New Ent-Kaurane-Type Diterpene Glycosides and Benzophenone from Ranunculus muricatus Linn.

Two new ent-kaurane diterpene glycosides, ranunculosides A (1) and B (2), and a new benzophenone, ranunculone C (3), were isolated from the aerial part of Ranunculus muricatus Linn. The chemical structures of compounds 1–3 were established to be (2S)-ent-kauran-2β-ol-15-en-14-O-β-d-glucopyranoside, (2S,4S)-ent-kauran-2β,18-diol-15-en-14-O-β-d-glucopyranoside, and (R)-3-[2-(3,4-dihydroxybenzoyl)-4,5-dihydroxy-phenyl]-2-hydroxylpropanoic acid, respectively, by spectroscopic data and chemical methods. The absolute configuration of 1 was determined by the combinational application of RP-HPLC analysis and Mosher’s method.


Introduction
Ranunculus muricatus Linn. (Ranunculaceae) is widely distributed in Asia, South America, Australia, and Europe. This plant mainly grows in the lower Yangtze River regions of China. It is used in folklore medicines in India for the therapy of tonsillitis diseases [1]. Although there is little documentation for folk and clinical use of the titled plant in China, other Ranunculus plants such as R. sceleratus Linn., R. japonicus Thunb., and R. termatus Thunb. have diverse clinical uses with a long history in traditional Chinese medicines and folk medicines, including those used for the treatment of lymphatic tuberculosis, malaria, swollen hemorrhoids, scrofula, and arthritis [2][3][4]. A series of simple lactone derivatives with 5-or 6-member rings, some steroids, and phenols was found in these plants, some of which indicated various biological activities, and they are responsible for their traditional or folk uses and clinical efficacies [5,6]. Our interest is in finding out the chemical characteristics and biological activities of the widely distributed plant R. muricatus. Wang et al.'s research led to the isolation and identification of eight known constituents from this plant, which are stigmasta-4-ene-3,6-dione, stigmasterol, anemonin, sitosterol, protocatechuic aldehyde, protocatechuic acid, and luteoin, and most of which belong to common compounds in plants [7]. Our previous study has revealed the presence of rich flavonoid glycosides in the plant [8]. Now, our further phytochemical investigation on the ethanolic extract from the aerial part of R. muricatus Linn. led to the isolation and identification of three new compounds (Figure 1), ranunculoside A (1), ranunculoside B (2), and ranunculone C (3). It is notable that ranunculosides A (1) and B (2) are the first two ent-kaurane diterpenoids from the genus plants and are present in glycoside. Herein we describe the isolation and structure elucidation of these compounds through spectroscopic analysis and chemical methods.

Results and Discussion
The ethanolic extract from the aerial part of R. muricatus Linn. was successively partitioned in an H2O suspension solution with petroleum ether, EtOAc, and n-BuOH, then applied to various column chromatography with silica gel, Octadecylsilyl (ODS), and Sephadex LH20, following by preparative reversed phase HPLC purification to yield compounds 1-3.
Compound 1 was obtained as an amorphous solid. Its HR-ESI-MS in positive ion mode showed a pseudomolecular ion peak at m/z 489.28287 [M + Na] + , suggesting its molecular formula as C26H42O7.

Results and Discussion
The ethanolic extract from the aerial part of R. muricatus Linn. was successively partitioned in an H 2 O suspension solution with petroleum ether, EtOAc, and n-BuOH, then applied to various column chromatography with silica gel, Octadecylsilyl (ODS), and Sephadex LH20, following by preparative reversed phase HPLC purification to yield compounds 1-3.
Comparison of the NMR data of 2 with those of 1 indicated that they were a pair of compounds extremely analogous in structure. The mere difference was that the methyl group (Me(18)) in 1 was replaced by a hydroxymethyl group in 2, because the hydroxymethyl signals at δ H 3.43/3.64 (dd, J = 10.5 Hz, 1H each) and δ C 71.9 (t) were observed in the 1 H-and 13 C-NMR spectra of 2. The complete structure of 2 was confirmed by HMBC and NOE correlations, which were similar to those of 1 (Figures 2 and 3). The acid hydrolysis of 2 under the same conditions mentioned in 1 also afforded the same sugar, D-glucose, which was also determined by the comparative HPLC analysis of the sugar derivative with that of standard sugars. Based on the consideration of the same biogeneric pathway, the absolute configurations of all the chiral centers except C-4 in the aglycone part of 2 should be identical to those of 1. The absolute configuration of the newly present chiral carbon C(4) due to the hydroxyl substitution was determined by careful analysis of its NOESY. Therefore, compound 2 was determined to be (2S,4S)-ent-kauran-2β,18-diol-15-en-14-O-β-D-glucopyranoside, which was named ranunculoside B.
Molecules 2015, 20, page-page 5 Compound 2 was obtained as an amorphous powder, and had a molecular formula of C26H42O8 according to the pseudomolecular ion peak at m/z 505.27707 [M + Na] + in HR-ESI-MS (positive mode), 16 mass units higher than that of 1, implying the presence of an additional oxygen atom in 2.
Comparison of the NMR data of 2 with those of 1 indicated that they were a pair of compounds extremely analogous in structure. The mere difference was that the methyl group (Me(18)) in 1 was replaced by a hydroxymethyl group in 2, because the hydroxymethyl signals at δH 3.43/3.64 (dd, J = 10.5 Hz, 1H each) and δC 71.9 (t) were observed in the 1 H-and 13 C-NMR spectra of 2. The complete structure of 2 was confirmed by HMBC and NOE correlations, which were similar to those of 1 (Figures 2 and 3). The acid hydrolysis of 2 under the same conditions mentioned in 1 also afforded the same sugar, D-glucose, which was also determined by the comparative HPLC analysis of the sugar derivative with that of standard sugars. Based on the consideration of the same biogeneric pathway, the absolute configurations of all the chiral centers except C-4 in the aglycone part of 2 should be identical to those of 1. The absolute configuration of the newly present chiral carbon C(4) due to the hydroxyl substitution was determined by careful analysis of its NOESY. Therefore, compound 2 was determined to be (2S,4S)-ent-kauran-2β,18-diol-15-en-14-O-β-D-glucopyranoside, which was named ranunculoside B.