Journal of Ethnopharmacology 287 (2022) 114919

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Syzygium cumini (L.) Skeels extracts; in vivo anti-nociceptive,

anti-inflammatory, acute and subacute toxicity assessment
Muhammad Qamar a, Saeed Akhtar a, Tariq Ismail a, Muqeet Wahid b, Sajed Ali c, Yasir Nazir d,
Shahid Murtaza e, Malik Waseem Abbas f, Zyta M. Ziora g, *
a
Institute of Food Science and Nutrition, Bahauddin Zakariya University, Multan, 60800, Pakistan
b
Department of Pharmacy, Bahauddin Zakariya University, Multan, 60800, Pakistan
c
Department of Biotechnology, University of Management and Technology, Sialkot, Pakistan
d
Faculty of Sciences, Department of Chemistry, University of Sialkot, Sialkot, 51300, Pakistan
e
Center of Excellence in Molecular Biology, 87-West Canal Bank Road, Thokar Niazbaig, University of the Punjab, Lahore, Pakistan
f
Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, 60800, Pakistan
g
Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia

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

Keywords: Ethnopharmacological relevance: Syzygium cumini (L.) Skeels has been extensively used in the ancient medical
Syzygium cumini system of Pakistan, India, Bangladesh, and Sri Lanka to combat diabetes, inflammation, and renal disorders.
Inflammation These health-promoting aspects of S. cumini are related to bioactive metabolites such as phenolic acids, antho­
Toxicity
cyanins, tannins, and flavonoids.
Hematology
Histopathology
Aim of the study: Earlier to this study, we have reported S. cumini extracts as potential sources of bioactive
Serum biochemistry compounds bearing antioxidant and anti-inflammatory properties. However, prior further suggesting S. cumini
HPLC fruit extracts for consumption against inflammatory disorders, it was mandatory to validate the claim and
Umbelliferone explore toxicity of the extracts. This study aims to determine the in vivo anti-nociceptive, anti-inflammatory,
Scopoletin acute, and subacute toxicity properties of S. cumini crude extracts, followed by identifying and quantifying
bioactive metabolites.
Material and methods: In the present study, the anti-nociceptive and anti-inflammatory potential of S. cumini
sequential crude extracts were evaluated using formalin and glutamate-induced paw licking method in mice. The
acute and sub-acute toxicity assessment of active extract was performed by oral administration in rats. An acute
toxicity trial was performed with two different doses, i.e., 2000 mg/kg and 3000 mg/kg for consecutive 14 days,
whereas a sub-acute toxicity study was conducted at doses of 750 mg/kg and 1500 mg/kg for the next 28 days.
Identification of bioactive compounds was performed using HPLC, and at the end, in silico docking calculations of
identified compounds were performed.
Results: The 100% methanolic extract (SCME) protected the mice from painful stimulation of formalin and
glutamate in a dose-dependent manner with the maximum effect of 49% and 67% at 200 mg/kg, respectively,
followed by moderate and non-influential effects of 50% methanolic extract and dichloromethane (DCM) extracts
when compared to control, i.e., normal saline. The results of acute toxicity recorded LD50 of SCME over 3000 mg/
kg, and no antagonistic effects were recorded during the subacute study when SCME dispensed at the rate of 750
mg/kg and 1500 mg/kg. SCME was found to induce no adverse effects to kidney, heart, liver, spleen, and paired
lungs examined by hematological, serum biochemical, histological analysis. HPLC analysis of S. cumini 100%
methanolic extracts revealed the presence of delphinidin 3-glucoside, peonidin-3,5-diglucoside, scopoletin, and
umbelliferone at the concentration of 127.4, 2104, 31.3, 10.4 μg/g whereas in 50% methanolic extract, the
quinic acid, catechin, and myricetin were present at the concentration of 54.9, 63.7, 12.3 μg/g, respectively.
Umbelliferone and scopoletin are newly reported compounds in the present study. In silico docking calculations
of these compounds indicated the potential of anti-nociceptive and anti-inflammatory activities.
Conclusions: These findings validate that S. cumini fruit extracts are a rich source of bioactive compounds that
needs to be considered to enhance biological activities with lesser side effects.

* Corresponding author.
E-mail address: z.ziora@uq.edu.au (Z.M. Ziora).

https://doi.org/10.1016/j.jep.2021.114919
Received 21 July 2021; Received in revised form 5 December 2021; Accepted 13 December 2021
Available online 5 January 2022
0378-8741/© 2021 Published by Elsevier B.V.
M. Qamar et al. Journal of Ethnopharmacology 287 (2022) 114919

treat gastric problems (Sharma et al., 2001).

Abbreviations list S. cumini leaves were also effective against renal problems, indiges­
tion, and hyperglycemia (de Albuquerque et al., 2007). S. cumini seeds
HPLC High performance liquid chromatography were regarded as an effective therapeutic agent to control hyperglyce­
COX-2 Cyclooxygenase 2 mia (Ratsimamanga, 1998), while S. cumini bark has been used to cure
LOX-5 Lipoxygenase 5 intestinal inflammation, hyperglycemia and relieve headaches. Also,
TRPV1 Transient receptor potential cation channel subfamily V bark juice was utilized in people medication for averting inflammation
member (Sudarsanam et al., 1995), and females with a history of repeating
DCM Dichloromethane miscarriages were also treated with bark juice (Chhetri et al., 2005).
OECD Organization for Economic Cooperation and S. cumini anatomical parts, including seed, pulp, skin, bark, and leaves
Development are reported for their radical scavenging ability (Zhang and Lin., 2009),
mg/kg milligram/kilogram anti-inflammatory properties (Modi et al., 2010), cytotoxic potential
LD50 Lethal dose (Aqil et al., 2012), and hypoglycemic effects (Ayyanar and Babu, 2012).
EDTA Ethylenediaminetetraacetic acid Literature is also available to support its chemopreventive, car­
RP-HPLC Reverse-phase high performance liquid dioprotective, antipyretic, hepatoprotective, and chemopreventive po­
chromatography tential (Das and Sarma, 2009; Syama et al., 2017; Arun et al., 2011;
PROPKA Predicts pKa values of ionizable groups in proteins Muruganandan et al., 2001).
ANOVA analysis of variance The present study was devised to assess in vivo anti-inflammatory
SCME Syzygium cumini methanolic extract potential of S. cumini fruit crude extracts. The study also emphasized
WBC White blood cells safety assessment from edible part of S. cumini 100% methanolic extract
RBC Red blood cell performing acute and subacute toxicity trails by oral administration
MCH Mean corpuscular hemoglobin using rat models followed by HPLC identification of bioactive com­
MCHC Mean corpuscular hemoglobin concentration pounds. In addition, in silico studies were also performed on HPLC
TFA Triflouroacetic acid identified bioactive compounds in S. cumini to predict ligand-protein
PDB Protein Data Bank interaction within the binding site of target inflammatory proteins
(cyclooxygenase 2, lipoxygenase 5 and transient receptor potential
cation channel subfamily V member 1 (TRPV1 receptor). The involve­
ment of physical energies terms (i.e. solvation energy) with suitable
1. Introduction force field, make docking calculations of isolated compounds from
S. cumini more acceptable with accuracy (Kuhn and Kollman, 2000;
Chronic inflammation is one of the most common disorder affecting Sirous et al., 2019). Molecular docking is a helpful tool to predict
millions of people around the globe (Rodriguez and Davoudian, 2016). possible mechanism of actions of the selected compounds of various
This statistic has diverted research to develop synthetic drugs; however, pharmacological studies. In present study, it was used to correlate and
the cost and safety issues, such as severe gastric lesions, digestive sys­ define anti-nociceptive and anti-inflammatory activities of identified
tem’s health, liver and kidney disorders are of significant concern compounds in S. cumini fruit extracts, and their interactions with target
(Kazemi et al., 2018). Consequently, inexpensive and nontoxic agents inflammatory proteins (cyclooxygenase 2, lipoxygenase 5 and transient
are required for treating inflammatory disorders. Bioactive metabolites receptor potential cation channel subfamily V member 1 (TRPV1)
present in many fruits and vegetables represent a safer, cost-effective, receptor.
and readily available alternative to synthetic drugs (Stankovic et al.,
2016). Plant-based products have been documented for 2. Material and methods
health-promoting properties since the start of human civilization (Cragg
and Newman, 2005). Bioactive compounds like anthocyanins, phenolic 2.1. Fruits collection and extraction
acids, flavonoids, and tannins have excellent anti-inflammatory and
antidepressant properties (Shah, Parveen and Khan, 2017). Conversely, S. cumini fruits were purchased from the local market of Pakistan in
there are only a few reports about the toxicity of plant-based natural April 2018, and a reference sample (1091/SC) was deposited at the
products that may attract the local communities owing to their safe, Herbarium located at the Institute of Food Science and Nutrition,
readily available, and cost-effective profile (Debjit et al., 2009). Bahauddin Zakariya University, Multan, Pakistan. Fruits were washed
Nevertheless, detailed scientific investigations on safety, with tap water with manual deseeding. Firstly, the pulp was dried under
cost-effectiveness, quality, and efficacy are the obligatory prior shade and then placed in an oven at 37 ◦ C. Dried pulp was ground into a
comprehensive application of plant-based products (Thelingwani and fine powder and stored in a cool place for extraction purpose. The ex­
Masimirembwa, 2014). tracts were obtained after successive extraction with n-hexane, DCM,
Syzygium cumini (L.) Skeels (Synonym; Eugenia jambolana Lam. or methanol, 50% methanol (50:50 v/v H2O: MeOH) and concentrated
Syzygium jambolana Dc or Eugenia cumini Druce), commonly known as under reduced pressure (800 mbars) in a rotary evaporator (Heidolph,
Jamun, Jambul, and black plum, belongs to the family Myrtaceae is Hei-Vap, Germany). Concentrated extracts were stored at -70 ◦ C in an
cultivated in various countries mainly in Pakistan, India, Indonesia, ultralow freezer (Sanyo, MDF-U32V, Japan) for further analysis.
Afghanistan, and Myanmar (Singh et al., 2015). S. cumini was considered
as an integral component of numerous ancient remedial systems inclu­
sive of Siddha, Tibetan, Unani, Sri Lankan, and Ayurveda to potentially 2.2. Solvents and reagents
treat diarrhea, menstrual disorders, obesity, hemorrhage, and vaginal
discharge (Ayyanar and Babu, 2012). Traditionally, hot water extract of Anthocyanins (peonidin-3,5-diglucoside, delphinidin 3-glucoside),
dried fruits is used to reduce stomach inflammation (Deka et al., 1983). phenolic acids (quinic acid, gallic acid), and flavonoids (umbellifer­
One glass of S. cumini fruit juice with half a teaspoon of stem bark one, catechin, myricetin, scopoletin) were supplied by the local vendor
powder is given daily to get relive from urinary tract inflammation of Sigma-Aldrich, USA. HPLC solvents (trifluoroacetic acid, methanol,
(Burkill, 1935). In addition, a blend of S. cumini leaves and cinnamon is water), inflammatory agents (formalin, glutamate), and anti-
considered beneficial against diarrhea in children (Nadkarni, 1976). The inflammatory agent (Indomethacin) were also purchased from the
juice of ripe fruits is stored for three days and administrated orally to same supplier.

2
M. Qamar et al.
2.3. Murine models
Swiss albino mice weighing 25–30 g and Sprague Dawley rats were
obtained from the Department of Pharmacy, Bahauddin Zakariya Uni­
versity, Multan (BZU). The animals (4 per cage) were kept in the animal
rearing facility, Institute of Food Science and Nutrition, BZU; under
normal temperature and specified laboratory conditions with ample
access to food and water. Animal trials were conducted by following the
guidelines set by the Institute of Laboratory Animal Resources, Com­
mission on Life Sciences, National Research Council (NRC, 1996),
Washington, USA. The Bioethical Committee of IFSN approved the
animal-use protocol entitled “In vivo anti-inflammatory activity of
Syzygium cumini fruit extracts” (approved protocol no. AEC-08-19).
2.4. Anti-inflammatory and anti-nociceptive potential
2.4.1. Formalin-induced paw licking
The formalin-induce paw licking method of Hunskaar and Hole
(1987) was adopted with slight modifications to determine the
anti-nociception and anti-inflammatory activity of S. cumini fruit ex­
tracts. Forty Swiss albino mice of both genders were divided into eight
groups (5/group i.e. control group that received normal saline), Indo­
methacin group (100 mg/kg), DCM, methanol, and 50% methanol
(treated with 100 and 200 mg/kg extract’s doses) (Fig. 1 A). After 30
min of intraperitoneal administration of test sample, 250 μL of 2.5%
formalin solution was injected into plantar aponeurosis surface of the
right hind paw of each mice and licking responses of animal was
observed at early neurogenic pain phase after 0–5 min and later
anti-inflammatory pain stage after 20–25 min.
2.4.2. Glutamate-induced hind paw licking in mice
The glutamate-induced paw licking described by Beirith et al. (2002)
was adopted with slight modifications to determine the anti-nociception
and anti-inflammatory activity of S. cumini fruit extracts. Forty Swiss
albino mice of both genders were divided into eight groups; control
(received the normal saline), Indomethacin (100 mg/kg), and 100
mg/kg and 200 mg/kg of respective DCM, methanol, 50% methanol
extracts (Fig. 1 B). After 30 min of intraperitoneal administration of the
test sample, 20 μL of glutamate (10 μmol/paw) was injected into the
Fig. 1. The anti-nociceptive and anti-inflammatory effect of the S. cumini on mice hind paw by Formalin (A) and Glutamate (B) induces method. Values are
expressed as Mean ± S.E.M, Data was analyzed by one-way ANOVA (Dunnett’s test) when compared to Control, and p < 0.05 was considered significant (*p < 0.05,
**p < 0.01, ***p < 0.001, ****p < 0.0001).
3
Journal of Ethnopharmacology 287 (2022) 114919
plantar aponeurosis surface of the right hind paw of each mice, and
licking responses were observed after 0–15 min as a sign of the time of
nociception.
2.5. Toxicity assessment tests
2.5.1. Acute toxicity (14-days study)
In the current study, rats were used conferring to the guidelines 407
and 423 of the Organization for Economic Cooperation and Develop­
ment (OECD) for acute oral toxicity tests (OECD 407, 2008; OECD 423,
2001). The rats were divided into two groups (n = 10), with 5 males and
5 females in each group. The animals were fasted for 12 h and weighed
before administering S. cumini 100% methanolic extract but provided
with easy access to water. Control (Group 1) received the normal saline
while the treated group received a dose of S. cumini 100% methanolic
extract 2000 mg/kg (Group 2) and 3000 mg/kg (Group 3). The animals
were then examined after the first half hour and 1 h. Then, the exami­
nation was performed after every hour for the next 5 h and finally
periodically for up to 48 h for behavioral changes, symptoms of acute
toxicity, and mortality for the first 30 min. Each animal was examined
daily and observed for changes in behavior and weight, toxicity signs,
and death for 14 days after treatment. The lethal dose (LD50) describes a
kill of 50% of the population, otherwise study animals should receive
greater than 3000 mg/kg of a dose of S. cumini 100% methanolic extract
if five or more rats survive. Animals were weighed and sacrificed by
dislocating the cervical, whereas organs were removed for necropsy at
the end of the experiment.
2.5.2. Sub toxicity (28-day study)
The experiment was performed following the OECD guidelines 407
programme (OECD 407, 2008) after slight changes. Thirty animals were
taken for the study, weighed, observed warily for general appearance
and behavior, and were divided into three groups wherein each group
had 10 animals of both sexes (n = 10; 5 males and 5 females). The
S. cumini 100% methanolic extract was dissolved in normal saline and
given orally for 28 consecutive days. Control (Group 1) was given
normal saline; Group 2 was administered with S. cumini extracts at the
rate of 750 mg/kg; Group 3 was administered with S. cumini extract at
the rate of 1500 mg/kg. The experimental rats were examined on a
M. Qamar et al.
routine basis for any change in the fur of the skin, posture, eyes,
behavior, mucous membrane, and mortality. After 28 days of S. cumini
extract administration, rats were fasted for 14 h but provided with
water. On the 29th day, rats were anesthetized with ether to take blood
samples to perform the hematological and biochemical examinations.
Subsequently, rats were sacrificed by cervical dislocation, different or­
gans, including heart, kidney, liver, spleen, and lungs were collected,
instantly washed with normal saline, studied macroscopically after
weighing individual rats, and the relative weight of the organs was
calculated.
2.5.3. Hematological and biochemical analysis
The blood samples collected earlier in EDTA tubes were subjected to
hematological examination employing an automated hematology
analyzer (ABX Pentra XL 80, France). After hematological staining, a
differential count of leukocytes was performed using light microscopy
(Optika, Italy). Likewise, blood samples collected in anticoagulant-free
tubes were centrifuged (3000 rpm) for 15 min, sera were separated
and then stored at -18 ◦ C for biochemical analysis using URIT 8021A
automated analyzer (URIT Medical Electronic Group Co., Ltd.).
2.5.4. Histological examination
Tissue samples of the heart, liver, and kidney were taken and fixed in
formalin (phosphate buffer; 10%; pH 7.0) for one day. After that, the
tissue samples were set in paraffin wax, stained with hematoxylin and
eosin (H&E) after cutting into sections of 2–3 μm. Olympus SP 350
digital camera and Stream Basic imaging software were used to take
photomicrographs (Olympus Corporation, Tokyo, Japan).
2.6. HPLC quantification of bioactive compounds
2.6.1. Method optimization
100 and 50% methanolic extracts were subjected to reverse-phase
high-performance liquid chromatography (RP-HPLC) by dissolving so­
lidified extracts in methanol (Cock, 2011). Separation was performed
using Agilent LC technology (C-18, 4.6 × 150 mm, 5 μm, Agilent, Ger­
many) and the column was maintained at 25 ◦ C. Two different gradient
elution systems were used to obtain a maximum separation: First con­
taining water (A) and methanol (B), both acidified with 0.1% TFA, and
with 53- and 50-min run time; Second containing water (A) and meth­
anol (B), both acidified with 0.1% FA, and with 40- and 60-min run time.
The detection was performed at different wavelengths, 210, 254, 380,
300, 330 nm, considering the nature of plant polyphenols absorbing at
varying wavelengths. The identification was performed by using
external standards and comparing the UV spectra with their known
retention time.
2.6.2. S. cumini sample preparation
100 and 50% methanolic S. cumini solidified extracts were dissolved
into 1 mL methanol to quantify the compounds. After that, samples were
centrifuged at 14000 rpm for 10 min. The supernatant was collected and
filtered using a syringe filter. 100 μL sample was injected into the HPLC
system for analysis.
2.7. In silico studies
2.7.1. Ligand preparation
The 2D structures compounds quantified on HPLC and standard anti-
inflammatory drug (indomethacin) were retrieved from PubChem (htt
ps://pubchem.ncbi.nlm.nih.gov) and prepared by LigPrep
(Schrödinger) in their neutral form and their conformation optimized in
the OPLS-3 force field. The prepared structures were employed for the
further grid and docking analysis.
2.7.2. Protein preparation
Molecular docking simulations were performed to determine
4
Journal of Ethnopharmacology 287 (2022) 114919
possible binding orientations of the identified compounds in the crystal
structure of inflammatory proteins (Cyclooxygenase 2, Lipoxygenase 5
and TRPV1 receptor). For this purpose, the highest resolution X-ray
structures (PDB ID: 5IKQ, 6N2W, and 3SUI) of target inflammatory
proteins were downloaded from the Protein Databank (RCSB PDB) (htt
ps://www.rcsb.org/) and prepared using the “Protein Preparation
Wizard” workflow in maestro Schrödinger for adding hydrogens and
setting protonation’s states appropriate for pH 7. The bond orders were
assigned in this module, and hydrogen atoms were added to the protein
molecule. The water molecules beyond 5Å were removed from the
protein structure followed by processing and optimization by using
PROPKA at pH 7.0, and OPLS3e force field was utilized to perform
restrained minimization for energy minimization and geometry opti­
mization of protein structure.
2.7.3. Molecular docking and receptor grid generation
For grid generation preparation, the active site of the target in­
flammatory protein is defined from the co-crystallized ligands from
Protein Data Bank and literature data (Sirous et al., 2019). Grid was
generated by specifying the residues involved in the catalytic site of
target protein. The receptor grid box was defined as 16 Å box. The
default docking setup parameters were employed for ligand docking
experiment (Friesner et al., 2004). Docking was performed with Glide
(Schrödinger) using XP-precision with default settings and glide scoring
function, reporting the 10 top-ranked poses for each ligand. The pre­
dicted binding energies (docking scores) and proper orientation of li­
gands isolated from S. cumini within the active region of target proteins
were also performed using Glide dock. XP. Finally, to predict the most
favorable binding mode of active compounds inside the active site of
proteins with suitable orientation in terms of docking score/binding
energy, visual inspection and 3D graphical images of the binding pose
and generation of figure of the best-scored docking complex was also
done with Maestro (Schrödinger).
2.8. Statistical analysis
Data derived from this study are expressed as mean ± Standard error
of means (SEM) for three measurements. Statistical differences between
the control and treatments were analyzed by analysis of variance
(ANOVA), Dunnett’s test using graph pad prism and p < 0.05 was
considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p <
0.0001).
3. Results and discussion
3.1. Formalin and glutamate-induced paw licking
SCME caused significant inhibition of 47.9% during 0–5 min (P <
0.001) and 67.3% during 15–30 min (P < 0.0001) at 200 mg/kg against
formalin induced paw licking in neurogenic and inflammation phase,
respectively when compared to control (normal saline), evident from
Fig. 1. A. It can be stated that SCME blocked the nociceptors, including
bradykinins, glutamate, and substances P, that are produced during the
first 5 min of formalin intoxication (Hunskaar and Hole, 1987). The
inhibition properties possessed by SCME during the second phase could
suppress the generation of prostaglandins, serotonin, bradykinins, and
histamine known as inflammatory agents (Tjolson et al., 1992). Earlier,
bark extract of S. cumini was reported to cause inhibition of 51.61%,
28.6%, and 28.6% against histamine, PGE2, and 5-HT induced paw
edema in rats when dispensed at the rate of 300 mg/kg (Muruganandan
et al., 2001). In another study, Muruganandan et al. (2001) found that
S. cumini bark extract at all administered dose levels (100, 300, 1000
mg/kg, p. o.) exhibited notable inhibition of formaldehyde induced hind
paw edema, further supporting the findings of the current investigation.
In contrast, S. cumini 50% methanolic and dichloromethane extracts
were found to exert moderate (35% at 200 mg/kg) to non-influential
M. Qamar et al. Journal of Ethnopharmacology 287 (2022) 114919

(16% at 200 mg/kg) inhibition against formalin induced paw licking as
compared to control. Indomethacin (standard drug) induced inhibition
of 84% at 100 mg/kg deliberated as notable (P < 0.0001)
anti-nociceptive and anti-inflammatory activity.
Glutamate plays a considerable role as an excitatory neurotrans­
mitter in charge of nociceptive signals and the activation of nociceptive
neurons. This mediation causes the release of various pain mediators
and neuropeptides, which might be intricated in nociceptive trans­
mission, both in the central and peripheral nervous system (Millan,
1999; Fundytus, 2001). The 100 and 50% methanolic extracts (200
mg/kg) significantly reduced painful stimulation of glutamate in a
concentration-dependent manner with a maximum effect of 67.17% (P
< 0.0001) and 57.77% (P < 0.0001) during 0–15 min at 200 mg/kg
respectively, as compared to control, a similar effect of the standard
drug (Indomethacin; 72.07% inhibition) and produced statistically sig­
nificant (P < 0.0001) anti-analgesic activity (Fig. 1. B). The activity
could be due to the presence of flavonoids, phenolic acids, anthocyanins,
and tannins reported to be present in S. cumini fruit (Qamar et al., 2021).
The findings are in agreement with the Quintans et al. (2014), wherein
S. cumini leaves hydromethanolic extract induced notable inhibition
against pain mediation by formalin and glutamate. SCME, even at a
higher dose of 200 mg/kg, did not exhibit the same inhibition as stan­
dard Indomethacin at a dosage of 100 mg/kg. The reasonable justifi­
cation could be that extract complexity with various compounds in the
mixture may enhance the activity, but occasionally, activity may decline
due to some antagonism occurring among the compounds (Chandran
et al., 2020).
3.2. Toxicity assessment of S. cumini methanolic extract (acute and
subacute)
As S. cumini methanolic extract demonstrated substantial anti-
nociceptive and anti-inflammatory activities was further subjected to
toxicity assessment. The analysis revealed no toxicity, behavioral
changes, and mortality among animals when S. cumini methanolic
extract was dispensed at the rate of 2000 and 3000 mg/kg during a 14-
day trial (Table 1A). There was no visible change in the body weight of
control and treated rats at the end of the trial. The weight of the heart,
kidney, lungs, spleen, and liver was also found within the normal range.
Moreover, no death was recorded throughout the 14-day trial; therefore,
it is inferred that LD50 of S. cumini methanolic extract was above 3000
mg/kg. S. cumini seed extract was reported to exert no mortality among
experimental rats when administered at 2000 mg/kg for 14 days during
acute toxicity studies (Kumar et al., 2007). The present investigation
findings are in alliance with Muruganandan et al. (2001), who reported
S. cumini bark extract administered at the rate of 10.12 g/kg with no
toxicity or morality observed among animals. Likewise, administration
of SCME at 750 mg/kg and 1500 mg/kg resulted in no mortality in the
subacute toxicity test throughout the 28 days. Moreover, no substantial
change in the behaviour of treated rats was observed as compared to rats
of the control group (received normal saline). The findings in Table 1A
showed no significant change in body weight in rats treated with 750
mg/kg and 1500 mg/kg SCME and rats of the control group (receiving
normal saline). The weight of the heart, kidney, paired lungs, spleen,
and liver of treated animals was found in accordance with the animals of
the control group. Previously, administration of hydro-alcoholic extract
of S. cumini leaves at a dose of 0.05 g/kg, 0.1 g/kg, 0.25 g/kg daily for

Table 1A
Acute and subacute toxicity assessment of Syzygium cumini 100% methanolic extract in rats.
Parameters Control group Acute toxicity (14 Days) Subacute toxicity (28 days)

Normal saline 2000 mg/kg SCME 3000 mg/kg SCME 750 mg/kg SCME 1500 mg/kg SCME

Body weight (g) 200 ± 5 195 ± 8 215 ± 8 190 ± 5 210 ± 8

Organ Weight
Heart (g) 0.62 ± 0.02 0.57 ± 0.2 0.63 ± 0.1 0.57 ± 0.05 0.63 ± 0.02
Paired Lungs (g) 2.1 ± 0.1 2.21 ± 1.2 2.44 ± 1.1 1.93 ± 0.2 2.36 ± 0.1
Liver (g) 7.8 ± 1 7.9 ± 0.1 8.7 ± 2.7 7.36 ± 0.8 8.5 ± 0.5
Spleen (g) 0.46 ± 0.2 0.44 ± 0.02 0.49 ± 0.03 0.41 ± 0.03 0.49 ± 0.03
Hematological Parameters
WBC’s (105/μL) 2.5 ± 0.1 2.9 ± 0.03 3.1 ± 0.01 3.2 ± 0.03 3.6 ± 0.02
Neutrophils (%) 40 ± 5 40.1 ± 3 39.8 ± 3 40.1 ± 7 40.3 ± 2
Lymphocytes (%) 45 ± 8 45.4 ± 4 46.7 ± 4.6 46.1 ± 5 44.8 ± 6
Eosinophils (%) 1 ± 0.5 0.91 ± 0.1 0.94 ± 0.1 0.92 ± 0.03 0.96 ± 0.01
RBC’s (106/μL) 8.6 ± 2 9.9 ± 2.1 9.5 ± 2.1 9.0 ± 0.7 9.1 ± 0.3
Hemoglobin (g/dl) 13 ± 2 14.7 ± 3.1 15.4 ± 1.8 14.2 ± 1 14.9 ± 1
Hematocrit (%) 45 ± 1 44.1 ± 4.2 45.8 ± 3.2 46.3 ± 3 44.9 ± 4
Mean corpuscular volume (MCV (f/L) 55.8 ± 10 56.2 ± 8.7 57.9 ± 6.3 56.3 ± 5 58.9 ± 5
Mean corpuscular hemoglobin (MCH (pg) 17.7 ± 2 17.4 ± 2.2 18.2 ± 1.1 17.2 ± 1 17.8 ± 1
MCHC (%) 31.6 ± 3 29.1 ± 0.9 30.9 ± 2.1 28.7 ± 2 30.2 ± 2
Platelets(105/μL) 5.8 ± 1 7.1 ± 1.1 6.4 ± 1.1 6.8 ± 0.2 6.5 ± 0.3
Serum Biological Parameters
Total Protein (g/dl) 3.8 ± 2 4.2 ± 1.1 3.97 ± 0.2 4.1 ± 0.3 3.99 ± 0.2
Albumin (g/dl) 1.8 ± 0.5 2.3 ± 0.4 2.0 ± 0.3 1.9 ± 0.1 1.86 ± 0.3
Albumin/Globulin ratio 2.1 ± 0.01 2.8 ± 0.5 2.5 ± 0.02 2.4 ± 0.02 2.2 ± 0.03
Lactate Dehydrogenase (U/L) 1546 ± 350 2145 ± 221 2032 ± 321 1849 ± 200 1615 ± 235
Asparate Transaminase (U/L) 98.2 ± 12 122 ± 11 128 ± 9.2 110.3 ± 16 112.3 ± 13
Alanine Transaminase (U/L) 25.5 ± 8 33.2 ± 7 36.4 ± 4 28.9 ± 5 30.8 ± 4
Alkaline Phosphatase (U/L) 280 ± 22 291 ± 13 293 ± 17 282 ± 20 286 ± 18
Total bilirubin (mg/dl) 0.2 ± 0.02 0.28 ± 0.02 0.32 ± 0.1 0.3 ± 0.01 0.2 ± 0.01
Creatinine (mg/dl) 1.3 ± 0.1 1.55 ± 0.03 1.68 ± 0.1 1.4 ± 0.5 1.45 ± 0.4
Uric Acid (mg/dl) 0.7 ± 0.03 1.07 ± 0.02 1.2 ± 0.1 0.9 ± 0.01 1.02 ± 0.01
Total Cholesterol (mg/dl) 57 ± 10 54.2 ± 4.6 53.1 ± 5.4 53.9 ± 2 53.5 ± 5
Triglycerides (mg/dl) 121 ± 20 117.9 ± 14.5 116 ± 4.3 118 ± 5 115.6 ± 17
Sodium (mmol/L) 107 ± 20 109 ± 17 120 ± 19 106 ± 10 110 ± 12
Chloride (mmol/L) 65 ± 25 81 ± 19 90 ± 21 70 ± 6 73 ± 10
Potassium (mmol/L) 2.8 ± 0.1 3.3 ± 0.02 3.5 ± 0.1 3.1 ± 0.3 3.4 ± 0.5

Table 1A: Values are expressed as Mean ± S.E.M, Data was analyzed by one-way ANOVA (Dunnett’s test) when compared to control, and p < 0.05 was considered
significant. (*p < 0.05, **p < 0.005). SCME, Syzygium cumini methanolic extract.

5
M. Qamar et al.
consecutive 30, 90, and 180 days in chronic toxicity model didn’t cause
any behavioural change, body weight change, and resulted with no
mortality (Silva et al., 2012), are in coherent with the findings of current
investigation.
3.3. Hematological parameters
The haematological parameters for acute and sub-acute studies are
presented in Table 1A. The rats received 2000 mg/kg and 3000 mg/kg of
SCME for 14 days (acute toxicity) resulted in a slight increase in the
haematological parameters, but lying within normal range, therefore it
is inferred that LD50 of S. cumini methanolic extract was above 3000 mg/
kg. Whereas oral administration of S. cumini methanolic extract for 28
days (subacute) did not disturb the haematological parameters. White
blood cells (WBC) slightly increases to 3.2 ± 0.03 (105/μL) and 3.6 ±
0.02 (105/μL) at SCME doses of 750 mg/kg and 1500 mg/kg, respec­
tively as compared to normal rat with WBC count of 2.5 ± 0.1 (105/μL).
Red blood cell (RBC) counts, platelets, and haemoglobin levels also
increased in both doses, indicative of erythropoietic potency of S. cumini.
The mean corpuscular hemoglobin (MCH) and mean corpuscular hae­
moglobin concentration (MCHC) values in rats at both doses of SCME
remained the same compared to normal rats.
Fig. 2. Histological examination after acute and subacute toxicity.
6
Journal of Ethnopharmacology 287 (2022) 114919
3.3.1. Serum biochemistry analysis
The serum profile for acute and sub-acute studies are presented in
Table 1A. After bolus oral administration of 2000 and 3000 mg/kg, no
difference was recorded between normal and treated rats. This indicated
that the higher bolus doses of SCME had no adverse effect on liver and
kidney. The liver parameters: albumin, albumin/globulin ratio, lactate
dehydrogenase, aspartate transaminase, alanine transaminase, alkaline
phosphatase and total bilirubin levels were high. The results of liver
parameters thus indicate no toxic effects of SCME on liver tissues in
acute and sub-acute studies. Also, kidney parameters, i.e., creatinine and
uric acid, showed the same behavior as liver. Neglectable rises in
creatinine level was recorded which may indicate presence of some
histological disturbance in kidney. Similarly, lipid profiling of treated
rats slightly decreased, describing the impact of SCME on body meta­
bolism. In sub-acute toxicity study, serum parameters had no difference
among normal and treated rats. Hence, consecutive oral administration
of S. cumini methanolic extract for 28 days had no toxic impact on liver
and kidney, whilst decrease in lipid profile in same manner as acute
toxicity.
3.3.2. Histopathological analysis
Histological sections of heart, liver, spleen and kidney were analyzed
(Fig. 2) and the microscopic observation revealed that there were no
M. Qamar et al.
remarkable pathological changes after oral administration of SCME,
except kidney where interstitial edema and vacuolar degenerations were
seen in sub-acute toxicity experiments. Though there were no significant
deviations in renal biochemical markers in sub-acute assay, yet the re­
sults warrant a comprehensive study on determining sub-acute renal
toxicity of S. cumini extracts. The architecture of the liver, heart, and
spleen was seen similar to the normal animals. The hepatocytes and
cardiomyocytes were found properly shaped with no signs of apoptosis
or clusters of neutrophils and lymphocytes, which may indicate that
SCME had no adverse effects on these organs. In kidney microscopic
observation, normal interstitium, glomeruli, and tubules were seen in
acute toxicity studies. Even in sub-acute study majority area of the
kidney had normal interstitium, glomeruli and tubules with reported
only one spot of interstitial edema and vacuolar degeneration. It in­
dicates renal epithelial cells inflammation was promoted by SCME.
Spleen had no significant difference by microscopic observations among
normal and treated animals.
3.4. Optimization of HPLC conditions and specificity validation
HPLC conditions were optimized in order to obtain clear isolation of
desired analytes. Maximum separation was observed with mobile phase
consisting of acidified (0.1% TFA) water (A) and acidified (0.1% TFA)
methanol (B) at flow rate of 1 mL/min with total run time of 35 and the
linear eluting gradient was as follows: 5% B in 0–2 min, 5%–10% B in
2–10 min, 10%–30% B in 10–25 min, 30%–80% B in 25–30 min, and
100% B in 30–35 min. Retention times of obtained peaks were compared
with known standards under the same conditions. External standards of
Fig. 3. HPLC chromatograms of external standards (A, C, E, G) and S. cumini extracts (B, D, F, H). A and B: 1. Scopoletin 2. Umbelliferone; C and D: 1. Delphinidin 3-
glucoside 2. Peonidin-3,5-diglucoside; E and F: 1. Catechin 2. Myricetin; G and H: 1. Quinic acid.
7
Journal of Ethnopharmacology 287 (2022) 114919
anthocyanins (delphinidin 3-glucoside, Peonidin-3,5-diglucoside) and
flavonoids (scopoletin, umbelliferone) gave better response at 520 nm
and 230 nm respectively. Similarly, clear peaks of flavonoids (scopole­
tin, umbelliferone) and anthocyanins (delphinidin 3-glucoside,
Peonidin-3,5-diglucoside) were obtained from 100% methanolic
extract of S. cumini fruit at 230 nm and 520 nm (Fig. 3). Catechin and
myricetin gave better response at 330 nm while quinic acid was iden­
tified at 254 nm from 50% methanolic extract of S. cumini fruit (Fig. 3).
3.5. Quantification analysis
Peonidin-3,5-diglucoside was quantified in the highest amount as
2104 μg/g obtained from the crude S. cumini 100% methanolic extract,
followed by delphinidin 3-glucoside, catechin, quinic acid, scopoletin,
myricetin, umbelliferone as 127.4, 63.7, 54.9, 31.3, 12.3 and 10.4 μg/g,
respectively, based on standard dilutions calibration curves as presented
in Table 1B. Previously, peonidin-3,5-diglucoside and delphinidin 3-
glucoside were also quantified (4.7 mg/100 g and 95.6 mg/100 g)
from ethanolic extract of S. cumini pulp by Faria et al. (2011). Catechin
was also quantified (14.78 μg/g) in ethanolic extracts of S. cumini fruit
by Shrikanta et al. (2015). Jampani et al. (2014) have identified del­
phinidin 3-glucoside in S. cumini fruit extracts, while the compound was
quantified (0.14 mg/g) in the same fruit by Reynertson et al. (2008).
Quinic acid and myricetin were previously identified using external
standards and HPLC-MS/MS analysis (Faria et al., 2011). The present
study reports the identification and quantification of scopoletin and
umbelliferone for the first time in S. cumini fruit extract. Previously,
scopoletin from leaf and root extracts of Hypochaeris radicata was
M. Qamar et al.
Table 1B
Quantification of bioactive compounds from S. cumini 50% and 100% methanolic extracts.
Sample Compounds name Wavelength LOD
100% MeOH Delphinidin 3-glucoside 520 3.02
Peonidin-3,5-diglucoside 1.36
Scopoletin 230 0.4
Umbelliferone 1.8
50% MeOH Quinic acid 254 0.6
Catechin 330 3.10
Myricetin 0.9
Table 1B: LOD, limit of detection. LOQ, limit of quantification. r2, regression coefficient. Rt min, retention time in minutes. MeOH, methanolic extract. 50% MeOH,
50% methanolic extract (50/50 H2O: MeOH).
reported to have anti-inflammatory and antioxidant activities (Jamuna
et al., 2015). Likewise, the findings of Sim et al. (2015) indicated that
umbelliferone protected the rats against alcohol-induced liver damage
by inhibiting the TLR4 signaling pathway and activating the antioxidant
system.
3.6. In silico studies
We have studied all the isolated compounds from S. cumini named
delphinidin 3-glucoside, peonidin-3,5-diglucoside, scopoletin, umbelli­
ferone, catechin, myricetin, quinic acid and standard anti-inflammatory
drug (indomethacin) using molecular docking simulations to determine
possible binding orientations and interactions of these compounds in the
crystal structure of inflammatory proteins, i.e., Cyclooxygenase-2, Lip­
oxygenase-5 and TRPV1 receptor.
3.6.1. Molecular docking for cyclooxygenase-2 enzyme
The selected compounds owing to their anti-nociceptive and ant-
inflammatory potential were docked in the enzymatic pocket of the
cyclooxygenase-2 enzyme (COX-2, PDB ID:5IKQ) for anti-inflammatory
activity (Table 2, Fig. 4 a). Among the selected ligands, Delphinidin 3-
glucoside was predicted with the lowest binding energy (ΔGbind:
45.78 kcal/mol), Van der Waals binding energy (ΔGvdW: 35.53 kcal/
mol) and lipophilic interactions (ΔGLipo: 10.64 kcal/mol), with a
hydrogen bond energy contribution as ΔGHbond: 2.94 kcal/mol as
compared with standard indomethacin (ΔGbind: 35.87 kcal/mol). Del­
phinidin 3-glucoside picked up four strong hydrogen bond interactions
with side-chain residues Arg121 (2.52 Å), Tyr356 (1.75 Å), Tyr386
(1.85 Å) and Met523 (2.01 Å). Scopoletin (ΔGbind: 25.16 kcal/mol) was
observed to pick up H-bond interaction with active site residue Ser531
(1.86 Å) with hydrophobic (ΔGvdW: 18.87 kcal/mol) and lipophilic
(ΔGLipo: 17.70 kcal/mol) energies. The scopoletin rings are further sta­
bilized by π-π stacking with side-chain residues Leu353, Phe519 and
Val524. It was found that Arg121, Tyr356, Tyr386, Leu353, Phe519,
Val524, Met523 and Ser531 are crucial residues in forming ligand
protein stable complex and is in accordance with the data reported by
Orlando and Malkowski (2016). Based upon docking score, the ranking
order of ligands with COX-2 is Myricetin (-10.99 kcal/mol) > Indo­
methacin (-10.44 kcal/mol) > Delphinidin 3-glucoside (-8.31 kcal/mol)
> Catechin (-7.76 kcal/mol) > Quinic acid (-7.68 kcal/mol) > Umbel­
liferone (-7.00 kcal/mol) > Scopoletin (-6.69 kcal/mol). The results are
not compared with other published molecular docking simulations due
to difficulties in finding the same PDB ID, the same applied software, and
the same standard.
3.6.2. Molecular docking for lipoxygenase-5 enzyme
The selected compounds were studied against lipoxygenase-5
enzyme (LOX-5, PDB ID:6N2W) for their anti-inflammatory activity
(Table 2, Fig. 4 b). The contribution of hydrophobic interactions in
ligand binding were more abundant within enzymatic pocket of LOX-5.
Peonidin 3,5-diglucoside revealed higher ligand binding energy (ΔGbind:
47.48 kcal/mol) with hydrophobic interactions ΔGvdW: 48.80 kcal/mol
and ΔGLipo: 19.58 kcal/mol. The investigations predicted three
Journal of Ethnopharmacology 287 (2022) 114919
LOQ Linear range (μg/ml) r2 Rt min Concentration (μg/g)
9.05 1.5–100 0.9973 7.98 127.4
4.04 3.9–250 0.9998 13.61 2104
1.19 7.8–500 0.9984 9.58 31.3
5.36 7.8–500 0.9992 10.36 10.4
1.5 5.8–93.7 0.9992 10.17 54.9
9.60 46.8–1500 0.9994 19.29 63.7
2.16 12.5–200 0.9985 24.1 12.3
hydrogen bond interactions with active site residues Arg596 (2.38 Å),
Trp599 (2.58 Å), and Glu417 (1.92 Å) with further stabilization by alkyl
hydrophobic π-π interactions with Ala410 and Leu607 within pocket of
LOX-5. Delphinidin 3-glucoside was next to peonidin 3,5-diglucoside in
binding energy (ΔGbind: 35.44 kcal/mol) with ΔGvdW (-44.96 kcal/mol)
hydrophobic and ΔGLipo (-17.93 kcal/mol) energies. Delphinidin 3-
glucoside picked up three hydrogen bond interactions with His367
(3.09 Å), Gln363 (2.83 Å) and Glu417 (2.20 Å) and become stable with
hydrophobic alkyl interaction with side chain Leu414 and Ala426
within clefts of LOX-5. It was observed that the active site residues
Gln363, His367, Ala410, Leu414, Glu417, Ala426, Arg596, Trp599, and
Leu607 are key to form stable inhibitor protein complexes as reported by
Gilbert et al. (2020). Based upon docking score, the following ranking
order of ligands with LOX-5 was observed, Peonidin 3,5-diglucoside
(-8.41 kcal/mol) > Delphinidin 3-glucoside (-7.26 kcal/mol) > Quinic
acid (-6.25 kcal/mol) > Myricetin > Scopoletin (-5.74 kcal/mol) >
Umbelliferone (-3.88 kcal/mol) > Catechin (-3.79 kcal/mol) > Indo­
methacin (-3.66 kcal/mol).
3.6.3. Molecular docking for TRPV1 receptor
Computational docking studies were performed to predict the most
favorable binding mode of the selected compounds isolated from
S. cumini inside the binding pocket of TRPV1 (PDB ID: 3SUI) for anti-
nociceptive activity (Table 2, Fig. 4c). Among the selected com­
pounds, Delphinidin 3-glucoside was predicted with lowest binding
energy (ΔGbind: 38.69 kcal/mol) and van Der Waals (ΔGvdW -30.90 kcal/
mol) and lipophilic interactions (ΔGLipo -18.05 kcal/mol). It was pre­
dicted to form three strong hydrogen bonds with residues Tyr511 (1.82
Å) and Thr550 (1.75 Å, 1.91 Å) within active site of TRPV1 receptor.
Scopoletin was second to delphinidin 3-glucoside in binding energy
(ΔGbind: 29.94 kcal/mol) with ΔGvdW (-24.63 kcal/mol) hydrophobic
and ΔGLipo (-14.45 kcal/mol) energies. Scopoletin picked up two H-bond
interactions with Ser512 (1.98 Å) and Thr550 (1.98 Å) and is stabilized
by hydrophobic alkyl interaction with Leu553,669, Ile573, and a π -π
interaction with Tyr511and Phe587 within pocket of TRPV1 receptor.
The active site residues that played a key vital role in stabilizing protein
inhibitor complex are Tyr511, Ser512, Thr550, Leu553, Ile573, Phe587
and Leu669 and are in accordance with Elokely et al. (2016). The
ranking order of ligands with TRPV1 on basis of docking score is Peo­
nidin 3,5-diglucoside (-9.86 kcal/mol) > Delphinidin 3-glucoside (-8.35
kcal/mol) > Myricetin (-8.11 kcal/mol) > Quinic acid (-7.63 kcal/mol)
> Catechin (-7.39 kcal/mol) > Scopoletin (-6.53 kcal/mol) > Indo­
methacin (-3.66 kcal/mol) > Umbelliferone (-6.24 kcal/mol).
4. Conclusions
SCME has shown a stronger anti-nociceptive and anti-inflammatory
potential in both in vivo assays in mice, i.e., formalin- and glutamate-
induced pain behavior, suggesting the potential for clinical application
of indigenous berry fruit to treat inflammation. Anti-inflammatory ac­
tivity is accredited to synergistic effects of anthocyanins, phenolic acids,
and flavonoids, identified and quantified in S. cumini fruit extracts by
HPLC method. The docking results support the anti-inflammatory
8
M. Qamar et al. Journal of Ethnopharmacology 287 (2022) 114919

Table 2
Binding energies (kcal/mol) of compounds with cyclooxygenase-2, Lipoxygenase 5 and TRPV1 calculated by Prime MMGBSA.
Name Docking ΔGBinding Log Ki ΔGCoulomb ΔGCovalent ΔGHbond ΔGLipophilic ΔGSolv ΔGvdW Residue-Ligand Interactions with Distance
score (μMolar) GB (Å)

Hydrogen Bonds Hydrophobic Bonds

Myricetin -10.99 -15.96 -5.7416 -37.41 19.63 -2.84 -25.09 31.65 -1.90 Arg121 (2.52), Alkyl Bond: Val350
Tyr356 (1.75), (4.93), Leu353 (5.20),
Tyr386 (1.85), Ala528 (3.55), Ala528
Met523 (2.01), (4.58),
C-H Bond:
Tyr356 (2.23),
Indomethacin -10.44 -35.87 -12.35 -39.56 3.24 -1.71 -27.96 66.80 -35.35 Arg121 (1.91), Electrostatic
Arg121 (2.21), attractive charge:
Ser531 (3.00), Arg121, (3.67), Alkyl
Leu353 (3.05), Bond: Ala528 (3.55),
Ser354 (2.54) Leu385 (3.78),
Met523 (4.27), Val350
(4.17), Leu532 (3.75),
Val524 (3.76)),
π-Alkyl Bond: his90
(4.57), trp388 (5.26),
trp388 (4.89), val350
(4.45), ala528 (3.67),
leu353 (5.39), val524
(4.05), ala528 (4.91),
ala528 (4.97)
Delphinidin 3- -8.31 -45.78 -27.6754 -34.00 3.55 -2.94 -10.64 33.79 -35.53 Ser354 (1.80),
glucoside Pro515 (1.95),
Pro515 (2.05),C-
H Bond: His90
(2.77), His352
(2.70), Asp516
(2.41), Gln193
(2.35), Gln193
(2.65), Tyr356
(2.67),
Catechin -7.76 -13.01 -3.5706 -13.31 17.36 -1.78 -23.52 23.73 -15.49 Arg121 (2.40), Alkyl Bond: Val350
Tyr356 (1.66), C- (5.06), Val350 (4.94),
H Bond: Ser354 Leu353 (5.30), Ala528
(2.92), Ala528 (3.30), Ala528 (4.32),
(3.07) Val117 (4.90), Val350
(5.09), Leu360 (4.95),
Leu532 (4.75)
Quinic acid -7.68 -23.60 -11.3620 -17.44 4.30 -1.32 -11.02 25.94 -24.06 Tyr386 (2.15), Alkyl Bond: Leu353
Met523 (1.74), (4.76)
C-H Bond:
Ala528 (3.10),
Met523 (2.86)
Umbelliferone -7.00 -23.37 -11.1927 -8.34 4.26 -0.52 -14.55 19.08 -23.31 Met523 (1.79), Alkyl Bond: Val350
C-H Bond: (4.96), Leu353 (5.45),
Val524 (2.58) Ala528 (3.78),
π-Alkyl Bond: Tyr386
(4.92), Trp388 (5.37)
Scopoletin -6.69 -25.16 -12.5067 -10.97 5.23 -0.52 -17.70 17.68 -18.87 Ser531 (1.86), C- Alkyl Bond: Leu353
H Bond: Tyr386 (4.84), Leu353 (4.80),
(2.97) Val524 (3.57), π
-Alkyl Bond: Phe519
(5.38)
Lipoxygenase-5
Peonidin 3,5- -8.41 -47.48 -28.93 -54.72 9.35 -3.26 -19.58 69.53 -48.80 Arg596 (2.38), Alkyl Bond: Ala410
diglucoside Trp599 (2.58), (4.85), Leu607 (5.34)
Glu417 (1.92),C-
H Bond: Leu414
(2.64), Thr427
(2.30), Glu417
(2.51)
Delphinidin 3- -7.26 -35.44 -20.07 -17.01 7.85 -2.39 -17.93 38.98 -44.96 His367 (3.09), Alkyl Bond: Leu414
glucoside Gln363 (2.83), (5.25), Ala426 (5.46)
Glu417 (2.20) C-
H Bond: His367
(3.07)
Quinic acid -6.25 -33.83 -18.89 -24.49 0.92 -1.64 -8.35 21.62 -21.89 His600 (2.21),
Thr427 (2.21),
Gln363 (1.76), C-
H Bond: Gln363
(2.66)
Myricetin -5.74 -33.28 -18.48 -31.09 2.37 -2.57 -14.37 38.52 -26.14
(continued on next page)

9
M. Qamar et al. Journal of Ethnopharmacology 287 (2022) 114919

Table 2 (continued )
Name Docking ΔGBinding Log Ki ΔGCoulomb ΔGCovalent ΔGHbond ΔGLipophilic ΔGSolv ΔGvdW Residue-Ligand Interactions with Distance
score (μMolar) GB (Å)

Hydrogen Bonds Hydrophobic Bonds

Gln363 (2.05), C- Alkyl Bond: Ala603

H Bond: His432 (5.18), Leu607 (5.13)
(2.40)Gln363
(2.47)
Scopoletin -4.17 -25.53 -12.78 -18.49 6.26 -1.69 -12.19 22.27 -21.69 Arg596 (2.21), Alkyl Bond: Ala426
Arg596 (2.63), (4.20), π-Alkyl Bond:
Pro569 (2.10), C- His432 (4.83)
H Bond: Gln363
(3.04), Gln363
(2.91)
Umbelliferone -3.88 -28.02 -14.62 -17.00 1.83 -1.78 -11.22 21.50 -21.35 Arg596 (1.94), Alkyl Bond: Ala426
Arg596 (2.59), (4.12), π-Alkyl Bond:
His600 (2.87), His432 (4.84), Trp599
Pro569 (2.05) (5.11)
Catechin -3.79 -16.61 -6.22 -26.84 6.59 -1.44 -15.59 43.22 -22.56 His600 (2.60), Alkyl Bond: Ala410
Ile673 (2.10), (5.45), Ala603 (4.53),
Gln363 (1.84), C- Leu607 (4.98),
H Bond: His367 π-Alkyl Bond: Trp599
(2.85), Gln363 (5.17)
(2.54)
Indomethacin -3.66 1.08 3.70 78.25 7.04 -0.89 -16.70 -33.59 -30.99 HIS367 (2.66), Electrostatic
HIS600 (2.40) attractive charge:
Arg596 (4.43),
Halogen Bond:
Asn407 (3.23), π - π T-
shaped:His372
(4.56), Alkyl Bond:
Ala410 (3.58), Ala603
(3.76), Leu368 (5.34),
π-Alkyl Bond: His372
(4.04), Trp599 (4.40),
Trp599 (5.12), Leu607
(5.20), Ala603 (4.76),
Leu607 (4.30), Leu368
(4.76), Ala410 (4.89)
TRPV1
Peonidin 3,5- -9.86 -25.94 -13.08 2.33 5.24 -0.05 -28.64 47.25 -52.07 C-H Bond: Alkyl Bond: Leu515
diglucoside Met547 (2.91), (5.43)
Tyr511 (2.74)
Delphinidin 3- -8.35 -38.69 -22.46 -24.28 3.99 -1.79 -18.05 32.34 -30.90 Tyr511 (1.82), Alkyl Bond: Leu577
glucoside Hr550 (1.75), (5.08)
Thr550 (1.91)
Myricetin -8.11 -17.91 -7.18 -7.57 3.67 -1.31 -16.13 33.19 -29.77 Thr550 (1.74), Alkyl Bond: Leu669
Thr550 (2.01) C- (5.22), Leu515 (5.46)
H Bond: Thr550
(2.52)
Quinic acid -7.63 -19.66 -8.47 -13.24 2.55 -1.93 -9.70 24.65 -21.99 Tyr511 (1.85), Alkyl Bond: Leu553
Ala566 (2.39), (5.14)
Glu570 (2.14),
Ser512 (2.20)
Catechin -7.39 -26.14 -13.23 -19.85 7.35 -1.44 -18.73 31.60 -25.07 Tyr511 (2.01), Alkyl Bond: Met547
Tyr511 (1.97), (5.36), Leu669 (4.93),
Thr550 (2.36) C- Met547 (4.18),
H Bond: Thr550 π-Alkyl Bond: Phe587
(3.00) (5.45), Phe591 (5.43)
Scopoletin -6.53 -29.94 -16.03 -13.78 1.44 -1.05 -14.45 22.52 -24.63 Ser512 (1.98), Alkyl Bond: Leu553
Thr550 (1.98) C- (5.19), Ile573 (5.30),
H Bond: Ala566 Leu669 (5.33) π-Alkyl
(2.49), Glu570 Bond: Tyr511 (5.49),
(2.63), Tyr511 Phe587 (5.34)
(2.63)
Indomethacin -6.33 -22.07 -6.36 -4.43 5.60 -0.01 -20.07 38.99 -41.71 – π- Sulfur Bond:
Met547 (4.10), π- π T
shaped: Tyr511
(5.77), Alkyl Bond
Ala546 (3.78), Leu515
(5.31), Leu553 (4.89),
Met547 (5.11),
π-Alkyl Bond: Phe543
(5.01), Phe591 (5.06),
Met547 (5.33),
Leu669 (5.48), Leu515
(4.69)
(continued on next page)

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M. Qamar et al. Journal of Ethnopharmacology 287 (2022) 114919

Table 2 (continued )
Name Docking ΔGBinding Log Ki ΔGCoulomb ΔGCovalent ΔGHbond ΔGLipophilic ΔGSolv ΔGvdW Residue-Ligand Interactions with Distance
score (μMolar) GB (Å)

Hydrogen Bonds Hydrophobic Bonds

Umbelliferone -6.24 -27.96 -14.57 -12.45 0.89 -1.02 -12.55 20.47 -23.29 Glu570 (2.06), Alkyl Bond:Leu553
Thr550 (1.90), C- (5.02), Leu669 (5.03),
H Bond: Glu570 π-Alkyl Bond: Phe587
(2.94), Tyr511 (5.0 8),
(2.58)

Fig. 4. Molecular docking: a COX-2; b LOX-5; c TRPV1.

capabilities of selected compounds, wherein Peonidin 3,5-diglucoside
and delphinidin 3-glucoside more potently interact with cyclo­
oxygenase 2, lipoxygenase 5 and TRPV1 to exert the activity. Myricetin
had strong binding affinity with all enzymes as compared to standard
marketed drug (indomethacin). Therefore, myricetin can be proposed as
a nature-derived drug alternative to combat inflammatory/nociceptive
disorders. Moreover, LD50 of SCME was recorded above 3000 mg/kg
depicts the practical safety or no toxicity of traditional fruit as no
behavioral changes and mortality was recorded during acute (14-days)
and subacute (28-days) toxicity studies in trial rats. However, more
detailed studies are required to elucidate the possible mechanisms of
action and pathways responsible for the anti-inflammatory capacities of
the plant or fruits extracts.
Declaration of competing interest
The authors declare no conflict of interest to this study.
Author contributions
Muhammad Qamar done the in vivo anti-nociceptive, anti-inflam­
matory activities and compiled the initial draft of manuscript; Saeed
Akhtar contributed in writing the paper and modified the formatted
manuscript text; Tariq Ismail designed the experiments and processed
the data; Muqeet Wahid performed in vivo toxicity and hematological
analysis; Sajed Ali and Shahid Murtaza assisted in in vivo toxicity model
regarding feeding and taking care of rats; Yasir Nazir performed docking
calculations: Malik Waseem Abbas performed serum biochemistry
analysis and histopathology. Zyta M. Ziora performed HPLC analysis and
coordinated the whole process of writing the manuscript. All the authors
have read the manuscript.
Declaration of competing interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Muhammad Qamar Saleem reports financial support was provided
by Higher Education Commission Pakistan.
Acknowledgement and funding source
Muhammad Qamar thanks the Higher Education Commission HEC,
Pakistan for providing funding to undertake research at IMB-UQ, Cooper
group, Australia under Award of scholarship “International Research
Support Initiative Program” and PIN # “IRSIP 43 Agri 06”. We are also
thankful to the University of Queensland, Brisbane, Australia for

11
M. Qamar et al.
accepting my research proposal and providing the Laboratory facilities.
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