Keyword focus (SEO variants): anti-aging herb, anti-aging herbal formula, Cistanche tubulosa extract, Cistanche deserticola echinacoside, longevity support, antioxidant defense, insulin/IGF-1 pathway, FoxO/DAF‑16, SKN‑1/Nrf2-like signaling, heat-shock response.

 

Abstract

This study investigated the anti-aging mechanism of a Traditional Chinese Medicine (TCM) multi-herb paste formula centered on Cistanche (a classic "longevity herb" in traditional practice), by combining network pharmacology with in vivo experiments in Caenorhabditis elegans (a widely used aging model organism). The aim was to provide experimental evidence supporting product development of an anti-aging herb-based formula.

A set of major active compounds within the formula was selected. Core targets were screened and a "herb–compound–target" network was built using STRING (v11.5) and Cytoscape (v3.9.1). GO functional annotation and KEGG pathway enrichment analyses were performed using the OmicsBean platform. In the biological validation, worms were divided into a control group and multiple dosage groups. Lifespan, reproduction, and locomotion were assessed. Enzymatic antioxidant activities-SOD, CAT, and GSH-Px-as well as oxidative damage markers ROS and MDA, were measured. qRT‑PCR was used to quantify changes in longevity- and stress-resistance–related genes.

Network pharmacology indicated that 36 active compounds may act on 325 aging-related targets, and topological analysis identified 18 core targets. KEGG enrichment suggested the formula may exert effects through 91 pathways, including cancer-related signaling, the insulin signaling pathway, PI3K‑Akt signaling, and estrogen signaling.

In vivo, the formula significantly extended mean lifespan and improved oxidative stress resistance in C. elegans. It increased SOD, CAT, and GSH‑Px activities, reduced ROS and MDA, downregulated insulin receptor-like genes daf‑2 and age‑1, upregulated daf‑16 and its downstream targets (sod‑3, mtl‑1, gst‑4, ctl‑1, ctl‑2), and activated skn‑1 and heat-shock related genes (hsf‑1, hsp‑16.1, hsp‑16.2).

Overall, this anti-aging herbal formula markedly extended lifespan and enhanced stress resistance in worms. Its mechanism is likely related to suppression of insulin signaling and strengthening of antioxidant/stress-response defenses, providing a scientific foundation for future development of Cistanche-based longevity products.

Keywords: multi-herb Cistanche paste; anti-aging herb; mechanism; oxidative stress; insulin signaling pathway.

 

CLICK THE PICTURE TO KNOW CISTANCHE FORMULA PRODUCTS FOR ANTI-AGING

 

1. Introduction: Why Anti-Aging Herbal Research Matters

Aging is a progressive, degenerative biological process associated with accumulating functional decline. As human lifespan increases, societies face expanding burdens from age-associated conditions including neurodegeneration, diabetes, cardiovascular disease, and cancer, whose incidence and mortality rise with age. Therefore, identifying interventions that support healthy aging and improve resilience to stressors is increasingly important.

Traditional Chinese Medicine has a long history of using multi-herb formulas to promote vitality. The formula studied here is a paste preparation developed based on classical principles and composed of eight herbs, including Cistanche, lily bulb (Baihe), and Polygonatum (Huangjing), among others. These herbs have been historically used and are also supported by modern pharmacology for activities such as antioxidant support, cognition support, immune modulation, and healthy aging.

This research used a two-level strategy:

Network pharmacology to systematically predict targets and pathways (reflecting TCM's multi-component, multi-target characteristics), and

Whole-organism validation in C. elegans, measuring lifespan, stress tolerance, and gene/protein markers.

 

2. Materials and Methods 

2.1 Instruments

Stereomicroscope (SMZ‑168, Motic); inverted fluorescence microscope (CKX41, Olympus); microplate reader (Multiskan FC, Thermo Fisher Scientific); PCR system (Mycycler, Bio‑Rad); qPCR system (MJ Mini™, Bio‑Rad).

2.2 Herbs and Reagents

Eight herbs were sourced as authenticated medicinal materials and met relevant pharmacopeial requirements (Chinese Pharmacopoeia 2025, Part I). Key reagents included a ROS detection kit (DCFH‑DA-based), Trizol and qPCR reagents, and enzymatic assay kits for SOD/CAT/GSH‑Px and MDA.

2.3 Network Pharmacology Workflow

Compound selection was based on:
(1) major components characterized previously (prior internal profiling) and content-based prioritization;
(2) effectiveness plus TCM role hierarchy (chief/deputy/assistant/envoy) and structural diversity;
(3) literature-confirmed absorbed ("blood-entry") components.

Target prediction used SwissTargetPrediction and TCMSP. Targets were standardized to gene symbols via UniProt.
Aging targets were collected from GeneCards and NCBI-Gene using "aging" as a keyword (GeneCards threshold ≥3% relevance), merged and de-duplicated, then intersected with compound targets. Cytoscape (v3.9.1) was used to build a herb–compound–target network.

PPI network: Intersected targets were submitted to STRING (species: Homo sapiens, confidence score: 0.900). Core targets were defined as nodes with degree, betweenness, and closeness above median values. GO and KEGG enrichment were performed using OmicsBean.

2.4 C. elegans Culture, Dosing, and Phenotype Assays

Worms were maintained at 20°C on NGM plates seeded with heat-inactivated E. coli OP50. Synchronization was done with alkaline hypochlorite. A 50 mg/mL stock solution of the paste was prepared and mixed into NGM to achieve final concentrations of 0, 25, 50, 75, 100, 200 μg/mL.

Lifespan: 100 worms/group; daily transfers; death recorded until all died.
Stress resistance: oxidative stress using H₂O₂-containing media; heat stress at 35°C.
Aging pigment (lipofuscin): blue autofluorescence quantified at days 7 and 11.
Mobility: body bends per 30 seconds measured at days 3 and 7.
Feeding: pharyngeal pumping counted at days 0, 3, 7.
Reproduction: egg laying and progeny counts tracked.
Biochemical assays: SOD, CAT, GSH‑Px, MDA; ROS fluorescence quantified by microscopy and ImageJ.
qRT‑PCR: expression of daf‑2, age‑1, akt‑1, daf‑16, sir‑2.1, hsf‑1, hsp‑16.1, hsp‑16.2, skn‑1, sod‑3, ctl‑1, ctl‑2, gst‑4, mtl genes, eat‑2; analyzed by 2−ΔΔCt2^{-\Delta\Delta Ct}2−ΔΔCt using act‑1 as reference.
Mutant validation: lifespan tested in daf‑16, skn‑1, hsf‑1, eat‑2 mutants.
Protein localization / reporter strains: DAF‑16 and SKN‑1 nuclear translocation, and SOD‑3/GST‑4/HSP‑16.2 reporter fluorescence were assessed.

Statistics: Kaplan–Meier survival with Log‑rank test; t-test/ANOVA with Dunnett post‑hoc; significance at P < 0.05.

3. Key Results (What Western Readers Can "See" Clearly)

3.1 Multi-Component, Multi-Target Anti-Aging Potential

36 candidate active compounds → 453 predicted targets (after de-duplication)

Aging-related targets: 4,083

Intersection: 325 shared targets

Network: 210 nodes and 593 edges (herbs, compounds, targets)

3.2 Core Targets and Pathways

18 core targets identified from the PPI network

GO analysis indicated enrichment in signaling regulation, stimulus/immune response regulation, and growth/development regulation; locations included cytosol and nucleus; functions included protein/enzyme binding and nucleotide binding.

KEGG enrichment: 91 pathways, prominently including insulin signaling and PI3K‑Akt-pathways strongly linked to metabolism, stress resistance, and longevity biology.

3.3 Lifespan Extension Shows a "Dose Window"

Compared with control (mean lifespan ~15.53 days), dosing at 25–75 μg/mL significantly extended mean lifespan to ~16.67–18.77 days.
Notably:

100 μg/mL did not extend lifespan

200 μg/mL reduced lifespan
This pattern suggests an optimal physiological window-common in bioactive natural products where too high a dose can introduce stress or toxicity.

3.4 Stronger Stress Resistance + Less Oxidative Damage

The formula increased antioxidant enzyme activities (SOD, CAT, GSH‑Px) and reduced oxidative markers (ROS, MDA), consistent with improved redox homeostasis and cellular protection under stress.

3.5 Gene & Protein Evidence Points to IIS / FoxO-Like Longevity Signaling

The formula:

Downregulated daf‑2 and age‑1 (upstream insulin/IGF‑1-like signaling)

Upregulated daf‑16 and its downstream protective genes (e.g., sod‑3, gst‑4, ctl genes)

Increased expression of skn‑1 and heat-shock genes (hsf‑1, hsp‑16.1, hsp‑16.2)

Increased DAF‑16 and SKN‑1 nuclear localization (functional activation)

Increased reporter signals for SOD‑3, GST‑4, HSP‑16.2

Mutant data indicated that lifespan extension was dependent on daf‑16, skn‑1, hsf‑1 (no significant right-shift in survival curves in these mutants), and not dependent on eat‑2 (dietary restriction pathway), supporting a primary mechanism via insulin signaling modulation and stress-response activation, rather than reduced food intake.

4. Discussion: Translating TCM "Efficacy" Into Modern Biology

From a modern systems-biology view, multi-herb formulas behave like network modulators rather than single-target drugs. The network pharmacology results in this study align with that reality: many compounds connect to overlapping targets and converge on conserved longevity biology.

Mechanistically, the findings can be grouped into four functional modules:

Longevity/stress transcription programs (FoxO-like / DAF‑16): activating downstream antioxidant, detoxification, and maintenance genes that reduce "wear-and-tear" over time.

Metabolic–growth signaling (PI3K/Akt/mTOR-related): balancing growth, survival, and maintenance processes, which are central to aging.

Oxidative stress and proteostasis: reduced ROS/MDA and enhanced heat-shock responses imply improved protein quality control and stress adaptation.

Aging–disease overlap: pathways intersecting cancer and metabolic regulation reflect the shared biology between aging and chronic disease risk.

In short: this formula did not simply "look good on paper." It produced whole-organism improvements (longer lifespan, better stress resilience, lower oxidative burden) with molecular validation (IIS suppression + DAF‑16/SKN‑1/HSF‑1 activation).

Limitations were acknowledged: pathway validation in mammals is still needed, and further safety/efficacy evaluation is required before clinical claims.

Marketing-Ready Add-On: Why Cistanche (Especially Cistanche tubulosa) Matters

For Western audiences, it helps to position Cistanche as an "anti-aging herb" (longevity-support botanical) whose best evidence often maps to:

oxidative stress defense

metabolic/insulin signaling balance

cellular stress resistance (heat-shock, detoxification)

Our Supply Advantage (WECISTANCHE / Factory Highlights You Can Cite)

Based on your company "About Us" page, the following factual points can be used to strengthen credibility:

 

CLICK THE PICTURE TO VISIT OUR FACTORY

Company: Chengdu Wecistanche Bio‑Tech Co., Ltd

Established: 2003

Location: headquartered in Luopu County, Xinjiang, China

Scale: integrated Cistanche industry chain including large cultivation bases and a GMP factory; large fresh herb collection/storage and processing capacity

Positioning: focus on high-quality Cistanche, especially Hotan/Xinjiang origin materials

Quality & certification mentions: GMP workshop and multiple certification claims are presented on the page

Academic collaboration: chief scientist and collaborations with multiple universities and research institutions are stated

Core product focus: Cistanche tubulosa (管花肉苁蓉) extract

Your key differentiation statement (to emphasize in product pages):
We specialize in Cistanche tubulosa extract, commonly favored in the market for its higher active-ingredient potential versus generic Cistanche material, making it a strong candidate for anti-aging herbal supplement development.