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Journal of Pharmacognosy and Phytochemistry 2016; 5(1): 206-210 E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2016; 5(1): 206-210 Received: 20-11-2015 Accepted: 23-12-2015

Lokadi Pierre Luhata Laboratoire de Phytochimie et contrôle qualité, ISAV-Kimwenza, 3724, Kinshasa, The Democratic Republic of Congo (DRC). Namboole Moses Munkombwe Department of Chemistry, University of Zambia, 32379, Lusaka, Zambia Hanzooma Hatwiko Department of Pharmacy, University of Zambia, 32379, Lusaka, Zambia Correspondence Lokadi Pierre Luhata Laboratoire de Phytochimie et contrôle qualité, ISAV-Kimwenza, 3724, Kinshasa, The Democratic Republic of Congo (DRC).

Isolation and 1H-NMR identification of a tiliroside from Odontonema strictum (Acanthaceae)

Lokadi Pierre Luhata, Namboole Moses Munkombwe, Hanzooma Hatwiko Abstract The dried leaves (310g) of Odontonema strictum (OSM) were poured and extracted in MeOH/DCM (1/1//v/v) for 48 hours to yield 25g of dry residue which were purified using chromatographic methods (vacuum liquid chromatography column (VLC), Sephadex LH-20 column and Thin Layer Chromatography (TLC)). Fractions containing flavonoids were mixed for further purifications. Compound 1, yellow amorphous powder (8 mg) was re-dissolved in MeOH and developed in EtAOc/MeOH (2:1) to give an Rf value of 0.27 and mp 301-303°C. The compound reacted positively to HCl-Mg and Molish reactions. The 1 H-NMR (400 MHz, CDCl3) spectrum displayed the typical signals for tilirosides. The presence of glycosidic flavonoids may possibly contribute to the pharmacological properties of OSM. Keywords: Odontonema strictum, identification, flavonoids, tilirosides, 1H NMR spectroscopy. Introduction OSM (Figure 1), a plant mostly found in tropical regions, is used as a folk medicine in Burkina-Faso to treat hypertension [1]. Felix Kini and his colleagues [2] have identified flavone glycosides (C-heterosides and O-heterosides in 0.37% and 1.13% respectively) as the active secondary metabolites. In our previous work, we have isolated stigmasterol and beta-sistosterol from OSM and confirmed the antibacterial properties of these phytosterols [3-4]. Recently, the hepatoprotective and antioxidant activity of OSM against CCl4-Induced Hepatic Injury in Rats have been confirmed by M. S. Refaey and his colleagues [5]. In the continuing study on this plant, we isolated and identified a compound which belongs to the group of tilirosides (TLDs). We are still working to elucidate the complete structure of the isolated compound. TLDs are glycosidic flavonoids which exhibit several pharmacological properties such as anti-diabetic and anti-hyperlipedemic [6], antiviral and cytotoxic activity [7], anti–inflammatory, anti-rheumatism [8], anti-microbial and antioxidant [9-10]. Kaempferol-3-O-β-D (6-O-transp-cinnamoyl) glucopyranoside (trans-tiliroside,) revealed significant anti-hyperglycemic effects when compared with phenethyldiguanide in alloxan mice. As a part of trans-tiliroside, kaempferol-3-O-β-D-glucopyranose and related analogues revealed weak anti-diabetes activity [11]. Structurally, Tilirosides are composed of three distinct parts: a flavonoid, a phenyl propanoid and a sugar (Figure 2).

Fig 1: Odontonema strictum

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‘

Fig 2: Example of a tiriloside

Experimental Plant Material Plant specimens (leaves) were collected in Lusaka (April 2014) and identified by Doctor Chuba and his team at the Department of Biological Sciences, University of Zambia. Voucher samples were prepared and deposited in the Herbarium of the Department of Biology. The plant samples were shade dried at room temperature and powdered into a fine powder in a blender. Extraction and Isolation The air-dried leaves of OSM (310 g) were extracted with 1.6L of Methanol (MeOH) and Dichloromethane (DCM), (1/1//v/v) for 24 hours to yield 25g of dry residue. The 25 grams were chromatographed over 40 g Si gel H in a vacuum liquid chromatography column (VLC) (13 x 4 cm). VLC was prepared using a sintered filter funnel and fine silica gel. The three quarters filled sintered funnel was placed of vacuum Erlemeyer flask and connected to water. The vacuum was applied to compact the silica and hexane flashed through twice for further compaction. The column was left under vacuum to dry. Once the VLC was dry, the slurry was introduced on top and filter paper was used to cover the slurry and the vacuum was applied using suction. Initially, 150 ml of mixture of n-HeX-EtOAc (1:1//v/v) was gradually added into the VLC and flashed through. The polarity was then increased by adding EtOAc (50% -100%) and later MeOH (4:1//v/v). In total, 10 fractions were collected (50 ml each). The total volume used for flashing through VLC was 200 ml. Fractions were mixed according to the chromatograms of TLCs and weight. The fractions were left to dry at room temperature. Fractions 1 and 2 (650 mg), fraction 3 (270 mg), fractions 4 and 5 (500 mg), fractions 6 and 7 (425 mg), fraction 8( 1500 mg) and fractions 9-10 (1700 mg) The Chromatogram of Fractions 8,9 and 10 gave an orange spot after sprayed with acidified vanillin followed by gentle heating (Figure 3b). These three fractions were mixed (and named fraction A) and left to dry at room temperature. Fraction A (3200 mg) was purified on a Sephadex LH-20 column (40 x 2 cm) using methanol and chloroform (1:2//v/v) as eluent to give Yellow amorphous powder (8 mg).

a. b.

Fig 3: Chromatograms of fraction A in two different systems. Results and Discussions The yellow crystals were re-dissolved in MeOH and developed in EtAOc/MeOH (2:1) to give an Rf value of 0.27 and mp 289-291 °C. The compound reacted positively to HCl-Mg and Molish reactions. For 1H NMR spectroscopy, the compound was dissolved in Chloroform (CDCl3). The 1 H-NMR δ (400 MHz, CDCl3) spectrum displayed (Figure 4) the typical signals of the kaempferol nucleus [12] (Table 1). A set of Meta coupled aromatic methines are present at [δ 6.27, d, (H6) and δ 6.38, d, (H8)]. Additionally, two A2B2 systems, each integrating for two protons, were found respectively at [δ 7.10, dd, 2H, (H2′/H6′) and δ 7.05, dd, 2H, (H3′/H5′)] and [δ 7.57, d, 2H, (H2′′′/H6′′′) and δ 6.95, d, 2H, J = 8.5 Hz (H3′′′/H-5′′′)]. Two olefinic methines [δ 7.61, d, (H7′′′) and δ 6.27, d, (H8′′′)] with trans coupling and finally evidence of a sugar moiety including five deshielded methines [δ 5.19, d, (H1′′) and those occurring between δ 3.91 and δ 3.38, (H2′′, H3′′, H4′′, H5′′)]. The spectrum showed also the presence of 5 peaks indicating the presence of five methyl groups at 0.896, 1.100, 1.158, 1.285, 1.535 ppm.

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Journal of Pharmacognosy and Phytochemistry Table 1: 1H NMR chemical shift values for the isolated compound recorded in CDCl3 (400 MHz) a

a-Chemical shift values are in δ (ppm).

Conclusion Fraction an extracted from OSM yielded a yellow powder. The structure of this compound was identified as belonging to the family of tiliroside (Fig. 2) based on its physical properties and 1H NMR spectra comparison with those of the reported compounds in the literature [13-14]. The identification of TLDs in OSM confirms the work done by Felix Kini and his colleagues in 2005. The presence of these phytochemicals could contribute to the bioactivity exhibited by the plant. In fact, TLDs are known as anti –inflammatory, anti-microbial, anti-hypertensive, anti-diabetic, anti-hyperlipedemic, anti-oxydant, antiviral and cytotoxic activity [15]. Further

investigations will lead to the characterization of the active TLDs of OSM. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgements The authors wish to thank Professor Kelly Chibale (University of Cape Town/ South Africa), FRSSAf, the Director of UCT Drug Discovery & Development Centre (H3-D) and MRC/UCT Drug Discovery and Development Research Unit for the use of his laboratory facilities.

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Fig 4: 1H NMR spectrum of compound 1

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11. Jin Zhu, Yanjun Zhang, Yi Liu, Hongwan Chu, Hongquan Duan. Synthesis and Biological Activity of trans-Tiliroside Derivatives as Potent Anti-Diabetic Agents. Molecules, 2010; 15:9174-9183.

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Lignan and phenylpropanoid glycosides from Fernandoa adenophylla. Phytochemistry, 2001; 57:1245-1248.

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14. Namboole Moses Munkombwe, Pelotshweu Galebotswe, Kabelo Modibesane, Nametso Morebodi. Phenylpropanoid glycosides of Gnidia polycephala Phytochemistry, 2001, 2003; 64:1401-1404.

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