Two New Phenolic Constituents from the Stems of Euphorbia griffithii

Abstract Phytochemical studies on MeOH extract of stems of Euphorbia griffithii led to the isolation of one new hydrolyzable tannin dimer, corilagiffithiin (1) and one new galloyl-glucoside (2), alongside six known ones (3–8). Their structures and absolute configurations were determined by in depth spectroscopic analyses and comparison of their 1D NMR and MS data with literature reported values. Configurations of sugar moieties were determined by acidic hydrolysis and subsequent GC analysis of their corresponding trimethylsilylated l-cysteine adduct. At a concentration of 50 μM, compounds 1–3 showed no anti-inflammatory activities. Graphic Abstract Electronic supplementary material The online version of this article (10.1007/s13659-019-00223-2) contains supplementary material, which is available to authorized users.

inflammations. In Angola, some species are used against skin ailments, gonorrhea, diarrhea, dysentery, asthma, tumors and coughs. In Nigeria, the exudates of the plant are employed as ear drops, in the treatment of boils, sores and enhancement of wound healing [1]. Spurges are also used as ornamental and household plants [1]. Phytochemical investigations have revealed that the Euphorbia genus contains mainly, triterpenoids, diterpenoids, flavonoids, tannins and polyphenols [1,3]. Pharmacologically, the diterpenoids of Euphorbia have exhibited cytotoxic activities, while the tripernoids and flavonoids have demonstrated possession of anti-inflammation and inhibition of virus replication effects [1,3,4].
E. griffithii is a robust rhizomatous perennial whose vertical stems dresses in small, red-tinged leaves and showy orange-red flowers in early summer; it is native to Himalayas and western Asia [5,6]. It has red stems and dark green leaves, which crop up in spring with a reddish tincture [7]. Exhaustive literature search did not show any evidence of previous phytochemical studies on E. griffithii. That notwithstanding, our study on the stems of E. griffithii disclosed the presence of one new hydrolyzable tannin dimer (1) and one new galloyl-glucoside (2), together with six known ones (3)(4)(5)(6)(7)(8). Compounds 1-3 were evaluated for their anti-inflammatory effects. Herein, we present the isolation, structural elucidation of the isolates, and bioassay tests of compounds 1-3.
The linkage position between the two units, a and b, was determined by the 13 C NMR chemical shift values and HMBC correlations. Firstly, HMBC correlations of H-6 and H-6″′ with C-7″′ in unit a, and H-1 and H-6′ with C-7′ in unit b confirmed the locations of aromatic rings C and D on O-6 of Glc-II and O-1 of Glc-I, respectively. The presence of five quaternary carbons [δ C 115.3, 143.6, 140.6, 135.8, and 139.8 (C-1′-5′, resp.) in aromatic ring D suggested that it was a galloyl group that had undergone oxidative coupling at C-2′, which was significantly downfield shifted to δ C 143.6, hence taking part in an ether linkage. Moreover, it was observed that the 13 C NMR resonance of C-5″′ of the HHDP moiety (ring C) was shifted further downfield (δ C 147.5), lower than the usual corresponding C-5″′ signal (δ C 145.6) of the corilagin [17,18]. This observation was attributed to formation of an ether linkage at the p-hydroxyl group of the galloyl unit [17]. The HMBC spectrum also showed correlations of H-6″′ (δ H 6.25) with C-5″′ (δ C 147.5) of the HHDP ring C. Eventually, the structure of compound 1, namely corilagiffithiin was determined as shown in Fig. 1.
Ethyl    H-2) to δ C 64.6 (C-1)] supported the existence of an ethoxyl group in 2. HMBC experiment showed that the protons at δ 5.33 (H-3′ of the glucose moiety) and 7.12 (H-2″ and 6″ of the galloyl group) correlated with δ C 168.5 (the carbonyl carbon of the galloyl group), connoting that the hydroxyl group at C-3′ of glucose was acylated by the galloyl group (Fig. 3). A closer look at the planar structure of compound 2 showed that it was constituted of a galloyl [21] and ethyl α-D-glucopyranoside [22]. Thus, compound 2 was identified as ethyl-O-(3′-O-galloyl)-α-d-glucopyranoside.
Compounds 1-3 were evaluated for their anti-inflammatory effects on NO levels in LPS-stimulated RAW 264.7 macrophages. The compounds were inactive under the concentration of 50 μM.

Extraction and Isolation
The air-dried stems of E. griffithii (10 kg) were extracted with MeOH (three times) under reflux at 60 °C. After removal of the organic solvent, the extract (4.

Acid Hydrolysis of Compounds 1-2 and GC Analysis
Compounds, 1-2 (3-5 mg), were separately dissolved in 5% HCl (2 mL) and heated (90 °C) for 2 h. HCl was then removed by evaporation in vacuum. The sugar mixtures were diluted with H 2 O and extracted with EtOAc. The aqueous layer was neutralized with 0.1 M NaOH and eventually dried to yield the monosaccharide mixture which was immediately dissolved in pyridine (2 mL). l-cysteine methyl ester hydrochloride (about 1.5 mg) was added in to the solution of the sugar mixtures in pyridine (2 mL), and the reaction kept at 60 °C for 1 h. Thereafter, trimethylsilylimidazole (2 mL) was added in to the mixtures and reaction kept further at 60 °C for 1 h and finally halted then submitted for GC analysis [23], run on Hewlett Packard (HP) 5890 series II gas chromatography equipped with flame ionization detector (FID) and thermal conductivity detector (TCD). The column used was HP-5: column temperature: 150-280 °C, increasing at the rate of: 3 °C/min; carrier gas: N2 (1.5 mL/min); injector and detector temperature: 250 °C; injection volume: 1.0 μL; and split ratio: 1/50. The retention times of the samples were compared with those of the derivatives of authentic sugars, under the same condition. The sugar moiety of 1 was determined to be d-glucose (t R : 28.537 min), 2 was also resolved to be d-glucose (t R : 28.398 min), by confirming with the standard d-glucose (t R : 28.418 min).

Anti-Inflammatory Activity
The NO production assay with L-N G -monomethyl arginine (L-NMMA) as a positive control was performed as described previously by Chen et al. [24].