Kuwait Journal of Science 50 (2023) 322–325

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Kuwait Journal of Science

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Full Length Article

Total phenolic and flavonoid contents and antioxidant, antimicrobial and

antiproliferative activities of Polycarpon tetraphyllum
Imran Uysal
Dept. of Food Processing, Bahçe Vocational School of Higher Education, Osmaniye Korkut Ata University, Osmaniye, Turkey

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

Keywords: Plants are organic materials that support a variety of biological processes. The biological activity of the Polycarpon
Antimicrobial tetraphyllum (L.) L plant was identified in this study. In this case, the Soxhlet apparatus was used to obtain ethanol
Antioxidant and methanol extracts of the plant's aerial portions. Total antioxidant status (TAS), total oxidant status (TOS), and
Caryophyllaceae
oxidative stress index (OSI) values for the plant were determined using Rel Assay kits. The antioxidant potential of
Complementary medicine
Polycarpon tetraphyllum
the plant extract was determined by the DPPH test. Antimicrobial activity was determined using the agar dilution
test against six bacterial and three fungal strains. The antiproliferative activity of the plant extract against A549
lung cancer cell line was determined by MTT assay. Phenolic compounds in the plant were screened with LC-MS/
MS device. As a result of the study, the DPPH activity of the plant was found to be higher in the methanol extract
(75.72
0.32) at a concentration of 2 mg/mL. TAS value of the plant was determined as 8.449
0.143 mmol/L,
TOS value as 12.129
0.200 μmol/L and OSI value as 0.144
0.004. It was determined that the plant extracts
showed the highest effect against Acinetobacter baumannii, Candida albicans and C. krusei at 50 μg/mL. The ethanol
extract of the plant was found to have stronger antiproliferative activity. In addition, the presence of acetohy-
droxamic acid, catechin hydrate, resveratrol, fumaric acid, 4-hydroxycinnamic acid, 4-gydroxybenzoic acid,
oleuropein, ellagic acid, quercetin, butein and luteolin were found in the plant. In this context, it was determined
that the plant has important biological activity.

Introduction potential of P. tetraphyllum species. In this case, it is essential to study

Natural remedies derived from plants have become more significant
in the field of supplementary and alternative medicine. For both their
medicinal and nutritional value, plants are an essential resource. They
include several bioactive compounds with important biological activities.
Biological activities of plants are due to these bioactive compounds
(Berestovoy et al., 2020). Antioxidant, anticancer, antiproliferative,
antibacterial, DNA protection, and hepatoprotective activities are only
few of the many biological activities discovered in plants (Maslennikov
et al., 2014; Mohammed et al., 2021; Unal et al., 2022).
Polycarpon tetraphyllum is in the family Caryophyllaceae. There are 16
species in this genus as Polycarpon. P. tetraphyllum is a species native to
the Mediterranean region in general. It also spreads in Africa, Asia,
Europe, Oceania, the Americas, and Colombia. Among its morphological
features, a long root system, 4–10 mm long internodes, leaves opposite or
verticillate round, oval or slightly obovate glabrous bases weakly oppo-
site and seeds semicircular (Fonsec-Cort
es and Sandoval-Ortega, 2022).
In this context, it was aimed to determine the antiproliferative, total
antioxidant, total oxidant DPPH scavenging activity and antimicrobial
plants in order to discover novel, natural products. The biological activity
potential of the plant P. tetraphyllum (L.) L was determined in this study.
Material and methods
Plant material and extraction
P. tetraphyllum samples were collected from Osmaniye (Turkey).
P. tetraphyllum sample was made into herbarium material and preserved
in Osmaniye Korkut Ata University laboratory (Herbarium number: IU-
MS-2787). The soil and muddy parts of the plant samples were washed
with distilled water in the laboratory environment. The aerial parts of the
plant were dried in a shaded and dry area. It was then pulverized in a
mechanical grinder. Methanol (Merck) and ethanol (Merck) were used as
solvents. Plant samples were extracted with methanol (20 g plant ma-
terial: 250 mL methanol) in a Soxhlet device at 50  C for 6 h. After the
extraction process, the methanol of the extract was evaporated in
Heidolph-Rotary TLR 1000 device and the crude extract was obtained.
This process was repeated in ethanol.

E-mail address: uysal-imran@hotmail.com.

https://doi.org/10.1016/j.kjs.2023.02.022
Received 17 November 2022; Received in revised form 27 January 2023; Accepted 1 February 2023
2307-4108/© 2023 The Author. Published by Elsevier B.V. on behalf of Kuwait University. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
I. Uysal
Antioxidant activity tests
The hydrogen atoms or electrons donation ability of the corre-
sponding extracts, and some pure compounds, were measured from the
bleaching of purple colored methanol solution of DPPH. Solutions were
prepared from plant extracts at concentrations of 0.25, 0.5, 1, and 2 mg/
mL. Added 1 mL of DPPH radical solution in methanol (final concen-
tration of DPPH was 0.2 mM). The mixture was shaken vigorously. It was
then left to stand for 30 min. The absorbance of the final solution was
read at 517 nm by a spectrophotometer (Shimadzu UV-1601, Kyoto,
Japan). Ascorbic acid (AA) was used as the control antioxidant (Shimada
et al., 1992). For the scavenging percentage of DPPH; % inhibition ¼
[(Abs control-Abs sample)\Abs control]x100 formula was used.
In addition, total antioxidant (TAS) and total oxidant (TOS) values of
plant sample were determined. Results were obtained using Rel Assay
TAS and TOS kits. The experiment was performed following the pro-
cedure specified by the manufacturer (Erel, 2004; Erel, 2005). The ratio
of the TOS value to the TAS value was used to calculate the OSI value,
also known as the oxidative stress index (Sevindik, 2019).
Antimicrobial activity test
The antimicrobial activity of the plant extracts was determined by the
agar dilution method. The lowest extract concentration that inhibited the
growth of the bacteria and fungus strains was determined. Staphylococcus
aureus ATCC 29213, S. aureus MRSA ATCC 43300, Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC
29212 and Acinetobacter baumannii ATCC 19606 are the microorganisms
that were employed. Three fungal strains Candida albicans ATCC 10231,
C. krusei ATCC 34135, and C. glabrata ATCC 90030 were used. The plant
extracts were altered to have concentrations of 800, 400, 200, 100, 50,
25, 12.5 and 6.25 μg/mL. In contrast to fungal strains, which were pre-
cultured in RPMI 1640 Broth medium, bacterial strains were pre-
cultured in Muller Hinton Broth medium (Sevindik, 2019).
Antiproliferative activity test
The MTT assay was used to assess the extracts' antiproliferative effects
on the A549 lung cancer cell line (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl-tetrazolium bromide). Trypsin–EDTA solution was used to
conduct separation following incorporation into cells (Sigma–Aldrich,
MO, USA). After being seeded on plates, the separated cells were cultured
for around 24 h. Following that, it was diluted to various quantities (25,
50, 100, and 200 μg/mL). After that, it was again incubated for around
24 h. After a total of 48 h, the supernatants were collected and added to
the growth medium. MTT 1 mg/mL was used in place of the growth
medium. After that, it was incubated at 37  C. Then, the MTT solution
was removed and DMSO was added to the cells. At 570 nm, it was read
finally (Bal et al., 2017).
Phenolic content analysis
The separation of phenolic was performed with a LC-MS-MS appa-
ratus of Nexera UHPLC (Shimadzu) with two LC-20AD pumps, DGU-
20A3R degasser, CTO-10ASVP column furnace, and SIL20AC auto
sampler. The column's temperature was held constant at 40  C. The
elution gradient was made using mobile phase A (water, 5 mM ammo-
nium formate, and 0.1% formic acid) and mobile phase B (methanol, 5
mM ammonium formate, and 0.1% formic acid). The gradient pro-
gramme of the B solvent was utilized to decide how to apply. B % (0.40)
(20.90) (23.99) (24.40) (29.40) (29.40) (29.40). The injection volume
was fixed at 4 L, and the solvent flow rate was employed. The optimal
Electrospray Ionization (ESI) settings are a heatsink temperature of 400

C, an interface temperature of 350  C, and a DL temperature of 250  C.
The drying gas flow was calculated to be the nebulizer gas flow plus 15 L/
min (K€ oksal et al., 2017).
323
Kuwait Journal of Science 50 (2023) 322–325
Table 1
TAS, TOS and OSI values of P. tetraphyllum.
Sample TAS (mmol/L) TOS (μmol/L) OSI (TOS/(TASx10))
P. tetraphyllum 8.449
0.143 12.129
0.200 0.144
0.004
Values are presented as mean
S.D.; n ¼ 6 (Experiments were made as 5
parallel).
Table 2
DPPH scavenging activity of P. tetraphyllum.
0.25 0.5 1 2 mg/mL
AA 76.94
1.17 89.88
0.86 95.04
0.45 97.03
0.44
EtOH 53.77
1.06 64.41
1.96 73.60
1.22 75.72
0.32
MeOH 41.36
0.64 49.63
0.63 60.22
1.72 68.15
0.10
AA: Ascorbic acid, EtOH: Ethanol extract, MeOH: Methanol extract.
Total phenolic and flavonoid tests
0.1 mL of methanol extracts was diluted to 1 mL with distilled water.
Then, Folin-Ciocalteu reagent (1 mL, 1:9, v/v) was added and vortexed.
To this solution, 0.75 mL of 1% Na2CO3 solution was added. Absorbance
was measured at 760 nm after incubation for 2 h at room temperature.
The amount of total phenolic content was calculated as mg GAE g1 from
calibration curve of gallic acid standard solution (Uysal et al., 2017).
In order to ascertain the plant's total flavonoid content, aluminium
chloride analysis was performed (Chang et al., 2002). 0.5 mL of Quer-
cetin, 0.5 mL of plant sample, 4.3 mL of methanol, 0.1 mL of 10% Al
(NO3)3, and 0.1 mL of 1 M NH4CH3COO were combined. Then it was
incubated for 40 min. At 415 nm, the absorbance was measured. The
amount of flavonoids was calculated as μg QE g1. All experiments were
performed in triplicate. The averages of the analysis results were taken,
and their standard deviations were calculated.
Results and discussion
Antioxidant activity
The antioxidant, oxidant, oxidative stress, and DPPH activity of
P. tetraphyllum used in our study were determined. The results obtained
are shown in Table 1 and Table 2.
Plants include bioactive substances that perform a variety of antiox-
idant functions (Dai and Mumper, 2010). In this study, we measured
P. tetraphyllum's TAS, TOS, and OSI values. The literature did not contain
any research on P. tetraphyllum's antioxidant capacity. According to
research on several plant species, the TAS values of Helianthemum sali-
cifolium (Mohammed et al., 2021), Rhus coriaria var. zebaria (Mohammed
et al., 2018), Allium calocephalum (Mohammed et al., 2019), Scorzonera
papposa (Mohammed et al., 2020), Gundellia tournefortii (Saraç et al.,
2019), Mentha longifolia subsp. longifolia (Sevindik et al., 2017) were
reported 9.490, 7.342, 5.853, 5.314, 6.831 and 3.628 mmol/L, respec-
tively. TOS values were 14.839, 5.170, 16.288, 24.199, 3.712 and 4.046
μmol/L, respectively, OSI values are reported as 0.157, 0.071, 0.278,
0.456, 0.054 and 0.112, respectively. The TAS value of P. tetraphyllum
was shown to be greater than R. coriaria var. zebaria, A. calocephalum, S.
papposa, M. longifolia subsp. longifolia, and G. tournefortii, and lower than
H. salicifolium in comparison to these studies. In comparison to R. coriaria
var. zebaria, G. tournefortii, and M. longifolia subsp. longifolia,
P. tetraphyllum had greater TOS and OSI values, whereas A. calocephalum,
S. papposa, and H. salicifolium had lower TOS and OSI values. High TAS
values indicate that the natural product is an important natural antioxi-
dant material. The TOS value serves as a gauge for all oxidant-active
substances found in natural products. It is anticipated that healthy nat-
ural goods will have low quantities of oxidant chemicals. The TOS value
of P. tetraphyllum employed in our investigation is at normal values when
compared to the data from the literature. The OSI score also illustrates
I. Uysal Kuwait Journal of Science 50 (2023) 322–325

Table 3
Antimicrobial effect of P. tetraphyllum.
Extracts S. aureus S. aureus MRSA E. faecalis E. coli P. aeruginosa A.baumannii C. albicans C. glabrata C. krusei

EtOH 100 100 100 200 400 50 50 100 50

MeOH 200 200 100 200 400 100 100 100 100

*50, 100, 200, 400 μg/mL represents the lowest concentration that inhibits the growth of microorganisms.

how much antioxidants reduce the amount of oxidant molecules that a

natural product's metabolic processes produce. The OSI values of
P. tetraphyllum used in this investigation were determined to be at normal
levels when compared to the data from the literature. The P. tetraphyllum
employed in our investigation was found to have high TAS values. As a
byproduct of their metabolic processes, living things generate potentially
harmful chemicals known as free radicals (Ushakova et al., 2021). To
counteract the harmful effects of an increase in these chemical levels, the
body deploys its antioxidant defence mechanisms. If the body's antioxi-
dant defences are weak, oxidative stress will set in. Alzheimer's, Par-
kinson's, cancer, heart difficulties, and tissue damage are just some of the
disorders that oxidative stress may cause in people. To mitigate the
potentially harmful effects of oxidative stress, using antioxidant supple-
ments is a sensible choice. Since synthetic antioxidants may have unin-
tended consequences, most individuals choose natural alternatives
(Mushtaq et al., 2020). Given its significant antioxidant activity, the Fig. 1. Antiproliferative effect of P. tetraphyllum.
P. tetraphyllum we employed in this study is likely to be a source of
natural antioxidants.
When the free radical scavenging potential of P. tetraphyllum used in Table 4
our study was evaluated, it was determined that EtOH extracts from plant Phenolic contents of P. tetraphyllum.
extracts showed higher activity in general. It is seen that ascorbic acid, Phenolics Retention time P. tetraphyllum (ppb)
which is a strong antioxidant and used as a standard, produces 97.03
Acetohydroxamic acid 0,406 2148.32
inhibition at 2 mg/mL concentration. It was determined that EOH ex- Catechin Hydrate 2532 111.3
tracts of P. tetraphyllum produced 75.75 inhibition and 68.15 inhibition Resveratrol 3606 1522.49
from MeOH extract at 2 mg/mL concentration. According to these results, Fumaric acid 0,809 11,254.72
it is seen that the plant extracts show lower activity than the standard 4-Hydroxycinnamic acid 3489 861.58
4-Hydroxybenzoic acid 3555 188.71
used. In addition, it was determined that the plant extracts exhibited
Oleuropein 3567 17.09
strong activity, although not as much as the standard. It was previously Ellagic acid 3644 1209.63
reported that plants have significant DPPH activity (Qader and Awad, Quercetin 3681 96.07
2014). According to the study findings, it was determined that Butein 3891 547.63
Luteolin 4069 749.15
P. tetraphyllum has a natural free radical scavenging potential.
ND: Not detected.
Antimicrobial activity
Antiproliferative activity
In our study, the effects of P. tetraphyllum on bacterial and fungal
strains were investigated. The results obtained are shown in Table 3. In this study, the effects of MeOH and EtOH extract of P. tetraphyllum
Numerous earlier investigations on plants have demonstrated that against A549 (lung cancer cell line) were investigated and the results are
they are significant antimicrobial agents (Korkmaz et al., 2021). When shown in Fig. 1.
the antibacterial properties of the extracts used in our experiment were We found that as the quantity of P. tetraphyllum extracts increased, so
examined, it was found that the EtOH extract had more activity overall did their antiproliferative effect. EtOH extract was shown to be more
than the MeOH extract. At a concentration of 400 g/mL, plant extracts active than MeOH extract in the investigation. It has been established
were efficient against P. aeruginosa. At 200 g/mL, plant extracts were that P. tetraphyllum has not been investigated for its potential anti-
efficient against E. coli. At a concentration of 100 g/mL for the EtOH proliferative effects in the scientific literature. Several researches have
extract and 200 g/mL for the MeOH extract, the plant was efficient indicated that plants may be used to effectively combat A459 cells
against S. aureus and S. aureus MRSA. At a concentration of 100 g/mL, (Mohammed et al., 2018; Korkmaz et al., 2021). Herbal medicine ther-
plant extracts were efficient against E. faecalis and C. glabrata. In com- apies have recently been recognized as an alternative to synthetic and
parison to A. baumannii, C. albicans, and C. krusei, EtOH extract was derivative pharmaceuticals in the treatment of cancer. It is extremely
efficacious at concentrations of 50 g/mL and MeOH extract at 100 g/mL. important for the drug pool to carry out new studies for this area of use. A
There are not enough antibiotics on the market because of the potential patient's health and the efficacy of a treatment plan both benefit greatly
negative effects of synthetic antibiotics employed in recent years and the from the usage of unadulterated natural goods (Nirmala et al., 2011). In
emergence of resistant microorganisms (Sevindik and Bal, 2021). Natural this context, P. tetraphyllum was shown to have potent antiproliferative
products with antimicrobial properties must be identified in this setting effects, leading us to thinking that it may serve as a natural anti-
so that novel agents can be developed (Bouarab Chibane et al., 2019). proliferative agent.
Plants are among the candidates for natural antimicrobial agents due to
their capacity to produce significant bioactive chemicals. The Phenolic content
P. tetraphyllum employed in our investigation was therefore determined
to be a potential natural antimicrobial agent against bacterial and fungal Plants are natural products rich in secondary metabolite content.
strains. These metabolites are non-nutritive but medically important compounds

324
I. Uysal
Table 5
Total Phenolic and Flavonoid contents of P. tetraphyllum.
Sample Total phenolic (mg GAE g1) Total Flavonoid (μg QE g1)
P. tetraphyllum 53.11
1.34 101.20
1.52
(Uysal et al., 2020). In this study, the phenolic contents of P. tetraphyllum
were determined. The results obtained are shown in Table 4.
No study has been found to determine the phenolic contents of
P. tetraphyllum before. In our study, 26 phenolic compounds were screened
in the LC-MS/MS device. According to the results obtained, the presence of
11 compounds was determined. The detected compounds are Acetohy-
droxamic acid, Catechin Hydrate, Resveratrol, Fumaric acid, 4-Hydroxy-
cinnamic acid, 4-Hydroxybenzoic acid, Oleuropein, Ellagic acid,
Quercetin, Butein and Luteolin. It has been discovered that P. tetraphyllum
may be a natural source in this case for the detected chemicals.
Total phenolic and flavonoid contents
Secondary metabolites known as phenolic acids and flavonoids are
frequently found in plants. It has a distinct flavour, fragrance, and health-
improving qualities, particularly found in vegetables and fruits eaten as
food (Ali and Neda, 2011). We measured P. tetraphyllum's total phenolic
and flavonoid content in this study. Table 5 displays the information that
was gathered.
There is no previous data on the total phenolic and flavonoid contents
of P. tetraphyllum. The plant's total phenolic content was found to be
53.11
1.34 mgGAE/g extract in our investigation. The plant has a total
flavonoid content of 101.20
1.52 mgQue/g dry weight. In the litera-
ture, the total phenolic contents of 51 different plant species were
determined as the lowest in Cicer aretinum with 2.8 and the highest in
Hypericum triquetrifolium with 70.3 (Tawaha et al., 2007). Compared to
the literature data, it is seen that the total phenolic content of
P. tetraphyllum is at high levels. In addition, in a different study, the total
phenolic content of the methanol extract of Polycarpon polycarpoides was
reported as 101.84 μg.GAE.mg1 and the total flavonoid content was
1.43 μg.GAE.mg1 (Arioua et al., 2020). In this context, it is seen that the
total phenolic and flavonoid contents of P. tetraphyllum used in our study
are high.
Conclusion
The phenolic content and biological activity of P. tetraphyllum were
examined in this study. The research revealed that the plant has a strong
capacity to scavenge free radicals. It was found that the plant has a high
level of antioxidant activity. Furthermore, it was found to have efficacy
against common bacterial and fungal strains. It was also discovered that
it has potent antiproliferative effects on the lung cancer cell line A549. It
was thus established that the plant may have significant biological value
as a natural resource.
Funding
This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
_ IK
I would like to express our gratitude to Dr. Mustafa SEVIND _ for
their contributions to the present study.
325
Kuwait Journal of Science 50 (2023) 322–325
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