Polyphenolic antioxidants capable of targeting the intestine and modulating the intestinal microbiota, while reducing intestinal inflammation, have been shown to influence memory, cognition, mood and behavior, thereby contributing to the prevention and treatment of various brain disorders [
25]. Polyphenols have garnered attention for their ability to stimulate certain gut bacteria involved in phenolic compound utilization and metabolism, making them a proposed treatment option for neurological disorders [
43,
44]. Polyphenols are the most ubiquitous phytochemicals in the human diet, with a total daily intake of approximately 1 g [
45]. These phenolic compounds, as secondary metabolites in plants, primarily protect them against various aggressions or infections caused by bacteria, insects or viruses [
43,
44]. These protective properties in plants are expected to be beneficial for humans as well. Polyphenolic compounds found in green and black tea are the most potent inhibitors of microbial growth.
Hence, considering the advantageous effects observed on the microbiota from polyphenols, including EGCG, a comprehensive mini-review was proposed. This review was based on a thorough scientific search of published studies in PubMed, without any limitations on publication date. The objective was to examine the potential advantages that EGCG might offer to individuals with ASD. The search was from January to April 2023 and utilized MeSH descriptors like "epigallocatechin gallate," "autism spectrum disorder," "metabolism," "microbiota," and "fatty acids," combined using the boolean operator "AND.".
3.1. Possible Role of EGCG in the Intestinal Microbiota of Patients with ASD
To date, there is a lack of published studies that have used EGCG in ASD. However, there are cellular and animal studies [
57,
58] that clearly demonstrate the potential of this antioxidant for clinical treatment of ASD. Additionally, in recent years several studies have been published employing other polyphenols that are structurally similar to EGCG, such as luteolin or quercetin, which have achieved evident clinical benefits in the disease [
59,
60,
61,
62]. Its consumption is linked to reduced psychological distress [
63], possibly by inhibiting the growth of specific pathogenic bacteria such as
Clostridium perfringens, Clostridium difficile [
64],
Bacteroides ovatus, and
enterobacteria (
Salmonella spp., Escherichia coli, Yersinia pestis, Klebsiella spp., Shigella spp. and
Eggerthella spp.) [
49,
65,
66,
67]. Furthermore, in the intestinal microbiota, EGCG increases the abundance of
Bifidobacterium spp. determined through in vitro studies [
65] or in Drosophila models of Parkinson’s disease [
66]. EGCG also increases in vitro bacteria of the Bacteroides genus (
Bacteroides uniformis,
Bacteroides stercoris, Bacteroides thetaiotaomicron and
Bacteroides cellulosilyticus), and
Lachnoclostridium spp. [65 in Male C57BL/6N mice
Akkermansia spp. [
67] and in ovariectomized (OVX) mice fed a high fat diet (HFD)
Prevotella spp. [
68].
A substantial portion of the vital microorganisms in the fecal microbiota of breastfed infants consists of species from the Bifidobacterium genus [
69].
Bifidobacterium spp. has psychobiotic effects that help reduce anxiety, stress, and other depressive behaviors, particularly strains
B. longum and
B. breve[
70,
71]. This bacterial genus is one of the initial colonizers of the newborn's intestines, and an imbalance in
Bifidobacterium spp. can affect infant neurodevelopment [
71]. Administering EGCG to increase
Bifidobacterium spp. has been found to enhance the intestinal microbiota, as supported by previous animal studies that showed an elevated abundance of
Bifidobacterium spp. in response to a diet enriched with EGCG[
72].
Bacteria within the Bacteroides genus play a crucial role breaking down complex molecules, particularly carbohydrates, and confer advantages to their hosts by impeding the colonization of potential pathogens in the digestive tract [
73,
74]. It has been seen that there are bacteria capable of modulating the immune system. For instance,
B. fragilis has been shown to restore the balance of T-cell populations in mice affected by ASD [
75], while
B. uniformis enhances the production of anti-inflammatory cytokines and improve metabolic and immune dysfunction [
65]. Moreover, this genus can improve social behaviors and physiological abnormalities in individuals with ASD [
76], indicating that an increase in
Bacteroides abundance caused by EGCG could enhance the quality of life in patients. Additionally, this polyphenol can reduce
B. ovatus, the main intestinal commensal responsible for a systemic antibody response in inflammatory bowel disease [
77]. These findings suggest that even within the same genus, EGCG may exert different effects on bacterial species.
Prevotella spp. are bacteria that exist in the human microbiota, and are responsible for degrading plant polysaccharides and participating in the synthesis of vitamin B1 [
78]. One specific species within the Prevotella genus is
Prevotella copri, which has been linked to enhanced glucose and insulin tolerance. It is commonly found in individuals who consume a diet rich in fiber, indicating a strong association between the effects of this bacterium and dietary habits. Within the Prevotella genus,
Prevotella copri can be found. This species has been associated with improved glucose and insulin tolerance and is commonly found in individuals who follow a fiber-rich diet, suggesting a strong connection between the effects of this bacterium and dietary patterns [
79,
80,
81,
82]. Consequently, both the bacteria within of the
Prevotella genus and their metabolic activity are significantly relevant to ASD: on one hand, children with ASD present higher risk of obesity, which implies insulin resistance [
83], an on the other hand, these children exhibit selective eating patterns, with notable preference for high amounts of simple carbohydrates and low intake of fiber [
84]. This dietary behavior is associated with lower levels of
Prevotella spp. within the microbiota. Additionally, it is worth noting that individuals with ASD have been reported to have low levels of vitamin B1, which plays a significant role in antioxidant activity [
85].
In relation to the Lachnoclostridium genus, it has been seen to possess anti-inflammatory properties and contribute to the maintenance of intestinal balance by producing butyric acid [
86]. This metabolite aids in the elimination of intestinal gases and its absence is associated with irritable bowel syndrome (IBS) [
87]. Therefore, the increase of
Lachnoclostridium spp. mediated by EGCG [
56] can be seen as a positive impact on the microbiota of ASD patients.
EGCG modulates intestinal microbiota and its metabolites, enriching the population of short-chain fatty acid (SCFA)-producing bacteria such as
Akkermansia spp., resulting in an improvement in the production of acetate, propionate and butyrate in a sodium dextran sulfate (SDS)-induced colitis mouse model, where levels of these metabolites were significantly reduced [
88,
89]; this promotes an anti-inflammatory and antioxidant state in the intestine. [
88].
Akkermansia spp. utilizes intestinal epithelial mucin as a source of energy. By degrading mucin, it releases nutrients such as monosaccharides, amino acids and SCFAs, which are used by other bacteria in the microbiota, stimulating their metabolic functions [
90].
Akkermansia spp. is involved in the regulation of glucose metabolism and adipose tissue homeostasis, therefore, the fact that EGCG increases the levels of
Akkermansia muciniphila improves intestinal dysbiosis and enhances barrier integrity [
67,
91,
92,
93].
Moreover, among the bacteria reduced by EGCG, Clostridia stand out as a group of Gram-positive bacilli that, when in excess, can lead to infection in the large intestine [
28]. Clostridia produce significant compounds such as butyrate or butyric acid, along with other SCFAs generated from the fermentation of dietary fiber. [
94]. Butyrate is an essential metabolite in the human colon as it is the preferred energy source for colonic epithelial cells. It contributes to maintaining intestinal barrier functions and has immunomodulatory and anti-inflammatory properties [
95]. However, excessively high levels of this metabolite in the intestinepromote intestinal permeability, which may allow the passage of toxic substances into the bloodstream, potentially leading to inflammation [
94]. Thus, EGCG's inhibitory effect on this bacterial group, as observed in rats and resulting in decreased levels of
Clostridium spp., balancing their levels, beneficial [
72]. This is particularly relevant for
Clostridium perfringens, as it significantly impacts disease symptomatology, including gastrointestinal issues. Both the bacterium itself and the gene that produces its toxin (CPB2) have been linked to gastrointestinal complications in ASD and are correlated with disease severity. Moreover, the isolated species from children with ASD show greater antibiotic resistance compared to healthy children [
96]. Conversely, the increase of
Clostridium difficile has a notably negative impact on ASD, and effective treatments against this bacterium using oral vancomycin have shown improvements in behavior and communication [
97,
98]. Therefore, the reduction of this strain mediated by EGCG could be beneficial for the clinical presentation of children affected by ASD.
EGCG treatment also leads to a decrease in enterobacteria, which are commonly present in the body but can cause infections when their growth is uncontrolled. Certain species have been linked to specific pathologies. Specifically,
Salmonella typhi is responsible for typhoid fever,
Shigella dysenteriae is the causative agent of bacillary dysentery and causes infantile gastroenteritis, and
Yersinia pestis causes plague [
99].
Lastly, it is important to highlight that specific bacteria increased by EGCG, such as
Lactobacillus acidophilus, Bifidobacterium longum, Akkermansia muciniphila and
Prevotella ruminicola, restore the balance of the intestinal microbiota when included to the diet, reducing oxidative stress in the intestine and the brain [
100] (
Table 1).
3.2. Outlook on the Anti-Inflammatory and Antioxidant Activity of EGCG in Autism. Neuroprotective Role
Immune system dysregulation, inflammation, and increased oxidative stress are significant factors in ASD [
101], which is directly linked to dysbiosis. Comorbid inflammatory conditions associated with immune dysregulation closely contribute to the emergence and progression of clinical features in children with ASD [
102,
103,
104]. Cytokines, small proteins regulating inflammation and neurological development. Increased levels of some of them, particularly, IL-4 o IL-10, have been seen in children with ASD[
105]. Notable changes in interferon-α (IFN-α), interleukin-7 (IL-7), IFN-γ-inducible protein-10, and IL-8 are particularly relevant to the disorder's pathogenesis [
106].
IL-1β and IL-10 levels produced by blood monocytes, along with the IL-1β/IL-10 ratio and miRNA expression, are relevant to innate immunity [
107,
108]. All these alterations closely associate with social behavioral impairments and cognitive development in children with ASD [
109], which could be due to a brain dysfunction associated with the proper production of growth factors and neurogenesis [
110]. Specifically, the brain-derived neurotrophic factor (BDNF) plays important roles in the formation, branching and connectivity of synaptic connections during development [
111,
112], as well as in synaptic plasticity, as it is involved in learning and memory [
113,
114].
Oxidative stress is directly linked to the intestinal microbiota, particularly in children with ASD [
115]. The fermentation of dietary fibers and resistant starch in the gut generates SCFAs [
116], with propionate being associated with gastrointestinal issues and neuroinflammation in ASD [
117]. Consequently, oxidative stress, closely tied to dysbiosis, plays a crucial role in the neuroinflammatory response and the development of ASD [
118,
119,
120,
121,
122]. Children with autism exhibit differences in antioxidant capacity and oxidative stress-related metabolites compared to healthy children [
123]. Imbalance in glutathione levels and decreased storage capacity contribute to increased susceptibility to oxidative stress in autistic children [
124]. Moreover, increased free radical production, influenced by amyloid precursor proteins (APP), is observed in various human disorders, including autism [
125,
126,
127].
Polyphenols, particularly EGCG, play a role as antioxidants and promote neurogenesis and plasticity in a Down syndrome mouse model [
128], showing potential in alleviating autistic symptoms. Other polyphenols such as cyanidin-3-glucosidereduce intestinal inflammation in human intestinal cells, affecting inflammatory cascades like nuclear factor kappa B (NF-kB), activator protein-1 (AP-1) and Janus kinase-signal transducer and activator of transcription (JAK/STAT) [
129]. Furthermore, resveratrol in an in vitro modulates inflammatory markers including nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), ionized calcium-binding adapter molecule 1 (Iba1), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and cyclooxygenase-(COX-2) [
130]
. Moreover, a study on rat pups with autistic traits found that a polyphenol-probiotic complex reversed autistic behaviors and modulated biochemical changes in IL-6, TNF-α, BDNF, 5-HT, AchE, and the granular layer, which is supported by all this evidence [
131].
Therefore, when analyzing the activity of various polyphenols in inflammation and oxidative stress, these molecules have demonstrated high efficacy in regulating APP levels [
132]. . Its ability to cross the BBB (based on the hBMEC model, which utilizes human-derived brain endothelial cells), plays a significant role in protecting cortical neurons against oxidative stress-induced cell death. Comparatively, EGCG demonstrated rapid BBB permeation, while cyanidin-3-glucoside (C3G) showed slower permeation, and quercetin did not cross the BBB. This makes EGCG particularly promising as a neuroprotector among various polyphenols [
133]. In relation to ASD, IL-8 stands out among the cytokines involved. The administration of oral EGCG reduces its levels through the inhibition of intracellular Ca2+ levels and the activation of ERK1/2 and NF-kappaB pathways. This is becauseit has been seen in the colon that the treatment of TNF-α-stimulated HT29 cells (human colon carcinoma cell lines) with EGCG inhibits IL-8 production by regulating genes in inflammatory pathways, suggesting its potential for preventing or mitigating colonic disorders in autism [
134].
Finally, it should be noted that elevated levels of reactive oxygen species (ROS), specifically hydrogen peroxide (H
2 O
2) and superoxide (O
2-), are reported in the intestine of children with ASD as a cause of damage to the epithelial tissue [
135,
136,
137], and EGCG negatively regulates the inflammatory response in inflamed intestinal epithelial cells, largely through a post-transcriptional regulatory mechanism [
138].
3.3. Role of EGCG in the Metabolic Activity Associated with Dysbiosis in ASD
EGCG regulates the production of SCFAs [
42], which are metabolites involved in the bidirectional communication between the gut and the brain. Succinate and butyrate, are particularly important and can be altered in individuals with autism due to microbial activity [
139]. An imbalanced ratio of succinate production/consumption can cause intestinal disturbances and impact the gut-brain axis. [
140]. Cheng et al. determined through a review that altered succinate levels have been linked to disrupted calcium homeostasis and dysregulated metabolic functions in autistic individuals [
41].
Regarding butyrate, it has been seen that children with ASD have lower levels of fecal butyrate, as well as a decrease in the abundance of taxa producing this metabolite [
141]. Butyrate is particularly relevant in ASD as its production mainly derives from the gut microbiome. Butyrate displays potent anti-inflammatory activity that contributes to gut health, regulates intestinal homeostasis , and modulates the expression of neurotransmitter genes [
142,
143]. Furthermore, it positively modulates the impaired mitochondrial function in ASD, characterized by reduced activity of complex IV in the electron transport chain (ETC)
[144], including improvements in oxidative phosphorylation and beta-oxidation. These observations have underscored the significant neuroprotective role of butyrate in children with ASD [
145]. In fact, it has recently been seen that maternal treatment with butyrate in the BTBR mouse model of ASD rescues social and partially repetitive behavior deficits in the offspring [
146]. Thus, proper butyrate production at adequate levels is crucial, and this depends on the correct development of the gut bacterial community. Oral EGCG delivery on the DSS-induced murine colitis model has been shown to increase both the abundance of
Akkermansia muciniphila and its butyrate production [
88], potentially reversing the adverse effects resulting from altered butyrate levels in ASD. On the other hand, among the altered fecal metabolite profiles in children with ASD (not present in neurotypical children), there is an increase in p-cresol, caprate, aspartate, and a reduction in GABA, nicotinate, glutamine, and thymine [
147]. Among all these metabolites, p-cresol is a uremic toxin produced by certain strains of
Clostridium spp., which has negative biological effects and appears to adversely affect the homeostasis of colonic epithelial cells in children with ASD. When present in excess, p-cresol induces DNA damage in vitro and negatively affects the integrity of colonic epithelial cells [
148]. In fact, EGCG may have beneficial effects by reversing the activity of this metabolite. In a recent study, it has been seen that an EGCG-enriched diet reduces plasma and urinary concentrations of p-cresol in mice by suppressing the abundance of Firmicutes at the phylum level and Clostridia at the order level [
149].
In conclusion, EGCG has an impact on both the intestinal microbiota and variables directly related to the microbiota such as metabolites, inflammation and oxidative stress, which could provide benefits for patients with ASD (
Figure 1).
Another noteworthy aspect is the impact of metabolites generated from the interaction between the polyphenol and the intestinal microbiota. Particularly, in rats, the metabolic pathway of EGCG leads to gallic acid (GA) and epigallocatechin (EGC) [
150]. EGC is degraded by colonic bacteria to yield microbial ring-fission metabolites, specifically converting to 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5). Then, the bacteria Adlercreutzia equolifaciens MT4s-5 and Flavonifractor plautii MT42 are capable of degrading EGC. The bacterium Adlercreutzia equolifaciens MT4s-5 catalyzes the conversion of EGC to 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (EGC-M1), and subsequently, Flavonifractor plautii MT42 converts the propan-2-ol to 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (EGC-M5) and 4-hydroxy-5-(3,4,5-trihydroxyphenyl)valeric acid (EGC-M4). Similarly, EGC-M5 is produced from 1-(3,5-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (EGC-M3), which is formed from EGC by Adlercreutzia equolifaciens MT4s-5 in the presence of hydrogen [
120]. A large portion of the formed EGC-M5 is absorbed and undergoes glucuronidation in the intestinal mucosa and/or liver to form EGC-M5 glucuronide (EGC-M5-GlcUA), which is distributed to various tissues through the bloodstream and ultimately excreted in urine [
151] (
Figure 2).
The microbial ring-fission metabolites of EGCG are found in plasma in both free and conjugated forms [
151], and in vitro studies have shown that they may reach the brain parenchyma through the BBB, promoting neuritogenesis and potentially exerting a relevant activity against the degenerative processes of neurodegenerative diseases [
151].
Specifically, when assessing the penetration capacity of these metabolites through the BBB, it has been seen that GA has higher permeability than EGCG and EGC, possibly due to its smaller molecular size [
153]. Moreover, comparing only the rif[ng-fission metabolites derived from ECG, EGC-M5 demonstrated greater permeability than its conjugate EGC-M5-GlcUA [
150]. Some of these ring-fission metabolites also exhibit anti-inflammatory activity [
154]. In particular, EGC-M5 has been found to have immunomodulatory properties, as it enhances the activity of CD4+ T cells and the cytotoxic activity of natural killer cells in BALB/c mice [
155]. Therefore, it seems evident that the ring-fission metabolites derived from the intestinal microbiota a of catechins demonstrate a protective capacity against various diseases, including neurodegenerative disorders.
3.4. Liposomal EGCG
However, it is crucial to understand the metabolic process and bioavailability of green tea catechins and EGCG, particularly in assessing their biological activity and comprehending their beneficial effects on human health. EGCG presents significantly lower bioavailability compared to other components of catechins [
156,
157]. Therefore, when assessing the activity of EGCG and its metabolites, it is essential to emphasize that their applications are greatly limited due to their low solubility, bioavailability and stability. The use of liposomal formulations may serve as an effective strategy for their administration in autism. Liposomal delivery aims to improve the poor stability of polyphenols against temperature, light, pH and oxygen [
28,
29], as well as their low permeability across intestinal membranes, which results in only a small proportion of these compounds remaining available for absorption in the human body after ingestion [
29,
30]. Both stability and oral bioavailability are enhanced through liposomal encapsulation, as it provides protection against degradation when passing through the gastrointestinal tract [
31]. Moreover, nanotechnology can promote controlled release of the polyphenol and modulating the interaction between polyphenols and the microbiota, which also represents an intriguing approach [
31], and it has been seen that EGCG has significantly improved stability when formulated as dual-drug loaded PEGylated PLGA nanoparticles (EGCG/AA NPs). Following oral administration in mice, EGCG accumulated in all major organs, including the brain, leading to increased synapse formation and reduction of neuroinflammation in Alzheimer's disease [
158]. Furthermore, concerning its metabolites and their administration using nanotechnology, Abbasalipour H.
et al. (2022) studied the neuroprotective effects of gallic acid on oxidative stress-induced cognitive impairment and the expression of the Nrf2/Keap1 gene in an autism model. Gallic acid-loaded nanophytosomes (GNP) were administered, and the results revealed improvements in learning and memory deficits by reducing oxidative stress, enhancing antioxidant enzyme activity and modulating the Keap1/Nrf2 gene expression [
159].