Successful Determination of Diverse Phenolic Compound Structures from Minute Alpine Plant Flower Samples Using a Newly Developed Method

Hyuga Hirano (United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology), a collaborative graduate student of the National Museum of Nature and Science (Director: Kenichi Shinoda), Takashi Kikuchi (Application Lab, Product Division, Rigaku Corporation, a Rigaku Holdings Group company), Futa Sakakibara (Technical Advisor, R&D Support Division, Asterism LLC), and Yoshinori Murai (Senior Researcher, Department of Botany), have successfully determined the structures of over 10 types of phenolic compound glycosides in alpine plants, which are difficult to secure as research samples due to their small size and regulatory/ethical considerations, by developing a trace component analysis method. Specifically, they succeeded in determining the structure of each contained component by isolating and crystallizing components from just 2 grams of flowers of the alpine plant Diapensia lapponica (Diapensiaceae) and appropriately utilizing analytical methods such as single-crystal X-ray diffraction (SC-XRD) and electron diffraction structural analysis (MicroED). Determining the structures of numerous components from such minute samples in wild plants, including alpine plants, is a pioneering research achievement. This technology can be applied not only to the chemical components of plants but also to exploratory research of untapped resources in a wide range of fields such as science, agriculture, and pharmacy. The results of this research were published online on February 22, 2026, in the international chemistry journal "Journal of Molecular Structure." Furthermore, research results using related technologies were also published in the international biochemical journal "Biochemical Systematics and Ecology" and the domestic agricultural industry journal "Agribio."

[Research Highlights] ・Established a method for isolating and crystallizing trace components. ・Successfully performed structural analysis by appropriately utilizing analytical methods such as single-crystal X-ray diffraction (SC-XRD) and electron diffraction structural analysis (MicroED) on the crystallized components. ・Successfully analyzed the structure of phenolic compounds in alpine plants with limited sample quantities, revealing that Diapensia lapponica flowers contain diverse phenolic compounds. ・During the development of the method, related research also discovered components related to the chemical adaptation mechanisms and phylogeny of plants.

1. Background and Achievements of the Research Alpine plants, distributed in Japan's alpine and subalpine zones, are known to adapt to harsh environmental stresses such as UV radiation and low temperatures by synthesizing and accumulating chemical components called phenolic compounds. Many of these phenolic compounds are also known to be potential natural resources, and research is particularly anticipated for alpine plants, where knowledge is limited compared to lowland areas. On the other hand, alpine plants are small due to their growth in harsh environments, and their distribution is limited to high-altitude regions, making them rare. Even for academic research, due to legal permits, ethical considerations, etc., it is necessary to minimize anthropogenic disturbance associated with collection, thus limiting the amount of sample available for structural analysis of the aforementioned components. This research team has been developing methods for component analysis from trace samples, and this time, using a minute amount of Diapensia lapponica flowers as a research sample, they developed a method to optimize the crystallization of each contained component after the isolation/purification by existing analytical methods such as High-Performance Liquid Chromatography (HPLC) and molecular weight measurement by Quadrupole Time-of-Flight Mass Spectrometry (QTOF-MS). Furthermore, by utilizing analytical methods such as single-crystal X-ray diffraction (SC-XRD) and electron diffraction structural analysis (MicroED), which can determine structures from crystals approximately 1/100 the size of conventional methods, they succeeded in structural analysis of trace components from very minute samples. This research revealed that Diapensia lapponica flowers, growing in harsh alpine environments, contain diverse phenolic compounds, including flavonoids such as quercetin glycosides, which have recently attracted attention as functional components with health benefits.

In the process of developing the aforementioned technology, numerous components were also isolated from Diapensia lapponica leaves, and their structural analysis was successful. Components contributing to UV protection and antioxidant activity were discovered, and it was further found that the accumulation of some of these components showed geographical differences from central Honshu to Hokkaido in the Japanese archipelago. These results were published in "Biochemical Systematics and Ecology." This current research is an improvement on that previous work. Additionally, an article in the domestic agricultural industry journal "Agribio" also summarizes and introduces the key points of this research.

Figure 1: Diapensia lapponica in bloom. It bears plum-like flowers in rocky alpine areas. In Japan, it is distributed from central Honshu to Hokkaido. Figure 2: Main analytical flow in this research.

2. Notes High-Performance Liquid Chromatography (HPLC): An apparatus that can efficiently separate and detect components in a liquid by utilizing differences in interaction between the mobile phase (solvent) and the stationary phase (column). The preparative HPLC system in Figure 2 is for preparative (separation) of each component. Quadrupole Time-of-Flight Mass Spectrometry (QTOF-MS): A mass spectrometer that combines different mass spectrometry technologies, quadrupole and time-of-flight, possessing high mass accuracy, resolution, and sensitivity. The LC-QTOF-MS in Figure 2 is a system connecting HPLC and QTOF-MS. Single-Crystal X-ray Diffraction (SC-XRD): A method that directly determines the three-dimensional structure of sample molecules from the pattern of diffracted X-rays obtained by irradiating a crystal sample with an X-ray beam. Electron Diffraction Structural Analysis (MicroED): A method that uses an electron beam instead of X-rays in SC-XRD, enabling the structural determination of sample molecules from smaller, sub-micrometer-sized crystals.

3. Future Prospects For the development of the analytical method in this research, Diapensia lapponica, which is relatively widely distributed among alpine plants, was used. However, this method is currently being applied to analyze more rare plants, including endemic Japanese species and endangered species. It is expected to reveal trace components of plants that were previously difficult to analyze and to be utilized in the search for useful resource materials. Furthermore, this technology is expected to be applicable in a wide range of fields such as science, agriculture, and pharmacy, and to become an important technology for obtaining fundamental information for basic and applied research.

4. Published Papers Title: Sustainable micro-scale identification of phenolic glycosides in alpine flower through single-crystal structure analysis Authors: Hyuga Hirano, Takashi Kikuchi, Futa Sakakibara, Yoshinori Murai Journal: Journal of Molecular Structure, 145740 (Published online February 22, 2026, scheduled for publication in July 2026)

[Related Papers] Title: Phenolic compound diversity reflecting phylogeographic structure of Diapensia lapponica subsp. obovata (Diapensiaceae) populations in Japan Authors: Hyuga Hirano, Toshiyo Kato, Keiichi Noguchi, Hisahiro Kai, Takuro Ito, Takashi Kikuchi, Futa Sakakibara, Yoshinori Murai Journal: Biochemical Systematics and Ecology, 125: 105168 (Published online November 20, 2025, scheduled for publication in April 2026)

[Related Article] Title: Structural Analysis of Trace Phenolic Glycosides in Alpine Plants Authors: Hyuga Hirano, Takashi Kikuchi, Futa Sakakibara, Yoshinori Murai Journal: Agribio, 10(3): 53-56 (Published in March 2026 issue) This research was conducted as part of JSPS KAKENHI (JP23K05503), Grant-in-Aid for JSPS Fellows (JP24KJ1011), and the National Museum of Nature and Science's comprehensive research "Science of Extreme Environments."