Kratom (Mitragyna speciosa) Leaf Ethanol Extract Showed In Vivo Analgesic Activity

Abstract

Analgesic drugs like morphine and other opioids exhibit several harmful effects. Thus, the exploration of a new and safer analgesic drug is necessary. Utilizing Indonesia's biodiversity richness, medicinal plants can serve as an alternative source of novel analgesic drugs. Here, we show the analgesic activity of Kratom (Mitragyna speciosa) leaf ethanol extract (KE) in formaldehyde-induced rat (Rattus norvegicus) models. The effect of KE was examined by observing the duration of spontaneous nociceptive behavior such as paw licking or limp leg. The result showed that rats treated with 70 mg/kg KE demonstrated significantly (p<0.01) decreased nociceptive behavior compared to those receiving vehicles. However, a higher KE dose (210 mg/kg) failed to increase the analgesic effect and showed a slight reduction (not significant) compared to the control group. These findings proved that kratom leaf ethanol extract has the potential to alleviate painful conditions.

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Pharmacology and Clinical Pharmacy Research ISSN:2527-7322 | e-ISSN: 2614-0020
Volume 8 No. 2 August 2023
Kratom (Mitragyna speciosa) Leaf Ethanol Extract
Showed In Vivo Analgesic Activity
Rahmad A. Prasetya1 and Tri P.L Sudarwati2
1Department of Clinical Pharmacy, Pharmacy Academy of Surabaya, Surabaya, Indonesia
2Department of Microbiology, Pharmacy Academy of Surabaya, Surabaya, Indonesia
Abstract
Analgesic drugs like morphine and other opioids exhibit several harmful effects. Thus, the
exploration of a new and safer analgesic drug is necessary. Utilizing Indonesia’s biodiversity
richness, medicinal plants can serve as an alternative source of novel analgesic drugs. Here,
we show the analgesic activity of Kratom (Mitragyna speciosa) leaf ethanol extract (KE) in
formaldehyde-induced rat (Rattus norvegicus) models. The effect of KE was examined by
observing the duration of spontaneous nociceptive behavior such as paw licking or limp leg.
The result showed that rats treated with 70 mg/kg KE demonstrated signicantly (p<0.01)
decreased nociceptive behavior compared to those receiving vehicles. However, a higher KE
dose (210 mg/kg) failed to increase the analgesic effect and showed a slight reduction (not
signicant) compared to the control group. These ndings proved that kratom leaf ethanol
extract has the potential to alleviate painful conditions.
Keywords: Mitragyna speciosa, kratom leaves, ethanol extract, opioid analgesic
Corresponding Author: Rahmad Aji Prasetya. Department of Clinical Pharmacy, Pharmacy Academy of Surabaya, Surabaya -
Indonesia. Email: prasetya.ra@akfarsurabaya.ac.id
Received: 14 April 2023 Revised: 17 Juli 2023 Published: 11 August 2023
doi: 10.15416/pcpr.v8i2.40727
Introduction
Pain is a distressing sensory and emotional
experience related to actual or potential tissue
damage or mimicking that experience. Pain
is a multidimensional sensory experience
and can be distinguished in intensity (mild,
moderate, severe), quality (blunt, burning,
sharp), duration (transient, intermittent,
persistent), and spread (supercial or deep,
localized or diffuse).1
Based on the 2013 Baseline Health Research
(Riskesdas) data, the citizen of East Java
Province, Indonesia, was the most users of
analgesic drugs in all provinces in Indonesia.
This shows the high prevalence of pain in
Indonesia, especially in East Java.2 Regarding
the treatment of mild pain, non-steroidal
anti-inammatory drugs (NSAIDs) are
still effective in blocking pain sensations.
However, for moderate to severe pain due to
cancer or post-surgery, more potent opioid
analgesics are needed, such as codeine,
tramadol, oxycodone, morphine, or fentanyl.
This class of drugs has proven effective
in treating severe pain by blocking opioid
receptors in the central nervous system (CNS).
On the other hand, these opioid analgesics
also possess concerning side effects, such
as constipation, respiratory depression,
addiction, and the potential for drug abuse.3
Therefore, it necessitates developing a new
and safer opioid analgesic drug. Utilizing

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Volume 8 No. 2 August 2023
Indonesia’s biodiversity richness, medicinal
plants are a viable option since they contain
various chemicals from which new analgesic
medications might be developed.
Mitragyna speciosa, known as kratom,
has several properties such as analgesic,
antinociceptive, sedative, antiobesity,
anticancer, anti-inammatory, antioxidant, and
antibacterial.4–8 Specically for its analgesic
activity, several studies have reported that the
content of kratom leaves can bind to opioid
receptors in the CNS, resulting in severe
pain cessation.4,9 This effect is mediated by
two major constituents, mitragynine and
7-hydromitragynine, which are isolated from
the methanol extract of kratom. Whereas
withdrawal symptoms and addiction were
evident in both animal models and regular
kratom users, indicating that mitragynine
possesses the risk which closely mimics those
of morphine.10,11 Therefore, the present study
aimed to explore antinociceptive activity of
kratom leaf ethanol extract in rats (Rattus
norvegicus).
Methods
Collection and preparation of plant extract
Fresh leaves of Kratom (Mitragyna speciosa)
were bought from a supplier in West
Kalimantan, Indonesia, and authenticated by
Tri Puji Lestari Sudarwati (Pharmacy Academy
of Surabaya, Indonesia). Leaves (around 30 g)
were then washed, shade dried, and crushed
into powder. Subsequently, dry leaves powder
(20 g) was macerated with 150 mL of 96%
ethanol for 24 hours at ambient temperature.
The mixture was stirred occasionally to
maintain homogeneity and then ltered.5 The
crude extract (3.38 g) was then concentrated
using a rotary evaporator and coded as KE
(Kratom Extract). The yield of the extract was
found to be 16.9% w/w.
Animals
The preclinical study protocol was approved
by the Institutional Ethical Committee of
the University of Surabaya, decree number:
97A/KE/VII/2022. Male Wistar rats used in
this study were purchased from the animal
house of Drh Rachmad Priyadi farm weighing
80–150 g. The animals were placed in
plastic cages in a room maintained at a room
temperature (21°C) and 12 h light: dark cycle,
with unlimited access to standard chow and
water, then acclimatized for seven days before
the study started.
All feasible measures were taken to minimize
animal suffering and limit the number of
animals utilized in research. On day one, they
were randomly placed in a group of three.
Later on day eight, each group was assigned to
receive different treatments as follows: Group
I was the negative control (vehicle-treated),
Group II was the positive control tramadol
0.9 mg/kg p.o, Group III received KE 70 mg/
kg p.o., and Group IV received KE 210 mg/
kg p.o. All animals were sacriced after the
treatment and measurement to avoid further
pain.
Formaldehyde-induced pain model
The test substances and controls were
administered to the subject animals according
to the previously outlined protocol. After
administering the treatments for one hour, 50
μL of a solution containing 2% formaldehyde
was injected subcutaneously into the left/right
hind paw of the Wistar rats and immediately
transferred to a transparent plastic cage
for better observation. The spontaneous
nociceptive behavior was determined
instantaneously by looking at the animal
behavior and measuring the duration every
time they were licking paws and limping
injected-leg.

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Volume 8 No. 2 August 2023
The paw licking and limping injected-leg
duration was examined from 0 to 5 minutes
(rst phase, neurogenic) to 15 to 30 minutes
(second-phase, inammatory).12 The inhibition
percentage (%) of nociceptive behavior was
also calculated following this formula:
Statistical Analysis
The results were reported as mean ± standard
error of the mean (SEM). The statistical
analysis was determined by One-way analysis
of variance (ANOVA) followed by Tukey’s
multiple comparison tests and performed
using GraphPad Prism version 8.0.1 for
Windows, GraphPad Software, San Diego,
California USA, www.graphpad.com; P<0.05
was considered as signicant.
Results and Discussion
In the past decade, kratom’s popularity has
soared in Western nations; recent estimates
suggest that about ve million Americans
regularly take kratom. This is supported by the
easy use of kratom leaves such as chewing,
brewing like tea, smoking as cigarettes, and
swallowing directly as compressed tablets
or capsules.13 In Indonesia, kratom obtained
from the West Kalimantan region has not been
widely studied for antinociceptive activity.
Only two articles have been published, the rst
examines the effect of the dichloromethane
fraction in mice, and the second examines
the water fraction in male mice.14,15 In the
present study, we opted to determine the
antinociception properties of kratom leaves
ethanol extract in Wistar albino rats.
Following the oral administration of test
substances, animals were induced with
formaldehyde to examine the analgesic
activity of kratom leaves ethanol extract
and compare it with positive (tramadol) and
negative (vehicle) control. Figure 1 depicted
the duration of paw licking or limping leg after
formaldehyde induction, while Table 1 showed
the inhibition percentage of rats’ nociceptive
behaviors. Compared to the control group, KE
Figure 1. Effect of KE at all the tested doses in both early neurogenic (0-5 min) and
late inammatory phases (15-30 min) estimated by formalin-induced pain models.
The duration of nociceptive behavior is presented as mean ± SEM (n = 3).
*** P < 0.001, ** P < 0.01 versus negative control using one-way ANOVA followed by
Tukey’s post hoc multiple-comparison test

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with the oral dose of 70 mg/kg signicantly
(P<0.01) reduced rats’ nociceptive behavior
in the late phase (54.7% reduction). Although,
the higher dose did not show a signicant
reduction, and tramadol (0.9 mg/kg) had more
signicant (P<0.001) reductions than tested
extracts. Furthermore, in the rst neurogenic
phase, kratom leaf ethanol extract did not
exhibit blockage to formaldehyde-induced
pain stimulation. This model is a biphasic pain
reaction.
The induced pain is mediated by glutamate
during the neurogenic phase of pain
transmission. While the second phase (15 to
30 minutes) of the inammatory pain response
is characterized by releasing inammatory
mediators such as prostaglandins, excitatory
amino acids, and histamine. Bradykinin has
the unusual ability to inuence both stages
simultaneously.16
Interestingly, another study reported that
methanol extract of kratom leaves with a
dose of 200 mg/kg could show signicant
reductions in both phases of the formalin test,
indicating that extract active compounds can
act in central and peripheral pain.8 In contrast,
our ndings suggest that active compounds
from 70 mg/kg ethanol extract tend to act
as antiinammatory pain, which inhibits
peripheral pain pathway. Meanwhile, a study
by Goh et al. (2021) revealed that 200 mg/
kg of kratom leaf ethanol extract possessed a
similar antinociceptive effect as morphine (5
mg/kg) in the tail-ick test.
This means ethanol extract of kratom leaves
also possesses central pain blockage. Yet,
Goh and co-workers used an accelerated
solvent extraction technique that increases
the interfacial interaction with the analyte by
driving the solvent into the sample matrix’s
pores, resulting in enhanced analyte recovery
and dry yield of extract (29.1% w/w).17 Taken
together, kratom leaves ethanol extract might
show antinociceptive and anti-inammatory
properties.
Its antinociceptive activity is inuenced by
the alkaloid content of mitragynine and the
active metabolite of 7-hidroxymitragynine
(7-HMG), which can bind to brain opioid
receptors.9,18 Within ethanol-dried extract of
kratom, it contains approximately 6.5% of
mitragynine, slightly lower than methanol
extract, which has more than 7%.17 Regarding
the pharmacokinetics prole, mitragynine is
a lipophilic, weak base that passively crosses
the intestinal and blood-brain barrier; thus,
it quickly permeates and is dispersed in the
brain.
The bioavailability was calculated to be
21%, and 85-95% of the drug is bound to
plasma proteins.19 Therefore, kratom and its
mitragynine demonstrate potential utility for
managing severe pain; however, abuse potential
and addiction risk hurdle their clinical usage.
According to Hemby et al. (2018), 7-HMG
possesses more abuse potential and induces
withdrawal than mitragynine,20 whereas
7-HMG is the product of phase I metabolism
Table 1. Average Percentage Inhibition of Nociceptive Behavior

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Volume 8 No. 2 August 2023
of mitragynine.21 Structural modication
to avoid the formation of 7-HMG during
metabolism might ease further development
of mitragynine as an opioid analgesic.
This study has limitations, such as the
number of animals and a single parameter
for antinociceptive activity measurement.
Furthermore, the formaldehyde-induced pain
model measurement relies on rats’ behavior
might result in observation bias. However, the
chosen method was benecial in exploring
the possibility of both antinociceptive
and antiinammatory activity of a certain
compound.
Conclusion
To conclude, we reported the analgesic activity
of kratom leaves ethanol extract based on
Wistar rats’ behavior following formaldehyde
induction. Prior to a human clinical study,
future researchers should pave the way to
examine the exact mechanism of kratom
alleviating pain and its safety prole.
Funding
This study was nanced by internal research
funding from Akademi Farmasi Surabaya,
Indonesia, decree number 080/AKFAR-SBY/
SK.01.8/2020.
Conict of Interest
The authors declared no conict of interest in
the manuscript.
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