Process-function-structure relationships in Zixue Powder: How traditional processing preserves more active constituents and improves therapeutic outcome.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Zixue Powder is a traditional herbal formulation used to treat febrile seizures and other related diseases. However, there are differences between the traditional and pharmacopoeial methods of preparation, and it remains unclear whether these variations affect the content and therapeutic efficacy of Zixue Powder. AIM OF THE STUDY: This study aimed to systematically compare traditional (ZXTP) and pharmacopoeial (ZXPP) Zixue Powder in terms of content, in vitro release, and therapeutic efficacy, and to explore the underlying reasons. MATERIALS AND METHODS: Powder properties were calculated. HPLC fingerprints combined with chemometric analysis identified chemical differences. In vitro release was measured by dialysis, and a febrile seizure rat model was used to evaluate efficacy. Western blotting assessed TNF-α, GluA1, and CaM. Structural features were characterized by FT-IR, DSC, and SEM. RESULTS: Significant differences were observed in powder characteristics between ZXTP and ZXPP, including five parameters specifically Carr's index and difference angle. Chromatographic fingerprint analysis indicated that the two preparations shared broadly similar chemical profiles, whereas quantitative analysis revealed that seven representative constituents, including agarotetrol, were present at significantly higher levels in ZXTP. In vitro release studies showed that the cumulative release rates of three components, including agarotetrol, within 0.25-8 h were significantly higher in ZXTP than in ZXPP. In a febrile seizure rat model, both preparations exerted antiepileptic effects; however, ZXTP was associated with a significantly prolonged seizure latency, shortened seizure duration, and reduced brain water content compared with ZXPP. Furthermore, ZXTP treatment resulted in greater attenuation of TNF-α, GluA1, and calmodulin levels, accompanied by improved preservation of hippocampal neuronal morphology. Scanning electron microscopy revealed a characteristic branched microstructural framework in ZXTP, whereas ZXPP exhibited an irregular block-like morphology. CONCLUSION: Traditional Zixue Powder showed higher constituent levels, faster in vitro release, and more pronounced neuroprotective effects than the pharmacopoeial preparation. These differences were accompanied by distinct microstructural features, suggesting that preparation procedures may influence the physicochemical and pharmacological properties of Zixue Powder.