Carbazole Alkaloids with Potential Neuroprotective Activities from the

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Carbazole Alkaloids with Potential Neuroprotective Activities from the Fruits of Clausena lansium Yan-Ping Liu,†,‡,§ Jia-Ming Guo,†,§ Yun-Yao Liu,‡ Shi Hu,†,§ Gui Yan,†,§ Lei Qiang,‡ and Yan-Hui Fu*,†,§ †

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Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, P. R. China ‡ State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China § Key Laboratory of Tropical Medicinal Plant Chemistry of Hainan Province, Hainan Normal University, Haikou 571158, P. R. China S Supporting Information *

ABSTRACT: Clausena lansium, also known as wampee, is a species of strongly scented evergreen trees belonging to the genus Clausena (Rutaceae), which is native to southern China. Its ripe fruits have been consumed as a very popular fruit and reported to possess a range of biological activities. To study the potential health-promoting constituents from the fruits of C. lansium, a chemical investigation on its fruits was thus carried out. In this study, 16 carbazole alkaloids (1−16), including six new carbazole alkaloids, clausenalansines A−F (1−6), were separated from the fruits of C. lansium. The molecular structures of these isolated new carbazole alkaloids (1−6) were ambiguously established on the basis of comprehensive spectroscopic methods. The known analogues (7−16) were determined via comparing their experimental data with those described in the literature, which were separated from C. lansium for the first time. All these isolated alkaloids were tested in vitro for their neuroprotective effects against 6-hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells. Carbazole alkaloids 1−16 displayed remarkable neuroprotective effects possessing the EC50 values ranging from 0.36 ± 0.02 to 10.69 ± 0.15 μM. These findings indicate that regular consumption of the fruits of C. lansium may help people prevent the occurrence of Parkinson’s disease. In addition, the separation and identification of these carbazole alkaloids possessing remarkable neuroprotective effects from the fruits of C. lansium could be extremely important to the discovery of new agents for the prevention and treatment of Parkinson’s disease. KEYWORDS: Clausena lansium, carbazole alkaloids, clausenalansines A−F, neuroprotective effect

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INTRODUCTION Parkinson’s disease is the second most common neurodegenerative disorder that affects people mainly in the later years of life and is usually characterized by the presence of Lewy bodies and the selective degeneration of nigrostriatal dopaminergic neurons, resulting in a clinical syndrome characterized by tremors, stiffness, slowness of movement, and postural instability.1 Treatment using levodopa is currently the most effective therapeutic option for Parkinson’s disease; however, it is only effective for symptomatic relief in the later stages of this disease. Therefore, there is a particularly urgent medicinal need for a new therapy for Parkinson’s disease. Though it is not completely understood about the mechanisms accountable for the death of the dopaminergic cell, increasing evidence obtained from human post-mortem studies and animal investigations has revealed that oxidative stress plays an important role in initiating this process of Parkinson’s disease.2−5 Thus, the inhibition of oxidative stress could be a feasible treatment method for Parkinson’s disease. At present, a lot of experimental models for Parkinson’s disease have been developed to assist in the evaluation of neuroprotective agents for this disease. Most of these experimental models are usually established using the experimental neurotoxins, such as 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6© XXXX American Chemical Society

hydroxydopamine (6-OHDA). Among these neurotoxins, 6OHDA is a hydroxylated analogue of dopamine commonly applied as a dopaminergic neuron degenerative agent to study neurotoxicity in the experimental models for Parkinson’s disease that can selectively damage dopaminergic neurons in vivo and in vitro and can cause oxidative stress.6−8 Therefore, 6-OHDA is a effective useful tool in establishing the experimental models for Parkinson’s disease and used for investigating the effectiveness and mechanism of action of potential therapeutic agents for Parkinson’s disease. The genus Clausena (Rutaceae) is comprised of approximately 30 species that are scattered throughout the subtropical and tropical regions. There are approximately 10 species as well as 2 varieties in China, appearing in Southern China.9 Most of them are famous for their fruits, which are usually very popular tropical, health-promoting fruits, while their roots, stems, leaves, and seeds have also been extensively applied in folk medicine or traditional Chinese medicine for the treatments of abdominal pain, malaria, cold, dermatopathy, Received: February 10, 2019 Revised: April 12, 2019 Accepted: May 7, 2019

A

DOI: 10.1021/acs.jafc.9b00961 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. 1H and

13

C-NMR Data of Clausenalansines A−C (1−3) clausenalansine A (1)

position 1 2 3 4 5 6 7 8 1a 4a 5a 8a 1′ 2′ 3′ 4′ 5′ 1-OMe 1-OH 2-OH 3-Me 3-CHO 5-OMe 6-CHO NH

δH a

8.20 (1H, s) 7.40 (1H, d, 2.4) 6.87 (1H, dd, 8.6, 2.4) 7.27 (1H, d, 8.6)

6.92 (1H, d, 10.0) 5.90 (1H, d, 10.0) 1.49 (3H, s) 1.49 (3H, s)

clausenalansine B (2) δC b 103.9 s 153.6 s 116.8 s 119.1 d 105.3 d 151.7 s 114.8 d 111.6 d 141.1 s 117.6 s 124.0 s 134.5 s 116.9 d 129.7 d 77.0 s 27.2 q 27.2 q

δH c

7.59 7.96 7.22 7.35 7.41

(1H, (1H, (1H, (1H, (1H,

s) d, 7.8) dd, 7.8, 7.6) dd, 8.0, 7.6) d, 8.0)

4.01 (3H, s)

clausenalansine C (3) δC d 131.2 145.2 117.8 116.9 119.5 119.6 124.5 110.6 131.0 117.9 124.0 139.6

δHa s s s d d d d d s s s s

7.26 (1H, d, 1.8) 8.23 (1H, d, 1.8)

8.50 (1H, d, 8.6) 7.12 (1H, d, 8.6)

δC b 143.2 109.7 131.0 115.9 162.1 108.6 129.5 104.8 133.1 123.5 118.0 138.7

s d s d s s d d s s s s

60.7 q 10.37 (1H, s)

8.04 (1H, s) 2.45 (3H, s) 10.33 (1H, s)

16.1 q

187.9 d

9.97 (1H, s) 4.03 (3H, s) 10.55 (1H, s) 11.55 (1H, s)

192.0 d 56.7 q 189.5 d

a

Measured in DMSO-d6 at 400 MHz. bMeasured in DMSO-d6 at 100 MHz. cMeasured in CDCl3 at 400 MHz. dMeasured in CDCl3 at 100 MHz.

and snake-bites.10 Among them, Clausena lansium, also known as wampee, is a species of strongly scented evergreen trees belonging to the genus Clausena of the family Rutaceae that is native to southern China. Its ripe fruits are consumed as a very popular tropical fruit and reported to possess a range of biological activities. C. lansium is very popular in China, Vietnam, Indonesia, Malaysia, and the Philippines, less frequently grown in India, Queensland, and Sri Lanka, and occasionally cultivated even in Hawaii and Florida not only for the nutritional value and pharmacological uses of its fruits but also as a medicinal and ornamental plant.10 In China, the fruits of C. lansium can be eaten raw or be salted or candied into a cool fruit which has the effects of improving digestion, smoothness, and heat removal, whereas its roots, stems, leaves, and seeds have been applied in folk medicine or as materials for Chinese medicine. Previous chemical investigations on this plant caused the separation and identification of a series of natural products, containing sesquiterpenes, alkaloids, glycosides, and coumarins, which displayed a wide variety of biological activities, such as neuroprotective, antitumor, hepatoprotective, anti-inflammatory, antifungal, antiobesity, nematicidal, antimicrobial, and hypoglycemic activities.11−23 Our preliminary findings revealed that the 90% ethanol extract of the fruits of C. lansium displayed pronounced neuroprotective effect against 6-hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells with an EC50 value of 8.08 ± 0.10 μg/mL in vitro. To study the potential healthpromoting constituents from the fruits of C. lansium, as a part of our ongoing research into the natural products possessing structural and biological diversity from the tropical fruits and medicinal plants in China,24−28 a systematic phytochemical study on the fruits of C. lansium was accordingly carried out.

The investigation resulted in the separation and characterization of six new carbazole alkaloids, clausenalansines A−F (1−6), together with 10 known carbazole alkaloids, clauraila E (7),29 clauszoline G (8),30 7-methoxymurrayacine (9),31 murrastinine B (10),32 murrayamine A (11),33 clauraila B (12),34 clauszoline N (13),35 clausine Q (14),36 clauraila A (15),34 and N-methoxy-3-formylcarbazole (16).37 The molecular structures of new carbazole alkaloids (1−6) were ambiguously established using comprehensive spectroscopic studies. The known analogues (7−16) were determined via comparing their experimental data with those described in the literature, which were separated from C. lansium for the first time. In addition, all these isolated carbazole alkaloids were examined in vitro for their neuroprotective effects against 6hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells. All isolated carbazole alkaloids (1−16) displayed remarkable neuroprotective effects, holding the EC50 values ranging from 0.36 ± 0.02 to 10.69 ± 0.15 μM. In this paper, we report the separation, structural elucidation, as well as neuroprotective effects of these carbazole alkaloids from the fruits of C. lansium.

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MATERIALS AND METHODS

General Experimental Procedures. A Beckman DU 640 spectrophotometer was applied to acquire the ultraviolet (UV) spectra of new carbazole alkaloids 1−6. A Nicolet 6700 spectrophotometer was used to obtain the infrared (IR) spectra of new carbazole alkaloids 1−6. A Bruker 400 MHz spectrometer was applied to acquire the NMR spectra of carbazole alkaloids 1−16. A quadrupoletime-of-flight (Q-TOF) Ultima Global GAA076 LC mass spectrometer was applied to record the high-resolution-electrospray ionizationmass spectrometry (HR-ESI-MS) spectra of carbazole alkaloids 1−16. A Dionex UltiMate 3000 LC series with a multiwavelength detector B

DOI: 10.1021/acs.jafc.9b00961 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of compounds 1−16 isolated from the fruits of C. lansium. i.d. 5 μm, 10 mm × 250 mm, 50% MeOH, 3.0 mL/min, tR 23.7, 32.9, and 36.6 min), respectively. Compounds 3 (11.5 mg), 4 (31.9 mg), 10 (22.8 mg), 12 (64.3 mg), and 16 (108.7 mg) were separated from Fr. 2D (5.7 g) via purification by Sephadex LH-20 gel CC and then prepared using semipreparative HPLC (Waters XBridge C18 column, 5 μm, i.d. 10 mm × 250 mm, 55% MeCN, 3.0 mL/min, tR 28.2, 32.7, 38.9, 45.8, and 52.5 min), respectively. Compounds 5 (31.5 mg), 6 (26.8 mg), 9 (45.2 mg), 11 (63.0 mg), and 14 (8.9 mg) were obtained from Fr. 2E (6.2 g) via purification by Sephadex LH-20 gel CC and then prepared using semipreparative HPLC (Waters XBridge C18 column, 5 μm, i.d. 10 mm × 250 mm, 70% MeOH, 3.0 mL/min, tR 18.5, 24.9, 29.7, 37.2, and 45.8 min), respectively. Clausenalansine A (1). White amorphous powder; UV (MeOH) λmax (log ε) 239 (4.72), 286 (4.26), 306 (3.82) and 343 (3.48) nm; IR (KBr) vmax 3368, 2969, 2847, 2732, 1728, 1618, 1509, 1468, 1383, 1239, 1168, 1073, and 802 cm−1; ESI-MS m/z 294 [M + H]+; HRESI-MS m/z 294.1128 [M + H]+ (calcd for C18H16NO3, 294.1125); 1 H and 13C NMR data (as shown in Table 1). Clausenalansine B (2). White amorphous powder; UV (MeOH) λmax (log ε) 228 (4.52), 278 (4.11), 286 (3.86) and 341 (3.53) nm; IR (KBr) vmax 3372, 2973, 1620, 1511, 1472, 1378, 1241, 1170, 1068, and 796 cm−1; ESI-MS m/z 228 [M + H]+; HR-ESI-MS m/z 228.1020 [M + H]+ (calcd for C14H14NO2, 228.1019); 1H and 13C NMR data (as shown in Table 1). Clausenalansine C (3). White amorphous powder; UV (MeOH) λmax (log ε) 237 (4.69), 287 (4.23), 298 (4.02) and 338 (3.62) nm; IR (KBr) vmax 3379, 2973, 2851, 2730, 1731, 1616, 1508, 1472, 1380, 1242, 1171, 1072, and 806 cm−1; ESI-MS m/z 270 [M + H]+; HRESI-MS m/z 270.0766 [M + H]+ (calcd for C15H12NO4, 270.0761); 1 H and 13C NMR data (as shown in Table 1). Clausenalansine D (4). White amorphous powder; UV (MeOH) λmax (log ε) 241 (4.72), 288 (4.16), 296 (3.88), and 337 (3.48) nm; IR (KBr) vmax 3402, 2973, 2846, 2727, 1726, 1612, 1503, 1466, 1383, 1239, 1168, 1059, and 789 cm−1; ESI-MS m/z 256 [M + H]+; HRESI-MS m/z 256.0971 [M + H]+ (calcd for C15H14NO3, 256.0968); 1 H and 13C NMR data (as shown in Table 1).

(MWD) was applied to perform semipreparative high-pressure liquid chromatography (HPLC), using an Waters XBridge C18 column (5 μm, 10 mm × 250 mm). Silica gel (200−300 mesh) was the product of Qing Dao Hai Yang Chemical Group Co. and was used for open column chromatography (CC). Precoated silica gel plates (G60, F254) were the product of Yan Tai Zi Fu Chemical Group Co. and were used for thin-layer chromatography. RP-18 gel (50 μm) was the product of YMC and was used for medium-pressure CC. Plant Material. The fresh ripe fruits of C. lansium were collected from Haikou City, Hainan Province, China, on August 1, 2017 and identified by Prof. Yan-Hui Fu, Key Laboratory of Southern Medicinal Plants Resources of Haikou City, Hainan Normal University. A voucher specimen (no. CLLA20170801) has been deposited at the Key Laboratory of Southern Medicinal Plants Resources of Haikou City, Hainan Normal University. Extraction and Isolation. The fresh ripe fruits of C. lansium (48.8 kg) were chopped using a multifunction food processor and extracted six times using 50 L of 90% ethanol at 25−38 °C, each time for 1 week. The extraction solvent was merged and concentrated under reduced pressure to afford a crude extract (4678.9 g). The obtained crude extract was then suspended in 15 L of distilled water and partitioned using petroleum ether (PE) five times (each time for 15.0 L) and EtOAc five times (each time for 15.0 L) to afford a PE extract and an EtOAc extract. The EtOAc extract (898.6 g) was chromatographed using silica gel CC and eluted by CHCl3/MeOH (95:5 to 50:50, v/v) to yield seven fractions (Fr. 1−Fr. 7). Fr. 2 (68.9 g) was further subjected to RP-18 gel medium-pressure CC eluted using MeOH/H2O (40:60 to 100:0 v/v) to afford eight fractions (Fr. 2A− Fr. 2H). Compounds 1 (23.7 mg), 7 (12.9 mg), as well as 13 (68.9 mg) were isolated from Fr. 2B (4.2 g) via being purified by Sephadex LH-20 gel CC and then prepared by semipreparative HPLC (Waters XBridge C18 column, i.d. 5 μm, 10 mm × 250 mm, 45% MeCN, 3.0 mL/min, tR 18.6, 35.7, and 40.9 min), respectively. Compounds 2 (12.2 mg), 8 (42.5 mg), and 15 (54.7 mg) were separated from Fr. 3C (2.8 g) via purification by Sephadex LH-20 gel CC and then prepared using semipreparative HPLC (Waters XBridge C18 column, C

DOI: 10.1021/acs.jafc.9b00961 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Selected 2D NMR correlations for clausenalansines A−C (1−3). Clausenalansine E (5). White amorphous powder; UV (MeOH) λmax (log ε) 238 (4.82), 286 (4.21), 299 (3.92), and 343 (3.52) nm; IR (KBr) vmax 3396, 2968, 1609, 1497, 1469, 1378, 1236, 1170, 1062, and 793 cm−1; ESI-MS m/z 228 [M + H]+; HR-ESI-MS m/z 228.1022 [M + H]+ (calcd for C14H14NO2, 228.1019); 1H and 13C NMR data (as shown in Table 1). Clausenalansine F (6). White amorphous powder; UV (MeOH) λmax (log ε) 237 (4.72), 286 (4.12), 296 (3.75) and 340 (3.39) nm; IR (KBr) vmax 2976, 1612, 1506, 1472, 1382, 1238, 1167, 1068, and 796 cm−1; ESI-MS m/z 242 [M + H]+; HR-ESI-MS m/z 242.1178 [M + H]+ (calcd for C15H16NO2, 242.1176); 1H and 13C NMR data (as shown in Table 1). Neuroprotective Activities Bioassays. In this bioassay, the human neuroblastoma SH-SY5Y cell line was used and acquired from the American Type Culture Collection. The cells were routinely cultured in complete Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 μg/mL streptomycin, and 100 U/mL penicillin and maintained at 37 °C with 5% CO2 in a humidified atmosphere. The neuroprotective activity assay was carried out in 96-well microplates using the MTT method. Briefly, the SH-SY5Y cells were cultured at a density of 2 × 104 cells/ well in 200 μL for 24 h in 96-well plates. The cells were treated using the positive control, 6-hydroxydopamine, at a concentration of 100 μM and were treated by the PE extract and the EtOAc extract of the fruits of C. lansium as well as carbazole alkaloids of various concentrations (0.0625, 0.32, 1.6, 8.0, and 40.0 μM) for an additional 24 h. Cell viability was assessed by treatment with MTT dissolved in 0.5 mg/mL of phosphate-buffered saline (PBS) at 37 °C for 4 h. Then, the PBS was carefully removed, and formazan crystals were dissolved in dimethyl sulfoxide. The absorbance of this solution was then measured at 540 nm by a microplate reader. The neuroprotective effect against 6-OHDA induced cell death was calculated via a semilogarithmic graph depicting the relationship between at least four different concentrations of carbazole alkaloids and their percentage effects of these concentrations on cell viability. All the samples were tested in triplicate, and the results were reported as the mean with standard deviation. All results were typically expressed as the EC50, and curcumin was used as a positive control.

Sephadex LH-20 CC, and semipreparative HPLC to afford six new carbazole alkaloids (1−6), together with 12 known analogues (7−16). The molecular structures of all these isolated carbazole alkaloids were as shown in Figure 1. Clausenalansine A (1) was isolated as a white amorphous powder. The molecular formula of 1 was identified as C18H15NO3 on the basis of its HR-ESI-MS with m/z 294.1128 [M + H]+ (calcd for C18H16NO3, 294.1125), requiring 12 degrees of unsaturation. Its IR spectrum exhibited the absorption bands at 3368, 2969, 2847, 2732, 1728, 1618, 1509, and 1468 cm−1 evidencing the presence of a hydroxy group, aldehyde group, double bond, and benzene ring. The UV absorptions in the UV spectrum of 1 at 236, 286, 297, and 338 nm were characteristic of carbazole alkaloids.31−35 Its 13C NMR data combined with distortionless enhancement by polarization transfer (DEPT) data (as shown in Table 1) suggested the presence of 18 carbons, which could be classified into two methyls, 15 sp2 carbons, along with one sp3 quaternary carbon. Furthermore, the 15 sp2 carbons were assigned to one carbazole ring, one aldehyde group, and one double bond which accounted for 11 out of 12 indices of hydrogen deficiency. Accordingly, the remaining one degree of unsaturation was due to one ring system in 1. The above data suggested that 1 displayed similar structural features to that of claulansine F,38 except that the methoxy group located at C-6 in claulansine F was replaced with a hydroxy group in 1, which was confirmed by the absence of a methoxy group at C-6 in claulansine F together with the heteronuclear multiple bond correlation (HMBC) correlations of H-5 to C-4a (δC 117.6), C-7 (δC 114.8), and C-8a (δC 134.5), H-8 to C-5a (δC 124.0) and C-6 (δC 151.7), also confirmed on the basis of the rotating-frame Overhauser spectroscopy (ROESY) correlation of H-4 with H-5. The chemical structure of 1 was further verified based on the detailed analyses of the heteronuclear single quantum coherence (HSQC), HMBC, 1 H− 1 H correlation spectroscopy (COSY) and ROESY spectra, as depicted in Figure 2. Accordingly, the chemical structure of 1 was elucidated as depicted in Figure 1. Clausenalansine B (2), a white amorphous powder, possessed a molecular formula of C14H13NO2 determined based on its HR-ESI-MS (m/z 228.1020, [M + H]+; calcd 228.1019), indicating nine indices of hydrogen deficiency. The IR spectrum of 2 revealed the presence of a phenyl group (1620, 1511, and 1472 cm−1) and hydroxy groups (3372 cm−1). The UV absorptions in the UV spectrum of 1 at 228,

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RESULTS AND DISCUSSION Phytochemical Investigation. The fresh ripe fruits of C. lansium were collected, chopped, and extracted with 90% ethanol. The extraction solvent was merged and concentrated under reduced pressure to afford a crude extract. The obtained crude extract was further suspended in distilled water and then partitioned successively using PE and EtOAc to yield the PE extract as well as the EtOAc extract, respectively. The EtOAc fraction was isolated and purified using silica gel, RP-18 gel, D

DOI: 10.1021/acs.jafc.9b00961 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 2. 1H and

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C-NMR Data of Clausenalansines D−F (4−6) clausenalansine D (4)

position 1 2 3 4 5 6 7 8 1a 4a 5a 8a 3-Me 3-CH2OH 3-CH2OMe 3-CH2OMe 5-OMe 6-CHO NH NOMe

δHa 7.12 (1H, s)

7.68 (1H, s)

8.14 (1H, d, 8.6) 6.86 (1H, d, 8.6)

2.23 (3H, s)

clausenalansine E (5) δC b

97.8 d 154.3 s 117.6 s 120.5 d 160.1 s 108.2 s 127.0 d 102.1 d 140.6 s 113.8 s 118.5 s 138.3 s 16.5 q

δH a 7.54 (1H, overlap) 7.53 (1H, overlap) 8.14 8.14 7.25 7.48 7.54

(1H, (1H, (1H, (1H, (1H,

overlap) overlap) dd, 7.8, 7.6) dd, 8.0, 7.6) overlap)

4.73 (2H, s)

3.95 (3H, s) 10.52 (1H, s) 11.36 (1H, s)

clausenalansine F (6) δC b 108.2 125.8 134.8 118.9 120.2 120.6 126.3 108.6 136.7 119.5 119.3 137.8

δH c d d s d d d d d s s s s

7.50 (1H, overlap) 7.50 (1H, overlap) 8.06 8.06 7.27 7.50 7.50

(1H, (1H, (1H, (1H, (1H,

overlap) overlap) dd, 7.8, 7.6) overlap) overlap)

δC d 108.1 126.1 129.9 120.0 120.2 120.4 126.4 108.3 137.2 119.9 119.8 137.9

d d s d d d d d s s s s

63.5 t 4.63 (2H, s) 3.45 (3H, s)

75.1 t 57.7 q

4.10 (3H, s)

63.3 q

56.4 q 189.1 d 4.08 (3H, s)

63.6 q

a

Measured in DMSO-d6 at 400 MHz. bMeasured in DMSO-d6 at 100 MHz. cMeasured in CDCl3 at 400 MHz. dMeasured in CDCl3 at 100 MHz.

aldehyde hydrogen resonating at δH 10.55 (1H, s) to C-5, C-6 (δC 108.6) and C-7 (δC 129.5), along with the ROESY correlations of the aldehyde hydrogen resonating at δH 10.55 (1H, s) with the methoxy hydrogens resonating at δH 4.03 (3H, s) and H-7. The structure of 3 was further verified on the basis of detailed analyses of the HSQC, HMBC, 1H−1H COSY, and ROESY spectra, as depicted in Figure 2. Thence, the chemical structure of 3 was elucidated as depicted in Figure 1. The molecular formula of clausenalansine D (4) was determined as C15H13NO3 based on its HR-ESI-MS (m/z 256.0971, [M + H]+; calcd 256.0968). The molecular weight of 4 is smaller than that of 3 by 14 mass units. Its 1H combined with 13C NMR data (as shown in Table 2) indicated that 4 held 15 carbons which displayed features resembling those of 3. Detailed comparisons of 1H and 13C NMR combined with DEPT data of 4 with 3 revealed three major differences between their chemical structures. First of all, the hydroxy group located at C-1 in 3 was replaced with a hydrogen atom in 3, which was confirmed on the basis of the presence of the hydrogen (H-1) resonating at δH 7.12 (1H, s), the HMBC correlation of H-1 to C-2 (δC 154.3), C-3 (δC 117.6), C-1a (δC 140.6), and C-4a (δC 113.8), along with the ROESY correlation of H-1 with the NH resonating at δH 11.36. Second, the hydrogen atom located at C-2 in 3 was replaced with one hydroxy group in 4, which was verified on the basis of the HMBC correlations of H-4 and the methoxy hydrogens resonating at δH 2.23 (3H, s) to C-2. Third, the aldehyde group located at C-3 in 3 was replaced with a methyl group resonating at δH 2.23 (3H, s) and δC 16.5 in 4, which was further verified based on the HMBC correlations of the methyl hydrogens resonating at δH 2.23 (3H, s) to C-2, C-3, and C-4 (δC 120.5), H-4 to the methoxy carbon resonating at δC 16.5, along with the ROESY correlation of the methyl hydrogens resonating at δH 2.23 (3H, s) with H-4. The chemical structure of 4 was further verified on the basis of detailed analyses of the HSQC, HMBC, 1H−1H COSY, and ROESY spectra, as

278, 286, and 341 nm were characteristic of a carbazole alkaloid.31−35 Its 13C NMR data combined with DEPT data (as shown in Table 1) suggested the presence of 14 carbons composed of two methyls as well as 12 sp2 carbons. Furthermore, the nine indices of hydrogen deficiency and 12 sp2 carbons were assigned to one carbazole ring. The above data suggested that 2 possessed a similar chemical structure with claulansine N.39 Careful comparison of the NMR spectroscopic data of 2 (as shown in Table 1) with those of claulansine N indicated that they both possessed the same structural skeleton, except that the molecular weight of 2 is smaller than that of claulansine N by 14 units, namely, the aldehyde group located at C-3 in claulansine N was replaced with a methyl group resonating at δH 2.45 (3H, s) and δC 16.1 in 2. The above elucidation was verified by the HMBC correlations of the methyl hydrogens resonating at δH 2.45 (3H, s) to C-2 (δC 145.2), C-3 (δC 117.8) and C-4 (δC 116.9), H-4 to the methyl carbon resonating at δC 16.1, along with the ROESY correlations of the methyl hydrogens resonating at δH 2.45 (3H, s) with 2-OH and H-4. The chemical structure of 2 was further confirmed on the basis of the detailed analyses of the HSQC, HMBC, 1H−1H COSY, and ROESY spectra, as depicted in Figure 2. Accordingly, the chemical structure of 2 was elucidated as depicted in Figure 1. Clausenalansine C (3), a white amorphous powder, had a molecular formula of C15H11NO4 established on the basis of its HR-ESI-MS (m/z 270.0766, [M + H]+; calcd 270.0761), indicative of 11 degrees of unsaturation. Its 1H and 13C NMR data (as shown in Table 1) revealed that 3 held 15 carbons displaying structural features resembling 3-formyl-1-hydroxycarbazole,40 except that the hydrogen atoms located at C-5 and C-6 in 3-formyl-1-hydroxycarbazole were substituted by a methoxy group resonating at δH 4.03 (3H, s) and δC 56.7, together with an aldehyde group resonating at δH 10.55 (1H, s) and δC 189.5 in 3, respectively. The above elucidation was confirmed based on the HMBC correlations of the methoxy hydrogens resonating at δH 4.03 (3H, s) to C-5 (δC 162.1), the E

DOI: 10.1021/acs.jafc.9b00961 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 3. Selected 2D NMR correlations for clausenalansines D−F (4−6).

resonating at δH 4.63 (2H, s) and δH 3.45 (3H, s) with H-2 and H-4. The chemical structure of 6 was further verified on the basis of detailed analyses of the HSQC, HMBC, 1H−1H COSY, and ROESY spectra, as depicted in Figure 3. Thus, the chemical structure of 6 was elucidated as depicted in Figure 1. Neuroprotective Activity. All isolated carbazole alkaloids were assessed for their neuroprotective activities against 6hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells using the MTT method in vitro, with curcumin as a positive control. As a result, all isolated carbazole alkaloids 1−16 displayed pronounced neuroprotective activities against 6-hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells in vitro, holding the EC50 values ranging from 0.36 ± 0.02 to 10.69 ± 0.15 μM (as shown in Table 3), which are comparable to that of curcumin, even

depicted in Figure 3. Thus, the chemical structure of 4 was elucidated as depicted in Figure 1. Clausenalansine E (5) was separated as a white amorphous powder. The molecular formula of 5, C14H13NO2, was determined on the basis of its HR-ESI-MS (m/z 228.1022, [M + H]+; calcd 228.1019), which could also be verified through 1H, 13C NMR combined with DEPT data. Its 1H as well as 13C NMR data (as shown in Table 2) reveled that 5 had 14 carbons which displayed extremely similar features to those of N-methoxy-3-formylcarbazole (16),37 except that the aldehyde group located at C-3 in N-methoxy-3-formylcarbazole (16) was replaced by a hydroxymethyl group in 5, which was confirmed by the presence of the hydroxymethyl group resonating at δH 4.73 (2H, s) and δC 63.5, along with the HMBC correlations of the hydroxymethyl hydrogens resonating at δH 4.73 (2H, s) to C-4 (δC 118.9), C-3 (δC 134.8) and C-2 (δC 125.8), together with the ROESY correlations of the hydroxymethyl hydrogens resonating at δH 4.73 (2H, s) with H-2 and H-4. The chemical structure of 4 was further verified on the basis of detailed analyses of the HSQC, HMBC, 1H−1H COSY, and ROESY spectra, as depicted in Figure 3. Thus, the structure of 5 was established as depicted in Figure 1. Although the structure of 5 is identical to that of the compound named as N-methoxy-3-hydroxymethyl-9H-carbazole proposed in the literature,41 based on the NMR data of the compound named as N-methoxy-3-hydroxymethyl-9H-carbazole described in the literature, we speculate that the chemical structure of the compound named as N-methoxy-3-hydroxymethyl-9H-carbazole in the literature should be identical to that of 3,3′[oxybis(methylene)]bis(9-methoxy-9H-carbazole), a dimeric carbazole alkaloid, in the literature.42 The molecular formula of clausenalansine F (6) was established to be C15H15NO2 using its HR-ESI-MS (m/z 242.1178, [M + H]+; calcd 242.1176). The molecular weight of 6 was larger than that of 5 by 14 mass units. Its 1H and 13C NMR data (as shown in Table 2) revealed that 6 held 15 carbons which exhibited features closely resembling those of 5, except that the hydroxymethyl group located at C-3 in 5 was replaced by a methoxymethyl group in 6, which was certified based on the presence of the methoxy group resonating at δH 3.45 (3H, s) and δC 57.7, together with the HMBC correlation of the methoxy hydrogens resonating at δH 3.45 (3H, s) to the hydroxymethyl carbon resonating at δC 75.1, along with the ROESY correlations of the methoxymethyl hydrogens

Table 3. Neuroprotective Activities of Compounds 1−16 cmpd 1 2 3 4 5 6 7 8 curcuminb

EC50 (μM)a

cmpd

± ± ± ± ± ± ± ± ±

9 10 11 12 13 14 15 16

0.86 0.36 1.12 2.16 0.69 2.58 1.29 2.65 5.82

0.06 0.02 0.15 0.09 0.06 0.05 0.07 0.11 0.12

EC50 (μM)a 3.26 8.62 10.69 5.56 4.68 8.53 6.31 2.73

± ± ± ± ± ± ± ±

0.10 0.16 0.15 0.11 0.12 0.15 0.16 0.07

a

EC50 was defined as the concentration giving half maximal protection against 6-OHDA induced cell death in human neuroblastoma SHSY5Y cells and expressed as the mean ± SD of triplicate determinations. bPositive control.

more remarkable neuroprotective activities than that of curcumin. The neuroprotective effects of all these isolates were found to possess some correlations with their chemical structures. Among these isolated compounds, compounds 1− 9, 12, 13, and 16 displayed more significant neuroprotective effects with the EC50 values below that of curcumin, while the other compounds 10, 11, 14, and 15 showed relatively weaker neuroprotective effects, holding the EC50 values above that of curcumin. It is worth mentioning that compound 2 displayed the most potent neuroprotective effect holding an EC50 value of 0.36 ± 0.02 μM, while the positive control, curcumin, possessed an EC50 value of 5.82 ± 0.12 μM. Compounds 1, 7− F

DOI: 10.1021/acs.jafc.9b00961 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry 9, and 12 showed more remarkable neuroprotective effects than curcumin, while their structural analogues 10 and 11 showed relatively weaker neuroprotective effects than curcumin. Detailed analysis of the structures and the neuroprotective effects of 1 and 7−12 suggested that these compounds with the aldehyde group at C-3 were more likely to possess significant neuroprotective effects. It was noteworthy that compounds 5, 6, and 16 exhibited remarkable neuroprotective effects, while compound 5 showed relatively stronger neuroprotective effect than its structural analogues 6 and 16 by about four times. Detailed analysis of the structures and the neuroprotective effects of 5, 6, and 16 revealed that the hydroxymethyl group at C-3 in the molecular structures of these compounds might play an important role in their neuroprotective effects. In addition, compound 13 possessed a pronounced neuroprotective effect, while their structural analogues 14 and 15 showed the moderate neuroprotective effects. Detailed analysis of the structures and the neuroprotective effects of 13−15 suggested that possessing a hydroxy group at C-6 in the molecular structures of these compounds might be more important in their neuroprotective effects. The structure−activity relationships and molecular mechanisms of action of these carbazole alkaloids with potential neuroprotective activities need to be further investigated. The present findings could provide a basis for further exploration of the fruits of C. lansium as a new source of natural neuroprotective agents. As a very popular fruit tree, C. lansium not only has provided people with their edible and palatable fruits as a popular health-promoting fruit but also provided us with their seeds, leaves, stems, and roots as weapons to fight various diseases. In our current study, systematic phytochemical study on the fruits of C. lansium was performed and resulted in the separation and identification of six new carbazole alkaloids, clausenalansines A−F (1−6) and 10 known carbazole alkaloids (7−16). In addition, the neuroprotective activities of all the isolated compounds against 6-hydroxydopamine induced cell death in human neuroblastoma SH-SY5Y cells were also tested and verified to be extremely significant. These findings could be used as an explanation of the health promoting functions of the fruits of C. lansium, which is usually considered to play an important role in preventing or reducing the occurrence of Parkinson’s disease. These findings also indicated that these isolated carbazole alkaloids possess remarkable neuroprotective effects from the fruits of C. lansium and could be considered as candidates for further research for therapeutic purposes in neural degenerative diseases such as Parkinson’s disease. It is worth noting that many carbazole alkaloids with structural and biological diversity were also previously isolated from C. lansium, which implies that the attraction of their fruits is not only due to their pleasant taste but also to their potential functions in promoting health. Therefore, as a resource of edible fruits and carbazole alkaloids possessing a range of biological activities, the fruits of C. lansium should draw increasingly more attention from medical and food scientists because they are consumed worldwide and have abundant pharmaceutical and nutritional values.

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Original 1D and 2D NMR spectra of clausenalansines A−F (1−6) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 898 6588 9422. E-mail: [email protected]. ORCID

Yan-Hui Fu: 0000-0003-1282-2178 Funding

This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 31660097 and 21662011), the Key Research and Development Project of Haikou (Grant No. 2017050), the Program for Innovative Research Team in University (Grant No. IRT-16R19), and the Special Project of the Central Government for the Development of Local Science and Technology (Grant No. ZY2018HN07). Notes

The authors declare no competing financial interest.

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H

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