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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Bioactive constituents from Croton sparsiflorus Morong a
a
a
Durre Shahwar , Naeem Ahmad , Asma Yasmeen , Muhammad a
ab
Akmal Khan , Sami Ullah
& Atta-ur Rahman
c
a
Research Lab. II, Department of Chemistry, Government College University, Lahore 54000, Pakistan b
Department of Chemistry, University of Gujrat, Hafiz Hayat Campus, Gujrat 50911, Pakistan c
HEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan Published online: 12 Aug 2014.
To cite this article: Durre Shahwar, Naeem Ahmad, Asma Yasmeen, Muhammad Akmal Khan, Sami Ullah & Atta-ur Rahman (2014): Bioactive constituents from Croton sparsiflorus Morong, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.947484 To link to this article: http://dx.doi.org/10.1080/14786419.2014.947484
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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Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.947484
SHORT COMMUNICATION Bioactive constituents from Croton sparsiflorus Morong Durre Shahwara*, Naeem Ahmada, Asma Yasmeena, Muhammad Akmal Khana, Sami Ullahab and Atta-ur Rahmanc
Downloaded by [Memorial University of Newfoundland] at 03:08 22 November 2014
a
Research Lab. II, Department of Chemistry, Government College University, Lahore 54000, Pakistan; Department of Chemistry, University of Gujrat, Hafiz Hayat Campus, Gujrat 50911, Pakistan; cHEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan b
(Received 22 April 2014; final version received 19 July 2014) Whole plant extracts of Croton sparsiflorus in methanol have shown significant enzyme inhibition and antioxidant activities. Bioassay-guided isolation of chloroform fraction at pH 3 resulted in the identification of crotsparinine (1) and crotsparine (2), while sparsiflorine (3) was purified from the chloroform fraction at pH 9. The structures of the compounds were confirmed through spectral analyses (EI-MS, 1H and 13C NMR). The isolated compounds 1 –3 exhibited remarkable enzyme inhibition activity with IC50 values 27.01 ^ 1.1, 22.26 ^ 1.0 and 18.02 ^ 1.3 mM in xanthine oxidase and 48.42 ^ 1.5, 48.05 ^ 1.4 and 7.42 ^ 1.0 mM in acetylcholine esterase assays, respectively. These compounds also showed potent radical scavenging and reducing properties in DPPH and FRAP assays, respectively. The present results suggest the validity of the traditional uses of C. sparsiflorus in rheumatism and gout. Furthermore, the isolated noraporphine alkaloids can be useful in the treatment of neurodegenerative diseases. Keywords: Croton sparsiflorus; enzyme inhibition; noraporphine alkaloids
1. Introduction Croton sparsiflorus Morong (family: Euphorbiaceae) is found growing as weed in Asia and South America. Although other species of genus Croton have been reported to be of significant ethnomedicinal use in Asia, very less research regarding biological activities has been reported on C. sparsiflorus (Salatino et al. 2007). Therefore, we carried out bioassay-guided fractionation of the aerial parts yielding noraporphine alkaloids, which represent the major class of compounds in C. sparsiflorus (Mehmood & Malik 2010; Damiano et al. 2011; Kumar et al. 2011). Free radicals are largely involved in oxidative stress that can initiate age-related and chronic diseases such as cardiovascular, diabetes, neurodegenerative, inflammation, Alzheimer’s and Parkinson’s (Marian et al. 2007). Alzheimer’s disease causes mental deterioration in elderly people. Current therapeutic strategy of this disease is based on the cholinergic hypothesis and specifically on acetylcholine esterase (AChE) inhibition (Krall et al. 1999; Terry & Buccafusco 2003). Xanthine oxidase (XO) oxidises into hypoxanthine/xanthine, which increases the level of uric acid in blood, leading to hyperuricaemia and ultimately to a painful inflammation known as gout (Carlos et al. 2006; Roger 2006). In view of the fact that the use of synthetic AChE inhibitors, e.g. donepezil and allopurinol for the cure of gout is associated with adverse side effects; the search of more safe and novel compounds with AChE and XO inhibitory activity has
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
2
D. Shahwar et al.
become a very important issue. This study was designed to evaluate in vitro XO, AChE inhibitory activity and antioxidant potential of extracts/fractions and the principal alkaloids crotsparinine (1), crotsparine (2) and sparsiflorine (3), extracted through bioassay-guided fractionation of C. sparsiflorus.
Downloaded by [Memorial University of Newfoundland] at 03:08 22 November 2014
2. Results and discussion Whole plant methanolic extract of C. sparsiflorus was dissolved in water and on further fractionation yielded CSH, CSCa, CSCb and CSE (Scheme S1). The results of AChE, XO and antioxidant activities of the extracts/fractions of C. sparsiflorus are shown in Table S1. Despite the exceptional results of the methanolic extract, further extraction of the crude extract was carried out at different pHs in order to separate the alkaloidal fraction and to determine their effectiveness in the bioassays. The fractions obtained at pH 3 and pH 9 were designated as CSCa and CSCb, respectively. Of the four fractions obtained by column chromatography (CC) of CSCa, CSCa-4 was rechromatographed using CC, yielding crotsparinine (1) and crotsparine (2), while sparsiflorine (3) was isolated through preparative TLC of CSCb. The structures of these compounds were assigned based on their spectral data (EI-MS, IH and 13C NMR) and comparison of the data reported in the literature (Bhakuni & Dhar 1968, 1969; Bhakuni et al. 1970). The purified compounds were assayed for the enzyme inhibition potential (XO and AChE) and antioxidant potential using DPPH (diphenyl picrylhydrazyle) and FRAP (ferric reducing antioxidant potential) assays. All the compounds showed remarkable DPPH radical scavenging activity with IC50 of 23.85 ^ 1.1 mM (1), 22.26 ^ 1.0 mM (2) and 19.78 ^ 1.9 mM (3), which showed that these compounds are more effective radical scavengers than BHT (IC50 ¼ 80.5 ^ 1.1 mM) taken as standard antioxidant. In the FRAP assay, compound 1 (185.5 ^ 1.6 mM equivalent to FeSO4·7H2O) more effectively reduced ferric ions (Fe3þ) than compound 2 (174.5 ^ 2.5 mM equivalent to FeSO4·7H2O) and compound 3 (147 ^ 1.2 mM equivalent to FeSO4·7H2O). The results are summarised in Table S2. It has been reported in the literature that semi-synthesised derivatives of natural boldine (a phanatherene-type aporphine alkaloid) showed antioxidant activity against reactive oxygen species (ROS) in the system of hypoxanthine-XO. The same compounds were inactive against XO. In these experiments, alkaloids with phenolic substituent displayed more powerful antioxidative activity than those containing methoxylated groups (Huang et al. 2009). Our results of XO-inhibition potential showed that all the three compounds inhibited the enzyme very strongly and greater than allupurinol (IC50 ¼ 64.19 ^ 1.5 mM). Sparsiflorine (3) was the most effective with IC50 of 18.02 ^ 1.3 mM, followed by crotsparine (2, IC50 ¼ 22.26 ^ 1.0 mM) and crotsparinine (1, IC50 ¼ 27.01 ^ 1.1 mM). Therefore, it can be suggested that compounds 1– 3 can become promising candidates for the development of novel and potent selective agents against gout. In the AChE inhibition assay, compound 3 was the most effective with IC50 of 7.42 ^ 1.0 mM followed by crotsparine (2, IC50 ¼ 48.05 ^ 1.4 mM) and sparsiflorine (1, IC50 ¼ 48.42 ^ 1.5 mM). Compound 3 demonstrated comparable inhibitory effect against AChE with the standard galanthamine (IC50 ¼ 2.7 ^ 1.5 mM). Remarkable results of sparsiflorine (3) indicated that the increase in the conjugation enhances p to p interaction, which is a primary requirement for aromatic pharmacophore to bind to peripheral anionic site (Milia´n et al. 2004). Structural resemblance of compounds 1 and 2 would be responsible for comparable results of the two compounds (Figure S3). In order to assess involvement of reduction of free radicals or ROS in the mechanism of AChE inhibition, the effects of the samples tested on the AChE activity were compared with their reducing abilities. The difference between ferric-reducing ability of compounds 1 –3 was
Natural Product Research
3
Downloaded by [Memorial University of Newfoundland] at 03:08 22 November 2014
remarkable as that between their enzyme inhibition potential. These results suggest that in the case of sparsiflorine (3), reducing potential towards Fe3þ ions has no correlation with the enzyme-inhibition potential and the inhibitory effects are possibly due to specific interaction of the compound with the enzyme. Our results suggest that the isolated compounds 1 – 3 from C. sparsiflorus possess significant antiradical potential and inhibited both the enzymes effectively comparable with standards. However, further research on these compounds can provide useful information about their active sites. Conclusion In this study, solvent extracts of C. sparsiflorus were analysed for their enzyme inhibition potential and antioxidant activity. The results of bioassay-guided purification of the active extracts suggested crotsparinine (1), crotsparine (2) and sparsiflorine (3) as the active components of this plant. However, further comprehensive investigations are required to determine structure – activity relationships of the active compounds. Supplementary material Experimental materials relating to this article are available online, alongside Scheme S1, Figures S1 –S3 and Tables S1 and S2. References Bhakuni DS, Dhar MM. 1968. Crotsparine, a new proaporphine alkaloid from Croton sparsiflorus Morong. Experientia. 24:10–11. Bhakuni DS, Dhar MM. 1969. Crotsparinine, a dihydroproaporphine alkaloid from Croton sparsiflorus. Experientia. 25:354. Bhakuni DS, Sheo S, Dhar MM. 1970. The alkaloids of Croton sparsiflorus. Phytochemistry. 9:2573–2580. Carlos DB, Maria JR, Isabel M, Jose GJM. 2006. Molybdenum and tungsten enzymes: the xanthine oxidase family. Curr Opin Chem Biol. 10:109–114. Damiano R, Alessandra G, Silvia M, Renato B, Guglielmo P, Ferruccio P, Laura S, Matteo R, Katia S, Gianni S. 2011. Chemical fingerprinting and bioactivity of Amazonian Ecuador Croton lechleri Mu¨ll. Arg. (Euphorbiaceae) stem bark essential oil: a new functional food ingredient. Food Chem. 126:837–848. Huang T, Yong-Biao W, Chi Z, Fang-Xian N, Wei Q, Shi-Liang H, Lin M, Zhi-Shu H, Lian-Quan G. 2009. Synthesis, biological evaluation and molecular modeling of oxoisoaporphine and oxoaporphine derivatives as new dual inhibitors of acetylcholinesterase/butyrylcholinesterase. Eur J Med Chem. 44:2523–2532. Krall WJ, Sramek JJ, Cutler NR. 1999. Cholinesterase inhibitors: a therapeutic strategy for Alzheimer disease. Ann Pharmacother. 33:441–450. Kumar RN, Murthy LJ, Sekar M, Jeevanandham S. 2011. Effects of ethanolic extracts of Croton sparsiflorus leaves on the liver of Swiss albino mice. Int J Pharm Biol Sci. 5:85–88. Marian V, Dieter JM, Mark TDC, Milan M, Joshua T. 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 39:44– 84. Mehmood R, Malik A. 2010. Isolation and characterization of crotosparsamide, a new cyclic nonapeptide from Croton sparsiflorus. Nat Prod Commun. 5:1885–1888. Milia´n L, Estelle´s R, Abarca B, Ballesteros R, Sanz MJ, Bla´zquez MA. 2004. Reactive oxygen species (ROS) generation inhibited by aporphine and phenanthrene alkaloids semi-synthesized from natural boldine. Chem Pharm Bull. 52:696–699. Roger HM. 2006. Xanthine oxidase: properties and physiological roles. Int Dairy J. 16:546–554. Salatino A, Maria L, Salatino F, Negri G. 2007. Traditional uses, chemistry and pharmacology of Croton species (Euphorbiaceae). J Braz Chem Soc. 18:11–33. Terry AVG, Buccafusco JJ. 2003. The cholinergic hypothesis of age and Alzheimer’s disease-related cognitive deficits: recent challenges and their implications for novel drug development. J Pharm Exp Ther. 306:821–827.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Bioactive constituents from Croton sparsiflorus Morong a
a
a
Durre Shahwar , Naeem Ahmad , Asma Yasmeen , Muhammad a
ab
Akmal Khan , Sami Ullah
& Atta-ur Rahman
c
a
Research Lab. II, Department of Chemistry, Government College University, Lahore 54000, Pakistan b
Department of Chemistry, University of Gujrat, Hafiz Hayat Campus, Gujrat 50911, Pakistan c
HEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan Published online: 12 Aug 2014.
To cite this article: Durre Shahwar, Naeem Ahmad, Asma Yasmeen, Muhammad Akmal Khan, Sami Ullah & Atta-ur Rahman (2014): Bioactive constituents from Croton sparsiflorus Morong, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.947484 To link to this article: http://dx.doi.org/10.1080/14786419.2014.947484
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 &
Downloaded by [Memorial University of Newfoundland] at 03:08 22 November 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.947484
SHORT COMMUNICATION Bioactive constituents from Croton sparsiflorus Morong Durre Shahwara*, Naeem Ahmada, Asma Yasmeena, Muhammad Akmal Khana, Sami Ullahab and Atta-ur Rahmanc
Downloaded by [Memorial University of Newfoundland] at 03:08 22 November 2014
a
Research Lab. II, Department of Chemistry, Government College University, Lahore 54000, Pakistan; Department of Chemistry, University of Gujrat, Hafiz Hayat Campus, Gujrat 50911, Pakistan; cHEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan b
(Received 22 April 2014; final version received 19 July 2014) Whole plant extracts of Croton sparsiflorus in methanol have shown significant enzyme inhibition and antioxidant activities. Bioassay-guided isolation of chloroform fraction at pH 3 resulted in the identification of crotsparinine (1) and crotsparine (2), while sparsiflorine (3) was purified from the chloroform fraction at pH 9. The structures of the compounds were confirmed through spectral analyses (EI-MS, 1H and 13C NMR). The isolated compounds 1 –3 exhibited remarkable enzyme inhibition activity with IC50 values 27.01 ^ 1.1, 22.26 ^ 1.0 and 18.02 ^ 1.3 mM in xanthine oxidase and 48.42 ^ 1.5, 48.05 ^ 1.4 and 7.42 ^ 1.0 mM in acetylcholine esterase assays, respectively. These compounds also showed potent radical scavenging and reducing properties in DPPH and FRAP assays, respectively. The present results suggest the validity of the traditional uses of C. sparsiflorus in rheumatism and gout. Furthermore, the isolated noraporphine alkaloids can be useful in the treatment of neurodegenerative diseases. Keywords: Croton sparsiflorus; enzyme inhibition; noraporphine alkaloids
1. Introduction Croton sparsiflorus Morong (family: Euphorbiaceae) is found growing as weed in Asia and South America. Although other species of genus Croton have been reported to be of significant ethnomedicinal use in Asia, very less research regarding biological activities has been reported on C. sparsiflorus (Salatino et al. 2007). Therefore, we carried out bioassay-guided fractionation of the aerial parts yielding noraporphine alkaloids, which represent the major class of compounds in C. sparsiflorus (Mehmood & Malik 2010; Damiano et al. 2011; Kumar et al. 2011). Free radicals are largely involved in oxidative stress that can initiate age-related and chronic diseases such as cardiovascular, diabetes, neurodegenerative, inflammation, Alzheimer’s and Parkinson’s (Marian et al. 2007). Alzheimer’s disease causes mental deterioration in elderly people. Current therapeutic strategy of this disease is based on the cholinergic hypothesis and specifically on acetylcholine esterase (AChE) inhibition (Krall et al. 1999; Terry & Buccafusco 2003). Xanthine oxidase (XO) oxidises into hypoxanthine/xanthine, which increases the level of uric acid in blood, leading to hyperuricaemia and ultimately to a painful inflammation known as gout (Carlos et al. 2006; Roger 2006). In view of the fact that the use of synthetic AChE inhibitors, e.g. donepezil and allopurinol for the cure of gout is associated with adverse side effects; the search of more safe and novel compounds with AChE and XO inhibitory activity has
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
2
D. Shahwar et al.
become a very important issue. This study was designed to evaluate in vitro XO, AChE inhibitory activity and antioxidant potential of extracts/fractions and the principal alkaloids crotsparinine (1), crotsparine (2) and sparsiflorine (3), extracted through bioassay-guided fractionation of C. sparsiflorus.
Downloaded by [Memorial University of Newfoundland] at 03:08 22 November 2014
2. Results and discussion Whole plant methanolic extract of C. sparsiflorus was dissolved in water and on further fractionation yielded CSH, CSCa, CSCb and CSE (Scheme S1). The results of AChE, XO and antioxidant activities of the extracts/fractions of C. sparsiflorus are shown in Table S1. Despite the exceptional results of the methanolic extract, further extraction of the crude extract was carried out at different pHs in order to separate the alkaloidal fraction and to determine their effectiveness in the bioassays. The fractions obtained at pH 3 and pH 9 were designated as CSCa and CSCb, respectively. Of the four fractions obtained by column chromatography (CC) of CSCa, CSCa-4 was rechromatographed using CC, yielding crotsparinine (1) and crotsparine (2), while sparsiflorine (3) was isolated through preparative TLC of CSCb. The structures of these compounds were assigned based on their spectral data (EI-MS, IH and 13C NMR) and comparison of the data reported in the literature (Bhakuni & Dhar 1968, 1969; Bhakuni et al. 1970). The purified compounds were assayed for the enzyme inhibition potential (XO and AChE) and antioxidant potential using DPPH (diphenyl picrylhydrazyle) and FRAP (ferric reducing antioxidant potential) assays. All the compounds showed remarkable DPPH radical scavenging activity with IC50 of 23.85 ^ 1.1 mM (1), 22.26 ^ 1.0 mM (2) and 19.78 ^ 1.9 mM (3), which showed that these compounds are more effective radical scavengers than BHT (IC50 ¼ 80.5 ^ 1.1 mM) taken as standard antioxidant. In the FRAP assay, compound 1 (185.5 ^ 1.6 mM equivalent to FeSO4·7H2O) more effectively reduced ferric ions (Fe3þ) than compound 2 (174.5 ^ 2.5 mM equivalent to FeSO4·7H2O) and compound 3 (147 ^ 1.2 mM equivalent to FeSO4·7H2O). The results are summarised in Table S2. It has been reported in the literature that semi-synthesised derivatives of natural boldine (a phanatherene-type aporphine alkaloid) showed antioxidant activity against reactive oxygen species (ROS) in the system of hypoxanthine-XO. The same compounds were inactive against XO. In these experiments, alkaloids with phenolic substituent displayed more powerful antioxidative activity than those containing methoxylated groups (Huang et al. 2009). Our results of XO-inhibition potential showed that all the three compounds inhibited the enzyme very strongly and greater than allupurinol (IC50 ¼ 64.19 ^ 1.5 mM). Sparsiflorine (3) was the most effective with IC50 of 18.02 ^ 1.3 mM, followed by crotsparine (2, IC50 ¼ 22.26 ^ 1.0 mM) and crotsparinine (1, IC50 ¼ 27.01 ^ 1.1 mM). Therefore, it can be suggested that compounds 1– 3 can become promising candidates for the development of novel and potent selective agents against gout. In the AChE inhibition assay, compound 3 was the most effective with IC50 of 7.42 ^ 1.0 mM followed by crotsparine (2, IC50 ¼ 48.05 ^ 1.4 mM) and sparsiflorine (1, IC50 ¼ 48.42 ^ 1.5 mM). Compound 3 demonstrated comparable inhibitory effect against AChE with the standard galanthamine (IC50 ¼ 2.7 ^ 1.5 mM). Remarkable results of sparsiflorine (3) indicated that the increase in the conjugation enhances p to p interaction, which is a primary requirement for aromatic pharmacophore to bind to peripheral anionic site (Milia´n et al. 2004). Structural resemblance of compounds 1 and 2 would be responsible for comparable results of the two compounds (Figure S3). In order to assess involvement of reduction of free radicals or ROS in the mechanism of AChE inhibition, the effects of the samples tested on the AChE activity were compared with their reducing abilities. The difference between ferric-reducing ability of compounds 1 –3 was
Natural Product Research
3
Downloaded by [Memorial University of Newfoundland] at 03:08 22 November 2014
remarkable as that between their enzyme inhibition potential. These results suggest that in the case of sparsiflorine (3), reducing potential towards Fe3þ ions has no correlation with the enzyme-inhibition potential and the inhibitory effects are possibly due to specific interaction of the compound with the enzyme. Our results suggest that the isolated compounds 1 – 3 from C. sparsiflorus possess significant antiradical potential and inhibited both the enzymes effectively comparable with standards. However, further research on these compounds can provide useful information about their active sites. Conclusion In this study, solvent extracts of C. sparsiflorus were analysed for their enzyme inhibition potential and antioxidant activity. The results of bioassay-guided purification of the active extracts suggested crotsparinine (1), crotsparine (2) and sparsiflorine (3) as the active components of this plant. However, further comprehensive investigations are required to determine structure – activity relationships of the active compounds. Supplementary material Experimental materials relating to this article are available online, alongside Scheme S1, Figures S1 –S3 and Tables S1 and S2. References Bhakuni DS, Dhar MM. 1968. Crotsparine, a new proaporphine alkaloid from Croton sparsiflorus Morong. Experientia. 24:10–11. Bhakuni DS, Dhar MM. 1969. Crotsparinine, a dihydroproaporphine alkaloid from Croton sparsiflorus. Experientia. 25:354. Bhakuni DS, Sheo S, Dhar MM. 1970. The alkaloids of Croton sparsiflorus. Phytochemistry. 9:2573–2580. Carlos DB, Maria JR, Isabel M, Jose GJM. 2006. Molybdenum and tungsten enzymes: the xanthine oxidase family. Curr Opin Chem Biol. 10:109–114. Damiano R, Alessandra G, Silvia M, Renato B, Guglielmo P, Ferruccio P, Laura S, Matteo R, Katia S, Gianni S. 2011. Chemical fingerprinting and bioactivity of Amazonian Ecuador Croton lechleri Mu¨ll. Arg. (Euphorbiaceae) stem bark essential oil: a new functional food ingredient. Food Chem. 126:837–848. Huang T, Yong-Biao W, Chi Z, Fang-Xian N, Wei Q, Shi-Liang H, Lin M, Zhi-Shu H, Lian-Quan G. 2009. Synthesis, biological evaluation and molecular modeling of oxoisoaporphine and oxoaporphine derivatives as new dual inhibitors of acetylcholinesterase/butyrylcholinesterase. Eur J Med Chem. 44:2523–2532. Krall WJ, Sramek JJ, Cutler NR. 1999. Cholinesterase inhibitors: a therapeutic strategy for Alzheimer disease. Ann Pharmacother. 33:441–450. Kumar RN, Murthy LJ, Sekar M, Jeevanandham S. 2011. Effects of ethanolic extracts of Croton sparsiflorus leaves on the liver of Swiss albino mice. Int J Pharm Biol Sci. 5:85–88. Marian V, Dieter JM, Mark TDC, Milan M, Joshua T. 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 39:44– 84. Mehmood R, Malik A. 2010. Isolation and characterization of crotosparsamide, a new cyclic nonapeptide from Croton sparsiflorus. Nat Prod Commun. 5:1885–1888. Milia´n L, Estelle´s R, Abarca B, Ballesteros R, Sanz MJ, Bla´zquez MA. 2004. Reactive oxygen species (ROS) generation inhibited by aporphine and phenanthrene alkaloids semi-synthesized from natural boldine. Chem Pharm Bull. 52:696–699. Roger HM. 2006. Xanthine oxidase: properties and physiological roles. Int Dairy J. 16:546–554. Salatino A, Maria L, Salatino F, Negri G. 2007. Traditional uses, chemistry and pharmacology of Croton species (Euphorbiaceae). J Braz Chem Soc. 18:11–33. Terry AVG, Buccafusco JJ. 2003. The cholinergic hypothesis of age and Alzheimer’s disease-related cognitive deficits: recent challenges and their implications for novel drug development. J Pharm Exp Ther. 306:821–827.
