Bioactive Phenylpropanoid Glycosides from Tabebuia avellanedae

Three novel phenylpropanoid glycosides 2, 5, 6 were isolated from water extract of Tabebuia avellanedae, together with three known phenylpropanoid glycosides 1, 3, 4. All compounds were identified on the basis of spectroscopic analysis and chemical methods and, for known compounds, by comparison with published data. All isolated compounds showed strong antioxidant activity in the DPPH assay, and compound 5 give the highest antioxidant activity among all compounds, with an IC50 of 0.12 µM. All compounds exhibited moderate inhibitory effect on cytochrome CYP3A4 enzyme.


Introduction
Tabebuia avellanedae Lorentz ex Griseb is a tree found in tropical rain forests in the northeast of Brazil.Its purple bark, known as "Taheebo", have been traditionally used for various ethnopharmacological applications.For example, the inner bark has been used as one of the primary medicines by the Callawaya tribe for over 1,000 years, and Taheebo was externally used as a poultice or concentrated tea for treating a variety of skin inflammatory diseases including eczema, psoriasis, and fungal infections, and even skin cancers [1][2][3][4].In the phytochemical work reported for the species, OPEN ACCESS naphthoquinones, furanonaphthoquinones, anthraquinones, benzoic acid derivatives, benzaldehyde derivatives, iridoids, coumarins and flavonoids were isolated and identified [5][6][7][8][9].In the course of our research for antioxidants ingredients from Taheebo, six phenylpropanoid glycosides (Figures 1−4) were obtained from water extract of it.This class of compounds showed antioxidant activities [10][11][12].The paper describes the isolation and structural determination of these compounds.All compounds were tested for antioxidant activity and inhibitory potency on CYP3A4 enzyme.3 1  2 1 1 1  4 11   1 11   3 11  2 11   9 111   8 111   7 111   6 111   5 111   4 111   3 111   2 111   1 111

Results and Discussion
Water extract of dried bark of Tabebuia avellanedae was successively partitioned into an EtOAc soluble and H 2 O soluble fraction.A combination of a series of flash chromatographic separations and preparative HPLC of the EtOAc soluble fraction afforded six phenylpropanoid glycosides.
The signals at δ 5.34 (H-1′′) correlated with signals at δ 79.9 (C-3′) in the HMBC spectrum of 2, suggesting fucose moieties linked at C-3′ of glucose.Meanwhile, the location of the caffeoyl at C-4′, and phenyethyl group at C-1′ of the glucose was confirmed by the HMBC spectrum, for the long range correlation peaks between signals at δ 168.3 (C-1′′′) and δ 4.95 (H-4′); δ 72.3 (C-1) and δ 4.37 (H-1′) were observed in the HMBC spectrum.Thus, compound 2 was deduced to be 1′-O-β-(3, 4-dihydroxyphenyl)-ethyl-4′-O-caffeoyl-α-L-fucopyranosyl-(l-3′)-D-glucopyranoside.  1C-NMR spectrum (Table 2) exhibited similar signals to compound 2, except that sugar group and the linkage position.Acid hydrolysis of 5 afforded D-galactose and L-rhamnose, identified by comparison with sugar standards by HPLC.The assignment of the data of the two sugars and their linkage position were determined by detailed analysis of 1 H-NMR, 13    The detailed analysis of the 1 H-NMR, 13 C-NMR, DEPT, HMBC and HSQC spectra gave the assignments of the two sugars and their linkage position, and the signals at

Bioactivity
The isolated compounds showed strong antioxidant activity and highly decreased DPPH free radical levels, and their antioxidant activity were stronger than that of positive control ascorbic acid (Table 3).Compound 5 showed the highest antioxidant activity among all tested compounds, with an IC 50 value of 0.12 µM.In addition, the antioxidant ability of compounds 3, 5, 6 was more than that of compounds 1, 2, 4, suggesting that apart from phenolic hydroxy groups, the galactose group also plays an important role in scavenging DPPH free radicals.Apart from their antioxidant activity, their inhibitory effects on CYP3A4 enzyme were further studied.The data (Table 4) showed phenylpropanoid glycosides had inhibitory effects on cytochrome CYP3A4 enzyme.Compound 6 was the most active, with an IC 50 value of 15.1 µM.As in the antioxidant assay, the inhibitory effect on CYP3A4 enzyme of compounds 3, 5, 6 was more than that of compounds 1, 2, 4, suggesting that the galactose group plays an important role in the inhibitory activity on CYP3A4 enzyme.

General
Optical rotations were determined with a Horiba SEPA-3000 high sensitivity polarimeter.UV spectra were measured on a Shimadzu UV-1600 visible spectrometer.IR spectra were recorded on a Shimadzu IR-8400 IR spectrophotometer.NMR spectra were obtained using a JEOL Delta 600 spectrometer in CD 3 OD with TMS as internal standard.Mass spectra were measured on a JEOL SX-102 mass spectrometer.Reversed-phase HPLC (5 µm, Waters) was performed.Silica gel (63-210 µm, Wako), ODS (63-212 µm, Wako) and Sephadex LH-20 (Sigma) were used for open column chromatography.TLC was performed on silica gel 60 F 254 and RP-18 F 254 (Merck).

Plant Material
Dried bark of Tabebuia avellanedae for the present investigation was kindly provided and taxonomically identified by Taheebo Japan Co., Ltd (Japan).A voucher specimen was deposited in a database at Kanazawa University Graduate School of Natural Science and Technology under registration numbers 2008-2010.
Compound 5 (3 mg) was subjected to acid hydrolysis as described for 2 to give a sugar fraction (1.3 mg).HPLC analysis of the sugar fraction under the same conditions as in the case of that of 2 showed the presence of D-galactose and L-rhamnose, t R (min): 13.41 (L-rhamnose, negative optical rotation); 18.23 (D-galactose, positive optical rotation).
Compound 6 (3 mg) was subjected to acid hydrolysis as described for 2 to give a sugar fraction (1.3 mg).HPLC analysis of the sugar fraction under the same conditions as in the case of that of 2 showed the presence of D-galactose and L-fucose, t R (min): 12.65 (L-fucose, negative optical rotation); 18.34 (D-galactose, positive optical rotation).

Antioxidant Assay
Test samples (100 µL) at different concentrations in MeOH and DPPH (200 µL, 6 × 10 −5 µM) in MeOH were added to 96-well microtiter plates.The plate was shaken for 1 min by a plate shaker, and incubated for 30 min at room temperature in the dark.After incubation, the absorbance was recorded at 517 nm.The tested samples at different concentrations without DPPH solution were used as blank control to eliminate the influence of the sample color.Ascorbic acid was used as positive control, and DPPH solution in MeOH served as a negative control.All tests were independently performed in triplicate.DPPH radical scavenging activity (%) = [1 − (Abs S − Abs B )/Abs C ] × 100%.

Inhibitory Activity on Cytochrome CYP3A4 Enzyme
Measurement of inhibitory activity on cytochrome CYP3A4 enzyme vivid substrate and fluorescent standards were reconstituted and a standard curve was prepared.40 µL of test samples at different concentrations diluted in 1% DMSO, positive inhibition control and solvent were added to each 96-well plate, then was added 50 µL of Master pre-mix consisting of P450 BACULOSOMES in vivid CYP 450, Reaction Buffer and Regeneration System (consisting of glucose-6-phosphate and glucose-6-phosphate dehydrogenase), and the plate was mixed and incubated for 20 min to allow samples to interact with the CYP3A4 enzyme.The reaction was started by addition of 10 µL reaction starting liquid including vivid substrate, NADP and Reaction Buffer mixture, and the fluorescence was recorded using a LAS-3000 luminescent image analyzer at a wavelength of 460 nm after 30 min duration of reaction.The inhibition rate was calculated using the equation: % inhibition = [1 − relative fluorescence units (rfu) in the presence of the test sample or positive inhibition control/rfu in the absence of the test compound or positive inhibition control] × 100.

Conclusions
The study identified six phenylpropanoid glycosides from the water extract of Taheebo.Phenylpropanoid glycosides displayed strong anti-oxidant activity and moderate inhibitory activity on CYP3A4 enzyme.It is reported that oxidative stress is implicated in a wide array of human diseases, including cancer, neurodegenerative diseases, diabetes, inflammatory joint diseases, cardiovascular dysfunctions, as well as ageing, so the presence of strong anti-oxidant phenylpropanoid glycosides in Taheebo may be a good illustration supporting the many biological activities of Taheebo displays and its use in folk medicine to treat many diseases for thousands of years.

Compound 6
was deduced to have the molecular formula C 29 H 36 O 15 based on a high resolution FABMS peak at m/z 625.2144 [M+H] + .The 1 H and 13 C-NMR spectra (Table 2) of 6 exhibited similar signals to compound 2, except for the sugar moieties.Acid hydrolysis of 6 afforded D-galactose and L-fucose, and identified by HPLC comparison with sugar standards.

δ 1 H δ 13 C δ 1 H δ 13 C δ 1 H δ 13 C
Compound 5 was suggested to have the molecular formula C 29 H 36 O 15 based on a high resolution FABMS peak at m/z 625.2135 [M+H] + .The 1 H-NMR and

Table 3 .
The scavenging activity of compounds 1-6 on DPPH free radicals.

Table 4 .
Inhibitory activity on CYP3A4 enzyme of compounds 1