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Hepatoprotective lignans and triterpenoids from the roots of Kadsura longipedunculata
Fitoterapia 142 (2020) 104487
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Hepatoprotective lignans and triterpenoids from the roots of Kadsura longipedunculata Si-Yuan Shao, Xin-Zhu Qi, Hua Sun, Shuai Li
T
⁎
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China
ARTICLE INFO
ABSTRACT
Keywords: Kadsura longipedunculata Tetrahydrobenzocyclooctabenzofuranone lignan Dibenzocyclooctadiene lignan Schiartane-type triterpenoid Hepatoprotective activity
Two new tetrahydrobenzocyclooctabenzofuranone lignans (1–2), a new dibenzocyclooctadiene lignan (3) and three new schiartane-type triterpenoids (4–6), together with six known compounds (7–12), were isolated from the roots of Kadsura longipedunculata. Their structures were elucidated by extensive NMR and HRESIMS spectroscopic data analysis. The absolute configurations of these compounds were determined by comparison of the experimental and calculated ECD spectra. Compound 12 exhibited moderate hepatoprotective activity against Nacetyl-p-aminophenol (APAP)-induced toxicity in HepG2 cells with cell survival rates of 53.04%.
1. Introduction
2. Experimental
Kadsura longipedunculata is a lianoid plant from the Magnoliaceae family and distributed widely in the south of China [1]. Its roots have been used as Chinese folk medicine for the treatment of rheumatoid arthritis, gastric, duodenal ulcer, dysmenorrhea, and traumatic injury [2]. Over the past ten years, a series of bioactive and novel lignans and triterpenoids have been isolated from the roots of genus Kadsura and Schizandra, which have attracted more attention of medical chemistry researchers [3–5]. In addition, pharmacological studies exhibited that those constituents possessed various bioactivities, including anti-inflammatory [6], hepatoprotective [7], antineoplastic [8], and anti-HIV activities [9]. In order to discover novel and bioactive structures from the plant, we have performed a long-time research and obtained many new and bioactive structures [10–14]. This study is a continuing and supplementary effort to find more bioactive constituents from the roots of K. longipedunculata. Finally, two new tetrahydrobenzocyclooctabenzofuranone lignans (1–2), a new dibenzocyclooctadiene lignan (3), and three new schiartane-type triterpenoids (4–6), together with six known compounds (7–12), were obtained from its ethanol fraction (Fig. 1). Their hepatoprotective activity against APAP-induced toxicity in HepG2 cells were evaluated. In the following paper, we reported the isolation, structural identification and hepatoprotective activity of these isolates.
2.1. General experimental procedures Optical rotations were measured with a JASCO P-2000 polarimeter. UV spectra were obtained on a JASCO V-650 spectrophotometer. IR spectra on a Nicolet 5700 spectrometer were used by FT-IR microscope transmission method. ECD spectra were obtained using a JASCO J-815 spectrometer. 1D and 2D NMR spectra were recorded on BRUKER AV500-III or INOVA-600 spectrometers with TMS as internal standard. Chemical shifts were given in ppm with reference to the solvent signals. HRESIMS were performed on an Agilent 6520 Accurate-Mass Q-Tof LCMS mass spectrometer. Analytical HPLC was performed on Shimadzu LC-20AT with an SPD-M20A detector, liquid chromatography with a COSMOSIL 5C18-ARII column (4.6 × 250 mm, 5 μm) eluted with water/methanol (flow rate, 1 mL/min, 210 nm). Semipreparative HPLC was performed on a Shimadzu LC-6 AD with an SPD-20A detector, liquid chromatography with COSMOSIL 5C18-PAQ column (10 × 250 mm, 5 μm) and Reprosil CHIRAL AM (4.6 × 250 mm, 5 μm). Column chromatography was performed with silica gel (200–300 silica gel, Qingdao Marine Chemical Factory, China), RP-C18 reversed-phase silica gel (120 Å 50 μm, YMC) and Sephadex LH-20 (GE Healthcare BioScience AB). Fractions were analyzed by TLC, and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in C2H5OH.
⁎ Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 2 Nan Wei Road, Xicheng District, Beijing 100050, PR China. E-mail address: [email protected] (S. Li).
https://doi.org/10.1016/j.fitote.2020.104487 Received 6 December 2019; Received in revised form 19 January 2020; Accepted 22 January 2020 Available online 24 January 2020 0367-326X/ © 2020 Elsevier B.V. All rights reserved.
Fitoterapia 142 (2020) 104487
S.-Y. Shao, et al.
Fig. 1. Structures of compounds 1–12.
2.2. Plant material
(2.5 mg). FrC.13.4.5 was subjected to semipreparative HPLC (CH3OH–H2O, 60:40, v/v) to produce 2 (3.0 mg) and 4 (2.5 mg). Kadlongilignan E (1): yellow amorphous powder, mp 181–182 °C; [α]D20 -67.0 (c 0.06, CH3OH); ECD (CH3OH) λmax (Δε) 206 (+8.55), 228 (−20.03), 318 (−7.22), 369 (+4.06) nm; UV (CH3OH) λmax (log ε) 217 (4.60), 331 (3.53) nm; IR (Microscope) νmax 3425, 2934, 1710, 1648, 1578, 1503, 1486, 1388, 1292, 1260, 1121, 1063 cm−1. 1H and 13 C NMR data see Table 1; HRESIMS (+) m/z 499.1964 [M + H]+ (calcd for C27H31O9, 499.1963). Kadlongilignan F (2): yellow amorphous powder, mp 127–128 °C; [α]D20 -8.0 (c 0.13, CH3OH); ECD (CH3OH) λmax (Δε) 221 (−7.92), 241 (+5.03), 317 (−10.92), 368 (+4.68) nm; UV (CH3OH) λmax (log ε) 221 (4.65), 331 (3.61) nm; IR (Microscope) νmax 3436, 2936, 2877, 1731, 1649, 1580, 1503, 1489, 1437, 1385, 1306, 1263, 1120, 1063, 1026 cm−1. 1H and 13C NMR data see Table 1; HRESIMS (+) m/z 515.2265 [M + H]+ (calcd for C28H35O9, 515.2276). Kadlongilignan G (3): yellow amorphous powder, mp 178–179 °C; [α]D20 +10.0 (c 0.02, CH3OH); ECD (CH3OH) λmax (Δε) 212 (−37.23), 240 (+30.34), 291 (+4.37) nm; UV (CH3OH) λmax (log ε) 209 (4.64), 251 (4.11), 287 (3.83) nm; IR (Microscope) νmax 3395, 2933, 1591, 1514, 1455, 1412, 1274, 1234, 1155, 1110, 996 cm−1. 1H and 13C NMR data see Table 1; HRESIMS (−) m/z 373.1654 [M - H]− (calcd for C21H25O6, 373.1657). Kadlongilactone A (4): white amorphous powder, mp 215–216 °C; [α]D20 +64.0 (c 0.1, CHCl3); ECD (CH3OH) λmax (Δε) 254 (+1.77) nm; UV (CH3OH) λmax (log ε) 204 (3.97) nm; IR (Microscope) νmax 3583, 2928, 2853, 1778, 1716, 1453, 1378, 1240, 1133, 1034 cm−1. 1H and 13 C NMR data see Table 2; HRESIMS (+) m/z 523.2669 [M + Na]+ (calcd for C29H40O7Na, 523.2666). Kadlongilactone B (5): white amorphous powder, mp 196–197 °C; [α]D20 +19.0 (c 0.1, CHCl3); ECD (CH3OH) λmax (Δε) 254 (+2.40) nm; UV (CH3OH) λmax (log ε) 203 (3.82) nm; IR (Microscope) νmax 3590, 3516, 2936, 1727, 1709, 1467, 1441, 1382, 1330, 1243, 1198, 1172, 1122 cm−1. 1H and 13C NMR data see Table 2; HRESIMS (+) m/z 539.3355 [M + Na]+ (calcd for C31H48O6Na, 539.3343). Kadlongilactone C (6): white amorphous powder, mp 190–191 °C; [α]D20 +63.0 (c 0.3, CHCl3); ECD (CH3OH) λmax (Δε) 254 (+2.33) nm; UV (CH3OH) λmax (log ε) 205 (3.83) nm; IR (Microscope) νmax 3553, 3513, 2911, 1737, 1699, 1446, 1382, 1358, 1198, 1153, 1107 cm−1. 1 H and 13C NMR data see Table 2; HRESIMS (+) m/z 539.3347 [M + Na]+ (calcd for C31H48O6Na, 539.3343).
The roots of K. longipedunculata were collected from Jiujiang County in Jiangxi province of China in March 2010. The plant was identified by Researcher Ce-Ming Tan, Institute of Biology Resources, Jiangxi Academy of Science. A voucher specimen (ID-S-2428) was deposited in the herbarium of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. 2.3. Extraction and isolation The air-dried and powdered roots of K. longipedunculata were extracted with 95% aqueous ethanol at room temperature. The solvent was evaporated under reduced pressure, producing an extract, which was chromatographed on a silica gel column with a gradient system of petroleum ether-acetone (50:1, 10:1, 5:1, 3:1, 1:1, v/v), acetone, and 80% aqueous ethanol as eluent, and 14 fractions (1–14) were collected according to the result of TLC analysis. Fraction 13 was subjected to a RP-C18 silica gel column chromatography eluted with a gradient of CH3OH–H2O (40:60, 60:40, 70:30, 90:10, v/v) to afford four subfractions FrC.13.1–13.4. Subfraction 13.2 was subjected to successive silica gel column chromatography (CHCl3–CH3OH, 60:1, 50:1, 30:1, 20:1 and 0:100, v/v) to give five subfractions FrC.13.2.1–13.2.5. FrC.13.2.1 was then separated on Sephadex LH-20 column (CHCl3–CH3OH, 1:1, v/v) to afford five subfractions FrC.13.2.1.1–13.2.1.5. FrC.13.2.1.2 was then purified by semipreparative HPLC (CH3OH–H2O, 55:45, v/v) to give 1 (3.4 mg), 12 (3.3 mg) and 11 (4.8 mg). FrC.13.2.1.5 (396 mg) was purified by semipreparative HPLC (CH3OH–H2O, 55:45, v/v) to give 7 (3.6 mg). FrC.13.2.2 was subjected to a RP-C18 silica gel column chromatography CH3OH–H2O (50:50, 55:45, 100:0, v/v) to give three subfractions FrC.13.2.2.1–13.2.2.3. FrC.13.2.2.1 was subsequently purified by semipreparative HPLC (CH3OH–H2O, 57:43, v/v) to give 3 (5.4 mg). FrC.13.2.2.2 was purified by semipreparative HPLC (CH3OH–H2O, 57:43, v/v) to give 5 (6.6 mg) and 9 (3.4 mg). FrC. 13.2.2.3 was then separated on semipreparative HPLC (CH3OH–H2O, 55:45, v/v) to give 8 (26.0 mg). Fraction 13.4 was separated on silica gel column chromatography eluted with a gradient of petroleum etherethyl acetate (5:1, 4:1, 2:1, 1:1, 1:2, v/v) to give five fractions, FrC.13.4.1–FrC.13.4.5. FrC.13.4.1 was separated on semipreparative HPLC (CH3OH–H2O, 64:36, v/v) to give 10 (4.5 mg). FrC.13.4.4 was subjected to semipreparative HPLC (CH3OH–H2O, 60:40, v/v) to yield 6 2
Fitoterapia 142 (2020) 104487
S.-Y. Shao, et al.
Table 1 1 H (500 MHz) and Pos.
13
1
2
δH 1 2 3 4 5 6a 6b 7 8 9a
δC
6.14 (d, 2.0) 2.58 (ddd, 16.0, 6.0, 2.0) 2.26 (dd, 16.0, 12.0) 1.85 (m) 2.02 (ddq, 7.0, 3.0, 7.0) 5.61 (d, 7.0)
195.3 132.3 156.6 120.1 148.0 40.3
31.8 42.3 78.9
δH
6.09 (d, 2.0) 2.55 (ddd, 16.0, 6.0, 2.0) 2.17 (dd,16.0, 2.0) 1.76 (m) 1.93 (ddq, 7.0, 3.0, 7.0) 5.99 (d, 7.0)
3 δC
4.49 (br s)
31.8 42.7
2.00 (m) 1.98 (m)
42.7 39.1
76.6
2.13 (dd, 13.0, 9.5) 2.01 (dd, 13.0, 5.0)
34.1
0.90 (d, 7.0)
0.84 (d, 7.0)
128.4 101.3 150.2 130.2 144.2 122.7 64.7 21.5
18
1.03 (d, 7.5)
9.7
0.88 (d, 7.0)
8.8
19a 19b 20a
6.03 5.96 4.58 9.0) 4.25 9.0)
102.1
6.02 (d, 1.5) 5.96 (d, 1.5) 4.57 (d, 9.0)
102.0
20b 1' 2' 3' 4'a 4'b 5' 6' 7' OCH3–1 OCH3–2 OCH3–3 OCH3–13
(d, 1.5) (d, 1.5) (ABq,
78.1
(ABq,
6.34 (td, 6.0, 1.0) 4.21 (m) 4.14 (m) 1.72 (d, 1.0)
3.71 (s) 4.01 (s)
6.30 (s)
δC 149.4 138.4 147.9 109.2 137.5 73.2
129.5 101.5 150.4 130.1 144.1 121.1 65.2 21.5
6.41 (s)
δH
195.8 132.1 157.1 120.7 148.0 40.3
9b 10 11 12 13 14 15 16 17
m, H-4'a), 4.14 (1H, m, H-4'b), and a methyl signal at δH 1.72 (3H, d, J = 1.0 Hz, H-5'). In addition, a cyclooctadiene moiety was evident from two secondary methyl signals at δH 1.03 (3H, d, J = 7.5 Hz, H-18) and 0.90 (3H, d, J = 7.0 Hz, H-17), a methylene at δH 2.58 (1H, ddd, J = 16.0, 6.0, 2.0 Hz, H-6a), and 2.26 (1H, dd, J = 16.0, 12.0 Hz, H6b), and two methines at δH 2.02 (1H, ddq, J = 7.0, 3.0, 7.0 Hz, H-8) and 1.85 (1H, m, H-7), and a oxymethine 5.61 (1H, d, J = 7.0 Hz, H-9). The 13C NMR data of 1 (Table 1) showed 27 carbon signals, including an α,β-unsaturated carbonyl (δC 195.3), an ester carbonyl (δC 167.9), 12 aromatic or olefinic carbons [δC 156.6, 150.4, 148.0, 144.1, 138.9, 132.3, 130.1, 129.5 (2×), 121.1, 120.1, 101.5], a methylenedioxy (δC 102.1), an oxymethine carbon (δC 78.9), two oxymethylene carbons (δC 78.1 and 59.3), a quaternary carbon (δC 65.2), two methoxy carbons (δC 59.1, 58.8), one methylene carbon (δC 40.3), two methine carbons (δC 42.3 and 31.8), and three methyl carbons (δC 21.5, 12.9 and 9.7). Analysis of the 1H NMR and 13C NMR data of 1 indicated that it was a dibenzocyclooctadiene lignan [12]. The presence of characteristic AB quartets at δH 4.58 and 4.25 (each d, J = 9.0 Hz, H-20) and the quaternary carbon signal at δC 65.2 (C-16) suggested that 1 is a modified dibenzocyclooctadiene-type lignan possessing a spirobenzofuranoid structure [13]. This was confirmed by the HMBC correlations of H-6 with C-5 and C-7, H-9 with C-7, C-8 and C-10, and H-20 with C-14 and C-16. In addition, the HMBC correlations of H-3' with C-1', H-4′ with C2', and H-5' with C-2', together with the correlation of H-9 with C-1 suggested a 4-hydroxyltigloyl group was located on C-9. The HMBC correlations of OCH3–2 with C-2, OCH3–3 with C-3, CH3–18 with C-8, CH3–17 with C-7, and H-19 with C-12 and C-13 assigned the positions of the substituents. The relative configuration of 1 was assigned by the ROESY correlations of H-11/H-9/H-8, indicating the β-configurations of H-9 and H-8 and α-configuration of CH3–18 [13] (Fig. 3). The α-configuration of CH3–17 was supported by the ROESY correlation of CH3–17/CH3–18 [13]. In addition, the ROESY correlation of H-4′/CH3–5′ suggested the configuration of the olefinic bond was E. The ECD spectrum of 1 showed negative Cotton effects at 228 and 318 nm and positive Cotton effects near 206 and 369 nm. The calculated ECD curve of 1 exhibited similar negative and positive Cotton effects except a positive Cotton effect near 250 nm (Fig. 4). It was speculated that the large hydroxyltigloyl group on C-9 influenced the dominant conformations of 1, so that the ECD curve of the 9-OH structure (1a) was also calculated and it led to a better result (Fig. 4). On basis of the above data and the ROESY correlations, the cyclooctene moiety of 1 was determined to possess a (7R, 8R, 9R, 16S) absolute configuration. Thus, the structure of 1 was established as shown in Fig. 1. Kadlongilignan F (2) was assigned as the molecular formula of C28H34O9 based on HRESIMS ion at m/z 515.2265 [M + H]+ (calcd for C28H35O9, 515.2276) and 13C NMR spectroscopic data. The 1H and 13C NMR spectra of 2 displayed that it was a tetrahydrobenzocyclooctabenzofuranone-type lignan, which was very similar with 1. Besides, one oxymethylene at δH 3.60 (2H, t, J = 6.5 Hz, H-6'), four methylenes at δH 2.05 (2H, m, H-2'), 1.52 (2H, m, H-3'), 1.53 (2H, m, H-5'), and 1.31 (2H, m, H-4') in the 1H NMR, and an ester carbonyl at δC 173.0, an oxymethylene carbon at δC 62.6, and four methylene carbons at δC 33.6, 24.4, 25.1 and 32.1 in the 13C NMR (Table 1), revealed the presence of a 6-hydroxycaproate group. The HMBC correlation of H-9 with C-1' indicated that the 6-hydroxycaproate group was connected on C-9. The ROESY correlations of H11/H-9/H-8 and CH3–17/CH3–18 suggested the β-configurations of H-9 and H-8 and α-configurations of CH3–17 and CH3–18 (Fig. 3). The ECD curve of 2 showed negative Cotton effects around 221 and 317 nm and positive Cotton effects at 241 and 368 nm, suggesting that 2 had the same stereochemistry with 1. Thus, the structure of 2 was determined as shown in Fig. 1. Kadlongilignan G (3) was assigned as the molecular formula of C21H26O6 by the HRESIMS ion at m/z 373.1654 [M - H]− (calcd for C21H25O6, 373.1657) and 13C NMR data. The UV spectrum with λmax
C (125 MHz) NMR Data of compounds 1–3 in CDCl3.
7.05 (s)
6.79 (s) 6.70 (s) 0.73 (d, 6.5) 1.02 (d, 6.5)
136.6 114.6 145.4 144.1 112.7 125.1 124.9 7.8 21.9
78.0
4.29 (d, 9.0) 167.9 129.5 138.9
2.05 (m) 1.52 (m)
173.0 33.6 24.4
59.3
1.31 (m)
25.1
12.9
1.53 (m) 3.60 (t, 6.5)
32.1 62.6
59.1 58.8
3.75 (s) 4.06 (s)
59.4 58.8
3.44 (s) 3.97 (s)
60.2 61.2
3.86 (s)
56.1
2.4. Hepatoprotective effects assay The biological evaluation of hepatoprotective activity was performed by established method [15]. 3. Results and discussion Kadlongilignan E (1) was assigned the molecular formula of C27H30O9 by the HRESIMS ion peak at m/z 499.1964 [M + H]+ (calcd for C27H31O9, 499.1963). The UV spectrum of 1 showed the aromatic and carbonyl moieties with λmax values at 217 and 331 nm. The IR spectrum of 1 displayed the presence of hydroxyl (3425 cm−1), α,βunsaturated carbonyl (1710 cm−1), olefinic bond (1648 cm−1), phenyl (1578, 1503 and 1486 cm−1) groups. The 1H NMR data of 1 (Table 1) showed the presence of two aromatic protons at δH 6.41 (1H, s, H-11) and 6.14 (1H, d, J = 2.0 Hz, H-4), one methylenedioxy group at δH 6.03 (1H, d, J = 1.5 Hz, H-19a) and 5.96 (1H, d, J = 1.5 Hz, H-19b), two methoxy groups at δH 4.01 (3H, s, OCH3–3) and 3.71 (3H, s, OCH3–2). A hydroxyltigloyl group was discovered by one olefinic proton at δH 6.34 (1H, td, J = 6.0, 1.0 Hz, H-3'), a methylene signal at δH 4.21 (1H, 3
Fitoterapia 142 (2020) 104487
S.-Y. Shao, et al.
Table 2 1 H and 13C NMR Data of compounds 4–6 in C5D5Na. Pos.
4
5
6
δH
δC
δH
δC
δH
1
4.28 (d, 4.8)
82.1
5.85 (dd, 9.0, 6.0)
126.3
2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15 16a 16b 17 18 19a 19b 20 21 22 23a 23b 24 25 26 27 28 29 30 OCH3–3 OH-4 OH-6 OH-9 OH-15
2.98 (dd, 18.0, 4.8) 2.75 (d, 18.0)
36.6
3.73 (dd, 18.0, 9.0) 3.28 (dd, 18.0, 6.0)
34.2
3.18 1.64 3.11 2.47
a
2.52 1.63 1.35 1.93 1.38 2.17
(dd, 13.2, 3.0) (overlapped) (overlapped) (m) (overlapped) (overlapped)
1.86 1.71 2.19 1.62
(overlapped) (dt, 13.8, 3.0) (overlapped) (overlapped)
3.33 1.91 1.32 1.36 0.88 2.09 2.09 1.91 0.88 4.37 2.05 1.85 6.41
(br s) (overlapped) (overlapped) (overlapped) (s) (s) (s) (overlapped) (d, 6.6) (dt, 13.2, 3.6) (m) (overlapped) (br d, 6.6)
175.4 84.9 59.4 27.1 24.1 44.9 73.9 99.4 38.5 31.2 41.5 72.6 54.5 31.3 44.3 14.6 46.4 37.3 13.5 79.7 23.4
1.92 (s)
140.0 128.0 166.1 17.2
1.12 (s) 1.26 (s)
23.0 29.5
4.47 (s)
2.81 2.14 1.64 1.41 1.41 1.60
(t, 8.4) (overlapped) (overlapped) (m) (m) (d, 10.0)
1.76 1.67 2.10 1.49
(m) (overlapped) (dd, 13.2, 4.8) (m)
1.25 1.70 1.25 1.54 0.78 2.21 2.82 2.00 0.95 4.44 2.15 1.95 6.49
(m) (overlapped) (overlapped) (m) (s) (d, 12.0) (d, 12.0) (m) (d, 6.6) (dt, 13.2, 3.0) (overlapped) (overlapped) (br d, 6.6)
1.93 1.12 1.36 1.38 3.59 5.43
(s) (s) (s) (s) (s) (s)
3.54 (d, 1.8)
173.5 73.4 49.9 29.8 23.2 54.4 70.4 141.0 37.7 30.8 46.5 49.6 34.1 26.8 46.7 15.2 50.7 39.4 13.6 80.5 23.7 140.1 128.0 166.3 17.2 19.5 27.7 29.5 51.7
δC (m) (overlapped) (m) (m)
2.15 (d, 4.5) 4.65 (d, 3.5) 1.36 (t, 13.0) 1.59 (overlapped) 2.39 (dd, 13.5, 3.0) 2.33 1.29 1.59 1.59
(m) (overlapped) (overlapped) (overlapped)
1.30 1.69 1.25 1.57 1.03 1.65 0.71 1.96 0.97 4.43 2.22 1.99 6.50
(overlapped) (overlapped) (overlapped) (overlapped) (s) (overlapped) (d, 3.5) (overlapped) (d, 5.5) (dt, 13.5, 3.0) (m) (overlapped) (br d, 6.0)
1.93 0.98 1.84 1.80 3.63 5.49 5.70
(s) (s) (s) (s) (s) (s) (d, 2.5)
31.6 32.7 174.8 75.7 48.0 66.9 34.5 39.3 22.4 26.0 26.7 33.3 45.9 48.4 36.2 27.2 48.6 18.8 33.6 39.5 13.1 80.5 23.6 140.1 128.1 166.3 17.2 20.2 29.4 31.4 51.3
600 MHz for compounds 4 and 5, 500 MHz for compound 6.
values at 209, 251 and 287 nm, and its IR spectrum with absorption bands at 1591, 1514 and 1455 cm−1 (aromatic moiety), indicated that 3 was a dibenzocyclooctadiene lignan [16]. The 1H NMR data of 3 (Table 1) displayed three aromatic protons at δH 7.05 (1H, s, H-4), 6.79 (1H, s, H-11) and 6.70 (1H, s, H-14) for a biphenyl moiety, three methoxy groups at δH 3.97 (3H, s, OCH3–2), 3.86 (3H, s, OCH3–13) and 3.44 (3H, s, OCH3–1). A cyclooctadiene ring was recognized from two methyls at δH 1.02 (3H, d, J = 6.5 Hz, CH3–18) and 0.73 (3H, d, J = 6.5 Hz, CH3–17), three methines at δH 2.00 (1H, m, H-7), 1.98 (1H, m, H-8), and 4.49 (1H, br s, H-6), and one methylene at δH 2.13 (1H, dd, J = 13.0, 9.5 Hz, H-9a) and 2.01 (1H, dd, J = 13.0, 5.0 Hz, H-9b). The HMBC correlations of H-6 with C-5 and C-7, H-9 with C-7, C-8, and C-10, CH3–17 with C-7, and CH3–18 with C-8 indicated the presence of a cyclooctadiene moiety. The HMBC correlations of OCH3–1 with C-1, OCH3–2 with C-2, and OCH3–13 with C-13 suggested that three methoxy groups were located at C-1, C-2 and C-13, respectively (Fig. 2). The ECD curve of 3 showed a negative Cotton effect around 212 nm and a positive Cotton effect near 240 nm, suggesting that 3 possessed an M-biphenyl configuration [17]. With the axial chirality defined, the ROESY experiment was used to establish the configurations of the remaining stereocenters. The ROESY correlations of H-4/H-6/H-9β and
H-9α/H-11/CH3–17/CH3–18 indicated that H-6 was β-oriented and CH3–17 and CH3–18 were α-oriented [18] (Fig. 3). The calculated ECD curve exhibited a negative Cotton effect around 210 nm and a positive Cotton effect near 250 nm, which matched very well with the experimental spectrum (Fig. 4). Therefore, the structure of 3 and its (M, 6S, 7S, 8S) absolute configuration was established as shown in Fig. 1. Kadlongilactone A (4) was isolated as a white powder. The HRESIMS provided a molecular formula of C29H40O7 by the quasi ion peak at m/z 523.2669 [M + Na]+ (calcd for C29H40O7Na, 523.2666) with 10 degrees of unsaturation. The 1H NMR data of 4 (Table 2) displayed an olefinic proton at δH 6.41 (1H, br d, J = 6.6 Hz, H-24), four methine proton signals (three oxygenated) at δH 4.37 (1H, dt, J = 13.2, 3.6 Hz, H-22), 4.28 (1H, d, J = 4.8 Hz, H-1), 3.33 (1H, s, H-15), and 2.52 (1H, dd, J = 13.2, 3.0 Hz, H-5), three methylene signals at δH 2.98 (1H, dd, J = 18.0, 4.8 Hz, H-2a), 2.75 (1H, d, J = 18.0 Hz, H-2b), and 2.09 (2H, s, H-19) and many methine and methylene proton signals between δH 1.35–2.19. In addition, five methyl proton signals at δH 1.92 (3H, s, H-27), 1.26 (3H, s, H-30), 1.12 (3H, s, H-29), 0.88 (3H, s, H-18), and 0.88 (3H, d, J = 6.6 Hz, H-21) were observed. The above proton signals suggested that 4 may be a triterpene. The 13C NMR data of 4 (Table 2) showed 29 carbon signals, including two ester carbonyls at δC 4
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Fig. 2. Key HMBC (H → C) of compounds 1–6 and key 1He1H COSY (—) correlations of compounds 4–6.
Fig. 3. Key ROESY (↔) correlations of compounds 1–6.
175.4 (C-3) and 166.1 (C-26), two olefinic carbons at δC 140.0 (C-24) and 128.0 (C-25), five quaternary carbons at δC 99.4 (C-10), 84.9 (C-4), 73.9 (C-9), 72.6 (C-14) and 41.5 (C-13), seven methine carbons at δC 82.1 (C-1), 79.7 (C-22), 54.5 (C-15), 59.4 (C-5), 44.9 (C-8), 44.3 (C-17), and 37.3 (C-20), eight methylene carbons at δC 46.4 (C-19), 38.5 (C11), 36.6 (C-2), 31.3 (C-16), 31.2 (C-12), 27.1 (C-6), 24.1 (C-7), and 23.4 (C-23), and five methyl carbons at δC 14.6 (C-18), 13.5 (C-21), 17.2 (C-27), 23.0 (C-29), and 29.5 (C-30). Combination the 1H and 13C NMR data with the unsaturation degrees suggested 4 was a nortriterpenoid with seven rings. The 1He1H COSY spectrum (Fig. 2) of 4 showed four proton spin systems involving H-1/H2–2, H-5/H2–6/H2–7/H-8, H2–11/H2–12, and H-15/H2–16/H-17/H-20/H3–21, H-22/H2–23/H-24. Then, the HMBC correlations (Fig. 2) of H-1 with C-4 and C-10, H-2 with C-3, H-30 with C-5 and C-29, H-29 with C-4, H-19 with C-5, C-8, C-9 and C-10, H-8 with C-14, H-11 with C-9, H-12 with C-13, H-15 with C-14, H-17 with C-13, H-22 with C-26 and H-27 with C-24, C-25 and C-26 established the main planar structure of 4. In addition, the HMBC correlation of H-
18 with C-13 suggested a methyl was attached on C-13. The ROESY correlations (Fig. 3) of H-1 with H-29, H-5 with H-30, H8 with CH3–18 and H-15, H-22 with H-17, and no correlations of H-15 with H-17, combined with the small coupling constant (3.6 Hz) between H-20 and H-22 suggested that H-1, H-8, H-15, CH3–18 and CH3–29 were β-oriented and H-5, H-17, H-22, CH3–21 and CH3–30 were α-oriented [10,19]. Comparison the NMR data of C-9, C-10 and C14 with the reported micrandilactone I [10] indicated that their substituents should be α-oriented. The ECD spectrum of 4 showed a positive Cotton effect near 254 nm, which suggested the absolute configuration of C-22 was R [10]. Then, the absolute configuration of 4 was determined as 1R, 5S, 8R, 9R, 10R, 13R, 14R, 15S, 17R, 20S and 22R. The result was finally confirmed by comparison of the experimental and calculated ECD curves (Fig. 4). Accordingly, the structure of compound 4 was established as shown in Fig. 1. Kadlongilactone B (5) was obtained as a white powder. Compound 5 was determined to have the molecular formula of C31H48O6 and eight unsaturation degrees via the positive HRESIMS ion at m/z 539.3355 5
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Fig. 4. Experimental (black) and calculated ECD spectra (red) in methanol of 1, 1a, 3 and 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
[M + Na]+ (calcd for C31H48O6Na, 539.3343). Comparison of the NMR data of 4 and 5 displayed that their structures were similar except a few differences. The 1H NMR data of 5 showed an extra olefinic proton signal at δH 5.85 (1H, dd, J = 9.0, 6.0 Hz, H-1), a methylene proton signal at δH 1.25 (2H, m, H-15), a methoxy signal at δH 3.59 (3H, s, OCH3–3), and a methyl group at δH 1.12 (3H, s, H-28), however, two oxygenated methine signals at δH 4.28 (1H, d, J = 4.8 Hz, H-1) and 3.33 (1H, s, H-15) were absent, which suggested 5 was a seco-triterpenoid [20]. The HMBC correlations of H-1 with C-10 and OCH3–3 with C-3 combined with no correlation of H-1 with C-4 suggested the A and B rings were split. The HMBC correlation of CH3–28 with C-14 suggested the extra methyl was connected on C-14. The ROESY correlations of CH3–28 with H-17 and 9-OH indicated that the CH3–28 was αoriented. The relative configurations of other chiral carbons were the same with 5 according to the ROESY spectrum. The ECD spectrum of 5 displayed a positive Cotton effect around 254 nm, which inferred the absolute configuration of C-22 was R. Combined with the relative configuration of 5, its absolute configuration could be determined as 5R, 8S, 9S, 13R, 14S, 17R, 20S and 22R. Therefore, the structure of 5 was determined as shown in Fig. 1.
Kadlongilactone C (6) was a white powder. The molecular formula of C31H48O6 was determined by the HRESIMS ion at m/z 539.3347 [M + Na]+ (calcd for C31H48O6Na, 539.3343), which was the same with 5. Comparison the NMR data of 5 and 6 showed that they were very similar except some differences. The 1H NMR data of 6 only showed one olefinic proton signal at δH 6.50 (1H, d, J = 6.0 Hz, H-24), which suggested the other olefinic bond was reduced. A methine proton signal at δH 4.65 (1H, d, J = 3.5 Hz, H-6) and the 1He1H COSY correlation of H-5 with H-6 suggested that the C-6 was oxygenated. In addition, the 1H NMR data displayed a shielded methylene proton signal at δH 0.71 (H-19b), together with the same eight unsaturation degrees, suggesting it may exist a cyclopropane moiety in 6 [21]. The HMBC correlations of H-19 with C-9 and C-10 assigned the position of the cyclopropane structure. The ROESY correlations of H-19/H-8/6-OH indicated they were all β-oriented. Its ECD curve displayed a positive Cotton effect at 255 nm, which indicated the absolute configuration of C-22 were R. Then, the absolute configuration of 6 was assigned as 5S, 6R, 8S, 9S, 10R, 13R, 14S, 17R, 20S and 22R. Thus, the structure of 6 was determined as shown in Fig. 1. Additionally, six known tetrahydrobenzocyclooctabenzofuranone 6
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Table 3 Hepatoprotective activity of compounds 1–12 (10 μM) against APAP-induced HepG2 cell damage. Groupa
Optical density
Cell survival rates (%)b
Control Model 1 2 3 4 5 6 7 8 9 10 11 12 Bicyclol
1.339 0.552 0.565 0.553 0.554 0.540 – – 0.548 0.608 0.633 – 0.574 0.710 0.653
100.00 ± 7.767 41.24 ± 13.587 31.09 ± 5.133 31.09 ± 4.702 41.40 ± 22.202 29.74 ± 2.963 – – 40.93 ± 3.285 45.41 ± 7.767 34.84 ± 2.467 – 42.87 ± 2.265 53.04 ± 4.507 48.77 ± 9.495
± ± ± ± ± ±
0.104 0.075⁎⁎⁎ 0.029 0.026 0.123 0.016
± 0.018 ± 0.015 ± 0.013 ± 0.013 ± 0.032## ± 0.062#
240 Section 1. [2] Compilation of Chinese Herb Medicine, 1 People’s Publishing House, Beijing, 1975, p. 581. [3] W.L. Xiao, R.T. Li, S.X. Huang, J.X. Pu, H.D. Sun, Triterpenoids from the Schisandraceae family, Nat. Prod. Rep. 25 (2008) 871–891. [4] J.S. Liu, M.F. Huang, Kadsuligans E–G from Kadsura Longipedunculata, Phytochemistry 31 (1992) 957–960. [5] H.T. Liu, B.G. Zhang, Y. Peng, Y.D. Qi, L.J. Xu, X.W. Yang, P.G. Xiao, New spirobenzofuranoid dibenzocyclooctadiene lignans from Kadsura oblongifolia, Fitoterapia 82 (2011) 731–734. [6] H.R. Li, L.Y. Wang, Z.G. Yang, S. Kitanaka, Kadsuralignans H–K from Kadsura coccinea and their nitric oxide production inhibitory effects, J. Nat. Prod. 70 (2007) 1999–2002. [7] W. Su, J.P. Zhao, M. Yang, H.W. Yan, T. Pang, S.H. Chen, H.Y. Huang, S.H. Zhang, X.C. Ma, D.A. Guo, I.A. Khan, W. Wang, A coumarin lignanoid from the stems of Kadsura heteroclita, Bioorg. Med. Chem. Lett. 25 (2015) 1506–1508. [8] Y.C. Shen, Y.C. Lin, Y.B. Cheng, Y.H. Kuo, C.C. Liaw, Taiwankadsurins A, B, and C, three new C19 homolignans from Kadsura philippinensis, Org. Lett. 7 (2005) 5297–5300. [9] J.X. Pu, L.M. Yang, W.L. Xiao, R.T. Li, C. Lei, X.M. Gao, S.X. Huang, S.H. Li, Y.T. Zheng, H. Huang, H.D. Sun, Compounds from Kadsura heteroclita and related anti-HIV activity, Phytochemistry 69 (2008) 1266–1272. [10] X.J. Wang, J.B. Liu, P. Pandey, J.B. Chen, F.R. Fronczek, S. Parnham, X.Z. Qi, R.J. Doerksen, D. Ferreira, H. Sun, S. Li, M.T. Hamann, Assignment of the absolute configuration of hepatoprotective highly oxygenated triterpenoids using X-ray, ECD, NMR J-based configurational analysis and HSQC overlay experiments, BBAGen. Subjects 1861 (2017) 3089–3095. [11] X.J. Wang, F.R. Fronczek, J.B. Chen, J.B. Liu, D. Ferreira, S. Li, M.T. Hamann, Assignment of absolute configuration of a new hepatoprotective schiartane-type nortriterpenoid using X-ray diffraction, Molecules 22 (2017) 65/1–65/5. [12] J.B. Liu, P. Pandey, X.J. Wang, X.Z. Qi, J.B. Chen, H. Sun, P.C. Zhang, Y.Q. Ding, D. Ferreira, R.J. Doerksen, M.T. Hamann, S. Li, Hepatoprotective dibenzocyclooctadiene and tetrahydrobenzocyclooctabenzofuranone lignans from Kadsura longipedunculata, J. Nat. Prod. 81 (2018) 846–857. [13] J.B. Liu, P. Pandey, X.J. Wang, K. Admams, X.Z. Qi, J.B. Chen, H. Sun, Q. Hou, D. Ferreira, R.J. Doerksen, M.T. Hamann, S. Li, Hepatoprotective tetrahydrobenzocyclooctabenzofuranone lignans from Kadsura longipedunculata, J. Nat. Prod. 82 (2019) 2842–2851. [14] X.Z. Qi, J.B. Liu, J.B. Chen, Q. Hou, S. Li, New seco-dibenzocyclooctadiene lignans with nitric oxide production inhibitory activity from the roots of Kadsura longipedunculata, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.06. 006. [15] S.Y. Shao, Z.M. Feng, Y.N. Yang, J.S. Jiang, P.C. Zhang, Eight new phenylethanoid glycoside derivatives possessing potential hepatoprotective activities from the fruits of Forsythia suspensa, Fitoterapia 122 (2017) 132–137. [16] K. Dong, J.X. Pu, H.Y. Zhang, X. Du, X.N. Li, J. Zou, J.H. Yang, W. Zhao, X.C. Tang, H.D. Sun, Dibenzocyclooctadiene lignans from Kadsura polysperma and their antineurodegenerative activities, J. Nat. Prod. 75 (2012) 249–256. [17] Y.C. Shen, Y.B. Cheng, T.W. Lan, C.C. Liaw, S.S. Liou, Y.H. Kuo, A.T. Khalil, Kadsuphilols A–H, oxygenated lignans from Kadsura philippinensis, J. Nat. Prod. 70 (2007) 1139–1145. [18] G.Y. Yang, Y.K. Li, R.R. Wang, X.N. Li, W.L. Xiao, L.M. Yang, J.X. Pu, Y.T. Zheng, H.D. Sun, Dibenzocyclooctadiene lignans from Schisandra wilsoniana and their antiHIV-1 activities, J. Nat. Prod. 73 (2010) 915–919. [19] C. Lei, S.X. Huang, J.J. Chen, L.B. Yang, W.L. Xiao, Y. Chang, Y. Lu, H. Huang, J.X. Pu, H.D. Sun, Propindilactones E–J, schiartane nortriterpenoids from Schisandra propinqua var. propinqua, J. Nat. Prod. 71 (2008) 1228–1232. [20] J.H. Yang, J. Wen, X. Du, X.N. Li, Y.Y. Wang, Y. Li, W.L. Xiao, J.X. Pu, H.D. Sun, Triterpenoids from the stems of Kadsura ananosma, Tetrahedron 66 (2010) 8880–8887. [21] Z.P. Mai, C. Wang, Y. Wang, H.L. Zhang, B.J. Zhang, W. Wang, X.K. Huo, S.S. Huang, C.Y. Wang, K.X. Liu, X.C. Ma, X.B. Wang, Bioactive metabolites of Schisanlactone E transformed by Cunninghamella blakesleana AS 3.970, Fitoterapia 99 (2014) 352–361. [22] L.N. Li, H. Xue, Dibenzocyclooctadiene lignans possessing a spirobenzo-furanoid skeleton from Kadsura coccinea, Phytochemistry 29 (1990) 2730–2732. [23] X.M. Gao, J.X. Pu, W.L. Xiao, S.X. Huang, L.G. Lou, H.D. Dong, Kadcoccilactones K–R, triterpenoids from Kadsura coccinea, Tetrahedron 64 (2008) 11673–11679. [24] Y. Lu, D.F. Chen, Kadsutherins A–C: three new dibenzocyclooctane lignans from the stems of Kadsura species, Helv. Chim. Acta 89 (2006) 895–901. [25] Y.C. Shen, Y.C. Lin, Y.B. Cheng, C.J. Chang, T.W. Lan, S.S. Liou, C.T. Chien, C.C. Liaw, A.T. Khalil, New oxygenated lignans from Kadsura philippinensis, Helv. Chim. Acta 91 (2008) 483–494.
a APAP (8 mM)-induced cells as the model group; Bicyclol as the positive control; the compounds were tested at 10 μM. bResults are expressed as the means ± SD (n = 3). ⁎⁎⁎p < .001 (vs control group). ###p < .001. ## p < .01. #p < .05 (vs model group). ‶-″: These compounds were not determined.
lignans and schiartane-type triterpenoids were isolated and identified as benzoyl oxokadsuranol (7) [22], propoxyl oxokadsuranol (8) [22], heteroclitin E (9) [23], kadsutherin C (10) [24], kadsuphilin J (11) [25], and micrandilactone I (12) [10]. All the compounds were tested for their hepatoprotective activity against APAP-induced toxicity in HepG2 cells. The cell survival rates of these compounds were given in Table 3. Compound 12 exhibited moderate hepatoprotective activity with cell survival rates of 53.04% at 10 μM (bicyclol as positive control). However, the other compounds only showed weak hepatoprotective activity. Declaration of Competing Interest We declare no conflict of interest for this study. Acknowledgments This work was financially supported by CAMS Initiative for Innovative Medicine (No. CAMS-2016-I2M-1-010). Author statment All the authors have seen and approved the final version of the manuscript being submitted. We warrant that the article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere. None Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2020.104487. References [1] China Flora Editing Group, Flora of China, Vol. 30 Science Press, Beijing, 1996, p.
7
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Hepatoprotective lignans and triterpenoids from the roots of Kadsura longipedunculata Si-Yuan Shao, Xin-Zhu Qi, Hua Sun, Shuai Li
T
⁎
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China
ARTICLE INFO
ABSTRACT
Keywords: Kadsura longipedunculata Tetrahydrobenzocyclooctabenzofuranone lignan Dibenzocyclooctadiene lignan Schiartane-type triterpenoid Hepatoprotective activity
Two new tetrahydrobenzocyclooctabenzofuranone lignans (1–2), a new dibenzocyclooctadiene lignan (3) and three new schiartane-type triterpenoids (4–6), together with six known compounds (7–12), were isolated from the roots of Kadsura longipedunculata. Their structures were elucidated by extensive NMR and HRESIMS spectroscopic data analysis. The absolute configurations of these compounds were determined by comparison of the experimental and calculated ECD spectra. Compound 12 exhibited moderate hepatoprotective activity against Nacetyl-p-aminophenol (APAP)-induced toxicity in HepG2 cells with cell survival rates of 53.04%.
1. Introduction
2. Experimental
Kadsura longipedunculata is a lianoid plant from the Magnoliaceae family and distributed widely in the south of China [1]. Its roots have been used as Chinese folk medicine for the treatment of rheumatoid arthritis, gastric, duodenal ulcer, dysmenorrhea, and traumatic injury [2]. Over the past ten years, a series of bioactive and novel lignans and triterpenoids have been isolated from the roots of genus Kadsura and Schizandra, which have attracted more attention of medical chemistry researchers [3–5]. In addition, pharmacological studies exhibited that those constituents possessed various bioactivities, including anti-inflammatory [6], hepatoprotective [7], antineoplastic [8], and anti-HIV activities [9]. In order to discover novel and bioactive structures from the plant, we have performed a long-time research and obtained many new and bioactive structures [10–14]. This study is a continuing and supplementary effort to find more bioactive constituents from the roots of K. longipedunculata. Finally, two new tetrahydrobenzocyclooctabenzofuranone lignans (1–2), a new dibenzocyclooctadiene lignan (3), and three new schiartane-type triterpenoids (4–6), together with six known compounds (7–12), were obtained from its ethanol fraction (Fig. 1). Their hepatoprotective activity against APAP-induced toxicity in HepG2 cells were evaluated. In the following paper, we reported the isolation, structural identification and hepatoprotective activity of these isolates.
2.1. General experimental procedures Optical rotations were measured with a JASCO P-2000 polarimeter. UV spectra were obtained on a JASCO V-650 spectrophotometer. IR spectra on a Nicolet 5700 spectrometer were used by FT-IR microscope transmission method. ECD spectra were obtained using a JASCO J-815 spectrometer. 1D and 2D NMR spectra were recorded on BRUKER AV500-III or INOVA-600 spectrometers with TMS as internal standard. Chemical shifts were given in ppm with reference to the solvent signals. HRESIMS were performed on an Agilent 6520 Accurate-Mass Q-Tof LCMS mass spectrometer. Analytical HPLC was performed on Shimadzu LC-20AT with an SPD-M20A detector, liquid chromatography with a COSMOSIL 5C18-ARII column (4.6 × 250 mm, 5 μm) eluted with water/methanol (flow rate, 1 mL/min, 210 nm). Semipreparative HPLC was performed on a Shimadzu LC-6 AD with an SPD-20A detector, liquid chromatography with COSMOSIL 5C18-PAQ column (10 × 250 mm, 5 μm) and Reprosil CHIRAL AM (4.6 × 250 mm, 5 μm). Column chromatography was performed with silica gel (200–300 silica gel, Qingdao Marine Chemical Factory, China), RP-C18 reversed-phase silica gel (120 Å 50 μm, YMC) and Sephadex LH-20 (GE Healthcare BioScience AB). Fractions were analyzed by TLC, and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in C2H5OH.
⁎ Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 2 Nan Wei Road, Xicheng District, Beijing 100050, PR China. E-mail address: [email protected] (S. Li).
https://doi.org/10.1016/j.fitote.2020.104487 Received 6 December 2019; Received in revised form 19 January 2020; Accepted 22 January 2020 Available online 24 January 2020 0367-326X/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. Structures of compounds 1–12.
2.2. Plant material
(2.5 mg). FrC.13.4.5 was subjected to semipreparative HPLC (CH3OH–H2O, 60:40, v/v) to produce 2 (3.0 mg) and 4 (2.5 mg). Kadlongilignan E (1): yellow amorphous powder, mp 181–182 °C; [α]D20 -67.0 (c 0.06, CH3OH); ECD (CH3OH) λmax (Δε) 206 (+8.55), 228 (−20.03), 318 (−7.22), 369 (+4.06) nm; UV (CH3OH) λmax (log ε) 217 (4.60), 331 (3.53) nm; IR (Microscope) νmax 3425, 2934, 1710, 1648, 1578, 1503, 1486, 1388, 1292, 1260, 1121, 1063 cm−1. 1H and 13 C NMR data see Table 1; HRESIMS (+) m/z 499.1964 [M + H]+ (calcd for C27H31O9, 499.1963). Kadlongilignan F (2): yellow amorphous powder, mp 127–128 °C; [α]D20 -8.0 (c 0.13, CH3OH); ECD (CH3OH) λmax (Δε) 221 (−7.92), 241 (+5.03), 317 (−10.92), 368 (+4.68) nm; UV (CH3OH) λmax (log ε) 221 (4.65), 331 (3.61) nm; IR (Microscope) νmax 3436, 2936, 2877, 1731, 1649, 1580, 1503, 1489, 1437, 1385, 1306, 1263, 1120, 1063, 1026 cm−1. 1H and 13C NMR data see Table 1; HRESIMS (+) m/z 515.2265 [M + H]+ (calcd for C28H35O9, 515.2276). Kadlongilignan G (3): yellow amorphous powder, mp 178–179 °C; [α]D20 +10.0 (c 0.02, CH3OH); ECD (CH3OH) λmax (Δε) 212 (−37.23), 240 (+30.34), 291 (+4.37) nm; UV (CH3OH) λmax (log ε) 209 (4.64), 251 (4.11), 287 (3.83) nm; IR (Microscope) νmax 3395, 2933, 1591, 1514, 1455, 1412, 1274, 1234, 1155, 1110, 996 cm−1. 1H and 13C NMR data see Table 1; HRESIMS (−) m/z 373.1654 [M - H]− (calcd for C21H25O6, 373.1657). Kadlongilactone A (4): white amorphous powder, mp 215–216 °C; [α]D20 +64.0 (c 0.1, CHCl3); ECD (CH3OH) λmax (Δε) 254 (+1.77) nm; UV (CH3OH) λmax (log ε) 204 (3.97) nm; IR (Microscope) νmax 3583, 2928, 2853, 1778, 1716, 1453, 1378, 1240, 1133, 1034 cm−1. 1H and 13 C NMR data see Table 2; HRESIMS (+) m/z 523.2669 [M + Na]+ (calcd for C29H40O7Na, 523.2666). Kadlongilactone B (5): white amorphous powder, mp 196–197 °C; [α]D20 +19.0 (c 0.1, CHCl3); ECD (CH3OH) λmax (Δε) 254 (+2.40) nm; UV (CH3OH) λmax (log ε) 203 (3.82) nm; IR (Microscope) νmax 3590, 3516, 2936, 1727, 1709, 1467, 1441, 1382, 1330, 1243, 1198, 1172, 1122 cm−1. 1H and 13C NMR data see Table 2; HRESIMS (+) m/z 539.3355 [M + Na]+ (calcd for C31H48O6Na, 539.3343). Kadlongilactone C (6): white amorphous powder, mp 190–191 °C; [α]D20 +63.0 (c 0.3, CHCl3); ECD (CH3OH) λmax (Δε) 254 (+2.33) nm; UV (CH3OH) λmax (log ε) 205 (3.83) nm; IR (Microscope) νmax 3553, 3513, 2911, 1737, 1699, 1446, 1382, 1358, 1198, 1153, 1107 cm−1. 1 H and 13C NMR data see Table 2; HRESIMS (+) m/z 539.3347 [M + Na]+ (calcd for C31H48O6Na, 539.3343).
The roots of K. longipedunculata were collected from Jiujiang County in Jiangxi province of China in March 2010. The plant was identified by Researcher Ce-Ming Tan, Institute of Biology Resources, Jiangxi Academy of Science. A voucher specimen (ID-S-2428) was deposited in the herbarium of Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. 2.3. Extraction and isolation The air-dried and powdered roots of K. longipedunculata were extracted with 95% aqueous ethanol at room temperature. The solvent was evaporated under reduced pressure, producing an extract, which was chromatographed on a silica gel column with a gradient system of petroleum ether-acetone (50:1, 10:1, 5:1, 3:1, 1:1, v/v), acetone, and 80% aqueous ethanol as eluent, and 14 fractions (1–14) were collected according to the result of TLC analysis. Fraction 13 was subjected to a RP-C18 silica gel column chromatography eluted with a gradient of CH3OH–H2O (40:60, 60:40, 70:30, 90:10, v/v) to afford four subfractions FrC.13.1–13.4. Subfraction 13.2 was subjected to successive silica gel column chromatography (CHCl3–CH3OH, 60:1, 50:1, 30:1, 20:1 and 0:100, v/v) to give five subfractions FrC.13.2.1–13.2.5. FrC.13.2.1 was then separated on Sephadex LH-20 column (CHCl3–CH3OH, 1:1, v/v) to afford five subfractions FrC.13.2.1.1–13.2.1.5. FrC.13.2.1.2 was then purified by semipreparative HPLC (CH3OH–H2O, 55:45, v/v) to give 1 (3.4 mg), 12 (3.3 mg) and 11 (4.8 mg). FrC.13.2.1.5 (396 mg) was purified by semipreparative HPLC (CH3OH–H2O, 55:45, v/v) to give 7 (3.6 mg). FrC.13.2.2 was subjected to a RP-C18 silica gel column chromatography CH3OH–H2O (50:50, 55:45, 100:0, v/v) to give three subfractions FrC.13.2.2.1–13.2.2.3. FrC.13.2.2.1 was subsequently purified by semipreparative HPLC (CH3OH–H2O, 57:43, v/v) to give 3 (5.4 mg). FrC.13.2.2.2 was purified by semipreparative HPLC (CH3OH–H2O, 57:43, v/v) to give 5 (6.6 mg) and 9 (3.4 mg). FrC. 13.2.2.3 was then separated on semipreparative HPLC (CH3OH–H2O, 55:45, v/v) to give 8 (26.0 mg). Fraction 13.4 was separated on silica gel column chromatography eluted with a gradient of petroleum etherethyl acetate (5:1, 4:1, 2:1, 1:1, 1:2, v/v) to give five fractions, FrC.13.4.1–FrC.13.4.5. FrC.13.4.1 was separated on semipreparative HPLC (CH3OH–H2O, 64:36, v/v) to give 10 (4.5 mg). FrC.13.4.4 was subjected to semipreparative HPLC (CH3OH–H2O, 60:40, v/v) to yield 6 2
Fitoterapia 142 (2020) 104487
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Table 1 1 H (500 MHz) and Pos.
13
1
2
δH 1 2 3 4 5 6a 6b 7 8 9a
δC
6.14 (d, 2.0) 2.58 (ddd, 16.0, 6.0, 2.0) 2.26 (dd, 16.0, 12.0) 1.85 (m) 2.02 (ddq, 7.0, 3.0, 7.0) 5.61 (d, 7.0)
195.3 132.3 156.6 120.1 148.0 40.3
31.8 42.3 78.9
δH
6.09 (d, 2.0) 2.55 (ddd, 16.0, 6.0, 2.0) 2.17 (dd,16.0, 2.0) 1.76 (m) 1.93 (ddq, 7.0, 3.0, 7.0) 5.99 (d, 7.0)
3 δC
4.49 (br s)
31.8 42.7
2.00 (m) 1.98 (m)
42.7 39.1
76.6
2.13 (dd, 13.0, 9.5) 2.01 (dd, 13.0, 5.0)
34.1
0.90 (d, 7.0)
0.84 (d, 7.0)
128.4 101.3 150.2 130.2 144.2 122.7 64.7 21.5
18
1.03 (d, 7.5)
9.7
0.88 (d, 7.0)
8.8
19a 19b 20a
6.03 5.96 4.58 9.0) 4.25 9.0)
102.1
6.02 (d, 1.5) 5.96 (d, 1.5) 4.57 (d, 9.0)
102.0
20b 1' 2' 3' 4'a 4'b 5' 6' 7' OCH3–1 OCH3–2 OCH3–3 OCH3–13
(d, 1.5) (d, 1.5) (ABq,
78.1
(ABq,
6.34 (td, 6.0, 1.0) 4.21 (m) 4.14 (m) 1.72 (d, 1.0)
3.71 (s) 4.01 (s)
6.30 (s)
δC 149.4 138.4 147.9 109.2 137.5 73.2
129.5 101.5 150.4 130.1 144.1 121.1 65.2 21.5
6.41 (s)
δH
195.8 132.1 157.1 120.7 148.0 40.3
9b 10 11 12 13 14 15 16 17
m, H-4'a), 4.14 (1H, m, H-4'b), and a methyl signal at δH 1.72 (3H, d, J = 1.0 Hz, H-5'). In addition, a cyclooctadiene moiety was evident from two secondary methyl signals at δH 1.03 (3H, d, J = 7.5 Hz, H-18) and 0.90 (3H, d, J = 7.0 Hz, H-17), a methylene at δH 2.58 (1H, ddd, J = 16.0, 6.0, 2.0 Hz, H-6a), and 2.26 (1H, dd, J = 16.0, 12.0 Hz, H6b), and two methines at δH 2.02 (1H, ddq, J = 7.0, 3.0, 7.0 Hz, H-8) and 1.85 (1H, m, H-7), and a oxymethine 5.61 (1H, d, J = 7.0 Hz, H-9). The 13C NMR data of 1 (Table 1) showed 27 carbon signals, including an α,β-unsaturated carbonyl (δC 195.3), an ester carbonyl (δC 167.9), 12 aromatic or olefinic carbons [δC 156.6, 150.4, 148.0, 144.1, 138.9, 132.3, 130.1, 129.5 (2×), 121.1, 120.1, 101.5], a methylenedioxy (δC 102.1), an oxymethine carbon (δC 78.9), two oxymethylene carbons (δC 78.1 and 59.3), a quaternary carbon (δC 65.2), two methoxy carbons (δC 59.1, 58.8), one methylene carbon (δC 40.3), two methine carbons (δC 42.3 and 31.8), and three methyl carbons (δC 21.5, 12.9 and 9.7). Analysis of the 1H NMR and 13C NMR data of 1 indicated that it was a dibenzocyclooctadiene lignan [12]. The presence of characteristic AB quartets at δH 4.58 and 4.25 (each d, J = 9.0 Hz, H-20) and the quaternary carbon signal at δC 65.2 (C-16) suggested that 1 is a modified dibenzocyclooctadiene-type lignan possessing a spirobenzofuranoid structure [13]. This was confirmed by the HMBC correlations of H-6 with C-5 and C-7, H-9 with C-7, C-8 and C-10, and H-20 with C-14 and C-16. In addition, the HMBC correlations of H-3' with C-1', H-4′ with C2', and H-5' with C-2', together with the correlation of H-9 with C-1 suggested a 4-hydroxyltigloyl group was located on C-9. The HMBC correlations of OCH3–2 with C-2, OCH3–3 with C-3, CH3–18 with C-8, CH3–17 with C-7, and H-19 with C-12 and C-13 assigned the positions of the substituents. The relative configuration of 1 was assigned by the ROESY correlations of H-11/H-9/H-8, indicating the β-configurations of H-9 and H-8 and α-configuration of CH3–18 [13] (Fig. 3). The α-configuration of CH3–17 was supported by the ROESY correlation of CH3–17/CH3–18 [13]. In addition, the ROESY correlation of H-4′/CH3–5′ suggested the configuration of the olefinic bond was E. The ECD spectrum of 1 showed negative Cotton effects at 228 and 318 nm and positive Cotton effects near 206 and 369 nm. The calculated ECD curve of 1 exhibited similar negative and positive Cotton effects except a positive Cotton effect near 250 nm (Fig. 4). It was speculated that the large hydroxyltigloyl group on C-9 influenced the dominant conformations of 1, so that the ECD curve of the 9-OH structure (1a) was also calculated and it led to a better result (Fig. 4). On basis of the above data and the ROESY correlations, the cyclooctene moiety of 1 was determined to possess a (7R, 8R, 9R, 16S) absolute configuration. Thus, the structure of 1 was established as shown in Fig. 1. Kadlongilignan F (2) was assigned as the molecular formula of C28H34O9 based on HRESIMS ion at m/z 515.2265 [M + H]+ (calcd for C28H35O9, 515.2276) and 13C NMR spectroscopic data. The 1H and 13C NMR spectra of 2 displayed that it was a tetrahydrobenzocyclooctabenzofuranone-type lignan, which was very similar with 1. Besides, one oxymethylene at δH 3.60 (2H, t, J = 6.5 Hz, H-6'), four methylenes at δH 2.05 (2H, m, H-2'), 1.52 (2H, m, H-3'), 1.53 (2H, m, H-5'), and 1.31 (2H, m, H-4') in the 1H NMR, and an ester carbonyl at δC 173.0, an oxymethylene carbon at δC 62.6, and four methylene carbons at δC 33.6, 24.4, 25.1 and 32.1 in the 13C NMR (Table 1), revealed the presence of a 6-hydroxycaproate group. The HMBC correlation of H-9 with C-1' indicated that the 6-hydroxycaproate group was connected on C-9. The ROESY correlations of H11/H-9/H-8 and CH3–17/CH3–18 suggested the β-configurations of H-9 and H-8 and α-configurations of CH3–17 and CH3–18 (Fig. 3). The ECD curve of 2 showed negative Cotton effects around 221 and 317 nm and positive Cotton effects at 241 and 368 nm, suggesting that 2 had the same stereochemistry with 1. Thus, the structure of 2 was determined as shown in Fig. 1. Kadlongilignan G (3) was assigned as the molecular formula of C21H26O6 by the HRESIMS ion at m/z 373.1654 [M - H]− (calcd for C21H25O6, 373.1657) and 13C NMR data. The UV spectrum with λmax
C (125 MHz) NMR Data of compounds 1–3 in CDCl3.
7.05 (s)
6.79 (s) 6.70 (s) 0.73 (d, 6.5) 1.02 (d, 6.5)
136.6 114.6 145.4 144.1 112.7 125.1 124.9 7.8 21.9
78.0
4.29 (d, 9.0) 167.9 129.5 138.9
2.05 (m) 1.52 (m)
173.0 33.6 24.4
59.3
1.31 (m)
25.1
12.9
1.53 (m) 3.60 (t, 6.5)
32.1 62.6
59.1 58.8
3.75 (s) 4.06 (s)
59.4 58.8
3.44 (s) 3.97 (s)
60.2 61.2
3.86 (s)
56.1
2.4. Hepatoprotective effects assay The biological evaluation of hepatoprotective activity was performed by established method [15]. 3. Results and discussion Kadlongilignan E (1) was assigned the molecular formula of C27H30O9 by the HRESIMS ion peak at m/z 499.1964 [M + H]+ (calcd for C27H31O9, 499.1963). The UV spectrum of 1 showed the aromatic and carbonyl moieties with λmax values at 217 and 331 nm. The IR spectrum of 1 displayed the presence of hydroxyl (3425 cm−1), α,βunsaturated carbonyl (1710 cm−1), olefinic bond (1648 cm−1), phenyl (1578, 1503 and 1486 cm−1) groups. The 1H NMR data of 1 (Table 1) showed the presence of two aromatic protons at δH 6.41 (1H, s, H-11) and 6.14 (1H, d, J = 2.0 Hz, H-4), one methylenedioxy group at δH 6.03 (1H, d, J = 1.5 Hz, H-19a) and 5.96 (1H, d, J = 1.5 Hz, H-19b), two methoxy groups at δH 4.01 (3H, s, OCH3–3) and 3.71 (3H, s, OCH3–2). A hydroxyltigloyl group was discovered by one olefinic proton at δH 6.34 (1H, td, J = 6.0, 1.0 Hz, H-3'), a methylene signal at δH 4.21 (1H, 3
Fitoterapia 142 (2020) 104487
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Table 2 1 H and 13C NMR Data of compounds 4–6 in C5D5Na. Pos.
4
5
6
δH
δC
δH
δC
δH
1
4.28 (d, 4.8)
82.1
5.85 (dd, 9.0, 6.0)
126.3
2a 2b 3 4 5 6a 6b 7a 7b 8 9 10 11a 11b 12a 12b 13 14 15 16a 16b 17 18 19a 19b 20 21 22 23a 23b 24 25 26 27 28 29 30 OCH3–3 OH-4 OH-6 OH-9 OH-15
2.98 (dd, 18.0, 4.8) 2.75 (d, 18.0)
36.6
3.73 (dd, 18.0, 9.0) 3.28 (dd, 18.0, 6.0)
34.2
3.18 1.64 3.11 2.47
a
2.52 1.63 1.35 1.93 1.38 2.17
(dd, 13.2, 3.0) (overlapped) (overlapped) (m) (overlapped) (overlapped)
1.86 1.71 2.19 1.62
(overlapped) (dt, 13.8, 3.0) (overlapped) (overlapped)
3.33 1.91 1.32 1.36 0.88 2.09 2.09 1.91 0.88 4.37 2.05 1.85 6.41
(br s) (overlapped) (overlapped) (overlapped) (s) (s) (s) (overlapped) (d, 6.6) (dt, 13.2, 3.6) (m) (overlapped) (br d, 6.6)
175.4 84.9 59.4 27.1 24.1 44.9 73.9 99.4 38.5 31.2 41.5 72.6 54.5 31.3 44.3 14.6 46.4 37.3 13.5 79.7 23.4
1.92 (s)
140.0 128.0 166.1 17.2
1.12 (s) 1.26 (s)
23.0 29.5
4.47 (s)
2.81 2.14 1.64 1.41 1.41 1.60
(t, 8.4) (overlapped) (overlapped) (m) (m) (d, 10.0)
1.76 1.67 2.10 1.49
(m) (overlapped) (dd, 13.2, 4.8) (m)
1.25 1.70 1.25 1.54 0.78 2.21 2.82 2.00 0.95 4.44 2.15 1.95 6.49
(m) (overlapped) (overlapped) (m) (s) (d, 12.0) (d, 12.0) (m) (d, 6.6) (dt, 13.2, 3.0) (overlapped) (overlapped) (br d, 6.6)
1.93 1.12 1.36 1.38 3.59 5.43
(s) (s) (s) (s) (s) (s)
3.54 (d, 1.8)
173.5 73.4 49.9 29.8 23.2 54.4 70.4 141.0 37.7 30.8 46.5 49.6 34.1 26.8 46.7 15.2 50.7 39.4 13.6 80.5 23.7 140.1 128.0 166.3 17.2 19.5 27.7 29.5 51.7
δC (m) (overlapped) (m) (m)
2.15 (d, 4.5) 4.65 (d, 3.5) 1.36 (t, 13.0) 1.59 (overlapped) 2.39 (dd, 13.5, 3.0) 2.33 1.29 1.59 1.59
(m) (overlapped) (overlapped) (overlapped)
1.30 1.69 1.25 1.57 1.03 1.65 0.71 1.96 0.97 4.43 2.22 1.99 6.50
(overlapped) (overlapped) (overlapped) (overlapped) (s) (overlapped) (d, 3.5) (overlapped) (d, 5.5) (dt, 13.5, 3.0) (m) (overlapped) (br d, 6.0)
1.93 0.98 1.84 1.80 3.63 5.49 5.70
(s) (s) (s) (s) (s) (s) (d, 2.5)
31.6 32.7 174.8 75.7 48.0 66.9 34.5 39.3 22.4 26.0 26.7 33.3 45.9 48.4 36.2 27.2 48.6 18.8 33.6 39.5 13.1 80.5 23.6 140.1 128.1 166.3 17.2 20.2 29.4 31.4 51.3
600 MHz for compounds 4 and 5, 500 MHz for compound 6.
values at 209, 251 and 287 nm, and its IR spectrum with absorption bands at 1591, 1514 and 1455 cm−1 (aromatic moiety), indicated that 3 was a dibenzocyclooctadiene lignan [16]. The 1H NMR data of 3 (Table 1) displayed three aromatic protons at δH 7.05 (1H, s, H-4), 6.79 (1H, s, H-11) and 6.70 (1H, s, H-14) for a biphenyl moiety, three methoxy groups at δH 3.97 (3H, s, OCH3–2), 3.86 (3H, s, OCH3–13) and 3.44 (3H, s, OCH3–1). A cyclooctadiene ring was recognized from two methyls at δH 1.02 (3H, d, J = 6.5 Hz, CH3–18) and 0.73 (3H, d, J = 6.5 Hz, CH3–17), three methines at δH 2.00 (1H, m, H-7), 1.98 (1H, m, H-8), and 4.49 (1H, br s, H-6), and one methylene at δH 2.13 (1H, dd, J = 13.0, 9.5 Hz, H-9a) and 2.01 (1H, dd, J = 13.0, 5.0 Hz, H-9b). The HMBC correlations of H-6 with C-5 and C-7, H-9 with C-7, C-8, and C-10, CH3–17 with C-7, and CH3–18 with C-8 indicated the presence of a cyclooctadiene moiety. The HMBC correlations of OCH3–1 with C-1, OCH3–2 with C-2, and OCH3–13 with C-13 suggested that three methoxy groups were located at C-1, C-2 and C-13, respectively (Fig. 2). The ECD curve of 3 showed a negative Cotton effect around 212 nm and a positive Cotton effect near 240 nm, suggesting that 3 possessed an M-biphenyl configuration [17]. With the axial chirality defined, the ROESY experiment was used to establish the configurations of the remaining stereocenters. The ROESY correlations of H-4/H-6/H-9β and
H-9α/H-11/CH3–17/CH3–18 indicated that H-6 was β-oriented and CH3–17 and CH3–18 were α-oriented [18] (Fig. 3). The calculated ECD curve exhibited a negative Cotton effect around 210 nm and a positive Cotton effect near 250 nm, which matched very well with the experimental spectrum (Fig. 4). Therefore, the structure of 3 and its (M, 6S, 7S, 8S) absolute configuration was established as shown in Fig. 1. Kadlongilactone A (4) was isolated as a white powder. The HRESIMS provided a molecular formula of C29H40O7 by the quasi ion peak at m/z 523.2669 [M + Na]+ (calcd for C29H40O7Na, 523.2666) with 10 degrees of unsaturation. The 1H NMR data of 4 (Table 2) displayed an olefinic proton at δH 6.41 (1H, br d, J = 6.6 Hz, H-24), four methine proton signals (three oxygenated) at δH 4.37 (1H, dt, J = 13.2, 3.6 Hz, H-22), 4.28 (1H, d, J = 4.8 Hz, H-1), 3.33 (1H, s, H-15), and 2.52 (1H, dd, J = 13.2, 3.0 Hz, H-5), three methylene signals at δH 2.98 (1H, dd, J = 18.0, 4.8 Hz, H-2a), 2.75 (1H, d, J = 18.0 Hz, H-2b), and 2.09 (2H, s, H-19) and many methine and methylene proton signals between δH 1.35–2.19. In addition, five methyl proton signals at δH 1.92 (3H, s, H-27), 1.26 (3H, s, H-30), 1.12 (3H, s, H-29), 0.88 (3H, s, H-18), and 0.88 (3H, d, J = 6.6 Hz, H-21) were observed. The above proton signals suggested that 4 may be a triterpene. The 13C NMR data of 4 (Table 2) showed 29 carbon signals, including two ester carbonyls at δC 4
Fitoterapia 142 (2020) 104487
S.-Y. Shao, et al.
Fig. 2. Key HMBC (H → C) of compounds 1–6 and key 1He1H COSY (—) correlations of compounds 4–6.
Fig. 3. Key ROESY (↔) correlations of compounds 1–6.
175.4 (C-3) and 166.1 (C-26), two olefinic carbons at δC 140.0 (C-24) and 128.0 (C-25), five quaternary carbons at δC 99.4 (C-10), 84.9 (C-4), 73.9 (C-9), 72.6 (C-14) and 41.5 (C-13), seven methine carbons at δC 82.1 (C-1), 79.7 (C-22), 54.5 (C-15), 59.4 (C-5), 44.9 (C-8), 44.3 (C-17), and 37.3 (C-20), eight methylene carbons at δC 46.4 (C-19), 38.5 (C11), 36.6 (C-2), 31.3 (C-16), 31.2 (C-12), 27.1 (C-6), 24.1 (C-7), and 23.4 (C-23), and five methyl carbons at δC 14.6 (C-18), 13.5 (C-21), 17.2 (C-27), 23.0 (C-29), and 29.5 (C-30). Combination the 1H and 13C NMR data with the unsaturation degrees suggested 4 was a nortriterpenoid with seven rings. The 1He1H COSY spectrum (Fig. 2) of 4 showed four proton spin systems involving H-1/H2–2, H-5/H2–6/H2–7/H-8, H2–11/H2–12, and H-15/H2–16/H-17/H-20/H3–21, H-22/H2–23/H-24. Then, the HMBC correlations (Fig. 2) of H-1 with C-4 and C-10, H-2 with C-3, H-30 with C-5 and C-29, H-29 with C-4, H-19 with C-5, C-8, C-9 and C-10, H-8 with C-14, H-11 with C-9, H-12 with C-13, H-15 with C-14, H-17 with C-13, H-22 with C-26 and H-27 with C-24, C-25 and C-26 established the main planar structure of 4. In addition, the HMBC correlation of H-
18 with C-13 suggested a methyl was attached on C-13. The ROESY correlations (Fig. 3) of H-1 with H-29, H-5 with H-30, H8 with CH3–18 and H-15, H-22 with H-17, and no correlations of H-15 with H-17, combined with the small coupling constant (3.6 Hz) between H-20 and H-22 suggested that H-1, H-8, H-15, CH3–18 and CH3–29 were β-oriented and H-5, H-17, H-22, CH3–21 and CH3–30 were α-oriented [10,19]. Comparison the NMR data of C-9, C-10 and C14 with the reported micrandilactone I [10] indicated that their substituents should be α-oriented. The ECD spectrum of 4 showed a positive Cotton effect near 254 nm, which suggested the absolute configuration of C-22 was R [10]. Then, the absolute configuration of 4 was determined as 1R, 5S, 8R, 9R, 10R, 13R, 14R, 15S, 17R, 20S and 22R. The result was finally confirmed by comparison of the experimental and calculated ECD curves (Fig. 4). Accordingly, the structure of compound 4 was established as shown in Fig. 1. Kadlongilactone B (5) was obtained as a white powder. Compound 5 was determined to have the molecular formula of C31H48O6 and eight unsaturation degrees via the positive HRESIMS ion at m/z 539.3355 5
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Fig. 4. Experimental (black) and calculated ECD spectra (red) in methanol of 1, 1a, 3 and 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
[M + Na]+ (calcd for C31H48O6Na, 539.3343). Comparison of the NMR data of 4 and 5 displayed that their structures were similar except a few differences. The 1H NMR data of 5 showed an extra olefinic proton signal at δH 5.85 (1H, dd, J = 9.0, 6.0 Hz, H-1), a methylene proton signal at δH 1.25 (2H, m, H-15), a methoxy signal at δH 3.59 (3H, s, OCH3–3), and a methyl group at δH 1.12 (3H, s, H-28), however, two oxygenated methine signals at δH 4.28 (1H, d, J = 4.8 Hz, H-1) and 3.33 (1H, s, H-15) were absent, which suggested 5 was a seco-triterpenoid [20]. The HMBC correlations of H-1 with C-10 and OCH3–3 with C-3 combined with no correlation of H-1 with C-4 suggested the A and B rings were split. The HMBC correlation of CH3–28 with C-14 suggested the extra methyl was connected on C-14. The ROESY correlations of CH3–28 with H-17 and 9-OH indicated that the CH3–28 was αoriented. The relative configurations of other chiral carbons were the same with 5 according to the ROESY spectrum. The ECD spectrum of 5 displayed a positive Cotton effect around 254 nm, which inferred the absolute configuration of C-22 was R. Combined with the relative configuration of 5, its absolute configuration could be determined as 5R, 8S, 9S, 13R, 14S, 17R, 20S and 22R. Therefore, the structure of 5 was determined as shown in Fig. 1.
Kadlongilactone C (6) was a white powder. The molecular formula of C31H48O6 was determined by the HRESIMS ion at m/z 539.3347 [M + Na]+ (calcd for C31H48O6Na, 539.3343), which was the same with 5. Comparison the NMR data of 5 and 6 showed that they were very similar except some differences. The 1H NMR data of 6 only showed one olefinic proton signal at δH 6.50 (1H, d, J = 6.0 Hz, H-24), which suggested the other olefinic bond was reduced. A methine proton signal at δH 4.65 (1H, d, J = 3.5 Hz, H-6) and the 1He1H COSY correlation of H-5 with H-6 suggested that the C-6 was oxygenated. In addition, the 1H NMR data displayed a shielded methylene proton signal at δH 0.71 (H-19b), together with the same eight unsaturation degrees, suggesting it may exist a cyclopropane moiety in 6 [21]. The HMBC correlations of H-19 with C-9 and C-10 assigned the position of the cyclopropane structure. The ROESY correlations of H-19/H-8/6-OH indicated they were all β-oriented. Its ECD curve displayed a positive Cotton effect at 255 nm, which indicated the absolute configuration of C-22 were R. Then, the absolute configuration of 6 was assigned as 5S, 6R, 8S, 9S, 10R, 13R, 14S, 17R, 20S and 22R. Thus, the structure of 6 was determined as shown in Fig. 1. Additionally, six known tetrahydrobenzocyclooctabenzofuranone 6
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Table 3 Hepatoprotective activity of compounds 1–12 (10 μM) against APAP-induced HepG2 cell damage. Groupa
Optical density
Cell survival rates (%)b
Control Model 1 2 3 4 5 6 7 8 9 10 11 12 Bicyclol
1.339 0.552 0.565 0.553 0.554 0.540 – – 0.548 0.608 0.633 – 0.574 0.710 0.653
100.00 ± 7.767 41.24 ± 13.587 31.09 ± 5.133 31.09 ± 4.702 41.40 ± 22.202 29.74 ± 2.963 – – 40.93 ± 3.285 45.41 ± 7.767 34.84 ± 2.467 – 42.87 ± 2.265 53.04 ± 4.507 48.77 ± 9.495
± ± ± ± ± ±
0.104 0.075⁎⁎⁎ 0.029 0.026 0.123 0.016
± 0.018 ± 0.015 ± 0.013 ± 0.013 ± 0.032## ± 0.062#
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a APAP (8 mM)-induced cells as the model group; Bicyclol as the positive control; the compounds were tested at 10 μM. bResults are expressed as the means ± SD (n = 3). ⁎⁎⁎p < .001 (vs control group). ###p < .001. ## p < .01. #p < .05 (vs model group). ‶-″: These compounds were not determined.
lignans and schiartane-type triterpenoids were isolated and identified as benzoyl oxokadsuranol (7) [22], propoxyl oxokadsuranol (8) [22], heteroclitin E (9) [23], kadsutherin C (10) [24], kadsuphilin J (11) [25], and micrandilactone I (12) [10]. All the compounds were tested for their hepatoprotective activity against APAP-induced toxicity in HepG2 cells. The cell survival rates of these compounds were given in Table 3. Compound 12 exhibited moderate hepatoprotective activity with cell survival rates of 53.04% at 10 μM (bicyclol as positive control). However, the other compounds only showed weak hepatoprotective activity. Declaration of Competing Interest We declare no conflict of interest for this study. Acknowledgments This work was financially supported by CAMS Initiative for Innovative Medicine (No. CAMS-2016-I2M-1-010). Author statment All the authors have seen and approved the final version of the manuscript being submitted. We warrant that the article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere. None Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2020.104487. References [1] China Flora Editing Group, Flora of China, Vol. 30 Science Press, Beijing, 1996, p.
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