin levels were measured. All these parameters were significantly decreased in treated animals [47
Despite the fact that zebrafish is widely used as a model for many human disorders, including metabolic diseases, there are only few studies analyzing the effect of saffron and/or its bioactive compounds on glucose management. In our study, we used zebrafish embryos to investigate the effect of the main bioactive compounds of Crocus sativus L. on the regulation of the glucose levels and the insulin secretion. We found that the administration of CRCs can significantly reduce glucose levels and in parallel increase insulin expression in developing embryos, suggesting a putative hypoglycemic role of this compound. Our study used a single application of CRCs in the water of developing embryos and more studies including adult feeding with CRCs would be needed to address long-term effects in zebrafish glucose homeostasis.
In general, various mechanisms have been proposed based on histopathologic observations and on measurement of blood glucose levels in several in vitro experimental models to explain the hypoglycemic and antidiabetic activity of saffron. Among them, enhanced stimulation of insulin secretion from β-cells, increased capacity of the peripheral tissues to use it properly, restoration of the β-cells of the endocrine pancreas, and inhibition of glucose production. In addition, the antioxidant activity of the stigmas seems to drastically influence its hypoglycemic effect [45
]. Here, we present elevated fluorescence levels of the pancreatic islets in the Tg
) transgenic fish and increased transcription of the endogenous insulin gene as a result of CRCs treatment of zebrafish larvae. These observations support the hypothesis that the hypoglycemic activity of crocins is at least in part, due to the enhanced insulin production of β-cells.
is a key gene in the process of gluconeogenesis. It is often used as an indicator of blood glucose levels since it is normally downregulated in cases of hypoglycemia and upregulated when blood glucose levels are elevated. In our study, lowering the levels of glucose were linked with a slight but significant increase in pck1
expression. This could be explained as a homeostatic, compensatory response of the zebrafish larvae to the reduced glucose levels induced by CRCs. Nevertheless, glucose production is simultaneously related to stimulated metabolism, an issue that needs to be further investigated taking into account that diabetes patients suffer metabolism complications. The same effect of low glucose levels and overexpression of pck1
was also in the Tg(pck1:Luc2
bioluminescence transgenic zebrafish line [18
]. Authors tested several chemical compounds for their capacity to reduce glucose levels and also studied their effect on pck1
expression. Among the tested compounds PK1195, a translocator protein 18 kDA (TSPO) ligand, induced pck1
expression simultaneously to a reduction in glucose levels. Authors concluded that compounds that belong to the TPSO family, regardless of their gluconeogenetic effect, can effectively interfere with levels of glucose in zebrafish larvae.
In summary, we employed zebrafish as a tool in order to explore the effect of crocins isolated from Crocus sativus L. on glucose metabolism. Our results suggest that the treatment of zebrafish larvae with non-toxic levels of crocins can significantly reduce the basal total glucose levels and increase insulin expression. These data provide some evidence that crocins are implicated in the glucose regulation, but further studies are needed in order to elucidate the exact mechanism of action and its potential as a putative agent on the antidiabetic field.
4. Materials and Methods
4.1. Extraction and Chemical Analysis of Crocins
Stigmas of Crocus sativus L. were cultivated at the Kozani prefecture and were kindly offered by the “Cooperative of Saffron producers, Kozani Greece”. Stigmas were collected in 2018.
L. is a male-sterile triploid lineage that ever since its origin has been propagated vegetatively. No wild population has been found so far and all the available plant material comes from cultivation. The crop is an autotriploid that evolved in Attica by combining two different genotypes of C. cartwrightianus
(a species endemic to Attica and some Aegean islands, Greece) [50
]. Triploid sterility and vegetative propagation prevented afterwards segregation of the favorable traits of saffron, resulting in worldwide cultivation of a unique clonal lineage. As a result, no infraspecific taxa have been recognized.
Five grams of dried stigmas were shattered and extracted with petroleum ether in order to remove lipid compounds, in an ultrasonic water bath (GRANT type, 300 × 140 × 150 mm internal dimensions), for 15 min at 25 °C and at the frequency of 35 kHz. The extraction was repeated 5 times and proceeded under the same conditions using diethyl ether. Extraction with petroleum ether and diethyl ether proceed prior to methanol extractions in order to eliminate the final extract from the presence of unwanted compounds such as lipids and safranal. The received powder was dried under nitrogen steam and the final extraction step took place as described above, using methanol. The organic solvent was evaporated in a rotary evaporator and a purified extract of CRCs was received. The final product that consisted of a red colored powder was kept at −4 °C for further analysis.
In order to ensure total evaporation of the organic solvent, an aqueous extract of CRCs was prepared and Raman spectroscopy was performed. No characteristic peaks that correspond to the presence of methanol were observed, indicating that the final product was methanol free.
The analysis of the extract was performed on an HPLC system (Agilent Series 1260-Agilent Technologies, Waldbronn, Germany) coupled to a 6530 Q-TOF mass spectrometer (Agilent Technologies, Singapore). The HPLC system consists of degasser, autosampler, quaternary pump, diode array detector, and a thermostatically controlled column oven. Chromatographic separation was performed at 40 °C on a Poroshell 120 EC-C18 4.6 × 50 mm, 2.7 μm reversed phase column. The following chromatographic conditions were applied: Flow rate 0.4 mL/min, injection volume 5 μL, mobile phase A (water LC/MS-0.1% formic acid), and mobile phase B (acetonitrile LC/MS-0.1% formic acid). The gradient elution program was applied as follows: 5–95% solvent B from 0 to 33 min and maintained at 95% up to 38 min. The sample was detected at 440 nm. The Q-TOF mass spectrometer was operated with a dual ESI source in the negative ionization mode and according to the following operating parameters: Capillary voltage 4000 V, gas temperature 300 °C, skimmer 65 V, octapole RF 750 V, drying gas 10 L/min, nebulizer pressure 450 psig, and fragmentor voltage 150 V. Scanning was performed from 100–1700. The Q-TOF-MS was calibrated on a daily basis using a reference mass solution (calibrant solution, Agilent Technologies) with internal reference masses at m/z 112.9856 and 1033.9881. The data acquisition and qualitative analysis were processed by MassHunter software (Agilent Technologies).
4.2. Determination of LC50
The toxicity of crocins on zebrafish embryos was evaluated according to OECD guidelines (OECD 2013 Toxicity test TG236, 2013). Briefly, fertilized embryos were collected at 3 hpf and treated with an aqueous solution of CRCs extract. A range of concentrations from 0–2 mg/mL was tested. The concentration of 0 mg/mL corresponds to the control group.
During the experiment the test media was not renewed, embryos were kept at 28 °C, and the plate was covered with aluminum foil to protect the crocins from degradation. Embryos were monitored daily up to 96 hpf and scored for lethality Three replicates were performed for each concentration tested. Results were calculated using the probit analysis (IBM SPSS Statistics v23). In order to ensure the stability of the extract, extracts of the same concentration were stored under the same conditions, and their UV-Vis spectra were recorded daily. The extracts remained stable during the experiment (data not shown).
4.3. Zebrafish Maintenance and Breeding
Zebrafish were raised under standard laboratory conditions at 28 °C on a 14/10 h day/night cycle in the animal facility of Biomedical Research Foundation of the Academy of Athens (EL25BIO003). Zebrafish are maintained according to the Recommended Guidelines for Zebrafish Husbandry Conditions [51
]. The genetic backgrounds used were the wild-type Ab strain for all toxicity, glucose level quantification and RT-PCR experiments, and the transgenic line Tg
) for visualizing pancreatic β-cells. The experimental protocols described in this study were completed by day 5 of the zebrafish embryo development and therefore, are not subject to the regulations of European animal protection guidelines, in accordance with the European Directive 2010/63 for the protection of animals used for scientific purposes.
4.4. Measurement of Glucose Levels
The measurement of glucose levels was performed on larvae zebrafish using a colorimetric/fluorimetric based enzymatic detection kit (Biovision-K606) and according to Jurczyk et al., 2011 [22
]. Zebrafish larvae at 72 hpf were placed in a 12-well plate and treated with the extract of crocins at a concentration of 0.2 mg/mL. Embryos were incubated at 28 °C for 48 h. The treatment was performed in triplicate.
The measurement of glucose was performed according to the protocol provided with slight modifications. Briefly, 20 embryos were placed in eppendorf tubes and the medium was removed. The collected embryos were frozen in liquid nitrogen and were kept at −80 °C for a minimum of 30 min. Then, 80 μL of phosphate-buffered saline (PBS) were added in each tube and embryos were homogenized using a pestle. Tubes were centrifuged and collection of the supernatant took place 8 μL of each sample were added in 42 μL of PBS in a 96-well plate. After that, 50 μL of the reaction mix containing one μL of glucose probe, one μL of glucose enzyme, and 48 μL of glucose buffer were added so as to adjust the final volume at 100 μL. To calculate the results, a standard curve of glucose was constructed (0–3.5 nmol/well). Reactions were incubated at 37 °C for 30 min and absorbance was measured at 570 nm.
4.5. RNA Isolation and cDNA Synthesis
Total RNA extraction from 120 hpf zebrafish larvae was carried out using TRIzol reagent (15596026-Invitrogen) and purification was performed with Turbo DNase (2238G2-Ambion), according to the manufacturers’ protocols. The RNA concentration and purity were determined by NanoDrop 2000c Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and cDNA was synthesized using the PrimeScript RT reagent kit (RR037A-Takara).
4.6. Quantitative Real-Time PCR
RT-PCRs were performed with a Roche cycler system (Light Cycler 96) using the KAPA SYBR FAST qPCR kit (KK4611-KAPA Biosystems) and gene-specific primers. The sequences of primers used are the following:
pck1f: TCTCCATCCCTCCGCTCATCA, pck1r: GGCCCAGCTGACTGCTCCT, insaf: TAAGCACTAACCCAGGCACA, insar: GATTTAGGAGGAAGGAAACC.
As a reference gene, the elongation factor was used with the following primers: efa1f:
TCTCTACCTACCCTCCTCTTGGTC and efa1r
: TTGGTCTTGGCAGCCTTCTGTG. The relative amounts of the different mRNAs were quantified with the ΔΔCt method [52
] and the fold-change ratio was calculated and expressed as mean ± SEM.
4.7. Monitoring Pancreas Development
Zebrafish larvae from the transgenic line Tg(ins:DsRed) were treated with 0.2 mg/mL of CRCs at 72 hpf. Embryos were incubated at 28 °C for 48 h and subsequently anesthetized using 0.4 mg/mL of Tricaine (BIA 1347-Apollo scientific) in order to proceed to imaging. To facilitate imaging, the embryos were mounted in a 1.2% low melting agarose. Fluorescent and brightfield images were captured using a Leica DMRA2 microscope (Leica, Switzerland) equipped with a HamamatsuORCA-Flash 4.0 V2 camera and analyzed using the Fiji software. Data are presented as mean ± SEM. Differences were analyzed using the two-tailed Student’s t-test. In addition, p was considered significant when less than 0.05.