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Chinese Science Bulletin, Volume 64, Issue 9: 935-947(2019) https://doi.org/10.1360/N972018-01176

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

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  • ReceivedJan 4, 2019
  • AcceptedJan 23, 2019
  • PublishedMar 6, 2019

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.


Funded by

名贵中药资源可持续利用能力建设项目(2060302)

中央级公益性科研院所基本科研业务费(ZZ10-008)


References

[1] Yang M, Zhang D K, Zhong L Y, et al. Study on culture and philosophy of processing of traditional Chinese medicines (in Chinese). China J Chin Mater Med, 2013, 38: 2223−2226 [杨明, 张定堃, 钟凌云, 等.对传统中药炮制文化与哲学的思考. 中国中药杂志, 2013, 38: 2223−2226]. Google Scholar

[2] Li W. Huangdi Neijing (in Chinese). Shenyang: Liaoning Ethnic Publishing House,1999 [李文. 黄帝内经. 沈阳: 辽宁民族出版社, 1999]. Google Scholar

[3] Shang Z Z. Notes on Medicine in “Fifty-two Patient Prescriptions” (in Chinese). Wuhu: Scientific Research Center of Wannan Medical College, 1985 [尚志钧.《五十二病方》药物注释. 芜湖: 皖南医学院科研科, 1985]. Google Scholar

[4] Zhang Z J. Annotation of Shang Han Lun (in Chinese). Changsha: Hunan Science and Technology Press, 1982 [张仲景. 伤寒论校注. 长沙: 湖南科学技术出版社, 1982]. Google Scholar

[5] He R. Annotation of Jinkui Yaolue (in Chinese). Beijing: People’s Medical Publishing House, 1990 [何任.《金匮要略》校注. 北京: 人民卫生出版社, 1990]. Google Scholar

[6] Huang L Q, Sun L Y, Zhang X B, et al. Brief introduction to progress of national census of Chinese medicine resources (pilot) (in Chinese). China J Chin Mater Med, 2017, 42: 4256−4261 [黄璐琦, 孙丽英, 张小波, 等. 全国中药资源普查(试点)工作进展情况简介. 中国中药杂志, 2017, 42: 4256−4261]. Google Scholar

[7] Jiang T X. Flora Dictionary (in Chinese). (Photocopy Edition). Volume I. Shanghai: Shanghai Literature & Art Publishing House, 1998 [蒋廷锡. 草木典. (影印版). 上册. 上海: 上海文艺出版社, 1998]. Google Scholar

[8] Compilation Committee of Chinese flora of the Chinese Academy of Sciences. Flora Reipublicae Popularis Sinicae (in Chinese). Beijing: Science Press, 1979. 67 [中国科学院中国植物志编辑委员会. 中国植物志. 北京: 科学出版社, 1979. 67]. Google Scholar

[9] Tang S W. Da guan Ben cao (in Chinese). Shang Z J collated. Hefei: Anhui Science & Technology Press, 2002. 180−181 [唐慎微. 大观本草. 尚志钧点校. 合肥: 安徽科学技术出版社, 2002. 180−181]. Google Scholar

[10] Liu M Q, Wang X Q, Chen S Q, et al. Histochemistry of Rehmannia glutinosa (in Chinese). J Chin Med Mater, 2013, 36: 1771−1773 [刘孟奇, 王小巧, 陈随清, 等. 地黄的组织化学研究. 中药材, 2013, 36: 1771−1773]. Google Scholar

[11] Wu Z C, Luo G M, Huo Y C. Extraction, isolation and TLC quantification of secretions from Rehmannia glutinosa secretory cells (in Chinese). Chin J Pharm Anal (Suppl), 1998, 18: 248−250 [吴子超, 罗干明, 霍永昌. 地黄分泌细胞分泌物的提取、分离和薄层扫描定量. 药物分析杂志(增刊), 1998, 18: 248−250]. Google Scholar

[12] Zhang B Y, Jiang Z Z, Wang Y F, et al. Analysis of chemical constituents in fresh, dried and prepared Rehmannniae Radix by UPLC/ESI-Q-TOF MS (in Chinese). Chin Trad Patent Med, 2016, 38: 1104−1108 [张波泳, 江振作, 王跃飞, 等. UPLC/ESI-Q-TOF MS法分析鲜地黄、生地黄、熟地黄的化学成分. 中成药, 2016, 38: 1104−1108]. Google Scholar

[13] Meng X Y. Studies on chemistry composition, standard of quality and anti-senescence activities of Rehmannia chingii Li (in Chinese). Master Dissertation. Yinchuan: Ningxia Medical University, 2015 [蒙雄裕. 天目地黄化学成分、治疗标准及其抗衰老活性研究. 硕士学位论文. 银川: 宁夏医科大学, 2015]. Google Scholar

[14] Li S L, Song J Z, Qiao C F, et al. A novel strategy to rapidly explore potential chemical markers for the discrimination between raw and processed Radix Rehmanniae by UHPLC-TOFMS with multivariate statistical analysis. J Pharm Biomed Anal, 2010, 51: 812-823 CrossRef PubMed Google Scholar

[15] Qi M, Xiong A, Li P, et al. Identification of acteoside and its major metabolites in rat urine by ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. J Chromatogr B, 2013, 940: 77-85 CrossRef PubMed Google Scholar

[16] Zeng G, Xiao H, Liu J, et al. Identification of phenolic constituents in RadixSalvia miltiorrhizae by liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom, 2006, 20: 499-506 CrossRef PubMed ADS Google Scholar

[17] Geng D D, Dong Q, Tan L, et al. On-line screening and identification of free adical scavenging compounds in Salvia miltiorrhiza Bunge and Salvia prattii Hemsl. by HPLC-DAD-ESI/MSn-DPPH (in Chinese). J Instrum Anal, 2015, 34: 314−320 [耿丹丹, 董琦, 谭亮, 等. HPLC-DAD-ESI/MSn-DPPH在线筛选与鉴别丹参和康定鼠尾草中抗氧化活性成分. 分析测试学报, 2015, 34: 314−320]. Google Scholar

[18] Pharmacopoeia of the People’s Republic of China. Pharmacopoeia Commission of the People’s Republic of China (in Chinese). Beijing: China Medical Science Press, 2015. 124 [国家药典委员会. 中华人民共和国药典. 一部. 北京: 中国医药科技出版社, 2015. 124]. Google Scholar

[19] Kang T G. Authentication of traditional Chinese medicine (in Chinese). Beijing: China Press of Traditional Chinese Medicine, 2016. 152−153 [康廷国. 中药鉴定学. 北京: 中国中医药出版社, 2016. 152−153]. Google Scholar

[20] Hu Z H. The relationship between the structure, development and medicinal components of medicinal plants (in Chinese). Shanghai: Shanghai Science and Technology Press, 2014. 352−354 [胡正海. 药用植物的结构、发育与药用成分的关系. 上海: 上海科学技术出版社, 2014. 352− 354]. Google Scholar

[21] Cheng Y L, Mei L T, Kuo H T. Effect of amylose content on the rheological property of rice starch. Cereal Chem, 1996, 73: 415−420. Google Scholar

[22] Ban G. History of the Han Dynasty (in Chinese). Beijing: Chinese Publishing House, 2016 [班固. 汉书. 北京: 中华书局, 2016]. Google Scholar

[23] Xu S. Origin of Chinese Characters (in Chinese). Beijing: Chinese Publishing House, 1985 [许慎. 说文解字. 北京: 中华书局, 1985]. Google Scholar

[24] Unknown. Plain Questions of Yellow Emperor’s Canon of Medicine (in Chinese). Beijing: China Medical Science and Technology Press, 2018 [佚名. 黄帝内经·素问. 北京: 中国医药科技出版社, 2018]. Google Scholar

[25] Sun X Y. The Shen Nong Ben Cao Jin (in Chinese). Beijing: People’s Medical Publishing House,1963. 13 [孙星衍辑. 神农本草经. 北京: 人民卫生出版社, 1963. 13]. Google Scholar

[26] Lei X. Lei Gong Pao Zhi Lun (in Chinese). Hefei: Anhui Science and Technology Publishing House, 1991. 24 [雷敩. 雷公炮炙论. 合肥: 安徽科学技术出版社, 1991. 24]. Google Scholar

[27] Jiangxi Institute of Archaeology, Nanchang museum, Nanchang Xinjian District museum. Haihunhou Han Tomb (in Chinese). Archaeol, 2016, (7): 45−62 [江西省文物考古研究所, 南昌市博物馆, 南昌市新建区博物馆. 南昌市西汉海昏侯墓. 考古, 2016, (7): 45−62]. Google Scholar

[28] Ji Q L. The History of Sucrose (in Chinese). Beijing: China Customs Publishing House, 2009 [季羡林. 蔗糖史. 北京: 中国海关出版社, 2009]. Google Scholar

[29] Zhang L. A Variorum of Prescriptions of Fifty-two Diseases of Mawangdui Han Tomb (in Chinese). Beijing: Ancient Books Publishing House of Traditional Chinese Medicine, 2017 [张雷. 马王堆汉墓帛书《五十二病方》集注. 北京: 中医古籍出版社, 2017]. Google Scholar

[30] Zhang Z J. Typhus Various Illnesses to Discuss (Guilin Guben) (in Chinese). Beijing: China Press of Traditional Chinese Medicine, 2014 [张仲景. 伤寒杂病论 (桂林古本). 北京: 中国中医药出版社, 2014]. Google Scholar

[31] Li S Z. Compendium of Materia Medica (in Chinese). Volume I. Beijing: Huaxia Publishing House, 1998. 706 [李时珍. 本草纲目. 上册. 北京: 华夏出版社, 1998. 706]. Google Scholar

  • Figure 1

    The Haihunhou tomb (M1) and the unearthed samples studied in this study. (a) The orthophoto map of M1; (b) the partitions of Haihunhou tomb; (c) the unearthed samples and lacquer box; (d) lacquer box for samples; (e) the samples were stacked on top of each other; (f1), (f2) characteristics of the front and the back of sample

  • Figure 2

    Micro-CT image of sample in wooden lacquer box unearthed from Haihunhou Han tomb

  • Figure 3

    The microstructure of sample in wooden lacquer box unearthed from Haihunhou Han tomb. (a) The sample cross section in freezing microtome, showing the excipients and plants, with white frozen glue; (b) the sample section under microscope, showing excipient layers and plants; (c1) fragments of excipient layers; (c2-1) starch granules in excipient layers (bright-field); (c2-2) starch granules in excipient layers (polarized-light); (c3) fragments of excipient layers; (c4-1) one of the masses containing crystals in the excipient layers (bright-field); (c4-2) one of the masses containing crystals in the excipient layers (polarized-light); (d1) periderm; (d2) secondary xylem containing vessels and parenchyma cells; (d3) secondary xylem containing vessels and rays; (d4) reddish brown cells; (d5) the reaction between plant tissues and iodine-potassium iodide test solution showing characteristic blue; (d6) vessels

  • Figure 4

    The roots and its microstructure of Rehmannia chingii. (a) Appearance characteristics; (b) transverse section; (c), (d) secretory cells; (e), (f), (g) vessels

  • Figure 5

    ESI-MS spectra of unearthed sample and Rehmannia chingii. (a) MS spectrum of unearthed sample; (b1) MS spectrum of m/z 623 in unearthed sample; (b2) MS/MS spectrum of m/z 623 in unearthed sample; (c) MS spectrum of R. chingii; (d1) MS spectrum of m/z 623 in R. chingii; (d2) MS/MS spectrum of m/z 623 in R. chingii; (e) TIC and SIM spectra (m/z 623 and m/z 179) of unearthed sample; (f1) MS spectrum of m/z 179 in unearthed sample; (f2) MS/MS spectrum of m/z 179 in unearthed sample

  • Figure 6

    UPLC-Q-TOF-MS total ion chromatograms and mass spectrograms. (a) The ion chromatograms of excipient layers aqueous extract; (b) the total ion chromatograms of glucose, sucrose, fructose, maltose; (c) mass spectrogram of m/z 341.11 peak in aqueous extract of excipient layers; (d) mass spectrogram of sucrose

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