A new linoleiyl arabinopyranoside from the bark of Bauhinia racemosa Lam and a new flavonoidal glycoside from the leaves of Cordia dichotoma Linn

Abstract Phytochemical investigation is very valuable for the ethnomedicinally important plants Bauhinia racemosa Lam (BR) and Cordia dichotoma Linn (CD) used for the cure of variety of ailments. This study was thus designed for phytochemical investigation of BR bark and CD leaves. Phytoconstituents were isolated from the methanolic extracts of the plants by column chromatography using silica gel as stationary phase. The structures had been established on the basis of their physicochemical and spectral data, i.e. IR, 1H NMR, 13C NMR and MS. Elution of the columns with different solvents furnished six compounds (1–6) from the methanolic extract of BR bark and three compounds (7–9) from the methanolic extract of CD leaves which were structurally elucidated. The present phytochemical investigation reported several new compounds useful in increasing the existing knowledge of phytoconstituents from BR bark and CD leaves which is very valuable, as these drugs are used in the Indian traditional systems of medicine.


Introduction
Bauhinia racemosa Lam (family Caesalpiniaceae) commonly named as mountain ebony in English, gul-e-anehnal in Urdu, kachnal in Hindi and Sona in Marathi, is used in different indigenous systems of medicine (Chopra et al. 1956;Gamble 1967;Garodia et al. 2007). Various parts like stem bark, leaves and roots of this plant are popular among the various ethnic groups in India for the therapy of several ailments and possess hypolipidemic, antioxidant and cytotoxic activities (Jain et al. 2004;Jagtap et al. 2006;Sashidhara et al. 2013;Miceli et al. 2015). The bark is reported to have analgesic, antipyretic, antifilarial, anthelmintic, antimalarial, abortifacient, anti-hepatoprotective, ulcerogenic and various other pharmacological activities (Sashidhara et al. 2012;. Previously reported phytochemical constituents from the bark of this plant are triterpenoids, sterols, octacosane, β-sitosterol and β-amyrin; from the root bark and the flowering buds are pentacyclic phenols and others (Sashidhara et al. 2012;Jain et al. 2013;Radha et al. 2016).
Cordia dichotoma Linn (family Boraginaceae) commonly named as Indian cherry in English, vadgundo in Gujarati and lasora in Hindi, is used in different indigenous systems of medicine like Ayurveda and Unani (kirtikar & Basu 1975). Various parts like stem, bark and leaves of this plant are popular among the various ethnic groups in India for the therapy of variety of ailments as demulcent, diuretic, anthelmentic, astringent, expectorant and anti-diabetic. The leaves are traditionally used for the therapy of jaundice at Dandakaranya area, Andhra Pradesh in India. It is reported to have antifertility, juvenomimetic, anti-inflammatory, antioxidant and various other pharmacological activities (Agnihotri et al. 1987;Choudhary et al. 1990;Neraliya & Srivastava 1997). Carotenoids are chiefly present in the leaves of this plant which have potent antioxidant activity (Valvi et al. 2011). Phytoconstituents like latifolinal, latifolidin and cordicinol were also isolated from the fruits and leaves of Cordia latifolia (Siddiqui et al. 2006(Siddiqui et al. , 2010. This paper describes the isolation and structural elucidation of potential phytoconstituents or compounds from the methanolic extracts of the plants B. racemosa bark and C. dichotoma leaves to explore and to increase the existing knowledge of their phytoconstituents which will be very valuable as these drugs are used in the Indian traditional systems of medicine.

Results and discussion
Structures of the phytoconstituents isolated from methanolic extract of B. racemosa (BR) bark 1-6 and phytoconstituents isolated from methanolic extract of C. dichotoma (CD) leaves 7-9 are shown in Figure 1.
Compounds 1-4, 6 isolated from BR bark and compounds 8 and 9 from CD leaves are the known compounds reported here for the first time in the respective plants. They were identified as α-d-glucopyranosyl- α-d-arabinose and β-d-arabinose, respectively by comparing their IR, 1 H NMR, 13 C NMR and MS spectral data with the literature values. The compounds 5 and 7 are the new compounds and are being reported here for the first time in the mentioned plants.
Compound 5 was obtained as a yellow sticky mass from methanol eluant and recrystallised from solvents, acetone:methanol (1:3). It responded to general tests of glycosides and showed Infrared (IR) absorption bands for hydroxyl group (3410, 3350 cm −1 ), ester (1723 cm −1 ) and doubly bonded unsaturation (1635 cm −1 ). On the basis of mass spectroscopy (MS) and Carbon nuclear magnetic resonance spectroscopy ( 13 C NMR) data, the molecular ion [M] + peak was determined at m/z 412 corresponding to molecular formula (MF) of an unsaturated acyl glycoside, C 23 H 40 O 6 . The ion peaks arising at m/z 279 [C 18 H 31 O 2 ] + and 149 [C 5 H 9 O 5 ] + suggested that the linoleiyl group was attached with a pentose moiety. The proton nuclear magnetic resonance ( 1 H NMR) spectrum displayed a one-proton doublet at chemical shift (δ) 5.38 (J = 4.8 Hz) assigned to anomeric H-1′ proton, one-proton multiplets from δ 4.21 to 3.58 due to other sugar protons and a two-proton doublet at δ 3.36 (J = 7.8 Hz) due to methylene H 2 -5′ protons, a two-proton triplet at δ 2.33 ascribed to methylene H 2 -2 adjacent to the ester function, two-proton multiplets at δ 2.08 due to methylene H 2 -8 protons adjacent to first double bond, two-proton multiplets at δ 2.71 due to methylene H 2 -11 protons in between two double bonds, two-proton multiplets at δ 1.92 due to methylene H 2 -14 protons adjacent to second double bond, one-proton multiplets between δ 5.95-5.28 due to vinylic-protons, signal brs at δ 1.29 due to other CH 2 protons and a three-proton triplet at δ 0.83 (J = 6.5 Hz) accounted to terminal C-18 methyl protons. The carbon nuclear magnetic resonance ( 13 C NMR) spectrum exhibited signals for ester carbon at δ 168.51 (C-1), anomeric carbon at δ 105.37 (C-1′), other sugar carbons from δ 75.29 to 65.26, vinylic carbons from δ 145.66 to 116.24, methylene carbon adjacent to ester group δ 38.41, methylene carbon between double bonds at δ 32.70, other methylene carbons between δ 31.33 and 22.68 and methyl carbon at δ 14.16 (C-18). On the basis of these evidences, the structure of 5 was elucidated and confirmed as linoleiyl-O-β-d-arabinopyranoside.

Plant materials
B. racemosa Lam bark was collected from Chittoor district of Andhra Pradesh and was authenticated by Dr k. Madhava Chetty (botanist), Department of botany, Sri Venkateswara University, Tirupati, India (authentication reference number: SVUBH/LE/1876).
C. dichotoma Linn leaves were collected from nearby region of kukrail forest and was authenticated by National Botanical Research Institute, Lucknow, India (authentication reference number: NBRI/CIF/306/2012).

Extraction and isolation
The CD leaves were shade dried and coarsely powdered. The powdered leaves (1.0 kg) were extracted exhaustively with methanol in a Soxhlet extractor. The methanolic extract was concentrated under reduced pressure to give a viscous mass . The methanolic extract was dissolved in a minimum amount of methanol and adsorbed on silica gel (60-120 mesh, Fisher Scientific) for preparation of slurry. It was air-dried and chromatographed over a silica gel column packed in petroleum ether. The column was eluted successively with petroleum ether, mixture of petroleum ether and chloroform (90:10, 75:25, 50:50 and 25:75), pure chloroform, and finally mixture of chloroform and methanol (99.5:0.5, 99:1, 98:2, 95:5), and methanol in the order of increasing polarity to isolate the compounds. Fractions were collected in 45-ml aliquots in fraction collector. It afforded several fractions. Homogeneity of column fractions was controlled by thin-layer chromatography (TLC). It was conducted on Whatman silica gel G-coated TLC plates with spots visualised under UV 254 nm illumination, by staining with iodine vapour and by spraying the plates with sulphuric acid-anisaldehyde reagent. Homogeneous column fractions (having same R f values) were combined and crystallised. Same methodology was performed for BR bark also. The isolated phytoconstituents were recrystallised to get the pure compounds. They were further processed for experimental procedure of structure identification (Ahmad et al. 2010).

Experimental procedure of structure identification
The chemical structures of the isolated phytoconstituents were elucidated by several instrumental analyses. Melting points (m.p.) were determined on a Perfit melting point apparatus in one end open capillary tubes and are uncorrected. The IR spectra were recorded in kBr pellets on an Agilent Cary 630 FTIR spectrometer in the range of 4000-450 cm −1 ; 1 H NMR spectra on a Bruker DRX-300 spectrophotometer using TMS (tetramethylsilane) as an internal standard and deuterated solvent to solubilise the phytoconstituent; 13 C NMR spectra on a Bruker DRX-300 spectrophotometer in 5-mm spinning tubes at 27 °C; the electrospray mass spectra by Waters UPLC-TQD Triple Quadrupole mass spectrometer (LC-MS/MS) (Ahmad et al. 2010). All the instruments for the spectral analysis are housed, operated and maintained in the Sophisticated Analytical Instrument Facility, Central Drug Research Institute, Lucknow, India.

Conclusion
This study characterised several new and known phytoconstituents isolated from the B. racemosa bark and C. dichotoma leaves. Compounds 1-4, 6 isolated from BR bark and compounds 8 and 9 from CD leaves are the known compounds reported here for the first time in the respective plants. The compounds 5 and 7, namely linoleiyl-O-β-d-arabinopyranoside and 5,7-dihydroxy-4′-methoxyflavone-7-O-β-d-glucopyranosyl-(Glc-6 → Glc-1′)-O-βd-glucopyranosyl-(Glc-6 → Glc-1′′)-O-β-d-glucopyranosyl-Glc-2′′-linolenate are the new compounds and are being reported here for the first time in the mentioned plants. Hence, the existing knowledge of their phytoconstituents may be increased by the present phytochemical investigation which is valuable as these drugs are used in the Indian traditional systems of medicine.