A New β -Carboline Alkaloid From the Aerial Part of Hedyotis capitellata

Abstract

A new β-carboline alkaloid, 10-hydroxy-capitelline (1) together with three known anthraquinones, hedanthroside B (2), hedanthroside C (3), and rubiadin (4) were isolated from the aerial part methanol extract of Hedyotis capitellata. Their structures were established by spectroscopic data (one-dimensional, two-dimensional nuclear magnetic resonance (1D, 2D-NMR) and high-resolution-electrospray ionization-mass spectrometry, HR-ESI-MS) and comparison with those reported in the literature. The anti-inflammatory activity of the isolated compounds is evaluated by their inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. At a concentration of 20 µM, compounds 1 to 4 showed weak inhibitory effects on NO production with inhibitory values ranging from 1.2% to 23.9% compared to a value of 74.5% for the positive control compound, N^G-monomethyl-L-arginine (L-NMMA).

Introduction

Hedyotis is a genus of the flowering plants belonging to the family Rubiaceae. ¹ The Hedyotis plants are widely distributed in tropical and subtropical regions with about 500 different species. ¹ The previous studies showed that they have diverse chemical constituents with mainly secondary metabolite classes of alkaloids,^2-5 anthraquinones,^5-8 flavonoids,^5,6 lignans, ⁹ iridoids, and triterpenoids.^5,6,10 The diversity in chemical composition has brought various important pharmacological activities such as antibacterial, antidiabetic, antioxidant, anti-inflammatory, and anticancer.^6,11,12 In Vietnam, Hedyotis capitellata is a typical species of the genus Hedyotis and is a traditional medicine as diuretic, anti-toxicity, and anti-inflammatory for the stomach, tongue, and throat. ¹⁰ In this study, we reported the isolation and structure elucidation of a new β-carboline alkaloid and three known anthraquinones from the methanol extract of the aerial part of H capitellata. The inhibition of nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 cells by compounds 1 to 4 was also evaluated.

Results and Discussion

Compound 1 was obtained as a yellowish-brown colloid, which reacted positively with the Dragendorff reagent, suggesting an alkaloid compound. Its molecular formula was deduced to be C₂₀H₂₄N₂O₃ from the high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) quasimolecular ion peak at m/z 341.1862 [M + H]⁺ (calcd for [C₂₀H₂₅N₂O₃]⁺, 341.1865, Δ = −0.9 ppm), indicating nine degrees of unsaturation (see Supplemental Figure S1). The proton nuclear magnetic resonance (¹H NMR) spectrum of 1 exhibited signals that are characteristic of a β-carboline skeleton (Table 1).^4,13 The two ortho-coupled signals at δ_H 8.20 (1H, d, J = 5.5 Hz) and 7.84 (1H, d, J = 5.5 Hz) were assigned to H-5 and H-6, respectively. One ABX-coupled olefinic proton pattern at δ_H 7.51 (d, J = 2.0 Hz), 7.12 (dd, J = 8.5, 2.0 Hz), and 7.44 (d, 8.5 Hz), one olefinic proton δ_H 6.68 (s), one methine carbinol proton δ_H 3.44 (dd, J = 10.5, 1.5 Hz), three methyl singlets at 1.22, 1.25, and 1.96 (each 3H) were identified. The carbon nuclear magnetic resonance ( ¹³ C NMR) spectrum of 1 indicated the presence of 20 carbon signals, including eight quaternary, seven methine, two methylene, and three methyl carbons, classified by the heteronuclear single quantum coherence (HSQC) spectrum (Table 1). The NMR data of 1 were similar to that of capitelline (1a), previously isolated from H capitellata, ⁴ except that there were some significant changes at C-9, C-10, and C-11 (Table 1), suggesting 1 was 10-hydroxycapitelline. This was further confirmed by the heteronuclear multiple bond correlation (HMBC) correlations as shown in Figure 1Figure 2. The other two hydroxyl groups were at C-5′ and C-6′ confirmed by the HMBC correlations from both H-7′ and H-8′ to the quaternary carbon at δ_C 73.9 and the methine carbinol carbon at δ_C 78.9, as well as by the ¹H-¹H correlation spectroscopy (COSY) cross peaks of H-3′/H-4′/H-5′. The double bond was at C-1′/C-2′ as indicated by the HMBC correlations from H-9 to C-1′/C-2′/C-3′ and from H-1′ to C-2 and C-3. In addition, the nuclear overhauser effect spectroscopy (NOESY) correlations of H-9/ H-6 and the HMBC correlations of H-9/C-7 and H-6/C-8 further confirmed the suggested structure of 1. The NOESY cross peaks between H-3′ (δ_H 2.65 and 2.43) and H-1 (δ_H 6.68) were observed, suggesting for E-geometric configuration of the C-1′/C-2′ double bond. The optical rotation of 1 was zero as well as no Cotton effects were observed in the electronic circular dichroism (ECD) spectrum, suggesting that 1 was racemic. From the above evidence, 1 was determined to be a new compound 10-hydroxy-capitelline (Supplemental Figures S1 to S7).

Figure 1.

Chemical structures of compounds 1 to 4 and capitelline (1a).

Figure 2.

Key HMBC and H-H COSY correlations of compound 1.

Table 1.

¹H NMR and ¹³ C NMR Data for Compounds 1 and Capitelline (1a).

Pos.	1		1a
	a , c δC, type	b , c δH (mult., J in Hz)	d δC, type
2	136.5, C	−	134.5, C
3	143.0, C	−	141.6, C
5	137.2, CH	8.20 (d, 5.5)	137.5, CH
6	114.1, CH	7.84 (d, 5.5)	113.0, CH
7	129.9, C	−	128.7, C
8	123.2, C	−	121.5, C
9	106.7, CH	7.51 (d, 2.0)	121.6, CH
10	152.2, C	−	119.6, CH
11	119.4, CH	7.12 (dd, 8.5, 2.0)	128.2, CH
12	113.5, CH	7.44 (d, 8.0)	111.8, CH
13	137.0, C	−	140.6, C
1′	121.0, CH	6.68 (s)	120.6, CH
2′	146.8, C	−	144.9, CH
3′	38.5, CH2	2.65 (m) and 2.43 (m)	37.4, CH2
4′	30.5, CH2	2.03 (m) and 1.63 (m)	29.2, CH2
5′	78.9, CH	3.44 (dd, 10.5, 1.5)	77.6, CH
6′	73.9, C	−	73.2, C
7′	26.0, CH3	1.25 (s)	26.1, CH3
8′	24.8, CH3	1.22 (s)	23.4, CH3
9′	18.6, CH3	1.94 (s)	18.1, CH3

Abbreviations: ¹H NMR, proton nuclear magnetic resonance; ¹³C NMR, carbon nuclear magnetic resonance.

^a

Measured at 125 MHz.

^b

Measured at 500 MHz.

^c

Measured in deuterated methanol.

^d

Measured in deuterated dimethyl sulfoxide. ⁴

Compounds 2 to 4 were identified to be hedanthroside B, hedanthroside C, ⁸ and rubiadin, ¹⁴ respectively, by the consistent ¹H and ¹³ C NMR data with those reported in the literature (Supplemental Figures S8 to S24). ¹⁴ The assignments of these data were confirmed by 2D NMR spectroscopic analysis (see Tables S1 and S2 in the Supplemental Material). Capitelline was previously isolated from H. capitellata (the sample was collected in March 1995 in Ninh Binh Province). However, we have not isolated this compound (the sample collected in August in Nam Dinh Province), which may be due to the differences of geographical location and sampling times. ⁴

β-Carboline alkaloid and anthraquinone compounds have been known for many interesting biological activities including anti-inflammatory activity.^6,11,12 So that, compounds 1 to 4 were then evaluated for their effects on NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Each compound was assessed at a concentration of 20 µM. Compounds 1 to 4 exhibited weak inhibitory effects on NO production with inhibitory values ranging from 1.2% to 23.9% compared to a value of 74.5% for the positive control compound, N^G-monomethyl-L-arginine (L-NMMA) (see Table S3 in the Supplemental Material). Therefore, compounds 1 to 4 were not further evaluated for their half-maximal inhibitory concentration (IC₅₀) values.

Material and Methods

General Experimental Procedures

Optical rotation was measured on a Jasco P-2000 polarimeter (589 nm). Circular dichroism (CD) spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics). NMR spectra were studied on a Bruker 500 MHz spectrometer, and HR-ESI-MS on an Agilent 6530 Accurate Mass quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS). Flash column chromatography was performed using either silica gel or reversed phase (RP-18, mesh) resins as adsorbent. Thin layer chromatography (TLC) was carried out on pre-coated silica gel 60 F₂₅₄ and/or RP-18 F_254S plates. TLC plates were visualized under ultraviolet irradiation (254 and 365 nm) or by spraying with H₂SO₄ solution (5%) followed by heating with a heat gun. High-performance liquid chromatography (HPLC) was carried out using an AGILENT 1100 HPLC system.

Plant Material

The plant H capitellata Wall. ex G. Don was collected in Nam Dinh in August 2018 and identified by Dr Nguyen The Cuong, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST). A voucher specimen (HC08.2018-1) was deposited at the Lab of Pharmaceutical Chemistry, VNU University of Science, Hanoi.

Extraction and Isolation

The dried and powdered aerial parts of H capitellata (3.6 kg) were ultrasonically extracted with hot methanol at 50^oC, (each batch: 0.6 kg, 2L MeOH, for 30 min, three times). After filtering, the filtrate was evaporated at 60^oC to give the methanol extract (HC, 170 g). This extract was suspended in water (2 L) and successively partitioned with hexane, CH₂Cl₂, and EtOAc to obtain corresponding extracts HCH (26.8 g), HCD (1.9 g), HCE (19.9 g), and the water layer. The HCE fraction (19.5 g) was chromatographed on a silica gel column (500 g, mesh) eluting with CH₂Cl₂-EtOAc (1:1.5) to obtain five subfractions (HCE1-HCE5). The HCE4 (2.1 g) was chromatographed on a silica gel column (70 g, mesh) eluting with CH₂Cl₂–MeOH (7:1) to yield compound 4 (22 mg). HCE5 (3.7 g) was chromatographed on a silica gel column (100 g, mesh) eluting with CH₂Cl₂-MeOH (6:1) to yield compound 1 (26 mg). The water fraction was chromatographed on a Diaion HP-20 column, eluted with a MeOH-H₂O gradient system (25:75, 50:50, 75:25, 100:0, each 1.5 L) to give four fractions, HCW1-HCW4, respectively. Fraction HCW2 (9.33 g) was separated on a silica gel column chromatography eluting with CH₂Cl₂:MeOH:H₂O (10:1:0.1) to give five smaller fractions, HCW2A-HCW2E. Fraction HCW2D (3.5 g) was separated by an RP-18 column (mesh) eluting with MeOH:H₂O (3:1) to obtain four fractions, HCW2D1-HCW2D4. Fraction HCW2D3 (0.8 g) was chromatographed on a silica gel column (30 g, mesh) eluting with CH₂Cl₂:MeOH:H₂O (3:1:0.2) to obtain compounds 2 (21.0 mg) and 3 (19.5 mg).

10-Hydroxy-Capitelline (1)

A yellowish-brown colloidal. $[\alpha ]$ _D^{25}$: 0.0 (c 0.1, MeOH); UV (MeOH) λ_max nm: 235, 267, 314; IR cm¹ (KBr): 3428, 2936, 1651, 1452. HR-ESI-MS m/z 341.1862 [M + H]⁺ (calcd. for C₂₀H₂₅N₂O₃⁺, 341.1865, Δ =  − 0.9 ppm). ¹H-NMR (CD₃OD, 500 MHz) and ¹³ C-NMR (CD₃OD, 125 MHz): see Table 1.

Nitric Oxide Assay

The detailed description of nitric oxide assay test is presented in Supplemental Material. ¹⁵

Conclusions

In summary, this report describes the isolation and structural elucidation of a new β-carboline alkaloid named 10-hydroxy-capitelline (1) and three known anthraquinones, hedanthroside B (2), hedanthroside C (3), and rubiadin (4) from the aerial part methanol extract of H capitellata. Compounds 1 to 4 showed weak inhibitory effects on NO production with inhibitory values ranging from 1.2% to 23.9% compared to a value of 74.5% for the positive control compound, L-NMMA.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211047705 - Supplemental material for A New β-Carboline Alkaloid From the Aerial Part of Hedyotis capitellata

Supplemental material, sj-docx-1-npx-10.1177_1934578X211047705 for A New β-Carboline Alkaloid From the Aerial Part of Hedyotis capitellata by Le Thi Huyen, Nguyen Thi Thu Hau, Hoang Son Vu, Nguyen Thi Lan Huyen, Bui Huu Tai, Nguyen Xuan Nhiem and Phan Van Kiem in Natural Product Communications