BIOMEDIKA
ISSN: 2085-8345.
E-ISSN: 2541-2582
Vol 18.
No.
1 Februari 2026 doi: 10.
23917/biomedika.
In Vitro Evaluation of the Antituberculosis Potential of Basil Leaf Ethanol Extract Using the Resazurin Microtiter Assay (REMA) Method Nurlina Setiadi1.
Humairah Medina Liza Lubis2.
Ikhfana Syafina3
AFFILIATIONS
Department of Biomedical.
Faculty of Medicine and Health Science Universitas Muhammadiyah Sumatera Utara Department of pathological anatomy.
Faculty of Medicine and Health Science Universitas Muhammadiyah Sumatera Utara Department of Pulmonology.
Faculty of Medicine and Health Science Universitas Muhammadiyah
Sumatera Utara
ABSTRACT
Background: Tuberculosis (TB) remains a global health challenge and is the second leading infectious cause of death.
Indonesia ranks among the top three countries with the highest TB burden.
Efforts to discover safer and more effective antituberculosis agents include exploring natural products such as basil (Ocimum basilicu.
, which contains antimicrobial bioactive compounds.
Objective: To determine the minimum inhibitory concentration (MIC) of basil leaf ethanol extract against Mycobacterium tuberculosis H37Rv using the Resazurin Microtiter Assay (REMA).
Methods: Basil leaves were extracted by maceration using 96% ethanol and tested at concentrations of 2.
5%, 1.
25%, and 0.
Antimycobacterial activity was evaluated using REMA, with rifampicin as the positive control and untreated media as the negative control.
Color change from blue to pink indicated bacterial growth.
Results: All concentrations of basil extract produced a pink color, indicating continued bacterial growth, whereas rifampicin remained blue, confirming assay validity.
The MIC of the extract was >2.
5 AAg/mL, indicating no inhibitory effect at the tested concentrations.
Conclusion: Basil leaf ethanol extract did not inhibit M.
tuberculosis H37Rv at the evaluated doses.
Further research using higher concentrations, phytochemical profiling (LCAeMS/GCAeMS), and fractionation of active compounds is recommended to explore its potential antimycobacterial KEYWORDS:
Basil Leaves.
Minimum Inhibitory Concentration.
Mycobacterium tuberculosis.
REMA This work is licensed under a Creative Commons AttributionNonCommercial 4.
International License.
CORRESPONDING AUTHOR:
Nurlina Setiadi nurlinasetiadi@gmail.
INTRODUCTION
after India, reporting an estimated 969,000 TB cases Tuberculosis (TB) remains a major global in 2021 .
per 100,000 populatio.
Within health problem and, in 2022, was the second leading Indonesia.
North Sumatra Province holds the third- cause of death from infectious diseases after COVID- highest TB burden after West Java and East Java, 19, causing twice as many deaths as HIV/AIDS.
with an estimated 83,949 cases.
More than 10 million people develop TB annually.
Drug-resistant tuberculosis (DR-TB) remains with 30 high-burden countries accounting for 87% one of the most significant challenges to global TB of global cases.
two-thirds of these occur in eight control efforts.
Globally, the annual number of countries, including India .
%).
Indonesia .
%).
MDR/RR-TB cases remained stable from 2020 to China .
1%), the Philippines .
%).
Pakistan 2023, with an estimated 400,000 cases in 2023.
5%).
Bangladesh .
6%), and the Democratic Drug-resistant TB cases occur annually, placing a Republic of Congo .
%).
Indonesia ranks second heavy burden on health systems, prolonging treatment duration, increasing treatment failures.
Avalaible online at https://journals2.
id/index.
php/biomedika In Vitro Evaluation of the Antituberculosis Potential of Basil A (Nurlina Setiad.
and contributing substantially to global antimicrobial One of the plants that has many benefits is In many high-burden and resource- basil leaves (Ocimum basilicu.
which has an abundant source of polyphenols and has long been consumes a large portion of healthcare budgets and known to be used to treat various types of diseases often exceeds available diagnostic and treatment through its function as an anti-inflammatory, anti- oxidant, immunomodulator, anti-microbial, analgesic drug-resistant These challenges highlight the urgent need to and diuretic.
identify new therapeutic candidates capable of In Indonesia, basil leaf plants are widely used combating Mycobacterium tuberculosis strains that on large islands, such as Sulawesi.
Sumatra, and no longer respond to conventional drugs.
One Kalimantan.
The tribe in Kolaka.
East Kolaka, promising approach is the exploration of natural Southeast Sulawesi, uses basil leaves to treat products with potential antimycobacterial properties.
In addition, basil leaves are also Medicinal plants are widely used in traditional used to treat acute lung diseases, including medicine across various regions and contain diverse bronchitis, cough, and sore throat, in Brazil, as well bioactive compounds that may serve as alternative as tuberculosis and acute lung diseases such as or complementary therapies for TB.
bronchitis in Ethiopia.
Previous studies reported that antimycobacterial antimycobacterial activity of plant extracts, the activity of the essential oil from African basil leaves Resazurin Microtiter Assay (REMA) has become an can damage microbial cell membranes, increase the established and reliable phenotypic method.
REMA is formation of ROS which causes oxidative damage to simple, rapid, cost-effective, and highly sensitive, microbes, damage microbial DNA and its replication making it especially suitable for screening natural wheels, denatured proteins essential for microbial product libraries, including herbal extracts.
This Through hydrodistillation methods and method uses resazurin as an indicator with the characterized using GC-MS, it showed antibacterial principle of oxidation-reduction.
Previous potential against S.
coli, and studies have shown that aqueous extracts of Ocimum sanctum.
Adhatoda vasica.
Leptedinia concentration (MIC) ranges from 2 to 4 g/mL, and reticulata, and Cocculus hirsutus exhibit strong the inhibitory zone is between 5 to 10.
antimycobacterial activity when evaluated using the REMA assay compared with the MTT assay.
BIOMEDIKA.
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No.
Februari 2026, 23 - 31 The By applying REMA, a sensitive, rapid, and costefficient phenotypic assay, this study provides new In Vitro Evaluation of the Antituberculosis Potential of Basil A (Nurlina Setiad.
and standardized evidence on the antimycobacterial concentrations and mixed thoroughly.
Rifampicin as potential of basil extract, offering a more reliable the positive control, was thawed and subsequently assessment compared with earlier approaches.
diluted in 7H9-S medium.
Twofold serial dilutions METHODS were prepared directly in 96-well plates by adding This study uses an experimental research the drug to 100 AAL of 7H9-S medium, yielding final design in vitro with a non-randomized pos test only concentrations ranging from 2.
0 to 0.
06 AAg/mL.
controlled group design method.
It was held from Wells containing medium without antibiotics served September 2024 to April 2025 at the Microbiology Laboratory of Faculty of Medicine.
Public Health and functioned as sterility controls.
The plates were then Nursing, sealed and incubated at 37AC.
After 7 days of Biochemistry Laboratory of Faculty of Medicine and incubation, 30 AAL of 0.
01% resazurin solution was Health Universitas Science.
Gadjah Universitas Mada Muhammadiyah Sumatera Utara.
On day 8, color changes from blue The stages of the procedure carried out are:
xidized for.
to pink .
educed for.
were first, the extraction of basil leaf simplicia is observed to determine bacterial growth.
The results macerated with 96% ethanol for 72 hours, filtered, were subsequently analyzed using a microplate and evaporated until a thick extract is obtained, then reader at 600 nm.
diluted with 10% DMSO to obtain a concentration of The experiment was performed in triplicate 5%, 1.
25%, 0.
Phytochemical tests were carried out on basil leaf extract using concentration values were reported as mean values.
qualitative methods to determine the content of MIC was defined as the lowest concentration of compounds such as flavonoids, alkaloids, tannins, extract or drug that prevented the change in saponins, triterpenoids and steroids.
The quantitative in vitro antimycobacterial Minimum .
isual Auno activity of basil leaf ethanol extract at concentrations mycobacterial growth,Ay whereas a pink color 5%, 1.
25%, and 0.
625% was evaluated using indicated Aumycobacterial growth.
Ay .
96-well microtiter plates with resazurin as an RESULT AND DISCUSSION indicator of bacterial viability.
A total of 100 AAL of Middlebrook 7H9-S medium was added to each well.
Tuberculosis remains one of the most challenging infectious diseases globally, particularly followed by 100 AAL of plant extract at the designated BIOMEDIKA.
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in low- and middle-income countries such as contained in it, such as saponins, tannins, alkaloids.
Indonesia.
The increasing incidence of resistance to both first-line and second-line anti-tuberculosis drugs underscores the urgent need for new, a minimum inhibitory concentration (MIC) ranging from 25-100 g/mL and 25-50 g/mL, respectively.
A study reported that the administration of flavonoids significantly decreased the survival of antimycobacterial agents.
Medicinal plants are intracellular M.
tuberculosis, increasing cell density, recognized as an important reservoir of structurally aggregation, and granuloma formation.
While diverse bioactive compounds, making them a saponins work by lowering the surface tension of the bacterial cell wall, thereby impairing the permeability non-toxic, antimycobacterial candidates.
of the cell membrane, which leads to lysis, making Based on the results of phytochemical enzymes and proteins out of the cell.
The release of screening of basil leaf ethanol extract, the following these cell contents causes the death of bacterial results were obtained:
Tannins have a role in forming complex Table 1.
Phytochemical Screening Results Compound Flavonoid Alkaloid Saponin Tanin Phenol Steroids Result Black Brownish-yellow Stable foam formation Blackish green Blackish blue Purple, blue or greenish Information screening presented in table 1, it was found that basil leaf extract contains flavonoid compounds, alkaloids, saponins, tannins, phenols and steroids.
if a hydrogen bond is formed between tannins and proteins then the protein will be denatured so that Based on the results of phytochemical The compounds with proteins through hydrogen bonds.
Meanwhile, alkaloids can inhibit DNA synthesis by inhibiting the topoisomerase enzyme in bacterial cells.
Alkaloids exhibit antibacterial activity while inhibiting the membrane-dependent ATP compounds aligns with previous phytochemical studies on basil leaves.
Previous research has shown that basil leaves have promising antimicrobial activity against .
the strains of M.
tuberculosis and M.
This activity is associated with the bioactive ingredients BIOMEDIKA.
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In these results, it was found that M.
tuberculosis bacteria grew in a control that was not given anything [C(B)], did not grow in the medium given rifampicin compound [C(M)], still grew in the control given aquadest solvent [C(S)], while in the .
control media given basil leaf ethanol extract compounds.
tuberculosis bacteria continued to grow at a concentration of 2.
25% and 625% after three repeats of testing.
Table 3.
Minimum Inhibitory Concentration Test Conce .
Grow Conce .
Grow
MIC
/mL Conce .
Grow Grow Grow Grow >2.
i Grow Grow Grow >2.
Figure 1.
Minimum inhibitory Concentration .
Extraction of compound extract solution with M.
tuberculosis H37Rv bacteria.
Addition of Resazurin to each group.
Color observation that occurs in each group >2.
Based on the image above, it was found that In the above results, the minimum inhibitory 25% and changed to red, while in the group given rifampicin it remained blue.
concentration of M.
tuberculosis was obtained after the administration of basil leaf ethanol extract at concentrations of 2.
5%, 1.
25% and 0.
625% which is >2.
5 g/mL, meaning that the concentration used Table 2.
Results of the test of the potency of basil leaf extract against M.
Test
C (B)
C (M)
C (S)
Grow Not Grow Concentration of ethanol extract of basil leaves (%) 1,25 0,625 Grow Grow Grow is not enough to inhibit the growth of bacteria.
The REMA method was chosen because it has advantages in terms of sensitivity, relatively short time, and visually interpretable results.
Based on Grow Not Grow Grow Grow Grow i Grow Not Grow Grow Grow Grow previous research, the methanol extracts of L.
sanguinolenta, and Z.
leprieurii inhibited the growth of M.
smegmatis as demonstrated by the Information:
C (B)
: Control Bacteria
C (M)
: Control media (Rifampici.
C (S) : Solvent Control REMA assay.
They exhibited greater activity against rifampicin-resistant strains.
The results of the study in Pakistan, that methanol extract of basil leaves showed up to 49% BIOMEDIKA.
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inhibition against M.
tuberculosis at a concentration methods, assay conditions, and phytochemical 25 mg/ml.
Based on the study, basil leaves that Previous research commonly used separate methanol extract into fractions and purify methanol extracts, essential oils, or purified fractions that concentrate bioactive compounds .
Ae.
, compounds exerted a maximum inhibition effect of whereas our study evaluated crude ethanol extract H37Rv at lower concentrations that may have contained concentration of 6.
25 g/mL, rather than total insufficient active constituents.
Although REMA is a .
highly sensitive and widely used assay .
, its In a previous study conducted in Ethiopia, an colorimetric nature may underestimate slow-acting extract of 80% methanol from basil leaves seeds compounds or be affected by extract coloration.
showed promising antimycobacterial activity against Phytochemical variability due to plant chemotype, strains of M.
tuberculosis (H37Rv.
SIT777.
SIT73, origin, and SIT26.
SIT37.
SIT1688.
SIT336.
SIT149.
SIT53 and contribute to inconsistent results.
SIT.
and M.
bovis (SB1176.
SB1953 and SB0.
CONCLUSION
bovis was shown with an average minimum inhibitory concentration of 6.
25 to 100 g/mL .
In previous studies, there was also a significant decrease in the growth rate of M.
tuberculosis using Ocimum sanctum alcohol extract with doses of 0.
25 L and 0.
5 L.
Another study reported the anti-tuberculosis effects of the essential oil Ocimum sanctum L.
(OsEO).
OsEO inhibited the growth of H37Rv with a MIC of 3 ml .
,931 m.
research at higher doses is needed to obtain the expected results, it is necessary to standardize and analyze the phytochemical profile of the extract before the activity test, for example with LC-MS or GC-MS as well as in vivo studies on the benefits of basil leaf extract against M.
LIMITATIONS
Only crude ethanol extract was tested, active components may have been too diluted to produce inhibitory effects.
isolates of M.
tuberculosis tested, with MICs ranging Differences between our findings and earlier antibacterial activity against M.
Further OsEO also inhibited the growth of all nine clinical 5 mL .
4655 m.
to 6 mL .
862 m.
Basil leaves ethanol extract concentrations In the study, using the REMA method, antimycobacterial activity against M.
tuberculosis and M.
extraction technique No chemical profiling (LC-MS/GC-MS) was performed to quantify active compounds.
studies likely arise from variations in extraction BIOMEDIKA.
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Concentration insufficient, as higher doses used in previous
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