Benefit of cinnamon (Cinnamomum burmannii) in lowering total cholesterol level after consumption of high-fat containing foods in white mice (Mus musculus) models [version 1; peer review: 1 approved, 1 approved with reservations]

Background: Hypercholesterolemia is a condition where cholesterol levels in the body exceed the normal range. In Indonesia, the development of traditional medicine is carried out by examining plants known for their medicinal qualities, including cinnamon ( Cinnamomum burmannii). There are many nutritional components contained in cinnamon, such as cinnamaldehyde. This has been suggested as a substance that can reduce cholesterol and triglyceride levels. This study was conducted to determine the efficacy of cinnamon in reducing total cholesterol levels of mice (Mus musculus) given high-fat feed. Methods: This is an experimental study with a pre-post control study design. The groupings were performed by a simple random sampling method. There were five groups (n=6/group): 1) Negative control (aquadest); 2) positive control of high-fat containing food (HFC; quail's yolk); 3) HFC + cinnamon extract (CE; dose 2mg/20g body weight (BW); 4) HFC + CE (dose 4mg/20gBW); 5) HFC + CE (dose 8mg/20gBW). The study was conducted for 28 days. The intervention of CE started on day 15 and ended on day 28. Measurement of total cholesterol and BW of mice was performed on days 0, 14 and 28. Results: A decrease in the total cholesterol of mice (p = 0.001) was found in the groups that consumed CE. However, there was not a significant change between groups in mice BW (p = 0.419). Conclusions: Providing cinnamon extract (Cinnamomum burmannii) for 28 days could decrease total cholesterol levels in mice compared to those not given cinnamon extract when consuming high-fat containing foods. Open Peer Review


Introduction
Hypercholesterolemia is a state where the cholesterol level in the body exceeds the normal range. Hypercholesterolemia can increase the risk of atherosclerosis, coronary artery disease, pancreatitis, diabetes mellitus, thyroid disorder, liver disease and renal disease 1 .
Data from the Indonesian Ministry of Health in 2019 reported it's estimated that coronary artery disease to be the first killer, 26.4% of all deaths in Indonesia 2 . Out of the 17 million the premature death (under the age of 70) due to non Communicable disease, most cases are in low-and middle income countries, and 37% are caused coronary heart disease and 6.7 million of these were triggered by stroke (WHO, 2015) 3 .
In Indonesia, the development of traditional medicine is performed by exploring herbs known to be beneficial for health, and this information is passed from generation to generation. One such herb is such as gambier (Uncaria gambir Roxb.) 4 , which has antioxidant effects and lowers blood glucose when treating type 2 diabetes mellitus. There is also bangun-bangun leaves (Coleus amboinicus), which are believed to be effective in relieving pain when distilled as ethanol and water extract 5 . Traditional medicine that comes from plants is used commonly by many people in Indonesia. One of these medicines is cinnamon (Cinnamomum burmannii,) which is often eaten daily with food. Cinnamon is a native plant in Indonesia and can be found abundantly in Central of Java, (Karanganyar), West Sumatra (Padang), Jambi (Kerinci), etc 6 . Cinnamon has been shown to reduce total cholesterol, low-density lipoprotein and triglyceride levels, as well as increasing high-density lipoprotein levels 7 . Cinnamon is a flavoring ingredient that has been used in daily routines as a spice. Data from various literature on cinnamon reveal that it mainly contains essential oils and important compounds such as cinnamaldehyde, eugenol, cinnamic acid, and cinnamate 6 .
This study was conducted to determine the efficacy of cinnamon in reducing total cholesterol levels of mice (Mus musculus) given high-fat feed. Mice were used in this study because of they have a similar biology to humans, and can therefore be a model for human hyperlipidemia [8][9][10] .

Methods
This is an experimental study in an animal model with a pre-post control study design. This study was conducted at the Pharmacology Laboratory of the Faculty of Medicine, Universitas Sumatera Utara, Indonesia.

Ethics
This study was approved by the Health Research Ethical Committee of Universitas Sumatera Utara (No:55/TGL/KEPK FK USU-RSUP HAM/2019).
This study used mice that were given high-fat containing food (HFC) to provide a hyperlipidemic mouse model. The intervention was carried out by giving different CE doses to find the dose that reduces the total cholesterol level of the mice. Cholesterol total levels were obtained from the blood by cutting the mice's tail. To ensure the experimental animals remained in a comfortable condition, the mice were given ketamine anesthesia intraperitoneally (80 mg/kgBW) before cutting of the tail 11 .

Experimental animals
The experiments took place at the Laboratory of Pharmacology -Universitas Sumatera Utara (USU). The animals were purchased from the Department Biology of Mathematics and Scientific Faculty USU. In total, 30 male white mice (Mus musculus), Swiss Webster strain, 10 to 12 weeks old, and weighing 25 -40 g were used. Before conducting the study, the mice were adapted to their cages (plastic (30 × 20 × 10 cm) and covered with fine wire mesh; base of the cages was covered with rice husks as thick as 0.5 -1 cm and replaced every day during the study) for 2 weeks before the experiment started. They had 12 hours of daylight (6:00 A.M. -6:00 P.M.) and 12 hours of dark (6:00 P.M. -6:00 A.M.). The mice were fed with standard feed (CP 551) from PT Charoen Pokphand-Indonesia and water was given ad libitum. Room temperature and humidity were kept at normal ranges. They were weighed once a week to avoid stress.

Allocation and treatment groups
After the adaptation period, the mice were randomly divided into five groups, with each group consisting of 6 mice.
The sample size was calculated according to Federer's formula 12 : (t-1) (n-1)>15 t = the number of groups n=the number of samples All 30 mice were given a number (1-30) using a SPIDOL marker pen, then randomized by putting the numbers in an envelope and dividing them into 5 groups according to the numbers are taken from the envelope 8 .
The study was conducted for 28 days.
Cinnamon extract. CE was given to the mice every morning at 8:00 am. Animals were kept during research in the mice laboratory in the Pharmacology laboratory. Before being given CE, it was dissolved with aquadest. The extract was given to the mice orally using feeding tubes. CE interventions began on the 15th day until the 28th day.
High-fat containing food. The quail egg yolk was used to induce hypercholesterolemia, because the concentration of the lipid in quail egg yolk is higher than chicken egg yolk 13 . A dosage of 0.5ml/day was given until day 28. The administration of quail egg yolk was done orally using feeding tubes as per the administration of CE. Vanessa et al. in 2013, who saw a decrease in total blood cholesterol levels in white rats (Rattus norvegicus) by administering instant cinnamon powder drink (Cinnamomum burmannii BI.) at a dose of 14.4 mg and 43.2 mg for 14 days. In their research, it was found that a dose of 14.4 mg can reduce cholesterol levels in rats. Therefore we used a dose of 14.4 mg, because it was effective and efficient 14 . We converted its dose to mice and increased it with variation doses of 2, 4 and 8 mg. The value converted from 200g rat to 20g mice is 0.14.

Laboratory analysis
Measurement of total cholesterol and BW of mice was performed on days 0, 14 and 28 (every two weeks). Ketamin 80 mg/kgBW mice intraperitoneally injected for anesthesia before the tail of the mice was cut 11 .
Mice tails were cut 1-2cm to draw blood, which was then assessed for total cholesterol using the digital autocheck ® cholesterol measurement tool. Mice were weighed using a digital scale.

Statistical analysis
Data were analyzed using SPSS 24. The number average of sample data presented as mean ± SD. The one-way ANOVA statistical analysis to indicate the effects of treatments for all group. If the result is significant (p < 0.05), then bootstrapping was performed using post-hoc Bonferroni for differentiating between each group. Figure 1 shows that groups K0 (negative controls), K1 (positive controls), K2 (CE 2 mg/20gBW), K3 (CE 4 mg/20gBW), and K4 (CE 8 mg/20gBW), had a decrease in total cholesterol levels. There is a significant difference in total cholesterol among the groups (p=0.001 between groups). A post-hoc Bonferroni test was performed to see the difference in total cholesterol averages within each group.

Results
It was seen that there was a difference in average total cholesterol levels between K0 (107.5 ± 1.87 mg/dl) vs K1 (120.3 ± 5.53 mg/dl), (p = 0.001). This shows that in the K1 group (positive control) given quail yolk succeeded in increasing cholesterol in mice with a significant difference compared to the K0 group (negative control), which was only given aquadest. In addition, differences in total cholesterol levels in the K1 group (120.3 ± 5.53 mg/dl) compared with K2 (107.3 ± 3.61 mg/dl), K3 (106.8 ± 4.57 mg/dl) and K4 (106.7 ± 0.51 mg/dl) groups showed that a significant decrease in total cholesterol levels (p = 0.001). This proves that CE is efficacious in reducing total cholesterol levels. Between groups K2, K3, and K4, which were all given CE, there was not a significant difference between total cholesterol levels (p > 0.05). Figure 2 shows that there were increases in BW in all five groups. The largest increase of BW was in group K2, which was the positive control group who were given HFC quail egg yolk at 0.5 ml/20gBW. The smallest increase in BW was found in group K5, which was the group provided with HFC quail egg yolk and CE with the dose of 8 mg/20gBW (highest dose in this study). One-way ANOVA showed that there was no significant difference in BW between groups (p=0.419), which could be inferred that the action of giving CE gave no effect in increasing BW in the mice.

Discussion
The aim of the study to investigated cholesterol levels was lowered in mice using CE and quail egg yolks for a high-fat diet for 28 days. In this study, it was proven that giving 0.5ml quail egg yolk for 28 days increased the total cholesterol level between the negative and positive control groups (post-hoc Bonferroni K0 vs K1; p=0.001).
There was a significant difference in the decrease of total cholesterol after treatment among all groups in this research. However, this is not consistent with Vanessa et al. (2013) who stated that there was a decrease, but the difference was not statistically significant. Their study was only conducted for 14 days, whereas our study was conducted for 28 days, leading to differing results 14 .
Cholesterol is formed by the action of HMG-CoA reductase enzyme (3-hydroxy-3-methylglutaryl-CoA) 15 . If hypercholesterolemia is left without implementing proper diet or treatment, it can cause occlusion in a blood vessel. To treat hypercholesterolemia, some medicines, such as simvastatin, could be given 15 . The use of statins will competitively block HMG-CoA reductase and efficiently reduce serum LDL cholesterol. But, treatment using simvastatin can cause rhabdomyolysis 16 . Thus, it needed research on traditional or herbal medicine, e.g. CE, that needs developed and have minimal adverse effects.
(2011) stated that cinnamon had the ability to be an antioxidant, antivirus, antifungal, antimicrobe, antitumor, and can lower cholesterol and blood pressure with low fat compounds. Cinnamon is believed to have a direct role in lipid metabolism and preventing hypercholesterolemia and hypertriglyceridemia, as well as in preventing free fatty acids with its strong lipolytic activity 17 . In the present study we believe that the increase of cholesterol levels due to quail egg yolk could be decreased with cinnamon by its ability to block HMG-CoA reductase enzyme and suppress lipid peroxidation through increased antioxidant enzyme activity 18 . In research by Pai et al. (2013), it was stated that cinnamaldehyde could lower cholesterol and triglyceride levels by the action of some enzymes secreted in certain amounts, which might contribute to bile acid synthesis 19 .
Our study showed that there was no difference in BW of the mice between groups (p = 0.419). This is not consistent with Vafa et al. (2012) who stated that consuming 3 grams of cinnamon for 8 weeks could decrease some biochemical and anthropometric variables compared to previous states significantly, i.e. a decrease of 1.19% body weight, 1.54% body mass index and 1.36% body fat. In addition to lowering BW variables, it could also decrease fasting blood glucose level by about 9.2%, and 6.12% of HbA1c and 15.38% of triglyceride levels 20 .
Leaf and Antonio (2017) stated that the higher the food intake, the higher the increase in BW 21 . In the present study, in addition to standard food intake, giving hypercholesterol and inter- ventional food should increase BW, because the mice had increased energy intake. The amount of food intake will affect the amount of energy intake, which will be saved as fat and impact on the mice's BWs, since energy intake is inversely proportional to physical activity. A high-fat diet group has low sensitivity to leptin, which results in increased appetite and food intake, thus increases the BW 21 . However, this was not the case in our study.

Conclusion
In the present study, a high-fat containing food used in combination with cinnamon extract (Cinnamomum burmannii) for 28 days could decrease total cholesterol levels in mice. Cinnamon extract is believed to have direct effects in lipid metabolism and prevent hypercholesterolemia and hypertriglyceridemia, as well as decreasing free fatty acids by its strong lipolytic activity. The smallest increase in BW was found in group K5 », K5 ??, you would like to say K4? 8.

Data availability
In the present study we believe that the increase of cholesterol levels due to quail egg yolk could be decreased with cinnamon by its ability to block HMG-CoA reductase enzyme and suppress lipid peroxidation through increased antioxidant enzyme activity18. » the mechanism involved in the antihypercholesterolemic effect must be declared with precaution, 'may be'.

9.
Our study showed that there was no difference in BW of the mice between groups (p = 0.419). This is not consistent with Vafa et al. (2012) 1 who stated that consuming 3 grams of cinnamon for 8 weeks could decrease some biochemical and anthropometric variables compared to previous states significantly, i.e. a decrease of 1.19% body weight, 1.54% body mass index and 1.36% body fat. In addition to lowering BW variables, it could also decrease fasting blood glucose level by about 9.2%, and 6.12% of HbA1c and 15.38% of triglyceride levels20. » model used by the study cited must be declared.

10.
In the present study, a high-fat containing food used in combination with cinnamon extract (Cinnamomum burmannii) for 28 days could decrease total cholesterol levels in mice. » this conclusion must be reformulated, high-fat containing food was used only to have the model for studying the effect of CE on cholesterol level, so the conclusion must be related only to CE.

11.
Reviewer Expertise: Ethnopharmacology, medicinal plants, diabetes, hypertension mean to homogenized (make smaller of the differences in each samples) for prevent BIAS. As we known, there are differences between male and female will increasing number of lipid and dysfunction metabolic, including of hyperlipidemia. It related to level of hormones.

Introduction
The aim of study should be better mentioned. 1.

Answer:
Thank you for your comment, we would confirm our statement on the aim of this study. The aim of the study to determine cinnamomum burmannii effective in reducing total cholesterol levels of mice (Mus musculus) given high fat feed.
It was not clear the question about the effect of plant on the lipid profile.

Answer:
We had explained about the effect of plant on the lipid profile related to few references. Please see in part of DISCUSSION (page 5 manuscript in F1000reseach). Cinnamon is believed to have a direct role in lipid metabolism and preventing hypercholesterolemia and hypertriglyceridemia, as well as in preventing free fatty acids with its strong lipolytic activity17. In the present study we believe that the increase of cholesterol levels due to quail egg yolk could be decreased with cinnamon by its ability to block HMG-CoA reductase enzyme and suppress lipid peroxidation through increased antioxidant enzyme activity18. In research by Pai et al. (2013), it was stated that cinnamaldehyde could lower cholesterol and triglyceride levels by the action of some enzymes secreted in certain amounts, which might contribute to bile acid synthesis19.
(This study needed continuos some research in the future to measured number of cinnamaldehyde in Cinnamon Extract Powder ® per 1 mg CE used. Then we'll do new research to prove how CE mechanism as anti hypercholesterolemia, Is it blocked by HMG-CoA reductase enzyme and suppress lipid peroxidation through increased antioxidant enzyme activity or dysfunction of bile acid synthesis or both mechanism) ?
In the past paragraph, the author described the lipid lowing effect of extract. Did this evidence extracted from traditional medicine reports? Answer: Yes, the explain about "this has been suggested as a substance……triglyceride levels" better moves to part of DISCUSSION, because we took from one of reference. We will add inform about the quail egg yolks as a high-fat diet that induces hypercholesterolemia in part of ABSTRACT. We can not add inform in the AIM of study. Because, if we added in the Aims of study, it was not consequence to the title of study. If we changed title "Benefit of cinnamon (Cinnamomum burmannii) in lowering total cholesterol level after consumption of the quail egg yolk as a high-fat diet that induces hypercholesterolemia". It will make the title very long and not in accordance to the rules of journals F1000Research.