Abstract

Objective

This study aimed to investigate the pharmacokinetic characteristics of two isomeric phenylethanoid glycosides-forsythiaside A (FTA) and acteoside (ACH)-in rats, and to analyze their interactions with the gut microbiota. The goal was to clarify how subtle structural differences affect metabolic behavior and microbiota regulation, providing evidence relevant to herbal extract for disease development and functional ingredient formulation.

Methods

Twelve Sprague–Dawley (SD) rats were randomly divided into two groups and administered FTA or ACH by intragastric gavage. Blood samples were collected at predefined time points. Plasma concentrations of the parent compounds and key metabolites-hydroxytyrosol (HT) and caffeic acid (CA)-were determined using high-performance liquid chromatography (HPLC).

Another 30 SD rats were randomly assigned to five groups: control, FTA 1-hour group (F_1), FTA 8-hour group (F_8), ACH 1-hour group (A_1), and ACH 8-hour group (A_8). Cecal contents were collected at corresponding time points and analyzed by 16S rRNA sequencing to evaluate microbiota structural shifts and their correlations with pharmacokinetic parameters.

Results

Overall exposure of both parent compounds was low after administration. ACH exposure was approximately half that of FTA. Although the pharmacokinetic curves of the phase I metabolite HT differed between FTA and ACH, their plasma levels were broadly comparable. In contrast, CA plasma levels were substantially lower than HT.

16S rRNA sequencing showed that both FTA and ACH altered gut microbiota structure and function to varying degrees. The relative abundance of Verrucomicrobia increased in A_1, while Lactobacillus was significantly enriched in F_1, and Bacteroides was significantly enriched in A_8 (P < 0.05). Differentially enriched species correlated with multiple KEGG pathways related to glycoside metabolism.

Conclusion

Differences in the rhamnose linkage positions between FTA and ACH contribute to their distinct pharmacokinetic behaviors. Both compounds induce measurable structural and functional changes in the gut microbiota following intragastric administration. Enriched bacterial taxa were associated with multiple glycoside metabolism pathways, supporting a drug–microbiota bidirectional interaction model.

Keywords: forsythiaside A; acteoside (verbascoside); pharmacokinetics; gut microbiota; phenylethanoid glycosides; herb extract for disease; glycoside metabolism

 

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1. Background and Rationale (for Herbal Health Users and Supplement Developers)

Forsythiaside A (FTA) is a phenylethanoid glycoside found in the fruit of Forsythia suspensa. Modern pharmacological research suggests FTA has multiple biological activities, including anti-inflammatory, antiviral, antibacterial, antioxidant, hepatoprotective, and neuroprotective effects. 111

Acteoside (ACH)-also widely known as verbascoside-is a naturally occurring water-soluble phenylethanoid glycoside present in many medicinal plants. It exhibits antioxidant, antitumor, anti-inflammatory, and neuroprotective activities, may improve learning and memory, and shows multi-target potential in depressive symptom relief. 2–32–32–3

FTA and ACH are structural isomers. Both are composed of caffeic acid (CA), hydroxytyrosol (HT), and rhamnose, linked to a central glucose unit by ester and glycosidic bonds; they differ only in the rhamnose attachment position.

From a product-development standpoint, this is a classic "same formula family, different behavior" issue: small structural changes can cause meaningful shifts in:

oral absorption and systemic exposure,

metabolic conversion rates,

and the direction and magnitude of gut microbiota modulation.

 

2. Key Scientific Context: Low Permeability, Low Exposure, and "Prodrug-like" Behavior

FTA and ACH have poor membrane permeability and are difficult to absorb through the intestine, resulting in low systemic exposure in vivo. Their oral pharmacokinetics often show poor absorption. Reported absolute bioavailability is approximately ~0.5% for FTA and ~1% for ACH. 4–64–64–6

In natural product pharmacology, both are often considered "prodrug-like": their in vivo activity may rely heavily on intestinal (especially microbiota-driven) metabolism that releases active aglycones or smaller phenolics. 7–87–87–8

After oral administration, FTA and ACH can undergo deglycosylation by microbial glycosidases to produce CA and HT, which then enter subsequent metabolic pathways and contribute to pharmacological effects. 9–109–109–10

This is directly relevant to "Herb Extract for Disease" product logic: if the active effect depends on microbiota conversion, then formulation design and target population microbiome variability become decisive factors for efficacy consistency.

 

3. Materials and Methods (Translated)

3.1 Reagents

2.5% tribromoethanol solution (Nanjing Aibei Biotechnology Co., Ltd.)

FTA (batch 230825) and ACH (batch 231015), purity ≥ 98% (Chengdu Zhibiaohua Pure Biotechnology Co., Ltd.)

Nimodipine as internal standard (IS) (Beijing Solarbio Science & Technology Co., Ltd.)

Methanol, acetonitrile, phosphoric acid, formic acid (Thermo Fisher Scientific, USA)

DNA purification and sequencing reagents (Vazyme, Invitrogen, Illumina, MP Biomedicals, NEB, etc.)

3.2 Instruments

Waters e2695-2489 HPLC; Mettler Toledo balance; nitrogen evaporator; vortex; tissue grinder; Eppendorf centrifuge; ultrasonic cleaner; electrophoresis; PCR system; Nanodrop spectrophotometer; CO2 euthanasia system.

3.3 Animals and Ethics

Forty-two SPF male SD rats (180–200 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Housing: 22 ± 2 °C, 60% ± 10% humidity, free access to food and water. Animals were euthanized using CO2 anesthesia. Animal procedures complied with the welfare standards of the Animal Ethics Committee of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences (approval: SLXD-20240625018).

3.4 Jugular Vein Cannulation

Rats were anesthetized with 2.5% tribromoethanol intraperitoneally until deep anesthesia was achieved. The right jugular vein was exposed and cannulated using polyethylene tubing (ID 0.5 mm, OD 1.0 mm), inserted 2.0–2.5 cm toward the heart, secured, tunneled subcutaneously to a dorsal exit site, and the wound sutured with iodine disinfection to reduce infection risk.

 

 

 

Note: A. Venn diagram for the FTA group; B. Venn diagram for the ACH group; C. Species composition at the phylum level; D. Species composition at the genus level; E. Relative abundance of Firmicutes in each group; F. Relative abundance of Bacteroidetes in each group;
G. Relative abundance of Proteobacteria in each group; H. Relative abundance of Actinobacteria in each group; I. Relative abundance of Verrucomicrobia in each group; J. Ratio of G- to G+ bacteria in each group; KB represents the control group (i.e., the 0 h group); **P < 0.01 compared to the control group.

 

3.5 Pharmacokinetic Study

Twelve SD rats were randomized into two groups (n = 6): FTA group and ACH group. Food was withheld for 12 hours before dosing (water allowed). Rats received FTA or ACH via gavage at 202.57 mg·kg⁻¹. Blood (0.3–0.5 mL) was collected at 5, 10, 15, 30, 45 min; 1, 2, 3, 6, 9, 12, and 24 h via the cannula. Samples were anticoagulated with heparin, centrifuged at 4 °C (4,680 r·min⁻¹ for 10 min; radius 10.1 cm). Plasma was stored at −80 °C.

3.6 HPLC Conditions

Column: XSelect HSS T3 (250 mm × 4.6 mm, 5 μm)

Mobile phase: 0.1% phosphoric acid in water (A) / acetonitrile (B)

Flow: 0.8 mL·min⁻¹; column temperature 30 °C

Injection: 20 μL; detection wavelength: 230, 235 nm

Gradient described as in the source text.

3.7 Plasma Sample Processing

200 μL plasma + 20 μL IS + 800 μL precipitation solvent (1% formic acid-acetonitrile). Vortex 3 min; centrifuge at 4 °C (12,700 r·min⁻¹, 10 min). Supernatant evaporated to dryness under nitrogen; residue reconstituted in 200 μL methanol, vortexed and centrifuged again; supernatant injected.

3.8 Method Validation

Solutions were prepared at specified concentrations; specificity, linearity, precision/accuracy (RSD and RE within 15%), repeatability, stability (room temperature up to 24 h), and recovery (approximately 97.87%–103.68% average; acceptable range 85%–115%) were assessed.

3.9 Cecal Content 16S rRNA Sequencing

Thirty rats were randomized into 5 groups: control, F_1, F_8, A_1, A_8. Cecal contents (0.2 g) were collected and DNA extracted using MagBeads FastDNA Kit for Soil. PCR amplification targeted 16S V3-V4 region using primers 338F and 806R. Products were purified, quantified, pooled, and sequenced on Illumina NovaSeq (2 × 250 bp) through a sequencing provider.

3.10 Bioinformatics and Statistics

QIIME2 (v2024.5) for demultiplexing, primer trimming, DADA2 denoising, ASV generation; annotation via Greengenes2. Beta diversity via Bray–Curtis and NMDS; PERMANOVA and ANOSIM for group differences; LEfSe for differential taxa; PICRUSt2 for KEGG functional prediction; SPSS for statistical tests.

 

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4. Results 

4.1 Analytical Method Performance

Standard curves were linear (r > 0.999). Specificity was acceptable with no interference from plasma matrix; precision and accuracy met bioanalytical requirements. Repeatability, stability, and recovery were all within required limits.

4.2 Pharmacokinetic Characteristics of FTA and ACH

After gavage, both parent compounds reached peak quickly and maintained low plasma concentrations. Key observations:

FTA plasma concentration was ~2× ACH, but both were below the exposure of their shared metabolite HT.

HT showed multi-peak and prolonged exposure in both groups, but curve shapes differed:

In the FTA group, HT peaked rapidly at ~0.08 h, fluctuated near peak, with additional peaks around ~3 h and ~12 h.

In the ACH group, HT showed a typical double-peak pattern: first peak ~0.08 h; main peak around ~3 h, followed by a high-concentration plateau.

CA concentrations were extremely low, near the limit of detection.

When parameters were computed (non-compartmental analysis, DAS2.0):

FTA AUC(0–t) was >4× that of ACH (P < 0.001)

FTA AUC(0–∞) was ~6× that of ACH (P < 0.01)

FTA Cmax was ~4× that of ACH (P < 0.001)

FTA had longer MRT and higher Vz/F than ACH (P < 0.05), suggesting longer residence and broader distribution.

R&D implication (supplement companies): Even for isomers in the same ingredient class, "dose equivalence" is not guaranteed. If a product targets outcomes linked to systemic exposure (e.g., neuroprotection-related signaling), FTA-like structures may deliver higher parent exposure; if effects depend mainly on metabolite HT and microbiota cycling, formulation aimed at stable HT generation might matter more than parent AUC alone.

4.3 Microbiota Diversity Changes

Sequencing depth was adequate; millions of reads produced ASVs of expected lengths.

Alpha diversity (Chao1):

ACH: 1 h increased significantly (+34.2%), returned to baseline at 8 h

FTA: 1 h decreased significantly (−19.6%), returned to baseline at 8 h

Shannon diversity changes were small overall, with a mild increase at 1 h after ACH.

Beta diversity (NMDS, Bray–Curtis):
Groups separated clearly, with significant differences between treatment groups (P < 0.001). Differences grew more pronounced with time.

4.4 Microbiota Composition (Phylum and Genus Shifts)

At the phylum level:

FTA: Firmicutes increased at 1 h then decreased by 8 h; Bacteroidetes decreased then increased.

ACH: Firmicutes decreased continuously; Bacteroidetes increased continuously.

Proteobacteria decreased then increased in both.

Verrucomicrobia rose then fell in ACH, stable in FTA.
The ratio (Proteobacteria+Bacteroidetes)/Firmicutes(Proteobacteria + Bacteroidetes)/Firmicutes(Proteobacteria+Bacteroidetes)/Firmicutes showed a "down then up" trend in both vs control.

At taxa enrichment level:

A_1: Verrucomicrobia increased (with Akkermansia driving some samples)

F_1: Lactobacillus enriched

A_8: Bacteroides enriched (P < 0.05)

Consumer-facing implication (herbal regulation): These shifts suggest different "microbiome response styles" within hours of ingestion. That may partly explain why people report different digestive and systemic responses to different PhG-rich herb extracts.

4.5 Co-occurrence Network and Functional Prediction

Microbial network analysis showed modularity (>0.5), and treatment changed the number of connections and nodes:

F_1/F_8: fewer nodes and connections → weakened interactions

A_1: more connections but higher negative correlations → potentially more competition

KEGG-related correlations suggested enriched taxa linked to glycan and glycoside metabolism pathways, supporting the idea that microbes participate in deglycosylation and downstream transformation, which then influences plasma metabolite profiles.

 

5. Discussion

Gut microbiota can influence drug absorption and pharmacokinetics through enzyme-mediated metabolism before absorption and via enterohepatic cycling. 11–1211–1211–12 Phenylethanoid glycosides are representative natural products whose oral bioavailability and efficacy can depend strongly on microbiota "activation." 13–1713–1713–17

This study showed:

Parent exposure differs markedly between FTA and ACH even though they are isomers.

HT displays multi-peak and prolonged exposure patterns, suggesting possible enterohepatic circulation and microbiota-mediated deconjugation/reabsorption cycles. 25,29–3025,29–3025,29–30

CA exposure is much lower than HT, potentially due to poor absorption and/or rapid conjugation (glucuronidation/sulfation) after absorption. 18–1918–1918–19

Microbiota changes differ between FTA and ACH, including enrichment of Lactobacillus (F_1) and Bacteroides (A_8), taxa often associated with glycosidase activities relevant to glycoside hydrolysis. 888

Why this matters for "Herb Extract for Disease" products:

If your product's intended benefits include anti-inflammatory, liver support, cognitive support, or microbiome support, then it is not enough to label "acteoside/verbascoside" as a generic active. The isomer, linkage type, and microbiome interaction can shift both pharmacokinetics and functional outcomes.

For companies, these data support building product differentiation around "microbiota-activated phenylethanoid glycosides" and designing formulations and claims with microbiome involvement in mind.

 

6. Strategic Integration: Where Cistanche Fits 

Cistanche (Cistanche tubulosa / Cistanche deserticola) is widely known in traditional use as "Desert Ginseng" and is positioned in modern wellness markets for benefits such as kidney-tonifying support, immunity support, anti-aging antioxidant support, constipation relief, cardiovascular and neurological health support, etc. (industry/brand education page content as provided via the WECISTANCHE URL).

From an ingredient science viewpoint, Cistanche is also rich in phenylethanoid glycosides, including acteoside/verbascoside and related compounds-placing it in the same broader class of microbiota-interacting glycosides discussed in this paper. This makes Cistanche a practical "bridge ingredient" for:

microbiota-friendly herb extract formulations,

"gentle daily regulation" products for digestion and vitality,

and stack designs combining Forsythia PhGs + Cistanche PhGs for multi-target positioning (e.g., antioxidant + intestinal comfort + vitality).

Example SEO keyword variants (for product pages / blogs):

Herb Extract for Gut Health & Constipation Support (Cistanche + PhGs)

Herb Extract for Anti-Inflammatory Support (Forsythiaside A + Acteoside)

Herb Extract for Cognitive & Neuroprotection Support (Acteoside/Verbascoside)

Herb Extract for Liver Protection (PhGs + microbiota–liver axis)

Herb Extract for Immune Regulation (PhG polysaccharide synergy positioning)

(Note: keep claims compliant with your target market regulations-e.g., structure/function claims vs disease treatment claims.)

 

7. Conclusion 

Although FTA and ACH are structural isomers, their pharmacokinetic behaviors in rats differ significantly. These differences suggest that gut microbiota may exhibit selective metabolic preferences due to different rhamnose linkage positions on the central glucose unit. Microbiome analysis further indicates that both FTA and ACH can differentially regulate gut microbial composition, enriching taxa with glycoside-metabolism potential. Functional prediction suggests that key enriched species in each group correlate with different KEGG pathways. This structural-difference-driven divergence in microbiota and functional pathways may be a deeper reason for the observed pharmacokinetic differences. Future studies should isolate and validate the functions of key taxa to identify probiotic resources with potential to optimize metabolism of such natural products.

 

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