A new glycosidic antioxidant from Ranunculus muricatus L. (Ranunculaceae) exhibited lipoxygenasae and xanthine oxidase inhibition properties

Abstract Phytochemical investigation of Ranunculus muricatus L. (Ranunculaceae) led to the isolation of a new metabolite named as ranuncoside from the ethyl acetate fraction of the plant. Structure of the novel compound was elucidated through detailed spectroscopic analyses, using UV, IR, 1H, 13C NMR and 2D NMR in combination with EIMS and HR EI-MS techniques. The compound was evaluated for antioxidant activity using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging assay. Its inhibitory potential was tested against lipoxygenase and xanthine oxidase enzymes. Ranuncoside potently scavenged the DPPH free radicals (IC50 = 56.7 ± 0.43 μM) and strongly inhibited the activities of lipoxygenase (IC50 = 63.9 ± 0.17 μM) and xanthine oxidase (IC50 = 43.3 ± 0.22 μM).

Reactive oxygen species (ROS) are well recognised for playing a dual role as both deleterious and beneficial species. Overproduction of ROS results in oxidative stress which is a harmful process that damages cell structures, including lipids, membranes, proteins and DNA. Oxidative stress has been culpably involved in various diseases including atherosclerosis, cancer, cardiovascular and neurodegenerative disorders. Much effort is devoted to developing rational drug therapy targeted at the 'oxidative stress component' of the cell death cycle (Valko et al. 2007). Leukotrienes (LTs) are pivotal lipid mediators of inflammation and allergy and play important roles in cardiovascular diseases, cancer and osteoporosis. 5-Lipoxygenase (5-LO) catalyses the first step in the biosynthesis of LTs from arachidonic acid, and based on the multiple potent pathophysiological actions of LTs, the pharmacological intervention with 5-LO is a challenge in the development of therapeutics (Pergola & Werz 2010). Xanthine oxidase (XO) is an important enzyme catalysing the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid which is excreted by kidneys. Excessive production and/or inadequate excretion of uric acid results in hyperuricemia and consequently the formation of gout with recurrent attacks of arthritis. Inhibitors of XO may be potentially useful for the treatment of gout or other XO-induced diseases (Kostić et al. 2015). Medicinal plants have historically proven their value as a source of molecules with therapeutic potential, and nowadays still represent an important pool for the identification of novel drug leads (Atanasov et al. 2015). In this study, a new metabolite was isolated from R. muricatus and was tested for antioxidant and inhibitory effect on lipoxygease and xanthine oxidase enzymes.
It is revealed from the DPPH free radical scavenging assay, that ranuncoside was able to scavenge free radicals and therefore possessed potent antioxidant activity. The standard antioxidant, butylated hydroxyanisole also showed a robust free radical scavenging activity as shown in Table 1. Antioxidants are widely used in dietary supplements and have been investigated for the prevention of various diseases. Studies suggested that antioxidant supplements might promote health. Plants have an innate ability to biosynthesise a wide range of non-enzymatic antioxidants capable of attenuating ROS-induced oxidative damage (Kasote et al. 2015).
Ranuncoside has the property of inhibiting the enzymatic activity of lipoxygenase as revealed from its IC 50 value. The standard inhibitor, baicalein also possessed a potent inhibitory potential against lipoxygenase as shown in Table 2. Lipoxygenases are a versatile class  of oxidative enzymes involved in the formation of arachidonic acid metabolites which are implicated in the activation of pro-inflammatory signal transduction pathways (Wisastra & Dekker 2014). Natural products are able to inhibit the lipoxygenase activity and therefore suppress the inflammatory response (Benrezzouk et al. 2001). The activity of XO was modified by ranuncoside and therefore demonstrated that it has the property of counteracting the chemical reactions catalysed by the enzyme, XO. The classical standard inhibitor, allopurinol also showed inhibitory potential towards XO but of higher magnitude as shown in Table 3. Activation of XO has been shown to produce oxidant species and subsequently oxidative stress (Desco et al. 2002). Inhibition of this enzyme reduces both vascular oxidative stress and circulating levels of uric acid. Plant extracts and their constituents show good inhibitory activity and therefore may have a positive impact on the prevention of diseases caused by increased activity of XO (Kostić et al. 2015).

Plant material
Whole plant (10 kg) of R. muricatus was collected in May and June, 2013, from Peshawar, Khyber Pakhtunkhwa, Pakistan. The plant was identified by Dr Muhammad Ibrar, Department of Botany, University of Peshawar, and a voucher specimen [Bot. 20112 PUP] 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 F 254 ,Merck,Darmstadt,Germany). Column chromatography 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 spectrophotometers (MA, USA) with tetramethylsilane as an internal standard. The chemical shifts (δ 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 EIMS and HR EI-MS in m/z (rel. %).

Extraction and isolation
The powdered plant material was macerated in methanol for extraction. The crude extract (1 kg) obtained after maceration, was fractionated in n-hexane, ethyl acetate, n-butanol and water. The ethyl acetate fraction (250 g) was subjected to column chromatography over silica gel. This fraction was eluted with gradient n-hexane-ethyl acetate and ethyl acetate methanol solvent systems. The column provided 400 fractions and based on TLC analysis, 35 major fractions were obtained. Fraction 9 (1 g) was further subjected to column chromatography over flash silica gel (ethyl acetate -n-hexane, 1:9), leading to isolation of compound 1 (20 mg) which was UV active.

DPPH free radical scavenging assay
The antioxidant activity was evaluated by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging assay. Briefly, 0.3 mM solution of DPPH was prepared in ethanol and 5 μL solution of isolated compound in different concentrations (62.5-500 μg) was mixed with 95 μL solution of DPPH and incubated at 37 °C for 30 min in plates. The absorbance was measured at 515 nm using microtitre plate reader (Spectramax plus 385 molecular Device, Union City, CA, USA). The activity was measured in comparison with dimethyl sulfoxide-treated control using butyl hydroxyl anisole as standard. The percent scavenging effect was calculated as: DPPH scavenging effect (%) = A c −A s /A c × 100, Where, A c is the absorbance of control (DMSO treated) and As is the absorbance of isolated compound. IC 50 values were calculated using GraphPad Prism® (version 4.0, Sandiego, CA).

Lipoxygenase inhibition assay
The lipoxygenase inhibitory activity was assessed according to a previous reported method (Tappel 1953) with slight modifications. A total volume of 200-mL assay mixture was prepared containing 160-mL sodium phosphate buffer (100 mM, pH 8.0), 10 mL of test compound, and 20-mL purified lipoxygenase. The contents were pre-incubated for 10 min at 25 °C. The reaction was initiated by addition of 10-mL linoleic acid (substrate solution). The change in absorbance was observed after 6 min at 234 nm. All reactions were performed in triplicates in 96-well microplate reader (Synergy HT, Biotek, Winooski, Vermont, USA). The positive and negative controls were included in the assay. The percent inhibition (%) was calculated as: Percent inhibition (%) = (control -test) × 100. Control is the total enzyme activity without inhibitor and test is the activity of the isolated compound. IC 50 values were calculated using GraphPad Prism® (version 4.0, Sandiego, CA).

Xanthine oxidase inhibition assay
The xanthine oxidase inhibitory activity was determined using a slight modification of the reference methods (Chan et al. 1994;Van Hoorn et al. 2002). Control solution was prepared by adding 7.0 μL of xanthine oxidase buffer solution (0.4 U/mL) to 0.1 M phosphate buffer (127.0 μL) and the mixture was incubated at 37 °C for 10 min. Then 66.0 μL of 400 μM xanthine buffer solution was added to the mixture and absorbance at 295 nm of reaction mixture was measured at 37 °C for 10 min by multi-detection microplate reader. The blank solution was prepared in an analogous way, but instead of the enzyme, it contained 7 μL of phosphate buffer solution. The test was performed in triplicate. For sample test, 7 μL of xanthine oxidase buffer solution (0.4 U/mL) was added to a solution consisting of 0.1 M phosphate buffer, pH 7.8 (77 μL) and 50 μL each of test samples which was treated in the same way as the control. 3.5 μL of phosphate buffer solution were used instead of xanthine oxidase solution for blank tests. Xanthine oxidase inhibitory activity was expressed as percent inhibition of xanthine oxidase, calculated as (1−B/A) × 100, where A is the change in absorbance of the assay without the test samples. (∆ absorbance with enzyme -∆ absorbance without enzyme), and B is the change in absorbance of the assay with the test sample (∆ absorbance with enzyme -∆ absorbance without enzyme). IC 50 values were calculated using GraphPad Prism® (version 4.0, Sandiego, CA).

Conclusion
Phytochemical analysis of R. muricatus and subsequent isolation and characterisation techniques revealed the presence of a new metabolite, named as ranuncoside which showed potent free radical scavenging activity. The novel isolated compound exhibited lipoxygenase and xanthine oxidase inhibitory properties and therefore may provide a natural source of new drug lead for the therapeutic management of diseases associated with exaggerated activities of these enzymes.