Sage Journals: Discover world-class research

Abstract

Background

Citrus fruits are among the most widely cultivated fruits worldwide, and contain bioactive compounds, including flavonoids, carotenoids, limonoids, coumarins, and vitamin C, some of which are significantly bioactive. Consequently, discovering new compounds from Citrus fruits is an important objective.

Methods

The seeds of C. × junos Siebold ex Tanaka were extracted with methanol, which afforded eleven constituent compounds whose chemical structures were elucidated using NMR and MS techniques. The anti-proliferative activities of the isolated compounds were evaluated using the WST-8 assay.

Results

Junosterpene, a new limonoid derivative, was isolated from the seeds of C. × junos, along with seven known limonoids, 21,23-dihydro-23-hydroxy-21-oxodeacetylnomilin, deacetylnomilin, 21,23-dihydro-21-hydroxy-23-oxonomilin, obacunoic acid, methyldeacetylnomilinate, limonin, and ichangin, and three known furanocoumarins, (−)-heraclenol, (−)-byak-angelicin, and (+)-oxypeucedanin hydrate. None of the isolated compounds exhibited significant anti-proliferative activity against the human glioblastoma cell lines T98G and U-251 MG.

Conclusions

The newly identified limonoid derivative is possible to contribute to the diverse bioactive profile of C. × junos and provides insight into its potential physiological or pharmacological properties.

Keywords

Rutaceae seeds limonoids coumarin

Introduction

Citrus fruits are among the most widely cultivated fruits worldwide.¹ Citrus, which is a genus in the Rutaceae family, encompasses 40 different species.² Mandarin-type Citrus is a heterogeneous group of East Asian Citrus with small fruit bodies.³ Indeed, mandarins have witnessed the greatest increase in global production among Citrus fruits,³ and contain bioactive compounds, including flavonoids, carotenoids, limonoids, coumarins, and vitamin C,⁴ with some compounds exhibiting significant bioactivities, including cytotoxic, antiviral, and antimutagenic properties.^5–7

Among mandarin-type Citrus fruits, C. × junos Siebold ex Tanaka is a sour orange that originated in Central China⁸ and whose fruit is not only consumed fresh and as juice, but also as traditional medicine.⁹ Previous studies have reported the beneficial health effects of C. × junos, including potent antidiabetic, anticancer, and anti-inflammatory properties in vitro and in vivo,¹⁰ with flavonoids,¹⁰ coumarins,¹¹ and limonoids⁸ having been isolated and identified. Coumarins, such as auraptene, and limonoids, such as limonin, have been reported to have antitumor properties.^12,13

In this paper, we report the isolation and structural determination of junosterpene (1), a new limonoid, and ten known compounds (2-11) from the methanol extract of the seeds of C. × junos.

Materials and Methods

General Experimental Procedures

Specific rotations were measured using a P-2200 digital polarimeter (l = 5 cm; JASCO, Tokyo, Japan). Fourier-transform infrared (FTIR) spectra were recorded on a JASCO FT/IR-4600 spectrometer. Ultraviolet–visible (UV-vis) spectra were acquired on a Shimadzu UV-1850 spectrophotometer (Shimadzu, Kyoto, Japan). High-resolution electrospray-ionization mass spectrometry (HR-ESI-MS) was performed using a JMS-T100LP AccuTOFLC-Plus 4G instrument (JEOL, Tokyo, Japan). ¹H (600 MHz), ¹³C (150 MHz), and 2D nuclear magnetic resonance (NMR) spectra were recorded using a JEOL JNM-ECZ 600R spectrometer. Normal-phase silica-gel column chromatography was performed on Wakogel^® 60N (FUJIFILM Wako Pure Chemical, Osaka, 63-212 µm). Reverse-phase silica-gel column chromatography was performed on C₁₈-OPN (Nacalai Tesque, Kyoto, Japan, 75 µm). TLC was performed using TLC plates pre-coated with 60F254 silica gel (Merck, Darmstadt, Germany; 0.25 mm, ordinary phase) and Merck RP-18 F254S silica gel (0.25 mm, reverse phase). High-performance liquid chromatography (HPLC) was performed using a SPD-10Avp UV-vis detector (Shimadzu, Kyoto, Japan), LC-10ADvp pump (Shimadzu), SCL-10Avp system controller (Shimadzu), and LabSolutions LC software (ver. 1.25, Shimadzu)with COSMOSIL 5C₁₈-AR-II (Nacalai Tesque, 250 × 4.6 mm i.d., 250 × 10 mm i.d.), and Luna Phenyl-Hexyl (250 × 4.6 mm i.d., 250 × 10 mm i.d.) (Phenomenex, CA, USA) columns used for analytical and preparative purposes, respectively. Solvent ratios are based on volume. IR, UV, and MS spectra were recorded using spectroscopic-grade methanol (Wako Pure Chemical, Japan).

Plant Material

C. × junos¹⁴ fruit was supplied by a local farmer and purchased from the Japan Agricultural Cooperative (JA) farmers’ market in Yamaguchi Prefecture (Japan) in November 2022 (voucher specimen number; SOCU-2022-22). Total DNA was extracted from plant samples, and PCR amplification and sequencing were performed by Rizo Inc. (Sample number; PD0152, Tsukuba, Japan).¹⁵ The obtained nucleotide sequences were analyzed using the BLAST tool available at the National Center for Biotechnology Information (NCBI) website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). BLAST analysis was also conducted by Rizo Inc. Sequences were queried against the NCBI nucleotide collection (nr/nt) database using default parameters. The top hits with the highest sequence identities and lowest E values were used for taxonomic identification. Plant species were identified based on sequence similarity. Voucher specimens were deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Yamaguchi, Japan. The plant material was also examined and identified by Prof. Hiroyuki Tanaka (PhD) (Department of Pharmacognosy and Kampo, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University). The deposited voucher specimens, including fruit, peels, flowers, and leaves, were dried in the shade and stored.

Extraction and Isolation

Dried seeds of C. × junos were extracted three times with methanol (MeOH) under reflux for 3 h, following a modified procedure based on the method reported.⁸ Solvent evaporation provided the MeOH extract. Additionally, an ethyl-acetate-soluble (EtOAc-soluble) fraction and an aqueous layer were obtained from the abovementioned MeOH extract. The aqueous layer was further extracted with n-butanol (n-BuOH) to give n-BuOH (6.5 g, 0.6%) and H₂O (62.5 g, 5.5%) fractions. The EtOAc-soluble fraction was subjected to normal-phase silica-gel column chromatography [1.0 kg, n-hexane:CHCl₃ (5:1→ 1:1→ 1:5) → CHCl₃→ CHCl₃:MeOH (1:0→ 200:1→ 100:1→ 50:1→ 10:1→ 7:1→ 5:1→ 1:1)]. C. × junos EtOAc-soluble fraction number five (CJEA5) (4.5 g) was further separated via reverse-phase silica-gel column chromatography [225.0 g, MeOH:H₂O (4:6→ 5:5→ 6:4→ 7:3→ 8:2→ 9:1→ 1:0)] to give eight fractions (CJEA5-1-5-8). Compounds 6 and 7 were obtained as white amorphous solids from fractions CJEA5-4 and CJEA5-6 following purification by reverse-phase silica-gel column chromatography. Compounds 2, 4, 5, 8, 9, 10, and 11 were obtained as white amorphous solids after purifying fraction CJEA5-3 (200.0 mg) by HPLC [H₂O:MeCN:AcOH) (75:25:0.3)]. Compound 1 was obtained as a white amorphous solid after purifying fraction CJEA5-3-7 (2.1 mg) by HPLC [H₂O:MeCN:AcOH (75:25:0.3)], while compound 3 was obtained as a white amorphous solid from fraction CJEA5-6 (700.0 mg) following HPLC [H₂O:MeCN:AcOH (60:40:0.3)]. See Figure 1 for compound structures. These fractionation and chromatography processes were performed with slight modifications to the protocol reported.⁸

Figure 1.

Skeletal (shorthand) structures of compounds isolated from C. × junos siebold ex tanaka seeds.

Junosterpene (1)

White amorphous solid; [α]_D²⁵ −28.8 (c 0.1, MeOH); FTIR (ATR) v_max 1655, 2833, 2949, and 3307 cm⁻¹; UV (MeOH) λ_max (logε) 204.5 nm (3.47); ¹H NMR (acetic acid-d₄, 600 MHz) and ¹³C NMR (acetic acid-d₄, 150 MHz) (Table 1); HR-ESI-MS m/z: 527.1908 (Calcd. for C₂₆H₃₂O₁₀Na [M + Na]⁺ m/z: 527.1888).

Table 1.

¹³C (150 MHz) and ¹H (600 MHz) NMR Data for Junosterpene (1) and Obacunoic Acid (5).

	1*		1**		5**
Position	δ C	δ H (J in Hz)	δ C	δ H (J in Hz)	δ C	δ H (J in Hz)
1	71.2	3.95 (br d, 7.6)	70.3	3.84 (br d, 6.6)	72.4	5.01 (br d, 8.4)
2	40.5^a	α3.01 (dd, 7.6, 15.6)	40.2	α2.86 (m)	36.1	α3.01 (m)
2	40.5^a	β 3.39 (br d, 15.6)	40.2	β 3.40 (br d, 6.6)	36.1	β 3.39 (br d, 16.8)
3	176.2		174.1		172.7
4	87.8		86.3		86.7
5	51.4	2.70 (m)	51.3	2.59 (dd, 3.6, 14.4)	52.5	2.60 (m)
6	40.5^a	α2.54 (m)	40.1	α2.46 (dd, 3.6, 14.4)	39.9	α2.52 (m)
6	40.5^a	β 2.90 (m)^a	40.1	β 2.99 (m)	39.9	β 3.05 (m)
7	210.6		210.1		209.4
8	54.9		54.4		54.7
9	45.8	2.86 (m)^a	45.4	2.83 (m)	45.6	2.50 (m)
10	46.4		46.1		45.7
11	19.1	α1.51 (m)	18.4	α1.63 (m)	17.9	α1.59 (m)
11	19.1	β 1.75–1.81 (m)	18.4	β 1.87 (m)	17.9	β 1.66 (m)
12	33.1	α1.60–1.67 (m)	32.4	α1.71 (m)	31.2	α1.29 (m)
12	33.1	β 1.91–1.94 (m)	32.4	β 1.92 (m)	31.2	β 2.12 (m)
13	39.6		39.1		39.4
14	67.5		67.0		67.0
15	54.4	3.80 (s)	54.1	3.76 (s)	54.1	3.87 (s)
16	168.8		168.2		168.6
17	80.6	5.34 (s)	79.7	5.37 (s)	77.1	5.37 (s)
18	22.1	1.13 (s)	21.1	1.14 (s)	20.6	1.15 (s)
19	18.0	1.28 (s)	16.9	1.28 (s)	16.3	1.39 (s)
20	165.5		163.0
21	100.0	6.10 (s)	101.0	6.58 (br s)	170.9
22	124.6	6.34 (s)	125.5	6.35 (s)		7.37 (br s)
23	172.0		171.4			6.21 (br s)
28	33.9	1.42 (s)	33.5	1.41 (s)	34.0	1.42 (s)
29	24.6	1.56 (s)	23.9	1.56 (s)	23.1	1.60 (s)
30	17.7	1.18 (s)	17.0	1.20 (s)	17.5	1.24 (s)
-OAc					20.8	2.05 (s)

*in acetic acid-d₄, ^** in methanol-d₃, ^aOverlapping signals.

Cell Culture

U-251 MG (IFO50288, Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan) and T98G (RCB1954, RIKEN BRC through the National Bio-Resource Project of the MEXT, Ibaraki, Japan) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with low glucose (FUJIFILM Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS: FUJIFILM Wako Pure Chemical Industries) and 5% penicillin-streptomycin solution (FUJIFILM Wako Pure Chemical Industries) under 5% CO₂ at 37 °C.¹⁶

WST-8 Assay

Cell proliferation was determined using a cell counting kit 8 (CCK-8: Wako Pure Chemical Industries) according to modifying the manufacturer's instructions and the method described previously.¹⁷ Cells were seeded at a density of 1.0 × 10⁴ cells/100 µL per well in 96-well cell culture plates (Coster 3596; Corning, NY, USA). After approximately 24 h, the cells were treated with adriamycin (Wako Pure Chemical Industries) or the isolated compounds (30 µM) for 24 h. CCK-8 solution containing WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt] (10 µL) was added to the plates and incubated in a CO₂ incubator for 2 h.¹⁷ Absorbances was measured at 450 and 620 nm using a microplate reader (Multiskan FC; Thermo Fisher Scientific, MA, USA).

Statistical Analysis

Statistical analyses were performed with GraphPad Prism 8.43 software using the Dunnett's test to analyze differences between treatment groups. Differences were considered significant at *P < 0.01 when compared against DMSO-treated cells.

Results

Isolating Limonoids from Citrus × junos

The MeOH extract (136.5 g, 12.0%) of the seeds of C. × junos (1.13 kg) was partitioned in 1:1 (v/v) EtOAc:H₂O to furnish an EtOAc-soluble fraction (67.5 g, 6.0%) and an aqueous layer. The EtOAc-soluble fraction was subjected to normal- and reverse-phase silica-gel column chromatography and repeated HPLC to give one new limonoid: junosterpene (1, 2.0 mg, 1.8 ppm), together with seven known limonoids: 21,23-dihydro-23-hydroxy-21-oxodeacetylnomilin (2, 1.1 mg, 1.0 ppm),¹⁸ deacetylnomilin (3, 20.0 mg, 17.6 ppm),¹⁹ 21,23-dihydro-21-hydroxy-23-oxonomilin (4, 0.3 mg, 0.3 ppm),²⁰ obacunoic acid (5, 0.6 mg, 0.5 ppm),²¹ methyldeacetylnomilinate (6, 100 mg, 88.2 ppm),¹⁹ limonin (7, 1.0 g, 881.8 ppm),²² ichangin (8, 105.9 mg, 93.4 ppm),²³ as well as three known furanocoumarins (FCs): (−)-heraclenol (9, 0.1 mg, 0.1 ppm),²⁴ (−)-byak-angelicin (10, 0.9 mg, 0.8 ppm),²⁵ and (+)-oxypeucedanin hydrate (11, 0.1 mg, 0.1 ppm)²⁶ (See Figure 1 for compound structures).

Structure of Junosterpene (1)

Junosterpene (1) was isolated as a white amorphous solid with a negative optical rotation ([α]²⁵_D −28.8 in MeOH). Its molecular formula (C₂₆H₃₂O₁₀) was determined by HR-ESI-MS; m/z: 527.1908 (Calcd. for C₂₆H₃₂O₁₀Na [M + Na]⁺ m/z: 527.1888) and ¹³C NMR spectroscopy. In addition to the parent ion, a peak at m/z 541.2080 was observed, corresponding to the sodium adduct of the methoxylated derivative, [M + Na]⁺ with a mass increase of 14 Da, suggesting nucleophilic substitution of the hemiacetal hydroxyl group by a methoxy from MeOH (solvent). The 1D NMR data (Table 1) showed that 1 possesses a carbon skeleton similar to those of 2¹⁸ and 3,¹⁹ with the presence of an acetoxy group at C-1 in 4²⁰ and 5²¹ as the major structural difference. The ¹H and ¹³C NMR spectra (acetic acid-d₄) exhibit characteristic signals for a limonoid group (Table 1), including five methyl groups [δ_H 1.13 (s, H-18), 1.28 (s, H-19), 1.42 (s, H-28), 1.56 (s, H-29), and 1.18 (s, H-30), each 3H], three oxymethines [δ_H 3.95 (br d, H-1), 3.80 (s, H-15), and 5.34 (s, H-17); δ_C 71.2 (C-1), 54.4 (C-15), and 80.6 (C-17)], α-β-substituted γ-hydroxybutenolide groups [δ_H 6.10 (br s, H-21) and 6.34 (s, H-22), each 1H, δ_C 165.5 (C-20), 100.0 (C-21), 124.6 (C-22), and 172.0 (C-23)], two ester carbonyl groups [δ_C 176.2 (C-3), 168.8 (C-16)], and a ketone group [δ_C 210.6 (C-7)]. To further support the proposed structure of compound 1, we compared the ¹³C NMR chemical shifts (in methanol-d₃) of C-16 and C-17 with those of structurally related limonoids bearing either intact or cleaved D-rings. In compound 1 (junosterpene), C-16 and C-17 resonate at δ_C 168.2 and 79.7, respectively (Table 1). Obacunoic acid (5),²¹ which also retains a closed A-ring, shows similar values (δ_C 168.6 and 77.1) (Table 1). In contrast, open-ring limonoids such as limonoate A-ring lactone²⁷ and nomilinoate A-ring lactone²⁷ exhibit downfield-shifted C-16 signals (δ_C 173.7 and 171.5) and upfield-shifted C-17 signals (δ_C 72.5 for both) (Table S1), consistent with ring cleavage and altered electronic environments. This trend is consistent with previous reports on ring-seco limonoids, where ring opening leads to deshielding at C-16 due to increased electron-withdrawing effects and shielding at C-17 due to loss of ring strain. This comparative data (Table 1, Fig. S1.16, and Table S1) provides additional evidence for the closed-ring structure of compound 1, independent of HMBC correlations. In compound 1, the hemiacetal carbon at C-21 exhibited a ¹³C NMR signal at δ_C 100.0 (C-21), consistent with a hemiacetal structure and further influenced by a γ-gauche effect arising from spatial proximity to the carbonyl group at C-23. As C-21 resides at the γ-position relative to C-23, their gauche arrangement induces electronic shielding, resulting in an upfield-shifted chemical shift. This effect supports the spatial orientation of C-21 and provides additional evidence for the proposed structure. The position of each functional group was determined using correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) NMR spectroscopy (Figure 2). In particular, long-range correlations were observed between the following proton and carbon pairs in 1: H-1/C-3 and 19; H-5/C-4, and 19; H-9/C-11 and 19; H-15/C-14 and 16; H-17/C-20 and 22; H-18/C-12, 13, 14, and 17; H-19/C-1, 5, 9, and 10; H-21/C-22 and 23; H-22/C-17, 21, and 23; H-28/C-4 and 29; H-29/C-4, 5, and 28; and H-30/C-7, 8, 9, and 14. Several limonoids bearing the same α-β-substituted-γ-hydroxybutenolide group, such as 4,²⁰ clauemargine F,²⁸ and sudachinoid C²⁹ have been reported. The relative configuration of 1 was determined by analyzing its ROESY spectrum (Figure 2). ROESY cross peaks between H-1 and H-6β; H-1 and H-12β; H-1 and H-19; H-2α and H-9; H-2β and H-6β; H-2β and H-19; H-2β and H-29; H-5 and H-6α; H-6α and H-28; H-6β and H-30; H-9 and H-6α; H-9 and H-18; H-15 and H-30; H-17 and H-12β indicate that 1-OH, H-2α, 5, 6α, 9, 11α, 12α, 18, and 28 are on one side of the molecule and that H-1, 2β, 6β, 11β, 12β, 15, 17, 19, 29, and 30 are on the opposite side. Although ROESY analysis supports this relative configuration, the absolute stereochemistry of compound 1 (1S, 5R, 8R, 9R, 10R, 13S, 14S, 15R, 17R) was established by comparison with those of structurally related limonoids (eg, 2¹⁸; [α]²⁵_D −87.9 in MeOH) and supported by its observed optical rotation ([α]²⁵_D −28.8 in MeOH). The chemical structure of 1 was established based on the evidence provided above (Figure 1). In addition to NMR analysis, the IR spectra of compounds 1–3 exhibit characteristic absorptions for hydroxyl (broad band around 3300 cm⁻¹) and carbonyl (bands around 1650 cm⁻¹) groups, further supporting the presence of hydroxyl moieties and ketone functionalities (Figures S1.12 and 13). 1 was an inseparable mixture of C-21 hemiacetal epimers.

Figure 2.

Important 2D NMR and ROESY correlations for junosterpene (1).

Evaluating Anti-Proliferative Effects

The anti-proliferative activities of the isolated compounds (1-11) were evaluated using the WST-8 assay in U-251 MG and T98G human glioma cells. None of the tested compounds exhibited significant anti-proliferative effects at a concentration of 30 µM for 24 h (Figures S1.14 and S1.15). In contrast, adriamycin (10 µM), as a positive control, reduced U-251 MG and T98G cell proliferation to 68.2% and 41.6%, respectively (Figures S1.14 and S1.15).

Discussion

Citrus fruits are rich sources of bioactive compounds, such as flavonoids, carotenoids, limonoids, sesquiterpenoids, and coumarins.⁴ While hundreds of limonoids have been isolated from various plants, their occurrence is largely limited to plant families within the order Rutales, predominantly Meliaceae and Rutaceae.¹³ Limonoids are unusual triterpenes owing to their extensively rearranged biosynthetic scaffolds.²⁹ At least two main scaffold modifications are proposed to be conserved in both plant families: a C-30 methyl shift of the protolimonoid scaffold (apo-rearrangement), and the conversion of the hemiacetal ring of melianol into a mature furan ring with the concomitant loss of a C4 carbon side-chain³⁰; additional modifications that are specific to Rutaceae hen yield nomilin-type intermediates.³⁰

However, the constituent components of the fruit of C. × junos have been less studied, although some studies on the bioactivities of its extracts have been reported.^10,31 The primary objective of this study was to isolate novel limonoids, which led to the isolation of junosterpene (1) from the seeds of C. junos. The new limonoid 1 is a nomilin-type limonid (seco-A, D ring scaffold) with a similar structure to that of 4,²⁰ as characterized by a C-30 methyl shift and the loss of four carbons (compared to a typical triterpene), which are structural features commonly observed in limonoids isolated from plants in the Rutaceae family. Chemophenetically, these findings support the classification of C. × junos within Rutaceae, particularly reflecting its biosynthetic capacity to generate nomilin-type limonoids, as evidenced by the isolation of such structures in this study^8,32 and consistent with established biosynthetic pathways in Rutaceae plants.³⁰ Citrus × junos Siebold ex Tanaka is recognized as an artificial hybrid,¹⁴ with its proposed parentage comprising C. cavaleriei × C. maxima × C. reticulata. Citrus limonoids are predominantly found in C. maxima,³¹ C. reticulata,^5,19,20 amd C. cavaleriei (C. ichangensis),³² which are well-established sources of these tetranortriterpenoids, reflecting its distinct biosynthetic profile.³³ Compared against previously reported concentrations of 3 and 7, our findings showed higher levels than those found in the peels analyzed by Kitagawa et al (3; 2.625 ppm),⁸ but lower than those reported by Minamizawa et al (3; 104.7 mg g⁻¹ and 7; 95.0 mg g⁻¹)³⁴ and Jo et al (3; 21.24 mg g⁻¹ and 7; 28.86 mg g⁻¹)³⁵ for the seeds. In addition to the limonoid components, the present study also isolated three known FCs (9-11) from the seeds of C. × junos. Previous phytochemical investigations of C. × junos identified nine FCs including 9 and 10.³⁶ FCs, although described in higher plants, are mainly distributed in four families: Apiaceae, Moraceae, Fabaceae, and Rutaceae.³⁷ The occurrence of such compounds in the seeds of C. × junos, together with limonoids, reflects the biosynthetic versatility typical of Rutaceae members, as evidenced by the co-occurrence of limonoids and FCs. This dual pathway reinforces the chemophenetic placement of C. × junos within the Citrus clade. These findings may provide chemophenetic markers that are not identifiable through classification systems based on volatile organic compounds (VOCs)^38,39 and flavonoids,⁴⁰ thereby enhancing our understanding of Citrus species diversity. Further comparative studies with other Rutaceae members are warranted to contextualize these biosynthetic capacities within phylogenetic frameworks. Further research into the biological activities of the isolated components is necessary to assess their potential pharmacological properties given the limited scope of the current study.

Conclusion

Junosterpene (1) was isolated as a new limonoid from the seeds of C. × junos and its chemical structure was elucidated using spectroscopic techniques. Compounds 2–11 were identified as 21,23-dihydro-23-hydroxy-21-oxodeacetylnomilin, deacetylnomilin, 21,23-dihydro-21-hydroxy-23-oxonomilin, obacunoic acid, methyldeacetylnomilinate, limonin, ichangin, (−)-heraclenol, (−)-byak-angelicin, and (+)-oxypeucedanin hydrate. The newly identified limonoid derivative may contribute to the diverse bioactive profile of C. × junos and provides insight into its potential physiological and pharmacological properties.

Supplemental Material

sj-doc-1-npx-10.1177_1934578X251401118 - Supplemental material for Limonoids and Furanocoumarins from a Cultivated Citrus × junos Siebold ex Tanaka

Supplemental material, sj-doc-1-npx-10.1177_1934578X251401118 for Limonoids and Furanocoumarins from a Cultivated Citrus × junos Siebold ex Tanaka by Daisuke Imahori, Takuya Muraoka, Miharu Kubota and Hiroyuki Tanaka in Natural Product Communications

Footnotes

Acknowledgements

This work was supported by the Japan Society for the Promotion of Science (JSPS): KAKENHI Grant Numbers 22K20720 and 23K16308. We would like to thank Editage () for English language editing.

ORCID iDs

Daisuke Imahori

Takuya Muraoka

Hiroyuki Tanaka

Ethical Approval

Not applicable.

Consent for Publication

Not applicable.

CRediT Authorship Contribution Statement

Daisuke Imahori: Writing–original draft, Project administration, Conceptualization. Takuya Muraoka: Data curation, Formal analysis. Miharu Kubota: Data curation. Hiroyuki Tanaka: Writing – review & editing, Supervision, Conceptualization.

Author Agreement Statement

We declare that this manuscript is original, has not been published previously, and is not currently under consideration for publication elsewhere.

We confirm that the manuscript has been read and approved by all named authors, and that there are no other persons who satisfy the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all authors.

We understand that the Corresponding Author is the sole contact for the Editorial process. He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs.

Funding

This work was supported by the Japan Society for the Promotion of Science (JSPS): KAKENHI Grant Numbers 22K20720 and 23K16308.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Statements and Declarations

There are no human subjects in this study, therefore informed consent is not required.

Statement of Human and Animal Rights

Not applicable.

Statement of Informed Consent

Not applicable.

Supplemental material

Supplemental material for this article is available online.

References

Terol

Ibanez

, et al. Genomics of the origin and evolution of Citrus. Nature. 2018;554:311–316. doi:10.1038/nature25447

Singh

Kaur

, et al. Insights into the chemical composition and bioactivities of citrus peel essential oils. Food Res Int. 2021;143:110231. doi:10.1016/j.foodres.2021.110231

Sugimoto

Kinjo

, et al. Diversification of mandarin citrus by hybrid speciation and apomixis. Nat Commun. 2021;12(1):4377. doi:10.1038/s41467-021-24653-0

Martí

Mena

Cánovas

, et al. Vitamin C and the role of citrus juices as functional food. Nat Prod Commun. 2009;4(5):677–700. doi:10.1177/1934578X090040050

Zheng

Yang

Chen

. The pharmacological and pharmacokinetic properties of obacunone from citrus fruits: a comprehensive narrative review. Fitoterapia. 2023;169:105569. doi:10.1016/j.fitote.2023.105569

Agrawal

Chandan

Gerald

. Pharmacological significance of hesperidin and hesperetin, two citrus flavonoids, as promising antiviral compounds for prophylaxis against and combating COVID-19. Nat Prod Commun. 2021;16(10):1–15. doi:10.1177/1934578X211042540

Santos Junior

Silva

SMC

Sales

, et al. Coumarins from Rutaceae: chemical diversity and biological activities. Fitoterapia. 2023;168:105489. doi:10.1016/j.fitote.2023.105489

Kitagawa

Matsumoto

Imahori

, et al. Limonoids isolated from the Fortunella crassifolia and the Citrus junos with their cell death-inducing activity on Adriamycin-treated cancer cell. J Nat Med. 2021;75:998–1004. doi:10.1007/s11418-021-01528-8

Lee

. Monitoring and risk assessment of pesticide residues in yuza fruits (Citrus junos Sieb. ex Tanaka) and yuza tea samples produced in Korea. Food Chem. 2012;135:2930–2933. doi:10.1016/j.foodchem.2012.06.111

10.

Shin

Park

Sung

, et al. Citrus junos Tanaka peel ameliorates hepatic lipid accumulation in HepG2 cells and in mice fed a high-cholesterol diet. BMC Complement Altern Med. 2016;16:499. doi:10.1186/s12906-016-1460-y

11.

Nakamura

Suzuki

Takagi

, et al. Stimulation of phosphorylation of ERK and CREB by phellopterin and auraptene isolated from Citrus junos. Nat Prod Commun. 2014;9(10):1491–1494. doi:10.1177/1934578X1400901021

12.

Shiran

Amani

Ajami

, et al. Antitumor effects of auraptene in 4T1 tumor-bearing BALB/c mice. Horm Mol Biol Clin Investig. 2021;42(3):245–252. doi:10.1515/hmbci-2020-0090

13.

Roy

Saraf

. Limonoids: overview of significant bioactive triterpenes distributed in plants kingdom. Biol Pharm Bull. 2006;29(2):191–201. doi:10.1248/bpb.29.191

14.

Citrus × junos Siebold ex Tanaka. Published on the Internet; Plants of the World Online, Royal Botanic Gardens, Kew. Accessed August 26, 2025. https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:771942-1.

15.

Tonosaki

Kudo

Kitashiba

, et al. Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants. Mol Breed. 2013;31:419–428. doi:10.1007/s11032-012-9799-3

16.

Wei

Zhai

Zeng

, et al. TRMT10A Regulates tRNA-ArgCCT m1G9 modification to generate tRNA-derived fragments influencing vasculogenic mimicry formation in glioblastoma. Cell Death Dis. 2025;16(1):209. doi:10.1038/s41419-025-07548-6

17.

Park

Song

Yoo

H-Y

, et al. Anti-aging effects and main chemical constituents of Spiraea salicifolia stem extracts. Nat Prod Commun. 2025;20(2):1–14. doi:10.1177/1934578X251322711

18.

Nakagawa

Takaishi

Tanaka

, et al. Chemical constituents from the peels of Citrus sudachi. J Nat Prod. 2006;69(8):1177–1179. doi:10.1021/np060217s

19.

Bennett

Hasegawa

. Limonoids of calamondin seeds. Tetrahedron. 1981;37(1):17–24. doi:10.1016/S0040-4020(01)97708-7

20.

Kikuchi

Ueno

Hamada

, et al. Five new limonoids from peels of satsuma orange (Citrus reticulata). Molecules. 2017;22(6):907. doi:10.3390/molecules22060907

21.

Makita

Ohta

Nakabayashi

. New limonoid from the seeds of Citrus natsudaidai. Agric Biol Chem. 1980;44(3):693–694. doi:10.1080/00021369.1980.10864015

22.

Breksa

APIII

Dragull

Wong

. Isolation and identification of the first C-17 limonin epimer, epilimonin. J Agric Food Chem. 2008;56(14):5595–5598. doi:10.1021/jf800473z

23.

Barrera

CAC

Barrera

EDC

Falla

DSG

, et al. Seco-limonoids and quinoline alkaloids from Raputia heptaphylla and their antileishmanial activity. Chem Pharm Bull. 2011;59(7):855–859. doi:10.1248/cpb.59.855

24.

Razdan

Kachroo

Harkar

, et al. Furanocoumarins from Heracleum canescens. Phytochemistry. 1982;21(4):923–927. doi:10.1016/0031-9422(82)80094-0

25.

Ishihara

Fukutake

Asano

, et al. Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection. J Chromatogr B Biomed Sci Appl. 2001;753(2):309–314. doi:10.1016/s0378-4347(00)00569-7

26.

Baek

Ahn

Kim

, et al. Furanocoumarins from the root of Angelica dahurica. Arch Pharmacal Res. 2000;23(5):467–470. doi:10.1007/BF02976574

27.

Zukas

Breksa

Manners

. Isolation and characterization of limonoate and nomilinoate A-ring lactones. Phytochemistry. 2004;65(19):2705–2709. doi:10.1016/j.phytochem.2004.08.016

28.

Xia

Yang

, et al. A, D-seco-limonoids from the stems of Clausena emarginata. J Nat Prod. 2014;77(4):784–791. doi:10.1021/np400797s

29.

Nakagawa

Duan

Takaishi

. Limonoids from Citrus sudachi. Chem Pharm Bull. 2001;49(5):649–651. doi:10.1248/cpb.49.649

30.

LPR

Hodgson

Liu

, et al. Complex scaffold remodeling in plant triterpene biosynthesis. Science. 2023;379(6630):361–368. doi:10.1126/science.adf1017

31.

Sapkota

Devkota

Poudel

. Citrus maxima (Brum.) Merr. (Rutaceae): bioactive chemical constituents and pharmacological activities. Evid Based Complement Alternat Med. 2022;2022:8741669. doi:10.1155/2022/8741669

32.

Herman

Hasegawa

Fong

, et al. Limonoids in Citrus ichangensis. J Agric Food Chem. 1989;37(4):850–851. DOI:10.1021/jf00088a003

33.

Heo

Lee

Kim

Lee

. Anti-obesity effects of ethanol extract of green Citrus junos peel enriched in naringin and hesperidin in vitro and in vivo. Nutr Res Pract. 2025;19(1):1–13. doi:10.4162/nrp.2025.19.1.1

34.

Minamisawa

Yoshida

Uzawa

. The functional evaluation of waste yuzu (Citrus junos) seeds. Food Funct. 2014;5(2):330–336. doi:10.1039/c3fo60440c

35.

Shin

Song

. Simultaneous determination of the flavonoids and limonoids in Citrus junos seed shells using a UHPLC-DAD-ESI/MS. Nat Prod Sci. 2020;26(1)(1):64–70. doi:10.20307/nps.2020.26.1.64

36.

Song

Shin

, et al. Anti-Inflammatory activities of isogosferol, a furanocoumarin isolated from Citrus junos seed shells through bioactivity-guided fractionation. Molecules. 2019;24(22):4088. doi:10.3390/molecules24224088

37.

Limones-Mendez

Dugrand-Judek

Villard

, et al. Convergent evolution leading to the appearance of furanocoumarins in citrus plants. Plant Sci. 2020;292:110392. doi:10.1016/j.plantsci.2019.110392

38.

González-Mas

Rambla

López-Gresa

Blázquez

Granell

. Volatile compounds in Citrus essential oils: a comprehensive review. Front Plant Sci. 2019;10:12. doi:10.3389/fpls.2019.00012

39.

Yang

Park

. Antioxidant effects of essential oils from the peels of Citrus cultivars. Molecules. 2025;30(4):833. doi:10.3390/molecules30040833

40.

Nogata

Sakamoto

Shiratsuchi

Ishii

Yano

Ohta

. Flavonoid composition of fruit tissues of citrus species. Biosci, Biotechnol, Biochem. 2006;70(1):178–192. doi:10.1271/bbb.70.178

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

4.55 MB