Research Article - Type running title here (8 words or less)

THE EFFECT OF NONI EXTRACT (MORINDA CITRIFOLIA) ON LEVELS OF

THE CYTOKINE INTERLEUKIN 6 IN WHITE RATS (Rattus norvegicus strain

Wistar) INDUCED BY PYRAZINAMIDE, ETHAMBUTOL AND LEVOFLOXACIN

Dhea Devika Wijaya1,3, Achmad Amir Solichudin2,3, Thontowi Djauhari1

1
Muhammadiyah Malang University, Bendungan Sutami st, Lowokwaru, Malang Indonesia
2
Brawijaya University, Veteran st, Ketawanggede, Malang, Indonesia
3
RSUD RA Basoeni, Pagerluyung st, Gedek, Mojokerto, Indonesia

*Corresponding Author. Email: dheadevikaww@gmail.com

Telp: +62-895403324848

Abstract

Background: The use of antibiotics pyrazinamide, ethambutol, and levofloxacin for TB

often causes side effects in the form of inflammation. This triggers the emergence of the pro-

inflammatory cytokine IL-6. Noni fruit (Morinda citrifolia) contains the active substance

scopoletin which is known to have anti-inflammatory potential. Objective: To determine the

effect of noni fruit extract (Morinda citrifolia) on levels of the cytokine interleukin 6 in white

rats of the Wistar strain induced with pyrazinamide, ethambutol and levofloxacin also to

know the effective dossage of noni fruit as anti inflamatory.

Method: The method used is True experimental research with a Post Test Control Group

Design approach. The samples used were male white mice. Mice were divided into 4 groups

(positive control, noni dose 20mg/200g, 40mg/200g, and 80mg/200g, each group contained 4

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mice. Mice were treated for 30 days. The positive control is given tuberculosis medication,

while the treatment group is given noni extract according to the dosage, then after 30 minutes

they are given tuberculosis medication. Blood is taken from the heart to determine IL-6

cytokine levels in mice using the Elisa test.

Results: It was found that decreased levels of the IL-6 cytokine were found in the treatment

group compared to the positive control group. The mean IL-6 cytokine levels in the positive

control were 1.013 pg/mL, noni 20mg/200gBW, 40mg/200gBW, and 80mg/200gBW were

0.198 pg/ml, 0.195 pg/ml, and 0.723 pg/mL, respectively. The antibiotics contained in

tuberculosis drugs will cause an inflammatory response in the human body. Noni has anti-

inflammatory potential, thereby reducing IL-6 levels in the treatment group to a lesser extent

than the positive control group.

Conclusion: It was found that there was a significant effect on IL-6 cytokine levels and the

effective dose of noni in reducing IL-6 cytokines.

Keyword: Tuberculosis medication, Noni, inflammation, Interleukin-6, effective dose.

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Introduction

Tuberculosis (TB) is a disease caused by the bacterium Mycobacterium tuberculosis and is one of the

main contributors to death in the world. According to the WHO Global TBC Report 2022, it is

estimated that there are 10.6 million people infected with TB in the world, an increase of 4.5%

compared to 2020. Indonesia is the second largest contributor and is estimated at 969,000 cases, an

increase of 17% compared to 2020.

One of the threats in controlling TB is drug-resistant tuberculosis (TB RO). Globally in 2021,

71% of bacteriologically confirmed TB patients were checked for rifampicin resistance and 166,991

were found to be resistant, an increase of 6.4% compared to 2020.

In Indonesia. It is estimated that TB RO is 2.4% of all new TB patients and 13% of TB who

have ever been treated with an estimate of 24,000 or 8.8/100,000 population. In 2019, around

11,500 RR TB patients were discovered and reported, around 48% of patients started second-line TB

treatment, with a treatment success rate of 45%.

Resistance of Mycobacterium tuberculosis bacteria to anti-tuberculosis drugs (OAT) is a

condition where the germs are immune so that they can no longer be killed by anti-tuberculosis

drugs. There are several types of resistance to OAT, including Monoresistance, Polyresistance,

Multidruf resistance (MDR), Pre-XDR, Extensive Drug Resistance (XDR) and rifampicin resistant TB.

Treatment with a combination of drugs can prevent resistance but can increase the

possibility of side effects. Side effects of medication will affect patient compliance with taking

medication. Bedaquiline, levofloxacin, clofazimine, ethionamide, high dose INH, pyrazinamide and

ethambutol are some of the drugs of choice for TB-RO. Administration of levofloxacin, clofazimine,

pyrazinamide and ethambutol itself takes a long time of 9-11 months. All OATs used to treat TB-RO

patients have the possibility of mild, moderate or severe side effects. Pyrazinamide has the side

effects of hepatitis, joint pain, reddish fever and other skin reactions. Ethambutol has the side effect

of decreasing eye sharpness while levofloxacin has the side effect of neuropsychiatric disorders, joint

disorders.

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Inflammation is the body's response to cell damage or infection. Inflammation can occur

without infection. When cells are damaged, they lose membrane integrity and intracellular molecules

are released into the surrounding environment, which activates pro-inflammatory signals. TNF-α, IL-6,

IL-1β, IL-10, TGF-β, IL-12, IL-18, AND IFN-γ are cytokines that play a role in the neuroinflammatory

process. TNF-α and IL-6 are the main pro-inflammatories, while the cytokine IL-10 acts as an anti-

inflammatory cytokine. Interleukin 6 is released by immune cells during inflammation and induces

proinflammatory effects, such as induction of acute phase reactions, as well as proliferation of B

lymphocytes.

Many herbs have been developed in the world, one of which is noni (morinda citrifolia). The

fruit, seeds, skin, leaves and flowers of the noni plant contain minerals such as potassium, iron, salt

and calcium as well as vitamins A, C, thiamine, niacin and riboflavin. Therefore, noni fruit is known

to have many health benefits for humans, including the presence of various chemicals which include

antibacterial, analgesic, antiviral, antifungal, antitumor, anti-inflammatory and antibiotic. In

Dussossoy et al's research, it was found that noni juice has anti-inflammatory and anti-oxidant

effects.

Due to the long treatment and administration of drugs in combination to TB-RO patients,

drugs can cause damage to some cells, which will induce sterile inflammation. In this study, noni

extract will be given which is expected to suppress the inflammation that occurs and reduce or

prevent side effects that will occur. arises from the administration of TB drugs.

Method

This research was carried out using the post test only control group design method with a

sample size of 16 mice. The test animal used was a white male Wistar strain rat (Rattus

norvegicus) aged 2-3 months, body weight 200 grams, the rat was in good health

characterized by its active movements, thick fur and clear eyes. The research was conducted

in the biomedical laboratory of FK UMM. This research was carried out for 37 days,

including 7 days of adaptation and 30 days of treatment.

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Stages of Making Noni Extract

The steps for making noni extract are as follows: Noni fruit is frozen for 30 hours and then

the juice is taken. Noni fruit is extracted using cold techniques because it is proven to be the best in

terms of chemical parameters. Next, the noni fruit is mashed with a blender, then soaked in 90%

alcohol in a ratio of 1:3 and shaken with an electric whisk (Strrrer) for 2 hours, then left for 24 hours.

The extract was filtered with the dregs soaked again in 90% alcohol 1:2, then shaken for 2 hours and

left for 24 hours. Third, the resulting filter (filtrate) is then evaporated using an electric evaporator

(evaporator) until a thick extract is obtained with a yield of + 7.65%.

Stage of Determining Noni Extract Dosage

Noni extract is divided into 3 different doses, namely dose 1 of 20 mg/200g BW/day, dose 2

of 40 mg/200g BW/day, dose 3 of 80 mg/200g BW/day and given for 30 days using a gastric tube. To

assess the level of effectiveness of noni extract in this experiment, 3 doses were oriented, namely:

100mg/kgBW, 200mg/kgBW, and 400mg/kgBW which were converted at a rat weight of 200g, to

20mg/200gBW, 40mg/200gBW, 80mg/200gBW.

1. Dosage I: 1g of noni extract is needed mixed with 50cc of Carboxyl Methyl Cellulose (CMC).

2. Dose II: 2g of noni extract is needed mixed with 50cc of Carboxyl Methyl Cellulose (CMC).

3. Dose III: 4g of noni extract required is mixed with 50cc of Carboxyl Methyl Cellulose (CMC).

Stage of Determining the Dosage of Combination OAT (Pyrazinamide, Ethambutol, and

Levofloxacin)

The drug dosage in the study refers to the Technical Guidelines for Management of Drug-

Resistant Tuberculosis in Indonesia.

Table 1. OAT Dosage Table

Body Weight (BB)
OAT
>70kg
Pirazinamid (Z) 1750-2000 mg
Ethambutol (E) 1600-2000 mg
Levofloksasin (Lfx) 750-1000 mg

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Dosage conversion from humans to mice using the conversion table is 0.018 for humans

70kg converted to mice 200gr. The conversion of OAT from humans to Wistar rats is:

Pirazinamid: 2000mgx0,018= 36mg

Ethambutol: 2000mgx0,018= 36mg

Levofloksasin: 1000mgx0,018= 18mg

Treatment Stage

Mice were divided into 4 groups, namely:

1. Positive control group (K+): Given BR-1 and OAT combined feed for 30 days without giving noni

extract.

2. Treatment group 1 (M1): Given BR-1 feed, combination OAT, and noni extract at a dose of

20mg/rat/day using the gastric sonde method for 30 days.

3. Treatment group 2 (M2): Given BR-1 feed, combination OAT, and noni extract at a dose of

40mg/rat/day using the gastric sonde method for 30 days.

4. Treatment group 3 (M3): Given BR-1 feed, combination OAT, and noni extract at a dose of 80

mg/rat/day using the gastric sonde method for 30 days.

IL-6 Level Measurement Stage

IL-6 levels were measured using the Human IL-6 Immunoassay Quantikine Elisa kit using

mouse blood specimens taken from the heart.

Results

Table 2.
Interleukin 6 levels (pg/mL) Average
Group
1 2 3 4 (pg/mL)
K+ 0.527 0.882 1.145 1.500 1.013
M1 0.327 0.127 0.218 0.118 0.198
M2 0.073 0.027 0.445 0.236 0.195
M3 0.955 0.618 0.391 0.927 0.723

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The table above shows that there is a decreasing trend in IL-6 levels in treatments 1 and 2.

The average IL-6 level in the positive control was 1,013 pg/mL. Treatment groups 1 and 2 had lower

IL-6 levels than the positive control, namely 0.198 pg/mL and 0.195 pg/mL. Meanwhile, treatment 3

experienced an increase in average compared to treatments 1 and 2, namely, 0.723 pg/mL.

Based on the Shapiro-Wilk test, it shows that each of these data is normally distributed

because it exceeds the significant value of p>0.05. Treatment M1 shows significance = 0.361, M2

shows significance = 0.544, M3 shows significance = 0.381. Because sig p (>0.05) in all groups, it can

be concluded that the data distribution is normal and continued with the Levene Test. The results of

the Levene Test homogeneity test show a significance of 0.095 (p>0.05), which means that the data

variance in interleukin levels is homogeneous so it can be concluded that the data distribution is

normal and homogeneous so that data analysis can be continued with the One Way ANOVA test.

The One Way Anova test shows a p value = 0.002, this shows that there is a significant

difference in interleukin 6 levels between the control and treatment groups. Significant value

p<0.05.

Based on the Post Hoc Bonferroni test analysis above, it can be concluded that the decrease

in IL-6 levels between the positive control groups with treatments 1 and 2 showed significant results.

Because p<0.05. Meanwhile, the decrease in IL-6 levels between the control group and treatment 3

showed no significant results. Because p>0.05.

Based on a simple linear test, a significance result of 0.000 (p<0.05) was obtained, which

shows that there is a relationship between the effect of administering noni extract on reducing IL-6

levels.

Discussion

This study used 16 male white rats (Rattus norvegicus strain Wistar) which were divided into 4

groups, namely the positive control group, the noni treatment group with a dose of 20 mg/200g

(MI), the noni treatment group with a dose of 40 mg/200g (MII). and the noni treatment group at a

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dose of 80 mg/200g (MIII). Each group consisted of 4 mice according to the inclusion criteria. This

research was conducted for 30 days. The research used mice that were given tuberculosis drugs

(ethambutol, pyrazinamide and levofloxacin) in the positive control group and the treatment group

was given tuberculosis drugs and noni extract according to the dose for each treatment group.

The aim of this study was to determine how much influence giving noni extract (Morinda

citrifolia) had on the levels of the cytokine Interleukin 6 in Wistar rats (Rattus norvegicus strain

Wistar) which were induced by tuberculosis drugs (ethambutol, pyrazinamide and levofloxacin).

Based on statistical tests that have been carried out using the Shapiro Wilk normality test

and homogeneity test using the Levene test, the data distribution results were normal and

homogeneous. The results of the One Way Annova test from this study showed p < 0.05, which

means there was a significant difference between the positive group given anti-tuberculosis drugs

(ethambutol, pyrazinamide and levofloxacin) and the treatment group given anti-tuberculosis drugs

and noni extract in various doses. . Because the data was distributed homogeneously, Bonferroni

Post Hoc test analysis was then carried out. The decrease in IL-6 levels between the positive control

groups with treatments 1 and 2 showed significant results. Because p<0.05. Meanwhile, the

decrease in IL-6 levels between the control group and treatment 3 showed no significant results.

Because p>0.05. Then, a simple linear statistical test is carried out to determine the influence

relationship. Based on the results of this simple linear test, it shows significant results between the

dose given and the decreased levels of IL-6. Based on a simple linear equation, administering noni

extract can reduce IL-6 levels by 82.6%. The remaining 17.3% was influenced by other factors that

researchers did not examine.

In the M3 research group, there was a trend of decreasing IL-6 levels compared to the

control group, however, IL-6 levels in M3 were higher than in M2 even though the noni dose was

greater. This could possibly happen because the dose of noni given to M3 has exceeded the effective

dose, causing another inflammatory reaction. So it can be seen that the effective dose of noni for

reducing IL-6 levels is at a dose of 20mg/200gr. Giving the lowest dose can reduce IL-6 levels by

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0.815 pg/ml. This is in accordance with research conducted by Andita, 2022 regarding the

effectiveness of administering doses of noni extract to reduce blood glucose levels in mice. This

research used noni extract at a dose of 2x1.3mg/20g for mice and 2x2.

Conclusion

There was an effect of noni fruit extract (Morinda citrifolia) on IL-6 cytokine levels, indicated

by a decrease in IL-6 cytokine levels in the M1, M2, M3 treatment groups compared to the positive

control group. It was found that there was a significant decrease in the M1 and M2 treatment

groups. The M3 treatment group saw an insignificant decrease. However, a trend was found to

decrease IL-6 cytokine levels in all treatment groups compared to the group. An effective dose was

found that was able to provide an effect, namely at a dose of 20mg/200gr.

Suggestion

Need done study more carry on about influence noni fruit extract (Morinda citrifolia) in

reducing interleukin-6 levels with varying dosesiWhichodifferent. Need done study about influence

Noni fruit extract (Morinda citrifolia) in reducing levels of proinflammatory interleukins others so

that the anti-inflammatory effectiveness of noni fruit extract (Morinda citrifolia) can be determined.

Acknowledgments

The authors thank Muhammadiyah Malang University for assisting during the research also

for biomedical laboratory Brawijaya University and Muhammadiyah Malang University.

Authors’ Contributions

DD, AA, TD, were involved in concepting and planning the research; DD and AA performed

the data acquisition/collection and dana analysis; DD and AA performed the result interpretation;

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DD and AA drafted the manuscript and designed the figures; DD, AA, and TD acquired the funding.

All authors took parts in giving critical revision of the manuscript.

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References

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