In-vitro and in-vivo anti-inflammatory properties of extracts and isolates of Pangdahai

Abstract

Background: : Pangdahai (matured, ripened, and dried seeds of Scaphium affine (Mast.) Pierre) is widely used in managing several diseases in countries like China, Vietnam, Japan, and India. This study evaluated the anti-inflammatory effects of the crude extracts (ethanol and aqueous) and isolated compounds of Pangdahai. Methods: : Xylene-induced ear edema in mice, carrageenan-induced paw edema in rats, and nitric oxide (NO) assay were used to evaluate and screen the crude extracts and isolated compounds from the ethanolic extracts of Pangdahai. TNF-α and IL-1β levels in the tissues of rat foot and ear were determined by ELISA. The cytotoxicity of the isolated compounds was also determined by MTT assay. Molecular docking studies using targets involved in the inflammatory process were also used to further evaluate the compounds. Results: : Both aqueous and ethanol extracts demonstrated significant anti-inflammatory effect and markedly attenuated vascular permeability in mice induced by acetic acid in a dose-independent manner. The ethanol extract also significantly inhibited levels of IL-1β and TNF-α. Four (4) compounds exhibited significant inhibitory effects on NO release without cytotoxicity on RAW 264.7 macrophage. These compounds also showed good binding affinities for COX-2, PLA2, IRAK-4 and NIK. Conclusions: This study validates, provides scientific evidence and justification for the use of the aqueous de-coctions of Pangdahai in pharyngitis traditionally. (+)-Pinoresinol, tiliroside, Z-caffeic acid, and 3,4-dihydrox-ybenzoic acid (protocatechuic acid) isolated from Pangdahai showed anti-inflammatory activities, which might be responsible for the actions of Pangdahai. Tiliroside showed high binding affinity comparable to the native ligands of inflammatory mediators. List of abbreviations COX-2 cyclooxygenase-2 DMSO Dimethylsulfoxide DMEM Dulbecco's modified eagle medium ELISA enzyme-linked immunosorbent assay FBS Fetal Bovine Serum IL Interleukin iNOS inducible nitric oxide synthase IRAK-4 Interleukin-1 Receptor-Associated Kinase-4 LPS Lipopolysaccharide NIK NF-κB-Inducing Kinase NO nitric oxide NSAIDs non-steroidal anti-inflammatory agents NTF Tumor necrosis factor OD Optical density PBS Phosphate buffered saline PDH Pangdahai

Full text

Phytomedicine Plus 4 (2024) 100533
Available online 13 February 2024
2667-0313/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
In-vitro and in-vivo anti-inammatory properties of extracts and isolates
of Pangdahai
Mahmood B. Oppong
a
,
b
,
*
, Shijie Cao
b
, Shi-Ming Fang
b
, Seth K. Amponsah
c
, Paul O. Donkor
d
,
Michael Lartey
a
, Lawrence A. Adutwum
a
, Kwabena F.M. Opuni
a
, Feng Zhao
e
, Qiu Feng
b
a
Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Sciences, University of Ghana, Legon, Ghana
b
Tianjin State Key Laboratory of Modern Chinese Medicine and School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 10 Poyanghu
Road, Jinghai District, Tianjin, 301617, China
c
Department of Medical Pharmacology, University of Ghana Medical School, Accra, Ghana
d
Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, College of Health Sciences, University of Ghana, Legon, Ghana
e
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of
Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, People’s Republic of China
ARTICLE INFO
Keywords:
Pangdahai
Anti-inammation
Cytotoxicity
Secondary metabolites
ABSTRACT
Background: : Pangdahai (matured, ripened, and dried seeds of Scaphium afne (Mast.) Pierre) is widely used in
managing several diseases in countries like China, Vietnam, Japan, and India. This study evaluated the anti-
inammatory effects of the crude extracts (ethanol and aqueous) and isolated compounds of Pangdahai.
Methods: : Xylene-induced ear edema in mice, carrageenan-induced paw edema in rats, and nitric oxide (NO)
assay were used to evaluate and screen the crude extracts and isolated compounds from the ethanolic extracts of
Pangdahai. TNF-
α
and IL-1β levels in the tissues of rat foot and ear were determined by ELISA. The cytotoxicity of
the isolated compounds was also determined by MTT assay. Molecular docking studies using targets involved in
the inammatory process were also used to further evaluate the compounds.
Results: : Both aqueous and ethanol extracts demonstrated signicant anti-inammatory effect and markedly
attenuated vascular permeability in mice induced by acetic acid in a dose-independent manner. The ethanol
extract also signicantly inhibited levels of IL-1β and TNF-
α
. Four (4) compounds exhibited signicant inhibitory
effects on NO release without cytotoxicity on RAW 264.7 macrophage. These compounds also showed good
binding afnities for COX-2, PLA2, IRAK-4 and NIK.
Conclusions: This study validates, provides scientic evidence and justication for the use of the aqueous de-
coctions of Pangdahai in pharyngitis traditionally. (+) – Pinoresinol, tiliroside, Z-caffeic acid, and 3,4-dihydrox-
ybenzoic acid (protocatechuic acid) isolated from Pangdahai showed anti-inammatory activities, which might
be responsible for the actions of Pangdahai. Tiliroside showed high binding afnity comparable to the native
ligands of inammatory mediators.
List of abbreviations
COX-2 cyclooxygenase-2
DMSO Dimethylsulfoxide
DMEM Dulbecco’s modied eagle medium
ELISA enzyme-linked immunosorbent assay
FBS Fetal Bovine Serum
IL Interleukin
iNOS inducible nitric oxide synthase
IRAK-4 Interleukin-1 Receptor-Associated Kinase-4
LPS Lipopolysaccharide
NIK NF-κB–Inducing Kinase
NO nitric oxide
NSAIDs non-steroidal anti-inammatory agents
NTF Tumor necrosis factor
OD Optical density
PBS Phosphate buffered saline
PDH Pangdahai
* Corresponding author at: Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Sciences, University of Ghana, P.O. Box LG 43, Legon,
Ghana.
E-mail address: mboppong@ug.edu.gh (M.B. Oppong).
Contents lists available at ScienceDirect
Phytomedicine Plus
journal homepage: www.sciencedirect.com/journal/phytomedicine-plus
https://doi.org/10.1016/j.phyplu.2024.100533

Phytomedicine Plus 4 (2024) 100533
2
PGE Prostaglandin E2
PMSF phenylmethylsulfonyl uoride
PLA2 Phospholipase A2
TCH Traditional Chinese Medicine
WHO World Health Organization.
1. Introduction
Inammation can be said to be one of the body’s reaction to tissue
damage or infection and is typically characterized by redness, swelling,
heat, and pain (Megha et al., 2021). In inammation, there can be
activation of inammatory mediators like chemokines and cytokines
(Zhu et al., 2018). There are three main phases of inammation. Phase
1is marked by an increase in vascular permeability leading to the
exudation of uids from the blood into the interstitial space; phase 2
involves the leukocytes inltration from the blood into the tissues, and
phase 3 is distinctively shown by granuloma formation and tissue repair
(Mukhopadhyay et al., 2019). Edema is also a characteristic feature of
the acute inammation (Li et al., 2021; Tian et al., 2021). Inammation
is a common symptom for most disease conditions, some of which
include pharyngitis, bowel diseases, arthritis, allergic rhinitis, and
atopic dermatitis (Zhu et al., 2018). Therefore, regulating mediators of
inammation could decrease disease severity or progression.
Conventionally, inammatory conditions are managed with anti-
inammatory agents. These agents could be steroidal or non-steroidal
in nature. The steroidal agents include glucocorticoids, such as predni-
sone, prednisolone, triamcinolone, methylprednisolone, and dexa-
methasone. Glucocorticoids can cause side effects like high blood sugar,
difculty responding to insulin, high blood pressure, muscle weakness,
vulnerability to infections, Cushing’s syndrome, stomach ulcers, and
mental health issues (Yang and Yu, 2021). The non-steroidal anti-in-
ammatory agents (NSAIDs), such as piroxicam, aspirin, aceclofenac,
ibuprofen, diclofenac, naproxen, indomethacin, and celecoxib, mainly
inhibit the synthesis of prostaglandin or cyclooxygenase. Despite their
clinical utility, NSAIDs are also known to cause gastric ulcers, liver and
kidney damage (Olry de Labry et al., 2021). NSAIDs elevate blood
pressure and increase the risk of myocardial infarction (Patrono, 2016).
The contribution of natural products to maintaining health and
wellbeing is underestimated. Natural products are the engine behind the
successes of traditional medicine and/or herbal medicine practices.
Medicinal plants are good sources of secondary metabolites which form
the basis for most commercially produced pharmaceuticals and herbal
remedies (Li et al., 2020). The use of medicinal plants in preventing and
treating/curing human diseases dates back to antiquity. The analgesic
and antipyretic properties of the bark of the willow tree have long been
documented by the Greeks and Romans (Montinari et al., 2019).
Empirical knowledge of these medicinal plants and their potential toxic
effects were passed on by oral tradition and sometimes recorded in texts
(Jansen et al., 2021). Monographs on specic herbs are accessible from
several sources, for example, the European Scientic Cooperative on
Phytotherapy and the World Health Organization (WHO, 2019).
Furthermore, Traditional Chinese Medicine (TCM) has attracted inter-
est, acceptability, and signicance in many countries. TCM continues to
play a major role in the management of diseases and is also an excellent
source in the discovery of natural bioactive compounds or lead com-
pounds (Wang et al., 2018).
Pangdahai (PDH) is the dried seeds of Scaphium afne (Mast.) Pierre,
of the family Malvaceae (Medicinal Plant Name Services, 2021). In the
Chinese Pharmacopoeia, it is recorded as Sterculia lychnophora Hance
Pierre (scientic synonym) (Chinese Pharmacopoeia Commission,
2015). PDH is famously used in traditional/folk medicine in Asia (China,
Japan, Vietnam, Thailand, and India). Decoctions of PDH are used for
treating pharyngitis, laryngitis, constipation, cough, menorrhagia, and
pain. The crude extracts and isolates of PDH have shown diverse phar-
macologic effects, including anti-inammatory, neuroprotective,
anti-microbial, anti-hypertensive, analgesic, antipyretic, anti-ulcer, and
anti-oxidative effects (Oppong et al., 2018). Clinically, PDH is notable
for treating chronic pharyngitis in China (Oppong et al., 2018). Data also
suggest that it contains many secondary metabolites such as lignans,
phenylpropanoids, avonoids, nitrogenous bases, phenolic acids, het-
erocyclic aromatic acids, phytosteroids, glycosides, sesquiterpenoids,
and nucleosides (Oppong et al., 2020). Indeed, continuous in-
vestigations must be done to ascertain and validate the traditional or
folkloric uses of plants and their extract. This work reports, the
anti-inammatory properties of the aqueous and ethanol extracts and
some isolated secondary metabolites of PDH for the rst time.
2. Methods
2.1. Chemicals and reagents
Dexamethasone acetate was purchased from Zhejiang Xianjun
Pharma Ltd., China. The water used was puried with Millipore Milli Q
plus purication system (Thermo Fisher Scientic, USA) Carrageenan,
xylene, physiological normal saline solution, 0.6 %
v
/
v
acetic acid solu-
tion, 0.5 %
w
/
v
Evans blue solution, Griess reagent were purchased from
Sigma, USA. Bacterial lipopolysaccharide (LPS) was purchased from
Sigma, USA. RAW 264.7 murine macrophage cell line was bought from
the American Type Culture Collection (USA). Fetal Bovine Serum (FBS)
was obtained from Hyclone (USA), Dimetylsulfoxide (DMSO) from
Solarbio (China), and Dulbecco’s modied eagle medium (DMEM) from
Thermo Fisher Scientic (USA). All other reagents used were of
analytical grade and commercially available.
2.2. Preparation of PDH extracts
The PDH was obtained in March 2016 from the Guangxi province
(China). A voucher specimen (No.: 20161205SL) was kept at the Tianjin
State Key Laboratory of Modern Chinese Medicine at Tianjin University
of Tradition Chinese Medicine, China.
Briey, 1 kg each of PDH was extracted separately with water and 95
%
v
/v ethanol, concentrated and dried in vacuo to yield 20.80 and 6.50
%
w
/
w
of aqueous and ethanol extracts, respectively as previously
described in our work Oppong et al., 2020.
2.3. Acquisition of animals
Male Sprague–Dawley rats, SPF grade (200–220 g) were obtained
from Shandong Yantai Raphael Biotechnology Co. (China). Male
Kunming mice (20 ±2 g) were obtained from Shandong Yantai Raphael
Biotechnology Co. (China). The experimental animals were kept in a
temperature- and humidity-controlled room (23 ◦C, 60 % air humidity).
They had unrestricted access to standard diet and water. They were kept
in separate metabolic cages with no food but unrestricted access to water
for 12 h before the experiment. All procedures conformed to the
Guidelines associated with Care and Use of Laboratory Animals (Na-
tional Institutes of Health).
Xylene–induced ear edema in mice
In brief, 35 male Kunming mice, weighing averagely 20 ±2 g, were
randomly grouped into 5: the model (negative control), dexamethasone
(positive control), low dose of PDH, medium dose of PDH and high dose
of PDH groups. With the aqueous extract of PDH, the low, medium, and
high dose groups were treated with 200, 400, and 800 mg/kg.d (bw) of
the extract. With the ethanol extract, the low, medium, and high dose
groups were treated with 20, 40, and 80 mg/kg.d (bw) of the extract. For
both extracts, 6 mg/kg.d (bw) of dexamethasone was used as a positive
control. The test agents (extracts and dexamethasone) were adminis-
tered directly into the stomach by oral gavage at a volume of 0.2 mL/10
g using normal saline as the vehicle.
The negative control group was given 2 mL of normal saline once
M.B. Oppong et al.

Phytomedicine Plus 4 (2024) 100533
3
daily. After the fth day of treatment, 0.1 mL of xylene was evenly
smeared on both the inner and outer sides of the right ear of each of the
mice (to induce edema), and the left ear was left as the control. The mice
were sacriced after 4 h. The left and right ears were cut along the ear
line. Ear discs were cut from both ears from the same part of each ear of
the same mouse with a stainless-steel perforator (diameter: 6 mm). The
ear discs were then weighed with an analytical balance (Zhao et al.,
2018). The degree of edema was evaluated by the difference in weight
between the right and left ear discs of the same mice. The degree of
edema inhibition was used as an index of the anti-inammatory activity
of the extracts.
Ear edema(mg) = weight of left ear disc − − weight of rightear disc
The ear tissues were stored in a refrigerator at -80 ℃.
2.5. Carrageenan-induced paw edema in rats
Male Sprague Dawley rats (SPF grade), weighing an average of 200
±20 g, were randomly put into 5 groups of 7 animals. One set of the
animals were assessed using the aqueous extract of PDH, and another set
of animals were assessed using the ethanol extract of PDH. For the rst
set (aqueous extract), the grouping included 7 rats put in 5 group:
negative control (2 mL normal saline), dexamethasone (positive control
- 6 mg/kg⋅day bw), low dose of aqueous extract of PDH (200 mg/kg⋅day
bw), medium dose PDH (400 mg/kg⋅day bw), and high dose PDH (800
mg/kg⋅day bw). The extracts were administered directly into the
stomach by oral gavage at a volume of 0.2 mL/10 g using normal saline
as the vehicle.
The ethanol extract of PDH followed the same procedure (7 rats in 5
groups) as done for the aqueous extract: low, medium, and high dose
groups received 20, 40, and 80 mg/kg⋅day (bw). Dexamethasone was
used as a positive control at 6 mg/kg⋅day (bw). The negative control
group received 2 mL of normal saline once daily.
Two (2) hours after the last treatment, the volumes of both hind-
paws up to the ankle joint of the rats were measured with a plethys-
mometer. Afterwards, the rats were injected with 1 %
w
/
v
carrageenan
solution (0.1 mL each) into the distal end of their left hind limbs. The
paw volumes were measured again after 1, 2, and 4 h. Each measure-
ment was done in triplicate (Rezq et al., 2021). The rats were then
sacriced by injecting 3 mL of 10 %
v
/
v
chloral hydrate solution into
their abdominal cavity. The paw tissues were removed and stored in a
refrigerator at -80 ℃.
The degree of edema in the rats was calculated as the difference in
weight between the paw volumes measured before carrageenan injec-
tion (basal volumes (V
B
)) and after carrageenan injection (pathological
volumes (V
A
)). Edema inhibition, relative to the percentage increase in
paw volume, was used as an index of the anti-inammatory activity of
the extracts.
Percentage increase in paw volume = {(VA−VB)/VB} × 100
Where
V
A
: Rat paw volume after carrageenan injection
V
B
: Rat paw volume before carrageenan injection
2.6. Acetic acid-induced vascular permeability in mice
Thirty-ve male Kunming mice, weighing averagely 20 ±2 g, were
randomly grouped into 5: the negative control, dexamethasone, low,
medium, and high groups. For the aqueous extract, mice in the low,
medium, and high dose groups were given 200, 400, and 800 mg/kg⋅day
(bw) of the extract. The extracts were administered directly into the
stomach by oral gavage at a volume of 0.2 mL/10 g using normal saline
as the vehicle. For the ethanolic extract, mice in the low, medium, and
high dose groups were given 20, 40, and 80 mg/kg⋅day (bw) of extracts.
The positive control was Dexamethasone at 6 mg/kg⋅day (bw) in both
cases. After the fth day of treatment, the tails of the mice were injected
with 0.5 %
w
/
v
Evans blue saline solution (0.2 mL), followed by an in-
jection of 0.2 mL 0.6 %
v
/
v
acetic acid 0.2 mL intraperitoneally. After
sacricing the mice, 10 mL saline solution was to wash their peritoneal
cavities (3x). The saline washings were pooled, ltered and centrifuged
(3000 rpm, 10 min) to obtain the supernatant (5 mL). The optical den-
sity (OD) values of the supernatant were measured at 590 nm with a
UV–Vis spectrophotometer (Rezq et al., 2021).
The intraperitoneal injection of dilute acetic acid causes an increase
in capillary permeability, and this can cause Evans blue to extrude into
the abdominal cavity. The amount of Evans blue represented the capil-
lary permeability, which was estimated by measuring the optical density
values of the supernatant.
2.7. Histo-pathological study of sections of mice edematous ear induced
by xylene
The ear tissues of the negative control group, dexamethasone group,
and PDH ethanol extract (low – high dose) groups were kept in 10 %
v
/
v
formaldehyde solution for 24 h to prepare parafn sections. The parafn
sections were dewaxed and then stained with hematoxylin and eosin.
The pathological changes of the local tissues of the mice auricles were
observed under a light microscope to ascertain the degree of inam-
mation (Huang et al., 2011).
2.8. Determination of the levels of TNF-
α
and IL-1β in the rat foot tissue
The rat foot tissues stored at -80 ◦C were obtained and crushed into
centrifuge tubes. Afterwards, 500
μ
L PBS and 5
μ
L PMSF (100 mM) were
added and placed on an ice water bath for 30 s and then centrifuged at 4
◦C, 13,000 r/min for 6 min. The supernatant was collected, and the
amount of protein was estimated with Bradford method kit. The TNF-
α
and IL-1β levels contained in 1 mg protein of rat foot tissue were
measured using enzyme-linked immunosorbent assay (ELISA) (Huang
et al., 2011). This was repeated for samples obtained from rats treated
with the ethanolic extract of PDH (showed the highest activity).
2.9. Determination of the levels of TNF-
α
and IL-1β in mouse ear tissue
The ear tissues of the mice stored at -80 ◦C were removed, crushed
and placed in centrifuge tubes. Afterwards, 500
μ
L PBS and 5
μ
L PMSF
(100 mM) were added and placed on an ice water bath for 30 s and then
centrifuged at 4 ◦C, 13,000 r/min for 6 min. The supernatant was
collected, and the amount of protein was estimated with Bradford
method kit. TNF-
α
and IL-1β levels in 1 mg protein of mice ear tissue
were measured using enzyme-linked immunosorbent assay (ELISA),
according to manufacturer’s protocol (Huang et al., 2011). This was
repeated for samples obtained from mice treated with the ethanolic
extract of PDH that showed the highest activity.
2.10. Isolation and characterization of compounds from Pangdahai
Compounds from PDH extracts were isolated and characterized using
various chromatographic and spectroscopic techniques described in our
previous work (Oppong et al., 2020).
2.11. In vitro anti-inammatory screening of isolated compounds
2.11.1. Cell culture and MTT assay
Complete DMEM media containing 10 %
v
/
v
FBS, 100 U/mL peni-
cillin, and 100 mg/mL streptomycin was used to culture RAW 264.7
macrophage cells. The culture was incubated in a humidied incubator
set at 5 % CO
2
and 37 ℃ with daily replacement of the culture media.
The cells were then seeded at 1 ×106 cells/well in a 96-well microtiter
plate. After overnight incubation, LPS (1
μ
g/mL) with or without the
isolated compounds from PDH (Uridine, Ethyl-3,4-dihydroxy benzoate,
(+) – Pinoresinol, Daucosterol, Vomifoliol, 2-(Hydroxymethyl)−
M.B. Oppong et al.

Phytomedicine Plus 4 (2024) 100533
4
5‑hydroxy pyridine, E – Caffeic acid, 1-O-Caffeoyl-β-d-glucopyranoside,
1-(β-d-Ribofuranosyl)−1H-1,2,4,-triazole, Tiliroside (Kaempherol-3-O-
β−6’’-p-hydroxycoumaroylglucose), 3-Cinnamoyltribuloside, β-Adeno-
sine, 3,4-Dihydroxybenzoic acid (Protocatechuic acid), Falandin B, Z-
Caffeic acid, Murratetra C, Uracil, p‑hydroxy benzoic acid, 5-hydroxy-
methyl-3-furoic acid, β-Sitosterol, 2-Furoic acid) serially diluted from
0 to 100
μ
M were then added and incubated further for 24 h. MTT re-
agent was then added to each well and incubated at 37 ◦C for further for
2.5 h. The formazan crystals formed in each well were sonicated for 15
min in 150
μ
L DMSO. Finally, a microplate reader was used to estimate
absorbance at 490 nm (Huang et al., 2011). Dexamethasone was used as
the control drug. The cell inhibition percentage was estimated using the
formula;
Percentage cell inhibition =100 − {(At−Ab)/(Ac−Ab)} × 100
Where
A
t
=Absorbance of test compound
A
b
=Absorbance of blank
A
c
=Absorbance of control
2.11.2. Nitric oxide assay
RAW 264.7 macrophage cells were treated with test compounds or
dexamethasone as described in Section 2.11.1. 100
μ
L of Griess reagent
was added to 100
μ
L of the cell culture supernatant. The mixture was
incubated at room temperature for 10 min, and the absorbance
measured at 540 nm. NaNO
2
solutions with concentrations of 10, 20, 40,
60, 80, and 100
μ
M were prepared, and their corresponding absorbance
values at 540 nm were measured. A standard calibration curve of con-
centration vs absorbance was plotted. The concentrations of nitrite in the
treated RAW 264.7 cells were calculated using the standard calibration
curve (Huang et al., 2011).
2.12. Data processing, statistical analysis and molecular docking
Origin Lab software (2018) (OriginLab, USA) was used to analyze the
data. Experimental data were reported as the mean value ±SD. Differ-
ences between groups (One-way ANOVA) at a P<0.05 were considered
signicant.
Four inammatory targets namely, Cyclooxygenase-2 (COX-2, Uni-
ProtID: Q05769), Phospholipase A2 (PLA2, UniProtID: P00624),
Interleukin-1 receptor-associated kinase-4 (IRAK-4, UniProtID:
Q9NWZ3) and NF-κB–inducing kinase (NIK, UniProtID: Q99558) were
obtained from RSCB PDB. Where the targets were ligand bound, the
coordinates of the ligand were removed. The structures were cleaned
using Discovery Studio version 21.1.0 (BIOVIA, San Diego). The
following compounds showing high anti-inammatory activity, namely,
(+) – pinoresinol, tiliroside, Z-caffeic acid, and 3,4-dihydroxybenzoic
acid (protocatechuic acid) were virtually screened using Python Pre-
scription Virtual Screening tool (PyRx 0.8, AutoDock Vina module). The
interactions between the protein-ligand were analyzed using Discovery
Studio version 21.1.0 (BIOVIA, San Diego). As a positive control, the
binding interactions of known ligands for each of the four targets were
also evaluated.
3. Results
3.1. Effect of aqueous and ethanol extracts of PDH on xylene–induced ear
edema in mice
The mice in the negative control group showed signicant edema
after xylene application for the aqueous extracts. Compared with the
negative control group, the dexamethasone treated group signicantly
inhibited ear edema induced by xylene (
##
P <0.01), which showed that
the experimental model was appropriately designed. The low and me-
dium doses of PDH extract had no signicant inhibitory effect. However,
the high dose had a signicant inhibitory effect equivalent to dexa-
methasone (**P <0.01). The effects of the aqueous extracts are pre-
sented in Fig. 1a.
The ethanolic extracts also exhibited similar results as the aqueous
extract. All the tested doses of the ethanol extract inhibited ear edema in
mice induced by xylene. The low dose and high dose exhibited signi-
cant inhibition of edema comparable to the dexamethasone group (**P
<0.01). The medium dose, however, did not show signicant inhibition.
The effects of the ethanol extracts are presented in Fig. 1d.
Fig. 1. : a. Effect of dexamethasone and aqueous extracts of PDH on xylene-induced ear edema in mice. b. Effect of Pangdahai aqueous extract on carrageenan-
induced paw edema in rats. c. Effect of aqueous extracts of Pangdahai on vascular permeability induced by acetic acid. d. Effect of dexamethasone and ethanol
extracts of Pangdahai on xylene-induced ear edema in mice. e. Effect of Pangdahai ethanol extract on carrageenan-induced paw edema in rats. f. Effect of ethanol
extracts of Pangdahai on vascular permeability induced by acetic acid. ** and
##
Statistically signicant at P < 0.01, *** statistically signicant at P < 0.001.
M.B. Oppong et al.

Phytomedicine Plus 4 (2024) 100533
5
3.2. Effect of aqueous and ethanol extracts of PDH on carrageenan-
induced rat paw edema
With the aqueous extract, the rats in the negative control group
showed increasing paw edema at 1, 2, and 4 h, while the positive control
(Dexamethasone) group signicantly inhibited rat paw edema induced
by carrageenan (
##
P <0.01). This indicated the appropriateness of the
experimental design. All tested doses of PDH aqueous extract signi-
cantly inhibited rat paw edema induced by carrageenan at 1 h after
compared to the negative control group (**P <0.01), but the effect was
not obvious at 2 h. However, the high dose demonstrated a signicant
(**P <0.01) inhibitory effect compared with the negative control group,
and the effect was most obvious at 4 h. Nevertheless, the degree of this
inhibitory effect was lower than that of the dexamethasone group. The
paw edema/swelling index of the experimental groups is shown in
Fig. 1b.
The ethanolic extracts also exhibited similar results as the aqueous
extract. For all the tested doses, the ethanol extract exhibited some de-
gree of inhibition of rat paw edema induced by carrageenan. The low
and medium doses exhibited signicant inhibition of edema comparable
to the dexamethasone group (**P <0.01) at 1 h. The high dose, how-
ever, showed no signicant inhibitory effects. The paw edema/swelling
index of the experimental groups is shown in Fig. 1e.
3.3. Effect of aqueous and ethanol extract of PDH on acetic acid-induced
mice vascular permeability
The vascular permeability was measured by the OD which repre-
sented the amount of Evans blue exuded into the peritoneal cavity. In
both aqueous - and ethanol-treated groups, the OD values of the negative
control groups signicantly increased following treatment with acetic
acid. Compared with the model group, the aqueous extract exhibited a
non-signicant reduction in the OD values at all tested doses (Fig. 1c).
The ethanol extracts, however, demonstrated a signicant (**P <0.01)
but dose-independent reduction of the OD values (Fig. 1f).
Fig. 2. Pathological changes in mice ears treated with Pangdahai ethanol extract (HE staining, ×100).
M.B. Oppong et al.

Phytomedicine Plus 4 (2024) 100533
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3.4. Histo-pathological study of sections of mice edematous ear induced
by xylene
HE staining was used to observe and conrm the changes in the
inamed cells induced by xylene. The normal group showed normal
tissues. Compared with the normal group, the negative control group
exhibited high degree of swelling which was marked by blistering of the
epithelial and conjunctival tissues, red-stained mesh-like collagen bers,
and signicant inltrated inammatory cells. Contrary to the xylene
groups previously given dexamethasone (positive control group) or PDH
extracts (low and medium dose groups), there was a reduction in edema
(slight edema), amount of red-stained mesh-like collagen bers and
inltration of inammatory cells (Fig. 2). These results collectively
indicate that ethanol extracts of PDH inhibited xylene-induced ear
edema and inltration of inammatory cells.
3.5. Effect of PDH ethanol extract on the levels of inammatory cytokines
The levels of TNF-
α
and IL-1β were signicantly (p < 0.01) inhibited
by dexamethasone in both in vivo acute inammation models. The
ethanol extracts of PDH exhibited signicant (p < 0.01) dose -depen-
dent inhibition of the expression of TNF-
α
and IL-1β in both models (as
shown in Fig. 3a–d). These results indicate that the anti-inammatory
properties of PDH ethanol extract were cognate to the inhibition of
TNF-
α
and IL-1β.
3.6. In-vitro anti-inammatory effects of some isolated compounds from
PDH
The results show that all tested compounds showed inhibition of NO
production with no obvious cytotoxicity at 100
μ
M. Among these com-
pounds, (+) - pinoresinol, tiliroside, 3-cinnamoyltribuloside, 3,4-dihy-
droxybenzoic acid, Z-caffeic acid, and 2-furoic acid showed signicant
inhibition (P <0.05) of NO-production in LPS stimulated RAW cells with
percentage inhibitions greater than 70 %. The percentage inhibitions
and IC
50
are shown in Table 1.
3.7. Binding afnities of selected compounds
The binding interactions between the four COX-2, PLA2, IRAK-4 and
NIK, which are known mediators of anti-inammatory process and four
of the isolated compounds were evaluated. As a positive control, known
ligands of these targets were used positive control. COX-2 and PLA2
were evaluated with celecoxib and Niumic acid, respectively. IRAK-4
and NIK on the other hand were evaluated using 1-(3-Hydroxypropyl)-
2-[(3-Nitrobenzoyl) amino]-1h-Benzimidazol-5-Yl Pivalate and Cdk1/2
Inhibitor III, respectively. The results of these studies are shown in
Table 2.
4. Discussion
The current study ascertained and validated the traditional or folk-
loric use of PDH as an anti-inammatory agent. Thus, we report the anti-
inammatory properties of the aqueous and ethanol extracts and some
isolated secondary metabolites of PDH.
Data from this study showed that extracts of PDH (ethanol and
aqueous) exhibited an inhibitory effect on xylene-induced ear edema in
mice. The aqueous extracts demonstrated a dose–independent inhibi-
tion, while the low and high doses of ethanol extracts signicantly
inhibited ear edema in mice induced by xylene. Xylene induces acute
neurogenic edema (Singsai et al., 2020) and cause swelling by increasing
vasodilation and vascular permeability when applied (Zhao et al.,
2018).
Furthermore, this study showed that the extracts of PDH inhibited
paw edema in rats induced by carrageenan. Carrageenan induces edema
in two phases with several mediators including histamine, serotonin, 5-
hydroxytryptamine, prostaglandins, bradykinin, cyclooxygenase, TNF-
α
, IL-1 and IL-6 involved (Umare et al., 2014; Karim et al., 2019; Patil
et al., 2019).
This study suggests that the ethanol extracts markedly attenuated
acetic acid-induced vascular permeability in a dose-independent
manner (Fig. 1c and f). Acute inammation is characterized by vasodi-
latation, exudation of plasma, increase in vascular permeability, and
Fig. 3. a. Effect of Pangdahai ethanol extract on the levels of TNF-
α
in paw tissues of rats induced by carrageenan. b. Effect of Pangdahai ethanol extract on the levels
of IL-β in paw tissues of rats induced by carrageenan. c. Effect of Pangdahai ethanol extract on the levels of TNF-
α
in ear tissues of rats induced by xylene. d. Effect of
Pangdahai ethanol extract on the levels of IL-1β in ear tissues of rats induced by xylene.
##
Compared with blank group p < 0.01; *Compared with model group p <
0.05; **Compared with model group p < 0.01.
M.B. Oppong et al.

Phytomedicine Plus 4 (2024) 100533
7
neutrophil migration into the site of inammation (Chen et al., 2018).
Exudation is a direct consequence of increased vascular permeability. In
acetic acid-induced vascular permeability assay, acetic acid causes the
level of mediators such as prostaglandins, serotonin, and histamine in
peritoneal uids to increase consequently, resulting in dilation of the
capillary vessels and an increase in vascular permeability (Dantas et al.,
2020; Rezq et al., 2021).
Data from this study showed that the anti-inammatory effects of
PDH ethanol extract could be related to TNF-
α
and IL-1β inhibition.
Though several cytokines are involved in inammation (Delgado et al.,
2003), TNF-
α
is the most signicant cytokine associated with local
and/or systemic inammation (Cuzzocrea et al., 1999). TNF-
α
stimu-
lates T cells and macrophages. It elevates levels of kinins and leukotri-
enes (Yun et al., 2008; Huang et al., 2011).
One of the of aims of this work was to assess the anti-inammatory
potentials of some isolated compounds from PDH. This was achieved
by measuring the degree of inhibition of nitric oxide (NO) in RAW 264.7
macrophages treated with Lipopolysaccharides (LPS). LPS activates
macrophages to release cytokines and inammatory mediators. These
include NO, cyclooxygenase-2, TNF-
α
and IL-6 (Saadat et al., 2019). NO
plays a signicant role in regulating physiological responses including
inammation (Doulias and Tenopoulou, 2020), and is used as a
biomarker of inammation in many biological samples (Rana, 2020).
Therefore, the ability of a compound to inhibit the production of nitric
oxide in LPS-stimulated RAW cells is indicative of its anti-inammatory
potentials (Rana, 2020).
In addition, molecular docking studies performed using known me-
diators of the inammatory process, i.e. COX-2, PLA2, IRAK-4 and NIK
demonstrated that (+) – pinoresinol, tiliroside, Z-caffeic acid and 3,4-
dihydroxybenzoic acid demonstrated good binding afnities. This
further supports our assertion that these agents could be the components
responsible for the observed anti-inammatory activity. It is interesting
to note that amongst the top four compounds, tiliroside demonstrated
binding afnities comparable and in some cases higher (PLA2 and NIK)
that those of the positive control as shown in Table 2.
Previous anti-inammatory studies on some of the compounds iso-
lated from PDH have shown that these compounds have anti-
inammatory properties. Tiliroside is reported to signicantly inhibit
mouse paw edema induced by phospholipase A2 and mouse ear edema
inammation induced by TPA (Sala et al., 2023). The work of Correa
et al., 2018 and Luhata et al., 2017 have also demonstrated the
anti-inammatory effects of tiliroside. Several studies (both in vitro and
in vivo) have established that protocatechuic acid possess
anti-inammatory effects (Semaming et al., 2015; Kakkar and Bais,
2014; Song et al., 2020; Hu et al., 2020). Anti-inammatory effects of
caffeic acid is also well demonstrated. Caffeic Acid is reported to
signicantly inhibit pro-inammatory cytokines, downregulated mRNA
expression of IL-1β, IL-6, and TNF-
α
(Gamaro et al., 2011; Wan et al.,
2021; Ehtiati et al., 2023), lymphocytes, polymorphonuclear neutro-
phils and, macrophages (Morones et al., 2016). Pinoresinol is reported to
exert potent anti-inammatory effects (Jang et al., 2022). Studies con-
ducted by Jung et al., 2010 and During et al., 2012 have demonstrated
that pinoresinol signicantly inhibits NO, PGE(2), TNF-
α
, IL-1β and IL-6
and attenuates mRNA and protein levels of inducible nitric oxide syn-
thase (iNOS), cyclooxygenase-2 (COX-2) and proinammatory cyto-
kines in LPS-activation. It is believed to exhibit the strongest
anti-inammatory properties by acting on the NF-κB signaling
pathway (During et al., 2012).
5. Conclusions
The aqueous and ethanol extracts of PDH have signicant anti-
inammatory effects in the animals used. This study validates and
provides scientic evidence for the traditional use of the aqueous de-
coctions of PDH in the treatment of inammatory-related conditions
such as pharyngitis. Additionally, (+) – pinoresinol, tiliroside, 3-
Table 1
Inhibitory activity of the compounds from Pangdahai on LPS-induced NO release
in RAW 264.7 cells.
Compound Concentration/
µM
NO
inhibition
(%)
IC
50
/
µM
Uridine 100 46.53 >100
Ethyl-3,4-dihydroxy benzoate 100 32.29 >100
*(+) – Pinoresinol 100 84.68 16.36
±0.79 50 83.06
25 79.26
12.5 36.94
Daucosterol 100 36.84 >100
Vomifoliol 100 46.65 >100
2-(Hydroxymethyl)−5‑hydroxy
pyridine
100 43.61 >100
E – Caffeic acid 100 34.86 >100
1-O-Caffeoyl-β-d-glucopyranoside 100 14.49 >100
1-(β-d-Ribofuranosyl)−1H-1,2,4,-
triazole
100 47.11 >100
* Tiliroside (Kaempherol-3-O-
β−6
′
’-p-
hydroxycoumaroylglucose)
100 89.47 17.58
±1.05 50 85.55
25 77.69
12.5 31.04
*
#
3-Cinnamoyltribuloside 100 102.65 27.78
±1.58 50 77.13
25 46.63
12.5 0.66
β-Adenosine 100 65.55 56.57
±2.55 50 48.27
25 23.24
12.5 18.98
*3,4-Dihydroxybenzoic acid
(Protocatechuic acid)
100 100.79 18.55
±1.35 50 75.40
25 62.88
12.5 37.92
3,6-Dihydroxy-5,11-epoxy-7E-
magastimaen-9-one (Falandin B)
100 43.34 >100
*Z-Caffeic acid 100 57.03 30.34
±2.00 50 56.46
25 48.27
12.5 26.98
2-Methoxy-benzoyl-β-d-
glucopyranoside (Murratetra C)
100 33.34 >100
Uracil 100 51.20 94.85
±4.13 50 40.61
25 33.46
12.5 14.73
p‑hydroxy benzoic acid 100 40.96 >100
5-hydroxymethyl-3-furoic acid 100 25.64 >100
β-Sitosterol 100 19.46 >100
*
#
2-Furoic acid 100 101.25 25.49
±1.73 50 99.58
25 50.99
12.5 22.09
*Signicant inhibitory effect on NO release at (P <0.05).
#
Some degree of cytotoxicity.
Table 2
Binding afnities of selected compounds with mediators of inammation.
Ligands Binding Afnities (kcal/mol)
COX-2 PLA2 IRAK-4 NIK
3,4-dihydroxybenzoic acid -5.1 -5.5 -6.3 -5.4
Caffeic acid -5.8 -6.6 -6.6 -6.6
(+) – Pinoresinol -7.7 -5.8 -7.4 -6.5
Tiliroside -8.2 -9.1 -7.8 -9.6
Native Ligand -8.4
a
-8.4
b
-9.4
c
-9
d
Native Ligands:
a
Celecoxib;
b
Niumic acid;
c
1-(3-Hydroxypropyl)-2-[(3-Nitro-
benzoyl) amino]-1h-Benzimidazol-5-Yl Pivalate;
d
Cdk1/2 Inhibitor III.
Targets: COX-2 - Cyclooxygenase-2; PLA2 - Phospholipase A2; IRAK-4 -
Interleukin-1 Receptor-Associated Kinase-4; NIK - NF-κB–Inducing Kinase.
M.B. Oppong et al.

Phytomedicine Plus 4 (2024) 100533
8
cinnamoyltribuloside and 3,4-dihydroxybenzoic acid (protocatechuic
acid) isolated from PDH showed in vitro anti-inammatory activities
supported by molecular docking studies, which could be the anti-
inammatory constituents of PDH.
Funding
This work was supported by the National Key Research and Devel-
opment Program of China (2019YFC1711000).
CRediT authorship contribution statement
Mahmood B. Oppong: Investigation, Formal analysis, Data cura-
tion, Writing – original draft. Shijie Cao: Investigation, Formal analysis,
Data curation. Shi-Ming Fang: Supervision. Seth K. Amponsah:
Writing – review & editing, Formal analysis, Data curation. Paul O.
Donkor: Writing – original draft, Formal analysis, Data curation.
Michael Lartey: Writing – review & editing, Formal analysis, Data
curation. Lawrence A. Adutwum: Writing – review & editing, Investi-
gation, Formal analysis, Data curation. Kwabena F.M. Opuni: Writing –
review & editing, Formal analysis, Data curation. Feng Zhao: Writing –
review & editing, Validation, Supervision, Methodology. Qiu Feng:
Supervision, Conceptualization, Project administration, Funding
acquisition.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
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