Anti-hypercholesterolemic effect of Zingiber montanum extract

Background: High cholesterol levels (hypercholesterolemia) has been recognized to cause various disease, most notably the cardiovascular disease. Unfortunately, most anti-hypercholesterolemic drugs deliver several side effects for patients, by which medicinal plants have begun to attract attention for treating hypercholesterolemia. Among others, Zingiber montanum (J.König) Link ex A.Dietr. has traditionally been taken for treating health problems caused by high cholesterol levels. Hence, this work aimed at investigating anti-hypercholesterolemic effects offered by the plant. Methods: This study was conducted on 30 male Wistar rats. During experiments, the subjects were divided into 6 groups (n=5), i.e. no treatment (Group 1, control); high-fat diet (Group 2, control); high-fat diet with simvastatin (Group 3); high-fat diet with plant extracts (Group 4-6 with 100, 200, and 400 mg/kg BW, respectively). After 4 weeks of treatments, blood samples were collected from each group. Then, plasma concentrations of triglycerides, total cholesterol, high density lipoproteins (HDL), and low density lipoproteins (LDL) were measured. Results: There were significant differences in total cholesterol (p=0.000), LDL (p=0.000) and triglycerides (p=0.001) for Groups 4-6 (high-fat diet treated with different plant extract doses) in comparison with Group 2 (high-fat diet, control). Meanwhile, there were no significant differences in HDL levels (p=0.830) between Group 2 (high-fat diet, control) and other groups. The results also showed significant differences in total cholesterol and LDL for subjects treated with plant extracts (Group 4, 100 mg/kg BW, p=0.000; Group 5, 200 mg/kg BW, p=0.000; Group 6, 400 mg/kg BW, p=0.000) compared to Group 2 (high-fat diet, control). Then, treatments with 400 mg/kg BW (Group 6) discovered significant reductions in total cholesterol, LDL, and triglycerides (p=0.030). Conclusion: Therefore, Z. montanum has been discovered to deliver anti-hypercholesterolemic effects to experimental subjects, making it potential to act as a natural source of anti-hypercholesterolemic agents.


Introduction
Hypercholesterolemia is a health condition characterized by a very high level of cholesterol in the blood 1 .If it is not well treated, hypercholesterolemia certainly increases coronary heart disease risk 2 .In current advances, various agents have been made available to treat hypercholesterolemia patients, including HMG CoA reductase inhibitors or statins (i.e.Simvastatin) 3,4 .
To avoid unintended side effects of artificially made antihypercholesterolemic agents, medicinal plants have begun to attract attention for treating hypercholesterolemia.In Indonesia, various locally growing plants have been used for traditional medicine.Among others, Zingiber montanum (J.König) Link ex A.Dietr., which belongs to the family Zingiberaceae, has been recognized to act as a traditional medicine in East Kalimantan, Indonesia, for treating health problems caused by high cholesterol levels [5][6][7][8] .This study, therefore, aimed at investigating anti-hypercholesterolemic effect of Z. montanum.

Plant extraction
In the laboratory, the rhizomes of Z. montanum were sliced and dried at room temperature for 3 days.After that, they were crushed and transferred into a glass container.Crushed rhizomes was soaked in absolute ethanol (9401-03 Alcohol, Anhydrous, Reagent, J.T. Baker) for 5 days.The mixture was shaken occasionally with a shaker (3525 Incubator Orbital Shaker, Lab-Line, US).After 5 days, it was filtered (Whatman Filter Paper 11µm, Sigma-Aldrich) and evaporated by using a rotary evaporator (RV06-ML Rotary Evaporator, IKA, Germany).In the end, dried extracts were obtained and stored at 4°C in a dark bottle.

Experimental model
In this study, experiments were designed to follow Federer's rule, with six groups of induction.For the experiments, 30 male Wistar rats (Rattus norvegicus, weighing 250-350g, aged 12-13 months) were obtained from Animal House of the Faculty of Medicine, Mulawarman University, Indonesia.They were randomly divided into 6 groups, i.e.Group 1 (no treatment, control), Group 2 (high fat diet, control), Group 3 (high fat diet with simvastatin), and Groups 4-6 (high fat diet with separate doses of Z. montanum extract; 100, 200, and 400 mg/kg, respectively).They were acclimatized for one week in a controlled room temperature (25°C) with a 12-hour light/dark cycle.Besides, they were provided with an access to food pellets, while filtered water was provided ad libitum to help them adapt to the new environment.During experiments, each test subject was separately housed in a wire cage (30×30×30 cm).In all treatments, high-fat diets were administered for all test subjects for 4 weeks, in which 10% chicken egg yolk and reused cooking oil were added to their standard pellet diets (JAPFA, Comfeed, Indonesia) with tap water ad libitum.

Biochemical analysis
After 4 weeks of treatment, blood samples were collected from each treatment group separately after an overnight fasting.All test subjects were anesthetized intraperitoneally with a ketamine injection (Hameln, Germany) at a 60 mg/kg BW dose before taking the blood samples.After the anesthetize, each test subjects was euthanized by applying cervical dislocation.Each blood sample was aspirated through the left ventricle of test subject's heart.Practically, two millilitres of blood were aspirated by using a 3 ml disposable syringe to later be filled into a vaccutainer tube with an anticoagulant.Then, plasma concentrations of triglycerides, total cholesterol, high density lipoproteins (HDLs), and low density lipoproteins (LDLs) were measured in three repetitions for each sample by utilizing an automatic analyzer system (BiOLis 24i; Boeki, Tokyo, Japan).

Data analysis
In this work, statistical analyses were performed in SPSS software version 16.0.Data normality was examined by applying the Shapiro-Wilk normality test.Then, parameter data were analyzed by using ANOVA and post hoc with Tukey test.The analyses set p-value of ≤ 0.05 as being significant.

Ethical considerations
All protocols taken in this study had been approved for Ethical Animal Care from the Medical and Health Research Ethics Commission, Faculty of Medicine, Mulawarman University with approval no.81/KEPK-FK/V/2018.All possible efforts had been ensured to ameliorate any suffering of animals treated as test subjects in this research.

Results
Looking at results of the statistical analyses, significant differences were found between total cholesterol (p=0.000),LDL (p=0.000) and triglycerides (p=0.001)(Figure 1) levels  achieved between Group 2 (high-fat diet, control) and Group 4-6 (treatments of Z. montanum extracts at different doses).Besides, there was no significant difference in HDL (p=0.830)levels between Group 2 and other groups.Meanwhile, post-hoc Tukey test revealed significant differences between total cholesterol (p=0.000) and LDL (p=0.000)levels of all Z. montanum treatments (Groups 4-6) with the high-fat diet control (Group 2).Then, results for Z. montanum treatment at 400 mg/kg BW doses (Group 6) particularly discovered significant reductions in total cholesterol, LDL and triglycerides (p=0.030)(Table 1).

Discussion
The Z. montanum (Supplementary File 1) has been widely taken as a medicinal plant in Asia.Pharmacological properties of Z. montanum include antimicrobial, antioxidant, insecticidal, anti-cancer, anticholinesterase, and anti-inflammatory [9][10][11] .In the literature, previous researches on anti-hypercholesterolemic effects of other Zingiber species mainly focused on Z. officinale (a.k.a.ginger) 18 .In general, a restoration of changes in lowdensity lipoprotein and HMG CoA reductase by Z. officinale administration with a high-fat diet has been suggested as an explanation for the effect of ginger in hyperlipidemia treatments 19 .
In fact, the rhizome extracts of Z. montanum showed the highest total curcuminoid content compared to other Zingiber species 7,12 .Curcumin as antioxidants would hence be able to efficiently prevent LDL oxidations 15 .The significant changes in LDL levels suggested Z. montanum to deliver an References effect on lipid metabolism 16 .Curcumin with other chemical compounds from Z. montanum was then suggested to offer antihypercholesterolemic effects.

Conclusion
Looking at all results in this study, Z. montanum extract have been discovered to reduce lipid profile levels.The medicinal plant could therefore deliver anti-hypercholesterolemic effects to experimental subjects, making it potential to act as a natural source of the anti-hypercholesterolemic agents.
Paper is well written and the experimental design is set up well .However, it is not the first study that is done on the beneficial characteristics of Z. montanum and statistical analysis should be better described.
However, I agree that Z. montanum has been discovered to deliver anti-hypercholesterolemic effects to experimental subjects, making it a potential to act as a natural source of antihypercholesterolemic agents.
Before being indexed, the article requires an English language edit.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Animal production 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.

Deny Susanti
Department of Chemistry, Faculty of Science, International Islamic University Malaysia, Kuantan, Malaysia 1.The manuscript needs to be sent for proofreading.There are a lot of grammatical mistakes and poor sentence construction, and there is no connection between sentences in paragraphs.
2. In the manuscript, the voucher of herbarium specimen of the plant should be stated, and also where the herbarium specimen is deposited and who is identifying the plant (botanist).
3. Please mention the replication that was conducted in each experiment.
4. Discussion: rewrite the discussion to discuss more details about the results of the experiments and compare the previous research or facts from the article or book, to support the results.
5. Check on how to write the species name of plants for the first and subsequent usage, and be consistent.

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

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Dataset 1 .
Effect of ethanol extract of Z. montanum and simvastatin in total cholesterol, triglycerides, high density lipoproteins (HDL), and low density lipoproteins (LDL) levels after 4 weeks of treatment in a high fat diet rat model https://dx.doi.org/10.5256/f1000research.16417.d221668

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 Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes 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:
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.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. Version 1
© 2018 Susanti 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.