Chemical Constituents From the Roots of Rubia oncotricha

Phytochemical investigation of the 70% ethanolic extract of the roots of Rubia oncotricha Hand.-Mazz. led to the isolation of 2 new anthraquinone glucosides named 1-hydroxy-2-hydroxymethyl-9,10-anthraquinone-11-O-β-D-glucopyranoside (1) and 1,3,6–trihydroxy-2-hydroxymethyl-9,10-anthraquinone-3-O-β-D-glucopyranoside (2), together with 5 known compounds (3-7). Their structures were elucidated by extensive spectroscopic data analysis (1-dimensional, 2-dimensional-nuclear magnetic resonance, and high resolution-electrospray ionization-mass spectrometry).


Introduction
Rubia oncotricha Hand.-Mazz., belonging to the plant family of Rubiaceae, is widely distributed in China as an endemic species. The dry roots and rhizomes of the plant have been used as Chinese folk herb for the treatment of cough, expectorant, jaundice, bronchitis, injuries, and so on by the local minorities in Guizhou province. 1,2 However, there have been few reports on the chemical constituents of this plant.
Previous phytochemical investigations of R oncotricha showed the isolation of anthraquinones, naphthoquinones, naphthohydroquinones, and triterpenoids, [3][4][5] some of which exhibited biological activities, such as antitumor and antinematodal activities. 6,7 During the course of searching for bioactive compounds, we performed a phytochemical study on the root and rhizome of R oncotricha. Previous studies mainly focused on the isolation and identification of low polar compounds, and there have been few reports on the highly polar chemical constituents of R oncotricha. Therefore, an n-butanol fraction of 70% EtOH extract of this species was investigated, which led to the isolation of 2 new anthraquinone glucosides (1-2) together with 5 known compounds (3-7) (Figure 1). Compounds (3)(4)(5)(6)(7) were isolated from genus Rubia for the first time. Herein, we described the structure elucidation of these new compounds.

Results and Discussion
Compound 1 was obtained as a yellow amorphous powder. The molecular formula of 1 was established as C 21 H 20 O 9 by its high-resolution-electrospray ionization-mass spectrometry  (Table 1)  Together, these data suggested that compound 1 revealed a structure of anthraquinone glucoside. The β-configuration of the sugar unit was assigned by high coupling constants (J = 7.6 Hz) of the anomeric proton. The absolute configuration of glucose was determined as D by gas chromatography (GC) analysis of its derivative of hydrolysate. In the 1 H detected heteronuclear multiple bond correlation (HMBC) spectrum, the correlations from H-11 to C-1, C-2, and C-1 ′ and from H-3 to C-1 and C-11 verified that the glucopyranosyl moiety was connected to C-11 ( Figure 2). Therefore, the structure of 1 was assigned as 1-hydroxy-2-hydroxymethyl-9,10-anthraquinone-11-O-β-D-glucopyranoside.

Conclusions
In the present work, 2 new anthraquinone glucosides (1-2), along with 5 known compounds (3-7) were isolated and identified from the roots of R oncotricha and characterized using 1D and two-dimensional (2D)-NMR and HR-ESI-MS spectroscopic analysis. Compounds 3-7 were isolated from the plants of genus Rubia for the first time. The current study provided more information on the chemical composition of R oncotricha, which lays a foundation for the development of this plant used as a Chinese folk herb.

Experimental
General HR-ESI-MS was measured on a Bruker Daltonics micro TOF-Q II mass spectrometer (Bruker Daltoniks GmbH). 1D and 2D NMR were recorded on a JEOL ECS 400 NMR spectrometer (Jeol). Ultraviolet (UV) spectra were acquired on a UV-2700 spectrometer (Shimadzu Corporation). Optical rotation values were measured on an AUTOPOL⍰ spectrometer (Rudolph Co., Ltd). IR spectra were recorded on an IR Tracer-100 spectrometer (Shimadzu Corporation). Column chromatography was performed with macroporous resin
Fr.3 (2.5 g) was subjected to silica gel column chromatography eluted with EtOAc-MeOH

Acid Hydrolysis and Determination of Absolute Sugar Configuration
Each compound (0.5 mg) was hydrolyzed with 2 M HCl (2.0 ml) at 95°C for 3 h. After cooling, the reaction mixture was partitioned between water and ethyl acetate 3 times. The aqueous layer was repeatedly evaporated to dryness with methanol, until neutral. The residue was dissolved in pyridine (0.4 mL), then L-cysteine methyl ester hydrochloride (1.0 mg) was added. The mixture was reacted at 60°C for 1 h, and then trimethylsilyl imidazole (0.15 mL) was added to the reaction mixture at 60°C for another 1 h, after the reaction was completed, the reaction solution was blown dry with nitrogen. The reaction residue was dissolved in water (1.0 mL), and then n-hexane (0.5 mL) was added for extraction 3 times, and the n-hexane layer was analyzed by GC. The absolute configurations of the monosaccharides were confirmed to be D-glucose by comparison of the retention times of the authentic sample [t R (D-glucose) 29.196 min, t R (L-glucose) 29.558 min)] (Supplemental material).