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Immunosuppressive withanolides from the flower of Datura metel L.
Journal Pre-proof Immunosuppressive withanolides from the flower of Datura metel L.
Yan Liu, Juan Pan, Yan-Ping Sun, Xin Wang, Yuan Liu, Bing-You Yang, Hai-Xue Kuang PII:
S0367-326X(19)32247-6
DOI:
https://doi.org/10.1016/j.fitote.2019.104468
Reference:
FITOTE 104468
To appear in:
Fitoterapia
Received date:
15 November 2019
Revised date:
21 December 2019
Accepted date:
27 December 2019
Please cite this article as: Y. Liu, J. Pan, Y.-P. Sun, et al., Immunosuppressive withanolides from the flower of Datura metel L., Fitoterapia (2019), https://doi.org/10.1016/ j.fitote.2019.104468
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© 2019 Published by Elsevier.
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Immunosuppressive Withanolides from the Flower of Datura metel L. Yan Liu1 , Juan Pan1 , Yan-Ping Sun, Xin Wang, Yuan Liu, Bing-You Yang* , Hai-Xue Kuang*
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang
These authors contributed equally to this article.
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1
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University of Chinese Medicine, Ministry of Education, Harbin 150040, China
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*Corresponding Author. E-mail: [email protected]; [email protected]
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Phone/Fax:+86-0451-87267038
ABSTRACT
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Thirteen new withanolide aglycones, baimantuoluolines L-X (1-13) and one new withanolide glycoside, baimantuoluoside J (14) were isolated from Datura metel L. flowers. The structures of the new compounds were elucidated by the detailed analysis of 1D and 2D NMR techniques and mass spectrometry, together with the closely related literatures.
Meanwhile,
all
isolated
compounds
were
evaluated
for
their
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immunosuppressive activities against mice splenocyte proliferation and antiproliferative
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activities against human gastric adenocarcinoma cells (SGC-7901), human hepatoma
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(HepG2), and human breast cancer (MCF-7) in vitro. It was found that compounds 1-14
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showed obvious immunosupressive effects and some of them have moderated
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antiproliferative activities. KEYWORDS
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Datura metel L.; withanolide; immunosupressive; antiproliferative
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1. Introduction Flos Daturae is the dry flowers of Datura metel L., which is a famous traditional Chinese medicine for centuries, belongs to the family of Solananeae. In recent years, many studies report that it plays a significant role in the treatment of psoriasis. According to these studies, the material basis compositions of treating p soriasis are
f
flavonoids and withanolides. Though much work on active constituents of Datura metel
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L. has been done [1-7], the traditional separation process is very complex and inefficient
pr
in separating the withanolides from the others. Together with other chromatographic
e-
columns, a series of withanolides, including fourteen new compounds (1-14) (Fig.1),
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were isolated and identified. Also, the immunosuppressive and antiproliferative
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activities of these isolates were evaluated and reported for the first time.
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2. Experimental 2.1. General. IR spectra were recorded on a Shimadzu FTIR-8400S. Optical rotations were measured on a JASCO P-2000 polarimeter. HR-ESI-MS was determined on Waters Xevo-TOF-MST M and Thermo Scientific Orbitrap FusionT M LumosT M TribridT M mass
13
C-NMR) at room temperature (25℃). D-941
oo
MHz for 1 H-NMR and 100 MHz for
f
spectrometer. The NMR spectra were recorded on a Bruker DPX 400 instrument (400
pr
macroporous resin was purchased from Amicogen (China) Biopharm Co. Ltd.. Samples
e-
were prepared in MeOD, chemical shifts were expressed in (ppm) and referenced to the
Pr
residual solvent signals. Coupling constants J were given in Hz. The UV spectra were recorded on Waters e2695-2998PDA. Semi-preparative HPLC (Waters 2535-2487) was
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performed on SunFire C18 (5 μm, 10×250 mm, Waters, American). Silica gel was used
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Qingdao Marine Chemical Ltd., China. The cancer cells were from Harbin Medical
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University. 2.2. Plant Material.
The dry flowers of D. metel L. were collected in August 2011 from Hainan Province, People’s Republic of China. It was identified by plant taxonomist Doc. Rui- feng Fan (Heilongjiang University of Chinese Medicine, P.R. China). A voucher specimen (No.2011020) was deposited in Heilongjiang University of Chinese Medicine. 2.3. Extraction and Isolation. The dried flowers (10 kg) of D. metel L. were extracted three times under reflux conditions with refluxing 70% ethanol (20 L) for 2.5 h, combined solution was filtered
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and evaporated under vacuum to be dilution, which was suspened in H2 O, and then passed D-941 macroporous resin, eluted by H2 O, 50% EtOH, 95% EtOH and 1%NaOH, successively. The 50% EtOH fraction (68.0 g) was subjected to column chromatography over silica gel, eluted with a stepwise gradient of CHCl3 -MeOH (1:0 to 0:1, gradient) to give seven major fractions: Fr.1 (7.6 g), Fr.2 (5.8 g), Fr.3 (8.1 g), Fr.4 (6.5 g), Fr.5 (5.2
f
g), Fr.6 (4.7 g) and Fr.7 (7.4 g). Fr.3 was subjected to additional silica gel
oo
chromatography, by elution with CHCl3 /MeOH (100:1 to 0:1, gradient), to afford
pr
sub- fractions A1-A6. Sub-fraction A2 (1.3 g) was further purified by ODS column
e-
chromatography with MeOH/H2 O (1:9 to 1:0, gradient) to afford 8 (5.3 mg) and 12 (8.0
Pr
mg). Fr.4 was purified by silica gel chromatography, eluted with CHCl3 /MeOH (100:1 to 0:1, gradient), to afford sub- fractions B1-B7. Sub-fraction B2 (1.0 g) was subjected
al
to ODS column chromatography with MeOH/H2 O (2:8 to 1:0, gradient) and finally
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purified by semi-preparative HPLC (MeOH/H2 O, 90-10; 3 mL/min) to afford 1 (7.8
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mg), 9 (8.5 mg) and 14 (12.5 mg). Sub-fraction B3 was purified in a similar treatment to that of Sub-fraction B6, to afford 7 (8.3 mg). Fr.5 (5.2 g) was also subjected to additional silica gel chromatography, by elution with CHCl3 /MeOH (50:1 to 0:1, gradient), to afford Sub-fractions C1-C5. Sub-fraction C2 (0.7 g) was subjected to Sephadex LH-20 column chromatography with MeOH/H2 O (2:8 to 1:0, gradient) to afford 6 (11.6 mg), 4 (23.7 mg) and 11 (10.4 mg). Fr.6 was purified by silica gel chromatography, eluted with CHCl3 /MeOH (100:1 to 0:1, gradient), to afford sub- fractions D1-D8. Sub-fraction D1 (0.8 g) was subjected to ODS column chromatography with MeOH/H2 O (2:8 to 1:0, gradient) to afford 3 (10.0 mg) and 10
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(15.2 mg). Sub- fraction D2 (0.4 g) was purified by semi-preparative HPLC (MeOH/H2 O, 90-10; 3 mL/min) to afford 5 (7.0 mg) and 13 (11.0 mg). Sub- fraction D3 (0.5 g) was purified by semi-preparative HPLC (MeOH/H2 O, 90-10; 3 mL/min) to afford 2 (6.8 mg). 2.3.1. Baimantuoluoline L (1) White, amorphous powder; D +32.2 (c 8.0, MeOH); UV (MeOH) λmax 226nm; 27
13
oo
f
IR (film) ν max 3327, 2915, 2868, 1699, 1214, 1122, 920, 832cm-1 ; 1 H and
C-NMR
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data, see Tab.1 and Tab.4; HRESIMS m/z 527.2612 [M+Na]+ (calcd for C28 H40O 8Na
2.3.2. Baimantuoluoline M (2)
e-
527.2621).
White, amorphous powder; D +7.0 (c 5.1, MeOH); UV (MeOH) λ max 223 nm;
Pr
27
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IR (film) ν max 3315, 2937, 2925, 1711, 1209, 1089, 1066, 896, 825 cm-1 ; 1 H and C-NMR data, see Tab.1 and Tab.4; HRESIMS m/z 555.2947 [M+Na]+ (calcd for
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C30 H44 O8Na 555.2934).
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13
2.3.3. Baimantuoluoline N (3)
White, amorphous powder; D +25.7 (c 4.0, MeOH); UV (MeOH) λmax 225 nm; 27
IR (film) ν max 3299, 2911, 2849, 1694, 1201, 930 cm-1 ; 1 H and
13
C-NMR data, see Tab.2
and Tab.4; HRESIMS m/z 571.2689 [M+K]+ (calcd for C30 H44 O8 K 571.2673). 2.3.4. Baimantuoluoline O (4) White, amorphous powder; D +8.9 (c 6.5, MeOH); UV (MeOH) λ max 226 nm; 27
IR (film) ν max 3376, 2933, 2868, 1703, 1188, 1124, 1058, 912, 883 cm-1 ; 1 H and 13
C-NMR data, see Tab.1 and Tab.4; HRESIMS m/z 515.3035 [M+H]+ (calcd for
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C30 H43 O7 515.3009). 2.3.5. Baimantuoluoline P (5) White, amorphous powder; 27 D +29.0 (c 34.8, MeOH); UV (MeOH) λ max 226 nm; IR (film) ν max 3451, 2937, 2924, 1705, 1611, 1238, 1007, 952, 881, 640 cm-1 ; 1 H and 13
C-NMR data, see Tab.1 and Tab.4; HRESIMS m/z 533.3102 [M+H]+ (calcd for
f
C30 H45 O8 533.3114).
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2.3.6. Baimantuoluoline Q (6)
White, amorphous powder; D +33.1 (c 7.2, MeOH); UV (MeOH) λ max 226 nm;
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27
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IR (film) ν max 3415, 2924, 2872, 1697, 1236, 1092, 955, 879, 791 cm-1 ; 1 H and C-NMR data, see Tab.1 and Tab.4; HRESIMS m/z 517.3150 [M+H]+ (calcd for
C30 H45 O7 517.3165).
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2.3.7. Baimantuoluoline R (7)
Pr
13
White, amorphous powder; D -89.6 (c 6.7, CHCl3 ); UV (MeOH) λmax 226 nm;
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27
13
C-NMR
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IR (film) νmax 3321, 2924, 2862, 1710, 1234, 1107, 902, 696 cm-1 ; 1 H and
data, see Tab.2 and Tab.4; HRESIMS m/z 499.3044 [M+H]+ (calcd for C30 H43 O6 499.3060).
2.3.8. Baimantuoluoline S (8) White, amorphous powder; D +29.6 (c 22.5, MeOH); UV (MeOH) λmax 226 27
nm; IR (film) ν max 3315, 2926, 2872, 1712, 1238, 1167, 1026, 879 cm-1 ; 1 H and 13
C-NMR data, see Tab.2 and Tab.4; HRESIMS m/z 517.3152 [M+H]+ (calcd for
C30 H45 O7 517.3165). 2.3.9. Baimantuoluoline T (9)
Journal Pre-proof White, amorphous powder; 27 D +48.3 (c 2.4, CHCl3 ); UV (CHCl3 ) λmax 226 nm; IR (film) ν max 3309, 2924, 2833, 1691, 1407, 1213, 1132, 1022, 908 cm-1 ; 1 H and 13
C-NMR data, see Tab.2 and Tab.4; HRESIMS m/z 457.2943 [M+H]+ (calcd for
C28 H41 O5 455.2797). 2.3.10. Baimantuoluoline U (10) White, amorphous powder; D +7.0 (c 5.1, MeOH); UV (MeOH) λmax 225 nm; 27
oo
f
IR (film) νmax 3206, 2924, 2872, 1707, 1058, 1022, 953, 881 cm-1 ; 1 H and
13
C-NMR
pr
data, see Tab.2 and Tab.4; HRESIMS m/z 499.3043 [M+H]+ (calcd for C30 H43 O6
2.3.11. Baimantuoluoline V (11)
e-
499.3060).
White, amorphous powder; D +8.9 (c 6.5, MeOH); UV (MeOH) λmax 226 nm;
Pr
27
13
C-NMR data, see
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IR (film) νmax 3316, 2912, 2863, 1702, 1209, 1127, 912 cm-1 ; 1 H and
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461.2668).
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Tab.3 and Tab.4; HRESIMS m/z 461.2659 [M+Na]+ (calcd for C28 H38O4Na,
2.3.12. Baimantuoluoline W (12) White, amorphous powder; D +7.9 (c 2.7, MeOH); UV (MeOH) λmax 225 nm; 27
IR (film) ν max 3303, 2894, 1712, 1201, 1099, 917 cm-1 ; 1 H and
13
C-NMR data, see Tab.3
and Tab.4; HRESIMS m/z 457.2949 [M-H]- (calcd for C28 H41 O5 457.2954). 2.3.13. Baimantuoluoline X (13) White, amorphous powder; D +12.1 (c 5.0, MeOH); UV (MeOH) λmax 224 nm; 27
IR (film) νmax 3310, 2900, 2867, 1681, 909 cm-1 ; 1 H and
13
C-NMR data, see Tab.3 and
Tab.4; HRESIMS m/z 501.3212 [M-H]- (calcd for C30 H45 O 6 501.3216).
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2.3.14. Baimantuoluoside J (14) White, amorphous powder; 27 D +10.9 (c 5.5, MeOH); UV (MeOH) λmax 226 nm; IR (film) ν max 3313, 2914, 2871, 1702, 1209, 1126, 915 cm-1 ; 1 H and 13 C-NMR data, see Tab.3 and Tab.4; HRESIMS m/z 687.3721 [M+Na]+ (calcd for C36 H56 O11Na 687.3720).
f
2.4. Splenocyte Proliferation of Mouse Bioassays.
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Single cell suspensions were prepared as previously described [8-9]. Cells were
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cultured in RPMI 1640 medium supplemented with 5% FBS containing test compounds
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with or without at a density of 1×10 6 in 96-well plates where 100 μL of 1-14 (0.01-10 μL/mg), and concanavalin A (Con A, final concentration 5 mg/L) were then added. The
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plate was incubated at 37◦ C under a humidified atmosphere of 5% CO 2 . After 44 h,
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50μL of MTT solution (2 g/L) were added to each well, followed by incubation for 4 h.
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The microtiter plates were centrifuged (1400×g, 5 min), and the untransformed MTT
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was removed carefully by pipetting. 200 μL of a Me2 SO working solution (192 μL Me2 SO with 8 μL HCl, 1 mol/L) was added to each well, and the absorbance was evaluated after 15 min in an ELISA reader at 492 nm. 2.5. Antiproliferative Bioassays. All compounds were tested for cytotoxicity against human gastric adenocarcinoma (SGC-7901), human breast cancer (MCF-7) and human hepatoma (HepG2) through the MTT method, in which 5-Fluorouracil (5-FU) was used as a positive control. Briefly, 100 μL of cell suspension (5 × 104 cells/mL) was seeded into 96-well microtiter plates and cultured for 24 h before the compound was added. Then, different concentrations of
Journal Pre-proof the compounds were added to the plates, the cells were cultivated for 48 h, and 20 μL of MTT (5 mg/mL) was added to each well. After 4 h, the culcompletely dissolved with 150 μL of DMSO in each well by vigorously shaking the ture medium was removed and the formazan crystals were plate. Finally, formazan absorbance was assessed by a PerkinElmer microplate reader at 570 nm [10].
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2.6. Result and discussion
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Baimantuoluoline L (1) was isolated as white amorphous powder with a molecular
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formula of C28 H40O8 as deduced by means of positive HRESIMS (m/z [M+Na]+ signal
e-
at m/z 527.2612). The UV absorption maximum at 226 nm and characteristic IR
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absorption bands observed at 3327 and 1699 cm-1 revealed the presence of an α, β-unsaturated ketone moiety, a hydroxyl and a carbonyl group, respectively. According 13
C NMR spectra are similar to baimantuoluoside F [3],
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to the literature, the 1 H and
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except that C-27 replaces β-D-glucopyranoside with a hydroxyl group. Additionally,
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C-20 of compound 1 was S configuration according to the cross peaks of Me-18/Me-21, H-20, Hβ-16, H-20/Hβ-16 and H-16/H-22 in the NOESY experiment (Fig.S1) [11]. The H-atom signal at δH 4.62 (dt, J = 13.4, 3.5 Hz) was attributed to H-22, revealing an (R) configuration at C-22 for common withanolides [11-13]. From these data, the structure of 1 was identified as 5α,6β,7α,12β,27-pentahydroxy-(20S,22R)-1-oxo-witha-2,24dienolide. baimantuoluoline M (2) was isolated as a white amorphous powder and possessed a molecular formula of C30 H44O8 based on the [M+Na]+ signal at m/z 555.2947 in the positive HRESIMS. The 1 H-NMR spectrum of 2 showed a distinct resemblance to 1,
Journal Pre-proof except for the presence of one ethyloxy signal at δH 3.50 (q, J = 7.2 Hz), 1.10 (q, J = 7.2 Hz) in 2 (Tab.1). Similarly, the
13
C-NMR spectrum showed an additional carbon signal
at δc 66.0, 15.5 in 2 (Tab.4). The HMBC correlations (Fig.S2) of C-27 of oxygenated methylene protons at δH 4.49 to C-29, and H-29 to C-30, confirmed that the ethoxy was located at C-27. The NOESY corrections in 2 suggested 6-OH and 12-OH were
f
β-oriented, 7-OH was α-oriented. δH 4.62 (dt, J = 13.4, 3.5 Hz) was attributed to H-22,
oo
revealing an (R) configuration at C-22. C-20 of 2 was R configuration according to the
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cross peaks of Me-18/Me-21, H-20, Hβ-16, Me-21/Hβ-16 and H-16/H-22 in the NOESY
e-
experiment [11]. Through the analysis, 2 was identified as 5α,6β,7α,12β-tetrahydroxy-
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27-ethoxy-(20R,22R)-1-oxowitha-2,24-dienolide.
Baimantuoluoline N (3) was isolated as a white amorphous powder and had a
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molecular formula of C30 H44 O8 as deduced by means of positive HRESIMS (m/z 13
C NMR spectra of 3 were extremely
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[M+K]+ signal at m/z 571.2689). The 1 H and
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similar to the compound 2, but subtle differences in shift values in 13 C NMR because of the relative configuration of 6-OH and 7-OH. The β-configuration of 7-OH and 12-OH as well as 5α-OH and 6α-OH was deduced by the NOESY enhancements. The absolute configurations of C-20 and C-22 according to the NOESY were identical to 2, R and R, respectively. Hence, the structure of 3 was identified as 5α,6α,7β,12β-tetrahydroxy-27ethoxy-(20R,22R)-1-oxo-witha-2,24-dienolide. Baimantuoluoline O (4) was completely analogous with 3, with no changes in the majority of the data. The subtle differences of 13 C-NMR spectrum of 4 were the signals at δC 57.1 and 57.0 replaced by the ones at δC 77.1 and 75.1 in 3, suggesting two
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hydroxyls at C-6 and C-7 dehydrated condensation, obviously. The chemical shifts and coupling constants of H-6 and H-7 indicated the presence of a 6α,7α-epoxy-5α-OH steroid moiety [18]. C-22 was R determined by coupling cleavage of H-22. According to the NOESY, the C-20 absolute configuration of 4 was determined as S. According to the literature, compound 4 is very similar to baimantuoluoside A [5], except that C-27
f
replaces β-D-glucopyranoside with an ethyloxy group. Compounds 4 was identified as
oo
5α,12β-dyhydroxy-27-ethyoxyl-6α,7α-epoxy-(20S,22R)-1-oxo-witha-2,24-dienolide.
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Baimantuoluoline P (5) was assigned a molecular formula of C30 H45O8 by
H-NMR data of 5 (Tab.1) displayed the presence of one ethyloxy signal at δH 3.54 (q, J 13
Pr
1
e-
HRESIMS and NMR experiments. Similar to those of withafastuosin F [6], the
= 7.0 Hz), 1.19 (t, J = 7.0 Hz). Similarly, the
C-NMR spectrum showed an additional
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carbon signal at δc 67.1, 15.5 in 5 (Tab.4). The HMBC correlations (Fig.S5) confirmed
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that the ethyloxy was located at C-27. The NOESY corrections in compound 5
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suggested 6-OH and 12-OH were β-oriented (Fig.S5). According to the NOESY, the absolute configurations of C-20 and C-22 were identical as S and R, respectively. Hence, the structure of 5 was identified as 5α,6β,12β,21-tetrahydroxy-27-ethoxy-(20S,22R)-1oxowitha-2,24-dienolide. Baimantuoluoline Q (6) was obtained as a white amorphous powder with the molecular formula of C30 H44 O7 , deduced by the HRESIMS, absence of an oxygen atom compared to 5. Its NMR data (Tab.2 and 4) greatly resembled to those of 5 except for the absence of one methylene (δH 1.98 and 1.48, 2H; δC 40.6) which were replaced with an oxygenated methine group (δH 3.59; δC 80.0) in 6. The NOESY cross-peaks between
Journal Pre-proof Me-19 and H-4a, H-4b and H-6 indicated 6-OH was β-oriented. Additionally, the absolute configuration of C-20 and C-22 were both R determined for their chemical shifts and coupling constant according to the literature [11-13]. From the above data, the structure of 6 was identified as 5α,6β-21-trihydroxy-27-ethoxy-(20R,22R)-1-oxo-witha2,24-dienolide. Baimantuoluoline R (7) was obtained as a white amorphous powder with the 13
f
oo
molecular formula of C30 H42 O6 , deduced by the HRESIMS. In
C NMR and DEPT
pr
spectra (Tab.4), nine methylenes (including an oxygenated methylene), eight methines
e-
(including an oxygenated methine group) and one characteristical oxo-saturated
Pr
quaternary carbon were observed. These data suggested compound 7 was a withanolide with an ether ring between C-21 and C-24, and structurally similar to daturilin [14]. The
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obvious differences between 7 and daturilin were the presence of two double bonds
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were between C-2 and C-3, C-4 and C-5 in 7 and C-6 was substituted by a hydroxy
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group, confirmed in the HMBC experiment which showed the correlation of H-6 to C-4 (Fig.S7). Its relative configuration was consistent with daturilin, and further verified by NOESY. The absolute configuration of C-20 was determined as R according to the NOE correlations of H-18 and H-20/H-22, and H-16β and H-20, and C-22 was authenticated as R [11-13]. The structure of 7 was identified as 6α-hydroxy-21,24epoxy-27-ethoxy-(20R,22R)-1- oxo-witha-2,4-dienolide. Baimantuoluoline S (8) was obtained as a white amorphous powder with the molecular formula of C30 H44 O7 , deduced by the HRESIMS. The 1 H-NMR data and 13
C-NMR indicated that 8 was structurally similar to baimantuoluoline D [6]. The only
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difference was that the methoxy in C-27 of baimantuoluoline D was replaced with the ethoxy in compound 8, and no hydroxy-substituted on C-12. The HMBC correlations of H-27/C-29/C-30 and H-6/C-4 confirmed that the ethoxy was located at C-27 and C-6 was attached by one hydroxy group. A 6β-hydroxy-5α-hydroxy-substituted steroid was confirmed by the chemical shifts and coupling constants of H-C(5) and H-C(6). The
f
absolute configuration of C-20 and C-22 were identical to 7 as R and R. The structure of
oo
8 was identified as 5α,6β-dihydroxy-21,24-epoxy-27-ethoxy-(20R,22R)-1-oxo-witha-2-
13
C NMR spectral data of 8 and baimantuoluoline T (9)
e-
Comparison of the 1 H and
pr
enolide.
Pr
indicated that they have the same substituent patterns and relative configurations in rings C-D and a side chain, and there is no ethoxy in C-27. A
13
C signal at δC 213.1
al
indicated the 1-oxo-withanolides with a 2,3-saturated in 9 [11]. A 1 H signal at δH 3.13 13
C signals for oxygenated quaternary and oxymethine
rn
(1H, br s, H-6) together with
Jo u
carbons at δC 64.3 (C-5) and 60.4 (C-6) suggested a 5β,6β-epoxy group in 9 [16]. Thus, from the above data, the structure of 9 was identified as 5β,6β;21,24-diepoxy-(20R, 22R)-1-oxo-lactone.
The NMR data of baimantuoluoline U (10) were closely comparable to those of the known withanolide jaborosalactone B [17]. The only difference is that the methoxy in C-27 of jaborosalactone B was replaced with the ethoxy in compound 10, and 10 had an hydroxy-substituted on C-21. The HMBC correlations of C-27 of oxygenated methylene protons at δ H 4.29 to C-29, and H-29 to C-30, confirmed that the ethoxy were located at C-27. And the HMBC correlations of H-2/C-10/C-4, H-3/C-5, and H-4/C-2/C-6,
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indicated that the double bonds were in C-2/C-3 and C-4/C-5. The NOESY cross-peaks of H-18/H-8, H-9/H-6 indicated 6-OH was β-oriented. And C-20 and C-22 were determined as R and R [11-13]. From the above data, the structure of 10 was identified as 6β,21-dihydroxy-27-ethoxy-(20R,22R)-1-oxo-witha-2,4,24-trienolide. In 1 H NMR spectrum of baimantuoluoline V (11), three olefinic proton signals (Tab.3) emerged at δH 5.63 (ddd, J = 9.8, 4.8, 2.2 Hz), 6.06 (dd, J = 9.8, 2.2 Hz) and
13
C NMR spectrum showed six olefinic carbons (Tab.4) at
pr
1.98 (3H, s). Meanwhile, its
oo
f
5.67 (t, J = 2.7 Hz), and four methyls at δH 0.80 (3H, s), 1.39 (3H, s), 1.85 (3H, s) and
e-
δC 122.8, 130.4, 142.6, 128.1, 153.4 and 122.0, and eight methylene groups including
Pr
one oxygenated methylene. From these data, in combination with HMBC experiments (Fig.S11), the structure of 11 was determined as 21-hydroxy-(20R,22R)-1-oxo-witha-
al
3,5,24-trienolide.
rn
In 1 H NMR spectrum of baimantuoluoline W (12), the signals at δH 0.78 (3H, s),
Jo u
1.02 (3H, s), 1.85 (3H, s) and 1.97 (3H, s) (Tab.3) manifested that C-21 was substituted by an hydroxyl group according to its dd splitting pattern; one olefinic protons signals δH 5.50 (br.d, J = 5.3 Hz) suggested compound 12 possessed 5-olefinic without 1-oxo-2-en structures. These data in
13
C NMR (Tab.4) suggested the A-D rings of
compound 12 were structurally similar to cilistol T [15] and the C-E rings were similar to 11. The positions of hydroxyl groups in C-1, C-3 and C-21 were further confirmed by HMBC experiment. Their relative configurations were determined by the data of
13
C
NMR and verified by NOESY. Hence, the structure of 12 was identified as (20R,22R)1α,3β,21-trihydroxy-witha-5,24-dienolide.
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The NMR spectra of baimantuoluoline X (13) were extremely similar to the compound 12, but there were subtle differences in 13 with an ethoxy signal. The relative configurations of compound 13 were in accordance with 12. The absolute configurations of C-20 and C-22 according to the NOESY were identical to 12, R and R, respectively. The structure of 13 was identified as (20R,22R)-1α,3β,21-trihydroxy-27-
f
ethoxy-witha-2,24-dienolide.
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Baimantuoluoside J (14) was isolated as an amorphous powder and showed
pr
positive results for the Molish reagent and the Liebermann-Burchard reaction, which 13
C NMR
e-
indicated that there may be a triterpenoid or steroid glycoside. Its 1 H and
Pr
spectra were extremely similar to the compound 13, the subtle differences between them were the presence of an additional β-D-glucopyranoside signal (δH 4.36 (d, J = 7.8 Hz);
al
δC 102.7, 75.1, 78.0, 71.7, 78.1, 62.9) in 14. The relative configurations of compound 14
rn
were in accordance with 13. The absolute configurations of C-20 and C-22 according to
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the NOESY were identical to 13, R and R, respectively. The structure of 14 was identified as 1α,3β,21-trihydroxy-27-ethoxy-(20R,22R)-witha-5,24-dienolide-3-O-β-Dglucopyranoside.
2.7 The data in vitro All isolated compounds were evaluated for their immunosuppressive activities against mice splenocyte proliferation. As shown in Tab.5, compounds 1, 3-8, 10, and 12 were found to show obvious immunosuppressive activity (IC50 <30μM) with the IC 50 values range from 14.8 to 28.1 μM. Compounds 1-14 were also tested for their antiproliferative activities against human gastric adenocarcinoma cells (SGC-7901),
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human hepatoma (HepG2) and human breast cancer (MCF-7) in vitro. Among them, some of compounds 1-14 exhibited moderate antiproliferative (IC50 <50μM), including compounds 3-8, 12 and 14 showed antiproliferative against SGC-7901 with IC 50 values of 21.8-40.2 μM; compounds 1-3, 5, 6, 10, and 11 showed antiproliferative against MCF-7 with IC50 values of 27.9-40.6 μM; compounds 1, 3, 4, 6, 7, 9, 11, 12 and 14
f
showed antiproliferative against HepG2 with IC50 values of 27.6-44.0 μM; and 13
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exhibited no cytotoxicity against the three cancer cells tested. From the preliminary
pr
immunosuppressive evaluation, it was observed that the regulations in terms of
e-
structure-activity relationship (SAR) were concluded as follows: i) withanolides with
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2-en-1-one systems in ring A were likely to show stronger acitivities than those none. ii) the comparison of the activities of 1, 5 with 7, 10, indicated withanolides with
systems.
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2.8 Conclusion
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2,4-dien-1-one systems in ring A were stronger than those substitued with 2-en-1-one
In the end, all the isolations were withanolides with some activities at some point. From the enrichment, a large of withanolides were obtained covering numerous new compounds. And, notably, some of them were observed possessing a same segment -OCH2 CH3 in C-27, including compounds 2-8, 10, 13 and 14. In addition to 27-OCH2 CH3 , these compounds compared with known withanolides, the novelty for compounds 3 and 8 had the presence of -OH in C-12 and compounds 10, 13 and 14 had the presence of -OH in C-21. However, compounds 2, 4-6 were possible to be the artifacts. A possibility was proposed: some etherifications were reacted between the
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compounds and elution solvent (ethyl alcohol) in the alkaline environment of D-941 macroporous resin which was anion exchange resin of polystyrene-type with alkalescence. These results reminded us that D-941 had potential enrichment applications in withanolides and even steroids. Further intensive removals at 27-OCH2 CH3 by a series of chemical methods and studies are currently undergoing in
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Pr
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pr
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our laboratory, and the results will be reported in due course.
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Associated content
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*Supporting information
13
C NMR, HR-ESI-MS, DEPT-135, 1 H- 1 H COSY, HSQC and
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Copies of 1 H NMR,
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Details for the key HMBC, 1 H- 1 H COSY and selected NOESY correlations of 1-14.
Pr
HMBC of 1-14. Notes
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Yan Liu and Juan Pan have contributed equally to this work.
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Acknowledgment
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The authors declare no competing financial interest.
This work was financed by National Natural Science Foundation of China (NSFC) (81773883; 81903781); China Postdoctoral Science Foundation (2018M631978); Excellent Youth Project of Heilongjiang Natural Science Foundation (YQ2019H029); Heilongjiang Postdoctoral Science Foundation (LBH-Z18245).
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REFERENCE [1] H. X. Kuang, B. Y. Yang, Y. G. Xia, W. S. Feng, Chemical Constituents from the Flower of Datura metel L, Arch. Pharm. Res. 31 (2008) 1094-1097. [2] H. X. Kuang, B. Y. Yang, Y. G. Xia, Q. H. Wang, Two New Withanolide Lactones from Flos Daturae, Molecules. 16 (2011) 5833-5839. [3] B. Y. Yang, Y. G. Xia, Q. H. Wang, D. Q. Dou, H. X. Kuang, Baimantuoluos ides D-G, four new withanolide glucosides from the flower of Datura metel L., Arch. Pharm. Res. 33 (2010) 1143-1148. [4] B. Y. Yang, Y. G. Xia, Q. H. Wang, D. Q. Dou, H. X. Kuang, Two new amide alkaloids from the flower of Datura metel L., Fitoterapia. 81 (2010) 1003-1005.
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f
[5] H. X. Kuang, B. Y. Yang, L. Tang, Y. G. Xia, D. Q. Dou, Baimantuoluosides A-C, Three New Withanolide Glucosides from the Flower of Datura metel L., Helv. Chim. Acta. 92 (2009) 1315-1323.
pr
[6] B. Y. Yang, Q. H. Wang, Y. G. Xia, W. S. Feng, H. X. Kuang. Baimantuoluolines D-F, Three New
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Withanolides from the Flower of Datura metel L., Helv. Chim. Acta. 91 (2008) 964-971. [7] B. Y. Yang, Q. H. Wang, Y. G. Xia, W. S. Feng, H. X. Kuang, Withanolide Compounds from the
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Flower of Datura metel L., Helv. Chim. Acta. 90 (2007) 1522-1528. [8] Y. Yin, F. Y. Gong, X. X. Wu, Y. Sun, Y. H. Li, T. Chen, Q. Xu, Anti-inflammatory and immunosuppressive effect of
flavones
isolated
from
Artemisia
vestita.
Journal of
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Ethnopharmacology. 120 (2008) 1-6.
[9] U. Wagner, E. Burkhardt, K. Failing, Evaluation of canine lymphocyte proliferation: comparison
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of three different colorimetric methods with the 3 H-thymidine incorporation assay, Vet Immunol Immunophathol. 70 (1999) 151-159.
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[10] S. B. Wu, J. J. Su, L. H. Sun, W. X. Wang, Y. Zhao, H. Li, S. P. Zhang, G. H. Dai, C. G. Wang, J. F. Hu, Triterpenoids and Steroids from the Fruits of Melia toosendan and Their Cytotoxic Effects on Two Human Cancer Cell Lines, J. Nat. Prod. 73 (2010) 1898-1906. [11] B. Y. Yang, Y. G. Xia, J. Pan, Y. Liu, Q. H. Wang, H. X. Kuang, Phytochemistry and biosynthesis of δ-lactone withanolides, Phytochem. Rev. 15 (2016) 771. [12] S. Minguzzi, L. E. S. Barata, Y. G. Shin, P. F. Jonas, H. B. Chai, E. J. Park, J. M. Pezzuto, G. A. Cordell, Cytotoxic withanolides from Acnistus arborescens, Phytochemistry. 59 (2002) 635-641. [13] B. Y. Yang, Y. G. Xia, Y. Liu, L. Li, H. Jiang, L. Yang, Q. H. Wang, H. X. Kuang, New antiproliferative and immunosuppressive withanolides from the seeds of Datura metel, Phytochemistry Letters. 8 (2014) 92-96. [14] S. Siddiqui, N. Sultana, S. S. Ahmad, S. I. Haider, A novel withanolide from Datura metel, Phytochemistry. 26 (1987) 2641-2643. [15] X. H. Zhu, J. Ando, M. Takagi, T. Ikeda, T. Nohara, Six new withanolide-type steroids from the leaves of Solanum cilistum, Chem Pharm. Bull. 49 (2001) 161-164.
Journal Pre-proof [16] B. N. Su, R. I. Misico, E. J. Park, B. D. Santarsiero, A. D. Mesecar, H. H. S. Fong, J. M. Pezzuto, A. D. Kinghorn, Isolation and characterization of biocative priciples of the leaves and stems of Physalis philadelphica, Tetrahedron. 58 (2002) 3453-3466. [17] H. Masao, G. Keiju, I. Nobuo, Synthetic of withanolide. 5.Synthesis of jaborosalactone A,B, and D, Jpurnal of American Chemical Society. 104 (1982) 3735-3737. [18] M. Manickam, S. Kumar, A. Sinha-Bagchi, S. C. Sinha, A. B. Ray, Withametelin H and Withafastuosin C, Two New Withanolides from the Leaves of Datura Species, J. Ind. Chem. Soc.
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71 (1994) 393-399.
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Dec 21, 2019
Dear editor:
Attached please find our manuscript entitled “Immunosuppressive Withanolides
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from the Flower of Datura metel L.” for publication in Fitoterapia. All authors have read and approved this version of the article, and due care has
pr
been taken to ensure the integrity of the work. This article or any one with similar
Sincerely,
al
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submission of this manuscript.
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content has not been submitted to any other journal. No conflict of interest exists in the
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Haixue Kuang, Ph.D. Professor Heilongjiang University of Chinese Medicine
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Fig.1. Structures of Compounds 1-14
Journal Pre-proof Tab.1 1 H-NMR Spectroscopic Data (400MHz) of compounds 1-5 1a
2b
3a
4a
5a
2
5.77 dd (10.2,2.4)
6.08 dd (10.4,2.4)
5.80 dd (10.4, 2.0)
5.74 dd (10.2,2.4)
5.78 dd (10.0,2.4)
3
6.65 ddd (10.1,5.0,2.2)
6.64 ddd (10.4,5.2,2.0)
6.64 ddd (10.4,5.2,2.4)
6.64 ddd (10.1,5.1,2.0)
6.66 ddd (10.0,5.1,2.1)
4
3.19 dt (19.6,2.4)
3.60 dt (19.6,2.4)
2.66 dd (20.0, 4.8)
2.76 dt (19.2, 2.4)
3.26 dt (19.9,2.4)
2.14 dd (19.6,5.0)
2.49 dd (19.6,5.2)
2.53 dt (20.0, 2.8)
2.45 dd (19.2,5.1)
2.04 dd (19.9,5.1)
6
3.50 d (3.2)
4.19 t (3.6)
3.35 d (8.8)
3.04 d (3.9)
3.51 t (2.3)
7
3.70 t (2.7)
4.31 m
3.38 t (9.2)
3.27 br.s
1.66 m
8
1.76 m
2.29 m
1.33 m
1.69 m
1.70 m
9
2.14 m
2.89 m
2.00 m
1.74 m
1.96 m
11
2.46 dt (12.8,4.0)
3.29 dt (12.4,3.6)
2.47 dt (12.4, 4.0),
2.90 dt (13.1,3.8)
2.45 dt (12.7,3.8)
No.
1.56 m
1.96 m
1.35 m
1.34 m
1.39 m
3.46 dd (11.1,4.4)
3.90 dd (6.6,4.2)
3.48 dd (11.2,4.4)
3.45 dd (10.9,4.6)
3.59 dd (11.2,4.1)
14
1.55 m
1.95 m
1.49 m
15
1.78 m
1.93 m
1.91 m
16
1.32 m
1.79 m
1.50 m
1.73 m
1.42 m
1.20 m
1.85 m
1.72 m
1.46 m
1.29 m
1.83 m
1.90 m
pr
1.31 m
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f
1.44 m 12
1.36 m
1.51 m
1.57 m
1.53 m
1.57 m
1.45 m
1.33 m
1.55 m
1.84 m
18
0.77 s
0.99 s
0.77 s
0.79 s
0.83 s
19
1.30 s
1.66 s
1.19 s
1.19 s
1.31 s
20
2.01 m
2.22 m
2.04 m
1.99 m
1.92 m
21
1.18 d (6.9,3H)
1.51 d (6.4)
1.17 d (6.8)
1.21 d (6.1)
3.92 dd (11.8,3.2)
22
4.62 dt (13.4, 3.5)
4.60 dt (13.2, 3.6)
4.60 dt (13.2, 3.6)
4.58 dt (13.2, 3.5)
4.56 dt (13.4,3.7)
23
2.55 dd (18.0,13.4)
2.42 dd (18.0,13.2)
2.56 dd (16.8, 14.5)
2.57 dd (18.0, 13.2)
2.83 dd (18.4,13.4)
2.24 dd (18.0, 2.8)
2.00 dd (18.0, 3.2)
2.25 dd (16.8, 3.0)
2.29 dd (18.0, 3.1)
2.40 dd (18.4,3.0)
4.38 d (11.8)
4.49 d (10.8)
4.28 d (10.4)
4.25 q (10.6)
4.29 d (10.6)
4.31 d (11.8)
4.32 d (10.4)
4.21 d (10.4)
4.20 d (10.6)
4.22 d (10.6)
2.00 s
2.09 s,
2.10 s
2.09 s
3.50 q (7.2)
3.53 q (6.8)
3.53 q (7.0)
3.54 q (7.0)
1.10 t (7.2)
1.18 t (6.8)
1.17 t (7.0)
1.19 t (7.0)
Pr
e-
1.59 m 17
28 29 30
2.10 s
rn
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27
al
3.85 dd (11.8,2.9)
*a: in CDOD3 ; b: in C5 D5 N; c: in CDCl3
Journal Pre-proof Tab.2 1 H-NMR Spectroscopic Data (400MHz) of compounds 6-10 No. 2
6a
7c
8a
9c
10a
5.76 dd (10.1,2.4)
5.97 dd (9.7,0.8)
5.77 dd (10.1,2.4)
2.64 m
5.98 dd (9.7,0.7)
2.36 m 3
6.64 ddd (10.1,5.0,2.1)
6.86 dd (9.7,5.9)
6.64 ddd (10.1,5.1,2.2)
1.88 m
7.08 dd (9.7,5.9)
4
3.25 dt (19.9,2.4)
6.09 d (5.9)
3.26 dt (19.9,2.4)
1.83 m
6.22 d (5.9)
2.04 dd (19.9,5.0)
2.04 m
3.51 br.s
4.52 t (2.3)
3.52 t (2.4)
3.13 br. s
4.53 t (2.5)
7
1.70 m
1.95 m
1.67 m
2.19 dt (14.6,3.0)
1.97 m
1.55 m
1.14 m
1.54 m
1.33 m
1.18 m
8
1.77 m
1.98 m
1.80 m
1.46 m
2.08 m
9
1.82 m
1.05 m
1.84 m
1.18 m
1.06 m
11
2.22 m
1.83 m
2.23 dt (12.9,2.6)
1.37 m
1.82 m
1.36 m
1.48 m
1.32 m
1.28 m
1.54 m
1.98 m
1.86 m
1.86 m
1.85 m
2.00 m
1.31 m
1.27 m
1.08 m
1.11 m
1.68 m
1.68 m
1.14 m
1.29 m
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12
f
6
1.20 m
1.47 dd (13.4,3.0)
1.25 m
1.02 m
1.24 m
15
1.66 m
1.62 m
1.73 m
1.23 m
1.17 m
1.22 m
1.79 m
1.65 m
1.82 m
1.72 m
1.80 m
1.44 m
1.32 m
1.36 m
1.44 m
1.45 m
17
1.73 m
1.58 m
1.70 m
1.71 m
1.66 m
18
0.81 s
0.68 s
0.75 s
0.63 s
0.86 s
19
1.30 s
1.41 s
1.30 s
1.16 s
1.46 s
20
1.86 m
1.73 m
1.84 m
1.73 m
1.84 m
21
3.92 dd (11.2,2.6)
3.79 d (13.6)
3.85 d (13.3)
3.86 d (13.6)
3.90 dd (11.3,2.7)
3.77 dd (11.2,4.3)
3.41 dd (13.6,2.5)
3.72 dd (13.3,3.8)
3.51 dd (13.6,2.8)
3.75 dd (11.3,4.2)
22
4.52 dt (13.4,3.5)
4.55 br. s
4.68 br. s
4.61 br. s
4.50 dt (13.3,3.6)
23
2.95 dd (18.4,13.4)
1.87 dd (13.9,4.4)
2.29 dd (13.9,4.4)
2.01 m
2.92 dd (18.1,13.3)
2.32 dd (18.4,3.1)
1.74 m
1.92 d (13.9)
1.84 m
2.28 dd (18.1,3.2)
2.44 dd (6.1,2.7)
2.60 br s
2.32 q (7.2)
3.99 dd (9.8,2.7)
3.88 dd (9.5,3.3)
1.33 d (7.2, 3H)
3.71 dd (9.8,6.1)
3.76 dd (9.5,2.5)
1.24 s
1.29 s
3.46 q (7.0)
3.42 q (7.0)
3.53 q (7.0)
1.13 t (7.0)
1.14 t (7.0)
1.18 t (7.0)
27
4.29 d (10.6) 4.22 d (10.6)
28
2.08 s
29
3.53 q (7.0)
30
1.18 t (7.0)
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25
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16
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1.48 m 14
*a: in CDOD3 ; b: in C5 D5 N; c: in CDCl3
4.28 d (10.6) 4.21 d (10.6)
1.20 s
2.07 s
Journal Pre-proof Tab.3 1 H-NMR Spectroscopic Data (400MHz) of compounds 11-14 No.
11a
1 2
12a
13a
14a
3.80 br.s
3.80 br. s
3.81 br. s
3.38 dd (19.8,2.1)
2.05 m
1.96 m
2.15 m
2.66 dd (19.8,4.7)
1.80 m
1.80 m
1.82 m
3
5.63 ddd (9.8,4.8,2.2)
3.91 m
3.91 m
4.04 m
4
6.06 dd (9.8,2.2)
2.24 m
2.29 m
2.46 dd (13.6,3.6)
2.28 m
2.34 m
2.30 m
6
5.67 t (2.7)
5.50 br. d (5.3)
5.50 br. d (5.2)
5.51 d (5.1)
7
2.15 m
1.97 m
1.97 m
1.99 m
1.62 m
1.62 m
1.55 m
1.60 m
1.60 m
1.60 m
1.48 m
9
1.75 m
1.75 m
1.74 m
1.72 m
11
1.77 m
1.77 m
1.77 m
1.61 m
1.35 m
1.35 m
1.37 m
2.01 m
1.96 m
2.00 m
1.93 m
1.43 m
1.24 m
1.27 m
1.24 m
14
1.17 m
1.17 m
1.15 m
1.12 m
15
1.72 m
1.72 m
1.70 m
1.70 m
1.19 m
1.19 m
1.29 m
1.15 m
1.79 m
1.78 m
16
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12
f
1.67 m 8
1.45 m
1.85 m
1.85 m
1.45 m
1.42 m
1.70 m
1.66 m
0.79 s
0.78 s
1.02 s
1.02 s
1.84 m
1.86 m
1.48 m
1.70 m
1.70 m
18
0.80 s
0.78 s
19
1.39 s
1.02 s
20
1.84 m
1.84 m
21
3.90 dd (11.2,2.8)
3.90 dd (11.2,2.8)
3.92 dd (11.3,2.7)
3.91 dd (11.2,2.6)
3.77 dd (11.2,4.3)
3.77 dd (11.2,4.4)
3.75 dd (11.3,4.7)
3.76 dd (11.2,4.2)
4.48 dt (13.2,3.4)
4.52 dt (13.2,3.4)
4.52 dt (13.2,3.4)
2.84 dd (18.4,13.2)
2.94 dd (18.1,13.4)
2.94 dd (18.4,13.2)
2.25 dd (18.4,3.2)
2.30 dd (18.1,3.7)
2.30 dd (18.4,3.2)
1.85 s
4.29 d (10.6)
4.28 d (10.6)
4.22 d (10.6)
4.21 d (10.6)
2.08 s
2.08 s
3.50 q (7.0)
3.50 q (7.0)
1.19 t (7.0)
1.18 t (7.0)
4.48 dt (13.4,3.6)
23
2.83 dd (18.6,3.4)
1.85 s
28
1.98 s
29
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2.22 dd (18.6,3.4) 27
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22
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1.48 m 17
1.97 s
30 1′
4.36 d (7.8)
2′
3.13 t (8.5)
3′
3.34 m
4′
3.25 m
5′
3.24 m
6′
3.84 dd (12.0,2.0) 3.64 dd (12.0,5.1)
*a: in CDOD3 ; b: in C5 D5 N; c: in CDCl3
Journal Pre-proof Tab. 4 13 C-NMR Spectroscopic Data (100MHz) of compounds 1-14 No.
1a
2b
3a
4a
5a
6a
7c
8a
9c
10a
11a
12a
13a
14a
1
207.0
204.9
205.9
205.5
207.3
207.6
205.1
207.5
213.1
208.1
212.9
73.6
73.6
73.6
2
128.9
128.8
128.9
129.3
129.0
129.0
126.9
129.1
35.1
126.7
40.7
39.2
39.2
37.7
3
143.4
142.0
143.6
142.4
144.1
143.9
139.7
143.9
31.8
142.9
122.8
67.0
67.0
74.9
4
36.6
36.6
36.2
38.0
36.6
36.6
118.0
36.6
35.1
118.6
130.4
42.5
42.5
39.1
5
80.1
79.4
79.6
74.7
78.2
78.3
157.8
78.3
64.3
160.3
142.6
139.4
139.4
139.1
6
75.6
75.7
77.1
57.1
75.2
75.3
74,0
75.2
60.4
74.7
128.1
125.2
125.1
125.5
7
73.0
72.9
75.1
57.0
33.9
34.1
40.5
31.4
20.6
42.0
32.1
32.9
32.9
32.9
8
35.0
34.8
41.3
36.4
30.5
31.5
30.6
34.1
29.2
32.1
33.1
33.4
33.4
33.4
34.6
34.2
40.1
34.7
41.0
42.6
49.5
42.6
42.9
51.5
42.6
42.6
42.6
42.6
53.6
53.2
52.6
52.2
52.8
53.0
54.0
53.0
52.3
55.8
53.4
42.7
42.7
42.8
34.6
35.0
35.3
33.3
33.6
24.6
21.3
24.5
21.9
22.5
23.6
21.3
21.3
21.3
78.4
77.2
78.5
78.1
80.0
40.6
39.4
41.3
39.2
39.8
39.9
39.9
40.0
39.9
13
49.2
48.5
50.2
49.7
49.6
44.1
42.8
44.1
42.7
43.8
43.9
43.8
43.8
43.8
14
49.8
49.1
54.2
50.9
55.4
57.0
55.5
56.9
55.7
56.9
57.4
57.6
57.6
57.6
15
23.9
23.4
26.9
23.8
25.0
25.3
24.2
25.1
24.0
25.5
25.2
25.5
25.5
25.5
16
27.5
26.8
27.8
27.6
28.9
28.1
26.3
27.4
26.3
28.2
28.1
28.2
28.2
28.2
17
54.9
54.2
55.5
54.5
49.3
48.4
47.5
49.4
47.5
48.0
48.4
48.3
48.3
48.3
12.9
13.5
8.3
8.0
8.4
13.1
16.7
15.3
15.2
16.2
16.3
20
39.1
38.1
38.8
38.9
47.6
46.9
21
15.5
15.3
15.6
15.5
60.6
60.1
22
80.9
79.3
81.0
80.8
79.6
32.4
31.3
32.6
32.1
33.6
24
158.0
156.5
159.9
159.8
160.3
25
126.4
124.1
124.0
123.9
26
168.6
166.1
168.5
168.3
27
56.4
64.2
64.6
28
20.3
20.3
20.5
29
66.0
67.1
30
15.5
15.4
2′ 3′
12.8
12.7
12.4
12.5
12.5
16.3
13.2
19.1
20.9
20.1
20.1
20.0
39.6
42.3
39.4
46.8
46.9
46.9
46.8
46.8
60.1
60.9
59.8
59.9
60.1
60.2
60.1
60.1
79.4
75.9
78.5
75.6
79.3
79.5
79.5
79.4
79.4
34.0
33.2
31.6
33.0
34.0
33.6
33.6
34.0
34.0
160.6
70.8
72.8
70.7
160.6
153.4
153.5
160.4
160.4
123.9
50.6
52.8
44.2
123.9
122.0
122.0
123.9
123.9
168.3
168.4
171.8
175.8
173.9
168.4
169.5
169.5
168.4
168.4
rn
123.9
64.5
64.6
64.6
67.0
71.3
9.4
64.6
12.4
12.5
64.6
64.6
20.6
20.6
20.5
27.0
25.1
26.6
20.5
20.4
20.4
20.5
20.5
67.0
67.1
67.0
66.4
67.9
67.0
67.0
67.0
15.3
15.5
15.5
15.2
15.3
15.4
15.4
15.4
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1′
12.5
19.1
al
23
pr
8.2
16.8
e-
8.0
19
Pr
18
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11 12
f
9 10
102.7 75.1 78.0
4′
71.7
5′
78.1
6′
62.9
*a: in CDOD3 ; b: in C5 D5 N; c: in CDCl3
Journal Pre-proof Tab.5 IC50 of 1-14 in Splenocyte Proliferation of Mouse Bioassays and Antiproliferative Bioassays. Growth Inhibition Constant (IC50 in μM )a
Compounds M ouse Splenocyte b
SGC-7901
-3
M CF-7
HepG2
-
-
8.9
32.7
1.4
1
19.5
>50
32.4
27.6
2
>30
>50
28.7
>50
3
24.3
26.3
27.9
34.8
4
27.1
35.5
>50
37.3
5
19.7
30.7
29.9
>50
6
21.0
29.4
35.8
40.1
7
14.8
21.8
>50
42.3
8
20.7
35.7
>50
9
>30
>50
10
16.9
>50
11
>30
>50
12
28.1
13
>30
14
>30
oo
40.6
>50
36.6
44.0
37.7
>50
33.9
>50
>50
>50
>50
37.5
pr
28.6
40.2
a
Jo u
rn
al
Pr
IC50 was defined as the concentration that resulted in a 50% decrease in cell number The IC50 >30 μM in Splenocyte Proliferation of Mouse Bioassays was deemed inactive. The IC50 > 50 μM in antiproliferative bioassays was deemed inactive. b Positive control. "-" means no experiment.
>50
>50
e-
5-FU
4.9×10
b
f
-
-
Cyclosporine
Journal Pre-proof
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rn
al
Pr
e-
pr
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f
Graphical Abstract
Yan Liu, Juan Pan, Yan-Ping Sun, Xin Wang, Yuan Liu, Bing-You Yang, Hai-Xue Kuang PII:
S0367-326X(19)32247-6
DOI:
https://doi.org/10.1016/j.fitote.2019.104468
Reference:
FITOTE 104468
To appear in:
Fitoterapia
Received date:
15 November 2019
Revised date:
21 December 2019
Accepted date:
27 December 2019
Please cite this article as: Y. Liu, J. Pan, Y.-P. Sun, et al., Immunosuppressive withanolides from the flower of Datura metel L., Fitoterapia (2019), https://doi.org/10.1016/ j.fitote.2019.104468
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
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Immunosuppressive Withanolides from the Flower of Datura metel L. Yan Liu1 , Juan Pan1 , Yan-Ping Sun, Xin Wang, Yuan Liu, Bing-You Yang* , Hai-Xue Kuang*
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang
These authors contributed equally to this article.
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1
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University of Chinese Medicine, Ministry of Education, Harbin 150040, China
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*Corresponding Author. E-mail: [email protected]; [email protected]
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Phone/Fax:+86-0451-87267038
ABSTRACT
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Thirteen new withanolide aglycones, baimantuoluolines L-X (1-13) and one new withanolide glycoside, baimantuoluoside J (14) were isolated from Datura metel L. flowers. The structures of the new compounds were elucidated by the detailed analysis of 1D and 2D NMR techniques and mass spectrometry, together with the closely related literatures.
Meanwhile,
all
isolated
compounds
were
evaluated
for
their
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immunosuppressive activities against mice splenocyte proliferation and antiproliferative
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activities against human gastric adenocarcinoma cells (SGC-7901), human hepatoma
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(HepG2), and human breast cancer (MCF-7) in vitro. It was found that compounds 1-14
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showed obvious immunosupressive effects and some of them have moderated
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antiproliferative activities. KEYWORDS
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Datura metel L.; withanolide; immunosupressive; antiproliferative
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1. Introduction Flos Daturae is the dry flowers of Datura metel L., which is a famous traditional Chinese medicine for centuries, belongs to the family of Solananeae. In recent years, many studies report that it plays a significant role in the treatment of psoriasis. According to these studies, the material basis compositions of treating p soriasis are
f
flavonoids and withanolides. Though much work on active constituents of Datura metel
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L. has been done [1-7], the traditional separation process is very complex and inefficient
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in separating the withanolides from the others. Together with other chromatographic
e-
columns, a series of withanolides, including fourteen new compounds (1-14) (Fig.1),
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were isolated and identified. Also, the immunosuppressive and antiproliferative
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activities of these isolates were evaluated and reported for the first time.
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2. Experimental 2.1. General. IR spectra were recorded on a Shimadzu FTIR-8400S. Optical rotations were measured on a JASCO P-2000 polarimeter. HR-ESI-MS was determined on Waters Xevo-TOF-MST M and Thermo Scientific Orbitrap FusionT M LumosT M TribridT M mass
13
C-NMR) at room temperature (25℃). D-941
oo
MHz for 1 H-NMR and 100 MHz for
f
spectrometer. The NMR spectra were recorded on a Bruker DPX 400 instrument (400
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macroporous resin was purchased from Amicogen (China) Biopharm Co. Ltd.. Samples
e-
were prepared in MeOD, chemical shifts were expressed in (ppm) and referenced to the
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residual solvent signals. Coupling constants J were given in Hz. The UV spectra were recorded on Waters e2695-2998PDA. Semi-preparative HPLC (Waters 2535-2487) was
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performed on SunFire C18 (5 μm, 10×250 mm, Waters, American). Silica gel was used
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Qingdao Marine Chemical Ltd., China. The cancer cells were from Harbin Medical
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University. 2.2. Plant Material.
The dry flowers of D. metel L. were collected in August 2011 from Hainan Province, People’s Republic of China. It was identified by plant taxonomist Doc. Rui- feng Fan (Heilongjiang University of Chinese Medicine, P.R. China). A voucher specimen (No.2011020) was deposited in Heilongjiang University of Chinese Medicine. 2.3. Extraction and Isolation. The dried flowers (10 kg) of D. metel L. were extracted three times under reflux conditions with refluxing 70% ethanol (20 L) for 2.5 h, combined solution was filtered
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and evaporated under vacuum to be dilution, which was suspened in H2 O, and then passed D-941 macroporous resin, eluted by H2 O, 50% EtOH, 95% EtOH and 1%NaOH, successively. The 50% EtOH fraction (68.0 g) was subjected to column chromatography over silica gel, eluted with a stepwise gradient of CHCl3 -MeOH (1:0 to 0:1, gradient) to give seven major fractions: Fr.1 (7.6 g), Fr.2 (5.8 g), Fr.3 (8.1 g), Fr.4 (6.5 g), Fr.5 (5.2
f
g), Fr.6 (4.7 g) and Fr.7 (7.4 g). Fr.3 was subjected to additional silica gel
oo
chromatography, by elution with CHCl3 /MeOH (100:1 to 0:1, gradient), to afford
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sub- fractions A1-A6. Sub-fraction A2 (1.3 g) was further purified by ODS column
e-
chromatography with MeOH/H2 O (1:9 to 1:0, gradient) to afford 8 (5.3 mg) and 12 (8.0
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mg). Fr.4 was purified by silica gel chromatography, eluted with CHCl3 /MeOH (100:1 to 0:1, gradient), to afford sub- fractions B1-B7. Sub-fraction B2 (1.0 g) was subjected
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to ODS column chromatography with MeOH/H2 O (2:8 to 1:0, gradient) and finally
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purified by semi-preparative HPLC (MeOH/H2 O, 90-10; 3 mL/min) to afford 1 (7.8
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mg), 9 (8.5 mg) and 14 (12.5 mg). Sub-fraction B3 was purified in a similar treatment to that of Sub-fraction B6, to afford 7 (8.3 mg). Fr.5 (5.2 g) was also subjected to additional silica gel chromatography, by elution with CHCl3 /MeOH (50:1 to 0:1, gradient), to afford Sub-fractions C1-C5. Sub-fraction C2 (0.7 g) was subjected to Sephadex LH-20 column chromatography with MeOH/H2 O (2:8 to 1:0, gradient) to afford 6 (11.6 mg), 4 (23.7 mg) and 11 (10.4 mg). Fr.6 was purified by silica gel chromatography, eluted with CHCl3 /MeOH (100:1 to 0:1, gradient), to afford sub- fractions D1-D8. Sub-fraction D1 (0.8 g) was subjected to ODS column chromatography with MeOH/H2 O (2:8 to 1:0, gradient) to afford 3 (10.0 mg) and 10
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(15.2 mg). Sub- fraction D2 (0.4 g) was purified by semi-preparative HPLC (MeOH/H2 O, 90-10; 3 mL/min) to afford 5 (7.0 mg) and 13 (11.0 mg). Sub- fraction D3 (0.5 g) was purified by semi-preparative HPLC (MeOH/H2 O, 90-10; 3 mL/min) to afford 2 (6.8 mg). 2.3.1. Baimantuoluoline L (1) White, amorphous powder; D +32.2 (c 8.0, MeOH); UV (MeOH) λmax 226nm; 27
13
oo
f
IR (film) ν max 3327, 2915, 2868, 1699, 1214, 1122, 920, 832cm-1 ; 1 H and
C-NMR
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data, see Tab.1 and Tab.4; HRESIMS m/z 527.2612 [M+Na]+ (calcd for C28 H40O 8Na
2.3.2. Baimantuoluoline M (2)
e-
527.2621).
White, amorphous powder; D +7.0 (c 5.1, MeOH); UV (MeOH) λ max 223 nm;
Pr
27
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IR (film) ν max 3315, 2937, 2925, 1711, 1209, 1089, 1066, 896, 825 cm-1 ; 1 H and C-NMR data, see Tab.1 and Tab.4; HRESIMS m/z 555.2947 [M+Na]+ (calcd for
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C30 H44 O8Na 555.2934).
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13
2.3.3. Baimantuoluoline N (3)
White, amorphous powder; D +25.7 (c 4.0, MeOH); UV (MeOH) λmax 225 nm; 27
IR (film) ν max 3299, 2911, 2849, 1694, 1201, 930 cm-1 ; 1 H and
13
C-NMR data, see Tab.2
and Tab.4; HRESIMS m/z 571.2689 [M+K]+ (calcd for C30 H44 O8 K 571.2673). 2.3.4. Baimantuoluoline O (4) White, amorphous powder; D +8.9 (c 6.5, MeOH); UV (MeOH) λ max 226 nm; 27
IR (film) ν max 3376, 2933, 2868, 1703, 1188, 1124, 1058, 912, 883 cm-1 ; 1 H and 13
C-NMR data, see Tab.1 and Tab.4; HRESIMS m/z 515.3035 [M+H]+ (calcd for
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C30 H43 O7 515.3009). 2.3.5. Baimantuoluoline P (5) White, amorphous powder; 27 D +29.0 (c 34.8, MeOH); UV (MeOH) λ max 226 nm; IR (film) ν max 3451, 2937, 2924, 1705, 1611, 1238, 1007, 952, 881, 640 cm-1 ; 1 H and 13
C-NMR data, see Tab.1 and Tab.4; HRESIMS m/z 533.3102 [M+H]+ (calcd for
f
C30 H45 O8 533.3114).
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2.3.6. Baimantuoluoline Q (6)
White, amorphous powder; D +33.1 (c 7.2, MeOH); UV (MeOH) λ max 226 nm;
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27
e-
IR (film) ν max 3415, 2924, 2872, 1697, 1236, 1092, 955, 879, 791 cm-1 ; 1 H and C-NMR data, see Tab.1 and Tab.4; HRESIMS m/z 517.3150 [M+H]+ (calcd for
C30 H45 O7 517.3165).
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2.3.7. Baimantuoluoline R (7)
Pr
13
White, amorphous powder; D -89.6 (c 6.7, CHCl3 ); UV (MeOH) λmax 226 nm;
rn
27
13
C-NMR
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IR (film) νmax 3321, 2924, 2862, 1710, 1234, 1107, 902, 696 cm-1 ; 1 H and
data, see Tab.2 and Tab.4; HRESIMS m/z 499.3044 [M+H]+ (calcd for C30 H43 O6 499.3060).
2.3.8. Baimantuoluoline S (8) White, amorphous powder; D +29.6 (c 22.5, MeOH); UV (MeOH) λmax 226 27
nm; IR (film) ν max 3315, 2926, 2872, 1712, 1238, 1167, 1026, 879 cm-1 ; 1 H and 13
C-NMR data, see Tab.2 and Tab.4; HRESIMS m/z 517.3152 [M+H]+ (calcd for
C30 H45 O7 517.3165). 2.3.9. Baimantuoluoline T (9)
Journal Pre-proof White, amorphous powder; 27 D +48.3 (c 2.4, CHCl3 ); UV (CHCl3 ) λmax 226 nm; IR (film) ν max 3309, 2924, 2833, 1691, 1407, 1213, 1132, 1022, 908 cm-1 ; 1 H and 13
C-NMR data, see Tab.2 and Tab.4; HRESIMS m/z 457.2943 [M+H]+ (calcd for
C28 H41 O5 455.2797). 2.3.10. Baimantuoluoline U (10) White, amorphous powder; D +7.0 (c 5.1, MeOH); UV (MeOH) λmax 225 nm; 27
oo
f
IR (film) νmax 3206, 2924, 2872, 1707, 1058, 1022, 953, 881 cm-1 ; 1 H and
13
C-NMR
pr
data, see Tab.2 and Tab.4; HRESIMS m/z 499.3043 [M+H]+ (calcd for C30 H43 O6
2.3.11. Baimantuoluoline V (11)
e-
499.3060).
White, amorphous powder; D +8.9 (c 6.5, MeOH); UV (MeOH) λmax 226 nm;
Pr
27
13
C-NMR data, see
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IR (film) νmax 3316, 2912, 2863, 1702, 1209, 1127, 912 cm-1 ; 1 H and
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461.2668).
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Tab.3 and Tab.4; HRESIMS m/z 461.2659 [M+Na]+ (calcd for C28 H38O4Na,
2.3.12. Baimantuoluoline W (12) White, amorphous powder; D +7.9 (c 2.7, MeOH); UV (MeOH) λmax 225 nm; 27
IR (film) ν max 3303, 2894, 1712, 1201, 1099, 917 cm-1 ; 1 H and
13
C-NMR data, see Tab.3
and Tab.4; HRESIMS m/z 457.2949 [M-H]- (calcd for C28 H41 O5 457.2954). 2.3.13. Baimantuoluoline X (13) White, amorphous powder; D +12.1 (c 5.0, MeOH); UV (MeOH) λmax 224 nm; 27
IR (film) νmax 3310, 2900, 2867, 1681, 909 cm-1 ; 1 H and
13
C-NMR data, see Tab.3 and
Tab.4; HRESIMS m/z 501.3212 [M-H]- (calcd for C30 H45 O 6 501.3216).
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2.3.14. Baimantuoluoside J (14) White, amorphous powder; 27 D +10.9 (c 5.5, MeOH); UV (MeOH) λmax 226 nm; IR (film) ν max 3313, 2914, 2871, 1702, 1209, 1126, 915 cm-1 ; 1 H and 13 C-NMR data, see Tab.3 and Tab.4; HRESIMS m/z 687.3721 [M+Na]+ (calcd for C36 H56 O11Na 687.3720).
f
2.4. Splenocyte Proliferation of Mouse Bioassays.
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Single cell suspensions were prepared as previously described [8-9]. Cells were
pr
cultured in RPMI 1640 medium supplemented with 5% FBS containing test compounds
e-
with or without at a density of 1×10 6 in 96-well plates where 100 μL of 1-14 (0.01-10 μL/mg), and concanavalin A (Con A, final concentration 5 mg/L) were then added. The
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plate was incubated at 37◦ C under a humidified atmosphere of 5% CO 2 . After 44 h,
al
50μL of MTT solution (2 g/L) were added to each well, followed by incubation for 4 h.
rn
The microtiter plates were centrifuged (1400×g, 5 min), and the untransformed MTT
Jo u
was removed carefully by pipetting. 200 μL of a Me2 SO working solution (192 μL Me2 SO with 8 μL HCl, 1 mol/L) was added to each well, and the absorbance was evaluated after 15 min in an ELISA reader at 492 nm. 2.5. Antiproliferative Bioassays. All compounds were tested for cytotoxicity against human gastric adenocarcinoma (SGC-7901), human breast cancer (MCF-7) and human hepatoma (HepG2) through the MTT method, in which 5-Fluorouracil (5-FU) was used as a positive control. Briefly, 100 μL of cell suspension (5 × 104 cells/mL) was seeded into 96-well microtiter plates and cultured for 24 h before the compound was added. Then, different concentrations of
Journal Pre-proof the compounds were added to the plates, the cells were cultivated for 48 h, and 20 μL of MTT (5 mg/mL) was added to each well. After 4 h, the culcompletely dissolved with 150 μL of DMSO in each well by vigorously shaking the ture medium was removed and the formazan crystals were plate. Finally, formazan absorbance was assessed by a PerkinElmer microplate reader at 570 nm [10].
f
2.6. Result and discussion
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Baimantuoluoline L (1) was isolated as white amorphous powder with a molecular
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formula of C28 H40O8 as deduced by means of positive HRESIMS (m/z [M+Na]+ signal
e-
at m/z 527.2612). The UV absorption maximum at 226 nm and characteristic IR
Pr
absorption bands observed at 3327 and 1699 cm-1 revealed the presence of an α, β-unsaturated ketone moiety, a hydroxyl and a carbonyl group, respectively. According 13
C NMR spectra are similar to baimantuoluoside F [3],
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to the literature, the 1 H and
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except that C-27 replaces β-D-glucopyranoside with a hydroxyl group. Additionally,
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C-20 of compound 1 was S configuration according to the cross peaks of Me-18/Me-21, H-20, Hβ-16, H-20/Hβ-16 and H-16/H-22 in the NOESY experiment (Fig.S1) [11]. The H-atom signal at δH 4.62 (dt, J = 13.4, 3.5 Hz) was attributed to H-22, revealing an (R) configuration at C-22 for common withanolides [11-13]. From these data, the structure of 1 was identified as 5α,6β,7α,12β,27-pentahydroxy-(20S,22R)-1-oxo-witha-2,24dienolide. baimantuoluoline M (2) was isolated as a white amorphous powder and possessed a molecular formula of C30 H44O8 based on the [M+Na]+ signal at m/z 555.2947 in the positive HRESIMS. The 1 H-NMR spectrum of 2 showed a distinct resemblance to 1,
Journal Pre-proof except for the presence of one ethyloxy signal at δH 3.50 (q, J = 7.2 Hz), 1.10 (q, J = 7.2 Hz) in 2 (Tab.1). Similarly, the
13
C-NMR spectrum showed an additional carbon signal
at δc 66.0, 15.5 in 2 (Tab.4). The HMBC correlations (Fig.S2) of C-27 of oxygenated methylene protons at δH 4.49 to C-29, and H-29 to C-30, confirmed that the ethoxy was located at C-27. The NOESY corrections in 2 suggested 6-OH and 12-OH were
f
β-oriented, 7-OH was α-oriented. δH 4.62 (dt, J = 13.4, 3.5 Hz) was attributed to H-22,
oo
revealing an (R) configuration at C-22. C-20 of 2 was R configuration according to the
pr
cross peaks of Me-18/Me-21, H-20, Hβ-16, Me-21/Hβ-16 and H-16/H-22 in the NOESY
e-
experiment [11]. Through the analysis, 2 was identified as 5α,6β,7α,12β-tetrahydroxy-
Pr
27-ethoxy-(20R,22R)-1-oxowitha-2,24-dienolide.
Baimantuoluoline N (3) was isolated as a white amorphous powder and had a
al
molecular formula of C30 H44 O8 as deduced by means of positive HRESIMS (m/z 13
C NMR spectra of 3 were extremely
rn
[M+K]+ signal at m/z 571.2689). The 1 H and
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similar to the compound 2, but subtle differences in shift values in 13 C NMR because of the relative configuration of 6-OH and 7-OH. The β-configuration of 7-OH and 12-OH as well as 5α-OH and 6α-OH was deduced by the NOESY enhancements. The absolute configurations of C-20 and C-22 according to the NOESY were identical to 2, R and R, respectively. Hence, the structure of 3 was identified as 5α,6α,7β,12β-tetrahydroxy-27ethoxy-(20R,22R)-1-oxo-witha-2,24-dienolide. Baimantuoluoline O (4) was completely analogous with 3, with no changes in the majority of the data. The subtle differences of 13 C-NMR spectrum of 4 were the signals at δC 57.1 and 57.0 replaced by the ones at δC 77.1 and 75.1 in 3, suggesting two
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hydroxyls at C-6 and C-7 dehydrated condensation, obviously. The chemical shifts and coupling constants of H-6 and H-7 indicated the presence of a 6α,7α-epoxy-5α-OH steroid moiety [18]. C-22 was R determined by coupling cleavage of H-22. According to the NOESY, the C-20 absolute configuration of 4 was determined as S. According to the literature, compound 4 is very similar to baimantuoluoside A [5], except that C-27
f
replaces β-D-glucopyranoside with an ethyloxy group. Compounds 4 was identified as
oo
5α,12β-dyhydroxy-27-ethyoxyl-6α,7α-epoxy-(20S,22R)-1-oxo-witha-2,24-dienolide.
pr
Baimantuoluoline P (5) was assigned a molecular formula of C30 H45O8 by
H-NMR data of 5 (Tab.1) displayed the presence of one ethyloxy signal at δH 3.54 (q, J 13
Pr
1
e-
HRESIMS and NMR experiments. Similar to those of withafastuosin F [6], the
= 7.0 Hz), 1.19 (t, J = 7.0 Hz). Similarly, the
C-NMR spectrum showed an additional
al
carbon signal at δc 67.1, 15.5 in 5 (Tab.4). The HMBC correlations (Fig.S5) confirmed
rn
that the ethyloxy was located at C-27. The NOESY corrections in compound 5
Jo u
suggested 6-OH and 12-OH were β-oriented (Fig.S5). According to the NOESY, the absolute configurations of C-20 and C-22 were identical as S and R, respectively. Hence, the structure of 5 was identified as 5α,6β,12β,21-tetrahydroxy-27-ethoxy-(20S,22R)-1oxowitha-2,24-dienolide. Baimantuoluoline Q (6) was obtained as a white amorphous powder with the molecular formula of C30 H44 O7 , deduced by the HRESIMS, absence of an oxygen atom compared to 5. Its NMR data (Tab.2 and 4) greatly resembled to those of 5 except for the absence of one methylene (δH 1.98 and 1.48, 2H; δC 40.6) which were replaced with an oxygenated methine group (δH 3.59; δC 80.0) in 6. The NOESY cross-peaks between
Journal Pre-proof Me-19 and H-4a, H-4b and H-6 indicated 6-OH was β-oriented. Additionally, the absolute configuration of C-20 and C-22 were both R determined for their chemical shifts and coupling constant according to the literature [11-13]. From the above data, the structure of 6 was identified as 5α,6β-21-trihydroxy-27-ethoxy-(20R,22R)-1-oxo-witha2,24-dienolide. Baimantuoluoline R (7) was obtained as a white amorphous powder with the 13
f
oo
molecular formula of C30 H42 O6 , deduced by the HRESIMS. In
C NMR and DEPT
pr
spectra (Tab.4), nine methylenes (including an oxygenated methylene), eight methines
e-
(including an oxygenated methine group) and one characteristical oxo-saturated
Pr
quaternary carbon were observed. These data suggested compound 7 was a withanolide with an ether ring between C-21 and C-24, and structurally similar to daturilin [14]. The
al
obvious differences between 7 and daturilin were the presence of two double bonds
rn
were between C-2 and C-3, C-4 and C-5 in 7 and C-6 was substituted by a hydroxy
Jo u
group, confirmed in the HMBC experiment which showed the correlation of H-6 to C-4 (Fig.S7). Its relative configuration was consistent with daturilin, and further verified by NOESY. The absolute configuration of C-20 was determined as R according to the NOE correlations of H-18 and H-20/H-22, and H-16β and H-20, and C-22 was authenticated as R [11-13]. The structure of 7 was identified as 6α-hydroxy-21,24epoxy-27-ethoxy-(20R,22R)-1- oxo-witha-2,4-dienolide. Baimantuoluoline S (8) was obtained as a white amorphous powder with the molecular formula of C30 H44 O7 , deduced by the HRESIMS. The 1 H-NMR data and 13
C-NMR indicated that 8 was structurally similar to baimantuoluoline D [6]. The only
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difference was that the methoxy in C-27 of baimantuoluoline D was replaced with the ethoxy in compound 8, and no hydroxy-substituted on C-12. The HMBC correlations of H-27/C-29/C-30 and H-6/C-4 confirmed that the ethoxy was located at C-27 and C-6 was attached by one hydroxy group. A 6β-hydroxy-5α-hydroxy-substituted steroid was confirmed by the chemical shifts and coupling constants of H-C(5) and H-C(6). The
f
absolute configuration of C-20 and C-22 were identical to 7 as R and R. The structure of
oo
8 was identified as 5α,6β-dihydroxy-21,24-epoxy-27-ethoxy-(20R,22R)-1-oxo-witha-2-
13
C NMR spectral data of 8 and baimantuoluoline T (9)
e-
Comparison of the 1 H and
pr
enolide.
Pr
indicated that they have the same substituent patterns and relative configurations in rings C-D and a side chain, and there is no ethoxy in C-27. A
13
C signal at δC 213.1
al
indicated the 1-oxo-withanolides with a 2,3-saturated in 9 [11]. A 1 H signal at δH 3.13 13
C signals for oxygenated quaternary and oxymethine
rn
(1H, br s, H-6) together with
Jo u
carbons at δC 64.3 (C-5) and 60.4 (C-6) suggested a 5β,6β-epoxy group in 9 [16]. Thus, from the above data, the structure of 9 was identified as 5β,6β;21,24-diepoxy-(20R, 22R)-1-oxo-lactone.
The NMR data of baimantuoluoline U (10) were closely comparable to those of the known withanolide jaborosalactone B [17]. The only difference is that the methoxy in C-27 of jaborosalactone B was replaced with the ethoxy in compound 10, and 10 had an hydroxy-substituted on C-21. The HMBC correlations of C-27 of oxygenated methylene protons at δ H 4.29 to C-29, and H-29 to C-30, confirmed that the ethoxy were located at C-27. And the HMBC correlations of H-2/C-10/C-4, H-3/C-5, and H-4/C-2/C-6,
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indicated that the double bonds were in C-2/C-3 and C-4/C-5. The NOESY cross-peaks of H-18/H-8, H-9/H-6 indicated 6-OH was β-oriented. And C-20 and C-22 were determined as R and R [11-13]. From the above data, the structure of 10 was identified as 6β,21-dihydroxy-27-ethoxy-(20R,22R)-1-oxo-witha-2,4,24-trienolide. In 1 H NMR spectrum of baimantuoluoline V (11), three olefinic proton signals (Tab.3) emerged at δH 5.63 (ddd, J = 9.8, 4.8, 2.2 Hz), 6.06 (dd, J = 9.8, 2.2 Hz) and
13
C NMR spectrum showed six olefinic carbons (Tab.4) at
pr
1.98 (3H, s). Meanwhile, its
oo
f
5.67 (t, J = 2.7 Hz), and four methyls at δH 0.80 (3H, s), 1.39 (3H, s), 1.85 (3H, s) and
e-
δC 122.8, 130.4, 142.6, 128.1, 153.4 and 122.0, and eight methylene groups including
Pr
one oxygenated methylene. From these data, in combination with HMBC experiments (Fig.S11), the structure of 11 was determined as 21-hydroxy-(20R,22R)-1-oxo-witha-
al
3,5,24-trienolide.
rn
In 1 H NMR spectrum of baimantuoluoline W (12), the signals at δH 0.78 (3H, s),
Jo u
1.02 (3H, s), 1.85 (3H, s) and 1.97 (3H, s) (Tab.3) manifested that C-21 was substituted by an hydroxyl group according to its dd splitting pattern; one olefinic protons signals δH 5.50 (br.d, J = 5.3 Hz) suggested compound 12 possessed 5-olefinic without 1-oxo-2-en structures. These data in
13
C NMR (Tab.4) suggested the A-D rings of
compound 12 were structurally similar to cilistol T [15] and the C-E rings were similar to 11. The positions of hydroxyl groups in C-1, C-3 and C-21 were further confirmed by HMBC experiment. Their relative configurations were determined by the data of
13
C
NMR and verified by NOESY. Hence, the structure of 12 was identified as (20R,22R)1α,3β,21-trihydroxy-witha-5,24-dienolide.
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The NMR spectra of baimantuoluoline X (13) were extremely similar to the compound 12, but there were subtle differences in 13 with an ethoxy signal. The relative configurations of compound 13 were in accordance with 12. The absolute configurations of C-20 and C-22 according to the NOESY were identical to 12, R and R, respectively. The structure of 13 was identified as (20R,22R)-1α,3β,21-trihydroxy-27-
f
ethoxy-witha-2,24-dienolide.
oo
Baimantuoluoside J (14) was isolated as an amorphous powder and showed
pr
positive results for the Molish reagent and the Liebermann-Burchard reaction, which 13
C NMR
e-
indicated that there may be a triterpenoid or steroid glycoside. Its 1 H and
Pr
spectra were extremely similar to the compound 13, the subtle differences between them were the presence of an additional β-D-glucopyranoside signal (δH 4.36 (d, J = 7.8 Hz);
al
δC 102.7, 75.1, 78.0, 71.7, 78.1, 62.9) in 14. The relative configurations of compound 14
rn
were in accordance with 13. The absolute configurations of C-20 and C-22 according to
Jo u
the NOESY were identical to 13, R and R, respectively. The structure of 14 was identified as 1α,3β,21-trihydroxy-27-ethoxy-(20R,22R)-witha-5,24-dienolide-3-O-β-Dglucopyranoside.
2.7 The data in vitro All isolated compounds were evaluated for their immunosuppressive activities against mice splenocyte proliferation. As shown in Tab.5, compounds 1, 3-8, 10, and 12 were found to show obvious immunosuppressive activity (IC50 <30μM) with the IC 50 values range from 14.8 to 28.1 μM. Compounds 1-14 were also tested for their antiproliferative activities against human gastric adenocarcinoma cells (SGC-7901),
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human hepatoma (HepG2) and human breast cancer (MCF-7) in vitro. Among them, some of compounds 1-14 exhibited moderate antiproliferative (IC50 <50μM), including compounds 3-8, 12 and 14 showed antiproliferative against SGC-7901 with IC 50 values of 21.8-40.2 μM; compounds 1-3, 5, 6, 10, and 11 showed antiproliferative against MCF-7 with IC50 values of 27.9-40.6 μM; compounds 1, 3, 4, 6, 7, 9, 11, 12 and 14
f
showed antiproliferative against HepG2 with IC50 values of 27.6-44.0 μM; and 13
oo
exhibited no cytotoxicity against the three cancer cells tested. From the preliminary
pr
immunosuppressive evaluation, it was observed that the regulations in terms of
e-
structure-activity relationship (SAR) were concluded as follows: i) withanolides with
Pr
2-en-1-one systems in ring A were likely to show stronger acitivities than those none. ii) the comparison of the activities of 1, 5 with 7, 10, indicated withanolides with
systems.
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2.8 Conclusion
rn
al
2,4-dien-1-one systems in ring A were stronger than those substitued with 2-en-1-one
In the end, all the isolations were withanolides with some activities at some point. From the enrichment, a large of withanolides were obtained covering numerous new compounds. And, notably, some of them were observed possessing a same segment -OCH2 CH3 in C-27, including compounds 2-8, 10, 13 and 14. In addition to 27-OCH2 CH3 , these compounds compared with known withanolides, the novelty for compounds 3 and 8 had the presence of -OH in C-12 and compounds 10, 13 and 14 had the presence of -OH in C-21. However, compounds 2, 4-6 were possible to be the artifacts. A possibility was proposed: some etherifications were reacted between the
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compounds and elution solvent (ethyl alcohol) in the alkaline environment of D-941 macroporous resin which was anion exchange resin of polystyrene-type with alkalescence. These results reminded us that D-941 had potential enrichment applications in withanolides and even steroids. Further intensive removals at 27-OCH2 CH3 by a series of chemical methods and studies are currently undergoing in
Jo u
rn
al
Pr
e-
pr
oo
f
our laboratory, and the results will be reported in due course.
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Associated content
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*Supporting information
13
C NMR, HR-ESI-MS, DEPT-135, 1 H- 1 H COSY, HSQC and
e-
Copies of 1 H NMR,
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Details for the key HMBC, 1 H- 1 H COSY and selected NOESY correlations of 1-14.
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HMBC of 1-14. Notes
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Yan Liu and Juan Pan have contributed equally to this work.
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Acknowledgment
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The authors declare no competing financial interest.
This work was financed by National Natural Science Foundation of China (NSFC) (81773883; 81903781); China Postdoctoral Science Foundation (2018M631978); Excellent Youth Project of Heilongjiang Natural Science Foundation (YQ2019H029); Heilongjiang Postdoctoral Science Foundation (LBH-Z18245).
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REFERENCE [1] H. X. Kuang, B. Y. Yang, Y. G. Xia, W. S. Feng, Chemical Constituents from the Flower of Datura metel L, Arch. Pharm. Res. 31 (2008) 1094-1097. [2] H. X. Kuang, B. Y. Yang, Y. G. Xia, Q. H. Wang, Two New Withanolide Lactones from Flos Daturae, Molecules. 16 (2011) 5833-5839. [3] B. Y. Yang, Y. G. Xia, Q. H. Wang, D. Q. Dou, H. X. Kuang, Baimantuoluos ides D-G, four new withanolide glucosides from the flower of Datura metel L., Arch. Pharm. Res. 33 (2010) 1143-1148. [4] B. Y. Yang, Y. G. Xia, Q. H. Wang, D. Q. Dou, H. X. Kuang, Two new amide alkaloids from the flower of Datura metel L., Fitoterapia. 81 (2010) 1003-1005.
oo
f
[5] H. X. Kuang, B. Y. Yang, L. Tang, Y. G. Xia, D. Q. Dou, Baimantuoluosides A-C, Three New Withanolide Glucosides from the Flower of Datura metel L., Helv. Chim. Acta. 92 (2009) 1315-1323.
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[6] B. Y. Yang, Q. H. Wang, Y. G. Xia, W. S. Feng, H. X. Kuang. Baimantuoluolines D-F, Three New
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Withanolides from the Flower of Datura metel L., Helv. Chim. Acta. 91 (2008) 964-971. [7] B. Y. Yang, Q. H. Wang, Y. G. Xia, W. S. Feng, H. X. Kuang, Withanolide Compounds from the
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Flower of Datura metel L., Helv. Chim. Acta. 90 (2007) 1522-1528. [8] Y. Yin, F. Y. Gong, X. X. Wu, Y. Sun, Y. H. Li, T. Chen, Q. Xu, Anti-inflammatory and immunosuppressive effect of
flavones
isolated
from
Artemisia
vestita.
Journal of
al
Ethnopharmacology. 120 (2008) 1-6.
[9] U. Wagner, E. Burkhardt, K. Failing, Evaluation of canine lymphocyte proliferation: comparison
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of three different colorimetric methods with the 3 H-thymidine incorporation assay, Vet Immunol Immunophathol. 70 (1999) 151-159.
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[10] S. B. Wu, J. J. Su, L. H. Sun, W. X. Wang, Y. Zhao, H. Li, S. P. Zhang, G. H. Dai, C. G. Wang, J. F. Hu, Triterpenoids and Steroids from the Fruits of Melia toosendan and Their Cytotoxic Effects on Two Human Cancer Cell Lines, J. Nat. Prod. 73 (2010) 1898-1906. [11] B. Y. Yang, Y. G. Xia, J. Pan, Y. Liu, Q. H. Wang, H. X. Kuang, Phytochemistry and biosynthesis of δ-lactone withanolides, Phytochem. Rev. 15 (2016) 771. [12] S. Minguzzi, L. E. S. Barata, Y. G. Shin, P. F. Jonas, H. B. Chai, E. J. Park, J. M. Pezzuto, G. A. Cordell, Cytotoxic withanolides from Acnistus arborescens, Phytochemistry. 59 (2002) 635-641. [13] B. Y. Yang, Y. G. Xia, Y. Liu, L. Li, H. Jiang, L. Yang, Q. H. Wang, H. X. Kuang, New antiproliferative and immunosuppressive withanolides from the seeds of Datura metel, Phytochemistry Letters. 8 (2014) 92-96. [14] S. Siddiqui, N. Sultana, S. S. Ahmad, S. I. Haider, A novel withanolide from Datura metel, Phytochemistry. 26 (1987) 2641-2643. [15] X. H. Zhu, J. Ando, M. Takagi, T. Ikeda, T. Nohara, Six new withanolide-type steroids from the leaves of Solanum cilistum, Chem Pharm. Bull. 49 (2001) 161-164.
Journal Pre-proof [16] B. N. Su, R. I. Misico, E. J. Park, B. D. Santarsiero, A. D. Mesecar, H. H. S. Fong, J. M. Pezzuto, A. D. Kinghorn, Isolation and characterization of biocative priciples of the leaves and stems of Physalis philadelphica, Tetrahedron. 58 (2002) 3453-3466. [17] H. Masao, G. Keiju, I. Nobuo, Synthetic of withanolide. 5.Synthesis of jaborosalactone A,B, and D, Jpurnal of American Chemical Society. 104 (1982) 3735-3737. [18] M. Manickam, S. Kumar, A. Sinha-Bagchi, S. C. Sinha, A. B. Ray, Withametelin H and Withafastuosin C, Two New Withanolides from the Leaves of Datura Species, J. Ind. Chem. Soc.
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al
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pr
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71 (1994) 393-399.
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Dec 21, 2019
Dear editor:
Attached please find our manuscript entitled “Immunosuppressive Withanolides
oo
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from the Flower of Datura metel L.” for publication in Fitoterapia. All authors have read and approved this version of the article, and due care has
pr
been taken to ensure the integrity of the work. This article or any one with similar
Sincerely,
al
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submission of this manuscript.
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content has not been submitted to any other journal. No conflict of interest exists in the
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Haixue Kuang, Ph.D. Professor Heilongjiang University of Chinese Medicine
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Fig.1. Structures of Compounds 1-14
Journal Pre-proof Tab.1 1 H-NMR Spectroscopic Data (400MHz) of compounds 1-5 1a
2b
3a
4a
5a
2
5.77 dd (10.2,2.4)
6.08 dd (10.4,2.4)
5.80 dd (10.4, 2.0)
5.74 dd (10.2,2.4)
5.78 dd (10.0,2.4)
3
6.65 ddd (10.1,5.0,2.2)
6.64 ddd (10.4,5.2,2.0)
6.64 ddd (10.4,5.2,2.4)
6.64 ddd (10.1,5.1,2.0)
6.66 ddd (10.0,5.1,2.1)
4
3.19 dt (19.6,2.4)
3.60 dt (19.6,2.4)
2.66 dd (20.0, 4.8)
2.76 dt (19.2, 2.4)
3.26 dt (19.9,2.4)
2.14 dd (19.6,5.0)
2.49 dd (19.6,5.2)
2.53 dt (20.0, 2.8)
2.45 dd (19.2,5.1)
2.04 dd (19.9,5.1)
6
3.50 d (3.2)
4.19 t (3.6)
3.35 d (8.8)
3.04 d (3.9)
3.51 t (2.3)
7
3.70 t (2.7)
4.31 m
3.38 t (9.2)
3.27 br.s
1.66 m
8
1.76 m
2.29 m
1.33 m
1.69 m
1.70 m
9
2.14 m
2.89 m
2.00 m
1.74 m
1.96 m
11
2.46 dt (12.8,4.0)
3.29 dt (12.4,3.6)
2.47 dt (12.4, 4.0),
2.90 dt (13.1,3.8)
2.45 dt (12.7,3.8)
No.
1.56 m
1.96 m
1.35 m
1.34 m
1.39 m
3.46 dd (11.1,4.4)
3.90 dd (6.6,4.2)
3.48 dd (11.2,4.4)
3.45 dd (10.9,4.6)
3.59 dd (11.2,4.1)
14
1.55 m
1.95 m
1.49 m
15
1.78 m
1.93 m
1.91 m
16
1.32 m
1.79 m
1.50 m
1.73 m
1.42 m
1.20 m
1.85 m
1.72 m
1.46 m
1.29 m
1.83 m
1.90 m
pr
1.31 m
oo
f
1.44 m 12
1.36 m
1.51 m
1.57 m
1.53 m
1.57 m
1.45 m
1.33 m
1.55 m
1.84 m
18
0.77 s
0.99 s
0.77 s
0.79 s
0.83 s
19
1.30 s
1.66 s
1.19 s
1.19 s
1.31 s
20
2.01 m
2.22 m
2.04 m
1.99 m
1.92 m
21
1.18 d (6.9,3H)
1.51 d (6.4)
1.17 d (6.8)
1.21 d (6.1)
3.92 dd (11.8,3.2)
22
4.62 dt (13.4, 3.5)
4.60 dt (13.2, 3.6)
4.60 dt (13.2, 3.6)
4.58 dt (13.2, 3.5)
4.56 dt (13.4,3.7)
23
2.55 dd (18.0,13.4)
2.42 dd (18.0,13.2)
2.56 dd (16.8, 14.5)
2.57 dd (18.0, 13.2)
2.83 dd (18.4,13.4)
2.24 dd (18.0, 2.8)
2.00 dd (18.0, 3.2)
2.25 dd (16.8, 3.0)
2.29 dd (18.0, 3.1)
2.40 dd (18.4,3.0)
4.38 d (11.8)
4.49 d (10.8)
4.28 d (10.4)
4.25 q (10.6)
4.29 d (10.6)
4.31 d (11.8)
4.32 d (10.4)
4.21 d (10.4)
4.20 d (10.6)
4.22 d (10.6)
2.00 s
2.09 s,
2.10 s
2.09 s
3.50 q (7.2)
3.53 q (6.8)
3.53 q (7.0)
3.54 q (7.0)
1.10 t (7.2)
1.18 t (6.8)
1.17 t (7.0)
1.19 t (7.0)
Pr
e-
1.59 m 17
28 29 30
2.10 s
rn
Jo u
27
al
3.85 dd (11.8,2.9)
*a: in CDOD3 ; b: in C5 D5 N; c: in CDCl3
Journal Pre-proof Tab.2 1 H-NMR Spectroscopic Data (400MHz) of compounds 6-10 No. 2
6a
7c
8a
9c
10a
5.76 dd (10.1,2.4)
5.97 dd (9.7,0.8)
5.77 dd (10.1,2.4)
2.64 m
5.98 dd (9.7,0.7)
2.36 m 3
6.64 ddd (10.1,5.0,2.1)
6.86 dd (9.7,5.9)
6.64 ddd (10.1,5.1,2.2)
1.88 m
7.08 dd (9.7,5.9)
4
3.25 dt (19.9,2.4)
6.09 d (5.9)
3.26 dt (19.9,2.4)
1.83 m
6.22 d (5.9)
2.04 dd (19.9,5.0)
2.04 m
3.51 br.s
4.52 t (2.3)
3.52 t (2.4)
3.13 br. s
4.53 t (2.5)
7
1.70 m
1.95 m
1.67 m
2.19 dt (14.6,3.0)
1.97 m
1.55 m
1.14 m
1.54 m
1.33 m
1.18 m
8
1.77 m
1.98 m
1.80 m
1.46 m
2.08 m
9
1.82 m
1.05 m
1.84 m
1.18 m
1.06 m
11
2.22 m
1.83 m
2.23 dt (12.9,2.6)
1.37 m
1.82 m
1.36 m
1.48 m
1.32 m
1.28 m
1.54 m
1.98 m
1.86 m
1.86 m
1.85 m
2.00 m
1.31 m
1.27 m
1.08 m
1.11 m
1.68 m
1.68 m
1.14 m
1.29 m
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12
f
6
1.20 m
1.47 dd (13.4,3.0)
1.25 m
1.02 m
1.24 m
15
1.66 m
1.62 m
1.73 m
1.23 m
1.17 m
1.22 m
1.79 m
1.65 m
1.82 m
1.72 m
1.80 m
1.44 m
1.32 m
1.36 m
1.44 m
1.45 m
17
1.73 m
1.58 m
1.70 m
1.71 m
1.66 m
18
0.81 s
0.68 s
0.75 s
0.63 s
0.86 s
19
1.30 s
1.41 s
1.30 s
1.16 s
1.46 s
20
1.86 m
1.73 m
1.84 m
1.73 m
1.84 m
21
3.92 dd (11.2,2.6)
3.79 d (13.6)
3.85 d (13.3)
3.86 d (13.6)
3.90 dd (11.3,2.7)
3.77 dd (11.2,4.3)
3.41 dd (13.6,2.5)
3.72 dd (13.3,3.8)
3.51 dd (13.6,2.8)
3.75 dd (11.3,4.2)
22
4.52 dt (13.4,3.5)
4.55 br. s
4.68 br. s
4.61 br. s
4.50 dt (13.3,3.6)
23
2.95 dd (18.4,13.4)
1.87 dd (13.9,4.4)
2.29 dd (13.9,4.4)
2.01 m
2.92 dd (18.1,13.3)
2.32 dd (18.4,3.1)
1.74 m
1.92 d (13.9)
1.84 m
2.28 dd (18.1,3.2)
2.44 dd (6.1,2.7)
2.60 br s
2.32 q (7.2)
3.99 dd (9.8,2.7)
3.88 dd (9.5,3.3)
1.33 d (7.2, 3H)
3.71 dd (9.8,6.1)
3.76 dd (9.5,2.5)
1.24 s
1.29 s
3.46 q (7.0)
3.42 q (7.0)
3.53 q (7.0)
1.13 t (7.0)
1.14 t (7.0)
1.18 t (7.0)
27
4.29 d (10.6) 4.22 d (10.6)
28
2.08 s
29
3.53 q (7.0)
30
1.18 t (7.0)
e-
Pr al
rn
25
Jo u
16
pr
1.48 m 14
*a: in CDOD3 ; b: in C5 D5 N; c: in CDCl3
4.28 d (10.6) 4.21 d (10.6)
1.20 s
2.07 s
Journal Pre-proof Tab.3 1 H-NMR Spectroscopic Data (400MHz) of compounds 11-14 No.
11a
1 2
12a
13a
14a
3.80 br.s
3.80 br. s
3.81 br. s
3.38 dd (19.8,2.1)
2.05 m
1.96 m
2.15 m
2.66 dd (19.8,4.7)
1.80 m
1.80 m
1.82 m
3
5.63 ddd (9.8,4.8,2.2)
3.91 m
3.91 m
4.04 m
4
6.06 dd (9.8,2.2)
2.24 m
2.29 m
2.46 dd (13.6,3.6)
2.28 m
2.34 m
2.30 m
6
5.67 t (2.7)
5.50 br. d (5.3)
5.50 br. d (5.2)
5.51 d (5.1)
7
2.15 m
1.97 m
1.97 m
1.99 m
1.62 m
1.62 m
1.55 m
1.60 m
1.60 m
1.60 m
1.48 m
9
1.75 m
1.75 m
1.74 m
1.72 m
11
1.77 m
1.77 m
1.77 m
1.61 m
1.35 m
1.35 m
1.37 m
2.01 m
1.96 m
2.00 m
1.93 m
1.43 m
1.24 m
1.27 m
1.24 m
14
1.17 m
1.17 m
1.15 m
1.12 m
15
1.72 m
1.72 m
1.70 m
1.70 m
1.19 m
1.19 m
1.29 m
1.15 m
1.79 m
1.78 m
16
oo
pr e-
12
f
1.67 m 8
1.45 m
1.85 m
1.85 m
1.45 m
1.42 m
1.70 m
1.66 m
0.79 s
0.78 s
1.02 s
1.02 s
1.84 m
1.86 m
1.48 m
1.70 m
1.70 m
18
0.80 s
0.78 s
19
1.39 s
1.02 s
20
1.84 m
1.84 m
21
3.90 dd (11.2,2.8)
3.90 dd (11.2,2.8)
3.92 dd (11.3,2.7)
3.91 dd (11.2,2.6)
3.77 dd (11.2,4.3)
3.77 dd (11.2,4.4)
3.75 dd (11.3,4.7)
3.76 dd (11.2,4.2)
4.48 dt (13.2,3.4)
4.52 dt (13.2,3.4)
4.52 dt (13.2,3.4)
2.84 dd (18.4,13.2)
2.94 dd (18.1,13.4)
2.94 dd (18.4,13.2)
2.25 dd (18.4,3.2)
2.30 dd (18.1,3.7)
2.30 dd (18.4,3.2)
1.85 s
4.29 d (10.6)
4.28 d (10.6)
4.22 d (10.6)
4.21 d (10.6)
2.08 s
2.08 s
3.50 q (7.0)
3.50 q (7.0)
1.19 t (7.0)
1.18 t (7.0)
4.48 dt (13.4,3.6)
23
2.83 dd (18.6,3.4)
1.85 s
28
1.98 s
29
al
Jo u
2.22 dd (18.6,3.4) 27
rn
22
Pr
1.48 m 17
1.97 s
30 1′
4.36 d (7.8)
2′
3.13 t (8.5)
3′
3.34 m
4′
3.25 m
5′
3.24 m
6′
3.84 dd (12.0,2.0) 3.64 dd (12.0,5.1)
*a: in CDOD3 ; b: in C5 D5 N; c: in CDCl3
Journal Pre-proof Tab. 4 13 C-NMR Spectroscopic Data (100MHz) of compounds 1-14 No.
1a
2b
3a
4a
5a
6a
7c
8a
9c
10a
11a
12a
13a
14a
1
207.0
204.9
205.9
205.5
207.3
207.6
205.1
207.5
213.1
208.1
212.9
73.6
73.6
73.6
2
128.9
128.8
128.9
129.3
129.0
129.0
126.9
129.1
35.1
126.7
40.7
39.2
39.2
37.7
3
143.4
142.0
143.6
142.4
144.1
143.9
139.7
143.9
31.8
142.9
122.8
67.0
67.0
74.9
4
36.6
36.6
36.2
38.0
36.6
36.6
118.0
36.6
35.1
118.6
130.4
42.5
42.5
39.1
5
80.1
79.4
79.6
74.7
78.2
78.3
157.8
78.3
64.3
160.3
142.6
139.4
139.4
139.1
6
75.6
75.7
77.1
57.1
75.2
75.3
74,0
75.2
60.4
74.7
128.1
125.2
125.1
125.5
7
73.0
72.9
75.1
57.0
33.9
34.1
40.5
31.4
20.6
42.0
32.1
32.9
32.9
32.9
8
35.0
34.8
41.3
36.4
30.5
31.5
30.6
34.1
29.2
32.1
33.1
33.4
33.4
33.4
34.6
34.2
40.1
34.7
41.0
42.6
49.5
42.6
42.9
51.5
42.6
42.6
42.6
42.6
53.6
53.2
52.6
52.2
52.8
53.0
54.0
53.0
52.3
55.8
53.4
42.7
42.7
42.8
34.6
35.0
35.3
33.3
33.6
24.6
21.3
24.5
21.9
22.5
23.6
21.3
21.3
21.3
78.4
77.2
78.5
78.1
80.0
40.6
39.4
41.3
39.2
39.8
39.9
39.9
40.0
39.9
13
49.2
48.5
50.2
49.7
49.6
44.1
42.8
44.1
42.7
43.8
43.9
43.8
43.8
43.8
14
49.8
49.1
54.2
50.9
55.4
57.0
55.5
56.9
55.7
56.9
57.4
57.6
57.6
57.6
15
23.9
23.4
26.9
23.8
25.0
25.3
24.2
25.1
24.0
25.5
25.2
25.5
25.5
25.5
16
27.5
26.8
27.8
27.6
28.9
28.1
26.3
27.4
26.3
28.2
28.1
28.2
28.2
28.2
17
54.9
54.2
55.5
54.5
49.3
48.4
47.5
49.4
47.5
48.0
48.4
48.3
48.3
48.3
12.9
13.5
8.3
8.0
8.4
13.1
16.7
15.3
15.2
16.2
16.3
20
39.1
38.1
38.8
38.9
47.6
46.9
21
15.5
15.3
15.6
15.5
60.6
60.1
22
80.9
79.3
81.0
80.8
79.6
32.4
31.3
32.6
32.1
33.6
24
158.0
156.5
159.9
159.8
160.3
25
126.4
124.1
124.0
123.9
26
168.6
166.1
168.5
168.3
27
56.4
64.2
64.6
28
20.3
20.3
20.5
29
66.0
67.1
30
15.5
15.4
2′ 3′
12.8
12.7
12.4
12.5
12.5
16.3
13.2
19.1
20.9
20.1
20.1
20.0
39.6
42.3
39.4
46.8
46.9
46.9
46.8
46.8
60.1
60.9
59.8
59.9
60.1
60.2
60.1
60.1
79.4
75.9
78.5
75.6
79.3
79.5
79.5
79.4
79.4
34.0
33.2
31.6
33.0
34.0
33.6
33.6
34.0
34.0
160.6
70.8
72.8
70.7
160.6
153.4
153.5
160.4
160.4
123.9
50.6
52.8
44.2
123.9
122.0
122.0
123.9
123.9
168.3
168.4
171.8
175.8
173.9
168.4
169.5
169.5
168.4
168.4
rn
123.9
64.5
64.6
64.6
67.0
71.3
9.4
64.6
12.4
12.5
64.6
64.6
20.6
20.6
20.5
27.0
25.1
26.6
20.5
20.4
20.4
20.5
20.5
67.0
67.1
67.0
66.4
67.9
67.0
67.0
67.0
15.3
15.5
15.5
15.2
15.3
15.4
15.4
15.4
Jo u
1′
12.5
19.1
al
23
pr
8.2
16.8
e-
8.0
19
Pr
18
oo
11 12
f
9 10
102.7 75.1 78.0
4′
71.7
5′
78.1
6′
62.9
*a: in CDOD3 ; b: in C5 D5 N; c: in CDCl3
Journal Pre-proof Tab.5 IC50 of 1-14 in Splenocyte Proliferation of Mouse Bioassays and Antiproliferative Bioassays. Growth Inhibition Constant (IC50 in μM )a
Compounds M ouse Splenocyte b
SGC-7901
-3
M CF-7
HepG2
-
-
8.9
32.7
1.4
1
19.5
>50
32.4
27.6
2
>30
>50
28.7
>50
3
24.3
26.3
27.9
34.8
4
27.1
35.5
>50
37.3
5
19.7
30.7
29.9
>50
6
21.0
29.4
35.8
40.1
7
14.8
21.8
>50
42.3
8
20.7
35.7
>50
9
>30
>50
10
16.9
>50
11
>30
>50
12
28.1
13
>30
14
>30
oo
40.6
>50
36.6
44.0
37.7
>50
33.9
>50
>50
>50
>50
37.5
pr
28.6
40.2
a
Jo u
rn
al
Pr
IC50 was defined as the concentration that resulted in a 50% decrease in cell number The IC50 >30 μM in Splenocyte Proliferation of Mouse Bioassays was deemed inactive. The IC50 > 50 μM in antiproliferative bioassays was deemed inactive. b Positive control. "-" means no experiment.
>50
>50
e-
5-FU
4.9×10
b
f
-
-
Cyclosporine
Journal Pre-proof
Jo u
rn
al
Pr
e-
pr
oo
f
Graphical Abstract