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Cytotoxic quinones from the aerial parts of Morinda umbellata L.
Phytochemistry 167 (2019) 112096
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Cytotoxic quinones from the aerial parts of Morinda umbellata L. Changkang Li, Xianming Su, Fenghua Li, Jia Fu, Hongqing Wang, Baoming Li, Ruoyun Chen, ⁎ Jie Kang
T
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 1 Xiannongtan Street, Beijing, 100050, China
ARTICLE INFO
ABSTRACT
Keywords: Morinda umbellata L. (Rubiaceae) Quinones Cytotoxicity
Although Morinda umbellata L. has been used in numerous folk medicines, there is a lack of phytochemical studies on this plant. Sixteen undescribed quinones, namely, ten anthraquinones (umbellatas A–J), one naphthohydroquinone (umbellata K), one naphthohydroquinone dimer (umbellata L), and four dinaphthofuran quinones (umbellatas M–P), were isolated from the aerial parts of Morinda umbellata L. (Rubiaceae). The structures of all the isolated quinones were elucidated based on spectroscopic methods. Four of the unknown quinones (umbellatas A, H, K and M) showed potent cytotoxic effects against A431, A2780, NCI-H460, HCT116, HepG2, and MCF-7 human cancer cell lines with IC50 values of 1.3–7.1 μM. These results reveal potential lead compounds for the development of new anticancer agents.
1. Introduction As a leading cause of death worldwide, cancer accounted for approximately 9.6 million deaths in 2018 (Ambekar and Kandasubramanian, 2019). Drug therapy is an effective treatment for cancer, and 65% of anticancer drugs (1981–2014) have been obtained from natural products or related derivatives (Newman and Cragg, 2016). In particular, various natural quinones have shown potent antitumor effects. For example, daunomycin, an anthraquinone-based anticancer drug obtained from Streptomyces peucetius (Ho and Nodwell, 2016), has been used in the treatment of leukemia and other neoplasms for many years (Newman and Cragg, 2007). In our previous study, the quinones isolated from Morinda parvifolia showed potent cytotoxic activities (Kang et al., 2016), which attracted us to further investigate cytotoxic quinones from the other medicinal plants in the same genus. Morinda umbellata L. (Rubiaceae) is a scandent shrub mainly distributed in the south of China (Luo et al., 1999). Its roots, bark, stems, and leaves have been used as folk medicines for the treatment of many diseases, including fever, coughing, stomach ache, and acute hepatitis (Ban et al., 2013; Chiou et al., 2014). However, the phytochemical and pharmaceutical properties of M. umbellata have not been extensively investigated. Only three papers have reported cytotoxic anthraquinones obtained from M. umbellata (Ban et al., 2013; Chiou et al., 2014; Pong and Chia-Fu, 1995). In our current study, the EtOAc fraction of the 95% EtOH extract of
the aerial parts of M. umbellata showed cytotoxic activity against human colorectal cancer cell line HCT116 with an IC50 value of 15.15 μg/mL. The bioactivity-guided isolation gave 16 undescribed quinones (umbellatas A–P) from the EtOAc fraction. Four of them (1, 8, 11, and 13) showed cytotoxic effects against A431, A2780, NCI-H460, HCT116, HepG2, or MCF-7 cancer cell lines with IC50 values of 1.3–7.1 μM. 2. Results The EtOAc fraction of the 95% EtOH extract of the aerial parts of M. umbellata yielded sixteen undescribed quinones, umbellatas A–P (1–16). The structures of 1–16 (Fig. 1) were elucidated by spectroscopic analysis. 2.1. Structural analysis Compound 1 was obtained as a yellow powder. Its molecular formula was established as C15H10O4 on the basis of HRESIMS data (m/z 253.0506 [M - H]-, calcd. for C15H9O4, 253.0506), indicating 11 degrees of unsaturation. The IR spectrum showed absorption bands that clearly indicated the presence of hydroxy groups (3426 cm−1), carbonyl groups (1668 cm−1), and aromatic rings (1588 and 1494 cm−1). The 1H NMR spectrum of 1 (Table 1) showed an oxygenated methylene at δH 4.69 (2H, s) and signals corresponding to two ABX systems [δH 8.13 (1H, br d), 8.11 (1H, d, J = 7.6 Hz), 7.79 (1H, dd, J = 7.6, 1.2 Hz)
Corresponding author. E-mail addresses: [email protected] (C. Li), [email protected] (X. Su), [email protected] (F. Li), [email protected] (J. Fu), [email protected] (H. Wang), [email protected] (B. Li), [email protected] (R. Chen), [email protected] (J. Kang). ⁎
https://doi.org/10.1016/j.phytochem.2019.112096 Received 30 April 2019; Received in revised form 10 August 2019; Accepted 11 August 2019 Available online 27 August 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
Phytochemistry 167 (2019) 112096
C. Li, et al.
Fig. 1. Structures of compounds 1–16.
and δH 8.07 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 2.4 Hz), 7.17 (1H, dd, J = 8.4, 2.4 Hz)]. The 13C NMR spectrum (Table 2) showed 15 carbon signals corresponding to six aromatic methines (δC 131.2, 129.9, 126.7, 123.9, 122.0, 112.7), six aromatic quaternary carbons (δC 165.4, 149.8, 135.3, 133.4, 131.8, 123.9), two carbonyl carbons (δC 182.8, 181.0), and one oxygenated methylene group (δC 62.3), consistent with an anthraquinone skeleton (Cai et al., 2005). The key HMBC correlations of H-4 (δH 8.07)/C-10 (δC 181.0), C-2 (δC 165.4), C-9a (δC 135.3); H-5 (δH 8.13)/C-10 (δC 181.0); and –CH2OH (δH 4.69)/C-7 (δC 131.2), C-5 (δC 123.9) (Fig. 2) suggested the presence of a hydroxy group at C-2 and a hydroxymethyl group at C-6. Based on the above evidence, compound 1 was identified as 2-hydroxy-6-hydroxymethylanthraquinone (umbellata A). Compound 2 was isolated as a yellow powder. Its molecular formula was deduced as C16H12O4 using HRESIMS (m/z 269.0805 [M + H]+, calcd. for C16H13O4, 269.0808). The 1H and 13C NMR spectroscopic data of 2 (Tables 1 and 2) were similar to those of 2-hydroxymethylanthraquinone (Park et al., 2006), but 2 indicated the presence of an extra methoxy group. The position of this group was confirmed by the key HMBC correlations (Fig. 2) of –OCH3 (δH 3.78)/C-6 (δC 165.0), H-8 (δH 8.41)/C-9 (δC 182.7), and C-6 (δC 165.0), C-10a (δC 136.5). Therefore, compound 2 was determined to be 2-hydroxymethyl6-methoxyanthraquinone (umbellata B). Compound 3 was obtained as a yellow powder. A molecular formula of C15H10O5 was determined from the HRESIMS spectrum, which showed a [M - H]- ion peak at m/z 269.0452 (calcd. for C15H9O5, 269.0456). The molecular formula of compound 3 is the same as that of 1,7-dihydroxy-2-hydroxymethylanthraquinone (Cai et al., 2005). The 1 H NMR signals (Table 1) included one ABX system [δH 8.06 (1H, d, J = 8.4 Hz), 7.38 (1H, d, J = 2.0 Hz), 7.14 (1H, dd, J = 8.4, 2.0 Hz)], a pair of ortho-coupled protons [δH 7.82 (1H, d, J = 7.6 Hz), 7.67 (1H, d, J = 7.6 Hz)], and an oxygenated methylene [δH 4.62 (2H, s)], which is similar to the 1H NMR data of 1,7-dihydroxy-2-hydroxymethylanthraquinone (Cai et al., 2005). They differ only in the position of one hydroxy group, which is located at C-6 in 3 but at C-7 in
the known compound (Cai et al., 2005). An oxymethylene proton signal at δH 4.62 correlated with the signal at δC 158.8 (C-1) and δC 133.0 (C3), H-5 (δH 7.38) correlated with C-10 (δC 182.8), C-8a (δC 123.3), and C-7 (δC 122.5), and H-8 (δH 8.06) correlated with C-9 (δC 187.6), C-6 (δC 167.1) and C-10a (δC 136.1) in the HMBC spectrum (Fig. 2) suggested that an oxymethylene group was at C-2, and two hydroxy groups were connected to C-1 and C-6, respectively. Accordingly, compound 3 was established as 1,6-dihydroxy-2-hydroxymethylanthraquinone (umbellata C). Compound 4 was separated as a yellow powder. It was determined to have a molecular formula of C16H12O5 from the [M - H]- ion peak at m/z 283.0613 (calcd. for C16H11O5, 283.0612). A comparison of the 1H and 13C NMR spectral data for compound 4 (Tables 1 and 2) and 1,3dihydroxyanthraquinone (Akrawi et al., 2011) demonstrated that these compounds had identical B- and C-rings, but different A-rings. A methoxymethyl group was connected to C-7 in the A-ring of 4, as determined by the HMBC correlations of –CH2OCH3 (δH 4.60) with C-6 (δC 132.2) and C-8 (δC 124.3). Based on the above analysis, compound 4 was determined to be 1,3-dihydroxy-7-methoxymethylanthraquinone (umbellata D). Compound 5, isolated as a yellow powder, had a molecular formula of C17H14O5, as determined based on HRESIMS (m/z 299.0907 [M + H]+, calcd. for C17H15O5, 299.0914), indicating 11 degrees of unsaturation. The 1H and 13C NMR data of 5 (Tables 1 and 2) were nearly identical to those of 4, except that 5 possessed a methoxy group instead of a hydroxy group at C-1, as confirmed by the HMBC correlations of –OCH3 (δH 3.86)/C-1 (δC 163.0) and H-4 (δH 7.13)/C-10 (δC 183.0), C9a (δC 112.0), C-2 (δC 105.2) as well as the 1D NOESY correlation between –OCH3 (δH 3.86) and H-2 (δH 6.74). Thus, compound 5 was established as 3-hydroxy-1-methoxy-7-methoxymethylanthraquinone (umbellata E). Compound 6, obtained as a yellow powder, had a molecular formula of C16H12O5, as established by HRESIMS (m/z 283.0616 [M - H]-, calcd. for C16H11O5, 283.0612). The NMR data indicated that the structure of 6 (Tables 1 and 2) was similar to that of 5. Notably, 5 and 6 differed in 2
7.17 dd (8.4, 2.4) 8.07 d (8.4)
8.13 br d
3
5
3
g
f
e
d
c
b
a
4.69 (2H, s)
3.78 (3H, s)
5.09 (2H, s)
7.33 br d (8.4) 8.41 d (8.4)
7.98 br d (8.0) 8.45 d (8.0) 7.89 br s
8.71 br s
2b
4.62 (2H, s)
13.25 br s
7.14 dd (8.4, 2.0) 8.06 d (8.4)
7.38 d (2.0)
7.67 d (7.6)
7.82 d (7.6)
3a
Recorded in DMSO‑d6 at 400 MHz. Recorded in pyridine-d5 at 400 MHz. Recorded in DMSO‑d6 at 600 MHz. Recorded in DMSO‑d6 at 500 MHz. Recorded in CD3OD at 400 MHz. Recorded in CDCl3 at 400 MHz. Recorded in pyridine-d5 at 600 MHz.
7-CH2OCH3
6-CH2OH 6-CH2OCH3 6-CH2OCH3 6-COOCH3 7-OCH3 7-CH2OCH3
2-COOCH3 3-COOCH3 3′-COOCH3 4-OCH3 4′-OCH3 5-OCH3 6-OCH3
2-CH2OH
11 5′ 6′ 7′ 8′ 1-OH 1-OCH3
10
8
7
6
7.79 dd (7.6, 1.2) 8.11 d (7.6)
7.43 d (2.4)
1 2
4
1a
Position
Table 1 1 H NMR data of compounds 1–16.
4.60 (2H, s) 3.38 (3H, s)
12.87 br s
8.08 br s
7.05 d (1.2) 8.10 d (8.0) 7.77 d (8.0)
6.46 d (1.2)
4a
4.60 (2H, s) 3.37 (3H, s)
3.86 (3H, s)
8.03 br s
8.07 d (7.8) 7.71 br d (7.8)
7.13 br s
6.74 br s
5c
3.93 (3H, s)
3.80 (3H, s)
7.52 d (2.4)
7.27 dd (8.4, 2.4)
8.02 d (8.4)
6.99 br s
6.58 br s
6c
4.60 (2H, s) 3.37 (3H, s)
7.76 dd (8.0, 1.2) 8.10 d (8.0)
8.04 d (1.2)
7.49 s
7.49 s
7a
3.80 (3H, s)
7.18 dd (8.8, 2.4) 8.02 d (8.8)
7.41 d (2.4)
7.88 d (8.4)
7.23 d (8.4)
8a
6.36 (2H, s)
7.20 dd (9.0, 2.5) 8.03 d (9.0)
7.46 d (2.5)
7.80 d (8.0)
7.33 d (8.0)
9d
6.30 (2H, s)
8.11–8.14 m
7.83–7.89 m
7.83–7.89 m
8.11–8.14 m
7.35 s
10d
3.91 (3H, s)
3.92 (3H, s)
7.14 dd (9.2, 2.8) 8.05 d (9.2)
7.45 d (2.8)
7.09 s
11e
3.59 3.59 4.02 4.02
(3H, (3H, (3H, (3H,
s) s) s) s)
8.17 d (8.0) 7.60–7.67 m 7.60–7.67 m 8.36 d (8.0)
8.36 d (8.0)
7.60–7.67 m
7.60–7.67 m
8.17 d (8.0)
12f
3.99 (3H, s)
3.96 (3H, s)
7.04 br d (8.4) 7.96 d (8.4)
7.30 br s
7.34 dd (9.2, 2.0) 8.11 d (9.2)
7.59 d (2.0)
13a
3.92 (3H, s)
7.14 dd (8.4, 2.4) 7.99 d (8.4)
7.39 d (2.4)
7.24 dd (9.2, 2.0) 8.26 d (9.2)
7.51 d (2.0)
14a
4.05 (3H, s)
4.01(3H, s)
7.17 br d (8.5) 8.03 d (8.5)
7.41 d (2.5)
8.28 d (8.0)
7.86 t (8.0)
8.49 d (8.0) 7.91 t (8.0)
15d
4.34 (3H, s)
4.17(3H, s)
7.42 dd (8.4,1.8) 8.02 d (1.8)
8.29 d (8.4)
8.29–8.33 (m)
7.68–7.71 (m)
8.29–8.33 (m) 7.68–7.71 (m)
16g
C. Li, et al.
Phytochemistry 167 (2019) 112096
Phytochemistry 167 (2019) 112096
C. Li, et al.
Table 2 13 C NMR data of compounds 1–16 (δ in ppm). Position
1a
2b
3a
4a
5c
6c
7a
8c
9d
10d
11e
12f
13c
14c
15d
16g
1 2 2a 3 3a 4 4a 5 6 6a 7 8 8a 9 9a 10 10a 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 3′a 4′ 4′a 5′ 6′ 7′ 8′ 8′a 1-OCH3 2-CH2OH 2-COOCH3 3-COOCH3 3′-COOCH3 4-OCH3 4′-OCH3 5-OCH3 6-OCH3 6-COOCH3 6-CH2OH 6-CH2OCH3 6-CH2OCH3 7-OCH3 7-CH2OCH3 7-CH2OCH3
112.7 165.4
125.2 151.2
158.8 138.8
165.2 107.7
163.0 105.2
163.3 105.1
112.8 152.1
147.7 158.7
148.4 154.9
149.7 140.5
147.8 110.4
102.9 158.6
102.7 158.8
121.2 129.5
122.7 129.5
122.0
132.1
133.0
168.3
166.3
NDh
152.1
120.9
112.6
147.4
152.9 116.7 168.3 108.3
120.5
119.1
128.8
129.1
129.9 123.9 123.9 149.8
127.9 133.2 111.1 165.0
118.9 132.0 113.8 167.1
110.0 134.8 127.0 132.2
107.5 136.5 126.1 130.9
109.3 136.5 128.4 118.4
112.8 126.7 124.6 144.8
125.0 125.0 111.4 162.5
124.1 127.6 113.0 164.5
112.4 127.9 126.6 133.7
149.2 131.9 105.6 158.5
124.3 167.5 148.2 129.0 122.8 128.1
126.1
126.9
123.9
124.6
131.2 126.7 131.8 182.8 135.3 181.0 133.4
121.2 130.3 128.0 182.7 134.6 183.4 136.5
122.5 130.3 123.3 187.6 115.2 182.8 136.1
145.6 124.3 133.4 184.7 108.2 182.0 132.0
145.3 124.5 135.0 178.6 112.0 183.0 131.0
163.9 109.9 137.8 177.4 110.2 182.5 125.5
132.1 126.6 132.4 181.4 126.7 181.6 133.3
121.1 129.6 126.7 181.2 126.6 181.4 134.7
122.2 130.1 125.8 180.2 116.8 181.8 136.0 104.6
134.2 126.3 133.0 179.3 109.5 181.4 133.0 103.6
120.0 126.4 124.9
127.5 123.8 126.5
152.1 115.8 166.1 180.8 114.2
153.5 124.2 167.6 180.3 113.4
151.6 118.2 165.9 180.5 113.5
153.0 116.9 167.2 180.3 130.5
166.8
164.3
164.4
121.8
121.0
120.3
120.7
165.4
129.5 172.7 154.5 147.5 123.4 120.8 NDh 114.7 135.5 123.4
129.2 172.9 158.8 145.5 123.7 119.8 NDh 116.6 135.9 123.1
129.6 173.0 154.5 148.5 121.5 127.0 NDh 115.3 135.4 123.5
114.2 175.4 154.2 149.9 122.1 128.7 NDh 119.9 125.8 135.5
64.1
64.6
52.7
53.3
a b c d e f g h
63.9
55.9
58.0
55.5
60.4
63.6 52.4
56.2
147.8 110.4 124.3 167.5 148.2 129.0 122.8 128.1 127.5 123.8 126.5
52.5 52.5 63.9 63.9
64.0 52.5
62.3
72.6 58.0
72.7 57.9
55.7
52.0
72.7 58.0
Recorded in DMSO‑d6 at 100 MHz. Recorded in pyridine-d5 at 100 MHz. Recorded in DMSO‑d6 at 150 MHz. Recorded in DMSO‑d6 at 125 MHz. Recorded in CD3OD at 100 MHz. Recorded in CDCl3 at 125 MHz. Recorded in pyridine-d5 at 150 MHz. ND: no detected.
the substituent at C-7, with the methoxymethyl group in 5 replaced by a methoxy group in 6. This was confirmed by the HMBC correlations of –OCH3 (δH 3.93)/C-7 (δC 163.9) and H-5 (δH 8.02)/C-10 (δC 182.5), C-7 (δC 163.9), C-8a (δC 137.8). Thus, compound 6 was determined to be 3hydroxy-1,7-dimethoxyanthraquinone (umbellata F). Compound 7, isolated as a yellow powder, was assigned the molecular formula of C16H12O5 based on its HRESIMS data (m/z 283.0609 [M - H]-, calcd. for C16H11O5, 283.0612). Compounds 7 and 4 are
isomers, but the positions of the hydroxy and methoxymethyl groups differed. In 1H NMR spectrum, one ABX system [δH 8.10 (1H, d, J = 8.0 Hz), 8.04 (1H, d, J = 1.2 Hz), 7.76 (1H, dd, J = 8.0, 1.2 Hz)], an aromatic singlet at δH 7.49 (2H, s), an oxygenated methylene [δH 4.60 (2H, s)] and one methoxy [δH 3.37 (3H, s)] were shown. The HMBC correlations of H-1 (δH 7.49)/C-9 (δC 181.4), C-3 (δC 152.1), C4a (δC 126.7); H-4 (δH 7.49)/C-10 (δC 181.6), C-2 (δC 152.1), C-9a (δC 126.7); –CH2OCH3 (δH 4.60)/C-7 (δC 132.1), C-5 (δC 124.6); H-8 (δH 4
Phytochemistry 167 (2019) 112096
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Fig. 2. Key HMBC correlations (blue arrows) and NOEs (red arrows) of compounds 1–16. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
8.10)/C-9 (δC 181.4), C-6 (δC 144.8), C-10a (δC 133.3), and H-5 (δH 8.04)/C-10 (δC 181.6), C-8a (δC 132.4), C-7 (δC 132.1) confirmed that two hydroxy groups and one methoxymethyl group were located at C-2, C-3, and C-6, respectively. Accordingly, compound 7 was identified as 2,3-dihydroxy-6-methoxymethylanthraquinone (umbellata G). Compound 8, isolated as a yellow powder, had a molecular formula of C15H10O5, as determined by HRESIMS (m/z 271.0605 [M + H]+, calcd. for C15H11O5, 271.0601), which indicated 11 degrees of unsaturation. The 1H and 13C NMR data of 8 (Tables 1 and 2) exhibited similarities to those of 1,2,6-trihydroxyanthraquinone (flavopurpurin) (Ruksilp et al., 2011), except that a hydroxy group at C-1 in flavopurpurin was replaced by a methoxy group in 8. The key HMBC correlations of –OCH3 (δH 3.80)/C-1 (δC 147.7) and H-3 (δH 7.23)/C-1 (δC 147.7), C-4a (δC 125.0) confirmed the position of the methoxy group in 8 (Fig. 2). Consequently, compound 8 was determined to be 2,6-dihydroxy-1-methoxyanthraquinone (umbellata H). Compound 9, obtained as a yellow powder, showed a molecular ion at m/z 291.0263 [M + Na]+ (calcd. for C15H8O5Na, 291.0264), indicative of a molecular formula of C15H8O5 and 12 degrees of unsaturation. The NMR data of compound 9 (Tables 1 and 2) were similar to those of morindaparvin A (Chang et al., 1982), but 9 contained an extra hydroxy group. The HMBC correlations of H-8 (δH 8.03)/C-9 (δC 180.2), C-6 (δC 164.5), C-10a (δC 136.0) (Fig. 2) demonstrated that the extra hydroxy group was located at C-6. Thus, compound 9 was determined to be 6-hydroxy-1,2-methylenedioxyanthraquinone (umbellata I). Compound 10 was obtained as a yellow powder. The [M - H]- ion peak at m/z 267.0296 (calcd. for C15H7O5, 267.0299) in the HRESIMS spectrum was attributed to a molecular formula of C15H8O5, which indicated compound 10 is an isomer of 9, but the position of the hydroxy group is different. The 1H NMR spectrum showed one AA′BB′ system [δH 8.11–8.14 (2H, m), 7.83–7.89 (2H, m)], one aromatic singlet at δH 7.35 (1H, s) and one aliphatic singlet at δH 6.30 (2H, s). The HMBC correlations of H-4 (δH 7.35)/C-10 (δC 181.4), C-2 (δC 140.5), and C-9a (δC 109.5), and H-11 (δH 6.30)/C-1 (δC 149.7) and C-2 (δC
140.5) (Fig. 2) indicated that a hydroxy group at C-3 and the group of –O–CH2–O– was connected at C-1 and C-2 in 10. Consequently, 10 was identified as 3-hydroxy-1,2-methylenedioxyanthraquinone (umbellata J). Compound 11 was obtained as a colorless powder. It was determined to have a molecular formula of C13H12O5 based on the [M + Na]+ ion peak at m/z 271.0575 (calcd. for C13H12O5Na, 271.0577), which indicated 8 degrees of unsaturation. The NMR data of compound 11 were closely related to those of methyl 4-hydroxy-1,6-dimethoxynaphthalene-2-carboxylate (morindaparvin C) (Kang et al., 2016), except that 11 had a hydroxy group at C-6 instead of the methoxy group in morindaparvin C. The HMBC correlations of H-8 (δH 8.05)/C-6 (158.5), C-1(152.9), C-4a (131.9), OCH3-1 (δH 3.92)/C-1 (152.9), and OCH3 (δH 3.91)/C-2a (C]O) (168.3) indicated the presence of the hydroxy group at C-6 and two methoxy groups at C-1 and C-2a (C]O), respectively. The NOE correlations of OCH3-1 (δH 3.92)/H-8 (δH 8.05) and OCH3 (δH 3.91)/H-3 (δH 7.09) further confirmed the positions of the two methoxy groups. Thus, compound 11 was determined to be methyl 4,6-dihydroxy-1-methoxynaphthalene-2-carboxylate (umbellata K). Compound 12, obtained as a red powder, had a molecular formula of C26H22O8, as determined from the HRESIMS data (m/z 461.1242 [M H]-, calcd. for C26H21O8, 461.1242), which indicated 16 degrees of unsaturation. The 1H NMR spectrum of 12 exhibited one AA′BB′ system [δH 8.36 (1H, d, J = 8.0 Hz), 8.17 (1H, d, J = 8.0 Hz), 7.60–7.67 (2H, m)] and two methoxy groups [δH 4.02 (3H, s), 3.59 (3H, s)]. The 13C NMR spectrum (Table 2) revealed 13 carbon resonances, including those corresponding to a carbonyl group (δC 167.5), four aromatic methines (δC 128.1, 127.5, 123.8, 122.8), and six aromatic quaternary carbons (δC 148.2, 147.8, 129.0, 126.5, 124.3, 110.4). These NMR data in combination with the molecular weight indicated that 12 was a symmetric dimer, similar to that of dimethyl 1,1′-dihydroxy-4,4′,7,7′tetramethoxy-2,2′-binaphthalene-3,3′-dicarboxylate (morindaparvin G) (Kang et al., 2016). The only difference between these compounds was the absence of two methoxy groups at C-7 and C-7′ in 12. The NOE 5
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(Table 2) spectroscopic data of 15 were very similar to those of 13, except that one hydroxy group was absent at C-2 in 15. This difference was in accordance with the HMBC correlations of H-4 (δH 8.28)/C-5 (δC 151.6), C-2 (129.5), C-15 (δC 121.5) (Fig. 2). Therefore, compound 15 was determined to be methyl 7,12-dihydro-9-hydroxy-5-methoxy-7,12dioxodinaphtho[1,2-b:2′,3′-d]furan-6-carboxylate (umbellata O). Compound 16 was found to have the same molecular formula as compound 15 (C23H14O7), as determined by HRESIMS (m/z 403.0819 [M + H]+, calcd. for C23H15O7, 403.0812), suggesting that compounds 16 and 15 are isomers. However, the position of the hydroxy group differed in these two compounds. The HMBC correlations of H-8 (δH 8.29)/C-7 (δC 180.3), C-10 (δC 165.4), C-20 (δC 135.5) and H-11 (δH 8.02)/C-12 (δH 175.4), C-19 (δC 125.8), C-9 (δC 121.8) (Fig. 2) confirmed that the hydroxy group was located at C-10 in 16 instead of C-9 in 15. Hence, compound 16 was identified as methyl 7,12-dihydro-10hydroxy-5-methoxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d]furan-6-carboxylate (umbellata P).
Table 3 The IC50 values (μM) for the cytotoxic effects of compounds 1, 8, 11, 13 and positive control (taxol). No.
A431
A2780
NCI-H460
HCT116
HepG2
MCF-7
1 8 11 13 taxol
23.9 NDa 48.5 5.9 0.004
NDa NDa 20.5 6.4 0.01
NDa NDa 14.7 7.1 0.004
NDa 2.4 1.3 4.1 0.009
NDa NDa 5.8 6.2 0.0005
4.8 NDa 4.9 16.5 0.08
A431(human skin squamous cancer cell lines); A2780 (ovarian cancer cell lines); NCI-H460 (human lung cancer cell lines); HCT116 (human colorectal cancer cell lines); HepG2 (human liver cancer cell lines); MCF-7 (human breast cancer cell lines). a ND: no detected (> 50 μM).
correlation of OCH3 (δH 4.02)/OCH3 (δH 3.59) and OCH3 (δH 4.02)/H-5 (H-5′) (δH 8.17) confirmed that the four methoxy groups were located at C-4 (C-4′) and C-3a (C-3′a) (C]O), respectively. Therefore, compound 12 was determined to be dimethyl 1,1′-dihydroxy-4,4′-dimethoxy-2,2′-binaphthalene-3,3′-dicarboxylate (umbellata L). Compound 13 was obtained as a yellow powder. Its molecular formula was established as C23H14O8 on the basis of HRESIMS data (m/z 417.0620 [M - H]-, calcd. for C23H13O8, 417.0616), indicating 17 degrees of unsaturation. The IR spectrum suggested the presence of hydroxyl groups (3244 cm−1), carbonyl groups (1723 and 1680 cm−1), and aromatic rings (1584 cm−1). The 1H NMR spectrum of 13 (Table 1) showed two methoxy protons at δH 3.99 (3H, s) and 3.96 (3H, s) as well as two ABX systems [δH 8.11 (1H, d, J = 9.2 Hz), 7.59 (1H, d, J = 2.0 Hz), 7.34 (1H, dd, J = 9.2, 2.0 Hz) and δH 7.96 (1H, d, J = 8.4 Hz), 7.30 (1H, br s), 7.04 (1H, br d, J = 8.4 Hz)]. The 13C NMR spectrum showed 22 carbon signals (should be 23, but one was not detected), including three carbonyl carbons (δC 180.8, 172.7, 166.1), six aromatic protonated carbons (δC 129.5, 126.1, 121.0, 120.5, 114.2, 102.9), eleven aromatic quaternary carbons (should be twelve, but one was not detected) (δC 166.8, 158.6, 154.5, 152.1, 147.5, 135.5, 123.4 × 2, 120.8, 115.8, 114.7), and two methoxy groups (δC 64.0, 52.5). The NMR data of 13 resembled those of methyl 7,12-dihydro-5hydroxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d]furan-6-carboxylate (Kang et al., 1999; Park et al., 2009), but there were two extra hydroxy groups in 13 at C-2 and C-9, and a methoxy group at C-5 in 13 replaced a hydroxy group in the known compound. These differences were confirmed by the key HMBC correlations of H-4 (δH 8.11)/C-2 (δC 158.6), C-5 (δC 152.1), C-15 (δC 123.4), H-11 (δH 7.96)/C-12 (δC 172.7), C-9 (δC 166.8), C-19 (δC 135.5) and OCH3-5 (δH 3.96)/C-5 (δC 152.1) (Fig. 2). The NOE correlation of OCH3 (δH 3.96)/H-4 (δH 8.11) further confirmed that the methoxy group was located at C-5. Based on the above evidence, compound 13 was identified as methyl 7,12-dihydro-2,9-dihydroxy-5-methoxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d]furan-6-carboxylate (umbellata M). Compound 14 was isolated as a yellow powder. A molecular formula of C22H12O8 was deduced from the HRESIMS data (m/z 403.0449 [M - H]-, calcd. for C22H11O8, 403.0459), implying 17 degrees of unsaturation. The 1H and 13C NMR data of 14 were nearly identical to those of 13. Furthermore, the molecular formula of 14 indicated that its molecular weight was 14 Da less than that of 13. This difference was caused by a hydroxy group at C-5 in 14 replacing a methoxy group in 13. The HMBC correlation between the methoxy group at δH 3.92 and the carbonyl carbon at δC 167.6 suggested that the methoxy group was connected to C-6a (C]O). Thus, compound 14 was identified as methyl 7,12-dihydro-2,5,9-trihydroxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d] furan-6-carboxylate (umbellata N). Compound 15, obtained as a yellow powder, displayed a molecular ion peak [M - H]- at m/z 401.0656 (calcd. for C23H13O7, 401.0667), indicating a molecular formula of C23H13O7. Thus, 15 has one less oxygen atom than compound 13. The 1H (Table 1) and 13C NMR
2.2. Cytotoxic effects The cytotoxic activities of compounds 1–16 were evaluated against a panel of six human tumor cell lines, including A431 (human skin squamous carcinoma cell line), A2780 (ovarian cancer cell line), NCIH460 (human lung cancer cell line), HCT116 (human colorectal carcinoma cell line), HepG2 (human liver carcinoma cell line), and MCF-7 (human breast carcinoma cell line), using the MTT method. Compounds 1, 8, 11, and 13 were found to exhibit potent cytotoxicity against the five cell lines with IC50 values ranging from 1.3 to 7.1 μM (Table 3). These results suggest that the quinones isolated from M. umbellata may be valuable drug candidates. 3. Discussion Bioactivity-guided isolation from the EtOAc fraction of the 95% EtOH extract of the aerial parts of M. umbellata gave sixteen undescribed quinones (umbellatas A–P). Structural elucidation of all the compounds was successfully achieved by spectroscopic analyses and comparisons with literature data. Because compounds 2, 4–8 and 11–16 having methoxy, methoxymethyl, or carbomethoxy groups in the structures, HPLC/DAD/(-)ESIMS was used to prove they are not artificial compounds produced during separation. Extracted ion chromatograms (EICs) of compounds 2, 4–8 and 11–16 were observed in the EtOAc fraction of 95% EtOH extract of Morinda umbellata. Thus, compounds 2, 4–8 and 11–16 are not artificial products. Among the isolated compounds, anthraquinones (1 and 8), naphthohydroquinone (11) and dinaphthofuran quinone (13) showed selective cytotoxic activities against human skin, ovarian, lung, colorectal, liver, and breast cancer cell lines. These findings indicate the potential of the quinones isolated from M. umbellata to act as lead compounds for developing cancer treatments based on natural remedies. 4. Experimental 4.1. General experimental procedures Optical rotations were measured on a JASCO P-2000 automatic digital polarimeter (Tokyo, Japan)). UV spectra were collected in MeOH on a JASCO V-650 spectrophotometer. IR spectra were recorded on a Nicolet 5700 spectrometer (Thermo Electron Corporation, Madison, WI, USA) using the FT-IR microscope transmission method. NMR spectra were acquired using a JEOL ECZ-400 spectrometer (JEOL, Tokyo, Japan), a Bruker Avance III-400 (or 500 or 600) spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), or an Agilent VNMRS 600 (600 MHz) spectrometer (Palo Alto, CA, USA). HRESIMS data were recorded on an Agilent 1200 SL series LC/6520 QTOF spectrometer 6
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(Böblingen, Germany) or a Thermo Scientific Q Exactive Focus Orbitrap mass spectrometer (Waltham, MA, USA). Column chromatography purification was performed using Sephadex LH-20 (GE Healthcare BioSciences AB, Uppsala, Sweden) and C-18 (50 mm, YMC, Kyoto, Japan). Semi-preparative HPLC separation was performed using a LC-6AD semipreparative HPLC with an Agilent column (XDB-C18, 5 μm, 9.4 × 250 mm). The cancer cell lines were purchased from Shanghai Fengshou Industrial Co., Ltd. (Shanghai, China).
4.4.3. 1,6-Dihydroxy-2-hydroxymethylanthraquinone (umbellata C) (3) Yellow amorphous powder; UV (MeOH) λmax (log ε) 219 (1.91), 269 (1.87), 409 (1.31) nm; IR vmax 3363, 1660, 1626, 1578, 1477 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 269.0452 [M - H]- (calcd. for C15H9O5, 269.0456). 4.4.4. 1,3-Dihydroxy-7-methoxymethylanthraquinone (umbellata D) (4) Yellow amorphous powder; UV (MeOH) λmax (log ε) 204 (2.12), 247 (1.99), 284 (1.98), 415 (1.38) nm; IR vmax 3343, 1666, 1633, 1599, 1459 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 283.0613 [M - H]- (calcd. for C16H11O5, 283.0612).
4.2. Plant materials The aerial parts of Morinda umbellata L. (Rubiaceae) were collected from Ganshiling Nature Reserve (GPS coordinates: N 18°39′-18°38′, E 109°66′-109°65′), Hainan Province, China in July 2014 (wet season), and were identified by Professor Lin Ma of the Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, China. A voucher specimen (ID-22679) was deposited at the Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, China.
4.4.5. 3-Hydroxy-1-methoxy-7-methoxymethylanthraquinone (umbellata E) (5) Yellow amorphous powder; UV (MeOH) λmax (log ε) 203 (1.97), 250 (1.89), 282 (1.72), 336 (0.95), 398 (0.97) nm; IR vmax 3394, 1644, 1588, 1418 cm−1; 1H NMR (DMSO‑d6, 600 MHz) and 13C NMR (DMSO‑d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 299.0907 [M + H]+ (calcd. for C17H15O5, 299.0914).
4.3. Extraction and isolation Air-dried, powdered aerial parts of M. umbellata (42 kg) were extracted with EtOH–H2O (95: 5, v/v, × 3) under reflux conditions for 2 h and concentrated under reduced pressure. The residue (2.6 kg) was suspended in H2O and successively partitioned with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract (190 g) was chromatographed on a silica gel (100–200 mesh) column (100 × 40 cm) eluted with a petroleum ether–EtOAc gradient system (95: 5 to 50: 50, v/v) to give fractions 1–28. Fractions 18–20 (12 g) were subjected to chromatography on a C18 medium-pressure column (46 × 6 cm) eluted with a MeOH–H2O gradient system (30:70 to 70: 30, v/v, 20 mL/min) to yield 82 sub-fractions (1a–82a). Sub-fraction 24a (60 mg) was further separated by semi-preparative HPLC eluted isocratically (MeCN–H2O = 2: 8, v/v, 4 mL/min) to give 11 (30 mg, tR = 24.6 min) and 12 (4 mg, tR = 43.4 min). Compounds 1 (10 mg, tR = 25.8 min) and 8 (3 mg, tR = 20.4 min) were isolated from sub-fraction 40a (40 mg) by semipreparative HPLC eluted with MeCN–H2O (2: 8, v/v, 4 mL/min). Subfraction 32a (100 mg) was subjected to a Sephadex LH-20 column eluted with MeOH–H2O (3: 7, v/v, 4 mL/min) to obtain 4 (8 mg, tR = 48.4 min), 5 (5 mg, tR = 38.2 min), 6 (1 mg, tR = 42.0 min), and 7 (10 mg, tR = 36.8 min). Purification of sub-fraction 69a (200 mg) by semi-preparative HPLC using MeCN–H2O (26: 74, v/v, 4 mL/min) afforded 2 (6 mg, tR = 29.7 min), 3 (4 mg, tR = 25.5 min), 9 (10 mg, tR = 40.8 min), and 10 (20 mg, tR = 46.7 min). Compounds 13 (20 mg, tR = 38.0 min), 14 (5 mg, tR = 45.8 min), 15 (5 mg, tR = 46.7 min) and 16 (1 mg, tR = 43.4 min) were obtained from sub-fraction 80a (500 mg) by Sephadex LH-20 column eluted with MeOH–H2O (4: 6, v/v, 4 mL/ min) and semi-preparative HPLC using MeCN–H2O (3: 7, v/v, 4 mL/ min).
4.4.6. 3-Hydroxy-1,7-dimethoxyanthraquinone (umbellata F) (6) Yellow amorphous powder; UV (MeOH) λmax (log ε) 205 (2.05), 276 (1.86), 379 (1.11) nm; IR vmax 3312, 1673, 1596, 1466 cm−1; 1H NMR (DMSO‑d6, 600 MHz) and 13C NMR (DMSO‑d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 283.0616 [M - H]- (calcd. for C16H11O5, 283.0612). 4.4.7. 2,3-Dihydroxy-6-methoxymethylanthraquinone (umbellata G) (7) Yellow amorphous powder; UV (MeOH) λmax (log ε) 205 (2.02), 242 (1.72), 288 (2.15) nm; IR vmax 3355, 1664, 1583, 1462 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 283.0609 [M - H]- (calcd. for C16H11O5, 283.0612). 4.4.8. 2,6-Dihydroxy-1-methoxyanthraquinone (umbellata H) (8) Yellow amorphous powder; UV (MeOH) λmax (log ε) 220 (1.78), 272 (1.81), 299 (1.60), 359 (1.26) nm; IR vmax 3191, 1650, 1563, 1468 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 271.0605 [M + H]+ (calcd. for C15H11O5, 271.0601). 4.4.9. 6-Hydroxy-1,2-methylenedioxyanthraquinone (umbellata I) (9) Yellow amorphous powder; UV (MeOH) λmax (log ε) 224 (1.88), 270 (1.89), 380 (1.36) nm; IR vmax 3377, 1666, 1578, 1450 cm−1; 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR (DMSO‑d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 291.0263 [M + Na]+ (calcd. for C15H8O5Na, 291.0264).
4.4. Compound characterization
4.4.10. 3-Hydroxy-1,2-methylenedioxyanthraquinone (umbellata J) (10) Yellow amorphous powder; UV (MeOH) λmax (log ε) 204 (1.91), 242 (1.86), 279 (1.86), 398 (1.20) nm; IR vmax 3234, 1672, 1632, 1574, 1453 cm−1; 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR (DMSO‑d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 267.0296 [M - H]- (calcd. for C15H7O5, 267.0299).
4.4.1. 2-Hydroxy-6-hydroxymethylanthraquinone (umbellata A) (1) Yellow amorphous powder; UV (MeOH) λmax (log ε) 210 (1.91), 271 (2.09), 334 (1.23), 382 (0.95) nm; IR vmax 3426, 1668, 1588, 1494 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 253.0506 [M - H]- (calcd. for C15H9O4, 253.0506).
4.4.11. Methyl 4,6-dihydroxy-1-methoxynaphthalene-2-carboxylate (umbellata K) (11) Colorless powder; UV (MeOH) λmax (log ε) 216 (1.85), 262 (1.99), 322 (1.29) nm; IR vmax 3302, 1701, 1621, 1491 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz), see Tables 1 and 2; HRESIMS m/z 271.0575 [M + Na]+ (calcd. for C13H12O5Na, 271.0577).
4.4.2. 2-Hydroxymethyl-6-methoxyanthraquinone (umbellata B) (2) Yellow amorphous powder; UV (MeOH) λmax (log ε) 209 (2.10), 268 (2.12), 333 (1.32) nm; IR vmax 3523, 1670, 1594, 1497 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 269.0805 [M + H]+ (calcd. for C16H13O4, 269.0808). 7
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4.4.12. Dimethyl 1,1′-dihydroxy-4,4′-dimethoxy-2,2′-binaphthalene-3,3′dicarboxylate (umbellata L) (12) Red amorphous powder; [α]20 D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (1.82), 253 (1.58), 342 (0.89) nm; IR vmax 3397, 1735, 1647, 1468 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Tables 1 and 2; HRESIMS m/z 461.1242 [M - H]- (calcd. for C26H21O8, 461.1242).
Inhibition Ratio (%) = (ODcontrol
4.4.13. Methyl 7,12-dihydro-2,9-dihydroxy-5-methoxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d]furan-6- carboxylate (umbellata M) (13) Yellow amorphous powder; UV (MeOH) λmax (log ε) 208 (1.72), 243 (1.75), 277 (1.91), 447 (1.00) nm; IR vmax 3244, 1723, 1680, 1584 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 417.0620 [M - H]- (calcd. for C23H13O8, 417.0616).
Acknowledgements
ODsample )/( ODcontrol
OD blank ) * 100% Declarations of interest There are no conflicts to declare.
This work was supported by the CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-010), the Fund of Jiansheng Fresh Herb Medicine Research, and the Drug Innovation Major Project (2018ZX09711-001-001-001 and 2018ZX09711-001-001-003). Appendix A. Supplementary data
4.4.14. Methyl 7,12-dihydro-2,5,9-trihydroxy-7,12-dioxodinaphtho[1,2b:2′,3′-d]furan-6-carboxylate (umbellata N) (14) Yellow amorphous powder; UV (MeOH) λmax (log ε) 208 (1.71), 245 (1.67), 281 (1.99), 454 (1.10) nm; IR vmax 3310, 1680, 1647, 1583 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 403.0449 [M - H]- (calcd. for C22H11O8, 403.0459).
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112096. References Akrawi, O.A., Hussain, M., Langer, P., 2011. Site-selective Suzuki-Miyaura reactions of the bis(triflate) of 1,3-dihydroxyanthraquinone. Tetrahedron Lett. 52, 1093–1095. https://doi.org/10.1016/j.tetlet.2010.12.098. Ambekar, R.S., Kandasubramanian, B., 2019. A polydopamine-based platform for anticancer drug delivery. Biomater. Sci. 7, 1776–1793. https://doi.org/10.1039/ C8BM01642A. Ban, N.K., Giang, V.H., Linh, T.M., Lien, L.Q., Ngoc, N.T., Thao, D.T., Nam, N.H., Cuong, N.X., Kiem, P.V., Minh, C.V., 2013. Two new 11-noriridoids from the aerial parts of Morinda umbellata. Phytochem. Lett. 6, 267–269. https://doi.org/10.1016/j.phytol. 2013.03.011. Cai, X.-H., Luo, X.-D., Zhou, J., Hao, X.-J., 2005. Quinones from Chirita eburnea. J. Nat. Prod. 68, 797–799. https://doi.org/10.1021/np049632f. Chang, P., Lee, K.-H., Shingu, T., Hirayama, T., Hall, I.H., Huang, H.-C., 1982. Antitumor agents 50. 1 Morindaparvin-A, a new antileukemic anthraquinone, and alizarin-1methyl ether from Morinda parvifolia, and the antileukemic activity of the related derivatives. J. Nat. Prod. 45, 206–210. https://doi.org/10.1021/np50020a017. Chiou, C.-T., Hsu, R.-Y., Lin, L.-C., 2014. Isolation and cytotoxic effect of anthraquinones from Morinda umbellata. Planta Med. 80, 1113–1117. https://doi.org/10.1055/s0034-1382956. Ho, L.K., Nodwell, J.R., 2016. David and Goliath: chemical perturbation of eukaryotes by bacteria. J. Ind. Microbiol. Biotechnol. 43, 233–248. https://doi.org/10.1007/ s10295-015-1686-6. Kang, J., Zhang, P., Gao, Z., Zhang, J., Zheng, Y., Wang, H., Chen, R., 2016. Naphthohydroquinones, naphthoquinones, anthraquinones, and a naphthohydroquinone dimer isolated from the aerial parts of Morinda parvifolia and their cytotoxic effects through up-regulation of p53. Phytochemistry 130, 144–151. https:// doi.org/10.1016/j.phytochem.2016.04.001. Kang, K.H., Huh, H., Kim, B.-K., Lee, C.-K., 1999. An antiviral furanoquinone from Paulownia tomentosa Steud. Phytother Res. 13, 624–626. https://doi.org/10.1002/ (SICI)1099-1573(199911)13:7%3C624::AID-PTR551%3E3.0.CO;2-A. Kuo, Y.-J., Hwang, S.-Y., Wu, M.-D., Liao, C.-C., Liang, Y.-H., Kuo, Y.-H., Ho, H.-O., 2008. Cytotoxic constituents from Podocarpus fasciculus. Chem. Pharm. Bull. 56, 585–588. https://doi.org/10.1248/cpb.56.585. Luo, X.R., Gao, Y.Z., Chen, W.Q., Ruan, Y.Z., 1999. Flora of China, vol. 71. Science Press, Beijing, pp. 190. Newman, D.J., Cragg, G.M., 2007. Natural products as sources of new drugs over the last 25 years. J. Nat. Prod. 70, 461–477. https://doi.org/10.1021/np068054v. Newman, D.J., Cragg, G.M., 2016. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661. https://doi.org/10.1021/acs.jnatprod.5b01055. Park, B.-S., Lee, H.-K., Lee, S.-E., Piao, X.-L., Takeoka, G.R., Wong, R.Y., Ahn, Y.-J., Kim, J.-H., 2006. Antibacterial activity of Tabebuia impetiginosa martius ex DC (taheebo) against Helicobacter pylori. J. Ethnopharmacol. 105, 255–262. https://doi.org/10. 1016/j.jep.2005.11.005. Park, Y., Kong, J.Y., Cho, H., 2009. A furanquinone from Paulownia tomentosa stem for a new cathepsin K inhibitor. Phytother Res. 23, 1485–1488. https://doi.org/10.1002/ ptr.2716. Pong, C., Chia-Fu, C., 1995. Isolation and characterization of antitumor anthraquinones from Morinda umbellata. Chin. Pharmaceut. J. 47, 347–353. https://www. researchgate.net/publication/292722762. Ruksilp, T., Sichaem, J., Khumkratok, S., Siripong, P., Tip-pyang, S., 2011. Anthraquinones and an iridoid glycoside from the roots of Morinda pandurifolia. Biochem. Syst. Ecol. 39, 888–892. https://doi.org/10.1016/j.bse.2011.07.003.
4.4.15. Methyl 7,12-dihydro-9-hydroxy-5-methoxy-7,12-dioxodinaphtho [1,2-b:2′,3′-d]furan-6- carboxylate (umbellata O) (15) Yellow amorphous powder; UV (MeOH) λmax (log ε) 206 (2.03), 255 (2.07), 274 (2.12), 443 (1.31) nm; IR vmax 3365, 1720, 1674, 1582 cm−1; 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR (DMSO‑d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 401.0656 [M - H]- (calcd. for C23H13O7, 401.0667). 4.4.16. Methyl 7,12-dihydro-10-hydroxy-5-methoxy-7,12-dioxodinaphtho [1,2-b:2′,3′-d]furan-6-carboxylate (umbellata P) (16) Yellow amorphous powder; UV (MeOH) λmax (log ε) 203 (2.25), 250 (2.15), 270 (2.13), 401 (1.25) nm; IR vmax 3368, 1702, 1674, 1578 cm−1; 1H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz), see Tables 1 and 2; HRESIMS m/z 403.0819 [M + H]+ (calcd. for C23H15O7, 403.0812). 4.5. Cytotoxicity assay 4.5.1. Cell cultures Six human cancer cell lines (A431, A2780, NCI-H460, HCT116, HepG2, and MCF-7) were cultured in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C under a humidified atmosphere containing 5% CO2. 4.5.2. Cell proliferation and cell viability assay Cytotoxic assays were performed using the MTT method (Kuo et al., 2008). Briefly, cells were grown in RPMI 1640 medium with 10% FBS, harvested using trypsin, and then seeded in 96-well cell culture microplates at 2500 cells per well for 24 h. Cells were divided into experimental and control groups. The experimental groups were treated with the extracted compounds at various concentrations and taxol (the positive control), whereas the control group received a same volume of culture medium. After incubating at 37 °C for 72 h, the supernatant was discarded and the cells were then incubated with the MTT reagent (0.5 mg/mL) at 37 °C for 4 h. The supernatant was discarded carefully and the cells were treated with DMSO (100 μL/well). The absorbance was measured at 570 nm with a microplate reader. The percentage of viable cells was calculated using the following equation.
8
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Cytotoxic quinones from the aerial parts of Morinda umbellata L. Changkang Li, Xianming Su, Fenghua Li, Jia Fu, Hongqing Wang, Baoming Li, Ruoyun Chen, ⁎ Jie Kang
T
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 1 Xiannongtan Street, Beijing, 100050, China
ARTICLE INFO
ABSTRACT
Keywords: Morinda umbellata L. (Rubiaceae) Quinones Cytotoxicity
Although Morinda umbellata L. has been used in numerous folk medicines, there is a lack of phytochemical studies on this plant. Sixteen undescribed quinones, namely, ten anthraquinones (umbellatas A–J), one naphthohydroquinone (umbellata K), one naphthohydroquinone dimer (umbellata L), and four dinaphthofuran quinones (umbellatas M–P), were isolated from the aerial parts of Morinda umbellata L. (Rubiaceae). The structures of all the isolated quinones were elucidated based on spectroscopic methods. Four of the unknown quinones (umbellatas A, H, K and M) showed potent cytotoxic effects against A431, A2780, NCI-H460, HCT116, HepG2, and MCF-7 human cancer cell lines with IC50 values of 1.3–7.1 μM. These results reveal potential lead compounds for the development of new anticancer agents.
1. Introduction As a leading cause of death worldwide, cancer accounted for approximately 9.6 million deaths in 2018 (Ambekar and Kandasubramanian, 2019). Drug therapy is an effective treatment for cancer, and 65% of anticancer drugs (1981–2014) have been obtained from natural products or related derivatives (Newman and Cragg, 2016). In particular, various natural quinones have shown potent antitumor effects. For example, daunomycin, an anthraquinone-based anticancer drug obtained from Streptomyces peucetius (Ho and Nodwell, 2016), has been used in the treatment of leukemia and other neoplasms for many years (Newman and Cragg, 2007). In our previous study, the quinones isolated from Morinda parvifolia showed potent cytotoxic activities (Kang et al., 2016), which attracted us to further investigate cytotoxic quinones from the other medicinal plants in the same genus. Morinda umbellata L. (Rubiaceae) is a scandent shrub mainly distributed in the south of China (Luo et al., 1999). Its roots, bark, stems, and leaves have been used as folk medicines for the treatment of many diseases, including fever, coughing, stomach ache, and acute hepatitis (Ban et al., 2013; Chiou et al., 2014). However, the phytochemical and pharmaceutical properties of M. umbellata have not been extensively investigated. Only three papers have reported cytotoxic anthraquinones obtained from M. umbellata (Ban et al., 2013; Chiou et al., 2014; Pong and Chia-Fu, 1995). In our current study, the EtOAc fraction of the 95% EtOH extract of
the aerial parts of M. umbellata showed cytotoxic activity against human colorectal cancer cell line HCT116 with an IC50 value of 15.15 μg/mL. The bioactivity-guided isolation gave 16 undescribed quinones (umbellatas A–P) from the EtOAc fraction. Four of them (1, 8, 11, and 13) showed cytotoxic effects against A431, A2780, NCI-H460, HCT116, HepG2, or MCF-7 cancer cell lines with IC50 values of 1.3–7.1 μM. 2. Results The EtOAc fraction of the 95% EtOH extract of the aerial parts of M. umbellata yielded sixteen undescribed quinones, umbellatas A–P (1–16). The structures of 1–16 (Fig. 1) were elucidated by spectroscopic analysis. 2.1. Structural analysis Compound 1 was obtained as a yellow powder. Its molecular formula was established as C15H10O4 on the basis of HRESIMS data (m/z 253.0506 [M - H]-, calcd. for C15H9O4, 253.0506), indicating 11 degrees of unsaturation. The IR spectrum showed absorption bands that clearly indicated the presence of hydroxy groups (3426 cm−1), carbonyl groups (1668 cm−1), and aromatic rings (1588 and 1494 cm−1). The 1H NMR spectrum of 1 (Table 1) showed an oxygenated methylene at δH 4.69 (2H, s) and signals corresponding to two ABX systems [δH 8.13 (1H, br d), 8.11 (1H, d, J = 7.6 Hz), 7.79 (1H, dd, J = 7.6, 1.2 Hz)
Corresponding author. E-mail addresses: [email protected] (C. Li), [email protected] (X. Su), [email protected] (F. Li), [email protected] (J. Fu), [email protected] (H. Wang), [email protected] (B. Li), [email protected] (R. Chen), [email protected] (J. Kang). ⁎
https://doi.org/10.1016/j.phytochem.2019.112096 Received 30 April 2019; Received in revised form 10 August 2019; Accepted 11 August 2019 Available online 27 August 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
Phytochemistry 167 (2019) 112096
C. Li, et al.
Fig. 1. Structures of compounds 1–16.
and δH 8.07 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 2.4 Hz), 7.17 (1H, dd, J = 8.4, 2.4 Hz)]. The 13C NMR spectrum (Table 2) showed 15 carbon signals corresponding to six aromatic methines (δC 131.2, 129.9, 126.7, 123.9, 122.0, 112.7), six aromatic quaternary carbons (δC 165.4, 149.8, 135.3, 133.4, 131.8, 123.9), two carbonyl carbons (δC 182.8, 181.0), and one oxygenated methylene group (δC 62.3), consistent with an anthraquinone skeleton (Cai et al., 2005). The key HMBC correlations of H-4 (δH 8.07)/C-10 (δC 181.0), C-2 (δC 165.4), C-9a (δC 135.3); H-5 (δH 8.13)/C-10 (δC 181.0); and –CH2OH (δH 4.69)/C-7 (δC 131.2), C-5 (δC 123.9) (Fig. 2) suggested the presence of a hydroxy group at C-2 and a hydroxymethyl group at C-6. Based on the above evidence, compound 1 was identified as 2-hydroxy-6-hydroxymethylanthraquinone (umbellata A). Compound 2 was isolated as a yellow powder. Its molecular formula was deduced as C16H12O4 using HRESIMS (m/z 269.0805 [M + H]+, calcd. for C16H13O4, 269.0808). The 1H and 13C NMR spectroscopic data of 2 (Tables 1 and 2) were similar to those of 2-hydroxymethylanthraquinone (Park et al., 2006), but 2 indicated the presence of an extra methoxy group. The position of this group was confirmed by the key HMBC correlations (Fig. 2) of –OCH3 (δH 3.78)/C-6 (δC 165.0), H-8 (δH 8.41)/C-9 (δC 182.7), and C-6 (δC 165.0), C-10a (δC 136.5). Therefore, compound 2 was determined to be 2-hydroxymethyl6-methoxyanthraquinone (umbellata B). Compound 3 was obtained as a yellow powder. A molecular formula of C15H10O5 was determined from the HRESIMS spectrum, which showed a [M - H]- ion peak at m/z 269.0452 (calcd. for C15H9O5, 269.0456). The molecular formula of compound 3 is the same as that of 1,7-dihydroxy-2-hydroxymethylanthraquinone (Cai et al., 2005). The 1 H NMR signals (Table 1) included one ABX system [δH 8.06 (1H, d, J = 8.4 Hz), 7.38 (1H, d, J = 2.0 Hz), 7.14 (1H, dd, J = 8.4, 2.0 Hz)], a pair of ortho-coupled protons [δH 7.82 (1H, d, J = 7.6 Hz), 7.67 (1H, d, J = 7.6 Hz)], and an oxygenated methylene [δH 4.62 (2H, s)], which is similar to the 1H NMR data of 1,7-dihydroxy-2-hydroxymethylanthraquinone (Cai et al., 2005). They differ only in the position of one hydroxy group, which is located at C-6 in 3 but at C-7 in
the known compound (Cai et al., 2005). An oxymethylene proton signal at δH 4.62 correlated with the signal at δC 158.8 (C-1) and δC 133.0 (C3), H-5 (δH 7.38) correlated with C-10 (δC 182.8), C-8a (δC 123.3), and C-7 (δC 122.5), and H-8 (δH 8.06) correlated with C-9 (δC 187.6), C-6 (δC 167.1) and C-10a (δC 136.1) in the HMBC spectrum (Fig. 2) suggested that an oxymethylene group was at C-2, and two hydroxy groups were connected to C-1 and C-6, respectively. Accordingly, compound 3 was established as 1,6-dihydroxy-2-hydroxymethylanthraquinone (umbellata C). Compound 4 was separated as a yellow powder. It was determined to have a molecular formula of C16H12O5 from the [M - H]- ion peak at m/z 283.0613 (calcd. for C16H11O5, 283.0612). A comparison of the 1H and 13C NMR spectral data for compound 4 (Tables 1 and 2) and 1,3dihydroxyanthraquinone (Akrawi et al., 2011) demonstrated that these compounds had identical B- and C-rings, but different A-rings. A methoxymethyl group was connected to C-7 in the A-ring of 4, as determined by the HMBC correlations of –CH2OCH3 (δH 4.60) with C-6 (δC 132.2) and C-8 (δC 124.3). Based on the above analysis, compound 4 was determined to be 1,3-dihydroxy-7-methoxymethylanthraquinone (umbellata D). Compound 5, isolated as a yellow powder, had a molecular formula of C17H14O5, as determined based on HRESIMS (m/z 299.0907 [M + H]+, calcd. for C17H15O5, 299.0914), indicating 11 degrees of unsaturation. The 1H and 13C NMR data of 5 (Tables 1 and 2) were nearly identical to those of 4, except that 5 possessed a methoxy group instead of a hydroxy group at C-1, as confirmed by the HMBC correlations of –OCH3 (δH 3.86)/C-1 (δC 163.0) and H-4 (δH 7.13)/C-10 (δC 183.0), C9a (δC 112.0), C-2 (δC 105.2) as well as the 1D NOESY correlation between –OCH3 (δH 3.86) and H-2 (δH 6.74). Thus, compound 5 was established as 3-hydroxy-1-methoxy-7-methoxymethylanthraquinone (umbellata E). Compound 6, obtained as a yellow powder, had a molecular formula of C16H12O5, as established by HRESIMS (m/z 283.0616 [M - H]-, calcd. for C16H11O5, 283.0612). The NMR data indicated that the structure of 6 (Tables 1 and 2) was similar to that of 5. Notably, 5 and 6 differed in 2
7.17 dd (8.4, 2.4) 8.07 d (8.4)
8.13 br d
3
5
3
g
f
e
d
c
b
a
4.69 (2H, s)
3.78 (3H, s)
5.09 (2H, s)
7.33 br d (8.4) 8.41 d (8.4)
7.98 br d (8.0) 8.45 d (8.0) 7.89 br s
8.71 br s
2b
4.62 (2H, s)
13.25 br s
7.14 dd (8.4, 2.0) 8.06 d (8.4)
7.38 d (2.0)
7.67 d (7.6)
7.82 d (7.6)
3a
Recorded in DMSO‑d6 at 400 MHz. Recorded in pyridine-d5 at 400 MHz. Recorded in DMSO‑d6 at 600 MHz. Recorded in DMSO‑d6 at 500 MHz. Recorded in CD3OD at 400 MHz. Recorded in CDCl3 at 400 MHz. Recorded in pyridine-d5 at 600 MHz.
7-CH2OCH3
6-CH2OH 6-CH2OCH3 6-CH2OCH3 6-COOCH3 7-OCH3 7-CH2OCH3
2-COOCH3 3-COOCH3 3′-COOCH3 4-OCH3 4′-OCH3 5-OCH3 6-OCH3
2-CH2OH
11 5′ 6′ 7′ 8′ 1-OH 1-OCH3
10
8
7
6
7.79 dd (7.6, 1.2) 8.11 d (7.6)
7.43 d (2.4)
1 2
4
1a
Position
Table 1 1 H NMR data of compounds 1–16.
4.60 (2H, s) 3.38 (3H, s)
12.87 br s
8.08 br s
7.05 d (1.2) 8.10 d (8.0) 7.77 d (8.0)
6.46 d (1.2)
4a
4.60 (2H, s) 3.37 (3H, s)
3.86 (3H, s)
8.03 br s
8.07 d (7.8) 7.71 br d (7.8)
7.13 br s
6.74 br s
5c
3.93 (3H, s)
3.80 (3H, s)
7.52 d (2.4)
7.27 dd (8.4, 2.4)
8.02 d (8.4)
6.99 br s
6.58 br s
6c
4.60 (2H, s) 3.37 (3H, s)
7.76 dd (8.0, 1.2) 8.10 d (8.0)
8.04 d (1.2)
7.49 s
7.49 s
7a
3.80 (3H, s)
7.18 dd (8.8, 2.4) 8.02 d (8.8)
7.41 d (2.4)
7.88 d (8.4)
7.23 d (8.4)
8a
6.36 (2H, s)
7.20 dd (9.0, 2.5) 8.03 d (9.0)
7.46 d (2.5)
7.80 d (8.0)
7.33 d (8.0)
9d
6.30 (2H, s)
8.11–8.14 m
7.83–7.89 m
7.83–7.89 m
8.11–8.14 m
7.35 s
10d
3.91 (3H, s)
3.92 (3H, s)
7.14 dd (9.2, 2.8) 8.05 d (9.2)
7.45 d (2.8)
7.09 s
11e
3.59 3.59 4.02 4.02
(3H, (3H, (3H, (3H,
s) s) s) s)
8.17 d (8.0) 7.60–7.67 m 7.60–7.67 m 8.36 d (8.0)
8.36 d (8.0)
7.60–7.67 m
7.60–7.67 m
8.17 d (8.0)
12f
3.99 (3H, s)
3.96 (3H, s)
7.04 br d (8.4) 7.96 d (8.4)
7.30 br s
7.34 dd (9.2, 2.0) 8.11 d (9.2)
7.59 d (2.0)
13a
3.92 (3H, s)
7.14 dd (8.4, 2.4) 7.99 d (8.4)
7.39 d (2.4)
7.24 dd (9.2, 2.0) 8.26 d (9.2)
7.51 d (2.0)
14a
4.05 (3H, s)
4.01(3H, s)
7.17 br d (8.5) 8.03 d (8.5)
7.41 d (2.5)
8.28 d (8.0)
7.86 t (8.0)
8.49 d (8.0) 7.91 t (8.0)
15d
4.34 (3H, s)
4.17(3H, s)
7.42 dd (8.4,1.8) 8.02 d (1.8)
8.29 d (8.4)
8.29–8.33 (m)
7.68–7.71 (m)
8.29–8.33 (m) 7.68–7.71 (m)
16g
C. Li, et al.
Phytochemistry 167 (2019) 112096
Phytochemistry 167 (2019) 112096
C. Li, et al.
Table 2 13 C NMR data of compounds 1–16 (δ in ppm). Position
1a
2b
3a
4a
5c
6c
7a
8c
9d
10d
11e
12f
13c
14c
15d
16g
1 2 2a 3 3a 4 4a 5 6 6a 7 8 8a 9 9a 10 10a 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 3′a 4′ 4′a 5′ 6′ 7′ 8′ 8′a 1-OCH3 2-CH2OH 2-COOCH3 3-COOCH3 3′-COOCH3 4-OCH3 4′-OCH3 5-OCH3 6-OCH3 6-COOCH3 6-CH2OH 6-CH2OCH3 6-CH2OCH3 7-OCH3 7-CH2OCH3 7-CH2OCH3
112.7 165.4
125.2 151.2
158.8 138.8
165.2 107.7
163.0 105.2
163.3 105.1
112.8 152.1
147.7 158.7
148.4 154.9
149.7 140.5
147.8 110.4
102.9 158.6
102.7 158.8
121.2 129.5
122.7 129.5
122.0
132.1
133.0
168.3
166.3
NDh
152.1
120.9
112.6
147.4
152.9 116.7 168.3 108.3
120.5
119.1
128.8
129.1
129.9 123.9 123.9 149.8
127.9 133.2 111.1 165.0
118.9 132.0 113.8 167.1
110.0 134.8 127.0 132.2
107.5 136.5 126.1 130.9
109.3 136.5 128.4 118.4
112.8 126.7 124.6 144.8
125.0 125.0 111.4 162.5
124.1 127.6 113.0 164.5
112.4 127.9 126.6 133.7
149.2 131.9 105.6 158.5
124.3 167.5 148.2 129.0 122.8 128.1
126.1
126.9
123.9
124.6
131.2 126.7 131.8 182.8 135.3 181.0 133.4
121.2 130.3 128.0 182.7 134.6 183.4 136.5
122.5 130.3 123.3 187.6 115.2 182.8 136.1
145.6 124.3 133.4 184.7 108.2 182.0 132.0
145.3 124.5 135.0 178.6 112.0 183.0 131.0
163.9 109.9 137.8 177.4 110.2 182.5 125.5
132.1 126.6 132.4 181.4 126.7 181.6 133.3
121.1 129.6 126.7 181.2 126.6 181.4 134.7
122.2 130.1 125.8 180.2 116.8 181.8 136.0 104.6
134.2 126.3 133.0 179.3 109.5 181.4 133.0 103.6
120.0 126.4 124.9
127.5 123.8 126.5
152.1 115.8 166.1 180.8 114.2
153.5 124.2 167.6 180.3 113.4
151.6 118.2 165.9 180.5 113.5
153.0 116.9 167.2 180.3 130.5
166.8
164.3
164.4
121.8
121.0
120.3
120.7
165.4
129.5 172.7 154.5 147.5 123.4 120.8 NDh 114.7 135.5 123.4
129.2 172.9 158.8 145.5 123.7 119.8 NDh 116.6 135.9 123.1
129.6 173.0 154.5 148.5 121.5 127.0 NDh 115.3 135.4 123.5
114.2 175.4 154.2 149.9 122.1 128.7 NDh 119.9 125.8 135.5
64.1
64.6
52.7
53.3
a b c d e f g h
63.9
55.9
58.0
55.5
60.4
63.6 52.4
56.2
147.8 110.4 124.3 167.5 148.2 129.0 122.8 128.1 127.5 123.8 126.5
52.5 52.5 63.9 63.9
64.0 52.5
62.3
72.6 58.0
72.7 57.9
55.7
52.0
72.7 58.0
Recorded in DMSO‑d6 at 100 MHz. Recorded in pyridine-d5 at 100 MHz. Recorded in DMSO‑d6 at 150 MHz. Recorded in DMSO‑d6 at 125 MHz. Recorded in CD3OD at 100 MHz. Recorded in CDCl3 at 125 MHz. Recorded in pyridine-d5 at 150 MHz. ND: no detected.
the substituent at C-7, with the methoxymethyl group in 5 replaced by a methoxy group in 6. This was confirmed by the HMBC correlations of –OCH3 (δH 3.93)/C-7 (δC 163.9) and H-5 (δH 8.02)/C-10 (δC 182.5), C-7 (δC 163.9), C-8a (δC 137.8). Thus, compound 6 was determined to be 3hydroxy-1,7-dimethoxyanthraquinone (umbellata F). Compound 7, isolated as a yellow powder, was assigned the molecular formula of C16H12O5 based on its HRESIMS data (m/z 283.0609 [M - H]-, calcd. for C16H11O5, 283.0612). Compounds 7 and 4 are
isomers, but the positions of the hydroxy and methoxymethyl groups differed. In 1H NMR spectrum, one ABX system [δH 8.10 (1H, d, J = 8.0 Hz), 8.04 (1H, d, J = 1.2 Hz), 7.76 (1H, dd, J = 8.0, 1.2 Hz)], an aromatic singlet at δH 7.49 (2H, s), an oxygenated methylene [δH 4.60 (2H, s)] and one methoxy [δH 3.37 (3H, s)] were shown. The HMBC correlations of H-1 (δH 7.49)/C-9 (δC 181.4), C-3 (δC 152.1), C4a (δC 126.7); H-4 (δH 7.49)/C-10 (δC 181.6), C-2 (δC 152.1), C-9a (δC 126.7); –CH2OCH3 (δH 4.60)/C-7 (δC 132.1), C-5 (δC 124.6); H-8 (δH 4
Phytochemistry 167 (2019) 112096
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Fig. 2. Key HMBC correlations (blue arrows) and NOEs (red arrows) of compounds 1–16. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
8.10)/C-9 (δC 181.4), C-6 (δC 144.8), C-10a (δC 133.3), and H-5 (δH 8.04)/C-10 (δC 181.6), C-8a (δC 132.4), C-7 (δC 132.1) confirmed that two hydroxy groups and one methoxymethyl group were located at C-2, C-3, and C-6, respectively. Accordingly, compound 7 was identified as 2,3-dihydroxy-6-methoxymethylanthraquinone (umbellata G). Compound 8, isolated as a yellow powder, had a molecular formula of C15H10O5, as determined by HRESIMS (m/z 271.0605 [M + H]+, calcd. for C15H11O5, 271.0601), which indicated 11 degrees of unsaturation. The 1H and 13C NMR data of 8 (Tables 1 and 2) exhibited similarities to those of 1,2,6-trihydroxyanthraquinone (flavopurpurin) (Ruksilp et al., 2011), except that a hydroxy group at C-1 in flavopurpurin was replaced by a methoxy group in 8. The key HMBC correlations of –OCH3 (δH 3.80)/C-1 (δC 147.7) and H-3 (δH 7.23)/C-1 (δC 147.7), C-4a (δC 125.0) confirmed the position of the methoxy group in 8 (Fig. 2). Consequently, compound 8 was determined to be 2,6-dihydroxy-1-methoxyanthraquinone (umbellata H). Compound 9, obtained as a yellow powder, showed a molecular ion at m/z 291.0263 [M + Na]+ (calcd. for C15H8O5Na, 291.0264), indicative of a molecular formula of C15H8O5 and 12 degrees of unsaturation. The NMR data of compound 9 (Tables 1 and 2) were similar to those of morindaparvin A (Chang et al., 1982), but 9 contained an extra hydroxy group. The HMBC correlations of H-8 (δH 8.03)/C-9 (δC 180.2), C-6 (δC 164.5), C-10a (δC 136.0) (Fig. 2) demonstrated that the extra hydroxy group was located at C-6. Thus, compound 9 was determined to be 6-hydroxy-1,2-methylenedioxyanthraquinone (umbellata I). Compound 10 was obtained as a yellow powder. The [M - H]- ion peak at m/z 267.0296 (calcd. for C15H7O5, 267.0299) in the HRESIMS spectrum was attributed to a molecular formula of C15H8O5, which indicated compound 10 is an isomer of 9, but the position of the hydroxy group is different. The 1H NMR spectrum showed one AA′BB′ system [δH 8.11–8.14 (2H, m), 7.83–7.89 (2H, m)], one aromatic singlet at δH 7.35 (1H, s) and one aliphatic singlet at δH 6.30 (2H, s). The HMBC correlations of H-4 (δH 7.35)/C-10 (δC 181.4), C-2 (δC 140.5), and C-9a (δC 109.5), and H-11 (δH 6.30)/C-1 (δC 149.7) and C-2 (δC
140.5) (Fig. 2) indicated that a hydroxy group at C-3 and the group of –O–CH2–O– was connected at C-1 and C-2 in 10. Consequently, 10 was identified as 3-hydroxy-1,2-methylenedioxyanthraquinone (umbellata J). Compound 11 was obtained as a colorless powder. It was determined to have a molecular formula of C13H12O5 based on the [M + Na]+ ion peak at m/z 271.0575 (calcd. for C13H12O5Na, 271.0577), which indicated 8 degrees of unsaturation. The NMR data of compound 11 were closely related to those of methyl 4-hydroxy-1,6-dimethoxynaphthalene-2-carboxylate (morindaparvin C) (Kang et al., 2016), except that 11 had a hydroxy group at C-6 instead of the methoxy group in morindaparvin C. The HMBC correlations of H-8 (δH 8.05)/C-6 (158.5), C-1(152.9), C-4a (131.9), OCH3-1 (δH 3.92)/C-1 (152.9), and OCH3 (δH 3.91)/C-2a (C]O) (168.3) indicated the presence of the hydroxy group at C-6 and two methoxy groups at C-1 and C-2a (C]O), respectively. The NOE correlations of OCH3-1 (δH 3.92)/H-8 (δH 8.05) and OCH3 (δH 3.91)/H-3 (δH 7.09) further confirmed the positions of the two methoxy groups. Thus, compound 11 was determined to be methyl 4,6-dihydroxy-1-methoxynaphthalene-2-carboxylate (umbellata K). Compound 12, obtained as a red powder, had a molecular formula of C26H22O8, as determined from the HRESIMS data (m/z 461.1242 [M H]-, calcd. for C26H21O8, 461.1242), which indicated 16 degrees of unsaturation. The 1H NMR spectrum of 12 exhibited one AA′BB′ system [δH 8.36 (1H, d, J = 8.0 Hz), 8.17 (1H, d, J = 8.0 Hz), 7.60–7.67 (2H, m)] and two methoxy groups [δH 4.02 (3H, s), 3.59 (3H, s)]. The 13C NMR spectrum (Table 2) revealed 13 carbon resonances, including those corresponding to a carbonyl group (δC 167.5), four aromatic methines (δC 128.1, 127.5, 123.8, 122.8), and six aromatic quaternary carbons (δC 148.2, 147.8, 129.0, 126.5, 124.3, 110.4). These NMR data in combination with the molecular weight indicated that 12 was a symmetric dimer, similar to that of dimethyl 1,1′-dihydroxy-4,4′,7,7′tetramethoxy-2,2′-binaphthalene-3,3′-dicarboxylate (morindaparvin G) (Kang et al., 2016). The only difference between these compounds was the absence of two methoxy groups at C-7 and C-7′ in 12. The NOE 5
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(Table 2) spectroscopic data of 15 were very similar to those of 13, except that one hydroxy group was absent at C-2 in 15. This difference was in accordance with the HMBC correlations of H-4 (δH 8.28)/C-5 (δC 151.6), C-2 (129.5), C-15 (δC 121.5) (Fig. 2). Therefore, compound 15 was determined to be methyl 7,12-dihydro-9-hydroxy-5-methoxy-7,12dioxodinaphtho[1,2-b:2′,3′-d]furan-6-carboxylate (umbellata O). Compound 16 was found to have the same molecular formula as compound 15 (C23H14O7), as determined by HRESIMS (m/z 403.0819 [M + H]+, calcd. for C23H15O7, 403.0812), suggesting that compounds 16 and 15 are isomers. However, the position of the hydroxy group differed in these two compounds. The HMBC correlations of H-8 (δH 8.29)/C-7 (δC 180.3), C-10 (δC 165.4), C-20 (δC 135.5) and H-11 (δH 8.02)/C-12 (δH 175.4), C-19 (δC 125.8), C-9 (δC 121.8) (Fig. 2) confirmed that the hydroxy group was located at C-10 in 16 instead of C-9 in 15. Hence, compound 16 was identified as methyl 7,12-dihydro-10hydroxy-5-methoxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d]furan-6-carboxylate (umbellata P).
Table 3 The IC50 values (μM) for the cytotoxic effects of compounds 1, 8, 11, 13 and positive control (taxol). No.
A431
A2780
NCI-H460
HCT116
HepG2
MCF-7
1 8 11 13 taxol
23.9 NDa 48.5 5.9 0.004
NDa NDa 20.5 6.4 0.01
NDa NDa 14.7 7.1 0.004
NDa 2.4 1.3 4.1 0.009
NDa NDa 5.8 6.2 0.0005
4.8 NDa 4.9 16.5 0.08
A431(human skin squamous cancer cell lines); A2780 (ovarian cancer cell lines); NCI-H460 (human lung cancer cell lines); HCT116 (human colorectal cancer cell lines); HepG2 (human liver cancer cell lines); MCF-7 (human breast cancer cell lines). a ND: no detected (> 50 μM).
correlation of OCH3 (δH 4.02)/OCH3 (δH 3.59) and OCH3 (δH 4.02)/H-5 (H-5′) (δH 8.17) confirmed that the four methoxy groups were located at C-4 (C-4′) and C-3a (C-3′a) (C]O), respectively. Therefore, compound 12 was determined to be dimethyl 1,1′-dihydroxy-4,4′-dimethoxy-2,2′-binaphthalene-3,3′-dicarboxylate (umbellata L). Compound 13 was obtained as a yellow powder. Its molecular formula was established as C23H14O8 on the basis of HRESIMS data (m/z 417.0620 [M - H]-, calcd. for C23H13O8, 417.0616), indicating 17 degrees of unsaturation. The IR spectrum suggested the presence of hydroxyl groups (3244 cm−1), carbonyl groups (1723 and 1680 cm−1), and aromatic rings (1584 cm−1). The 1H NMR spectrum of 13 (Table 1) showed two methoxy protons at δH 3.99 (3H, s) and 3.96 (3H, s) as well as two ABX systems [δH 8.11 (1H, d, J = 9.2 Hz), 7.59 (1H, d, J = 2.0 Hz), 7.34 (1H, dd, J = 9.2, 2.0 Hz) and δH 7.96 (1H, d, J = 8.4 Hz), 7.30 (1H, br s), 7.04 (1H, br d, J = 8.4 Hz)]. The 13C NMR spectrum showed 22 carbon signals (should be 23, but one was not detected), including three carbonyl carbons (δC 180.8, 172.7, 166.1), six aromatic protonated carbons (δC 129.5, 126.1, 121.0, 120.5, 114.2, 102.9), eleven aromatic quaternary carbons (should be twelve, but one was not detected) (δC 166.8, 158.6, 154.5, 152.1, 147.5, 135.5, 123.4 × 2, 120.8, 115.8, 114.7), and two methoxy groups (δC 64.0, 52.5). The NMR data of 13 resembled those of methyl 7,12-dihydro-5hydroxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d]furan-6-carboxylate (Kang et al., 1999; Park et al., 2009), but there were two extra hydroxy groups in 13 at C-2 and C-9, and a methoxy group at C-5 in 13 replaced a hydroxy group in the known compound. These differences were confirmed by the key HMBC correlations of H-4 (δH 8.11)/C-2 (δC 158.6), C-5 (δC 152.1), C-15 (δC 123.4), H-11 (δH 7.96)/C-12 (δC 172.7), C-9 (δC 166.8), C-19 (δC 135.5) and OCH3-5 (δH 3.96)/C-5 (δC 152.1) (Fig. 2). The NOE correlation of OCH3 (δH 3.96)/H-4 (δH 8.11) further confirmed that the methoxy group was located at C-5. Based on the above evidence, compound 13 was identified as methyl 7,12-dihydro-2,9-dihydroxy-5-methoxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d]furan-6-carboxylate (umbellata M). Compound 14 was isolated as a yellow powder. A molecular formula of C22H12O8 was deduced from the HRESIMS data (m/z 403.0449 [M - H]-, calcd. for C22H11O8, 403.0459), implying 17 degrees of unsaturation. The 1H and 13C NMR data of 14 were nearly identical to those of 13. Furthermore, the molecular formula of 14 indicated that its molecular weight was 14 Da less than that of 13. This difference was caused by a hydroxy group at C-5 in 14 replacing a methoxy group in 13. The HMBC correlation between the methoxy group at δH 3.92 and the carbonyl carbon at δC 167.6 suggested that the methoxy group was connected to C-6a (C]O). Thus, compound 14 was identified as methyl 7,12-dihydro-2,5,9-trihydroxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d] furan-6-carboxylate (umbellata N). Compound 15, obtained as a yellow powder, displayed a molecular ion peak [M - H]- at m/z 401.0656 (calcd. for C23H13O7, 401.0667), indicating a molecular formula of C23H13O7. Thus, 15 has one less oxygen atom than compound 13. The 1H (Table 1) and 13C NMR
2.2. Cytotoxic effects The cytotoxic activities of compounds 1–16 were evaluated against a panel of six human tumor cell lines, including A431 (human skin squamous carcinoma cell line), A2780 (ovarian cancer cell line), NCIH460 (human lung cancer cell line), HCT116 (human colorectal carcinoma cell line), HepG2 (human liver carcinoma cell line), and MCF-7 (human breast carcinoma cell line), using the MTT method. Compounds 1, 8, 11, and 13 were found to exhibit potent cytotoxicity against the five cell lines with IC50 values ranging from 1.3 to 7.1 μM (Table 3). These results suggest that the quinones isolated from M. umbellata may be valuable drug candidates. 3. Discussion Bioactivity-guided isolation from the EtOAc fraction of the 95% EtOH extract of the aerial parts of M. umbellata gave sixteen undescribed quinones (umbellatas A–P). Structural elucidation of all the compounds was successfully achieved by spectroscopic analyses and comparisons with literature data. Because compounds 2, 4–8 and 11–16 having methoxy, methoxymethyl, or carbomethoxy groups in the structures, HPLC/DAD/(-)ESIMS was used to prove they are not artificial compounds produced during separation. Extracted ion chromatograms (EICs) of compounds 2, 4–8 and 11–16 were observed in the EtOAc fraction of 95% EtOH extract of Morinda umbellata. Thus, compounds 2, 4–8 and 11–16 are not artificial products. Among the isolated compounds, anthraquinones (1 and 8), naphthohydroquinone (11) and dinaphthofuran quinone (13) showed selective cytotoxic activities against human skin, ovarian, lung, colorectal, liver, and breast cancer cell lines. These findings indicate the potential of the quinones isolated from M. umbellata to act as lead compounds for developing cancer treatments based on natural remedies. 4. Experimental 4.1. General experimental procedures Optical rotations were measured on a JASCO P-2000 automatic digital polarimeter (Tokyo, Japan)). UV spectra were collected in MeOH on a JASCO V-650 spectrophotometer. IR spectra were recorded on a Nicolet 5700 spectrometer (Thermo Electron Corporation, Madison, WI, USA) using the FT-IR microscope transmission method. NMR spectra were acquired using a JEOL ECZ-400 spectrometer (JEOL, Tokyo, Japan), a Bruker Avance III-400 (or 500 or 600) spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), or an Agilent VNMRS 600 (600 MHz) spectrometer (Palo Alto, CA, USA). HRESIMS data were recorded on an Agilent 1200 SL series LC/6520 QTOF spectrometer 6
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(Böblingen, Germany) or a Thermo Scientific Q Exactive Focus Orbitrap mass spectrometer (Waltham, MA, USA). Column chromatography purification was performed using Sephadex LH-20 (GE Healthcare BioSciences AB, Uppsala, Sweden) and C-18 (50 mm, YMC, Kyoto, Japan). Semi-preparative HPLC separation was performed using a LC-6AD semipreparative HPLC with an Agilent column (XDB-C18, 5 μm, 9.4 × 250 mm). The cancer cell lines were purchased from Shanghai Fengshou Industrial Co., Ltd. (Shanghai, China).
4.4.3. 1,6-Dihydroxy-2-hydroxymethylanthraquinone (umbellata C) (3) Yellow amorphous powder; UV (MeOH) λmax (log ε) 219 (1.91), 269 (1.87), 409 (1.31) nm; IR vmax 3363, 1660, 1626, 1578, 1477 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 269.0452 [M - H]- (calcd. for C15H9O5, 269.0456). 4.4.4. 1,3-Dihydroxy-7-methoxymethylanthraquinone (umbellata D) (4) Yellow amorphous powder; UV (MeOH) λmax (log ε) 204 (2.12), 247 (1.99), 284 (1.98), 415 (1.38) nm; IR vmax 3343, 1666, 1633, 1599, 1459 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 283.0613 [M - H]- (calcd. for C16H11O5, 283.0612).
4.2. Plant materials The aerial parts of Morinda umbellata L. (Rubiaceae) were collected from Ganshiling Nature Reserve (GPS coordinates: N 18°39′-18°38′, E 109°66′-109°65′), Hainan Province, China in July 2014 (wet season), and were identified by Professor Lin Ma of the Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, China. A voucher specimen (ID-22679) was deposited at the Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, China.
4.4.5. 3-Hydroxy-1-methoxy-7-methoxymethylanthraquinone (umbellata E) (5) Yellow amorphous powder; UV (MeOH) λmax (log ε) 203 (1.97), 250 (1.89), 282 (1.72), 336 (0.95), 398 (0.97) nm; IR vmax 3394, 1644, 1588, 1418 cm−1; 1H NMR (DMSO‑d6, 600 MHz) and 13C NMR (DMSO‑d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 299.0907 [M + H]+ (calcd. for C17H15O5, 299.0914).
4.3. Extraction and isolation Air-dried, powdered aerial parts of M. umbellata (42 kg) were extracted with EtOH–H2O (95: 5, v/v, × 3) under reflux conditions for 2 h and concentrated under reduced pressure. The residue (2.6 kg) was suspended in H2O and successively partitioned with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract (190 g) was chromatographed on a silica gel (100–200 mesh) column (100 × 40 cm) eluted with a petroleum ether–EtOAc gradient system (95: 5 to 50: 50, v/v) to give fractions 1–28. Fractions 18–20 (12 g) were subjected to chromatography on a C18 medium-pressure column (46 × 6 cm) eluted with a MeOH–H2O gradient system (30:70 to 70: 30, v/v, 20 mL/min) to yield 82 sub-fractions (1a–82a). Sub-fraction 24a (60 mg) was further separated by semi-preparative HPLC eluted isocratically (MeCN–H2O = 2: 8, v/v, 4 mL/min) to give 11 (30 mg, tR = 24.6 min) and 12 (4 mg, tR = 43.4 min). Compounds 1 (10 mg, tR = 25.8 min) and 8 (3 mg, tR = 20.4 min) were isolated from sub-fraction 40a (40 mg) by semipreparative HPLC eluted with MeCN–H2O (2: 8, v/v, 4 mL/min). Subfraction 32a (100 mg) was subjected to a Sephadex LH-20 column eluted with MeOH–H2O (3: 7, v/v, 4 mL/min) to obtain 4 (8 mg, tR = 48.4 min), 5 (5 mg, tR = 38.2 min), 6 (1 mg, tR = 42.0 min), and 7 (10 mg, tR = 36.8 min). Purification of sub-fraction 69a (200 mg) by semi-preparative HPLC using MeCN–H2O (26: 74, v/v, 4 mL/min) afforded 2 (6 mg, tR = 29.7 min), 3 (4 mg, tR = 25.5 min), 9 (10 mg, tR = 40.8 min), and 10 (20 mg, tR = 46.7 min). Compounds 13 (20 mg, tR = 38.0 min), 14 (5 mg, tR = 45.8 min), 15 (5 mg, tR = 46.7 min) and 16 (1 mg, tR = 43.4 min) were obtained from sub-fraction 80a (500 mg) by Sephadex LH-20 column eluted with MeOH–H2O (4: 6, v/v, 4 mL/ min) and semi-preparative HPLC using MeCN–H2O (3: 7, v/v, 4 mL/ min).
4.4.6. 3-Hydroxy-1,7-dimethoxyanthraquinone (umbellata F) (6) Yellow amorphous powder; UV (MeOH) λmax (log ε) 205 (2.05), 276 (1.86), 379 (1.11) nm; IR vmax 3312, 1673, 1596, 1466 cm−1; 1H NMR (DMSO‑d6, 600 MHz) and 13C NMR (DMSO‑d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 283.0616 [M - H]- (calcd. for C16H11O5, 283.0612). 4.4.7. 2,3-Dihydroxy-6-methoxymethylanthraquinone (umbellata G) (7) Yellow amorphous powder; UV (MeOH) λmax (log ε) 205 (2.02), 242 (1.72), 288 (2.15) nm; IR vmax 3355, 1664, 1583, 1462 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 283.0609 [M - H]- (calcd. for C16H11O5, 283.0612). 4.4.8. 2,6-Dihydroxy-1-methoxyanthraquinone (umbellata H) (8) Yellow amorphous powder; UV (MeOH) λmax (log ε) 220 (1.78), 272 (1.81), 299 (1.60), 359 (1.26) nm; IR vmax 3191, 1650, 1563, 1468 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 271.0605 [M + H]+ (calcd. for C15H11O5, 271.0601). 4.4.9. 6-Hydroxy-1,2-methylenedioxyanthraquinone (umbellata I) (9) Yellow amorphous powder; UV (MeOH) λmax (log ε) 224 (1.88), 270 (1.89), 380 (1.36) nm; IR vmax 3377, 1666, 1578, 1450 cm−1; 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR (DMSO‑d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 291.0263 [M + Na]+ (calcd. for C15H8O5Na, 291.0264).
4.4. Compound characterization
4.4.10. 3-Hydroxy-1,2-methylenedioxyanthraquinone (umbellata J) (10) Yellow amorphous powder; UV (MeOH) λmax (log ε) 204 (1.91), 242 (1.86), 279 (1.86), 398 (1.20) nm; IR vmax 3234, 1672, 1632, 1574, 1453 cm−1; 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR (DMSO‑d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 267.0296 [M - H]- (calcd. for C15H7O5, 267.0299).
4.4.1. 2-Hydroxy-6-hydroxymethylanthraquinone (umbellata A) (1) Yellow amorphous powder; UV (MeOH) λmax (log ε) 210 (1.91), 271 (2.09), 334 (1.23), 382 (0.95) nm; IR vmax 3426, 1668, 1588, 1494 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz), see Tables 1 and 2; HRESIMS m/z 253.0506 [M - H]- (calcd. for C15H9O4, 253.0506).
4.4.11. Methyl 4,6-dihydroxy-1-methoxynaphthalene-2-carboxylate (umbellata K) (11) Colorless powder; UV (MeOH) λmax (log ε) 216 (1.85), 262 (1.99), 322 (1.29) nm; IR vmax 3302, 1701, 1621, 1491 cm−1; 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz), see Tables 1 and 2; HRESIMS m/z 271.0575 [M + Na]+ (calcd. for C13H12O5Na, 271.0577).
4.4.2. 2-Hydroxymethyl-6-methoxyanthraquinone (umbellata B) (2) Yellow amorphous powder; UV (MeOH) λmax (log ε) 209 (2.10), 268 (2.12), 333 (1.32) nm; IR vmax 3523, 1670, 1594, 1497 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 269.0805 [M + H]+ (calcd. for C16H13O4, 269.0808). 7
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4.4.12. Dimethyl 1,1′-dihydroxy-4,4′-dimethoxy-2,2′-binaphthalene-3,3′dicarboxylate (umbellata L) (12) Red amorphous powder; [α]20 D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (1.82), 253 (1.58), 342 (0.89) nm; IR vmax 3397, 1735, 1647, 1468 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz), see Tables 1 and 2; HRESIMS m/z 461.1242 [M - H]- (calcd. for C26H21O8, 461.1242).
Inhibition Ratio (%) = (ODcontrol
4.4.13. Methyl 7,12-dihydro-2,9-dihydroxy-5-methoxy-7,12-dioxodinaphtho[1,2-b:2′,3′-d]furan-6- carboxylate (umbellata M) (13) Yellow amorphous powder; UV (MeOH) λmax (log ε) 208 (1.72), 243 (1.75), 277 (1.91), 447 (1.00) nm; IR vmax 3244, 1723, 1680, 1584 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 417.0620 [M - H]- (calcd. for C23H13O8, 417.0616).
Acknowledgements
ODsample )/( ODcontrol
OD blank ) * 100% Declarations of interest There are no conflicts to declare.
This work was supported by the CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-1-010), the Fund of Jiansheng Fresh Herb Medicine Research, and the Drug Innovation Major Project (2018ZX09711-001-001-001 and 2018ZX09711-001-001-003). Appendix A. Supplementary data
4.4.14. Methyl 7,12-dihydro-2,5,9-trihydroxy-7,12-dioxodinaphtho[1,2b:2′,3′-d]furan-6-carboxylate (umbellata N) (14) Yellow amorphous powder; UV (MeOH) λmax (log ε) 208 (1.71), 245 (1.67), 281 (1.99), 454 (1.10) nm; IR vmax 3310, 1680, 1647, 1583 cm−1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 403.0449 [M - H]- (calcd. for C22H11O8, 403.0459).
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4.4.15. Methyl 7,12-dihydro-9-hydroxy-5-methoxy-7,12-dioxodinaphtho [1,2-b:2′,3′-d]furan-6- carboxylate (umbellata O) (15) Yellow amorphous powder; UV (MeOH) λmax (log ε) 206 (2.03), 255 (2.07), 274 (2.12), 443 (1.31) nm; IR vmax 3365, 1720, 1674, 1582 cm−1; 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR (DMSO‑d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 401.0656 [M - H]- (calcd. for C23H13O7, 401.0667). 4.4.16. Methyl 7,12-dihydro-10-hydroxy-5-methoxy-7,12-dioxodinaphtho [1,2-b:2′,3′-d]furan-6-carboxylate (umbellata P) (16) Yellow amorphous powder; UV (MeOH) λmax (log ε) 203 (2.25), 250 (2.15), 270 (2.13), 401 (1.25) nm; IR vmax 3368, 1702, 1674, 1578 cm−1; 1H NMR (pyridine-d5, 600 MHz) and 13C NMR (pyridine-d5, 150 MHz), see Tables 1 and 2; HRESIMS m/z 403.0819 [M + H]+ (calcd. for C23H15O7, 403.0812). 4.5. Cytotoxicity assay 4.5.1. Cell cultures Six human cancer cell lines (A431, A2780, NCI-H460, HCT116, HepG2, and MCF-7) were cultured in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C under a humidified atmosphere containing 5% CO2. 4.5.2. Cell proliferation and cell viability assay Cytotoxic assays were performed using the MTT method (Kuo et al., 2008). Briefly, cells were grown in RPMI 1640 medium with 10% FBS, harvested using trypsin, and then seeded in 96-well cell culture microplates at 2500 cells per well for 24 h. Cells were divided into experimental and control groups. The experimental groups were treated with the extracted compounds at various concentrations and taxol (the positive control), whereas the control group received a same volume of culture medium. After incubating at 37 °C for 72 h, the supernatant was discarded and the cells were then incubated with the MTT reagent (0.5 mg/mL) at 37 °C for 4 h. The supernatant was discarded carefully and the cells were treated with DMSO (100 μL/well). The absorbance was measured at 570 nm with a microplate reader. The percentage of viable cells was calculated using the following equation.
8