Journal of Medicinal Plants for Economic Development
ISSN: (Online) 2616-4809, (Print) 2519-559X
Page 1 of 5
Original Research
Effect of lupeol from Vernonia glaberrima (Asteraceae)
on pain and inflammation
Authors:
Amina J. Yusuf1
Musa I. Abdullahi1
Aisha M. Umar1
Fatima Musa2
Affiliations:
1
Department of
Pharmaceutical and
Medicinal Chemistry, Usmanu
Danfodiyo University, Sokoto,
Nigeria
Department of Biochemistry,
Usmanu Danfodiyo
University, Sokoto, Nigeria
2
Corresponding author:
Amina Yusuf,
amynajega@gmail.com
Dates:
Received: 15 May 2020
Accepted: 27 June 2020
Published: 15 Sept. 2020
How to cite this article:
Yusuf, A.J., Abdullahi, M.I.,
Umar, A.M. & Musa, F.,
2020, ‘Effect of lupeol from
Vernonia glaberrima
(Asteraceae) on pain and
inflammation’, Journal of
Medicinal Plants for
Economic Development 4(1),
a84. https://doi.org/10.4102/
jomped.v4i1.84
Copyright:
© 2020. The Authors.
Licensee: AOSIS. This work
is licensed under the
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Background: Steroids have been reported to possess analgesic and anti-inflammatory
activities, and Vernonia glaberrima also possesses analgesic and anti-inflammatory properties.
Aim: The aim of the study was to evaluate the effect of lupeol isolated from the n-hexane
soluble fraction of the methanol leaf extract of V. glaberrima on pain and inflammation.
Method: Lupeol was re-isolated from the leaf of V. glaberrima by using chromatographic
procedures; it was subjected to analgesic and anti-inflammatory studies by using acetic acidinduced writhing test in mice and formalin-induced pain and inflammation in rats,
respectively. The intraperitoneal lethal dose (LD50) of lupeol was determined by using Lorke’s
method.
Results: The results of the study showed that lupeol significantly (p < 0.05) decreased
writhing response at doses 12.5 mg/kg, 25.0 mg/kg and 50.0 mg/kg corresponding to
percentage inhibition of 83.60%, 83.63% and 80.02%, respectively. This was higher than
piroxicam, the standard drug (73.8%), at 10 mg/kg. The compound was also able to
significantly (p < 0.05) reduce nociceptive response in both phases of the formalin test, and
there was a remarkable reduction of oedema by the compound at the second, third
and fourth hours. The median LD50 of the compound was estimated to be greater than
5000 mg/kg.
Conclusion: The findings of this study indicated that lupeol from the leaf of V. glaberrima has
good analgesic and anti-inflammatory activity that validates the ethnomedicinal use of the
plant in the treatment of pain and inflammatory conditions.
Keywords: Vernonia glaberrima; lupeol; analgesic; anti-inflammatory; Lorke’s method.
Introduction
Pain is the most common reason for a physician consultation in the most advanced countries
(Conaghan 2012), and it is a major symptom in many medical conditions and significantly
interferes with a person’s quality of life and general functioning (Howland & Mycek 2006).
Analgesics are drugs that selectively relieve pain by acting in the central nervous system (CNS) or
peripheral pain mechanisms without significantly altering consciousness (Howland & Mycek
2006), but the drugs are accompanied by serious side effects such as euphoria, tolerance,
respiratory depression, dependence, renal damage and gastrointestinal irritation to mention but
a few (Emily & Gari 2008; Howland & Mycek 2006).
Vernonia glaberrima Welw. Ex.O. Hoffm., an erect shrub commonly known as bitter leaf of the
red clay soil, belongs to the Asteraceae family; it is mostly found on hillside grassland of West
and Central Africa (Burkill 1995). Vernonia glaberrima is commonly known as Shiwáákáár
(shìwaákár)-ján-gágárií in Hausa-Northern Nigeria (Burkill 1995). The plant is used in
ethnomedicine to treat different diseases such as malaria, migraine, diabetes, psora and
dysmenorrhea (Burkill 1995). It is also employed in the treatment of pain, inflammation,
vertigo, microbial infections, body pain, skin cancer and other skin-related disorders (Abdullahi
et al. 2015a; Alhassan et al. 2018). Chemical investigations on the leaf of the plant have been
confined to the isolation and characterisation of fatty acids, steroids and coumarins (Emily &
Gari 2008). Pharmacological studies ranging from anticancer, analgesic, anti-inflammatory,
antimalarial, anti-diabetic, antimicrobial and antiviral activities have also been reported
(Abdullahi et al. 2015b, 2015c, 2015d, 2017; Alhassan et al. 2018; Ananil et al. 2000). This study
was conducted to evaluate the analgesic and anti-inflammatory activities of lupeol from
V. glaberrima leaf.
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Open Access
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Materials and methods
Original Research
cages at room temperature. All experimental procedures
were approved by the Animal Right and Ethics Community
of the University.
Study area
All experiments were conducted in the Laboratory of
Department of Pharmaceutical and Medicinal Chemistry
in collaboration with Department of Pharmacology and
Toxicology, Faculty of Pharmaceutical Sciences, Usmanu
Danfodiyo University, Sokoto, Nigeria, from August 2017 to
October 2017.
The intraperitoneal lethal dose (LD50) of lupeol from
V. glaberrima was determined according to the method
described by Lorke (1983).
Chemicals, reagents and standard drug
Analgesic studies
All chemicals and reagents used in this study were of
analytical grade. Piroxicam capsules (U.S.P, Yangzhou
Pharmaceutical Co. LTD, Jiangsu, China) and pentazocine
(injection BP; Alpha Laboratories LTD, India) were used.
Isolation and structure elucidation of lupeol
Lupeol (Figure 1) was re-isolated from the n-hexane fraction
of the methanol leaf extract of V. glaberrima as white
crystalline substance by using a combination of vacuum
liquid and low-pressure column chromatography. The
structure of the compound was established by using oneand two-dimensional nuclear magnetic resonance (NMR)
spectroscopic analysis and by direct comparison of data
obtained with those reported in the literature (Alhassan
et al. 2018).
Experimental animals
Forty Swiss albino mice and 50 adult Wister rats of either sex
weighing 18 g – 22 g and 175 g – 190 g, respectively, were
obtained from the Animal House Facility of the Department
of Pharmacology and Toxicology, Usmanu Danfodiyo
University, Sokoto, Nigeria. They were fed with laboratory
diet and water ad libitum and maintained under standard
conditions (12-h light and 12-h dark cycle) in propylene
H
H
H
H
FIGURE 1: Structure of lupeol.
http://www.jomped.org
Acute toxicity studies
Acetic acid-induced abdominal constrictions in mice
The method described by Koster, Anderson and Beer (1959)
was adopted; 25 albino mice were divided into five groups
of five mice each. Group 1 was injected with 10 mL/kg i.p.
of normal saline (negative control). Groups 2–4 were
injected i.p. with 12.5 mg/kg, 25 mg/kg and 50 mg/kg of
lupeol, respectively, and group 5 received piroxicam
10 mg/kg (positive control). Thirty minutes later, each
mouse was injected with 1 mL/kg of aqueous solution of
acetic acid (0.6%). The number of abdominal constrictions
for each mouse was counted 5 min after injection of acetic
acid for a period of 10 min. The percentage inhibition
of abdominal constrictions was calculated by using the
following formula:
Mean no. of writhes (negative control) −
Mean no. of writhes (test)
Inhibition (%) =
× 100.
Mean no. of writhes (negative control)
[Eqn 1]
Formalin test in rats
The test was conducted in accordance with the method
described by Dubuisson and Dennis (1977). Twenty-five
rats were used. The rats were divided into five groups,
each containing five rats. Group 1 was injected with
1 mL/kg of normal saline i.p. (negative control). Groups
2–4 were injected i.p. with 12.5 mg/kg, 25 mg/kg and
50 mg/kg of lupeol, respectively, and group 5 was injected
with pentazocine 10 mg/kg i.p. (positive control). Thirty
minutes after this treatment, 50 µL of a freshly prepared
2.5% solution of formalin was injected subcutaneously
under the plantar surface of the left hind paw of each rat.
The rats were placed individually in an observation
chamber and monitored for 1 h. The severity of pain
response was recorded for each rat based on the following
numerical scale: (0) rat walked or stood firmly on the
injected paw; (1) the injected paw was favoured or partially
elevated; (2) the injected paw was clearly lifted off the
floor; (3) the rat licked, chewed or shook the injected paw.
Anti-nociceptive effect was determined in two phases. The
early phase (phase 1) was recorded during the first 5 min,
whereas the late phase (phase 2) was recorded during the
last 45 min with a 10 min lag period in-between both
phases (Dubuisson & Dennis 1977).
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Anti-inflammatory studies
Original Research
significant (p < 0.05) reduction of abdominal constrictions
by 73.84% (Table 2).
Formalin-induced inflammation in rats
Formaldehyde 2.5% v/v was used as inflammogen
(Pateh et al. 2011). Rats were divided into five groups of
five rats each. Thirty minutes before injection of 2.5% v/v
formalin (50 µL volumes in the subplantar region of the
left hind paw of the rat), the groups were treated i.p. as
follows:
• Group 1 received 1 mL normal saline per kg, as negative
control.
• Groups 2, 3 and 4 received lupeol, at 12.5 mg/kg, 25 mg/
kg and 50 mg/kg, respectively.
• Group V received 10 mg piroxicam per kg, as positive
control. The paw diameter (cm) was measured by using
Vernier calliper at an interval of 1 h for 4 h.
Statistical analysis
All data were expressed as mean ± standard error of mean
(SEM). The mean values of control groups were compared
with the mean values of treated groups by using one-way
analysis of variance (ANOVA) followed by post hoc test by
using the Statistical Package for Social Sciences (SPSS)
software (version 22). Values were considered significant at
p < 0.05.
Ethical consideration
Formalin test in rats
The pre-treatment of rats with lupeol significantly (p < 0.05)
reduced nociceptive response induced by formalin in both
phases (Table 3). There was a remarkable reduction of pain
by the compound at the graded doses (12.5 mg/kg,
25.0 mg/kg and 50 mg/kg) with 60.67%, 74.52% and
80.93% inhibition in the first phase, whereas in the second
phase, the compound had 57.82%, 79.65% and 82.53%
inhibition of pain; the standard drug, piroxicam (at 10 mg/
kg), had 53.73% and 67.07% at both phases.
Formalin-induced inflammation in rats
Lupeol exhibited significant (p < 0.05) anti-inflammatory
activity at the graded doses employed (Table 4). The highest
dose (50 mg/kg) was able to inhibit oedema induced by
formalin with 47.62%, 49.35%, 52.86% and 58.21% at the
first, second, third and fourth hours, respectively. The
standard drug, piroxicam (10 mg/kg), had 31.75%, 48.05%,
47.14% and 50.75% inhibition.
TABLE 2: The effect of lupeol from Vernonia glaberrima on acetic acid-induced
writhing in mice.
Treatment (mg/kg)
Mean no. of writhes
% inhibition
Normal saline
21.67 ± 4.7
-
Lupeol (12.5)
3.67 ± 0.3*
83.60
Lupeol (25.0)
3.33 ± 1.8*
83.63
Lupeol (50.0)
4.33 ± 1.8*
80.02
Piroxicam (10)
5.67 ± 3.7*
73.84
Ethical approval to conduct this study was received from
the Animal Right and Ethics Community of the Usmanu
Danfodiy University (UDUS/UREC/2020/001).
Note: Each value represents mean ± standard error of mean.
*, p < 0.05 compared with normal saline (one-way analysis of variance); n = 5.
Results
TABLE 3: The effect of lupeol from Vernonia glaberrima on formalin-induced
pain in rats.
Toxicity studies
Mean pain scores
The intraperitoneal LD50 of lupeol was found to be greater
than 5000 mg/kg (Table 1). No death was recorded after
administration of lupeol at the different doses in both
phases.
Treatment (mg/kg)
First phase
% inhibition
Second phase
Normal saline
5.77 ± 0.1
-
11.45 ± 0.6
-
Lupeol (12.5)
2.27 ± 0.2*
60.67
4.83 ± 0.1
57.82
Lupeol (25.0)
1.47 ± 0.2*
74.52
2.33 ± 0.2*
79.65
Lupeol (50.0)
1.10 ± 0.1*
80.93
2.00 ± 0.1*
82.53
Pentazocine (10)
2.67 ± 2.7*
53.73
3.77 ± 2.3*
67.07
Note: Each value represents mean ± standard error of mean.
*, p < 0.05 compared with normal saline (one-way analysis of variance); n = 5.
Analgesic studies
Acetic acid-induced writhing test
Lupeol at the graded doses (12.5 mg/kg, 25.0 mg/kg
and 50 mg/kg) significantly (p < 0.05) decreased the
number of writhes in a dose-dependent manner. The
reference drug, piroxicam (at 10 mg/kg), exhibited a
TABLE 4: The effect of lupeol from Vernonia glaberrima on formaldehydeinduced inflammation in rats.
Treatment (mg/kg)
First
Second
Dose (mg/kg)
No. of mice used
Mortality
10
3
0/3
100
3
0/3
1000
3
0/3
1600
1
0/1
2900
1
0/1
5000
1
0/1
Note: The intraperitoneal LD50 of lupeol from V. glaberrima were calculated as follows:
LD50 of lupeol ≤ 5000 mg/kg i.p.
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Mean paw diameter (cm) (% inhibition)
1h
2h
3h
4h
Normal saline
0.63 ± 0.1
0.77 ± 0.2
0.70 ± 0.2
0.67 ± 0.1
Lupeol (12.5)
0.57 ± 0.0
0.60 ± 0.0
0.53 ± 0.1
0.48 ± 0.0
(9.52)
(22.08)
(24.29)
(28.36)
Lupeol (25.0)
0.40 ± 0.2
0.36 ± 0.1*
0.33 ± 0.0
0.30 ± 0.1*
(36.51)
(53.25)
(52.86)
(55.22)
Lupeol (50.0)
0.33 ± 0.2
0.39 ± 0.2*
0.33 ± 0.0*
0.28 ± 0.0*
(47.62)
(49.35)
(52.86)
(58.21)
Piroxicam (10)
0.43 ± 0.0
0.40 ± 0.0*
0.37 ± 0.3
0.33 ± 0.1
(31.75)
(48.05)
(47.14)
(50.75)*
TABLE 1: Median lethal dose (LD50) of lupeol from Vernonia Glaberrima.
Phase
% inhibition
Note: Each value represents mean ± standard error of mean.
*, p < 0.05 compared with normal saline (one-way analysis of variance); n = 5.
Values in parenthesis indicate percentage inhibition of inflammation.
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Discussion
Lupeol from V. glaberrima has showed significant (p < 0.05)
analgesic and anti-inflammatory effects. The median LD50 of
lupeol was found to be greater than 5000 mg/kg, which
indicates that the compound is generally non-toxic (Lorke
1983) (Table 1); previous studies conducted on the methanol
leaf extract of V. glaberrima and the n-hexane soluble fraction
showed a relatively lower LD50 values of 2154 mg/kg and
1265 mg/kg, respectively (Abdullahi et al. 2015c, 2016). The
possible explanation for this might be related to the presence
of other secondary metabolites in the extract and fraction that
contributes to the toxicity level of the plant.
Acetic acid-induced writhing test was used to assess the
peripheral nociceptive effect of the lupeol (Gené et al. 1998).
Writhings generated by administration of acetic acid in mice
are because of profound pain of endogenous nature that
recurs for a prolonged period of time. Because of irritant
nature, these principles are also prone to induce lesions
(Warden 2010). The intense pain is characterised by episodes
of retraction of abdomen and stretching of hind limbs; the
signals transmitted to central nervous system in response to
pain because of irritation cause release of mediators such as
prostaglandins, which contribute to the increased sensitivity
to nociceptors (Buer 2014).
Lupeol at the graded doses significantly (p < 0.05) decreased
writhing response at doses 12.5 mg/kg, 25 mg/kg and
50 mg/kg corresponding to percentage inhibition of 83.60%,
83.63% and 80.02%, respectively (Table 2). This was higher
than piroxicam, the standard drug (73.84%), at 10 mg/kg.
Pain inhibition by lupeol was significantly higher than the
effect observed by the reference drug, piroxicam. The findings
are in close agreement with what was reported for the
n-hexane fraction of V. glaberrima (Abdullahi et al. 2016).
Formalin test in mice involves biphasic responses categorised
into early phase (neurogenic pain) and the late phase
(inflammatory reactions). Lupeol was able to significantly
(p < 0.05) diminished the nociceptive response induced by
formalin in a dose-dependent manner (Table 3); it exhibited
60.67%, 74.52% and 80.93% inhibition of pain at the graded
doses (12.5 mg/kg, 25 mg/kg and 50 mg/kg) employed in
the early phase; thus, the effect was higher than the standard
drug piroxicam with 53.73% inhibition. A similar trend was
observed for lupeol in the second phase; thus, the lowest
dose exhibited the highest inhibition of pain. Studies have
indicated that central acting drugs can inhibit both early and
last phases, whereas peripheral acting drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) can only inhibit
the last phase, which suggests that lupeol may possess central
acting effects (Derardt et al. 1980; Maria et al. 1997).
Inflammation is a physiological body response against any
harmful stimulus, leading to the activation of inflammatory
cells that secrete increased amounts of nitric oxide (NO),
nitric oxide synthase (iNOS), prostaglandin E2 (PGE2),
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Original Research
cyclooxygenase-2 (COX-2) and cytokines (Derardt et al. 1980;
Gepdiremen et al. 2005). To evaluate the effect of lupeol from
V. glaberrima on inflammation, formalin test was used, which
led to the development of oedema after injection of the
inflammatory agent (formalin) in the left hind paw; this was
accompanied by the release of inflammatory mediators; and
lupeol significantly (p < 0.05) reduced the oedema level
induced by formalin at the graded doses (Table 4). The highest
dose (50 mg/kg) indicated a higher inhibition of inflammation
compared with the control with 49.35%, 52.86% and 58.21%
inhibition at the second, third and fourth hours, respectively.
The ability of lupeol to suppress the level of oedema suggests
it to be a good anti-inflammatory agent (Gallo & Sarachine
2009; Siddique & Saleem 2011), and the mechanism of action
of lupeol has been postulated to act via inhibition of tissue
response to the induced nociception (Chen et al. 2012; Geetha
& Varalakshmi 2001), especially through the involvement of
cytokines (De Lima et al. 2013). In addition, it has recently
been shown that lupeol acetate acts as an anti-inflammatory
agent by regulating tumour necrosis factor (TNF)-alpha and
IL-2-specific mRNA, besides up-regulating the synthesis of
IL-10 mRNA (Ashalatha et al. 2010).
Conclusion
Lupeol from V. glaberrima have showed significant analgesic
and anti-inflammatory effects with the highest dose
(50 mg/kg) having a strong effect. This further validates the
ethnomedicinal claim of the use of the plant in the treatment
of pain and inflammatory conditions. To the best of our
knowledge, this is the first report that validated the
analgesic and anti-inflammatory activities of lupeol from
V. glaberrima leaf.
Significance statement
This study discovers the analgesic and anti-inflammatory
effects of lupeol from V. glaberrima that can be beneficial to
researchers and pharmaceutical industries in drug discovery
and development. This study will help the researchers to
uncover the critical areas of natural product research that
many researchers were not able to explore. Thus, a new
theory on natural product research may be arrived at.
Acknowledgements
We acknowledged the efforts of Mal. Abdullahi of
Pharmacology and Toxicology & Mal Hamza of
Pharmaceutical & Medicinal Chemistry, Usmanu Danfodiyo
University, Sokoto, for assisting in animal handling and
extraction.
Competing interests
The authors have declared that no competing interest exists.
Authors’ contributions
M.I. Abdullahi performed research concept and design;
A.M. Umar and A.J. Yusuf conducted the research and
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Page 5 of 5
collected the data; A.M. Umar, A.J. Yusuf and F. Musa
performed data analysis and interpretation; A.J. Yusuf wrote
the article; and all authors were responsible for the critical
revision and final approval of the article.
Original Research
Ashalatha, K., Venkateswarlu, Y., Priya, A.M., Lalitha, P., Krishnaveni, M. &
Jayachandran, S., 2010, ‘Anti-inflammatory potential of Decalepis hamiltonii
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Funding information
This research received no specific grant from any funding
agency in the public, commercial or not-for-profit sector.
Chen, Y.F., Ching, C., Wu, T.S., Wu, C.R., Hsieh, W.T. & Tsai, H.Y., 2012, ‘Balanophora
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Data availability statement
Data sharing is not applicable to this article as no new data
were created or analysed in this stud.
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Disclaimer
The views and opinions expressed in this article are those of
the authors and do not necessarily reflect the official policy or
position of any affiliated agency of the authors.
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analgesic effects of morphine, meperidine and brain stem stimulation in rats and
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