Two Novel Quassinoid Glycosides with Antiviral Activity from the Samara of Ailanthus altissima

Phytochemistry investigations on Ailanthus altissima (Mill.) Swingle, a Simaroubaceae plant that is recognized as a traditional herbal medicine, have afforded various natural products, among which C20 quassinoid is the most attractive for their significant and diverse pharmacological and biological activities. Our continuous study has led to the isolation of two novel quassinoid glycosides, named chuglycosides J and K, together with fourteen known lignans from the samara of A. altissima. The new structures were elucidated based on comprehensive spectra data analysis. All of the compounds were evaluated for their anti-tobacco mosaic virus activity, among which chuglycosides J and K exhibited inhibitory effects against the virus multiplication with half maximal inhibitory concentration (IC50) values of 56.21 ± 1.86 and 137.74 ± 3.57 μM, respectively.


Extraction, Isolation and Sructure Elucidation
The air-dried and milled samara of A. altissima was extracted with methanol to afford a crude extract, which was resuspended in water and fractioned by liquid-liquid partition. From the trichloromethane partition, a total of ten lignans were isolated, while compounds 1 and 2, together with 4, 6, 9 and 16, were purified from the n-butyl alcohol partition ( Figure 1).

Extraction, Isolation and Sructure Elucidation
The air-dried and milled samara of A. altissima was extracted with methanol to afford a crude extract, which was resuspended in water and fractioned by liquid-liquid partition. From the trichloromethane partition, a total of ten lignans were isolated, while compounds 1 and 2, together with 4, 6, 9 and 16, were purified from the n-butyl alcohol partition ( Figure 1).  Compound 1 was isolated as a colorless crystal, and its molecular formula was established as C 26 Figure S1). Its IR spectrum ( Figure S2) displayed absorption bands indicating the presence of hydroxyl (3427 cm −1 ), δ-lactone (1731 cm −1 ) and double bond (1640 cm −1 ). The 1 H-NMR ( Figure S3) Figure S4) and distortionless enhancement by polarization transfer (DEPT) ( Figure S5) NMR spectra revealed that compound 1 has 26 carbons including two carbonyl (δ C 207.7 and 169.1), two olefinic carbons (δ C 134.6 and 124.2), one hemiketal carbon (δ C 106.9), six saccharide-type carbons (δ C 105.2, 76.7, 76.3, 74.2, 70.0 and 61.1), as well as three methyl, three methylene, seven methine, and two quaternary carbons. The above 1 H and 13 C-NMR data were similar to those of the quassinoid glycosides isolated from the same plant materials, as reported previously in our paper [13]. Specifically, a keto group was attached at C-12 position, as indicated by the observed HMBC correlations ( Figure 2 Compound 1 was isolated as a colorless crystal, and its molecular formula was established as C26H36O12 by high resolution ionization mass spectroscopy (HRESIMS) (m/z 564.2175 [M + Na + H] + , calcd for C26H37O12Na 564.2177) ( Figure S1). Its IR spectrum ( Figure S2) displayed absorption bands indicating the presence of hydroxyl (3427 cm −1 ), δ-lactone (1731 cm −1 ) and double bond (1640 cm −1 ). The 1 H-NMR ( Figure S3 1), as well as three methyl, three methylene, seven methine, and two quaternary carbons. The above 1 H and 13 C-NMR data were similar to those of the quassinoid glycosides isolated from the same plant materials, as reported previously in our paper [13]. Specifically, a keto group was attached at C-12 position, as indicated by the observed HMBC correlations (  Compound 2 was purified as a colorless crystal with a molecular formula of C32H46O17 as shown by a sodiated molecular ion peak at m/z 725.2656 (calcd for C32H46O17Na 725.2627) observed with HRESIMS ( Figure S10). The IR spectrum ( Figure S11) displayed absorption bands indicating the presence of hydroxyl (3398 cm −1 ), δ-lactone (1718 cm −1 ) and double bond (1648 cm −1 ). Its 13 C-NMR and DEPT spectra (Figures S13 and S14) showed 32 carbon resonances, including two methyl, six methylene, eighteen methine, and six quaternary carbons. Compound 2 was also a quassinoid glycoside as deduced from a comparison of its 1 H and 13 C-NMR data with those of compound 1, as well as the analysis of its DEPT, COSY, HSQC, HMBC, and NOESY spectra (Figures S13-S18). Signals Compound 2 was purified as a colorless crystal with a molecular formula of C 32 H 46 O 17 as shown by a sodiated molecular ion peak at m/z 725.2656 (calcd for C 32 H 46 O 17 Na 725.2627) observed with HRESIMS ( Figure S10). The IR spectrum ( Figure S11) displayed absorption bands indicating the presence of hydroxyl (3398 cm −1 ), δ-lactone (1718 cm −1 ) and double bond (1648 cm −1 ). Its 13 C-NMR and DEPT spectra (Figures S13 and S14) showed 32 carbon resonances, including two methyl, six methylene, eighteen methine, and six quaternary carbons. Compound 2 was also a quassinoid glycoside as deduced from a comparison of its 1 H and 13 C-NMR data with those of compound 1, as well as the analysis of its DEPT, COSY, HSQC, HMBC, and NOESY spectra (Figures S13-S18). Signals of a terminal double bond [δ H 5.22 (br s, 2H, H-21); δ C 120.3 (C-21)] were observed in its 1 H-NMR and 13 C-NMR spectra (Figures S12 and S13), and the HMBC correlations ( Figure 3) observed between H-12 [δ H 3.89 (1H, s)] and C-21, between H-14 [δ H 2.80 (dd, J = 13.5, 5.4 Hz, 1H) and C-21, between H-21 and C-12 (δ C 81.0), as well as HMBC correlations between H-21 and C-14 (δ C 48.0). Two anomeric protons appearing at δ H 4.60 (d, J = 7.8 Hz, 1H, H-1 ) and 4.56 (d, J = 7.9 Hz, 1H, 1 ) in the 1 H-NMR spectra indicated the presence of two glucopyranosyl units, both of which must be β-anomer as suggested by their coupling constant. Acid hydrolysis of 2 afforded only d-glucose, which was identified by thin-layer chromatography (TLC) comparisons with sugar standards, and the HMBC correlations between the anomeric proton H-1 and C-2 (δ C 84.1), between H-2 [δ H 4.19 (1H, m)] and C-1 (δ C 105.0), and between the anomeric proton H-1 and C-3 (δ C 88.1), between H-3 [δ H 3.58 (1H, t, J = 8.9 Hz)] and C-1 (δ C 105.2) confirmed that two glucopyranosyl units were connected via a (1→3) linkage, and the saccharide moiety was attached at the C-2 position. The NOESY cross-peaks (

Antiviral Activities against the Replication of Tobacco Mosaic Virus
The isolated compounds were tested for their inhibitory activities against the replication of tobacco mosaic virus using the leaf-disc method. The lignans (3-16) obtained showed only weak or no inhibitory effect at a concentration of 0.5 mM ( Table 1). Both of the quassinoid glycosides obtained, chuglycoside J (1) and K (2), exhibited antiviral activities with IC50 values determined as 56.21 ± 1.86 and 137.74 ± 3.57 μM, while the commercial antiviral agents, ningnanmycin and ribavirin, possessed an IC50 of 183.31 ± 4.26 and 255.19 ± 4.57 μM, respectively.

Antiviral Activities against the Replication of Tobacco Mosaic Virus
The isolated compounds were tested for their inhibitory activities against the replication of tobacco mosaic virus using the leaf-disc method. The lignans (3-16) obtained showed only weak or no inhibitory effect at a concentration of 0.5 mM ( Table 1). Both of the quassinoid glycosides obtained, chuglycoside J (1) and K (2), exhibited antiviral activities with IC 50 values determined as 56.21 ± 1.86 and 137.74 ± 3.57 µM, while the commercial antiviral agents, ningnanmycin and ribavirin, possessed an IC 50 of 183.31 ± 4.26 and 255.19 ± 4.57 µM, respectively.

Discussion
Quassinoids, one kind of degraded triterpenoid derivative with multiple bioactivities such as anticancer, antimalarial, antimicrobial, antidiabetic, antiviral, and anti-inflammatory effects, are widely distributed in the family Simaroubaceae and the secondary metabolites characteristic of this family [26][27][28]. Quassinoids can generally be classified into five groups according to the basic features, including C 18 , C 19 , C 20 , C 22 and C 25 types. By the year 2004, more than 200 natural quassinoids obtained from 34 species in 14 genera were reported, and the structural characteristics of 190 quassinoids were reported between the year 2004 and 2018 [27,28]. Thus far, more than 50 quassinoids have been isolated from A. altissima, most of which belong to the C 20 class bearing a δ-lactone moiety [13,26]. Pharmacological and clinical investigations have revealed that C 20 quassinoids from Ailanthus genus plant are very promising for their medical use, such as antitumor, antimalarial, antiviral, antiparasitic properties etc. [4][5][6]. We have previously reported the identification of eighteen C 20 quassinoids including nine new quassinoid glycosides, named chuglycosides A-I, from the samara of A. altissima. The generated name chuglycoside arises from 'chu', the Chinese phonetic alphabets of one of the variant names of A. altissima, which is commonly used in the classics of traditional Chinese medicine. The quassinoids previously obtained from the samara of A. altissima can generally be classified as the C-6 or C-15 substituted derivatives of chaparrione and ailanthone, as well as their monoglycosides [13]. Ailanthone is the best known bioactive secondary metabolite isolated from A. altissima, which displays multiple pharmacological properties, in particular significant antitumor effects against a variety of cancer cell lines in vitro [29,30]. Two more C 20 quassinoid glycosides were reported based on our current findings, among which chuglycoside J wears a keto group in C-12, which, in the case of quassinoids from A. altissima, usually appears as a hydroxy substituted group, while chuglycoside K was the only one diglycoside we obtained from the extract of the samara of A. altissima. These findings suggest that phytochemical investigations to reveal the structural diversity of quassinoids synthesized by A. altissima are still worth undertaking.
In the field of modern agriculture, plant diseases caused by viruses are one of the major causes of biological disasters in agriculture and horticulture, which result in dramatic losses every year all over the world. The research and development of efficient antiviral agents with characteristics of low pesticide resistance, eco-friendliness and a novel mechanism is urgently and continuously needed [31,32]. Tobacco mosaic virus, the type member of the Tobamovirus group, is a positive-strand RNA virus that infects more than 400 plant species belonging to 36 families, such as tobacco, tomato, potato, and cucumber [33][34][35][36][37]. The tobacco mosaic virus/tobacco system is employed as a useful model in studies designed to clarify antiviral properties and the mechanism of action of novel antiviral agents. Our continuous efforts regarding the discovery of novel anti-phytopathogen viruses from natural products and studies of the mechanisms of antiviral action have proven that quassinoids from A. altissima could inhibit the coat protein expression and systemic spread of tobacco mosaic virus in tobacco [13] and this evidence has proven that quassinoids from A. altissima could be considered as lead structures for antiviral agent design and development.

Extraction and Isolation
The air-dried samara of Ailanthus altissima was collected in October 2013 at Muyang, Jiangsu, China. The voucher specimen was deposited at the Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, under the accession number MF131001.
The plant material was extracted and partitioned as described previously in our paper [13]. In brief, the milled air-dried samara of A. altissima (sample MF131001, 7500 g) was extracted three times with a total of 25 L methanol at room temperature. The dried extract was then resuspended in water and successively partitioned with n-hexane, trichloromethane, and n-butyl alcohol.
The n-butyl alcohol partition (90 g) was fractioned by silica gel column chromatography and eluted with mixtures of 0 to 100% methanol in chloroform to give fourteen fractions (fraction C1-C14). Fraction C2 was subjected to MCI gel column chromatography and eluted with a gradient of 15-100% water in MeOH to afford fractions C10a-C10e. Fraction C10e was chromatographed over a silica gel column and eluted with CHCl 3 -MeOH (v/v 97:3) to yield 16 (5.5 mg). Fraction C3 (3.5 g) was separated by MCI gel column chromatography and eluted with a gradient of 15-100% methanol in water to afford fractions C3a-C3p. Fraction C3i was chromatographed over a silica gel column using a mixture of CHCl 3 -MeOH (96:4) as eluent to yield 9 (22.0 mg). Fraction C5 (2.2 g) was chromatographed using a MCI gel column and eluted with a gradient of 15-100% methanol in water to afford fractions C5a-C5e. Fraction C5c was purified using RP-18 gel column chromatography with a solvent of 30% methanol in water as eluent to give 2 (5.0 mg). Fraction C8 (10.1 g) was subjected to MCI gel column chromatography and eluted with a gradient of 5-100% water in MeOH to afford fractions C8a-C8k. Fraction C8c was subjected to RP-18 gel column chromatography and eluted with 30% MeOH in water in to give 4 (70.9 mg). Fraction C10 (10.1 g) was subjected to MCI gel column chromatography and eluted with a gradient of 5-100% methanol in water to afford fractions C10a-C10h. Fraction C10e was further purified using RP-18 gel column chromatography and eluted with 30% methanol in water in to afford 6 (11.5 mg). Fraction C11 (10.3 g) was chromatographed using a MCI gel column and eluted with a gradient of 5-100% methanol in water to afford fractions C11a-C11h. Fraction C11e was then separated using RP-18 gel column chromatography and eluted with a mixture of 45% MeOH in water and finally chromatographed over a silica gel column and eluted with CHCl 3 -MeOH-H 2 O (v/v/v 80:20:2) to yield 1 (

Acid Hydrolysis of Compounds 1 and 2
Compound 1 or 2 (each 2 mg) was hydrolyzed at 95 • C for 2 h in 2 mL of 1 M HCl (dioxane-H 2 O, v/v 1:1), respectively. After being evaporated to dryness, the reaction mixtures were diluted in water and extracted with 2 mL ethyl ether three times. The aqueous layer was neutralized with NaHCO 3 and evaporated under vacuum to furnish a neutral residue for thin-layer chromatography (TLC) analysis, which indicated the presence of only d-glucose (Rf 0.40; eluted with MeCOEt-isoPrOH-Me 2 CO-H 2 O, v/v 20:10:7:6).

Antiviral Assay
The isolated quassinoids and lignans were dissolved in dimethyl sulfoxide (DMSO) and diluted to the required concentration before the test. Two commercial agents, ningnanmycin and ribavirin, were used as positive control agents, while a solution of 0.01 M phosphate-buffered saline (PBS) containing 1% DMSO was used as negative control. Purified tobacco mosaic virus (TMV) U1 strain was obtained from the Institute of Plant Virology, Fujian Agriculture and Forestry University. Nicotiana tabacum cv. K 326 , which were cultivated to 5-6 leaf stage in an insect-free greenhouse, were used for the anti-tobacco mosaic virus (TMV) assay.
The antiviral assay was conducted using the leaf-disc method as previously described in our paper [5,7,13,38,39]. In brief, the growing leaves of tobacco were mechanically inoculated and infected with the target virus. Six hours later, leaf discs of 1 cm diameter were punched and floated on the test solutions, while leaf discs from the healthy leaves were used as mock. Six replicates were carried out for each sample. The test solutions with leaf discs were kept in a Petri dish and incubated for 48 h at 25 • C in a culture chamber, and then the leaf discs were grounded with the addition of 0.01 M pH 9.6 carbonate coating buffer (500 µL for each leaf disc) and centrifuged. The supernatant of each sample (200 µL) was transferred to a 96-well plate, which was then used to perform a standard indirect enzyme-linked immunosorbent assay as described in the literature [34,35]. The virus concentration was calculated according to a standard curve constructed based on the optical density at 405 nm (OD 405 ) values of a series of the diluted solutions of purified TMV.