Isolation and characterization of some flavonoids from the leaf of Tapinanthus globiferus growing on Vitex doniana

. Tapinanthus globiferus (A. Rich) Tiegh (Loranthacae) is a semi-parasitic plant growing on several plant species such as Vitex doniana . it is used in ethno-medicine for the treatment of fungal infection, itching, hypertension, ulcers, epilepsy, diabetes and cancer. The aim of this study was to isolate bioactive compound(s) from the leaf of T. globiferus . The powdered plant material was extracted with 90% methanol using maceration method and the resulting crude methanol leaf extract was partitioned into n -hexane, chloroform, ethylacetate and n -butanol fractions. The ethylacetate fraction was chromatographed on a silica gel and sephadex LH-20 column which led to the isolation of two flavonoids, and the identity of the compounds was determined on the basis of chemical test and NMR analysis. Based on the 1D and 2D NMR data, the compounds were 2-(3’4’-dihydroxyphenyl)-3,5,7-trihydroxy-4 H -chromane-4-one (quercetin) and 2-(2,4-dihydroxyphenyl)-5,7-dihydroxy-3-(3-methylhexyl)-4 H -chromen-4-one. This is the first report of isolation of these compounds from globiferus growing on Vitex doniana


Introduction
Tapinanthus globiferus (A. Rich) Tiegh belonging to the Loranthaceae Family is a semi-parasite with glabrous pendulous stems up to 1.2 m long with roots that mostly grows on the branches of a large number of tree species including Vitellaria paradoxa, Kola, Citrus, Combretum, Acacia, Aloe and Terminalia as host trees (Waterberg et al., 1989;Polhill and Wiens, 1998). It is commonly known as mistletoe (English), Kauchi (Hausa), afomo (Yoruba), and Osisi/Okwuma osa (Igbo) in Nigeria. T. globiferus is used in ethnomedicine to treat itching (Burkill, 2000), tumour (Yineger and Yewhalaw, D., 2007) and removal of placenta after parturition (Sher and Alyemeni, 2011). The plant is also used to treat diseases such as hypertension, ulcers, epilepsy, diabetes, weakness of vision and promoting muscular relaxation before delivery (Bassey, 2012). Ogunbolude et al. (2014) reported the identification and quantification of quercetin and some phenolic acids from T. globiferus growing on other host. Biological studies of T. globiferus growing on other hosts have been documented. Antitrypanosomal (Abedo et al., 2013) and anticonvulsant (Abubakar et al., 2016) activities of the plant were also reported. Extensive literature search revealed that there is no report yet on the isolation and characterization of any compound from the plant T. globiferus growing on Vitex doniana.

Materials and methods
General procedures NMR data were recorded on a Bruker AVANCE spectrometer (600 MHz) with residual solvent as internal standard. Melting point was determined on an Electro thermal melting point apparatus. Thin layer chromatography (TLC) was carried out using silica gel 60 GF254 pre-coated aluminium sheets by Sigma Aldrich, Germany. Low pressure column and Vacuum liquid chromatography were conducted using LOBA Cheme silica gel (60-200) mesh in a sintered glass funnel while gel filtration chromatography was performed using sephadex LH-20 (Sima, Spruce Street, St. Louis, MO, USA). Spots on TLC plates were visualized by spraying with 10% H2SO4 followed by heating at 105 o C for 10 min.

Plant sample
Plant sample of T. globiferus growing on Vitex doniana was collected from Dange Shuni Local Government Area of Sokoto State, Nigeria in December 2016. It was identified and authenticated by Namadi Sanusi of the Herbarium Section, Department of Biological Sciences, Ahmadu Bello University Zaria, with a voucher (No. 900107). The plant material was air dried, pulverized, labelled and stored in a polythene bag for further use.

Isolation of compounds
The powdered leaf of T. globiferus (2.0 kg) was exhaustively extracted with 3 L of 90% methanol for 6 days. The extract was filtered using Whatman No. 1 filter paper and the filtrate was evaporated to dryness using rotary evaporator at 40 ℃ to afford crude methanol leaf extract (140 g). Some part of the extract (120 g) was partitioned into n-hexane, chloroform, ethylacetate and n-butanol fractions. The ethylacetate fraction (3 g) was subjected to purification using VLC with n-hexane: ethylacetate mixtures as solvent systems. Twenty (20 mL) of a total of 60 collections were made and combined based on their TLC profile to afford four major fractions coded A-D. Fraction D was further purified using a combination of low pressure column and sephadex LH-20 column to afford a yellow amorphous substance, 10 mg (Compound 1). A portion of the ethylacetate fraction (4 g) was chromatographed on a silica gel column with n-hexane: ethylacetate mixtures as solvent systems and a total of seventy six collections were made and merged based on their TLC profile to afford seven major fractions coded E1-E7. Repeated gel filtration of fraction E3 led to the isolation of a yellow amorphous substance, 8 mg (Compound 2).
The HSQC spectrum established direct correlation between protons and their respective carbons. Aliphatic side chain was further substantiated by the correlations observed between the proton at δH 1.36 and δc 28.9 # 1.36, and δH 1.34 and 29.3 among others.

Study limitation
Two compounds were isolated from the ethylacetate fraction of Tapinanthus globiferus, however, testing the efficacy of the isolated compounds on different ailments is recommended for possible drug development.