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Kanyanga et al. World Journal of Pharmacy and Pharmaceutical Sciences
IN VIVO ANTI-INFLAMMATORY AND ANALGESIC PROPRIETIES
OF EXTRACTS, FRACTIONS AND POLYSACCHARIDES FROM
BRUCEA SUMATRANA ROXB. (SIMAROUBACEAE) LEAVES IN
EXPERIMENTAL ANIMALS
Tshodi Ehata M.1, Nsaka Lumpu S.
1, Lami Nzunzu J.
1, Cimanga Kanyanga R.*
1,2
Vlietinck A. J.2 and Pieters L.
2
1Department of Medicinal Chemistry and Pharmacognosy, Laboratory of Pharmacognosy and
Phytochemistry, Faculty of Pharmaceutical Sciences, University of Kinshasa, P. O. Box 212,
Kinshasa XI, Democratic Republic of Congo.
2Department of Pharmaceutical Sciences, Natural Products & Food Research and Analysis
(Natura), University of Antwerp, Universiteitsplein1, B-2610, Antwerpen, Belgium.
ABSTRACT
The anti-inflammatory and analgesic activities of extracts, fractions
and polysaccharides from B. sumatrana leaves collected in Mai-
Ndombe in Democratic Republic of Congo (DR-Congo) were
evaluated in Wistar rats and reported for the first time in this study.
Results indicated that, when tested against carrageenan-induced
increase of liquid volume of animal foot (paw edema), lyophilized
aqueous extract and its soluble fractions chloroform, ethylacetate, n-
butanol and residual aqueous fractions, 80% methanol and total
alkaloids extracts, crude and pure polysaccharide fractions,
administered at oral doses of 50 and 100 mg/kg body weight
respectively, induced significant reduction of paw edema development
in dose-dependent manner. At the highest oral dose of 100 mg/kg body
weight, they produced percentage inhibitions of carrageenan effects
more than 82% for lyophilized aqueous extract, from 65.71±0.02 to
77.42±0.04% for soluble fractions, more than 85% for 80% MeOH and total alkaloids
extracts, and ranging between 71.42±0.00 and 91.42±0.01% for polysaccharides compared to
negative control (0% inhibition of paw edema). Diclofenac used as anti-inflammatory
reference product caused more than 97.14±0.03% inhibition of paw edema development. In
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.632
Volume 9, Issue 11, 401-434 Research Article ISSN 2278 – 4357
*Corresponding Author
Cimanga Kanyanga R.
Department of
Pharmaceutical Sciences,
Natural Products & Food
Research and Analysis
(Natura), University of
Antwerp,
Universiteitsplein1, B-2610,
Antwerpen, Belgium.
Article Received on
14 September 2020,
Revised on 05 October 2020,
Accepted on 26 October 2020
DOI: 10.20959/wjpps202011-17662
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analgesic test, acetic acid-induced pains was used to determine the analgesic activity. Results
from this assay revealed that, the oral administration of lyophilized aqueous extract and its
fractions, 80% methanol and total alkaloids extracts, crude and pure polysaccharide fractions
from Brucea sumatrana leaves at 50 and 100 mg/kg body weight caused marked reduction of
the number of acetic acid-induced abdominal writhes of treated Wistar rats in dose-dependent
manner compared to negative control, indicating significant peripheral antinociceptive
activity. All extracts including lyophilized aqueous, 80% methanol and total alkaloids
significantly reduced abdominal writhes of the treated animals with percentage reductions
ranging from 83 to 86% with the 80% methanol extract as the most active (86.32±0.02%).
Chloroform, ethylacetate, n-butanol and residual aqueous soluble fractions also showed good
reduction of acetic acid-induced abdominal writhes in treated animals with percentage
inhibitions between 67 to 76% with ethylacetate fraction as the most active (76.83±0.03%).
Crude and isolated pure polysaccharide fractions provoked prominent reduction of abdominal
writhes of treated animals with percentage reductions ranging from 82 to 83% with crude
polysaccharide extract as the most active (85.15±0.01%). Aspirin used as reference analgesic
drug reduced the number of abdominal writhes by 89.27±0.001%. A number of
phytochemical compounds associated with anti-inflammatory and analgesic activities were
observed to be present in all tested samples of B. sumatrana leaves. This present study
therefore, scientifically confirmed the traditional use of the studied medicinal plant part in the
management of various pains in traditional medicine including rheumatism. These results
showed that all tested samples from B. sumatrana leaves possessed good and appreciable
anti-inflammatory and analgesic effects. They can thus, support the use of the plant part in
traditional medicine in painful conditions acting centrally and peripherally in both evaluated
biological activities.
KEYWORDS: Brucea sumatrana, leaves, extracts, fractions, polysaccharides, in vivo anti-
inflammatory and analgesic activities, paw edema, writhes, Wistar rats.
INTRODUCTION
Medicinal plants and their secondary metabolites were progressively used in the treatment of
diseases as complementary medicines. Inflammation was a pathologic condition including a
wide range of diseases such as rheumatic, immune-mediated conditions, diabetes,
cardiovascular accidents, etc. Inflammation was a defense response of the body to hazardous
stimulus such as allergens and/or injuries to the tissues. On the other hand, uncontrolled
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inflammatory responses were the main cause of a vast continuum of disorders including
allergies, cardiovascular dysfunctions, asthma, rheumatism, metabolic syndromes, cancers,
autoimmune diseases and other imposing a huge economic burden on individuals and
consequently to the society (Bagad et al., 2013).
Inflammation (from Latin: inflammatio or Inflammare.) was a part of the complex biological
responses of body tissues to harmful stimulus, such as pathogens, damaged cells, or irritants.
It was a protective response involving immune cells, blood vessels, and molecular mediators.
Figure 1: Foot inflammation.
The functions of inflammation were to eliminate the initial causes of cell injuries, to clear out
necrotic cells and tissues damaged from the original insults, the inflammatory processes, and
to initiate tissue repairs (https://en.wikipedia.org/wiki/Inflammation, 2019).
When inflammation occurred, various chemicals from the body's white blood cells were released
into the blood and affected tissues to protect the body from foreign substances. The release of
divers types of chemicals increased the blood flow to the area of injuries or infections. They
caused leak of fluid into the tissues and may result in redness, warmth irritations, swelling and
eventually wearing down of cartilage. (https://www.webmd.com/arthritis/about-inflammation,
2019).
The five classical signs of inflammation were heat, pain, redness, swelling, and loss of
functions (Latin calor, dolor, rubor, tumor, penuria and functio laesa) (Abbas et al., 2009;
https://en.wikipedia.org/wiki/Inflammation, 2019). Moreover, inflammation can be classified
as either acute (short-lived) or chronic (long-lasting).
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Acute inflammation was the initial response of the body to harmful stimulus and was
achieved by the increased movements of plasma and leukocytes (especially granulocytes)
from the blood into the injured tissues. There was five key signs of acute inflammation:
Pain: This may occur continuously or only when a person touched the affected area,
Redness: This happened because of an increase in the blood supply to the capillaries in
the area,
Loss of function: There may be difficulty moving a joint, breathing, sensing smell, and
so on,
Swelling: A condition call edema can develop if fluid builds up,
Heat: Increased blood flow may leave the affected area warm to the touch.
These signs were not always present. Sometimes, inflammation was “silent,” without
symptoms. A person may also feel tired, generally unwell, and had fever. Symptoms of acute
inflammation lasted a few days. (https://www.medicalnewstoday.com/articles/248423, 2019).
Initial treatment for acute inflammation in the foot or ankle consists of RICE therapy:
●- Rest: Stayed off the foot or ankle. Walking may cause further injury.
●- Ice: Applied an ice pack to the injured area, placing a thin towel between the ice and the
skin. Use ice for 20 minutes and then wait at least 40 minutes before icing again.
●- Compression: An elastic wrap should be used to control swelling.
●- Elevation: The foots or ankles should be raised slightly above the level of your heart to
reduce swelling (https://www.foothealthfacts.org/conditions/acute-inflammation 2019).
Subacute inflammation was the period between acute and chronic inflammation and may last
2 to 6 weeks. (https://www.ncbi.nlm.nih.gov/books/NBK493173/, 2019).
The chronic inflammation was prolonged inflammation and can lead to a progressive shift in
the type of cells present at the site of inflammation, such as mononuclear cells. It was
characterized by simultaneous destruction and healing of the tissues from the inflammatory
processes (https://en.wikipedia.org/wiki/Inflammation, 2019). The common symptoms of
chronic inflammation included: fatigue, fever, mouth sores, rashes, abdominal and chest
pains (https://www.healthline.com/health/chronic-inflammation#symptoms, 2019).
In addition, inflammation was not a synonym for infection. Infection described the interaction
between the action of microbial invasion and the reaction of the body's inflammatory
response, the two components were considered together when discussing infection. The word
was used to imply microbial invasive caused for the observed inflammatory reaction.
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Inflammation on the other hand, described purely the body's immunovascular responses,
whatever the cause may be. It included a number of events which can be considered under
three phases viz. acute transient, delayed sub-acute and chronic proliferate phases (Abbas,
2009; Danya, 2017; https://en.wikipedia.org/wiki/Inflammation, 2019).
During inflammation, the liberation of endogenous mediators like serotonins, kinins,
bradykinins, proteases, lysosomes, histamines and prostaglandins occurred as substances that
indicated and modulated cells and tissue responses involved in inflammation. They were in
small quantity eliciting pain responses (Danya, 2017). Some enzymes such as
cyclooxygenase (COX) and lipoxygenase were involved in inflammation, pains and platelet
aggregation. They were the key in the synthesis of prostaglandins and throxanes. In addition,
inflammation was a severe response by living tissues to any kind of injuries, cell death, some
diseases as cancer and ischemia, etc. and can give six primary indicators as pain, redness,
heat, heat, warmness and swelling. (Waisman et al., 2015; Azab et al, 2016). Proteolytic
enzymes such as bromelain, papain, pancreatin, trypsin, chymotrypsin and the flavonoid
rutin, were also essential regulators and modulators of the inflammatory responses (Azab et
al., 2016).
In general, for the treatment of inflammation, nowadays, steroids, nonsteroidal anti-
inflammatory drugs, corticosteroids such as prednisolone, immunosuppressants, antimalarial
medications such as hydroxychloroquine were currently used. Other drugs known as disease-
modifing antirheumatic drugs stop (MDARDs) including methotrexate, sulfasalazine,
lefleluomide, azathioprine and cyclophosphamide, and biologic drugs such as infliximab,
anercept and andabulimab, were usually used, or prescribed in special or particular cases for
the relief of inflammatory diseases and required long-term of treatment
(https://www.webmd.com/arthritis/about-inflammation, 2020). Their use was often associated
of the occurring of serious side effects such as bleeding gastro-intestinal and pectic ulcers
(Oukacha et al., 2018).
On the other hand, analgesic products refered to a group of drugs used to temporally relieved
various pains. They were sometimes known as painkillers. They blocked pain signals by
changing how the brain interested the signals and showed olown the central nervous system.
Analgesics were drugs which relieved pains without altering sensory awareness and
consciousness or blocking the conduction of nerve impulses. They were also known as anti-
inflammatory drugs, due to their actions to reduce local inflammatory responses. Most anti-
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inflammatory-analgesic drugs were derived from two compounds: salicylic
acid and phenacetin (Henna and Znad, 2018).
For treatment various pains, analgesics compounds were those with multiple active
ingredients and included many of the stronger prescription analgesics. Active analgesic
ingredients that had been commonly used included: aspirin , ibuprofen, caffeine,
codeine or oxycodone, paracetamol or acetaminophen (Dafalgan), phenacetin and the new
drugs tramadol chlorhydrate (Tradonal) and oxycodone chlorhydrate/naloxone chlorhydrate
(Traginact) used to treat severe pains. Several formulations had disappeared from over-the-
counter status in drug store aisles and other retail outlets. One example was APC (aspirin,
phenacetin and caffeine) common compound tablets from the 1940s to 1983 which was less
used or abandoned because of harmful side effects of phenacetin. Anacin in the United States
was reformulated to eliminate it while Vincent's APC was no longer sold. Bex was
analgesic compound popular in Australia for much of the 20th
century. It came in the form of
APC (aspirin-phenacetin-caffeine) tablets or powder, containing 42% aspirin and 42%
phenacetin plus caffeine and was no longer sold
(https://en.wikipedia.org/wiki/Bex_(compound_analgesic, 2020). The United States Food and
Drug Administration also now required that manufacturers of analgesic compounds
unequivocally stated each ingredient's purpose
(https://en.wikipedia.org/wiki/Compound_analgesichttps://en.wikipedia.org/wiki/Compound_
analgesic, 2020). Combining analgesic compounds with alcohol prescription or illegal other
drugs, can create dangerous and unpredictable effects and can in some cases lead to death.
Mixing alcohol with painkillers, opioids/opiates can be a deadly combinations
(https://www.alcohol.org/mixing-with/painkillers/). Even, low doses can impair olwing
ability. Therefore, new analgesic drugs from medicinal plants with low toxicity and without
side effects, were need to replace the known synthetic drugs.
In recent years, there had been an increase interest to find new anti-inflammatory and
analgesic drugs with possibly fewer side effects and safe from natural sources , particularly
from medicinal plants (Shojali et al., 2015).
Many medicinal plants were studied in this field having as aim to prove that they were
endowed with these two biological activities in animal models for further use in humans after
other scientific studies (Onzago et al., 2013; Bahmani et al., 2014; Hemanyet et al., 2014;
Adebayo et al., 2015; Nworu and Akah, 2015; Azab et al., 2016; Maione et al., 2016;
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Rajamanickam and Rajamanickam, 2016; Hijazi et al., 2017; Koech et al., 2017; Oguntibeju
et al., 2018; Toghueo, 2019). Active natural anti-inflammatory and analgesic products were
isolated from some medicinal plant extracts and reported (Serafini et al., 2010; Fürst et al.,
2014; Abdel-Rhaman, 2016; Rauf et al., 2017, Khan et al., 2020).
2. MATERIALS AND METHODS
2.1. Plant material
Plant material was constituted with leaves of B. sumatrana Roxb. (Simaroubaceae) collected
in Mai-Ndombe in Democratic Republic of Congo (DRCongo). The plant was identified at
the Institut National d’Etudes et de Recherches en Agronomie (INERA), Department of
Biology, Faculty of Sciences, University of Kinshasa. A voucher specimen of the plant had
been deposited in the herbarium of this institute and in the laboratory of Pharmacognosy and
Phytochemistry of the faculty of Pharmaceutical Sciences of the same university. Leaves
were dried at room temperature and reduced to powder using an electronic blender and kept
in brown bottles hermetically closed.
Figure 2: Brucea sumatrana leaves and immature fruits.
2.2. Preparation of extracts and partition of lyophilized aqueous BSLAE-1 extract
60 g of powdered leaves were mixed with 300 ml distilled water and boiled on hotplate for 15
minutes. The mixture was cooled and filtered on a filter paper F001 grade (CHLAB GROUP,
08205, Barcelona, Spain). Next, the filtrate was evaporated in vacuum to reduce volume to 10
ml, which was further lyophilized to give a dried lyophilized aqueous extract denoted as
BSLAE-1 (45.84 g). The partition of 20 g of lyophilized aqueous extract BSLAE-1 was
carried out using solvents of different polarities like chloroform, ethylacetate, n-butanol
together with the resulting residual aqueous soluble fraction. All fractions were treated as
described above to yield corresponding dried extracts named as BSLAE-1.1 (4.51 g),
BSLAE-1.2 (5.75 g), BSLAE- 1.3 (4.15 g) and BSLAE-1.4 (5.07 g) (Harborne, 1998; Trease
and Evans, 2000).
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On other hand 30 g of plant material were macerated with 80% methanol for 24 h. After
filtration in the same conditions described above, the marc was exhaustively percolated with
the same solvent. The macerate et percolate were combined and evaporated in vacuum to
give a dried extract denoted as BSME (24.02 g).
2.3. Extraction and purification of polysaccharides
2.3.1. Water-soluble polysaccharides
Methods proposed by Liu et al., (2014) and Wang et al., (2018) were used for the extraction,
separation and purification of polysaccharides. About 20 g of lyophilized aqueous BSLAE-1
extract, was dissolved in 100 ml distilled water, filtered, and the volume of the filtrate was
reduced to 10 ml with rotative rotavapor. After, about a five-fold volume of ethanol 95% was
added into the filtrate for polysaccharides precipitation. The precipitated solution was then
placed in a refrigerator at 4°C for 24 hours giving a high white precipitate after this period.
This last was filtrated and dried to give dried white extract denoted as PBSLAEc (crude
polysaccharides : 16.63 g). This extract gave positive test with phenol/H2SO4 (red-violet)
colour for polysaccharides (Sonialmol et al., 2011). The polysaccharide concentration was
determined with the phenol-sulfuric acid method at 481 nm (Jiang et al., 2010).
2.2. Purification of polysaccharide
The crude polysaccharide sample PBSLAEc (10 g) was purified by gel column
chromatography DEAE (dietylaminoethyl)-cellulose put in deionized water and dumped the
clarity supernatant liquid. After, 500 ml of NaOH 0.5 mol/L were added for 30 minutes,
bathing the cellulose with water until neutral. It was then soaked in 500 ml of HCl 0.5 mol/L
for 30 minutes and treated as described above for bathing, and the process was repeated three
times at which DEAE-cellulose was ready for use. The crude polysaccharide PBSLAEc was
dissolved in deionized water and centrifuged, and the supernatant was loaded onto a new
DEAE-cellulose column (40g, 60 × 2.5 cm = length x internal diameter), which was eluted
with deionized water and NaCl 0.1 M solution in order. The elution (3 ml) was collected and
carbohydrate content determined based on the phenol-sulfuric acid method at 481 nm
absorbance (Jiang et al., 2010). The crude polysaccharide PBSLAEc was separated on
Sephadex LH-20 (40g, 60 x 2.5 cm) with the same eluents into chromatographically pure 4
fractions, which were then freeze-dried, and coded as PBSLAE-1 (2.25 g), PBSLAE-2 (2.11
g), BSLAE-3 (2.38 g) and PBSLAE-1.4 (2.67 g). (Liu et al., 2014; Zhao et al., 2015).
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2.3. Anti-inflammatory activity assay for extracts, fractions and polysaccharides from
B. sumatrana leaves
Methods of Ouedrago et al., (2015) and Amri et al., (2018) were used to evaluate the anti-
inflammatory effects of lyophilized aqueous BSLAE-1, its soluble fractions CHCl3, EtOAC,
n-BuOH and residual H2O2 BSLAE-1.1 to -1.4, 80% methanol BSME and total alkaloids
BSTA extracts, and polysaccharides PBSLAEc, PBSLAE-1 to-4 from B. sumatrana leaves in
animal model. Wistar rats with 155-160 g body weight (bw) were used and divided into
groups with 5 rats for each oral dose of test sample administered at oral doses of 50 and 100
mg/kg bw,
- Group I : received 5 ml distilled water as negative control,
- Group II received 5 mg/kg bw of diclofenac as positive control,
- Groups IIIa and IIIb were administered lyophilized aqueous extract BSLAE-1,
- Groups IVa and b to VIIa and b received extracts of chloroform, ethylacetate, n-butanol and
residual aqueous extracts of soluble fractions respectively (BSLAE-1.1 to BSLAE-1.4),
- Groups VIIIa and b, IXa a an b, were administered 80% MeOH BSME and total alkaloids
BSTA extracts respectively,
- Groups Xa and b to XIIIa and b were given polysaccharides PBSLAEc and PBSLAE-1 to -4
respectively,
One hour after separately administration of test samples, inflammation was induced by
administration of 50 μl of carrageenan 1% in NaCl 5% in cousin-plantar of right foot of each
experimental treated animal. The measurement of the paw edema was performed by
displacement technique using a vernier caliper to find out the circumference of paw edema
immediately before and after 1, 3, and 5 hours respectively, followed carrageenan injection.
Volume of each animal foot treated was measured 1, 3 and 5h respectively after
administration of carrageenanm solution. The percentage inhibitions of carrageenan-induced
paw edema by samples were calculated using the following formula:
(Vt-Vo)nc - (Vt-Vo)ts
(Vt-Vo)ncx 100% Inhibition of carrageenan activity =
where Vt was the foot volume at time t after injection of carrageenan and Vo the volume of
foot before the injection of carrageenan in negative and treated animals respectively.
LVNc -LVTa
LVNc
X 100% inhibition of inflammation =Briefly,
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where LVNc was the liquid volume in negative control and LVTa the liquid volume in
animal foot after treatment.
Eliminated liquid volume in treated animal foot = liquid volume in negative control - liquid
volume in treated animal foot (after treatment).
2.4. Determination of analgesic activity of extracts, fractions and polysaccharides from
B. sumatrana leaves
2.4.1. Acetic acid induced writhing method
The determination of analgesic effects of extracts, fractions and polysaccharides of B.
sumatrana leaves was carried out through acetic acid-induced writhes (pains) in experimental
animals (Wistar rats of 155-160 g bw) following procedures described by Shojali et al.,
(2015); Yougbaré-Ziébrou et al., (2016) and Hijazi et al., (2017). Wistar rats were divided
into 6 groups with 5 rats for each oral dose for each group and orally administered test
samples at doses of 50 and 100 mg/kg bw respectively:
- Group I received intraperitoneally acetic acid administered to the experimental animals to
create pain sensations as negative control,
- Group II received Aspirin 5 mg/kg bw was administered 15 minutes prior to acetic acid
injection as positive control,
- Groups IIIa and b received lyophilized aqueous extract BSALE-1,
- Groups IVa and b to VIIa and b received respective extracts of fractions chloroform,
ethylacetate, n-butanol and residual aqueous phase BSLAE-1.1 to BSLAE-1.4 respectively,
- Groups VIIIa an b, IXa and b were given 80% MeOH BSME and total alkaloids BSTA
extracts respectively,
- Groups Xa and b to pure polysaccharide fractions PBSLA-1 to -4. All test samples and
vehicle were administered orally 30 minutes prior to intraperitoneal administration of 3% v/v
acetic acid solution.
Prior to the induction pains and administration of the oral doses of test samples, all the
experimental animals were fasted for 12 hours and were allowed access to water ad libitium.
Pains were induced by injecting intraperitoneally 3% acetic acid solution at a dose of 10
ml/kg bw into the left side of the abdomen of each experimental Wistar rat. Immediately,
after acetic acid injection, abdominal muscle constrictions in the abdomen and turning of
body trunk of the laboratory animals were seen as an indication of pains (Shojali et al., (2015)
and Hijazi et al., (2017). All samples of B. sumatrana leaves (50 and 100 mg/kg bw) and
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reference products Aspirin and Diclofenac (5 mg/kg bw), were separately administered after
30 minutes. The numbers of writhes (writhings: muscular contractions) were counted 5 min
after acetic acid injection over a period of 20 min and after 1, 3 and 5 h respectively. The
number of writhes in each group was compared with the negative control group and the
percent reductions or inhibitions of writhe counts were calculated using the following
formula:
% Inhibition = Wnc -WtsX 100
Wnc
Where Wnc are number of writhes (writhings) of negative control group and Wts writhes of
the tested sample in treated animals group (Shojali et al., 2015; Hijazi et al., 2017).
2.4.2. Hot plate test
The hot plate test was a test of the pain responses in animals similar to the tail-flick test. Both
hot plate and tail-flick methods were generally used for centrally acting analgesic (Calsson et
al., 1987) while peripherally acting drugs were ineffective in these tests, but sensitive to
acetic acid-induced writhes test (Matera et al., 2014). The hot plate test was used in the
present study to measure response latency times. Procedures described by Muhammad et al.,
(2012) and Hiyazi et al. (2017) were used. The hot plate was provided by UGO BASILE
HOT PLATE (Model 7280, Germany). Elapsed times between placement of the animals on
the hot plate and the occurrence of the licking of the hind paws, shaking, or jump off from the
surface were recorded as response latency times in seconds. Wistar rats of either sex
weighing 155-160 g were used. Anti-nociceptive effects of B. sumatrana leaves extracts,
fractions and polysaccharides were investigated using hot plate test in selected rats which
were divided into 6 groups with 5 rats for each oral dose of each group and orally
administered test samples at the doses of 50 and 100 mg/kg bw respectively:
- Group I received 5 ml saline solution (0.9% NaCl) as negative control group,
- Group II received 5 mg Aspirin as positive control,
- Groups IIIa and b, IVa and b were administered lyophilized aqueous BSLAE-1,
- Groups Va and b, and VIa ad b were given extracts of chloroform, ethylacetate, n-butanol
and aqueous residual soluble fractions (BSLAE-1.1 to BSLAE-1.4) respectively,
- Groups VII a and b, VIIIa and b were administered 80% methanol BSME and total
alkaloids extracts respectively,
- Groups IXa and b, X and b, XI and b, XIIa and b received polysaccharide PBSLAEc and
PBSLAE-1 to -4 respectively.
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Figure 3: Hot plate apparatus.
Animals were subjected to pre-testing on a hot plate (Harvard apparatus) maintained at
30.0 ± 0.1°C at room temperature and those having latency times greater than 15 sec on hot
plate during pre-testing were excluded. Pains were induced by placing experimental animals
on the hot plate meter. The latency times for responses were measured at different time
intervals. After 30 min of dose administrations, all rats were dropped inside individual
cylinders onto the hot plate and the latency times (time for which animals remained on the
hot plate without licking or flicking of hind limb or jumping) were recorded. In order to
prevent the tissue damages, the cut-off times of 30 sec were set for all animals. Only rats that
showed initial nociceptive response within 30 seconds were selected and used for the study.
The latency times were recorded for each group at 0, 30, 60, 90 and 120 min respectively.
Percent analgesia produced was calculated using the following formula:
% Analgesia =Test latency - Control latency
Cut-off time - Control latencyx 100
(Muhammad et al., (2012 and Hiyazi et al. (2017)).
The percentage analgesic activity (PAA) was calculated by using the following formula:
PAA= (La-Lb) / Lb ×100
La =Latency time after treatment with drug or extract
Lb=Latency time before treatment with drug or extract.
2.10. Statistical analysis
Statistical analysis of results for animal experiments were expressed as mean ± SEM and
were evaluated by ANOVA followed by Dunnet’s multiple comparisons. The results obtained
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were compared with the vehicle control group. The p value < 0.05 was considered as
significant.
3. RESULTS AND DISCUSSION
3.1. Anti-inflammatory activity of extracts, fractions and polysaccharides from B.
sumatrana leaves
The inflammation was a common phenomenon occurring in the human body. Inflammation
(from Latin: inflammatio) was a part of the complex biological responses of body tissues to
harmful stimulus, such as pathogens, damaged cells or irritants. It was also a protective
response involving immune cells, blood vessels, and molecular mediators. Anti-inflammatory
agents as steroidal compounds by injections destroyed and possibly induced the redistribution
of blood peripheric lymphocytes (Ejebe et al., 2010; Saleem et al., 2015). They can have
beneficial effects in reducing particularly inflammation and relieving various pains in the
human body (Saleem et al., 2015).
The induction of paw edema by injection of carrageenan in animal foot was well established.
The assessment of anti-inflammatory activity of medicinal plant extracts, isolated natural and
synthetic products in vivo was currently performed using this method. The treatment of liquid
volume increased by carrageenan injection in animal foot (paw edema) provoked a biphasic
anti-inflammatory responses for which the initial phase lasted about 1h30 minutes after
injection. It showed the release of serotonins, histamines, kinins and bradykinins or again the
development of paw edema in animal foot after carrageenan solution injection due to the
release of these biochemical mediators (Georgewill et al., 2010a,b; Saleem et al. 2015; Singh
et al., 2016). The second taking place after, was due to the biosynthesis of prostaglandins, and
release of proteases and lysosomes (Sonialmol et al., 2011; Livia de Paulo et al., 2012;
Yougbaré-Ziébrou et al., 2015; Singh et al., 2016).
In the present study to appreciate the level of anti-inflammatory and analgesic activity,
following criteria were taken in account: 90 < IAi, IAa ≤ 100%; pronounced activity, 70 ≤
IAi, IAGa < 90: good activity, 50 ≤ IAi, IAa ≤ 70: moderate activity; 40 ≤ IAi, IAa < 50,
weak activity, IAi, IAa < 40%: inactive. (IAi: inhibition of anti-inflammatory and IAa;
inhibition of analgesic activity respectively). The ingestion of carrageenan solution in animal
foot induced increase of liquid volume in treated animal foot which known a swelling called
paw edema.
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Results obtained in this pharmacological model, indicated that samples of B. sumatrana
leaves were found to cause significantly (p < 0.05) the decrease of paw edema in treated
animal foot in dose-dependent manner by inhibiting the action of carrageenan. The liquid
volume of treated animal foot measured after treatment (liquid again present in foot after
treatment) were presented in Table 1.
Table 1: Anti-inflammatory effects of extracts, fractions and polysaccharides extracts
from B. sumatrana leaves: foot volume (ml) produced by samples in the presence of
carrageenan after treatment.
Samples and
groups TTT
Foot volume (ml) in treated
rats after treatment
% inhibition of inflammation
after 5 h of treatment
1h 3h 5h
N. control I 5 ml DW 0.27 0.31 0.35 0.00±0.00
Diclofenac II 5 0.5 0.3 0.1 97.14±0.03
BSLAE-1 IIIa 50 0.15 0.11 0.9 74.28±0.01
IIIb 100 0.12 0.9 0.6 82.85±0.03
BSLAE-1.1 IVa 50 0.20 0.18 0.16 54.28±0.02
IVb 100 0.16 0.14 0.11 65.71±0.02
BSLAE-1.2 Va 50 0.17 0.15 0.10 71.42±0.01
Vb 100 0.15 0.12 0.8 77.42±0.04
BSLAE-1.3 VIa 50 0.14 0.21 0.18 48.57±0.01
VIb 100 0.18 0.17 0.13 62.85±0.02
BSLAE-1.4 VIIa 50 0.19 0.16 0.14 60.00±0.01
VIIb 100 0.14 0.12 0.10 71.42±0.03
BSAT VIIIa 50 0.16 0.10 0.4 88.57±0.03
VIIIb 100 0.14 0.9 0.2 94.28±0.02
BSME IXa 50 0.16 0.9 0.5 85.71±0.04
IXb 100 0.13 0.10 0.4 88.57±0.02
PBSLAEc Xa 50 0.11 0.7 0.5 85.57±0.03
Xb 100 0.9 0.6 0.3 91.42±0.01
PBSLAE-1 XIa 50 0.10 0.13 0.6 82.85±0.04
XIb 100 0.12 0.8 0.5 85.74±0.02
PBSLAE-2 XIIa 50 0.14 0.11 0.9 74.28±0.01
XIIb 100 0.12 0.9 0.7 80.00±0.03
PBSLAE-3 XIIIa 50 0.15 0.13 0.10 71.42±0.01
XIIIb 100 0.13 0.11 0.8 77.14±0.02
PBSLAE-4 XIVa 50 0.14 0.12 0.10 71.42±0.00
XIVb 100 0.11 0.9 0.6 82.85±0.02
N. control: negative control, TTT: treatment, DW: distilled water, BSLAE-1: lyophilized
aqueous extract, BSLAE-1.1 to -1.4: chloroform, ethylacetate, n-butanol and aqueous soluble
fractions respectively, BSAT and BSME: total alkaloids and 80% methanol extracts
respectively, PBSLAEc: crude polysaccharide, PBSLAE 1 - 4: isolated pure polysaccharides.
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By use of difference of foot liquid volume in negative control group to that in foot of treated
animals, the liquid volume again present in treated animal foot was found. In this way, it was
observed that high amount of liquid volume in treated animals was eliminated as a sign of
foot paw edema decrease. Results of this effect indicated that paw edema induced by
carrageenan solution injection was automatically reduced by the oral administration of all
samples from B. sumatrana leaves. By this effect, they exerted their anti-inflammatory
activity by producing significant (p < 0.05) reduction of treated animal foot liquid volume
(liquid after treatment) from 0.15 to 0.33 ml after 1, 3 and 5 h, compared to negative control
presenting 0.27, 0.31 and 0.35 ml of foot volume liquid respectively at the same time of
treatment (Table 1). At the highest oral dose of 100 mg/kg bw, lyophilized aqueous extract
BSLAE-1 caused 0.29 ml reduction of liquid volume of treated animal foot corresponding to
82.85% reduction of paw edema compared to negative control showing 0.35 ml after 5h of
treatment with 0% reduction of paw edema. At the same oral dose, total alkaloids BSTA and
80% methanol BMSE extracts acted in the same manner by reducing treated animal foot
liquid volume of 0.33 and 0.31 ml compared to negative control with 0.35 ml and
corresponded to 94.28 and 88.57% reduction of paw edema respectively after the same time,
with BSTA extract as the most active compared to BSLAE-1 and BSME extracts (Table 1).
These three extracts showed their pronounced anti-inflammatory activity and the decreasing
order of their activity can be established as BTSA > BME > BSALE-1 > NC.
Moreover, all soluble fractions from the partition of lyophilized aqueous BSLAE- extract also
showed good reduction of paw edema in treated animal foot. At the highest oral dose of 100
mg/kg bw, they produced significant (p < 0.05) reduction of treated animal foot liquid
volume from 0.11 to 0.8 ml compared to negative control with 0.35 ml in untreated animal
foot. These diminutions corresponded to 65.71 to 77.42% reduction of paw edema
development with ethylacetate BSLAE-1.2 rich in flavonoids as the most active compared to
remaining soluble fractions (Table 1). The decreasing order of their effects can be established
as BSLAE-1.2 (77.42%) > BSLAE-1.4 (71.42%0 > BSLAE-1.1 (65.71%) > BSLAE-1.3
(62.85%) > NC (0%).
Figures 4 and 5showed the reduction of treated animal foot liquid volume increased by the
administration of carrageenan solution, provoked by the oral administration of lyophilized
aqueous extract BSLAE-1 and its soluble fractions BSLAE-1.1 to BSLAE-1.4, 80% MeOH
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Figure 4: Eliminated liquid volume of treated animal foot produced by lyophilized
aqueous BSLAE-1, MeOH BSME and total alkaloids BSAT extracts at oral dose of 100
mg/kg bw, diclofenac (Diclof) and negative control (NC).
BSME and total alkaloids extracts respectively. Extracts BSLAE-1, BSME and BSAT
produced 82.85, 88.57 and 91.42% reduction of treated animal foot paw edema respectively
follow-up to the elimination of treated animal foot liquid volume of 0.29, 0.31 and 0.32 ml
respectively, at the administered highest oral dose of 100 mg/kg bw after 5h of treatment
(Fig. 4).
All soluble fractions BSLAE-1.1 to BSLAE-1.4 acted in the same manner as the parent
extract by producing also 60.00 to 78.14% reduction of treated animal foot paw edema with
ethylacetate soluble fraction as the most active at the same highest oral dose of 100 mg/kg bw
due to the elimination of treated animal foot liquid volume from 0.22 to 0.27 ml compared to
negative control (0.35 ml) (Fig. 5).
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Figure 5: Eliminated liquid volume of treated animal foot produced by soluble fractions
BSLAE-1.1 to BSLAE-1.4 from the partition the lyophilized aqueous extract BSLAE-1
and diclofenac sodium.
Polysaccharides from B. sumatrana leaves were also assessed for their potential anti-
inflammatory activity against carrageenan induced paw edema of rats foot. Results indicated
that crude polysaccharide PBSLAEc and pure polysaccharide fractions exhibited markedly
anti-inflammatory activity by reducing significantly (p < 0.05) treated animals liquid volume
foot at the highest oral dose of 100 mg/kg from 0.27 to 0.32 ml compared to negative control
with 0.35 ml after 5 h of treatment. This percentage reduction of paw edema in treated animal
foot brought, was from 77.14 to 91.42%. PBSLAEc extract showed high elimination (0.32
ml) that corresponded to 91.42% reduction of paw edema as the most active compared to four
pure polysaccharide fractions PBSLAE-1 to -4.
The four chromatographically pure isolated polysaccharides had also the same effects by
causing significant reduction liquid volume of treated animal foot (p < 0.05) from 0.27 to
0.30 ml compared to negative control with 0.35 ml. This reduction corresponded to 0.77.14 to
0.85% reduction of paw edema development of treated animal foot. The decreasing order of
their activity can be presented as PBSLAEc (91.42±0.01%) > PBSLAE-1 (85.74±0.02%) >
PBSLAE-4 (82.85±0.02% > PBLAE-2 (80.00±0.03%) > PBSLAE-3 (77.14±0.02%) > NC
(0%). The activity of pure isolated polysaccharides was weak compared to crude
polysaccharide. This finding suggested that these chemicals will act in synergistic manner to
restore the activity level of the parent extract. They can partly be considered as active anti-
inflammatory principles of B. sumatrana leaves extract.
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Figure 6 showed the eliminated volume of liquid in treated animal foot after 5 h of treatment
with polysaccharides. This liquid volume was eliminated slowly from 1 h and continued to
significantly increase until 5 h to reach the high eliminated volume of 0.32 ml showed by
crude polysaccharide PBSLAEc, followed by pure polysaccharide fractions PBSLAE-1 with
0.30 ml, PBSLAE-4 with 0.29 ml, PBSLAE-2 with 0.28 ml and PBSLAE-3 with 0.27 ml
corresponding to different percent reductions of paw edema in treated rats foot already
mentioned above.
Figure 6: Eliminated liquid volume of treated animal foot by polysaccharides,
diclofenac sodium and negative control (NC).
Diclofenac had carried out the reduction of liquid volume of treated animal foot to 0.34 ml
corresponding to 97.14% of paw edema reduction compared to negative control (0.35 ml, %
reduction) and showed high anti-inflammatory activity compared to all samples from B.
sumatrana leaves (Fig.4,5 and 6).
Other studies have postulated that the antinociceptive activity of plant extracts may be due to
inhibition of interleukin-1β and interleukin-8 release by resident peritoneal cells or to
suppression of prostaglandins and bradykinins (Jamaluddin et al., 2011). Therefore, it can be
assumed that the inhibitory effect of the all samples from B. sumatrana leaves on
carrageenan-induced inflammation could be due also to the inhibition of the enzyme
cyclooxygenase and lipoxygenase, leading to the inhibition of prostaglandin synthesis as also
previously reported by Patel et al., (2014) for the combination extracts from Vitex negundo
and Murraya koenigii and by the inhibition of the release of inflammatory mediators cited
above. Our results are in good agreement with other studies reporting previously the anti-
inflammatory activity of other isolated polysaccharides in various medicinal plant species
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(Soniamol et al., 2011; Hur et al., 2012; Wang et al., 2013, Ibrahim et al., 2014; Zhou et al;.,
2014, Iluri et al., 2015; Cheng et al., 2016, Rjeibiek et al., 2020).
The investigation of plant extracts and natural phytochemical products presenting more than
70% declining of paw edema provoked by various medicinal plant extracts empirically used
to treat rheumatism and other pains in traditional medicine, can be used to treat rheumatism
and other pains. They can also lead to the discovery of promising new anti-inflammatory
agents with interesting effects in animal models without significant side effects and toxicity
as alternative to known anti-inflammatory medicines having many sides effects in humans
(Sarpong et al., 2019).
Many studies have reported that anti-inflammatory activities of herbal extracts and herb-
derived compounds were mainly due to their inhibition of arachidonic acid (AA) metabolism,
cyclooxygenase (COX), lipoxygenase (LOX), pro-inflammatory cytokines, inducible nitric
oxide, and transcription activation factor (NF-êB). Some anti-inflammatory medicinal herbs
were reported to stabilize lysosomal membrane and some caused the uncoupling of oxidative
phosphorylation of intracellular signalling molecules. Many had also been shown to possess
strong oxygen radical scavenging activities (Nworu and Akah, 2015). In addition to the above
mentioned justifications, it can be suggested that the observed anti-inflammatory effects of
samples from B. sumatrana leaves may also be due to their positive actions or effects
(inhibition) on these anti-inflammatory mediators, on the state of some membranes and other
pathways implicated in inflammation.
3.3. Analgesic activity of extracts, fractions and polysaccharides from B. sumatrana
leaves
Analgesia was defined as a state of reduced awareness to pains, and analgesic compounds
were substances, which decreased pain sensations and also called painkillers acting by
increasing threshold of painful stimulus. Analgesic drugs were also known as anti-
inflammatory drugs, due to their actions to reduce local inflammatory responses. Most anti-
inflammatory-analgesic drugs were derived from two compounds: salicylic acid and
phenacetin (Henna and Znad, 2018). Those usually used were Aspirin, Paracetamol (non -
narcotic type), Caffeine and Morphine (narcotic type) and other. Painful reactions in
experimental animals can be produced by applying noxious or unpleasant stimulus such as
thermal (radiant heat as a source of pains), chemicals (irritants such as acetic acid and
bradykinin) and physical pressures (tail compression) (Shashank et al., 2013).
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Table 2 presented effects of extracts, fractions and polysaccharides from B. sumatrana leaves
on acetic acid-induced writhes in tested animals. Results revealed that all tested samples from
B. sumatrana leaves exhibited prominent reduction (p < 0.001) of acetic acid-induced writhes
in treated animals in a dose-dependent manner (Table 2). At the highest oral dose of 100
mg/kg bw, lyophilized BSLAE-1, 80% methanol BSME and total alkaloids BSTA extracts
produced more than 84% writhe inhibitions with BSME extract as the most active
(86.32±0.03%), followed by total alkaloids BSTA extract (85.68 ±0.03% inhibition) and
lyophilized aqueous BSLAE-1 extract (84.71±0.01%inhibition). A significant difference (p <
0.05) was observed between the activity of BSME compared to BSLAE-1, but not between
BSME and BSTA, and BSTA compared to BSLAE-1 (p > 0.05) (Table 2).
Table 2: % Inhibition of treated animal writhes by extracts, fractions and
polysaccharide extracts from B. sumatrana leaves (Analgesic activity)
Samples Treatment at oral
dose, mg/kg bw)
Number of writhe
movements after 120
minutes
% inhibition of writhe
movements after 120
minutes
Acetic acid I 10 ml 18.65±0.01 -
Aspirin II 5 2.00±0.00 89.27±0.01
BSLAE-1 IIIa 50 3.05±0.01 83.64±0.02
IIIb 100 2.85±0.00 84.71±0.01
BSLAE-1.1 IVa 50 4.95±0.00 73.45±0.01
IVb 100 5.85±0.01 74.00±0.03
BSLAE-1.2 Va 50 4.70±0.03 74.80±0.0
Vb 100 4.32±0.02 76.83±0.01
BSLAE-1.3 VIa 50 4.62±0.02 75.22±0.02
Vib 100 4.55±0.02 75.60±0.00
BSLAE-1.4
VIIa 50 4.85±0.00 74.00±0.03
VIIb 100 4.41±0.00 76.35±0.01
BSTA
VIIIa 50 2.85±0.02 84.78±0.00
VIIIa 100 2.67±0.02 85.68±0.03
BSME
IXa 50 2.75±0.01 85.25±0.01
IXb 100 2.55±0.03 86.32±0.03
PBSLAEc
Xa 50 3.25±0.02 82.57±0.00
Xb 100 2.75±0.01 85.25±0.01
PBSLAE-1
XIa 50 3.40±0.03 81.77±0.02
XIb 100 3.30±0.03 82.30±0.01
PBSLAE-2 50 3.28±0.01 82.41±0.02
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XIIa
XIIa 100 3.12±0.02 83.27±0.01
PBLAE-3
XIIIa 50 3.50±0.01 81.23±0.00
XIIIb 100 3.40±0.03 81.77±0.03
PBSLAE-4
XIVa 50 3.35±0.02 82.03±0.03
XIVb 100 3.15±0.00 83.11±0.01
See Table 1
Figure 7 showed the effects of lyophilized aqueous BSLAE-1, 80% methanol extract BSME
and total alkaloids BSTA, and fractions BSLAE-1.1 to -1.4 on writhe movements of treated
animals respectively. It was observed that all extracts induced significant reduction (p < 0.05)
of writhe movements of treated animals from 2.25 to 2.67 compared to negative control
showing 18.65.
Figure 7: Effects of extracts of B. sumatrana leaves on writhe movements of treated
animal foot after treatment.
Soluble factions from the partition of lyophilized aqueous extract BSLAE-1 acted also in the
same manner as the parent extract by reducing significantly writhe movements from
4.32±0.02 to 6.25±0.02 with ethylacetate BSLAE-1.2 showing high effect (4.32±0.02)
followed by residual aqueous (4.41±0.00), n-butanol, chloroform (4.55±0.000) and BSLAE-
1.3 (4.95±0.000,. These reduction effects of writhe movements corresponded to percentage
reductions of writhe developments from 67.02±0.00 to 76.83±0.01%. The most active soluble
fraction being ethylacetate BSLAE-1.2 (76.83±0.01) followed by residual aqueous BSLAE-
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1.4 (76.33±0.000), n-butanol BSLAE-1.3 (75.60±0.01) and chloroform BSLAE-1.1
(74.00±0.01) with good activity. No significant difference (p > 0.05) was observed between
the activity of BSLAE-1.2 compared to BSLAE-1.3 and BSLAE-1.4, but this existed when
BSLAE-1.2 and BSLAE-1.4 were compared to BSLAE-1.1 and BSLAE-1.3 in pairs or
individually state.
Figure 8 showed the effects of soluble fractions on writhe movements of treated animals and
indicated that these samples significantly reduced this parameter from 4.32 to 5.85 compared
to negative control showing 18.65.
Figure 8: Effects of soluble fractions BSLAE-1.1 to -1.4 of B. sumatrana leaves on writhe
movements of treated animal foot after treatment
On the other hand, at the highest oral dose of 100 mg/kg bw, crude PBSLAEc and pure
polysaccharide fractions PBSLAE-1 to -4 exerted markedly and pronounced analgesic effects
by producing percentage reduction of writhes by 81.77±0.03 to 85.25±0.01% with the crude
polysaccharide PBSLAEc as the most active (85.25±0.01%) followed by pure polysaccharide
fractions PBLAE-2 (83.27±0.03%) and PBSLAE-4 (83.11±0.01%) showing almost the same
level of activity since no significant difference (p > 0.05) was deduced, PBSLAE-1
(82.30±0.01) and PBSLAE-3 (81.27±0.03) with comparable analgesic effects. Significant
difference (p < 0.05) in activity was observed between polysaccharide extract PBLSAEc
compared to their respective pure polysaccharide fractions PBSLAE-1 to -4. In addition the
activity displayed by all pure ploysaccharide fractions PBSLAE-1 to -4 were weak compared
to crude extract polysaccharide PBSLAEc. This finding suggested that these pure
polysaccharide fractions will act in synergistic manner to reproduce the activity level of the
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parent extract. Aspirin, used as a reference analgesic product declined writhe production by
89.27±0.03% as pronounced activity compared to all samples from B. sumatrana leaves.
Figure 9 presented the effects of polysaccharide of with movements of treated rats at the
highest oral dose f 100 mg/kg bw and indicated that these samples were able to induced
significant reduction of writhe movement during the treatment from 2.75 to 3.40 compared to
negative control with 18.65. Aspirin as analgesic reference (5 mg/kg bw) product reached
significant reduction to 2 and showed high analgesic activity compared to B. sumatrana
leaves samples.
Figure 9: Effects of polysaccharides PBLAEc and PBSLAE-1 to -4 of B. sumatrana
leaves on writhe movements of treated animal foot after treatment.
Acetic acid-induced writhe movements had been associated with increased level of some
inflammatory mediators like PGE2 and PGF2α in peritoneal fluids as well as lipoxygenase
products and release of prostangladins (Shoiab et al., 2016). The writhing method was found
effective to evaluate peripherally and centrally active analgesics as it was found in the present
study for B. sumatrana samples and other previously studies already cited above. In addition,
acetic acid performed its action by release of endogenous mediators like prostaglandins from
arachidonic acid through cyclooxygenase enzymes. These prostaglandins stimulated the
nociceptive neurons with induction of pain sensations (Shoiab et al., 2016). Thus, the
analgesic effects of samples from B. sumatrana leaves found in the present study by this
assay, may be due probably to the inhibition of prostaglandin synthesis, which was
considered as a peripheral mechanism of pain inhibitions (Zulfiker et al., 2010) and the
release of endogenous mediators, the decrease activity of some inflammatory mediators like
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PGE2 and PGF2α in peritoneal fluids as well as cyclogenase and lipoxygenase products, and
other mechanisms of inflammation mentioned above. In other words, the significant pain
reductions demonstrated in the present study by selected plant extracts, fractions and
polysaccharides might be due to the presence of analgesic principles acting with the
prostaglandin pathways (Zulfiker et al., 2010).
The abdominal constriction responses induced by acetic acid were a sensitive procedure to
evaluate the potential analgesic activity of drugs. It had been suggested that acetic acid acted
by releasing endogenous mediators that stimulated the nociceptive neurons (Arciniega et al.,
2015). It was sensitive to non-steroidal anti-inflammatory drugs (NSAIDs), narcotics and
other centrally acting drugs (Bagda et al., 2013; Arciniega et al., 2015; Azab et al., 2016).
Recently, it was found that the nociceptive activity of acetic acid may be due to the release of
cytokines, such as TNF-α, interleukin-1β and interleukin-8, by resident peritoneal
macrophages and mast cells (Jamaluddin et al. 2011). Results reported in the present study
might also indicated that the antinociceptive action of B. sumatrana samples from the leaves
in this pharmacological model test could be due to inhibition of the release of these mediators
as also reported by Jamaluddin et al. (2011) for different extracts of the whole plant
Amaranthus spinosus L. and must have central and peripheral activity perhaps acting in a
similar manner as conventionally used therapeutic drugs that reduced pain perceptions in
nociceptors by inhibiting production of prostaglandins (Koech et al., 2017). It also then acted
in contrary to acetic acid effects to manifeste its analgesic effects.
3.4. Hot Plate method
The hot plate test was a test of the pain responses in animals, similar to the tail-flick test.
Both hot plate and tail-flick methods were used generally for centrally acting analgesic
Table 3: Latency times in seconds.
Samples TTT
(mg/kg bw) Latency times in seconds
% Analgesic
effects after
120 min
0 min 30 min 60 min 120 min
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NC 5ml DW 9.35±0.03 9.41±0.01 9.38±0.02 9.34±0.00 -
Aspirin
I 5 9.78±0.00 27.06±0.01 29.15±0.02 38.68±0.01 75.85±0.03
Diclofenac
II 5 9.87±0.09 27.69±0.00 29.36±0.01 39.12±0.01 76.12±0.00
BSLAE-1
IIIa 50 14.25±0.00 16.35±0.03 22.85±0.01 38.15±0.01 75.51±0.02
IIIb 100 16.20±0.03 18.85±0.03 23.54±0.01 38.95±0.03 76.02±0.01
BSLAE-1.1
IVa 50 11.21±0.00 21.05±0.00 25.25±0.01 31.35±0.02 70.20±0.01
IVb 100 13.02±0.02 23.17±0.02 28.03±0.03 33.02±0.01 71.71±0.03
BSLAE-1.2
Va 50 13.42±0.01 15.85±0.02 19.85±0.00 33.94±0.03 72.48±0.00
Vb 100 9.37±0.00 16.02±0.00 21.02±0.00 35.05±0.00 73.35±0.03
BSLAE-1.3
VIa 50 10.25±0.02 18.35±0.02 23.24±0.02 29.13±0.02 67.93±0.03
VIb 100 13.07±0.00 21.78±0.02 24.94±0.00 31.01±0.02 69.88±0.02
BSLAE-1.4
VIIa 50 9.28±0.01 15.92±0.00 20.06±0.00 31.92±0.02 70.74±0.03
VIIb 100 11.30±0.03 16.12±0.01 22.35±0.03 33.68±0.02 72.26±0.01
BSME
VIIIa 50 9.39±0.04 16.25±0.01 21.85±0.03 37.02±0.03 74.77±0.01
VIIIb 100 14.45±0.02 17.95±0.03 24.78±0.03 41.25±0.01 77.35±0.00
BSTA
IXa 50 9.43±0.01 16.95±0.03 23.86±0.01 38.12±0.00 75.50±0.02
IXb 100 16.51±0.01 18.25±0.02 25.24±0.00 43.15±0.03 78.35±0.03
PBSLAEc
Xa 50 16.58±0.00 18.02±0.03 23.78±0.01 40.15±0.01 76.73±0.01
Xb 100 20.12±0.03 24.03±0.01 26.24±0.00 42.05±0.03 77.78±0.03
PBSLAE-1
XIa 50 15.26±0.02 24.25±0.01 27.05±0.03 35.85±0.01 73.94±0.02
XIb 100 17.23±0.01 20.55±0.01 23.85±0.01 38.05±0.02 75.45±0.02
PBSLAE-2
XIIa 50 20.85±0.01 23.58±0.02 25.64±0.01 37.95±0.01 75.38±0.01
XIIb 100 22.58±0.02 25.69±0.00 29.06±0.03 39.75±0.00 76.50±0.00
PBSLAE-3
XIIIa 50 21.23±0.02 23.56±.002 26.35±0.01 35.65±0.03 73.80±0.03
XIIIb 100 23.06±0.02 25.65±0.02 29.85±0.03 37.65±0.02 75.19±0.02
PBSLAE-4
XIV a 50 22.56±0.03 26.35±0.01 28.69±0.02 36.68±0.03 74.53±0.01
XIVb 100 25.63±0.01 28.92±0.03 30.25±0.01 38.15±0.00 75.51±0.03
TTT: treatment, DW: distilled water
(Calsson et al., 1978) while peripherally acting drugs were ineffective in these tests, but
sensitive to acetic acid-induced writhing test (Matera et al., 2014). The method measured the
complex response to a non-inflammatory and acute nociceptive input. It was one of the
models normally used for studying central antinociceptive activity. It was an established fact
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Kanyanga et al. World Journal of Pharmacy and Pharmaceutical Sciences
that any agent that caused a prolongation of the hot plate latency times using this test may act
centrally and peripherally (Ibironke and Ajiboye, 2007). In the present study, various plant
extracts, fractions and polysaccharides from B. sumatrana leaves when given in doses of 50
and 100 mg/kg, p.o elicited a significant analgesic activity in the hot plate as evidenced by
the increase of latency times expressed in seconds (Table 3) as compared to vehicle control at
the end of 120 min. The increase in latency times was dose-dependent. Different extracts
including lyophilized aqueous BSALAE-1, 80% methanol BSME and total alkaloids BSTA
as well soluble fractions BSALE-1.1 to BSLAE-1.4, chloroform, ethylacetate, n-butanol and
residual aqueous phase from the partition of BSALAE-1 extract, demonstrated markedly
analgesic activity on hot plate by the increase of the latency times in treated animals (Table
3). With the administration of the highest oral dose of 100 mg/kg bw, extracts from B.
sumatrana leaves gave percent inhibitions of latency times of 76.02±0.01, 77.35±0.03% and
78.35±0.00 for lyophilized aqueous, 80% MeOH and total alkaloids extracts respectively,
with the total alkaloids BSTA extract as the most active extract compared to the two other
(Table 3).
In the same manner, soluble fractions also exhibited analgesic activity by significant increase
of latency times from 31.01±0.02 to 35.05±0.02 sec. Among these soluble fractions,
ethylacetate BSLAE-1.2 showed high activity with 35.05±0.02 latency time (Lt) and
73.35±0.03% analgesic effect (Ae), followed by residual aqueous BSLAE-1.4 (33.68±0.02 Lt
and 2.26±0.01% Ae), chloroform BSLAE-1.1 (33.02±0.02 Lt and 71.71±0.02% Ae) and n-
butanol BSLAE-1.4 (31.01±0.02 Lt and 69.88±0.02 Ae). Significant difference (p < 0.05)
was observed between the activities of these soluble fractions compared between them.
At the highest oral dose of 100 mg/kg bw, polysaccharides from B. sumatrana leaves also
caused significant increase of latency times (p < 0.05) from 37.65 to 42.05 seconds after 120
minutes of treatment and corresponded to 75.19±0.02 to 77.78±0.03% production of
analgesic effects. The most active polysaccharide was crude polysaccharide PBSLAEc with
42.65±0.03 sec latency time (Lt) and 77.78±0.03% analgesic effect (Ae), followed by pure
polysaccharide fraction PBSLAE-2 (39.75 Lt and 776.50% Ae), PBSLAE-4 (38.15 Lt and
75.51±0.02% Ae), PBSLAE-1 (38.05 Lt and 75.45±0.02 Ae) and PBSLAE-3 (37.65 Lt and
75.19±0.002% Ae) compared to negative control (9.34, 0% Ae). Their effects were
considered as good, promising and appreciable since many samples showed a percentage
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production of analgesic effects more than 70% by the increase of latency times compared to
negative control (Table 3).
The results clearly indicated all samples from B. sumatrana leaves exhibited potent analgesic
effects without causing any gastric damages supporting previous claims of their anti-
inflammatory and analgesic effects as also reported for Withania somnifera which may be a
useful contribution to highlight the action mechanism as arthritic drug in heathly human
volunteers using mechanical pain model (Murthy et al., (2019). Much attention on screening
analgesic of natural substances activities was made on acetic acid-induced writhes in tested
animal model (Hasan et al., 2010). The analgesic effect displayed by Aspirin (75.85%) used
as reference product was weak compared to lyophilized, 80% methanol and total alkaloids
extracts (76.02 -78.35%), crude polysaccharide (77.78%) and pure polysaccharide fraction
PBSLAE-2 (76.50±0.00%), but high compared to all soluble fractions BSLAE-1.1 to -1.4
(71.71-73.35%), and comparable to pure polysaccharide fractions PBSLAE-1-to -3 (75.19-
75.90%). From these results, samples of B. sumatrana leaves suppressed abdominal
constriction responses induced by acetic acid and inhibited pain sensations in a pattern
similar to Aspirin used as a reference drug to manifeste their analgesic effects.
The literature had reported secondary metabolites and phytochemical groups with anti-
inflammatory activity in animal models. They included polysaccharides (Soniamol et al,
2011; Livia de Paulo et al., 2012), terpenes and steroids (Arciniega et al., 2015, Vega et al.,
2017), flavonoids and biflavonoids (Sarafini et al., 2010, Pascoal et al., 2014), essential oils
(Menezes et al., 1990, Bourkhiss et al., 2010; Nguyen et al., 2020), alkaloids (Bribi et al.,
2015; Marya and Khan, 2017; Wen et al., 2018), saponins (Grabowska et al., 2018). Those
reported as analgesics included alkaloids (Hayfaa et al., 2013; Bribi et al., 2015;
Tunsunkhadjaeva et al., 2015; Shoiab et al., 2016), polysaccharides (Livia et al., 2012;
Ibrahim et al., 2014; Iluri et al., 2015; Cheng et al., 2016) , tannins, flavonoids, steroids and
terpenes (Bagdad et al., 2013; Abdel-Rahman, 2016). Flavonoids were reported to have a role
in analgesic activity primarily by targeting prostaglandins. Annegowda et al., (2010) reported
that flavonoids showed analgesic action by enhancing the endogenous serotonins level or
interact with 5-HT2A and 5-HT3 receptors. Alkaloids were well known for their ability to
inhibit pain perceptions (Zulfiker et al., 2010) and other reports insisted on the role played by
tannins in anti-nociceptive and anti-inflammatory activities of medicinal plant extracts
(Annegowda et al., 2010; Hemayet et al., 2014; Nguyen et al.,2020). The presence of some
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phytochemical groups previously identified in B. sumatrana leaves (Tshodi et al., 2012;
Cimanga et al., 2015) may account for the observed both evaluated biological activities of
extracts and fractions of the studied plant part. Isolated polysaccharide fractions can be in
part, considered as the ones of active anti-inflammatory and analgesic principles of this
studied medicinal plant.
In general, the anti-inflammatory and analgesic effects showed by B. sumatrana leaves
samples were interesting, encouraging and promising. Obtained results can support and
justify the use of the plant part in traditional medicine to treat rheumatism and other pains
without significant side effects in humans.
CONCLUSION
The present study reported for the first time the anti-inflammatory and analgesic activities of
extracts and fractions as well as polysaccharides of B. sumatrana leaves collected in Mai-
Ndombe in Democratic Republic of Congo. Results indicated that extracts, fractions and
polysaccharides exhibited good and interesting both evaluated biological activities in animal
model. Thus, the traditional use of B. sumatrana leaves to treat rheumatism and other pains in
popular medicine in various African countries can be supported and justified by these
reported results in the present study.
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