Abstract Kombucha is a fermented beverage increasingly consumed for its reported functional properties, yet information on its developmental effects during pregnancy remains limited. This exploratory study evaluated prenatal developmental outcomes following exposure to Phaleria macrocarpa fruit infusion and kombucha during organogenesis in mice and characterized the fermented preparation using GC–MS. Twenty pregnant mice were allocated to four groups (n = 5 litters per group) and treated orally from gestation day (GD) 6 to GD 15. Maternal body weight, litter size, fetal viability, fetal body weight, crown–rump length, and external, visceral, and skeletal findings were assessed on GD 18. Maternal body-weight gain did not differ significantly among groups. Mean litter size was lower in the treated groups than in the control group, while fetal deaths were infrequent and occurred only in kombucha-treated litters. Mean fetal body weight and crown–rump length were significantly lower in treated groups, with the lowest values in the higher-exposure kombucha group. No gross external, visceral, or skeletal malformations were identified among the fetuses examined. GC–MS profiling tentatively identified several volatile and semi-volatile constituents, including phenolic derivatives, alcohol-related compounds, fatty-acid-related constituents, and 5-hydroxymethylfurfural. Given the limited number of litters and restricted maternal and reproductive assessments, these findings should be interpreted as preliminary growth-associated observations rather than definitive evidence of developmental toxicity or safety. Larger, adequately powered studies with comprehensive reproductive endpoints and quantitative chemical characterization are warranted.

Keywords: Phaleria macrocarpa, Kombucha, Prenatal developmental evaluation, Fetal development, GC–MS

Funding: The authors express their gratitude to Politeknik Kesehatan Kementerian Kesehatan Jambi for supporting this research through the PKPT grant under the Inter-University Collaboration Program (Grant Number: HK.02.03/F.XXXVII/1715/2025).

Citation:  Rusdi, M.S., Efendi, M.R., Rahmah, Sofiyetti, and Sari, D.N. 2027. Prenatal developmental evaluation of Phaleria macrocarpa kombucha: An experimental study in pregnant mice with GC–MS-based metabolite profiling. Natural and Life Sciences Communications. 26(1): e2027002.

Graphical Abstract:

INTRODUCTION

Kombucha is a traditional fermented beverage prepared from green or black tea leaves and sucrose, fermented with a symbiotic culture of bacteria and yeasts (SCOBY) for 7–21 days (Jayabalan et al., 2014; Júnior et al., 2022). During fermentation, SCOBY produces a diverse array of metabolites, including polyphenols, organic acids (acetic, gluconic, and lactic acids), vitamins, amino acids, and residual sugars (de Miranda et al., 2022). These bioactive compounds contribute to reported antioxidant and antimicrobial properties, and studies have demonstrated that kombucha exhibits enhanced antioxidant capacity compared to unfermented tea (Vázquez-Cabral et al., 2017). Despite its functional benefits, kombucha is a biologically active fermented product whose metabolite composition varies depending on substrate and fermentation conditions, potentially altering its safety profile in vulnerable populations.

P. macrocarpa (Scheff.) Boerl., commonly known as mahkota dewa, is a medicinal plant native to Papua, Indonesia, and widely used in traditional medicine (Quattrocchi, 2016). Various parts of the fruit have traditionally been used for the management of pain, metabolic disorders, and inflammatory conditions (Lay et al., 2014; Ahmad et al., 2023). Phytochemical investigations have identified flavonoids, lignans, benzophenones, and xanthones as major constituents contributing to its antioxidant and antihyperglycemic activities (Lay et al., 2014; Easmin et al., 2017).

Fermentation may modify the phytochemical composition of plant substrates through microbial biotransformation, potentially enhancing bioavailability and biological activity. Our previous metabolomic profiling demonstrated that P. macrocarpa kombucha contains diverse polyphenolic and xanthone derivatives associated with antioxidant potential. In vivo studies further indicated enhanced antihyperglycemic activity in fermented preparations compared with unfermented infusion (Rusdi et al., 2025).

Despite its growing popularity as a functional beverage, systematic evaluation of the safety of herbal-based kombucha preparations during pregnancy remains limited. Fermentation-derived metabolites and bioactive phytochemicals may exert biological effects that differ from those of the original plant material (Dutta and Paul, 2019). However, controlled prenatal developmental studies assessing such preparations are scarce. Therefore, the present exploratory study aimed to characterize prenatal developmental outcomes following exposure to P. macrocarpa fruit kombucha during organogenesis in mice and to describe its metabolite profile using GC–MS analysis. The study was intended to generate preliminary observations and was not designed as a definitive OECD TG 414 prenatal developmental toxicity study.

MATERIALS AND METHODS

Plant material

The sample used in this research was collected from Desa Bahar Mulya, Kec. Bahar Utara, Muaro Jambi. A taxonomist at the Universitas Padjdjaran identified and authenticated the plants with specimen number 48/HB/06/2023.

Preparation of P. macrocarpa fruit infusion and kombucha

The infusion of P. macrocarpa fruit was prepared by weighing 10 g of dried fruit powder and extracting it with 100 mL of distilled water at 90°C for 15 minutes. The mixture was filtered, and the volume was adjusted to 100 mL by rinsing the plant residue with additional distilled water to maximize the extraction yield. For kombucha preparation, 10 g of sucrose (10% w/v) and 3 g of SCOBY culture were added to the freshly prepared infusion. Fermentation was conducted at room temperature (25 ± 2 °C) in sterile glass jars covered with gauze to allow air exchange while preventing contamination, and the preparations were incubated for 14 days without agitation. At the end of fermentation, the kombucha was filtered to remove microbial biomass and stored at 4°C until use (Rusdi et al., 2025).

GC–MS Analysis

Metabolite profiling of P. macrocarpa kombucha was performed using an Agilent 5977 GC–MS system equipped with an HP-5MS column (30 m × 250 µm × 0.25 µm). The kombucha sample was analyzed directly without derivatization or additional extraction. One microliter of the sample was manually injected in split mode (split ratio 50:1), with helium as the carrier gas at a flow rate of 1.0 mL/min. The injector temperature was maintained at 290°C, and the oven temperature was programmed from an initial 60°C (hold 0 min) to 290°C at a rate of 15°C/min, with a final hold of 30 minutes. The MSD transfer line was set at 290°C. Mass spectrometric detection was performed in scan mode over an m/z range of 35–600. The ion source and quadrupole temperatures were set at 230°C and 150°C, respectively. A solvent delay of 2.5 minutes was applied to avoid solvent interference. Data acquisition and processing were conducted using MassHunter software, and compound identification was based on comparison of mass spectra with the NIST (National Institute of Standards and Technology) library. All compound assignments were considered tentative (Nikolova et al., 2022; Likittrakulwong et al., 2023).

Animal grouping and treatment

Twenty healthy female Swiss Webster mice (Mus musculus), aged 2–3 months and weighing approximately 20–30 g, were used in this study. All females were experimentally naïve and exhibited normal behavior and general condition. The animals were acclimatized to the laboratory environment for 7 days before the experiment. During acclimatization, standard feed and drinking water were provided ad libitum. Four healthy male Swiss Webster mice aged 2–3 months were used for mating. Prior to mating, female mice were monitored daily to identify the estrus phase based on external genital characteristics. Estrus was determined by visual observation of vaginal opening, vulvar swelling, and pinkish coloration of the external genital area, which are indicative of sexual receptivity in mice. Females identified in the estrus phase were paired with proven fertile males at a ratio of 1:4 (one male to four females) and housed overnight. Successful mating was confirmed the following morning by the presence of a vaginal plug, which was designated as gestation day (GD) 0.

Females without evidence of vaginal plug formation were returned to the mating cage and re-evaluated in subsequent cycles until successful copulation occurred. Only plug-positive females were included in the study and subsequently randomized into experimental groups (n = 5 pregnant dams per group).

Treatments were administered orally once daily from gestation day (GD) 6 to GD 15, corresponding to the critical window of organogenesis in mice as defined by OECD Test Guideline 414 (OECD, 2018). All experimental procedures were conducted in accordance with institutional animal care and use guidelines and were approved by the Ethics Committee of Politeknik Kesehatan Kementerian Kesehatan Jambi (Approval No. LB.02.06/2/717/2025).

The control group received distilled water (0.2 mL/20 g body weight), while the infusion group received P. macrocarpa fruit infusion at 1% body-weight equivalent (0.2 mL/20 g). The kombucha groups received low and high exposure levels equivalent to 1% and 2% of body weight (0.2 and 0.4 mL/20 g, respectively). These exposure levels were selected for exploratory assessment, considering reported human kombucha consumption patterns (Fraiz et al., 2025), without assuming direct human-equivalent dosing. Maternal body weight and general clinical condition were monitored daily throughout gestation.

Prenatal developmental assessment

The prenatal developmental assessment was adapted from the method described by Rusdi et al. (2023). On GD 18, pregnant animals were euthanized by cervical dislocation, and laparotomy was performed. The uterus was examined to record the number of live fetuses, dead fetuses, and grossly visible resorptions. All fetuses were initially examined macroscopically for external abnormalities, including abnormalities of the tail, ears, eyelids, and digits. Approximately one-third of the fetuses from each litter were fixed in Bouin’s solution for 14 days for visceral examination. After fixation, the fetuses were removed from the fixative, and the craniofacial structures were examined by careful sectioning of the head, including inspection of the palate for clefts or other visible abnormalities. The remaining two-thirds of the fetuses were subjected to skeletal examination by immersion in Alizarin Red solution for 2–3 days with occasional gentle agitation until the soft tissues became sufficiently transparent and the stained skeletal structures were visible. Skeletal specimens were then examined for abnormalities in bone configuration and number. Findings from the treated groups were compared with those of the control group (Hayes and Kobets, 2023).

Data analysis

Data were analyzed at the dam or litter level, as appropriate. Normality and homogeneity of variance were assessed prior to parametric analysis. Continuous outcomes meeting these assumptions were analyzed using one-way ANOVA followed by Duncan’s multiple range test. Because fetal deaths occurred infrequently, fetal mortality outcomes were summarized descriptively at the litter level. For fetal growth endpoints, the litter was considered the experimental unit. Statistical significance was set at P < 0.05.

RESULTS

GC–MS Profiling of P. macrocarpa Kombucha

GC–MS analysis of the P. macrocarpa kombucha preparation revealed multiple volatile and semi-volatile constituents. Compound assignments were based on comparison with the NIST mass spectral library and were considered tentative. The chromatographic profile included phenolic derivatives, alcohol-related compounds, fatty-acid-related constituents, and 5-hydroxymethylfurfural (5-HMF) (Table 1).

Table 1. GC–MS metabolite profile of P. macrocarpa fruit Kombucha.

Note: Duplicate peaks representing the same compound were combined and reported as total area percentage.

Effect of P. macrocarpa fruit kombucha on maternal body weight

Maternal body weight increased progressively from gestation day (GD) 1 to GD 18 across all experimental groups, consistent with normal physiological gestational progression. Initial body weights ranged from 25.00 to 27.60 g, while final body weights on GD 18 ranged from 43.80 to 44.60 g (Table 2). Statistical analysis demonstrated that administration of P. macrocarpa fruit infusion or kombucha did not significantly affect maternal body weight or total gestational weight gain compared with the control group (P > 0.05). Mean maternal weight gain ranged from 16.60 ± 1.95 g to 18.80 ± 1.30 g, with minor intergroup variation that was not statistically significant (Figure 1; Table 2).

Figure 1. Vaginal plug-positive females (mated females) (A), observation after laparotomy (B).

Table 2. Maternal body weight and weight gain in pregnant mice treated with P. macrocarpa fruit infusion and kombucha.

Note: 1% and 2% refer to administration volumes equivalent to 1% and 2% of body weight, respectively. Data are expressed as mean ± SD (n = 5 dams per group). Different superscript letters within the same column indicate statistically significant differences (P < 0.05).

Effect of P. macrocarpa fruit infusion and kombucha on fetal parameters

Mean litter size was lower in all treated groups than in the control group (P < 0.05). The mean number of fetuses per litter was 8.40 ± 1.51 in the control group, 6.80 ± 1.30 in the infusion group, 6.20 ± 0.44 in the lower-exposure kombucha group, and 6.40 ± 1.14 in the higher-exposure kombucha group (Table 3).

Table 3. Litter-based fetal viability analysis in mice treated with P. macrocarpa fruit infusion and kombucha.

Fetal viability is summarized in Table 3. Fetal deaths were absent in the control and infusion groups and occurred sporadically in the kombucha-treated groups. One litter in the lower-exposure kombucha group contained one dead fetus, whereas two litters in the higher-exposure group each contained one dead fetus (Figure 2). No grossly visible uterine resorption sites were identified in any experimental group at GD 18.

Table 4. Litter-based analysis of fetal growth parameters in pregnant mice treated with P. macrocarpa fruit infusion and kombucha.

Note: Values are presented in mean ± SD; values with different letters in the columns indicate statistically significant differences (P < 0.05) based on the Duncan Multiple Range Test.

Fetal body weight was significantly influenced by treatment (Table 4). When analyzed at the litter level (n = 5 dams per group), the control group exhibited the highest mean fetal weight (1.60 ± 0.13 g), followed by the P. macrocarpa fruit infusion group (1.18 ± 0.10 g). The 1% kombucha group showed a mean fetal weight of 1.06 ± 0.14 g, whereas the 2% kombucha group presented the lowest mean value (0.88 ± 0.24 g). Statistical analysis indicated significant differences among groups (P < 0.05).

A progressive reduction in mean fetal weight was observed across treatment groups, with lower values in kombucha-treated litters compared with controls. Similarly, fetal crown–rump length showed significant differences among groups (P < 0.05). The control group exhibited the greatest mean length (2.99 ± 0.36 cm), followed by the infusion group (2.30 ± 0.12 cm), the 1% kombucha group (2.09 ± 0.17 cm), and the 2% kombucha group (1.94 ± 0.17 cm).

Figure 2. Live (A)and Dead fetuses (B).

Effect of P. macrocarpa fruit infusion and kombucha on morphological defects external morphological observations

No external malformations were observed in fetuses from any treatment group when compared with the controls. No gross external abnormalities were identified among the fetuses examined in any group. However, minor hemorrhagic findings were noted in a few fetuses: one case of abdominal hemorrhage in the 1% kombucha group and two cases in the 2% kombucha group (Figure 3).

For visceral examination, fetuses were fixed in Bouin’s solution to facilitate detailed assessment of soft tissues. Across all groups, no abnormalities were detected, including cleft palate, eyelid fusion defects, or auricular malformations. Skeletal preparations stained with Alizarin Red showed no gross differences in bone number or configuration between treated and control groups. No gross visceral or skeletal abnormalities were identified among the specimens examined (Figure 3). However, the limited number of litters restricts the ability to detect uncommon developmental abnormalities.

Figure 3. Hemorrhagic, skeletal, and visceral observations.

DISCUSSION

The present exploratory study provides preliminary evidence that exposure to P. macrocarpa fruit infusion and kombucha during organogenesis was associated with differences in fetal growth parameters at the litter level. Mean fetal body weight and crown–rump length were lower in the treated groups than in the control group, with the lowest values observed in the higher-exposure kombucha group. A progressive reduction in fetal growth parameters was observed across the tested exposure groups. Importantly, these differences were not accompanied by statistically significant changes in maternal body weight gain or by gross external, visceral, or skeletal abnormalities among the fetuses examined. Thus, under the present experimental conditions, the developmental response was expressed primarily through growth-related parameters rather than through readily detectable structural abnormalities.

The GC–MS profile provides chemical context for the biological findings and illustrates the chemical complexity of the fermented P. macrocarpa preparation. Several volatile and semi-volatile constituents were tentatively identified, including phenethyl alcohol, 3-ethylphenol, 5-hydroxymethylfurfural, 2-methoxy-5-vinylphenol, nonanoic acid, tryptophol, and fatty-acid-related compounds. Similar constituents and chemical classes have been reported in conventional and alternative-substrate kombucha preparations. For example, phenethyl alcohol and related volatile compounds have been frequently detected during kombucha fermentation, while 5-hydroxymethylfurfural has also been reported in kombucha produced from several herbal substrates (Dechakhamphu et al., 2023; Suffys et al., 2023; Nam et al., 2025; Tangapo et al., 2025; Wulansari et al., 2026). These similarities suggest that the chemical profile observed in the present preparation is broadly consistent with those reported for other kombucha products.

The final chemical composition of kombucha is influenced by the original plant substrate, processing conditions, and changes occurring during SCOBY fermentation (de Miranda et al., 2022; Tran et al., 2022; Suffys et al. 2023). Accordingly, some compounds detected in the present preparation may have originated from the P. macrocarpa substrate, whereas others may have been formed or modified during fermentation. However, because the unfermented infusion and fermented preparation were not comparatively profiled by GC–MS, the specific origin of individual compounds cannot be determined. Furthermore, the present analysis was qualitative and compound assignments were tentative; therefore, the GC–MS findings should primarily be regarded as chemical characterization of the tested preparation rather than evidence linking individual metabolites to the observed fetal growth differences.

Maternal body weight increased progressively throughout gestation in all groups, and no statistically significant differences in total gestational weight gain were detected. This finding indicates that the fetal growth-associated differences were not accompanied by overt maternal weight loss or obvious clinical deterioration. Maternal condition is important in developmental studies because alterations in maternal nutritional or physiological status may secondarily influence fetal development (OECD, 2018). Nevertheless, maternal toxicity assessment was limited to body weight and general clinical observations. Food consumption, water intake, gravid uterine weight, maternal organ weights, and clinical pathology parameters were not evaluated. Therefore, subtle maternal physiological, nutritional, or metabolic effects cannot be excluded, and the present design does not allow direct fetal effects to be distinguished from potentially maternally mediated effects.

The treated groups also showed lower mean litter sizes than the control group. Although the observed litter sizes were broadly consistent with values reported for laboratory mice, litter size is known to vary according to strain and maternal factors. Therefore, overlap with reported ranges does not negate the between-group difference observed in the present study. Fetal deaths occurred sporadically in the kombucha-treated groups, while no grossly visible uterine resorption sites were identified in any group. However, because corpora lutea and total implantation sites were not recorded, implantation-related losses could not be comprehensively assessed. Consequently, the lower litter size cannot be attributed to a specific reproductive mechanism and should be interpreted as a preliminary finding. In addition, because only five litters were included per group and fetal deaths were infrequent, the available data are insufficient to exclude a treatment-associated effect on fetal mortality. 

A notable finding of the present study was the progressive reduction in litter-level mean fetal body weight from the control group to the infusion, 1% kombucha, and 2% kombucha groups. The mean fetal body weights were 1.60 ± 0.13, 1.18 ± 0.10, 1.06 ± 0.14, and 0.88 ± 0.24 g, respectively, indicating that the largest difference occurred in the 2% kombucha group. Crown–rump length showed a comparable overall pattern, with lower values in all treated groups than in the control group. These findings suggest that fetal growth may represent a sensitive developmental endpoint under the present exposure conditions. Reduced fetal body weight and crown–rump length may reflect altered prenatal growth; however, these parameters alone are insufficient to establish intrauterine growth restriction or developmental toxicity. Although the recorded fetal weights and crown–rump lengths overlapped with values previously reported for late-gestation laboratory mice, such overlap does not negate the significant between-group differences or establish their biological insignificance. These findings should therefore be regarded as preliminary growth-associated signals requiring confirmation in an adequately powered study (Theiler, 1989).

No gross external, visceral, or skeletal abnormalities were identified among the fetuses examined. Skeletal preparations showed no apparent deviations in vertebral configuration, or bone number, while visceral examination did not reveal gross craniofacial or soft-tissue abnormalities. These observations suggest that the growth-associated differences were not accompanied by overt structural disruption under the tested conditions. Minor hemorrhagic findings were observed in a small number of fetuses from the kombucha-treated groups. Given their low frequency and the limited number of litters, their biological relevance and relationship to treatment cannot be determined reliably. Moreover, the absence of gross abnormalities among the fetuses examined should not be interpreted as definitive evidence of developmental safety, because uncommon malformations or subtle developmental alterations may not be detected in a study of this size (Al-Gubory, 2017).

Several limitations should be considered when interpreting the present findings. The study included five pregnant dams, and therefore five litters, per group, which is substantially lower than the number generally recommended for a definitive prenatal developmental toxicity study under OECD Test Guideline 414. The limited number of litters reduces statistical power, increases uncertainty around the estimated effects, and restricts the ability to detect uncommon developmental outcomes. Maternal evaluation was restricted to body weight and general clinical observations, while food consumption, water intake, maternal organ weights, and clinical pathology parameters were not assessed. Therefore, subtle maternal physiological, nutritional, or metabolic influences on fetal growth cannot be completely excluded.

In addition, the ethanol concentration generated during kombucha fermentation was not quantified in the present study. Because ethanol exposure during pregnancy may represent a potential contributing factor to fetal developmental outcomes,  the relative contribution of ethanol to the observed fetal growth-associated findings cannot be evaluated. Future investigations should therefore include quantitative determination of ethanol levels in fermented preparations and consider appropriate controls to distinguish the effects of ethanol from those of plant-derived and fermentation-related metabolites.

Furthermore, corpora lutea and total implantation sites were not recorded, preventing calculation of pre-implantation loss and comprehensive assessment of post-implantation loss. Consequently, the reduction in litter size cannot be attributed to a specific reproductive mechanism. Finally, GC–MS analysis provided tentative metabolite identification based on spectral-library matching rather than quantitative confirmation using authentic reference standards. Therefore, the detected compounds should be interpreted as chemical features of the tested preparation rather than definitive bioactive constituents responsible for the observed developmental effects.

 These limitations define the exploratory nature of the present investigation and should be considered when interpreting the biological significance of the observed differences. Future studies should incorporate larger experimental groups, comprehensive maternal and reproductive assessments, quantitative ethanol determination, appropriate ethanol-controlled designs, and targeted chemical characterization using validated analytical approaches to clarify the contribution of fermentation-derived constituents to fetal developmental outcomes.

CONCLUSION

This exploratory study evaluated prenatal developmental outcomes following exposure to P. macrocarpa fruit infusion and kombucha during organogenesis in mice. Mean litter size, fetal body weight, and crown–rump length were lower in the treated groups, with the lowest fetal growth values observed in the higher-exposure kombucha group. Fetal deaths were infrequent and occurred only in kombucha-treated litters, while no grossly visible uterine resorptions or gross external, visceral, or skeletal malformations were identified. Given the limited number of litters and restricted maternal and reproductive assessments, statistical power was limited and maternally mediated contributions to the observed fetal findings cannot be excluded. Accordingly, these findings should be interpreted as preliminary growth-associated observations rather than definitive evidence of developmental toxicity or safety. GC–MS profiling provided tentative chemical characterization of the fermented preparation. Larger, adequately powered studies incorporating comprehensive maternal, reproductive, and developmental assessments together with quantitative chemical characterization are warranted.

ACKNOWLEDGEMENTS

The authors express their gratitude to Politeknik Kesehatan Kementerian Kesehatan Jambi for facilitating this research.

AUTHOR CONTRIBUTIONS

Mesa Sukmadani Rusdi: Conceptualization (Lead), Methodology (Lead), Formal Analysis (Lead), Validation (Lead), Resource (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Lead), Investigation (Equal), Supervision (Lead), Project administration (Lead); M. Rifqi Efendi: Conceptualization (Equal), Methodology (Lead), Formal Analysis (Equal), Data Curation (Equal), Validation (Lead), Investigation (Equal), Resource (Equal), Writing – Original Draft (Lead), Writing – Review & Editing (Equal); Dea Novita Sari: Methodology (Supporting), Data Curation (Supporting), Formal Analysis (Equal), Validation (Supporting), Writing – Original Draft (Supporting);  Sofiyetti: Methodology (Supporting), Data Curation (Equal), Formal Analysis (Equal), Validation (Supporting), Writing – Original Draft (Equal), Writing – Review & Editing (Equal), Investigation (Lead); Rahmah: Methodology (Supporting), Data Curation (Supporting), Formal Analysis (Equal), Validation (Supporting), Writing – Review & Editing (Supporting). 

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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