**1. Introduction**

The rhizome of *Atractylodes macrocephala* Koidz (RAM)—called Baizhu in traditional Chinese medicine (TCM)—is a well-known herbal medicine. It was documented in the earliest existing book on TCM—"Shen Nong's Materia Medica" written during the Han Dynasty (A.D. 25-220). RAM has been traditionally used for the treatment of various disorders, such as loss of appetite, diarrhea, limb weakness, gastrointestinal dysfunction, immune dysfunction, diabetes, and some chronic inflammatory diseases [1]. It was reported that RAM is used in more than 835 TCM preparations, as well as an integral part of more than 4340 classic prescriptions for treating chronic diseases [1]. Indeed, RAM is considered a functional food, a tonic, and a constituent of various health products purported for promoting digestion, alleviating fatigue, improving sleeping, enhancing immunity, and treating alimentary anemia [2]. RAM is traditionally used in its processed form which is commonly achieved by stir-frying

raw RAM with wheat bran [3,4]. This type of processing technique, known as Pao-Zhi in TCM, has been widely used for the preparation of Chinese Materia Medica [5] with a long history. It is believed that after processing, property and function of remedies can be changed, medical potency can be increased, and/or toxicity and side effect can be reduced. The practice of processing RAM can date back to the Tang dynasty (A.D. 618–907) [6], and the earliest processing protocol for RAM by stir-frying with wheat bran was recorded in the famous Sheng Ji Zong Lu, the TCM prescription book written in A.D. 1117. This processing method is also currently documented in the Chinese Pharmacopeia [7]. Although processed RAM is commonly used as an ingredient of various Chinese medicines and health products, so far, the chemical compositional change as a result of processing has not been clearly delineated.

Extensive phytochemical and pharmacological studies have been conducted on raw RAM, revealing the presence of volatile organics, sesquiterpenoids, triterpenoids, polyacetylenes, coumarins and phenylpropanoids, flavonoids and their glycosides, steroids, benzoquinones, and polysaccharides [1]. Among them, sesquiterpene lactones (i.e., atractylenolids I, II, and III) are considered to be the characteristic and bioactive constituents of RAM [8,9]. Efforts have been previously undertaken to investigate the changes occurring in the chemical components of RAM before and after processing by utilizing HPLC, GC/MS, and other analytical techniques. Substantial changes in the concentration of constituents, including those in essential oil, as well as the eudesmane-type sesquiterpenoids (such as atractylon, atractylnolides I, II, and III, etc.), were observed [3,10–13]. These changes can be explained as the result of evaporation, conversion, and/or degradation by heating, due to their physicochemical properties such as volatility and instability [13,14]. Nevertheless, in continuation of the quest to probe the overall integrity of botanical preparations by using NMR-based metabolomics to investigate the change of chemical composition which occurred during processing, we observed that some new characteristic signals appeared in the NMR spectra of the processed RAM, which suggested that some unknown compounds could be generated during processing. Herein, the discovery of five new compounds isolated from the RAM processed by stir-frying with wheat bran is reported. Their chemical structures were determined by both spectroscopic and spectrometric analyses. In addition, a plausible mechanism for the generation of these new compounds was proposed. Furthermore, their cytotoxic, antimicrobial, and antileishmanial activities were evaluated.
