Prenylated Phenolic Compounds from the Leaves of Sabia limoniacea

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Prenylated Phenolic Compounds from the Leaves of Sabia limoniacea and Their Antiviral Activities against Porcine Epidemic Diarrhea Virus Hyo-Moon Cho,† Thi-Kim-Quy Ha,† Lan-Huong Dang,† Ha-Thanh-Tung Pham,† Van-On Tran,‡ Jungmoo Huh,† Jin-Pyo An,† and Won-Keun Oh*,†

Downloaded via TULANE UNIV on March 20, 2019 at 01:50:33 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea ‡ Department of Botany, Hanoi University of Pharmacy, Hanoi, Vietnam S Supporting Information *

ABSTRACT: Porcine epidemic diarrhea virus (PEDV), a serious swine epidemic, has been rampant in Asia since the 1990s. Despite the widespread use of PEDV vaccines, the occurrence of PEDV variants requires the discovery of new substances that inhibit these viruses. During a search for PEDV inhibitory materials from natural sources, seven new sabphenosides (1−7) and a new flavonoid (8), as well as eight known phenolic compounds (9−16), were obtained from the leaves of Sabia limoniacea. The structural determination of the new phenolic derivatives (1−8) was accomplished using comprehensive spectroscopic methods. Their absolute configurations were assigned by a combination of the ECD exciton chirality method following selective reduction and calculation of their ECD spectra. The bioactivities of the isolated compounds were measured based on their abilities to inhibit viral replication of PEDV. Among the test compounds, 15 and 16 exhibited the most promising antiviral activities, with IC50 values of 7.5 ± 0.7 μM and 8.0 ± 2.5 μM against PEDV replication. This study suggests that compounds 15 and 16 could serve as new scaffolds for the treatment of PEDV infection through the inhibition of PEDV replication.

C

livestock industry.6 The recent PEDV outbreak in the United States, Canada, and Mexico caused serious damage and resulted in an approximately 10% reduction in the number of pigs raised.7,8 However, current commercial PEDV vaccines are not sufficient, and the use of the antiviral agents used in humans for the treatment of pigs is limited due to concerns about the occurrence of resistant CoVs in humans. Thus, the discovery of a natural product-derived viral therapeutic substance that acts on PEDV is a good approach for avoiding PEDV damage in swine husbandry. Sabia limoniacea Wall. ex Hook. f. & Thomson (www.theplantlist.org) (synonym Androglossum reticulatum Champ. ex Benth), a tree belonging to the Sabiaceae family, has broad leaves and grows approximately 10 m high. S. limoniacea is commonly found throughout tropical and southern Asia, such as in Vietnam, India, and China. However, only a few previous

oronaviruses (CoVs) are envelope viruses belonging to the Coronaviridae family, and they have a positive strand RNA genome and a nucleocapsid with helical symmetry.1 CoVs have the largest genome among known RNA viruses, and their genomes range from approximately 26 to 32 kilobases.2 CoV infections occur in a broad range of hosts, including mammalian and avian species, and impact the gastrointestinal, upper respiratory, hepatic, and central nervous systems.3 In humans and birds, CoVs primarily cause upper respiratory tract infections, while in pigs and bovines, coronaviruses are a major cause of enteric infections that result in serious economic losses.4 Porcine epidemic diarrhea virus (PEDV), the pathogenic CoV that infects the epithelial cells of porcine intestine, causes severe acute enteritis, blocking absorption and causing serious mucous membrane degeneration, which results in severe watery diarrhea in young pigs.5 Infection by PEDV is one reason for high mortality in piglets (almost 100% mortality is observed when this infection occurs in one-week-old piglets), which can cause serious economic losses in the © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 30, 2018

A

DOI: 10.1021/acs.jnatprod.8b00435 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

studies on the genus Sabia have reported biologically inactive flavonoids and prenylated phenolic compounds as chemical constituents.9,10 Although there is a record in the Chinese Ethnic Pharmacopeia of this species being used to treat icteric hepatitis, hemostasis, and inflammation, there have been no scientific papers on the biological activity and secondary metabolites from S. limoniacea. Herein, the purification and structural elucidation of seven new prenylated phenolics, 1−7, sabphenosides E−K, a new flavonoid, 8, and eight known compounds, 9−16, from S. limoniacea leaves are reported. The purified compounds were also evaluated for their antiviral activities against PEDV replication. Among the tested compounds, sabphenols A (15) and B (16) showed the strongest PEDV inhibitory activities. To examine their PEDV inhibitory activities in cells, the cytopathic effects (CPEs) of the selected compounds were also measured using PEDVinfected Vero cells.

molecular formula C24H34O11. The presence of hydroxy (3350 cm−1) and carbonyl (1730 cm−1) groups was confirmed by its IR spectrum. The 1H and 13C NMR data (Tables 1 and 3) displayed an aromatic methine (δH 6.62, 1H, s; δC 118.0), an oxymethine (δH 4.96, 1H, s; δC 69.9), two oxymethylenes at [δH 3.33 (1H, dd, J = 16.8, 8.0 Hz) and 3.44 (1H, d, J = 16.8, 8.0 Hz); δC 24.6] and [δH 3.32 (1H, dd, J = 15.2, 7.2 Hz) and 3.57 (1H, d, J = 15.2, 7.2 Hz); δC 27.8], two vinylic groups at (δH 5.08, 1H, t, J = 5.6 Hz; δC 123.5) and (5.44, 1H, t, J = 7.2 Hz; 122.4), and three methyl groups (δH 1.62, 1.63, 1.70; δC 25.6, 13.7, 17.8). A carboxylic carbon at δC 174.7 and an oxygenated secondary carbon at δC 69.9 were also observed. A β-glucopyranosyl moiety was identified by the large anomeric J-coupling (δH 4.37, 1H, d, J = 8.0 Hz; δC 106.3) and five proton resonances in the aliphatic region. Collectively, these results suggested that compound 1 is a prenylated phenolic glucoside.10 Further examination of the 2D NMR data of compound 1 (Figure 1) indicated that it is structurally similar to known compound 12.10 The only difference in the NMR spectra of 1 and 12 is that the 5″-methyl group of 12 is replaced by a hydroxymethyl group in 1. These conclusions were confirmed via the HMBC cross-peaks from H-1⁗ to C-4 (δC 142.7) and from H-5″ to C-3″ (δC 132.1) and C-4″ (δC 13.7) in 1. The Δ2″(3″) olefin geometry in 1 was defined as E based on the ROESY correlation of H-2″ with H-5″. The absolute configuration at the benzylic C-7 was elucidated by

■

RESULTS AND DISCUSSION Seven new sabphenosides, 1−7, and a new flavonoid, 8, were obtained from a 70% ethanol extract of S. limoniacea leaves using various chromatographic techniques. Compound 1 was obtained as a pale yellow, amorphous powder with [α]25 D −68 (c 0.1, MeOH). Its negative HRESIMS data showed an [M − H]− ion at m/z 497.2036 (calcd for C24H33O11, 497.2028), which is in accordance with the B



Article

Table 1. 1H NMR Spectroscopic Data for Compounds 1−4 pos. 1 2 3 4 5 6 7 8 1′

2′ 3′ 4′ 5′ 1″

2″ 3″ 4″ 5″ 5″COH HMG 1‴ 2‴ 3‴ 4‴

1a

2a

3a

4c

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

6.62, s 4.96, s

6.59, s 4.75, s

6.56, s 4.64, s

pos. 6‴ Glc 1⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗

6.60, s 4.79, s

3.33, dd (16.8, 3.31, dd (15.2, 3.28, dd (16.0, 8.0)e 8.0)e 6.5)e 3.44, dd (16.8, 3.60, dd (15.2, 3.67, dd (16.0, 8.0)e 8.0)e 6.5)e 5.08, t (5.6) 5.10, t (7.2) 5.12, br t

3.31, dd (14.3, 8.5)e 3.55, dd (14.3, 8.5)e 5.12, t (7.0)

1.62, s 1.70, s 3.32, dd (15.2, 7.2)e 3.57, dd (15.2, 7.2)e 5.44, t (7.2)

1.62, s 1.70, s 3.35, dd (15.2, 8.0)e 3.57, dd (15.2, 8.0)e 5.61, t (7.2)

1.62, s 1.69, s 3.20, dd (16.0, 6.4)e 3.53, dd (16.0, 6.4)e 5.38, t (7.0)

1.62, s 1.70, se 3.23, dd (15.6, 8.5)e 3.69, dd (15.6, 8.5)e 5.44, t (7.0)

1.63, s 3.78, s

1.70, s 4.51, s

1.60, s 3.76, s

1.70, se 3.86, d (11.0) 4.06, d (11.0)

6⁗COH Glc 1′′′′′ 2′′′′′ 3′′′′′ 4′′′′′ 5′′′′′ 6′′′′′ Rha 1′′′′′′ 2′′′′′′ 3′′′′′′ 4′′′′′′ 5′′′′′′ 6′′′′′′

8.24, s

1a

2a

3a

4c

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

d (8.0) t (8.8)e t (8.8)e me me d (11.5)

4.36, d (7.2) 3.31, t (8.8)e 3.25, t (8.0)e 3.18, me 3.18, me 3.48, d (12.0)e

3.68, d (11.5)e

3.67, d (12.0)e

4.42, d (8.0) 3.33e 3.30e 3.19e 3.48e 4.22, br dd (10.4) 4.36, dd (12.0, 6.4) 8.23, s

4.37, 3.32, 3.25, 3.18, 3.18, 3.50,

4.34, 3.31, 3.25, 3.18, 3.18, 3.50,

d (7.6) t (8.5)e t (8.0)e me me d (11.5)e

3.67, d (11.5)e

4.09, d (7.6) 2.96e 3.22e 2.96e 3.14e 3.41, d (10.2) 3.80, d (10.2) 4.59, br s 3.62e 3.43e 3.16e 3.43e 1.13, d (5.9)

a Recorded in DMSO-d6 at 800 MHz. bRecorded in DMSO-d6 at 500 MHz. cRecorded in DMSO-d6 at 850 MHz. dRecorded in methanold4 at 400 MHz. eOverlapped.

the ECD exciton chirality method. These results may facilitate the assignment of the absolute configuration of various other sabphenosides. Compound 1 was assigned the trivial name sabphenoside E. Compound 2 was isolated as a pale yellow, amorphous gum with [α]25 D −28 (c 0.1, MeOH). This compound showed an [M − H]− ion at m/z 525.2 by ESIMS, and its HRESIMS data exhibited a molecular ion at m/z 497.2037 [M − H − CO]− (calcd for C24H33O11, 497.2028) suggesting a molecular formula of C25H34O12. The presence of hydroxy (3372 cm−1), carbonyl (1748 cm−1), and olefinic (1631 cm−1) moieties was indicated by the IR spectrum. The NMR data indicate that 2 is structurally similar to compound 1, except that the 5″-methyl group in 1 is replaced with the formyl ester group in compound 2. This was confirmed via the HMBC correlations (Figure 1) from −CHO (8.24, 1H, s) to C-5″ (δC 68.9). The (2″E) olefin geometry was defined by the ROESY cross-peak, and the (7R) absolute configuration was assigned from the ECD spectrum of compound 2 (Figure 3C). Compound 2 was assigned the trivial name sabphenoside F. Compound 3, with [α]25 D −19 (c 0.1, MeOH), was acquired as a pale yellow, amorphous gum. It showed an [M − H]− ion at m/z 525.2 in its ESIMS data, and a molecular formula of C25H34O12 was deduced from its negative HRESIMS ion at m/ z 497.1996 [M − H − CO]− (calcd for C24H33O11, 497.2028) and 13C NMR data. The IR spectrum had absorption peaks characteristic of hydroxy (3302 cm−1) and carbonyl (1704

the electronic circular dichroism (ECD) exciton chirality method and confirmed by comparison of the calculated and experimental ECD spectra. The hydroxycarbonyl unit of compound 1 was reacted with (S)-1-phenylethylamine and subsequently reduced to obtain a derivative B1 of compound 1 with two strong chromophores, and this product was used in the ECD exciton chirality analysis (Figure 2). The UV spectrum of derivative B1 (Figure 2) exhibited a strong absorption at λmax 218 nm due to the π → π* excitation of the phenyl chromophores, and the ECD spectrum indicated a negative Cotton effect (CE) at 218 nm. Therefore, compound 1 was defined as having negative chirality (Figure 3A). Additionally, this chirality was supported by comparison of the experimental 1H NMR spectrum (Figure S58, Supporting Information) with that obtained through molecular modeling (Figure S59, Supporting Information). These results suggest an absolute configuration of (7R) for 1, and this was confirmed by comparison analysis of its experimental and calculated ECD spectra (Figure 3B). After the absolute configuration at the benzylic position of compound 1 was elucidated, the ECD spectra of compounds 1−6 and 12−14 were compared with the experimental ECD data of compound 1 (Figure 3C). Although sabphenosides A−D have been reported from S. japonica,10 the absolute configurations of these compounds have not been defined. Therefore, the absolute configurations of sabphenosides 1−6 and 12−14 were defined based on their experimental and calculated ECD curves in conjunction with C



Article

Table 2. 1H NMR Spectroscopic Data for Compounds 5−8 pos. 1 2 3 4 5 6 7 8 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″

HMG 1‴ 2‴ 3‴ 4‴ 6‴ Glc 1⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗ Glc 1′′′′′ 2′′′′′ 3′′′′′ 4′′′′′ 5′′′′′ 6′′′′′ Rha 1′′′′′′ 2′′′′′′ 3′′′′′′ 4′′′′′′ 5′′′′′′ 6′′′′′′

5b

6b

δH (J in Hz)

δH (J in Hz)

6.62, s 5.84, s

6.64, s 5.85, s

3.29e 3.38e 5.01, t (6.5)

3.35e 3.46e 5.09, t (6.5)

1.60, s 1.69, s

1.62, s 4.03, d (12.0) 4.13, d (12.0) 3.68e 3.28e 5.43, t (7.0)

3.68e 3.28e 5.48, t (7.0) 1.68, s 3.86, d (14.0)e 4.06, d (14.0)e

1.64, s 3.78, s

2.58, d (13.5) 2.68, d (13.5)

2.57, d (13.5) 2.68, d (13.5)

2.50−2.54e 1.29, s

2.50−2.54e 1.30, s

4.41, 3.33, 3.26, 3.20, 3.20, 3.51, 3.67,

4.42, d (8.0) 3.33e 3.26, t (8.0)e 3.19, me 3.20, me 3.48, d (11.5)e 3.69, d (11.5)e

d (7.5) d (11.5)e t (8.5)e me me d (11.5)e d (11.5)e

7a pos. 1 2 3 4 5 6 7 8 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″ Glc 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ Api 1⁗ 2⁗ 3⁗ 4⁗ 5⁗

δH (J in Hz)

6.40, s 3.27e 3.25e 3.51e 5.02, br t 1.61, s 1.72, s 3.26e 3.26e 5.44, br te 1.64, s 3.80, s 4.45, d (8.0) 3.98e 3.59e 3.22e 3.15e 3.46e 3.63e 5.44, br s 3.43, br s

8d pos. 1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ Rha 1″ 2″ 3″ 4″ 5″ 6″ Api 1‴ 2‴ 3‴ 4‴ 5‴

δH (J in Hz)

6.21, d (2.0) 6.37, d (2.0)

7.33, d (2.1)

6.93, d (8.3) 7.30, dd (8.3, 2.1) 5.40, 4.18, 3.85, 3.30, 3.58, 0.99,

d (2.5) dd (3.2, 1.7) dd (9.6, 3.3) t (9.6)e d (9.5, 6.1) d (6.2)

5.12, br s 3.92, d (2.4) 3.68, d (9.7) 3.82, d (9.7) 3.54, br se

3.56e 3.62e 3.37e 3.39e

4.11, d (8.0) 2.97e 3.24e 2.99e 3.15e 3.44, d (11.5) 3.80, d (11.5) 4.60, br s 3.62e 3.43e 3.17e 3.43e 1.13, d (6.0)

a Recorded in DMSO-d6 at 800 MHz. bRecorded in DMSO-d6 at 500 MHz. cRecorded in DMSO-d6 at 850 MHz. dRecorded in methanol-d4 at 400 MHz. eOverlapped.

cm−1) functional groups. The structure of 3 was similar to that of 2, except that the formyl ester group was at a different position in 3. In the 13C NMR data of 3, the C-6⁗ signal was

shifted downfield by 2.3 ppm, suggesting that the formyl ester group was attached at C-6⁗ of glucose.11 These conclusions were confirmed by the HMBC cross-peak (Figure 1) from HD


Journal of Natural Products Table 3.

13

Article

C NMR Spectroscopic Data for Compounds 1−8 1a

2a

3a

4c

5b

6b

pos.

δC

δC

δC

δC

δC

δC

pos.

δC

pos.

1 2 3 4 5 6 7 8 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″ 5″-COH HMG 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ Glc 1⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗ 6⁗-COH Glc 1′′′′′ 2′′′′′ 3′′′′′ 4′′′′′ 5′′′′′ 6′′′′′ Rha 1′′′′′′ 2′′′′′′ 3′′′′′′ 4′′′′′′ 5′′′′′′ 6′′′′′′

136.2 129.8 147.1 142.7 135.4 118.0 69.9 174.7 24.6 123.5 125.0 25.6 17.8 27.8 122.4 132.1 13.7 66.4

129.4 129.3 146.9 142.1 135.1 117.7 70.2 172.9 24.7 123.9 125.2 25.5 17.8 28.1 128.8 130.6 13.9 68.9 162.1

129.2 129.1 146.6 141.7 135.0 117.6 69.8 171.5 24.1 124.2 125.0 25.5 17.8 27.8 122.7 131.1 13.6 66.3

131.3 129.5 146.8 142.2 135.2 117.9 69.8 171.9 24.6 123.9 125.1 25.6 18.0 28.0 127.3 131.4 14.1 73.8

131.2 130.6 147.5 143.7 134.1 119.3 71.6 170.9 24.7 122.8 126.2 25.6 18.0 28.0 126.2 132.3 14.1 74.0

130.7 135.1 147.5 143.5 135.8 119.2 71.7 170.5 24.1 123.9 125.6 21.2 59.7 27.8 122.0 132.3 13.7 66.3

131.5 130.0 146.8 141.2 135.7 121.6 40.4 174.6 25.5 123.0 125.2 25.6 17.7 27.1 122.3 130.0 13.7 66.5

170.2 45.5 69.2 45.5 172.6 27.2

170.0 45.5 69.2 45.6 172.6 27.2

106.4 73.9 76.2 69.6 77.3 60.9

106.3 74.0 76.2 69.7 77.4 60.9

106.3 73.9 76.1 69.6 77.3 60.8

1 2 3 4 5 6 7 8 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″ Glc 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ Api 1⁗ 2⁗ 3⁗ 4⁗ 5⁗

1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ Rha 1″ 2″ 3″ 4″ 5″ 6″ Api 1‴ 2‴ 3‴ 4‴ 5‴

101.2 73.3 75.4 70.5 76.7 67.0

101.8 73.4 75.5 70.5 76.7 67.1

101.0 69.8 70.5 72.2 68.4 17.9

101.0 70.4 70.7 72.1 68.4 17.8

106.3 73.9 76.2 69.6 77.4 60.8

106.3 73.9 76.2 69.6 77.4 60.8

106.1 73.9 75.8 69.6 73.9 63.1 162.1

7a

104.0 85.5 76.1 69.9 77.0 60.8 110.5 76.7 78.8 74.3 64.8

8d δC 158.5 136.4 179.7 163.3 99.8 165.9 94.7 159.3 105.8 122.7 116.8 149.8 146.5 116.4 122.9 102.7 79.4 71.7 73.6 71.9 17.8 112.2 79.4 80.4 75.0 65.5

a

Recorded in DMSO-d6 at 200 MHz. bRecorded in DMSO-d6 at 125 MHz. cRecorded in DMSO-d6 at 212.5 MHz. dRecorded in methanol-d4 at 100 MHz. eOverlapped.

ion [M − H]− at m/z 805.3117 (calcd for C36H53O20, 805.3136) and 13C NMR data. The infrared absorption bands at 3394 and 1639 cm−1 were characteristic of hydroxy and carbonyl groups, respectively. The NMR data suggested that it is highly similar to compound 1, but the anomeric protons of a β-glucose (δH 4.09, 1H, d, J = 7.6 Hz) and an α-rhamnose (δH 4.59, 1H, br s) were present. The skeletal connectivity was deduced from the HMBC cross-peak of H-1′′′′′ with C-5″ (δC

6⁗ to −CHO (δC 162.1). The ECD spectra and ROESY correlations indicated that the absolute configuration of the secondary carbon and the olefin geometry were (7R) and (2″E), respectively. Compound 3 was assigned the trivial name sabphenoside G. Compound 4 was acquired as a pale yellow gum with [α]25 D −38 (c 0.1, MeOH). The molecular formula of 4 was determined to be C36H54O20 based on its HRESIMS molecular E



Article

Figure 1. Key HMBC (from H to C) and ROESY correlations of compounds 1−8.

Figure 2. Absolute configuration of the HMG group in compounds 5, 6, 13, and 14. These reactions were also used in part to determine the absolute configurations of the benzylic positions of sabphenosides. All procedures were described based on the reaction of the hydroxycarbonyl unit of compound 1 and (S)-1-phenylethylamine. The amination of the compound 1 with (S)-1-phenylethylamine gave compound A. Compounds B1 and B2 were generated by the reduction of compound A with LiAlH4. The acetylation of compound B2 after isolation of compounds B1 and B2 afforded (3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide (C). The chirality of the HMG group of the conjugate was determined to be S by comparing the 1H NMR data of the product with reference values (Table S2 and Figure S57, Supporting Information).

F



Article

Figure 3. Determination of the C-7 absolute configuration of sabphenosides (1−6 and 12−14). (A) Chemical modification of compound 1 to generate two chromophores for ECD exciton chirality analysis. The derivate B1 was synthesized by amination and reduction of the carboxylic acid of compound 1 with (S)-1-phenylethylamine. The arrow represents the electronic transition dipole of compound B1 with two chromophores. (B) Experimental ECD (in MeOH) and calculated ECD (R, S) spectra of compound 1 (σ = 0.1 eV; shift = +7, Table S1 and Figure S68, Supporting Information). (C) Experimental ECD (in MeOH) spectra of compounds 1−6 and 12−14. After the absolute configuration of compound 1 was determined, the ECD spectra of compounds 1−6 and 12−14 were compared with the experimental ECD data of compound 1.

group was determined by comparing its experimental 1H NMR data with reference values after a series of reactions including amination, reduction, and acetylation (Figure 2). Compound 5 was assigned the trivial name sabphenoside I. Compound 6 was acquired as a pale yellow, amorphous powder with [α]25 D −49 (c 0.1, MeOH). The molecular formula of 6 was determined to be C30H42O16 from its negative HRESIMS [M − H]− ion at m/z 657.2406 (calcd for C30H41O16, 657.2400) and the 13C NMR data. The infrared absorption bands suggested the presence of hydroxy (3307 cm−1) and carbonyl (1714 cm−1) groups. The 1H and 13C NMR data suggest that its structure is similar to that of 5, except that two of the sugar units are absent and an additional hydroxymethyl group is present in 6. This conclusion was confirmed by the HMBC correlations from H-5′ [δH 4.03 (1H, d, J = 12.0 Hz) and 4.13 (1H, d, J = 12.0 Hz)] to C-2′ (δC 123.9) and C-4′ (δC 21.2). Following a procedure similar to what was used for compound 5, the geometry of the two olefinic groups and the absolute configuration of C-7 and C-3‴ were assigned as (2′E,2″E,7R,3‴S). Compound 6 was assigned the trivial name sabphenoside J. Compound 7 was acquired as a pale yellow, amorphous powder with [α]25 D −26 (c 0.1, MeOH). Its molecular formula was assigned as C29H42O14 from its HRESIMS data, which showed an [M − H]− ion at m/z 613.2499 (calcd for

73.8). The ECD spectrum and ROESY cross-peaks indicate a (7R) absolute configuration and a (2″E) olefin geometry. Compound 4 was assigned the trivial name sabphenoside H. Compound 5 was acquired as a pale yellow gum with [α]25 D −67 (c 0.1, MeOH), and its molecular formula was determined to be C42H62O24 from its negative HRESIMS [M − H]− ion at m/z 949.3540 (calcd for C42H61O24,949.3558) and 13C NMR data. The infrared absorption bands at 3393 and 1715 cm−1 were characteristic of hydroxy and carbonyl functionalities, respectively. The comparison of the NMR data of 5 with those of 4 showed that the structures of the two compounds are similar. However, the NMR data of compound 5 displayed signals for a 3-hydroxy-3-methylglutaryl (HMG) unit, which was identified from its two methylenes at [δH 2.58 (1H, d, J = 13.5 Hz) and 2.68 (1H, d, J = 13.5 Hz); δC 45.5] and [2.50− 2.54 (2H, overlapped); 45.5], methyl group (δH 1.29, 3H, s; δC 27.2), an oxygenated tertiary carbon (δC 69.2), an ester carbonyl carbon (δC 170.2), and carboxylic carbon (δC 172.6). The presence of an HMG residue in compound 5 was confirmed by the HMBC correlations from H-2‴ to C-1‴ and C-3‴ (δC 69.2), from H-4‴ to C-3‴ and C-5‴ (δC 172.6), and from H-6‴ to C-3‴. The (2′′E) olefin geometry was confirmed by the ROESY cross-peaks, and the (7R) absolute configuration was based on the negative CE at 218 nm in its ECD spectrum (Figure 3C). The (3S) configuration of the HMG G



Article

Figure 4. Cytopathic effect of isolated compounds from S. limoniacea on PEDV. The CPE inhibition assay was performed to evaluate the potential of the compounds isolated from S. limoniacea on PEDV-infected Vero cells at 20 μM. Compounds 15 and 16 showed the strongest activity on PEDV-infected cells. In addition, compounds 7, 8, 11, and 12 also showed moderate inhibitory activities on PEDV. The results are shown as the means ± SD of three independent experiments, and statistical significance was determined by Student’s t test with *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the virus-injected control group.

Figure 5. (A) The immunofluorescence assay showed that compounds 15 and 16 inhibited PEDV replication in a dose-dependent manner. PEDVinfected cells were exposed to azauridine as a positive control and the test compounds at various concentrations (10, 20, and 30 μM). After 24 h of incubation, the cells were fixed, stained, and visualized under a fluorescence microscope. (B) Dose-dependent inhibitory effect of compound 16 on PEDV N and S protein synthesis as determined using a Western blot assay. (C) Relative intensities (target protein/β-actin) are represented in a bar graph, and each bar depicts the means ± SD from three different analyses; *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the virusinjected N protein and control group, while #p < 0.05, ##p < 0.01, and ###p < 0.001 are compared with the virus-injected S protein and control group. (D) 3D docking simulation and 2D diagram of the ligand interactions of compound 16 with PEDV 3C-like protease (PDB accession code 5GWZ). (E) 3D docking simulation and 2D diagram of the ligand interactions of 16 with SARS 3C-like protease (PDB accession code 3V3M).

presence of a β-apiofuranosyl moiety that was not present in 1. The 13C NMR signal of C-7 was shifted from δC 69.9 in 1 to δC 40.4 in 7 (Table 3), suggesting that the hydroxyacetic acid moiety in 1 was replaced with an acetic acid group in 7. The

C29H41O 14, 613.2502). The infrared absorption bands indicated the presence of hydroxy (3337 cm−1) and carbonyl (1700 cm−1) functionalities. The NMR data of 7 were highly similar to those of 1. However, the data of 7 showed the H



Article

presence of the β-apiofuranosyl group was suggested by the HMBC cross-peak of H-1⁗ to C-2‴ (δC 85.5). The (2″E) geometry of the olefinic group was assigned from its ROESY data. Compound 7 was assigned the trivial name sabphenoside K. Compound 8 was acquired as a pale yellow, amorphous powder with [α]25 D −41 (c 0.1, MeOH). The molecular formula of compound 8 was assigned as C26H28O15 from its negativeion peak for [M − H]− at m/z 579.1319 (calcd for C26H27O15, 579.1355) and 13C NMR data. The IR spectrum showed bands characteristic of hydroxy and carboxyl moieties at 3337, 2916, 2850, 2348, 1700, 1590, and 1338 cm−1. The 1D NMR data (Tables 2 and 3) exhibited an ABX spin system involving H-2′ (δH 7.33, 1H, d, J = 2.1 Hz; δC 116.8), H-5′ (6.93, 1H, d, J = 8.3 Hz; 116.4), and H-6′ (7.30, 1H, dd, J = 8.3, 2.1 Hz; 122.9) and meta coupling between H-6 (δH 6.21, 1H, d, J = 2.0 Hz; δC 99.8) and H-8 (6.37, 1H, d, J = 2.0 Hz; 94.7). Two anomeric protons appeared in the 1H NMR data of 7 at δH 5.40 (1H, d, J = 2.5 Hz) and 5.12 (1H, br s). The 13C NMR data of 7 showed signals for 26 carbons, including 15 carbons that were attributed to the flavonoid structure, the two anomeric carbons at δC 102.7 and 112.2, and the nine remaining carbons (δC 17.8−80.4) of the rhamnopyranosyl and apiofuranosyl units. The sugar linkage was determined from the HMBC correlations from H-1‴ to C-2″ (δC 79.4) and from H-1″ to C-3 (δC 136.4), which revealed that the apiofuranosyl-(1→2)rhamnopyranosyl moiety was located at C-3, similar to what was observed in compound 11.12 Compound 8 was defined as quercetin 3-O-(2-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside. Based on the spectroscopic analysis and comparison with literature values, the remaining compounds were identified as rutin (9),13 quercetin 3-O-(2″-O-β-apiofuranosyl-6″-O-αrhamnopyranosyl-β-glucopyranoside) (10),14 kaempferol 3O-(2-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside (11),15 sabphenoside A (12),10 sabphenoside B (13),10 sabphenoside D (14),10 sabphenol A (15),10 and sabphenol B (16).10 In this study, the absolute configurations of sabphenosides A, B, and D (12−14) are reported for the first time, and they were determined by comparing the experimental ECD data of the known compounds to those of compound 1 (Figure 3C). The abilities of compounds 1−16 and a positive control, azauridine, to inhibit viral replication were evaluated. All isolated compounds were tested for their PEDV inhibitory effects on Vero cells at a concentration of 20 μM, as no cytotoxicity was observed at this concentration (Figure S60, Supporting Information). Compounds 7, 8, 11, and 12 showed moderate inhibitory activities, and 15 and 16 exhibited the strongest inhibitory activities (Figure 4). In addition, compounds 15 and 16 showed concentration-dependent inhibitory activities when their inhibitory activities against PEDV were measured at concentrations of 5, 10, and 20 μM (Figure S61, Supporting Information). Spike and nucleocapsid proteins play an important role in the viral life cycle, especially during the replication of PEDV. Thus, compounds 15 and 16 were further studied for their inhibitory effects on PEDV replication at concentrations of 10, 20, and 30 μM in an immunocytochemistry assay (Figure 5A) with the nucleocapsid antibody. Compared with Vero cells (mock) that were not infected with PEDV, the infected cells showed observable fluorescence from the nucleocapsid protein of the virus. The amount of virus detected clearly decreased after treatment with compounds 15 and 16 at different concentrations or with the positive control, azauridine. We also measured the synthesis of

the GP2 spike and GP6 nucleocapsid proteins to confirm the viral inhibitory activity of compound 16. Based on Western blot analysis (Figure 5B and C), compound 16 showed a considerable inhibitory effect on viral spike and nucleocapsid protein synthesis. These results indicate that the inhibitory activity of compound 16 is more potent than that of azauridine at 20 μM. The 3C-like protease (3CL protease) of coronaviruses is essential for the proteolytic processing of polyproteins in viral replication.16 The SARS virus is a human coronavirus in the same coronaviridae family, and the important active site (His 41) is similar to that of PEDV.16−18 Therefore, 3D docking modeling of compound 16 in the active sites of PEDV 3CL protease and SARS-CoV 3CL protease was conducted using DS 4.0 software (Discovery studio 4.0 software, Accelrys). The results showed that compound 16 formed several interactions with His 41 present in the active site of PEDV 3CL protease (Figure 5D) and SARS-CoV 3CL protease (Figure 5E). The CDOCKER interaction energies were −39.8582 and −34.7148 kcal/mol, respectively. Consequently, the important interactions between the amino acids of 3CL-protease with 16 suggested its mode of action is the inhibition of 3CL protease and elucidated the potential of sabphenol derivatives as inhibitors of SARS-CoV and PEDV.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined with a JASCO P-2000 polarimeter (JASCO International Co. Ltd., Tokyo, Japan). A Nicolet 6700 FT-IR spectrometer (Thermo Electron Corp., Waltham, MA, USA) was used to measure the IR spectra. The ECD experiments were conducted using a Chirascan ECD spectrophotometer, and the ECD spectra were analyzed and visualized using Pro-Data Viewer software version 4.4.2.0 (Applied Photophysics, Leatherhead, UK). The conformational analysis was carried out with a Conflex 8 instrument (Conflex Corp., Tokyo, Japan). The molecular geometry analysis was simulated and visualized with TmoleX 4.3 and Turbomole (COSMOLogic GmbH, Leverkusen, Germany). 1D and 2D NMR data were acquired with Bruker Advance 400, 500, 800, and 850 MHz spectrometers (Bruker, Rheinstetten, Germany) at the College of Pharmacy, Seoul National University, Korea. An Agilent 6530 Q-TOF spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to collect the HRESIMS data. The ESIMS spectra were obtained on an Agilent 1100 series LC/MSD TRAP (Agilent Technologies, Waldbronn, Germany). For column chromatography (CC), Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan) and mediumpressure liquid chromatography (MPLC) with a COSMOSIL 40 C18PREP column (Nacalai Tesque Inc., Kyoto, Japan) were used. Sephadex LH-20 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Silica gel (40−63 μm) and RP-C18 (40−63 μm) from Merck (Darmstadt, Germany) were used. TLC separations were conducted on RP-18 F254 and silica gel 60 F254 plates from Merck (Darmstadt, Germany). An HPLC system equipped with a Gilson System and an Optima Pak C18 column (10 × 250 mm, 5 μm, RS Tech, Seoul, Korea) with UV detection at 205 and 254 nm was used. Extrapure grade solvents were used for fractionation and isolation processes (Daejung Chemicals & Metals Co. Ltd., Siheung, Korea). The authentic reference sugars were purchased from Sigma-Aldrich, and Lcysteine methyl ester hydrochloride and o-tolyl isothiocyanate were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Plant Material. Sabia limoniacea leaves were purchased from Ba Vi district, Hanoi City, Vietnam, in June 2015. The samples were identified based on their morphological characteristics by one of the authors (V.O.T.). A voucher specimen was deposited in the Medicinal Herbarium of Hanoi University of Pharmacy with the accession number HNIP.18510/15. I



Article

Sabphenoside F(2): pale yellow, amorphous powder; [α]25 D −28 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (2.90) 284 (1.69) nm; ECD (c 1 × 10−4 M, MeOH) λmax (Δε) 215 (−9.96), 230 (−4.06), 258 (0.04) nm; IR νmax 3272, 2976, 1748, 1688, 1608, 1447, 1338, 1288, 1078 cm−1; 1H and 13C NMR data, see Tables 1 and 3; negative-ion ESIMS m/z525 [M − H]−; negative-ion HRESIMS m/z 497.2037 [M − H − CO]− (calcd for C24H33O11, 497.2028). Sabphenoside G(3): pale yellow, amorphous powder; [α]25 D −20 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (2.80), 284 (1.65) nm; ECD (c 1 × 10−4 M, MeOH) λmax (Δε) 215 (−17.5), 230 (−8.02), 258 (0.01) nm; IR νmax 3302, 2998, 2960, 1867, 1704, 1658, 1500, 1314, 1026 cm−1; 1H and 13C NMR data, see Tables 1 and 3; negative-ion ESIMS m/z525 [M − H]−; negative-ion HRESIMS m/z 497.1996 [M − H − CO]− (calcd for C24H33O11, 497.2028). Sabphenoside H(4): yellowish, amorphous powder; [α]25 D −38 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (2.98), 282 (1.83) nm; ECD (c 1 × 10−4 M, MeOH) λmax (Δε) 216 (−6.3), 259 (−0.1) nm; IR νmax 3394, 2928, 2851, 1639, 1439, 1061 cm−1; 1H and 13C NMR data, see Tables 1 and 3; negative-ion HRESIMS m/z 805.3117 [M − H]− (calcd for C36H53O20, 805.3136). Sabphenoside I (5): pale yellow, amorphous powder; [α]25 D −67 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (2.47), 280 (1.30) nm; ECD (c 1 × 10−4 M, MeOH) λmax (Δε) 217 (−5.92), 230 (−3.72), 263 (0.37) nm; IR νmax 3393, 2996, 2929, 1715, 1447, 1288, 1078 cm−1; 1H and 13C NMR data, see Tables 2 and 3; negative-ion HRESIMS m/z 949.3540 [M − H]− (calcd for C42H61O24, 949.3558). Sabphenoside J (6): pale yellow, amorphous powder; [α]25 D −49 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 215 (2.00), 285 (1.00) nm; ECD (c 1 × 10−4 M, MeOH) λmax (Δε) 216 (−12.8), 230 (−8.11), 260 (−0.67) nm; IR νmax 3307, 2998, 2929, 1714, 1447, 1279, 1088 cm−1; 1H and 13C NMR data, see Tables 2 and 3; negative-ion HRESIMS m/z 657.2406 [M − H]− (calcd for C30H41O16, 657.2400). Sabphenoside K (7): pale yellow, amorphous powder; [α]25 D −26 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (2.13), 280 (0.90) nm; IR νmax 3337, 2916, 2850, 2348, 1700,1590, 1338 cm−1; 1H and 13C NMR data, see Tables 2 and 3; negative-ion HRESIMS m/z 613.2499 [M − H]− (calcd for C29H41O14, 613.2502). Quercetin-3-O-(2″-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside (8): brown, amorphous powder; [α]25 D −41 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (2.75), 255 (2.56), 348 (2.39) nm; IR νmax 3337, 2916, 2850, 2348, 1700, 1590, 1338 cm−1; 1H and 13C NMR data, see Tables 2 and 3; negative-ion HRESIMS m/z 579.1319 [M − H]− (calcd for C26H27O15, 579.1355). ECD Calculations. Conformational analysis of compound 1 was simulated using molecular mechanics force-field (MMFF94s) calculations with a search limit of 1.0 kcal/mol in Conflex 7 (Conflex Corp., Tokyo, Japan). TmoleX 4.3 and Turbomole (COSMOLogic GmbH, Leverkusen, Germany) at the def-SV(P) basis set for all atoms and the B3LYP functional level in the gas phase were used to optimize the ground-state geometry of the meaningful conformers. The computational ECD spectra of the optimized conformers were represented by time-dependent density functional theory using 631G/B3LYP according to the Boltzmann distributions. The ECD data were generated using Gaussian functions for each transition (σ is the width of the band at a height of 1/e). The value of σ was calculated with 0.10 eV, and the excitation energies and rotatory strengths for transition i were defined as ΔEi and Ri, respectively.20

Extraction and Isolation. The dried leaves of S. limoniacea (3.6 kg) were extracted with 70% EtOH at 25 °C (3 × 5 L, for 2 days each). The solvent was evaporated under reduced pressure to afford a concentrated residue (349 g). The crude extract was suspended in H2O (2.5 L) and continuously partitioned with EtOAc (2.5 L) to give the deionized water extract (265 g). The EtOAc-soluble fraction (84 g) was further separated by CC using silica gel (from 10:1 to 1:20) and RP-C18 silica gel (70% MeOH) to give compounds 15 (4.0 mg; tR = 37.9 min) and 16 (12.0 mg; tR = 29.0 min). The water-soluble fraction (265 g) was subjected to Diaion HP-20 column chromatography and sequentially eluted with water, 30% MeOH, and 100% acetone. The 30% MeOH fraction (22.5 g) was separated by MPLC eluting with MeOH/H2O (from 2:98 to 100:0) to afford four subfractions (SB1−SB4) based on TLC analysis. Fraction SB3 (12.1 g) was purified by silica gel CC using EtOAc/MeOH/H2O/ acetic acid (from 50:1:1:0.1 to 1:1:1:0.1) to give six subfractions (SB3.1−SB3.6). Subfraction SB3.2 (200 mg) was chromatographed on a Sephadex column with 50% MeOH to provide compound 12 (8.0 mg; tR = 35.4 min). Fraction SB3.3 (4.3 g) was purified on a Sephadex column with 50% MeOH to afford eight subfractions (SB3.3.1−SB3.3.8). Subfraction SB3.3.2 (591.5 mg) was chromatographed over RP-C18 gel eluting with 30% MeOH to afford five subfractions (SB3.3.2.1−SB3.3.2.5). Subfraction SB3.3.2.3 (245.3 mg) was purified by HPLC [mobile phase: CH3CN/H2O (20:80)] to give compounds 4 (2.3 mg; tR = 15 min) and 5 (11 mg; tR = 30 min). Subfraction SB3.3.4 (315.3 mg) was subjected to RP-C18 CC using 30% MeOH to afford six subfractions (SB3.3.4.1−SB3.3.4.6). Compounds 14 (10 mg; tR = 17.8 min) and 13 (35 mg; tR = 34.5 min) were obtained by HPLC [mobile phase: CH3CN/H2O (20:80)] from subfraction SB3.3.4.5 (100 mg). Three subfractions (SB3.3.5.1− SB3.3.5.3) were obtained from fraction SB3.3.5 (158.3 mg) following Sephadex LH-20 CC with 70% MeOH. Compounds 1 (5.0 mg; tR = 24 min), 2 (2.3 mg; tR = 30.1 min), and 3 (2.0 mg; tR = 31 min) were isolated from subfraction SB3.3.5.1 [50 mg; HPLC conditions: mobile phase: CH3CN/H2O (20:80)]. Fraction SB3.4 (2.41 g) was further purified by Sephadex CC with 50% MeOH to provide six subfractions (SB3.4.1−SB3.4.6) and compound 6 (6.0 mg; tR = 26 min). Compound 7 (2.0 mg; tR = 33 min) was isolated from fraction SB3.3.8 [30 mg; HPLC mobile phase: CH3CN/H2O (10:80)]. The 100% acetone fraction (98.0 g) was separated by RP-C18 CC with MeOH/H2O (from 4:6 to 100% MeOH) to give five subfractions (SBA1−SBA5). Subfraction SBA1 (150.5 mg) was subjected to preparative HPLC using CH3CN/H2O (20:80, v/v) to give 10 (5.0 mg; tR = 20.1 min) and 9 (10 mg; tR = 28.2 min). Fraction SBA2 (180.7 mg) was subjected to preparative HPLC using CH3CN/H2O (20:80, v/v) to give 8 (20 mg; tR = 30.1 min) and 11 (7.0 mg; tR = 28.2 min). Absolute Configuration of Sugars in Compounds 1−8.19 Compounds 1−8 (1.0−3.0 mg) were acid-hydrolyzed with 1 N HCl (1.0 mL) at 95 °C for 12 h. The reaction solution was neutralized with Na2CO3 by checking with a pH paper, and the solution was partitioned with EtOAc. The H2O layer was dried in vacuo, and the residue was reacted with L-cysteine methyl ester hydrochloride (2.0 mg) in anhydrous pyridine (0.8 mL) at 65 °C for 1 h. The resulting mixture was subsequently reacted with o-tolyl isothiocyanate (1.8 μL) at 60 °C for 1 h. The final solution was analyzed by LC-MS (column: an INNO C18 column, 5 μm, 120 Å, 4.6 × 250 mm; CH3CN/H2O mobile phase (33:73, v/v); diode array detector; detection wavelength: 254 nm; flow rate: 0.6 mL/min). The retention times of sugar derivatives of compounds 1−8 were compared with those of the derivatives of authentic D-glucose (tR = 20.5 min), D-apiose (tR = 32.2 min), and L-rhamnose (tR = 33.8 min). As a result, it proved the Dconfiguration of the glucose and apiose and L-configuration of rhamnose moieties in compounds 1−8. Sabphenoside E(1): pale yellow, amorphous powder; [α]25 D −68 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (2.92), 280 (2.25) nm; ECD (c 1 × 10−4 M, MeOH) λmax (Δε) 217 (−6.40), 230 (−4.13), 262 (0.00) nm; IR νmax 3350, 2997, 2929, 1730, 1446, 1244, 1078 cm−1; 1H and 13C NMR data, see Tables 1 and 3; negative-ion HRESIMS m/z 497.2036 [M − H]− (calcd for C24H33O11, 497.2028).

Δε(E) =

1 2.297 × 10−39

1 2πσ

A

2

2

∑ ΔEiR ie[−(E −ΔEi) /(2σ) ] i

The Boltzmann-averaged calculated ECD spectra of the representative structures were in excellent agreement with the measured ECD spectrum of 1, which showed two strong negative CEs at 217 and 276 nm (Figure 3B). These results confirmed the (7R) absolute configuration of 1. Therefore, the absolute structures of 1−6 and 12−14 could be determined by comparative analysis of their experimental ECD spectra (Figure 3C). Assignment of the Absolute Configuration of HMG in Compounds 5, 6, 13, and 14. Et3N (23.4 μmol), (S)-1J



Article

phenylethylamine (23.4 μmol), (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP, Sigma-Aldrich) (11.7 μmol), and hydroxybenzotriazole (HOBt, Sigma-Aldrich) (15.6 μmol) were reacted with the isolated compound in N,Ndimethylformamide (DMF, Sigma-Aldrich). The solution was gently mixed at 25 °C for 9 h, and the reaction was quenched with 1 N HCl and concentrated under N2 gas. The resulting residue was chromatographed using a silica gel Sep-Pak Plus Long cartridge (CH2Cl2/MeOH, 10:1) to give amide A, and the purity was confirmed by LC-MS analysis. LiAlH4 (22.2 μmol) was mixed with a solution of amide A in tetrahydrofuran (THF) (300 μL) at low temperature. The solution was gently stirred at 25 °C, and the reaction was quenched with 1 N HCl. After the resulting mixture was extracted three times with EtOAc, the concentrated colorless oil (B2) was analyzed by LC-MS to determine the purity of the product. Compound B2 was subsequently acetylated with Ac2O (12.5 μmol) in pyridine (30 μL) at 25 °C for 24 h. After the reaction, the solution was added to EtOAc and washed with H2O. The EtOAc-soluble part was dried under vacuum to afford a colorless oil. This material was further purified by HPLC with an ODS column (Optima Pak C18, 10 × 250 mm) eluting with CH3CN/H2O (4:6) to give compound C (1.1 mg) as a colorless oil. The 1H NMR spectrum of compound C was the same as that of (3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide, instead of the (3S)-configuration reported in the reference (Table S2, Supporting Information). The absolute configurations of the HMG group in sabphenosides B (13) and D (14) and in new compounds 5 and 6 (each 5.0 mg) were assigned using the same process, and the purified final products were found to be (3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide by comparing their 1H NMR spectra to those in previous papers (Figure S57, Supporting Information).21,22 (3R)-5-O-Acetyl-1-[(S)-phenylethyl]mevalonamide (C): colorless oil; 1H NMR (CDCl3, 800 MHz) δ 7.26−7.36 (5H, m, C6H5-1′), 6.02 (1H, br s, NH), 5.16 (1H, br s, H-1′), 4.24 (2H, br s, H-5), 2.41 and 2.28 (each 1H, d, J = 12.8 Hz, H-2), 2.04 (3H, s, CH3COO), 1.85 (2H, m, H-4), 1.52 (3H, d, H-2′), 1.24 (3H, s, CH3); HRESIMS m/z 294.1701 [M + H]+. Cell Culture and Virus Stock. Vero cells (African green monkey kidney cell line; ATCC CCR-81) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, U0SA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, Logan, UT, USA) with 100 μg/mL streptomycin, 100 U/mL penicillin (Gibco, Grand Island, NY, USA), and 10% fetal bovine serum (HyClone) and maintained in a 5% CO2 incubator at 37 °C. PEDV was supplied from Choong Ang Vaccine Laboratory (Daejon, Korea). Cytotoxicity Assay. The viability of the Vero cell line was assessed with the 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at 1 × 105 cells per well and grown for 24 h before use. The cells were treated with the test compounds for 48 h. The final concentration of DMSO in the culture medium of the treated cells was adjusted to less than 0.5% (v/v) to prevent a solvent effect. After 20 μL of 2 mg/mL MTT solution was added to each well, the samples were incubated for an additional 4 h. The supernatant was carefully discarded before adding 100 μL of DMSO to each well to dissolve the formazan crystals. The optical density was measured at 550 nm. The statistical significance was calculated and compared with the vehicle. Cytopathic Effect Inhibition Analysis. Vero cells were grown in a 96-well plate with 1 × 105 of confluence for 24 h, and then PEDV with a multiplicity of infection (MOI) of 0.01 was used for 2 h. The cells were treated with various concentrations of the isolated compounds and incubated at 37 °C for an additional 72 h under a 5% CO2 atmosphere. After incubation, the cell viability was estimated by the MTT assay. The statistical significance was calculated and compared with the null group. Immunofluorescence Assay. Vero cells were maintained on sterilized glass coverslips (SPL Life Science, Korea) for 1 day. After infection with PEDV (0.01 MOI) for 2 h, the cells were washed twice with phosphate-buffered saline (PBS) and transferred to DMEM

containing the test compounds at different concentrations. Cells were incubated at 37 °C and 5% CO2 for 1 day and washed twice with cold PBS. The coverslip was fixed in 4% paraformaldehyde solution for 30 min, and the membrane was permeabilized using 0.2% Triton X-100 solution. Cells were blocked with 1% bovine serum albumin solution for 1 h at room temperature. Subsequently, the monoclonal antibody against nucleocapsid (AbFrontier Co., Ltd., Seoul, Korea) was added, and the samples were incubated overnight. Anti-rabbit fluorescein isothiocyanate (FITC) (Abcam, Cambridge, UK) was added, and the samples were incubated for an additional 2 h. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, USA) solution (500 nM) for 5 min at room temperature and washed again with cold PBS. The cells were mounted and observed under a fluorescence microscope (Olympus ix70, Olympus Corporation, Tokyo, Japan). Western Blot Assay. The Vero cell line was grown to approximately 90% spreading in a six-well plate (1 day), and the cells were infected with PEDV and incubated for an additional 2 h. After the medium was changed to DMEM without serum, the compounds of interest were added at different concentrations. After 24 h, the cells were washed with chilled PBS and lysed with EBC lysis buffer [1 mM EDTA, 0.5% NP-40, 50 mM NaF, 120 mM NaCl, and 50 mM Tris-HCl (pH 7.6)]. A protein assay kit (Bio-Rad Laboratories, Inc., Hercules, MA, USA) was used to measure the protein concentrations. SDS-polyacrylamide electrophoresis was performed using a gel electrophoresis kit from Bio-Rad. The transferred PVDF membranes (0.45 μm Immobilon-P, USA) were incubated with the antibodies against β-actin (ab6276, Abcam, UK), nucleocapsid (N), and spike (S) proteins (Abfrontier Co., Ltd., Seoul, Korea) overnight at 4 °C and with the secondary antibodies at room temperature for 1 or 2 h in tris buffered saline (TBS). Finally, the samples were evaluated using an enhanced chemiluminescence Western blotting detection kit (Thermo Fisher Scientific). Statistical Analysis. Statistical analysis was conducted using oneway analysis of variance with Student’s t-tests, and SPSS (version 20.0; IBM Corporation, USA) was used. Each experiment was carried out in triplicate. All data are shown as the means ± standard deviation, and *p < 0.05, **p < 0.01, and ***p < 0.001 were considered to indicate statistically significant differences. The significance presented as #p < 0.05, ##p < 0.01, and ###p < 0.001 was determined relative to the virus-injected S protein and control group.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00435. HRESIMS, 1D and 2D NMR, and infrared absorption spectra of compounds 1−8; 1H NMR spectrum of HMG, and 1H NMR spectrum of the amination derivative; 3D structural models, relative free energies, and populations for computationally calculated conformers of compound 1; cytotoxicity assay, MTT assay, and CPE inhibition assay results (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel and Fax (W. K. Oh): +82-02-880-7872. E-mail: wkoh1@ snu.ac.kr. ORCID

Won-Keun Oh: 0000-0003-0761-3064 Notes

The authors declare no competing financial interest. K



■

Article

ACKNOWLEDGMENTS This work was supported financially in part by grants from the Marine Biotechnology Program of the Ministry of Oceans and Fisheries (PJT200669) and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Animal Disease Management Technology Development Program funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (318031031SB010).

■

REFERENCES

(1) Xing, Y.; Chen, J.; Tu, J.; Zhang, B.; Chen, X.; Shi, H.; Baker, S. J. Gen. Virol. 2013, 94, 1544−1567. (2) Park, S. J.; Song, D.-S.; Park, B.-K. Arch. Virol. 2013, 158, 1533− 1541. (3) Yang, J.; Li, Ha; Quy, T. K.; Dhodary, B.; Pyo, E.; Nguyen, N. H.; Cho, H.; Kim, E.; Oh, W. K. J. Med. Chem. 2015, 58, 1268−1280. (4) Pensaert, M. B.; de Bouck, P. Arch. Virol. 1978, 58, 243−247. (5) Li, C.; Li, Z.; Zou, Y.; Wicht, O.; van Kuppeveld, F. J. M.; Rottier, P. J. M.; Bosch, B. J. PLoS One 2013, 8, 1−9. (6) Mole, B. Nature 2013, 499, 388. (7) Cima, G. J. Am. Vet. Med. Assoc. 2013, 243, 467−470. (8) Jung, K.; Saif, L. Vet. J. 2015, 204, 134−143. (9) Deng, Y.; Zheng, Y.; Chen, B.; Zhang, G. L.; Wu, F. E. J. Asian Nat. Prod. Res. 2005, 7, 741−745. (10) Juan, Y.; Zhang, H. H.; Qiang, Yu.; Xuan, L. Helv. Chim. Acta 2009, 92, 1880−1888. (11) Kim, K. H.; Park, Y. J.; Chung, K. H.; Richard Yip, M. L.; Clardy, J.; Senger, D.; Cao, S. J. Nat. Prod. 2015, 78, 320−324. (12) Lin, H. C.; Tsai, S. F.; Lee, S. S. J. Chin. Chem. Soc. 2011, 58, 555−562. (13) Guvenalp, Z.; Ozbek, H.; Unsalar, T.; Kazaz, C.; Demirezer, L. O. Turk. J. Chem. 2006, 30, 391−400. (14) Rune, S.; Torgils, F.; Michel, J. V. J. Agric. Food Chem. 2008, 56, 2436−2441. (15) Yi-Shan, L.; Shoei-Sheng, L. Helv. Chim. Acta 2014, 97, 1672− 1682. (16) Ye, G.; Deng, F.; Shen, Z.; Luo, R.; Zhao, L.; Xiao, S.; Fu, Z. F.; Peng, G. Virology 2016, 494, 225−235. (17) Shi, Y.; Lei, Y.; Ye, G.; Sun, L.; Fang, L.; Xiao, S.; Fu, Z. F.; Yin, P.; Song, Y.; Peng, G. Vet. Microbiol. 2018, 213, 114−122. (18) Kim, J. W.; Ha, T. K.; Quy; Cho, H.; Kim, E.; Shim, S. H.; Li Yang, J.; Oh, W. K. Bioorg. Med. Chem. Lett. 2017, 27, 3019−3025. (19) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899−901. (20) Srebro-Hooper, M.; Autschbach, J. Annu. Rev. Phys. Chem. 2017, 68, 399−420. (21) Eom, H. J.; Lee, D.; L, S.; Noh, H. J.; Hyun, J. W.; Yi, P. H.; Kang, K. S.; Kim, K. H. J. Agric. Food Chem. 2016, 64, 7171−7178. (22) Komo, T.; Hirai, N.; Matsumoto, C.; Ohigashi, H.; Hirota, M. Phytochemistry 2004, 65, 2517−2520.

L


Prenylated Phenolic Compounds from the Leaves of Sabia limoniacea

Recommend Documents