←確認用doi （左上Y座標：－17.647 pt） 2 Chem. Pharm. Bull. 69, 2–10 (2021) Vol. 69, No. 1 Current Topics Pharmaceutical Research for Quality Evaluation and Characterization of Foods and Natural Products Review Isolation and Characterization of Neuroprotective Components from Citrus Peel and Their Application as Functional Food Yoshiko Fur ukawa,*,a Satoshi Okuyama,a Yoshiaki Amakura,b Atsushi Sawamoto,a Mitsunari Nakajima,a Morio Yoshimura,b Michiya Igase,c Naohiro Fukuda,d Takahisa Tamai,d and Takashi Yoshidab,e a Department of Pharmaceutical Pharmacology, College of Pharmaceutical Sciences, Matsuyama University; 4–2 Bunkyo-cho, Matsuyama, Ehime 790–8578, Japan: b Department of Pharmacognosy, College of Pharmaceutical Sciences, Matsuyama University; 4–2 Bunkyo-cho, Matsuyama, Ehime 790–8578, Japan: c Department of Geriatric Medicine and Neurology, Ehime University Graduate School of Medicine; 454 Shizugawa, To-on, Ehime 791–0295, Japan: d Ehime Institute of Industrial Technology; 487–2 Kumekubota, Matsuyama, Ehime 791–1101, Japan: and e Department of Pharmaceutical Sciences, Okayama University; Okayama 701–1152, Japan. Received March 22, 2020 The elderly experience numerous physiological alterations. In the brain, aging causes degeneration or loss of distinct populations of neurons, resulting in declining cognitive function, locomotor capability, etc. The pathogenic factors of such neurodegeneration are oxidative stress, mitochondrial dysfunction, inflamma- tion, reduced energy homeostatis, decreased levels of neurotrophic factor, etc. On the other hand, numerous studies have investigated various biologically active substances in fruit and vegetables. We focused on the peel of citrus fruit to search for neuroprotective components and found that: 1) 3,5,6,7,8,3,4-heptamethoxy- flavone (HMF) and auraptene (AUR) in the peel of Kawachi Bankan (Citrus kawachiensis) exert neuroprotec- tive effects; 2) both HMF and AUR can pass through the blood–brain barrier, suggesting that they act di- rectly in the brain; 3) the content of AUR in the peel of K. Bankan was exceptionally high, and consequently the oral administration of the dried peel powder of K. Bankan exerts neuroprotective effects; and 4) intake of K. Bankan juice, which was enriched in AUR by adding peel paste to the raw juice, contributed to the prevention of cognitive dysfunction in aged healthy volunteers. This review summarizes our studies in terms of the isolation/characterization of HMF and AUR in K. Bankan peel, analysis of their actions in the brain, mechanisms of their actions, and trials to develop food that retains their functions. Key words auraptene; heptamethoxyflavone; Kawachi Bankan; neuroprotection; extracellular signal-regulat- ed kinase 1/2; antiinflammation 1. Introduction relatively easily pass through the blood–brain barrier (BBB) Citrus is one of the most familiar types of fruit, and is and act directly in the brain. However, the biological abilities cultivated and eaten worldwide. It has long been known that of citrus components have been conventionally revealed using citrus fruit are a rich source of nutrients and bioactive com- peripheral tissues or cells, and there have been few studies on pounds including vitamin C, folic acid, dietary fiber, pectin, their effects on the central nervous system (CNS). Second, the and minerals.1,2) Abundant epidemiological data and experi- various bioactive components are present in all parts of citrus mental studies revealed that the intake of citrus fruit is ben- plants, but the peel is an especially rich source.6,7) Citrus peel eficial to health.3,4) In the last two to three decades, intensive is generally discarded along with the seeds because the fruit investigations have clarified that citrus is abundant in bioac- is usually consumed as fresh products or juice. The use of tive secondary metabolites such as flavonoids, coumarins, citrus peel as a neuroprotectant might therefore represent an limonoids, and carotenoids.5) The biological activities of these effective use of waste. Third, citrus species are highly vari- citrus compounds are known to cover a wide range including able.8) The number of species in Tanaka’s classification sys- antibacterial, antitumor, antiinflammatory, antioxidant, anti- tems is 156, although it is generally accepted that there were diabetic, and antimetabolic disorder activity.3) only 3 (or possibly as many as 5) original citrus species. The There are three reasons why we tried to identify neuro- members of the citrus family can be readily hybridized and protective compounds in citrus peel. First, many bioactive grafted, that is, most citrus species were created as hybrids. In secondary metabolites in citrus fruit have low molecular Ehime prefecture, Japan, an eminent citrus-growing district, weight with lipophilicity, suggesting the possibility that they more than 40 types of citrus fruit are harvested. These facts * To whom correspondence should be addressed. e-mail: [email protected] © 2021 The Pharmaceutical Society of Japan Vol. 69, No. 1 (2021) Chem. Pharm. Bull. 3 suggested the high possibility of identifying new neuroprotec- ability. The ethanol extract of K. Bankan was then parti- tive components in citrus plants. tioned into n-hexane-, ethyl acetate-, and water-soluble por- tions. When the neurons were treated with each extract, the 2. Screening of Neuroprotective Components from n-hexane extract had the ability to phosphorylate ERK1/2. Citrus Peel These results suggest that some nonpolar compound(s) of K. As test samples, we selected representative citrus species Bankan is responsible for this activity. We then isolated and grown in Ehime prefecture and prepared ethanol extracts characterized the active compound(s) in the hexane extract from the peel of both unripe and ripe fruit. As the screening of K. Bankan. Chromatographic separation of the hexane method, we analyzed the ability of citrus ethanol extracts to extract revealed 4 known compounds, which were charac- activate (i.e., phosphorylate) extracellular signal-regulated terized by spectroscopic analysis as a coumarin derivative kinase (ERK)1/2 of neurons cultured from the rat embryonic and 3 polymethoxyflavones (PMFs). The coumarin deriva- cortex.9) ERK1/2 is one of the most common signal transduc- tive was auraptene (7-geranyloxycoumarin; AUR), a simple tion molecules by which extracellular stimuli are propagated coumarin bearing a geranyloxyl moiety at C-7 (Fig. 1a). The from the cell surface to cytoplasmic and nuclear effectors. In PMFs are 5,6,7,8,4′-pentamethoxyflavone (tangeretin; TGT) the nervous system, activated (phosphorylated) ERK1/2 regu- with 5 methoxy groups at the 5,6,7,8, and 4′ positions (Fig. lates synaptic plasticity and development of long-term poten- 1b), 5,6,7,8,3′,4′-hexamethoxy flavone (nobiletin; NBT) with 6 tiation (LTP)10) through cellular changes including the activa- methoxy groups at the 5,6,7,8,3′, and 4′ positions (Fig. 1c), and tion (phosphorylation) of the cAMP response element-binding 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF) with 7 methoxy protein (CREB), which is a transcription factor located within groups at the 3,5,6,7,8,3′, and 4′ positions (Fig. 1d). the nucleus.11) Activated (phosphorylated) CREB appears to be Before our research, the neuroprotective abilities in the a critical step in the signaling cascade that leads to the struc- CNS of TGT and NBT had been reported. The neuroprotec- tural changes underlying the development of LTP.12) tive ability of TGT was investigated in Parkinson’s disease Table 1 shows the levels of 8 ripe samples to activate (PD) model rats induced by 6-hydroxydopamine; subchronic ERK1/2, indicating that the extracts prepared from the peel treatment (oral administration) with high doses of TGT mark- of Kawachi Bankan (Citrus kawachiensis) have the greatest edly reduced the loss of tyrosine hydroxylase-positive cells in the substantia nigra (SN) and decreased the striatal dopamine content.13) The neuroprotective ability of NBT was investigat- Table 1. Summary of ERK 1/2 Activation by the Peel Extracts and the ed in Alzheimer’s disease (AD) model rats induced by chronic Contents of Auraptene/3,5,6,7,8,3′,4′-Heptamethoxyflavone in the Peel of 14) Citrus Fruits amyloid-β (1–40) infusion, learning-impaired mice induced by the administration of MK-801 (dizocilpine maleate), a po- 15) ERK1/2 Content of Content of D Conventional name tent N-methyl- -aspartate (NMDA) receptor antagonist, and activationa) AUR (%)b) HMF (%)c) AD model mice induced by olfactory bulbectomization.16) We Ougonkan n.d. 0.008 focused on AUR and HMF thereafter, because their effects Unshu Mikan + n.d. 0.010 in the brain had not yet been reported. As expected, we con- 9) 17) Ponkan + n.d. n.d. firmed that HMF and AUR had the ability to phosphory- Kanpei n.d. 0.016 late CREB in cultured neuronal cells. Benimadonna n.d. n.d. Harehime n.d. 0.038 3. Analysis of Citrus Components Marihime n.d. 0.007 When we analyzed the constituents in the peel of more Setoka n.d. n.d. than 20 species of citrus (Ougonkan, Unshu Mikan, Ponkan, Haruka n.d. 0.013 Kanpei, Benimadonna, Harehime, Marihime, Setoka, Haruka, Harumi n.d. Harumi, Kiyomi, Iyokan, blood orange (Tarocco), blood or- Kiyomi n.d. 0.012 ange (Moro), mandarin orange (Kara), Dekopon, Hassaku, Iyokan − n.d. 0.008 Amanatsu, An seikan, Buntan, grapefruit, K. Bankan, Lemon, Blood Orange (Tarocco) ± n.d. 0.011 Hirami Lemon, and Buttekan) using HPLC, we observed that Blood Orange (Moro) + the peel of K. Bankan contains 15 constituents, including Mandarin Orange (Kara) + AUR, TGT, NBT and HMF.18) As shown in Table 1, AUR is Dekopon 0.006 detected only in the peel of a kind of Citrus maxima (Has- Hassaku 0.028 0.011 saku, Amanatsu, Anseikan, Buntan, K. Bankan), and its level Amanatsu 0.005 n.d. in K. Bankan was about three-Fold greater than that in Has- Anseikan 0.004 n.d.