Interactions between turnover rate and bioactivity of atractylenolide III in Atractylodes macrocephala rhizome in the theory of crude drugs’ pairs and prescriptions in traditional Chinese and Japanese Kampo medicine

The dried rhizome of Atractylodes macrocephala Koidz. (AM) is widely utilized in traditional Chinese and Japanese Kampo medicine for its therapeutic effects including digestive improvement, spleen strengthening, and dampness reduction. Atractylenolide III (AIII), an anti-inflammatory and gastric-protective sesquiterpene of AM, serves as a critical chemical reference substance (CRS) of AM, essential for its pharmacological validation. However, its content in the decoction of traditional formula varied due to its chemical properties. This study aimed to investigate the impact of the combinations of AM and other crude drugs in traditional medicinal drug-pair theory by assessing the relationship of extracting conditions, phytochemical profiles, and the bioactivities of the decoctions prepared using various crude drug combinations. AM was decocted with the dried sclerotium of Wolfiporia cocos (WE), the dried root and rhizome of Glycyrrhiza uralensis (GU), the dried root of Panax ginseng (PG), the dried twig of Cinnamomum cassia (CC), or the dried rhizome of Zingiber officinale (ZO), respectively, with varying ratios. The extraction parameters investigated included the ratio of crude drugs-to-water, heating times, and pH of the decoction. The contents of AIII and other CRS were analyzed using HPLC. Bioactivities, including anti-inflammatory effects in RAW 264.7 cells, gastroprotective effects in gastric mucosal cells, and antioxidative effects in SH-SY5Y neuronal cells, were also evaluated. The best extracting yield of AIII from AM was achieved with the ratio of crude drugs to water 1:60 (w/v) for 30-90 min of heating time at pH 5. The combinations of crude drugs significantly affected AIII turnover rates, with the highest yield obtained from formulations containing 25% AM, particularly the Lingguizhugantang (ryokeijutsukanto, LGZGT) that contains AM, WE, GU, and CC. The combination of crude drugs enhanced pharmacological activities, with LGZGT notably improving gastric mucosal protection and anti-inflammatory effects. In contrast, Shenzhetang (jinchakuto, SZT) that contains AM, WE, GU, and ZO, exhibited notable anti-inflammatory effects attributed to increased 6-gingerol content despite a reduced AIII concentration. These findings provide critical insights into optimizing AM-based formulations in traditional Chinese or Japanese Kampo medicinal theory for improving clinical efficacies and standardized phytochemical quality control.