BIOMOLECULAR AND HEALTH SCIENCE JOURNAL 2021 OCTOBER, VOL 04 (02)
Antibacterial mechanism of Kratom (Mitragyna
speciosa) methanol extract on Streptococcus
pneumoniae and Eschericia coli bacteria
Hotimah Masdan Salim1* , Marinda Dwi Puspitarini2, Yuani Setiwati3, Michio
1Department of Biochesmitry medicine and Biomolecular science, Faculty of Medicine, Universitas Nahdlatul Ulama
2Department of Microbiology, Faculty of Medicine, Universitas Nahdlatul Ulama Surabaya, Indonesia
3Division of Pharmacology and Therapy, Department of Anatomy, Histology and Pharmacology, Faculty of Medicine,
Universitas Airlangga, Surabaya, Indonesia
4Department of Diabetes, Endocrinology and Metabolism.Fukushima Medical University, Japan
A R T I C L E I N F O A B S T R A C T
Received 24 August 2021
Received in revised form 22
Accepted 19 October 2021
Available online 30 June 2021
*) Corresponding author:
Introduction: Kratom (Mitragyna speciosa) is a tropical herbal plant native to the North Malay
Peninsula, Malaysia and Thailand. In Indonesia, kratom us a typical plant of West Kalimantan,
especially in Putussibau. Kratom leaf has many pharmacological eects, one of them is
antioxidant. However, the antioxidant and antimicrobial activities of Mitragyna speciosa leaf
extracts are lacking. This study was to examine the eect of the methanol extract of Mitragyna
speciosa (Rubiaceae Family) leaves on microorganisms Escherichia coli and Strepococcus
Methods: Kratom leaves were extracted with methanol as solvent. In this study, S. pneumoniae
and E. coli bacteria have been used. Antibacterial activity tests were carried out at concentrations
of 0.78%, 1.56%, 3.125%, 6.25%, 12.5%, 25%, 50% and 100%. Insilico was used to search for
secondary metabolites and metabolite interactions in Mytragina speciosa.
Results: Mitragyna speciosa leaf methanol extract was eective in inhibiting the growth of E.
coli and S. pneumoniae signicantly (P<0.001). The growth of e.coli was not identied from a
concentration of 25% to 100%. However, there was no bacterial growth in S. pneumoniae from a
concentration of 6.25% to 100%. In the insilico study it was found that the secondary metabolite
of Mitragyna speciosa is quercetin, which interacts with the ATP1 protein as a predictor in the
mechanism of Mitragyna speciosa as an antibacterial.
Conclusion: In conclusion, there is no correlation between nutritional status and remission
outcome of patients with ALL in the induction phase of therapy. However, high percentage of
underweight patients shows nutrition needs special attention to improve therapy outcomes.
Indonesia has a lot of forests which are very wide with
a variety of types of plants. Diversity of plant species in
Indonesia, not much is known about its optimal use as a
medicinal plant.1 Medicinal plants have species diversity
to thousands of species. Where it is known that there are
about 40,000 species of medicinal plants that have been
known in the world, and 30.000 of them are suspected to
be located in Indonesia. This number represents 90% of
the plant drugs found in Asia. Of these, 25% of them or
about 7.500 species are known to have herbal properties
or medical plants. However, only 1.200 types of plants
have been used for raw materials for herbal medicines
or herbs.2 One of the herbal plants is Kratom which is an
herbal plant native to North Malay Peninsula, Malaysia ,
Thailand and Indonesia.3 In Indonesia, kratom is a plant
that grows in West Kalimantan, especially in Putussibau
district, which kratom leaves are known as “purik”
leaves. In general, people consume kratom leaves by
chewing, brewing like tea or smoking.4
Kratom is one of the traditional medicinal plants that
produces stimulant (at low doses) and sedative eects
(at high doses), diarrhea, smoothing blood circulation,
increase endurance and stamina, prevent constipation,
Biomolecular and Health Science Journal
Available at https://e-journal.unair.ac.id/BHSJ ; DOI: 10.20473/bhsj.v4i2.28933
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
BIOMOLECULAR AND HEALTH SCIENCE JOURNAL 2021 OCTOBER, VOL 04 (02) 100
treatment for diabetes and reduce sugar levels.5 Previous
studies on kratom leaves shows pharmacological
eect activity, among those; analgesic and stimulants,
antidepressant, anti-inammatory and antinociceptive,
antioxidant and antibacterial.6 The eectiveness of
kratom as an antibacterial has been carried out on several
types of bacterial, including the bacterial that cause acne
namely Propionibacterium acnes, in Salmonella typhi
and Bacillus subtilis.7
The activity of kratom as an antimicrobial cannot be
separated from the composition contained in the kratom
leaves, including the high content of phenolics and
avonoids in the methanol extract of kratom leaves.8
Phytochemical studies have shown that there are
several chemical constituents of Mitragyna speciosa
plants including indole alkaloids, including mitragynine,
speciocilliatine, and paynantheine,9 and also contain
secondary metabolites such as avonoids, saponin,
monoterpenes, triterpenoids, secoirioids and
The antimicrobial potential of kratom is also
expected to have an eect on the bacteria that cause
respiratory and digestive tract infections. Therefore, this
study aimed to examine the eect of kratom leaf as an
antimicrobial on Staphylococcal pneumonia and E. coli
The plant of leaves of Mitragyna speciosa were collected
from Putussibau state, West Kalimnatan, Indonesia.
Mitragyna speciosa leaves are washed with water to
remove dirt before the drying process. Then the leaves
are cut and dried in direct sunlight for 2-3 days. After
cutting the leaves and grounded into powder.
The dried leaves of M. speciosa were ground in powder
form. A total of 2.08 kg of Mitragyna speciosa powder
was extracted using 96% methanol as solvent. The
solvent was changed every 1x24 hours and macerated
for 7x24 hours. After maceration, it was concentrated
using a rotary evaporator and a water bath to obtain
a thick extract of 20 g of crude methanol extract and
stored at -20°C.
Test microorganisms and growth media
We collected S. pneumoniae and E. coli provided by the
Surabaya Health Laboratory Center (BBLK). Bacterial
colonies were taken by means of inoculum suspension
for 24 hours of culture, then suspended in sterile 0.9%
The antibacterial activity of Mitragyna speciosa leaf
extract was tested by agar diusion method using NA
media. Bacterial suspension of 0.02 ml 1:40 dilution was
mixed with 10 ml of NA medium in a diluent bottle, then
shaken unti l homogeneous.Take the inoculant in the
tube and scratch it on a petri dish, and incubate at 37°C
for 18-24 hours, then observe the growth of bacteria.
Testing the antibacterial activity of kratom leaf extract
against S. pneumoniae and E. coli as carried out with
8 (eight) variations of concentration namely 0.78%,
1.56%, 3.72%, 6.25%, 12.5%, 25%, 50% and 100%. The
whole test tube incubated in an incubator at 37°C for
18-24 hours. Then make observations of the whole tube
against tube clarity by viewing control.
Screening of metabolite secondary of Mitragyna speciosa
was carried out from http://www.swissadme.ch/index.
php. The 3D structure of the active ingredient, a search
was carried out from the PubChem (http://pubchem.
ncbi.nlm.nih.gov) server with the recorded CID. Then
to nd out the interaction of secondary metabolites with
proteins in the body, a search was carried out on the
STITCH (http://stitch.embl.de) database server.
This research was repeated three times. All results
are expressed as mean ± SEM. Dierences between
groups were analyzed by one-way analysis of variance
(ANOVA), followed by Tukey's post hoc analysis.
Dose-response curve comparisons were made with a
two-factor repeated-measurement ANOVA, followed by
Tukey's post hoc test for comparison between groups.
P<0.05 was considered signicant. analysis using SPSS
version 21 program.
Minimum Inhibitory Concentration of Methanol
Extract of Mytragina Speciosa of E.Coli
Minimum Inhibitory Concentration of extract ethanol
Mytragina Speciosa of E. coli showed that the activity
decreased at a concentration of 25%, 50% and 100%
(gure.1A), where there was no visible bacterial growth
compared to the control group. The bacterial count also
showed that the lower the extract concentration, the
more bacterial growth (Figure.1B)
Figure.1 Antimicroba activity (MIC) of Mytragyna
speciosa extract in E. coli
Minimum Inhibitory Concentration of Methanol
Extract of Mytragina Speciosa in Strepococcus
Minimum Inhibitory Concentration of Extract Methanol
Mitragyna Speciosa of S. pneumoniae showed that the
activity decreased at a concentration of 6,25%12,5%,
25%, 50% and 100% (gure.2A), where there was no
visible bacterial growth compared to the control group.
The bacterial count also showed that the lower the extract
concentration, the more bacterial growth (Figure.2B)
Figure.2 Antimicroba activity (MIC) of Mytragyna
speciosa extract in Strep.penumonia
Interaction of kratom secondary metabolite
Based on previous research, the composition of the
methanol extract of Mytragina speciosa has been
obtained. In this study we examine the interaction of
the active compound from the methanolic extract of
mitragyna speciosa, namely quercetin which is the
equivalent of the avonoid content. (Figure.3)
BIOMOLECULAR AND HEALTH SCIENCE JOURNAL 2021 OCTOBER, VOL 04 (02) 101
Figure 1. Minimum Inhibitor Concentration of Methanol Extract of Mytragina Speciosa in E.Coli. Ctrl; Control,
MS; Mitragyna Speciosa. **; P<0.01, ***;P<0.001 vs control group
Figure 2. Minimum Inhibitory Concentration of Methanol Extract of Mytragina speciosa in Streptococcus penumonie.
Ctrl; Control, MS; Mitragyna Speciosa. **; P<0.01, ***;P<0.001 vs control group
Figure.3 prediction of interactions between chemicals (quercetin) and proteins in the body insilico.
BIOMOLECULAR AND HEALTH SCIENCE JOURNAL 2021 OCTOBER, VOL 04 (02) 102
Quercetine is a avonoid that is widely found in plants.
In this insilico quercetine has an interaction bond with
atp1. ATP synthase (F(1)F(0) ATP synthase or Complex
V) generates ATP from ADP in the presence of a proton
gradient across the membrane generated by the electron
transport complex of the respiratory chain. This quercetin
interaction is predictive of its antimicrobial activity
by inhibiting ATP1 synthesis at the cellular level.
While osl and xd1 are proteins that do not have special
In this study, it was found that the methanolic extract of
Mitragyna speciosa (Kratom) had an antimicrobial eect
on bacteria that cause respiratory tract infection and
bacteria in the digestive tract. The results of this study
showed that Mytragina speciosa methanol extract was
eective as an antimicrobial at higher concentrations.
Where in this study found at a concentration of more
than 12.5% Escherichia coli bacteria were not found, and
at concentrations > 3.72% Streptococcus pneumoniae
bacteria. In line with this study, previous studies have
shown the eectiveness of Mytragina speciosa as
an antimicrobial of several types of extract solvents.
Antimicrobial activity was found to be eective in
MeoH and Alkaloid extracts, which have the ability to
inhibit growth and kill larger bacteria Salmonella typhi
and Bacillus subtilis.8 Other studies have also found that
kratom has an antimicrobial eect that has an inhibitory
eect on Aeromonas hydrophylla.7
Antibacterial activity test shows that increasing the
concentration of extract given can increase the killing
power of the extract against bacteria. However, if the
concentration of the extract continues to increase, it can
reduce the killing power. This nding suggested that
the increasing concentration of Kratom can lead to an
increase in viscosity of the extract, thus aecting the rate
of diusion of extract in agar media. In addition to the
concentration factor, the type of antimicrobial material
can also determine the ability to inhibit bacterial growth.11
The eectiveness of kratom as an antimicrobial
has been known for a long time, based on the results
of phytochemicals in kratom leaves, it has been found
that kratom leaves contain alkaloid, avonoids, steroid,
saponin and tannins and have antioxidant eect.12 It
has been found in previous studies that compounds
derived from plants have antibacterial activity based
on phytochemical data. The components of the active
substances in mitragyna speciosa extract have been
proven, including alkaloids, avonoids, saponin, tannins,
phenols and other active compounds.13
In nature, avonoid compounds are products extracted
from plants and they are found in several parts of the
plant. Flavonoids play a variety of biological activities
in plants, animals and bacteria. Flavonoids are associated
with a wide spectrum of health-promoting eects and are
indispensable components in a wide range of nutraceutical,
pharmaceutical, medicinal and cosmetic applications.
This is due to its antioxidant, anti-inammatory, anti-
mutagenic and anti-carcinogenic properties coupled
with its capacity to modulate the function of key cellular
enzymes.14 In previous studies, it was found that the active
substance of the avonoid class is quercetin in methanol
extract of Mitragyna speciosa.15
The quercetin content in the MeOH Kratom extract is
quite high. The presence of quercetin as a sub-avonoid
gives hope that the content of kratom has a good eect
for use as herbal medicine. In this study we found that
quercetin has a high pharmacokinetic eect on absorption
in the gastrointestinal tract. In addition, to nd out of
mechanism target of quercetin, we examine in insilico
studies. this study showed predictions of quercetine's
target of action on enzymes, oxidoreductase, G-protein
receptors, enzymes, proteases, lyase and cytochrome
p450. Based on the results of this study, it is possible
that the quercetin content in the Methanol kratom extract
has the ability as an antibacterial against Streptococcus
pneumoniae and Eschericia coli. This tudy is supported by
the results of previous studies which found that quercetin
inhibits the formation of streptococcus pneumoniae
In addition, other studies also found that quercetine in
surgacane bagasse extract showed bacteriostatic activity
against the growth of S. aureus, L. monocytogenes, E.
coli, and S. typhimurium,17 and could change ATP activity,
thus eecting the growth of E.coli.18 In an in vivo study,
it was found that quercetin can protect mice from s.aureus
infection by inhibiting thrombin activity.19 To conrm the
interaction of quercetine with proteins in the body, we
performed an insilico screening. In this study it was found
that quercetine can interact with ATP synthase FO subunit
1 (ATP1) in humans. As we know that ATP is the main
energy compound in biological synthesis, and most of it
is provided by the F1FO-ATP synthase which is located in
the membrane and can be found in bacteria, mitochondria
The ndings in this study can be used as a basis
for explaining the mechanism that methanol extract of
Mitragina speciosa (kratom) leaves has an antibacterial
eect on Streptococcus pneumoniae and Eschericial coli,
through the mechanism of inhibition of ATP synthase Fo
subunit 1 (ATP1).
Mytragina speciosa (kratom) methanol extract has an
antimicrobial eect on streptococcus pneumonia and e.coli
through the eect of quercetine contained in kratom. This
antimicrobial mechanism is thought to be by inhibiting ATP
synthesis through the ATP synthase F0 subunit 1 (atp1)
pathway. The broad spectrum antimicrobial properties of
quercetin can be used in the prevention and treatment of
various infectious bacterial diseases and may provide a
treatment option to reduce the use of antibiotics.
We would like to thanks LPPM Universitas Nahdlatul
Ulama Surabaya for providing nancial support and to
Miss Gardina as sta in the microbiology laboratory for
guiding us in the laboratory.
Conict of Interest
The author stated there is no conict of interest
BIOMOLECULAR AND HEALTH SCIENCE JOURNAL 2021 OCTOBER, VOL 04 (02) 103
1. Wiryono, Puteri, V. N. U. & Senoaji, G. The diversity of plant
species, the types of plant uses and the estimate of carbon stock
in agroforestry system in Harapan Makmur Village, Bengkulu,
Indonesia. Biodiversitas 17, 249–255 (2016).
2. PT Industri Jamu Dan Farmasi Sido Muncul Tbk. Inspiring for a
Better Life. (2019).
3. Hassan, R. et al. Mitragynine Attenuates Morphine Withdrawal
Eects in Rats-A Comparison With Methadone and Buprenorphine.
Front. psychiatry 11, 411 (2020).
4. Henningeld, J. E., Fant, R. V & Wang, D. W. The abuse potential
of kratom according the 8 factors of the controlled substances act:
implications for regulation and research. Psychopharmacology
(Berl). 235, 573–589 (2018).
5. Fluyau, D. & Revadigar, N. Biochemical Benets, Diagnosis, and
Clinical Risks Evaluation of Kratom. Front. psychiatry 8, 62 (2017).
6. Cheaha, D. et al. Eects of alkaloid-rich extract from Mitragyna
speciosa (Korth.) Havil. on naloxone-precipitated morphine
withdrawal symptoms and local eld potential in the nucleus
accumbens of mice. J. Ethnopharmacol. 208, 129–137 (2017).
7. Juanda, E., Andayani, S. & Maftuch, M. Phytochemical Screening
and Antibacterial Activity of Kratom Leaf (Mitragyna speciosa
Korth.) Against Aeromonas hydrophilla. J. Exp. Life Sci. 9, 155–
8. Parthasarathy, S. et al. Evaluation of antioxidant and antibacterial
activities of aqueous, methanolic and alkaloid extracts from
Mitragyna speciosa (Rubiaceae family) leaves. Molecules 14,
9. Basiliere, S., Bryand, K. & Kerrigan, S. Identication of ve
Mitragyna alkaloids in urine using liquid chromatography-
quadrupole/time of ight mass spectrometry. J. Chromatogr. B
1080, 11–19 (2018).
10. Raa, R. B. Kratom and other mitragynines: the chemistry and
pharmacology of opioids from a non-opium source. (CRC Press,
11. Li, J. et al. Antimicrobial Activity and Resistance: Inuencing
Factors. Front. Pharmacol. 8, 364 (2017).
12. Yuniarti, R. et al. Characterization, Phytochemical Screenings
and Antioxidant Activity Test of Kratom Leaf Ethanol Extract
(Mitragyna speciosa Korth) Using DPPH Method. J. Phys. Conf.
Ser. 1462, (2020).
13. León, F. et al. Phytochemical Characterization of the Leaves
of Mitragyna Speciosa Grown in USA. Nat. Prod. Commun. 4,
14. Khalid, M., Saeed-ur-Rahman, Bilal, M. & HUANG, D. Role of
avonoids in plant interactions with the environment and against
human pathogens — A review. J. Integr. Agric. 18, 211–230 (2019).
15. Pandey, B. P., Pradhan, S. P. & Adhikari, K. LC_ESi_QTOF_MS
for the Proling of the Metabolites and in Vitro Enzymes Inhibition
Activity of Bryophyllum pinnatum and Oxalis corniculata Collected
from Ramechhap District of Nepal. Chem. \& Biodivers. 17, (2020).
16. Wang, J. et al. Quercetin increzases the antioxidant capacity of the
ovary in menopausal rats and in ovarian granulosa cell culture in
vitro. J. Ovarian Res. 11, 51 (2018).
17. Zhao, Y., Chen, M., Zhao, Z. & Yu, S. The antibiotic activity and
mechanisms of sugarcane (Saccharum ocinarum L.) bagasse
extract against food-borne pathogens. Food Chem. 185, 112–118
18. Yang, D., Wang, T., Long, M. & Li, P. Quercetin: Its Main
Pharmacological Activity and Potential Application in Clinical
Medicine. Oxid. Med. Cell. Longev. 2020, 8825387 (2020).
19. Wang, L. et al. Quercetin protects rats from catheter-related
Staphylococcus aureus infections by inhibiting coagulase activity.
J. Cell. Mol. Med. 23, 4808–4818 (2019).
20. Walker, J. E. The ATP synthase: the understood, the uncertain and
the unknown. Biochem. Soc. Trans. 41, 1–16 (2013).