**4. Conclusions**

Pao-Zhi (frying and cooking of herbs) is an ancient pharmaceutic technique in TCM to facilitate the use of herbal medicines for specific clinic needs [31]. Traditionally, most Chinese herbal medicines undergo elaborate processing in order to become ingredients that are prescribed or utilized in the manufacturing of TCM proprietary drugs [32]. Although the practice of processing has a long history, the underlying mechanisms largely remain unclear for most Chinese herbal medicines. In the present study, through the characterization of chemical profiles coupled with NMR metabolomics approach, the chemical changes resulting from the traditional processing protocol associated with RAM preparation were investigated. For the first time, five new chemical adducts, which were formed during processing *A. macrocephala* with wheat bran, were isolated and their structures were identified. The findings allowed us to gain valuable insights into the chemical reactions which occur during the processing procedures. Processed RAM is widely used in various formulations of TCM drugs and health care products. Stir-frying with wheat bran is one of the most widely used traditional processing methods for RAM in TCM [12]. Although the processed RAM is listed as an item in the Chinese Pharmacopoeia, there are currently no modern standardized processing protocols or quality control standards for the processed RAM products. The findings of this study may provide useful information for developing such standards with a scientific basis. Furthermore, as the change of chemical profile will inevitably influence the associated pharmacological properties of processed RAM, further investigations of the bio-activities of the newly generated compounds are needed.

**Supplementary Materials:** The supplementary information is available online. Figure S1.1: 1H-NMR spectrum of compound **1**, Figure S1.2: 13C-NMR spectrum of compound **1**, Figure S1.3: HSQC spectrum of compound **1**, Figure S1.4: COSY spectrum of compound **1**, Figure S1.5: HMBC spectrum of compound **1**, Figure S1.6: HR-APCI-MS spectrum of compound **1**, Figure S1.7: IR spectrum of compound **1**, Figure S1.8: UV spectrum of compound **1**, Figure S2.1: 1H-NMR spectrum of compound **2**, Figure S2.2: 13C-NMR spectrum of compound **2**, Figure S2.3: HSQC spectrum of compound **2**, Figure S2.4: COSY spectrum of compound **2**, Figure S2.5: HMBC spectrum of compound **2**, Figure S2.6: HR-APCI-MS spectrum of compound **2**, Figure S2.7: IR spectrum of compound **2**, Figure S2.8: UV spectrum of compound **2**, Figure S3.1: 1H-NMR spectrum of compound **3**, Figure S3.2: 13C-NMR spectrum of compound **3**, Figure S3.3: HSQC spectrum of compound **3**, Figure S3.4: COSY spectrum of compound **3**, Figure S3.5: HMBC spectrum of compound **3**, Figure S3.6: HR-APCI-MS spectrum of compound **3**, Figure S3.7: IR spectrum of compound **3**, Figure S3.8: UV spectrum of compound **3**, Figure S4.1: 1H-NMR spectrum of compound **4**, Figure S4.2: 13C-NMR spectrum of compound **4**, Figure S4.3: HSQC spectrum of compound **4**, Figure S4.4: COSY spectrum of compound **4**, Figure S4.5: HMBC spectrum of compound **4**, Figure S4.6: HR-APCI-MS spectrum of compound **4**, Figure S4.7: IR spectrum of compound **4**, Figure S4.8: UV spectrum of compound **4**, Figure S5.1: 1H-NMR spectrum of compound **5**, Figure S5.2: 13C-NMR spectrum of compound **5**, Figure S5.3: HSQC spectrum of compound **5**, Figure S5.4: COSY spectrum of compound **5**, Figure S5.5: HMBC spectrum of compound **5**, Figure S5.6: HR-APCI-MS spectrum of compound **5**, Figure S5.7: IR spectrum of compound **5**, Figure S5.8: UV spectrum of compound **5**.

**Author Contributions:** Conceptualization, C.Z. and J.Z.; methodology, J.Z.; extraction, isolation, purification and identification, C.Z. and Y.M.; formal analysis, M.W.; writing—original draft preparation, J.Z., Y.M., and A.G.C.; writing—review and editing, M.W. and A.G.C.; project administration, I.A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by "Discovery & Development of Natural Products for Pharmaceutical & Agricultural Applications" funded by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6060-6-015.

**Acknowledgments:** The authors sincerely thank Shabana I. Khan and Bharathi Avula, at the NCNPR, University of Mississippi, for bioactivity testing and HR-APCI mass spectral recording, respectively.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
