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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Urease inhibitory constituents from Daphne retusa a
a
b
b
Farrukh Mansoor , Itrat Anis , Ajmal Khan , Bishnu P. Marasini , b
b
Muhammad Iqbal Choudhary & Muhammad Raza Shah a
Department of Chemistry, University of Karachi, Karachi, 75270, Pakistan b
International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, 75270, Pakistan Published online: 25 Nov 2013.
To cite this article: Farrukh Mansoor, Itrat Anis, Ajmal Khan, Bishnu P. Marasini, Muhammad Iqbal Choudhary & Muhammad Raza Shah (2014) Urease inhibitory constituents from Daphne retusa, Journal of Asian Natural Products Research, 16:2, 210-215, DOI: 10.1080/10286020.2013.837457 To link to this article: http://dx.doi.org/10.1080/10286020.2013.837457
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 2, 210–215, http://dx.doi.org/10.1080/10286020.2013.837457
Urease inhibitory constituents from Daphne retusa Farrukh Mansoora, Itrat Anisa*, Ajmal Khanb, Bishnu P. Marasinib, Muhammad Iqbal Choudharyb and Muhammad Raza Shahb a Department of Chemistry, University of Karachi, Karachi 75270, Pakistan; bInternational Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
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(Received 26 May 2013; final version received 20 August 2013) The bioassay-guided fractionation of Daphne retusa Hemsl. has led to the isolation of a new aryl tetrahydronaphthalene lignan derivative named as daphnretusic acid (1), along with six new source compounds such as 5,7-dihydroxyflavone (2), 7hydroxyflavone (3), 6-methoxyflavone (4), (þ) pinoresinol (5), (þ ) sesamin (6), and b-sitosterol-3-O-b-D -glucopyranoside (7). Their structures were elucidated by 1H NMR, 13C NMR, 1D, 2D NMR, UV, IR, and EIMS analyses. All the fractions (n-hexane, CHCl3, AcOEt, CH3OH, and water) and pure compounds (1 – 7) were subjected to the assay of urease and a-chymotrypsin inhibitory activities. Chloroform and methanol soluble fractions showed moderate urease inhibition. Compound 2 exhibited significant urease inhibition with IC50 value 60.4 ^ 0.72 mM, whereas compounds 1 and 3 –7 remained inactive during urease inhibition and a-chymotrypsin bioassays. Keywords: Daphne retusa; Thymeleaceae; flavones; urease inhibitors; a-chymotrypsin enzyme
1.
Introduction
Plant family Thymeleaceae comprises 500 species assigned to 50 genera, and one of them is Daphne [1]. Almost all species of this genus are poisonous but are reported to be effective against diuretic, purgative, hemostatic fever, aches, rheumatism, toothache, ulcers, abortifacient, and emetic disorders [2,3]. Previously coumarins [4,5], flavonoids, diterpenoids [6], and coumarinolignans [7] have been reported from this genus. Daphne retusa is one of the important species of the genus Daphne and is widely distributed in the East Asia. In Pakistan, it is found in northern areas [1]. Daphne retusa is used in folk medicine because of its detumescence and acesodyne effects [8], and its ethanolic extract (75%) was found to be anti-inflammatory and anti-analgesic [9]. So far only few
coumarins have been isolated from this species [4,5]. Urease enzyme occurs throughout plant and animal kingdoms, utilizing urea as nitrogen source especially in plants at germination stages [10,11] and also acts as their defense protein. Urease is a reputed cause of infection by stone formation contributing to the pathogenesis of urolithiasis and urinary catheter encrustation [10 – 12]. In agriculture, high urease concentration causes economic and environmental problems by releasing large amounts of ammonia in the environment during urea fertilization, hence leading to undue loss of fertilizers. This process deprives plant from their essential nutrients, and the toxicity of ammonia leads to increase the soil pH [13,14]. Taking into account our interest in the
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Downloaded by [University of Sussex Library] at 09:12 28 October 2014
Journal of Asian Natural Products Research chemotaxonomic and ethnopharmacological importance of the medicinal plants of Pakistan [15] and therapeutical role of secondary plant metabolites in urease inhibition, Atta-ur-Rahman and Choudhary [16] prompted us to carry out bioassayguided phytochemical studies on D. retusa. Investigations resulted in the isolation of a new aryl tetrahydronaphthalene lignan derivative named as daphnretusic acid (1), along with six new source compounds 5,7dihydroxyflavone (2), 7-hydroxyflavone (3), 6-methoxyflavone (4), (þ) pinoresinol (5), (þ) sesamin (6), and b-sitosterol 3-Ob-D -glucopyranoside (7). All the isolated compounds and water, methanol, ethyl acetate, chloroform, and hexane soluble fractions were subjected to the urease inhibition. Compound 2 has shown a significant urease inhibitory activity with IC50 value 60.4 ^ 0.72 mM, while water, chloroform, and hexane soluble fractions were moderately active and methanolic extract was also significantly active. 2.
Results and discussion
The methanolic extract of the whole plant of D. retusa was suspended in water and successively extracted with n-hexane, chloroform, and ethyl acetate. The chloroform and ethyl acetate-soluble fractions were subjected to silica gel column chromatography as described in the experimental part. The six new source compounds (2–7) were identified on the basis of their respective spectral data (Figure 1). Daphnretusic acid (1) was obtained as an amorphous powder, having a molecular formula C19H20O8 deduced from the molecular ion peak appeared at m/z 376.1164 in HREIMS. The 1H NMR spectrum showed signals of five aromatic protons [d 6.67 (s, H5), 6.53 (s, H-8), 6.88 (d, J ¼ 8.1 Hz, H-50 ), 6.78 (dd, J ¼ 8.1, 1.7 Hz, H-60 ), 6.75 (d, J ¼ 1.7 Hz, H-20 )]; one methoxyl and two methylenic protons (H-1 and H-3a); and two methine protons at d 4.02 (s, H-4) and 2.50 (m, H-2), which indicated that the compound
Figure 1.
211
Structure of daphnretusic acid (1).
is a lignan derivative. The 13C NMR, DEPT and HSQC experiments displayed signals of 19 carbons including downfield signals of carbonyl group C-2a (d 179.1), one oxygenated quaternary carbon (d 76.7), one oxygenated methylene (d 69.5), nine quaternary carbons, one methoxyl group, one aliphatic methylene, and two methine carbons, which further supported the aryltetrahydronaphthalene lignan skeleton. All the assignments of protons were further validated by 1H– 1H COSY analysis which showed correlations of multiplet protons at d 2.50 (H-2) with double-doublet protons at d 2.92 (1H, J ¼ 13.8, 4.5 Hz, H-1a) and triplet at d 3.04 (1H, J ¼ 13.8 Hz, H-1b), while the resonance at d 3.88 (1H, J ¼ 12.0 Hz, H-3ax) and 4.23 (1H, J ¼ 12.0 Hz, 3ay) appeared as two doublets, therefore suggesting that the hydroxymethylene (H-3a) was attached to quaternary carbon (C-3) but methylene protons (H-1) were connected with one methine proton (H-2) and one quaternary carbon (C-9). This confirmed that the compound is different from previously reported compounds [17,18]. These assignments (1) were also confirmed through HMBC correlations, in which the methylene protons at d 2.92 and 3.04 showed correlations with the carbons at d 179.1 (C-2a), 43.4 (C-2), 76.7 (C-3), 114.2 (C-8), 123.0 (C-9), and 130.5 (C-10), whereas hydroxymethylene protons at d 4.23 (H-3ax) and 3.88 (H-3ay) showed cross-peaks with C-3 at d 76.7 and C-4 at d 48.34, and a methine singlet at d 4.02 (H-4) showed HMBC correlations with the carbons at d 76.7 (C-3),
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123.0 (C-9), 133.3 (C-10 ), 113.0 (C-20 ), and 122.8 (C-60 ). These correlations confirmed that the hydroxyl group attached at C-3 while free carboxylic group occurred at C-2 position. The presence of methoxyl group at C-30 was confirmed through HMBC interactions (see Figure 2). The relative configuration of C-4 could be established on the basis of previously reported compounds in which H-4 (d 4.02) showed singlet only when dihedral angle of 908 between H-3 and H-4 [17,19] or C-4 is adjacent to quaternary carbons (C-3 and C-10). Stereochemistry of C-2 was further confirmed through Nuclear Overhauser enhanced and exchange spectroscopic (NOESY) correlations of H-2 with H-3a, which suggested that they were the same stereoisomer like tetralin [17,20]. Finally, on the basis of the above evidence, the structure of (1) was a new compound daphnretusic acid. The known isolated compounds are 5,7-dihydroxyflavone (2) [21], 7-hydroxyflavone (3) [21], 6-methoxyflavone (4) [22], (þ ) pinoresinol (5) [23], (þ ) sesamin (6) [24], and b-sitosterol 3-O-b-D -glucopyranoside (7) [25], respectively. All the physical and spectral data of these compounds were in close agreement with the already reported literature. The bioassay-guided fractionation of Daphne retusa has led to the isolation of potent urease inhibitors. The water, methanol, ethyl acetate, chloroform, and n-hexane soluble fractions of plant (airdried, powdered twigs of Daphne retusa) were tested in vitro for their urease
Figure 2.
Important HMBC correlations of 1.
inhibition activity. Compounds 2– 4 are flavonoids, but only compound 2 showed moderate activity (IC50 60.4 ^ 0.72 mM). The moderate activity of 2 might be associated with the hydroxyl group at C-5, facilitating its interaction with nickel via hydrogen bonding. Compound 4 has a methoxy group at C-6 hence lacking interactions with nickel; similarly hydroxyl groups found at C-7 in compound 3 remained recessive due to the absence of hydroxyl group at C-5 position. The a-chymotrypsin is a protease enzyme and digests protein debris of the ulcer; also it had been used for the treatment of peptic ulcer [16,26]. All compounds were inactive in case of a-chymotrypsin. 3. 3.1
Experimental General experimental procedures
Optical rotation was measured on a JASCO DIP-360 digital polarimeter (Maryland, USA). The melting points were recorded on Gallenkamp apparatus (Bristol, UK) and are uncorrected. The UV spectra were recorded on a Hitachi UV3200 spectrometer (Tokyo, Japan) with chloroform and methanol as solvent. IR spectra were recorded on Shimadzu IR460 spectrophotometer (Tokyo, Japan) using KBr disk. Mass spectra were recorded on a JEOL JMS-HX 110 spectrometer (Japan) with data system. The 1H NMR and 13C NMR spectra were recorded on Bruker AMX-400 instruments (France) in CDCl3 (ARMAR, Switzerland) and CD3OD by using tetramethyl silane as an internal standard. The chemical shift values are reported in ppm (d) units and the scalar coupling constants (J) are in Hz. Column chromatography was carried out by using silica gel of 70 – 230 and 230 – 400 mesh sizes. Aluminum sheets precoated with silica gel 60 F254 (20 £ 20 cm, 0.2 mm thick; E-Merck) were used for thin layer chromatography analysis to check the purity of compounds and were visualized under UV light (254 and
Journal of Asian Natural Products Research 366 nm) followed by ceric sulfate as spraying reagent (heating). All organic solvents and chemicals were obtained from Merck Scientific or otherwise mentioned in its respective portion.
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3.2
Plant material
The plant Daphne retusa Hemsl. (whole plant) was collected from Gilgit (Northern areas), Pakistan, in the summer season of 2005. The plant was identified by Mr Manzoor Ahmed, Plant Taxonomist, Department of Botany, Government Post Graduate College, Abbottabad. A voucher specimen (DR-004) was deposited in the herbarium of the department. 3.3
Extraction and isolation
The freshly collected plant material (whole plant, 22 kg) of D. retusa was shade dried (9 kg), ground, and extracted with methanol (M tedia-USA, 3 £ 15 liters, each for 10 days). The combined methanolic extract was evaporated under reduced pressure at room temperature to yield a crude residue (535 g). The whole extract was suspended in water and successively extracted with n-hexane (158 g), chloroform (M tedia-USA, 95 g), ethyl acetate (109 g), and water (85 g). The urease-active, chloroform-soluble fraction was subjected to column chromatography over silica gel and eluted with n-hexane, nhexane – chloroform, chloroform, and chloroform – methanol with a gradient increase in polarity to afford 25 subfractions. The sub-fractions (F1 – F5) were a binary mixture of two compounds that was re-chromatographed over silica gel and eluted with CHCl3:n-hexane (9.6:0.4) to afford compounds 2 –4 in pure form. The sub-fractions (F6 – F8) were re-chromatographed over silica gel and eluted with chloroform:MeOH (9.8:0.2) to afford compounds 5, 6, and 1, and then compound 7 was eluted with chloroform: MeOH (9.5:0.5).
213
3.3.1 Daphnretusic acid 1 Amorphous powder; m.p. 164 – 1668C. [a]D 2 78 (MeOH; c 1.02). UV: lmax (MeOH) 206 (4.16), 226 (4.56), 278 (3.92) nm; IR (KBr) y max cm21: 3427 (OH), 3011 (CH), 1700, and 1620 –1400. For 1H NMR and 13C NMR spectral data, see Table 1. EIMS m/z (%): 376 [M]þ(15), 358 (7), 328 (14), 165 (8), 151 (29), 137 (100). HREIMS: m/z 376.1164 [M]þ (calcd for C19H20O8, 376.1158). 3.4
Urease inhibition assay
A reaction mixture containing 1 unit of urease enzyme (Jack bean urease) solution and 55 ml of buffers having 100 mM urea was incubated in the presence of 5 ml of test compounds (0.5 mM concentration) at 308C for 15 min in 96-well plates. Urease activity was evaluated by measuring ammonia production as described in the indophenol method [14]. Briefly, 45 ml each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 ml 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 Device, Sunnyvale, CA, USA). All reactions were performed three times in a final volume of 200 ml. The data (change in absorbance per minute) were processed with the help of Softmax Pro software (Molecular Device). All the assays were carried out at pH 8.2 (0.01 M K2HPO4 3H2O, 1 mM BDTA, and 0.01 M LiCl2). Percentage inhibitions were calculated from the formula 100 2 (OD testwell / ODcontrol) £ 100. Thiourea was used as the standard inhibitor of urease. 3.5
Chymotrypsin inhibition assay
Chymotrypsin (Beijing, China, 12 units/ml prepared in Tris– HCl buffer, pH 7.6) was pre-incubated along with test compounds (prepared to a final concentration of 7%
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Table 1. 1H NMR (CD3OD, 400 MHz) (d ppm) and spectral data for daphnretusic acid (1).
C NMR (CD3OD, 100 MHz) (d ppm)
Multiplicity
dC
dH
1
t
38.3
2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 2a 3a OCH3
d s d d s s s s s s d s s d d s t
43.4 76.7 48.3 111.6 145.3 144.2 114.2 123.0 130.5 133.3 113.0 147.8 145.2 116.7 122.8 179.1 69.5 55.7
2.92 (1H, dd, J ¼ 13.8, 4.5 Hz, H-1a), 3.04 (1H, t, J ¼ 13.8 Hz, H-1b) 2.50 (1H, m, H-2)
Carbons
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13
4.02 (s, H-4) 6.67 (s, H-5) 6.53 (s, H-8)
6.75 (d, J ¼ 1.7 Hz, H-20 ) 6.88 (d, J ¼ 8.1 Hz, H-50 ) 6.78 (dd, J ¼ 8.1, 1.7 Hz, H-60 ) 3.88 (d, J ¼ 12.0 Hz, H-3ax), 4.23 (d, J ¼ 12.0 Hz, H-3ay) 3.94 (3H, s, OCH3-30 )
dimethylsulfoxide) for 25 min at 308C. The solution of substrate (N-succinyl-phenylalanine-p-nitroanilide of Sigma-Aldrich, 0.4 mM) was added to the reaction mixture in order to initiate the enzymatic reaction. The observed absorbance was due to the release of p-nitroaniline, which was continuously monitored at 410 nm until a significant color change occurred [26].
3.6 Determination of IC50 values The concentrations of the test fractions and compounds, which retarded the hydrolysis of substrates by 50% (IC50), were evaluated by monitoring the effect of various concentrations of them in the assays on inhibition values. The IC50 values were then calculated using the EZ-fit Enzyme Kinetics program (Perrelia Scientific Inc., Amherst, USA).
4. Conclusion The development of new and safe enzyme inhibitors has led to the discovery of drugs.
Imidazoles, hydroxamic acids, thiols, and phosphazenes are established synthetic urease inhibitors, but very little knowledge is available on the role of natural products as urease inhibitors [15,16]. Owing to the widespread application of this enzyme, its inhibition by potent and specific sources could provide an invaluable addition for treatment of infections. Moreover, screening of natural products also contributes chemically.
Acknowledgments Authors of manuscript greatly acknowledge the financial support of the Dean, Faculty of Science, University of Karachi for this research.
References [1] E. Nasir, and S.I. Ali, Flora of West Pakistan (Fakhri Printing Press, Karachi, 1971), pp. 1 – 10. [2] S.R. Baquar, Medicinal and Poisonous Plants of Pakistan (Printas Press, Karachi, 1989), p. 161.
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Journal of Asian Natural Products Research [3] H.E. Connor, The Poisonous Plants in New Zealand (E. C. Keating, Government Printer, Wellington, 1977), p. 172. [4] Y.Z. Lin, C.R. Ji, L.S. Yang, and W.S. Feng, Chin. Bull. Bot. 11, 41 (1994). [5] X.J. Hu, H.Z. Jin, S.U. Jaun, W. Zhang, W.Z. Xu, S.K. Yan, R.H. Liu, J.Q. Li, and W.D. Zhang, Chin. J. Nat. Med. 7, 34 (2009). [6] S.H. Li, L.J. Wu, and H.Y. Yin, China J. Chin. Nat. Med. 27, 401 (2000). [7] N. UIIah, S. Ahmed, P. Mohammad, Z. Ahmed, H.R. Nawaz, and A. Malik, Phytochemistry 51, 103 (1999). [8] M.S. Wang, M. Yu, and Y.J. Zhang, Chin. Herb. Med. Comm. 7, 13 (1976). [9] X. Hu, H. Jin, W. Xu, W. Zhang, X. Liu, S. Yan, M. Chen, J. Li, and W.D. Zhang, J. Ethnopharmacol. 120, 118 (2008). [10] K.M. Khan, Z.S. Saify, M.A. Lodhi, N. Butt, S. Perveen, G.M. Maharvi, M.I. Choudhary, and Atta-ur-Rahman, Nat. Prod. Res. 20, 523 (2006). [11] H.L.T. Mobley and R.P. Hausinger, Microbiol. Rev. 53, 85 (1989). [12] H.L.T. Mobley, M.D. Island, and R.P. Hausinger, Microbiol. Rev. 59, 451 (1995). [13] J.M. Bremner, Fert. Res. 42, 321 (1995). [14] M.W. Weatherburn, Anal. Chem. 39, 971 (1967).
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[15] A.K. Jan, M.R. Shah, I. Anis, and I.K. Marwat, J. Enzy. Inhib. Med. Chem. 24, 192 (2009). [16] Atta-ur-Rahman, and M.I. Choudhary, Pure Appl. Chem. 73, 555 (2001). [17] A. Pelter, R.S. Ward, R. Venkateswarlu, and C. Kamakshi, Tetrahedron 45, 3451 (1989). [18] F. Kawamura, S. Kawai, and H. Ohashi, Phytochemistry 44, 1353 (1997). [19] A.S. Chawla, A.K. Sharma, S.S. Handa, and K.L. Dhar, Phytochemistry 39, 4378 (1992). [20] H.Z. Li, G.J. Luo, H.M. Li, X.L. Li, and R.T. Li, Chin. Chem. Lett. 22, 85 (2011). [21] D.W. Aksnes, A. Standnes, and O.M. Anderson, Magn. Res. Chem. 34, 820 (1996). [22] P.W. Freeman, S.T. Murphy, J.E. Nemorin, and W.C. Taylor, Aust. J. Chem. 34, 1779 (1981). [23] N.R. Guz and F.R. Stermitz, Phytochemistry 54, 897 (2000). [24] W.Y. Tsui and G.D. Brown, Fitoterapia 68, 479 (1996). [25] I. Rubinstein, L.J. Goad, A.D.H. Clague, and L.J. Mulheirn, Phytochemistry 15, 195 (1976). [26] A. Coblentz, J. Am. Geriatr. Soc. 16, 1039 (1968).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Urease inhibitory constituents from Daphne retusa a
a
b
b
Farrukh Mansoor , Itrat Anis , Ajmal Khan , Bishnu P. Marasini , b
b
Muhammad Iqbal Choudhary & Muhammad Raza Shah a
Department of Chemistry, University of Karachi, Karachi, 75270, Pakistan b
International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, 75270, Pakistan Published online: 25 Nov 2013.
To cite this article: Farrukh Mansoor, Itrat Anis, Ajmal Khan, Bishnu P. Marasini, Muhammad Iqbal Choudhary & Muhammad Raza Shah (2014) Urease inhibitory constituents from Daphne retusa, Journal of Asian Natural Products Research, 16:2, 210-215, DOI: 10.1080/10286020.2013.837457 To link to this article: http://dx.doi.org/10.1080/10286020.2013.837457
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 2, 210–215, http://dx.doi.org/10.1080/10286020.2013.837457
Urease inhibitory constituents from Daphne retusa Farrukh Mansoora, Itrat Anisa*, Ajmal Khanb, Bishnu P. Marasinib, Muhammad Iqbal Choudharyb and Muhammad Raza Shahb a Department of Chemistry, University of Karachi, Karachi 75270, Pakistan; bInternational Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
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(Received 26 May 2013; final version received 20 August 2013) The bioassay-guided fractionation of Daphne retusa Hemsl. has led to the isolation of a new aryl tetrahydronaphthalene lignan derivative named as daphnretusic acid (1), along with six new source compounds such as 5,7-dihydroxyflavone (2), 7hydroxyflavone (3), 6-methoxyflavone (4), (þ) pinoresinol (5), (þ ) sesamin (6), and b-sitosterol-3-O-b-D -glucopyranoside (7). Their structures were elucidated by 1H NMR, 13C NMR, 1D, 2D NMR, UV, IR, and EIMS analyses. All the fractions (n-hexane, CHCl3, AcOEt, CH3OH, and water) and pure compounds (1 – 7) were subjected to the assay of urease and a-chymotrypsin inhibitory activities. Chloroform and methanol soluble fractions showed moderate urease inhibition. Compound 2 exhibited significant urease inhibition with IC50 value 60.4 ^ 0.72 mM, whereas compounds 1 and 3 –7 remained inactive during urease inhibition and a-chymotrypsin bioassays. Keywords: Daphne retusa; Thymeleaceae; flavones; urease inhibitors; a-chymotrypsin enzyme
1.
Introduction
Plant family Thymeleaceae comprises 500 species assigned to 50 genera, and one of them is Daphne [1]. Almost all species of this genus are poisonous but are reported to be effective against diuretic, purgative, hemostatic fever, aches, rheumatism, toothache, ulcers, abortifacient, and emetic disorders [2,3]. Previously coumarins [4,5], flavonoids, diterpenoids [6], and coumarinolignans [7] have been reported from this genus. Daphne retusa is one of the important species of the genus Daphne and is widely distributed in the East Asia. In Pakistan, it is found in northern areas [1]. Daphne retusa is used in folk medicine because of its detumescence and acesodyne effects [8], and its ethanolic extract (75%) was found to be anti-inflammatory and anti-analgesic [9]. So far only few
coumarins have been isolated from this species [4,5]. Urease enzyme occurs throughout plant and animal kingdoms, utilizing urea as nitrogen source especially in plants at germination stages [10,11] and also acts as their defense protein. Urease is a reputed cause of infection by stone formation contributing to the pathogenesis of urolithiasis and urinary catheter encrustation [10 – 12]. In agriculture, high urease concentration causes economic and environmental problems by releasing large amounts of ammonia in the environment during urea fertilization, hence leading to undue loss of fertilizers. This process deprives plant from their essential nutrients, and the toxicity of ammonia leads to increase the soil pH [13,14]. Taking into account our interest in the
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Downloaded by [University of Sussex Library] at 09:12 28 October 2014
Journal of Asian Natural Products Research chemotaxonomic and ethnopharmacological importance of the medicinal plants of Pakistan [15] and therapeutical role of secondary plant metabolites in urease inhibition, Atta-ur-Rahman and Choudhary [16] prompted us to carry out bioassayguided phytochemical studies on D. retusa. Investigations resulted in the isolation of a new aryl tetrahydronaphthalene lignan derivative named as daphnretusic acid (1), along with six new source compounds 5,7dihydroxyflavone (2), 7-hydroxyflavone (3), 6-methoxyflavone (4), (þ) pinoresinol (5), (þ) sesamin (6), and b-sitosterol 3-Ob-D -glucopyranoside (7). All the isolated compounds and water, methanol, ethyl acetate, chloroform, and hexane soluble fractions were subjected to the urease inhibition. Compound 2 has shown a significant urease inhibitory activity with IC50 value 60.4 ^ 0.72 mM, while water, chloroform, and hexane soluble fractions were moderately active and methanolic extract was also significantly active. 2.
Results and discussion
The methanolic extract of the whole plant of D. retusa was suspended in water and successively extracted with n-hexane, chloroform, and ethyl acetate. The chloroform and ethyl acetate-soluble fractions were subjected to silica gel column chromatography as described in the experimental part. The six new source compounds (2–7) were identified on the basis of their respective spectral data (Figure 1). Daphnretusic acid (1) was obtained as an amorphous powder, having a molecular formula C19H20O8 deduced from the molecular ion peak appeared at m/z 376.1164 in HREIMS. The 1H NMR spectrum showed signals of five aromatic protons [d 6.67 (s, H5), 6.53 (s, H-8), 6.88 (d, J ¼ 8.1 Hz, H-50 ), 6.78 (dd, J ¼ 8.1, 1.7 Hz, H-60 ), 6.75 (d, J ¼ 1.7 Hz, H-20 )]; one methoxyl and two methylenic protons (H-1 and H-3a); and two methine protons at d 4.02 (s, H-4) and 2.50 (m, H-2), which indicated that the compound
Figure 1.
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Structure of daphnretusic acid (1).
is a lignan derivative. The 13C NMR, DEPT and HSQC experiments displayed signals of 19 carbons including downfield signals of carbonyl group C-2a (d 179.1), one oxygenated quaternary carbon (d 76.7), one oxygenated methylene (d 69.5), nine quaternary carbons, one methoxyl group, one aliphatic methylene, and two methine carbons, which further supported the aryltetrahydronaphthalene lignan skeleton. All the assignments of protons were further validated by 1H– 1H COSY analysis which showed correlations of multiplet protons at d 2.50 (H-2) with double-doublet protons at d 2.92 (1H, J ¼ 13.8, 4.5 Hz, H-1a) and triplet at d 3.04 (1H, J ¼ 13.8 Hz, H-1b), while the resonance at d 3.88 (1H, J ¼ 12.0 Hz, H-3ax) and 4.23 (1H, J ¼ 12.0 Hz, 3ay) appeared as two doublets, therefore suggesting that the hydroxymethylene (H-3a) was attached to quaternary carbon (C-3) but methylene protons (H-1) were connected with one methine proton (H-2) and one quaternary carbon (C-9). This confirmed that the compound is different from previously reported compounds [17,18]. These assignments (1) were also confirmed through HMBC correlations, in which the methylene protons at d 2.92 and 3.04 showed correlations with the carbons at d 179.1 (C-2a), 43.4 (C-2), 76.7 (C-3), 114.2 (C-8), 123.0 (C-9), and 130.5 (C-10), whereas hydroxymethylene protons at d 4.23 (H-3ax) and 3.88 (H-3ay) showed cross-peaks with C-3 at d 76.7 and C-4 at d 48.34, and a methine singlet at d 4.02 (H-4) showed HMBC correlations with the carbons at d 76.7 (C-3),
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123.0 (C-9), 133.3 (C-10 ), 113.0 (C-20 ), and 122.8 (C-60 ). These correlations confirmed that the hydroxyl group attached at C-3 while free carboxylic group occurred at C-2 position. The presence of methoxyl group at C-30 was confirmed through HMBC interactions (see Figure 2). The relative configuration of C-4 could be established on the basis of previously reported compounds in which H-4 (d 4.02) showed singlet only when dihedral angle of 908 between H-3 and H-4 [17,19] or C-4 is adjacent to quaternary carbons (C-3 and C-10). Stereochemistry of C-2 was further confirmed through Nuclear Overhauser enhanced and exchange spectroscopic (NOESY) correlations of H-2 with H-3a, which suggested that they were the same stereoisomer like tetralin [17,20]. Finally, on the basis of the above evidence, the structure of (1) was a new compound daphnretusic acid. The known isolated compounds are 5,7-dihydroxyflavone (2) [21], 7-hydroxyflavone (3) [21], 6-methoxyflavone (4) [22], (þ ) pinoresinol (5) [23], (þ ) sesamin (6) [24], and b-sitosterol 3-O-b-D -glucopyranoside (7) [25], respectively. All the physical and spectral data of these compounds were in close agreement with the already reported literature. The bioassay-guided fractionation of Daphne retusa has led to the isolation of potent urease inhibitors. The water, methanol, ethyl acetate, chloroform, and n-hexane soluble fractions of plant (airdried, powdered twigs of Daphne retusa) were tested in vitro for their urease
Figure 2.
Important HMBC correlations of 1.
inhibition activity. Compounds 2– 4 are flavonoids, but only compound 2 showed moderate activity (IC50 60.4 ^ 0.72 mM). The moderate activity of 2 might be associated with the hydroxyl group at C-5, facilitating its interaction with nickel via hydrogen bonding. Compound 4 has a methoxy group at C-6 hence lacking interactions with nickel; similarly hydroxyl groups found at C-7 in compound 3 remained recessive due to the absence of hydroxyl group at C-5 position. The a-chymotrypsin is a protease enzyme and digests protein debris of the ulcer; also it had been used for the treatment of peptic ulcer [16,26]. All compounds were inactive in case of a-chymotrypsin. 3. 3.1
Experimental General experimental procedures
Optical rotation was measured on a JASCO DIP-360 digital polarimeter (Maryland, USA). The melting points were recorded on Gallenkamp apparatus (Bristol, UK) and are uncorrected. The UV spectra were recorded on a Hitachi UV3200 spectrometer (Tokyo, Japan) with chloroform and methanol as solvent. IR spectra were recorded on Shimadzu IR460 spectrophotometer (Tokyo, Japan) using KBr disk. Mass spectra were recorded on a JEOL JMS-HX 110 spectrometer (Japan) with data system. The 1H NMR and 13C NMR spectra were recorded on Bruker AMX-400 instruments (France) in CDCl3 (ARMAR, Switzerland) and CD3OD by using tetramethyl silane as an internal standard. The chemical shift values are reported in ppm (d) units and the scalar coupling constants (J) are in Hz. Column chromatography was carried out by using silica gel of 70 – 230 and 230 – 400 mesh sizes. Aluminum sheets precoated with silica gel 60 F254 (20 £ 20 cm, 0.2 mm thick; E-Merck) were used for thin layer chromatography analysis to check the purity of compounds and were visualized under UV light (254 and
Journal of Asian Natural Products Research 366 nm) followed by ceric sulfate as spraying reagent (heating). All organic solvents and chemicals were obtained from Merck Scientific or otherwise mentioned in its respective portion.
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3.2
Plant material
The plant Daphne retusa Hemsl. (whole plant) was collected from Gilgit (Northern areas), Pakistan, in the summer season of 2005. The plant was identified by Mr Manzoor Ahmed, Plant Taxonomist, Department of Botany, Government Post Graduate College, Abbottabad. A voucher specimen (DR-004) was deposited in the herbarium of the department. 3.3
Extraction and isolation
The freshly collected plant material (whole plant, 22 kg) of D. retusa was shade dried (9 kg), ground, and extracted with methanol (M tedia-USA, 3 £ 15 liters, each for 10 days). The combined methanolic extract was evaporated under reduced pressure at room temperature to yield a crude residue (535 g). The whole extract was suspended in water and successively extracted with n-hexane (158 g), chloroform (M tedia-USA, 95 g), ethyl acetate (109 g), and water (85 g). The urease-active, chloroform-soluble fraction was subjected to column chromatography over silica gel and eluted with n-hexane, nhexane – chloroform, chloroform, and chloroform – methanol with a gradient increase in polarity to afford 25 subfractions. The sub-fractions (F1 – F5) were a binary mixture of two compounds that was re-chromatographed over silica gel and eluted with CHCl3:n-hexane (9.6:0.4) to afford compounds 2 –4 in pure form. The sub-fractions (F6 – F8) were re-chromatographed over silica gel and eluted with chloroform:MeOH (9.8:0.2) to afford compounds 5, 6, and 1, and then compound 7 was eluted with chloroform: MeOH (9.5:0.5).
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3.3.1 Daphnretusic acid 1 Amorphous powder; m.p. 164 – 1668C. [a]D 2 78 (MeOH; c 1.02). UV: lmax (MeOH) 206 (4.16), 226 (4.56), 278 (3.92) nm; IR (KBr) y max cm21: 3427 (OH), 3011 (CH), 1700, and 1620 –1400. For 1H NMR and 13C NMR spectral data, see Table 1. EIMS m/z (%): 376 [M]þ(15), 358 (7), 328 (14), 165 (8), 151 (29), 137 (100). HREIMS: m/z 376.1164 [M]þ (calcd for C19H20O8, 376.1158). 3.4
Urease inhibition assay
A reaction mixture containing 1 unit of urease enzyme (Jack bean urease) solution and 55 ml of buffers having 100 mM urea was incubated in the presence of 5 ml of test compounds (0.5 mM concentration) at 308C for 15 min in 96-well plates. Urease activity was evaluated by measuring ammonia production as described in the indophenol method [14]. Briefly, 45 ml each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 ml 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 Device, Sunnyvale, CA, USA). All reactions were performed three times in a final volume of 200 ml. The data (change in absorbance per minute) were processed with the help of Softmax Pro software (Molecular Device). All the assays were carried out at pH 8.2 (0.01 M K2HPO4 3H2O, 1 mM BDTA, and 0.01 M LiCl2). Percentage inhibitions were calculated from the formula 100 2 (OD testwell / ODcontrol) £ 100. Thiourea was used as the standard inhibitor of urease. 3.5
Chymotrypsin inhibition assay
Chymotrypsin (Beijing, China, 12 units/ml prepared in Tris– HCl buffer, pH 7.6) was pre-incubated along with test compounds (prepared to a final concentration of 7%
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Table 1. 1H NMR (CD3OD, 400 MHz) (d ppm) and spectral data for daphnretusic acid (1).
C NMR (CD3OD, 100 MHz) (d ppm)
Multiplicity
dC
dH
1
t
38.3
2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 2a 3a OCH3
d s d d s s s s s s d s s d d s t
43.4 76.7 48.3 111.6 145.3 144.2 114.2 123.0 130.5 133.3 113.0 147.8 145.2 116.7 122.8 179.1 69.5 55.7
2.92 (1H, dd, J ¼ 13.8, 4.5 Hz, H-1a), 3.04 (1H, t, J ¼ 13.8 Hz, H-1b) 2.50 (1H, m, H-2)
Carbons
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4.02 (s, H-4) 6.67 (s, H-5) 6.53 (s, H-8)
6.75 (d, J ¼ 1.7 Hz, H-20 ) 6.88 (d, J ¼ 8.1 Hz, H-50 ) 6.78 (dd, J ¼ 8.1, 1.7 Hz, H-60 ) 3.88 (d, J ¼ 12.0 Hz, H-3ax), 4.23 (d, J ¼ 12.0 Hz, H-3ay) 3.94 (3H, s, OCH3-30 )
dimethylsulfoxide) for 25 min at 308C. The solution of substrate (N-succinyl-phenylalanine-p-nitroanilide of Sigma-Aldrich, 0.4 mM) was added to the reaction mixture in order to initiate the enzymatic reaction. The observed absorbance was due to the release of p-nitroaniline, which was continuously monitored at 410 nm until a significant color change occurred [26].
3.6 Determination of IC50 values The concentrations of the test fractions and compounds, which retarded the hydrolysis of substrates by 50% (IC50), were evaluated by monitoring the effect of various concentrations of them in the assays on inhibition values. The IC50 values were then calculated using the EZ-fit Enzyme Kinetics program (Perrelia Scientific Inc., Amherst, USA).
4. Conclusion The development of new and safe enzyme inhibitors has led to the discovery of drugs.
Imidazoles, hydroxamic acids, thiols, and phosphazenes are established synthetic urease inhibitors, but very little knowledge is available on the role of natural products as urease inhibitors [15,16]. Owing to the widespread application of this enzyme, its inhibition by potent and specific sources could provide an invaluable addition for treatment of infections. Moreover, screening of natural products also contributes chemically.
Acknowledgments Authors of manuscript greatly acknowledge the financial support of the Dean, Faculty of Science, University of Karachi for this research.
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