Successful Structural Determination of Diverse Phenolic Compounds from Minute Alpine Plant Flower Samples Using Newly Developed Method

A research team including Hyuga Hirano (a collaborative graduate student at the United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, National Museum of Nature and Science, Director: Ken-ichi Shinoda), Takashi Kikuchi (Application Lab, Product Division, Rigaku Corporation, a group company of Rigaku Holdings Corporation), Futa Sakakibara (Technical Advisor, R&D Support Division, Asterism LLC), and Yoshinori Murai (Senior Researcher, Department of Botany) has successfully determined the structures of over 10 types of phenolic glycosides in alpine plants. This was achieved through the development of a trace component analysis method, which is particularly challenging for alpine plants due to their small size and difficulties in securing research samples from a regulatory and ethical standpoint. Specifically, components were isolated and crystallized from just 2 grams of flowers of the alpine plant Diapensia lapponica (Diapensiaceae). By appropriately utilizing analytical techniques such as single-crystal X-ray diffraction (SC-XRD) and electron diffraction structural analysis (MicroED), the structures of each contained component were successfully determined. This is a pioneering research example in determining the structures of numerous components from such minute samples in wild plants, including alpine species. 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 pharmacology. The results of this research were published online in the international chemistry journal "Journal of Molecular Structure" on February 22, 2026. 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 techniques such as single-crystal X-ray diffraction (SC-XRD) and electron diffraction structural analysis (MicroED) on the crystallized components. ・Successfully analyzed the phenolic compounds of 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 on alpine plants is particularly anticipated, as knowledge is limited compared to lowland species. However, alpine plants grow in harsh environments, making their plant bodies small, and their distribution is limited to high-altitude regions, making them rare. Even for academic research, it is necessary to minimize human disturbance associated with collection due to legal permits and ethical considerations, thus limiting the sample quantity available for structural analysis of the aforementioned components. Our 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, we developed a method to optimize the crystallization of each contained component after isolation and purification using existing analytical techniques such as high-performance liquid chromatography (HPLC) and molecular weight measurement using quadrupole time-of-flight mass spectrometry (QTOF-MS). Furthermore, by utilizing analytical techniques such as single-crystal X-ray diffraction (SC-XRD) and electron diffraction structural analysis (MicroED), which can determine structures from crystals approximately 1/100th the size of conventional ones, we succeeded in analyzing the structures of trace components from very small samples. This research revealed that the flowers of Diapensia lapponica, which grow in harsh alpine environments, contain diverse phenolic compounds, including flavonoids such as quercetin glycosides, which have recently attracted attention as functional components offering health benefits. In the process of the aforementioned technology development, numerous components were also isolated and their structures analyzed from the leaves of Diapensia lapponica, leading to the discovery of components contributing to UV protection and antioxidant activity. Furthermore, it was found that the accumulation of some of these components shows geographical differences from central Honshu to Hokkaido in the Japanese archipelago, and these results were published in "Biochemical Systematics and Ecology." This current research is a further improvement of that study. The domestic agricultural industry journal "AgriBio" also summarized and introduced the key points of this research.

2. Notes High-Performance Liquid Chromatography (HPLC): An apparatus that can highly 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 an HPLC for preparative separation of each component. Quadrupole Time-of-Flight Mass Spectrometer (QTOF-MS): A mass spectrometer that combines different mass spectrometry technologies, quadrupole and time-of-flight, offering high mass accuracy, resolution, and sensitivity. The LC-QTOF-MS in Figure 2 is an HPLC connected to a QTOF-MS. Single-Crystal X-ray Diffraction (SC-XRD): A technique that directly determines the three-dimensional structure of sample molecules from the diffraction X-ray pattern obtained by irradiating a sample crystal with an X-ray beam. Electron Diffraction Structural Analysis (MicroED): A technique that uses an electron beam instead of X-rays in SC-XRD, enabling structural determination of sample molecules from smaller, sub-micrometer-sized crystals.

3. Future Prospects For the development of the analytical method in this research, we utilized Diapensia lapponica, which is relatively widely distributed among alpine plants. Currently, we are applying this method to analyze more rare plants, such as Japanese endemic species and endangered species. This is expected to reveal trace components of plants that were previously difficult to analyze and to be utilized for exploring useful resource substances. Furthermore, this technology can be applied to a wide range of fields such as science, agriculture, and pharmacology, and is expected 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 (Online publication on 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 (Online publication on 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."