Rotenoid and isoflavone metabolites from an antioxidant seed extract of Dalbergia lanceolaria subsp. paniculata (roxb.) thoth

Abstract A new rotenoid named 12-O-methylrotenolol along with five known rotenoid and isoflavone metabolites were isolated from the seeds of Dalbergia lanceolaria subsp. paniculata, collected from Egypt. The structures of these compounds were identified by physical and spectroscopic data measurements ([α]D, UV, 1D- and 2D-NMR and MS). The methanol extract of the seeds exhibited strong antioxidant activity with IC50 value 0.7 µg/µl against DPPH radical, in respect to quercetin as antioxidant reference (IC50 1.5 μM), while the tested compounds from this extract showed weak activities with IC50 values ranged from 19.6 to 33.0 µM.

Isoflavonoids and their subgroup rotenoids are naturally occurring plant metabolites, mainly distributed in Leguminosae family (Deyou et al. 2015). Rotenoids are structurally isoflavonoids having an extra carbon at the C-2 position connected to the C-2 0 position through an ether linkage to form a tetracyclic ring system. They have shown potential as anti-inflammatory (Tewtrakul et al. 2009), antiviral (Phrutivorapongkul et al. 2002) and anticancer candidate agents (Deng et al. 2010;Mittraphab et al. 2018). In our continuing search for natural antioxidants, the MeOH extract of the seeds of D. paniculata was found to exhibit strong scavenging DPPH free radicals. The extract was subjected for isolation of the flavonoid constituents as antioxidant candidates. In this paper, we report the isolation and the structure elucidation of 12-O-methylrotenolol as a new rotenoid and five known rotenoid and isoflavone compounds. The isolated compounds were evaluated for their antioxidant compared to its active extract.
Compound 1 was isolated as an amorphous powder with a molecular formula of C 24 H 26 O 7 as determined from HRESI-MS positive spectrum at m/z 449.1761 [M þ Na] þ , in conjunction with 13 C-NMR data. 1D-NMR spectra ( 1 H, 13 C and DEPT-135) of 1 with the aid of HSQC experiment displayed resonances for a rotenoid unit including four aromatic methines CH-1 (d C 111.1; d H 6.84, s), CH-4 (d C 100.9; d H 6.37, s), CH-10 (d C 101; d H 6.25, d, J = 8.04 Hz), CH-11 (d C 130.6; d H 6.99, d, J = 8.04 Hz); olefinic methylene CH 2 -7 0 (d C 111.8; d H 4.98 & 4.84); three sharp singlet signals of methoxy groups at d C 56.8, 56.4 and 55.7, bearing protons at d H 3.62, 3.65 and 3.38, respectively. The most downfield singlet assigned for aliphatic 8 0 -CH 3 (d C 17.5; d H 1.67). The NMR spectra additionally revealed methylene carbons C-6 (d C 64.6) and C-4 0 (d C 31.6) bearing in HSQC spectrum pairs of non equivalent protons, where oxy H 2 -6 protons resonated at d H 4.38 (dd, J = 2.4&12 Hz, H-6b) and 4.36 (d, J = 12 Hz, H-6a), while H 2 -4 0 protons resonated at d H 3.09 (dd, J = 9.6 Hz, H-4 0 a) and 2.77 (d, J = 7.8 Hz, H-4 0 b). These methylene protons correlated in COSY spectrum with H-6a at d H 4.43 (d, J = 1.08 Hz), and H-5 0 at d H 5.14 (t, J = 9.0), respectively. However, the 13 C-NMR spectrum of 1 revealed the carbon resonances of rotenoid derivative as in rotenolone (Magalhaes et al. 1996) with absence of carbonyl carbon (C-12), and instead OCH 3 and aliphatic oxy-methin (d C 77.0; HSQC with d H 4.71, s) were observed, suggested that the carbonyl group at position 12 was reduced to secondary alcoholic OH and then methylated. This further confirmed by the HMBC data (Table S2) through the correlations from H-12 to C-11, C-6a (d C 70.7), C-12a (d C 64.4), C-11a (d C 112.7), C-1a (d C 112.0) and C-7a (d C 150.2), while C-12 received correlations from protons 6a, 11 and OCH 3 (d H 3.38). Also, the later OCH 3 protons showed strong cross peak only with C-12. The HMBC correlations (Table S2)  (d H 5.43, br) at C-12a. Other HMBC correlations ( Figure S1) assigned the quaternary carbons located on the ring junctions and also confirmed the assignments of all proton and carbon resonances in 1. The relative configuration of the new compound was cis-rotenoid structure (cis coupled B/C ring system) deduced from the chemical shift of the epimer proton H-1 at d H 6.97 (cis about $d H 7.00), whereas trans-analogues with a trans linked C/D ring unit (trans rotenoids), it resonates at about $d H 8.00) (Unai et al. 1973). The chemical shift of H-6a and its small J value (1.08 Hz) coupling with H 2 -6, suggested its b-configuration as an equatorial proton, correlated in ROESY spectrum with methylene proton (H-6b) at d H 4.38 Moreover, the J value of H-5 0 with the upfield 4 0 -H 2 (in the ABX system) and upfield 8 0 -H 3 were determined the configuration of the E-dihydrofuran ring and 6aa/12aa positions with 5'b configuration in the cis-rotenoid 1 (Kostova and Ognyanov 1986). The ROESY correlation between protons H-12 and H-6a indicated a cis relationship. The absolute configuration of C-6a and C-12a was suggested R,R, in comparison the optical rotation of 1 with those of 12a-hydroxyrotenoids (Wu et al. 2015). Thus, the structure of compound 1 was identified as 12-Omethylrotenolol. The antioxidant activities of the crude extract of D. paniculata seeds and the isolated rotenoid and isoflavone compounds (1-6) were evaluated using DPPH free radical in vitro assay (Abou El- Kassem et al. 2012). With this method, the radical scavenging activity of the total extract exhibited a strong antioxidant with IC 50 value 0.7 mg/ml compared to quercetin as antioxidant reference (IC 50 value 0.45 mg/ml). However, the tested compounds here lack hydroxyl groups and therefore displayed weak activities with IC 50 values ranged from 19.6 to 33.0 mM (Table 1), in respect to the potent antioxidant activity of their crude extract (Rice-Evans et al. 1996). The activity of the extract may be attributed to a possibly synergistic effect of these compounds or may be due to the presence of unidentified compounds in the extract.   (2) 19.5 ± 0.78 cis-12a-Hydroxyrot-2-enonic acid (3) 26.8 ± 0.50 2-Methoxygliricido (4) 27.6 ± 0.39 Pseudobaptigenin (5) 22.4 ± 0.31 Dalpatein (6) 20.7 ± 0.43 Quercetin (antioxidant)

Experimental
1.5 ± 0.02 (0.45 mg/ml) layer). NMR spectra were obtained on Bruker 600 MHz NMR spectrometer with standard pulse sequences operating at 600 MHz for 1 H and 150 MHz for 13 C NMR, respectively. DMSO was used as solvent. UV-visible absorption spectra were taken on UV-2550 UV-vis spectrometer (Shimadzu, Kyoto, Japan). Optical rotations were detected by Jasco P-1010 Polarimeter. UV spectra were obtained using Shimadzu UV 240 spectrometer (Tokyo, Japan). ESI-MS (Electron Spray Ionization mass spectrometry) spectra were recorded on LC-MS TOF (Bruker, Germany).

Plant material
The seeds of Dalbergia lanceolaria subsp. paniculata (Roxb.) Thoth were collected from Zoo Garden, Giza, Egypt in October 2014. The recent accepted denomination of the species is Dalbergia lanceolaria subsp. paniculata (Roxb.) Thoth with a synonym Dalbergia paniculata Roxb. The plant was identified by Dr. Mohammed El-Gibaly, Consultant of Plant Taxonomy, Cairo University, and the respective voucher specimen (No. 23-4-2015) has been deposited in Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

Extraction and isolation
The air-dried seeds of D. paniculata (1 kg) were exhaustively extracted with methanol over a period of 5 days at room temperature. Removal of solvent under reduced pressure provided MeOH crude extract (45 g). The resulting total extract was defatted by n-hexane and the residue (15 g) was further fractionated by column chromatography over polyamide 6S (Merck) eluted with water followed by percentage increasing MeOH (up to 100%). All fractions obtained were combined according to their PC (1 MM in BAW eluent) analysis to give 7 fractions. Each of these fractions was subjected to preparative PC, where the compounds appeared as separated bands under UV. Each paper band was cutted and eluted with 70% aqueous MeOH to extract the absorbed compound. Finally, column Sephadex LH-20 eluted with aqueous MeOH was used to obtain pure compounds 1 (7 mg), 2 (36 mg; R f : 0.0.82; [a] D 20 -12.9, c 0.1, MeOH), 3 (11 mg; R f : 0.84; [a] D 20 -0.75, c 0.01, MeOH), 4 (9 mg, R f : 0.81), 5 (4 mg, R f : 0.92) and 6 (5 mg, R f : 0.90).

Antioxidant assay
The antioxidant activity of MeOH extract and isolated compounds 1-6 against 1,1-diphenyl-2-pycrylhydrazyl (DPPH) free radicals was carried out using modified method of (Abou El-Kassem et al. 2012). Briefly, 190 ml DPPH (80 mg/ml in EtOH) was added to a 10 ml sample solution (100 mg/ml in DMSO) in a 96-well plate and mixed by shaking. After incubation for 30 min at room temperature the absorbance was recorded using an ELISA reader at 517 nm. The inhibition percentage (%) of the radical scavenging activity was calculated using the following equation: Where A 0 is the absorbance of the control and As is absorbance of the sample at 517 nm. The experiment was carried out in triplicate, using quercetin as an antioxidant reference.