Introduction
Polygalae Radix (PR), derived from the dried roots of Polygala tenuifolia Willd., is a renowned herbal medicine in traditional Chinese medicine (TCM) systems. Known as "Yuanzhi" in China and "Onji" in Japan, PR is widely used for its calming, phlegm-dispelling, and mind-enlightening properties. It is also recognized as a safe ingredient in health foods, listed among the 114 TCMs approved by China’s National Health Commission for dietary use.
Processing—known as Pao-Zhi in TCM—is a critical step that enhances therapeutic efficacy and reduces inherent toxicity. The primary forms of processed PR include:
- Polygalae Cortex (PC): Removal of the xylem (woody core)
- Licorice-processed (LP): Frying PC with licorice juice
- Honey-processed (HP): Frying PC with honey
While these methods are traditionally believed to improve safety and effectiveness, scientific validation of their impact on chemical composition and biological activity remains essential. This study leverages UPLC-Q-TOF-MS/MS combined with multivariate statistical analysis to compare the chemical profiles, bioactive compound levels, and antioxidant capacities of crude and processed PR.
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Methodology Overview
Sample Preparation and Processing
Raw P. tenuifolia was sourced from a certified cultivation base in Yulin, Shaanxi Province. Four sample types were prepared:
- Crude PR: Dried roots
- PC: Xylem removed manually
- HP: PC fried with honey at 180°C
- LP: PC decocted with licorice juice
Each sample was extracted using 70% methanol and analyzed via UPLC-Q-TOF-MS/MS in negative ion mode for comprehensive metabolite profiling.
Analytical Techniques
- Chromatography: Waters ACQUITY UPLC BEH C18 column, gradient elution with acetonitrile and formic acid
- Mass Spectrometry: ESI source, m/z 50–2000 range, high-resolution detection
Bioactivity Assays:
- Total phenolic content (TPC) via Folin-Ciocalteu method
- Total flavonoid content (TFC) using aluminum nitrate colorimetry
- Total saponin content (TSC) with vanillin-perchloric acid assay
- Antioxidant capacity assessed via ABTS, FRAP, CUPRAC, and DPPH assays
Statistical analysis included PCA, PLS-DA, HCA, and Pearson correlation to identify key compositional differences and bioactivity trends.
Chemical Profiling of Crude and Processed PR
A total of 131 chemical compounds were identified across all samples, classified into six major groups:
- 41 saponins (31.3%)
- 45 oligosaccharide esters (34.35%)
- 25 xanthones (19.08%)
- 2 organic acids
- 3 carbohydrates
- 15 exogenous compounds from processing agents
Key Compound Classes
Saponins: The Primary Active Constituents
Saponins are the main pharmacologically active components in PR, known for their neuroprotective, sedative, and anti-inflammatory effects. Diagnostic fragment ions at m/z 455 and 425 were used to identify triterpenoid saponins like Tenuifolin, a marker compound.
Processing significantly altered saponin levels:
- HP and LP showed reduced saponin content due to thermal degradation and glycosidic bond cleavage
- Some hydrolysis products may contribute to detoxification effects
Oligosaccharide Esters: Neuroprotective Powerhouses
These compounds exhibit strong antioxidant and neuroprotective activities. With sucrose as a core structure, they are esterified with various organic acids. Examples include Tenuifoliside A and Sibiricose A5.
Thermal processing led to partial degradation, but certain derivatives remained stable or even increased in concentration.
Xanthones: Bioactive Phenolics
Xanthones contribute to PR’s antidepressant and anti-gout properties. Identified through neutral loss patterns (e.g., 162 Da for glucose), compounds like Polygalaxanthone III showed variable responses to processing.
Exogenous Compounds from Processing Aids
- HP samples contained 8 honey-derived compounds (e.g., sucrose, diosmetin)
- LP samples introduced 7 licorice components (e.g., liquiritin, isoliquiritin), known for their neuroprotective and anti-inflammatory benefits
This demonstrates how processing not only modifies existing compounds but also introduces new bioactive elements.
Multivariate Analysis Reveals Distinct Chemotypes
Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) clearly separated crude and processed samples:
- PC1 and PC2 explained 81.4% of variance
- Crude PR clustered distinctly from all processed forms
- HP and LP formed separate clusters due to auxiliary ingredient signatures
Hierarchical Cluster Analysis (HCA) confirmed four distinct groups corresponding to PR, PC, HP, and LP.
VIP (Variable Importance in Projection) analysis identified top discriminators:
- Peak No. 7 (unknown oligosaccharide ester): VIP = 2.58
- Peak No. 11 (1,7-dihydroxy-2,3-dimethoxyxanthone): VIP = 2.57
These findings validate that processing induces significant chemical reorganization.
Bioactive Compound Levels and Antioxidant Activity
Total Phenolics, Flavonoids, and Saponins
| Sample | TPC (mg GAE/g DW) | TFC (mg RE/g DW) | TSC (mg TE/g DW) |
|---|---|---|---|
| PC | 12.345 ± 0.107 | 78.467 ± 0.440 | 19.205 ± 0.068 |
| PR | 9.459 ± 0.014 | 52.338 ± 0.281 | 18.548 ± 0.096 |
| HP | 9.381 ± 0.009 | 63.052 ± 0.280 | 14.435 ± 0.012 |
| LP | 7.443 ± 0.019 | 72.800 ± 0.608 | 17.065 ± 0.120 |
Key Insight: PC exhibited the highest levels across all three metrics, indicating superior bioactive potential compared to both crude PR and further processed forms.
In Vitro Antioxidant Capacity
All four antioxidant assays consistently ranked samples as follows:
DPPH Radical Scavenging (% Inhibition)
- PC: 51.56%
- HP: 48.16%
- PR: 45.73%
- LP: 43.39%
ABTS⁺ Activity (μM/g DW)
- PC: 23.98
- PR: 21.44
- HP: 21.34
- LP: 21.22
FRAP & CUPRAC Reducing Power
Both assays showed:
- Highest activity in PC
- Lowest in LP
- Strong correlation between TFC and antioxidant power (r > 0.95)
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Frequently Asked Questions (FAQ)
What is the main difference between crude and processed Polygalae Radix?
The primary difference lies in chemical composition and safety profile. Processing removes the xylem (which may contain irritants), alters saponin structures through hydrolysis, and introduces beneficial compounds from licorice or honey, enhancing therapeutic effects while reducing gastrointestinal side effects.
Why does Polygalae Cortex (PC) show higher antioxidant activity?
PC retains high levels of phenolics, flavonoids, and saponins without thermal degradation from frying. The removal of the xylem concentrates active compounds in the cortex, leading to superior antioxidant performance compared to both raw roots and heat-treated products.
Does honey or licorice processing improve medicinal value?
Yes, but differently:
- Honey processing (HP) enhances calming effects and improves palatability.
- Licorice processing (LP) introduces neuroprotective flavonoids like liquiritin and reduces pharyngeal irritation.
However, both methods reduce overall saponin content due to heat exposure.
How reliable is UPLC-Q-TOF-MS/MS for herbal analysis?
Extremely reliable. This technique offers high resolution, accurate mass measurement, and broad metabolite coverage, making it ideal for identifying hundreds of compounds in complex plant matrices—essential for quality control and standardization in herbal medicine.
Can processed Polygalae Radix be used in functional foods?
Absolutely. Given its approval as a health food ingredient in China and demonstrated antioxidant properties—especially in PC form—it holds strong potential for inclusion in brain health supplements, anti-aging formulations, and natural nootropics.
Is there a risk of losing active compounds during processing?
Yes, particularly with high-heat methods like frying. Saponins and oligosaccharide esters are sensitive to heat and moisture, leading to partial degradation. Optimizing processing parameters (time, temperature, humidity) is crucial to balance detoxification with bioactivity preservation.
Conclusion
This study provides a comprehensive chemical and functional comparison of crude and processed Polygala tenuifolia. Using UPLC-Q-TOF-MS/MS and multivariate analysis, we identified 131 compounds and demonstrated that processing significantly reshapes the phytochemical landscape.
Key findings:
- Polygalae Cortex (PC) exhibits the highest levels of total phenolics, flavonoids, saponins, and antioxidant activity
- Heat-based processing (HP, LP) reduces certain native compounds but introduces beneficial exogenous ones
- Multivariate models clearly distinguish processing types based on chemical fingerprints
These results offer a scientific basis for optimizing PR processing methods to maximize health benefits while ensuring safety. They also support the development of standardized extracts for use in pharmaceuticals, nutraceuticals, and cosmeceuticals targeting cognitive health and oxidative stress-related conditions.
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