Characterization of Phenolic Constituents from Ephedra Herb Extract

Nine known compounds: trans-cinnamic acid, catechin, syringin, epicatechin, symplocoside, kaempferol 3-O-rhamnoside 7-O-glucoside, isovitexin 2-O-rhamnoside, herbacetin 7-O-glucoside, and pollenitin B and a new flavonoid glycoside, characterized as herbacetin 7-O-neohesperidoside (1) on the basis of spectroscopic analysis and chemical evidence, were isolated from a traditional crude drug, “Ephedra herb extract”. Compound 1 had no effects on HGF-induced motility, whereas herbacetin, which is an aglycone of 1, significantly inhibited it.


Introduction
Ephedra herb (Japanese name: "Mao"), is defined in the Japanese Pharmacopoeia (JP) as the terrestrial stem of Ephedra sinica Staf, E. intermedia Schrenk et C.A. Meyer or E. equisetina Bunge (Ephedraceae), [1]. It is one of the most important crude drugs of Kampo prescriptions such as Maoto, Kakkonto, Makyokansekito, and Syoseiryuto, which have been used as anti-tussive, expectorant, anti-pyretic analgesic, and bronchodilator agents [2]. It is well known that the traditional biological properties of Ephedra herb are attributable in a large part to ephedrine-type alkaloids such as ephedrine, pseudoephedrine, and norephedrine [3].
Recently, our research group reported that the extract product of Ephedra herb ("Ephedra herb extract") inhibits the hepatocyte growth factor (HGF)-induced motility of human breast cancer MDA-MB-231 cells through the suppression of c-Met tyrosine phosphorylation [4]. In that paper, we discussed that the active components in the extract might be non-alkaloid substances because ephedrine had no effect on HGF-induced motility. Reports on the constituents in the extract besides ephedrine-type alkaloids are limited, although condensed tannins [5] and flavonoids were found in some Ephedra species [6,7]. In order to explore the active components associated with the antitumor expression shown by Ephedra herb extract, we have investigated the phenolic compounds in the extract, and characterized 10 compounds, including a new flavonoid, which is the subject of this paper.

Results and Discussion
The Ephedra herb extract, purchased from Tsumura & Co., was dissolved in water and partitioned with n-hexane, ethyl acetate (EtOAc), and n-butanol (BuOH) to give the respective n-hexane, EtOAc, BuOH, and water extracts. The EtOAc and n-BuOH extracts, which showed the presence of polyphenolics in HPLC, were chromatographed using Diaion HP-20, Sephadex LH-20, Toyopearl HW-40, MCI-gel CHP-20P, and/or YMC GEL ODS-AQ with aqueous methanol (MeOH) in a stepwise gradient mode. The fractions showing similar HPLC patterns were combined and further purified by column chromatography with aqueous MeOH, to afford a new compound 1, together with the nine known compounds trans-cinnamic acid (2) [8], syringin (3) [9], catechin (4) [10], epicatechin (5) [10], symplocoside (6) [11], pollenitin B (7) [12], herbacetin 7-O-glucoside (8) [6], kaempferol 3-O-rhamnoside 7-O-glucoside (9) [13], and isovitexin 2-O-rhamnoside (10) [14] (Figure 1), which were identified by direct comparison with authentic specimens or by spectral comparisons with data reported in the literature.  [12]. The presence of a herbacetin skeleton in 1 was indicated by A 2 B 2 -type doublets at δ 6.90 and 8.20 (2H, each d, J = 9.0 Hz) and a singlet at δ 6.62 (1H, s) in the 1  protons of sugar units were observed, together with 10 mostly overlapped proton signals at δ 3.3-3.9, suggesting the presence of two sugar residues. The chemical shifts of the sp 3 -carbon signals in the 13 C-NMR spectrum were consistent with those of rhamnose and glucose in compound 9 or other analogs [13]. Compound 1 was thus presumed to be herbacetin rhamnoglucoside, which was verified by production of herbacetin (11) [15] as the aglycone upon acid hydrolysis. The sugar components liberated in the hydrolysis were identified as D-glucose and L-rhamnose according to the previously reported method [16]. Glycosidic linkages in 1 were determined as β-and α-configuration for D-glucose and L-rhamnose, respectively, based on the coupling constants of each anomeric proton signal. The linking position of each unit was confirmed by cross-peaks between glucose H-1 (δ 5.20) and C-7 (δ 151.9) of herbacetin, and rhamnose H-1 (δ 5.29) and C-2 (δ 80.5) of glucose in HMBC ( Figure 2). Therefore, 1 was established as herbacetin 7-O-neohesperidoside.   The effect of 1 on the HGF-induced motility of MDA-MB-231 cells was estimated with a method similar to that in the previous report [4]. However, the tested compound had no effects on HGF-induced motility. In general, it is reported that glycosides in Kampo medicines are metabolized to aglycones by intestinal flora [17]. We thus assessed the effects of 11, which is an aglycone of 1, together with 4 and 5 on the HGF-induced motility. The HGF-induced motility was significantly reduced by the addition 4 μg/mL of 11; however, 4 and 5 had no effects on the HGF-induced motility (Figure 3). Thus, it is suggested that 11 is one of the active components associated with the antitumor effect shown by the Ephedra herb extract. Further investigation of other compounds, including other fractions in the Ephedra herb extract, is currently in progress.

Samples and Reagents
Ephedra herb (from the aerial parts of E. sinica) extract was obtained from Tsumura & Co. (Tokyo, Japan). L-Cysteine methyl ester hydrochloride and o-tolyl isothiocyanate were purchased from Wako Pure Chemical Industries (Osaka, Japan). All other reagents were of analytical grade.

Determination of Sugar Configuration
The sugar configuration was determined using previously described methods [16]. Compound 1 (1.0 mg) was hydrolyzed by heating in 0.5 M HCl (0.2 mL) and neutralized with Amberlite IRA400. After evaporation and drying, the residue was dissolved in pyridine (0.2 mL) containing L-cysteine methyl ester hydrochloride (1.0 mg) and heated at 60 °C for 1 h. After cooling, o-tolyl isothyocyanate (1.0 mg) in pyridine (0.2 mL) was added to the mixture and heated again at 60 °C for 1 h. The reaction mixture was directly analyzed by RP-HPLC. The peaks coincided with those of derivatives similarly prepared from D-glucose and L-rhamnose.

Acid Hydrolysis of 1
A solution of 1 (1.5 mg) in 0.1 M HCl (0.5 mL) was heated in a boiling water bath for 5 h. After cooling, the reaction mixture was adsorbed on the Sep-Pak tC18 Plus cartridge (900 mg). After washing with water, the product was eluted with MeOH, and the concentrated solution was analyzed by reversed-phase HPLC to detect herbacetin (11, R t 27.3 min), which was identified by co-chromatography with an authentic sample [15].

Transwell Migration Assay
The MDA-MB-231 cells, obtained from the American Type Culture Collection (Manassas, VA, USA), were suspended at 5 × 10 4 cells in 100 μL of DMEM containing 4 or 10 μg/mL of the tested compounds, and poured into the upper well of the Transwell permeable support system (Corning Incorporated, Acton, MA, USA). Six hundred microliters of DMEM medium containing 50 ng/mL of HGF (R&D Systems, Minneapolis, MN, USA) was added to the lower well. The transwell was then incubated for 20 h at 37 °C, and the number of cells that had migrated to the lower well was counted. Motility (%) is {(the number of migrated cells with the tested compound/the number of migrated cells without it) × 100}. Each assay was performed in triplicate, and the error bars represent the standard deviation. The significance was determined using Dunnett's test.