Open Veterinary Journal, (2026), Vol. 16(7): 4558-4565

Research Article

10.5455/OVJ.2026.v16.i7.39

Effect of betel leaf water extract in drinking water on pathogenic bacteria and blood lipid profile of broilers

Desak Putu Mas Ari Candrawati^*, I Gusti Nyoman Gde Bidura, Ni Wayan Siti and Eny Puspani

Faculty of Animal Husbandry, Udayana University, Badung, Indonesia

*Corresponding Author: Desak Putu Mas Ari Candrawati. Faculty of Animal Husbandry, Udayana University, Badung, Indonesia. Email: dsk_candrawati [at] unud.ac.id

Submitted: 02/03/2026 Revised: 06/06/2026 Accepted: 19/06/2026 Published: 17/07/2026

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Abstract

Background: Broilers have certain disadvantages, such as high levels of fat and cholesterol in their meat and susceptibility to disease infection. Furthermore, the ban on the use of Antibiotic growth promoters, which can cause side effects, presents a challenge. Safe natural feed additives are sought, one of which uses betel leaf water extract (BLWE).

Aim: This study aimed to evaluate the effect of BLWE administration via drinking water on pathogenic bacterial populations and blood lipid profiles in broilers.

Methods: A completely randomized design was used with 4 treatments, 5 replications, and each unit consisted of 6-day-old chicks. Treatments: A (control group, without BLWE), B (drinking water with 2% BLWE), C (drinking water with 4% BLWE), and D (drinking water with 6% BLWE). Blood lipid profiles and pathogenic bacteria were observed in the broiler intestine.

Results: Triglycerides and low-density lipoprotein (LDL) significantly decreased (p < 0.05) with BLWE administration, up to 6%. Meanwhile, high-density lipoprotein significantly increased (p < 0.05) at the 6% BLWE level compared with the control treatment (A). The total cholesterol significantly (p < 0.05) decreased with 6% BLWE administration. Total pathogenic Escherichia coli bacteria decreased significantly (p < 0.05) with 6% BLWE administration, and Coliform bacteria decreased significantly (p < 0.05) at BLWE levels of 4% and 6%.

Conclusion: This study indicates that administering BLWE at up to 6% can improve blood lipid profiles and reduce pathogenic E. coli and Coliform bacteria.

Keywords: Broiler performance, Gut health, Natural feed additives, Piper betle, Phytogenic feed additives.

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Introduction

Optimal broiler performance can be achieved through good feed quality and maintenance management. However, digestive health challenges, such as susceptibility to infections caused by pathogenic bacteria such as Escherichia coli, can reduce feed efficiency and inhibit broiler growth (Candrawati et al., 2024). Kulnanan et al. (2021) reported that E. coli is a pathogenic bacterium that can cause economic losses in poultry worldwide. However, high levels of fat and cholesterol in broiler meat can reduce meat quality and potentially harm consumers. Attia et al. (2017) stated that broilers have a disadvantage in that their meat has high fat and cholesterol content. In addition, Regar et al. (2019) found that broiler meat contains 200 mg/dl of cholesterol.

Antibiotic growth promoters (AGP) were banned in the European Union in 2006 under Regulation (EC) No. 1831/2003, and in Indonesia under Law No. 18/2009 in conjunction with Law No. 41/2014 and Minister of Agriculture Regulation No. 14/2017, owing to concerns over antimicrobial resistance and residues in animal products. AGP have traditionally been used to suppress pathogenic bacteria such as E. coli and Coliform; however, their use has been banned due to their adverse effects. Mir et al. (2017) stated that the use of antibiotics in feed can cause antibiotic residues in livestock products and increase the resistance of pathogenic bacteria to antibiotics. The ban on the use of AGP demands innovation in the use of natural feed additives that are harmless to livestock and function not only as antibacterials but also to improve the blood lipid profiles of broilers.

One subject of interest for this study is betel leaf water extract (BLWE). The mechanism of action of betel leaf decoction is the same as that of commercial antibiotics (Hermanto et al., 2023). Several phytochemical compounds and nutritional components in betel leaves are known as bioactive compounds with pharmacological properties, including antimicrobial, anti-inflammatory, antimutagenic, and antioxidant effects (Gupta et al., 2023). The essential oil and flavonoid content in betel leaves can be used as antibiotics and antioxidants, and saponins can function as immunostimulants to increase immunity (Rokhmana et al., 2013). Furthermore, Taukoorah et al. (2016) stated that essential oils from betel leaves have significant antimicrobial activity against a broad spectrum of microorganisms, including E. coli, P. aeruginosa, S. aureus, and Acinetobacter.

Antibacterial and antioxidant compounds in betel leaves can naturally lower blood fat levels. Betel leaves contain flavonoids, which are natural phenolic compounds (Singh et al., 2023; Singh et al., 2024). Flavonoids can lower cholesterol levels by increasing bile acid excretion, thereby reducing fat deposition in blood vessels (Carvajal-Zarrabal et al., 2005). Moreover, betel leaves can lower cholesterol levels by inhibiting cholesterol biosynthesis in the liver and reducing intestinal fat absorption (Gramza and Korczak, 2005). The oral administration of betel leaves can reduce total cholesterol levels in rats (Thirumalai et al., 2014). In addition, betel leaf meal at 1% in feed can reduce cholesterol levels, and administration at 0.7% provided the lowest cholesterol levels in broiler meat (Hardiyansya et al., 2017).

Although the use of betel leaf meal in feed has been explored, research on the efficacy of its water extract, particularly when administered via drinking water, on the combined outcomes of gut health and lipid metabolism in broilers is limited. A water-based extract was selected in preference to dietary betel leaf meal for several practical and physiological reasons. First, aqueous extraction is simple, inexpensive, and avoids the use of organic solvents, yielding a safe product for direct livestock consumption. Second, extraction concentrates the water-soluble bioactive compounds (such as flavonoids, tannins, and phenols) into a more readily available form than intact leaf meal, in which these compounds remain bound within the plant matrix. Third, administration through drinking water offers distinct advantages over feed incorporation: it ensures more uniform intake, allows rapid dosage adjustment without reformulating the feed, and maintains delivery of the additive even when feed consumption declines, as commonly occurs during heat or disease challenge. Therefore, the use of BLWE in drinking water is expected to be a practical strategy to support digestive health, suppress pathogenic bacteria, and improve blood lipid profiles, thereby supporting healthier, lower cholesterol, and safer broiler meat consumption. Therefore, this study was conducted to evaluate the effect of BLWE on pathogenic bacterial populations and blood lipid profiles in broilers. It was hypothesized that the administration of BLWE via drinking water would reduce intestinal pathogenic bacterial populations (Escherichia coli and Coliform) and improve blood lipid profiles [lower total cholesterol, triglycerides, and low-density lipoprotei (LDL), and higher high-density lipoprotein (HDL)] in broilers in a concentration-dependent manner, with the most pronounced effects expected at the highest inclusion level.

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Materials and Methods

Study design

The research was conducted for 5 weeks at the Sesetan Farm, Faculty of Animal Husbandry, Udayana University, Denpasar, Bali, Indonesia. Blood lipid profile analysis and observation of pathogenic bacteria were performed at the Animal Product Technology Laboratory, Faculty of Animal Husbandry, Udayana University, Denpasar, Bali, Indonesia. Battery colony cages equipped with feed and water troughs were used in this study. The materials used were 120 one-day-old chicks (DOC) with a homogenous body weight of 49.52 ± 2.46 g. The betel leaves used were fresh, green betel leaves. The 4 treatments were administered to the CP 511B ration from PT. Charoen Pokphand Indonesia Rations and drinking water were provided ad libitum.

Observations of blood lipid profiles included total cholesterol, triglycerides, HDL, and LDL, while observations of pathogenic bacteria included E. coli and Coliform. Eosine methylene blue agar medium was used to identify E. coli and Coliforms. Distilled water (Aquades), RI reagent (Monoreagent), and a cholesterol standard of 200 mg/dl were used for the observation of blood lipid profile. The equipment used included analytical scales, syringes, centrifuges, micropipettes, test tubes, spectrophotometers, Petri dishes, colony counters, Bunsen burners, blue tips, and yellow tips.

The study used a completely randomized design (CRD) using 4 treatments and 5 replications, with each treatment unit consisting of 6 DOCs. Treatments given were as follows: A (control group, without BLWE), B (drinking water with 2% BLWE), C (drinking water with 4% BLWE), and D (drinking water with 6% BLWE). Blood lipid profiles and pathogenic bacteria were observed when the broilers were 35 days old.

Making and administering betel leaf water extract

Fresh, green betel leaves were used, which were then cleaned. The betel leaf to water ratio was 1:1 (w/v); the mixture was homogenized and then filtered. The obtained BLWE was ready for treatment administration. The manufacture of the BLWE is shown in Figure 1. The phytochemical composition of the resulting extract was determined at the Integrated Service Laboratory, Faculty of Agricultural Technology, Udayana University (Table 1). The concentrations of the major bioactive compounds and antioxidant activity of the BLWE used in this study are shown in Table 1. The extract contained flavonoids (74.34 mg/100 g), tannins (66.80 mg/100 g), and phenols (40.85 mg/100 g), with an IC₅₀ of 2712.43 mg/100 g, confirming the presence of the bioactive compounds discussed in relation to the lipid-lowering and antibacterial effects observed in this study.

Fig. 1. BLWE manufacturing method.

Table 1. Phytochemical content of betel leaf water extract.

The administration of 2% BLWE (B) consisted of 20-ml BLWE in 980 ml of drinking water, 4% BLWE (C) was 40 ml in 960 ml of water, and 6% BLWE (D) was 60 ml in 940 ml of water.

Observation of the blood lipid profile

Blood lipid profile was observed using the method described by Shintawati et al. (2011). Blood sampling was performed on the broiler’s brachial vein (wing section). Blood was collected in a tube, incubated for 15 minutes, and then centrifuged for 20 minutes at approximately 3,000 rpm. Finally, the serum was ready for cholesterol analysis. The absorbance was measured at 500 nm using a spectrophotometer calibrated with distilled water. Total cholesterol, triglycerides, and high-density lipoprotein levels were calculated directly from relative absorbance values relative to the standard. Meanwhile, LDL levels were determined indirectly using the Friedewald formula: LDL=Total Cholesterol—HDL—(Triglycerides/5) (Friedewald et al., 1972).

Observation of pathogenic bacteria

The pathogenic bacteria E. coli and Coliform were observed using the standard plate count method on eosin methylene blue agar medium. Incubated samples were observed after 24 hours at 37°C in the incubator. Colony counting was performed using a Quebec colony counter (Devenish et al., 1984).

Statistical analysis

Data were analyzed using the Statistical Package for the Social Sciences software version 25. Data were arranged according to a CRD with 4 treatments and 5 replicates, with the replicate (cage) serving as the experimental unit. Before analysis, the assumptions of normality and homogeneity of variance were tested using the Shapiro–Wilk and Levene’s tests, respectively. All variables satisfied these assumptions (p > 0.05) and were, therefore, analyzed using one-way analysis of variance, where a significant treatment effect was detected (p < 0.05); means were separated using Tukey’s honestly significant difference test (Boulgouris et al., 2009).

Ethical approval

The broilers used in this study were maintained in accordance with animal care standards, and the Animal Ethics Commission of the Faculty of Veterinary Medicine, Udayana University, Indonesia, approved the study (approval number B/156/UN 14.2.9/PT.01.04/2025; 21 September 2025).

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Results

Blood lipid profile

Total cholesterol

Total cholesterol levels in broilers receiving 2% (B) and 4% (C) BLWE in drinking water were 0.16% and 2.16% lower than the control treatment (A), respectively; however, these reductions were not statistically significant (p > 0.05). On the contrary, administration of 6% BLWE (D) resulted in a 10.59% reduction compared with the control, which was statistically significant (p < 0.05) (Table 2).

Table 2. Effect of betel leaf water extract in drinking water on the blood lipid profile of 5-week-old broilers.

Triglycerides

Triglyceride levels in the control group (A) were 262.14 mg/dl. Broilers receiving 2%, 4%, and 6% BLWE showed reductions of 7.12%, 7.63%, and 11.44%, respectively, compared to the control. All BLWE treatments significantly reduced triglycerides (TG) levels (p < 0.05) relative to the control (Table 2). A progressive decrease in TG levels was observed with increasing BLWE concentration (Fig. 2).

Fig. 2. Effect of betel leaf water extract (BLWE) in drinking water on the blood lipid profile of 5-week-old broilers. A=control (without BLWE); B=2% BLWE; C=4% BLWE; D=6% BLWE.

High-density lipoprotein

HDL levels ranged from 40.05 to 58.04 mg/dl. Administration of 2% BLWE (B) resulted in HDL levels 10.86% lower than the control (A); however, this difference was not statistically significant. Treatment with 4% BLWE (C) increased HDL levels by 14.49% compared with the control, although not significantly. The 6% BLWE treatment (D) increased HDL levels by 29.18% relative to the control, and this increase was statistically significant (p < 0.05) (Table 2).

Low-density lipoprotein

LDL levels in the control group (A) were 81.27 mg/dl. Administration of 2%, 4%, and 6% BLWE reduced LDL levels by 15.68%, 15.98%, and 25.75%, respectively. All BLWE treatments significantly lowered LDL levels (p < 0.05) compared with the control (Table 2).

Intestinal pathogenic bacteria

Escherichia coli

The E. coli count in the control treatment (A) was 2.0 × 10⁶ CFU/g. Treatments with 2% (B) and 4% (C) BLWE resulted in lower counts than the control; however, these reductions were not statistically significant (p > 0.05). The 6% BLWE treatment (D) significantly reduced E. coli counts compared with the control (p < 0.05) (Table 3).

Table 3. Effect of betel leaf eater extract in drinking water on total pathogenic bacteria in the small intestine of 5-week-old broilers.

Coliform

The Coliform count in the control treatment (A) was 1.85 × 10⁸ CFU/g. Treatment with 2% BLWE (B) showed a nonsignificant reduction compared with the control (p > 0.05). However, 4% (C) and 6% (D) BLWE significantly reduced Coliform counts compared with the control (p < 0.05) (Table 3).

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Discussion

Effect of BLWE on lipid metabolism

The significant reduction in total cholesterol at 6% BLWE suggests a dose-dependent hypolipidemic effect. Higher BLWE concentrations likely increase the availability of bioactive compounds, such as flavonoids, tannins, polyphenols, essential oils, vitamin C, and saponins, which are known to influence lipid metabolism.

Flavonoids inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key enzyme in cholesterol biosynthesis (Mazroatul et al., 2016; Klau and Mufaddilah, 2023). Inhibition of this enzyme reduces the production of endogenous cholesterol. In addition, flavonoids may suppress ACAT activity, thereby reducing cholesterol esterification and intestinal cholesterol absorption (Hai and Smith, 2021).

Essential oils present in betel leaves may stimulate bile secretion and pancreatic lipase activity, thereby enhancing lipid digestion and excretion (Elbaz et al., 2022). Increased bile acid excretion forces the liver to convert more cholesterol into new bile acids, further reducing circulating cholesterol levels (Patra et al., 2022; Sakinah et al., 2020).

The observed decrease in TG levels may be attributed to flavonoid-induced enhancement of lipoprotein lipase activity, which promotes TG hydrolysis (Hayudanti, 2018). Furthermore, antioxidant compounds such as flavonoids, tannins, and polyphenols may reduce oxidative stress and inhibit hepatic TG synthesis.

The significant increase in HDL levels at 6% BLWE indicates improved reverse cholesterol transport. HDL facilitates cholesterol transport from peripheral tissues to the liver, thereby reducing the risk of atherosclerosis (Vergeer et al., 2010). Flavonoids may enhance lecithin-cholesterol acyltransferase activity, promoting free cholesterol conversion into esterified cholesterol and contributing to HDL formation (Faadlilah and Ardiaria, 2017).

The reduction in LDL levels across all BLWE treatments suggests that lipid transport is modulated. Flavonoids may exert statin-like effects by inhibiting HMG-CoA reductase (Oktavelia and Kusuma, 2022; Zaelani et al., 2024). In addition, saponins can form insoluble complexes with cholesterol, thereby reducing its absorption and circulating LDL levels (Zaelani et al., 2024).

Effect of BLWE on intestinal pathogenic bacteria

The significant reduction of E. coli and Coliform populations at higher BLWE concentrations demonstrates the antimicrobial potential of BLWE. Although these bacteria are part of the normal intestinal flora, excessive proliferation may lead to pathogenic conditions.

Betel leaves contain approximately 4.2% essential oils (Dewi et al., 2023), as well as phenylpropanoids, tannins, and flavonoids, which exhibit strong antimicrobial activity (Neelam et al., 2020; Neumann et al., 2022). Water and ethanol extracts of green betel leaves have demonstrated antibacterial effects against E. coli (Tilarso et al., 2022).

Phenolic-rich essential oils can disrupt microbial cell walls, cause protein denaturation, increase membrane permeability, and ultimately lead to cell damage (Hernandi et al., 2019). Chavicol, found in betel leaves, reportedly exhibits bactericidal activity 5 times stronger than phenol. These mechanisms likely explain the dose-dependent reduction in intestinal pathogenic bacteria observed in broilers receiving 4% and 6% BLWE.

Study limitations

This study has several limitations that should be acknowledged. First, we did not measure growth performance parameters such as body weight gain, feed conversion ratio (FCR), and mortality rate; their inclusion would strengthen the practical relevance of the findings for commercial broiler production. Second, we did not record feed and water intake, which is particularly relevant because high concentrations of BLWE may reduce water palatability due to bitterness, potentially affecting consumption and the interpretation of physiological responses. Third, the mechanistic explanations proposed in this study (for example, inhibition of HMG-CoA reductase and enhanced bile acid excretion) are based on the existing literature and were not directly verified through measurement of liver enzyme activity, oxidative stress markers, or gene expression. Fourth, the experiment used a negative control only, without a positive (antibiotic) control group, which limits the ability to directly compare the extract’s efficacy against commercial AGP. Fifth, the small number of birds per replicate may limit the statistical power for detecting smaller treatment effects, as reflected in the nonsignificant differences observed for some parameters at lower inclusion levels. Finally, the water-based extraction method, while practical and safe for direct administration, may yield lower concentrations of lipophilic bioactive compounds compared with solvent-based extraction, and the stability of volatile compounds in drinking water was not quantified. Future studies should address these limitations to confirm and extend the present findings.

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Conclusion

The administration of BLWE in drinking water exerted concentration-dependent effects on broilers, with significant improvements in blood lipid profiles, and significant reductions in pathogenic E. coli and Coliform populations observed primarily at the 6% inclusion level. These findings indicate that BLWE delivered through drinking water may serve as a promising plant-based feed additive to support gut health and produce lower cholesterol, safer broiler meat, offering a potential natural alternative to synthetic additives in poultry production. These results reinforce the role of betel leaf bioactive compounds (flavonoids, tannins, and phenols) in modulating lipid metabolism and inhibiting pathogenic bacteria. However, these conclusions should be interpreted with appropriate caution because significant effects were largely confined to the highest concentration and were not accompanied by production performance or direct mechanistic data. Future research should incorporate growth performance indicators (body weight gain, FCR, and mortality), feed and water intake measurements, a positive antibiotic control group, standardized and quantified extract preparation, direct mechanistic markers (liver enzyme activity, oxidative stress markers, and gene expression), and molecular microbiological techniques to confirm efficacy and determine the optimal and economically viable BLWE inclusion level for commercial broiler production.

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Acknowledgments

None.

Conflict of interest

The author(s) declare(s) that there are no conflicts of interest.

Funding

This study received no specific grant.

Authors' contributions

DPMAC: Conceptualization, Methodology, Investigation, Data Curation, Formal Analysis, Writing–Original Draft, Visualization, Project Administration. IGNGB: Conceptualization; Methodology; Supervision; Validation; Writing–Review and Editing; Funding Acquisition. NWS: Investigation, Laboratory Analysis, Data Curation, Resources, Writing, Review, and Editing. EP: Formal analysis, validation, supervision, writing, review, and editing. All authors have read and approved the final version of the manuscript.

Data availability

The datasets generated and analyzed during the current study are available upon reasonable request from the corresponding author.

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