Abstract Hypercholesterolemia is often considered a major cause of death globally, including in Malaysia, resulting in fatalities due to heart attack or stroke. Recent research indicates that consuming coffee may lower the risk of heart failure and stroke by reducing cholesterol level. The present study investigated the efficacy of consuming Liberica coffee pulp yoghurt in female rats with hypercholesterolemia. 30 rats were divided into six different groups: a control group (GC), negative control groups (NC), a positive control group treated with simvastatin (20 mg/kg) (PC), a low dosage treatment group (8.6 mg/kg) (T1), and a high dosage treatment group (25.7 mg/kg) (T2). All rats were fed a high-cholesterol diet and received treatment daily for 8 weeks. At the end of week 8, all rats were culled. The blood and organs were obtained for biochemistry tests. The results showed that the body and organ weights of GC, PC, T1 and T2 groups show less compared to NC group. The lipid profile shows a significant (P <0.05) increase in NC group compared to other groups. Total cholesterol (TC), triglycerides (TG) and low-density lipoprotein (LDL) levels in NC group show higher among all the groups. T2 group shows significantly (P <0.05) the lowest in TC, TG and LDL levels. Liver and kidney profiles showed no significant changes. The liver histology of the NC groups showed the presence of ballooning cells compared to the other groups. Visceral fat also showed significant hyperplasia in NC group. Overall, this study demonstrated the effectiveness of L.coffee pulp yoghurt at the highest dosage able to reduce hypercholesterolemia.

Keywords: Hypercholesterolemia, Lipid profile, Simvastatin, Coffee pulp yogurt, Liberica coffee

Funding: The authors are grateful for the research funding provided by the Management and Science University, Malaysia (SG-004-022022-FHLS and MPCG-001-022023-FHLS).

Citation: Abidin, A. Z., Bahari, H., Othman, F., Norkhairani, N. E., Balan, S. S. 2025. The effectiveness of consuming Liberica coffee pulp yoghurt on hypercholesterolemia induced male Sprague Dawley rats. Natural and Life Sciences Communications. 24(2): e2025031.

INTRODUCTION

Coffee is widely consumed across the globe and is one of the most popular beverages worldwide. According to data from the International Coffee Organization, the total world coffee exports amounted to approximately 11.4 million bags in February 2022, reflecting a rise of about 0.2 million bags of coffee (International Coffee Organization, 2022). This surge in coffee demand can be attributed to recent studies demonstrating that coffee has favorable effects on several chronic diseases, such as cardiovascular diseases, obesity, and even cholesterol or lipid levels. The beneficial effects of coffee are due to its chemical content, which includes approximately 1000 bioactive compounds and minerals such as Ca, K, Fe, P, Ni, Mg, and Cr. Among these compounds, caffeine, chlorogenic acids, diterpenes, and trigonelline are the most prominent (Nuhu,  2014). There are four main species of coffee available on the market, with arabica coffee, robusta coffee, and liberica being the most consumed. Liberica coffee, also known as Liberian coffee, originated in Liberia in West Africa and was later transported and cultivated in other parts of the world such as Malaysia, Indonesia, and the Philippines. It is renowned for its floral and fruity aroma, along with a slight hint of woody taste when converted into coffee (Ablan Lagman, 2023).

Elevated cholesterol levels, known as hypercholesterolemia, are a significant risk factor for cardiovascular disease (CVD), which remains the primary cause of death and illness globally. According to the National Health and Morbidity Survey (NHMS) (2023) findings, 33.3% of adults in Malaysia suffer from hypercholesterolemia. This condition has multiple contributing factors, with genetic predisposition being the main cause. Secondary factors include poor dietary habits, tobacco use, and underactive thyroid function (Abdul Murad et al., 2023)

A variety of factors contribute to increased cholesterol levels, including an unhealthy or fatty diet that primarily consists of animal products such as meat and dairy, which are high in saturated fats and sugar. These foods contain substances that stimulate the liver to produce excess cholesterol, leading to hypercholesterolemia (Cunha et al., 2021). Elevated cholesterol levels can result in accumulation of cholesterol in the blood, leading to coronary heart disease and stroke. The build-up of fats or plaques on arterial walls, a condition known as atherosclerosis, can cause the narrowing of blood flow, eventually leading to the blockage of blood flow and the bursting of blood capillaries. This reduction in blood flow can cause chest pain, whereas complete blockage of blood flow can result in a heart attack or stroke (Feyisa et al, 2019).

Yoghurt is a popular fermented milk product that has gained considerable popularity in recent times, owing to its nutritional benefits. It is considered a nutrient-dense food because of its high protein, vitamin, mineral, and probiotic contents. Yoghurt is an excellent source of protein, with an average of 8.5 grams per cup, which is sometimes higher than the protein content of milk. Most proteins in yoghurt are caseins, which are rich in essential amino acids and absorb minerals such as calcium and phosphorus (Holt et al., 2013). Yoghurt also contains a variety of vitamins and minerals, with the highest amounts being vitamin B12, which helps maintain healthy blood and nerve cells; calcium, which is essential for strong bones; and phosphorus, which plays a vital role in many biological processes. Yoghurt is a probiotic carrier food because it delivers significant amounts of probiotic bacteria to the body. Probiotics are live bacteria that provide many health benefits, including enhancing the immune system, lowering cholesterol, aiding in vitamin synthesis, improving digestive health by protecting against diarrhea and constipation, and even improving lactose digestibility (He  et al, 2008; Tumbarski et al, 2024). Recently, coffee-flavored yoghurt and yoghurt coffee have been developed to enhance the functional properties of yoghurt and coffee. Research by Jonah Mbae has shown that adding different concentrations of coffee extract to yoghurt increases lactic acid bacteria counts, total phenolic compounds, and antioxidant activity (Mbae et al., 2022). The goal of this research project was to demonstrate the potential benefits of incorporating coffee extract into yoghurt, specifically in terms of its ability to lower hypercholesterolemia. This study specifically examined the use of waste pulp from L.coffee, a sustainable product, as a source of the extract.

MATERIAL AND METHODS

Extraction of Liberica coffee pulp

The Federal Agricultural and Marketing Authority (FAMA) in Banting, Selangor, provided the Liberica coffee pulp. Then Liberica coffee pulp was washed with flowing tap water to eliminate any foreign materials.

In this experiment, a Moka pot was used to extract Liberica coffee pulp. Moka coffee pot was used because it can optimally extract high amounts of caffeine, chlorogenic acid, and other coffee components. After extraction, all L. coffee pulp extracts were freeze-dried and stored at -20°C for further analysis (dePaula and Farah 2019).

LC yoghurt preparation

To formulate coffee yoghurt, 100 ml of full cream (Dutch lady Purefarm UHT) was used along with a starter culture containing Lactobacillus delbrueckii subsp. Bulgaricus ATCC 11842 and Streptococcus thermophilus APC151 were purchased from New England Cheesemaking Supply (UK). Yoghurt was processed by pre-warming milk to 60°C, adding the bacterial culture, and incubating the mixture using a yoghurt maker. The temperature increased to 90°C for 30 min to achieve complete pasteurization. The mixture was incubated for 6 h at a pH of 4.6. Yoghurt was then transferred to a refrigerator overnight for 12 h (Mbae 2022). After preparing the yoghurt solution at a low dosage, the recommended dosage of LC yoghurt was prepared by dissolving LC extract. The amount of powder prepared based on the weekly weight of the rats was 8.6 mg/100 g for low dose and high dose at 25.7 mg/ 100 g. The dosage of L. coffee yogurt is based on previous study conducted by Mahmoud et al. (2013).

High-cholesterol diet preparation

A high-cholesterol diet (HCD) was prepared using the protocol described by Balan et al. (2021) with some modifications. To this 50% normal rat pellets (Gold Coin Feed mills) were mixed with 20% egg yolk, 24% ghee (Crispo), and 6% corn oil (Vecorn). All the mixed ingredients were baked in an oven at 60°C for 60 min. The baked HCD were cut into pieces and stored in a freezer at 2–4 °C before being fed to the rats. Standard chow and water were provided daily to all rats. The negative group and treatment groups received a high-fat diet.

Ethical approval

All experiments were conducted after obtaining approval from the Animal Ethics Committee of Management and Science University (ethics code AE-MSU-073). In this study, 30 female Sprague-Dawley rats was used weighing between 150-200g.

Experimental animals

There was a total of 5 group in this study, each group consist of n=6 female rats weighing between 150-200g (age 5-6 weeks). All rats were acclimatized for one week at 22 ± 3 °C with proper regulation of a 12/12 h light/dark cycle and fed normal chow food purchased from Gold Coin Feed mills (M) Sdn Bhd, Selangor, Malaysia. After one week, the rats were divided into five groups: control group (GC), negative control (NC), positive control with 20 mg/kg simvastatin (PC), treatment 1 with 8.6 mg/100g LC extract (T1) and treatment 2 with 25.7 mg/100g LC extract (T2).  Hypercholesterolemia was induced and treatment was administered for 8 weeks. Bodyweight were recorded every week. In this study female rats was used based on the previous study done by Wang et al. (2019). In his previous study, female rats showing prominent increase in lipid profile compare to male rats.

Plasma biochemistry analysis

Prior to blood collection, the rats were fasted for 12 hours. They were euthanized, and 5 ml blood was collected in a plain tube to obtain the serum. Serum was collected to analyze the lipid, liver, and kidney profiles. The plasma biochemistry was analyzed using an Alere Cholestech LDX Analyzer at Pantai Hospital, Petaling Jaya Malaysia.

Organ weight and histological analysis

Organ weights, such as liver, kidney, spleen, retroperitoneal white adipose tissue (RpWAT), and visceral fat, were recorded and fixed using 10% natural buffered formalin (NBF). Tissue processing was performed for all liver, kidney and fat samples. Sectioning of the block tissue was maintained at a thickness of 6-8 µm. The sections were stained with hematoxylin and eosin (H and E) staining protocol. The well-stained slides were mounted and observed under a microscope for further verification (Galay et al., 2014).

Statistical analysis

All data are presented as the mean ± Standard Error Mean (SEM) of three independent experiments performed in triplicates. One-way analysis of variance (ANOVA) was performed for several comparisons. All analyses were conducted using IBM SPSS Statistics for windows, version 22.0 (IBM Corp., Armonk, N.Y., USA). A P-value < than 0.05 (P <0.05) was considered statistically significant.

RESULTS

Changes of body weight of rats from week 1 through to week 8

At week 8, the body weight of rats in the high cholesterol diet (HCD) group was significantly (P <0.05) higher than that of rats in the normal group. The rats treated with L. coffee pulp yoghurt at a dosage of 8.6 mg/100 g for low dose and high dose at 25.7 mg/100 g showed a significant (P <0.05) decrease by 16% and 19%, respectively, compared to the HCD group. Nevertheless, HCD with simvastatin (drug) also decreased body weight by 18% and 6%, respectively, compared to the HCD group.

Figure 1. Effect of LC pulp yogurt on weekly body weight.

Data are expressed as mean ± SEM and were analyzed by one-way ANOVA, followed by post hoc LSD. Significant level set at P <0.05.

Abbreviation: GC, control group; NC, Negative control; PC, rat treated with 20mg/100g simvastatin drug; T1, rats supplemented with L. coffee pulp yoghurt at 8.6 mg/ 100g; T2, rat supplemented with L. coffee pulp yoghurt at 25.7 mg/ 100g.

Effect of LC pulp yoghurt on weight

Table 1 indicates the organ weight of rats.  Table shows that the liver weight of NC group is higher compared to other groups, however treatment group 2 shows significantly less weight compared NC group.  This may be due to possible treatment-related effects on liver function and metabolism.  The weights of the spleen, heart, and retroperitoneal white adipose tissue (RpWat) however, seem to be constant among the various therapy groups, with only slight variations noted.  For the kidney weight, GC group show slightly higher weight compared other group and T2 show significant reduction (P <0.05) compared NC group. There were differences in the relative weights of visceral fat between the treatment groups. It seems that visceral fat had greater relative weights in some groups GC and NC group than in others. T1 and T2 groups significant (P <0.05) reduction.  This may indicate possible therapeutic effects on the distribution and metabolism of adipose tissue.

Table 1. Effect of LC pulp yoghurt on weight of liver, spleen, kidney and heart.

Note: Data are expressed as mean ± SEM and were analyzed by one-way ANOVA, followed by post hoc LSD. Significant level set at P <0.05. Different superscript letters a, b. c denotes significant differences at P <0.05

Abbreviation: GC, control group; NC, Negative control; PC, rat treated with 20mg/100g simvastatin drug; T1, rats supplemented with L. coffee pulp yoghurt at 8.6 mg/ 100g; T2, rat supplemented with L. coffee pulp yoghurt at 25.7 mg/ 100g.

Effect of L. coffee pulp yoghurt on lipid profile

The total cholesterol (TC) levels in NC group show significantly (P <0.05) higher compared to other groups. PC and T2 group show lower level of TC compared to GC and NC as well. The level of high-density lipoprotein (HDL) showed that T1 is higher compared to other groups of rats. Similar tendencies, with G2 and G5 showing significantly higher levels than the other groups. No significant changes among all the group for HDL, however for low density lipoprotein (LDL) it shows NC has significantly (P <0.05) higher compared to other groups of rat. Triglyceride (TG) level in NC group is significantly higher (P <0.05) compared to other groups.

Figure 2. Effect of L. coffee pulp yoghurt on lipid profile.

Data are expressed as mean ± SEM and were analyzed by one-way ANOVA, followed by post hoc LSD. Significant level set at P <0.05. Different superscript letters a, b. c denotes significant differences at P <0.05.

Abbreviation: GC, control group; NC, Negative control; PC, rat treated with 20mg/100g simvastatin drug; T1, rats supplemented with L. coffee pulp yoghurt at 8.6 mg/100g; T2, rat supplemented with L. coffee pulp yoghurt at 25.7 mg/100g. TC, Total cholesterol: TG, Triglyceride; LDL, Low-density lipoprotein; HDL, High-density-lipoprotein.

Effect of L. coffee pulp yoghurt on renal profile

For renal profile, sodium level shows relatively minor variation across all the group between 136.33 mmol/L and 137.67 mmol/L. These fluctuations are within the normal physiological range and may not indicate significant alterations in sodium homeostasis due to treatment. The potassium levels in NC were the lowest (7.30 mmol/L), while those in T1 were the highest values (8.03 mmol/L). The potassium concentration in the blood plasma fell within the normal range, indicating that the treatment had little effect on the potassium levels. Minor variations in the groups' chloride levels are also evident; the values range from 100.37 mmol/L to 103.50 mmol/L. Because these deviations are within the expected range, there may not have been any notable changes in chloride homeostasis. The urea levels in NC were the lowest at 3.43 mmol/L compared to other groups. This varying amount of urea may indicate differing effects of treatment on urea metabolism, as urea levels reflect renal function and metabolic activity. Group T2 had the greatest creatinine level (36.00 mmol/L), compared to other groups, however this only shows minor variation among the group. Since creatinine levels are a good indicator of renal function, discrepancies in creatinine levels between the treatment groups may be due to variations in renal function.

Table 2. Effect of L. coffee pulp yoghurt on renal profile.

Note: Data are expressed as mean ± SEM and were analyzed by one-way ANOVA, followed by post hoc LSD. Significant level set at P <0.05. Different superscript letters a, b. c denotes significant differences at P <0.05.

Abbreviation: GC, control group; NC, Negative control; PC, rat treated with 20mg/100g simvastatin drug; T1, rats supplemented with L. coffee pulp yoghurt at 8.6 mg/ 100g; T2, rat supplemented with L. coffee pulp yoghurt at 25.7 mg/ 100g.

Effect of L. coffee pulp yoghurt on liver profile

Table 3 shows the biochemical parameters of the liver profile for total protein (TP), albumin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Total protein from GC was significantly (P <0.05) higher than that from the other groups. However, the TP levels in the other groups showed no significant differences among themselves. The albumin level in GC was significantly higher than other groups and show minor variation only with any significant changes. ALP levels in GC show lover among other groups and NC show higher level of ALP.  The PC and T1 group had lower ALP levels. AST levels in T1 were significantly higher than those in the other groups, with GC and NC levels.  The levels of ALT in T2 were also significantly higher than those in the other groups. However, GC, NC, PC and T1 show variation in ALT level.

Table 3. Effect of L. coffee pulp yoghurt on renal profile.

Note: Data are expressed as mean ± SEM and were analyzed by one-way ANOVA, followed by post hoc LSD. Significant level set at P <0.05. Different superscript letters a, b. c denotes significant differences at P <0.05.

Abbreviation: GC, control group; NC, Negative control; PC, rat treated with 20mg/100g simvastatin drug; T1, rats supplemented with L. coffee pulp yoghurt at 8.6 mg/ 100g; T2, rat supplemented with L. coffee pulp yoghurt at 25.7 mg/ 100g. TP, total protein; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Effect of L. coffee pulp yoghurt on histology changes

The histological analysis was performed on visceral fat (VF), liver, and kidneys. Photomicrographs are shown in Figure 3. Based on the histology of visceral fat, GC, PC, T1, and T2 showed normal adipocyte size compared with histological sections of visceral fat from NC. NC groups showed hypertrophy of visceral fat cell adipocytes. Liver histology of GC, PC, T1, and T2 showed normal strands of hepatocytes (H), sinusoids, and central veins. This showed that 0% of the hepatocytes were affected. The liver grading score was 0 because of the absence of lobular inflammation, steatosis cells, or ballooning cells. For NC liver cells, ballooning cells (BC) were present, with no steatosis or lobular inflammation. A liver grading score of 1 with few ballooning cells was present. The histology of kidney cells shows no changes among all the groups.

Figure 3. Histology of visceral fat, liver and kidney of all groups.

Histology of visceral fat (VF), liver and kidney of rats. Panel (A) represents the visceral fat histology, (B) represent the liver histology, and (C) represent the renal histology. The arrow in the picture shows the changes in the histology of cells.

Abbreviation: GC, control group; NC, Negative control; PC, rat treated with 20mg/100g simvastatin drug; T1, rats supplemented with L. coffee pulp yoghurt at 8.6 mg/ 100g; T2, rat supplemented with L. coffee pulp yoghurt at 25.7 mg/ 100g. a, Adipocyte; H, hepatocyte; BC, ballooning cell; G, glomerulus: magnification bar: 50µm.

DISCUSSION

Coffee has a multifaceted impact on human health, and the previous studies have demonstrated its potential advantages and drawbacks. Coffee consists of biologically active compounds, including chlorogenic acids, which have been linked to health benefits, such as a decreased risk of type 2 diabetes mellitus (T2DM), cardiovascular diseases (CVD), and specific types of cancer (van Dam and Feskens 2002; Cornelis, 2019). However, excessive coffee consumption can lead to negative health effects including impaired glucose tolerance, reduced insulin sensitivity, and adverse effects on sleep quality (van Dam and Feskens, 2002; Poole, 2017). Notably, the preparation method of coffee and the presence of compounds, such as diterpenes, can influence its health effects, particularly in relation to blood cholesterol levels (Cornelis, 2019). Although coffee peel has not been explicitly mentioned, the focus remains on the beverage and its components. The significance of coffee in human health appears to be considerable, with the recommendation that coffee consumption should be within an optimal range to maximize benefits and minimize risks (Poole et al., 2017).

Based on the current study using coffee peel (husk), the results showed changes in body and organ weights. It shows that coffee peel is also able to reduce body weight and organ weight as well. Previous research has indicated that coffee and its components may lead to a reduction in body weight in mice through various pathways. Shimoda et al. (2006) showed that green coffee bean extract (GCBE), which is rich in chlorogenic acid and caffeine, can decrease visceral fat content and body weight in mice. This suggests that GCBE may inhibit fat absorption and activate fat metabolism in the liver, with chlorogenic acid being partly responsible for these effects (Shimoda et al., 2006). Current studies also show the coffee peel also similar metabolism which can be able to reduce the fat absorption in the body. Muhammad et al. (2019) supported the idea that coffee intake is negatively correlated with adiposity, and this relationship may be influenced by genetic factors, such as the UCP gene variant (Muhammad et al., 2019). Icken et al. (2015) found that weight loss maintainers consumed more caffeinated beverages, indicating a potential role for caffeine in weight loss maintenance (Icken et al., 2015). Mangubat et al. (2011) reported that nicotine, a compound also found in coffee, reduced body weight gain and abdominal fat in mice, particularly when consuming a high-fat diet (Mangubat et al, 2011). Additionally, the effect of coffee on weight loss has not been thoroughly investigated in humans (Icken et al., 2015). In conclusion, Liberica coffee peel and its components, particularly caffeine and chlorogenic acid, may contribute to weight reduction by inhibiting fat absorption and enhancing fat metabolism in mice.

According to Cowan et al. (2014), high-fat-fed rats, which serve as a model for hypercholesterolemia, display a reduction in body weight, adiposity, liver triglycerides, and energy intake when they consume coffee (Cowan et al., 2014). This suggests that coffee may decrease calorie intake or alter metabolism, leading to a decline in these parameters. However, it is crucial to note that this study also found that coffee peel consumption resulted in reduction of lipid profile. On the other hand, Lebeau et al. (2022) did not directly address hypercholesterolemia, but examined the effects of a high-calorie diet on tumour-bearing rats, indicating that energy intake is regulated in these animals, suggesting that factors other than coffee consumption may significantly influence calorie intake. Moreover, they emphasized the importance of diet manipulation in inducing hypercholesterolemia in animal models but did not specifically address the impact of coffee on calorie intake (Esegbue et al., 2017). Current studies also reveal that LC yoghurt causes changes in the lipid profile, which are correlated with body weight and organ weight. The reduction in cholesterol levels was particularly pronounced in rats that consumed a higher amount of LC pulp yoghurt. The reason for this marked decrease in cholesterol levels, especially in T2, was the high caffeine content in the yoghurt dose administered to T2. As previously discussed in the caffeine has a similar effect or property as the statin or simvastatin drugs used in this study. Consuming LC pulp yoghurt daily caused caffeine in the yoghurt to block PCK9 expression. Inhibition of PCK9 expression prevented SREBP activation in rat hepatocytes. Since SREBP was not activated, there was no cholesterol synthesis, resulting in more LDL-c being absorbed into the liver due to the lack of cholesterol production. Additionally, the absence of cholesterol causes the liver to produce more HDL cholesterol, which is responsible for transporting cholesterol from the blood into the liver to produce essential enzyme bile, automatically reducing cholesterol levels in the blood. This is also because caffeine blocks PCK9 expression in LDL-r, allowing LDL-r to remain longer and accepting more LDL into the liver for excretion (Cowan et al., 2014; Lebeau et al., 2022; Esegbue, et al., 2016).

Furthermore, the weights of the livers for T1 and T2 showed a reduction compared to the other treatment groups, such as PC. According to previous studies, organ weight is a sensitive indicator of drug toxicity (Piao et al., 2013). A significant increase or decrease in organ weight relative to that in the normal group indicated drug toxicity during treatment. However, T1 and T2 did not exhibit a significant increase in liver weight compared with the other groups. In a similar study, Mohamed (2013) observed that yoghurt with plant extracts had therapeutic effects and decreased liver weight, and an increase in liver weight was closely related to liver lipid increase. This suggests that T1 and T1 had lower liver weights because the treatment used plant LC pulp extract and caused less stress on the liver compared to drug or PC group. Although drugs or PC can reduce cholesterol levels, they are not a good long-term treatment, as they cause liver stress and may result in liver problems. This is supported by histological findings. The liver histology of NC showed the presence of ballooning cells compared with the other groups. Based on these results, it can be concluded that caffeine has a significant impact on cholesterol regulation within the body. Specifically, consuming a considerable amount of caffeine through yoghurt resulted in the lowest lipid profile levels, which included a reduction in cholesterol levels, body weight, and organ weight (Mahmoud et al., 2013; Gavrieli et al., 2013; Stevens, L.M et al, 2021).

CONCLUSION

In this study, we investigated the usage of L. coffee pulp yoghurt to reduce hypercholesterolemia in rats. Although statins drug is available to reduce cholesterol levels in the blood, they are only a temporary solution and can cause adverse effects such as liver failure. Therefore, alternative methods to reduce cholesterol levels have been explored, specifically the consumption of coffee or caffeine, which has recently been shown to reduce cholesterol levels in the body. This highlights the significant potential benefits of L. coffee pulp (husk of used coffee bean) as an alternative method for reducing high cholesterol levels in addition to consuming drugs. However, this study has limitations, primarily due to its reliance on an animal model, which many not accurately reflect human physiological responses. To corroborate these findings and elucidate the mechanism underlying the cholesterol effect, further study on molecular and human should prioritize. 

ACKNOWLEDGEMENTS

The authors thank the Management and Science University and Universiti Puta Malaysia for support this project.

AUTHOR CONTRIBUTIONS

Nor Elianis Norkhairani and Azrina Zainal Abidin assisted in conducting the experiments, performed the statistical analysis and data visualization. Santhra Segaran Balan wrote the manuscript. Hasnah Bahari and Fezah Othman designed and conducted all the experiments. All authors have read and approved of the final manuscript.

CONFLICT OF INTEREST

The authors declare that they hold no competing interests.

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