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Short communication
Urease inhibitory activity of ursane type sulfated saponins from the aerial parts of Zygophyllum fabago Linn Saleha Suleman Khan a , Ajmal Khan a , Afsar Khan b,∗ , Abdul Wadood c , Umar Farooq b , Amir Ahmed d , Aqib Zahoor a , Viqar Uddin Ahmad a,∗∗ , Bilge Sener e , Nurgun Erdemoglu e a
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan c Department of Biochemistry, Shankar Campus, Abdul Wali Khan University, Mardan, Pakistan d Pharmaceutical Division, Pakistan Council of Scientific and Industrial Research Laboratories Complex, Karachi 75280, Pakistan e Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara 06330, Turkey b
a r t i c l e
i n f o
Article history: Received 17 June 2013 Received in revised form 6 August 2013 Accepted 19 September 2013 Keywords: Zygophyllum fabago Urease inhibitory activity Sulfated saponins Ursane Molecular docking
a b s t r a c t Five ursane type sulfated saponins have been isolated from the aerial parts of Zygophyllum fabago Linn. (locally called Chashum). The urease inhibitory effects of these compounds have been investigated for the first time as well as their molecular docking studies have also been carried out to check the structure–activity relationship. The IC50 values of these compounds could not be found due to paucity of the samples. The molecular docking studies were performed only for the most active compound mono sodium salt of 3,23-di-O-sulfonyl-23-hydroxyurs-20(21)-en-28-oic acid 28-O-[-d-glucopyranosyl] ester (Zygofaboside A; 1). © 2013 Elsevier GmbH. All rights reserved.
Introduction Many plants belonging to the genus Zygophyllum have been shown to cause various biological effects (Shabana et al. 1990; Rimbau et al. 1999) and are used in folk medicine in Mediterranean countries. The plant Zygophyllum fabago is one of the important herbs with known anti-rheumatic, anthelminthic, cathartic, and anti-asthmatic properties (Bay 1999). It is also used as a part of drug for rheumatism and gout; also used externally as poultice to cure skin diseases, external wounds, septic, and injuries. In China Z. fabago is used as antitussive, expectorant, and as anti-inflammatory agent (Feng et al. 2008). The plant collected from Pakistan showed very strong antifungal activity (95% at 200 g) against the human pathogen Candida albicans (Zaidi and Crow 2005). Zygophyllum fabago extracts may also contain
Abbreviations: NMR, nuclear magnetic resonance; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond correlation; COSY, correlation spectroscopy; NOESY, nuclear overhauser effect spectroscopy; MOE, molecular operating environment; IC50 , 50% inhibitory concentration; EtOH, ethanol; min, minutes; NaOH, sodium hydroxide; NaOCl, sodium hypo chloride. ∗ Corresponding author. Tel.: +92 992 383591/5; fax: +92 992 383441. ∗∗ Corresponding author. Tel.: +92 2134819020; fax: +92 2134819018/9. E-mail addresses: saleha [email protected] (S.S. Khan), [email protected], [email protected] (A. Khan), [email protected] (V.U. Ahmad).
antiviral photosensitizers but the activities were only seen at high concentrations (Hudson et al. 2000). Some of the extracts of Z. fabago from Turkish origin exhibited much higher activity against BChE (Orhan et al. 2004). In an on going search for the bioactive compounds from Z. fabago, the EtOH extract of this plant was selected for investigation. From previous studies, several unusual disulfated ursane derivatives with a double bond at C20 C21 have been reported which might have good potential invarious biological activities. Compounds isolated in the present study (Fig. 1) include five known compounds; mono sodium salt of 3ˇ,23-di-O-sulfonyl-23-hydroxyurs-20(21)-en-28-oic acid 28-O-[-d-glucopyranosyl] ester (Zygofaboside A; 1), (3,4␣)3,23,30-trihydroxyurs-20-en-28-al 3,23-di(sulfate) sodium salt (2), 3-O-[-d-2-O-sulfonylglucopyranosyl] quinovic acid 28-O[-d-glucopyranosyl]ester (Zygophyloside G; 3), 3-O-[2-O-sulfo-d-quinovopyranoside] quinovic acid 28-O-[-d-glucopyranosyl] ester (Zygophyloside E; 4), and 3-O-[-d-quinovopyranoside] quinovic acid 28-O-[-d-glucopyranosyl] ester(5). Compounds 1 and 2 have already been reported by us as new natural products from the same plant (Ahmad et al. 2007; Khan et al. 2010b). While compounds 3, 4 and 5 are new source compounds from Z. fabago. Urease (urea amidohydrolase EC 3.5.15) catalyzes the hydrolysis of urea to ammonia and carbon dioxide (Sumner 1926). Urease is the enzyme responsible for an organism to use urea as a nitrogen source. In plants urease also acts as a defense protein
0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.09.009
Please cite this article in press as: Khan, S.S., et al., Urease inhibitory activity of ursane type sulfated saponins from the aerial parts of Zygophyllum fabago Linn. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.09.009
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2
30 20
29 12 25 2 3 R1O 24
1 4
11
10 5
9 6
26 8 7
19 13 14 27
R2 21
18
17 H 16 15
22 28 R3
R2
1 R1 =SO 3Na,R 2 =H,R 2 R1 =SO 3Na,R 2 =OH,R
OO
OH
23
OH
OSO3H
= O-β -D-glucopyranosyl 3 =H 3
O
H
O
O COOH CH2OH O OH OH
R1
OH 3 R1 =OSO 3Na,R 2 =CH 2OH 4 R1 =OSO 3H,R 2 =CH 3 5 R1 =OH,R 2 =CH 3
Fig. 1. Chemical structures of compounds 1–5.
in systemic nitrogen transport pathways. Urease is known to be one of the major causes of pathologies induced by H. pylori, thus allowing them to survive at low pH of the stomach and, therefore, plays an important role in the pathogenesis of gastric and peptic ulcer, apart from cancer as well. Urease is directly involved in the formation of infection stones in kidney and contributes to the pathogensis of urolithiasis, pyelonephritis, ammonia, hepatic encephalopathy, hepatic coma, and urinary catheter encrustation (Mobley et al. 1995; Arfan et al. 2010). In agriculture, by contrast, hydrolysis of fertilizer urea by soil urease, if too rapid, results in unproductive volatilization of nitrogen and may cause ammonia toxicity or alkali-induced plant damage. Urease also has a role in the inactivation of complement, which is a component of host defense mechanism (Krajewska 2002). Due to the diverse functions of this enzyme, its inhibition by potent and specific compounds could provide an invaluable addition for the treatment of infections caused by urease-producing bacteria. In the present study we assessed the urease inhibition activity of five known ursane type sulfated saponins. Materials and methods Plant material The aerial parts of Z. fabago Linn. were collected from Ankara, Turkey, in June 2002. The plant was identified by one of us (B.S.). A voucher specimen (GUE # 2312) was deposited in the herbarium of the Faculty of Pharmacy, Gazi University, Ankara, Turkey.
(Zygophyloside G; 3) (Pöllmann et al. 1997), 3-O-[2-O-sulfo-d-quinovopyranoside] quinovic acid 28-O-[-d-glucopyranosyl] ester (Zygophyloside E; 4) (Ahmad et al. 1993), and 3-O-[d-quinovopyranoside] quinovic acid 28-O-[-d-glucopyranosyl] ester (5) (Aquino et al. 1989). These compounds were identified by their physical and spectroscopic (1 H and 13 C NMR, and mass spectra) data, including HMQC, HMBC, COSY, and NOESY experiments that allowed the unequivocal assignment of their structures. Urease inhibition assay Reaction mixtures comprising 25 l of enzyme (Jack bean urease) solution and 55 l of buffers containing 100 mM urea were incubated with 5 l of test compounds (each 0.5 mM) at 30 ◦ C for 15 min in 96-well plates. Urease inhibition activity was determined by measuring ammonia production using the indophenol method as described by Weatherburn (Weatherburn 1967). Briefly, 45 l of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 l of alkali reagent (0.5% w/v NaOH and 0.1% active chloride, NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min using a microplate reader (Molecular Devices, USA). All reactions were performed in triplicate in a final volume of 200 l. The results (change in absorbance per min) were processed by using SoftMax Pro software (Molecular Devices, USA). The entire assays were performed at pH 6.8. Percentage inhibitions were calculated from the formula 100 − (Atestwell /Acontrol ) × 100. Where “A” is the absorbance of the “test well” as well as the “control”. Thiourea was used as a reference inhibitor of urease (Khan et al. 2010a).
Preparation of the crude plant extracts The EtOH extract was prepared from aerial parts (12 kg) of Z. fabago by maceration. The dark-green residue (450 g) was dissolved in H2 O and partitioned between n-Hexane (50 g), EtOAc (100 g), and water (300 g). Identification of compounds The previously reported compounds mono sodium salt of 3,23di-O-sulfonyl-23-hydroxyurs-20(21)-en-28-oic acid 28-O-[-dglucopyranosyl] ester (Zygofaboside A; 1) (Ahmad et al. 2007), 3,23-di(sulfate) (3,4␣)-3,23,30-trihydroxyurs-20-en-28-al sodium salt (2) (Khan et al. 2010b), 3-O-[-d-2-O-sulfonylglucopyranosyl] quinovic acid 28-O-[-d-glucopyranosyl]ester
Molecular docking The molecular docking was performed by using MOE-Dock implemented in molecular operating environment using the default parameters (MOE 2008).The X-ray crystallographic structure of Bacillus pasteurii (PDBcode 4UBP) was downloaded from the protein data bank. All the water molecules were removed from the protein structure, then hydrogens were added and energy minimization was carried out using default force field. The compound’s structure was modeled using the Builder program implemented in MOE. The compound’s structure was also energy minimized and partial charges were calculated before docking. The molecular interactions were visualized using LIGPLOT implemented on MOE.
Please cite this article in press as: Khan, S.S., et al., Urease inhibitory activity of ursane type sulfated saponins from the aerial parts of Zygophyllum fabago Linn. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.09.009
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3
Fig. 2. Predicted binding mode of the most active compound 1 in the active site of Bacillus pasteurii urease.
Results and discussion
Molecular docking
In vitrourease inhibition
In order to understand the binding mode of these compounds the most active compound 1 was docked into the active site of Bacillus pasteurii urease (PDBcode 4UBP) (Benini et al. 2000). From the docking studies it was observed that strong interactions of oxygen atoms of the sulfate moiety of the compound with both the nickels in the active site of the enzyme were established at a distance of ˚ respectively. Furthermore, two metal ion interac1.87 and 1.66 A, tions were found between Na ion of the compound and Asp 363/His 139, respectively. A hydrogen bond between oxygen atom of the compound and active site residue His 222 was also found. Besides hydrogen bond, several hydrophobic interactions were observed between the compound and active site residues (Ala 170, Ala 279, Met 318, Leu 319, Ala 366, and Met 367) (Fig. 2). These interaction patterns, especially of sulfate moiety, might be one of the reasons for the activities of these compounds which help to anchor the molecules in the binding pocket of urease.
Organic compounds isolated from higher plants have been used extensively in the treatment of many diseases in past and present. Natural compounds provide a good pharmacophore template for new drugs. A number of synthetic compounds including imidazoles, hydroxamic acids, and phosphazenes are effective urease inhibitors, but limited studies have been conducted on natural products (Ahmad et al. 2003; Rahman and Choudhary 2001). The isolated compounds 1–5 were first time evaluated for in vitrourease inhibition assay. The compounds showed significant urease inhibition at 0.5 mM concentration. The results are collected in Table 1. The IC50 values of these compounds could not be found due to paucity of the samples. The proposed structure–activityrelationship shows that the activity of these compounds may be due to the sulfate moieties in these compounds.The compound 1 was found the most active showing 87% inhibition at 0.5 mM (having two sulfate moieties). The second most active compound 2 having 75% inhibition also contains two sulfate moieties. The significant structural difference between compounds 1 and 2 is the aldehyde group at position 28 instead of glucose moiety in compound 2 which may be responsible for the low activity of the compound. The compounds 3 and 4 also showed 60% and 40% inhibition, respectively. Both of these compounds contain a single sulfate moiety. The highest activity of compound 3 may be due to the presence of sodium atom in the sulfate moiety. The compound 5 showed very low inhibition (only 7%) because it contains no sulfate group. The results demonstrate that sulfate moieties are the active constituents in these compounds.
Table 1 Urease inhibitory activity of compounds 1–5. Compounds
Inhibition at 0.5 mM concentration (%)
1 2 3 4 5 Reference (Thiourea)
87.0 75.8 60.0 40.0 07.0 98.0
Conclusion The results suggest that the activity of the plant Zygophyllum fabago Linn. may be due to synergetic effect of active compounds including those investigated in the present studies; thus this plant is a potential candidate for obtaining future new remedy against the infectious diseases caused by urease producing bacteria. The SAR studies show that the sulfate moieties are the active constituents in these compounds. References Ahmad, V.U., Khan, S.S., Ahmed, A., Khan, A., Farooq, U., Arshad, S., Sener, B., Erdemoglu, N., 2007. Sulfated triterpene glycosides from Zygophyllum fabago. Natural Product Communications 2, 1085–1088. Ahmad, V.U., Ghazala, Uddin, S., Ali, M.S., 1993. Saponins from Zygophyllum propinquum. Phytochemistry 33, 453–455. Ahmad, V.U., Hussain, J., Hussain, H., Jassbi, A.R., Ullah, F., Lodhi, M.A., Yasin, A., Choudhary, M.I., 2003. First natural urease inhibitor from Euphorbia decipiens. Chemical & Pharmaceutical Bulletin 51, 719–723. Arfan, M., Ali, M., Ahmad, H., Anis, I., Khan, A., Choudhary, M.I., Shah, M.R., 2010. Urease inhibitors from Hypericum oblongifolium Wall. Journal of Enzyme Inhibition and Medicinal Chemistry 25, 296–299. Aquino, R., de Simone, F., Pizza, C., Conti, C., Stein, M.L., 1989. Plant metabolites. Structure and in vitro antiviral activity of quinovic acid glycosides from Uncaria tomentosa and Guettarda platypoda. Journal of Natural Products 52, 679–685.
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Benini, S., Rypniewski, W.R., Wilson, K.S., Miletti, S., Ciurli, S., Mangani, S., 2000. The complex of Bacillus pasteurii urease with acetohydroxamate anion from X-ray data at 1.55 A˚ resolution. Journal of Biological Inorganic Chemistry 5, 110–118. Bay, T.T., 1999. Turkiye’deBitkilerle Tedavi-Gdemisten Bugume (Therapy with Medicinal Plants in Turkey: Past and Present), second ed. Nobeltip Kitabevleri, Istanbul. Feng, Y.-L., Wu, B., Li, H.-R., Li, Y.-Q., Xu, L.-Z., Yang, S.-L., Kitanaka, S., 2008. Triterpenoidal saponins from the bark of Zygophyllum fabago L. Chemical & Pharmaceutical Bulletin 56, 858–860. Hudson, J.B., Lee, M.K., Sener, B., Erdemoglu, N., 2000. Antiviral activities in extracts of Turkish medicinal plants. Pharmaceutical Biology 38, 171–173. Khan, I., Ali, S., Hameed, S., Rama, N.H., Hussain, M.T., Wadood, A., Uddin, R., Ul-Haq, Z., Khan, A., Ali, S., Choudhary, M.I., 2010a. Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole derivatives. European Journal of Medicinal Chemistry 45, 5200–5207. Khan, S.S., Khan, A., Ahmed, A., Ahmad, V.U., Farooq, U., Arshad, S., Bader, S., Zahoor, A., Siddiqui, I.N., Sener, B., Erdemoglu, N., 2010b. Two new disulfated triterpenoids from Zygophyllum fabago. Helvetica Chimica Acta 93, 2070–2074. Krajewska, B., 2002. Ureases: Roles, properties and catalysis. Wiadomo´sci Chemiczne 56, 223–253. Mobley, H.T., Island, M.D., Hausinger, R.P., 1995. Molecular biology of microbial ureases. Microbiological Reviews 59, 451–480.
Molecular Operating Environment MOE, 2008.10; C.C.G.I.M.: Quebec, Canada. Orhan, I., Sener, B., Choudhary, M.I., Khalid, A., 2004. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal plants. Journal of Ethnopharmacology 91, 57–60. Pöllmann, K., Gagel, S., Elgamal, M.H.A., Shaker, K.H., Seifer, K., 1997. Triterpenoid saponins from the roots of Zygophyllum species. Phytochemistry 44, 485–489. Rahman, A.U., Choudhary, M.I., 2001. Bioactive natural products as a potential source of new pharmacophores. A theory of memory. Pure and Applied Chemistry 73, 555–560. Rimbau, V., Cerdan, C., Vila, R., Iglesias, J., 1999. Anti-inflammatory activity of some extracts from plants used in traditional medicine of North-African countries (II). Phytotherapy Research 13, 128–132. Shabana, M.M., Mirhom, Y.W., Genenah, A.A., Aboutabl, E.A., Amer, H.A., 1990. Study into wild Egyptian plants of potential medicinal activity, ninth communication: hypoglycemic activity of some selected plants in normal fasting and alloxanised rats. Archivfür Experimentelle Veterinarmedizin 44, 389–394. Sumner, J.B., 1926. The isolation and crystallization of the enzyme urease. Journal of Biological Chemistry 69, 435–441. Weatherburn, M.W., 1967. Phenolhypochlorite reaction for the determination of ammonia. Analytical Chemistry 39, 971–974. Zaidi, M.A., Crow Jr., S.A., 2005. Biologically active traditional medicinal herbs from Balochistan, Pakistan. Journal of Ethnopharmacology 96, 331–334.
Please cite this article in press as: Khan, S.S., et al., Urease inhibitory activity of ursane type sulfated saponins from the aerial parts of Zygophyllum fabago Linn. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.09.009
ARTICLE IN PRESS Phytomedicine xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.de/phymed
Short communication
Urease inhibitory activity of ursane type sulfated saponins from the aerial parts of Zygophyllum fabago Linn Saleha Suleman Khan a , Ajmal Khan a , Afsar Khan b,∗ , Abdul Wadood c , Umar Farooq b , Amir Ahmed d , Aqib Zahoor a , Viqar Uddin Ahmad a,∗∗ , Bilge Sener e , Nurgun Erdemoglu e a
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan c Department of Biochemistry, Shankar Campus, Abdul Wali Khan University, Mardan, Pakistan d Pharmaceutical Division, Pakistan Council of Scientific and Industrial Research Laboratories Complex, Karachi 75280, Pakistan e Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara 06330, Turkey b
a r t i c l e
i n f o
Article history: Received 17 June 2013 Received in revised form 6 August 2013 Accepted 19 September 2013 Keywords: Zygophyllum fabago Urease inhibitory activity Sulfated saponins Ursane Molecular docking
a b s t r a c t Five ursane type sulfated saponins have been isolated from the aerial parts of Zygophyllum fabago Linn. (locally called Chashum). The urease inhibitory effects of these compounds have been investigated for the first time as well as their molecular docking studies have also been carried out to check the structure–activity relationship. The IC50 values of these compounds could not be found due to paucity of the samples. The molecular docking studies were performed only for the most active compound mono sodium salt of 3,23-di-O-sulfonyl-23-hydroxyurs-20(21)-en-28-oic acid 28-O-[-d-glucopyranosyl] ester (Zygofaboside A; 1). © 2013 Elsevier GmbH. All rights reserved.
Introduction Many plants belonging to the genus Zygophyllum have been shown to cause various biological effects (Shabana et al. 1990; Rimbau et al. 1999) and are used in folk medicine in Mediterranean countries. The plant Zygophyllum fabago is one of the important herbs with known anti-rheumatic, anthelminthic, cathartic, and anti-asthmatic properties (Bay 1999). It is also used as a part of drug for rheumatism and gout; also used externally as poultice to cure skin diseases, external wounds, septic, and injuries. In China Z. fabago is used as antitussive, expectorant, and as anti-inflammatory agent (Feng et al. 2008). The plant collected from Pakistan showed very strong antifungal activity (95% at 200 g) against the human pathogen Candida albicans (Zaidi and Crow 2005). Zygophyllum fabago extracts may also contain
Abbreviations: NMR, nuclear magnetic resonance; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond correlation; COSY, correlation spectroscopy; NOESY, nuclear overhauser effect spectroscopy; MOE, molecular operating environment; IC50 , 50% inhibitory concentration; EtOH, ethanol; min, minutes; NaOH, sodium hydroxide; NaOCl, sodium hypo chloride. ∗ Corresponding author. Tel.: +92 992 383591/5; fax: +92 992 383441. ∗∗ Corresponding author. Tel.: +92 2134819020; fax: +92 2134819018/9. E-mail addresses: saleha [email protected] (S.S. Khan), [email protected], [email protected] (A. Khan), [email protected] (V.U. Ahmad).
antiviral photosensitizers but the activities were only seen at high concentrations (Hudson et al. 2000). Some of the extracts of Z. fabago from Turkish origin exhibited much higher activity against BChE (Orhan et al. 2004). In an on going search for the bioactive compounds from Z. fabago, the EtOH extract of this plant was selected for investigation. From previous studies, several unusual disulfated ursane derivatives with a double bond at C20 C21 have been reported which might have good potential invarious biological activities. Compounds isolated in the present study (Fig. 1) include five known compounds; mono sodium salt of 3ˇ,23-di-O-sulfonyl-23-hydroxyurs-20(21)-en-28-oic acid 28-O-[-d-glucopyranosyl] ester (Zygofaboside A; 1), (3,4␣)3,23,30-trihydroxyurs-20-en-28-al 3,23-di(sulfate) sodium salt (2), 3-O-[-d-2-O-sulfonylglucopyranosyl] quinovic acid 28-O[-d-glucopyranosyl]ester (Zygophyloside G; 3), 3-O-[2-O-sulfo-d-quinovopyranoside] quinovic acid 28-O-[-d-glucopyranosyl] ester (Zygophyloside E; 4), and 3-O-[-d-quinovopyranoside] quinovic acid 28-O-[-d-glucopyranosyl] ester(5). Compounds 1 and 2 have already been reported by us as new natural products from the same plant (Ahmad et al. 2007; Khan et al. 2010b). While compounds 3, 4 and 5 are new source compounds from Z. fabago. Urease (urea amidohydrolase EC 3.5.15) catalyzes the hydrolysis of urea to ammonia and carbon dioxide (Sumner 1926). Urease is the enzyme responsible for an organism to use urea as a nitrogen source. In plants urease also acts as a defense protein
0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.09.009
Please cite this article in press as: Khan, S.S., et al., Urease inhibitory activity of ursane type sulfated saponins from the aerial parts of Zygophyllum fabago Linn. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.09.009
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2
30 20
29 12 25 2 3 R1O 24
1 4
11
10 5
9 6
26 8 7
19 13 14 27
R2 21
18
17 H 16 15
22 28 R3
R2
1 R1 =SO 3Na,R 2 =H,R 2 R1 =SO 3Na,R 2 =OH,R
OO
OH
23
OH
OSO3H
= O-β -D-glucopyranosyl 3 =H 3
O
H
O
O COOH CH2OH O OH OH
R1
OH 3 R1 =OSO 3Na,R 2 =CH 2OH 4 R1 =OSO 3H,R 2 =CH 3 5 R1 =OH,R 2 =CH 3
Fig. 1. Chemical structures of compounds 1–5.
in systemic nitrogen transport pathways. Urease is known to be one of the major causes of pathologies induced by H. pylori, thus allowing them to survive at low pH of the stomach and, therefore, plays an important role in the pathogenesis of gastric and peptic ulcer, apart from cancer as well. Urease is directly involved in the formation of infection stones in kidney and contributes to the pathogensis of urolithiasis, pyelonephritis, ammonia, hepatic encephalopathy, hepatic coma, and urinary catheter encrustation (Mobley et al. 1995; Arfan et al. 2010). In agriculture, by contrast, hydrolysis of fertilizer urea by soil urease, if too rapid, results in unproductive volatilization of nitrogen and may cause ammonia toxicity or alkali-induced plant damage. Urease also has a role in the inactivation of complement, which is a component of host defense mechanism (Krajewska 2002). Due to the diverse functions of this enzyme, its inhibition by potent and specific compounds could provide an invaluable addition for the treatment of infections caused by urease-producing bacteria. In the present study we assessed the urease inhibition activity of five known ursane type sulfated saponins. Materials and methods Plant material The aerial parts of Z. fabago Linn. were collected from Ankara, Turkey, in June 2002. The plant was identified by one of us (B.S.). A voucher specimen (GUE # 2312) was deposited in the herbarium of the Faculty of Pharmacy, Gazi University, Ankara, Turkey.
(Zygophyloside G; 3) (Pöllmann et al. 1997), 3-O-[2-O-sulfo-d-quinovopyranoside] quinovic acid 28-O-[-d-glucopyranosyl] ester (Zygophyloside E; 4) (Ahmad et al. 1993), and 3-O-[d-quinovopyranoside] quinovic acid 28-O-[-d-glucopyranosyl] ester (5) (Aquino et al. 1989). These compounds were identified by their physical and spectroscopic (1 H and 13 C NMR, and mass spectra) data, including HMQC, HMBC, COSY, and NOESY experiments that allowed the unequivocal assignment of their structures. Urease inhibition assay Reaction mixtures comprising 25 l of enzyme (Jack bean urease) solution and 55 l of buffers containing 100 mM urea were incubated with 5 l of test compounds (each 0.5 mM) at 30 ◦ C for 15 min in 96-well plates. Urease inhibition activity was determined by measuring ammonia production using the indophenol method as described by Weatherburn (Weatherburn 1967). Briefly, 45 l of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 l of alkali reagent (0.5% w/v NaOH and 0.1% active chloride, NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after 50 min using a microplate reader (Molecular Devices, USA). All reactions were performed in triplicate in a final volume of 200 l. The results (change in absorbance per min) were processed by using SoftMax Pro software (Molecular Devices, USA). The entire assays were performed at pH 6.8. Percentage inhibitions were calculated from the formula 100 − (Atestwell /Acontrol ) × 100. Where “A” is the absorbance of the “test well” as well as the “control”. Thiourea was used as a reference inhibitor of urease (Khan et al. 2010a).
Preparation of the crude plant extracts The EtOH extract was prepared from aerial parts (12 kg) of Z. fabago by maceration. The dark-green residue (450 g) was dissolved in H2 O and partitioned between n-Hexane (50 g), EtOAc (100 g), and water (300 g). Identification of compounds The previously reported compounds mono sodium salt of 3,23di-O-sulfonyl-23-hydroxyurs-20(21)-en-28-oic acid 28-O-[-dglucopyranosyl] ester (Zygofaboside A; 1) (Ahmad et al. 2007), 3,23-di(sulfate) (3,4␣)-3,23,30-trihydroxyurs-20-en-28-al sodium salt (2) (Khan et al. 2010b), 3-O-[-d-2-O-sulfonylglucopyranosyl] quinovic acid 28-O-[-d-glucopyranosyl]ester
Molecular docking The molecular docking was performed by using MOE-Dock implemented in molecular operating environment using the default parameters (MOE 2008).The X-ray crystallographic structure of Bacillus pasteurii (PDBcode 4UBP) was downloaded from the protein data bank. All the water molecules were removed from the protein structure, then hydrogens were added and energy minimization was carried out using default force field. The compound’s structure was modeled using the Builder program implemented in MOE. The compound’s structure was also energy minimized and partial charges were calculated before docking. The molecular interactions were visualized using LIGPLOT implemented on MOE.
Please cite this article in press as: Khan, S.S., et al., Urease inhibitory activity of ursane type sulfated saponins from the aerial parts of Zygophyllum fabago Linn. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.09.009
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Fig. 2. Predicted binding mode of the most active compound 1 in the active site of Bacillus pasteurii urease.
Results and discussion
Molecular docking
In vitrourease inhibition
In order to understand the binding mode of these compounds the most active compound 1 was docked into the active site of Bacillus pasteurii urease (PDBcode 4UBP) (Benini et al. 2000). From the docking studies it was observed that strong interactions of oxygen atoms of the sulfate moiety of the compound with both the nickels in the active site of the enzyme were established at a distance of ˚ respectively. Furthermore, two metal ion interac1.87 and 1.66 A, tions were found between Na ion of the compound and Asp 363/His 139, respectively. A hydrogen bond between oxygen atom of the compound and active site residue His 222 was also found. Besides hydrogen bond, several hydrophobic interactions were observed between the compound and active site residues (Ala 170, Ala 279, Met 318, Leu 319, Ala 366, and Met 367) (Fig. 2). These interaction patterns, especially of sulfate moiety, might be one of the reasons for the activities of these compounds which help to anchor the molecules in the binding pocket of urease.
Organic compounds isolated from higher plants have been used extensively in the treatment of many diseases in past and present. Natural compounds provide a good pharmacophore template for new drugs. A number of synthetic compounds including imidazoles, hydroxamic acids, and phosphazenes are effective urease inhibitors, but limited studies have been conducted on natural products (Ahmad et al. 2003; Rahman and Choudhary 2001). The isolated compounds 1–5 were first time evaluated for in vitrourease inhibition assay. The compounds showed significant urease inhibition at 0.5 mM concentration. The results are collected in Table 1. The IC50 values of these compounds could not be found due to paucity of the samples. The proposed structure–activityrelationship shows that the activity of these compounds may be due to the sulfate moieties in these compounds.The compound 1 was found the most active showing 87% inhibition at 0.5 mM (having two sulfate moieties). The second most active compound 2 having 75% inhibition also contains two sulfate moieties. The significant structural difference between compounds 1 and 2 is the aldehyde group at position 28 instead of glucose moiety in compound 2 which may be responsible for the low activity of the compound. The compounds 3 and 4 also showed 60% and 40% inhibition, respectively. Both of these compounds contain a single sulfate moiety. The highest activity of compound 3 may be due to the presence of sodium atom in the sulfate moiety. The compound 5 showed very low inhibition (only 7%) because it contains no sulfate group. The results demonstrate that sulfate moieties are the active constituents in these compounds.
Table 1 Urease inhibitory activity of compounds 1–5. Compounds
Inhibition at 0.5 mM concentration (%)
1 2 3 4 5 Reference (Thiourea)
87.0 75.8 60.0 40.0 07.0 98.0
Conclusion The results suggest that the activity of the plant Zygophyllum fabago Linn. may be due to synergetic effect of active compounds including those investigated in the present studies; thus this plant is a potential candidate for obtaining future new remedy against the infectious diseases caused by urease producing bacteria. The SAR studies show that the sulfate moieties are the active constituents in these compounds. References Ahmad, V.U., Khan, S.S., Ahmed, A., Khan, A., Farooq, U., Arshad, S., Sener, B., Erdemoglu, N., 2007. Sulfated triterpene glycosides from Zygophyllum fabago. Natural Product Communications 2, 1085–1088. Ahmad, V.U., Ghazala, Uddin, S., Ali, M.S., 1993. Saponins from Zygophyllum propinquum. Phytochemistry 33, 453–455. Ahmad, V.U., Hussain, J., Hussain, H., Jassbi, A.R., Ullah, F., Lodhi, M.A., Yasin, A., Choudhary, M.I., 2003. First natural urease inhibitor from Euphorbia decipiens. Chemical & Pharmaceutical Bulletin 51, 719–723. Arfan, M., Ali, M., Ahmad, H., Anis, I., Khan, A., Choudhary, M.I., Shah, M.R., 2010. Urease inhibitors from Hypericum oblongifolium Wall. Journal of Enzyme Inhibition and Medicinal Chemistry 25, 296–299. Aquino, R., de Simone, F., Pizza, C., Conti, C., Stein, M.L., 1989. Plant metabolites. Structure and in vitro antiviral activity of quinovic acid glycosides from Uncaria tomentosa and Guettarda platypoda. Journal of Natural Products 52, 679–685.
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G Model PHYMED-51524; No. of Pages 4 4
ARTICLE IN PRESS S.S. Khan et al. / Phytomedicine xxx (2013) xxx–xxx
Benini, S., Rypniewski, W.R., Wilson, K.S., Miletti, S., Ciurli, S., Mangani, S., 2000. The complex of Bacillus pasteurii urease with acetohydroxamate anion from X-ray data at 1.55 A˚ resolution. Journal of Biological Inorganic Chemistry 5, 110–118. Bay, T.T., 1999. Turkiye’deBitkilerle Tedavi-Gdemisten Bugume (Therapy with Medicinal Plants in Turkey: Past and Present), second ed. Nobeltip Kitabevleri, Istanbul. Feng, Y.-L., Wu, B., Li, H.-R., Li, Y.-Q., Xu, L.-Z., Yang, S.-L., Kitanaka, S., 2008. Triterpenoidal saponins from the bark of Zygophyllum fabago L. Chemical & Pharmaceutical Bulletin 56, 858–860. Hudson, J.B., Lee, M.K., Sener, B., Erdemoglu, N., 2000. Antiviral activities in extracts of Turkish medicinal plants. Pharmaceutical Biology 38, 171–173. Khan, I., Ali, S., Hameed, S., Rama, N.H., Hussain, M.T., Wadood, A., Uddin, R., Ul-Haq, Z., Khan, A., Ali, S., Choudhary, M.I., 2010a. Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole derivatives. European Journal of Medicinal Chemistry 45, 5200–5207. Khan, S.S., Khan, A., Ahmed, A., Ahmad, V.U., Farooq, U., Arshad, S., Bader, S., Zahoor, A., Siddiqui, I.N., Sener, B., Erdemoglu, N., 2010b. Two new disulfated triterpenoids from Zygophyllum fabago. Helvetica Chimica Acta 93, 2070–2074. Krajewska, B., 2002. Ureases: Roles, properties and catalysis. Wiadomo´sci Chemiczne 56, 223–253. Mobley, H.T., Island, M.D., Hausinger, R.P., 1995. Molecular biology of microbial ureases. Microbiological Reviews 59, 451–480.
Molecular Operating Environment MOE, 2008.10; C.C.G.I.M.: Quebec, Canada. Orhan, I., Sener, B., Choudhary, M.I., Khalid, A., 2004. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal plants. Journal of Ethnopharmacology 91, 57–60. Pöllmann, K., Gagel, S., Elgamal, M.H.A., Shaker, K.H., Seifer, K., 1997. Triterpenoid saponins from the roots of Zygophyllum species. Phytochemistry 44, 485–489. Rahman, A.U., Choudhary, M.I., 2001. Bioactive natural products as a potential source of new pharmacophores. A theory of memory. Pure and Applied Chemistry 73, 555–560. Rimbau, V., Cerdan, C., Vila, R., Iglesias, J., 1999. Anti-inflammatory activity of some extracts from plants used in traditional medicine of North-African countries (II). Phytotherapy Research 13, 128–132. Shabana, M.M., Mirhom, Y.W., Genenah, A.A., Aboutabl, E.A., Amer, H.A., 1990. Study into wild Egyptian plants of potential medicinal activity, ninth communication: hypoglycemic activity of some selected plants in normal fasting and alloxanised rats. Archivfür Experimentelle Veterinarmedizin 44, 389–394. Sumner, J.B., 1926. The isolation and crystallization of the enzyme urease. Journal of Biological Chemistry 69, 435–441. Weatherburn, M.W., 1967. Phenolhypochlorite reaction for the determination of ammonia. Analytical Chemistry 39, 971–974. Zaidi, M.A., Crow Jr., S.A., 2005. Biologically active traditional medicinal herbs from Balochistan, Pakistan. Journal of Ethnopharmacology 96, 331–334.
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