Two new defensive constituents from potato tubers (Solanum tuberosum)

1 Introduction

Potato (Solanum tuberosum L.) is one of the most important staple crops for direct and processed consumption in both developed and developing countries. It is the second most important crop in Europe and fourth on a global scale after wheat, rice and maize [1]. China with an annual yield of about 80 million tons is the largest potato producer in the world, accounting for 26.3% and 22.2% of the global total area and yield, respectively [2]. However, the potato production is seriously affected by the late blight disease (LBD), which is caused by pathogenic fungus (Phytophthora infestans) infection. The LBD can cause productivity losses of 20~30%, even up to 50~70%, and thus, the prevalence of LBD will lead to drastic consequences to the world food safety [3, 4]. Chemical fungicides (e.g. metalaxyl mancozeb and oxadixyl mancozeb) are widely used to prevent the pathogenic fungus; however, their application is limited because of the high toxicity and contamination to the environment. Defensive metabolites from natural sources are promising candidates for searching modern anti-LBD agents for the advantage of environmental friendliness [5, 6].

In order to clarify the defensive constituents of LBD-infected potatoes, phytochemical investigation on the tubers of S. tuberosum resulted in two new compounds, solatuberenol A (1) and 3-O-β-d-glucopyranosyl stigmasta-5(6),24(28)-diene (2). Herein, we reported their isolation, structural elucidation and antifungal activity against P. infestans.

2 Results and discussion

Compounds 1 and 2 were obtained from the tubers of S. tuberosum by repeated column chromatography (CC) on D101 macroporous adsorption resin, silica gel, sephadex LH-20. Their structures were identified to be solatuberenol A (1) and 3-O-β-d-glucopyranosyl stigmasta-5(6),24(28)-diene (2) (Fig. 1) by physical and spectroscopic data (HR-ESIMS, [α]_D, 1D and 2D NMR and ECD).

Fig. 1:

Structures of compounds 1 and 2.

Compound 1 was obtained as white powder with a molecular formula of C₁₃H₂₀O₃ (Ω=4), which was deduced from the negative HR-ESIMS at m/z=247.1289 ([M+Na]⁺, −1.6 mDa). The UV absorption peak at 237 nm indicated an α,β-unsaturated carbonyl group. In accordance with the molecular formula, all the 13 carbons were well resolved in the ¹³C NMR spectrum, including four methyls, one methylene, four methines and four quaternary carbons. In the ¹H NMR spectrum, the singlet at δ_H=5.87 ppm suggested a tri-substituted double bond; two coupled protons at δ_H=5.74, (d, J=15.7 Hz) and 5.82 ppm (dd, J=15.7, 5.2 Hz) verified a E-form double bond. The ¹H ¹H COSY correlations of 7-H/8-H/9-H/10-H established the buten-2-ol moiety, which was further determined to be linked with C-6 by the HMBC correlations from 7-H and 8-H to C-6 (Fig. 2). The NMR data of 1 were similar to that of (R)-4-hydroxy-4-((S)-5-hydroxy-2-methylhexan-2-yl)-3-methylcyclopent-2-en-1-one [7], except for an additional double bond and the absence of two methylenes (C-6 and C-7). In the CD spectra, the negative Cotton effect at λ=320 nm (Δε_max=−1) for n→π* and the positive Cotton effect at λ=241 nm (Δε_max=+22) for π→π* indicated that the configuration at C-4 was S. The configuration at C-9 was assigned by quantum chemical calculations at the B3LYP/6-31G(d,p)//6-31G(d,p) levels. The calculated [α]_D values for the two epimers were −54 (4S,9S) and +149° (4S,9R), respectively, and thus, the configuration at C-9 was determined as R by comparing to the experimental [α]_D value (+193). On the basis of the above analyses, compound 1 was deduced to be (E,S)-4-hydroxy-4-((R)5-hydroxy-2-methylhex-3-en-2-yl)-3-methylcyclopent-2-en-1-one, and named as solatuberenol A (1).

Fig. 2:

Key 2D correlations of compounds 1 and 2.

Compound 2 was isolated as white powder whose molecular formula was determined to be C₃₅H₅₈O₆ by the prominent [M+H]⁺ ion peak at m/z=647.3341 (+0.1 mDa) in positive HR-ESIMS. In accordance with the molecular formula, all the 35 carbons were well recognized in the ¹³C NMR spectrum. Six carbons at δ_C=103.0, 75.8, 72.1, 79.1, 79.0 and 63.3 ppm were characteristic for the presence of glucosyl group, which was assigned as β-configuration by the anomeric proton at δ_H=5.10 ppm (J=7.7 Hz). In addition to the glucosyl moiety, the residual 29 carbons including six methyls (δ_C=12.4, 13.4, 19.5, 19.9, 21.66 and 21.7 ppm) suggested a steroid aglycone. Two pairs of tri-substituted double bonds at δ_C=141.3, 122.4, 146.3 and 117.5 ppm indicated a stigmasta-5(6),24(28)-diene skeleton, which was further verified by the HMBC correlations from 6-H to C-4, C-8 and C-10, and from 28-H to C-23 and C-25. The Δ^24,28 double bond was designated to be Z-form from the correlations of 29-H/26, 27-H and 3-H/29-H in the ROESY spectrum [8]. In the HMBC spectrum, the correlations from 3-H to C-1′ and from 1′-H to C-3 assigned the glucosidation at C-3 position. Similarly, 3-H was proposed to occupy β-configuration from the chemical shift at δ_H=3.99 ppm (m) and the ROESY correlations between 3-H and 19-H₃. Acidic hydrolysis of 2 yielded d-glucose, which was confirmed by the [α]_D value of +51.3° (c=0.1, MeOH). From the above analyses, the structure of 2 was determined as 3-O-β-d-glucopyranosyl stigmasta-5(6),24(28)-diene.

Compounds 1 and 2 were assayed for their anti-LBD potency against the mycelia growth of P. infestans by fungal poisoned food technique. Compounds 1 and 2 showed moderate activity against P. infestans, with mycelia-growth inhibition of 30.1% and 52.4%, respectively, at the concentration of 500 ppm.

3 Experimental

4 Supporting information

1D and 2D NMR, UV, CD, [α]_D, HRMS, computational details and other supporting data associated with this article can be found in the online version (DOI: 10.1515/znb-2016-0234).