In vitro anti-oxidant potential of new metabolites from Hypericum oblongifolium (Guttiferae)

Phytochemical investigations on Hypericum oblongifolium led to the isolation of a flavone named folicitin (1) and a bicyclic conjugated lactone, folenolide (2) from the ethyl acetate fraction of methanolic extract. Both metabolites were characterised as new compounds based on detailed spectroscopic analyses. In vitro anti-oxidant potential of both the compounds was evaluated by the DPPH radical scavenging assay. Compound 1 exhibited significant antioxidant activity while compound 2 was found inactive.

Phytochemical investigations on Hypericum oblongifolium led to the isolation of a flavone named folicitin (1) and a bicyclic conjugated lactone, folenolide (2) from the ethyl acetate fraction of methanolic extract. Both metabolites were characterised as new compounds based on detailed spectroscopic analyses. In vitro anti-oxidant potential of both the compounds was evaluated by the DPPH radical scavenging assay. Compound 1 exhibited significant antioxidant activity while compound 2 was found inactive.

Introduction
Hypericum, a large genus of herbs and shrubs, grows widely in the temperate regions throughout the globe. Hypericum oblongifolium belongs to the family Guttiferae which comprises of 50 genera and 1200 species (Cakir et al. 2003). There are about 400 species of genus Hypericum and they are used as traditional medicinal plants in various parts of the world. These plants are the richest sources of flavones and xanthones (Cakir et al. 2003). The genus is represented by nine species in Pakistan. Useful biologically active compounds, dyes, pigments, timbers, gums and resins have been isolated from members of this family (Mabberly 1987).
H. oblongifolium Wall., is an evergreen shrub, found at an altitude of 5000 -6000 ft, and is common on Khasia Hill in China and in the Himalayas. It is used for the treatment of hepatitis, nasal haemorrhage, bacterial diseases and as a remedy for the sting of bees and dog bites (Dulger & Gonuz 2005). Most of these species have been used as a treatment of gastric ulcer, external wounds, sedative, antispasmodic and also as antiseptic in folk medicine (Ferheen et al. 2006). The crude extract of H. oblongifolium has been found to possess respiratory, gastrointestinal and cardiovascular inhibitory effects . The aqueous and n-butanol fractions of H. oblongifolium possess significant in vitro antiglycation, antioxidant and anti-lipid peroxidation activities with no toxic effects (Abbas et al. 2013). The hexane fraction is reported to possess relatively potent anti-proliferative activity (Ali et al. 2011b). Two new antiinflammatory xanthones, hypericorin A and hypericorin B from the twigs of H. oblongifolium were isolated (Ferheen et al. 2005;Ali et al. 2011a). Bioassay guided fractionation of the plant has also led to the isolation of three potent urease inhibitors (Ferheen et al. 2005;Arfan et al. 2010).
Free radicals are produced due to oxidation inside the body and lead to the initiation of chain reactions. These reactions can be harmful for cells and can cause death of cells leading to various human health disorders, such as atherosclerosis, cardiovascular and neurodegenerative diseases (Witztum & Steinberg 1991;Esterbauer et al. 1992). These chain reactions can be stopped by the radical scavenging potential of antioxidants (Sies 1997). Antioxidants are commonly found in fruits, vegetables, tea, coffee and cacao (Gülc in 2012). Hypericum species contain various pharmacologically active compounds such as flavonoids, naphthodianthrones, phloroglucinols and xanthones (Robson 1990). Flavonoids are the most common class of plant phytochemicals presenting a wide range of biochemical properties, including anti-inflammatory (Owoyele et al. 2008), antibacterial (Hnatyszyn et al. 2003), antifungal (Li et al. 2005), antioxidant (Bernardi et al. 2007) and anti-cancer activities (Seelinger et al. 2008). Flavonoids have been recorded to act as scavengers of several oxidising species (Tournaire et al. 1993). The aim of the present study was the phytochemical investigation of H. oblongifolium and the evaluation of the antioxidant potential of the isolated metabolites.

Results and discussion
Phytochemical investigation of H. oblongifolium led to the isolation of two new metabolites.
The COSY spectrum showed correlations between H-5 0 and H-6 0 , thus meaning that these are attached to adjacent carbon atoms. CZH protons at d 4.21 (H-3 00 ) showed COSY cross peaks with the proton at d 3.39 (H-4 00 ) which in turn showed COSY cross peaks with H-5 00 (d 3.73). H-5 00 also showed COSY interactions with H-6 00 (d 3.42) which in turn showed interaction with the methyl protons (H-7 00 ). A relay of the COSY cross peaks between these methine and methyl protons suggested that all of them are in the same cycle and that the methyl group is also in the same ring.
The COSY spectrum showed correlations between H-2 and H-8, thus meaning that these are attached to adjacent carbon atoms. Methine proton at d 3.98 (H-2) showed COSY cross peaks with the proton at Based on the above interactions and other spectral analyses, the structure of compound 2 was elucidated as 3,4-dihydroxy-6-oxabicyclo[3.2.1]oct-1-en-7-one (Figure 1). To the best of our knowledge this compound has not been reported earlier and is thus regarded as a new compound.
Compounds 1 and 2 were evaluated for their anti-oxidant potential using the DPPH radical scavenging assay (Table 1).

Experimental 3.1. Plant material
The whole plant (12 kg) was collected in the month of April and May, 2011, from Bara Gali, District Abbottabad, Khyber Pakhtunkhwa, Pakistan. The plant was identified by Dr. Muhammad Ibrar, Department of Botany, University of Peshawar, and a voucher specimen (08823) was deposited in the herbarium of the same department.

General experimental conditions
Thin layer chromatography (TLC) was carried out on pre-coated silica gel plates (G-60, F 254 , Merck, Darmstadt, Germany). Column chromatography (CC) was performed on silica gel (G-60, 70 -230 mesh). 1 H and 13 C NMR spectra were recorded in deuterated methanol on Bruker Avance-NMR spectrometers (MA, USA) with tetramethylsilane as an internal standard. The chemical shifts (d values) are given in parts per million (ppm), and the coupling constants (J values) in Hertz. JEOL JMS-600H Mass spectrometer (Tokyo, Japan) was used for recording EI-MS and HR EI-MS; in m/z (rel. %). Visualisation of chromatograms was achieved under UV (254 and 365 nm) before using spraying reagents (exposure to ammonia vapour or aluminium chloride spray).

Extraction and isolation
The powdered plant material was macerated in 20 % methanol for extraction. The crude extract (2.5 kg) obtained after maceration, was fractionated in n-hexane, ethyl acetate, n-butanol and water, respectively. The ethyl acetate fraction (490 g) was subjected to column chromatography over silica gel. This fraction was eluted with gradient n-hexane-ethyl acetate and ethyl acetatemethanol solvent systems. The column provided 300 fractions were made and pooled based on TLC analysis, resulting in 16 major fractions. Fraction 14 (2 g) was further subjected to column chromatography over flash silica gel (ethyl acetate-chloroform, 1:1) leading to the isolation of compound 1 (40 mg). The fraction 16 (ethyl acetaten-hexane, 1:3) was re-chromatographed and eluted with acetone -chloroform (2:3) solvent system to get compound 2 (30 mg) as a white powder.

DPPH radial scavenging assay
The free radical scavenging activity was determined by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) using the method described by Gülc in et al. (2005). The solution of DPPH of 0.3 mM was prepared in ethanol. Five mL of each sample of different concentrations (62.5 -500 mg) was mixed with 95 mL of DPPH solution in ethanol. The mixture was dispersed in 96-well plates and incubated at 378C for 30 min. The absorbance at 515 nm was measured by microtitre plate reader (Spectramax plus 384 Molecular Device, Union City, CA, USA) and percent radical scavenging activity was determined in comparison with the dimethyl sulfoxide (DMSO) treated control. Butyl hydroxyl anisole was used as a standard. DPPH scavenging effect ð%Þ ¼ Ac 2 As Ac £ 100 ; where, Ac ¼ Absorbance of control (DMSO treated). As ¼ Absorbance of sample.

Conclusion
In conclusion, the phytochemical investigation of H. oblongifolium was carried out which yielded two new metabolites, a flavone, folicitin (1) and a bicyclic conjugated lactone, folenolide (2). DPPH radical scavenging assay proved that compound 1 is a potential anti-oxidant compound.

Supplementary material
Supplementary material relating to this paper is available online, alongside Figures S1 -S4 and Tables S1 -S2.