Screening and Validating of 1, 1‑Diphenyl‑2‑Trinitrophenylhydrazine Scavengers from Danshen‑Honghua Herbal Pair

Background: Salvia miltiorrhiza (Danshen [DS]) and Carthamus tinctorius (Honghua [HH]) are commonly used traditional Chinese medicines for activating blood and removing stasis, and they were usually used as DS‑HH (Danshen‑Honghua [DH]) herbal pair in clinical applications. Characterizing the antioxidant active ingredients in DH herbal pair may be helpful for understanding their curative effect on cardiovascular diseases. Objective: The objective of the study is to screen the 1, 1‑diphenyl‑2‑trinitrophenylhydrazine (DPPH) scavenging active compounds in DH herbal pair by spectrum‑effect relationship analysis. Materials and Methods: First, the water extracts of DH herbal pair with different ratios (DS: HH = 1:0, 0:1, 1:1, 2:1, 3:1, 5:1, 1:5, 1:3, and 1:2) were prepared. Then, the clearance effects of DH herbal pair and single drugs on DPPH were compared, meanwhile, high performance liquid chromatography was applied for chemical analysis of DH extracts. Finally, DPPH scavengers in DH herbal pair were predicted and identified by spectrum‑effect relationship analysis and liquid chromatography‑mass spectrometry (LC‑MS) analysis. Results: Compared with single drugs, the extracts of herbal pairs had higher clearance rate to DPPH. Eighteen potential active compounds (peaks) in the extract of DH herbal pair were predicted, and 13 of them were tentatively identified by LC‑MS analysis. Furthermore, antioxidant activities of eight pure compounds from the DH herbal pair were validated by DPPH radical scavenging assay with Vitamin C as positive control drug. Among them, six compounds including danshensu, protocatechuic acid, coffee acid, chlorogenic acid, rutin, and salvianolic acid A were found to have high antioxidant activity. Conclusion: DH herbal pair showed strong clearance effect on DPPH, and danshensu, protocatechuic acid, coffee acid, chlorogenic acid, rutin and salvianolic acid A are the active components.