A New Erythrinan Alkaloid Glycoside from the Seeds of Erythrina crista-galli

A new Erythrina alkaloid glycoside, named erythraline-11β-O-glucopyranoside, was isolated from the seeds of Erythrina crista-galli L., together with five known Erythrina alkaloids and an indole alkaloid. The structure of the new alkaloid glycoside was elucidated by spectroscopic methods, and all of the compounds were evaluated for their antiviral activity against tobacco mosaic virus.


Introduction
The Erythrina genus, including approximately 200 species, is mainly distributed in tropical and subtropical areas. Four local and six introduced Erythrina species are distributed in China [1]. Erythrina crista-galli L. (Fabaceae), known as cockspur coral tree, is a popular ornamental plant in South America and tropical and subtropical regions of South Asia. Pharmacological investigations have demonstrated that E. crista-galli seed extracts possess sedative, hypertensive, laxative, and diuretic activities [2]. Phytochemical studies on this plant showed the presence of Erythrina [3,4] and benzylisoquinoline alkaloids [5,6]. The alkaloids of the Erythrina type, which possess a unique spirocyclic structure, are responsible for these activities. Great efforts have been made to reveal the biosynthetic pathway and mechanism for the Erythrina alkaloid biosynthesis in the past few decades [2]. Furthermore, there are chemical studies on its non-alkaloidal constituents, in which flavonoids, cinnamylphenols and pterocarpans were reported functioning as phytoalexins and possessing antimicrobial activity [7][8][9][10][11]. In this paper, we describe the isolation and structural characterization of a new Erythrina alkaloids glucoside, erythraline-11β-O-glucopyranoside (1), together with six known alkaloids (2-7) (Figure 1) from the seeds of Erythrina crista-galli, and these isolated compounds were evaluated for their inhibitory activity against tobacco mosaic virus (TMV) using the leaf-disc method.

Introduction
The Erythrina genus, including approximately 200 species, is mainly distributed in tropical and subtropical areas. Four local and six introduced Erythrina species are distributed in China [1]. Erythrina crista-galli L. (Fabaceae), known as cockspur coral tree, is a popular ornamental plant in South America and tropical and subtropical regions of South Asia. Pharmacological investigations have demonstrated that E. crista-galli seed extracts possess sedative, hypertensive, laxative, and diuretic activities [2]. Phytochemical studies on this plant showed the presence of Erythrina [3,4] and benzylisoquinoline alkaloids [5,6]. The alkaloids of the Erythrina type, which possess a unique spirocyclic structure, are responsible for these activities. Great efforts have been made to reveal the biosynthetic pathway and mechanism for the Erythrina alkaloid biosynthesis in the past few decades [2]. Furthermore, there are chemical studies on its non-alkaloidal constituents, in which flavonoids, cinnamylphenols and pterocarpans were reported functioning as phytoalexins and possessing antimicrobial activity [7][8][9][10][11]. In this paper, we describe the isolation and structural characterization of a new Erythrina alkaloids glucoside, erythraline-11β-O-glucopyranoside (1), together with six known alkaloids (2-7) (Figure 1) from the seeds of Erythrina crista-galli, and these isolated compounds were evaluated for their inhibitory activity against tobacco mosaic virus (TMV) using the leaf-disc method.
Compound 1 was isolated as a white amorphous powder. It was assigned with a molecular formula of C 24 H 29 NO 9  . Its 13 C-NMR and DEPT spectra showed 24 carbon resonances, including one methoxy, five methylenes, twelve methines, and six quaternary carbons (including one olefinic). The 13 C-NMR data of 1 were similar to those of erythraline (2), except for the additional signals for a glucopyranosyl group at δ C 105.9 (C-1'), 78.2 (C-3'), 78.0 (C-5'), 75.4 (C-2'), 71.6 (C-4 ), and 62.8 (C-6'), as well as an oxygenated methine appearing at δ C 74.5 instead of a methylene carbon appearing at δ C 25.3 (C-11) in that of 2. The data above suggested a glucose moiety attached through an oxygen to C-11. The glucoside must have a β attachment, since the anomeric proton H-1 (δ H 4.61) exhibits a diaxial coupling (J = 7.8 Hz) with H-2' (δ H 3.23). The HMBC and 1 H-1 H COSY correlations ( Figure 2) confirmed that 1 was a typical Erythrinan alkaloid having a characteristic spiro-carbon positioned in the center of rings A, B, and C with two olefins of ∆ 1,2 and ∆ 6,7 and a dioxy-methylene group attached to the C-15 and C-16. The presence of a glucose moiety at C-11 was further confirmed by HMBC correlation observed from the anomeric proton H-1' to C-11. The HMBC correlation from methoxy protons H-18 (δ H 3.32) to C-3 (δ C 77.4) indicated that C-3 was substituted with a methoxy group. The relative configuration of 1 was determined by NOESY experiment. The observed cross-peaks ( Figure 3) between H-3/H-14, H-4β, and correlations between H-4β/H-11, H-10α implied that the protons at C-3 and C-11 were placed at βand α-orientation, respectively. Therefore, the structure of 1 was established as erythraline-11β-O-glucopyranoside. Biogenetic considerations on Erythrina alkaloids and the positive optical rotation value suggested that 1 has an S configuration at C-5 [6,14,17].

General Experimental Procedures
Optical rotations were measured on a JASCO-1020 polarimeter (JASCO, Tokyo, Japan). UV spectra were measured using a Perkin Elmer Lambda 800 UV/VIS spectrometer (PerkinElmer, Waltham, MA, USA). IR spectra were obtained with a Thermo Nicolet Avatar 360 FT-IR spectrometer (Thermo Nicolet Corporation, Madison, WI, USA). 1 H-and 13 C-NMR spectra were obtained with a Bruker AVANCE III 500 spectrometer (Bruker BioSpin, Rheinstetten, Germany) using tetramethylsilane as an internal standard. HRESIMS were obtained with a Thermo Scientific™ TSQ Quantum Access MAX triple stage quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA).

General Experimental Procedures
Optical rotations were measured on a JASCO-1020 polarimeter (JASCO, Tokyo, Japan). UV spectra were measured using a Perkin Elmer Lambda 800 UV/VIS spectrometer (PerkinElmer, Waltham, MA, USA). IR spectra were obtained with a Thermo Nicolet Avatar 360 FT-IR spectrometer (Thermo Nicolet Corporation, Madison, WI, USA). 1 H-and 13 C-NMR spectra were obtained with a Bruker AVANCE III 500 spectrometer (Bruker BioSpin, Rheinstetten, Germany) using tetramethylsilane as an internal standard. HRESIMS were obtained with a Thermo Scientific™ TSQ Quantum Access MAX triple stage quadrupole mass spectrometer (Thermo

Plant Materials
The seeds of Erythrina crista-galli L. were collected in July 2016 at the Fujian Agriculture and Forestry University (Fuzhou, China), and identified by Professor Chun-Mei Huang. Herbarium specimens were deposited at the Key Laboratory of Bio-Pesticide and Chemistry-Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, China (Specimen number: EC16701S).

Virus and Host Plant
Purified TMV (strain U1) was obtained from Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China, whose concentration was determined as 15 mg/mL using an ultraviolet spectrophotometer method (virus concentration = (A 260 × dilution ration) / E 0.1%, 260 nm 1 cm ). The purified virus was kept at −20 • C and was diluted to 30 µg/mL with 0.01 M PBS before use. Nicotiana tabacum cv. K326, which were cultivated and grown to a 5-6-leaf stage in an insect-free greenhouse, were used as an anti-TMV assay, as a systemic TMV infection host. Purified compounds were dissolved in DMSO and diluted with 0.01 M PBS to a certain concentration for test. The final concentration of DMSO in the test solution (≤2%) showed no adverse effect on the plants.

Leaf-Disc Method
Growing leaves of N. tabacum cv. K326 were mechanically inoculated with TMV (30 µg/mL in 0.01 M PBS). After 6 h, leaf discs (1 cm diameter) were punched and floated on solutions for test. Discs of healthy and inoculated leaves floated on a solution of 0.01 M PBS with 2% DMSO were used as a mock and negative control, respectively. Three replicates were carried out for each sample. After incubating for 48 h at 25 • C in a culture chamber, the leaf discs were grounded in 0.01 M carbonate coating buffer (pH 9.6), and OD 405 values were measured using TAS-ELISA method. TAS-ELISA was performed as described in the literature [18,19]. Virus concentration was calculated from a standard curve constructed using OD 405 values of purified TMV at concentrations of 1.0, 0.5, 0.25, 0.125, and 0.0625 µg/mL. The inhibition of test solutions on TMV was calculated as follows: inhibition rate = [1 − (virus concentration of treatment) / (virus concentration of negative control)] × 100%.

Conclusions
A new Erythrina alkaloid glycoside together with five known Erythrina alkaloids and an indole alkaloid have been isolated from the MeOH extract of the seeds of Erythrina Crista-galli L. All the isolated alkaloids showed noticeable inhibition against the replication of TMV but in a relatively higher concentration as compared with that of the positive control agent. Further investigations are needed to reveal the biological action mechanism of Erythrina alkaloids.

Supplementary Materials:
The following are available online. The 1 H-and 13 C-NMR of all the isolated compounds, and the IR, UV/Vis, HRESIMS, DEPT, HSQC, HMBC, and NOESY of Compound 1.