Isolation and structural characterization of phytoconstituents from leaves of Bignonia binata

Abstract Phytochemical investigation of Bignonia binata leaves led to the isolation of three new compounds: including a glycoside of simple alcohol, namely binatoside (2), 3,4-dihydroxy-N-methyl piperidin-2-one (7), and a phenyl ethanoid glycoside, namely bignanoside C (8), alongside with five known compounds; including a glycoside of simple alcohol; (2S) propane-1,2-diol 1-O-(6-O-caffeoy1)-β-D-glucopyranoside (1), phenyl ethanoids; leucosceptoside A (3) and plantainoside C (4), and iridoids; ipolamiide (5) and strictoloside (6). The structure of the isolated compounds was elucidated by various spectroscopic methods, including 1 D and 2 D NMR experiments, HR-ESI-MS as well as by comparison with the literature. Graphical Abstract


Introduction
Bignonia is a genus of flowering plants belonging to family Bignoniaceae which have been distributed from USA to South America (Mahmoud et al. 2019a(Mahmoud et al. , 2020a(Mahmoud et al. , 2020b)).
Bignonia binata Thunb.(syn: Clytostoma binatum Thunb.) is recorded from Mexico to Argentina (Zuntini et al. 2015).Our previous phytochemical investigations of B. binata revealed the isolation of a variety of secondary metabolites, including one neolignan, two iridoids, two lignans, one triterpene, one sterol, two phenylethanoids, four flavonoids and one nucleoside, in addition, the cytotoxic, antimicrobial, antimalarial, antitrypanosomal, hepatoprotective and nephroprotective, DPPH radical scavenging activities have been evaluated (Mahmoud et al. 2020a;2020b;Hamed et al. 2021;Samy et al. 2022).Additionally, the chemical profile was investigated by LC-HR-ESI-MS, where it revealed the presence twelve compounds related to phenylethanoids, flavonoidal glycosides and two iridoids (Samy et al. 2022).The present study deals with isolation and structural characterization of eight compounds, including three new ones (Figure 1).
The 13 C-NMR of compound 7 showed the presence of six carbon signals, including two methylene carbon signals at d C 34.4 (C-5) and 56.3 (C-6), two oxymethine carbon signals at d C 80.7 (C-3) and 75.8 (C-4).Additionally, one quaternary carbon signal at d C 171.2 and one tertiary methyl carbon signal at d C 40.5, which resonated as a singlet proton signal at d H 2.46 (3H) in 1 H-NMR.The 1 H-1 H COSY spectrum of compound 7 (Figure S12) revealed the presence of cross peaks between H-5 at d H 2.08 with H-6a and H-6b at d H 3.86 and 3.33, respectively and with H-4 at d H 4.61, additionally cross  peaks between H-3 at d H 3.77 and H-4 at d H 4 at d H 2.46 and C-3 and C-6 at d C 80.7 and 56.3, respectively, indicated the attachment of the CH 3 to the nitrogen atom.From the above 1 H-1 H COSY and HMBC of compound 7, it was concluded that it was tentatively identified as 3,4-dihydroxy-N-methyl piperidin-2-one.

General experimental procedures
Optical rotation of the compounds was obtained on a JASCO P-1030 polarimeter. 1 H and 13 C NMR spectra were recorded on Bruker Avance 400 MHz, 500 MHz and 600 MHz instruments in DMSO-d 6 and CD 3 OD.HR-ESI mass spectrum was taken on a LTQ Orbitrap XL mass spectrometer.Solvents used in this work, e.g., petroleum ether (pet.ether; B.p. 60-80 C), dichloromethane (DCM), ethyl acetate (EtOAc), methanol (MeOH), and ethanol (EtOH), were purchased from El-Nasr Company for Pharmaceuticals and Chemicals, Egypt and were distilled before use.Acetonitrile (CH 3 CN) and MeOH of high performance liquid chromatography (HPLC) grade were used for HPLC separations and purifications and were obtained from SDFCL sd Fine-Chem Limited, India.Deuterated solvents (Sigma-Aldrich, Germany), including methanol (CD 3 OD) and dimethyl sulfoxide (DMSO-d 6 ), were used for nuclear magnetic resonance (NMR) spectroscopic analyses.Column chromatography (CC) was performed using silica gel 60 (El-Nasr Company for Pharmaceuticals and Chemicals, Egypt; 60-120 mesh) or sephadex LH-20 (0.25-0.1 mm, GE Healthcare, Sweden), RP 18 (Nacalai Tesque, Kyoto, Japan), and Diaion HP-20 (Mitsubishi Chemical Industries Co., Ltd.Japan), while silica gel GF 254 for thin layer chromatography (TLC) (0.25-0.1 mm, El-Nasr Company for Pharmaceuticals and Chemicals, Egypt) was employed for vacuum liquid chromatography (VLC) (6 Â 30 cm, 90 g) at room temperature and then the sample was loaded as solute and the elution produced by the aid of water vacuum pump.
Thin layer chromatography (TLC) analyses were carried out using pre-coated silica gel 60 GF254 plates (E.Merck, Darmstadt, Germany; 20 Â 20 cm, 0.25 mm in thickness).Ultraviolet lamp (UVP, LLC, USA) was used for visualization of spots on thin layer chromatograms at 254 and/or 365 nm.Spots were visualized by spraying with 10% sulfuric acid in methanol followed by heating at 110 C on a hot plate.

Plant material
The leaves of B. binata were collected in December 2015, from El-Zohria botanical garden, Giza, Egypt.Authentication of the plant was identified by Prof. Nasser Barakat, Department of Botany, Faculty of Science, Minia University, Minia, Egypt.A voucher specimen (Mn-ph-Cog-033) has been deposited in the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Minia University, Minia, Egypt.

Extraction and isolation
The air dried powdered (2.7 kg) of B. binata leaves were extracted by maceration with 95% ethanol till exhaustion at room temperature with frequent agitation.The ethanolic extract was concentrated under reduced pressure to give a viscous green residue (415 g), which was suspended in 400 ml of distilled water, transferred to a separating funnel and defatted with successive portions of pet.ether (300 ml each, Â6).The combined pet.ether fractions were concentrated under reduced pressure to yield the pet.ether fraction (Fr.I, 75 g).The mother liquor was repartitioned with successive portions of EtOAc (300 ml each, Â6).The combined EtOAc fractions were concentrated under reduced pressure to yield the EtOAc fraction (Fr.II, 125 g).Finally, the remaining mother liquor was concentrated under reduced pressure to afford the aqueous fraction (Fr.III, 200 g).
The EtOAc fraction (Fr.II, 125 g) was subjected to fractionation using VLC-technique.Elution was performed initially using EtOAc and then EtOAc-MeOH gradient mixtures in order of increasing the polarity gradually in 10% by MeOH till EtOAc-MeOH 70:30.The effluents were collected in fractions (3 L each).Each polarity was concentrated under reduced pressure affording four subfractions (Fr.II 1 -Fr.II 4 ).

Conclusion
Medicinal plants are considered as resources for bioactive secondary metabolites.Our study aimed to investigate the phytoconstituents of B. binata leaves, resulting in the isolation and structural characterization of three compounds, together with five known compounds, using various spectroscopic techniques.The identified compounds belong to different classes, indicating the variety of plant-derived secondary metabolites.

Disclosure statement
No potential conflict of interest was reported by the authors.

Figure 1 .
Figure 1.Chemical structures of the isolated compounds.