Five New Limonoids from Peels of Satsuma Orange (Citrus reticulata)

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May 31, 2017 - Satsuma orange (Scientific name: Citrus reticulata Blanco; Synonym: ...... T.; Naoki, H.; Hase, T. The structure of graucin A, a new bitter limonoid.
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Five New Limonoids from Peels of Satsuma Orange (Citrus reticulata) Takashi Kikuchi, Yasuaki Ueno, Yoshino Hamada, Chika Furukawa, Takako Fujimoto, Takeshi Yamada and Reiko Tanaka * Faculty of Pharmaceutical Sciences, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1142, Japan; [email protected] (T.K.); [email protected] (Y.U.); [email protected] (Y.H.); [email protected] (C.F.); [email protected] (T.F.); [email protected] (T.Y.) * Correspondence: [email protected]; Tel.: +81-72-690-1084 Academic Editor: Derek J. McPhee Received: 21 April 2017; Accepted: 26 May 2017; Published: 31 May 2017

Abstract: Five new: 21,23-dihydro-21-hydroxy-23-oxonomilin (1), 21,23-dihydro-23-methoxy-21oxonomilin (2), 21,23-dihydro-21-hydroxy-23-oxonomilinic acid methyl ester (3), 21,23-dihydro-23methoxy-21-oxolimonin (4), and 21,23-dihydro-21-oxolimonin (5), and seven known limonoids were isolated from peels of satsuma orange (Citrus reticulata). The isolated compounds were evaluated for their inhibitory effects on macrophage activation by an inhibitory assay of nitric oxide (NO) production. Among them, compound (2) exhibited NO inhibitory activity without cytotoxicity. Keywords: limonoid; Rutaceae; satsuma orange; Citrus reticulata; inhibitory activity on nitric oxide production

1. Introduction Satsuma orange (Scientific name: Citrus reticulata Blanco; Synonym: Citrus unshiu Marcov.; Japanese name: unshu mikan), belongs to Rutaceae, and is cultivated in Japan. The peels of its fruits, called chinpi, have been used as an aromatic stomachic, cold medicine, expectorant, and antitussive [1]. Limonoids [2,3], flavonoids [4–7], tocopherol analogues [8,9] have been isolated from peels of C. reticulata, phenyl glycosides from flower buds [10], and a limonoid [11], flavonoids [11], and a cyclic peptide from its fruits [12]. Moreover, some bioactivities of constituents from C. reticulata have been reported, such as the suppression of adipogenesis by a limonoid [3], the inhibitory activity on histamine release from rat peritoneal mast cells [4], hypotensive activity [5] and antioxidant activity [6] of flavonoids, the radical-scavenging activity [8] and hepatoprotective and neuroprotective activities [9] of tocopherol analogues, as well as antimicrobial activities of essential oil [13]. In the present study, we isolated five new limonoids 1–5; 21,23-dihydro-21-hydroxy-23-oxonomilin (1), 21,23-dihydro-23-methoxy-21-oxonomilin (2), 21,23-dihydro-21-hydroxy-23-oxonomilinic acid methyl ester (3), 21,23-dihydro-23-methoxy-21-oxolimonin (4), and 21,23-dihydro-21-oxolimonin (5), along with known compounds (6–12). Compounds 1–12 were evaluated for inhibitory effects of limonoids on nitric oxide (NO) production by macrophages. 2. Results and Discussion Five new (1–5), and seven known compounds (6–12) were isolated from the C. reticulata peels (Figure 1). The known compounds were identified as limonin (6) [14,15], shihulimonin A (7) [16], limonexic acid (8) [17], evolimorutanin (9) [18], kihadanin A (10) [19], deacetylnomilin (11) [20], and ichangin (12) [21] by comparison of their spectroscopic data with those previously reported.

Molecules 2017, 22, 907; doi:10.3390/molecules22060907

www.mdpi.com/journal/molecules

Molecules 2017, 22, 907

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Molecules 2017, 22, 907

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Figure 1. Structures of limonoids isolated from peels of C. reticulata. Figure 1. Structures of limonoids isolated from peels of C. reticulata.

21,23-Dihydro-21-hydroxy-23-oxonomilin (1) was obtained as colorless crystals. Its molecular

21,23-Dihydro-21-hydroxy-23-oxonomilin (1) was as colorless Its molecular [M obtained + H]+, calcd.: 547.2179) crystals. by the HRFABMS. formula was established as C28H34O11 (m/z 547.2178 + , calcd.: −1H] −1) groups. The was IR spectrum showed the presence of hydroxy (3466 cm ) and carbonyl (1733 cm The formula established as C H O (m/z 547.2178 [M + 547.2179) by the HRFABMS. 28 34 11 1H- and 13C-NMR spectra indicated the presence of six methyl − 1 − 1 groups observed as singlets The IR spectrum showed the presence of hydroxy (3466 cm ) and carbonyl (1733 cm ) groups. methyls (δH 1.11 (s), 1.16the (s), presence 1.34 (s), 1.48 (s), 2.10 (s)), three oxymethines The 1including H- and 13acetyl C-NMR spectra indicated of (s), six1.56 methyl groups observed as singlets (δ H 3.70 (s), 5.05 (brd), 5.34 (brd); δC 52.6 (d), 70.7 (d), 78.1 (d)), two unprotonated oxycarbons (δC including acetyl methyls (δH 1.11 (s), 1.16 (s), 1.34 (s), 1.48 (s), 1.56 (s), 2.10 (s)), three oxymethines 65.0 (s), 84.4 (s)), a hemiacetal (δH 5.99 (brs); δC 97.4 (d)), a trisubstituted olefin (δH 6.30 (dd); δC 123.2 (δH 3.70 (s), 5.05 (brd), 5.34 (brd); δC 52.6 (d), 70.7 (d), 78.1 (d)), two unprotonated oxycarbons (d), 162.5 (s)), a ketone (δC 206.5 (s)), four ester carbonyls (δC 165.5 (s), 168.9 (s), 169.1 (s), 169.6 (s)) (δC 65.0 (s), 84.4 (s)), a hemiacetal (δH 5.99 (brs); δC 97.4 (d)), a trisubstituted olefin (δH 6.30 (dd); (Table 1). In the HMBC experiment, the following correlations were observed: Me-18 (δH 1.11 δC 123.2 (d), 162.5 (δ and 206.5 four(d)); ester carbonyls (s), 169.1 C 165.5 (s))/C-12, C-13, (s)), C-14 a(δketone C 65.0 (s)), C C-17(s)), (δC 78.1 Me-19 (δH 1.34 (δ (s))/C-1 (δC(s), 70.7168.9 (d)), C-5, C-9, (s), 169.6 and (s)) C-10; (Table 1). In the HMBC experiment, the following correlations were observed: Me-18 Me-28 (δH 1.48 (s)) and Me-29 (δH 1.56 (s))/C-4 (δC 84.4 (s)) and C-5; Me-30 (δH 1.16 (s))/C-7 (δH 1.11 (s))/C-12, C-13, (δC C-14; 65.0 (s)), and C-17 (δC 78.1(δ(d)); Me-19 (δH1-OCOCH 1.34 (s))/C-1 (δC 70.7 (δC 206.5 (s)), C-8,C-14 C-9, and H-1 (δ H 5.05 (brd))/C-3 C 169.1 (s)) and 3 (δC 169.6 (s)); (d)), H-15and (δH 3.70 (s))/C-16 (δH 6.30(δ (dd))/C-21 (δC 97.4(δ (d)) (Figure In C-5; addition, a (δH C-5, C-9, C-10; Me-28(δ (δCH165.5 1.48(s)); (s)) H-22 and Me-29 (s)) 2). and Me-30 H 1.56 (s))/C-4 C 84.4 trisubstituted olefin(s)), existed C-20and andC-14; C-22,H-1 and(δ a Hhemiactal carbon existed at C-21 HMBC 1.16 (s))/C-7 (δC 206.5 C-8,atC-9, 5.05 (brd))/C-3 (δC 169.1 (s)) since and 1-OCOCH 3 correlations were from H-17 H 5.34 (brd)) to the acetal carbon (δC 97.4 (d)) and olefin (δC 169.6 (s)); H-15 (δHobserved 3.70 (s))/C-16 (δC (δ 165.5 (s)); H-22 (δH 6.30 (dd))/C-21 (δC 97.4 (d)) (Figure 2). carbonsa(δ C 123.2 (d), 162.5 (s)). The above data suggested that compound 1 had a similar structure In addition, trisubstituted olefin existed at C-20 and C-22, and a hemiactal carbon existed at C-21 since to nomilin [22] except for the lack of a furan ring and the presence of a β-substituted HMBC correlations were observed from H-17 (δH 5.34 (brd)) to the acetal carbon (δC 97.4 (d)) and olefin γ-hydroxybutenolide group. The following correlations were also observed in the 1H-1H COSY carbons (δC 123.2H-1/H (d), 162.5 (s)). The above data suggested that compound 1 had a similar structure to experiment; 2-2; H-5/H2-6; H-9/H2-11; H2-11/H2-12. In the NOESY experiment, the following nomilin [22] except for the lack a furan ring and the presence a β-substituted correlations were observed;ofMe-28/H-6α; Me-18/H-9α, H-22,of and 1-OCOCH3; γ-hydroxybutenolide H-2β/Me-19 and 1 H-1 H COSY experiment; H-1/H -2; group. The following correlations were also observed in the Me-29; H-6β/Me-19 and Me-30; H-12β/H-17 (Figure 3). Therefore, the structure of 1 was established, 2 H-5/H -6; H-9/H Inan the NOESY experiment, the following correlations were as 2shown in Figure Compound 1 was inseparable mixture of C-21 hemiacetal epimers. 2 -11;1.H 2 -11/H2 -12. observed; Me-28/H-6α; Me-18/H-9α, H-22, and 1-OCOCH3 ; H-2β/Me-19 and Me-29; H-6β/Me-19 and Me-30; H-12β/H-17 (Figure 3). Therefore, the structure of 1 was established, as shown in Figure 1. Compound 1 was an inseparable mixture of C-21 hemiacetal epimers.

(CD CO) and and 33 (in (in CDCl CDCl33 ++ 11 drop drop CD CD33OD) OD) aa.. (CD33))22CO) 11 22 33 δδHH Mult. δδCC δδHH Mult. δδCC δδHH Mult. δδCC Mult. (J (J in in Hz) Hz) Mult. (J (J in in Hz) Hz) Mult. (J (J in in Hz) Hz) 1β 5.05 brd 70.7 4.97 dd (7.1) 72.0 dd 6.54 brs 76.3 1β 5.05 brd (6.4) (6.4) 70.7 dd 4.97 (7.1) 72.0 6.54 brs 76.3 dd 22 αα 3.11 dd 35.3 tt αα 2.98 dd 36.0 tt A m 35.4 tt 3.11 dd (6.4, (6.4, 15.8) 15.8) 35.3 2.98 dd (7.1, (7.1, 15.8) 15.8) 36.0 A 2.35 2.35 m 35.4 Molecules 2017, 22, 907 3 of 10 ββ 3.24 brd ββ 3.50 dd BB 2.82 m 3.24 brd (15.8) (15.8) 3.50 dd (1.2, (1.2, 15.8) 15.8) 2.82 m 33 169.1 169.5 172.0 169.1 ss 169.5 ss 172.0 ss 44 84.4 84.7 ss 74.1 84.4 ss 84.7 74.1 ss 1 H-2017, Molecules 22, 907 15.8) of 10 3 )253.0 Table 1.2.62 (600 MHz) and 13 C-NMR MHz) spectra (in CDCl3 ), 2m (in3 (CD CO) 55 dd (3.9, 51.1 dd 14.7) 51.7 dd 12.01 2.62 dd (3.9, 15.8) 51.1 dd(1502.69 2.69 dd (3.5, (3.5,data 14.7) of compounds 51.7 2.01 m 53.0 6α αα 2.60 15.8) 38.6 dd 39.6 tt αα 2.46 dd 38.9 tt 6α 2.60 dd 15.8) 38.6 a . tt αα 2.51 2.51 dd (3.5, (3.5, 14.7) 14.7) 39.6 2.46 dd (5.3, (5.3, 14.9) 14.9) 38.9 and 3 (in CDCl3dd +1 (3.9, 1(3.9, drop CD3 OD) Table 1. H(600 MHz) and 13C-NMR (150 MHz) ttspectra (in CDCl3),tt (14.9) 2(14.9) (in 6β ββ 2.78 tt (15.8) ββ 3.10 (14.7) 6β 2.78 (15.8) 3.10 (14.7) data of compoundsββ12.80 2.80 a. 3 + 1 drop (CD3)2CO) and 3 (in CDCl206.5 77 ss CD3OD) 208.3 210.0 206.5 208.3 ss 210.0 ss 1 2 3 Position 88 53.1 ss 53.2 ss 52.5 53.1 53.2 52.5 ss 1 2 3 δH Mult. (J in Hz) δC δH Mult. (J in Hz) δC δH Mult. (J in Hz) δC Position 99 2.44 44.3 2.61 dd 11.8) 2.14 44.4 δbrd H Mult. (J in Hz) δH Mult. (J in Hz) δ(11.4) C 2.44 brd (11.2) (11.2) 44.3 δC dd 2.61 dd (2.4, (2.4, 11.8) δC 44.8 44.8 δdHd Mult. 2.14 (J in Hz)brd brd (11.4) 44.4 dd 1β 5.05 brd (6.4) 70.7 d 4.97 d (7.1) 72.0 d 6.54 brs 76.3 d 1β 5.05 brd (6.4) 44.1 70.7 ss d 4.97 d (7.1) 72.0 45.1 d 6.54 brs 76.3 d 10 10 2 44.1 45.1 46.1t ss α 3.11 dd (6.4, 15.8) 35.3 t α 2.98 dd (7.1, 15.8) 36.0 t ss A 2.35 m 35.4 46.1 2 α 3.11 dd (6.4, 15.8) 35.3 t α 2.98 dd (7.1, 15.8) 36.0 t A 2.35 m 35.4 t αα 1.70 m 17.3 tt ααβ1.63 m 17.44 tt Bαα 2.49 19.0 tt β 3.24 brd 3.50 dd (1.2, 2.82 m m 1.70 m(15.8) 17.3 1.63 m 15.8) 17.44 B 2.82 2.49m m 19.0 β 3.24 brd (15.8) β 3.50 dd (1.2, 15.8) 11 11 3 169.1 s 169.5 s 172.0 s ββ 1.66 m m ββ 1.72 m 1.66 m β 1.73 1.73 m 1.72 m 3 169.1 s β 169.5 s 172.0 s 4 84.4 s 84.7 s 74.1 s αα 1.29 m 32.0 t α 1.16 m 30.6 t αα 1.61 m tt 4 2.62 s s 53.0 31.6 1.29 m 15.8) 32.0 m 14.7) 84.751.7 30.6 1.61 m 31.6 dd (3.9, 51.184.4 td s α 1.16 2.69 dd (3.5, d t 2.01 m 74.1 12 12 5 5α 2.60 dd 2.62 dd (3.9, 15.8) 38.651.1 t d β 2.69 (3.5, 14.7) 53.0 ββ 2.02 (7.9, 13.0) m 2.01 m 6α (3.9, 15.8) 2.51 dddd (3.5, 14.7) 51.739.6 d t2.01 αββ 2.46 38.9 t 2.02 dddd (7.9, 13.0) βα2.10 2.10 m 2.01m dd (5.3, 14.9) m 6α dd (3.9, 15.8) 38.6 t αβ 2.51 14.7) 39.6 t α 2.46 β 2.80 dd (5.3, 14.9)t (14.9) 38.9 t β 2.78 α 2.60 t (15.8) 3.10 dd (3.5, t (14.7) 13 37.7 39.0 ss 37.5 13 6β 37.7 ss 39.0 β 2.80 37.5 ss 6β β 2.78 t (15.8) 206.5 β 3.10 t (14.7) t (14.9) 7 s 208.3 s 210.0 s 14 65.0 s 66.8 s 14 8 65.0 66.8 65.0s ss 7 208.353.2 s 210.0 s 52.5 65.0 53.1206.5 ss s s s 15 3.70 ss (11.2) 52.6 m 54.5 3.64 ss 8 2.44 s 52.5 s 44.4 52.5 15 9 3.70 52.6 dd s 3.95 3.95 m 11.8) 53.244.8 54.5 d 2.14 3.64 52.5d dd brd 44.353.1 d 2.61 dd (2.4, dd brd (11.4) 9 2.44 brd (11.2) 165.5 2.61 dd (2.4, 11.8) 44.845.1 d brd (11.4) 44.4 d 46.1166.3 44.144.3 sss d s2.14 16 166.9 16 10 165.5 166.9 166.3s ss 10 45.117.44s s 19.0 α 1.70 m 17.344.1 t s α 1.63 m t m 46.1 t 17 5.34 brd 78.1 tt (1.1) 76.2 dd α 2.49 5.33 dd (1.5) 78.6 17 11 5.34 brd (1.7) (1.7) 78.117.3 dd t 5.32 5.32 (1.1) 76.2 5.33 (1.5) t 78.6 dd β 1.66 α 1.70 m m 1.73 m 19.0 αβ 1.63 mm 17.44 t α 2.49 β 1.72 m 11 18 1.11 s 21.4 q 1.20 s 20.4 q 1.10 s 21.4 18 1.11 21.4 qt 1.20 s 20.4 βt 1.72 q α 1.61 1.10 m α 1.29 β 1.66 s m m 32.0 1.16 30.6 m s 31.6 21.4t qq βα1.73 mm 12 β 2.02 α 1.29 dd (7.9, 2.10 m m 31.6 19 1.34 ss 13.0) 16.64 qq β 2.01 1.31 ss 16.7 m 32.0 qq t 1.47 αβ 1.16 mm 30.6 16.1 t α 1.61 m t 19 1.34 16.64 1.47 m 16.1 1.31 16.7 qq 12 13 37.7 39.0 37.5163.6s β 2.02 dd (7.9, 13.0)162.5 β 2.10 m βs 2.01 m 20 sss 134.0 ss ss 20 14 162.5 134.0 163.6 65.037.7 s s s s 13 39.066.8 s 37.5 s 65.0 21 5.99 brs 97.4 d 169.80 s 6.00 brs 98.2 d 21 15 5.99 brs 97.4 d 169.80 s 6.00 brs 98.2 d s 52.665.0 d s 3.95 m d 3.64 s 65.0 d 143.70 66.854.5 s s 52.5 22 6.30 dd 123.2 tm 150.9 dd 6.29 brs 165.552.6 d 22 16 6.30 dd (0.9, (0.9, 1.7) 1.7)s 123.2 ds d 7.48 7.48 t (1.1) (1.1) 150.9 6.29 s brs d 166.3122.7 122.7s dd 15 3.70 3.95 54.5166.9 d 3.64 52.5 5.34 brd (1.7) 78.1165.5 sd s 6.02 5.32 (1.1) 76.2 d (1.5) 78.6169.8d s 23 168.9 ttt(1.1) 16 166.9103.5 166.3 s 23 17 168.9 s 6.02 (1.1) 103.5 d dd 5.33 169.8 s 18 1.11 s 21.4 q 1.20 s 20.4 q 1.10 s 21.4 q 17 5.34 brd (1.7) 78.1 d 5.32 t (1.1) 76.2 d 5.33 d (1.5) 78.6 d 28 1.48 ss s 33.4 1.39 ss 33.9 1.35 28 19 1.48 33.4 qqq 1.39 33.9 q qq 1.31 1.35 33.6q qq 1.34 16.64 1.47 m 16.1 s ss 16.7 33.6 18 1.11 s 21.4 q 1.20 s 20.4 q 1.10 s 21.4 q 29 1.56 ss 23.4 ss 23.3 1.34 ss 162.5 134.0 s qq 163.627.5 29 20 1.56 23.416.64 qqs q 1.64 1.64 23.3 1.34 s 27.5s qq 19 1.34 s 1.47 m 16.1 q 1.31 16.7 q 169.80 s qq 6.00 brs ss 98.2 16.3 5.99 97.4 d 30 1.16 ssbrs 16.66 ss 1.14 30 21 1.16 16.66 1.28 17.49 1.14 16.3d qq 20 162.5 qq s 1.28 134.017.49 s 163.6 s 22 6.30 dd (0.9, 1.7) 123.2 d 7.48 t (1.1) 150.9 d 6.29 brs 122.7 d 1′ 169.6 s 2.00 ss 20.7 qq 21 5.99 brs 169.80 s brs 98.2 d 169.8170.8 1′ 23 169.6 20.7 170.8s ss 168.997.4 ss d 2.00 6.02 t (1.1) 103.5 d6.00 22 6.30 dd (0.9, 1.7) 123.2 d 7.48 t (1.1) 150.9 d 6.29 brs 122.7 d 2′ 2.10 ss s 20.9 qqq 169.82 2.08 1.48 33.4 1.39 s 33.9 s ss 33.6 21.1 2′ 28 2.10 20.9 169.82 q ss 1.35 2.08 21.1q qq 231.56 6.02 t (1.1)s 103.523.3 d s 27.5 2933 s 23.4168.9 q s 1.64 q 1.34 s 169.8 3-OCH 3.67 ss 52.3 3-OCH 3.67 s 52.3q qq 28 1.48 s 33.4 q 1.39 s 33.9 q 1.35 33.6 q 30 1.16 s 16.66 q 1.28 s 17.49 q 1.14 s 16.3 q 33 3.45 ss 56.9 qq 23-OCH 3.45 56.9 23-OCH 0 29 1.56 s 23.4 q 1.64 s 23.3 q 1.34 s 27.5 q 1 169.6 s 2.00 s 20.7 q 170.8 s Position Position

302.10 1.16 were s 16.66 1.28 s 17.49 q s q 11H20 aa Assignments s 20.9 q1q 169.82 2.08 spectroscopic s 16.3 21.1 1H based on HMQC, HMBC, and NOESY data. Assignments were based on HH COSY, COSY, HMQC, HMBC, ands1.14 NOESY spectroscopic data. 1′ 169.6 s 2.00 s 20.7 q 3-OCH3 3.67 s 170.8 s 52.3 2′ 2.10 s 20.9 q 169.82 s 2.08 s 21.1 q 23-OCH3 3.45 s 56.9 q 3-OCH 3 3.67 s 52.3 q a Assignments 1 H-1 H COSY, HMQC, (A) (B) were based on HMBC, and NOESY spectroscopic data. (A)23-OCH (B) 3 3.45 s 56.9 q a

q q

Assignments were based on 1H-1H COSY, HMQC, HMBC, and NOESY spectroscopic data.

(A)

(B)

1H1 11 111H2. Key HMBC ) and COSY ( (( 1H Key (( ( )) and 2. and Figure Key HMBC HMBC Figure 2. 2.Figure and HHH COSY COSY compound 1. compound compound 1. 1.

) (A) and and NOENOE ( )) (A) (A) and NOE

of )) (B) (( ) (B) correlations of (B) correlations correlations of

21,23-dihydro-23-methoxy-21-oxonomilin (2) was obtained as colorless crystals. Its molecular

21,23-dihydro-23-methoxy-21-oxonomilin (2) colorless crystals. Its molecular 21,23-dihydro-23-methoxy-21-oxonomilin (2) was obtained as colorless crystals. Its molecular molecular formula was established as C29H32O11 (m/z 561.2346 [M +obtained H]+, calcd.: as 561.2336) by the HRFABMS. 21,23-dihydro-23-methoxy-21-oxonomilin (2) was was obtained as colorless crystals. Its + + Compound 2 was suggested to have a similar structure to 1 except for the absence of a β-substituted formula was established as H 561.2346 [M 561.2336) the formula was was established established as asCC C2929H H3232O O1111 (m/z (m/z 561.2346 [M H] calcd.: 561.2336) by the HRFABMS. formula (m/z 561.2346 [M+++H] H],+, ,calcd.: calcd.: 561.2336)by bygroup the HRFABMS. HRFABMS. 29 and 32 O11 γ-hydroxybutenolide group the presence of an α-substituted γ-methoxybutenolide Compound 2 was suggested to have a similar structure to 1 except for the absence of a β-substituted Compound 2 was suggested to have a similar structure to 1 except for the absence of a β-substituted Compound 2 was suggested to have a similar structure to 1 except for the absence of a β-substituted γ-hydroxybutenolide group and presence an γ-methoxybutenolide group γ-hydroxybutenolidegroup groupand and the presence of an α-substituted α-substituted γ-methoxybutenolide group γ-hydroxybutenolide thethe presence of anof α-substituted γ-methoxybutenolide group because of the HMBC correlations from H-17 (δH 5.32 (t)) to an ester carbonyl carbon (δC 169.8 (s)) and a trisubstituted olefin (δC 134.0 (s), 150.9 (d)). Therefore, the structure of 2 was established, as shown in Figure 1. The configuration at C-23 was not determined. 21,23-dihydro-21-hydroxy-23-oxonomilinic acid methyl ester (3), an amorphous solid, possessed the molecular formula C29 H38 O12 (m/z 601.2264 [M + Na]+ , calcd.: 601.2261). The 1 H- and 13 C-NMR spectra showed three methyls observed as singlet including acetyl and methoxy groups (δH 1.10 (s),

2

α 3.11 β 3.24

3 4 5 6α 6β 7 8 9 10

2.62 α 2.60 β 2.78

2.44 α 1.70 β 1.66 α 1.29 β 2.02

11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 1′ 2′ 3-OCH3 23-OCH3

3.70 5.34 1.11 1.34 5.99 6.30 1.48 1.56 1.16 2.10

a

Molecules 2017, 22, 907 dd (6.4, 15.8) brd (15.8)

35.3

t

α 2.98 β 3.50

dd (7.1, 15.8) dd (1.2, 15.8)

36.0

t

A 2.35 B 2.82

m m

35.4

t

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s correlations from H-17 169.5 s 172.0 carbon s because of the169.1 HMBC (δH 5.32 (t)) to an ester carbonyl (δC 169.8 (s)) 84.4 solefin (δC 134.0 (s), 150.9 84.7 and a trisubstituted (d)). sTherefore, the structure74.1 of 2 swas established, as dd (3.9, 15.8) 51.1 d 2.69 dd (3.5, 14.7) 51.7 d 2.01 m 53.0 shown Figure at C-2339.6 was not dd (3.9, in 15.8) 38.61. The t configuration α 2.51 dd (3.5, 14.7) t determined. α 2.46 dd (5.3, 14.9) 38.9 t Molecules 2017, 22, 907 of 10 t (15.8) β 3.10 t (14.7) 21,23-dihydro-21-hydroxy-23-oxonomilinic acidβ 2.80 methyl t (14.9) ester (3), an amorphous 4solid, s s 210.0 s possessed the 206.5 molecular formula C29H38O12 208.3 (m/z 601.2264 [M + Na]+, calcd.: 601.2261). The 1H- and 53.1 s 53.2 s 52.5 s 13C-NMR spectra showed three methyls observed as singlet including acetyl and methoxy groups (11.2) d 3.67 2.61 (s)),dda(2.4, 11.8) propyl 44.8 d H 1.34 2.14 (s), 1.35 brd (11.4) 44.4 (s)), d three oxymethine 1.14brd (s), 1.31 (s),44.3 2.08 (s), hydroxy (δ (s); δC 74.1 44.1 s 45.1 s s δC 74.1 (s)), three (δH 3.64 1.10 (s), (s), 5.33 1.14 (s), 1.31 (s), 2.08 (s), 3.67 (s)), a hydroxy propyl (δH 1.34 (s),(δ46.1 1.35 (s); (δ (d), 6.54 (brs); δ 52.5 (d), 76.3 (d), 78.6 (d)), a hemiacetal 6.00 (brs); δC 98.2 (d)), H C m H m 17.3 t α 1.63 17.44 t α 2.49 m 19.0 t oxymethine (δ H 3.64 (s), 5.33 (d), 6.54 (brs); δC 52.5 (d), 76.3 (d), 78.6 (d)), a hemiacetal (δH 6.00 (brs); an unprotonated oxycarbon olefin (δHm6.29 (brs); δC 122.7 (d), 163.6 (s)), m β 1.73 (δC 65.0 m(s)), a trisubstituted β 1.72 98.2m (d)), unprotonated (δC 65.0 a αtrisubstituted olefin (δH(s), 6.29 (brs);(s)). δC 122.7 (d), 32.0 t α four 1.16 oxycarbon 30.6 t 1.61 31.6 t 172.0 aδCketone (δCan210.0 (s)), ester mcarbonyls (δ(s)), 166.3 (s), 169.8m(s), 170.8 In the C dd (7.9, 13.0) β 2.10 (s)), four m ester carbonyls (δβC2.01 m 163.6 (s)), a ketone (δ C 210.0 166.3 (s), 169.8 (s), 170.8 (s), 172.0 (s)). In the HMBC experiment, the following correlations were observed: Me-18 (δH 1.10 (s))/C-12, C-13, 37.7 sthe following correlations 39.0 were s observed: Me-18 (δH 37.5 s HMBC 1.10 (s))/C-12, C-13, C-14 C-14 (δCexperiment, 65.0 (s)), (δC 78.6 (d)); Me-19 66.8 (δH 1.31 (s))/C-1 (δC 76.3 (d)), 65.0 C-17 s s 65.0C-5,s C-9, C-10; Me-28 (δ C 65.0 (s)), C-17 (δC 78.6 (d)); Me-19 (δH 1.31 (s))/C-1 (δC 76.3 (d)), C-5, C-9, C-10; Me-28 (δH 1.35 (s)) s (s)) and 52.6Me-29 d 3.95 m s (δH 1.14 52.5(s))/C-7 d (δH 1.35 (δH 1.34 (s))/C-4 (δC54.574.1 d(s)),3.64 C-5; Me-30 (δC 210.0 (s)), s(s))/C-4 (δC 74.1 (s)), C-5; 166.9 s and Me-29 (δ H165.5 1.34 Me-30 (δ H 1.14 (s))/C-7 (δC 166.3 210.0 (s)), C-8, C-14; C-8, C-9, C-14; H-2B (δH 2.82 (m)), 3-OCH3 (δH 3.67 (s))/C-3 (δC 172.0 (s)); H-15 (δH 3.64 C-9, (s))/C-16 brd (1.7) 78.1 d 5.32 t (1.1) 76.2 d 5.33 d (1.5) 78.6 d H-2B (δH (s)); 2.82 H-17 (m)), 3-OCH 3 (δH 3.67 (s))/C-3 (δC 172.0 (s)); H-15 (δH 3.64 (s))/C-16 (δC 166.3 (s)); H-17 (δ s 21.4 (δ q H 5.33 1.20 (s))/C-20 s (δC 163.6 20.4 (s)), q C-22 1.10 (δC 122.7 s (d)); H-22 21.4 (δqH 6.29 (brs))/C-21 C 166.3 (δ H 5.33 (s))/C-20 (δC 163.6 (s)), C-22 (δC 122.7 (d)); H-22 (δH 6.29 (brs))/C-21 (δC 98.2 (d)), C-23 s (d)), C-23 16.64 (δ q C 169.8 1.47 16.1 qcorrelations 1.31 s q (δC 98.2 (s)). mThe following were also 16.7 observed in the 1 H-1(δ HC 1 1 162.5 s 134.0 s 163.6 s 169.8 (s)). The following were also observed in the -11–H H- H COSY experiment: H-1 (δH COSY experiment: H-1 (δcorrelations H-5–H -6; H-9–H From the above data, 2brs 2 -12. H 6.54 (brs))–H-2A; brs 97.4 H-5–H d 169.80 s 2 6.00 98.2 d 6.54 (brs))–H-2A; 2-6; H-9–H2-11–H2-12. From the above data, compound 3 was similar to compound to 3-O-methyl acid [23] except dd (0.9, 1.7) 3 was 123.2similar d 7.48 t (1.1) 21,23-dihydro-23-hydroxy-21-oxonomilinic 150.9 d 6.29 brs 122.7 d 3-O-methyl 21,23-dihydro-23-hydroxy-21-oxonomilinic acid [23] for the of an 168.9 6.02 t (1.1) 103.5 d 169.8 s of aabsence for the absence of ansα-substituted γ-hydroxybutenolide group and except the presence β-substituted s 33.4 q 1.39 sgroup and 33.9the qpresence 1.35 s 33.6 q α-substituted γ-hydroxybutenolide a β-substituted γ-hydroxybutenolide group. The relative configuration was of determined by theγ-hydroxybutenolide NOESY experiment s The relative 23.4 q configuration 1.64 s was determined 23.3 q 1.34 the NOESY s 27.5 q group. by experiment (Figure 3). The (Figures 3). The16.66 configuration S because of q 1.28 of C-1 swas determined 17.49 q as 1.14 s the following 16.3 q NOE correlations: configuration of C-1 was determined as S because of the following NOE correlations: (δH 6.54 169.6 s 2.00 s Me-19 20.7 q (s)), Me-28 (δH 1.35 (s)), 170.8 H-1 (δH 6.54 (brs))/H-2B (δH 2.82 (m)), (δH 1.31 andsMe-29 H-1 (δH 1.34 (s)); s 169.82 s (δ2.08 s 21.1(δHq 1.34 (s)); H-2A (δH (brs))/H-2B (δH20.9 2.82 q(m)), (δ Me-19 (δH 1.31 (s)), Me-28 H0 1.35 (s)), and Me-29 H-2A (δH 2.35 (m))/H-9 the structure of H 2.14 (brd)); Me-19/Me-23.67(δH 3.67 s(s)). Therefore, 52.3 q (δH 2.14 (brd)); Me-19/Me-2′ H 3.67 (s)). Therefore, the structure of 3 was established 32.35 was(m))/H-9 established as shown 1. (δ Compound 3 was also an inseparable mixture of C-21 3.45 in Figure s 56.9 q as shown inepimers. Figure1 1. 1Compound 3 was also an inseparable mixture of C-21 hemiacetal epimers. hemiacetal

Assignments were based on H- H COSY, HMQC, HMBC, and NOESY spectroscopic data.

(A)

(B) 23 22

20

12

19

10

9

8

1 2

4

16

18

7 5

29

28

17 15

14

30 2'

21 13

11

1'

6

3

3-OMe

Figure 2. Key HMBC ( compound 1.

) and 1H-1Figure H COSY ( NOE ) (A)correlations and NOE ( 3. Key Figure 3. Key NOE correlations (

) of (B)compound correlations 3. of ) of compound 3.

21,23-dihydro-23-methoxy-21-oxolimonin (4) was obtained as an amorphous solid. Its molecular 21,23-dihydro-23-methoxy-21-oxolimonin (4)aswas obtained as an amorphous solid. Its molecular 21,23-dihydro-23-methoxy-21-oxonomilin (2) was obtained colorless crystals. Its molecular + , calcd.: formula was established as C27 H32 O10 (m/z 539.1891 [M + Na] 539.1894) by the HRFABMS. +, calcd.: 27 H 32 O 10 (m/z 539.1891 [M + Na] 539.1894) by the The formula was established as C + formula was established as C29H32O11 (m/z 561.2346 [M + H] , calcd.: 561.2336)−1by the HRFABMS. HRFABMS. The absorbance in the IR spectrum indicated hydroxy (3434 −1 cm ) and carbonyl (1750−1cm−1 ) groups. absorbance in the IR spectrum indicated hydroxy (3434 cm ) and carbonyl (1750 cm ) groups. The Compound 2 was suggested have a similar structure to 1 except for the absence of a β-substituted 1 H- and to 13 C-NMR The spectra indicated five methyl groups observed as singlets including methoxy 1H- and 13C-NMR spectra indicated methyl groups observed as singlets including methoxy γ-hydroxybutenolide group and the presence of anfive α-substituted γ-methoxybutenolide group group (δH 1.09 (s), 1.18 (s), 1.18 (s), 1.29 (s), 3.60 (s)), an oxymethylene (δH 4.46 (d), 4.74 (d); group (δH 1.09 (s), 1.18 (s), 1.18 (s), 1.29 (s), 3.60 (s)), an oxymethylene (δH 4.46 (d), 4.74 (d); δC 65.1 (t)) δC 65.1 (t)) three oxymethines (δH 4.03 (brd), 4.12 (s), 5.43 (t); δC 53.8 (d), 75.2 (d), 79.2 (d)), two three oxymethines (δH 4.03 (brd), 4.12 (s), 5.43 (t); δC 53.8 (d), 75.2 (d), 79.2 (d)), two unprotonated unprotonated oxycarbons (δC 65.7 (s), 80.3 (s)), an acetal (δH 5.77 (t); δC 102.5 (d)) a trisubstituted oxycarbons (δC 65.7 (s), 80.3 (s)), an acetal (δH 5.77 (t); δC 102.5 (d)) a trisubstituted olefin (δH 7.25 (t); olefin (δH 7.25 (t); δC 133.8 (s), 149.1 (d)), a ketone (δC 206.1 (s)), and three ester carbonyls (δC 166.0 (s), δC 133.8 (s), 149.1 (d)), a ketone (δC 206.1 (s)), and1 three ester carbonyls (δC 166.0 (s), 168.8 (s), 168.9 (s)) 1 H COSY correlations (Figure 4) suggested that 168.8 (s), 168.9 (s)) (Table 2). The HMBC and H1 1 (Table 2). The HMBC and H- H COSY correlations (Figure 4) suggested that compound 4 had a compound 4 had a similar structure to limonin [15] except for the lack of a furan ring and presence of similar structure to limonin [15] except for the lack of a furan ring and presence of an α-substituted an α-substituted γ-methoxybutenolide group. In the NOESY experiment, the following correlations γ-methoxybutenolide group. In the NOESY experiment, the following correlations were observed; were observed; Me-29/H-19β; H-19β/H-6β, Me-30; H-12β/H-17; H-6α/Me-28; H-5/H-9; H-9/Me-18; Me-18/23-OMe; Me-30/H-19α; H-19α/H-2α (Figure 4). Therefore, the structure of 4 was established, as shown in Figure 1. The configuration at C-23 was not established.

2′ 2.10 ss 2′ 2.10 3-OCH 3-OCH33 Molecules 2017, 22, 907 33 23-OCH 23-OCH

20.9 20.9

qq 3.45 3.45

ss

169.82 169.82

ss

56.9 56.9

qq

2.08 2.08 3.67 3.67

ss ss

21.1 21.1 52.3 52.3 5 of 10

qq qq

Assignments Assignments were were based based on on HH- H H COSY, COSY, HMQC, HMQC, HMBC, HMBC, and and NOESY NOESY spectroscopic spectroscopic data. data. Me-29/H-19β; H-19β/H-6β, Me-30; H-12β/H-17; H-6α/Me-28; H-5/H-9; H-9/Me-18; Me-18/23-OMe; Me-30/H-19α; Molecules 2017, 22, 907 H-19α/H-2α (Figure 4). Therefore, the structure of 4 was established, as shown in5 of 10 (A) (B) (A) (B) Figure 1. The configuration at C-23 was not established. aa

11

(A)

11

(B)

1 111H-111HFigure 4. Key HMBC ) and COSY(( ( 2. HMBC (( ( )) and COSY and Figure 4. Key HMBC Figure 2. Key and H-1H HH COSY compound 4. compound 1. compound 4. 1.

(A) and and NOE NOE ((( )) )(A) (A) and NOE

) (B) correlations of of )) (B) correlations (B) correlations of

Table 2. 1H- (600 MHz) and 13C-NMR (150 MHz) spectra data of compounds 4 (in CDCl3) and 5 (in

21,23-dihydro-23-methoxy-21-oxonomilin (2) obtained as colorless Its 21,23-dihydro-23-methoxy-21-oxonomilin (2) was was obtained ascompounds colorless crystals. crystals. Its3molecular spectra data of 4 (in CDCl )molecular and Table 2. 1 H- (600 MHz) and 13 C-NMR (150 MHz) (CD3)2CO) a. a. ++, calcd.: 561.2336) by the HRFABMS. formula as C 29 H 32 O 11 (m/z 561.2346 [M + H] 5 (in was (CD3established )established CO) formula was as C 29 H 32 O 11 (m/z 561.2346 [M + H] , calcd.: 561.2336) by the HRFABMS. 2

4 5 Compound aa similar to for of Position Compound 22 was was suggested suggestedδHto to have have similar structure structure toδ11H except except for the the absence absence of aa β-substituted β-substituted Mult. (J in Hz) δC Mult. (J in Hz) δC 4 5 γ-hydroxybutenolide group and the presence of an α-substituted γ-methoxybutenolide group γ-hydroxybutenolide group and the presence of an α-substituted γ-methoxybutenolide group Position 1β 4.03 brd (4.2) 79.2 d 4.27 brd (4.1) 80.1 d δH 2α

a

t

δH 2.87 4.27 2.73 2.87 2.73

(J in Hz)36.5 dd Mult. (1.5, 16.7) (4.1) dd (4.1,brd 16.7) dd (1.5, 16.7)170.0 dd (4.1, 16.7) 80.7 dd (3.6, 15.2) 59.8 dd (3.6, 15.2) 37.1 dd (3.6, 15.2) t (15.2) dd (3.6, 15.2) t (15.2) 208.2 51.7 m 48.2 46.7 m m 18.8 m m m m 29.0 m m m 39.9 67.6 s s 55.2 167.3 d (1.2) d (1.2) 76.6 s s 19.7 d (13.5) 65.7 d (13.5) d (13.5) d (13.5)

t δC 80.1 s 36.5 s d170.0 80.7 t 59.8 37.1 s s208.2 d51.7 s 48.2 t 46.7 18.8

4.032β 2.66 3 s 2.98 4 s 5 d 2.60 6α t 2.40 2.22 2.60 6β 3.16 2.47 2.40 s 3.16 2.84 7 8 s 9 d 2.83 s 2.83 2.5010 11α t 1.96 1.7711β 1.96 2.07 2.07 12α 1.40 ddd (7.3, 9.1, 14.4) 28.7 t 1.44 t 1.4012β ddd (7.3, 9.1, 14.4)m 28.7 t 1.44 29.0 2.24 2.06 2.24 m 2.06 13 38.6 s s 38.6 s 39.9 14 65.7 s s 65.7 s 67.6 4.12 s s d55.2 4.1215 53.8 53.8 d d 4.19 4.19 16 s167.3 166.0 166.0s s 5.43t (1.5) t (1.5) 75.2 75.2 d d 5.35 d76.6 5.4317 5.35 1.18 s s q19.7 1.1818 20.0 20.0 q q 1.27 1.27 4.4619α d (13.2) d (13.2) 65.1 65.1 t t 4.65 4.65 4.46 t 65.7 4.7419β d (13.2) d (13.2) 4.97 4.74 4.97 133.8 133.8s s 20 129.6 s129.6 168.8 168.8s s 21 173.1 s173.1 7.25 t (1.5) 149.1 d 7.85 dd (1.7, 2.9) 154.4 22 7.25 t (1.5) 149.1 d 7.85 dd (1.7, 2.9) 154.4 d 5.77 t (1.5) 102.5 d 4.99 dd (1.7, 3.5) 71.9 23 5.77 t (1.5) 102.5 d 4.99 dd (1.7, 3.5) 71.9 t 1.29 s 30.2 q 1.13 s 21.8 1.29 s s s s 21.8 q30.3 1.1828 21.2 30.2 q q 1.13 1.24 1.18 s s s s 30.3 q18.2 1.0929 17.8 21.2 q q 1.24 1.18 1.09 s s s 18.2 q 3.6030 57.8 17.8 q q 1.18 s1 57.8 q 23-OCH3 3.60 1 Assignments were based on H- H COSY, HMQC, HMBC, and NOESY spectroscopic data.

1β 2α 2β 3 4 5 6α 6β 7 8 9 10 11α 11β 12α 12β 13 14 15 16 17 18 19α 19β 20 21 22 23 28 29 30 23-OCH3 a

Mult. Hz)(1.7, 16.8)δC 2.66 (J indd 35.6 brd (4.2)dd (4.2, 16.8)79.2 d 2.98 dd (1.7, 16.8) 35.6 168.9t dd (4.2, 16.8) 80.3 168.9 60.4 s 2.22 m 80.3 s 2.47 dd (3.5, 14.6) 36.3 m 60.4 d 2.84 dd (14.6, 15.8) dd (3.5, 14.6) 36.3 t 206.1 dd (14.6, 15.8) 206.1 51.1 s 2.50 dd (3.3, 12.7)51.1 48.0 s dd (3.3, 12.7) 48.0 45.8 d 1.77 (2H) 45.8 18.5 s (2H) 18.5 t

d t s s d t s s d s t t s s d s d q t s s d t q q q

Assignments were based on 1H-1H COSY, HMQC, HMBC, and NOESY spectroscopic data.

21,23-Dihydro-21-oxolimonin (5) was obtained as an amorphous solid. Its molecular formula was established as C26 H30 O9 (m/z 487.1967 [M + H]+ , calcd.: 487.1968). Compound 5 was similar to 4 except for the absence of an acetal carbon and the presence of an oxymethylene at C-23. The structure was confirmed by 2D NMR spectra including HSQC, HMBC, 1 H-1 H COSY, and NOESY experiments. Therefore, the structure of 5 was established, as shown in Figure 1. Macrophages may be a potential therapeutic target for inflammatory diseases [24]. Activated macrophages release pro-inflammatory mediators, such as NO, reactive oxygen species, interleukin-1β,

Molecules 2017, 22, 907

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tumor necrosis factor-α, and other inflammatory mediators, which play important roles in biological defense. However, the overexpression of these mediators had been implicated in diseases such as osteoarthritis, rheumatoid arthritis, and diabetes because the increased production of pro-inflammatory mediators induces severe or chronic inflammation [24]. Isolated compounds (1–12), and NG monomethyl-L-arginine, acetate (L-NMMA), which is a NO synthase inhibitor and was used as a positive control, were evaluated for macrophage activation by the inhibitory assay of NO production in RAW264.7 mouse macrophages stimulated by lipopolysaccharide (LPS). Compounds 1–7, and 9–12 did not exhibit cytotoxicity at 1–30 µM (Table 3). Of these, compound 2 (IC50 25.4 µM) showed a comparable inhibitory effect on NO production to L-NMMA (IC50 23.9 µM) (Table 3). Compounds 3, 5, 8, 11 and 12 showed some inhibitory activities (produced NO ratio 3: 80.3%; 5: 83.2%; 8: 76.0%; 11: 78.1%; 12: 73.9% at 30 µM). The other limonoids did not exhibit inhibitory effects on NO production. These results suggested that compound 2 has potential as an anti-inflammatory disease agent. Table 3. Inhibitory effects of NO production by limonoids from peels of Citrus reticulata. Inhibitory Ratio of NO % (Cell Viability %) a,b Compound

1 µM

3 µM

10 µM

30 µM

IC50 (µM)

1

109.9 ± 2.8 (99.1 ± 1.3)

110.9 ± 1.7 (102.5 ± 1.8)

104.8 ± 2.2 (103.7 ± 0.7)

106.6 ± 2.5 (99.3 ± 1.3)

>30

2

95.2 ± 1.6 (101.3 ± 1.5)

89.8 ± 2.1 * (98.7 ± 1.4)

80.4 ± 2.1 ** (99.1 ± 0.3)

39.3 ± 0.5 ** (94.5 ± 0.4)

25.4

3

99.4 ± 0.8 (96.8 ± 0.2)

99.4 ± 2.3 (94.5 ± 0.3)

94.1 ± 1.9 (93.2 ± 0.4)

80.3 ± 1.2 ** (93.4 ± 0.6)

>30

4

96.2 ± 7.9 (102.6 ± 0.4)

94.0 ± 2.7 (97.0 ± 0.3)

100.8 ± 1.2 (95.1 ± 0.2)

95.2 ± 3.4 (91.7 ± 0.5)

>30

5

99.5 ± 5.6 (97.9 ± 1.9)

100.6 ± 4.7 (97.9 ± 0.2)

93.8 ± 1.6 (99.5 ± 0.5)

83.2 ± 5.6* (96.6 ± 0.2)

>30

6

102.3 ± 4.4 (100.1 ± 0.3)

93.4 ± 5.7 (95.6 ± 0.5)

90.9 ± 3.1 (97.2 ± 0.9)

86.0 ± 5.5 (99.3 ± 0.9)

>30

7

99.8 ± 1.4 (102.3 ± 1.5)

99.0 ± 3.0 (106.3 ± 2.2)

96.8 ± 2.5 (104.2 ± 1.4)

93.9 ± 4.0 (100.2 ± 1.4)

>30

8

97.2 ± 1.1 (98.1 ± 2.4)

94.7 ± 2.4 (97.6 ± 0.7)

90.9 ± 1.6 ** (92.0 ± 0.5)

76.0 ± 2.2 ** (89.3 ± 0.5)

>30

9

94.3 ± 4.3 (101.4 ± 0.7)

85.2 ± 6.4 (97.7 ± 0.6)

91.3 ± 2.8 (101.3 ± 1.2)

89.9 ± 3.2 (97.6 ± 2.1)

>30

10

95.0 ± 4.2 (104.7 ± 2.3)

96.8 ± 1.3 (101.8 ± 1.0)

97.7 ± 2.2 (98.9 ± 0.5)

86.6 ± 1.6 (98.6 ± 1.8)

>30

11

103.6 ± 3.1 (95.9 ± 0.1)

93.2 ± 3.2 (100.1 ± 0.4)

84.7 ± 3.2 * (99.3 ± 1.5)

78.1 ± 3.4 ** (100.4 ± 0.6)

>30

12

106.6 ± 4.1 (100.7 ± 0.3)

92.6 ± 3.2 (98.1 ± 1.0)

88.4 ± 5.5 (98.3 ± 1.6)

73.9 ± 3.4 ** (98.3 ± 1.2)

>30

L -NMMA c

93.3 ± 2.2 (101.5 ± 0.9)

91.4 ± 0.8 (101.9 ± 0.4)

68.9 ± 4.5 ** (98.5 ± 0.9)

43.1 ± 1.1 ** (109.4 ± 0.5)

23.9

a Each value represents the mean ± standard error (S.E.) of four determinations; b Significant differences from the vehicle control group shown as * p < 0.05 and ** p < 0.01; c Positive control.

3. Experimental 3.1. General Experimental Procedure The following chemicals and reagents were purchased: fetal bovine serum (FBS) from Invitrogen Co. (Carlsbad, CA, USA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) from Sigma-Aldrich Japan Co. (Tokyo, Japan), Dulbecco’s modified Eagle’s medium (D-MEM), and antibiotics from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals and reagents were of analytical grade. Melting points were determined on a Yanagimoto micro-melting point apparatus and were uncorrected.

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Optical rotations were measured with a JASCO DIP-1000 digital polarimeter. IR spectra were recorded on a Perkin-Elmer 1720X FTIR spectrophotometer. UV spectra were measured on a HITACHI U-2000 spectrometer. 1 H- (600 MHz) and 13 C- (150 MHz) NMR spectra were recorded on an Agilent vnmrs600 with tetramethylsilane as the internal standard. HR-FAB-MS was recorded on a JEOL JMS-7000 mass spectrometer. Silica gel (70–230 mesh, Merck) and silica gel 60 (230–400 mesh, Nacalai tesque, Inc., Kyoto, Japan) were used for column chromatography and medium-pressure liquid chromatography, respectively. HPLC was carried out on an ODS column (Cosmosil 5C18 -MS-II column (Nacalai Tesque, Inc., Kyoto, Japan), 25 cm × 20 mm i.d., 5 µm particle size) at 35 ◦ C with MeCN/H2 O (1:1 (System I), 45:55 (System III), 2:3 (System IV), 3:7 (System V), flow rate 4.0 mL/min), Cosmosil PAQ (Nacalai Tesque, Inc., Kyoto, Japan), 25 cm × 20 mm i.d., 5 µm particle size) at 35 ◦ C (MeCN/H2 O, flow rate 4.0 mL/min, (1:1) (System II)). 3.2. Plant Material The fruits of C. reticulata were produced in Wakayama prefecture (Japan) in 2013. A voucher specimen of the peels of Citrus reticulata was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences. 3.3. Extraction and Isolation The peels of Citrus reticulata fruits (dry weight 4952 g), produced in Wakayama, were subjected to extraction with MeOH under reflux (15 L, 3 days, 3 times). The MeOH extract (280 g) was then partitioned between AcOEt and H2 O (9 L/9 L, 4 times). The AcOEt-soluble fraction (280 g) was subjected to SiO2 column chromatography (CC) (SiO2 (3.5 kg); hexane/AcOEt (5:1, 1:1, and 0:1), and AcOEt:MeOH (1:1, and 0:1) in increasing order of polarity) resulting in twenty fractions (Fr. A–T). Fr. J (11 g), eluted with AcOEt, was subjected to SiO2 CC to yield 10 fractions (SiO2 (300 g); hexane/AcOEt (5:1, 3:1, 1:1, 1:5, 1:10, 1:20, and 0:1), and AcOEt:MeOH (5:1, and 0:1) in increasing order of polarity), J1–J10. Preparative HPLC (System III) of J6 (237 mg), eluted with hexane/AcOEt (1:10), gave 5 (4.5 mg; tR 24.6 min). Fr. K (11 g), eluted with AcOEt, was subjected to SiO2 CC to yield 11 fractions (SiO2 (270 g); hexane/AcOEt (1:1 and 0:1), and AcOEt:MeOH (1:1, and 0:1) in increasing order of polarity), K1–K11, followed by SiO2 CC of K5 (1 g), eluted with AcOEt, to yield 8 fractions (SiO2 (60 g); AcOEt and MeOH), K5-1–K5-8. Preparative HPLC (System IV) of K5-2 (215 mg), eluted with AcOEt, gave 12 fractions, K5-2-1–K5-2-12. K5-2-9 was identified as 6 (26 mg; tR 47.4 min). Preparative HPLC (System V) of K5-2-2 (11 mg) gave 7 (5.4 mg; tR 47.7 min). Preparative HPLC (System V) of K5-2-3 (20 mg) gave 8 (3.0 mg; tR 58.6 min) and 9 (1.3 mg; tR 49.0 min). Preparative HPLC (System V) of K5-2-6 (10 mg) gave 10 (1.9 mg; tR 91.8 min). Preparative HPLC (System V) of K5-2-8 (7.9 mg) gave 3 (1.1 mg; tR 92.0 min). Preparative HPLC (System IV) of K5-3 (397 mg) gave 8 (3.7 mg; tR 58.8 min). Fr. K6 (3 g), eluted with AcOEt, was subjected to SiO2 CC, to yield 8 fractions (SiO2 (60 g); AcOEt and MeOH), K6-1–K6-8. Preparative HPLC (System I) of K6-2 (376 mg), eluted with AcOEt, gave 10 fractions, K6-2-1–K6-2-10. Preparative HPLC (System V) of K6-2-1 (135 mg) gave 1 (14 mg; tR 104.5 min) and 2 (2.4 mg; tR 186.8 min). Preparative HPLC (System IV) of K6-2-3 (4.2 mg) gave 4 (2.5 mg; tR 51.6 min).Preparative HPLC (System II) of K6-2-3 (1120 mg), eluted with AcOEt, gave 15 fractions, K6-3-1–K6-3-15. K6-3-9 was identified as 11 (42 mg; tR 27.8 min). Preparative HPLC (System V) of K6-3-4 (59 mg) gave 1 (12 mg) and 12 (5.5 mg; tR 92.6 min). 21,23-Dihydro-21-hydroxy-23-oxonomilin (1). Colorless crystals, m.p. 200–201 ◦ C; [α]21 D −100.0 (c = 0.083, EtOH); IR νmax KBr cm−1 : 3466, 2921, 2852, 1747, 1733, 1717, 1596, 1378, 1232, 1025; UV λmax EtOH nm (logε): 211.5 (3.82); FAB-MS m/z: 547 [M + H]+ , 569 [M + Na]+ ; HR-FAB-MS m/z: 547.2178 (calcd. for 547.2179: C28 H35 O11 ). 21,23-Dihydro-23-methoxy-21-oxonomilin (2). Colorless crystals, m.p. 208–210 ◦ C; [α]12 D −15.7 (c = 0.075, EtOH); IR νmax KBr cm−1 : 3425, 2925, 1751, 1716, 1374, 1120, 1027; UV λmax EtOH nm (logε): 212.5

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(3.72); FAB-MS m/z: 561 [M + H]+ , 583 [M + Na]+ ; HR-FAB-MS m/z: 561.2346 (calcd. for 561.2336: C29 H37 O11 ). 21,23-Dihydro-21-hydroxy-23-oxonomilinic acid methyl ester (3). Amorphous solid; [α]25 D −54.7 (c = 0.16, KBr − 1 EtOH); IR νmax cm : 3453, 2924, 2853, 1743, 1596, 1442, 1382, 1261, 1028; UV λmax EtOH nm (logε): 203.5 (3.80); FAB-MS m/z: 601 [M + Na]+ ; HR-FAB-MS m/z: 601.2264 (calcd. for 601.2261: C29 H38 NaO12 ). KBr 21,23-Dihydro-23-methoxy-21-oxolimonin (4). Amorphous solid; [α]23 D −15.7 (c = 0.12, EtOH); IR νmax cm−1 : 3434, 2922, 2852, 1750, 1713, 1632, 1456, 1384, 1034; UV λmax EtOH nm (logε): 206.5 (3.43); FAB-MS m/z: 539 [M + Na]+ ; HR-FAB-MS m/z: 539.1891 (calcd. for 539.1894: C27 H32 NaO10 ). KBr cm−1 : 2969, 21,23-Dihydro-21-oxolimonin (5). Amorphous solid; [α]25 D −53.9 (c = 0.10, EtOH); IR νmax EtOH 1749, 1712, 1457, 1361, 1265, 1095; UV νmax nm (logε): 206.0 (3.76); FAB-MS m/z: 487 [M + H]+ ; HR-FAB-MS m/z: 487.1967 (calcd. for 487.1968: C26 H31 O9 ).

3.4. Cell Culture RAW264.7 cells were grown in D-MEM medium supplemented with 10% FBS and antibiotics (100 units/mL penicillin G sodium salt and 100 µg/mL streptomycin sulfate). Cells were incubated at 37 ◦ C in a 5% CO2 humidified incubator. 3.5. Cytotoxicity Assay Cytotoxicity assay was performed according to a method reported previously [25] Briefly, RAW264.7 cells (5 × 104 cells in 100 µL) were seeded onto a 96-well microplate and incubated for 24 h. DMEM containing test samples (100 µL total volume, final concentration of 30, 10, 3, or 1 µM) dissolved in DMSO (final concentration 0.2%) was added. After treatment for 24 h, MTT solution was added. After a 3-h incubation, 20% sodium dodecyl sulfate in 0.1 M HCl was added to dissolve the formazan produced in the cells. The absorbance of each well was read at 570 nm using a microplate reader. The optical density of vehicle control cells was assumed to be 100%. 3.6. Inhibitory Assay of NO Production An inhibitory assay of NO production was performed according to a method reported previously [26] with slight modifications. Briefly, RAW264.7 cells (5 × 104 cells in 100 µL) were seeded into a 96-well microplate and incubated for 24 h. DMEM containing test samples (100 µL total volume, final concentration of 30, 10, 3, or 1 µM) dissolved in DMSO (final concentration 0.2%), and LPS (final concentration of 5 µg/mL), was added. After treatment for 24 h, the supernatant of culture medium was transferred to another 96-well microplate, and then 50 µL of 0.15% N-(1-naphtyl)ethylenediamine in H2 O, and 1.5% sulfanilamide in 7.5% phosphoric acid were added. After incubation for 30 min, the absorbance of each well was read at 570 nm using a microplate reader. The optical density of vehicle control cells was assumed to be 100%. 4. Conclusions In this study, we isolated five new compounds and elucidated their structures. They were limonoids having β-substituted γ-hydroxybutenolide (1, 3), α-substituted γ-methoxybutenolide (2, 4), and α-substituted butenolide (5) groups. Compounds 1 and 3 were inseparable mixtures of C-21 hemiacetal epimers. In NO inhibitory assay, compound 2 (IC50 25.4 µM) possessed a comparable inhibitory effect on NO production to L-NMMA (IC50 23.9 µM) without cytotoxicities. These results suggested that compound 2 has potential as an anti-inflammatory disease agent. Supplementary Materials: Supplementary materials are available online. Acknowledgments: We are grateful to M. Fujitake and K. Minoura of this university for MS and NMR measurements.

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Author Contributions: T.K., T.Y. and R.T. conceived and designed the experiments; T.K., Y.U., Y.H., C.F, T.F. and R.T. performed the experiments; T.K., Y.U., Y.H. and C.F analyzed the data; T.K. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Not available. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).