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Molecules 2019, 24(22), 4033; https://doi.org/10.3390/molecules24224033

Article
Alkaloids with Nitric Oxide Inhibitory Activities from the Roots of Isatis tinctoria
1
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2
Institute of TCM International Standardization of Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
3
Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 201203, China
*
Correspondence: [email protected] (Y.L.); [email protected] (R.W.); Tel.: +86-21-5132-2191 (Y.L.); +86-21-5132-2181 (R.W.); Fax: +86-21-5132-2193 (Y.L. & R.W.)
These authors contribute equally to this work.
Received: 22 October 2019 / Accepted: 6 November 2019 / Published: 7 November 2019

Abstract

:
As our ongoing research project on Ban Lan Gen (Isatis tinctoria roots), a total of 23 alkaloids were obtained. Compounds 1 and 2 contain an unusual C–C bond between the 2(1H)-quinolinone moiety and the phenol moiety and between the 2(1H)-quinolinone moiety and the 1H-indole moiety, respectively. Compound 3 possesses an unusual carbon skeleton and its putative biosynthetic pathway was discussed, and Compound 23 was deduced as a new indole alkaloid glycoside. Compounds 4–7 were identified as four new natural products by extensive spectroscopic experiments. Additionally, the anti-inflammatory activity was assessed based on nitric oxide (NO) production using Lipopolysaccharide-stimulated RAW264.7 macrophages. Compounds 9, 15, and 17 showed inhibitory effects with IC50 values of 1.2, 5.0, and 74.4 μM.
Keywords:
Isatis tinctoria roots; alkaloids; structure identification; anti-inflammatory activity

1. Introduction

Isatis tinctoria L. (synonym, Isatis indigotica Fort.), named Ban Lan Gen in the Chinese Pharmacopoeia, belongs to the gene Isatis (Brassicaceae family), which is widely distributed and cultivated in the North of the Yangtze River, China [1,2,3,4]. Alkaloids were considered as one of the characteristic constituents of this plant, which possess diverse bioactivities such as anti-inflammatory, antiviral, antibacterial, antitumor, and antioxidant activities [5,6,7]. Up to now, more than 100 alkaloids have been isolated from I. tinctoria, such as indole alkaloids, quinazolone alkaloids, quinoline alkaloids, and so on [1,2,3,4,5]. As our ongoing phytochemical and pharmacological research project on this plant [8,9,10,11,12], four new alkaloids and four new natural products, along with 15 known analogues, were obtained, and their structures and absolute configurations were determined by extensive spectroscopic data analysis, including one-dimensional and two-dimensional-NMR, HRESIMS, and IR, specific rotation data, and electronic circular dichroism (ECD) experiments. The known compounds (422, Figure 1) were identified by comparison of their spectroscopic and optical rotation data with those in the reported literature as 4-p-hydroxyphenyl-2(1H)-quinolinone (4) [13], 2-(1H-indol-2-yl)-6-methoxy-4(3H)-quinazolinone (5) [14], 2-(2-hydroxyphenyl)-4(3H)-quinazolinone (6) [15], 2-(but-3-en-1-yl)-4(3H)-quinazolinone (7) [16], 2-(1H-indol-2-yl)-4(3H)-quinolinone (8) [17], tryptanthrin (9) [18], 3-(2,4-dioxo-1,2- dihydroquinazolin-3(4H)-yl)propanoic acid (10) [19], indiforine C (11) [3], 4-(2,4-dioxo-1,2- dihydroquinazolin-3(4H)-yl)butanoic acid (12) [20], methyl 4-(2,4-dioxo-1,2-dihydroquinazolin- 3(4H)-yl)butanoate (13) [21], 3-(2-hydroxyphenyl)-4(3H)-quinazolinone (14) [22], 3-(2-carboxyphenyl)-4(3H)-quinazolinone (15) [23], 4-methyl-1,2-dihydro-2-oxoquinazoline (16) [24], 2-methyl-4(3H)-quinazolinone (17) [25], 4-hydroxy-3-methyl-2(1H)-quinolinone (18) [26], 2-amino-4-quinolinecarboxylic acid (19) [27], 4(1H)-quinolinone (20) [28], 4(1H)-quinolone-3- carboxylic acid (21) [29], and 1,2,3,4-tetrahydro-4-hydroxy-quinolinecarboxylic acid (22) [30]. The NO inhibitory activities of the isolates (123) were also evaluated against the LPS-stimulated RAW264.7 macrophages. In the present paper, we report the isolation and structure determination, putative biosynthetic pathway, and the NO inhibitory activities of these alkaloids.

2. Results and Discussion

Isatisindigoticanine E (1) was obtained as a yellow amorphous powder. The molecular formula was assigned as C15H11NO3 on the basis of the negative ion HRESIMS peak at m/z 252.0666 [M − H] (calculated 252.0666 [M − H]), together with its one-dimensional-NMR data (Table 1). The 1H-NMR spectrum displayed signals of a 1,2,4-trisubstituted benzene ring [31] at [δH 7.20 (1H, d, J = 2.2 Hz, H-5), 6.63 (1H, dd, J = 8.3, 2.2 Hz, H-7), and 6.66 (1H, d, J = 8.3 Hz, H-8)], a 1,4 disubstituted benzene ring at [δH 7.57 (2H, d, J = 8.5 Hz, H-2’,6’) and 6.90 (2H, d, J = 8.5 Hz, H-3’, 5’) and also showed a trisubstituted double bond [9] at δH 7.48 (1H, s, H-3) and three exchangeable protons at δH 10.19 (1H, brs, NH-1), 10.12 (1H, brs, OH-6), and 8.96 (1H, brs, OH-4’). The 13C-NMR spectrum showed 15 carbon signals, among which 7 × C carbons at δC (169.5, 159.6, 152.2, 135.4, 126.1, 125.4, 122.5) and 8 × CH carbons at δC (136.7, 132.1, 132.1, 116.5, 116.1, 116.1, 110.7, 110.1) were found based on the DEPT 135 experiment. The two-dimensional-NMR spectroscopic features confirmed the inference above. The proton and protonated carbon resonances in the NMR spectra of 1 were unambiguously assigned by the HSQC experiments [32,33]. The 1H-1H COSY correlations (Figure 2) of H-2’,6’/H-3’,5’, along with HMBC correlations (Figure 2) of H-2’/C-4’ and H-3’/C-1’, indicated a phenol moiety in 1 [31]; 1H-1H COSY correlations of H-7/H-8, along with the HMBCs of NH-1/C-2, C-8, and C-8a, H-3/C-2, and C-4, and H-5/C-4 and C-7, indicated a 6-hydroxy-2(1H)-quinolinone moiety in 1 [34]; HMBCs of H-3/C-1’ and H-2’,6’/C-4 confirmed the 6-hydroxy-2(1H)-quinolinone moiety connected with the phenol moiety via a C-4-C-1’ bond. The structure of 1 was then determined, as depicted in Figure 1.
Isatisindigoticanine F (2) was obtained as a yellow amorphous powder. The molecular formula was assigned as C18H14N2O2 by the one-dimensional-NMR data and the HRESIMS positive ion peak at m/z 291.1125 [M + H]+ (calculated 291.1128 [M + H]+). The 1H-NMR spectrum (Table 1) of 2 showed signals of a 1,2,3-trisubstituted benzene ring at [δH 6.67 (1H, d, J = 7.5 Hz, H-6), 7.15 (1H, overlap, H-7) and 6.85 (1H, d, J = 7.3 Hz, H-8)], an ortho-disubstituted benzene ring at [δH 7.50 (1H, d, J = 7.5 Hz, H-4’), 7.15 (1H, overlap, H-5’), 7.00 (1H, dd, J = 7.5, 7.4 Hz, H-6’) and 7.14 (1H,overlap, H-7’)] [35]; two trisubstituted double bonds at δH 8.63 (1H, s, H-3) and 9.54 (1H, s, H-3’), as well as two exchangeable protons at δH 12.06 (1H, brs, NH-1) and 10.51 (1H, brs, NH-1’) and a methoxy group at δH 4.04 (3H, s, 5-OMe). After analysis of the 13C-NMR, DEPT 135 and HSQC data (Table 1), a 1H-indol-2-yl moiety (112.5, C; 133.5, CH; 126.3, C; 117.9, CH; 121.1, CH; 127.0, CH; 109.5, CH; 139.4, C) [8,10] and a 5-methoxy-2(1H)-quinolinone moiety (168.3, C; 130.6, CH; 118.8, C; 116.8, C; 155.0, C; 102.6, CH; 123.9, CH; 106.2, CH; 138.0, C; 55.9, CH3) were observed [34]. HMBCs of H-3/C-2’ and H-3’/C-4 indicated the 1H-indol-2-yl moiety connected with the 5-methoxy-2(1H)-quinolinone moiety via a C-4-C-2’ bond. These inferences were confirmed by detailed analysis of the two-dimensional-NMR data including HSQC, HMBC (Figure 2), and 1H–1H COSY (Figure 2) experiments. The structure of 2 was thus deduced, as depicted in Figure 1.
Isatisindigoticanine G (3), a yellow amorphous powder, possessed the molecular formula of C20H15N3O based on the positive HRESIMS ion at m/z 314.1297 [M + H]+ (calculated 314.1288 [M + H]+) and one-dimensional-NMR data. The 1H-NMR spectrum (Table 1) of 3 showed signals of two ortho-disubstituted benzene rings at [δH 8.13 (1H, d, J = 8.0 Hz, H-5), 7.47 (1H, dd, J = 8.0, 7.2 Hz, H-6), 7.82 (1H, dd, J = 8.1, 7.2 Hz, H-7) and 7.72 (1H, d, J = 8.1 Hz, H-8)] and [δH 7.87 (1H, d, J = 7.5 Hz, H-4’),7.22 (1H, dd, J = 8.1, 7.5 Hz, H-5’), 7.25 (1H, dd, J = 8.1, 7.5 Hz, H-6’) and 7.50 (1H, d, J = 7.5 Hz, H-7’)], two trisubstituted double bonds at δH 7.84 (1H, d, J = 2.2 Hz, H-2’) and 8.13 (1H, s, H-1’’), as well as an exchangeable proton at δH 11.98 (1H, brs, NH-1’) [35]. The 13C-NMR and the DEPT 135 spectra (Table 1) displayed 8 × C carbons at δC (160.5, 156.8, 148.8, 136.4, 127.4, 125.6, 120.3, 112.5), 10 × CH carbons at δC (134.9, 128.3, 126.3, 126.2, 126.0, 123.0, 122.6, 120.9, 118.5, 112.6), and 2 × CH2 carbons at δC (44.7, 26.2). The two-dimensional-NMR spectra (Figure 2) of 3 showed the 1H-1H COSY correlations of H-5/H-6/H-7/H-8, H-3’’/H-4’’ and HMBCs from H-5/C-4 from H-1’’/C-2 and C-3’’ and from H-4’’/C-2 and C-4, which indicated a 8H-pyrido[2,1-b]-11(9H)-quinazolinone moiety in 3 [36]; 1H-1H COSY correlations of H-4’/H-5’/H-6’/H-7’ and the HMBCs from NH-1’/C-2’, C-3’, C-3’a, and C-7’a indicated a 1H-indol-3-yl moiety in 3 [10]. HMBCs from NH-1’/C-9 and C-6, and from H-2’/C-2’’ and H-1’’/C-3’ determined the 8H-pyrido[2,1-b]-11(9H)-quinazolinone moiety connected with the 1H-indol-3-yl moiety via a C-2’’-C-3’ bond. The structure of 3 was thus determined, as depicted in Figure 1.
Isatindigoside D (23) was isolated as a red amorphous powder with [α] D 20 + 12.1° (c 0.19, MeOH). Its molecular formula of C23H22N2O7 (14 IHD) was deduced from the NMR data and the HRESIMS positive ion peak at m/z 490.1592 [M + Na]+, (calculated 490.1585 [M + Na]+). When comparing the one-dimensional (Table 2) and two-dimensional-NMR data (Figure 2) with the reported bisindoloside of isatindigobisindoloside C [35], they showed almost identical NMR spectroscopic features except for the differences around C-2 (downfield of C-2’, C-3’, and C-3’’, upfield of C-2’’). These differences, along with the optical rotation data ([α] D 20 + 12.1, c 0.19 in MeOH) supported Compound 23, would be the C2-epimer of isatindigobisindoloside C ([α] D 20 − 33.9, c 0.11 in MeOH) [35]. The experimental and calculated ECD curves of (2S)-23 matched well (Figure 3), which confirmed the S absolute configuration of 23 [35,37], and the calculation details are listed in the Supporting Information (Figures S33 and S34). Acid hydrolysis of 23 resulted in the product of d-glucose, which was confirmed by GC analysis of the acetylation derivative of the hydrolysate of 23 and the authentic sugars (tR d-glucose 45.23 min, tR l-glucose 45.38 min) [8,9]. The large coupling constant of Glc-H1 (J = 7.8 Hz) revealed the β-glucopyranosyl linkage in 23 [38,39]. Accordingly, the structure of isatindigoside D (23) was elucidated as depicted (Figure 1).
NO is a messenger molecule that is widespread in cells and can affect a variety of physiological and pathological processes. The production of NO causes tissue damage and can trigger a variety of inflammatory diseases. LPS induces the release of NO from RAW264.7 cells by detecting the release of NO widely used to investigate the anti-inflammatory effects of the compounds [2,10,40]. As our ongoing phytochemical and pharmacological research project on I. tinctoria [8,9,10,11,12], Compounds 123 were obtained and were evaluated for their anti-inflammatory activity based on NO inhibitory effects in the LPS-activated RAW 264.7 cells [40]. The cytotoxicity of Compounds 123 were tested at three different concentrations (25, 50, and 100 μM), and the results showed that only Compound 9 showed cytotoxicity above 25 μM, while the other compounds were above 100 μM. The results of NO production showed that Compounds 9, 15, and 17 exhibited inhibitory activities with IC50 values of 1.2, 5.0, and 74.4 μM (Table 3).
Isatisindigoticanine G (3) is the first example of a 8H-pyrido[2,1-b]-11(9H)-quinazolinone moiety connected with a 1H-indol-3-yl moiety via a C–C bond of C-2’’–C-3’. For its unusual structural features, a plausible biosynthetic pathway is discussed in Figure 4. First, myrosinase catalyzed hydrolysis of progoitrin and epiprogoitrin to give 3a [1]. 3a was connected with 2-aminobenzoic acid moiety by steps of dehydration to give 3b [10], and then 3c was obtained via a cyclization reaction of 3b [2,11]. 3c was connected with 1H-indole moiety by enzyme-catalyzed reaction to give 3d [5] and was then changed via a dehydration reaction to give 3 [9,10,11].

3. Experimental Section

The General Experimental Procedures, Extraction and Isolation, Plant Materials, Inhibitory Assay of NO Production and ECD Calculation sections are listed in the Supporting Information.

3.1. Physical and Spectroscopic Data of Isatisindigoticanines E–G and Isatindigoside D

Isatisindigoticanine E (1), a yellow amorphous powder; IR (KBr) νmax: 3406, 2923, 1647, 1609, 1556, 1517, 1466, 1383, 1273, 1093, 745 cm−1; m/z 356.1398 [M + H]+ (calculated 356.1394 [M + H]+); 1H-NMR (DMSO-d6, 600 MHz) and 13C-NMR (DMSO-d6, 150 MHz); see Table 1.
Isatisindigoticanine F (2), a yellow amorphous powder; IR (KBr) νmax: 3456, 1679, 1621, 1516, 1461, 1319, 1206, 1135, 1021, 952, 749 cm−1; m/z 291.1125 [M − H] (calculated 291.1128 [M + H]+); 1H-NMR (DMSO-d6, 600 MHz) and 13C-NMR (DMSO-d6, 150 MHz); see Table 1.
Isatisindigoticanine G (3), a yellow amorphous powder; IR (KBr) νmax: 3404, 2919, 1708, 1601, 1468, 1400, 1384, 1092, 745 cm−1; m/z 314.1297 [M + H]+ (calculated 314.1288 [M + H]+); 1H-NMR (DMSO-d6, 600 MHz) and 13C-NMR (DMSO-d6, 150 MHz); see Table 1.
Isatindigoside D (23), a red amorphous powder; [α] D 20 + 12.1 (c 0.19, MeOH); IR (KBr) νmax: 3420, 2939, 1722, 1598, 1514, 1461, 1261, 1069, 1025, 859, 813 cm−1; HRESIMS: m/z 490.1592 [M + Na]+, (calculated 490.1585 [M + Na]+); 1H and 13C-NMR (600 and 150 MHz in DMSO-d6); see Table 2.

3.2. Absolute Configuration Determination of Sugar

Compound 23 (2 mg) was hydrolyzed in 2 M hydrochloric acid (4 mL) at 80 °C for 2 h. After cooling, the solution was concentrated under vacuum, dissolved with water, and extracted twice with dichloromethane (CH2Cl2). The residue was dissolved in distilled water and reduced with NaBH4 for 3 h at room temperature. After neutralization with AcOH and evaporation to dryness, the residue was acetylated with Ac2O for 1 h at 100 °C. The resulting alditol acetate was subjected to GC analysis under the following conditions: capillary column, HP-5ms (60 m × 0.25 mm × 0.25 μm); detector, FID; detector temperature, 280 °C; injection temperature, 280 °C; initial temperature 140 °C, subsequently increased to 240 °C at a rate of 5 °C/min, and then 1 min to increase to 260 °C, finally, subsequent increase to 280 °C at a rate of 2 °C/min; carrier, N2 gas [8,9]. The D glucose moiety in 23 was confirmed by the comparison of their retention times (tR) with those of authentic sugars (tR d-glucose 45.23 min, tR l-glucose 45.38 min).

4. Conclusions

In this paper, a total of 23 alkaloids were reported, including four new ones: isatisindigoticanines E–G (13) and isatindigoside D (23). Four new natural products and 15 known analogues were isolated from Ban Lan Gen. Isatisindigoticanine G possesses an unusual carbon skeleton of an 8H-pyrido[2,1-b]-11(9H)-quinazolinone moiety connected with a 1H-indole moiety via a C–C bond of C-2’’–C-3’. Compounds 9, 15, and 17 showed NO inhibitory effects with IC50 values of 1.2, 5.0, and 74.4 μM in the LPS-stimulated RAW264.7 macrophages. This study is important as it explains the chemical and biological diversity of Ban Lan Gen. Furthermore, the new structures need more biocativity experiments to discover their more meaningful uses, which may stimulate us to better develop and utilize these compounds.

Supplementary Materials

The following are available online https://www.mdpi.com/1420-3049/24/22/4033/s1, Copies of IR, HREIMS, 1H-NMR, 13C-NMR, DEPT 135, HSQC, HMBC, and 1H-1H COSY of 13 and 23. Experimental and calculated ECD spectra of 23.

Author Contributions

R.W., Y.L., and K.C. conducted the experiments; D.R. and J.L. carried out the anti-inflammatory activity experiments; Y.S., Q.J., and W.Z. analyzed the MS, ECD, and NMR data; D.Z. did the isolation, confirmed the structures, and wrote the paper; R.W. oversaw the research project and drafted the paper.

Funding

This work was supported by the National Natural Science Foundation of China (81573571, 81673570), the Excellent Academic Leaders Program of Shanghai (16XD1403500), the programs of the High Level University Innovation Team, the Shanghai E-Research Institute of Bioactive Constituents in Traditional Chinese Medicine, and the Shanghai Scientific and Technological Innovation Program (18401931100).

Conflicts of Interest

No competing financial interests were declared by the authors.

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Sample Availability: Samples of the Compounds 1–23 are available from the authors.
Figure 1. Structures of Compounds 123.
Figure 1. Structures of Compounds 123.
Molecules 24 04033 g001
Figure 2. Key 1H-1H COSY and HMBC correlations of Compounds 13 and 23.
Figure 2. Key 1H-1H COSY and HMBC correlations of Compounds 13 and 23.
Molecules 24 04033 g002
Figure 3. Experimental and calculated ECD spectra of 23.
Figure 3. Experimental and calculated ECD spectra of 23.
Molecules 24 04033 g003
Figure 4. Putative biosynthetic pathway of 3.
Figure 4. Putative biosynthetic pathway of 3.
Molecules 24 04033 g004
Table 1. 1H-NMR (600 MHz in DMSO-d6) and 13C-NMR data (150 MHz in DMSO-d6) of 13.
Table 1. 1H-NMR (600 MHz in DMSO-d6) and 13C-NMR data (150 MHz in DMSO-d6) of 13.
No.123
δHδCδHδCδHδC
110.19, s 12.06, brs
2 169.5 168.3 156.8
37.48, s136.78.63, s130.6
4 126.1 118.8 160.5
4a 122.5 116.8 120.3
57.20, d (2.2)110.1 155.08.13, d (8.0)126.3
6 152.26.67, d (7.5)102.67.47, dd (8.0, 7.2)126.0
76.63, dd (8.3, 2.2)110.77.15, overlap123.97.82, dd (8.1, 7.2)134.9
86.66, d (8.3)116.56.85, d (7.3)106.27.72, d (8.1)126.2
8a 135.4 138.0 148.8
1’ 125.410.51, s 11.98, brs
2’7.57, d (8.5)132.1 112.57.84, d (2.2)128.3
3’6.90, d (8.5)116.19.45, s133.5 112.5
3’a 126.3 127.4
4’ 159.67.50, d (7.5)117.97.87, d (7.5)118.5
5’6.90, d (8.5)116.17.15, overlap121.17.22, dd (8.1, 7.5)120.9
6’7.57, d (8.5)132.17.00, dd (7.5, 7.4)127.07.25, dd (8.1, 7.5)123.0
7’ 7.14, overlap109.57.50, d (7.5)112.6
7’a 139.4 136.4
1’’ 8.13, s122.6
2’’ 125.6
3’’ 3.17, 2H, m26.2
4’’ 4.25, 2H, t (7.0)44.7
OMe 4.04, s55.9
6-OH10.12, s
4’-OH8.96, s
Table 2. 1H-NMR (600 MHz in DMSO-d6) and 13C-NMR data (150 MHz in DMSO-d6) of 23.
Table 2. 1H-NMR (600 MHz in DMSO-d6) and 13C-NMR data (150 MHz in DMSO-d6) of 23.
No.23No.23
δH (J in Hz)δCδH (J in Hz)δC
1a7.45, brs172.93’’ 112.1
1b7.14, brs3’’a 126.7
25.68, s38.74’’7.56, d (8.0)118.4
1’10.35, brs 5’’6.93, dd (8.0, 7.1)118.9
2’ 126.86’’7.04, dd (8.1, 7.1)120.5
3’ 133.07’’7.32, d (8.1)111.4
3’a 121.27’’a 136.0
4’7.77, d (8.0)118.1Glc-14.63, d (7.8)106.6
5’6.92, dd (8.0, 7.2)118.423.38, overlap74.1
6’6.97, dd (8.1, 7.2)120.833.26, m76.8
7’7.26, d (8.1)111.643.28, m69.8
7’a 133.353.14, m77.2
1’’10.94, brs 6a3.56, dd (10.8, 5.6)61.0
2’’7.38, s123.96b3.67, dd (10.8, 1.8)
Table 3. NO inhibitory activities of Compounds 123 in RAW 264.7 cell line.
Table 3. NO inhibitory activities of Compounds 123 in RAW 264.7 cell line.
CompoundsIC50 aCytotoxicityCompoundsIC50 aCytotoxicity
1>100>10013>100>100
2>100>10014>100>100
3>100>100155.0 ± 1.3 >100
4>100>10016>100>100
5>100>1001774.4 ± 3.8>100
6>100>10018>100>100
7>100>10019>100>100
8>100>10020>100>100
91.2 ± 0.9>2521>100>100
10>100>10022>100>100
11>100>10023>100>100
12>100>100AG b22.7 ± 0.4>100
a IC50 values were expressed as mean ± SD (n = 3). b AG = aminoguanidine hydrochloride was used as the positive control.

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