The earliest excipient products of Traditional Chinese Medicine: Identification and analysis of samples from wooden lacquer box unearthed from Haihunhou tomb in the Western Han Dynasty

Abstract

The processing of traditional Chinese medicine is an integral part of Chinese medical heritage. The recent discovery of the earliest processed drugs may provide strong evidence for the origins of traditional Chinese medicine processing. The remains were found in wooden lacquer boxes, unearthed from Haihunhou tomb in Nanchang, Jiangxi Province. By analyzing the appearance of the unearthed samples, it was found that the outer layers of the samples were composed of an auxiliary material and the inside was composed of plant material. As can be seen from the three-dimensional image taken using nuclear magnetic resonance screening, the sample had a hollow irregular rod structure. The outer layers were composed of a substance with weak signal strength, and the inside was similar to the fiber structure of plants with high water contents. Microscopic analysis showed that periderm, secondary xylem, and broad wood rays could be seen in the transverse section of the plants inside the samples. It can be further speculated that the plants inside the samples were derived from the roots or stems of dicotyledonous herbs. Additionally, the putative roots or stems of the dicotyledonous herbs had secondary structures and contained one to three clustered, red-brown cells. No starch granules, calcium oxalate crystals, or stone cells were observed in the transverse section. According to the microscopic database of the National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, it is speculated that the plants inside the samples were derived from the roots of Rehmannia species. A comparative study of the microscopic characteristics of wild Rehmannia chingii roots and the unearthed samples was carried out. It was found that both displayed periderm, broad secondary phloem, wood rays, similar vessel morphologies, and red-brown cells. The unearthed samples and R. chingii roots were analyzed by electrospray ionization mass spectrometry (ESI-MS) in negative ion mode. The results showed that the mass spectrum peak of m / z 623 in the unearthed samples agreed with that in the roots of R. chingii , and was putatively identified as acteoside or forsythiaside. Moreover, the peak at m / z 179 in the unearthed samples was identified as caffeic acid based on the molecular weight using high resolution tandem mass spectrometry (MS/MS) data, and was usually identified as a hydrolysate of acteoside or forsythiaside. There were about two to three outer layers of excipient on the samples. They were distributed unevenly, thicker at the top and thinner at the bottom. The plants and excipient layers were observed by microscope, and starch grains were found in the cross section and powder of the excipient layers, while no starch grains were found in the cross section or powder of the plant material inside. However, the reaction between the plant tissues and iodine-potassium iodide test solution produced the characteristic blue color. This phenomenon was related to the gelatinization of starch granules. Under a polarized light microscope, a large number of crystals were seen in the excipient layers. An aqueous extract of the excipient layers was analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and an ion fragment peak at m / z 341.11 was detected, indicating a disaccharide. When compared to the reference substances glucose, sucrose, fructose, and maltose, the retention times and mass spectra were very similar to that of sucrose. According to ancient literary and historical records, the tomb owner suffered from severe rheumatism before his death, which is consistent with the efficacy of R. chingii . It is further speculated that the samples in the wooden lacquer boxes are the remains of processed ancient traditional Chinese medicine products. The origin of the excipients in the processed medicines might be related to improving the taste, which could provide a scientific basis for further understanding the processing and application of ancient traditional Chinese medicines.