Moringa oleifera fruit extract as a potential antioxidant against liver injury by 2-Nitropropane induction in obese male mice model: pre-clinical study

Background: Moringa oleifera fruit extract contains beneficial chemical compounds. This study was conducted to observed the power of antioxidant against liver injury by 2-Nitropropane induction in an obese male mice model. Methods: This research was in vivo laboratory experimental study with a post-test control design group only. The population was obese male mice models, Swiss strain, aged 6–8 weeks, weighing between 60–80 gr. The research sample was determined by Federer's formula for a complete randomized design experimental test, group N (control), O1 (induced by 2-Nitropropane intraperitoneal (i.p) once), O2 (induced by 2-Nitropropane i.p twice), P1 (induced by 2-Nitropropane i.p. once and gavage with M. oleifera fruit extract 500mg/kg bodyweight (BW) once a day), P2 (induced by 2-Nitropropane i.p. twice and gavage of M. oleifera fruit extract 500mg/kg BW once a day), and P3 (induced by 2-Nitropropane i.p. twice and gavage of vitamin C 500mg/kg BW once a day). Antioxidant potential parameters were measured by levels of malondialdehide (MDA), glutation (GSH), 8-hydroxy-2'-deoxyguanosine (8-OHdG), catalase activity, manganese superoxide dismutase (MnSOD), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT). This research was held at the Biochemistry laboratory of Medicine Faculty, UPN Veteran Jakarta in May–September 2020. Analysis was carried out using SPSS version 20.0. The parameters were tested using ANOVA. Results: MDA levels decreased, GSH increased, 8-OHdG decreased, catalase activity increased, MnSOD activity increased and SGOT, SGPT levels decreased. M. oleifera fruit extract was statistically proven to be a candidate for potential antioxidant against liver injury of 2-Nitropropane induction in obese male mice model. Conclusions: M. oleifera fruit extract was statistically evident as an antioxidant substance that reduces oxidative stress in acute liver injury caused by 2-Nitropropane induction.


Introduction
Indonesia has abundant natural wealth, spread from the west to the east. 1 One of the natural resources is plants as a source of traditional medicine.Moringa oleifera fruit is the Moringa plant's fruit, and contains beneficial chemical compounds. 2he vitamin content in M. oleifera includes vitamin C, vitamin A, vitamin B1 and B2.The antioxidant content includes flavonoids, quercetin, isothiocyanates and glucosinolates. 3,4[7][8][9] Antioxidants are chemical ingredients that can donate one or more electrons to free radicals so that free radicals can be mutated. 7,8There are two antioxidants: natural antioxidants and synthetic antioxidants.[12] Natural antioxidants can protect the body against damage caused by reactive oxygen species that can inhibit the occurrence of degenerative diseases and can inhibit lipid peroxidase in the body. 13Vitamin C as an antioxidant exogen can be used to reduce oxidative stress.It can be an effective antioxidant in inhibiting free radicals with a suitable dose and its chemically capable of reacting with most radicals free and oxidants in the body. 14,15In 2016, obesity was estimated to exceed 250 million people worldwide. 16Obesity was the third leading cause of cancer after tobacco and alcohol. 15xperts estimate that obesity causes about 20% of all cancer cases. 14,15,17idemiological studies show that obesity was associated with an increased risk of cancer. 16,18One of the cancers thought to occur due to obesity was hepatocellular carcinoma. 13,17,191][22] Obesity causes angiogenesis, tumorigenesis and chronic inflammation. 15,23Experts suspect obesity is also associated with the incidence of hepatocellular cancer. 15,24,25The chemical 2-Nitropropane can induce hepatocellular. 262-Nitropropane (2NP) can trigger the mechanism of hepatocarcinogenesis. 17,24,27 Research by Metwally et al. (2017) in obese female rats given M. oleifera at a dose of 600 mg/kgBW orally for 12 weeks is a good candidate for therapy for symptoms of metabolic syndrome. 8ere have been many studies reporting on M. oleifera fruit.However, only a few studies have reported on the potential of M. oleifera fruit extract as a potent antioxidant against liver injury in the early events of chemically induced carcinogenesis, such as 2-Nitropropane in obese male mice models.Can M. oleifera fruit extract treat liver injury by 2-Nitropropane induction in an obese male mice model?This research observed the compound antioxidant of M. oleifera fruits extract against liver injury by 2-Nitropropane induction in an obese male mice model.M oleifera fruits extract can treat the liver injury by 2-Nitropropane induction in an obese male mice model. 3,5

Ethical considerations
This study protocol was reviewed and approved by The Health Research Ethics Committee (HREC) Pembangunan Nasional Veteran University Jakarta, approval number B/1822/4/2019/KEPK on April 23 th 2019.This study was held May-September 2020, and all authors provide a statement indicating that all efforts were made to ameliorate harm to animals.Researchers agreed to use the minimum number of experimental animals using the Ferderer formula.The experimental animals were treated according to the 5 F principle.The experimental animals were terminated with ketamine xylazine anesthesia, then buried.

REVISED Amendments from Version 1
We have improved this article with version 2 according to the reviewer's feedback, including: adding a statistically significant was 0.05.
We correct the typo rat to mice.Some studies that was in line with this research are Nafi, Fakhri and Susantiningsih's study that the benefits of Moringa oleifera fruits were high potentially as exogenous antioxidants.
Any further responses from the reviewers can be found at the end of the article

Research design
The research design was an experimental laboratory study with a post-test control group only design.The indicators seen on antioxidant potential were by seeing malondialdehyde (MDA), glutathione (GSH), 8-Hydroxy-2 0 -deoxyguanosine (8-OhdG), catalase activity, manganese superoxide dismutase (MnSOD), serum glutamic pyruvic transaminase (SGPT), and serum glutamic oxaloacetic transaminase (SGOT).The research location was in the Biochemistry Laboratory Faculty of Medicine Universitas Pembangunan Nasional Veteran Jakarta.The population was male obese mice models Swiss strain aged 6-8 weeks weighing between 60-80gr obtained from the Animal Laboratory of University Institut Pertanian Bogor, Bogor West Java.Determination of the research sample using the Federer formula for a completely randomized design experimental test.

Research procedure
Mice were obtained from the Animal Laboratory of University IPB Bogor, the health of the mice was monitored every day, and their body weight was weighed before conducting the study.After going through a laboratory adaptation process for seven days, obesity was induced in mice aged 4-5 weeks by feeding them high-fat diets for 4 weeks until the average body weight of obese mice was 60-80g.Then the mice were placed in a plastic cage with a lid made of ram wire and covered with husk, fed with high-fat feed pellets and drinking water was given ad libitum.The cage environment was made so that it was not humid, with adequate ventilation and adequate lighting where the light time is 14 hours and the dark was 10 hours.
This study using obesity models of mice; a possible thing that can happen is the sudden death of mice which causes the need to use new mice so the number of mice increases.Each mouse that has undergone surgery and liver removal will be buried.The mice were well cared for, and fed according to the standard.After completing the research, the mice were necropsied and buried.During the study there were no sick mice or mice that had adverse effects, so no mice were excluded.
After 10 weeks, the mice were fasted, then necroption under anesthesia: xylazine 0.1 mg/kgBW + ketamine 0.03 mg/kgBW by i.p., then euthanized.Prior to necropsy, the mice were anesthetized using ketamine xylazine until completely asleep and then dissected.The blood samples were taken from the heart's left ventricle with a 3cc syringe by cardiac puncture.The blood was placed in a vacutainer, and the serum was stored at -80°C.The liver was taken and weighed then examined for biochemical markers.The dissected mice will be put into a special animal bag, then buried properly.The liver tissue will be measured for MDA, GSH, 8-OHdG, Catalase activity, MnSOD, SGPT, and SGOT levels.

The preparation of induction 2-Nitropropane
The dose of 2-Nitropropane (130263-5ml Aldrich ® ) of 0.02 mg/kgBW was given intraperitoneally to induce oxidative stress and liver injury in male obese mice models. 27

M. oleifera fruits extraction
The research used M. oleifera fruit extract from Lampung Province was a concentrated preparation obtained by removing the active ingredients of M. oleifera fruits.A total of 500 g of dried simplicia M. oleifera fruits with 70% ethanol solvent maceration got 55.2 g of M. oleifera fruits to extract.

Liver homogenate using a homogenizer
A homogenate was made by putting 100 mg of mice liver tissue into a 1.5 ml Eppendorf tube, then adding 1 ml of phosphate buffered saline (PBS) (0.01 M pH 7.4).This was crushed using a homogenizer for 90 seconds (maximum time) at a speed of 3500 rpm (the crushed samples were placed in the ice floes until they are all crushed) using Tokyo Rikaikai homogenizer.Next, the Eppendorf tube was inserted and centrifuged for 10 minutes at 3500 rpm.To obtain the supernatant from liver tissue, the supernatant was taken and transferred to a new Eppendorf tube.After that, the Eppendorf tube was put in the refrigerator with a minimum temperature of -20°C overnight.

MDA Level examined
Examination of liver MDA levels using the Will method 28 from Assay kit Elabscience ® at the Biochemistry Laboratory, Faculty of Medicine, University of Indonesia.A total 10 μL MDA Color Reagent solution was added into each well of MDA Standard, Blank Control, and Test Samples.The reaction mixture was incubated at room temperature for 10-30 minutes and 40 μL of Reaction Solution was added to make the total assay volume to 100 μL/well.The final reaction mixture was incubated at room temperature for 30-60 minutes.The end-point absorbance was measured with an absorbance plate reader with path-check correction at optical density (OD) 695-700 nm.Absorption readings were taken using a spectrophotometer (Thermo scientific ® ab233471 Lipid Peroxidation (MDA) Assay Kit (Colorimetric)).Furthermore, to get the concentration results is to plot the absorbance data of the sample into a standard curve. 23H levels were examined Homogenate supernatant as much as 40 μL entered a 1.5 ml tube, then 210 μL of 5% trichloroacetic acid was added and mixed using a 3-5 second vortex.Then the solution was centrifuged at 5000 rpm for 10 minutes, then take the supernatant and transferred to a 2 ml Eppendorf tube.Then 800 μL of PBS pH 8.0 was added and mixed by vortex until homogenous.After that, 25 μL 5,5 0 -Dithiobis 2-nitrobenzoic acid (DTNB) reagent was added so that the yellow-colored compound benzoate would be formed due to the binding of the hydryl sulfur (SH) group by DTNB.This was then incubated at room temperature for 1 hour in a dark room, then the absorbance was read using a spectrophotometer (Thermo scientific ® ) at the wavelength (λ) = 412 nm.All samples were measured by using the Duplo principle (measurement twice); to avoid errors in calculations. 23,28OHdG level examined We took 10 mg of homogenate dissolved in 0.1M phosphate buffer pH 7.5 was homogenized and then centrifuged at 1000 Â g for 10 minutes.The supernatant was purified using a Deoxyribonucleic acid (DNA) (Quick-DNATM MiniPrep Plus) extraction kit.The DNA was digested using nuclease P1 following the manufacturer's instructions.The pH was adjusted to 7.5-8.5 using 1 M Tris buffer.One unit per 100 g of Alkaline phosphatase DNA was added and incubated at 37°C for 30 minutes.[31] MnSOD specific enzyme activity measurement MnSOD activity was determined biochemically using the RanSOD ® kit.It determined the total SOD activity utilizing the degree of inhibition of this formazan color as measured by a 505 nm spectrophotometer A. The sample was diluted 20 times using PBS 0.01 M pH 7. 25 μL of standard/diluted sample was put into the cuvette, then 20 μL of mixed substrate mixture was added, mixed well, and the xanthine oxidase enzyme was added.The researcher read the light absorption with a spectrophotometer at a wavelength of 505 nm in the first 30 seconds after the enzyme was added (A1) and 3 minutes later (A2). 32,33talase enzyme activity measurement The liver of obese mice were weighed with an analytical balance of 100 mg and then inserted into a tube.Then PBS was added to as much as 1 mL/100 mg of the liver. 32,34e liver and PBS tube were crushed using a homogenizer for 90 seconds.After thrashing, the tube was put into a centrifuge with 3500 rotations per minute for 10 minutes.The supernatant was then taken and moved it into a different tube.The tube was stored for one night at a temperature of -20 o C. Catalase enzyme activity was expressed in U/mL.One unit means the amount of enzyme that catalyzes the reaction of 1 μM substrate per minute.Sample absorption value with formula:

SGPT and SGOT level measurement
The Eppendorf tube was taken from the refrigerator and 0.1 ml of the supernatant + 1 ml of the SGOT/SGPT reagent Tris was inserted into the cuvette and placed in the spectrophotometer (λ = 340 nm, temperature = 37°C) then mixed using a micropipette (the timer runs when the SGOT/SGPT reagent enters the first time).The start button on the spectrophotometer was clicked and the absorbance was read after 1 minute and 3 minutes. 2alysis of data Analysis was carried out using SPSS version 20.The tests were started with tests of normality and homogeneity.Parametric tests followed data that were normally distributed and homogeneous.Parametric test with one-way analysis of variance (ANOVA) and if different significantly, was followed by Bonferroni's post hoc test for multiple comparisons.

Results
The results of the phytochemical from Balai Penelitian Tanaman Rempah dan Obat (BALITRO) Bogor showed the content of extract substances are alkaloids, tannins, flavonoids, and glycosides.The extracts have quercetin content, saponin and triterpenoid substances.The statistical analysis was conducted using SPSS version 20.Parametric test with ANOVA and Bonferoni's post hoc test for multiple comparation.

MDA and GSH levels
Figure 1 shows that the M. oleifera fruits extract in groups P1 and P2 had an average MDA level of 1.729 nmol/mL and 1.559 nmol/mL, which was lower than group O1 and O2; the P3 group given vitamin C has an average MDA level of 1.766 nmol/mL, which was increased compared to the control group N and group O1, with an average MDA level of 1.541 nmol/mL and 1.825 nmol/mL.The GSH level of group O1 was decreased to 0.654 nmol/mL compared to the N group, O2 group.The group that was given M. oleifera fruits extract had an average GSH level of 0.765 nmol/mL, compared to group P1 and the group P2 of 0.865 nmol/mL.The GSH level of the P1 and P2 groups decreased compared to the O2 group.These data on the MDA level have been analyzed further using Post Hoc Test.

8-OHdG level, catalase activity and MnSOD activity
The average liver 8-OHdG levels in groups O1 and O2 was increased (12.763 ng/mL and 15.249 ng/mL) compared to group N 7.832 ng/mL (Figure 2).In group P1, P2 and P3 the average liver 8-OHdG was decreasing to 10.233 ng/mL, 8.012 ng/mL and 8.534 ng/mL.The catalase enzyme activity in the male obese mice model in group N shows 1.98 u/mL higher than all O1, O2, P1 P2 and P3 groups.In the O1 and O2 groups, there was a decrease in catalase activity of 1.782 u/mL and 1.436 u/mL compared to the other groups.The group given M. oleifera fruits extract had an average catalase activity enzyme level of 1.762 u/mL and 1.824 u/mL.The MnSOD activity level in group O1 was decreased compared to group N, and then more decreased in group O2, as seen on Figure 2, than other groups.The MnSOD activity enzyme level increased in the P1 (0.876 u/mL) and P2 (0.882 u/mL) groups which were given M. Oleifera fruits extract.The MnSOD activity enzyme level also increased in the P3 group (0.681 u/mL) which was given Vitamin C. SGOT and SGPT levels SGOT and SGPT levels in the liver of each group showed a decrease in SGOT and SGPT levels in P1, P2 and P3 compared to O1 and O2 (Figure 3).This figure showed a significant decrease in SGOT and SGPT levels in groups that were given M. oleifera fruits extract than groups N. M. oleifera fruit extract as a candidate for potential antioxidant against for liver injury induced 2-Nitropropane in male obese mice models.

Discussion
Moringa fruit obtained from the Moringa plant is a fruit with many benefits because it contains beneficial chemical compounds.[4] M. oleifera fruits extract contains flavonoids as antioxidants. 2,35The antioxidant activity of flavonoids comes from their ability to donate hydrogen atoms and the ability to chelate metal. 18M. oleifera fruits extract was also anti-obesity because it can down-regulate leptin and resistin mRNA expression while at the same time up-regulating the expression of adiponectin mRNA. 8Vitamin C as an antioxidant agent given as a positive control in group P3 can be compared with M. oleifera fruits extract.The results of the research show that M. oleifera fruits extract have a significant difference as a candidate for antioxidant properties for decreasing oxidative level of induction of 2-Nitropropane in male obese mice models.
The results of this study showed that there was a decrease in MDA levels in P1 and P2 groups that were given M. oleifera fruits extract.The level of GSH increased in the groups O1 and O2.So, it can be concluded that M. oleifera fruits extract can inhibit oxidative stress due to the induction of 2-Nitropropane.As a positive control in group P3, vitamin C also does the same as an antioxidant exogen.So, this study proved that M. oleifera fruits extract has the ability as an exogenous antioxidant.
The level of 8-OHdG also decreased when groups P1 and P2 were given M. oleifera fruits extract compared to groups O1 and O2.So, we can see that the administration of M. oleifera fruits extract can repair damaged cells caused by 2-Nitropropane induction.Likewise, MnSOD and catalase activity increased when groups P1, P2 and P3 were given M. oleifera fruits extract.These data indicate an increase in the activity of MnSOD 36 and catalase enzymes assisted by the presence of antioxidant candidates and expenses from the M. oleifera fruit extract. 6,32,33duction of 2-Nitropropane can cause liver cell damage and acute liver injury, which was characterized by increased levels of SGOT and SGPT. 18,27When given M. oleifera fruits extract, there was a decrease in liver SGOT and SGPT levels in groups P1 and P2 compared to groups O1 and O2.These findings indicate that M. oleifera extract can be a potential antioxidant candidate in liver damage due to 2-Nitropropane induction. 18is study was in line with Nafi, 37 Bilqis, 19 Anis, 18 Fiqih, 21 Rahmi 38 and Susantiningsih's 38 research that M. oleifera fruit extract has high antioxidant levels and deserves to be called a potential antioxidant candidate in 2-Nitropropane-induced obese male mice through the parameters.The findings of increased levels of MDA, SGOT and SGPT in liver tissue indicate that 2-Nitropropane induction causes acute liver injury.This research answers the objectives, to observe the contains of antioxidants M. oleifera fruits extract against liver injury by 2-Nitropropane induction in an obese male mice model through pre-clinical study.

Conclusion
M. oleifera fruit extract was statistically proven to be used as a candidate for potential antioxidant against liver injury of induction 2-Nitropropane in obese male mice model by looking at the levels of MDA, GSH.This project contains the following underlying data: • Data Moringa oleifera fruit.xlsx.(Moringa oleifera fruit as a candidate for potential antioxidant against liver injury in the early events of chemically induced carcinogenesis, such as 2-Nitropropane in male obese mice models.) • Raws Data DokMar.xlsx(The data of MDA and GSH levels, 8-OHdG, MnSOD levels, SGPT and SGOT level).
• Raw Data yang diminta Reviewer oleh MST TS.xlsx (Raw data for Moringa oleifera fruit as a candidate for potential antioxidant against liver injury in the early events of chemically induced carcinogenesis, such as 2-Nitropropane in male obese mice models.)

Taghred Saber
Zagazig University, Zagazig, Egypt I found that this manuscript is totally relevant for the field and brings interesting results that deserve consideration.However, certain points need to be revised before the manuscript could be considered for indexing.
The laboratory conditions of the animal house should be provided.

○
The solvent of moringa oleifera extract should be provided.

○
The level of statistical significance (P value) should be mentioned under the title of Statistical Analysis.
○ Statistical comparison should be provided in the results and foot note of the figures.

○
The authors should mention the result values as mean ± standard error.

○
Foot note of all figures should include the value of P.

Indonesia
The researchers mentioned using obese mice with a body weight of 40-60 grams.What data indicates that these mice were obese?Certain indicators are frequently used to assess the level of obesity and the impact of anti-obesity drugs.These variables include blood pressure, body mass index, insulin tolerance test, total body fat, adiposity index, Lee index, fat pad mass, and magnetic resonance imaging and dual-energy X-ray absorptiometry. 1.
This study employed 2-Nitropropane to induce oxidative stress and liver injury in male obese mouse models.The study involved two groups, O1 and O2, which were given 2-Nitropropane once and twice.It is advisable to include the dosing schedule because it is not clear how much time passed between the two doses and how long after 2-Nitropropane and M. oleifera extract were administered. 2.
2-Nitropropane can induce liver injury.Is there data indicating that this induction was successful?Can the authors provide a histopathological description of the liver?

3.
The results of this study, both from the graphs and the result descriptions, do not show which data is significantly different between groups.No p-values are mentioned.How can the authors claim that there are significant results?

4.
What is the basis for administering a dose of 500mg/kgBW?Have there been any previous studies on this?The method section does not cite any previous research.

5.
The GSH level decreased more in group P2 compared to O2.However, in group P1 with O1, this decrease was not as pronounced.Why did this happen?The same pattern also occurred in the levels of MDA, SGOT, SGPT, and 8oHDG.Please explain this in the discussion.This raises the question of whether M. oleifera might be more effective when given in cases of more severe liver injury.

6.
need to be included for each result obtained and the level of significance needs to be denoted by the use of "*, **, or ***" on the graphs.
Once the appropriate statistical significance is shown, the correct conclusions can be drawn.Statistical significance is required to verify the current conclusions made by the authors.

3.
There are also many spelling and grammatical errors.The authors should have the manuscript proofread by an English language expert.

4.
Mice and rats are not the same.Yet, it is used interchangeably in the manuscript.This needs to be corrected.

5.
In the discussion, the authors merely repeat their results.The results need to be discussed in the context of other previously published literature.Are the findings in this study similar or different from other published studies? 6.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Biochemistry and molecular biology, including oxidative stress, DNA damage, and epigenetics.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Figure 1 .
Figure 1.The malondialdehide (MDA) and glutathione (GSH) level.M. oleifera fruit extract as a candidate for potential antioxidant against for liver injury induced 2-Nitropropane in male obese mice models.The orange bars show MDA levels, the yellow bars show GSH levels.

Figure 3 .
Figure 3. Serum glutamic oxaloacetic (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels.M. oleifera fruit extract as a candidate for potential antioxidant against for liver injury induced 2-Nitropropane in male obese mice models.
8-OHdG, Catalase activity, MnSOD activity and SGOT and SGPT levels.Antioxidant substances in M. oleifera fruit extract can significantly reduce oxidative stress in acute liver injury caused by 2-Nitropropane induction.The limitation of this research is the induction of 2-Nitropropane which requires special skills.Further clinical research on healthy human subjects is needed.

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
Figure legends are short and incomprehensible.It is necessary to expand and rewritten the legend for these figures.No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The author(s) is/are employees of the US Government and therefore domestic copyright protection in USA does not apply to this work.The work may be protected under the copyright laws of other jurisdictions when used in those jurisdictions.Faculty of Medicine, Gunadarma University, Depok, West Java, Indonesia2Program Doktor Ilmu Biomedik, Faculty of Medicine, Universitas Indonesia, Depok, West Java, https://doi.org/10.5256/f1000research.133590.r204636© 2023 Likhayati Septiana W.