HPLC-Based Quantification of Flavonoids in Bushukan Peel Reveals Species-Specific Variability in Citrus Functional Compounds

Citrus peels are often used in health foods, but their benefits are usually assumed based on the type of fruit rather than confirmed by analyzing what they contain. A paper recently published in Metabolites¹ presents a case study demonstrating that Japanese-grown bushukan samples contain low or undetectable levels of two widely expected citrus flavonoids: hesperidin and nobiletin. “Rather than identifying novel compounds,” write the authors of the paper, “the purpose of this work is to emphasize a practical consideration for health food development.” Dried whole-fruit powder samples of bushukan, prepared by freeze-drying and hot-air drying at 50 °C, were analyzed, and the contents of hesperidin and nobiletin were quantified using high-performance liquid chromatography (HPLC) following methanol reflux extraction.

What is Bushukan and Why Are its Peels Being Studied for their Potential Health Benefits?

Bushukan is a type of sour citrus fruit known for its strong fragrance and bright yellow color when ripe. It has a long shape with segments that look like fingers, which is why it is often called “Buddha’s hand.”^2,3

Citrus peels have long been considered beneficial for health. Earlier research showed that two compounds from citrus peel, hesperidin and nobiletin, helped reduce anxiety-related behaviors in older dogs, such as howling due to fear. Based on this, a citrus peel-based product was developed for senior dogs, and testing showed it was safe even when given repeatedly at high doses.^4,5

“When citrus peels are intended for food use,’ write the authors of the study,¹ “their chemical composition should be evaluated with careful consideration of species, cultivation area, and harvesting conditions, as these factors can substantially influence component profiles. In this context, bushukan was selected as a suitable case material because it lacks a developed edible pulp; consequently, the usable portion consists almost entirely of peel tissue, and the entire fruit was dried, pulverized, and analyzed to evaluate peel-specific functional components.’

What Were the Findings?

The researchers detected hesperidin at 75 mg/100 g under both freeze-drying and hot-air drying conditions, whereas nobiletin was below the practical limit of quantification (approximately 1 mg/100 g). No reduction in hesperidin content was observed after drying at 50 °C. These levels were markedly lower than those reported for commonly used citrus peels, such as satsuma mandarin, in previous studies.¹

What Does This Say About Assumed Health Benefits?

“The findings,” write the authors of the paper,¹ “highlight that functional value should not be presumed based solely on the basis of botanical classification or traditional reputation. Comprehensive, species- and origin-specific compositional analysis, together with appropriate safety evaluation, is essential when utilizing citrus peels as raw materials for health food applications. This commentary does not seek to generalize functional properties across citrus species; rather, it provides an evidence-based, practice-oriented illustration of the need for compositional verification.”

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References

1. Nakahigashi, J.; Kobayashi, E. Citrus Peels in Health Foods: A Case Study of Pulp-Free Japanese-Grown Bushukan (Citrus medica var. sarcodactylis). Metabolites 2026, 16 (4), 254. DOI: 10.3390/metabo16040254
2. Chen, J.; Wu, G.; Zhu, C. et al. Structurally Diverse Flavonoids from Citrus medica L. var. sarcodactylis Swingle and Their Multiple Bioactivities: A Review. J. Food Nutr. Res. 2022, 10, 81–87. DOI: 10.12691/jfnr-10-2-1
3. Lim, T. K. (Ed.) Citrus medica var. sarcodactylis. In Edible Medicinal and Non-Medicinal Plants;, volume 4 Springer: 2012; 690–694. DOI: 10.1007/978-94-007-4053-2_45
4. Nakahigashi, J.; Kurikami, M.; Iwai, S. et al. Exploring the Pharmacokinetics and Gut Microbiota Modulation of Hesperidin and Nobiletin from Mandarin Orange Peel in Experimental Dogs: A Pilot Study. Metabolites 2024, 15 (1), 3. DOI: 10.3390/metabo15010003
5. Yonezawa, T.; Lei, Y.; Marasigan, C. N. B. B. et al. Preliminary Safety Assessment for Mandarin Orange Peel Administration to Dogs Based on Physical Conditions and Blood Examination Parameters. Metabolites 2026, 16 (3), 213. DOI: 10.3390/metabo16030213