Analgesic and Anti-Inflammatory Activities of 80% Methanol Extract and Solvent Fractions of Ehretia cymosa Thonn (Boraginaceae) Leaves in Rodents

Abstract

Background
Ethnobotanical studies in various districts of Ethiopia reported that Ehretia cymosa (E. cymosa) is used for the management of headache, abdominal pain, arthritis and rheumatism. However, there is no scientific investigation done so far to confirm these traditional claims. Thus, the aim of this study was to assess the analgesic and anti-inflammatory effects of the 80% methanol extract and fractions of E. cymosa leaves.

Methods
The dried and pulverized leaves of E. cymosa were soaked with 80% methanol to obtain a crude extract. Fractionation was done using chloroform, ethyl acetate and water by a soxhlet apparatus. The analgesic effects of the crude extract and solvent fractions were assessed using acetic acid-induced writhing and hot plate tests whereas anti-inflammatory activities were investigated using carrageenan-induced paw edema and cotton-pellet-induced granuloma models.

Results
In all the tested doses, the 80% methanol extract and solvent fractions revealed substantial (p < 0.001) analgesic activities in acetic acid induced writhing test. In the hot plate method, all the tested doses of E. cymosa crude extract and the solvent fractions produced significant analgesic activities (p < 0.05). In the carrageenan-induced acute inflammation model, all tested doses of the crude extract and solvent fractions resulted in a significant decline in paw edema. The 80% methanol extract and solvent fractions of E. cymosa at all the tested doses significantly reduced inflammatory exudates and granuloma mass formations (p < 0.001).

Conclusion
From the results of this investigation, it can be stated that 80% methanol extract, aqueous, ethyl acetate and chloroform fractions of E. cymosa exhibited considerable analgesic and anti-inflammatory activities, supporting the plant’s traditional use as a remedy for a variety of painful and inflammatory conditions.

Full text

ORIGINAL RESEARCH
Analgesic and Anti-Inammatory Activities of 80%
Methanol Extract and Solvent Fractions of Ehretia
cymosa Thonn (Boraginaceae) Leaves in Rodents
Getachew Ashagrie
1
, Abiy Abebe
2
, Shemsu Umer
3
1
Department of Pharmacy, College of Health Sciences, Woldia University, Woldia, Ethiopia;
2
Biomedical Research Team, Traditional and Modern
Medicine Research Directorate, Ethiopian Public Health Institute, Addis Ababa, Ethiopia;
3
Department of Pharmacology and Clinical Pharmacy, School
of Pharmacy, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia
Correspondence: Getachew Ashagrie, Tel +251927368467, Email Getachewashagrie16@gmail.com
Background: Ethnobotanical studies in various districts of Ethiopia reported that Ehretia cymosa (E. cymosa) is used for the
management of headache, abdominal pain, arthritis and rheumatism. However, there is no scientic investigation done so far to
conrm these traditional claims. Thus, the aim of this study was to assess the analgesic and anti-inammatory effects of the 80%
methanol extract and fractions of E. cymosa leaves.
Methods: The dried and pulverized leaves of E. cymosa were soaked with 80% methanol to obtain a crude extract. Fractionation was
done using chloroform, ethyl acetate and water by a soxhlet apparatus. The analgesic effects of the crude extract and solvent fractions
were assessed using acetic acid-induced writhing and hot plate tests whereas anti-inammatory activities were investigated using
carrageenan-induced paw edema and cotton-pellet-induced granuloma models.
Results: In all the tested doses, the 80% methanol extract and solvent fractions revealed substantial (p < 0.001) analgesic activities in
acetic acid induced writhing test. In the hot plate method, all the tested doses of E. cymosa crude extract and the solvent fractions
produced signicant analgesic activities (p < 0.05). In the carrageenan-induced acute inammation model, all tested doses of the crude
extract and solvent fractions resulted in a signicant decline in paw edema. The 80% methanol extract and solvent fractions of
E. cymosa at all the tested doses signicantly reduced inammatory exudates and granuloma mass formations (p < 0.001).
Conclusion: From the results of this investigation, it can be stated that 80% methanol extract, aqueous, ethyl acetate and chloroform
fractions of E. cymosa exhibited considerable analgesic and anti-inammatory activities, supporting the plant’s traditional use as
a remedy for a variety of painful and inammatory conditions.
Keywords: analgesic, hot plate, anti-inammatory, Ehretia cymosa, carrageenan-induced paw edema, cotton pellet granuloma
Background
Pain is an unpleasant sensory and emotional sensation connected to or similar to actual or potential tissue injury.
1
Although it is an unpleasant sensation, it has protective benets through providing warning signal about the existence of
a problem or threat.
2
Pain is a dynamic and intricate phenomenon involving the interaction of numerous receptors,
neurotransmitters, neural bers, neural pathways and both distinct and dispersed anatomical sites. It begins physiologi-
cally with the stimulation of high-threshold primary sensory neurons (the nociceptor that detect noxious stimuli) and by
particular transmission mechanism in their peripheral terminals and transmitted to the CNS via specialized neuronal
networks.
3
Drugs used to manage both chronic and acute pain are opiates, non-opioid analgesics (primarily non-steroidal
anti-inammatories), antidepressants, anticonvulsants, cannabinoids, and topical agents.
4
Pain could be due to inam-
matory or non-inammatory responses to tissue damage.
Inammation is one of the immune system’s initial defense responses to an infection or cellular/tissue damage. This
reaction may also be directed against irritants, invading infections as well as autoimmune or neurodegenerative disorders.
The changes that occur during an acute inammatory event are crucial in the survival of the host, despite the fact that it
Journal of Experimental Pharmacology 2023:15 63–79 63
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Open Access Full Text Article
Received: 6 December 2022
Accepted: 15 February 2023
Published: 23 February 2023

can also cause numerous illnesses like cancer, rheumatoid arthritis, and cardiovascular dysfunctions.
5,6
Varieties of cells
are involved in the process of inammation, and the four primary cell types that trigger inammation are macrophages,
neutrophils, lymphocytes, and mast cells. Once they have been attracted to the damaged area, they emit a variety of
inammatory mediators, including cytokines, histamine, nitric oxide (NO), prostaglandins, leukotrienes, and other
substances.
7
As a result, plasma proteins can enter into the tissue due to modication of the endothelial cell junctions
in the blood vessel wall.
8
These vascular actions are the cause of the three typical signs of inammation; the increased
blood perfusion provides the redness and heat, whereas the leakage of uid into the tissue promotes swelling. Non-
steroidal anti-inammatory drugs (NSAIDs) are useful to diminish the detrimental effects of inammation.
9
Corticosteroids also prevent several mechanisms involved in inammation. Glucocorticoids are useful drugs to reduce
inammation and immune activation in a variety of disorders including asthma, allergy, rheumatoid, collagen, vascular,
dermatological, inammatory bowel, and other systemic disorders.
10–12
Currently available conventional medications for
pain relief and management of inammation-related disorders are challenging, especially as they cause numerous adverse
effects including gastrointestinal irritation, cardiovascular toxicities, tolerance and dependency.
13
In addition, most
patients fail to achieve sufcient pain relief, even with the use of various analgesics and anti-inammatory agents.
14
Thus, there is a requirement for steeping up research on medicinal plants that are useful for the treatment of painful and
inammatory conditions.
In many parts of the world, medicinal plants have been utilized for thousands of years as traditional therapies for
a wide range of human ailments.
15
They are crucial for maintaining the wellbeing of the societies by possessing a variety
of compounds with promising biological activities that alter biological process in the body.
16,17
Secondary metabolites of
medicinal plants, including polyphenols, avonoids, terpenoids and alkaloids, are indispensable bases for developing
analgesic and anti-inammatory drugs.
18
Ethiopia has a diversied heritage of traditional medicinal practices, known for
using plants to prepare more than 90% of the remedies.
19
According to reports, up to 80% of the people rely on
traditional medicines as a main source of health care.
20
E. cymosa is a deciduous shrub or small tree that grows 2–9 m tall and is mainly present in the Savanna and
secondary jungle of Africa. The leaves are oval shaped, while the fruits are black, ovoid to globose drupe of 2–6 mm
long.
21
It is an indigenous plant widely distributed in various parts of Ethiopia where it is locally called “Game” in
Amharic and “Hulaga, Ulaga, Garmi” in Afan Oromo. Experimental studies revealed that different extracts and solvent
fractions of E. cymosa have a number of activities, including antioxidant, antihyperglycemic, antidiarrhea, for skin
wounds, for paralysis, antiepileptic and antimicrobial.
21,22
Ethnobotanical survey in different parts of Ethiopia indicated
that E. cymosa is used in rheumatism,
23
headache,
24
and febrile illness (mich).
25
Even though the leaves of E. cymosa
were claimed for its analgesic and anti-inammatory activities by traditional practitioners, as far as our knowledge no
research has been done on the antinociceptive and anti-inammatory activities of the plant. Therefore, it is crucial to
conduct scientic research to the analgesic and anti-inammatory effects of this plant.
Materials and Methods
Drugs and Chemicals
Carrageenan (Sigma Aldrich, Germany), Ketamine (India), Acetyl Salicylic Acid (Bayer Germany), Morphine,
Indomethacin (Cadila, Ethiopia), Methanol (Carlo Erba, Italy), Chloroform & Ethyl Acetate (Bulex Laboratory, India),
Sodium Phosphate, Sodium Hydroxide, Sodium Carbonate, Distilled Water (DW), Normal Saline (Addis Pharmaceutical
Factory, Ethiopia), Tween 80 (Sigma Aldrich, Germany), and Glacial Acetic Acid (Basell, India) were used in the
experiment.
Materials and Instruments
Lyophilizer (OPERON, OPR-FDU-5012, Korea), UV Spectrophotometer (Jenway Model 6500, England), Digital
Plethysmometer (Ugo Basile-Cat no 7140, Italy), Hot plate (Ugo Basile 720), Rotary Evaporator (Heidolph,
Germany), Soxhlet Apparatus, Electronic Balance (KERN-ALJ 220-4, Germany), tissue drying oven (Medite -
Medizin technik, Germany).
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Plant Material Collection and Authentication
The fresh leaves of E. cymosa were collected from Shashamane, Oromia, Ethiopia, in February 2021. The plant material
was identied and authenticated by Mr. Melaku Wondaferash at the National Herbarium, Department of Plant Biology
and Biodiversity Management, Addis Ababa University. For future use, a voucher specimen was placed with the voucher
number GA 001.
Preparation of the Extract
Three-hundred grams (300g) of the dried and pulverized leaves of E. cymosa were extracted with 80% methanol by
maceration for 72 hr (three times) and shaking sometimes using mini orbital shaker at 120 rpm. The resulting liquid was
ltered rst with muslin cloth and then with Whatman No. 1 lter paper. After extraction, methanol was vaporized under
vacuum by a rotary evaporator at 40°C. The resultant solution was then frozen at −20°C until it solidied, and the
residual solvent was dried by a lyophilizer. The dried powder residue/extract was weighed, placed into a glass vial,
appropriately labeled and kept in a desiccator on silica gel until use.
Fractionation
The dried and powdered leaves of E. cymosa (400g) were packed into a thimble and sequentially extracted with
chloroform, ethyl acetate and water using a soxhlet apparatus. The solvents were evaporated under reduced pressure
and the fractions were then dried in a drying oven at a temperature not exceeding 40°C. The nal semisolid mass of each
fraction was kept in a desiccator until use under the same manner as the crude extract.
Experimental Animals
Healthy Swiss albino mice (6–8 weeks of age) weighing 25–35 g and Wistar albino rats (6–8 weeks of age) weighing
200–250 g were involved in the procedure. The animals were kept under standard environmental conditions in plastic
cages at normal temperature and on a 12 hr light–dark cycle with unlimited access to standard pelleted diet and water ad
libitum. To minimize stress, animals were acclimatized to laboratory settings before the start of the experiment in all
procedures. The animals used in this study were treated according to internationally recognized standard guidelines for
the use of laboratory animals.
26
The School of Pharmacy, College of Health Sciences, Addis Abeba University’s ethical
review committee granted its approval under protocol number ERB/SOP/180a/13/2020.
Acute Toxicity Study
Acute oral toxicity study was performed according to the internationally accepted protocol of OECD Guideline 425.
26
For toxicity investigation fasted female albino mice, which were between 6 and 8 weeks old were used. A single female
mouse was given 2000 mg/kg of the extract as a single dose in the initial screening test to determine the starting dose.
Since no death was noticed within 24 hr, another four mice were given the same dose of extract. The animals were
watched for the rst four hours at intervals of 30 minutes, and then for the following 14 days at intervals of 24 hours.
They were monitored for general toxic signs and symptoms like changes in skin and fur, somatomotor activities and
behavioral patterns, eyes and mucous membranes, diarrhea and salivation, convulsions and tremor, food and water intake,
weight loss, lethargy, paralysis and mortality.
Animal Grouping and Dosing
Swiss albino mice of both sex weighing 25–35 g (for analgesic activities) and Wistar albino rats weighing 200–250 g (for anti-
inammatory activities) were divided randomly into ve groups of six animals each. Group I was allocated as negative control
and received vehicles. Group II was assigned as a positive control and was given standard drugs such as morphine (20 mg/kg)
for hot plate test, aspirin (150 mg/kg) for acetic acid induced writhing test, indomethacin (10 mg/kg) for carrageenan-induced
paw edema and cotton-pellet-induced granuloma model of inammation. The other three groups (test groups) received
different doses (100, 200 and 400 mg/kg) of the 80% methanol extract orally.
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For testing the solvent fractions, the animals were randomly assigned into eleven groups of six animals each. Group
I was used as a negative control and received 2% tween 80 at a dose of 10 mL/kg. Group II served as a positive control
and received morphine (10 mg/kg) for hot plate test, aspirin (150 mg/kg) for acetic acid induced writhing test, and
indomethacin (10 mg/kg) for carrageenan-induced paw edema and cotton-pellet-induced granuloma model of inamma-
tion. The next three test groups (group III–V) were given three different doses (100, 200 and 400 mg/kg) of the aqueous
fraction. Another three test groups (Group VI–VIII) received ethyl acetate fraction at three dose levels, whereas the
remaining three test groups (Group IX–XI) received the same three dose levels of the chloroform fraction.
Evaluation of Analgesic Activities of the Extract
Acetic Acid-Induced Writhing Test
The method reported by Yimer et al was used to demonstrate the peripheral analgesic effects of the extract (or solvent
fractions).
14
This procedure was carried out by randomly grouping mice that had been fasted overnight with free access
to water. Mice of either sex were administered different doses of the crude extract (or solvent fractions), a vehicle
(negative control) and a standard drug aspirin 150 mg/kg (positive control) 1 hr prior to acetic acid (0.6% v/v) (10 mL/
kg, i.p) administration based on their particular groups. The analgesic effect of the extract (or solvent fractions) was
determined ve minutes after the acetic acid injection by counting the numbers of writhing, which involves contraction
of the abdominal muscle along with stretching of the hind limbs for 30 minutes. The percentage protection of the number
of writhes compared to the control group was served as an index of analgesia and was determined by the subsequent
formula.
14
Hot Plate Method
Hot plate test was performed to assess the anti-nociceptive activities of the extracts (or solvent fractions) by placing the
mouse into an open-ended cylindrical chamber with a oor containing a metallic plate heated by a thermode. A plate was
maintained at a constant temperature of 55°C ± 1°C and result in two behavioral responses that are described in terms of
their response times that is paw licking and jumping which are supraspinally-integrated responses. Mice of either sex
received different doses of the crude extract (or solvent fractions), a vehicle (negative control, P.O.) and standard drug
morphine (20 mg/kg oral) based on their respective groups. They were placed individually on a hot plate with a 15-
second cutoff period to prevent injuries to the animals’ paws. The latency to lick the paw or jump off the hot plate was
measured at 0, 30, 60, 90, and 120 minutes to determine the reaction time. The percentage increase in reaction time or
inhibition of pain threshold was calculated using the formula:
27
Evaluation of Anti-Inammatory Activity of the Extract
Carrageenan Induced Paw Edema
Acute inammation was induced by injection of carrageenan (1% w/v carrageenan in normal saline, 100 µL) into the
right hind paw of the rats. Before induction of inammation, the paw was labeled with ink at the lateral malleolus.
Carrageenan were injected into each group of rats 1 hr after oral administration of the crude extract (or solvent fractions),
the vehicle and the standard drug. Inammation was measured in mL by which displacement of water by edema with
a digital plethysmometer at time 0, 1, 2, 3, 4, 5 and 6hrs after carrageenan injection. The percentage protection of edema
was determined in comparison to the control rats using the formula.
14
where PEC paw edema in control group, PET paw edema in test group.
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Cotton Pellet Granuloma Method
This method is used to determine the transudative and proliferative (granulomatous) features of chronic inammation.
28
Male albino Wistar rats (200–250 g) were fasted for 12 h with free access to water prior to the beginning of the
experiment. 2% tween 80, indomethacin (10 mg/kg P.O.) and crude extract (or solvent fractions) were given to the
control, standard and test groups of rats, respectively. By rolling a piece of cotton weighing 10 mg and sterilizing it in an
autoclave for 30 minutes at 120°C and 15 lbs of pressure, sterile cotton pellets weighing 10±1 mg were prepared. The
rats were sedated with ketamine (50 mg/kg), and using forceps, a subcutaneous tunnel was created aseptically on both
parts of the recently shaved groin region of each rat 20 minutes after administration of the standard drug and extracts (or
solvent fractions). Then, a chromic catgut (0/4metric-1/2 Circle) was used to stitch two sterilized cotton pellets into
a subcutaneous tunnel on either side. The standard drug (indomethacin 10 mg/kg) and extracts (or solvent fractions) were
given for seven days (P.O., once a day). On the 8th day, the rats were sacriced with anesthesia, subsequently; the pellets
enclosed by granuloma tissue were carefully dissected and cleared from extraneous tissue. The wet weight of the cotton
was measured and then dried at 60°C for 24 hrs and by subtracting the weight of the cotton pellets the net dry weight was
recorded. The exudate amount (mg), granulation tissue formation (mg), and percent inhibition of exudate and granuloma
tissue formation was calculated by the formula:
Where: Measure of exudates formation = immediate wet weight of pellet - Constant dry weight of pellet
Measure of granuloma tissue formation = Constant dry weight - Initial weight of cotton pellet.
Determination of Total Phenol, Flavonoid and Alkaloid Contents
Determination of Total Phenolic Content
The total phenolic content of leaf extract and solvent fractions of E. cymosa were estimated according to the Folin
Ciocalteu colorimetric method.
29
To construct the calibration curve different concentrations of the standard (gallic acid)
solutions (100, 50, 25, 12.5, 6.75 μg/mL) were prepared in methanol. One mL of the standard was then moved into test
tubes and 5 mL of methanol and 0.5 mL of Folin–Ciocalteu’s reagent were added into the test tubes. After 5 minutes,
1.5 mL of Na2CO3 (20%) was added and the volume was adjusted to 10 mL with methanol. Reactions were incubated
for 90 min at room temperature and kept under the dark condition. Using a UV spectrophotometer, the solution’s
absorbance was determined at 760 nm. The experiments were performed in triplicates to give more precise results.
A similar procedure was performed for the extract or solvent fractions (100 µg/mL) and the blank solutions. The total
phenolic content was estimated using a standard curve of gallic acid (y = 0.0055x - 0.195, R
2
= 0.9767). Total phenolic
contents were described as mg of gallic acid equivalent per g of extract or fractions.
Determination of Total Flavonoid Content
The total avonoid content of the extract or solvent fractions of E. cymosa were determined using aluminum chloride
colorimetric assay.
30
Different concentrations of the standard (Quercetin) (1, 0.50, 0.25, 0.125 and 0.065 mg/mL) were
prepared in methanol to obtain calibration curve. One mL of the standard was then transferred into test tubes. 0.3 mL 5%
NaNO2 was added and left for 5 minutes. An additional 0.3 mL of 10% AlCl3 was mixed with the solution and left for 5
minutes. Then, 2 mL solution of 1M NaOH was added into the solution and lled with methanol to make nal volume to
10 mL. Finally, the solutions were allowed to stand for 30 minutes in the dark at room temperature. The absorbance of
the solution was measured at 510 nm using a UV spectrophotometer. A similar procedure was performed for the extract
or solvent fractions (1 mg/mL) and the blank solutions. The total avonoid content was calculated using a standard curve
of quercetin (y=0.4321x +0.0656, R
2
= 0.9783). The total avonoid contents were described as mg of quercetin
equivalent per 100 g of extracts. All of the steps were done in triplicate.
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Determination of Total Alkaloid Contents
The total Alkaloid Content of the extract or solvent fractions of E. cymosa were determined by Bromocresol green (BCG)
method.
31
To construct a calibration curve, different concentrations of atropine (120, 60, 30, 15 and 7.5 g/mL) were made
in methanol. One mL of the atropine solution was placed into separatory funnels. Then, 5 mL of phosphate buffer (PH,
4.7) and 5mL BCG solution were added to the ltrates and the mixture was shaken with 4 mL of chloroform two times.
The chloroform extracts were then collected in a test tube and its nal volume was adjusted to 10 mL with chloroform.
The plant extract or solvent fractions (1 mg/mL) prepared in methanol were dissolved in 2N HCl solution and then
ltered. One mL of the ltrate was added into a separatory funnel and extracted with 5 mL of chloroform 2 times. The
chloroform extract was removed and the PH of the remaining solution was adjusted to neutral with 0.1 M NaOH solution.
To the neutralized solution 5 mL of BCG and 5 mL of buffer solution (PH, 4.7) were added and shaken. The complex
was washed twice with 4 mL of chloroform using vigorous shaking. The nal volume of the extract was then set to
10 mL and collected in a test tube. The absorbance of the chloroform extracts of the atropine and extract/or solvent
fractions were determined at a wavelength of 470 using UV-Spectrophotometer. The blank solution was treated in the
same way. All procedures were carried out three times. The average absorbance of the blank solution was subtracted from
the standard and sample solutions and the total alkaloid content was estimated using the standard curve of Atropine
(y=0.00378x+0.0474, R
2
=0.9896) (Figure 1).
Statistical Analysis
The data were analyzed using statistical package for social science (SPSS) software version 25. The results were
expressed as mean ± standard error of the mean (S.E.M.) and statistical signicance was assessed by using One-way
A. B.
C.
y = 0.4321x + 0.0656
R² = 0.9783
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.5 1 1.5
Absorbance
Concentration of quercetin (mg/ml)
Figure 1 Calibration curve of standards ((A) for gallic acid, (B) for quercetin, (C) for atropine).
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Analysis of Variance (ANOVA) followed by a Tukey post hoc test to compare variations among groups and the results
were considered signicant at p < 0.05. The analyzed data were then reported using tables and graph as necessary.
Results
Acute Toxicity Study
The results of acute oral toxicity test of 80% methanol extract of leaves of E. cymosa at a dose of 2000 mg/kg revealed
that the extract did not produce any substantial alterations in behavior, respiration, cutaneous effects, sensory nervous
system responses or gastrointestinal effects within 24 hr and in the next 14 days. There was no recorded mortality or
hazardous reactions including convulsions, ataxia, diarrhea, or excessive urination. Based on the Limit Test at 2000 mg/
kg of OECD guideline 425 (2008), it can be proposed that the oral LD50 of crude 80% methanol extract is higher than
2000 mg/kg in mice. Accordingly, the nding indicates that the extract has a low toxicity prole in mice.
Effect of 80% Methanol Leaves Extract and Solvent Fractions on Acetic Acid-Induced
Writhing Test
The results of the acetic acid writhing test in mice are shown in Table 1. At all tested doses, the 80% methanol leaves
extract of E. cymosa signicantly (p < 0.001) reduced the writhing reactions of the mice produced by the intra-peritoneal
injections of acetic acid in a dose-dependent manner. This was checked by performing a linear regression analysis (R
2
=
0.878). The higher (400 mg/kg) dose of the extract displayed a signicant analgesic effect (p < 0.001) compared to the
lower and middle (100 and 200 mg/kg) doses of the extracts. Similarly, acetyl salicylic acid exhibited a signicant effect
(p < 0.001) compared to the 100 and 200 mg/kg dose of the extracts. However, the effect of the extract at 400 mg/kg did
not differ signicantly from that of aspirin (Table 1).
Similarly, at all the tested doses, the solvent fractions exhibited signicant analgesic activities (p < 0.001) compared
to the control. Ethyl acetate and chloroform fraction demonstrated better reduction in the number of writhes in mice
compared to the control. The ethyl acetate and chloroform fraction, in higher doses, decreased the number of abdominal
constrictions signicantly with 64% and 57.2% inhibition, respectively. Higher dose of ethyl acetate fraction demon-
strated comparable analgesic activity with that of acetyl salicylic acid (65.5%) (Table 2).
Effects of the 80% Methanol Extract and Solvent Fractions on Hot Plate Test
In this model, all the tested doses of E. cymosa extract demonstrated substantial analgesic activity (p < 0.05) at 90 and 120
minutes of observation when compared with the negative control (Table 3). The standard drug morphine resulted in signicant
analgesic activity (p < 0.001) at these intervals of observations. The latency delayed by the three doses of the extract was
considerably lower (p < 0.05) than that of the standard treatment at these intervals of observations. Throughout the observation,
there was no signicant difference between the middle and lower dose levels of the extract. The higher dose of the extract
revealed a signicantly different analgesic effect compared the lower and middle dose of the extract (p < 0.05) at 90 and 120
minutes of observations. The 80% methanol extract of E. cymosa at 100, 200 and 400 mg/kg doses exerted maximum analgesic
Table 1 Effect of 80% Methanol Leaves Extract of E. cymosa Against Acetic Acid
Induced Writhing Model in Mice
Treatment Groups No. of Writhes (Mean ± SEM) % Analgesic Activity
2% TW 54.33 ± 0.98 –
ASA 150mg/kg 20.83±1.14 (acd)* 61.6
EC100 38.67±1.25 (abe)* 28.8
EC200 36.16±1.25 (abe)* 33.4
EC400 24.50±0.99 (acd)* 54.9
Notes: a, as compared to negative control; b, compared to ASA 150 mg/kg; c, compared to EC100mg/kg;
d compared to EC200mg/kg; e, compared to EC400mg/kg, where, TW= Tween 80, ASA= acetyl salicylic
acid (150 mg/kg), EC= Ehretia cymosa, *p < 0.001.
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effect at 120 minutes with respective values of 60.2 65 and 77.2%. The analgesic effect at this moment was shown to be dose-
dependently increased. (R
2
= 0.88) (Table 3).
All the tested doses of the fractions (except 100 mg/kg of aqueous fraction at 30 minutes) resulted in signicant (p <
0.001) delay in reaction times during 30, 60, 90 and 120 minutes of observation compared to the control group (Table 4).
Higher dose of ethyl acetate fraction (400 mg/kg) and the standard drug morphine exhibited signicant (p < 0.05)
analgesic activities compared to the other fractions at these intervals of observations. Higher doses of aqueous, chloro-
form and ethyl acetate fractions showed higher increments in latency time at 120 minutes, with respective values of
63.8%, 64.6%, and 72.1% (Table 4).
Effect of 80% Methanol Extract and Fractions on Carrageenan Induced Paw Edema
In the carrageenan-induced paw edema, the 80% methanol extract at all tested doses demonstrated a signicant reduction
in paw edema that starts from 1 hr and continued for 6 hr after induction (p < 0.001 from 1st −6thhr) as compared to the
control group (Table 5). Intergroup comparison among doses of the E. cymosa showed a statistically signicant effect
between 100 and 400 mg/kg (p < 0.05). However, no signicant differences were observed between 100 mg and 200 mg/
kg and 200 and 400 mg/kg dose groups. The standard drug indomethacin (10 mg/kg) revealed a signicant (p < 0.05)
anti-inammatory activity compared to the lower and middle doses of the extracts.
The maximum anti-inammatory effect of 100, 200, and 400 mg/kg E. cymosa was recorded 6 hours after induction,
with values of 28.6%, 30.4%, and 36%, respectively, and the effect at this hour increased dose-dependently (R
2
= 0.92).
Table 2 Effects of the Solvent Fractions of E. cymosa Against Acetic Acid Induced
Writhing Model in Mice
Treatment Groups No. of Writhes (Mean ± S.E.M) % Analgesic Activity
2%TW 60.33 ± 1.05 –
ASA 19.5 ± 1.01 (cdefgij)*h
#
65.5
AF100 49.16 ± 0.60 (abe)* 18.5
AF200 45.0 ± 0.81 (abe)* 25.4
AF400 32.66 ± 1.45 (abcd)* 45.8
CF100 40.50 ± 1.11 (abh)* 32.8
CF200 37.83 ± 1.40 (abh)* 37.3
CF400 25.83 ± 0.60 (af)* b
#
57.2
EAF100 39.66 ± 0.88 (abjk)* 34.2
EAF200 33.0 ± 0.96 (abik)* 45.3
EAF400 21.66 ±1.05 (aij)* 64.0
Notes: a, compared with negative control; b, compared with ASA 150 mg/kg; c, compared with AF100;
d, compared with AF200; e, compared with AF400; f, compared with CF100; g, compared with CF200; h,
compared with CF400; i, compared with EAF100; j, compared with EAF200 and k, compared with
EAF400. *P < 0.001,
#
P < 0.05. Where; TW= Tween 80, AF= Aqueous Fraction; CF= Chloroform
Fraction and EAF= Ethyl Acetate Fraction.
Table 3 Effects of 80% Methanol Extract of E. cymosa on Hot Plate Latency Time in Mice
Hot Plate Latency Time in Second (Mean ± SEM) (% of Inhibition)
GROUP M0 M30 M60 M90 M120
2%TW 2.11±0.59 1.23±0.59 1.81±0.59 1.72±0.17 1.75±.37
MP 3.60±1.04 (41.4) 6.63±1.06 (81.4)a*c
#
d* 8.25±0.81 (78.0) a*c*d*e** 9.38±0.91 (81.6) a*c*d*e
#
10.97±1.05 (84.0) a*c*d*e
#
EC100 2.63± 0.32 (19.8) 2.68 ± 0.43 (54.1) b
#
3.22±0.28 (43.7) b*e
#
4.06±0.32 (57.6) a
#
b*e** 4.40±0.23 (60.2) a
#
b*e**
EC200 2.52 ± 0.07 (16.3) 2.98 ± 0.14 (58.7) b
#
3.94±0.28 (54.0) a
#
b* 4.45±0.39 (61.3) a**b*e
#
5.02±0.44 (65.0) a**b*e
#
EC400 2.78 ± 0.24 (24.1) 4.55 ± 0.44 (72.9) a
#
5.68± 0.51 (68.1) a*b**c
#
6.80±0.47 (74.7) a*b**c**d
#
7.70±0.47 (77.2) a*b**c**d
#
Notes: a, compared to negative control; b, compared to morphine 20mg/kg; c, compared to EC 100mg/kg; d compared to EC 200mg/kg; e, compared to EC
400mg/kg, *p < 0.001, **p < 0.01,
#
p < 0.05. Where; TW= Tween 80, EC= Ehretia cymosa; MP, Morphine 20 mg/kg; M (0, 30, 60, 90 and 120), at 0, 30, 60, 90
and 120 minutes of observations.
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Higher dose of the crude extract (400 mg/kg) exhibited a comparable anti-inammatory activity with 10 mg/kg of
indomethacin throughout the observation period (Table 5).
All tested doses of the solvent fractions as well as the standard drug (indomethacin 10 mg/kg) exhibited a signicant
reduction of paw edema starting from 1hr and the effect continued for 6 hrs after induction compared with negative control (p <
0.001). Indomethacin 10 mg/kg exhibited a statistically signicant anti-inammatory activity compared to all fractions (except
higher dose of ethyl acetate fraction) at 6 hr post induction. Maximum anti-inammatory effect was detected with higher doses of
aqueous, chloroform and ethyl acetate fractions at 6 hr post induction with corresponding values of 35.3%, 35.9% and 40.8%
(Table 6). Although all three fractions displayed signicant reduction of paw edema as compared to the negative control, ethyl
acetate fraction was the most active fraction in terms of anti-inammatory effects on carrageenan-induced rat paw edema
(Table 6).
Effect of 80% Methanol Extract and Solvent Fractions of E. cymosa on Cotton Pellet
Induced Granuloma
Subcutaneous implantation of cotton pellets in the groin region of rats caused granulomatous inammation with a highest
granuloma weight and exudates recorded in the 2% tween 80 treated controls as shown in Table 7. The 80% methanol
extract of E. cymosa at all tested doses signicantly prevented inammatory exudates and granuloma mass formation (p
Table 4 Effects of Solvent Fractions of E. cymosa on Hot Plate Latency Time in Mice
Hot Plate Latency Time in Second (Mean±SEM) (% of Inhibition)
Group M0 M30 M60 M90 M120
2% TW 1.55 ± 0.23 1.95 ± 0.24 2.53 ± 0.13 2.80 ± 0.17 3.15 ± 0.14
MP 4.36 ± 0.33 (64.4) 6.6 ± 0.20 (70.4) (acdefghij)* 8.75 ± 0.66 (71.0) (acdefgij)*h** 10.05 ± 0.54 (72.1) (acdfgij)*e**h** 12.02 ± 0.62 (73.7) (acdefgij)*h**
AF100 2.28 ± 0.297 (32.0) 3.13 ± 0.26 (37.7) b* 4.55 ±0.40 (44.4) a
#
b* 4.88 ±0.21 (42.6) a
#
b*e** 6.03 ± 0.42 (47.7) a**b*e
#
AF200 2.53 ± 0.24 (38.7) 3.65 ± 0.31 (46.5) (ab)* 5.03 ± 0.22 (49.7) a**b* 6.23 ± 0.37 (55.0) (ab)* 7.13 ± 0.27 (55.8) (ab)*
AF400 2.72 ± 0.18 (43.0) 3.58 ± 0.09 (45.5) a**b* 5.46 ± 0.44 (53.1) (ab)* 7.54 ± 0.72 (62.8) a*b**c** 8.70 ± 0.27 (63.8) a*b*c
#
CF100 2.55 ± 0.23 (39.2) 3.40 ± 0.25 (42.6) a**b* 4.42 ± 0.44 (42.6) b*h
#
5.18 ± 0.58 (45.9) a**b*h** 5.95 ± 0.54 (47.0) a**b*h**
CF200 2.82 ± 0.36 (44.8) 3.62 ± 0.14 (45.9) (ab)* 5.03 ± 0.35 (49.7) a**b* 5.53 ± 0.38 (49.4) a*b*h** 6.62 ± 0.42 (52.3) (ab)*
CF400 3.42 ± 0.21 (54.5) 4.51 ± 0.31 (56.6) a*b* 6.52 ± 0.62 (61.1) a*b**f
#
7.65 ± 0.44 (63.4) a*b**f**g
#
8.91 ± 0.39 (64.6) a*(bf)**
EAF100 2.66 ± 0.27 (41.7) 3.70 ± 0.32 (47.3) (abk)* 5.60 ± 0.33 (54.8) (abk)* 6.78 ± 0.28 (58.7) (abk)* 8.1 ± 0.56 (61.0) (abk)*
EAF200 3.20 ± 0.19 (51.5) 4.12 ± 0.26 (52.5) (abk)* 6.0 ± 0.24 (57.8) (abk)* 6.65 ± 0.32 (57.9) (abk)* 7.78 ± 0.52 (59.5) (abk)*
EAF400 4.08 ± 0.15 (52.7) 6.13 ± 0.27 (67.9) (aij)* 8.63 ± 0.23 (70.6) (aij)* 9.58 ± 0.18 (70.7) (aij)* 11.30 ± 0.79 (72.1) (aij)*
Notes: a, compared with negative control; b, compared with Morphine 20mg/kg; c, compared with AF100; d, compared with AF200; e, compared with AF400; f, compared
with CF100; g, compared with CF200; h, compared with CF400; i, compared with EAF100; j, compared with EAF200 and k, compared with EAF400. *P < 0.001, **P < 0.01,
#
P < 0.05. Where; TW= Tween 80, AF= Aqueous Fraction; CF=Chloroform Fraction and EAF= Ethyl Acetate Fraction.
Table 5 Effects of 80% Methanol Extract of E. cymosa Against Carrageenan Induced Paw Edema Model in Rats
Increase in Paw Volume (mL) Mean±SEM (% of Inhibition)
Group 0 hr 1 hr 2 hr 3 hr 4 hr 5 hr 6 hr
2%TW 1.09 ± 0.01 1.19 ± 0.02 1.3 ± 0.02 1.41 ± 0.01 1.5 ± 0.02 1.56 ± 0.01 1.61 ± 0.01
INDO 0.98 ± 0.01
(10.1)
1.04 ± 0.02
(12.6) a*c
#
d
#
1.12 ± 0.02
(13.8) a*c**
1.16 ± 0.01
(15.6) a*c*d**
1.20 ± 0.01
(20.0) a*c*d**
1.12 ± 0.02
(28.2) a*c*d**
1.02 ± 0.02
(37.3) (acd)*
EC100 1.04 ± 0.02
(4.6)
1.12 ± 0.02
(5.9) a*b
#
e
#
1.19 ± 0.01
(8.5) a*b**e**
1.24 ± 0.01
(12.0) a*b*e**
1.31±0.01
(12.7) (abe)*
1.19 ± 0.01
(23.7) a*b*e
#
1.15 ± 0.01
(28.6) (abe)*
EC200 1.05 ± 0.02
(3.7)
1.11 ± 0.01
(6.7) a*b
#
1.17 ± 0.01
(10.0) a*
1.22 ± 0.01
(13.5) a*b**
1.27 ± 0.01
(15.9) a*b**
1.19 ± 0.01
(23.7) a*b**
1.12 ± 0.01
(30.4) a*b*
EC400 1.01 ± 0.01
(7.3)
1.05 ± 0.01
(11.8) a*c
#
1.12 ± 0.01
(13.8) a*c**
1.20 ± 0.01
(15.9) a*
1.22 ± 0.01
(18.6) a*c*
1.14 ± 0.01
(26.8) a*c**
1.03 ± 0.02
(36.0) a*c*
Notes: a, as compared with negative control; b, compared to indomethacin 5 mg/kg; c, compared to EC100; d, compared to EC200; e, compared to EC400.
*P < 0.001, **P < 0.01,
#
P < 0.05. Where, EC =Ehretia cymosa, INDO, Indomethacin 10 mg/kg; TW, Tween 80.
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Table 6 Effects of Solvent Fractions of E. cymosa Against Carrageenan Induced Paw Edema Model in Rats
Increase in Paw Volume (mL) (Mean±SEM) (% of Inhibition)
Group 0 hr 1hr 2 hr 3 hr 4 hr 5 hr 6 hr
2%TW 1.07 ± 0.02 1.17 ± 0.02 1.32 ± 0.02 1.44 ± 0.012 1.51 ± 0.02 1.58 ± 0.012 1.64 ± 0.012
INDO 0.85 ± 0.019 (20.6) 0.92 ± 0.021 (21.4) a*c* 1.00 ± 0.022 (24.2) a*c* 1.10 ± 0.016 (23.6) a*c*d**i
#
1.17±0.01 (22.5) a*c* 1.07 ± 0.015 (32.2) a*c*d**f
#
i** 0.94 ± 0.018 (42.7) (acdfgi)* e
#
h
#
j**
AF100 0.92± 0.034 (14.0) 1.04 ± 0.012 (11.0) a*b* 1.21 ± 0.022 (8.3) a** 1.28 ± 0.025 (11.1) a*b*e
#
1.34 ± 0.018 (11.2) (abe)* 1.26 ± 0.024 (20.2) (abe)* 1.22 ± 0.024 (25.6) (abe)*
AF200 0.92 ± 0.021 (14.0) 1.01 ± 0.014 (13.6) a* 1.15 ± 0.02 (12.8) a
#
1.25 ± 0.015 (13.2) a*b** 1.29 ± 0.013 (14.6) a*e
#
1.19 ± 0.011 (21.2) a*b** 1.13 ± 0.01 (32.3) (abk)*
AF400 0.89 ± 0.019 (16.8) 0.95 ± 0.033 (18.8) a* 1.05 ± 0.04 (20.4) a* 1.15 ± 0.046 (20.1) a*c
#
1.16 ± 0.057 (23.2) a*c*d** 1.11 ± 0.032 (29.7) a*c* 1.06 ± 0.019 (35.3) a*b
#
c*
CF100 0.88 ± 0.023 (17.7) 0.89 ± 0.014 (23.9) a* 1.06 ± 0.04 (19.6) a* 1.18 ± 0.03 (18.0) a* 1.22 ± 0.035 (19.2) a* 1.17 ± 0.014 (25.9) a*b
#
1.15 ± 0.006 (29.8) (ab)*
CF200 0.87 ± 0.024 (18.7) 0.90 ± 0.019 (23.1) a* 1.07 ± 0.051 (18.9) a* 1.18 ± 0.029 (18.0) a* 1.22± 0.036 (19.2) a* 1.14 ± 0.035 (27.8) a* 1.08 ± 0.031 (34.1) a*b*
CF400 0.87 ± 0.018 (18.7) 0.91 ± 0.02 (22.2) a* 1.05 ± 0.029 (20.4) a* 1.14 ± 0.027 (20.8) a* 1.19 ± 0.014 (21.2) a* 1.13 ± 0.012 (29.8) a* 1.05 ± 0.021 (35.9) a*b
#
EAF100 0.86 ± 0.017 (19.6) 0.90 ± 0.015 (23.1) a* 1.10 ± 0.042 (16.6) a* 1.23 ± 0.013 (14.6) a*b
#
1.26 ± 0.01 (16.5) a* 1.19 ± 0.008 (21.1) a*b** 1.19 ± 0.042 (27.4) a*b*j
#
k*
EAF200 0.85 ± 0.036 (20.5) 0.89 ± 0.025 (23.9) a* 1.04 ± 0.037 (21.2) a* 1.16 ± 0.012 (19.4) a* 1.22 ± 0.013 (19.2) a* 1.15 ± 0.019 (27.2) a* 1.07 ± 0.018 (34.7) a*b**i
#
EAF400 0.82 ± 0.029 (23.3) 0.86 ± 0.025 (26.4) a* 1.01 ± 0.013 (23.5) a* 1.15 ± 0.008 (20.1) a* 1.17 ± 0.012 (22.5) a* 1.11 ± 0.009 (29.7) a* 0.97 ± 0.016 (40.8) (ai)*
Notes: a, compared with negative control; b, compared with 10mg/kg indomethacin; c, compared with AF100; d, compared with AF200; e, compared with AF400; f, compared with CF100; g, compared with CF200; h, compared with
CF400; i, compared with EAF100; j, compared with EAF200 and k, compared with EAF400. *P < 0.001, **P < 0.01,
#
P < 0.05. Where; TW= Tween 80, AF= Aqueous Fraction; CF= Chloroform Fraction and EAF= Ethyl Acetate Fraction.
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< 0.001) in contrast to the control. The anti-inammatory activities of the E. cymosa increased in a dose-dependent
manner (R
2
= 0.99). The standard drug (indomethacin 10 mg/kg) signicantly reduced both exudate and granuloma
formation compared to the negative control group, lower and medium doses of the extract (p < 0.05). The percentage
inhibition of formation of exudate by the crude extract was 14.6, 22 and 30.2% whereas the percentage inhibition of
formation of granuloma was 38.5, 41.2 and 50.6% for granuloma at 100, 200 and 400 mg/kg, respectively (Table 7).
All the tested concentrations of the three solvent fractions signicantly reduced the formation of inammatory
exudate and granuloma mass compared with the control group (p < 0.001) (Table 8). Higher dose of the ethyl acetate
fraction revealed a considerable anti-inammatory activity in both exudate and granuloma inhibition (except 400 mg CF)
compared with the other fractions. The highest percentage suppression of exudate and granuloma formation was
exhibited by 400 mg/kg of EAF (36.4% and 48.7%), respectively, compared to all other doses of the EAF, CF, and
AF (Table 8).
Determination of Total Phenol, Flavonoid and Alkaloid Contents
The total phenolic content is expressed as mg of gallic acid equivalent (GAE) per gram of sample using the formula
derived from the calibration curve y=0.0055x+0.195. The highest total phenolic content was observed in the ethyl acetate
fraction (281.8 mg GAE/g). This was followed by the aqueous fraction (245.5 mg GAE/g) and 80% methanolic extract
(214.5 GAE/g).
The total avonoid content is described as mg of quercetin per gram of sample (mg QE/g) using the equation from the
calibration curve y=0.4321x+0.0656. The total avonoid contents in 80% methanolic extract, chloroform and ethyl
Table 7 Effects of 80% Methanolic Extract of E. cymosa on Cotton Pellet Induced Granuloma in Rats
Group of
Mice
Mean Weight of Exudates in mg
(Mean ±S.E.M)
% Exudate
Inhibition
Mean Weight of Granuloma in mg
(Mean ±S.E.M)
% Granuloma
Inhibition
2%TW 120.6 ± 1.44 – 50.52 ± 1.29 –
INDO 81.82 ± 1.37 (acd)* 32.1 22.13 ± 0.66 a*c*d** 56.2
EC100 102.96 ± 1.72 (abde)* 14.6 31.05 ± 1.18 a*b*e
#
38.5
EC200 94.2 ± 1.10 (abce)* 22.0 29.70 ± 1.35 a*b** 41.2
EC400 84.18 ± 1.23 (acd)* 30.2 24.93 ± 1.58 a*c
#
50.6
Notes: a, compared with negative control; b, compared with indomethacin 10 mg/kg; c, compared with EC100; d, compared with EC200; e, compared with EC400. *P <
0.001, **P < 0.01,
#
P < 0.05. Where TW; Tween 80, INDO; Indomethacin 10mg/kg, EC; Ehretia cymosa.
Table 8 Effects of Solvent Fractions of E. cymosa on Cotton Pellet Induced Granuloma in Rats
Treatment Group Mean Weight of Exudates
(Mean ± SEM)
% Inhibition Mean Weight of Granuloma
(Mean ± SEM)
% Inhibition
2%TW 120.83 ± 1.41 – 42.70 ± 1.73 –
INDO 74.17 ± 0.99 (acdefgij)* 38.6 20.25 ± 0.39 (acdefgi)*j
#
52.5
AF100 111.99±1.39 (be)*a
#
d** 7.3 34.83 ± 1.06 (ab)*e** 18.4
AF200 100.76 ± 1.09 (abe)* 16.6 31.28 ± 0.92 (ab)* 26.7
AF400 88.08 ± 1.71 (abcd)* 27.1 28.08 ± 0.72 a*b*c** 34.2
CF100 108.45 ± 2.02 (ab)*g** 10.2 31.07 ± 1.55 (ab)*h
#
27.2
CF200 98.10 ± 2.18 (abh)*f** 18.8 30.87 ± 1.90 (ab)*h
#
27.7
CF400 80.25 ± 2.12 (afg)* 33.6 25.24 ± 1.15 a*(fg)
#
40.9
EAF100 100.87 ± 1.26 (abjk)* 16.5 29.73 ± 0.84 (abk)* 30.4
EAF200 87.21 ± 2.19 (abi)*k** 27.8 27.43 ± 0.99 a* 35.7
EAF400 76.88 ± 2.51 (ai)*j** 36.4 21.90 ± 0.79 (aij)* 48.7
Notes: a, compared with negative control; b, compared with 10mg/kg indomethacin; c, compared with AF100; d, compared with AF200; e,
compared with AF400; f, compared with CF100; g, compared with CF200; h, compared with CF400; i, compared with EAF100; j, compared with
EAF200 and k, compared with EAF400. *P < 0.001, **P < 0.01,
#
P < 0.05 Where; TW; = Tween 80, AF= Aqueous Fraction; CF= Chloroform
Fraction and EAF= Ethyl acetate Fraction.
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acetate fractions are 109.6 mg QE/g, 112 mg QE/g and 125.9 mg QE/g, respectively. The total alkaloid content of the
extracts/or solvent fractions were derived from the regression formula of the calibration curve y=0.0037x+0.0474 and
described as mg of atropine equivalents per gram of sample (mg AE/g). The ethyl acetate fraction revealed the highest
total alkaloid content of 192.8 mg AE/g, followed by the chloroform fraction (147.6 mg AE/g) and the 80% methanolic
extract (144.9 mg AE/g) (Table 9).
Discussion
The results of the acute oral toxicity study revealed that the extract did not cause any substantial alterations in the
behavior and respiration, cutaneous effects, sensory nervous system reactions or gastrointestinal effects within 24 hr and
the follow up period of 14 days. Moreover, no death or any noxious responses like convulsion, ataxia and diarrhea were
recorded at the limit dose of 2000 mg/kg of crude extract of E. cymosa. Thus, the oral LD50 of the 80% methanolic
extract of the plant was predicted to be higher than 2000 mg/kg and the experimental doses were calculated on the basis
of this nding.
The 80% methanolic extract and solvent fractions of the plant were assessed for analgesic potential using hot plate
and acetic acid induced writhing test in mice. The acetic acid-induced abdominal constriction method is the preferred
assay for assessing the peripheral anti-nociceptive activities of drugs or medicinal plants.
32
It is reported that adminis-
tration of acetic acid enhances the level of PGE2, PGE2α and lipoxygenase products in the peritoneal uid.
32
Moreover,
it triggers the production of a number of unpleasant endogenous mediators, including histamine, serotonin, and
bradykinin, which are responsible for the hallmarks of inammation.
33,34
Acetic acid induced writhing test mimics
visceral pain and the writhing that results from the test is typically characterized by the contraction of the abdominal
muscles, the expansion of the forelimbs, and the lengthening of the body.
34,35
This elongation is supposed to be produced
by the activation of local peritoneal receptors and prostaglandin pathways in the experimental animal model. Peritoneal
muscles may be partially responsible for abdominal writhing.
36
The crude extract of E. cymosa produced signicant
analgesia (p < 0.001) at the given doses (100, 200, 400 mg/kg) in mice. The number of writhing was reduced by the
extract dose-dependently as compared with the control (R
2
= 0.878). When compared to the control, all tested doses of
the three fractions demonstrated signicant analgesic activities (p < 0.001). Another similar study revealed that,
triterpenes isolated from Ehretia microphylla exhibited signicant analgesic activity against acetic acid-induced abdom-
inal constriction.
37
The result of this study indicated that the extract and solvent fractions of the plant signicantly inhibited abdominal
contractions similar to that of aspirin. Thus, the potential mechanism for peripheral analgesic activities of the extract/
solvent fractions might be related to suppression of the production and release of several endogenous inammatory
mediators (prostaglandin pathway in the pain perception cascade) and inhibition of sensitivity of peripheral nociceptors.
Previous reports have proven that drugs which block the cyclooxygenase enzyme pathway reduce writhing, which is
a marker of pain in experimental animal models.
38
The analgesic action of the plant is probably attributed to its
phytochemical constituents. Studies conrmed that phenols, avonoids, terpenoids and steroids all acted as inhibitors
of prostaglandin synthesis.
39
Heat induced nociceptive pain in the paw of mice is particularly a sensitive model to assess the analgesic activities of
the medicinal agents. Hot plate test is frequently used in illuminating the centrally mediated anti-nociceptive effects of
the test substances due to its sensitivity to potent analgesics, limited tissue damage with a cutoff time of 15 sec that is
normally carried out to limit the time by which the mouse exposed to the hot plate.
14,32
Additionally, the model takes less
Table 9 Total Phenolic, Flavonoid and Alkaloid Content of 80% Methanol Extract and Fractions of E. cymosa
Phenol (mg of GAE/g) Flavonoid (mg of QE/g) Atropine (mg of AE/g)
Crude extract 214.5 109.6 144.9
Aqueous Fraction 245.5 84.2 120.98
Ethyl acetate fraction 281.8 125.9 192.8
Chloroform fraction 192.7 112 147.6
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time, and measurements are typically precise. In this test, jumping and paw licking are both regarded supraspinally
integrated behavioral responses, and the time of the latency to the onset of this reaction following injection is an indicator
of the analgesic activity.
40
The plate was kept at 55±1°C, and only opioid-like agents are active at this temperature. All
the test doses of E. cymosa extract displayed signicant analgesic activity (p < 0.05) at 90 and 120 minutes of
observation compared to control. All the test doses of the solvent fractions (except 100 mg/kg aqueous fraction at 30
minutes) resulted in signicant (p < 0.001) delay in reaction times during 30, 60, 90 and 120 minutes of observation
compared to the control. In consistence with this, morphine provides signicant analgesia at all observations. In line with
this study, methanol extract of the leaf, fruit, and stem bark of Ehretia serrata and Ehretia obtusifolia exhibit pronounced
analgesic effect in mice by reducing pain.
41
The probable analgesic activity of extract and solvent fractions of E. cymosa
might be a central mechanism via stimulation of the opioid system. It has been demonstrated that phenols like ellagic
acid have a central analgesic effect that may act through the opioid system.
42
In the current study, carrageenan-induced paw edema for acute inammation and cotton pellet-induced granuloma for
chronic inammation were used as models to assess the anti-inammatory effects of plant extract and solvent fractions.
Carrageenan is a phlogistic, non-antigenic substance and is without obvious systemic effect. Additionally, it is thought
that this experimental model showed excellent reliability for acute phase inammation.
14
Carrageenan-induced paw
edema is an extensively used primary assay for examining potential novel anti-inammatory agents and is thought to be
biphasic.
43
The initial phase starts within 1–2 hr after the injection of carrageenan and is brought on by the release of
serotonin, histamine, and bradykinin from mast cells into the nearby injured tissues. Meanwhile, the second phase starts
3–6 hr following carrageenan injection, which is associated with the production and release of prostaglandins, leuko-
trienes, and various cytokines such as IL-1β, IL-6, IL-10, and TNF-α.
43
In the brain, cyclooxygenase (COX)-2 is in
charge of mediating prostaglandin synthesis, which results in peripheral inammation and, in turn, induces hyperalgesia
and allodynia. In mast cells, PGD2, PGF2α and PGE2 are products of cyclooxygenase pathway that enhance vascular
permeability and vasodilatation, resulting in edema.
44
The 80% methanolic extract of E. cymosa signicantly (p < 0.001)
inhibits carrageenan-induced paw edema over a period of 1 to 6 hr compared to the control group. All tested doses of the
fractions also signicantly inhibited paw edema starting from 1hr and the effect lasted until 6hrs post-induction compared
with the control group (p < 0.05). In agreement with this study, the chloroform, methanolic and aqueous extract of
Ehretia laevis showed effective anti-inammatory activity by decreasing paw volume at different doses and the plant has
phenolic acids, avonoids, fatty acids, steroids and alkaloids.
45,46
The late phase of carrageenan-induced inammation is
an intensifying phase of swelling owing to increased vascular permeability and edema associated prostaglandins and can
be prevented by NSAIDs.
47
Similarly, the extracts and solvent fractions had reduced paw edema during the late phases,
so the anti-inammatory activity of the extract and solvent fractions may be a mechanism that entails blockage of COX
related to the inammatory cascade triggered by carrageenan. Natural compounds such as phenols, avonoids, alkaloids,
terpenoids, glycosides prevent the formation of prostaglandins in the late phase of inammation.
39,44
Thus, the presence
of these phytochemical constituents in the extract and fractions might inhibit the production of prostaglandins and
bradykinin which are involved in the anti-inammatory activity.
Cotton pellet-induced granuloma is a common method to assess anti-inammatory potential in transudative and
proliferative constituents of chronic inammation. In this model, inammatory responses involve inltration of mono-
cytes, proliferation of broblasts, angiogenesis and exudation.
48
In the cotton pellet-induced granuloma formation, the
responses can be divided into three phases. The initial, transudative phase, which occurs 0–3 hr following cotton pellet
implantation, characterized by the leakage of uid from blood vessels produced by an increase in vascular permeability.
The second, exudative phase, which occurs 3–72 hr after cotton pellet implantation, is associated with protein leakage
from the bloodstream around the granuloma as a result of the thorough preservation in vascular permeability change. The
formation of granulomatous tissues as a result of ongoing release of pro-inammatory mediators is known as the last,
proliferative phase, which lasts for three to six days.
49,50
An increase in broblasts, the production of collagen and
mucopolysaccharide, and the penetration of proliferating broblasts into exudate are the characteristics of granuloma
tissue formation. This process nally results in the formation of a vascularized mass.
50
The process of granuloma
formation arises owing to the discharge of pro-inammatory mediators and oxygen-derived free radicals, as well as
lysosomal enzymes like activating protein, which results in tissue injury. The transudative phase of inammation is
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represented by an increase in the cotton pellet’s wet weight, whereas the proliferative phases of inammation is measured
by an increase in the cotton pellet’s dry weight. The amount of uid (exudate) absorbed by the pellet has a substantial
effect on the granuloma’s wet weight, whereas the amount of granulomatous tissue generated correlates well with the
granuloma’s dry weight.
51
All the tested doses of 80% methanol extract and the three fractions of E. cymosa signicantly
reduced inammatory exudates and granuloma mass formations (p < 0.001) as compared to the control. As a non-
steroidal anti-inammatory drug, indomethacin produces its inhibitory effect in cotton pellet-induced granuloma experi-
ment by preventing granulocyte inltration to the cotton pellet and inhibiting the generation of collagen bers.
52
Indomethacin also caused a reduction in weight gain by suppressing the inammation, this mechanism was due to the
inhibition of the prostaglandin synthesis in the site of inammation that was induced by the cotton pellet.
53
It can be
assumed that the extract also acted similarly and the mechanism was assumed to be the same as compared to the
indomethacin which is the inhibition of the synthesis of prostaglandin. The study also revealed a reduction in granuloma
formation and these might be attributed to the ability of E. cymosa extract and solvent fractions to decrease the number of
broblasts and production of collagen and mucopolysaccharide, which are natural proliferative indicators of granulation
tissue formation.
The total phenolic content is described as mg GAE per gram of sample using the equation obtained from the calibration curve.
It can be seen that the total phenolic content was highest with ethyl acetate fraction (281.8 mg GAE/g). This was followed by the
aqueous fraction (245.5 mg GAE/g) and 80% methanolic extract (214.5 GAE/g), respectively. In previous study conducted
elsewhere, the total phenolic content of ethyl acetate and methanol fractions of E. cymosa was 25.27 and 27.44 mg GAE/g,
respectively, which is signicantly lower than the present study.
54
This difference might be due to the fact that phenolic content of
plants depends on a number of intrinsic (genetic) and extrinsic factors (maturity at harvest and storage condition). The higher
analgesic and anti-inammatory effects of ethyl acetate fraction corresponded to its highest phenolic contents.
The total avonoid content is expressed as mg QE/g using the equation from the calibration curve. The total avonoid
contents in 80% methanolic extract, chloroform and ethyl acetate fractions are 109.6 mg QE/g, 112 mg QE/g and
125.9 mg QE/g, respectively. The nding of this study is corroborated with other study in which the total avonoid
content of ethyl acetate and methanol fractions of E. cymosa was 221.44 and 235.31 mg QE/g, respectively.
54
The high
concentration of avonoids in plant extract correlated with its analgesic and anti-inammatory activities.
The total alkaloid content of the extracts/or solvent fractions were obtained from the regression formula of the calibration
curve and described as mg of atropine equivalents per gram of sample (mg AE/g). The ethyl acetate fraction showed the
maximum total alkaloid content of 192.8 mg AE/g, followed by the chloroform fraction (147.6 mg AE/g) and 80% methanolic
extract (144.9 mg AE/g). This is the rst report on the determination of the total alkaloid content of E. cymosa.
In general, the anti-inammatory activities of E. cymosa extract and fractions in this study are consistent with
previous reports that indicated plants which contain predominantly alkaloids, avonoids, saponins, tannins, Phenols,
glycosides, and triterpenoids showed powerful anti-inammatory effects.
7
Conclusion
From the nding of this study, it can be concluded that the 80% methanolic extract, aqueous, ethyl acetate and
chloroform fractions of E. cymosa exhibited signicant analgesic and anti-inammatory activities which support the
traditional use of the plant for the treatment of various painful and inammatory conditions. The plant extract or solvent
fractions attained peripheral analgesic activity and central pain inhibition potential. It also demonstrated an anti-
inammatory activity, in both acute and chronic phases of inammation.
Abbreviations
NO, nitric oxide; NSAID, non-steroidal anti-inammatory drugs; COX, cyclooxygenase; IL, interleukin; AE, atropine
equivalent; GAE, gallic acid equivalent; QE, quercetin equivalent; OECD, Organization for Economic Cooperation and
Development.
Data Sharing Statement
The data is available on the hand of the corresponding author and provided on reasonable request.
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Ethics Approval and Consent to Participate
This study was approved by the School of Pharmacy, College of Health Sciences, Addis Ababa University, ethical review
committee with protocol number ERB/SOP/180a/13/2020. OECD guideline 25 was used for the welfare of the laboratory
animals.
Acknowledgments
The authors would like to thank Ethiopian Public Health Institute (EPHI) and Department of Pharmacology and Clinical
Pharmacy, Addis Ababa University for providing laboratory animals and chemicals.
Author Contributions
All authors made a signicant contribution to the work reported, whether that is in the conception, study design,
execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically
reviewing the article; gave nal approval of the version to be published.
Funding
The study is supported by Addis Ababa University.
Disclosure
The authors declare that they have no conicts of interest in this work.
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