Urease inhibitory profile of extracts and chemical constituents of Pistacia atlantica ssp. cabulica Stocks

The current study was designed to evaluate the urease inhibitory profile of extract and fractions of Pistacia atlantica ssp. cabulica Stocks followed by bioactivity-guided isolated compounds. The crude extract was found significantly active with urease inhibitor (95.40% at 0.2 mg/mL) with IC50 values of 32.0 ± 0.28 μg/mL. Upon fractionation, ethyl acetate fraction displayed 100% urease inhibition with IC50 values of 19.9 ± 0.51 μg/mL at 0.2 mg/mL. However, n-hexane and chloroform fractions exhibited insignificant urease inhibition. Similarly, the isolated compound, transilitin (1) and dihydro luteolin (2) demonstrated marked urease attenuation with 95 and 98% respectively, at 0.15 mg/mL. Both the isolated compounds showed marked potency with IC50 values of 8.54 ± 0.54 and 9.58 ± 2.22 μg/mL, respectively. In short, both the extract and fractions and isolated compounds showed marked urease inhibition and thus a useful natural source of urease inhibition. Graphical abstract showing the image Pistacia atlantica, chemical structure of isolated compounds and urease enzyme.


Introduction
Anacardiaceae is a large family having 70 genera and more than 600 species with great economic importance. The species of the genus Pistacia are evergreen or deciduous resin-bearing shrubs and trees which have height of 8-10 m (Yi et al. 2008;Bozorgi et al. 2013).
The genus Pistacia includes 13 species (Ozden-Tokatli et al. 2010). Among these, three are important species of pistacia including Pistacia atlantica ssp. cabulica Stocks, P. khinjuk Stocks and P. vera which wildly grow in Iran. The trees of wild Pistacia are spread in central Asia, near the borders of Afghanistan, north-east of Iran and Turkmenistan. The three subspecies of P. atlantica has been reported, Cabulica, Kurdica and Mutica. The species of P. atlantica is considered to have an Irano Turanian species with three subspecies: Asiatic (subsp. cabulica), Mediterranean (subsp. atlantica) and Asiatic Mediterranean (subsp. mutica) (Karimi et al. 2009).
Pistacia atlantica ssp. cabulica Stocks is a tree up to 7 m tall and it is widely distributed in S. Iran, Pakistan and Afghanistan. Some species of Pistacia have been used as a folk medicine and these plants are used as anti-inflammatory, antipyretic, antibacterial, antiviral, throat infection and in the treatment of diarrhoea (Villar et al. 1987;Kordali et al. 2003;Benhammou et al. 2008). The plant crude extracts exhibited broad spectrum of antimicrobial particularly antifungal activities (Kordali et al. 2003). The different parts of plant show the presence of flavanoids, and other parts are used as a folk medicine for the treatment of different diseases like dysentery, cough, jaundice and gas trouble. The leaves are boiled in water and the extraction is used for dysentery and cough. The gum is cooked in a pot with water, dried and powder is made out of it, which is used for jaundice and gas trouble (Tareen et al. 2010).
The discovery of effective and safe urease inhibitors has been an important area of pharmaceutical research due to the association of ureases with several pathological conditions, as well as for agriculture applications . A number of synthetic compounds including thiazoles, triazoles, isocoumarin, keto acids and thiobarbituric acids are effective urease inhibitors, but limited studies have been conducted on natural products (Amtul et al. 2004;Abid et al. 2010). Previously, our research group reported that flavonoids showed good urease inhibition (Rauf et al. 2011); therefore, in the current study the bioassay-guided isolation strategy was employed and as a result two flavonoids were isolated as potential urease inhibitors.

Results and discussion
Molecular-docking studies were carried out to study the binding interaction of compound 1-2 against the urease enzyme. Both showed the reliable docking score as comparison with standard thiourea (Table 1). The superimposition and docking poses of the standard thiourea, compound 1-2 and co-crystallized ligand are shown in the Figure S1 (see Figure S1 online only). The interaction analysis revealed (see Figure S2 online only) that there are three hydrogen bond formed by compound 1. Compound 2 has also predicted satisfactory docking result, but actually it does not show hydrogen bonding with the binding site of urease enzyme (see Figure S3 online only). All interactions were observed of hydrophobic in nature.
The results of extracts and isolated compounds from P. atlantica are displayed in Figure 1. The crude extract showed 95.40% at 0.2 mg/mL with IC 50 values of 32 μg/mL. Upon fractionation, ethyl acetate fraction displayed 100% urease inhibition IC 50 values of 19.9 μg/mL (Table 2). However, n-hexane and chloroform fractions exhibited insignificant inhibition.  The isolated compounds, 1 and 2 demonstrated marked urease attenuation with 95 and 98% respectively, as compared to standard thiourea (98%) at 0.15 mg/mL (Figure 1). Both the isolated compounds showed marked potency with IC 50 values of 8.54 and 10.49 μg/mL, respectively. When we compared the activity of these compounds 1 and 2 to standard compound thiourea (IC 50 = 1.59 ± 0.11 μg/mL), both compound showed comparable activities Table 2. The activity of these compound may be due the dihydroxyl moieties (-OH) at ring C of flavoniods, which may interact with the residues near to the nickel atoms in the active site of urease in similar fashion as thiourea interacting. The active pharmacophore in the both compounds can be the dihydroxyl moieties. The literature reported that polyphenol and flavoniods showed significant urease activity, and our results are close agreement with the literature data, therefore our results will be provided significant contribution in this regards Uddin et al. 2011;Muhammad et al. 2012).
Urease (urea amidohydrolase) is typically observed in different bacteria, fungi, algae and plants; it is an enzyme that catalyses the hydrolysis of urea to ammonia and carbamate, which is the final step of nitrogen metabolism in living organisms . Carbamate rapidly and spontaneously decomposes, yielding a second molecule of ammonia. These reactions may cause significant increase in pH and are responsible for negative effects of urease activity in human health and agriculture (Khan et al. 2007;Lateef et al. 2012). From the medical view point, infections induced by these bacteria such as Helicobacter pylori and Proteus mirabilis usually have a high urease activity. Urease is vital to H. pylori metabolism and virulence, as necessary for its colonisation of the gastric mucosa, and is a potent immunogen that elicits a vigorous immune response. This enzyme is used for taxonomic identification and for diagnosis and follow-up after treatment, and is a vaccine. Urease represents an interesting model for metalloenzyme studies. H. pylori contribute in urinary tract and gastrointestinal infections, probably augmenting the severity of several pathological conditions like peptic ulcers and stomach cancer. Ureases are also involved in the development of urolithiasis, pyelonephritis, hepatic encephalopathy, hepatic coma and urinary catheter encrustation (Kuwahara et al. 2000;Zhu-Ping et al. 2007). Targeting urease for treating pathogenic disorders may open a new line of treatment for infections caused by urease-producing bacteria. The results of our study showed profound inhibition of urease (Jack Bean) by the extract and subsequent fraction of P. atlantica. The bioactivity-guided isolation when led to the isolation of various two compound, 1 and 2, both strongly supported the urease inhibitory effects of extract and fractions of the plant.
The stable complex of receptor ligand was analysed which is based upon the hydrogen bond and hydrophobic interactions. Both compound 1-2 predicted reliable docking score comparison with standard thiourea. Generally, most of the potent compounds can show hydrogen bond and hydrophobic interactions with the enzyme active site. If these interactions are present in the compound then it will be responsible for mediating the biological activity. The superimposition and docking poses of the standard thiourea, compound 1-2 and co-crystallized ligand are shown in the Figure 2. As shown from the Table 1, the docking result of compound 1 shows the binding energy −5.8 kcal/mol (Autodock vina score) and the total energy −99 kcal/mol (iGEMDOCK score) which is reliable than the scoring function of the standard thiourea.
The interaction analysis revealed that there are three hydrogen bond formed by compound 1. In that one hydrogen bond formed by Gly549 residue with a distance of 3.10 Å while other two hydrogen bond formed by Ser633 with a distance of 2.47 and 2.42 Å. Five hydrophobic contacts were observed from the residues Ala439, Thr441, His491, Ala635 and Met636. Hence it is concluded that these interactions of compound 1 are responsible for such good docking score. Compound 2 was also docked in the binding site of urease enzyme but interaction analysis revealed that it does not shows hydrogen bonding with urease enzyme. All interactions were observed of hydrophobic in nature.

Conclusion
It can be concluded that both the extract and fractions and isolated compounds of P. atlantica evoked profound urease inhibition in in vitro assay that equally sported by the docking simulation compounds. It is, therefore, believed that it could be a useful natural therapeutic tool when urease is involved.