Food Chemistry 363 (2021) 130259

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Assessing the ethnobotanical potential of Carissa opaca berries by merging

outcomes from metabolomics profiling, enzyme assays, and in silico
docking studies
Kashif Bashir a, Sadia Naz b, Umar Farooq b, Fazli Wahid c, Abdul Jabbar Shah a,
Erin P. McCauley d, e, Phillip Crews d, Taous Khan a, *
a
Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus 22060, Pakistan
b
Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus 22060, Pakistan
c
Department of Biomedical Sciences, Pak-Austria Fachhochschule: Institute of Applied Sciences and Technology, Mang, Khanpur Road, Haripur, Pakistan
d
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, United States
e
Department of Chemistry and Biochemistry, California State University Dominguez Hills, Carson, CA 90747, United States

A R T I C L E I N F O A B S T R A C T

Keywords: The phytochemical profile of Carissa opaca fruit extract and fractions was established through dereplication
Carissa opaca fruit strategies employing LC-MS/MS and global natural product social molecular networking (GNPS). Crude extract
α-Glucosidase inhibition and fractions were evaluated for their potential to inhibit α-glucosidase and urease in vitro. Flavonoid-O-
LC–MS/MS-based molecular networking
glycosides, flavonoid-C-glycosides, flavonoids, proanthocyanidin B2, phenolics, and triterpenoids were anno­
Molecular docking
Polyphenolics
tated as the major classes of secondary metabolites present in the extract and fractions. α-Glucosidase inhibition
Urease inhibition was associated with n-butanol and ethyl acetate fractions comparable to acarbose (IC50 = 120.43 µg/mL) with
IC50 values of 123.67 and 131.72 µg/mL, respectively. The ethyl acetate fraction showed good urease inhibition
comparable with thiourea (IC50 = 103.71 µg/mL) with an IC50 value of 109.14 µg/mL. Molecular docking studies
of compounds observed in the crude extract and bioactive fractions had significant binding scores, which sup­
ported results for enzyme inhibition in vitro. This study provided a detailed phytochemical profile of C. opaca
fruit and its enzyme inhibition potential.

1. Introduction polyphenolic constituents in edible berries including strawberries,

raspberries, blueberries, rowanberries, blackcurrants, and red grapes
Polyphenolics constitute a large class of phytochemicals with more exhibiting α-glucosidase inhibition (Berger, Ostberg-Potthoff, Bakur­
than 8,000 compounds identified from various plant species. These adze, Winterhalter, & Richling, 2020; Boath, Stewart, & McDougall,
compounds are abundant in edible fruits, which contribute many 2012; Sulaiman & Ooi, 2013). Similarly, polyphenolic-rich extracts from
beneficial effects to human health with regular consumption (Pandey & mung bean, pineapple, cinnamon, avocado peel, and guava revealed
Rizvi, 2009; Tungmunnithum, Thongboonyou, Pholboon, & Yangsabai, interesting inhibitory activity against Jack bean urease, which is
2018). The presence of these health-promoting constituents either pre­ involved in gastric ulcers and gastric cancer (Shabana, Kawai, Kai,
vents or delays the onset of various diseases including inflammation, Akiyama, & Hayashi, 2010).
oxidative stress, hypertension, diabetes, aging, neurodegeneration, and Carissa opaca Stapf ex Haines (Apocynaceae) is an evergreen and
cancer (Nile & Park, 2014). Polyphenolics have been extensively studied wild thorny shrub found in dry parts of Pakistan, Sri Lanka, India, and
for their inhibitory potential against several enzymes associated with Burma. The ripened fruit (purple-black, oblong berry) of C. opaca is
various diseases especially diabetes, hypertension, Alzheimer, inflam­ edible and has a sweet–sour taste. In Pakistan, the ripe fruit is sold in
mation, gastric ulcer, gastric cancer, and HIV-1 infections (Choi et al., some local markets and streets from October to February. The fruit is
2016; Deo et al., 2016; Umesalma & Sudhandiran, 2010; Yener et al., used in the preparation of pickles (Izhar & Ahmed, 2016). The fruit
2020). A number of researchers have demonstrated the richness of possesses good quantities of various mineral elements including

* Corresponding author.
E-mail address: taouskhan@cuiatd.edu.pk (T. Khan).

https://doi.org/10.1016/j.foodchem.2021.130259
Received 14 March 2021; Received in revised form 24 May 2021; Accepted 30 May 2021
Available online 1 June 2021
0308-8146/© 2021 Published by Elsevier Ltd.
K. Bashir et al. Food Chemistry 363 (2021) 130259

potassium, magnesium, iron, zinc, copper, and chromium (Ahmed et al., purpose, the crude extract (900 g) was suspended in distilled water and
2011). The fruit is a rich source of carbohydrates, lipids, proteins, and fractionated with organic solvents including n-hexane, chloroform, ethyl
fibers. The nutritional value of the fruit is 333.84 cal/100 g (Izhar & acetate, and n-butanol based on increasing order of polarity. All the
Ahmed, 2016). Preliminary quantitative phytochemical studies showed fractions were concentrated using Büchi Rotavapor® R-300 (BÜCHI
that fruit is rich in polyphenolics and exhibited strong antioxidant, Labortechnik AG, Flawil, Switzerland). Fractions including n-hexane
antimicrobial, antitumor, and anticancer activities (Sahreen, Khan, & (130 g), chloroform (11 g), ethyl acetate (9 g), n-butanol (50 g), and
Khan, 2010; Sahreen, Khan, Khan, & Shah, 2013). The methanolic water (650 g) were obtained and stored at − 20 ◦ C until further use.
extract from fruit has been shown to protect the kidney against oxidative
trauma due to high levels of antioxidants (Sahreen, Khan, Khan, & 2.4. Sample preparation for LC–MS/MS analysis
Alkreathy, 2015). The consumption of the fruit has shown beneficial
health effects in various ailments including diabetes and gastrointestinal Samples of C. opaca fruit methanolic extract and fractions were
problems (Izhar & Ahmed, 2016; Kaunda & Zhang, 2017) prepared for LC–MS/MS analysis by dissolving in HPLC grade methanol
C. opaca fruit extracts have been studied for the estimation of total (1 mg/mL), vortexed, sonicated, and filtered through a 0.45 µm mem­
phenolic and flavonoid contents and associated biological activities like brane filter (VWR, Radnor, PA, USA). A 100 µL volume for each sample
antioxidant, antitumor, and antimicrobial. However, the literature to was transferred to the HPLC autosampler vial and stored at − 20 ◦ C in a
date lacks any detailed phytochemical profiling of this plant. Therefore, refrigerator until analysis.
the current research was aimed at establishing a comprehensive
phytochemical profile of C. opaca fruit. For this purpose, LC-MS/MS and 2.5. Phytochemical analysis and dereplication
global natural product social molecular networking (GNPS) were
applied to comprehensively annotate the secondary metabolites present Metabolomics profiling of extract and fractions was carried out using
in C. opaca fruit. Quantitative phytochemical analysis for determining Dionex UltiMate 3000 ultra-high-performance liquid chromatography
total phenolic and flavonoid contents was also carried out. Additionally, (UHPLC) coupled to a Velos Pro hybrid linear trap quadrupole (LTQ)
in vitro and in silico α-glucosidase and urease inhibitory activities were mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The
performed to provide a scientific rationale for the health benefits of this chromatographic system was composed of Dionex Ultimate 3000
plant in associated problems. UHPLC comprising of RS (rapid separation) quaternary pump and RS
autosampler for separation of compounds while the detection part was
2. Material and methods equipped with a photodiode array (PDA) detector operated by Chro­
meleon software. The ESI technique was used along with tandem mass
2.1. Chemicals MS/MS in both positive and negative ion modes under similar chro­
matographic conditions. For chromatographic separation, a UHPLC
Solvents including n-hexane, ethyl acetate, chloroform, n-butanol, Luna Omega reverse-phase column C18 (50 mm × 2.1 mm, 1.6 µm)
methanol, and chemicals including aluminium chloride and potassium (Phenomenex, Torrance, CA, USA) was used. The gradient program was
acetate were purchased from Daejung Chemicals and Materials set using 0.1% formic acid in water (A) and 0.1% formic acid in aceto­
(Gyeonggi-do, Shiheung, Korea). Quercetin was obtained from Alfa nitrile (B). The gradient elution profile was as follows: 5% B (0.5 min),
Aesar (Ward Hill, MA, USA). Folin-Ciocalteu reagent and gallic acid 5–95% B (0.5–6.5 min), 95% B (6.5–8.5 min) and 95–5% B (8.5–10
were procured from Scharlau Chemicals (Barcelona, Spain). α-Glucosi­ min). The column temperature was maintained at 30 ◦ C. A flow rate of
dase from Saccharomyces cerevisiae was acquired from Sigma-Aldrich (St. 0.5 mL/min was maintained while injection volume was kept as 10 µL.
Louis, MO, USA). Jack bean urease was procured from SERVA Electro­ Spectra were recorded between the m/z range of 110 to 2000, collision-
phoresis GmbH (Heidelberg, Germany). HPLC grade solvents, including induced dissociation energy of 35 V, and resolution of 30,000 FWHM
acetonitrile and methanol, were bought from Thermo Fisher Scientific (full width and half maximum). Thermo XCalibur software was used for
(Waltham, MA, USA). A Milli-Q Water Purification System (Millipore, data acquisition, analysis, and interpretation of LC-MS/MS results.
Bedford, MA, USA) was used to obtain water for HPLC. Formic acid was These parameters include m/z, MS-MS fragmentation pattern, molecular
purchased from Acros Organics (Morris Plains, NJ, USA). formula, unsaturation equivalent, and m/z error in ppm.
Molecular networking of the C. opaca fruit crude extract and frac­
2.2. Plant material tions was performed using the global natural product social molecular
networking (GNPS) platform (https://gnps.ucsd.edu/ProteoSAFe/stat
The fresh ripened fruit of C. opaca was collected from Rawalpindi, ic/gnps-splash.jsp). All MS/MS data were converted to GNPS sup­
Punjab, Pakistan in March 2016. The plant specimen was authenticated ported “.mzML” format files using MSConvert package from the Pro­
by plant taxonomist, Dr. Abdul Nazir, Assistant Professor, COMSATS teoWizard 3.0.20360 (Proteowizard Software Foundation, Palo Alto,
University Islamabad (CUI), Abbottabad Campus, with voucher spec­ CA, USA). The files were uploaded on the GNPS platform using recom­
imen number (CUHA113). The plant specimen was placed at the her­ mended FTP client WinSCP. The spectral networks were imported into
barium of the Department of Environmental Sciences, CUI, Abbottabad Cytoscape 3.8.2 and were visualized for various features. Furthermore,
Campus for future record. the advanced molecular networking tool MolnetEnhancer was applied to
identify the major chemical classes of compounds present. The results of
2.3. Extraction and fractionation the GNPS analysis tools were carefully matched with results obtained
through manual annotation (Wang et al., 2016).
The fruit was garbled, washed thoroughly with tap water to remove The total phenolic content (TPC) was determined by adopting Folin-
dust particles, and subjected to drying under shade. The dried fruit was Ciocalteu reagent (FCR) method described by Madaan, Bansal, Kumar,
pulverized into a fine powder using a laboratory mill. The powdered and Sharma (2011). Briefly, stock solutions of crude extract and frac­
plant material (7.5 kg) was extracted thrice with methanol (20 L) for 21, tions were prepared in methanol at a concentration of 5 mg/mL. Then,
7, and 3 days, successively at room temperature. The extracted material 0.5 mL of the stock solution was mixed with 1.5 mL of FCR and incu­
was filtered using muslin cloth followed by Whatman 42 filter paper and bated for 5 min. Then, 4 mL of Na2CO3 solution (7.5%) was added to the
was concentrated at 40 ◦ C under reduced pressure using Büchi Rota­ reaction mixture and the final volume was made up with distilled water
vapor® R-300 (BÜCHI Labortechnik AG, Flawil, Switzerland) to obtain to 25 mL. Finally, the solution was incubated for 30 min after which the
crude methanol extract (950 g). Fractions of the methanol extract were absorbance was measured with a UV–visible spectrophotometer (PG
prepared using the solvent–solvent extraction technique. For this Instruments, Leicestershire, UK) at 765 nm. The standard curve for

2
K. Bashir et al. Food Chemistry 363 (2021) 130259

Table 1
Details of the identified constituents in C. opaca fruit crude extract and fractions using HPLC-DAD-ESI-MS/MS analysis.
C. opaca fruit crude extract

Compound Retention UV [M + MS2 fragmentation [M− H]− MS2 fragmentation Dereplication result Molecular Exact
No. time (min) λmax H]+ m/z ions [M + H]+ m/z ions [M− H]− Formula Mass
(nm) (Calc.)

1 0.53 194, – – 191.057 173.024, 127.041, quinic acid C7H12O6 192.063

211 111.046, 85.091
2 1.30 259, – – 153.018 109.067 protocatechuic acid C7H6O4 154.026
293
3 1.50 240 – – 195.051 177.026, 159.056, gluconic acid C6H12O7 196.058
129.022
4 2.1 300, 355.175 337.130, 193.051, 353.090 191.022 chlorogenic acid C16H18O9 354.095
325 163.041
5 3.40 310, 339.108 321.116, 177.226, 337.094 191.064 p-coumaroylquinic acid C16H18O8 338.100
369 147.036, 119.012
6 3.6 266, 565.156 547.186, 529.202, 563.143 545.087, 503.086, isoschaftoside C26H28O14 564.147
332 499.176, 481.169, 473.068, 443.061,
457.162, 427.175, 383.076, 353.059
409.146
7 3.70 260, 449.108 431.131, 413.128, 447.095 429.091, 357.066, orientin C21H20O11 448.100
295 383.125, 353.111, 327.074
329.111, 287.084
8 3.85 255, 433.113 415.160, 397.158, 431.100 341.087, 311.064, apigenin-8-C-glucoside C21H20O10 432.105
351 367.128, 337.150, 269.139 (Vitexin)
313.139, 271.125
9 3.95 266, 611.162 465.155, 303.070 609.141 301.082 Rutin C21H20O12 610.153
344
10 4.00 255, 465.103 303.076 463.084 301.072 isoquercitrin C21H20O12 464.095
351
11 4.09 254, 625.177 479.118, 317.066 623.552 315.059, 300.648 isorhamnetin-3-O-rutinoside C28H32O16 624.169
291
12 4.20 266 479.119 317.090 477.994 314.150 isorhamnetin-3-O-glucoside C22H22O12 478.111
13 5.07 269, 271.060 186.406 269.043 225.097, 201.101, apigenin C15H10O5 270.052
282 183.102, 149.074
369
14 6.26 223 524.372 506.393, 184.050 – – lysophosphatidylcholine C26H54NO7P 523.363
(18:0)
15 6.51 222 520.340 502.360, 184.101 – – lysophosphatidylcholine C26H50NO7P 519.332
(18:2)
16 6.88 223 522.356 504.384, 184.074 – – lysophosphatidylcholine C26H52NO7P 521.348
(18:1)
17 7.21 194, – – 595.291 415.236, 315.043, phosphatidylinositol (18:2) C27H49O12P 596.296
223 279.245, 241.033
18 8.01 206 457.368 439.398, 411.412, 455.354 407.346, 391.331, ursolic acid C30H48O3 456.360
393.406 367.454

n-Hexane fraction of C. opaca fruit extract

14** 6.26 223 524.372 506.393, 184.050 – – lysophosphatidylcholine18:0 C26H54NO7P 523.363
15** 6.51 222 520.340 502.360, 184.101 – – lysophosphatidylcholine 18:2 C26H50NO7P 519.332
19 6.68 223 496.340 479.461, 184.069 – – lysophosphatidylcholine PC C24H50NO7P 495.332
16:0
16** 6.88 223 522.356 504.384, 184.074 – – lysophosphatidylcholine 18:1 C26H52NO7P 521.348
17** 7.21 194, – – 595.291 415.236, 315.042, phosphatidylinositol (18:2) C27H49O12P 596.296
223 279.244, 241.032
20 7.3 218 – – 597.289 417.257, 315.043, phosphatidylinositol (18:1/ C27H51O12P1 598.311
281.249, 241.028 0:0)
21 7.52 195, 281.291 264.257, 246.263 279.234 261.230 linoleic acid C18H32O2 280.240
224
22 7.90 193, 283.281 265.312, 247.298 281.249 281.248. 263.247, oleic acid C18H34O2 282.255
225 141.306

Chloroform fraction of C. opaca fruit extract

23 5.2 269 317.096 302.052, 285.074 315.058 300.032 isorhamnetin C16H12O7 316.058
24 4.78 253, 303.050 285.066, 257.093, 301.032 179.067, 151.117 quercetin C15H10O7 302.043
369 229.084, 165.050
**
18 8.01 206 457.368 439.398, 411.412, 455.354 407.346, 391.331, ursolic acid C30H48O3 456.360
393.406 367.454

Ethyl acetate fraction of C. opaca fruit extract

2** 1.30 259, – – 153.018 109.068 protocatechuic acid C7H6O4 154.026
293
**
4 2.1 300, 355.175 337.131, 193.052, 353.090 191.022 chlorogenic acid C16H18O9 354.095
325 163.042
5** 3.40 310, 339.108 321.116, 177.226, 337.094 191.064 p-coumaroylquinic acid C16H18O8 338.100
369 147.036, 119.012
7*** 3.70 260, 449.108 447.095 429.091, 357.066, orientin C21H20O11 448.100
295 327.074
(continued on next page)

3
K. Bashir et al. Food Chemistry 363 (2021) 130259

Table 1 (continued )
C. opaca fruit crude extract

Compound Retention UV [M + MS2 fragmentation [M− H]− MS2 fragmentation Dereplication result Molecular Exact
No. time (min) λmax H]+ m/z ions [M + H]+ m/z ions [M− H]− Formula Mass
(nm) (Calc.)

431.132, 413.128,
383.125, 353.111,
329.111, 287.084
8** 3.95 255, 433.113 415.160, 397.158, 431.010 341.087, 311.064, apigenin-8-C-glucoside C21H20O10 432.105
351 367.129, 337.150, 269.139 (Vitexin)
313.140, 271.125
10** 4.00 255, 465.103 303.076 463.084 301.072 Isoquercitrin C21H20O12 464.095
351
**
11 4.09 254, 625.177 479.1187, 317.066 623.552 315.059, 300.650 isorhamnetin-3-O-rutinoside C28H32O16 624.169
291
12** 4.20 266 479.119 317.090 477.099 314.150 isorhamnetin-3-O-glucoside C22H22O12 478.111
25 4.27 290 579.113 561.157, 439.104, 577.101 559.036, 435.073, proanthocyanidin B2 C30H26O12 578.142
425.149, 285.079 423.019, 417.007,
279.021
24** 4.78 253, 303.050 285.067, 257.093, 301.032 179.067, 151.117 quercetin C15H10O7 302.043
369 229.084, 165.050
**
13 5.07 369 271.060 186.407 269.043 225.097, 201.101, apigenin C15H10O5 270.052
183.102, 149.074
23** 5.2 269 317.096 302.052, 285.074 315.058 300.032 isorhamnetin C16H12O7 316.058
18*** 8.01 206 457.368 439.398, 411.412, 455.354 407.346, 391.331, ursolic acid C30H48O3 456.360
393.406 367.454

n-Butanol fraction of C. opaca fruit extract

6** 3.6 266, 565.156 547.186, 529.202, 563.143 545.087, 503.086, isoschaftoside C26H28O14 564.147
332 499.176, 481.169, 473.068, 443.062,
457.162, 427.175, 383.077, 353.060
409.146
7*** 3.70 260, 449.108 431.132, 413.128, 447.095 429.091, 357.066, orientin C21H20O11 448.100
295 383.125, 353.111, 327.074
329.111, 287.084
9** 3.95 266, 611.162 465.155, 303.070 609.141 301.082 rutin C27H30O16 610.153
344
10*** 4.00 255, 465.103 303.076 463.084 301.072 isoquercitrin C21H20O12 464.095
351
***
11 4.09 254, 625.177 479.119, 317.066 623.552 315.059, 300.650 isorhamnetin-3-O-rutinoside C28H32O16 624.169
291
***
12 4.20 266 479.119 317.090 477.091 314.150 isorhamnetin-3-O-glucoside C22H22O12 478.111
24** 4.78 253, 303.050 285.067, 257.093, 301.032 179.067, 151.117 quercetin C15H10O7 302.043
369 229.084, 165.050
13*** 5.07 269, 271.060 186.407 269.043 225.097, 201.101, apigenin C15H10O5 270.052
282 183.102, 149.074
369

Aqueous fraction of C. opaca fruit extract

2** 0.0.53 211 – – 191.057 173.024, 127.041, quinic acid C7H12O6 192.063
111.046, 85.091
3** 1.50 212 – – 195.051 177.026, 159.057, gluconic acid C6H12O7 196.058
129.022
6*** 3.6 266, 565.156 547.186, 529.202, 563.143 545.087, 503.086, isoschaftoside C26H28O14 564.147
332 499.176, 481.170, 473.068, 443.062,
457.162, 427.175, 383.076, 353.060
409.146
a
Base peak in bold; b **, *** represents compounds observed in crude extract and also observed in one or two subsequent fractions, respectively; and c the “exact mass”
is the calculated mass of the protonated/deprotonated molecule.

estimation of TPC was constructed using gallic acid as standard (0–250 2.6. Enzyme inhibition assays
µg/mL). The results were calculated as µg of gallic acid equivalents
(GAE) per mg of sample. The α-glucosidase inhibitory potential of C. opaca fruit extract and
The total flavonoid content (TFC) was measured by aluminium fractions was evaluated according to the chromogenic method (Moradi-
chloride colorimetric method as reported by Chang, Yang, Wen, and Afrapoli et al., 2012). The test samples were prepared by dissolving the
Chern (2002). In brief, the stock solution of the extract and fractions extract and fractions at a concentration of 200 µg/mL in DMSO. The
were prepared in methanol at a concentration of 5 mg/mL. Then, 500 µL enzyme solution was composed of 20 μL of α-glucosidase (0.5 units/mL)
of each sample was taken from the stock solution and diluted with 1.5 and 120 μL of phosphate buffer (0.1 M, pH 6.9). Test samples (10 μL)
mL of methanol. To this, 100 µL each of aluminium chloride (10%) and were added to the enzyme solution in 96-well plate and incubated at
potassium acetate (1 M) was added. The final volume was made up to 5 37 ◦ C for 15 min. After this, 20 μL of substrate (p-nitrophenyl-α-D-glu­
mL with distilled water. The reaction mixture was incubated for 30 min copyranoside) solution (5 mM) was added to this solution and incubated
and the absorbance was measured with a UV–visible spectrophotometer for a further 15 min. Finally, the reaction was terminated by the addition
(PG Instruments, Leicestershire, UK) at 415 nm. The standard curve for of 80 μL of Na2CO3 solution (0.2 M), and absorbance of the resulting
TFC was constructed using quercetin as standard (0–250 µg/mL). TFC solution was recorded at 410 nm with SpectraMax microplate reader
was calculated as µg of quercetin equivalents (QE) per mg of sample. (Molecular Devices, Sunnyvale, CA, USA). The system without extract

4
K. Bashir et al. Food Chemistry 363 (2021) 130259

Fig. 1. Employing the molecular network of C. opaca fruit crude extract with the focus on selected nodes to provisionally dereplicate for the presence of known
flavonoid-C-glycosides and flavonoid-O-glycosides. The values are m/z [M− H]− of compounds identified by tandem-mass-based molecular networking present in
C. opaca fruit.

and fractions was used as negative control while the blank consisted of IC50 values.
the system without α-glucosidase. Acarbose was used as a standard in­
hibitor. The α-glucosidase inhibition (%) was calculated with the help of 2.7. Molecular docking studies
the following formula:
Molecular docking studies were performed to evaluate the binding
Absorbance of Control − Absorbance of Sample
Inhibition(%) = × 100 interaction of compounds observed in bioactive fractions into the active
Absorbance of Control
pocket of α-glucosidase and urease (Fig. S2). The 3D-structure of both
The % α-glucosidase inhibition was plotted against the various enzymes were retrieved from the Protein Data Bank with PDB ID: 2JKE
concentrations of test samples and IC50 values were calculated using (Resolution: 1.7 Å) (Gloster, Turkenburg, Potts, Henrissat, & Davies,
regression curve analysis. 2008) and 3LA4, (Resolution: 2.0 Å) (Balasubramanian & Ponnuraj,
The urease inhibition potential of extract and fractions were inves­ 2010) for α-glucosidase and urease, respectively. Docking experiments
tigated against Jack bean urease enzyme using the indophenol method were performed via Molecular Operating Environment (MOE) docking
as reported by Weatherburn with modifications (Khan et al., 2014). The program version 2016.08 (Vilar, Cozza, & Moro, 2008).
reaction mixture comprising of urease solution (25 μL) and 10 μL of each Protein structures were prepared using a multistep process involving
extract at 200 μg/mL, prepared in DMSO. The reaction mixture was put (i) removing the water molecules, (ii) addition of polar hydrogen atoms,
in each well and incubated at 30 ◦ C for 15 min. Then, 50 μL urea (100 and (iii) energy optimization using default parameters of MOE energy
mM) in phosphate buffer was placed in each well and followed by in­ minimization algorithm [gradient: 0.05, Force Field: MMFF94X]. The
cubation for 30 min. Then, a mixture consisting of 45 μL of phenol re­ geometrical parameters were optimized, and partial charges were
agent (1% w/v phenol and 0.005% sodium nitroprusside) and 70 μL calculated. Finally, the active site for both the enzymes was defined
alkali reagent (0.5% w/v NaOH and 0.1% NaOCl) was put in each well. within 10 Å of the native ligand, and 10 best-docked conformations were
The rate of change in absorbance was recorded at 630 nm after 50 min generated in each case. The complex with the lowest binding score was
using a SpectraMax microplate reader (Molecular Devices, Sunnyvale, selected and used for further analysis in each case. Ligand-interaction
CA, USA). Thiourea was used as the reference inhibitor. The formula module of MOE was used to calculate the 2D ligand-enzyme in­
mentioned below was employed to calculate the percent urease teractions while the view of the docking results and analysis of their
inhibition: surface with graphical representations were done using MOE and Dis­
Absorbance of Control − Absorbance of Sample covery Studio Visualizer. The structures of test compounds were pre­
Inhibition(%) =
Absorbance of Control
× 100 pared using Chemdraw ultra and optimized before molecular docking
studies.
The % urease inhibition was plotted against various concentrations
of the samples and a regression curve was established to calculate the

5
K. Bashir et al. Food Chemistry 363 (2021) 130259

2.8. Statistical analysis Table 2

Total phenolic and flavonoid contents (expressed as equivalents) of C. opaca fruit
All experiments were performed in triplicate and the results were crude extract and fractions.
expressed in terms of mean ± standard deviation. The inhibitory activ­ Entry Extract Total phenolic content (µg Total flavonoid content
ities, expressed as IC50 values, were calculated with GraphPad Software No. GAE/mg) (µg QE/mg)
(Version 5, California Corporation, CA, USA). Microsoft Excel version 1. Crude 52.4 ± 0.3 17.2 ± 0.2
2013 was used for constructing of standard calibration curve for TPC 2. n-Hexane 23.1 ± 1.7 9.6 ± 0.2
and TFC. 3. Chloroform 25.9 ± 2.5 12.0 ± 0.4
4. Ethyl 299.3 ± 1.6 92.5 ± 0.6
acetate
3. Results and discussion 5. n-Butanol 47.1 ± 0.1 26.6 ± 0.3
6. Aqueous 30.5 ± 1.3 10.6 ± 0.1
3.1. UHPLC-DAD-ESI-MS/MS analysis and dereplication a
Gallic acid equivalent = GAE, bQuercetin equivalent = QE

HPLC-MS/MS is routinely employed in the separation and tentative

fragmentation. Flavonoid-C-glycosides containing apigenin as aglycone
identification of complex mixtures from plant sources. Therefore, this
were identified as isoschaftoside and apigenin-8-C-glucoside (vitexin)
technique was used to carry out a comprehensive and detailed phyto­
while luteolin was the aglycone part of C-glycoside, orientin. Derivatives
chemical analysis of crude extract and fractions of C. opaca fruit. A total
of hydroxycinnamic acid include caffeoylquinic acid and coumar­
of 25 compounds were observed in crude extract and fractions. These
oylquinic acid and were annotated in crude extract and ethyl acetate
compounds belonged to various groups of phytochemicals including
fraction. The n-hexane fractions showed molecular network nodes cor­
phenolic acids, simple flavonoids, flavonoid-O-glycosides, flavonoid-C-
responding to phospholipids, the ethyl acetate fraction contained nodes
glycosides, polyols, triterpenoids, fatty acids, phosphatidylcholines, and
corresponding to flavonoid glycosides and phenolic acid derivatives
phosphatidylinositol. The various constituents observed in the crude
(Link 1, supplementary material), and the n-butanol fraction contained
extract and fractions are listed in Table 1 along with their m/z values in
nodes corresponding to flavonoid glycosides (Link 2, supplementary
both positive/negative ion modes, MS/MS fragmentation patterns,
section). The molecular networks of flavonoid-O-glycosides and
retention time, and absorbance wavelengths. The additional details
flavonoid-C-glycosides observed in C. opaca fruit are shown in Fig. 1. In
about LC–MS/MS parameters of compounds observed in the crude
this study, a total of 14 polyphenolic compounds were identified from
extract and fractions are provided in Table S1. Phospholipids and fatty
C. opaca fruit, indicating that this fruit is rich in phenolic constituents
acids including lysophosphatidylcholine (18:2), lysophosphatidylcho­
including phenols, flavonoids, and proanthocyanidin. To the best of our
line (18:1), lysophosphatidylcholine (18:0), lysophosphatidylcholine
knowledge, all the polyphenolics identified through LC–MS/MS GNPS
(16:0), phosphatidylinositol (18:0), phosphatidylinositol (18:2), linoleic
molecular networking are being reported for the first time from C. opaca
acid and oleic acid were observed in n-hexane fraction. The observed
fruit, while most of them have been firstly identified in genus Carissa.
constituents in chloroform fraction include isorhamnetin, quercetin, and
ursolic acid. Similarly, protocatechuic acid, chlorogenic acid, p-cou­
maroylquinic acid, orientin, apigenin-8-C-glucoside, isoschaftoside, 3.2. Total phenolic and flavonoid contents
isoquercitrin, isorhamnetin-3-O-rutinoside, isorhamnetin-3-O-gluco­
side, proanthocyanidin B2, quercetin, apigenin, isorhamnetin, and Colorimetric assays are widely applied to measure TPC and TFC in
ursolic acid were annotated in ethyl acetate fraction. Furthermore, iso­ plants (Aryal et al., 2019). TPC and TFC of C. opaca fruit extract and
schaftoside, orientin, rutin, isoquercitrin, isorhamnetin-3-O-rutinoside, subsequent fractions were determined from the calibration curve of
quercetin, and apigenin were observed in the n-butanol fraction. gallic acid (y = 0.0016x − 0.0431, R2 = 0.9918) and quercetin (y =
Aqueous fraction contained quinic acid, gluconic acid, and isoschafto­ 0.0065x − 0.0853, R2 = 0.9968), respectively (Figs. S3 and S4). The TPC
side. The structures of observed compounds in crude extract and frac­ and TFC of the extract and fractions of C. opaca fruit are shown in
tions of C. opaca fruit are given in Fig. S1. Flavonoid-O-glycosides were Table 2. The ethyl acetate fraction of C. opaca showed the highest TPC
fragmented with loss of sugar moieties 132, 146, and 162 Da indicating and TFC with 299.3 µg GAE/mg of extract and 92.5 µg QE/mg of extract,
loss of pentose, rhamnose, and glucose (Hassan, Abdelaziz, & Al Yousef, respectively. The lowest TPC and TFC were present in n-hexane fraction
2019). Flavonoid-C-glycosides showed major fragments with the loss of with a value of 23.1 µg GAE/mg and 9.6 µg QE/mg of extract,
60, 90, and 120 Da as characteristic ions (Wang et al., 2019). Previously, respectively.
quercetin and rutin were reported from the fruit of C. carandus using LC- Secondary metabolites, including phenolic acids, flavonoids, fla­
MS/MS analysis in positive ion mode (Patil et al., 2012). Ursolic acid vones, flavonoid glycosides, flavone glycosides are responsible for free
was reported from the root of C. carandus, leaves of C. macrocarpa, radical scavenging properties and related health benefits (Pandey &
C. spinarum, and C. bispinosa (Kaunda & Zhang, 2017). Rizvi, 2009). Polyphenolics from plant sources are considered safer for
In molecular networking analysis, each node represents one metab­ long-term use than synthetic antioxidants due to the associated side
olite, labeled according to its m/z value. Metabolites are grouped based effects (Lourenço, Moldão-Martins, & Alves, 2019). Quantitative anal­
on similarity in their fragmentation patterns, which indicated similarity ysis showed the abundance of polyphenolic compounds in the ethyl
in their core chemical structure. A total of 251 nodes were observed acetate and n-butanol fraction of C. opaca fruit extract (Table 2), which
upon the investigation of molecular networking outcomes of C. opaca was well supported by qualitative analysis of the same fractions
fruit crude extract (Fig. 1). The major clusters observed include flavo­ (Table 1). Weerawatanakorn and Pan (2017) reported high polyphenolic
noid glycosides (C and O), hydroxycinnamic acid derivatives, simple contents in C. carandas fruit, which displayed nitric oxide synthase and
flavonoids, glycoglycerols, glycerophosphoinositols, linoleic acid de­ cyclooxygenase-2 inhibition. The results of the current study showed
rivatives, and fatty acids. The ethyl acetate and n-butanol fractions that C. opaca fruit is rich in polyphenolic compounds, which may be
contained flavonoid-O-glycosides (cluster-1), flavonoid-C-glycosides responsible for its enzyme inhibition activities (Table 3) observed in the
(cluster 1), flavonoids (as single nodes), and hydroxycinnamic acid de­ current study.
rivatives (cluster-1) as the major class of secondary metabolites. The
ethyl acetate fraction showed the presence of the flavonoid-O-glycosides 3.3. Enzyme inhibition activity
quercetin and isorhamnetin. Rutin and isoquercitrin released the agly­
cone quercetin upon fragmentation. Isorhmentin-3-O-glucoside and 3.3.1. α-Glucosidase inhibition
isorhamnetin-3-O-rutinoside released the aglycone isorhamnetin upon Enzyme targets for treating diabetes, a chronic and serious metabolic

6
K. Bashir et al. Food Chemistry 363 (2021) 130259

Table 3 Prakash, 2011). Currently, various oral hypoglycemic agents including

α-Glucosidase and urease inhibitory activities of C. opaca fruit crude extract and acarbose, miglitol, and voglibose are prescribed for the management of
fractions. diabetes. However, due to their side effects, like abdominal discomforts
Entry Extract α-Glucosidase inhibition Urease Inhibition and flatulence, research has been diverted to safer and effective alter­
No. IC50 (μg/mL) IC50 (μg/mL) natives especially from natural sources (Zengin et al., 2015). Various
1. Crude 125.81 ± 0.3 130.68 ± 0.3 fruit extracts, pulps, and juices from edible plants, which are rich in
2. n-Hexane NA NA polyphenolic constituents have demonstrated promising α-glucosidase
3. Chloroform 142.50 ± 0.7 135.79 ± 0.6 inhibitory activities (Assefa et al., 2020; Berger et al., 2020; McDougall
4. Ethyl acetate 131.72 ± 0.3 109.14 ± 0.3
et al., 2005; Sulaiman & Ooi, 2013). The results obtained in the current
5. n-Butanol 123.67 ± 0.8 NA
6. Aqueous NA NA study for α-glucosidase inhibitory activity of C. opaca fruit extract and
7. acarbose 120.43 ± 0.3 Not tested fractions are given in Table 3. The fruit extract displayed a strong
(standard) inhibitory activity against α-glucosidase with an IC50 value of 125.81
8. thiourea Not tested 103.71 ± 0.9 μg/mL that is almost equivalent to acarbose (IC50 of 120.43 μg/mL).
(standard)
Effective inhibition was also shown by n-butanol, ethyl acetate, and
a
NA represents samples with less than 50% or no inhibition. chloroform fractions with IC50 values of 123.67, 131.72, and 142.50 μg/
mL, respectively. Aqueous and n-hexane fractions were devoid of any
disorder, is one of the major strategies for the development of antidia­ inhibitory activity against α-glucosidase. Various chemical constituents
betic drugs. The inhibition of α-glucosidase, which is responsible for identified in C. opaca fruit extract and subsequent fractions (Table 1) by
hydrolytic cleavage of oligosaccharides into absorbable mono­ GNPS molecular networking have been previously reported for
saccharides, is a key approach to decrease the rise in blood glucose levels α-glucosidase inhibition. These include (with IC50 values) isoschaftoside
and suppress postprandial hyperglycemia (Kumar, Narwal, Kumar, & (910.50 µg/mL), quercetin (0.53 mmol/L), isoquercitrin (0.24 mmol/L),

Fig. 2. 2D structural view of the binding interaction with (A) isorhamnetin-3-O-rutinoside, (B) apigenin-8-C-glucoside, (C) isoschaftoside, and (D) chlorogenic acid
inside active pocket of α-glucosidase.

7
K. Bashir et al. Food Chemistry 363 (2021) 130259

Fig. 3. 2D structural view of binding interaction pattern with (A) proanthocyanidin B2, (B) chlorogenic acid, (C) isorhamnetin-3-O-glucoside, (D) p-coumaroylquinic
acid inside active pocket of urease.

vitexin (0.42 mmol/L), isorhamnetin-3-O-rutinoside (0.51 mmol/L) and The ethyl acetate fraction displayed the highest urease inhibitory ac­
chlorogenic acid (2.99 mM) (Yin, Zhang, Feng, Zhang, & Kang, 2014). tivity with IC50 value of 109.14 μg/mL followed by chloroform fraction
Hence, these identified compounds may be responsible for the observed with IC50 value of 135.79 μg/mL. In the current study, the crude extract
α-glucosidase inhibitory activity of C. opaca fruit extract and fractions. of C. opaca fruit, ethyl acetate, and chloroform fractions exhibited good
Moreover, inhibition of α-glucosidase by the C. opaca fruit extract and inhibition against urease. Both ethyl acetate and chloroform fractions
fractions might be a probable mechanism towards its antidiabetic contain flavonoids and triterpenoids as identified by LC–MS/MS GNPS
potential. molecular networking analysis (Table 1). Additionally, the ethyl acetate
fraction is rich in phenolic acids, flavonoids, and flavonoid glycosides,
3.3.2. Urease inhibition which might be responsible for the observed urease inhibitory activity.
Urease, a metalloenzyme, is an important target in drug discovery for Polyphenolic constituents from various plant sources have been re­
antiulcer and antigastric cancer agents. Several urease inhibitors have ported to possess potent urease inhibitory activities (Hassan & Žemlička,
been developed in the past but most of those displayed severe side ef­ 2016). Furthermore, various constituents identified in C. opaca fruit
fects, toxicity, and instability. To overcome these limitations, the search extract, and the ethyl acetate and chloroform fractions (Table 1) have
for new natural sources of urease inhibitors especially from plants is shown in vitro urease inhibition in previous studies. These include (with
important (Hassan & Žemlička, 2016). The edible part of various plants IC50 values) quercetin (11.2 µM), rutin (67.6 µM), orientin (28 µM), and
including mung bean, pineapple, cinnamon, guava, and peel from avo­ vitexin (35 µM) (Kafarski & Talma, 2018; Xiao et al., 2012), which may
cado have showed inhibition activity (70–100%) against Jack bean be responsible for urease inhibition of C. opaca fruit.
urease (Shabana et al., 2010). In this scenario, the current study was
undertaken to evaluate the urease inhibitory activity of C. opaca fruit
extract and fractions, and the results obtained are given in Table 3. The 3.4. Molecular docking studies
extract demonstrated significant urease inhibition with an IC50 value of
130.68 μg/mL compared to standard thiourea (IC50 = 103.71 μg/mL). Bioactive compounds observed through mass spectrometry were
subjected to computer-based molecular docking studies to evaluate their

8
K. Bashir et al. Food Chemistry 363 (2021) 130259

binding interaction pattern with the active pocket of α-glucosidase and exhibited inhibition against urease. Results of computational studies
urease. The good inhibition potential of the n-butanol, ethyl acetate, and showing strong binding potential from many of the compounds identi­
chloroform fraction of C. opaca fruit crude extract against α-glucosidase fied in the extracts by LC–MS/MS GNPS molecular networking to
may be attributed to the presence of bioactive compounds listed in α-glucosidase and urease further strengthened and supported the in vitro
Table 1. All the identified compounds except isorhamnetin-3-O- enzyme inhibition activity results of the crude extract and bioactive
glucoside, rutin, and ursolic acid showed the binding interaction with fractions.
an active pocket of α-glucosidase having binding scores in the range of
− 5.386 to − 10.391 kcal/mol. Isorhamnetin-3-O-rutinoside showed the
Declaration of Competing Interest
best binding score of − 10.391 kcal/mol having H-bonding interaction
with Trp331, Glu391, Lys467, Glu194, and Ser217 as shown in Fig. 2.
The authors declare that they have no known competing financial
Apigenin-8-C-glucoside represented good inhibition with a binding
interests or personal relationships that could have appeared to influence
score of − 10.371 kcal/mol having H-bonding interaction with Glu 194,
the work reported in this paper.
Glu508, Glu532, and Lys467. Isoschaftoside showed a binding score of
− 9.693 kcal/mol having H-bonding interaction with Ile335, Glu439,
Acknowledgments
Lys467, and Glu532. Similarly, chlorogenic acid showed a binding score
of − 9.453 kcal/mol interacted through H-bonding with Glu391, Tyr533,
We are highly thankful to Higher Education Commission (HEC),
Glu194, and Ser217. The binding scores of all identified compounds are
Pakistan, for funding this project under Phase II, Batch III, Indigenous
listed in Table S2.
Ph.D. Fellowships Program for 5000 Scholars, and the International
The results of molecular studies against urease showed that all the
Research Support Initiative Program (IRSIP) Scholarship at the Uni­
ligands exhibited binding interaction with the active binding pocket of
versity of California, Santa Cruz, CA, USA.
urease having binding scores in the range from − 4.978 to − 14.381 kcal/
mol. Proanthocyanidin B2 with the highest binding score of − 14.381
kcal/mol showed H-bonding interaction with Asp494, Arg439, His545, Appendix A. Supplementary data
and Asp633 along with π-cation interaction with His594 and Arg439 and
a π-π interaction with His593 (Fig. 3). Isorhamnetin-3-O-glucoside was Supplementary data to this article can be found online at https://doi.
determined to have a binding score of − 11.726 kcal/mol showed H- org/10.1016/j.foodchem.2021.130259.
bonding interaction with His593, Arg609, Arg439, and His492 along
with a π-cation interaction with His593 similar to proanthocyanidin B2. References
Chlorogenic acid had a binding score of − 11.337 kcal/mol showing the
Ahmed, D., Waheed, A., Chaudhary, M. A., Khan, S. R., Hannan, A., & Barkaat, M.
H-bind interaction with Gln635, Arg439, Asp633, His545, and His492. (2011). Nutritional and antimicrobial studies on leaves and fruit of Carissa opaca
The p-coumaroylquinic acid exhibited a binding score of − 10.915 kcal/ Stapf ex Haines. Asian Journal of Chemistry, 23(5), 2072–2076.
mol having H-bonding interaction with Arg609 and three histidine Aryal, S., Baniya, M. K., Danekhu, K., Kunwar, P., Gurung, R., & Koirala, N. (2019). Total
phenolic content, flavonoid content and antioxidant potential of wild vegetables
residues (i.e., His492, His519, and His593) along with a π-cation inter­ from western Nepal. Plants, 8(4), 96. https://doi.org/10.3390/plants8040096.
action with Arg439 of the active pocket. The binding scores of com­ Assefa, S. T., Yang, E.-Y., Chae, S.-Y., Song, M., Lee, J., Cho, M.-C., & Jang, S. (2020).
pounds observed in bioactive fractions are listed in Table S2. Alpha glucosidase inhibitory activities of plants with focus on common vegetables.
Plants, 9(1), 2. https://doi.org/10.3390/plants9010002.
The use of computational approaches for the identification of various Balasubramanian, A., & Ponnuraj, K. (2010). Crystal structure of the first plant urease
bioactive compounds from the natural origin as drug leads is well from Jack bean: 83 years of journey from its first crystal to molecular structure.
documented (Rosdi, Arif, Bakar, Razali, & Zulkifli, 2018). In silico mo­ Journal of Molecular Biology, 400(3), 274–283. https://doi.org/10.1016/j.
jmb.2010.05.009.
lecular docking studies facilitated the rationalize binding poses and
Berger, K., Ostberg-Potthoff, J. J., Bakuradze, T., Winterhalter, P., & Richling, E. (2020).
interaction pattern of compounds in the current study and suggested Carbohydrate hydrolase-inhibitory activity of juice-based phenolic extracts in
them as possible inhibitors against α-glucosidase and urease. Most of correlation to their anthocyanin/copigment profile. Molecules, 25(22), 5224. https://
doi.org/10.3390/molecules25225224.
these compounds showed good binding scores and blocked the active
Boath, A. S., Stewart, D., & McDougall, G. J. (2012). Berry components inhibit
site that led to inhibition of both enzymes and loss of catalytic activity. α-glucosidase in vitro: Synergies between acarbose and polyphenols from black
In silico predictions of these bioactive compounds as inhibitors of urease currant and rowanberry. Food Chemistry, 135(3), 929–936. https://doi.org/10.1016/
and α-glucosidase are further suggested to be confirmed through in vitro j.foodchem.2012.06.065.
Chang, C.-C., Yang, M.-H., Wen, H.-M., & Chern, J.-C. (2002). Estimation of total
enzyme inhibition studies that will aid to identify the possible mecha­ flavonoid content in propolis by two complementary colorimetric methods. Journal
nism behind their inhibitory activity. Molecular docking studies of the of Food and Drug Analysis, 10(3), 178–182. https://doi.org/10.38212/2224-
identified compounds present in the crude extract and bioactive frac­ 6614.2748.
Choi, J. S., Bhakta, H. K., Fujii, H., Min, B.-S., Park, C. H., Yokozawa, T., & Jung, H. A.
tions of C. opaca fruit against α-glucosidase and urease showed strong (2016). Inhibitory evaluation of oligonol on α-glucosidase, protein tyrosine
bonding scores and further strengthened the in vitro results of our study. phosphatase 1B, cholinesterase, and β-secretase 1 related to diabetes and
Alzheimer’s disease. Archives of Pharmacal Research, 39(3), 409–420. https://doi.
org/10.1007/s12272-015-0682-8.
4. Conclusions Deo, P., Hewawasam, E., Karakoulakis, A., Claudie, D. J., Nelson, R., Simpson, B. S.,
Smith, N. M., & Semple, S. J. (2016). In vitro inhibitory activities of selected
The present research provides a detailed phytochemical profile of Australian medicinal plant extracts against protein glycation, angiotensin converting
enzyme (ACE) and digestive enzymes linked to type II diabetes. BMC Complementary
C. opaca fruit. The major secondary metabolites included phenolic acids, and Alternative Medicine, 16(1), 435. https://doi.org/10.1186/s12906-016-1421-5.
flavonoids, flavonoid glycosides, proanthocyanidin B2, and triterpe­ Gloster, T. M., Turkenburg, J. P., Potts, J. R., Henrissat, B., & Davies, G. J. (2008).
noids, which were annotated by LC–MS/MS GNPS molecular Divergence of catalytic mechanism within a glycosidase family provides insight into
evolution of carbohydrate metabolism by human gut flora. Chemistry & Biology, 15
networking. A total of 25 compounds were observed by LC–MS/MS (10), 1058–1067. https://doi.org/10.1016/j.chembiol.2008.09.005.
analysis GNPS molecular networking, of which 15 secondary metabo­ Hassan, W. H. B., Abdelaziz, S., & Al Yousef, H. M. (2019). Chemical Composition and
lites belonged to the bioactive ethyl acetate, n-butanol, and chloroform Biological Activities of the Aqueous Fraction of Parkinsonea aculeata L. Growing in
Saudi Arabia. Arabian Journal of Chemistry, 12(3), 377-387. DOI:10.1016/j.
fractions. Most of these compounds are being reported for the first time
arabjc.2018.08.003.
from C. opaca fruit and genus Carissa as well. The crude extract and Hassan, S. T. S., & Žemlička, M. (2016). Plant-derived urease inhibitors as alternative
fractions from C. opaca fruit exhibited inhibitory activity against chemotherapeutic agents. Archiv der Pharmazie, 349(7), 507–522. https://doi.org/
α-glucosidase and urease almost equivalent to standard inhibitors. The 10.1002/ardp.201500019.
Izhar, S., & Ahmed, D. (2016). Carissa opaca: A plant with great potential for future drugs
ethyl acetate, chloroform, and n-butanol fractions strongly inhibited for degenerative and infectious diseases. ChemistrySelect, 1(12), 3005–3011. https://
α-glucosidase, while the ethyl acetate and chloroform fractions doi.org/10.1002/SLCT.201600462.

9
K. Bashir et al. Food Chemistry 363 (2021) 130259

Kafarski, P., & Talma, M. (2018). Recent advances in design of new urease inhibitors: A Shabana, S., Kawai, A., Kai, K., Akiyama, K., & Hayashi, H. (2010). Inhibitory activity
review. Journal of Advanced Research, 13, 101–112. https://doi.org/10.1016/j. against urease of quercetin glycosides isolated from Allium cepa and Psidium guajava.
jare.2018.01.007. Bioscience, Biotechnology, and Biochemistry, 74(4), 878–880. https://doi.org/
Kaunda, J. S., & Zhang, Y. J. (2017). The Genus Carissa: An Ethnopharmacological, 10.1271/bbb.90895.
phytochemical and pharmacological review. Natural Products and Bioprospecting, 7 Sulaiman, S. F., Ooi, K. L., & Supriatno. (2013). Antioxidant and α-glucosidase inhibitory
(2), 181–199. https://doi.org/10.1007/s13659-017-0123-0. activities of cucurbit fruit vegetables and identification of active and major
Khan, S. S., Khan, A., Khan, A., Wadood, A., Farooq, U., Ahmed, A., … Erdemoglu, N. constituents from phenolic-rich extracts of Lagenaria siceraria and Sechium edule.
(2014). Urease inhibitory activity of ursane type sulfated saponins from the aerial Journal of Agricultural and Food Chemistry, 61(42), 10080-10090. DOI:10.1021/
parts of Zygophyllum fabago Linn. Phytomedicine, 21(3), 379–382. https://doi.org/ jf4031037.
10.1016/j.phymed.2013.09.009. Tungmunnithum, D., Thongboonyou, A., Pholboon, A., & Yangsabai, A. (2018).
Kumar, S., Narwal, S., Kumar, V., & Prakash, O. (2011). α-Glucosidase inhibitors from Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical
plants: A natural approach to treat diabetes. Pharmacognosy Reviews, 5(9), 19–29. and medical aspects: An overview. Medicines, 5(3), 93. https://doi.org/10.3390/
https://doi.org/10.4103/0973-7847.79096. medicines5030093.
Lourenço, S. C., Moldão-Martins, M., & Alves, V. D. (2019). Antioxidants of natural plant Umesalma, S., & Sudhandiran, G. (2010). Differential inhibitory effects of the polyphenol
origins: From sources to food industry applications. Molecules, 24(22), 4132. https:// ellagic acid on inflammatory mediators NF-kappaB, iNOS, COX-2, TNF-alpha, and IL-
doi.org/10.3390/molecules24224132. 6 in 1,2-dimethylhydrazine-induced rat colon carcinogenesis. Basic & Clinical
Madaan, R., Bansal, G., Kumar, S., & Sharma, A. (2011). Estimation of total phenols and Pharmacology & Toxicology, 107(2), 650–655. https://doi.org/10.1111/j.1742-
flavonoids in extracts of Actaea spicata roots and antioxidant activity studies. Indian 7843.2010.00565.x.
Journal of Pharmaceutical Sciences, 73(6), 666–669. https://doi.org/10.4103/0250- Vilar, S., Cozza, G., & Moro, S. (2008). Medicinal chemistry and the molecular operating
474X.100242. environment (MOE): Application of QSAR and molecular docking to drug discovery.
McDougall, G. J., Shpiro, F., Dobson, P., Smith, P., Blake, A., & Stewart, D. (2005). Current Topics in Medicinal Chemistry, 8(18), 1555–1572. https://doi.org/10.2174/
Different polyphenolic components of soft fruits inhibit alpha-amylase and alpha- 156802608786786624.
glucosidase. Journal of Agricultural and Food Chemistry, 53(7), 2760–2766. https:// Wang, M., Carver, J. J., Phelan, V. V., Sanchez, L. M., Garg, N., Peng, Y., … Bandeira, N.
doi.org/10.1021/jf0489926. (2016). Sharing and community curation of mass spectrometry data with Global
Moradi-Afrapoli, F., Asghari, B., Saeidnia, S., Ajani, Y., Mirjani, M., Malmir, M., … Natural Products Social Molecular Networking. Nature Biotechnology, 34(8),
Hamburger, M. (2012). In vitro α-glucosidase inhibitory activity of phenolic 828–837. https://doi.org/10.1038/nbt.3597.
constituents from aerial parts of Polygonum hyrcanicum. DARU Journal of Wang, Y., Liang, Z., Liao, X., Zhou, C., Xie, Z., Zhu, S., … Huang, Y. (2019). Identification
Pharmaceutical Sciences, 20(1), 37. https://doi.org/10.1186/2008-2231-20-37. of C-glycosyl flavones by high performance liquid chromatography electrospray
Nile, S. H., & Park, S. W. (2014). Edible berries: Bioactive components and their effect on ionization mass spectrometry and quantification of five main C-glycosyl flavones in
human health. Nutrition, 30(2), 134–144. https://doi.org/10.1016/j. Flickingeria fimbriata. BMC Chemistry, 13(1), 94-94. DOI:10.1186/s13065-019-
nut.2013.04.007. 0616-5.
Pandey, K. B., & Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidants in human Weerawatanakorn, M., & Pan, M.-H. (2017). Phytochemical components of Carissa
health and disease. Oxidative Medicine and Cellular Longevity, 2(5), 270–278. https:// carandas and the inhibitory effects of fruit juice on inducible nitric oxide synthase
doi.org/10.4161/oxim.2.5.9498. and cyclooxygenase- 2. Journal of Food Biochemistry, 41(3), e12343. https://doi.org/
Patil, R. P., Pai, S. R., Pawar, N. V., Shimpale, V. B., Patil, R. M., & Nimbalkar, M. S. 10.1111/jfbc.12343.
(2012). Chemical characterization, mineral analysis, and antioxidant potential of Xiao, Z.-P., Wang, X.-D., Peng, Z.-Y., Huang, S., Yang, P., Li, Q.-S., … Zhu, H.-L. (2012).
two underutilized berries (Carissa carandus and Eleagnus conferta) from the Western Molecular docking, kinetics study, and structure– activity relationship analysis of
Ghats of India. Critical Reviews in Food Science and Nutrition, 52(4), 312–320. https:// quercetin and its analogous as Helicobacter pylori urease inhibitors. Journal of
doi.org/10.1080/10408398.2010.500227. Agricultural and Food Chemistry, 60(42), 10572–10577. https://doi.org/10.1021/
Rosdi, M. N. M., Arif, S. M., Bakar, M. H. A., Razali, S. A., Zulkifli, R. M., & Ya’akob, H. jf303393n.
(2018). Molecular docking studies of bioactive compounds from Annona muricata Yener, I., Kocakaya, S. O., Ertas, A., Erhan, B., Kaplaner, E., Oral, E. V., … Kolak, U.
Linn as potential inhibitors for Bcl-2, Bcl-w and Mcl-1 antiapoptotic proteins. (2020). Selective in vitro and in silico enzymes inhibitory activities of phenolic acids
Apoptosis, 23(1), 27-40. DOI:10.1007/s10495-017-1434-7. and flavonoids of food plants: Relations with oxidative stress. Food Chemistry, 327,
Sahreen, S., Khan, M. R., & Khan, R. A. (2010). Evaluation of antioxidant activities of Article 127045. https://doi.org/10.1016/j.foodchem.2020.127045.
various solvent extracts of Carissa opaca fruits. Food Chemistry, 122(4), 1205–1211. Yin, Z., Zhang, W., Feng, F., Zhang, Y., & Kang, W. (2014). α-Glucosidase inhibitors
https://doi.org/10.1016/j.foodchem.2010.03.120. isolated from medicinal plants. Food Science and Human Wellness, 3(3), 136–174.
Sahreen, S., Khan, M. R., Khan, R. A., & Alkreathy, H. M. (2015). Protective effects of https://doi.org/10.1016/j.fshw.2014.11.003.
Carissa opaca fruits against CCl4-induced oxidative kidney lipid peroxidation and Zengin, G., Guler G. O., Aktumsek, A., Ceylan, R., Picot, C. M. N., & Mahomoodally, M. F.
trauma in rat. Food & Nutrition Research, 59(1), 28438. https://doi.org/10.3402/fnr. (2015). Enzyme inhibitory properties, antioxidant activities, and phytochemical
v59.28438. profile of three medicinal plants from Turkey, Advances in Pharmacological and
Sahreen, S., Khan, M. R., Khan, R. A., & Shah, N. A. (2013). Estimation of flavoniods, Pharmaceutical Sciences, vol. 2015, Article ID 410675, 8 pages, DOI:10.1155/2015/
antimicrobial, antitumor and anticancer activity of Carissa opaca fruits. BMC 410675.
Complementary and Alternative Medicine, 13(1), 372. https://doi.org/10.1186/1472-
6882- 13-372.

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