Urease inhibition potential of Di-naphthodiospyrol from Diospyros lotus roots

Abstract The dimeric napthoquione 5,8,4′-trihydroxy-1′-methoxy-6, 6′-dimethyl-7,3′-binaphtyl-1,4,5′,8′-tetraone (1) was isolated from the chloroform fraction of Diospyros lotus extract. Compound 1 was screened for its inhibitory effects against four enzymes: urease, phosphodiesterase-I, carbonic anhydrase-II and α-chymotrypsin, and showed selective activity against urease enzyme with an IC50 value of 254.1 ± 3.82 μM as compared to the standard thiourea (IC50 = 21 ± 0.11 μM). Furthermore, in silico docking study was carried out to explain the molecular mechanism of compound 1 against the target receptor.


Introduction
Diospyros lotus (date-plum) belonging to the family Ebenaceae comprises about 750 species and is distributed across subtropical/tropical regions of Asia (Japan) and USA (Uddin et al.   . It grows up to 9 m in height in semi-shaded region (Chittendon 1956). different parts of genus Diospyros species are important medicinal plants which have been traditionally used for fungal infection, lumbago hiccup, for internal haemorrhage, for bed-wetting in children, insomnia, hypertension, dyspnea, antimicrobial, anticancer, etc. (Rauf et al. 2016achildren, insomnia, hypertension, dyspnea, antimicrobial, anticancer, etc. (Rauf et al. , 2016b. Diospyros are used as carminative, sedative, astringent, febrifuge vermicide and vermifuge. (Tezuka et al. 1973;Ganapaty et al.2006). Various classes of secondary metabolites including tri-terpenoids of lupane, oleanane, dimeric napthoquione, phenolic compounds and ursane series have been isolated from Diospyros; which showed antipyretic properties (Watt et al.1932;Chopra & Nayar 1956;Ullah et al. 2015a). D. lotus extract and isolated compounds have been reported to exhibit excellent anti-inflammatory, muscle relaxant and sedative activities (Uddin et al. 2011;Rauf et al. 2015a;Ullah et al., 2015b). Urease belongs to urea amidohydrolase class with an EC number 3.3.1.5 which hydrolyses urea into ammonia and carbamate. The carbamate at the physiological pH catabolises spontaneously to carbonic acid and yields another molecule of ammonia (Sumner 1926). Urease is known to play crucial role in pathologies induced by Helicobacter pylori (peptic ulcers and gastric cancer) by facilitating survival of the pathogen in the acidic environment of stomach. In addition, ureases have been reported to cause urolithiasis, a type of infection, caused by Yersinia enterocolitica and Proteus mirabilis (Mobley et al. 1995). Moreover, urease is also responsible for the development of infection-induced reactive arthritis and acute pyelonephritis (Mobley et al. 1995;Arfan et al. 2010).
The main nitrogenous waste product of biological systems is urea which is quickly metabolized by the action of micro-organisms. Urease enzymes are present in a number of fungi, bacteria and plants that play a vital function in nitrogen cycle; these enzymes supply nitrogen for the growth of micro-organisms by catalysing urea degradation (Bremner 1996). In agriculture, the high level of urease activity is associated with several environmental and economical hazards. Globally, urea is commonly used as fertilizer. In agriculture, the high activity of ureolytic bacteria increases the amount of ammonia in the soil via fast urea degradation. Consequently, plants are damaged due to lack of necessary nutrients and by the toxicity of ammonia and carbon dioxide which are released from urea degradation (Bremner 1996). during the seed germination process, urease plays a vital role in the metabolism of nitrogen (Mulvaney & Bremner 1981;Khan et al. 2010).
The discovery of effective and safe urease inhibitors have been an important area of pharmaceutical research due to the association of ureases with several pathological conditions, as well as for agriculture applications (Amtul et al. 2004). A number of synthetic constituents such as triazoles, thiazoles, keto acids, isocoumarin and thiobarbituric acids are active urease inhibitors. On the other hand, partial studies have been conducted on natural products as urease inhibitors (Abid et al. 2010;Akhtar et al. 2010;Khan et al. 2010;Rauf et al. 2011). In the present work, a new source dimeric anthraquinone was isolated from the chloroform fraction of D. lotus extract. The compound was identified as 5,8,4′-trihydroxy-1′-methoxy-6, 6′-dimethyl-7,3′-binaphtyl-1,4,5′,8′-tetraone (1) . Furthermore, its inhibitory effect against different enzymes was undertaken along with in silico docking to explain the molecular mechanism of action of compound 1 against the target receptor.

Results and discussion
Organic compounds isolated from myriad plants have extensive usage in health care. Also, the phytochemicals furnish good pharmacophore templates for new drugs. In this context, compound 1 was evaluated against the enzyme urease, phosphodiesterase-I, carbonic anhydrase-II and α-chymotrypsin through enzyme inhibition assays. Results revealed that compound 1 was significantly active against urease enzyme with an IC 50 value of 254.1 ± 3.82 μM. Interestingly, compound 1 did not show significant inhibition of phosphodiesterase-I, carbonic anhydrase-II and α-chymotrypsin. Selectivity of the compound towards urease suggests that the activity might be due to the keto moiety at C-4 along with hydroxyl groups at C-5, which may bind with residues near to Ni atoms in the active site of urease enzyme (Abid et al. 2010). Results are presented in Table S1 (See supplementary Table S1 online only).
Computational docking studies were carried out to analyse the binding pattern of compound 1 in the active site of urease enzyme. Two softwares were employed for the purpose of docking, Autodock Vina and i-GEM dOCK followed by optimisation of docking procedures.
Generally, docking studies show that, if a compound contributes less interaction energies then it has better activities against the targeted enzyme. Additionally, docking can predict if there are specific features in the investigated compound mediating their potency. docking details pertaining to compound 1 and reference thiourea are presented in Table S2 (See  supplementary Table S2 online only).
Results show that docking energies of compound 1 are better than the reference thiourea, which might have biological relevance. docked confirmations of compound 1 and reference thiourea are displayed in Figure S1 (See supplementary Figure S1 online only). docking statistics observed from compound 1 are −7.9 kcal/mol (generated by Autodock vina) and −108 kcal/mol (generated by i-GEMdOCK). In both cases, the energy of compound 1 is better than that of standard thiourea (See supplementary Table S2 online only). The best docked confirmation of compound 1 based on hydrogen and hydrophobic interactions was analysed. Interaction analysis revealed that there is one hydrogen bond (2.34 Å) formed by compound 1 in the binding site of urease enzyme, observed from His592 with the -OH group of compound 1 ( Figure S2) (See supplementary Figure S2 online only). Furthermore, we investigated hydrophobic interactions of compound 1 (Figure Se, see online only) and observed contact with the binding site surrounding residues (Thr440, His491, Glu492, His518, His544, Ala548, Gly549, Met587, leu588, Arg608, Asp632 and Ala635) of urease enzyme.

Supplementary material
Supplementary data related to this manuscript including experimental sections, Table S1-S2 along with Figures S1-S2 are available online.

Disclosure statement
No potential conflict of interest was reported by the authors.