Comparative Evaluation of Different Fruit Parts of Phyllanthus emblica L. for Functional Food Applications: Nutritional Composition, Safety, and Gut Microbiota-Associated Aging Benefits

TL;DR

This study compares different parts of the Phyllanthus emblica fruit to determine which is best for functional foods that support healthy aging.

Contribution

The study provides a comparative evaluation of P. emblica fruit parts for their nutritional, safety, and anti-aging properties.

Findings

P. emblica juice powder (PJP) showed the best nutritional and antioxidant properties.

PJP improved aging-related outcomes and enriched beneficial gut bacteria like Akkermansia and Alistipes.

The seed-containing core caused hepatotoxicity and safety concerns.

Abstract

Population aging has increased interest in food-based strategies to support healthy aging. Phyllanthus emblica L. (P. emblica) is an edible fruit widely used in functional food products; however, the nutritional characteristics, safety, and functional differentiation of its distinct fruit parts generated during processing remain unclear. In this study, four anatomical parts of P. emblica (the whole fruit, P), juice powder (PJP), pomace (PP), and seed-containing core (PC) were systematically evaluated for nutritional composition, antioxidant activity, safety, and anti-aging potential using a D-galactose-induced aging mouse model. Results show that the P. emblica juice powder (PJP) retained the most favorable nutritional characteristics and exhibited superior antioxidant capacity. A clear efficacy gradient was observed (PJP > P > PP > PC), with PJP showing the most pronounced improvements in aging-associated functional outcomes and redox and inflammatory status. Given the role of the gut–brain axis in aging-related regulation, gut microbiota composition and short-chain fatty acids (SCFAs) production were further analyzed. PJP selectively enriched beneficial bacteria, including Akkermansia and Alistipes, and increased SCFA levels, particularly propionic acid and isovaleric acid. In contrast, the seed-containing core raised safety concerns, characterized by hepatotoxicity and elevated serum aminotransferases. Overall, this study establishes a clear hierarchy of efficacy and safety among P. emblica fruit parts and supports the rational selection of juice powder for functional food development targeting healthy aging.

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1. Introduction

Population aging has become a global issue, driving increasing demand for dietary strategies that support healthy aging and cognitive maintenance [1,2]. Accumulating evidence indicates that aging is closely associated with oxidative stress, chronic low-grade inflammation, and alterations in gut microbiota composition, collectively contributing to functional decline and increased disease susceptibility [3]. In this context, foods rich in bioactive compounds, particularly polyphenols and dietary fibers, have attracted considerable attention for their potential to modulate aging-related physiological changes through diet-based interventions [4,5].

Phyllanthus emblica L. (P. emblica) is a widely consumed edible fruit with long-standing applications in traditional medical systems [6,7], including Traditional Chinese Medicine [8], Tibetan medicine [9,10], and Ayurveda [11], where it has been valued for maintaining vitality and promoting longevity. The fruit is rich in vitamin C, polyphenols, flavonoids, and polysaccharides, which have been linked to antioxidant, anti-inflammatory, and gut-modulating properties [12,13]. From a food science perspective, P. emblica is typically processed into juice powders, extracts, or concentrates, generating multiple by-products such as pomace and seed-containing cores [8,14]. These different fruit parts vary substantially in physicochemical properties, nutritional composition, and potential health effects, yet systematic comparative evaluations remain limited. Most existing studies have focused on extracts or isolated compounds from P. emblica, with less attention paid to the functional differentiation and safety of whole food parts relevant to industrial processing [15,16]. In particular, juice powder represents the primary ingredient used in functional food formulations, while pomace is often discarded despite its potential as a fiber- and polyphenol-rich by-product. Conversely, the seed-containing core is sometimes retained during processing, although its suitability for long-term dietary intake has not been adequately assessed from a food safety perspective.

The gut–brain axis has emerged as a key pathway linking diet, microbial metabolites, and cognitive function [17,18]. Short-chain fatty acids (SCFAs), generated through microbial fermentation of dietary components, are critical for maintaining intestinal barrier integrity, regulating inflammatory responses, and supporting neural function [19]. Consequently, assessing the effects of distinct P. emblica parts on gut microbiota composition and SCFAs production is vital for identifying safe and effective functional food candidates.

We systematically evaluated four anatomical parts of P. emblica fruit, namely, the whole fruit (P), juice powder (PJP), pomace (PP), and seed-containing core (PC), for their nutritional composition, antioxidant properties, safety profile, and anti-aging effects. These assessments were conducted using a well-established D-galactose (D-Gal)-induced aging mouse model, which is commonly employed to simulate aging-related oxidative stress and cognitive decline. Recognizing the central role of gut microbiota and microbial metabolites in modulating cognitive and physiological functions, we further investigated how these fruit parts influence gut microbiota composition and SCFAs production. This integrative approach, combining food composition, physiological outcomes, and microbial metabolite analyses, allows us to (i) identify the most appropriate part for functional food applications, (ii) evaluate the reutilization potential of by-products generated during processing, and (iii) provide a rigorous assessment of food safety, particularly concerning the potential toxicity of the seed-containing core.

2. Materials and Methods

2.1. Materials and Reagents

Rutin, gallic acid, anhydrous glucose, Trolox, and cellulase were obtained from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Sodium hydroxide, sodium nitrite, aluminum nitrate, phenol, disodium hydrogen phosphate, and sodium carbonate were of analytical grade and purchased from commercial suppliers in China. A standard mixture of 11 short-chain fatty acids (SCFAs) was obtained from Zhenzhun Biotechnology (Shanghai, China). ELISA kits for TNF-α: 1217202 and IL-6: 1210602 were purchased from Dakewe Biotech (Beijing, China), and assay kits for total superoxide dismutase (SOD: G4306-96T) and malondialdehyde (MDA: G4302-95T) were purchased from Servicebio (Wuhan, China), tannin (BC1390), vitamin C (E-BC-K034-S), Alanine Aminotransferase (ALT: BC1555); Aspartate Aminotransferase (AST: BC6435) were from commercial sources (Servicebio, Solarbio, and Elabscience, Wuhan and Beijing, China). The Morris water maze (MWM) test was performed using the VisuTrack system (XinRuan, Shanghai, China).

2.2. Materials and Sample Preparation

Fresh P. emblica fruits (15 kg) were collected from Baoshan, Yunnan Province, China, and authenticated by Prof. Baozhong Duan (Dali University). The fruits were washed, manually deseeded, and the pulp was processed using a slow juicer (Joyoung Co., Ltd., Jinan, China; Model Z11-LZ868) to separate the juice from the pomace. The obtained juice was then subjected to stepwise centrifugation to remove residual insoluble particles: first at 109× g for 10 min, followed by 977× g for 20 min, and finally at 10,850× g for 40 min using an HC-3018R refrigerated centrifuge (Anhui Zhongke Zhongjia, Hefei, China) equipped with a 6 × 50 mL fixed-angle rotor. The clarified supernatant was lyophilized to obtain P. emblica juice powder (PJP). The pomace (PP) and fruit cores (PC) were separately freeze-dried, ground, and stored at 4 °C. These preparations allowed comparison of bioactivities among different fruit parts, in accordance with the traditional concept of “different parts, different functions” in Chinese medicine.

2.3. Nutrition Ingredient Analysis

The total phenolic content (TPC) of the fig pulp extract powder (PJP) was quantitatively determined using the Folin–Ciocalteu colorimetric method. Simultaneously, rutin, gallic acid, and anhydrous glucose were used as calibration standards to measure the total flavonoid content (TFC) and total sugar content, respectively. Accordingly, the results were expressed as rutin equivalents, gallic acid equivalents, and glucose equivalents. Additionally, the contents of ellagic acid and vitamin C were determined using commercial assay kits following the manufacturers’ instructions.

2.4. Antioxidant Activity Assays

2.4.1. DPPH Radical Scavenging Assay

The free radical scavenging activity of P. emblica fractions was assessed using the DPPH assay [20]. Briefly, 100 μL of sample solution at varying concentrations (0.1–2 mg/mL) was mixed with 100 μL of 0.1 mM DPPH solution in methanol. The reaction mixture was incubated in the dark at room temperature for 30 min, after which the absorbance was measured at 517 nm using a microplate reader. Radical scavenging activity was expressed as the percentage inhibition relative to a control, with Trolox serving as a positive control.

2.4.2. ABTS Radical Cation Scavenging Assay

ABTS radical cation (ABTS^+^) was generated by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate, followed by incubation in the dark at room temperature for 16 h. The resulting ABTS^+^ solution was diluted with ethanol to achieve an absorbance of 0.70 ± 0.02 at 734 nm. Subsequently, 100 μL of sample solution at varying concentrations (0.1–2 mg/mL) was mixed with 100 μL of the diluted ABTS^+^ solution and incubated at room temperature for 6 min. Absorbance was then measured at 734 nm, and the results were expressed as percentage inhibition and Trolox equivalents (μmol TE/g of sample).

2.5. High-Performance Liquid Chromatography

HPLC analysis was performed using an Agilent ZORBAX SB-C18 analytical column (4.6 mm × 250 mm, 5 μm) maintained at 25 °C. Separation was carried out with a binary gradient system consisting of methanol (mobile phase B) and 0.1% phosphoric acid in water (mobile phase D) at a constant flow rate of 1.0 mL·min^−1^. The effluent was monitored at 270 nm, and a 10 μL aliquot was injected for each analysis. It should be noted that this HPLC analysis was intended for qualitative fingerprinting to compare the overall chromatographic profiles among fractions; no quantitative determination or calibration curves were performed.

2.6. Scanning Electron Microscopy and Particle Size Distributions

The microstructure of PJP was examined using a scanning electron microscope (SEM; JEOL, Akishima City, Tokyo Metropolitan Area, Japan, TM4000plus). Powder samples were mounted on aluminum stubs using double-sided conductive carbon tape and sputter-coated with a thin layer of gold to enhance conductivity. Images were captured at various magnifications to observe surface morphology and particle structure. Hydrodynamic particle size distribution was measured by dynamic light scattering (DLS) using a particle size analyzer (Litesizer DLS 500, Graz, Austria). Samples were appropriately diluted with ultrapure water before measurement, and all measurements were performed in triplicate at 25 °C.

2.7. Animal Experiment Design

Seventy-nine male C57BL/6J mice (6 weeks old) were obtained from SPF Biotechnology (Beijing, China). After one week of acclimatization, mice were randomly assigned to six groups: CON (control), MODEL (D-Gal, 400 mg/kg, i.g.), P (D-Gal + P, 200 mg/kg, i.g.), PJP (D-Gal + PJP, 200 mg/kg, i.g.), PP (D-Gal + PP, 200 mg/kg, i.g.), and PC (D-Gal + PC, 200 mg/kg, i.g.). The CON group included 14 mice, while each of the remaining groups contained 13 mice. The experiment lasted nine weeks. During the first week, mice were allowed to adapt to housing conditions. From weeks 2 to 9, D-Gal administration and treatment with the respective P. emblica fractions were performed simultaneously once daily, generally between 9:00 and 11:00 AM (Beijing time) to ensure consistency in metabolic conditions. Behavioral assessments, including the Morris Water Maze, were conducted during the final week. All treatments were administered via intra-gastric gavage (i.g.), with doses determined from preliminary tests to ensure efficacy and safety. All procedures were conducted in accordance with the Institutional Animal Care and Use Committee guidelines of Dali University (Approval No. 2024-P2-132).

2.8. Behavioral Testing

Cognitive abilities were studied with the Morris water maze (MWM) [21]. The MWM test was conducted in a circular pool filled with water maintained at 22 ± 1 °C and divided into four quadrants (Shanghai Xinruan Open Field Experimental Module). This protocol was designed based on a previous study.

2.9. Enzyme-Linked Immunosorbent Assay (ELISA)

At the end of the experiment, blood samples were collected from the retro-orbital sinus, allowed to clot at room temperature for 30 min, and then centrifuged at 3000 rpm for 20 min. The resulting serum was collected and stored at −20 °C until analysis. Total superoxide dismutase (T-SOD) activity and malondialdehyde (MDA) content were measured using commercial assay kits (T-SOD: G4306-96T; MDA: G4302-95T; Servicebio, Wuhan, China). Serum TNF-α and IL-6 levels were quantified using commercial ELISA kits (TNF-α: 1217202; IL-6: 1210602; Dakewe Biotechnology, Shenzhen, China) following the manufacturers’ protocols. For ELISA measurements, the serum volume added to each well adhered to kit instructions, and all samples were analyzed in triplicate. Absorbance was measured with a microplate reader, and cytokine concentrations were calculated using standard curves provided with the kits. Detection limits were defined according to the manufacturers’ specifications.

2.10. Hematoxylin and Eosin (H&E) Staining

Heart, liver, spleen, lung, kidney, and brain tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin (H&E) for histopathological evaluation.

2.11. Nissl Staining

Nissl staining was performed to assess neuronal morphology in the hippocampus. Briefly, brain tissues were fixed in 4% paraformaldehyde at 4 °C for 24 h and subsequently embedded in paraffin. Coronal sections, 5 μm thick, were prepared using a microtome (Leica, Wetzlar, Germany) and rehydrated through a graded ethanol series. The sections were then stained using a commercial Nissl staining kit (Servicebio, Wuhan, China). Images were captured under a light microscope at 200× magnification (Leica, Wetzlar, Germany), and neuronal numbers in the hippocampus were quantified using ImageJ software (ImageJ 1.54p).

2.12. Golgi Staining

Golgi–Cox staining was performed to visualize dendritic spines, which were quantified in the dentate gyrus using light microscopy. Briefly, images were analyzed with ImageJ software, and dendritic morphology parameters were measured according to standardized procedures. Neurons were randomly selected, and all analyses were conducted in a blinded manner to minimize bias. Quantified data were averaged for each animal prior to statistical analysis.

2.13. Quantitative Real-Time PCR (qPCR) Analysis

Quantitative PCR (qPCR) was performed to validate the abundance of specific cognition-related gut bacteria, particularly Akkermansia muciniphila, which exhibited the most pronounced change following PJP treatment in the 16S rRNA sequencing analysis. Total DNA was extracted from fecal samples using a commercial kit (TianGen, Beijing, China). qPCR was conducted with SYBR Green Master Mix (Servicebio, Wuhan, China) using primers synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Primers were designed to specifically target the 16S rRNA gene of Akkermansia muciniphila (forward: 5′-CAGCACGTGAAGGTGGGGAC-3′; reverse: 5′-CCTTGCGGTTGGCTTCAGAT-3′) [22]. Standard curves were generated by serial dilution of reference DNA to determine bacterial abundance.

2.14. 16S rRNA Gene Sequencing

Fecal microbial DNA was isolated using the E.Z.N.A.^®^ Stool DNA Kit (Omega Bio-tek, Norcross, GA, USA). The V4 region of the 16S rRNA gene was amplified (primers 515F/806R), and PCR products were sequenced on the Illumina MiSeq platform (2 × 300 bp, Biozeron, Shanghai, China). Quality-filtered reads were clustered into operational taxonomic units (97% similarity), and α/β-diversity analyses were performed using QIIME 2.

2.15. Short-Chain Fatty Acid Analysis

Fecal short-chain fatty acids (SCFAs), including acetic, propionic, butyric, isobutyric, valeric, and isovaleric acids, were quantified using gas chromatography (GC). Briefly, approximately 300 mg of fecal material was homogenized in 1.5 mL of deionized water and centrifuged at 12,000× g for 10 min at 4 °C. An aliquot of the supernatant (300 μL) was acidified with 30 μL of formic acid and subsequently filtered through a 0.45-μm polysulfone membrane prior to GC analysis. Standard solutions of SCFAs (Sigma-Aldrich, St. Louis, MO, USA) at a series of concentrations were prepared in deionized water to generate external calibration curves. GC analysis was conducted using an Agilent 7820A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID). Chromatographic separation was achieved under the following oven temperature program: an initial temperature of 50 °C, increased to 120 °C at a rate of 15 °C/min, followed by an increase to 170 °C at 5 °C/min, and finally raised to 210 °C at 15 °C/min, where it was held for 3 min. The injector and detector temperatures were maintained at 240 °C and 200 °C, respectively. Helium was used as the carrier gas at a flow rate of 1 mL/min, and the injection volume was 1 μL. Quantification of SCFAs in fecal samples was performed using external standard calibration curves.

2.16. Statistical Analysis

All tests are repeated three times or more and the results are shown as mean ± SEM. All statistical analyses were performed using GraphPad Prism 9.0 software. For comparison of two groups, the two-tailed independent Student’s t-test was used. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey’s post hoc test for multiple comparisons. Statistical significance: p < 0.05 (), p < 0.01 (), p < 0.001 (), p < 0.0001 (****), ns: no significant difference.

3. Results

3.1. The Physicochemical Characteristics and Antioxidant Properties in Different Parts of P. emblica

To compare the bioactivity potential of different anatomical parts of P. emblica, we analyzed their phytochemical profiles, antioxidant activities, and microstructural characteristics (Figure 1A). Among all the fractions, PJP exhibited the highest levels of total phenolic compounds, flavonoids, and vitamin C, as well as the greatest ABTS and DPPH values. In both ABTS and DPPH assays, PJP demonstrated the strongest free radical scavenging activity, expressed as milligrams of Trolox equivalents per gram of dry weight (Table 1) [23,24]. The P ranked second, PP and PC showed lower bioactive contents and significantly weaker antioxidant activities [24]. Scanning electron microscopy of the dry samples revealed distinct surface morphologies: PJP particles appeared smooth and uniform, facilitating rapid dissolution and compound release, whereas P, PP, and PC powders exhibited rough and irregular surfaces (Figure 1B–E). Particle size analysis in aqueous solution confirmed that PJP had the smallest mean particle diameter, followed by P, PP, and PC. (Figure 1F–I). In order to preliminarily compare the chemical composition characteristics of different parts of the P. emblica, we conducted HPLC analysis, and the fingerprint is shown in Figure 2. According to previous research findings [25,26,27], they are: 1. gallic acid; 2. epigallocatechin; 3. chebulinic acid; 4. ellagic acid. Collectively, these results suggest that the compositional richness and favorable microstructural properties of PJP synergistically contribute to its superior antioxidant activity.

3.2. Safety and Tolerability Assessment of Different P. emblica Fruit Fractions During Dietary Intervention

To evaluate the safety profiles of different P. emblica parts, a D-Gal-induced aging mouse model was established and treated for 8 weeks (Figure 3A). Throughout the experiment, mice in the model group exhibited progressive weight loss, whereas intragastric administration of PJP effectively reversed this trend (Figure 3B) [28,29]. Organ-level safety assessments revealed no significant abnormalities in organ indices for the PJP, P, and PP groups (Figure 3C). In contrast, the PC group showed a significantly increased liver index, indicative of mild hepatomegaly (Figure 3D). Histological evaluation of other major organs (heart, spleen, lung, kidney and, brain) showed no detectable damage across all treatment groups (Figure 3E). Histopathological examination of the liver further confirmed distinct injury in the PC group, characterized by hepatocellular ballooning, cytoplasmic rarefaction, and nuclear loss (Figure 3F). As shown by the blue arrow, the green arrows in the other groups represent normal liver cells. These morphological findings were supported by serum biochemical analyses, with markedly elevated ALT and AST enzyme activities observed in the PC group (p < 0.01), while levels remained normal in the PJP, P, and PP groups (Figure 3G,H). In summary, dietary intake of the seed-containing core was associated with marked hepatic structural alterations and elevated liver enzyme levels, indicating impaired liver function and potential safety concerns. These data demonstrate that 8-week administration of PJP, P, and PP is safe, while the PC part induces potential adverse effects on liver health.

3.3. The Effects of Different Parts of P. emblica on the Cognitive Abilities in Aging Mice

Cognitive function was assessed using the MWM test (Figure 4A) [21]. During the training phase, all groups showed progressive reductions in escape latency; however, mice treated with PJP, P, or PP exhibited significantly shorter latencies compared to the D-Gal model group (p < 0.01; Figure 4D). In the subsequent probe trial, the PJP, P, and PP groups demonstrated increased platform crossings and spent more time in the target quadrant, indicating improved spatial memory retrieval (Figure 4C). In contrast, the PC group performed comparably to the D-Gal model, showing no significant cognitive improvement. Notably, the swimming speed of the PC group was significantly slower (Figure 4B). Additionally, Nissl staining of the hippocampus revealed no neuronal abnormalities in the PJP, P, or PP groups, whereas significant neuronal shrinkage was observed in the model group (Figure 5A). To investigate underlying neural mechanisms, hippocampal neuronal morphology was examined using Golgi-Cox staining [30]. D-Gal exposure caused dendritic atrophy and spine loss in the hippocampus, whereas PJP and P treatment preserved dendritic architecture and spine density at levels comparable to controls (Figure 5B,C). The PP group showed moderate recovery, while the PC group exhibited minimal effects. Collectively, these behavioral and morphological results demonstrate that PJP and P effectively ameliorate D-Gal-induced learning and memory impairments, likely by preserving hippocampal synaptic integrity.

3.4. The Effects of Different Parts of P. emblica on Oxidative Stress and Neuroinflammation in Aging Mice

Systemic oxidative stress and chronic inflammation are key hallmarks of aging [31]. In this study, D-Gal exposure significantly increased serum malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity, confirming the induction of oxidative stress. Oral administration of both PJP and P effectively counteracted these changes, significantly normalizing MDA and SOD levels (Figure 6C,D). Concurrently, D-Gal induced a pro-inflammatory state, as evidenced by elevated serum levels of TNF-α and IL-6 (Figure 6E,F). Consistent with the oxidative stress findings, PJP markedly suppressed this cytokine surge, while PP and PC failed to elicit a significant anti-inflammatory effect. To determine if systemic inflammation translated to neuroinflammation, immunofluorescence staining for ionized calcium-binding adaptor molecule 1 (IBA1) was performed, revealing pronounced microglial activation in D-Gal-treated mice, a hallmark of neuroinflammation. This activation was substantially attenuated by PJP and P treatments (Figure 6A,B). Collectively, these results highlight PJP and P as the most effective parts in mitigating systemic oxidative stress, inflammation, and associated neuroinflammation.

3.5. The Selective Remodeling of the Gut Microbiota by Different Parts of P. emblica in Aging Mice

Gut microbiota dysbiosis is a key contributor to aging-related cognitive decline [32]. To assess the effects of different P. emblica parts, we conducted 16S rRNA sequencing on colon contents. Our analysis revealed that both D-Gal-induced aging and subsequent treatments with P. emblica parts significantly altered the microbial community structure, as reflected by changes in alpha and beta diversity (Figure 7A,B). At the phylum level, the abundance of Firmicutes was higher in the P, PP, and PC groups compared to the model group. Notably, Verrucomicrobiota abundance significantly increased in the PJP group, while Bacteroidota levels showed no significant differences across groups. At the genus level, the P group exhibited significant increases in Lactobacillus and Lachnoclostridium, both associated with enhancing intestinal barrier function and reducing chronic inflammation [33]. In the PJP group, Akkermansia and Alistipes, genera linked to maintenance of the intestinal mucosal barrier, were enriched [34]. The PP and PC groups also showed elevated levels of Lactobacillus and Lachnoclostridium relative to the model group (Figure 7C). Overall, these findings demonstrate that different parts of P. emblica possess distinct capacities to modulate the gut microbiota [35].

Simultaneously, LEfSe analysis (LDA score > 4) was performed to identify microbial taxa significantly enriched in each group [36]. In the PJP group, Verrucomicrobiota was predominantly represented by Akkermansia, along with several genera associated with preservation of the intestinal mucus layer and metabolic regulation. This cluster emerged as a key biomarker shaping the gut microbiota of elderly mice treated with PJP [12]. In contrast, Roseburia dominated the microbiota in the P group, while the PP group was characterized primarily by Alistipes and Blautia. The PC group showed predominance of Lachnoclostridium (Figure 8A–C). To validate the 16S rRNA sequencing results at the species level, quantitative PCR specific for Akkermansia muciniphila was conducted, confirming a significant increase in A. muciniphila abundance in the PJP group (Figure 8D), consistent with sequencing data [37]. Overall, these results suggest that among the P. emblica parts tested, PJP most effectively modulates the gut microbiota by selectively enriching beneficial bacteria such as A. muciniphila and Alistipes, thereby reversing D-Gal-induced gut microbiota dysbiosis.

3.6. The Effects of Different Parts of P. emblica on SCFAs Profiles and Microbial Correlations in Aging Mice

SCFAs, key products of microbial fermentation, play vital roles in maintaining intestinal and metabolic homeostasis [38]. Dysregulation of SCFAs metabolism is increasingly linked to gut dysbiosis and cognitive decline during aging [39]. To assess whether different P. emblica parts modulate SCFAs production in D-Gal-induced aging mice, 11 SCFAs were quantified in colon contents, with eight consistently detected (Figure 9C) [5]. D-Gal treatment significantly reduced several neuroprotective SCFAs, including acetic, propionic, isovaleric, and valeric acids, indicating impaired microbial metabolic activity. Intragastric administration of PJP significantly restored these metabolites, particularly isovaleric and propionic acids, while PP and PC had only modest effects. KEGG enrichment analysis revealed that altered metabolites were primarily involved in propanoate and butanoate metabolism, glycolysis/gluconeogenesis, and pyruvate metabolism, with notable enrichment in nicotinate and nicotinamide pathways related to NAD^+^ biosynthesis (Figure 9A). Pearson correlation analysis identified distinct associations between SCFAs and bacterial genera (Figure 9B): Alistipes correlated positively with acetic, propionic, and butyric acids; Lachnoclostridium correlated with butyric acid; and Roseburia correlated with isobutyric acid. Lactobacillus showed a positive correlation with isovaleric acid but a negative correlation with several other SCFAs, consistent with bacterial abundance changes at the genus level. These findings suggest that enrichment of Alistipes and Lachnoclostridium in PJP-treated mice contributes to enhanced SCFAs production. Overall, PJP most effectively remodeled microbial metabolism by increasing neuroprotective SCFAs, potentially improving gut–brain axis integrity and cognitive function, whereas PP and PC exhibited weaker effects.

4. Discussion

In this study, we systematically compared four freeze-dried parts of P. emblica, whole fruit (P), juice powder (PJP), pomace (PP), and core (PC), using a D-Gal-induced aging mouse model. PP demonstrated moderate protective effects, while PC showed limited efficacy and induced adverse effects on liver health. In contrast, our results consistently revealed a clear efficacy gradient (PJP > P > PP > PC). Notably, PJP and P provided comprehensive benefits, including enhanced cognitive function, preservation of dendritic spine integrity, restoration of redox and inflammatory balance, and remodeling of gut microbiota and SCFAs metabolism. The observed neuroprotective and anti-aging effects of PJP may be attributed, at least in part, to the abundant phenolic and flavonoid compounds present in P. emblica, which are well-known for their antioxidant activity. To our knowledge, this is the first systematic comparison of the anti-aging potential among distinct P. emblica parts. Our comparative investigation highlights two major findings that clarify traditional controversies and offer new mechanistic insights.

I.Re-evaluating the seed-containing core from a food safety and long-term consumption perspective.

One of the most important findings of this study is the unfavorable hepatic response associated with the PC of P. emblica, raising concerns regarding its suitability as a food-derived ingredient for long-term consumption. Histopathological analysis revealed evident hepatic alterations, including hepatocellular ballooning, cytoplasmic rarefaction, and nuclear loss, suggesting substantial hepatocellular stress following prolonged exposure [40]. It is noteworthy that fatty acid constituents present in the seed have been reported to exhibit antioxidant properties, which may partially explain the modest reductions in systemic oxidative stress markers observed in this study, consistent with previous reports [41,42]. However, such limited antioxidative benefits do not offset the pronounced hepatic safety risks identified. From a food science and nutrition perspective, safety considerations are particularly critical for edible materials intended for frequent and long-term intake, as is often the case for medicinal edible fruits such as P. emblica [43]. Plant-derived food materials, especially seeds and cores, may contain anti-nutritional factors or potentially harmful constituents that are not suitable for chronic consumption when concentrated or insufficiently processed [44]. This issue is particularly relevant for anti-aging and preventive dietary applications, where cumulative exposure may impose an increased hepatic burden over time [45]. Similar part-specific safety concerns have been reported in other edible plants, underscoring the need to distinguish edible and non-edible fractions during food processing and product formulation [46,47]. Collectively, these findings suggest that the seed-containing core of P. emblica should be excluded from functional food development and long-term dietary applications. This study therefore provides practical and evidence-based guidance for the rational selection of safe fruit fractions, supporting the removal of the PC during processing to ensure consumer safety and sustainable utilization of P. emblica as a functional food resource.

II.PJP exerts superior anti-aging effects via unique microbial and metabolite interactions

PJP demonstrated the most potent anti-aging efficacy, attributable not only to its systemic effects but, importantly, to its distinctive modulation of the gut–brain axis. A key finding was the synergistic enrichment of Akkermansia muciniphila (phylum Verrucomicrobiota) and Alistipes (phylum Bacteroidetes), a signature unique to the PJP group [48]. A. muciniphila is known to enhance gut barrier integrity and maintain immune-metabolic homeostasis, while Alistipes plays a significant role in restoring levels of acetate and propionate, key SCFAs with established neuroprotective properties [32]. This dual modulation suggests a complementary mechanism: A. muciniphila strengthens intestinal barrier function, whereas Alistipes supplies beneficial microbial metabolites [49]. Moreover, the PJP-induced enrichment of Alistipes alongside increased acetic and propionic acid levels directly links this genus to improved microbial metabolite profiles and neuroprotection, an association rarely emphasized in previous anti-aging studies [19,50]. This synergistic microbial remodeling, targeting multiple facets of gut–brain communication, advances our understanding of how P. emblica mediates its anti-aging effects and highlights the critical role of targeted microbial regulation.

This study has two primary limitations. First, the specific hepatotoxic components within the PC remain unidentified. Notably, no toxicity was observed with the whole fruit (P), which may be due to the relatively low content of PC in the whole fruit, insufficient to cause toxicity, or the presence of detoxification mechanisms within P that mitigate the potentially harmful components of PC. Clarifying these mechanisms is essential to ensure the safety of P. emblica products. Second, the precise mechanism by which PJP-induced remodeling of the intestinal microbiota improves cognitive impairment in aging mice via the gut–brain axis has yet to be elucidated. Future research should address these gaps by: (1) employing modern separation and structural identification techniques to isolate and characterize the hepatotoxic constituents in PC, and investigating potential detoxification pathways within the whole fruit (P) to support the safe use of P. emblica formulations; (2) utilizing multi-omics approaches to deeply explore the molecular mechanisms underlying PJP’s modulation of the gut–brain axis, including regulation of relevant signaling pathways and neurotransmitter alterations, to provide a robust theoretical foundation for P. emblica-based anti-aging therapies.

5. Conclusions

This study systematically evaluated different anatomical parts of P. emblica and, innovatively, identified potential liver-related safety risks associated with long-term consumption of the seed-containing core (PC), underscoring the critical need for site-specific safety assessments of natural products. Concurrently, we identified distinct anti-aging effects among the parts in a D-Gal-induced aging model, establishing a clear efficacy gradient, PJP > P > PP > PC, with PJP emerging as the most promising candidate for further development. Mechanistically, the superior efficacy of PJP is closely linked to its unique enrichment of beneficial gut bacteria such as Akkermansia and Alistipes, enhancement of SCFAs metabolism, and subsequent improvement of the gut–brain axis function. In summary, our findings confirm that the efficacy and safety profiles of P. emblica are highly dependent on its specific anatomical parts. These insights not only highlight PJP as a targeted part for developing anti-aging functional foods and therapeutics while issuing safety warnings regarding PC, but also provide a solid theoretical foundation for future precise research and rational utilization of P. emblica.