Two New Compounds Isolated from Liriope muscari

Two new compounds, (2S,3R)-methyl 7-hydroxy-2-(4-hydroxy-3-methoxy-phenyl)-3-(hydroxymethyl)-2,3-dihydrobenzofuran-5-carboxylate (1) and (4R,5S)-5-(3-hydroxy-2,6-dimethylphenyl)-4-isopropyldihydrofuran-2-one (2), tentatively named norcurlignan and limlactone, respectively, were isolated from Liriope muscari, together with the known compound (−)-pinoresinol (3). The structures of these compounds were elucidated and characterized on the basis of 1D NMR, 2D NMR, CD and MS data. The in vitro antioxidant activities of compounds 1–3 were assessed by the DPPH and ABTS scavenging methods.


Introduction
Liriope muscari (Decne.) Bailey (Liliaceae) is called duantingshanmaidong in China. Due to the lack of Radix Ophiopogonis (maidong in Chinese) resources and the similar pharmacological activities of these two herbal medicines, L. muscari is used locally in Fujian province as a substitute for maidong [1]. Maidong is a traditional herbal medicine widely used in China as a tonic agent.

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Pharmacology studies also showed that this herbal medicine has a positive effect on various inflammation-related diseases [2], however, its antioxidant activity was seldom reported [3].
The absolute stereochemistry at C-2 and C-3 of compound 1 was determined by NOE experiments and its CD spectrum. In the NOE spectrum, irradiation at H-2 signal caused the enhancement of the H-9 signal while H-3 signal was not enhanced, which indicated that H-2 and H-3 were trans-oriented. The absolute stereochemistry at C-2 and C-3 of compound 1 was determined to be 2S,3R by comparison of its CD curve with that of curlignan [13], both showing one negative and one positive Cotton effect (CE) around 230 and 280 nm, respectively. These data established the structure of compound 1 as (2S,3R)-methyl 7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydrobenzofuran-5-carboxylate. This novel natural product is tentatively named norcurlignan after its homologue curlignan.   (Table 2), DEPT and HSQC spectra of compound 2 allowed the assignment of 15 carbon signals to one secondary, five tertiary, five quaternary carbons, and four methyls, further suggesting 2 be a 4-phenyldihydrofuran-2-one.
In the HMBC spectrum (Figure 3), the observation of diagnostic correlations from H-5 to C-2' and C-6', allowed the tetrasubstituted phenyl group to be attached to C-5. The isopropyl group was assigned to C-4, based on the correlations of H-7 and H-8 with C-4, and the correlations of H-3 and H-5 with C-6. The ester carbonyl group was assigned to C-2, based on the correlations of H-4 and H-3 with C-2. The positions of the phenyl group substituents were established by the HMBC spectrum. These data pooled together would suggest compound 2 to be a 5-(3-hydroxy-2,6-dimethylphenyl)-4isopropyldihydrofuran-2-one. The assigned structure for compound 2 was confirmed by the analysis of the 1 H-1 H COSY spectrum, which further revealed the presence of a CH (H-5)-CH (H-4)-CH 2 (H-3), CH 3 -CH (H-6)-CH 3 fragment ( Figure 3).
The absolute stereochemistry at C-4 and C-5 was determined on the basis of the coupling constants and the CD spectrum. According to the coupling constant between H-4 and H-5 (J = 9.0 Hz), H-4 and H-5 was determined to be trans-oriented [14], so there were two possible configurations which were (4R,5S) or (4S,5R). According to literature on the CD spectra of γ-lactone rings [15], the C-CO-O-C group tends to form a coplanar conformation. When observed from a specific location, if β-C (C-4) is above the plane, the CE associated with the lactone n→π* transition should be positive, as it is observed in the CD spectrum at 224 nm. Through Chembio 3D simulation, it was found that only if the configuration is (4R,5S) (Figure 4), is it possible that β-C (C-4) is above the plane in the minimum energy state. In summary, the absolute configuration was determined to be (4R,5S). These data established the structure of compound 2 as (4R,5S)-5-(3-hydroxy-2,6-dimethylphenyl)-4-isopropyldihydrofuran-2-one. This novel natural product is tentatively named limlactone.

In Vitro Antioxidant Activity
DPPH and ABTS radical scavenging assays were carried out to evaluate the antioxidant activities of compounds 1-3 using vitamin C (VC) and butylated hydroxytoluene (BHT) as positive controls. The results are shown in Table 3. In both methods, compounds 3 showed potential activity, which was consistent with the reported results [16]. Compounds 1 and 2 showed potential activity in the ABTS assay. The DPPH and ABTS assay were carried out using the same methods reported in the previous paper [12]. . All other chemicals were of analytical reagent grade and used without any further purification.

Plant Material
Fresh fibrous roots of L. muscari. were collected from Quanzhou City, Fujian Province, China, in May 2010. The species was identified by Professor Zhang J. (National Institutes for Food and Drug Control, NIFDC for short). The voucher specimens were deposited at the herbarium of NIFDC. The roots were air-dried and ground to a powder using a grinding mill (Retsch Muhle, Haan, Germany).