Content uploaded by Bratati Sikdar
Author content
All content in this area was uploaded by Bratati Sikdar on Jul 25, 2022
Content may be subject to copyright.
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
59
Exploration of antibacterial and antioxidant potential of
a few members of the family Piperaceae
Sikdar Bratati, Raj Adarsha and Roy Sudipta*
Department of Botany, University of Kalyani, Kalyani, Nadia, 741235 West Bengal, INDIA
*dr.sudiptaroy@gmail.com; sroy_bot@klyuniv.ac.in
Abstract
Piperaceae is a highly diverse and large family
composed of five genera of which Piper and Peperomia
are the most abundant. The current study endeavours
five Piper species (Piper betle, Piper nigrum, Piper
longum, Piper chaba and Piper retrofractum) and one
species of Peperomia (Peperomia pellucida)
concerning their phytochemical contents, antioxidant
and antibacterial properties. Besides their economic
uses, these plants also possess curative properties that
have been exploited ethnomedicinally since the
primaeval days. The methanolic extracts of both fresh
(FL) and shade dried (SDL) leaves of these plants
showed the presence of various phytochemicals.
Among the studied plants, polyphenols like total
phenolics (TPC), total flavonoids (TFC) and total
tannins (TTC) contents were maximum in P. betle FL
extract (TPC:39.50±0.99 mg GAE/ g extract, TFC:
19.40±0.57 mg QE/ g extract and TTC 11.08±0.11 mg
GAE/g extract) and significantly higher than the
others. Antioxidant efficacies of the extracts by total
antioxidant activity (TAA), ferric reducing antioxidant
power (FRAP) and ability to scavenge different
radicals (DPPH, ABTS, NO and SO), were also highest
in P. betle.
The study also highlighted the strong antibacterial
activities of the extracts against both Gram-positive
and Gram-negative bacteria. P. betle FL extract
showed the highest activity representing the maximum
zone of inhibition (24.65±0.21 mm) and lowest
MIC/MBC values (0.58±0.04/0.65±0.07 mg/ml)
against E. coli. These findings exhibit the potential of
these plant extracts, especially P. betle, in the
prospective exploration of plant-derived antioxidants
and therapeutic uses of these plants for developing
novel antibacterial drugs.
Keywords: Piperaceae, Piper, Peperomia, Polyphenolics,
Antibacterial, Antioxidant.
Introduction
The beneficial roles of plants were marked from primeval
periods and the conventional acquaintance has been
disseminated over the eras till modern days. A large variety
of plant-derived secondary metabolites like phenolics,
flavonoids, alkaloids, tannins, terpenoids and saponins are
known as effective antimicrobics and are of pharmacological
significance.12 The medicinal properties of such compounds
have been explored due to their compelling pharmacological
behaviour, minimal toxicity and cost effectivity.16 Plants
remained important bio-resources for traditional medicine
systems over the centuries and the bioprospecting of plant
resources led to the development of folk medicines,
nutraceuticals, food supplements, pharmaceutics and
modern medicines.12
Plants produce a wide range of natural antioxidant molecules
that can scavenge reactive oxidative species (ROS)
responsible for oxidative damage during oxidative stress.
Antioxidants from plants like flavonoids, tannins,
coumarins, anthocyanins, chromones, lignans, stilbenes,
carotenoids and vitamins exhibit varied properties including
anti-inflammatory, anti-analgesic, antiviral, antibacterial,
anticancer, anti-ageing etc.19 The increase in multi-drug
resistance in pathogenic microorganisms poses a grave
threat to mankind due to the random use of synthetic
antimicrobial drugs. To contend with this situation, new
antimicrobials are required and plants with medicinal
principles are of great choice for novel antimicrobial agents.6
Considering these facts, the study intended to explore two
diverse bioactivities namely antioxidant and antibacterial
efficacies in a few members of Piperaceae.
Piperaceae is a large family of angiosperm, generally known
as the pepper family consisting of 5 genera and about 3600
species. However, most of the species are clustered within
the two main genera, which are Piper and Peperomia.37
Piper nigrum L., a source plant of black and white pepper,
is a well-known member of the family. The Piper betle L.
leaves, consumed in various ways in South Asian countries,
are considered a trifling stimulant and are used by different
communities during religious occasions as well. Betel leaves
are also known for their vast ethnomedicinal properties since
ancient times. In addition, other Piper species like P. longum
L., P. chaba Trel. & Yunck. and P. retrofractum Vahl. are
reported for their distinct bioactivities.37 Peperomia
pellucida (L) Kunth, another representative of the family
Piperaceae has also been reported for its ethnobotanical uses
and pharmacological activities.31
Although the fruits of P. nigrum and P. longum have been
known significantly for their pharmacological and
economical values37, studies on their leaves are gaining
importance in recent days due to the presence of a wide
spectrum of phytochemicals. 6,17 Along with fresh leaves,
dried leaves are also beneficial in various ways if the
phytoconstituents remain unaltered. However, contradictory
reports exist regarding the loss of bioactivities during drying
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
60
methods.33 Keeping this view, we have used two types of
leaf samples- fresh leaf (FL) and shade dried leaf (SDL) in
the study.
The present study emphasises a comparative investigation of
six members of Piperaceae namely: P. betle, P. nigrum, P.
longum, P. chaba, P. retrofractum and Peperomia pellucida
about their phytoconstituents, varied antioxidant activities
and antibacterial properties using their leaf methanolic
extracts. This endeavour aims to designate effective
antioxidants and potent antibacterials from these important
members of the family Piperaceae, which can be useful in
the forthcoming period for the development of natural
antioxidants and plant-derived antimicrobials.
Material and Methods
Plant materials: Piper betle (paan, betel) leaves were
procured from a ‘paan boroj’ of Shimurali, Nadia, West
Bengal (23.044364º N, 88.512731º E, 12m above sea level).
Leaves of Piper nigrum (golmorich, black pepper), Piper
longum (pipul, Indian long pepper, Pippali), Piper
retrofractum (Javanese long pepper), Piper chaba (chui jhal
or piper chilli) were acquired from the Spice Garden, Bidhan
Chandra Krishi Viswavidyalaya, Kalyani (22.989893º N,
88.449395º E, 11m above sea level). We also collected the
leaves of Peperomia pellucida (luchi pata, shining bush
plant) from the University of Kalyani campus, Kalyani, West
Bengal (22.989133º N and 88.447411º E, 11m above the sea
level). The plants were identified with the help of a
taxonomic manual29 and the voucher specimens were
preserved at the departmental repository at the University of
Kalyani.
Methanolic extract preparation: Mature leaves of
different plant species were collected, cleaned and air dried
to remove surface moisture. Leaf samples (20g) were
trimmed into small pieces and extracted using 90% methanol
(v/v) by maintaining the sample to solvent ratio at 1:20
(w/v), kept for 48h at room temperature (RT; 30±2º C) with
agitation (45 rpm) and eventually were filtered to obtain
fresh leaf filtrates. Meanwhile, fresh leaves (20g) were shade
dried for 15 days, pulverized to form a powdered sample and
extracted similarly to obtain shade dried leaf filtrates. The
fresh and shade dried leaf filtrates were then concentrated
under reduced pressure in a rotary evaporator (Büchi,
Switzerland) at 40º C to obtain the fresh leaf extract (FL) and
shade dry leaf extract (SDL) respectively. These crude
extracts were preserved at 4º C until further use.
Phytochemical analyses of the extracts
Qualitative tests: Qualitative detections of different
phytochemical groups like sugars, phenolics, flavonoids,
tannins, alkaloids, terpenes, iridoid glycosides and saponins
were done following standard methods as described by Raj
et al.32
Quantitative tests for polyphenolics: Determination of
different polyphenolic components in the plant samples was
done following similar methods described by Ojha et al.23
Briefly, the extracts were mixed with Folin Ciocalteu (FC)
reagent and sodium carbonate, kept for incubation at dark for
45 min and the absorbances were recorded at 765 nm.
Phenolic contents were assessed from a calibration curve (r2=
0.982) of gallic acid and represented as gallic acid equivalent
(mg GAE/ g).
Total flavonoid contents (TFC) were performed by
successive addition of 5% sodium nitrite and alkaline
aluminium chloride (10%) in the extract samples and after
incubating at dark for 10 min, the absorbances were taken at
510 nm. The quantifications were done with the use of a
standard curve of quercetin (r2= 0.984) and expressed as
quercetin equivalent (mg QE/ g).
For total tannin content (TTC) estimation, extracts were
mixed consecutively with 0.1M ferric chloride and 8mM
potassium ferricyanide and incubated for 10 min at RT and
measured at 720 nm. A standard curve of gallic acid (r2=
0.993) was prepared to quantitate the tannin contents as
gallic acid equivalent (mg GAE/ g).
Determination of antioxidant activities: Antioxidant
activities were measured by total antioxidant activity (TAA),
ferric reducing antioxidant power (FRAP) and radical
scavenging assays like 2,2-diphenyl-1-picryl-hydrazyl-
hydrate (DPPH), 2,2´-azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid)(ABTS), superoxide (SO) and nitric oxide
(NO) following previously described methods.32
In brief, TAA was determined by the phospho-molybdenum
method in which extracts were mixed with sulfuric acid,
sodium phosphate and 4 mM ammonium molybdate
sequentially and incubated for 90 min at 90±2º C. The
reaction mixtures were measured at 695 nm and the
quantitation was done using a standard curve (r2= 0.983) of
ascorbate and denoted as ascorbic acid equivalent (mg AAE/
g).
For FRAP assay, extract samples were mixed with FRAP
reagent, incubated for 10 min at 37º C and measured at 594
nm. The estimates were calculated from a standard curve of
ferrous sulphate (r2= 0.994) and represented as mM Fe2+
equivalent/mg of extract.
The DPPH scavenging assay was performed by mixing
extracts (in varying concentrations: 0.025-1.0 mg/ml) and
DPPH solution (6 x 10-5 M). After incubation (15 min; RT;
at dark), the absorbances were measured at 517 nm. In ABTS
assay, extracts in different dilutions were mixed with
appropriately diluted ABTS reagent (formulated by mixing
2.45 mM potassium persulfate and 7 mM ABTS in deionized
water, set aside at dark condition for 16h which was then
diluted to an optical density of 0.70±0.02) and recorded at
734 nm. The NO radical scavenging property was assessed
by mixing the extracts with an equal volume of sodium
nitroprusside under illumination for 2h.
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
61
The mixtures were then treated with Greiss reagent, kept for
10min at RT and the absorbances were taken at 542 nm. In
SO assay, different concentrations of extracts were added in
0.05 M phosphate buffer (pH 7.5) which were mixed
sequentially with 0.018 mM riboflavin, 0.32 mM
ethylenediaminetetraacetic acid (EDTA) and 0.04 mM nitro
blue tetrazolium (NBT). The mixtures were then subjected
to illumination at RT for 1.5h and absorbances were taken at
590 nm.
In all the scavenging assays, the activity in percentage was
calculated by:
Scavenging activity (%) =
The 50% scavenging of the radicals (IC50 values) was
calculated from the percent scavenging of a sample
employing different concentrations.
Evaluation of antibacterial activity
Bacterial strains in the study: The antibacterial assays were
performed using three gram-positive (Bacillus subtilis
MTCC 121, Staphylococcus aureus ATCC 25923 and
Staphylococcus epidermidis MTCC 3086) and three gram-
negative (Escherichia coli MTCC 443, Vibrio cholerae
N16961, Salmonella enterica serovar Typhi C-6953)
bacteria. They were cultured aerobically either in nutrient
broth (NB; HiMedia) or in tryptone soy broth (TSB;
HiMedia) at 37º C with agitation (45 rpm) and whenever
required, agar plates of the said media were used. Mueller-
Hinton broth (MHB) and Mueller-Hinton agar (MHA) plates
were used during the antibacterial assays with incubation at
37º C for 24 h.
Bacterial inhibitory assay: The inhibitory assay was
performed following the agar-plate-based disc diffusion
assay of Kirby-Bauer3 with modifications.32 Bacterial
suspensions (100 µl, comprising around 2×108 colony
forming units/ml) were swabbed over MHA plates to obtain
uniform bacterial growth. Meanwhile, methanolic leaf
extracts of different dilutions (3, 6 and 12 mg) were made
from the stock solutions (200 mg/ml; in methanol) and
applied on sterile filter paper discs (5 mm). Methanol (60 µl)
and ampicillin (6 µg/ disc) were used as negative and positive
control respectively. The dried paper discs were aseptically
positioned over the bacterial smear, incubated overnight at
37º C and the bacterial inhibition zones were measured.
Estimation of the minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC):
The MIC and MBC of the extracts against different bacteria
were determined based on the micro-well dilution method13
elaborated by Raj et al.32 The extracts were diluted serially
(0.25-15 mg/ml) in MHB and poured into the wells (190 µl)
of a microtiter plate (Tarsons, India). Bacterial inoculums (10
µl, 1×107 CFU/ml) were applied to the wells to attain the
resulting volume of 200 µl in each well. Appropriate controls
like a positive control (ampicillin), extract control (extracts
without inoculum) and inoculum control (growth medium
only with inoculum, excluding extract) were also examined
in parallel with the extract treatments.
Subsequently, the microwell plates were incubated overnight
at 37°C with mild shaking (35 rpm) and the absorbances were
recorded at 620 nm to monitor bacterial growth with the aid
of a microtiter plate reader (BioTek, Switzerland). The MICs
were determined by evaluating the minimum concentration
of the sample where no growth of the bacteria was evident.
The MBC was estimated using a loopful of bacterial
suspension (approximately 5 µl) from each well of the MIC
plate and the suspensions were streaked on MHA plates. The
plates were kept overnight at 37°C and the lowest
concentration, at which no bacterial colonies appeared, was
determined as the MBC of the sample.
Statistical analyses: The data were illustrated as mean ±
standard deviation (SD) of three replications. One-way
Analysis of Variance (ANOVA) followed by Tukey’s
multiple comparison test (p<0.05) was used to compare the
results of phenolic estimates and antioxidant assays. In the
disc diffusion assay, significant variations (p<0.05)
between/among the different extract concentrations were
determined by comparing critical differences (CD).
Results and Discussion
Phytochemical analyses: Our study revealed the presence
of different phytochemicals in the six plant species detected
by qualitative analyses as represented in table 1. The
phytochemical groups like sugars, phenolics, flavonoids,
tannins, alkaloids, terpenes, iridoids, saponins were detected
at high to moderate levels in all the plants with higher
amounts of phenolic components in P. betle and P. nigrum
while higher terpene contents were detected in P. nigrum, P.
longum and Peperomia pellucida. Iridoid glycosides and
saponins were detected in P. betle, P. retrofractum and
Peperomia pellucida. A host of studies by various
researchers revealed the extraction of wide-ranging
phytochemicals by methanol in different plants.27,46
Methanol is a widely used solvent for extraction as its polar
nature helps to extract the polar compounds easily,41
however, the extraction of many non-polar compounds is
also facilitated by the solvent.15,39 Methanolic extraction of
different phytochemical classes in the leaves of different
Piper spp.9,44 and Peperomia pellucida25 was also
documented by others. Among the different plant parts,
leaves are known to contain rich sources of phytochemicals,
particularly phenolics,21,47 presumably due to their higher
photosynthetic efficacies.47 As such distinctions between
phytochemical contents in FL and SDL were mostly absent
following qualitative analyses which may be due to the
feeble sensitivity of the employed assays.
(Acontrol – Asample)
Acontrol
× 100
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
62
Table 1
Qualitative estimation of phytochemicals in methanolic extracts of a few members of Piperaceae.
Plant Species
Phytochemicals
Sugars
Phenolics
Flavonoids
Tannins
Alkaloids
Terpenes
Iridoids
Saponins
PB
FL
+
++
++
++
++
+
+
+
SDL
+
++
++
++
++
+
+
+
PN
FL
+
++
++
++
+
++
-
-
SDL
+
++
++
++
-
++
-
-
PL
FL
+
+
+
+
+
++
-
-
SDL
-
++
+
+
+
++
-
-
PR
FL
+
++
+
+
+
+
++
+
SDL
-
++
+
+
-
+
+
+
PC
FL
+
++
+
+
-
+
+
-
SDL
-
++
+
-
-
+
-
-
PP
FL
++
+
++
-
-
++
++
+
SDL
++
+
++
-
-
++
+
+
‘++’ High, ‘+’ Moderate, ‘−’ Absent; PB: Piper betle, PN: Piper nigrum, PL: Piper longum, PR: Piper retrofractum, PC: Piper
chaba, PP: Peperomia pellucida; FL- Fresh leaf extract, SDL- Shade-dried leaf extract.
Figure 1: Quantitative estimation of Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and
Total Tannin Content (TTC) in different leaf extracts.
PB: Piper betle, PN: Piper nigrum, PL: Piper longum, PR: Piper retrofractum, PC: Piper chaba, PP: Peperomia pellucida;
FL- Fresh leaf extract, SDL- Shade-dried leaf extract; GAE: Gallic acid equivalent, QE: Quercetin equivalent.
Different alphabets (uppercase for FL and lowercase for SDL) used in the figure represent significant differences (p<0.05)
calculated by Tukey’s Post-hoc test.
The quantitative estimates of polyphenolic components in
the leaf extracts were depicted in figure 1. All the studied
plants possessed substantial TPC, TFC and TTC contents
and among them, P. betle represented the highest yield in
every component. In comparison between the leaf samples,
FL demonstrated a better yield than SDL in most cases. The
highest TPC (mg GAE/g extract) was observed in P.
betle (FL: 39.50±0.99; SDL: 34.70±0.71)
significantly (p<0.05) differing from others while the lowest
yield was noticed in Peperomia pellucida (FL: 28.70±0.99;
SDL: 21.20±0.28). The total phenolics of other plants were
placed in between these two plants. In flavonoid contents
(TFC; mg QE/g extract), the superiority was attained by P.
betle (FL: 19.40±0.57; SDL: 17.65±0.35) with
significant (p<0.05) variations from other plants and among
them, the lowest flavonoid yield was observed in P.
longum (FL: 15.96±0.42; SDL: 14.05±0.36).
The flavonoid content in Peperomia pellucida was
remarkably higher (FL: 17.98±0.32; SDL: 16.08±0.53) but
fell short to reach the highest value. The TTC yields (mg
GAE/g extract), however, followed a nearly similar trend to
TPC with the highest in P. betle (FL: 11.08±0.11;
SDL: 8.33±0.11) and lowest in Peperomia
pellucida (FL: 3.93±0.07; SDL: 3.93±0.25) with
significant (p<0.05) variations in most of the cases. The
predominance of phenolic compounds in the leaf tissues was
also documented by various studies21,47 and also from our
previous studies.24 The better yield of phenolics using either
FL or SDL remained conflicting as several reports indicated
higher yields with FL26,30 while superior phenolic yields with
SDL were also reported by many researchers.24,35
However, in our study, the TPC, TFC and TTC contents
were greater with FL than SDL pointing towards the
inactivation or degradation of certain phytochemicals during
drying. The proposition is strengthened by the earlier
observations revealing the loss of various essential oils and
other compounds during drying of Arum palaestinum30 and
P. betle33 leaves. Various studies estimated different
phenolic components in Piper spp.3,40,43,45 or Peperomia
pellucida22 individually.
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
63
Figure 2: Antioxidant properties of fresh (FL) and shade dried (SDL) leaf extracts by
total antioxidant activity (TAA), ferric reducing antioxidant potential (FRAP) and radical scavenging
[DPPH, ABTS, Nitric oxide (NO) Superoxide (SO)] assays.
PB: Piper betle, PN: Piper nigrum, PL: Piper longum, PR: Piper retrofractum, PC: Piper chaba, PP: Peperomia pellucida;
AA: Ascorbic acid, Q: Quercetin.
Different alphabets (uppercase for FL and lowercase for SDL) used in the figure represent significant differences (p<0.05)
calculated by Tukey’s Post-hoc test
However, this study presented a comparative investigation
of phenolic components of six important members of the
family Piperaceae. Among the six plants, P. betle leaf
extracts exhibited the highest TPC, TFC and TTC values
while in other members, the estimates varied substantially.
Such variations among the plants may depend on various
factors like plant genotype, tissue types, growth stages and
environmental factors among others.18
Antioxidant activities: The antioxidant activities of
different plant extracts employing several assays were
illustrated in figure 2. The higher estimates of TAA and
FRAP are positively correlated with their antioxidant
activities while inversely proportional relationships exist
between IC50 values in the radical scavenging assays
(DPPH, ABTS, NO and SO) and their bioactivities.
The antioxidant activities of these plants on an individual
basis were reported by various studies,3,22,34,40,43,45 however,
the study made a comparative evaluation among the plants
in determining the most efficient one. Our study also
encompasses a number of antioxidative assays of different
principles as it has been evinced that the effectiveness of
antioxidants is better arbitrated through diverse analyses.28
In all the antioxidant assays, FL extracts showed higher
antioxidant activities than SDL and the observation was also
in agreement with several studies performed on different
plants.28,35 Such a decrease in activities was attributed to the
degradation or modification of antioxidant phytochemicals
during drying.5
The TAA activity (mg AAE/gm extract) was maximum in
FL extract of P. betle (69.05±0.21) followed by Peperomia
pellucida (43.10±0.14), P. nigrum (42.70±0.48), P.
chaba (42.63±0.26), P. longum (41.28±0.30) and P.
retrofractum (40.70±0.14). A similar trend was also
observed in SDL extracts but at a lesser amount. In both FL
and SDL, P. betle activities were significantly (p<0.05)
higher than the others. In other reports, the TAA activities at
varying degrees were also documented in Piper spp.3,34,39
and in Peperomia pellucida.22 The higher TAA activity in
the studied plants was corroborated by their rich phenolic
contents and that was also evidenced in previous
reports.3,34,42
The FRAP assay also displayed maximum activity (mM
Fe2+/g) in P. betle (FL: 36.42±2.96, SDL: 15.77±0.86) with
significant variations (p<0.05) than the other plants.
However, in both FL and SDL extracts, the other plants
showed near-identical activities with a nonsignificant
(p>0.05) relationship excepting the FL extract of P. nigrum.
Among the different plants, FRAP activity of P. betle FL
extract was much closer to the reference (positive control)
ascorbic acid, though differing significantly (p<0.05). The
FRAP activity was deduced in Piper spp. using different
solvent extracts and better activity was shown by many in
hydroalcoholic fractions3,34 while better activity with ethyl
acetate was documented by few.1
The leading antioxidant activity of P. betle was again
documented in the radical scavenging assays represented by
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
64
their lower IC50 values. Results also revealed greater
antioxidant activities in FL than SDL. In the DPPH assay,
the lowest IC50 values (mg/ml) were observed in P. betle
(FL: 40.77±1.91; SDL: 47.29±2.08), almost approaching the
activities of standard ascorbic acid (33.38±3.35) showing
nonsignificant (p>0.05) variations. Such a strong activity of
P. betle points toward its greater potential as a source of
antioxidants having more antioxidant molecules in the
extract. The other leaf extracts manifested weaker activities
with much higher IC50 values showing significant variations
(p<0.05) to P. betle as well as ascorbic acid.
Similar trends were observed in the ABTS assay with a
minimum IC50 value in P. betle (FL: 72.38±4.50; SDL:
79.73±6.41) with non-significant relations to the positive
control ascorbic acid. The other Piper spp. demonstrated
higher IC50 values differing significantly (p<0.05) from P.
betle, however, variations between themselves in SDL
extracts were nonsignificant. The highest IC50 values were
demonstrated by Peperomia pellucida (FL: 278.64±20.70;
SDL: 318.29±42.45), significantly (p<0.05) differing from
other samples.
In NO and SO assays, the lowest IC50 value among the plants
was also observed in P. betle FL extract (NO: 100.80±4.98
and SO: 290.04±14.22) followed by P. nigrum FL extract
(NO: 159.22±35.15 and SO: 327.96±18.64) and the
estimates were not statistically significant with the standard
quercetin (NO: 90.01±5.72 and SO: 282.51±6.37
respectively).
Assessment of all antioxidant assays revealed the superiority
of P. betle over the other studied members of the family
Piperaceae. The highest activities of P. betle in all the
antioxidant assays can be interrelated with their higher
polyphenolic contents as polyphenols are regarded as one of
the major contributors of antioxidants in plants.42
Table 2
Growth inhibition zones (mm) showing antibacterial activity of the extracts against different bacterial strains
Plant
Extract
Conc.
(mg/
disc)
B. subtilis MTCC 121
S. aureus ATCC
25923
S. epidermidis MTCC
3086
E. coli MTCC 443
V. cholerae N16961
S. Typhi C-6953
FL
SDL
FL
SDL
FL
SDL
FL
SDL
FL
SDL
FL
SDL
PB
3
19.50±
1.70
11.5±
0.85
14.85±
0.07
8.95±
0.21
17.35±
1.63
13.20±
0.71
20.85±
1.48
11.95±
0.78
15.60±
2.40
8.65±
0.78
15.90±
1.41
11.00±
1.27
6
23.20±
1.41
17.35±
0.64
16.90±
0.14
13.55±
0.35
19.35±
0.78
14.90±
1.41
22.85±
1.06
14.35±
0.49
17.35±
0.78
12.70±
1.41
17.90±
1.41
13.95±
1.20
12
24.45±
0.92
19.80±
1.56
18.85±
1.34
14.70±
0.28
20.45±
0.64
15.80±
1.56
24.65±
0.21
16.60±
0.99
18.70±
1.56
16.25±
0.49
20.20±
0.42
15.20±
1.41
PN
3
8.30±
0.71
6.80±
0.08
8.05±
0.35
ND
7.45±
0.64
5.40±
0.57
8.35±
0.78
7.27±
0.75
7.45±
3.46
5.27±
0.46
6.85±
1.34
5.45±
0.07
6
9.45±
0.35
7.85±
0.89
9.23±
1.07
ND
8.95±
1.20
7.40±
2.12
9.7± 0.28
9.02±
2.37
7.45±
3.46
6.10±
1.49
8.30±
1.84
7.25±
0.92
12
10.55±
0.78
8.70±
0.92
9.47±
0.38
6.17±
0.55
10.1±
0.71
8.47±
1.42
10.80±
0.42
9.90±
3.37
9.90±
4.10
7.17±
1.52
9.40±
0.71
9.45±
0.78
PL
3
7.20±
0.99
6.58±
0.64
6.97±
0.47
ND
9.15±
3.18
ND
6.30±
0.71
6.15±
0.79
6.45±
2.05
6.40±
0.72
6.85±
1.48
5.87±
0.28
6
7.85±
1.34
7.80±
1.36
7.90±
0.95
ND
10.55±
3.32
ND
7.15±
0.92
7.35±
0.30
7.70±
2.69
6.93±
0.49
7.35±
0.64
6.95±
1.34
12
8.95±
1.34
8.93±
1.53
8.47±
1.07
5.53±
0.47
11.95±
1.48
6.30±
0.20
7.95±
0.92
8.85±
0.66
9.05±
2.47
7.37±
0.42
9.40±
0.71
7.25±
0.49
PR
3
6.26±
0.21
ND
ND
ND
ND
ND
6.53±
0.35
ND
ND
ND
ND
ND
6
7.86±
0.21
ND
6.07±
0.38
6.37±
0.74
7.35±
0.78
6.40±
0.46
7.70±
0.56
6.52±
0.50
7.25±
0.42
ND
6.32±
0.33
ND
12
8.23±
0.28
7.03±
0.71
8.13±
0.35
7.67±
0.67
8.25±
0.07
7.80±
0.60
8.30±
0.66
7.50±
0.45
8.11±
0.63
ND
7.49±
0.52
ND
PC
3
6.95±
0.64
5.93±
1.14
6.20±
1.44
ND
5.40±
0.57
ND
7.95±
2.76
7.07±
0.21
5.95±
1.35
5.70±
0.36
5.95±
0.21
5.67±
0.38
6
8.55±
1.06
7.63±
1.14
6.70±
2.00
ND
7.35±
2.05
5.77±
0.35
9.80±
2.83
8.63±
1.42
6.85±
1.48
6.00±
0.95
6.65±
0.92
5.75±
0.21
12
9.15±
0.07
8.90±
2.63
7.80±
1.56
6.36±
0.55
8.10±
1.70
7.37±
1.32
11.85±
2.19
11.23±
2.99
8.65±
2.33
7.00±
1.11
7.85±
2.76
7.55±
0.49
PP
3
5.85±
0.07
5.65±
0.90
5.90±
0.26
ND
5.85±
0.07
ND
5.95±
0.21
5.67±
0.86
ND
ND
5.50±
0.71
ND
6
7.00±
0.14
6.25±
1.95
7.90±
0.61
ND
7.10±
0.57
ND
7.20±
0.14
6.42±
0.62
7.70±
0.14
6.70±
0.28
6.45±
2.05
ND
12
9.35±
0.64
7.45±
2.53
9.57±
0.35
6.17±
0.55
7.45±
0.64
6.33±
0.47
8.60±
0.42
7.75±
1.91
9.55±
0.49
8.85±
1.48
6.95±
2.76
ND
CD value at 0.5%
level
0.504
1.005
0.654
0.292
0.766
0.534
0.695
0.988
0.787
1.285
0.777
0.587
Amp
0.006
41.00± 1.10
41.80± 1.20
31.10± 0.60
22.00± 0.60
10.80± 0.20
21.80± 0.80
PB: Piper betle, PN: Piper nigrum, PL: Piper longum, PR: Piper retrofractum, PC: Piper chaba, PP: Peperomia pellucida; FL- Fresh
leaf extract, SDL- Shade-dried leaf extract; Amp: Ampicillin; ND = Not Detected
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
65
A positive correlation between the polyphenol contents and
antioxidant activities was also established by several
previous studies.23,36 The noticeable activities of P. nigrum
leaf extract in NO and SO scavenging assays also highlight
its potential in combating oxidative damage and warrant
further exploration.
Antibacterial activities: The inhibitory activities of
bacterial growth by methanolic extracts (3, 6 and 12 mg) of
the members of Piperaceae against different bacterial strains
following disc diffusion assay, are presented in table 2. The
different plant extracts manifested sufficient antibacterial
activity in a dose-dependent manner showing maximum
activities at the highest concentration (12 mg). The
variations between the extract concentrations were
significant (p<0.05) in most cases. It is well-known that
concentration-dependent increments in activities are the
hallmark of the specific mode of action by any bioactive
compounds. The concentration-dependent increments in
antibacterial activities in our study established the specificity
of these extracts. Similar observations were noticed in
various plant extracts showing distinct bioactivities. 14,32
The FL extracts were much more efficacious than the SDL
extract types in all the studied plants and P. betle FL extract
(12mg) unveiled the highest inhibitory activity with the
inhibition zones (mm) against E. coli (24.65±0.21) followed
by B. subtilis (24.45±0.92), S. epidermidis (20.45±0.64), S.
Typhi (20.20±0.42), S. aureus (18.85±1.34) and V. cholerae
(18.70±1.56). Among the SDL extracts, P. betle exhibited
the highest activities against the bacterial strains, though
failed to reach their FL counterparts. There are divergent
reports about the efficacy of FL and SDL in different plant
types.2,10
In our study, FL extracts were superior to SDL and that was
also evidenced by various studies.8,10 It was demonstrated
that several metabolites were either degraded or inactivated
during drying5,30 and in P. betle, the loss of essential oils
was reported in dried leaves.33 However, various other plant
species showed better antimicrobial activity and
phytochemical yield in SDL.2 In both FL and SDL extracts
of P. betle, the inhibitory effect was noticeable from the
initial concentration (3 mg) and displayed a significant
(p<0.05) upsurge in activity with increments in extract
concentration (Figure 3, Table 2).
The antibacterial efficacy of P. betle was documented by
several solvent extracts including methanol and the putative
phytochemicals in such activities were postulated by various
researchers.2,44 Our study also affirmed the effectiveness of
P. betle in inhibiting pathogenic bacteria of which few are
multidrug-resistant. The greater efficiency of the species can
be interrelated with its higher phytochemical contents,
particularly polyphenols, as phenolics are implicated in
various bioactivities.44
Figure 3: Disc diffusion assay showing antibacterial activities of different members of Piperaceae
against different bacterial strains.
PB: Piper betle, PN: Piper nigrum, PL: Piper longum, PR: Piper retrofractum, PC: Piper chaba, PP: Peperomia pellucida;
Each plate represents fresh leaf extracts (FL) at upper half and shade-dried leaf extracts (SDL) at the lower half; increments in
extract concentrations (3, 6 and 12 mg/ disc) are shown in the clockwise direction; c: vehicle control
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
66
Although various solvent extracts showed antibacterial
efficacy to different degrees, hydroalcoholic solvents were
proven to be a better choice3,34 and our study also
corroborated with that. The remarkable inhibitory effects of
P. nigrum (inhibition zones ranging from B.
subtilis:10.55±0.78 to S. aureus:9.47±0.38 in FL extract and
E. coli:9.90±3.37 to S. aureus:6.17±0.55 in SDL extract) are
worth mentioning, albeit failed to reach the level of P. betle.
The antibacterial efficacy of P. nigrum was evaluated by
many workers and found promising activity in its
leaves.24,42,47
The other three Piper species (P. longum, P. retrofractum,
P. chaba) and Peperomia pellucida demonstrated relatively
weaker inhibitory activities than P. betle and on many
occasions, the lower extract concentrations of these plants,
SDL, in particular, failed to produce any inhibition zones.
The lowest activities among the six plants were observed in
Peperomia pellucida in both FL and SDL. The antibacterial
activities of the above Piper spp.11,36,43 and Peperomia
pellucida25 were reported discretely following various
solvent extracts.
The antibacterial efficacies of the plant extracts against
different bacteria were determined by their MICs and MBCs
(Table 3). Results revealed that FL extracts were with lower
MIC and MBC estimates than SDL indicating greater
antibacterial potency of the FL extracts.
Among the six plants, P. betle demonstrated highest
activities against E. coli (MIC: 0.58±0.04; MBC: 0.65±0.07)
followed by B. subtilis (MIC: 0.59±0.04; MBC: 0.67±0.00),
S. epidermidis and S. aureus (MIC: 0.63±0.04; MBC:
0.70±0.00), V. cholerae (MIC: 0.73±0.04; MBC: 0.80±0.00)
and S. Typhi (MIC: 0.90±0.00; MBC: 0.95±0.07) using FL
extracts. The SDL extracts of the species were also effective
but to a lesser degree than FL. The other studied members of
Piperaceae documented much higher MICs and MBCs
reflecting their weak antibacterial efficacies, significantly
(p<0.05) differing from P. betle. Ampicillin, used as a
positive control, demonstrated the lowest MICs and MBCs
among all the treatments with significant (p<0.05)
differences from all the plant extracts. The lower MIC and
MBC values of P. betle corroborating its higher antibacterial
efficacy were also evidenced by many reports.3,44
Table 3
Minimum Inhibitory Concentration (MIC; mg/ml) and Minimum Bactericidal Concentration (MBC; mg/ml) of fresh
(FL) and shade dried (SDL) leaf extracts of different members of Piperaceae.
Plant
Extracts
Gram-positive
Gram-negative
B. subtilis
MTCC 121
S. aureus
ATCC 25923
S. epidermidis
MTCC 3086
E. coli MTCC
443
V. cholerae
N16961
S. Typhi C-6953
FL
SDL
FL
SDL
FL
SDL
FL
SDL
FL
SDL
FL
SDL
PB
0.59±
0.04
0.85±
0.07
0.63±
0.04
1.75±
0.35
0.63±
0.04
1.75±
0.35
0.58±
0.04
1.50±
0.71
0.73±
0.04
1.50±
0.00
0.90±
0.00
3.00±
0.00
0.67±
0.00
0.90±
0.07
0.70±
0.00
2.50±
0.71
0.65±
0.07
2.00±
0.00
0.65±
0.07
1.75±
0.35
0.80±
0.00
2.00±
0.00
0.95±
0.07
3.75±
0.35
PN
4.50±
0.71
7.25±
0.35
7.25±
0.35
9.50±
0.71
6.50±
0.35
9.25±
0.35
5.25±
0.35
7.25±
0.35
9.25±
0.35
12.25±
0.35
12.00±
1.41
12.50±
0.71
5.75±
0.35
7.50±
0.71
8.00±
0.00
10.75±
0.35
7.25±
0.35
9.50±
0.71
6.00±
0.00
8.00±
0.71
10.00±
0.00
12.50±
0.71
13.00±
1.41
14.75±
0.35
PL
6.50±
0.71
10.25±
0.35
7.50±
0.71
11.25±
0.35
7.25±
0.35
9.25±
0.35
7.25±
0.35
11.00±
0.00
10.25±
0.35
12.25±
0.35
12.50±
0.71
13.75±
0.35
7.25±
0.35
11.50±
0.71
8.00±
0.71
12.50±
0.35
8.25±
0.35
10.00±
0.00
8.50±
0.35
12.50±
0.71
11.00±
0.00
13.50±
0.71
14.75±
0.35
14.00±
0.71
PR
8.25±
0.35
11.25±
0.71
9.00±
0.71
ND
9.25±
0.35
ND
8.50±
0.71
11.00±
0.35
11.25±
0.35
ND
12.50±
0.71
ND
9.25±
0.35
12.50±
0.71
10.75±
0.35
ND
9.50±
0.71
ND
9.25±
0.71
12.25±
0.35
12.50±
0.71
ND
14.00±
0.71
ND
PC
7.25±
0.35
10.25±
0.35
9.25±
0.35
14.25±
0.53
9.25±
0.35
13.25±
0.35
8.00±
0.00
10.00±
0.00
10.25±
0.35
12.75±
0.35
13.25±
0.35
13.75±
0.35
8.25±
0.35
11.50±
0.71
9.75±
0.35
15.75±
0.35
9.50±
0.71
14.00±
0.00
8.75±
0.35
11.50±
0.71
11.50±
0.71
13.50±
0.71
14.75±
0.35
14.75±
0.35
PP
5.25±
0.35
8.25±
0.35
8.75±
0.35
ND
7.25±
0.35
ND
5.50±
0.71
8.00±
0.00
9.50±
0.71
13.00±
0.00
13.00±
0.71
ND
6.00±
0.00
9.00±
0.00
9.50±
0.71
ND
8.25±
0.35
ND
6.25±
0.35
8.75±
0.35
10.50±
0.71
14.50±
0.71
14.25±
0.35
ND
Amp
0.012±0.01
0.013±0.03
0.011±0.01
0.013±0.01
0.021±0.02
0.028±0.01
0.015±0.00
0.014±0.01
0.013±0.03
0.013±0.01
0.023±0.03
0.040±0.00
PB: Piper betle, PN: Piper nigrum, PL: Piper longum, PR: Piper retrofractum, PC: Piper chaba, PP: Peperomia pellucida; Amp:
Ampicillin; ND= Not Detected; for each plant extract the upper and lower row represent MIC and MBC respectively.
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
67
To summarize, the study made a comparison between
different members of Piperaceae to provide a broader
perspective of the antibacterial and antioxidant efficacies of
these members among which P. betle leaf methanolic extract
showed its supremacy over other studied plants.
Conclusion
Plant-derived antioxidants and antimicrobial agents are
occupying the centre stage day by day as they are safe and
reliable. The study presented a comparative assessment of
the polyphenol contents, antioxidative and antibacterial
efficacies of six plants belonging to the family Piperaceae
using leaf methanolic extracts. In addition, a comparison
between fresh and shade dried samples was done to get an
idea about their use. Findings from the study revealed
significant antioxidant and antibacterial activities in all the
plants at varying degrees and fresh leaves manifested better
activities than shade dried leaves.
Among these plants, Piper betle fresh leaf extract showed
the highest antioxidant and antibacterial activities that were
corroborated by their rich polyphenolic contents. It is
envisioned from the study that the potent antioxidant and
antibacterial properties of P. betle leaves can be exploited
for the isolation of phytochemicals to be used as dietary
antioxidants and in therapeutics as antimicrobial(s) of plant
origin.
Acknowledgement
The authors are thankful for the facilities provided by the
University of Kalyani and its DST-PURSE programme. BS
and AR were supported by fellowships from UGC. The
authors are grateful to Dr. Sudha Gupta, Department of
Botany, University of Kalyani and Prof. Gaurab
Gangopadhyay, Division of the Plant Biology, Bose
Institute, Kolkata for their valuable suggestions during the
manuscript preparation.
References
1. Abrahim N.N., Kanthimathi M.S. and Abdul-Aziz A., Piper
betle shows antioxidant activities, inhibits MCF-7 cell proliferation
and increases activities of catalase and superoxide dismutase, BMC
Complement Altern. Med., 12(1), 1-11 (2012)
2. Alabri T.H.A. et al, Comparative study of phytochemical
screening, antioxidant and antimicrobial capacities of fresh and dry
leaves crude plant extracts of Datura metel L., J. King Saud Univ.
Sci., 26(3), 237-43 (2014)
3. Annegowda H.V., Tan P.Y., Mordi M.N., Ramanathan S.,
Hamdan M.R., Sulaiman M.H. and Mansor S.M., TLC–
Bioautography-Guided Isolation, HPTLC and GC–MS-Assisted
Analysis of Bioactives of Piper betle Leaf Extract Obtained from
Various Extraction Techniques: in vitro Evaluation of Phenolic
Content, Antioxidant and Antimicrobial Activities, Food Anal.
Methods, 6(3), 715-26 (2013)
4. Bauer A.W., Kirby W.M.M., Sherris J.C. and Turck M.,
Antibiotic Susceptibility Testing by a Standardized Single Disk
Method, Am. J. Clin. Pathol., 36, 493-6 (1966)
5. Chan E.W.C., Lim Y.Y., Wong S.K., Lim K.K., Tan S.P., Lianto
F.S. and Yong M.Y., Effects of Different Drying Methods on The
Antioxidant Properties of Leaves and Tea of Ginger species, Food
Chem., 113(1), 166-72 (2009)
6. Dey P., Goyary D., Chattopadhyay P., Kishor S., Karmakar S.
and Verma A., Evaluation of Larvicidal Activity of Piper longum
Leaf Against the Dengue Vector, Aedes aegypti, malarial vector,
Anopheles stephensi and filariasis vector, Culex quinquefasciatus,
S. Afr. J. Bot., 132, 482-90 (2020)
7. Fair R.J. and Tor Y., Antibiotics and Bacterial Resistance in the
21st Century, Perspect. Med. Chem., 6, 25-64 (2014)
8. Francine U., Jeannette U. and Pierre R.J., Assessment of
antibacterial activity of neem plant (Azadirachta indica) on
Staphylococcus aureus and Escherichia coli, J. Med. Plants
Stud., 3(4), 85-91 (2015)
9. Gong Y., Li H.X., Guo R.H., Widowati W., Kim Y.H., Yang
S.Y. and Kim Y.R., Anti-allergic Inflammatory Components from
the Leaves of Piper crocatum Ruiz & Pav., Biol. Pharm. Bull.,
44(2), 245-50 (2021)
10. Habbal O., Hasson S.S., El-Hag A.H., Al-Mahrooqi Z., Al-
Hashmi N., Al-Bimani Z., Al-Balushi M.S. and Al-Jabri A.A.,
Antibacterial activity of Lawsonia inermis Linn (Henna) against
Pseudomonas aeruginosa, Asian Pac. J. Trop. Biomed., 1(3), 173-
6 (2011)
11. Jamelarin E.M. and Balinado L.O., Evaluation of Antibacterial
Activity of Crude Aqueous, Ethanolic and Methanolic Leaf
Extracts of Piper retrofractum Vahl. and Piper betle L., Asian J.
Biol. Life Sci., 8(2), 63-7 (2019)
12. Kabera J.N., Semana E., Mussa A.R. and He X., Plant
Secondary Metabolites: Biosynthesis, Classification, Function and
Pharmacological Properties, J. Pharm. Pharmacol., 2(7), 377-92
(2014)
13. Klančnik A., Piskernik S., Jeršek B. and Možina S.S.,
Evaluation of Diffusion and Dilution Methods to Determine the
Antibacterial Activity of Plant Extracts, J. Microbiol. Methods,
81(2), 121-6 (2010)
14. Kora A.J. and Arunachalam J., Assessment of antibacterial
activity of silver nanoparticles on Pseudomonas aeruginosa and its
mechanism of action, World J. Microbiol. Biotechnol., 27(5),
1209-16 (2011)
15. Lago J.H.G., Brochini C.B. and Roque N.F., Terpenoids from
Guarea guidonia, Phytochemistry, 60(4), 333-8 (2002)
16. Lautie E., Russo O., Ducrot P. and Boutin J.A., Unravelling
Plant Natural Chemical Diversity for Drug Discovery
Purposes, Front. Pharmacol., 11(397), 1-37 (2020)
17. LE Mballa D. et al, Cafeteria Diet-Induced Metabolic and
Cardiovascular Changes in Rats: The Role of Piper nigrum Leaf
Extract, Evid. Based Complement Altern. Med., 2021, 1-14 (2021)
18. Li Y., Kong D., Fu Y., Sussman M.R. and Wu H., The Effect
of Developmental and Environmental Factors on Secondary
Metabolites in Medicinal Plants, Plant Physiol. Biochem., 148, 80-
9 (2020)
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
68
19. Mucha P. et al, Overview of the Antioxidant and Anti-
Inflammatory Activities of Selected Plant Compounds and Their
Metal Ions Complexes, Molecules, 26(16), 1-52 (2021)
20. Nakagami T., Nanaumi-Tamura N., Toyomura K., Nakamura
T. and Shigehisa T., Dietary Flavonoids as Potential Natural
Biological Response Modifiers Affecting the Autoimmune
System, J. Food Sci., 60, 653-6 (1995)
21. Nemzer B., Al-Taher F. and Abshiru N., Phytochemical
Composition and Nutritional Value of Different Plant Parts in Two
Cultivated and Wild Purslane (Portulaca oleracea L.) Genotypes,
Food Chem., 320, 1-9 (2020)
22. Ng Z.X., Than M.J.Y. and Yong P.H., Peperomia pellucida
(L.) Kunth., Herbal Tea: Effect of Fermentation and Drying
Methods on The Consumer Acceptance, Antioxidant and Anti-
inflammatory Activities, Food Chem., 344, 1-9 (2021)
23. Ojha S., Raj A., Roy A. and Roy S., Extraction of Total
Phenolics, Flavonoids and Tannins from Paederia foetida L.
Leaves and their Relation with Antioxidant Activity, Pharmacogn.
J., 10(3), 541-7 (2018)
24. Ojha S., Raj A., Roy A., Sikdar B. and Roy S., Efficacy of
Aqueous and Ethanolic Extraction on Phenolics and Antioxidant
Activity of Paederia foetida L. and Spermacoce stricta L.F., J.
Plant Dev. Sci., 8(3), 131-5 (2016)
25. Oloyede G.K., Onocha P.A. and Olaniran B.B., Phytochemical,
Toxicity, Antimicrobial and Antioxidant Screening of Leaf
Extracts of Peperomia pellucida from Nigeria, Adv. Environ. Biol.,
5(12), 3700-9 (2011)
26. Osman H., Rahim A.A., Isa N.M. and Bakhir N.M.,
Antioxidant Activity and Phenolic Content of Paederia foetida and
Syzygium aqueum, Molecules, 14(3), 970-8 (2009)
27. Parveen S., Shahzad A., Upadhyay A. and Yadav V., Gas
Chromatography-Mass Spectrometry Analysis of Methanolic Leaf
Extract of Cassia angustifolia Vahl., Asian J. Pharm. Clin. Res.,
9(3), 111-6 (2016)
28. Pisoschi A.M., Pop A., Cimpeanu C. and Predoi G.,
Antioxidant Capacity Determination in Plants and Plant-derived
Products: A Review, Oxid. Med. Cell Longev., 2016, 1-36 (2016)
29. Prain D., Bengal Plants, Botanical Survey of India, Calcutta,
India (1903)
30. Qneibi M., Jaradat N., Zaid A.N., Abu-Khalaf N., Natsheh A.R.
and Hussen F.M., Evaluation of Taste, Total Phenols and
Antioxidant for Fresh, Roasted, Shade Dried and Boiled Leaves of
Edible Arum palaestinum Bioss., Marmara Pharm. J., 22(1), 52-8
(2018)
31. Raghavendra H.L. and TR P.K., Ethnobotanical Uses,
Phytochemistry and Pharmacological Activities of Peperomia
pellucida (L.) Kunth (Piperaceae)-A Review, Int. J. Pharm.
Pharm. Sci., 10(2), 1-8 (2018)
32. Raj A., Sikdar B., Roy A., Mukhopadhyay A. and Roy S.,
Antioxidant and Antibacterial Activities of Phytochemicals in
Methanolic Extracts of Five Underutilized Leafy Vegetables, Res.
J. Biotechnol., 16(8), 1-10 (2021)
33. Ramalakshmi K., Sulochanamma G., Rao J.M.L., Borse B.B.
and Raghavan B., Impact of Drying on Quality of Betel Leaf (Piper
betle L.), J. Food Sci. Technol., 39(6), 619-22 (2002)
34. Rameshkumar K.B., Aravind A.A. and Mathew P.J.,
Comparative Phytochemical Evaluation and Antioxidant Assay of
Piper longum L. and Piper chaba Hunter. Used in Indian
Traditional Systems of Medicine, J. Herbs Spices Med.
Plants, 17(4), 351-60 (2011)
35. Roshanak S., Rahimmalek M. and Goli S.A.H., Evaluation of
Seven Different Drying Treatments in Respect to Total Flavonoid,
Phenolic, Vitamin C Content, Chlorophyll, Antioxidant Activity
and Color of Green Tea (Camellia sinensis or C. assamica)
Leaves, J. Food Sci. Technol., 53(1), 721-9 (2016)
36. Roy A.C., Islam M.S., Haque M.M., Hossain M.S. and Sayeed
M.A., Microbiological investigation of Piper chaba Hunter., Int. J.
Sci. Eng. Res., 6(4), 879-84 (2015)
37. Saeed N., Khan M.R. and Shabbir M., Antioxidant activity,
total phenolic and total flavonoid contents of whole plant extracts
Torilis leptophylla L., BMC Complement Altern. Med., 12(1), 1-12
(2012)
38. Salehi B., Zakaria Z.A., Gyawali R., Ibrahim S.A., Rajkovic J.,
Shinwari Z.K., KhanT., Sharifi-Rad J., Ozleyen A., Turkdonmez
E. and Valussi M., Piper Species: A Comprehensive Review on
Their Phytochemistry, Biological Activities and Applications,
Molecules, 24(7), 1-118 (2019)
39. Sannigrahi S., Mazumder U.K., Pal D. and Mishra S.L.,
Terpenoids of Methanol Extract of Clerodendrum infortunatum
Exhibit Anticancer Activity Against Ehrlich’s Ascites Carcinoma
(EAC) in Mice, Pharm. Biol., 50(3), 304-9 (2012)
40. Shanmugapriya K.S.P.S., Saravana P.S., Payal H., Mohammed
S. and Williams B., Antioxidant Potential of Pepper (Piper nigrum
Linn.) Leaves and its Antimicrobial Potential Against Some
Pathogenic Microbes, Indian J. Nat. Prod. Resour., 3(4), 570-7
(2012)
41. Snyder H.L. and Kirkland J.J., Introduction to Modern Liquid
Chromatography, 2nd Edition, John Wiley & Sons, Inc., Canada
(1979)
42. Souza J.N., Silva E.M., Loir A., Rees J.F., Rogez H. and
Larondelle Y., Antioxidant Capacity of Four Polyphenol-rich
Amazonian Plant Extracts: A Correlation Study Using Chemical
and Biological in vitro Assays, Food Chem., 106(1), 331-9 (2008)
43. Sultana N.A., Hasan Zilani M.N., Taraq K.T.M. and Al-Din
M.K., Phytochemical, Antibacterial and Antioxidant Activity of
Piper longum Leaves, Pharmacology Online, 1, 27-35 (2019)
44. Syahidah A., Saad C.R., Hassan M.D., Rukayadi Y., Norazian
M.H. and Kamarudin M.S., Phytochemical Analysis, Identification
and Quantification of Antibacterial Active Compounds in Betel
Leaves, Piper betle Methanolic Extract, Pak. J. Biol. Sci., 20(2),
70-81 (2017)
45. Takahashi M., Oe M., Arakaki M. and Wada K., Effect of Leaf
Growth on The Taste and Aroma Functions and Antioxidant
Characteristics of Hihatsumodoki (Piper retrofractum Vhal)
Leaf, J. Food Meas. Charact., 14(2), 1002-11 (2020)
Research Journal of Biotechnology Vol. 17 (8) August (2022)
Res. J. Biotech
69
46. Truong D.H., Nguyen D.H., Ta N.T.A., Bui A.V., Do T.H. and
Nguyen H.C., Evaluation of The Use of Different Solvents for
Phytochemical Constituents, Antioxidants and in vitro Anti-
inflammatory Activities of Severinia buxifolia, J. Food
Qual., 2019, 1-10 (2019)
47. Zhang T.J., Zheng J., Yu Z.C., Huang X.D., Zhang Q.L., Tian
X.S. and Peng C.L., Functional Characteristics of Phenolic
compounds Accumulated in young Leaves of two Subtropical
Forest Tree species of different Successional Stages, Tree Physiol.,
38(10), 1486-501 (2018).
(Received 14th March 2022, accepted 17th May 2022)
