Lianqiaoxinoside B, a Novel Caffeoyl Phenylethanoid Glycoside from Forsythia suspensa

Chemical investigation of the 70% ethanol extract of the unripe fruits of Forsythia suspensa resulted in the isolation of a novel caffeoyl phenylethanoid glycoside, lianqiaoxinoside B, together with the known compound forsythoside H. The new compound was elucidated to be 1'',2''-[β-(3,4,-dihydroxylphenyl)-α,β-dioxoethanol]-3''-O-caffeoyl-O-α-rhamnopyranosyl-(1→6)-O-β-glucopyranoside by extensive spectroscopic and chemical studies. Lianqiaoxinoside B and forsythoside H showed strong antioxidant and antimicrobial activities in vitro by the 2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonate (ABTS) radical-scavenging assay and plate method. This study can be further extended to exploit for the possible application of caffeoyl phenylethanoid glycosides as the alternative antioxidants and antimicrobial agents of natural origin.


Introduction
Forsythia suspensa (Thunb.) Vahl, a small tree widely distributed in China, Korea, Japan and many European nations, belongs to the family Oleaceae [1]. The fruit of F. suspensa is also called in Chinese "Lianqiao" and has been used for antiinflammatory, diuretic, drainage and antidotal purposes [2]. The

OPEN ACCESS
herb is listed in the Chinese Pharmacopoeia 2010 as an important crude drug and an aqueous extract is also used as a medicine. In previous papers, we reported the isolation of two phenylethanoid glycosides -forsythiaside and lianqiaoxinside A -and four lignans -phillygenin, (+)-isolariciresinol, phillyrin and (+)-pinoresinol-β-D-glucoside -from F. suspensa [2,3]. In this paper we report the isolation of lianqiaoxinoside B and forsythoside H from a 70% EtOH extract of unripe F. suspensa fruits. Their chemical structures ( Figure 1) were elucidated by HRESIMS, 1D and 2D NMR techniques and chemical methods. The high antioxidant and antimicrobial activities of these two caffeoyl phenylethanoid glycosides were also examined.

Structure Elucidation of 1
Compound 1 was obtained as a white amorphous powder and showed positive results for the Molish reagent. The UV spectrum of 1 showed absorption band at 222, 246 (sh.), 287 (sh.) and 333 nm, respectively, which was considered to correspond to a caffeoyl phenylethanoid glycoside. Its molecular formula was established as C 29    All the hydrogen and carbon signals of 1 have been fully assigned by combination of DEPT, 1 H-1 H COSY, HSQC, HMBC and NOESY experiments and comparison of spectral data with caffeoyl phenylethanoid glycosides reported in the literature [4]. The shift value for C-2 (δ 74.3) is found at a remarkably high field when compared with similar compounds [4], however, we considered that it is possibly due to the shielding effect of the caffeoyl group in the C-3 oxygen atom. The structure of 1 was thus assigned as 1'',2''-[β(3,4,-dihydroxylphenyl)-α,β-dioxoethanol]-3''-Ocaffeoyl-O-α-rhamnopyranosyl-(1→6)-O-β-glucopyranoside, and was named lianqiaoxinoside B.

In Vitro Antimicrobial Activity
When studying the influence of the concentration of lianqiaoxinoside B and forsythoside H on the antimicrobial activities against bacterial strains we used two-fold microdilution broth method. The MIC data of the two compounds were presented in Table 1

Antioxidant Activity
Recently, the significance of phenolic compounds as dietary antioxidants has been highlighted by experts [5][6][7]. The amount of phenol hydroxyls and amount of intramolecular H-bonding in a molecule play a key role in its antioxidant activity [5]. Many researchers have reported the strong antioxidant activity of rutin and chlorogenic acid. They are common natural phenolic antioxidants and they all have ortho-substituted hydroxyl structures [6]. The compounds lianqiaoxinoside B and forsythoside H exhibited significant ABTS radical scavenging ability, evidencing IC 50 values of 15.6 and 17.7 μg/mL (Figure 3), compared with a positive control, Vc (IC 50 6.8 μg/mL). Recently many researches have demonstrated that forsythiaside, which is the marker and major constituent in this plant, possesses strong antioxidant activities [7]. Moreover, lianqiaoxinoside B and forsythoside H yielded nearly the same antioxidant activities. Obviously, all of these phenylethanoid glycosides have two ortho-substituting hydroxyl groups in both the caffeoyl and phenylethanoid moieties, which could be an important factor for their high antioxidant activity.

General
IR spectra were recorded on a Shimadzu FTIR-8400S spectrometer. NMR spectra were recorded on a Bruker DPX 400 NMR instrument (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR). Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as internal standard, and coupling constants are given in Hz. HRESIMS were carried out on Waters Xevo QTOF mass spectrometer. Preparative HPLC (Waters, Delta 600-2487) was performed on a Hypersil-ODS II (10 m, 20 × 300 mm, Yilite, Dalian, People's Republic of China).

Plant Material
The unripe fruits of

Determination of the in-Vitro Antimicrobial Activity
The experimental strains Staphylococcus aureus, Escherichia coli, Beta-hemolytic streptococci, Bacterium vulgare, Aeruginosus bacillus, Micrococcus pneumoniae, S. albus and B. dysenteriae were supplied by the Molecular Microbiology Laboratory of Heilongjiang University of Chinese Medicine. All the strains were tested for purity by Gram staining and by biochemical tests. The strains were kept at −70 °C in LB agar, activated by transferring into nutritive agar, and incubating at 37 ± 1.0 °C for 18 h.
The antimicrobial activity against eight bacterial strains under different concentrations was determined by the plate method. A two-fold microdilution broth method was used to determinate the minimum inhibitory concentration (MIC) value. Details how to determine the antimicrobial activity were provided in a previously published paper [3].

ABTS Radical-Scavenging Assay
The radical scavenging capacity of antioxidant for the ABTS (2,2'-azinobis-3-ethylbenzothiazoline-6-sulphonate) radical action was determined as previously described [5][6][7]. ABTS was generated by mixing 7 mM of ABTS at pH 7.4 (5 mM NaH 2 PO 4 , 5 mM Na 2 HPO 4 and 154 mM NaCl) with 2.5 mM potassium persulfate (final concentration) followed by storage in the dark at room temperature for 16 h before use. The mixture was diluted with ethanol to give an absorbance of 0.70 ± 0.02 units at 734 nm using spectrophotometer (Helios, Unicam, Cambridge UK). For each sample, the diluted methanol solution of essential oil (100 μL) was allowed to react with fresh ABTS solution (900 μL), and then the absorbance was measured 6 min after initial mixing. Ascorbic acid was used as a standard. The capacity of free radical scavenging was expressed by IC 50 (mg/L) value, which represents the concentration required to scavenge 50% of ABTS radicals. The free radical scavenging activity of each solution was then calculated as percent inhibition according to the following equation: %inhibition = 100 (A (blank)-A (sample))/A (blank).

Acid Hydrolysis and Derivatization with PMP Reagent
An aliquot of each sample (1.0 mg) was dissolved in 2M TFA (2 mL) in a 5 mL ampoule. The ampoule was sealed under a nitrogen atmosphere and kept in boiling water bath to hydrolyze samples into aglycone part and monosaccharides for 10 h. After the ampoule was cooled to room temperature, the reaction mixture was centrifuged at 3,000 rpm for 5 min. The supernatant was collected and methanol (1.5 mL) was added into it for the reaction mixture to be evaporated to dryness under a reduced pressure. Then the same amount of methanol was again added and dried by the same method as above, and the procedure was repeated thrice for TFA to be removed. The hydrolyzed and dried sample solutions were diluted with distilled water (2 mL) for the following experiments.
Two hundred μL of the hydrolyzed above sample was placed in 2.0 mL centrifuge tubes, then 0.5 M methanol solution (100 μL) of PMP and ammonia (200 μL) were added to each. Each mixture was allowed to react for 30 min in a 70 °C water bath, then cooled to room temperature and neutralized with formic acid (200 μL). The resulting solution was separated by liquid-liquid extraction using a volume of isoamyl acetate (two times) and chloroform (one time), respectively. After being shaken vigorously and centrifuged, the organic phase was carefully discarded to remove the excess reagents. Then the aqueous layer was filtered through a 0.22 μm membrane and diluted with water before UPLC analysis. The glucose and rhamnose were identified in 1 and 2 (rhamnose, t R 2.03 min; glucose t R 4.08 min). Details of the UPLC procedure can be seen our previous published paper [8].

Conclusions
Caffeoyl phenylethanoid glycosides are a category of important botanical ingredients which are extracted from many herb plants. As a part of our chemical investigation on the unripe fruits of F. suspensa, we have isolated a novel caffeoyl phenylethanoid glycoside with a 7,2′′-epoxy moiety and one known compound, forsythoside H. Their structures were established on the basis of spectroscopic and chemical evidence. It is also the first time the NMR data of forsythoside H in CD 3 OD is reported, which showed obvious difference with the spectrum in DMSO-d 6 recorded in reference [9]. The pharmacological tests showed that they both exhibited high antioxidant and antimicrobial activities. This study can be further extended to exploit for the possible application of caffeoyl phenylethanoid glycosides as the alternative antioxidants and antimicrobial agents from natural origin.