Received: 25 November 2022 | Revised: 30 May 2023 | Accepted: 27 June 2023

DOI: 10.1002/efd2.107

RESEARCH ARTICLE

LC‐MS based metabolite profiling, in‐vitro antioxidant and

in‐vivo antihyperlipidemic activity of Nigella sativa extract

Amit Kumar Shrivastava1 | Laxmi Shrestha1 | Buddhi Raj Pokhrel2 |

Bishal Joshi3 | Gopal Lamichhane4 | Bojana Vidović5 | Niranjan Koirala6

1
Department of Pharmacology, Universal Abstract
College of Medical Sciences, Bhairahawa,
Rupandehi, Nepal The aim of this study was to identify the bioactive phytoconstituents present in the
2 aqueous extract of Nigella sativa and also, to evaluate the antioxidant and
Department of Biochemistry, Universal
College of Medical Sciences, Bhairahawa, antihyperlipidemic activity in Wistar rats. The LC‐MS/MS analysis was assessed for
Rupandehi, Nepal the determination of different bioactive compounds present in N. sativa extract. Total
3
Department of Physiology, Universal College phenolic and flavonoid content were determined by using validated Folin‐Ciocalteu
of Medical Sciences, Bhairahawa, Rupandehi, and Aluminum chloride colorimetric methods, respectively. The in‐vitro antioxidant
Nepal
and in‐vivo antihyperlipidemic activity in Wistar rats were also evaluated. Preliminary
phytochemical screening of the extract showed the presence of alkaloids, flavonoids,
4
Department of Oriental Pharmacy, Wonkwang‐
Oriental Medicines Research Institute,
Wonkwang University, Iksan, South Korea phenols, glycosides, and amino acids in the aqueous extract. The bioactive compounds
5 of the aqueous extract were identified through LC‐MS/MS analysis. The in‐vitro
Department of Bromatology, Faculty of
Pharmacy, University of Belgrade, Belgrade, antioxidant activity of N. sativa showed the highest free radical scavenging capacity in
Serbia DPPH, H2O2, and OH radical scavenging assays with IC50 values 11.916 ± 2.828,
6
Research Laboratory, Gandaki Province 30.294 ± 13.790, and 12.048 ± 2.828 µg/mL, respectively. Evaluation of antihyperlipi-
Academy of Science and Technology, Pokhara, demic activity of extract in Wistar rats showed that a high dose (800 mg/kg) of
Gandaki Province, Nepal
extract significantly decreased total cholesterol (TC) 71.76 ± 6.91 mg/dL, TG
83.6 ± 8.09 mg/dL, low‐density lipoproteins (LDL‐c) 33.86 ± 6.05 mg/dL, very low‐
Correspondence
Amit Kumar Shrivastava and Laxmi Shrestha,
density lipoproteins (VLDL‐c) 16.72 ± 1.61 mg/dL level in blood. However, the
Department of Pharmacology, Universal HDL‐C level was significantly improved (21.18 ± 1.80 mg/dL) as compared to HFD‐
College of Medical Sciences, Bhairahawa, induced control rats (11.76 ± 1.14 mg/dL) after 28 days of treatment. Also, at the same
Rupandehi, Nepal.
Email: [email protected] and
dose, animal body weight was also decreased to 162.6 ± 16.40 g compared with control
[email protected] 184.4 ± 10.24 g. The aqueous extract of N. sativa was found to be an effective natural
Niranjan Koirala, Research Laboratory, source of antioxidant and hypolipidemic agents. This activity was attributed to the
Gandaki Province Academy of Science and presence of diverse bioactive compounds in it.
Technology, Pokhara, Nepal.
Email: [email protected]
KEYWORDS
Funding Information antihyperlipidemia activity, antioxidant activity, DPPH, dyslipidemia, Nigella sativa
None

1 | INTRODUCTION lipoprotein cholesterol (HDLc) are measured due to their

clinical significance (Ahn & Choi, 2015). Several studies
Dyslipidemia is the major risk factor and predictor of revealed that hyperlipidemia promotes atherosclerosis in
cardiovascular diseases (CVD) and balancing lipid profiles in blood vessels that may lead to increases the risk of cardiac
specified range can protect one from its consequences. ischemia/reperfusion injury, myocardial infarction, and other
In general, serum triglyceride (TG), total cholesterol CVDs (Balakumar & Babbar, 2012; Pathak et al., 2015; Yao
(TC), low‐density lipoprotein cholesterol (LDLc), very low‐ et al., 2020). The retention of low‐density lipoprotein into the
density lipoprotein cholesterol (VLDLc), and high‐density arterial wall promotes oxidation of reactive oxygen species

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
© 2023 The Authors. eFood published by John Wiley & Sons Australia, Ltd on behalf of International Association of Dietetic Nutrition and Safety.

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2 of 19 | LC‐MS BASED METABOLITE PROFILING AND ACTIVITY OF NIGELLA SATIVA EXTRACT

(ROS) which initiates the formation of complexes through 2.1 | Plant material and authentication
biochemical and inflammatory reactions. The oxidized
lipoproteins affect microphases to form foam cell through The seed of N. sativa was purchased from the local
activation of scavenger receptors which results in promoting market of Bhairahawa, Rupendehi, Nepal. The seeds
cholesterol biosynthesis (Delgado et al., 2012). In addition, were authenticated and reconfirmed by Pratikshya
an increase in the production of ROS plays a role in Chalise, Research Superintend, National Herbarium
development of atherosclerosis. ROS also interact with and Plant Laboratories, Godavari, Lalitpur, Kathmandu
different types of biomolecules like lipids, carbohydrates, (Registration number 165).
proteins, and so forth in the body and alters cellular
functions (Yang et al., 2008). Therapeutic intervention
available for dyslipidemia nowadays can be grouped as; 2.2 | Chemicals
statins, bile‐acid sequestrants, fibrates, ezetimibe, niacin,
omega‐3 fatty acids, apo‐B antisense oligonucleotides, The high‐fat diet (vanaspati ghee and coconut oil 3:1
proprotein convertase subtilisin/kexin type 9 (PCSK‐9) ratio) were purchased from the local market. All the
inhibitors and, microsomal triglyceride transport protein chemicals were used in the present study are ACS grade.
inhibitors (Ahn & Choi, 2015; Zodda et al., 2018). They have
either limited efficiency or cannot be used due to incidence of
adverse reactions. To address these issues, inexpensive and 2.3 | Preparation of aqueous extract of
nontoxic natural phytochemicals that were traditionally used N. sativa (AENS)
as remedies have found insurgence in current trends of
pharmacological research. In search for a newer group of The N. sativa seeds were grounded into coarse powder
drugs, pharmacological research toward traditional herbal and weighed quantity were placed in maceration flask
plants have been widely exploited (Ahn & Choi, 2015; and sufficient amount of distilled water (1:5 ratio) was
Balbaa et al., 2016; Bensiameur‐Touati et al., 2017; Modi added. It was then kept at room temperature with
et al., 2021; Panthi et al., 2020; Sultan et al., 2014). occasional shaking. The complete maceration was done
Nigella sativa is an annual plant commonly grown in for about 72 h. After the maceration was completed, the
Europe, the Middle East, and Western Asia (Bamosa menstruum was collected, evaporated and the extract
et al., 2010; El‐Dakhakhny et al., 2002). This herb, also was obtained. The percentage yield was calculated and
known as black seed or black cumin, has been used extract was stored at 4°C in an air‐tight container
since ancient times for the treatment of various illnesses (Benlafya et al., 2014).
like bronchial asthma, headache, dysentery, infections,
hypertension, and diabetes mellitus (Bamosa et al., 2010). (Weight of extract) × 100
Recently, N. sativa has garnered much interest in the %yeild = . (1)
(Weight of powder)
scientific community owing to its ability to decrease
blood glucose and improve lipid profile (Daryabeygi‐ 2.4 | Preliminary phytochemical screening
Khotbehsara et al., 2017; Salama, 2011; Sultan
et al., 2014). Studies on hyperlipidemia‐induced rat The preliminary phytochemical screening of aqueous
models have also shown similar promising results of extract of N. sativa was carried out to detect presence
N. sativa extracts on lipid profiles. Decrease in serum different bioactive secondary metabolites (Asaduzzaman,
TG, TC, LDLc, and increase in HDLc were achieved to Nahar, Rahman, Hasan, Khatun, 2015).
varying degrees in various studies (Balbaa et al., 2016;
El‐Dakhakhny et al., 2002; Le et al., 2004). Similar findings
were reported in human studies as well (Daryabeygi‐ 2.5 | Liquid chromatography (LC)‐mass
Khotbehsara et al., 2017; Heshmati et al., 2015). spectroscopy (MS)/MS analysis
The present study aims to determine bioactive
compounds, measure antioxidant activity and lipid The MS analysis was performed by Central Drug
profile in hyperlipidemic rats. Research Institute (CDRI), Lucknow, Uttar Pradesh,
India.

2 | M A T E R I A L S AN D M E T H O D S
2.5.1 | Preparation of sample
The study was conducted in the Department of
Pharmacology, Universal College of Medical Sciences The N. sativa extract was dissolved in water to prepare
(UCMS), Bhairahawa, Nepal from August 2019 to stock solution of 1 mg/mL concentration. Again, the
April 2020 after the approval of Institutional Review primary stock solution was dissolved in acidified
Committee (IRC) (Registration number UCMS/IRC/ methanol (1% glacial acetic acid), further, the sample
186/18). was dissolved in methanol and was prepared 50 ng/mL
SHRIVASTAVA ET AL.
| 3 of 19

concentration of working sample. The working sample linear equation of gallic acid standard. The result was
aliquot was again dissolved in acetonitrile and stan- expressed as mg(GAE)/g of dry weight (Alhakmani
dard calibration curve was determined (Wahajuddin et al., 2013; Shrivastava et al., 2022).
et al., 2016).

2.7 | Determination of total flavonoid content

2.5.2 | Chromatographic techniques
The total flavonoid content was determined by using
The high‐performance liquid chromatography (HPLC) the aluminum chloride with colorimetric method based
system contains 200 series pump and auto sampler on the quantification of yellow‐orange color formation.
attached with temperature‐controlled device. The 10 µL The 0.5 mL (dilute in extraction solvent) of extract
of N. sativa sample was introduced into a column mixed with 2 mL of distilled water and 0.15 mL (5%)
specification such as Waters Atlantis C18 (4.6 × 50 mm, sodium nitrite and kept for 6 min. The 0.15 mL of
5.0 µm). The HPLC system was performed in isocratic (10%) AlCl3 (Aluminum chloride) and 1 mL of (1 M)
mode by using fixed ratio of mobile phase 47.5:47.5:5 NaOH (Sodium hydroxide) was added and adjust
consisting of acetonitrile: methanol: ammonium volume up to 5 mL with distilled water then, absorb-
formate buffer (10 mM, pH 4.5). Before use of mobile ance was measured at 510 nm. The flavonoid content
phase, it was filtered with 0.22 µm Millipore filter and was expressed as mg of quercetin equivalent (QE)/g
aired ultrasonically for 20 min. The separation was (Sen et al., 2013).
performed at room temperature and auto‐sampler was
carried out and determined the calibration standard by
injecting blank sample into the column (Wahajuddin 2.8 | In‐vitro antioxidant activity
et al., 2016).
The in‐vitro antioxidant activity of aqueous extract of
N. sativa was done by previously explained methods with
2.5.3 | MS analysis minor modification. DPPH● scavenging activity (More &
Makola, 2020; Shrivastava et al., 2021), nitric oxide
The MS analysis was performed by using API 4000 (NO●) scavenging activity (Kamble et al., 2020),
mass spectrometer equipped with electrospray ioniza- hydrogen peroxide (H2O2) scavenging activity (Kamble
tion (ESI) system. For the ESI system 5500‐volt electric et al., 2020), Hydroxyl (OH●) radical scavenging activity
supply, and instrument parameters like nebulizer gas, (Ujowundu, 2017), Metal chelating activity (Benchikh
curtain gas, auxiliary gas, and collision gas were set et al., 2022), Ferric reducing power (FRAP) activity
at 40, 13, 50, and 10, respectively. Also, the sample (Kamble et al., 2020), total antioxidant activity (Kamble
parameters decluttering potential (DP), collision energy et al., 2020).
(CE), entrance potential (EP), and collision exit
potential (CXP) were 110, 42, 10, 10 V; 50, 30, 4, 10 V
and 90, 33, 6, 8 V were set for N. sativa extract. 2.9 | Experimental animals
Whereas nitrogen was used as curtain and collision gas
similarly zero air was used as source gas. The mass Adult Wistar rats weighing about (150–200 g) were
spectrometer was run and performed multiple reaction purchased from Department of Plant Resources,
monitoring mode. The mass charge and atomic transi- Kathmandu, Nepal. All animals were housed in animal
tion was determined through m/z value (Wahajuddin house facility of Universal College of Medical Sciences
et al., 2016). (UCMS), Bhairahawa, Rupandehi, Nepal and were
maintained at a standard room temperature of 25 ± 1°C
and relative humidity of 45%–55%. The “12 h” light and
2.6 | Determination of total phenolic content the dark cycle was maintained for 7 days with free access
to standard diet and water under hygienic condition.
Total phenolic content was estimated using the Folin‐ After 1‐week of adaptation, the animals were used for the
Ciocalteu reagent method. The 0.5 mL of extract study.
(0.1–2 mg/mL) was mixed with 2 mL of Folin‐Ciocalteu
reagent (diluted 1:10 with distilled water) and added
Na2CO3 (7.5%) to neutralize the solution. The solution 2.10 | Experimental designs
mixture was incubated for 30 min at room temperature
with occasional shaking for the development of color. The animals were divided into five groups including
The resultant blue color was measured at 765 nm. The normal control, negative control (3 mL/kg HFD induced
reference gallic acid was used for plotting the calibration hyperlipidaemia), Atorvastatin (40 mg/kg), low dose
curve. The total phenolic content was calculated from the N. sativa extract (400 mg/kg) and high dose of N. sativa
4 of 19 | LC‐MS BASED METABOLITE PROFILING AND ACTIVITY OF NIGELLA SATIVA EXTRACT

extract (800 mg/kg), each group containing five (n = 5) was determined on different time intervals before and
animals. Before conducting the experiment, the ethical after starting the treatment (Rai et al., 2013).
approval was taken from Institutional Review Commit-
tee (IRC), UCMS, Bhairahawa, Nepal (Registration
number: UCMS/IRC/186/18). 2.13 | Blood and biochemical analysis
Before the induction of hyperlipidaemia, the rats were
2.11 | Acute toxicity study fasted for 6 h and anesthetized by diethyl ether. Then, the
blood sample from all groups were collected in a
The acute toxicity study was assessed previously heparinized tube from retro‐orbital plexuson initial day
described by Asif et al. (2015). In their study, an albino (0th day) of the treatment. After that same procedure
mouse of 18–25 g of body weight was used. The animals was followed 7, 14, and 28th days of treatment for
were divided into five groups, each group containing five measuring cholesterol and TG levels as described by
(n = 5) animals. The animals were treated orally with (Chen et al., 2000; Davooth et al., 2015). The sample was
aqueous extract of N. sativa up to 5000 mg/kg. After oral sent to the laboratory for determination of cholesterol,
administration of N. sativa extract, no visible symptoms TG, LDL, HDL, VLDL by spectrophotometric tech-
of toxicity like ptosis, writhing, convulsion, tremors, nique (Huma Star 600, Catalog no. 16660/001)
salivation, behavioral abnormalities, and so forth, were
TG
observed (Asif et al., 2015). Thus obtained safe dose, 400 VLDL = , (2)
and 800 mg/kg of N. sativa extract were selected for 5
anti‐hyperlipidaemic activity.
LDL [totalcholesterol‐ (HDL + VLDL)]. (3)

2.12 | Induction of hyperlipidemia 2.14 | Statistical analysis

2.12.1 | High‐fat diet‐induced All the grouped data were statistically evaluated, and the
hyperlipidemia in Wistar rat results are expressed as the mean ± standard error means.
One‐way analysis of variance (ANOVA) followed by
The high‐fat diet was prepared by mixing vanaspati Newman Keulus multiple comparison tests was per-
ghee and coconut oil (3:1) v/v ratio and was given to formed. p Value of <0.05, <0.01, <0.001 was considered
Wistar rats at the dose 3 mL/kg/day for 28 days to be statically significant. All analysis was performed by
(Figure 1) (Munshi et al., 2014). The body weight of using Graph Pad Prism (5.01 GraphPad Software Inc.).
both normal control and HFD‐induced hyperlipidaemia LDL and VLDL were calculated by using formula.

F I G U R E 1 Schematic diagram representing the timeline of hyperlipidemia experiment. Mice were fed with high‐fat diet (HFD) and control diet
for 28 days and the same diet throughout the study was maintained.
SHRIVASTAVA ET AL.
| 5 of 19

3 | RESULTS were verified with estimated atomic mass. In this study,

crude extract was introduced for LC‐MS/MS analysis thus
3.1 | Preliminary phytochemical screening multiple peaks were elucidated and analyzed. All the peaks
were determined through m/z value and classified the
Total 100 g of N. sativa seed powder was added in compounds according to their class. The present MS study
500 mL of distilled water for three days. Then the solvent revealed the presence of total 73 compounds. The phenolic
was filtered and dried. The semisolid content was and flavonoid containing compound were high in numbers
weighed accurately. The total weight of extract was 8 g. followed by alkaloid, fatty acid, saponin, vitamins, and so
The preliminary phytochemical screening of aqueous forth. Among phenolic compounds, rosmanol m/z 347.2,
extract showed the presence of tannins, steroids, terpe- thymol m/z 151.2, thymol‐o‐sophoroside m/z 473.4, fleuric
noids, and saponins. Among these, the presence of acid m/z 193.2, thermoquinol glucosidase m/z 332.2, and so
terpenoids was highest (Table 1). forth, were identified, and flavonoid such as myricetin m/z
319.4, quercetin m/z 302.23, apigenin m/z 270.3, kaempferol
m/z 387.3, biochanin‐A m/z 285.2 also observed. Saponins:
3.2 | Determination of total phenolic and sapindoside B m/z 881.2, tauroside E m/z 749.5, alkaloids:
flavonoid content manogoflorine m/z 342.3, thymoquinone m/z 165.3 also
detected in the extract. Also, aqueous extract of N. sativa
The total phenolic and flavonoid content of aqueous found to contain fatty acid such as eicosanoid acid m/z
extract of N. sativa was expressed in mg equivalent of 309.2, eicosadienoic acid m/z 307.3, palmitic acid m/z 255.2,
GAE/g and quercetin equivalent/g respectively. The total and some vitamins including ascorbic acid m/z 652.5,
phenolic and flavonoid content was concentration depen- vitamin A m/z 329.3, vitamin E m/z 431.4. Other classes of
dent. The mean total phenolic and flavonoid content of metabolites observed in extract were summarized in
aqueous extract of N. sativa was 173.190 ± 4.875 mg Table 3. This study indicated that aqueous extract
GAE/g and 129.761 ± 6.151 mg quercetin equivalent/g of of N. sativa contains many phytoconstituents which are
extract, respectively. The result are expressed in Table 2. responsible of different biological activity like quercetin
for antioxidant activity, thymoquinone for anti‐
hyperlipidaemic, antidiabetic activity, and so forth.
3.3 | Determination of different chemical
composition of aqueous extract of N. sativa by
LC‐MS/MS analysis 3.4 | Antioxidant activity

In this analysis raw extract of N. sativa was subjected for The antioxidant activity of N. sativa was determined
the determination of chemical composition. The obtained using standard ascorbic acid BHT and BHA as expressed
spectra are shown in supplementary figures. The spectral in Table 4. The antioxidant activity of aqueous extract of
peaks were determined with respective to retention time, N. sativa was expressed with IC50 value. The DPPH●
fragmentation pattern of individual components with scavenging activity showed that N. sativa extract had
previously published studies (Table 3). The spectral peaks good IC50 value which was 11.916 ± 2.828 µg/mL.
Whereas in comparison of ascorbic acid and BHT, the
BHT has highest inhibition as compared with ascorbic
T A B L E 1 Preliminary phytochemical screening of aqueous extract
acid which was 3.049 ± 1.086 and 7.012 ± 2.499 µg/mL,
of Nigella Sativa seeds.
respectively. It also revealed that N. sativa showed
Name of phytochemical Presence almost similar inhibition activity as compared with
S.N. constituents (+)/Absences (−)
ascorbic acid.
1 Alkaloids − Hydrogen peroxide plays a crucial role for production
2 Flavonoids − of ROS. The unusual accumulation of hydrogen peroxide
promotes oxidative stress and inflammation into biological
3 Phenols −
system. The H2O2 scavenging activity was concentration
4 Tannins + dependent. The H2O2 scavenging activity of N. sativa
5 Glycosides − extract IC50 value was 30.294 ± 13.790 µg/mL which was
expressed in Table 4. In comparison between two standard
6 Amino acids − compound BHT and Ascorbic acid, BHT have higher
7 Steroids + radical inhibition IC50 value 2.215 ± 0.789 µg/mL as
compared with ascorbic acid IC50 9.360 ± 3.336 µg/mL.
8 Terpenoids ++
In Table 4 represents various in‐vitro antioxidant
9 Saponin + activities such as nitric oxide, hydroxyl radical scavenging
Abbreviations: +, presences of phytochemical constituents; ++, presence of high and metal chelating activity revealing potent inhibitory
quantity of phytochemical constituents; −, absence of phytochemical constituent. potential of N. sativa with IC50 value of 209.848 ± 49.884,
6 of 19 | LC‐MS BASED METABOLITE PROFILING AND ACTIVITY OF NIGELLA SATIVA EXTRACT

TABLE 2 The total phenolic and flavonoid content of aqueous extract of N. sativa was determined with respective to gallic acid and quercetin.
Aqueous extract of N. sativa
The mean absorbance of total phenolic content at
765 nm The mean absorbance of total Flavonoid content at 510 nm
Concentration Abs. of Conc. of gallic TPC (mg Abs. of Conc. of TFC (mg of
(mg/mL) extract acid (g/mL) GAE/g) extract quercetin (g/mL) quercetin/g)
0.2 0.157 11.119 111.190 0.123 8.619 86.190

0.4 0.194 13.714 137.142 0.139 9.761 97.619

0.8 0.243 16.285 162.85 0.170 11.976 119.761

1 0.278 20.714 207.140 0.230 16.214 162.142

2 0.348 24.742 247.619 0.259 18.309 183.095

Note: The mean total phenolic and flavonoid content of N. sativa was 173.190 ± 4.875 mg GAE/g equivalent, 129.761 ± 6.151 mg quercetin/g equivalent, respectively. The
value was expressed in mean ± SD.
Abbreviations: TFC, total flavonoid content; TPC, total phenolic content.

12.048 ± 2.828, and 219.861 ± 50.870 µg/mL, respectively. 28 days except normal control. During the 28 days of
The ascorbic acid showed significant inhibition in hydroxyl treatment time frame the blood sample was collected of
radical scavenging activity (IC50 2.733 ± 0.974 µg/mL). individual animal groups at 7, 14, and 28 days of final
While BHA have highest inhibition in nitric oxide(NO−) treatment termination. The lipid profiles of different days
scavenging activity (IC50 0.938 ± 0.273 µg/mL), and BHT in of treatment were expressed in Table 6. The result
hydroxyl and metal chelating activity (IC50 1.513 ± 0.539 showed that after 28 days of treatment with low and
and 0.902 ± 0.293 µg/mL), respectively. high dose of 400 and 800 mg/kg N. sativa was signifi-
In ferric ion reducing power assay (FRAP) and total cantly decreased the TC 73.44 ± 6.92, 71.76 ± 6.91 mg/dL
antioxidant activity, increases in absorbance showed the (p < 0.05) and TG 87.5 ± 6.67, 83.6 ± 8.09 mg/dL
antioxidant activity. Both FRAP and total antioxidants (p < 0.001, p < 0.01) as compare with negative control
showed significant absorbance (2.038 ± 0.373 and group was 248.34 ± 16.07, 224.3 ± 23.14 mg/dL (p < 0.05,
1.438 ± 0.215 nm), respectively. The ascorbic acid in both p < 0.001), respectively. The VLDL‐c and LDL‐c level
activities expressed almost similar absorbance was also significantly increased in 28 days (44.86 ± 4.62,
(1.950 ± 0.149 and 1.836 ± 0.339 nm), respectively. The 191.70 ± 15.86 mg/dL) of HFD administration in nega-
result indicated that N. sativa extract also had similar tive control group as compared with initial day
antioxidant activity (Table 4). (25.28 ± 4.05, 43.96 ± 8.63 mg/dL), respectively. The
The most significant correlation coefficient of differ- low and high doses of N. sativa was significantly reducing
ent antioxidant activity was determined between total the VLDL‐c (17.5 ± 1.33, 16.72 ± 1.61) and LDL‐c
phenolic content (TPC) and DPPH● (R2 = 0.997), H2O2● (35.87 ± 5.51, 33.86 ± 6.05) level in Wistar rats at 28 days
(R2 = 0.993), NO● (R2 = 0.973), respectively. Whereas in of treatment. Whereas at 28 days of treatment, the HDL‐
correlation with total flavonoid content (TFC) and c level was increased 20.06 ± 1.75, 21.18 ± 1.80 mg/dL as
DPPH● (R2 = 0.986), H2O2● (R2 = 0.981), NO● compared with negative control 11.76 ± 1.14 mg/dL,
(R2 = 0.993), respectively. The correlation of total respectively. The result revealed that high dose of
phenolic and flavonoid content with different antiox- N. sativa showed highest antihyperlipidaemic activity.
idant activity strongly suggested that presence of TPC
and TFC promote the free radical scavenging activity.
The correlation coefficient values were reported in 3.6 | Effect of aqueous extract of N. sativa
Table 5. on body weight of high‐fat diet induced
hyperlipidemia in Wistar rats
3.5 | Lipid profile of high‐fat diet‐induced The increases in body weight were observed at the end of
hyperlipidemia in Wistar rat 28th days in the high‐fat diet induced hyperlipidaemia
group as compared with control group. The body weight
In the present study, the lipid profile was determined in increased from 185.8 ± 8.79 to 199.6 ± 15.83 g at 28th
different time frame. On the initial day without days in high‐fat induced hyperlipidaemia group. After
administration of drug and high‐fat diet the blood 7 days of treatment with low and high dose of N. sativa
sample was collected of random selection of experimental extract group, the weight was reduced from 200.2 ± 24.01 to
animals from individual animal group. After that the 188.8 ± 17.29 g and 184.4 ± 10.24 to 184.6 ± 8.52 g, respec-
animals were fed high‐fat diet and drugs every day for tively, whereas there was increase in body weight in the
SHRIVASTAVA ET AL.
| 7 of 19

TABLE 3 Identification of phytoconstituent from aqueous extract of N. sativa seed extract by LC‐MS/MS analysis.
Ionization
mode
λ‐max Molecular [M + H]+/
Class (nm) Phytochemical Compound RT (min) formula m/z value Mass [M−H]− Biological activity
−
Phenolic 288 Rosmanol 5.61 C20H26O5 347.2 346.4 [M−H] Antitumor

Anti‐infective

Anti‐inflammatory

Antioxidant
+
550 Thymol 1.15 C10H14O 151.2 150.221 [M−H] Antianxiety

Antitumor

Antibacterial

Antioxidant

Mosquito repellents

Anticholinesterase

N/A Thymol‐O‐sophoroside 1.72 C22H33O11 473.4 473 [M−H]+ Antimicrobial

activity

285 P‐coumaroyl acid 1.95 C9H8O3R1 278.3 278.134 [M−H]+ Antioxidant activity
derivative
Anti‐inflammatory
activity

425 2″‐O‐pentoxide‐8‐C 2.13 C21H19O11 579.3 447.4 [M−H]+ Antioxidant activity

hexoside luteolin
Hepatoprotective
+
290 Gentisic acid dipentoside 1.59 C7H6O 417.3 154.12 [M−H] Antiulcer activity

Antioxidant activity

Hepatoprotective
effect

Cardioprotective
effect

Skeletal muscle
relaxant

N/A 1,1‐diphenyl‐4‐ 2.33 C22H20OS 332.1 332.5 [M−H]− Anti‐inflammatory

phenylthiobut‐3‐en‐ activity
1‐ol

578.3 P‐Coumaroyl glucose 2.66 C15H18O8 326.2 327.30 [M−H]− Antioxidant activity

Anti‐inflammatory
activity

765 Thermoquinol 6.18 C15H17O8 325.2 325.093 [M−H]+ Antioxidant activity

glucosidase

540 Jaceosidin 2.76 C17H13O27 431.2 329.073 [M−H]+ Anti‐inflammatory

activity

215 Fleuric acid 2.33 C10H10O4 193.2 194.18 [M−H]‐ Anticancer activity

Angiogenesis activity

Antimutagenic
activity

Flavonoids 328 Myricetin 10.04 C15H10O8 319.4 318.237 [M−H]+ Antioxidant activity

(Continues)
8 of 19 | LC‐MS BASED METABOLITE PROFILING AND ACTIVITY OF NIGELLA SATIVA EXTRACT

TABLE 3 (Continued)

Ionization
mode
λ‐max Molecular [M + H]+/
Class (nm) Phytochemical Compound RT (min) formula m/z value Mass [M−H]− Biological activity
Anti‐inflammatory
activity

Anticancer activity
–
557 Quercetin 9.91 C15H10O7 301.1 302.23 [M−H] Antioxidant activity

Anti‐inflammatory
activity

Antiulcer activity

Immunosuppression
+
337 Apigenin 20.46 C15H10O5 270.3 270.24 [M−H] Antihyperglycemic
activity

Anticancer activity

Antidepressant
activity

240 Myricetin‐3‐O‐glucoside 5.29 C21H20O13 281.2 480.4 [M−H]− Antioxidant activity

+
253 Kaempferol 1.61 C15H10O6 387.3 287 [M−H] Antioxidant activity

Antiepileptic activity

Anti‐inflammatory
activity

340 Biochanin‐A 11.23 C16H12O5 285.2 284.263 [M−H]+ Anti‐inflammatory

activity

Hepatoprotective
effects

Neuroprotective
effect

Anticancer activity
+
250 Kaempferol‐3‐O‐ 11.04 C27H20O11 449.3 449 [M−H] Antioxidant activity
glucoside
Anti‐inflammatory
activity

Antimicrobial
activity

265 Kaempferol‐3‐O‐[β‐D‐ 6.18 C33H39O21 771.4 771 [M−H]+ Anticancer activity

glucopyranosyl‐
(1 → 2)‐β‐D‐ Antioxidant activity
galactopyranosyl‐ Anti‐inflammatory
(1 → 2)‐β‐D‐ activity
glucopyranoside]
Antihyperglycemic
activity

Cardioprotective
effect

335 Kaempferol‐3‐O‐hexose‐ 1.16 ‐ 581.3 281 [M−H]+ Anti‐inflammatory

O‐pentoside activity

560 Quercetin‐2‐O‐rutinoside 27.60 C27H30O16 611.4 611 [M−H]+ Anticancer activity

Antioxidant activity
SHRIVASTAVA ET AL.
| 9 of 19

TABLE 3 (Continued)

Ionization
mode
λ‐max Molecular [M + H]+/
Class (nm) Phytochemical Compound RT (min) formula m/z value Mass [M−H]− Biological activity
−
450 Kaempferol‐3‐O‐ 2.13 C32H53O30 917.2 918 [M−H] Unknown
sophorotrioside‐7‐O‐
rhamnoside

734 6″‐O‐Acetylgenistin 2.76 C23H22O11 475.0 474.4 [M−H]+ Antioxidant activity

+
325.9 Irilone 4.55 C16H10O6 299.1 298.25 [M−H] Antioxidant activity
+
Alkaloids 366 Norargemonine 6.15 C20H22NO4 342.1 341 [M−H] Antimalarial activity
+
241 Nigellimine 12.83 C12H13NO2 203.0 203 *M+H] / Antibacterial activity
[M−H]−
Antifungal activity
+
280 2‐(4‐nitrobutyryl) 12.83 C12H19NO4 241.1 241 [M−H] Antitumor activity
cyclooctan one

227 Damascenine 8.24 C10H13NO3 196.0 195.21 [M−H]+ Anti‐inflammatory

activity

Antipyretic activity
−
232 Magnoflorine 4.03 C20H24NO4 342.3 342.4 [M−H] Anti‐anxiety activity

Anti‐inflammatory
activity

Anticancer activity
+
264 Nigellidine 5.32 C18H19N2O2 295.3 295 [M−H] Anti‐SARS‐CoV‐2
activity

Antioxidant activity
+
298 Rotenone 9.65 C23H22O6 395.3 394.4 [M−H] Neuroprotective
effect

Anti‐inflammatory
activity

263 3‐O‐methylorobol 5.29 C16H12O6 299.3 300.26 [M−H]+ Antioxidant activity

Antiallergic activity
+
245 Daidzein 9.68 C15H10O4 419.3 254.23 [M−H] Antioxidant activity

Antihistaminic (H1)
activity

254 Thymoquinone 19.53 C10H12O2 165.3 164.204 [M−H]+ Antioxidant activity

Antibacterial activity

Anticancer activity

Neuroprotective
effect

292 Hederin 2.13 C41H66O12 751.7 751 [M−H]+ Anti‐inflammatory

activity

Anticancer activity

Antileishmanial
activity

Saponins 765.44 Sapindoside B 3.99 C46H74O16 881.2 883.1 [M−H]− Antiprotozoal

activity

(Continues)
10 of 19 | LC‐MS BASED METABOLITE PROFILING AND ACTIVITY OF NIGELLA SATIVA EXTRACT

TABLE 3 (Continued)

Ionization
mode
λ‐max Molecular [M + H]+/
Class (nm) Phytochemical Compound RT (min) formula m/z value Mass [M−H]− Biological activity
Anti‐inflammatory
activity

Antiplatelet
aggregation
activity

N/A Tauroside E 6.18 C41H66O12 749.5 751.0 [M−H]− Antidepressant

activity

Antimicrobial
activity

Anthocyanins 518 Delphinidin‐3‐O‐ 5.74 C27H31O17 626.0 627.156 [M−H]− Anticancer activity
glucosyl‐glucoside
Anti‐inflammatory
activity

Neuroprotective
activity

299 Cynidin 3,5‐O‐ 26.58 C27H31O16 610.1 611.5 [M−H]− Antioxidant activity
diglucoside
Cardioprotective
activity

Antihyperlipidemic
activity

525 Peonidin‐3‐O‐ 11.23 C33H41O20 756.3 757.7 [M−H]− Anti‐inflammatory

sambabioside‐5‐O‐ activity
glucoside
Antioxidant activity
−
490 Pelargonidin‐3‐O‐ 4.16 C26H29O14 564.0 565.5 [M−H] Antiarthritic activity
sambubioside
Antioxidant activity
−
Flavones 348 Luteolin 7‐O‐glucuronide 25.07 C21H18O12 461.0 464.4 [M−H] Hepatoprotective
activity

Antioxidant activity

Antimicrobial
activity

275 Chrysoeriol 7‐O‐ 2.13 C22H22O11 463.0 462.4 [M−H]+ Antihyperglycemic

glucoside activity

Antimicrobial
activity

230 Apigenin 7‐O‐apiosyl‐ 12.83 C26 H28O14 565.2 564.14 [M−H]+ Antifungal activity
glucoside
Anti‐inflammatory
activity

280 Gardenin‐B 14.42 C19H18O7 359.0 358.3 [M−H]+ Neuroprotective

activity

Anticancer activity

Gastroprotective
activity

Antiasthmatic
activity
SHRIVASTAVA ET AL.
| 11 of 19

TABLE 3 (Continued)

Ionization
mode
λ‐max Molecular [M + H]+/
Class (nm) Phytochemical Compound RT (min) formula m/z value Mass [M−H]− Biological activity
−
Flavanones 229 6‐geranylnaringenin 20.46 C25H28O5 407.0 408.1937 [M−H] Antioxidant activity

Antimicrobial
activity

Lignans 400 Lariciresinol sesquiligans 8.24 C30H36O10 555.3 556.23 [M−H]− Anticarcinogenic
activity

Antioxidant activity

Antidiuretic activity
+
241 Cyclolariciresinol 4.55 C20H24O6 361.2 360.157 [M−H] Gastroprotective
activity

Estrogenic activity
−
Tyrosols 230 Oleuropein 22.35 C25H32O13 539.2 540.1843 [M−H] Antimicrobial
activity

Antioxidant activity

Anticancer activity

Anti‐inflammatory
activity

Cardioprotective
activity

517 Ligstroside‐aglycone 4.55 C19 H22O7 361.2 362.1366 [M−H]− Anti‐inflammatory

activity

Anticancer activity
+
N/A P‐HPEA‐AC 22.35 C10 H12 O3 181.1 180.078 [M−H] Antioxidant activity

Anticancer activity
+
Hydroxycoumarins 344 Scopoletin 22.16 C10 H8O4 193.0 192.0423 [M−H] Antitubercular
activity

Hepatoprotective
activity

Antifungal activity

Hydroxy benzo 254 2,3, dihydroxy‐1‐ 15.08 C10H12O5 213.1 212.201 [M−H]+ Antioxidant activity
quinones guaiacylpropanone
Anticancer activity
+
Naphthoquinone 330 1,4‐napthoquinone 26.58 C10H6O2 159.1 158.15 [M−H] Antibacterial activity

Antimalarial activity
+
423 Juglone 12.83 C10H6O3 175.0 174.15 [M−H] Antioxidant activity

Antiviral activity

Anticancer activity
+
Curcuminoids 421 Demethoxycurcumin 1.96 C20H18O5 339.2 338.115 [M−H] Antiarthritic activity

Antihyperlipidemic
activity

Anti‐inflammatory
activity

(Continues)
12 of 19 | LC‐MS BASED METABOLITE PROFILING AND ACTIVITY OF NIGELLA SATIVA EXTRACT

TABLE 3 (Continued)

Ionization
mode
λ‐max Molecular [M + H]+/
Class (nm) Phytochemical Compound RT (min) formula m/z value Mass [M−H]− Biological activity
+
Terpenes 570 Limonen‐6‐ol‐pivalate 27.60 C15H24O2 236.0 236.35 *M+H] / Antimicrobial
[M−H]− activity

Antioxidant activity
+
Sesquiterpenes N/A Longifolene 1.59 C15H24 204.2 204.36 *M+H] / Antifungal activity
[M−H]−
Anti‐inflammatory
activity

Di‐sacchrides 351 D‐glucose 6‐O‐α‐D‐ 1.76 C12H24O12 342.3 360.31 [M−H]− Antifungal activity
galactopyranosyl
Antibacterial activity
−
Fatty acids N/A Eicosanoic acid 10.04 C20H40O2 309.2 312.538 [M−H] Anti‐inflammatory
activity

N/A Trihydroxy 4.10 C18H34O5 329.1 330.5 [M−H]− Antibacterial activity

octadecenoic acid
Anti‐inflammatory
activity

Antihyperlipidemic
activity

N/A Dihydroxy 3.89 C18H34O4 313.2 314.5 [M−H]− Antibacterial activity

octadecanoic acid
Antioxidant activity
−
N/A Hydroxy 7.77 C18H32O3 295.2 296.4 [M−H] Cardioprotective
octadecadienoic acid activity

Antioxidant activity
−
N/A Hexadecenoic acid 2.13 C16H30O2 253.2 254.41 [M−H] Antiarthritic activity

Anti‐inflammatory
activity

N/A Palmitic acid 6.34 C16H32O2 255.2 256.424 [M−H]− Antihyperlipidemic

activity

Anti‐inflammatory
activity

N/A Octadecenoic acid 5.29 C18H34O2 281.2 282.47 [M−H]− Anti‐amyloidosis

activity

Antibacterial activity
−
N/A Eicosadienoic acid 10.03 C20H36O2 307.2 308.5 [M−H] Anti‐inflammatory
activity

Antioxidant activity
+
Vitamins N/A 1‐(+) ascorbic acid 2,6‐ 19.78 C38H68O8 652.5 652.9 *[M+H] / Antidiuretic activity
dihexadecanoate [M−H]−
Antiseptic activity

Antimicrobial
activity

325 Vitamin A 19.92 C20H30O 329.3 Immune stimulants

+
265 Vitamin D2 9.68 C28H44O 397.4 396.6 [M−H] Neuroprotective
activity

Cardioprotective
activity
SHRIVASTAVA ET AL.
| 13 of 19

TABLE 3 (Continued)

Ionization
mode
λ‐max Molecular [M + H]+/
Class (nm) Phytochemical Compound RT (min) formula m/z value Mass [M−H]− Biological activity
+
N/A Vitamin K2 25.07 C31H40O2 445.4 444.6 [M−H] Antiosteoporosis
activity

Cardioprotective
activity

295 Vitamin E 21.02 C29H50O2 431.4 430.7 [M−H]+ Antiosteoarthritis

activity

Antioxidant activity
+
Others N/A Pyrrolidine‐2‐one‐3β 20.20 C16H25NO5 311.4 311.17 *[M+H] / Anti‐inflammatory
(propanoic acid [M−H]− activity
methyl ester),5‐
methylene 4α

Source: Farag et al. (2014), Hadi et al. (2016), Kadam and Lele (2017), Servi et al. (2022).

negative control group. At the end of 28th days of treatment perform the MS analysis. Several studies reported that
with low and high dose of N. sativa extract group reached the spectral analysis of N. sativa extract contains variety
171.8 ± 14.98 g and 162.6 ± 16.40 g, respectively (Table 7). of bioactive phytoconstituent. In present study, the m/z
value of different LC‐MS/MS spectra was determined
through previous studies (Farag et al., 2014; Kadam &
4 | DISCUSSION Lele, 2017; Servi et al., 2022).
In the present study, different antioxidant activities of
Seeds of N. sativa (black cumin) have been traditionally aqueous extract of N. sativa was determined. The DPPH
used for centuries in the treatment of many clinical scavenging activity of N. sativa extract was found to be
conditions including diabetes, hypertension, hyperlipidae- IC50 11.916 ± 2.828 µg/mL. Several previous studies of
mia, depression, and so forth. This study was designed to DPPH scavenging capacity N. sativa extract were found
observe the in‐vitro antioxidant and in‐vivo antihyperlipi- to be IC50 24.69 ± 2.17, 8.00 ± 1.34 µg/mL, respectively
demic effects of N. sativa seed extract. In the previous study (Mazhar, 2022; Thilakarathne et al., 2018). The present
done by Ishtiaq et al., preliminary phytochemical screening study of N. sativa had shown highest DPPH scavenging
of aqueous extract of N. sativa showed presence of tannins, activity as compared with previously reported studies.
flavonoids, coumarins, and diterpenes. Whereas in the Another previous the N. sativa extract showed significant
present study tannins, saponins, steroids and terpenoids DPPH scavenging activity was 2.46 µg/mL as compared
were present. In comparison with previous study the result with present study it showed highest antioxidant capacity
of preliminary phytochemical screening showed similar (Sandhya et al., 2020).
composition (Ishtiaq et al., 2013). The hydrogen peroxide and nitric oxide both are highly
The presence of phenolic and flavonoid content in the reactive molecules which is significantly increases the
extract expressed that highest radical scavenging activity. oxidative stress into the biological system. Nitric oxide
The previous study represented that the aqueous extract of are synthesized into the body but excessive production
N. sativa contains higher TPC and TFC content was increases oxidative stress (Zhao, 2007). In the present study
494.86 ± 3.62, 127.25 ± 2.6 in comparison with the present was to determine the antioxidant capacity of N. sativa
study was 173.190 ± 4.875 mg GAE/g equivalent, 129.761 ± extract. Previously several studies were done to determine
6.151 mg quercetin/g equivalent, respectively (Taroq the H2O2● and NO● scavenging activity Hassane et al.
et al., 2018). It was clearly indicated that the TPC content (2022) and Sandhya et al. (2020) showed that N. sativa seed
of the previous study was almost two times higher while in extract significantly inhibit the H2O2● radicals was IC50
comparison between both studies expressed similar TFC 1.03 ± 0.05 µg/mL (**p < 0.01), and 95.14 µg/mL, respec-
content. tively. Similarly, previous study done by Hassane et al. of
The chemical composition of N. sativa seed extract N. sativa IC50 0.08 ± 0.02 µg/mL (**p < 0.01) significantly
varied that may be related to geographical distribution. showed nitric oxide scavenging activity (Hassane
At present different species of N. sativa are available et al., 2022). Several previous studies showed highest
which contains different kinds of chemical compounds H2O2● and NO● scavenging capacity compared with the
(Hassan et al., 2016). Due to presence of many bioactive present study. Another previous study done by Hassan
chemical compound in N. sativa extract, so it needs to et al., determined hydroxyl radical scavenging activity and
14 of 19 | LC‐MS BASED METABOLITE PROFILING AND ACTIVITY OF NIGELLA SATIVA EXTRACT

metal chelating assay. The result showed that N. sativa at

Total antioxidant mean 2000 mg/mL concentration had the highest scavenging
absorbance (nm) capacity was 71 ± 5% and 59 ± 4%, respectively (Hassan
1.438 ± 0.215
et al., 2016).

1.836 ± 0.339
In the present study, the Wistar rats were divided
into five groups containing five rats in each group.
Among these, a control group in which only a normal
–

– diet was given during the experiment. Negative control

group; atorvastatin group, low dose N. sativa and high
dose N. sativa group. All these groups except normal
absorbance (nm)

control group were given a high‐fat diet respectively.

2.038 ± 0.373

1.950 ± 0.149
FRAP mean

The lipid profile including TG, TC, HDL, VLDL, and

LDL was estimated at different time intervals. The
results showed the increase in level of different types of
–

cholesterol because of a high‐fat diet. The aqueous

extract of N. sativa was administered to the test groups
Ferrous ion chelating

of Wistar rats. The results of the present study showed

219.861 ± 50.870

decrease in TC, TG, and LDLc level and increase in

0.902 ± 0.293

5.850 ± 1.903
activity (IC50)

HDLc level compared with control and negative

control groups.
The antihyperlipidemic activity of N. sativa may
possibly be due to two mechanisms; (1), removal of LDL
–

from circulation by Apo A1 lipoprotein, Apo‐B100

lipoprotein, and LDL receptor genes from liver cells,
Hydroxyl scavenging

decreases in absorption of dietary cholesterol and

12.048 ± 2.828

1.513 ± 0.539

2.733 ± 0.974
activity (IC50)

enhance bile acid synthesis. (2) the phytochemical

constituent of N. sativa regulates cholesterol synthesis
through the activation of HMG‐CoA reductase (Ibrahim
et al., 2014; Kaatabi et al., 2012).
–
Determination of antioxidant activity of aqueous extract of N. sativa seed extract.

A previous study done by Davooth et al., reported that

chloroform extract of N. sativa at 400 and 800 mg/kg
Nitric oxide scavenging

showed significant decreases the TC level was 102.56 ± 5

(p < 0.01), 92.06 ± 7.53 mg/dL (p < 0.001) as compared with
209.848 ± 49.884

0.938 ± 0.273

14.522 ± 4.226

control group 139.32 ± 19.72 mg/dL, respectively. Also,

activity (IC50)

N. sativa decreases the TG level 133.28 ± 8.01, and

128.83 ± 15.82 mg/dL although it increases the HDL level,
but the result expressed that 400 mg/kg of extract showed
–

more significant effect (59.88 ± 1.80 mg/dL) compared

with 800 mg/kg extract (51.21 ± 6.33 mg/dL) (Davooth
et al., 2015). In the present study, the aqueous extract of
H2O2 scavenging

30.294 ± 13.790

N. sativa at 400 and 800 mg/kg showed highest significant

2.215 ± 0.789

9.360 ± 3.336
activity (IC50)

inhibition of TC 73.44 ± 6.92 and 71.76 ± 6.91 mg/dL

(p < 0.05) but less effectively increases the HDL level was
20.06 ± 1.75, 21.18 ± 1.80 mg/dL as compared with previ-
ous study after 28 days of treatment, respectively. The
–

Note: The results are presented as mean ± SD.

mean body weight of hyperlipidaemic animals after 28 days

of treatment showed that significantly decreases in the
DPPH scavenging

previous study at 400 and 800 mg/kg of N. sativa extract

11.916 ± 2.828

3.049 ± 1.086

7.012 ± 2.499
activity (IC50)

was 220.83 ± 7.36, and 228.00 ± 7.90 g, whereas in the

present study at the same dose of extract also showed the
significant decreases the body weight 171.8 ± 14.98, and
162.6 ± 16.40 g in comparison with both studies showed
–

that aqueous extract of N. sativa was more effective as

Ascorbic acid

compared with previous study (Davooth et al., 2015).

Compound

extract
TABLE 4

N. sativa

Another study done by Asaduzzaman, Nahar,

BHA
BHT

Rahman, Hasan, Khatun, Tamanna, et al. (2015)

showed that administration of 300 mg/kg of N. sativa
SHRIVASTAVA ET AL.
| 15 of 19

TABLE 5 Carl's Pearson correlation between phytochemicals and antioxidant activities.

TFC TPC DPPH H2O2 NO OH
TPC 0.975*

DPPH 0.986** 0.977*

H2O2 0.981** 0.993*** 0.984**

NO 0.993*** 0.973* 0.996*** 0.976*

OH 0.963* 0.968* 0.972* 0.989** 0.957*

Metal chelating 0.959* 0.935* 0.912* 0.918* 0.939* 0.867

Note: Significance of correlation between antioxidant activity.

Abbreviations: TFC, total flavonoid content; TPC, total phenolic content.
*p < 0.05, significant; **p < 0.01, very significant; ***p < 0.001, highly significant correlation.

TABLE 6 Lipid profile of high‐fat diet induced hyperlipidaemia in Wistar rat.

S.N. Parameter Group 0th day 7th day 14th day 28th day
1 TC Normal control 80 ± 5.17 84.34 ± 4.98 83.76 ± 5.45 84 ± 5.30

High‐fat diet 82.3 ± 6.30* 209.28 ± 11.61* 230.38 ± 12.68* 248.34 ± 16.07*

Atorvastatin (40 mg/kg) 73.2 ± 8.99* 157.92 ± 14.26* 90.28 ± 8.08* 62.82 ± 6.68*

Low dose (400 mg/kg) 76.68 ± 4.86* 163.76 ± 12.59* 89.06 ± 6.70* 73.44 ± 6.92*

High dose (800 mg/kg) 84.44 ± 10.36* 161.78 ± 9.17* 84.76 ± 7.75* 71.76 ± 6.91*

2 TG Normal control 106 ± 6.81 107.7 ± 8.89 101.96 ± 6.28 100.26 ± 3.87

High‐fat diet 126.46 ± 20.21*** 168.94 ± 13.44*** 195.68 ± 13.63*** 224.3 ± 23.14***

Atorvastatin (40 mg/kg) 114.78± 1 4.30*** 99.8 ± 9.17*** 82.6 ± 8.69*** 69.6 ± 6.69***

Low dose (400 mg/kg) 115.06 ± 20.39*** 108.1 ± 22.09*** 99.9 ± 15.83*** 87.5 ± 6.67***

High dose (800 mg/kg) 118.7 ± 18.51** 107 ± 15.13** 97.3 ± 12.71** 83.6 ± 8.09**

3 HDL‐C Normal control 10.4 ± 1.06 12.66 ± 1.30 12.70 ± 1.67 13.04 ± 1.30

High‐fat diet 13.06 ± 1.17 18.32 ± 1.03 12.34 ± 1.55 11.76 ± 1.14

Atorvastatin (40 mg/kg) 12.96 ± 1.97 16.48 ± 1.18 18.3 ± 1.29 18.80 ± 2.0

Low dose (400 mg/kg) 12.30 ± 1.98 14.64 ± 1.06 17.92 ± 1.70 20.06 ± 1.75

High dose (800 mg/kg) 12.75 ± 1.60 14.48 ± 0.96 17.46 ± 2.39 21.18 ± 1.80

4 VLDL‐C Normal control 21.2 ± 1.36 21.54 ± 1.77 20.38 ± 1.26 20.04 ± 0.77

High‐fat diet 25.28 ± 4.05*** 33.78 ± 2.68*** 39.12 ± 2.71*** 44.86 ± 4.62***

Atorvastatin (40 mg/kg) 22.94 ± 2.87*** 19.96 ± 1.83*** 16.52 ± 1.73*** 13.92 ± 1.33***

Low dose (400 mg/kg) 23 ± 4.09*** 21.7 ± 4.52*** 19.98 ± 3.16*** 17.5 ± 1.33***

High dose (800 mg/kg) 23.74 ± 3.07** 21.4 ± 3.02** 19.46 ± 2.54** 16.72 ± 1.61**

5 LDL‐C Normal control 48.4 ± 4.0 50.14 ± 7.31 50.67 ± 5.60 50.92 ± 5.55

High‐fat diet 43.96 ± 8.63 157.18 ± 11.0 178.92 ± 12.34 191.70 ± 15.86

Atorvastatin (40 mg/kg) 37.3 ± 10.37 121.48 ± 16.63 53.36 ± 7.55 30.09 ± 4.13

Low dose (400 mg/kg) 41.37 ± 2.76 127.5 ± 15.96 51.16 ± 8.07 35.87 ± 5.51

High dose (800 mg/kg) 47.94 ± 8.04 125.9 ± 8.57 47.84 ± 6.92 33.86 ± 6.05

Note: All values were expressed as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001 in one‐way analysis of variance (ANOVA) with Newman Keuls multiple comparison
test was used for between group differences.
Abbreviations: HDL‐C, high‐density lipoprotein cholesterol; LDL‐C, low‐density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride; VLDL‐C,
very low‐density lipoprotein cholesterol.
16 of 19 | LC‐MS BASED METABOLITE PROFILING AND ACTIVITY OF NIGELLA SATIVA EXTRACT

TABLE 7 Effect of aqueous extract of Nigella sativa on body weight of high‐fat diet‐induced hyperlipidaemia in Wistar rats.
Initial body Body weight in Body weight in Body weight in
S.N. Groups weight 7 days 14 days 28 days
1 Control 180 ± 12.34 181.2 ± 12.85 179.2 ± 8.5 178.8 ± 8.9

2 High‐fat diet 185.8 ± 8.79 196.4 ± 11.97 196.6 ± 14.55 199.6 ± 15.83

3 Atorvastatin (40 mg/kg) 204.4 ± 21.98 191.4 ± 14.10 180.8 ± 12.68 174.6 ± 11.74

4 Low dose (400 mg/kg) 200.2 ± 24.01 188.8 ± 17.29 179.8 ± 13.54 171.8 ± 14.98

5 High dose (800 mg/kg) 184.4 ± 10.24 184.6 ± 8.52 175.8 ± 9.78 162.6 ± 16.40

Note: All values are expressed as mean ± SEM n = 5 each treatment was compared with negative control.

extract for 28 days significantly improved TC N. sativa seed in reducing the disease complications.
4.469 ± 0.05 mg/dL, TGs 1.943 ± 0.163 mg/dL, LDL Previous study total 88 subjects with age above 18‐year‐
2.953 ± 0.109 mg/dL, VLDL 1.368 ± 0.074 mg/dL, old and TC >200 mg/dL were included. According to the
and HDL 0.49 ± 0.014 mg/dL (**C p < 0.05) respec- subject condition 500 mg of N. sativa seed powder
tively. Whereas in present study, we found containing capsule was prescribed four times a day for
significant improvement in TC 73.44 ± 6.92 mg/dL, 4 weeks. During this study initial biochemical laboratory
TG 87.5 ± 6.67 mg/dL, LDL 35.87 ± 5.51 mg/dL, VLDL parameters were analyzed and repeated after the
17.5 ± 1.33 mg/dL, and HDL 20.06 ± 1.75 mg/dL at termination of the study. After 4 weeks of treatment,
400 mg/kg of N. sativa extract. In both studies, they observed N. sativaseed to significantly decreases TC
N. sativa extract was significantly decreasing the TC, by 4.78%, LDL by 7.6%, and TG by 16.65%, respec-
TG, LDL, and VLDL while HDL level was increased. tively. This study revealed that N. sativa are effective to
It was indicated that N. sativa is effective for reduces the hyperlipidaemic condition (Sabzghabaee
hyperlipidaemic condition. Iqbal et al. (2017), reported et al., 2012). Another randomized control trial of
that 150 mg/kg N. sativa extract significantly decreases N. sativa on dyslipidaemia was conducted in India, total
the TG level to 132.02 ± 0.69 mg/dL and 40 participants were included and equally divided into
119.06 ± 0.44 mg/dL on first day and termination day two groups control and test group, respectively. At
of the experiments. In another previous study done by 500 mg of N. sativa seed powder containing capsule was
Saxena et al. (2020), the ethanol extract of N. sativa at prescribed four times a day for 60 days. After the
500 mg/kg showed that feeding of extract significantly treatment N. sativa effects on dyslipidaemia was
reduces the TC 23%, TG 25%, LDL 22%, VLDL 23%, compared with baseline and control group. The result
respectively. Whereas this study concluded that N. revealed that test group significantly decreased the level
sativa showed effective antihyperlipidaemic activity in of TC 213.1 ± 11.16 mg/dL, TGs 175.5 ± 23.43 mg/dL,
Wistar rats. Also, similar response was observed in the and LDL 133.7 ± 9.32 mg/dL, respectively. Whereas
present study. HDL level was 45.1 ± 2.74 mg/dL slightly increased
For evaluating the weight‐reducing effects of (Rasheed et al., 2014).
N. sativa, we measured the body weight of Wistar rats
at a different time interval (7th, 14th, and 28th day)
and found a significant decrease in body weight in 5 | CONCLUSIONS
Wistar rats treated with AENS 400 and 800 mg/kg
compared with control and negative control group. In this study, it was found that administration of aqueous
However, no significant decrease in body weight was extracts of N. sativa seeds in Wistar rats has a favorable
observed in Wistar rats treated with 800 mg/kg AENS effect on the lipid profile as it reduced TG, LDL
when compared with 400 mg/kg AENS. The decrease cholesterol, TC, and increased the HDL cholesterol level.
in body weight by N. sativa can be correlated with its This work shows the evidence to support ethnopharmaco-
effect on the suppression of appetite (Brennan & logical use of medicinal plant for improving lipid metabo-
Mantzoros, 2006). lism. In conclusion, the promising effects of aqueous extract
N. sativa is a known spice due to its medicinal of N. sativa indicate that it can be a potent alternative for
properties and use in culinary dishes. The main strength the treatment of hyperlipidaemia in the future.
of N. sativa is seeds, which contains high amount of
omega‐3 fatty acid and linolic acid, shows many AUTHOR CONTRI BUTI ONS
pharmacological effect but it mostly preferred in obesity Amit Kumar Shrivastava: Conceptualization; formal
and hyperlipemia condition (Gupta et al., 2022). Many analysis; investigation; writing—original draft. Laxmi
clinical trials were conducted to determine the effect of Shrestha: Data curation; formal analysis; investigation.
SHRIVASTAVA ET AL.
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Buddhi Raj Pokhrel: Formal analysis; methodology; antiperoxidative activities of M. communis berries methanol
resources; validation. Bishal Joshi: Data curation; formal extract and its fractions. Turkish Journal of Agriculture ‐ Food
analysis; investigation; software; visualization. Gopal Science and Technology, 10(6), 1089–1094.
Benlafya, K., Karrouchi, K., Charkaoui, Y., El Karbane, M., &
Lamichhane: Resources; software; validation; writing— Ramli, Y. (2014). Antimicrobial activity of aqueous, ethanolic,
review and editing. Bojana Vidović: Proof reading and methanolic, cyclohexanic extracts and essential oil of Nigella
editing. Niranjan Koirala: Data analysis, writing original sativa seeds. Journal of Chemical and Pharmaceutical Research,
draft, proof reading and editing. 6(8), 9–11.
Bensiameur‐Touati, K., Kacimi, G., Haffaf, E. M., Berdja, S., &
Aouichat‐Bouguerra, S. (2017). In vivo subacute toxicity and
AC KNOWLE DGMENTS antidiabetic effect of aqueous extract of Nigella sativa. Evidence‐
The authors are grateful to the working faculty Based Complementary and Alternative Medicine, 2017, 1–13.
Mr. Chandrajeet Kumar Yadav, B. Pharmacy student Brennan, A. M., & Mantzoros, C. S. (2006). Drug Insight: The role of
Miss Pramila Thapa Magar for their effort and Dr. leptin in human physiology and pathophysiologyemerging clinical
Koirala Research Institute for Biotechnology and applications. Nature Clinical Practice. Endocrinology &
Metabolism, 2(6), 318–327.
Biodiversity. All the experiments were performed in Chen, S. J., Rader, D. J., Tazelaar, J., Kawashiri, M., Gao, G., &
departmental facilities. Wilson, J. M. (2000). Prolonged correction of hyperlipidemia in
mice with familial hypercholesterolemia using an adeno‐
CONFLI CT OF I NTE RES T STAT EME NT associated viral vector expressing very‐low‐density lipoprotein
The authors declare no conflicts of interest. receptor. Molecular Therapy, 2(3), 256–261.
Daryabeygi‐Khotbehsara, R., Golzarand, M., Ghaffari, M. P., &
Djafarian, K. (2017). Nigella sativa improves glucose homeostasis
ET HI CS ST ATE MENT and serum lipids in type 2 diabetes: A systematic review and meta‐
None declared. analysis. Complementary Therapies in Medicine, 35, 6–13.
Davooth, M. S., Sarojini, R., Maheswaran, A., & Parameswari, R.
(2015). Study on anti‐hyperglycaemic and hypolipidemic activity
ORCID
of Nigella sativa seeds. Indian Journal of Pharmacy and
Amit Kumar Shrivastava http://orcid.org/0000-0002- Pharmacology, 2(4), 191–196.
8915-9186 Delgado, R. L., Acosta, M. E., Fraga, P. Á., Bécquer, V. M. A.,
Laxmi Shrestha http://orcid.org/0000-0002-2489-4278 Soto, L. Y., Falcón, C. V., Vázquez, L. A. M., Martínez, S. G., &
Gopal Lamichhane https://orcid.org/0000-0003- Fernández, S. E. (2012). Lipofundin‐induced hyperlipidemia
1487-7578 promotes oxidative stress and atherosclerotic lesions in New
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Zodda, D., Giammona, R., & Schifilliti, S. (2018). Treatment strategy

for dyslipidemia in cardiovascular disease prevention: Focus on How to cite this article: Shrivastava, A. K.,
old and new drugs. Pharmacy, 6(1):10.
Shrestha, L., Pokhrel, B. R., Joshi, B., Lamichhane,
G., Vidović, B., & Koirala, N. (2023). LC‐MS based
SUPPORTING I NFORMATION metabolite profiling, in‐vitro antioxidant and in‐vivo
Additional supporting information can be found online antihyperlipidemic activity of Nigella sativa extract.
in the Supporting Information section at the end of this eFood, 4(4), e107. https://doi.org/10.1002/efd2.107
article.