Determination of Mineral Composition and Phenolic Content and Investigation of Antioxidant, Antidiabetic, and Antibacterial Activities of Crocus sativus L. Aqueous Stigmas Extracts

The aim of this study is to investigate the in vitro antioxidant, antidiabetic, and antibacterial activities of Moroccan and Italian Crocus sativus (L.) stigmas extracts. The antioxidant activity was evaluated by DPPH radical scavenging assay, and the results showed that the Moroccan extract has a powerful antioxidant activity with an IC50 of 0.32 ± 0.059 μg/mL compared to the Italian extract (IC50 of 3.14 ± 0.021 μg/mL). Additionally, the antidiabetic activity was evaluated by using alpha-amylase and alpha-glucosidase inhibition assay, and both extracts showed significant antidiabetic activity. However, the antibacterial activity was evaluated by the disc diffusion method to determine the inhibitory diameters and microplate dilutions method to determine the minimum inhibitory concentration. Our findings revealed that both Moroccan and Italian extracts were more effective against Gram-positive than Gram-negative bacteria. From this study, we can conclude that the studied extracts of C. sativus are rich in natural compounds and could have a broad application in the pharmaceutical, food, and medical fields.


Introduction
Natural products have been used since antiquity as remedies for the treatment of several diseases, as well as in traditional food preparations. Nowadays, the use of natural products, especially medicinal plants and spices, takes a major place in the research of new alternative or precursors to chemical drugs that are used in the treatment of several chronic diseases. Indeed, many studies have approved the antioxidant, anti-inflammatory, and anticarcinogenic, antidiabetic, and antimicrobial activity of plants extracts and spices [1][2][3][4][5].
Crocus sativus L. is an herbaceous species belonging to the Iridaceae family, commonly known as saffron. Harvest, processing, handling, drying, packing, and storage are the stages involved in its production [6].
e flowering of C. sativus occurs, in an extremely short period, only once a year, and its harvest must be done manually [7], and for the production of 1 kg of saffron (dry stigmas), nearly 158,000 flowers are needed [8], which makes red dried stigmas of C. sativus one of the most expensive culinary herbs in the world [9].
Saffron has been cultivated in Morocco for centuries in the Taliouine region precisely over an area of 565 ha, and more recently in the region of Taznakht (province of Ouarzazate) on an area of 105 ha [10,11]. Its cultivation is considered an important source of employment and income for the local population. However, its aroma, flavor, and good quality have given it national and international appreciation [12,13].
Since ancient times, saffron has been widely used as a spice for culinary purposes and food coloring, especially in the food industries, and it has been used in traditional medicine, textiles, and cosmetics. Due to its healing properties, saffron has been used to treat various disorders such as insomnia, liver disease, digestive disorders, and menstruation disorders, and it was also used as an aphrodisiac, antidepressant, and stimulant [13,14]. However, the biological activities of Moroccan C. sativus are still not well studied.
us, in order to fill this gap, we evaluated the chemical and biological activities of Moroccan and Italian C. sativus extracts.

Plant Material.
e plant of C. sativus was obtained from Taliouine region in Morocco during flowering stage and from Italy. After harvest, the plant material (stigma) was dried at room temperature (22°C) in the open air and protected from light and heat. en, they were crushed for extraction.

Preparation of the Saffron Extracts.
Five grams of the dried plant material was infused in 100 ml of boiling distilled water for 15 min, then filtered, and evaporated under vacuum at 40°C using a rotary evaporator. e extracts were cooled and stored in the refrigerator at 4°C until use.

Determination of Total Phenolic Compounds.
e amount of total phenolic contents was determined according to Folin-Ciocalteu method [15], with some modifications. Briefly, a volume of 100 μl of each extract was introduced using a micropipette into test tubes, followed by the addition of 1000 μl of the Folin-Ciocalteu reagent (0.2 N). After 10 min of incubation at room temperature, 800 μl of sodium carbonates (Na 2 CO 3 ) (75 g/l) has been added, and the solutions were kept in the dark for 30 minutes at room temperature. e absorbance of each solution was measured by spectrophotometer at 765 nm. e total phenolic content was expressed as milligrams gallic acid equivalents per Gram of dry weight of extract (mg GAE/g extract).

Determination of Flavonoid Contents.
e total flavonoids were determined using a colorimetric method [16] with few modifications. Briefly, 250 μl of the extract was mixed with 1000 μl of distilled water, and then 75 μl of a 15% sodium nitrite (NaNO 2 ) solution was added. After 6 minutes of incubation at room temperature, 75 μl of 10% aluminum chloride (AlCl 3 ) was added. After 6 minutes of incubation at room temperature, 1000 μl of sodium hydroxide (NaOH) (4%) was added. e total volume is made up to 2500 μl of distilled water. e solution was mixed and left to stand for 15 min. e absorbance was measured at 510 nm against a blank. Total flavonoid contents (TFCs) were expressed as mg Catechin equivalent per Gram of dry weight of extract (mg CE/g extract).

Determination of Mineral Content by ICP-AES.
Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for rapid and precise determinations of minor and major mineral content in digested powder of studied plants. Briefly, 1 g of C. sativus stigma was digested by heating in an oven at 500°C, and the rest of the powder was totally solubilized in 5 ml of concentrated nitric acid (HNO 3 ) + 15 ml of hydrochloric acid (HCl). After complete digestion, the mixed samples were cooled at room temperature and made up to a final volume 100 ml with ultrapure water. Digestion was then analyzed in duplicate, and concentrations of trace metal elements were measured directly using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES, ermo Scientific). e optimal instrumental conditions are maintained at 12 l/min for the stable plasma gas flow rate, and the auxiliary and the nebulizer gas flow rate were kept at 1 and 0.7 l/min, respectively. Indeed, the sample flow rate was 1.5 ml/min, and the Radio Frequency (RF) power was 1200 W.

Determination of Total Antioxidant Capacity.
e antioxidant activity was measured using the DPPH method. e purplish hue of the free and stable radical DPPH (2,2diphenyl-picryl-hydrazyl) is absorbed at 517 nm. is radical is reduced and turns yellow in the presence of antioxidant molecules. Briefly, 0.5 ml of each sample concentration was mixed with 0.5 ml of methanolic solution of DPPH (0.04 g/l) and then incubated in the dark at room temperature for 30 min [17]. Absorbance was measured at λ � 517 nm using a spectrophotometer.
e calculation of the percentage inhibition of DPPH is carried out by applying the following equation: A blank: absorbance value of the DPPH blank sample A sample: absorbance value of the extract solution. Ascorbic acid was used as a positive control, and the percent inhibition versus extract concentration curve was used to measure concentrations of 50% inhibition (IC 50 ).

α-Amylase Inhibition Assay.
e α-amylase inhibition assay was conducted by reacting different concentrations of C. sativus stigma aqueous extracts with α-amylase enzyme and starch solution, as previously described [18], with slight modifications. Briefly, 250 µl of extract was mixed with 250 µl of α-amylase (240 U/ml, in 0.02 M phosphate buffer solution (PBS), pH 6.9, with 0.006 M NaCl) and incubated at 37°C for 10 min. After incubation, 250 µl of 1% (w/v) soluble starch (in 0.02 M PBS, pH 6.9) was added, and the obtained mixture was subjected to a second incubation at 37°C for 30 min. en, 250 µl of DNS color reagent was added, and the reaction was stopped by heating in a boiling water bath for 10 min. After cooling to room temperature, the mixture has been diluted with 2 ml of PBS, and the absorbance was measured at 540 nm.

α-Glucosidase Inhibition
Assay. Different concentrations of extracts were prepared in distilled water. However, the α-glucosidase enzyme (0.1 U/ml) and substrate p-Nitrophenyl-α-D-glucopyranoside (p-NPG, 1 mM) were dissolved in PBS (0.1 M, pH 6.7). Afterward, 150 μl of each extract concentration was preincubated with 100 μl of enzyme at 37°C for 10 min, and then 200 μl of substrate was added to the reaction mixture. e enzymatic reaction was conducted at 37°C for 30 min and stopped by adding 1 ml of Na 2 CO 3 (1 M). All samples were analyzed in triplicate with different concentrations to determine the IC 50 values, and the absorbance was measured at 405 nm. e inhibition percentage (%) was calculated by the following equation for both assays: Absorbance was abbreviated as follows: AC (control), AC b (control blank), AS (sample), and AS (sample blank).

Determination of Antibacterial Activity.
e antibacterial activity of C. sativus extracts was evaluated against four Gram-negative bacteria, including E. coli ATCC 25922, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 15442, and Klebsiella pneumonia ATCC 43816, and two Gram-positive bacteria, including Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 13932. Before conducting experiments, the bacterial cultures were inoculated on Tryptone Soya Agar (TSA) (Biokar, Beauvais, France) from the frozen stock and incubated at 37°C during 24 h.

Disc Diffusion Assay.
e antibacterial activity of C. sativus extracts was evaluated by disc diffusion method according to the protocol described previously [19], with a few modifications. Briefly, a bacterial suspension equivalent to 0.5 McFarland (10 8 cfu/mL) was prepared and swabbed on the surface of Mueller-Hinton agar (MHA) (Biokar, Beauvais, France) plates. Afterwards, a 6 mm diameter filter disc (Whatman No. 4) soaked in 20 µl of each extract (1 g/ml) was deposited on the surface of MHA plate. Sterile distilled water (10 µl) and gentamicin (30 µg) were used, respectively, as negative and positive controls of this study. e plates were left for 15 minutes until the absorption of extracts, and then, they were incubated at 37°C for 18-24 h. After incubation, the inhibition diameter of each extract was measured in millimeters (disk included).

Determination of MIC and MBC. Determination of Minimum Inhibitory Concentration (MIC) and Minimum
Bactericide Concentration (MBC) was conducted according to the microplate method following the protocol described previously [20]. Briefly, decreasing concentration of extracts was prepared, inoculated by bacterial inoculum, and incubated at 37°C for 24 h. After incubation, the wells correspond to the lower concentration and do not present visible growth of bacteria were considered as the MIC. e MBC was determined by subculturing samples from the wells on TSA (Biokar, Beauvais, France) and incubated at 37°C for 24 h. e lower concentration that did not present any growth on TSA was considered as the MBC.
2.9. Statistical Analysis. Microsoft Excel (New York, USA) was used for data analysis. Experiments were performed in triplicate, and the results were presented as means ± standard error. Student's test (P < 0.05) was used to evaluate the statistical difference between the studied extracts.

Determination of Total Phenolic and Flavonoid Contents.
e total phenolic contents (TPC) and the total flavonoid contents (TFC) of the aqueous extracts of C. sativus are presented in Table 1. e results showed that the Moroccan extract contained a high TPC (72.47 ± 1.62 mg GAE/g extract) compared to the Italian extract (31.47 ± 0.18 mg GAE/ g extract). Similarly, flavonoids content showed higher amounts in Moroccan extract (51.47 ± 0.32 mg CE/g extract) than Italian extract (22.61 ± 1.25 mg CE/g extract). Statistical analysis showed that the difference in TPC and TFC between the Moroccan and Italian extracts is statistically significant (P < 0.05). ese can be explained by the difference in ecological conditions, such as soil type, microclimatic conditions, geographic position, sampling site, age, and vegetative stage of plants and fruits [21][22][23]. Phenolic compounds are natural antioxidants occurring in green vegetables, fruits, and oils [24,25]. Some phenolic compounds have been reported to possess anti-inflammatory, antiviral, and anticancer activities [24,26]. ese compounds have gained attention because of the protective effect played by antioxidants in many pathological diseases, in which oxidative stress is implicated [4].

Determination of Mineral Content.
Mineral elements are involved in important biological functions of the cells. e mineral content, including the macroelements (Ca, P, K, Mg, and Na), the microelements (S, Co, Fe, B, Cu, Zn, Mn, and V), and the heavy metals (Cd, Pb, Ni, Mo, Cr, and AS), was determined in both Moroccan and Italian C. sativus extracts, and the results are presented in Table 2. Our results indicate that potassium (K) was the most abundant macroelement in both extracts, with concentration ranging from 55.32 g/kg in the Moroccan extract to 53.021 g/kg in the Italian extract. Sulfur was the most abundant microelement with a concentration of 5.4 g/kG for the Moroccan extract and 4.8 g/Kg for the Italian extract. For the other macro-and microelements, interesting differences were recorded between the Moroccan and Italian extracts; these differences can be explained by climatic conditions, pedologic condition, crop cycle length, drying method, irrigation, corm planting, and dimension [27].

Antioxidant Activity.
In a preliminary investigation of the antioxidant potential of C. sativus extracts, we investigated the radical scavenging activity by DPPH. e property of the DPPH• radical, which is remarkably stable, is due mainly to the steric hindrance around the divalent nitrogen and, to a lesser extent, to the "push-pull" effect of electron donor and acceptor (diphenylamino and picryl group, respectively) onto the divalent N [28]. During the reaction, free radicals accept a single pair of electrons or hydrogen atoms and converge from purple to yellow color. e scavenging effect of C. sativus extracts and the ascorbic acid on DPPH radical was expressed by IC 50 , which is presented in Table 3. Our results showed that the Moroccan extract presents the higher antioxidant power (IC 50 � 0.32 ± 0.059 μg/ml) compared with the Italian extract (IC 50 � 3.14 ± 0.021 μg/ml). Interestingly, the antioxidant activity of Moroccan extract showed a comparable IC 50 with Ascorbic acid (0.29 ± 0.003 µg/ml). In fact, previous studies have investigated the DPPH radical scavenging effect of flowers, petals, and leaves of C. sativus [12,24,29], while few papers have described the antioxidant activity of stigmas [30]. Additionally, the difference between the antioxidant activity of Moroccan and Italian extracts could be due to the difference in their phenolic and flavonoid contents. Indeed, many studies previously mentioned that phenolic and flavonoid compounds have remarkably antioxidant properties in both in vitro and in vivo systems [28,31,32].

Antidiabetic Activity.
e inhibitory activity of both Moroccan and Italian extracts against enzymes α-glucosidase and α-amylase was evaluated, and the obtained results are summarized in Table 4. e results showed that the Moroccan extracts exhibited the higher inhibitory activity against α-glucosidase and α-amylase, with an IC 50 of 75.25 ± 1.05 μg/ml and 687.12 ± 0.21 μg/ml, respectively, while the Italian extract exhibited the lower inhibitory activity with and IC 50 of 97.83 ± 1.03 μg/ml against α-glucosidase and an IC 50 of 720.05 ± 1.01 μg/ml against α-amylase.
e potency of C. sativus aqueous extracts to inhibit the enzymes α-glucosidase and α-amylase suggests that the compounds possessing antidiabetic activity are extracted in water, supporting the traditional use of aqueous extract of C. sativus in the treatment of diabetes. In fact, many previous studies have demonstrated the hypoglycemic effect of phenolic compounds, by improving postprandial blood glucose levels, acute insulin secretion, and insulin sensitivity, since limiting the rate of glucose absorption from the intestines into the bloodstream was considered the only strategy that could help prevent diabetes [33].

Antibacterial Activity.
e antibacterial activity of aqueous extracts of C. sativus was performed against six bacterial strains belonging to Gram-negative and Grampositive bacteria, by disc diffusion assay and broth dilution method.
e results of disc diffusion assay showed that Moroccan C. sativus extract creates an inhibition diameter ranging from 21.3 ± 0.9 mm to 22.8 ± 1.2 mm against Grampositive bacteria and from 9.1 ± 0.1 mm to 18.2 ± 0.3 mm against Gram-negative bacteria (Figure 1), while Italian extract creates an inhibition zone ranged from 18.4 ± 0.4 mm to 22.8 ± 1.2 mm against Gram-positive bacteria and from 8.0 ± 0.1 mm to 15.1 ± 0.1 mm against Gram-negative bacteria ( Figure 1). However, results of broth dilution method showed that Moroccan C. sativus extract had a MIC value of 3.9 mg/ml for Gram-positive bacteria, and MIC ranged from 15.62 mg/ml to 62.5 mg/ml for Gram-negative bacteria (Table 5). Likewise, Italian C. sativus extract had a MIC value of 7.81 mg/ml for Gram-positive bacteria, and the value was between 31.25 mg/ml and 125 mg/ml for Gram-negative bacteria (Table 5). In addition, our results demonstrated that both C. sativus extracts had a bactericidal effect with a ratio of MBC/MIC<4. So far, some worldwide studies have reported the antimicrobial activity of C. sativus extracts. In a study performed previously in Morocco, Jadouali and colleagues showed that leave extracts of C. sativus had a great antibacterial activity against Listeria sp. [29]. Also, Vahidi and his group studied the antimicrobial activity of C. sativus  Data are expressed as mean ± SD (n � 3). TFC: total flavonoid content; TPC: total phenolic content; mg CE/g extract: mg catechin equivalent per gram of extract; mg GAE/g extract: mg Galic Acid equivalent per gram of extract. Different letters in the same row represent significant differences at P < 0.05. 4 Advances in Pharmacological and Pharmaceutical Sciences extracts harvested from Khorasan in Iran and prepared them with different solvents against various strains of bacteria and fungi [34]. However, Asgarpanah et al. reported the antimicrobial effect of methanol extract prepared from different parts of C. sativus harvested from southern Khorasan province in Iran against various strains of bacteria and fungi [35]. In Turkey, ethanol and aqueous extracts of C. sativus showed interesting antibacterial effect against mastitis pathogens [36]. And, Muzaffar et al. showed the high antibacterial activity of methanolic and petroleum ether extracts of C. sativus collected from India [37].
Our results showed that aqueous extract of C. sativus is more effective against Gram-positive bacteria than Gramnegative. is difference could be due to the difference in the cell wall structure of the tested bacteria. Indeed, the susceptibility of Gram-positive bacteria to C. sativus extract in comparing with Gram-negative bacteria might be explained by the higher susceptibility of its cell wall to the chemical components containing extracts. Nevertheless, the lower sensitivity of Gram-negative bacteria can be explained by the difficulty of the extract components to diffuse through the hydrophilic barrier of the cell wall [34]. Additionally, our results showed that the antibacterial activity of C. sativus extract originating from Morocco is higher than that originating from Italy. is difference could be linked to the difference in the chemical composition of the studied extracts. In fact, the richness of Moroccan extract with flavonoids and phenolic compounds may strengthen its antibacterial activity [35,38].

Conclusion
Morocco is a country that has rich plant resources with a high diversity of medicinal plants that are used in the treatment and prevention of several illnesses. From this study, we conclude that Moroccan C. sativus stigmas extract has a great antioxidant, antimicrobial, and antidiabetic properties and is rich in mineral elements. erefore, it can be used as a source of bioactive compounds and in the treatment of infectious diseases caused by resistant bacteria, in addition to the broad applications in food and medical fields. However, based on the results of this study, further in vivo and ex vivo confirmatory tests for effective extracts and subfractions of C. sativus are recommended.

Data Availability
All used data in this study are included within the manuscript.    Data are expressed as mean ± SD (n � 3). Different letters in the same row represent significant differences at P < 0.05. Data are expressed as mean ± SD (n � 3). Different letters in the same row represent significant differences at P < 0.05.

Conflicts of Interest
Advances in Pharmacological and Pharmaceutical Sciences 5