Cycloartane-type triterpenoid derivatives and a flavonoid glycoside from the burs of Castanea crenata

Phytochemistry 158 (2019) 135–141

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Cycloartane-type triterpenoid derivatives and a flavonoid glycoside from the burs of Castanea crenata

T

Nanyoung Kima,1, SeonJu Parkb,1, Nguyen Xuan Nhiemc, Jae-Hyoung Songd, Hyun-Jeong Kod, Seung Hyun Kimb,∗ a

National Institute of Food & Drug Safety Evaluation Herbal Medicinal Products Division, Ministry of Food and Drug Safety, Chungcheongbuk-do, 28159, South Korea College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, 21983, South Korea c Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Hanoi, Viet Nam d Laboratory of Microbiology and Immunology, College of Pharmacy, Kangwon National University, Chuncheon, 24341, South Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Castanea crenata Fagaceae Cycloartane-type triterpenoids Flavonoid glycosideChemical compounds studied in this article: Astragalin (PubChem CID: 5282102) Tiliroside (PubChem CID: 5320686) cis-Tiliroside (PubChem CID: 10175330) Helichrysoside (PubChem CID: 44259193)

Five undescribed cycloartane-type triterpenoids, which were isolated for the first time from the genus, and a flavonoid glycoside together with 11 known compounds were isolated from the burs of Castanea crenata. The structures were elucidated based on the spectroscopic analysis of 1D and 2D NMR and MS data. All isolated compounds were evaluated for antiviral activities against HRV1B-, CVB3-, and PR8-infected cells. Most kaempferol derivatives showed statistically significant antiviral activities against HRV1B-infected cells. Among the tested compounds, kaempferol-3-O-[2″,6″-di-O-Z-p-coumaroyl]-β-D-glucopyranoside exhibited the most consistent and effective antiviral activities against all infections.

1. Introduction Human enteroviruses, which are small, single-stranded, positivesense RNA viruses, belong to the Enterovirus genus of the family Picornaviridae. Human enteroviruses have been classified into four species (A–D), including the most common viruses such as human rhinovirus 1B (HRV1B) and coxsackievirus B3 (CVB3). HRV1B is a major cause of the common cold, which may lead to pneumonia and bronchiolitis (Shim et al., 2016). While CVB3 is also the most common human pathogen for viral myocarditis, which may leads to dilated cardiomyopathy and causes unexpected death in children and adolescents or end-stage congestive heart failure in adults (Yajima, 2011). Influenza viruses, which are negative-sense RNA viruses of the family Orthomyxoviridae, are classified into three serotypes, A, B, and C. Among them, influenza A/PR/8 virus (PR8) can cause severe respiratory disease and cause much of the morbidity and mortality observed annually (Shi et al., 2007). For people with immune deficiencies, the viral infection frequently leads to severe and sometimes lethal cases. Due to these reasons, serious efforts have been put into investigating an efficient treatment or prevention of these viral infections. However, control over these viral infections still remains a global public

health epidemic even in the era of potent antiviral drugs. In our previous studies, we isolated and tested phytochemical antiviral activities and reported that labdane-type diterpenoids from Vitex limonifolia and compounds from Lindera glauca were potent antiviral phytochemicals (Ban et al., 2018; Park et al., 2018). In a continuing attempt to identify natural products that exhibit antiviral effects, a phytochemical investigation of the burs of Castanea crenata Siebold & Zucc. (Fagaceae) was performed. C. crenata, a type of chestnut tree native to South Korea and Japan, has been used in traditional medicine for the treatment of gastroenteritis, bronchitis and regurgitation (Zhao et al., 2011). Chestnut processing generates two waste products: the bur and the shell. While the shell is separated in the peeling process and is used as fuel, the bur usually remains in the woodland, facilitates the proliferation of insect larvae, and causes damages to crops. As an oriental medicinal material, chestnut burs, called involucre, have been used for the treatment of furuncle, pertussis, and bronchitis. Previous phytochemical studies of C. crenata burs identified diverse chemical components such as tannins, phenolic acids, flavonoids, coumarins, phenylpropanoids, and steroids. These components were proved to have antiviral activities (Bermejo et al., 2002; Buzzini et al., 2008; Özçelik et al., 2011; Park et al., 2018). In addition, there is a report on

∗

Corresponding author. E-mail address: [email protected] (S.H. Kim). 1 These two authors contributed equally to the work. https://doi.org/10.1016/j.phytochem.2018.11.001 Received 19 September 2018; Received in revised form 1 November 2018; Accepted 1 November 2018 0031-9422/ © 2018 Elsevier Ltd. All rights reserved.

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Fig. 1. Chemical structures of compounds 1–5, 8, 9 and 12–16.

Compound 1 was obtained as a yellow amorphous powder and its molecular formula was determined as C27H42O6 by HR-ESI-MS [M–H]– ion at m/z 461.2903 (calcd for C27H41O6, 461.2909). The 1H NMR spectrum of 1 displayed the signals of two cyclopropane methylene protons at δH 0.21 (d, J = 4.4 Hz) and 0.70 (d, J = 4.4 Hz), two oxymethine protons at δH 3.91 (dd, J = 4.2, 11.6 Hz) and 3.43 (ddd, J = 6.4, 10.2, 12.0 Hz), one secondary methyl group at δH 0.81 (d, J = 6.2 Hz), and three tertiary methyl groups at δH 0.86 (s), 0.91 (s), and 1.00 (s). These were similar to those of the other cycloartane-type triterpenes. The 13C NMR and DEPT spectra of compound 1 revealed the signals of 27 carbons including four methyls, 10 methylenes, six methines, and seven quaternary carbons (Table 1), which were assigned as a trinorcycloartane-type triterpene. The NMR spectroscopic data of 1 were similar to those of norquadrangularic acid A (Banskota et al., 2000), except for the presence of a hydroxyl group at C-7, and the disappearance of a hydroxyl group at C-1. The HMBC correlations between H-29 (δH 1.00) and C-3 (δC 76.2)/C-4 (δC 55.4)/C-5 (δC 43.6)/C28 (δC 180.6) suggested the position of a hydroxyl group at C-3 and carboxylic group at C-28. The HMBC correlations between H-7 (δH 3.43)/H-6 (δH 1.28) and C-7 (δC 70.6) and between H-22 (δH 1.74)/H23 (δH 2.12) and C-24 (δC 178.4) confirmed the position of a hydroxyl group at C-7 and carboxylic acid at C-24, respectively (Fig. 2). The large vicinal coupling constant for Hα-2 and H-3 (JHα-2/H-3 = 11.6 Hz) indicated its trans di-axial orientation. The axial orientations of H-7 and H-8 were also deduced by their large coupling constant value (JH-7/H8 = 7.6 Hz) (Toume et al., 2011). Moreover, the observations of NOESY

C. crenata extracts, which showed inhibitory activity against human immunodeficiency virus (HIV)-1 protease (Park et al., 2003). Therefore, phytochemical exploration of chestnut burs is a meaningful contribution towards the discovery of previously undescribed natural antiviral sources.

2. Results and discussion The methanol extract of the C. crenata burs was suspended in water and partitioned successively with CH2Cl2 and EtOAc to obtain three layers. By means of various chromatographic and isolation techniques, six undescribed compounds and 11 known compounds were isolated (Fig. 1). These structures were elucidated by extensive spectroscopic methods, including 1D and 2D NMR experiments, and by HR-ESI-MS analysis. The remaining 11 known compounds were compared with those previously reported literature data and identified as stachyanthuside B (6) (Yan and Guo, 2004), 3,3′-di-methylellagic acid 4′O-β-D-xylopyranoside (7) (Nono et al., 2014), astragalin (8) (Kim et al., 1994), tiliroside, kaempferol-3-O-[2″-O-E-p-coumaroyl]-β-D-glucopyranoside, kaempferol-3-O-[2″,6″-di-O-E-p-coumaroyl]-β-D-glucopyranoside, kaempferol-3-O-[2″,6″-di-O-Z-p-coumaroyl]-β-D-glucopyranoside and kaempferol-3-O-[4″-acetyl-2″,6″-di-E-p-coumaroyl]-β-D-glucopyranoside (9, 11 and 13–15) (Zhang et al., 2007), cis-tiliroside (10) (Liao et al., 2014), kaempferol-3-O-[3″,6″-di-O-E-p-coumaroyl]-β-D-glucopyranoside (12) (Liu et al., 1999), and helichrysoside (17) (Alves et al., 2012). 136

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433.2959). The 1H NMR and 13C NMR spectra of 3 also showed the typical signal for a cycloartane-type triterpene: a methylene of cyclopropane ring, two oxymethine, one oxymethylene, three tertiary methyl groups, and one secondary methyl group. Additionally, the analysis of NMR data indicated the structure of 3 was similar to those of 2 except for the replacement of the pentanol group into a butanol group at C-20. The HMBC correlations from H-22 (δH 1.66) to C-23 (δC 60.1) verified the positions of a hydroxyl group at C-23 (Fig. 2). Based on the above evidence, the structure of compound 3 was established as 23-norcycloartane-28-oic acid and named castaartancrenoic acid C. Compound 4 was isolated as a yellow amorphous powder and its molecular formula was confirmed as C30H48O5 by HR-ESI-MS [M–H]– ion at m/z 487.3422 (calcd for C30H47O5, 487.3429). The 1H NMR spectrum suggested that compound 4 is a cycloartane-type triterpene: the methylene protons of cyclopropane ring at δH 0.36 (d, J = 4.2 Hz) and 1.03 (m), three oxymethine protons at δH 3.99 (d, J = 10.2 Hz), 4.43 (m), and 4.78 (dd, J = 3.8, 11.8 Hz). In addition, exo-olefin protons at δH 5.05 (s) and 5.28 (s), and four tertiary methyl groups at δH 1.12 (s), 1.14 (s), 1.71 (s), and 2.00 (s) along with one secondary methyl group at δH 1.06 (d, J = 6.0 Hz) were shown. The 13C NMR and DEPT spectra of compound 4 displayed the presence of 30 carbons including five methyls, 11 methylenes, eight methines, and six quaternary carbons (Table 2). The NMR data of 4 were similar to those of combretic acid B (Toume et al., 2011), except for the substitution with hydroxyl groups at C-7 instead of acetyl groups. The HMBC correlations were similar to those of compounds 1–3 (Fig. 2). In addition, the HMBC correlations between H-24 (δH 4.43) and C-26 (δC 110.9)/C-27 (δC 17.5) as well as between H-23 (δH 2.00) and C-22 (δC 32.5)/C-24 (δC 76.1)/C-25 (δC 148.8) proved the locations of the hydroxyl group at C24 and the exo-olefin group at C-26 (Fig. 2). The configuration of hydroxyl group at C-24 as α was deduced from the NOESY correlations H21 (δH 1.06) and H-22 (δH 1.22); Hβ-23 (δH 2.00) and H-24 (δH 4.43) (Fig. 3). Consequently, the structure of compound 4 was characterized as 3β,7β,24α-trihydroxycycloart-25,26-ene-28-oic acid and named castaartancrenoic acid D. The molecular formula of compound 5 was also determined as C30H48O5 by HR-ESI-MS [M–H]– ion at m/z 487.3429 (calcd for C30H47O5, 487.3429). The 1H and 13C NMR spectroscopic data of 5 was similar to those of compound 4 except for the configuration of a hydroxyl group at C-24. The β configuration of the hydroxyl group at C-24 was determined based on the NOESY correlations between H-21 (δH 1.05) and Hα-22 (δH 1.74); Hα-22 (δH 1.74) and H-24 (δH 4.44). In addition, Toume et al. suggested that the configuration of C-24 can be determined by comparing 13C NMR signals of C-24 to C-27 (Toume et al., 2011). 24-Epiquadrangularic acid M, which has a 24α hydroxyl group, showed δC 76.1 (C-24), 149.6 (C-25), 110.4 (C-26), and 17.7 (C27), while quadrangularic acid M, which has a 24β hydroxyl group, exhibited δC 75.6 (C-24), 149.6 (C-25), 110.0 (C-26), and 18.2 (C-27). The 13C-NMR chemical shift of C-24 to C-27 in 4 and 5 were δC 76.1 (C24), 148.8 (C-25), 110.9 (C-26), and 17.5 (C-27); 75.6 (C-24), 149.0 (C25), 110.4 (C-26) and 17.8 (C-27) and the configuration at C-24 was determined to be 24α and 24β, respectively. Based on the above data, the structure of compound 5 was identified as 3β,7β,24β-trihydroxycycloart-25,26-ene-28-oic acid and named castaartancrenoic acid E. Compound 16 was isolated as a yellow amorphous powder and its molecular formula was confirmed as C41H34O16 determined by HR-ESIMS [M–H]– ion at m/z 781.1799 (calcd for C41H33O16, 781.1769). 1H NMR spectrum of 16 showed the signals of two trans-p-coumaroyl moiety at δH 6.21 (d, J = 16.0 Hz) and 7.30 (d, J = 16.0 Hz); 6.00 (d, J = 16.0 Hz) and 7.58 (d, J = 16.0 Hz). Two aromatic proton signals at δH 5.98 (s) and 6.17 (s) along with 6.71 (d, J = 7.2 Hz) and 7.82 (d, J = 7.2 Hz) confirmed the kaempferol aglycone. In addition, the βlinkage of the glucosyl moiety was deduced from the coupling constant (J = 7.8 Hz) of the anomeric proton signal at δH 5.61 (Table 3). 13C NMR spectrum of 16 revealed 41 carbon signals, including 16 quaternary, 23 methine, one methylene, and one methyl group (Table 3).

Table 1 NMR spectroscopic data for compounds 1–3. Pos

1 δC

2 a,b

δH

a,c

(J in Hz)

δC

3 (J in Hz)

δCa,b

δHa,c (J in Hz)

32.1

70.7

1.32 (m, α) 1.59 (m, β) 1.52 (m, β) 1.67 (m, α) 3.92 (dd, 4.2, 11.6, α) – 2.05 (dd, 3.8, 13.0, α) 1.06 (m, β) 1.30 (m, α) 3.45 (m, α)

1.28 (m, α) 1.56 (m, β) 1.53 (m, β) 1.65 (m, α) 3.90 (d, 11.2, α) – 2.02 (d, 10.0, α) 1.05 (m, β) 1.30 (m, α) 3.42 (d, 7.6, α) 1.63 (d, 7.6, β) – – 1.33 (m, α) 1.70 (m, β) 1.52 (m) – – 1.38 (m) 1.49 (m) 1.24 (m) 1.85 (m) 1.47 (m, α) 0.91 (s, β) 0.19 (d, 4.4, α) 0.69 (d, 4.4, β) 1.46 (m) 0.81 (d, 5.4, α) 1.10 (m) 1.66 (m) 3.46 (m) 3.52 (m)

a,b

δHa,c

1.29 (m, α) 1.55 (m, β) 1.53 (m, β) 1.66 (m, α) 3.91 (dd, 4.2, 11.6, α) – 2.03 (dd, 6.6, 13.0, α) 1.05 (m, β) 1.28 (m, α) 3.43 (ddd, 6.4, 10.2, 12.0, α) 1.64 (d, 7.6, β)

32.1

54.7

1.66 (d, 7.6, β)

54.7

– – 1.34 1.70 1.52 – – 1.39 1.50 1.26 1.84 1.48 0.91 0.21 0.70

21.6 27.0 28.2

– – 1.35 1.71 1.53 – – 1.39 1.52 1.24 1.84 1.50 0.90 0.22 0.72

21.5 27.0 28.2

1

32.1

2

30.4

3

76.2

4 5

55.4 43.6

6

33.7

7

70.6

8

54.7

9 10 11

21.5 26.9 28.2

12 13 14 15

34.0 46.9 50.0 37.1

16

29.3

17 18 19

53.0 17.5 27.7

20 21

37.2 18.7

1.35 (m) 0.81 (d, 6.2, α)

37.5 19.2

1.35 (m) 0.83 (d, 5.6, α)

34.5 19.3

22

32.8

33.6

32.4

24 28 29 30

178.4 180.6 9.8 19.4

0.99 (m) 1.43 (m) 1.34 (m) 1.54 (m) 3.43, d (7.4) – 0.98 (s, β) 0.84 (s, α)

40.3

23

1.20 1.74 2.12 2.25 – – 1.00 0.86

(m, α) (m, β) (m)

(m) (m) (m) (m) (m, α) (s, β) (d, 4.4, α) (d, 4.4, β)

(m) (m) (m) (m)

(s, β) (s, α)

30.4 76.3 55.4 43.6 33.7

34.1 46.8 50.0 37.1 29.4 53.1 17.4 27.7

30.6 63.7 180.7 9.8 19.4

(m, α) (m, β) (m)

(m) (m) (m) (m) (d, 9.2, α) (s, β) (d, 4.4, α) (d, 4.4, β)

30.4 76.3 55.4 43.6 33.8 70.7

34.1 46.9 50.0 37.1 29.5 53.4 17.4 27.7

60.1

180.2 9.9 19.4

– 0.99 (s, β) 0.84 (s, α)

a

Measured in pyridine-d5. 200 MHz. c 800 MHz, assignments were done by HSQC, HMBC, COSY and ROESY experiments. b

correlations between H-3 (δH 3.91) and H-5 (δH 2.03); H-29 (δH 1.00) and Hβ-19 (δH 0.70); H-7 (δH 3.43) and H-30 (δH 0.86) suggested the configuration of hydroxyl groups at C-3 and C-7 as β and carboxylic group at C-4 as α (Fig. 3). Based on the above data, the structure of compound 1 was determined as 3β,7β-dihydroxy-24-norcycloartane24,28-dioic acid and named castaartancrenoic acid A. The HR-ESI-MS of compound 2 showed [M–H]– ion at m/z 447.3108 (calcd for C27H43O5, 447.3116), suggesting molecular formula of C27H44O5. The 1H NMR spectrum of 2 showed signals of a cycloartanetype triterpene. The NMR data indicated that the structure of 2 was similar to that of 1, except for the deletion of a carbonyl group at C-24. The COSY experiment established the location of a hydroxymethylene group at C-23 by establishing the spin system of –CH2–CH2–CH–CH (CH3)–CH2–CH2–CH2–. Based on the above data, the structure of compound 2 was determined to be 3β,7β,24-trihydroxy-24-norcycloartane-28-oic acid and named castaartancrenoic acid B. The molecular formula of compound 3 was confirmed as C26H42O5 by HR-ESI-MS [M–H]– ion at m/z 433.2950 (calcd for C26H41O5, 137

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Fig. 2. The key HMBC and COSY correlations of compounds 1, 3, 4 and 16.

Fig. 3. The key NOESY correlations of compounds 1–2 and 4–5.

The NMR data analysis of 16 showed it was similar to those of kaempferol-3-O-[2″,6″-di-O-E-p-coumaroyl]-β-D-glucopyranoside (Zhang et al., 2007), except for the addition of an acetyl group. The sugar moiety connected to C-3 was identified by the HMBC correlation

between H-1″ (δH 5.61) and C-3 (δC 134.6). The HMBC correlation between H-3″ (δH 5.12) and carbonyl group (δC 172.2) suggested the position of an acetyl group connected to C-3″. Moreover, the linkages of the E-p-coumaroyl groups were determined by the HMBC correlation 138

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Table 2 NMR spectroscopic data for compounds 4–5. Pos

4

5

Pos

a,b

δHa,c

(J in Hz)

δC

31.2

1.46 1.81 2.08 2.25 4.78 – 2.68 1.77 2.21 3.99 2.16 – – 1.48 1.86 1.66 – – 1.85 1.99 1.41 2.00 1.67 1.14 0.36 1.03 1.53 1.06 1.83 2.17 1.84 2.00 4.43 – 5.05 5.28 2.00 – 1.71 1.12

(m, α) (m, β) (m, β) (m, α) (dd, 3.8, 11.8, α)

31.1

δC 1

Table 3 NMR spectroscopic data for compound 16.

2

30.1

3 4 5 6

75.2 54.8 42.7 33.5

7 8 9 10 11

69.3 53.8 20.3 25.9 27.1

12 13 14 15

32.9 45.7 49.0 36.3

16

28.4

17 18 19

51.8 17.0 26.9

20 21 22

36.3 18.7 32.5

23

32.1

24 25 26

76.1 148.8 110.9

27 28 29 30

17.5 180.4 10.1 18.9

(d, 12.8, α) (m, α) (m, β) (d, 10.2, α) (d, 7.6, β)

(m) (m) (m)

(m) (m) (m) (m) (m, α) (s) (d, 4.2, α) (m, β) (m) (d. 6.0) (m, α) (m, β) (m, α) (m, β) (m, β) (s) (s) (s) (s, β) (s)

a,b

30.0 75.1 54.7 42.6 33.4 69.2 53.7 20.2 25.8 27.0 32.8 45.5 48.8 36.2 28.4 51.7 16.8 26.7 36.2 18.6 32.2 32.4 75.6 149.0 110.4 17.8 180.1 10.0 18.8

δH

a,c

1.45 1.80 2.07 2.24 4.78 – 2.68 1.74 2.20 3.97 2.15 – – 1.47 1.85 1.65 – – 1.85 1.98 1.41 2.01 1.67 1.12 0.36 1.02 1.53 1.05 1.74 2.03 1.43 1.74 4.44 – 5.05 5.32 1.99 – 1.71 1.11

(J in Hz) (m, α) (m, β) (m, β) (m, α) (dd, 3.8, 11.8, α)

Agly 2 3 4 5 6 7 8 9 10 1′ 2′, 6′ 3′, 5′ 4′ Glc 1″ 2″ 3″ 4″ 5″ 6″

(d, 12.8, α) (m, α) (m, β) (m, α) (d, 8.2, β)

(m) (m) (m)

(m) (m) (m) (m) (m, α) (s) (d, 4.2, α) (d, 4.2, β) (m) (d, 6.0) (m, α) (m, β) (m, β) (m, α) (t, 6.1, α)

16 δCa,b

δHa,c (J in Hz)

159.0 134.6 179.2 163.2 100.1 165.8 94.8 158.4 105.9 122.8 132.2 116.3 161.6

– – – – 5.98 – 6.17 – – – 7.82 6.76 –

100.4 73.8 76.9 70.1 75.9 63.9

5.61 (d, 7.8) 5.05d 5.12d 3.53d 3.56d 4.15 (d, 11.8) 4.27 (d, 11.8)

(s) (s)

(d, 7.2) (d, 7.2)

Pos 3″-OAc COCH3 COCH3 2″-coumaroyl α″′ β″′ γ″′ 1″′ 2‴, 6″′ 3‴, 5″′ 4″′ 6″-coumaroyl α‴′ β‴′ γ‴′ 1‴′ 2‴′, 6‴′ 3‴′, 5‴′ 4‴′

δCa,b

δHa,c (J in Hz)

172.2 21.0

– 1.93 (s)

146.8 114.5 168.1 127.2 131.4 116.9 161.4

7.30 6.21 – – 7.22 6.71 –

147.9 114.7 168.8 127.2 131.6 116.9 161.6

7.58 6.00 – – 7.37 6.71 –

(d, 16.0) (d, 16.0)

(d, 8.2) (d, 8.2)

(d, 16.0) (d, 16.0)

(d, 8.2) (d, 8.2)

a

Measured in methanol-d4. 100 MHz. c 400 MHz. d Overlapped signals, assignments were done by HSQC, HMBC and COSY experiments. b

Table 4 Antiviral activity of active compounds against HRV1B, CVB3, and PR8 viruses. Compound

(s) (s) (s)

HRV1B 4 5 8 9 12 13 14 15 16 Rupintrivir CVB3 14 Rupintrivir PR8 13 14 Oseltamivir

(s, β) (s)

a

Measured in pyridine-d5. 200 MHz. c 800 MHz, assignments were done by HSQC, HMBC, COSY and ROESY experiments. b

between H-2″ (δH 5.05) and C-γ‴ (δC 168.1), between H-6″ (δH 4.15 and 4.27) and C-γ‴' (δC 168.8) (Fig. 2). Thus, the structure of 16 was identified as kaempferol-3-O-[3″-acetyl-2″,6″-di-E-p-coumaroyl]-β-Dglucopyranoside. To search for effective antiviral phytochemicals, all of the isolated compounds were evaluated for antiviral activities by employing the SRB method using cytopathic effect (CPE) reduction assay in HeLa or Vero cells. Rupintrivir against both HRV1B and CVB3 and oseltamivir against PR8 were used as positive controls (Shim et al., 2016). Among the isolated compounds, most of kaempferol derivatives showed statistically significant antiviral activities against HRV1B-infected cells (Table 4). Several flavonoids have been reported for their antiviral effects against RNA viruses such as influenza A, HIV, rotavirus, murine norovirus, feline calicivirus, and porcine epidemic diarrhea virus (Choi et al., 2009; Özçelik et al., 2011; Seo et al., 2016). Among the tested compounds, kaempferol-3-O-[2″,6″-di-O-Z-p-coumaroyl]-β-D-glucopyranoside (14) exhibited the most consistent and effective antiviral activity against HRV1B-, CVB3-, and PR8-infected cells. Thus, these compounds could be previously undescribed effective antiviral compounds and further studies of their antiviral effects are in progress.

TIc

CC50a

IC50b

> 50 > 50 > 50 > 50 28.37 > 50 28.19 > 50 > 50 > 50

6.32 ± 5.58 ± 19.4 ± 34.1 ± 6.14 ± 1.22 ± 5.89 ± 7.46 ± 5.51 ± < 0.04

0.46 0.24 3.63 3.34 0.06 0.01 0.21 0.52 0.31

> 7.91 > 8.96 > 2.57 > 1.46 4.62 > 40.9 4.78 > 6.70 > 9.07 ND

> 50 > 50

43.6 ± 1.97 7.88 ± 0.94

> 1.14 > 6.34

> 50 > 50 > 50

37.1 ± 3.62 28.2 ± 0.69 2.19 ± 0.55

> 7.91 > 8.96 > 22.8

Results are presented as the mean IC50 values ± SD obtained from three independent experiments carried out in triplicate. a Concentration required to reduce cell growth by 50% (μM). b Concentration required to inhibit virus-induced CPE by 50% (μM). c Therapeutic index = CC50/IC50.

3. Conclusions Phytochemical investigation of the burs of C. crenata resulted in isolation of five cycloartane-type triterpenes, castaartancrenoic acids A–E, and one flavonoid, kaempferol-3-O-[3″-acetyl-2″,6″-di-E-p-coumaroyl]-β-D-glucopyranoside, together with 11 known compounds. Cycloartane triterpenoids were isolated for the first time from the genus. Therefore, these compounds could be potential chemotaxonomic markers for the species. To identify antiviral phytochemicals, isolated compounds were evaluated for their bioactivities against HRV1B-, 139

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461.2903 [M–H]– (calcd for C27H41O6, 461.2909); for 1H (CD3OD, 800 MHz) and 13C NMR (CD3OD, 200 MHz) spectroscopic data, see Table 1.

CVB3-, and PR8-infected Vero or HeLa cells. The antiviral assays demonstrated that the compounds isolated from C. crenata could be effective antiviral agents to treat viral infection caused by HRV1B, CVB3, and PR8.

4.5. Castaartancrenoic acid B (2)

4. Experimental

Yellow amorphous powder; [α ]20 D : +18.0 (MeOH, c = 2.0); IR (KBr) νmax: 3340, 1708, 1448, 1114, 1021 cm−1; C27H44O5, HR-ESI-MS m/z: 447.3108 [M–H]– (calcd for C27H43O5, 447.3116); for 1H (CD3OD, 800 MHz) and 13C NMR (CD3OD, 200 MHz) spectroscopic data, see Table 1.

4.1. General The chemical shifts are expressed in parts per million from TMS. The H and 13C NMR spectra were operated at 800 and 200 MHz measured with an Agilent 800-MR-NMR spectrometer, respectively. Data processing was carried out with the MestReNova ver. 6.0.2 program. HRESI-MS experiment were conducted using an Agilent 6550 iFunnel QTOF LC/MS system. Optical rotations were determined on a Jasco DIP370 automatic polarimeter. Preparative HPLC was performed on an Agilent 1260 HPLC system. Column chromatography (CC) was carried out with silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) and RP-silica gel (150 μm, Fuji Silysia Chemical). Thin layer chromatography (TLC) was carried on a pre-coated silica-gel 60 F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm, Merck) plate. The FT-IR spectra (KBr) were recorded on a Nicolet 380 spectrometer. 1

4.6. Castaartancrenoic acid C (3) Yellow amorphous powder; [α ]20 D : +55.0 (MeOH, c = 2.0); IR (KBr) νmax: 3345, 1650, 1420, 1114, 1021 cm−1; C26H42O5, HR-ESI-MS m/z: 433.2950 [M–H]– (calcd for C26H41O5, 433.2959); for 1H (CD3OD, 800 MHz) and 13C NMR (CD3OD, 200 MHz) spectroscopic data, see Table 1. 4.7. Castaartancrenoic acid D (4)

4.2. Plant material

Yellow amorphous powder; [α ]20 D : +24.2 (MeOH, c = 2.0); IR (KBr) νmax: 3363, 1707, 1437, 1010, 950 cm−1; C30H48O5, HR-ESI-MS m/z: 487.3422 [M–H]– (calcd for C30H47O5, 487.3429); for 1H (pyridine, 800 MHz) and 13C NMR (pyridine, 200 MHz) spectroscopic data, see Table 2.

The burs of Castanea crenata Siebold & Zucc. (Fagaceae) were collected from Seokjang farm in Deoksan-myeon, Jincheon-gun, Chungcheongbuk-do, Republic of Korea during the dry season in April 2014 and identified by Prof. Jong Hee Park in College of Pharmacy, Pusan National University, Busan, Korea. A voucher specimen (CC201404) was deposited at the Herbarium of College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea.

4.8. Castaartancrenoic acid E (5) Yellow amorphous powder; [α ]20 D : +40.0 (MeOH, c = 2.0); IR (KBr) νmax: 3440, 1672, 1437, 1093, 1012 cm−1; C30H48O5, HR-ESI-MS m/z: 487.3429 [M–H]– (calcd for C30H47O5, 487.3429); for 1H (pyridine, 800 MHz) and 13C NMR (pyridine, 200 MHz) spectroscopic data, see Table 2.

4.3. Extraction and isolation Pulverized C. crenata burs (9.7 kg) were extracted with 80% methanol (20 L × 3 times) using sonication for 15 h to yield 532 g extract after evaporation of the solvent. The extract was suspended in water and successively partitioned with dichloromethane and ethyl acetate to obtain the CH2Cl2 (CB1, 123.21 g), EtOAc (CB2, 75.37 g) and water (CB3, 98.06 g) residues after removal of the solvents in vacuo. The CH2Cl2 residue was subjected to silica gel CC with a gradient of CHCl3: MeOH (40: 1 → 1: 1, v/v) to obtain six sub-fractions (CB1A–CB1F). CB1C fraction was applied to a silica gel CC eluted with CHCl3: MeOH (10: 1, v/v) solvent system to obtained CB1C1–CB1C6 smaller fractions. CB1C3 was subjected to HPLC, using J'sphere ODS H-80 250 mm × 20 mm column, eluted with 43% MeCN at a flow rate of 3 mL/min to yield 15 (9.6 mg) and 16 (12 mg), while CB1C4 yield 1–3 (12.6, 4.3, and 5.9 mg) by HPLC eluting with 40% aqueous MeCN. In addition, CB1C5 fraction was also subjected to HPLC eluting with 65% aqueous MeCN to yield 4 (3.3 mg) and 5 (4.5 mg). EtOAc soluble fraction was also divided into six sub-fractions (CB2A–CB2F) using CHCl3: MeOH gradient elution (40: 1 → 1: 1, v/v) on silica gel column chromatography. Using silica gel CC eluted with CHCl3: MeOH (5: 1, v/ v), CB2C fractions were allowed to obtain 6 (41.3 mg), 17 (24.2 mg) along with four smaller fractions (CB2C1–CB2C4). The CB2C1 fraction was applied to a RP-silica gel CC eluted with MeOH: H2O (1: 1, v/v) to give 7 (6.6 mg), 8 (81.3 mg), 9 (5.8 mg) and 10 (11.6 mg). CB2C2 fraction was further purified by HPLC using 25% aqueous MeCN to obtain 11 (7.0 mg), 12 (8.9 mg), 13 (3.2 mg), and 14 (4.8 mg).

4.9. Kaempferol-3-O-[3″-acetyl-2″,6″-di-E-p-coumaroyl]-β-Dglucopyranoside (16) Yellow amorphous power; [α ]20 D : +25.0 (MeOH, c = 2.0)); IR (KBr) νmax: 3320, 1653, 1605, 1411 cm−1; C41H34O16, HR-ESI-MS m/z: 781.1789 [M–H]– (calcd for C41H33O16, 781.1769); for 1H (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) spectroscopic data, see Table 3. 4.10. Cell culture The HRV1B, CVB3, and PR8 were obtained from the division of vaccine research of the Korea Centre Disease Control and Prevention and were propagated at 37 °C in HeLa and Vero cells (ATCC, Manassas, VA, USA). CVB3 was expanded in Vero cells while HRV1B and PR8 was expanded in HeLa cells. These cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 0.01% antibiotic–antimycotic solution. The antibiotic–antimycotic solution, trypsin–EDTA, FBS, and MEM were purchased from Gibco BRL (Invitrogen Life Technologies, Karlsruhe, Germany). Tissue culture plates were purchased from Falcon (BD Biosciences, San Jose, CA, USA). SRB was purchased from SigmaAldrich (St. Louis, MO, USA). The cells were incubated in MEM supplemented with 10% FBS and 0.01% antibiotic–antimycotic solution in a humidified atmosphere of 5% CO2 at 37 °C. Cells were detached using trypsin-EDTA. Five subsequent cycles of subculturing were done before the cells were incubated with isolated compounds (Choi et al., 2009).

4.4. Castaartancrenoic acid A (1) Yellow amorphous powder; [α ]20 D : +10.0 (MeOH, c = 2.0); IR (KBr) νmax: 3300, 1653, 1445, 1165, 1017 cm−1; C27H42O6, HR-ESI-MS m/z: 140

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4.11. Antiviral assay

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Antiviral activity was assayed by employing the SRB method using cytopathic effect (CPE) reduction as recently reported (Choi et al., 2009). Briefly, 2 × 104 cells/well of Vero or HeLa cells were seeded using MEM supplemented with 10% FBS and 0.01% antibiotic–antimycotic solution onto a 96-well culture plate and incubated for 24 h. The next day, medium was removed and cells were washed with 100 μL of PBS (Invitrogen Life Technologies). Subsequently, the diluted virus suspension (0.09 mL), containing a 50% cell culture infective dose (CCID50) of the virus, was added to Vero cells to produce the 50% CPE within 48 h after infection. Next, MEM containing 10 μM of the compounds (0.1 mL) was added. The culture plates were incubated at 37 °C in 5% CO2 for 2 days until the 50% CPE was achieved. Subsequently, the 96-well plates were washed once with 200 mL of PBS. Ice-cold 70% acetone in water (100 μL) was added to each well and incubated for 30 min at −20 °C. After removing the 70% acetone, plates were dried in a drying oven at 55 °C for 30 min. 0.4% (w/v) SRB in 1% acetic acid solution (100 μL) was added to each well and left for 30 min at room temperature. The SRB solution was then removed and the plates were washed 5 times with 1% acetic acid in water before oven-drying at 55 °C. Bound SRB was then solubilized with 10 mM unbuffered Tris-base (Sigma) solution (100 μL). After 30 min, the absorbance was read at 540 nm using a VERSAmax microplate reader (Molecular Devices, Palo Alto, CA, USA) with a reference absorbance at 620 nm. The antiviral activity of each test compounds in HRV1B-, CVB3-, and PR8-infected cells was calculated as a percentage of the corresponding untreated control. Acknowledgment This research was supported by the National Research Foundation of Korea (NRF) grant [NRF-2016R1A2B4006742] funded by the Ministry of Education, Science and Technology, Republic of Korea. References Alves, C.Q., David, J.M., David, J.P., Villareal, C.F., Soares, M.B., Queiroz, L. P. d., Aguiar, R.M., 2012. Flavonoids and other bioactive phenolics isolated from Cenostigma macrophyllum (Leguminosae). Quím. Nova 35, 1137–1140. Ban, N.K., Thoa, N.T.K., Linh, T.M., Van Kiem, P., Nhiem, N.X., Tai, B.H., Van Minh, C., Song, J.-H., Ko, H.-J., Kim, S.H., 2018. Labdane-type diterpenoids from Vitex limonifolia and their antivirus activities. J. Nat. Med. 72, 290–297. Banskota, A.H., Tezuka, Y., Tran, K.Q., Tanaka, K., Saiki, I., Kadota, S., 2000. Thirteen

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