Abstract Butterfly pea (Clitoria ternatea L.) is widely used in Indonesia as a natural food and beverage colorant and in traditional medicine; its anthocyanins and flavonoids possess antioxidant and anti-inflammatory properties that may support gastrointestinal mucosal (for example in duodenum/colon) integrity, although its histological safety remains understudied. This study evaluated acute and subchronic toxicity of butterfly pea flower infusion on the histological structure of the duodenum and colon in male albino rats aged 8-12 weeks. Forty rats were orally administered the infusion in acute (2,500 and 5,000 mg/kg body weight for 14 days) and subchronic (250, 500, and 1,000 mg/kg body weight for 28 days) regimens. Duodenal and colonic tissues were processed using the paraffin method and stained with Hematoxylin-Eosin for structural and histomorphometric analysis, and with Periodic Acid Schiff-Alcian Blue for mucus characterization. Data were analyzed using independent t-tests, one-way ANOVA, Mann-Whitney, and Kruskal-Wallis tests (α = 0.05), with mucus evaluated descriptively. No significant changes observed in villus length, crypt depth, or tunica thickness in either organ. Goblet cell profile and area decreased after acute exposure but increased subchronic treatment. Histopathological examination revealed very mild leukocyte infiltration (<2.5%) in both organs and minimal villus fusion (<2%) in the duodenum. Duodenal mucus shifted to mixed type in acute high-dose groups but remained predominantly acidic under subchronic exposure, while colonic mucus remained acidic. These findings indicate that butterfly pea flower infusion did not induce significant histological alterations in the duodenum and colon under the tested experimental conditions, although dose regulation is recommended.

Keywords: Butterfly pea flower infusion, Colon, Duodenum, Histomorphometry, Histopathology, Rattus norvegicus

Funding: This research received funding from Hibah Rekognisi Tugas Akhir (RTA Grant), Universitas Gadjah Mada 2024 (Number: 4971/UN1.P1/PT.01.01/2024).

Citation:  Aeniah, S., Apriliyani, T., Pusparini, N.A.O., Retnoaji, B., and Nuriliani, A. 2026. Evaluation of acute and subchronic toxicity of butterfly pea (Clitoria ternatea L.) infusion on histological structure of duodenum and colon in Albino rats Rattus norvegicus (Berkenhout, 1769). Natural and Life Sciences Communications. 25(4): e2026085.

Graphical Abstract:

INTRODUCTION

Degenerative diseases constitute the leading cause of global mortality and continue to increase annually. The World Health Organization (WHO) (2023), reported that more than 70% of deaths worldwide are attributable to non-communicable diseases, such as cardiovascular disease, diabetes mellitus, hypertension, renal failure, and cancer, which are generally associated with age-related organ functional decline, unhealthy lifestyle, oxidative stress, and genetic factors (Hafsah et al., 2022; Zhou, 2022). In Indonesia, the prevalence of degenerative diseases is relatively high, with hypertension affecting 34.1% of adults and diabetes mellitus approximately 10.9% (Ministry of Health of the Republic of Indonesia, 2020). In addition, the prevalence of cancer has increased from 1.4 to 1.8%, stroke from 7 to 10.9%, and chronic kidney disease from 2 to 3.8% (P2P, 2023). These data indicate that degenerative diseases represent a serious public health problem and require sustained and comprehensive management.

Efforts to control degenerative diseases are generally focused on the management of risk factors, including smoking cessation, dietary improvement, increased physical activity, and limitation of alcohol consumption (WHO, 2016). In addition, conventional medical therapy using drugs such as antihypertensives, insulin, and statins remains the mainstay of treatment. However, long term use of these medications may lead to adverse effects and requires relatively high costs. This condition has encouraged the public to seek alternative treatments based on natural or traditional products, which are perceived to be safer, more accessible, and more sustainable (Balkrishna et al., 2024). The use of herbal plants is also considered to carry a lower risk of side effects compared with the continuous use of synthetic drugs (Inggi, 2023). Although herbal medicines are widely regarded as safe because of their natural origin, studies, and case reports have demonstrated that certain herbal products may induce adverse effects, including gastrointestinal disturbances, hepatotoxicity, nephrotoxicity, cardiotoxicity, and other toxic responses following prolonged or inappropriate use. Therefore, scientific toxicological evaluation is necessary to ensure the safety of herbal products, particularly those consumed orally and continuously (Jităreanu et al., 2022). 

As a megabiodiversity country, Indonesia possesses abundant floral resources, including traditional plants such as butterfly pea (Clitoria ternatea L.), which are cultivated and utilized by the community as natural colorants for foods and beverages as well as in traditional medicine, owing to their reported antidiabetic, antioxidant, anti-inflammatory, and antimicrobial activities (Jeyaraj et al., 2021; Aryal et al., 2022). The consumption of butterfly pea in the form of an infusion or aqueous decoction is the most common practice, either as an herbal tea, a natural food colorant, or a functional beverage for digestive health (Hasanah et al., 2023; Setiati, 2024). In line with the national program on the self sufficiency of medicinal raw materials based on natural resources, comprehensive scientific investigations on the safety aspects of widely consumed medicinal plants are required, including butterfly pea, for which toxicological data remain limited to date.

Previous studies by Pusparini et al. (2026) investigated the acute and subchronic toxicity of butterfly pea flower infusion on the liver and kidneys. The results demonstrated that administration of the infusion in rats induced very slight histological alterations in the liver, characterized by leukocyte infiltration, cellular degeneration, vacuolization, and hemorrhage, as well as very mild histological changes in the kidneys, indicated by leukocyte infiltration and tubular hemorrhage. In addition, acute toxicity testing using ethanol extracts of butterfly pea flowers has also been conducted; for instance, Hidayati et al. (2024) reported that oral administration of a 70% ethanol extract at doses exceeding 5,000 mg/kg body weight in albino rats did not produce toxic effects.

However, previous studies have primarily focused on systemic organs such as the liver and kidneys as major detoxification sites, whereas the gastrointestinal tract, which represents the primary site of exposure to orally administered butterfly pea infusion, has not yet been thoroughly investigated. The duodenum represents the initial site of contact following gastric emptying and is highly active in absorption, enzymatic activity, and pH regulation, making it particularly susceptible to chemical or osmotic disturbances. The colonic mucosa is exposed to residual compounds after transit through the gastrointestinal tract, where prolonged contact time, microbial interactions, and water reabsorption processes may enhance the local effects of potentially toxic substances. These physiological characteristics make both regions biologically plausible targets for detecting mucosal alterations induced by water infusion (Jensen et al., 2023). Therefore, the present study offers novelty by employing butterfly pea flower infusion that reflects common daily consumption practices and by extending previous toxicological assessments through a specific focus on the duodenum and colon to determine the presence of any direct toxic effects on the gastrointestinal tract. This study addresses a toxicological gap by evaluating the effects of aqueous butterfly pea infusion on intestinal mucosa, which remains underexplored in previous studies. Consequently, the findings of this study are expected to provide a more comprehensive evaluation of the safety of oral consumption of butterfly pea flower infusion and to support the evidence-based development of phytopharmaceuticals.

MATERIALS AND METHODS

Materials

The materials used in this study included fresh butterfly pea flowers (Clitoria ternatea L.) obtained from the Martani Butterfly Pea Garden, Prambanan, Yogyakarta, Indonesia. The authenticity of the plant material was confirmed through taxonomic identification at the Laboratory of Plant Systematics, Faculty of Biology, Universitas Gadjah Mada, under document number 0057/S.Tb./II/2024. A total of 40 male albino rats (Rattus norvegicus), aged 8-12 weeks with a body weight of approximately 200 g, were obtained from the Animal House of the Faculty of Biology, Universitas Gadjah Mada. Additional materials included reverse osmosis water (ad libitum), standard RatBio feed (PT Citra Ina Feedmill; 20 g per rat per day), ketamine-xylazine anesthetic solution, 10% neutral buffered formalin (NBF) (Merck) as a fixative, graded ethanol (Merck), paraffin (paraplast Surgipath Leica), xylene (Merck), toluene (Merck), and staining reagents for Hematoxylin-Eosin (HE) (Merck) and Periodic Acid-Schiff-Alcian Blue (PAS-AB) (Merck). The main equipment comprised experimental animal cages, an oral gavage set, surgical instruments, a rotary microtome (Microm GmbH HM 310), an oven (Memmert), a hot plate (Iwaki HPS-2002), and a Leica DM750 light microscope.

Preparation of butterfly pea flower infusion

Fresh butterfly pea flowers were washed with running water to remove adhering impurities and subsequently sun-dried for 1-2 days, followed by ovendrying at 50°C for 1 hour until a constant dry weight was obtained in two consecutive weighings. The dried butterfly pea flowers were ground into powder and extracted in distilled water to obtain infusion concentrations corresponding to the required experimental doses. For acute toxicity testing, 25 g and 50 g of powdered flowers were each extracted in 100 mL distilled water to obtain concentrations of 250 mg/mL (1:4, w/v) and 500 mg/mL (1:2, w/v), which corresponding to doses of 2,500 and 5,000 mg/kg body weight, respectively. For subchronic testing, a stock infusion was prepared by extracting 10 g of powdered flowers in 100 mL distilled water to obtain a concentration of 100 mg/mL (1:10, w/v), corresponding to a dose of 1,000 mg/kg body weight. Lower concentrations corresponding to doses of 500 mg/kg and 250 mg/kg body weight were obtained by diluting the stock infusion with distilled water at ratios of 1:1 and 1:3 (v/v), respectively. All mixtures were heated at 90°C for 15 min in accordance with the Indonesian Food and Drug Authority Guideline (2010), filtered using filter paper, and freshly prepared daily to ensure stability and prevent contamination.

Grouping of experimental albino rats

The experimental protocol was approved by the Ethics Committee of the Faculty of Veterinary Medicine, Universitas Gadjah Mada, under approval number 127/EC-FKH/Eks./2023.

Sample size calculation

The sample size was determined using the resource equation approach for exploratory animal studies (Arifin and Zahiruddin, 2017). For one-way ANOVA designs, an acceptable error degree of freedom (DF) ranges from 10 to 20, which corresponds to approximately five to seven animals per group depending on the number of treatment groups. Based on this calculation, five rats were included in each experimental group for both acute and subchronic toxicity studies.

Prior to treatment, the experimental animals were acclimatized for 7 days and housed in plastic cages measuring 40 x 50 x 30 cm³, covered with wire mesh (Dewi et al., 2017). Wood shavings were used as bedding and were replaced every three days. The experimental animals were divided into two main groups, namely acute toxicity and subchronic toxicity tests, in accordance with the guidelines issued by Indonesian Food and Drug Authority Guidelines (2022) and Organisation for Economic Co-operation and Development (2001), with stepwise dosing starting at 2,500 mg/kg body weight (BW). Each group consisted of five replicates and was observed for 14 days. In the initial stage, the acute control I group received distilled water, whereas the acute treatment I group was administered butterfly pea flower infusion at a dose of 2,500 mg/kg BW. If no mortality or only one animal death was observed at this dose level, the dose was increased to 5,000 mg/kg BW. This subsequent stage also comprised two groups with five replicates each, namely acute control II receiving distilled water and acute treatment II receiving butterfly pea flower infusion at a dose of 5,000 mg/kg BW, and both groups were observed for 14 days.

After completion of the acute toxicity test, the subchronic toxicity test was conducted for 28 days using four experimental groups, each consisting of five replicates. These groups comprised a control group (administered distilled water) and three treatment groups receiving graded doses of butterfly pea flower infusion, namely 250, 500, and 1,000 mg/kg BW. The infusion was administered orally by gavage at a volume of 2 mL per animal. Prior to treatment, the rats were fasted for 12 h with free access to drinking water. Toxic signs were intensively observed during the first 30 min following infusion administration.

Preparation of histological specimens

The rats were euthanized using a ketamine (100 mg/kg BW) and xylazine (10 mg/kg BW) mixture at a dose ratio of 10:1, administered intramuscularly at an injection volume of 0.1 mL per 100 g BW, followed by cervical dislocation. Histological preparations were processed using the paraffin method according to Cook (2003) with modifications. The duodenum and colon were collected, weighed, and fixed in 10% neutral buffered formalin (NBF) for 24 h. Dehydration was carried out using graded ethanol, and clearing was performed with toluene. The paraffin blocks were sectioned at a thickness of 5 µm and stained with Hematoxylin-Eosin (H&E) and Periodic Acid-Schiff-Alcian Blue (PAS-AB).

Histological structure observation 

In this study, each experimental group consisted of five animals, except for the acute toxicity group at 2,500 mg/kg BW, which consisted of four animals. One histological slide was prepared from each animal and each slide contained 6 sections obtained at intervals of every 25^th serial section (Merzel and Leblond, 1969). For each section, histological observations were conducted in three fields of view using a Leica DM750 light microscope. Histomorphometric measurements were performed using a 4x objective lens and a 10x ocular lens. In the duodenum, approximately 10-12 measurements (villi, crypts, etc.) per field of view were obtained, resulting in a total of approximately 180-216 measurements per animal. In the colon, approximately 3-6 measurements per field of view were obtained, resulting in a total of approximately 54-108 measurements per animal. The mean value from these measurements was used for statistical analysis. The analyzed parameters included histomorphometry, mucus characteristics, and the degree of tissue damage.

Histomorphometric measurements of the duodenum comprised villus length, crypt depth, thickness of the mucosa, submucosa, muscularis, and serosa, as well as the profile and area of goblet cells. In the colon, the observed parameters included the depth of the crypts of Lieberkühn, thickness of the mucosa, submucosa, muscularis, and serosa, as well as the profile and area of goblet cells. Quantitative histological data were obtained using ImageJ software (National Institutes of Health, USA; version 1.54g) (Setiawan et al., 2018). The histomorphometric measurements are presented in Figure 1.

Figure 1. Histomorphometric parameters of the duodenum and colon of albino rats. Duodenum (A) Colon (B). (VL = villus length, TMC = tunica mucosa, TSU = tunica submucosa, TMS = tunica muscularis, TSR = tunica serosa, CL = crypts of Lieberkühn, GC = goblet cells) (H&E staining, scale bar: 500 µm).

Mucus characteristics were analyzed based on the distribution of acidic mucus (blue), neutral mucus (magenta), and mixed mucus (bluish-purple) within the tissue structures. Histological damage was assessed using a scoring system for leukocyte infiltration and villus fusion, referring to Erben et al. (2014) with modifications.

Statistical analysis

Histomorphometric data from the acute study were analyzed using the independent samples t-test, whereas data from the subchronic study were analyzed using one-way ANOVA, with a significance level of P ≤ 0.05, followed by Duncan’s multiple range test when significant differences were detected. Histopathological scores of the duodenum and colon were analyzed using the Mann-Whitney U test for the acute treatment and the Kruskal-Wallis test for the subchronic treatment (P ≤ 0.05), followed by Dunn’s post hoc test when significant differences were observed. Mucus characteristics based on PAS-AB staining were analyzed descriptively and comparatively according to differences in the distribution of neutral, acidic, and mixed mucus among the treatment groups.

RESULTS

Butterfly pea flowers contain various phytochemical compounds with beneficial pharmacological activities; however, consumption at high doses or over prolonged periods may potentially induce toxic effects that are not always clinically detectable (Pusparini et al., 2026). Therefore, the present study was conducted to evaluate the acute and subchronic toxicity of butterfly pea flower infusion on the histological structures of the duodenum and colon of rats, in accordance with established guidelines and previous studies (Indonesian Food and Drug Authority Guidelines, 2022; Apriliyani, 2024; Pusparini, 2024).

Acute and subchronic administration of butterfly pea flower infusion does not induce histomorphometric alterations in the duodenum of albino rats

The results showed that acute administration of butterfly pea flower infusion at doses of 2,500 and 5,000 mg/kg BW. for 14 days did not cause significant differences (P > 0.05) in villus length compared with the control group. Likewise, crypt depth and the thickness of the mucosal, submucosal, muscularis, and serosal layers did not differ significantly among groups (P > 0.05) (Figure 2). These findings are consistent with the study by Putri et al. (2024), who reported that oral administration of butterfly pea flower ethanol extract at a dose of 90 mg/animal/day for 14 days did not alter duodenal morphometric parameters, including villus length, crypt depth, and mucosal thickness, in rats (P > 0.05).

However, a significant decrease (P ≤ 0.05) in the profile of goblet cells was observed at the dose of 2,500 mg/kg BW, with 57 cells/500 µm compared with 110 cells/500 µm in the control group. In addition, the goblet cell area was significantly reduced (P ≤ 0.05) in both treatment groups. At a dose of 2,500 mg/kg BW, the goblet cell area was 72.27 ± 5.135 µm², which was lower than that of the control group (138.87 ± 23.659 µm²), whereas at a dose of 5,000 mg/kg BW, the goblet cell area further decreased to 61.08 ± 5.518 µm² compared with the corresponding control value of 137.52 ± 20.717 µm² (Figure 2).

Furthermore, in the subchronic treatment at doses of 250, 500, and 1,000 mg/kg BW, butterfly pea flower infusion also did not produce significant differences in villus length, crypt depth, or the thickness of the mucosal, submucosal, muscularis, and serosal layers of the duodenum compared with the control group (P > 0.05). However, both the profile and area of goblet cells increased significantly (P ≤ 0.05). In the subchronic control group, the profile of goblet cells was 54 cells/500 µm with a mean area of 93.65 ± 2.819 µm². Administration of 250 mg/kg BW increased the profile of goblet cells to 68 cells/500 µm with an area of 119.05 ± 13.306 µm², 500 mg/kg BW increased them to 96 cells/500 µm with an area of 147.33 ± 15.898 µm², and 1,000 mg/kg BW increased them to 147 cells/500 µm with an area of 150.10 ± 32.547 µm² (Figure 2).

This increase reflects an adaptive mucosal mechanism through enhanced mucus production as a protective response to repeated exposure to bioactive compounds (Knoop and Newberry, 2018; Hall and Hall, 2020). The elevated profile of goblet cells observed in the subchronic treatment is consistent with the findings of Erhunmwunse and Odiase (2014), who reported that oral administration of aqueous extract of Hibiscus sabdariffa calyces at doses of 250, 500, and 1,000 mg/kg BW for 31 days significantly increased the profile of goblet cells and mucin secretion in the colonic mucosa compared with controls, which was interpreted as a protective and anti-inflammatory effect. Conversely, a decrease in goblet cell profile has been reported by Refai (2014), who showed that administration of chamomile extract at 100 mg/kg BW for 12 days induced deterioration of the intestinal histological structure in rats, characterized by villus shortening and disruption, disorganization of the epithelial layer, reduced goblet cell density, epithelial cell degeneration and necrosis, and increased infiltration of inflammatory cells, indicating a cytotoxic effect on the intestinal mucosa. Refai (2014) reported cytotoxic intestinal effects following chamomile extract administration, characterized by epithelial degeneration and necrosis, which are generally associated with irreversible cellular injury. In contrast, the histopathological changes observed in the present study were limited to mild leukocyte infiltration and alterations in goblet cell profile without evidence of epithelial necrosis or marked tissue destruction. Such mild mucosal alterations may represent an adaptive and potentially reversible response to repeated exposure, reflecting the capacity of intestinal tissue to maintain mucosal homeostasis under mild chemical stress conditions. Nevertheless, prolonged uncontrolled exposure may still exceed this adaptive capacity and contribute to more pronounced mucosal injury (Miller and Zachary, 2017). The histomorphometric measurements of the duodenum of albino rats following acute and subchronic toxicity treatments are presented in Figure 2.

Figure 2. Duodenal histomorphometry of albino rats following acute and subchronic administration of butterfly pea flower infusion. Acute treatment at a dose of 2,500 mg/kg BW (A), acute treatment at a dose of 5,000 mg/kg BW (B), subchronic treatment at doses of 250, 500, and 1,000 mg/kg BW (C). Histomorphometric data of the duodenum in the acute study were analyzed using the independent samples t-test, whereas those in the subchronic study were analyzed using one-way ANOVA. Data are presented as mean ± SD (standard deviation), acute control I and acute treatment at 2,500 mg/kg BW, n = 4; all other groups, n = 5. Different letters (a-c) above the histograms indicate significant differences among treatment groups (P ≤ 0.05).

Acute and subchronic toxicity of butterfly pea flower infusion induces very mild leukocyte infiltration in the histological structure of the duodenum

Histopathological evaluation of the duodenum was performed to detect structural alterations following butterfly pea flower infusion treatment. The results showed that no histopathological lesions were observed in either the acute or subchronic control groups (score 0 for all parameters). In the acute treatment groups at doses of 2,500 and 5,000 mg/kg BW, as well as in the subchronic treatment groups at doses of 250, 500, and 1,000 mg/kg BW, only very mild leukocyte infiltration with a score of 1 was observed, whereas other pathological parameters showed no lesions (score 0).

According to Çikler et al. (2023), the presence of mild leukocyte infiltration in the intestinal mucosa represents a protective and reversible tissue response, particularly in the presence of antioxidant compounds. In a water-avoidance stress rat model, epithelial damage, a reduction in goblet cell profile, and increased infiltration of inflammatory cells were observed; however, administration of fulvic acid was able to restore mucosal structure, increase goblet cell profile, and reduce oxidative stress. These findings indicate that mild leukocyte infiltration and transient reductions in goblet cell density are reversible and reflect an adaptive response rather than permanent toxic injury. The histological damage scores of the duodenum are presented in Table 1, Figure 3, and Figure 4.

Table 1. Histopathological damage of the duodenum in albino rats following acute and subchronic administration of butterfly pea flower infusion.

Note:  Data are presented as mean ± SD (standard deviation), with n = 5; # indicates n = 4. Values within the same column for each variable followed by different superscript letters (a-b) indicate significant differences among treatment groups (P ≤ 0.05). Description: = normal, 1 = very mild (1-10% damage), 2 = mild (10-25% damage), 3 = moderate (26-50% damage), 4 = severe (>50% damage).

Figure 3. Butterfly pea flower infusion induces very mild villus fusion in the duodenum. Acute control I (n = 4) (A), acute treatment at 2,500 mg/kg BW (n = 4) (B), acute control II (n = 5) (C), acute treatment at 5,000 mg/kg BW (n = 5) (D), subchronic control (n = 5) (E), subchronic treatment at 250 mg/kg BW (n = 5) (F), subchronic treatment at 500 mg/kg BW (n = 5) (G), subchronic treatment at 1,000 mg/kg BW (n = 5) (H). (H&E staining, scale bars: 500 µm and 200 µm). (Blue ovals indicate villus fusion).

Figure 4. Butterfly pea flower infusion induces very mild leukocyte infiltration in the duodenum. Acute control I (n = 4) (A), acute treatment at 2,500 mg/kg BW (n = 4) (B), acute control II (n = 5) (C), acute treatment at 5,000 mg/kg BW (n = 5) (D), subchronic control (n = 5) (E), subchronic treatment at 250 mg/kg BW (n = 5) (F), subchronic treatment at 500 mg/kg BW (n = 5) (G), subchronic treatment at 1,000 mg/kg BW (n = 5) (H). (H&E staining, scale bars: 200 µm and 50 µm). (Black circles indicate leukocyte infiltration).

Acute and subchronic administration of butterfly pea flower infusion induces changes in duodenal mucus characteristics in albino rats

Periodic Acid-Schiff-Alcian Blue staining revealed that, in both acute and subchronic control groups, goblet cell mucus was predominantly acidic. In the acute treatment groups at doses of 2,500 and 5,000 mg/kg BW the mucus characteristics shifted to mixed (acidic-neutral) mucus. In contrast, in all subchronic treatment groups, mucus characteristics remained predominantly acidic. The duodenal mucus characteristics in each treatment group are presented in Table 2 and Figure 5.

Table 2. Duodenal mucus characteristics of albino rats following acute and subchronic administration of butterfly pea flower infusion.

Note: Description: Duodenal mucus characteristics were analyzed using descriptive comparative analysis with n = 5; # indicates n = 4.

Figure 5. Duodenal mucus characteristics of albino rats following acute and subchronic administration of butterfly pea flower infusion. Acute control I (n = 4) (A), acute treatment at 2,500 mg/kg BW (n = 4) (B), acute control II (n = 5) (C), acute treatment at 5,000 mg/kg BW (n = 5) (D), subchronic control (n = 5) (E), subchronic treatment at 250 mg/kg BW (n = 5) (F), subchronic treatment at 500 mg/kg BW (n = 5) (G), subchronic treatment at 1,000 mg/kg BW (n = 5) (H). (PAS-AB staining, scale bar: 500 µm). (Black circles indicate mucus staining).

Acute and subchronic administration of butterfly pea flower infusion does not induce histomorphometric alterations in the colon of albino rats

Administration of butterfly pea flower infusion at doses of 2,500 and 5,000 mg/kg BW did not induce significant changes in most colonic histomorphometric parameters, including crypt depth and the thickness of the tunica mucosa, submucosa, muscularis, and serosa, compared with the control groups (P > 0.05). However, both the profile and area of goblet cells at these acute doses were significantly reduced compared with the controls (P ≤ 0.05) (Figure 6). In the acute control I group, the profile of goblet cells was 135 cells/500 µm with a mean area of 88.48 ± 8.617 µm², whereas at a dose of 2,500 mg/kg BW, these values decreased to 77 cells/500 µm and 55.73 ± 5.372 µm², respectively. In the acute control II group, the profile of goblet cells was 133 cells/500 µm with an area of 79.09 ± 22.264 µm², whereas at a dose of 5,000 mg/kg BW, these values declined to 89 cells/500 µm and 53.75 ± 6.411 µm², respectively (Figure 6). The reduction in both the profile and area of goblet cells is supported by the study of Kynkaanniemi et al. (2024) which reported that a diet containing 10% birch wood fiber extract for 4 weeks rich in flavonoids and tannins with anti-inflammatory, antibacterial, and antioxidant properties significantly decreased the profile of goblet cells in the distal colon compared with controls

Administration of butterfly pea flower infusion at doses of 250, 500, and 1,000 mg/kg BW did not result in significant changes in crypt depth or in the thickness of the colonic wall layers (P > 0.05). However, a significant increase in the profile of goblet cells was observed (P ≤ 0.05) (Table 3). In the subchronic control group, the profile of goblet cells was 100 cells/500 µm with a mean area of 44.72 ± 2.419 µm². Treatment with 250 mg/kg BW increased the profile of goblet cells to 128 cells/500 µm with an area of 91.29 ± 17.351 µm², a dose of 500 mg/kg BW increased them to 134 cells/500 µm with an area of 93.79 ± 25.205 µm², and a dose of 1,000 mg/kg BW increased them to 135 cells/500 µm with an area of 114.96 ± 21.294 µm² (Figure 6). The increase in both the profile and area of colonic goblet cells in the subchronic treatment is supported by the study of Erhunmwunse and Odiase (2014), who reported that oral administration of aqueous extract of Hibiscus sabdariffa calyces with anti-inflammatory activity at doses of 250, 500, and 1,000 mg/kg BW for 31 days significantly increased the profile of goblet cells in the colonic mucosa compared with controls. The histomorphometric measurements of the colon of albino rats following acute and subchronic toxicity treatments are presented in Figure 6.

Figure 6.  Colonic histomorphometry of albino rats following acute and subchronic administration of butterfly pea flower infusion. Acute treatment at a dose of 2,500 mg/kg BW (A), acute treatment at a dose of 5,000 mg/kg BW (B), subchronic treatment at doses of 250, 500, and 1,000 mg/kg BW (C). Data are presented as mean ± SD (standard deviation), acute control I and acute treatment at 2,500 mg/kg BW, n = 4; all other groups, n = 5. Different superscript letters (a-c) within the same column for each variable indicate significant differences among treatment groups (P ≤ 0.05).

Acute and subchronic toxicity of butterfly pea flower infusion induces very mild leukocyte infiltration in the histological structure of the colon of albino rats

All acute and subchronic control groups exhibited a score of 0 for all damage parameters, indicating the absence of histological lesions in the colon. In both the acute and subchronic treatment groups, only one parameter showed alteration, namely leukocyte infiltration with a score of 1 (very mild). The occurrence of leukocyte infiltration is supported by Refai (2014), who reported that oral administration of chamomile extract, which is rich in flavonoids, at a dose of 100 mg/kg BW for 12 days induced histological damage in the intestinal tract and colon of rats, characterized by increased infiltration of inflammatory cells, indicating cytotoxic effects on the colonic mucosa. The histopathological damage scores of the colon are presented in Table 3 and Figure 7.

Table 3. Histopathological damage of the colon in albino rats following acute and subchronic administration of butterfly pea flower infusion.

Note: Data are presented as mean ± SD (standard deviation), with n = 5; # indicates n = 4. Values within the same column for each variable followed by different superscript letters (a-b) indicate significant differences among treatment groups (P ≤ 0.05). 0 = normal, 1 = very mild (1-10% damage), 2 = mild (10-25% damage), 3 = moderate (26-50% damage), 4 = severe (>50% damage).

Figure 7. Butterfly pea flower infusion induces very mild histopathological alterations in the colon. Acute control I (n = 4) (A), acute treatment at 2,500 mg/kg BW (n = 4) (B), acute control II (n = 5) (C), acute treatment at 5,000 mg/kg BW (n = 5) (D), subchronic control (n = 5) (E), subchronic treatment at 250 mg/kg BW (n = 5) (F), subchronic treatment at 500 mg/kg BW (n = 5) (G), subchronic treatment at 1,000 mg/kg BW (n = 5) (H). (H&E staining, scale bars: 200 µm and 50 µm), (Black circles indicate leukocyte infiltration).

Acute and subchronic administration of butterfly pea flower infusion does not induce changes in colonic mucus characteristics in albino rats

Periodic Acid-Schiff-Alcian Blue staining of the colon revealed that mucus in all groups, both control and treated, was predominantly acidic. No differences in colonic mucus characteristics were observed between the treatment and control groups at any of the tested doses. Thus, the colon maintained an acidic mucus profile across all treatment groups, in contrast to the duodenum, which at high acute doses exhibited a shift toward mixed mucus. This finding reflects physiological and functional differences among intestinal segments, as the colon consistently produces acidic mucus to preserve mucosal barrier function and maintain microbial homeostasis.

The increase in goblet cell profile without accompanying changes in mucus type indicates an adaptive response characterized by enhanced production of protective mucus, without qualitative alterations in the glycoprotein composition of the mucus (Zhang et al., 2022). The colonic mucus characteristics for each treatment group are presented in Table 4 and Figure 8.

Table 4. Colonic mucus characteristics of albino rats following acute and subchronic administration of butterfly pea flower infusion.

Note: Description: Colonic mucus characteristics were analyzed using descriptive comparative analysis with n = 5; # indicates n = 4.

Figure 8. Colonic mucus characteristics of albino rats following acute and subchronic administration of butterfly pea flower infusion. Acute control I (n = 4) (A), acute treatment at 2,500 mg/kg BW (n = 4) (B), acute control II (n = 5) (C), acute treatment at 5,000 mg/kg BW (n = 5) (D), subchronic control (n = 5) (E), subchronic treatment at 250 mg/kg BW (n = 5) (F), subchronic treatment at 500 mg/kg BW (n = 5) (G), subchronic treatment at 1,000 mg/kg BW (n = 5) (H). (PAS-AB staining, scale bar: 500 µm), (Black circles indicate mucus staining).

DISCUSSION

Acute and subchronic administration of butterfly pea flower infusion does not alter duodenal histomorphometry in albino rats         

Villus length and crypt depth of the duodenum in all treatment groups did not differ significantly from those in the control group. This indicates that administration of butterfly pea flower infusion did not alter villus architecture, and thus the absorptive capacity of the mucosa remained within normal limits. Optimal villus length reflects an adequate absorptive surface area (Ziegler et al., 2018). The absence of changes in villus length suggests that the duodenal mucosa did not undergo structural disturbances that could affect absorptive function. Morphological alterations of villi, such as shortening or elongation, are generally associated with adaptive responses, irritation, or tissue injury induced by exposure to certain substances (Nciri et al., 2015; Ziegler et al., 2018). In the present study, such alterations were not observed, indicating that butterfly pea flower infusion at the tested doses did not exert toxic effects on the villus structure of the duodenum. The absence of significant histological damage in the duodenum and colon following acute and subchronic administration of butterfly pea flower infusion is consistent with previous studies reporting tissue-protective effects of Clitoria ternatea in experimental animal models. Umbar et al. (2025) demonstrated that Clitoria ternatea administration attenuated histological damage and preserved tissue integrity in a rotenone-induced rat model, attributed to its antioxidant and anti-inflammatory phytochemical constituents.

Crypt depth in all treatment groups also did not differ significantly from that of the control group, indicating that the crypt architecture remained within normal limits. The stability of crypt depth together with relatively constant villus length reflects a preserved villus to crypt ratio. This condition indicates a balanced state of epithelial cell proliferation and differentiation without excessive proliferative stress (Zheng and Duan, 2023). The growth and maintenance of villus and crypt structures are regulated by the Wnt and Notch signaling pathways, which control the proliferation, migration, and differentiation of intestinal stem cells (Choi and Augenlicht, 2024; Zhang et al., 2025). In the present study, no disruption of these pathways was evident, as reflected by the maintained integrity of villus and crypt morphology.

The thickness of all layers of the duodenal wall, including the tunica mucosa, submucosa, muscularis, and serosa, did not differ significantly in any treatment group compared with the control. This finding indicates that administration of butterfly pea flower infusion did not induce structural alterations such as edema, fibrosis, muscular hypertrophy, or mucosal atrophy, which are commonly observed following exposure to toxic compounds (Matar et al., 2024). The ability of butterfly pea flower infusion to maintain duodenal mucosal homeostasis and structural integrity may be attributed to its anthocyanin, flavonoid, and phenolic contents, which possess antioxidant and anti-inflammatory activities capable of attenuating oxidative stress and local inflammatory responses (Nair et al., 2015; Jeyaraj et al., 2021). Anthocyanins have also been reported to enhance and preserve the expression of tight junction proteins such as ZO-1, claudins, and occludin, thereby maintaining epithelial barrier integrity and preventing increased mucosal permeability (Yu et al., 2023). With reduced oxidative damage and preserved intercellular junctions, the regenerative processes of villi and crypts remain unaffected, resulting in the maintenance of structural and functional homeostasis of the duodenal mucosa.

Alterations were observed in goblet cell parameters. In the acute treatment at doses of 2,500 and 5,000 mg/kg BW, both the profile and area of goblet cells decreased significantly. This reduction may be associated with mucus depletion due to increased secretion as an initial response to luminal stress exposure (Johansson and Hansson, 2016; Song et al., 2023). In contrast, in the subchronic treatment, the profile and area of goblet cells increased significantly, indicating enhanced mucosal secretory activity as an adaptive response to repeated exposure (Sarkar et al., 2016; Yang and Yu, 2021). The distinct patterns of goblet cell responses between acute and subchronic treatments are consistent with the characteristics of the duodenal mucosa, which exhibits a high rate of epithelial turnover and rapid regenerative capacity (Johansson and Hansson, 2016).

The differential goblet cell responses between acute and subchronic exposure are associated with phytochemical mediated modulation by butterfly pea constituents, particularly anthocyanins and flavonols, which are known to enhance goblet cell differentiation, maturation, and the expression of mucin genes such as MUC2 (Yu et al., 2023; Wang et al., 2025). The increase in goblet cell profile under subchronic exposure is supported by inhibition of the TLR4-NF-κB inflammatory pathway, improvement of short-chain fatty acid (SCFA)-producing microbiota that promote goblet cell maturation, and attenuation of oxidative stress, thereby creating a mucosal environment favorable for secretory cell function (Jeyaraj et al., 2021; Yang and Yu, 2021; Yu et al., 2023). This pattern is consistent with the concept of hormesis, in which repeated low-to-moderate exposure to antioxidant and anti-inflammatory compounds elicits adaptive responses, manifested as enhanced goblet cell profile and functional capacity.

Previous research conducted by Kovitvadhi et al. (2024) demonstrated that supplementation with Clitoria ternatea crude extract at 0.5 g/kg BW in rabbits improved nutrient digestibility without significantly affecting gut histology. Similarly, Mujnisa et al. (2025) reported that supplementation with 70% ethanolic extract of Clitoria ternatea flowers at doses of 4-10 mL/L drinking water in broilers improved carcass quality but did not show a substantial effect on intestine morphometrics. However, these studies were conducted under nutritional supplementation conditions rather than toxicity assessment. In contrast, the present study employed a high-dose aqueous butterfly pea infusion under acute and subchronic toxicity conditions in rats, which may explain the mild biological responses observed, including alterations in goblet cell profile, mucus characteristics, and very mild leukocyte infiltration. Similar mild histological alterations following high dose butterfly pea administration have also been reported previously in the liver and kidneys, indicating that high-dose exposure may induce adaptive tissue responses despite the absence of severe histopathological damage (Pusparini et al., 2026).

Acute and subchronic toxicity of butterfly pea flower infusion induces very mild leukocyte infiltration in the histological structure of the duodenum            

Very mild leukocyte infiltration reflects a minimal inflammatory response as a physiological reaction to exposure to foreign substances (Sabir and Jan, 2025). This response is associated with activation of the innate immune system through the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) expressed on epithelial cells and mucosal immune cells (Li and Wu, 2021). The flavonoid constituents of butterfly pea flower infusion are known to suppress the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, thereby maintaining the inflammatory response at a mild level and preventing its progression to structural damage of the mucosa (Nair et al., 2015).

The absence of other structural lesions indicates that the integrity of the duodenal mucosa was preserved, as such parameters are typically associated with disruption of intercellular junction complexes, vascular impairment, cellular homeostasis dysfunction, and chronic mucosal injury (Liu et al., 2025; Zhang et al., 2025). Accordingly, the histological alterations observed were limited to very mild leukocyte infiltration, which represents a functional adaptive response and does not indicate significant histopathological toxicity. This finding is consistent with the protective properties of butterfly pea phytochemicals, which are rich in flavonoids, anthocyanins, tannins, and saponins (Jeyaraj et al., 2021).

The phytochemical profile of butterfly pea flower infusion, particularly ternatin anthocyanins and flavonol glycosides, plays a crucial role in explaining the minimal histopathological changes through their potent antioxidant and anti-inflammatory activities. These compounds inhibit the activation of major inflammatory pathways, including nuclear translocation of NF-κB, expression of iNOS/NO, COX-2 activity, and TLR4-MyD88-TRAF6 signaling, thereby reducing the production of pro-inflammatory cytokines such as TNF-α and IL-6 while enhancing IL-10 levels and strengthening endogenous antioxidant defenses (CAT, SOD, GPx) (Nair et al., 2015; Yu et al., 2023; Wang et al., 2025; Pramudya et al., 2026). The attenuation of pro-inflammatory responses, together with the preservation of the mucosal barrier, allows controlled involvement of immune cells in host defense and tissue repair without inducing tissue injury. Therefore, the presence of mild leukocyte infiltration reflects a regulated adaptive mucosal response to exposure to the bioactive compounds of butterfly pea rather than evidence of histological toxicity. To support these findings, a proposed mechanism illustrating how the bioactive compounds of butterfly pea flower infusion maintain gastrointestinal mucosal integrity through antioxidant and anti-inflammatory pathways is presented in Figure 9.

Figure 9. Proposed mechanism of bioactive compounds in butterfly pea flower infusion in maintaining gastrointestinal mucosal integrity through antioxidant and anti-inflammatory pathways. Bioactive compounds such as polyphenols, flavonoids, and anthocyanins reduce oxidative stress by decreasing reactive oxygen species (ROS) and suppressing the nuclear factor-kappa B (NF-κB) signaling pathway. This leads to reduced production of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), as well as decreased expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). These mechanisms contribute to the maintenance of mucosal integrity, increased mucus production through mucin 2 (MUC2) expression, and enhanced epithelial protection in the gastrointestinal tract. Some graphical elements in this figure were created using Canva.

Acute and subchronic administration of butterfly pea flower infusion induces changes in mucus characteristics of the duodenum in albino rats

Intestinal mucus consists of mucin glycoproteins and other components that play a crucial role in mucosal protection, in which the characteristics of acidic and neutral glycoconjugates reflect goblet cell function and the quality of the mucosal barrier (Yao et al., 2021; Li et al., 2025). In the present study, the control groups exhibited a predominance of acidic mucus, whereas high-dose acute treatment resulted in mixed mucus, indicating alterations in glycoconjugate composition associated with goblet cell stress responses and modifications in glycosylation processes (Bergstrom et al., 2020). In contrast, under subchronic treatment at all doses, mucus characteristics remained predominantly acidic despite a significant increase in goblet cell profile, suggesting preservation of mucus quality and functional mucosal adaptation. This pattern is consistent with the concept of hormesis, whereby repeated exposure to low-to-moderate doses induces adaptive tissue responses without eliciting pathological changes (Calabrese et al., 2016; Knoop and Newberry, 2018).

The predominance of acidic mucus in the subchronic groups may be attributed to the effects of butterfly pea anthocyanins, which enhance MUC2 expression and support mucin production through modulation of short-chain fatty acid (SCFA)-producing microbiota and attenuation of inflammation (Yu et al., 2023; Wang et al., 2025). Acidic mucins rich in sialomucins and sulfomucins possess greater stability and protective capacity due to their negative charge, rendering them more resistant to enzymatic and microbial degradation and more effective in maintaining mucosal barrier function (Jeyaraj et al., 2021; Wang et al., 2025).

Acute and subchronic administration of butterfly pea flower infusion does not alter colonic histomorphometry in albino rats

The depth of colonic crypts in all treatment groups, both acute and subchronic, did not differ from that of the control group. This finding indicates that the proliferative activity of intestinal stem cells and progenitor cells remained within the normal range, with no evidence of a hyperproliferative response that typically accompanies mucosal injury (Sequetto et al., 2014; Zheng and Duan, 2023). The thickness of the colonic tunica mucosa also did not differ significantly among groups, suggesting that epithelial mass, immune cell infiltration, and local inflammatory status remained stable. The absence of changes in mucosal thickness indicates that neither mucosal atrophy nor hypertrophy occurred as a result of exposure to butterfly pea flower infusion (Nickel, 2022; Matar et al., 2024). Similarly, the thicknesses of the tunica submucosa, muscularis, and serosa remained unchanged, indicating the absence of severe inflammation or extensive tissue damage in the colonic wall layers (Roulis and Flavell, 2016; Neurath et al., 2025).

The stability of colonic morphometric parameters observed in this study may be attributed to the protective effects of butterfly pea anthocyanins and flavonols, which possess potent antioxidant and anti-inflammatory activities, thereby safeguarding the mucosa against oxidative stress and inflammatory processes that could otherwise induce structural alterations. In addition, these compounds have been reported to enhance the expression of tight junction proteins and to preserve colonic mucosal integrity under inflammatory conditions (Yu et al., 2023), which may explain the unchanged thickness of the tunica mucosa, submucosa, muscularis, and serosa across all treatment groups.

 Under acute exposure, the profile and area of goblet cells decreased, which is associated with a transient cellular stress response and increased mucus secretion as an initial protective mechanism, resulting in depletion of intracellular mucus stores (Arike and Hansson, 2016; Song et al., 2023). In contrast, subchronic exposure led to a significant increase in both the profile and area of goblet cells, reflecting an adaptive mucosal response characterized by enhanced differentiation and maturation of goblet cells, supported by the activity of polyphenols and metabolites such as short-chain fatty acids (SCFAs) (Bergstrom et al., 2020; Joyce et al., 2022).

The differential goblet cell responses between acute and subchronic exposure indicate mucosal adaptation to the bioactive compounds of butterfly pea flower infusion, consistent with the concept of hormesis, namely a protective tissue response elicited by repeated low-to-moderate doses of a stimulus (Sequetto et al., 2014; Calabrese, 2016). In the colon, the subchronic increase in goblet cells is associated with phytochemical modulation by butterfly pea constituents, particularly anthocyanins and polyphenols, which inhibit the TLR4/NF-κB signaling pathway, thereby promoting goblet cell differentiation and upregulating MUC2 expression (Wang et al., 2025). The antioxidant activities of anthocyanins and flavonols reduce oxidative stress and proinflammatory cytokine levels, thereby supporting secretory cell function and maintaining mucosal integrity (Nair et al., 2015; Jeyaraj et al., 2021). Accordingly, the reduction in goblet cell profile and area observed under acute exposure reflects an early response due to enhanced mucus secretion, whereas the increase observed under subchronic exposure represents a compensatory mucosal mechanism to restore and augment mucus production capacity, mediated by the actions of polyphenols and SCFA metabolites (Bergstrom et al., 2020; Joyce et al., 2022).

Acute and subchronic toxicity of butterfly pea flower infusion induces very mild leukocyte infiltration in the histological structure of the colon

The colonic lamina propria physiologically contains resident leukocytes that play essential roles in antigen surveillance and the maintenance of mucosal immune homeostasis (Shi, 2021; Di Sabatino et al., 2023). A slight increase in leukocyte infiltration in the absence of epithelial damage reflects a controlled adaptive immune response to exposure to bioactive compounds. Flavonoids and anthocyanins present in butterfly pea flowers, after being metabolized by the gut microbiota, contribute to the maintenance of immune tolerance and mucosal environmental balance. Accordingly, polyphenols have been reported to enhance intraepithelial lymphocyte infiltration without inducing tissue injury (Mantel et al., 2023; Moon et al., 2023).

In addition, flavonoids and anthocyanins exert anti-inflammatory effects through inhibition of the nuclear factor kappa B (NF-κB) and Toll-like receptor 4 (TLR4) signaling pathways, thereby suppressing the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), and consequently limiting oxidative stress and destructive inflammation (Al-Khayri et al., 2022; Aghababaei and Hadidi, 2023). The absence of other histopathological alterations in all treatment groups indicates that the observed leukocyte infiltration represents an adaptive response in the colon rather than a manifestation of pathological tissue injury.

Acute and subchronic administration of butterfly pea (Clitoria ternatea L.) flower infusion does not alter mucus characteristics in the colon of albino rats

Colonic mucus is primarily composed of the mucin MUC2, which forms a protective layer over the epithelial surface and is biochemically classified into acidic and neutral mucus (Yang and Yu, 2021; Yao et al., 2021). In the present study, mucus characteristics in the colon of all groups were dominated by acidic mucus, reflecting a high content of sialomucins and sulfomucins. This pattern is consistent with the physiological condition of the colon, in which acidic mucus exhibits greater protective capacity than neutral mucus because the negative charges of sulfate and carboxyl groups enhance gel stability and resistance to enzymatic and bacterial degradation (Grondin et al., 2020; Tonetti et al., 2024; Li et al., 2025).

The predominance of acidic mucus is also associated with the acidic colonic environment resulting from microbial fermentation that produces short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate (Louis and Flint, 2017; Facchin et al., 2024). The stability of acidic mucus is closely linked to the activity of the gut microbiota, particularly SCFA-producing bacteria such as Faecalibacterium and Bacteroides (Chen et al., 2025). These metabolites, especially butyrate, serve as an energy source for colonocytes and have been shown to stimulate MUC2 expression and improve mucosal barrier function (Ottria et al., 2026). In addition, recent studies indicate that SCFAs can stimulate mucus growth through activation of specific receptors, thereby strengthening the mucus barrier and maintaining epithelial integrity (Holmberg et al., 2024). This mechanism contributes to epithelial protection, limits the growth of pathogenic bacteria, and plays an important role in regulating mucosal immune responses (Bilotta and Cong, 2019). The consistency of acidic mucus characteristics across all treatment groups indicates that administration of butterfly pea flower infusion did not disrupt goblet cell secretory function or the integrity of the colonic mucosa (Johansson and Hansson, 2016; Yao et al., 2021). This condition is associated with enhanced MUC2 expression, modulation of the gut microbiota, and attenuation of inflammation by the bioactive compounds of butterfly pea, thereby maintaining stable production of negatively charged acidic mucins that effectively preserve mucosal barrier function (Jeyaraj et al., 2021; Yang and Yu, 2021; Yu et al., 2023; Wang et al., 2025).

CONCLUSION

Based on the results of this study, acute and subchronic administration of butterfly pea flower infusion did not induce changes in the histomorphometric parameters of the duodenum and colon and did not cause histological damage to these organs; however, very mild inflammation characterized by leukocyte infiltration was observed. The infusion also affected mucus characteristics, particularly in the duodenum at high acute doses, which exhibited a shift in mucus composition as an adaptive mucosal response. In contrast, the mucus characteristics of the colon remained predominantly acidic across all treatment groups, indicating preservation of mucosal barrier homeostasis. Overall, these findings indicate that butterfly pea flower infusion did not produce overt histological injury in the duodenum and colon under the tested experimental conditions. Nevertheless, dosage considerations remain important to minimize potential toxic effects.

ACKNOWLEDGEMENTS

The authors would like to express their gratitude to the Laboratory of Animal Structure and Development, Faculty of Biology, Universitas Gadjah Mada, for providing facilities, equipment, and technical assistance throughout the conduct of this study.

AUTHOR CONTRIBUTIONS

Siti Aeniah: Conceptualization (Lead), Study Design (Equal), Materials (Equal), Data Curation (Lead), Formal Analysis (Lead), Investigation (Lead), Literature Search (Lead), Writing - Original Draft (Lead); Tia Apriliyani: Conceptualization (Equal), Study Design (Equal), Data Curation (Supporting), Investigation (Equal); Nur Ainun Oktavia Pusparini: Conceptualization (Equal), Study Design (Equal), Data Curation (Supporting), Investigation (Equal); Bambang Retnoaji: Conceptualization (Equal), Study Design (Supporting), Supervision (Equal), Materials (Supporting), Formal Analysis (Supporting), Literature Search (Supporting), Writing – Review & Editing (Equal); Ardaning Nuriliani: Conceptualization (Equal), Study Design (Lead), Supervision (Lead), Resources (Lead), Materials (Lead), Formal Analysis (Equal), Literature Search (Equal), Writing - Review & Editing (Equal), Critical Review (Lead).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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