Zanthoxylum nitidum (Roxb.) DC (Rutaceae) is a traditional medicine used for the treatment of various diseases like toothache, gingivitis, fever, colic vomiting, diarrhea, and cholera. Three new alkaloids, zanthocadinanine C (1), 7-methoxy-8-demethoxynitidine (2), and zanthonitiside I (3) were isolated from the stems and twigs of Z. nitidum. Their structures were determined on the basis of extensive spectroscopic, including 1-dimensional and 2-dimensional nuclear magnetic resonance and mass spectroscopy data. Compounds 1–3 were evaluated for cytotoxic activity against 5 human cancer cell lines, KB, MCF-7, LNCaP, HepG-2, and LU-1. Compound 2 showed significant cytotoxic activity against all tested human cancer cell lines with IC50 values ranging from 10.3 to 12.6 µM.
Zanthoxylum nitidum (Roxb.) DC. (Rutaceae) is a scandent prickly shrub distributed in the Molucca Islands, New Guinea, South China, and Vietnam. In folk medicine, its roots have been used to promote blood circulation, dissipate blood stasis, and cure snake bite; leaves and stems have been used to treat toothache, sore throat, cough, and fever. Previous phytochemical investigations of this plant led to the isolation of phenanthridine alkaloids,1–3 quinolines,4 phenylpropanoids,5 lignans, and coumarins.4–6 These compounds exhibited anti-virus,7 anti-inflammatory,8,9 and anti-cancer activities.4–10 This paper reports three new alkaloids from Z. nitidum. In addition, these compounds were also evaluated for cytotoxic activity against 5 human cancer cell lines, KB, MCF-7, LNCaP, HepG2, and LU-1.
Compound 1 was obtained as a yellowish amorphous powder. Its molecular formula was deduced to be C36H41O4 by high resolution-electrospray ionization mass spectrometry (HR-ESI-MS) analysis from the ion at m/z 552.3108 [M + H]+ (calcd. for [C36H42O4]+, 552.3090). The 1H nuclear magnetic resonance (NMR) spectrum of 1 exhibited the signals of 6 aromatic protons, including two sets of ortho-protons at δH 6.93 (d, J = 8.5 Hz)/7.54 (d, J = 8.5 Hz) and 7.46 (d, J = 8.5 Hz)/7.71 (d, J = 8.5 Hz), two para-protons at δH 7.09 (s) and 7.53 (s), one benzylic proton at δH 4.61 (dd, J = 4.0, 10.5 Hz), one methylenedioxy group at δH 6.01 (2H, s), two methoxy groups at δH 3.92 (3H, s) and 3.94 (3H, s), and one N-methyl group at δH 2.60 (s). These suggested the partial structure of 1 as a 6-substituted hydrochelerythrine moiety,3 which was further supported by a characteristic HR-ESI-ESI mass fragmentation pattern with an ion at m/z 348.1220 (calcd. for C21H18O4N, 348.1236). In addition, the 1H NMR spectrum showed signals of one olefinic proton at δH 4.95 (s), three methyl groups at δH 0.61 (d, J = 7.0 Hz), 0.68 (d, J = 7.0 Hz), and 1.72 (s), suggested the presence of a cadinane-type sesquiterpene moiety. The heteronuclear multiple bond correlation (HMBC) (See Figure 2, Supplementary data) between H-1 (δH 7.09) and C-3 (δC 147.7)/C4a (δC 127.6); H-4 (δH 7.53) and C-2 (δC 147.4)/C-12a (δH 131.1) indicated the location of a methylenedioxy group at C-2 and C-3. The HMBCs between H-6 (δH 4.61) and C-7 (δC 145.9); between H-9 (δH 6.93)/H-10 (δH 7.54) and C-8 (δC 152.1); between methoxy (δH 3.94)/(δH 3.92) and C-7 (δC 145.9)/C-8 (δC 152.1) proved the positions of the two methoxy groups to be at C-7 and C-8. The position of the N-methyl group was also confirmed by the observation of HMBC cross peaks from N-methyl (δH 2.60) to C-4b (δC 140.2) and C-6 (δC 55.8). Thus, this part of the alkaloid was determined to be dihydrochelerythrine.3 The HMBCs from H-11′ (δH 2.00 and 2.07) to C-3′ (δC 30.1)/C-4′ (δC 135.3)/C-5′ (δC 126.3) and from H-15′ (δH 1.72) to C-1′ (δC 130.2)/C-9′ (δC 32.3)/C-10′ (δC 124.1) suggested the positions of the two double bonds to be at C-1′/C-10′ and C-4′/C-5′. The position of the isopropyl at C-7′ was proved by HMBCs between H-13′ (δH 0.68)/H-14′ (δH 0.61) and C-7′ (δC 44.5)/C-12′ (δC 26.4). The correlation spectroscopy (COSY) of H-2′ (δH 1.92 and 2.77) and H-3′ (δH 2.10 and 2.28); H-7′ (δH 0.90) and H-12′ (δH 1.52) and H-13′ (δH 0.68)/H-14′ (δH 0.61) confirmed the C-2′/C-3′ and C-7′/C-12′/C-13′ (C-14′) bonds. The α-configuration of the isopropyl group at C-7′ was based on the nuclear Overhauser effect observations of H-6′ (δH 2.36) and H-7′ (δH 0.90)/H-14′ (δH 0.61). All these proved the to be cadinane-type sesquiterpene, 1Hα,7Hβ-cadina-1′(10′),4′(5′)-diene-11-yl. The 1Hα,7Hβ-cadina-1′(10′),4′(5′)-diene-11-yl moiety connection with dihydrochelerythrine at C-6 was confirmed by HMBCs between H-11′ (δH 2.00 and 2.07) and C-6 (δC 55.8)/C-6a (δC 130.5), as well as by COSY of H-6 (δH 2.36)/H-11′ (δH 2.00 and 2.07). The CD spectrum of 1, showing one negative Cotton effect at ([θ]224,rel – 0.52), and three positive Cotton effects [θ]208,rel + 0.40, [θ]252,rel + 1.00, [θ]286,rel + 0.32, was similar to that of epi-zanthocadinanine B, which proved the configuration at C-6 as S.3 Based on the above evidence, the structure of 1 was elucidated and the compound named as zanthocadinanine C.
Compound 2 was obtained as a yellowish powder. Its molecular formula, C21H18O4N+, was established on the basis of the HR-ESI-MS ion at m/z 348.1257 [M]+ (calcd. for [C21H18O4N]+, 348.1230). In the 1H NMR spectrum, two ortho protons at δH 8.28 (1H, d, J = 8.0 Hz) and 8.31 (1H, d, J = 8.0 Hz), two meta protons at δH 8.83 (1H, d, J = 2.0 Hz) and 8.85 (1H, d, J = 2.0 Hz), two para protons at δH 7.78 (1H, s) and 8.30 (1H, s), one methylenedioxy at δH 6.35 (2H, s), one N-methyl group at δH 5.00 (3H, s), and two methoxy groups at δH 4.11 (3H, s) and 4.18 (3H, s) showed the presence of benzo[c]phenanthridine. The 13C NMR and distortionless enhancement by polarization transfer spectra of 2 exhibited the signals of 10 non-protonated carbons, 7 methines, 1 methylene, and 3 methyl carbons. Analysis of the 1H- and 13C-NMR data indicated the structure of 2 to be similar to that of nitidine, except for the movement of the methoxy group from C-8 to C-7.11 In the HMBC spectrum, methylenedioxy protons (δH 6.02) showed long-range correlations with C-2 (δC 148.7) and C-3 (δC 148.8) indicating that the methylenedioxy was linked to C-2 and C-3. The HMBCs between two methoxy groups (δH 4.18)/(δH 4.11) and C-7 (δC 145.4)/C-9 (δC 150.6), respectively, proved the position of the two methoxy groups at C-7 and C-9. The position of the N-methyl group was confirmed by HMBCs from methyl (δH 5.00) to C-4a (δC 120.1) and C-6 (δC 150.8) (Figure 2). Consequently, the new structure 2 was assigned to 7-methoxy-8-demethoxynitidine.
The molecular formula of 3 was determined to be C21H27O8N by the HR-ESI-MS ion at m/z 422.1776 [M + H]+ (calcd. for [C21H28O8N]+, 422.1809). The 1H NMR spectrum of 3 showed the proton signals of ortho-substituted benzene at δH 7.25 (1H, dd, J = 7.6, 8.0 Hz), 7.32 (1H, d, J = 8.0 Hz), 7.49 (1H, dd, J = 7.6, 8.0 Hz), and 7.77 (1H, d, J = 8.0 Hz), 1 olefinic proton at δH 5.47 (t, J = 6.8 Hz), 2 methyl groups at δH 1.78 and 3.95 (each 3H, s), and 1 anomeric proton at δH 4.33 (1H, d, J = 7.6 Hz). The 13C NMR and HSQC spectra of 3 showed the signals of 21 carbons, including 6 non-protonated carbons at δC 118.3, 122.8, 133.9, 138.9, 164.1, and 166.2; 10 methines at δC 71.8, 75.2, 77.9, 78.2, 102.8, 116.7, 123.6, 124.1, 127.8, and 131.5; 3 methylenes at δC 23.9, 62.9, and 68.2; and 2 methyl carbons at δC 21.9 and 62.8. The signals in the 13C NMR at δC 62.9, 71.8, 75.2, 77.9, 78.2, and 102.8, and the multiplicity of anomeric protons in the 1H NMR at δH 4.33 (1H, d, J = 7.6 Hz), were attributed to a β-glucopyranosyl; the remaining signals were attributed to an isoprene quinoline alkaloid.12 In addition, 2D-NMR experiments completed the structure elucidation (Figure 1). The HMBC from the methoxy group (δH 3.95) to C-4 (δC 164.1) proved the position of the methoxy group at C-4. The HMBCs from H-10 (δH 3.44) to C-2 (δC 166.2)/C-3 (δC 122.8)/C-4 (δC 164.1)/C-11 (δC 127.8)/C-12 (δC 133.9) and from H-14 (δH 1.78) to C-11 (δC 127.8)/C-12 (δC 133.9)/C-13 (δC 68.2) suggested the positions of the isoprenyl group at C-3 and the double bond at C-11/C-12. Acid hydrolysis of 3 revealed D-glucose (identified by comparison with standard D-glucose). Moreover, the sugar moiety was located at C-13 by HMBCs from H-1′ (δH 4.33) to C-13 (δC 68.2) (Figure 2). The Z configuration of the double bond at C-11/C-12 was confirmed by comparing the 13C NMR chemical shift of the methyl group at this double bond (δC 21.9) with those of a compound having a Z configuration, 7,8-secoeranthin β-d-glucopyranoside (δC 13.1),13 and one having an E configuration, hydroxyostholeepoxide (δC 21.8).14 Consequently, the structure of 3 was elucidated and named zanthonitiside I.
Chemical structures of alkaloids 1–3.
Key heteronuclear multiple bond correlations of compounds 1–3.
Compounds 1–3 were evaluated for cytotoxic activity against 5 human cancer cell lines, KB, MCF-7, LNCaP, HepG-2, and LU-1. Significant cytotoxic activity was shown against all 5 with IC50 values ranging from 10.3 to 12.6 µM, compared with those of the positive control, ellipticine (IC50 ranging from 1.3 to 1.7 µM). Compounds 1 and 3 did not show any cytotoxic activity (IC50 > 100 µM) (Table 1). Thus, alkaloid 2 may become a target molecule for the treatment of cancers.
Effects of 1–3 on the Growth of Human Cancer Cells.
Compounds
IC50 (µM)
KB
MCF-7
LNCaP
HepG2
LU-1
1
>100
>100
>100
>100
>100
2
11.1 ± 2.7
12.6 ± 2.4
11.7 ± 0.6
10.3 ± 0.3
10.7 ± 0.4
3
>100
>100
>100
>100
>100
Ellipticine (positive control)
1.7 ± 0.1
1.5 ± 0.2
1.5 ± 0.2
1.4 ± 0.2
1.3 ± 0.2
Experimental
General
Optical rotations were determined on a Jasco DIP-370 automatic polarimeter. The NMR spectra of compounds 1 and 2 were recorded using a Bruker DRX 500 spectrometer; NMR spectra of compound 3 were recorded using a 400-MR spectrometer. The HR-ESI-MS were obtained using an Agilent 6550 iFunnel QTOF LC/MS system. Preparative HPLC was carried out using an AGILENT 1200 HPLC system. Column chromatography was performed on either silica-gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) or RP-18 resins (30-50 μm, Fuji Silysia Chemical Ltd.). For thin layer chromatography (TLC), pre-coated silica-gel 60 F254 (0.25 mm, Merck) and RP-18 F254S (0.25 mm, Merck) plates were used.
Plant Materials
The stems and twigs of Z. nitidum were collected at Nahang, Tuyen Quang, Vietnam in February 2016, and identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (XT-01/NaHang) was deposited at the Institute of Natural Products Chemistry, VAST.
Extraction and Isolation
The dried powdered stems and twigs of Z. nitidum (5.0 kg) were extracted with hot methanol 3 times (each 10 L, 2 days) at 50°C and then the solvent was removed in vacuo to yield the methanol extract (250.0 g). This was suspended in H2O (1.5 L) and successively partitioned with n-hexane, chloroform, and ethyl acetate (EtOAc) to yield n-hexane (ZN1, 80.0 g), chloroform (ZN2, 65.0 g), and EtOAc (ZN3, 26.0 g) residues, and a water layer (ZN4). ZN1 was chromatographed on a silica gel column and eluted with a solvent system of n-hexane:EtOAc (100:0, 70:1, 50:1, 30:1, 10:1, and 1:1, v/v) to give 6 fractions, ZN1A-ZN1F. ZN1C was repeatedly chromatographed on a silica gel column, eluting with n-hexane:EtOAc (10:1, v/v) to give 5 smaller fractions, ZN1C1-ZN1C5. Compound 1 (10 mg) was obtained from ZN1C3 fraction by using a RP-18 column and eluting with acetone:water (4:1, v/v). ZN1C5 was chromatographed on a silica gel column with dichloromethane/methanol (20:1, v/v), and then a RP-18 column eluting with methanol: water (4: 1, v/v) to yield compound 2 (7 mg). ZN1E was loaded onto a silica gel column and eluted with dichloromethane: methanol (10:1, v/v) to give 3 fractions, ZN1E1-ZN1E3. Compound 3 (4.0 mg) was obtained from ZN1E2 using an HPLC column (J’sphere, ODS H-80, 250 × 20 mm) with a flow rate of 3 mL/min eluting with 25% acetonitrile.
HR-ESI-MS m/z: 422.1776 [M + H]+ (calcd. for [C21H28O8N]+, 422.1809).
Acid Hydrolysis
A solution of compound 3 (2 mg) in 3% HCl (4 mL, each) was heated under reflux for 4 hours. Then, the solution was evaporated under reduced pressure and the residue subjected to TLC analysis. d-Glucose was identified by comparing with the standard D-glucose on TLC [Silica gel, developed with CHCl3:MeOH:H2O (6:4:1, v/v/v)].
Cytotoxic Assays
The KB (human oral carcinoma), MCF-7 (human breast carcinoma), LNCaP (human prostate cancer), HepG-2 (human hepatocellular carcinoma), and LU-1 (human lung carcinoma) cell lines were obtained from Milan University, Italy and Hawaii University, USA. Effects of compounds 1–3 on the growth of human cancer cells were determined by measuring the cytotoxic activity using the sulforhodamine B (SRB) assay. The cancer cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C in a humidified 5% CO2 atmosphere. Exponentially growing cells were used throughout the experiments. The SRB assays were performed as follows: human cancer cells (1 × 105 cells/mL) were treated for 2 days with 1, 10, 20, 50, and 100 µM of each compound and ellipticine. All the experiments were performed 3 times. The inhibitory concentration of 50% (IC50) was determined for each compound.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Authors' Note
Thi Hong Van Nguyen, Thi Tuyen Tran, Thi Inh Cam, Minh Quan Pham, and Quoc Long Pham are also affiliated with Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research is funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.01-2015.44.
Supplemental Material
References
1.
CuiXG.ZhaoQJ.ChenQLet al. Two new benzophenanthridine alkaloids from Zanthoxylum nitidum. Helv Chim Acta. 2008;91(1):155–158.doi:10.1002/hlca.200890006
2.
HuJ.ZhangWD.LiuRHet al. Benzophenanthridine alkaloids from Zanthoxylum nitidum (ROXB.) DC, and their analgesic and anti-inflammatory activities. Chem Biodivers. 2006;3(9):990–995.doi:10.1002/cbdv.200690108
3.
YangCH.ChengMJ.ChiangMY.KuoYH.WangCJ.ChenIS. Dihydrobenzo[c]phenanthridine alkaloids from stem bark of Zanthoxylum nitidum. J Nat Prod. 2008;71(4):669–673.doi:10.1021/np700745f
4.
YangCH.ChengMJ.LeeSJ.YangCW.ChangHS.ChenIS. Secondary metabolites and cytotoxic activities from the stem bark of Zanthoxylum nitidum. Chem Biodivers. 2009;6(6):846–857.doi:10.1002/cbdv.200800107
5.
IshikawaT.SekiM.NishigayaKet al. Studies on the chemical constituents of Zanthoxylum nitidum (Roxb.) D. C. (Fagara nitida Roxb.). III. The chemical constituents of the wood. Chem Pharm Bull. 1995;43(11):2014–2018.doi:10.1248/cpb.43.2014
6.
ZuoGY.WangCJ.HanJ.LiYQ.WangGC. Synergism of coumarins from the Chinese drug Zanthoxylum nitidum with antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA). Phytomedicine. 2016;23(14):1814–1820.doi:10.1016/j.phymed.2016.11.001
7.
YangG.ChenD. Alkaloids from the roots of Zanthoxylum nitidum and their antiviral and antifungal effects. Chem Biodivers. 2008;5(9):1718–1722.doi:10.1002/cbdv.200890160
8.
ChenJJ.LinYH.DaySH.HwangTL.ChenIS. New benzenoids and anti-inflammatory constituents from Zanthoxylum nitidum. Food Chem. 2010;125(2):282–287.doi:10.1016/j.foodchem.2010.09.069
9.
WangZ.JiangW.ZhangZ.QianM.DuB. Nitidine chloride inhibits LPS-induced inflammatory cytokines production via MAPK and NF-kappaB pathway in RAW 264.7 cells. J Ethnopharmacol. 2012;144(1):145–150.doi:10.1016/j.jep.2012.08.041
10.
WangCF.FanL.TianMet al. Cytotoxicity of benzophenanthridine alkaloids from the roots of Zanthoxylum nitidum (Roxb.) DC. var. fastuosum How ex Huang. Nat Prod Res. 2015;29(14):1380–1383.doi:10.1080/14786419.2014.1002090
LiuJ.LiCJ.NiLet al. Anti-inflammatory alkaloid glycoside and quinoline alkaloid derivates from the stems of Clausena lansium. RSC Adv. 2015;5(98):80553–80560.doi:10.1039/C5RA14173G
ZhaoJ.ZhouM.LiuY.ZhangG.LuoY. Chromones and coumarins from the dried fructus of Cnidium monnieri. Fitoterapia. 2011;82(5):767–771.doi:10.1016/j.fitote.2011.03.008
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
