New Method Enables Structural Determination of Diverse Phenolic Compounds from a Tiny Alpine Flower Sample

Hyuga Hirano (United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, and collaborative graduate student at the National Museum of Nature and Science (President: Makoto Manabe)); Takashi Kikuchi (Application Laboratories, Global Product Unit, Rigaku Corporation, a group company of Rigaku Holdings); Futa Sakakibara (Technical Advisor, R&D Support Division, Asterism G.K.); Yoshinori Murai (Senior curator, Department of Botany, National Museum of Nature and Science); and colleagues have successfully determined the structures of more than ten phenolic glycosides from a tiny sample of an alpine plant flower by developing a trace analysis method. This achievement is particularly significant because alpine plants are typically small and difficult to collect due to legal, ethical, and environmental considerations, resulting in extremely limited sample availability. Determining the structures of numerous chemical components from such a small sample represents a pioneering research accomplishment.

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Figure 1: Pincushion plant (Diapensia lapponica) in bloom, growing in alpine rocky areas. Its distribution in Japan extends from the Chubu region of Honshu to Hokkaido.

The method developed by the research team can be applied not only to the analysis of plant chemical constituents, but also to the exploration of underutilized resources across a wide range of fields, including physics, agriculture, and pharmaceutical sciences.

The research results were published online on February 22, 2026, in the online Journal of Molecular Structure. Related studies employing similar techniques have also been published in Biochemical Systematics and Ecology.

Research Summary:

• A method of isolating and crystallizing trace chemical components was established.
• Structural analysis of crystallized components was successfully achieved using analytical techniques such as single-crystal X-ray diffraction (SC-XRD) and microcrystal electron diffraction (MicroED).
• Using this approach, structural determination of phenolic compounds was successfully conducted in alpine plants for which research samples are difficult to obtain, revealing a diverse range of phenolic compounds present in the flowers of Diapensia lapponica.
• During the development of this method, related studies also led to the discovery of components associated with chemical adaptive mechanisms and phylogenetic characteristics in plants.

1. Background and Results

Alpine plants are distributed throughout Japan’s alpine and subalpine zones. To withstand the environmental stresses characteristic of alpine environments, such as intense ultraviolet radiation and low temperatures, these plants adapt by synthesizing and accumulating chemical compounds known as phenolic compounds. Many phenolic compounds are also recognized as potential natural resources. As alpine plants remain less well studied than lowland plants, further research in this field is particularly anticipated. At the same time, the harsh environments in which alpine plants grow result in small plant size. In addition, their distribution is restricted to high-altitude regions, making them relatively rare. Even in academic research, legal, permitting, and ethical considerations require that plant collection be strictly limited to minimize human disturbance to alpine ecosystems. Consequently, the amount of sample material available for structural analysis is extremely limited.

The research team had been developing a method for analyzing the components of trace samples. In this study, using a very small sample of flowers of the alpine plant Diapensia lapponica, the team isolated and purified individual components using high-performance liquid chromatography (HPLC) and determined their molecular weights by quadrupole time-of-flight mass spectrometry (QTOF-MS). The team then developed a method to optimize the crystallization of each component.

By applying analytical techniques such as single-crystal X-ray diffraction (SC-XRD) and microcrystal electron diffraction (MicroED), which enable structural determination from crystals approximately one hundredth the size required by conventional methods, the team successfully determined the structures of trace components from extremely small samples. The results demonstrated that D. lapponica, which thrives in harsh alpine environments, contains flavonoids and other phenolic compounds, including quercetin glycosides. These compounds have attracted attention in recent years as functional ingredients with potential health benefits.

In related research published in Biochemical Systematics and Ecology, the research team also succeeded in isolating and determining the structures of numerous components from the leaves of the same plant, discovering compounds that contribute to ultraviolet protection and antioxidant activity. The study further revealed that the accumulation of some components differs geographically, from the Chubu region of Honshu to Hokkaido. The present study represents an advancement of these earlier findings.

2. Notes

High-performance liquid chromatography (HPLC): An analytical technique that separates and detects components based on differences in their interactions with a mobile phase (solvent) and a stationary phase (column). The preparative HPLC system shown in Figure 2 was used to isolate each component.

Quadrupole time-of-flight mass spectrometry (QTOF-MS): A mass spectrometry technique that combines quadrupole and time-of-flight analyzers to achieve high mass accuracy, resolution, and sensitivity. The LC-QTOF-MS system shown in Figure 2 integrates HPLC with QTOF-MS.

Single-crystal X-ray diffraction (SC-XRD): A method in which sample crystals are irradiated with an X-ray beam, and the resulting diffraction pattern is used to directly determine the three-dimensional molecular structure.

Microcrystal electron diffraction (MicroED): A structural analysis technique that uses an electron beam instead of X-rays, enabling structure determination from much smaller, submicrometer-sized crystals.

3. Future Directions

In developing the analytical method described here, the research team used samples from the pincushion plant, an alpine species with a relatively wide geographical distribution. The team is currently applying this method to the analysis of rarer plant species, including species endemic to Japan and endangered species.

This approach is expected to facilitate the identification of trace components in plants that have previously been difficult to analyze, as well as the exploration of novel and underutilized biological resources. Furthermore, the method has broad applicability across diverse fields, including physics, agricultural science, and pharmaceutical sciences, and is expected to serve as an important source of foundational information for both basic and applied research.

4. Presented 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
Publication: Journal of Molecular Structure, 145740 (online February 22, 2026)

Related paper
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
Publication: Biochemical Systematics and Ecology, 125: 105168 (online November 20, 2025; scheduled for print release in April 2026)

This research was supported by a Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP23K05503 and JP24KJ1011, and “Integrated Research on Extreme Environments” of the National Museum of Nature and Science, Japan.

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