Metabolite and gut microbiota co-biomarkers in Danggui Shaoyao San: insights into a shared therapeutic approach

Based on the tryptophan metabolic pathway, some studies have found that the ratio of plasma kynurenine to tryptophan is also directly correlated with insulin resistance (Oxenkrug et al., 2013) and T2DM (Rebnord et al., 2017), indicating that tryptophan metabolites may plays an important role in the pathogenesis of T2DM. Indolepropionic acid is also a deamination product of amino acid tryptophan (Menni et al., 2019), which may plays a protective role in T2DM by protecting β cell's function (de Mello et al., 2017). Dietary restriction of tryptophan has been shown to modulate metabolic hormones in obese rats. A low-tryptophan diet reduced fasting circulating levels of glucose, insulin, C-peptide, and leptin, while concomitantly increasing levels of glucagon, pancreatic polypeptide, and glucagon-like peptide-1 (Zapata et al., 2018). This interplay between tryptophan intake and metabolic regulation is further supported by clinical observations. For instance, Roux-en-Y gastric bypass (RYGB) surgery reduces serum levels of tryptophan and its downstream metabolite kynurenine in patients with (T2DM), an effect potentially associated with improved glycemic control (HbA1c) and reduced body mass index (BMI). Importantly, obesity-driven chronic systemic inflammation is a key setting in which tryptophan-kynurenine (TRP-KYN) pathway metabolites are believed to play a mechanistic role, contributing to the development of obesity-related comorbidities including T2DM (Yeung et al., 2022). After clinical administration of metformin, insulin sensitivity increased along with downregulation of Kynurenine pathway (Muzik et al., 2017). These findings suggest that pancreatic 5-HT plays an important role in insulin secretion by acting on different 5-HT receptors expressed on different cell types. In the serotonin pathway of tryptophan, human beta cells produce and secrete serotonin in response to increased glucose concentration.5-HT decreased glucagon secretion and cAMP levels in adjacent α cells. This suggests that pharmacological activation of the 5-HT1F receptor by 5-HT reduces glucagon secretion, thereby exerting a hypoglycemic effect in diabetic mice (Almaça et al., 2016).

Impaired clearance of amyloid-beta (Aβ) peptide is a major pathophysiological factor in AD (Murphy and LeVine, 2010). In this context, endogenous tryptophan metabolites are increasingly recognized for their regulatory roles. For instance, 5-hydroxyindoleacetic acid (5-HIAA) and kynurenic acid (KYNA) can stimulate the activity/expression of neprilysin (NEP), an Aβ-degrading enzyme, thereby counteracting Aβ-induced neurotoxicity. Furthermore, various tryptophan metabolites modulate brain Aβ levels under both normal and pathological conditions by interacting with the aryl hydrocarbon receptor (AhR) and regulating downstream metalloproteinases (Maitre et al., 2020). It is noteworthy that alterations in tryptophan metabolism may be compartmentalized; LC-MS/MS quantification has revealed that changes in peripheral levels of serotonin (5-HT) and norepinephrine (NE) do not uniformly reflect those in the brain (Wang et al., 2019). Collectively, these mechanisms through which tryptophan metabolism influences Aβ dynamics contribute significantly to the pathogenesis and progression of AD.

Previous studies have demonstrated abnormalities in tryptophan catabolism in PCOS (Zhao et al., 2012; Refaey et al., 2017; Onesti et al., 2019; Min et al., 2020; Zangeneh et al., 2020). These findings suggest that the tryptophan-kynurenine pathway may be activated in PCOS pathogenesis. Specifically, the urinary kynurenine-to-tryptophan ratio showed a significant positive correlation with levels of key reproductive hormones:including luteinizing hormone (LH), the LH/follicle-stimulating hormone (FSH) ratio, anti-Müllerian hormone (AMH), and dehydroepiandrosterone sulfate (DHEAS), but not with FSH alone. Concurrently, metabolites such as 3-hydroxykynurenine (3-OH-KYN), quinolinic acid, the kynurenine-to-tryptophan ratio, and 3-hydroxyanthranilic acid (3-OH-AA) were positively correlated with measures of glucose metabolism and insulin resistance: fasting blood glucose (FBG), fasting serum insulin (FSIns), and the homeostatic model assessment of insulin resistance (HOMA-IR). These associations indicate a close link between tryptophan-kynurenine metabolism and both reproductive endocrine and metabolic dysregulation in PCOS (Yang et al., 2022; Han et al., 2025; Belenkaia et al., 2019).

As Kynurenine pathway is positively correlated with BMI, there is still an interaction between the pathophysiological mechanism of PCOS and abnormal tryptophan metabolism after excluding the influence of obesity. The levels of tryptophan, canurine and uric acid in all subjects were positively correlated with LH and AMH. Therefore, abnormal activation of the tryptophan-canurine pathway may affect neuroendocrine feedback in patients with PCOS, which may be a potential therapeutic target for PCOS. The study showed an increase in C-reactive protein (CRP), and an increase in IL-6 and TNF-α, in women with PCOS compared with the control group, and this increase was not associated with obesity (Rostamtabar et al., 2021). There is little information on the relationship between plasma CRP and tryptophan catabolism. To investigate this mechanism, a study employed an experimental endotoxemia model induced by intravenous lipopolysaccharide injection to examine the kynurenine pathway (KP). Enzyme activity analyses revealed that the activity of kynurenine 3-monooxygenase (KMO) and kynureninase (KYNU), along with kynurenine aminotransferase (KAT), increased within 3–6 h post-injection, leading to the depletion of tryptophan and kynurenine (Millischer et al., 2021). More relevant to the pathophysiology under discussion, the activity of indoleamine 2,3-dioxygenase 1/tryptophan 2,3-dioxygenase (IDO-1/TDO2) was upregulated at 24–48 h. This increase coincided with a peak in both the serum kynurenine-to-tryptophan (KYN/TRP) ratio and C-reactive protein (CRP) levels. Since IDO activity is known to be induced by low-grade inflammation and psychological stress, and given a recent finding of its negative correlation with hormone receptor activity (Onesti et al., 2019), we hypothesize that elevated pro-inflammatory cytokines, such as CRP, may drive the observed increase in IDO activity. Supporting this, plasma CRP levels strongly correlate with other key inflammatory markers like interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), reliably reflecting systemic inflammatory activity (Felger et al., 2020).

Beyond branched-chain amino acids, the aromatic amino acid phenylalanine has emerged as a significant biomarker in the pathogenesis of T2DM. Epidemiological studies consistently identify elevated circulating phenylalanine as a robust predictor of future insulin resistance and T2DM, an association observed across diverse ethnicities and largely independent of obesity measures (Würtz et al., 2013; Tillin et al., 2015; Tai et al., 2010). Clinically, the close link between phenylalanine and diabetic metabolism is further evidenced by the finding that its plasma levels decrease in response to hypoglycemic agents like metformin (Preiss et al., 2016).

The pathophysiological role of phenylalanine extends beyond a mere biomarker. It actively contributes to insulin resistance through multiple, converging mechanisms. Experimental evidence indicates that phenylalanine and its metabolites can impair insulin signaling in skeletal muscle, potentially via pathways involving mTOR, JNK, and IRS1, leading to inhibited glucose transport/phosphorylation and reduced glycogen synthesis (Krebs et al., 2002; Dornhorst, 2001). Furthermore, phenylalanine-induced oxidative stress may inhibit phenylalanine hydroxylase activity, creating a feed-forward loop that elevates phenylalanine while reducing its conversion to tyrosine (Stephens et al., 2015). This metabolic dysregulation is compounded by hormonal crosstalk; for instance, hyperglucagonemia, common in T2DM,can shift phenylalanine metabolism toward oxidation, altering its bioavailability (Tessari et al., 1996).

This mechanistic understanding illuminates potential therapeutic strategies. Interventions that lower phenylalanine levels or modulate its metabolism show promise. For example, the gut-microbiota-modulating agent berberine alleviates diabetic symptoms partly by reducing aromatic amino acid metabolism (Yao et al., 2020). Interestingly, endogenous exercise-induced metabolites like N-lactoyl-phenylalanine (N-lac-phe) can deplete phenylalanine, offering a molecular explanation for physical activity's protective effect against T2DM (Newgard et al., 2009; Robinson et al., 2014). Even pharmaceutical approaches utilize this pathway, as demonstrated by the phenylalanine-derived drug nateglinide, which enhances glucose-stimulated insulin secretion (Li VL. et al., 2022). Collectively, these insights position phenylalanine not only as a diagnostic marker but also as a node linking metabolic dysfunction, hormonal imbalance, and potential therapeutic intervention in T2DM.

Emerging evidence positions phenylalanine at the intersection of metabolic dysregulation and protein aggregation in AD. Metabolomic analyses of postmortem human brain tissues reveal that phenylalanine levels are significantly upregulated in AD patients compared to controls, suggesting a systemic metabolic alteration associated with the disease (Liu et al., 2021). Beyond its role as a metabolite, phenylalanine residues within the amyloid-β (Aβ) peptide play a critical structural role in driving its pathogenic aggregation. Mechanistic studies demonstrate that the aromatic side chain of phenylalanine is crucial for Aβ self-assembly. For instance, substituting phenylalanine residues in Aβ with cyclohexylalanine (Cha)—a non-aromatic analog—effectively inhibits the formation of mature Aβ fibrils (Genji et al., 2017). Conversely, strategic mutation of Phe20 to Cha in Aβ-derived peptides can promote and stabilize the formation of neurotoxic oligomers, which are considered key pathogenic species in early AD (Haerianardakani et al., 2020). Collectively, these findings indicate a dual pathogenic link: aberrant elevation of brain phenylalanine may reflect or contribute to a metabolic environment conducive to AD pathogenesis, while the intrinsic properties of phenylalanine residues directly facilitate the aggregation of Aβ into toxic assemblies.

Elevated circulating phenylalanine levels are not only a metabolic hallmark of PCOS but also implicated in its reproductive and neuroendocrine dysfunction. As a potential "warning sign" for compromised oocyte developmental competence (Huang et al., 2021), high phenylalanine may reflect a suboptimal follicular fluid environment, aligning with the established view that amino acid balance is crucial for blastocyst development (Gardner, 1998). More broadly, phenylalanine metabolism intersects with PCOS pathogenesis through its link to insulin resistance, a key driver of the syndrome. Intriguingly, phenylalanine is directly incorporated into arginine-phenylalanine-amide (RFamide)-related peptide-3 (RFRP3), a neuropeptide encoded by the Rfrp gene. RFRP3 acts in the mammalian hypothalamus to inhibit gonadotropin-releasing hormone (GnRH) secretion (Shaaban et al., 2018). Thus, perturbations in phenylalanine availability could theoretically influence the synthesis or function of RFRP3, potentially contributing to the dysregulated GnRH pulsatility and subsequent hormonal imbalances (e.g., elevated LH) characteristic of PCOS. This integrative perspective positions phenylalanine as a metabolic node connecting systemic insulin resistance, local ovarian follicle health, and central neuroendocrine regulation in PCOS.

In recent years, Japanese researchers have demonstrated in zebrafish that intestinal dysbiosis is linked to the pathogenesis of T2DM (Okazaki et al., 2019), a finding subsequently corroborated in rat models (Peng et al., 2019). Collectively, these studies indicate that the gut microbiota and its metabolites play a pivotal role in the initiation and progression of T2DM.

Concurrently, the composition and abundance of gut microbes in T2DM patients are markedly altered. Metagenomic analyses by Junjie Qin et al. revealed that, although changes within the phylum Firmicutes are heterogeneous, butyrate-producing genera—such as Faecalibacterium, Roseburia, and Coprococcus—are significantly reduced, whereas Proteobacteria expanded and Bacteroidetes decreased (Qin et al., 2012), The number of Bifidobacterium (Li et al., 2023) decreased, while randomized studies suggested an increase in Bacteroides (Zhang et al., 2025). These characteristic microbial shifts can serve as biomarkers for monitoring T2DM development.

Recent scholarly investigations indicated that dysbiosis of the gut microbiota may facilitate the aggregation of amyloid-β, neuroinflammatory responses, oxidative stress, and insulin resistance, which are implicated in the pathogenesis of AD (Vogt et al., 2017). Different bacterial genera and species can generate various metabolites, such as aminobutyric acid (GABA), serotonin (5-HT), histamine, and dopamine, etc., which are implicated in a series of emotions, behaviors, and cognitive functions as neurotransmitters or precursors of neurotransmitters (Di Benedetto et al., 2017). In the metabolic process, the host and its gut microbiota jointly produce a series of metabolites, such as short-chain fatty acids, which can directly or indirectly mediate microbiota-gut-brain interactions, which is vital to the health of the host (Chen et al., 2021). Intestinal flora dysbiosis induces a reduction in beneficial substances such as short-chain fatty acids (SCFAs) and hydrogen (H₂), alongside an increase in harmful substances like amyloid and trimethylamine N-oxide (TMAO). This imbalance leads to increased permeability of both the intestinal mucosal barrier and the blood-brain barrier, activates the peripheral immune response, and elevates peripheral and central oxidative stress (OS) levels. Finally, intestinal disorders promote the pathological progression of AD by increasing amyloid plaque formation, neuroinflammation and IR (Vogt et al., 2018). Mouse models have confirmed that the altered gut microbiota in AD is characterized by an expansion of Proteobacteria and Bacteroidetes, along with a marked decrease in Firmicutes—especially Bifidobacterium (Manfredi et al., 2025).

As a prevalent metabolic disorder, PCOS is intrinsically characterized by insulin resistance, hepatic steatosis, and chronic low-grade inflammation (Dumesic et al., 2015). Consequently, its pathogenesis mirrors that of another metabolic disease:T2DM, and is likewise shaped by the gut microbiota. In women with PCOS, the gut microbial profile exhibits a marked increase in the phylum Bacteroidetes; within this phylum, Bacteroides species attenuate short-chain fatty acid (SCFA) production, thereby fostering obesity and insulin resistance (Kelley et al., 2016). Notably, model-based comparative analyses have further documented a significant decrease in the abundance of Lactobacillus and Bifidobacterium (Zhang et al., 2019).

Based on these findings, we constructed a gut microbial marker map for the comorbidity of T2DM, AD, and PCOS (seen Figure 2), highlighting the consistent reduction of Bacteroides across all three conditions. While the primary drivers of this dysbiosis may differ among diseases:ranging from direct metabolic derangement in T2DM to brain-gut axis signaling in AD, emerging intervention studies suggest that the resulting microbial shift itself may actively contribute to a shared physiopathologic mechanism. Crucially, a fecal microbiota transplantation study demonstrated that transferring gut microbiota from AD patients to animal models exacerbated cognitive decline and neuropathology, directly proving the pathogenic potential of disease-associated dysbiosis (Jia et al., 2025). Furthermore, a large-scale meta-analysis of gut microbiomes across multiple diseases revealed a significant compositional similarity between AD and type 2 diabetes, underscoring the existence of a common microbial basis for these comorbidities (Jin DM. et al., 2024). Therefore, the consistent alteration of Bacteroides highlighted in our Figure 2 may represent more than a correlative biomarker; it may reflect a common, functionally consequential microbial disturbance that actively participates in the intertwined pathophysiology of metabolic and neuroendocrine disorders. This perspective strengthens the rationale for investigating Bacteroides not only as a diagnostic marker but also as a potential therapeutic target for modulating the shared disease susceptibility underlying T2DM, AD, and PCOS.

Angelica sinensis polysaccharide, the main active constituent of Angelica, exhibits anti-inflammatory (Du et al., 2020), antioxidant, anti-apoptotic (Du et al., 2023), and hepatoprotective (Wen et al., 2022) effects, which are likely mediated by the modulation of specific signaling pathways. Zhou et al. demonstrated that Angelica polysaccharide exerts anti-inflammatory effects by inhibiting the TLR4/MyD88/NF-κB signaling pathway, and notably, the serum metabolite 5-methyl-dl-tryptophan (5-MT) was restored by Angelica polysaccharide treatment (Zou et al., 2023). Paeoniflorin, another major blood-entry metabolite of DSS, has also been shown to activate the TRPA1 channel and the PLC-γ1/PIP2 signaling pathway to promote 5-HT release, thereby modulating tryptophan metabolism (Zhan et al., 2021). Additionally, paeoniflorin was reported to reduce the expression of the L-tryptophan-catabolizing enzyme tryptophan-2,3-dioxygenase in the liver of stress-induced depressive mice, thereby increasing the 5-hydroxytryptamine/tryptophan ratio and decreasing the kynurenine/tryptophan ratio (Liang et al., 2023). Tetramethylpyrazine (ligustrazine), an alkaloid and one of the main metabolites of Ligusticum chuanxiong in DSS, possesses diverse physiological functions, including antioxidant, anti-inflammatory, anti-apoptotic, autophagy modulation, vasodilation, angiogenesis regulation, mitochondrial damage inhibition, endothelial protection, and neuroprotection (Lin et al., 2022). Studies have found that ligustrazine can modulate the mRNA expression levels of tryptophan and 5-hydroxytryptamine indoleacetic acid, increase 5-HT concentration, and exert anxiolytic-like effects (Lee et al., 2018). Senkyunolide I, a natural phthalide widely distributed in umbelliferous plants, exhibits analgesic, anti-inflammatory, antioxidant, and antithrombotic pharmacological effects (Huang et al., 2023). Notably, senkyunolide I is also considered to alter levels of 5-hydroxytryptamine (5-HT), 5-hydroxytryptophan (5-HTP), 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), and dopamine (DA) in blood and brain, thereby exerting analgesic and anti-migraine effects (Wang et al., 2011).

Ferulic acid, an active constituent widely found in traditional Chinese medicines such as Angelica sinensis and Ligusticum chuanxiong, exhibits biological activities in oxidative stress, inflammation, vascular endothelial injury, fibrosis, apoptosis, and platelet aggregation (Li et al., 2021). Since ferulic acid is derived from the metabolism of phenylalanine and tyrosine (Jin P. et al., 2024), changes in blood ferulic acid levels are accompanied by corresponding alterations in phenylalanine. Apart from ferulic acid, paeoniflorin is also involved in phenylalanine metabolism, such as modulating phenylalanine metabolism in rats with rheumatoid arthritis (Liu et al., 2022d) and endometriosis (Wu et al., 2019), and restoring biomarker levels (phenylalanine). Yuan et al. employed metabolomics combined with network pharmacology to reveal that ligustrazine can regulate phenylalanine metabolism in rats with neuropathic pain, indicating a close relationship between ligustrazine and phenylalanine (Yuan et al., 2025).

Currently, studies on the relationship between blood-entry constituents and Bacteroidetes are scarce. Tang et al. reported that the abundance of Bacteroidetes in type 2 diabetic mice could be restored by Angelica polysaccharide, and the Firmicutes/Bacteroidetes ratio could also be reversed following Angelica polysaccharide treatment (Tang et al., 2023). Similar findings were observed with paeoniflorin, where administration of paeoniflorin to rats with irritable bowel syndrome significantly restored the Firmicutes/Bacteroidetes ratio (Wang et al., 2023).