molecules

Article
Inter-Varietal Variation in Phenolic Profile, Antioxidant,
Anti-Inflammatory and Analgesic Activities of Two
Brassica rapa Varieties: Influence on
Pro-Inflammatory Mediators
Nida Nazar 1 , Abdullah Ijaz Hussain 1,2, * and Hassaan Anwer Rathore 3, *

1 Department of Chemistry, Government College University Faisalabad, Faisalabad 38000, Pakistan;

nidanazar786@gmail.com
2 Hi-Tech Lab, Government College University Faisalabad, Faisalabad 38000, Pakistan
3 Department of Pharmaceutical Sciences, College of Pharmacy, QU Health, Qatar University, Doha 2713, Qatar
* Correspondence: abdullahijaz@gcuf.edu.pk (A.I.H.); hrathore@qu.edu.qa (H.A.R.);
Tel.: +92-3007631058 (A.I.H.); +974-50147988 (H.A.R.)

Abstract: The present research study aims to appraise the potential of polyphenol-rich extracts
from two Brassica rapa varieties on antioxidant, anti-inflammatory and analgesic activities using
carrageenan-induced paw edema model in rats. Methanol extracts of peels and pulps of Brassica rapa
yellow root (BRYR) and Brassica rapa white root (BRWR) were prepared using the soxhlet extraction
technique. All four extracts were analyzed by reversed-phase high-pressure liquid chromatography
(RP-HPLC) for the polyphenols, and results showed that 10 phenolic acids and 4 flavonoids were
detected. Gallic acid was the major phenolic acid (174.6–642.3 mg/100 g of dry plant material) while
catechin was the major (34.45–358.5 mg/100 g of dry plant material) flavonoid detected in the extracts.
The total phenolic contents (TPC) of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp extracts
were in the range of 1.21–5.01 mg/g of dry plant material, measured as GAE, whereas the total
flavonoid contents (TFC) were found in the range of 0.90–3.95 mg/g of dry plant material, measured
Citation: Nazar, N.; Hussain, A.I.;
as QE. BRYR peel extract exhibited the best DPPH radical scavenging activity (IC50 , 3.85 µg/mL)
Rathore, H.A. Inter-Varietal Variation
and reducing potential as compared with other extracts. The in vivo anti-inflammatory potential
in Phenolic Profile, Antioxidant,
Anti-Inflammatory and Analgesic
was assessed by carrageenan-induced rat paw edema, and the analgesic potential was investigated
Activities of Two Brassica rapa by a hot plate test. Suppression of biochemical inflammatory biomarkers including C-reactive
Varieties: Influence on protein (CRP), rheumatoid factor (RF) and tumor necrosis factor (TNF-α), and interleukin-6 (IL-6)
Pro-Inflammatory Mediators. concentration were also determined. Results showed that BRYR peel extracts reduced paw edema
Molecules 2023, 29, 117. https:// and suppressed the production of TNF-α, IL-6, CRP and RF most significantly, followed by BRWR
doi.org/10.3390/molecules29010117 peel, BRYR pulp and BRWR pulp extracts. In addition, histopathology observation also supports the
Academic Editors: Francesco Epifano anti-inflammatory effect of peel extracts as being greater than that of root pulp extracts. Moreover, it
and Zhi Na was observed that the analgesic effect of the root-peel extracts was also more pronounced as compared
with root-pulp extracts. It can be concluded that BRYR peel extract has higher phenolic contents and
Received: 16 September 2023
showed higher suppression of TNF-α, IL-6, CRP and RF, with strong antioxidant, anti-inflammatory
Revised: 22 October 2023
and analgesic effects.
Accepted: 24 October 2023
Published: 24 December 2023
Keywords: polyphenols; carrageenan; C-reactive protein; TNF-α; IL-6; rheumatoid factor

Copyright: © 2023 by the authors.

Licensee MDPI, Basel, Switzerland. 1. Introduction
This article is an open access article
Inflammation is the main protective response of the primary immune system against
distributed under the terms and
damage and is characterized by both acute and chronic inflammations [1,2]. Acute in-
conditions of the Creative Commons
flammation results in the formation of edema, leukocyte infiltration, and macrophage
Attribution (CC BY) license (https://
infiltration in the damaged muscle [3]. Whereas, in chronic inflammation, prolonged in-
creativecommons.org/licenses/by/
filtration of these immune system components results in more serious conditions such as
4.0/).

Molecules 2023, 29, 117. https://doi.org/10.3390/molecules29010117 https://www.mdpi.com/journal/molecules

Molecules 2023, 29, 117 2 of 18

arthritis, cancer, asthma, atherosclerosis, and autoimmune ailments [4]. The majority of
health issues only appear when the inflammation persists for a long time and is irreversible.
Inflammation triggers cytokine production, including immunosuppressive interleukin (IL-)
4 and IL-10, or the pro-carcinogenic IL-6 and TNF-α [5]. Furthermore, C-reactive protein
(CRP) is a highly inflammatory protein that increases 1000 times at the site of injury or
inflammation [6]. Therefore, CRP shows a vital impact on inflammatory processes and
stimulates macrophages to secrete pro-inflammatory cytokines including tumor necrosis
factor-α (TNF-α) and interleukin (IL) [7]. The eruption of pro-inflammatory cytokines
(TNF-α and IL-6) from active inflammatory pathways tangled in pathological pain pro-
cesses is essential for autoimmune disorders. Imbalance of pro-inflammatory cytokines
and anti-inflammatory cytokines causes inflammation [5–7].
Non-steroidal anti-inflammatory drugs (NSAIDs) belong to an important class of pain-
relieving drugs and suppress pro-inflammatory cytokines (CRP, TNF-α, and IL-6) with anti-
inflammatory and analgesic effects [8]. However, existing NSAIDs and cyclooxygenase-2
(COX-2) inhibitors have also been linked with several adverse effects such as cardiovascular
and abdominal complications [9]. Certain dietary polyphenols such as quercetin and catechin
are natural antioxidants and exhibit the effect of balancing the generation of pro- and anti-
inflammatory cytokines, and increase IL-10 secretion while inhibiting TNF-α [10,11]. Thus,
anti-inflammatory drugs derived from natural sources and used in traditional medicine
have attracted the attention of health professionals and the pharmaceutical industry due
to their effectiveness and fewer side effects [1,2,4]. Polyphenols are a class of natural
compounds that are under consideration due to many reasons [8,12,13].
Brassica rapa (B. rapa), commonly known as turnip, is an edible root consumed as a
vegetable, which belongs to the Brassicaceae family [14–16]. B. rapa is a rich source of
bioactive compounds including isothiocyanates, glucosinolates, anthocyanins, carotenoids,
flavonoids, sulforaphane, organic acids and phenolic compounds [17,18]. The whole plant
has been used not only for nourishment, but also as a traditional remedy against several
diseases, including inflammation [2]. B. rapa ethanol extract has been reported to display an
anti-arthritis effect against complete Freund’s adjuvant-induced inflammation and exhibits
incredible anti-inflammatory effect and analgesic potential [2,19].
Inter-varietal comparative study of polyphenols-rich extracts of root-peel and root-
pulp of yellow and white varieties of Brassica rapa with antioxidant, anti-inflammatory and
analgesic effects on suppression of TNF-α, IL-6, CRP and RF are not reported yet. Therefore,
the current study is designed to investigate the suppression of TNF-α, IL-6, CRP and RF
levels in rats with antioxidant, anti-inflammatory and analgesic effects of polyphenol-rich
extracts of root-peel and root-pulp of yellow and white varieties of B. rapa. Moreover, the
extracts were analyzed for phenolic profile using reverse-phase high performance liquid
chromatography (RP-HPLC).

2. Results and Discussion

2.1. Extract Yield
Table 1 shows the extract yields of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp.
The extracts’ yields ranged from 6.72 to 13.35 g/100 g of dry plant material. Peel extracts of
B. rapa reported a lower yield compared with root pulp extracts. Overall, the yield of BRYR
pulp extract was higher (13.35 g/100 g) than that of the other extracts. This study reported
a significant difference (p ≤ 0.05) in B. rapa peel and root pulp extract yields, which may be
due to differences in the composition of extractable constituents in different plant materials.
Sultana et al. [20] reported a higher extract yield of yellow and white Brassica rapa than our
findings, and the variation may be due to the variation in the extraction process. Extract
yield depends on various factors, including the solvent polarity, method of extraction and
time of the extraction procedure [12,20].
Molecules 2023, 29, 117 3 of 18

Table 1. Extract yield and antioxidant activity of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
methanol extracts.

Extracts Yield (g/100 g) TPC (mg GAE/g) TFC (mg QE/g) DPPH, IC50 (µg/mL)
BRYR peel 8.25 ± 0.41 b 5.01 ± 0.25 d 3.95 ± 0.20 d 3.85 ± 0.19 b
BRWR peel 6.72 ± 0.34 a 3.21 ± 0.16 c 2.28 ± 0.11 c 4.64 ± 0.23 c
BRYR pulp 13.35 ± 0.67 d 2.16 ± 0.11 b 1.30 ± 0.07 b 5.08 ± 0.25 c
BRWR pulp 10.63 ± 0.53 c 1.21 ± 0.06 a 0.90 ± 0.08 a 5.76 ± 0.29 d
BHA -------- -------- -------- 1.24 ± 0.06 a
Values are mean ± standard deviation of triplicate experiments. Superscripted different letters indicate the
significant difference (p ≤ 0.05) between different extracts. BRYR: B. rapa yellow root; BRWR: B. rapa white root;
BHA: butylated hydroxyanisole; TPC: mg gallic acid equivalent (GAE)/g of dry material; TFC: mg quercetin
equivalent (QE)/g of dry material.

2.2. Quantification of Phenolic Acids and Flavonoid by RP-HPLC

The phenolic acid and flavonoid compositions of BRYR peel, BRWR peel, BRYR
pulp, and BRWR pulp extracts were determined by RP-HPLC using a multi-wavelength
detector (MWD). Chromatograms showing the separation of phenolic acids and flavonoids
from the BRYR peel extract at 250, 270, 290, 350 and 380 nm are presented in Figure 1.
The peak numbers mentioned are the compounds detected in the BRYR peel extract.
Chromatograms of other extracts are presented in Supplementary Data. Qualitative and
quantitative data from all the extracts are represented in Table 2. Ten phenolic acids were
detected: gallic acid, chlorogenic acid, p-hydroxyl benzoic acid, syringic acid, vanillic acid,
p-coumaric acid, sinapic acid, ferulic acid, cinnamic acid, and benzoic acid. Gallic acid
(174.6–642.3 mg/100 g) and chlorogenic acid (84.3–267.1 mg/100 g) were the major phenolic
acids present in abundance in all extracts. The BRYR peel extract showed the highest
concentration of gallic acid, syringic acid, cinnamic acid and ferulic acid. Chlorogenic acid
was in highest concentration in the pulp extracts of both BRWR and BRYR. Four flavonoids—
catechin, kaempferol, quercetin and rutin—are being reported as the major polyphenols
in B. rapa extracts. Catechin was reported to have the highest concentration in BRYR
extract (358.5 mg/100 g) and the lowest (34.45 mg/100 g) in BRYR extract. Quercetin and
kaempferol were reported in all the B. rapa extracts. The highest quercetin and kaempferol
contents were reported in the BRWR peel extract, i.e., 11.38 and 34.21 mg/100 g, respectively.
Rutin (4.63 mg/100 g) was only detected in BRYR pulp extract. The different B. rapa
extracts retained altered phenolic acid and flavonoid concentrations, and a significant
(p ≤ 0.05) difference was observed. Most polyphenol compounds have the ability to act as
antioxidants and are polar in nature, and methanol and other polar solvents are the efficient
solvents for the extraction of these phenolic acids and flavonoid compounds [21]. Therefore,
the high concentrations of gallic acid, chlorogenic acid and p-coumeric acid in the analyzed
extracts might be due to the polarity of the organic extraction solvent [12,13,20]. RP-HPLC
investigation of BRYR peel, BRWR peel, BRYR pulp, and BRWR pulp extracts agrees
well with findings stated by previous researchers [22–28]. The glycosylated derivatives
of quercetin, kaempferol, and isorhamnetin have been reported in turnip top [22,24]. In
another study, ferulic acid, sinapic acid, 3-p-coumaroylquinic and derivatives of kaempferol
and isorhamnetin were identified in aqueous extracts of different parts of turnip [23].
Quercetin, kaempferol, hydroxybenzoic acid and hydroxycinnamic acid derivatives have
also been reported in microgreens of Brassica rapa [27].
Molecules
Molecules2023, 29,28,117
2023, x FOR PEER REVIEW 4 of4 of
19 18

Figure1.1.Chromatograms
Figure Chromatograms showing the the separation
separationof
ofphenolic
phenolicacids
acidsand
andflavonoids
flavonoidsinin
B.B.
rapa yellow
rapa yellow
peelextract
peel extract at different
at different wavelengths.
wavelengths. PeakPeak numbers
numbers represents
represents the specific
the specific compoundscompounds as
as mentioned
mentioned
in Table 2. in Table 2.
Molecules 2023, 29, 117 5 of 18

Table 2. Composition of phenolic acids and flavonoids from methanol extracts of BRYR peel, BRWR
peel, BRYR pulp and BRWR pulp by RP-HPLC.

Concentration (mg/100 g of Dry Plant Material)

Peak No. Compounds
BRYR Peel BRWR Peel BRYR Pulp BRWR Pulp
1 Gallic acid 642.3 ± 32.1 a 174.6 ± 8.7 d 228.1 ± 11.4 c 330.5 ± 16.5 b
2 p-hydroxyl benzoic acid 43.25 ± 2.16 bc 26.20 ± 1.31 c 46.51 ± 2.33 b 65.33 ± 3.27 a
3 Chlorogenic acid 84.3 ± 4.2 d 196.3 ± 9.8 c 237.2 ± 11.9 b 267.1 ± 13.3 a
4 Vanillic acid - - 9.31 ± 0.47 -
5 Syringic acid 104.0 ± 5.2 - - -
6 p-coumaric acid 186.3 ± 9.3 b 108.4 ± 5.4 c - 207.3 ± 10.3 a
7 Sinapic acid 1.43 ± 0.07 c 0.41 ± 0.02 c 19.03 ± 0.95 a 3.43 ± 0.17 b
8 Ferulic acid 18.60 ± 0.93 a 3.13 ± 0.16 b 0.99 ± 0.05 bc 0.39 ± 0.02 c
9 Rutin - - 4.63 ± 0.23 -
10 Cinnamic acid 78.77 ± 3.94 a 21.24 ± 1.06 b 8.15 ± 0.41 b 3.23 ± 0.16 b
11 Benzoic acid 1.10 ± 0.06 b 3.05 ± 0.15 a - -
12 Catechin 358.5 ± 17.9 a 138.0 ± 6.9 c 34.45 ± 1.72 d 196.8 ± 9.83 b
13 Quercetin 7.24 ± 0.36 b 11.38 ± 0.57 a 5.74 ± 0.29 b 7.24 ± 0.36 b
14 Kaempferol 16.39 ± 0.82 c 34.04 ± 1.72 a 31.5 ± 1.57 b -
Superscripted different letters indicate the significant difference (p ≤ 0.05) between different extracts. BRYR:
B. rapa yellow root; BRWR: B. rapa white root; BHA: (Butylated hydroxyanisole).

2.3. TPC, TFC and Antioxidant Activity

Total phenolic contents (TPC), total flavonoid contents (TFC), and DPPH radical
scavenging activity of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp extracts were
determined and are presented in Table 1. Total phenolic contents were measured in the
range of 1.21–5.01 mg/g of dry material as gallic acid equivalent (GAE) in all the B. rapa
extracts. Generally, peel extracts showed the highest concentration of TPC as compared
to pulp extract. Similarly, BRYR peel and BRWR peel extracts showed TPC 3.95 and
2.28 mg/g of dry material, measured as quercetin equivalents (QE). Overall, significantly
(p ≤ 0.05) higher TPC (5.01 mg/g, GAE) and TFC (3.95 mg/g, QE) were observed in the
BRYR peel extract, but these were lowest in the BRWR pulp extract (Table 1). The function
of phenolic acids and flavonoid compounds include defense against free radicals, ROS,
allergies, microorganisms, viruses, ulcers, tumors and inflammation [9]. The current results
show that Brassica rapa extracts have higher TPC and TFC values as compared with those
stated by Sultana et al. [20]. However, in another finding, aqueous extract of Brassica rapa
root extract was reported to have TPC (9.41, GAE/mg) and TFC (1.01 µg QE/mg) values
which were in partial agreement with our findings [29]. Yucetepe [30] also investigated
the TPC (169.29 mg/g, GAE dry weight) in the purple peel of an extract of Brassica rapa,
extracted with 80% methanol using ultrasonic extraction. These changes in TPC and
TFC values can be due to different geographical, agricultural and climatic conditions of
the regions.
A stable free radical, 2-2′ -diphenyl-1-picrylhydrazyl (DPPH), was utilized to assess
free radical scavenging ability of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
extracts. Different concentrations of the extracts of B. rapa showed different radical scav-
enging activity, and the results are reported in terms of IC50 (µg/mL) in Table 1. IC50
values of the BRYR peel, BRWR peel, and BRYR pulp and BRWR pulp ranged between
3.85 and 5.76 µg/mL, as presented in Table 2. Antioxidant compounds found in plants,
such as phenols and flavonoids, have been shown to protect against ROS and free radical
damage [31]. A lower IC50 value of extract indicates a greater antioxidant activity of DPPH
assay, and vice versa [21]. The BRYR peel extract exhibited the highest antioxidant poten-
tial (IC50 , 3.85 µg/mL), while BRWR pulp showed the lowest antioxidant activity (IC50 ,
5.76 µg/mL). The synthetic antioxidant BHT showed the best DPPH radical scavenging
activity (IC50 = 1.24 µg/mL). Therefore, the current study demonstrated that peel and pulp
extracts of both yellow and white varieties extracts displayed potent antioxidant activity.
 activity (IC50 = 1.24 µg/mL). Therefore, the current study demonstrated that peel and pulp
Molecules 2023, 29, 117 extracts of both yellow and white varieties extracts displayed potent antioxidant activity.
6 of 18
The results of the present study on DPPH and reducing power tests were comparable to
those of Saeed et al. [15]. Fernandes et al. [23] revealed that aqueous root extract of Brassica
rapa was shown to have poor antioxidant capacity as compared to leaves, stems and
The results of the present study on DPPH and reducing power tests were comparable
flowers. However, these results are inconsistent with other studies showing that aqueous
to those of Saeed et al. [15]. Fernandes et al. [23] revealed that aqueous root extract of
root extract of B. rapa has a be er DPPH screening activity than the turnip green aqueous
Brassica rapa was shown to have poor antioxidant capacity as compared to leaves, stems and
extract
flowers.[32]. According
However, theseto results
Berdja are
et al. [29], aqueous
inconsistent withroot
other extract of showing
studies turnip was thatshown to
aqueous
have significantly lower DPPH antioxidant activity (2100 µg/mL) as compared
root extract of B. rapa has a better DPPH screening activity than the turnip green aqueous to synthetic
antioxidants. The difference
extract [32]. According in DPPH
to Berdja et al.values compared
[29], aqueous rootto previous
extract ofstudy
turnipresults may be
was shown to
due to adopting altered extraction methods and differences in seasonal,
have significantly lower DPPH antioxidant activity (2100 µg/mL) as compared to synthetic agro-climatic and
geographical
antioxidants.conditions.
The difference in DPPH values compared to previous study results may be
due The reducing
to adopting potential
altered of BRYR
extraction methodspeel, and
BRWR peel, BRYR
differences pulp and
in seasonal, BRWR pulp
agro-climatic and
extracts were examined,
geographical conditions. and the results are presented in Figure 2. The different extracts
of B. rapa
The(concentration
reducing potential up toof10.0
BRYR mg/mL) exhibited
peel, BRWR a concentration-dependent
peel, BRYR pulp and BRWR pulp reducing
extracts
power. Moreover,and
were examined, it is
theobserved that
results are differentinmethanol
presented Figure 2. extracts at 10 extracts
The different mg/mL of showed
B. rapa
some variation in
(concentration upreducing power that
to 10.0 mg/mL) showed
exhibited significant difference (p ≤ reducing
a concentration-dependent 0.05). A higher
power.
reducing power indicates the greater antioxidant potential of reducing
Moreover, it is observed that different methanol extracts at 10 mg/mL showed some power assay [21].
Among all the extracts, BRYR peel extract was be er in terms of reducing
variation in reducing power that showed significant difference (p ≤ 0.05). A higher reducing power followed
by BRWR
power peel, BRYR
indicates pulp and
the greater BRWR pulp
antioxidant extracts.
potential The reducing
of reducing power power results
assay [21]. Amongof this
all
study were comparable
the extracts, BRYR peel with those
extract wasof previous
better studies
in terms investigated
of reducing power byfollowed
other researchers
by BRWR
[15,20,33].
peel, BRYR Phytochemical
pulp and BRWR characterization
pulp extracts.ofThe peelreducing
extracts,power
especially yellow
results peel
of this extracts,
study were
revealed
comparable the with
occurrence
those ofofprevious
more phenolic and flavonoid
studies investigated by compounds.
other researchersFor reducing
[15,20,33].
efficiency,
Phytochemicalthe higher reduction power
characterization of peel is extracts,
due to the high phenolic
especially yellowcontents [15]. revealed
peel extracts,
the occurrence of more phenolic and flavonoid compounds. For reducing efficiency, the
higher reduction power is due to the high phenolic contents [15].

2 BHA BRWR peel BRYR pulp BRYR peel BRWR pulp

1.8 a

1.6
Absorbance (700 nm)

1.4
1.2
1 b

0.8 c

0.6 d

0.4
0.2
0
0 2 4 6 8 10
Concentration (mg/mL)
Figure 2. The reducing power assay of B. rapa extracts. Values are mean ± standard deviation of
Figure 2. The reducing power assay of B. rapa extracts. Values are mean ± standard deviation of
triplicate experiments. Superscripted different letters indicate the significant difference (p ≤ 0.05)
triplicate experiments. Superscripted different le ers indicate the significant difference (p ≤ 0.05)
between different extracts. BRYR peel: B. rapa yellow root peel; BRWR peel: B. rapa white root peel;
between different extracts. BRYR peel: B. rapa yellow root peel; BRWR peel: B. rapa white root peel;
BRYR pulp:B.B.rapa
BRYRpulp: rapayellow
yellowroot
rootpulp;
pulp;BRWR pulp:B.B.rapa
BRWRpulp: rapawhite
whiteroot
rootpulp.
pulp.

2.4. Acute Inflammatory Model

2.4. Acute Inflammatory Model
The effect of polyphenol-rich BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
extracts and indomethacin on carrageenan-induced paw edema is presented in Table 3.
B. rapa extracts significantly reduced carrageenan-induced inflammation by decreasing the
paw diameter of rats (Figure 3). In the present study, maximum inhibition of paw diameter
Molecules 2023, 29, 117 7 of 18

was observed at 3 h after carrageenan injection (Figure 4). During the third observation
hour (h), BRYR-peel- and BRWR-peel-extract-treated rats were reported to have higher
inhibition (44.32–71.83%) than those treated with BRYR pulp and BRWR pulp extracts
(43.65–55.63%), when compared with normal control (NC). At that time, indomethacin was
reported to have a 63.26% inhibition value. In addition, BRYR peel extract was reported
to have the most significant anti-inflammatory effect after 1 h of carrageenan injection
among all other extracts, which was sustained throughout the experiment. Moreover, it
Molecules 2023, 28, x FOR PEER REVIEW 7 of of
was also found that the anti-inflammatory potential of BRYR peel was superior to that 19
indomethacin during 3–4 h of observation.

3. Effect
TableThe effectof polyphenol-rich B. rapaBRYR
of polyphenol-rich extracts andBRWR
peel, indomethacin on % inhibition
peel, BRYR pulp and of BRWR
inflammation
pulp
in carrageenan-induced paw edema in rats.
extracts and indomethacin on carrageenan-induced paw edema is presented in Table 3. B.
rapa extracts significantly reduced carrageenan-induced inflammation by decreasing the
% Inhibition of Inflammation
Groups paw diameter of rats (Figure 3). In the present study, maximum inhibition of paw
1 diameter
h 2h
was observed 3h
at 3 h after carrageenan injection 4 h(Figure 4). During 5 hthe third
# # ± 3.42 # to
BRYR-Peel 57.82 ±observation
2.89 hour ± 2.99
59.83(h), BRYR-peel- 71.83and ± 70.86 ± 3.54 rats were
BRWR-peel-extract-treated
5.59 68.49reported
BRWR-Peel 41.69 ±have # 47.84 ± 2.39
2.08 higher inhibition # ± 2.22
44.32than # 54.17 ± 1.71 # #
± 1.42 pulp
48.37BRWR
(44.32–71.83%) those treated with BRYR pulp and
#
35.47 ±extracts #
38.83 ± 1.94 when compared #
55.63 ± 2.78 with normal 48.41 ±control # #
BRYR-Pulp 1.77 (43.65–55.63%), 2.42 At ±
(NC).37.66 1.88 time,
that
BRWR-Pulp 30.57 ± 1.53 # 35.60 ± 1.78 # 43.65 ± 2.18 # 37.93 ± 1.90 # 30.04 ± 1.50 #
indomethacin was reported to have a 63.26% inhibition value. In addition, BRYR peel
PC 73.93 ± 3.70 68.30 ± 3.42 63.26 ± 3.16 66.37 ± 3.32 80.57 ± 4.03
extract was reported to have the most significant anti-inflammatory effect after 1 h of
Results inhibition percentage ± standard deviation (n = 6) when compared with the positive control group
carrageenan injection among all other extracts, which was sustained throughout the
(Indomethacin). BRYR-Peel: B. rapa yellow root peel, BRWR-Peel: B. rapa white root peel, BRYR-Pulp: B. rapa
experiment.
yellow root pulp, BRWR-Pulp:itB.was
Moreover, rapa also
white found that
root pulp, PC:the anti-inflammatory
Positive potential
control. # symbolizes significantof BRYR
difference
(p ≤ 0.05)
peel wasassuperior
comparedto to PC.
that of indomethacin during 3–4 h of observation.

Figure
Figure 3.
3. Representative
Representative images
images of
of rat’s
rat’s paws
paws after
after carrageenan
carrageenan injection.
injection. (a)
(a) Normal
Normal control
control group;
group;
(b) BRYR-Peel group (B. rapa yellow root peel); (c) BRWR-Peel group (B. rapa white root peel); (d)
(b) BRYR-Peel group (B. rapa yellow root peel); (c) BRWR-Peel group (B. rapa white root peel);
BRYR-Pulp group (B. rapa white root pulp); (e) BRWR-Pulp group (B. rapa white root pulp); (f)
(d) BRYR-Pulp group (B. rapa white root pulp); (e) BRWR-Pulp group (B. rapa white root pulp);
Positive control group (Indomethacin).
(f) Positive control group (Indomethacin).

90 According to# study, paw edema is associated with increased NC vascular permeability,
80 cellular infiltration and fluid leakage from sites of inflammation [4]. Paw diameter mea-
BRYR-Peel
#
surement is a tool used to assess # the effectiveness of anti-inflammatory remedies [14]. A
Paw Diameter (mm)

70 BRWR-Peel
reduction in paw diameter indicates a decline in the release of inflammatory mediators
60 # *#
(IL-6 and TNF-α), *#
which
BRYR-Pulp
is a vital indicator# for anti-inflammatory potential of drugs or
50 *# plant extracts
*# [2]. In*#the present study, it is found that B. rapaBRWR-Pulp
extracts exert a dose-reliant
*# *# PC
40 anti-inflammatory
*# potential by reducing
*# the paw diameter
*# of inflamed rats. The greater in-
*# hibition *#
*# of carrageenan-induced paw edema by BRYP-extract-treated rats can be elucidated
30 *# * *# * *
*#
*
20 *
*#
*
10
0
1h 2h 3h 4h 5h
Molecules 2023, 29, 117 8 of 18

by the existence of more polyphenols. Several pharmacological effects of polyphenols have

been documented. In addition, polyphenols are acknowledged to retain anti-inflammatory
potential [9]. For example, gallic acid possesses anti-inflammatory properties that could be
due to restraint of pro-inflammatory mediators (IL-6 and TNF-α) [34]. Many studies have
also been reported on the diverse therapeutic potential, including the anti-inflammatory
and immunomodulatory potential, of chlorogenic acid [35]. Kaempferol is a flavonoid and
exhibits anti-inflammatory properties [36]. Besides abundance of these polyphenols, higher
TPC and TFC values of peel extracts also authenticated their anti-inflammatory potential.
Therefore, it was suggested that the anti-inflammatory potential of B. rapa probably asso-
ciated with
Figure its phenolic compounds.
3. Representative images of rat’sThe
pawsfindings of the current
after carrageenan study
injection. are comparable
(a) Normal control group;
with results of the study investigated by Semwal et al. [2]. In which Complete
(b) BRYR-Peel group (B. rapa yellow root peel); (c) BRWR-Peel group (B. rapa white root Freund’s
peel); (d)
Adjuvant (CFA)-injection-induced rat paw edema was significantly reduced
BRYR-Pulp group (B. rapa white root pulp); (e) BRWR-Pulp group (B. rapa white root by the ethanol
pulp); (f)
extract of Brassica
Positive rapa (dose
control group 200 mg/kg) [2].
(Indomethacin).

90 #
NC
80 BRYR-Peel
#
#
Paw Diameter (mm)

70 BRWR-Peel
60 # *# *# #
BRYR-Pulp
50 *# *# *# BRWR-Pulp
*# *# PC
40 *# *# *#
*# *#
*#
30 *# * *# * *
*#
*
20 *
*#
*
10
0
1h 2h 3h 4h 5h
Time (h)
Figure
Figure 4. Effect
4. Effect of polyphenol-rich
of polyphenol-rich B. extracts
B. rapa rapa extracts and indomethacin
and indomethacin on paw ondiameter
paw diameter after
after car-
carrageenan
rageenan injection.
injection. NCNormal
NC group: group:control;
NormalBRYR-Peel
control; BRYR-Peel group:
group: B. rapa yellowB.root
rapapeel;
yellow root peel;
BRWR-Peel
BRWR-Peel
group: group:
B. rapa white B.peel;
root rapa BRYR-Pulp
white root peel; BRYR-Pulp
group: group:
B. rapa yellow B.pulp;
root rapa yellow root pulp;
BRWR-Pulp BRWR-Pulp
group: B. rapa
white root pulp; PC: Positive control group. * symbolizes significant difference (p ≤ 0.05) as compared
to NC. # symbolizes significant difference (p ≤ 0.05) as compared to PC.

2.4.1. Suppression of Biochemical Inflammatory Biomarkers

The effect of polyphenol-rich BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
extracts and indomethacin on suppression of TNF-α, IL-6, CRP and RF in blood serum is
given in Figure 5 and Table 4. The TNF-α level was higher in the normal control rat group
(80.35 pg/mL). However, the TNF-α level was observed significantly (p ≤ 0.05) suppressed
(ranged, 35.40–43.21 pg/mL) in B. rapa peel extracts compared to root pulp extracts (ranged,
63.64–70.92 pg/mL). Similarly, B. rapa peel extracts also showed lower concentration
(3070–3580 pg/mL) IL-6 levels than those of root pulp extracts (5810–6350 pg/mL). More-
over, the normal control group showed a maximum level of CRP (6.91 mg/L) and RF
(16.21 IU/mL) in the blood serum, showing the inflammation in the animals. Reduction
in the raised levels of CRP and RF of treatment groups showed the effectiveness of the
extracts. The maximum decrease in CRP (2.93 mg/L) and RF (10.93 IU/mL) levels was
observed in the BRYR pulp group, which is analogous with the PC group. Overall, the most
effective B. rapa extract, with significantly decreased levels of TNF-α, IL-6, CRP and RF in
blood serum, was reported for BRYR peel. Thus, a significant anti-inflammatory effect of
all the extracts, especially peel extracts, was observed in the acute inflammatory model by
suppression of pro-inflammatory cytokines (TNF-α, IL-6, CRP, and RF).
 most effective B. rapa extract, with significantly decreased levels of TNF-α, IL-6, CRP and
RF in blood serum, was reported for BRYR peel. Thus, a significant anti-inflammatory
Molecules 2023, 29, 117 effect of all the extracts, especially peel extracts, was observed in the acute inflammatory
9 of 18
model by suppression of pro-inflammatory cytokines (TNF-α, IL-6, CRP, and RF).

# *# A
80
*#
TNF-α (pg/mL)

60
*#
*#
40 *
20

0
NC BRYR-Peel BRWR-Peel BRYR-Pulp BRWR-Pulp PC
Treated Groups
9000 #
8000 B
*#
IL-6 (pg/mL)

7000 *#
6000
5000 *#
4000 *#
*
3000
2000
1000
0
NC BRYR-Peel BRWR-Peel BRYR-Pulp BRWR-Pulp PC
Treated Groups
Figure 5.
Figure 5. Effect
Effectofofpolyphenol-rich
polyphenol-rich B. rapa extracts
B. rapa and and
extracts indomethacin on suppression
indomethacin of TNF-α,
on suppression and
of TNF-α,
IL-6 in blood serum: (A) tumor necrosis factor (TNF)-α, and (B) IL-6. Results are presented as mean
and IL-6 in blood serum: (A) tumor necrosis factor (TNF)-α, and (B) IL-6. Results are presented as
± standard deviation (n = 6) and analyzed by one-way ANOVA (p ≤ 0.05). NC group: Normal control;
mean ± standard deviation (n = 6) and analyzed by one-way ANOVA (p ≤ 0.05). NC group: Normal
BRYR-Peel group: B. rapa yellow root peel; BRWR-Peel group: B. rapa white root peel; BRYR-Pulp
control;
group: B.BRYR-Peel
rapa yellow root B.
group: rapaBRWR-Pulp
pulp; yellow root peel; BRWR-Peel
group: rootB.pulp;
group:
B. rapa white rapa white root peel;
PC: Positive BRYR-
control
Pulp group: B. rapa yellow root pulp; BRWR-Pulp group: B. rapa white root pulp; PC:
group. * symbolizes significant difference (p ≤ 0.05) as compared to NC. # symbolizes significantPositive control
group. * symbolizes
difference (p ≤ 0.05) assignificant
compareddifference
to PC. (p ≤ 0.05) as compared to NC. # symbolizes significant
difference (p ≤ 0.05) as compared to PC.
Table 4. Effect of polyphenol-rich B. rapa extracts and indomethacin on suppression of CRP and RF
in blood serum of rat groups.
Table 4. Effect of polyphenol-rich B. rapa extracts and indomethacin on suppression of CRP and RF in
Groups blood serum of rat groups.CRP (mg/L) RF (IU/mL)
NC 6.91 ± 0.35 # 16.21 ± 0.81 #
Groups CRP (mg/L) RF (IU/mL)
BRYR-Peel 2.93 ± 0.15 * 10.93 ± 0.55 *
NC 6.91 ± #
0.35± 0.20 * 16.21 ± 0.81 #*
BRWR-Peel 3.96 12.36 ± 0.62
BRYR-Peel 2.93 ± 0.15 * 10.93 ± 0.55 *#
BRYR-Pulp 4.52 ± 0.23 *# 13.97 ± 0.70 *
BRWR-Peel 3.96 ± 0.20 * 12.36 ± 0.62 *#
BRWR-Pulp
BRYR-Pulp 5.61 ±
4.52 ± 0.23 *#
0.28 *# 14.32 ± 0.72 *
13.97 ± 0.70 *#
PC
BRWR-Pulp 3.14 ±
5.61 ± 0.28 * 0.16
# * 14.32 ±± 0.72
10.80 0.61**#
PC Statistical significance3.14
according
± 0.16 to
* ANOVA (p ≤ 0.05). NC: Normal control
10.80(NC); BRYR-Peel:
± 0.61 * B. rapa
yellow root peel; BRWR-Peel: B. rapa white root peel; BRYR-Pulp: B. rapa yellow root pulp; BRWR-
Statistical significance according to ANOVA (p ≤ 0.05). NC: Normal control (NC); BRYR-Peel: B. rapa yellow
Pulp:
root B. rapa
peel; white root
BRWR-Peel: pulp;
B. rapa PC:root
white Positive control. * symbolizes
peel; BRYR-Pulp: B. rapa yellowsignificant
root pulp;difference (p B.
BRWR-Pulp: ≤ 0.05) as
rapa white
compared
root pulp; PC:to Positive
NC. # symbolizes significantsignificant
control. * symbolizes differencedifference
(p ≤ 0.05)(pas≤compared to PC. to NC. # symbolizes
0.05) as compared
significant difference (p ≤ 0.05) as compared to PC.
Previous studies have shown that TNF-α is a significant element in the inflammatory
response. It produces
Previous an intrinsic
studies have shown protective
that TNF-αresponse by stimulating
is a significant macrophages
element in and
the inflammatory
releasing leukotrienes
response. It producesand kinins, which
an intrinsic furtherresponse
protective activate the
by release of other
stimulating inflammatory
macrophages and
mediators [37]. Interleukin 6 (IL-6) is another important mediator which is released
releasing leukotrienes and kinins, which further activate the release of other inflammatory in the
infected areas
mediators [37].[38]. TNF-α and
Interleukin IL-6isappear
6 (IL-6) anothertoimportant
be major pro-inflammatory
mediator which ismediators
released in
the infected areas [38]. TNF-α and IL-6 appear to be major pro-inflammatory mediators
tangled in the pathophysiology of inflammation. The decrease in these pro-inflammatory
mediators in groups treated with BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
extracts demonstrated the immune-regulatory function of B. rapa. Previous research has
shown a scientific correlation between TNF-α and IL-6 stimulation of CRP transcription
and TNF-α and IL-6 involvement in inflammation [7]. The literature also reports that acute
Molecules 2023, 29, 117 10 of 18

phase CRP reaction is the first acute protein that is described as a known sign of sensitive
inflammatory biomarker and symptom of tissue deterioration [6]. It has been documented
that CRP exhibits a significant contribution to the development of inflammation [3]. CRP
and RF levels were elevated in inflamed rats, while standard drugs and all the extracts
reduced these parameters, indicating a protective effect of the B. rapa extracts. In the present
study, BRYR peel, BRWR peel, BRYR pulp and BRWR pulp extracts reduce the levels of these
factors, thereby improving inflammation. These results have been confirmed by previous
findings where B. rapa seed oil treatment regulates the level of TNF-α mRNA in rats with
osteoporosis [39]. Furthermore, suppression of these pro-inflammatory cytokines is due
to the existence of phenolic compounds [9]. Phenolic acids include sinapic acid, caffeic,
cinnamic, and ferulic acids, identified in BRYR peel, BRWR peel, BRYR pulp and BRWR
pulp extracts, and exhibit anti-inflammatory effects by decelerating TNF-α expression [40].
Flavonoids like catechin, quercetin, and kaempferol, also found in all B. rapa extracts, have
the ability to inhibit IL-6 production as well as reduce CRP level and clinical signs of
inflammation [10,11,36]. Therefore, these observations deliver a viable indication that this
herbal product could be effective in the cure of inflammatory infections.

2.4.2. Histopathology Study

The histopathology analysis data of the NC, BRYR-peel, BRWR-peel, BRYR-pulp and
BRWR-pulp PC groups is shown in Figure 6. It has been well characterized that neu-
trophil infiltration plays a significant character in carrageenan-induced paw inflammation.
Microscopic examination of rats’ paws inflamed with carrageenan displayed connective
tissue infiltration along with acute edema in the epidermis and dermis layers. Micro-
graphs show that major edema and neutrophils were observed in normal control rats
along with the deterioration of collagen tissue and high leukocyte infiltration in the dermis
layer compared with the treated groups. Less inflammation was observed in the treated
BRYR-Pulp and BRWR-Pulp groups than in the NC group. However, the BRYR-Peel and
BRWR-Peel groups, and the PC (indomethacin-treated rat) group did not display any kind
of inflammation or neutrophil cells. This means that the PC group, as well as the BRYR-Peel
and BRWR-Peel groups, showed decreased leukocyte and collagen degradation than the
BRYR-Pulp and BRWR-Pulp groups. Therefore, the histopathology study revealed that
B. rapa extracts diminished leukocyte and collagen deterioration and improved vascularity
by reducing inflammation. According to Li et al. [4], rats treated with alkaloid leaf extract
of E. cuneatum managed to reduce infiltration of collagen and leukocyte tissue interruption
in a dose-dependent manner, and these outcomes mirrored our current results.

2.5. Analgesic Activity

Table 5 represents the effect of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
extracts and indomethacin on analgesic activity. The hot plate method was used to assess
thermal stimulation in the skin and measures the time for the rat to jump or lick to analyze
its potential effects [41]. Administration of B. rapa extracts (200 mg/kg) triggered an
important stimulatory effect in the rat by elongating the latency time. At 90 min after
the administration of the dose, BRYR peel and BRWR peel extracts (200 mg/kg) achieved
maximum thermal stimulation latency. However, the PC group observed a maximum delay
in heat stimulation at 60 min (min). BRYR pulp and BRWR pulp extracts were also observed
with maximum thermal stimulation at 90 min after dose administration, compared to the
control group. Figure 7 demonstrates the analgesic potential of indomethacin- and extract-
treated groups as maximum possible analgesia (MPA). The MPA of the BRYR-peel group
was found to be 40.28%, which is better than that of the PC group (39.10%). The BRWR-
Peel, BRYR-Pulp and BRWR-Pulp groups displayed a significant (p ≤ 0.05) reduction in
MPA. Overall, the BRYR-Peel group displayed an outstanding analgesic potential at 90 min
(min) and so BRYR-Peel was found to be effective in relieving pain. According to the results
of the present investigation, the analgesic potential of these BRYR peel, BRWR peel, BRYR
pulp and BRWR pulp extracts might be exerted by the central nervous system [42]. A
Molecules 2023, 29, 117 11 of 18

study on polyphenol-rich plants found that the plant’s polyphenols can relieve pain [11,43].
Another study reported the analgesic effect of phenolic and flavonoids compounds [43].
Moreover, RP-HPLC characterization of peel and pulp extracts of B. rapa varieties revealed
the presence of gallic acid, quercetin and ferulic acid, which have exhibited strong analgesic
potential in vivo studies [34,44,45]. These results are confirmed by previous findings where
Molecules 2023, 28, x FOR PEER REVIEW
treatment with an alcoholic extract of B. rapa root provided significant pain11reliefof 19 in a

dose-reliant manner [19].

Figure 6. Effect of polyphenol-rich B. rapa extract on carrageenan-induced rat paw tissue by

Figure 6. Effect of polyphenol-rich B. rapa extract on carrageenan-induced rat paw tissue by
histopathological analysis. (A) Normal control group; (B) BRYR-Peel group (B. rapa yellow root peel);
histopathological analysis.
(C) BRWR-Peel group (A)white
(B. rapa Normal control
root peel); (D)group; (B) BRYR-Peel
BRYR-Pulp group
group (B. rapa rapa
(B.root
white yellow
pulp); (E) root
peel); (C) BRWR-Peel
BRWR-Pulp group (B.group
rapa white rapa pulp);
(B. root white(F)
root peel);control
Positive (D) BRYR-Pulp rapaDermis;
group (B. Dr:
group (Indomethacin). white root
Ep: Epidermis;
pulp); HF: Hair
(E) BRWR-Pulp Follicles;
group BV:white
(B. rapa Bloodroot
Vessels. Arrows
pulp); mark inflammation.
(F) Positive control group (Indomethacin). Dr:
Dermis; Ep: Epidermis; HF: Hair Follicles; BV: Blood Vessels. Arrows mark inflammation.
2.5. Analgesic Activity
Table 5 represents the effect of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
Table 5. Effect of polyphenol-rich B. rapa extracts and indomethacin on analgesic activity.
extracts and indomethacin on analgesic activity. The hot plate method was used to assess
thermal stimulation in the skin and measures the time for the rat to jump or lick to analyze
Reaction Time (min)
Groups its potential effects [41]. Administration of B. rapa extracts (200 mg/kg) triggered an
Start Time
important stimulatory 30 effect in the rat by 60 elongating the latency90 time. At 90 min after 120 the
NC administration
3.50 ± 0.20 3.52of±the
0.21dose, BRYR3.53
# peel± and
0.22 BRWR peel
# extracts
3.51 ± 0.20 (200 mg/kg)
# 3.55achieved
± 0.22 #
BRYR-Peel maximum
3.60 ± 0.27 thermal
6.19 ± 0.31 * #
stimulation latency. However,
6.53 ± 0.33 * the PC group
6.93 ± 0.35 *observed 6.29 ± 0.31 *#
a maximum
BRWR-Peel delay
3.58 ± 0.23 in heat stimulation
5.81 ± 0.29 * # at 60 min (min). BRYR
6.17 ± 0.31 * # pulp and BRWR pulp
6.76 ± 0.34 * extracts were
6.14 also*
± 0.31
BRYR-Pulp 3.57 ± 0.22
observed with ± 0.41 * thermal
5.63maximum ± 0.31 *# at 90 6.46
6.11stimulation min±after 5.93 ± 0.30 *
0.32 *dose administration,
BRWR-Pulp 3.55 ± 0.21
compared to5.43 the± control
0.28 * group.5.47 ± 0.277*#demonstrates
Figure 5.89 ±the *#
0.29analgesic 5.43 ± 0.27of*
potential
PC 3.52 ± 0.25
indomethacin- 6.41
and± 0.32 *
extract-treated 6.91 ± 0.35
groups as* maximum 6.83 ± 0.34 analgesia
possible * 5.79 ± 0.29
(MPA). The*
MPA of the BRYR-peel group was found to be 40.28%, which is be er than that of
Reaction time ± standard deviation (n = 6) when compared with the positive control group (Indomethacin). the PC
group significance
Statistical (39.10%). according
The BRWR-Peel,
to ANOVABRYR-Pulp
(p ≤ 0.05). NC:and BRWR-Pulp
Normal groups
control (NC); displayed
BRYR-Peel: B. rapa ayellow
rootsignificant
peel; BRWR-Peel: B. rapareduction
(p ≤ 0.05) white root peel; BRYR-Pulp:
in MPA. B. rapa
Overall, theyellow root pulp;
BRYR-Peel BRWR-Pulp:
group displayed B. rapa
an white
root pulp; PC: Positive control. * symbolizes significant difference (p ≤ 0.05) as compared to NC. # symbolizes
outstanding analgesic potential at 90 min (min) and so BRYR-Peel was found to be
significant difference (p ≤ 0.05) as compared to PC.
effective in relieving pain. According to the results of the present investigation, the
analgesic potential of these BRYR peel, BRWR peel, BRYR pulp and BRWR pulp extracts
might be exerted by the central nervous system [42]. A study on polyphenol-rich plants
found that the plant’s polyphenols can relieve pain [11,43]. Another study reported the
 analgesic effect of phenolic and flavonoids compounds [43]. Moreover, RP-HPLC
characterization of peel and pulp extracts of B. rapa varieties revealed the presence of gallic
acid, quercetin and ferulic acid, which have exhibited strong analgesic potential in vivo
studies [34,44,45]. These results are confirmed by previous findings where treatment with
Molecules 2023, 29, 117 an alcoholic extract of B. rapa root provided significant pain relief in a dose-reliant manner
12 of 18
[19].

45.00
Maximum Possible Analgesia (%)
40.00 #
35.00
#
30.00
25.00
20.00
15.00
10.00
5.00
0.00
BRYR-Peel BRWR-Peel BRYR-Pulp BRWR-Pulp PC
Treated Groups
Figure 7. Effect of polyphenol-rich B. rapa extracts on analgesic activity as maximum possible
Figure 7. (MPA)
analgesia Effect compared
of polyphenol-rich B. rapa extracts
to indomethacin. NC group:on Normal
analgesic activity
control; as maximum
BRYR-Peel group:possible
B. rapa
analgesia (MPA) compared to indomethacin. NC group: Normal control; BRYR-Peel
yellow root peel; BRWR-Peel group: B. rapa white root peel; BRYR-Pulp group: B. rapa group:
yellowB. root
rapa
yellow root peel; BRWR-Peel group: B. rapa white root peel; BRYR-Pulp group: B. rapa yellow root
pulp; BRWR-Pulp group: B. rapa white root pulp; PC: Positive control group. # symbolizes significant
pulp; BRWR-Pulp group: B. rapa white root pulp; PC: Positive control group. # symbolizes
difference (p ≤ 0.05) as compared to PC.
significant difference (p ≤ 0.05) as compared to PC.
3. Materials and Methods
Table 5. Effect of polyphenol-rich B. rapa extracts and indomethacin on analgesic activity.
3.1. Collection, Pretreatment and Storage of Samples
Reaction
Two varieties of B. rapa (yellow andTime (min)
white) were collected from the controlled agricul-
Groups
Start tural
Timefields of the Ayyub
30 Agriculture Research
60 Institute, Jhang
90 Road, Faisalabad,120Pakistan.
NC Samples
3.50 ± 0.20 were validated
3.52 ± 0.21 and
# verified (voucher
3.53 ± 0.22 # no. 248-bot-21 and
3.51 ± 0.20 # 249-bot-21)
3.55 by the#Tax-
± 0.22
BRYR-Peel onomist,
3.60 ± 0.27 Botany Department,
6.19 ± 0.31 * # Government College
6.53 ± 0.33 * University Faisalabad.
6.93 ± 0.35 * The samples
6.29 ± 0.31 *#
BRWR-Peel were
3.58 ± 0.23 rinsed thoroughly with
5.81 ± 0.29 *# distilled water. The
6.17 ± 0.31 *# peel and root pulp
6.76 ± 0.34 * were separated,
6.14 ± 0.31sliced,
*
and shaded to dry at room temperature, and ground into semi-powder (mesh size 80) with
BRYR-Pulp 3.57 ± 0.22 5.63 ± 0.41 * 6.11 ± 0.31 * # 6.46 ± 0.32 * 5.93 ± 0.30 *
an electric blender (AG-640, ANEX, Karachi, Pakistan). The samples were then transferred
BRWR-Pulp 3.55 ± 0.21 5.43 ± 0.28 * 5.47 ◦± 0.27 *# 5.89 ± 0.29 *# 5.43 ± 0.27 *
to sealed plastic packets and kept at 4 C.
PC 3.52 ± 0.25 6.41 ± 0.32 * 6.91 ± 0.35 * 6.83 ± 0.34 * 5.79 ± 0.29 *
Reaction
3.2. timeand
Chemical ± standard
Reagentsdeviation (n = 6) when compared with the positive control group
(Indomethacin). Statistical significance according to ANOVA (p ≤ 0.05). NC: Normal control (NC);
All reference and standard chemicals including gallic acid, ferulic acid, p-hydroxybenzoic
BRYR-Peel: B. rapa yellow root peel; BRWR-Peel: B. rapa white root peel; BRYR-Pulp: B. rapa yellow
acid, chlorogenic
root pulp; acid, B.
BRWR-Pulp: syringic acid,root
rapa white vanillic
pulp;acid,
PC: sinapic
Positive acid, p-coumaric
control. acid,significant
* symbolizes salicylic
acid, cinnamic acid, benzoic acid, rutin, kaempferol, catechin, quercetin, 2,2-diphenyl-
difference (p ≤ 0.05) as compared to NC. # symbolizes significant difference (p ≤ 0.05) as compared to
1-picrylhydrazyl
PC. radical (DPPH•), carrageenan, Folin-Ciocalteu reagent and butylated
hydroxyanisole (BHA) were acquired from Sigma-Aldrich (St Louis, MO, USA). ELISA kits
from Elabscience
3. Materials and all other chemicals and reagents used in this study, such as ferric
and Methods
chloride, trichloroacetic acid, sodium hydrogen phosphate, sodium dihydrogen phosphate
3.1. Collection, Pretreatment and Storage of Samples
and methanol, were of analytical grade unless otherwise specified and acquired from Merck
Two varieties
Co. (Darmstadt, of B. rapa (yellow and white) were collected from the controlled
Germany).
agricultural fields of the Ayyub Agriculture Research Institute, Jhang Road, Faisalabad,
Pakistan.
3.3. Samples
Extract were validated and verified (voucher no. 248-bot-21 and 249-bot-21) by
Preparation
Brassica rapa yellow root (BRYR) peel, Brassica rapa white root (BRWR) peel, Brassica rapa
yellow root (BRYR) pulp, and Brassica rapa white root (BRWR) pulp extracts were prepared
by a reported method [12]. Briefly, ground root peel and root pulp samples (50 g per
sample) were extracted using methanol (300 mL) by a soxhlet extractor (500 mL capacity)
for 6–8 h. The extracts were filtered with Whatman filter paper no. 1 and concentrated by a
rotary evaporator (EYELA, SB-651, Rikakikai Co., Ltd., Tokyo, Japan) at reduced pressure.
Molecules 2023, 29, 117 13 of 18

The concentrated and desiccated extracts were weighed, and the following formula was
used for estimation of yield.
The extracts were kept in a freezer (−4 ◦ C) up until used for further studies.
 
g Dry extract weight
Yield = ×100
100 g Dry plant material weight

3.4. Quantification of Phenolic and Flavonoids Compounds by RP-HPLC

3.4.1. Sample Preparation
For the quantitative and qualitative investigation of phenolic and flavonoid com-
pounds from BRYR peel, BRWR peel, BRYR pulp and BRWR pulp extracts, a previously
reported method was followed [12]. Briefly, 100 mg extract was dissolved in methanol
(10 mg/mL), filtered through a 45-micron (um) nylon syringe filter, and kept at 4 ◦ C. Fresh
stock solutions of standards were made by dissolving authentic compounds in methanol
(10 mg/mL). The standard solutions were diluted with methanol for further processing to
obtain the requisite concentration (0.4–10 mg/mL). Standard concentration versus the area
of peak was plotted to draw a calibration curve of each standard.

3.4.2. Chromatographic Conditions

The Agilent 1260 infinity HPLC system (Agilent, Santa Clara, CA, USA) with a C-18
column (150 × 4.6 mm internal diameter, 2.7 micrometer (µm) particle size) equipped with a
gradient binary pump system (G7112B), 1260 autosampler (G7129A) and multiwavelength
detector (G7165A) was used. HPLC analysis data is given as a Supplementary Materials.
The non-linear gradient with methanol was acetonitrile (30:70 as solvent A) and 0.1% acetic
acid in distilled water as solvent B. The following gradient program was developed: 10% A
from 0 to 5 min; 10–30% A from 5 to 25 min; 30–40% A from 25 to 40 min; 40–90% A from 40
to 60 min and kept at 90% A from 60–65 min. The MWD detector settings were 250, 270, 290,
310, 330, 350, 370 nm 1.2 nm resolution and 10 points/s sampling rate. Qualitative analysis
was performed using the matching of retention times with the authentic standards and
spiking of standards in the samples, while for quantitative analysis, the standard addition
method was applied.

3.5. In Vitro Antioxidant Potential

3.5.1. Total Phenolic Contents
The total phenolic contents (TPC) of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
extracts were estimated by the Folin–Ciocalteu reagent as reported by Singleton et al. [46]. Gal-
lic acid was used as standard (0.1–0.8 mg/mL) to prepare the standard curve (y = 0.9478x + 0.1097,
R2 = 0.9965), and the findings were reported as mg/g of dried material as gallic acid equivalent.

3.5.2. Total Flavonoid Contents

The total flavonoid contents (TPC) of BRYR peel, BRWR peel, BRYR pulp and BRWR
pulp extracts were calculated as performed by Zhishen et al. [47]. The quercetin was used as
standard (0.1–0.8 mg/mL) to prepare the standard curve (y = 0.7159x + 0.0342, R2 = 0.9973),
and the findings were reported as mg/g of dried material, as quercetin equivalent.

3.5.3. DPPH Radical Scavenging Assay

The antioxidant activity of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp
extracts, in term of potential to neutralized 2,2-diphenyl-1-picrylhydrazyl free radicals
(DPPH), was examined by a published method [30]. The extract (10 µg/mL) was mixed
in methanol with the same amount of DPPH solution (90 µmol/L in methanol). After
incubating the solution for 30 min at ambient temperature (30 ◦ C), the absorbance was
noted at a wavelength of 517 nm. Butylated hydroxyanisole (BHA) was used as a positive
standard. Radical scavenging percent concentrations were determined by the mean of the
following formula:
Molecules 2023, 29, 117 14 of 18

Absorbance of DPPH solution − Absorbance of sample solution

Radical Scavenging(%) = × 100
Absorbance of DPPH solution
The 50% inhibition (IC50 ) of extract concentration was determined from the graph
plotting extract concentration against radical scavenge percentage.

3.5.4. Reducing Power

The procedure reported by Hussain et al. [12] was used to determine the reduc-
ing power of BRYR peel, BRWR peel, BRYR pulp and BRWR pulp extracts. Different
concentrations (2–10 mg/mL) of extracts were used to determine the reducing poten-
tial. Absorbance was taken at 700 nm of wavelength nm by spectrophotometer (Lambda
25 UV/VIS, L600000B, Perkin Elmer Singapore, Singapore).

3.6. In Vivo Anti-Inflammatory and Analgesic Potentials

Healthy male Wistar Kyoto (WKY) rats (140–160 g) were purchased from the Animal
House, Faculty of Pharmaceutical Sciences, Government College University Faisalabad.
Before starting the experimental work, these animals were kept for 5 days in an animal
house for acclimatization. The animal house maintained all the standard requirements,
such as an ambient temperature (25 ± 3 ◦ C), 12 h dark/light cycle and humidity range
(30–70%), which were essential to the rats’ survival. The rats were fed normal rat chow
and water ad libitum. One week after acclimatization, the rats were randomly distributed
in different cages with six rats per group/cage. All animal-based experimental work was
conducted with the Review Board approval at Government College University Faisalabad,
Pakistan with an ethical permit, GCUF/ERC/26.

3.6.1. Acute Inflammatory Model

Animal Grouping
For evaluating the anti-inflammatory potentials of BRYR peel, BRWR peel, BRYR pulp
and BRWR pulp extracts, animals were divided into following groups, with 6 rats in each
group: Normal Control (NC) group (received only standard pellet diet and normal saline
orally), BRYR-peel group (received BRYR peel extract (200 mg/kg) along with pellet diet
and normal saline orally), BRWR-Peel group (received BRWR peel extract (200 mg/kg)
along with pellet diet and normal saline orally), BRYR-Pulp group (received BRYR pulp
extract (200 mg/kg) along with pellet diet and normal saline orally), BRWR-Pulp group
(received BRWR pulp extract (200 mg/kg) along with pellet diet and normal saline orally),
Positive Control (PC) group (received Indomethacin (10 mg/kg) along with pellet diet and
normal saline orally).

Study Design
To investigate acute inflammation, a carrageenan-induced paw edema model was
performed using the described method [14]. The experimental groups (BRYR-Peel, BRWR-
Peel, BRYR-Pulp and BRWR-Pulp) were treated with an extract dose of 200 mg/kg/day for
14 days, whereas the positive control group was given an indomethacin dose of 10 mg/kg
only on the 14th day. On the 14th day, each rat was injected with 0.1 mL of 1% of freshly
prepared carrageenan to induce inflammation below the plantar fascia of the right hind paw.
A digital vernier caliper (Ugo Basile, model 7140, Gemonio, Italy) was used to measure paw
diameter (PVo ) before and 1, 2, 3, 4 and 5 h after carrageenan injection (PVt ) [48]. Edema
inhibition in the treated groups vs. the control group was calculated using the following
equation:

(P Vt − PV◦ )control − (P Vt − PV◦ )treated

% Inhibition of Inflammation = × 100
(P Vt − PV◦ )control

where PVt represents the diameter of inflammation in the rat treated after carrageenan and
PVo represents the diameter of inflammation in the rat treated before carrageenan.
Molecules 2023, 29, 117 15 of 18

Suppression of Biochemical Inflammatory Biomarkers

After experiment completion, rats of each group were euthanized by cervical decapita-
tion. Blood samples and paw tissues were collected. Blood was collected in a vacuum vessel
containing no anticoagulant, centrifuged it at 3000 rpm for 4 min (min), and a serum sample
was obtained. Further, serum samples were analyzed by an automatic hemocytometer to
examine suppression of biochemical biomarkers C-reactive protein (CRP) and rheumatoid
factor (RF). The ELISA (Enzyme-Linked Immunosorbent Assay) was also performed using
a kit protocol (Elabscience, E-EL-R0019, and E-EL-R0015 catalog numbers) to calculate
tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) concentration in blood serum [49].

Histopathology Study
For the histopathology study, each paw tissue sample was immersed in neutral forma-
lin solution (10%), retained in paraffin wax, weighed, and stained by hematoxylin-eosin.
Histopathological changes in the paw tissue were photographed under an optical micro-
scope [4].

3.6.2. Analgesic Activity

Animal Grouping
For evaluating analgesic activity of BRYR peel, BRWR peel, BRYR pulp and BRWR
pulp extracts, animals were divided into following groups with 6 rats in each group:
Normal Control (NC) group (received only standard pellet diet and normal saline orally),
BRYR-Peel group (received BRYR peel extract (200 mg/kg) along with pellet diet and
normal saline orally), BRWR-Peel group (received BRWR peel extract (200 mg/kg) along
with pellet diet and normal saline orally), BRYR-Pulp group (received BRYR pulp extract
(200 mg/kg) along with pellet diet and normal saline orally), BRWR-Pulp group (received
BRWR pulp extract (200 mg/kg) along with pellet diet and normal saline orally), Positive
Control (PC) group (received Indomethacin (10 mg/kg) along with pellet diet and normal
saline orally).

Study Design
Analgesic potential was estimated by the hot plate process as described by Fan et al. [41]
with slight modifications. The experimental groups (BRYR-Peel, BRWR-Peel, BRYR-Pulp
and BRWR-Pulp) were given an extract dose of 200 mg/kg for 7 days, whereas the positive
control group was treated with indomethacin 10 mg/kg. On the 7th day, analgesic activ-
ity was performed on all experimental group rats after 1 h of administration of extracts,
and indomethacin. Each rat of all groups was placed on a hot plate in a glass beaker
at 55 ± 0.5 ◦ C. The reaction time of the rat against pain stimuli, and distress behavior
including jumping and paw licking, was monitored. The latency time period or duration of
the pain response was recorded by stopwatch in seconds. Before dosing, the first reading
was observed at 0 time, while the remaining readings were observed at intervals of 30, 60,
90 and 120 minutes (min) after the extracts and drug intake. The cutoff time to prevent any
injury was fixed at 12 seconds (s). This was considered as a control for pain response time.
Maximum possible analgesia (MPA) was determined by the following formula:

Response time for treatment − Response time for saline

MPA (%) = × 100
12 − Response time for saline

3.7. Statistical Analysis

A Q-test was applied on each rat of each group to find the outlier values for both
activities, but no outlier values were noticed. The sample of each variety of B. rapa was
analyzed separately in triplicate. The data were represented as mean ± standard deviation
(SD). One-way analysis of variance (ANOVA) was applied with the statistical package
(Minitab, Version 17, Minitab Inc., State College, PA, USA) and the Tukey test was applied
Molecules 2023, 29, 117 16 of 18

to compare the differences between mean values; differences were considered statistical
significant if the probability value (p) was ≤0.05.

4. Conclusions
It can be concluded that the root peel extracts of both varieties of B. rapa exhibited more
antioxidant potential than the root pulp extracts due to the presence of more polyphenols,
and the best antioxidant activity was recorded with BRYP extract. Ten phenolic acids
and four flavonoids were detected from the B. rapa extracts. Gallic acid and chlorogenic
acid were the phenolic acids present in abundance in all extracts, whereas catechin was
reported in the highest concentration in BRYR extract. A reduced rate of inflammation
and suppressed level of pro-inflammatory biomarkers were observed in the BRYR-Peel
and BRWR-Peel groups as compared with the BRYR-Pulp and BRWR-Pulp groups. TNF-α,
IL-6, CRP and RF levels were lowest in the BRYR-Peel group, followed by the BRWR-Peel
and root-pulp-extract-treated rat groups. Additionally, a decrease in leukocyte infiltration
and collagen degradation were observed in rats treated with root-peel extracts rather than
root pulp extracts by histopathology study. Analgesic activity results also showed that
the rats given the peel extracts experienced less pain compared with the rats given the
root pulp extracts. The plant extracts follow an extract-dependent suppression of TNF-α,
IL-6, CRP and RF levels to reduce the development of inflammation and pain. BRYP at
a dose of 200 mg/kg is the best anti-inflammatory extract with the greatest suppression
potential of pro-inflammatory cytokines (TNF-α, IL-6, CRP and RF) along with antioxidant
and analgesic activities. Further study can be planned to isolate specific compounds from
B. rapa responsible for the suppression of pro-inflammatory cytokines (TNF-α, IL-6, CRP
and RF), free radicals and pain-causing agents. The transduction pathways to treat acute
and chronic inflammation and various pains should also be tested.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules29010117/s1.
Author Contributions: Conceptualization: H.A.R. and A.I.H.; methodology: H.A.R. and A.I.H.;
investigation: A.I.H. and N.N.; data collection: N.N.; Resources: H.A.R. and A.I.H.; Supervi-
sion: H.A.R. and A.I.H.; Project administration: A.I.H.; Writing—original draft preparation: N.N.;
Writing—review and editing: H.A.R. and A.I.H. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by Qatar University.
Institutional Review Board Statement: All animal-based experimental work was conducted with
the Institutional Review Board approval at Government College University Faisalabad, Pakistan with
an ethical permit GCUF/ERC/26.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data included in this study are available upon request by contacting
the corresponding authors.
Acknowledgments: Qatar University funded the publication of this project. The support and
resources provided by the College of Pharmacy, QU Health, Qatar University to the corresponding
author, HA Rathore is acknowledged greatly. The authors acknowledge the Central Hi-Tech Lab,
Government College University Faisalabad, Pakistan for providing assistance in characterization of
compounds and the Department of Pharmaceutics for supporting in-vivo study.
Conflicts of Interest: The authors declare no conflict of interest.

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