Medicinal plants, vegetables and food products are considered to be rich sources of natural compounds that may play an important role in human health, by maintaining it, or preventing and curing diseases. In medicinal plants, these compounds are considered as bioactive natural compounds that can be subsequently developed as new drugs. In foods, they would be defined as phytonutrients, without having therapeutic claims but with important health benefits that can be ultimately used in disease prevention [1
L. (black cumin) is an annual herbaceous plant belonging to the Ranunculaceae
family, widely grown in the Mediterranean countries, Middle East, Eastern Europe and Western Asia [3
]. The black cumin seeds taste hot-peppery and have been used as a spice in several foods, such as bread, yogurt, pickles, sauces and salads [3
], but also in the preparation of black cumin paste [4
], being considered as a valuable functional food [5
]. They are extensively used in traditional medicines in Pakistan, India, China, Saudi Arabia and the countries bordering the Mediterranean region for the treatment of asthma, cough, bronchitis, headache, rheumatism, fever, kidney and liver disorders, influenza, eczema, and as a diuretic, lactagogue, carminative and vermifuge [6
]. Recent scientific investigations on Nigella sativa
seeds and its oil indicate a number of bioactivities for the plant, which include anticarcinogenetic [7
], antiulcer [8
], antibacterial and antifungal [9
], antihypertensive [11
], hepatoprotective [12
], anti-inflammatory, antipyretic and analgesic [13
], as well as antioxidant activities such as quenching reactive oxygen species [14
], prevention of rheumatoid arthritis in rat models [15
], and antihyperlipidemic [16
L., commonly known as lady-in-a-mist or ragged lady, is an annual plant belonging to the buttercup family Ranunculaceae
and widespread throughout temperate regions of Europe [17
]. N. damascena
is grown as an ornamental plant. Its seeds are used in traditional medicine because of their analgesic, anti-edematous and antipyretic effects and, due to their sweet scent of strawberry, to prepare food [18
The two Nigella
species have been used since ancient times for medicinal purposes but also as a food or preservative for foods [20
]. Due to its adaptability, N. sativa
had a higher commercial value and a larger growing area than N. damascena
has had, and as a consequence there has also been more investigation into the chemical composition and biological activity of N. sativa
than that of N. damascena
. Black cumin produces a wealth of phytochemicals including fixed and volatile oils, proteins, flavonoids, glycosides, alkaloids (mainly magnoflorine), and saponins [21
]. N. damascena
seeds contain fatty and essential oils, proteins, alkaloids, flavonoids and saponins [17
The full complement of bioactive compounds from Nigella
species has yet to be elucidated, a step necessary in order to explain their medicinal and alimentary uses. Although N. sativa
itself is a very competitive subject for phytochemical studies, little is known on the comparative chemical composition of black cumin grown in different regions, and even less on that of other taxa in the genus Nigella
, in this case, N. damascena.
However, as one can notice after a literature review, the phytochemical investigations related to Nigella
species are mostly concerned on volatile or fatty oils composition and their bioactivities and neglect, for instance, their phenolic composition. Dietary phenolic compounds received tremendous attention among nutritionists, food scientists and consumers due to their roles in human health. Research in the past years strongly supports a role for polyphenols in the prevention of degenerative diseases, particularly cancers, cardiovascular and neurodegenerative diseases [23
]. Also, in recent time, new diuretic drugs have been proposed mainly from natural products and among these, the polyphenolic compounds have received special attention from scientists. Regarding the bioactivities of N. sativa
, only isolated publications deal with the diuretic activities of essential oil and dichloromethane extract of black cumin’s seeds [25
], but no data was found related to the N. damascena
To overcome the lack of information regarding the polyphenolic composition and diuretic potential of N. sativa and N. damascena seeds, the aim of this study was to set a comparative study between these two taxa, in order to bring valuable new data regarding the nutritional value of Nigella seeds related to their polyphenolic compounds (phenolic acids, flavonoids and flavonols), antioxidant and diuretic effects. To increase our understanding of the pharmacological and nutraceutical properties of N. sativa and N. damascena we employed a rapid, highly accurate and sensitive HPLC method in tandem with mass-spectrometry for the simultaneous determination of the phenolic compounds. Furthermore the antioxidant potential of the two species was tested by several assays pointing the different antioxidant facets of Nigella extracts. Finally, the diuretic properties of black cumin and lady-in-a-mist seeds were tested using a rat experimental model.
3. Experimental Section
3.1. Plant Materials and Extraction Procedure
The vegetal material from N. sativa and N. damascena seeds was harvested from Arad County, Romania, in the summer of 2014. Voucher specimens (Voucher Nos. 10/2014 and 11/2014) were deposited in the Herbarium of the Pharmacognosy laboratory of the Vasile Goldiş Western University of Arad, Romania. The plant material was air dried at room temperature in the shade, separated and ground to a fine (≤300 µm) powder and then extracted. One gram of each sample was weighed and macerated with 5 mL of 70% ethanol, for 10 days. The samples were then centrifuged at 4000 rpm for 30 min, and then the supernatant was recovered. In order to obtain more accurate data on flavonoid glycosides and aglycones concentration, each sample was analyzed before and after acid hydrolysis. Extractive solution (2 mL) was treated with 2 M hydrochloric acid (2 mL) and ascorbic acid solution (0.2 mL, 100 mg∙mL−1), and the mixtures were heated at 80 °C on a water bath for 30 min, ultrasonicated for 15 min, and heated for another 30 min at 80 °C. During the heating, methanol (1 mL) was added to the extraction mixture every 10 min, in order to ensure the permanent presence of methanol. The mixtures were centrifuged at 4000 rpm and the solutions were diluted with distilled water in a 10 mL volumetric flask and filtered through a 0.45 μm filter before injection.
3.2. Chemical and Instrumentation
Chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercetin, isoquercitrin, quercitrin, hyperoside, kaempferol, myricetin, fisetin from Sigma (St. Louis, MO, USA), ferulic acid, sinapic acid, gentisic acid, patuletin, luteolin from Roth (Karlsruhe, Germany), cichoric acid, caftaric acid were from Dalton (Toronto, ON, Canada). HPLC grade methanol, ethanol, hydrochloric acid, ferric chloride, copper chloride and Folin-Ciocalteu reagent were purchased from Merck (Darmstadt, Germany), while 2,4,6-tris(2-pyridyl)-s-triazine radical (TPTZ), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,9-dimethyl-1,10-phenantroline (Neocuproine), sodium molybdate dihydrate, sodium nitrite, sodium hydroxide, sodium carbonate, sodium acetate trihydrate, and anhydrous aluminum chloride were from Sigma-Aldrich (Steinheim, Germany). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Alfa-Aesar (Karlsruhe, Germany). All spectrophotometric data were acquired using a Jasco V-530 UV-VIS spectrophotometer (Jasco International Co., Ltd., Tokyo, Japan).
3.3. HPLC-UV/MS Analysis of Polyphenols
3.3.1. Apparatus and Chromatographic Conditions for the Analysis of Polyphenols
The identification and quantification of polyphenolic compounds was carried out using an Agilent Technologies 1100 HPLC Series system (Agilent, Santa Clara, CA, USA) equipped with G1322A degasser, G13311A binary gradient pump, column thermostat, G1313A autosampler and G1316A UV detector. The HPLC system was coupled with an Agilent 1100 mass spectrometer (LC/MSD Ion Trap SL). For the separation, a reverse-phase analytical column was employed (Zorbax SB-C18 100 × 3.0 mm i.d., 3.5 μm particle) and the work temperature was set at 48 °C. The detection of the compounds was performed on both UV and MS mode. The UV detector was set at 330 nm until 17.5 min, then at 370 nm. The MS system operated using an electrospray ion source in the negative mode. ChemStation and DataAnalysis software from Agilent were used for processing the chromatographic data. The mobile phase was a binary gradient: methanol and acetic acid 0.1% (v/v). The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol, for 35 min; then 42% methanol for the next 3 min [34
]. The flow rate was 1 mL∙min−1
and the injection volume was 5 µL.
The MS signal was used only for qualitative analysis based on the specific mass spectra of each polyphenol. The MS spectra obtained from a standard solution of polyphenols were integrated in a mass spectra library. Later, the MS traces/spectra of the analyzed samples were compared to spectra from library, which allows positive identification of compounds, based on spectral match. The UV trace was used for quantification of identified compounds from MS detection [34
3.3.2. Identification and Quantification of Polyphenols
Using the chromatographic conditions described above, the polyphenols eluted in less than 40 min. The detection and quantification of polyphenols was performed in UV-assisted mass spectrometry detection. Due to peak overlapping, four polyphenol-carboxylic acids (caftaric, gentisic, caffeic, and chlorogenic) were determined only based on MS spectra, whereas for the rest of the compounds the linearity of the calibration curves was very good (R2
> 0.998), with detection limits in the range of 18 to 92 ng∙mL−1
. The detection limits were calculated as the minimal concentration yielding a reproducible peak with a signal-to-noise ratio greater than three. Quantitative determinations were performed using an external standard method; retention times were determined with a standard deviation ranging from 0.04 to 0.19 min. Calibration curves in the 0.5–50 µg∙mL−1
range with good linearity (R2
> 0.999) for a five point plot were used to determine the concentration of polyphenols in plant samples. For all compounds, the limit of quantification was 0.5 µg∙mL−1
, and the limit of detection was 0.1 µg∙mL−1
. The detection limits were calculated as minimal concentration producing a reproductive peak with a signal-to-noise ratio greater than three. The accuracy was between 94.13% and 105.3%. Accuracy was checked by spiking samples with a solution containing each polyphenol in a 10 µg∙mL−1
concentration. In all analyzed samples the compounds were identified by comparison of their retention times and recorded electrospray mass spectra with those of standards in the same chromatographic conditions [34
3.4. Determination of Total Polyphenols and Flavonoids Content
The total phenolic content (TPC) of the extracts was measured using the Folin-Ciocalteu method with some modifications [34
]. Two milliliters from each ethanolic extract were diluted 25 times and then mixed with Folin-Ciocalteu reagent (1 mL) and distilled water (10.0 mL) and diluted to 25.0 mL with a 290 g∙L−1
solution of sodium carbonate. The samples were incubated in the dark for 30 min. The absorbance was measured at 760 nm, using a JASCO UV-VIS spectrophotometer. Standard curve was prepared by using different concentrations of caffeic acid and the absorbances were measured at 760 nm. TPC values were determined using an equation obtained from the calibration curve of caffeic acid (R2
= 0.999). Total polyphenolic content was expressed as mg caffeic acid/g dry material plant (mg CAE/g plant material).
The total flavonoids content was calculated and expressed as rutin equivalents after the method described in the Romanian Pharmacopoeia (Xth Edition) [38
]. Each extract (5 mL) was mixed with sodium acetate (5.0 mL, 100 g∙L−1
), aluminum chloride (3.0 mL, 25 g∙L−1
), and made up to 25 mL in a calibrated flask with methanol. Each solution was compared with the same mixture without reagent. The absorbance was measured at 430 nm. The total flavonoids content values were determined using an equation obtained from calibration curve of the rutin graph (R2
3.5. In Vitro Antioxidant Activity Assays
3.5.1. DPPH Bleaching Assay
The free radical scavenging activity of the ethanolic extracts was measured in terms of hydrogen donating or radical scavenging ability using this method. Trolox was chosen as a standard antioxidant. The DPPH solution (25 mM) in ethanol was prepared and 5.0 mL of this solution was added to 5.0 mL of extract solution (or standard) in ethanol at different concentrations (4–200, respectively, 5–20 µg∙mL−1
for extracts and standard). After 30 min of incubation at 40 °C in a thermostatic bath, the decrease in the absorbance (n
= 3) was measured at 517 nm. The percent of DPPH discoloration was calculated as: DPPH scavenging ability = (Acontrol
) × 100, where Acontrol
is the absorbance of DPPH radical + ethanol (containing all reagents except the sample) and Asample
is the absorbance of DPPH radical + sample extract. The control solution was prepared by mixing 70% vol. ethanol (5.0 mL) and DPPH radical solution (5.0 mL). Afterwards, a curve of % DPPH scavenging capacity vs.
concentration was plotted and IC50
values were calculated. IC50
denotes the concentration of sample required to scavenge 50% of DPPH free radicals [37
]. The lower the IC50
value is the more powerful the antioxidant capacity [37
]. For all the samples, the determinations were made in triplicate and the mean values were reported.
3.5.2. Trolox Equivalent Antioxidant Capacity (TEAC) Assay
In the Trolox equivalent antioxidant capacity (TEAC) assay, the antioxidant capacity is reflected in the ability of the natural extracts to decrease the color, reacting directly with the ABTS cation radical. The latter was obtained by oxidation of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) with potassium persulfate. Original extracts were diluted 5 times, and 3 µL from the diluted extract were added to 997 µL ABTS solution. The amount of ABTS radical consumed by the tested compound was measured at 734 nm, after 30 min of reaction time. The evaluation of the antioxidant capacity was obtained using the total change in absorbance at this wavelength; all determinations being made in triplicate [34
3.5.3. Ferric Reducing Antioxidant Power (FRAP) Method
FRAP method is based on the change in color of a complex with iron of the TPTZ radical, 2,4,6-tris(2-pyridyl)-s-triazine and on reduction of the ferric ion to the ferrous iron in this complex [39
]. The color of the complex is turned from light yellowish-green to blue. This color change can be easily correlated with the antioxidant power by a spectrophotometric determination. A 2.5 mL, 10 mM TPTZ solution in 40 mM hydrochloric acid are added to 2.5 mL, 20 mM ferric chloride solution and 25 mL acetate buffer (pH 3.6). This mixture is the FRAP reagent. A 0.2 mL sample were added 0.6 mL water and 6 mL FRAP reagent. Each extract was diluted 2 to 10 mL with water. Also, a blank solution using water instead of the samples, was prepared in the same way. Trolox was used as standard antioxidant. The spectrophotometric determination was performed at 593 nm. For all the samples the determinations were made in triplicate and the mean values were reported.
3.5.4. Cupric Reducing Antioxidant Capacity (CUPRAC) Assay
CUPRAC method is based on the change in color of a complex with copper of the Neocuproine, 2,9-dimethyl-1,10-phenantroline and on the reduction of copper ion (II) to the copper ion (I) from this complex. The color of the complex is turn from light green to reddish-orange. This color change can be easily correlated with the antioxidant power by a spectrophotometric determination. A 1 mL, 7.5 mM neocuproine solution, 1 mL, 10 mM copper chloride solution, and 1 mL ammonium acetate buffer at pH = 6.8 are added. This mixture is the CUPRAC reagent. A 0.1 mL sample was added to 1 mL water and 3 mL CUPRAC reagent. The mixture was incubated at room temperature for 30 min. The Nigella
extracts were diluted 1:10 mL (v
) with water before the analysis. Trolox, (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), was used as standard antioxidant in a range of 10–50 µg∙mL−1
and the spectrophotometric determination was performed at 450 nm [31
]. All the determinations were made in triplicate and the mean values were reported.
3.6. In Vivo Studies
Experiments were performed on Wistar Bratislava male rats (ca. 200 g each). All animals were maintained in standard conditions and were selected by checking their diuresis with water. The experiments were realized according to the guidelines of the European Community Council Directive 86/609 and after being approved by the Ethic Committee of University of Medicine and Pharmacy ‘Iuliu Hatieganu’ from Cluj-Napoca, Romania.
Furosemide (Sigma-Aldrich Co.) was used as a reference diuretic drug.
3.6.3. Evaluation of Diuretic Activity
The evaluation of diuretic activity test was performed on male rats as already described by Olah et al.
and Compaore et al
]. Male Wistar rats were divided into four groups, of six animals each, in laboratory cages. They were fed with standard laboratory diet ad libitum
and allowed free access to drinking water. The animals were deprived of food and water for 24 h prior to the experiment and were divided into four groups of six rats each. Then, each animal received 2.5 mL 100 g−1
p.o. 0.9% natrium chloride solution for hydration. After 1 h, the following substances were administrated orally.
The first group of animals, serving as control, received 1 mL/rat of distilled water, the second group received furosemide (30 mg kg−1, p.o.) in distilled water (as standard), and the third and fourth groups received the ethanolic extracts of N. sativa and N. damascena, respectively, at a dose of 100 mg kg−1 in distilled water.
3.6.4. Analytical Procedure
The diuresis was expressed in mL/kg/24 h, and the diuretic index was calculated as a ratio of the values in the treated groups compared to the control group. The concentration of sodium and potassium ions were measured in urine by a potentiometric method, using a VITROS 250 Chemistry System auto-analyzer (Johnson and Johnson Clinical Diagnostic, Cluj-Napoca, Romania) and were expressed in mM/kg/24 h. Saluretic index was calculated for sodium and potassium as a ratio of their concentrations in the treated groups compared to the control group [42
]. Uric acid concentration was determined using a colorimetric method at λ = 670 nm, through an enzymatic reaction which transforms uric acid in a colored compound, and finally was expressed in mg∙kg−1
3.7. Statistical Analysis
The experiments were designed and the experimental data evaluated using one-way analysis of variance (ANOVA), with p < 0.05 as threshold for statistical significance. The statistical results confirm the hypothesis that the differences between the results are either not significant (p > 0.05), significant (0.001 < p < 0.05) or highly significant (p < 0.001). The average of multiple measurements (triplicates or more) was listed in the Tables together with the standard deviations. Statistical analysis was performed using Excel software package.
The results of the present study reveal important data regarding the phenolic composition, antioxidant and diuretic effects of seed extracts from two medicinal plants that are also considered to be important functional foods, black cumin and lady-in-a-mist. Referring to phytochemical investigations, the differences between the two species are both qualitative and quantitative. Quercetin and hyperoside were quantified only in N. damascena seeds extract, meanwhile, kaempferol was found only in N. sativa. In the hydrolyzed extracts, both quercetin and kaempferol were found in higher amounts in N. sativa. Nevertheless, the presence of p-coumaric and ferulic acids in the hydrolyzed extracts reveal the existence of several glycoside conjugates of these acids. In terms of total phenolics and flavonoids, N. damascena extract contains higher amounts. The antioxidant potential of the two species was tested through several electron transfer assays, which indicated N. damascena as exhibiting a higher free radical scavenging activity. Administration of the ethanolic extract of N. sativa (100 mg∙kg−1) resulted in a significant increase in urine volume, although less than found with the reference drug, but N. damascena extract did not present a diuretic effect. In reference to the elimination of Na+, K+ and uric acid, the extract of N. sativa showed a greater natriuretic than kaliuretic effect and a similar uricosuric effect with control and N. damascena. For N. damascena, the Na+/K+ was sub unitary, but not due to an increase of the kaliuretic effect, but mostly to a decrease of Na+ excretion.