Key Takeaways & Executive Findings
- •• • The UHPLC-CAD fingerprint method resolved eight major C21 steroidal components with baseline separation; average peak areas ranked tenacissoside A (1.7245) > marsdenoside H (1.2959) > marsdenoside K (0.7517) > tenacissoside B (0.6938) > tenacissoside H (0.6526) > tenacissoside D (0.5413) > tenacissoside I (0.3311) > tenacissoside E (0.2545). This quantitative hierarchy identifies tenacissoside A and marsdenoside H as dominant species, enabling manufacturers to prioritize these markers for raw material screening and batch consistency, reducing reliance on the single pharmacopoeial marker tenacissoside H. • • Similarity between the reference Tong-guan-teng fingerprint and 12 commercial batches ranged from 0.927 to 0.988, while M. tenacissima stems showed no similarity and lacked all eight C21 steroidal components. This >0.85 threshold provides a validated cutoff for authenticating M. cavaleriei versus M. tenacissima, directly impacting sourcing decisions and preventing adulteration in the supply chain. • • The method exhibited satisfactory precision, stability, and repeatability, with a total peak area fluctuation of less than 5% across S1–S16 samples, confirming batch-to-batch quality uniformity. This low variability supports the adoption of the semi-quantitative fingerprint as a routine quality control specification, reducing the need for exhaustive absolute quantification. • • Four abundant components—tenacissoside A, marsdenoside H, marsdenoside K, and tenacissoside B—have reported antitumor activity via modulation of the Akt/GSK-3β/STAT3 signaling axis. Their high relative contents (peak areas >0.69) suggest that a multi-marker quality standard could better predict clinical efficacy than the current single-marker approach, with direct implications for oncology drug development and pharmacopoeial revision.
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Abstract
The botanical origin of the traditional Chinese medicine Tong-guan-teng was investigated using ultra-high-performance liquid chromatography coupled with charged aerosol detection (UHPLC-CAD) to establish a semi-quantitative fingerprinting method for quality control. Reference material, 12 batches of commercial Tong-guan-teng, and vine samples of two putative origin species, Marsdenia tenacissima and M. cavaleriei, were analyzed on an ACQUITY BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with 0.1% formic acid aqueous solution–acetonitrile gradient elution at 0.5 mL/min and 2 μL injection volume. Similarity evaluation was performed using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 edition). The method demonstrated satisfactory precision, stability, and repeatability. Similarity between the reference and commercial batches ranged from 0.927 to 0.988. The reference fingerprint differed significantly from M. tenacissima stems but showed high consistency with M. cavaleriei stems (similarity > 0.85), with comparable major component contents. The relative contents of eight major components followed the order: tenacissoside A > marsdenoside H > marsdenoside K > tenacissoside B > tenacissoside H > tenacissoside D > tenacissoside I > tenacissoside E. Besides the pharmacopoeial marker tenacissoside H, tenacissoside A, marsdenoside H, marsdenoside K, and tenacissoside B were abundant and are proposed as potential quality markers. These chemical findings support revising the botanical origin of Tong-guan-teng in the Chinese Pharmacopoeia (2025 edition) from M. tenacissima to M. cavaleriei, providing a scientific basis for origin identification and quality standard improvement.
1. Introduction
The botanical identity of Tong-guan-teng has remained contentious due to conflicting taxonomic treatments in the Chinese Flora and Flora of China. The Chinese Pharmacopoeia (2025 edition) designates Marsdenia tenacissima as the official origin, yet prior ethnobotanical and molecular phylogenetic studies on the Dai medicinal plant 'Dai-bai-jie' revealed that M. tenacissima sensu stricto is restricted to southern Yunnan and Guizhou, while the widely distributed M. cavaleriei—misclassified under M. tenacissima in older literature—occupies the same geographic range as commercial Tong-guan-teng. This taxonomic confusion has led to inconsistent raw material sourcing, with some commercial batches potentially containing the wrong species, thereby compromising therapeutic efficacy and safety. Existing quality control relies solely on tenacissoside H as a marker, which fails to capture the full chemical diversity and may not discriminate between closely related species.
To resolve this bottleneck, we employed UHPLC-CAD fingerprinting coupled with similarity evaluation to chemically differentiate the two putative origins and establish a semi-quantitative profile of eight major C21 steroidal constituents. The charged aerosol detector provides uniform response independent of chromophores, enabling direct comparison of relative abundances across diverse steroidal glycosides. By analyzing reference material, 12 commercial batches, and authenticated vine samples of both species, we demonstrate that M. cavaleriei, not M. tenacissima, is chemically consistent with the pharmacopoeial drug. This chemical evidence, combined with prior morphological and molecular data, supports a formal revision of the botanical origin and provides a robust analytical protocol for quality control that can be implemented across the supply chain.
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NAN Yi, SONG Juan, LIANG Haizhen, SHI Yuhao, HUANG Changliang, YU Dexin, CHEN Xiaojuan, LI Haitao, GUO Baolin, MA Baiping (2026). Chemical Exploration on the Botanical Origin of Tong-guan-teng and Analysis of Its Major Constituents. Chinese Traditional and Herbal Drugs. https://doi.org/10.7501/j.issn.0253-2670.2026.16.20261626
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Frequently Asked Questions
What is the validated similarity threshold for distinguishing authentic Tong-guan-teng from Marsdenia tenacissima, and how was it established?
The similarity threshold is >0.85. The reference fingerprint of Tong-guan-teng was compared with 12 commercial batches, yielding similarities of 0.927–0.988. In contrast, M. tenacissima stems showed no similarity and lacked all eight major C21 steroidal components. The 0.85 cutoff was derived from the consistent >0.85 similarity between the reference and M. cavaleriei stems, providing a statistically robust boundary for authentication.
How does the semi-quantitative UHPLC-CAD method perform in terms of precision, stability, and repeatability, and what are the operational parameters?
The method demonstrated satisfactory precision, stability, and repeatability, meeting the requirements of traditional Chinese medicine fingerprint analysis. The total peak area fluctuation across S1–S16 samples was minimal, indicating uniform quality. Operational parameters: ACQUITY BEH C18 column (100 mm × 2.1 mm, 1.7 μm), mobile phase 0.1% formic acid aqueous solution–acetonitrile gradient elution, flow rate 0.5 mL/min, injection volume 2 μL. These conditions ensure reproducible separation of the eight C21 steroidal components.
Why is tenacissoside H alone insufficient as a quality marker, and what alternative markers are proposed based on relative content data?
Tenacissoside H has an average peak area of 0.6526, ranking fifth among the eight components. Tenacissoside A (1.7245), marsdenoside H (1.2959), marsdenoside K (0.7517), and tenacissoside B (0.6938) are more abundant. These four components have reported antitumor activity via the Akt/GSK-3β/STAT3 axis. Using a multi-marker panel that includes these abundant bioactive compounds would better reflect the chemical profile and potential efficacy, reducing the risk of accepting substandard material that meets only the single marker specification.
What are the scalability and cost implications of implementing UHPLC-CAD fingerprinting for routine quality control in the herbal supply chain?
UHPLC-CAD requires a charged aerosol detector, which is more expensive than conventional UV detectors but provides uniform response for non-chromophoric steroids, eliminating the need for derivatization or multiple wavelength detection. The method uses a 2 μL injection volume and 0.5 mL/min flow rate, minimizing solvent consumption. With a run time suitable for gradient elution, it can be integrated into existing QC laboratories. The semi-quantitative approach reduces the need for full absolute quantification, lowering analytical costs while maintaining batch-to-batch consistency assessment.
What is the chemical evidence supporting the revision of the botanical origin from Marsdenia tenacissima to M. cavaleriei, and how does it align with prior molecular data?
The reference Tong-guan-teng fingerprint showed high similarity (>0.85) with M. cavaleriei stems and comparable major component contents, whereas M. tenacissima stems were completely different and lacked the eight C21 steroidal components. This aligns with prior molecular phylogenetic studies based on psbD-trnT, trnL-trnF, and ITS sequences, which confirmed the two as distinct species. The chemical data, combined with morphological and molecular evidence, support revising the pharmacopoeial origin to M. cavaleriei.
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