Urease from Canavalia ensiformis (Jack bean) (SLBB0100V Sigma Aldrich), thiourea (CAS 62-56-6, Sigma Aldrich), sodium nitroprusside (CAS 13755-38-9, Sigma Aldrich CHEMIE GmbH USA), ceftriaxone (Geltis, Shaigan Pharmaceuticals, Pakistan), sodium carbonate (Merck, USA), 2,4,6-tri(2-pyridyl)-1,3,5-triazine Alfa Aesar, CAS (3682-35-7, Germany) and solvents of analytical grade were purchased from distributor of Sigma Aldrich in Pakistan.

Whole plant of P. hydropiper was acquired from Talash Valley, Dir (L), Khyber Pakhtunkhwa (KP), Pakistan in the month of July, 2013 as we reported previously (Ayaz et al., 2014a, 2016b). The identification of plant was done by Dr. Gul Rahim, and subsequently a voucher specimen was maintained with Voucher No. H.UOM.BG.107 at the University of Malakand, herbarium for future record purpose. The plant material was dried in shade with subsequent pulverization and fractionation. The whole process was carried following standard protocol as we reported previously (Ayaz et al., 2014b; Ullah et al., 2016; Ali et al., 2017).

Crude saponins were isolated from dried plant powder as we reported for this plant and other natural products (Ayaz et al., 2014a, 2016b; Zeb et al., 2014; Ahmad et al., 2015). Briefly, 60 g of powder plant material was transferred to conical flask and 100 ml of 20% ethanol was added to it. The resultant mixture was slowly heated for 4 h at 55°C using water bath. The mixture was filtered and again extracted with 200 ml of ethanol. The volume from both extractions were combined and concentrated with the help of water bath. Finally, 40 ml was transferred to separating funnel and 20 ml of diethyl ether was added to it with vigorous shaking. The Diethyl ether layer was discarded and to the aqueous fraction, 60 ml of n-butanol was added. The resultant aqueous-butanol mixture was passed through 5% NaCl solution and solvents were evaporated to obtain crude saponins (Zeb et al., 2016).

We previously reported isolation of essential oils from P. hydropiper (Ayaz et al., 2015a). Briefly, fresh collected leaves of P. hydropiper were washed with distilled water and subsequently hydrodistilled with the help of a Clevenger apparatus (Clevenger, 1928). The resultant oils were condensed, collected, and any water content was removed via anhydrous sodium sulfate (Bassole et al., 2011; Ayaz et al., 2017). The oils were transferred to glass vials, tightly sealed and refrigerated at −30°C prior to further analysis.

Samples were analyzed via gas chromatograph (Agilent USB-393752) equipped with FID detector and capillary column as per specifications we published previously (Ayaz et al., 2015a; Ahmad et al., 2016).

Components of the samples were identified by comparing their retention times with previously known compounds in the literature and from the spectral data acquired from NIST and Wiley and NIST libraries. Moreover, the fragmentation pattern of the mass spectra was also compared with already published data for further authentication (Stein et al., 2002; Adams, 2007).

Urease inhibitory potentials of the test samples were evaluated following previously reported procedure (Weatherburn, 1967). In brief, 25 μl enzyme solution and 55 μl of buffer (containing 100 mM urea) were incubated with 5 μl of test solution (1 mM concentration) in micro plate reader (room temperature for 15 min). Enzyme inhibitory potentials of our test samples were assessed from the rate of ammonia production. After 50 min of incubation, absorbance was recorded at 630 nm using Uquant Micro plate reader (BioTek Instruments Highland Park Winooski SN 213541 USA) using thiourea as control drug. Percent enzyme inhibition was calculated as;

Concentrations of test samples which exhibited 50% inhibition against the test enzymes (IC₅₀) were calculated from dose-response curve for urease inhibition assay.

All assays were repeated three times and results obtained presented as means ± SEM. One way ANOVA followed by multiple comparison Dunnett's test was applied to the data for the comparison of test samples with standard drugs using GraphPad software. P < 0.05 were considered as statistically significant.

An IC₅₀ based cluster analysis was performed and dendrogram was developed for test samples via SPPS software version 16.0 using Ward's procedure to make hierarchical clusters.

GC followed by GC-MS analyses were performed to identify major compounds in various fractions and essential oil isolated from P. hydropiper. In the phytochemical analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, Ph.EtAc, and Ph.Lo the total number of identified compounds were 126, 124, 181, 131, 164, and 141, respectively. Among the identified compounds in Ph.Cr, 2,3-dihydro benzofuran, neophytadiene, 3,7,11,15-tetramethyl-2-hexadecen and 1-dodecanol, 3,7,11-trimethyl-hexa-hydro-farnesol were found in highest concentrations of 7.89, 25.2, 10.71, and 8.14%, respectively (Table 1). In analysis of Ph.EtAc, coumaran (34.05%), p-vinylguaiacol (16.94%), and alpha santolina alcohol (13.37%) were found in major concentrations. Similarly, in analysis of Ph.Hex, humulene oxide (13.79%), 9,12,15-octadecatrienoic acid, methyl ester (8.85%), methyl palmitate (7.68%), drimenol (7.26%), and caryophyllene oxide (7.7%) were the compounds found abundantly. Methyl linolenate, methyl palmitate, and 9,12-octadecadienoic acid methyl ester were present in higher concentrations of 10.47, 10.46, and 7.23%, respectively in Ph.Chf. Furthermore, coumaran (53.32%), p-vinylguaiacol (17.44%), borneol (14.21%), alpha-santolina alcohol (4.45%), and aristolone (4.01%) were major identified phyto-constituents in Ph.Bt. Among the identified compounds of Ph.Lo, decahydronaphthalene, and bicyclo [2.2.2]oct-2-ene, 1,2,3,6-tetramethyl were highly abundant compounds with 38.29 and 36.33% composition, respectively (Figure 1).

Ureases are implicated in the development of numerous pathological conditions of human being. They are major contributors in pathogenesis caused by H. pylori by allowing this bacteria to survive at acidic pH of the stomach thus leading to gastric, peptic ulcer and even cancer (Mobley and Hausinger, 1989). They are also involved in the formation of infectious stones and play a part in the pathogenesis of pyelonephritis, urolithiasis, hepatic encephalopathy, hepatic coma, and urinary catheter encrustation (Mobley et al., 1995). Similarly, in agriculture elevated urease activity may lead to considerable environmental and economic problems through the release of abnormally large quantity of ammonia into the atmosphere during urea fertilization. This persuade plant damage principally by depriving them from their vital nutrients, cause ammonia toxicity and raise pH of the soil (Mobley et al., 1991; Zonia et al., 1995). Consequently, the inhibition of urease producing organisms is among the important strategies for the treatment of infections caused by these bacteria, to diminish environmental pollution and augment efficiency of urea nitrogen uptake by plants (Andrews et al., 1986). So, the study of urease inhibition has medical, environmental and agronomic significance.

In our current study, Ph.Lo and Ph.Sp exhibited highest urease inhibition property causing 79.33 and 75.83% enzyme inhibitions at 1 mg/ml concentration with IC₅₀ of 90 and 98 μg/ml, respectively. Standard drug thiourea showed 87.40% inhibition at the same concentration and IC₅₀ of 80 μg/ml. Activity of Ph.Lo and Ph.Sp were comparable with positive control at the same concentration, which warrant further purification and characterization for possible utilization in gastric disorders. Ph.Chf and Ph.EtAc have also showed 62.16 and 52.16% enzyme inhibitions and IC₅₀ of 520 and 880 μg/ml, respectively (Table 2). These fractions may be subjected to column chromatography for isolation of novel bioactive compounds.

The Bacterium P. mirabilis is a frequent cause of urinary tract infections, especially in the elderly catheterized individuals, and is the second leading cause of urinary tract infections after Escherichia coli (Mohammed et al., 2014). Ureolytic activity of numerous microorganisms including P. mirabilis, Proteus vulgaris, and Ureaplsma urealyticum is implicated in urolithiasis which cause chronic pelvic and kidney inflammation (Williamson, 2001). The ammonia produced by urease is responsible for the raise in urine pH followed by formation of struvite and apatite stones which are characteristic features of P. mirabilis infection. Excessive ammonia fabrication by these microorganisms may contribute to ammonia encephalopathy or hepatic coma and facilitate H. pylori survival in the stomach followed by infections (Sujoy and Aparna, 2013). Additionally, elevated level of ammonia disrupt mucosal permeability, especially hydrogen ions transport via mucosal surface and thus cause peptic ulcers (Suzuki et al., 1992). Consequently, research is focused on urease inhibitor compounds for the development of drugs useful against urease mediated bacterial infections (Mobley and Hausinger, 1989; Mobley et al., 1995; Estiu and Merz, 2004). In the current study, Ph.Chf, Ph.Hex, Ph.EtAc, and Ph.Sp fractions were found most active against P. mirabilis causing inhibitory zones of 28.25, 28, 26, and 26 mm, respectively at 100 μg/ml concentrations in disc diffusion assay (Figure 2). In well diffusion assay, each of Ph.Chf, Ph.Hex, and Ph.Sp produced inhibitory zone of 25 mm at 100 μg/ml concentration (Figure 3)”) Further, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were found most active exhibiting MICs of 25, 30, 32.25, and 40.50 μg/ml, respectively (Figure 4).

The aspirin induced ulcerogenesis in pylorus ligated rat model was associated with significant changes in the mean ulcer score [F_{(5, 30)} = 7.141, P = 0.0002], gastric juice volume [F_{(5, 30)} = 8.245, P < 0.0001], gastric juice pH [F_{(5, 30)} = 5.715, P = 0.0008], free acidity [F_{(5, 30)} = 4.544, P = 0.0033], total acidity [F_{(5, 30)} = 2.740, P = 0.0373], and pepsin concentration [F_{(5, 30)} = 2.335, P = 0.0664]. As shown in Table 3, treatment with aspirin at a dose of 200 mg/kg (Group II) for 4 days produced ulcerative changes in the gastric mucosa which were observed as drastic changes in the ulcerative biochemical profile in which aspirin significantly increased the ulcer score (P < 0.001), increased the volume of gastric juice (P < 0.001), decreased the pH of gastric juice (P < 0.001), increased the free acidity (P < 0.01), total acidity (P < 0.05), and pepsin concentration (P < 0.05) in the gastric juice as compared to vehicle treated controls (Group I). The prophylactic treatment with the Ph.Cr extract exhibited marked gastroprotective effect in the aspirin induced pyloric ligation ulcerogenesis model as the tested doses of 100 mg/kg (Group IV), 200 mg/kg (Group V), and 400 mg/kg (Group VI) afforded significant anti-ulcerogenic proclivity evidenced by a non-significant changes in the ulcerative biochemical parameters except at a dose of 100 mg/kg (Group IV) in which a transient increase in the ulcer score (P < 0.05) and a decrease in the gastric juice pH (P < 0.05) was observed compared to the vehicle treated controls (Group I). The positive control, ranitidine at a dose of 50 mg/kg (Group III) exhibited a robust gastroprotective profile as it is devoid of any aspirin like gastric ulceration observed as no obvious changes in the ulcerogenicity parameters.

Treatment with aspirin (200 mg/kg) was associated with severe histopathological changes evidenced by numerous microscopic ulcers throughout the gastric mucosa. The pathological changes significantly altered the normal histoarchitecture of the glandular portion of the stomach and were observed as disruption and erosion of the mucosal epithelium. There was shredding of mucosal epithelial cells with necrotic debris observed in the lumen of the ulcers. The underlying blood vessels supplying the superficial mucosa were heavily congested with red blood cells. Severe infiltration of lymphocytes was observed in the connective tissues above the muscularis mucosae and throughout the damaged mucosa. There were marked edematous changes in the submucosal layer. In some areas, the mucosal layer was superficially eroded while in other areas the ulcer extended to the submucosa and down to the muscularis externa producing severe inflammatory changes in these tissue layers. A representative photomicrograph of gastric mucosa from a rat treated with aspirin (Group II) is shown in Figure 6B.

The histological examination of the gastric mucosa following treatment with vehicle (Group I; Figure 6A), ranitidine (Group II; Figure 6C) and the Ph.Cr extract at the tested doses of 200 mg/kg (Group V; Figure 6E) and 400 mg/kg (Group VI; Figure 6F) revealed normal histoarchitecture with no surface epithelial disruption, deep mucosal damage, edema, necrotic lesion, or leukocyte infiltration except for the 100 mg/kg dose of the Ph.Cr extract (Group IV) at which mild shredding of the mucosal surface epithelium was observed (Figure 6D).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.