Advances in Traditional Medicine 
https://doi.org/10.1007/s13596-020-00541-8
RESEARCH ARTICLE
Effect of Anthonotha macrophyla (P. Beauv) leaf extract 
on carrageenan‑induced paw oedema, oxidative stress makers 
and hyperalgesia in murine models
Emmanuel Kofi Kumatia1 · Frederick Ayertey1 · Regina Appiah‑Opong2 · Ebenezer Ofori‑Attah2 · Peter Bolah1 · 
Stephen Antwi3 · Alfred Ampomah Appiah1 · Augustine Ocloo3,4 
Received: 1 September 2020 / Accepted: 15 December 2020 
© Institute of Korean Medicine, Kyung Hee University 2021
Abstract
Anthonotha macrophylla leaf (AML) is employed in the management of pain, inflammation, and some diseases associated 
with oxidative stress in African traditional medicine. This study investigated the anti-inflammatory and analgesic activi-
ties, the acute toxicity and anti-oxidant properties of AML, characterised the phytochemical constituents, and quantified 
its total phenolic and flavonoid contents. Anti-inflammatory and analgesic activities of AML extract were evaluated using 
carrageenan-induced oedema assay in rats’ paw and acetic acid-induced writhing assays respectively. Anti-oxidant effect of 
the extract was assessed in vivo by measuring liver antioxidant enzymes and in vitro by using DPPH radical scavenging assay 
and the acute toxicity of the extract was investigated in vivo in mice. The phytochemicals were characterised using basic 
phytochemical screening and total phenolics and flavonoids were quantified using established methods. The AML extract 
exhibited significantly high anti-inflammatory activity and moderate analgesic activity compared to diclofenac sodium. The 
extract exhibited moderate DPPH scavenging activity, significantly increased Superoxide Dismutase (SOD) and Glutathione 
peroxidase (GPX) activities, and did not have any effect on lipid peroxidation. AML was found to contain high phenolic 
and low flavonoids. The L D50 of the extract was above 5000 mg/kg body weight. In conclusion, the AML extract possessed 
significant anti-inflammatory and moderate analgesic activities, which may be mediated through its strong antioxidant prop-
erties due to its phenolic contents. The  LD50 value showed that the extract was safe in the short-term utilization. The extract 
may therefore serve as a source of anti-inflammatory, analgesic and antioxidant agents.
Keywords Anthonotha macrophylla leaf extract · Anti-inflammatory · Analgesic activity · Acute toxicity · Antioxidant 
activity
Introduction
Over one-third of the world’s population is plagued with 
chronic pain and about US$100 billion is utilized every year 
on health care, litigations and compensations as a result of 
*  Augustine Ocloo pain in the United States of American alone (Loeser et al. 
 aocloo@ug.edu.gh 2001). Pain is associated with nearly all human diseases. 
1 Acute pain is implicated in common conditions such as  Department of Phytochemistry, Centre for Plant Medicine 
Research, Mampong-Akwapim, Ghana inflammation, injuries or headache, whereas chronic pain 
2 is associated with conditions such as diabetic neuropathy,  Department of Clinical Pathology, Noguchi Memorial 
Institute of Medical Research, University of Ghana, temporomandibular joint syndrome, and cancers (Stucky 
Legon, Accra, Ghana et al. 2001).
3 Department of Pharmacology, Centre for Plant Medicine Inflammation is the body’s direct response to damage 
Research, Mampong-Akwapim, Ghana to its tissues and cells in order to heal the affected part 
4 Department of Biochemistry, Cell and Molecular Biology, (Weiss 2008). Inflammation may occur as a result of infec-
University of Ghana, Legon, Ghana tion, painful incisive injury, or immunologically modulated 
Vol.:(012 3456789)
 E. K. Kumatia et al.
injury (Vinay et al. 2004). The inflammatory reaction is ini- anti-inflammatory, analgesic, and anti-oxidant activities of 
tiated and sustained by the release of inflammatory media- A. macrophylla leaf and in addition, quantify the flavonoid 
tors such as histamine, prostaglandins (PGs), leukotrienes and phenolic contents of the leaf.
(LTB4), nitric oxide (NO), platelet-activation factor (PAF), 
bradykinin, serotonin, lipoxins, cytokines and growth fac-
tors from the cells and proteins of the complement, kinins 
and coagulation systems from the plasma when the body is Materials and methods
injured (Sarulkah et al. 2008; Vogel 2002). These inflamma-
tory mediators also cause sensitization of nociceptors in the Chemicals and other reagents
tissues of the affected area, hence, resulting in pain.
Moreover, the human body, as a biological machine, car- Carrageenan and acetic acid were purchased from Sigma 
ries out constant metabolism to generate substances that are Chemical Co. (St. Louis, USA). Diclofenac sodium salt was 
beneficial to the body. During this process, Reactive Oxygen purchased from Sigma-Aldrich (St. Louis, USA). Normal 
Species (ROS) free radicals such as hydroxyl ( OH.–), super- saline was supplied by Otsuka Pharmaceuticals India Pri-
oxide anion  (O –2 ), hydroperoxyl ( OOH–), peroxy  (RO2–) vate Limited (Vasana-Chakawadi, India). Analytical grade 
and alkoxy  (RO–) are also produced (Cai et al. 2004). These ethanol was used to extract the leaf.
free radicals are useful in receptor activation, gene expres-
sion and signal transduction in biological systems (Ajith and 
Janardhanan 2007). Nonetheless, excess free radicals lead Collection and identification of plant leaves
to the development of oxidative stress which is the princi-
pal cause of chronic ailments like diabetes, cardiovascular A. macrophylla leaves were collected in October, 2016 from 
diseases, cancers, degenerative diseases and inflammation Fumesua in the Ashanti Region of Ghana. The leaves were 
(Cai et al. 2004; Tang et al. 2004). Antioxidants are mol- authenticated by Mr. Ntim Gyakari, a botanist. The sam-
ecules, which interrupts the vicious actions of free radicals ples of the leaves (CPMR 4937) were deposited in the her-
by disrupting the initiation, propagation or termination of barium of Centre for Plant Medicine Research, Mampong-
the oxidative stress processes generated by free radicals (Cui Akwapim, Ghana.
et al 2004). Plant based antioxidants compounds are known 
to inhibit the production of free radicals and relieve ailments 
caused by oxidative stress (Cai et al. 2004; Dragland et al. Processing and preparation of plant extract
2003; Akinmoladun et al. 2010; Ozen et al. 2010). Addi-
tionally, several investigations have shown that plant-based The leaves were sun-dried for 6 days. It was then milled and 
products with high flavonoids and phenolic contents also stored in air tight plastic bags and kept in a locker until its 
have high antioxidant effects (Cai et al. 2004; Hendra et al. usage. The leaves (200 g) were extracted with 2 L of 70% 
2011; Ravipati et al. 2012; Zhang et al. 2011). The body, on ethanol for 5 days. The extract was decanted and filtered 
its own, also produces anti-oxidant enzymes such as super- and the residue was re-extracted with 2 L of 70% ethanol for 
oxide dismutase (SOD), catalase (CAT), glutathione perox- 5 days and filtered. The filtrates from the extractions were 
ide (GPx) to counter the effect of oxidative stress and lipid combined and the ethanol removed by rotary evaporation at 
peroxidation in response to free radicals. This function of 42 ℃. The aqueous extract obtained was lyophilized to yield 
the body is enhanced by the intake of exogenous antioxidant 9.43 g (4.72% w/w) of greenish powder.
agents including some plant products.
Anthonotha macrophylla is an indigenous African 
medicinal plant whose leaf is used to treat headache, diar- Experimental animals
rhoea, dysentery, skin infections, and as a remedy for poi-
sonous stings and bites. The bark is used as a vermifuge The laboratory animals used for this work were obtained 
and panacea against venereal diseases. The root is used to from the Animal Experimentation Unit of the Centre for 
relieve intestinal distress (Mshana et al. 2000; Keay 1960; Plant Medicine Research (CPMR), Mampong-Akwapim 
Burkill 1985). Literature available show that only toxicity in the Eastern Region of Ghana. The animals were fed ad 
studies and aphrodisiac activity have been conducted on libithum on powdered feed supplied by Ghana Agro Food 
the leaf of A. macrophylla (Essien and Sam 2018; Yakubu Company (GAFCO), Tema, Ghana. The animals were also 
and Abdulquadri 2016). Although, the leaf of the plant is allowed free access to sterilized water. Permission was 
used in traditional medicine to treat pain, inflammatory and granted by CPMR for this study to be conducted and all 
oxidative disorders, the plant has not been evaluated for national and international protocols were followed to ensure 
these activities. This study aimed therefore, to evaluate the that any discomfort suffered by the animals was minimal.
1 3
Effect of Anthonotha macrophyla (P. Beauv) leaf extract on carrageenan‑induced paw oedema,…
Basic phytochemical screening of the AML extract Acute toxicity assay of the AML extract
The crude ethanolic extract (50 ml) was measured into The safety or toxicity of AML extract in the short-term uti-
a ceramic evaporating dish. The dish was heated over lization was assessed in male ICR mice (21–28 g of body 
the water bath until all the ethanol evaporated from the weight). The test was performed on a single group of mice 
extract. The aqueous extract obtained was screened for the (N = 6) with each given 5, 000 mg of AML extract recon-
presence or absence of ten (10) classes of phytochemical stituted in 10 mL of distilled water at a concentration of 
compounds as previously described by Trease and Evans 100 g/mL per their respective body weight. The mice were 
(1989). observed for a total of 14 days for signs of toxicity such as 
changes in eye colour, salivation, lachrymation, coma, and 
eventual death. The mice were observed on an hourly basis 
Quantification of total phenolic content of the AML within the first 12 h of administration, then on daily basis 
extract for the remaining 13 days.
Folin–Ciocalteau method was employed to evaluate the 
total phenolic content of the 70% ethanol extract of AML Anti‑inflammatory activity of the AML extract
extract (Singleton et  al. 1999). Briefly, 100 μL of 5% 
Folin–Ciocalteau reagent was combined with 20 μL of Anti-inflammatory activity of AML extract was evaluated 
0–5 mg/mL of the extract in increasing order of concen- using the carrageenan-induced paw oedema model in rats 
tration. This was followed by addition of 80 μL of 7.5% as described by Winter et al. (1962). Female Wistar rats 
sodium carbonate to the mixture in a 96-well plate. The (197–212 g) were separated into five (5) groups of four 
resultant mixture was thoroughly mixed and incubated in (4) rats each. Group 1 served as control and received the 
the dark at room temperature for 1 h. The absorbance of corresponding volume of distilled water. Group 2 received 
the mixture was then measured with an infinite M200Pro diclofenac (DS) at a dose of 4 mg/kg p.o. as the standard 
micro-titer plate reader (Tecan, Austria) at 765 nm. The drug. Groups 3–5, which served as the test groups, received 
experiment was performed in triplicate. A standard cali- reconstituted lyophilized leaf extract of AML at doses of 5, 
bration curve using Gallic acid under the same experimen- 50, and 300 mg/kg p.o., respectively. The drug and extracts 
tal conditions was plotted from which the concentration of were administered via oral gavage.
phenol in the plant extract was estimated. The total phe- The baseline line paw volume of each rat was determined 
nolic content of the extract was expressed as microgram of by an average of 2 measurements before administration 
Gallic acid equivalent (GAE) per milligram of dry sample. of any drug using plethysmometer (UGO Balise, 7140). 
Oedema was then induced by the sub-plantar injection of 
0.1 ml carrageenan in normal saline (1% w/v) in the right 
Quantification of total flavonoids content hind paw of each animal. The paw volume of the right hind 
of the AML extract limb of each animal was measured again at an hourly inter-
val for 5 h after the administration of the drug/extract. The 
Aluminum colorimetric method (Chang et al. 2002; Stank- total oedema induced during the 4-h duration of the experi-
ovic 2011) with slight modifications was employed to ment was measured as Area Under the time course Curves 
quantify the total flavonoid content of the crude ethanolic (AUC). Anti-inflammatory activity (AI) was calculated as:
extract of the AML. Various concentrations (0.0391–5 mg/ AI =((Mean AUC of CG −Mean AUC of TG))
mL) of the extract in distilled water were prepared by 
serial dilution. Equal volume (100 μL) of the extract was ∕(Mean AUC of CG) × 100 %
combined with equal volume of aluminum chloride (100 where: CG = Control Group and TG = Treated Group.
μL) and then incubated at room temperature for 10 min 
in the dark. The absorbance of the resultant mixture was Analgesic activity of the AML extract
determined at a wavelength of 415 nm with Infinite micro-
titer plate reader (Tecan, Austria). A standard calibration Analgesic activity of AML was assessed using acetic acid—
curve was plotted using quercetin as the reference com- induced writhing assay (Koster et al. 1959). Male ICR mice 
pound. The concentration of flavonoids in the extract was (16–24 g) were segregated into seven (5) groups with each 
then determined from the calibration curve. The total fla- group containing four (4) mice. The drug and extracts were 
vonoid present in the extract was expressed as milligram administered as follows; Group 1 serve as control and were 
of quercetin equivalents (QE) per gram of extract. Water given equivalent volume of distilled water. Group 2 was 
was used as a blank.
1 3
 E. K. Kumatia et al.
given an oral dose of 10 mg/kg diclofenac sodium (DS) as Preparation of liver samples for anti‑oxidant enzymes 
a standard non-steroidal anti-inflammatory drug (NSAID). determination
Group 3–5 were also given the test extract (AML) at 5, 50, 
and 300 mg/kg orally. Pain was induced in each mouse by The rats were sacrificed by cervical dislocation 4 h after 
intraperitoneal injection with 1% acetic acid 30 min after induction of inflammation and dissected immediately. Their 
administration of the extract or standard drug. Each mouse livers were removed and washed in ice-cold normal saline 
was then placed into a separate plastic cage and its number (0.9%) and kept at  − 70 °C. Each liver sample (1 g) was 
of writing movements counted for 20 min. Analgesic activ- homogenized in 10 mL of 250 mM sucrose solution, 1 mM 
ity was calculated by relating the mean writing number of EDTA, 1 mM DL-dithiothreitol and 15 nM Tris HCl at pH 
the extract/drug with that of the control using the formula: of 7.4. Each individual homogenate was centrifuged for 
30 min at 9000 rpm. The supernatant portion obtained was 
%AE = ((MRc −MRt)∕MRc) × 100 collected and used to determine the activities of the antioxi-
where: MRc = mean writhing numbers of the vehicle-treated dant enzymes and effect on lipid peroxidation as reported 
control group. MRt = mean writhing numbers of treated previously by Appiah-Opong et al. (2015).
groups.
Determination of superoxide dismutase (SOD) activity
DPPH free‑radical scavenging activity of the plant 
extract Quercetin was used as substrate for the determination of 
SOD activity after the test sample was appropriately diluted 
The free radical scavenging activity of AML extract was as described previously by Tietez (1969). The test mixture, 
evaluated using a previously described method (Kostyuk which has a total volume of 1 mL, constitutes 0.08 mmol/L 
et al. 1990). Approximately 100 µl of 2, 2-diphenyl-1-pic- EDTA; 1 mol/L sodium phosphate buffer (pH 7.8) in a ratio 
rylhydrazyl (DPPH) in methanol (0.5 mM) was added to of 1:1; 0.1 mL of the diluted supernatant (diluted in the 
100 µl each of varying concentrations of AML extract and ratio of 1: 1000) and 2.3 mL of distilled. The absorbance 
allowed to stand for 20 min at room temperature (25 ºC) of the mixture was measured at 0 and 20 min at 406 nm in 
in the dark. The absorbance of the resultant mixture was a microplate reader (Tecan, Austria) to determine the rise 
measured at 517 nm. Ascorbic acid was used as standard in absorbance of the analyte due to oxidation of quercetin. 
and butylated hydroxytoluene (BHT) as a positive control. SOD activity was determined as the amount of the enzyme 
The percentage antioxidant activity of the test samples was that inhibited the oxidation of quercetin by 50% under the 
calculated using the equation: experimental conditions.
Antioxidant = ((A − B)∕A) × 100 % Determination of glutathione peroxidase (GPx) activity
where A is optical density of the blank, and B is the optical 
density of the test sample. The procedure used involved the reduction of 5, 5-dithio-
The concentration of AML extract or positive control bis (2-nitrobenzoic acid) (DTNB) with reduced glutathione 
BHT at 50% antioxidant activity was determined to be the (GSH) to yield a yellow substance (chromogen). The con-
Effective concentration at 50% value ( EC ). The experiment centration of chromogen obtained was determined by meas-50
was carried out in triplicate. uring absorbance at 405 nm spectrophotometrically. The concentration of the yellow substance is directly propor-
In vivo antioxidant activity of the AML extract tional to the concentrations GSH and GPx (Di Rosa 1972).
The in vivo anti-oxidant activity of the extract analysed Determination of the effect of AML extract on lipid 
in rats that have been induced with inflammation. Briefly, peroxidation
three groups of three rats each (N = 3) were used. Group 1 
received 1 mL of distilled water as negative control. Group The effect of the extract on lipid peroxidation was evalu-
2 received DS (10 mg/kg, p.o.) and group 3 AML extract ated using thiobarbituric acid reactive substances (TBARS) 
300 mg/kg p.o.. Inflammation was induced in the right hind method (Draper and Hadley 1990). Test mixture was made 
paw of the rats as described above in the anti-inflammatory by mixing 100 μL of the liver homogenate and 100 μL of 
assay. Superoxide dismutase (SOD) and Glutathione per- 5% trichloroacetic acid (TCA) and centrifuged at 4000 g for 
oxidase (GPx) activities were measured in the hepatocytes. 10 min. Thereafter, 100 μL of the supernatant and 200 μL of 0.67%) thiobarbituric acid reagent (TBA) were mixed and 
incubated for 15 min in a boiling water bath. The resultant 
mixture was allowed to cool and its absorbance measured at 
1 3
Effect of Anthonotha macrophyla (P. Beauv) leaf extract on carrageenan‑induced paw oedema,…
532 nm. The degree of lipid peroxidation was evaluated as Total phenolic content (TPC) and total flavonoids 
thiobarbituric acid reactive substances (TBARS), which was content (TFC)
expressed as malondialdehyde (MDA) formation in nmoles/
mg of tissue. The results for total phenolic and total flavonoid contents of 
the crude ethanolic AML extract are shown in Table 2. The 
Statistical analysis standard calibration curves drawn for gallic acid and quercetin 
from which the total phenolics and total flavonoids were calcu-
GraphPad Prism 5 statistical software was used for all lated gave R 2 values of 0.9981 and 0.9999 respectively. These 
analyses. Each point on the time course curve or column in values are almost equal to 1 (100%). This shows that there is 
the bar chart represents the mean ± S.E.M. The data in the linearity over the calibration range during the experiments. 
time-course curves experiment was analysed using Two Way Total phenolic and flavonoids content of AML were calculated 
ANOVA followed by Bonferroni’s Post hoc test. In addition, to be 29.94 ± 1.67 mg GAE / g and 1.34 ± 0.26 mg QE / 100 g 
One-way ordinary ANOVA repeated measures test was also of dry weight of the extract respectively.
used to analyse data for the bar charts followed by Dunnett’s 
Post hoc test. The results were considered statistically signif- Acute toxicity of crude ethanolic AML extract
icant when p < 0.05 by comparing the mean of test subjects 
to the control group. The mice that were given the single dose of 5000 mg of AML 
extract each did not show any sign of toxicity or death neither 
in the first 24 h of extract administration nor in the remaining 
Results 13 days of observation.
Basic phytochemical screening Anti‑inflammatory activity of the crude ethanolic 
AML extract
The results of the phytochemical screening of the crude 
ethanolic AML extract are shown below in Table 1. Four The result of the anti-inflammatory study on AML extract is 
(4) classes out of the ten (10) phytochemical constituents shown below in Fig. 1. The AML (5–300 mg/kg, p.o.) pro-
analysed, namely; saponins, flavonoids, phytosterols, and duced a slight dose-dependent inhibition of the formation 
free reducing sugars were detected. of oedema in the carrageenan-induced paw oedema assay 
in rats on the time course curve (Fig. 1a) and on the overall 
anti-inflammatory response calculated as the Area Under the 
Curve (AUC) (Fig. 1b). The highest dose of AML (300 mg/kg, 
Table 1  The results of the basic phytochemical screening of the crude p.o.) produced the highest overall anti-inflammatory response 
ethanolic AML extract which was statistically significant (p < 0.05) compared to the 
Phytochemical constituent Present/absent untreated control and was comparable to that of the diclofenac 
sodium salt (DS) (10 mg/kg p.o.) (Fig. 1b). Moreover, the 
Saponins  + highest dose of AML also produced statistically significant 
Flavonoids  + (p < 0.05) inhibition of oedema formation on the third hour on 
Phytosterols  + the time course curve (Fig. 1a) compared to the untreated con-
Phenolic compounds  + trols. The anti-inflammatory activities calculated for the AML 
Free reducing sugars  + extract and the DS were 66.14% and 63.29%, respectively.
Alkaloids −
Triterpenes − Analgesic activity of the crude ethanolic AML 
Polyuronides − extract
Anthracenosides −
Cyanogenic glycosides − The results of the analgesic effect of AML are shown below 
 + means present; − means absent in Fig. 2. AML at doses of 50 – 300 mg/kg p.o. reduced 
Table 2  Total phenolic and total Analyte Equation R2 TPC (mg/100 mg) TFC (mg/100 mg)
flavonoid contents of the crude 
ethanolic AML extract Gallic acid y = 0.384x + 0.0521 0.9981 29.94 ± 1.67 –
Quercetin y = 14.733x + 0.0015 0.9999 – 1.34 ± 0.26
N = 3; TPC = total phenolic content; TFC = total flavonoid content
1 3
 E. K. Kumatia et al.
B
4
A
2.0
Control 3
1.5 Diclo n.s. n.s.
AML 5 2
1.0 AML 50 + +
AML 300 1
0.5
*
0.0 0
1 2 3 4 5 Control Diclo 10 5 50 300
-0.5 Time (h)
    AML
Fig. 1  Inhibitory effect of AML (5–300 mg/kg, p.o.) and diclofenac (untreated) control group analysed with two-way ANOVA followed 
sodium (10  mg/kg, p.o.) on carrageenan-induced paw oedema in by Bonferroni’s post hoc test. +p < 0.05 or p > 0.05 (n.s. = not signifi-
rat’s paw on time course curve (a) and total oedema response cal- cant) compared to vehicle-treated control (untreated) group analysed 
culated as Area Under the Curve (b). Each point or column repre- with one-way ANOVA followed by Dunnett’s post hoc test
sents mean ± SEM (n = 4). *p < 0.05 compared to vehicle-treated 
150 DPPH scavaging activity
The DPPH scavaging activity of the extract  (EC50 value of 
100 0.06 ± 0.00 mg/ml) (Fig. 3b) was not statistically signifi-n.s. n.s. cantly different from that of Ascorbic acid (0.03 ± 0.00 g/
+
+++ ml) (Fig. 3a).
50
The effect of crude ethanolic AML extract on SOD 
and GPx activities
0
Control DS 10  5 50 300 The effects of AML extract on SOD and GPx activities in rat 
liver after 4 h of carrageenan-induced paw oedema as com-
AML pared to the diclofenac sodium salt (DS) is shown in Fig. 4. 
Both AML extract and DS significantly (p < 0.01) elevated 
Fig. 2  The effect of the crude ethanolic AML extract (5–300  mg/ SOD activity in response to carrageenan-induce inflamma-
kg, p.o.) and diclofenac sodium (DS) (10 mg/kg, p.o.) on acetic acid-
induced writhing counts in mice. Each column denotes mean ± SEM tion (Fig. 4a) compared to the control. However, only the 
(N = 4). +p < 0.05; +++p < 0.001 or p > 0.05 (n.s. = not significant) extract significantly (p < 0.05) elevated GPx activity and not 
compared to vehicle-treated (untreated) control group (One-way the DS (p > 0.05) (Fig. 4b).
ANOVA followed by Dunnett’s multiple comparisons test)
The effect of the crude ethanolic AML extract 
acetic acid-induced writhing counts in mice compared to the on lipid peroxidation activity
untreated control with the 300 mg/kg p.o. eliciting significant 
(p < 0.05) analgesic effect. However, the analgesic activity of The results obtained in the lipid peroxidation assay as shown 
300 mg/kg p.o. AML extract (31.0%) was slightly lower than below (Fig. 5), show that both AML extract and DS had 
the standard non-steroidal anti-inflammatory drug, DS (10 mg/ no significant effect (p > 0.05) on lipid peroxidation in vivo.
kg, p. o.), which was 50.0%.
1 3
increase in oedema (ml)
Mean writhing counts
Total Area Under Time Corse Curve
Effect of Anthonotha macrophyla (P. Beauv) leaf extract on carrageenan‑induced paw oedema,…
A B
Fig. 3  DPPH free radical scavenging activity of Ascorbic acid (a) and AML (b) showing their  EC50 and  R2 values using regression analysis. 
N = 3
A B
800 0.020 +
600 ++ ++ 0.015
n.s.
400
0.010
.
200
0.005
0
Negative DS AML 0.000
Control (10 mg/kg) (300 mg/kg) Negative DS AML
Control (10 mg/kg) (300 mg/kg)
Fig. 4  The effect of AML extract (300 mg/kg, p.o.) and DS (10 mg/ +p < 0.05, ++p < 0.001 or p > 0.05 (n.s. = not significant) compared to 
kg, p.o.) on SOD (a) and GPx (b) activities in rat liver. Each point vehicle-treated control group (One-way ANOVA followed by Dun-
represents mean SEM (n = 3; Test was also performed in triplicate 3). nett’s multiple comparisons test)
Discussion polyphenols found in plants which aid the human body to 
combat diseases (Stankovic 2011). The present study inves-
Polyphenols are essential class of phytochemical compounds tigated the anti-inflammatory, analgesic, and anti-oxidant 
found in plants because they act as antioxidant agents to activities of AML extract and quantified its total phenolic 
eradicate free radicals from biological systems. Polyphenols and flavonoid contents.
have hydroxylated aromatic benzene ring(s) in their struc- The results from the basic phytochemical analysis showed 
ture and act as antioxidants by mopping up free radicals that the crude ethanolic AML extract contains four general 
and chelate metal ions which intiates the formation of ROS groups of phytochemicals, namely, saponins, flavonoids, 
(Stankovic 2011). Flavonoids are an important group of phytosterols and reducing sugars. The extract was found 
to contain high amounts of phenols and low quantities of 
1 3
% SOD activity
GSH (mg/ml)
 E. K. Kumatia et al.
8×10-7 of AML may therefore, contribute significantly to its high 
n.s. n.s. antioxidant and anti-inflammatory activities observed in this 
6×10-7 study.
DPPH is nitrogen centered free radical which has an odd 
4×10-7 electron that absorbs strongly at 517 nm. In the presence of 
free radical scavenger (antioxidant), the DPPH radical accept 
hydrogen and becomes reduced (DPPH-H) (Cai et al. 2004)). 
2×10-7 The antioxidant activity of a substance in the DPPH assay 
is given by the effective concentration ( EC50) value of the 
0
Negative Diclofenac AML substance. The Effective concentration ( EC50) is the concen-
Control (10 mg/kg) (300 mg/kg) tration of the antioxidant that can reduce half the amount of 
DPPH radical present in the test solution to DPPH-H. The 
Fig. 5  The effect of AML (300  mg/kg, p.o.) and diclofenac sodium  EC50 obtained for AML in this study is low and is compara-
(10 mg/kg, p.o.) on lipid peroxidation activity in rat liver. Each point ble to that of the standard, indicating that AML has a high 
represents mean SEM (n = 3; Test was performed in triplicate 3). antioxidant capacity in vitro.
p > 0.05 (n.s. = not significant) compared to vehicle-treated control 
group (One-way ANOVA followed by Dunnett’s multiple compari- The production of reactive oxygen species (ROS), which 
sons test) are key contributors to the initiation of oxidative stress, 
was found to take place in the carrageenan induced paw 
inflammation process (Di Rosa 1972). Furthermore, the 
flavonoids. The phenols and flavonoids present in the extract liver was reported to be the major organ which undertakes 
may partly be responsible for its significant anti-oxidant and cellular homeostasis, by the production of different types 
anti-inflammatory activities. of endogenous antioxidant enzymes which eliminates ROS 
None of the mice that were administered with 5000 mg/ (Ighodaro and Akinloye 2018). The liver also performs 
kg body weight of the extract died or displayed any sign of crucial functions in the breakdown of drugs and other sub-
toxicity within the 14 days period of observation, indicating stances ingested into the body. Hence, its oxidative state 
that the  LD50 of AML extract is above 5000 mg/kg body might be affected by the intake of various exogenous sub-
weight in male ICR mice. Hence, AML extract may be safe stances (Ighodaro and Akinloye 2018). SOD and GPx are 
in the short-term usage even at a very high dose of 5000 mg/ known as first-line anti-oxidant enzymes which prevent the 
kg. Based on the results of the acute toxicity assay, three generation of free radicals and ROS in cells (Ighodaro and 
doses of the extract which were lower than its  LD50 values Akinloye 2018). Moreover, GPx also belongs to a second 
were selected for the other assays. group of endogenous antioxidant enzymes called the free 
Carrageenan-induced oedema in rat paw model of inflam- radical scavengers which mop up active radicals to obstruct 
mation has been shown to be a tri- phasic process which is chain initiation and disrupt chain proliferation reactions 
governed by production of pro-inflammatory mediators (Di (Vasilaki and McMillan 2011). The result from the current 
Rosa, 1972; Koster et al. 1959). The first Phase occurs from study shows that administration of AML extract (300 mg/kg 
0 to 1.5 h after carrageenan is injected. This phase involves p.o.) in carrageenan induced paw inflammation in rats after 
and is controlled by the production of histamine and sero- 5 h significantly elevated the production of SOD and GPx 
tonin, followed by the second phase which occurs from 1.5 enzymes in rat liver. On the other hand, DS (10 mg/kg p.o.) 
to 2.5 h, which is mediated by the release of bradykinins only significantly increased SOD activity but has no effect 
(Cordova et al. 2011). The last phase is controlled by the on GPx activity. This result indicates that AML extract is 
production of prostaglandins from 2.5 to 6 h after induction able to protect against damage from oxidative stress by pre-
(Ding et al. 2009). The extract significantly (p < 0.05) inhib- vention of free radical formation, obstruction of free radical 
ited oedema formation at the 3 h on the time course curve. chain initiation and propagation reactions. Whereas, DS is 
This period corresponds to the third Phase of inflammation. only able to protect against free radical formation. Therefore, 
Hence, AML extract may be exerting its anti-inflammatory AML extract possessed in vivo antioxidant activity.
action by suppressing the production or action of prostaglan- Lipid peroxidation is a metabolic process in which ROS 
dins. Previous studies have shown that high total flavonoids causes oxidative destruction of lipids in cell membranes and 
and total phenolic content of plant extracts leads to high anti- thereby substantially distort the integrity of the structure 
oxidant and anti-inflammatory activities (Antonio and Souza and function of the cell membrane. These actions of ROS 
Brito 1998; Chohan et al. 2012; Pan et al. 2010; Khedir et al. may also result in damage of important bio-proteins such as 
2016). Since phenolic compounds and flavonoids eradicate enzymes that repair DNA and polymerases in addition to the 
free radicals which initiate oxidative stress and inflammatory production of aldehyde by-products like malondialdehyde 
reactions in biological systems, the high phenolic content and 4-hydroxy-2-nonenal (Escalante et al. 2002). The result 
1 3
[TBARS] moles/mg protein
Effect of Anthonotha macrophyla (P. Beauv) leaf extract on carrageenan‑induced paw oedema,…
from this study shows that AML extract and DS did not Funding This work was supported by Centre for Plant Medicine 
reduce the production of TBARS in the treated groups as Research, Mampong-Akwapim, Ghana. There is no external funding 
compared to the control group. Therefore, AML extract and received for this article.
DS could not protect against lipid peroxidation. Data availability The data generated from the research to support these 
It has been reported that intraperitoneal injection of findings can be obtained from the corresponding author upon reason-
acetic acid into mice abdominal cavity leads to increased able request.
prostaglandins production in the peritoneal fluid after about 
30 min (Kaefer and Milner 2008). Thus, the stretching and Code availability Not applicable.
abdominal contortion produced in the writhing assay corre-
late with sensitization of pain receptors to the generation of Compliance with ethical standards 
prostaglandins. The result from this study indicate that AML 
(300 mg/kg, p.o.) significantly (p < 0.05) blocked writhing Ethical statement Permission for the animal study was granted by the Ethical Review Committee of the Centre for Plant Medicine Research, 
movement in the mouse acetic acid-induced writhing assay. Ghana, (Reference No: CPMR-Et/M.03/2016) and all national and 
This indicates that the analgesia produced by AML may be international protocols for care of animals were followed to ensure 
due to the ability of the extract to suppress the production that any discomfort suffered by the animals was minimal.
or action of the cyclooxygenase enzyme and prostaglandins.
Plant secondary metabolites such as saponins, flavonoids, Conflict of interest Emmanuel Kofi Kumatia has no conflict of interest. 
and phytosterols are known to possess varieties of biological Frederick Ayertey has no conflict of interest. Regina Appiah-Opong has no conflict of interest. Ebenezer Ofori-Attah has no conflict of in-
activities which make the plants medicinal. For example, terest. Peter Bolah has no conflict of interest. Stephen Antwi has no 
saponins are known to possess anti-inflammatory and anti- conflict of interest. Alfred Ampomah Appiah has no conflict of inter-
allergic, cytotoxic, and anti-viral activities (Lacaille-Dubois est. Augustine Ocloo has no conflict of interest.
and Wagner 2000; Levy 1969; Matsuda et al. 1990; Mishra 
et al. 2013). Flavonoids were reported for their anti-inflam-
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