-Ethoxy-6

: Isoﬂavonoids possess a 3-phenylchroman skeleton and are the biologically active secondary metabolites of various plants that are used for different health promoting and restoring effects through a variety of mechanisms. Chromatographic separation of the n -hexane extract from the stems of Placolobium vietnamense led to the isolation of a new isoﬂavone derivative, placovinane ( 1 ), together with four known compounds ( 2 – 5 ). The structures of isolated compounds were identiﬁed from their spectroscopic data and by comparison with the literature. All isolated compounds were evaluated for their α -glucosidase inhibition. They all exhibited potent α -glucosidase inhibition with IC 50 values ranging from 11.0 to 87.3 µ M, which was signiﬁcantly less than the positive control acarbose (IC 50 179 µ M). The cytotoxicity of 1 was evaluated against KB, Hep G2, and MCF7 cell lines, and displayed weak cytotoxicity toward KB and Hep G2 cell lines, with the IC 50 values of 89.6 and 93.8 µ M, respectively.


Introduction
Flavonoids are polyphenolic compounds that are ubiquitous in nature.Flavonoids have a variety of therapeutic properties, including anticancer, antioxidant, anti-inflammatory, and antiviral effects [1,2].Isoflavonoids are a subclass of flavonoids derived from the phenylpropanoid and polyketonic chain pathway, which is established both enzymatically and genetically.There are many biological properties associated with isoflavones, including antioxidant, anticancer, antimicrobial, and anti-inflammatory properties [3].Placolobium vietnamense N.D.Khoi & Yakovlev is distributed in primary forests in the Central Highlands of Vietnam.It is a perennial tree with brown bark, straight, and cylindrical trunk.Its fruit is a small pod with one seed.This plant is used in folk medicine to treat debility, snake bites, and to recover after childbirth [4].As part of our research program focusing on new isoflavonoids from P. vietnamense, an indigenous species in Vietnam, we report herein the isolation and structural characterization of a new isoflavonoid derivative, placovinane (1), along with four known compounds, daidzein (2) [5], 8-O-methylretusin (3) [6], isorhamnetin (4) [7], and vanillic acid (5) [8] (Figure 1) from the stems of P. vietnamense.The structure of 1 was elucidated by the spectroscopic data interpretation and comparison with those reported in the literature.All isolated compounds were assayed for their α-glucosidase inhibition, and the new compound 1 was tested for its cytotoxicity against the KB, Hep G2, and MCF7 cell lines.
It should be noted that 1 has an ethoxy group at the C-1″.Since the ethoxy group is relatively rare in nature, it seems probable that this new compound is an artifact of the isolation process, which involves the use of EtOH as a solvent.
It should be noted that 1 has an ethoxy group at the C-1″.Since the ethoxy group is relatively rare in nature, it seems probable that this new compound is an artifact of the isolation process, which involves the use of EtOH as a solvent.It should be noted that 1 has an ethoxy group at the C-1 .Since the ethoxy group is relatively rare in nature, it seems probable that this new compound is an artifact of the isolation process, which involves the use of EtOH as a solvent.

α-Glucosidase Inhibitory Activity
All isolated compounds were evaluated for their α-glucosidase inhibitory activity, with acarbose used as a reference compound.The resulting IC 50 values, as listed in Table 2, indicated that all the compounds showed stronger α-glucosidase inhibitory activity than acarbose (IC 50 179 µM).Compound 2, especially, displayed the most highly potent αglucosidase inhibition with an IC 50 value of 11.0 µM.The results suggested that compounds 1-5 from the stems of P. vietnamense were active α-glucosidase inhibitors that could be used as effective hypoglycemic drugs for diabetes therapy.

Cytotoxicity against Human Cancer Cell Lines
Further evaluation of 1 for its in vitro cytotoxicity against three human cancer cell lines (KB, Hep G2, and MCF7) was performed using the MTT assay, which is detailed in the Materials and Methods.The resulting IC 50 values displayed in Table 3 were compared to ellipticine as a positive control.Compound 1 was shown to have mild cytotoxicity against KB and Hep G2 cell lines, with IC 50 values of 89.6 and 93.8 µM, respectively.

General Experimental Procedures
The NMR spectra were recorded on Bruker AvanceNEO 600 MHz and Bruker Avance III™ HD 500 MHz NMR spectrometers in DMSO-d 6 (Merck, Darmstadt, Germany).Optical rotations were measured on a A.KRÜSS Optronic P8000 polarimeter (KRÜSS, Hamburg, Germany).The IR data were obtained with a Jasco 6600 FT-IR spectrometer using an ATR technique (Jasco, Japan).The HRESIMS spectral data were generated with a X500 R QTOF model mass spectrometer (Sciex, Framingham, MA, USA) and Dionex Ultimate 3000 HPLC system hyphenated with a QExactive Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific, Waltham, MA, USA).Silica gel 70-230 mesh (Merck) and Sephadex LH-20 gel (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography.

Plant Material
The stems of P. vietnamense were collected in Dak Nong province, Vietnam, in February 2017.This plant species was identified by botanist Vo Van Chi.A voucher specimen (No.SGU-A001) was deposited in the Herbarium of the Laboratory of Chemistry-Biology-Environment, Sai Gon University, Vietnam.

α-Glucosidase Inhibitory Assay
The in vitro α-glucosidase inhibitory evaluation of 1-5 was performed according to the previously described protocol [12].Then, 100 mM pH 6.9 sodium phosphate buffer was used as a solvent to dissolve the substrate (5.0 mM p-nitrophenyl-D-glucopyranoside) and α-glucosidase (0.2 U/mL).The substrate (40 µL) was added to the reaction mixture after the inhibitor (50 µL) had previously been preincubated with α-glucosidase.The reaction was stopped after 20 min of enzymatic activity at 37 • C by adding 130 µL of 0.2 M Na 2 CO 3 .The amount of enzyme activity was determined by calculating absorbance at 405 nm.After each sample was tested in triplicate at five different concentrations around the IC 50 values, the mean values were retained.The inhibition percentage (%) was determined as follows: Inhibition (%) = [1 − (A sample /A control )] × 100.

-5. Compound IC 50 (µM) a
a Results were expressed as the means ± SD of three replicates.b Positive control.

Table 3 .
Cytotoxic activity of 1 against three human cancer cell lines.
a Results were expressed as the means ± SD of three replicates.b Positive control.