Review Series to Celebrate Our 100th Volume
Pursuing the identity of a natural medicine licorice by Dr. Shoji Shibata that he firstly investigated half a century before
2025 Volume 101 Issue 5 Pages 239-248
Details
2025 Volume 101 Issue 5 Pages 239-248
This article is an explanatory review on a study of licorice stored in Shosoin Repository reported in this journal by Dr. Shoji Shibata in 2003 (Proc. Jpn. Acad. Ser. B 79, 176–180). The study using new technologies at that time for identification of plant species was a follow-up research of his own study performed half a century before. The study revised previous results and elucidated that the licorice stored in Shosoin Repository was derived from Glycyeehiza uralensis.
This is a review explaining an article previously published in this journal; the article was elaborated by Dr. Shoji Shibata in 2003 describing the identification of the species of origin of the licorice roots stored in the Shosoin Repository.1) From 1948 to 1949, the remedial natural products maintained in the Repository, including the licorice roots of interest here, were scientifically investigated for the first time. The results of identification of their species of origin were published in 1955. As a result of technological advancements enabling more accurate investigation, Dr. Shoji Shibata, who was one of team members of the scientific investigation of Shosoin Repository, published a study in 2003 and corrected the previous conclusion [hereinafter, referred to as “the 2003 article (or study)”].
After serving as a faculty member at the University of Tokyo Department of Pharmaceutical Sciences and the Meiji Pharmaceutical University, Dr. Shibata led the Shibata Laboratory of Natural Medicinal Materials, which is affiliated with a pharmaceutical company well known for its commercial products containing glycyrrhizin, the most characteristic ingredient of licorice roots. The present author started her medicinal plant research after Dr. Shibata retired from academic position, though, Dr. Shibata’s presence was conspicuous in many academic meetings and conferences that the author attended early in her career. The author had few opportunities to speak to the famed scholar, and admired him only from afar. The author recalls finding Dr. Shibata seated in the front row of the auditorium in many scientific meetings related to her area of expertise. When the academic society that the author was affiliated with was planning to launch large events or undergo major reforms, the professors in charge felt it was important to consider Dr. Shibata’s thoughts and comments. Dr. Shibata was an influential and demanding figure in the fields of herbal medicine and natural product chemistry. According to one of Dr. Shibata’s students, who is now a professor emeritus, Dr. Shibata was a serious, rigorous scientist in the laboratory, but had a genuine smile and a laid-back manner when he was outside of that setting.
Licorice roots and stolons (hereinafter, collectively referred to as “roots”) are one of the most widely used natural medicinal products in Japan, with an annual consumption exceeding 2,000 tons.2) Per the Japanese Pharmacopoeia, licorice roots are derived from two species of the genus Glycyrrhiza. Licorice plants are native to the entire Eurasian continent, but not to Japan. The G. glabra plant, whose Japanese nomenclature is “supein kanzo” (Spanish licorice), grows naturally from the eastern part of Kazakhstan to Spain. Currently, approximately 20 species in the genus Glycyrrhiza have been identified. Plants of the two aforementioned species and several others have sweet-tasting roots, and their extract has been used for flavoring and therapeutic purposes.
In Japan, licorice extracts are more widely used as a flavoring or cosmetic ingredient than as a medicine. More than 16,000 tons of licorice roots are used annually by the flavoring and cosmetic industries.
Licorice roots have commonly been employed as a component in many traditional medicine formulas in Japan, such as Kakkonto and Shoseiryuto. Shakuyakukanzoto, a formula to treat leg cramps, bears the Japanese name of licorice “Kanzo” in its title. Licorice may have various pharmacological properties, such as antitussive, expectorant, analgesic, and detoxicating effects, depending on its combination with other components.3) An additional advantage of licorice is that its sweet flavor masks the otherwise bitter or acrid taste of traditional medicines. Hypokalemia is a well-known adverse reaction caused by excessive licorice consumption. The proper use of licorice is one of the most important basic lessons that pharmacy students must learn when they study traditional Japanese herbal medicine.
Licorice has also been used for treating ailments not only in Japan and China but also in many other parts of the Eurasian continent. When the author had the opportunity to interview Kazakh nomads long ago, they showed her licorice plants that were used as an ingredient in herbal medicines. The author also found that licorice was sold at an herbal medicine shop in Iran. The time-tested utility of this plant in many parts of the world underscores its therapeutic benefits.
Apart from the medicinal use, licorice is frequently used as the source of a sweetening agent. Gummy candies flavored with licorice extract are not well known in Japan, but are very common in Europe and North America. They are black and have a distinct smell and taste. In Japan, licorice extract is used in confections, toothpaste, soy sauce, soybean paste, fermented pickles, and many other items. One reason to use licorice extract as a commercial sweetener is that it is much sweeter than sugar.
The sweet taste of the licorice root is attributable to glycyrrhizin, one of its characteristic ingredients. The chemical structure of glycyrrhizin is distinctly different from that of sugar. Research has revealed that glycyrrhizin exerts important pharmacological effects. The biological effects of licorice root are attributable not only to glycyrrhizin but also to other various chemical ingredients. Consequently, the biological effects of licorice should not be discussed based on glycyrrhizin alone. In fact, other compounds commonly present in licorice extract such as glabridin and liquiritin have specific biological effects and are used as active ingredients in commercial products.
It is surprising that the underground part of the plant tastes sweet whereas its overground part does not. Morphologically, licorice roots and stolons are slender and straight, apparently showing nothing special. However, licorice is very vigorous, mildly invasive and often extends into open habitats. The author often observed that some licorice plants she cultivated in the medicinal botanical garden of her university reached beyond the fence that had been erected to delineate the licorice area. In the Central Asian region, licorice sometimes grows in semi-wild conditions, covering a massive amount of ground.
To understand the background of the 2003 article, two previous publications, cited as Refs. 14) and 45) therein, deserve note. In the first investigations of Shosoin’s medicinal products, Dr. Mitiiti Fujita morphologically analyzed the licorice roots and concluded that they belonged to the species G. glabra var. glandulifera, which was considered as a source plant of a herbal medicine of Ural licorice. Dr. Shibata quantitatively determined the glycyrrhizin contents of several specimens using chemical (not chromatographic) methods. Along with the analytical results of other products, these were reported in the 1955 publication “Shosoin Medicinals”, which was cited as Ref. 1 in the 2003 article.
Here, a brief explanation of the Shosoin Treasury and the pharmacognostic methods will help the reader to better understand the nature of the research. Shosoin is a timber-framed structure built approximately 1300 years ago in Nara, Japan. It housed the treasures and daily life objects of an Emperor, which were donated to the temple to commemorate his passing away. These items were preserved in excellent condition until now. The collection included approximately 60 kinds of natural medicinal products, which were stored along with an inventory ledger describing their names and amounts, indicating that they were used for therapeutic purposes. As described earlier, these products were pharmacognostically studied between 1948 and 1949.
According to the standard pharmacognostic methods at that time, the species of herbal samples were mostly determined based on their region of origin. The geographical region of origin was the primary determinant of the species, and vice versa. The determination of plant species requires morphological analyses of the flowers, fruits, and other reproductive organs. However, herbal medicinal products were typically derived from the root, bark, prickles, and other parts of the plant, making it impossible to perform a morphological observation of the reproductive organs. Presently, most herbal products are obtained from the cultivation of plants of known identity, and geographical information on the location of their growth does not contribute much to the identification of their species. Until several decades ago, however, herbal products were mostly obtained from wild plants, and the data on their region of origin provided a key insight into the species they belonged to.
The Shosoin Repository did not have records on the geographical origins of its medicinal products, and the investigation team was allowed to analyze only small samples (Fig. 1 of the 2003 article1)). Access to the repository items was strictly controlled by the Imperial Household Agency, which managed the heritage of the Royal Family. Current technologies such as X-ray computed tomography enable three-dimensional analysis of the internal tissue structure in a nondestructive manner. However, light microscopy of prepared specimens was the only morphological analysis approach available to the investigation team at that time. Dr. Fujita compared the Shosoin specimens with several commercial licorice products of different origins. He assigned the Shosoin licorice root to G. glabra var. glandulifera, the species known to yield top-quality products in the Nara era.
Dr. Shibata quantitatively determined the glycyrrhizin content of the specimens using chemical reactions because sophisticated analytical devices such as high-performance liquid chromatography (HPLC) were not common then. The hydrolysis of glycyrrhizin yields two molecules of glucuronic acid. He collected the glucuronate precipitate obtained after glycyrrhizin hydrolysis, and converted it to furfuralthiobarbituric acid. He estimated the glycyrrhizin content under the assumption that 16.2 grams of furfuralthiobarbituric acid corresponds to 100 grams of glycyrrhizin. Using specimens of Shosoin licorice items No. 1, No. 2, and No. 3, he determined that their glycyrrhizin contents were 11.7%, 13.3%, and 12.3%, respectively. For comparison, the glycyrrhizin contents of commercially available products were between 3.6% and 8.9%, indicating that the Shosoin products had higher glycyrrhizin concentrations. These results also suggested that glycyrrhizin contained in the licorice roots was resistant to decomposition and denaturation over a very long period.
The commercial licorice products analyzed for comparison were of Spanish (Tortosa), Ural, and Chinese origin. In their report, the Ural root was attributed to the species G. glabra var. glandulifera. Textbooks typically assigned Ural licorice products to this species until recently,1),6) as the author learned when she was an undergraduate student. However, DNA-based determination showed that G. glabra var. glandulifera is not the source plant of Ural licorice and Ural licorice in fact belongs to G. uralensis.7),8) This long-time misunderstanding emphasizes the difficulty of identifying the species based on the morphology and anatomy of the underground plant segment alone. G. glabra var. glandulifera is now classified as a synonym of G. glabra.9)
Here, the author wishes to address the long-lived taxonomical confusion relating to the licorice plant of Ural origin and its root product in Japan. The Japanese name of the plant G. ularensis is “uraru kanzo” which pronunciation is the same as the Japanese name of Ural licorice (“uraru kanzo”), which source plant was assigned to G. glabra var. glandulifera. This led to an inconsistency wherein the root product called “uraru kanzo” did not originate from the plant with the same name.
The analytical methodologies of natural product chemistry have advanced greatly over time. Research has identified many ingredients of licorice roots other than glycyrrhizin, including several species-specific flavonoids. Advances in HPLC have made it possible to detect these compounds in very small samples. Using this technique, Dr. Shibata attempted to find species-specific compounds contained in the sample shown in Fig. 1 of the 2003 article.1) The results were reported in a 1991 journal publication, which was cited as Ref. 4 in the 2003 article.5)
Disappointingly, no species-specific chemicals were detected, but six flavonoid and glycoside derivatives, including glycyrrhizin, were quantitatively identified and reported in the 1991 publication. Dr. Shibata also investigated the origin species of different types of commercial licorice products imported to Japan. Based on these results, he argued that the Shosoin licorice roots could be assigned to G. uralensis.
Later in 1994 to 1995, the second investigation of Shosoin medicaments was performed, and Dr. Shibata was a representative of the researchers group conducting the investigation.10) Licorice was one of the object herbs of the investigation, and Dr. Shibata mentioned it at the last paragraph of introduction section of the 2003 publication.
The 2003 publication covered two subjects, namely, genetic and chemical analyses. Using the polymerase chain reaction (PCR) technique, the DNA sequences useful for differentiating higher plant species were compared between the Shosoin licorice (ID Code ML-44, N-99) and plant samples of three representative species (G. glabra, G. uralensis, and G. inflata) appropriately identified by morphological characteristics of their fruits and leaves.
For DNA sequence analysis, maternally inherited chloroplast DNA, which is frequently used for species identification were chosen and tried; however, Shosoin licorice did not give a good result. That was probably because the chloroplast genome is more susceptible to decomposition than nuclear genome because it contains a greater proportion of weakly interacting adenine–thymine rich domains and is smaller in size, so that the chloroplast genome from the aged Shosoin samples might be too damaged to function as a template for PCR. Instead, the internal transcribed spacer sequence of the nuclear ribosomal DNA was good enough for PCR and the amplicons were compared. The results are shown in Fig. 3 of the 2003 article.1) The Shosoin sample contained DNA sequences specific to G. uralensis (sites 1 and 2 shown in Fig. 3), indicating that this species was the botanical origin of the Shosoin licorice roots. This was confirmed by further analysis using 205 commercial samples of known identity.
In the chemical analysis, attempts were made to detect the following species-specific compounds: glycycoumarin (G. uralensis), glabridin (G. glabra), and licochalcone A (G. inflata). However, these experiments yielded negative results. Next, HPLC was used to quantify the six major ingredients of licorice root extract: glycyrrhizin, liquiritin, liquiritin apioside, isoliquiritin, isoliquiritin apioside, and isoliquiritigenin. Using principal component analysis (a type of multivariate analysis), the profiles of these chemicals were compared between the Shosoin licorice and 117 genetically characterized commercial samples. The results are presented in Fig. 4 of the 2003 article.1) In the scatter plot of the first and second principal components, the pattern of distribution of these chemicals of the Shosoin licorice, which is indicated by an open circle, is most similar to that of G. uralensis.
Overall, these genetic and chemotaxonomic analyses demonstrate that the botanical origin of the Shosoin licorice was G. uralensis.
The 2003 article was co-authored by Dr. Shibata and two of the researchers from a company. These researchers were scientists at Japan’s leading herbal extract manufacturer, and contributed to the analysis of a large number of comparative licorice samples. Genetic analyses by the research group of this company showed that G. uralensis was the botanical origin of the Ural licorice products.7),8) Dr. Shibata approached these scientists and asked for their cooperation when he was impressed by their academic presentation, according to one of the two other authors of the 2003 study.
Dr. Shibata was well known for his work on lichen and fungal metabolites and secondary metabolite biosynthesis. His expertise and interest actually covered a much wider area of biology and chemistry, as showcased here. Half a century after the start of the scientific investigation into the Shosoin licorice, Dr. Shibata finally elucidated its true identity in a convincing manner. Over this period, he maintained a strong interest in a rarely accessible object of research that was the property of the Imperial Family and a plant that has been commonly employed as a medication in various parts of the world.
Edited by Takao SEKIYA, M.J.A.
Correspondence should be addressed to: M. Ito, Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan (e-mail: michiho_ito@nihs.go.jp).
This paper commemorates the 100th anniversary of this journal and introduces the following paper previously published in this journal. Shibata, S., Kondo, K. and Terabayashi, S. (2003) Identification of the licorice root stored in Shosoin based on the sequences of internal transcribed spacer (ITS) on nrDNA and the chemotaxonomic consideration. Proc. Jpn. Acad. Ser. B 79 (6), 176–180 (https://doi.org/10.2183/pjab.79B.176).
[From Proc. Jpn. Acad. Ser. B, Vol. 79 No. 6, pp. 176–180 (2003)]
;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/PJA10105B-01figA01.jpg%22>');%0A%09%09%09newWindow.document.close();%0A%09%09)
;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/PJA10105B-01figA02.jpg%22>');%0A%09%09%09newWindow.document.close();%0A%09%09)
;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/PJA10105B-01figA03.jpg%22>');%0A%09%09%09newWindow.document.close();%0A%09%09)
;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/PJA10105B-01figA04.jpg%22>');%0A%09%09%09newWindow.document.close();%0A%09%09)
;%0A%09%09%09newWindow.document.open();%0A%09%09%09newWindow.document.write('<img src=%22./Graphics/PJA10105B-01figA05.jpg%22>');%0A%09%09%09newWindow.document.close();%0A%09%09)
Dr. Michiho Ito is the Head of Division of Pharmacognosy, Phytochemistry, and Narcotics, National Institute of Health Sciences, Ministry of Health, Labour and Welfare, Japan.
She took this position in April 2022, having previously worked at Kyoto University as an Associate Professor in the Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences. She graduated from the Faculty of Pharmaceutical Sciences, Kyoto University in 1992 and was appointed as an Assistant Professor at the Faculty of Pharmaceutical Sciences, Kyoto University in 1996. She received her Ph.D. from Kyoto University (Pharmaceutical Sciences) in 1999. She was a visiting researcher in Professor Rodney Croteau’s lab at the Institute of Biological Chemistry, Washington State University, U.S.A., from 2002 and returned to Kyoto University in 2003, and she was appointed as an Associate Professor in 2003. Her areas of specialty are pharmacognosy, pharmaceutical botany, phytochemistry, and regulatory sciences. She has authored more than 200 scientific publications since 1993. She has a license as a pharmacist, being a member of the International Pharmaceutical Federation (FIP) since 2009, and was awarded an FIP Fellow Award in 2016. She has been a collaborating member of the Science Council of Japan since 2011. She has been an expert member of many pharmaceutical regulatory discussion groups such as a committee for the safety and discrimination between foods and drugs of the Japanese Ministry of Health, Labour and Welfare, and several drafting committees for the Japanese Pharmacopoeia organized by the Pharmaceuticals and Medical Devices Agency of Japan.
