Isolation and identification of compounds with dioxin-induced AhR transformation inhibitory activity from the leaves of Mallotus japonicus ( Thunb . ) Muell . Arg .

1 Department of Nutrition, Faculty of Health Science, Hyogo University, Hyogo, Japan. 2 National Institutes of Biomedical Innovation, Health and Nutrition, Japan. 3 Department of Agrobioscrince, Graduate School of Agricultural Science, Kobe University, Kobe, Hyogo, Japan. 4 Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agricultural Science, Hokkaido University, Hokkaido, Japan.


INTRODUCTION
Dioxins are some of the most toxic substances known to humans, and cause serious health problems, such as birth defects, cancer promotion, immunosuppression, and weight loss upon ingestion.The mechanism of toxicity for dioxins involves binding to cytosolic aryl hydrocarbon receptors (AhR) and subsequent receptor transformation *Corresponding author.E-mail: khosokaw@hyogo-dai.ac.jp.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (De Vito and Birnbaum, 1994).
Moreover, the initial step of the toxic action has been shown to be AhR dependent (Craig and Gustafsson, 1997).Thus, drugs or food components that suppress AhR transformation can be used to treat or prevent dioxin toxicity.The extracts of 368 plant parts from 191 species were previously screened for activity against dioxin-induced AhR transformation (Nishiumi et al., 2006).As a result, the extract from the leaves of Mallotus japonicus strongly suppressed AhR transformation.M. japonicus is a deciduous tree that sprouts red-colored buds and is widely distributed in Japan, where the bark is used as a natural remedy.There have been several reports on the chemical constituents of the different parts of M. japonicus, that is, the seeds, leaves, bark, fruits, and pericarp.
Furthermore, the compounds identified were found to exhibit a range of biological activities, including cytotoxicity (Arisawa et al., 1990a), antiherpetic activity (Arisawa et al., 1990a), xanthine oxidase inhibition (Arisawa et al., 1990b), and antitumor effects (Arisawa et al., 1990b).However, no other biologically active compounds from this plant have been identified.On the other hand, the extract screening results led to the isolation and structural elucidation of an active AhR transformation inhibitor from the leaves of M. japonicus using preparative high-performance liquid chromatography (HPLC).
The present study isolated and identified AhR transformation inhibitors from M. japonicus leaves and evaluated their activities.While the majority of AhR transformation inhibitors previously reported were polyphenols (Ashida et al., 2000), the present study revealed a hydrolysable tannin as one of the AhR transformation inhibitors from M. japonicus leaves, which exhibited high activity and has not been reported previously.Thus, it is proposed that some tannins have the ability to suppress dioxin-induced AhR activity.

Plant material
M. japonicus was deposited at the Tsukuba Division, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation, Health and Nutrition (NIBIOHN) in Japan.Its accession number is 0095-79TS.Plants were cultivated and the leaves were collected in the experimental field of the Tsukuba Division of NIBIONH.The leaves were dried at about 55 to 60°C and used for the extraction.

Extraction and isolation
Dried leaves (440 g) were extracted using 70% aqueous acetone (1.3 L) three times.The combined extracts (3.9 L) were concentrated to 1 L under reduced pressure using a rotary evaporator.The precipitate was then filtrated, and the supernatant was extracted using n-hexane (300 mL, 3 times), ethyl acetate (300 mL, 3 times) and 1-butanol (300 mL, 3 times) in order of increasing polarity.The resultant extracts were concentrated to dryness, and 26 mg, 4.2 g, and 20.6 g were obtained, respectively.Finally, a 45.7g extract was obtained by evaporating the water layer.Active components were isolated from the ethyl acetate and 1-butanol fractions using preparative HPLC (SHIMADZU 10A system; Shimadzu Co., Kyoto, Japan), with a Cadenza 5CD-C18 column (20 × 150 mm; Imtakt Co., Kyoto, Japan).The mobile phase for the ethyl acetate fraction was 10 to 40% CH3CN in water (0 to 30 min), and 40 to 90% CH3CN in water (30 to 60 min), at a flow rate of 4.0 mL/min and detection at 300 nm.Four fractions were obtained and all fractions were re-chromatographed by preparative HPLC.The mobile phase for the fractions was 10% CH3CN in water (0 to 20 min), 10 to 40% CH3CN in water (20 to 30 min), and 40 to 90% CH3CN in water (30 to 60 min).Further, the obtained fractions were re-chromalograhed by preparative HPLC.The mobile phase for the fractions was 10% CH3CN in water (0 to 20 min), 10 to 90% CH3CN in water (20 to 50 min), at a flow rate of 4.0 mL/min and detection at 300 nm.As a result, five compounds (1, 22.9 mg; 2, 4.9 mg; 3, 2.7 mg; 4, 6.2 mg; and 5, 3.1 mg) were isolated.The mobile phase for the 1-butanol fraction was 10 to 90% MeOH in water (0 to 60 min), at a flow rate of 2.0 mL/min and detection at 254 or 400 nm.Three fractions were obtained and each fraction was re-chromatographed by preparative HPLC.The mobile phase for each fraction was 10 to 70% MeOH in water (0 to 60 min), at a flow rate of 2.0 mL/min and detection at 254 or 400 nm).As a result, three compounds (4, 14.0 mg; 5, 204.8 mg; and 6, 37.6 mg) were isolated.

Recording of NMR spectra
NMR spectra of each compound were recorded on a Bruker AMX500 instrument ( 1 H, 500 MHz; 13 C, 125 MHz).Chemical shifts were determined relative to residual signals for methanol-d 4 solvent (δH 3.3 ppm, δC 49.0 ppm).Field desorption and fast atom bombardment (FAB) mass spectra were obtained using a JEOL SX102A instrument.

Measurement of AhR transformation by SW-ELISA
The antagonistic effects of the isolated compounds on AhR transformation were assessed using the in vitro cell-free system of Nishiumi et al. (2006).Briefly, a rat hepatic cytosolic fraction containing AhR was pre-incubated with various concentrations (0.1 to 50 μM) of each compound dissolved in methanol.After 20 min, the cytosolic fraction was treated with 1 nM 2,3,7,8-tetrachlorodibenzi-p-dioxin or dimethylsulfoxide (10 μL/mL) as a vehicle control and incubated at 20°C for 2 h in the dark.AhR transformation was measured by SW-ELISA as described previously (Fukuda et al., 2004).Briefly, the reaction mixture consisted of 10 μL of HEDG buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 1.5 mM EDTA, 1.0 mM dithiothreitol and 10%(v/v) glycerol) containing 750 mM KCl (final concentration of 150 mM) and 40 μL of the cytosolic fraction after incubation as described earlier.The reaction mixture (50 μL) containing the transformed AhR was plated on a dioxin responsive element probe-bound 96-well microtiter plate, and AhR transformation was measured.

RESULTS AND DISCUSSION
As reported previously in Nishiumi et al. (2006), the leaves of M. japonicus strongly suppressed AhR transformation.The dried leaves of M. japonicus were  extracted with 70% aqueous acetone, and the crude extract was fractionated using different solvents and then subjected to preparative HPLC to yield six compounds (1-6, Figure 1) that exhibited antidioxin activity.Compounds 1-5 and 4-6 were obtained from the ethyl acetate and 1-butanol extracts, respectively.Thus, compounds 4 and 5 were isolated from both fractions.The FAB-mass spectrum of compound 1 showed [M -H] -and [M + Na] + ion peaks at m/z 991 and 1015, respectively, which is in good agreement with the mass calculated for C 44 H 32 O 26 (Table 1).Analysis of the 1 H NMR and 13 C NMR spectra indicated the presence of glucose, and four kinds of gallic acid derivatives (ring-A to D, Table 2) were observed.These data were almost identical to those of Yoshida et al. (1992).Accordingly, compound 1 was determined to be phyllanthusiin D. Yoshida et al. (1992) raised the possibility that phyllanthusiin D (1) found in Phyllanthus flexuosus leaves could be an artifact.As the phyllanthusiin D (1) isolated in the present experiment was not detected in the initial stage of the extraction, it may also be an artifact.
The FAB-mass spectrum of compounds 2 to 6 showed C NMR spectra of the aglycones in compounds 2 to 5 were highly similar to each other.On the other hand, both spectra of the positions 2' (δ H 8.06, δ C 132.4), 3' (δ H 6.89, δ C 116.2) and 6' (δ H 8.06, δ C 132.4) in compound 6 were different from those of compounds 2 to 5. It was revealed that the aglycone of compounds 2 to 5 was quercetin and that of compound 6 was kaempferol.The sugar moiety in compounds 3 and 4 was a monoglycoside and that in compounds 5 and 6 was a diglycoside.From the spectral analysis of compounds 3 to 6, the sugar moieties were identified as rhamnose, glucose, rutinose and rutinose, respectively.Ultimately, the isolated compounds were identified by comparison of their analytical data (Tables 1  and 3) with those in the literature (2, quercetin (Awaad et al., 2006); 3, quercitrin (Mendez et al., 1995); 4, isoquercitrin (Mendez et al., 1995); 5, rutin (Slimestad et al., 2008); and 6, kaempferol-3-rutinoside (Kazuma et al., 2003).
The antidioxin activity of the isolated compounds (1-6) is shown in Table 4. Phyllanthusiin D (1) showed the highest activity (IC 50 0.12 µM), quercitrin (3) and isoquercitrin (4) exhibited moderate activities (IC 50 0.45 and 0.97 µM, respectively), and rutin (5) exhibited weak activity (IC 50 16 µM).The two other compounds (quercetin (2) and kaempferol-3-rutinoside (6)) showed low activities.The types of compounds previously reported to exhibit antidioxin activity were mainly flavonoids and catechins (Ashida et al., 2000;Xue et al., 2017).It is known that the antagonistic effect of flavonoids is attributable to its action as a ligand of AhR and the important moiety of structure are the non-polar or less polar molecules (Ashida et al., 2000), although the important structures of flavonoids for AhR activation are number of hydroxy groups (Jin et al., 2018).
On the other hand, catechins act as antagonists by binding to the AhR chaperone protein, hsp90 (Palermo et al., 2005;Yin et al., 2009).The important structure of catechins as antagonist is phenolic groups of the A-ring (Khandelwal et al., 2013).Although phyllanthusiin D (1) has many hydroxy groups and does not have the aromatic ring like A-ring of catechins, the strong activity was exhibited.The antagonistic effect of phyllanthusiin D (1) may be expressed by binding to both AhR and hsp90, since it is known that tannins have several biological and pharmacological activities (Okuda, 2005).A main property of tannins is protein precipitation, which is termed

Conclusion
Six compounds (phyllanthusiin D, quercetin, quercitrin, isoquercitrin, rutin and kaempferol-3-rutinoside) were isolated and identified from the leaves of M. japonicus as antagonists of dioxin.Phyllanthusiin D exhibited the highest activity.The mechanism of action of the antagonists should be Activity that was measured between 0 and 100 μM was very low, so IC50 value was not indicated.
further investigated.This work shows that the tannin phyllanthusiin D has antidioxin activity and could be applied to the development of functional foods.

Table 4 .
IC50 values against TCDD-induced AhR transformation of six compounds isolated from M. japonicas leaf extract.