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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
Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots a
a
b
a
Abdur Rauf , Ghias Uddin , Haroon Khan , Mohammad Arfan & Bina S. Siddiqui
c
a
Institute of Chemical Sciences, University of Peshawar, K.P.K Peshawar, Pakistan b
Department of Pharmacy, Abdul Wali Khan University, Mardan 23200, Pakistan
Click for updates
c
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan Published online: 23 Feb 2015.
To cite this article: Abdur Rauf, Ghias Uddin, Haroon Khan, Mohammad Arfan & Bina S. Siddiqui (2015): Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1013957 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1013957
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,
Downloaded by [Universite Laval] at 16:19 04 March 2015
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1013957
SHORT COMMUNICATION Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots Abdur Raufa*, Ghias Uddina, Haroon Khanb, Mohammad Arfana and Bina S. Siddiquic a
Institute of Chemical Sciences, University of Peshawar, K.P.K Peshawar, Pakistan; bDepartment of Pharmacy, Abdul Wali Khan University, Mardan 23200, Pakistan; cH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
Downloaded by [Universite Laval] at 16:19 04 March 2015
(Received 17 December 2014; final version received 28 January 2015)
HO Beta-Sitosterol (3) O O O Isolation
OH
OH OH O
O O OH Hydroxydiospyrin (7)
7 methyl juglone (2)
HO Lupeol (1)
O OH O OH OH
O
O O
HO
HO Betulinic acid (5)
Stigmasterol (4)
Diospyrin (6)
The aim of this study was to explore the extract/fractions and compounds of Diospyros lotus against various Gram-positive and Gram-negative bacteria strain. The results showed marked susceptibility of extract and its fractions against test pathogens. Among them, chloroform fraction was most dominant and effective against all tested bacteria. The chloroform fraction was subjected to column chromatography which led to the isolation of lupeol (1), 7-methyljuglone (2), b-sitosterol (3), stigmasterol (4), betulinic acid (5), diospyrin (6) and 8-hydroxyisodiospyrin (7). Among the isolated compounds, betulinic acid (5) showed significant activity against most of the tested pathogen. In conclusion, our study validated the traditional uses of the plant in the treatment of infectious diseases which was also strongly supported by the isolated compound, betulinic acid (5). Keywords: Diospyros lotus; Ebenaceae; phytoconstituents; antibacterial activity
1. Introduction The genus Diospyros (Ebenaceae) consists of woody shrubs and trees distributed in the tropical and subtropical regions of the world. Around 500 species are known worldwide of which 24 species are native to India (Uddin et al. 2013). Diospyros lotus L. is a deciduous tree, growing in China and Asia. D. lotus has been cultivated for its edible fruits. The fruits of D. lotus is used as a sedative, astringent, nutritive, antiseptic, antidiabetic, antitumour, astringent, laxative, nutritive and as a febrifuge and for the treatment of constipation (Uddin et al. 2011, 2014). D. lotus fruits are used to for the treatment of diarrhoea, dry coughs and hypertension.
*Corresponding author. Emails: [email protected], [email protected] q 2015 Taylor & Francis
2
A. Rauf et al.
Phytochemical studies have been previously carried out on many Diospyros species and have revealed the widespread presence of naphthoquinones and naphthalene derivatives, dimeric naphthoquinones and lupane triterpenes (Tezuka et al. 1973; Rauf et al. 2014). Chemical investigation of the fruits led to the identification of some fatty acids, sugars phenolic compounds and non-volatile acids in D. lotus (Loizzo et al. 2009; Rashed et al. 2012). This article deals with the antibacterial activity of crude extract and subsequent solvent fractions of D. lotus against various bacteria. The chloroform fraction of the plant led to the isolation of seven different compounds followed by their activity against the same pathogens.
Downloaded by [Universite Laval] at 16:19 04 March 2015
2. Results and discussion 2.1. Effect of extract/fractions against test bacteria The results of crude extract and subsequent solvent fractions of D. lotus against tested bacteria are displayed in Table S1 (S1 online only). The extract/fractions of the plant were susceptible against various Gram-positive and Gram-negative bacteria. The crude extract showed antibacterial activity against all tested bacterial, however, most against Bacillus subtilis. Upon fractionation, chloroform was the most active fraction with marked activity against all Grampositive bacteria (B. subtilis, Staphylococcus aureus and Staphylococcus epidermidis). With the exception of Escherichia coli, the ethyl acetate fraction of the plant followed the similar trend of sensitive. However, hexane fraction did not show impressive activity. 2.2. Effect of isolated compounds (1 – 7) against test bacteria The antibacterial activity of isolated compounds (1– 7) is shown in Table S2 (S2 online only). According to the results, compound 5 (betulinic acid) was active against most of the tested bacteria. However, the rest of the compounds exhibited selectively showed activity one or two bacteria. This study revealed broad-spectrum activity of the extract/fractions of D. lotus against various pathogenic bacteria that was followed by the isolated compounds (1– 7) from the chloroform-soluble fraction. It has been extensively recognised globally that irrational usage of antibiotics is responsible for the proliferation of pathogenic bacteria with increasing resistance to various classes of antibiotics. Untimely, this has led to a progressive loss in the therapeutic effectiveness of antibiotic at a rate that varies with the complexity of the required mutation(s), the ease of clonal spread or the rate of horizontal gene transfer (Walter et al. 2011; Khan et al. 2012). The prevalence of infections due to multidrug-resistant pathogens has shown dramatic increase around the globe (Espinoza et al. 2008; Eom et al. 2013). Many new antibiotics have been introduced to combat with such bacterial infections but multiple-resistance genes have appeared rapidly among bacteria which developed resistance to those antibiotics. Nature has been a source of medicinal agents for thousands of years. An impressive number of modern drugs has been isolated from medicinal plants including antimicrobial (Habtemariam 2003; Israr et al. 2012). It is also assumed that these plants-based antibacterial may possibly have the novel and different mechanism(s) of action and thus could be more effective especially in the reduction/prevention of antimicrobial resistance. In our study on the crude extract/fractions of D. lotus, we observed strong antibacterial action on various Gram-positive and Gram-negative tested bacteria. Similarly, the isolated compounds were also subjected to the same test in which compound 5 (betulinic acid) exhibited marked sensitivity against the tested pathogens. Based on the results, we confidently recommend this plant species for the further isolation of secondary metabolites as well as the synthesis of various derivatives of betulinic acid in order to discover molecules of clinical applications.
Natural Product Research
3
3. Conclusion In summary, the crude extract/fractions of D. lotus possess profound antibacterial activity against different Gram-positive and Gram-negative bacteria and therefore validated the traditional uses of the plant in the treatment of various infections. The study was also complimented by broad-spectrum activity of isolated compound, betulinic acid. Thus, it is a strong candidate for further detailed studies to get more effective lead compound(s) against such pathogens. Supplementary material Experimental details relating to this paper along with Tables S1 – S4 and Figure S1 are available online.
Downloaded by [Universite Laval] at 16:19 04 March 2015
Acknowledgements The authors are grateful for the financial support provided by Higher Education Commission of Pakistan [grant number 112-26510-2PS1-258].
Conflict of interest We declare that we have no conflict of interest.
References Eom S-H, Kim D-H, Lee S-H, Yoon N-Y, Kim JH, Kim TH, Chung Y-H, Kim S-B, Kim Y-M, Kim H-W, et al. 2013. In vitro antibacterial activity and synergistic antibiotic effects of phlorotannins isolated from Eisenia bicyclis against methicillin-resistant Staphylococcus aureus. Phytother Res. 27:1260–1264. doi:10.1002/ptr.4851. Espinoza C, Viniegra G, Loera O, Heredia G, Trigos A. 2008. Antibacterial activity against plant pathogens by crude extracts and pure compounds from Idriella sp. Revista Mexicana De Micologia. 26:9–15. Habtemariam S. 2003. In vitro antileishmanial effects of antibacterial diterpenes from two Ethiopian Premna species: P. schimperi and P. oligotricha. BMC Pharmacol. 3:6–10. doi:10.1186/1471-2210-3-6. Israr F, Hassan F, Naqvi B, Azhar I, Jabeen S, Hasan S. 2012. Report: studies on antibacterial activity of some traditional medicinal plants used in folk medicine. Pak J Pharm Sci. 25:669–674. Khan H, Khan Murad A, Abdullah. 2012. Antibacterial, antioxidant and cytotoxic studies of total saponin, alkaloid and sterols contents of decoction of Joshanda: components identification through TLC. Toxicol Ind Health. doi:10. 1177/0748233712468023. Loizzo MR, Said A, Tundis R, Hawas UW, Rashed K, Menichini F, Frega NG, Menichini F. 2009. Antioxidant and antiproliferative activity of Diospyros lotus L. extract and isolated compounds. Plant Food Hum Nutr. 64:264–270. doi:10.1007/s11130-009-0133-0. Rashed K, Zhang X, Luo M, Zheng Y. 2012. Anti-HIV-1 activity of phenolic compounds isolated from Diospyros lotus fruits. Phytopharmacology. 3:199–207. Rauf A, Uddin G, Siddiqui BS, Muhammad N, Khan H. 2014. Antipyretic and antinociceptive activity of Diospyros lotus L. in animals. Asian-Pac J Trop Biomed. 4:S382–386. Tezuka M, Takahashi C, Kuroyanagi M, Satake M, Yoshihira K, Natori S. 1973. New naphthoquinones from Diospyros. Phytochem. 12(1):175–183. doi:10.1016/S0031-9422(00)84643-9. Uddin G, Rauf A, Siddiqui BS, Arfan M, Rahman IU. 2013. Proximate chemical composition and antimicrobial activities of fixed oils from Diospyros lotus L. Med Chem. 3:282–285. Uddin G, Rauf A, Siddiqui BS, Muhammad N, Khan A, Shah SUA. 2014. Anti-nociceptive, anti-inflammatory and sedative activities of the extracts and chemical constituents of Diospyros lotus L.. Phytomedicine. 21:954–959. doi:10.1016/j.phymed.2014.03.001. Uddin G, Rauf A, Siddiqui BS, Shah SQ. 2011. Preliminary comparative phytochemical screening of Diospyros lotus Stewart. Middle-East J Sci Res. 10:78–81. Walter C, Shinwari ZK, Afzal I, Malik RN. 2011. Antibacterial activity in herbal products used in Pakistan. Pak J Bot. 43:155–162.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots a
a
b
a
Abdur Rauf , Ghias Uddin , Haroon Khan , Mohammad Arfan & Bina S. Siddiqui
c
a
Institute of Chemical Sciences, University of Peshawar, K.P.K Peshawar, Pakistan b
Department of Pharmacy, Abdul Wali Khan University, Mardan 23200, Pakistan
Click for updates
c
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan Published online: 23 Feb 2015.
To cite this article: Abdur Rauf, Ghias Uddin, Haroon Khan, Mohammad Arfan & Bina S. Siddiqui (2015): Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1013957 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1013957
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,
Downloaded by [Universite Laval] at 16:19 04 March 2015
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1013957
SHORT COMMUNICATION Bioassay-guided isolation of antibacterial constituents from Diospyros lotus roots Abdur Raufa*, Ghias Uddina, Haroon Khanb, Mohammad Arfana and Bina S. Siddiquic a
Institute of Chemical Sciences, University of Peshawar, K.P.K Peshawar, Pakistan; bDepartment of Pharmacy, Abdul Wali Khan University, Mardan 23200, Pakistan; cH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
Downloaded by [Universite Laval] at 16:19 04 March 2015
(Received 17 December 2014; final version received 28 January 2015)
HO Beta-Sitosterol (3) O O O Isolation
OH
OH OH O
O O OH Hydroxydiospyrin (7)
7 methyl juglone (2)
HO Lupeol (1)
O OH O OH OH
O
O O
HO
HO Betulinic acid (5)
Stigmasterol (4)
Diospyrin (6)
The aim of this study was to explore the extract/fractions and compounds of Diospyros lotus against various Gram-positive and Gram-negative bacteria strain. The results showed marked susceptibility of extract and its fractions against test pathogens. Among them, chloroform fraction was most dominant and effective against all tested bacteria. The chloroform fraction was subjected to column chromatography which led to the isolation of lupeol (1), 7-methyljuglone (2), b-sitosterol (3), stigmasterol (4), betulinic acid (5), diospyrin (6) and 8-hydroxyisodiospyrin (7). Among the isolated compounds, betulinic acid (5) showed significant activity against most of the tested pathogen. In conclusion, our study validated the traditional uses of the plant in the treatment of infectious diseases which was also strongly supported by the isolated compound, betulinic acid (5). Keywords: Diospyros lotus; Ebenaceae; phytoconstituents; antibacterial activity
1. Introduction The genus Diospyros (Ebenaceae) consists of woody shrubs and trees distributed in the tropical and subtropical regions of the world. Around 500 species are known worldwide of which 24 species are native to India (Uddin et al. 2013). Diospyros lotus L. is a deciduous tree, growing in China and Asia. D. lotus has been cultivated for its edible fruits. The fruits of D. lotus is used as a sedative, astringent, nutritive, antiseptic, antidiabetic, antitumour, astringent, laxative, nutritive and as a febrifuge and for the treatment of constipation (Uddin et al. 2011, 2014). D. lotus fruits are used to for the treatment of diarrhoea, dry coughs and hypertension.
*Corresponding author. Emails: [email protected], [email protected] q 2015 Taylor & Francis
2
A. Rauf et al.
Phytochemical studies have been previously carried out on many Diospyros species and have revealed the widespread presence of naphthoquinones and naphthalene derivatives, dimeric naphthoquinones and lupane triterpenes (Tezuka et al. 1973; Rauf et al. 2014). Chemical investigation of the fruits led to the identification of some fatty acids, sugars phenolic compounds and non-volatile acids in D. lotus (Loizzo et al. 2009; Rashed et al. 2012). This article deals with the antibacterial activity of crude extract and subsequent solvent fractions of D. lotus against various bacteria. The chloroform fraction of the plant led to the isolation of seven different compounds followed by their activity against the same pathogens.
Downloaded by [Universite Laval] at 16:19 04 March 2015
2. Results and discussion 2.1. Effect of extract/fractions against test bacteria The results of crude extract and subsequent solvent fractions of D. lotus against tested bacteria are displayed in Table S1 (S1 online only). The extract/fractions of the plant were susceptible against various Gram-positive and Gram-negative bacteria. The crude extract showed antibacterial activity against all tested bacterial, however, most against Bacillus subtilis. Upon fractionation, chloroform was the most active fraction with marked activity against all Grampositive bacteria (B. subtilis, Staphylococcus aureus and Staphylococcus epidermidis). With the exception of Escherichia coli, the ethyl acetate fraction of the plant followed the similar trend of sensitive. However, hexane fraction did not show impressive activity. 2.2. Effect of isolated compounds (1 – 7) against test bacteria The antibacterial activity of isolated compounds (1– 7) is shown in Table S2 (S2 online only). According to the results, compound 5 (betulinic acid) was active against most of the tested bacteria. However, the rest of the compounds exhibited selectively showed activity one or two bacteria. This study revealed broad-spectrum activity of the extract/fractions of D. lotus against various pathogenic bacteria that was followed by the isolated compounds (1– 7) from the chloroform-soluble fraction. It has been extensively recognised globally that irrational usage of antibiotics is responsible for the proliferation of pathogenic bacteria with increasing resistance to various classes of antibiotics. Untimely, this has led to a progressive loss in the therapeutic effectiveness of antibiotic at a rate that varies with the complexity of the required mutation(s), the ease of clonal spread or the rate of horizontal gene transfer (Walter et al. 2011; Khan et al. 2012). The prevalence of infections due to multidrug-resistant pathogens has shown dramatic increase around the globe (Espinoza et al. 2008; Eom et al. 2013). Many new antibiotics have been introduced to combat with such bacterial infections but multiple-resistance genes have appeared rapidly among bacteria which developed resistance to those antibiotics. Nature has been a source of medicinal agents for thousands of years. An impressive number of modern drugs has been isolated from medicinal plants including antimicrobial (Habtemariam 2003; Israr et al. 2012). It is also assumed that these plants-based antibacterial may possibly have the novel and different mechanism(s) of action and thus could be more effective especially in the reduction/prevention of antimicrobial resistance. In our study on the crude extract/fractions of D. lotus, we observed strong antibacterial action on various Gram-positive and Gram-negative tested bacteria. Similarly, the isolated compounds were also subjected to the same test in which compound 5 (betulinic acid) exhibited marked sensitivity against the tested pathogens. Based on the results, we confidently recommend this plant species for the further isolation of secondary metabolites as well as the synthesis of various derivatives of betulinic acid in order to discover molecules of clinical applications.
Natural Product Research
3
3. Conclusion In summary, the crude extract/fractions of D. lotus possess profound antibacterial activity against different Gram-positive and Gram-negative bacteria and therefore validated the traditional uses of the plant in the treatment of various infections. The study was also complimented by broad-spectrum activity of isolated compound, betulinic acid. Thus, it is a strong candidate for further detailed studies to get more effective lead compound(s) against such pathogens. Supplementary material Experimental details relating to this paper along with Tables S1 – S4 and Figure S1 are available online.
Downloaded by [Universite Laval] at 16:19 04 March 2015
Acknowledgements The authors are grateful for the financial support provided by Higher Education Commission of Pakistan [grant number 112-26510-2PS1-258].
Conflict of interest We declare that we have no conflict of interest.
References Eom S-H, Kim D-H, Lee S-H, Yoon N-Y, Kim JH, Kim TH, Chung Y-H, Kim S-B, Kim Y-M, Kim H-W, et al. 2013. In vitro antibacterial activity and synergistic antibiotic effects of phlorotannins isolated from Eisenia bicyclis against methicillin-resistant Staphylococcus aureus. Phytother Res. 27:1260–1264. doi:10.1002/ptr.4851. Espinoza C, Viniegra G, Loera O, Heredia G, Trigos A. 2008. Antibacterial activity against plant pathogens by crude extracts and pure compounds from Idriella sp. Revista Mexicana De Micologia. 26:9–15. Habtemariam S. 2003. In vitro antileishmanial effects of antibacterial diterpenes from two Ethiopian Premna species: P. schimperi and P. oligotricha. BMC Pharmacol. 3:6–10. doi:10.1186/1471-2210-3-6. Israr F, Hassan F, Naqvi B, Azhar I, Jabeen S, Hasan S. 2012. Report: studies on antibacterial activity of some traditional medicinal plants used in folk medicine. Pak J Pharm Sci. 25:669–674. Khan H, Khan Murad A, Abdullah. 2012. Antibacterial, antioxidant and cytotoxic studies of total saponin, alkaloid and sterols contents of decoction of Joshanda: components identification through TLC. Toxicol Ind Health. doi:10. 1177/0748233712468023. Loizzo MR, Said A, Tundis R, Hawas UW, Rashed K, Menichini F, Frega NG, Menichini F. 2009. Antioxidant and antiproliferative activity of Diospyros lotus L. extract and isolated compounds. Plant Food Hum Nutr. 64:264–270. doi:10.1007/s11130-009-0133-0. Rashed K, Zhang X, Luo M, Zheng Y. 2012. Anti-HIV-1 activity of phenolic compounds isolated from Diospyros lotus fruits. Phytopharmacology. 3:199–207. Rauf A, Uddin G, Siddiqui BS, Muhammad N, Khan H. 2014. Antipyretic and antinociceptive activity of Diospyros lotus L. in animals. Asian-Pac J Trop Biomed. 4:S382–386. Tezuka M, Takahashi C, Kuroyanagi M, Satake M, Yoshihira K, Natori S. 1973. New naphthoquinones from Diospyros. Phytochem. 12(1):175–183. doi:10.1016/S0031-9422(00)84643-9. Uddin G, Rauf A, Siddiqui BS, Arfan M, Rahman IU. 2013. Proximate chemical composition and antimicrobial activities of fixed oils from Diospyros lotus L. Med Chem. 3:282–285. Uddin G, Rauf A, Siddiqui BS, Muhammad N, Khan A, Shah SUA. 2014. Anti-nociceptive, anti-inflammatory and sedative activities of the extracts and chemical constituents of Diospyros lotus L.. Phytomedicine. 21:954–959. doi:10.1016/j.phymed.2014.03.001. Uddin G, Rauf A, Siddiqui BS, Shah SQ. 2011. Preliminary comparative phytochemical screening of Diospyros lotus Stewart. Middle-East J Sci Res. 10:78–81. Walter C, Shinwari ZK, Afzal I, Malik RN. 2011. Antibacterial activity in herbal products used in Pakistan. Pak J Bot. 43:155–162.
