To identify the chemical constituents of different fractions of the water extract of Chaixian Huashen decoction (CXHSD) and to evaluate the antipyretic and analgesic activities of each fraction in order to improve the extraction process.

The water extract of CXHSD was obtained via water reflux extraction (CXHSD-W). An alcohol - water extract was obtained via 70% ethanol reflux extraction once, followed by water reflux extraction twice (CXHSD - EW). The water extract was separated using macroporous adsorption resin into four elution fractions: water (CXHSD-00), 30% ethanol (CXHSD-30), 60% ethanol (CXHSD-60), and 95% ethanol (CXHSD-95). The constituents of each fraction were identified using UPLC-Q-TOF-MS technology against a self-built compound library. The contents of total flavonoids, total saponins, and total alkaloids in the water extract, the four elution fractions, and the alcohol-water extract were determined by ultraviolet spectrophotometry. The antipyretic and analgesic activities of each fraction were evaluated using a lipopolysaccharide (LPS)-induced fever model and an acetic acid-induced writhing pain model in mice.

A total of 114 compounds were identified from the four fractions of the water extract. The main active components, including flavonoids, saponins, and alkaloids, were primarily distributed in CXHSD-30 and CXHSD-60. The results of ultraviolet spectrophotometric determination for total flavonoids, total saponins, and total alkaloids were consistent with the above findings. Animal experiments showed that CXHSD-60 significantly inhibited LPS-induced fever (P<0.001). The inhibition rate of CXHSD-30 was the highest (76.68%, P<0.001) against acetic acid-induced pain. CXHSD-EW contained higher levels of total flavonoids and total saponins than CXHSD-W while possessing superior antipyretic and analgesic activities compared to CXHSD-W.

The addition of 70% ethanol extraction in CXHSD preparation increases the extraction yield of active substances and enhances its antipyretic and analgesic activities. The optimized extraction process for this formula is determined to be ethanol extraction once, followed by water extraction twice.